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Biological Laboratory Library

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Presented by

Prentice-Hall, Inc.
He-K York City

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GENERAL BIOCHEMISTRY

PRENTICE-HALL CHEMISTRY SERIES
Wendell M. Latimer, Ph.D., Editor

GENERAL
BIOCHEMISTRY

by

WILLIAM H. PETERSON, Ph.D.

Emeritus Professor of Biochemistry
University of W isconsin, Madison

FRANK M. STRONG, Ph.D.

Professor of Biochemistry
University of Wisconsin, Madison

r V

New York PRENTICE-HALL, INC. 1953

Copyright, 1953, by Prentice-Hall, Inc., 70 Fifth Avenue, New ■
York. All rights resei-ved. No part of this book may be re-
produced in any form, by mimeograph or any other means, ,
without permission in writing from the publishers. Library of ]
Congress Catalog Card Number : 53-8022. j

Printed in the United States op America

PREFACE

This book considers the chemical activities not only of animals but
alsi) those of plants and microorganisms. It aims to be a complete,
though brief, treatise on the whole field of biochemistry, stressing the
most important features of the subject.

The first part deals with the materials of the cell, and the second with
tiie functions of the cell. Emphasis, however, has been placed on the
dynamic aspects of biochemistry as well as on its material features.
This purpose inevitably leads to a consideration of complex phenomena.
To make such phenomena understandable is no easy task, but the attempt
has been made.

The subject matter is by no means beyond the comprehension of the
reader with only a general chemistry background, though best appre-
ciated and understood by the reader with a knowledge of organic chem-
istry. In view of the increased coverage (chapters on Nucleic Acids,
Hormones, and Biological Energetics) and the particular emphasis which
has been placed on metabolic reactions (chapters on Plant Metabolism,
Animal INIetabolism, and Metabolism of Microorganisms) , the present
work is well suited to more advanced readers. By careful selection of
chapters, the book should also prove useful to those interested in agri-
culture and home economics.

The authors are indebted to their colleagues. Professors Casida, John-
son, Lardy, Meyer, Plant, Potter, Stahmann, and Williams for reading
one or more chapters of the manuscript and making many valuable sug-
gestions and criticisms of the book. They are doubly indebted to Pro-
fessor Burris for his chapter on Plant Metabolism, and to Professor
Plant for the two chapters on Digestion and Enzymes. The authors are
grateful to Dr. Mary Shine Peterson for the preparation of Tables 3-1,
4-2, 5-1, A-1, A-2, and A-3, and for critical reading of many of the
chapters in the book.

In making these acknowledgments, the authors in no sense imply
that errors of omission and commission are to be charged to those named.
We apply to ourselves alone Byron's apostrophe to the ocean, "Upon
the watery plain, the wrecks are all thy deed."

\y. H. Peterson

F. M. Strong

ACKNOWLEDGMENTS

The authors gratefully acknowledge permissions granted to
reproduce illustrations appearing in this book, as follows:

Color plates I and II and Figures 7-2, 8-1, 8-2, 8-3, 8-4,
8-5, 15-9, and 15-10 reproduced from Hunger Signs in
Crops, revised edition, i)ublished by the American Society
of Agronomy and The National Fertilizer Association,
Washington, D. C, 1949.

Color plate III reproduced from Clinical Nutrition, by
JoUiffe, Tisdall, and Cannon and published by Paul B.
Hoeber, Inc., New York, 1950.

Color plate IV reproduced from Crystalline Vitainin Bj^
U.S.P., published by Merck & Co., Inc., Rahway, N. J.,
1951.

TABLE OF CONTENTS

1. Introduction 1

2. Water 8

3. Carbohydrates ■ 19

4. LiPiDES (Fats and Related Substances) 71

5. Proteins 103

6. Nucleoproteins, Nucleic Acids and Related

Substances 150

7. Acidity 161

8. Biochemically Important Mineral Elements 176

9. Vitamins 200

10. Enzymes 260

11. Hormones 286

12. Digestion 311

13. Animal Metabolism 323

14. Metabolism of Microorganisms 357

15. Plant Metabolism 387

16. Biological Energetics J^13
Appendix: Composition and Energy Value of Foods 433
Index 4^9

67621

IX

Chapter 1 /S^ >0^s~^^O>

tV^^fCTiN?!

INTRODUCTION \5 \ ^^S^^ ^ I ^

The living world

Biochemistry, as the name implies, means the chemistry of living
things. Obviously such a meaning includes the chemistry of plants and
microorganisms, as well as animals. The first two groups are indispensa-
ble to a living world, but the third is not. Although a living world com-
posed only of plants and microorganisms would be unfamiliar to us, it
would be adequate to maintain a balance between the synthetic processes
of the plant and the degradative processes of microorganisms. Put in
other terms, the carbon and nitrogen cycles in nature could be kept in
balance without the help of animals. The, latter are superimposed upon
the plants and microorganisms; and man, because of his dominant posi-
tion in the living world, places himself at its center.

The brief phrase, "chemistry of living things," covers a vast field of
subject matter. It includes, in the first place, the chemical make-up of all
the individual substances of which living tissues are composed. These
substances are extraordinarily numerous. A single cell of the simj^lest
type contains scores, probably hundreds, of different chemical substances
— no one knows how many in any particular organism. Furthermore,
many of these substances, or compounds as the chemist prefers to call
them, are extremely complex. Whole classes of biological compounds
are so involved that, even today, the exact structural formula of no
single member is known; prime examples are the proteins and nucleic
acids. Quantitatively, the most important single constituent is water.
Everything else is classified as dry matter or solids, which consist mostly
of organic compounds (substances containing carbon), although many
inorganic substances are present in small amounts.

Secondly, the "chemistry of living things" includes whatever chemical
changes the above substances undergo as the organism grows, reproduces,
absorbs and uses food, excretes waste products, and in general carries
out the activities incidental to being and remaining alive. The sum
total of all these chemical processes and the chemical compounds involved
in them is the living organism. The individual at any moment is a
dynamic balance between opposing processes of building up and break-
ing down, of taking in and throwing off, just as a lake is the resultant
of the inflow and outflow of its waters.

1

INTRODUCTION

Objectives and methods of biochemistry

The ultimate objectives of the science of biochemistry are a complete
knowledge of the structure and properties of all chemical compounds
present in living things and a complete understanding of the chemical
reactions they undergo both in health and disease. Usually, knowledge
of materials must first be obtained before much can be learned about
their function. At the present time the chief types of organic substance
in most biological materials are fairly well known. These major com-
ponents are the carbohydrai,es, fats and proteins. However, it has be-
come increasingly clear during the last few decades that many compounds,
e.g., vitamins and hormones, normally present in living cells in only
very small amounts often play important physiological roles. An im-
pressive number of these compounds is now known, but many more cer-
tainly remain to be studied. The development of our knowledge of
metabolism is even more recent and incomplete. Some of the processes
involving food utilization and energy production are emerging into focus,
but as yet only the barest beginning has been made in finding out what
chemical reactions occur during the normal functioning of living things.

Biochemical research is being intensively pursued in hundreds of labora-
tories throughout the world. The methods of study are drawn mainly
from the older sciences such as chemistry, physics, mathematics, biology,
physiology, etc., of which biochemistry is an outgrowth and descendant.

Isolation Methods. Efforts to ascertain the chemical nature of bio-
logical materials ordinarily start with an extraction or purification process
by which one constituent is isolated, i.e., separated in pure form from
all the others. The isolation of a pure biological substance is often a
difficult feat because most biological materials are complex mixtures
containing hundreds of different individual chemical substances, many
of which frequently are closely similar in composition or properties and,
therefore, difficult to separate. In addition, the particular substance
sought may be present in very low concentrations, perhaps only one
part in many million parts of the source material. For example, Doisy
and co-workers extracted and processed the equivalent of four tons of
sow ovaries to obtain about 10 mg. of the sex hormone, estradiol (p. 292).
This small yield represented about half of the hormone originally present,
since its normal concentration in the ovary of the sow is only one part
in 150,000,000! This isolation of estradiol represents an achievement
on a par with the famous work of the Curies in obtaining radium from
pitchblende and illustrates some of the difficulties which confront the
biochemical investigator studying the composition of living things.

There are many kinds of procedure used in isolating biochemical sub-
stances, and only a brief indication of their nature can be attempted

INTRODUCTION o

here. Frequently large classes of substances can be separated from
each other because of their different degrees of solubility. For example,
a mixture of fat and sugar can easily be separated by shaking with
ether and water. These two solvents, being immiscible, on standing form
layers, one of which contains the sugar and the other the fat. Two types
of materials bcith dissolved in the same solvent may be separated by
causing one to precipitate. For example, a boiling water extract of a
fresh fruit or vegetable will contain, among other things, both sugars
and proteins. This mixture can be separated by adding a soluble salt
of a heavy metal such as lead acetate and filtering, since the proteins
are thereby rendered insoluble. Again, some substances are held on
the surface of adsorbents, e.g., activated charcoal, while others are not;
certain substances can be volatilized (distilled) leaving others behind.
By progressively applying such fractionation procedures, a particular sub-
stance can be gradually separated from the compounds which are origi-
nally mixed with it in the living material, and thus brought nearer
to a state of purity.

Once an individual chemical substance has been isolated, it can be
analyzed by standard chemical methods, broken down into simpler frag-
ments, which are also analyzed, and in general examined to see just how
it is constituted chemically. If the compound is not of too great com-
plexity, e.g., has a molecular weight of a few hundred or less, its structure
is usually established within a few years. The results of this work are
expressed as a structural formula, which shows just what the substance
is and how it may be expected to react with other substances. Since
most compounds isolated from living things are organic (carbon) com-
pounds, such studies fall into the realm of organic chemistry.

Nutrition. A large part of biochemical research for the past fifty
years has been concerned with the nutrition of animals, plants, and micro-
organisms. The objectives of this work have been to find out just what
chemical substances are needed in the food of living organisms to nourish
them properly and to determine w^iat purpose each nutrient serves. In
the earlier days of the twentieth century, attention was focused mainly
on the energy-yielding and body-building materials which constitute the
bulk of the food, namely the carbohydrates, fats and proteins. More
recently, substances required in smaller amounts such as the mineral
elements, vitamins, and other gro^\'tll factors have been intensively studied.
These remarks apply particularly to animals and microorganisms, as
plants need only mineral elements besides carbon dioxide and water for
nourishment.

The experimental methods of investigating these questions are similar
in principle regardless of the type of organism being studied, and may
be illustrated for the case of animals. The general approach has been
to feed animals a diet prepared from purified ingredients and to observe

'* INTRODUCTION

whether they grew normally and remained healthy. Usually growth
declined, but by supplementing the purified diet with some natural food
material, such as yeast, whole milk, or liver extract, growth was restored.
Such experiments showed that the supplement must have contained some
essential food factor lacking from the purified experimental (basal) diet.
The next step was to isolate this substance, determine its chemical struc-
ture, and add it as a pure compound to the basal diet for further feeding
trials. Whenever this was done, it usually was found that extra supple-
ments were again needed, or in other words, that the supplement first
used must have been contributing more than one essential food factor.

By such methods it has now been shown that a long list of chemical
substances is required to fulfill the dietary needs of animals. In the
case of rats and chickens most, if not all,, of the essential food factors
have been discovered, since rapid growth and apparently normal develop-
ment can be obtained on diets composed exclusively of pure chemicals.
However, when such a "synthetic diet" is fed to other animals such
as guinea pigs, they respond so poorly that other still-unknown food
substances are obviously needed. In fact the use of many different
species of animals for nutritional studies has been a fruitful source of
information, for, although many requirements are similar, many differ-
ences have also been found. Not only animals, but plants and micro-
organisms have been extensively studied as to their nutritional require-
ments, and the latter especially, because of their small size and rapid
growth, have served as admirable test subjects.

Study of Metabolic Reactions. The study of the chemical reactions
that take place in living organisms is regarded by many biochemists as
the most significant and fundamental aspect of the science. As pointed
out above, relatively little progress along this line was made until recently,
but since emphasis is now shifting strongly in this direction, the rate of
discovery of new information has sharply increased, and extensive addi-
tions to our knowledge may be expected in the relatively near future.
In studying metabolic reactions one approach has been to investigate
the composition of the food consumed and the waste eliminated by an
organism in order to attempt to deduce what must have happened inside
the organism to convert the one into the other. This method has yielded
some information, but obviously suffers from severe limitations.

A more fruitful approach has been to transfer the reactions being
studied from the organism to the test tube. In several instances it has
been possible to duplicate cellular reactions in the absence of the cells
themselves. For example, many of the intermediates, such as succinic
acid, involved in carbohydrate metabolism are oxidized by molecular
oxygen to carbon dioxide and water when added to suitable tissue
preparations. Finely ground suspensions of liver tissue in an aqueous
buffer are suitable for this purpose. Similarly, cell-free yeast prepara-
tions can ferment glucose to carbon dioxide and alcohol. Once such a

INTRODUCTION «>

system that is able to reproduce typical metabolic reactions in vitro ^
is discovered, the way will be open for experimental study. The chemical
compounds involved in each step of the process can be isolated in pure
form, and the effect of removing them from the system or replacing
them at various concentrations can be observed. The catalysts (enzymes
and coenzymes) which make the whole process possible can similarly be
studied one at a time, and, in general, each step can be subjected to
detailed examination. It is in this way that much of our knowledge
of metabolism has been acquired.

A still newer technique, which promises to be of major significance in
unraveling the chemistry of metabolism, is based on the use of isotopes.
The widespread use of this method was made possible by the atomic
energy development. Eventually, this may well prove to have been
one of the most constructive and valuable results of that program. Iso-
topes of the common elements — C, H, 0, N, S, P, and others — are used
as metabolic "tracers" by incorporating <^e or more of them into some
substance normally involved in metabolism. The "labeled" metabolite
is then administered to the test organism. After a suitable interval the
distribution of the isotope in the various tissues or tissue components
of the organism is determined. Thus if a rat is fed glycine containing
N^^ in the amino group, and the purine compounds in the animal's tissues
are later found to contain N^^ in comparable amounts, it may be con-
cluded that glycine is concerned in the biosynthesis of purines, and
specifically that one of the nitrogen atoms in the purine ring came from
the amino group of glycine. Other examples of the use of isotopes
will be encountered throughout the text.

Information about metabolic processes can also be obtained by block-
ing some particular process and then searching for a way to remove the
block. The desired effect can be obtained, for example, with antimetab-
olites, substances so similar to certain normal metabolites that they
get in the way of the latter but yet are unable to carry out their func-
tions. Again, it is often possible to produce mutants of lower organisms
{e.g., the mold, Neurospora) , which lack the power to carry out certain
metabolic reactions. In such cases it has frequently been observed that
the effect [e.g., gro^\i:h failure) of the block may be removed by ad-
ministering some apparently unrelated chemical. This indicates that
the counteracting agent may be the substance normally formed by the
blocked reaction. As an illustration, suppose an organism needs substance
A to serve as a catalyst for the transformation of B into C:

B " C

An antimetabolite of A would probably inhibit the growth of this organ-
ism, but this inhibition would be counteracted by C.

^ In vitro means, literally, in glass and implies occurring outside an.v living thing.

O INTRODUCTION

Relation of biochemistry to biology

Biologists have traditionally studied living organisms on the basis of
the cell, as the smallest intact living unit. The cell occupies much the
same relative position in biology as the molecule does in chemistry.
The smaller components of living cells have come under scrutiny, as the
biologist, equipped with ever more powerful microscopes, has probed
deeper and deeper into the mysteries of living matter. The main parts
of a typical cell are the cell wall, nucleus, and cytoplasm. The living
material making up the nucleus and cytoplasm is termed protoplasm;
it is a grayish, translucent, jelly-like material, which under the micro-
scope can be seen to consist of a meshwork filled with fluid. The nucleus
contains chromosomes, and these in turn, under very high magnification,
reveal structural irregularities which may have functional significance.
Thus the biologist studies and interprets life mostly in terms of its
smallest visible fragments. %

From the chemical viewpoint, protoplasm is an aqueous, colloidal
solution containing protein as the chief solid ingredient, but with appre-
ciable amounts of fatty substances, nucleic acids, and other compounds
present. The metabolic reactions occurring in the cell take place in
this solution, and are studied and interpreted by the biochemist in terms
of molecules of the reacting substances. Most molecules are far too small
to be seen in any microscope, and their actual existence can only be sur-
mised from indirect evidence. However, the giant molecules of proteins
and nucleic acids are large enough so that they can actually be "seen,"
that is, photographed, with the help of the electron microscope, an instru-
ment that makes possible 50,000-100,000 fold magnification.

It seems most probable that the merging of biochemistry and biology
will continue in the future to an even greater extent, as the functional
activities of living things come more to be studied and explained in
chemical terms. However, it will not suffice to regard metabolism merely
as a group of chemical processes occurring at random in the same solu-
tion. Each living cell is a miniature "chemical factory" where food
molecules pass in an orderly fashion through a long series of interrelated
chemical reactions. A highly organized physical structure, with each
catalyst (enzyme) in a definite position in relation to the others, must
exist to accomplish this end. The study of such levels of organization
can probably be more properly classified as biology rather than as bio-
chemistry, although it must be obvious that the borderline is indefinite.

Study of biochemistry

The first task of the beginning student must be to learn something
of the materials of the cell in order to provide a basis for subsequent

INTRODUCTION '

study of metabolic processes. At least an elementary knowledge is
needed, not only of the major cellular components (water, carbohydrates,
fats, and proteins), but also of the minerals, vitamins, hormones, and
enzymes, which, although present in smaller amounts, arc equally vital
to the living organism.

Relatively little time can be devoted, at first, to discussion of detailed
evidence for various facts and how this evidence was obtained, since
major emphasis must be given to the facts themselves. In other words,
the results rather than the methods of biochemical research form the
chief subject matter of the beginning course. It is for this reason that
the methods have been briefly outlined in this introductory chapter. As
in all elementary studies, the student is asked to accept great masses
of inform.ation more or less on faith, with the clear understanding, how-
ever, that each fact is firmly supported by experimental evidence which
he can review and assess for himself, if he so desires. References and
suggested readings are listed at the end of each chapter for this purpose.

REFERENCES AND SUGGESTED READINGS

Green, D. E. (editor), Cunenif; in Biochejnical Research, Interscience Publishers,

Inc., New York, 1946.
Haurowitz, F., Progress in. Biochemistry, Interscience Publishei-s, Inc., New York,

1950.
MacCorquodalc, D. W., Thayer, S. A., and Doisy, E. A., "The Isolation of the

Principal Estrogenic Substance of Liquor FolHcuH,"' J. Biol. Chcrn., 115, 435 (1936).
Needham, J. and Baldwin, E., Hopkins and Biuchcmistry, W. Hpfi'er and Sons, Ltd.,

Cambridge, 1949.

Chapter 2

WATER

Occurrence and importance

Water is the most abundant substance in living matter. The great
physiologist, Claude Bernard, said, "All living matter lives in water."
In his Outline of History, Wells put it this way, "We talk of breathing
air, but what all living beings really do is to breathe oxygen dissolved
in water."

Table 2-1 gives the water content of some typical animal, plant, and
microbial materials. The human body is about 65 per cent water, a corn
plant about 75 per cent, and a bacterial cell about 80 per cent. The
amount of water varies not only with the type of material but also with
its period of development. Two examples, for which we have adequate
data, will show the variation with age. The pig embryo at 15 days of
development consists of 97 per cent water and 3 per cent solids, and
at birth the young pig is made up of about 89 per cent water and 11
per cent solids. The water content continues to decrease as the pig
grows, being about 67 per cent at 100 lbs. weight and 43 per cent for
a very fat animal weighing 300 lbs. The same relationship between
water content and age holds for other farm animals and also for man.

The water content of the corn plant remains practically constant, about
88 per cent, during the actively growing period from the seedling to the
tassel state, decreases rapidly to around 70 per cent at the time the
kernels begin to glaze, and falls to about 52 per cent when the plant is
mature and ready to harvest.

A high water content is characteristic of youth and activity and a
lowered figure is associated with old age and inactivity. The relation
between water content and activity of tissues is further demonstrated by
comparison of different tissues in the same individual. The metabolically
active tissues of the body, e.g., brain and liver, contain much more water
than the relatively inactive portions such as bones and fatty tissues.

WATER ^

Table 2-1
Water conlenl of some important biological miiterials

Water
Material [per cent]

Human body 65

Brain, gray matter 84

Liver 76

Muscle 73

Blood 80

Milk 87

Saliva : . . 99.5

Bone 10-40

Adipose tissue (mainly fat) 10-30

Larvae of clothes moth 58

Pig embiyo, 15 days old 97

Pig at birth 89

Pig at maturity, depending on fatness 40-50

Cora i)lant, seedling to tassel period 85-90

Com plant, kernels glazed 68-72

Corn plant, maturity 50-60

Bacteria 73-90

Yeasts 68-83

Molds 75-85

It is all too common a fallacy to limit the meaning of "foods" to the
energy-yielding materials — carbohydrates, fats, and proteins — with the
inclusion perhaps of mineral elements. If the term food be considered
to include all substances that are essential for the growth and repair
of body tissue, as most certainly it should, then water likewise is truly
a food. This error in thinking has arisen from the fact that in the
past most biologists have treated water as if it were an inert material
and have looked upon the solids of plant and animal tissues as the im-
portant part of the organism. Gortner has pointed out how mistaken
is this view; he illustrates his argument by citing the composition of
the tadpole, 95 per cent water and 5 per cent solids. "It would be
ridiculous to speak of this organism as being composed of only 5 per
cent of vital materials. The water is as much a part of the tadpole as
arc the fats, proteins, etc., which serve to form the gel structure, and the
biochemical and biophysical reactions which take place within the cells
and tissues of the tadpole are determined probably more by the water
which is present than by any or all of the other constituents."

Free and bound water

The term "bound water" has come into use to designate water that has
been adsorbed by the colloids of the living cell, in contrast to "free water,"

10

WATER

which is not an integral part of the plant or animal tissue with which
it is associated. The major part of bomid water is held probably by
proteins, but other classes of compounds are known to retain relatively
large amounts of water. Thus adipose tissue contains considerable
water, certain of the compound lipides, such as lecithin, emulsify readily in
water, and the polysaccharides of plant tissues are decidedly hydrophilic.

It is not certain just how bound water is held by colloidal material.
One explanation applied to proteins is that sharing of electrons between
the protein molecule and the water molecule sets up a binding force that
holds the water to the protein. Such a force is called a hydrogen bond
or bridge and consists of an electropositive hydrogen atom standing
between two electronegative atoms, e.g., N and 0, thus — N : H : 0 — .
The hydrogen shares its electron with both the N and 0.

Proteins contain many groups such as — NH2, — COOH that can form
a hydrogen bond with water. A protein molecule may contain several
thousand binding groups. For example, gelatin, a rather small protein
having a molecular weight of about 35,000, is calculated to have 960
molecules of water bound to each molecule of gelatin when a gel is
formed. There is much difference of opinion as to the quantity of water
held by proteins in solution, but 0.3 g. of water per gram of protein is
a commonly suggested figure.

Bound water, especially that bound by the protoplasm of the cell,
appears to be one of the several important factors involved in frost and
drought resistance. Plants that are exposed to low temperatures in
winter increase the proportion of bound water and the concentration of
water-soluble protein in the cell sap, thus developing what is called
winter hardiness. Plants, such as cactus, that live under arid or semi-
arid conditions hold their water largely in the bound state. Insects also
increase the percentage of bound water under conditions of cold or
drought.

Yeast cells furnish another example of the intimate association of
residual water and life processes. A commercial product known as active
dry yeast contains only about 8 per cent moisture, but the cells are
still alive and will survive for many months. If soaked in warm water
for a few minutes, the yeast promptly starts producing carbon dioxide
and can be used in place of baker's press yeast for bread-making. How-
ever, if the cells are dried to around 5 per cent moisture, they die, and
will not revive when placed in water.

On the other hand, cultures of most microorganisms if lyophilized
(dried from the frozen state) can be kept in this condition for years and
still grow when placed in a suitable medium.

WATER 11

Necessity for water

The demands of tlic body for water arc far more imperative than
those for food. An animal can live for 100 days or more without food
but dies within five to ten days if no water is supplied. With a Scotch
collie, Hawk conducted two experiments in which the dog was main-
tained for 105 and 117 days, respectively, without food, but with an
abundance of water. At the end of each period the animal was still in
a fair condition of health.

In the life of the plant enormous quantities of water arc transpired.
From three hundred to four hundred pounds of water are involved in
the manufacture of one pt)und of dry matter. ^A'ater is one of the great
raw materials from which the plant produces sugars, fats, proteins, and
all the other substances which go to make up the plant cell.

Function of water

Since all chemical changes involved in digestion are of a hydrolytic
nature, water is concerned in the first step toward utilization of fats,
proteins, and the higher carbohydrates. Water functions as a medium
for the transportation of food materials during digestion, absorption, and
circulation; and of waste prcxlucts, as well, in the process of elimination.
Hawk has demonstrated that digestion by saliva is more rapid if the
saliva is diluted with about seven times its volume of water. Gastric
digestion and pancreatic digestion also are aided by liberal quantities of
water. Carbohydrates, fats, and proteins are more completely absorbed
when large quantities of water are ingested with the food. The growth
of bacteria, and consequent putrefactive processes, are decreased by the
use of liberal quantities of water with food.

AVater also plays an important role as a heat regulator. Because of
the high specific heat of water, oxidation in the body can proceed without
greatly increasing the temperature at the site of oxidation. Water is
a good conductor of heat and thus aids in the transfer of heat from the
interior to the surface of the body. Finally, because of its high latent
heat of vaporization, water carries off heat by vaporization in the expired
breath and evaporation from the surface of the body. It is estimated
that about 25 per cent of the heat produced in the body is carried off
by way of the breath and by evaporation from the skin. In animals that
do not perspire freely — dogs, swine, cattle — excess heat is dissipated by
increasing the respiration (panting).

Finally, water performs an important function as a lubricant in the
many movements of the muscles, joints and organs of the body.

12 WATER

Water requirement

The water requirement varies with different conditions. For an adult
doing no manual work, and at the time of year when the weather does not
induce visible perspiration, it is estimated that an intake of about 3 1. of
water per day is needed for good health. Of this quantity, about 2 1. will
be contained in the food, leaving 1 1. to be drunk as water. Many health
authorities advocate drinking from 5 to 8 glasses of water daily, which
is more than most people manage to do.

Individuals who work under conditions which induce much perspiration,
for example, blast furnace workers, may lose many liters of sweat per day
and hence must drink large quantities of water. Since the sweat carries
with it salts, depletion must be avoided by taking salt tablets or by adding
salt to the drinking water. About 0.1 per cent of sodium chloride in the
water is scarcely perceptible, and such water quenches thirst about as
well as unsalted water.

The water requirement would be even higher than it is except for the
fact that the ingested water is reused several times for different purposes
before it is finally lost from the body. Water withdrawn from the blood
stream for such secretions as saliva, gastric juice, pancreatic juice, in-
testinal juice, and bile is salvaged during absorption and made available
for further use. The volume of these secretions amounts to from 3.7 to
9.8 1. daily, which means an average reuse of two to three times.

Formally it was thought that water should not be drunk with meals,
but as a result of Hawk's work it appears thaat this idea is erroneous.
Under certain conditions such as dropsy, heart and kidney disturbances
large quantities of water may be objectionable, but for the normal in-
dividual it is probable that too little water is usually consumed.

Metabolic water

Water is invariably .one of the end products of oxidation of carbo-
hydrates, fats, and proteins. Such water is called "metabolic water."
To be specific, let us refer to the equation for complete oxidation of
glucose in the body:

CeHioOe + 6O2 = 6CO2 + 6H2O + 683 Cal.i

During oxidation of 180 g. (1 mole) of glucose, 108 g. (6 moles) of water
is produced with accompanying liberation of 683 Cal. of heat. By the
same proportion, 15.9 g. of water would be formed for every 100 Cal.
derived by oxidation of glucose.

^ For a definition of calorie see p. 413.

WATER 13

From fats, proteins, and starch the yield of water per 100 Cal. is some-
what less than the amount calculated for glucose. Approximately 12.5 ml.
of metabolic water is formed during oxidation of a 100 Cal. portion of
a diet in which i)roteins, fats, and carbohydrates contribute 15, 35, and 50
per cent, respectively, of the total calorics. On this basis, a person con-
suming 3000 Cal. daily would derive 375 ml. of water from oxidation
of these energy-yielding foods, an amount approximating one-tenth of
the intake of water as a drink.

Some species of insect, e.g., clothes moth and grain weevil, obtain nearly
all of their water from metabolic processes. The clothes moth feeds on
wool, which contains about 5-10 per cent of absorbed water. Foi- each
gram of dry wool (protein) consumed, ai)proximately 0.4 ml. of watei- is
formed by the chemical processes involved in metabolism.

Water balance

For a normal man the intake and output of water are so regulated
that the amount in the body remains fairly constant. If the intake is
increased without any other change in external conditions, the output
in the urine is very promptly increased. If the atmospheric temperature
rises, or if muscular effort is increased, then more water is eliminated
through the skin as insensible or sensible (visible) perspiration and less
is put out in the urine. With increased muscular effort there is also more
water eliminated through the lungs, but the greatest variation occurs in
the volume of urine and perspiration. A typical water balance for an
average-sized man of sedentary occupation is about as follows:

Water intake ml. Water output ml.

Drinking water 850 In urine 1450

Water in coffee, milk, soup, and In feces 150

other fluids 600 Evaporated from the skin 600

Water in solid foods 700 Vaporized through the lungs 350

Metabolic water 350

Total: 2500 2550

On this particular day there is a negative balance of 50 ml.; on a succeed-
ing day the balance might be positive by that much or more.

Water supplies

The importance of a pure water supply cannot be overestimated. To
determine the potability of a water requires careful chemical and bac-
teriological analyses. Pure water in the chemist's sense of the term is
not required to furnish a sanitary water supply. All ground and surface
waters dissolve more or less salts and other materials. It is only when

14

WATER

such waters contain harmful organisms or acquire offensive odors or tastes
that they become objectionable. A good drinking water should be clear,
colorless, odorless, have a cool and refreshing taste, and be free from harm-
ful organisms.

In the chemical examination of water the different forms of nitrogen
are regarded as the best index of the quality of the water. The water
is analyzed for its content of albuminoid ammonia, ammonia, nitrites,
and nitrates. These different forms of nitrogen are closely related to one
another. The albuminoid ammonia really represents protein nitrogen and
is readily converted by organisms into ammonia. Other organisms then
oxidize the ammonia to nitrites and nitrates. The nitrogen of the nitrates
may then be utilized by plants that grow in the water and be again built
up into proteins. These processes are called ammonification and nitri-
fication and form a part of what is known as the nitrogen cycle in nature.
The relation of the different forms of nitrogen may be expressed by the
following equations in which glycine is taken to represent protein:

Ammonification:

2CH0NH0COOH + 3O2 = 2NH3 + 4CO2 + 2H2O
Nitrification :

2NH3 + 30o = 2HNO2 + 2HoO
2HNO0 + O. = 2HNO3
2HNO3 + CaC03 = Ca(N03)o + HoO + COo

These compounds of nitrogen are not toxic in the quantities in which
they occur in water. Far more ammonia, for example, is produced as a
result of metabolism than is consumed in the drinking of water. How-
ever, the presence of a constant and continuing supply of albuminoid
ammonia, ammonia, and nitrates may indicate that the water is being
contaminated by sewage, since these forms of nitrogen are particularly
high in sewage. A state of change is regarded as a state of danger and
calls for careful investigation as to the origin of these forms of nitrogen.
Care must be observed in interpreting the data obtained by analyzing
water. The normal nitrogen content of such waters must be known be-
fore judgment can be passed. Deep well water would not have the same
nitrogen content as a surface or spring water. It is necessary to know
something of the topographical features surrounding the water supply.

The chloride content of water is often also of value in determining the
character of the water. If it has been polluted by sewage, the chloride
content will be abnormally high. Here again, however, it is necessary
to observe great caution in passing judgment. Waters from along the
coast or from regions where salt deposits exist may be very high in
chlorides and still be perfectly safe. It is not the absolute amount, but

WATER 15

the departure from the normal in that vicinity that is significant. Sewage,
which contains a very high content of salt, will raise the chlorine content
very quickly if a little of it gets into the water.

Although sedimentation, flocculation with chemicals, and filtration
through layers of sand and gravel I'emove nuich suspended matter and
hence lessen the bacterial content of water, these methods alone cannot
be relied upon for purification of a public water supply. The agent most
commonly employed to destroy bacteria in water is liquid chlorine.
Penfield and Gushing state that 85 per cent of the public water supplies
of the United States are sterilized with chlorine. According to these
authors the average typhoid death rate in 77 important cities of the
United States has decreased from 20.54 deaths per 100,000 in 1910 (when
the use of chlorine was initiated) to only 0.76 in 1937. Better water
supplies, and the use of sulfa drugs, antibiotics, and other therapeutic
measures, have decreased the typhoid death rate to 0.2 per 100,000 in 1948.

Hardness of water

From the household and industrial viewpoint, the hardness of water is
an economic question that involves the cost of large quantities of soap and
water-softening materials. The hardness of water is due to the presence
of bicarbonates, sulfates, chlorides, and silicates of calcium, magnesium,
and iron. These salts form insoluble precipitates with soap and therefore
give what is called hardness to the water. New types of cleaning agents,
called "synthetic detergents" (p. 87) , have been developed in recent
years. They form soluble calcium and magnesium soaps and hence can
be used in hard water.

Hardness of water is spoken of as being temporary or permanent de-
pending upon whether it is due to bicarbonates or other salts. If the
hardness is due to bicarbonates, heating or boiling the water will, to a large
extent, precipitate the calcium or magnesium bicarbonates as insoluble
carbonates. On the other hand, boiling has no effect upon water that
contains sulfates or other salts of calcium and magnesium. Such waters
are said to be permanently hard. Many different methods for softening
hard water can be used such as boiling, or addition of lime, washing soda,
phosphates, or other precipitants. In all cases a precipitation of the
calcium and magnesium is the end to be desired. The action of these
precipitants may be represented by the following equations:

Heat :

Ca(HC03)o = CaCOa + HoO + COo

• Lime :

Ca(HC03)o + Ca(0H)2 = 2CaC03 + 2H2O

16 WATER

Washing soda:

Ca(HC03)2 + NaoCOa = CaCOg + 2NaHC03
CaS04 + NagCOs = CaCOg + Na2S04

Phosphates :

3Ca(HC03)2 + 2Na3P04 = Ca3(P04)2 + GNaHCOa
3CaS04 + 2Na3P04 = Ca3(P04)2 + 3Na2S04

The use of various water-softening materials in the kitchen and laundry
of the average household is not a very satisfactory procedure. In most
cases little or no softening of the water, and consequent saving of soap,
is effected. At best it is a temporary expedient and to a large extent means
waste of money. A satisfactory method of softening water is by means
of a zeolite. Silicates of this character are sold under such trade names
as Permutit, Refinite, and Bormite. Water treated in this way is soft.
In fact, it requires less soap than distilled water because of the presence
of a small amount of sodium bicarbonate. The softening of water by
means of a silicate may be represented by the following equation:

Ca(HC03)2 + NaoO-Alo03-2Si02-6H20 =
CaO-Al203-2Si02-6H20 + 2NaHC03

After a time the silicate will have taken up all the calcium or magnesium
that it will hold and must then be regenerated if the water is to be softened.
This is done by means of a strong solution of salt that displaces the
absorbed calcium or magnesium and again forms the sodium silicate.
This operation is called the regeneration of the silicate and is indicated
by the following equation:

CaO'Al203-2Si02-6H20 + 2NaCl = Na20-Alo03-2Si02-6H20 + CaClg

A more recent method for softening water is by means of cation exchange
resins. Such resins are extremely insoluble, high polymer organic com-
pounds that contain many acidic groups, e.g., sulfonic, — SO3H; carboxyl,
— COOH; etc. A phenol sulfonic acid resin is made by polymerizing
m-phenol sulfonic acid, C6H4 (OH) SO3H, and formaldehyde. Commercial
products of this type are Amberlite IR-100 and Dowex-50. The removal
of a cation such as Ca + + from water by the resin may be represented
by the following equation:

Ca(HC03)o or CaS04 + 2NaR = CaRs + 2NaHC03 or Na2S04

In the above equation NaR represents the sodium form of the ion exchange
resin. Similar equations can be set up to show the removal of magnesium,
iron, and other cations. When the resin becomes loaded with cations, it
is regenerated by washing with sodium chloride brine. The reaction is
illustrated by the equation

CaR. + 2NaCl = 2NaR + CaCIg

WATER 17

An ion exchange resin that removes anions can be synthesized by con-
densing a phenol amine, e.g., CcH4(OH)CHoNHCH3 and formaldehyde.
Amberlite IR4B is a commercial product of this type. The removal of
anions may be represented by the equation

CaS04 + 2R0H = Ca(OH). + R0SO4

The alkalinity thus produced may be neutralized by using the anion
exchanger in series with an acid form of cation exchanger (HR), in which
case the water is completely demincralized, thus

Ca(0H)2 + 2HR = CaRo + 2H2O

The anion exchanger can be regenerated with sodium hydroxide, and the
cation exchanger with sulfuric acid.

A desalting kit for the production of potable water in case of forced
landings in overseas flying consists of silver zeolites (AgZ), silver oxide,
and barium hydroxide compressed into briquets and placed in a suitable
container for filtering sea water. The reactions involved are as follows:

AgZ + NaCl = NaZ + AgCl
Ba(OH)o + MgS04 = Mg(0H)2 + BaS04

The sodium, chloride, magnesium, and sulfate ions, the chief constituents
of sea water, are removed as the insoluble compounds NaZ, AgCl,
Mg(0H)2, and BaS04. The capacity of the zeolite material is about ten
times its volume of sea water.

The above discussion of ion exchangers is merely a bare outline of the
subject, and the interested student is referred to the books listed at the
end of the chapter for complete information on this complex and rapidly
developing field of work.

REVIEW QUESTIONS ON WATER

1. Would you expect the young leaves of a growing plant to be higher or lower
in water content than the fully developed leaves? Explain.

2. Arrange these materials in ascending order of water content: milk, blood, cab-
bage, saliva, strawberries, fish. (Consult Tables 2-1 and A-1.)

3. Explain the terms "bound water" and "free water."

4. Explain the term hydrogen bond. With which other elements is it associated?
Consult the index.

5. What changes in composition would you expect to find in wheat plants ana-
lyzed in September and again in December?

6. What is the daily water requirement of an adult? Name some of the chief
functions of water in the body.

7. Explain how clothes moth lar\ae get their water. Can you name another
insect or living organism that gets its water in the same way?

8. What is meant by "metabolic water"? What is the approximate contribution
of this Vv'ater to the total intake?

18 WATER

9. Discuss the advisability of drinking liberal quantities of water at meal time.

10. Write an equation for the reaction between soap and hard water.

11. Write an equation for the softening of water by zeolites (silicates).

12. Explain the terais (1) temporaiy and (2) permanent hardness of water.

13. Define the term ion exchange resin. Find the name of one resin other than
those given in the text and the name of the company that produces it.

14. Write equations for the removal of MgCla from water by ion exchange resin.

REFERENCES AND SUGGESTED READINGS

Adolph, E. F., "The Metabolism and Distribution of Water in Body Tissues,"

Physiol. Rev., 13, 336 (1933).
Gortner, R. A., Outlines oj Biochemistry , 3rd ed., John Wiley and Sons, Inc., New

York, 1949.
Hawk, P. B., Oser, B. L., and Summerson, W. H., Practical Physiological Chemistry,

12th ed., P. Blakiston's Son and Company, Inc., Philadelphia, 1947.
Kunin, R. and Myers, R. J., Ion Exchange Resins, John Wiley and Sons, Inc., New

York, 1951.
Mason, W. P. and Buswell, A. M.. Examination oj Water, John Wiley and Sons,

Inc., New York, 1931.
Moulton, C. R., "Age and Chemical Development in Mammals," J . Biol. Chem.,

57, 79 (1923).
Nachod, F. C, Ion Exchange Theory and Application, Academic Press, Inc., New

York, 1949.
Penfield, W. and Gushing, R. E., "Bathing the Green Goddess, Purification of

Chlorine," J. Ind. Eng. Chem., 31, 377 (1939).
Thresh, J. C, Beale, J. F., and Suckling, E. V., The Examination oj Water and

Water Supplies, P. Blakiston's Son and Company, Inc., Philadelphia, 1933.

Chapter 3

CARBOHYDRATES

Composition and definition

The word "carbohydrate" hterally means carbon combined with water
and originates in the fact that many carbohydrates have the composition
Ca^HoO) j;. Tlie values of x and y may range from three to many thousand.
Although several carbohydrates do not have this composition, and some
other substances do, e.g., lactic acid (CsHgOs) , the term nevertheless fits
the great majority of carbohydrates and is in common use.

Carbohydrates may be cliemically defined as simple sugars, or more
complex substances which yield simple sugars on hydrolysis.^ Simple
sugars are either aldehydes or ketones which contain at least two, and
usually several, hydroxyl groups. The aldehyde-alcohol type is called
an aldose, e.g., glucose, and the ketone-alcohol type a ketose, e.g., fructose.

Occurrence and importance

The plant world is the great source of the carbohydrates. The dry
matter of plants (excepting certain oily seeds) is from 60 to 90 per cent
carbohydrate. These compounds are constituents of most materials that
satisfy the primary needs of human life.

Our food is made up principally of carbohydrates — approximately 70
per cent by weight of the food in the average diet. Much of our clothing
is made from carbohydrates — cotton, rayon, and linen. In the United
States probably more houses are built of wood than of all other materials
combined. Even in many buildings of brick and stone, wood enters into
the construction of walls, floors, stairways, and windows. The great fuel
materials, wood and coal, are either carbohydrates or derived from carbo-
hydrates. The carbohydrates are at the very foundation of the economic
structure of society.

The importance of the carbohydrates is shown by Table 3-1. The
carbohydrate industries listed employ nearly as many people and turn
out products of greater value than the combined machinery and chemical
and drug industries.

^ Hydrolysis consists in the cleavage of a complex molecule into smaller fragments
with the simultaneous addition of water.

19

20 CARBOHYDRATES

Table 3-1

Economic importance of some industries based on carbohydrates *

Wage Value of prod-
Industry earners ucts shipped

1. Lumber and products (except furniture) 635,708 $4,691,931,000

2. Cotton and rayon fabrics, yarns, threads 604,469 5,496.299,000

3. Grain mill and bakery products 392,585 8,183,849,000

4. Paper and products 449,833 7,051,485.000

5. Confectionery 75,165 944,925,000

6. Sugar (cane and beet) 35,423 1,141,437,000

7. Starch, dextrin, sirups 12,324 459,978,000

8. Fermentation products (beverages, vinegar, yeast,

etc.) 91,754 1,671,712,000

9. Canvas products and textile bags 24,453 447,089.000

10. Cordage and twine 15.950 167,648,000

2,337,664 $30,256,353,000

* Compiled from the 1947 Census of Manufacturers, Bureau of the-^ensus, 1950,
and from the Statistical Abstract of the United States, 1951, published by the Depart-
ment of Commerce.

For comparison, figures for some other industries from the same source,
for the same year, are as follows:

Wage Value of prod-
Industry earners ucts shipped

Machineiy, including electrical 2,346,682 $19,667,327,000

Industrial chemicals, and drugs 353,445 5,145,963,000

An abridged classification of carbohydrates

I. Monosaccharides

A. Trioses, CaHsOs

1. Aldotriose : D-glyceraldehyde

2. Ketotriose : dihydroxy acetone

B. Tetroses, C4H804

1. Aldotetroses : o-erythrosc, o-threose

C. Pentoses

1. Aldopentoses, C5H10O5: L-arabinose, D-arabinose, o-xylose, D-ribose

2. Ketopentose, CgHiuO.-, : L-xylulose

3. Desoxypentose,^ CoHioO*: 2-desoxy-D-ribose (desoxyribose)

D. Hcxoses

1. Aldohexoses, CeHi-Ou: o-glucose, n-galactose , n-niamiose

2. Ketohexoses, CuHi206: n-fructose, ^-sorbose

3. Desoxyhexoses, C0H12O5: Q-desoxy-h-mamwsc (h-rhamnose), Q-desoxy-i.-
galactose (L-fucose)

4. Aminohexoses, CsHisOsN: 2-amino-i>-glucose {^-glucosamine) , 2-amino-D-
galactose (galactosamine)

^ The prefix "desoxy" means "lacking oxygen." Note that the formula of the desoxy-
pento.se is C5Hio04, whereas the other pentoses are CsHioO^.

CARBOHYDRATES 21

5. Hexuronic acids, CoHioO? : u-glucuronic acid, D-mannurordc acid, D-galac-
turonic acid
E. Heptoses

1. Ketoheptoses, CtH^Ot : u-mannoheptulose, sedoheplulose
II. Disaccharides, dsH^iiOn

A. Anhydrides ^ of glucose and galactose : lactose, melibiose

B. Anhydride of jrlucose and fructose: sucrose

C. Aniiydrides of glucose and glucose: maltose, iso-77ialtose, cellobiose, gen-
tiobiose, trehalose

D. Anhydrides of a hexose and a hexuronic acid (aldobiuronic acids) : cello-
biuronic acid, geutiubiuronic acid

III. Ti-isaccharides, CisHaaOio

A. Anhydride of galactose, glucose, and fructose: raffinose

B. Anhydride of glucose, fructose, and glucose : melizitose

IV. Polysaccharides

A. Homopolysaccharides (anhydrides of single monosaccharides)

1. Pentosans, (C5H804)j: xylan, araban

2. Hexosans

a. Glucosans, (CcHioOs)^ : cellulose, starch, dextrin, glycogen, bacterial

dex trait.
h. Fructosans, (CeHioOs)^: inulin, bacterial levari

c. Galactosans, (CuHioOs)*: snail galactogen

d. Mannosans, (CeHioOs)^: vegetable nut manjian, salep mavnan

e. Polyglucosamine : ^ chilin

f. Polyuronides,' (C«H808)x

(1) Polygalacturonic acid: pectic acid

(2) Polymannuronic acid : alginic acid

B. Heteropolysaccharides (anhydrides of several monosaccharides)

1. Hemicelluloses: alkali-soluble polysaccharides associated with cellulose in
wood, straw, cornstalks, and other fibrous plant tissues. On hj'drolysis,
form mainly D-xylose, together with L-arabinose, D-glucose, uronic acids,
and other sugars.

2. Plant gums: gums from injured trees or bushes, mostly water-soluble, and
forming D-glucuronic acid and other sugars on hydrolysis. Examples:
gum arable, viesquitc gum, cherry gum.

3. Plant mucilages: polysaccharides extractable from the seeds, roots, leaves,
and bark of various plants, forming colloidal solutions. Examples: gum
gatto, linseed mucilage, agar-agar.

4. Mucopolysaccharides: water-soluble polysaccharides found in animals and
often associated with protein. Usually form aminohexoses and (or) hexu-
ronic acids, as well as other sugars when hydrolyzed. Examples: hya-
luronic acid, heparin, choiidroitin sulfate, pneumococcus polysaccharides,
blood-group polysaccharides.

V. Substances Related to Carbohydrates
A. Sugar alcohols, open chain

1. Four carbon type, C4Hio04: erythntol

2. Five carbon type, Cr-Hi-Oi: xylitul, arabitol, nbilul

3. Six carbon type, (hexitols), CoHiiOe: mannitol, sorbitol, dulcitol

An anhj-dride is a product formed by removing the elements of water from another
substance or substances. Frecjuently an H atom is split out of one moleculi' and an
(JH fjroup from another, and tlie residues unite to form the anliydrido.

-polysaccharide giving glucosamine and acetic acid on hydrolysi.s.

^ Polysaccharide giving a uronic a«'id on hydrolysis.

22 CARBOHYDRATES

B. Cyclic polyalcohols: inositol

C. Sugar acids (other than uronic acids)

1. Aldonic acids, C6H12O7: D-gluconic acid, n-mannonic acid, D-galactonic acid.

2. Saccharic acids, CeHioOs : niucic acid, saccharic acid.

3. Ketoaldonic acids, CgHiuOt: 2-Ketogluconic acid, 5-ketogluconic acid.
4. Ascorbic acids, CeHioOtt". ascorbic acid.

MONOSACCHARIDES

Simple sugars, or monosaccharides, containing 3, 4, 5, 6, and 7 carbon
atoms occur in nattire. They are called trioses, tetroses, 'pentoses, hexoses,
and heptoses, respectively. Those of the aldose type which contain 5 and
6 carbon atoms {aldopentoses and aldohexoses) are most common.

Most of the monosaccharides may be represented by a formula of one
of the following types:

CH2OH

I y

CHO CO

I I

(CHOH)„ (CHOH)„

CH2OH CH2OH

Aldoses Ketoses

(w = l,2,3,4, or 5) (n =0,1,2,3, or 4)

Note that all the carbons are attached to each other and that each holds
an oxygen. Carbon 1 of the aldoses and 2 of the ketoses are especially
important because the most significant chemical reactions of the mono-
saccharides involve these points.

Stereoisomerism of monosaccharides

Organic molecules which contain an asymmetric carbon, i.e., a carbon
atom attached to four different atoms or groups, can exist in "right hand"
or ''left hand" forms. These are called dextro or d- and levo or L-forms,
respectively. The simple sugars all contain carbon atoms of this asym-
metric type, and hence can exist in the left- or right-handed patterns.
For example, the triose, giycer aldehyde, has one asymmetric carbon
(carbon 2, the center one) :

(1) CHO CHO

I I

(2) H-C— OH HO-C— H

I I

(3) CH2OH CH2OH

Carbon D-Form . L-Form

number Glyceraldehyde

CARBOHYDRATES

23

The two forms in which this substance can exist are indicated by writing
the — OH of carbon 2 on the right or on the left. When the aldehyde
group is placed at the top of the formula, the D-form is the one with this
— OH on the right. Such substances which differ from each other only
in the way certain components of the molecule are arranged in space are
called stereoisomers. The exact arrangement of one particular isomer
is called its configuration.

"When several asymmetric carbons are present in a molecule, d- and l-
arrangemcnts about each must be considered. The total number of
stereoisomers in such cases equals (2)"^, where n is the number of different
asymmetric carbon atoms. Therefore, there are 8 possible aldopentoses
and 16 aldohexoses (see formulas below).

It is convenient to represent sugar isomers by means of diagrams which
are related to the structural formulas of the sugars as shown:

(1) CHO

I

(2) HCOH

(3) HCOH

I

(4) HCOH

I

(5) CH2OH

Carbon Formula Diagram
number

An aldopentose

(1)

(2)

cno

1

HCOH

(3) HCOH

I

(4) HCOH

I

(5) HCOH

(G)

Carbon
number

CH.OH
Formula Diagram

An aldohexose

The small circle represents the aldehyde group (carbon 1), and the short
side lines indicate which way the — OH on each asymmetric carbon ex-
tends from the chain. Aldopentoses have three asymmetric carbon atoms
(2, 3, 4), while aldohexoses have four (2, 3, 4, 5).
The different aldopentoses and aldohexoses have the following formulas:

00 9? 99 9?

I>L DL DL DL

Ribose Arabinose Xylose Lyxose

Configuration of stereoisomeric aldopentoses

24

CARBOHYDRATES

OO OO OO YO

D L

AUose

D L

Altrose

D L

Mannose

0 0

D L

Glucose

D L

Gulose

D L

Galactose

D L

Idose

D L

Talose

Configuration of stereoisomeric aldohexoses

Note that the D-forms are those in which the configuration of the asym-
metric carbon farthest from the aldehyde group, i.e., carbon 4 in pentoses
and 5 in hexoses, is the same as that in D-glyceraldehyde.

Optical rotation

Substances containing asymmetric carbons can also rotate polarized
light. ^ If such light is passed through a solution of D-glyceraldehyde, for
example, the emergent beam will be twisted a certain number of degrees.
The amount of twisting is measured in an instrument called a polarimeter.
This effect is called optical rotation, and substances which show it are
said to be optically active. When measured under specified conditions,
the angle of rotation is called the specific rotation and is a characteristic
property of the optically active substance.

It is important to note that there is no necessary relationship between
configuration and the sign of optical rotation. The D-forms may show
either positive or negative rotation.-

Cyclic formulas of monosaccharides

The aldose formulas have been written above in the *'open-chain" or
"free aldehyde" form. Certain properties of the aldoses indicate that an
aldehyde group is present (combination with phenylhydrazine; for ex-
ample, to form hydrazones or osazones). However, other properties

^ This light vibrates in only one plane, as contrasted with ordinary light which vi-
brates in all possible planes. The student ma.v visualize a polarized light beam as a
Hat, narrow ribbon of light.

** Positive rotation is clockwise rotation when you look toward the light source.

CARBOHYDRATES

25

l)uint to the absence of aldehyde groups {e.g., failure to bind bisulfite or
give the usual Schiff test^). The reason for this apparent contradiction
is that most of the sugar at any one time exists in a cyclic or "oxide-ring"
structure, which is derived by interaction of the aldehyde group with
one of the — OH groups, usually at carbon 5:

HCOH

,0H

O

IIOCH o

(3) -^c-

(2) /C = 0

HCOH OH

1(5) (6)

HC— CHoOH

HO^(i)/H

(2) /C^

HCOH ^O

(4)

H

II 1(5) (G)

HOCH o HC— CH2OH

H

(S) (6)

Alpha ring form

HOCH o HC-CH,OH

(3) \p^

Beta ring form

Free aldehyde or
oiDcn chain form

Open-chain and ring formulas of D- glucose

Note that carbon 1 has now become asymmetric so that there are two
stereoisomers of the ring structure. The alpha-forms of D-sugars have
the — OH of carbon 1 on the right when the formulas are written as shown
with carbon 1 on top. For sugars belonging to the L-series (see diagrams,
pp. 23, 24) the alpha-ring forms have this OH on the left.

The oxide ring forms of the sugars are often represented diagrammati-
cally. For example, for the above alpha-ring form:

H-^OH

H

(2)

HO

(3)

OH O

-H H H ,

0 /(5)

(6)
CH2OH

(4)

H

A B

Diagrams representing a-D-glucose

In A the diagram is drawn with carbon 1 at the top, but in B the molecule
has been moved into a different position. The student should realize
that the essential features of such formulas lie in the number and kind
of atoms present, and in what order they are linked together. Whether
the formulas are written with a particular part {e.g., carbon 1 in the
formulas above) at the top, side, or bottom, is incidental and merely
a matter of convenience.

In the diagram B above, — OH groups shown pointing downward have

^ Restoration of the pink color to Schiff's reagent, a dilute solution of rosaniline
which has been decolorized with sulfurous acid.

26 CARBOHYDRATES

the same configuration as those on the right in the open-chain formulas
with carbon 1 on top.

The ring structures shown above are of the 1,5-oxide, or pyranose type,
and are formed by aldohexoses, aldopentoses, and ketohexoses. Oc-
casionally a 1,4-oxide or furanose ring is formed, as for example in the
fructose component of sucrose (see p. 44).

Pentoses

There is no clear-cut evidence to show that pentoses occur free in
plants. No free pentoses or characteristic derivatives of free pentoses
have ever been isolated from seeds or green plants, or from any other
natural source. Qualitative tests and quantitative data which were
formerly attributed to free pentoses are now thought to be due to other
compounds, such as glucuronic acid. A good test for pentoses depends
on their conversion into furfuraldehyde by heating with fairly concen-
trated solutions of mineral acids:

H^ /OH H

HOCH "^0 H* HC=^ ^0

I I — -* I I -}-3H,0

HC C-CH2OH ^ HC=C— CHO

0 H
H

An aldopentose Furfuraldehyde

(furanose ring form)

This product produces a brilliant rose-red color when warmed with
aniline acetate, and therefore indicates the presence of pentoses. Hex-
uronic acids also give this test (p. 39) but hexoses do not, since they are
converted by the acid treatment into levulinic acid, CH3COCH2CH2COOH,
which gives no color with aniline acetate.

Anhydrides of some of the pentoses are very abundant in plant materials,
however, and therefore the corresponding pentoses can be easily prepared.

jy-Xylose. Xylose is sometimes called wood sugar, as it can be made
readily from wood, straw, seed hulls, and other fibrous materials. It is
easily prepared from corn cobs by hydrolysis and crystallization. Corn
cobs contain about 35 per cent of pentosans and yield about 12-15 g. of
xylose per 100 g. of cob. The pure sugar sells for about $25 per pound,
largely because there is not enough demand for it to make large-scale
production worth while. It has been estimated that on a large scale it
could be made for 5 cents per pound. Its use is limited almost entirely
to bacteriological laboratories, where it is of considerable aid in the
classification of bacteria.

-L-Arabinose. This pentose is found in the form of complex poly-
saccharides in wheat and rye brans, in pectins, and in gummy materials

CARBOHYDRATES

27

such as cherry gum, mesquite gum, and gum arable. Such gums are
frequently found associated with pectin in phmt materials. Arabinose
has also been obtained from peas and beans. In tlie laboratory it is
generally made from sugar beet pulp or mesquite gum. The latter re-
sembles gum arabic, being produced by a shrub which grows abundantly in
Arizona and other states of the Southwest. Yields of arabinose amounting
to 20 per cent can be readily obtained from the gum. Arabinose, like
xjdose, finds its chief use in bacteriological laboratories.

D-Ribose. Although from the standpoint of obtaining it in quantity,
D-ribose is an exceedingly rare and expensive sugar (about $400 per
pound); yet from the standpoint of its occurrence and functions in
living organisms, it is one of the most common and important of the
carbohydrates. It is present in all living cells as a component of ribose-
nucleic acids (see Chap. 6) and also as a part of several coenzymes
(p. 273) . Furthermore, two of the key substances involved in the process
of muscle contraction, adenosine diphosphate (ADP) and adenosine tn-
phosphate (ATP), are D-ribose derivatives.

The pure sugar may be obtained by hydrolysis of yeast nucleic acid,
or prepared synthetically from D-arabinose, The formula is indicated
by the diagram on p. 23.

2-Desoxy-D-ribose. This sugar has been found only as a component
of desoxyribonucleic acids, which are present in the nuclei of all living
cells, specifically in the chromosomes of the nucleus (see Chap. 6) .
Therefore, it is of great interest as one of the chemical substances in-
volved in the transmission of hereditary characteristics from one genera-
tion to the next.

As its name implies, the substance has the formula of an aldopentose,
except that the oxygen on carbon 2 is missing, and has the configuration
of D-ribose:

CHO CHjOH

1 I

CH: CO

I I

HCOH HC— OH

I I

HCOH HO-CH

I I

CH2OH CH2OH

2-Desoxy- D-ribose L-Xylulose

It is much more reactive and less stable than the ordinary aldoses or
ketoses and is particularly distinguished by giving a positive aldehyde
test with the Schiff reagent. This property of desoxyribose is the basis
for the Feulgen and diphcnylamine tests for desoxyribosenucleic acids.

28 * CARBOHYDRATES

jj-Xylulose. This sugar, which has also been called L-xyloketose, is
a ketopentose excreted in cases of human pentosuria.^ From one to
several grams may be excreted daily by a patient. L-Xylulose is so
difficult to crystallize that it has been obtained only in the form of
sirups. Crystalline derivatives are known, however, which serve to
characterize and identify the sugar. It is an unusually strong reducing
agent, as is shown by its ability to reduce Benedict's solution even at
room temperature, whereas most other sugars give a positive result only
upon heating.

Hexoses

n-Glucose. This sugar is also called dextrose. From the biological
standpoint it is the most important carbohydrate in nature both because
of its wide distribution and because of its prominence in physiological
processes. It is the circulating carbohydrate of animals. Glucose is the
sugar into which all the available carbohydrates of food are converted
before oxidation in the body.

In the free state it occurs in practically all fruits, being especially
abundant in grapes, figs, dates, and raisins. The blood contains about
0.08 per cent; in normal urine the amount may vary from traces to
about 0.2 per cent. In diabetic urine the sugar sometimes rises to 10
per cent.

In the combined state it forms a part, or the whole composition, of
many other sugars such as sucrose, lactose, and maltose. Starch, glycogen,
and true cellulose yield glucose on complete hydrolysis. Certain sub-
stances known as glucosides yield on hydrolysis glucose together with
some nonsugar compound often of characteristic odor or taste. An
example of such a glucoside is amygdalin, the substance that gives the
almond its peculiar flavor. Mustard owes its strong taste and odor
to an oil produced from the glucoside, sinigrin. The following equation
illustrates the action of the enzyme myrosin upon sinigrin:

C10H16NS2KO9 + H2O = CeHisOg + C3H5NCS + KHSO4

Sinigrin Glucose Mustard Potassium

oil acid sulfate

Formation of Glucose in Nature. The plant is the factory in which
the world's food supply is manufactured. All animal life depends ulti-
mately upon the vegetable world for its sustenance. Even carnivorous
animals are indirectly supported by the plant; they prey on animals
that feed upon plants. Man being an omnivorous creature receives a

^An abnormal condition characterized by the presence of a pentose sugar in the
urine.

CARBOHYDRATES 29

large part of his nourishment directly from plant sources. The synthesis
of food from simi)le compounds is, therefore, a most fundamental opera-
tion, and it is the peculiar function of plants. The formation of glucose
may be taken as typifying this synthesis, although recent investigations
reveal that various sugar phosphates and sucrose are formed before
glucose (see p. 397).

Carbon dioxide from the air and water from the soil are converted
in the leaves of plants into the various carbohydrates. Since sunlight
furnishes the energy required for the synthesis of carbohydrates, this
process is known as photosynthesis. The net result of the process is
often represented by the following equation :

6C0o + 6H0O + 717.6 Cal. = CgHioOg + 60.

In this equation C6H12O6 stands for a hexose sugar such as glucose.
Additional details are given in Chap. 15.

Tiie most important point to note in connection with photosynthesis is
that energy, 717.6 Cal. for a gram molecule of hexose sugar (180 g.), is
required to cause the reaction to take place. The energy thus stored
becomes available to man and other animals when the carbohydrate is
oxidized in the body:

Oxidation or respiration

CgHioOe + 6O2 = 6CO0 + 6H2O + 683 Cal.i

The supply of carbon dioxide furnished by animals and microorgan-
isms enables the plant to continue its life processes. Plants, animals,
and microorganisms are so interdependent that no one class could func-
tion in its normal manner without the activities of the other two.

Preparation of Glucose. Commercial glucose is made from starch,
corn starch in the United States and potato starch in Europe. To pre-
pare a glucose sirup the starch is suspended in water containing a small
amount of hydrochloric acid (0.6 per cent) and is heated under pressure
until the solution fails to give a red color with iodine, at which point
the solution still contains a large proportion of partially hydrolyzed car-
bohydrate. The acid is neutralized, and the liquid is decolorized with
powdered adsorptive charcoal and concentrated to a thick sirup contain-
ing about 80 per cent solids. Large quantities of this flat sirup are
used in making candy. For table use, cane sirup is added to the fiat
sirup to give it a better flavor; this is the so-called "corn" sirup. Owing
to certain peculiarities that it possesses, glucose is, in certain respects,

^ The ai)paront discfppancy between 717.6 and GS3 Cal. is due to differences in the
concentration and state of the reactauts and products in the two processes (see pp.
395 and 414).

30 CARBOHYDRATES

superior to sucrose for the making of fondants, creams, fancy candies,
chewing gum, doughnuts, and other products.

To prepare crystalhne glucose the hydrolysis is carried to completion;
the sugar solution is then concentrated in an evaporator to a density
of 1.36 to 1.45. The concentrated sugar solution is then introduced into
crystallizing vessels that contain some of the crystals from the preceding
batch. This practice of seeding the liquor is an essential step in obtain-
ing crystals of approximately the desired size and uniformity. The
crystals of hydrated glucose are separated from the mother liquor by
means of centrifugal machines, washed in the same machines, and then
sent through a drier. In 100-pound bags it now sells for about 8 cents
per pound.

The sweetness of glucose is approximately 75 per cent of that possessed
by our common sugar, sucrose. Its calorific value, however, is about
equal to that of sucrose. Glucose is readily jermented by practically
all microorganisms. The spoilage of fruits and vegetables is accom-
panied by a destruction of glucose. The manufacture of alcoholic bever-
ages is based upon the fermentation of glucose by yeast.

The most characteristic chemical property of glucose is its reduction
of solutions of copper salts with the formation of a precipitate, cuprous
oxide. The color of the precipitate varies from yellow to brick red,
depending upon the fineness of the particles of oxide. Some of the most
common copper reagents used in sugar tests are Fehling's (copper sulfate,
sodium potassium tartrate, and sodium hydroxide) , Benedict's (copper
sulfate, sodium citrate, and sodium carbonate), and Barfoed's (copper
acetate and acetic acid) solutions.

Glucose reduces these reagents because it is oxidized by the cupric
ion (Cu + + ) present. The process is dependent on the presence of the
aldehyde group in the glucose molecule or, in general, on the presence
in the sugar tested of an aldehyde or ketone group not attached to other
atoms in the form of a glycoside (p. 40). However, it is immaterial
whether the sugar is in the open chain or oxide ring form. In the latter,
the aldehyde (or ketone) group is apparently covered up, but it is still
a -potential aldehyde group because of the easy interconversion of the
chain and ring forms in solution.

Many other aldehydes such as formaldehyde, acetaldehyde, and chloral
also have reducing power. Reduction of Fehling's solution and similar
reagents must, therefore, be recognized, not as the peculiar attribute of
sugars, but rather as a general property common to many substances.

The chemical changes which reducing sugars undergo during the Fehl-
ing's test are very complex. Certainly one main reaction is oxidation
of the aldehyde group to a carboxyl with the formation of the correspond-
ing aldonic acid:

CARBOHYDRATES 31

CHO COOH

! I

(CHOH)„ + 2CuO *► (CHOH)„ + Cu^O

CH.OH CHjOH

An aldose Fehling's An aldonic Cuprous

reagent acid oxide

The copi)cr of Fehling's solution is actually present as a copper com-
pound of sodium potassium tartrate, NaKCuC4H20r„ but CuO more
clearly indicates the oxidizing character of the solution.

The above reaction accounts for less than half of the cuprous oxide
actually produced during the Fehling's reaction. The rest is produced
indirectly by the oxidation of simpler substances into which the reducing
sugars are converted by the strong alkali (sodium hydroxide) in the
reagent. Decomposition by alkali is a characteristic property of reducing
sugars generally. Nef isolated 93 substances from the decomposition
of sugars in alkaline solution.

Benedict's solution is a less sensitive reagent for reducing sugars than
Fehling's solution because it contains a weaker alkali, sodium carbonate.
This is an advantage, since it is used to test for sugar in urine, which
commonly contains small amounts of nonsugar reducing substances.
These materials are less apt to give a false result with Benedict's than
with Fehling's solution. Barfoed's reagent is not alkaline at all, but
rather is acidic; hence it requires a very strong reducing agent to pro-
duce a positive result. It is for this reason that the Barfoed's reagent
can be used to distinguish simple sugars from other carbohydrates (see
p. 42).

Any sugar capable of reducing Fehling's solution is called a reducing
sugar. All monosaccharides are reducing sugars, as are also the common
disaccharides, maltose, lactose, and cellobiose (but not sucrose). Fehl-
ing's reaction forms the basis of a useful method of analyzing foods for
their sugar content. A weighed sample is extracted with hot water,
and a portion of the solution so obtained, after treatment to remove
interfering substances, is hydrolyzed and allowed to react with Fehling's
solution under carefully standardized conditions. The precipitate of
cuprous oxide is collected and weighed, and from the weight found the
percentage of sugar in the sample may be calculated.

In the analysis of fruits, vegetables, sirups, candies, blood, urine, and
so on, total reducing sugar is generally expressed as glucose. No attempt
is made to distinguish between glucose or fructose as both have the
same nutritive value and approximately the same reducing power.

Glucose reacts with an excess of phenylhydrazine to form an insoluble
precipitate known as glucosazone according to the following equation:

32

CARBOHYDRATES

CHO

1

HCOH

I
HOCH

I
HCOH

I
HCOH

I
CH2OH

D-Glucose

CH=NNHC6H5

I
C=NNHC6H6

+ SCeHjXHNHi
(phenylhydrazine)

HOCH

I
HCOH

I
HCOH

+ CeHsNHj + 2H2O + NH3

Aniline

CH2OH

D-Glucosazone

This precipitate is made up of long yellow needles, usually arranged in
a broom-like or sheaf structure. The osazone is frequently of great
help in establishing the presence of glucose in a digestion product, fruit
juice, or other saccharine substance. It cannot be relied upon alone, how-
ever, because all reducing sugars give osazones. In fact, D-fructose and
D-mannose produce the same osazone as D-glucose does. In many cases,
however, the crystalline form, and more particularly the optical rota-
tion and melting point of the osazones, are of great help in identifying
individual sugars.

D-Galactose

Galactose does not occur free in nature but is found combined with
other sugars in many carbohydrates and related compounds. Each of
the sugars, lactose and raffinose, gives one molecule of galactose on
hydrolysis, and certain polysaccharides, the galactans, yield galactose
as the chief hydrolytic product. Legumes, impure pectin, agar, and
Douglas fir and other coniferous woods are other galactose-yielding ma-
terials. Galactose is also a constituent of certain galactosides found in
brain and nerve tissue, and of many animal proteins. Its occurrence
in these physiologically important tissues gives to galactose added sig-
nificance and importance.

Galactose is generally made from lactose by hydrolysis and crystalliza-
tion. Aside from the small amount required by bacteriological labora-
tories for the study of the fermentation characteristics of bacteria, there
is no particular demand for it.

Most bread yeasts do not ferment galactose, but many wild yeasts
ferment it readily. Bacteria, generally speaking, attack it more slowly
than either glucose or fructose. Being an aldose, galactose reduces
Fehling's solution and gives a characteristic osazone with phenylhydra-
zine.

The most distinctive property of galactose is the formation, when

CARBOHYDRATES 33

oxidized with nitric acid, of an insoluble dibasic acid, mucic acid. The
formation of mucic acid is used both as a qualitative and quantitative
test for the presence of galactose-yielding compounds. It is of use in
showing that milk has been used in the preparation of milk chocolate,
infant foods, and other preparations. The following equation indicates
the nature of the reaction:

CH20H(CHOH)4CHO + 30 = C00H(CH0H)4C00H + HoO
Galactose Mucic acid

The occurrence of L-galactose among the hydrolysis products of flax-
seed mucilage has been reported recently. Galactose is one of few
sugars (arabinose is another) which, thus far, has been found to occur
in nature in both d- and L-forms.

D'Mannose

Mannose does not occur in the free state in nature. However, it is
widely distributed in mannans, polysaccharides that yield mannose on
hydrolysis — compare fructosan, galactan, pentosan. That mannose may
play an important role in animal physiology is indicated by relatively
recent observations. Mannose is a constituent of egg albumin (1.77
per cent), serum albumin (0.45 per cent), and many other proteins
(0.3^.0 per cent) .

The hexahydric alcohol mannitol, C6H8(OH)6, corresponding to man-
nose, is also widely distributed in nature. It has been found in the
pineapple, onion, green bean, cauliflower, olive, mushroom, and in the
bark and leaves of many trees. It is the chief constituent of Sicilian
manna, a sweet exudate produced by a certain species of ash when
incisions are made in the bark. Many other trees and shrubs produce
mannas of varying composition as a result of the sting of certain insects.
It is supposed that the manna upon which the Israelites subsisted during
their wanderings in the wilderness was an exudate secreted by a species
of tamarisk tree. In Australia, India, and other countries manna from
different species of trees is used as a food by the natives. Many differ-
ent kinds of sugar have been isolated from these mannas.

D-Fructose

Fructose, also called levulose, is widely distributed ia nature, and in
the free state is generally associated with glucose and sucrose. It is
particularly abundant in fruit juices, whence comes the name fruit sugar.
Vegetables, the nectar of flowers, and the sap of green leaves and stalks
also contain fructose and glucose. Honey contains about equal quantities

34

CARBOHYDRATES

(40 per cent each) of these two sugars. The two occur frequently in
nearly equal amounts, and since they both are formed by hydrolysis
of sucrose, it is supposed that the two originate from the action of the
enzyme sucrase on sucrose. In some fruits such as apples and pears,
fructose seems to be more abundant than glucose, however.

Rafiinose and melezitose are two other sugars that yield a molecule
of fructose on hydrolysis. The polysaccharide inulin gives only fructose
on hydrolysis and thus stands in the same relation to fructose as starch
does to glucose. Fructose may be prepared from either sucrose or inulin,
but more easily from the latter.

In recent years considerable effort has been expended in an attempt
to produce fructose, or levulose as it is called in trade, on a commercial
scale. There would be a great demand for levulose at a reasonable price
because of its marked sweetening power — nearly twice that of sucrose.
The most promising source is the Jerusalem artichoke, a plant which
grows well in temperate climates and yields a high tonnage of tubers
per acre. The tubers are sliced and the sugars extracted in much the
same way as sucrose is extracted from the sugar beet. After hydrolysis
of the juice, levulose is precipitated as the calcium compound. This is
removed, decomposed by carbon dioxide, and the free sugar is obtained
either in the form of a sirup or, by careful concentration and cooling
of the sirup, as the crystallized product.

Neither glucose nor fructose crystallizes readily, but fructose has a
particularly strong tendency to remain in a sirupy condition. This is
well illustrated by honey, in which the glucose generally crystalUzes after
two or three months storage, while the fructose remains in a sirupy state.
Browne states that "the granulation of honey was known to the ancients
and crystallized glucose as thus observed was probably the first sugar
knowTi to mankind."

If fructose and glucose are present in the sirup from cane or sugar
beet, they interfere with the crystallization of the sucrose. This property
is used to advantage in the preparation of cane sirup from sucrose. Su-
crase, an enzyme obtained from yeast, is added to the warm sirup and
allowed to hydrolyze the sucrose for about 12 hours. At the end of
this time the sirup is further concentrated and may be stored without
danger of crystallizing. A similar effect is produced by the partial
hydrolysis of sucrose in making jelly and fondant. If sufficient fruc-
tose and glucose are present, the unhydrolyzed sucrose is prevented
from crystallizing and a smooth even texture results. Whenever
sucrose crystallizes, it imparts a rough gritty texture to the candy or
jelly.

D-Fructose is a ketohexose with the same configuration as D-glucose
about carbon atoms 3, 4, and 5:

CARBOHYDRATES

35

CHjOH

I

CO

I

IIOCII

I

HCOH

I

HCOH
I
CH2OII

Open-chain formula

CH.OH

^C-OH
HOCH 0

I H I
HCOHg CH,

H

Pyranose ring formula

D-Fructose

Many of the properties of glucose tliat have been noted are possessed
by fructose also. It is readily fermented by yeast and bacteria. By
the action of certain mannitol-forming bacteria (for example, L. pento-
aceticus) fructose is reduced to the hexahydric alcohol, mannitol. This
change takes place in the making of sauerkraut, silage, and certain wines.

Fructose and other ketoses reduce Fehling's solution and give the other
tests associated with reducing power fully as well as do aldoses. The
structure responsible for this reducing power is the ketone group situated
next to an alcohol group, thus: C = 0. In fact, this same structure is

HCOH
present in aldoses, as is illustrated below:

H2COH

— I--
C=0

I
HCOH

---[- — -
HCOH

I
HCOH

I
H2COH

Ketohexose

H

c=o

. .. I

HCOH

--H--

HOCH

I
HOCH

I
HCOH

H2COH
Aldohexose

As in the case of glucose, the sodium hydroxide in Fehling's solution also
brings about the decomposition of fructose into many simpler substances,
which become oxidized and contribute to the formation of the cuprous
oxide precipitate.

With phenylhydrazine, D-fructose forms an osazone, which is identical
with D-glucosazone and D-mannosazone (note identical structure and
configuration of carbons 3 to 6 in the formulas of these three sugars).

36

CARBOHYDRATES

The optical rotation of D-fructose, however, is very different from that
of D-ghicose, being —92.4° as compared to +52.7° (see Table 3-2).

Fructose and other ketoses can be distinguished from aldoses by the
resorcinol test, which consists in the production of a blood-red color
when a ketose is boiled with a solution of resorcinol in hydrochloric acid.
Since other ketoses are not common, a positive resorcinol test is a' good
indication of the presence of D-fructose or of other carbohydrates which
produce D-fructose on hydrolysis (sucrose, raffinose, melezitose, inulin,
etc.).

Table 3-2

Melting points and optical rotations of common sugars

Sugar
D-Xylose

Melting
point [°C]*
145

Optical Melting Optical

rotation [a] v'f Sugar point [°C] rotation [a]n

+ 18.8 L-Sorbose 161 —43.4

— 104.5 D-Glucosamine .. 110 +70 tt

+ 104.5 D-Glucuronif acid 156 y +36.3

—23.7 Lactose 202 +52.6

+52.7 Sucrose 188 +66.5

+80.2 Maltose 103 +130.4

+ 14.2 Cellobiose 225 +34.6

—92.4 Trehalose 97 +178.3

lJ—^\.^ IWOC Xll/

D-Arabinose 160

L-Arabinose 160

D-Ribose 87

D-Glucose 146

D-Galactose 167

D-Mannose 163

D-Fructose 104

* Of form obtained most commonly.

t Specific rotation of the equilibrium mixture of a and (3 forms (if any) measured
in water at or near 20°C.
tt Hydrochloride.

Ketoses are also differentiated from aldoses by greater resistance to
mild oxidative treatments. For example, under proper experimental con-
ditions aldoses can be converted almost quantitatively into the corre-
sponding aldonic acids by oxidation with iodine in alkaline solution
according to the equation:

CHO
I
(CHOH)^ + I2 + 3NaOH

CH.OH

Aldose

COONa

I
(CHOH)^ + 2NaI + 2H2O

CH2OH

Aldonic acid
(sodium salt)

Under identical conditions ketoses remain practically unaffected.

h'Sorbose

This ketohexose is found in nature only in the fermented juice of
mountain ash berries, where it undoubtedly arises as a result of bacterial
oxidation of D-sorbitol. As an industrial product, however, it has acquired

CARBOHYDRATES

37

considerable importance in recent years because it is an intermediate
in the synthesis of vitamin C. It is produced on a commercial scale
by selective bacterial oxidation of D-sorbitol with Acetobacter suboxydans.
The necessary sorbitol is produced by the chemical reduction of D-glucose:

CHO

1

CHjOH
HCOH

CH2OH

HCOH

HC-OH
1

HOCH
1

H:

HOCH
HCOH

1
0 HOCH

HCOH

1

(chemicai)

i. 1

(.4. suboxydans) TTpnTT
1

HCOH

1

HCOH
1

1

CO

1

CHaOH

CH2OH

1

OH, OH

D-Giucose

D-Sorbitol

L^orbose

Hexosamines

The two amino sugars found in nature are related to common aldo-
hexoses and in each instance bear the amino group on carbon 2:

CHO

I
HC— NH2

I
HOCH

I
HCOH

I
HCOH

CHO

I
HC— NH2

I
HOCH

I
HOCH

I
HCOH

CH2OH
D-Glucosamine

CH2OH
D-Galactosamine

D-Glucosamine (chitosamine) is the sole constituent sugar formed by
hydrolysis of chitin; it is also a component of mucin and several other
animal and bacterial polysaccharides. An unusual derivative, N-methyl-
L-glucosamine, is one of the components of streptomycin, an important
antibiotic. The chief natural occurrence of D-galactosamine (chondro-
samine) is as a component of chondroitin in cartilage (p. 67).

Both sugars show the reactions of aldohexoses (reducing power, osa-
zone formation) and, in addition, have the basic properties of the amino
group.

Desoxyhexoses

These sugars, which are also known as methyl pentoses, lack, the
oxygen atom on carbon 6:

38

CARBOHYDRATES

CHO

CHO

HC— OH

HOCH

HO— CH

HCOH

HC— OH

HCOH

HC— OH

HOCH

CH3

CH3

6-Desoxy-D-glucose

6-Uesoxy-L-galactose
(L-fucose)

CHO

I
HCOH

I
HCOH

I
HOCH

I
HOCH

I
CH3

6-Desoxy-L-mannose
(L-rhamnose)

They are found in many plant species, particularly in the form of
glycosides. These substances show the usual properties of monosaccha-
rides, except that on heating with strong acids they yield 5-methyl
furfural (contrast aldopentoses, aldohexoses). L-Rhamnose is probably
the most abundant of these three sugars in nature. It is' one of the
component sugars in several plant gums and mucilages (p. 66) , in the
important heart stimulant drugs known as cardiac glycosides, and is
present in at least two antibiotics of bacterial origin. These particular
antibiotics represent an interesting chemical type because they consist
of the sugar, L-rhamnose, attached to a fatty acid, beta hydroxy decanoic
acid.

Vronic acids

Those simple sugar derivatives which have both an aldehyde and a
carboxyl group are termed uronic acids. Three occur in nature, all re-
lated to aldohexoses:

CHO

CHO

CHO

HCOH

HOCH

HCOH

HOCH

HOCH

HOCH

HCOH

HCOH

HOCH

HCOH

HCOH

HCOH

COOH

COOH

COOH

D-Glucuronic acid

D-Mannuronic acid

D-Galacturonic acid

D-Glucuronic acid is found in the animal body as a component of
mucopolysaccharides (p. 67) such as heparin and chondroitin. It is
utilized by the body to detoxify various harmful drugs which may be
ingested. For example, if dogs are fed borneol, it is excreted in the

CARBOHYDRATES

39

urine as borncol glucuronidc. A number of bacterial carbohydrates,
particularly the innnunopolysaccharides, form D-glucuronic acid on
hydrolysis. It is also a component of nearly all of the plant gums such
as gum arabic and cherry gum.

D-Galacturonic acid is pei'haps best known as the fundamental build-
ing block t)f pectic acid, although it is also one of the components of
many plant mucilages. AMien hydrolyzed, alginic acid, from sea weeds,
yields D-mannuronic acid as the only primary product.

The most characteristic chemical projierty of the hexuronic acids is
the ease with which they lose carbon dioxide (decarboxylation) on heat-
ing with mineral acids. The carbon dioxide production is essentially
quantitative, being used both for detecting uronic acids and for determin-
ing their quantity. It is possible that pentoses arise in nature from
hexuronic acids, since members of each group with corresponding con-
figurations often occur together, e.g., D-galacturonic acid and L-arabinose:

CHO CHO

I I

HCOH HCOH

I I

4-CO2

HOCH

?

HOCH

HOCH

HOCH

HCOH

CH2OH

COOH

D-Galacturonic acid

L-Arabinose

However, pentoses cannot be isolated from the products of the chemical
decarboxylation of the uronic acids. Furfural is formed as from pen-
toses, but in smaller yields up to about 40 per cent, wdiereas pentoses
give 70-80 per cent of the theoretically possible amount.

A qualitative test for hexuronic acids consists in boiling with hydro-
chloric acid and naphthoresorcinol. A blue pigment is formed which can
be extracted with the organic solvents, ether or benzene. Pentoses and
a few other sugars give a similar test. In fact pentoses and uronic acids
in general tend to show similar properties, except for the carbon dioxide
evolution already mentioned.

DISACCHARIDES

Glycosides

Simple sugars have a marked ability to combine with other molecules
which contain —OH groups. The combination always involves the —OH
group on carbon 1 of the simple sugar if it is an aldose, or 2 if a ketose,

40

CARBOHYDRATES

both being in the oxide ring form. The process may be illustrated by
the combination of glucose with methyl alcohol:

CHjOH

CH2OH

+ CH3OH

OH

HO

H OH

a-D-Glucose

+ H2O

OCH3

H OH

Methyl a-D-Glucoside

As a class such substances are termed glycosides, but individual members
are named from the component parts, as indicated in the above example.
Both a- and ^-glycosides may be formed from the corresponding a- and
y8-sugars.

The glycosides do not show the simple sugars' characteristic proper-
ties of reducing power or osazone formation because the aldehyde or
ketone group is covered up. Glycosides are rather stable to alkalies
but are hydrolyzed by acids to form the original components.

If the second molecule with which a monosaccharide combines happens
to be that of another monosaccharide, the product is a disaccharide.
A disaccharide may therefore be defined as a glycoside formed from two
simple sugar molecules by removing one molecule of water.^ The second
simple sugar may be either the same kind or a different kind from the
first. For example, two molecules of glucose may combine as follows:

CH2OH

CH2OH

HO

H OH

a-D-Glucose

H OH

a-D-Glucose

CHjOH CH2OH

a XT Tx J^-— 0.

+ n,o

H OH H OH

4-D-Glucosyl-o;-D-glucose

The product in this case is an a-D-glucoside with the second glucose unit
attached through its carbon 4; it is therefore called 4-D-glucosyl^a-

^ This statement is intended to be a definition only. Disaccharides probably are
not actually produced in living cells by removing water from simple sugars (see
p. 399).

CARBOHYDRATES

41

D-glucose. This particular disaccharidc is maltose. The "disaccharide
hnkage" connecting the two simple sugar units in this case is an a-type
and goes from carbon 1 of one unit to 4 of the other. This is often
abbreviated to an "a-l,4-Unkage."

Any alteration in the nature of the disaccharidc linkage, or in the
component sugars, results in a different disaccharidc. For example, two
glucose units combined by a /?-l,4 linkage form the disaccharidc, cello-
biose, which is a substance distinctly different from maltose. The chem-
ical make-up of the more common disaccharides is shown in Table 3-3.

Common name and
component sugars

Sucrose :

D-glucose

D-fructose
Lactose:

D-glucose

D-galactose
Melibiose :

D-glucose

D-galactose
Maltose:

D-glucose

D-glucose
i.so-Maltose :

D-glucose

D-glucose
Cellobiose:

D-glucose

D-glucose
Gentiobiose:

D-glucose

D-glucose
Trehalose :

D-glucose

D-glucose

* For simplicity,
from these names,
structural formulas

Table 3-3

Chemical constitution of disaccharides

Disacchaiide
linkage

a,P-l,2

P-1,4

a-1,6

a-1,4

a-1,6

P-1,4

P-1,6

a, a-1, 1

Chem.ical
name*

1-a-D-glucosyl-P-D-
fructose

4-D-glucosyl-(3-D-
galactose

6-D-glucosyl-a-D-
galactose

4-D-glucosyl-a-D-
glucose

6-D-glucosyl-a-D-
glucose

4-D-glucosyl-P-D-
glucose

6-D-glucosyl-P-D-
glucose

1-a-D-glucosyl-a-D-
glucose

the designation of furanose and pyranose rings has been omitted
All are pyranose except for the fructose unit in sucrose (see
for the individual disaccharides).

The disaccharides may or may not have reducing properties, depending
on whether the disaccharide linkage involves the aldehyde (or ketone)
group of only one of the component simple sugars, or of both. In the
latter case since no free, or potentially free, aldehyde or ketone group
remains in the disaccharide, it therefore gives no osazone and does not
respond to the Fehling's or other similar tests. The structures of sucrose
and trehalose are of this nonreducing type.

42

CARBOHYDRATES

On the other hand such disaccharides as maltose, lactose, and the
others in Table 3-3 do contain a potential aldehyde group and show
the characteristic reactions of reducing sugars, although to a smaller
degree than the monosaccharides. This lowered reducing power is not
surprising when it is remembered that even in the reducing disaccharides
one reducing group has been covered up in forming the disaccharide
linkage. The Barfoed test for monosaccharides is' based on the stronger
reducing power of the simple sugars as compared to the disaccharides.

Like other glycosides, the disaccharides can be hydrolyzed, whereupon
they take on a molecule of water and form the corresponding simple
sugars. This hydrolysis may be brought about by heating the disac-
charide with dilute acid, or by the action of certain enzymes. Such hydro-
lytic enzymes are found in the digestive tracts of animals, in yeasts, bac-
teria, and molds, and in many higher plants. The enzymes are named
according to the sugar upon which they act; sucrase (also called invertase)
acting on sucrose, maltase on maltose and lactase on lactose. The equa-
tion of hydrolysis is as follows:

CioHooOu + HoO = CeHjoOe + CeHioOe

Sucrose Glucose Fructose

Maltose Glucose Glucose

Lactose Glucose Galactose

The mixture of glucose and fructose formed by hydrolysis of sucrose is
called "invert sugar." Obviously it consists of equal parts of glucose
and fructose.

Sucrose

This sugar is known also as saccharose, cane sugar, beet sugar, or
simply "sugar." As already stated in connection with glucose and fruc-
tose, sucrose is generally associated with these monosaccharides in flowers,
fruits, roots, and seeds of plants. It is especially abundant in sugar
cane, sugar beet, sorghum, and the sap of the maple and palm. The
first two plants, which contain 16-20 per cent sucrose, are the chief
commercial sources of this sugar. Sorghum contains an abundance of
sucrose, but it has not as yet been possible to produce sugar successfully
from this plant.

Annual world production of raw sugar during the last 5 years has been
30-35 million tons, and is still rising. About two-thirds of the total
is produced from sugar cane and nearly all of the rest from sugar beets.
The United States and its island possessions, together with the Philippine
Islands, produce about one-sixth of the world total. Louisiana and
Florida are the leading cane sugar states; California and Colorado, the
beet sugar states.

The annual per capita consumption of sugar in the United States is

CARBOHYDRATES

43

very high, running to over 100 lbs. in most years. The desirabiUty of
such a Large consumption of sugar is doubtfuL Sugar suppUes about one-
sixtli of the cahiric intake and hence displaces the consumption of less-
refined foods that would carry minerals and vitamins as well as energy,
Sherman, who has given much thought to this matter, suggests that,
instead of devoting so much land, labor, and money to the production
of sugar, it would be a wiser policy to increase the production and con-
sumption of foods which furnish needed nutrients as well as calories.
However, it should be pointed out that sucrose is one of the very cheapest
sources of food energy. A comparison of various low-cost foods from
this standpoint is given in Table 3-4.

— The manufacture of sucrose is an excellent example of a chemically
controlled industry. From the determination of the sugar content of
the raw beet to the analysis of the finished product, it is an application
of the principles involved in the preparation of any pure chemical. Ex-
traction, clarification, evaporation, and crystallization are the important
steps involved. Because of the ease w'ith which sucrose crystallizes, it
lends itself readily to this method of purification.

Sucrose is sweeter than glucose but not so sweet as fructose. It is
claimed by the majority of investigators that invert sugar, which is formed
when sucrose is hydrolyzed, is sw-eeter than sucrose, but there is consider-
able difference of opinion on this point. It is difficult to determine the
comparative sw^eetening power of sugars owing to the fact that small
differences in concentration cannot be detected by the sense of taste.
For example, sucrose solutions differing by less than 1.5 per cent cannot
be readily distinguished. Some of the sugars have other tastes tlian
that of sweetness, which complicates the comparison. The compara-
tive sweetness of sugars, giving sucrose a value of 100, has been rated
as follows: lactose 16, raffinose 23, galactose 32, rhamnose 33, mal-
tose 33, xylose 40, glucose 74, sucrose 100, invert sugar 130, fructose

173.

In cooking operations, such as the making of jelly where sucrose is
hydrolyzed, it would seem that the proper time to add the sucrose is
at the beginning of the cooking. This insures the maximum hydrolysis
of sucrose, and consequent maximum sweetening power. Moreover, the
hydrolysis products, glucose and fructose, prevent crystallization (grain-
ing) of unhydrolyzed sucrose and give the best conditions for producing
a jelly of smooth texture. Approximately 50 per cent of the added
sucrose is converted into invert sugar by the usual methods of jelly
making. Any cooking operation that involves the use of sucrose and
acid, such as the canning of fruit and making of jams and of many
kinds of pie, will bring about a considerable hydrolysis of sucrose. It
is probable that in many other cooking operations sucrose undergoes
slight hydrolysis as a result of the effect of salts and other food con-
stituents.

44

CARBOHYDRATES

Table 3-4

Cost of food calories as provided by various low-cost foods

Price per Calories per - Cost per 100

Food pound [cents] * pound calories [cents]

Potatoes 5 377 1.32

Bread, white 14 1250 1.12

Macaroni 17 1710 1.00

Rice 15 1670 0.90

Beans, dry 13 1530 0.85

Flour, patent 9 1650 0.55

Sucrose 10 1750 0.57

* Retail prices at Madison, Wisconsin, January, 1952.

The chemical constitution of sucrose is expressed by the following
formula:

H,COH

0. j^ H2COH ^O^ H2COH

H OH OH H

a-GIucoside part ^-Fructoside part

Sucrose

Note that the disaccharide linkage is a,(3-l,2 and involves the original
reducing groups of each of the component simple sugars. Hence sucrose
does not reduce Fehling's solution or give an osazone with phenylhydra-
zine. It is fermented by yeast and by most bacteria. Strictly speaking,
sucrose is not fermented because it is first hydrolyzed; the resulting
glucose and fructose undergo the fermentation. Likewise, in the utiliza-
tion of sucrose by the body, hydrolysis precedes absorption.

Sucrose may be estimated either by chemical means or through the
aid of a saccharimeter.^ (See optical rotation.) If reducing sugar is
determined before and after hydrolysis, the increase in reducing sugar
furnishes a means of calculating sucrose. Since a molecule of water is
added during the hydrolysis, 95 per cent of the invert sugar (increase
in reducing sugar) is equivalent to the sucrose in the sample. For
€xample:

per cent

Reducing sugar before hydrolysis 4.36

Reducing sugar after hydrolysis 9.28

Invert sugar 4.92

Sucrose, .95 X 4.92 4.67

^ This is a polarimeter especially calibrated to read percentage of sucrose in the
solution tested rather than the angle of rotation of the polarized light.

CARBOHYDRATES

45

The figure 95 per cent is obtained from the hydrolysis equation of
sucrose and is the ratio of tlie niolccuhnr weight of sucrose to the sum
of the molecular weights of glucose and fructose, the sugars of which
invert sugar is composed (342 -^ 360 = 0.95).

Optical Rotation of Sucrose. The rotation of polarized light is the
basis for determining sucrose by means of a saccharimcter. This instru-
ment enables the beet sugar manufacturer to determine what he should
pay for his beets and the custom house official to decide what should be
the import duty on a cargo of sugar. It is as important to tlie sugar
industry as the "Babcock Tester" is to the dairy industry and serves
as an outstanding example of an abstract physical property becoming
of great economic value.

Sucrose is dextrorotatory (+66.5), but invert sugar is levorotatory
( — 19.85) because fructose rotates polarized light more to the left
(—92.4) than glucose does to the right (+52.7). Because the rotation
is reversed (inverted) when sucrose is hydrolyzed, the hydrolysis of
sucrose is called "inversion." The change in the direction of rotation
is also the reason for the terms "invert sugar" and "invertase" — the
name of the enzyme that effects the hydrolysis. Sucrase is a better name
for this enzyme because it denotes which sugar is hydrolyzed. The term
"inversion" can be properly applied only to the hydrolysis of sucrose
because the hydrolysis of other sugars is not accompanied by a change
in the direction of optical rotation. By determining the rotation of a
sugar solution, for example, from cane, beets, fruits, and so on, before
and after hydrolysis, the percentage of sucrose may be determined because
the change in rotation is directly proportional to the quantity of sucrose
present. The saccharimcter enables the analyst to determine in a few
minutes the percentage of sucrose and thus puts all operations in the
sugar industry on an exact basis.

Malt

ose

This disaccharide is widely distributed in leaves and young seedlings
and is especially abundant in germinating seeds. It is the principal
sugar formed by the action of the digestive enzymes ptyalin and amylopsin
on starch and glycogen. In the germination of seeds a starch-splitting
enzyme, diastase, is produced and brings about the conversion of the
insoluble starch into a soluble sugar, maltose, which is utilizable by the
plant cells. Additional information on starch-splitting enzymes is given
on p. 58.

Malt sirups can be made from the water-soluble material of germinated
barley. Also a sirup can be prepared from the sweet potato' by steeping
the finely cut potato in water at 40°C. for a few hours. After being
filtered from insoluble material, the solution is concentrated to a thick
sirup, having the flavor of the sweet potato.

46

CARBOHYDRATES

Malted milks and certain infant foods contain the water-soluble ma-
terial of germinated barley. The water extract, evaporated to dryness
and mixed with the other ingredients, imparts the peculiar malt flavor
to these products. Pure maltose can be prepared by digestion of starch
with diastase, followed by evaporation and crystallization from 60 per
cent alcohol. It is not much used in the crystalline form.

As is indicated by its formula, maltose is a reducing type disaccharide:

CH,OH

CH2OH

OH

OH H

Maltose

Note that carbon 1 of the right-hand glucose unit, as the formula is
written above, is an aldehyde group in the oxide-ring form (compare with
the formula of a-D-glucose, p. 25). It therefore gives a positive test
with Fehling's solution and an osazone with phenylhydrazine. The
osazone is rather soluble in water but usually separates on cooling in
the form of daisy-like crystals (Fig. 3-1). Note also that the two
glucose units are held together in an a-l,4-linkage. This same type of
linkage is present in several of the more common polysaccharides such
as starch and glycogen.

Yeasts, bacteria, and other microorganisms ferment maltose with
about the same ease that they ferment glucose. It is assumed that the
maltose is first hydrolyzed and then fermented.

Cellobiose

Like maltose, this disaccharide, which does not occur free in nature,
is composed of two glucose units attached through the 1 and 4 positions,
but unlike maltose, the disaccharide linkage is the /3-type:

CH2OH

CH2OH

OH

Cellobiose

Note that in the left-hand glucose unit, as the formula is written above,
the configuration of carbon 1 is jS, whereas in maltose it is a. This
is the only structural difference between cellobiose and maltose. Cello-

CARBOHYDRATES

47

Fig. 3-1. Ciy.stalliue form of derivatives of various sugars: (a) glucosa-
zonc, (b) lactosazone, (c) .xylosazone, id) maltosazone, (e) galactosazone,

(f) mucic acid.

biose is of interest particularly in connection with the chemistry of
cellulose, whicii aL-^o is built \\\) from tilncosc unit.>< attached to each
other through /?-l,4-linkages. Cellobiose, in fact, bears the same rela-
tionship to cellulose as maltose does to starch and can be obtained from
cellulose by partial hydrolysis.

48 CARBOHYDRATES

Lactose

This sugar has been found only in the milk of mammals. It varies
from 1.5 to 8 per cent depending upon the species. Human milk con-
tains from 4 to 6.3 per cent and cow's milk about 5 per cent. Based
on the annual production of milk (more than 120,000,000,000 lb.), it
is estimated that the consumption of lactose in the United States is equiva-
lent to more than one-third that of sucrose. Lactose is obtained from
skim milk by removing the casein with acid or rennet and purifying
the resultant whey by heating and liming. The clear liquor from these
treatments is concentrated in a vacuum pan until crystallization begins.
The hot sirup is then transferred to cooling pans and stirred until crystalli-
zation is complete. The mush of yellow crystals is dropped into a
centrifuge, freed of excess sirup, and washed with cold water. The crude
sugar is refined by dissolving, bone-blacking, and recrystallizing. The
refined sugar is dried and ground to pass a 200 mesh sieve. The yield
of refined sugar averages about one-half the lactose contained in the
whey. Lactose production in 1949 amounted to 19,025,000 lb., but this
is only a small part of what could be produced if there were sufficient
demand for it. Smith and Claborn estimate that at least 2,700,000,000
lb. could be made from available skim milk, buttermilk, and whey.
Much of the highly purified lactose is used in infant feeding, and in the
manufacture of infant foods and pharmaceutical preparations. Large
quantities of pure lactose are also used in the production of penicillin.

Lactose is not very soluble and is almost tasteless. It gives a faint
suggestion of sweetness, but this is slight in comparison with the sweet-
ness of glucose or sucrose. A more soluble and sweeter form of lactose
can be made by crystallizing the sugar at a temperature above 95°C.
This sugar is known as anhydrous beta lactose. The milk sugar of
commerce is hydrated alpha lactose. The anhydrous beta lactose appears
to be stable for a considerable time at ordinary conditions of tempera-
ture and moisture. Because of its greater solubility and sweetening
power, it appears that there should be a demand for this product. A
more general use of lactose has been advocated for the reason that the
ingestion of lactose helps to maintain a healthy condition of the intestinal
tract. Although many bacteria are unable to use lactose, others ferment
it readily and thus are favored in their development. Among the latter
are L. acidophilus, a lactic acid-producing microorganism, which is more
or less abundant in the intestinal tract. The growth of this desirable
organism is favored by an abundance of lactose, and its development
results in an acid, reaction that checks the growth of the troublesome
proteolytic bacteria. Lactose is not fermented by ordinary yeasts.

Lactose reduces Fehling's solution and gives a characteristic osazone,
both of which tests indicate the presence of an aldehyde group in its
structure. This compound is shown in the following formula:

CARBOHYDRATES

49

CH2OH

CH2OH

H OH H OH

/3-Galactoside part Glucose part

a -Lactose

Note that lactose is a galactoside, not a glucoside, and that the disac-
charide Hnkage is a /?-l,4-typc. The (potential) aldehyde group is at
carbon 1 of the glucose part. In the formula above, the configuration
of this reducing group is written as a (—OH down). In j8-lactose the
configuration around this carbon is reversed, but the structure is other-
wise identical to that of ordinary a-lactose.

The mucic acid test is positive with lactose because of the galactose
component which is set free by hydrolysis during the test.

THE TRISACCHARIDES, CisH^aOie

Raffinose

Rafiinose is the most important trisaccharide. It is found in small
quantities (2-5 per cent) in cottonseed, beet molasses, and manna, and
to a less extent in barley, wheat, and other cereals. The amount of
raffinose in sugar beets increases considerably as a result of abnormal
climatic conditions such as drought and freezing. The sucrose from
such beets is hard to crystallize properly as it tends to take on an
elongated pointed form. Raffinose is not readily fermented, does not
reduce Fehling's solution, and on hydrolysis gives one molecule each
of fructose, glucose, and galactose.

Melezitose

Another trisaccharide that has attracted some notice because of its
occurrence in the exudate of the Douglas fir and other coniferous trees
is melezitose. In dry seasons the trees become laden with an exudate
very rich in this sugar. At such times honey bees gather the material
and incorporate it into the honey, where it soon crystallizes and may
suggest adulteration. Upon hydrolysis it yields two molecules of glu-
cose and one of fructose.

POLYSACCHARIDES

The polysaccharides are the most complex, as well as the most numer-
ous and abundant, of the carbohydrates found in nature. They are

50

CARBOHYDRATES

made up of many molecules of one or more simple sugars combined
by glycosidic linkages. For example, a molecule of the polysaccharide
amylose, a form of starch, consists of about 200-300 glucose units
attached to each other by a-l,4-linkages as shown by the following
formula:

CH2OH

CH,OH

CH.OH

H

OH

CH2OH

jAo^V_i

H

Amylose

Only four glucose units are shown in this formula, the rest merely being
indicated to save space by the n outside the brackets. This abbreviation
means that the part inside the brackets is repeated n times in the com-
plete formula, n being about 100-150 maltose units in this particular
ease. Thus amylose, like all polysaccharides, is a very large molecule,
far bigger than the mono-, di-, and trisaccharides so far considered.
The molecular weight of amylose is in the range of 10,000-100,000 (no
exact value can be determined), whereas sucrose, for example, has a
molecular weight of only 342. Polysaccharides in general have molecular
weights ranging from a few thousands to several millions.

The example given above, amylose, represents the simplest type of
polysaccharide structure, i.e., a long series of simple sugar residues, all
of the same kind attached to each other in a single long chain. A second
type consists of a branching structure rather than a single chain. Gly-
cogen is an example of this type of polysaccharide. Its structure is
indicated by the following diagram, in which each small circle repre-
sents a glucose unit:

o
o

o 000 O OOOo

%

o
o

o

0°

o o

o o

o o

o o

o
o

o

o

o

o

o

00000000000000000000

ooooooo 0000000000

o o

o o

o o

0000000000000 00 00 00 000 o o

o 2

o nOO°°° ooo

OOO ^0° O .aO°

0^0

o
o
o
o
o

o"Oo o^

•oo

000

Diagram of glycogen, a branched polysaccharide

CARBOHYDRATES 51

A third, and still more complex, type of polysaccharide is made up
of several different kinds of simple sugar units, which may be arranged
either in a single chain or in u branched structure. These are called
heteropolysacchaiides, whereas those containing only one sugar are
classed as homopoly saccharides. The chemical formulas of homopoly-
saccharides are often written in a still more abbreviated form than that
of amylose given above. Since one molecule of water is taken away
when each glycoside linkage is formed, most of the simple sugar units
in the polysaccharide structure (in fact all except those at chain ends)
must have the composition of the simple sugar concerned, less one
oxygen and two hydrogen atoms. Thus the formula of a pentosan
(polysaccharide made up of pentose units) may be written as (C5H804)a,
and that of a hexosan as (CeHioOy)^;. These formulas are commonly
used because they are compact and easy to write, but they are not
precisely correct.

Another important feature of polysaccharide structure is the glycosidic
linkage between the monosaccharide residues. This linkage always
extends from the reducing group of one simple sugar unit to one of the
other carbons of the next unit. This second unit is attached through
its reducing group to a third, and so on. Thus no uncombined reducing
groups are present in the polysaccharide molecule except the one at the
end of the chain (see formula for amylose above). Even branched poly-
saccharides like glycogen have only one reducing group per molecule.
Consequently, polysaccharides as a rule have practically no reducing
power.

As a class the polysaccharides are noncrystalline, white solids, which
are insoluble or only slightly soluble in water. Probably as a result
of this limited solubility they have no appreciable sweetening power.
On boiling with dilute solutions of strong acids they are all hydrolyzed,
although at greatly differing rates, into the component monosaccharides.

Pentosans iC5H804)j.

Polysaccharides giving D-xylose or L-arabinose on hydrolysis, that is,
the pentosans, are very common in nature, especially in the plant kingdom.
Most of them, however, arc not comjiosed exclusively of pentose residues,
but also contain various hexoses, or hexuronic acids, or both, atid thus
belong to the mixed type of polysacciiaridcs.

The total amount of pentosans contained in various plants is shown
in Table 3-5. It will be noted that the largest percentages are found
in two main types of plant materials, the plant gums, and the woody
or fibrous tissues. Xylan occurs chiefly in wood, straw, leaves, seeds,
and vegetables, whereas araban is commonly found in gums and mu-
cilaginous materials. Xylan is frequently associated with glucose in

52

CARBOHYDRATES

a double anhydride as gluco-xylan; arabinose may be paired with galac-
tose as a galacto-araban.

Table 3-5
Pentosans in plant materials

[Undried basis]

per cent

Navy bean 8.4

Com meal 5.0

Corn (whole) 7.4

Dried peas 7.2

Barley (whole) 11.1

Cottonseed flour 5.6

Beets 1.7

Spinach 1.0

Cabbage 1.0

Wheat bran 22.0

Wheat straw 27.1 ,

Corn fodder 21.8

Com cobs 35.0

Gum arabic 26.0

Cherry gum 52.0

White pine wood 7.0

Maple wood 21.7

Although the physiological function of pentosans is still obscure, it
is doubtful that they are merely the result of an accumulation of waste
material. Their very general occurrence in plant material probably
indicates that they perform an important function in the life of the plant.
Their close relation to cellulose suggests the possibility that they are
particularly concerned with structural requirements. Pentosans may
also serve as a reserve carbohydrate in the metabolism of the apple
tree and thus play an important part in the bearing of fruit.

On hydrolysis, pentosans give pentoses, as is represented by the follow-
ing equation:

(C5H8O4), + a:HoO = a:C5Hio05

When substances containing pentosans are boiled with relatively con-
centrated solutions of mineral acids (HCl, H2SO4, or H3PO4), the pen-
tosans fire hydrolyzed to pentoses, and the pentoses are converted into
furfural, as already explained. The red color obtained when furfural
reacts with aniline acetate (p. 26) thus serves as a good qualitative
test for pentosans. The presence of pentosans in vegetables and whole
cereals may be easily demonstrated by holding a piece of filter paper
moistened with aniline acetate in the vapors which are evolved when
the sample is boiled with 20 per cent hydrochloric acid. By condensing
the vapors containing the furfural and adding phloroglucinol, a precipitate

CARBOHYDRATES

53

is formed that may be weighed. From this weight the quantity of
pentosans can be calculated.

Furfural is also an interesting and important chemical for other reasons.
It offers a means of utilizing agricultural waste products such as oat
hulls, corn cobs, etc., because these residues contain large amounts of
pentosans which can be converted into furfural by a simple, cheap process.
The crude furfural so produced is a brownish, oily liquid, which sells
for about 10 cents per pound. Large amounts have been used in petroleum
refining, and more recently as the starting material for the making of
nylon.

The nutritive value of the pentosans is still an unsolved problem. In
passing through the digestive tract, considerable quantities disappear.
In herbivora from 50-75 per cent of the pentosans, and in man about
15 per cent, appear to be utilized. This utilization must be an indirect
one for no digestive enzymes that bring about hydrolysis of pentosans
are known to occur in higher animals. It is assumed that the bacteria
of the intestine break down the pentosans into soluble products such as
acetic and lactic acids, which are then absorbed and utilized. Consider-
ing the large amount of pentosans consumed by herbivorous animals, it
seems that such a fermentation must be very rapid.

Hexosans (CeHioOs)^,

Starch is the most important food carbohydrate. It contributes more
calories to the usual diet of human beings than any other single sub-
stance. Ordinary starch, as it is found in plants, is a mixture of amylose
and amylopectin. Usually there is a much greater proportion of the
amylopectin. For example, corn starch and potato starch each contain
about four-fifths amylopectin and one-fifth amylose. The so-called waxy
corn starch is almost all amylopectin. The two fractions can be separated
by dispersing the crude starch in hot water saturated with butyl alcohol.
On cooling slowly, the amylose separates as a semicrystalline precipitate,
which is easily removed. The amylopectin can then be recovered from
the solution.

Both amylose and amylopectin are polysaccharides made up of anhydro-
glucose units attached to each other by a-l,4-linkages. Amylose, as
explained on p. 50, is a linear-type polysaccharide, consisting of a long,
unbranched chain of about 200-300 glucose units. On the other hand,
amylopectin has a branched structure somewhat similar to that of gly-
cogen (see diagram on p. 50). At the point of branching, a-l,6-linkages
are present. The molecular weight of amylopectin is thought by many
investigators to be at least 500,000, which corresponds to 2000-3000
glucose units in the molecule.

Amylose is less soluble in water than amylopectin. It gives a clear

54 CARBOHYDRATES

blue color with iodine, whereas amylopectin gives a violet-blue color.
This color is attributed to the branched structure of amylopectin.
Glycogen, which is even more highly branched than amylopectin, gives
only a red-brown color in the iodine test. Neither component of starch
shows any reducing power unless very refined tests are employed. Thus
the usual Fehling's test is entirely negative with native starch. How-
ever, soluble starch, which is made by subjecting starch to a mild acid
and heat treatment, does give a positive Fehling's test. This effect indi-
cates that the process of making the starch soluble has also resulted in
some decomposition and liberation of aldehyde groups.

Starch usually contains a few hundredths of a per cent of phosphorus,
probably as a result of the fact that it is formed in plants from glucose- 1-
phosphate. Fatty acids (for example, oleic, linoleic, and palmitic) have
been found in various cereal starches, but it is probable that they are
present as impurities rather than as actual constituents of the starch
molecules since they can be removed by extraction with boiling methyl
alcohol.

Starch is found in almost all chlorophyll-bearing plants. It is es-
pecially abundant in the common cereals (wheat, rye, oats, and rice) ;
it makes up from 60 to 80 per cent of the seed. Also peas and beans
may contain 50 per cent of starch. In certain oily seeds {e.g., cottonseed,
flaxseed, and soybeans) fats, instead of starch, form the storage ma-
terial. As a general rule seeds grown in the tropical regions are oil
bearing, whereas those of the temperate regions are high in starch. Many
tubers, such as the potato, are made up largely of starch. When unripe,
the apple and banana contain considerable quantities of starch. While
these fruits ripen, the starch is converted into sugar. The changes in
cereals during the ripening period are just the opposite of those that
occur in the apple and banana. Sweet corn is a striking example of a
plant that contains an abundance of sugar when the kernals are young,
but only a little sugar and much starch when the seed is mature.

In nature the starch molecules are built up to form a larger aggregate
called a granule. Every plant has its own characteristic starch granules,
with or without particular markings (see Figs. 3-2 to 3-5). For ex-
ample, the potato starch granule is large, oval, and marked by concentric
lines arranged around a point called the hilum, at one end of the granule.
That of wheat starch, on the other hand, is smaller and spherical in
shape, without any particular markings. Oat starch is made up of a
number of particles and forms what is known as a compound starch
granule. The different fragments fit together in the form of a mosaic.
Because of this distinctiveness in appearance, it is comparatively easy
to determine the kind of starch that is present in a food material.
Microscopic examination of spices and flours is of great help in determin-
ing whether or not these materials have been adulterated by the addition

From Reichert, The DifferenfUition and Sppcificitii of Starchex.
Courtfsy of tlic ("ariifK'f InstitutiDii of \Va.sliin}j;fon.

Fig. 3-2. Stai'ch granules from potato.

From Reichert, The l)i Ifcn iit inl i(/ii and Specifieitu nf sianln
Courtesy of the ("arnoKif Insritutinn of Washington.

Fig. 3-3. Starch granules from wheat.

55

From Keichert, The Differeiitiatiott and Specificity of Starches,
Courtesy of the Carnejiie Institution of Washinston.

Fig. 3-4. Strach granules from corn.

■"?«ig^, IV V m «v...'^''.A-

«*.^;,6

From Reichert, The Differentiation and Specificity of Starches.
Courtesy of the Carnegie Institution of Washington.

Fig. 3-5. Starch granules from rice.

56

CARBOHYDRATES

57

of foreign substances whose starch granules are different from those of
the pure materials.

Conmiercial starch is usually made from corn or wheat in the United
States and from jiotatoes in Europe. Other commercial starches are sago,
tapioca, and arrowroot. The preparation of starch in the United States
is associated with the manufacture of numerous other products such as
corn oil, gluten feed, and glucose sirup. For details concernmg the proc-
ess of manufacture, see the industrial chemistries listed.

Starch is insoluble in cold water, but at higher temperatures (52° to
72°C., varying with the kind of starch) the starch grains absorb water,
swell, and finally form a sticky paste or opalescent semisolution. The
absorption of water and swelling of starchy material on heating is well
illustrated by the changes in volume and viscosity that rice undergoes
when it is boiled.

Like other polysaccharides, starch is hydrolyzed by boiling in dilute
mineral acid solutions. If the boiling is continued long enough, the starch
is converted entirely into glucose, as shown in the following equation:

(CeHioOs)^ + a;H20 -^^ xCeHijOs
Starch Glucose

However, the large starch molecule does not split up all at once into
glucose but passes through a number of intermediate stages. At first
only a few of the glucosidic linkages are hydrolyzed so that large frag-
ments of the original molecule are formed. This renders the starch water-
soluble. More hydrolysis leads to smaller fragments of the starch mole-
cule, which are called dextrins. These in turn are broken down into
maltose and finally glucose.

The manufacture of sirup from starch involves its hydrolysis by acid
with glucose and maltose as the principal products, together with a con-
siderable quantity of dextrin. The hydrolysis is commonly carried to
the point at which iodine no longer gives a color with the hydrolysis
mixture. The composition of commercial corn sirup, as calculated from
a number of analyses reported by Fetzer, Evans, and Longenecker, is
as follows:

per cent

Water 18.48

Dextrins 28.11

Maltose 36.33

Dextrose 16.78

Crude protein 0.05

Ash 0.25

Starch, dextrins, and glycogen are also hydrolyzed by various starch-
splitting enzymes called amylases. This process differs from acid hy-
drolysis in that the chief product formed is maltose rather than glucose:

58

CARBOHYDRATES

amylase

(CeHioOs)^. +^H20 *■ -2Ci2H2iOii

Starch , dextrins, Maltose

or glycogen

This conversion, however, is generally not complete because the amylases
can attack only a-l,4-linkages between glucose units. Thus amylose is
completely hydrolyzed into maltose, but the breakdown of amylopectin
stops when a-l,6-linkages (branch points) are reached. This discon-
tinuance results in the formation of limit dextrins which consist mostly
of glucose tri- and tetrasaccharides, each containing at least one a-1,6-
linkage. The amount of limit dextrins formed is usually about 10 to 20
per cent of the amount of starch hydrolyzed.

The amylase enzymes have received various common names according
to the place where they are found. Thus the amylase of saliva is called
ptyalin; that in pancreatic juice, amylopsin; and the very active amylases
present in sprouting cereal grains and other plant sources are frequently
named diastase. Takadiastase is another amylase preparation, obtained
from a mold fungus, Aspergillus oryzae, w^hich has long been used in the
Orient for making certain fermented foods. However, in all of these
variously named preparations there are only two basically different types
of amylases, which are designated merely as alpha and beta amylase,
respectively. The manner in which each of these attacks starch is ex-
plained in detail in Chap. 10.

The removal of starch from textiles and starched goods by means of
amylase is an application of enzyme action to an industrial problem.
The fabric is not attacked as when alkali or acid is used, and hence the
enzyme is to be preferred to other means of starch removal. Many textile
mills now make use of such commercial amylase preparations.

Dextrins

As already noted, dextrins are intermediate products in the hydrolysis
of starch to maltose. They differ from starch by being soluble in cold
water and from maltose by being insoluble in alcohol. They are also
found as native products in the roots, stems, and leaves of many plants.
Starchy seeds in the resting stage contain a small percentage of dextrin
and when germinating, a large amount.

Dextrins are formed from starch in many household operations re-
quiring heat: baking of bread, cake, etc., and ironing of starched clothes.
The toasted breakfast foods, corn flakes, shredded wheat, puffed rice, and
so on, contain considerable quantities of dextrin produced by the heating
of these foods.

Commercial dextrins are made by heating starch with or without the
addition of acid. If acid is used, a lower temperature is sufficient to

CARBOHYDRATES ^^

bring about the partial hydrolysis of the starch. "British Gum" is one
of the important commercial dextrins. Dextrins are widely used as
adhesives on postage stamps, envelopes, and textiles. Mucilage and
other industrial pastes are composed largely of dextrin. Tn the manu-
facture of cotton clotli. the material is sized with dextrin to make possible
the printing of the pattern. The candy industry uses large quantities of
dextrin to give a smooth texture to the product.

The starch-splitting enzymes, ptyalin, amylopsin, and diastase, act also
on dextrins and form the same end product as from starch, namely,
maltose. Since dextrins include a number of degradation products of
starch, it is not surprising that they differ in their response to the iodine
test. Some are colored blue-violet, others red-brown, and yet others are
not colored at all by iodine.

Glycogen

Glycogen, or as it is sometimes called, animal starch, is tlie chief form
in which carbohydrates are stored in the animal body. It is found most
abundantly in the liver and muscles, but has also been isolated from bone,
blood, skin, and many other tissues. It seems to be present in all animal
cells. The amount fluctuates within wide limits. Hunger and severe
muscular work greatly deplete the supply of glycogen, whereas liberal
feeding with carbohydrate foods greatly increases it. By feeding, the
glycogen content of the rabbit's liver has been raised to 27 per cent of
the total weight of the liver. In the dog, under the same conditions, a
17 per cent glycogen content of the liver has been found. In man it is
estimated to reach as high as 10 per cent on a high carbohydrate diet.
Under usual conditions the liver of an animal contains from 1.5-4.0 per
cent. Other animal tissues have been found to contain the following
percentages of glycogen: muscle, 0.5-0.9; skin, 0.1-1.7; bone, 0.2-1.9;
blood, 0.007-0.016. The percentage of glycogen varies in the same kind
of tissue of different animals, in the different muscles of t'he same animal,
and in the different parts of the same muscle.

Like amylopectin, glycogen is believed to consist of branched chains
that form a macromolecule containing about 2400 glucose residues. Such
a molecule would have a molecular weight of about 400,000. The length
of the individual chains in glycogen appears to be shorter than in starch
and is thought to contain from 12 to 18 glucose units instead of 24 to 30
units as reported for starch.

A so-called plant glycogen has been found in several plants, molds,
yeasts, etc., which possesses many of the chemical properties of animal
glycogen, for example, iodine reaction, but is unlike it in certain other
aspects, such as oj^tical activity.

Glycogen is a snow-wliitc powder readily soluble in water, with which

60 CARBOHYDRATES

it forms an opalescent colloidal solution. With iodine, glycogen gives
a red-brown color, which is made somewhat more pronounced by the
addition of sodium chloride. It does not reduce Fehling's solution. It
is hydrolyzed by the action of dilute acids to maltose, and finally to
glucose. Like starch, glycogen is not fermented by yeast, but is readily
hydrolyzed by starch-splitting enzymes. Diastase, ptyalin, and amy-
lopsin convert it into maltose.

Bacterial dextrans

The dextrans ^ are polysaccharides produced by several species of
bacteria, notably Leucanostoc mesenteroides. Composed entirely of
glucose, they are high molecular weight (several million) , water-soluble
substances, which can be precipitated from aqueous solutions by adding
an equal volume of alcohol. The glucose units are attached to each other
by a-l,6-linkages in chains which have many branches. At the branch
points, a-l,4-linkages occur. The structure is thus the reverse of that
of glycogen and amylopectin, where the chains are held together by
a-l,4-linkages, and a-l,6-links are found only at branch points.

Bacterial dextrans, like other glucosans, can be hydrolyzed with acids
to form glucose. Dextran degraded by. partial hydrolysis to an average
molecular weight of about 100,000 has been used in the form of a 6 per
cent solution in 0.9 per cent saline solution as a substitute for plasma in
blood transfusions. Although by no means a complete substitute for
plasma or whole blood, such solutions do have considerable value for body
fluid replacement in cases of severe burns, shock, blood loss, and the
like. One of the main problems in such cases is to prevent further loss
of fluid from the body, and this can only be done if the fluid used for the
transfusion contains a nondiffusible solute which gives it an osmotic
pressure similar to that normally caused by the blood serum proteins.
The dissolved substance must have about the right molecular weight,
must remain in the blood for a day or two, must not cause too great
viscosity, and must not be toxic or produce any undesirable side effects.
Partly hydrolyzed dextran is one of the most satisfactory materials of
a number which have been investigated for this purpose.

Cellulose

Cellulose consists of an unbranched chain of D-glucose units joined by
y8-l,4-linkages. Thus it closely resembles amylose except for the /3-
linkage and a much higher molecular weight. Many efforts have been
made to determine the number of glucose units in the chain, and values
ranging all the way from a few hundred to several thousand have been

^ Do not confuse with dextrins.

