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THE BIOCHEMISTRY OF

B VITAMINS

Roger J. Williams

Robert E. Eakin

Ernest Beerstecher, Jr.

William Shive

University of Texas, Austin, Texas

BOOK DIVISION

REINHOLD PUBLISHING CORPORATION

330 West Forty-second St., New York 18, U. S. A.

Printed in the United States of America by

THE HADDON CKAfTSMHX, INC., SCRANTON, PA.

GENERAL INTRODUCTION

American Chemical Society's Series of
Chemical Monographs

By arrangement with the Interallied Conference of Pure and Applied
Chemistry, which met in London and Brussels in July, 1919, the American
Chemical Society was to undertake the production and publication of
Scientific and Technologic Monographs on chemical subjects. At the same
time it was agreed that the National Research Council, in cooperation with
the American Chemical Society and the American Physical Society, should
undertake the production and publication of Critical Tables of Chemical
and Physical Constants. The American Chemical Society and the National
Research Council mutually agreed to care for these two fields of chemical
progress. The American Chemical Society named as Trustees, to make
the necessary arrangements for the publication of the Monographs,
Charles L. Parsons, secretary of the Society, Washington, D. C; the late
John E. Teeple, then treasurer of the Society, New York; and the late
Professor Gellert Alleman of Swarthmore College. The Trustees arranged
for the publication of the ACS Series of (a) Scientific and (b) Techno-
logical Monographs by the Chemical Catalog Company, Inc. (Reinhold
Publishing Corporation, successor) of New York.

The Council of the American Chemical Society, acting through its Com-
mittee on National Policy, appointed editors (the present list of whom
appears at the close of this sketch) to select authors of competent
authority in their respective fields and to consider critically the manu-
scripts submitted.

The first Monograph of the Series appeared in 1921. After twenty-three
years of experience certain modifications of general policy were indicated.
In the beginning there still remained from the preceding five decades a
distinct though arbitrary differentiation between so-called "pure science"
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differentiation was fast becoming nebulous. Research in private enterprise
had grown apace and not a little of it was pursued on the frontiers of
knowledge. Furthermore, most workers in the sciences were coming to
see the artificiality of the separation. The methods of both groups of
workers are the same. They employ the same instrumentalities, and
frankly recognize that their objectives are common, namely, the search
for new knowledge for the service of man. The officers of the Society
therefore combined the two editorial Boards in a single Board of twelve
representative members.

Also in the beginning of the Series, it seemed expedient to construe
rather broadly the definition of a Monograph. Needs of workers had to be

iv THE BIOCHEMISTRY OF B VITAMINS

recognized. Consequently among the first hundred Monographs appeared
works in the form of treatises covering in some instances rather broad
areas. Because such necessary works do not now want for publishers, it is
considered advisable to hew more strictly to the line of the Monograph
character, which means more complete and critical treatment of relatively
restricted areas, and, where a broader field needs coverage, to subdivide
it into logical sub-areas. The prodigious expansion of new knowledge
makes such a change desirable.

These Monographs are intended to serve two principal purposes: first,
to make available to chemists a thorough treatment of a selected area in
form usable by persons working in more or less unrelated fields to the end
that they may correlate their own work with a larger area of physical
science discipline; second, to stimulate further research in the specific
field treated. To implement this purpose the authors of Monographs are
expected to give extended references to the literature. Where the literature
is of such volume that a complete bibliography is impracticable, the
authors are expected to append a list of references critically selected on
the basis of their relative importance and significance.

AMERICAN CHEMICAL SOCIETY

BOARD OF EDITORS

William A. Hamor, Editor of Monographs

Associates

L. W. Bass S. C. Lind

T. H. Chilton C. H. Mathewson

Barnett Cohen W. T. Read

Farrington Daniels Walter A. Schmidt

J. Bennett Hill E. R. Weidlein

E. H. Huntress W. G. Whitman

This volume is dedicated to

The Clayton Foundation For Research

which has generously and continuously supported research
dealing with the B vitamins.

ACKNOWLEDGEMENTS

We wish to express our thanks to Dr. R. R. Williams and to our former
colleague Professor E. E. Snell for critically reading substantial portions
of the manuscript. We also acknowledge gratefully the help and en-
couragement received from all of the members of the Biochemical Insti-
tute staff. Individual members have read portions of the manuscript,
helped in its final preparation and aided in numerous ways to make the
book possible. To all of these we express our sincere gratitude and thanks.