CARBOHYDRATES 61

reported (compare with amylose, p. 50). Probably the higher figures arc
more nearly correct for intact cellulose as it actually exists in plants.

The woody and fibrous tissues which provide strength and rigidity for
plants, as bones do for animals, are composed of a mixture of cellulose
with several other polysaccharides [hemicelluloses and cellulosans) and a
noncarbohydrate material, lignin. Cotton fibers are an exception to this
statement since they consist of practically pure cellulose (over 98 per
cent) .

From an industrial and economic standpoint cellulose is the most im-
portant of all the carbohydrates (see Table 3-1, p. 20). Cotton and
linen goods, rayon, paper and pulp products, rope, twine, and other cordage
materials are composed almost entirely of cellulose. The largest single
source is wood, cither in its natural form or in the form of paper and pulp
products. Wood contains, on the dry basis, about 60-70 per cent of
carbohydrates and 20-30 per cent of lignin. About half of the carbo-
hydrate fraction consists of true cellulose. The process of paper-making
is essentially a matter of separating the cellulose from the lignin, hemi-
cellulose, and other constituents of wood. Pressure-cooking the wood,
in the form of chips, at 130-175°C., with water containing such chemicals
as calcium bisulfite plus sulfur dioxide (sulfite process) or sodium hy-
droxide plus sodium sulfide (Kraft process) , dissolves the lignin and most
of the hemicelluloses. The insoluble fiber or "pulp," consisting of most
of the cellulose plus smaller amounts of other resistant polysaccharides, is
separated from the water solution, called "waste liquor," and either rolled
into sheets to make paper or used as a source of crude cellulose for other
industrial purposes (see below). During the years 1947-1950 wood pulp
was produced in the United States at the rate of about 12,000,000 to
15,000,000 tons annually.

Disposal of the enormous quantities of waste liquors, produced as a
by-product of the pulp and paper industry, is still an unsolved challenge
to chemists. The sugars present can be fermented to produce alcohol,
lactic acid, or yeast, but only a small portion of the total is so utilized.
Heating with strong alkali converts 10 to 20 per cent of the lignin into
vanillin, a component of vanilla. Unfortunately the use of vanillin for
flavoring offers a market for only a tiny fraction of the lignin available.

CHO

Mercerized cloth, named after John Mercer who originated the process,
is obtained by treating cotton cloth with alkali and subsequently washing
and drying the cloth. The individual fibers become thicker and shorter.

62 CARBOHYDRATES

Their strength becomes approximately 20 per cent greater, their affinity
for dyes is greatly increased, and a smooth glossy surface is produced.

Synthetic yarn, also known as rayon or artificial silk, is a cellulose
product that has achieved great importance since World War I. In 1949
nearly 500,000 tons were produced in America, while world production
in 1947 was twice this figure. The rapid progress of this industry in the
United States is indicated by the fact that only 63,500 tons of rayon were
produced in 1930. Synthetic yarns are made from xanthate, acetate,
nitrate, and cuprammonium compounds of cellulose. Of the four, the
most important is the type produced by the xanthate or viscose process,
which uses sodium hydroxide and carbon disulfide as the chemicals for
dissolving the cellulose. Although the term "rayon" was originally
applied solely to the product of this process, the Federal Trade Commis-
sion has ruled that all manufactured textile fibers of cellulosic origin
shall be included in the term.

The cellulose products, instead of being spun as a thread, may be
produced in the form of a sheet or film. Cellophane, a colorless trans-
parent material which is extensively used for wrapping purposes, is made
by the viscose process, in which the cellulose is regenerated in the form
of a sheet of varying thickness. Motion picture films and glass substi-
tutes, which allow the ultraviolet rays of the sunlight to pass through, are
other examples of sheet cellulose products. ''Safety glass" used in auto-
mobiles usually consists of a sheet of glass on each side of a layer of
cellulose acetate. Cotton lacquers, such as Duco, which in recent years
have come into extensive use for the surfacing of automobiles and furni-
ture, contain as one of their essential constituents some ester of cellulose,
usually the nitrate or acetate.

Nitrate esters of cellulose are used for many purposes. Cellulose
trinitrate [C6H702(N03)3]a; is the well-known explosive, guncotton. The
less completely nitrated cellulose is known as pyroxylin and is extensively
used in the manufacture of plastics, such as celluloid. Celluloid is a
mixture of two parts pyroxylin and one part camphor. The strongly
combustible nature of all celluloid materials is due to the presence of
the nitrate groups. Collodion is a solution of pyroxylin in alcohol and
ether. AVhen this solution is painted over a wound, the alcohol and
ether evaporate leaving a thin membrane, "new skin." When used in
manufacture of artificial silk, cellulose nitrate must be denitrated to
render it noninflammable. This is accomplished by treatment with an
acid sulfide, for example, sodium acid sulfide (NaHS) .

Cellulose is not acted upon by the enzymes of the digestive tract of
vertebrates. However, certain snails and insects secrete an enzyme,
cellulase, which is capable of digesting cellulose. Cows, sheep, and
horses consume large quantities of cellulosic material, and a large pro-
portion of this material (50 to 85 per cent) disappears from the digestive

CARBOHYDRATES

63

tract. Since no cellulose-digesting enzyme is known to be secreted by
these animals, it is assumed that the hydrolysis is brought about by the
action of bacteria. The products formed by such bacteria — acids, or
possibly even glucose — may be absorbed and thus serve as sources of
energy to the animal. Although cellulose is of no importance to man
as a source of energ>', its presence in the digestive tract may serve a
useful purpose in giving bulk to the food and may assist in the elimina-
tion of food residues. However, there is considerable difference of opinion
regarding the value of bulk in the diet.

On complete hydrolysis pure cellulose gives only glucose. Soft woods
(spruce, pine, fir, etc.) give about 50 per cent of glucose and about 10
per cent of xylose and other sugars. However, it is not practicable to
make glucose from wood and other cellulosic materials because of the
difficulties encountered in purifying the sugar. In Europe, wood sugar
solutions are fermented for production of ethyl alcohol and yeast on a
commercial scale. Such liquors have also been evaporated and used
as feed for cattle.

Fructosans

Polysaccharides that upon hydrolysis yield fructose are reported to
be fairly widespread in plants. Eight fairly well-defined members of
this group of polysaccharides have been described. The best known
are inulin and the bacterial levari produced by Bacillus subtilis. Inulin
consists of about 30 D-fructose residues linked from carbon 2 of one residue
to 1 of the next. It is abundant in the roots of the Jerusalem artichoke,
dahlia, sunflower, dandelion, and many other plants. The amount varies
with the season of the year. It has been proposed that inulin be manu-
factured from the Jerusalem artichoke, since this plant produces a very
large tonnage per acre, and that the inulin be converted into levulose
by acid hydrolysis. However, up to the present time this process has
not been attempted on a commercial scale.

Inulin is a white powder readily soluble in hot water but only slightly
soluble in cold water. It gives no color with iodine solution and is
easily hydrolyzed with dilute acids. It is not acted upon by diastase,
ptyalin, amylopsin, or any known body enzyme. It is not fermented
by ordinary yeast, but is easily broken down by many bacteria.

The bacterial levan of B. subtilis is similar to inulin in its properties,
but is made up of D-fructose residues united at the 2 and 6 positions.

Galactans

Anhydrides of the sugar, galactose, are known as galactans. They are
frequently found in combination with arabinose as a double compound,

64 CARBOHYDRATES

galactoaraban. Galactans are found in peas, beans, and certain other
legumes. They are not hydrolyzed by the enzymes of the digestive tract;
consequently their nutritive value can be only indirect.

Mannosans

These polysaccharides both in the simple form and in combination with
the anhydrides of other sugars, for example, fructose and galactose, have
been found in yeast, mushrooms, seeds, nuts, fruits, berries, leaves, and
practically all plant tissues. They are especially abundant in the ivory
nut, coffee bean, and carob bean. The best known members of this
class are ivory nut mannan and salep mannan. Wlien salep, a meal
obtained from the dried tuberous roots of various orchids, is extracted
with water and alcohol added to the extract, the mannan precipitates
as a white powder. On acid hydrolysis only D-mannose is formed. The
polysaccharide is made up of the D-mannose residues attached by /3-l,4-
linkages in a single, unbranched chain. Ivory nut mannan Js similarly
constituted, but is insoluble in water.

Although certain molluscs and crustaceans secrete enzymes that hydro-
lyze mannans, no digestion of these carbohydrates is brought about by
the digestive enzymes of higher animals.

Chitin

The polysaccharide, chitin, is widely distributed in nature, being found
in the exoskeletons of many invertebrate animals, as well as in certain
plants and fungi. Typical examples of its occurrence are the cuticle
of insects and the shells of crabs and lobsters, where it makes up about
one-fourth to one-half of the dry weight. It functions as a highly re-
sistant protective substance and, together with protein and mineral matter,
gives strength and rigidity to the organism. Chitin is remarkably in-
soluble in all ordinary solvents and resistant to alkaline hydrolysis,
although it can be hydrolyzed by long heating wdth strong acid.

Chemically, chitin consists of N-acetyl-D-glucosamine residues linked
through the 1,4-positions into a linear chain several hundred units long.
It is thus one of the very few major carbohydrates in nature which con-
tains nitrogen.

Pectin

This carbohydrate is contained in the water extract of many fleshy
fruits. On addition of acid and sugar in proper concentrations, pectin
forms a gelatinous mass well known as jelly. The mother substance
existing in the plant, designated as protopectin, is considered by many

CARBOHYDRATES

65

investigators to be a member of the group of hemicelluloses. It is very
widely distributed in nature, being found in varying quantities in most
fruits, vegetables', and roots. Ripening of the fruit, or action of acid
and heat, converts the insoluble protopectin into soluble pectin. This
change is well illustrated in jelly making, where boiling the fruit is
necessary to get the maximum amount of pectin. However, prolonged
boiling converts the pectin into hydrolysis products that do not have
the property of jelling. In the ripening of fruits, enzymes bring about
the hydrolysis of pectin, and, hence, overripe fruits are not suitable for
making jelly.

Structurally, pectin is a polysaccharide consisting of a long chain of
D-galacturonic acid units (pp. 38, 39) in which some of the carboxyl groups
are united with metliyl alcohol through an ester linkage (— COOCH3).
The galacturonic acid units are joined through carbons 1 and 4, as are
the glucose units in starch, glycogen, and cellulose. Opinions differ, but
it appears that any arabinose or galactose obtained by hydrolysis of
pectin preparations comes from associated polysaccharides rather than
from the pectin itself.

The manufacture of commercial pectin to aid the housewife in com-
pelling unwilling jellies to jell, or in making jellies from fruits that
contain little or no pectin, has become an industry of considerable pro-
portions. A well-known product of this kind is "Certo," which is made
from apple pomace. Dry pectin has recently been developed from
apples and lemons. On the basis of dry matter, apple pomace and
lemon pulp contain about 20 and 35 per cent, respectively, of pectin.
Rinds of "cull" lemons are used for this purpose and furnish a much
larger supply of raw material than can be utilized at the present time.
Sugar beet pulp contains on the dry basis about 25 per cent of pectin and
offers an almost unlimited supply of raw material for tho manufacture
of pectin.

Sugar, acid, and pectin are necessary to form a gel. These three in-
gredients may be varied within rather wide limits, but a jelly of good
texture contains about 60-70 per cent sugar, 1-2 per cent acid (ex-
pressed as tartaric and equivalent to pH 3.2-3.5) and 0.5-1.0 per cent
pectin.

Closely related to pectin is the acidic polysaccharide, alginic acid, which
is obtained from marine algae. Like pectin, it has the property of holding
large amounts of water in a colloidal gel. For this reason it is used,
in the form of its sodium salt, as a stabilizer in ice cream and other foods,
and in cosmetics. Because it is capable of forming hard, resistant, surface
films, it is also used in making special grades of paper, cloth, and printer's
ink. Chemically, alginic acid is composed of D-mannuronic acid residues
attached by /3-1,4-linkages in an unbranched chain structure. It is
remarkably resistant to hydrolysis, even when exposed to,. strong acid

66

CARBOHYDRATES

or alkali. Since no body enzymes can digest alginic acid or pectin, they
have no food value.

HETEROPOLYSACCHARIDES

Most of the carbohydrates in this group are too complex and too im-
perfectly known to be included in an elementary book. Examples of
several types have been given in connection with the classification of
carbohydrates (p. 21), and several others have been mentioned briefly
in the sections on wood (p. 61), pentosans (p. 52), and galactans (p.
63). A few heteropolysaccharides of special importance are discussed
in more detail below.

Heteropolysaccharides from plants

The hemicelluloses are one of the most important subgroups of this
large, rather poorly-defined, class of carbohydrates. As indicated on
p. 61, they are present in fibrous and woody plant tissues, where they
are combined with cellulose and lignin to form the cell walls. The hemi-
celluloses are distinguished from cellulose by the facts that they are
acidic substances and are made up quite largely of D-xylose units, although
other sugars (D-galactose, L-arabinose, D-glucose, D-mannose) may also
be present in smaller amounts. Their acidic properties arise from the
presence of a hexuronic acid, probably D-glucuronic acid, which is also
one of the component units. Ordinary wood pulp contains considerable
amounts of hemicelluloses.

The plant gums such as cherry gum, mesquite gum, gum arabic, and
gum tragacanth are neutral salts of complex polysaccharide acids com-
posed of residues of hexoses, pentoses, methyl pentoses and uronic acids.
The uronic acid in nearly all plant gums is D-glucuronic acid, and the
sugars commonly present include D-galactose, D-mannose, L-arabinose,
D-xylose, L-rhamnose, and L-fucose. The complete structures have not
been worked out. Plants produce such gums when they are injured, no
doubt as a protective mechanism.

Another group of mixed-type polysaccharides, widely distributed in
plants, form viscous, colloidal solutions in water and hence are called
mucilages. These are roughly divided into neutral, acidic, and sulfate-
containing groups. An example of a neutral mucilage is gum ghatti,
which on hydrolysis gives rise to 16 per cent of D-galactose and 84 per cent
of D-mannose. The majority of the mucilages in seeds are of the acidic
type, with the acidity being due in all cases to D-galacturonic acid resi-
dues. Sea weeds contain a large number of mucilages, nearly all of
which contain sulfate groups {i.e., some of the hydroxyl groups of the
sugar residues present are esterified with sulfuric acid). D-Galactose

CARBOHYDRATES

67

is the chief sugar obtained on hydrolysis. The best known sea weed
mucilage is agar, which is widely used in bacterial culture media because
of its property of forming gels.

Hetero polysaccharides of animals

Several carbohydrates of this type occur in small amounts in the animal
body. Hyaluronic acid, a polysaccharide composed of equimolar portions
of D-glucosamine acetate and D-glucuronic acid residues, forms a viscous,
gel-like material present in connective tissues, eyes (aqueous and vitreous
humor), joints (synovial fluid), and various other organs. It functions
as a cementing substance between the cells of connective tissue (so-called
"ground substance") and, because of its viscosity, resists penetration by
foreign matter, i.e., infection by bacteria. Hyaluronic acid is attacked
and liquefied by an enzyme, hyaluronidase, which is present in some
bacteria, in certain animal tissues, and in the poisonous secretions of
many reptiles and other animals. This enzyme, to the extent that it
is present, contributes to the rapid spread of toxic agents throughout
the body ; it is therefore of great medical interest.

Chondroitin sulfate, a major component of cartilage, is a heteropoly-
saccharide made up of D-glucuronic acid and D-galactosamine acetate
residues, with some of the hydroxyl groups esterified by sulfuric acid.
In the living animal it is probably attached through its carboxyl and
sulfate groups to the amino groups of proteins. Mucoitin sulfate, present
in mucosa (e.g., stomach mucosa and gastric juice), is similarly consti-
tuted except that it contains D-glucosamine acetate residues.

Another animal polysaccharide of considerable importance is heparin,
a natural anticoagulant (inhibitor of blood coagulation) , which occurs
in the liver, muscles, and other organs of the body. The component
building blocks of heparin, as shown by their formation on complete
hydrolysis, are D-glucuronic acid, D-glucosamine, and sulfuric acid. It
is noteworthy that no acetic acid is involved since in the other animal
polysaccharides, which contain amino sugars, the amino group is acety-
lated {i.e., combined with acetic acid to form the acetylamino group,
— NHCOCH3). Furthermore, heparin contains more sulfate residues
than most of the other sulfate-containing carbohydrates discussed above.
According to Wolfrom and co-workers the repeating unit in heparin is
a tetrasaccharide composed of two residues of glucosamine and two of
glucuronic acid plus five sulfate radicals. An unusual feature of the
heparin structure is the union of the amino group of each glucosamine
unit with a sulfate radical to form a sulfamic acid group, — NHSO3H.
The sulfuric acid here takes the place of acetic acid in other animal
polysaccharides.

68 CARBOHYDRATES

Immuno'polysaccharides

Certain polysaccharides, unknown until fairly recently, doubtless play
a role of greater importance in our lives than many of the related com-
pounds with which we are more familiar. These are the immuno-poly-
saccharides, which pneumococci, streptococci, tubercle bacilli, and many
other types of bacteria synthesize and transfer to the solution, blood as
well as culture media, in which they grow. Each type produces a char-
acteristic chemical compound or "specific soluble substance," the presence
of which in the blood stream of an individual stimulates production
of antibodies, and thus builds up immunity to a given disease. Since
these polysaccharides are "type specific," it is apparent that each must
differ chemically from the other. D-Glucose, D-glucosamine, and various
sugar acids have been identified among the hydrolysis products of these
immuno-polysaccharides. A recent contribution to immuno-chemistry
is the discovery that appropriate synthetic organic compounds-containing
glucuronic and galacturonic acids, as w^ell as the specific polysaccharides
of bacterial origin, may evoke production of antibodies and thus establish
immunity to a particular disease in an experimental animal. These results
show the great importance of the sugar acids and throw new light on
the structure of the specific polysaccharides.

REVIEW QUESTIONS ON CARBOHYDRATES

1. Define: carbohydrate, simple sugar, uronic acid, heteropolysaccharide, asym-
metric carbon atom, optical rotation, desoxysugar, glycoside.

2. How many substances of each of the following types can theoretically exist:
aldopentose, 2-ketohexose, aldohexose? Explain.

3. Name four disaccharides made up of glucose units only and explain how they
differ from each other.

4. Give two commercial sources of (1) sucrose, (2) cellulose, (3) starch; one
commercial source of (4) glucose, (5) lactose. Briefly outline the procedure in the
manufacture of sucrose from one of the above sources. Outline the steps in the
manufacture of glucose.

5. Explain the terms: (1) invert sugar, (2) hydrolysis, (3) sucrase, (4) pentosan,
(5) mercerization, (6) celluloid.

6. Write equations and name the products in: (1) the photosynthesis of glucose,
(2) the hydrolysis of sucrose, (3) the digestion of starch by saliva.

7. What are the chief carbohydrates in (1) honey, (2) fruits, (3) liver, (4) blood,
(5) milk, (6) conden.sed milk, (7) cereals? Approximately what is the percentage
of the carbohydrate named in each case?

8. Explain the terms: (1) pentose, (2) photosynthesis, (3) pectin, (4) dextrin,
(5) beta lactose, (6) cellophane, (7) rayon.

9. Write equations and name the products in: (1) the hydrolysis of starch by
acid, (2) the mucic acid test for galactose, (3) a positive Fehling's test.

10. By means of graphic formulas explain how glucose, galactose, and fructose may
all have the same molecular formula, CoHiiOe, and still be different chemical com-
pounds. Explain why sucrose does not reduce Fehling's solution, while maltose does.

CARBOHYDRATES

69

11. Give another example of two substances that have the same molecular formula
and explain the differences in their structure.

12. Name three carbohydrates found in animal material, telling where each is found
and approximately how nuicli of each is present.

13. Discuss the occurrence of pentose-yielding substances in nature. What be-
comes of these sub.stances? What commercial value do they have?

14. Explain the changes that occur when fruits are boiled to make jelly. What
happens when the fruit is boiled too long?

15. Name some important commercial products made from cellulose.

16. What is raj'on? Name the principal cellulose compounds used in its manu-
facture.

17. Name all the carbohydrates that are suitable for food purposes. Name those
not digested by the digestive enzymes of higher animals. Name an animal that
can digest cellulose.

REFERENCES AND SUGGESTED READINGS

Bates, F. J., Polarinietry, Saccharimetry, and the Sugars, National Bureau of Stand-
ards Circular C440, U. S. Government Printing Office, Washington, D. C, 1942.

Bell, D. J., Introduction to Carbohydrate Biochemistry, University Tutorial Press
Ltd., London, 1940.

Browne, C. A., and Zerban, F. W., Physical and Chemical Methods oj Sugar Analysis.
3rd ed., John Wiley and Sons, Inc., New York, 1941.

"Dextran as a Plasma Substitute" (editorial), J. Am. Med. Assoc, 139, 850 (1949).

Fetzer, W. R., Evans, J. W., and Longenecker, J. B., "DeteiTnination of Dextrin,
Maltose, and Dextrose in Corn Symp," Ind. Eng. Chcm., Anal. Ed., 5, 81 (1933).

Furnas, C. C. (editor), Rogers Manual oj Industrial Chemistry, 6th ed., D. Van
Nostrand Company, Inc., New York, 1942, vols. I and II.

Gortner, R. A., Gortner, R. A., Jr., and Gortner, W. A., Outlines of Biochemistry,
3rd ed., John Wiley and Sons, Inc., New York, 1949, pp. 517-764.

Honeyman, John, An Introduction to the Chemistry of the Carbohydrates, The
Clarendon Press, Oxford, 1948.

Klein, G. (editor), Handbuch dcr Pflanzenanalyze, Julius Springer, Vienna, 1932,
vol. 2, pp. 764-788.

Levene, P. A., Hexosamines and Mu/:oproteins, Longmans, Green and Company,
London, 1925.

McCance, R. A., and Widdowson, E. M., The Chemical Composition of Foods, 2nd
ed., Chemical Publishing Company, Inc., New York, 1947.

Norman, A. G., The Biochemistry of Cellulose, the Polyuronides, Lignin, etc., Oxford
University Press, London, 1937.

Percival, E. G. V., Structural Carbohydrate Chemistry, Prentice-Hall, Inc., New
York, 1950.

Pigman, W. W., and Goepp, R. M., Jr., Chemistry of the Carbohydrates, Academic
Press Inc., New York, 1948.

Pigman, W. W., and Wolfrom, M. L. (editors). Advances in Carbohydrate Chem-
istry, Academic Press Inc., New York, vols. 1-5, 1945-1950.

Sherman, H. C, Chemistry of Food and Nutrition, 7th ed.. The Macmillan Com-
pany, New York, 1946, Chap. II.

Shreve, R. Norris, The Chemical Process Industries, McGraw-Hill Book Company,
Inc., New York, 1945.

Smith, L. T., and Clabora, H. V., "Utilization of Lactic Acid," Ind. Eng. Chem.,
News Ed., 17, 370 (1939).

70

CARBOHYDRATES

Tollens, B., and Eisner, H., Kurzes Lehrbuch der Kohlenhydrate, 4th ed., Barth

Publishing Company, Leipzig, 1935.
Wolfrom, M. L., Montgomery, R., Karabinos, J. V., and Rathgeb, P., "The Structure

of Heparin," J. Am. Chem. Soc, 72, 5796 (1950).

Chapter 4

LIPIDES (FATS AND RELATED
SUBSTANCES)

In every-day use the term fat has a fairly definite meaning. It sug-
gests such famihar substances as butter, lard, tallow, olive oil, cotton-
seed oil, and so on. However, if the distribution of fats in nature is
studied more closely, it soon becomes apparent that fats exist in less ob-
vious and less easily characterized combinations. Although the common
fats are essentially combinations of glycerol and fatty acids, many other
constituents are contained in fats and fat-like substances. Because of
this heterogeneity no very satisfactory classification of the fats has yet
been worked out. Perhaps the best one yet developed is that by Bloor
who bases his classification on three points: (1) solubility {e.g., insoluble
in water, soluble in ether, chloroform), (2) structure, i.e., esters of the
fatty acids, either actual or potential, (3) utilization by living organisms.
From Bloor's classification it is evident that fats or Hpides are essentially
ester combinations that yield various products on hydrolysis. The table
on page 73 gives examples of the different classes of lipides and the prod-
ucts formed by hydrolysis. This table gives a general view of the
lipides as a whole and should be used as a guide to which additions are
to be made as each class of lipides is studied more intensively.

ESTERS

Definition

Since the lipides consist, for the most part, of esters, it is important
for the student to understand clearly just what is meant by the term
ester. An ester is a substance formed by the chemical reaction of an
alcohol with an acid, whereby a molecule of water is eliminated. The
formation of a simple ester is represented by the following equation:

O O

II 11

CHsOH + HOCCH, ^=^ C2H5OCCH3 + H,0

Ethyl alcohol Acetic acid Ethyl acetate Water

71

72 LIPIDES (fats and RELATED SUBSTANCES)

The underlined H of the alcohol and HO of the acid become separated
from their previous points of attachment and unite to form H2O, while
the remaining portions of the acid and alcohol combine to form the
ester as indicated. The chemical formula of any ester therefore follows
directly from the formulas of the alcohol and acid of which it is composed.
The name of any pai'ticular ester is derived similarly. Thus in the above
equation the product is called ethyl acetate. An ester prepared from
methyl alcohol and lactic acid would be called methyl lactate, and so on.

Preparation

The actual preparation of esters in the laboratory is carried out, in
general, by warming the chosen alcohol and acid together with a small
amount of a strong mineral acid, such as sulfuric or hydrochloric, which
serves as a catalyst. The process is called esterification. Since esterifica-
tion reactions are, in general, reversible, removal of the water as it is
formed often helps to secure a good yield of the desired ester. It is
obvious that a very large number of different esters may be prepared from
the various alcohols and acids (particularly organic acids) that are
known and are available.

Properties of esters

Many of the simpler esters are liquids that possess pleasant, fruity-
odors and hence are used to some extent as artificial flavoring essences.
More complex esters are found very abundantly and widely distributed
in nature, e.g., in fats, waxes, and other lipides, as explained below. By
far the most important industrial use of synthetic esters is based on their
properties as solvents. Automobile lacquers, for example, are prepared
by dissolving pyroxylin, a pigment, and certain other ingredients in a
suitable ester such as butyl acetate:

O

II
C4HSOCCH3

Butyl acetate

All esters may be broken down into their acid and alcohol components
by hydrolysis:

0 O

II II

C4H9OCCH3 + H2O > CHgOH + HOCCH3

Butyl acetate Water Butyl alcohol Acetic acid

It will be noted that this reaction is the reverse of esterification. The
hydrolysis may be brought about with the aid of an enzyme, if one is

LIPIDES (fats and RELATED SUBSTANCES)

73

Table 4-1
Classes of lipides and their hydrolysis products

Lipide

I. tjimple lipides:
1. True fats

(in butter, lard, oils)

2. Waxes

(in beeswax)

II. Compound lipides:

1. Phospholipides, e.g.,
lecithins (in egg yolk,
brain)

Hydrolysis Products'

Alcohol
(name and formula)

Glycerol,
C3H.(0H)3

Cetyl,

CisHcxCH^OH

Glycerol,

C8H5(0H)3

Glycolipides, e.g.,
kerasin (in brain)

III. Derived lipides:

1. Fatty acids '
(in fats)

2. Sterols

(in fats, waxes)

Galactose,

Ga-UiaUe

Acid and other products
(name and formula)

Stearic,

CnH^-. COOH
Oleic,

C17H33 COOH
Cerotic,

C^sHsi COOH

Phosphoric,

H.PO4
Oleic,

CnH33 COOH
Palmitic,

Cr.H3i COOH
Choline,

(CH3)aX(0H)C.H4 0H
Lignoceric,

C23H47 COOH
Sphingosine.

Ci8H»(0H)2 NH.

Oleic,
CitHss COOH

Cholesterol,

C27H450H

* For a siven fat, e.g., butter, the typical rather than the total products of
hydrolysis are listed. Fatty acids, wax alcohols, and sterols may occur free or
combined. They are also classed as derived lipides.

available that acts on the ester to be hydrolyzed, or by means of strong
acids or superheated steam. A more convenient and widely used method
consists in subjecting the ester to the action of a strong alkali such as
sodium hydroxide, whereby the alcohol and a salt of the acid are produced:

0

II
C4H9OCCH3 + NaOH

Butyl Sodium

acetate hydroxide

O

II
-* C4H90H + NaOCCHs

Butyl Sodium

alcohol

acetate

This process is called saponification. If it is desired to obtain the organic
acid itself, the solution of the sodium salt may be treated with a strong
mineral acid:

74

LIPIDES (fats and RELATED SUBSTANCES)
0 0

NaOCCHa + H2SO4
Sodium Sulfuric
acetate acid

NaHS04 + HOCCH3
Sodium Acetic

acid sulfate acid

TRUE FATS

Definition

The true fats may be defined as esters of the trihydroxy alcohol, glycerol,
and the higher fatty acids. The esters of glycerol are called glycerides,
a natural fat being a mixture of various glycerides in different propor-
tions. The terms fat and oil are used to distinguish between solid and
liquid fats. If the substance is solid at 20°C., it is spoken of as a fat.
If it is a liquid at this temperature, it is regarded as an oil. The
use of the term oil in this connection must not be confused with its use
as applied to so-called mineral oils, such as kerosene oil which is a
hydrocarbon, and not related to fats.

Occurrence and importance

The fats are abundant in both plant and animal materials such as
cottonseed, peanut, coconut, olive, milk, butter, cheese, and meats. The
cereals as a rule are comparatively low in fat, since starch takes the

Table 4r-2
Economic importance of some industries based on fats *

Indiistry

1. Oils (vegetable and animal)

2. Butter

3. Soap and glycerine

4. Grease and tallow

5. Shortenings (vegetable) . . . .

6. Oleomargarine

Wage
earners
30,959
30,131 1
27,660
12,472
8,003
2,567

111,792

Value of
products
shipped
$1,741,238,000
1,037,000,000
1,085,789,000
304,535,000
884,713,000
214,598,000

$5,267,873,000

* Compiled from the 1947 Census of Manufactures, Bureau of the Census, 1950,
and from the Statistical Abstract of the United States, 1951, published by the Depart-
ment of Commerce.

t Estimated in part.

place of fat in the composition of such seeds. It is noteworthy that the
seeds of the tropical regions of the earth are generally characterized by

LIPIDES (fats and RELATED SUBSTANCES)

75

a high fat content, while those of the temperate zone are usually high
in starch. This is only a general rule since there are many exceptions;
rice, which is a tropical product, contains little fat, and soybean, which
contains much fat, grows in temperate regions. The fats of commerce
are removed from the material in which they occur either by mechanical
means or by extraction with suitable solvents. The mechanical methods
arc more generally used. They may vary widely — churning in the making
of butter, heating and filtering in the rendering of lard and tallow,
pressing, with or without the aid of heat, in obtaining olive, cottonseed,
and other oils.

Elementary composition

The fats are comparatively high in carbon and low in oxygen. This
is in sharp contrast to the carbohydrates, which contain a high percentage
of oxygen. A comparison of the elementary composition of fat, starch,
and protein is as follows:

Physiological

fuel value

Calories

per cent :

Carbon

Hydrogen

Oxygen

Nitrogen

per gram

Fat

76.5

12.0

11.5

—

9.0

Starch

44.4

6.2

49.4

—

4.0

Protein

53

7

23

16

4.0

From the above table it is evident that fats have a considerably higher
heat value than either proteins or carbohydrates. On oxidation in the
body, fats give two and one-fourth times as much heat as the other
foodstuffs. This is because of the higher content of carbon and hydrogen
in fats.

Lipides such as lecithin and cephalin, closely related to the true fats,
contain, in addition, the elements phosphorus and nitrogen. As a class,
however, lipides (and carbohydrates) do not contain nitrogen and thus
are sharply differentiated from proteins.

Products on hydrolysis

Some idea regarding the nature of a fat may be obtained by breaking
down the fat into its constituent parts — that is, by hydrolyzing it. When-
ever a natural fat is hydrolyzed by acids or enzymes, glycerol and a
number of fatty acids are obtained. The fatty acids are divided into
two series, the saturated and the unsaturated. A study of these acids
is indispensable to a proper understanding of the fats themselves.

76 LIPIDES (fats and RELATED SUBSTANCES)

Glycerol, C3H5(OH)3

During and after World War I, considerable quantities of glycerol,
or glycerine as it is commonly called, were produced by a yeast fermenta-
tion process. Today, however, it is obtained mainly as a by-product of
the soap industry. The liquors that remain after the soap has been
removed are distilled in a vacuum, and by a series of fractionations
glycerol is obtained free from impurities. In many cases the glycerol
is not removed from the soap. If left in, it tends to make the soap
transparent and of better quality. A process for the manufacture of
glycerol from certain petroleum fractions has also been developed.

Glycerol is a viscous, colorless liquid that has a sweet taste and no
odor. It is extensively used in the manufacture of nitroglycerine, an
ester of glycerol and nitric acid, C3H5(N03)3, which is the basis for a
large number of explosives such as dynamite, blasting gelatin, etc. Nitro-
glycerine also finds use as a drug for alleviation of the severe pain asso-
ciated with some types of heart disease. Glycerol is also widely used
as a solvent in many technical operations. It is extensively used in
the manufacture of cosmetics and toilet and pharmaceutical prepara-
tions. It can be used both internally and externally with perfect safety
and, in fact, can be utilized as a food by human beings.

The saturated fatty acids

Saturated fatty acids contain all the hydrogen with which they are
capable of uniting, whereas unsaturated fatty acids contain carbon atoms
joined together by double bonds and hence can unite with more hydrogen.

Thus it may be seen from the following graphic formulas that butyric
acid is saturated and crotonic acid is unsaturated:

H H H O
I I I II
H— C— C— C— C-O-H

I I I
H H H

Butyric acid

H H H O

I I I II
H— C— C=C— C— 0-H

I
H

Crotonic acid

Saturated acids have a higher melting point than unsaturated fatty
acids with the same number of carbon atoms. Hard fats, such as tallow,
give a high percentage of saturated acids, and soft fats, a low percentage.

LIPIDES (fats and RELATED SUBSTANCES) 77

Since natural fats are mixtures of various glycerides, a large number
of fatty acids is obtained on hydrolysis. The following table gives the
principal fatty acids of the saturated series:

Table 4-3

Saturated fatty acids

A' awe Formula Typical Occurrence

(C„H2„.iC00H)

Formic HCOOH Not in fats

Acetic CH3COOH Not in fats

Propionic CILCH.COOH Not in fats

Butyric CHsCCHJ.COOH Butter

Caproic CH3(CH2)4C00H Butter, coconut and palm oils

Capry^lic CH3(CH.)«C00H Coconut and palm oils, butter

Capric CH3(CH.),sC00H Coconut and palm oils, butter

Laurie CH3(CHo)]oCOOH Laurel, coconut and palm oils

Myristic CH3(CH2)ioC00H Butter, wool fat, spermaceti

Palmitic* CH3(CH=)i4C00H All animal and vegetable fats, notably

lard

Stearic * CH3(CH2)i6C00H Animal and vegetable fats, notably tal-
low

Arachidic CH3(CH2)i8C00H Peanut oil

* Most abundant fatty acids. See Table 4-5.

An examination of the table shows that only fatty acids containing an
even number of carbon atoms are obtained from natural fats. Although
there are several important exceptions to this statement, it is nevertheless
true for the great majority of fats. This rule is rather suggestive of the
way in which the fats must be built up in nature. It is probable that
fatty acids are formed in nature by addition of units containing two
carbon atoms, giving rise only to acids with an even number of carbon
atoms. The saturated acid obtained in greatest amount from fats is
palmitic. Stearic acid is also obtained in large quantities but not to
the same extent as palmitic. The fatty acids, such as butyric, of lower
molecular weight are found to a considerable extent as glycerides in
butter and coconut oil, but except in these two fats they occur in com-
paratively small amounts. Some of the lower fatty acids such as formic,
acetic, and propionic acid belong to the series of saturated fatty acids,
but no glycerides of these acids are found in natural fats. Small amounts
of these acids, and other fatty acids of low molecular weight, are found
free in perspiration and urine. Salts and esters of fatty acids of "both
low and high molecular weight are contained in the feces.

Butyric acid is a colorless, mobile liquid boiling at 162^C. and is com-
pletely miscible with water. Caproic and caprylic acids are also liquids
at room temperatures; capric acid is a semisolid, but lauric acid is defi-
nitely a solid. The change from liquid to solid is thus associated with

78 LIPIDES (fats and RELATED SUBSTANCES)

an increase in the number of carbon atoms in the molecule. The change
in physical state is accompanied by a decrease in solubility. Stearic
acid, which melts at 70°C., is practically insoluble in water.

The fatty acids up to and including capric are easily removed from
solutions by distillation with steam and hence are known as volatile fatty
acids. The determination of the volatile fatty acids is a matter of con-
siderable importance in the analysis of fats, as it aids in chstinguishing
one type of fat from another. Butter fat, for example, gves a higher
proportion of volatile acids than any other fat or oil.

The volatile fatty acids likewise have a decided odor. Butyric acid
has a strong odor similar to that of rancid butter. Caproic, caprylic,
and capric acids have a pronounced animal odor and are sometimes spoken
of as the goat acids. When fats become rancid, a small amount of these
volatile fatty acids is formed and gives to the fats a particularly objec-
tionable odor and taste. Thus the objectionable odor of rancid butter
is due largely to the presence of free butyric acid.

y

Unsaturated fatty acids

If a fatty acid contains a pair of carbon atoms that are joined together
by two bonds instead of one, that is, a double bond, it is said to be
unsaturated. Such an acid can take up hydrogen, iodine, bromine, oxy-
gen, or other elements by the breaking open of one of these two bonds.
This leaves an open position on each carbon atom. Therefore, for each
double bond, two hydrogens can combine with the compound, which would
then be regarded as a saturated compound. This condition of unsatura-
tion has a unique relation to the physical state of the fatty acid and
likewise to the glycerides of the fatty acid. For example, oleic acid
(C17H33COOH), which contains one double bond, and therefore two
fewer hydrogens than stearic acid, is a liquid, whereas stearic acid
(Ci7H35COOH)is a solid. Oleic acid melts at 14°, whereas stearic acid
melts at 70°. If the fatty acid contains more than one double bond,
it will have a correspondingly lower melting point; linoleic acid, which
contains two double bonds, has a melting point of —18°.

The physical state of the fatty acids is carried over to the glycerides
of these acids. Oleic acid and olein are liquids, and stearic acid and
stearin are solids. Linolein and linolenin, as expected, are liquid glycer-
ides. Since fats are mixtures of glycerides, a fat will be soft or hard
depending upon the proportion of liquid glycerides it contains. Oils
differ from fats in that they contain a larger proportion of liquid glycer-
ides. As a general statement, it might be said that oils contain about
80 per cent of unsaturated glycerides, whereas solid fats do not contain
more than 40 to 50 per cent. Unsaturation is a fundamental property
and, in most fats, is the key to the whole question of their physical state.

LIPIDES (fats and RELATED SUBSTANCES) ^9

If this is kept in mind, an understanding of many physical and chemical
properties of fats is easily acquired.

Unsaturated acids have the ability to exist in different isomeric forms,
which are called geometric isomers. These are designated by the prefixes
CIS- and trans-. This type of isomerism, a consequence of the presence
of carbon-to-carbon double bonds, may be illustrated by the formulas of
oleic acid and its ^ra/is-isomer, elaidic acid:

HC(CHj)7CH, H.CCCHOtCH

HC(CHs)7C00H HC(CH07COOH

Oleic acid Elaidic acid

(cis- isomer) (frans- isomer)

Acids like linoleic with two double bonds can exist in four geometric
isomers, corresponding to the cis-trans arrangement about each ; in general,
the number of isomers possible is (2)", where n is the number of double
bonds present. Generally the natural fatty acids occur in the cis form,
although vaccenic appears to be a trans acid. Where trans forms do not
occur naturally, they may readily be produced by treating the cis acids
with nitrous acid or certain other reagents. This reaction has come
to be spoken of as ''elaidinization" from the circumstance that oleic acid
is thus partially converted into elaidic. The trans acids are higher
melting and less soluble than the corresponding cis forms.

The most important unsaturated fatty acids, together with their for-
mulas and occurrence, are listed in Table 4-4. Many other acids with
varying numbers of carbon atoms and different degrees of unsaturation
have been reportedly obtained from brain, liver, and other tissues. The
chemistry of these acids and their function in the animal organism are
not yet clearly defined. Their presence in some of the most important
organs of the body leaves little room for doubt that their role is an
important one.

Although it is rather well established that the animal body can de-
saturate fats, certain limitations to this process apparently exist in
many, if not all, species. Rats kept on diets devoid of unsaturated fats
develop a scahness of the skin, lesions in the kidneys, sterility, and
loss of weight, and eventually die. This nutritional deficiency can be
prevented by including either linoleic or arachidonic acid in the diet.
These particular unsaturated fatty acids have therefore come to be
called "essential fatty acids." No one has demonstrated a need of the
human body for these acids, but even though they may be required, their
widespread occurrence in foodstuffs renders any disease in man resulting
from their deficiency quite unlikely.

Quantitative Relations of the Fatty Acids. ISlany of the statements
made in the preceding pages regarding the fatty acids become more

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80

LIPIDES (fats and RELATED SUBSTANCES) 81

clear if a study is made of their quantitative distribution. The com-
position of the mixture of fatty acids obtained by hydrolysis of some
common fats and oils is given in Table 4-5.

Butterfat and coconut oil are unique in that they yield such a large
number of fatty acids, many of which are lower members of the saturated
fatty acid series. Note the small number of fatty acids obtained from
lard and the large percentage of unsaturated acids given by the oils.

Nonsaponifiable matter

In addition to glycerol and fatty acids, natural fats contain another
type of material called nonsaponifiablc matter or "nonsap." This is
customarily separated after saponification by extracting the alkaline
soap solution with ether. The "nonsap" left after evaporation of the
ether consists of fat-soluble pigments, sterols, vitamins, antioxidants,
and other miscellaneous substances. Although the nonsaponifiablc com-
ponents constitute only a small part (1-2 per cent) of most natural fats,
they are often of great importance in relation to the flavor, color, keeping
qualities, and nutritional value of the fat.

GLYCERIDES OF COMMON FATS

Although the percentages of different fatty acids given by hydrolysis
of natural fats are fairly accurately known, much less information exists
as to the particular glycerides from which these fatty acids are obtained.
By crystallizing the fats from acetone and other solvents, a partial separa-
tion of the individual glycerides in a number of fats has been made. The
separation is a long and laborious procedure, and in no sense complete.
All the data accumulated show that the number of glycerides is very
great and that they are more complex than w-as previously supposed.

Since glycerol, C3H5(OH)3, contains three hydroxyl groups, it can be
esterified with one, two, or three molecules of acid to give monoglycerides,
diglycerides, and triglycerides, respectively. It is this last type which
is found in fats. The three acid radicals in a triglyceride may be all
alike, in which case the substance is called a simple glyceride; if more
than one kind of radical is present, the compound is called a mixed glycer-
ide. The glycerides are named according to the fatty acids involved in
their formation.

Thus the ester formed from glycerol and three molecules of palmitic
acid is called tripalmitin, or simply palmitin. Its structural formula is
written below. Other typical simple glycerides are triolein, tristearin,
and tributyrin:

to

a.

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82

LIPIDES (fats and RELATED SUBSTANCES)

83

o

II

H,COC(CH,)uCH,
O

ii

HCOC(CHj)uCH,

0

II
H2COC(CH5)uCH,

Tripalmitin

O

II
HsC0C(CHj).6CH,

0

II
HCOC(CH2),6CH,

O

II
H2COC(CH3)i6CH,

Tristearin

O

II
HjCOC(CH2)7CH=CH(CKj)7CH,

O
II
HC0C(CH,),CH=CH(CH2),CH,

O
II
H2COC(CH5),CH=CH(CH2)7CH,

Triolein

O

II

H2COC(CIl2)2CH3

O

II
HCOC(CH,)2CH3

O

II
H2COC(CH2)2CH3

Tributyrin

The ester formed from glycerol combined with two molecules of stearic
acid and one of palmitic acid is called distearo-palmitin. This is a typical
mixed glyceride. Another is oleo-stearo-palmitin:

O

II
HjCOC(CH2)i6CH3

O
II
HCOC(CH2),6CH,

O

II
HjCOCCCHOmCH,

Distearo-palmitin

O

II
H2COC(CH2)vCH=CH(CH2)7CH,

0
II
HCOC(CH2),6CH3

0

II
HaCOCCCHOuCH,

Oleo-stearo-palmitin

IVIore condensed formulas are also frequently written. Thus, for example,
the oleic acid radical (oleyl radical) may be abbreviated from

O

II

o

— C(CH2)7CH=CH(CH2)7CIl3 to — CCuHj,

and triolein may be written

0

II

C3H5(OCCi7H33)j

84 LIPIDES (fats and RELATED SUBSTANCES)

However, the more detailed structural formulas should be used by the
beginning student until famiharity with them has been gained. It is
evident that a great many different individual glycerides might be
formed by suitably combining glycerol with the various fatty acids. It
has been calculated that ten fatty acids can produce 550 possible com-
binations.

Formerly it was customary to regard the natural fats as consisting
chiefly of simple glycerides, but more recent work shows that they con-
sist largely of mixed glycerides. It is impossible to say with certainty
just how much of any given fatty acid goes to make up simple or mixed
glycerides in a fat. In the case of butyric acid, it is known that the
acid does not exist as a simple glyceride in butter, but is present in a
mixed combination. Tributyrin is a bitter substance; obviously, it cannot
be present in butter. It is probably more nearly correct to say that
natural fats consist essentially of mixtures of mixed glycerides.

PHYSICAL PROPERTIES OF FATS

As already explained, fats may be either solids or liquids at room tem-
perature (20°C.). The common animal fats are solids at this tempera-
ture, and the majority of vegetable fats (oils) are liquids. Fats and
oils are lighter than water and, as a rule, have a specific gravity of about
0.8. As usually seen they are noncrystalline, although many fats can
be made to crystallize under suitable conditions. (See Fig. 4-1.) They
are poor conductors of heat, therefore serving a useful purpose in the
insulation of the body.

Fats are colorless when they are obtained in a pure state. The more
or less yellow color common to many fats is due to the presence of a
pigment, and not to the fat itself. Butter, for example, varies in color
with the season. In June, when cows feed on grass, the butter is highly
colored, while in January, when the animals receive a dry ration, the
butter is paler in color. The yellow pigment, therefore, is contained in
the feed of the animals. This is largely carotene, C40H56, which occurs
in all green plants, in the petals of many flowers, such as the narcissus,
and in many vegetables such as carrots, squash, etc. In grass it is not
evident because the green pigment, chlorophyll, masks the carotene.
Xanthophyll, C40H56O2, is another yellow pigment which is widely dis-
tributed in plant materials. This is the chief coloring pigment found
in egg yolk. It is also contained in butterfat, but in a much smaller
percentage than is carotene. The egg yolk likewise varies in color with
the feed of the poultry. Lard contains no coloring matter, probably
because swine are fed on rations largely free from green material.

The coloring of fats, which is of importance commercially, has been
brought into considerable prominence in connection with the vitamin A

LIPIDES (fats and RELATED SUBSTANCES)

85

content of food. In 1919 Steenbock called attention to the occurrence
of vitamin A in close association with the yellow pigmentation of fats
and foods; for example, yellow corn was found to be rich in vitamin A,
while white corn was deficient in this vitamin. The same correlation
between vitamin and pigment was found with respect to carrots, squash,

From Hawk and Bersjeim, Practical Phi/siolopical CheuiMnj.
Courtesy of P. Blakiston's Son & Co., Inc.

Fig. 4-1. Crystals of beef fat.

cabbage, and other vegetables. The work of von Euler, Moore, and
others has demonstrated that carotene of plants is the precursor of
vitamin A in the animal body. Carotene is transformed in the body,
apparently in the liver, from an intensely yellow pigment into an almost
colorless compound. This transformation is accompanied by changes in
structure and other properties of the pigment (see p. 207).

In the pure state, fats have no taste, but by a curious anomaly natural
fats are the chief materials that give flavor to food. Food prepared with-
out the addition of fat is considered by many people unpalatable; liberal
additions of butter make food particularly appetizing. Butter owes its
taste largely to diacetyl (CH3 CO CO CH3) and acetylmethyl-carbinol
(CH3 CO CHOH CH3), compounds produced by bacteria in the ripen-
ing of tlie cream.

The same condition obtains with respect to odor. When purified, fats
have no odor; yet natural fats frequently have marked odors. This
apparent contradiction is explained by the readiness with which fats
take up odors. The housewife carefully avoids i)utting onions and
butter together in the refrigerator. The absorption of odors by fats is

86

LIPIDES (fats and RELATED SUBSTANCES)

used to advantage in the extraction of delicate perfumes from flowers.
Rose petals are spread on glass plates covered with a thin layer of lard
and tallow and left in contact with the fat for 24-72 hours. At the
end of this time the old flowers are removed and a new lot is added.
Finally the fat is extracted with cold alcohol to remove the essence,
and the alcoholic solution is concentrated or bottled directly. This proc-
ess yields perfumes of the finest quality.

The substances that often are present in natural fats and, as explained
above, impart to them characteristic colors, flavors, and odors are not
chemically related to the fats themselves, but are merely associated
with them on account of being "fat-soluble," that is, easily soluble in
fat. Thus carotene is a fat-soluble pigment. When the cream is churned
nearly all of the carotene remains in the butter rather than in the
buttermilk, which of course is largely an aqueous medium. Substances
that are fat-soluble are usually insoluble in water, and vice versa.

y
CHEMICAL PROPERTIES OF FATS

Hydrolysis and saponification

The most important chemical reaction of fats is hydrolysis, with the
production of glycerol and fatty acids. This process may be brought
about by means of acids, superheated steam, or enzymes. The fat-
splitting enzymes are known as lipases. They occur in many tissues of
the body and in plant material, especially in oily seeds. A well known
fat-splitting enzyme is steapsin, which is secreted by the pancreas and
is involved in the digestion of fats. Many bacteria, molds, and other
microorganisms produce fat-splitting enzymes. An equation illustrat-
ing the hydrolysis of a fat, for example, palmitin, is:

0 0

II steapsin II

C3H5(OCCuH3l)3 + 3H2O > C3H5(OH)3 + 3HOCC15H31

Palmitin Glycerol Palmitic acid

If the hydrolysis is brought about by means of alkali, a soap is formed
instead of a fatty acid, and the process is then called saponification:

0 O

II II

CsHiCOCCuHaOa + 3NaOH > C3H5(OH)3 + SNaOCCuHji

Palmitin Glycerol Sodium palmitate

(soap)

In this equation the formula of sodium palmitate is written in such a
manner as to show how it is produced by the action of the NaOH on
the palmitin. It might also be written CisHsiCOONa. This substance

LIPIDES (fats and RELATED SUBSTANCES) 87

is a typical soap, and the above equatit)n illustrates the commercial manu-
facture of soaps.

Soaps

A soap is defined chemically as a metallic salt of a fatty acid contain-
ing ten or more carbon atoms. All commercial soaps, however, are mix-
tures of several individual "soaps" because they are made from natural
fats which are mixtures of glycerides. The glycerides are all saponified
at once, and each fatty acid radical is converted into the corresponding
soap. Thus the product is a mixture corresponding in composition to
the fatty acid make-up of the original fat.

Sodium and potassium soaps, being fairly soluble in water, are useful
washing agents. Soaps of other metals, although too insoluble in water
to form a lather, are very valuable for other purposes, as described below.
The consistency and washing qualities of soaps depend partly on the
metal and on the fatty acid radicals of which they are composed. Thus
from a given fat, sodium hydroxide will tend to produce the harder and
potassium hydroxide, the softer soap. On the other hand, if the same
alkali is used throughout, a liquid fat, containing unsaturated or low
molecular weight fatty acid radicals, will tend to produce a soft or liquid
soap, whereas a hard fat like tallow will make a hard soap. Other things
being equal, the soaps of capric, lauric, and myristic acids, that is the
saturated fatty acids containing 10, 12, and 14 carbon atoms, lather
better, and of these the lauric soaps are the best. It is for this reason
that fats such as palm and coconut oils, yielding a large amount of these
particular fatty acids on hydrolysis, are so valuable in soap making.

Water-soluble soaps are classified as detergents, substances which, when
dissolved in water, lower the surface tension of the water and help to
loosen and wash away particles of grease and dirt. Other kinds of
detergents are also produced more or less directly from fats and have
become so popular that over 1.2 million lb. were produced in 1950.
These so-called "synthetic detergents," which should not be called soaps,
are of many types, but all of them consist of a water-soluble, salt-like
group attached to a long-chain, fat-like residue. A typical example is
sodium alkyl sulfate, ROSOsNa, where R represents alkyl groups cor-
responding to various fatty acids such as lauric, myristic, palmitic, and
stearic. These synthetic detergents differ mainly from ordinary soaps by
having a sulfate in place of the carboxyl group. Since they form soluble
calcium and magnesium compounds, which are not precipitated by the
minerals in hard water, they are as effective washing agents in hard water
as in soft water. Their aqueous solutions are also practically neutral,
whereas those of ordinary soaps are quite strongly alkaline and have a
pH of about 9.

88 LIPIDES (fats and RELATED SUBSTANCES)

Various water-insoluble soaps of metals other than sodium or potassium
have important industrial uses. Soaps of the higher saturated or slightly
unsaturated fatty acids such as stearic, palmitic, and oleic acids, with
such metals as aluminum, calcium, lead, barium, lithium and others, are
used in making lubricating greases. When combined with lubricating
oils, these soaps produce semisolid gels, or greases. More highly un-
saturated acids such as linoleic or eleostearic are combined with lead,
manganese, or cobalt to produce ''driers" for use in paints, varnishes,
and enamels. These soaps catalyze the oxidation processes, which cause
the films to "dry" or harden. Zinc oleate and stearate are used as
antiseptics and astringents in medicinal preparations. Of the above soaps,
aluminum and zinc stearates are quantitatively the most important, being
produced annually to the extent of some 10 million lb. each.

Acrolein test

When glycerol is heated strongly, and especially if a deliydrating agent
such as potassium bisulfate is present, it decomposes into water and
acrolein:

CsHjCOH), ^^gQ^ > CH2=CHCH0 + 2H2O

The unsaturated aldehyde, acrolein, has a characteristic sharp, irritating
odor and is partially responsible for the smell of burnt fat.

Fats likewise give the acrolein test since on heating to a sufficiently
high temperature the glycerides in the fat are partially broken down with
the eventual formation of acrolein.

Iodine number

The unsaturation of a fat is determined by means of iodine, which gives
the so-called iodine number of a fat. The iodine number is the per-
centage of iodine by weight that the fat will absorb; for example, if a
fat has an iodine number of 100, one gram of the fat will absorb one
gram of iodine. The following table shows how the iodine number gener-
ally varies with the physical state of the fats. It will be noted that
the hard fat, tallow, has a low iodine number, 35^5, whereas lard, a
soft fat, has an iodine number of 50-70, and the oils have iodine numbers
ranging from 80 to 200. If judged by the low iodine number, butter,
and especially coconut oil, should be hard fats. The low melting point
of coconut oil (about 25°) as compared with that of tallow (about 45°) is
due to the presence of large quantities of glycerides of the lower saturated
fatty acids. The softness of butter is caused by two factors, unsaturation
and glycerides of low molecular weight. In most fats and oils only the
first of these two factors plays a part.

LIPIDES (fats and RELATED SUBSTANCES) 89

Table 4-6

Iodine niiinber of some ronimon oils and fats

Coconut oil 5-10

Buttcrfat 26-38

Beef tallow 35-45

OIoo oil from beef tallow 40- 55

Lard 50-70

Olive oil 79-90

Peanut oil 87-100

Cottonseed oil 104-1 16

Corn oil 111-124

Soybean oil 137-143

Linseed oil 170-200

Linseed oil has the highest iodine number of any known fat. It is a
highly unsaturated oil and takes up atmospheric oxygen very readily
to form a hard tough film. For this reason it is peculiarly well adapted
for paint purposes. When paint is spread over a surface, the linseed
oil takes up oxygen from the air and forms a thin, hard, watertight coat.
No other oil has ever been found which is equal to linseed oil in this
respect. Tung and soybean oils come nearer to it than any other oils and
are used to supplement linseed oil in the paint industry. At the present
time the demand for linseed oil is far greater than can be met by the
supply. Were it possible to desaturate other oils such as cottonseed
and peanut oil so that they would have the same drying capacity as
linseed oil, it would be of enormous benefit to the paint industry. Un-
fortunately no method for doing this has yet been discovered, although
the reverse process of saturating an unsaturated oil can be easily ac-
complished. The term "drying oil" is applied to liquid fats, like linseed
oil, which have iodine numbers in the range 150 to 200 and form hard,
dry films when spread over a surface and exposed to the air.

The drying oils are unsuited for lubricating purposes because they
tend to become gummy and sticky. The same property of unsaturation
operates, but in lubricating oils it is an undesirable property rather than
a desirable one.

Hydrogenation of oils

The saturating or hardening of fats has become an important com-
mercial process. If an oil or soft fat is exposed .to the action of hydrogen
in the presence of finely divided nickel (a catalyst) at a moderately
high temperature (150° to lOOT..) and pressure (25 lb. per sq. in.),
it combines with the hydrogen and is converted into a solid fat. This
process is applied annually to several hundred thousand tons of un-
saturated fats in the United States. Although not all of this hydrogenated

90 LIPIDES (fats and RELATED SUBSTANCES)

material is converted into edible fats, great quantities of well known
commercial products, e.g., "Crisco" and ''Snowdrift/' are prepared in
this manner from peanut, cottonseed, and other oils. These fats are
more stable to heat than natural fats, such as lard, and are, therefore,
peculiarly well adapted to certain cooking operations, such as deep-fat
frying. The natural fats tend to decompose at higher temperatures,
owing, it is assumed, to the presence of small amounts of free fatty
acid. The more free acid present in a fat, the more readily it is decom-
posed by heat.

Rancidity of fats

When fats are kept for a long time, they develop objectionable odors
and tastes, a condition which is known as rancidity. Many different
factors such as heat, light, moisture, air, enzymes, bacteria, and metals
are involved in the decomposition of fats. The principal chemical changes
are hydrolysis and oxidation. The former is particularly important in
the case of butter and other dairy products because the free fatty acids,
produced when butter fat is hydrolyzed, include several of the lower,
saturated series {e.g., butyric, caproic, etc., see Table 4—5), which have
very sharp, unpleasant odors. Rancidity due to oxidation develops par-
ticularly in moderately unsaturated fats and oils, a group which includes
the bulk of the common food fats. Atmospheric oxygen slowly reacts
to produce hydroperoxides, fats or fatty acids having an — OOH group
attached to a carbon atom next to a double bond. Once formed, the
hydroperoxides serve as catalysts for further oxidation. As a result,
lower fatty acids, ketones, peroxides, and other substances are formed,
and the glycerol disappears. The unpleasant odors and flavors of these
products make the fat rancid. Rancidity is a term which applies to
any objectionable odor or taste in fats, no matter how brought about.

There exist a number of different substances, not themselves fats,
which have a remarkable power of slowing down the development of
oxidative rancidity. Such substances are called antioxidants. They are
present naturally in many fats, which have not been too extensively
refined, and have a large influence on the keeping qualities of fats. Ex-
amples of antioxidants are crude lecithin, hydroquinone (HOC6H4OH),
vitamin C, and the tocopherols (vitamins E, p. 216). Only small
amounts, of the order of 1 per cent of the fat, or less, are sufficient to
delay the onset of oxidative rancidity for extended periods. Antioxidants
appear to function by interfering with the catalytic effect of the hydro-
peroxides mentioned above. In so doing they are themselves slowly
oxidized, so that their effect eventually wears off. Antioxidants are
deliberately added to many food fats to improve their keeping qualities.
They are also thought, by many investigators, to play an important
biological role in preventing unwanted oxidations from occurring in vivo.

LIPIDES (fats and RELATED SUBSTANCES)

91

Determination of fat

The percentage of fat in a foodstuff is determined by extracting the
fat with etlier and weighing it. In tables of analyses this is generally
spoken of as fat, or more correctly, ether extract. ' It is not necessarily
all fat, since ether will dissolve many other substances such as waxes,
resins, fatty acids, and coloring matter, all of which may be contained
in natural fats. The ether extract of cereals is mainly fat, whereas a
large proportion of that obtained from vegetables consists of fatty acids,
phospholipides, nonsaponifiable matter, etc. The following table shows
how variable is the comi)osition of ether extract:

Table 4-7
Composition of ether extract

Material

Neutral

Free fatly

Nonsaponifiable

extracted

fats

acids

"Lecithin"

matter

(per cent)

(per cent)

(percent)

(per cent)

Potatoes

16.3

56.9

3.1

10.9

Beets

23.0

35.3

10.7

Com

88.7

6.7
14.0

3.7

Barley

73.0

6.1

Oats .

59.2

35.4

0.8

2.7

Peas .

58.6

112

27.4

7.4

Soybeans

95.5

12

1.8

1.5

A special test for the determination of fat in certain dairy products,
particularly milk, was introduced in 1890 by Stephan Moulton Babcock.
The "Babcock test," as it is universally called, has been of decisive im-
portance to the growth of dairying in this country, since it made possible
a quick, practical means of judging the butter fat production of individual
cows, permitting selection of the best producers for breeding. The test
is made by treating a definite amount of milk with an equal volume of
90 per cent sulfuric acid and warming the mixture gently. On cen-
trifuging this mixture, the fat separates as a distinct layer, which is
measured in the neck of a special flask cahbrated to read directly the
percentage of butter fat.

WAXES

Definition

"Waxes are classified as simple lipides, together with the true fats,
but unlike fats they contain higher monohydroxy (sometimes dihydroxy)
alcohols in place of glycerol. These alcohols exist in the- wax in com-

92 LIPIDES (fats and RELATED SUBSTANCES)

bination with fatty acids, that is, as esters. An example of an individual
wax ester is cetyl palmitate:

O

II
CHalCHO hCHjOCCCH.) uCHi

The natural waxes are mixtures of many such esters and often contain
hydrocarbons as well.

Occurrence and importance

Waxes are widely distributed in nature, both in plant and animal ma-
terial. They are generally found on the external surfaces, where they
serve as a protective coating and prevent undue evaporation of moisture.
They keep the feathers of birds and the wool and hair of animals soft
and pliable. Some of the important natural waxes are as follows: bees-
wax, secreted by the honey bee; lanolin (wool wax or degras), a waxy
material obtained from wool; spermaceti and sperm oil, saHd and liquid
waxes, respectively, which are separated from the oily liquid in the head
of the sperm whale ; Chinese wax, the secretion of an insect ; and carnauba
wax, a coating found on the leaves of the carnauba (Brazil) palm. Waxes
are important commercial materials and are extensively used in the
manufacture of candles, floor polishes, and varnishes. Many industrial
waxes, however, are synthetic products made from higher chlorinated
hydrocarbons, etc.

Properties

The waxes are, as a rule, solid materials that have a melting point
between 60° and 80°C. They are insoluble in water and are poor con-
ductors of heat. Waxes are much more resistant to saponification than
fats or oils. Light, air, bacteria, molds, and other agents that readily
bring about changes in the fats have little or no effect upon the waxes.
Because of these properties it is evident that they are particularly suitable
for the protection of the surfaces of animals and plants.

Composition

Waxes can be separated into their constituents by saponification with
alkali, which slowly forms soaps of the acidic components, and extrac-
tion of the aqueous soap solution with ether. The ether dissolves the
nonsaponifiable material (higher alcohols and hydrocarbons), which in
the case of waxes amounts to about 50 per cent of the original weight
(note difference from fats, p. 81). A list of the chief components of
several natural waxes is given in Table 4-8.

^3
d

S3

eo

H

o3

<A

o

J3

«

3

4-3

03

3

e

S3

o

V

to

o

X

a

a

crt to

u

^

o

^ C3

»-

03

a>

J^

O fe X

X

53

<c

^

c3

<U '^ 03

03

CO

tT

&

• 1-4

•4-^

§ -= fs

^ m

3

is

o5
(3

O
03

a

II 1

s3 <U

CO

C

S 03

03

- <u

9

.— 1

- ci

^

^ O

-

o

;>< >

!»:

X =J X

XX X

cS

5

r/1

S =*

03

53 ^ 03

ca 03 <rt

s

g

<u

& *:::

^

^ -S P

^ & &

t_t

p*

cj

CO 03

CO

to ? to

lO to to

0)

dJ

■fH

dj O

(D

0) ^ o

O) <U 0)

p,

O.

-a

OJ — '

(D

O ^ <D

<U QJ 0)

OQ cc O

W M W

pq

pq pq pq
K ffi K HH Ph nq

pq pq pq

(0

^^ ^i \M

M^ u^ M^ M^

a

q q q q q q

o o o o o o

o o o o

"2

S

o

6,

a w w a a m

o U U O o O

j^ ^ ^ ^ ^ ^

:■ f-H 71 <^) -■ ^)

K >^ ffi K HH Ph

o P. u u o o

o o o o o o
u o o o o o
^ ^ ^ J^ A A

^ J; ^ ^

M ?) ri N
pH hM HH PH

3
S

N Tl CI CI M ?1

^^1 ^^^^ ^^ '^^ "^^ "^^^

MH iJh l-M MH H-l HH

o o o u o o

o o o o

P5 r; CO eo
Ph Ph Ph pH

0

3

u

o
II

r; « r: «

p^ Ph hM Ph

u u o o

^ JO n rs « 00

K ffi W ffi W W
u o o u o o

u u u o

w

s

u

0

a
s

0

t-

t:3

o

3

o

•

J3

u

V

j:

60

H

1 §

5 C

CO
1— 1

00

T— (

GO o C^
(M CO CO

CD 00 O C^ •*

M (M CO CO CO

t>. (32 T^ eo

IM (M CO CO

o
O

O O <^ <5

a

o

<;

H
H

H 'So

."S •-- ^ to

S -^ -1-^ to

c p a :^

-^3-0 0

oa w tp-t ^-(

PL, O ^ '^

iz;

S

i

s

s

c
o

a
a

o

o

o

93

94 LIPIDES (fats and RELATED SUBSTANCES)

Note particularly the high carbon number of most of the components
listed, and the fact that the hydrocarbons all contain odd rather than
even numbers of carbon atoms. Although lanolin is one of the most
common and important waxes, it is not included in Table 4-8 because
of its unique composition. It contains over 20 unusual branched chain
acids and hydroxyacids combined as esters with a variety of alcohols,
among which are ceryl alcohol, cholesterol, other sterols, and at least
two alcohols of the triterpene type. Another unusual type of wax con-
stituent, reportedly present in Chinese urushi wax, is represented by two
of the higher dibasic acids, H00C(CHo)i8C00H, and HOOC(CH2)2o-
COOH.

STEROLS

Sterols are solid, cyclic alcohols usually containing 27-29 carbon atoms,
17 of which are arranged in a characteristic ring system consisting of
three six-membered rings and one five-membered ring (sjee Fig. 4-2).
Over thirty sterols have been found in nature. They occur in the tissues
of animals, in plants, abundantly in yeasts and molds, but apparently not
at all in bacterial cells. Some are saturated; others contain one, two
or three double bonds. Various side chains and one or more hydroxyl
groups complete the sterol structure (see formulas of cholesterol below,
and ergosterol, p. 213). The sterols may occur free, or combined with
fatty acids as esters, or with carbohydrates as glycosides.

Fig. 4-2. Diagram and numbering of the steroid ring system.

Several types of substance closely related to the sterols are of particular
biological importance. They include the bile acids, sex hormones (p.
292), adrenal cortical hormones (p. 290), vitamins D (p. 210), several
heart-stimulating drugs {e.g., digitalis) , saponins, and others. The sterols
and their related substances are collectively designated as steroids. All
contain the characteristic steroid ring system (Fig. 4-2).

Cholesterol

This substance is the characteristic sterol of higher animals. Although
present in every cell of the body, it is most abundant in egg yolk, animal
fats like cream and butter, cod liver oil, and especially in nerve and brain
tissue. In fact, the concentration in the human brain may reach the

LIPIDES (fats and RELATED SUBSTANCES) 95

surprisingly high value of 17 per cent of the dry weight. Cholesterol
is also the chief component of human gallstones, deriving its name from
this circumstance (Greek: chole, bile + stereos, solid). It has the
formula:

CH3 CH,

CH(CH,)3-CH-CH,

H2 CH3 I
C I C

H2 CH3 I
^C I ^C. H /C CH,

H, H

Cholesterol

The general distribution of cholesterol in the body indicates that it
must perform some important function there. It has been found that
cholesterol protects the blood corpuscles from the dissolving action of
certain poisons (saponins) . It also checks the tendency of the bile salts
to dissolve the blood corpuscles and inhibits the action of the fat-splitting
enzymes (lipases). A closely related compound, 7-dehydrocholesterol,
is the precursor of one of the important D vitamins.

In some types of heart disease (atherosclerosis) so much cholesterol
and other lipides are deposited on the inner walls of the heart arteries
that the blood supply to the heart muscle is seriously impaired. Since
this condition is associated with a high level of cholesterol in the blood,
it has been suggested that diets rich in cholesterol should be avoided.
Unfortunately this would eliminate the use of eggs, milk, butter, pork,
liver, and other highly nutritious foods. Furthermore, even if no cho-
lesterol is ingested, large amounts are synthesized in the body from other
substances. It has been shown that the blood cholesterol level is more
definitely associated with other factors such as age and obesity than
it is wath dietary intake.

Sitosterol

Vegetable oils such as corn, wheat, cottonseed, and linseed oils contain
sitosterol and other sterols. Sitosterol is found in the nonsaponifiable
fraction of the oil. Some investigators have maintained that the plant
sterols are converted into cholesterol and other zoosterols in the animal
body. There is abundant evidence, however, that cholesterol is syn-

96

LIPIDES (fats and RELATED SUBSTANCES)

thesized in the body from two-carbon fragments produced during carbo-
hydrate and fat metabolism.

Ergosterol

This sterol has acquired great importance because of the discovery
that it is the parent substance of one of the D vitamins. AVhen ergosterol
is exposed to ultra violet light for a few minutes, several new compounds
are formed. One of these, calciferol, has, besides chemical properties
that differentiate it from its parent substance, marked antirachitic
potency. (See discussion on vitamin D, p. 212, for details regarding
these changes.)

Ergosterol is a white, crystalline compound, which has a melting point
of 160-161^0. It is obtained commercially from yeast and mold cells,
where it occurs in concentrations up to 2 per cent of the dry weight of
the fungal tissue. The dry yeast or mold is extracted with alcohol and
benzene, and on concentration of the extract, crystals of crude ergosterol
separate. Its chief commercial use is in the manufacture of vitamin D
preparations.

BILE SALTS

Bile aids in fat digestion (p. 317) because most of its solid matter con-
sists of bile salts, strong emulsifying agents which form water-soluble
complexes with fatty materials. These substances are sodium salts of
peptide-like combinations of glycine or taurine (NH2CH2CH2SO3H) with
bile acids, steroids bearing a carboxyl and one or more hydroxyl groups.
The commonest and most abundant bile acid is cholic acid, and the
glycine and taurine conjugates of it are glycocholic and taurocholic acids,
respectively.

CH3 CH3

HO H3CH(CH2)2COOH HO H3CH(CH2)2CONHCH..CH2S03Na

HO'

OH HO
Cholic acid

Sodium salt of taurocholic acid
(a typical bile salt)

PHOSPHOLIPIDES

Phospholipides, or phosphatides, are fat-like substances that contain
phosphorus and nitrogen. Upon hydrolysis they are broken down into

LIPIDES (fats and RELATED SUBSTANCES) 97

their components, which inchide fatty acids or aldeliydes, phosphoric
acid, a nitrogenous base, and a polyhydroxy alcohoh PhosphoHpidcs
may be divided into three subgroups according to the alcoholic com-
ponent, as follows:

glycerol: lecithin, ccphalin, plasmalogen

inositol: lipositol, mono- and diphosphoinositides

sphingosine : sphingomyelin

The lecithins and cephalines arc the best known and most important
members of the entire group.

Phospholipides are believed to occur in every cell and are particularly
abundant in some of the most important and active tissues of the body:
brain, liver, and mammary gland. They appear to be an essential part
of the actual cell structure, and not merely stored-up food as are the
true fats in adipose tissue. This essential character is indicated by the
fact that the amount of phospholipides in the tissues is not materially
reduced during extreme starvation. For this reason these substances are
often called "essential lipides," or the "nonvariable component" of tissue
lipides, in contrast to the true fat, or "variable component."

Lecithins

The structure of a lecithin may be illustrated by the formula:

O

II
H2C — 0 — C — CijHsi

o

11

HC — O — C — C17H33

o

II

H2C-O— P— OCH^CH^NCCH,),
I +

o-

This formula shows that the substance is a glyceride in which one fatty
acid radical has been replaced by a group consisting of phosphoric acid
combined as an ester with choline (p. 162). Since the phosphate residue
is strongly acidic and the choline nitrogen strongly basic, the two neu-
tralize, each other and form an inner salt, or zwitterion, which is indicated
in the formula by the + and — signs. The fatty acids in lecithins tend
to be rather highly unsaturated, although some saturated acids are usually
present. A hydrolecithin, in which bot-li fatty acids (palmitic) are satu-
rated, has also been found in brain and lung tissue. It is much less
soluble in the usual fat solvents than unsaturated lecithins. It is theo-
retically possible to have lecithins in which the phosphoric acid-choline

98

LIPIDES (fats and RELATED SUBSTANCES)

group is attached to the center, or beta, carbon atom of glycerol. How-
ever, it seems very doubtful if such beta lecithins exist in nature.

Many different lecithins have been found in brain, nerves, liver, pan-
creas, heart, blood, and other active tissues of the body. Egg yolk and
soybeans are especially rich (2 to 5 per cent) in lecithins and serve as
starting materials for commercial preparation. Soybean lecithin, pro-
duced in ton quantities, is used as an emulsifying agent in many food
products; for example, the addition of 0.2 per cent soybean lecithin to
oleomargarine gives a consistency that closely resembles that of butter.
To obtain the crude lecithin, the fat extracted from the original source
material is treated with acetone. True fats are soluble; lecithins and
cephaline, being insoluble, precipitate out. The precipitation can be
made more complete by adding magnesium chloride or cadmium chloride,
which form sparingly soluble addition compounds with lecithins and
cephalins. The cephalins are separated from lecithins by virtue of their
lower solubility in alcohol.

Highly purified lecithins are colorless, greasy, paraffin-like substances.
Colorless preparations, however, are seldom seen, since they are difficult
to obtain and quickly darken on exposure to air. The outstanding char-
acteristic of the lecithins is their high chemical reactivity. They are
easily oxidized, easily hydrolyzed, and have a great capacity for com-
bining with other substances such as water, salts, proteins, and carbo-
hydrates. As indicated above they are also excellent emulsifying agents,
probably because their structure includes both the water-attracting salt
group and the long carbon chains of the fatty acid residues (compare
soaps and detergents, p. 87) .

Hydrolysis of lecithins, with moderately strong acid or alkali, readily
breaks them down into the constituent fatty acids, choline, and a-glycero-
phosphoric acid. The attachment between the glycerol and the phos-
phoric acid is very strong. It is only broken by long boiling with strong
acid.

0

CHaOH CH^OC-CsHj,

I I

CHOH CHOH

0 O
II II

CHjOP-OH CH^O-P-OCHoCHzNCCH,),

1 I •*■
OH 0-

a-Glycerophosphoric acid A lysolecithin

Enzymatic hydrolysis also occurs in living tissues as a result of the ac-
tion of lecithinases. One type of lecithinase (type B) splits off' both fatty
acid residues. Another (called type A) removes only one, leaving a
product, lysolecithin, which is poisonous because of its power to hemolyze

LIPIDES (fats and RELATED SUBSTANCES)

99

(dissolve) red blood cells. Cobra snake venom contains Iccithinase A,
and presumably owes its deadly effect, at least in part, to the production
of lysolecithin. Still another hydrolysis i)roduct of lecithins (or cepha-
lins) are the phosphatidic acids, which are found in the form of metallic
salts in various plant and animal tissues.

O

II

CH2 — 0 — C — CisHai

0

II

CH— O-C-CnHaj

0

'I 0
CHaO— PC^r^Ca

A phosphatidic acid
(calcium salt)

Cephalins

The cephalins are found closely associated with lecithins in many
tissues, but particularly in brain tissue. A phospholipide preparation ob-
tained from ether-soluble brain lipides, by precipitation with alcohol,
has been called "brain cephalin." It was thought to differ from lecithin
only in containing ethanolamine, or cholamine (HOCHoCHoNHo), as
the basic constituent in place of choline. However, it has been demon-
strated that "brain cephalin" is actually a mixture of several phospho-
lipides, only one of which corresponds to the above structure. This com-
ponent of "brain cephalin" is now designated by the more specific name,
phosphatidyl ethanolamine. A second component is very similar in
structure but contains the amino acid serine, HOCHoCHCOOH, in place

NHo

of ethanolamine. It is named phosphatidyl serine. When obtained
from brain tissue, it contains oleic and stearic acids, as the only fatty
acids. The chemical formulas of these substances are as follows:

O

II
CH2 — 0 — CC17H34

o

II

CH — 0 — CCnHas

I 0

CH2-0— p— OCH2CH2NH3

I

0-

Phosphatidyl ethanolamine
(inner salt formula)

O

II
CHj — 0 — CCkHss

0

II

CH-0— CCnHjj
0

II

CHj-O-P— OCH2CHCOOH
1 I

ONa NH2

Phosphatidyl serine
(sodium salt)

100

LIPIDES (fats and RELATED SUBSTANCES)

Phosphatidyl serine is a strong acid, existing at physiological pH values
combined as a salt with sodium or potassium. It is alcohol-insoluble,
whereas phosphatidyl ethanolamine is easily soluble in alcohol. The
''cephalin" of beef brain, and of pig heart and liver, is made up mostly
of phosphatidyl ethanolamine (50-75 per cent), mixed with smaller
amounts of phosphatidyl serine (13-35 per cent) . According to Chargaff,
egg yolk cephalin is entirely of the ethanolamine type. Cephalins are
thought to play a part in the process of blood clotting.

Sphingomyelins

This type of phospholipide differs from most others in being insoluble
in ether. It is also insoluble in acetone, but forms a colloidal solution
in water. It can be extracted from tissues with hot alcohol, but on
cooling crystallizes out as a white solid. First discovered in brain tissue,
it is now known to occur in many organs, as well as in blood and milk.
Enormously increased amounts are present in all the organs and tissues
(except brain) of patients suffering from Niemann-Pick's disease.

Sphingomyelin on hydrolysis gives rise to an amino alcohol (sphingo-
sine) , plus a fatty acid (usually lignoceric) , phosphoric acid, and choline.
The fatty acid is attached to the amino group to form the amide, ligno-

CH3(CH2),2CH=CH-CH-CH— CH2

I I I

OH NH2 OH

Sphingosine

ceryl sphingosine. This substance is a normal component of pig spleen
and liver and can also be obtained by partial hydrolysis of sphingomyelin.

CH3(CHo)nCH=CH-CH-CH— CH2

I I I

OH NH OH

I

CO
I

(CH2)22
CH3

Lignoceryl sphingosine

The phosphoric acid and choline are presumably present in sphingomyelin
as a phosphorylcholine, inner-salt group (see formula of lecithin, p. 97) ,
attached to one of the hydroxyl groups of sphingosine.

Other phospholipides

Almost nothing is known of the biological significance of the phospho-
lipides other than lecithins and cephalins, but the fact that they occur

LIPIDES (fats and RELATED SUBSTANCES) 101

in large amounts in brain and nerve tissue undoubtedly means that they
have an important function there. An unusual lipide type is represented
by the ylastnnlogens, substances first found in beef muscle, but also present
in brain. These lipidcs make up about 10 per cent of the total phos-
phatides of these tissues. Plasmalogen on hydrolysis gives rise to chola-
mine, a-glyccrophosphoric acid, and a higher aldehyde corresponding to
a common fatty acid, e.g., palmitaldehyde (C15H31CHO) or stearaldehyde
(C17H30CHO). Therefore, plasmalogens most probably have the follow-
ing formula, in which R represents the alkyl radical of a higher fatty acid.

I ^CHR

CHO^ O

I II

CH2— O— P— OCHoCHjNH,

I ^

0-

Plasmalogen

Phospholipides containing inositol have been found in soybeans, as well
as in nerve tissue. One of the components of "brain cephalin" has been
found to be an alcohol-insoluble phospholipide which on hydrolysis gives
rise to equimolar portions of glycerol, a fatty acid, and inositol meta-
diphosphate:

O

H II

^Cc::<)-P— (0H)2

HCOH HCOH Q

I I II

HCOH„ HC-0-P(0H)2

Inositol meta-diphosphate

CEREBROSIDES (GLYCOLIPIDES)

Compound lipidcs containing a sugar residue but no phosphorus are
present in relatively large amounts (7 per cent of the solid matter) in
brain tissue and probably occur in all organs of the body. These sub-
stances, called cerebrosides, are insoluble in ether and water, but dissolve
in pyridine or in hot alcohol. They are white solids which on hydrolysis
yield one molar erjuivalent each of a higher fatty acid {e.g., lignoceric) ,
sphingosine or dihydrosphingosine, and galactose. Such a combination
of a carbohydrate, fatty acid, and base shows what unexpected com-
pounds exist in the body and demonstrates that the line between carbo-
hydrates, fats, and nitrogenous compounds is not nearly as sharp as
classifications might indicate. All the fatty acids which have been found

102 LIPIDES (fats and RELATED SUBSTANCES)

to occur in cerebrosides and sphingomyelin are saturated, or have only-
one double bond, and contain a large number of carbon atoms (mostly C24
or C26)-

REVIEW QUESTIONS ON LIPIDES

1. Explain the terms: (1) fat, (2) phospholipide, (3) wax, (4) sterol, (5) soap,
(6) saponification, (7) hydrogenation of oils, (8) detergent, (9) antioxidant.

2. Explain chemically why tallow is a solid and olive oil is a liquid. Name the
chief fatty acids obtained from each.

3. Give (1) the elements in (a) carotene, (b) cholesterol, (2) the equation for the
saponification of triolein with potassium hydroxide, (3) the chemical groups con-
tained in lecithin, (4) the graphic formula of glycerol, (5) the formula for one con-
stituent in a wax.

4. Discuss the factors that operate to make a fat rancid.

5. Explain the differences in the chemical composition of the ether extracts (fat)
of various food materials. Compare, for example, the ether extracts of oatmeal and
spinach.

6. Explain the terms (1) simple glyceride, (2) mixed glyceride, aad discuss their
occurrence in a natural fat.

7. Explain how an oil is made commercially. What is the source of (1) linseed
oil, (2) tallow, (3) "Crisco"?

8. Explain the significance and the limitations of the term "fat" as used in tables
of food analyses.

9. Why is January milk so pale in color as compared with June milk? Can you
think of any way by which winter milk could be improved in color?

10. Generally compare the biological roles of true fats, waxes, and complex lipides.

11. Correct the following statements if incorrect: (1) "Dreft" is the trade name of
a cleaning agent that does not form an insoluble precipitate with hard water. Chem-
ically, it is the sodium salt of sulfated aliphatic alcohols, chiefly lauryl. (2) Hydro-
genation of coconut oil will produce a solid fat. (3) Glycerine and glycerol are dif-
ferent names for the same compound. (4) Stearin and sterol also mean the same
thing. (5) Linseed oil is obtained from the linen seed.

REFERENCES AND SUGGESTED READINGS

Bloor, W. R., Biochemistry of the Fatty Acids, Reinhold Publishing Corporation,

New York, 1943.
Bull, H. B., Biochemistry of the Lipides, John Wiley and Sons, Inc., New York,

1947.
Chargaff, E., Ziff, M., and Rittenberg, D., "A Study of the Nitrogenous Constituents

of Tissue Phosphatides," J. Biol. Chem., 144, 343 (1942).
Hilditch, T. P., Chemical Constitution of Natural Fats, John Wiley and Sons, Inc.,

New York, 1940.
Jamieson, G. S., Vegetable Fats and Oils, Reinhold Publishing Corporation, New

York, 1932.
Markley, K. S., Fatty Acids, Interscience Publishers, Inc., New York, 1947.
Thannhauser, S. J., and Schmidt, G., "Lipins and Lipidoses," Physiol. Rev., 26, 275

(1946).
Wittcoff, H., The Phosphatides, Reinhold Publishing Corp., New York, 1951,

Chapter 5

PROTEINS

Proteins are organic compounds containing nitrogen, which, together
with the carbohydrates and the fats, form the principal part of the solids
of living matter. The name comes from a Greek word, proteios, mean-
ing first. It was originated in 1839 by a Dutch chemist, Mulder, because
of his belief in the widespread occurrence and great importance of the
proteins. In nutritiori, the proteins are used principally for body-building
and maintenance rather than for providing energy. Protein materials
are derived from both plant and animal sources, although in the average
American diet animal proteins appear to predominate.

Like the polysaccharides and fats, proteins can be broken down into
their unit structures. These units are amino acids, the "building stones"

Table 5-1

Economic Importance of some industries based on proteins *

Value of products

Industry Wage earners shipped

1. Leather and its products 383,175 $3,673,849,000

2. Meat products, including poultry 274,441 8,766,322,000

3. Wool, felt, and hair products 230,524 2,432,355,000

4. Furs and their products 37,561 564,660,000

5. Sea foods (canned) 20,153 226.519,000

6. Glue and gelatin 5,372 99,260,000

951,226 $15,762,965,000

* Compiled from the 1947 Census of Manufactures, Bureau of the Census, 1950, and
from the Statistical Abstract of the United States, 1951, published by the Department
of Commerce.

from which the proteins are constructed. In the common proteins there
are about twenty different amino acids; many hundred molecules of these
amino acids are combined to form the larger aggregates called proteins.
Smaller aggregates of the order of 100 units or less are usually classed
as peptides.

Although proteins do not play as large a role in our economic life as
carbohydrates, their importance is very great. They are the distinguish-
ing constituents of many essential foods (e.g., meats, fish, poultry, eggs,

103

104

PROTEINS

beans, peas, etc.) as well as important clothing and furnishing materials
(e.g., woolens, felts, furs, silks, leather, etc.). Table 5-1 gives data
for some industries utilizing protein materials.

Comparison wdth Table 3-1 (p. 103) shows that the protein industries
approach the machinery industry in monetary value of products, though
they do not give employment to as many workers. Since Table 5-1 is
not exhaustive, it does not include any nonindustrial business based on
protein materials; for example, eggs, which had a value in 1950 of
$1,811,387,667 at the farms where they were produced.

Several pure proteins are prepared and sold — gelatin for food and film
industries; insulin for the treatment of diabetes; pepsin, trypsin, and
other enzymes; and the peptide antibiotics, bacitracin and tyrothricin.
Other purified protein materials are marketed as vaccines, toxins, and
antitoxins.

Occurrence and preparation .

Every kind of cell contains its own special proteins, and, therefore, the
number of individual proteins must be enormous. About 700 have been
isolated and examined. Perhaps 200, contained in the most important
foodstuffs and biological materials, have been studied in some detail.
Since there are about 40 known amino acids, each one of which may be
used many times, it is evident that an enormous number of proteins is
possible. Perhaps comparison with the number of words in the English
language will make the possibilities more evident. We have 26 letters,
each of which may be used several times in a given word. They comprise
about 600,000 words listed in an unabridged dictionary. Many new
words are added yearly, just as many new proteins are discovered each
year. There are probably thousands of new proteins in the many species
of plants, animals, and microorganisms that have not been investigated.

The general method of preparing a protein is to dissolve it in its
particular solvent, water, salt solution, or alcohol, and then alternately
precipitate impurities or protein by changing one or more factors such as
pH, salt concentration, or temperature. Solution and reprecipitation are
repeated many times until the protein is obtained as pure as possible,
and preferably in crystalline form. Some proteins, for example, egg
albumin, can be obtained crystalline after only one or two operations,
but others, such as muscle phosphorylase, require about ten different
treatments before they will crystallize. A twentyfold purification is
usual, but in the case of botulinum toxin A, purification increases the
potency more than 200 times that of the crude material.

Isolation of proteins challenges the skill and resourcefulness of the
most experienced investigators. Great progress, however, has been made
in the last twenty years. To date, more than 150 proteins have been

From Hawk and Berseim. Practical Phjif^iolngical Chemistry.
Courtesy of I'. P.laldstoii's Son & ("o.. Inc.

Fig. 5-1. Crystalline egg albumin.

From Hawk and BeiKciin, Practical Phi/siolof/ical Chemistry.
Coiirte.sy of I". Blakiston's Soii & Co., Inc.

Fig. 5-2. Oxyhemoglobin of the horse.

105

From Reicliert and Brown, The CrvstaUooraphy of Hemoqlo
litis. Courtesy of the Carnegie Institution of Wasliinsrton

gton.

Fig. 5-3. Oxyhemoglobin of the squirrel.

From Keichert and Brown, The Cri/xtaUooraphy of Hemoglo-
bins. Courte.s.v of the Carnegie Institution of Washington.

Fig. 5-4. Oxyhemoglobin of the guinea pig.

106

PROTEINS

107

obtained in a crystalline state. Crystallinity, however, is no sure sign
of homogeneity (purity). The best tests of homogeneity are electro-
phoresis, ultracentrifuge sedimentation, solubility measurements, and
maximum biological activity (if tiie protein has such a property).

If a solution of protein is placed in an electrophoresis cell, and an
electric current is passed througii the solution, the protein moves toward
the cathode or anode, depending upon the polarity of the electric charge
carried by the protein. The position of the protein in the solution is
marked by a change in the refractive index of the solution and shows
up as a boundary which can be determined by suitable optical means.
The migration of the protein, during a given period of time, can be
followed and its electrophoretic mobility calculated. If the protein
consists of only one component, there will be only a single boundary in
the solution. The protein is then said to be pure or homogeneous. If
the solution contains a second component, this will usually migrate at
a different rate of speed than the first so that it will be revealed in the
optical pattern. Electrophoresis can be used to determine not only the
number of components but also the amount of each component in the
solution. An example of its usefulness is found in the extensive study
being made of the proteins of blood in health and disease.

In the ultracentrifuge, the protein solution is subjected to a centrifugal
force about 250,000 times as great as the force of gravity. Heavier par-
ticles settle faster than lighter ones. If the solution contains two or
more proteins, there will be either two or more boundaries, or else a
diffuse boundary in the optical pattern. A single protein in solution
shows only one boundary when it is sedimented.

The solubility test of homogeneity depends upon the fact that the
quantity of dissolved protein, until saturation is reached, is directly pro-
portional to the weight of sample taken. After saturation there is no
increase in dissolved protein, irrespective of the weight of sample. How-
ever, if the sample contains more than one protein, the solubility curve
does not show a sharp break but continues to rise until the saturation
point is reached for each component.

Few of the proteins isolated have been subjected to all of these tests.
Most of the proteins, e.g., casein, gliadin, that have been prepared from
our common foodstuffs appear to be mixtures of several components.

The names and amounts of the principal proteins in some common food-
stuffs are given in Table 5-2. Tlie percentages given are, in most cases,
the amounts actually isolated. The totals, in general, agree well with
the figures for crude protein (N X 6.25), and hence, it may be assumed
that the kinds and amounts of protein in many of our staple foods are
well established.

From Table 5-3 it is apparent that proteins have great importance
other than as food constituents. Some of our most imjiortant articles

108

PROTEINS

Table 5-2

Principal proteins of some common foodstuffs

(Undried basis)

Foodstuff
Barley .

Beans

Blood

Com

Eggs

Lean meat

Milk

Oats

Proteins

Classification *

Hordein

Alcohol-soluble

Hordenin

Glutelin

Leucosin

Albumin

Edestin

Globulin \
Proteose J

Proteose

Total

Phaseolin

Globulin

Phaselin

Globulin

Total

Serum albumins

Albumin

Serum globulins

Globulin

Fibrinogen

Globulin

Hemoglobin

Chromoprotein

Total

Zein

Alcohol-soluble

Zeanin

Glutelin

May sin

Globulin

Globulin

Globulin

Proteose

Proteose

Total

Egg albumin

Albumin

Vitellin

Phosphoprotein

Total

Myogen

Albumin

Myoalbumin

Albumin

Myosin

Globulin

Globulin

Globulin

Total

Casein

Phosphoprotein

Lactalbumin

Albumin

Globulin

Globulin

Total

Avenalin

Globulin

Glutelin

Glutelin

Prolamin

Alcohol-soluble

Percentage in
foodstuff

4.0
4.5
0.3

1.95

10.7S

21.5
2.0

23.5

4.0
3.3

0.3
14.0

21.6

5.0

3.15

0.25

0.14

0.06

8.60

8.2
5.2

13.4

2.0

0.2

13.6

4.2

20.0

2.74
0.60

Trace

Total

3.34

1.5

11.2

1.3

14.0

* See p. 110 for basis of classification.

PROTEINS

109

Table S-2 — (Continued)

Foodstuff
Peas . . .

Rice

Rye

Wheat

Proteins

Classification *

Legumilin

Albumin

Legumin

Globulin
Globulin .

Vicillin

Proteose

Proteose

Undetermined

.

Total

Or\'zenin

Glutelin

Globulin

Globulin \
Ale. sol. J

Ale. sol.

Total

Gliadin

Alcohol-soluble

Soealenin

Glutelin

Edestin

Globulin
Proteose .

Proteose

Leucosin

Albumin

Total

Gliadin

Alcohol-soluble

Glutenin

Glutelin

Globulin

Globulin

Leucosin

Albumin

Proteose

Proteose

Miscellaneous

Percen tage in

foodstuff

2.0

10.0

1.0
11.5

24.5

4.0
5.9

9.9 .

4.0
2.44

1.76

0.43

8.63

3.91
4.17
0.63
0.36
0.43
1.10

Total

10.60

of clothing are protein in character. Probably all enzymes and some
hormones are proteins. The discovery of the existence of proteins as
viruses, toxins, and poisons, deadly to man, has emphasized their vital
importance and greatly increased the impetus to research.

Elementary composition

All proteins contain carbon, hydrogen, oxygen, and nitrogen. Most
proteins contain sulfur, some contain phosphorus, and a few contain iroij,
copper, or manganese. Phosphorus is not really a constituent of the
protein part of the molecule. Only the conjugated proteins — proteins
united with a prosthetic (additional) group — contain phosphorus, the
phosphorus being in the form of a phosphoric acid ester. Iron, likewise,
is not found in any amino acid. Hence it is not in the protein molecule
proper but is contained in the prosthetic group, e.g., hematin in hemo-
globin.

110

PROTEINS

Table 5-3
Examples of enzyme proteins and nonfood proteins

I*R0TEIN Classification Source

Keratin Albuminoid Hair, wool, feathers,

hide, nails, etc.

Fibroin Albuminoid Silk

Sericin Albuminoid Silk

Spongin Albuminoid Sponge

Amylopsin Albumin Pancreas

Pepsin Albumin Stomach

Trypsin Albumin Pancreas

Papain Albumin Latex of papaya tree

Urease Globulin? Jack bean

Catalase Chromoprotein Liver, etc.

Flavoprotein Chromoprotein Yeast, heart, etc.

Insulin Globulin? Pancreas

Mosaic virus Nucleoprotein Diseased tobacco plants

Tuberculin Unclassified Tubercle bacillus

Avidin Albumin? Egg white

Crotoxin Albumin Rattlesnake venom

Bacitracin Polypeptide Antibiotic from

Bacillus licheniformis

Botulinum toxin A . Globulin Toxin from CI. botulinum

The elementary composition of proteins varies within wide limits,
but the average figures show C, 53 per cent; H, 7 per cent; 0, 23 per
cent; N, 16 per cent and S, 1 per cent. Nitrogen shows the greatest varia-
tion, ranging from about 10 per cent for the glycoproteins to 30 per cent
for the protamines. In the common food proteins it varies within much
narrower limits, 15.5-18.7 per cent, and, hence, an average figure of 16
per cent is taken. On the other hand, proteins from entirely different
sources, and different in character, may have the same elementary com-
position. Except in a very general way elementary composition is of
but little value in the differentiation of proteins.

CLASSIFICATION

Because of their number and complexity, proteins are difficult to
classify. The basis for classification is mainly (1) products on hydrolysis,
(2) solubility, (3) coagulability, and (4) precipitability. It is probable
that many compounds quite unlike in structure fall into the same group,
and it is certain that many so-called proteins are not chemical entities.
Even when crystallized, the proteins are not always homogeneous. How-
ever, even though imperfect, a classification is indispensable for study
and discussion. The official classification, with slight modifications,
follows. For examples of many of the classes see Tables 5-2 and 5-3.

PROTEINS 111

Simple proteins

These are naturally occurring proteins that on hydrolysis yield only
a-amino acids. This definition is not strictly correct because many of
the albumins contain small quantities (1-2 per cent) of carbohydrate,
e.g., mannose, galactose. Simple proteins are subdivided as follows:

Albumins. Soluble in pure water and dilute salt solutions, coagulable
by heat, precipitated by saturation with ammonium sulfate.

Globulins. Insoluble in pure water but soluble in neutral salt solutions
{e.g., 5 per cent NaCl) , coagulable by heat, precipitated by half-satura-
tion with ammonium sulfate.

Glutelins. Insoluble in water or salt solution, but soluble in dilute
acids or alkalies (e.g., 0.1 per cent).

Prolamins (Alcohol-soluble Proteins). Insoluble in water, dilute salt
solutions, or absolute alcohol, but soluble in 70-80 per cent alcohol.

Albuminoids. Insoluble in the reagents given for the preceding pro-
teins. A heterogeneous group of simple proteins found usually in the
skeletal structures and protective coatings of animals; examples are kera-
tin from horn, hide, hoof, hair, feathers, and wool, elastin from ligaments,
collagen from hide and tendons, and fibroin and sericin from silk. Gela-
tin, although it does not fit into this group, is classed as an albuminoid
because it is obtained from collagen by boiling with water. It is more
properly a derived protein.

Histones. Proteins (having basic properties) coagulable by heat, solu-
ble in water, dilute acids, or alkalis, but insoluble in dilute ammonia.
They form precipitates with other proteins and yield on hydrolysis large
quantities of the basic amino acids. Typical examples are globin from
hemoglobin and histones from the thymus gland and leucocytes.

Protamines. These are the simplest natural proteins and contain only
a small number of amino acids, among which arginine predominates —
in some cases comprising 85 per cent or more of the protein. The pro-
tamines are strongly basic, soluble in water, and not coagulable by heat.
They form crystalline salts with mineral acids and precipitates with other
proteins, e.g., insulin (protamine-insulin). They are found in ripe sperm
cells. The most studied compounds have been obtained from fish sperm,
e.g., salmine from salmon, sturine from sturgeon, and clupeine from
herring.

Conjugated proteins

These proteins are combinations in which a simple jirotein is united
with a characteristic nonprotein group. The nonprotein group is called
a prosthetic group. The subdivisions are as follows:

112 PROTEINS

Nucleoproteins. These are basic proteins, e.g., histones, in combination
with nucleic acids. They are obtained most readily from thymus and
other glands, yeast, and wheat germ.

Glycoproteins or Glucoproteins. The prosthetic group is carbohydrate
in character, and makes up a large proportion (25-35 per cent) of the
glycoprotein. As a consequence, the nitrogen content of glycoproteins
is low, 9-13 per cent. Example^ are: proteins from saliva (mucin),
vitreous humor, gastric mucosa, and jellyfish.

Phosphoproteins. The prosthetic group is phosphoric acid, linked as
an ester to the protein through a hydroxyamino acid, e.g., serine. Two
of the best known proteins, casein and vitellin, belong to this class.

Chromoproteins. The prosthetic group is colored. For example:
hematin of hemoglobin is red, cyanin of hemocyanin (the respiratory pig-
ment in the blood of the lobster and other molluscs) is blue, melanin of
hair proteins is black, riboflavin phosphate of flavoproteins (respiratory
enzymes) is red, and retinene (aldehyde of vitamin A) of rhodopsin (the
chromoprotein involved in vision) is yellow. -^

Lipoproteins. The prosthetic group is fatty acid, lecithin, or a phos-
pholipide other than lecithin. Lipoproteins are a poorly defined group
occurring in egg yolk, brain tissue, lungs, etc.

Derived proteins

This division includes denatured proteins and cleavage products formed
by partial hydrolysis of naturally occurring proteins with acids or en-
zymes. The hydrolysis products are polypetides of varying size, and
the subdivisions have little chemical justification. The principal classes
still in use are:

Proteoses. Hydrolytic products of proteins that are soluble in water,
not coagulable by heat, and precipitated by saturating the solution con-
taining them with ammonium sulfate.

Peptones. These polypeptides are probably of smaller molecular
weight than the proteoses since they are found in the filtrate of the
ammonium sulfate precipitation of proteoses. So-called "peptones," used
in the preparation of bacteriological media, are mixtures of polypeptides,
mainly proteoses.

Peptides. Combinations of two or more amino acids. They are called
di-, tri-, tetra-, etc., peptides, according to whether they contain two,
three, four, or more amino acid residues in the niolecule. Some peptides
occur naturally, e.g., glutathione, pteroylglutamic acid (a vitamin), peni-
cillins, etc. Large numbers of peptides have been synthesized.

PROTEINS

113

PRODUCTS ON HYDROLYSIS— AMINO ACIDS

Number and kind of amino acids

\Mien a protein is iiydrolyzed by means of acid, alkali, or enzymes,
alpha amino acids, usually called amino acids, are obtained as products.
If the protein is a conjugated protein, such as casein, nucleoprotein,
etc., other products are obtained in addition. Only the amino acids,
however, come from the protein molecule proper; the additional com-

Courtesy ol' W. C. Ko.se and Joiiniul of liioloijicul Chemistry.
Fig. 5-5. Threonine.

pounds originate in the prosthetic group. The number of amino acids
that have been definitely obtained as a result of hydrolysis is about
twenty-five; about fifteen more have been reported, but their presence
in protein is either not well established or they occur only in unusual
protein materials, e.g., djencolic acid of the djencol bean and a,y-diamino-
butyric acid in the polymyxin antibit)tics (pp. 118, 119). Several others,
citrulline, homocysteine, and cystathionine, do occur free in the body
tissues and play an important role in intermediary metabolism. Amino
acids are classified according to the number of amino groups, carboxyl
groups, and other characteristics as shown on pp. 116-120.

rroni Hawk and Beigeiiu, Practical Phyniological Chemistry.
Courtesy of P. Blakiston's Son & Co., Inc.

Fig. 5-6. Phenylalanine.

From Hawk and Bergeim. Practical Physiological Chemistry.
Courtesy of P. Blakiston's Son & Co., Inc.

Fig. 5-7. Tyrosine.

114

~1

1

From Schmidt's Chemistry of the Amino Acids and Proteins.
Courtesy of Charles C. Thomas, publisher, Springfield, Illinois.

Fig. 5-8. Cystine.

From lluwk and I'.crgeim, Practical I'hysioloyical Chemistry.
Courtesy of P. Blakiston's Son & Co.. Inc.

Fig. 5-9. Histidine.

115

116

PROTEINS

Photo by Horst G. Schneider.

Fig. 5-10. Trytophan.
Classification and formulas of amino acids

I. MONOAMINO-MONOCARBOXYLIC ACIDS

A. Aliphatic acids:

COOH
I
HoN— C— H

H

COOH
I
H,N-C— H

CH,

COOH
I
HjN— C— H

I
CH2

CH3

COOH
I
H,N-C— H

CH

H3C ^CHj

L-Glycine or L- Alanine or L-Aminobutyric acid L- Valine or

aminoacetic acid a-aminopropionic a-aminoisovaleric

acid acid

COOH
I
H2N— C— H
I
CH2

I

CH
/ \
H3C CH3

COOH

I
H2N— C— H
I

H3C CzHs

COOH

I
H2N— C— H

I
CH2

I
CH2

I
CH2

COOH

I
H2N-C— H

CH2OH

L- Leucine or L-Isoleucine or

a-aminoisocaproic /3-methyl-a-amino-
acid valeric acid

CH3
L-Norleucine or L-Serine or

a-aminocaproic a-amino-/3-hydroxy-
acid propionic acid

COOH
I
H2N— C— H
I

CH2
I
CH:OH

L-Homoserine or

a-amino-7-hydroxy-

butyric acid

COOH

I
H2N— C— H
I
H— C— OH
I

CH3
L-Threonine or
a-aniino-/3-hydroxy-
butyric acid

PROTEINS

117

B. Acids in'th aromatic nuclei:

COOH
I
H2N-C-H
I

I

HC^ CH

I II

H

L-Phenylalanine

COOH
I
H2N— C— II
I

CH2
I

I II

1
OH

L-Tyrosine or
p-hydroxyphenylalanine

C. Acids containing sulfur:
COOH

HjN-C— H

CH2
I
SH

L-Cysteine or
^-thiolalanine

COOH
I
H,N— C— H

CH2

\

S
I

s

I

CH2

1

H— C— NH2

I
COOH

L-Cystine (dicysteine) or
di-/3-thiolalanme

COOH

I
H2N-C— H

I
CH2

I
S

I
CH2

I
H-C— NH2

I
COOH

L-Lanthionine

COOH
I
H2N-C-H

CH2

I
CH2

I

s

I

CH3

L-Methionine or
a-amino-7-methylthiol-
butyric acid

COOH
I
H2N— C— H

CH2
I

CH2
I
SH

L- Homocysteine

COOH COOH
I I

H2N— C— H H2N— C— H

CH2 CH2

I I

CH2 CHi

I I

s s

L-Homocystine
(dihomocysteine)

118

PROTEINS

COOH
I

COOH
I

H,N-C— H HjN— C— H

COOH
I

COOH
I

I
CHj

S —

-CH,

I
CHj

I

H,N— C— H HjN— C— H

L-Djencolic acid

v»

CHj
I
CHr

CH,

-A-

(Horiiocysteine part) (Cysteine part)
L-Cystatliionine

COOH
I
H— C— NH2
I
H3C — C — CH3

SH

D-Penicillamine
OS-thiolvaline)

D. Adds containing iodine.

COOH

I
H2N-C— H

CH2
I

HC^ ^CH

I II

IC^^^CI

I
OH

L-Diiodotyrosine

COOH
I
H2N— C— H

CH2
1

HC^ ^CH

I II

IC^^^CI

I

0

I

HC^ ^CH

I II

I
OH

L- Thyroxine

PROTEINS

119

II. MONOAMINO-DICARBOXYLIC ACIDS AND RELATED AMIDES

COOH
I
H2N-C-H

I

CH,

I

COOH.

COOH

I
H,N— C— H

I
CHj

I
CO-NHz

COOH
I
H2N-C-H
I

CH,
I
CHj

COOH
L-Glutamic acid or

COOH

I
H2N-C— H

I
CHj

I
CH,

CONH,

L-Glutamine

L-Aspartic acid or L-Asparagine
a-aminosuccinic acid (amide of aspartic a-aminoglutaric acid (amide of glutamic

acid)

acid)

III. Basic amino acids

COOH

I
H-C— NH,

I
CH,

I
CH,

I
NH,

COOH

I
H2N-C— H

I
CH,

I
CH,

I
O

I

NH,

D-a, 7-Diaminobutyric acid

L-Canaline;

COOH

I
H,N-C— H

I
CH,

I
CH,

I
0

I
NH

I
C=NH

I
NH,

L-Canavanine

COOH

I
H,N-C-H

I
CH2

I
CH,

I
CH,

I
CH,

I
NH,

L-Lysine or
a, e-diaminocaproic acid

COOH

I
H,N-C-H

I
CH

I
CH,

I
CHOH*

I
CH,

I
NH,

L-Hydroxylysine
'(position of OH , whether
D or L, is not known)

120

PROTEINS

COOH
I
H2N-C— H

HC =

I I

H

CH2

I
C

L-Histidine or
jS-imidazolealanine

COOH

I
H2N-C— H

I
CH2

I
CH2

I
CH2

NH

I
C=NH

I
NH2

L-Arginine or
5-guanidino-a-aminovaleric acid

COOH

HjN-C— H

I
CH2

I
CHj

CH2

NH,

COOH

I
H2N— C— H
I
CH2

I
CHj

CH2

NH
I
C=0

CH3

COOH

L-Ornithine

IV. Heterocylic amino acids

COOH
I
NH2-CH

NH2

L-Citrulline

H
HC*^ C-

CH2
I
C

HC^^^C^^^CH

H H

L-Tryptophan or
indolealanine

H-C— NH-C— H

I I

COOH CH.

CHa
CH2

NH
I
C=NH

I
NH2

L-Octapine

H,C

CH,

HjC^^^^CH-COOH
N
H

L-Proline or
a-pyrrolidine-carDoxylic acid

PROTEINS 121

HO— HC CH,

HjCs^^^^CH-COOH

N
H

L-Hydroxyproline

Every amino acid contains at least one amino (NHo) group and one
carboxyl (COOH) grouj). (Proline and hydroxyproline may be re-
garded as modified amino acids, in which the amino group has been
linked to a second carbon, thus forming a ring compound.) One of the
amino groups in the above list is always attached to the alpha carbon,
hence the name alpha amino acids. Acids with the amino group attached
to carbons other than the alpha carbon occur in nature (e.g., beta-alanine
in pantothenic acid, beta-lysine in certain antibiotics, and gamma-
aminobutyric acid in biological fluids) . Other types of amino acids will
probably be found as more plants and microorganisms are investigated.

The part of the formula other than NHl.CH-COOH is called the side
chain and is represented by the letter R. Amino acids differ with respect
to their side chains, and, hence, it is this part of the molecule that im-
parts distinctive features to the compound. Since the most important
chemical and physiological properties of amino acids are attributable to
the side chains, the student should note these carefully and become familiar
with the groups contained therein. Some of these distinguishing groups
are:

1

HC^^^CH

1 II

1

HC^ CH

1 II

«? ?

HC5%. ^CH
C
H

HC^^ ^CH

1

OH

HN., ^N
H

Phenyl

Phenol

H

Imidazole

NH
II H
HjN-C-N—

HC^^^C
1 II
HC^ C

C—

II
s^p^^CH

H

H

Guanidino

Indole

It is evident that the alpha carbon is asymmetric in all of the amino
acids except glycine; hence there are two structural forms of the acids.
The two forms may be represented by the general formulas where R

COOH COOH

I I

HjN-C-H H-C-NH,

I I

R R

L-Forrn D-Form

122

PROTEINS

denotes the remainder of the formula, e.g., CH3 for alanine, etc. Formerly
the small letters d and I were used to denote the two forms, but since
these letters are also used to indicate optical rotation, the American and
British chemical societies have adopted small capital letters to show
configuration. The reference compounds, d and l serine, correspond to
those used in sugar chemistry, viz., d and l glyceraldehyde. Optical
rotation is indicated by the words dextro and leva, or by plus ( + ) and
minus ( — ) signs.

Most of the amino acids are of the l type, but small amounts of the d
acids have been reported in several common proteins. Cancerous tissue
has been reported to be much higher in D-glutamic acid than normal
tissue, but the weight of evidence seems to be against this conclusion.
Several antibiotics, e.g., gramicidin, tyrocidine and actinomycin, contain
large amounts of D-phenylalanine, D-leucine, and D-valine. Other anti-
biotics, e.g., penicillin and polymyxin, contain the previously unknown
amino acids, D-penicillamine and D-a,y-diaminobutyric acid, respectively.
The presence of unusual structures of amino acids, sugars, etc., seems to
be a general characteristic of antibiotics.

Some of the amino acids listed have not yet been found in proteins.
For a long time a number of these were assumed not to occur in pro-
teins, but recently several have been found in polypeptides, e.g., or-
nithine in gramicidin and lanthionine in subtilin (an antibiotic). Homo-
cysteine, as yet unreported in any protein, is an intermediary product in
the conversion of methionine to cysteine. Cystathionine is also an inter-
mediate in this conversion. The steps are

+ serine

methionine > homocysteine > cystathionine

> cysteine and homoserine (in neurospora) or

a-ketobutyric acid (in animals)

Amino acid composition of proteins

To determine the amino acid content of a protein, it must first be
hydrolyzed. This is usually done with about twenty per cent hydrochloric
acid at 100°C. for 10 to 20 hours. The acid is then removed, and the
amount of each amino acid is determined quantitatively. This analysis
is one of the most difficult tasks in analytical chemistry, having defied
the efforts of some of the world's ablest chemists for the past seventy-
five years. Only within the last decade have methods been perfected to
such an extent that all the nitrogen or sulfur in a protein can be accounted
for in the amino acid figures. The most promising methods at present
seem to be microbiological and chromatographic. Bacteria are most
widely used in microbiological assays, and the procedures are the same
as those used in analyzing for vitamins (p. 234) . Chromatographic pro-

PROTEINS 123

cedures are based on differences in the degree of adsorption of amino acids
by solids, in the reaction of amino acids with ion exchange materials, and
in the solubility of amino acids in organic solvents. For details regard-
ing the operation of these methods, the original papers of Moore and Stein
and the book by Block and Boiling should be consulted.

Table 5-4 gives the percentages of amino acids found in some typical
proteins. The table includes proteins representative of foods, enzymes,
hormones, viruses, antibiotics, and fibers. In a given protein the figures
vary greatly. In egg albumin, for example, there is about 14 times as
much glutamic acid as there is tryptophan. In another common food
protein, gliadin, the glutamic acid exceeds the tryptophan more than one
hundredfold.

If all the proteins are considered, glutamic and aspartic acids and
leucine are seen to be the most abundant amino acids, while tryptophan,
histidine, and methionine are least abundant. The amino acids present
in largest amounts are those most closely related to the intermediary
compounds of carbohydrate metabolism, e.g., glutamic acid and a-keto-
glutaric acid form a pair, and aspartic acid and oxalacetic acid make up
a second pair (p. 331). The least abundant amino acids such as tryp-
tophan and methionine are the most complex in structure, probably in-
volving also the largest number of steps in synthesis.

Proteins that contain considerable amounts of all of the amino acids,
e.g., egg albumin and casein, are called "complete"; those that are lacking
or very high in certain amino acids, e.g., gelatin and zein, are said to be
"incomplete." A better term to denote the uneven composition of pro-
teins is disproportionate.

Certain highly specialized proteins such as fibroin and salmine are
conspicuously disproportionate in make-up. However, other proteins
such as pepsin, insulin, and botulinum toxin A having marked biological
properties show no unusual features in amino acid content. Their bio-
logical properties must be related to the structure of the molecule as a
whole and not to the kind or amount of amino acids that are found in
the molecule.

In most cases the sum of the figures for the amino acids amounts to
more than 100 per cent. Because of the water taken up in hydrolysis,
the total should be about 115 per cent of the starting material. With
the improved methods now available, the total nitrogen of the protein
can usually be accounted for in the individual amino acids. Likewise,
the extent of carbon and sulfur recoveries are useful criteria in judging
the validity of the analytical data.

From a practical viewpoint, data on the amino acid composition of
foods are more useful than those on the amino acid content of individual
proteins. Such data are gradually becoming available, and figures for
some of our staple foods are given in Table 5-5. However, we need many

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126

PROTEINS

IZ

Table 5

-5

Amino

arid

i-untent

of some foods

(Pe

r cent.

on undried basis) *

Milk

Muscle

Peas

(en-

(ani-

Muscle

and

MiNO Acids

Cheese

Com

Liver

tire)

mal)

(fish)

Oats

beans

Alanine

0.48

0.94
1.32

0.15

1.34
1.40

1.18
1.25

0.98

O.IS

Arginine

. . 0.86

1.58

Aspartic acid . . .

1.38

1.41

Cystine

. . . 0.09

0.15

0.28

0.04

0.22

0.20

0.26

0.29

Glutamic acid . .

2.12

0.75

2.80

Glycine

0.25

1.6
0.5

0.08
0.09

0.91
0.60

0.44

0.33

1.78

Histidine

. . 0.78

0.50

Isoleuc'ine . .

L73

0.04
1.50

0.96
1.68

0.26
0.39

1.09
1.45

1.10
1.60

0.71
1.15

1.24

Leucine

. . . 2.1

1.58

Lysine

. . 2.0

0.25
0.31

1.40
0.64

0.30
0.11

1.81
0.60

1.52
0.54

0.52
0.29

1.46

Methionine ....

. . . 0.83

0.45

Phenylalanine . .

. .. 1.52

0.50

1.22

0.19

0.91

0.74

0.79

1.12

Proline

1.09

0.51

1.55

Serine

0.85
0.37

1.48
1.06

0.15
0.16

1.09
0.91

0.68
0.79

0.52

0.95

Threonine

. . . 0.88

0.88

Tr>'ptophan ....

. . . 0.38

0.06

0.30

0.05

0.25

0.20

0.19

0.18

Tyrosine

. .. 1.62

0.60
0.53

0.78
1.20

0.14
0.19

0.73
1.00

0.64
1.01

0.65
0.78

0.63

Valine

. . . 1.85

1.24

(Per cent on undried basis) *

Soy Sweet

bean po- White Wheat

Amino Acids Rice meal tatoes potatoes flour

Alanine 116

Arginine 0.54 2.56 0.06 0.10 0.41

Aspartic acid

Cystine 0.10 0.67 0.20

Glutamic acid 6.44

Glycine 0.74

Histidine 0.13 1.02 0.03 0.04 0.23

Isoleucine 0.39 2.10 0.07 0.07 0.45

Leucine 0.62 2.80 0.10 0.19 0.74

Lysine 0.24 2.38 0.09 0.17 0.20

Methionine 0.23 0.60 0.03 0.05 0.21

Phenylalanine 0.38 1.86 0.09 0.12 0.58

Proline 1-75

Serine 1.47 0.46

Threonine 0.29 1.37 0.08 0.14 0.29

Tryptophan 0.10 0.49 0.04 0.04 0.09

Tyrosine 0.43 1.40 0.40

Valine 0.46 1.86 0.11 0.11 0.44

* Calculated from data in Block and Rolling, 2iid ed.

Whole Whole
wheat eggs

0.56 0.87

Yeasts

0.59

0.24

0.32

0.14

3.81

0.35

1.93

0.28

0.32

0.39

0.53

1.01

0.79

0.92

1.21

0.99

0.35

0.92

0.99

0.33

0.53

0.26

0.67

0.83

0.59

0.56

0.43

0.57

0.72

0.16

0.20

0.17

0.53

0.59

0.47

0.56

0.95

0.76

128

PROTEINS

more such data before the amino acid content of the diet can be cal-
culated with any assurance of being correct. It is still not certain
that the ratio of an individual amino acid to the total nitrogen in a food
remains constant from sample to sample. In a standard product such
as milk the variation is probably small, but in a food such as white
potatoes large fluctuations are to be expected.

In recent work Brand has obtained a yield of 116.3 per cent for /?-lacto-
giobulin and a recovery of 100 per cent of the nitrogen. Since all the
nitrogen, and also all the surfur, in the protein was accounted for in
the percentages of amino acids, it was possible to calculate the number
of molecules (actually residues equivalent to the molecular weights minus
the molecular weight of water) of each amino acid in the protein. For
example, the number of alanine residues is calculated as follows:

The alanine content of (3-lactoglobulin is 6.2 per cent (Table 5-4) and the molecular
weight is 41,500.

6.2% alanine (m.w. 89) =4.95% alanine residue (m.w. 71).
.0495 X 41,500 = 2054 g. alanine residues per gram molecular weight of^-lactoglobulin.
2054 -^ 71 =: 28.9, or in round numbers 29 residues.

The calculations gave a total for all the amino acids of 370 residues.
Adding together the molecular weights of all the residues, a total of
42,020 was obtained, which is in good agreement with that obtained by
ultracentrifuge measurements (41,500). From these data Brand pro-
posed an amino acid formula for y8-lactoglobulin as follows:

Gly8Ala29Val2iLeu5oIleu27Proi5Phe9CySH4(CyS— )8Met9Try4Arg7His4Lys33

Asp36Glu24 ( GluNHs ) saSeraoThroiTyrg ( HoO) 4

In this formula the amino acids are shown by the first three letters
of the name, and the number of residues by the figure following the
abbreviation. (CyS — )8 means that 8 half molecules or 4 whole mole-
cules of cystine are present. Glutamic acid residues with the second
carboxyl group neutralized as an amide, i.e., glutamine, are represented
as (GluNH2)32- The four molecules of water come from the H and
OH of terminal NH^ — and COOH groups, respectively, in polypeptide
chains. ^-Lactoglobulin is believed to contain 4 polypeptide chains per
molecule.

In a similar manner the number of amino acid residues present can
be calculated for the proteins listed in Table 5-4, for which the data are
sufficiently complete.

Linkage of amino acids to form protein

As indicated below in the formulas, the amino acids contain at least
one amino group and one carboxyl group. The amino group gives basic

PROTEINS

129

character to the compound, while the carboxyl group gives it acidic
properties. AVhen two amino acids are joined together, water is elimi-
nated, and the acids are linked tlirough the carboxyl group of one and
the amino group of the other. TJiis is a fundamental point and should
be clearly noted. For example, the combination of glycine and alanine
may be represented thus:

NH2'CH-C0:0H + HjNH-CHCOOH
H CH3

NH2-CH-';C0-NH:-CHC00H -f H2O

I ■ " I

H CH3

Glycine Alanine Glycyl-alanine

The linkage between the carboxyl group of the glycine and the amino
group of the alanine is a peptide linkage. In the formula it is enclosed
by (lotted lines. A combination of two amino acids is known as a
dipcptide. If three amino acids are joined together, a tripeptide is ob-
tained; four amino acids give a tetrapeptide.

There is obviously another dipeptide of these two amino acids in which
the carboxyl group of the alanine is linked to the amino group of the
glycine:

NH2-CH-;C0-NH:CHC00H
I I

CH3 H

Alanyl-glycine

If two molecules of glycine or two of alanine are combined, two more
peptides, glycyl-glycine and alanyl-alanine, respectively, are obtained.
Thus with two amino acids there are four possible peptides.