Permission to use the following material is acknowledged with thanks:
Table 2, pp. 250-251, Table 3, p. 253 and Table 4, p. 253, from Williams
and Spies: Vitamin Bt and Its Use In Medicine, copyright 1938, by the
Macmillan Company. Table 6, p. 257, Journal of Nutrition, L. J. Roberts,
Figure 3, p. 313, Indian Journal of Medical Research, P. S. Sarma. Table
20, pp. 324-325, Food and Nutrition Board, National Research Council.
Tables 29 and 30, pp. 366-367, Academic Press, C. W. Denko. Table 34,
p. 389, Proceedings of the Society for Experimental Biology and Medi-
cine, F. G. Brazda. Figure 17, p. 405, Journal of Nutrition, J. Salcedo, Jr.
and L. E. Holt, Jr. Figures 18-20, pp. 416-418, Lancet, T. D. Spies.

PREFACE

We have sought in this volume to fulfill, with respect to the chosen
field, the two fundamental purposes set forth at the time the American
Chemical Society Monograph Series was instituted.

First, we have attempted to present the material "in readable form,
intelligible to those whose activities may be along a wholly different line"
to the end that they may gain perspective and interest and appreciation
of the fundamental unity existing in the broad area involving physical
as well as biological science.

Second, we have sought to promote research in the field of the B vita-
mins "by furnishing a well digested survey of the progress already made
in that field and by pointing out directions in which investigation needs
to be extended." In connection with this latter purpose we have had in
mind two groups: the oncoming advanced students who from year to
year must have a means of becoming acquainted with the field, and the
ever increasing number of chemists whose fundamental training has been
in some other branch but who are turning to biochemistry as an attractive
field of investigation.

In keeping with the title of the volume and the purposes set forth, we
have not included full discussions of the organic chemistry or technology
of the substances involved, nor have we given a historical treatment of
their discovery and identification. To trace all the misconceptions and
inevitable blunderings which have entered into the development of our
present knowledge and at the same time to do justice to the numerous
investigators would be an impossible task, and from the standpoint of
our purposes an attempt would not be worthwhile.

We agree with the late G. N. Lewis' statement, "a monograph of this
sort belongs to the ephemeral literature of science. The studied care
which is warranted in the treatment of the more slowly moving branches
of science would be out of place here. Rather with the pen of a journalist
we must attempt to record a momentary phase of current thought, which
may at any instant change with kaleidoscopic abruptness."

Although the four authors have found themselves to be in substantial
agreement on most points, in the presentation of the material in the four
sections they have used their respective judgments, expressed their own

vii

vm PREFACE

individual opinions and organized the material in accordance with their
own thinking on the subject. One of our primary aims has been to present
constructive and suggestive viewpoints, and in so doing we have had to
run the risk of being in error. This risk could have been avoided by com-
piling an uncritical review with none of the reviewers' opinions expressed,
but to have done this would have effectively defeated the purpose of
the book.

We wish to beg the indulgence of our readers for mistakes and imper-
fections that may be found, and if they are such as can be remedied in
later printings or editions we will appreciate having them called to our
attention. There has been some unavoidable duplication in different areas
of the discussion, but this has been retained whenever doing so would
contribute materially to the unity and understandability of the particular
section. In view of the magnitude of the task, which turned out to be even
greater than we had anticipated, and because the preparation of the
manuscripts had to be superimposed upon active research and/or teaching
programs, we hope that our readers will be charitable in judging the
product of our labors.

It has not been feasible to synchronize our efforts completely and in
the respective sections the literature has not been completely reviewed
up to exactly the same date. Even within sections the up-to-dateness may
vary slightly from topic to topic. For example, the citation of individual
crucial papers up through April or May, 1950, does not mean that all of
the literature has been covered up to that point. We cannot hope that the
material which we have presented will remain up-to-date for long. Indeed,
a sincere desire to promote research is equivalent to hoping that this
volume will rapidly become out-of-date. But, like Professor Lewis, we
can hope that necessary changes will involve "matters of detail rather
than of essence."