A tripeptide consists of three amino acids joined together by two
peptide linkages, as is illustrated by the tripeptide, tyrosyl-lysyl-aspartic
acid:

NH2-CH-iC0-NH-;CH-:C0-NH-:CHC00H
I ■-- I I

CH2 (0112)3 CH2

I I I

C6H4-OH CHj-NHz COOH

Tyrosyl-lysyl-aspartic acid

There are 27 possible tripeptides with three amino acids, provided each
acid is used once, twice, or three times in a given combination. The
number of combinations (polypeptides) that can be obtained by use of
25 different amino acids is almost unlimited. Sherman stated, "If a
protein be imagined made up of 30 molecules of 18 different amino acids,
one taken twice, one three times, another 3, one 4, one 5 times and 13

130 PROTEINS

taken once each, there would be 10^^ isomers." There is an adequate
mathematical basis for the existence of literally billions of proteins —
many more than probably occur in nature. The surprising thing is that
with so many possibilities, cells through countless generations produce
proteins of identical chemical and physical characteristics.

Attention is called to the occurrence of a free amino and a free car-
boxyl group at the left and right ends, respectively, in the formulas of
the above peptides. Because of these groups, peptides possess both basic
and acidic properties. The side chains may also contribute to the basicity
and acidity of the peptide because of free amino and carboxyl groups
contained therein. Lysine and aspartic acid are examples of amino acids
containing basic and acidic groups in the side chains. There are several
more such dicarboxylic and basic amino acids. (See formulas of the
amino acids.) In long peptides such as proteins it is the side chains,
and not the end groups, that contribute most to the basicity or acidity
of the molecule. Note also that the phenol group of the tyrosine is free
in the above peptide, and, hence, it will have properties characteristic
of this group, e.g., positive Millon and xanthoproteic tests. In proteins
there will be many such distinctive groups free, e.g., phenol, indole,
imidazole, to impart their characteristic properties to the molecule.

Hundreds of peptides have been made in the laboratory. Fischer,
one of the most famous investigators of the "composition of the proteins,
made a large number of polypeptides. One of these contained 3 leucine
and 15 glycine radicals, which gives a molecular weight of 1213 — one
of the largest molecules that has ever been produced synthetically. These
synthetic polypeptides possessed many of the properties of native pro-
teins such as solubility, color tests, and hydrolysis by enzymes. Although
they are far from being as complex as the native proteins, their synthesis
is a considerable step toward an understanding of the way in which a
protein molecule is put together.

In the preceding discussion of peptides no consideration has been given
to the possibility of the second carboxyl group of aspartic acid and
glutamic acid being involved in the linkages. Glutathione, an important
constituent of all cells, is a tripeptide in which glutamic acid is linked
to the next amino acid through the y-carboxyl instead of the a-carboxyl.
Thus,

CO— NH-CHCONHCH-COOH
I I I

CH2 CH2 H

I I

CH2 SH

I

HCNH

2

COOH
Glutathione (7,1- glutamyl- L-cysteyl-glycine)

PROTEINS l-^l

Glutamic acid is also linked through its y-carboxyl in several peptides
of bacteriological origin. In the vitamin, pteroyltriglutamic acid (one
of the several forms of folic acid), the second and third glutamic acid
residues are linked to the preceding by a y-linkage. Thus,

P— NH-CH-(CHj).-CO.NH-CH(CH2)2-CO-NHCH(CHj),-COOH

' * I 1 I

COOH COOH

COOH

7-Linkage 7-Linkage

P stands for the pteroyl group. For the complete formula see p. 247.
The corresponding triglutamate with a-glutamic acid linkages has also
been synthesized, but it has no vitamin potency.

A still more unusual polypeptide is the capsular substance produced
by Bacillus mithrncis. This substance is a long chain polypeptide con-
taining only D-glutamic acid residues, 50 to 100 in number, joined together
mainly through y-linkages. There is some indication that small numbers
of y-glutamic acid linkages occur in such common proteins as casein,
edestin, hemoglobin, etc. To 'date, the authors have found no reports
regarding the occurrence of second or /?-carboxyl linkages for aspartic
acid in naturally occurring polypeptides.

Sequence of the amino acids in the protein molecule

Data are now available for the number of amino acid residues present
in many proteins, and information concerning the order in which the
amino acids occur in the chain is gradually accumulating. Data have
been obtained by partially hydrolyzing the protein and isolating some
of the peptides that have been formed, then determining the order of
the amino acids in the peptides. Examples of such peptides are as
follows: glycyl-alanine, alanyl-glycine, and glycyl-tyrosine from silk
fibroin; prolyl-phenylalanine and leucyl-glutamic acid from ghadin;
phosphoseryl-glutamic acid from casein.

Applying this technique to the antibiotic gramicidin S, Consden et al.
isolated four dipeptides and one tripeptide from the partial hydrolysis
products. These pieces :ontained all of the five amino acids found in
gramicidin S, and, hence, the order of the amino acids was worked
out to be:

(-a- (L-valyl) -L-ornithyl-L-leucyl-D-phenylalanyl-L-proline-) 1 or 2

Gramicidin S then appears to be a cyclic penta- or decapeptide. It is
obviously a very simple compound in comparison to the common pro-
teins. An open chain peptide having the same sequence of amino acids
has been synthesized by Harris and Work, but its antibiotic activity
was small as compared to that of the natural compound. These results

132

PROTEINS

suggest that the antibiotic activity of gramicidin S is related largely to
its cyclic structure.

A very extensive piece of work has been done recently by Sanger and
associates on insulin. This protein consists of two types of polypeptide
chains. One type has glycine as the N-terminal (free NHo group) residue,
and the other chain is headed by phenylalanine. Each molecule of
insulin contains two glycine chains and two phenylalanine chains held
together by — S— S— bridges of cystine residues (p. 117). The bridges
can be broken by oxidation with performic acid to — SO3H groups, thus
setting free the chains. The two types- of chains can then be separated
and the sequence of the amino acids in them determined. This was done
by partial hydrolysis with hydrocliloi'ic acid and enzymes and isolation
of the peptides split off from the chain. More than 60 peptides ranging
from dipoptides to hexapeptides were isolated from the hydrolyzate of
the phenylalanine chain, and their structures determined by chromatog-
raphy. From all these fractions the sequence of the amino acids in
the whole chain was deduced to be: ■^'

Phe.Val.Asp.Glu.His.Leu.CySO3H.Gly.Ser.His.Leu.Val.Glu.Ala.Leu.Tyr.Leu.VaL
CySO3H.Gly.Glu.Arg.Gly.Phe.Phe.Tyr.Thr.Pro.Lys.Ala.

The total number of amino acid residues in the cliain is 30.

The sequence of the amino acids in the glycine chain was worked out
in the same way. The chain consists of 21 amino acid residues arranged
as follows:

Gly.Ileu.Val.Glu.CySO3H.CySO3H.Ala.Ser.Val.CySO3H.Ser.Leu.Tyr.Glu.Leu.
Glu.Asp.Ty r.CySOsH .Asp .

In the phenylalanine chain the last amino acid residue in the chain,
that is, the residue with a free carboxyl group, is alanine. In the glycine
chain the carboxyl terminal residue is designated as Asp., i.e., an aspartic
acid residue. In the intact insulin it is an asparagine residue. During
acid hydrolysis the amide group is split to give aspartic acid. It is be-
lieved that two other aspartic acid residues in the chain are in reality
asparagine residues, and six of the glutamic acid residues are actually
glutamine residues. The exact location of these amide groups is not
known.

The relation of the four chains to one another is still to be determined.
Several arrangements are possible, but the presence of four cysteic acid
residues in the glycine chain and two in the phenylalanine chain suggests
that the two glycine chains lie between the phenylalanine chains, A
diagram of such an arrangement for the intact insulin follows:

PROTEINS

133

Chain I

(1)
HiNPhe-

Chain II HsNGly

Chain III H2NGly

Chain IV H^NPhe-

(7)

s

s

CO) I (7)

•Cys-Cys-

S

I (6) (7)

•Cys-Cys •

S
I

S

1(7)

■Cys-

(11)
•Cys-
I
S

I

s

I (11)
Cys-

(19)

■r

s

I

s

1(20)

• Cys -

(20)

•Cys-
I

S
I

s

1(19)

•Cys-

(30)

•Ala-COOH

(21)

-AspNH2

(21)

-AspNHj

(30)

•Ala-COOH

This diagram shows the four chains headed by phenylalanine and
glycine and ending in alanine and asparagine, AspNHo. The cystine
residues with one-half in one chain and the other half in the adjacent
chain form the — S— S— bridges that hold the chains together. The
numbers above the chains indicate the order of the amino acid residues
in the chains. The intervening amino acid residues given on p. 132
have been omitted because of limitations of space. The chains are prob-
ably not strung out in a long line, as shown in tlie diagram, but may be
coiled to form layers, as will be apparent from the discussion in the
next section.

Even though the sequence of the amino acids in the chains is known
and the relation of the chains to one another may be as assumed in the
diagram, the question as to what there is about this arrangement that
gives insulin its hormone property still remains unansw^ered. In time
there may be an answer even to that question.

Structure of the protein molecule

A protein of moderate size sucli as egg albumin, having a molecular
weight of 40,000 and an average amino acid residue weight of 120, con-
tains about 350 amino acid residues. The residues are joined together
to form a number of chains of varying lengths. For example, egg
albumin and /^-lactoglobulin contain four chains, edestin — six, lactalbumin
— nine, and insulin — four. The average number of amino acid resi-
dues in the chains of these proteins is calculated to be 89 for egg albumin,
92 for /3-lactoglobulin, 16 for lactalbumin, and 25 for insulin.

Since a protein has three-dimensional form, the chains must be ar-
ranged so as to provide such a structure. Many theories, based mainly

134 PROTEINS

on X-ray data, have been advanced regarding the arrangement of the
chains in the protein molecule. Since the carbon and nitrogen atoms
in the backbone of the chain are at angles to one another, the chain has
a zig-zag appearance with hydrogens, oxygens, and "tails" (side chains
of the amino acid residues) sticking out at various angles with the back-
bone carbons and nitrogens. (See hydrogen bonding, p. 135.)

If such a chain is folded back on itself, or if two chains are arranged
in the right order, the result is a puckered two-dimensional structure
or layer. Such a layer may be compared to a piece of lace crocheted
from a single thread. Several such layers may be superimposed on
one another to build a three-dimensional structure, as in a layer cake.
Layers may also be folded back and forth in some such fashion as a
road map is folded. When a protein is spread on a water surface, the
layers unfold and form a film only one layer thick. In such cases the
polar groups {e.g., amide, carboxyl, hydroxyl, phenol, etc.) are drawn
into the water surface while the nonpolar groups {e.g., hydrogen, paraffin,
benzene, etc.) are repelled and extend upward from the water surface.

A somewhat different arrangement of the chains has been proposed by
Pauling for the structure of fibrous proteins, for example, a-keratin of
wool. According to this view, the chains are arranged in the form of
a helix (a spiral spring is an example of a helix) to give a hollow cylinder-
like structure. For collagen, Pauling concludes that the molecule con-
sists of three chains twisted about one another to give a rope-like effect.

Theories on the structure of proteins are constantly changing as more
X-ray, infra-red, and other physical measurements are made. To quote
Bernal, ''The problem of the protein structure is now a definite and
not unattainable goal."

One of the difficult problems in protein structure is to find a satisfac-
tory explanation for the manner in which the folds in a molecular chain,
or the layers in a molecule, are held together. Several types of forces
have been postulated, three of which will be mentioned here.

Salt Linkage. Basic groups of one side chain (the second amino group
of lysine, the imadazole group of histidine, and the guanidino group of
arginine) may be united to acidic groups (the second carboxyl group of
aspartic and glutamic acids) of another chain to form an electrostatic
bond, that is, a salt.

Chain 1 NH-CH-CO NH-CHCO---

I I

(CHO4 (CH,)4

NH, »- NH;

+ .

COOH *■ COO-

I I

CH2 CH2

Chain2 OC-CH-NH--- ••OC-CHNH-

Salt linkage

PROTEINS

135

Sulfide Linkage. One-half of a cystine molecule may be part of one
chain; the other half may be located in a second chain to form an
— S— S— linkage.

Chain 1

•NH-CHCO"

I
CH2

Chain 2

I

S

I
CH2

I
CO-CHNH

Hydrogen Bonding. A third, and more important, type of binding is
the hydrogen bond, in which electrons are shared between the hydrogen
of an imino group ( — NH— ) located in one chain and the oxygen of a
carboxyl group ( — CO—) in another chain.

Chain 1

Chain 2

H H

1 R 1

^c l^c

> II H 11

Q 9

R

1

?^c^

H II
0

: <

H H

H

> 1 H 1 H 1

^N^ 1 N^ 1 N^

R II R II
0 0

Shared electrons forming a
hydrogen bond or bridge

There are approximately as many — NH — and — CO — groups in a
protein molecule as there are amino acid residues. Hence the number
of hydrogen bonds set up would be in the hundreds. Although a single
hydrogen bond constitutes only a weak chemical linkage, a dozen to-
gether provide about as much strength as a covalent (e.g., — C — C — )
bond.

Proteins are classified on the basis of their shape as fibrous or cor-
puscular. ("Globular" was formerly used as the descriptive term, but
"corpuscular" seems now to be the preferred designation.) As the term
implies, the fibrous proteins are long and slender, or unsymmetrical. In
some cases the length is 30 times the cross section. The corpuscular
proteins are more nearly symmetrical. Many of these have a long axis
(length) only twice as great as the short axis (thickness). Methemo-
globin is reported to be 64 A long, 48 A wide, and 36 A thick.^

^ One millimeter equals 10,000,000 A (angstrom units).

136 PROTEINS

CONJUGATED PROTEINS AND THEIR
PROSTHETIC GROUPS

The conjugated proteins (protein plus a prosthetic group) will, of
course, give other products than amino acids on hydrolysis. Some
important compounds that either constitute the prosthetic group or arise
from it on hydrolysis follow.

Nucleo proteins

Nucleic acids occur either in the free state or in combination with
proteins to form nucleoproteins. Because of their number, complexity,
and importance, the nucleoproteins and the nucleic acids will be discussed
in a separate chapter.

Phospho proteins

The phosphoproteins also give phosphoric acid on hydrolysis. Phos-
phoric acid is linked to serine through the hydroxy group of the amino
acid to form a phosphoric acid ester that can unite with bases, e.g.,
calcium, to form salts. The serine phosphoric acid esters seem to account
for most of the phosphorus in the phosphoproteins.

Glycoproteins

These are ill-defined proteins containing carbohydrate complexes
(chondroitin sulfuric acid or mucoitin sulfuric acid) as prosthetic groups.
Typical examples of these proteins occur in salivary mucin, gastroin-
testinal mucus, and the vitreous humor of the eye. They are slippery
materials and, hence, serve as useful lubricants. They facilitate the
movement of the food through the intestinal tract and, since they are not
digestible, protect the tract against the proteolytic enzymes.

On hydrolysis, chondroitin sulfuric acid gives one mole each of glu-
curonic acid, galactosamine, acetic acid, and sulfuric acid. Mucoitin
sulfuric acid gives glucosamine instead of galactosamine, but the other
hydrolysis products are the same as from chondroitin sulfuric acid.
There is still some uncertainty as to how the several products are bound
together. These carbohydrate complexes account for 25-35 per cent of
the glycoprotein. In consequence the nitrogen content is low, 9-13
per cent.

PROTEINS

137

Chromo proteins

This group of proteins probably includes a larger number of important
proteins than any other subdivision. They arc functional rather than
structural, playing a role of the first order as carriers of gases, mediators
in the oxidation process, and intermediates in the phenomenon of vision.

Hemoglobin. The chromoproteins contain colored prosthetic groups.
Hemoglobin, tlie red pigment of the blood cells, is the best known protein
of this type. Heme is the prosthetic group and globin, a histone, is the
protein j^art of the combination. Heme accounts for only 3.8 per cent
of the hemoglobin; globin comprises the other 96.2 per cent. Heme con-
tains four atoms of iron, which amounts to 0.33 per cent of the hemoglobin.
There are four heme units attached to one globin i)ai't to make up hemo-
globin.

The exact nature of the attachment is not known, but it is generally
considered to be either an ionic or covalent bond between the iron of the
heme and the histidine of the globin. The union is weak and can be
easily broken by warming with acetic acid and sodium chloride. A salt,
hemin (C34H32N404FeCl), is formed and this crystalhzes readily. The
structural formula of hemin is

CH=CH2
H

CH3

II

V V

0)/ I

CH3--C

(S)/C — N
CH3— C^x IV III

-N,

N=

FcCl
\

N-

,C— CH=CH2

\

CH

,C-CH3

(5)

H

CH2
CH2
COOH

CH3

CH2
I
COOH

Heme

It consists of four pyrrole groups (I-IV) joined together through four
methene groups (a,/3,y,8). To the pyrrole groujis are also attached four
methyl groups (1,3,5,8), two vinyl groups (2 and 4), and two propionic
acid residues (6 and 7). In the center of the formula is a trivalent atom

138

PROTEINS

of iron Fe+++ (the iron is ferrous Fe ++ in the original hemoglobin and
is oxidized to ferric Fe + + + in the process of isolating hemin) which is
joined to the four nitrogens of the pyrrole rings by partial valences and
to a chlorine atom to form the chloride salt. The four pyrrole groups,
without the side chains and iron, form a unit known as porphin, and
derivatives thereof are called porphyrins. Many porphyrins occur in
nature, e.g., coproporphyrin of feces, uroporphyrin of urine, and chloro-
phyll (p. 389) . Chlorophyll, the green pigment of plants, contains an ad-
ditional ring, differs from heme in its side chains, and has Mg at the center
instead of Fe. One of the propionic side chains in chlorophyll is linked
as an ester to the unsaturated alcohol phytol. That the most important
animal pigment and the most important plant pigment should have
related structures is something to be noted carefully.

The outstanding chemical feature of heme (hemoglobin) is its ability
to combine with oxygen and thus serve as a transport agent in the blood.
Each heme combines with one molecule of oxygen. Since there are four
heme units in each molecule of hemoglobin, the reaction between hemo-
globin and oxygen may be represented thus:

Hb-h402^Hb(02)4
Hemoglobin Oxyhemoglobin

more conveniently, but less exactly,

Hb + O2 ^ HbOa

This reaction takes place in the lungs; in the tissues it is reversed.

Hemoglobin also reacts with carbon monoxide to form a combination
that is several hundred times stronger than that with oxygen. It there-
fore takes much oxygen to displace the carbon monoxide from the hemo-
globin complex and makes breathing of carbon monoxide a very dan-
gerous matter. Hemoglobin also combines with carbon dioxide, and a
considerable part of the carbon dioxide contained in the blood is combined
with it. Hemoglobin is thus a carrier of gases both in going from the
lungs and in returning to them.

If hemoglobin is exposed to mild oxidizing agents, for example, potassium
ferricyanide, the iron is converted from the ferrous state Fe ++ to the
ferric form Fe + + + . The resulting hemoglobin is called methemoglobin.
It can carry only one-half as much oxygen as hemoglobin and does not
readily release oxygen.

The hemoglobins of different species differ in the globin part of the
molecule, but the heme part is the same in all. Note the difference in
crystalline structure of hemoglobins from different species (Figs. 5-1 to
5-4).

A form of hemoglobin has recently been reported to occur in the

PROTEINS 139

nodules of leguminous plants and is believed to play a role in the fixa-
tion of atmospheric nitrogen by these nodules.

Erythrocruorin and Chlorocruorin. Many proteins other than hemo-
globin contain iron porphyrins as prosthetic groups. The basic structure
of these iron porphyrins is the same as that of the heme in hemoglobin,
and in many forms of life they also serve as the oxygen-carrying agent.
An example of this type of compound is erythrocruorin, the respiratory
pigment of the common earthworm and other worms. Chlorocruorin, a
green pigment, serves the same purpose for marine worms, e.g., Spiro-
graphis. These pigments are dissolved in the blood, not contained in
blood cells as are the hemoglobins. They are usually of large molecular
weight, several million, and contain many heme groups, e.g., 190 in the
chlorocruorin of Spirographis. The side chains of the hemes differ from
those in hemoglobin, but the iron is in the ferrous state as in hemoglobin.

Cytochromes. There are at least three cytochromes, a, b, and c, that
differ from one another in solubility, reaction to cyanide, and other
properties. The cytochrom.es occur in all oxygen-using cells and, hence,
are the most widely distributed heme proteins in nature. They form an
oxidation-reduction system and serve as carriers of hydrogen in the oxi-
dation scheme of cells. (See pp. 283 and 333.)

Cytochrome c is the best known cytochrome and has been obtained
in a crystalline and homogeneous state. It is a small protein, molecular
weight 13,000, and contains only one heme per molecule. The iron con-
tent is 0.43 per cent and is present either in the ferrous or ferric state,
according to whether the cytochrome is in the reduced or oxidized state.
The heme part of the molecule is bound not only by an iron-to-histidine
bonding, as in hemoglobin, but, in addition, is joined by two covalent
linkages between vinyl groups of the heme and cysteine residues of the
globin. Thus,

NH-CH'CO NH'CH-CO

Globin part

Heme part

These bonds are very stable, and on hydrolysis the cysteine residues go
with the heme. In other words, the — S — C — linkage is more stable
than the peptide linkage — CO — N — .

Heme- containing Enzymes. Catalases occur widely in plant and ani-
mal tissues. Beef liver catalase has been crystallized and contains

140

PROTEINS

approximately 0.1 per cent of iron. On the basis of 225,000 for the
molecular weight, the iron content corresponds to four atoms per mole-
cule. The iron appears to be in the ferric state, and the heme unit is
the same as that in hemoglobin.

Two peroxidases, horse radish peroxidase II and cytochrome c peroxi-
dase from yeast, have been obtained in crystalline, or highly purified,
form and found to contain the same heme as hemoglobin. Two other
peroxidases, myeloperoxidase from leucocytes and lactoperoxidase from
milk, contain green-colored hemes and, hence, are also called verdo-
peroxidases. Both have hemes containing iron, but the structures of
these hemes have not yet been determined.

Other M etal- containing Proteins. Ferritin is a brown-colored protein
containing up to 23 per cent of iron. The iron is present as colloidal
iron oxide or phosphate and is very loosely bound to the protein. Ferritin
occurs in the liver, spleen, and bone marrow and is believed to serve as
a storage form of iron.

Hemocyanins serve as respiratory proteins for the lobster, octopus,
and other marine animals. The blood of the lobster becomes blue when
aerated, hence the term hemocyanin, literally blue blood (a term that man
has applied to himself as a mark of distinction, without considering its
connotations). Hemocyanin contains copper (about 0.35 per cent), but
the nature of the prosthetic group, if any, carrying the copper is still
an unsettled question. The molecular weights ascribed to the hemo-
cyanins are enormous, 2 to 5 million.

Additional copper-containing proteins are : hemocuprein from red blood
corpuscles, hepatocuprein from the liver and several oxidizing enzymes,
e.g., tyrosinase, ascorbic acid oxidase, etc. Other metals forming com-
plexes with proteins, but not having color, are magnesium in carboxylase,
zinc in insulin and carbonic anhydrase, and manganese in arginase.

Other Colored Proteins. Flavoproteins or "yellow enzymes" have ribo-
flavin phosphate, or a dinucleotide of riboflavin and adenine, as the pros-
thetic group and are yellow in color, hence the name "yellow enzyme"
given to them. About a dozen such enzyme proteins have been reported.
They play an important role as hydrogen transport agents and can exist
in either a reduced or oxidized state (see Fig. 10-4).

Rhodopsin is a red, light-sensitive pigment found in the retina of land
and marine animals, e.g., man, cattle, squid, and plays an important
role in vision. It is a chromoprotein that consists of the prosthetic group,
cis-retinene 1, and a protein called opsin. Retinene 1 is the aldehyde
(C19H07CHO) corresponding to vitamin Aj (C19H27CH2OH). In fresh
water vertebrates, such as fish, a somewhat different chromoprotein called
porphyropsin takes the place of rhodopsin. Porphyropsin contains
retinene 2, which corresponds to vitamin Ao. Opsins from different sources

PROTEINS

141

combine with either retinene, but it is not certain that they are identical
proteins (p. 204) .

Lipoproteins

As a class, these proteins are not well-defined and are regarded by some
investigators as mixtures of lipides and proteins rather than as chemical
entities. They occur in cell nuclei, blood, milk, bacteria, etc. Lipovitellin
of egg yolk, thromboplastic protein (functioning in blood clotting) of
lung tissue, and the polymyxin antibiotics are examples of such proteins.
The prosthetic groups that have been obtained from lipoproteins include
lecithin, cephalin, and fatty acids.

GENERAL PROPERTIES OF THE PROTEINS

Form

As usually obtained, proteins are amorphous, but, under carefully con-
trolled conditions, they can be made to ciystallize. Great progress has
been made in the preparation of crystalline proteins in recent years,
especially of those that are enzymes. About 150 proteins have been
crystallized and, of these, approximately 40 are enzymes, respiratory
pigments, toxins, viruses, or hormones. The most commonly prepared
crystalline proteins are the animal albumins and the plant globulins.

Size of the protein molecule

From several lines of evidence it is known that the protein molecule
is large. A minimum molecular weight can be determined in various
ways: from the percentage of some element, such as sulfur, phosphorus,
or iron, which occurs in small cjuantities; from the percentage of some
amino acid found in the protein. The true molecular weight is obtained
by physical methods such as ultracentrifuge measurements and osmotic
pressure determinations.

The following example illustrates the method of calculating the mini-
mum molecular weight of hemoglobin from the iron content, 0.33 per
cent. Assuming one atom of iron, atomic weight 56, a proportion is
set up as follows:

0.33 : 56 = 100 : a;; x= 17,000 minimum m.w.

Since there are four atoms of iron in hemoglobin, the true molecular weight
becomes 68,000, which is in excellent agreement with ultracentrifuge
data.

142

PROTEINS

If all the nitrogen of a protein is accounted for in the amino acid
analysis, the number of amino acid residues can be calculated, and the
sum of the molecular weights of the residues probably equals the molecular
weight of the protein. The molecular weight of ^-lactoglobulin obtained
in this way is 42,020, and that given by the ultracentrifuge method is
41,500.

Protamines are small proteins, m.w. 3000 to 5000, and hemocyanins
are very large, m.w. several million. Many viruses have apparent
molecular weights up to several hundred miUion. Home authorities
prefer to call these values particle weights rather than molecular weights,
giving proteins probable molecular weights approximating 17,600, or a
multiple thereof; that is, 2, 4, 6, 8, 16, 24 ... , 384 times 17,600. For
example, zein and hemoglobin are reported to have molecular weights
of about 35,000 (2 units) and 68,000 (4 units), respectively. The
molecular weight of the proteoses is supposed to range from 4000 to
5000 and that of the peptones from 800 to 1000.

Color reactions

Many color tests have been proposed by different investigators to
detect the presence of proteins. The most common are given below.

The Xanthoproteic Test. With concentrated nitric acid, most proteins
give a yellow color that becomes more pronounced if the solution is made
alkaline. The familiar stain that is formed if nitric acid comes in
contact with the skin is due to the action of this acid upon the proteins
of the skin. The cause of the test is the formation of a nitro-phenyl-
derivative somewhat similar, perhaps, to picric acid. A modified phenyl
grouping such as is contained in tyrosine and tryptophan seems to be
necessary to the test. Tryptophan gives a better test than tyrosine,
whereas phenylalanine, although it contains the phenyl group (CeHs),
does not give the test at all. All the common proteins give the test,
but considerable quantities of protein are required. Although the test
is general, it is not very sensitive.

The Millon Test. A brick-red color is developed when some proteins
are heated with the Millon reagent (mercury dissolved in nitric acid).
The reaction is due to the presence of the phenol group (C6H4OH),
which is contained in the amino acid, tyrosine. Proteins that contain
no tyrosine will therefore fail to give a Millon test. Gelatin gives a faint
Millon test either because it contains a minute quantity of tyrosine or
because it has not yet been freed of other tyrosine-containing proteins.
Carbolic acid and salicylic acid, which are not amino acids, likewise
give this test because they contain the phenol group.

The Biuret Test. A pink to purple color is obtained when proteins
are treated with alkali and minute quantities of copper sulfate. The

PROTEINS

143

color is due to the presence of two peptide groups, — CO'NH — . When
three amino acids are joined, two such groups are formed, for example,

HNH-CH-CO-NH'CH'CO-NH-CH'COOK

I I I

Ri xvt Rt

Tripeptides, with the exception of glycyl-glycyl-glycine, give the test,
as do also peptones, proteoses, and all native proteins. It is one of the
most general of the protein color tests.

The Hopkins-Cole Test. A purplish color is developed when a protein
containing tryptophan radicals is treated with the Hopkins-Cole reagent
(magnesium glyoxylate). The cause of the color development is the
indole group, which exists in the amino acid, tryptophan. It has been
assumed that the color is due to the formation of indigo by the action
of the reagents on indole groups.

The Ninhydrin Test. All amino acids (except proline and hydroxy-
proline) and, hence, all proteins give a blue to purple color with ninhydrin.

CO
CeH. C(OH)j
CO

Only a free amino and a free carboxyl group are required for the test.
All amino acids except the two mentioned possess such groups. The blue
color results from a condensation of two molecules of the reagent with
ammonia, which splits off from the amino group of the amino acid.
The color compound is the anion of a salt and has the following formula:

CeH. C— N=C ^CeH*
C-0- CO

Precipitation

The precipitatin of proteins varies considerably with the reagent that
is used. Some reagents precipitate only the globulins, whereas others
precipitate a larger number of proteins but do not precipitate the pep-
tones. Some reagents precipitate not only the proteins and peptones
but also carry down certain amino acids. Among the most effective
precipitants are ammonium sulfate, magnesium sulfate, mercury salts,
trichloracetic acid, phosphotungstic acid, tungstic acid, tannic acid, col-
loidal iron, and strong solutions of alcohol. Phosphotungstic acid appears
to be the reagent which precipitates the largest percentage of nitrogen,
whether this is in the form of proteins, peptones, amino acids, or other
nitrogenous compounds.

144

PROTEINS

The theory for the precipitation of proteins is based on the amphoteric
character of the proteins, i.e., the presence of both basic and acidic groups
in the molecule. If the protein is more basic than the reagent, the precipi-
tate is a protein salt of that reagent. If it is more acidic than the reagent,
the precipitate comes down as proteinate; that is, the protein behaves as
an acid and the precipitant acts as a base. For each protein there is
a pH value called the iso-electric point, on one side of which the protein
acts as a base and on the other side as an acid; for example, the iso-
electric point of gelatin is at the pH value of 4.7. Below 4.7, gelatin
acts as a base and is precipitated by acids as a gelatin salt such as gelatin
hydrochloride. Above 4.7, gelatin behaves as an acid and is precipitated
as a gelatinate such as sodium gelatinate. The two types of precipita-
tion may be represented in simplified form by the following equations.

By acids : RNH. + HCl -^ RNHo-HCl
By bases : R'COOH + NaOH -^ R'COONa

In place of hydrochloric acid in the acid precipitation, we may have
acids such as picric, tannic, tungstic, phosphotungstic, etc., which form
more insoluble compounds with proteins. Instead of sodium hydroxide,
calcium or barium hydroxide may be used to give the corresponding
calcium or barium salt. The metallic salt of the protein may also be
formed by adding a soluble salt of the metal to the sodium hydroxide-
protein solution. For example, if lead acetate, one of the best protein
precipitants, is added to a solution, lead proteinate is formed and, since
this is insoluble, a precipitate is produced.

The above theory of precipitation and salt formation assumes the
formation of an excess of positive charges on the protein molecule

RNHo + H2O -> RNH3+ + OH-

below the iso-electric point and an excess of negative charges on the
protein molecule

R'COOH -^ R'COO- + H+

above the iso-electric point. Under the first condition, the protein reacts
with negative ions, e.g., Cl~, to form a protein salt; under the second,
it reacts with positive ions, e.g., Na + , to form a proteinate. At the
iso-electric point the positive charges equal the negative charges; hence
the protein combines with neither acid nor base. Above or below the
iso-electric point the protein is unbalanced and therefore combines with
oppositely charged ions.

Denaturation

Neurath and associates define protein denaturation as "any nonpro-
teolytic modification of the unique structure of a native protein, giving

PROTEINS 145

rise to definite changes in chemical, physical, or biological properties."
Modifications of structure by addition of a group such as acetylation,
which obviously changes the properties of the protein, are not included
in the term.

Denaturation, or more properly coagulation, of protein has been ob-
served by man ages before he knew about proteins. It is a phenomenon
that must have come to his attention soon after he began to prepare his
food by heating it. Despite its apparent nature, a clear understanding
of coagulation is still lacking.

A most striking example of coagulation is the conversion of egg white
from a liquid to a solid when an egg is boiled. This change appears
first to involve denaturation of the egg white followed by aggregation
of the denatured protein into floes and then into a solid coagulum. The
evidence for these stages is the fact that if a solution of egg white is
heated in a salt-free medium below or above the isoelectric point of
the protein, about pH 4.7, the solution remains clear. However, the
increases in viscosity and SH groups, e.g., in cysteine, show that the
egg white has been denatured. If the pH of the clear solution is adjusted
to 4.7, coagulation takes place without any further heating.

A decrease in solubility is only one of the changes that occur when a
protein is denatured. A greater susceptibility to the action of enzymes
is another effect. The digestibility of egg white, for example, is much
increased by heating. A third effect is a partial or complete loss of
biological activity, if the native protein possesses such a property. En-
zymes, antibodies, and viruses lose their potency when completely de-
natured. Loss of crystallizability, changes in viscosity, and an increase
in the number of reactive groups {e.g., sulfhydryl (— SH), disulfide,
phenol, and indole) are other changes brought about by denaturation.
Not all of these changes are equally apparent and, if measured quantita-
tively, do not run parallel to one another. These differences are prob-
ably an indication that different structural arrangements in the molecule
are affected to a varying degree by the denaturing agent. It is apparent
that no single criterion is adequate as an index of denaturation. If the
denaturation has not gone too far, the process may be reversed under
suitable conditions, and much of the protein recovered in the original
state.

Denaturation may be brought about not only by heat but also by
freezing, irradiation with ultraviolet light, acid, alkali, alcohol, urea,
guanidine salts, and some of the new type of detergents (p. 87). The
last three compounds are the reagents ordinarily used in experimental
work to produce denaturation.

The measurement most commonly used to detect denaturation is titra-
tion of the SH and S — S groups with a mild oxidizing agent. The S— S
groups are first reduced with cyanide to SH and then titrated. The

146

PROTEINS

increase in SH groups after reduction is a measure of the S— S groups
present. The reagents commonly used in titrating are potassium ferri-
cyanide and a dye, porphydrindin blue. The end point of the former
is determined with sodium nitroprusside as indicator. It gives a red
color with SH groups. On reduction the dye becomes a colorless com-
pound.

Phenol and indole groups have weak reducing properties in alkaline
solution toward ferricyanide and can be determined after previous oxida-
tion of the SH groups at a different pH.

The question naturally arises as to what occurs in the structure of
the molecule when a protein is denatured. Denaturation is undoubtedly
a disruption of the highly complex and precisely organized folding of
the peptide chains and the interrelation of these chains to one another.
In the disruption of this organization, groups {e.g., SH) that were previ-
ously buried deep in the molecule become exposed and reactive. Bio-
logical activity, which depends upon very specific arrangements in the
chain structures, is lost with the disappearance of these arrangements.
The regeneration of the crystalline form and partial recovery of biological
activity that has been observed in some instances may be ascribed to an
only partial disorganization of the chains. If the pattern still exists, the
chains may refold themselves into the original structure.

QUANTITATIVE DETERMINATION OF PROTEIN
(KJELDAHL METHOD)

Crude protein

The protein content of any food material is obtained by determining
the total nitrogen and multiplying the result by 6.25. This value is
called the crude protein content of the food material. Two assumptions
are made in determining protein in this way:

1. All nitrogen is assumed to be present in the substance as protein.
This is not necessarily the case, as food materials frequently contain a
large proportion of the nitrogen in other than protein form. Examples
of compounds containing nitrogen that are not protein are amino acids,
amides, alkaloids, cyanates, purines, pyrimidines, creatine, and creatinine.
From this list it is seen that in foodstuffs there are many compounds that
contain nitrogen but are not proteins. In finished products such as seeds
the major portion of the nitrogen is in the form of protein, while in
actively metabolizing tissue such as partly formed seeds and vegetables,
particularly string beans and green peas, the larger part of the nitrogen
may be in other than protein form. In muscle and other animal tissue
there is a considerable proportion of so-called meat extractives that
contain nitrogen but are not protein.

PROTEINS

147

2. The second assumption is that all proteins contain 16 per cent of
nitrogen, in which case the ratio of the protein to nitrogen is 100:16
or 6.25. Since the nitrogen content varies from 15.5 to 18.7 per cent,
the use of the factor 6.25 may give too high or too low results, depending
upon the protein under consideration. In the case of the cereals, the
nitrogen content is higher than 16 per cent, and the factor 5.7 more
nearly represents the ratio between the protein and the nitrogen. With
milk the factor 6.37 is used. In general, however, the factor 6.25 is used
and is the one that is employed in the calculation of general tables of
analysis for the composition of food materials.

It should be observed that the term "crude protein" has nothing to
do with the purity of the protein, but merely j-epresents results obtained
by laboratory procedure.

Purpose of reagents used in determining total nitrogen

In the Kjeldahl determination of total nitrogen, carbon and hydrogen
must be oxidized and the nitrogen converted into ammonium sulfate.
Sulfuric acid is the oxidizing reagent that is used. The material first
becomes black and charred, but after prolonged heating it clears up and
becomes free from carbon. The action of the sulfuric acid may be repre-
sented by the following equation:

H0SO4 -» H.O + SOo + O

The sodium or potassium sulfate added raises the boiling point of
the solution and thus aids in the oxidation of the material. A small
amount of copper selenite is also added and acts as a catalyst to promote
oxidation. The oxidation of protein may be represented by the oxida-
tion of a fragment of a protein, for example, glycine.

CH2(NH2)-COOH + 30-> 2CO2 + HgO + NH3

2NH3 + H0SO4 -^ (NH4)2S04

Through the addition of concentrated sodium hydroxide, ammonia is
liberated and collected by distilling it into a known amount of standard
acid. From the amount of acid neutralized by the ammonia, the nitrogen
and, hence, the crude protein (N X 6.25) are obtained by a simple
calculation.

REVIEW QUESTIONS ON PROTEINS

1. Name two of the chief proteins in (1) milk, (2) eggs, (3) blood, (4) wheat;
one protein in (5) com and (6) peas. Which of these proteins probably contain (a)
phosphorus? (b) sulfur?

148 PROTEINS

2. Give the graphic formula for (1) cystine, (2) tyrosine, (3) tryptophan, (4)
lysine, (5) uric acid. Write out the graphic formula for (a) a dipeptide, (b) a
tripeptide. Give the elements contained in hematin.

3. Name eight chemical groups contained in amino acid molecules. Which of
the above are responsible for color tests, and which reagent is used to show the
presence of the particular group?

4. What is meant by the term "incomplete protein"? Name two proteins that
are incomplete, and show in what respect they are incomplete.

5. Explain the use of the following reagents ia the Kjeldahl determination of
crude protein, (1) concentrated H2SO4, (2) Na2S04, (3) concentrated NaOH. What
is meant by the term "crude protein"? As usually determined, what assumptions are
involved?

6. Which three proteins are the most abundant in the food that you have eaten
in the last three meals? Give data on which you base your conclusions.

7. Approximately how many grams of tryptophan are there in a glass of milk?

8. Which two amino acids are found most abundantly in proteins?

9. Name two kinds of reagents that may be used to precipitate proteins. In each
case explain how the reagent brings about the precipitation, and name the product
formed.

10. Give the name and source of (1) three proteins that are enzyines, (2) three
proteins contained in industrial nonfood products, (3) one protein that is a hormone,
(4) two proteins that are respiratory pigments, (5) two proteins other than those
named in (1) to (4) that have been crystallized.

11. Which protein color tests will be positive with the tripeptide, tyrosyltryptophyl-
cystine? Give reasons for conclusion in case of each test.

12. Correct the following statements if incorrect:

(1) Milk and eggs are proteins that belong in every diet.

(2) In calculating crude protein it is assumed that all the protein of the sample
is in the form of nitrogen.

(3) In practical nutrition the deficiencies of incomplete proteins are remedied
by the addition of individual amino acids.

13. If the tyrosine content of a protein is 3.78 per cent and the molecular weight is
42,000, calculate the number of moles of tyrosine per mole of protein.

14. From the data given in the text calculate the number of residues of glycine,
alanine, and valine in (3-lactoglobulin. Do 3'our figures in round numbers check with
those of the text?

15. In which two ways does cystathionine appear to be split in S-transfer?

16. What is the least number of dipeptides that would have to be isolated and
identified to prove the sequence of anjino acids in p-amicidin S?

17. For outside reading: How would you determine whether a dipeptide contain-
ing alanine and leucine is alanyl-leucine or leucyl-alanine?

18. For outside reading: How many isomers of pteroyltriglutamic acid are due to
the arrangement of the glutamic acid residues.

REFERENCES AND SUGGESTED READINGS

Anson, M. L. and Edsall, J. T., Advances in Protein Chemistry, Vols. I-VII. Aca-
demic Press Inc., New York, 1945-51.

Baldwin, E., Dynamic Aspects of Biochemistry, 2nd ed.. The University Press, Cam-
bridge, England, 1952.

Bemal, J. D., "Structure of Proteins," Proc. Roy. Inst. Gt. Brit., 30, 541 (1939).

PROTEINS 149

Block, R. J. and Boiling, D., The Amino Acid Composition of Proteins and Foods,

2nd ed., Charles C. Thomas, Springfield, Illinois, 1951.
Brand, E., Saidel, L. J., Goldvvater, W. H., Kassell, B., and Ryan. F. J., "The Em-
pirical Formula of (3-Lactoglobulin," J. Am. Chem. Soc, 67, 1524 (1945).
Cohn, E. J. and Edsall, J. T., Proteins, Amino Acids and Peptides, Reinhold Publish-
ing Corp., New York, 1943.
Consden, R., Gordon, A. H., Martin, A. J. P., and Synge, R. L. M., "Gramicidin S:

the Sequence of the Amino Acid Residues," Biochem. J., 41, 596 (1947).
Gortner, R. A. and Gortner, W. A., Outlines of Biochemistry, 3rd ed., John Wiley

and Sons, Inc., New York, 1949.
Greenberg, D. M., Amino Acids and Proteins, Charles C. Thomas, Springfield, Illinois,

1951.
Harris, J. I. and Work, T. S., "The Synthesis of Peptides Related to Gramicidin S

and the Significance of Optical Configuration in Antibiotic Peptides," Biochem. J.,

46,582 (1950).
Haurowitz, F., Chemistry and Biology of Proteins, Academic Press Inc., New York,

1950.
Hawk, P. B., Oser, B. L., and Summerson, W. H., Practical Physiological Chemistry,

12th ed., The Blakiston Company, Philadelphia, 1947.
Hubbard, R. and Wald, G., "Cis-trans Isomers of Vitamin A and Retincne in

Vision," Science, 115, 60 (1952).
Luck, J. M., Loring, H. S., and Mackinney, G., Annual Revieio of Biocheynistnj, Vols.

I-XXI, Annual Reviews Inc., Stanford, California, 1932-52.
Moore, S. and Stein, W. H., "Chromatography of Amino Acids on Starch Columns,"

J. Biol. Chem., 176, 337 (1948), 178, 53 (1949).
Neurath, H., Greenstein, J. P., Putnam, F. W., and Erickson, J. O., "The Chemistry

of Protein Denaturation," Chem. Rev., 34, 157 (1944).
Pauling, L., "The Configuration of Polypeptide Chains in Proteins," Record of Chem-
ical Progress, 12, 155 (1951).
Roughton, F. J. W. and Kendrew, J. C, Haemoglobin, Interscience Publishers Inc.,

New York, 1949.
Sanger, F. and Thompson, E. O. P., "The Amino-acid Sequence in the Glycyl Chain

of Insulin," Biochem. J., 52, iii (1952).
Sanger, F. and Tuppy, H., "The Amino Acid Sequences in the Phenylalanyl Chain

of Insulin," Biochem. J., 49, 463, 481 (1951).
Schmidt, C. L. A., The Chemistry of the Amino Acids and Proteins With Addendum,

2nd ed., Charles C. Thomas, Springfield, Illinois and Baltimore, 1945.
Sherman, H. C, Chemistry of Food and Nutrition, 7th ed.. The Macmillan Company,

New York, 1946.
Wald, G., "The Chemistry of Rod Vision," Science, 113, 287 (1951); 115, 60 (1952).
West, E. S. and Todd, W. R., Text Book of Biochemistry, The Macmillan Company,

New York, 1951.

Chapter 6

NUCLEOPROTEINS, NUCLEIC ACIDS
AND RELATED SUBSTANCES

Introduction

As already stated in the previous chapter, nucleoproteins are conjugated
proteins having nucleic acids as prosthetic groups. The literature on
nucleoproteins and nucleic acids is extensive and increasing at a rapid rate.
Hundreds of papers and more than a dozen reviews dealing with various
phases of the subject have appeared in the last five years. The great
activity in this field can be attributed to the widespread occurrence, in-
triguing chemical nature, and metabolic importance of these substances.

One compelling reason for the attention being given to nucleoproteins
and nucleic acids is a growing belief that these compounds are closely
associated with the reproductive processes and may furnish the physical
basis of heredity. Chromosomes, the constituents of cells carrying the
hereditary characters, or genes, are largely, if not wholly, nucleoproteins.
Whatever chemical compounds' make up the genes, it is obvious that such
compounds must be sufficiently diverse in character to permit the almost
infinite number of combinations that occur in nature. In the nucleic
acids there is adequate diversification to meet this requirement.

NUCLEOPROTEINS

The term nucleoprotein arose because nucleic acids and the associated
protein, protamine, were first obtained from the highly nucleated material
of pus cells and fish sperm. Other nuclear cells such as thymus, liver,
spleen, and yeast are rich in nucleoproteins, but some nonnuclear cells,
for example, red blood corpuscles, also are a good source of nucleoprotein.
In fish sperm cells nucleoprotein makes up 50-80 per cent of the solid
material and over 90 per cent of the defatted nucleus. In the cell sap
of tobacco plants infected with virus, the nucleoprotein which makes up
the virus may amount to 2 g. per liter of sap. In yeast cells the nucleo-
protein amounts to only about 0.15 per cent of the dry matter. Bacteria
are much higher than yeast in nucleoproteins, e.g., 2-3 per cent of the dry
matter in Escherichia coli cells,

150

NUCLEOPROTEINS, NUCLEIC ACIDS, RELATED SUBSTANCES 151

Preparation

Nueleoproteins are labile substances; hence to obtain them from cells
onl3^ mild reagents and low temperatures can be used.

An example of present day methods is the procedure IVIirsky and Ris
used for the preparation of chromosomes from calf spleen. The tissue was
broken up in a Waring blender and soluble matter removed with 0.14M
sodium chloride, in which the chromosomes were insoluble. Unbroken
cells and other coarse materials were separated by filtering through finely
woven cloth and sedimenting the chromosomes in a centrifuge at 3500
rpm. The suspension, filtration through cloth, and centrifugation were
repeated several times until the phosphorus figure, which was taken
as a measure of the nucleic acid content, became constant. Under the
microscope the material showed the characteristic coiled pattern of
chromosomes. Differential centrifugation methods are rapid, and 10 to
15 g. of purified material can be obtained in the course of a morning's
work.

Tobacco bushy stunt virus, which is destroyed by almost any chemical,
was separated by Stanley from the other constituents of ground tobacco
leaves by means of differential centrifugation and purified still further
by crystallization.

Separation of components

The nucleic acid-protein complex is usually a very loose one, and the
combination can be broken up and the two components separated in
various ways. Some of these procedures are as follows:

The protein may be denatured either by heating or by treating with
urea to give an insoluble compound, this then being removed by filtration.
Alternatively, the protein may be obtained by extraction with chloroform
and octyl alcohol, leaving the nucleic acid in solution. A third method
is to destroy the nucleic acid with the enzyme, ribonuclease, and then
separate the unchanged protein from the digested material.

If the nucleic acid is the component wanted, it may be obtained from
the solution after the protein has been removed by one of the methods
described above. The protein may also be destroyed by trypsin and
the nucleic acid recovered. If the protein is a histone, separation may
be accomplished by dialysis against IM sodium chloride. Histone passes
through the membrane and leaves the nucleic acid behind.

Linkage between protein and nucleic acid

The bond between protein and nucleic acid is in some cases electro-
static, or salt-like, since the two parts may be separated by the passage of

152 NUCLEOPROTEINS, NUCLEIC ACIDS, RELATED SUBSTANCES

an electric current through the solution. The positively charged protein
moves to the cathode and the negatively charged nucleic acid goes to the
anode. Thymus nucleoprotein is an example of this type. The protein
is histone, and, since this is a strongly basic substance, it forms a salt
with nucleic acid. The two components are probably joined together
through the basic groups of arginine, histidine and lysine, and the phos-
phoric acid groups of the nucleic acid. Histones and protamines are
particularly high in arginine. If a sample of histone or protamine dis-
solved in 0.14il/ sodium chloride is added to a solution of nucleic acid
having the same strength of sodium chloride, the two components react
and form a precipitate. Such precipitates are probably the result of
interaction of the molecules as a whole, and not arginine alone. The same
quantity of arginine, histidine, and lysine as is contained in the protamine
forms no precipitate at the same pH.

There may be a second type of nucleoprotein in which the protein and
nucleic acid are bound together by nonpolar linkages. Some nucleopro-
teins migrate as single entities, and the protein cannot be separated from
the nucleic acid until it has first been denatured. However, some investi-
gators do not regard this as conclusive evidence of a nonsalt type of
bonding because the structure of the native protein is quite different from
that of the denatured protein, and the configuration may modify the
strength of the bonding groups.

Quantitative data on components

In Table 6-1 are given examples of nucleoproteins, the kinds of pro-
teins contained therein, and the proportion of protein to nucleic acid.
Histones and protamines are the common type, but lipoproteins occur
frequently in the nucleoproteins of animal tissues. Chromosomes seem to
contain two kinds of nucleoprotein, histone and nonhistone types.
Mirsky, from whose papers these data are taken, differentiates the two
types on the basis of the insolubility of the nonhistone protein in
HgS04-H2S04 solution (histone is soluble) and its greater tryptophan
content (nonhistone contains 1.36 per cent and histone only 0.14 per
cent) .

Nucleolipoprotein, containing lipoprotein in combination with nucleic
acid, indicates a protein having two prosthetic groups, lipide and nucleic
acid. Several viruses, e.g., vaccinia virus, have been found to be nucleo-
lipoprotein complexes.

The nucleic acid portion of the nucleoproteins may range from a small
fraction, 5 per cent, to m-ore than half of the total. The desoxyribo-
nucleic acid (DNA) seems to make up a larger percentage of the nucleo-
protein than the ribose form (RNA). Both types of nucleic acid often
occur in the same cell. In chromosomes the DNA type predominates,
but in yeast cells the RNA form is in excess.

NUCLEOPROTEINS, NUCLEIC ACIDS, RELATED SUBSTANCES 153

Table 6-1

Types of protein and nucleic acid found in some typical nucleoproteins

Composition of nucleoproteix *
Source of nucleoprotein Protein, % Nucleic acid, %

Calf thymus Histone, 40 DNA, 60

Sperm heads of fish Protamine, 40 DNA, 60

Liver Lipoprotein, 95 DNA, 5

Chromosomes of calf thymus:

Soluble fraction, 90% Histone, 47 DNA, 45

fRNA 11
Residual fraction, 10% Nonhistone, ? ■ „,, . ' „

IDNA, 2

Tobacco mosaic virus Not classified, 94 RNA, 6

Tobacco ring-spot virus .... Not classified, 60 RNA, 40

Tuberculin from tubercle

bacillus Not classified, 60 DNA, 40

Yeast Not classified, 90-105 RNA, 5-10

DNA, ?

Bacteria Not classified, 80-85 RNA, 15-20

* DNA denotes desoxyribonucleic acid ; RNA means ribonucleic acid. The nature
of the different types of nucleic acid will be discussed later. Where no figures regard-
ing the amounts are available, this is indicated by a (juestion mark (?) in the second
and third columns.

The viruses of the tobacco plant are well-characterized. They have
been obtained in crystalline form and their properties carefully deter-
mined. The protein part varies from 60 to 94 per cent of the nucleo-
protein in the two viruses listed in Table 6-1. The amino acids of tobacco
mosaic virus account for 106 per cent of the virus. (See Table 5-4).
The nucleic acid is of the ribose type, which is the most abundant type
found in plant material.

The molecular weights reported for nucleoproteins are large, 2 million
for calf thymus nucleohistone and 40 million for tobacco mosaic virus.

NUCLEIC ACIDS

Component units

The nucleic acids are themselves complex structures with molecular
weights ranging from 17,000 for yeast nucleic acid to more than a million
for the acid from the thymus gland. The particle size varies with the
method of preparation, hence, the smaller weights may represent split
products of the larger units. The molecules appear to be rod-like in
shape, with the length of the particles 40 to 400 times that of their
diameter.

Nucleic acids are divided into two classes depending upon the kind of
hydrolysis products. This will be evident from an inspection of the
following tabulation.

154

NUCLEOPROTEINS, NUCLEIC ACIDS, RELATED SUBSTANCES

Products from ribo-
nucleic acid

Adenine, guanine
Cytosine, uracil
v-Ribose
Phosphoric acid

Products from desoxy-
ribonucleic acid

Adenine, guanine

Cytosine, 5-methylcystosine, thymine

D-Desoxyribose

Phosphoric acid

Classification of
products
Purine
Pyrimidine
Pentose
Acid

Four of the products are found in both kinds of nucleic acid. The dis-
tinguishing products are uracil and D-ribose for ribonucleic acid and
thymine, 5-methylcytosine and D-desoxyribose for the other type. The
sugars are the products from which the terms ribonucleic acid (RNA)
and dcsoxyribonucleic acid (DNA) are derived.^ It was at one time
believed that ribonucleic acid was found only in plants and dcsoxyribo-
nucleic acid only in animal cells. This view is incorrect, and it now
appears probable that all cells contain both types. The dcsoxyribonucleic
acid seems to be most abundant in the nucleus of the cell, and the ribo-
nucleic type to be preponderant in the cytoplasm surrounding the nucleus.

Purines and pyritnidines

The structural formulas of the purines are given below:

(1) N=CH (6)

I I
(2)HC(5)C— NH (7)

CH(8)
//

(3) N— C-N

(4) (9)

Purine
(synthetic base)

N=C— NHj

I i

HC C-NH
\

//

N— C-N

Adenine

CH

N=C— OH

I I
HoNC C-NH
\

CH
//
N— C-N

Guanine

N=C— OH
I I
HC C-NH

C-N

Hypoxan thine

N=C— OH
I I
HO-C C-NH
\

//

N— C-N

Xanthine

CH

^Research workers use the terms pentosenucleic acid (PNA) and desoxypentose-
nucleic acid (DNA) as general terms and limit the more specific names to nucleic
acids where the sugars have been definitely established as ribose (in yeast, liver, and
tobacco mosaic virus) and desoxy ribose (in calf thymus). This cautious attitude
is probably desirable for the research worker, but since ribose and desoxyribose are
the usual sugars found in nucleic acids, it is less complicated for the beginning student
to start with the particular name and proceed to the general term Avhen it becomes
necessary.

NUCLEOPROTEINS, NUCLEIC ACIDS, RELATED SUBSTANCES

N=C— OH HN— 0=0

II II

HO-C C-NH 0=C C-NH

155

C— OH
//

\

C=0

/

N— C-N HN— C-NH

Uric acid
(hydroxy form) (carbonyl form)

The naturally occurring purines may be referred to the synthetic base,
purine. The various atoms in the rings are numbered to denote the
position in the structure. Thus adenine is designated 6-aminopurine, and
guanine is 2-amino-6-hydroxypurine. In solution the hydroxy compounds
exist in two tautomeric forms, as is shown in the formulas for uric acid.

Adenine and guanine are constituents of native nucleic acids, and the
other three compounds are products derived from the first two as a result
of metabolism. Hypoxanthine is formed in the body by deamination of
adenine, and on oxidation this product forms xanthine, which may also
originate from deamination of guanine. Oxidation of xanthine gives
uric acid, which is the end product of purine metabolism in man.

Three other purines occur in our common beverages. Caffeine (1,3,7-
trimethylxanthine) is found in the coffee bean to the extent of about
1 per cent and in tea leaves to about 2 per cent. Theobromine (3,7-di-
methylxanthinc) occurs in the cocoa bean (about 2 per cent) and theo-
phylline (l,3-dimeth3''lxanthine) is found is small quantities in tea leaves.
Caffeine, removed from the coffee bean in the making of decaffeinized
coffee, is used in the manufacture of cola drinks. However, this supply
is not sufficient for the purpose, and much of the caffeine used in soft
drinks is made synthetically.

The pyrimidines have the following structural formulas:

(1) N=CH(6) N=C— NHj N=C— NH2

II II II

(2)HC CH(5) HO— C CH HO— C C— CHj

II II II II II II

(3) N— CH(4) N— CH N— CH

Pyrimidine Cytosine 5-Methylcytosine

(synthetic base)

N=C— OH N=C— OH

II II

HO— C CH HO— C C— CH3

II II II II

N-CH N-CH

Uracil Thymine

The pyrimidine ring forms a part of the purine structure and is numbered
in the same way. Thus cytosine is 2-hydroxy-6-aminopyrimidine, uracil
is 2,6-hydroxydipyrimidine, and thymine is 2,6-dihydroxy-5-methyl-
pyrimidine. 5-Methylcytosine, reported many years ago as occurring

156

NUCLEOPROTEINS, NUCLEIC ACIDS, RELATED SUBSTANCES

in the tubercle bacillus, has now been found in small quantities in desoxy-
ribonucleic acids from cattle spleen, fish sperm, and wheat germ, but
not in the desoxyribonucleic acids from bacteria and viruses. To date,
none has been found in ribonucleic acids. The vitamin, thiamine, is a
pyrimidinethiazole combination. Its pyrimidine can be designated as
2,5-dimethyl-6-aminopyrimidine.

Nucleosides and nucleotides

If the hydrolysis of a nucleic acid is done under suitable conditions,
the breakdo\Mi may be stopped before it is complete and nucleosides and
nucleotides obtained. A nucleoside is a purine or pyrimidine-pentose
combination, and a nucleotide is a nucleoside-phosphoric acid complex.
The various bases, and the corresponding well-known nucleosides and
nucleotides, are listed in the following tabulation:

Base

Nucleoside

M ononucleotide

Adenine

Adenosine

Adenylic acid

Guanine

Guanosine

Guanvlic acid

Cytosine

Cytidine

Cytidylic acid

Uracil

Uridine

Uridylic acid

Thymine

Thymidine

Thymidylic acid

The nucleosides are designated, according to the sugar contained in
them, as ribosides (adenosine, guanosine, cytidine and uridine) or as
desoxyriboside (thymidine) . The corresponding mononucleotides contain
the same sugars as the nucleosides. There are obviously other nucleosides
and nucleotides of desoxyribose, but, to date, these have not been given
specific names. They are often designated by prefixing the term desoxy
to the i>ames of the ribose-containing compounds: desoxyadenosine, des-
oxyadenylic acid, etc.

The structural formula of one of the mononucleotides, adenylic acid,
will be given to show the order of the components and the linkages that
join the parts together.

(6)

0) N=C— NHj

HC C-

(7)

■N
\\

OH

I
0— P=0

I

OH

CH

(3) N— C-N-

(4) (9)

Adenine
Adenosine

— 0
OH

-C— C— C— C— CH2OH
H H H H

(!') (20 (30 (40 (5')

R

i

b
o

s
-e

Ribose phosphoric acid
>-i

Adenosine-3-phosphoric acid

Adenylic acid (adenosine monophosphate)

NUCLEOPROTEINS, NUCLEIC ACIDS, RELATED SUBSTANCES 1^7

Adenine and ribose are joined by a j8-glycosidic linkage from the
nitrogen of position-9 of the adenine to carbon- 1 of the ribose. The
adenosine and phosphoric acid are united by an ester hnkage. Adenosine
and adenylic acid have now been synthesized by Todd and co-workers,
so that there is no doubt remaining as to their structure. These workers
have also synthesized a number of other nucleosides and nucleotides.

A second type of adenylic acid has been obtained from yeast nucleic
acid. In this type the phosphoric acid is thought to be linked to carbon-2'
instead of 3'. If this proves to be correct, it provides strong support for
the view that nucleotides are linked together through phosphoric acid,
which is joined to one nucleotide at carbon-2' and to the other nucleotide
at carbon-3'. These structures are complicated and difficult to deter-
mine, but distinct progress is being made toward their final solution.

In the pyrimidines the y8-glycosidic linkage is between the nitrogen
at number 3 position and carbon-l' of the ribose. The phosphoric acid
is located at carbon-3', as in the purine nucleotides. These structures
have been established beyond doubt by synthesis of cytidine and uridylic
acid.

The nucleosides and nucleotides of desoxyribose are believed to have
the same linkages between base, sugar, and phosphoric acid as those of
ribose, but the data are not so conclusive as for the ribose compounds.

Polynucleotides

Nucleic acids found in nature are usually polynucleotides, consisting
of many purine and pyrimidine nucleotides joined together to form a
single structure. Estimates ranging from 60 nucleotides for yeast nucleic
acid to 4000 for thymus nucleic acid have been given. Such estimates
are in accord with the large molecular weights obtained for these nucleic
acids. Formerly, it was believed that these large molecules were made up
of many tetranucleotide units, but this view is now generally abandoned.

The molar ratios of the different purines and pyrimidines to one another
do not bear out the idea of a regularly occurring tetranucleotide unit.
For example, Chargaff and co-workers found that the desoxynucleic acid
of salmon sperm gave molar ratios of the constituents as follows: Adenine
to guanine, 1.43; thymine to cytosine, 1.43; adenine to thymine, 1.02;
guanine to cj^tosine, 1.02; purines to pyrimidines, 1.02. Adenine oc-
curred in excess of guanine, and thymine was more abundant than
cytosine. Oddly enough the ratios are the same in both cases, and the
total purine is equal to the total pyrimidine content. In other desoxy-
nucleic acids Chargaff found adenine exceeded guanine, and thymine out-
weighed cytosine, but the ratios were different than in the salmon nucleic
acid.

The nucleotides are joined together through phosphoric acid groups,
but just how is not known. One possibility is a linkage from carbon-2'

158 NUCLEOPROTEINS, NUCLEIC ACIDS, RELATED SUBSTANCES

of one ribose to phosphoric acid and a second linkage of this to carbon-3'
of the next nucleotide Such an arrangement for three ribonucleotides
can be represented as follows:

O

II

Adenine-ribose

(3')

(2')

Cytosine-ribose

(2')

Guanine-ribose •

0— P— OH

■oJ

O

II
■0— P— OH

_l

■0

0

■0— P— OH
■0^

I —

etc.

The numbers 2' and 3' denote the carbon atoms in the ribose to which
the phosphoric acid group is linked. y

Obviously, there are other ways of joining the guanine nucleotide to
the other two ribonucleotides. For example, the ribose part of the
guanine nucleotide could be linked to the adenine nucleotide, instead of
to the phosphoric acid in the cytosine nucleotide. The result would be
a triester structure, instead of the diester form given by the first type
of combination. The number of possibilities would increase as the
number of nucleotides joined together became larger. A more complicated
branching structure would be the result. There is no information as
to the sequence of the nucleotides in the nucleic acid structure.

In the desoxynucleotides, carbon-2' of the sugar can not serve as a
point of linkage because it has no hydroxyl group. It is generally assumed
that the desoxynucleotides are joined together by way of carbons-3' and
5' of their respective sugars.

Substances related to nucleosides and nucleotides

Adenosine Phosphates. A nucleotide with the phosphoric acid at car-
bon-5' of the ribose, instead of at carbon-3', is the well-known muscle-
adenylic acid. It is also called adenosine monophosphate (AMP).

N=C-NH,

EC C-N

-C-l/-

CH

0-

HO OH

H

O

0

O

- C-C-C-C-C-0-P-O-P-O-P-OH
H H H H H I

(1') (2') (3') (4') (5') OH

Adenosine monophosphate -

Adenosine diphosphate

Adenosine triphosphate

OH

OH

NUCLEOPROTEINS, NUCLEIC ACIDS, RELATED SUBSTANCES

159

Besides the monophosphate, adenosine forms a diphosphate, ADP, and
a triphosphate, ATP. The three derivatives of adenosine play an out-
standing role in enzyme chemistry and intermediary metabolism.

Coenzymes I and II. These compounds are dinucleotides of adenine
and the base, nicotinamide. Coenzyme I is also known as diphospho-
pyridine nucleotide (DPN) , and coenzyme II as triphosphopyridine nu-
cleotide (TPN). The make-up of DPN can be seen from the following
designation:

O

II
Adenine-ribose — 0 — P — OH

I

O
I
Nicotinamide-ribose — 0 — P — OH

II
O

Note that the two nucleotides are joined through the phosphoric acid
molecules, instead of the pentose-phosphoric acid-pentose structure found
in nucleic acids. The phosphoric acid groups are linked to carbon-S'
of the ribose units.

TPN is like DPN except that it has a third phosphoric group attached
to carbon-2' of the ribose found in the adenine nucleotide. For the struc-
tural formulas of DPN and TPN see p. 276.

Flavin Nucleotides. There is a so-called mononucleotide, riboflavin
phosphate, and a dinucleotide of riboflavin phosphate and adenylic acid.
The riboflavin phosphate is not a true nucleotide because the ribose part
is replaced by the sugar alcohol corresponding to ribose, viz., ribitol. The
difference in structure is evident from the formula on p. 278. The flavin
nucleotides are coenzymes, and a discussion of their function will be
given in the chapter on enzymes.

5,Q-Dimethylbenzimidazole Riboside. This nucleotide and the corre-
sponding nucleoside have been obtained as degradation products of vita-
min Bi2. The structural formula is

OH
I
O— P=0

B.zC^((i)

(1)

HaC (5)

<WA

(4)

N-

<3)

/

CH

0-

OH

OH

-C— C— C— C-CH2OH
H H H H

a') (2') (3') (4') (5')

Note that the ribose has a furanose, instead of a pyranose, structure.
In the formula, the phosphoric acid is attached to carbon 3' of the ribose,
but the point of attachment is uncertain. It may be at carbon 2'.

160 NUCLEOPROTEINS, NUCLEIC ACIDS, RELATED SUBSTANCES

REVIEW QUESTIONS ON NUCLEOPROTEINS

1. Which types of protein have been found in nucleoproteins? How much is
protein and how much is nucleic acid in a few typical nucleoproteins?

2. Name eight compounds that may be obtained from nucleic acids on hydrolysis?
To what class of compoimds does each belong? Name some other examples of each
class.

3. Define nucleoside and nucleotide, givirig an example of each. Which nucleo-
tides are associated with (1) vitamins, (2) enzymes?

4. Compare the probable structure of the dinucleotide, adenylic acid — cytidylic
acid with the pyridine and flavin dinucleotides. Point out similarities and differ-
ences.

5. Catalog the linkages that join together the parts of a nucleic acid.

REFERENCES AND SUGGESTED READINGS

Chargaff, E., Lipshitz, R., Green, C, and Hodes, M. F., "The Composition of Des-
oxyribonucleic Acid of Salmon Sperm," J. Biol. Chcm., 192, 223 (1951).

Cold Spring Harbor Symposia on Quantitative Biology, Nucleic Acids and Nucleo-
proteins, Vol. XII, The Biological Laboratory, Cold Spring Harbor, N. Y., 1947.

Davidson, J. N., The Biochemistry of the Nucleic Acids, John Wiley and Sons, Inc.,
New York, 1950.

Green, H. N. and Stoner, H. B., Biological Actions of the Adenine Nucleotides,
H. K. Lewis and Co., Ltd., 1950.

Mirsky, A. E. and Ris, H., "Isolated Chromosomes," J. Gen. Physiol, 31, 1 (1948).

Schlenk, F., "Chemistry and Enzymology of Nucleic Acids," Advances in Enzymol-
ogy, 9, 455 (1949).

Stanley, W. M., "Purification of Tomato Bushy Stunt Virus by Differential Centrifu-
gation," J. Biol. Chem., 135, 437 (1940).

Symposia of the Society for Experimental Biology, No. 1, Nucleic Acid, The Uni-
versity Press, Cambridge, England, 1951.

Symposium on Biochemistry of Nucleic Acids, J. Cellular Comp. Physiol., 38,
Supplement, 1951.

Tamm, C, Hodes, M. E., and Chargaff, E., "The Formation of Apurinic Acid from
the Desoxyribonucleic Acid of Calf Thymus," J. Biol. Chem., 195, 49 (1952).

Chapter 7

ACIDITY

So many acids and bases, both organic and inorganic, occur in living
organisms that only a few of them can be considered here. Some of
the more common organic acids are citric in citrus fruits, tartaric in
grapes, malic in apples, and oxalic in rhubarb and spinach. In some
cases these acids are present in the plant tissues in the free condition.

CH2COOH

1

COOH
1

COOH
1

HO-C— COOH

HOCH
1

HOCH
j

COOH
1

CH2— COOH

HCOH
1

CH2

COOH

COOH

COOH

Citric acid

L-Tartaric acid

L-Malic acid

Oxalic acid

but often they occur as salts. Thus lemon juice contains about 5 per cent
of free citric acid, but much of the tartaric acid in grape juice has had
its acidity partially neutralized by potassium. Crystals of potassium
acid tartrate, "cream of tartar," often are deposited from grape juice
or wine on long standing. Similarly, the oxalic acid in some plants is
free, and in others exists as calcium oxalate. This is a matter of con-
siderable consequence because free oxalic acid, as well as its water-soluble
salts, is a strong poison. The halogeton weed, which grows in Nevada
and several other Western states, contains up to 18 per cent of soluble
oxalates on the dry weight basis. Livestock, particularly sheep, have
been killed by the thousands by eating this weed (see Fig. 7-1). Fortu-
nately, the oxalic acid in rhubarb and spinach is present largely in the
form of the very insoluble calcium oxalate which is nontoxic. Rhubarb
leaves, however, are said to contain harmful concentrations of soluble
oxalates.

A whole series of organic acids is involved in the normal metabolism
of carbohydrates and fats in the animal body (Chap. 13). Among the

COOH
1

COOH

1

COOH
1

COOH
1

CH3

HOCH
1

1

CO
1

CH2
1

CH3

CH3

CH2

1
COOH

Lcetic acid

L- Lactic acid

161

Pyruvic acid

Succinic acid

162

ACIDITY

simpler members are acetic, lactic, pyruvic, and succinic acids. In addi-
tion, living cells contain many organic phosphates (Chap. 13) which are
strongly acidic because they contain the phosphate group.

By Life photographer Carl Iwasaki (c) Ti)iie Inc.
Fig. 7-1. Sheep eating the halogeton weed, which is poisonous because
of its high content of sokible oxalates. One animal has ah-eady died from
eating the weed.

Alkaline substances encountered in biological materials are somewhat
less numerous than the acids. Methylamine and trimethylamine are weak
bases, which are present in decayed fish and contribute to the unpleasant
"dead-fish" odor. The strong base, choline, is found in many tissues,
usually as a salt such as the chloride, or combined in more complex forms
(phospholipides, p. 97). Other amines found in decaying animal matter
are formed through decomposition of amino acids (p. 321).

CH3NH,

(CH3)3N

(CH3)3NCH2CH20H

CI

Methylamine Trimethylamine Choline chloride

All of the bases and acids contained in a tissue contribute to the acidity
or alkalinity of that tissue. Acidity (or alkalinity) in biological ma-
terials is of two kinds, "total" and "active."

ACIDITY 163

Active acidity refers only to the concentration of hydrogen ions present
in the material. Hydrogen ions, it will be remembered, are produced by
ionization of an acid, for example,

CH3COOH ^ CH3COO- + H +

The hydrogen-ion concentration is expressed in terms of pH, which will
be considered later.

Total acidity, on the other hand, refers to the total amount of acid
present, both ionized and nonionized. It is usually expressed as per
cent by weight ; for example, vinegar contains 4 to 5 per cent acetic acid.

TOTAL ACIDITY

Determination of total acidity or alkalinity is important when one
needs to know how much of some material is required to react with the
acid or alkali in another material. The determination usually is accom-
plished by titration with standard solutions, that is, by measuring the
volume of base or acid that is required to react with a given amount
of the sample. The first requisite for such an analysis, therefore, is a
standard solution, which is simply a solution of known concentration.
The concentration of standard solutions is usually expressed in terms
of molarity or normality.

Molar solutions

By definition a molar solution is of such concentration that one liter
of the solution contains exactly one gram molecular weight, or one 7nole,
of the solute. Any given fraction of a liter of such a solution, there-
fore, would contain an equivalent portion of a gram molecular weight.
Hence, withdrawal of aliquots from such a standard solution offers a
speedier method of obtaining a known weight of reagent than can be
effected by the process of weighing. Moreover, the accuracy is much
greater than that which can be attained by weighing minute quantities
of material.

Normal solutions

Since a mole of one compound may react with one, two, or more moles
of another compound, or with only a fraction — one-half, one-third, and
so on — of a mole of the second compound, molar solutions are seldom
convenient to use. This is not true of normal solutions, which are so
prepared that a given volume of a solution of one compound is equivalent
to exactly the same volume of solution of any other compound. This
is very clearly evident in the case of acids and bases. In displaying

164

ACIDITY

their most outstanding property, their ability to react to form salts,
the molecules react according to the number of replaceable hydrogens of
acids and hydroxyl groups of bases. Note in the following equations
that the reacting powers per mole of HCl, H2SO4, and H3PO4 are in
the ratio 1:2:3.

NaOH + HCl -^ NaCl + HoO
2NaOH + H2SO4 -> NaoS04 + 2HoO
3NaOH + H3PO4 -^ Na3P04 + SHoO

Hence the complete reaction of one mole of sodium hydroxide requires
one mole of hydrochloric acid, but only one-half mole of sulfuric or one-
third mole of phosphoric acid. If these amounts of the respective acids
be diluted to a common volume, for example, 1000 ml., the resulting
solutions are of equivalent concentrations so far as their ability to react
with alkalies is concerned. It is upon such a basis that normal solu-
tions are prepared.

By definition a normal solution is of such concentration that one liter
of the solution contains exactly one gram equivalent weight of the solute.
The gram equivalent weight is that weight of a compound that contains
one gram of replaceable (acid) hydrogen, or will react with one gram
of replaceable hydrogen, or is in any way equivalent to this weight of
hydrogen. To calculate the gram equivalent weight of acids divide the
gram molecular weight by the number of replaceable hydrogen atoms
in the molecule. Since one hydroxyl group requires one acid hydrogen
for its neutralization, it follows that the gram equivalent weight of
bases is obtained by dividing the gram molecular weight by the number
of hydroxyl groups in the molecule. For a salt the divisor is the number
of hydrogens that have been replaced in the formation of the salt from
the corresponding acid. Obviously, one obtains a like result in the last
two instances by dividing the gram molecular weight by the valence of
the metal contained therein. Thus the divisor for NaOH is 1, but for
CaS04 it is 2. The divisor in each case is termed the hydrogen equiva-
lent. The use of these values in calculations involving normal solutions
is illustrated in Table 7-1.

One gram equivalent weight of a chemical substance is frequently
called simply an equivalent of that substance. One-thousandth of this
amount similarly is termed a milliequivalent (abbreviation m.e.q.),
which, if expressed in milligrams, is the same numerical figure as an
equivalent expressed in grams. For example, in the case of acetic acid
an equivalent is 60 g. (Table 7-1) and a milliequivalent is 60 mg. One
liter of a normal solution always contains one equivalent of the solute,
one milliliter containing one milliequivalent. Analogous fractions of a
mole are also frequently used in biochemical work. Thus, one-thousandth
of a mole is a milli^nole, and one-millionth is a micromole. Molarity

ACIDITY

165

of solutions is indicated by a number followed by M, and normality
by a number followed by A''.

Table

7-1

Weights

of typical reagents in re

presentative

standard solutions

Gr\ms of

REAGENT

Per liter

Per ml.

Molecular

Hydrogen

Equivalent

of normal

oj normal

Reagent

weight

equivalent

weight

solution

solution

HCl

36.5

1

36.5

36.5

0.0365

HC2H3O2

60

1

60

60

0.060

H.SO4

98

2

49

49

0.049

H2C=04-2H=0

126

2

63

63

0.063

H=C,H,Oo

150

2

75

75

0.075

H3PO4

98

3

32.7

32.7

0.0327

H3C«Hr,0r

192

3

64

64

0.064

NaOH

40

1

40

40

0.040

Ca(0H)2

74

2

37

37

0.037

NH4OH

35

1

35

35

0.035

XaCl

58.5

1

58.5

58.5

0.0585

BaCNOs)^

261.4

2

130.7

130.7

0.1307

AU(S04)3

342

6

57

57

0.057

K2C4H4O6

226.2

2

113.1

113.1

0.1131

KHC.aOa

188.1

1

188.1

188.1

0.1881

NaHCOa

84

1

84

84

0.084

Standardization of solntions

It is not always possible to prepare standard solutions by weighing
out the amount of reagent theoretically required, because many substances
are not obtainable in sufficient purity, and others take up water or
carbon dioxide when exposed to the air during the time required for weigh-
ing. Solutions of such substances can, however, be "standardized" by
titration against a solution of known concentration prepared from a
"primary standard," that is, a substance that can be obtained in a high
state of purity and conveniently weighed. Frequently, normal solutions
are too concentrated for accurate measurement of the limited amount
of acid or base in the substance that is being analyzed. In general prac-
tice, 0.1 normal solutions are quite satisfactory.

Titration is a process of measuring the volume of one solution that is
required to react exactly with a definite amount of a second solution.
In titrating acids and bases the point at which the reaction is completed
is revealed by the color of an "indicator," which is added before the
titration is started. The selection of a suitable indicator is explained
below. In 'practical work equal volumes of solutions of the same
normality are considered to react exactly with one another. If the two
solutions do not have the same normality, it takes proportionately more

166 ACIDITY

of the less concentrated to titrate a given quantity of the more concen-
trated. If solution A is titrated against solution B, the following rela-
tionship holds:

volume of A X normality of A = volume of B X normality of B

Thus if the normality of A is known, that of B may be calculated from
the titration data, since from the above equation it may be seen that

volume of A X normality of A

normality of B

volume of B

As an example of a typical standardization let us assume that 21.4
ml. of a solution of sodium hydroxide are required to neutralize 25 ml.
of tenth-normal (O.LV) oxalic acid. The normality of the sodium hy-
droxide solution will then be equal to

25X0.1 ^,,^
-^j;^ = 0.117

We should say, therefore, that the sodium hydroxide solution is 0.117
normal, which means simply that it is 0.117 times as concentrated as a
normal solution.

It is obvious that if solution A is one-tenth as concentrated as solution
B, a given volume of A is equivalent to one-tenth of that volume of
B. Likewise in the example just considered, 1000 ml. of the solution
that is 0.117 normal is equivalent to only 117 ml. of normal solution.
The volume of a solution used in a titration when multiplied by its
normality gives the equivalent volume of normal solution. In other
words, by means of this calculation one determines the volume that
the solution would occupy if it were exactly normal.

Analysis of biological materials

In order to determine the percentage of a given constituent in any
material, two things must be known, namely, the weight of sample taken
and the amount of standard solution needed to titrate it. The follow-
ing calculation of the citric acid content of lemon juice is typical:

Weight of sample 5-0 g.

Volume of alkali, normality 0.103, for titratioa 34.6 ml.

Molecular weight of citric acid (HsCgHsOt) 192

Hydrogen equivalent of citric acid 3

Equivalent weight of citric acid 64

Weight of citric acid per ml. of normal reagent 0.064 g.

Volume of normal citric acid equivalent to alkali used for

titration (34.6 X 0.103) 3.56 ml.

Weight of citric acid in sample (3.56 X 0.064) 0.2278 g.

(0.2278 X 100 \
I 4.56 per cent

ACIDITY 167

Indicators

The ability to determine when sufficient reagent has been added in
titration of an acid or base depends upon the sensitivity of certain dyes
to changes in acidity. Such compounds are called indicators. Many
dyes that are used as indicators change color in either slightly acidic
or basic media rather than at exact neutrality. This is a desirable
characteristic, as may be seen by a study of the salts formed through
interaction of the respective acids and bases. Salts of strong bases
and weak acids, e.g.;, sodium carbonate, undergo hydrolysis when dis-
solved in water, producing basic solutions, whereas those salts formed
by union of weak bases and strong acids, like ammonium sulfate, are
somewhat acidic for a similar reason. Therefore when titrating an acid
with a base, or vice versa, it is essential that the standard solution be
added until the same degree of acidity or alkalinity is produced that
would result by dissolving the corresponding salt in water. Choice of
indicators is made accordingly rather than with the idea of determining
the point of exact neutrality. Methyl orange, methyl red, bromthymol
blue, and phenolphthalein are examples of indicators in common use.
The first two are suitable for titration of weak bases, and the last one
for weak acids.

HYDROGEN-ION CONCENTRATION

"Active" acidity as contrasted to "total" acidity is due solely to that
portion of the total replaceable hydrogen that, under prevailing condi-
tions, exists in the ionic state. As a commonplace illustration one may
liken acidity of a solution to the wealth of an individual. Total acidity
corresponds to total wealth, which includes currency, real estate, personal
property, notes, bonds, and so on. Active acidity, on the other hand,
is comparable only to currency, and just as the response of a ticket sales-
man is conditioned by the currency in the hand of a prospective pur-
chaser, so the behavior of a cell is conditioned by the active hydrogen
in the aqueous medium surrounding it. It is true that other forms of
wealth are convertible into currency, and, likewise, acids tend to dis-
sociate further as some of their hydrogen ions are used up by chemical
reaction.

The hydrogen-ion concentration has much more to do with enzyme
action and the maintenance of a normal colloidal structure in cells than
has total acidity. A fatigued muscle may contain as much as 0.4-0.5
per cent lactic acid for a time without undergoing injury, but a like
concentration of hydrochloric or sulfuric acid would result in death to
the tissue. Consider also the supply of carbon dioxide — potentially car-
bonic acid — carried by the blood stream. Introduction into the blood

168

ACIDITY

stream of an equivalent amount of other common acids, even such acids
as citric and acetic, which the body normally oxidizes for energy, would
doubtless be fatal. In these two instances it is not the concentration
of total acid that determines whether or not injury results; it is the
concentration of hydrogen ions. Figure 7-2 shows the effect of various
hydrogen-ion concentrations on plants. Growth is poor when the active
acidity is too high (pH too low).

Courtesy of Illinois Agricultural Experiment Station. Reproduced from Hiinper
Signs in Crops, a publication of the American Society of Agronomy and the
National Fertilizer Association, Washington, D. C.

Fig. 7-2. The effect of increasing acidity (left to right) on growth of red
clover. The poor growth results from calcium starvation, because high
acidity interferes with the absorption and retention of calcium by the plant
roots.

For these reasons measurement of the hydrogen-ion concentration fre-
quently is of more significance than determination of titratable acidity
or alkalinity of a given biological fluid or extract. But one must not
conclude that active acidity is always of prime importance. A familiar
illustration involves the use of soda and sour milk as a leavening agent
in the making of corn bread. If the housewife should add only enough
soda to react with the hydrogen ions initially present in the sour milk,
most of the lactic acid, i.e., the nonionized part, would not be neutralized,
and sour bread would be the inevitable result. Sufficient soda to react
with all of the acid must be added.

Water is a neutral substance because it yields an equal, though rela-
tively small, number of hydrogen and hydroxyl ions. The concentra-
tion is known to be 0.0000001 (also expressed 10"'^) gram-ion per liter,
which is equivalent to 0.0000001 g. of hydrogen ions and 0.0000017 g. of
hydroxyl ions. It must be borne in mind that hydrogen ions exist even
in basic solutions. Their concentration is reduced as basicity increases,
but, theoretically, all are never entirely removed from a solution.

ACIDITY 169

Concentration of hydrogen ions may be expressed directly in gram-ions
per liter as above (comparable to moles per liter when considering a
given reagent). Usually, however, when dealing with biological ma-
terials this necessitates the use of relatively small decimal fractions
with attendant possibility of an error in writing. A more convenient
method is that of pH, by which one merely expresses the logarithm of
the reciprocal of the hydrogen-ion concentration. Thus the reciprocal
of 0.0000001, the hydrogen-ion concentration of water, is 10,000,000 or
10^, and the logarithm of this number is 7. The pH of pure water there-
fore is 7, and all solutions of such pH are said to be neutral.

That acidic solutions have pH values less than 7 is apparent in view
of the fact that any concentration greater than 0.0000001 will have a
correspondingly smaller reciprocal and consequently a smaller logarithm
(pH value). Conversely, all basic solutions have pH values greater than
7. To those unfamiliar with this system of expressing active acidity,
it may seem that small differences in pH correspond to unbelievably great
differences in actual hydrogen-ion concentration. This can best be
realized by a comparison of several hydrogen-ion concentrations and
corresponding pH values simultaneously:

Concentration in

Reciprocal of

gram-ions per liter

concentration

VH

0.1

10

1

0.001

1,000

3

0.000001

1,000,000

6

0.00000001

100.000.000

8

0.0000000001

10,000,000,000

10

Thus far only a tenfold (or some power thereof) increase or decrease
in hydrogen-ion concentration has been considered, but it is obvious
that between any two such values, e.g., 0.1 and 0.01 gram-ions per liter,
are countless possible concentrations with corresponding pH values. To
make interpretation of pH values easy Table 7-2 has been included.
Column 1 merely gives the approximate logarithms of some appropriate
numbers between 1 and 10 (listed in column 2) .

Table 7-2
Comparison of pH values

Approximate equivalent
Change in pH • change in acidity

0.1 1.25 times

0.2 1.6 time.s

0.3 2.0 times

0.6 4.0 times

0.9 8.0 times

1.0 10.0 times

170 ACIDITY

Example. Compare pH 6.6 and 5.1.

The difference between pH 6.6 and 5.1 is 1.5 units, which can be broken down into
units found in the table, namely, 1.0, 0.3, and 0.2.

A difference of 1.0 equals 10 times.

A difference of 0.3 equals 2 times.

A difference of 0.2 equals 1.6 times.
Hence a difference of 1.5 equals 10 X 2 X 1.6 = 32.
Therefore pH 5.1 is 32 times as acid as pH 6.6.

Buffers

Although addition of a minute amount of hydrochloric acid, or any
other strong acid, to water produces relatively a great change in pH,
biological fluids, in general, do not undergo a comparable change when
strong acid or base is added to them, because of the presence of certain
compounds in these fluids. Such compounds which resist change in
acidity or basicity are known as buffers.

In general, a buffer consists of a weak acid (or base) and its salt.
The buffer is the mixture of the two substances. Examples are acetic
acid — sodium acetate, carbonic acid — sodium bicarbonate, ammonium
hydroxide — ammonium chloride. Frequently the second hydrogen of a
di- or tri-basic acid serves as the weak acid, as in the buffer NaHoPOi —
Na^HPOi. Other metals, such as potassium, are equally satisfactory
in buffers provided they form water-soluble salts with the acids concerned.

Buffers exert their effect through chemical reactions that use up most
of the hydrogen or hydroxyl ions that are added. This action depends,
fundamentally, on the fact that the weak acid (or base) is only slightly
ionized. A weak acid HA ionizes according to the equation:

HA?^H++A-

The A~ here represents the acid radical. Since this ionization is a
reversible process, the addition of extra hydrogen ions shifts the reaction
back to the left (law of mass action) and thereby converts most of
the added H+ into undissociated HA molecules. On the other hand, if
a strong base is added to the buffer, the 0H~ ions react with H+ to form
water, and more of the HA molecules ionize to replace most of the H +
ions used. In either case the pH remains relatively constant.

The exact pH of any individual buffer solution and the pH change
resulting from the addition to it of a certain quantity of strong acid
or alkali may be calculated readily from a knowledge of the dissociation
constant of the weak acid or base in the buffer. The mathematical
expression for the dissociation constant Ka of a weak acid is based on the
equation for its ionization. It is:

r. _ [H+] ' [A-]

[HA]

ACIDITY

171

The brackets indicate concentrations expressed on a molar basis. Since
weak acids ionize to only a slight degree, the numerical values of[H+]
and [A-] are small, whereas [HA] is large. Consequently K^ for weak
acids is a small number, for example, 0.000018 in the case of acetic
acid. The weaker the acid, the smaller is its Ka value, and vice versa.
Strong acids like hydrochloric are considered to be completely ionized
in water solution and, therefore, have no, or more exactly an infinitely
large, Ka value.

Considerations exactly similar to those set forth above apply also to
weak bases. The corresponding expressions are:

BOH^B+ + OH-

and

^ [B + ] • [0H-]
•^ [BOH]

To illustrate how pH values of particular buffers may be calculated,
several typical problems will be worked out.

Problem 1. What is the pH of a O.IM acetate buffer solution?

The phrase "O.IM acetate bulfer" means that both acetic acid and sodium
acetate are present in O.IM concentration. The ionization equation for acetic
acid is:

CH3COOH ?^ H+ + CH3COO-

and its dissociation constant has the numerical value 1.8 X 10~^. The pH may
be found from the expression for the dissociation constant :

[H + ] • [CH3COO-]

A% = 1.8 X 10-s

[CH3COOH]

by inserting the proper values for [CH3COO-] and [CH3COOH], and solving
for [H+].

Let X=[H+]. Then [CH3COOH] = (0.1 - X), since the original acetic
acid concentration was O.IM. The concentration of acetate ions is the sum
of the concentrations resulting from the ionization of both acetic acid and sodium
acetate. That from the acetic acid is obviously X, while that from sodium
acetate is 0.1, because such salts are strong electrolytes and are completely ionized
in aqueous solution. Therefore, [CH3COO-] = U^l + A').

Substituting these values in the expression for K^we have:

, X ' (0.1 + A^)

1.8x10-== ^p,_^^'

Now X is very much smaller than 0.1, since we are dealmg with a slightly
ionized acid, so (0.1 + A) and (0.1 - A") are both very nearly equal to 0.1.
Making this substitution, we have as a close approximation :

, 0.1 A
1.8x10-5 = -^

or A = 1.8 X 10-5

172 ACIDITY

Since the pH is the negative logarithm ^ of the molar H+ concentration,

pH = - log (1.8 X 10-5)

= - log 1.8 4- (-log 10-5)

= - 0.26 + 5

= 4.74 (Answer to Problem 1)

Note that in this problem [H+] = K^, or pH = pKa. This relation
holds for any buffer where the acid and salt are present in equal amounts.
When different amounts are present, the pH may be calculated from
the equation:

In the case of a base-type buffer, the corresponding equation is:

[base]

[OH-]=E:bX

[salt]

If the composition of the buffer and the numerical value of Kb are known,
[0H-] and, hence, pOH can be calculated. From this result the cor-
responding pH value can easily be found from the relation:

pH + pOH = 14

which holds for any aqueous solution at room temperature.

The use of the above equations in buffer calculations is illustrated
below.

Problem 2. What is the pH of 40 ml. of O.lM acetate buffer to which has
been added 10 ml. of O.liV HCl?

The HCl added amounts to 1 m.e.q. (10 X 0.1), and the buffer originally con-
tained 4 m.e.q. (40 X 0.1) each of acetic acid and sodium acetate. For purposes
of calculation it may be assumed that the 1 m.e.q. of HCl reacts with 1 m.e.q.
of sodium acetate to form 1 m.e.q. of additional acetic acid":

HCl + CHsCOONa > NaCl + CH3COOH

Consequently, the acid : salt ratio of the buffer has been changed from 1 {i.e.,
4 : 4) to 5 : 3. The pH may therefore be calculated by substituting known values
in the equation above:

[H+] = 1.8 X 10-5 x-1

= 3X 10-5
whence pH = 4.52 (Answer to Problem 2)

1 The small letter "p" is used to mean "the negative logarithm of."

2 More precisely, since the HCl, NaCl, and CHgCOONa do not exist as such in
water solution but are 100 per cent ionized at all times, the only reaction which
actually occurs is :

H+ + CH3COO- -» CH3COOH

ACIDITY l'^3

Note that the original pH of the buffer has dropped only 0.22 unit. This
answer shows very clearly the effect of the buffer because if 10 ml. of 0.1 A'' HCl
are added to 40 ml. of plain water, without any buffer present, the resulting H +
concentration is 1 m.e.q. in 50 ml., or 0.02M . Therefore the pH is — log 0.02,
or 1.7, a very much greater drop.

The salt-acid combination is most effective when the salt and acid arc
present in equal molecular proportions. Of course, there is a limit to the
capacity of the buffer to take up hydrochloric acid or sodium hydroxide.
For example, when about 85 per cent of the sodium acetate has been
converted into acetic acid, or vice versa, the limit is close at hand. On
adding more acid or alkali, the pH of the solution changes rapidly, and,
hence, there is little buffer action.

The most widely used buffers are mixtures of sodium or potassium salts
of relatively weak acids and the corresponding free acid. For most
purposes, acids such as phosphoric, carbonic, acetic, and other organic
acids are used. Carbonates, bicarbonates, and phosphates, together
with proteins, form the most important buffers in the body. These main-
tain the pH within very narrow limits even though considerable acid or
base is added.

Measurement of pH

Each hydrogen ion bears an electric charge, and the concentration of
these ions can be measured most accurately by electrometric means.
This method, however, requires the use of relatively expensive apparatus
and an experienced operator. For many dyes there is a particular degree
of acidity at which there is a very definite change in color, and fortu-
nately the various dyes, or indicators, change color at different .hydrogen-
ion concentrations. This fact serves as a basis for a colorimetric method
that is quite simple, as well as fairly accurate. The method consists of
matching the color produced by an appropriate indicator in the unknown
solution with the color of a standard solution of known hydrogen-ion
concentration to which the same indicator has been added. If the color
of the unknown is the same as that of the standard, the hydrogen-ion
concentration likewise must be the same. Standard color charts and
glass discs of appropriate colors that may be substituted for the standard
solutions possess the added advantage of being more permanent than
the solutions.

In Table 7-3 are given the approximate pH values of a number of
biological materials.

174 ACIDITY

Table 7-3

pH values of representative biological materials

Matenal pH Value

Blood, normal limits 7.3-7.5

Blood, extreme limits 7.0-7.8

Enzymes, activity range of

Amylopsin, optimum 7.0

Erepsin, optimum 7.8

Invertase, optimum 5.5

Lipase, pancreatic 7.0-8.0

Malta.se, optimum 6.1-6.8

Pepsin, optimum 1 .5-2.4

Trypsin, optimum 8-9

Fruit juices

Apple 3.8

Banana 4.6

Grapefruit 3.0-3.3

Orange 3.1-4.1

Tomato ^' 4.2

Ga.stric juice, adult 1.6-1.8

Milk, cows, limits 6.2-7.3

Milk, human 7.0-7.2

Muscle juice : 6.8

Plants (extracted juice)

Alfalfa tops 5.9

Carrot 5.2

Cucumber 5.1

Peas, field 6.8

Potato 6.1

Rhubarb; stalks 3.4

String beans 5.2

Saliva 6.2-7.6

Sweat 4.5-7.1

Tears : 7.2

Urine, human, limits* 4.2-8.0

REVIEW QUESTIONS ON ACIDITY

1. Explain the difference in meaning of "active" and "total" acidity. In what
terms are concentrations of the two usually expressed?

2. Define: (1) molar solution, (2) normal solution,^ (3) hydrogen equivalent, (4)
gram equivalent weight, (5) indicator.

3. What is the normality of a solution of NaOH if 25 ml. of it are required to
neutralize 20 ml. of O.IA'^ oxalic acid?

4. What is the normality of a solution of acetic acid which contains 0.3 g. of this
reagent in 50 ml.?

5. What volume of 0.5iV NaOH would be required to neutralize the acetic acid
mentioned in the preceding question?

6. What is a hydrogen ion? Represent by equations the ionization of (1) HNO3,
(2) NaCl, (3) ClicHOHCOOH, (4) NaOH, (5) H2SO..

ACIDITY 175

7. What is a buffer? Write equations to illustrate the reaction of a buffer with
HCI and XaOH, respectively.

8. What do pH 5, 7, and 9 mean with respect to acidity, neutrality, and alkalinity?

9. How much more acid is the first member of the following pairs than the second
member: pH 6 vs. 7, pH 4 vs. 7, pH 4.2 vs. 6.3, pH 4 vs. 8.3, pH 2 vs. 10?

10. Give the approximate pH values of six representative biological materials.

11. Give the names and structural formulas of ten organic acids (other than fatty
acids) and five organic bases commonlj' found in biological materials.

REFERENCES AND SUGGESTED READINGS

Bonner, James, Plant Biochemistry, Academic Press, Inc., New York, 1950.

Clark, W. M., The Dctermiimtioi of Hydrogen Ions, 3rd ed.. The Williams and
Wilkins Company, Baltimore, 1928.

Kolthoff, I. M. and Laitinen, H. A., pH and Electrotitratiovs, 2nd ed., John Wiley
and Sons, Inc., New York, 1941.

La Motte, F. L., Kenny, W. R., and Reed, A. B., pH a)td Its Practical Application,
The Williams and Wilkins Company, Baltimore, 1932.

Pierce, W. C. and Haenisch, E. L., Quantitative Analysis, 3rd ed., John Wiley antl
Sons, Inc., New York, 1948.

Schmidt, C. L. A. and Allen, F. W., Fundamentals of Biochemistry, 1st ed., McGraw-
Hill Book Company, Inc., New York, 1938.

Chapter 8

BIOCHEMICALLY IMPORTANT MINERAL

ELEMENTS

Definition

The mineral elements of biochemical interest are all those chemical
elements, except carbon, hydrogen, oxygen, and nitrogen, which are, or
may be, present in the tissues of living organisms. They are frequently
called inorganic, or ash elements, since, as a rule, they remain in the
ash when biological materials are burned. Those which have been
proved to be essential constituents of living tissues are listed below.
They are classified as major and minor (or trace) elements on the basis
of the amounts usually present in biological samples.

Major mineral elements

Metals

Nonmetals

Sodium

Sulfur

Potassium

Chlorine

Calcium

Phosphorus

Magnesium

Minor mineral elements or trac

Metals

Nonmetals

Iron

Iodine

Copper

Boron

Cobalt

Zinc

Manganese

Molybdenum

Most of these elements are required by living organisms, generally.
However, boron is needed only by plants; sodium, chlorine, iodine, and
cobalt, only by animals. Thus plants require 15 chemical elements in
all (counting C, H, 0, and N), and animals 18.

Aluminum, vanadium, arsenic, bromine, fluorine, silicon, and other
mineral elements are widely distributed in living cells and may have
important biological functions, but as yet their essential nature has not
been demonstrated, except in a few isolated cases {e.g., vanadium is
apparently a normal, necessary constituent of a respiratory pigment in

176

BIOCHEMICALLY IMPORTANT MINERAL ELEMENTS 1'^'^

certain marine worms). Still other elements, for example, selenium, are
found only in the tissues of plants or animals grown in certain restricted
localities. Some of these elements which are of interest for one reason
or another are discussed in more detail below.

Ashing

When it is desired to examine a biological sample for mineral elements,
the first step is to dry the sample, then burn it to remove organic matter,
and convert the mineral elements present into simple inorganic com-
pounds. From the chemical standpoint the process of burning or ashing
is essentially a very vigorous oxidation, which is carried out in the air
at a temperature of about 600-800°C. The organic substances present
are decomposed as the sample is heated and turn black on account of the
formation of free carbon. As the heating continues this carbon is oxi-
dized to carbon dioxide, which escapes. Disappearance of the black
color, therefore, indicates that the ashing is complete. The hydrogen in
the original organic matter is converted to water vapor, and the nitrogen
escapes in tlie form of nitrogen gas.

The mineral elements are contained in biological materials partly in
complex organic combinations such as sulfur in methionine, phosphorus
in lecithin, iron in hemoglobin, etc. (see Table 8-2). As these organic
substances are destroyed during the ashing process, the mineral elements
in them combine with each other — metals with nonmetals — and fre-
quently also with oxygen to form inorganic salts such as the chlorides,
sulfates, phosphates and silicates of sodium, potassium, calcium, and
magnesium. The ash, then, consists largely of these salts.

If the sample happens to contain relatively more metals than non-
metals, as in vegetables, fruits, milk, only a part of the metals present
can be converted into such salts because there will not be enough
nonmetals to go around. In this case the excess metals combine with
oxygen or carbon dioxide, which is always available from the burning
organic matter, to form oxides and carbonates. Sodium and potassium
form the carbonates, and calcium and magnesium the oxides, since their
carbonates are unstable at the high temperatures used. Such ash,
therefore, is strongly alkaline and, because of the carbonates present,
effervesces when dissolved in mineral acid.

On the other hand, if the sample contains a larger amount of nonmetals
than metals, as in meats, cereals, eggs, the excess nonmetals will be
converted into the corresponding oxides, most of which are volatile, and
therefore escape {e.g., SO2) . Thus, 99 per cent of the sulfur in rice and in
corn meal is lost during burning. Silicon is an exception since its oxide,
SiOo, is very nonvolatile. This loss may be prevented by adding to the
sample before ashing a reagent which will shift the balance of metals

1'78 BIOCHEMICALLY IMPORTANT MINERAL ELEMENTS

versus nonmetals in favor of the metals. The oxides, peroxides, hy-
droxides, carbonates, or nitrates of sodium, potassium, calcium, or
magnesium are suitable for this purpose. The peroxides or nitrates are
especially useful because they are also strong chemical oxidizing agents
and help to complete the ashing in a shorter time or at a lower tem-
perature.

Once the ash has been obtained it is usually taken up in an acid solu-
tion such as nitric, the insoluble silica filtered off, and the solution tested
for metallic and nonmetallic ions by the usual methods of qualitative
analysis.

Occurrence

The amount of inorganic material contained in foodstuffs varies with
the material, and in the case of plants with the type of soil, fertilizer,
etc. Approximately 4.4 per cent of the total weight of the body consists
of inorganic compounds. The bones contain from 22 to 82 per cent,
whereas the muscles and the body fluids contain about 1 per cent. Plant
material varies in ash content from 1 to 10 per cent. More is contained
in the stems and leafy portions of the plants than in the seeds.

Although the concentration of any element in a given food material
may vary markedly, depending upon the conditions under which it is
produced, the table given below and the tables in the Appendix afford
a fairly accurate estimate of the relative concentrations of these elements
in various foodstuffs. In Table &-1 note the low calcium content of
cereals in comparison with the large demands for this element in the
animal body. Observe that the milling process results in a concentra-
tion of the mineral elements in the bran and, hence, a depletion of the
same in the flour.

Table 8-1

Mineral composition of some typical foodstuffs

(Fresh basis)

percentage of: Ca Mg K Na P CI S Fe

Wheat, whole 0.055 0.163 0.409 0.106 0.342 0.088 0.175 0.006

Wheat, flour, white .021 .021 .137 .053 .096 .079 .155 .0012

Wheat, bran 065 .420 1.25 .007 1.43 .042 .245 .014

Corn meal 016 .084 .213 .039 .152 .146 .111 .0011

Cabbage 054 .016 .217 .038 .031 .034 .074 .00066

Turnips .042 .019 .193 .104 .032 .054 .048 .00061

Cow's milk 123 .019 .129 .047 .088 .114 .031 .00024

Animal body (Ox). 1.24 .030 .117 .089 .682

More extensive tables are given in the Appendix. The table on trace
elements (p. 443) shows the small amounts contained in foodstuffs. Of
these elements iodine is the least abundant. It should be noted that

BIOCHEMICALLY IMPORTANT MINERAL ELEMENTS

179

Element
Potassium

Calcium

Table 8-2

Some specific organic compounds of mineral elements
known to exist in plant or animal materials

Compound name and formula

Acid salt of tartaric KHC4H4OU

acid

Salts of citric acid K.HCbH.Ot

Salts of malic acid KHCJH4O5

Acid salt of tartaric Ca(HC4H40o)2

acid

Salts of phytic acid C6Ha(CaP04)a

Magnesium

Iron

Sulfur

Phosphorus

Chlorine

Iodine

Copper

Cobalt
Zinc

Calcium caseinate Not known

Salts of phytic acid CflH6(MgP04)6

Chlorophyll

Hemin

Ferratin

Cystine

Glutathione

Insulin

C55H72N4Mg05

C34H3.N4Fe04Cl

Not known

C6H12N2S.O4

CioHirNsSOe

(C45H69NuS0l4)n

Thiamine chloride C12H17N4SCI

Allyl isothiocyanate
Allyl sulfide

C3ILNCS

(CsIDsS

Lecithins

Cephalins
Nucleic acids

e.g., C44H88NPO9

e.g.,
e.g.,

Phosphoproteins
Hexosemonophos-

phate
Hexosediphosphate
Phytic acid

Creatine phosphate
Cliloromycetin

Thyroxine

Hemocyanins

Vitamin B12
Carbonic anhydrase

C41H80NPO8

(_/2[>xl45a\ EPU26

Not known

CeHn05(H2P04)

CcH4„04(H.oP04)2

C«Hc(H2P04)«

C4H10N3PO5
CnHisOoNsCU

Ci6HnL04N

Not known

C61-«4ll86-92N 14O13PCO

Not known

Contained in
Grapes, cucumbers

Fruits, vegetables
Fi-uits, vegetables
Grapes

Bran of wheat, rye,

etc.
Milk
Bran of wheat, rye,

etc.
Green plants
Hemoglobin of blood
A protein of spleen

and intestinal wall
Proteins
Animal tissues
A hormone, secreted

by Isles of Langer-

hans
Yeast, pork muscle,

etc.
Mustard, onions
Garlic, radishes, cab-
bage, turnips, etc.
Egg yolk, brain,

nerves, etc.
Blood
Nuclear tissue, e.g.,

thymus
Egg yolk, milk

Yeast, muscle

Yeast

Bran of wheat, rye,
etc.

Muscle

Streptomyces Vene-
zuela

A hormone secreted
by thyroid gland

Respiratoiy protein in
lower animals {e.g.,
lobster)

Animal tissues

Red blood cells

180

BIOCHEMICALLY IMPORTANT MINERAL ELEMENTS

the unit (micrograms) in which the iodine is expressed is only one-thou-
sandth as large as that used for the other elements. Cereals, as a class,
contain relatively high percentages of all these elements, but it must be
borne in mind that these high values are due largely to the high per-
centage of dry matter in cereals. Conversely, vegetables do not rank
as high on a percentage basis because of the large amount of water
contained in them. Perhaps a more correct basis for comparison would
be percentages of the foods as eaten, that is, in the cooked condition.
Differences resulting from water content would then be largely elimi-
nated.

A second consideration that must be kept in mind is the quantity of
a particular food that is eaten. A food may be conspicuously high in
some element, but if it is eaten only occasionally, it contributes very little
toward the actual supply of that element in the diet. Lobster, for ex-
ample, is high in copper (1.5 mg. per 100 g.), but since lobster is eaten
rarely or not at all by the majority of people, it is a relatively unimportant
factor in supplying copper to the average diet. Another -example will
illustrate the converse situation. Although milk is low in iron, it is, be-
cause of the large quantity consumed, one of the largest contributors of
iron in the diet of a small child.

On the basis of both composition and consumption, cereals and vege-
tables are the chief sources of supply of the trace elements.

The mineral elements exist in living tissues partly in the form of in-
organic ions (K+, Na+, Ca+ + , Mg+ + , Fe+ + , C1-, SO4 — , HPO4 — ,
H2P04~, etc.) dissolved in tissue fluids, and partly as components of
various organic molecules. Many of the latter are of particular bio-
logical importance and are therefore given special attention. In Table
8-2 are a few organic compounds that are known to exist in plant or
animal materials.

Unusually large or small amounts of particular mineral elements occur
in the soil in various regions of the world, and this distribution often
causes the vegetation and drinking water in these areas to contain
correspondingly high or low amounts of the elements concerned. As a
result, domestic animals and human beings consume abnormal quantities,
frequently with serious, and even fatal, consequences. The best known
cases involve deficiencies of copper, cobalt, zinc, boron, and iodine in
some localities, and dangerously high concentrations of selenium, molyb-
denum and fluorine in others. These cases will be discussed below in
greater detail in connection with the individual elements. In general,
quantities of the mineral elements much larger than the physiologically
required amounts are likely to be toxic. This is particularly true of the
trace elements.

BIOCHEMICALLY IMPORTANT MINERAL ELEMENTS

181

General functions

Considerable importance has been attached by certain investigators
to a proper balance in the diet between basic and acidic elements. Such
emphasis is based on the idea that an excess of either type is not desir-
able. As a result of metabolism the basic and acidic elements will be
combined to form salts in much the same way that these elements com-
bine when the foodstuff is burned to form salts in the ash. To illustrate
this result more clearly: the sulfur of cystine and the magnesium of
chlorophyll may, as a result of metabolism, be combined and excreted as
magnesium sulfate. Since in the body a number of organic acid radicals,
e.g., the citrate and malate, may be oxidized completely to carbon dioxide
and water, the corresponding metallic salts frequently contribute only
to the supply of basic elements in the diet. It is for this reason that
most fruit juices, in spite of their actual acidity, exert a basic effect in
the body. A few organic acids, however, are not so oxidized and, hence,
contribute to the total acidity (which normally is caused by the three
elements, chlorine, phosphorus, and sulfur). In this class are benzoic and
quinic acids. The presence of these acids in cranberries, plums, and
prunes is responsible for the acidic effect of these fruits in the body.

In order to determine whether there is an excess of basic or acidic
mineral elements in foodstuffs, the quantities of each are expressed in
terms of the equivalent volume of O.LV base or acid per 100 g. of the
food. If the basic elements together are equal to a larger volume of
O.LV solution than the total of the acidic elements, the foodstuff is said
to be basic, and vice versa; e.g., 100 g. of beef will contain 120 ml. of O.LV
acid in excess of the basic elements. Milk, on the other hand, contains
22.5 ml. excess of base over acidic elements. Fruits, vegetables, milk,
and legumes contain an excess of basic elements, while cereals, meat, and
eggs have a preponderance of the acidic elements. Certain investigators
have assumed that people naturally combine foodstuffs high in basic
material with foodstuffs high in acidic compounds. It has been suggested
that perhaps we eat vegetables with meat for this reason. To have an
exact balance between the two may not be as important as has been
assumed, but it is probable that a large excess of one over the other is
not a desirable condition.

The remarkable constancy of the pH of blood, 7.3-7.5, is made possible
through the systems of buffers contained therein. In addition to the
proteins, especially hemoglobin, the chief buffer systems are the car-
bonates, H2CO3 and NaHCOs, and the phosphates of potassium and
sodium, e.g., NaH2P04 and Na2HP04.

The osmotic pressure existing between body cells is due in part to the
presence of salts of the mineral elements. The movement of liquids from

182

BIOCHEMICALLY IMPORTANT MINERAL ELEMENTS

one part of the cell to the other or through the walls of a cell may be
brought about by the presence of these salts. If a high concentration of
salts exists in a given locality, water tends to flow in that direction to
dilute the solution, while the salts tend to move in the direction of the
water, thus equalizing conditions. Similarly, the water-holding capacity
of the blood is in part due to this action of its inorganic salts.

Irritability of muscles and nerves, that is their ability to respond to
physiological stimulation in a normal manner, is dependent upon the
proper kind and amount of inorganic ions in the body fluids which bathe
them. Many enzymes are inactive unless some particular inorganic ion
is present. Examples are given below and in Chap. 10.

Functions of specific elements

Sodium and Potassium. These elements exist in living tissues almost
exclusively in the form of Na+ and K+ ions. They make up the basic
portion of several buffer systems which maintain the physiological pH
values not only of the blood, as mentioned above, but also of such body
fluids as saliva and the pancreatic and intestinal juices. Potassium
ions constitute the main base inside the cells of the body, whereas sodium
is characteristically more concentrated in the blood plasma and in-
terstitial fluids, that is, tissue fluids outside the actual cells. Sodium
ions, in fact, make up over 90 per cent of all the cations (positively
charged ions) of these fluids (Table 8-3) and thus, together with Cl~
ions, are mainly responsible for their osmotic pressure.

Table 8-3
Approximate electrolyte distribution in human blood plasma *

Cations Anions

(Milliequivalents per liter of plasma water)

Sodium (Na+)

154

Chloride (CI")

106

Potassium (K*)

5

Bicarbonate (HCO3-)

28

Calcium (Ca*^)

5

Protein

17

Magnesium (Mg*"^)

3

Others

17

* Data from Hawk, Oser, and Summerson.

Sodium and potassium ions play a vital role in the process by which
carbon dioxide is carried by the blood stream from the muscles, where
it is produced, to the lungs, where it is eliminated from the body (see
p. 187). Both tend also to promote muscle relaxation.

Common salt, sodium chloride, supplies most of the sodium and
chlorine in the diet. The daily salt requirement for normal adult per-
sons is in the neighborhood of 5 g., but the exact amount needed depends
on the water intake, because salt is carried out of the body in the urine

BIOCHEMICALLY IMPORTANT MINERAL ELEMENTS

183

and, especially, in sweat. The average American diet furnishes 10-15 g.
of salt a day, which is more than adequate, except during profuse sweat-
ing. An extra gram of salt (above the 5 g. minimum) should be con-
sumed for each liter of water intake in excess of 4 1. per day. This may
amount to as much as 20-30 g. in extreme cases. So-called heat prostra-
tion after hard work in hot weather is often merely the result of un-
compensated salt losses and may be prevented by proper attention to
salt intake.

Among the symptoms characterizing potassium deficiency in rats are
lethargy, distension of the abdomen, edematous kidneys, discoloration of
the skin owing to improper blood supply, failure of growth, and early
death. Pathological changes may be detected in the intestines, pan-
creas, kidneys, and hearts of such animals.

The potassium requirement for human beings is unknown, but the
element is present in nearly all foods in such large amounts that the
daily intake appears to be entirely adequate.

Calcium and Phosphorus. Both of these elements are essential con-
stituents of all living cells. Calcium is present in the animal body in
larger amounts than any other mineral element. About 99 per cent of
the total is in the bones and teeth, which are made up of approximately
one-half moisture and organic matter and one-half inorganic or mineral
matter. The latter consists essentially of calcium phosphate together
with smaller amounts of calcium carbonate. IMagnesium and other ele-
ments are also present in minor amounts.

The remainder of the calcium exists mostly as Ca++ ions in the body
fluids, where it is of fundamental importance for the normal activity of
nerves, muscles, and heart, for the clotting of blood, and for maintaining
the permeability of cell membranes. Thus the clotting of freshly drawn
blood may be prevented, or greatly retarded, by the addition of a
reagent, such as sodium oxalate or citrate, which removes the Ca+ +
ions by forming an insoluble (oxalate) or nonionized (citrate) product.
Coagulation of milk also requires calcium, and the cementing substances
which hold cells together in tissues appear to involve this element.
Normally, the total calcium content of man's blood ranges from 9 to 11
mg. per 100 ml. of plasma. In the young it is slightly higher. About
half the total is inorganic Ca++ ions. If the blood calcium falls below
certain levels, depending upon the species of animal, tetany (generalized
spasmodic muscle contractions) results, and death may follow unless
restorative measures are employed. Both vitamin D and the secretion
of the parathyroid gland operate in controlling the calcium content of
the blood, the former through an increased "net absorption" of food
calcium and the latter by mobilization of the calcium in certain labile
structures of the skeleton.

The bulk of the phosphorus of the body, about 80 per cent, is also

184 BIOCHEMICALLY IMPORTANT MINERAL ELEMENTS

contained in the bones and teeth. About half the remainder is combined
with the organic constituents of the muscles, while the rest is distributed
throughout the blood and other tissues of the body. Mention was made
above of the buffer action of the alkali phosphates contained in the blood,
and it will be recalled that phosphorus is also a constituent of various
organic compounds previously studied, such as lecithins, certain proteins,
and nucleic acids, as well as a whole series of substances involved in
intermediary carbohydrate and fat metabolism and in muscle contrac-
tion (Chaps. 10 and 16). AVithout a supply of phosphorus none of these
essential materials could be formed by the living cell, so it is easy to
understand why this element is vital to normal health and development.

The amounts of calcium and phosphorus needed daily by human beings
depend on many factors, one of which is the Ca : P ratio in the food
eaten. When either element is consumed in a large excess, the excretion
of the other is increased, so it is desirable that this ratio be about 0.7-1.0.
In other words, the food eaten should contain about equal quantities of
calcium and phosphorus, or slightly more of the phosphorus. Further-
more, unless enough vitamin D is furnished (see p. 211) absorption of
calcium through the intestinal wall is greatly reduced.

A third factor affecting calcium requirements is the presence in the
food eaten of oxalic acid, or soluble salts of oxalic acid, which produce
insoluble calcium oxalate. This compound is not utilized as a source
of calcium in the body. The concentration of soluble oxalates in some
plants reaches toxic levels (see p. 161).

For these and other reasons there is a tremendous variation among
individuals in their ability to make use of dietary calcium, some ab-
sorbing as little as 5 per cent of the amount eaten. There is evidence,
however, that the efficiency of utilization increases when the calcium
content of the food is low and the needs of the body are acute.

The amounts of various dietary essentials that should be supplied
daily by a good diet have been carefully studied by the Food and
Nutrition Board of the National Research Council. Their recommended
daily allowances, which are designed to provide an excess over the bare
minimum requirements for life, were announced in 1941 and revised in
1948. For calcium the recommended amounts are: adults 1.0 g., preg-
nancy (latter half) 1.5 g., lactation 2.0 g., children up to 10 years 1.0 g.,
adolescents 1.0 to 1.4 g. The phosphorus intake should be at least
equal to that of calcium for children, adolescents, and women during
pregnancy and lactation, and for other adults about 1.5 times the calcium
intake.

Calcium is the one essential mineral element which is most likely to
be supplied in inadequate amounts by the average American diet. The
reasons for this situation are the facts that two of the principal types
of foodstuffs, meats and cereals, are notably deficient in this element and

Courtesy of O. H. Sears, Univ. of Illinois
A. S\inpU)nis oi nitrogen deficiency appear in nninoculated soylicans growini; in a soil which
does not contain the appropriate nitrogen-fixing nodule bacteria. Note the pale green to
vellowish color and the lower height of the plants in the nninoculated check strip.

V>. Coltoii boil showing potassinm-deficicnc \ sMuplonis. Left, normal, large, well oj)encd boll.
Riglu. small, inniialure, ])artlv o[)enecl Ixill icsniiing liom a deficiency of potassium.

Plate I. Nitrogen and polassjnm deficiencies in planis.

^-^^■'■^

Plate II. Phosplioriis luiiigcr causes purpling of ihe leaves of many strains of corn.

BIOCHEMICALLY IMPORTANT MINERAL ELEMENTS

185

that foods, in general, contain far less calcium than the animal body
(Table 8-1). Milk is one of the best food sources, a quart supplying
approximately one gram of both calcium and phosphorus. Cheese, egg
yolk, and green leafy vegetables are also excellent sources; most other
vegetables contain moderate amounts. Ground bone is well utilized by
the animal body as a source of calcium and phosphorus, and, in fact,
bone meal is commonly used as a supplement to the feed of domestic
animals. The bones in most types of canned fish should be regarded as
valuable food rather than being carefully picked out and thrown away
as is usually the case.

On the other hand, the phosphorus supply needs no special attention.
A diet providing the recommended amounts of protein and calcium will
almost certainly contain sufficient phosphorus, at least for human beings.
Cattle, however, occasionally become phosphorus deficient, but only
when their feed consists almost entirely of forage grown in certain areas
where the soil is low in this element (e.g., parts of Wisconsin, Montana,
Texas, and Florida).

Diets inadequate or imbalanced in their calcium, phosphorus, or vita-
min D contents lead to rickets in children and to osteomalacia (softening
of the bones) in adults (see chapter on Vitamins). All three factors are
also obviously essential for sound, well-formed teeth, although many other
influences likewise play important roles in dental health.

Magnesium. This element is also an indispensable constituent of all
living cells. Since it is a part of the chlorophyll molecule (p. 388),
magnesium is essential for photosynthesis and, hence, indirectly respon-
sible for the production of all our foodstuffs. About three-fourths of
the magnesium in the animal body is contained in the skeleton, the rest
being present in the blood and other body fluids as Mg++ ions. The
normal concentration of Mg++ in human blood serum is 2-3 mg. per
100 ml. Marked increases of up to 10-20 mg., resulting, for example,
from the injection of soluble magnesium salts, lead to generalized anes-
thesia, complete muscle relaxation, and eventual death. If animals or
human beings are deprived of magnesium until the blood level falls to
0.6-0.8 mg. per 100 ml., or less, symptoms of magnesium deficiency
appear. These include dilation of capillaries, extreme nervousness, con-
vulsions (tetany) , and death.

Magnesium ions are necessary for the normal activity of several
enzymes in the body, particularly peptidase, carboxylase, enolase, hexo-
kinase, and others. In some cases the Mg++ may be replaced by other
ions, such as Mn+ + . However, for many biological functions there
exists marked antagonism between various inorganic ions. Calcium and
magnesium offer an outstanding illustration of this antagonism. Thus
the enzyme adenosine triphosphatase, which is activated by Ca++ ions,
is inhibited by Mg+ + . Likewise Ca++ is the best antidote for the

186 BIOCHEMICALLY IMPORTANT MINERAL ELEMENTS

anesthesia and paralysis of animals caused by excess Mg++ ions in the
body fluids.

The magnesium requirement of human beings is not known with
certainty, but it has been estimated to lie in the range of 0.2 to 0.4 g.
per day for a normal adult. Studies of American dietaries indicate that
the daily magnesium intake per 3000 calories varies from 0.17 to 0.53 g.
This amount evidently meets the normal needs since cases of human
magnesium deficiency are almost never encountered.

Iron and Copper. The iron contained in the animal and human body
is mostly present as a component of certain conjugated proteins, of
which the best known is hemoglobin. The iron is contained in the
prosthetic group of hemoglobin, which is called heme. Like chlorophyll,
heme belongs to the porphyrin class of substances and carries its iron
atom in the center of the porphyrin ring.

Iron is also an essential component of several physiologically important
enzymes such as catalase, peroxidase, the cytochromes, and cytochrome
oxidase. Like hemoglobin, these enzymes are conjugated proteins with
an iron-porphyrin type of prosthetic group. The iron content of hemo-
globin and of the above enzymes lies in the range of 0.1 to 0.4 per cent.
There is also present in the animal body another iron containing protein,
ferritin, which, in contrast to the above materials, contains as much as
23 per cent of iron. Ferritin is present in the spleen and in the intestinal
wall, where it is probably involved in the metabolism of iron in the body,
particularly in absorption and storage.

Copper also is known to be associated with certain proteins in living
tissues. It is an essential component of several enzymes and of a re-
spiratory pigment, hemocyanin. This substance is present in the blood
of certain lower animals, for example, the lobster, snail, and other in-
vertebrates, and acts as an oxygen carrier just as hemoglobin does in
higher forms. Hemocyanins from various species contain about 0.2 to 0.4
per cent of copper and range in molecular weight from 350,000 to several
milhon. The copper is easily removed on acidification, being fully
utilized as a source of food copper by animals. It has not been estab-
lished whether the copper is attached to a prosthetic group of the
porphyrin type. Although this might be expected by analogy with
hemoglobin, the easy removal of the metal argues against this possibility,
and no porphyrin derivative has been obtained from hemocyanin. How-
ever, turacin, a feather pigment of the turaco bird (South Africa), is a
copper-porphyrin derivative.

The copper-containing enzymes include ascorbic acid oxidase, poly-
phenol oxidase, laccase, and several other oxidases. Each of these en-
zymes is a protein which contains a small amount of copper, ranging
from 0.15 to 0.34 per cent, as an integral part of the molecule. The
blood of the ox, sheep, and horse has been found to contain another

BIOCHEMICALLY IMPORTANT MINERAL ELEMENTS

187

copper protein, hemocuprein, which has been purified and obtained in
the form of blue crystals containing 0.34 per cent of copper. The copper
in hemocuprein and in a similar substance, called hepatocuprein, in the
liver accounts for nearly all the copper in the bodies of higher animals and,
presumably, also in human beings.

The function of iron in the body is to serve as one of the essential raw
materials for the various physiologically important iron-containing sub-
stances listed above. Given an adequate dietary supply of iron, the
body can synthesize whatever amounts of hemoglobin and the iron-
porphyrin enzymes it may need, provided, however, that sufficient amounts
of copper are also supplied by the food intake. Just how copper func-
tions in this regard is not understood, but it is an observed fact that
copper is needed for the proper absorption and utilization of iron. It
is not surprising, therefore, that diets low in either copper or iron lead
to the development of nutritional anemia, that is, an abnormally low
amount of hemoglobin in the blood, which is corrected by consuming a
complete diet. An average family diet contains about 2 mg. of copper
per person per day.

The tissues of the body are dependent almost entirely upon hemo-
globin for their oxygen supply. In the lungs, where relatively large
amounts of oxygen are available from the inhaled air, hemoglobin is
converted into oxyhemoglobin. The oxygen is held in a rather loose
combination and is easily given off' whenever the oxyhemoglobin reaches
a place w4iere the prevailing oxygen pressure is low, that is, where
little oxygen is present. This occurs normally, of course, in the muscles.

Hemoglobin also functions indirectly in the transportation of carbon
dioxide to the lungs. Oxyhemoglobin is a stronger acid than hemoglobin
itself and when it is formed in the lungs combines with a certain amount
of potassium. As the oxyhemoglobin changes to hemoglobin in the
muscles, this potassium is released and combines with carbon dioxide to
form potassium bicarbonate. This substance is then carried back to
the lungs where the potassium is taken up by freshly produced oxyhemo-
globin, and the bicarbonate radical becomes free carbonic acid. The last
step in the process of eliminating carbon dioxide involves the decomposi-
tion of this newly formed carbonic acid. Here still another mineral
element, namely zinc, plays an essential role, since it is a component of
carbonic anhijdrase, an enzyme, present in red blood cells, which greatly
speeds up the breakdown of carbonic acid:

carbonic

H2CO3 < * H2O + CO2

anhydrase

As a result of the action of this enzyme, carbon dioxide is released in
gaseous form and exhaled as rapidly as it is brought to the lungs.

The iron-containing enzymes, such as cytochrome, are very widely

188

BIOCHEMICALLY IMPORTANT MINERAL ELEMENTS

distributed in both plant and animal cells, and in the various lower forms
of life, where they are concerned with biological oxidation processes (see
Chap. 13). A good daily allowance of iron for human beings has been
estimated by the Food and Nutrition Board to be as follows: normal
adult, 12 mg.; woman during pregnancy and lactation, 15 mg.; infants,
6 mg.; children 1 to 12 years old, 7 to 12 mg.; and adolescents 13 to 15
years old, 15 mg. The need for iron is, of course, greatest whenever
hemoglobin is being formed in the body in relatively large amounts, as
during rapid growth and after the loss of blood. For this reason women
should have more iron than men to compensate for blood lost during
menstruation. In fact, there is evidence that adult men, and women
after the menopause, get along quite satisfactorily even when they receive
much less than the above amounts of iron.

An adequate copper intake is provided by an amount equal to about
one-tenth that of iron. For adults, this is in the range of 1 to 2 mg. per
day. A good diet containing enough of the other essential food factors
may be depended upon to contain sufficient copper, except in a few areas
where the copper content of the soil is abnormally low. Such copper-
deficient areas have been reported in Holland, Florida, New Zealand,
and parts of Great Britain and Australia.

Iron, on the other hand, is one of the mineral elements which is apt to
be supplied in too small amounts by ordinary dietaries. This is partly
due to the modern process of refining cereals, which removes much of
the iron (see Table 8-1 ) , and partly to the fact that the iron contained
in many foods is not well assimilated by the body. Thus the iron in
the iron-porphyrin substances listed above is not utilized, although most
simple, inorganic iron salts, and even metallic iron itself if finely divided,
are able to meet bodily needs very well. It is important, therefore, to
consider not only the total iron content of various foods, but also the
proportion of it which is physiologically available. The best food sources
of iron are liver and egg yolk. Muscle meats, fish, green leafy vegetables,
and dried peas and beans are also good sources, since they are high in
total iron, and about half of it is available. White flour is low in iron,
but is now being enriched approximately to whole grain levels so that
it probably contributes materially to the total intake. Milk is deficient
not only in iron, but also in copper and manganese.

Cobalt. That cobalt also plays a role in hemoglobin formation is
evidenced by the fact that cattle and sheep in certain areas of New
Zealand, Australia, and Florida sometimes suffer from a nutritional
anemia that can be cured only by administration of small amounts of
this element. Cobalt deficient areas have also been reported in Wisconsin,
Michigan, New Hampshire, North Carolina, Great Britain, and Scotland.
The occurrence of deficiency symptoms is limited to cows, sheep, and
other ruminants. Feeding trials with cobalt-low rations have failed to

BIOCHEMICALLY IMPORTANT MINERAL ELEMENTS

189

demonstrate a need for this element in rats or rabbits but showed that
the requirement, if any, of the rat for cobalt is less than 0.6 /xg. per day.

However, vitamin B12 has recently been found to contain cobalt, and
since it plays a vital role in hemoglobin formation in many species, in-
cluding man, the need for cobalt is evident. Since vitamin B12 contains
only slightly more than 4 per cent of cobalt, and only a few micrograms
of the vitamin are needed daily, it is not surprising that requirement for
this element was difficult to demonstrate.

Manganese. This element seems to be essential for both plant and
animal life, although as yet no specific manganese-containing substance
of biological importance is known. Since plants deprived of manganese
become chlorotic (contain too little chlorophyll), it is probable that the
element plays some role in chlorophyll synthesis (Fig. 8-1). Lack of
iron has a similar result (Fig. 8-2). In animals, manganese deficiency
leads to poor growth, failure of reproduction, and- abnormal bone de-
velopment. Chickens suffer from a condition known as perosis (slipped
tendon), which results from poorly shaped leg bones and is cured by
manganese plus choline. Eggs produced by hens on a low-manganese
ration hatch poorly.

Although the detailed manner in which lack of manganese brings about
these difficulties is not known, it seems most probable that the explanation
will be found to lie in the effect of Mn++ ions on various enzymes.
Arginase is activated by a number of metal ions, of which Mn++ is
probably the most important under natural conditions. Carboxylase
requires either Mg++ or Mn++ for activity. Certain peptidases and
phosphatases also are known to be activated by Mn+ + .

The human requirement for manganese is not definitely known, but it
is estimated to be in the neighborhood of a few mg. per day for the adult.
Whole cereals, and especially cereal brans, are high in manganese. Tea
is an outstanding source (150-900 parts per million in the dry leaves),
contributing several milligrams daily to those who use this beverage.
The daily intake on ordinary American diets has been calculated to be
at least 2.5 mg., which evidently meets all bodily needs, since human
manganese deficiency does not occur, as far as is known.

Zinc. The essential nature of zinc for animal life has been demon-
strated by feeding young rats a ration very low in this element (Hove,
Elvehjem, and Hart). Poor growth and various abnormalities resulted.
It has also been found that about 0.3 per cent of zinc is present in the
enzyme carbonic anhydrase, which occurs in red blood cells, as well as
in the pancreas and stomach lining of animals. This enzyme plays a
vital role in the elimination of carbon dioxide from the body, as ex-
plained above (p. 187) . It has been suggested that carbonic anhydrase
also functions in the secretion of hydrochloric acid by the stomach lining.
Zinc likewise is a constituent of crystalline insulin, although zinc in-

190

BIOCHEMICALLY IMPORTANT MINERAL ELEMENTS

Courtesy of New Jersey Agricultural Experiment Station. Reproduced from
Hunger Sigtis in Crops, a publication of the American Society of Agronomy and
the National Fertilizer Association, Washington, D. C.

Fig. 8-1. Right, manganese deficient leaves showing diminished chloro-
phyll content. Left, normal leaf.

Courtesy of California Agricultural Experiment Station. Reproduced from
Hunger Signs in Crops, a publication of the American Society of Agronomy and
the National P^ertilizer Association, Washington, D. C.

Fig. 8-2. Iron deficiency symptoms on lemon leaves. Left, normal leaf.
The other leaves show increasing lack of iron from left to right.

sulinate is apparently not the active form of this hormone. A combina-
tion of zinc and protamine with insulin is widely used because it acts
much longer than insulin alone.

Plants also require zinc. Fertile soils contain 1 to 5 parts per million

BIOCHEMICALLY IMPORTANT MINERAL ELEMENTS

191

or more. Zinc-deficient soils are well known in the southern United
States, where the lack of zinc causes "rosette" disease of pecan and
cherry trees, "mottled leaf" of citrus fruits, and "bronzing" of tung oil
trees. The stunting effect of zinc deficiency on grapefruit is shown in
Fig. 8-3.

Courtesy of California Agricultural Kxperiment Station. Reproduced from
Hunger Signs in Crops, a publication of the American Society of Agronomy and
the National Fertilizer Association, Washington, D. C.

Fig. 8-3. Grapefruit showing, on the left, the effects of acute zinc defi-
ciency; center, fniit from similar tree two months after treatment, showing
discolored tough areas impregnated with gum in the thick rind; and, on the
right, fruit from a similar tree treated 15 months previously.

The amount of zinc needed by human beings is not well established, but
it has been estimated to be 0.3 mg. per kilogram of body weight for
growing children. Adult persons on an average diet in this country
probably consume about 12 mg. per day, and this amount appears to be
sufficient for all bodily needs. Foods high in zinc include wheat bran,
wheat germ, oysters, liver, egg yolk, cocoa, and others (see Appendix,
Table A-3). In human tissues the highest concentrations (ca 200 ppm.)
are found in the nails and hair.

Sulfur. Sulfur is present in more organic compounds of biochemical
importance than any other mineral element, with the possible exception
of phosphorus. A partial list is given in Table 8-2. Plants have the
ability to manufacture from inorganic sulfates all the organic sulfur

192

BIOCHEMICALLY IMPORTANT MINERAL ELEMENTS

compounds needed for their tissue structures and metabolism. Animals,
on the other hand, must be supplied with several preformed sulfur-con-
taining organic substances. These include methionine, thiamine, biotin,
and possibly others.

Glutathione, a tripeptide of glutamic acid, cystine, and glycine, is
apparently universally present in living tissues, and by virtue of the
ability of the cystine constituent to change from the oxidized to the
reduced state, cysteine, and vice versa, the compound may function in the
oxidation-reduction processes of the tissues.

Chlorine. Chlorine, in the form' of hydrochloric acid, imparts to the
gastric juice the proper acidity for the reaction of the digestive enzymes
found therein. Only a very few organic chlorine compounds have been
discovered in nature. One example is Chloromycetin, an antibiotic.
However, thousands of organic chlorine compounds have been produced
synthetically, and many have important industrial uses.

The chlorine required by animals and human beings is supplied almost
exclusively by common salt. So much salt is ordinarily consumed that
the need for chlorine is amply filled.

Iodine. This element is essential to the proper development and
functioning of the thyroid gland. A lack of iodine results in an enlarge-
ment of the thyroid, a condition known as simple goiter. McLendon
made an extensive study of the distribution of iodine in food and water.
He showed that in the areas where the iodine content of the materials
was low, simple goiter was prevalent. In some regions, over 70 per cent
of the girls of high school age had goiter. It might almost be said that
goiters were as common there, and as lightly regarded, as freckles.
Prophylactic measures — use of iodized salt, addition of iodides to drink-
ing water, distribution of iodine tablets to school children — are now
being adopted in many parts of the United States and are rapidly de-
creasing the incidence of this disfiginung disease. The ocean is the great
source of iodine and its derivatives. Sea foods and foods grown near
the sea commonly contain relatively large amounts, whereas foods coming
from far inland are low in iodine.

A low iodine content in plants and water affects, not only human
beings, but also farm animals. Swine, sheep, and cattle develop en-
larged thyroid glands. The young may be born with little or no hair, and,
if alive, usually die shortly after birth.

It is estimated that the normal human adult requires from 0.15 to
0.30 mg. of iodine daily. Fortunately, the body is able to store this
element quite readily, and the administration of iodide for two to four
weeks twice yearly is effective in preventing the development of simple
goiter. Iodized salt, which also is employed as a prophylactic measure,
normally contains 0.015 per cent of iodine.

BIOCHEMICALLY IMPORTANT MINERAL ELEMENTS

193

Fluorine. Fluorine has not been proved to be an essential element for
either plant or animal life, although it is normally present in many living
tissues. The element tends to be accumulated in the bones and teeth of
animals in the form of the mineral, fluoroapatite, [Ca3(P04)2]3 * CaF2.
Normal bones of animals and human beings contain about 0.02-0.1 per
cent of fluorine, but much higher percentages (up to 1 per cent or over)
have been found in cases of excessive fluorine intake. Much interest has
been aroused in fluorine because of its effect on the skeleton of animals
and man and because of its ability to reduce human caries (tooth decay).

Fluorine is unevenly distributed in the soils of various parts of the
world, being found particularly in phosphate rock and in cryolite (a
sodium aluminum fluoride). The amount in drinking water is likewise
variable, ranging from none to 15 parts per million (ppm.), or more.
In those areas where the water supply contains 1 to 2 ppm., or over, many
people are afflicted with "mottled" teeth, a condition characterized by
soft, chalky areas in the tooth enamel which gradually become discolored
and pitted. The threshold level for mottling appears to be near 1 ppm.,
since this amount of fluorine in the water supply produces mild mottling
in about 10 per cent of the growing children. The first teeth are not
affected in this condition, but the damage is done to the permanent teeth
before eruption.

Larger fluorine intakes, corresponding to about 5 ppm. or more in the
water, lead to thickening and other malformation of the bones — both in
animals and in human beings. Severe malformation of the spine has been
reported among people living in certain areas in India, where about 5 ppm.
of fluorine is naturally present in the water, and has been attributed to
abnormally liigh fluorine intake.

It has been well established that increasing amounts of fluorine (or
more exactly, soluble fluorides) in drinking water up to about 2 ppm.
result in decreased tooth decay in children, provided they have received
such water since infancy. In order to avoid, insofar as possible, any
danger of mottling, the amount of fluorine added to the water supplies
of communities experimenting with this method of controlling tooth decay
is ordinarily such as to raise the level to 1 ppm., but no higher. It is
probable that fluorine acts to reduce caries by inhibition of bacterial
enzymes, possibly by combining with and rendering unavailable such
metals as zinc, cobalt, manganese, copper, calcium, and others, which
activate various enzymes. The fact that fluoroapatite is considerably
harder than normal calcium phosphate may also be a factor in the reduc-
tion of tooth decay by fluorine.

Boron, Molybdenum, and Silicon. The essential nature of boron for
plant growth was discovered in rather recent times. It was first shown to
be needed by corn in 1915, broad beans in 1923, tomatoes and potatoes

194

BIOCHEMICALLY IMPORTANT MINERAL ELEMENTS

in 1928, and still more recently by a long list of agriculturally important
plants. It is now generally recognized as an essential component of
commercial fertilizers for use on low-boron soils. Various plants need
from about 0.1 to 0.5 ppm. of available boron in the soil, and many soils,
particularly in the more humid regions, have less than this amount. As
a result, such crops as sugar beets and alfalfa have frequently suffered
from "diseases," which are now known to be due simply to boron de-
ficiency and are easily controlled by proper fertilization. Some effects of
boron deficiency are shown in Figs. 8-4 and 8-5.

Courtesy of South Carolina Asricultural Experiment Station. Reproduced from
Iluuf/er .S'(V/)i.s in Crops, a publication of tlie American Society of Agronomy and
tlie National Fertilizer Association, Washington, D. C.

Fig. 8-4. Left to right: Ear from healthy boron-treated corn plant, and

three ears from boron-deficient plants. Note one-sided shriveling of kernels

on the deficient ears.

Molybdenum was not recognized as an essential element for plants
until 1939 (Arnon and Stout), but it is now known to be required, for
example, by tomatoes, oats, and barley. It is also needed by such lower
forms as molds (Aspergillm niger) and the nonsymbiotic (free-living)
nitrogen-fixing bacteria {e.g., Azotobacter chroococcum) .

Most soils contain not more than 5 ppm. of molybdenum, but a few
areas are known (in Scotland, California, and Wyoming) where the
amount is as high as 20-200 ppm. Livestock grazing these areas re-
ceive enough of the element from the forage to cause serious poisoning,

BIOCHEMICALLY IMPORTANT MINERAL ELEMENTS

195

which, however, can be alleviated by feeding small amounts of soluble
copper salts.

Although silicon is a normal constituent of many plants, no absolute
requirement for it has been well enough established to be generally
accepted. Silicon tends to be concentrated particularly in fibrous plant
tissues such as wheat straw, cereal brans, rice hulls, timothy hay, and
the like. Such plant materials have a high ash content (3-7 per cent
or more), and from one-fourth to two-thirds or more of the ash consists
of silicates and silicon dioxide.

Courtesy of A. B. Burrell, Cornell University. Reproduced from Hunger Signs
in Crops, a publication of the American Society of Agronomy and the National
Fertilizer Association, Washington, D. C.

Fig. 8-5. Oldenburg apples about five weeks after petal fall, showing
symptoms of both internal and external cork due to boron deficiency.

Selenium. Although this element is not among those needed by living
things, it has attracted much attention because of its presence in the
plants grown in certain localities and its toxic effect on animals consuming
such plants. The so-called "alkali disease" of horses and cattle, known
since pioneer days in parts of Kansas, Nebraska, South Dakota, and
Wyoming, is a chronic form of selenium poisoning. The disease, which
also affects sheep, pigs, dogs, rats, and poultry, follows consumption of
a diet containing 5-10 ppm. or more of selenium. Symptoms include
soreness and sloughing of the hoofs. Fig. 8-6, loss of hair, stiff joints, and
atrophy of the heart and liver. Poor hatching of eggs is one of the
earliest signs in poultry. A more acute form of the disease known as
"blind staggers," resulting from higher selenium intake, is quickly fatal.
The most practical antidote or control measure for range cattle, sur-

196

BIOCHEMICALLY IMPORTANT MINERAL ELEMENTS

prisingly enough, is the feeding of arsenic in the salt supply at a con-
centration of 25 ppm.

Availability of organic and inorganic combinations

Much work has been done to determine whether inorganic forms of
the mineral elements can supply the needs of the body for these elements.
With respect to phosphorus and iron the answer today seems to be clearly
in the affirmative. Moreover, inorganic phosphorus and iron not only

Courtesy of A. L. Moxoii and the South Dakota Auiieultuial Kxperiincut Station.
Fig. 8-6. Hind feet of a cow suffering from selenium poisoning.

can supply the requirements of the body but seem actually to be more
useful than some organic forms of the elements. The iron of hemin
and similar porphyrin compounds is unavailable to the body. In the
case of sulfur it is clearly established that some of the sulfur must be
present in the form of methionine, thiamine, and biotin. These sub-
stances cannot be synthesized from inorganic sulfur, and, hence, a part
of the sulfur supply must be furnished to the body in organic form.
Sulfates and other forms of sulfur, however, are utilized. The observa-
tion that milk is the best available source of calcium probably can be
attributed to the high digestibility of this food rather than to the presence
of a particular calcium compound. Extremely poor utilization of the
calcium of spinach is due, doubtless, to its reaction with the oxalic acid
in this foodstuff to give insoluble calcium oxalate. In general, it may be
said that, with the exception of sulfur as noted above, and probably
cobalt which is needed in the form of vitamin B12 (p. 249), the bodily
needs for the various mineral elements may be met entirely satisfactorily
by simple inorganic salts of these elements.

BIOCHEMICALLY IMPORTANT MINERAL ELEMENTS

197

Excretion

Sodium, potassium, calcium, magnesium, phosphorus, and chlorine are
excreted mainly as inorganic salts in both urine and feces. Those salts
that are readily soluble are excreted chiefly in the urine ; those salts that
are less readily dissolved are excreted largely in the feces. Iron, copper,
zinc, manganese, and cobalt are excreted mainly in the feces.

The excretion of sulfur is almost entirely by way of the kidneys. In
this respect it resembles the excretion of nitrogen. Several forms of
sulfur are contained in the urine. From 60 to 90 per cent of the total
sulfur is excreted in the form of sulfates; from 5 to 15 per cent as ethereal
sulfates; and 5 to 20 per cent as neutral or unoxidized sulfur. Taurine
and cystine are forms of unoxidized sulfur contained in the urine.

Variations and losses of mineral elements in foods

Unless the food that is eaten is analyzed, it is practically impossible to
say whether or not the requirements listed above have been satisfied.
Different samples of food vary so greatly in their mineral content that
any calculation based on existing data is extremely uncertain. For
example, Peterson, Elvehjem, and Jamison found that 18 samples of
cabbage from various parts of the United States showed variations
(from the lowest to the highest) of 96 per cent for calcium, 118 per cent
for phosphorus, and 246 per cent for iron. Such extreme variations
would probably not appear in a mixed diet over a long period of time.

A second difficulty in the calculation of the mineral content of the diet
results from losses incurred in the cooking of the food. Such losses vary
with the kind of food, the method of cooking, and the length of time of
cooking. If cabbage is boiled and the water is discarded, 72 per cent of
the calcium, 60 per cent of the phosphorus, and 66 per cent of the iron
may be lost. The average losses for 16 vegetables reported by Peterson
and Hoppert were:

Calcium
Method of cooking percent

Steaming 10.7

Pressure-cooking 12.0

Boiling 20-32

Differences in the requirements of individuals, variations in the vitamin
D content of the diet, variations in the composition of the food, and
losses in the preparation of food make it imperative that a liberal allow-
ance be made in excess of any calculated figures.

hosphorus

Iron

Protein

per cent

per cent

per cent

16.7

21.3

16.0

19.4

17.4

19.1

30-46

40-48

31-43

198 BIOCHEMICALLY IMPORTANT MINERAL ELEMENTS

REVIEW QUESTIONS ON MINERAL ELEMENTS

1. Give the names and sources of two organic forms of (1) phosphorus, (2) cal-
cium, (3) sulfur; one organic and one inorganic form of (4) magnesium, (5) iron,
(6) iodine, (7) copper, (8) zinc, (9) cobalt.

2. Discuss the mineral element calcium with reference to (1) its function in the
body, (2) the daily requirement of the body, (3) its distribution in food materials.
Repeat the above discussion for phosphorus and iron.

3. Discuss the relative availability to the body of organic and inorganic forms of
the mineral elements important in nutrition.

4. Name five important trace mineral elements. Are any of these essential to the
body? If so, in what way do they serve it?

5. Calculate the acid-base balance between 0.210 gram Ca and 0.168 gram S.
Ca = 40, S = 32.

6. About how much copper and manganese are contained in the food eaten daily
by an adult on an average diet? Name three foods rich in copper; three rich in
manganese.

7. How much calcium is contained in a quart of milk; a one-pound- loaf of bread;
a five-pound head of cabbage?

8. How much calcium may be lost if the water in which vegetables are boiled is
discarded? Name some practical means by which the mineral elements extracted
in the cooking of vegetables may be conserved and utilized.

9. Which are the main types of chemical change that occur during ashing?

10. Of what does the ash from biological material consist? List five typical sub-
stances that you would expect to be present in wood ashes.

11. How could the loss of sulfur or chlorine during ashing be prevented?

12. List all the elements now thought to be essential for the normal nutrition of
higher animals; of higher plants.

13. Which of the nutritionally important mineral elements are associated with the
action of certain enzymes? List the enzymes concerned.

14. Discuss the effect of abnormal concentrations of specific mineral elements in
the soil of certain areas on the animals and people living in those areas.

REFERENCES AND SUGGESTED READINGS

Amon, D. I. and Stout, P. R., "Molybdenum as an Essential Element for Higher

Plants," Plant Physiology, 14, 599 (1939).
Berger, K. C, "Boron in Soils and Crops," Advances in Agroiiomy, 1, 321 (1949).
Elvehjem, C. A., "The Biological Significance of Copper and Its Relation to Iron

Metabolism," Physiol. Rev., 15, 471 (1935).
Food and Nutrition Board, "Recommended Daily Dietary Allowances, Revised

1948," Nutrition Reviews, 6, 319 (1948).
Hambidge, G., Hunger Signs in Crops, 2nd ed., American Society of Agronomy,

Washington, D. C, 1949.
Hart, E. B. and Elvehjem, C. A., "Mineral Metabolism," Ann. Rev. Biochem., 5,

271 (1936).
Hawk, P. B., Oser, B. L., and Summerson, W. H., Practical Physiological Chemistry,

12th ed., The Blakiston Company, Philadelphia, 1949, p. 1002.
Heilbrunn, L. V., An Outline of General Physiology, 2nd ed., W. B. Saunders Com-
pany, New York, 1943.

BIOCHEMICALLY IMPORTANT MINERAL ELEMENTS 199

Hoagland, D. R., Inorganic Nutrition of Plants, Chronica Botanica Company, Wal-

tham, Mass., 1948.
Hove, E., Elvehjem, C. A., and Hart, E. B., "The Physiology of Zinc in the Nutrition

of the Rat," Am. J. Physiol., 119, 768 (1937).
Lunde, G., "The Geochemistry of Iodine and Its Circulation in Nature," Chem. Rev.,

6, 45 (1929).
McClendon, J. F., "The Distribution of Iodine with Special Reference to Goiter,"

Physiol. Rev., 7, 189 (1927).
Monier- Williams, G. W., Trace Elements in Food, John Wiley and Sons, Inc., New

York, 1949.
Moxon, A. L., "Alkali Disease, or Selenium Poisoning," South Dakota Agricultural

Experiment Station, Bulletin 311 (1937).
Peterson, W. H., Elvehjem, C. A., and Jamison, Lida A., "Variations in the Mineral

Content of Cabbage and Sauerkraut," Soil Science, 20, 451 (1925).
Peterson, W. H. and Hoppert, C. A., "The Loss of Minerals and Other Constituents

from Vegetables by Various Methods of Cooking," /. Home Econ., 17, 265 (1925).
Salter, W. J., The Endocrine Function oj Iodine, Harvard University Press, Cam-
bridge, Mass., 1940.
Sherman, H. C, Chemistry oj Food and Nutrition, Chapters XII to XVI, 7th ed.,

The^Macmillan Company, New York, 1946.
Shohl, A. T., Mineral Metabolism, Reinhold Publishing Corporation, New York, 1939.
Stiles, W., Trace Elements in Plants and Animals, The Macmillan Company, New

York, 1946.
Underwood, E. J., "The Significance of the Trace Elements in Nutrition," Nutrition

Abstracts and Reviews, 9, 515 (1940).

Chapter 9

VITAMINS

The various vitamins differ so greatly in composition that it is impossible
to define them in terms of their chemical structure, as is done with other
classes of compounds such as carbohydrates, fats, and proteins. From
the physiological viewpoint, which is probably the best for an accurate
characterization, vitamins may be defined as organic compounds that
are essential constituents of the diet but are required in only minute
amounts for the normal functioning of the body. Thus they may be
seen to differ from hormones in that the body cannot synthesize them,
from trace elements in that they are organic compounds, and from the
main structural and energy-yielding materials of the diet (the carbo-
hydrates, fats, and proteins) in that they are required in extremely
minute amounts. For example, an average adult person consumes about
600 g. (dry basis) of the major food materials daily, whereas his total
vitamin requirements are only about 0.1-0.2 g. per day. The reason why
such small amounts are effective is that their role in the chemistry of
life processes is essentially catalytic. Several such as thiamine, ribo-
flavin, nicotinic acid, pantothenic acid, and pyridoxine have already been
shown to form a part of, or to act with, various enzymes, and in all
probability the remainder will be found to function in a similar manner.

The development of our knowledge of the vitamins has come from
two main lines of investigation: the study of nutritional diseases, mainly
beriberi, scurvy, rickets, and pellagra, and the feeding of "simpHfied"
or "purified" diets to experimental animals.

Until about 1900 it was rather generally believed that an adequate
diet for animals or man need contain only purified protein, carbohydrate,
fat, and mineral salts. However, earlier work of Lunin (1881) and,
particularly, the work of Hopkins (1906) demonstrated conclusively that
animals on such diets did very poorly unless small amounts of certain
additional foods, such as milk, were also given them. Thus the idea
arose that besides the four major constituents of the diet "accessory food
factors" were needed in small amounts for normal nutrition.

What are now known to be vitamin deficiency diseases have been
studied for centuries, but with relatively little progress until recent times.
The first and one of the most difficult steps was to prove that a disease
was caused by faulty diet and not by some infection or other agent.

Some of the observations which gradually led to the acceptance of this

200

VITAMINS 201

idea were the curing of scurvy by fresh fruits and vegetables (Lind 1757,
Budd 1841), the prevention of beriberi by using other foods in place of
polished (white) rice, and the demonstration by Eijkman (1897) that a
disease of fowls similar to human beriberi could be produced or pre-
vented at will by feeding the birds various diets. Eijkman thus intro-
duced the use of experimental animals as test subjects for such studies,
without which the modern development of the vitamin field would prob-
ably have been impossible.

Funk (1912) was the first investigator to formulate and state clearly
the concept that each of the then known deficiency diseases was caused
by the absence from the diet of a separate definite chemical substance.
He had been trying to obtain in pure form from rice polishings the sub-
stance capable of preventing or curing beriberi, and since it appeared
to have the chemical properties of an amine, he proposed as a name for
all such essential substances the term "vitamine," a contraction of 'Vital
amine."

At approximately the same time McCollum and other investigators
discovered that certain fats such as butter and cod-liver oil contained a
substance that in small amounts was capable of promoting the growth
of young rats and preventing the development of an eye disorder known
as xerophthalmia. Since the beriberi preventing substance was soluble
in water, McCollum concluded there were two "accessory dietary factors."
He designated the one "fat soluble A" and the other ''water soluble B."
Several years later (1920), when it had become evident that there were
at least three factors, two of which were in no way related to amines,
Drummond suggested that the terms of Funk and jNIcCollum be combined
by dropping the letter "e" from the word "vitamine" and adding the
letters A, B, and C to give the terms "vitamin A," "vitamin B," and
"vitamin C." As new vitamins have been discovered they have been
designated by letters added to the class name such as vitamin D, or by
use of subscript numbers, e.g., vitamin Bi, Bg, etc.

The number of known vitamins has grown rapidly from the early
abbreviated list composed of vitamins A, B, and C to one comprising
some fifteen to twenty factors that apparently deserve to be included in
this category. Only those substances that have been generally accepted
by research workers in this field are considered in this brief treatment.
For convenience they may be classified as either fat-soluble or water-
soluble. In the former class belong A, D, E, and K, and in the latter,
ascorbic acid (or vitamin C) and the vitamin B group. This group
includes thiamine (vitamin Bi), riboflavin, nicotinic acid (or niacin),
pantothenic acid, pyridoxine (vitamin Be), biotin, pteroylglutamic acid
(abbreviated PGA; also called folic acid), vitamin B12 (antipernicious
anemia vitamin), choline, inositol, and para-aminobenzoic acid (PABA).

Unfortunately, in the earlier work the experimental diets lacked more
than one vitamin with the result that symptoms ascribed to a deficiency

202 VITAMINS

of one vitamin were often in reality due to a lack of several. To illus-
trate, the original vitamin B has been found to consist of some ten or
more factors. This accounts, in part, for the lack of a systematic nomen-
clature, which is so apparent in the list just given. Now that each of
the above vitamins is well known as a distinct chemical substance, much
confusion can be avoided if they are designated by their scientific names
rather than by mere letters. All of the vitamins listed have been isolated
in pure form, and the chemical structures have been worked out, except
for vitamin B12. In addition, all the vitamins are produced commercially,
mainly by synthetic processes, and hence are available in practically
unlimited amounts at much lower prices than formerly.

Since chemical processes in widely differing organisms are frequently
closely related, one might be led to assume that in all higher animals
the vitamin requirements would be practically the same. That such an
assumption is without foundation, however, is evidenced by the fact
that the rat synthesizes its own supply of ascorbic acid. So far as this
animal is concerned, therefore, this compound is not a vitamin. Skin
disorders resembling those of pellagra in man can be produced in the
rat and chick, but they do not respond to treatment with nicotinic acid,
as in the case of man, and neither is pellagra in man relieved by the
vitamins that effect cures in the above-named species. Other ex-
amples of such differences in dietary requirements may be noted in dis-
cussions of the various vitamins.

The relative prevalence of vitamin and other dietary deficiencies in
the United States is difficult to estimate closely, but is probably much
greater than has been supposed. Sebrell has commented on the situation
as follows:

. . . Today it is recognized that all of the known nutritional diseases probably exist
to some extent in the United States. Those that appear to be of most importance
are anemia, due to iron or cobalt deficiency; nutritional edema, due to protein
deficiency ; hyperkerataosis ^ and night blindness, due to vitamin A deficiency ; beriberi
and peripheral neuritis," due to thiamine (vitamin Bi) deficiency, frequently secondaiy
to such conditions as alcoholism, pregnancy and diabetes; lip lesions, seborrhea" and
keratitis,^ due to riboflavin deficiency; pellagra or encephalopathy,^ due to nicotinic
acid deficiency; swollen bleeding gums, skin and subperiosteal hemorrhages,'' due to
ascorbic acid deficiency; rickets and osteomalacia,'^ due to vitamin D deficiency;
hemorrhagic disease of the newborn, due to vitamin K (phthiocol) deficiency; tetany,
due to hypocalcemia,* and probably many other at present less well identified condi-
tions with a nutritional background. . . .

^ JJxcessive growth of the cornea.

^ Inflammation of the nerve endings.

^ A chronic disease of the sebaceous, or oil-secreting glands of the skin.

* Inflammation of the cornea.
^Any disease of the brain.

* Bleeding under the periosteum, the fibrous membrane covering bones.
'' Softening of the bones.

* Abnormally small amounts of calcium in the blood.

VITAMINS 203

An interesting sidelight on this question is furnished by the estimate
that during 1948-1950 the people of the United States spent over $500,-
000,000 annually for vitamin preparations. This figure does not include
the value of vitamins used in enriched foods and food supplements, which
is also estimated to approximate $500,000,000 a year.

VITAMIN A
Physiological function

Vitamin A was first recognized and studied as a growth-promoting
substance, but it was soon discovered that it plays other equally im-
portant roles in the body. In the absence of adequate amounts of vitamin
A the epithelial cells of the mucous membranes lose their ability to secrete
normally and, as a consequence, become dry and hardened. This harden-
ing process is known technically as cornification or keratinization (pro-
duction of a horny layer). In many species the eye seems to be par-
ticularly susceptible to this change, and a disease known as xerophthalmia
is produced. When the tear glands cease to secrete, the eyes are not
cleansed properly, they become noticeably irritated, and infection ensues.
As the exuding pus dries, movement of the eyelids becomes increasingly
difficult until eventually they become closed tightly. In the later stages
ulcers form on the cornea. When these ulcers rupture, the lens pops
out, thus permanently destroying sight. Keratinization likewise occurs
extensively in the respiratory tract (including the lungs), the alimentary
tract, the urino-genital tract, and the salivary and ductless glands. In-
fants suffering from xerophthalmia also exhibit a dry, scaly skin.

Because keratinization of the mucous membranes opens those parts of
the body to bacterial invasion, some have ascribed to vitamin A an anti-
infective role, particularly valuable in warding off colds and other respira-
tory diseases. However, vitamin A does not appear to be toxic to
organisms that cause these diseases. Only to the extent that it helps main-
tain a healthy condition of the mucous surfaces and increases the general
resistance of the body is it "anti-infective."

In addition to keratinization of epithelial tissue there are a number
of other symptoms of vitamin A deficiency. Urinary calculi and deposits
of calcium phosphate have been observed in rats suffering from vitamin
A deficiency. Although xerophthalmia seldom develops in the chick,
immense deposits of urates accumulate in the kidney and renal tubules
as a result of vitamin A deficiency. The vitamin is also essential to
reproduction, for on diets deficient in this vitamin, female rats cease
to ovulate normally, and males show testicular degeneration, even though
the vitamin E intake may be adequate. Sherman has shown that fer-
tility and longevity of rats is greater on a ration rich in vitamin A than
on one that is low in this factor.

204 VITAMINS

Vitamin A plays a vital role in connection with vision. The rods in
the retina of the eye contain a rose-colored pigment, rhodopsin, which
is a conjugated chromoprotein. When light falls on the retina, rhodopsin
is broken down into a simple protein, opsin, plus trans-retinene, a yellow
substance now known to be vitamin A aldehyde. Associated with this
chemical change is the production of a stimulus which, being imparted
to the optic nerve, results in vision. These changes are very rapid and
are probably completed within a few hundredths of a second.

Subsequently, a slower process, not dependent on light, brings about
the regeneration of rhodopsin so that an adequate supply for normal
vision is continuously present in the retina. Rhodopsin is formed from
opsin plus cts-retinene, a geometric isomer of the ^rans-retinene released
by the action of light on rhodopsin.^ The m-retinene needed may be
obtained by isomcrization of the trans form, or from cis-vitamin A through
the action of alcohol dehydrogenase and DPN (p. 275) :

alcohol

C19H27CH2OH + DPN <^ ^ , '• CnH^vCHO + DPN-Hj

dehydrogenase

CIS-Vitamin A cis-Retinene

Since the breakdown and resynthesis of rhodopsin are probably not per-
fectly efficient processes, some retinene derived from vitamin A no doubt
must be continually supplied.

The retinas of fresh-water fish do not contain rhodopsin but a closely
similar, light-sensitive pigment, porphyropsin. This substance fulfills the
same functions for the vision of these fish as rhodopsin does for higher
animals and man. The outstanding difference lies in the retinene and
vitamin A associated with it, which are different from those derived from
rhodopsin. To distinguish the two varieties of the vitamin, that from
mammals is designated Ai and the other, A2. The corresponding retinenes
are similarly designated retmenei and retinene2. Fresh-water fish have
vitamin A2, not only in the retina, but also in the liver and other organs.

Since vitamin A is needed for rhodopsin formation, it would be expected
that persons deficient in this vitamin would have subnormal amounts of
rhodopsin in the "retina. In any event, it has been observed that such
individuals cannot see as well in dim light or adapt themselves to changes
in light intensity as readily as those whose supplies of vitamin A are
ample for immediate resynthesis of the hght-sensitive pigment. By means
of the biophotometer it is possible to measure accurately one's ability
to adapt to darkness after the eyes are exposed to a bright light for
a few minutes. Adaptation is determined by the amount of light neces-
sary for the individual to recognize the number of openings in a screen

^ For a brief explanation of cis- and irans-isomers see p. 79. Since vitamin A and
retinene each contain five double bonds, theoretically (2)5 such isomers of each can
exist. It is not known which particular cis-isomer is involved in rhodopsin formation.

VITAMINS 205

through which dim light is transmitted. Pronounced inabiUty to see in
dim light is termed "night-blindness."

Prevalence of vitamin A deficiency

Because the young of mammals, including man, are born with limited
stores of vitamin A, we find them more susceptible to a deficiency than
adults. Consequently, xerophthalmia is most often observed in young
children of the extremely poor classes, or in children at times of great
food shortage. Although considered to be a rare disease in the United
States, 13 cases were reported in 1931. Of every five children of the
poor classes in Yucatan one is said to be suffering from xerophthalmia.
Reports from Ceylon state that the disease is very prevalent there, espe-
cially in asylums and charity boarding schools. If, as is reported, the
diets in the latter institutions furnish only 30 per cent of the estimated
minimum requirement of vitamin A, one can readily understand why
two-thirds of the children are suffering to some extent. In China, India,
Java, and Sumatra the disease is said to be very common. Biophotometer
tests reveal that diets of the poorer classes in our country frequently
are inadequate with respect to vitamin A, and some indicate that diets
of those in the higher economic brackets are likely to show suboptimal
amounts of vitamin A.

Chemical nature

The vitamins A are closely related to a group of yellow, orange, and
red pigments called carotenoids, present in many plants and animals. The
group name is derived from one of the best known and most important
members, carotene, the orange coloring matter of carrots. Lycopene of
the red tomato, zeaxanthine of yellow corn, and xanthophyll of egg yolk
are other common carotenoids. There are several isomers of carotene, but
the one most closely related to vitamin Ai is beta-carotene, of which the
formula is given below:

HaC .CH3 CHa CH, CH3 CHa Haa .CH,

/C:; II II ^cC

U^C^ ^C-CH:CH-C:CH-CH:CH-C:CH-CH:CH-CH:CCH:CH-CH:CCH:CHC^ ^CH,

I II II I

H.C^^X:-CH, CHa~G,^^CH»

Hi Hj

Beta-carotene, C^oHi*

A high degree of unsaturation, in which the double bonds occur alternately
with single bonds along the carbon chain, is characteristic of carotene, as
well as of vitamin A and the whole carotenoid group. Pure beta-carotene
is a dark red, crystalline solid (Fig. 9-1).

The relationship of vitamin A to carotene was first established by

f

m

Courtesy of L. Zechmei«te/.
Fig. 9-1. Carotene.

"^

Fig. 9-2. Vitamin A (with one molecule of solvent of crystallization).
Yellow crystals first isolated by Prof. Harry N. Holmes and Dr. Ruth E.
Corbet, Oberlin College, 1936.

206

VITAMINS 207

animal feeding experiments, which proved that carotene is converted into
the vitamin in the animal body. Later, pure vitamin Ai was obtained
from fish liver oils in the form of lemon-yellow crystals (Fig. 9-2).
When the chemical formula of the purified vitamin had been established,
its relation to carotene became even more apparent:

H3C CH3 CH3 CH3

HiC C— CH:CHC:CHCH:CHC:CHCH20H
H2C.^ ^C — CH3

Vitamin Ai, C19H27CH2OH

It may be seen that the carbon structure of vitamin Ai is exactly half
that of the beta-carotene molecule and that the vitamin is a primary
alcohol containing five double bonds. The exact chemical formula of
vitamin Ao has not yet been determined.

Of the many carotenoids of known chemical structure only four (alpha-,
beta-, and gamma-carotene, and cryptoxanthin) of common occurrence in
foodstuffs are convertible into vitamin A. It was long supposed that
this transformation occurred in the liver, but more recent information
tends to discredit this view. Carotene injected into the blood stream of
rats deprived of vitamin A does not prevent the animals from dying of
vitamin A deficiency, even though the livers may contain considerable
amounts of carotene at the time of death. Increased vitamin A contents
in the intestinal wall and in the lymph, following the injection of carotene,
make it appear most probable that the conversion occurs during absorp-
tion of the carotene through the intestinal wall.

The fact that vitamin Ai is almost exactly one-half of a beta-carotene
molecule suggests that two molecules of the vitamin might arise from the
conversion of one molecule of carotene. This, however, does not seem
to be the case, since on a weight basis beta-carotene has only about one-
half (or less) of the biological value of vitamin A. Therefore, the con-
version is probably not a splitting of the carotene molecule in the middle,
but rather a degradation of one end of the molecule until one molecule
of vitamin A is produced.

Other related substances which possess vitamin A activity have been
prepared synthetically, as has the vitamin itself. These include the
corresponding aldehyde (C19H27CHO, retinene), acid (C19H27COOH),
and methyl ether (C19H27CH2OCH3), In a natural state the primary
alcohol group of vitamin Ai is frequently involved in ester linkage, and
such esters as the acetate

O

II
C19H27CH2OCCH3

208 VITAMINS

and palmitate

0

II

Ci9H27CH20C(CH2)l4CH3

have also been prepared artificially. They are fully active, and yet con-
siderably more stable than the free vitamin.

The vitamin A value of a given food sample may be due either to the
actual vitamin itself, an ester of it, one of the carotenoids listed above,
or to a mixture of these. The carotenoids which are capable of conversion
to vitamin A are often designated as provitamins. Unless otherwise
specified, the collective meaning, that is, precursors as well as the true
vitamin, is implied in the subsequent discussion.

Preparations of vitamin A rapidly lose their potency in the presence
of oxidizing agents. Atmospheric oxygen at room temperature or above
brings about noticeable destruction of vitamin A unless the vitamin is
protected by antioxidants. These antioxidants, or inhibitors, are asso-
ciated with lipidcs in nature and hence stabilize the vitamin in food-
stuffs. Vitamin E is one of the most important of these antioxidants,
which act to minimize the destruction of vitamin A by oxidation. Cooked
foods retain the greater part of their original vitamin A potency. Since
vitamin A is fat-soluble, it is extracted from foodstuffs along with the
lipides by the common fat solvents. Saponification of these lipides does
not destroy the vitamin.

The vitamin A value of biological samples may be determined by
animal feeding tests, but this laborious method has been almost entirely
supplanted by quicker chemical methods. However, the animal assay
measures the precursors as well as true vitamin A, whereas each chemical
test measures either one or the other, but not both. The chemical meas-
urement of true vitamin A depends on reacting it with antimony trichlo-
ride in dry chloroform (Carr-Price reaction). A clear, deep blue color
is produced which is compared with the intensity of the color from known
amounts of the pure vitamin to estimate the potency of the sample.
The carotenoid pigments, many of which have no vitamin A value, all
respond to some extent to this same test, and so interfere unless a suitable
correction is applied. This method is widely used for determining the
true vitamin A content of butter, cheese, fish oils, pharmaceuticals, and
many animal tissues such as blood and liver.

The vitamin A value of plant samples is usually determined by measur-
ing the carotene content. This is accomplished by extracting with a fat
solvent, purifying the extract to remove interfering pigments, and then
comparing the yellow color of the solution with that of known concen-
trations of carotene in the same solvent.

VITAMINS 209

Occurrence

The carotenoid pigments possessing vitamin A activity are synthesized
in the plant, where they are found associated with such pigments as
xanthophyll and chlorophyll, which have no vitamin A potency. From
available information it appears that all animals are ultimately depend-
ent upon the carotenoid pigments for their supply of vitamin A, for in
no instance has the real vitamin been reported to occur in plant prod-
ucts. Neither has any animal been observed to possess the ability to
synthesize the vitamin from other compounds. The potency of animal
products may be due either to stored carotenoids or to the vitamin that
the body has formed from these ingested pigments.

The most potent sources of vitamin A are the fish-liver oils. Of these
oils, that of the cod has been most widely used. The oil from the liver
of the halibut has been shown to possess 75 to 125 times the potency
of cod-liver oil. Foods containing relatively high concentrations of
vitamin A include: butter, cream, cheese, milk, egg yolk, liver, the green
vegetables such as spinach, escarole, string beans, and leaf lettuce, apri-
cots, bananas, oranges, peaches, prunes, pumpkins, sweet potatoes, and
tomatoes. The green outer leaves of lettuce are reported to contain 30
times as much vitamin A as the crisp inner portion. The potency of
cow's milk has been found to vary with the breed and ration. ]\Iilk
remains a good source of the vitamin, even in winter, since the cow is
able to draw upon her reserves when confined to feeds deficient in vitamin
A. In some milk {e.g., Guernsey) the larger part of the potency is due
to carotene, while in others {e.g., Holstein) there is more vitamin A than
carotene. Since the vitamin A activity of colostrum (the first milk after
parturition) is 10-100 times that of ordinary milk, it is apparent that
nature has provided for immediate supplementation of the meager supply
with which the young are provided at birth. Human milk is 3-5 times
richer in this factor than cow's milk. Glandular organs, like liver, con-
tain much more vitamin A than muscles. Cereals and cereal grains (with
the exception of yellow corn) , roots, and tubers are generally conspicuously
deficient in the vitamin.

Requirements

According to the Food and Nutrition Board of the National Research
Council, the vitamin A requirement of the average adult or adolescent
is satisfied by the consumption of approximately 5000 international units
daily. The needs of smaller children are somewhat less. Pregnancy
may increase the demand for vitamin A to 8000 international units per
day. Quantities of representative foods that would supply approximately

210 VITAMINS

5000 international units are: ^ lb. of butter, % lb. of carrots, 1 to 1^/^
oz. of spinach, or 1-2 teaspoonfuls of cod-liver oil.

The international unit (I.U.) is defined as the vitamin A potency of
0.6 microgram (/xg.) of pure beta-carotene. Pure vitamin A has a bio-
logical potency of one unit in about 0.25 /xg. Thus 5000 I.U. would
correspond to only 1.2 mg. of true vitamin A, but 3 mg. of beta-carotene
would be needed to supply this number of units. The other carotenoid
precursors of vitamin A are only about one-half as effective as beta-
carotene, and so about 6 mg. would be required if the total vitamin A
supply were to come from this source. The above recommended daily
allowances are based on the assumption that approximately two-thirds
of the total vitamin A value of the diet will be contributed by one or
more of the precursors and one-third by vitamin A itself.

A greatly excessive intake, for example, several million units taken
over a period of a few days or weeks, is apt to produce symptoms of
toxicity such as nausea and vomiting, headache, peeling of the skin, and
general prostration. The amounts necessary to cause such sickness, how-
ever, are so much greater than the normal requirements of the body
that in the ordinary course of events no such disorder should ever arise.

VITAMIN D

Physiological function

Lack of vitamin D manifests itself largely in the form of a disease
known as rickets, and for this reason it is sometimes called the antirachitic
vitamin. The most obvious symptoms of rickets are bowed legs, enlarged
joints, and malformation of the head and chest. These abnormalities
result from improper calcification of the growing bones, as may be readily
detected by comparing X-ray photographs of rachitic and normal bones.
Analyses of the bones of rachitic rats show that the ash content on
the fat-free dry basis may be as low as 25-30 per cent as compared with
approximately 50 per cent for the bones of normal rats of the same age.
Such bones are necessarily weak and eventually assume the character-
istically bowed shape. Enlargements occur also where the rib bones join
the cartilages, and to this chain of bead-like formations has been given
the name "rachitic rosary." Weak abdominal muscles, together with
malformation of the chest, are responsible for a pot-bellied appearance.
Since bones and teeth are so closely related chemically, it is not surpris-
ing that rachitic children also have defective teeth. It is hardly necessary
to state that growth is retarded with the onset of rickets and eventually
ceases. The disease is seldom fatal, but it predisposes the individual to
other diseases, of which broncho-pneumonia is said to be most com-
mon.

VITAMINS

211

The physiological action of vitamin D in preventing or curing rickets
is thought to be due largely to the fact that it promotes the absorption
of calcium from the food through the intestinal wall and into the blood
stream. In addition, some investigators feel that it has some direct
action, at present of an obscure nature, in promoting the actual deposi-
tion of calcium and phosphorus in the growing bone. It may be that
these beneficial effects result from an interaction of the vitamin with

1

Courtesy of the Wisconsin Alumni Kesearch Foundation.
Fig. 9-3. Rickets.

alkaline phosphatase, a type of enzyme which is concerned with phos-
phorus absorption in the intestine and reabsorption in the kidney (a
process which reduces loss of phosphates in the urine). This enzyme
is especially active in bone where it breaks up organic phosphates of
the blood and thus provides inorganic phosphate (PO4-) for bone
formation. A water-soluble derivative of vitamin D has recently been
shown to stimulate the action of alkaline phosphatase very markedly,

212

VITAMINS

and this observation, if confirmed, would go a long way toward explaining
the action of vitamin D in rickets.

Prevalence of vitamin D deficiency

No other deficiency disease has been as noticeable in the United
States as that due to lack of vitamin D. Infants from 3 to 24 months of
age are most susceptible. Not only is rickets seasonal in appearance, but
it is observed more frequently in the North than in the South, where
more sunshine is available throughout the year. In fact, it is almost
inevitable during the cold Northern winters unless the diets of the young
are supplemented with vitamin D.

Rickets is said to be most prevalent in Great Britain, France, Belgium,
Germany, European Russia, Austria, Hungary, and the United States.
Sebrell cites reports of the U. S. Census Bureau to the effect that 244
deaths resulted from rickets in 1938. Before vitamin D therapy was
used (1928) the Ministry of Health reported 87 per cent of London ele-
mentary school children as giving evidence of having had some degree
of rickets. A survey of children in Sydney, Australia, revealed that
about one-half of them either were or had been rachitic. In 1943 Follis
and co-workers reported the results of examining 230 consecutive cases
of children from 2 to 14 years of age who had died from various causes
in Baltimore, Maryland. No less than 46.5 per cent were found on
autopsy to have had some degree of rickets. It will be noted that coun-
tries of the extreme north are not included in the above list, a fact which
is probably due to the important place that fish and glandular organs
assume in the diets of these peoples. Young girls of the upper Moham-
medan classes in India suffer from rickets as a result of religious tenets,
now rapidly disappearing, that require the women to remain in seclusion
after becoming 12 years of age. Lower classes fare better, even though
their diet is less satisfactory, because they live more in the open air.

Chemical nature

Although the existence of as many as ten different antirachitic com-
pounds has been rather well established, only two of the D vitamins have
been proved to be of any practical importance so far as antirachitic
medicines and foods are concerned. These may be produced in the
laboratory from ergosterol and from 7-dehydrocholesterol, respectively,
by irradiation with ultraviolet light. The principal structural changes
associated with acquisition of antirachitic potency are shown by com-
parison of the formulas of ergosterol and calciferol. Ring B is opened,

VITAMINS

213

and a methylene group (CH2=) is formed from the methyl group attached
to rings A and B. Similar changes are wrought in the structure of
7-dehydrocholesterol and quite likely in all other sterols during their
activation. Vitamin ^2, or calciferol as the irradiation product of
ergosterol is known, is a white crystalline substance that differs from

(2i)^cH

(IS) I (20)

(22)%^

CH2 I

CHa^^jj

CH2

1(2)
1(3)

HO-CH

<^(i)

(19)
CH3

1(11)

(13)

CH

(9)

(10) (S)

B

(14)

117)^
(16)

D

(23TCH

!

(24)CH— CHj

I (25)

CH2 (26)CH — CH3

(27)

CH-

— CH ^^^^^^'

(5)^
(4)x/'"V)

CH2 CH

C

^^^CH

(21)
H3C,

Ergosterol, C28H43OH

^CH(20)

(18)

CH2 ^^'cn

^12)\/,^17)\

(16)

,CH

(22)-^5^«^^

(ll^CH

1 (25)

(24) CH— CH3

CH2 (13)^
(19) |(11) C

.CH2 ?f ^ CH2 (^^ICH-
CH2 <^(io) (s)C

D

CH2 (26)CH— CH3
I (27)

^CH2 (28)CH3

1(2)

1(3) (5)

CH2

'<(6)>H
^CH

Calciferol, C28H43OH

the parent sterol in several properties such as melting point, optical rota-
tion, solubility, and precipitability.

It seems quite probable that the three double bonds between carbons
10 and 19, 5 and 6, and 7 and 8, respectively, (designated by * in the
above formula) are necessary for vitamin activity, since this grouping
is present in each of the D vitamins whose chemical structures have
been accurately determined. In spite of the rather large number of
D vitamins that have been produced in the laboratory, only two of
natural occurrence (D2 and D3) have been isolated thus far. Vitamin
D3, which is obtained from fish-liver oils, is identical with the vitamin
formed by the action of ultraviolet light upon 7-dehydrocholesterol.
Vitamins D2 and D3 have approximately the same potency toward mam-
mals, including man, but D3 is about 30 times as effective as D2 is for
fowls. The D vitamins are so resistant to high temperatures that prac-
tically no loss of activity is suffered when a food is cooked.

214 VITAMINS

Relationship of light and mineral content of diet to vitamin
D requirements

Sunlight prevents rickets by the action of its Ultraviolet rays on some
provitamin in the skin, presumably 7-dehydrocholesterol. Practically
none of the effective ultraviolet rays penetrate ordinary window glass, and,
hence, much transmitted light possesses little or no antirachitic value.

Calcification of growing bone, the process in which vitamin D is so
vitally concerned, consists primarily in the deposition of calcium and
phosphorus as a complex calcium phosphate in the organic matrix. No
amount of vitamin D in the diet, therefore, can take the place of these
inorganic constituents. Not only must the two elements be present in
adequate amounts, but their relative proportions influence greatly the
need for vitamin D. Rations employed in producing rickets experi-
mentally in rats are made especialty effective through the addition of
large amounts (three per cent usually) of calcium carbonate. Rickets
so produced is said to be of the low phosphorus type (common clinically)
since the inorganic phosphate content of the blood serum of animals on
such a ration is abnormally low. In human beings the normal amount
of inorganic phosphorus in the blood plasma is approximately 4 mg. per
100 ml., and the calcium content is 10 mg. per 100 ml. If either or
both of these values becomes low enough so that their product (milligrams
inorganic P X milligrams Ca per 100 ml.) drops to 30 or less, rickets
is almost certain to develop. A value of 40 or above is considered
normal. A low calcium type of rickets also occurs, and a third type in
which serftm calcium and phosphorus are both low is observed occasionally.

Occurrence

The most potent natural sources of D vitamins are the fish-liver oils.
Fish in which considerable oil is distributed throughout the body, e.g.,
sardines and salmon, are the richest food sources. The concentration
in salmon averages approximately 12 /xg. per 100 g., while several other
fish are nearly as high.

Next in order of concentration are egg yolk and butter with 4.6 and
2.3 fig. per 100 g., respectively. Although milk normally contains only
small amounts of the vitamin — reports vary from 0.008 to 0.106 fig. per
100 g. — it may be enriched by irradiation so as to contain 0.3-1.1 fig.
per 100 g., which is equivalent to about 135-400 LIT. per quart. Milk
produced by cows fed irradiated yeast also contains about 400 I.U.
per quart. Direct irradiation of the animals is without effect in increasing
the concentration of vitamin D above the normal level in milk. The
amount of vitamin D in a number of typical foods is indicated in the

VITAMINS

215

following table. It will be noted that, except for vitamin D milk and
certain fish, most native foods contribute only negligible amounts of
this vitamin.

Table 9-1

Vitamin D content of foods

(The values are expressed as micrograms of vitamin D per 100 g. of edible portion,

fresh basis.)

Food Vitamin D

Egg yolk 4.6

Halibut-liver oil 3500

Lamb liver 0.43

Mackerel 4-6

Food
Beef liver

Vitamin D
0.72

Beef steak

0.33

Beet greens

Bread, vitamin D

0.004

1.7

Butter

2.3

Cabbage

0.005

Carrots

Carrot tops

0.004

0.075

Cheese ....

0.83

Cod-liver oil

Corn oil

250

0.22

Cream

0.42

Crisco

0.22

Milk, whole 0.11

Milk, vitamin D, irradiated. 1.1
Milk, vitamin D, fortified. . . 1.1

Pork liver 1.1

Salmon 12

Sardines 6-8

Spinach 0.005

Tuna 5-8

Veal liver 0.24

Precursors of vitamin D are widely distributed in plant and animal
materials. Therefore, if a food is subjected for a few seconds to the
light from an ultraviolet lamp, it becomes potent as an antirachitic agent
through the conversion of its provitamin into vitamin D. Certain brands
of cereals, cookies, evaporated milk, yeast, and bread containing vitamin
D are now commercially available. In some cases the potency is due
to the addition of small amounts of irradiated ergosterol rather than to
irradiation of the food.

Since foodstuffs can be rendered antirachitic through treatment with
light from an ultraviolet lamp, one might assume that plant products
would possess considerable potency as a result of the effect of the ultra-
violet rays of the sun. Such has not been found to be true. At least
by the time foods are prepared for consumption little, if any, vitamin
D is present.

In addition to the fish-liver oils and foods that contain the D vitamins,
extremely potent preparations such as (1) liver oils fortified with addi-
tional vitamin D, (2) Hver oil concentrates, (3) calciferol dissolved in
an inert substance such as corn oil are on the market. Viosterol is a
general name for preparations of irradiated ergosterol. Cod-liver oil
contains on the average about 250 /^g. per 100 g., a concentration which
is exceeded by the liver oils of a number of other species. Thus halibut-
liver oil is 10-20 times richer, and the liver oil of one species of tuna
fish has been reported to contain 150,000 iJ.g. per 100 g. Since with
massive doses of vitamin D, calcification may occur in tissues other

216 VITAMINS

than bone, e.g., in the aorta, kidneys, S,nd intestines, the extremely potent
preparations of vitamin D should be used with care and preferably in
accordance with the advice of a competent physician.

Requirements

Because of the various factors that affect the individual's need for
vitamin D, it is difficult to determine exactly the required amount. The
Food and Nutrition Board of the National Research Council recommends
10 /Jig. per day for infants under one year and for women during lactation
and the latter half of pregnancy. This is equivalent to 400 I.U. since
0.025 fjLg,. provides one unit. Older children should also receive 10 [xg.
(400 units) daily, but adults probably require no vitamin D in tKe diet
unless they are seldom or never exposed to direct sunlight. This amount,
10/xg., would be contained in approximately 1-2 teaspoonfuls of cod-
liver oil, 2 lb. of butter, 2.5 gal. of whole milk, or 1 doz. eggs. It should
be emphasized that no ordinary diet can be relied upon to meet the require-
ments of a growing child for vitamin D unless special precautions are
taken to include adequate amounts of a fish-liver oil, or other suitable
source.

One fortunate aspect of the problem of supplying adequate amounts
of vitamin D to growing children is that if more than the day's require-
ment is consumed at one time, the extra amount is stored in the body
(mostly in the liver) and prevents the development of any deficiency
for a correspondingly longer time. Thus a single dose of 20,000 I.U.
of vitamin D per kilogram of body weight was found to protect puppies
from rickets for as long as 12-14 months.

This ability of vitamin D to be stored extensively in the body is also
shared by the other fat-soluble vitamins (A, E, and K), but not by
the water-soluble B vitamins or vitamin C.

VITAMIN E

Physiological function

Because the outstanding function of vitamin E seems to be that of
promoting reproduction, it has become known as the "antisterility factor"
or "vitamin of reproduction." Such a term is somewhat misleading in
view of the fact that the vitamin performs other functions in the body,
including that of promoting growth. Moreover, it was noted previously
that vitamin A also is essential to reproduction.

The most detailed investigations regarding the functions of vitamin E
have been carried out with rats. In the male rat on a diet deficient in
vitamin E the germ cells undergo a permanent degeneration, as evidenced

VITAMINS

217

by failure to recover even when the animal is fed large amounts of the
vitamin. The females, on the other hand, continue to ovulate normally,
mating occurs, and the foetuses grow until about the twelfth or thirteenth
day of gestation, at which time they die and are resorbed by the mother's
body. Injury to the female is less severe than to the male, for normal
young may be born in the next gestation period if an abundance of
vitamin E is supplied. However, repeated resorptions may lead to
paralysis and other serious injury. Similar experiments with mice indi-
cate that females respond to vitamin E deficiencies as do rats, but the
males do not show testicular damage after prolonged lack of the vitamin.

Deficiency of vitamin E produces muscle dystrophy in several species.
This is a condition of impaired nutrition of the cell that results in
degenerative changes in the muscle fibers. Eventually the fibers atrophy
and are replaced by connective tissue and fat. Such changes necessarily
result in paralysis, although the two do not always correspond in degree.
The chemical composition of the involved muscles is altered in that lipide
phosphorus and cholesterol increase greatly, whereas there is a marked
decrease in glycogen and creatine. Abnormally large amounts of creatine
are excreted in the urine. Muscle dystrophy has been observed in dogs,
rabbits, guinea pigs, and rats, and cures have been effected through ad-
ministration of vitamin E. Muscle dystrophy is also a disease of man,
but unfortunately human patients are not cured by vitamin E. The
vitamin E content of eggs coincides closely with their hatchability and
with the vitality of the young chicks. Because of the function of hor-
mones in reproduction, it has been suspected that a relationship exists
between tissue concerned with their production, for instance, the pituitary
gland, and vitamin E supply. Experimental data to support such a
view, however, are still lacking. Ability to store vitamin E is pronounced.

Evidence is accumulating which indicates that the chief physiological
function of vitamin E may be associated with its pronounced antioxidant
properties. The muscles of vitamin E deficient animals show a greatly
increased respiration (oxygen uptake) as compared with normal muscles,
and the respiration is dramatically reduced by administration of vitamin
E to such animals. It has recently been found, also, that feeding ade-
quate amounts of vitamin E greatly reduces the oxidation of carotene
and vitamin A in the intestinal tracts and body tissues of animals so
that they are able to remain in good condition on much less vitamin A
than would otherwise be required.

Chemical nature

Vitamin E activity in foodstuffs has been traced to four phenols
(hydroxy derivatives of benzene), which are known as alpha-, beta-,
gamma-, and delta-tocopherol, respectively. The alpha form has much

218 VITAMINS

greater vitamin E activity than the others; it is the form usually meant
when the term vitamin E is used. Alpha-tocopherol has the structure
represented by the following formula:

CH3

HO-C^ "C^^^CH, CH3 CH3 CH3

CH3— C^ ^C>, /C- (CH2) 3— CH- (CH,) 3— CH (CH2) 3-CH— CHj

I CH3

CH3

a-Tocopherol, C29H60O2

The E vitamins are remarkably stable to heat, alkali, and many ordinary
chemical reagents, but they are unstable to ultraviolet light and oxidizing
agents. The potency of food materials containing these vitamins is
rapidly destroyed by oxidation in the presence of certain fats (notably
lard) and iron salts. The E vitamins are also destroyed in rancid fats.

Occurrence

The amounts of alpha-tocopherol and of all the various tocopherols
present in some common food materials are listed in Table 9-2. Certain

Table 9-2

Tocopherol (vitamin E) content of foods

Tocopherol content *

Food Total Alpha

Bread, white 0-23

Butter 2.40

Cheese, American 1-00

Chocolate, unsweetened 11-1 5.3

Corn oil 87 7'

Cottonseed oil 90 56

Eggs, hen's 2.00 1.16

Fruits, various 0.24-0.74 0.23-0.72

Lard 2.7 2.3

Margarine 54 28

Meat, fish, and poultry, various 0.25-1.40 0.21-1.40

Milk, cow's, whole 0.12

Oatmeal 2.10 1.94

Peanuts 9.30 4.60

Rice, brown 2.40 1.20

Rice, polished 0.57 0.35

Soybean oil 140 10

Vegetables, various 0.06-4.0 0.1^.0

Wheat, whole 2.20

Wheat germ oil 200-300 130-195

Yeast, brewer's, dried 0 0

* Milligrams per 100 g.

VITAMINS

219

vegetable oils, particularly wheat germ oil, arc the richest sources. Whole
grain cereals, eggs, and peanuts are other rather good sources of vitamin
E. Fruits and vegetables as a class are rather poor sources, as are most
meats, fish, poultry and other animal products, except butter. Harris,
Quaife, and Swanson estimate that the average daily intake of alpha-
tocopherol in the United States is about 14 mg. per capita, of which nearly
8 mg. are obtained from fats and oils in the diet.

Prevalence of vitamin E deficiency

Various breeding troubles in farm animals such as horses and sheep
have been thought to result from inadequate intake of vitamin E, but it
is now practically certain that this is not the cause. Vitamin E deficiency
most probably does not occur except when the vitamin is deliberately
excluded from the diet.

Claims have been made that vitamin E deficiency occurs quite com-
monly in women during pregnancy and that miscarriages, sterility, and
similar reproductive disorders of human beings have been helped by
administration of extra vitamin E. Other reports, however, contradict
these claims, so that at present the matter is still unsettled.

VITAMIN K

Physiological function

When chicks or other fowls are fed a ration deficient in vitamin K,
eventually the blood ceases to clot within normal time with the result
that there is a marked tendency for hemorrhages to occur. This condi-
tion is characterized by bleeding from the pinfeathers and hemorrhages
into the subcutaneous tissues and muscles. The concentration of pro-
thrombin, one of the factors involved in clotting of blood, invariably is
found to be reduced in this deficiency disease. Just how vitamin K
influences the formation of prothrombin remains to be determined. Since
the sequence of changes involved in clotting of blood very likely is the
same for all species, it will not be surprising if further investigations
reveal a universal need of vitamin K. Experiments have demonstrated
that man, rabbits, mice, and perhaps cattle require this vitamin, although
deficiencies do not normally occur because adequate amounts usually
are provided by the food intake, or are synthesized by intestinal bacteria.

Chemical nature

Several substances exhibiting vitamin K activity have been found in
nature, and many more have been synthesized in the chemical laboratory.

220 VITAMINS

The K vitamins are derivatives of 1,4-naphthoquinone, and this structure,
or one readily convertible into it in the body, is essential for vitamin K
activity. In the accompanying formula of vitamin Ki the 1,4-naphtho-
quinone structure consists of rings A and B without the side chains

H
1

0
II

C^

/C^

HC^(*)^C

A

-CHs

H

H

H

.„. B

1

1

1

Hc'4;(5)/C

-CHs

CH:C-(CH03

•C-(CH,)3

■C-(CH2)3

•C-CHs

1

1

11

CH3

CH,

CH3

CH3

H

0

Vitamin Ki, C31H46O2

attached at positions 2 and 3. Synthetic 2-methyl-l,4-naphthoquinone
is even more active, gram for gram, than vitamin K, but the two are
of about equal activity when compared on a molecular weight basis. It
may be, as some have suggested, that in the body the K vitamins are
degraded to 2-methyl- 1,4-naphthoquinone or, as others believe, perhaps
the latter is used in the body to synthesize these vitamins. A number
of bacteria have been found to synthesize compounds possessing vitamin
K activity. Phthiocol (2-methyl-3-hydroxy- 1,4-naphthoquinone), a yel-
low pigment found first in the tubercle bacillus, is one such compound.

Water-soluble derivatives of vitamin K (e.g., the sodium salt of
2-methyl- 1,4-naphthohydroquinone diphosphate) have been prepared
synthetically and are medically useful in certain cases (see below) . Both
menadione, as 2-methyl- 1,4 naphthoquinone is called, and the water-
soluble derivatives are in common use.

The K vitamins are fat-soluble and, therefore, are dissolved by the
usual fat solvents such as ether and petroleum ether. They are stable
to heat, but are destroyed by alkalies, strong acids, oxidizing agents, and
sunlight. Solutions of vitamin K gradually lose their activity when sub-
jected to light from an ordinary electric light bulb.

Prevalence of vitamin K deficiency

The general distribution of vitamin K in foodstuffs doubtless is re-
sponsible for the fact that deficiencies seldom are encountered in the
adult. The vitamin apparently is also produced in the intestinal tract
through the agency of the bacteria normally present. Vitamin K prepara-
tions were used on man first in treatment of obstructive jaundice, a con-
dition in which a low concentration of prothrombin in the blood always
prevailed. Deficiency of vitamin K in this instance is related to lack
of bile, without which the vitamin is poorly absorbed from the intestinal
tract. Surgical removal of the obstruction was formerly accompanied

VITAMINS

221

by severe hemorrhages, but this is now very effectively prevented by
prior administration of vitamin K, either by injection or by use of a
water-soluble derivative which is easily absorbed. The deficiency of
prothrombin associated with certain intestinal conditions — for example,
obstruction and severe diarrheal diseases such as ulcerative colitis, sprue,
and celiac disease — also responds to treatment with compounds possess-
ing antihemorrhagic activity.

Since the concentration of prothrombin in the blood of a newborn
baby is quite low, a large percentage of deaths during the first few days
of life are due to hemorrhages resulting from injuries suffered at birth.
Fortunately, however, investigations have revealed that prothrombin
levels may be markedly increased within a short time by administra-
tion of vitamin K to the newborn. Administration of vitamin K to the
mother prior to childbirth has been shown to raise the level of prothrombin
in the foetus. These treatments should effect a drastic reduction in
mortality of infants due to hemorrhage. The hereditary disease, hemo-
philia, does not respond to treatment with vitamin K.

A substance, dicumarol, which has a specific antagonistic effect toward
vitamin K has been found in improperly cured sweet-clover hay. If
freshly cut, partially dried, sweet clover is piled up for a few days, it
undergoes a fermentation during which coumarin, a normal component
of the hay, is converted into dicumarol. The chemical formulas of these
substances are indicated below:

OH OH

H H
C C

HC^ ^C^ ^CH

II 1 1
HC^^^C^^/C=0

H

HI 1 H

c c c c

HC^ '^^C^ ^C — CH2 C^ '^Q^ ^CH

II 1 1 1 1 II
HC^^^C^^/C=0 0=C>,^^C^^^CH

H H

Coumarin

Dicumarol

The blood of cattle eating such hay soon loses its normal clotting power,
with the result that any injury is followed by severe and often fatal
bleeding. Dicumarol was discovered by Link and co-workers.

The administration of dicumarol to experimental animals or man inter-
feres with the formation of prothrombin, so that the result is similar to
that of vitamin K deficiency. In fact, it is now well established that
extra doses of the vitamin will counteract the effect of dicumarol, and
vice versa. Neither substance affects the clotting power of blood in vitro,
that is, if added after the blood is removed from the body. Dicumarol
has been put to good use clinically to reduce the danger of the forma-
tion of blood clots inside the blood vessels following surgery.

A chemically related substance, sold under the trade name "Warfarin,"
is an effective poison for rats and mice. It acts by causing fatal hemor-
rhages. Warfarin's advantages for this purpose are that the effects are

222 VITAMINS

SO slow that the rats do not become "bait-shy" and that higher animals
are proportionately less sensitive to it than are rodents.

0=C-CH3
^ I

O f=

L2

H I c H

H H

"Warfarin"

Occurrence

Vitamin K is particularly concentrated in green tissues of plants. Al-
falfa, kale, spinach, carrot tops, chestnut leaves, and oat sprouts are
some of the most potent sources of the vitamin. It also occurs in soy-
bean and other vegetable oils. Cereals and seeds, however, are generally
poor sources.

ASCORBIC ACID (VITAMIN C)
Physiological function

A pronounced lack of vitamin C in the diet leads to a deficiency disease
known as scurvy. The onset is gradual and is characterized by loss of
weight, a sallow or pallid complexion, tendency to fatigue, and shortness
of breath. The gums become swollen, bleed easily, and ulcers may form.
In later stages the teeth loosen and may drop out. Hemorrhages into
the mucous membranes, skin, joints, limbs, and bone marrow, together
with fragility of the bones are noted upon autopsy. These hemorrhages
into the skin are quite obvious during life because blue-black spots occur
after trivial injury, or they may appear spontaneously. The joints be-
come swollen, and fleeting pains are noted in them. Death may eventu-
ally follow the headache, convulsions, and delirium that are seen in the
later stages, or it may be caused directly by such complications as heart
failure or pneumonia. Only two species other than man, namely, guinea
pigs and monkeys, have been found susceptible to scurvy. Other animals
seem to possess the power to synthesize vitamin C. However, the amount

Plate III. Clinical symptoms of vitamin C deficiency (scurvy). A. Inflammation
and congestion about hair follicles. B. Petechiae (multiple small hemorrhages under
the skin) in a scorbutic patient after application of blood pressure cuff. Increased
pressure below the cuff has caused rupture of weakened capillaries. C. Purpura
(purple blotches caused by bleeding under the skin) in a scorbutic patient, from the
pressure of shoes. D. Purpura and petechiae in a scorbutic patient. E. Bleeding
gums in an infant with scurvy. F. Scorbutic gums in an adult. (Reproduced with
permission of Paul Hoeber, Inc., from Clinical Nutrition by Jolliffe, Tisdall, and
Cannon, New York, copyright 1950.)

^'i

*^

.. «(1 .V V

BriORK TREAIMENT

.%

o

0

^^Jf

# ^#

C^^Bf

e> c?^

#/"■)#,

C*fe

18 HOURS AFTER VITAMIN B,.^

A. Hciii<)])<iicii( (blocxl (cll loimiiig) re-
sponse oi hone marrow after injection of
25 miirooianis of \itaniiii R in a patient
Asilli petniiioiis anemia in relapse. The
large blue and pink siinetnres are red
blood cells whose de\(lopinent has been
arrested hv lack of \ilamin li Note the
formation of normal cells (small pink
bodies) after tieatment.

m

•

"i^*«

f"!.

#

90 HOURS AFTER VITAMIN IJ^.,

B. Regeneration of lingnal papillae (normal tongne snrface) after vitamin B^^

administration.

Plate 1\ . Response oi pernicious anemia patients to treatment \\ilh \iiamin B^.,.

VITAMINS 223

synthesized or the rapidity with which the synthesis is carried out may
not always be great enough to supply all the physiological needs of the
animal. This fact has been strikingly demonstrated by the discovery
that injections of vitamin C are of marked value in overcoming various
breeding difficulties of cattle that have long been a source of loss to the
dairy industry.

Since the average human dietary is not likely to be completely lacking
in vitamin C, the symptoms of the earlier stages of scurvy, subacute or
latent scurvy as it has been termed, are of more significance to us. The
formation of intercellular substances which tend to acquire properties
resembling a gel seems to be under the influence of vitamin C. In the
absence of adequate amounts of the vitamin these intercellular substances,
which normally act as a cementing medium, are not formed properly or
may even be resorbed. Such changes may be observed microscopically
in connective tissue, bones, and teeth, and are beheved to occur in blood
vessels as well. Since the tooth changes are rather pronounced and readily
observed, a study of the extent of such changes offers a method for deter-
mining vitamin C. Hemorrhagic tendencies are noticeable even in sub-
acute scurvy. In fact, the "capillary resistance" test, which has been
devised to detect the beginning stages of scurvy, has as its basis the
resistance to development of hemorrhagic spots, through rupture of the
surface capillaries, when a tourniquet is used to produce intravascular
pressure. This condition and other symptoms of scurvy are shown in
Plate III, opposite p. 222.

Prevalence of vitamin C deficiency

Although scurvy in its worst form was quite prevalent in early explora-
tion days, its occurrence at the present time is rather limited. It is said
to be fairly common among the natives in South Africa, and certain tribes
of aborigines in central Australia are reported to suffer considerably
from it. The subacute form seems to be more prevalent than had been
suspected prior to development of the "capillary resistance" test. For
example, students in the university at Upsala, Sweden, showed a decreased
resistance to hemorrhage in the spring corresponding to a proportionate
decrease in vitamin C in the diet. The test has not been employed so
extensively in the United States. However, American investigators have
shown that administration of large quantities of vitamin C is beneficial
in promoting normal tooth formation in the growing child. Such results
suggest a probable deficiency in the diets of many American people.
Some experimental results even point to the probability that certain
rheumatic pains, particularly noticeable in late winter, may actually have
their origin in a lack of vitamin C. A rather interesting observation,
reported in 1939, was the marked improvement that vitamin C produced
in men suffering from lead poisoning.

224

VITAMINS

Chemical nature

Vitamin C isolated in crystalline form from natural sources has been
named L-asc6rbic acid. Its chemical constitution is shown by the struc-
tural formula in the following equation, which also indicates how it is
converted into an oxidized form:

0

II

C

I
COH

COH

I
HC

O

-2(H)

L-ascorbic

acid
^ oxidase
* +2(H)

0

II

c

I

c=o

I

c=o

I

HC

0

HOCH

I
H2COH

L-Ascorbic acid
(reduced form)

HOCH

H,COH

L-Dehydroascorbic acid
(oxidized form)

A close resemblance to the formulas of hexose sugars is evident.

Vitamin C is readily oxidized by mild oxidizing agents, and, in fact,
its tendency to lose hydrogen atoms is so great that it is actually a strong
reducing agent. The vitamin may function in living tissues by virtue of

Courtesy ol' Merck &. Co., Inc.

Fig. 9-4. L-Ascorbic acid.

VITAMINS 225

its reversible oxidation and reduction, as shown in the equation above.
These reactions are catalyzed by ascorbic acid oxidase, which, however,
has been found so far only in plant tissues. The reducing power of vita-
min C has also been utilized in determining by chemical means the amount
of ascorbic acid in a given material. This is accomplished by use of a
dye, 2,6-dichlorophcnolindophcnol, w4iich is reduced by vitamin C to a
colorless form. Hence the quantity of dye that is reduced is a measure
of the vitamin potency of the food. The chemical method for determin-
ing vitamin C agrees well with the results obtained with feeding experi-
ments, and since it takes but a few minutes to make a determination,
chemical analysis has largely superseded biological assay. Atmospheric
oxygen reacts quite readily with ascorbic acid to destroy it, especially at
slightly elevated temperatures and in alkaline solution.

Occurrence

Although paprika exhibits the highest individual concentration of vita-
min C hitherto observed in nature, as a class, the citrus fruits such as
oranges, limes, lemons, and grapefruit, probably deserve to be ranked
first in vitamin C content. Other good sources that find extensive use
in the diet include tomatoes, strawberries, raw cabbage, fresh spinach, leaf
lettuce, turnips, rutabagas, carrots, bananas, apples, and potatoes. The
large consumption of potatoes in the average American dietary rather
than a high content of vitamin is responsible for its contribution to the
total intake of vitamin C. IMilk is too low in vitamin C to furnish an
adequate supply, and, hence, pediatricians recommend administration of
fruit juices early in the life of a baby to compensate for the deficiency.
The bottle-fed baby receives only about one-fourth as much vitamin C
from cow's milk as it would from its mother's milk. Meat and eggs con-
tain very little vitamin C. Cereal grains likewise contain little, if any,
although considerable amounts are formed during germination.

Because of the ease with which vitamin C is destroyed by oxidation and
also because it is water-soluble and therefore lost if the cooking water
is discarded, cooked foods are apt to contain much less than the original
raw materials. Studies made in a Canadian army camp during World
War II revealed that of every 100 mg. of vitamin C in the food brought
into the kitchens only 15 mg. reached the table! Boiling cabbage for
20-30 minutes and discarding the water may cause the loss of over 80
per cent of its vitamin C potency. Even when vegetables such as broccoli
or spinach are allowed to remain at room temperature for 24 hours, 50
to 70 per cent vitamin destruction occurs. The principles to be followed
if loss of this sensitive but vital food factor is to be avoided or minimized
may be summarized thus: (1) eat the food raw if possible, (2) if the
food is to be cooked, use the minimum amount of water, heat quickly to

226 VITAMINS

the boiling point (to destroy ascorbic acid oxidase) and cook no longer
than necessary, (3) use fresh fruits and vegetables immediately after
harvesting, (4) if the food must be stored, keep it cold and protected
from air as much as possible {e.g., wrapped in paper, with the original
husks or skins on, not cut or sliced up, etc.), (5) avoid the use of ma-
terials, especially copper, which catalyze the oxidation of vitamin C.
Many of these same principles apply equally well to the preservation of
other sensitive vitamins. In general, if food is so handled that its vitamin
C is largely retained, all other food factors will most probably be pre-
served also.

Certain tissues of the body are rich in vitamin C. It is particularly
concentrated in the cortex of the adrenal glands, in the lens, aqueous
humor, and vitreous humor of the eye, and in the pituitary gland. In
fact, concentration seems to be correlated with metabolic activity. Al-
though liver contains more than other animal tissues used for food, this
organ does not seem to function so prominently in the storage of vitamin
C as it does in retaining vitamins A and D, and riboflavin.

Requirements

The needs of various persons for this vitamin are shown in Table 9-3,
from which it can be seen that relatively large amounts are required.
Relying upon one food source for the day's supply, one would need to
consume three-fourths cup of orange juice, four to five quarts of milk,
one-fourth pound of raw cabbage, or three-fourths pound of raw toma-
toes.

THIAMINE (VITAMIN B^)

Physiological function

An inadequate intake of this vitamin eventually leads to the onset
of a disorder known in human beings as beriberi and in the experimental
animal as polyneuritis. There are two distinct forms of beriberi: (1)
the dry type, in which there is excessive muscular atrophy, anaesthesia
of the skin, and paralysis of the legs, arms, diaphragm, and intercostal
muscles; (2) the wet type, which is characterized by swelling of the arm,
leg, and trunk muscles; by effusion of fluid into the body cavities; by
congestion of the liver; and by dilatation of the heart with resultant
heart failure. The wet type is somewhat more common in infants, but
symptoms of both types frequently appear in the same patient. In
general, the symptoms appear first in the feet and lower parts of the
legs, and progress upward toward the trunk.

It is now generally agreed that thiamine functions in the animal body

VITAMINS

227

in the form of its pyrophosphoric acid ester, which is known as cocar-
boxylase. This substance is required for the action of various enzymes
(for example, carboxylase and pyruvic oxidase), which function in the
breakdown of pyruvic acid formed during normal carbohydrate metab-
olism. Thus when the body is deprived of an adequate amount of thi-
amine, pyruvic acid accumulates in the blood, heart, and nervous tissues
until toxic concentrations are reached, and the visible symptoms of beri-
beri result. Degeneration of nerve tissue formerly attributed to a lack
of thiamine is apparently the result of other dietary deficiencies. Animals
that receive adequate amounts of vitamin A and riboflavin but an in-
sufficient amount of thiamine show no degeneration of nerve tissue.

Another characteristic symptom in thiamine deficient animals is ano-
rexia, or loss of appetite. Administration of the vitamin to such animals

Courtesy of the Dept. of Biochemistry, University of Wisconsin.
Fig. 9-5. Thiamine deficiency in the chick. The posture is typical of poly-
neuritis in birds.

results in a phenomenal stimulation of the desire to eat, and the animals
may consume as much food in the succeeding twenty-four hours as they
had previously in an entire week. The growth of young animals on thi-
amine deficient diets is subnormal long before the severe symptoms of
polyneuritis become apparent. Thiamine has also been used with fair
success as a stimulant to the appetite in human beings. However, it
should be emphasized that many other factors also affect the appetite
and that thiamine is probably only effective in those cases where the
anorexia is actually due to a lack of this particular vitamin. Decreased

228 VITAMINS

motility of the stomach and intestinal tract with resultant constipation
are also caused by this deficiency.

Perhaps because of its intimate relation to carbohydrate metabolism,
thiamine plays an important role in the life of plants and microorganisms,
as well as higher animals. Many microorganisms cannot be grown with-
out it, and others, although able to synthesize it, make a much faster
growth if it is contained in the nutrient medium. The need for thiamine
seems to be universal, and it probably plays a part in the activities of
all living cells. Plants, however, seem able to synthesize adequate
amounts of thiamine to supply their own needs. Claims regarding the
beneficial effect of thiamine and other B vitamins when applied to grow-
ing plants as yet are unsubstantiated, and the evidence is conflicting.
It seems quite certain that these claims have been grossly exaggerated.

Prevalence of thiamine deficiency

Historically, beriberi has been associated with the Orient, where polished
rice constitutes such a large part of the diet. In spite of the knowledge
for many years that it is a deficiency disease, beriberi is still prevalent
in India, China, Japan, the Philippine Islands, and other Oriental coun-
tries. According to Salcedo and co-workers, beriberi is second only to
tuberculosis as a cause of death in the Philippines. Mortality figures for
1947 were 132 per 100,000 population, and 13 per cent of the population
showed evidence of thiamine deficiency.

Although the disease in its worst form has been considered rare in the
United States, recent studies of various forms of neuritis have revealed
that a number of these exhibit a clinical picture comparable to that of
classical beriberi. Diets in the United States are seldom restricted to
foods so low in thiamine as is polished rice, yet it now seems fairly cer-
tain that the food ordinarily consumed by many people in this country
fails to furnish an "optimal" amount of this vitamin and that a slight
degree of thiamine deficiency is much more widespread than was formerly
supposed. Furthermore, a number of conditions which either increase
the thiamine requirement or in some way interfere with the utilization
of the normal supply probably contribute to the appearance of this de-
ficiency. Excessive consumption of alcohol is an important factor in
this regard, for the caloric value of the alcohol leads to a diminished
food intake, and, in addition, gastrointestinal disorders incident to exces-
sive use of alcohol may reduce the absorption of the vitamin. In one
of our large hospitals 61.6 per cent of 1000 consecutive male patients
admitted to the alcoholic ward in 1935 were suffering from polyneuritis.
Whereas during pregnancy the requirements of the body for thiamine
are materially increased, the nausea and vomiting frequently associated
with this condition may actually curtail the food intake and, with it, the

VITAMINS 229

vitamin supply, thereby inducing a form of neuritis. Since infectious
diseases likewise increase the needs of the body for this vitamin, they may
be causal agents in the production of thiamine deficiencies.

Chemical nature

This vitamin, which has been synthesized by two distinctly different
methods, contains the familiar pyrimidine ring (cytosine, thymine, and
uracil of the nucleic acids are pyrimidines) together with the thiazole ring,
in which the sulfur is contained. In addition to these two rings note
in the formula below that this vitamin is an amine.

CH3
I
N=C— NH2— HCl .C=C-CH2-CH20H

I I >^
CH3-C C— CH2— nC

II II I \
N— CH CI ^C— S

H

. Thiamine chloride hydrochloride, C12H18N4SCI2

In addition to being water-soluble, thiamine dissolves in acids and
dilute alcohol. It is fairly stable to heat in acid solution, but in an
alkaline medium it is rapidly destroyed by heat. Although the evidence
is somewhat conflicting, it appears to be fairly well established that ap-
preciable destruction of thiamine occurs during the cooking of food.
Thus it has been reported that up to 57 per cent of the thiamine may
be lost by stewing meat, about an equal amount by roasting or baking,
and 10 to 30 per cent by frying. Baking bread causes about 15 to 20
per cent loss. The destruction of thiamine by boiling vegetables has
been reported to amount to as much as 22 per cent, with an additional
15 per cent present in the cooking water. The amount of destruction
increases rapidly at temperatures above 100° C. Pressure cooking, there-
fore, causes considerably more destruction of thiamine than cooking at
ordinary pressure.

An enzyme (thiaminase) in certain fish, mostly of fresh water origin,
has the specific property of destroying thiamine. Foxes fed diets contain-
ing raw carp develop a paralysis which was found to be actually a thiamine
deficiency, brought about through the agency of thiaminase. Cooking
the carp, or feeding extra amounts of thiamine to the animals prevented
the disease.

In the last few years several relatively quick chemical methods of
assaying foods for their thiamine content have been proposed. Perhaps
the most widely used is the "thiochrome method," which is based on the
oxidation of thiamine, in an extract of the food, to thiochrome, which
shows a characteristic bluish fluorescence in ultraviolet light. The in-

230

VITAMINS

tensity of this fluorescence serves as a measure of the amount of thiamine
that was present in the food. Such assay methods are now used much
more extensively than the longer animal feeding methods, although the
latter are still relied upon as the final test.

Courtesy of ]\Iei'<'k & Co., Inc.

Fig. 9-6. Thiamine.

Occurrence

The richest known source of thiamine is brewer's yeast. It has been
found, however, by Kingsley and Parsons that the greater part of the
thiamine (and riboflavin) in yeast is not utilized by human beings unless
the yeast is given some previous treatment such as cooking which kills
the yeast cells. Next in order of concentration follow pork muscle, rice
polishings, and bran of grains. Obviously, cereals can constitute a good
source of this vitamin only when the entire grain is used. If considered
on the dry basis, most vegetables and fresh fruits are fairly potent
sources. Eggs and most meats contain appreciable amounts of the vita-
min. Synthetic thiamine is being employed to an increasing extent for
the fortification of commercial foods, particularly white flour.

Requirements

The amount of thiamine needed daily by a normal person depends on
the amount and kind of food that he consumes. As indicated above,
this vitamin functions in the metabolism of carbohydrate in the body,

VITAMINS 231

and it probably also is involved in the utilization of excess dietary pro-
tein as a source of energy. It is not apparently needed for the metab-
olism of fat, and the inclusion of a large amount of fat in the diet exerts
a thiamine-sparing action. In short, it may be said that the thiamine
requirement is related to the nonfat calories provided by the food con-
sumed. Abnormal conditions affecting the thiamine requirement of the
body have been considered on pp. 228 and 229.

Table 9-3

Daily human requirement for water soluble vitamins*

Nicotinic Ascorbic

Thiamine Riboflavin acid acid

Subject mg. mg. mg. mg.

Man (154 lb., 70 kg.)

Moderately active 1.5 1.8 15 75

Very active 1.8 1.8 18 75

Sedentary \2 1.8 12 75

Woman (123 lb., 56 kg.)

Moderately active 1.2 1.5 12 70

Very active 1.5 1.5 15 70

Sedentary 1.0 1.5 10 70

Pregnancy (latter half) 1.5 2.5 15 100

Lactation 1.5 3.0 15 150

Children

Under 1 year 0.4 0.6 4 30

1-3 years (27 lb., 12 kg.) 0.6 0.9 6 35

4-6 years (42 lb., 19 kg.) 0.8 1.2 8 50

7-9 years (58 lb., 26 kg.) 1.0 1.5 10 60

10-12 years (78 lb., 35 kg.) 1.2 1.8 12 75

Girls

13-15 years (108 lb., 49 kg.) 1.3 2.0 13 80

16-20 years (122 lb., 55 kg.) 1.2 1.8 12 80

Boys

13-15 years (108 lb., 49 kg.) 1.5 2.0 15 90

16-20 years (141 lb., 64 kg.) 1.7 2.5 17 100

* As recomended by the Food and Nutrition Boai'd of the National Research Council,
1948.

Detailed estimates of the amount of thiamine needed daily by various
persons are shown in Table 9-3. For most people approximately 1-2
mg. daily constitute a safe intake. Quantities of representative foods
that would supply an amount within these limits are: 1 lb. brown rice,
4 lb. cabbage, 2 lb. asparagus, i/4 lb. lean pork chop, 2 qt. whole milk,
or 5 lb. white flour. If desired, the above amounts of thiamine may be
expressed as international units, on the basis that 3 /^g. of the pure vita-
min correspond to one unit.

One of the largest dietary sources of thiamine, as well as of niacin and
riboflavin, would be bread and other grain products, except for the great
losses of these vitamins which occur on milling (Table 9-4). To com-

232

VITAMINS

pensate for these losses white flour and bread are now usually enriched
with the three pure vitamins, at least to the levels.

Table 9-4

B vitamins in bread and flour

Whole wheat Enriched white Enriched white

Vitamin flour* White flour* flour *1i bread *t

Thiamine 0.55 0.066 0.44 0.33

Riboflavin 0.12 0.033 0.26 0.26

Niacin 5.56 0.77 3.52 2.75

* Milligrams per 100 g. t Minimum standards.

RIBOFLAVIN

Physiological function

The outstanding symptom in young animals or birds fed on a ration
low in riboflavin is retarded growth. In poultry, deprivation of this
vitamin leads to diminished egg production and especially to a failure
of the eggs to hatch. Continued deficiency in the chick causes a condi-
tion known as "curled toe paralysis," in which the bird is unable to walk
and eventually dies. Examination of such chickens reveals extensive
nerve degeneration.

The necessity of riboflavin has also been demonstrated for rats, dogs,
pigs, and man. In a series of outstanding studies Sebrell and his co-
workers of the United States Public Health Service have shown that
when adult persons subsist for an extended period of time on a diet low
in riboflavin, they contract an illness of which characteristic symptoms
are soreness and inflammation of the tongue (glossitis), and cracks and
sores on the lips and at the corners of the mouth (cheilosis). Further-
more, such patients nearly always suffer from various disorders of the
eye. These include abnormal sensitivity to light, dimness of vision, and
inflammation and development of blood vessels in the cornea. All of
these symptoms respond promptly to the daily administration of 5 to
10 mg. of pure riboflavin. It is probable that human riboflavin deficiency
often accompanies pellagra, although masked somewhat by the more
striking symptoms of the latter disease, and that it is rather widespread
among the population of America. It has also been reported in India,
the West Indies, and many parts of Africa. A children's disease known
as "perleche," occasionally" seen in the southern United States, is actually
a riboflavin deficiency.

In living tissues riboflavin is built up into more complex substances,
namely, riboflavin phosphate and flavin-adenine-dinucleotide (abbreviated
FAD, p. 277), which serve as coenzymes for a series of enzymes involved
in metabolism. These enzymes, which are called flavoproteins or "yellow

VITAMINS

233

enzymes" because of the color imparted to them by the flavin group,
have the function of catalyzing the removal of hydrogen atoms from
certain metabolites and passing the hydrogen to some acceptor, such as
cytochrome c or molecular oxygen. Xanthine oxidase, d- and L-amino
acid oxidases, and cytochrome c reductase are examples of flavoproteins.
As a result of its participation in the make-up of these enzymes, ribo-
flavin fulfills a vital role on the "main line" of the oxidation processes
by which food energy is made available to living tissues. Like the other
B vitamins, it apparently is needed by all living cells.

\ * V

-mrn^

&

fc. ^ -:.

Courtesy ol' Meixk & Co., lac.

Fig. 9-7. Riboflavin.

Chemical nature

Riboflavin, which previously has also been called vitamin B2 or vitamin
G, is a water-soluble pigment widely distributed in plant and animal
materials, although in very minute concentrations. Dilute solutions of
it have a greenish yellow color and in ultraviolet light show a charac-
teristic greenish yellow fluorescence. It is, for example, the substance
mainly responsible for the rather faint but distinct color of whey and
of egg white It was first obtained in the pure state in 1933 and was
synthesized in 1935. The pure vitamin, which is an orange-red powder,
is now readily available It is produced mainly by fermentation with
special riboflavin-producing microorganisms, for example, Eremothedum
ashbyii.

234 VITAMINS

The structural formula of riboflavin is:

H CHj— (CH0H)3— CH2OH
CHa— C^ \r ^C^ ^C=0

I A II B I C I

J II

H O

Riboflavin, C17H20N4O6

Note the benzene A and pyrimidine C rings, as well as the sugar-like side
chain, which is related to the rare pentose sugar, ribose.

Riboflavin is very stable to heat, acids, and oxidizing agents, but is
easily destroyed by light and by alkalies. Losses attendant on the cook-
ing of food may range from 0 to 60 per cent. A quart of milk in an
ordinary clear glass bottle, if set in direct sunlight, may lose as much
as 50 per cent of its riboflavin content in two hours. Brown glass bottles
or paper cartons prevent this destruction. Although riboflavin is a water
soluble vitamin, surprisingly little — perhaps 10 to 20 per cent — is removed
by boiling vegetables in water. This is probably attributable to the
fact that in tissues it exists largely combined with various proteins.

Occurrence

Riboflavin is formed primarily in green leaves of actively growing
plants; hence, green leafy vegetables constitute a good source of this
vitamin. Brewer's yeast is a higlily potent source. Of the various meats,
liver contains the greatest concentration of the vitamin and is followed
closely by kidney. Tlie muscle meats likewise contain appreciable
amounts. Milk and eggs are quite satisfactory sources, and in the well-
planned diet the former contributes largely to the total riboflavin
intake.

At present several fairly reliable methods other than animal assay are
available for determining the amount of riboflavin in foodstuffs. Chem-
ical methods depend on the measurement of the characteristic color or
fluorescence in suitably clarified extracts of the food. Still another
method of assay has been based on the fact that certain lactic acid bac-
teria require riboflavin for normal growth and acid production. An
aqueous suspension of the sample is fermented by the organism, and the
amount of lactic acid produced (as determined by titration of the entire
culture) serves as a measure of the riboflavin present. Similar assay
methods are used for most of the other B vitamins and for amino
acids.

VITAMINS

235

Requirements

Detailed information regarding the riboflavin requirement of man is
given in Table 9-3. In general, a daily intake of 1.5-3.0 mg. would
appear to be adequate. However, Sherman concludes, from a series of
experiments on rats carried out over a long period of time, that the
optimum intake, as indicated by a higher level of health and general
well-being, may be several times as great as the minimum amount needed
to prevent deficiency symptoms. It does not seem likely that harmful
effects will result from too large an intake of riboflavin, since excessive
doses are rapidly excreted in the urine.

Amounts of representative foods that would supply approximately 2
mg. of riboflavin are: l^/^ lb. lean beef, 2 oz. liver, 1 lb. kale, 1 qt. whole
milk, 1 lb. eggs, 13 lb. white flour.

NICOTINIC ACID (NIACIN)

Physiological function

Extreme deficiency of nicotinic acid produces a disease in man known
as pellagra. This disease is characterized by roughness and pigmentation

Courtesy of J. M. Ruffln, D. T. Smith, and The Southern Mediwl Journal.
Fig. 9-8. Typical dermatitis of pellagra.

236

VITAMINS

of the skin, particularly on the hands, arms, face, and back of the neck.
Apparently the sun exerts an irritating effect, causing exposed areas of
the skin to exhibit first this abnormality. The mouth becomes sore be-
cause of lesions which develop on the mucous membrane, and the tongue
may become sore, red, and swollen. Indigestion and diarrhea are asso-
ciated conditions, and in severe cases insanity may result. The symptoms
of pellagra have been summed up by the "three D's": dermatitis, diar-

Courtesy of J. M. Ruffln, D. T. Smith, and The Southern Medical Journal.
Fig. 9-9. Effect of nicotinic acid on dermatitis of pellagra.

rhea, and dementia. In dogs the disease is known as black-tongue. Pigs
and monkeys have also been shown to develop a similar disease when
deprived of nicotinic acid. Rats, on the other hand, apparently do not
require this vitamin in the diet.

Nicotinic acid, like riboflavin, is an essential constituent of several en-
zyme systems. In fact, it is a definite part of triphosphopyridine nucleo-
tide (TPN, p. 275) , without which one cytochrome reductase cannot func-
tion. In addition to the amide of nicotinic acid this coenzyme contains
adenine, two molecules of ribose, and three molecules of phosphoric acid.
A compound differing from this only in that it contains one less phos-
phoric acid molecule is diphosphopyridine nucleotide (DPN), which is
essential to the breakdown of glucose by animals and to fermentation
by yeast. Its function as a part of oxidation-reduction systems is the

VITAMINS

237

important role played by nicotinic acid, or nicotinamide, in many, if not
all, living cells.

Prevalence of nicotinic acid deficiency

Next to rickets, pellagra is probably the most common vitamin de-
ficiency disease in the United States. Although doubtless occurring prior
to the twentieth century, it was not recognized here until about 1907 or
1908. The disease is confined largely to the southern states where the
diet of the poorer classes is derived chiefly from corn meal, molasses, and
fat pork. The United States Public Health Service estimated that in
1929 there were 200,000 cases of pellagra in the country. In 1930 pellagra
was reported to have caused 7146 deaths, of which 98 per cent were
in the cotton belt. In contrast to the above figures, there were reported
in 1940 only 8688 cases of pellagra. Pellagra also occurs in Egypt,
Rumania, and South Africa, always in areas where corn is a main article
of diet. Over 1000 cases annually were reported in South Africa during
1944-1947.

Symptoms of niacin deficiency in animals are relieved by administer-
ing tryptophan, and it is now known that tryptophan can be converted
into nicotinic acid in the animal body (p. 347). Since corn contains
little tryptophan, this relationship helps to explain why pellagra develops
especially among corn-eating populations. It has been suggested also
that corn may contain an antivitamin (p. 256) antagonistic to niacin.
In addition to these complicating factors, it is probable that pellagra
also involves deficiencies of other vitamins besides niacin. Nearly all
cases of pellagra require treatment with thiamine and riboflavin, in addi-
tion to nicotinic acid, in order to clear up all of the symptoms, and some
have also been reported to benefit from the use of pyridoxine. For this
reason it is very probable that the pellagra problem cannot be solved
by the use of nicotinic acid alone, but that the answer lies much more
in a general improvement of the diet. It is probable that this deficiency
disease will not be eradicated in the South until the prevailing diet of
corn bread, molasses, and fat meat is properly supplemented. This con-
stitutes an economic problem, since practically all potent sources of
the vitamin are relatively expensive foods, which the poorer classes are
unable to buy.

Chemical nature

Nicotinic acid is a relatively simple derivative of pyridine and possesses
a structure represented by the accompanying formula. The correspond-
ing amide is also effective as a vitamin:

238

VITAMINS

H 0

^C, II
HC^ ^C—C—OU

II I

Nicotinic acid, C6H5NO2
(Niacin)

H O

II

HC^ ^C— C— NHj
II I

Nicotinamide, CeHjONj
(Niacinamide)

As its name implies, the compound is closely related to nicotine, from
which it can be easily formed by oxidation in the laboratory. This
vitamin is soluble in dilute alcohol and in water; it is very stable to
heat, light, acids, alkalies, and oxidizing agents. In fact, it is more
resistant to chemical attack than any of the other vitamins. Nicotin-
amide is preferred for therapeutic use since it is less likely to cause
a burning sensation of the face that is often noted after doses of nicotinic
acid.

Occurrence

Nicotinic acid is a substance that was known to organic chemists for
many years before its usefulness as a vitamin was discovered. It is now
prepared synthetically on a rather large scale and is among the most
easily obtainable and cheapest of the vitamins. An amount sufficient
to cure a pellagrin costs only about ten cents. The synthetic material is
now being used in the enrichment of white flour. The term "niacin" has

-Z^r^

?,.j»f.

I; .*i

'^-

Courtesy of Merck & Co., Inc.
Fig. 9-10. Nicotinic acid.

VITAMINS

239

been recommended as a more suitable name for nicotinic acid in com-
mercial products.

Good food sources of this vitamin are liver, lean meat, and yeast. Fair
sources are certain whole cereals, legumes, and wheat germ. Milled
cereals, fats, and molasses are low in nicotinic acid. Several procedures
have been suggested for assaying foodstuffs for their nicotinic acid content.
Of these the most widely used are the dog assay based on the cure of
black-tongue, a chemical method based on the development of a yellow
color when an extract of the sample is treated with cyanogen bromide
and aniline, and a bacterial method similar to the procedure described
for riboflavin.

Requirements

Estimates of the amount of nicotinic acid (or its equivalent in the
form of nicotinamide, or other related substances such as DPN and
TPN) needed daily by various persons are summarized in Table 9-3.
Notice how much larger amounts are required than in the case of other
B vitamins. Quantities of representative foods which will probably
supply 10 to 20 mg. of nicotinic acid are: 1 oz. dried yeast, 3 oz. pork
liver, 1/2 lb. lean beef or pork, 4I/2 lb. spinach, or S^^ lb. tomatoes.

PANTOTHENIC ACID

Physiological function

The existence of pantothenic acid was first suggested by Williams and
associates in 1933, as a result of their work on the stimulation of yeast
growth by extracts of various biological materials. The active substance
present has been found to be identical with the dietary factor that pre-
vents chick dermatitis, a disease that was for a time thought to be
analogous to pellagra in man.

When young chicks are placed on a ration deficient in this vitamin,
crusty scabs form at the corners of the mouth and gradually enlarge
until the skin around the nostrils and underneath the lower mandible
is affected. Growth ceases and feathering is retarded. Death may result
within two or three weeks after these symptoms become apparent. Ad-
ministration of pure calcium pantothenate causes resumption of growth
and disappearance of the dermatitis.

Rats, dogs, and swine have also been found to require this vitamin.
The black portions of the fur of rats and foxes kept on diets low in
pantothenic acid have been observed to turn gray. They have been found
to regain their normal color when the vitamin was administered (Fig.
9-15. p. 255). Pantothenic acid has also been shown to be essential
in the nutrition of a number of lower organisms, especially yeasts and
lactic acid bacteria. There is fairly definite evidence also that panto-

240

VITAMINS

thenic acid is involved in human nutrition, although clinical experience
in this direction is still rather meager.

Pantothenic acid functions as part of a coenzyme (coenzyme A or Co yl)
in a system which brings about the condensation of acetic and oxalacetic
acids to form citric acid, one of the steps of the citric acid cycle (p. 330).
In fact Co A is probably needed for all metabolic reactions of the "two
carbon fragment" (acetic acid or some closely related substance) pro-
duced during the oxidation of fats and carbohydrates in the body. Since
this fragment is also used for the biological production of fats, steroids,
acetyl choline, and probably many other products, the indispensable nature
of pantothenic acid for living organisms is easily understandable.

Chemical nature

Pantothenic acid is a peptide-like compound composed of y8-alanine
united through an amide linkage to an hydroxy acid. The complete
structural formula is:

T' ?

CH2— C— CH— C— NH— CH, — CHj— COOH

III
HO H3C OH

Pantothenic acid, C9H17O5N

The details of this formula were worked out in 1940, and the synthesis
of the vitamin was also accomplished in the same year. The substance

■ ■ ' ■■■' ■' >'/ A: ^1

1r J<"

<•■'. _„„'**_.// .-^

Courtesy of Merck & Co., Inc.
Fig. 9-11. Pantothenic acid.

VITAMINS 241

occurs in two forms, dextrorotatory and levorotatory, and of these only
the dextrorotatory form has biological activity. Since free pantothenic
acid can readily be obtained only as a sirupy, gummy mass, it is usually
converted to the calcium salt, which is a w^iite powder and the form in
which the synthetic product is supplied.

Since it is an amide, pantothenic acid is readily hydrolyzed by heating
in either acid or alkaline solution. Hydrolysis results in complete de-
struction of the vitamin activity. It is rather stable to boiling in neutral
aqueous solutions, although it is destroyed by long heating at 120°C.

It appears that pantothenic acid is not" extensively destroyed by ordi-
nary cooking of food. Losses of approximately 50 per cent may, however,
occur if the cooking water from vegetables is discarded.

Occurrence

Yeast, liver, egg yolk, and rice polishings are very rich sources of
pantothenic acid, while dairy products, whole cereals, muscle meats,
green leafy vegetables, and certain other vegetables like cauliflower and
sweet potato, may be classed as good sources. Fruits and egg white are
low in pantothenic acid.

The assay of foods for this vitamin is based on the growth response
of chicks when fed the test material. A bacterial method very similar
to the one described above for riboflavin has also been developed. The
human requirement for pantothenic acid has not yet been determined,
but it has been suggested that about 10 mg. per day is adequate.

PYRIDOXINE (VITAMIN B^)

Physiological function

Rats receiving an inadequate supply of this vitamin develop a derma-
titis, which makes its appearance in a characteristic manner. The paws
and tips of the ears and nose are first affected, becoming red and swollen.
The area immediately surrounding the nostrils becomes bare, and there
may be a nasal discharge. The administration of pure pyridoxine ma-
terially improves the condition of the rat, but even more striking improve-
ment results from the use of certain fats, especially those which supply
the so-called "essential fatty acids." The relation between the physio-
logical action of these fatty acids and pyridoxine is not yet clear. It
may well be that both are required for the normal nutrition of the rat.
Neither black-tongue, pellagra, nor chick dermatitis is cured by pyri-
doxine. It has been shown, however, that pyridoxine is required by
dogs, swine, pigeons, and chickens, and several reports indicate that it
is also important in human nutrition. Deficiency symptoms that have

242

VITAMINS

been encountered in various animals include a type of anemia and fits
resembling epileptic seizures in human beings. Pyridoxine deficiency in
man has been observed in a number of cases of pellagrins who still were
not completely well after receiving nicotinic acid, thiamine and riboflavin.
Symptoms noted in such patients were nervousness, irritability, abdominal
pain, weakness, and difficulty in walking. These symptoms were quickly
relieved by the use of synthetic pyridoxine.

Chemical nature

Pyridoxine, or vitamin Be, was first isolated as a pure chemical sub-
stance in 1938, and during the next year it was prepared synthetically.

('ouite.sy of MeiL'k & Co., Inc.
Fig. 9-12. Pyridoxine.

The chemical nature of this vitamin is best expressed by its structural
formula:

CH2OH

I

II (2) (6) I

CH3— C^(i)^CH

Pyridoxine, CsHuNOa

Note that it is related to nicotinic acid in that it is a pyridine derivative.
The name pyridoxine is derived from the chemical name for this substance,

VITAMINS 243

which is 2-methyl-3-hydroxy-4,5-di- (hydroxymethyl) -pyridine.

Two closely related substances, pyridoxal and pyridoxamine, are repre-
sented by the following formulas:

CHO CH2NH2

I I

c c

HO— C-^ "==0— CH2OH HO— C^ ^C— CH2OH

HaC-C^^^CH HsC-G^^^CH

Pyridoxal, CsHgOsN Pyridoxamine, C8H12O2N2

These substances have about the same vitamin Be activity for animals and
for yeast cells as pyridoxine does, but are several thousand times more
effective for certain bacteria. A phosphorylated derivative, pyridoxal
phosphate, functions as a coenzyme for enzyme systems present in many
bacteria, which break down amino acids into the corresponding amines
by removing carbon dioxide from the carboxyl group of the amino acid
(p. 321) . It is therefore called a codecarboxylase. Both pyridoxal phos-
phate and pyridoxamine phosphate function as coenzymes in certain trans-
amination reactions (p. 343) and may, therefore, be called cotransami-
nases.

CHO CH2NH2

I I

HO— C^ ^C— CH2O-PO3H2 HO— C^ >C— CH2O— PO3H2

I II I II

H3C— C^^.^H H3C— C*^^,^CH

Pyridoxal phosphate Pyridoxamine phosphate

Pyridoxal phosphate also serves as a coenzyme for the enzyme system
involved in the synthesis of tryptophan by a certain mold species {Neuro-
spora crassa) . It is, therefore, quite clear that the Be vitamins play
important roles in both the decomposition, inter conversion, and synthesis
of amino acids in living cells.

Pyridoxine is stable to heat, alkalies, and strong acids, but is rather
easily attacked by oxidizing agents. As yet little work has been done
on its destruction during the cooking of food. No reliable figure for the
human requirement is available, but a tentative value of 1.5 mg. per
day has been suggested.

Occurrence

Pyridoxine is present in yeast, bran and embryo of cereal grains, meats,
milk, and leafy vegetables. The amount of this vitamin in a number
of common foods is given in Table 9-5.

244

VITAMINS

Table 9-5
Pyridoxine content of common foods

Milligrams per 100 g.
edible portion

Beef, lean 0.40

Beef, liver 0.73

Bread, white 0.30

Bread, whole wheat 0.70

Cabbage 0.29

Carrot 0.19

Chicken, dark meat 0.20

Lamb, leg of 0.38

Milk, whole '. 0.20

Oatmeal 0.25

Pork loin 0.60

Potatoes, white 0.16

Yeast, dried brewer's 5.5

BIOTIN

This member of the vitamin B complex is a substance which has been
variously known as "coenzyme R," "vitamin H," "biotin," and the "anti-
egg white injury factor." It was first obtained in pure form and given
the name biotin in 1936 by Kogl, who was studying it as one of the
vitamin-like substances required for normal yeast growth.

Physiological function

The feeding of biotin brings about the cure of a nutritional disease
which develops when rats, chickens, or human beings consume large
amounts of raw egg white. This "egg white injury" disease is primarily
a dermatitis, characterized in the rat by swelling and inflammation of
the skin, especially around the mouth, and by loss of hair. The disease
is actually an induced biotin deficiency caused by the combination of
the biotin normally present in the food with a particular protein, avidin,
present in raw egg white. When so combined, biotin cannot be absorbed
and utilized by the animal organism. Cooked egg white on the other
hand is perfectly safe in the diet, since heating to 100°C. destroys the
ability of avidin to combine with the vitamin.

Although the above facts demonstrate that biotin is an indispensable
nutrient, it has not been possible to produce the "egg white injury" dis-
ease in rats by feeding them diets extremely low in biotin. Apparently
a sufficient supply of the vitamin to meet the needs of the animal is
synthesized by bacteria in the intestinal tract. However, this deficiency
can be produced in the chick without the use of raw egg white. Like-

VITAMINS

245

wise many of the lower organisms such as yeasts, bacteria, and fungi
do require biotin for normal development. No biotin deficiency has been
observed in human beings consuming their customary diets. The daily
intake of biotin on an average diet ranges from 25 to 50 ixg., and the
urine and feces together may contain from two to five times these
quantities.

Fig. 9-13.

Courtesy of the S.
Biotin.

M. A. Corporation.

Biotin appears to function (possibly in the form of a coenzyme, al-
though none has yet been identified) as a catalyst for one of the reac-
tions of the citric acid cycle (p. 330) :

COo + CH3— CO— COOH
Pyruvic acid

-> HOOC— CHo— CO— COOH
Oxalacetic acid

Further, certain lactobacilli which normally require biotin grow well with-
out it if oleic acid is supplied instead. This observation indicates some
kind of a metabolic relationship between these two substances, perhaps
participation of biotin in the biosynthesis of oleic acid. The vitamin is
also required for deamination by bacterial cells of serine, threonine, and
aspartic acid.

Chemical nature

Biotin has a two ring structure with a side chain attached to one of
the rings. It is an acid, as is indicated by the carboxyl group in the
side chain. Note the urea-like structure in one of the rings {A) and the

246 VITAMINS

presence of sulfur in the other ring (B) . Biotin and thiamine are the only
vitamins that contain sulfur.

0

II

c

NH "^NH

A

CH CH

I B I
CH2 CH-(CH,)4-C00H

S

Biotin, C10H16O3N2S

Although it is readily destroyed by such oxidizing agents as hydrogen
peroxide, biotin is, in general, a very stable substance. It is not affected
by light, strong acids such as normal HCl or H2SO4, nor by exposure to a
degree of heat greater than that encountered during ordinary cooking
operations. However, it is destroyed by strong alkali. ^ In many tissues
it appears not to exist in a free state, but in combination with some cell
constituent, presumably protein. This view is supported by the recent
isolation from autolyzing yeast of biocytin, a peptide-hke combination
of biotin and the amino acid, lysine. Note that the linkage is through
the evsilon amino group of lysine.

0

II

I I

HC CH

I I

H2C CH(CH2)4CONH— (CH2)4— CH-COOH

S I

NHi

Biocytin

Nothing is known as yet regarding the amount of biotin needed by
human beings. However, the quantities required by various lower organ-
isms are so extremely minute that it must be regarded as one of the
most highly active substances known. Its effect on yeast growth, for
example, can still be detected at dilutions of 1:300,000,000,000.

PTEROYLGLUTAMIC ACID

This vitamin was first observed in connection with studies on the nutri-
tional requirements of lactic acid bacteria. An impure preparation from
liver, designated as the "norite eluate factor," was shown to be necessary,

VITAMINS

247

in addition to previously known vitamins, for the normal growth of these
organisms. The effective substance present in such preparations was
later found to be identical with "factor U" and "vitamin M," which at
that time were still unidentified, but were recognized as dietary essentials
for chicks and monkeys, respectively. Other investigators, working with
various experimental animals, proposed still other names for vitamin-
like substances which eventually turned out to be pteroylglutamic acid,
or closely related compounds. These names included "vitamin Be,"
"factor R," "factor S," "folic acid," "S. lactis R factor or SLR factor,"
"liver L. casei factor." The term folic acid is still in use, but should
now be replaced by the proper chemical names (see below).

Chemical nature

Pteroylglutamic acid is a complex substance made up of three parts,
glutamic acid, para-aminobenzoic acid (p. 254), and a pterin, chemically
linked together:

H

_N N C

H2N— C^')"^C^'^*)^CH HC^^^'^'^C-CONHCHCHiCHiCOOH

1(2) II (^) I II (5') (l')| I

N5^)i^(^)Sc-CH.NH-ci:;;3')|^tH COOH

I H

OH

Pteroylglutamic acid

This substance is identical with the "liver L. casei factor," vitamin Be,
and folic acid. The name folacin was proposed in 1949 by the American
Institute of Nutrition as a synonym for folic acid. The "fermentation
L. casei factor" is very similarly constituted except that three glutamic
acid residues are present. In this form, also called teropterin, the second
and third glutamic acid residues are linked to the preceding one through
the gamma carboxyl group rather than through the alpha carboxyl (see
p. 131). This is the same type of peptide linkage as is found in gluta-
thione. Still another form, vitamin Be conjugate, contains seven glutamic
acid residues. The SLR factor, or rhizopterin, contains no glutamic acid
at all but bears an aldehyde or formyl group on the nitrogen atom in
position 10:

H,N-G^(^^C-^S^CH CHO C-C

1(3) II el I / W

n4:(4) ^C^ (5)!^^C-CH,-N-C c-cooh

C N (9) (10) \ /

I HC=CH

OH
Formylpteroic acid (or rhizopterin)

248 VITAMINS

The glutamic acid derivative of rhizopterin, formylpteroylglutamic acid,
or formyl folic acid, has been prepared synthetically and found to possess
the typical vitamin activity of other members of this group. It probably
also occurs naturally. Rhizopterin itself, however, does not relieve the
symptoms of folic acid deficiency in higher animals.

Very recently a substance needed for normal growth of the bacterium
Leuconostoc citrovorum (the so-called "citrovorum factor") has been
found to be closely related to formylpteroylglutamic acid, from which
it can be obtained by reducing and heating. The product, named folinic
acid by one group of investigators and leucovorin by another, has been
shown (Consulich, et al, Pohland, et al.) to have the following formula:

.^

H
N^ /N. H H

HsN-C^ C" XH2 C — C COOH

.L.

// \

^ '^ H(i-CH,HN-C C-CONHCHCHaCH^COOH

I I C=C

OH CHO ^ ^

Folinic acid (or leucovorin)

In many tests folinic acid possesses higher activity than other members
of the folic acid group. It may be the metabolically active (coenzyme)
form of this vitamin, or at least it may be more closely related to the
coenzyme than pteroylglutamic acid itself.

Physiological function

This vitamin is essential for a wide variety of living organisms, and,
in fact, is probably needed by all living cells. The outstanding deficiency
symptoms in higher forms (mammals, birds) are anemia, leucopenia (a
reduced number of white blood cells) , weight loss, oral lesions, and diar-
rhea. In the chick the deficiency also results in abnormally poor
feathering.