Roger J. Williams
Robert E. Eakin
Ernest Beerstecher, Jr.
William Shive
Austin, Texas
June 15, 1950

Chapter

IIA

Chapter

IIIA

Chapter

IVA

Chapter

VA:

CONTENTS

General Introduction iii

Preface vii

Section A: Characterization, Distribution, Assay and Biogenesis of
B Vitamins

B Vitamins: What They Are 3

Distribution of B Vitamins 18

Combined Forms — Extraction 30

Assay Methods 45

Biogenesis of the B Vitamins 78

Section B: The Catalytic Functions of the B Vitamins

Chapter IB : Biochemical Reactions and Their Catalysts 95
Chapter IIB: Coenzymes Derived from B Vitamins 123

Chapter IIIB : The Functions of the B Vitamins in Meta-
bolic Processes 216

Section C: The Role of the B Vitamins in Animal and Plant
Organisms

Chapter IC: Methods of Assessing B Vitamin Require-
ments 243

Chapter IIC: Factors Influencing B Vitamin Require-
ments 264

Chapter IIIC: The B Vitamin Requirements of Animals

and Plants 306

Chapter IVC: Metabolism of the B Vitamins 336

Chapter VC: Physiological, Pharmacological, and Tox-

icological Effects 377

Chapter VIC: B Vitamin Deficiency States 395

Section D: The Comparative Biological Activities of the B Vitamins
and Related Compounds

Chapter ID: Introduction and Theoretical Considera-
tions 443
ix

CONTENTS

Chapter IID:

Chapter

HID

Chapter

IVD

Chapter

VID

Chapter

VIID

Chapter

VIIID

Chapter

IXD

Chapter

XID

Utilization of Competitive Analogue-
Metabolite Inhibition in the Elucida-
tion of Biochemical Processes Involv-
ing Vitamins 458
P-Aminobenzoic Acid 481
Biotin 542
The Folic Acid Group 565
The Nicotinic Acid Group 604
Pantothenic Acid 620
The Vitamin B6 Group 652
Riboflavin 669
Thiamine 684
Biological Activities of Other Nutritional
Factors of Doubtful Status 703

Section A

CHARACTERIZATION, DISTRIBUTION, ASSAY
AND BIOGENESIS OF B VITAMINS

Roger J. Williams

Chapter IA

B VITAMINS: WHAT THEY ARE

Historically the term "vitamin B" was applied to the water-soluble
organic material present in yeast, wheat germ, protein-free milk, etc.,
which was found to be necessary in small amounts for the nutrition of
young animals. At the time this designation came into general use the
dietary importance of minerals, proteins (amino acids), carbohydrates,
fats, "vitamin A," and vitamin C was recognized, and "vitamin B" meant
something distinct from these other recognized food materials.

When it became evident that vitamin B was not a single substance but
several, the designations Bi, B2, B3, etc., were introduced. These have
more recently given way in most cases to names for the specific chemical
substances involved: thiamine, riboflavin, etc. We shall discuss later
specific cases of substances which according to one's point of view may
or may not be included among the "B vitamins."

The time may well arrive when the term "B vitamin" will be abandoned,
and each specific chemical substance will be considered entirely as a
separate entity. At the present time, however, there is good reason for
retaining the term, because B vitamins appear to have common attributes
which set them apart from all other vitamins.

Microbiological assay methods have made it possible to learn that the
specific compounds commonly considered as members of the B family
are universally distributed in all living cells, whether of plant, animal or
bacterial origin. Since this appears not to be true of any of the other
vitamins, it was suggested elsewhere that B vitamins may be tentatively
defined as those which occur as indispensable constituents of all living
matter.1 If this suggestion is valid, their fundamental importance is self-
evident.

Studies relating to the functioning of individual members of the B
family of vitamins have demonstrated that they are integral parts of
biological catalytic systems and that they constitute essential factors
in the metabolic machinery of widely diverse forms. It seems probable
that this is true of all of the B vitamins, and an amended definition may
include this idea. In this case, we may say that B vitamins are those
which enter into the catalytic systems of all living cells.

Since the water solubility or fat solubility of a compound is not absolute

4 THE BIOCHEMISTRY OF B VITAMINS

but rather a matter of degree, we have not included the loose term "water
solubility" as an essential characteristic of a B vitamin. Indeed, if the
idea of the existence of a distinctive group of vitamins which function
catalytically in all living cells is a valid one, it is conceivable that we
may eventually come to include some of the "fat-soluble" compounds
among the B vitamins. Of such compounds already recognized, possibly
the most likely candidates for inclusion are the vitamins E, which appear
to be widely distributed in diverse organisms and tissues.2 If these
vitamins should be found to be part of the catalytic machinery of all
cells, there would seem to be no very valid reason for excluding them
from the B vitamins. The fact that a separate letter designation has been
used for them would not preclude this possibility. Biotin, which was early
called "vitamin H," is now recognized as a member of the B family. The
question of the universal occurrence of B vitamins in relation to other
vitamins is discussed further in Chapter IIA.