That several hurhan diseases are the result of a lack of pteroylglutamic
acid or related substances is indicated by the improvement which follows
their administration. The best example is sprue, a disease characterized
by macrocytic anemia (enlarged red blood cells), leucopenia, glossitis
(inflammation of the tongue) , diarrhea with large amounts of fatty ma-
terial in the feces, weight loss, and poor absorption of food from the intes-
tine. Daily doses of 10 mg. of pteroylglutamic acid or of the triglutamate,
teropterin, result in prompt relief of these symptoms. Related conditions
described as nutritional macrocytic anemia and macrocytic anemia of
pregnancy are similarly benefited. Pernicious anemia patients are bene-
fited somewhat, but the improvement is temporary and incomplete, in
contrast to the effects of vitamin B12 (see below).

VITAMINS 249

In all of the above diseases the administration of relatively large daily-
doses (about 4 g.) of a simple pyrimidine compound, namely thymine
(p. 155), has an almost equally beneficial result. From this and other
evidence it seems probable that the biological function of pteroylglutamic
acid is concerned with the biosynthesis of thymine and other components
of nucleic acids. Teropterin has been claimed to relieve pain in advanced
cases of human cancer and to retard the growth of tumors in experimental
animals., '„€)

Ir

Food sources and requirements fe^nl^,

The pteroylglutamic acids are rather sensitive substances which may
be quite largely destroyed during the cooking of foods. Losses of 50
to 90 per cent have been reported in meats cooked in different ways.
Vegetables kept for three days at room temperature lost 20 to 80 per
cent, and large losses occurred during canning. When a solution of the
pure vitamin was placed in bright daylight for 8 hours, 88 per cent was
destroyed. 'i^^ t>

.^According to Toepfer ^H co-workers a number of common foods may
be^rouped as follows, oA4he basis of the milligrams of folic acid which
they contain per 100 g. of dry weight: Over 1,0:^. brewer's yeast, chicken
liver, _^sparagus, broadleaf endive, broccoJi, l^^'lettuce, spinach; 0.4-1.0,
most of the other leafy greens, liver, blackey'e peas, dried beans, soy
flour; O.l-d.4, other vegetables except root vegetables and a few fruits;
0.03-0.1, root vegetables, most fresh fruits, grains and grain products,
nuts, lean beef; 0.03 or less, eggs, milk, meats (other than beef), poultry.

The amount of pteroylglutamic acid normally required by human
beings has not been established. Various animal species need 0.005 to
0.06 mg. per kilogram of body weight per day.

VITAMIN B^o

It has long been recognized that liver and suitable extracts prepared
from liver contain some substance which is effective in the treatment
of pernicious anemia, a serious, wasting disease of man, which if untreated
is invariably fatal. IMany efforts to isolate and identify the "antiper-
nicious anemia factor" in liver have been made. With the discovery
of pteroylglutamic acid and the observation that it is effective in curing
certain pathological blood conditions, it seemed that the long-sought
substance might have been found. However, continued treatment of per-
nicious anemia patients with pteroylglutamic acid proved disappointing,
since the initial improvement did not last and was often followed by
severe neurological complications.

Finally, in 1948, a red crystalline substance was isolated from liver

250

VITAMINS

which proved to be effective against pernicious anemia in amazingly
small doses (Fig. 9-14). The new material, designated vitamin Bio,
contained 4.4 per cent cobalt, 2.3 per cent phosphorus and had the formula
C61-64H86-92N14O13PC0 (molecular weight about 1350). Although the

Courtesy of Abbott Laboratories.
Fig. 9-14. Vitamin Bio crystals (x 200).

complete structure is not yet known, several fragments of the molecule,
including 5,6-dimethyl benzimidazole, have been identified after acid
hydrolysis. Surprisingly, this substance itself showed full vitamin B12
activity for rat growth when tested in 5 mg. daily doses. Other hydrolysis

HoCC

^

H

,N,

\

CH

X'^ N

H H

5,6-Dimethyl benzimidazole

products identified are propanolamine (CH3CHOHCH2NH2) and a phos-
phorylated derivative of the 5,6-dimethylbenzimidazole (ribose-3-phos-
phate attached to the N at position 1). Vitamin Bio also contains a

VITAMINS 251

cyanide group (CN) bound in a coordination complex with the cobalt
atom, which can be replaced by CI, SO4, OH, SCN, or other groups to
produce analogs of the natural substance. The analog containing the
water molecule has been called vitamin Bi2a and is apparently identical
with another preparation provisionally designated Bi^b- Brink and co-
workers have suggested that the B12 molecule, except for the cyanide
group, be called cohalamin. By this nomenclature, vitamin B12 would
be named cyano-cobalamin and Bi2a, hydroxo-cobalamin. All of these
various forms of the vitamin have approximately the same kind and
amount of biological activity.

Physiological function

In the short period since its isolation vitamin B12 has acquired excep-
tional practical importance because of its demonstrated usefulness in
pernicious anemia and related diseases, in livestock feeding, and in human
nutrition. Its absence from the tissues of the body is apparently the
specific cause of pernicious anemia. Injection of as little as 1 fig. per
day dramatically alleviates the symptoms of this disease. It is less
effective when given by mouth because pernicious anemia patients lack
some substance ("intrinsic factor") in the gastric juice which protects
vitamin B12 and favors its absorption. Small doses of vitamin B12 are
also effective in sprue and other macrocytic anemias. See Plate IV op-
posite p. 223.

It has been known for many years that animal protein supplements
{e.g., meat scraps, dried whey, etc.) used in livestock feeding contain some
factor necessary for growth of animals fed only plant proteins. This un-
known substance was called the animal protein factor (APF). Vitamin
B12 is certainly the chief and, perhaps, the only component of APF.
Because of its high APF potency, it is now widely used in animal feeds.
Availability of vitamin Bio has made possible the use of larger proportions
of the relatively cheap plant protein concentrates (soybean, linseed,
cottonseed meals) , which are more plentiful than those from animal
sources, and has thus been a boon to livestock production.

The vitamin B12 used in feeds is obtained almost exclusively from
fermentation sources, and especially as a by-product of the fermentations
which produce such antibiotics as aureomycin, terramycin, and strepto-
mycin. It was noted that crude B12 concentrates from these sources gave
greater growth responses in some species than could be accounted for by
their B12 content. The extra effect was traced to the antibiotics still
present as impurities in the concentrates. This discovery has opened
new vistas in the science of nutrition, since by use of this combination
faster growth rates have been achieved than had previously been con-
sidered optimal on the best mixtures of natural foods. The effect is

252

VITAMINS

shown by a wide variety of antibacterial agents and is probably due to
destruction of intestinal microorganisms which otherwise compete with
the animal for essential food factors.

Very recently Wetzel et al. have reported that doses of 10 /Ag. of vita-
min Bi2 given daily by mouth to a group of malnourished school children
resulted in definite stimulation of growth in 5 of the 11 cases treated.
These results establish the existence of human vitamin B12 deficiency
other than that of pernicious anemia. How extensive this may be remains
to be determined by further study, but present indications are that vita-
min B12 may well prove to have wide applications in human nutrition.

The metabolic function of vitamin B12 in the animal body is evidently
closely related to that of pteroylglutamic acid (for example, both are
effective in certain types of anemia). Specifically, vitamin B12 appears
to take part in the biosynthesis of nucleic acids and in the formation
and use of active methyl groups in the body (for example, in the forma-
tion of methionine from homocystine) .

Food sources and requirements

As already indicated, vitamin B12 is more concentrated in foods of
animal origin than in plant products, and relatively large amounts are
formed during the growth of many microorganisms. The distribution of
this vitamin in various foods, as determined by Elvehjem and co-workers
by means of a rat assay method, is shown in Table 9-6. No figure for
the normal human requirement for vitamin Bio has been established, but
1 ixg. per day, if injected, is sufficient to maintain pernicious anemia
patients in good condition. This amount is much less than the minimum
human requirement of any other vitamin or trace element.

Table 9-6
Vitamin B12 content of foods

(Micrograms per 100 g., fresh basis)

Food

Barley

Beans

Beef, liver

Beef, kidney

Beef, round, cooked

Beef, tongue

Cabbage

Cheddar cheese ....

Chicken liver

Cow's milk

Minimum vitamin

Bi2 content
*

*

15
20
2-3

3

*

1.4
11

Trace

Food

Egg yolk

Goat's milk

Green peas

Horse meat, canned

Mutton

Pork, shoulder

Pork, ham

Potatoes

Tomato juice

Veal

Minimum vitamin

B12 content

1.4

*

*

3.4
3

1.1-2

1.2

*

2

* No measureable amount.

VITAMINS

CHOLINE

253

Physiological function

A lack of choline in the diet of young, rapidly growing rats results in
the accumulation of excessive amounts of fat in the liver. There may
also be damage to the kidneys, which become discolored from internal
hemorrhage. The "fatty livers" are restored to normal by feeding small
amounts of choline or of methionine. On the other hand, feeding choles-
terol aggravates the condition. Older rats are much less likely to suffer
from the symptoms of choline deficiency.

It is supposed that the fatty deposits in the liver are caused partly
by a failure of fat transport and partly by a decrease in the normal
rate of fat catabolism (that is, transformation into other simpler ma-
terials) in the liver. The evidence at present available is consistent with
the assumption that neutral fat (that is, glycerides) must be converted
into phospholipides before it is transported elsewhere in the body or, if
it remains in the liver, before it is catabolized. Since choline is one
component of the lecithin type of phospholipides, it would obviously be
needed for these purposes. In fact, it has been possible with the aid of
radioactive phosphorus to follow the rate of "phospholipide turnover"
in the liver, that is, the rate at which phospholipide molecules are formed
and removed, and to demonstrate that choline increases this rate. The
effect was observed within one hour and was proportional to the amount
of choline fed.

Choline is also required for the normal nutrition of chicks and of
young turkeys. In conjunction with manganese it prevents the develop-
ment of a disease of chickens known as perosis, in which the leg tendon
slips off from the hock joint as a result of malformation of the bone, and
the bird is consequently unable to walk. Normal egg production by
chickens is also impaired by a lack of sufficient choline in the diet.

One of the main metabolic functions of choline is to supply "labile"
methyl groups for various transmethylation reactions. These are de-
scribed in Chap. 13.

Chemical nature

Choline is a very strong base, with the followmg structural formula:

CH3
HOCH2-CH2— N— CH3
HOCH5

Choline

254 VITAMINS

Like nicotinic acid it had been known to organic chemists and had been
obtained synthetically long before its usefulness as a vitamin was dis-
covered. It is very soluble in water and is quite stable to boiling in
dilute aqueous solution. Hot alkalies, however, decompose it with the
formation of trimcthylamine.

Bound choline in the form of lecithin is present in every living cell,
and free choline is likewise very widely distributed in biological materials.
At present, no information is available regarding the human requirement
for this dietary factor.

OTHER DIETARY FACTORS

There are a number of other factors that have been reported as essential
in the diet of experimental animals, but to discuss them in any detail
would be beyond the scope of this book. However, two definite chemical
substances in addition to those already considered have been shown quite
conclusively to belong to the vitamin B complex. These are para-amino-
benzoic acid and inositol:

COOH

1 i

1

NH2

i-aminobenzoic

acid

H
O

/§\
HOCH ^ HCOH

HOCH „ HCOH

c
o

H

Inositol

The former is probably used for the biosynthesis of pteroylglutamic acid
and owes its vitamin-like activity in certain species to this circumstance.
Inositol is required by mice and rats for normal growth and the avoidance
of dermatitis and loss of hair. It is not known to be required by human
beings.

Another vitamin-like substance needed by certain microorganisms is
lipoic acid, which has recently been obtained in pure form and found to
have the following structure:

CH2CH2CH(CHj)4— COOH

s s

a-Lipoic acid or thioctic acid

According to Reed and De Busk it is combined in the living cell with
thiamine and phosphoric acid to form lipothiamide pyrophosphate, which
appears to be a necessary coenzyme for the oxidative decarboxylation of
a-keto acids, such as pyruvic acid, during metabolism.

VITAMINS

255

N=CH

I I /

H3C— C C— CH — N

N-C g ^

CH3 O O

I II II

,C=C— CH2CH2OP— 0— P- OH

I 1

OH OH

NH-CO(CH2)4-CH— CH^CH,
I I

s s

Lipothiamide pyrophosphate

Still another compound, carnitine, has recently been shown by Carter
and co-workers to function as a vitamin for a lower animal organism,
namely, the larva of the yellow meal worm, Tenebrio niolitor. These

(CH3)3N+CHoCHOHCH,COO-
Carnitine

larvae will not grow on synthetic diets containing all the previously
known vitamins, but require the addition of supplements such as liver
or whey. The effective substance was named vitamin Bt. When iso-

1

L .,

^

^.,

1'

^

r .

i ' - ^

m

Courtesy of the S. M. A. Corporation.
Fig. 9-15. Pantothenic acid deficiency in the rat. These animals were
reared on identical diets except that the one on the left received an ade-
quate supply of pantothenic acid, while the diet of the other was deficient
in this vitamin.

lated in pure form, it proved to be identical with carnitine, a compound
which had long been known as a constituent of meat extract. It is
possible that carnitine functions in the larvae as a source of labile methyl
groups (p. 344).

The so-called "antigray-hair factor" may or may not be a definite
substance different from the other known vitamins. It is well established
that graying of the hair does result from certain nutritional deficiencies
in various species of animals, particularly the rat, mouse, dog, and fox.

256 VITAMINS

Deficiencies of pantothenic acid, para-aminobenzoic acid, copper, and
biotin have each been reported to cause such graying. However, there
is at the present time no acceptable scientific evidence that gray hair in
human beings can be restored to its original color by the dietary use
of any of these materials, or of any other "gray hair factor."

Other less well-defined factors are vitamin P, which has been reported
to correct bleeding caused by weakened capillaries in human beings,
vitamin B13, and vitamin B14. A large number of other vitamin-like sub-
stances are apparently needed for the normal nutrition of various species
of animals, and particularly of microorganisms, but knowledge of their
nature and biologica'l significance is too limited to warrant their con-
sideration here.

Antivitamins

Substances chemically related to certain vitamins interfere with their
normal physiological functioning and are therefore called antivitamins.
For example, mice fed pyrithiamine (a thiamine analog, see formula)
develop typical symptoms of thiamine deficiency. Similarly, pyridine-3-

CH3 CH2CH2OH
N=C— NH2 C— C^

II / w

CH3— C C— CH2— N CH

II II "V /

, Pyrithiamine

sulfonic acid and glucoafecorbic acid act as antagonists of nicotinic acid
and vitamin C, respectively. In each case, administration of the vitamin
concerned corrects the deficiency, and it appears that the response of
the organism depends on the relative amount of the vitamin and anti-
vitamin present.

An explanation for behavior of this sort was advanced by Woods and
Fildes who found that p-aminobenzoic acid (PABA) can counteract the
antibacterial effect of the drug, sulfanilamide. They suggested that
PABA is an essential metabolite for the bacteria and that sulfanilamide
exerts its effect by acting as an inhibitor of the bacterial enzymes con-
cerned with the metabolic use or functions of PABA (e.g., conversion to

COOH SO2NH2

C C

p-Aminobenzoic acid Sulfanilamide

VITAMINS 257

pteroylglutamic acid). According to this view antivitamins are com-
petitive enzyme inhibitors (see p. 272 for the analogous case of malonate
versus succinate).

Substances are also known which act against other types of essential
metabolites. For example, methionine sulfoximine, a substance produced
in fiour by a formerly used bleacliing agent (nitrogen trichloride) has been

0- NH2

CH3— S-CHjCHj— C-COOH

I I

HN- H

L-Methionine sulfoximine

found to cause "running fits" in dogs by acting as an antagonist of the
essential amino acid methionine. In general, such materials are called
antimetabolites. Many additional examples of antimetabolites are listed
by Woolley.

The concept of competition for enzyme surfaces offers a reasonable
explanation for the action of antivitamins and other antimetabolites and
furthermore may well serve as a guiding principle in the search for new
drugs to combat disease. In theory, it should be possible selectively to
poison any unwanted organism with a drug patterned after the chemical
structure of some metabolite essential for that organism. Injury to the
host would be avoided if the metabolite were peculiar to the parasite
only.

REVIEW QUESTIONS ON VITAMINS

1. What are vitamins? Name those about the existence of which there is no
controversy.

2. Discuss for each of the commonly accepted vitamins: (1) occurrence; (2)
symptoms caused by lack of the vitamin.

3. Which vitamins have been obtained in ciystalline form? Give briefly the
chemical nature of each. Which have been synthesized in the laboratory?

4. Discuss the anti-infective properties of vitamin A.

5. Account for differences in need for vitamin D supplements in northern and
southern regions. Why is sunlight transmitted through an ordinary window pane
ineffective in preventing rickets?

6. In addition to man, which animals suffer from scur^jy? How is the nonsus-
ceptibility of other animals explained?

7. Discuss incidence of the various deficiency diseases in the United States.

8. Which vitamin is formed from a plant pigment? Which from sterols? Which
one is particularly susceptible to oxidation?

9. Explain the following terms: (1) "Viosterol," (2) ascorbic acid, (3) pro-vitamin
A, (4) riboflavin, (5) calciferol, (6) nicotinic acid, (7) pantothenic acid, (8) folacin.

10. What effect is produced by ingestion of massive doses of vitamin D?

11. What is the nature of the tissue changes responsible for noticeable respiratory
trouble in A-deficient animals?

12. List the factors that influence the vitamin D requirement of au aaimal.

258

VITAMINS

13. Which vitamins are known to function as parts of enzyme systems?

14. Account for the fact that pellagra is much more prevalent in the southern
states of America than elsewhere.

15. Correct the following statement: If two samples of milk have the same amount
of color, they have the same vitamin A potency.

16. Which vitamins contain N, S, P, Co?

REFERENCES AND SUGGESTED READINGS

Brink, N. G., Kuehl, F. A., Jr., and Folkers, Karl, "Vitamin B12: The Identification of

Vitamin B12 as a Cyano-Cobalt Coordination Complex," Science, 112, 354 (1950).
Carter, H. E., Bhattacharyya, P. K., Weidman, K. R., and Fraenkel, G., "Chemical

Studies on Vitamin Bt Isolation and Characterization as Carnitine," Arch. Biochem.

and Biophys., 38, 405 (1952).
Consulich, D. B., Roth, B., Smith, J. B., Jr., Hultquist, M. E., and Parker, R. P.,

"Chemistry of Leucovorin," /. Am. Chem. Soc, 74, 3252 (1952).
Drummond, J. C, "The Nomenclature of the So-Called Accessory Food Factors

(Vitamins)," Biochem. J., 14, 660 (1920).
Eddy, W. H., What Are the Vitamins? Reinhold Publishing Corp., New York,

1941.
Eddy, W. H. and Dalldorf, G., The Avitaminoses, 2nd ed.. The Williams and Wilkins

Company, Baltimore, 1941.
Eijkman, C, "An Experiment in Combatting Beri-Beri," Virchow's Archiv fur patho-

logische Anatomic und Physiologic, 149, 187 (1897).
Follis, R. H., Jackson, D., Eliot, M. M., and Park, E. A., "Prevalence of Rickets in

Children between Two and Fourteen Years of Age," Am. J. Diseases Children, 66,

1 (1943).
Food and Nutrition Board, "Recommended Daily Dietary Allowances, Revised,

1948," Nutrition Rev., 6, 319, (1948).
Funk, C, "Etiology of Deficiency Diseases — Beri-Beri, Polyneuritis in Birds, Epi-
demic Dropsy, Scurvy in Animals (Experimental), Infantile Scurvy, Ship Beri-Beri,

Pellagra," J. State Med., 20, 341 (1912).
Gordon, E. S., Nutntional and Vitamin Therapy in General Practice, 3rd ed.. The

Year Book Publishers, Inc., Chicago, 1947.
Gyorgy, P. (editor), Vitamin Methods, vols. 1 and 2, Academic Press, Inc., New

York, 1951.
Harris, L. J., Vitamins and Vitamin Deficiencies, P. Blakiston's Son and Company,

Philadelphia, 1938.
Harris, P. L., Quaife, M. L., and Swanson, J., "The Vitamin E Content of Foods,"

J. Nutiition, 40, 367 (1950).
Harris, R. S. and Thimann, K. V. (editors), Vitamins and Hormones, Advances in

Research and Applications, vols. 1-9, Academic Press, Inc., New York, 1943-1951.
Hopkins, F. G., "The Analyst and the Medical Man," Analyst, 31, 385 (1906).
Kingsley, H. N. and Parsons, H. T., "The Availability of Vitamins from Yeast, IV,"

J. Nutrition, 34, 321 (1947).
Kogl, F. and Toennis, G., "Isolation of Crystalline Biotin from Egg Yolk," Z. Physiol.

Chem., 242, 43 (1936).
Lewis, U. J., Register, U. D., Thompson, H. T., and Elvehjem, C. A., "Distribution of

Vitamin B^ in Natural Materials," Proc. Soc. Exptl. Biol. Med., 72, 479 (1949).
Link, K. P., "The Anticoagulant from Spoiled Sweet Clover Hay," T/ie Harvey Lec-
ture Series, 39, 162 (1944).
Lunin, N., "Concerning the Significance of Inorganic Salts in Animal Nutrition,"

Z. Physiol. Chem., 5, 31 (1881).

VITAMINS 259

McCollum, E. V. and Kennedy, C, "The Dietary Factors Operating in the Production

of Polyneuritis," J. Biol. Chem., 24, 491 (1916).
Pohland, A., Flynn, E. H., Jones, R. C, and Shive, W, "The Structure of Folinic

Acid-SF, a Growth Factor Derived from Pteroyl-Glutamic Acid," Abstracts oj the

119th Meeting, Am. Chem. Soc, Boston, 1951, p. 18 M.
Reed, C. I., Struck, H. G., and Steck, I. E., Vitamin D, The University of Chicago

Press, Chicago, 1939.
Reed, L. J. and De Busk, B. G., "Lipothiamide Pyrophosphate : Coenzyme for Oxida-
tive Decarboxylation of a-Keto Acids," J. Am. Chem. Soc, 74, 3964 (1952).
Robinson, F. A., The Vitamin B Complex, John Wiley and Sons, Inc., New York,

1951.
Rosenberg, H. R., Chemistry and Physiology of the Vitamins, revised reprint. Inter-
science Publishers, Inc., New York, 1945.
Salcedo, J., CaiTasco, E. O., Jose, F. R., and Valenzuela, R. C, "Studies on Beriberi

in an Endemic Subtropical Area," J. Nutrition, 36, 561 (1948).
Sebrell, W. H., "Nutritional Diseases in the United States," J. Am. Med. Assoc., 115,

851 (1940).
Sherman, H. C, Chemistry of Food and Nutrition, 7th ed., The Macmillan Com-
pany, New York, 1946.
Spies, T. D., Experiences with Folic Acid, The Year Book Publishers, Inc., Chicago,

1947.
Sure, B., The Little Things in Life, D. Appleton-Centuiy Company, New York, 1937.
du Vigneaud, V., chapter on "Biotin," in The Biological Action of the Vitamins, The

University of Chicago Press, Chicago, 1942.
Toepfer, E. W., Zook, E. G., Orr, M. L., and Richardson, L. R., Folic Acid Content

of Foods, Agriculture Handbook No. 29, United States Department of Agriculture,

U. S. Government Printing Office, Washington, D. C, 1951.
Wetzel, N. C, Fargo, W. C, Smith, I. H., and Helikson, J., "Growth Failure in School

Children as Associated with Vitamin B12 Deficiency-Response tO' Oral Therapy,"

Science, 110,651 (1949).
Williams, R. J., Eakin, R. E., Beerstecher, E., Jr., and Shive, W., The Biochemistry of

B Vitamins, Reinhold Publishing Corp., New York, 1950.
Williams, R. J., Lyman, C. M., Goodyear, G. H., Truesdail, J. H., and Holaday, D.,

"Pantothenic Acid, A Growth Determinant of Universal Biological Occurrence,"

J. Am. Chem. Soc, 55, 2912 (1933).
Williams, R. R. and Spies, T. D., Vitamin Bi and Its Use in Medicine, The Macmillan

Company, New York, 1939.
Woods, D. D. and Fildes, P., "The Antisulfanilamide Activity (In Vitro) of p-Amino-

benzoic Acid and Related Compounds," Chemistry and Industry (London) 18,

133 (1940).
Woolley, D. W., A Study of Antimetabolites, John Wiley and Sons, Inc., New York,

1952.

Chapter 10

ENZYMES

by G. W. E. PL ALT

Assistant Professor, Institute for Enzyme Research
University of Wisconsin

Enzymes may be defined as thermolabile organic catalysts elaborated
by living cells and capable of exerting their effects independently of
these cells. Certain topics, especially those concerned with digestion and
metabolism (Chaps. 11-16) , will necessitate mention of these biocatalysts ;
but nothing will be said there regarding their chemical nature, their
mode of action, factors affecting their rate of action, and their other
properties.

Occurrence

Great numbers of enzymes can be detected in all hving cells. If one
considers the quantity and diversity of enzymes present in a cell, it be-
comes evident that the cell contents must consist largely of enzymes.
Enzymes, such as oxidative enzymes, functioning normally within the
cell are usually called endo-enzymcs. If the usual site of action is out-
side the cell, as is the case with those involved in digestion, the enzymes
are designated exo-enzymes.

Chemical nature

All enzymes that have been obtained in a high degree of purity are
proteins. Many such enzymes have been obtained in the crystalline
state, e.g., urease, catalase, pepsin, trypsin, carboxypeptidase, a- and
^-amylases, yellow enzyme, ribonuclease, aldolase, and alcohol-, lactic-,
and phosphoglyceraldehyde dehydrogenases. Such enzymes are similar
to other proteins in elementary composition, amino acid content, and prop-
erties, e.g., color tests, solubility, isoelectric point, thermolability, etc.
For example, aldolase recrystallized four to six times was found by Velick
and Ronzoni to consist of 18 amino acid residues. Complete accounting
of the nitrogen was obtained in the amino acid residues. The number
of residues per mole of aldolase (1267) was calculated, and from these
data the amino acid formula of aldolase could be expressed as Glyios

260

ENZYMES

261

Courtesy of Drs. R. M. Heiriolt and J. H. Northrop aud Tlie Journal oj (Jeneral

PJnjsiologij.

Fig. 10-1. Pepsin.

Keproduced by courtesy of Drs. M. Kunitz and J. H. Northrop and The Journal

of General Physiology.

Fig. 10-2. Trypsinogen.

C^ I

Alai35 Valgg Leui23 Ileu84 (Cys-) 13 Metn Sergg Thrye Argsi Hisgs Lysgi
Pro69 Phe26 Tyr4i Tryie Aspio- Gkuoo. Glutamic acid is low and valine
is high as compared with most proteins, but otherwise there is nothing
distinctive about the amino acid content.

Some enzymes are simple proteins, e.g., pepsin, and others are con-
jugated proteins, e.g., lactic dehydrogenase. The latter has a nicotin-
amide-containing compound as its prosthetic group. When the enzyme

262

ENZYMES

\

Courtesy of Drs, M. Kunitz and J. H. Northrop and The Journal of General

Physiology.

Fig. 10-3. Trypsin.

consists of a specific protein and a prosthetic group, as in the case of
lactic dehydrogenase, the protein part is called the apoenzyme, and the
prosthetic group is called the coenzyme of the complete enzyme. See
Table 10-1 for examples of other coenzymes.

Classification

Enzymes have been named on the basis of occurrence {e.g., pepsin),
the substance (substrate) upon which they act, the products formed by
their action, the nature of the linkages broken, or a particularly char-
acteristic type of reaction they may perform [e.g., oxidases). For ex-
ample, the enzyme which catalyzes the hydrolysis of sucrose has three
names; (1) sucrase, a name derived from the substrate, (2) invertase, so
named because the hydrolysis product, an equimolar mixture of glucose
and fructose, is called invert sugar, and (3) a-glucopyrano-^-fructofuran-
osidase, a term that indicates the type of linkage broken by the enzyme.
The variety in enzyme terminology will be apparent upon examination
of Table 10-1. Since enzymes catalyze such an enormous variety of
reactions, it is difficult to catalogue them in an exact manner. The classi-
fication used in Table 10-1 is empirical, but will serve as a guide to the
student for- the organization of the material. Like all classifications it
is imperfect and subject to change with advancing knowledge.

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269

270 iENZYMES

ISature of enzyme action

It has been stated that enzymes are biological catalysts. This means
that they are agents which affect the rates of metabolic reactions. How-
ever, although they greatly affect the speed of reactions, they do not
influence the extent of the chemical change concerned, that is, they do
not influence the final position of chemical equilibrium. The latter is
determined by the nature (particularly the energy content) of the react-
ing substances and the products formed (see Chap. 16). The rates of
metabolic reactions, however, are all-important for living organisms, since
they must be able to utilize foods fast enough to keep up with their
metabolic needs. For example, the same amount of glucose can be ob-
tained from starch as from cellulose on chemical hydrolysis; yet, while
the former will support growth in man, the latter will not. The explana-
tion is that enzymes are present in the human digestive tract which accel-
erate the hydrolysis of starch, whereas there are none that attack cellulose.
The uncatalyzed breakdown of cellulose to glucose is much too slow
to be of use to the body. Cattle and other ruminants, however, have in
their paunch vast numbers of bacteria which contain enzymes that can
break down cellulose to organic acids and thus provide the animal with
utilizable food. It follows from the above that any agent or condition
which affects the catalytic ability of one or more of the enzymes involved
in the metabolism of a vital food will have a profound effect on the
development of the whole organism.

Many of the chemical reactions which occur easily in living organisms
are difficult to reproduce in the laboratory in good yields and require
drastic conditions of pressure, temperature, or pH to proceed at adequate
speed; yet these reactions take place under much milder conditions in
living cells. Enzymes accomplish this end, since by virtue of their
specificity they guide reactions to the desired products, and because they
can lower the energy of activation of the reaction {i.e., the energy neces-
sary to get it started).

Mechanism of action

According to the most widely accepted theory an enzyme functions
through union with its substrate to form a labile intermediate compound
or "enzyme-substrate complex," which in turn decomposes with forma-
tion of the end products of the reaction and regeneration of the enzyme.
This mechanism can be schematically represented as follows:

enzyme -|- substrate ^ enzyme-substrate — > product (s) + enzyme

complex

ENZYMES

271

Since the enzyme-substrate complex is a very labile product and is
present for only a very short time, it is difficult to demonstrate its exist-
ence. However, in the case of catalase it has been possible to provide
evidence for the existence of such an intermediate compound with the
aid of very speedy, automatically recorded, electrophotometric measure-
ments.

Factors affecting activity

The speed with which a given reaction proceeds in the presence of
an enzyme is influenced by many variables, which include the following:
(1) concentration of substrate, (2) concentration of enzyme, (3) specific
activators such as coenzymes and metalHc ions, (4) temperature, (5) pH,
(6) oxidation-reduction potential, (7) ionic strength, and (8) products
of the reaction.

When the influence of substrate concentration on the speed of an en-
zymatic reaction is studied, it is observed that the rate of the reaction
increases with substrate concentration up to a certain point beyond
which there is no increase in activity. This occurs because all the
enzyme eventually is converted into the intermediary compound by mass
action, and the limiting speed of the reaction then becomes that of the
decomposition of this complex. If under these conditions one doubles
the enzyme concentration, the rate of reaction will also double, since
twice as much enzyme-substrate complex will be available for decomposi-
tion.

The rate of most enzyme-catalyzed reactions is increased about 1.2-4
fold by a 10° rise in temperature. This temperature effect is much lower
than that observed in the case of many uncatalyzed chemical reactions.
For this reason enzymatic reactions proceed at higher speeds at low
temperatures than the corresponding uncatalyzed ones. Most enzymes
are thermolabile and will lose activity when exposed to high tempera-
tures, e.g., 60°C., over prolonged periods of time.

Acidity also has a profound effect on enzyme activity. Each enzyme
in the presence of a certain substrate has a characteristic pH at which
its activity is highest. Some enzymes, e.g., pepsin, require an acid
medium; others, e.g., trypsin, need alkaline conditions for maximum
activity. Most enzymes work best under conditions which are neither
strongly oxidizing nor reducing, and, in fact, are frequently inactivated
by strong oxidizing or reducing agents.

The effectiveness of some enzymes is influenced by the ionic strength
(concentration of ions) of the solution in which they act; this is in addi-
tion to the specific effects of various anions and cations. In most cases,
enzymes are inhibited by the end products of the reactions which they
catalyze.

272 ENZYMES

Specificity

In the study of digestion (Chap. 12) it is noted that fat-splitting en-
zymes are without effect on carbohydrates or proteins. Neither does an
enzyme that hydrolyzes one of the latter attack fats. Even the common
disaccharides require different enzymes to effect their hydrolysis. Spe-
cificity is frequently due to type of linkage rather than to individual
compounds, as is evidenced by the fact that trypsin digests various pro-
teins that differ markedly in composition and size of molecule. Further-
more, emulsin, which causes hydrolysis of many ^-glucosides, has no
effect on the isomeric a-glucosides ; the reverse is true of maltase.

Inhibition

Enzymes are inhibited by a variety of conditions, which have already
been indicated under factors affecting activity. In the present discussion
attention is focused on the types of inhibition that can be obtained with
chemical reagents. There are two main types: competitive and noncom-
petitive. If, for example, succinic dehydrogenase is inhibited by malonate
{i.e., a soluble salt of malonic acid) , a substance which is similar in struc-
ture to succinate (the normal substrate of this enzyme), the inhibition
can be competitively reversed by increasing the substrate concentration.
This means that the amount of inhibition produced depends primarily
on the relative amounts of malonate and succinate present. On the
other hand, if this enzyme is inhibited by quinone, for example, the
activity cannot be restored by an increase in succinate concentration.
This is termed noncompetitive inhibition. These phenomena can be
visualized if one considers that the enzyme surface has specific points of
attachment which fit snugly against groups of the substrate molecule.
If an inhibitor is used that is so similar in structure to the substrate that
it also can fit into the "mold" on the enzyme surface, it can compete
with the substrate for position. However, if an agent is used that changes
the enzyme in some way, the substrate can no longer attach to the surface
regardless of the amount used. The relation between substrate and in-
hibitor then becomes noncompetitive.

Various inhibitors have been used successfully for the study of met-
abolic reactions. If it is desired to study the conversion of a-keto-
glutarate to succinate in the presence of other enzymes of the Krebs cycle
(see Chap. 13) , one can prevent the further metabolism of succinate by
the addition of malonate. For additional examples concerned with vita-
mins, see p. 256.

Some drugs are known to exert their action by inhibition of enzymes.
For example, eserine, an alkaloid (C15H21N3O2) that stimulates the
parasympathetic nervous system, inhibits the enzyme choline esterase

ENZYMES

273

which decomposes acetylcholine; as a result, the latter accumulates and
causes increased stimulation.

Activation

Zymogens. A number of enzymes are secreted in the form of inactive
precursors known as zymogens. For each zymogen there is some reagent
that can change it into the active enzyme. To illustrate, pepsinogen, the
zymogen of pepsin, is slowly converted into active pepsin by hydrogen
ions, but it is rapidly activated by pepsin itself; that is, the activation
is autocatalytic. Chymotrypsinogen is converted into chymotrypsin by
trypsin. The conversion of trypsinogen to the active form is autocata-
lytic, i.e., by trypsin itself.

Ions. Certain enzymes can be separated into two fractions by dialysis.
Either fraction alone is inactive, but upon recombination the activity is
restored. The portion of the enzyme that can pass through the membrane
has a much smaller molecular weight than the remaining part. This
dialyzable portion is considered as a cofactor which is necessary for the
activity of the total enzyme. In some cases more drastic conditions
than simple dialysis must be employed to separate the cofactor from
the apoenzyme; for example, treatment with acid in ammonium sulfate
solution in the case of certain flavo-proteins. In some enzymes the
cofactor is so tightly bound that it has not been yet possible to remove
it without destroying the enzyme.

In many cases the cofactor is simply a metallic ion. For example,
Mn++, Co + + , or Zn + + have been found to be activators for certain
peptidases. The theory has been proposed that the metal ions form
coordination compounds and act as bridges to bring substrate and enzyme
together. Certain enzymes have a characteristic anion requirement, e.g.,
salivary amylase is activated by chloride.

Coenzymes. Another group of cofactors are organic compounds which
are called coenzymes. The study of coenzymes has received much atten-
tion by biochemists for the past 20 years, and the chemical structures
of many of them have been determined. The cofactors required by
several enzymes are given in Table 10-1.

1. Cocarboxylase. It has been pointed out previously (p. 227) that
thiamine is required for the metabolism of carbohydrates, and particularly
of pyruvic acid. The reason for this requirement is that the enzyme which

CHa Q O

Is=C-NH2 c=C-CH5CH— OP-0— P-0-

/ I I

o o

H3C-C C-CH,-+N

N-CH Y ^ H H

H

Fig. 10-4. Thiamine pyrophosphate (cocarboxylase).

274 ENZYMES

cleaves pyruvic acid into carbon dioxide and a two carbon fragment
(acetaldehyde in yeast) contains thiamine pyrophosphate (cocarboxy-
lase) (Fig. 10-4) as a coenzyme. The degradation of fatty acids to
two carbon compounds (Fig. 13^) does not require the presence of thi-
amine pyrophosphate. In animals on a low-thiamine, high-fat diet the
supply of two carbon fragments from carbohydrate is limited owing to
the small quantity of cocarboxylase in the tissues, but this deficiency
is compensated by the generation of these metabolic intermediates in
adequate amounts from fat.

A compound of cocarboxylase and lipoic acid, lipothiamide (LTPP),
acts as a coenzyme for the oxidative decarboxylation of pyruvic acid
and a-ketoglutaric acid by certain bacteria, e.g., E.coli. In an enzyme
system obtained from this organism the following series of reactions has
been demonstrated:

pyruvate (a-ketoglutarate) -|- LTPP + DPN^

acetyl LTPP (succinyl LTPP) + COo + DPNHa
acetyl LTPP (succinyl LTPP) +CoA-^ acetyl Co A (succinyl Co A) + LTPP

2. Coenzyme A. Lipmann and co-workers discovered that a coenzyme
is necessary for the acetylation of sulfanilamide. Subsequent studies
demonstrated that the same substance is required for the metabolic forma-
tion of acetylcholine from choline and for the condensation of oxalacetic
acid with the two carbon fragments from fat or carbohydrate metabolism
to produce citric acid (Fig. 13-4). Since in each case acetic acid or an
acetyl group seemed to be involved, the coenzyme was named coenzyme
A {Co A), a "coenzyme for acetylation." Chemical investigations re-

^HjJ>0,H,

9 ?l 9. ^ OH

■ I I I II II I

CHCHCH-CHCH,OP-0-PO-CH,C(CH,),CHCONH-CH,CH,CO-NH-CH,CH,SH

NHr *•

Fig. 16-5. Structure of coenzyme A suggested by Baddiley and Thain.
It is possible that this formula will require some revision as fuller informa-
tion becomes available.

vealed that Co A was a derivative of pantothenic acid, thus providing
an insight into the metabolic functions of this B vitamin. The Co A
molecule also appears to contain adenine, ribose, ^-thioethylamine, and
two or three phosphate radicals. Although the exact chemical formula
is not yet known, a suggested structure is given in Fig. 10-5. The sub-
stance seems to function by accepting acetyl groups from one metabolite

ENZYMES 275

and then donating them to another; in other words, it serves as an acetyl
carrier. This is illustrated in the following scheme:

apoenzyme 1

acetyl X -\- Co A >■ X + acetyl Co A

acetyl Co A -t Y ^^"^"'^"^'i. acetyl Y + Co A

The apoenzymes 1 and 2 are specific for X and Y; for example, different
apoenzymcs are required for the formation of citric acid than for acetyla-
tion of choline.

3. Pijridino Coenzymes. Several coenzymes have been found necessary
for the numerous dehydrogenation reactions which constantly occur in
all living cells. Warburg and co-workers demonstrated the need for one
such substance for the enzymatic dehydrogenation of glucose-6-phosphate
to phosphogluconic acid. The coenzyme was isolated in pure form and
shown to contain three molecules of phosphoric acid, two of pentose,
one of adenine, and one of nicotinamide (later identified as the pellagra-
curing vitamin) . This substance was called "coenzyme II," but now is
preferably designated as triphosphopyridine nucleotide (TPN, Fig. 10-6) .
It has been shown to be a component, for example, of the dehydrogenases
that act on glucose-6-phosphate and on isocitrate, and for the enzyme
system that converts malate to pyruvate and carbon dioxide (reaction
15, Fig. 13-4).

Another coenzyme in this group is called cozymase, coenzyme I, or
preferably diphosphopyridine nucleotide (DPN) . It has exactly the same
chemical structure as TPN except that it contains only two phosphate
groups, as is indicated by the name. The extra phosphate group in
TPN is the one on the second carbon of the ribose residue in the
adenylic acid half of the molecule. Among dehydrogenases which require
DPN are those involved in the oxidation of D-glyceraldehyde-3-phosphate,
lactate, ethanol, malate, L-a-glycerophosphate, and glucose.

DPN and TPN are called "pyridino" coenzymes because of the pyridine
ring in the nicotinamide component. It is also the pyridine ring which
undergoes chemical reaction when the coenzymes function in oxidation-
reduction reactions. The exact nature of this important change, which
is the same for both DPN and TPN, may be understood by studying
the structural formulas given in Fig. 10-6. In the oxidized form the
pyridine nitrogen has a valence of five and exists as the basic ion of a
quaternary ammonium salt. This positive charge is neutralized by one
of the negatively (acidic) charged phosphate groups of the molecule.
In Fig. 10-6 these groupings are starred and appear as follows:

^ ^CH I

+N* -0— P—

' 1

♦O

+

+

c <

■a

c5

o

o
_o

o

O

o

I I

o— o

^^

o
o

o

:0

o— o— u— o-
Ui W K K

o

K I

-O — O— CL,=

^— o=

/^

o

// \* o o

K— o ^— o— o— o— o

\ / K W ffi M

o=o

-O— O— Oh

=o

o

I

V

i

1

K

c3

as

N

M

+

a

a
p

1

amuapY

o

<u

-a
• ^^
*^
o
_a>

"o

3
C
O

c
o

I 1 =

o— O^

r^

X

-O:

o o
■o— o-

o

-o-

•o

(U

T)

c3

(3

03

m

n

K

^

15

PL,

CD

O

H

3

-a

>.

f.

OJ

J3

t~^

Tl

o

T3

CD

a

-^

T3
<V

D

(U

a

2J

-S

•r-l

1

X

si

O

a

-^

^

<D

9

-t^

J3

o

H

3

c

,

T-i

«

■-S

IS

O ^

CI

— o— o— &H=o

-a
S

03

o

O

I X

o — o

/s £\\*

S— OS _3^— o

\ "? 2 / X

X X
o o

-O — 0;:^0

■o— o— f^
o

12;

Oh

H

a

o

■a
a>

N

• «^

-a

'S

o

rs

o
"o

3

a

S .-^ S

« q; Qj

^ "^ "3

t< 3 O)

o fl S

O CD

+^ o

o3 (D

;= o

ft

=o«

apiui'Bin^oot^

-^ ^

o

o3

CD

o

O *

o -^

TO t-t

s ^

CD CC

+i CD
-3 CD

9soqi^

1 1 ■

276

ENZYMES

277

When the oxidized coenzyme is reduced, the nitrogen changes to a
valence of three, the double bond between the nitrogen and the adjacent
carbon atom is reduced, and a hydrogen ion is formed. These changes
may be represented as follows:

+N* +2(H) ^^=^ N* +H+

I I

The trivalent nitrogen is much less basic and no longer neutralizes a
phosphate group. Consequently the hydrogen ion formed might add to
this group:

-0— P— + H+ :^=^ HO— P—

II II

O O

However, the phosphate group is strongly acidic and in the physiological
pH range is almost completely dissociated {i.e., the equilibrium point of
the above reaction lies far to the left). What actually happens is that
the newly formed hydrogen ion is picked up by the buffer systems of
the cell and is used later in the reaction of reduced cytochrome c with
oxygen (see below) .

It should be remembered in this connection that an atom of hydrogen
consists of a proton (hydrogen ion, H + ) and an electron. The electron
corresponding to the proton set free in the reduction of DPN or TPN
becomes attached to the coenzyme, neutralizing the positive charge on
the nitrogen atom. The reduced coenzyme thus actually carries one
hydrogen atom and one electron, the proton of the second hydrogen being
carried in the cell buffers.

4. Riboflavin Coenzymes. These substances are also coenzymes of
oxidation-reduction reactions. There are two of these: the so-called ribo-
flavin mononucleotide (FMN) , which is more accurately named riboflavin-
5'-phosphate, and riboflavin adenine dinucleotide (FAD) . The abbrevia-
tions start with "F" because riboflavin is often called simply "flavin."
The formulas of these compounds are given in Fig. 10-7. Since the union
between the isoalloxazine and ribitol (alcohol corresponding to ribose)
residues is not glycosidic, neither substance strictly speaking is a nucleo-
tide, but the above names are in common use and are likely to be retained.
These coenzymes act by taking up and giving off two hydrogens. In
each case the hydrogen atoms are attached to positions 1 and 10 in the
flavin part of the molecule (Fig. 10-7) . FMN is a coenzyme for TPN-
cytochrome reductase and L-amino acid oxidase. FAD is required by
xanthine oxidase and glycine oxidase.

278

ENZYMES

H

0

1

II

c

N=C— NH.

^C^ N*

|(1XG)|

HsC-

^'''>^

HC„^ C— N

||(2) ||(5)(7)^

(2)

(8)CH

H5C-

1 1

tl.

1

H CH,

1

ilL/

HCOH

HCOH

(2')|

(2')| 0

HCOH

HCOH

(3')|

-

(3')|

HCOH

110

(4')|

(4')|

CH,

CH2

(5')|

(5')|

0
1

0
1

HO-P

0

P-OH

II

II

0

0

Riboflavin adenine dinucleotide (FAD, oxidized form)

H

1

V

f ? If

H3C-

C N*

H3C-

.C. ^N* ^C^
C^ ^C^ ^C^ ^NH

(7)

(2}

1 1 1

HaC-

-C^(S)/C\(9)/C:

C N

H 1

CHi

HCOH

VO/C-0

N*

H3C-

^^ /C^ /C>. ^C=0
^C^ ^N^ ^^

1 1 1

H CH, H

1
HCOH

(2')|

+ 2(H)

1

HCOH

>•

HCOH

(3')|

,^

-2(H)

1

HCOH

HCOH

(4')|

1

CH,

CH,

(5')j

1

0
1

0

1

HO-P— OH

HO-P-OH

II

II

0

0

Oxidized form

Reduced form

Riboflavin mononucleotide (FMN)

Fig. 10-7. Riboflavin coenzymes. The atoms which acquire
hydrogens on reduction are starred.

5. Iron Porphyrin Compounds. A number of enzymes such as catalase
and peroxidase have iron porphyrin compounds as the prosthetic group.
The cytochromes, a group of pigments present in a large number of
organisms and tissues, are also of this type, since they consist of a char-

ENZYMES

279

acteristic protein and a heme compound. There are at least three cyto-
chromes, designated as a, b, and c. The heme portion is more firmly
attached to the protein in these pigments than the corresponding func-
tional groups of the pyridino- or flavo-proteins. The cytochromes are
concerned with oxidation-reduction reactions, and their concentration in
aerobic organisms bears a direct relationship in many instances to the
respiratory activities of the cell. The best characterized of tliese respira-
tory pigments is cytochrome c. It contains 0.43 per cent of iron and
is believed to have a molecular weight of 13,000. The most probable
formula for the heme component of cytochrome c, according to the evi-

PROTEIN

S
I

CH — CH3 CHj
I H I

COOH

Fig. 10-8. Cytochrome c. The heme component (prosthetic group) is
shown and also its attachment to the protein part of the molecule by two
sulfur linkages and the iron atom.

dence available at present, is shown in Fig. 10-8. Cytochrome c (abbre-
viated Cyt. c) functions as an electron carrier in cellular oxidation-
reduction reactions by virtue of its iron atom which alternately changes
its valence from 2 to 3:

Cyt. c (Fe+ + + ) + e ?=^ Cyt. c (Fe+ + )

6. Pyridoxal Phosphate. Enzymes which catalyze the decarboxylation
of histidine, tyrosine, lysine, and glutamic acid to form carbon dioxide
and the corresponding primary amine require pyridoxal phosphate as a
coenzyme. This coenzyme is also a necessary cofactor for transaminase

280

ENZYMES

(Table 10-1, class G.l.a.). Its chemical formula has been given on
p. 243.

7. Other Coenzymes. Three definite chemical substances are known
to serve as coenzymes for the interconversion of several organic phos-
phates during carbohydrate metabolism. Glucose-l,6-diphosphate is a
coenzyme for phosphoglucomutase, which catalyzes the migration of a
phosphate group between the 1 and 6 positions of glucose. Glyceric acid-
2,3-diphosphate acts in an entirely analogous manner in catalyzing the
migration of phosphate groups between the 2 and 3 positions of
glyceric acid. A coenzyme necessary for the enzymatic conversion of
galactose-1-phosphate to glucose-1-phosphate (reaction II, Fig. 13-1) has
been purified by Caputto and co-workers. The suggested formula is
given below:

N=C-OH
I I
0=C CH

I II
N— CH

•0-

H H

I I
0 0

H

0

0

■0-

H H

I I

OHO

-C— C— C— C— C— 0— P— 0— P— 0— C— C — C— C— C-CH2OH

III I I I I I I I I

H H H H OH OH H H OH H H
^ ^ ■'

Uridine part Glucose part

Coenzyme for conversion of galactose-1-phosphate to glucose-1-phosphate

A number of other compounds or their derivatives are suspected to be
coenzymes on the basis of their chemical properties or their gross metabolic
effects. The tripeptide glutathione (GSH, p. 130) can be oxidized to
form a double molecule, the parts of which are held together by a disulfide
( — S — S — ) linkage. Specific pyridinoproteins have been studied which
catalyze this reaction. Although the role of glutathione in oxidation-
reduction reactions is not fully understood, it is known to be a cofactor
in the glyoxalase reaction (Table 10-1, class D.l.a.) and to be a func-
tional part of glyceraldehyde phosphate dehydrogenase. Ascorbic acid
is also capable of undergoing alternate oxidation and reduction, but the
mechanism of its metabolic function has not been explained. Biotin has
been implicated in certain carbon dioxide-fixation reactions, e.g., the con-
densation of carbon dioxide and pyruvate to form oxalacetate (reaction 14,
Fig. 13-4) , but to date no enzyme has been purified which has been proven

ENZYMES 281

to require a biotin containing coenzyme. Vitamins containing para-
aminobenzoic acid, e.g., folic acid and the "citrovorum factor," seem to be
concerned with the transfer of formyl or formaldehydo groups in the
organism (Chap. 9). The family of B12 vitamins has an effect on the
metabolism of methyl groups (Chap. 9) and on the synthesis of desoxy-
ribonucleotides.

Role of enzymes in tissue oxidation

It has already been noted in the discussion of coenzymes and in Table
10-1 (section F) that a large group of enzymes is concerned with oxida-
tion-reduction reactions. There are three general groups of enzymes in
this class, the oxidases, peroxidases, and dehydrogenases. In the 1920's
there were two concepts concerned with the oxidation of substrates in the
organism. The advocates of the Warburg school contended that sub-
stances were oxidized because of activation of oxygen by iron. In model
experiments with iron-containing charcoal and enzyme preparations, it
was shown that the oxidation of substrate was accompanied by a reduction
of iron from the ferric to the ferrous state.

However, Wieland and co-workers demonstrated that an organic sub-
stance in the reduced form could be oxidized in the presence of palladium
black. Palladium is known to have a strong affinity for hydrogen, and
the process was termed "dehydrogenation." The idea was advanced that
biological oxidations occurred more as a result of activation of hydrogen
than of oxygen. This view was greatly advanced by the work of Thun-
berg who demonstrated that the removal of hydrogen from succinate, for
example, could be accomplished in the absence of oxygen by methylene
blue and a specific enzyme. Methylene blue is a dye which is readily
reduced and is thereby decolorized:

HOOC— CH0CH2— COOH + MB

Succinic acid Methylene

blue

enzyme

HOOC— CH=CH— COOH + MB-H^

Fumaric acid Leuco

methylene

blue
(colorless)

Ih will be seen later that both principles apply to oxidation processes in
living organisms.

Oxidases are enzymes which lead to oxidation of a substrate by molecu-
lar oxygen. Thus cytochrome c is converted from the ferrous to the

282 ENZYMES

ferric state by molecular oxygen under the influence of cytochrome oxi-
dase (Table 10-1, class F.II.a.). Tyrosinase is another example of an
oxidase.

Peroxidases lead to the oxidation of substrates by hydrogen peroxide.
Catalase also activates hydrogen peroxide and decomposes it to water and
oxygen in the absence of added substrates. However, in the presence of
certain oxidizable substrates, catalase can act as a peroxidase. To illus-
trate :

2H2O2 *■ 2H20H-02 (H2O 2 decomposition)

H2O2 + CH3CH2OH '^^^^"'^ * 2H2O + CH3CHO (peroxidation)

or peroxidase

Dehydrogenases result in a removal of hydrogen ions and an equal
number of electrons from an organic molecule. Examples of various
dehydrogenases have already been encountered in the discussion of
pyridino and flavin coenzymes. The nature of the coenzymes of certain
other important dehydrogenases, e.g., succinic dehydrogenase (Table
10-1, class r.1.3.), has not been completely elucidated to date. When
the biocatalyst is concerned with the oxido-reduction of a compound from
which electrons, but not hydrogens, are removed, the enzyme is called a
reductase, e.g., cytochrome reductase (Table 10-1, class F.I.2.a.).

The basic mechanism of all oxidation-reduction reactions involves the
transfer of electrons, and the enzymes or coenzymes concerned can be
considered as carriers of electrons. To illustrate schematically:

(1) HOOC— CHj— CH2— COOH

Succinic acid dehydrogenase^

•<-

HOOC— CH=CH— COOH + 2H+ + 2e
Fumaric acid

(2) 2Cytochrome c (Fe+++) + 2e

reductase 2Cytochrome c (Fe+''")

(3) Oxidase

(a) 2Cytochrome c (Fe++) ^ 2Cytochrome c (Fe+++) -|- 2e

(b) 2H+ + 2e + §02 > H.O

If one considers the oxido-reduction enzymes in relation to each other,
a certain pattern emerges. For example, a dehydrogenase containing
TPN or DPN can extract hydrogen and electrons from a given substrate,
reduced TPN or DPN can react with flavo-protein, and this then can
react with cytochrome c, which can be oxidized by oxygen in the presence
of cytochrome oxidase. This relationship can be summarized in the
following series of equations:

(1) DPN

-.2c

E

NZYMES

». DPNH

R + or

TPNH

Substrate
(oxidized)

RH2 + or

dehydrogenase

+ H+

TPN

Substrate I

(reduced) !
1

+ 2c

(2) DPN-H

+ flavin
1

1
- 2c ^ DPN

or + H+

dehydrogenase or

+ flavin ■2H

TPXH

+ 2e TPN

(3) flavin •2H + 2cytochrome c(Fe+++)

^ - 2f

reductase

+ 2e

283

flavin + 2cytochrome c (Fe++) + 2H+

(4) 2cytochrome c (Fe++) + 2H+ + IO2

oxidase

- 2e

2cytochrome c (Fe+++) + H2O

(1) + (2)
+ (3) + (4)

RH2

Substrate
(reduced)

+ \0,

2e

H2O + R

Substrate
(oxidized)

It will be observed that the net result of the oxidation of the substrate
to product is the removal of two hydrogen atoms [two hydrogen ions
plus two electrons, since H (atom) = H+ (ion) + e (electron)] from the
substrate, which combine with oxygen to form water. It should also be
noted that the coenzyme that becomes reduced in one reaction is reoxi-
dizcd in the next and thus is ready for the first reaction again; this is
indicated by the broken arrows in the scheme.

In subsequent chapters (13, 16), it will be explained that the degrada-
tion of foods is a stepwise process which proceeds through a series of inter-
mediary compounds before the end products are excreted by the organism.
The above scheme suggests that this situation also applies to oxidation-
reduction processes. It should be realized, furthermore, that a certain
increment of energy becomes available at each of the steps of the oxida-
tion-reduction chain and that this energy can be transferred to functions
useful to the organism by appropriate, stepwise mechanisms, such as
the formation of high energy phosphate linkages (Chap. 16).

Although some tissues contain all the electron transferring systems
indicated in the scheme, it should be remembered that not all of the
steps are necessary for every process. For example, the oxidation-reduc-

284 ENZYMES

tion reactions of anaerobic glycolysis (Chap. 13) proceed through DPN-
linked enzymes only.

REVIEW QUESTIONS ON ENZYMES

1. Explain the terms (1) apoenzyme, (2) coenzyme, (3) zymogen, (4) activator,
(5) carrier.

2. How general is the occurrence of enzymes in nature? Distinguish between endo-
and exo-enzymes.

3. (1) Name five of the enzymes that have been obtained in the crystalline state.
(2) What has proved to be the chemical nature of all crystallized enzymes?

4. (1) What is the essential nature of enzyme action? (2) What is the effect of
an enzyme on the chemical equilibrium of a reaction?

5. (1) List the factors that influence the rate of enzyme action and discuss eacli
briefly. (2) What is meant by specificity of enzymes?

6. What is probably the mechanism of enzyme action?

7. Define the following: (1) dehydrogenase, (2) catalase, (3) oxidase, (4) peroxidase.

8. What is the cliemical nature of the cytochromes, and to what substance previ-
ously studied are they therefore related?

9. Explain wherein the structure of (1) glutathione, (2) DPN, (3) riboflavin phos-
phate, and (4) cytochrome c could make it possible for these compounds to function
in tissue oxidation.

REFERENCES AND SUGGESTED READINGS

Baldwin, E., Dynamic Aspects of Biochemistry, 2nd ed., Cambridge University Press,

Cambridge, 1952.
Baddiley, J. and Thain, E. M., "Coenzyme A. Part III. Synthesis of Pantothenic

Acid-2':4' Phosphate and Further Structural Considerations," J. Chem. Sac, 3421

(1951).
Caputto, R., Leloir, L. F., Trucco, R. E., Cardini, C. E., and Paladini, A. C, /. Biol.

Chem., 179,497 (1949).
Haldane, J. B. S., Enzymes, Longmans, Green and Company, New York, 1930.
Lardy, H. A. (editor) Respiratory Enzymes, Burgess Publishing Company, Min-
neapolis, 1949.
Lardy, H. A., "Vitamins and Carbohydi-ate Metabolism," J. Chem. Ed., 25, 262 (1948).
Lipmann, F., Kaplan, N. 0., Novelli, G. D., Tuttle, L. C, and Guirard, B. M.,

"Coenzyme for Acetylation, a Pantothenic Acid Derivative," J. Biol. Chem., 167,

869 (1947).
McElroy, W. D. and Glass, B., Phosphorus Metabolism, Vol. I, The Johns Hopkins

Press, Baltimore, 1951.
Northrop, J. H., Kunitz, M., and Herriot, R. M., Crystalline Enzymes, Columbia

University Press, New York, 1948.
Peters, R. A. and Thompson, R. H. S., "Pyruvic Acid as an Intermediary Metabolite

in the Brain Tissue of Avitaminous and Normal Pigeons," Biochem. J., 28, 916

(1934).
Sumner, J. B. and Myrback, K., Tlie Enzymes, Academic Press Inc., New York, 1951.
Thunberg, T., "Action of Animal Tissues on Methylene Blue," Skand. Arch. Physiol.,

35, 163 (1917).
Velick, S. F. and Ronzoni, E., "The Amino Acid Composition of Aldolase and

D-Glyceraldehyde Phosphate Dehydrogenase," J. Biol. Chem., 173, 627 (1948).

ENZYMES 285

Warburg, O., Die Katalytischen Wirkungen der lebendigen Substanz, Julius Springer,

Berlin, 1928.
Warburg, 0. and Christian, W., "Activation of the Robison Hexosemonophosphoric

Acid Ester in the Red Blood Cells and the Method for Preparation of Activating

Enzyme Solutions," Biochem. Z., 242, 206 (1931).
West, E. S. and Todd, W. R., Textbook of Biochemistry, Macmillan Company, New

York, 1951.
Wieland, H.. 'Tber den Mechanismus der Oxydationsvorgiinge," Ergcbnisse Physiol.,

20, 477 (1922).

Chapter 11

HORMONES

The hormones have been defined by Houssay as "specific chemical
substances produced by an organ or tissue which, after being discharged
into the circulating fluids, may reach all parts of the organism and in
small amounts markedly influence the functions of other organs or
systems without themselves contributing important quantities of matter
or energy." Thus they resemble the vitamins very closely, differing only
by being produced in the body rather than having to be supplied ready-
made in the food. The hormones are produced by specialized organs
called the glands of internal secretion, or endocrine glands, such as the
pancreas, thyroid, ovaries, and others. Hormone manufacture and secre-
tion is the physiological function of these glands, and the effects which
follow their removal or alteration are merely the result of too little or
too much hormone production. Often it is possible to overcome the
effect of glandular lack by supplying the necessary hormone from an
outside source (for example, insulin). The various hormones were dis-
covered by showing that the effects of removing certain endocrine glands
could be counteracted with extracts of the same glands from other
individuals.

Chemical types

The known hormones of higher animals are sometimes grouped roughly
into three chemical types. Those of the pancreas and pituitary, plus a
few others, are proteins or -peptides. The sex hormones and adrenal cortex
hormones are steroids. The third group is made up of adrenalin and
thyroxine, which are classified together as phenolic compounds, although
they are otherwise quite dissimilar. Most of the plant and insect hor-
mones do not fit into any of these classes. Some of the former are con-
sidered briefly in Chap. 15.

Control of hormone production

The various glands of internal secretion and the hormones they produce
make up a closely interrelated system, which is delicately balanced and
responsive to many influences. The functioning of this system helps

286

HORMONES

287

the organism to adjust its metabolic activities so as to cope with changes
in the outside environment and to maintain a stable internal condition.
The system operates for the most part automatically. Thus, for example,
an increase in the blood glucose level stimulates the pancreas to secrete
more insulin, which promotes utilization of the sugar and hence brings
the concentration down again. The pituitary produces, among others,
hormones which stimulate gro-wth and activity of the thyroid, ovaries,
testes, and adrenal cortex. The characteristic hormones of these glands
depress pituitary function. In general, the rate of hormone production
is controlled either by other hormones, by various other chemical sub-
stances in the body, or to a lesser degree by nervous stimulation originat-
ing in the external environment.

Hormone metabolism and function

The smooth operation of various bodily processes often involves the
concerted action of a whole series of hormones. For example, carbo-
hydrate metabolism cannot proceed normally without the help of hor-
mones from the pancreas, pituitary, thyroid, and adrenal cortex. Sexual
reproduction in mammals depends on the hormones of the ovaries, testes,
pituitary, adrenal cortex, and, to some extent, the thyroid.

Just how the observed effects are brought about is, in most cases, not
known. Presumably, since they are active in very small amounts, the
hormones must act through certain enzyme systems {e.g., effect of
epinephrine on phosphorylase, p. 289). Likewise the metabohc fate of
the hormones, that is, what becomes of them after being secreted into
the blood, is still largely unknown, although some of the steroid hormones
have been found recently to be converted into modified products and
excreted in the urine.

With this general introduction, attention will now be directed to the
individual endocrine organs and the hormones they produce.

NONPROTEIN HORMONES

Hormones of the adrenal medulla

Epinephrine (adrenalin) and norepinephrine (noradrenalin or arterenol)
are the hormones produced by the adrenal medulla, the inner part of a
small endocrine gland located just above each kidney. About five to
six times as much of the former substance is normally formed by the
adrenal as of the latter. These two hormones are almost certainly syn-
thesized from phenylalanine in the body. The biosynthetic pathway used
is not entirely known, but is probably somewhat as follows:

288

COOH

H2N— CH
I

I

,^^-^

COOH
I

H2N— CH
I
CH,

HORMONES

COOH
I
H.,N— CH

i
CH,

NH2

CH2
I
CH2

I
C.

C C

HC=^ XH (ox) HC^ ""CH (ox) HC^ ^CH — CO2 HC^^^CH

II * I II * I II * I II

HC^ XH HC=:, C-OH HC^ C-OH

c c c

HC

•^^,^CH

C
H

L-Phenylalanine

OH
L-Tyrosine

OH
L-Dihydroxy-
IDhenylalaninc

OH
Hydroxytyramine

(ox)

NH2
I
CH2

I
HOCH
I

HC^ ""CH

HC^ ^C— OH
C

I
OH

^Norepinephrine

metliylation

HNCH3

CH2
I

HOCH
I

HC=^ CH

I II

HC^ ,/C— OH

I
OH

i-Epinepiirine

It has been demonstrated that slices of the adrenal medulla convert hy-
droxytyramine in vitro into a substance with the biological properties
of epinephrine and that norepinephrine is methylated in vivo according
to the reaction shown. Furthermore, Gurin and Delluva injected into
rats D L-phenylalanine labeled with C^'* (radioactive) in the —COOH
group and (or) in the alpha position (carbon atom next to the — COOH).
They showed that radioactive epinephrine with all of its C^"^ located
in the terminal carbon of the side chain was formed. This is good evi-
dence that epinephrine is actually made from phenylalanine by the
living animal. There is no direct proof that dihydroxyphenylalanine
takes part in the biosynthesis, but some such dihydroxy substance must
obviously be involved at some stage of the process.

Both hormones contain an asymmetric carbon atom and are therefore
optically active. The levorotatory or i-forms are the naturally occurring
isomers and are about fifteen times more effective than the corresponding
c?-forms. Both isomers of each hormone have been prepared by chemical
synthesis, and the pure Z-forms have been isolated from the adrenals
of various animals.

Injection of epinephrine is followed by a rapid rise of blood pressure

HORMONES 289

due to increased heart rate and contraction of the arteriols (small arter-
ies) . The blood pressure falls again rather quickly unless additional
doses are given. The hormone also brings about contraction of the iris,
relaxation of bronchial muscles, increased salivary secretion, and other
effects. These responses, in general, are the same as those caused by
stimulation of the sympathetic nerves going to the same tissues or organs.
In fact, it appears that epinephrine, or norepinephrine, is necessary for
transmission of nerve impulses in the sympathetic (or "adrenergic")
nerve system. Some sympathetic nerve cells produce epinephrine, and
others, norepinephrine. According to Tainter and Luduena, these hor-
inones probably pass through the nerve trunks to the endings, are released
when the nerve is stimulated, and act on the particular tissue concerned
to produce the effect finally seen.

Epinephrine also causes a rise in the amounts of glucose and lactic acid
in the blood stream and increases the basal metabolic rate (p. 424).
Blood sugar is derived from glycogen by the following reactions (see
Fig. 13-1):

1 TT T-,/^ (phosphorvlase) ,11 (phosphogluco-mutase)

glycogen + H3F04 ' »■ glucose- 1-phosphate *-

(phosphatase')

glucose- 6-phosphate > glucose + H3PO4

It is the first of these steps which is stimulated by epinephrine. Norepine-
phrine has only about one-eighth the effect of epinephrine in raising
the blood sugar level.

The secretion of the adrenal medulla is under nervous control and is
increased in times of stress or intense emotion (suffocation, rage, fear,
etc.). The net result is a general mobilization of the resources of the
individual to meet the crisis. Aside from its direct natural functions,
epinephrine has also found a number of medical applications, particularly
as a vasoconstrictor (blood vessel constrictor), and for relief of the
bronchial spasms of asthma and hay fever. Injected together with a
local anesthetic such as Novocaine, it contracts small blood vessels and
reduces blood flow through the area affected. It thus permits a longer
and more intense response from a given dose of the anesthetic. It also
has antihistamine action and usually gives relief in a variety of allergic
conditions thought to be due to the liberation of a histamine-like substance.

Histamine is the amine formed by decarboxylation of histidine (p. 321) .
Even small doses of it can produce symptoms of allergy. A number of
drugs intended to destroy or counteract histamine have been developed.
They are called antihistamines, and their effect is called an antihistamine
action. Benadryl and Pyribenzamine are two widely used antihistamine
drugs:

290

HORMONES

^^HC— 0— CHsCHoNCCHs)^

Benadryl

\ /

^ /-N— CH2CH,N(CH3)2
Pyribenzamine

Hormones of the adrenal cortex

The outer part, or cortex, of the adrenal gland produces a series of
hormones which are essential for life. In this respect the cortex differs
from the medulla, for the latter can be removed from animals without
causing death. However, in 1930 Hartman and Brownell found that
adrenalectomized animals ^ could be kept alive if injected at regular
intervals with material extracted from the adrenals of other animals
of the same or different species. The life-maintaining principle was
contained in the nonsaponifiable part of the extract from the cortex and
was eventually found to consist of a group of closely related steroids.
At least 28 individual steroids have been separated from such extracts
as pure crystalline substances, of which six possess marked adrenal cor-
tical activity.^ The chemical structures of these six compounds are as
follows (see p. 94 for diagram and numbering of the steroid ring system) :

or ^^ ^ 0

1 l-Desoxycorticosterone,
C21H30O3

Corticosterone,
C21H30O4

1 1-Dehydrocorticosterone,
C21H28O4

17-Hydroxy-ll-desoxy- 17-Hydroxycorticosterone, Cortisone, C21H28O5
corticosterone, C21H30O4 C21H30O5 (17-Hydroxy-ll-dehydro-

corticosterone)

^ Animals with both adrenals completely removed.

^ In addition, there is a noncrystalline residue which still contains one-fourth to one-
half the biological activity of the original extracts.

HORMONES 291

Note that the six substances differ chiefly in the presence or absence
of oxygen on carbons 11 and 17. They were first obtained as pure
chemical substances during the period 1935 to 1939.

That the normal adrenal cortex has a number of functions has been
revealed by study of adrenalectomized animals and of human victims
of Addison's disease, a fatal illness caused by insufficient secretion of
adrenal hormones. In such cases there is a marked decrease in the ability
of the organism to work and to withstand stresses of any sort (for ex-
ample, fasting or exposure to cold). Sodium and chloride ions are ex-
creted in the urine in such excessive amounts that bodily supplies are
depleted, whereas excretion of potassium and urea are subnormal. Glyco-
gen disappears from the liver, great muscular weakness develops, and
growth ceases.

The cortical hormones listed above differ in their ability to counteract
these symptoms. 11-Desoxycorticosterone is the most active member
of the group for regulating sodium, potassium, and chloride metabolism,
and for maintaining the life of adrenalectomized animals. Cortisone,
on the other hand, is relatively inactive in these respects, but it is the
most potent member of the group for increasing the ability to work and
resist stress and for stimulating glycogen formation. The other four
hormones have effects similar to one or the other of these two, or both.

Addison's disease was for a time (and to some extent still is) treated with
cortical extracts following the work of Hartman and Brownell in 1930.
This treatment prolongs the life of the patients, but it is prohibitively
expensive and only partly successful. The use of 11-desoxycorticosterone,
after it became available about 1940, resulted in a major gain in life
expectancy, but even this hormone did not fully replace the missing
adrenal secretion. Most patients so treated lack normal vigor, and about
half die within seven years. The additional injection of cortisone may
quite possibly make up the deficiency, but sufficient amounts of cortisone
for clinical use have been produced only recently, and some years will
be needed to decide this question definitely. As might be expected from
the nature of their disturbed electrolyte metabolism, Addison's disease
patients are greatly benefited by diets low in potassium and high in
common salt.

The opposite situation — oversecretion of adrenal cortical hormones —
is also a serious clinical condition. This may result from tumor growth
of the pituitary gland, which causes an increased secretion of a pituitary
hormone (adrenocorticotropic hormone, or ACTH) that stimulates the
adrenal cortex. In this condition (Cushing's syndrome) blood levels of
sodium are high and those of potassium low,, that is, just opposite from
the situation in Addison's disease.

Tumor growth on the adrenal itself leads to secretion of male sex
hormones. If the patient happens to be an adult female, this results in
sex inversion (virilism) , which manifests itself by deepening of the voice,

292 HORMONES

growth of a beard, atrophy of the breasts, cessation of menstruation, and
development of a mascuhne-type musculature.

In the last few years several of the most common and distressing human
diseases of previously unknown origin have been found to respond to
treatment with adrenal hormones. The outstanding example is rheuma-
toid arthritis, a painful crippling disease characterized by swelling and
inflammation of the joints. Arthritic patients had sometimes been ob-
served to show improvement under stress (late pregnancy, starvation,
major surgery, etc.). Since it was known that adrenal function also is
increased by stress, Hensch and Kendall reasoned that some of the adrenal
hormones might be of value in arthritis. Their report describing the
beneficial effect of cortisone and ACTH appeared in April 1949 and was
quickly followed by confirmatory findings elsewhere.

Great efforts have been made by chemical and pharmaceutical firms
to produce these substances in adequate amounts. Cortisone has been
obtained synthetically by an involved process starting with cholic acid
from cattle bile (p. 96). Recently, certain plant steroids have been
found which can be used as starting materials and greatly simplify the
synthetic process. As a result, prices have been reduced and supplies
increased, although larger amounts are still needed. ACTH, on the other
hand, has not been synthesized and can only be obtained from animal
pituitary glands. This is a limited and costly source. Investigations
of the structure suggest that only part of the ACTH protein is needed
for the biological effect and that the activity may, in fact, reside in a
rather small peptide portion of the molecule. If the exact structure of
this peptide can be established, there will be a possibility of producing
it synthetically in any desired amount.

Sex hormones

This term is applied to hormones secreted by the gonads (ovaries and
testes), although several other glands are equally essential for reproduc-
tion. Whether or not the adrenals normally produce sex hormones is
uncertain, although the effect from abnormal production is well known
(see above).

Functions. The periodic sexual cycles of female mammals are con-
trolled largely by the ovarian hormones, estradiol and progesterone.
These substances are made in the ovary as a result of stimulation by
two gonadotropic hormones from the pituitary, namely the follicle-stimu-
lating, or follicle-ripening (FSH), and luteinizing (LH) hormones.
Figure 11-1 shows how these substances function during a normal human
menstrual cycle.

On the first day of the cycle (first day of menstruation) secretion of
FSH begins and causes one of the primitive, undeveloped egg sacs, or

HORMONES

293

j^rimary follicles, in the cortex of the ovary to start growing. After about
13 to 15 days the mature, or Graafian, follicle bursts, releasing an egg
cell or ovum (ovulation). The bursting of the Graafian follicle is caused
by LH. The egg finds its way into one of the Fallopian tubes and thence
to the uterus. The developing follicle, and to a lesser extent the entire
ovary, secrete estradiol in increasing amounts after about the fourth day.
Estradiol causes the endometrial cells in the mucosa of the uterus to
start dividing so that by the time of ovulation the mucosa is actively
growing. A similar effect is produced on the cells lining the vagina.
Any substance which produces these effects is called an estrogenic hor-
mone, or an estrogen. In female animals, estrogens also produce sexual
heat or estrus; hence, the name.

After discharging its ovum the remainder of the follicle changes into
another structure called the corpus luteum. The corpus luteum develops
into a mature state under the influence of LH, which starts to be secreted

Pituitary

Pituitary
Hormone
Factors

Ovary

Anterior
Lobe

Follicle

Stimulating

Ilnrnioni'

>'

4b

Po^^tcrior
Lobe

LutoinizinR Lutcotrophic

Jlonnone Hormone

|l'riiiiordial Developing Maturing
Follicle Follicle Follicle

Ovulation

Do\elo|)ing
T'orpus I^utrnni

.Mature
r|ius Liiliuni

Degenerating
( 'ni|]iis Luteum

Ovarian

Hormone

Factors

Uterine »

Endometrium

Phases Repair IMohlVratmn Srcrrlmy Menstrual idii

Days I 1 I .J I 6 17 I .S I !l Hul 1 1112 11311 111. JllGl I7llsl I'jl2ul.'ll22l23l 24l2ol2(il27l2Sl i\2\S\

Courtesy of J. C Stucki and the Department of Zoology, University of Wisconsin.
Fig. 11-1. Hormonal control of the menstrual cycle. This is a simplified
diagram which does not take into account interactions between the three
pituitaiy hormones nor the action of the ovarian hormones on the pituitary
gland. The curves indicate approximate variations in the urinary excre-
tion of ovarian hormones during the cycle. For further explanation see
text.

294

HORMONES

by the pituitary gland about the twelfth to fourteenth day of the cycle
(see Fig. 11-1), and is maintained in that condition partly by LH and
partly by the luteotropic hormone. The corpus luteum, in turn, pro-
duces progesterone, which has a further profound effect on the lining of
the uterus, causing the lining and especially the tiny glands present in
it to develop enormously (Fig. 11-2) and to secrete a nutritive fluid. In

Courtesy oi W. G. Black and the D(_pditment ot Genetics, University of Wisconsin.
Fig. 11-2. Effect of progesterone on the uterus. Spayed rabbits were
given 10 daily injections of 1 mg. each of progesterone in oil, and killed
24 hours after the last injection. Left, untreated control ; right, treated with
progesterone.

this state the uterus is prepared to receive the ovum, in case fertilization
has occurred during its passage through the Fallopian tube. If fertiliza-
tion does not occur, the ovum dies a few hours after ovulation, and in a
few days the corpus luteum stops functioning and degenerates. Secre-
tion of progesterone consequently diminishes, and as a result much of
the lining of the uterus sloughs off, and in primates it is discharged as
the menstrual flow.

In case the ovum has been fertilized, it becomes embedded in the spe-
cially prepared uterine lining and begins to grow. The corpus luteum
continues to secrete progesterone and thus to maintain the uterus in a
condition favorable for the developing fetus. Any deficiency or interrup-
tion in the supply of progesterone results in death of the embryo and
consequent miscarriage. This is one of the reasons why certain women
suffer repeated miscarriages and are unable to bear a living child. Thi§

HORMONES

295

difficulty has been completely overcome in many cases by the clinical
use of progesterone.

After about the third month of pregnancy the placenta also begins to
function as an endocrine organ and partially takes over the production
of progesterone. It also secretes hormones with FSH and LH action,
and probably others. The urinary excretion of various hormones during
pregnancy is shown in Fig. 11-3. Toward the later months estradiol is
again produced in increasing amounts. This renders the muscles of
the uterus more responsive to the oxytocic hormone of the pituitary (a
"contracting" hormone, see p. 303) and eventually brings about the onset
of labor. Delivery is facilitated by still another hormone, relaxin. This
substance has been studied mainly in animals, where it appears to be
produced by the placenta under the influence of estradiol and progesterone.
As the name implies, relaxin loosens the pelvic ligaments and thus facili-
tates birth. Chemically it appears to be a peptide.

The final hormone cooperating in the reproductive process in the female
is the lactogenic hormone, which is produced immediately following de-
livery (Tig. 11-3) and stimulates milk production. It is probable that
the luteotropic hormone is, in fact, the same substance as the lactogenic
hormone.

4 5 6

Lunar Months of Pregnancy
Reproduced by courtesy of Parke, Davis & Company's Therapeutic Notes.
Fig. 11-3. Hormone production during pregnancy.

In addition to the roles described above, the female sex hormones exert
a profound inffuence on the development of the secondary sex character-
istics such as body shape, body hair distribution, and voice tone, and on
the growth of the reproductive organs.

The male sex hormones, testosterone and androsterone, control the
development of the male genital organs, the production of spermatozoa,

296

HORMONES

and the appearance of male characteristics pecuHar to the species. In
man these include a typical distribution of body hair, deep voice, mascu-
line shape of body and muscular development, and growth of the beard.
Substances producing these effects are called androgens, or androgenic
hormones. A typical effect is shown by androgens on the growth of the
comb and wattles of the chick (Fig. 11-4). This response has been

Courtesy of University of Wiscon.sin, Department of Poultry Husbandry.
Fig. 11-4. Effect of androgen on growth of comb and wattles of imma-
ture cockerels. Both birds were 19 days old when photographed, but the
one on the right had been treated with 15 mg. of testosterone propionate
9 days previously.

!■

made the basis for a quantitative assay method.

Chemical Nature. The chemical formulas of the more important sex
hormones obtained from natural sources are given below:

Estrogenic hormones

HORMONES

297

Progesterone

Testosterone

Androsterone

Androgenic hormones

Estradiol, progesterone, and testosterone appear to be the true, primary
hormones, since they are considerably more active, weight for weight,
than the others. The chemical structures of all the steroid hormones
appear to be remarkably similar for substances having such widely
different biological properties (compare p. 290). The structures of the
estrogens and androgens can be modified considerably, however, without
marked loss of potency. For example, stilbestrol and doisynolic acid
are about as effective as estrone, and the former, in fact, is even more
active than estrone when given orally (Table 11-1). On the other hand,

H,^ H

II I H

C H U
H ^ Hs

Stilbestrol

Doisynolic acid

almost any change in the progesterone molecule produces an inactive
product.

Thyroid hormone

The thyroid gland is a small mass of specialized tissue — about 30 g.
in the human adult — located in the neck near the larynx. It produces
a hormone which greatly stimulates metabolic activity, particularly the
oxidative, energy-yielding processes. Iodine has long been recognized
as related to thyroid function (for example, in goiter), but it was not
until 1919 that the isolation of an active substance, thyroxine, from thyroid
tissue was reported by Kendall. A related substance, diiodotyrosine
or iodogorgoic acid, has also been isolated from thyroid, and, in fact,
accounts for about two-thirds of the total iodine content of the gland.

298 • HORMONES

Table 11-1

Estrogenic potency of various substances

Effective dose for rats

Substance Injected (jig.) Oral {\ig.)

Natural estrogenic hormones

Estradiol 0.08-0.13 50

Estrone 0.7-0.8 50-60

Estriol 10 10

Synthetic products

Stilbestrol 0.3-0.4 0.7-1.0

Hexestrol 0.2

Doisynolic acid 0.8-0.9

I I NH2 I NH2

/ \_0-/ \_CR„— C— COOH ho/ \

HO— ( / ^ \ V-CH,-C— COOH HOC V-CHj-C— COOH

l"" l"" H l"" H

L-Thyroxine L-Diiodotyrosine

Thyroxine contains no less than 65.4 per cent of iodine by weight.
It was obtained from thyroid glands only after drastic alkaline hydrolysis,
which, incidentally, converted the natural L-form into the corresponding
DL-mixture. Thyroxine and diiodotyrosine are not present in the living
gland in the free state but are contained in a protein, thyroglobulin.
This substance has a high molecular weight, estimated at 700,000, and
is consequently nondiffusible. It contains a rather constant total amount
of tyrosine, diiodotyrosine, and thyroxine residues amounting to 3-4
per cent, but the relative proportions of the three vary with the iodine
intake. Iodine ingested with food is quickly absorbed by the thyroid
and is used to convert more of the tyrosine of thyroglobulin into diiodo-
tyrosine and thyroxine residues. Thus the gland acts both as a factory
and storehouse for bound thyroid hormone.

When the hormone is needed in other parts of the body, some of the
thyroglobulin is broken down by proteolytic enzymes so that either
free thyroxine or small, water-soluble, diffusible peptides containing
thyroxine as one of the component amino acids are liberated and carried
away by the blood stream. While circulating in the blood the thyroxine
is bound rather loosely to one of the plasma proteins. Just what happens
when these substances reach the tissues ultimately affected is not known,
but free thyroxine apparently is not involved. Nevertheless, pure
L-thyroxine, either natural or synthetic, does produce the effects of whole
thyroid when administered to animals or to human patients. The term
"thyroid hormone" applies to any substance capable of causing the char-
acteristic physiological effects and thus includes both free thyroxine and
the various bound forms of it existing in the animal body. Diiodotyrosine
has no appreciable thyroid hormone activity.

The mechanism of thyroid hormone synthesis in the body is not known

HORMONES

299

with certainty, but very likely involves addition of iodine to tyrosine
followed by self-condensation of the diiodotyrosine produced to form
thyroxine. Similar reactions, at any rate, take place quite readily out-
side the body. Thus, when proteins such as casein or egg albumin are
treated with iodine for several hours in the presence of a mild alkali
(sodium carbonate) , iodine becomes incorporated into the protein molecule
and thyroxine is formed. The amount of hormone formed is closely
correlated with the tyrosine content of the protein used; gelatin, which
does not contain tyrosine, fails to yield thyroxine under this treatment,
lodinated casein has been used extensively as a source of thyroid hormone,
for example, to feed dairy cows. Milk production is thereby increased,
but higher feed costs plus possible injury to the animals make the prac-
tice of doubtful economic value.

Thyroid Disorders. Common goiter is an enlargement of the thyroid
resulting from low iodine intake. Where the iodine supply is not greatly
deficient, the enlarged gland is able to maintain normal function. The
only serious result is pressure on surrounding organs; for example, pres-
sure on the windpipe may be sufficient to cause difficulty in breathing.
The condition can usually be corrected by supplying adequate iodine (see
p. 192) , or in extreme cases by surgically removing a part of the gland.

A more serious condition arises whenever the thyroid is damaged in any
way (for example, by an infection) so that it can no longer produce an
adequate supply of hormone. This may occur at any time from fetal
life onward. In adults the condition (called myxedema) is character-
ized by a general slowing down of metabolic activities, slow heart rate,
low blood pressure, easy fatigue, increased blood cholesterol levels, dimin-
ished urinary excretion of 17-kctosteroids, and decreased amounts of
"plasma-bound iodine." This plasma-bound iodine, which represents the
amount of circulating thyroid hormone, may drop from the normal range
of 4^6 to below 2 ;u,g. per 100 ml. of blood plasma. Replacement therapy
with thyroxine or whole thyroid substance (dried, ground animal thyroids)
is effective but must be continued throughout life.

Thyroid deficiency in early life has all the above effects and, in addi-
tion, retards both mental and physical growth. The aff'ected individuals
often survive to adulthood, but are dwarfs and idiots. This condition is
called cretinism, and the sufferers from it, cretins. Treatment with
thyroid hormone is effective, but only if started before normal develop-
ment has been stunted.

Overactivity of the thyroid (Grave's disease) is likewise most injurious
to health. Excessive production of the hormone leads to high basal
metabolism, excess energy production, restlessness, tremor of extremities,
excessive flushing and perspiration, loss of weight, low blood cholesterol,
high excretion of 17-ketosteroids, and frequently exopthalmos (protrud-
ing eyeballs). Psychic disturbances often accompany Grave's disease
and may be a factor in causing it. Treatment is designed, of course, to

300

HORMONES

counteract the excessive hormone production. This has been accom-
plished by partial or complete removal of the gland, or, more recently,
by the use of antithyroid drugs and chemicals. Many common foods
including spinach, cabbage, turnips, walnuts, lima beans, peas, carrots,
grapes, grapefruit, and others have distinct goitrogenic (goiter-producing)
or antithyroid effects. The responsible substance was obtained in pure
form from yellow turnips and was shown to be l-b-vinyl-2-thiooxazoli-
done. Various synthetic drugs such as 2-thiouracil and 6-propyl-2-
thiouracil also have marked antithyroid action. These substances do
not prevent absorption of iodine by the thyroid, but interfere with the
incorporation of it into the thyroid hormone. Some of them, e.g., propyl-
thiouracil, have proved to be very valuable in the treatment of Grave's
disease.

H^a- NH

I (4) (3) I

^5-Viny 1- 2- thiooxazolidone
Nf===C-OH N===C-OH

I (3) (4) I j (3) (4) I

HS— C(2) (5)CH HS— C(2) (5)CH

N^^L^CH N^-^^-^C-CH.CH.CH3

2-Thiouracil 6-Propyl-2-thiouracil

Radioactive iodine compounds containing the I^^^ isotope have also
found application in the diagnosis and treatment of excessive thyroid
activity. A small test dose of the radioactive iodine, for example, in
the form of potassium iodide, is given the patient, and the accumulation
of the iodine in the thyroid followed with a Geiger counter placed near
the throat. If an abnormally high fraction of the test dose enters the
gland, excessive thyroid activity is indicated. In this case, larger doses
of I^^^ are given, and as the iodine becomes lodged in the gland, the radia-
tions emitted by it kill a part of the thyroid tissue. Surrounding tissues
are essentially unaffected, and the extent of thyroid destruction can easily
be controlled by regulating the dosage.

PROTEIN AND PEPTIDE HORMONES

Hormones of the pancreas

The pancreas is a pale pink organ about ten inches long in an adult
person, which produces a group of digestive enzymes and discharges them
into the small intestine. It also has the function of producing at least
one hormone, insulin, which is secreted directly into the blood stream.

HORMONES 301

The name insulin comes from the Latin insula, meaning island, and
refers to the fact that the hormone is formed by small groups of special-
ized cells called the "islets of Langerhans." There are about 250,000 to
2,500,000 of these islets in man. Lack of insulin causes diabetes ■mellitus,
a fatal human disease characterized by excessive urinary excretion and by
the presence of large amounts of glucose in the urine. After this fact
was established around 1890, many efforts were made to prepare active
extracts from animal pancreas glands. They were without success be-
cause the hormone was destroyed by the proteolytic enzymes present.
The first really effective extracts were obtained in 1922 by Banting, Best,
Collip, and IMacLeod through the use of acidified alcohol, which inacti-
vated the enzymes. Because of the great demand for insulin for the
treatment of diabetes, commercial production was soon undertaken, and
many workers studied methods of purifying the crude extracts. Pure
crystalline insulin was finally isolated by Abel and co-workers in 1926.

Chemical Nature. Insulin is a water- and alcohol-soluble protein,
having its isoelectric point at pH 5.3. The molecular weight has been
estimated at 36,000 to 48,000, but it is now known that these values repre-
sent aggregations of smaller units. The true molecular weight is gener-
ally considered to be 12,000.^ Insulin has a sulfur content of 3.1 per
cent, which is much higher than that of most other proteins. In spite
of much searching no organic prosthetic group or structure other than
the usual amino acids has ever been found as a component of insulin.
Crystalline insulin contains about 0.3 per cent of zinc, but this is of
doubtful significance as essentially zinc-free amorphous preparations
have equal biological activity. As a result of a brilliant series of re-
searches carried out during the last few years, chiefly by Sanger, the
structure of insulin is known in greater detail than is that of any other
protein (p. 132).

Physiological Function. The appetite and thirst of the untreated
diabetic are enormous, but in spite of the great quantities of food and
drink consumed, the body weight becomes progressively less. Blood
glucose levels rise so much above the renal threshold that large amounts
are excreted in the urine (p. 327). Bodily glycogen stores are depleted.
Urinary excretion of nonprotein nitrogen compounds is increased as a
result of increased conversion of deaminated amino acids into carbohy-
drate. Excessive oxidation of fat occurs, and ketosis develops (p. 338).
These symptoms have usually been interpreted as being due to decreased
utilization of carbohydrates by the body, but some authorities believe
that the disease is more a result of increased formation of carbohydrates
(from fat and protein) than of decreased utilization. The over-produc-

^ However, Fredericq and Neurath have recently obtained evidence that the actual
value may be only 6,000. Larger apparent molecular weights result from reversible
aggregation in solution, the extent of which depends on the pH, concentration*, tempera-
ture, and kind and amount of inorganic ions pi'esent.

302 HORMONES

tion theory is supported by the observation that removal of the liver
from either diabetic or normal animals is followed by an equally rapid
decline of blood glucose levels in both. Under these conditions at least,
the diabetic animal utilizes carbohydrate as. well as the normal one.

All the symptoms of the disease are relieved by injection of insulin.
The hormone is not effective by mouth because it is destroyed by digestive
enzymes, as mentioned above. Excessive doses are sometimes adminis-
tered accidentally to diabetic patients. In such cases, as might be
expected, the blood sugar level falls sharply and may go so low as to
result in coma and, if not treated, death. This condition, insulin shock,
is counteracted quickly and completely by injection of glucose. Symp-
toms of insulin deficiency can be produced in animals by feeding alloxan,
and it has been suggested, but not proved, that alloxan may be in some
way connected with the development of diabetes. When present in the
body, its effect is to destroy the islet cells of the pancreas.

HN C=0

I I

o=c c=o

I I

HN C=0

Alloxan

A condition closely resembling diabetes mellitus can be produced in
experimental animals by feeding the drug, ])hlorhizin, a glucoside present
in the root and bark of apple, pear, and plum trees. It has the follow-
ing chemical structure:

CO-CH2— CH2-< /"OH

OH

Phlorhizin

The blood sugar level in phlorhizin diabetes, however, is lower than
normal. The drug produces the symptoms of diabetes by preventing
the resorption of glucose by the tubules of the kidney, thus, in effect,
lowering the renal threshold and causing urinary excretion of sugar even
while the blood sugar level is in or below the normal range.

The mechanism by which insulin acts is not definitely known. It
has been claimed by Cori and co-workers that insulin counteracts the
inhibition of hexokinase by hormones of the pituitary and adrenal glands.
However, the experimental results have been interpreted differently by
other workers, and the status of this suggestion, therefore, is still in
doubt.

HORMONES

303

Hyperglycemic Factor of the Pancreas. Certain commercial prepara-
tions of both amorphous and crystalline insulin have been found to con-
tain an impurity which, surprisingly enough, causes an increase in blood
sugar levels. This substance, the hyperglycemic factor, is present in the
pancreas and also in the stomach lining. It appears to be a protein and
to act by causing breakdown of glycogen (glycogenolysis). Its physio-
logical role has not been clarified.

Lipocaic. Animals which have been rendered diabetic by removal of
the pancreas have been observed to accumulate great amounts of fat in
the liver. Since the development of such fatty livers can be prevented
by the feeding of raw pancreatic tissue, but not by insulin, the existence
of another hormone in the pancreas was postulated. This substance,
which was named lipocaic, is probably not a hormone, since the effect
can be produced by pancreatic juice {i.e., the external secretion of the
pancreas) and can also be duplicated by choline or methionine. Preven-
tion of fatty livers by raw pancreas is apparently due to the presence of
proteolytic enzymes which make methionine more readily available from
food proteins.

Hormones of the posterior pituitary

The pituitary or hypophysis, a small endocrine organ located in the
center of the head, is the master gland of the whole hormone system of
higher animals. Its special importance is due to the large number of
hormones it produces and to the fact that several of these have the
particular function of stimulating other glands to secrete their character-
istic hormones. Thus the pituitary directly or indirectly influences a
great number of bodily processes. The gland consists of anterior and
posterior lobes and a small center section. Extracts of the posterior
lobe have three well-defined effects on the animal, namely, those of raising
the blood pressure (pressor effect) , stimulating the contraction of uterine
muscle and, to a smaller extent, of smooth muscles generally (oxytocic
effect), and suppressing urinary secretion (antidiuretic effect).

The antidiuretic effect is caused by the same substance that produces
the pressor effect (vasopressin). Lack of it causes diabetus insipidus,
a disease in which the volume of urine excreted is enormously increased,
although sugar is not present.

Oxytocin. The substance responsible for the oxytocic effect, oxytocin,
has been very highly purified, and the best preparations of it seem to be
substantially one substance. It is a white, amorphous, water-soluble
powder with the properties of a basic polypeptide. According to Pierce
and du Vigneaud it yields on hydrolysis one molecule each of leucine,
isoleucine, tyrosine, proline, glutamic acid, aspartic acid, glycine, and
cystine, and three molecules of ammonia. The molecular weight is close
to 1,000. Thus it is an eight-membered peptide. The sequence of the

304 HORMONES

amino acid residues has not been determined. One physiological role
of oxytocin seems to be to expedite labor by increasing uterine contrac-
tions. Partially purified preparations have found chnical application
for this purpose and for control of hemorrhage after delivery. The
hormone is ineffective orally. Oxytocin also plays a role in milk secre-
tion in that it stimulates contraction of the smooth muscles in the walls
of the milk ducts.

Vasopressin. The pressor hormone of the posterior lobe has been vari-
ously called vasopressin, pressin, or pitressin. Like oxytocin it is a basic
octapeptide, which has a molecular weight of about 1,000. It has been
obtained as a white, amorphous, water-soluble powder. On hydrolysis
it gives the same products as does oxytocin, except that arginine and
phenylalanine are present, but leucine and isoleucine are not. When
injected into animals vasopressin causes a pronounced but temporary
rise in blood pressure. Presumably it must function normally to help
regulate blood pressure, along with thyroxine, epinephrine, and other
substances.

Abnormally high blood pressure (hypertension) is a common human
disease and a major cause of death. Attempts to learn its cause have
uncovered several other substances of natural occurrence which greatly
influence blood pressure. One of these is renin, a protein secreted into
the blood stream by the kidney. Renin is a proteolytic enzyme. It
acts on a particular protein, one of the globulins in the blood plasma,
which therefore is called renin substrate. Renin itself does not have
pressor activity, but the product of its action on the substrate, a peptide
called angiotonin or hypertensin, is highly active. The elevated pressure
is caused by contraction of small arteries combined with increased heart
action. The effect from a single dose lasts only a few minutes because
angiotonin is quickly destroyed in normal individuals by another enzyme,
angiotonase. Pathological hypertension could conceivably be produced
by overproduction of vasopressin or renin, or by lack of angiotonase, but
these possibilities have not been proved correct, and no really satisfac-
tory treatment for the disease has yet been found.

Still another pressor substance normally present in the animal body
is serotonin. This substance was isolated from blood serum in the form
of a crystalline product which proved to be a mixed sulfate of serotonin
and creatinine. The effective component, serotonin itself, is 5-hydroxy
tryptamine :

CH2CH2NH2

HORMONES

3()S

Hormones of the anterior pituitary

The anterior lobe produces six well-recognized hormones, which have
been named according to their biological effects. All six have been
extensively purified and found to be proteins of relatively low mo-
lecular weight. The functions of several have been considered briefly
in previous sections of this chapter. Additional properties are listed
in Table 11-2.

One of the most interesting and important of the group is the adreno-
corticotropic hormone (ACTH, p. 291). The active protein can be
hydrolyzed with pepsin or hydrochloric acid until about half of the
peptide bonds have been broken without diminishing the activity. This
indicates that the effective substance most probably is a peptide com-
posed of far fewer amino acids than the ACTH protein. This peptide
has not been isolated in pure form, but partly purified products have
been obtained which are reported to have molecular weights in the range
1000-2000 and to be much more active than ACTH protein on a weight
basis.

The growth hormone (GH) has the power of stimulating growth, both
of the skeleton and soft tissues of the animal body. Normally the long
bones are "closed off" at the ends and stop developing shortly after the
attainment of sexual maturity. This cessation of bone elongation is
probably caused by the sex hormones produced at that time. However,
in some cases this does not occur, and the continued production of excess
GH, leads to gigantism. Heights of eight and nine feet have occurred
in human beings. If extra secretion of GH occurs after full maturity,
some parts of the body are still able to grow and others are not. This
results in distorted growth, which causes a gorilla-like appearance. This
condition is called acromegaly (Fig. 11-5). Conversely, a deficiency of
GH causes one type of dwarfism. Such dwarfs have normal intelligence
but are physically small, delicately formed, and doll-like. The diabeto-
genic activity of the pituitary (p. 325) probably is also the result of
GH, or of a substance closely associated with it.

The lactogenic hormone of the anterior pituitary, sometimes called pro-
lactin, is another very interesting substance, not only because it stimulates
milk production by mammals, but also because it seems to influence mental
attitudes. Its effects have been described by R. G. Hoskins as follows:

"In addition to its effect on milk production, the anterior-lobe product prolactin
has a striking influence on animal behavior. It induces broodinessi in the fowl and
modifies the nesting behavior in certain fish (Riddle). Its influence on the instinctual
behavior of rats has been studied by Wiesner and Sheard and by Riddle.

"The method of procedure is to place young female rats in cages with materials for
nest building. They are then tested as to the strength of their maternal urge by

306

HORMONES

being offered new-born baby rats for adoption. In most instances the females remain
indifferent to the intruders. But if, to these nonchalant misses, a few doses of pro-
lactin are administered, not only are their mammary glands stimulated but a remark-
able change in their behavior takes place. They will now eagerly adopt as many
babies as may be offered, build elaborate nests for them, and eagerly mother them.

Iteproduced by permission from TuriUT, (Iciicidl i:niluciin(j}ii(nj, W. B. Saunilers

('omi)an.v.

Fig. 11-5. Acromegaly.

The yearning seems to be universal. Their maternal reactions are not confined to
infants of their own kind but are extended to baby mice, baby rabbits, or even
helpless squabs. For a normal, vigorous rat to do other than promptly make a feast
of a proffered squab is proof positive that something fundamental has happened
to her instincts. What part prolactin may play in the determination of human
instincts and emotions is as yet unknown, but the stimulus to imagination is
tempting."

Gastrointestinal hormones

The secretion of digestive juices and the movements of the stomach
and intestines incidental to the digestion of food are partly controlled
and regulated by several gastrointestinal hormones. These processes
are also influenced markedly by nervous stimulation, and it has been diffi-
cult to sort out the two types of effect. At present four gastrointestinal
hormones have been quite definitely proved to exist, and a number of
others are suspected. None of them are definitely known to be essential
for life or to cause any disease if produced in abnormally large or small
amounts.

Before considering these substances in detail, a distinction should be

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HORMONES

made between them and "secretogogues." The latter are chemical sub-
stances in the food, or derived from food during digestion, which directly
or indirectly stimulate the secretion of digestive juices. They are not
classified as hormones because they are not produced by the body.

Gastrin. This hormone is produced by the mucosa of the lower, or
pyloric, end of the stomach and to a lesser extent by the mucosa of the
duodenum (that portion of the small intestine immediately beyond the
stomach). It is secreted into the blood stream (though not directly)
as a result of stimulation by secretogogues and has the effect of increasing
the flow of gastric juice. The juice so formed is high in hydrochloric acid
but low in pepsin.

Gastrin can be extracted from suitable mucosa and has been purified
considerably, although not completely. It is destroyed by proteolytic
enzymes or by boiling in one-tenth normal sodium hydroxide solution
and is precipitated by trichloracetic acid. It is probably a peptide or
low molecular weight protein. Histamine in very small doses has the
same effect as gastrin, and it may be that histamine actually is the hor-
mone. This has not been proved, however, mainly because the effective
level of histamine is too low to be detected in the blood stream by the
analytical methods at present available. An observable gastric response
is produced in human beings by the injection of only 0.004 /xg. of hista-
mine per kilogram body weight per minute.

Secretin. This substance stimulates the secretion of water by the pan-
creas and of bile by the liver. The .increased flow of pancreatic juice
is relatively poor in enzyme content.^ Secretin exists in an inactive
form (prosecretin) in the mucosa of the upper small intestine, or duode-
num, and is released by the action of dilute hydrochloric acid from the
stomach (pH ca. 4.6).

Two crystalline secretin preparations have been isolated, both in the
form of salts with picrolonic acid. One appears to be a peptide, while
the other is a compound of low molecular weight. The peptide, however,
can be extensively hydrolyzed by aminopolypeptidase without loss of
secretin activity. It is suggested that the two products may be related
in much the same way as thyroxine and thyroglobulin. The exact
formula of secretin is not known.

C holecystokinin. The name of this hormone (literally ''gall bladder
mover") indicates its physiological function which is to stimulate con-
traction and emptying of the gall bladder. Like secretin, it is produced
in the mucosa of the duodenum. It is secreted indirectly into the blood
stream whenever fat, fatty acids, peptone, or dilute hydrochloric acid
enter the intestine. It has been only partially purified, and its chemical

^ Another gastrointestinal hormone, the existence of which is very probable but not
conchisively proved, stimulates the secretion of enzijmes by the pancreas. Purifipd
prei)a rations of this substance, pancreozymin, have no effect on the volume of pan-
creatic secretion.

HORMONES

309

constitution is therefore not known. However, it tends to follow secre-
tin in extraction and purification procedures and may be a peptide.

Entcrogastrone. This hormone is also secreted by the duodenal mucosa,
but it has the effect of inhibiting the movements of the stomach, as well
as the stomach's secretion of hydrochloric acid. Secretion of entero-
gastrin into the blood stream is brought ab(jut by the presence in the
small intestine of fatty acids, especially oleic acid, soaps, neutral fat,
or relatively concentrated solutions of sucrose, glucose, or lactose. The
hormone has not been isolated in pure form, but the best preparations
contain amino acids and have the properties of peptides. If sufficiently
pure preparations to avoid undesirable side effects were available, entcro-
gastrone would be of value for the treatment of gastric ulcers in human
patients.

REVIEW QUESTIONS ON HORMONES

1. Distinguish between hormones and vitamins.

2. Name two hormones which are derived from amino acids in the animal body,
and outline the process by which this conversion takes place.

3. Define the terms: vasoconstrictor, secretogogue, androgen, estrogen, endocrine
organ, gastrin, oxj'tocin.

4. Point out the similarities and differences between the types of diabetes caused
by alloxan, phlorhizin, pancreatic deficiency, and posterior pituitary deficiency.

5. Gi\-e examples to illustrate three different ways in which the quantity of hor-
mones secreted by various glands is controlled in the body.

6. Make a list of hormones known to participate in the process of reproduction
in mammals. Indicate briefly the function oi each.

7. Outline the mechanisms by which the blood calcium level is controlled, explain-
ing the influence of each factor. What are the consequences of abnormal blood
calcium levels?

8. To which chemical classes do the majority of hormones belong? Give examples.

9. List diseases caused by abnormal hormone production in animals, and name
the hormone associated with each.

10. Why is the pituitary sometimes called the "master gland" of the animal or
human body?

REFERENCES AND SUGGESTED READINGS

Abel, J. J., Ceiling, E. M. K., Rouiller, C. A., Bell, F. K., and Wintersteiner, O.,
"Ciystalline Insulin," J. PharmacuL, 31, 65 (1927).

Banting, F. G., Best, C. H., Collip, J. B., and MacLeod, J. J. R., "Preparation of
Pancreatic Extracts Containing Insulin," Trans. Roy. Soc. Canada, 16, Sect. V, 1
(1922).

Colowick, S. P., Coi-i, G. T., and Slein, M. W., "The Effect of Adrenal Cortex and
Anterior Pituitaiy Extracts and In.sulin on the Hexokinase Reaction," J. Biol.
Chem., 168, 583 (1947).

Cori, C. F., "Enzymatic Reactions in Carbohydrate Metabolism," The Harvey Lec-
tures (1945-46), p. 253.

310 HORMONES

Fieser, L. F. and Fieser, M., Natural Products Related to Phevanthrene, Reinhold

Publishing Corporation, New York, 1949.
Fredericq, E.-and Neurath, H., "The Minimum Molecular Weight of Insulin, J. Am.

Chem.Soc, 72, 2684 (1950).
Gurin, S. and Delluva, A. M., "The Biological Synthesis of Radioactive Adrenalin

from Phenylalanine," J. Biol. Chem., 170, 545 (1947).
Harris, R. S., Marrian, G. F., and Thimann, K. V., Vitamins and Hormones, vols.

1-10, Academic Press, Inc., New York, 1943-1952.
Hartman, F. A. and Brownell, K. A., "The Hormone of the Adrenal Cortex," Science,

72, 76 (1930).
Hensch, P. S., Kendall, E. C, Slocumb, C. H, and Polley, H. F., "The Effect of a

Hormone of the Adrenal Cortex and of Pituitary- Adrenocorticotrophic Hormone

on Rheumatoid Arthritis," Proc. Staff Meet., Mayo Clinic, 24, 181 (1949).
Hoskins, R. G., Eitdocnnology, W. W. Norton and Company, New York, 1941.
Houssay, B. A., Chapter 13 on Hormones, in Currents in Biochemical Research, ed.

by D. E. Green, Interscience Publishers, Inc., New York, 1946.
Kendall, E. C, "Isolation of the Iodine Compound Which Occurs in the Thyroid,"

J. Biol. Chem., 39, 125 (1919).
Lesh, J. B., Fisher, J. D., Bunding, I. M., Koc&is, J. J., Walaszek, L. J., White, W. F.,

and Hays, E. E., "Studies on Pituitary Adrenocorticotropin," Science, 112, 43

(1950).
Pierce, J. G. and du Vigneaud, V., "Studies on High Potency Oxytocic Material

from Beef Posterior Pituitaiy Lobes," J. Biol. Chem., 186, 77 (1950).
Pincus, G., editor. Recent Progress in Hormone Research, vols. 1-7, Academic Press,

Inc., New York, 1947-1952.
Pincus, G. and Thimann, K. V., The Hormones, vols. I and II, Academic Press, Inc.,

New York, 1948.
Roche, J. and Michel, R., "Natural and Artificial lodo-Proteins," Advances in Pro-
tein Chemistry, 6, 253 (1951).
Sanger, F., "The Arrangement of Amino Acids in Proteins," Advances in Protein

Chemistry, 7, 1 (1952).
Soskin, S. and Levine, R., Carbohydrate Metabolism, University of Chicago Press,

1946.
Stephens, G. A., Hormones and Vitamins, Geo. Newnes, Ltd., London, 1947.
Tainter, M. L. and Luduena, F. P., "Sympathetic Hormonal Transmission," Recent

Progress in Hormone Research, 5, 3 (1950).
Turner, C. D., General Endocrinology, W. B. Saundei-s Company, Philadelphia, 1948.

Chapter 12

DIGESTION

by G. W. E. PLAUT

Assistant Professor, Institute for Enzyme Research,
University of Wisconsin

Most foods have to be converted to the proper physical and chemical
state before they can be utilized by the body. Digestion is the series of
mechanical and chemical processes which accomplishes this result.

SALIVARY DIGESTION

Composition of saliva

Food particles are reduced to smaller size by the mechanical action of
the teeth. While in the mouth they are moistened and mixed with saliva,
the secretion of the submaxillary, sublingual, and the parotid glands.
The sublingual glands secrete a thick fluid which is rich in the glycoprotein,
mucin. When mucin is hydrolyzed it yields, in addition to protein, sul-
furic acid, acetic acid, glucuronic acid, and glucosamine. Mucin serves
to lubricate the food for its subsequent passage through the esophagus
to the stomach. A thin watery fluid, low in organic matter (serous
secretion) , is produced by the parotid gland. The submaxillary gland
contributes a mixture of the two types of secretion.

The saliva contains inorganic constituents found in blood. A small
amount of thiocyanate is also present. Some organic compounds char-
acteristic of blood such as uric acid, urea, and creatinine are also present.
An a-amylase called ptyalin is present in saliva. It catalyzes the hy-
drolysis of starch and glycogen to maltose and polysaccharides of lower
molecular weight. The action of the amylase on starch persists on the
way from the mouth to the stomach. The activity stops in the stomach
when the acidity becomes too unfavorable. This enzyme has been crys-
tallized from human saliva. It is inactivated upon dialysis against dis-
tilled water, but the activity can be restored by the addition of chloride
ions. Human saliva has a neutral reaction (about pH 6-8).

311

312 DIGESTION

Secretion of saliva under natural conditions

The secretion of saliva appears to be mainly controlled by the nervous
system. There are two general modes of nervous stimulation of salivary
flow. (1) The presence of materials in the mouth leads to the secretion
of saliva. There is a remarkably purposeful variation in the composition
of saliva depending on the nature and mechanical state of the material
present in the mouth. Thus a watery secretion is produced in the pres-
ence of dry powder, whereas acid leads to the secretion of a fluid high
in mucin, which would tend to neutralize the acid. (2) Stimulation of
other organs of sense, aside from that of taste, also leads to salivation.
We are all familiar with the experience of our mouths watering when we
smell or see a particularly tasty food. It is obvious that one must have
had the experience of tasting the particular food at one time and that
the stimulation due to smell or sight must have been acquired then.
Such an acquired stimulation is known as a conditioned reflex.

"When the body is exposed to a situation of water loss, the salivary
secretion is depressed. As a result the mouth becomes dry, and the person
experiences the sensation of thirst. A normal human adult secretes
1-1.5 1. of saliva per day.

After the food has been prepared in the mouth for further digestion, it is
swallowed and passes through the esophagus to the stomach.

GASTRIC DIGESTION

In the stomach the foods are mixed with the gastric juice. The gastric
movement renders the food creamy and semifluid in consistency. This
mass is then known as the chyme. It passes out of the stomach through
the pyloric opening into the duodenum.

Gastric juice is secreted by three main types of cells. The secretion
of mucous cells is high in mucin, the parietal cells contribute hydrochloric
acid, and the zymogenic cells supply the zymogen, pepsinogen. In con-
trast to other fluids of the body, gastric juice has a very acid reaction,
e.g., pH 1.5-1.9, in the case of man. It has been estimated that the
secretion of parietal cells is 0.16A^ hydrochloric acid. If only hydrochloric
acid were secreted in the stomach," the pH would be around 1 ; however,
the other secretions contain substances such as proteins and sodium bi-
carbonate which partially neutralize the hydrochloric acid. The unneu-
tralized portion of the acid can be determined by titration with alkali
and is known as the free acid of the gastric juice (0.05-0. lA^ HCl) , while
the sum of the neutralized and the free acid is called the total acid.
When food is mixed with the gastric juice still more of the free acid is
neutralized, and the acidity of the chyme is pH 3-5, depending on the
nature of the food.

DIGESTION

313

The hydrochloric acid of gastric juice is made from blood which has
an approximately neutral reaction. The hydrogen ion of hydrochloric
acid comes from carbonic acid and the chloride ion originates from
sodium chloride. These ions are selectively absorbed from the blood
by the parietal cells, and the hydrochloric acid thus formed is secreted
into the stomach. The loss of hydrogen ions from the blood is evidenced
by an increase in the alkalinity of the blood which has passed through
the gastric mucosa of the stomach during a period of active hydrochloric
acid secretion. The precise 'mechanism by which hydrogen and chloride
ions are concentrated by parietal cells to form an acid from the almost
neutral blood has not been completely elucidated. It should be realized
that a great deal of energy is required to raise the hydrogen ion concen-
tration from 4X lO'^M (pH 7.4) in blood to O.IQM (pH 0-1), the
hydrogen ion concentration of parietal secretion. This transformation
constitutes an approximate 4,000,000 fold increase in the concentration
of H + .

The relationship of some components of the blood to the secretion of
hydrochloric acid by the parietal cells is pictured in the following scheme.

Incoming Blood

Outgoing Blood

COo + HoO

H2CO3

H+ + HCO3-
XaCl

CI- + Xa+

(Parietal membrane)

-\

-^ HCOr

+ UXaHCOa

:o+

Na

J

H+ + Cl-

:^HC1

(Appears in parietal cell secretion)

The high acidity of gastric juice has a bactericidal effect on the micro-
organisms ingested with the food and is ideal for the action of some
of the digestive enzymes present in this fluid. Most of the enzymes of
gastric juice work best at pH 2-4.

The principal proteolytic enzyme of gastric juice is pepsin. It attacks
proteins and reduces them to smaller fragments such as proteoses, pep-

314 DIGESTION

tones, and some amino acids. The enzyme is secreted by the zymogenic
cells in an inactive form, pepsinogen. Pepsinogen is converted to pepsin
by hydrogen ions and pepsin (see p. 273). Rennin is present in par-
ticularly high concentrations in the stomachs of young mammals. This
enzyme is involved in the curdling of milk. Rennin catalyzes the con-
version of casein to soluble paracasein. Paracasein combines with cal-
cium ion to form insoluble calcium paracaseinate. Rennin obtained from
calf stomach is used commercially to curdle milk in cheese making.
Pepsin can also curdle milk, but accomplishes this result by a process
different from that of rennin.

The flow of gastric juice is regulated by several factors. Nervous im-
pulses due to various stimuli, such as food in the mouth, or even the
odor or sight of food, cause secretion. Fear or worry have been shown
to suppress secretion. Mechanical pressure inside the stomach has a
slight effect, but the presence of certain foods {e.g., meat extract, peptone)
in the stomach causes a tremendous increase in gastric flow. This effect
of food seems to be independent of the nervous system, and some evidence
has been obtained to show that a substance may be present in gastric
mucosa which reacts with a food component to form a hormone, gastrin
(p. 308). This substance is liberated into the blood and causes gastric
secretion. The injection of histamine, a compound present in gastric
mucosa as well as in other body tissues, causes the secretion of a gastric
juice which is high in hydrochloric acid but low in pepsin, in contrast
to the normal composition. The composition of chyme leaving the
stomach for the duodenum has an effect on gastric digestion. Thus when
fat or acid are placed into the duodenum, gastric secretion and motility
are inhibited. A material extracted by l\'y from intestinal mucosa, when
injected into the blood stream, produces the same type of depression
of gastric activity. It is a hormone called enter ogastr one (p. 309) . Uro-
gastrone, isolated from urine, has a similar effect.

Various chemicals have a profound effect on the secretion and composi-
tion of gastric juice. Ethyl alcohol leads to a secretion high in hydro-
chloric acid and mucin and low in pepsin; liver, meat, and vegetable
extracts are powerful stimulants of normal gastric juice secretion, while
acid depresses secretion. The concentration of hydrochloric acid is
chronically lowered or raised in certain pathological conditions. The
hydrochloric acid is completely absent (achlorhydria) , e.g., in pernicious
anemia, and is produced in excessive amounts (hyperacidity) in most
cases of duodenal ulcers.

Bland diets, for example, milk, are used in treating ulcer patients to
prevent excessive gastric secretion. The high buffering capacity of such
diets helps to neutralize the free acid of the gastric secretion.

DIGESTION

315

INTESTINAL DIGESTION

When the chyme enters the duodenum it is mixed with the secretions
of the pancreas and bile.

Pancreatic secretion

Pancreatic juice has an alkaline reaction, pH 7.1-8.2, and contains
a variety of very active enzymes which can attack carbohydrates, pro-
teins, and fats. Trypsin and chymotrypsin hydrolyze proteins and poly-
peptides to smaller polypeptides and amino acids. The action of the pro-
teolytic enzymes depends in part on the sequence of amino acids in the
protein or peptide which they attack. Peptides that are the products
of one of the proteinases can therefore be hydrolyzed further by another
proteinase of different specificity. In model experiments with the syn-
thetic peptide, carbobenzoxy-L-glutamyl-L-tyrosyl-glycinamide, it was
found that pepsin split the amino linkage of tyrosine, and chymotrypsin
split the carboxyl linkage of tyrosine. In the following formulation the
site of cleavage is indicated by dotted lines:

CeHi-CH^-O-CO-NH-CH-CO-

Carbobenzoxy -

■NH-CHCO-

CH2
I

I
COOH

t Pepsin

Glutamyl-

-NH'CH2-CO-NH2
Glycinamide

CH2

C6H4OH

t

Chymotrypsin

Tyrosyl-

From experiments with other synthetic peptides, it was found that
phenylalanine can be substituted for tyrosine. From studies on native
proteins, e.g., insulin, it appears that pepsin splits linkages other than
those involving the amino group of tyrosine and phenylalanine. For
example, the Leu. Val. bond is readily hydrolyzed, and the Ala. Leu.
bond to a considerable degree. Pepsin appears to have a much wider
range of specificity than chymotrypsin or trypsin.

Trypsin splits peptides at the carboxyl linkage of either lysine or
arginine. Thus

Ri-NHCH-C(

r

)— i— NH-R2

1.
Trypsin

CH2
NH2

Ri and R2 denote the
remainder of the chain

Lysyl (or arginyl)

316 DIGESTION

The proteinases are therefore complementary in action and can break
down the large protein molecules to smaller and smaller units. Carboxy-
peptidases attack polypeptides at the carboxyl end, liberating the ter-
minal amino acid of the chain. In contrast, the aminopeptidases (mainly
secreted in the intestines) attack the peptide linkage at the free amino
end of the chain. To illustrate:

HOlH HOjH

NH,CHC04nH-CHC0 NH-CHCO-r-NHCHCOOH

Ill 1 i '

R' j R^ R' I R^

Aminopeptidase Carboxypeptidase \

Neither trj^psin, chymotrypsin, nor carboxypeptidase are secreted as
the active enzyme by the pancreas, but rather as an inactive precursor,
called a zymogen. Trypsinogen, the precursor of trypsin, is converted
to the latter by the action of enterokinase (an enzyme present in intestinal
juice) or by trypsin itself. Chymotrypsinogen goes to chymotrypsin in
the presence of trypsin. Trypsin is also instrumental in the conversion
of procarboxypeptidase to the active enzyme. Trypsin, trypsinogen,
chymotrypsin, chymotrypsinogen, and carboxypeptidase have been iso-
lated in crystalline form.

The pancreatic juice also contains lipases, enzymes that catalyze the
hydrolysis of fat, and amylases, enzymes that catalyze the hydrolysis of

CH^O-CO-CCHOigCHs HOCH2

CHO-CO-CCHOieCHs + HoO -^^^^ + HOCH 3CH3(CH2)i6COOH

CH20-CO-(CHo)i6CH, HOCH2

Tristearin Glycerine Stearic acid

starch to lower molecular weight polysaccharides (dextrins) and maltose.
Pancreatic amylase has been crystallized and appears to be identical
in chemical, physical, and enzymatic properties with ptyalin.

The flow of pancreatic juice is regulated in part by the nervous system;
however, it has also been found that the injection into the blood stream
of an extract of duodenal mucosa results in copious secretion of pancreatic
juice. This extract is a hormone called secretin. Another hormone from
duodenal mucosa, pancreozymin, has no effect on the volume of pancreatic
secretion, but it does effect an increase in the trypsin, amylase, and lipase
content of the juice (p. 308). About 600 ml. of pancreatic juice are
secreted daily by an adult man.

DIGESTION

317

Bile

Bile is continuously produced by liver cells. It is collected by a series
of ducts from these sources and stored in the gall bladder (some animals,
e.g., the rat, do not have a gall bladder, and consequently the bile is
also stored in the liver cells). The pH of liver bile is about 8-8.6, while
that of the gall bladder is around 7. The main components of bile are
bile salts, bile pigments, cholesterol, and lecithin.

An inspection of the formulas of the principal bile acids reveals that

CONHCH.COOH
^^ . '

Glycine

Cholic acid

Glycocholic acid

they are related to the sterols (p. 95). The prefix glyco or tauro is

Cholic acid

Taurocholic acid

used to show that the sterol portion of the bile acid (cholic acid in the
above structures) is combined by a peptide linkage with glycine or
taurine, respectively. Glyco and taurodesoxycholic acids have been iso-
lated (the hydroxyl group in carbon 7 of cholic acid is replaced by a
hydrogen in desoxy cholic acid) from bile. Similar ccmjugates of cheno-
desoxycholic acid (hydroxyl of cholic acid in position 12 replaced by
hydrogen) and lithocholic acid (hydroxyls of cholic acid in carbons 7
and 12 replaced by hydrogens) have been demonstrated to be present
in bile. The sterol portion of the bile acids have a great affinity for

318 DIGESTION

nonpolar substances, e.g., fat, while the carboxyl and hydroxyl groups of
the molecule have a great affinity for polar solvents, such as water. Bile
acids, therefore, have the properties of a detergent, and their mode of
action is akin to that of soaps.

As a result of these chemical properties the bile salts have the ability
to increase the water solubility of lipides such as fats and cholesterol,
and vitamins A, D, E, and K. The increased water solubility of these
otherwise practically water-insoluble materials facilitates their passage
through the intestinal wall into other body fluids. The speed of hydrolysis
of fats to fatty acids and glycerol in the presence of lipase is increased
in the presence of bile salts.

Once the bile salts have been secreted they are reabsorbed in the in-
testines and transported via the bloodstream to the liver, where they
are used over again. However, it has been shown with isotopically labeled
compounds that the sterol portion of the bile salts can be formed new
from administered cholesterol.

The principal pigments of bile are bilirubin and biliverdin, which are

Ml iV Mfi nP Pi iM Mi iV M=CH3—

11 I 1 A 1 Jl V = CH2=CH—

HO^^ n'^c'^N^^^N^OH P = -CH.CH.COOH
H H H H

Biliverdin

products of degradation of heme. The color of feces is mainly due to

Ml

V Ml

P Pn nMMr==iV

VI I iV Mr nr rn jiMiVir==| v

H H H2 H H

Bilirubin

the products of bacterial reduction of bile pigments, e.g., stercobilin and
stercobilinogen.

The discharge of bile into the duodenum is regulated in part by the
nervous system and by secretin. Another substance, cholecystokinin,
has been implicated in the contraction of the gall bladder. The presence
of the bile salts themselves in the duodenum exerts a powerful stimula-
tion on the flow of bile.

Intestinal secretion

The intestinal juice is secreted by a large number of glands in the
mucosa. This fluid has a reaction of pH 7-8.5. It contains a large
number of enzymes; among them are enterokinase (converts trypsinogen
to trypsin) , peptidases (hydrolyze peptides to free amino acids), nucleases

DIGESTION

319

(hydrolyze nucleic acids to polynucleotides and nucleotides) , nucleotidases
(hydrolyze nucleotides to the corresponding nucleoside and phosphate,
e.g., adenylic acid + HoO -^ adenosine + phosphoric acid) , nucleosidases
(split nucleosides into purine or pyrimidines and pentose, e.g., adenosine +
H20-> adenine + ribose), phosphatases (hydrolyze phosphate esters into
the corresponding alcohol and phosphoric acid, e.g.,

O

!l
HO-POCH2— CHNHo— COOH + H,0

xjQ ^*\phosphatase

Serine phosphate

HOCHo— CHNH2-COOH + H3PO4

Serine

sucrase (hydrolyzes sucrose to glucose and fructose), maltase (hydrolyzes
maltose to two molecules of glucose), and lactase (lactose + HoO— >
glucose + galactose). JNIucin is secreted by epithelial cells of the small
and large intestines; it lubricates the movement of material through this
portion of the intestinal tract. The flow of intestinal juice is markedly
stimulated by the application of mechanical pressure to the intestinal
wall; therefore, the mere physical presence of food will stimulate secre-
tion.

Schematic representation of enzymatic degradation of major foodstuffs

a amj'lases
starch or fi amylases maltases ,

glycogen ^ "^^^^^^^ *■ g^"^°^®

sucrase r , . •

sucrose *• fructose + glucose

, . pepsin, trypsin polypeptides + carboxypeptidase amino

chymotrypsin, etc. ammo aClUS aminopeptidase aCldS

I dipeptidase, etc. ^

fat — ]P^:^ ^ fatty acids + glycerol

ABSORPTION FROM THE GASTROINTESTINAL TRACT

Fairly large quantities of ethanol, methanol, and water are absorbed
in the stomach, and hydrocyanic acid is rapidly taken up at this site
in fatal amounts. The mucosa of the small intestines, however, is the
most important location for the absorption of foodstuffs. The intestinal
wall is covered with a large number of microscopic, protruding processes
known as villi. Each of the villi contains a small blood vessel and a lymph
vessel (lacteal). The villi are the principal absorbing units of the small
intestines. The materials that can be absorbed are transported across
the membranes which separate the intestinal content from the blood and

320

DIGESTION

lymph vessels. These breakdown products of foods enter the general
circulation from the smaller vessels and are taken up by the various
organs of the body. Amino acids and monosaccharides pass into the
capillaries and from there into the portal blood. Some authorities claim
that small quantities are transported into the lymph. Over 90 per cent
of the fatty acids of fat absorbed in the intestines of the rat have been
demonstrated to be transferred to intestinal lymph. It is doubtful if the
fatty acids of fats can be transported directly into the portal blood.

Calcium and iron are absorbed mainly from the upper part of the
small intestines. The intestinal content has a profound effect on the
extent of calcium absorption. Soluble calcium salts such as the gluconate,
lactate, and chloride are readily available, but the phosphate is not.
Cereals reduce calcium absorption since they contain phytic acid (inositol
hexaphosphoric acid), which binds calcium; spinach has a similar effect
because of its oxalic acid content. As has been mentioned previously,
vitamin D enhances calcium absorption (p. 211). Iron absorption is
notoriously inefficient. For example, if a normal child is fed 5 mg. of iron,
only about 12 per cent of the iron is absorbed. However, the greater the
need of the body for iron, the greater is tlie increase in the uptake of
this element. It has been shown that in cases of iron-deficiency anemia,
absorption increases many times over normal. Most of the other inor-
ganic salts and the bulk of the water are absorbed in the colon.

As a result of water removal in Ihe colon, the materials there acquire
a semisolid consistency. A large number of bacteria act in the colon
on materials which have been passed there from the small intestines or
which are secreted by the walls of the colon. It has been estimated that
10-90 per cent of the weight of the feces is derived from bacteria. The
characteristic odor of feces is due mainly to indole and skatole. These
are products of degradation, presumably of tryptophan. Part of the

NH2
I
CH.— CH— COOH

Tryptophan

indole and skatole are reabsorbed into the blood and are mainly con-
verted into the sulfate esters (indoxyl sulfate and skatoxyl sulfate)

DIGESTION

321

Ou

OSO,OK

N'
H

Indican
(potassium indoxyl sulfate)

in the liver and are excreted in this form in the urine. Tyramine, hista-
mine, putrescine, and cadaverine occur in feces; they are probably the
products of bacterial decarboxylation of the amino acids tyrosine, histi-
dine, ornithine, and lysine, respectively:

HO—/ \— CH.CHNH2COOH
Tyrosine

-* HO-

r^

CH2CH2NH2+ CO2

Tyramine

HN-CH

N-C— CH:CHXH,COOH

Histidine

HN-CH
-> HC^ +CO2

N-C— CH2CH2NH2
Histamine

NH

II
NH2-C— NH-CH2- CH2— CH2— CHNH2- COOH

Arginine

O

NH2-C— NH2 + KH2CH2CH2CH2CHNH2COOH -

argmase
+ H2O

Urea

Ornithine

NH2CH2CH2CH2CH2NH2 + CO2
Putrescine

NH2CH0CH2CH2CH2CH2NH2 + CO2
Cadaverine

NH0CH2CH2CH2CH2CHNH2COOH -

Lysine

Hydrogen sulfide and methane are among the gaseous products of putre-
faction in the colon. A typical analysis of the intestinal gases of swine
gave 25 per cent methane, 50 per cent carbon dioxide, and 25 per cent
hydrogen. In herbiverous animals large quantities of gas are produced
in the paunch, in addition to those in the intestines. The decomposition
of foods by bacteria, leading to gas formation in the paunch, may account
for as much as 25 per cent of the energy loss from the food during the
digestive process.

The nutritional significance of the intestinal bacterial flora has been

322

DIGESTION

thoroughly appreciated only in the last few years. It was observed in
earlier nutritional experiments that some rats receiving a diet deficient
in B complex vitamins would recover spontaneously from the deficiency
without supplementation of the diet with the missing vitamins. Although
receiving a B vitamin deficient diet, other rats which were prevented
from eating their feces (coprophagy is a common practice in the animal
world) required much more supplementation with B vitamins than those
consuming feces. When rats are fed a purified diet to which all the
vitamins except folic acid have been added, they will develop normally;
however, when succinylsulfathiazole (which is not absorbed from the
intestines) is added to this ration, typical symptoms of folic acid de-
ficiency develop, and the amount of this vitamin in the cecal content
and various tissues decreases. It is thought that the sulfa drugs under
these conditions depress the bacterial synthesis of folic acid in the in-
testines. In contrast to the depression of vitamin production by an anti-
bacterial agent described above, it has been found more recently that
the addition of certain antibiotics, e.g., penicillin, aureomycin, terramycin,
and streptomycin, to the ration will increase the rate of growth of animals
usually 10-20 per cent, even under farm conditions. The quantity fed
is small (2-5 mg. per pound of feed). It is thought that the antibiotics
inhibit the growth of those organisms which assimilate large quantities
of certain vitamins present in the intestinal tract and which therefore
reduce the supply for absorption by the animal.

*

REVIEW QUESTIONS ON DIGESTION

1. Discuss the digestion of (1) proteins, (2) starch, (3) fat.

2. Define (1) conditioned reflex, (2) chyme, (3) secretin.

3. Which reactions are catalyzed by the following enzymes: (1) pepsin, (2) rennin,
(3) ptyalin, (4) lipase, (5) carboxypeptidase, (6) amino peptidase, (7) nucleotidases?

4. Persons with obstructed bile ducts hemorrhage easily, even when their diets con-
tain large quantities of vitamin K. What may be the reason for this condition?

REFERENCES AND SUGGESTED READINGS

Best, C. H. and Taylor, N. B., Physiological Basis of Medical Practice, Williams and
Wilkins Company, Baltimore, 1950.

Elvehjem, C. A., "Nutritional Significance of the Intestinal Flora," Federation Pro-
ceedings, 7, 410 (1948).

Grossman, M. I., "Gastrointestinal Hormones," Physiol. Rev., 30, 33 (1950).

Hawk, P. B., Oser, B. L., and Summerson, W. H., Practical Physiological Chemistry,
12th ed., The Blakiston Company, Philadelphia, 1947.

Northrop, J. H., Kunitz, M., and Herriot, R. M., Crystalline Enzymes, Columbia
University Press, New York, 1948.

Chapter 13

ANIMAL METABOLISM

METABOLISM OF CARBOHYDRATES

The chief function of carbohydrate in the animal body is to provide
energy in a form which the animal can use. Like all fuels, it must be
burned, or oxidized, for the energy to be released. The end result of
the burning process is the conversion of the sugar into carbon dioxide
and water, which is the reverse of photosynthesis:

CgHioOe + 60o -^ 6CO2 + 6H0O + 683 Cal.

When carried out by living organisms, this process is called respiration.
This term is often used in a broader sense to include all metabolic proc-
esses by which gaseous oxygen is used to oxidize organic matter chiefly
to carbon dioxide and water. This type of metabolism is most pro-
nounced in animals, but is also carried out by plants and by a few
microorganisms.

If the carbohydrate is burned directly in a flame, all the energy is
released in the form of heat. Some heat is produced also in the animal
body, but much of the energy released is stored up in the form of certain
chemical by-products, particularly adenosine triphosphate (ATP) , which
can later be used for muscle contraction or other useful purposes. Fur-
thermore, the energy is released in small portions and at temperatures
low enough so the living tissues are not injured. Direct burning of
carbohydrate material {e.g., wood, paper) of course never takes place
except at a temperature fatally high to all living things ; yet the same net
result is accomplished rapidly and continuously in all living animals.
Obviously, nature must have devised some very special and effective
method of "low-temperature, biological burning."

After a very great deal of painstaking research, many of the details
of this complicated process have now been discovered. In brief, what
happens is that the carbohydrate undergoes a long series of chemical
changes, each altering it slightly, so that it is gradually converted into
the final end products, carbon dioxide and water. Each chemical reaction
involved in the process is catalyzed by a particular enzyme, without
which the reaction will not proceed fast enough to be of any use to the
organism. Many of these essential enzymes in turn require the presence

323

324

ANIMAL METABOLISM

of coenzymes and (or) activators in order to function properly (Chap.
10). This whole sequence of linked chemical changes forms a pathway
over which each molecule of carbohydrate passes, as need for energy
arises.

All of these changes, and any others which carbohydrates undergo in
body tissues, are referred to collectively as intermediary carbohydrate
7netabolism. Although the major part of carbohydrate metabolism has
to do with breakdown into simpler substances, the process is in large
part reversible, and certain carbohydrates {e.g., glycogen, lactose) are
formed in the body from other carbohydrates or from intermediate break-
down products. The "building-up" aspects of metabolism are called
anabolis7n; break-down processes are termed catabolism.

Interconversion of digested carbohydrates

Formation of Glycogen. Carbohydrate metabolism starts when the
products of carbohydrate digestion pass through the intestinal wall and
enter the blood stream. These products, from a normal diet, are D-glu-
cose, D-fructose, and D-galactose. If mannose is eaten, it can also be
metabolized. All four sugars are interconvertible in the animal body
and give rise to glycogen by means of the metabolic reactions shown
in Fig. 13-1.

At first, each sugar combines with a phosphate radical taken from
ATP (reaction I, Fig. 13-1).^ This is an irreversible- reaction catalyzed
by hexokina.se and Mg++ ions. It may be represented by the usual type
of equation. For example,

„ , ■ , , m-r, (hexokinase)

D-glucose + ATP — ^^ > D-glucose-6-phosphate + ADP

or more concisely by the scheme used in Fig. 13-1:

ADP

D-glucose-6-phosphate

The ATP here serves as the biological equivalent of a match used to
light a fire. ATP is a concentrated storehouse of chemical energy.
When one of its three phosphate radicals is transferred to the sugar,
some of the energy is transferred too. This activates the sugar so that

^ Well established chemical reactions occurring in the animal body have been num-
bered for easy reference throughout the chapter.

^ Although this reaction, as such, is irreversible, the glucose-6-phosphate can easily
be hydroly^ed back to free glucose (see p. 325).

ANIMAL METABOLISM

325

it can begin to undergo "biological burning," just as a match heats a
piece of paper to its kindling point so that it will burn.

The other reactions shown in Fig. 13-1 are reversible, equilibrium reac-
tions (note double arrows) . Such reactions go either in one direction or
the other, depending on the relative amounts of the various reacting
substances present. Thus after a meal, when a large amount of sugar
comes into the blood stream, a considerable part is converted into glycogen,
but when the sugar phosphates are consumed during exercise, the glycogen
is broken down again.

Glycogen may also be formed from a variety of other substances which
are involved in the further metabolism of carbohydrates (see below).
Consequently, the amount of glycogen present in the body at a given
time reflects a balance between the intake of all glycogen-forming food
materials and the metabolic consumption of carbohydrate as energy
sources.

The amount of glycogen which can be stored, however, is limited. In
a normal human adult, the top level of glycogen is seldom over 6 per
cent in the liver and 0.7 per cent in the muscles. These percentages cor-
respond to a total quantity of about 110 g. in the liver and 250 g. in
the muscles. Consumption of additional amounts of food above those
needed to maintain this amount of glycogen in the body leads, as is
well-known, to the formation of fat. . '

Blood Sugar Level. There is also a close interrelationship between gly-
cogen, blood sugar, and the action of several hormones. The only sugar
which is present in appreciable amounts in the general blood circulation
is D-glucose, which for this reason is often called blood sugar. The
blood glucose supply is furnished partially by direct absorption from the
intestine, but mainly by hydrolysis of D-glucose-6-phosphate coming from
glycogen: ^

(phosphatase)

H2O + D-glucose-6-phosphate > D-glucose + H3PO4

The hormone adrenalin acts to increase the amount of glucose in the
blood-stream (p. 289) . Adrenalin is secreted by the adrenal gland in
response to intense emotions such as rage or fear. It is usually assumed
that this secretion represents a physiological preparation for intense
muscular activity to cope with the situation which aroused the emo-
tion.

Two other hormones, insulin from the islets of Langerhans in the
pancreas and the diabetogenic hormone from the anterior pituitary gland,
also affect the blood sugar level. It is claimed that the latter hormone is

^ Blood sugar directly absorbed from the intestine may also be formed by hydrolysis
of a glucose phosphate, since phosphorylation probably occurs during absorption.

w w

w

o— o-

J

^^

/

o

\

J

o

W r?

\

^^Q

o

o

o

03

tin

n.

"7

\°

s

w-^,

o

K^

i— o ^

w

1

o

m

o

w

a

u-J

k"

°7^

c3

o^

^M

O

1

w

O

bc a

326

ANIMAL METABOLISM

327

a powerful inhibitor of hexokinase, whereas insulin counteracts this in-
hibition. The effect of the diabetogenic hormone therefore is to raise
the blood sugar level by preventing the phosphorylation essential for
the utilization of blood glucose. Insulin has the opposite effect. The
disease, diabetes, may be caused either by too much diabetogenic hor-
mone or too little insulin.

The normal blood sugar level in man varies between 0.07 and 0.10 per
cent (70 to 100 mg. per 100 ml. of blood) during fasting, but rises to
0.12-0.15 per cent after a meal. Some of the controlling influences which
operate to maintain this level have been listed above and are presented
diagrammatically in Fig. 13-2. Another factor which sets an upper
limit to the blood sugar concentration is excretion in the urine. Normally

Controlling Factors Blood sugar Descriptive

Increase Decrease percentage terms

a
c

a

o
o

o

o

a;
O

o3

0.18

0.15

r ^ ^

Renal
threshold

J
\

Normal

0.07

0.02

Physiological
effect

Excretion
in urine

Hyperglycemia

Normal
well-being

range

J

^

Insulin
" shock

Listlessness,
Hypoglycemia fatigue

Coma,
death

0.00 •- ^ ^

Fig. 13-2. Blood sugar level and its control.

only traces are excreted (an average of only 142 mg. in the urine of a
normal man during 24 hours), but whenever the blood sugar level rises
to a certain point, called the renal threshold, urinary excretion occurs.
Thus in cases of diabetes the urine usually contains 3-5 per cent of
glucose (about 50-100 g. excreted per day). The renal threshold varies
with the individual, but ordinarily it is about 0.15-0.18 per cent: Levels
of blood sugar much below 0.07 per cent lead to unconsciousness and

323

ANIMAL METABOLISM

death, and even values only slightly below the normal range result in
feelings of listlessness and fatigue.

Glycolysis. The catabolism of carbohydrate in the animal body may
be divided for purposes of study into two main phases, the anaerobic
and the aerobic. The anaerobic phase, which is called glycolysis, pre-
cedes the aerobic part and consists in the conversion of glycogen into
pyruvic acid and (or) lactic acid. The metabolic reactions which make
up glycolysis are shown in Figs. 13-1 and 13-3.^ The whole process is
often called the Embden-Meyerhof scheme.

Starting with glucose, two moles of phosphoric acid or inorganic phos-
phate, are converted into "organic phosphate" (reaction 8, Fig. 13-3),^
and two moles of ATP are used up, being converted into ADP (reactions
I and 4, Fig. 13-1). However, four moles of ATP are again formed
from ADP (reactions 9 and 12) so there is a net gain of two moles of
ATP for each mole of glucose used. In reaction 7, hydrogen is removed
from glyceraldehyde-3-phosphate and is held in the form of a reduced
coenzyme, DPN • Ho (p. 332). Four atoms of hydrogen are thus pro-
duced per mole of glucose. The net result of glycolysis, under condi-
tions of mild exercise, can be summarized by the equation:

CgHioOe + 2H3PO4 + 2ADP + 2DPN -»

2CH3COCOOH + 2ATP + 2H2O + 2DPN • H2

During mild exercise, the hydrogen of the DPN • H2 is converted into
water by combining with oxygen via the cytochrome system (p. 332).
However, when exercise is very violent, oxygen cannot be carried by the
blood stream to the muscles quickly enough to reoxidize the DPN • Ho
as fast as it is formed. When this situation occurs, pyruvic acid is
reduced to lactic acid (reaction 13, Fig. 13-3) so that lactic acid becomes
the end product of anaerobic glycolysis. This process gives the organism
an extra burst of energy for a short time, but the muscles soon become
loaded Avith lactic acid, and exhaustion results. During rest, about four-
fifths of the lactic acid is converted back into glycogen, and the re-
mainder is oxidized to carbon dioxide and water.

Lactic acid formation (reaction 13) therefore is essentially an offshoot
from tlie main line of carbohydrate metabolism. The main pathway
leads to pyruvic acid, ATP, and DPN • Ho, as given in the equation
above. These products are disposed of during the aerobic phase of car-
bohydrate metabolism.

It should be noted that each carbon atom of the pyruvic acid comes

• ^ The glyceraldehyde and glyceric acid phosphates, appearing in these charts, are
frecjuently called phospho-glyeeraldehydes and phospho-glyceric acids, respectively.

= ()nly one molecule of HaPOi is shown in Fig. 13-3, but two C-3 fragments are
formed from each C-6 unit (reaction 5) so that the products shown subsequently
(reactions 7-13) represent only one-half of the molecules coming from one mole of
glucose.

OPOJI,

CHO-OP03H,

I

CO

I

HOCIi

,j- ■* — (point of cleavage)

h:coii

OH 11
(ring form)

D-Fructosc-1 ,G-diphosphate

^9%

ricoH
I

CH,-0P03H.,
(open chain form)

I

11 %l (5)

(aMnlasc)

/^

CHoOPOsH.
CO

I

CH.OH

Dihydroxyacetone-
phosphate

4%

(ij)

90 Tc

(tiioseplio»|)liate

isomeiase)

+ 211

Hal'U,

lIV,
V)

- 211

(D-i

3

del

CHO

I
HCOH
I
CHoOPO,Ho

D-Glyceraklcliyde-3-
pliospliate

vcciaMeliydL--! |/r DPN
-phosphate I (7) (
>yam,ena-se) | Nj3p>^..H^

C3H5(OH)3

Clycerol

COOH
I
HCOH
1
CH.OPOsH,

D-Glyceric acid 3-phosphate

[Enzj-me-glyccric acid-3- phosphate complex]
+ H,PO,l is)T- IIjPOi

ATP ADP

(9)

>•

(trail jphospliuryluM',
Mg++ or Mn++)

■1

\-

iiTcKio) y-'/" , , , ,

' (phosphoglyceromutase)

COOH

I
HC-O— PO3H2

- H.O

(11)

CH2OH

D-Glyceric acid 2-phosphate

COOH

I
HOCH

+ H,0
(enolase)

DPN DPXHo

(13)

coopajH, .

HCOH

I

CH.OPOjH,
D-Glyceric acid 1 ,3-diphosphate

COOH

I
COPO.H,

li
CIL

Pyruvic acid ciiol phospliate
ADPt^I a

jl (12) (tiaiispliosphorylasc,

COOH

I
CO

CH3

L-Lactic acid

(lactic acid
dehydrogenase)

CH3
Pyruvic acid

(aerobic metabolism)
Fig. 13-3. Reactions of glycolysis {continued). Labeling as in Fig. 13-1.

329

330

ANIMAL METABOLISM

from a definite part of the original glucose molecule (see Fig. 13-3, espe-
cially reactions 5 and 6). This may be pictured as follows:

(1)

CHO
I
HCOH

I
(3) HOCH

(2)

(3)

(4) HCOH

(5) HCOH

I

(6) H2COH

Glucose

CHj

CO

I

COOH

+
COOH

I
CO

I

CH3 (3)
Pyruvic acid

(2)

(1)
(1)
(2)

The carbon atoms for the methyl groups of the two pyruvic acid molecules
(carbon 3 of the pyruvic acid) come from carbons 1 and 6 of the glucose,
those for the CO groups from 2 and 5, and those for the COOH groups
from 3 and 4. The correctness of these relationships has been well estab-
lished by studies with compounds containing isotopic carbon atoms in
known positions.

Oxidation of pyruvic acid

The Citric Acid Cycle. Pyruvic acid is metabolized by the reactions
shown in Fig. 13-4.^ Although oxygen does not appear in this scheme,
the process is an aerobic one because the hydrogen atoms produced at
several points are constantly being combined with oxygen by the cyto-
chrome system. The outstanding feature of the process is its cyclic
nature. Oxalacetic acid and acetic acid combine to form citric acid,
which then goes back to oxalacetic again (reactions 18-25). This is
called the citric acid cycle.^ The main sequence of reactions, normally
followed by the bulk of the pyruvic acid metabolized, is indicated in
Fig. 13-4 by heavy arrows. Reverse reactions are shown with light
arrows, and various associated processes by broken lines.

The result of the operation of the citric acid cycle is that the original
molecule of pyruvic acid is completely broken down into carbon dioxide
and hydrogen, which later becomes water (see below). This may be
seen by reading clockwise around the cycle and noting what is added
or subtracted in each step. Starting with pyruvic acid, four moles of
water are added (reactions 16, 19, 22, 24) and one removed (reaction

^ In this figure, and throughout this chapter, the two-carbon substance arising from
the metabolism of pyruvic acid and from fats is shown for simplicity as free acetic acid.
It is almost certain, however, that this intermediate is actually an acetyl group
(CH3CO— ), which is taken up by a coenzyme {Co A, p. 274) as fast as it is formed
and later transferred to some other substance {e.g., oxalacetic acid).

''Also called the tricarboxylic acid cycle, or Krebs cycle.

(Glycolysis)

11

CO2,

•2H

■*■ CH3COCOOH .^ H,NCHCOOH

(15)

-CO:

+ COj,
+ 2H

(H)

CH3

L- Alanine

+ COj

Pyruvic acid

I+H^o
- CO2
-2H

CH3COOH ^. Fatty acid

Acetic acid oxidation

COCOOH

\ (17) ^

CH.COOH
Oxalacetic acid

CHjCOOH

HOCCOOH
I
CH2COOH

Citric acid

HOCHCOOH

I
CH.COOH

L-Malic acid

H2NCHCOOH

CH.COOH
L-Aspartic acid

+ H2O

(24)

- H2O

CHCOOH

II
CHCOOH

Fumaric acid

CHCOOH

CCOOH
I
CHjCOOH

cts-Aconitic acid
- HjO (19) |+H,a

HCOHCOOH

I
HCCOOH

I

CH^COOH

d-Isocitric acid

+ 2H

CHjCOOH

CH2COOH
Succinic acid

H2NCHCOOH
CHj

CH2COOH

L-Glutan\ic
acid

A

- HjO
+ CO2
+ 2H
+ H.O'
-CO2
-2H

COCOOH

I
CHj

COCOOH
I
HCCOOH
I
CHiCOOH

Oxalosuccinic acid

- COa

Fig. 13-4.

CHjCOOH
alpha-Ketoglutaric acid

The citric acid cycle and related metabolic reactions.

331

332

ANIMAL METABOLISM

18). Ten atoms of hydrogen (two each in reactions 16, 20, 22, 23, 25)
and three moles of carbon dioxide (reactions 16, 21, 22) are also removed.
The net result, therefore, is:

CH3COCOOH + 3HoO -> 10(H) + SCOo

Since at the end of the cycle another molecule of oxalacetic acid is formed,
more pyruvic acid can at once be catabolized. The citric acid cycle
may be regarded as a sort of machine for metabolizing pyruvic or acetic
acids, or any other substance which can be converted into one of the
compounds involved in the cycle (e.g., glutamic acid, p. 343).

Oxalacetic acid occupies a position of special importance, since it is
the substance with which the incoming stream of acetic acid molecules
must combine in order to set the cycle in operation. Although oxalacetic
acid is regenerated at each "turn of the wheel," it is obvious that at
least a small amount must be present before the cycle can start at all.
In other words, there must be some source of oxalacetic acid other than
that regenerated by the cycle itself. This other source is pyruvic acid,
which can combine with carbon dioxide to give oxalacetic acid directly
(reaction 14) or with carbon dioxide and hydrogen (from TPN • Ho) to
form malic acid, which then goes to oxalacetic (reactions 15 and 25).
It is probable that the latter pathway is quantitatively the more im-
portant in animal tissues.

Cytochrome System. The only oxidative processes shown in Fig. 13-4
are indirect ones consisting of the addition of water and removal of
hydrogen. Thus succinic acid, for example, is converted into oxalacetic
acid, which contains one more oxygen atom. This indirect method of
oxidation is a very common biochemical process.

The hydrogen so produced is never present in the free state in the
tissues. It forms reduced coenzymes [e.g., DPN • H2) and from them
is passed through the cytochrome system to combine with the oxygen
brought to the muscles by the blood stream. It is important to note
that, of the two metabolic end products — carbon dioxide and water —
only the latter comes from a direct union with the inhaled oxygen. The
carbon of the original sugar is never oxidized directly to carbon dioxide.
Likewise, the bulk of the energy derived from the metabolism of fats
and carbohydrates comes from the oxidation of hydrogen (p. 422).
, The most important coenzymes which receive hydrogen from metabo-
lites and transfer it to cytochrome are the pyridine nucleotides, DPN
and TPN, and the flavin nucleotides, FAD and FMN (p. 277). In most
cases the hydrogen from the metabolite first passes to one of the pyridine
coenzymes, which is thereby converted into the reduced form, DPN • H2^

^ These abbreviations are used merely for convenience. In reality, one of tlie two
extra hydrogens is ionized :

DPN-Ho^ (DPX.H)- + H^

ANIMAL METABOLISM

333

or TPN • H2 (see Chap. 10 for exact formulas) . Next, the hydrogen is
most probably transferred to one of the flavin nucleotides. This may
be represented, for example, as follows:

TPN -Hi + FAIN

(26)

TPN + FMN-Hi

Note that the pyridine nucleotide is returned to its original condition,
ready to take up more hydrogen. The reduced flavin coenzyme then
hands its hydrogen to cytochrome c (Cyt. c) :

FMN-Hj + 2Cyt. c Fe+++

(27)

FMN + 2Cyt. c Fe++ + 2W

The final reaction is the reoxidation of the reduced cytochrome c by mo-
lecular oxygen (from oxyhemoglobin) under the influence of cytochrome
oxidase, with the formation of water:

2Cyt. c Fe++ + 2H+ + ^Os

(28) cytochrome
oxidase

2 Cyt. c Fe+++ + H2O

The transport of hydrogen through the cytochrome system may be
represented by the scheme shown in Fig. 13-5. The light curved arrows
are used to indicate the alternate reduction and reoxidation of the hydro-

Hydrogen transport system

^

FMN-H.

FMN

2Cyt c Fe+++

,2Cyt c Fe++
2H+

HoO

Net result

H
I
HOOC— C-OH

HC— COOH

I
HjC— COOH

Isocitric acid
(metabolite)

+ 10,

0

II

C— COOH

I +H,0

HC-COOH

I
H.C-COOH

Oxalosuccinic acid
(oxidized metabolite)

Fig. 13-5. Transport of hydrogen through the cytochrome system. Al-
ternate oxidation and reoxidation of the hydrogen carriers is indicated by
light curved arrows. Heavy arrows show path of hydrogen from metabolite
to o.'cygen. >

334

ANIMAL METABOLISM

gen carriers, TPN, FMN, and cytochrome c. Note that the two hydrogen
atoms from one molecule of the metabolite are passed from one coenzyme
to another before they are finally combined with oxygen (heavy arrows).
This seems like an unwieldy and roundabout method of bringing hydrogen
and oxygen together. Apparently, the purpose of this procedure is to
release energy in small steps (p. 420) rather than in a sudden burst, which
probably would injure living tissues. Through the action of the cyto-
chrome system all the hydrogen released from pyruvic acid by the reac-
tions of the citric acid cycle is converted into water.

Cytochrome c is an iron-containing protein (p. 279). The enzyme,
cytochrome oxidase, is poisoned by cyanide. The importance of the cyto-
chrome system to higher animals is apparent from the fact that cyanide
inhibits the respiration of animal tissue preparations to the extent of
80 per cent or more.

Summary of carbohydrate metabolism.

The conversation of food carbohydrates into glycogen and their oxida-
tion to carbon dioxide and water have been considered above under the
headings, glycolysis, citric acid cycle, and cytochrome system. It must
be emphasized that these phases of carbohydrate metabolism are not
in any way separate from each other but operate continuously and
simultaneously in the living animal. In order to gain a clearer over-all
picture, the result of these processes as applied to a molecule of glucose
may be summarized as follows:

CfiHuOs * 2CH3COCOOH + 4(H)

2CH3COCOOH + 6H2O > 20(H) + 6CO2

24(H) + 6O2 > 12H2O

Sum: CeHioOe + 6O2 *■ 6H2O + 6CO2

At various stages of the process, energy is stored up by conversion of
ADP into ATP, and some energy is released as heat. These aspects will
be considered in Chap. 16.

METABOLISM OF LIPIDES

The fat which is poured into the blood stream by way of the lymph
system, following a fatty meal, can be used by the animal organism in
four different ways. These are storage, excretion, oxidation, or con-
version into essential lipides.

ANIMAL METABOLISM

335

Fat storage

The main purpose of fat metabolism is to provide energy by oxidation
of the fat. However, before this occurs a large part of the fat eaten
is temporarily deposited in the fatty tissues of the body. This deposit
provides a reserve of energy for the organism far greater than that in
the form of glycogen for not only is a much greater quantity of fat
deposited, but it has an energy value of 9 Calories per gram as compared
to only 4 for carbohydrate. A certain amount of stored fat is also
desirable as a protective covering for certain organs, especially the kidney.

Dynamic State of Stored Fat. Until rather recently it was supposed
that stored fat was more or less inert metabolically — excess food laid
away and left undisturbed until needed. This viewpoint was entirely
changed by the experiments of Schoenheimer, who fed animals fatty
acids containing deuterium, an isotope of hydrogen, in place of some
of the hydrogen atoms ordinarily present. He found that after four
days about half of the deuterium was present in the stored fats and that
much of the isotope had been shifted to several other fatty acids besides
the one fed. Also, when water containing deuterium was injected into
mice, much of the isotopic hydrogen quickly appeared in the body fats.
He concluded that the stored fat was normally in a constant state of
flux, even in adult animals having a substantially constant weight and
total fat content. About one-half of the body fat is synthesized and one-
half broken down each week.

Nature of Stored Fat. In general, each animal species tends to lay
down a type of depot fat characteristic of the species, but the nature
of this fat is also greatly influenced by the kind of food eaten. This is
true because the animal possesses only a hmited ability to transform
one fatty acid into another.

With the aid of isotopic tracers, chiefly deuterium, it has been demon-
strated that the animal can shorten or lengthen the chain of saturated
fatty acids. Thus stearic acid, for example, can be converted into pal-
mitic and myristic acids, and palmitic can be changed back into stearic
again. Animal tissues also contain enzymes which can change saturated
acids into certain unsaturated ones, for example, stearic into oleic acid.
This process, however, is limited to the introduction of one double bond
at the 9,10-position. Desaturation at the a,/?-position also probably
occurs during beta oxidation (p. 336).

That the animal cannot synthesize more highly unsaturated fatty acids
such as linoleic or linolenic is shown by the fact that these are essential
components of the diet (p. 79) .

The tissues of animals are able to bring about a saturation of a,^-un-
saturated acids. However, if the food fats are more highly unsaturated

336 ANIMAL METABOLISM

than the body fats normally are, the latter will become more unsaturated
also. This is a matter of considerable economic importance in the
feeding of hogs, where a very soft fat is undesirable in the pork. When-
ever the body fats are produced by the feeding of carbohydrates, a
type of fat characteristic of the animal results. Hogs fed on soybean
or peanut meals are "finished" on corn for this reason.

Fat transport

The blood stream serves as the vehicle for carrying fats to various
organs of the body. The blood normally contains simple fat (triglycer-
ides) only for a few hours after a meal. These absorbed glycerides are
carried in the form of tiny fat droplets called chylomicrons to the fat-
storage tissues {e.g., under the skin). Later, the fat to be oxidized is
carried to the liver, apparently in the form of phospholipides.

A number of conditions are kno^\Ti which bring about a greatly increased
amount of fat in the liver. For example, interruption of the normal flow
of pancreatic juice in dogs was found experimentally to cause an accumu-
lation of over 300 g. of fat in the liver, whereas the liver of a normal dog
of similar size contains only 10-15 g. At the same time, the blood phos-
pholipide level fell from 60 to about 30 mg. per 100 ml. This "fatty
liver" condition is prevented or corrected by feeding choline, which pre-
sumably acts by way of forming more phospholipide and thus promoting
the transport of fat away from the liver. Methionine also shows lipo-
tropic action {i.e., prevents accumulation of fat in the liver) , probably
because it can be used in the metabolic synthesis of choline (p. 345) .

Metabolic oxidation of fat

Whether or not the fat is stored, eventually it becomes oxidized to
carbon dioxide and water with the Uberation of energy. This oxidative
catabolism of fat is an aerobic process, which is started chiefly in the
liver and finished in the muscles and kidneys.

The glycerol part of the fat is most probably dehydrogenated and phos-
phorylated to form D-glyceraldehyde-3-phosphate (Fig. 13-3) , which may
then be metabolized by the carbohydrate pathways already discussed.
Thus it may either be converted into glycogen or oxidized to carbon
dioxide and water.

Beta Oxidation. The fatty acids cannot enter the sugar metabolism
pathway so simply because of their widely different chemical nature.
It now appears quite certain that these long chain acids are chiefly broken
down according to Knoop's theory of beta oxidation. Briefly, Knoop's
theory states that two carbon pieces, which appear to be molecules of
acetic acid or some closely related substance, are broken off from the

ANIMAL METABOLISM 337

— COOH end of the fatty acid. These are then further oxidized to carbon
dioxide and water. The exact details of how the two-carbon piece is
broken off have not been completely worked out, but are probal)ly some-
what as follows:

CHoCHnCHsCH.COOH ~|^ CH2CH,CH=CHC00H

Carboxyl end of saturated Corresponding a, /3-

"fatty acid molecule unsaturated acid

O

+ H2O II + HoO

^ CH2-CH,-C— CH2COOH •

-2(H) ^"^ ^"^•- ^ -.xxov.v.w.x ^3jj

(30) Corresponding /3-keto acid

CH2CH2COOH + CH3COOH

Saturated fatty Acetic acid

acid with two less
carbon atoms

Note that it is the beta carbon atom (second from the — COOH group)
which is oxidized. This process is then thought to occur over and over
until the original fatty acid molecule has been broken down entirely
to acetic acid and hydrogen. For example, stearic acid, containing
eighteen carbon atoms, is split in 8 places to yield 9 molecules of acetic
acid as follows:

CH3(CH2)i6COOH + 16HoO^ 9CH3COOH + 32(H)

Block and Rittenberg have estimated the normal acetic acid production
in rats to be about 1 g. per 100 g. of body weight per day. The exact
amount will of course be influenced by the proportion of fat in the ration.
Thus acetic acid, and possibly also acetoacetic acid, represent the end
products of fat catabolism in the liver. These products are transported
by the blood to the muscles and kidneys, where the oxidation is completed.

Other Theories of Fat Oxidation. There is evidence that the methyl
group of fatty acids (the "omega" carbon atom at the opposite end of
the chain from the — COOH) may be oxidized to a second carboxyl group.
This oxidation would produce a dibasic acid which could then undergo
)8-oxidation from each end. The "omega oxidation" probably occurs to
a minor extent, and only with fatty acids of intermediate chain length
(about 8-12 carbon atoms) . That it can occur, however, has been shown
by feeding dogs triglycerides of such fatty acids as undecanoic (saturated,
C-11 acid). The urine of these dogs was found to contain dibasic acids
of 11, 9, and 7 carbon atoms. Omega oxidation is probably not a major
pathway of normal fat catabolism.

A third type of fat catabolism is 7nidtiple alternate oxidation. Accord-
ing to this idea the fatty acid is oxidized at the )8-carbon and at each

338 ANIMAL METABOLISM

alternate carbon beyond the y8-position toward the methyl end of the
chain. This results in a polyketo acid of the type, . . .COCH2COCH2-
COCH2COCH2COOH, which breaks down all at once to form acetic
(or acetoacetic) acid. It is still uncertain whether this type of oxida-
tion occurs extensively during fat metabolism in animals.

Oxidation of Acetic Acid. The two-carbon fragment produced during
fat catabolism, as described above, can be further metabolized in a variety
of ways. Probably the bulk of it, under normal circumstances, is com-
pletely oxidized to carbon dioxide and water. This oxidation occurs
chiefly in the muscles and kidneys, the main energy-using organs. The
acetic acid condenses with oxalacetic acid to form citric acid (reaction
17, Fig. 13-4), which is then further metabolized by the reactions of the
citric acid cycle. This condensation, therefore, is a connecting link be-
tween fat and carbohydrate metabolism. Following the reactions from
acetic acid back to oxalacetic acid (Fig. 13-4), it may be seen that
8(H) and 2CO2 are removed, while 2H2O have been added. This means
that the acetic acid has been completely broken down:

CH3COOH + 2H20-> 8(H) + 2CO2

Since stearic acid was shown above to form nine acetic acid molecules,
the complete catabolism of this acid can now be represented as follows:

CH3(CH2)i6COOH + 34H20^ 104(H) + ISCOo

The hydrogen atoms, of course, are united with coenzymes as fast as they
are produced and are immediately transferred through the cytochrome
system to oxygen, thereby being converted into water.

Ketosis. One of the most important features of fat metabolism is
the fact that fat is not oxidized efficiently to carbon dioxide and water
unless carbohydrate is also being oxidized at the same time. The reason
probably is that the supply of oxalacetic acid, which is formed from
pyruvic acid and carbon dioxide (reactions 15 and 25, Fig. 13-4), is
low when carbohydrates, and hence pyruvic acid, are not being metab-
olized. The essential relationships involved may be illustrated by an
hypothetical case. Suppose only one molecule of oxalacetic acid is pres-
ent in a cell which needs to oxidize three molecules of acetic acid. Three
separate "turns" of the citric acid cycle, one after another, will be required
to complete the job. However, if two molecules of pyruvic acid are
also present, they can be converted into two extra molecules of oxalacetic
acid, and hence all of the acetic acid — as well as the pyruvic — can be
metabolized at one turn of the cycle. At any rate, when carbohydrates
are not being metabolized, the acetic acid coming from fats is not
oxidized as fast as it is produced. Instead it piles up and is recombined
into acetoacetic acid:

(32)

CHjCOOH + CH3COOH ^-^ CH3COCH2COOH + H2O

ANIMAL METABOLISM 339

From this, in turn, are formed acetone and ^S-hydroxybutyric acid:

+ 2(H) _

CHaCHOHCHjCOOH (34)^ CHsCOCH.COOH -^^ CH3COCH,
|3-Hydroxy butyric acid " ^^^ Acetoacetic acid Acetone

These three substances collectively are called "ketone bodies." When
fat, but not carbohydrate, is metabolized, the ketone bodies accumulate
in the blood and are excreted in the urine. This condition is called
ketosis. Since two of the ketone bodies are acids, ketosis also involves
a condition of acidosis, which if not relieved, leads to coma and death.

Ketosis may be caused by eating a diet high in fat and low in carbo-
hydrate. For most people a diet having over 75 per cent of the calories
in the form of fat and less than 15-20 per cent as carbohydrate is keto-
genic (i.e., produces ketosis). However, Eskimos, for example, can tol-
erate even higher amounts of fat. Ketosis may also develop during
starvation or after long-continued vomiting, because in such cases the
main food material being metabolized is the stored fat. Diabetics are
very apt to develop ketosis because of their lowered ability to metabolize
sugars. The excretion of ketone bodies in cases of ketosis in human
beings often amounts to 15-20 g. per day and has been reported in
extreme cases to be more than 100 g. per day.

The exact manner in which acetoacetic acid is formed in ketosis has
been the subject of much dispute. Formerly, it was thought to arise
only from the four carbon atoms at the methyl end of fatty acid mole-
cules, i.e., the last to be degraded by normal yi9-oxidation. However, it
was later found that enzymatic oxidation of caprylic acid (the saturated
C-8 acid) gave rise to tivo moles of acetoacetate. The /^-oxidation theory,
of course, could account for only one mole from one mole of the fatty
acid. It was found further that if the caprylic acid were labeled with
C^^, a heavy isotope of carbon, in the — COOH group, the C^^ appeared
in both the — COOH and CO groups of the acetoacetic acid. These
experimental findings showed that, at least in this case, the caprylic
acid was first oxidized to C2 fragments, which then recombined as indi-
cated in reaction 32 above.

Other metabolic reactions of acetic acid

Fat Synthesis. It is a matter of common observation that consump-
tion of excess food leads to fatness. Fat can be synthesized in the animal
body from either carbohydrates or proteins, although the carbohydrates
are the only important source.

The production of fat from carbohydrate is a process of reduction
and requires energy. Part of the sugar must be oxidized in order that
the rest may be converted into fat. Although the mechanism of the
conversion is not positively known, the sugar is probably broken down

340

ANIMAL METABOLISM

in the usual way to pyruvic acid, which in turn forms acetic acid or a
related C2 substance. The long chain fatty acids are then most prob-
ably produced by uniting a number of these C2 units. This plan
accounts for the fact that nearly all the natural fatty acids contain
an even number of carbon atoms.

Furthermore, it has been shown by the isotope tracer technique that
acetic acid does form fatty acids in the animal body. Acetic acid labeled
with deuterium in the methyl group and C^^ in the — COOH was given
to mice and rats, and the body fats examined after a few days. Both
deuterium and C^^ were present in the fatty acids, and in the same amounts
relative to each other as in the acetic acid fed. The isotopes were present
in all parts of the fatty acid molecules. Other animals were fed deu-
terium oxide ("heavy water") in place of ordinary water, and the body
fats were found to have taken up the deuterium. These facts are all
consistent with the idea that the fatty acids are synthesized by con-
densation of Co fragments, followed by reduction with hydrogen derived
from water in the body tissues.

Thiamine is required for fat synthesis, possibly because it is a part
of cocarboxylase which is required for the oxidative decarboxylation of
pyruvic acid to form acetic acid (reaction 16, Fig. 13-4). Recently it
has been found that another vitamin, namely biotin, is involved in the
synthesis of oleic acid, particularly in microorganisms.

Steroid Synthesis. Acetic acid has also been found to serve as a
metabolic precursor of cholesterol in the animal body. At least half,
and probably more, of the carbon and hydrogen atoms in cholesterol
are derived from this source. Several other substances such as ethyl
alcohol, leucine, and butyric acid can also take part in cholesterol syn-
thesis, but probably only because they are first converted into acetic
acid.

Other important animal steroids are known to be formed, in turn, from
cholesterol. Thus the transformation of cholesterol into cholic acid and
pregnanediol has been demonstrated with isotopic compounds.

Acetylation of Amines. When amines, e.g., sulfanilamide, not normally
present in the body are given to animals, they often are converted at
least partially into acetyl derivatives, which are excreted. This repre-
sents a bodily mechanism for throwing off foreign and possibly toxic
materials. As a rule, the acetylated products are less toxic than the
original amines.

Acetylation also occurs in the case of normal tissue constituents {e.g.,
amino acids, choline) and is, in fact, a very common metabolic reaction.
It has been amply demonstrated that the acetyl groups come from acetic
acid. Acetylcholine, produced by acetylation of choline, is an essential
substance for nerve functioning.

The participation of acetic acid in the metabolic production of por-
phyrins and uric acid is discussed under protein metabolism (p. 351).

ANIMAL METABOLISM 341

In general, it must be concluded that acetic acid, or some closely related
Co substance, is a very active material metabolically and enters into
many of the catabolic and anabolic activities of the living animal
cell.'

METABOLISM OF PROTEINS

Synthesis and interconversion of amino acids in animal tissues

Essential Amino Acids. The metabolism of proteins in the animal
body is largely a matter of the transformations of amino acids. At least
20 of these "building blocks" are present in animal proteins and must
therefore be supplied to the animal, either directly from food proteins
or indirectly by synthesis from other food constituents. Those acids which
cannot be synthesized by the animal at measurable rates are called nutri-
tionally essential amino acids. Those which can be synthesized, but at
rates which are sometimes inadequate {e.g., during rapid growth) may be
called semiessential. Presumably all other amino acids present in body
proteins must be capable of being synthesized in adequate amounts (and
fast enough) to meet all requirements. Of course, these "nonessential"
amino acids also may be present in the food, and, in fact, the main supply
normally comes from this source.

Lists of essential and semiessential amino acids are given in Table 13-1
for several species. These lists are based mainly on studies of the growth
of young animals and on nitrogen balance studies with adults. The
latter method, which is the one used for experiments with human beings,
involves a comparison of the total intake and output of nitrogen when
only certain amino acids are given the subjects. If the lack of a par-
ticular amino acid results in a negative nitrogen balance (output larger
than intake), this is evidence that the acid is needed and cannot be syn-
thesized in the body. For the best nutrition the diet should supply not
only the essential and semiessential amino acids, but a good selection
of the nonessential ones as well. Although the latter can be produced
in the tissues from other materials (see below), it is probably more
efficient to consume them ready-made in the form in which they are
needed.

It will be noted in Table 13-1 that some of the essential amino acids
can be replaced by certain closely related substances, namely, the
D-isomers, or the alpha-keto or alpha-hydroxy analogs. This situation
probably results from the fact that these amino acids can enter into
the process of transamination (see below) . In these cases it is the carbon
chain of the amino acid which is the essential feature, and not the alpha-
amino group. It has also been shown that a-amino adipic acid can
replace lysine for the rat, that is, it acts as a physiological precursor of
this essential amino acid.

342

ANIMAL METABOLISM

Table

13-1

Null

ritionally essential

and

semiessential amino

acids

Classification

Man

Rat

Chick

Essential amino

isoleucine

histidine *t

arginine

acids

leucine

isoleucine *t

histidine

(no synthesis)

lysine

leucinet

isoleucine

methionine *

lysine

leucine *

phenylalanine *

methionine *t

lysine

threonine

phenylalanine *t

methionine *

tryptophan

threonine

phenylalanine *

valine

tryptophan *t
valine t

threonine

tryptophan

valine

Semiessential

arginine *

arginine

cystine

amino acids

histidine

cystine

glutamic acid

(synthesis

glutamic acid

glycine

sometimes

proline

proline

inadequate)

tyrosine

* Can be replaced by D-isomer.

t Can be replaced by corresponding alpha-keto or alpha-hydroxy acid.

Amination. The process of oxidative deamination, which all the amino
acids undergo (p. 351), is reversible in the case of glutamic acid. This
reversibility makes possible the synthesis of glutamic acid from a-keto-
glutaric acid and ammonia, as follows:

COOH

I
CO

I

CHs + NH3

CH2
I
COOH

a-Ketoglutaric acid

- H2O

(35) *"
^+H20

COOH

■

"I
C=NH

I
CH2

I
CH2

+ 2(H)
(36)*'

COOH

I
NH.CH

I
CH2

- 2(H) I

. (L-Glutamic acid ^^2

COOH dehydrogenase) ^^^^

a-Iminoglutaric acid

L-Glutamic acid

This process takes place mainly in the liver and kidneys.

The necessary hydrogen is obtained from the reduced forms of either
DPN or TPN, which of course are always available in the body as a
result of fat and carbohydrate metabolism. Since the a-ketoglutaric
acid is produced from carbohydrates, this process constitutes a link
between the metabolism of proteins and sugars.

The ammonia must be provided from some dietary source, which
normally comes from the deamination of other amino acids. This means
that the above process, which may be called amination, does not result
in a net increase in the total supply of amino acids. Its value lies, rather,

ANIMAL METABOLISM

343

in the fact that, in conjunction with transamination, it enables the body-
to convert one amino acid into another.

Transamination. Two enzymes have been found in animal tissues
which catalyze the transfer of an amino group from an amino acid to a
keto acid. Each of these transamination reactions requires glutamic
acid as the amino group donor or a-ketoglutaric acid as the acceptor,
and pyridoxal phosphate as a coenzyme:

COOH

COOH

COOH

COOH

HjNCH

CO

CH,

+ CH,

CH2

COOH

COOH

L-Glutamic

Oxalacetic

acid

acid

COOH

COOH

H2NCH

CO

CH,

+ CHa

CHo

COOH

L-Glutamic

Pyruvic

acid

acid

(oxalacetic

(37)

transaminase)

(pyruvic

(3S)

transaminase)

CO

HoNCH

CH,

1

+

CH,

CH,

COOH

COOH

a-Ketoglutai

•ic

L-Aspartic

acid

acid

COOH

COOH

CO

H,NCH

CH,

+

CH,

CH,

COOH

a-Ketoglutaric

L-Alanine

aiud

The presence of these highly active transaminases in nearly all animal
tissues suggests that transamination is a major metabolic reaction. Two
additional amino acids, aspartic acid and alanine, are thus obtained from
sugar metabolism intermediates. There are indications that other amino
acids can take part in transamination also, but the importance of the
reaction in these cases is doubtful as far as normal metabolism is
concerned.

Transmethylation. Many of the organic substances present in the
tissues of higher animals contain methyl groups attached to nitrogen or
to sulfur (examples: creatine, choline, methionine). Other substances
(for example, pyridine), not normally present, when fed to animals are
converted into methylated derivatives and are excreted in that form.
A clearer understanding of this process of methylation was obtained by
du Vigneaud from a study of methionine in relation to rat growth. He
found that this essential amino acid could be replaced by choline plus
homocysteine and proved, by using deuterium as a tracer, that methionine

344

ANIMAL METABOLISM

was formed in the animal by the transfer of methyl groups from choline
(reaction 39, Fig. 13-6) . '

COOH

I
H2NCH

I

I

CH2

I

SCH3

L-Methionine

+ (CHs)! (39) I- (CH3)

COOH

•I
H2NCH

CH2

I
CH2

SH

L-Homocysteine
I

(CHa),

N+
I
CHj

CHj

I

OH

Choline

A
(46)

NH2
I

CH2

I

OH

Ethanolamine

+ (CH3)

COOH

I
CHo

I

NH2

Glycine

V

(CH3)

- COa
(45)

- H,0

COOH

(40)

H,NCH

COOH

CH, H.NCH

I I

CHo— S— CH,

L-Cystatliionine

(41)

COOH

I
CO

I

CH2

I

CH3

+ H,0, - NH3
(Ug** or Zn+*)

COOH

I
H2N— CH

CH,

I

SH

L-Cysteine

(43)

HCOOH

Formic acid

(44)

COOH

H2NCH

I
CH2

I
OH

L-Serine

- 2(H)^
(42)
+ 2(H)

COOH COOH

I I

H.NCH H2NCH

" I I

CH,— S— S— CH2

a-Ketobutyric acid L-Uysteine L-Cystine

Fig. 13-6. Metabolic interrelationships of glycine, serine, methionine,
and cystine, and some methylation reactions in animal tissues.

Without choline in the diet, homocysteine was unable to replace methio-
nine for rat growth. It was concluded that the animal was unable to
synthesize methyl groups needed for certain methylation reactions, but
could transfer them, by the process of transmethylation, from other
methylated substances, such as choline. Such substances are called
methyl donors and are said to contain labile methyl groups. An adequate

ANIMAL METABOLISM ^'^^

source of labile methyl groups is one of the essential components of a
complete diet for higher animals. However, this requirement can be
met indirectly if the diet contains certain vitamins (see below) .

Methionine itself is also a methyl donor and has been shown to pro-
vide methyl groups for the formation of both choline and creatine (p.
348) . Choline is produced in the animal body by the addition of methyl
groups from methionine to ethanolamine (reaction 46, Fig. 13-6) , which
in turn is derived from serine (reaction 45) . Five other substances have
now been found which can serve as methyl donors in biological systems.
Two of them, betaine [ (CH3)3N+CH2COO-] and dimethyl-propiothetin
[(CH3)2S+CH2CH2COO-] occur in nature and probably take .part in
methylation reactions in living cells.

Recent studies have proved that when adequate supplies of folic acid
and vitamin B12 are present in the diet, rats can synthesize labile methyl
groups from glycine, serine, acetone, or formic acid and hence do not
require a methyl donor for growth. This was established by isotopic
tracer studies which showed that carbon atoms from these substances
appeared in the methyl groups of choline and thymine. Also, when rats
were fed a diet containing all the known vitamins including folic acid
and vitamin B12, but without any methyl donor, and with homocysteine
as the only sulfur-containing amino acid, good growth occurred. Pre-
sumably the rats converted the homocysteine into methionine under these
conditions.

Other Metabolic Interconversions oj Amino Acids. As a result of recent
investigations, based almost entirely on the use of isotopic tracers, several
other metabolic relationships among amino acids have been discovered.
For example, L-cystine is synthesized in the animal body from L-serine
and L-methionine. The intermediate steps, which involve homocysteine
and cystathionine, are shown in Fig. 13-6. In some still obscure manner
the cleavage of cystathionine (reaction 41) results in the formation
of a-ketobutyric acid as the other product besides cysteine. Note that
only the sulfur of the cystine is derived from methionine and that the
rest of the molecule comes from serine.

These reactions provide a reasonable explanation for the fact that
cystine is not a nutritionally essential amino acid and that it has a
"sparing action" for methionine. That is, when the diet contains plenty
of cystine, no methionine has to be diverted to cystine synthesis so that
less methionine is needed. Another substance which can give rise to
cystine in the body of the rat is L-lanthionine (p. 117).

Another metabolic relationship, which is now well established, is the
formation of serine from glycine. The most probable route of this syn-
thesis is shown in Fig. 13-6, reactions 43 and 44. One molecule of glycine
is changed into formic acid, which then combines with a second molecule
of glycine to form serine. The reverse conversion of serine into glycine
also occurs readily in the animal body.

346

ANIMAL METABOLISM

Higher animals also are able to convert glutamic acid into proline,
hydroxyproline, and ornithine, as follows:

COOH

I
H,NCH

1

CHj
1

(47)

H2C-

-CH2

(48)

*■

H

HOC CH2

1

CH2
COOH

HjC. /CHCOOH
H

H2C. /CHCOOH
H

-Glutamic acid

L-Proline

1 t

L-Hydroxyproline

\

(49)

k

COOH

H2NCH

I

CH2

I

CH2

I

CH2

I

NH2

L-Ornithinc

The ornithine produced is readily converted into two additional amino
acids, citrulline and arginine (see urea formation, p. 352). Although
arginine can thus be synthesized in the animal, the rate of production
is often too slow to meet bodily needs. It has been shown, for example,
that the growth of rats fed a ration lacking arginine is greatly stimu-
lated by adding this amino acid.

Tyrosine is another amino acid which is synthesized in the animal
body, the precursor being phenylalanine:

COOH

COOH

H2NCH
CH,

H2NCH

(50)

L-Phenylalanine

OH
L-Tyrosine

Accordingly, it has been found that tyrosine has a sparing action fur
phenylalanine, just as cystine has for methionine.

ANIMAL METABOLISM

347

Utilization of amino acids

The main use for amino acids in the animal body is the synthesis of
tissue proteins. Such synthesis is not only necessary for young, growing
animals, but it is also essential for full-grown adults, because tissue
proteins are continually being broken down and resynthesized. Borsook
gives 10 days as the half-life (period in which one-half of a substance
is decomposed) of the proteins in the internal organs of man and 158
days for those in other tissues (mainly the muscles).

The amount of dietary protein needed to supply the normal require-
ments of human beings depends on age (stage of growth) and on the
amino acid composition of the proteins consumed. Assuming good
quality food proteins, satisfactory allowances per kilogram of body
weight are: men, 1 g.; women, 1-1.8 g.; children, 1.5-3 g. ; infants, 3.5 g.

Conversion of amino acids into other metabolites

In addition to protein synthesis, amino acids are used as raw materials
for the synthesis of a series of essential substances by animal tissues.
The formation of nicotinic acid from tryptophan {p. 237) probably follows
the pathway* indicated below, although some details remain unproved:

I

H

NH2
I
C— CH.— C— COOH

II " H

H H

L- Tryptophan

HC

^^

H

HC

^

H

NH2

I
^C-COCH^-C— COOH
H

.C— NH,

Kvnurenine

H NH2

/C. I

HC^ C— CO-CH2-C-COOH

H

HC

^ /C-NH2
C
I
OH

3-Hydroxy kvnurenine

HC

^

H
C.

HC

*^

C— COOH

II

€— NH2

I
OH

3-Hydroxy anthranilic
acid

HC^
I

H

C.

HC

^

N'

C— COOH

II

C— COOH

Quinolinic acid

HC^
I

H
C.

HC

^

C— COOH

II
CH

N
Nicotinic acid

348

ANIMAL METABOLISM

It has been established that nicotinic acid can be formed from trypto-
phan in the actual tissues of the animal body, although the same con-
version can also be brought about by intestinal microorganisms.

H2N— CH— COOH
I

CH2
I

I
CONH2

L-Glutamine

Glutamic acid is extensively converted into glutamine in animal tissues.
In fact, a large part of the glutamic acid in the body, both free and
combined, probably exists in the form of glutamine.

Creatine and creatinine are produced from three amino acids, glycine,
arginine, and methionine. The guanidine group of arginine first com-
bines with glycine to form guanidoacetic acid, which then is methylated
by methionine :

H^NCH^COOH + I

NH2

I
C=NH

I
NH

I
(CH,)3

I
HCNH2

Glycine

COOH

L-Arginine

(51)

NH2

I
C=NH

NH

I
CH.,

I
COOH

+

NH,

I
(CH,)3

HCNH2

I
COOH

Guanidoacetic acid Ornithine

NH,

CH3

NH,

H

C=NH

1

S
1

C=NH

S

NH +

(CH,)2

(52)

>

NCH3

+

(CH,),

CH2

HCNH2

CH2

HCNH2

COOH

COOH

COOH

COOH

Guanidoacetic acid

L-Methionine

Creatine

L-

-Homocysteine

The formation of ornithine in the first reaction has not been estab-
lished, but would certainly be expected. Choline can also furnish the
methyl group for the second reaction, but only indirectly by first trans-
ferring it to methionine. The creatine so formed is converted into the

ANIMAL METABOLISM

349

anhydride, creatinine, through the intermediary formation of creatine
phosphate.

(53a) /

HN=C COOH ..L > HN=C,

\ /

N— CHj

CH,

Creatine

+ H3PO4

H

N— PO3H2

(53b)

N— CH2

CH3

Creatine phosphate

H

N

HN=C CO

\ /

N— CH2

CH3

Creatinine

Tyrosine, itself produced from phenylalanine, is the starting point for the
biosynthesis of melanin, the dark-colored pigment of the hair and skin,
and for the hormones, adrenalin, nor adrenalin, and thyroxine (see Chap.
11). Melanin is produced by the enzymatic oxidation of tyrosine to an
unstable intermediate product which polymerizes (reacts with itself) to
form the high molecular weight pigment. According to Mason the most
probable course of the process is as follows:

NH2

CH2— C— COOH
I H

HC^ XH

(0)

NH2

CH2— C— COOH
I H

HC^ XH

HC

^.

.CH

OH

L-Tyrosine

tyrosinase

rearrangement

HOC:^ XH

X

I
OH

Dihydroxyphenyl
alanine, or "Dopa'

350

ANIMAL METABOLISM

H

HOC^ /C. .CHCOOH
H H

(0) ^

tyrosinase

0=C'

H

.C

^

c

o=c.

c

H

,^

c.

-CH2

XHCOOH

H

2-Carboxy-2,3-dihydro-
5,6-dih5'droxyindoIe

Hallochrome

- CO2

rearrangement

HOC

^

H

C.

H

C-

CH

(O)

HOCi::^ ^C^ ^CH

c

H

H

5,6-Dihydroxyindol('.

tyrosinase
polymerization

Melanin

0=C'
0=C,

■c

H

CH

II
.CH

H

Indole-5,6-quinone

Abnormal metabolism of tyrosine and phenylalanine is exhibited by
some people. In tlie condition known as alcaptonuria, tyrosine is con-
verted into homogentisic acid. This substance is excreted in the urine

CHoCOOH

CHoCOCOOH

HC

^^

^COH

HOC

^

.CH

HC

HC

^

C,

■^CH

0'
H

=^

C^
H

.CH

Homogentisic acid

Phenylpyruvic acid

and causes it to turn black on standing exposed to air. In another con-
dition, phenylketonuria, large amounts of -phenylpyruvic acid are formed
from phenylalanine and are excreted in the urine. This apparently
results from an inability to convert phenylalanine to tyrosine. Both of
these conditions represent hereditary abnormalities of amino acid
metabolism.

Certain amino acids, particularly glycine, have also been demonstrated
to be among the building blocks used by the animal in the biosynthesis
of purines and porphyrins. The various carbon and nitrogen atoms of
the purine skeleton come from the following sources: carbons 4 and 5,

ANIMAL METABOLISM

351

(i)N=C(6)

I 1(5)

(2)C C— N
'')\

(*)C

(9) /

(3)N— C — N
(^)

Purine skeleton

and nitrogen 7 from glycine; carbons 2 and 8 from acetic acid; carbon
6 from carbon dioxide; and nitrogens 1, 3, and 9 from ammonia. In the
porphyrin ring system,

A

C(4)

>— N

/ (2) (1)

(3)\ /^a\ /(4)^
(2)^ ^

C(3)

D

(5),

c.

0) (5r%

(2) ^

Porphyrin ring system^

carbons A-2, B-2, C-2, D-2, a, 13, y, and 8 come from CHo of glycine,
while A-4:, 5-4, C-4, and D-4 are derived from the CH3 of acetic acid.
Also the four nitrogen atoms come from the NH2 group of glycine.
The mechanism by which these components are put together to form
purine and porphyrin compounds in the animal body is still un-
known.

Breakdown of amino acids in the animal body

Deamination. In the average American diet more protein is con-
sumed than is needed for synthesis of tissue proteins and the other essen-
tial substances derived from amino acids. Consequently, the body re-
ceives an excess of amino acids which must be disposed of in some manner.
Direct excretion would be wasteful and, in fact, occurs to only a small
extent (in the urine). Most of the excess amino acids seem to be broken
down by the process of oxidative deamination. Two steps are involved ;
first, dehydrogenation of the amino acid forms an imino acid:

^ The four individual pyrrole rings are sometimes designated as I, II, III, and IV,
respectively, rather than by A, B, C, and D (p. 137).

352 ANIMAL METABOLISM

H,N-CH-COOH -"""0 acid oxidase- ^ H N^C-COOH + 2(H)

I (54) I

R R

Amino acid Corresponding imino acid

which in the second step is hydrolyzed to the corresponding keto acid
and ammonia:

HN=C-COOH + H2O -^ 0=C-COOH + NH3

R . R

A number of enzymes present in animal tissues catalyze the dehydrogena-
tion of various amino acids (see Chap. 10). The hydrogen split off in
the first step is transferred directly to an acceptor, which differs accord-
ing to the enzyme involved, but frequently is oxygen. Assuming that
oxygen is the acceptor, the net result of oxidative deamination may be
summarized by the following equation:

HaN-CH-COOH + O2 ±^^ 0==C— COOH + NH3 + H2O2

R R

The hydrogen peroxide is decomposed by catalase, or used to oxidize other
metabolites. The other products are further metabolized as described
below.

Formation of Urea. Ammonia is a toxic substance that cannot be
tolerated by animal tissues in large amounts and therefore must be
eliminated as fast as it is formed. For most higher animals it is com-
bined with carbon dioxide to form the waste product, urea. A summary
equation indicating the net result of this combination is as follows:

2NH3 + COo -^ H2N - CO - NH2 + HoO

Urea

However, such a direct union does not occur in the body. Urea is formed
instead by a cyclic process involving several intermediate substances.
Almost certainly, the immediate source of the urea is arginine, which is
broken down into urea and ornithine by the enzyme, arginase: '

NH2 NH2 NH2

C=NH (CHo)3 + CO

I +H.0

(CH=)a COOH

NH ,,gi„,3e HC-NH2 NH2

HC— NH2

I
COOH

L-Arginine L-Ornithine Urea

ANIMAL METABOLISM

353

Arginase is present in the livers of those species which excrete urea as the
main end product of nitrogen metabolism, but is absent from others
such as birds, reptiles, and insects which excrete uric acid instead of urea.
Evidently there must be some way by which ammonia is incorporated
into arginine in the body, in preparation for urea formation. A possible
method, suggested in 1932 by Krcbs and Henseleit, is known as the
ornithine cycle:

NH2

NH2

1

CO

(CH,)3

(57)
+ NH3

NH

HCNH2

+ C0;

(CH,)3

COOH

HCNH2
COOH

L-Ornithine

L-CitruUine

+ NH3

(58)

NH2

I

C=NH

I
NH

I

(CH2)3

+ H20

HCNH2

COOH
L-Arginine

Splitting of the arginine which was formed would then give the end
product, urea, plus another molecule of ornithine and start the cycle
over again.

Most investigators still regard this scheme as essentially correct,
although later work has shown that aspartic and glutamic acids are also
involved in the process and also that an energy source (probably ATP)
is necessary to drive the reactions in the direction indicated. According
to Ratner and Pappas the conversion of citrulline to arginine (reaction
58) probably does not occur simply by addition of ammonia, as shown
in the above equation, but rather by interaction with aspartic acid:

KH2

1
C=0

I
NH

I

(CHo)3

HCNH2

I
COOH

L-CitruUine

COOH

H,N— CH

+

CH,

I
COOH

L-Aspartic acid

(An

intermediate

condensation

product)

NH2

I

C=NH COOH

I I

NH HOCH

-I +1

(CH2)3 CH2

HCNH2 COOH

COOH

L-Arginine

L-Malic acid

This indicates that the nitrogen of certain amino acids may be converted
into urea in the body without ever having existed as free ammonia.

Another modification of the Krebs-Henseleit cycle was proposed by
Cohen and Grisolia. They presented evidence that the carbon dioxide

354 ANIMAL METABOLISM

needed for reaction 57 does not react directly with ornithine, but first
combines with ghitamic acid to form an intermediate substance, which
then transfers its carbon dioxide to ornithine. Glutamine probably is
not involved in urea formation ; but it does serve as the source of urinary
ammonia (i.e., ammonium salts) in mammals.

Metabolism of deaminated amino acid residues

The products resulting from deamination of amino acids (usually a-keto
acids) are in most cases utilized as a source of energy by the animal
body, that is, oxidized to carbon dioxide and water. The metabolic
pathways by which this oxidation occurs are not fully known, but are
certainly different for each amino acid.

The keto acids from the deamination of alanine, aspartic acid, and
glutamic acid are normal intermediates in carbohydrate metabohsm and
can be either broken down to carbon dioxide and water or built up into
glycogen or glucose by the reactions already considered (Figs. 13-1,
13-3, and 13-^). In diabetic dogs several other amino acids also are
convertible, wholly or in part, into glucose. Included in this group are
glycine, serine, arginine, and proline.

Three of the amino acids, phenylalanine, tyrosine, and leucine, produce
acetoacetic acid when fed to diabetic dogs. This substance, it will be
recalled, is one of the compounds resulting from oxidation of fatty acids
in the animal body. These amino acids therefore appear to be oxidized,
after being deaminated, in the manner of fatty acids. Amino acids which
give rise to acetoacetic acid in the body are said to be ketogenic (form
ketone bodies), whereas those convertible into carbohydrate are anti-
ketogenic.

In some cases ring structures of the more complex amino acids are
nonutilizable. Tryptophan, for example, is partly degraded into indole
and skatole, which are excreted in the feces.

H H

C C

HC^ ^C CH HC^ ""C C-CHa

HC^ /C. XH HC^ ^C^ ^CH
^C N'^ C N

H H H H

Indole Skatole

REVIEW QUESTIONS ON ANIMAL METABOLISM

L Define metabolism, catabolism, anabolism.

2. What is the main purpose of carbohydrate metabolism in the animal body? In
which respects does this process resemble actual burning of carbohydrate in a flame?

ANIMAL METABOLISM

355

3. Summarize the main factors operating to control the blood sugar level.

4. Is lactic acid one of the intermediate substances formed on the main pathway
of normal carbohydrate metabolism? Under which conditions is it formed in con-
siderable amounts? Why is it formed? What becomes of it?

5. Show by balanced equations the net result of glycolysis, starting with glucose;
of the combined operation of the citric acid cycle and the cytochrome system, starting
with pj-ruvic acid.

6. List four phosphorylated and six nonphosphorylated intermediate substances
involved in carbohydrate metabolism.

7. Summarize the evidence as to whether stored fat is motabolically active or
inert.

8. What is meant by "beta" oxidation of fatty acids; by "omega" oxidation?
Which is most important in animal tissues?

9. List eight substances which may be more or less directly produced from acetic
acid in animal tissues.

10. Discuss the relationship between the ability of animal tissues to carry out
synthetic reactions and the need for amino acids or proteins in an animal's food.

11. Which metabolic reactions ser\'e to link together the metabolism of carbo-
hydrates and proteins; of carbohydrates and fats?

12. What is transmethylation? Name the substances now known to serve as
methyl group donors, and list several substances produced in the body as a result
of methylation.

13. Name 5 substances produced in the animal body wholly or partially from
glycine.

14. Which amino acids are ketogenic; antiketogenic?

REFERENCES AND SUGGESTED READINGS

Baldwin, E.. Dynamic Aspects of Biocheniistnj, 2nd ed., Cambridge University

Press, Cambridge, 1952.
Bloch, K., "The Metabolism of Acetic Acid in Animal Tissues," Physiol. Rev., 27,

574 (1947).
Bloch, K. and Rittenberg, D., "An Estimation of Acetic Acid Formation in the Rat,"

J. Biol. Chem., 159, 45 (1945).
Bloor, W. R., Biochemistry of the Fatty Acids, Reinhold Publishing Corporation,

New York, 1943.
Borsook, H., "Protein Turnover and Incorporation of Labeled Amino Acids into

Tissue Proteins in Vivo and in Vitro," Physiol. Rev., 30, 206 (1950).
Breusch, F. L., "The Biochemistry of Fatty Acid Catabolism," Advances in Enzymol-

ogy, 8,343 (1948).
Chaikoff, I. L. and Entenman, C, "Anti-Fatty Liver Factor of the Pancreas,"

Advances in Enzymology, 8, 171 (1948).
Cohen, P. P, and Grisolia, S., "The Intermediate Role of Carbamyl-L-Glutamic Acid

in Citndline Synthesis," J. Biol. Chem., 174, 389 (1948).
Duel, H. J. and Morehouse, M. G., "The Interrelation of Carbohydrate and Fat

Metabolism," Advances in Carbohydrate Chemistry, 2, 119 (1946).
du Vigneaud, V., Ressler, C, and Rachele, J. R., "The Biological Synthesis of 'Labile

Methyl Groups,'" Science, 112, 267 (1950).
Gortner, R. A. Jr. and Gortner, W. A., Outlines of Biochemistry, 3rd ed., John Wiley

and Sons, Inc., New York, 1949, Chapters 19, 20, 26, and 32.
Greenberg, D. M. (editor) Amino Acids and Proteins, Charles C. Thomas, Publisher,

Springfield, 1951, Chapters X, XIII.

356 ANIMAL METABOLISM

Kleinzeller, A., "Synthesis of Lipids," Advances in Enzymology, 8, 299 (1948).
Knoop, F., "Catabolism of Aromatic Fatty Acids in the Animal Body," Beitr. Chem.

Physiol. Path., 6, 150 (1905).
Krebs, H. A. and Henseleit, K., "Urea Formation in the Animal Body," Z. Physiol.

Chem., 210, 33 (1932).
Lardy, H. E. (editor) Respiratory Enzymes, Burgess Publishing Company, revised

ed., Minneapolis, 1949.
Martins, C. and Lynen, F., "Problem des Citronensaure cyklus," Advances in

Enzymology, 10, 167 (1950).
Mason, H. S., "The Chemistiy of Melanin," J. Biol. Chem., 172, 83 (1948).
Medes, G., "Fat Metabolism," Annual Rev. Biochem., 19, 215 (1950).
Ratner, S. and Racker, E., "Carbohydrate Metabolism," Annual Rev. Biochem., 19,

187 (1950).
Ratner, S. and Pappas, A., "Biosynthesis of Urea," J. Biol. Chem., 179, 1183, 1199

(1949).
Schoenheimer, R., The Dynamic State of Body Constituents, Harvard University

Press, Cambridge, 1942.
Swanson, P. P. and Clark, H. E., "Metabolism of Proteins and Amino Acids," Annual

Rev. Biochem., 19, 235 (1950).

Chapter 14

METABOLISM OF MICROORGANISMS

Interrelations of microorganisms, animals and plants

Microorganisms form an integral and indispensable part of a living
world. Some consideration of their chemical activities is not only de-
sirable but essential if an over-all view of biochemistry is to be obtained.
Chlorophyll-containing plants are the ''factories" in which organic matter
is made and energy is stored. Many of the organic compounds produced
in nature contain nitrogen, and in the fixation of atmospheric nitrogen,
bacteria, such as rhizobia, are undoubtedly the most important agents.
The building of organic matter is balanced by its destruction; here again
microorganisms seem to play the leading role. Dead plant and animal
materials are converted by microorganisms into simple compounds such
as carbon dioxide, ammonia and nitrates, which are used again by
plants. Although animals contribute to the breakdown of organic matter,
in the over-all effect microorganisms undoubtedly are the principal agents.
The balance between construction and destruction of organic matter is
often spoken of in connection with single elements and designated as
the carbon cycle and the nitrogen cycle in nature.

The intimate association of microorganisms and animals is of course
obvious from the fact that animals act as hosts to vast numbers of bac-
teria in the intestinal tract. The importance of bacteria to the host is
conspicuous in the case of ruminants, where they probably are indis-
pensable. They break down cellulose and other resistant plant materials
to compounds that can be utilized by the animal, synthesizing all the
B vitamins needed by the ruminant. Even nonruminants appear to
derive a large part of their supply of certain vitamins, e.g., biotin, from
the synthetic action of bacteria.

On the debit side of the association account is the production of dis-
ease by infectious microorganisms in animals and plants. The practical
problem then is to promote the development of useful microorganisms
and to retard the growth of harmful types.

From a scientific viewpoint, the study of the metabolism of micro-
organisms has been a most fruitful effort. A first insight into inter-
mediary metabolism came from a study of yeast. This has been extended
to animals and bacteria, and from these studies a diversified but also a
unified pattern of metabolism is emerging.

357

358

METABOLISM OF MICROORGANISMS

Growth requirements

Energy and Carbon. Because of the thousands of species of micro-
organisms, it is much more difficult to state their growth requirements
than it is those of higher animals, where only a few species have to be
considered. Perhaps the only general statement one can make is that
all require some source of energy. A few species of microorganisms can,
like plants, use light as a source of energy, but the vast majority obtain
their energy from chemical elements or compounds. Merely listing a
few examples shows the diversity of sources: elemental H, C, and S,
simple compounds such as HoS and NH3, carbon compounds ranging from
carbon dioxide and methane through the carbohydrates, lipides, and
proteins to such resistant materials as lignin and paraffin.

Stephenson cites an example from the work of den Dooren de Jong
to illustrate the amazing synthetic powers of microorganisms. The
bacterium, Pseudomonas putida, can meet all its carbon requirements
from 77 different carbon compounds out of 200 tested. The utilizable
compounds included 6 carbohydrates, 10 alcohols, 13 fatty acids, 17
amino acids, 9 amides, and 7 amines. It would probably be a safe
statement to make that there is no form of combined carbon in the
world that cannot be utilized by some microorganism.

Nitrogen. Since all living cells contain protein, some form of nitrogen
must be supplied. In some cases atmospheric nitrogen is utilized {e.g.,
Azotobacter vinelandii) ; in others, inorganic nitrogen, such as nitrates
and ammonia, is adequate {e.g., yeasts, molds, and autotrophic bacteria) ;
but in many others only amino acids can meet the needs of the cell.
Lactic acid bacteria are conspicuous examples of cells that require pre-
formed amino acids for growth. One of these, Leuconostoc mesenteroides,
requires 17 amino acids, many more than are required by higher ani-
mals.

The requirement for certain amino acids may depend upon the absence
of a vitamin. For example, some strains of propionic acid bacteria
require riboflavin if ammonium sulfate is the source of nitrogen, but if
amino acids are supplied, no riboflavin is needed. Another example is
Lactobacillus arabinosus, which grows without tryptophan if vitamin Be
is present, and without Be if tryptophan is present. In both cases it
synthesizes the compound that is omitted. Hence there is not an absolute
requirement for either compound, but the cell cannot make both com-
pounds simultaneously.

The interesting phenomenon of imbalance among amino acids exists
in microorganisms, perhaps more markedly than with higher animals.
Thus a certain strain of Escherichia coli will grow in the absence of
tyrosine, but not in its presence unless phenylalanine is also present. In

METABOLISM OF MICROORGANISMS

359

this instance tyrosine and phenylalanine are antagonistic to one another.
There are many other examples of imbalance between amino acids.

Growth of some bacteria requires, or is increased by, purines and
pyrimidines in the culture medium (e.g., Staphylococcus aureus and
Clostridium tetani) . Some bacteria use uric acid and other purines as
their sole source of carbon and nitrogen (e.g., Clostridium acidiurici) .

Growth Factors (Vitamins, etc.). The growth factor requirements of
microorganisms vary over a wide range of compounds. For example,
E.coli can grow in a medium containing no B vitamins, whereas Lacto-
bacillus casei requires at least seven of these vitamins and, in addition,
some growth factors of undetermined nature. In some cases only a part
of the vitamin molecule is required preformed in the medium; e.g., the
^-alanine jiart of pantothenic acid by yeast and the pantoyl part of it
by Acetobacter suboxydans. In other cases, a combined form of the
vitamin is required; e.g., nicotinamide riboside by Hemophilus parain-
jluenzae and pantetheine (pantothenic acid-/3-aminoethanethiol) by
Lactobacillus bulgaricus. An example of a progressively more complex
series of compounds, and bacteria requiring them, follows:

Bacteria Compounds required

Clostridium acetobutylicum p-aminobenzoic acid

Streptococcus fecalis pteroic acid

Lactobacillus casei pteroyl glutamic acid

Lactobacillus citrovorwn formyltetrahydropteroj'lglutamic acid

The probable explanation of this series is that the last compound in
the series is either the one that functions in metabolism or is nearer
to it than the earlier members. The bacteria that need only the simpler
compounds probably synthesize the complex compound from the simpler
ones. For example, CI. acetobutylicum can perform all the syntheses
between p-aminobenzoic acid and formyltetrahydropteroylglutamic acid,
and L. citrovorum cannot perform some, if any, of them.

Another such progressively complex series starts with pantothenic
acid, proceeds to pantetheine, and ends with coenzyme A. A third series
can be formed from pyrimidine (or thiazole), thiamine, and lipothiamide.
The existence of these series of compounds suggests that the compound
actually functioning in the metabolism of the cell is the complex com-
pound and not the siinple one.

The fat-soluble vitamins (A, D, E, and K) , so essential for animals,
are not required by microorganisms. Many microorganisms synthesize
K and carotene and ergosterol, the precursors of A and D. Ascorbic
acid stimulates the growth of some bacteria, but it seems to act as a
reduction-oxidation compound rather than as a vitamin. On the other
hand, many other compounds not required in the diet of animals serve
as growth factors for microorganisms. Examples of such compounds,
in addition to those already named, and the associated microorganisms

360 METABOLISM OF MICROORGANISMS

follow: hemin (Hemophilus injiuenzae) ; putrescine, NHo(CH2)4NH2
{H. parainfluenzae) ; a-lipoic acid, also called thioctic acid, p. 254 {Strep-
tococcus lactis and Tetrahymena geleii) ; coprogen, an organic-iron com-
pound (Philobolus kleinii) .

Inorganic Elements. The requirement of K, Mg, Mn, Fe, S, and P
for the growth of microorganisms is well established, and numerous reports
indicating the need for Ca, Cu, Zn, Mo, and Co have appeared. The
reason for the uncertainty regarding the need for some of these elements
is the small amount that is required and the difficulty of removing traces
of these elements from the medium. An example of the difficulties that
exist is illustrated by a study of the vitamin B12 requirements of bacteria.
This vitamin is synthesized by many bacteria, and since it contains cobalt,
this element must have been present in the medium. While the vitamin
can be shown to have been formed, the presence of cobalt in the medium
is not easily demonstrated. The quantity of cobalt required for the
synthesis of all the vitamin needed by the bacteria is of the order of
0.4 m^ug Co per liter ^ of medium — a quantity that is not easily detected.

On the other hand, microorganisms that synthesize large quantities
{e.g., 2-3 jug per milliliter) of vitamin B12 must be supplied with cobalt
salts if maximal yields of vitamin B12 are to be obtained.

In practical work the needs of microorganisms for all the inorganic
elements are met by adding phosphate or sulfate salts of potassium,
magnesium, manganese, and iron to the medium. These salts usually
carry enough impurities to meet the needs, if any, of the microorganism
for other elements.

Growth efficiency

Cells differ greatly as to the efficiency with which they convert nutrients
into cell material. The comparison is best made on the basis of dry
matter of food converted into dry matter of cells, in order to eliminate
the effect of varying moisture contents on the calculations. If the
figures are expressed on a percentage basis, the efficiency of the cell is
obtained. The efficiency varies under different conditions, but if those
giving optimal values are taken, the conversion of nutrients into cell
dry matter is about as follows:

Group Example Percent

Aerobic bacteria Azolohacter vivelandii 14

Anaerobic bacteria Clostridium acetobutylicum I

Yeast Saccharomyces cerevisiae, aerobic growth 50

Yeast Saccharomyces cerevisiae, anaerobic growth 5

Molds Penicillium chrysogenum, in penicillin production. 40

Molds Aspergillus niger, in citric acid production 5

^ One milligram is equivalent to 1,000 micrograms (^,g) and one microgram is equiva-
lent to 1,000 millimicrograms (m|j,g).

METABOLISM OF MICROORGANISMS 361

Young of some common higher animals:

Cattle 4

Swine 7

Chicken 10

Fish 10

Yeast when grown under aerobic conditions is probably the most
efficient of the microbial cells ; molds come next and, from the meager data
available, bacteria are third. Animals, even the most efficient, are far
below aerobic microorganisms in their ability to convert food into living
cells.

Slow-growing animals are less efficient than animals that attain ma-
turity in a short time. This is to be expected since cells that grow
slowly use up a larger proportion of the food for maintenance than rapidly
growing cells.

Perhaps the most noteworthy figures are those comparing aerobic
and anaerobic growth. For example, the same species of yeast gives
about 10 times more weight of cells under aerobic than under anaerobic
conditions. Producers of baker's yeast understand this fact and blow
enormous quantities of air through the medium to obtain high yields
of yeast. Under anaerobic conditions the nutrients, e.g., glucose, are
converted mainly into ethyl alcohol and carbon dioxide instead of into
yeast cells. It is impossible to have high yields of cells and alcohol
in the same fermentation. The situation is analogous to that with
cattle; the farmer obtains a high production of either beef t)r milk, but
not both from the same animal.

The effect of aerobic conditions in stimulating the growth of cells is
seen also by comparing the two bacteria, A. vinelandii and CI. aceto-
hutylicum. The first is an aerobe and gives high yields of cells. The
second, an anaerobe, gives a low yield of cells but large amounts of
products such as acetone, ethyl alcohol, and butyl alcohol.

Molds are aerobic microorganisms. If allowed to grow under favor-
able conditions, e.g., penicillin fermentation, they are about as efficient
as yeast in converting nutrients into cells. In citric fermentation, growth
is deliberately restricted in order to promote the yield of citric acid.

Metabolic rate

Microorganisms transform matter at a much faster rate than do animals.
It takes about 100 days for an adult human being to consume his own
weight of food. Cattle accomplish this in 40 days and swine do it in
20 days. Yeast cells take about 30 minutes to metabolize their weight
of nutrients. The mold A. niger converts its own weight of glucose to
gluconic acid in about 2 minutes, and a urea-splitting bacterium trans-
forms its weight of urea to ammonia in a few seconds. One reason why

362 METABOLISM OF MICROORGANISMS

some microorganisms work so fast is that they derive very little energy
from the chemical changes that they bring about. Hence, of necessity,
they must work over a large amount of matter in order to meet their
energy needs.

Comparison of microorganisms with respect to end products

The term fermentation may be defined as the chemical process by
which organic compounds are converted into new compounds by micro-
organisms or by enzymes obtained from microorganisms. With modi-
fying words it is used in a broad sense to designate products formed,
materials utilized, or agents involved. When dealing with products
formed, we have such phrases as alcohol fermentation, citric acid fer-
mentation, penicillin fermentation, tea fermentation, etc. To feature
the materials utilized, such terms as glucose-, xylose-, cellulose-fermenta-
tion are used. In designating the microbiological agent such expressions
as yeast fermentation, bacterial fermentation, and mold fermentation are
employed. In this chapter the word fermentation is used in all three
of these ways. No distinction is made among processes that are
anaerobic and produce gas, e.g., alcoholic fermentation, those that are
anaerobic and produce no gas, e.g., lactic acid fermentation, and those
that are aerobic and produce gas (carbon dioxide), e.g., citric acid and
penicillin fermentations.

In a restricted sense the term fermentation is used to denote an
anaerobic type of metabolism. Associated with this usage is the term
respiration, brought over from animal metabolism to denote what in
effect is complete oxidation of the substrate to carbon dioxide and water.
Attempts to separate microbial metabolism into fermentation and respira-
tion processes seem highly artificial, since the yeast cell, for example,
may operate on either an aerobic system or an anaerobic system, and at
times even on both systems simultaneously.

In Table 14-1 are listed the products that are characteristic of bacteria,
yeasts, and molds. The percentage given for the glucose or fructose
converted into the corresponding product is maximal, or nearly so. It
is believed that figures showing such a performance of a microorganism
are more meaningful than data obtained under conditions that do not
permit the cells to function at or near their optimal capacity.

An inspection of this table shows that there are about a half dozen
products that are common to all three groups. Carbon dioxide is the
compound that is not only common to all three, but is also produced
in large amounts by many members of each group. It is probably a
universal product of cell metabolism. In certain cases a product is
more generally found in one or two of the groups rather than in the

METABOLISM OF MICROORGANISMS 363

others. Lactic acid, for example, is produced by many bacteria, but
only a few molds make it, and yeasts produce it only in traces or under
special conditions.

Products that are limited to bacteria are hydrogen, the lower fatty
acids, butylene glycol, butyl alcohol, acetone, and isopropyl alcohol.
Several of these products are closely related, as will be shown later.

Di- and tri-basic acids are characteristic of molds. Succinic acid is
the only one of this type common to all three groups. Although it has
been found in yeast fermentations, it appears to originate from oxida-
tion and decarboxylation of glutamic acid rather than from glucose.

If a fourth column to include animals were tabulated, it would be
found to be mostly negative, except for carbon dioxide. Lactic acid,
acetic acid, and acetone are found at times in the urine, but they are
not normal end products of animal metabolism.

The high yields obtained with some microorganisms have made possible
a number of industrial fermentations. The mechanism of the formation
of these products will be discussed later.

Table 14-1

Characteristic end products of carbohydrate metabolism formed by
bacteria, yeasts, and molds *

Compound Bacteria Yeasts Molds

Carbon dioxide CI. acetobutylicum, 55 S. cerevisiae, 45 P. chrysogenum, 70

Lactic acid L. delbrilckii, 90 S. cerevisiae, trace R. oryzae, 75

Ethyl alcohol T. mobile, 45 S. cerevisiae, 45 F. avenaceum, 40

Acetic acid L. gayonii, 10 S. cerevisiae, 1-3 A. nigcr, trace

Glycerol L. lycopersici, 20 S. cerevisiae, 1-40 Aspergillus sp., 10

Mannitol L. gayonii, 65 None Aspergillus sp., 35

Gluconic acid A. suboxydans, 95 None A.niger,95

Propionic acid P. pentosaceum, 60 None ?

Formic acid E. typhi, 10 None None

Hydrogen E. coli, 0.5 None None

Succinic acid E. coli, 12 Doubtful Rhizopus,sp., small

Acetoin A. aerogenes, 0.5 Trace None

Butylene glycol A. aerogenes, 45 ? None

Butyric acid CI. saccharobutyricum. None None

40

Butyl alcohol CI. acetobutylicum, 2Q None None

Acetone CI. acetobutylicum,, 7 None None

Isopropyl alcohol . . . .CI. butylicum, 6 None None

Oxalic acid None None A. niger, 80

Fumaric acid None None R. nigricans, 60

Citric acid None None A. niger, 90

* The microorganisms named are typical of the best producers of the compounds.
The figures denote the maximum percentage of product, based on glucose or fructose
fermented, that has been found in the literature. The yield of product, usually less,
will vary with other substrates, microoi-ganisms, and fermentation conditions.

364 METABOLISM OF MICROORGANISMS

AEROBIC METABOLISM OF CARBOHYDRATES

The conventional and most convenient method of classifying the varied
types of metabolism performed by microorganisms is on the basis of
utilization of oxygen. If oxygen is used, the metabolism is called aerobic ;
and if not, it is designated as anaerobic. This is an arbitrary classifica-
tion, as many organisms have both an aerobic and an anaerobic system
and operate on one or the other as circumstances require. Examples of
such microorganisms are E. coli and ordinary baker's yeast, *S. cerevisiae.
Bacteria that can grow either in the presence or absence of air are termed
facultative aerobes or facultative anaerobes depending upon which con-
dition appears more favorable.

By bacteria

In general, aerobic bacteria oxidize sugars to carbon dioxide and water,
but there are certain bacteria that are exceptions to this rule. Acetobacter
suboxydans, for example, oxidizes the carbons along the chain of a
polyhydroxy substance like glucose, but cannot cut the chain into shorter
pieces. From glucose it forms gluconic acid and 5-ketogluconic acid,
and from sorbitol it makes sorbose. The oxidizability of a compound
is very specific and depends upon the structure of the molecule. For
example, sorbitol and mannitol are oxidized to the corresponding keto-
sugars, sorbose and fructose, respectively; but ducitol, the alcohol cor-
responding to galactose, is not attacked. From a study of these and
other sugar alcohols, Bertrand concluded that two alcohol groups adjacent
to the primary alcohol must be cis to one another for oxidation to take
place. Dulcitol and xylitol do not have such a structure and are not
oxidized. However, as more polyhydric alcohols and bacteria have been
studied, it has been found that the requirements are both less specific
and more complex than is expressed by Bertrand's rule.

Aerobic bacteria are very important in the production of vinegar, anti-
biotics, and enzymes; in the retting of flax; and in the disposal of sewage
by the activated sludge process. Antibiotics are of special interest and
recent development. An antibiotic is generally defined as an organic
compound produced by microorganisms which in small concentrations
inhibits or kills other microorganisms, usually pathogenic in character.
The definition is admittedly arbitrary, as it excludes inhibitory com-
pounds produced by higher plants, such as quinine, and purely synthetic
compounds, such as sulfa drugs. The term as thus defined, however, is
useful and convenient for practical purposes.

The number of antibiotics reported in the literature runs into several
hundreds. Most of these are poorly characterized chemically, but ap-
proximately 60 have been obtained sufficiently pure to permit determina-

METABOLISM OF MICROORGANISMS

36;

tion of their elementary composition. Some of these antibiotics are well-
characterized structurally, but most of them are poorly defined chemical
compounds. One reason why so little is known regarding the chemical
nature of many antibiotics is the fact that they are too toxic for clinical
use. Toxicity removes one of the strong incentives for determining
their structure. This is unfortunate, because a knowledge of the struc-
ture of a toxic substance is perhaps as important as an understanding
of the make-up of the less toxic substance. There is a fine opportunity
for qualified chemists in this field.

The best known antibiotic in actual use is of course penicillin, but
since it is a mold product it will be discussed in the section on molds.
The other antibiotics are produced either by bacteria or streptomycetes.
The latter are classified as bacteria, although they have mold-like char-
acteristics, for example, growth in long thread-like filaments. Each of
these antibiotics will be discussed separately.

Streptomycin. This antibiotic was discovered by Waksman and asso-

H NH

I II
N— C— NH2

HO I

I /C-H/H

H.

0

C— H

H2N— C— N— C>^ ^C— H
II ^ C \

Streptidine moiety
(gives basic property)

O

H— C

I
0=C— C— OH

H C-H

O-

C— H

CH3

Streptose moiety
(reducing property)

CH3-N— C-H

H^ I
O H— C-OH

I
HO— C— H

I

C-H

I
CHoOH

N-Methyl-L-glucosamine

' — Streptobiosamine ■
moiety

Streptomycin
(free base)

ciates in 1944 and is produced by the microorganism Streptoinyces griseus.
Like most microorganisms, S. griseus produces several other antibiotics,
namely, mannosidostreptomycin, a combination of mannose and strepto-
mycin; actidione, an antifungal compound (C15H23NO4) ; and grisein
(C40H61N10O00SFC) . By selection, strains of S. griseus have been obtained

366

METABOLISM OF MICROORGANISMS

which give mainly streptomycin, and in high yields — about 2-3 g. per
liter of medium. This antibiotic is produced by many companies in the
United States and foreign countries. In 1952 the production in the
United States was 440,000 lb., and the market value at the plants was
about $50,000,000.

Its use is attended with some drawbacks. The tubercle and other
bacteria become resistant to streptomycin. It also has toxic effects on
the eighth cranial nerve which may lead to deafness on prolonged use.
However, in spite of much effort, no other antibiotic has been found
equal to streptomycin in the treatment of tuberculosis. See Fig. 14-1,

Courtesy of Abbott I^aboratoiies.
(a)

Courtesy of The .Squibb Institute for
iNIedical IJesearfli.
(b)

Fig. 14-1. Tubercle bacillus and streptomycin, (a) Photomicrograph of
infected limg tissue of mouse. The rod-like particles are Mycobacterium
tuberculosis, the microorganism that causes tuberculosis. Other dark areas
show accompanying cellular lung tissue, (b) Crystals of streptomycin tri-
chloride, the most useful antibiotic in the treatment of tuberculosis.

Streptomycin, as can be seen from the structural formula, is a complex
organic base consisting of three parts: streptidine, streptose, and a methyl
derivative of glucosamine. The streptidine base contains two guanidino
groups (compare arginine, p. 120) which give streptomycin its salt-forming
characteristics. Commercial preparations of streptomycin are usually
the trihydrochloride or trihydrosulfate. Streptidine is clearly a deriva-

METABOLISM OF MICROORGANISMS

367

tive of inositol, a vitamin which is found in many plant and animal
materials.

Streptose is an unusual type of sugar. It has an aldehyde group
attached along the carbon chain to give a branching structure. The
aldehyde group can be reduced chemically to give the corresponding
alcohol. This derivative was thought for a time to be superior to strepto-
mycin clinically, but later work indicated that it had the same disad-
vantages as streptomycin.

It has been suggested that streptomycin interferes with nucleic acid
metabolism. Since it is a basic substance, it may react with the acidic
groups of nucleic acid and form complexes that are not metabolized.
According to Umbreit, the mode of action of streptomycin is through
interference with pyruvic acid metabolism. This is a complex phenome-
non involving many enzymes, coenzymes, and chemical changes. If
streptomycin interferes with the pyruvate metabolism of microorganisms,
the question naturally arises as to why it does not affect the same metab-
olism in the animal? The explanation usually given is that streptomycin
does not penetrate the animal cell but remains in the extracellular fluid.
The bacteria infecting the animal cannot prevent the entrance of strepto-
mycin into their cells, hence their metabolism becomes deranged and
they die.

Bacteria rapidly acquire resistance to streptomycin, and in such cells
Umbreit has found that the oxalacetate-pyruvate relation has disap-
peared. The bacteria have apparently been able to develop a new me-
tabolic pathway with which streptomycin does not interfere. A still more
puzzling phenomenon is the development of dependent bacteria, that is,
bacteria that will not grow unless streptomycin is added to the medium.
No satisfactory explanation has as yet been found for the development
of dependent strains. A possible explanation that has been advanced
is that the dependent strain produces so much of some metabolite that
it kills itself when no streptomycin is present to counteract the effect of
this metabolite. Some support for this theory is found in the increased
production of the metabolite para-aminobenzoic acid by certain bacteria
when sulfanilamide is added to the medium.

Aureomycin and Terramycin. These two antibiotics are very similar

CH3

^H^ H C ?^ H V^ H N-CH3

H. tlz"^ XT \ I TT \l

ll-C^ ^C ^C^ ^C"^ X— OH

H-C^ C C^ .ci*"^ .C-CONH

(7)^ i^)\ (S)Y OH f')

OH O OH 0

Terramycin, C22H24N209

2

368

METABOLISM OF MICROORGANISMS

biologically and chemically. The structural formula for terramycin was
worked out first and is given above. Aureomycin, CooHosNoOsCl, has
the same basic structure as terramycin. Both are derivatives of naphtha-
cene and have many groups in common. Aureomycin contains a chlorine
atom and one less hydroxyl group than terramycin, but the chlorine and
hydroxyl group are not interchangeable. They occupy different places
in the molecule, as indicated by the CI and H in parentheses in the struc-
tural formula. Chlorine replaces a hydrogen at position 10, and a hydro-
gen takes the place of the hydroxyl at 12 in the terramycin formula.
There seems also to be some doubt as to the location of the dimethylamino
group in aureomycin; it may be interchanged with the hydroxyl group
attached at 4a. Both compounds form yellow-colored salts and have
many other similar physical and chemical properties. For further details
the many papers on these antibiotics that have appeared recently should
be consulted.

As would be expected from their close chemical relationship, they
are much alike in their bacterial spectrum. Both act on gram-positive ^
and gram-negative bacteria, on organisms producing rickettsial diseases,
e.g., typhus fever, and are potent in certain virus infections, such as
virus pneumonia. Neither antibiotic is effective against the tubercle
bacillus, bacteria of the proteus and pseudomonas types, or fungi. Aureo-
mycin and terramycin are given by mouth in clinical treatments.

Aureomycin and terramycin are produced commercially by fermenta-
tion. The microorganism producing aureomycin is called Streptomyces
aureofaciens because of the golden yellow appearance of the colonies of
the microorganism on agar plates and also because the antibiotic is yellow.
The terramycin organism is named Streptomyces rimosus because of the
cracked appearance of the colonies on agar plates.

Since each antibiotic is produced by a single company, no official
figures are available as to the yearly production. Judging from the
widespread use of these antibiotics, their production must be several
hundred thousand pounds per year and their market value must run
into millions of dollars.

Chloromycetin (Chloram,phenicol). Chloromycetin is the name com-

H H

C=C H HN-CO-CHCU

O.N-C C-C C-CH,OH

\ / I I

C— C OH H

I I
H H

Chloromycetin

^ Bacteria that take the gram-stain are called gram-positive and those that do not
are said to be gram-negative. Consult a book on bacteriology for the reagents {e.g.,
gentian violet, etc.) used in making the stain and the method of performing it.

METABOLISM OF MICROORGANISMS •^69

monly used for this antibiotic, but it is also known by tlie more chemical
name chloramphenicol. The compound contains chlorine and is pro-
duced by a streptomyces, hence it is quite apparent how the word
Chloromycetin came to be devised. The microorganism producing
Chloromycetin is called Streptomyces venezuelae, an obviously poor name
since it denotes geographical origin and not an inherent characteristic.
The microorganism was obtained from a sample of soil from Venezuela.
It has also been found in soils from Illinois and Japan and is probably
widely distributed in nature.

It is produced industrially both by fermentation and by synthesis.
To date it is the only commercial antibiotic that is produced synthetically
as well as by fermentation.

The most distinctive feature about the chemical structure of Chloro-
mycetin is the nitro group. Few organic compounds in nature contain
a nitro group. It also contains chlorine, which, though not common,
occurs in aureomycin and a number of mold products, e.g., erdin. The
presence of an amide linkage relates it to peptides and explains its
hydrolysis by enzymes found in cells of Proteus vulgaris. Chloromycetin
contains no acidic or basic groups, hence it does not form salts. It is
a neutral compound that crystallizes as colorless needles or elongated
plates.

Chloromycetin is relatively inactive against gram-positive bacteria,
but is very potent against the gram-negative bacteria associated with
intestinal diseases such as typhoid fever and dysentery. It is active
against the same rickettsial and viral diseases as aureomycin and terra-
mycin.

Chloromycetin is relatively stable to acids and alkali, is rapidly ab-
sorbed from the gastro-intestinal tract, and hence is usually given by
mouth. Liver and kidney tissues reduce the — NO2 group to an — NHo
group. About 90 per cent of the administered dose is excreted as an
inactive compound and 10 per cent as unchanged Chloromycetin in 24
hours.

Terramycin, aureomycin, and Chloromycetin are alike in bacterial spec-
trum and appear to be similar in their mode of action; they interfere
strongly with protein synthesis but are much less effective in stopping
nucleic acid synthesis. A more specific reaction in protein metabolism
has been observed for Chloromycetin. It acts as an antagonist against
phenylalanine, but the antagonism is noncompetitive. Only low con-
centrations of Chloromycetin can be overcome by addition of phenyl-
alanine. At higher concentrations its effect cannot be reversed by
adding more phenylalanine. This makes Chloromycetin a particularly
effective antimetabolite since the inhibited organism cannot counteract
the Chloromycetin by producing more phenylalanine.

Bacitracin, Polymyxin, and Tyrothricin. These three antibiotics are
all polypeptides and are produced respectively by Bacillus licheniformis,

370 METABOLISM OF MICROORGANISMS

Bacillus poly my xa, and Bacillus brevis. The amino acid content of
bacitracin is given in Table 5^ and presents no unusual features. There
are several polymyxins, A, B, C, D, and E, and each one contains large
amounts (more than 50 per cent) of the unusual amino acid, L-a,y-di-
aminobutyric acid. A second distinctive feature is the presence in the
molecule of a nine-carbon fatty acid, probably 6-methyloctanoic acid.

Tyrothricin is not a homogeneous substance but consists mainly of
gramicidin, a neutral cyclic polypeptide. On hydrolysis gramicidin gives
five amino acids and ethanolamine, NHoCH^CHoOH.

Bacitracin resembles penicillin in being most active against gram-posi-
tive bacteria. It causes kidney damage (evidenced by albumin in the
urine) . Because of this toxic effect, its use is limited to combating local
infections.

The polymyxins are very potent against gram-negative bacteria, in-
cluding the very resistant Proteus and Pseudomonas bacteria. Unfortu-
nately, the polymyxins cause more or less kidney damage, so their use
will probably be limited to refractory infections that do not respond to
other treatments.

Tyrothricin is the oldest commercial antibiotic, but probably the least
used of the commercial products. It acts on gram-positive bacteria
but is not suitable for injection or oral administration. It is used only
for topical purposes, tluit is, where it can be brought into direct contact
with the infecting oi;ganism, e.g., surface abscesses.

Because of the millions of gallons of media that must be used for the
production of antibiotics, the fermentation is done in deep tanks of
5-15,000 gallon capacity. Sterile air is forced through the medium at
the rate of about one-half volume of air per volume of medium per minute.
The medium is also stirred vigorously to increase aeration. Deep tank
fermentation was first developed for the production of penicillin and
later applied to the production of other antibiotics and vitamins.

Vitamin B12 is produced simultaneously with streptomycin, aureomycin,
and terramycin. Hence producers of these antibiotics obtain a second
valuable product in the same fermentation. Vitamin B12 is also pro-
duced commercially by a mixed aerobacter-proteus type of fermentation.
The vitamin is formed by many different kinds of bacteria and molds.
Yeasts produce little, if any, of it.

By yeast

Baker's yeast can grow under both aerobic and anaerobic conditions.
If an abundance of air and a low concentration of sugar [e.g., 0.5 per
cent) are supplied, the end products of metabolism are mainly carbon
dioxide and yeast (50 per cent of the weight of sugar is obtained as dry
weight of yeast) ; there is practically no alcohol. See Fig. 14-2.

METABOLISM OF MICROORGANISMS

371

If the sugar content of the medium is raised to 5 per cent, the yield of
yeast is markedly reduced, and much alcohol is formed. In other words,
an anaerobic type of metabolism comes to the front, although an abun-
dance of air is present. The explanation for this is that ordinary yeast

Courtesy of Dr. Charles N. Frcy, Fieiscbmauu Laboratories.
Fig. 14-2. Budding veast cells.

has a weak aerobic enzyme system and a strong anaerobic system. If
more sugar is present than can be metabolized aerobically, the anaerobic
system begins to operate.

By molds

Molds cannot grow in the absence of air; carbon dioxide and water are
the usual products of metabolism. However, many species convert a
large percentage of the sugar in the medium into other carbon products.
Examples of products that make up more than 40 per cent by weight of
the sugar consumed and the molds producing them are given in Table
14—1. The highest yields of compounds in the table are for gluconic
acid. Actually over 100 per cent has been obtained, if allowance is
made for glucose going to mycelium. This yield is possible since one
oxygen is added per mole of glucose, which amounts to 196 g. of gluconic
acid from 180 g. of glucose, or 109 per cent.

Citric Acid. This acid has been obtained many times in yields of
80-90 per cent, but the usual yields, without allowing for glucose going
to mycelium, are around 70 per cent. Very special conditions have to
be maintained to keep the mold growth low. Such conditions are low
concentrations of metals, particularly manganese, high concentrations
of sugar, and low pH in the medium. An explanation for the effect of
manganese is that this metal serves as a cofactor for some enzyme system
that functions in the breakdown and oxidation of citric acid. If there
is a deficiency of manganese, the enzyme cannot operate, and then citric

372

METABOLISM OF MICROORGANISMS

acid accumulates. Associated with citric acid production is sparse spore
formation. The mycelium presents a beaded or braided appearance, and
this appearance supports the idea that accumulation of citric acid is an
abnormal type of metabolism.

One of the theoretical problems connected with citric acid production
is how to harmonize the high yield with the conventional system of inter-
mediary metabolism that operates in yeast and animals (p. 331). This
system would require three 2-carbon pieces, or one and one-half moles
of glucose per mole of citric acid. On a percentage basis only 71 per
cent of citric acid could be obtained. Many theories have been proposed
to account for higher yields. The current and best explanation is the
uptake of carbon dioxide by pyruvic acid to form oxalacetic acid (Wood-
Werkman reaction) and condensation of this acid with acetic acid to form
citric acid:

CO2 + CH3 • CO • COOH-^ HOOC • CHo • CO • COOH

HOOC • CH2 • CO • COOH + CH3 • COOH-^ HOOC- CHo • C(OH) • COOH

I
CH2 • COOH

Uptake of isotopic carbon dioxide has been shown to take place, but
whether this is adequate to account for the high yields is still not certain.
The mechanism of citric acid formation is under active investigation in
both animal and mold studies, and many of the questions now unanswered
should be cleared up in the near future.

Penicillin. Besides the major products mentioned in Table 14-1,
molds produce hundreds of other compounds in amounts from a fraction
of a per cent to 10 per cent. The best known of these products is peni-
cillin. Approximately 20 tons of penicillin are produced every month
in the United States alone. Yields of 1 g. of penicillin per liter of medium
are usual, and about 70 per cent of the penicillin in the broth is recovered
as finished product. A typical medium is: lactose, 3 per cent; corn
steep solids (the concentrate of the water extract obtained in the indus-
trial manufacture of starch, gluten, and other corn products), 3 per cent;
calcium carbonate, 0.5 per cent; sodium sulfate, 0.1 per cent; phenyl-
acetic acid, 0.3 per cent. The medium is sterilized, inoculated with a
high-yielding strain of Penidllium chrysogenum, and aerated and stirred
vigorously during the fermentation period, 3-4 days. The penicillin is
extracted from the acidified broth with amyl acetate, transferred to a
buffer, purified, and finally crystallized as the sodium, potassium, or
procaine salt. Since penicillin is an acid, many different salts can be
made, but the above three are those in commercial use. More than a
dozen companies are producing penicillin in this country, and the market
value of the yearly product has been more than 100 million dollars for
several years. See Fig. 14-3.

Courtesy of Abbott Laboratories.

(b)

Courtesy of Myron P. Backus,
(a)

<_'ouil('s\ ot Alibott Laboratuiics.

"(c)

Fig. 14-3. Production of penicillin, (a) Colony of the high-yielding
penicillin mold, Penicillium chrysogenum, Wis. Q176. This culture was
used industrially for many years to produce penicillin, (b) Fermentation
tanks of 6000 gal. capacity used in the submerged production of penicillin,
(c) Crystals of the sodium salt of penicillin.

373

374 METABOLISM OF MICROORGANISMS

The yield of penicillin has been increased about a thousandfold over
that obtained in the beginning of its production. The high yield has
been attained largely by selection of better cultures. The best of these
have been obtained by treating the mold spores with x-ray, ultraviolet
light, or N-mustard gas to give high-yielding mutants.

Other factors in obtaining high yields have been the use of better
media and better methods of aeration and agitation of the media. The
improvement in penicillin yields is strikingly similar to the development
in wheat-raising. Penicillin production might be called factory farming,
for the principles operating are the same as in wheat production.

Molds produce at least a half dozen different types of penicillin in
the same medium. These differ only in the R group part of the mole-
mule. Today, only one type of penicillin is wanted in commerce, that
is the benzyl or G penicillin, which has the formula

CeH-CH,-C-NH-CH-C^ C-(CH3)2

O 0=C N- CH-COOH

R group Penicillin G

If a suitable precursor, e.g., phenylacetic acid, is added to the medium,
the mold obligingly responds by incorporating this compound into the mol-
ecule. Other R groups are: in F penicillin, pentenyl (CH3 • CH2 * CH =
CH • CH2-) ; in K penicillin, heptyl (CH3 • CHo • CHo • CHo • CHo • CH2 •
CH2 — ). More than 20 penicillins have been obtained by addition of
the appropriate precursors.

Penicillin acts on gram-positive bacteria, and in exceedingly low con-
centrations. For example, 0.03 units per milliliter will inhibit the growth
of the assay organism Micrococcus pyogenes var. aureus (formerly called
Staphylococcus aureus). Since a unit of penicillin is 0.6 /xg., 0.03 unit
is less than 0.02 fxg. per milliliter or 2 mg. per 100 1. of medium. A
clinical dose of 100,000 units is only 60 mg. Unfortunately, strains of
microorganisms that are resistant to penicillin are beginning to appear.
These resistant strains probably come from patients who have been re-
cently treated with penicillin.

The most obvious effect of penicillin on the microbial cell is that al-
though the cell grows larger, it does not divide. This shows that formation
of cell constituents, e.g., proteins and nucleic acids, continues for some
time after the penicillin enters the cell. Eventually the enlarged cell
bursts.

Interference with absorption of amino acids, protein synthesis, nucleic
acid synthesis, and phosphorylation reactions have all been attributed
to penicillin. It is difficult to determine which of these are primary
effects and which are secondary manifestations. Metabolism is a series
of events, and interference at one place will show up in all subsequent

METABOLISM OF MICROORGANISMS

375

places and eventually reflect back to the original point of interference.
It would probably be more correct to think of metabolism as a cycle
rather than a chain of events.

Penicillin is specifically and irreversibly bound by gram-positive bac-
teria. For example, 0.49 units of penicillin per gram of dry weight
are bound by cells of Bacillus cereus. Extracts of the cells also bind
the penicillin, and it should be possible to identify the substance in the
extract that possesses binding power. It may be that the reaction between
penicillin and this cell constitutent is the primary reaction and other
effects are secondary.

A well-defined effect of penicillin on nucleic acid metabolism is reported
by Park. This is the accumulation of uridine-5'-pyrophosphate complexes
in cells of Staphylococcus aureus that have been treated with penicillin.
In addition to uracil, ribose, and phosphoric acid, one of these complexes
contains an N-acetyl amino sugar. A second complex contains L-alanine
in addition to the other four components, and a third has attached to
it a peptide made up of DL-alanine, L-lysine, and D-glutamic acid. Prob-
ably the accumulation of these complexes is a secondary effect caused
by the blocking of some reaction that utilizes the uracil compounds.

From the discussion given, it is evident that the specific effect of
penicillin is still undetermined. However, since many able investigators
are attacking the problem, distinct progress toward its solution may be
expected.

Tetronic Acids. The production of a series of compounds closely
related in structure is a characteristic feature of mold metabolism. Be-
sides the penicillins, another such series is the tetronic acids.

HO-C==C-H(R')

(R)II-CH.-C^ C=0
H

1. y-Methyl tetronic acid, Penicillium charlesii.

2. Carolinic acid, P. charlesii:

R' = CO(CHo)oCOOH (succinyl group).

3. Carolic acid (+HoO), P. charlesii:

R' = C0(CHo)2CH.0H (y-hydroxybutyryl).

4. Terrestric acid (+HoO), P. terrestre :
R' = CO(CHo)2Ch6h • C0H5

(an ethyl derivative of the R' group in carolic acid) .

5. Dehydrocarolic acid (+H2O), Pe?iza7/iwm cinerascens:
CH3— of carolic acid is replaced by CH2=

6. CarHc acid, P. charlesii:

R = HOOC; R' = CO(CH2)2CH20H (y-hydroxybutyryl)

7. Carlopic acid, P. charlesii:

R = HOOC; R' = CO- CH0CH2CH3 (butyryl).

376 METABOLISM OF MICROORGANISMS

Formulas with (+H0O) mean that their peculiar structure is present
only in water. These compounds crystallize as anhydrides. y-Methyl
tetronic acid may be regarded as the parent substance, and the others,
as substitution products. Carolinic acid has a succinyl group in place
of the hydrogen on the a-carbon. Carolic acid has a carbinol group
instead of a carboxyl group in the side chain. In carlosic acid the end
group in the R' side chain is methyl. These three compounds clearly
represent different degrees of oxidation. In carlosic acid and carlic acid
there are also carboxyl groups replacing the hydrogen at R in the formula.
All of these compounds are produced in only small amounts, in the order
of 1 to 2 per cent of the sugar fermented. It is of special interest that
five of the compounds are produced by the same mold. These must be
interrelated in the metabolism of P. charlesii.

Many other such series of compounds have been described by Raistrick
as characteristic of mold metabolism, e.g., a citric, an anthraquinone,
and a tropolone series. Over 200 mold products have been isolated in
more than a quarter century of research work by Raistrick and co-workers.
From this wealth of material many interesting features of mold metabolism
have been discovered. For more information see his review paper listed
in the references at the end of this chapter.

Another noteworthy aspect of mold metabolism is the formation of
organic chlorine compounds. Examples of such compounds are erdin
(C16H10O7CI2), and geodin (C17H12O7CI2). These two compounds are
produced by the same mold, Aspergillus terreus, and are closely related
in structure.

Spoilage of such commercial products as wood, paper, leather, hay,
grain, bread, etc., constitutes a debit side of mold activities. An
inhibitor of mold growth, propionic acid, is widely used in the bread
industry. For use on nonfood materials, there are a number of mold
inhibitors, e.g., pentachlorphenol, CeClsOH.

ANAEROBIC METABOLISM OF CARBOHYDRATES

By bacteria

The anerobic metabolism of bacteria is probably more diversified than
the aerobic and probably results in a larger number of products. (See
Table 14-1.) The principal types of anaerobic fermentations can be
classified by their major end products, as follows:

1. The homolactic type of fermentation {e.g., by S. lactis) accounts for
more than 90 per cent of the glucose as lactic acid. Thus

CgHioOe -^ 2CH3 • CHOH • COOH

METABOLISM OF MICROORGANISMS 377

2. The heterolactic fermentation {e.g., by L. pentoaceticus) turns about
half of the gkicose into lactic acid and converts the other half mainly
into carbon dioxide and ethyl alcohol. The equation is

C6Hi206-» CH3 • CHOH • COOH + COo + CH3 • CHoOH

Sometimes considerable amounts of acetic acid and small quantities of
glycerol are formed.

These two types of lactic acid fermentation, homolactic and hetero-
lactic, are important in the industrial production of lactic acid and in
the making of cheese, sauerkraut, pickles, and silage.

3. The propionic fermentation {e.g., by P. pentosaceum) gives propionic
acid, acetic acid, succinic acid, and carbon dioxide as major products,
but under certain conditions considerable amounts of lactic acid are
formed. The propionic fermentation may be regarded as superimposed
upon a homolactic fermentation, but it does not seem probable that lactic
acid is an intermediate in the production of propionic acid. In this and
the following fermentations the reactions are too complicated to be readily
expressed by simple equations.

4. The colon-aerogenes-typhoid bacteria, not only produce all of the
compounds formed by the mixed lactics except glycerol, but in addition
make formic acid, hydrogen, and butylene glycol. This is a very hetero-
geneous group of organisms, and the proportion of the products to one
another varies greatly with the species of bacteria. Perhaps the most
distinctive products are: formic acid by Eberthella typhi, acids and
hydrogen by Escherichia coli, and acetoin and butylene glycol by Aero-
bacter aerogenes.

5. The butyric acid fermentation {e.g., by CI. acetobutylicum) is char-
acterized by the almost complete absence of lactic acid and the appear-
ance of acetic acid, butyric acid, carbon dioxide, hydrogen, butyl alcohol,
ethyl alcohol, and acetone. Isopropyl alcohol may replace acetone
wholly or in part in certain butyric fermentations. Some bacteria in
this group do not form the last four compounds, collectively called
solvents, while others produce them in large amounts.

6. The naturally occurring methane fermentation {e.g., by Methano-
bacterium omelianskii) involves a unique type of metabolism. The two
extremes of oxidized and reduced carbon products, carbon dioxide and
methane, meet here. This apparent contradiction did not appear so
strange when it was discovered that methane arose, at least partially,
by reduction of carbon dioxide with hydrogen. Methane (also called
marsh gas) occurs extensively in coal mines, stagnant waters, sewage,
certain types of plants, and in the intestinal tract of animals.

7. Fairly well-characterized polysaccharides have been obtained from
more than 60 species of bacteria. Some of these give unusual products

378 METABOLISM OF MICROORGANISMS

on hydrolysis such as gkicuronic acid, D-arabinose, and inositol. Some
of the most notable polysaccharides, their important characteristics, and
the bacteria producing them are as follows :

(a) Cellulose. This is true cellulose, identical in chemical and physical
properties with that found in higher plants. It is produced by Acetobacter
xylinum and other members of this genus.

(b) Polysaccharides with marked physiological and chemical proper-
ties are produced by many pneumococci. The polysaccharide produced
by Type III consists of alternate glucose and glucuronic acid units bound
together through oxygen by a /i^-linkage from carbon 4 of the glucose
to carbon 1 of the glucuronic acid and a second ^S-linkage between carbon
3 of the glucuronic acid and carbon 1 of the glucose. There appear to
be over 600 units each of glucose and glucuronic acid in the polysaccharide
chain. The polysaccharide has marked antigenic properties; when in-
jected into a rabbit, it evokes production of antibodies and immunity
against infection with Type III pneumococcus.