One of the interesting observations which in a measure appears to
differentiate the B vitamins from the members of the so-called fat-soluble
group is the fact that in the case of the fat-soluble vitamins there are
in every instance several naturally occurring and distinct chemical struc-
tures which possess the specific vitamin activity, whereas among the
B vitamins the physiological activity is more specifically associated with
a single chemical structure. It is true that in several instances among
the B vitamins, modified structures possess specific vitamin activity, but
in general each B vitamin is represented by a single substance or at
least by a few very closely related structures. As an instance of the
latter, in case a specific vitamin {e.g., nicotinamide, riboflavin) is in-
volved as a catalytic hydrogen carrier, it is reasonable that both the
oxidized and reduced forms should have physiological activity; likewise,
if a vitamin is a catalyst for ammonia transfer (e.g., pyridoxal), it is
not surprising that an aminated form (pyridoxamine) should possess
physiological activity.

We may now profitably consider in a critical manner the meaning and
significance of the word "vitamin" — a term the meaning of which we
have so far in this discussion taken for granted. It involves historically
a nutritional concept and has come to be applied almost exclusively to
certain organic substances which function in the nutrition of higher
animals. While a considerable number of the B vitamins were discovered
and isolated using yeasts and bacteria as test organisms, they have not
been admitted to the family of vitamins unless they have been found to
be nutritionally effective for higher animals.

A substance which counteracts a vitamin deficiency is not, however,
necessarily designated a vitamin. If this were so, thiamine pyrophosphate,

B VITAMINS: WHAT THEY ARE 5

coenzymes I and II, riboflavin nucleotides, and even flavoproteins, etc.,
would be considered as vitamins because they are capable of counteract-
ing respectively thiamine, niacin and riboflavin deficiencies. Actually,
they are not designated as vitamins. The easiest way out of the difficulty
in a specific case is to consider as a vitamin only the simplest compound
capable of performing the specific nutritional function. In cases where
two or more compounds of about the same complexity function alike
nutritionally, each may conveniently be called a vitamin. Nicotinic acid
and nicotinamide on the one hand, and pyridoxal, pyridoxamine and
pyridoxine on the other, are examples.

The importance of some of the compounds commonly designated as
vitamins does not rest, moreover, solely upon their functioning in nutri-
tion. Nicotinamide from the nutritional standpoint may not be essential
for animals if tryptophan is abundantly supplied, yet it is a nutritional
substance and is important in that it constitutes a part of the metabolic
machinery in every cell. Even though mammals generally, including
human beings, are probably capable of synthesizing nicotinamide in their
bodies from tryptophan, it is nonetheless a compound of great biochemical
interest and importance. Likewise, the importance of thiamine, riboflavin,
pantothenic acid and other members of the B family of vitamins does
not depend only upon the fact that they cannot be synthesized by higher
animals. As essential parts of the metabolic machinery, they are most
fundamental, regardless of their nutritional importance. Their nutritional
functioning may even be considered of secondary significance.

Looking at the matter with these facts in mind we may suspect that
the B vitamins actually belong to a larger group of organic catalytic
units which are indispensable to all cells, but which may or may not be
vitamins in the nutritional sense. Some of these indispensable units may
be uniformly synthesized by higher animals. We have no name for this
inclusive group of catalytic substances,* if such exists, and it appears
premature to discuss them at present. Until the time arrives when we
fully recognize the existence of such a group, it will be well to retain the
term "vitamin" and the nutritional concept which underlies it. A sub-
stance therefore cannot be classed as a vitamin unless it functions nutri-
tionally for higher animals.

Following this line of reasoning we may expand our definition of a B
vitamin to include those organic substances which act catalytically in
all living cells and which junction nutritionally for at least some of the
higher animals. We cannot guarantee, of course, that this delineation of
B vitamins will remain valid indefinitely. If it should be found that some
of the typical B vitamins lack a catalytic function or that some of them

* The name "catalins" has, however, occurred to the author as an appropriate one.

6 THE BIOCHEMISTRY OF B VITAMINS

are absent from certain types of cells, then our definition might have to
be modified immediately. At present such modification does not appear
likely.

Chemically Recognized B Vitamins

There are a number of specific chemical substances of known structure
which are universally recognized as B vitamins. We may, therefore, gain
a more specific idea of what is meant by the term "B vitamin" by dis-
cussing briefly these individual compounds.

Thiamine (aneurin) earlier received the designation "Bi" in keeping
with the fact that it was the first B vitamin to be discovered and isolated.
There is substantially no confusion resulting from identifying vitamin

CH3
N=C— NH2HC1 C=C— CH2— CH2OH

CH3— C C— CH2 N

II II + \

N— CH CI- C— &

Thiamine chloride hydrochloride

Bx as thiamine, because thiamine is the only naturally occurring structure
which is capable of performing the vitamin functions (p. 684). More
complex structures containing the thiamine unit as a part may function
nutritionally, as may also products formed by reversible oxidation and
reduction, if such exist. The chemistry of thiamine has been reviewed
by R. R. Williams.3

Riboflavin, earlier called vitamin B2, has the structure indicated below
and is the only naturally occurring structure possessing the characteristic
vitamin activity. Several synthetic flavins have lesser biological activity;

CH2OH

HO— C— H

CH,

H I

CNN

CH3— C C C C=0

I II I I

CH3— C C C NH

V V V

Riboflavin

B VITAMINS: WHAT THEY ARE 7

esters and other simple derivatives of riboflavin may possess full activity,
and conjugated forms such as the nucleotides or flavoproteins may be
nutritionally effective in proportion to their content of combined ribo-
flavin. The (reversibly) reduced form of riboflavin and its conjugates
are also physiologically active. Since the establishment of its constitution
and its synthesis in 1935 by Kuhn and Karrer and their co-workers, there
have been few advances in the organic chemistry of riboflavin. A com-
plete review of this topic may be found in the literature.4

Nicotinic Acid, Nicotinamide. These two compounds interchangeably
possess vitamin activity and the only other naturally occurring com-
pounds which can function nutritionally in the same manner are more
complicated derivatives which may act because they contain the essen-

H H

C C

HC C— COOH HC C— CONH2

II 1 II i

HC CH HC CH

\ X \ •

N N

Nicotinic acid Nicotinamide

tial structure in combined form. Combined forms, even naturally occurring
ones, are not necessarily wholly effective. The chemistry of nicotinic
acid is too old a topic in the field of organic chemistry to require com-
ment here.

Pantothenic Acid. Probably this compound was mainly responsible for
what was first designated "vitamin B3"; it is the only naturally occurring
one capable of performing the nutritional function. Conjugated forms

CH3 OH O

HO— CH2— C^— CH— C— NH— CH2— CH2— COOH

CH3

Pantothenic acid

may possess vitamin activity because they contain the fundamental
structure in combination. The chemistry of pantothenic acid has been
reviewed 5 and detailed material on the subject will be found in the
literature cited in the review. Other material dealing with the chemistry
of pantothenic acid will be found elsewhere in this volume (p. 464).
Pyridoxal, Pyridoxamine, Pyridoxine. For animals these three forms
are nutritionally interchangeable and are often thought to be in equilib-
rium in vivo. Lactic acid bacteria often show incomparably greater
response to pyridoxal or pyridoxamine or their phosphates 6 than to
pyridoxine, so these forms of the vitamin which were discovered later

8 THE BIOCHEMISTRY OF B VITAMINS

appear to be fundamentally more important biocatalytically than
pyridoxine. The three members of the group possess for animals what
has been called "vitamin B6" activity. The chemistry of these compounds

CHO CH2NH2 CH2OH

A A A

HO— C C— CH2OH HO— C C— CH2OH HO— C C— CH2OH

H3C— C CH H3C— C CH H3C— C CH

\/ \/ V

N N N

Pyridoxal Pyridoxamine Pyridoxine

is discussed in a series of articles by Heyl et at. and in earlier contributions
from the same laboratory.7- 8- 9- 10

Biotin. This substance is the only naturally occurring one (except its
conjugates and possibly oxybiotin discussed below) known to be capable
of counteracting the deficiency induced by feeding raw egg white or
avidin, its active constituent, to animals. It is an example of a B vitamin
which appears to be of little or no practical importance nutritionally

II
C

HN NH

HC CH

H2C CH— CH2— CH2— CH2— CH2— COOH

Biotin

(because it is so readily produced by intestinal organisms) but is none-
theless most interesting as a universal cellular constituent which prob-
ably acts catalytically. It is effective in unusually low concentrations.