(c) Dextrans. Many bacteria produce dextrans, but L. mesenteroides
is the best known dextran-producer. One reason for the current interest
in this bacterium is that it produces a dextran that is now being manu-
factured as a substitute for blood plasma. L. mesenteroides is found as
a contaminant in sugar factories, and the dextran produced interferes
seriously with manufacturing operations. A 10 per cent sucrose solu-
tion is fermented in about 24 hours and gives a yield of 25-35 per cent
dextran, based on sucrose used. The dextran comes from the glucose
part of the sucrose molecule, but glucose itself does not give any dextran,
although the microorganism grows well on this sugar. On a glucose
medium the microorganism behaves as a heterolactic. The dextran
appears to be formed from sucrose according to the following equation:

n(CioHooOn) -> (CgHioOs)?! + /^(CeHisOs)
Sucrose Dextran Fructose

Some of the glucose and most of the fructose are fermented to lactic acid,
acetic acid, ethyl alcohol, carbon dioxide, and mannitol. Potent enzyme
preparations which bring about the rapid formation of dextran and
fructose from sucrose have been obtained from the culture solution.

The dextran has a branching structure with apparently a-l,6-linkages in
the main chain and a-l,4-linkages at the branching points (pp. 50 and 60) .
The molecular weights of dextrans from different strains of L. mesenter-
oides are enormous, e.g., 25 to 80 millions. These dextrans are too large
for use directly as blood plasma substitutes. They are partly degraded
by controlled acid hydrolysis and fractionated to give products of suitable
size, e.g., molecular weights of about 75,000. Only about 10 per cent
of the original dextran is obtained as material suitable for clinical use.
An extensive search is now in progress for microorganisms that will pro-

METABOLISM OF MICROORGANISMS

379

duce more suitable polysaccharides than those obtained from L. mesen-
teroides.

(d) Levans. Polysaccharides of this type are produced from sucrose
by several microorganisms, e.g., Bacillus subtilis. Yields of levan up
to 30 per cent, based on the sucrose used, have been obtained. Enzyme
preparations give approximately the same yields, and the reaction seems
to follow the same equation as for dextrans, but the polysaccharide and
free sugar are reversed. Thus:

nlCi.HooOn) -^ (C6HioO,)„ + niC^Br^O^)
Sucrose Levan Glucose

By yeast

In the absence of air, ethyl alcohol and carbon dioxide account for about
90 per cent of the sugar fermented, as indicated by the following equation:

CeHioOo -> 2C0H5OH + 2C0o

Small amounts of acetic acid and glycerol are also produced. If the
medium is kept alkaline, pH about 8.5, large quantities of acetic acid
and glycerol are formed. The metabolism of the yeast is shifted so
that a minor product, glycerol, becomes a major product. The equation
for the fermentation may be represented as

2C,;Hi,06 + H2O -^

2CO2 + CHoCOOH + CH,,CHoOH + 2CHoOH • CHOH • CHoOH

This theoretical distribution of products is not realized, since more alcohol
and less glycerol are usually formed.

Glycerol production may also be increased by adding sulfites to the
medium. This fixes the intermediate acetaldehyde as CH3CHOII • 0-
•S02Na and prevents its reduction to ethyl alcohol. A corresponding
amount of another intermediate, dihydroxyacetone phosphate, is con-
verted to glycerol. The sulfite process for production of glycerol was
used by the Germans in World War I. It is still under consideration, but
to date has not been operated successfully. The fermentation equation
may be written as

CeHioOe -^ CH2OH • CHOH • CH2OH + CH3CHO + CO2

Only about one-half of this yield of glycerol is obtained in practical
operations because some of the acetaldehyde escapes fixation and instead
goes to ethyl alcohol.

Under anaerobic conditions the yield of yeast (dry weight) is around
5 per cent of the sugar fermented, about one-tenth as much as is pro-
duced under aerobic conditions.

380 METABOLISM OF MICROORGANISMS

SYSTEMS OF INTERMEDIARY METABOLISM

Aerobic metabolism

Microorganisms that use oxygen have a glycolysis and oxidizing system
such as animals possess and metabolize glucose via pyruvic acid and the
citric acid cycle to carbon dioxide and water. However, another route,
by which glycolysis is side-stepped and possibly also the citric acid cycle,
appears to function in yeast and bacteria, and to some extent in liver.
This route lias been known for a long time but has not received much
attention until recently. It has been called "the hexose monophosphate
shunt," but this is a poor name since the route appears to be more than
a detour around glycolysis. It is a direct oxidation of glucose in which
a number of entirely new compounds appear as intermediates in tiie
following sequence:

Glucose ■ -^ glucose-G-PO* • > gluconic acid-G-POi

(4)
-^ 2-ketogluconic acid-6-P04 ^ ribulose-5-P04

(6)

C. compound (X 2) *■ tetiose|

(tn

(7)

j^l " >^-^ sedoheptulose-7-P04

fiose-3-P04 *- '

(9)

(glucose-6-P04 *■ recycled

(8)

+ triose-3-P04 '^ 1 ^ , _,^ (lO)^ „ , . o T,n _l rn

( tetrose-4-P04 *" ? triose-S-PO^ + COj

The nature of the Co compound in step 5 is still unknown; it does not
appear to be a glycolic aldehyde. The ketopentose, ribulose-5-P04, is
in equilibrium with the aldopentose, ribose-5-P04, but the predominant
form seems to be the ketose. The occurrence of a C7 sugar, sedoheptulose,
in the metabolism of a Cq sugar is an unexpected and noteworthy phenom-
enon. The disposition of the tetrose-4-P04 (step 10) is still uncertain,
but it appears to go to a triose-phosphate and a one carbon compound,
which may be carbon dioxide. All details of the direct oxidation pathway
have not yet been worked out, but the main outlines of the route are
evident.

Since some cells are equipped with both the glycolysis-citric acid cycle
mechanism and the direct oxidation system, the question naturally arises
as to the relative importance of the two systems. Investigators are
cautious about expressing an opinion, because sufficient data are not
yet available for answering this question. However, judging from the

METABOLISM OF MICROORGANISMS

381

number of papers that are appearing, some information on this problem
should soon be forthcoming.

A second type of direct oxidation that does not involve phosphoryla-
tion operates in the metabolism of glucose by certain aerobic bacteria,
e.g., Pseudomonas aeruginosa. Oxidation of glucose leads to gluconic
acid, 2-ketogluconic acid, pyruvic acid, and the formation of large amounts
of a-ketoglutaric acid. Yields of this keto acid up to 0.55 mole per
mole of glucose have been obtained. This provides a convenient method
for the preparation of a-ketoglutaric acid.

ANAEROBIC METABOLISM

Yeast

Hexose diphosphate and pyruvic acid, so prominent as intermediary
products in animal metabolism, were first observed in yeast. The steps
in glycolysis are the same as far as pyruvic acid in both animal and
yeast metabolism. In yeast fermentation the whole process is anaerobic;
the pyruvic acid is decarboxylated to acetaldehyde, and this is then
reduced to ethyl alcohol. The hydrogen necessary for the last step
comes from the dehydrogenation (oxidation) of phosphoglyceraldehyde
to phosphoglyceric acid via DPN • H2 as carrier. If this reduction is
blocked, for example, by fixing the acetaldehyde with sulfite, the hydrogen
is used to reduce dihydroxyacetone to glycerol. Glycerol production
always occurs to a slight extent (3-5 per cent of the glucose), but with
the main route of fermentation blocked, the yeast makes the side line
a main route. The alternative pathway is a very neat and convenient
device for continuing metabolism under adverse conditions.

If alcoholic fermentation is studied with labeled glucose, it is found
that carbon 1 appears in the methyl group, 2 in the carbinol group of
ethyl alcohol, and carbon 3 in the carbon dioxide. This accords with
the Embden-Meyerhof scheme of intermediary metabolism. (See Chap.
13.)

Bacteria

The so-called ''mixed" lactic fermentation shows some unexpected
departures from the alcoholic fermentation of yeast. Carbon 1 of glucose
appears in the carbon dioxide. Carbons 2 and 3 are found in the methyl
and carbinol (or carboxyl) groups, respectively, of ethyl alcohol (or
acetic acid). Carbon 4 comes out in the carboxyl group of the lactic
acid. The two halves of the glucose molecule are metabolized differently.
Various intermediary compounds have been found. The first series of

382 METABOLISM OF MICROORGANISMS

compounds appears to be the same as in the oxidative pathway of aerobic
organisms :

Glucose *■ glucose-6-phosphate *• gluconic acid-6-phosphate

(CO2

2-ketogluconic acid-6-phosphate

I ribulose-5-phosphate

{C2 compound *■ ethyl alcohol or acetic acid
triose-3-phosphate *" lactic acid (+ H3PO 4)

The fermentation of glucose becomes a pentose fermentation after the
loss of carbon 1. Tlie latter part of this formulation fits well for the
fermentation of pentoses by the same bacteria. A main difference be-
tween the two is that ethyl alcohol is a major product in the fermentation
of glucose; it is absent from that of pentose. On the other hand, acetic
acid is usually a minor product from glucose and a major product from
pentoses. The explanation for these differences probably lies in the
accumulation of a reduced coenzyme, perhaps DPN • Ho, in the glucose
fermentation. The accumulated coenzyme may then be used for the
reduction of the C2 intermediate to give ethyl alcohol. In the fermenta-
tion of the pentoses there is no such accumulation of reduced coenzyme.

The discussion just given applies to the aldohexoses (glucose, galactose,
and mannose) but does not fit the fermentation of fructose. Fructose
gives approximately 1 mole each of carbon dioxide, acetic acid, and lactic
acid, no ethyl alcohol, and 2 moles of mannitol. IMannitol is evidently
formed by reduction of other molecules of fructose and appears to be
paired with acetic acid as a reduction product. The interrelations of the
two may be seen from the following equations:

CgHisOe + H2O -> COo + CH3COOH + CH3CHOHCOOH + 4H
2C6H10O6 + 4H -^ 2C6H14O6
Fructose Mannitol

Usually less than 2 moles of mannitol are obtained per 3 moles of fruc-
tose fermented.

The reasons for the differences between fermentation of glucose and
fructose will probably appear when labeled fructose can be used and
data are obtained regarding the intermediary compounds that are formed.
To date, there are no such data.

The mechanisms of other fermentations have been studied extensively,
but space in this chapter does not permit a detailed discussion of the
various steps leading from substrate to product. Only a few short state-
ments and overall equations will be given.

Propionic acid appears to be either a reduction product of lactic acid

METABOLISM OF MICROORGANISMS

383

or, more probably, a decarboxylation product of succinic acid. The latter
may be formed by uptake of carbon dioxide with pyruvic to form oxal-
acetic acid, and subsequent reduction of this to succinic acid.

CO2 + CH3-C0-C00H > HOOC-CH.CO-COOH >

HOOC-CHo-CH,COOH + H2O

The uptake of carbon dioxide by propionic acid bacteria was first demon-
strated by AVood and Werkman, and tliis mechanism is therefore desig-
nated as the Wood- Werkman reaction.

Succinic acid is also assumed to be formed by oxidative condensation
of two molecules of acetic acid. The overall equation is

HOOC CH3 + CH3COOH -^ HOOC CHoCHoCOOH + 2H

The two hydrogens are not released as gas but are used for the reduction of
other compounds.

Acetoin (acetylmethylcarbinol) is formed in both yeast and bacterial
fermentations. In yeast it is formed by condensation of pyruvic acid
and acetaldehyde, and in bacteria by condensation of two moles of
pyruvic acid. a-Acetolactic acid is an intermediate in each case. Equa-
tions for the formation by yeast may be expressed as follows:

CH3CO • COOH + CH3CHO -> CHo • COH • COOH

I-

CO • CH3
CH3CO • COH • CH3 -^ CH3 • CO • CHOH • CH3 + COo

COOH

In the biosynthesis by bacteria the first equation involves both con-
densation and decarboxylation.

2CH3CO • COOH -> CH3 • COH • COOH + CO.

CO • CH3

In the aerogenes fermentation, acetoin is reduced to give a major
product, 2,3-butylene glycol.

CH3 • CO • CHOH • CH3 -f 2H -» CH3 • CHOH • CHOH • CH3

In the colon-aerogenes fermentation, formic acid and hydrogen are
conspicuous products. These come from the hydrolytic cleavage of
pyruvic acid

CH3CO • COOH + HoO^ CH3COOH + HCOOH

and subsequent breakdown of formic acid by the enzyme, hydrogenlyase.

HCOOH -> H., + CO.,

384 METABOLISM OF MICROORGANISMS

Butyric acid, butyl alcohol, and acetone all appear to arise from con-
densation of acetic acid by way of acetoacetic acid.

CH.3 • COOH + CH3 • COOH -^ CH3 • CO • CHo • COOH + HoO
CH3 • CO • CHo • COOH + 4H ^ CH3 • CHo • CHo • COOH + HoO
CH3 • CHo • CHo • COOH + 4H -> CH3 • CHo • CHo • CH2OH
CH3 • CO • CHo • COOH -^ CH3 • CO • CH3 + COo

The hydrogen for these reductions probably comes from the oxidative
steps in the glycolysis scheme that lead to pyruvic acid. There is also
hydrogen available from the breakdown of pyruvic acid to acetyl phos-
phate and carbon dioxide.

CH3 • CO • COOH + H3PO4 -> CH3 • CO • 0 • PO3H2 + CO2 + Ha

Methane arises, in part at least, by reduction of carbon dioxide with
hydrogen :

CO2 + 4H2 -^ CH4 + 2HoO

A puzzling situation exists in respect to conversion of acetic acid to
methane. Isotope studies show that the methyl carbon, represented by
(*), is found in the methane, and the carboxyl carbon, indicated by
(**), appears in the carbon dioxide; hence methane, in this case,
does not arise by reduction of carbon dioxide by hydrogen. The direct

C*H3C**00H -^ C*H4 + C**02

decarboxylation as shown in the above equation is probably not the
actual mechanism.

REVIEW QUESTIONS ON METABOLISM OF MICROORGANISMS

1. Name some present-day foods that are the result of fermentation processes.
Name the class of microorganisms involved in each case.

2. Define the terms "baker's yeast," "press yeast," "active dry yeast." What is
the scientific name for these types of yeast? Are any other species of yeast pro-
duced commercially? (Consult Chemical Abstracts for references to some of these
questions.)

3. Give the major and minor products in alcoholic fermentation of glucose. How
can the ratio of the products be varied?

4. Make a table of all the microorganisms listed in this chapter, the products
formed by them from sugar, and, where given, the percentage of sugar recovered
as product.

5. Compare the fermentation of glucose by yeast with the metabolism of glucose
in the animal body. Give similarities and differences.

6. What determines whether yeast or bacteria will develop in a food that is not
sterilized? For example, fmit juices undergo an alcoholic fermentation, whereas
milk becomes sour. Cabbage when shredded and packed into vats undergoes a
mixed lactic acid fermentation. (Consult a book on bacteriology.)

7. What products, if any, would you expect to be formed by A. suhoxydans
from (1) glycerol, (2) dulcitol, (3) sorbitol, (4) 2,3-butylene glycol, (5) gluco.«e?

METABOLISM OF MICROORGANISMS

385

8. Name the direct precursor of each of tlie following intermediates in glucose
fermentation by yeast: (1) fructose 1,6-diphosphate, (2) 3-phosphoglyceric acid, (3)
carbon dioxide, (4) fructose-6-phosphate.

9. What products would you expect from the fermentation of 5-ketogluconic
acid by a lactic organism?

10. If carbons 1 and 2 in glucose are labeled, where would you expect them to
appear in the products from (1) a yeast fermentation, (2) a heterolactic fermenta-
tion? Explain answer.

11. In the production of baker's yeast, sugar is added slowly to the medium during
fermentation, while in production of alcohol by yeast it is all added at the beginning.
Why the difference in procedure?

12. Write out structural formulas for all the assumed intermediates in the fer-
mentation of glucose by heterolactic bacteria.

13. Compile a list of references to original papers dealing with the direct oxida-
tion (hexose monophosphate shunt) of glucose by microbial and animal cells.

14. Name some of the chemical and physiological characteristics of bacterial poly-
saccharides.

15. Compile a table of all the commercial antibiotics, giving the name, formula,
and microorganism producing each and two infectious organisms against which it
acts.

REFERENCES AND SUGGESTED READINGS

Annual Review of Biochemistry, Vol. 11 to date, Annual Reviews Inc., Stanford,

California.
Animal Review of Microbiology, Vol. 1 to date, Annual Reviews Inc., Stanford,

California.
Baron, A. E., Handbook of Antibiotics, Reinhold Publishing Corp., New York, 1950.
Evans, T. H. and Hibbert, H., "Bacterial Polysaccharides," Advances in Carbohydrate

Chemistry, 2, 204 (1946).
Gale, E. F., The Chemical Activities of Bacteria, 2nd ed.. Academic Press Inc., New

York, 1951.
McElroy, W. D. and Glass, B., Phosphorus Metabolism, The Johns Hopkins Press,

Baltimore, 1951.
Park, J. T., "Isolation and Structure of the Uridine-5'-Pyrophosphate Derivatives

which Accumulate in Staphylococcus aureus when Grown in the Presence of

Penicillin," Symposium on Mode of Action of Antibiotics, Second International

Congress of Biochemistiy, Paris, 1952.
Pratt, R. and Dufrenoy, J., Antibiotics, J. B. Lippincott and Company, Philadelphia,

1949.
Prescott, S. C. and Dunn, C. G., Industrial Microbiology, 2nd ed., McGraw-Hill Book

Company, Inc., New York, 1949.
Raistrick, H., "A Region of Biosynthesis," Proc. Roy. Soc, A, London, 199, 141

(1949).
Schatz, A., Bugie, E., and Waksman, S. A., "Streptomycin, a Substance Exhibiting

Antibiotic Activity Against Gram-positive and Gram-negative Bacteria," Proc.

Soc. Exptl. Biol. Med., 55, 66 (1944).
Stephenson, M., Bacterial Metabolism, 3rd ed., Longmans, Green and Company,

New York, 1949.
Stephens, C. R., Conover, L, H., Hochstein, F. A., Regna, P. P., Pilgrim, F. J.,

386 METABOLISM OF MICROORGANISMS

Brunings, K. T., and Woodward, R. B., "Tenamycin VIII. Structure of Aureo-

mycin and Terramycin," ,/. Am. Chem. Soc, 74, 4976 (1952).
Umbreit, W. W., "The Mode of Action of Streptomycin," Sijmposium on Mode of

Action of Antibiotics, Second International Congress of Biochemistry, Paris, 1952.
Underkofler, L. A. and Hickey, R. J., Industrial Fermentations, Chemical Publishing

Company, Inc., New York. In press.
Werkman, C. H. and Wilson, P. W., Bacterial Physiology, Academic Press Inc., New

York, 1951.

;

Chapter 15

PLANT METABOLISM

by PROFESSOR R. H. BVRRIS

Department of Biochemistry, University of Wisconsin

PHOTOSYNTHESIS

Man's existence on this planet is directly dependent upon plants, for
they furnish him nearly all his food, fuel, and fiber. About a billion
tons of organic matter are oxidized on the earth each day. It is obvious
that such oxidation would exhaust the world's supply of organic matter
in a few years if there were no process to balance the oxidation. Photo-
synthesis provides such a balance by reducing carbon dioxide, and the
energy for the reduction comes from sunlight. The earth intercepts only
one-half billionth part of the energy dissipated by the sun, and by photo-
synthesis only a fraction of one per cent of the radiant energy reaching
the earth is stored as chemical energy, but this is sufficient to maintain
a balance in the carbon cycle of nature (Fig. 15-1). One is forced to
the conclusion that, from the standpoint of man's welfare, no chemical
reactions surpass the photosynthetic reactions in importance.

Respiration,
burning

Photosynthesis

Animals
Respiration

CO,

Green plants

Microorganisms

Decay

XPhotoreduction,
chemautotrophic
and heterotrophic
fixation of COj

Fossil fuels,
wood, etc.

Burning

CO2

CO2

Fig. 15-1. Diagram of the carbon cycle in nature.

The chief reaction for fixing atmospheric carbon dioxide is photosyn-
thesis, and carbon dioxide is released to the atmosphere mainly through

387

388 PLANT METABOLISM

the action of microorganisms. In addition to tlie reactions shown, carbon
dioxide may be added to the cycle from volcanoes and hot springs, and
removed from the cycle as carbonates.

In addition to reducing carbon dioxide, photosynthesis releases oxygen,
and this oxygen is used by animals and plants for their energy-releasing
respiratory processes. This oxidation destroys the substances produced
in photosynthesis, but the energy made available in this way is essential
for maintenance of animal and plant life, as is discussed in Chap. 16.
Life is dynamic, having both its anabolic and catabolic phases, but only
the plants are capable of the net synthesis essential for keeping the overall
life processes in balance.

The simplest formulation of the photosynthetic reaction is:

n(CO0 + 2n(H,0) ,,,i,lte"ne.gy * (CH2O), + n(0,) + n(H,0) + n(119.6) Cal.

Carbon dioxide is reduced to an end product with the empirical formula
of a carbohydrate (CH20)„. The energy stored in this reduced com-
pound is supplied the plant as radiant energy. The oxygen evolved all
originates from water, so it is necessary to designate 2 molecules of water
on the left side of the equation.

Photosynthesis can be described as a sensitized, photochemical, oxida-
tion-reduction reaction. The sensitizer is chlorophyll, for it captures
light and functions in the transformation of radiant into chemical energy.
The reaction is in part a photochemical one, for light energy is required
to drive it. It is an oxidation-reduction with carbon dioxide serving as
the oxidant and water as the reductant. The carbon dioxide is reduced
to the level of carbohydrate, that is, to substances containing as many
oxygen atoms as carbons and twice this number of hydrogens. This
description applies only to monosaccharides; polysaccharides have the
same reduction level, but water has been removed from them.

Chlorophylls

The green pigment chlorophyll serves as the photosensitizer in the
photosynthetic reactions. The first requirement in the utilization of light
is that the light be absorbed, and chlorophyll accomplishes this. From
the absorption spectrum for chlorophyll a, shown in Fig. 15-2, it is evi-
dent that the pigment absorbs strongly in the blue and red regions of
the spectrum and transmits much of the green portion of white light.

Chlorophyll in the cells of higher plants is concentrated in chloroplasts.
The chloroplasts usually are discs about 5 microns in diameter, and within
them the chlorophyll is further concentrated in minute bodies called
grana. Although lacking chloroplasts, the blue-green algae do contain
grana, which are distributed throughout the cells.

The chlorophyll molecule apparently does not occur free in the cell
but is bound to proteins and lipides. It is suggested that aqueous organic

PLANT METABOLISM

389

solvents, such as methanol plus water, effectively extract chlorophyll
from plants because the water breaks the linkage of the protein, and
the organic solvent, the linkage of the lipide to the chlorophyll molecule.

40U ^ 500 600

Blue Wave Length, millimicrons Red

From Zscheile and Comar (1941).

Fig. 15-2. Extinction curve for chlorophyll a in ethyl ether.

The exact nature of the native chlorophyll complex is unknown, but
apparently this lipoprotein has a molecular weight of 19,000 and contains
two molecules of chlorophyll, about 40 per cent lipide and 8.5 per cent
nitrogen.

CH=CH2 CHj
I H I
,C. .C. C..

HaC-C^) I

.c-

H3C— C.(s) IV
H

" (4)C-CH2CH3

N N=^C
\ .' \

■N N — a

III (5)C— CHj

CII c c

I

c=o

H.,C HC

"I I

H.C C=0

H3oC,o-0-C-=0 OCHj

Fig. 15-3. Chlorophyll a.

390 PLANT METABOLISM

The green pigment from higher plants can be separated into chlorophyll
a and chlorophyll h by repeated distribution between immiscible solvents
or by adsorption chromatography. The first method was used by AVill-
statter in his classic investigations of the chemistry of the chlorophylls.
Tswett's separation of plant pigments in 1906 introduced the useful
tool of adsorption chromatography ^ to chemists. Chlorophylls other than
a and h have been isolated from diatoms and algae, but their structures
have not been determined in detail. In addition, bacteriochlorophyll
has been isolated from purple sulfur bacteria and characterized. It differs
from chlorophyll a (Fig. 15-3) only in having an acetyl group (— COCH3)
at position 2 and extra hydrogens at 3 and 4.

When seeds are germinated in the dark, the plants produced are termed
etiolated plants, being devoid of chlorophyll. However, some proto-
chlorophyll is present in etiolated plants, and when they are illuminated
the protochlorophyll is rapidly converted to chlorophyll a by a process
of reduction. It is suggested that chorophyll h then arises from chloro-
phyll a. Most plants have about 3 times as much chlorophyll a as h,
and the sum of these chlorophylls usually constitutes 0.7 to 1.3 per cent
of the dry weight of leaves.

In addition to chlorophylls, the chloroplasts contain other pigments
that may function indirectly in photosynthesis. Some of the light that
they absorb can be utilized, but there has been no demonstration that
they can function photosynthetically in the complete absence of chloro-
phylls.

The determination of the structure of chlorophyll by Willstatter, Stoll,
Conant, Hans Fischer, and their co-workers constitutes a brilliant con-
tribution to organic chemistry. The empirical formula for chlorophyll
a is C55H7205N4Mg, and for chlorophyll 5 is C55H7o06N4Mg. The cur-
rently accepted structural formula for chlorophyll a is shown in Fig. 15-3.
There remains some question whether the semi-isolated double bond -
occurs in pyrrole ring II or is in ring III; if it were in ring III, the Mg
would be bound between rings I and II rather than between I and III
as shown.

There are a number of characteristics of the chlorophyll molecule
which should be noted. First, much of the stability of the molecule can
be attributed to the system of conjugated double bonds,^ designated
by the bold lines in the outline formula of Fig. 15-4. Second, the

1 The procedure for adsorption chromatography is briefly as follows : a finely
powdered adsorbent such as magnesium oxide is padded in a glass tube to form a
column, and a solution of the mixture to be separated is poured on the top. When
a suitable solvent is passed through the column, the individual components of the
mixture move at different rates and separate into distinct bands.

= This is the double bond that is not a part of the conjugated system, shown between
carbons 3 and 4 in Fig. 15-3.

^ Alternate single and double bonds are said to be conjugated.

PLANT METABOLISM

391

molecule contains 4 pyrrole rings (Fig. 15-5) labeled I, II, HI and
IV. Third, the molecule contains an atom of ]\Ig held by primary bonds
to two nitrogen atoms of the pyrrole nuclei and by secondary bonds to
the other nitrogen atoms. Fourth, the pyrrole rings are connected by

Fig. 15-4. System of conjugated double bonds
in chlorophyll shown by bold lines.

HC-

-CH

^W

I
H

Fig. 15-5. Pyrrole.

methene bridges (— CH=). Fifth, pyrrole ring I carries a vinyl group
(— CH^CHo). Sixth, attached to pyrrole ring III is a homocylic ring
bearing a carbonyl group and a carboxyl esterified with methanol. Sev-
enth, pyrrole ring IV carries a propionic acid side chain esterified with
phytol; phytol is a higher alcohol (C20II39OH) with a double bond be-
tween its a and (3 carbons. The phytyl group gives chlorophyll its solu-
bility in fat solvents. Eighth, chlorophyll is optically active because
of the presence of asymmetric carbon atoms. In chlorophyll b the — CH3
of pyrrole ring II is replaced by — CHO. Protochlorophyll is like chloro-
phyll a but is dehydrogenated at carbon atoms 7 and 8.

Primary photochemiral reaction

Photosynthesis consists, not of a single reaction, but of a sequence of
reactions. Much interest attaches to the first reaction involving light,
or the primary photochemical reaction. Photosynthesis was described
as a sensitized, photochemical oxidation-reduction, and water was desig-
nated as the reductant. Water is not normally an effective reducing
agent ; somehow, energy must be added to it to make it effective. Much
evidence suggests that the primary photochemical reaction is a photolysis
of water, a reaction in which light energy is used for the splitting of water

392 PLANT METABOLISM

with the release of hydrogen to some hydrogen acceptor, which can func-
tion in subsequent reductions. This active hydrogen may be passed from
one acceptor to another before it is finally used for the reduction of
carbon dioxide.

Release of oxygen

The reaction releasing oxygen is dependent upon, but apparently not
identical with, the primary photochemical reaction. If the photolysis
of water is considered as an oxidation-reduction reaction, the hydrogen
acceptor mentioned is reduced, and some other acceptor of the hydroxyl
radical remaining is oxidized. This oxidized product releases O2, and
it may be assumed that an enzymatic mechanism is concerned in this
release.

It has been implied throughout the discussion that all the oxygen
formed in photosynthesis comes from water, but this fact has only been
accepted in recent years. Early workers suggested that it came from
carbon dioxide, or part from carbon dioxide and part from water. The
work of van Niel with the photosynthetic bacteria . suggested that, if
bacterial photoreduction were analogous to the photosynthesis of higher
plants, water should supply all the oxygen released in photosynthesis.
This hypothesis was verified experimentally with the stable isotopic
tracer O^^, when it was shown that the O^^ level of photosynthetic O2
corresponded exactly with the 0^^ level of HoO^*, in which photosynthe-
sizing algae were suspended.

Carbon dioxide

Normal air contains 0.03 per cent carbon dioxide, and upon this plants
must depend for their source of carbon. Air is taken in through the
stomatal openings on the leaf and is dissolved in the leaf sap. Carbonic
anhydrase is an enzyme in the leaves which speeds the formation of car-
bonic acid from the carbon dioxide and water. There is no clear experi-
mental answer to the question whether carbon dioxide is used in photo-
synthesis as the dissolved gas or as the carbonate and bicarbonate
ions.

Evidently the carbon dioxide entering a leaf is bound in a loose com-
plex on some large molecules, a binding which may be compared roughly
to the binding of oxygen by hemoglobin. Many lines of evidence indicate
that the carbon dioxide is utilized next in a carboxylation reaction. A
general formulation of such a reaction is:

RCHO + CO2 -» RCCOOH

II
0

PLANT METABOLISM 393

A carboxylation involves no reduction and requires relatively little energy.
JNIuch energy is needed for reduction of the carboxylated compound to
the level of a carbohydrate, a reaction which can be pictured as:

RCCOOH + 2H -^ RCHOHCOOH

II
0

RCHOHCOOH + 2H -^ RCHOHCHO + HoO

It is evident that 4 hydrogens are required to reduce one carbon dioxide
molecule to the oxidation-reduction level of carbohydrate and that a
molecule of water is produced.

Shortly after the turn of the twentieth century, it was realized that
photosynthesis could be divided into distinct light and dark reactions,
i.e., reactions which do, or do not, require light in order to proceed.
There is no evidence that the initial fixation of carbon dioxide, and its
subsequent reduction, requires light directly. In fact, all evidence shows
that the reactions of carbon dioxide in photosynthesis are dark reactions.
Some of the most convincing experiments are based upon the use of
specific inhibitors at varying light intensities. If an inhibitor docs not
decrease the rate of photosynthesis at low light intensities, but does at
high intensities- (where the dark reactions are the ones which limit the
rate of the overall process) , it is an inhibitor for a dark reaction. Cyanide,
for example, acts in this fashion, and since it inhibits carboxylation, the
conclusion is drawn that carboxylation does not require light.

The rate of photosynthesis is dependent on the partial pressure of
carbon dioxide, and though it is difficult to generalize for many plants
at different light intensities, it may be said that raising the concentra-
tion of carbon dioxide from its usual level of 0.03 per cent to 0.1 per
cent will usually double the rate of photosynthesis. Most plants are
not inhibited by carbon dioxide up to 10 per cent, but their growth is
reduced by concentrations greater than this.

Partial reactions

As pointed out, photosynthesis is a complex of reactions rather than
a single reactit)n. The study of such an involved process is obviously
much easier if it can be first separated into its individual steps. One
of the most notable achievements of recent years has been the recon-
struction of partial reactions in cell-free preparations; that is, certain
ground-up plant tissues have been found to carry out parts of the photo-
synthetic process. This has permitted the study of individual reactions
or reaction series in a system much simpler than that encountered in
intact cells.

In 1937, Hill found tliat a suspension of chloroplasts plus leaf extract

394 PLANT METABOLISM

would liberate small amounts of oxygen upon illumination. Substitution
of potassium ferric oxalate solution for the leaf extract increased the
rate of oxygen evolution, and later workers have further improved the
system by substituing p-benzoquinone. The potassium ferric oxalate or
p-benzoquinone serves in place of carbon dioxide, which is the oxidant
in normal photosynthesis. The Hill reaction with p-benzoquinone is:

O OH

II I

9

II +2H.0 ,, "„^,, > 2 I II +0.-52Cal.

chlorophist
suspension

HCv. ^CH suspension HC^vv^ ^CH

O OH

Benzoquinone Hydroquinone

Oxygen is liberated, the energy of light is used in the reduction of the
quinone, and energy is stored in the hydroquinone formed. The Hill
reaction should more properly be called the Hill reactions, for both
photochemical and dark reactions apparently are involved. This isolated
system again emphasizes the close relationship of the photochemical and
oxygen-liberating reactions of photosynthesis.

Success in carrying out light-sensitive partial reactions concerned in
the reduction of carbon dioxide without the use of intact cells was not
achieved until 1951, when Vishniac and Ochoa reported that light energy
could be used in the reductive carboxylation of pyruvic acid to malic
acid (reaction 15, Fig. 13-4). Grana from spinach leaves used the
energy captured from light for the reduction of TPN. The reduced TPN,
in the presence of the proper enzymes, then caused the reductive car-
boxylation of pyruvic to malic acid. In a similar manner DPN, reduced
in the light, could effect the reduction of pyruvic to lactic acid (reaction
13, Fig. 13-3). The overall reactions did not occur in the dark, but it
should be noted that the reactions directly involving the carbon dioxide
were dark reactions. Illuminated chloroplasts, or grana, which we al-
ready have seen can liberate Oo, also can transfer the hydrogen pro-
duced in the photolysis of water to oxidized DPN or TPN. The co-
enzymes in turn can reduce pyruvate to lactate or function in the reductive
carboxylation of pyruvate to malate. These observations have been con-
firmed by Tolmach, and Arnon has reconstructed a system for the light-
dependent reductive carboxylation of pyruvate to malate, in which all the
parts (except TPN) were of plant origin.

It is justifiable to say that the Hill reaction represents a true partial
reaction of photosynthesis, but there is some question whether or not
this can be said about the reactions described by Vishniac and Ochoa.

PLANT METABOLISM 395

The important consideration is that the reactions involve the capture of
radiant energy by chlorophyll and use of the energy in the reduction
of coenzymes, which can serve in further reductions. These should be
viewed as model reactions useful in the study of portions of the photo-
synthetic process rather than as proved partial reactions of photosynthesis
as they actually take place in normal plants.

Storage of energy

The formulation of the photosynthetic reaction (p. 388) indicated that
119.6 Cal. were stored for each mole of carbon dioxide reduced to the
level of carbohydrate; this is equivalent to 717.6 Cal. per mole of glu-
cose formed. These values are in terms of change in free energy (aF,
p. 414), when glucose, at molar concentration, is formed in aqueous solu-
tion at 25°C. from liquid water and carbon dioxide at a concentration
of 0.03 per cent. That the value for AF differs from that given for
glucose oxidation in Chap. 16 (683 Cal.) can be attributed to the differ-
ence in concentration of reactants and products in photosynthesis and
in animal respiration.

The energy stored in photosynthetic products represents radiant energy
which has been captured and converted to chemical energy. Light
energy is absorbed or radiated in discrete units, or quanta, which are
equal to hv, when h is Planck's constant (6.547 X 10~-^ ergs per sec),
and V is the frequency [frequency is calculated by dividing the wave-
length of light (cm.) into the velocity of light, 3 X 10^*^ cm. per sec.].
The total amount of energy in 6.06 X 10^^ quanta is called one Einstein.
It is evident that the higher the frequency (shorter the wavelength),
the greater is the energy of each quantum of light. Einstein's theory
of photochemical equivalence indicates that a photochemical reaction
is induced when one molecule absorbs on© quantum of light of charac-
teristic frequency. The question of the quantum efficiency of photosyn-
thesis may be stated as follows: How many quanta are required for the
reduction of one molecule of carbon dioxide to the level of carbohydrate?

In 1922 Warburg and Negelein determined the quantum efficiency of
photosynthesis by the alga Chlorella in light of various frequencies.
They found that 4 to 5 quanta were required for each molecule of carbon
dioxide. The value of 4 quanta was readily accepted, because it was
attractive to think that one quantum was required to activate each of
the 4 hydrogens necessary to reduce carbon dioxide to the carbohydrate
level. Some 16 years later. Manning, Stauffer, Duggar, and Daniels
employed a variety of methods but were unable to reproduce the high
efficiency reported by Warburg and Negelein. Several other investigators
likewise were unsuccessful and found that 8 to 12 quanta were required
rather than 4. Recently Warburg has repeated his earlier experiments

396 PLANT METABOLISM

and has obtained essentially the same results as before. Warburg and
Burk have consistently observed quantum efficiencies of about 4 with
several modified techniques.

In red light with 43 Cal. per Einstein, a quantum efficiency of 4 repre-
sents almost 70 per cent efficiency ^ in storing the energy of the incident
light. This is a higher efficiency than that of any known photochemical
reaction which can be performed under controlled conditions. Even a
quantum efficiency of 8 to 12 is good in terms of known photochemical
reactions, and if photosynthesis actually has a quantum efficiency of 4,
it is unique. It is impossible at present to make a categorical state-
ment of the quantum efficiency of photosynthesis, for the matter remains
highly controversial.

Intermediates and products of photosynthesis

One of the most interesting problems of photosynthesis is the determina-
tion of the organic compounds produced by the sequence of reactions.
What substances are formed between the first reduction of carbon dioxide
and the final appearance of the finished sugar molecule? There is an
extensive literature on this subject, much of which has centered around
the formaldehyde hypothesis. In 1870, Baeyer suggested formaldehyde
as the first product of photosynthesis, and though there never has been
substantial evidence to support it, the hypothesis has been perpetuated
in textbooks of biochemistry and botany to the present.

Current interest centers around phosphoglyceric acid as the first demon-
strable product of photosynthesis. Pioneering work from 1939 to 1942
with the short-lived radioisotope C^^ resulted in the introduction of a
number of new concepts and the discard of certain old ideas on photo-
synthetic intermediates. Since 1946, the use of the long-lived (5,568 ± 30
year half life) radioactive isotope C^^ has led to real advances in our
knowledge of intermediates. Although it remains incomplete, a general
picture of the reaction sequence has now emerged. Calvin and co-
workers have made particularly notable contributions. By skillful use
of a combination of C^'*, paper chromatography,^ and "radioautography" ^

1 Calculation : /^^'^^ x 100 = 69.5%.
4 X 43

^ In paper chromatography a small drop of a mixture of compounds in solution is
placed near one end of a long strip of filter paper and is dried. The end near the
point of application is immersed in a suitable solvent. As this solvent moves by
capillary action past the added mixture of compounds and on toward the other end
of the paper, it carries with it the individual components of the mixture varying dis-
tances and thus achieves their separation.

^ "Radioautography" is a procedure for locating radioactive substances separated
by paper chromatography. After the chromatographic separation is completed, the
paper is dried and allowed to "take its own picture" by placing it on a photographic
film in the dark. Black areas on the developed film correspond to the location of
radioactive substances on the paper strip.

PLANT METABOLISM 397

they have unraveled many of the complexities of the early photosynthetic
reactions and have revealed the importance of phosphoglyceric acid as
an intermediate.

Figure 15-6 gives reactions postulated by Calvin and co-workers as
occurring between the initial fixation of carbon dioxide and its final
reduction to sugar. ^ The initial reaction is the carboxylation of the
hypothetical vinyl phosphate (1) to 2-phosphogly eerie aeid (2), which
is then converted to 3-phosphoglyeerie acid (3). In extremely short
periods (5 seconds) of photosynthesis by algal cells in the presence of
C^'^Oo, it has been shown that up to 85 per cent of the C^"' fixed appears
in 2- and 3-phosphoglyccric acids. By reversing the glycolytic scheme
(see Figs. 13-1 and 13-3), 3-phosphoglyceric acid can be converted to
sugar. It first is reduced to 3-phosphoglyceraldehyde (4) , which is in
equilibrium with diliydroxyacetone phosphate (5). Condensation of
these compounds yields fructose-l,6-diphosphate (6) , which in turn yields
fructose-6-phosphate (7) and glucose-6-phosphate (8). Free fructose
and glucose can be obtained from these by the removal of phosphate.
The method by which plants form sucrose is unknown, and the sucrose
phosphate shown is a hypothetical intermediate. However, all the gly-
colytic steps included in Fig. 15-6 have been demonstrated to occur in
plants (preparations from pea seedlings).

The left part of the scheme represents a suggested method for regen-
erating the Co fragment, which must serve as the acceptor of carbon
dioxide when phosphoglyceric acid is formed; this remains the least well-
defined and most controversial portion of the scheme. In the first formu-
lation shown, one molecule of 2-phosphoglyceric acid is used in sugar
formation, while water is removed from the other molecule to yield
2-phosphoenolpyruvic acid (9). Addition of another molecule of carbon
dioxide yields enol-oxalacetic acid (10). This is converted to an unde-
fined C4 acid, w'hicli splits to give 2 molecules of glyoxylic acid (11) ;
upon reduction this would yield glycolic acid (12). Several Co com-
pounds have been suggested between glycolic acid and the hypothetical
vinyl phosphate (1).

An alternative method, postulated by Calvin and co-workers, for form-
ing a triose to serve in the reversed glycolytic mechanism and for regen-
erating the C2 fragments is shown in skeleton form in the lower portion
of Fig. 15-6. A C4 and a C3 compound are condensed to form the C7
sugar, sedoheptulose. This compound is split to yield a Co fragment
which recycles and a C5 keto-sugar, ribidose. The ribulose in turn is
split to a triose and a Co compound, which also recycles. The complete
cycle, involving 3 carboxylations and subsequent reductions, yields a
molecule of triose. Most of the specific intermediates in this reaction
sequence remain to be defined.

^ In Fig. 15-6 "P" indicates either a phosphate grouj) or a molecule of phosphoric acid.

o— o

I

o— o

a
o

o

o

I

w
o-

o-

-o

-o

a

o
w
o

I

W

-o

O-

-o

0=0

I

0—0
I ;

0—0
0—0

0—0

I

0=0

I

o-

-o

o-

-o

0 — 0

W M
0—0

K 04
0—0

a a,
0—0

a
o
o
o

I

a Oh

0—0

a a
0—0

o

a

0

0

a

0

0,

a 1

0

0

0 a<

0—0

II

■* a

0

1

1

0-

II

-5 3

+ 1

a

u

0

0
0

a

a— 0—0

a
o

n

O

M

o

a

o
o

o

a I
0—0

a

+

O

a
o
o
(J

0=0
I
a

o
a
o

I

a
u-

I

a
o-

I

a
o-

I

a
o

a
-o

a
-o,

a"
-o

— o

y y

o

3
to

a
o-

-o

o

1

O 3

O

o

o

Id

^;

O

J

o-

OJ

dJ

;^
O

I

o

o

-d

CI

03

u
>,

OJ
Oi

fct)

3
CO .

=3 9
OJ OJ

Pi

_d

d
o

;-i

03
O

%-)
O

i/i

03

• ^H

CO

cc
O

«5
I

to

be

398

PLANT METABOLISM 399

Carbohydrates. Although information is incomplete on the early prod-
ucts of photosynthesis and their mode of formation, the total materials
of the plant are the net final products of photosynthesis after modifica-
tion by other phases of plant metabolism. The carbohydrates are quanti-
tatively the most important group of compounds, and cellulose, starches,
and sugars are particularly abundant.

Glucose and fructose commonly occur as free and combined sugars in
plants, whereas mannose and galactose are found only in combined form.
Among the disaccharides, sucrose is especially important. Maltose and
cellobiose do not occur free in demonstrable concentrations but arise
from the hydrolysis of starch and cellulose, respectively. Plants also
may yield the disaccharides gentiobiose, trehalose, and melibiose, but
they do not contain lactose. Raffinose, gentianose, and melizitose are
the most common plant trisaccharides, and stachyose is a tetrasaccharide
which has been isolated from lupine seeds.

Particular interest is attached to the method by which the plant syn-
thesizes sucrose because the sugar is important to plants and man. It
has not as yet been synthesized chemically. Sucrose can be formed by
detached leaves when they are infiltrated with glucose, fructose, mannose,
galactose, or glyceraldehyde; the synthesis will not take place in the
absence of oxygen. The only controlled synthesis of sucrose by a cell-
free preparation has been achieved with an enzyme system from the
bacterium, Pseudamonas saccharophila. This organism produces a
sucrose phosphorylase (more properly termed a transglucosidase) , which
by phospliorolysis of sucrose produces fructose plus glucose-1-phosphate.
The equilibrium of the reaction is such that by a reversal of the reaction
a synthesis of about 5 per cent of sucrose from glucose- 1 -phosphate plus
fructose can be achieved. Though higher plants accumulate much sucrose,
there has been no success to date in demonstrating their synthesis of
sucrose by sucrose phosphorylase or by any other isolated enzyme system.

Even less is known concerning the synthesis of cellulose. Although
bacterial preparations have been induced to form cellulose from a number
of precursors, some as simple as Co compounds, there is no detailed in-
formation on the pathway of its formation.

Knowledge of starch synthesis is much more nearly complete. Peat,
Bourne, and co-workers (see Bernfeld for discussion) have shown that
potato tubers and other plant tissues contain a starch phosphorylase
which by phospliorolysis converts starch to glucose-1-phosphate. The
reversal of this reaction to yield starch from glucose-1-phosphate is
readily demonstrable. The first preparations used, synthesized only
amylose, the straight chain starch, but later preparations also yielded
amylopectin, the branched chain starch. The enzyme which synthesizes
amylose from glucose- 1 -phosphate is termed the P-enzyme. A separate
enzyme, termed the Q-enzyme, is needed to form the linkages at the

400

PLANT METABOLISM

branch points in the amylopectin structure. The Q-enzyme, in conjunc-
tion with the P-enzyme, will form amylopectin:

P-enzyme .-,

glucose-1-phosphate < — amylose ■^'""' — > amylopectin

Lipides. Lipides are the chief storage materials in seeds. Much more
energy is stored in a gram of lipide than in a gram of protein or carbo-
hydrate (see p. 423). When oily seeds mature, their fat content builds
up rapidly, chiefly at the expense of carbohydrate. Upon maturation,
the fat increases in unsaturation, and the constituent fatty acids increase
in average chain length. These changes in seeds are reversed when the
seeds germinate. The fact that almost all the fatty acids have an even
number of carbon atoms suggests that they originate from condensation
of C2 units.

NITROGENOUS COMPOUNDS AND THEIR METABOLISM

The animal requires an external supply of several amino acids (or
their keto acid analogues) to survive, but the plant can synthesize its
needed amino acids from inorganic sources of nitrogen, carbon dioxide,
and water. Lack of an adequate supply of nitrogen in the soil, more
frequently than the lack of any other element, limits the growth of
plants. As a result, nitrogenous fertilizers, and crop rotations including
nitrogen-fixing legumes, are of great importance in our agricultural
economy.

Nitrogen nutrition

Nitrate has been used as a fertilizer for centuries. Because it can
be employed in higher concentrations than ammonia, it often has been
considered inherently superior to ammonia for the nutrition of plants.
Actually, when ammonia is supplied in nontoxic concentrations, it nor-
mally supports a rate of growth in plants equal to or superior to that for
nitrate. The conditions for optimum utilization of the two compounds
differ, however; nitrate is used best at a pH below, and ammonia above,
neutrality.

Urea and calcium cyanamide (CaCN2) are widely used organic fer-
tilizers which readily yield ammonia. Manures and composts serve as
sources of nitrogen for plants, and it is generally suggested that their
utilization by the plant is preceded by ammonifi cation and nitrification}
In the process of ammonification, bacteria convert the nitrogen of pro-
teins and other compounds to ammonia. This is followed by the action

^ Nitrification is the process of converting ammonia to nitrites, or nitrites to nitrates,
or botli.

PLANT METABOLISM

401

of the autotrophic ^ nitrifying bacteria which oxidize ammonia to nitrite
{Nitrosomonas and Nitrosococcus) and nitrite to nitrate (Nitrobacter) .
Nitrification is an important process in the soil, but it is unreasonable
to assume that plants can use only nitrate, the end product of the process,
for their nutrition. We have already seen that they can use ammonia
directly, and nitrite also is an excellent source of nitrogen for plants when
it is present in subtoxic concentrations. Even complete ammonification
may be unnecessary before the nitrogen of organic residues is used. In
such material the nitrogen is present chiefly as protein, and upon hydroly-
sis this protein will yield peptides and free amino acids. For plants
grown in the absence of bacteria (to avoid complications from bacterial
action on the nitrogenous compounds) certain single amino acids can
serve adequately as the sole source of nitrogen. Peptone, a mixture of
peptides and free amino acids, also supports good growth. Evidently
plants can use the products at all stages of protein breakdown: peptides,
amino acids, ammonia, nitrites, and nitrates.

Synthesis of amino acids

A plant supplied nitrate or nitrite must reduce these compounds to
the reduction level of ammonia for the synthesis of amino acids. Whether
tlie reduction yields free ammonia or reduced nitrogen bound in a more
complex form has not been established. The best defined amino acid
synthesis from ammonia is the reductive amination of a-ketoglutaric
acid to glutamic acid by way of a-iminoglutaric acid:

COOH COOH COOH

I I I

CO ^ C=NH HcNXH

I + NH3 ^=^ I + R2O ^ ^^^ "^ > I

(CH2)2 (CH,)2 * glut, acid (CH,),

I I clehydrogena-ie I

COOH COOH '^^^ COOH

a-Ketoglutaric acid a-Iminoglutaiic acid Glutamic acid

This reaction sequence has been accomplished with cell-free enzyme
preparations from plants when reduced DPN has been added. Compara-
ble reductive aminations may result in the formation of other amino
acids, although they have not been demonstrated.

Glutamic acid is a key compound in the nitrogen metabolism of animals,
plants, and microorganisms. It is probable that many amino acids in
plants are formed by transamination from glutamic acid to various a-keto
acids. By transamination the amino group of an amino acid is trans-

^ Autotrophic bacteria are those microorganisms which can derive all their energy
from the oxidation of a simple inorganic compound or element (e.g., oxidation of S
to 80^=) and are able to use carbon dioxide as their sole source of carbon.

402

PLANT METABOLISM

ferred to an a-keto acid, and a new a-amino acid and a new a-keto
acid are formed (see Chap. 13, reactions 37 and 38) :

0 NH2 NH, O

II I I II

R-C-COOH + R'-CH-COOH ^izzi R-CH-COOH +R'-C-COOII

Free ammonia does not function in the reaction. Transaminations from
glutamic acid to pyruvic and oxalacetic acids to form alanine and aspartic
acid, respectively, have been demonstrated in plants, and other trans-
aminations occur but at considerably slower rates.

Metabolism of amides by seedlings

Germinating seeds may produce large quantities of the amides, aspara-
gine and glutamine. Asparagine is the yS-amide of aspartic acid (HOOC—
CHNH2 — CHo — CONHo), and glutamine has a comparable structure
(HOOC— CHNH2-(CHo)o— CONH,). When a high-protein seed such
as a lupine seed germinates, there is a demand upon the i^rotein as an
energy yielding material for growth before the seedling establishes its
synthetic capacity. AVhen protein is broken down and respired, ammonia
accumulates; high levels of ammonia would be toxic, but by forming
amides the plant detoxifies it. As much as 85 per cent of the protein
nitrogen disappearing in a germinating lupine seed may appear in the
single compound, asparagine. Later in the growth of the plant the
amide nitrogen is used in the resynthesis of protein, and when the plant
matures, its amide content is very low.

Biological fixation of nitrogen

The nitrogen cycle in nature is pictured in Fig. 15-7, in a form which
is simplified by omitting the marine cycle of nitrogen. Nitrogen is added
to the cycle from the vast reservoir of atmospheric N2 by chemical fixa-
tion,^ by the symbiotic nitrogen fixation of leguminous plants, and by
the nonsymbiotic nitrogen fixation of free-living bacteria and blue-green
algae. Nitrogen is lost to the sea by leaching, erosion, and sewage dis-
posal, and to the atmosphere by bacterial denitrification." Man's in-
stallations for the chemical fixation of No are impressive, but their con-
tribution is minor compared to that of biological nitrogen fixation in
maintaining the nitrogen cycle in balance. Chiefly because of improper

^"Fixation" is the conversion of gaseous nitrogen (N„) into chemically combined
forms such as ammonia, nitric acid, protein, etc.

* Denitrification is the release of free nitrogen (Ng) from nitrogenous compounds,
or the opposite of fixation.

3

PLANT METABOLISM 403

Chemical
N fixation

Soil ^ > Loss of N to sea by

leachuig and erosion

_ , . . / \ Nonsymbiotic

u- , ^"'V xT°r .■ r \ biolosical N fixation
biological N fixation ^ -,. . ^ !! -.j

V, >■ Plants < * iNIicroorganisms » JNj

Denitrification

Animals

Loss of N to sea
as sewage

Fig. 15-7. The nitrogen cycle in nature.

land use and wasteful disposal of sewage, the land area of the earth
yearly increases its nitrogen debt, despite nitrogen fixation by plants
and bacteria.

Agents Capable of Fixing No. Leguminous plants such as clover, al-
falfa, peas, and beans carry nodules on their roots. In these nodules,
No can be fixed into forms usable by the plant. The nodules are caused
by the invasion of the roots by bacteria, the rhizohia, which cause the
root tissue to grow at the site of invasion. Nodules have a characteristic,
well-organized structure, and certain of the plant cells are packed with
bacteria. It is interesting to note that a hemoglobin much like mam-
malian hemoglobin is present in nodules; this is the only reported occur-
rence of hemoglobin in the plant kingdom. Neither the leguminous plant
alone nor the rhizobia alone can fix N2, but in symbiotic association ^
they can. Symbiotic nitrogen fixation is quantitatively the most impor-
tant means on the land surface of the earth for fixing No.

The nonsymbiotic nitrogen fixers, that is, those organisms capable of
fixing No by themselves, include the aerobic Azotobacter, the anaerobic
Clostridium and Desulfovibrio, the photosynthetic bacteria Rhodospiril-
luni, Chromatium, Chlorobacterium, and Rhodomicrobium, and repre-
sentatives from 3 families of blue-green algae. It is very difficult to
appraise the quantitative importance of the No fixation by these organ-

^ Symbiotic association, or si/mhiosls, is a relationship in whicli two different livinj;
organisms exist in close association with each other in such a manner that each
derives benefit fi'om the other's existence.

404

PLANT METABOLISM

isms. Azotobacter and Clostridium are important under normal soil
conditions, and the blue-green algae function in maintaining the nitrogen
supply under the wet conditions encountered in rice paddies. The other
organisms probably function most extensively in aquatic habitats.

Mechanism of N2 Fixation. Although the intermediates arising di-
rectly from the fixation of N2 are unknown, these intermediates are
quickly converted to ammonia before its incorporation into organic com-
pounds. The reduction to ammonia logically should pass through hy-
droxylamine as an intermediate, but formation of amino acids from
hydroxylamine via their oximes does not appear to be an important path-
way of synthesis. Ammonia is the key compound in the sense that it
is the compound which combines with carbon chains to yield organic
nitrogenous substances in the plants or bacteria. The primary pathway
of No fixation involves conversion of No to NH3, through unknown inter-
mediates, and the formation of glutamic acid by reductive amination
of a-ketoglutaric acid. Glutamic acid in turn can form new amino
acids by transferring its amino group by transamination.

The evidence that ammonia is the key intermediate in biological nitro-
gen fixation has been accumulated largely by investigating the fixation
with the aid of the stable isotopic tracer, N'^. These studies, and tests
with specific inhibitors of nitrogen fixation, indicate a unity in the mech-
anism of nitrogen fixation in all the diverse organisms investigated.

RESPIRATION OF PLANTS

Although plants are most notable for their photosynthetic capacities,
they carry on respiration both in the light and in the dark. Their
respiration per unit weight is less intense than that of most animals
because many of the structural materials of the plant such as cellulose
and lignin are metabolically inert. However, when activity is expressed
per unit of nitrogen in the tissues, the respiratory activity of plants and
animals is comparable.

The activity of cytochrome oxidase has been clearly demonstrated in
plants, and cytochrome c has been isolated from plant materials. Plant
cytochrome c and cytochrome oxidase are very similar to these constitu-
ents of the cytochrome system of animal origin. Other oxidases of im-
portance in plants are ascorbic acid oxidase and tyrosinase (catecholase,
dopa oxidase, and laccase are plant enzymes similar to tyrosinase). It
is characteristic of the true oxidases that they use O2,' produce HoO rather
than H0O2, and have a heavy metal component. Both ascorbic acid
oxidase and tyrosinase contain copper, and though cytochrome oxidase
never has been purified, it apparently contains iron. A dominant posi-
tion of the true oxidases characterizes plant respiration, in contrast to

PLANT METABOLISM

405

animal respiration where cytochrome oxidase is the only oxidase assum-
ing an important role.

Peroxidases are widely distributed in plants, but their function is not
well defined. They use H0O2 for the oxidation of a variety of phenols
and aromatic amines. The peroxidase from horse radish has been
crystallized and shown to be a hematin compound. Catalase, which con-
verts hydrogen peroxide to water and Oo, and has peroxidatic activity,
likewise is a hematin enzyme widely distributed in plants. Hemoglobin,
cytochrome c, peroxidase, and catalase each has hematin as its prosthetic
group.

Many organic acids are relatively abundant in plants, and they serve
as substrates for their respective dehydrogenases. Malic, citric, and
oxalic acids are quantitatively the most important plant acids. Among
these, malic acid is a particularly active metabolite; citric acid is inter-
mediate in activity; and oxalic acid is rather inert. Glycolic acid and
lactic acid are rapidly oxidized in plants by glycolic and lactic acid
dehydrogenases.

The formation, interconversion, and oxidation of organic acids in plants
are competing processes which give rise to spectacular changes in the
organic acid levels from day to day. For example, the succulent plants
accumulate high concentrations of organic acids at night and convert
them to starch during the day (isocitric acid, a rare acid, may constitute
18 per cent of the dry weight of the leaves of the succulent plant, Bryo-
phyllum calycinum) . In the dark, a large share of the malic acid of
tobacco leaves is converted to citric acid.

Glycolysis in plants and animals yields pyruvic acid, which may be
reduced to lactic acid or oxidized by aerobic processes. The oxidation
of pyruvate via the Kreb's tricarboxylic acid cycle has been described for
animal tissue in Chap. 13. Although information is much less complete
for plants, there is good evidence that oxidation can occur by the same
pathway outlined for animal tissue. Early work depended upon evidence
that in the presence of inhibitors, such as malonate, intermediates in
the tricarboxylic acid cycle accumulated. Now it has been possible to
show that preparations of washed mitochondria ^ from mung bean and
lupine seedlings can oxidize all the intermediates of the tricarboxylic
acid cycle and can use some of the energy so obtained for converting
ADP to ATP.

^ Mitochondria are small, discrete particles existing within cells which can be
separated from the rest of the cell contents by breaking the cell walls mechanically,
suspending the mixture in a suitable liquid medium (e.g., 0.4.1/ sucrose), and cen-
trifuging. The mitochondria settle out only after a certain centrifugal force has been
reached. Larger particles settle first and are discarded, and smaller particles remain
in suspension. Washing is accomplished by resuspending in fresh liquid and recen-
trifuging.

406

PLANT METABOLISM

PLANT GROWTH SUBSTANCES

The growth of plants is regulated by a variety of chemical entities
included under the broad heading, plant growth substances.^ These com-
pounds by being transported within the plant may effect changes at a
distance from their point of formation. The most studied and versatile,
naturally occurring plant growth substance is 3-indoleacetic acid:

HC

^

HC

^

H

c

^C-

-c-

CH2-

-COOH

^./C\-./^H

c

r^

H

H

3-Indoleacetic acid

H
UC^ ^C

HC

'^

H

C— CHoCN

II
CH

N
H

3-Indoleacetonitrile

This compound, variously known as p-indoleacetic acid (lAA) and hetero-
auxin, and the structurally similar 3-indoleacetonitrile, have been isolated
directly from plants, as has traumatic acid (HOOC — CH=CH — (CHo)s-
— COOH), but evidence for most of the other growth substances reported
to occur in plants lacks substantial chemical verification. Indoleacetic
acid at a concentration of 0.01 mg. per liter will cause oat coleoptiles - to
bend and will increase streaming of their prott)plasm; 2.0 mg, per liter
will inhibit the growth of oat roots by 50 per cent and will stimulate
the respiration of oat seedlings; higher concentrations will inhibit both
respiration and stem growth.

In addition to indoleacetic acid, many chemically synthesized substances
will influence the growth of plants. For a compound to be effective it
must as a rule contain an unsaturated ring and a side chain on the ring
carrying a carboxyl group, or a group readily converted to a carboxyl;
the carboxyl must be at least one carbon atom removed from the ring
and must have a proper spatial relationship with the ring.

To the activity of growth substances are attributed such responses of
plants as phototropism (bending toward light) , geotropism (bending of
roots toward and stems away from gravity), bending resulting from in-
jury (traumatic acid is active), initiation of flowering, and epinasty
(downward bending of leaves without wilting) . Plant growth substances
have been used in a large number of practical applications. They are
employed to prevent premature dropping of blossoms and fruits. The

^ "Plant growth substances" is a broad term covering the compounds often referred
to as plant hormones, phijtohoymones, groirth regulating suhatances, and auxins.
^ The sheath around the first leaf sent out by the germinating oat seed.

PLANT METABOLISM 407

methyl ester of naphthalene acetic acid and tnaleic hydrazide will keep
stored potatoes and carrots from sprouting.

H H

C C

HC^ ^C^ "^C-CH.-COOCHs CH-CONH

HC^ ^C\ ^CH CH-CONH

C C

H H

Naphthalene acetic acid, Maleic hydrazide

methyl ester

Growth substances will hasten the rooting of cuttings (Fig. 15-8) and
will inhibit the development of buds. By application of growth substances
to flowers, seedless fruits can be produced.

The most important use of plant growth substances is as herbicides, or
weed killers, and this application has greatly modified current agricultural
practice. General herbicides such as oils or acids have been employed
for many years, but the plant growth substances have the great advan-
tage of acting as selective herbicides. For example, much lower con-
centrations of 2, 4:-dichlorophenoxy acetic acid ("2,4-D") are required to

0-CHo-COOH 0-CH,COOH

I " I

c c

HQ^'''"^,CC1 HC^ ^CCl

I II I II

HCW4)/CH CIC^ XH

C X

CI CI

"2,4-D'' "2,4,0-T"

2,4-Dichlorophenoxyacetic acid 2,4,5-Trichlorophenoxyacetic acid

kill dicotyledonous plants (most broad-leafed plants such as beans and
clover) than to kill monocotyledonous ones (cereals, other grasses, lilies,
etc.). Thus broad-leafed weeds can be destroyed in a field of grain
without damaging the grain. The closely related substance "2,4,5-T,"
especially in the form of an ester dissolved in an oily carrier such as
kerosene, is very effective for killing weedy brush. Both compounds are
very useful for the eradication of such weeds as poison ivy. The way
in which selective herbicides act is still obscure.

MINERAL NUTRITION

The essential elements for the nutrition of plants include the macro-
nutrient elements: C, H, 0, N, P, K, S, Mg, Ca, and Fe; and the micro-
nutrients: Cu, Zn, B, Mn, and Mo. As was pointed out in the discussion

11

■J!

Courtesy of Dr. W. C. Cooper and the American Society of Plant Pliysiologists.

Fig. 15-8. Effect of indoleacetic acid on the rooting of lemon cuttings.

Upper, untreated. Lower, treated for eight hours with a solution of 0.5

mg. of indoleacetic acid per ml. Photograph taken two and one-half weeks

after treatment.

408

PLANT METABOLISM

409

Reproduced from Hiinfjer .S'l'.r/ns in Crops, a publication of tlie American Society
of Agronomy and tlie National Fertilizer Association, Washington, D. C.

Fig. 15-9. Tobacco plants suffering from various mineral deficiencies —
B, nitrogen; C, phosphorus; D, potassium; E, boron; F, calcium; G,
magnesium. Reduction of growth has occurred in all cases. A is a normal
plant.

of the general function of mineral elements in Chap. 8, animals require
all of these elements except boron.

The cells of the root have a capacity for selective intake of ions. For
example, from a solution high in sodium and low in potassium, the plant
may absorb much more potassium than sodium. From soil the plant
obtains its cations, such as Ca + + , by base exchange (that is, by a
process by which the plant exchanges metabolic hydrogen ions for a
cation adsorbed on a soil colloid). In contrast, the plant absorbs its
anions, such as SO4"", from the soil solution around the roots.

Some effects of certain mineral deficiencies are illustrated in Figs.
15-9 and 15-10.

410

PLANT METABOLISM

Courtesy of Purdue University Agricultural Experiment Station. Reproduced
from Hiiuf/er Signs in Orops, a publication of the American Society of Agronomy
and the National Fertilizer Association, Washington, D. C.

Fig. 15-10. Oats in Crosby silt loam of low fertility. Pot 16 (NPK),
treated with complete fertilizer, serves as check; plants healthy and vigor-
ous. Pot 13 (PK), nitrogen starvation; plants spindling, yellowish green,
slightly purplish stems. Pot 14 (NK), phosphorus starvation; plants dark
green, stems weak, slightly purplish tinged. Pot 15 (NP), potassium de-
ficiency; dark-green, weak plants, with oldest leaves brown and tip ends
deadened.

REVIEW QUESTIONS ON PLANT METABOLISM

1. What is the prime source of most of the energy used by man?

2. Which process is of greatest importance in fixing carbon dioxide in the carbon
cycle of nature; of greatest importance in releasing carbon dioxide?

3. How are photosynthesis and respiration interrelated?

4. What are the chemical differences between chlorophyll a, chlorophyll b, proto-
chlorophyll, and bacteriochlorophyll?

5. What is the evidence that all oxygen released in photosynthesis arises from
water?

6. Describe a light reaction and a dark reaction in photosynthesis. How can they
be differentiated?

7. Why is a quantum efficiency of 4 attractive from a theoretical standpoint?
Why is such a high efficiency questioned?

8. What is the first demonstrable organic intermediate in photosynthesis? Which
products may it in turn yield?

9. How does the plant synthesize starch? What is the difference between amylose
and amylopectin?

10. Do plants require any preformed amino acids?

11. Which are the most important sources of fixed nitrogen for maintaining the
nitrogen cycle in balance?

PLANT METABOLISM 411

12. Which groups of organisms can fix No? What is the key intermediate in the
fixation process?

13. What differences can you cite in the respiration of plants and animals? What
similarities ?

14. Why are plant growth substances often much supcMior to oils and acids as weed
killers?

15. It is stated that growth substances arc responsible for i)hototropism and geo-
tropism. How can they cau.se such responses in plants?

REFERENCES AND SUGGESTED READINGS

Arnon, D. I., "Extracellular Photosynthetic Reactions," Nature, 167, 1008 (1951).

Baeyer, Adolph, '•Concerning Dehydration and Its Significance for Plant Life and
Fermentation," Ber. dcut. chcm. Ges., 3, 63-75 (1870).

Bernfeld, P., "Enzymes of Starch Degradation and Synthesis," Advances in Enzy-
mo/o^iy, 12, 379 (1951).

Bonner, J., Plant Biochemistry , Academic Press Inc., New York, 1950.

Burk, D., Cornfield, J., and Schwartz, M., "The Efficient Transformation of Light
into Chemical Energy in Photosynthesis," Sci. Monthly, 73, 213 (1951).

Calvin, M., "The Path of Carbon in Photosynthesis, VI," J. Chem. Ed., 26, 639
(1949).

Chibnall, A. C, Protein Metabolism in the Plant, Yale University Press, New Haven,
1939.

Fischer, H., "Chlorophyll," Chem. Rev., 20, 41 (1937).

Franck, J. and Loomis, W. E. (editors). Photosynthesis in Plants, Iowa State College
Press, Ames, 1949.

Hassid, W. Z. and Doudoroff, M., "Synthesis of Disaccharides with Bacterial En-
zymes," Advances in Enzymology, 10, 123 (1950).

Hill, R., "Oxidoreduction in Chloroplasts," Advances in Enzymology, 12, 1 (1951).

Lardy. H. A. (editor). Respiratory Enzymes, Burgess Publishing Company, Minne-
apolis, 1949.

Manning, W. M., Stauffer, J. F., Duggar, B. M., and Daniels, F., "Quantum Efficiency
of Photosynthesis in Chlorella," J. Am. Chem. Soc, 60, 266 (1938).

Rabinowitch, E. I., Photosyyithesis and Related Processes, Interscience Publishers
Inc., New York, Vol. I (1945) Vol. II, part 1 (1951).

Skoog, F. (editor), Plant Growth Substances, University of Wisconsin Press, Madi-
son, 1951.

Tolmach, L. J., "Effects of Triphosphopyridine Nucleotide upon Oxygen Evolution
and Carbon Dioxide Fixation by Illuminated Chloroplasts," Nature, 167, 946
(1951).

Truog, E. (editor), Mineral Nutntion oj Plants, University of Wisconsin Press,
Madison, 1951.

Tswett, M., "Adsorption Analysis and the Chromatographic Method. Application
to the Chemistry of the Chlorophylls," Ber. dent, botan. Ges., 24, 384-393 (1906).

van Niel, C. B., "On the Morphology and Physiology of the Purple and Green Sulfur
Bacteria," Arch. Mikrobiologie, 3, 1 (1931).

Vishniac, W. and Ochoa, S., "Photochemical Reduction of Pyridine Nucleotides by
Spinach Grana and Coupled Carbon Dioxide Fixation," Nature, 167, 768 (1951).

Warburg, 0. and Burk, D., "The Maximum Efficiency of Photosynthesis," Arch.
Biochem., 25, 410 (1950).

412 PLANT METABOLISM '

Warburg, 0. and Negelein, E., "The Transformation of Energy During the Assimila- I

tion of Carbon Dioxide," Naturwissenschajten, 10, 647 (1922). .

Willstatter, R. and Stoll, A., Investigations on Chlorophyll (Translation by Shertz,

F. M. and Merz, A. R.), Science Press, Lancaster, Pa., 1928.
Wilson, P. W., The Biochemistry of Symbiotic Nitrogen Fixation, University of

Wisconsin Press, Madison, 1940.
Wilson, P. W. and Burris, R. H., "The Mechanism of Biological Nitrogen Fixation,"

Bactenol. Rev., 11, 41 (1947).
Zscheile, F. P. and Comar, C. L., "Influence of Preparative Procedure on the Purity

of Chlorophyll Components as Shown by Absorption Spectra," Botan. Gaz., 102,

463 (1941).

i

Chapter 16

BIOLOGICAL ENERGETICS

All living organisms must have a continuous supply of energy in a
usable form. The study of energy sources, utilization, and quantitative
requirements is called biological energetics. The great bulk of all food
consumed goes to meet this need. In all cases the energy is derived from
chemical reactions carried out by the living cell, whereby the foodstuffs
are converted into products of lower energy content. The difference
between the energy content of the foods eaten and waste products excreted
represents approximately the energy which may be used (with greater
or lesser efficiency) by the organism.

The chemical reactions on which living things depend for their energy
supply are many and varied. Lower forms frequently live under anaer-
obic conditions, carrying out reactions which do not involve oxygen.
Thus for example, glucose is converted into carbon dioxide and alcohol
by yeast, or into lactic acid by lactic acid bacteria. Such conversions
yield relatively little energy, and the yeast or bacteria accordingly are
forced to metabolize large amounts of the foodstuff (here glucose) . The
higher animals and man, on the other hand, are aerobic organisms and
oxidize their foodstuffs to the stage of carbon dioxide and water. Since
this represents complete combustion, much larger amounts of energy are
liberated, and less food per unit weight of living tissue is needed.

The energy used by living things appears partly in the form of heat,
partly as muscular work, and partly in many other forms such as elec-
trical, chemical, and light energy. It has become customary, however,
to express all of these forms in terms of heat units, or calories. A calorie
(cal.) is the amount of heat needed to raise the temperature of one gram
of water one degree Centigrade, specifically from 14.5 to 15.5°C. The
kilocalorie (Cal.) is one thousand times larger.^ The energy difference
between foods and waste products may be expressed quantitatively by
means of these units. For example, the combustion of one mole (180 g.)
of glucose gives 673,000 cal. The heat change accompanying a reaction
is represented by the symbol. A//' (A = change; H = heat). It is given

^ The British Thermal Unit (BTU) is the amount of heat re<iuired to raise the
temperature of one pound of water 1°F. One BTU equals 252 cal., or 1 Cal. equals
very nearly 4 BTU. Another energy unit is the foot-pound, the amount of work
done in lifting one pound through one foot. One calorie equals slightly more than
three foot-pounds.

413

414 BIOLOGICAL ENERGETICS

a negative sign if heat is released when the reaction proceeds from left
to right. For example, in the case above, AH is —673,000 cal.

The energy released from chemical processes which can be used for
doing useful work is called the free energy change of the reaction and is
represented by AF. For any particular process, \F may be either larger
or smaller than AH. If AF happens to have a larger negative value than
AH, more useful work can theoretically be obtained from the process
concerned than corresponds to the amount of heat liberated. This rather
surprising situation is caused by changes in entropy, which occur when-
ever chemical or physical transformations are carried out. The relation
between the three quantities, for processes occurring at constant tempera-
ture and pressure — as is approximately true in biological systems — is
given by the equation :

af = ah-tas

where AS is the entropy change, and T the absolute temperature at which
the process occurs. The entropy change, AS, is expressed in calories
per degree. It is difficult to define exactly, but may be thought of as
heat flowing into or out of the system from the surroundings while a
process is going on. For a more complete discussion of entropy consult
a textbook of physical chemistry or thermodynamics, such as those listed
at the end of the chapter.

An illustration may help to make these abstract concepts more under-
standable. For the complete oxidation of glucose, AH is —673,000 cal.
as stated above, whereas AF is about — 683^,000 cal. The exact AH and
aF values depend on the state of the reactants and products, that is,
whether solid, liquid, or gaseous, and at what concentration, temperature,
pressure, etc. The value — 683,000 cal. is based on approximately physi-
ological concentrations, viz., glucose 0.05M, carbon dioxide 0.1 atmosphere,
oxygen 0.01 atmosphere, and on a temperature of 37°C. Useful work
equivalent to 683,000 cal., therefore, could be obtained from burning one
mole of glucose, if the machine or living tissue doing the work was able
to operate with 100 per cent efficiency. Actual machines and tissues,
of course, never reach this peak of perfection, but the efficiency which
they do achieve may be determined by comparison with the free energy
change of the process being used.

The aF value is also an indication of whether a particular chemical
reaction can take place (if properly started) and to what extent it will
proceed. Reactions which release free energy (Ai^ negative) occur readily
and are called exergonic. The opposite type, called endergonic, will not
take place unless energy is supplied (AF positive). The change in free
energy is the driving force of the reaction, and the larger the amount of
free energy release, the more complete the reaction will be.

The completeness of equilibrium reactions is represented mathematically

BIOLOGICAL ENERGETICS

41:

by the equilibrium co7istant, K. For a reaction of the type A + B^
C + D, A' equals [C] X [D] divided by |AJ X IB], where the brackets
indicate molar concentrations at equilibrium (review the law of mass
action in a textbook of general chemistry) . Thus if K is one, the equi-
librium point is reached when half of the starting materials have been
converted into products, whereas if K is ten, the reaction is about 90
per cent complete.

The relation between AF and K is given by the equation,

-^F = RT\nK

where R is the gas constant (1.987 cal. per degree per mole), T the abso-
lute temperature, and In K the natural logarithm of K. Substituting
numerical values and converting to ordinary logarithms, this equation
becomes at body temperature (37°C) :

- A/'' =1419 log X

This equation enables one to calculate the equilibrium point of any reac-
tion for which the free energy change is known, and vice versa. For
example, if ^F is zero, K is one, but if aF equals —10,000, K is 1.22 X 10'.
The latter value indicates an equilibrium point very far to the right.

Muscle contraction

The free energy available from oxidation of foodstuffs is not usable
by animals and human beings in the form of heat. In other words, the
body is not simply a heat engine,^ although it is an engine or a machine
in the sense that it converts energy from one form into others. Some
heat of course is needed for warmth, but except under the most severe
conditions of cold and exposure, more than enough heat for this purpose
is always available. Heat production is primarily a consequence of the
fact that the body, like other machines, operates at much less than 100
per cent efficiency. In fact, excess heat is an important waste product
which must be ehminated to maintain health and vigor.

The question therefore arises as to just how the body does convert
the chemical energy of foodstuffs into muscular work and other useful
processes. A partial answer to this question has been obtained from
studying the chemistry of muscle contraction. It was discovered around
1930 that muscle contains two substances which are readily hydrolyzed
with the liberation of large amounts of energy. These compounds are
adenosine triphosphate (ATP, p. 158) and creatine phosphate (CrP,
p. 349). The latter was observed to decrease in amount as contraction
occurred and to be completely used up if the muscle was stimulated to

^ A heat engine is a macliine wliicli operates by virtue of temperature and pressure
changes.

416 BIOLOGICAL ENERGETICS

exhaustion. Thus it appeared quite possible that the breakdown of
CrP into free creatine and phosphoric acid might furnish the energy for
contraction. However, it was demonstrated by Lohmann in 1934 that
muscle contains no enzyme capable of catalyzing the direct hydrolysis
of CrP, but that the substance is used up instead by reacting with adeno-
sine diphosphate (ADP) :

CrP + ADP ^Ct + ATP

The Lohmann reaction, as this is called, has a small AF and can there-
fore proceed readily in either direction, depending on changes in pH
and concentrations of reactants. It obviously will go to the right and
consume CrP only when ADP is available, ADP is present only in
very small amounts in resting muscle, but can be formed from ATP by
hydrolysis :

ATP + H:0 — ^^^"'' > ADP4-H3P04

This breakdown of ATP into ADP is catalyzed by adenosine triphospha-
tase (ATP-ase) and has a AF value of —11,500 cal. From the above
considerations it is evident that this breakdown of ATP must take place
before CrP can be used up by the Lohmann reaction. ATP, therefore,
is most probably the immediate energy source for muscle contraction.

Just how the chemical energy in ATP is converted into the mechanical
energy of contraction is not well understood, but it is known that ATP-ase
is present in muscle fibers in large amounts. In fact, ATP-ase probably
makes up a part of the fiber, being itself a long, thread-like protein,
molecules of which are arranged lengthwise along the fiber. When ATP
breakdown occurs, some of the side-chain groups in these or other pro-
tein molecules in the muscle probably become altered in such a way
that they have an attraction for other groups in the same molecules.
This would cause a puckering and shortening of the molecules so affected,
and consequently a contraction of the whole fiber.

High energy phosphate bonds

The energy released when the terminal phosphate group of ATP is split
off (Ai^ = — 11,500 cal.) must have been contained in the particular
valence bond which held this group to the rest of the molecule. Numer-
ous other phosphate derivatives are also involved in metabolic reactions

0

II
(see Chap. 13) . All are of the type (HO)oP— X, where X may be either

an oxygen or nitrogen atom, which is attached in turn to another phos-
phate radical or to some organic structure. These phosphorus com-
pounds have been found to fall roughly into two main groups according

BIOLOGICAL ENERGETICS 417

to the amount of free energy liberated on hydrolysis of the P — X bond.
A number of more important ones are collected in Table 16-1. For the
low energy group, AF is about — 2000 to — 4000 calories per mole, whereas
for the high energy compounds it amounts to around — 11,000 to — 15,000

Table 16-1

Free energies of hydrolysis of some phosphoric acid derivatives *

Compound AF, cal. pK Temp., °C

Glucose-1-phosphate — 4,750 8.5 38

Glucose-6-phosphate —3,000 8.5 38

Fructo?e-6-phosphate —3,350 8.5 38

Glycerol-l-phosphate —2,200 8.5 38

ATP (terminal group) —11,500 7.5 20

Acetyl phosphate —14,500 6.3 37

Pyruvic acid enol phosphate — 15,900 ? 20

Creatine phosphate —13,000 7.7 20

Arginine phosphate — 11,800 7.7 20

* Reproduced from Avison and Hawkins, "The Role of Phosphoric Esters in Bio-
logical Reactions," Quart. Rev., 5, 171 (1951) by permission of the authors and the
Chemical Society (London).

calories. The latter substances are said to contain a high energy phos-
phate bond, which is written as "-^P". It is only the chemical energy of
such bonds which can be transformed directly into useful work by living
organisms, and so far as known, only ATP serves as the immediate source
of such energy, both for muscular work and for all other purposes. The
metabolic breakdoum of foodstuffs, so far as energy requirements are
concerned, is a matter of generating high energy phosphate bonds and
of synthesizing ATP.

Phosphagens

The presence in CrP of a high energy bond, taken together with the
facts already presented, indicates that CrP serves as a '--'P storehouse
in muscle. When contractions begin, ATP starts to be used. It would
be quickly exhausted except for the Lohmann reaction, which starts func-
tioning as soon as some ADP is formed. ATP is thereby resynthesized
at the expense of CrP, and the ATP level is kept up until most of the
CrP is used, and the muscle becomes exhausted. This situation is reached,
however, only during very severe work, because during moderate exer-
cise the metabolism of glycogen soon starts, and '^P compounds are pro-
duced as fast as needed (see below). When muscular work stops, gly-
cogen breakdown continues for a time, ATP is resynthesized, and the
Lohmann reaction goes into reverse until the normal amount of CrP
characteristic of resting muscle is restored.

This arrangement gives the muscle a much greater supply of quickly

418

BIOLOGICAL ENERGETICS

available energy than the ATP alone could provide, since considerably-
larger amounts of CrP are present (Table 16-2) . In view of its metabolic
role, CrP has been called a phosphagen. Another phosphagen, arginine
phosphate, takes the place of CrP in the muscles of most invertebrates.

Table 16-2
Relation of muscular activity to concentration of various substances in muscle

Muscle

Concentration t when

muscle is:

Substance

species and type

Resting

Fatigued

In rigor

Adenosine triphosphate

Average mammal,

striated

5.0

2.5-4.5*

0.005*

Adenosine triphosphate

Average mammal,

cardiac

1.5

trace or none

Adenosine diphosphate

Average mammal.

striated

0.005=^

0.5-2.5*

5

Creatine phosphate

Average mammal,

striated

20

10-15

trace or none

Creatine phosphate

Average mammal,

cardiac

2

trace or none

Creatine phosphate

Rat, smooth

1

trace or none

Arginine phosphate

Crab, striated

32

18

trace or none

Lactic acid

Average mammal,

striated

1.7

45

68

Inorganic phosphate

Average mammal,

striated

0.1*

10

30

* Values estimated from probable ATP-ADP-inorganic P ratios as calculated from
energy relations of aerobic and anaerobic metabolism (see M. J. Johnson, Chap. XII,
in Respiratory Enziimes, by Lardy : Burgess Publishing Company, Minneapolis, 1949).

t Millimoles per kilogram, fresh weight.

Still another phosphagen of unknown composition has been detected
in certain lower organisms. The amounts of phosphagens in various
tissues are shown in Table 16-2. By far the largest concentrations are
present in those muscles which are capable of the greatest work output
(striated muscle) .

Generation of high energy phosphate bonds

Glycolysis. The reactions of glycolysis, which result in the formation
of high energy bonds, are now known in detail. They have been presented
in Chap. 13 (Figs. 13-1 and 13-3, reactions 9 and 12) . In all, four '--'P
bonds are so generated for each hexose unit, that is, four molecules of
ADP are converted to ATP. However, two molecules of ATP are used
up along the way (reactions I and 4) so that the "net yield" to the
organism is two moles of ATP per mole of glucose metabolized to lactic
acid. Since each mole of ATP gives up 11,500 cal. when used for work
{i.e., hydrolyzed to ADP), this figure represents an energy yield from
glycolysis of 23,000 cal. per mole of glucose. The free energy change

BIOLOGICAL ENERGETICS

419

for the conversion of glucose to lactic acid is not known with certainty,
but is probably close to 40,000 cal. per mole. If this figure is correct,
the efficiency of glycolysis is nearly 60 per cent.

It is perhaps not realistic to discuss the energy relationships of gly-
colysis in terms of the conversion of glucose to lactic acid, since this acid
represents only an offshoot from the main pathway of carbohydrate
metabolism and is not produced at all except during severe work (review
p. 328) . Even then, it is reconverted to pyruvic acid during rest. How-
ever, if carbohydrate breakdown is to be divided for purposes of study
into anaerobic and aerobic phases, the anaerobic part must be treated
as ending with lactic acid, even though discussion of the aerobic phase
begins with pyruvic acid. Allowance for the energy released in con-
verting lactic acid to pyruvic will be made below.

Aerobic Metabolism. Complete combustion of glucose to carbon di-
oxide and water releases about 683,000 cal. under physiological conditions.
From the above figures it is obvious that only a small fraction of this
total appears during anaerobic glycolysis. Approximately 94 per cent
of the energy of the glucose remains to be released through the operation
of the citric acid cycle. It is of great interest to discover what portion
of this remaining energy becomes fixed in a biologically usable form (pre-
sumably ATP) , and to learn just how the reactions of the citric acid
cycle result in the formation of the necessary /^P bonds. During the
oxidation of one molecule of pyruvic acid by one "turn" of the cycle, 10
atoms of hydrogen are released (2 in each of five steps, namely, reactions
16, 20, 22, 23, and 25, Fig. 13-4). The energy from the whole cycle is
actually produced by the combination of these hydrogen atoms with the
oxygen of the inhaled air, and ^P bonds are evidently formed at the
same time. Before discussing this subject in greater detail, it seems
desirable to consider briefly the nature of oxidation and the quantitative
relations between oxidation and energy changes.

Oxidation is often defined as addition of oxygen or removal of hydrogen,
but cases are also common in which oxidation occurs without either
oxygen or hydrogen being directly involved. The most exact and gen-
eral definition states that oxidation is a loss of electrons. For example,
Fe++-^Fe+ + + -f e, where e stands for an electron, the unit negative
charge of electricity. The tendency of substances to give up electrons
and become oxidized is expressed in terms of volts as an electrical po-
tential, called the oxidation-reduction or redox -potential. Strong oxi-
dizing agents have positive potentials ranging up to about -\-2 volts,
while reducing agents go down to about — 1 volt, and even lower in a
few cases. These relations provide a scale of oxidizing power, much
as the pH scale measures active acidity. When the oxidized and reduced
forms of an oxidizing agent are in equal concentrations, that is, when
the oxidizing agent is half reduced, its redox potential is called by defini-

420 BIOLOGICAL ENERGETICS

tion the normal potential, Eq. If a substance of high Eo reacts with
one of lower £'0, the potential of the former (the oxidizing agent) drops
as more and more of it becomes changed to its reduced form, and the
potential of the latter (the reducing agent) rises as it is converted into
its oxidized form. Finally, the two potentials become equal and no
further reaction occurs. The free energy released depends on AE, the
difference between the two Eq values. This relationship is given by the
following equation:

— AF = nF AE

where n is the number of electrons involved in the reaction, and F is
the Faraday.^

These principles may now be used to explore the possibilities of '-'P
generation during the biological oxidation of metabolites. As explained
above, the energy released comes from hydrogen atoms split off at various
stages. Each pair of hydrogens passes through a system of coenzymes
or carriers before finally being united with oxygen (review cytochrome
system, p. 333). A series of oxidation-reduction systems is therefore
involved, each having its own characteristic £'0 value. The biological
oxidation, for example, of lactic to pyruvic acid probably involves the
various coenzymes and redox potentials shown in Fig. 16-1.

The two hydrogens are split off at a potential of — 0.18 volt and, at
first, combine with DPN:

CH3CHOHCOOH + DPN ^ CH3COCOOH + DPN • H2

Since the AF of this reaction is +4600 cal., the equilibrium point lies
far to the left, and the lactic acid will not be oxidized unless the DPN • Ho
is removed. If the amount of DPN • H2 is in some manner kept very
low, the equilibrium point is displaced to the right in accordance with
the law of mass action. Of course, the hydrogens are, in fact, immediately
transferred from the DPN • Ho through the remaining steps shown in
Fig. 16-1, and for each of these steps AF has a large negative value.
This means that their equilibrium points lie far to the right, and the
hydrogens are therefore pulled along until they unite with oxygen. They
have then traveled over a potential span of 0.99 volt ( — 0.18 to +0.81),
an interval which corresponds to a free energy change of — 45,700 cal.^
This energy, how^ever, is not released in a single burst, but in three suc-
cessive smaller portions, as shown in Fig. 16-1. One of these is near
10,000 cal., about the amount needed for a /^P bond, while the others are

^ In any chemical process associated with electron transfer, a certain definite quantity
of electricity is always needed to bring about the transformation of one gram equivalent
weight of the reacting substance (examples, electrolysis of water, electroplating of
metals). This quantity, the Faraday, is 96,500 coulombs, which is equivalent to
23,060 calories per volt.

2 Calculation : — AF = nF AE = 2x 23,060 x 0.99 = 45,700,

BIOLOGICAL ENERGETICS

421

considerably larger. Therefore, approximately the right amount of
energy is made available in about the right sized "packages" for the
generation of three high energy bonds, when one mole of lactic acid is
oxidized to pyruvic acid and water.

Overall reaction

CH3CHOHCOOH + iO, 7-^ CH3COCOOH + HjO
Lactic acid Pyruvic acid

Corresponding redox potentials
and energy changes
Eo AE AF

Inlermediate stages volts volts cal.

1. Lactic acid < * pyruvic acid + 2H —0.18) ^ ,^ , . ^^«

f —0.10 +4,600
2.DPN+2H ^ DPNH+H* -0.28 _

3. rAD+2H 5=^ FADH, -0.06 _u70n

4. 2Cy,,. . Fe*- + 2H ^ 2Cyt. o Fe- + 2H* +0.26 ^^^ l^'

5. iOj + 2H :i=^ HjO +0.81 ^

Fig. 16-1. Intermediate stages and energy relationships in the biological
oxidation of lactic to pyruvic acid.

The five pairs of hydrog-ens that split out when pyruvic acid is com-
pletely oxidized by the citric acid cycle likewise pass through the hydro-
gen transport system. Each pair originates at a definite redox potential,
which is given in Table 16-3. Calculations of the energy released as each
pair becomes united with oxygen indicate that sufficient energy should
be available to generate the number of '~P bonds shown in the last
column. The total is 16 such bonds per mole of pyruvic acid. Since
three more could have been formed in the conversion of lactic into pyruvic
acid, the total for the aerobic phase of carbohydrate metabolism would
be 2 X (16 + 3) or 38 per mole of glucose. It must be emphasized that
these figures are only estimates based on the information now available.

Table 16-3

Redox potentials at which hydrogen is released during oxidation of pyruvic

acid via fhe citric acid cycle

Nu

mheroj ~'P

Acid

Acid

Corresponding Eo

bo

nds possibly

dchydrogenaled

produced

value *at pH 7

formed

Pyruvic

Acetic

—0.63

4

Isocitric

Oxalosuccinic

0.13

3

a-Ketoglutaric

Succinic

0.60

4

Succinic

Fumaric

0.00

2

Malic

Oxalacetic

0.10

Total

3

: 16

* Voits.

422 BIOLOGICAL ENERGETICS

Esterification of Inorganic Phosphate. Another line of evidence has
been uncovered which bears on this question of the number of ~P bonds
formed during the aerobic phase of carbohydrate metabohsm. Ground-up
preparations from tissues such as liver and kidney are able to take up
molecular oxygen and use it to oxidize pyruvic acid, or any other acid
involved in the citric acid cycle, to carbon dioxide and water. Inorganic
phosphate is needed for this oxidation, and as the oxidation proceeds,
some of the phosphorus becomes esterified, that is, united with organic
substances. Lehninger has demonstrated with the aid of the isotope,
P^-, that the newly formed organic phosphate has the properties of ATP.
It is very probable that one molecule of ATP is produced for every atom
of phosphorus esterified during the oxidation. The number of P atoms
taken up for each atom of oxygen used is difficult to measure accurately
because of side reactions which break down the new '— -P bonds even as
others are being formed. The best results, however, show values ap-
proaching those given in the last column of Table 16-3. This evidence,
then, also tends to indicate that phosphorylations occur and '^P bonds
are formed each time hydrogen atoms, from whatever source, are passed
from one hydrogen carrier to the next. In fact, this hydrogen transport
system is almost certainly the chief energy transformer of aerobic
organisms.

Efficiency of Energy Metabolism. The above discussion deliberately
goes somewhat beyond the bounds of present well-established knowledge
in order to estimate the efficiency of energy metabolism in animals. If
38 -^P bonds are produced during the conversion of lactic acid to carbon
dioxide and water and two more are produced during glycolysis, a total
of 40 moles of ATP could be formed from the metabolism of one mole
of glucose. If these figures are correct, the efficiency of the overall process
would be

40X11,500^
683,000
This is a very high value in comparison with other types of machines.
The maximum efficiency of a steam engine, for example, is around 25
per cent and that of a diesel engine about 40 per cent. As a matter of
fact, direct work measurements show that animals also have maximum
efficiencies of about 30-40 per cent, but usually work at only about 15-20
per cent efficiency. This is not surprising since the above value of 67
per cent applies only to ATP formation. No information is available
regarding the efficiency with which the chemical energy of ATP can be
converted into muscular work by the animal.

Physiological Fuel Value of Foods. Until quite recently the study
of energy metabolism in animals and man was conducted almost ex-
clusively from the standpoint of the total heat produced by combustion
of various foods and the total energy needs of metabolism under various
ponditions. Although thi§ sort of knowledge does not provide as much

BIOLOGICAL ENERGETICS

423

insight into energy metabolism as the direct chemical approach described
above, it nevertheless has important practical applications.

The heat of combustion of a food is determined by use of the bomb
calorimeter. This is a heavy steel cylinder or "bomb" surrounded by
water in an insulated container. The sample plus oxygen at high pres-
sure is placed in the tightly closed cylinder and ignited by a spark. The
amount of heat liberated as the sample burns is determined by noting
the exact rise in the temperature of the water and making suitable cor-
rections for heat taken up by the bomb itself. The results are expressed
as calories liberated per mole or per gram of substance burned.

When pure chemicals are examined in this way, the heats of com-
bustion are found to vary according to the composition of the sample.
The figures given in Table 16-4 show that higher percentages of carbon
and hydrogen are associated with higher heat values, whereas the opposite
is true for oxygen. The reason for this, of course, is simply that sub-
stances like glucose with a high oxygen content are in effect already
partly oxidized.

The fuel values of foods likewise depend on their elementary composi-
tion, but information of this sort is not usually available. Instead,
foods are usually analyzed for their contents of carbohydrate, fat, pro-
tein, minerals, and moisture. These percentages constitute the proxitnate
composition. The heat value of a given food can be calculated easily
from its proximate composition, if the heat contributed by each of the
major components is known. Minerals and moisture, of course, contribute
nothing in this regard. Carbohydrates, proteins, and fats in the bomb
calorimeter give about 4.1, 5.7, and 9.5 Cal. per gram, respectively (see
Table 16-4). However, not all of this energy is available to the animal
body partly because foods are not completely digested and absorbed and
partly because they are not always oxidized completely in the body.
Proteins in particular are oxidized to carbon dioxide, water, and free
nitrogen in the calorimeter, but in the body the nitrogen is converted
into excretory products (urea, creatine, etc.), which are themselves or-
ganic substances capable of being burned, and releasing additional heat.

Table 16-4

Relation of chemical composition to heat of combustion of various substances

Substance Composition Heat of combustion

C% H% 0% N7o (Cal. per gram)

Glucose 40.0 6.7 53.3 3.73

Sucrose 42.1 6.4 5L5 3.96

Starch 44.4 6.2 49.4 4.22

Alanine 40.4 7.9 35.9 15.7 4.35

Casein* 53.1 7.0 22.5 15.8 5.85

Stearic acid 76.0 12.8 11.2 9.53

Animal fats (av.) 76.5 12.0 11.5 9.50

* Also contains 0.8% S and 0.8% P,

424

BIOLOGICAL ENERGETICS

When allowance is made for these facts, the average physiological fuel
value of carbohydrates, proteins, and fats becomes, in round numbers,
4, 4, and 9 Cal. per gram, respectively. Note that these values are ex-
pressed in kilocalories and that they refer to the total energy obtained
by the organism, both in the form of heat and in the form of ATP or
other ~P compounds. However, no allowance for entropy changes is
included, and as explained previously these changes are likely to pro-
vide additional energy. The entropy changes involved in the metabolism
of most foodstuffs are not known.

The physiological fuel values of a list of common foods are given in
the Appendix (Table A-1, p. 434). A study of this list emphasizes the
tremendous effect of two components, namely fat and moisture, on the
calorific value. Watery foods like fresh fruits and vegetables contribute
very few calories, whereas concentrated, dried foods, especially those high
in fat such as nuts, chocolate, vegetable oils, etc., have very high energy
value. Sherman has pointed out that an ounce of olive oil is equal in
fuel value to over three pounds of lettuce!

Energy requirements

Basal Metabolism. The most conspicuous use for food energy is in
the performance of muscular work. However, even at rest, energy is
required by a living animal or human being to keep various vital func-
tions in operation. These include, not only such obvious processes as
breathing, heart action, and blood circulation, but also the maintenance
of a certain minimum muscle tension or tonus (even when lying down
and completely "relaxed") and tlie normal operation of the organs of
digestion, secretion, and excretion. The kidneys, for example, use energy
to excrete waste products, and energy is needed to cause digested foods
to pass through the intestinal wall. Even the synthesis and maintenance
of the protein molecules which make up body tissues require energy,
since the equilibrium point of the reaction

protein + water ^ amino acids

lies far to the right, and energy must be supplied to shift it to the left.

The minimum rate of energy metabolism to provide for such functions
is called the basal metabolism, and the energy thereby consumed is con-
sidered to be the minimum energy requirement. The basal metabolism
is measured about 12 hours after eating so that no digestion of food is
taking place, and with the subject lying down in a room of comfortable
temperature. Under these conditions all the energy being used by the
body appears directly in the form of heat since no external work of any
sort is being done.

The basal metabolism can be measured by direct calorimetry, that is,

BIOLOGICAL ENERGETICS 425

by placing the subject in a large calorimeter and detcrniining the heat
output as described above for the bomb calorimeter. This actually
has been done in many cases, but is so cumbersome and expensive that
a shorter, indirect method has been devised. This method depends
on the fact that heat output is closely related to oxygen consumed and
carbon dioxide liberated by the subject. Thus for the oxidation of
glucose,

CeHioOg + 60o -» 6CO2 + 6H2O + 678 Cal.

it is apparent that six moles of oxygen are used for the oxidation of one
mole of glucose and the production of 678 Cal.^ Six moles of a gas
occupy 6 X 22.4 or 134.4 1. at standard temperature and pressure. There-
fore each liter of oxj^gen used in this reaction results in the production
of 678 -^ 134.4 or 5.047 Cal. For fat oxidation the relations are some-
what different. Taking tristearin as an example,

2(Ci7H35COO)3C3H5 + 1630o-^ 114C0o + llOHoO + 17,060 Cal.

it is seen that in this case one liter of oxygen produces 17,060 -f- (163 X
22.4) or 4.67 Cal. Oxidation of average mixed food fats yields 4.69
Cal. and of mixed proteins, 4.82 Cal. per liter of oxygen consumed.

An indication of which type of foodstuff is being oxidized by the body
at a given time is provided by the respiratory quotient (R.Q.), which
is the ratio of carbon dioxide given off to oxygen used :

_ CO2 given off
O2 used

The amounts of the two gasses may be expressed in moles or in volumes
(measured at the same temperature and pressure) . It follows from the
above equations that the R.Q. for oxidation of glucose is 1.00 and of
tristearin 0.70 (114-^163). In general, the R.Q. for average mixed
carbohydrates is 1.00, for fats 0.71, and for proteins 0.81. It has been
found that the R.Q. for both animal and human subjects under the con-
ditions of the basal metabolism test is close to 0.82. For this R.Q.
value each liter of oxygen consumed produces 4.825 Cal.- All that is
necessary, therefore, to find the metabolic rate is to measure the liters
of oxygen consumed per unit time and multiply by 4.825.

The basal metabolism is a fundamental characteristic of the living
animal. Its magnitude depends on the body weight, body surface area,
sex, age, and other factors. However, for healthy individuals of a given

^ The current value.s of heat output per liter of oxygen are based on 678 Cal. as the
heat of combustion of glucose, although 073 Cal. probably is more nearly correct.

" Calculated on the assumption that only fats and carbohydrates are being oxidized.
The amount of protein oxidized, wliich can be estimated from the urinary nitrogen
excretion, does not have to be considered since proteins also yield 4.82 Cal. per liter
of oxygen.

426

BIOLOGICAL ENERGETICS

size and age there is a normal rate of basal metabolism, and any marked
deviations therefrom indicate an abnormal, perhaps diseased, condition.
Brody has emphasized the relation between the body size and basal
metabolism of a large number of animal species. He found that the
formula

Cal. per day = 70 X (body weight in kg.)"-'^^

holds very well for a tremendous range of body sizes (Fig. 16-2). Note
that the smallest animals have the highest basal metabolism per kilogram

0.1 1.0 10

cal. /day i ' ' '""i — ■ ■ ■ '"i-i — ■ > i

&

cal./kg./day
10.000

I- Cal. = 39.5 1b."""

B.T.U. = 156.8 lb.""*
: Cal. = 70.5kg.''"*

s

■^ 1,000

o

XI
(A

<u
^.

1 100

10

Body Weight, lbs.

100 1,000 10,000

'";;;, B.T.U./day

100,000

Rabbits
Ducks

Guinea
Pigeons
Canaries

Elephants -C^'^

Horses-. ^fe

Dairy bulls
Beef cows
Beef steers "^^^^ Beef steers
Ponies -tr<i^^ Dairy cows
Humans -j^^ ^ Swine

Sheep
Dogs
20% deviation lines
Domestic fowl

10,000

-. 1,000

100

20

a

CO

"o

JO
oj

<A

0.01 0.1 1.0 10 100 1,000 5,000

Body Weight, kgs.
From Brody, Bioenergetics and Growth, Reinhold Publishing Corporation.
Fig. 16-2. Relation of body weight to basal metabolism of mature ani-
mals of various species. Tlie rising curve, showing basal metabolism per
animal per day, is represented very well by the equation, Cal. per day =
70.5 X (body weight in kg.) °'"*. However, from the nature of the data the
numerical values 0.734 and 70.5 are somewhat doubtful and probably should
be rounded off to 0.73 and 70, respectively. The declining curve, showing
metabolism per kg. of body weight per day, similarly corresponds to the
equation, Cal. per kg. body weight = 70.5 X (body weight in kg.)""-"".

of body weight, although the largest animals, of course, have the highest
total metabolism. More detailed formulas have been worked out for
human beings, which take account of age, sex, height, and body surface
area, as well as weight. For example, a 30 year old man who is 175 cm.
(5 ft. 9 in.) tall and weighs 70 kg. (156 lb.) should have a basal metabolic
rate (BMR) close to 1630 Cal. per day.^

Total Energy Requirements of Human Beings. The amount of food

^Calculated from Harris and Benedict's formula (see Sherman, Chemistry of Food
and Nutrition, 7th ed., p. 164).

BIOLOGICAL ENERGETICS 427

needed above the basic metabolic level depends almost exclusively on
muscular activity. Representative figures for various types of activity ^
are given in Table 16-5. An approximate idea of the total daily energy

Table 16-5

Total energy expenditure under dilTerent conditions of musriilar activity *

Calories per hour
Per 70 kg.

Form OF ACTivriY (Av. Man) Per kg. Per lb.

Sleeping 65 0.93 0.43

Awake, lying still 77 1.10 0.50

Sitting at rest 100 1.43 0.65

Standing relaxed 105 1.50 0.69

Dressing and undressing ' 118 1.69 0.77

Typewriting rapidly 140 2.00 0.91

Walking, 2.6 mph 200 2.86 1.30

Carpentry work '. 240 3.43 1.56

Walking, 3.75 mph 300 4.28 1.95

Sawing wood 480 6.86 3.12

Swimming 500 7.14 3.25

Running, 5.3 mph 570 8.14 3.70

Walking upstairs 1100 15.8 7.18

* Reproduced by permission from Sherman, Chemistry of Food and Nutrition, 7th
ed., The Macmillan Company, New York, 1946, p. 189.

requirement might be obtained from these figures by estimating the time
spent in various ways. However, more accurate estimates can be made
by other methods. If, for example, an exact record of the total food
intake of an individual is kept for a period of several months, and the
body weight does not change appreciably during that time, the number
of calories consumed per day must have been very nearly equal to the
energy requirement. Another method is to measure oxygen consumption
while the subject is engaged in various activities. Recommended allow-
ances based on these and other types of measurements have been made
by the Food and Nutrition Board of the National Research Council and
are listed in Table 16-6. Note the relatively high allowances for growing
children and the increased requirements caused by pregnancy and lacta-
tion and by heavy muscular work.

Obesity. The problem of obesity, or excessive fatness, is unfortunately
a very common and serious one in many of the more prosperous, indus-
trialized countries. The implications for health, longevity, and general
well-being are too well known to require comment. However, the basic
facts regarding control of body weight are apparently not well under-
stood by many people.

^ Note fliat til? values in Table 10-5 are for total energy expenditures under various
conditions, i.e., they include the basal enerjiy requirement.

4-^ BIOLOGICAL ENERGETICS

Table 16-6
Recommended daily calorie allowances *

Calories

Men (154 lb, 70 kg.):

Sedentaiy 2,400

Physically active 3,000

With heavy work 4,500

Women (123 lb, 56 kg.):

Sedentaiy 2,000

Moderately active 2,400

Very active 3,000

Pregnancy (latter half) 2,400

Lactation 3,000

Children :

Under 1 yr 110/2.2 lb. (1 kg.)

1-3 yrs. (27 lb., 12 kg.) 1,200

4-6 yrs. (42 lb., 19 kg.) 1,600

7-9 yrs. (58 lb., 26 kg.) 2,000

10-12 yrs. (78 lb., 35 kg.) 2,500

Girls, 13-15 yrs. (108 lb.) 2,600

Girls, 16-20 yrs. (122 lb.) 2,400

Boys, 13-15 yrs. (108 lb.) 3,200

Boys, 16-20 yrs. (141 lb.) 3,800

* "Calorie allowances must be adjusted up or down to meet specific needs. The
calorie values in the table are, therefore, not applicable to all individuals but, rather,
represent group averages. The proper calorie allowance is that which over an ex-
tended period will maintain body weight or rate of growth at the level most conducive
to well-being." '

There is only one possible cause of obesity, namely, a greater aver-
age calorie intake over extended periods of time than is balanced by
energy expenditures. No matter what the other circumstances may be,
if the body weight is increasing (beyond the bounds of normal growth),
the food intake must be excessive. Frequently this simple and obvious
fact is either disbelieved or ignored. Perhaps one reason for this is that
the relation between the calories eaten and the amount of work needed
to use them up is not clearly appreciated. Some specific examples may
help to emphasize this relationship. Suppose a young woman has a
light snack, consisting of one ounce of shelled peanuts, in addition to
regular meals sufficient for normal energy requirements. One ounce of
peanuts has a physiological fuel value of about 180 Cal.^ (Appendix,
Table A-1), which is equivalent to slightly over 540,000 foot-pounds of
work.^ Assuming that this energy is converted into work with 20 per
cent efficiency, a 56 kg. (123 lb.) woman would have to climb a vertical

^ Most of the popular five-cent candy bars also contribute about this same number of
calories.

^ One Cal. equals slightly more than 3000 foot-pounds.

BIOLOGICAL ENERGETICS

429

distance of (540,000 X 0.20) -:- 123, or 878 feet in order to use it com-
pletely. If tliis very considerable exertion, or its equivalent in other
work, is not made, the extra calorics provided by the snack will inevitably
go to form fat.

As another example, suppose a person wishes to exercise enough to
remove one pound of fat. The fat tissues of the body contain about
one-fifth moisture and four-fifths actual fat. One pound of such tissue,
therefore, represents 454 X 0.8 X 9, or 4080 Cal. According to Table
16-5, walking at the rate of 3.75 miles per hour involves a total energy
expenditure of 300 Cal. per hour for a 70 kg. man. Therefore, the man
would have to walk 13.6 hours (4080 -=- 300) , or a distance of 51 miles
(13.6 X 3.75) to "burn off" the pound of fat. Of course, considerably
more weight might be lost in the form of perspiration, but this weight
would be quickly regained through increased water intake. These ex-
amples show that it is very difficult, and usually impractical, to counteract
excessive food intake by increased muscular activity.

Another source of confusion is the fact that some individuals gain
weight on a food intake which would not be excessive for others. This
may be due to differences in the efficiency with which the food is digested
and absorbed, or to differences in basal energy requirements. Other
things being equal, older people need fewer calories than younger ones,
and a short stocky person probably has a lower basal metabolism than
a tall gangling one.

The solution to the problem of overweight is very simple in theory,
but, of course, in practice it is complicated by the appetite, which unfor-
tunately is not an accurate guide to the energy requirements of the body.
However, only a slight reduction of the calorie intake, if persistently
continued, is capable of restoring normal weight. If as calculated above,
one pound of fatty tissue corresponds to 4080 Cal., a reduction of only
100 Cal. a day would amount to the equivalent of 8 pounds a year. Ad-
justments of this magnitude can usually be made merely by intelligent
food selection. The principles to be observed are to avoid fat insofar
as possible and to increase the consumption of high protein foods such
as fish, poultry, and lean meat. A high protein intake satisfies hunger
with the consumption of relatively few calories. Bulky foods like fresh
vegetables are also indicated. Severe dieting or use of drugs for reducing
are at best undesirable and often actually dangerous to health. The most
satisfactory procedure is a rearrangement of one's eating habits such that
a gradual but steady weight reduction occurs.

REVIEW QUESTIONS ON BIOLOGICAL ENERGETICS

1. What is a calorie; a foot-pound? How many feet would a 150 lb. weight have
to be raised to use up energy equivalent to 500 calories?

2. If the heat of combustion of ethyl alcohol is 326 Cal., how many calories would

430

BIOLOGICAL ENERGETICS

be provided by the alcohol in a glass of beer (assume 250 ml. per glass and 4 per
cent alcohol in the beer) ?

3. What is meant by a high energy phosphate bond? Name three substances that
contain high energy and three that contain low energy phosphate bonds.

4. Which type of energy is used as the immediate source of energy for performing
biological processes of various kinds? How docs the animal body differ from a
heat engine in its functioning?

5. List two reactions of glycolysis in which ~P bonds are formed. Which parts
of the aerobic oxidation process in the body are probably mainly responsible for the
liberation of energy and the formation of ~P bonds?

6. What is the Lohmann reaction? Explain the importance of this reaction for
muscle contraction.

7. What is free energy, and how is it related to the heat of a chemical reaction?
What is the relation between the free energy change of a reaction and the extent
to which the reaction proceeds.

8. Define basal metabolism, and explain the general relationship between the basal
metabolic rates of various animals.

9. What is there about the chemical nature of fats, proteins, and carbohydrates,
and about the way they are metabolized in the body, which accounts for the differ-
ence in their phj'siological energy values?

10. Calculate the number of calories provided by a light lunch consisting of a
glass of milk (250 ml.) and a sandwich made up of the following ingredients:

white bread 56.2 g.

lettuce 32.7 g.

butter 8.0 g.

oil dressing 2.1 g.

American cheese 50.7 g.

n. About how far could a person run by using the energy from the lunch itemized
in Question 10?

12. What is the respiratory quotient? Is it possible to have an R.Q. greater than
1.0? Explain.

REFERENCES AND SUGGESTED READINGS

Avison, A. W. D. and Hawkins, J. D., "The Role of Phosphoric Esters in Biological

Reactions," Quart. Ret). Chem. Soc. London, 5, 171 (1951).
Baldwin, E., Dynamic Aspects of Biochemistry , 2nd ed.. The University Press, Cam-
bridge, 1952.
Brody, S., Bioenergetics and Groivfh, Chapters 6, 12, 13, Reinhold Publishing Corp.,

New York, 1945.
Bull, H. B., Physical Biochemistry, 2nd ed., John Wiley and Sons, Inc., New York,

1951.
Daniels, F., Outlines of Physical Chemistry, John Wiley and Sons, Inc., New York,

1948. ^,

Food and Nutrition Board, National Research Council, "Recommended Daily Dietary Ip'

Allowances," revised 1948, Nutrition Rev., 6, 319 (1948).
Glasstone, S., Thermodynamics for Chemists, D. Van Nostrand Company, New York,

1947.
Johnson, M. J., Chapters 3 and 12 in Respiratory Enzymes, ed. by H. A. Lardy,

Burgess Publishing Company, Minneapolis, 1949.
Lehninger, A. L. and Smith, S. W., "Efficiency of Phosphorylation Coupled to Electron

BIOLOGICAL ENERGETICS 431

Transport between Dihydrodiphosphopyridine Nucleotide and Oxygen, J. Biol.

Chem., 181, 415 (1949).
Lipmann, F., "Metabolic Generation and Utilization of Phosphate Bond Energy,"

Advances in Enzynwlogy , 1, 99 (1941).
Lipmann, F., "Metabolic Process Patterns" in Currents in Biochemical Research,

ed. by D. E. Green, Interscience Publishers, Inc., Now York, 1946.
Lohmann, K., "Enz.ymic Disruption of Creatinephosphoric Acid; the Chemistry of

Muscle Contraction," Biochem. Z., 271, 264 (1934).
Mommaerts, W. F. H. M., Muscular Contraction, Interscience Publishers, Inc., New

York, 1950.
Oesper, P., "Source of the High Energy Content in Energy-Rich Phosphates," Arch.

Biochem., 27,255 (1950).
Ogston, A. G. and Smithies, O., "Some Thermodynamic and Kinetic Aspects of

Metabolic Phosphorylation," Physiol. Rev., 28, 283 (1948).
Parks, G. S. and Huffman, H. M., Free Energies oj Some Organic Compounds,

Chemical Catalog Company, New York, 1932.
Peterson, W. H. and Wilson, P. W., "Energetics of Heterotrophic Bacteria," Chem.

Rev., 8,427 (1931).
Sherman, H. C, Chemistry of Food and Nutrition, Chapters 8, 9, 10, 7th ed.. The

Macmillan Company, New York, 1946.

Appendix

COMPOSITION AND ENERGY VALUE

OF FOODS

The four tables making up this appendix are revisions of the tables
contained m the authors' previous book, Elements of Food Biochemistry .
The new material in these tables is taken largely from AVatt and Merrill,
Composition of Foods, U.S.D.A. Agricultural Handbook No. 8, 1950. A
small number of new entries come from Sherman, Chemistry of Food
and Nutrition, 7th ed., McCanse and Widdowson, The Composition of
Foods, and Morrison, Feeds and Feeding, 21st ed. The tables include
data for most of our raw food materials and for representative canned
foods. By comparing these data an idea can be obtained of the differ-
ences in composition resulting from commercial canning operations. More
comprehensive data on canned and cooked foods are contained in Watt
and Merrill and in INIcCanse and AViddowson. No data on farm rough-
ages have been included because of space limitations. Such data are
found in Morrison.

In using these tables the student should bear in mind that the com-
position of any given sample that may be analyzed does not neces-
sarily correspond to the figures recorded here. Some variations are
found in the proximate composition of different samples, but greater
variations occur in figures for the mineral and vitamin contents. Dif-
ferences as great as 100 per cent may be encountered among a series
of plant samples. These differences are due chiefly to soil and climate
conditions, but they are also related to varieties and maturity of the
plant. From the dietary viewpoint, such variations are not serious be-
cause variations in different foods cancel each other. The actual intake
during a period of time will probably correspond closely to that cal-
culated from the figures of the table.

Places marked with a question mark in Tables A-2 and A-3 indicate
that no quantitative data have been as yet found. A fair idea of the
probable amount present may be obtained by noting the figures given
for a similar material. For example, the magnesium and other figures
missing for buckwheat are probably in the same range as those given
for another cereal, say, barley or rye. From a nutritional viewpoint,
the important mineral elements are calcium, phosphorus, and iron, and
these elements are given for all the foods in these tables. Values for
sodium and chlorine are omitted as indicated by (a) , where a consider-
able amount of sodium chloride is added in making the product, such as
butter.

433

Table A-1

Proximate composition of foods

(Edible portion)

Carbohydrate Food

Mois- Pro- Avail- energy

Food ture Ash tein Fat Total* a5?et (Cal. per

% % % % % % 100 g.)

Almonds 4.7 3.0 18.6 54.1 19.G 16.9 597

Apples, fresh S4.1 0.3 0.3 0.4 14.9 13.9 58

dried 23.0 1.4 1.4 1.0 73.2 69.3 277

Apricots, fresh 85.4 0.6 1.0 0.1 12.9 12.3 51

dried 24.0 3.5 • 5.2 0.4 66.9 63.7 262

canned, sirup 77.3 0.6 0.6 0.1 21.4 21.0 80

Artichokes 83.7 1.1 2.9 0.4 11.9 8.7 63

Asparagus, fresh 93.0 0.7 2.2 0.2 3.9 3.2 21

canned 93.6 1.3 1.9 0.3 2.9 2.4 18

Avocados 65.4 1.4 1.7 26.4 5.1 3.3 245

Bacon, medium fat 20.0 4.3 9.1 65.0 (1.1) 1.1 630

Bananas 74.8 0.8 1.2 0.2 23.0 22.4 88

Barley, grain 10.2 2.1 12.8 2.1 72.8 70.1 356

pearled 11.1 0.9 8.2 1.0 78.8 78.3 349

Beans, Lima, dried 12.6 3.8 20.7 1.3 61.6 57.3 333

fresh 6(i.5 1.7 7.5 0.8 23.5 22.0 128

navy, dried 10.5 3.9 22.0 1.5 62.1 58.2 338

string, fresh 88.9 0.8 2.4 0.2 7.7 6.3 35

baked, canned 70.0 2.0 5.8 3.0 19.2 18.3 125

green, canned 93.5 1.2 1.0 0.1 4.2 3.6 18

Beef, medium fat 60.0 0.9 17.5 22.0 0 0 273

chuck, medium fat (;5.0 0.9 18.6 16.0 0 0 224

sirloin, medium fat . . . 62.0 0.9 17.3 20.0 0 0 254

rib, medium fat 59.0 0.8 17.4 23.0 0 0 282

round, medium fat 69.0 1.0 19.5 11.0 0 0 182

corned, medium fat . . 54.2 5.0 15.8 25.0 0 0 293

Beets S7.6 1.1 1.6 0.1 9.6 8.7 42

Beet greens 90.4 1.7 2.0 0.3 5.6 4.2 27

Blackberries 84.8 0.5 1.2 1.0 12.5 8.3 57

Blueberries 83.4 0.3 0.6 0.6 15.1 13.9 61

Brains (all kinds) 78.8 1.4 10.4 8.6 0.8 0.8 125

Bran — see wheat

Brazil nuts 5.3 3.4 14.4 65.9 11.0 8.9 646

Bread, rye 35.3 2.0 9.1 1.2 52.4 52.0 244

white, commercial 34.7 1.8 8.5 3.2 51.8 51.6 275

whole wheat 36.6 2.5 9.3 2.6 49.0 47.5 240

Broccoli 89.9 1.1 3.3 0.2 5.5 4.2 29

Brussels sprouts 84.9 1.3 4.4 0.5 8.9 7.6 47

Buckwheat, grain 12.0 1.9 10.3 2.3 73.5 62.8 356

Butter 15.5 2.5 0.6 81.0 0.4 0.4 716

Buttermilk (culfd skim

milk) 90.5 0.8 3.5 0.1 5.1 5.1 36

Butternuts 3.8 2.9 23.7 61.2 8.4 6.4 679

Cabbage 92.4 0.8 1.4 0.2 5.3 4.3 24

celery 95.4 0.7 1.2 0.3 2.4 1.9 14

Cantaloupe 94.0 0.6 0.6 0.2 4.6 4.0 20

Carrots 88.2 1.0 1.2 0.3 9.3 8.2 42

canned 92.2 0.9 0.5 0.4 6.1 5.5 28

Cashew nuts 3.6 2.7 18.5 48.2 27.0 25.7 578

Cauliflower 91.7 0.8 2.4 0.2 4.9 4.0 25

Celery 93.7 1.1 1.3 0.2 3.7 3.0 18

Chard, leaves 91.0 1.2 2.6 0.4 4.8 4.0 27

Cheese, cheddar,

American 37.0 3.7 25.0 32.2 2.1 2.1 398

cottage, from skim milk 76.5 1.5 19.5 0.5 2.0 2.0 95

Swiss 39.0 3.8 27.5 28.0 1.7 1.7 870

Cherries 83.0 0.6 1.1 0.5 14.8 14.5 61

* Difference between 100 per cent and the sum of the percentages of water, ash. pro-
tein, and fat. Figures in parentheses were not obtained in this way.
t Carbohydrate exclusive of fiber,

434

Table A-1 (Continued)

Proximate composition of foods

(Edible portion)

Carbohydrate Food

Mois- Pro- Avail- encrgu

Food ture Ash tein Fat Total* ahlef ((^il. per

% % % % 7o % 100 g.)