Oxybiotin, the compound corresponding to biotin with oxygen replac-
ing the sulfur, is especially interesting as a substance which almost falls
within our definition of a B vitamin. Certain microorganisms, at least,
can use it in place of biotin and do not convert it into biotin.11 Oxybiotin
in these cells can act as a part of the metabolic machinery, but since it
appears ordinarily not to be a constituent of cells, and certainly there
is no evidence that it is present in all cells, its inclusion in the group of
B vitamins is not justified.

The organic chemistry of biotin and its derivatives has been reviewed
by Hofmann 12 and by Melville,13 and further discussion of its chemistry
will be found in Section D of this volume.

B VITAMINS: WHAT THEY ARE 9

Folic Acid. The vitamin activity which characterizes this substance
most clearly is its ability to prevent a specific type of anemia in chicks.
No other naturally occurring structure (except its conjugates) is known
to be capable of performing this function.

OH
I H H

C N C=C

N C C-CH2-NH-C C-C-NH-CH-CHr-CHj-COOH

NH2-C C CH C— C O COOH

\ / \ / H H

N N

Folic acid (Pteroylglutamic acid)

More complicated structures occur naturally and their physiological
significance will be discussed in later chapters, but the simplest com-
pound capable of performing the vitamin function is the one pictured
above, and in accordance with our previous discussion it is the only one
which will be considered as a B vitamin. There is no serious doubt, on
the basis of microbiological evidence, but that it functions universally
in living matter. The fact that it is effective in very minute amounts
strongly suggests a catalytic role.

A series of articles dealing with the chemistry of folic acid and its
derivatives treat this subject rather fully.14, 15' 16, 17, 18, 19

Structurally Known Compounds of Somewhat Doubtful Status

In addition to the seven chemically characterized vitamins with dis-
tinct nutritional functions listed above, there are three other well recog-
nized compounds of nutritional signficance: inositol, choline, and
p-aminobenzoic acid, which merit consideration because they possess at
least some of the characteristics of B vitamins.

We have used three criteria in our previous discussions. To belong in
the B group a compound must: (1) function nutritionally for higher
animals, (2) be universally present in living cells, and (3) act or be
presumed to act catalytically. These criteria are listed, in our opinion,
in the approximate order of their importance. The status of each of the
three substances mentioned above can be questioned on the basis of at
least one of these criteria.

Inositol appears to function nutritionally for various experimental
animals (though contrary evidence has been obtained20), and evidence
for its universal occurrence is perhaps as good as for other members of
the group, but its relative abundance in tissues and its occurrence as a
structural unit in recently discovered lipides, makes one question its
inclusion. It is not uncommon for inositol to be present in tissues in

10 THE BIOCHEMISTRY OF B VITAMINS

amounts 100 or even 1000 times that of the more typical B vitamins.
Favoring the inclusion of inositol as a member of the group is the finding
that it constitutes a functioning portion of pancreatic amylase.21* This
indicates a catalytic role. It may be that inositol does indeed act cat-

OH OH

OH/|

V H

l\ H

H OH/|

OH H
Inositol

alytically like the other B vitamins and that its relative abundance is
explained by the fact that it also is a constituent of certain widely
occurring lipides.

The relatively recent determination of the configuration of inositol is
alluded to in a later chapter (p. 18) .

Choline is without question of great nutritional importance and has
some of the earmarks of a B vitamin. If any one of a number of mammals
and fowls is fed a diet deliberately made up so as to be low in choline
content, a serious deficiency results which can be counteracted by the
addition of pure choline salts to the diet. However, methionine and
CH3
H3C— N+— CH2— CH2OH
CH3 Cl-
Choline chloride
betaine (as well as other substances which do not occur naturally) are
also effective under these conditions, and it is clear that they may per-
form at least part of the function of choline. It appears likely that they
may replace choline entirely, especially in the presence of some other
unidentified substance.22 Ethanolamine can evidently be methylated by
Type III pneumococcus 23 to produce choline which in combined form
(phospholipides) is probably always present in living cells. Furthermore,
evidence involving mutant strains of Neurospora shows clearly that the
synthesis of choline takes place through the intermediate formation of
ethanolamine.24

It appears clear that the naturally occurring substances, choline, betaine
and methionine alike, are able to furnish animals with transferable
methyl groups which are essential to life, and that in addition choline

* Contradictory evidence has been reported.21"

B VITAMINS: WHAT THEY ARE 11

itself is an essential cell constituent and must be furnished in the food
or built up by animals. Just what is required for this building up process
is not known. If ethanolamine is the simplest substance which in conjunc-
tion with methionine can overcome the lack of choline in the diet, it
instead of choline should be designated as the vitamin, according to our
previous discussion. From the practical nutritional standpoint, however,
choline is more important than ethanolamine, but its status as a vitamin
is complicated by the existence of other natural food substances which at
least partially replace it.

From the standpoint of universality in cells, choline appears to be like
the typical B vitamins. Its catalytic functions have not been demonstrated
and its relative abundance and the high "requirements" of animals may
be cited against this possibility. On the other hand, it may (like inositol?)
be both a catalytic unit and a constituent of certain phospholipides.
There is some resistance to designating choline as a vitamin because it
was known to be a common constituent of natural foods (combined in
phospholipides) long before the typical vitamins were discovered.

p-Aminobenzoic acid is unique among the vitamins or vitamin-like
substances so far considered in that it makes up an integral part of one
of the typical B vitamins, namely folic acid. Its nutritional functioning

COOH

/ \
HC CH

h£ in

NH2

p-Aminobenzoic acid

has been demonstrated, but not in connection with a diet already con-
taining an adequate supply of folic acid. Furthermore, relatively large
quantities have been used. It is possible, since p-aminobenzoic acid
makes up a part of the folic acid molecule (p. 9), that its nutritional
value is dependent upon its use as a building unit out of which folic
acid may be made by intestinal bacteria or otherwise. Even before the
presence of the p-aminobenzoic acid residue in folic acid was established,
there was evidence that feeding p-aminobenzoic acid to chicks promoted
intestinal production of folic acid.25

The crucial questions which have not been conclusively answered at
the present writing are whether p-aminobenzoic acid is required nutri-
tionally in addition to folic acid, and whether it has catalytic functions
independent of folic acid. There are strong indications of a positive

12 THE BIOCHEMISTRY OF B VITAMINS

answer to the second question, and these will be discussed in later chap-
ters. p-Aminobenzoic acid occurs universally in living matter in its com-
bination in folic acid and without much doubt in other forms also (p. 41) .
It is a highly important catalytic unit and appears to constitute a unique
example of a "vitamin within a vitamin."

B Vitamins and Related Principles Not Completely Characterized
Chemically

In the infancy of vitamin investigations vitamin B was thought to be
a single entity. Since that time, one after another well defined chemical
substance has been found to contribute to the physiological activity which
resides in crude extracts of yeast, liver, etc. How much further this dis-
covery of new organic components possessing vitamin activity will go,
no one can say.

When the time arrives that the list is complete, it will be possible to
feed baby chicks and young rats completely synthetic diets, raise them
to maturity in a healthy condition, have them reproduce normally
generation after generation, and raise young as they do when fed natural
diets. Furthermore, it will be possible to accomplish this feat when the
animals are kept under sterile conditions, free from the symbiotic aid
of microorganisms. Until this latter is possible, one cannot be sure but
that some essential vitamins of bacterial origin are as yet unrecognized.
Biotin is an example of a vitamin which might have been overlooked in
non-sterile feeding experiments were it not for the experimental use of
raw egg white in diets and the presence in it of avidin, which combines
quantitatively with biotin, rendering it inactive.

Many of the B vitamins have been discovered and concentrated by
the use of microorganisms, but historically they were not admitted to the
family of B vitamins until their functioning in animal nutrition had
been demonstrated. The study of microbial nutrition therefore constitutes
an important means whereby hitherto unrecognized nutrilites may be
discovered. Their acceptance as vitamins may depend upon the demon-
stration of their functioning in the nutrition of higher animals.

There is the point of view among some active vitamin investigators,
particularly those who have approached the subject from the standpoint
of enzyme chemistry or microbial nutrition, that catalytic substances of
relatively small molecular dimensions may properly be regarded as
vitamins whether or not they are nutritionally required by higher animals.
This point of view has merit and may eventually be adopted. An alterna-
tive suggestion has already been made, namely, that the term "vitamin"
retain its historical nutritional connotation and that perhaps a new, more
inclusive term (e.g., catalins, p. 5) will be needed to designate all

B VITAMINS: WHAT THEY ARE 13

biocatalysts of low molecular weight, regardless of their nutritional
significance or insignificance.

The search for substances which are potentially B vitamins is an active
one at the present time. Advance in this field is so rapid that this mono-
graph cannot hope to carry up-to-the-minute information. It is not sur-
prising that the author of this section should be in possession of a certain
amount of unpublished information which is pertinent to this discussion.
The only safe procedure in view of the activity in this field, however,
seems to be to treat all unpublished information which may be in the
writer's possession as though it did not exist. The discussions which
follow will, therefore, be based almost entirely upon results which have
received general publication.