Chicken, broilers 71.2 1.1 20.2 7.2 0 0 151

roaster-s 6G.0 1.0 20.2 12.6 0 0 200

hens 55.9 1.1 IS.O 25.0 0 0 802

Chocohite, bitter 2.3 3.2 5.5 52.0 29.2 2().6 501

sweetened 1.4 1.4 2.0 29.8 ()2.7 61.3 471

Clam.s ,S0.3 2.1 12.8 1.4 3.4 3.4 81

Cocoa, plain 3.9 5.0 8.0 23.8 48.9 44.3 293

Coconut, dried, .shredded. 3.3 0.8 3.6 39.1 53.2 49.1 556

fresh 46.9 1.0 3.4 34.7 14.0 10.8 359

Collard.s 8(i.6 1.7 3.9 0.(; 7.2 6.0 40

Corn, grain 11.0 1.3 10.0 4.3 73.4 71.3 372

popcorn, popped 4.0 1.6 12.7 5.0 76.7 74.5 386

sweet, fresh 73.9 0.7 3.7 1.2 20.5 19.7 92

canned 80.5 0.9 2.0 0.5 16.1 15.3 67

Corn flakes 3.6 2.9 8.1 0.4 85.0 84.4 385

Corn meal, whole 12.0 1.2 9.2 3.9 73.7 72.1 355

Cotton.seed, whole 7.3 3.5 23.1 22.9 43.2 26.3 471

Cowpeas, fresh 65.9 1.4 9.4 0.6 22.7 22.7 130

dried, mature 10.6 3.5 22.9 1.4 61.6 57.4 342

Crab 80.0 1.7 16.1 1.6 0.6 0.6 86

Crackers, graham 5.5 2.2 8.0 10.0 74.3 73.5 393

soda 5.7 2.4 9.6 9.6 72.7 72.5 420

Cranberries 87.4 0.2 0.4 0.7 11.3 9.9 48

Cream, cofifee 72.5 0.6 2.9 20.0 4.0 4.0 204

whipping 59.0 0.5 2.3 35.0 3.2 3.2 330

Cucumber 96.1 0.4 0.7 0.1 2.7 2.2 12

Currants, red 84.4 0.6 1.2 0.2 13.6 9.6 55

Dandelion greens 85.8 2.0 2.7 0.7 8.8 7.0 44

Dates 20.0 1.8 2.2 0.6 75.4 73.0 284

Doughnuts 18.7 1.0 6.6 21.0 52.7 52.5 425

Duck 54.3 1.0 16.0 28.6 0 0 321

Eel 71.6 1.0 18.6 9.1 0 0 162

Eggplant 92.7 0.5 1.1 0.2 5.5 4.6 24

Eggs, hen 74.0 1.0 12.8 11.5 0.7 0.7 162

white only 87.8 0.6 10.8 0 0.8 0.8 50

yolk only 49.4 1.7 16.3 31.9 0.7 0.7 361

duck 70.8 1.0 13.1 14.3 0.8 0.8 184

goose 70.4 1.1 13.9 13.3 1.3 1.3 180

Endive or escarole 93.3 0.9 1.6 0.2 4.0 3.2 20

Farina 10.5 0.4 10.9 0.8 77.4 77.0 370

Figs, dried 24.0 2.4 4.0 1.2 68.4 62.6 270

fresh 78.0 0.6 1.4 0.4 19.6 17.9 79

Fish, various (av.) 72.1 1.4 18.0 8.5 0 0 129

Flour, buckwheat, light. . 12.0 0.9 6.4 1.2 79.5 79.0 348

rye, medium 11.0 1.1 11.4 1.7 74.8 73.8 350

wheat, whole 12.0 1.7 13.3 2.0 71.0 68.7 333

patent 12.0 0.4 10.5 1.0 76.1 75.8 364

Frog's legs 81.9 1.1 16.4 0.3 0 0 73

Gelatin, plain, dry 13.0 1.3 85.6 0.1 0 0 335

Goose 51.1 0.9 16.4 31.5 0 0 349

Gooseberries 88.9 0.4 0.8 0.2 9.7 7.8 39

Grapefruit 88.8 0.4 0.5 0.2 10.1 9.8 40

juice, canned, unsweet-
ened 89.2 0.4 0.5 0.1 9.8 9.7 38

Grapes 81.9 0.4 1.4 1.4 14.9 14.4 70

Grape juice 81.0 0.4 0.4 0 18.2 18.2 67

* Difference between 100 per cent and the sum of the percentages of water, ash, pro-
tein, and fat.

t Carbohydrate exclusive of fiber,

435

Table A-1 (Continvied)

Proximate composition of foods

(Edible portion)

Mois-

FooD ture

%

Heart, beef 77.0

pork 76.8

Hominy, dry 12.0

Honey 20.0

Ice cream, plain ()2.1

Jellies 84.5

Kale 80.0

Kidney, beef 74.9

pork 77.1

sheep 77.8

Kohlrabi 90.1

Lamb, medium fat 55.8

leg, mediimi fat 03.7

rib, medium fat 51.9

shoulder, medium fat. 58.3

Lemons 89.3

Lentils, dried (entire

seeds) 11.2

Lettuce 94.8

Limes 80.0

Liver, beef 09.7

calf 70.8

pork 72.3

Lobster 79.2

Loganberries 82.9

Macaroni, dry 8.6

Milk, fresh, whole 87.0

skim 90.5

canned, evaporated,

unsweetened 73.7

condensed, sweetened 27.0

dried, whole 3.5

skim 3.5

goat, fresh 87.4

human, fresh 87.5

Molasses, cane, medium. 25.4

Mulberries 82.8

Mushrooms 91.1

Mustard greens 92.2

INIutton — see lamb

Nectarines 82.9

Oats, grain 9.8

Oatmeal or rolled oats . . 8.3

Okra 89.8

Oleomargarine 15.5

Olives, pickled, green . . . 75.2

ripe, pickled 75.8

Onions, mature 87.5

green 87.6

Oranges 87.2

Orange juice, fresh or

canned 87.5

Oysters, fresh, solids . . . 80.5

Pancreas (all kinds) ... 63.3

Papayas 88.7

Parsley 83.9

Carlo)

hydrate

Food

Pro-

Avail-

energi/

ish

tein

Fat

Total*

ahlef

(Cal. per

%

%

%

%

%

100 g.)

1.1

10.9

3.7

0.7

0.7

108

1.1

16.9

4.8

0.4

0.4

117

0.4

8.7

0.8

78.1

77.7

362

0.2

0.3

0

79.5

79.5

294

0.8

4.0

12.5

20.0

20.6

207

0.3

0.2

0

05.0

65.0

252

1.7

3.9

0.6

7.2

6.0

40

1.1

15.0

8.1

0.9

0.9

141

1.2

10.3

4.6

0.8

0.8

114

1.3

10.0

3.3

1.0

1.0

105

1.0

2.1

0.1

0.7

5.6

30

0.8

15.7

27.7

0

0

317

0.9

18.0

17.5

0

0

235

0.8

14.9

32.4

0

0

350

0.8

15.6

25.3

0

0

295

0.5

0.9

0.6

8.7

7.8

32

3.3

25.0

1.0

59.5

55.8

337

0.9

1.2

0.2

2.9

2.3

15

0.8

0.8

0.1

12.3

11.4

37

1.4

19.7

3.2

0.0

6.0

136

1.3

19.0

4.9

4.0

4.0

141

1.5

19.7

4.8

1.7

1.7

134

2.2

16.2

1.9

0.5

0.5

88

0.5

1.0

0.6

15.0

13.0

62

0.7

12.8

1.4

76.5

70.1

377

0.7

3.5

3.9

4.9

4.9

68

0.8

3.5

0.1

5.1

5.1

36

1.5

7.0

7.9

9.9

9.9

138

1.7

8.1

8.4

54.8

54.8

320

6.0

25.8

26.7

38.0

38.0

492

7.9

35.6

1.0

52.0

52.0

302

0.7

3.3

4.0

4.6

4.0

67

0.2

1.4

3.7

7.2

7.2

68

3.2

2.1

0

09.3

69.3

286

0.8

1.2

0.6

14.6

12.6

69

1.1

2.4

0.3

4.0

3.1

16

1.2

2.3

0.3

4.0

3.2

22

0.5

0.5

0.1

16.0

15.6

67

4.0

12.0

4.6

69.6

58.6

368

1.9

14.2

7.4

68.2

67.0

390

0.8

1.8

0.2

7.4

6.4

32

2.5

0.0

81.0

0.4

0.4

720

5.8

1.5

13.5

4.0

2.8

132

2.6

1.5

17.3

2.9

L3

160

0.6

1.4

0.2

10.3

9.5

45

0.6

1.0

0.2

10.6

8.8

45

0.5

0.9

0.2

11.2

10.6

45

0.4

0.8

0.2

11.1

11.0

44

2.0

9.8

2.1

5.6

5.6

84

1.3

15.8

19.2

0

0

236

0.6

0.6

0.1

10.0

9.1

39

2.4

3.7

1.0

9.0

7.2

50

* Difference between 100 per cent and the sum of the percentages of water, ash, pro-
tein, and fat.

t Carbohydrate exclusive of fiber.

436

Table A-1 (Continued)

Proximate composition of foods

(Edible portion)

Mois-

FooD ture

%

Parsnips 7S.6

Peaches, fresh 86.9

dried 24.0

canned (sirup packed) 80.9

Peanuts 2.6

Peanut butter 1.7

Pears 82.7

Peas, fresh 74.3

Peas, canned 82.3

dried, mature 11.6

Pecans 3.0

Peppers, green 92.4

Persimmons, native .... 64.4

Pineapple 85.3

canned (sirup packed) 7S.0

juice, canned 86.2

Plums 85.7

Pork, fresh, medium fat. 42.0

fresh, ham, medium fat 53.0

loin, medium fat 58.0

cured, ham, medium fat 42.0

Potatoes 77.8

Potato chips 3.1

Prunes, dried 24.0

Pumpkin 90.5

Quinces 85.3

Rabbit 70.5

Radishes 93.6

Raisins 24.0

Raspberries, red 84.1

Rhubarb 94.9

Rice, brown 12.0

white 12.3

puffed 3.5

Rutabagas 89.1

Rye, grain 11.0

Salmon, Pacific, raw . . . 63.4

canned, red 67.3

Sardines, canned in oil . . 57.4

Sauerkraut, canned .... 93.2

Scallops 80.3

Shrimp, canned 75.6

Sirup, table blends 25.0

Soybeans, whole 7.5

Soybean flour, low fat. . . 11.0

Spinach 92.7

S(iuash, summer 95.0

winter 88.6

Strawberries 89.9

Sugar, granulated 0.5

Sweet potatoes 68.5

Tangerines 87.3

Tapioca 12.6

Tomatoes 94.1

canned 94.2

Tomato juice, canned . . . 93.5

* Difference between 100 per cent and the sum of the percentages of water, ash, pro-
tein, and fat.

t Carbohydrate exclusive of fiber.

437

Carhoi

hydrate

Food

Pro-

Avail-

energy

ish

tein

Fat

Total*

able t

(Cal. per

%

%

%

%

%

100 g.)

1.2

1.5

0.5

18.2

16.0

78

0.5

0.5

0.1

12.0

11.4

46

3.0

3.0

0.6

69.4

65.9

265

0.4

0.4

0.1

18.2

17.8

68

2.7

26.9

44.2

23.6

21.2

559

3.4

26.1

47.8

21.0

19.0

576

0.4

0.7

0.4

15.8

14.4

63

0.9

6.7

0.4

17.7

15.5

98

1.0

3.4

0.4

12.9

11.5

68

3.0

23.8

1.4

60.2

54.8

339

1.6

9.4

73.0

13.0

10.8

696

0.5

1.2

0.2

5.7

4.3

25

0.9

0.8

0.4

33.5

32.0

141

0.4

0.4

0.2

13.7

13.3

52

0.4

0.4

0.1

21.1

20.8

78

0.4

0.3

0.1

13.0

12.9

49

0.5

0.7

0.2

12.9

12.4

50

0.6

11.9

45.0

0

0

457

0.8

15.2

31.0

0

0

344

0.9

10.4

25.0

0

0

296

5.4

16.9

35.0

0.3

0.3

389

1.0

2.0

0.1

19.1

18.7

83

4.0

6.7

37.1

49.1

48.0

544

2.1

2.3

0.6

71.0

69.4

268

0.8

1.2

0.2

7.3

6.0

31

0.4

0.3

0.1

13.9

12.1

58

1.0

2t).9

7.6

0

0

175

1.0

1.2

0.1

4.2

3.5

20

2.0

2.3

0.5

71.2

71.2

268

0.5

1.2

0.4

13.8

9.1

57

0.7

0.5

0.1

3.8

3.1

16

1.1

7.5

1.7

77.7

77.1

360

0.4

7.6

0.3

79.4

79.2

362

2.3

5.9

0.6

87.7

S7.2

392

0.8

1.1

0.1

8.9

7.6

38

1.8

12.1

1.7

73.4

71.4

321

1.0

17.4

16.5

0

0

223

3.0

20.2

9.6

0

0

173

4.7

25.7

11.0

1.2

1.2

214

2.1

1.1

0.2

3.4

2.7

16

1.4

14.8

0.1

3.4

3.4

78

4.5

18.7

0.9

0.3

0.3

89

0.6

0

0

74.0

74.0

286

4.7

34.9

18.1

34.8

29.8

331

5.5

44.7

1.1

37.7

35.4

228

1.5

2.3

0.3

3.2

2.6

20

0.4

0.6

0.1

3.9

3.4

16

0.8

1.5

0.3

8.8

7.4

38

0.5

0.8

0.5

8.3

6.9

37

0

0

0

99.5

99.5

385

1.1

1.8

0.7

27.9

26.9

123

0.7

0.8

0.3

10.9

9.9

44

0.2

0.6

0.2

86.4

86.3

360

0.6

1.0

0.3

4.0

3.4

20

0.7

1.0

0.2

3.9

3.5

19

1.0

1.0

0.2

4.3

4.1

21

Table A-1 (Continued)

Proximate composition of foods

(Edible portion)

Carbohydrate Food

Mois- Pro- Avail- energy

Food ture Ash tein Fat Total* ahle-f (Cal. per

% % % % % % 100 g.)

Tongue, beef (IS.O 0.9 16.4 15.0 0.4 0.4 207

Tuna, canned, solids 00.0 2.7 29.0 8.2 0 0 198

Turkey 5S.3 1.0 20.1 20.2 0 0 268

Turnips 90.9 0.7 1.1 0.2 7.1 6.0 32

Turnip tops S9.5 1.8 2.9 0.4 5.4 4.2 30

A'eal, medium fat 68.0 1.0 19.1 12.0 0 0 190

Walnuts, English 3.3 1.7 15.0 64.4 15.6 13.5 654

Watermelons 92.1 0.3 0.5 0.2 6.9 6.3 28

Watercress 93.6 1.1 1.7 0.3 3.3 2.8 18

Wheat, grain 11.0 1.6 13.0 2.0 72.4 70.6 360

bran, crude 10.1 6.1 16.6 3.7 63.5 53.2 354

germ 11.0 4.3 25.2 10.0 49.5 47.0 361

puffed 3.8 3.6 10.8 1.6 80.2 78.5 355

shredded 5.6 1.7 10.1 2.5 80.1 77.8 360

Yams 72.6 1.0 2.1 0.2 24.1 23.3 107

Yeast, compressed 70.9 2.4 13.3 0.4 13.0 12.7 86

dried, brewer's 7.6 7.9 46.1 1.6 36.8 36.0 273

* Difference between 100 per cent and the sum of the percentages of water, ash, pro-
tein, and fat.

t Carbohydrate exclusive of fiber.

438

Table A-2

Major mineral elements in the edible portion of foods

(Grams per 100 g.)

Cal- Mag- I'otas- Phos- Vhlo-

FoOD cium nesium sium Sodium phorus rine Sulfur

Almonds 254 .275 .756 .024 .475 .0.37 ACA

Apples, fresh OCX) .000 .110 .015 .010 .004 .004

dried 019 .029 .557 .072 .048 .019 .019

Apricots, fresh OKI .012 .370 .021 .023 .004 .000

dried 086 .002 1.924 .109 .1 19 .021 .031

canned in sirup 010 .007 .250 .001 .015 .002' .001

Asparagus 021 .015 .200 .008 .0(52 .047 .051

Bacon, medium fat 013 ? V V .108 ? ?

Bananas 008 .024 .412 .023 .028 .163 ,013

Barley, grain 058 .126 .495 .070 .343 .139 .152

Beans, navy, dried 103 .165 1.284 .189 .437 .007 224

Lima, fresh 063 .067 .606 .089 .158 .009 .068

dried 0.68 .181 1.899 .282 .381 .025 .156

string or green 066 .032 .288 .012 .044 .045 .024

green, canned 027 ? ? ? .019 ? ?

baked, canned 049 .037 .344 (a ) .113 (a) .051

Beef, medium fat Oil .032 .382 .066 .l(i1 .056 .221

Beets 027 .027 .235 .053 .043 .040 .01 7

Beet greens 118 .097 .390 ? .045 ? .035

Brains Oil .016 .269 .160 .338 .155 .130

Bread, white 079 ,034 .110 .517 .092 .602 ,083

whole wheat 096 ? ? ? .263 ? ?

rye 072 ? ? ? .147 ? ?

Broccoli 130 .024 .352 .030 .076 .076 .126

Brussels sprouts 034 .015 .375 ? .078 ? .098

Buckwheat, grain 090 ? .450 ? .310 ? ?

Butter 020 .002 .019 (a) .016 (a) .009

Cabbage 046 .016 .217 .038 .031 .034 .074

celery 043 .011 .400 .028 .041 .023 .013

Cantaloupe 017 ,016 ,243 ,048 ,016 ,048 .016

Carrots 039 .020 .219 .050 ,037 .035 .019

canned 022 ? ? ? .024 ? ?

Cashew nuts 046 .267 ? ? .428 ? ?

Cauliflower 022 .023 .292 .048 .072 .038 .074

Celery 050 .025 .320 .101 .040 .225 .021

Chard 105 ,053 .318 .086 .036 .039 .124

Cheese, cheddar 725 .031 ,116 ,900 ,495 ,972 ,214

cottage 096 ? ? ? ,189 ? ?

Swiss 925 ? ? ? .563 ? ?

Cherries 018 ,012 .125 .015 .020 .004 .018

Chestnuts 029 .048 .415 .037 .081 .010 .049

Chicken 014 .047 .402 .054 .200 .034 .303

Chocolate 098 .082 .400 .019 .446 .009 .114

Clams 096 .090 ,172 ,603 .139 1.065 .219

Cocoa 125 .192 .534 .060 .712 .050 .197

Coconut, fresh 021 .040 .360 .040 .098 .120 .044

dried, sweetened 043 .077 .693 .053 .191 .225 .076

Collards 249 .017 ? ? .058 ? ?

Corn, grain 010 .142 .300 .036 .256 .041 ,124

sweet, fresh 009 ,047 ,278 .040 ,120 .014 .037

sweet, canned 004 ? ? ? .051 ? ?

Cornflakes Oil ? ? V .058 ? ?

Cottonseed, wliole 140 .320 1.110 .290 .070 ? .240

Cowpeas, dried 077 .265 1.305 .036 .451 ,019 .250

Crabs 126 .117 .271 .366 .261 ,570 .255

Crackers, graham 020 ? ? .203 ? ? ?

soda 020 ? ? .096 ? ? V

Cranberrips 014 .005 .056 .002 .011 .004 .008

Cream 097 .006 .112 .031 .077 .067 .033

Cucumbers 010 .020 .170 .026 .021 .028 .011

Currants fresh 036 .031 .208 .015 .033 .010 .021

dried 180 .155 1.040 .075 .220 .050 .105

439

Table A-2 (Continvied)
Major mineral elements in the edible portion of foods

(Grams per 100 g.)

Cal- Mag- Potas- Phos- Chlo-

FooD cium nesiiim slum Sodium phorus rine Sulfur

Dandelion greens 187 .036 .461 .168 .070 .099 .170

Dates 072 .065 .580 .040 .060 .253 .048

Duck 015 ? ? ? .188 ? ?

Eel 018 .018 .241 .032 .202 .035 .133

Eggplant 015 .015 .260 .026 .037 .063 .020

Eggs 054 .009 .149 .111 .210 .100 .233

Egg white 006 .011 .149 .175 .017 .131 .211

Egg yolk 147 .013 .110 .078 .586 .067 .214

Endive 079 .013 .381 .060 .056 .071 .032

Figs, fresh 054 .020 .205 .043 .032 .037 .017

dried 186 .068 .709 .151 .111 .126 .060

Fish, various (a V.) 024 .024 .375 .064 .225 .137 .199

Flour, patent 016 .021 .137 .053 .087 .079 .155

whole wheat 041 .122 .324 .160 .372 .177 .124

rye, medium 027 ? ? ? .262 ? ?

Frog 018 .024 .308 .055 .147 .040 .163

Garlic 006 .008 .130 .009 .090 .004 .318

Goose 015 .031 .406 ? .188 ? .326

Gooseberries 022 .009 .150 .010 .028 .009 .015

Grapefruit 022 .007 .164 .006 .018 .007 .005

Grapefruit juice, canned . . . .008 ? ? ? .013 ? ?

Grapes 017 .004 .267 .011 .021 .002 .009

Heart 009 .035 .329 .102 .203 .204 .151

Honey 005 .004 .051 .006 .016 .015 .003

Horseradish 160 .028 .550 .094 .059 .013 .234

Kale 225 .055 .486 .050 .062 .120 .160

Kidney 014 .019 .240 .238 .233 .376 .148

Kohlrabi 046 .052 .370 .050 .050 .050 .039

Lamb (mutton) 010 .033 .260 .070 .212 .069 .187

Leeks 091 .037 .380 .036 .049 .110 .056

Lemons O40 .006 .152 .009 .022 .006 .012

Lentils, dried 059 .082 .662 .754 .423 .062 .123

Lettuce 042 .015 .256 .028 .023 .085 .014

Liver 009 .021 .255 .021 .333 .091 .258

Lobster 061 .022 .258 ? .184 ? ?

Macaroni 022 .038 .054 .010 .165 .077 ,119

Milk, cow, fresh 118 .019 .129 .047 .093 .114 .031

evaporated 243 .038 .258 .094 .195 .228 .067

powder 949 .118 .955 .348 .728 1.029 .229

goat 129 ? ? .026 .106 .163 ?

human 032 .005 .055 .011 .017 .058 .011

Molasses, medium 290 ? ? ? .069 ? ?

Mushrooms 009 .012 .280 .013 .115 .026 .025

Mustard greens 220 .016 .330 .020 .038 .090 .142

Oatmeal (rolled oats) 053 .143 .365 .072 .405 .027 .207

Oats, grain 094 .150 .450 .090 .318 .089 .187

Okra 082 .038 ? ? .062 ? .014

Oleomargarine 020 ? ? ? .016 ? ?

Olives, pickled 087 .012 .809 (a) .017 (a) .032

Onions 032 .016 .200 .020 .044 .053 .065

Oranges 033 .011 .177 .014 .023 .006 .011

Orange juice 019 .014 .200 .006 .016 .008 .005

Orange juice, canned 010 ? ? ? .018 ? ?

Oysters 094 .039 .204 .470 .143 ? .180

Parsley 193 ? ? ? .084 ? ?

Parsnips 057 .038 .396 .010 .080 .038 .025

Peaches, fresh 008 .015 .174 .012 .022 .006 .005

dried 044 .087 1.009 .070 .126 .035 .029

canned in sirup 005 .006 .151 .001 .014 .004 .001

Peanuts 074 .169 .706 .052 .393 .040 .27(5

Pears 013 .005 .110 .010 .016 .004 .010

Peas, green 022 .035 .259 .024 .122 .049 .035

440

Table A-2 (Continued)

Major mineral elements in the edible portion of foods

(Grams per 100 g.)

» Cal- Mag- Potas- Phos- Chlo-

FOOD cium nesium sium Sodium phorus rine

canned 025 .024 .201 (a ) .067 (a )

mature 057 .121 .948 .072 .388 .034

Peppers, green Oil .025 .270 .015 .025 .031

Peppers, red 035 .013 .120 .000 .042 .014

Persimmons 000 .005 .170 .013 .020 .019

Pineapple 01 fi .014 .230 .008 .011 .038

canned in r.iriip 029 .008 .057 .001 .007 .004

Pineapple juice, canned ... .015 V ? ? .008 V

Plums 017 .010 .212 .003 .020 ,002

Pork 007 .027 .415 .081 .117 .040

Potatoes Oil .027 .498 .030 .056 .048

Prunes, dried 054 .032 .845 .101 .085 .004

Pumpkins 021 .021 .198 .011 .044 .025

Rabbit 018 .029 .415 .047 .244 .051

Radishes 037 .014 .160 .083 .031 .056

Raisins 078 .017 .796 .120 .129 .068

Raspberries 040 .018 .141 .007 .037 .010

Rhubarb 051 .015 .392 .010 .025 .070

Rice, brown 079 .141 .334 .068 .310 .066

white 024 .033 .046 .012 .136 .056

puffed 021 ? ? ? .116 ?

Rutabagas 055 .015 .210 .052 .041 .031

Rye, grain 075 .130 .477 .060 .376 .043

Salmon, canned 184 .030 .320 (a) .292 (a)

Sardines, canned 386 .035 .433 (a) .550 (a)

Sauerkraut, canned 036 ? ? (a) .018 (a)

Shrimps, canned 115 .105 .404 (a) .263 (a)

Soybeans, mature 227 .287 1.693 .280 .586 .007

Spaghetti — see macaroni

Spinach 081 .048 .416 .093 ,055 .118

Squash, winter 019 .006 .161 .011 .028 .018

summer 015 .008 .150 .002 .015 ?

Strawberries 028 .019 .205 ? .027 ?

Sugar beets 030 .041 .440 .130 .049 .180

Sweet potatoes 030 .035 .381 .031 .049 .022

Tomatoes Oil .016 .277 .013 .027 .048

canned Oil ? ? ? .027 ?

Tomato juice, canned 007 ? ? ? .015 ?

Tuna, canned 008 ? ? ? .224 ?

Turkey 023 .028 .367 .130 .320 .123

Turnips 040 .019 .193 .104 .034 .054

Turnip greens 259 .079 .300 .260 .050 .390

Veal Oil .030 .380 .086 .193 .073

Venison 010 .029 .336 .070 .249 .041

Walnuts 083 .132 .606 .013 .380 .030

Watercress 195 .010 .100 .031 .046 .059

W\atermelon 007 .006 .071 .012 .012 .006

Wheat, grain 040 .163 .409 .060 .383 .088

bran 094 .420 1.252 .007 1.312 .042

germ 084 ? ? ? 1.096 ?

puffed 046 ? ? ? .329 V

shredded 047 ? ? ? .360 ?

Yams 041 .015 .290 .015 .042 .037

Yeast, dried 054 .145 2.230 .149 1.570 ?

Sulfur

.044
.178
.030
.030
.011
.003
.003

V

.004
.216
.033
.024
.016
.184
.038
.043
.012
.008
.121
.114

.069
.152
.235

.283

V

.340
.269

.027
.029

V

.013
.021
.014

.017
")

7

.234
.048
.051
.199
.211
.120
.071
.005
.175
.245

V

•)

V

.013
.015

441

Table A-3

Trace elements in foods

(Fresh basis unless otherwise indicated)

Food Fe

Ahnonds 4.4

Apples 3

Apricots, fresh 5

dried 4.9

Artichokes 2.2

Asparagus 9

Avocados 6

Bacon 8

Bananas 6

Barley, grain S.9

l)earled l.,S

Beans, navy, dried 6.9

kidney, dried G.9

Lima, dried 7.5

Lima, fresh 2.3

string 1.1

Beef, chuck 2.8

heart 4.6

kidney 7.9

liver 6.6

"lean" 4.2

loin 2.6

steak 2.5

sweetbreads 6.0

Beets 1.0

Beet greens 3.2

Blackberries 9

Blueberries 8

Brazil nuts 3.4

Bread, rye 1.6

white 6

whole wheat 2.2

Broccoli 1.3

Brussels sprouts 1.3

Butter 0

Buckwheat, grain 4.0

Buttermilk 1

Butternuts 6.8

Cabbage / .5

Calf's liver 10.6

Cantaloupe 4

Carrots 8

Cauliflower 1.1

Celery 5

Celery cabbage 9

Chard 2.5

Cheese, hard 1.0

cottage 3

Cherries 4

Chestnuts 2.2

Chicken 1.5

Chocolate 3.0

Citron 8

Clams 4.3

Cocoa 11.6

Coconut, dried 3.6

fresh 2.0

Cod-liver oil V

Coffee, beans 5.4

water extract 46

Collards 1.6

Milligrams

per 100 g

Micrograms per 100

of edible

portion

of edihle portion

Cu

Mil

Zn

Iodine

L2

1.2

1.9

9

.1

.11

.07

6.6

.15

?

.04

9

.32

.28

?

?

.32

.38

?

?

.11

.19

.34

6.9

.21

.29

?

?

.41

.08

•>

5.0

.21

1.1

.26

20.0

1.2

1.6

2.3

9.1

.26

V

?

9

.98

1.9

3.1

4.8

.92

1.6

5.2

1.8

.86

1.1

->

9

.53

.6

1.5

9

.13

.37

.09

6.9

.1

•>

*?

9

•>

•>

f

30.0

.11

1.0

2.4

9.0

2.0

.32

3.5

14.0

.05

.02

1.5

3.5

.1

■ii

-)

?

.11

.02

?

9.1

.08

.07

2.0

9

.12

.62

.65

3.3

.12

1.2

.02

8.0

.15

.57

9

9

.11

3.4

7

?

1.3

.94

?

9

.28

1.3

9.0

.25

.42

3.3

11.3

.33

3.2

9

11.0

.20

.26

•?

15.0

.11

.30

")

6.2

.04

.04

9

8.6

.9

3.3

9

?

.05

V

?

9

1.2

")

9

9

.11

.21

.20

2.3

6.3

.37

3.0

9

.05

.05

.09

2.3

.12

.37

.35

4.4

.27

.15

.22

1.6

.12

.17

.21

12.3

.06

.12

?

9

.11

.8

V

11.0

.09

.11

V

10.0

V

.05

V

6.4

.13

.03

.15

.6

.39

1.7

.19

9

.54

V

.46

9

2.1

3.2

2.6

?

.57

7

9

2.1

0

M

3.6

124.0

2.4

3.5

2.6

9

.62

?

?

9

.53

1.3

.84

1.8

•>

0

.9

860.0

1.3

V

.5

8.6

•)

")

?

4.0

V

2.0

9

1.0

442

Table A-3 (Continued)

Trace elements in foods

(Fresh basis unless otherwise indicated)

Milligrams per 100 g.
of edible portion

Food Fe Cu Mn Zn

Corn, gram 3.0 .71 .7 2.2

germ 25.0 .91 3.0 J).4

meal, yellow 1.1 .19 .22 1.8

sweet 5 .08 .31 ?

Cornflakes 1.3 ? ? ?

Cottonseed 14.0 5.0 1.2 ?

Cowpeas 2.5 .17 1.5 ?

Crab 2.0 1.3 0.3 2.5

Crackers, graham 1.9 ? ? ?

soda 1.1 ? ? ?

Cranberries G .11 .38 ?

Cream 1 .15 ? ?

Cucumber 31 .13 .13 .12

Currants, dried 3.3 .8 .31 ?

fresh 9 .13 ? .2

Dandelion greens 3.1 .17 .34 1.2

Dates 2.1 .23 2.0 .32

Duck l.S .46 .03 .34

Eggplant 4 .09 .23 .28

Eggs, hen 2.7 .17 .04 1.3

Egg white 2 .04 ? .01

Egg yolk 7.2 .25 .11 3.8

Endive 1.7 .09 .23 .12

Escarole 1.1 .14 ? .19

Figs, dried 3.0 ..34 .34 .36

fresh (') .00 ? .12

Filberts 4.1 1.2 ? 1.0

Fish, general 61 .33 .02 .80

Flour, buckwheat 1.0 .72 2.1 1.0

whole wheat 3.3 .47 4.3 1.9

rye, medium 2.0 .43 2.0 ?

patent 8 .14 .54 1.2

Garlic ? .26 .46 .92

Goose 1.8 .33 .05 ?

Gooseberries 5 .10 .05 .1

Grapefruit 2 .45 .01 ?

Grapes 0 .11 .08 .17

Grape juice 3 .02 ? ?

Hazelnuts 4.3 1.2 3.6 .97

Hickory nuts 2.0 1.4 ? ?

Hominv 1.0 .18 .11 ?

Honey 9 .15 .03 ?

Huckleberries — see blueberries

Kale 2.2 .52 .86 ?

Kidney — see beef, lamb

Kohlrabi 6 .14 .12 ?

Kumquats 55 .09 .07 ?

Lamb, general 2.4 .42 ? ?

chop 2.2 .42 .04 ?

kidney 9.2 .31 ? 1.9

Lard 0 .02 ? ?

Leeks 1.3 .17 ? .23

Lemons <> .04 .35 ?

Lemon juice 1 .13 ? .17

Lentils (dried) 7.4 .59 3.3 5.4

Lettuce, head 8 .11 1.0 .39

leaf 2.0 .14 .82 .44

Liver — see beef. etc.

Lobster 6 1.5 .04 .24

443

Micrograms per 100 g.

of edible portion

Iodine

12.0

3.3

V
V

5.'7
30.2

.V

3.3
5.7

.83

"j
?
?

'.8

12.0

6.8

16.0

3.7

•)
?

1.5

y

60.5 (saltwater)
7.0 (fresh water)
y

y

2.3
3.0

2.7

y
y

1.3
y

.9
1.4

y

y
2.3

y
y
y

15.0
y

9.3

y

.5

5.2
y

2.9
2.7

80.1

Table A-3 (Continued)

Trace elements in foods

(Fresh basis unless otherwise indicated)

Food Fe

Loganberries 1.2 .14

Macaroni 1.5 .07

Mangoes 2 .04

Milk, cow's 1 .02

Milk powder 6 .34

Milk, human 1.3 .03

Molasses, cane, light 4.3 1.4

Mushrooms 1.0 1.0

Muskmelon — see cantaloupe

Mussels ? .35

Mustard greens 2.9 .12

Mutton, leg 4.8 .4

chop 1.0 .16

liver ? 1.6

Nectarines 46 .06

Oatmeal 4.5 .38

Oats, grain 7.2 1.4

Okra 7 .14

Oleomargarine 0 .04

Olives 1.6 .25

Onions 5 .11

Oranges 4 .18

Oi'ange juice 2 .05

Oysters 5.6 3.4

Oyster plant — see salsify

Parsley 13.0 .23

Parsnips 7 .12

Peaches 6 .07

dried 6.9 .27

Peanuts 1.9 1.1

Pears 3 .16

Peas, dried 4.7 1.1

fresh 1.9 .23

Pecans 2.4 1.4

Peppers, green 4 .11

red 6 V

Pimentos 1.5 .60

Pineapple 3 .09

Pistachio nuts 7.9 1.2

Plums 5 .14

Pork, general 1.8 1.5

chop 2.5 .31

liver 18.0 1.3

Potatoes 7 .17

Prunes, dried 3.9 .29

Pumpkin 8 .07

Quinces 85 .13

Radishes 1.0 .22

Raisins 3.3 .23

Raspberries 9 .16

Rhubarb 5 .09

Rice, brown : 2.0 .26

white 8 .2

puffed 1.8 ?

Rutabagas 4 .12

Rye, grain 3.7 .63

Salsify (oyster plant) 1.4 .3

Salmon 1.2 .23

Sardines 3.5 .04

Scallops 1.8 .23

444

MlUigrams per 100 g.
of edihle portion

Cu Mn Zn

.03

.44
.12

.46
1.2
?

?

?

1

3.3
3.9

.56

.12
.38
.03

.13

1.2

.04

'.68
.86
.05

1.8
.3

3.5
.15
.19
'j

1.5
.67
.11
?

.06
.38
.41
.16
.04
.04
.17
.34
.07
.16

1.9

1.1

.12

3.8
.41

?

.26
3.9

.45

.36

?
?
1
A

4.5

2.2

4.1

9

2.9
?

.3

1.3

.17
?

46.0

•)

.02

1.6

.16
4.0
1.1

V

!o6

?
.23

.28
?

.03
1.4

.79
.31
.05
.21

7

.16
.20
.35
.16
2.1
.22
7

.30

1.8
.22

.8
.94

f

Micrograms per 100
of edihle portion
Iodine

2.7

7

1.6

3.8

32.0

7

7
0

80.2
5.4

1.8
7

3.3

7

4.2
5.2
5.6
7.4
7

3.6

.6

1.5

74.2

7

3.6
1.3

7

.7

.4
7

2.1

7

7

2.3

16!o
7

4.7
7.6

7

14.0
3.9

.12
1.4

7

6.4
7

1.0
26.0
25.0

5.1
7

6.7

6.7

7

29.1
27.0
47.5

Table A-3 (Continued)

Trace elements in foods

(Fresh basis unless otherwise indicated)

Food Fe

Shrimp 3.1

Sirup 4.1

Soybeans S.O

Soybean flour 13.0

Spinach 3.0

Squash, summer 4

winter 6

Strawberries 8

Sweet potatoes 7

Tangerines 4

Tapioca 1.0

Tea extract 72

Tomatoes 6

Tuna fish 1.2

Turkey 3.8

Turnips 5

Turnip gi-eens 2.4

Veal, medium, lean 2.9

Vinegar 5

Walnuts, black 0.0

English 2.1

Watercress 2.0

Watermelon 2

W^heat, grain 3.1

bran 10.3

germ 8.1

puffed 3.0

shredded 3.5

Yams 7

Yeast, dried 13.0

lillignims per 100

ff.

Micrograms per 100 g

of edible

portion

of edible portion

Cu

Mn

Zn

Iodine

1.2

.23

1.4

35.5

.09

•)

9

">

1.1

2.9

1.8

6.3

1.2

3.2

7

V

.11

.73

.62

41.0

.08

.14

•?

2.3

.10

0'>

.21

•f

.07

.23

.09

•>

.15

.3

.23

2.4

.09

.04

V

•?

.07

•)

.04

V

•>

•)

V

16.0

.09

.13

.24

1.5

.5

?

•)

30.5

.17

.03

V

•)

.08

.16

.08

7.5

.08

1.9

.28

2.4

.20

.03

3.5

5.0

.04

1.0

V

9

3.2

•)

'>

9

.88

2.4

2.3

9

.1

.42

.56

3.6

.07

.02

9

9

.8

3.7

5.4

7.6

1.3

11.0

12.0

9

2.7

13.0

14.3

?

">

V

-)

V

•?

V

•>

9

•)

.05

'}

4.7

3.0

.5

9.0

9

445

Table A-4
Vitamin content of common foods

(Amount per 100 g., edible portion)

Panto-
Vitamin A Ascorhic Ribo- thenic Nicotinic
value acid Thiamine flavin acid acid
Food I.U. mg. mg. mg. mg. mg.

Almonds 0 trace 0.25 0.67 0.40 4.6

Apples, fresh 90 5 0.04 0.03 0.05 0.2

dried 0 12 0.10 0.10 0 1.0

Apricots, fresh 2.790 7 0.03 0.05 0 0.8

dried 7,430 12 0.01 0.16 0 3.3

canned * 1.350 4 0.02 0.02 0.09 0.3

Asparagus 1,000 33 0.16 0.19 0.50 1.4

Bananas 430 10 0.04 0.05 0.07 0.7

Barle.v, entire 70 0 0.50 0.20 0.95 6.0

dr.v pearled 0 0 0.12 0.08 0 3.1

Beans, dried 0 2 0.67 0.23 0.60 2.2

Lima, fresh 280 32 0.21 0.11 0 1.4

dried 0 2 0.48 0.18 1.2 2.0

string or green 630 19 0.08 0.11 0.15 0.5

green, canned t 500 5 0.04 0.05 0.06 0.4

baked, canned 80 2 0.05 0.04 0.09 0.5

Beef, medium fat 0 0 0.08 0.16 0.65 4.4

Beets 20 10 0.02 0.05 0.2 0.4

Beet greens 6.700 34 0.08 0.18 0.5 0.4

Brains 0 18 0.23 0.26 0 4.4

Bread, white 0 0 0.05 0.11 0.4 0.9

white enriched 0 0 0.24 0.15 0.4 2.2

whole wheat 0 0 0.30 0.13 0.8 3.0

rye (33%) 0 0 0.18 0.08 0.5 1.5

Broccoli . 3,500 118 0.10 0.21 1.2 1.1

Brussels sprouts 400 94 0.08 0.16 0.6 0.7

Buckwheat, entire ... 0 0 0.46 0.15 1.3 2.0

Butter 3.300 0 trace 0.01 0 0.1

Cabbage SO 50 0.06 0.05 0.23 0.3

celerv 260 31 0.03 0.04 0 0.4

Cantaloupe 3,420 33 0.05 0.04 0 0.5

Carrots 12,000 3 0.06 0.06 0.21 0.5

canned t 17.570 3 0.02 0.02 0.13 0.3

Cashew nuts 0 0 0.63 0.19 0 2.1

Cauliflower 90 69 0.11 0.10 0.80 0.6

Celerv 0 7 0.05 0.04 0.3 0.4

Chard 2,800 38 0.06 0.07 0 0.4

Cheese, cheddar 1,400 0 0.02 0.42 0.28 trace

cottage 20 0 0.02 0.31 0.25 0.1

Swiss 1,450 0 0.01 0.40 0.35 0.1

Cherries 620 8 0.05 0.06 0 0.4

Chicken 0 0 0.08 0.16 0.60 8.0

Chocolate, bitter 60 0 0.05 0.24 0 1.1

Clams 110 0 0.10 0.18 0 1.6

Cocoa 30 0 0.12 0.38 0 2.3

Coconut, fresh 0 2 0.10 0.01 0 0.2

dried, sweetened ... 0 0 trace trace 0 trace

Collards 6.870 100 0.11 0.27 0 2.0

Corn, entire, yellow 810 0 0.54 0.24 0.80 1.7

sweet, fresh 390 J 12 0.15 0.12 0 1.7

sweet, canned 230 $ 5 0.03 0.06 0.20 0.9

Cottonseed, meal 44 0 0.4 0.5 1.0 3.0

Cowpeas, dried 30 2 0.92 0.16 1.8 2.2

Crabs 0 0 0.14 0.06 0 2.7

Cranberries 40 12 0.03 0.02 0 0.1

Cream, heavy 1,440 1 0.02 0.11 0 0.1

Cucumbers 0 8 0.03 0.04 0.39 0.2

Currants, fresh 120 36 0.04 0 0 0

Solids and liquid.

t Drained solids.
446

I Yellow sweet corn.

Table A-l (Conlimied)
Vitamin content ol" cuniinon foods

(Amount per 100 g., edible portion)

V it(n>i ill A
value

Food I. IT.

Dandelion, greens .... VA.CiiA)

Dates m

Eels l.'OO

Eggplant ^^0

Eggs 1,140

Egg white 0

Egg yolk 3.210

Endive 3,000

Figs, fresh SO

dried 80

Fish, various (av.) .. 375

Flour, white, enrielied 0

wliole wheat 0

rye (light) 0

Frog legs, raw 0

Gooseberries 200

Grapefruit trace

Grapefruit juice,

canned trace

Grapes 80

Haddock 0

Heart 30

Honev 0

Kale 7,540

Kidney 1,150

Kohlrabi trace

Lamb (mutton) 0

Lemons 0

Lentils (dried) 570

Lettuce (head) 540

Liver 43,900

Lobster 0

Macaroni, enriched . , 0

Milk, cow, fresh 100

evaporated 400

powder 1,400

goat 160

human 80

Mushrooms 0

Mustard greens 6,460

Oatmeal (rolled oats) 0

Oats, entire 83

Okra 740

Olives 300

Onions 50

Oranges 190

Orange juice 190

Orange juice, canned . 100

Oysters 320

Parsley 8,230

Parsnips 0

Peaches, fresh 880

dried 3,250

canned * 450

Peanuts 0

Pears 20

Peas, green 680

canned t 670

* Solids and liquid. t Drained solids.

Panto-

[scorhic

Ribo-

thenic

Nicotinic

acid

Thiamine

flarin

acid

acid

mg.

mg.

mg.

mg.

mg.

36

0.19

0.14

0

0.8

0

0.09

0.10

0

2.2

0

0.28

0.37

0

1.4

5

0.04

0.05

0

0.6

0

0.10

0.29

2.7

0.1

0

0

0.26

0.13

0.1

0

0.27

0.35

6.0

trace

11

0.07

0.12

0.23

0.4

2

0.06

0.05

0

0.5

0

0.16

0.12

0

1.7

9

0.08

0.15

0

8.2

0

0.44

0.26

1.0

3.5

0

0.48

0.07

1.0

5.5

0

0.15

0.07

0

0.6

0

0.14

0.25

0

1.2

33

0

0

0

0

40

0.04

0.02

0

0.2

35

0.03

0.02

0.12

0.2

4

0.06

0.04

0

0.2

0

0.05

0.08

0

2.4

6

0.58

0.89

0

7.8

4

trace

0.04

0

0.2

115

0.10

0.26

0.40

2.0

13

0.37

2.55

0

(5.4

61

0.06

0.05

0.16

0.2

0

0.14

0.20

1.0

4.5

50

0.04

trace

0

0.1

5

0.56

0.24

0

2.2

8

om

0.08

0.13

0.2

31

0.26

3.33

5.5

13.7

0

0.13

0.06

0

1.9

0

0.88

0.37

0

6.0

1

0.04

0.17

0.30

0.1

1

0.05

0.36

0

0.2

6

0.30

1.46

2.5

0.7

1

0.04

0.11

0

0.3

3

0.01

0.04

0.2

0.2

5

0.10

0.44

0

4.9

102

0.09

0.20

0

0.8

0

0.60

0.14

1.3

1.0

0

0.62

0.17

L3

1.4

30

0.08

0.07

0

1.1

0

trace

0

0

0

9

0.03

0.04

0.15

0.2

49

0.08

0.03

0.07

0.2

49

0.08

0.03

0

0.2

42

0.07

0.02

0.13

0.2

0

0.15

0.20

0

1.2

193

0.11

0.28

0.6

1.4

18

0.08

0.12

0

0.2

8

0.02

0.05

0

0.9

19

0.01

0.20

0

5.4

4

0.01

0.02

0.05

0.7

0

0.30

0.13

3.4

16.2

4

0.02

0.04

0

0.1

26

0.34

0.16

0.58

2.7

9

0.12

0.06

0.20

1.0

447

Table A-4 (Continued)

Vitamin content of common foods

(Amount per 100 g., edible portion)

Vitamin A
value

Food I.U.

mature 370

Peppers, green 630

red 0

Persimmons 2,710

Pineapple, fresh 130

canned * 80

Plums 350

Pork, fresh 0

Potatoes, white 20

Prunes, dried 1,890

Pumpkins 3,400

Radishes 30

Raisins 50

Raspberries, red 130

Rhubarb 30

Rice, entire 0

polished 0

Rutabagas 330

Rye, entire 0

Salmon, red, canned * 230

Sardines, canned f... 0

Sauerkraut, canned t • 40

Shrimp, canned t • • • • 60

Soybeans, mature .... 110
Spaghetti —

see macaroni

Spinach 9,420

Squash, winter 4,950

summer 260

Strawberries 60

Sweet potatoes 7,700

Tomatoes, raw 1,100

canned 1,050

Tomato juice, canned . 1,050

Tuna, canned f 80

Turkey trace

Turnips trace

Turnip greens 9,540

Veal 0

Walnuts, English 30

Watercress 4,720

Watermelon 590

Wheat, entire 0

Wheat bran 0

Wheat germ 0

Yeast, brewer's, dried. 0

* Solids and liquid. t Drained solids.

Panto-

iscoriic

Rilo-

thenic

Nicotinic

acid

Thiamine

flarin

acid

acid

mg.

mg.

mg.

mg.

mg.

2

0.77

0.28

1.8

3.1

120

0.04

0.07

0.12

0.4

350

0

0

0

0

11

0.05

0.05

0

trace

24

0.08

0.02

0

0.2

9

0.07

0.02

0.10

0.2

5

0.06

0.04

0

0.5

0

0.58

0.14

0.70

3.1

17

0.11

0.04

0.4

1.2

3

0.10

0.16

0

1.7

8

0.05

0.08

0

0.6

24

0.03

0.02

0

0.3

trace

0.15

0.08

0

0.5

24

0.02

0.07

0

0 3

9

0.01

0

0

0.1

0

0.32

0.05

0

4.6

0

0.07

0.03

0.8

1.6

36

0.07

0.08

0

0.9

0

0.36

0.18

0

1.1-

0

0.04

0.16

0.75

7.3

0

0.01

0.17

0.60

4.8

IG

0.03

0.06

0

0.1

0

0.01

0.03

0.29

2.2

trace

1.07

0.31

1.6

2.3

59

0.11

0.20

0.20

0.6

8

0.05

0.12

0

0.5

17

0.05

0.09

0

0.8

60

0.03

0.07

0

0.3

22

0.09

0.05

1.0

0.6

23

0.06

0.04

0.08

0.5

16

0.06

0.03

0.23

0.7

16

0.05

0.03

0.25

0.8

0

0.05

0.12

0.34

12.8

0

0.09

0.14

0

8.0

28

0.05

0.07

0.25

0.5

136

0.09

0.46

0

0.8

0

0.14

0.25

1.3

6.4

3

0.48

0.13

0.88

1.2

77

O.OS

0.16

0.15

0.8

6

0.05

0.05

0

0.2

0

0.57

0.12

1.3

5.9

0

0.68

0.25

3.0

33

0

2.05

0.80

1.0

4.6

0

9.69

5.45

20

36.2

448

INDEX

Absorption, intestinal, 319
Absorption of fatty acids, 320
Accessory food factors, 200
Acetic acid :

bacterial formation of, 363. 377

in fat metabolism, 337

in porpbyrin syntbesis, 351

in steroid synthesis, 340

in vinegar, 163

metabolic reactions of, 339

yeast formation of, 363, 379
Acetoacetic acid :

formation in ketosis. 339

from amino acids, 354

from fat catabolism. 338, 339
Acetohacier suboxijdans :

growth factors for, 359

oxidation of glucose Ijy, 364

oxidation of sorbitol by, 37

products of, 363
Acctobacter xijlinum, 378
Acetoin, formation of, 383
Acetolactic acid, 383
Acetone :

formation of, 377, 384

from fat catabolism, 339
Acetylation of amines, 340
Acetylcholine, 340
Acetylmethyl carbinol, 85
Achlorhydria, 314
Acid-base balance of foods, 181
Acidity, active, 167
Acidity, active vs. total, 162, 163
Acidity, total :

determination in biological materials,
166

measurement of, 163
Acidosis, during ketosis, 339
Aconitase. 266
c'(.s-Aconitic acid, 331
Acrolein test, 88
Acromegaly, 305
ACTH, 291, 305

peptide nature, 292

properties of, 307
Actidione, 366

Addison's disease, symptoms of, 291
Adenase, 265
Adenine :

formula of, 154

metabolism of, 155

nucleic acids and, 154-159
Adenosine, 156
Adenosine diphosphate (ADP), 158

amount in muscles, 418

enzymes and, 267

relation to energetics, 416—418

Adenosine diphosphate (Cont.) :

role in carbohydrate metabolism, 324-
326
Adenosine monophosphate, 158
Adenosine tripliosphate (ATP) :

amount in muscles, 418

energy from, 415-417

enzymes and, 267

formation during hydrogen transport,
420-422

formation in plants, 405

formula of, 158

relation to carbohydrate metabolism,
323-329
Adenylic acid :

linkages in, 156

muscle, 158

with nucleic acids, 156, 157
ADP (see Adenosine diphosphate)
Adrenal cortex, hormones of, 290
Adrenal cortical hormones :

deficiency of, symptoms, 291

excess of, symptoms, 291
Adi-enal medulla, 287
Adrenal steroids, 290
Adrenaline (see Epinephrine)
Adrenocorticotropic hormone, 291, 305
Adsorption chromatography, 390
Aeration, effect on yeast growth, 361,

370
Aerobacter aerogenes, 364, 377
Agar, 67

Alanine, formula of, 116
P-Alanine, 359
Alanyl-glycine, 129, 131
Albumins :

amino acid content, 124

crystalline, 105

in common foods, 108

properties of, 110
Alcaptonuria, 350
. Alcohol dehydrogenase, 204, 268
Alcoholism, with polyneuritis, 228
Aldehyde groups, Schiff test for, 25, 27
Aldobiuronic acids, 21
Aldohexoses, stereoisomers of. 24
Aldolase, 266
Aldolase, amino acid composition, 125,

260
Aldonic acids, formation of, 36
Aldopentoses, stereoisomers of, 23
Aldoses. 19, 22
Aldotetrose, 20
Aldotriose, 20
Alginic acid, 65
Alkali disease, 195
Allose, 24
Alloxan, 302
Altrose, 24
449

450

INDEX

Aluminum, in living organisms, 176

Aluminum stearate, 88

Amidases, 264

Amide nitrogen, in plants, 402

Amination, 342

Amino acid decarboxylation, 321

Amino acid metabolism, abnormalities of,

350
Amino acid oxidases, 268
Amino acids :

absorption, 320

antiketogenic, 354

classification. 116

color tests, 142

content of in proteins, 124

content of foods. 127

D and L forms, 121

determination, 122

distinguishing groups in. 121

essential, 341-342

formulas, 116

gl.vcogeu-forming, 354

ketogenic, 354

microorganisms and, 358

nutritionally essential, 341, 342

semiessential, 341, 342

sequence in proteins, 131

synthesis by plants, 401, 402

utilization by plants, 401

utilization of related compounds, 341,
342
a-Aminoadipic acid, as lysine precursor,

341
p-Aminobenzoic acid. 254. 256, 359, 367
Aminobut.yric acid, formula, 116
Amino peptidases, 264

specificity of, 316
Ammonia :

as nitrogen source for plants, 400, 401

conversion to urea in vivo. 352

detoxification by plants, 402
Ammonification, 14, 400
Amygdalin, 28
Amylases :

action on starch, dextrin, and glycogen,
58

alpha and beta, 58

classification. 263
Amylopectin. 53
Amylopsin, 58, 263
Amylose, 53

molecular weight, 50

structure of, 50
Anabolism, 324
Androgens, 296
Androsterone. 295-297
Anemia, 248, 251
Anemia, nutritional, 187
Angiotonase. 304
Angiotonin, 304
Anhydrides, 21

Aniline acetate test for pentoses, 26
Animal protein factor, 251
Anions, of blood plasma, 182
Anorexia, relation of thiamine to, 227
Anterior pituitary, hormones of, 305, 307

Antibiotics (see Aureomycin, Penicillin,
etc.) :
as stimulants of animal growth, 251,
322

production methods, 370
Anticoagulant, heparin as, 67
Antidiuretic, effect of posterior pituitary,

303
Antieggwhite injury factor (see Biotin)
Antigray-hair factor, 255, 256
Antihistamines, 289
Antimetabolites, 257
Antioxidants, 90

Antipernicious anemia factor, 249
Antithyroid drugs, 300
Antivitamins, 256
Apoenzyme, 262

Appetite, stimulation by thiamine, 227
Arabinose, 26
Arabitol, 21
Arachidic acid. 77
Arachidonic acid, 80
Arginase, 266

relation to urea formation, 352, 353
Arginine :

bacterial decarboxylation, 321

biosynthesis, 346, 352

formula, 120
Arginine phosphate, in invertebrate mus-
cle, 418
Arsenic :

as antidote for selenium, 196

in living cells, 176
Arterenol (see Norepinephrine)
Arthritis, effect of cortisone and ACTH,

292
Ascorbic acid :

formula, 224

in foods, table, 447-448

microorganisms and, 359

physiological function. 222
Ascorbic acid oxidase, 224
Ash:

composition of, 177

content of foods, table, 434

in normal and rachitic bone, 210
Ashing of biological materials, 177
Asparaginase, 265
Asparagine :

formula, 119

in plants, 402
Aspartase, 265
Aspartic acid:

formula, 119

relation to urea formation. 353
Aspergillus niger, 360, 361, 363
Aspergillus sp., 363
Aspergillus terreiis, 376
Asthma, use of epinephrine in, 289
Asymmetric carbon atoms, 22
ATP (see Adenosine triphosphate)
ATP-ase, role in muscle contraction, 416
Aureom.vcin :

formula of, 367

mode of action, 369

range of activity, 368

INDEX

451

Autotrophic bacteria, 401

Avidin, 244

Azotobacter, nitrogen fixation by, 403,

404
Azotobacter vinelandii, 358, 360, 361

B

Babcock test, 91
Bacilliia anthracis, 131
BaciUus brevis, 370
BacillKS cereus, 375
Bacillus licheniformis, 369
BaciUus j)oh/miixa. 370
Bacillus subtilis, 379
Bacitracin. 124, 370

Bacteria, 357-386 (see specific bacteria
like BaciUus anthracis, etc.)

biotin s.vnthesis by, 244

intestinal flora, 321

synthesis of vitamins by. 220. 233
Bacteriochlorophyll, 390
Barfoed's solution, 30
Barfoed's test. 42
Basal metabolism :

definition, 424

factors influencing, 426

measurement of. 425

of various species, 426
Beeswax, 92. 93
Beet sugar, 42
Benadryl :

as antihistamine drug, 289, 290

formula, 290
Benedict's solution, 30, 31
Benzoquinone, Hill reaction with, 394
Beri-beri, 226
Bertrand's rule, 364
Beta oxidation, 336, 337
Betaine, 345
Bile, 317
Bile acids, 96
Bile pigments, 318
Bile salts, 90, 318
Bilirubin, 318
Biliverdin, 318
Biochemistry :

objectives and methods, 2

relation to biology, 6

scope of, 1
Biocytin, 246
Biophotometer. 204
Biotin, 244-246

formula, 246

in fat synthesis, 340

metabolic function, 280
Black-tongue, 236, 239
"Blind staggers," caused by selenium poi-
soning, 195
Blood clotting :

and calcium, 183

and dicoumarol, 221

and heparin. 67

and vitamin K. 219, 221

and Warfarin, 222
Blood plasma :

calcium content of, 214

Blood plasma (Cont.) :

cations and anions in, 182

inorganic phosphate of, 214

pH of, 174
Blood pressure :

regulation of, 304

with adrenaline, 288, 289
Blood sugar :

effect of epinephrine on. 289

factors affecting level of, 327

hormonal control of, 325

levels in diabetes, 301

sources of, 325
Body fat, effect of diet on nature of, 336
Bomb calorimeter, 423
Bones :

ash content of normal and rachitic, 210

composition of, 183

mineral content of, 17S
Borneol giucuronide, 39
Boron deficiency :

effect on apples, 195

effect on corn, 194

effect on tobacco plant, 409
Boron, essential for plants, 193, 194
Botulinum toxin A, 124
British thermal unit (BTU), 413
Bromeliii, 265

Bromine, in living cells, 176
Buffer calculations, 171, 172
Buffers :

capacity of. 173

definition, 170

in blood, 173

pH of, 170
Butter flavor, 85
Butterfat composition. 82
Butvl alcohol, 363. 377, 384
Butylene glycol, 363, 383
Butyric acid :

bacterial formation of, 363, 377, 384

in butter, 77

C

Cadaverine, 321
Caffeine, 155
Calciferol. 96, 213
Calcification of bone, 214
Calcium :

absorption, 320

content of foods, table, 439

food sources, 185

functions in body. 183

in animal body. 183

in blood plasma, 183, 214

in bones and teeth, 183

requirement, 184
Calcium cyanamide as nitrogen fertilizer,

400
Calcium deficiency, effect on tobacco

plants, 409
Calcium oxalate, 161
Calorie allowances, for men, women, and

children, 428
Calorie, definition, 413
Calorie requirement, relation to age. 429

452

INDEX

Calorific value of foods, 422, 423

Calorimeter, 423

Canaline, formula, 119

Canavanine, formula, 119

Cane sirup, 34

Cane sugar, 42

Capillary fragility, relation to vitamin

C. 223
Capillary resistance test, 223
Capric acid, 77
Caproic acid, 77
Caprylic acid, 77
Carbohydrases, 263

Carbohydrate content of foods, table, 434
Carbohydrate metabolism :

linkage to protein metabolism, 342, 343

summary, 334
Carbohydrates :

action of acids on, 26, 30, 42, 51, 57. 64

classification of, 20

definition of, 19

economic importance. 19. 20

formation in plants, 399

interconversion in body, 324

occurrence of, 19

physiological fuel value of. 423, 424
Carbon cycle in nature, 387
Carbon dioxide :

amount in atmosphere, 392

balance in nature, 387

sources of in carbon cycle, 387, 388

transport by blood. 187
Carbon dioxide fixation :

as a dark reaction, 394

function of biotin in, 245

in citric acid formation, 372

in oxalacetic acid formation, 332

in photosynthesis, 392, 393

in propionic acid formation. 383
Carbon, requirements of microorganisms,

358
Carbonic anhydrase, 179, 187. 269, 392
Carboxylases, 266
Carboxypeptidases, 264, 316
Carboxylation :

function of biotin in, 245

in photosynthesis, 392

use of light energy for. 394
Caries, relation to fluorine intake. 193
Carlic acid, 325
Carlosic acid, 375
Carnauba wax, 92, 93
Carnitine. 255
Carolic acid, dehydro, 255
Carolinic acid, 375
Carotene :

conversion to vitamin A, 207

crystals, 206

determination of, 208

formula of, 205

in butter, 84

synthesis by bacteria, 359
Carotenoids, 205
Carr-Price reaction, 208
Casein, 108, 124
Catabolism. 324

Catalase, 268, 282, 405

Cations of blood plasma, 182

Cell wall, cellulose and lignin in, 66

Cellobiose, 41, 46

Cellobiuronic acid, 21

Cellophane. 62

Cells, components of, G

Cellulases, 263

Celluloid, 62

Cellulose, 60

from wood, 61

industrial products from, 62

production by bacteria, 378
Cellulose nitrate, 62
Cephalins, 99, 100
Cereals, calcium in, 178
Cerebrosides. 101
Cerotic acid in waxes, 93
Ceryl alcohol, 93
Cetyl alcohol, 93
Chenodesoxycholic acid, 317
Chinese insect wax, 92, 93
Chitin, 64

Cliitosamine (see D-Glucosamine")
Chloramphenicol (see Chloromycetin)
Chlorella, photosynthesis by. 395
Chlorine, biochemical importance. 192
Chlorine compounds in biological mate-
rials. 192
Chlorine content of foods, table, 439
Chlorocruorin, 139
Chloromycetin :

formula of, 368

mode of action of. 369

range of activity, 369
Chlorophvll a, formula. 389
Chlorophvll h, 300
Chlorophylls. 388-391

amount in leaves. .390

esterase, 264
Chloroplasts, 388
Chlorotic plants, manganese in relation

to, 189
Cholamine. 99
Cholecystokinin, 308, 318
Cholesterol :

in heart disease. 95

occurrence, 94, 95

synthesis in body, 95
Cholesterol esterase, 264
Cholic acid, 96, 317

for cortisone synthesis, 292

formation from cholesterol, 340
Choline, 253

as source of methyl groups, 343-345

biosynthesis of, 345

prevention of fatty livers by. 336
Choline chloride, 162
Chondroitin sulfate, 67
Chondrosamine, 37

Chromatium and nitrogen fixation. 403
Chromatography, 390
Chromoproteins, 137
Chromosomes, 150, 152
Chylomicrons, 336
Chyme, 312

I

I

INDEX

453

Chyniotrypsin :

amino acid composition, 124

classification, 265

in pancreatic secretion, 315
Chymotrypsinofien, conversion to chymo-

trypsin, '^10
Vis- and trans- isomers, 79
Citric acid, 161

mechanism of formation, 372

])roduction of. 363
Citric acid cycle, 330. 332, 380

enerjiy from, 410. 421
Citrovaiiiin factor, 248
('itrnlline, formula. 120
Clostiidiiint aceiohHfiflicinii :

butyric acid formation by, 377

fermentation pi-oducts of. 36.3

growth efficiency of, 123, 360, 361

growth factors for, 359
Clostridium acidiurici, 359
Clostridium buti/licum, 363
Clostridium, nitrogen fixation bv, 403,

404
Clostridium snccharohutyricum. 363
Clostridium tetani. 359
Clupanadonic acid, SO
Cobalamin, 251
Cobalt :

in hemoglobin formation, 188

in vitamin Bjo. 189
Cobalt deficient areas, 188
Cocarboxylase, 227, 273
Cocoanut oil. composition, 82
Codecarboxylase, 243

Coenzyme I {see Diphosphopyridine nu-
cleotide)
Coenzyme II (see Triphosphopyridine nu-
cleotide)
Coenzyme A, 240

growth factors and, 359

structure of, 274
Coenzyme R {see Biotin)
Coenzymes :

as activators, table. 266-268

as i)rosthetic groups, 262

ex.imples of, 273-280

hydrogen carrying, 332, 333

reduction of by light energy, 394, 395
Coleoptiles, bending of by plant hormones,

406
Comb growth, effect of androgens on, 296
Composition of food :

variations in. 433

tables, 433-448
Configuration of stereoisomers, 23
Conjugases, 265

Conjugated double bonds, 390, 391
Constipation, in thiamine deficiency, 228
Copper :

compounds in living cells, 179, 186

destruction of vitamin C by. 226

enzymes containing. 186

human requirement for, 188

in foods, table, 442

role in hemoglobin formation, 187
Coprogen, 360

Coprophagy, 322

Cori ester {see D-Glucose-1-phosphate)

Corn sirup, 29

composition of, 57
Corpus luteum, 293
Corticosterone, formula, 290
Cortisone :

formula, 290

physiological effects of, 291

synthesis of, 292
Coumarin, 221
Cream of tartar. 161
Creatine, biosynthesis of, .348, 349
Creatine phosphate (CrP) :

amount in muscles, 418

relation to muscle contraction, 415, 416

structure of, 349
Creatine transphosphorylase, 267
Cretinism, 299
Crotonic acid, 76
Cryptoxanthine, 207
Cushing's syndrome. 291
Cyanide group, in vitamin Bjo. 250, 251
Cyanocobalamin, 251
Cystathionine, .344, 345

formula, 118

in cystine synthesis, 122
Cysteine, formula. 117
Cystine, 115

biosynthesis of, 345

formula. 117
Cytidine. 156
Cytidylic acid, 156

Cvtochrome c, as electron carrier, 279,
282, 283

formula, 279

iron content of, 139, 279

protein nature of, 139

role in hydrogen transport, 333, 334

role in oxidation, 282-283
Cytochrome oxidase, 268, 334
Cytochrome system, 332, 333, 404
Cytochromes, 139
Cytosine, 154-158

D

2, 4-D, formula of, 407
Dark adaptation, with vitamin A, 204
Dark reactions of photosynthesis, 393
Deamination of amino acids. 351
Decarboxylases. 266
Decarboxylation, bacterial, 321
Dehydroascorbic acid, formula, 224
Dehydrocarolic acid, 375
7-Dehydrocholesterol, 95
11-Dehvdrocorticosterone, formula, 290
Dehydrogenases, 2(i2, 282
Denaturation of proteins, 144
Denatured proteins, properties of, 145
Denitrification, 402
Dental caries, 193
Derived proteins, 112
Dermatitis, of pellagra, 235, 236
Desmolases, 266
Desoxycholic acid, 317
11-Desoxycorticosterone, formula, 290

454

INDEX

Desoxy-D-glucose, 38
Desoxyhexoses, 37

Desoxy nucleic acid (DNA), 152-154
Desoxyribose. 27

and nucleic acids, 154, 156-158
Desoxysugars, 20

Desiilfovihrio, nitrogen fixation and, 403
Detergents, synthetic, 15, 87
Detoxication, 340
Deuterium, as metabolic tracer, 335, 340,

343
Dextrans, 60, 378
Dextrin, limit, 58
Dextrins, 57
Dextrose, 28
Diabetes. 327

ketone bodies and, 339

urinarv glucose in, 327

with alloxan, 302

with phlorizin, 302
Diabetes insipidus, 303
Diabetes mellitus. 301
Diabetic animals, utilization of carbohy-
drate by. 302
Diabetogenic hormone, 305. 325. 327
Diacetyl, 85

Diaminobutyric acid, 119. 122, 370
Diastase, 58
Dibasic acids :

from fat catabolism, 337

in waxes, 94
2-6-Dichlorophenolindophenol, use in de-
termining vitamin C, 225
2, 4-Dichlorophenoxyacetic acid. 407
Dicumarol. 221

Diethylstilbestrol (see Stilbestrol)
Dieting, dangers of, 429
Digestion, 311-322
Digestion, gastric, 312

intestinal, 315

salivary, 311
Dihydrosphingosine, in cerebrosides, 101
Dihydroxyacetone phosphate, 329
Dihydroxvphenylalanine, 288
Diiodotyrosine. 118, 297, 298
5, 6-Dimethvlbenzimidazole, from vitamin

Bi„, 250
Dimethylbenzimidazole riboside, 159
Dimethylpropiothetin, 345
Dipeptidases, 265
Diphosphopyridine nucleotide (DPN) :

forms of, 275

function in cytochrome system, 332, 333

relation to nucleotides, 158

role in tissue oxidation, 282

structure of, 275
Diphosphothiamine (see Thiamine pyro-
phosphate)
Disaccharide linkage, 41
Disaccharides, composition of, 40

hydrolysis of, 42

reducing power of, 41, 42
Diseases, nutritional, 200
Dissociation constants, definition, 170
Djencolic acid, formula, 118
Doisynolic acid, 297, 298

"Dopa," formula, 349

Double bonds, con.iugated, 390, 391

DPN (see Diphosphopyridine nucleotide)

Drving oils, 89

Dulcitol, 21

Dwarfism, 305

E

Eating habits, relation to body weight,

429
Eberthella typhi., fermentation products

of, 128. 363, 377
Edestin. 108

Eggwhite injury disease. 244
Einstein, energy unit, 395
Elaidic acid, 79
Electrolyte metabolism, effect of cortical

hormones on. 291
Electrolytes, in blood plasma. 182
Electrophoresis of proteins, 107
Eleostearic acid, 80
Embden-Meyerhof scheme, 326, 329
Emulsifying agent, lecithin as, 98
Emulsin, 263

Endergonic reactions, definition, 414
Endocrine glands, 286
Endoenzymes, 260
Energetics, biological definition, 413
Energy, forms of in living things, 413

content of foods, table, 434

for muscular work, immediate source of,
416

phosphate bond, 416^22

sources of, for bacteria, 358
Energy metabolism, efficiency of, 422
Energy production in tissues, 334
Energy requirements, for various muscu-
lar activities, 427

of animals, 424-427

of human beings, 426^28
Energv units, interrelationships between,

413
Engines, efficiency of, 422
Enolase, 266
Enriched bread, 232
Enriched flour, 232
Enterogastrone, 309, 314
Enterokinase, 316
Entropy (As), 414
Entropy changes. 424
Enzymes, 260-285

activation, 273

amino acids in, 124-126, 260

chemical nature, 260

classification, table, 262, 263-269

crystalline, 260

definition of, 260

effect of ions on, 273

effect on energy of activation, 270

endo and exo, 260

factors affecting activity, 271

inhibition of. 272

mechanism of action, 270

nature of action, 270

occurrence, 260

prosthetic groups of, 261, 278

INDEX

455

Enzymes (Cont.) :

role in tissue oxidation, 281

specificity, 272

substrate complex, 270

table, 2G3-2G9
Epinasty, 406
Epinephrine. 287-289

biosynthesis of, 2S8

physiological effects, 288. 289

relation to nerve transmission. 289
Epithelial tissues, vitamin A and, 203
Equilibrium constant, relation to A^, 415
Equivalent, chemical, 164
E(iuivalent weights, 164
Erdin. 369. 37(>
Ergosterol, 96, 359

formula, 213
Erucic acid. 80
Erythritol. 21
Erythrocruorin, 139
D-Erythrose, 20
Escherichia coli, 358. 359

fermentation products of, 363, 377
Essential amino acids, 341, 342
Essential fatty acids, 335

in pyridoxine deficiency, 241
Essential lipides, 97
Esterases, 264
Esterification, 72
Esters, 71

hydrolysis of, 72

industrial use as solvents, 72

properties of, 72

saponification of, 73
Estradiol :

formula, 296

physiological functions of, 292-295
Estriol, formula, 296
Estrogens. 293, 296
Estrogenic hormones, 293, 296
Estrone, formula, 296
Estrus. 293
Ethanolamine, 99, 344
Ether extract, composition of. 91
Ethyl alcohol, formation of. 363. 367. 379
Exercise, relation to body weight, 429
Exergonic reactions, definition, 414
Exoenzymes, 260
Eyes, effect of vitamin A deficiency. 203

F

Factor R, 247

Factor S, 247

Factor U, 247

Faraday, definition of, 420

Fat content of foods, table, 434

Fat oxidation by animals :

relation of citric acid cycle to, 338, 339

theories of, 336-338
Fat-soluble substances, 86
Fat-soluble vitamins, storage of, 216
Fat synthesis, 339. 340
Fat transport, 336
Fats :

absorption of, 318. 320

chemical properties of, 86

Fats (Cont.) :

definition of true, 74

desaturation of, by animals, 335

determination of, 91

dynamic state of, 335

elementary composition, 75

fatt.v acid comjiosition of. 82

industrial importance of. 74

iodine number of, 89

modification of by animals. 335

occurrence of. 74

physical pi-operties of, 84

l)hysiological fuel value of, 423. 424

raTicidity of, 90

removal by exercise. 429

saturation of. 89

storage in body, 335
Fatty acids :

distribution, 80. 82

essential in nutrition, 79

occurrence in various fats, 82

omega oxidation of, 337
Fatty acids, saturated. 76, 77

formulas. .SO

occurrence in fats. 77

odors of lower, 77

rancidity due to lower. 78

table of. 77

volatility of lower, 78
Fatty acids, unsaturated:

formulas, 80

geometric isomers of, 79

melting points of, 78

occurrence, 80

table of. SO
Fatty aldehydes, in plasmalogens, 101
Fatty livers. 253, 336

choline and, 253

methionine and, 253

prevention by raw pancreas, 303
Feces, composition, 320
Fehling's solution, 30, 31
Fehling's test, 30, 31

structure responsible for, 35
Fermentation, alcoholic, 379, 381

butyric, but.vl. acetone, 377

colon-aerogenes-typhoid, 377

definition of, 362

heterolactic. 377, 381, 382

homolactic. 376

methane, 377

products of microorganisms, table. 363

propionic, 377
Fermentation products, related series of,

374, 375
Ferritin, 140, 186
Fibrinogen, 108, 124
Fibroin, of silk, 110, 124
Ficin. 265

Fixation of nitrogen. 402, 403
Flavin adenine dinut'leotide (FAD). 277
Flavin mononucleotide (FMX). 159. 277,
278

function in cytochrome system, 332. 333
Flavin nucleotides, as hydrogen carriers,
332, 333

456

INDEX

Flavoproteins, 140, 268
Fluorine :

in bones, 193

relation to tooth decay, 193

toxicity of. 193
Fluoroapatite, in bones, 193
FMN (see Flavin mononucleotide)
Folacin. 247
Folic acid, 247

food sources, 249

metabolic function, 281

relation to methvlation in v'wo, 345
Folinic acid. 248
Follicle-stimulatins hormone (FSH). 292

properties of. 307
Food calorie utilization, relation to work

done, 428. 429
Food intake, relation to body weight, 429
Foods :

amino acid content. 127

cost of food calories, 43, 44

mineral composition of, tables, 439-445

proximate composition of. table. 434-
438

vitamin content of, table, 447—448
Foot-pound (energy unit), definition. 413
Formic acid, 77. 363. 377, 383

from glycine in animal body, 344
Formyliiteroic acid. 247
Formylpteroylglutamic acid, 248
Formyltetrahydropteroylglutamic acid,

359
Fragility test {see Capillary resistance

test )
Free energy change :

in oxidation-reduction reactions, 420

of glycolysis, 418, 419

relation to chemical eijuilibrium. 414,
415
Free energy (A-P'), definition, 414

equilibrium constants and, 415

of glucose combustion. 414

relation to entropy and heat change, 414
Free HCl, gastric juice, 312, 313
Fructosans, 63

Fructosazone (see Glucosazone)
Fructose, 33, 34

metabolism of. 324. 326

ring forms of, 26, 35
Fructose-1, 6-diphosphate, in glycolysis,

326
Fructose-6-phosphate, 336
Fruit drop, control of premature, 406, 407
Fucose, 38
Fuel value, of various food materials, 422,

423
Fumarase, 266
Fumaric acid, 331
Furanose ring forms of sugars, 26
Furfural, 53

Furfuraldehyde from pentoses, 26
Fusarium avenaceum, 370

G

Galactans, 63
Galacto-araban, 52

Galactosamine, 37
Galactosazone crystals, 47
D-Galactose. 32

metabolism of. 324. 326
L-Galactose, 33

Galactose-1-phosphate, 280, 326
Galactosidases, 263
Galactosides, 32
Galacturonic acid, 38, 39
Gallstones, 95

Gases, exchange in lungs, 187
Gastric digestion, 312
Gastric iuice :

HCl in. 312. 313

pH of, 312

regulation of flow, 314

secretion of, 308
Gastrin, 308

Gastrointestinal hormones, 306
Gelatin, 124
(Jentiobiose. 41
Gentiobiuronic acid, 21
Geodin. 376
Geometric isomers, 79
Geotropism. 406
Germinating seeds, formation of amides

by. 402
Gigantism, 305
Gliadin. 109. 125
Globulins, 108, 109, 110, 111, 125
Glucoascorbic acid, antagonist of vitamin

C, 256
Glucokinase, 267
Gluconic acid, 22, 129, 364, 380
Glucosamine, 37

Glucosamine, N-acetyl. in chitin, 64
Glucosamine, N-methyl, 37
Glucosans, 21
Glucosazone, 31, 32
Glucosazone crystals, 47
Glucose :

blood levels of, 325, 327

commercial preparation of, 29

formation in nature, 28

from amino acids, 354

metabolism of, 236, 324

occurrence, 28

oxidation of by cupric ion, 20

phosphorylation and absorption, 325

ring forms of, 25
Glucose-l,6-diphosphate, 280
Glucose-1-phosphate, 280

in glycolysis, 326
Glucose-6-phosphate, from hexokinuse re-
action, 324
Glucosidase, 263
Glucosides, 28
Gluco-xylan, 52
Glucuronic acid, 38, 378
Glutamic acid :

formula of, 119

in plant metabolism, 401, 402

in transamination, 342. 343

role in nitrogen fixation, 404

y-linkage, 131
Glutamic decarboxylases, 206

INDEX

457

Glutamic dehydrogenase, 208
Glutamic-oxalacetic transaminase, 2G8
(ilutaniinase, 2G5
Glutainiiie, 34S

fornnila, 119

in plants, 402

source of urinary ammonium salts, 354
Glutatliione, 192

formula, 130

in oxidation-reduction, 280
Glutelins. 108, 109, 111
Glutenin, 109, 125

Glyceraldehyde, dcxiro and leva forms, 22
Glyceraldehyde-S-phosphate, 329
Glyceric acid-2.3-diphosphate, 2S0
Glyceric acid phosphates, 329
Glycerides, formulas of, 83

in natural fats, 81, 84

mixed, 81, 83 .

physical state in relation to fatty acid
composition, 78

simple, 81, S3
Glycerol ( glycerine ) , 76

metabolic oxidation of, 329, 336

production by yeast. 377, 379, 381
a-Glvcerophosphoric acid, 98
Glycine :

as purine and porphyrin precursor, 350,
351

as serine precursor, 345

formula, 116

in bile salts, 96

in creatine biosynthesis, 348
Glycocholic acid, 96, 317
Glycogen, 59

amounts present in liver and muscles,
325

branched structure of, 50

depletion of body stores in diabetes, 301

formation in body, 325

from amino acids, 354

from glycerol, 336

with adrenalin, 289

with insulin, 301
Glycogen metabolism, relation to muscu-
lar work, 417
Glycogen phosphorylase, 267
Glycolipides, 73, 101
Glycolysis :

ATP formation during, 418

efficiency of, 419

energy yield from, 418

equation for, 328

in plants, 405

in yeast, 381

reactions of, 326, 329
Glycolytic mechanism, in photosynthesis,

397
Glycoproteins, 136
Glycosidases, 263
Glycosides, 39, 40
Glycyl-alanine, 129, 131
Glvoxalase, 267
Glyoxylic acid, 397, 398
Goiter, 192, 299
Gonadotropic hormones, 292

Graafian follicle, 293

Gramicidin, 122, 131

Grana, 388

Grapefruit, effect of zinc deficiency on,

191
Grave's disease, symptoms of, 299
Graying of hair, relation to vitamin in-
take, 239
Grisein, 366
Growth efficiency, effect of oxygen on, 360,

361
Growth factors, for microorganisms, 359
Growth hormone, 305

properties of, 307
Growtii regulating substances in plants,

406
Guanidinoacetic acid, 348
Guanine :

formula of, 154

metabolism of, 155

nucleic acids and, 154-158
Guanosine, 156
Guanylic acid, 156
Gulose, 24
Gum arable, 66
Gum ghatti, 66
Gun cotton, 62

H

Hair, zinc content of, 191
Hallochrome, 350

Halogeton weed, oxalates in, 161, 162
Hardening, of fats or oils by hydrogena-

tion, 89
Harden-Young ester (see Fructose-1. 6-

diphosphate)
Heat change {/S,H), definition, 41.'!
Heat engine operation, difference from

muscle contraction, 415
Heat of combustion :

of food materials, 423

of glucose, 414
Heat production in body, relation to ef-
ficiency of energy use, 415
Heat prostration, cause of, 183
Heat regulation of body, 11
Heavy water, use in metabolic tracer ex-
periments, 340
Hemicelluloses, 21, 66
Hemin, 137, 360
Hemocuprein, 140, 187
Hemocyanins, 140, 186
Hemoglobin :

amine acid content. 125

classification, 112

components of, 137

in carbon dioxide transport, 138, 187

in oxygen transport, 138

in root nodules, 103, 403
Hemophilia, 221
Hemophilus influenzae, 360
Hemophilus parainfluenzae, 359, 360
Hemorrhage :

control of by oxytocine, 304

in vitamin C deficiency, 222, 223

in vitamin K deficiency, 221

458

INDEX

Hemorrhagic disease of infants, 221
Heparin, 67

Hepatocupreiu, 140, 187
Herbicides, selective, 407
Heteropolysaccharides, 51, 66

of animals, 67

of plants. 66
Hexokinase inhibition :

by diabetogenic hormone, 325, 326

effect of insulin on, 302
Hexokinase reaction, 324
Hexosamines, 37
Hexosans, 53
Hexosediphosphate, 179
Hexosemonophosphate, 179
Hexuronic acids :

furfural from, 37

relation to pentoses, 39
Hill reaction, 393, 394
Hippuricase, 265
Histamine, 321

effect on secretion of gastric juice,
308

relation to allergy, 289
Histidine :

bacterial decarboxylation of, 321

formula, 115, 120
Histidine decarboxylases, 266
Histones, 111, 153
Homocysteine :

for rat growth, 343, 344

formula, 117

intermediary product, 122
Homogentisic acid, 350
Homopolysaccharides, 51
Homoserine, formula, 116
Honey, sugars in, 33
Hopkins-Cole test, 143
Hordein, 108, 125
Hormones :

adrenal, 287-290, 290-202

anterior pituitary, 305-307

antidiuretic, 303

cortical, 290-292

definition, 286

estrogenic, 296

follicle-stimulating (FSH), 307

gastrointestinal, 306-309

gonadotropic, 292

growth, 305, 306

lactogenic, 295, 305-307

luteotropic, 293, 294

metabolic function of, 287

of plant growth, 406, 407

ovarian, 292

pancreatic, 300-303

parathyroid, 182

posterior pituitary, 303

progestational, 294, 297

testicular, 295, 296

thyroid-stimulating, 307
Hyaluronic acid, 67
Hyaluronidase, 67, 263
Hydrases, 266

Hydrocarbons, higher paraffin in waxes,
93

Hydrochloric acid, gastric secretion of,

312-313
Hydrogen, bacterial formation of, 377,

383
Hydrogen bonds, 10
Hydrogen carriers, coenzymes as, 334
Hydrogen equivalent, 164
Hydrogen ion concentration :

biological importance of, 167

pH and, 169
Hydrogen sulfide, in colon, 321
Hydrogen transport system, 333

as energy generator, 420-422
Hydrogenation of oils, 89
Hydrolases, 263
Hydrolecithin, 97
Hydrolysis, 19

Hydroperoxides in fat oxidation, 90
Hydroquinone, 394

as antioxidant, 90
3-Hydroxyanthranilic acid, 347
P-Hydroxybutyric acid, 339
17-Hydroxycorticosterone formula, 290
17-Hvdroxv-ll-desoxycorticosterone for-
mula, 290
3-Hydroxykynurenine, 347
Hydroxylysine, formula, 119
Hydroxyproline :

biosynthesis of, 346

formula, 121
5-Hydroxytryptamine, 304
Hydroxytyramine, 288
Hyperacidity, 314
Hyperglycemia, 327
Hyperglycemic factor of pancreas, 303
Hypertension, 304
Hypoglycemia, 327
Hypophysis, hormones of, 303
Hypoxanthine :

formula of, 154

metabolism of, 155

Idose, 24
Imino acids, 352
a-Iminoglutaric acid, 342
Immuno-polysaccharides, 68
Indican, 321
Indicators :

for pH measurement, 173

use of for acid-base titrations, 167
Indole, 320. 354
Indoleacetic acid, 406

effect on rooting, 408
Indoleacetonitrile, 406
Indole-5,6-quinone, 350
Inorganic elements, required by micro-
organisms, 360
Inorganic phosphate, formation of ATP

from, 422
Inositol, 254, 367, 378
Inositol meta diphosphate in phospholi-

pides. 101
Insulin, 300-302

amino acid content, 125

mechanism of physiological action, 302

INDEX

459

Insulin (Cont.) :

se(|uenoe of nmino acids in, 110, 132

zinc in, IS!), 190
Insulin shock, 302. 327
Intermediary metabolism, 3S0
Intestinal absorption, 319, 320
Intestinal digestion. 315
Intestinal juice, enzymes in, 318, 319
Intestinal secretion, 31S
Intrinsic factor in pernicious anemia, 251
Inulin, 63

Inversion of sucrose, 42, 43, 45
Invert su^ar. 42, 45
Invertase, 202, 263
lodinated proteins as source of thyroid

hormone, 299
Iodine :

human requirement for, 192

relation to goiter, 192
Iodine content of foods, table, 442
Iodine number of fats, 88, 89
Iodine, radioactive, use in Grave's dis-
ease, 300
Iodized salt, 192
lodoijorgoic acid, 297, 29S
Ion antagonism, 185
Ion exchange resins, 16-17
Iron :

absorption of, 320

availability of various forms, 188

compounds of in living cells, 179, 186

content of foods, table, 442

food sources of, 188

human requirement for, 188

storage of in body, 186
Irradiated milk, 214
Islets of Langerhans, 301
Isocitric acid, 331
Isocitric acid dehydrogenases, 268
Isocitric acid, dehydrogenation of, 333
Isocitric acid, oxidation energy from, 421
Isoelectric pH, 144
Isoleucine, formula, 116
Isopropyl alcohol, 377
Isotopic carbon as metabolic tracer, 339,

340
Isotopic nitrogen (N^^) as metabolic

tracer, 404
Isotopic tracer studies, 335, 345

Jaundice, use of vitamin K in treatment

of, 220
Jelly, pectin in, 65

K

Keratin, 110, 125

a-Ketobutyric acid, from cystathionine,

345
Ketogenic diets, 339
Ketogluconic acid. 304, 380
a-Ketoglutaric acid, 331, 342

role in nitrogen fixation, 404
a-Ketoglutaric acid oxidation, energy

from, 421
Ketoglutaric oxidase, 266

Ketoheptoses, 21
Ketone bodies, 339
Ketose, definition, 19
Ketoses, 22

differentiation from aldoses, 36

Fehling's reaction with, 35

test for. 30
Ketosis, 338, 339

in diabetes, 301
17-Ketosteroids, 299
Ketotriose, 20

Kidney, effect of phlorizin on, 302
Kilocalorie, definition, 413
Kjeldahl method, 146
Knoop's theory of fat catabolism, 336,

337
Krebs cycle, 330

in plants, 405
Krebs-Henseleit urea cycle, 352-354
Kynurenine, 347

Lacteal, 319
Lactic acid :

amount in muscles, 418

bacterial production of, 363, 376. 377

formation during violent exercise, 328
Lactic acid dehydrogenase, 268
Lactobacillus arahinosus, 358
Lactohacillus hulgaricus, 359
Lactohacillits casei, 359
Lactobacillus citrovorum, 359
Lactobacillus delbruckii, 363
Lactobacillus gaijonii, 363
Lactobacillus lijcopersici, 363
Lactobacillus pentoaceticus, 377
Lactogenic hormone, 125, 295

properties of, 307
Lactoglobulin, 125
Lactosazone crystals, 47
Lactose, 48, 49, 263

alpha and beta forms of, 49

food value, 48
Lanolin, 94
Lanthioniiie :

as source of cystine, 345

formula of, 117, 122

in subtilin, 122
Laurie acid, 77
Lead poisoning, treatment with vitamin

C, 223
Lecithinases A and B, 98
Lecithins, 97

with fat transport, 253
Legumin, 109, 125
Leucine, formula, 116
Leuconostoc citrovorum, growth factor

for, 248
Leuconostoc niesenteroides, 378
Leucosin, 108, 109
Leucovorin, 248
Levans, 63, 379

Levulinic acid, from hexoses, 26
Levulose, 33

Light reactions of photosynthesis, 393
Lignin, 61

460

INDEX

Lignocerylsphingosine, 100
Limit dextrins. 58
Linoleic acid, 80, 82
Linseed oil as paint oil, 89
Lipases, 86, 263
Lipides :

classes and hydrolysis products, 71, 73

compound, 73

definition, 71

derived, 73

essential, 97

in plant seeds. 400

metabolism of, 334-341

simple, 73
Lipocaic, 303
Lipoic acid, 254, 274, 360
Lipoproteins, 112, 141
Lipothiamide, 274, 359
Lipothiamide pyrophosphate, 254, 255
Lipotropic action, 336
Lithocholic acid, 317
Liver, fatty, 253
Lohman reaction, 416. 417
Luteinizing hormone, 292

properties of, 307
Luteotropic hormone, 293, 294
Lycopene, 205
Lysine :

formula. 119

bacterial decarboxylation, 321
Lysolecithin, 98, 263
Lysozyme, 263
Lyxose, 23

M

Macrocytic anemia, 248, 251

Magnesium :

content of in American dietary, 186
role in animal body, 185

Magnesium content of foods, table, 439

Magnesium deficiency, effect on tobacco
plants, 409

Magnesium ions, enzymes activated by,
185

Maleic hydrazide, as plant growth sub-
stances, 407

Malic acid, 161, 331

Malic acid formation, use of light energy
for, 394

Malic acid oxidation, energy from, 421

Malt sirup, 45

Maltase, 263

Maltosazone crystals, 47

Maltose, 45

structural formula, 46

iso-Maltose, 21, 41

Manganese content of foods, table, 442

Manganese deficiency, effect on leaves, 190

Manna, 33

Mannans, 33, 64

Mannitol, 33, 35

Mannoheptulose, 21

Mannosazone (see Glucosazone)

Mannose, 33

Mannose, metabolism of. 324, 326

Mannose-6-phosphate, 326

Mannosidostreptomycin. 365
Mannuronic acid, 38, 39, 65
Maternal instincts, relation of lactogenic

hormone to, 305, 306
Melanin, 349, 350
Melezitose, 49
Melibiose, 21, 41
^lelissic acid, in waxes, 93
Melissyl alcohol. 93
Menadione. 220

Menstrual cycle, hormonal control of. 298
Menstruation, 292
Mental attitudes of animals, effect of

hormones on, 305
INIercerized cloth, 61
Mesquite gum, 27

arabinose from, 26
Metabolic rate of microorganisms, 361
Metabolism :

antimetabolites in study of, 5
inborn errors of, 350
lipide, 334-341
methods of study, 4
of amino acids. 341-354
of carbohydrates, 323
of protein. 341-354
of sugars, 324-330
use of isotopes in, 5
Metabolism of microorganisms, 357-386
aerobic, 364
anaerobic, 376
comparison of products, 362
intermediary, 380
Methane, bacterial formation of, 377. 384
Methane in colon, 321
Methanohacierium omelianskii, 377
Methemoglobin, 138
Methene bridges, in chlorophyll molecule,

391
Methionine :

conversion to cystine, 122
effect on fatty livers, 336
formula of, 117
in choline synthesis, 345
in creatine synthesis, 345, 348, 349
methylation and, 343-345
Methionine sulfoximine, 257
Methyl donors, 344
Methyl-a-D-glucoside, 40
Methylamine, 162

Methylation. as a metabolic reaction, 343
Methvlcvtosine, 154
5-Methylfurfural, 38
Methylpentoses, 37
Methyltetronic acid, 375
Microbiological assay of riboflavin, 2.')4
Micrococcus pyogenes, 374
Micromole, definition, 164
Microorganisms :

growth efficiency, 360
growth requirements of, 358
interrelations of animals, plants, and,
357
Milk :

mineral deficiencies of, 188
vitamin D enriched, 214, 215

INDEX

461

Milk production :

effect of iodinated casein on. 200

effect of lactogenic hormone on,
205
Milk secretion, stimulation by oxytocin,

304
Millie(]uivalent. definition, 164
Millimole, definition, 1(54
Jlillins of grains, loss of thiamine during,

231, 232
IVIillon test. 142
Mineral elements :

abnormal distribution, 180

absorption of, by plants, 400

availability of various forms, lOG

excretion of, 107

general biological functions, 181

in various organic compounds, 170

loss from foods on cooking, 107

loss on ashing, 177, 178

major, 176

minor, 176

needed by animals, 176

needed by plants, 176

occurrence, 3 78, 179

testing for, 178
Minerals :

in animal body, 178

in foodstuffs, 178
^Miscarriage, prevention by progesterone,

204
Mitochondria, from plant tissue, 405
]Moisture content of foods, table, 434
Molar solutions, 163, 164
Molarity of solutions, 163-165
IMold spoilage, 376
]Mole, definition, 163
Molybdenum, needed by plants, 104
Molybdenum toxicity, 194
Monosaccharides, 20, 22

absorption, 320

cyclic and open chain forms, 24
Montanic acid in waxes, 93
Montanvl alcohol, 93
Mottled teeth, 193
Mucic acid, 33

crystals of, 47
Mucilages, 66
Mucin, 311
Mucoitin sulfate, 67
Mucous cells, 312
Muscle contraction, 415-416

stimulation of smooth, by oxytocin,
304
Muscle dystrophy, 217
Muscular activities :

energy expenditures for, 427

relation to composition of muscle,
418
Mustard oil, 28
Mutases, 266
Myogen, 108, 125
Mvokinase, 267
Myosin, 108, 126
INIyristic acid, 77
Myxedema, 209

N

Naphthalene acetic acid, as plant growth

regulator, 407
Naphthoquinone derivatives, vitamin K

activity of. 220
Naphthoresorcinol, test for uronic acids,

39
Nerve impulse, transmission of, 280
Neuberg ester {see D-Fructose-6-phos-

phate)
Neuritis, in thiamine deficiency, 228
Niacin (see Nicotinic acid)

in foods, table, 447-448
Nicotinamide, 238
Nicotinamide riboside, 359
Nicotinic acid :

biosynthesis from tryptophan, 347

food sources, 239

formula, 238

human retpiirements, 239

in foods, table. 448

metabolic antagonist of, 256

physiological function, 235, 236

stability of, 238
Nieman-Picks disease, 100
Night blindness, 205
Ninhydrin test, 143
Nitrate fertilizers. 400
Nitrification, 14, 400
Nitrifying bacteria, 401
Nitrogen balance studies, 341
Nitrogen cycle in nature, 402, 403
Nitrogen deficiency, effect on plants, 409,

410
Nitrogen fixation :

by bacteria. 402. 403

by leguminous plants, 402, 403

by soil bacteria, 403, 404

mechanism of. 404
Nitrogen metabolism, of plants, 400-

402
Nitrogen requirements of microorganisms,

358
Nitrogen trichloride as flour bleach, 257
Nitroglycerine, 76

Nitrosococctis and nitrification, 401
Nitrosomo7ias and nitrification, 401
Nonsaponifiable matter, 81
Norepinephrine, 287, 288
Norleucine. formula. 116
Normal potential (Eo), definition of, 420
Normal solutions. 163. 164
Normality of solutions, 103-165
Nucleases, in digestion, 318
Nucleic acids :

amount in nucleoproteins, 152, 153-160

classes of. 153

molecular weight of, 153, 157

products on hydrolysis, 154

structure of, 157
Nucleoproteins, 112. 150-160

components of, 151, 152, 153

importance, 150

linkage between components, 151

molecular weight, 153

462

INDEX

Nucleoproteins (Cont.) :

occurrence of, 150

preparation of, 151

proteins of, 153

relation to chromosomes, 150, 151

types of, 152

virus, 153
Nucleosidases in digestion, 319
Nucleoside phosphorylases, 207
Nucleosides, 156
Nucleotidases in digestion, 318
Nucleotides, 156-158

structure of, 158
Nutrition, objectives and methods, 3
Nylon from pentosans via furfural, 53

O

Obesity, 427-429

cause of, 428

relation to varying food needs, 429
Octapine, formula, 120
Oils, fatty, hardening of, 89
Oleic acid :

cis-trans isomers of, 79

formula, 79, 80
Oleyl alcohol, 93

Omega oxidation of fatty acids, 337
Opsin, 204
Optical activity, 24
Optical rotation, 24
Organic acids in plants, metabolism of,

405
Ornithine, 321

bacterial decarboxylation, 321

formula, 121

from glutamic acid, 346
Ornithine cycle, 352-354
Osazone crystals, 47
Osazones, 31, 32
Osmotic pressure, 181, 182
Osteomalacia, 202
Ovarian hormones, 292, 293
Overweight :

cause of, 428

correction of. 429
Ovovitellin, 108, 126
Ovulation, 293
Oxalacetate carboxylase, 266
Oxalacetic acid :

in citric acid cycle, 330, 331

source of, for citric acid cycle, 332
Oxalacetic transaminase, reaction cata-
lyzed by, 343
Oxalates, soluble, toxicity of, 161, 162
Oxalic acid, occurrence in foods, 161
Oxalosuccinic acid, 331
Oxalosuccinic carboxylase, 266
Oxidases, 268

in plants, 404
Oxidation, definition of, 419

metabolic, by hydrogen removal, 332

multiple alternate, of fatty acids, 337,
338

of fatty acids, 336-339

of glucose, direct, 380

of glucose, nonphosphorylative, 381

Oxidation (Cont.) :

of organic matter on earth, 387
of sugar alcohols, 364
omega, of fatty acids. 337
Oxidation-reduction carriers, 275-279
Oxidation reduction potential, definition

of, 419
Oxidation-reduction, principles of, 419.

420
Oxidative deamination, 351, 352
Oxide-ring forms of sugars, response to

reducing sugar tests, 30
Oxide-ring formulas of sugars, alpha and

beta forms, 25
Oxygen isotope, use as tracer, 392
Oxygen production, in photosynthesis,

388, 392
Oxyhemoglobin, 105, 138
Oxytocic hormone, 295
Oxytocin, 303, 304

PABA (see Para-aminobenzoic acid)
Paint, unsaturated oils for, 89
Palmitaldehyde, 101
Palmitic acid, in waxes, 93
Palmitoleic acid, 80
Pancreas, 300

hyperglycemic factor of. 303

in carbohydrate metabolism, 300-.'i03,
327
Pancreatic amylases, 263, 316
Pancreatic digestion. 315
Pancreatic lipase, 316
Pancreatic secretion, hormonal control of,

308
Pancreozymin, 308, 316
Pantatheine, 359
Pantathenic acid, 359

formula, 240

in foods, table, 447-448

physiological function, 239
Papain, 265

Paper chromatography, 396
Paper making, 61
Paper pulp. 61
Para-aminobenzoic acid (see p-Amino-

benzoic acid)
Parathyroid secretion, effect on blood

calcium, 183
Parietal cells. 312
Pectin, 64, 65
Pectinase, 263
Pellagra, 235, 236, 237, 238

relation to corn as food, 237
Penicillamine, 122

formula, 118
Penicillin, 122

activity of, 374

formula of, 374

industrial production of, 372

mode of action of, 374

precursors of, 374

range of activity, 374

resistance of bacteria to. 374
Penicillinase, 265

INDEX

463

Penicillium charlesii, 375
Penicillin in chrysogenum :

growth efficiency of, 360

penicillin production by, 372

products of, 363
Penicillium cinerascens, 375
Penicillium terrestre, 375
Pentosans, 51

function of, in plants, 52

furfural fi'om, 53

hydrolysis of, 52

nutritive value, 53

occurrence, 51, 52
Pentoses, 26
Pentosuria. 26. 28
l»-enzyme, 399
Pepsin. 110. 126, 265, 313
Pepsinogen, 314
Peptidases, 264
Peptides :

linkage. 129

side chains. 130

utilization by plants, 401
Pernicious anemia, 248, 249, 251, 314
Perosis. 253

relation of manganese to, 189
Peroxidases, 26S, 282

in plants. 405
pH :

definition, 168, 169

effect on plant growth, 168

measurement of, 173

of salt solutions, 171, 172

regulation of body, 173
pH changes, relation to changes in H-ion

concentration, 169
pH value of biological materials, 174
Phaseolin. 108, 126
Phenylacetic acid, 374
Phenylalanine, 114

formula, 117
Phenylhydrazine, reaction with sugars,

31. 32, 35
Phenylpyruvic acid, 350
Philoholus kleinii, 360
Phlorhizin, 302

Phosphagens, definition, 417, 418
Phosphatases, 264

activation by vitamin D, 211

in digestion. 319
Phosphate bond energies, table of. 417
Phosphate bonds. IS.F values of, table, 417
Phosphate bonds of high energy :

definition, 416. 417

formation of, 420-422
Phosphate, transfer enzymes. 267
Phosphates, bond energy of, table. 417

in glycolysis. 326-329

organic, in metabolism, 326-329, 415-
421
Phosphatides, 96
Phosphatidic acids, 99
Phosphatidyl ethanolamine, 99
Phosi)hatidyl serine, 99
Phosphocreatine (see Creatine phos-
phate)

Phosphodiesterases, 264
2-Phosphoenolpyruvic acid, 397
Phosphoglucomutase, 267
Phosphoglyceraldehyde dehydrogenases,

268
Phosphoglyceric acid transphosphorylase,

267
Phosphoglyceric acids, in photosynthesis,

396-398
Phosphoglyceromutase, 267
Phosphohexoisomeras^, 267
Phosphohexokinase, 267
Phospholipides :

classification, 97

in fat transport, 336
Phosphoproteins. 136

Phosphopyruvate transphosphorylase, 267
Phosphorus :

areas low in, 185

requirement, 184

role in animal body, 184
Phosphorus content of foods, table, 439
Phosphorus deficiency, effect on plants,

409, 410
Phosphorylases, 267

stimulation by epinephrine, 289
Phosphoserine, 319
Photochemical reaction, primary. in

photosynthesis, 391
Photolvsis of water, in photosynthesis,

391
Photosynthesis, 29, 387^00

dark reactions in, 393

definition of, 388

efficiency of, 395, 396

energy relations of, 395, 396

energy stored by, 387

equation for, 388

formaldehyde theory of, 396

Hill reaction in. 393

importance of, 387

intermediates of, 396^00

light reactions in, 393

mechanism of, 397, 398

partial reactions of, 393, 394

products of. 396^00

quantum efficiency of, 395, 396

rate of, 393
Phototropism. 406
Phthiocol. 220
Phytase, 264
Phytic acid, calcium and magnesium salts

■ of, 179
Phvtohormones, 406
Phytol, 391

Pitressin (see Vasopressin)
Pituitary gland, relation to menstrual

cycles, 292-294
Pituitary hormones. 303-306
Pituitarv, hormones of posterior lobe,

303
pKa, relation to acid strengths, 171
Placenta, hormones from, 295
Planck's constant, 395
Plant growth substances, 406, 407
Plant gums, 66

464

INDEX

Plant hormones, 406, 407
Plant metabolism :

glycolysis in, 405

Krebs cycle in, 405

organic acids in, 405
Plant nutrition :

effects of mineral deficiencies, 409, 410

mineral elements for, 407
Plant respiration, true oxidases in, 404
Plants :

nitrogen nutrition* of, 400-402

respiration of. 404-^05
Plasmalogens, 101
Polarimeter, 24
Polarized light, 24

Polished rice, relation to thiamine de-
ficiency. 228
Polymyxin, 122

bacterial spectrum of, 370

components of. 370

toxicity of, 370
Polyneuritis, 227
Polyphosphatases, 264
Polysaccharides :

molecular weights of, 50

production by bacteria, 378

properties of, 51

structure of. 50, 51
Polyuronides, 21
Porphyrins :

biosynthesis of, 351

prosthetic groups of enzymes, 278
Porphyropsin, 204

Potassium content of foods, table, 439
Potassium deficiency, effect on plants,

409, 410
Potassium, in animals, 183
Pressin {see Vasopressin)
Progesterone :

effect on uterus, 294

physiological functions, 292-295
Prolactin, 305
Proline :

biosynthesis from glutamic acid, 346

formula, 120
Propanolamine, from vitamin Bjo, 250
Propionibacterium pentosaceum, 363, 377
Propionic acid, 77

Propionic acid fermentation, 363, 377, 382
6-Propyl-2-thiouracil. 300
Prosthetic group. 111, 136
Protamines :

nucleoproteins and, 153

properties of. Ill
Protein-bound iodine. 298. 299
Protein content of foods, table, 434
Protein foods, use in correcting over-
weight, 429
Protein, human reciuirements for, 347
Protein metabolism, 341-354

link to carbohydrate metabolism, 342,
343
Proteinases, 265

of bacteria, 265

specificity, 315-316
Proteins, 103-149

Proteins (Cont.) :

amino acid composition, 122, 124

classification, 110

coagulation, 145

color tests, 142

commercial, 103

conjugated. 111. 136

criteria of purity, 107

crude, definition, 146, 434

crystalline, 104

denaturation of, 144

derived. 111

determination, 146

economic importance, 103

elementary composition, 75. 109

half-life period in tissues, 347

hydrolysis, 122

isolectric pH. 144

linkages in. 131

molecular weights, 141

number. 104

occurrence, 104, 108

physiological fuel value of, 423, 424

precipitation, 143

preparation. 104

products on hydrolysis, 111, 113

simple. 111

solubility. 111

structure, 133

table of common, 108
Proteus vulgaris. 369
Prothrombin, 219, 220, 221
Protochlorophyll. 390

formula, 391
Protomone (iodinated casein), 298
Protoplasm, 6

Proximate composition, definition of, 423
Pseudomonas aeruginosa, 381
Pseudomonas putida, 358
Pseudomonas saccharophila, 399
Pseudomonas sp., 370
Pteroic acid, 359
Pteroylglutamic acid, 246-249, 359

stability of, 249
Pterovlglutamic acid deficiency, symptoms

of, 248
Pteroyltriglutamic acid, 131
Ptyalin, 58, 263
Purines, 154-157

biosynthesis of, 351

formula of, 154

list of. 154
Putrescine, 260, 321
Pyranose ring forms of sugars, 26
Pyribenzamine :

antihistamine drug, 289, 290

formula, 290
Pyridine nucleotides, as hydrogen car-
riers, 332, 333
Pyridine-3-sulfonic acid, as nicotinic acid

antagonist, 256
Pyridino coenzymes, 275
Pvridino proteins, 268
Pyridoxal, 243
Pyridoxal phosphate. 243. 279

coenzyme for transamination, 343

i

INDEX

465

Pyridoxamine, 243
Pyridoxamine phosphate, 243
Pyridoxine :

food sources, 244

formula. 242

physiol()5;ical function, 241
Pyrimidines, 154-157

formuhi of, 155

list of, 154
Pyrithianiine, 256
I'yroxylin, G2
Pyrrole. 390. 391
Pyi-uvic acid :

conversion to oxalacetic acid, 332

formation during glycolysis, 32S. 329

from glucose, 330

in thiamine deficiency, 227

metabolic oxidation of, 330-334
Pyruvic acid enolphosphate, 329
Pyruvic acid oxidation, energy from. 421
Pyruvic oxidase, 266, 274
Pyruvic transaminase, reaction catal.vzed
by, 343

Q

Q-enzyme, 399
Quinolinic acid, 347
R

Rachitic rosary, 210

Radioactive carbon isotopes as metabolic

tracers, 288, 396, 397
Radioautography, 396
RafBnose, 49
Rancidity, 90

hydrolytic, 90

oxidative, 90
Rayon, 62

Redox potential, definition of, 419
Reducing sugars, 31

determination in foods, 31

effect of alkali on, 31
Reductive amination. 401
Relaxin, 295
Renal threshold, effect of phlorizin on,

302
Renin, 265, 304, 314
Reproduction, relation of vitamin E to,

216, 217, 219
Resorcinol test for ketoses. 36
Respiration :

definition of, 323

of animals, effect of cyanide on. 334

of plants, 404. 405
Respiratory quotient, 425
Retinene, 204
Rhamnose, 38
Rheumatoid arthritis, effect of cortisone

and ACTH. 292
Rhizobia, in root nodules, 403
Rhizopterin, 247
Nhi::opi(i!i nigricans, 363
Rhizopus orijzae, 363
Rhizopus sp., 363
Rhodo»iicrohium and nitrogen fixation,

403
Rhodopbin, 140, 204

Rhodospirillum and nitrogen fixation, 403
Ribitol, 21
Riboflavin :

chemical properties, 233, 234

determination of, 234

food sources, 234

formula, 234

human requirements, 235

in foods, table, 447^48

light destruction of, 234

physiological functions, 232, 233
Riboflavin adenine dinucleotide (FAD),

277
Riboflavin coenzymes. 277
Riboflavin mononucleotide (FMX), 277

role in tissue oxidation. 282

structure of, 276
Riboflavin-5'-phosphate, 159, 277
Ribonuclease, 126
Ribonucleic acid, 154
Ribose, 27

Ribose, nucleic acids and, 154. 156-158
Ribose-3-phosphate. in vitamin Bja, 250
Ribose-5-phosphate. 380
Ribulose, 397, 398
Ribulose-5-phosphate, 380
Ricinoleic acid. 80
Rickets :

occurrence of. 212

relation of sunlight to, 212

symptoms, of, 210
Robison ester (see D-Glucose-6-phosphatp)
Root nodules, 403
Running fits in dogs, 257

S

Saccharic acid, 22

Saccharimeter, determination of sucrose

with, 45
Haccharom ijces cerevisiae :

fermentation products of, 363

growth efficiency of, 360
Saliva :

amylase in, 311

composition of, 311

secretion, 312
Salivary amylase, 263
Salmine. 111. 126
Salt depletion, 12
Salt metabolism, effect of cortical steroids

on, 291
Salt, i-equirement, 1S2
Saponification, 73

of fats, 86
Schiff's reagent, 25. 27
Scurvy, 223
Secretion, 308, 316
Secretogogues, 308
Sedoheptulose, 21. 380. 397. 398
Selenium poisoning. 195, 196
Semiessential amino acids, 341, 342
Serine :

formation from glycine, 344, 345

formula, 116

metabolic reactions involving, 344, 345
Serine phosphate, 319

466

INDEX

Serotonin, 304

Sex hormones, 292-297

male, 295-297
Silicon :

in living cells, 176

in plants, 195
Simple sugars. 19, 22

distinguishing from other carbohydrates,
31
Sinigrin, 28
Sitosterol, 95
Skatole, 320, 354
Slipped tendon in chicks, 189
Snake venoms, lecithinase in, 99
Soaps, 86, 87

water-insoluble, uses of, 88
Sodium :

functions in body, 182

in body fluids, 182

with Addison's disease, 291

with adrenals. 291
Sodium chloride, in nutrition, 182, 183
Sodium content of foods, table, 4.39
Sodium palmitate, 86
Solar energy, fraction intercepted by

earth, 387
Solutions :

molar, 163
, normal, 163

standard, 163

standardization of, 165
Sorbitol, production from glucose, 37
Sorbose, 36
Specific rotation, 24
Sperm oil. 92
Spermaceti, 92. 93
Sphingomyelins, 100
Sphingosine, 73, 100

dihydro-, in cerebrosides. 101
Sprouting of stored vegetables, chemical

control of, 407
Sprue, 248, 251
Standard solutions, 103
Standardization of solutions, 165. 166
Standards, primary, for titrations, 165
Staphylococcus aureus:

effect of penicillin on, 375 *

nitrogen requirements of, 358
Starch :

acid hydrolysis of, 29

elementary composition, 75

granules, 54, 55, 56

iodine test, 54

occurrence, 54

phosphorus in. 54

synthesis of in plants, 399, 400
Steapsin, 86
Stearaldehyde, 101
Stearic acid, 77
Stercobilin, 318
Stercobilinogen, 318
Stereoisomerism, 23
Stereoisomers. 23
Sterility, relation of vitamin E to. 216,

217, 219
Steroid ring system, 94

Steroids, 94

biosynthesis of, 340
Sterols. 94
Stilbestrol, 297, 298
Stored fat, dynamic state of, 335
Streptidine, 306
Streptococcus fecalis. 359
Streptococcus lactis, 360, 376
Streptomyces aureofaciens, 368
Streptomyces griseus, 365
Streptomyces rimosus. 368
Streptomyces venezuelae, 369
Streptomycin :

bacteria dependent on, 367, .368

formula of, 365-367

mode of action, 367

production of, .366

resistance to, .367

toxicity of, 366
Streptose, 365, 366, 367
Substrate, 270
Subtilin, 122
Succinic acid, bacterial formation of, .363,

377, 383
Succinic acid oxidation, energy from, 421
Succinic dehydrogenase, 268
Sucrase, 262, 263
Sucrose :

advantages of hydrolysis of, 34

cost of food calories from, 43

determination in foods, 44, 45

food value, 43

formation in plants. 397-399

human consumption, 42. 43

inversion of, 42, 43, 45

occurrence, 42

optical rotation of, 45

jii-eparation of, 43

production, 42

structural formula. 44
Sucrose phosphorylase, 267
Sucrose synthesis, 399
Sugar alcohols, 21
Sugars :

action of acids on, 26, 39, 52

action of alkalies on. 31

D- and L- forms of, 22

desoxy, 20, 27

formation in plants, 397, 398

interconversion in body, 324

melting points of, table, 36

optical rotations of, table, 36

reducing power of, 30, .31, 35, 41

ring structures of, 24-26

sweetness of, 43
Sulfa drugs, effect on intestinal bacteria,

322
Sulfanilamide. 256
Sulfatase. 264
Sulfur compounds :

in mustard, garlic, etc., 179

required by animals, 192
Sulfur content of foods, table, 4.39
Sweet clover, toxicity of fermented, 221
Sweetening power of sugars, 43
Symbiosis, 403

I

.J

i

INDEX

467

2.4.5-T, formula of, 407
Tagatose, 24
Takadiastase. 58
Tartaric acid, 161
Taurine, in urine, 197
Tauroeholic acid, 96, 317
Tea. manganese in, 189
Teeth, fluorides and. 193
Teeth, mottled. 193

relation of vitamin C to, 223

relation of vitamin D to, 210
TcrDiohactcriitiii mohile, 363
Teropterin. 247, 249
Terramycin :

formula of, 367, 368

mode of action. 369

range of activity, 368
Terre.strie acid. 375
Testosterone. 295, 297
Tetan.v. relation to calcium. 183
Tetrahymena geleii, 360
Tetronic acid, 375
Tetroses. 20
Theobromine, 155
Theophylline, 155
Thiamiiiase, 229, 266
Thiamine :

destruction during food preparation, 229

determination of, 229, 230

food sources, 230

formula. 229

human requirements, 230. 231

in fat synthesis. 340

in foods, table. 447--448

physiological function, 226, 227. 228

required by microorganisms. 356

requirements, factors modifying. 228
Thiamine deficiency, prevalence of, 228
Thiamine pyrophosphate, 227
Thiazole. 359
Thiochrome method, for thiamine assay,

229
Thioctic acid, 360
Thiouracil, as antithyroid drug, 300
Threonine, 113

formula. 116
Threose, 20
Thymidine, 156
Thymidylic acid, 156
Thymine. 154-157

pterovlglutamic acid activity of, 249
Thyroglobulin, 298
Thvroid deficiency. 299
Thyroid hormone. 297-300
Thyrotropic hormone, properties of, 307
Thyroxine. 297-300

formula, 118
Titration, 163, 165
Tobacco mosaic virus, 110, 126. 153
Tocopherol, apha, formula, 218
Tocopherols. 217

as antioxidants. 90

content of foods. 218
TPN (see Triphosphopyridine nucleotide)

TPN-cytochrome c. reductase, 268
Trace elements, 176
Transaminases, 268
Transamination, 343

in plants, 401, 402
Transglucosidases, 269
Transmethylation, 343
Transphosphorylases, 267
Traumatic acid, 406
Trehalose, 41
Tributyrin, 83, 84
Tricarboxylic acid cycle, 330
2.4.5-Trichlorophenoxyacetic acid, 407
Trimethylamine. 162
Triolein, 83

Triosephosphate isomerase, 267
Trioses, 20
Tripalmitin. 83
Triphosphopyridine nucleotide (TPN) :

forms of, 275

function in cytochrome system, 332,
333

relation to nucleotides, 158

role in tissue oxidation. 282

structure of, 276
Tristearin, 83
Trypsin, 285

in pancreatic secretion, 315
Trypsinogen, conversion to trypsin, 316
Tryptophan :

bacterial degradation of, 320

conversion to niacin in vivo, 347

formula, 120
Tuberculin, 153
Turacin, 186
Tyramine, 321
Tyrosinase, 268
Tyrosine :

as hormone precursor. 349

bacterial decarboxylation of, 321

biosynthesis, 346

crystals of, 114

formula. 117
Tyrothricin :

components of, 370

toxicity of, 370

U

Ulcers, 314

Ultraviolet light and vitamin D, 212, 215

Units :

of penicillin, 374

of vitamin A, 210

of vitamin D. 216
Unsaturated fatty acids, in nutrition, 79
Uracil, 154-156
Urea :

as nitrogen fertilizer, 400

synthesis of, 352
Urease. 265

Uric acid, as nitrogenous waste product,
353

formation of, 155

formula of, 155
Uridine, 156
Uridine diphosphate glucose, 280

468

INDEX

Uriflylic acid, 156
Urine :

excretion of glucose in, 301

excretion of liormones in, 295

pH of, 174
Urogastrone, 314
Uronic acids, 38, 39

tests for, 39

Vaccenic acid. 80

Valine, formula, 116

Vanadium, in respiratory pigment, 176

Vanillin, from lignin, 61

Vasoconstrictor, epinephrine use as,

289
Vasopressin. 304
Villi, 319

Vinyl groups in chlorophyll, 391
Vinyl phosphate, 308
l-5-Vinyl-2-thiooxazolidone. 300
Viosterol, 215
Virilism, 291
Vision, chemistry of, 204
Visual purple {see Rhodopsin)
Vitamin A, 203-210

food sources, 209

human requirements for. 209

international unit of, 210

measurement of. 208

oxidation of. 208

relation to yellow color of foods, 84, 85

sources of, 209

toxicity due to overdoses, 210
Vitamin A acetate, 207
Vitamin A crystals, 206
Vitamin A deficiency, prevalence of, 205
Vitamin A derivatives, 207, 208
Vitamin A palmitate, 208
Vitamin A value, 208

of foods, table. 447-448
Vitamin Aj, formula, 207
Vitamin A^, 204
Vitamin B^, 247
Vitamin Be conjugate, 247
Vitamin Bj {see Thiamine)
Vitamin Bg {see Riboflavin)
Vitamin Bg {see Pyridoxine)
Vitamin Bj^, 249-252

food sources, 252

human requirements, 252

metabolic function of, 252, 281

production of, 370

relation to methylation in vivo, 345
Vitamin B]2 deficiency in humans. 252
Vitamin Bj^a, 251
Vitamin B12,,. 251

Vitamin business, annual value. 203
Vitamin C :

antagonist for. 256

biosynthesis of, 222

determination of, 225

human requirements, 226, 231

in foods, table. 447-448

loss in preparing foods. 225

occurrence and food sources of, 225

Vitamin O (Cont.) :

prevention of losses from foods, 225,
226

reducing power of, 224, 225
Vitamin C deficiency :

prevalence of, 223

symptoms of, 222, 223

tests for, 223
Vitamin D :

chemical nature, 212

concentrates of. 215

human requirement, 216

mechanism of action. 211

physiological function, 210

precursors of, 215

relation to calcium absorption, 183

requirements, 214

sources of, 214, 215

storage of in body, 216

toxicity of overdose, 215, 216
Vitamin D deficiency, prevalence of, 212
Vitamins D^ and D3. 213
Vitamin deficiency diseases, existence in

United States, 202
Vitamin E :

as antioxidant, 217

distribution, table, 218

food sources, 218, 219

physiological functions, 216, 217, 219

protection of vitamin A by. 208
Vitamin E deficiency, symptoms of, 216,

217
Vitamin G {see Riboflavin)
Vitamin H {see Biotin)
Vitamin K :

occurrence and food sources, 222

physiological function, 219
Vitamin Kj, formula, 220
Vitamin M. 247
Vitamins :

classification. 201

definition. 200

history of, 200, 201

in foods, table. 447-448

microorganisms and, 359

relation to enzymes, 200
A^olatile fatty acids, 78

W

Warfarin. 221, 222

Water :

balance, 13

chlorination of water supplies, 15

conservation of in vivo, 12

content and age of cells, 8-9

content and survival of cells, 10

content of biological materials, table, 9

demand in animals and plants, 11

free and bound, 9-10

function of in metabolism, 11

hardness of, 15

in photosynthesis. 392

indices of pollution, 14

metabolic, 12, 13

need of. 11

occurrence and importance, 8, 9

INDEX

469

Water (Cont.) :

potable, 13

purification of water supplies, 15

re(|uirement in humans, 12

softening, 15-17
Wax alcohols, 91, 93
Wax esters. 92
Waxes, natural :

biological role, 92

composition of, 92-94

occurrence, 92

properties of, 92
Weed killers, 407
Weight reduction :

by muscular activity, 429

principles of, 429
Work, relation to free energy changes, 414

X

Xanthine :

formula of, 154

metabolism of, 155

oxidases, 268
Xanthophyll, 84. 205, 209
Xanthoiiroteic test, 142
Xerophthalmia, 203, 205
Xylan, 51

Xylitol, 21

Xylosazone crystals, 47
D-Xvlose, 20
L-Xylulose, 27, 28

Yeast, production, 361. 371
Yellow enzymes, 140

Zeaxanthine, 205

Zein, 108, 126

Zeolites. 16, 17

Zinc :

biological role, 189-101

in carbonic anhydrase, 187, 189

content in average diet, 191

content of food, table, 442

food sources of, 191

in fertile soils, 191

in insulin, 301

Zinc deficienc.v, effect on plants, 191

Zinc oleate, 88

Zinc stearate, 88

Zwitterion, 97

Zymogenic cells, 312

Zymogens, 273

1.

"t