If we retain the nutritional point of view with respect to the meaning
of the term "vitamin," then before any new substance can be accepted
as a member of the family, evidence must be available with respect to
its nutritional need by animals. We shall therefore discuss first those
substances for which such need has been demonstrated.

Second, before any growth factor can be discussed intelligently, its
unitary nature and its existence as a chemical entity must be reasonably
well established. In order to meet this criterion, the substance in question
must have been characterized chemically or concentrated to a relatively
high degree.

Before we inquire what is meant by "concentrating a vitamin to a
high degree," it will be well to look at some historical data with respect
to the vitamins which are already well recognized.

Rice polish was the starting material used for the first isolation of
thiamine. It contains in round figures 33 /tg of thiamine per gram.
Thiamine is therefore about 30,000 times as active physiologically as rice
polish, and this figure represents the extent to which concentration had
to be carried to yield the active principle. Using the same type of calcu-
lation, we arrive at the values for the typical B vitamins listed below.

Degree of Concen-

Vitamin

Source

tration Required

Thiamine

Rice polish

30,000 times

Riboflavin

Whey solids

40,000 times

Liver

13,000 times

Nicotinic acid

Liver

3,000 times

Pantothenic acid

Liver

8,000 times

Folic acid

Spinach

160,000 times

Liver

90,000 times

Biotin

Egg yolk

1,400,000 times

Liver

400,000 times

From these data we may infer that before a new vitamin is obtained in
pure form, it is probable that it will have to be concentrated several
thousand times at least, starting with any rich natural source as a refer-

14 THE BIOCHEMISTRY OF B VITAMINS

ence point. Concentration may have to be carried up to a millionfold or
more before the substance is pure. In view of these facts it apears unde-
sirable to refer to a "vitamin concentrate" or to a vitamin having been
obtained "in concentrated form" unless its concentration has been carried
to a point at least 100 times that of a rich natural source. In the literature
in the past numerous "concentrates" have been so designated when they
were not substantially more active than crude extracts of liver or yeast.
In the writer's own experience, the difference between having a "concen-
trate" which is a crude extract and one which is several thousand times
as active as the starting material may involve the expenditure of many
thousands of man-hours of effort. To designate all such products "con-
centrates" regardless of how far the process has been carried shows a
lack of proper discrimination.

Anti-Pernicious Anemia Principle (Vitamin Bio)- Of the vitamins
which have not as yet been completely characterized chemically, by far
the most interesting one at the present writing is the anti-pernicious
anemia principle which has been isolated approximately simultaneously
in crystalline form both in England26 and in the United States.27

It is a red crystalline substance which does not melt before decomposi-
tion ; it contains cobalt, phosphorus and nitrogen, and is reported to have
a molecular weight of about 1500. It is active in clinical pernicious anemia
in doses of about 1 /xg per day, and is thought to be identical with the
"animal protein factor" 28 as well as the "cow manure factor" 29 which have
been reported to be required by chickens.

The physiological activity of the substance as judged by its effect on
human subjects is much greater, weight for weight, than that of any of
the vitamins discussed. Its purification from liver represents several
millionfold concentration.

The effectiveness of this principle in extremely small doses suggests its
catalytic functioning, which has been studied by inhibition analysis
(p. 475). Its relationship to normal nutrition is not entirely clear, but
the requirement of chickens and its relationship to macrocytic anemias of
nutritional origin make its status as a vitamin relatively secure. While
its universal presence in living cells has not been explored, it appears to
be widely distributed in nature and to be produced by various bacteria
and molds.30

It is interesting and significant to note that, although numerous labora-
tories have engaged in studying and attempting to concentrate the anti-
pernicious anemia principle, in terms of chronology no outstanding prog-
ress was reported until after the development of a microbiological test.
Actually, workers in England, however, appear to have used the red color
obtained chromatographically as a basis for clinical testing. This vitamin

B VITAMINS: WHAT THEY ARE 15

extends further the list of B vitamins for the elucidation of which micro-
biological investigations have proved most valuable. It is also worthy of
note that every vitamin isolated or concentrated by using microbiological
tests has fallen into the category of "B vitamins" by common consent and
on the basis of the criteria which we have outlined (p. 5).

Thymidine is worthy of note in this connection because of its functional
relation to the anti-pernicious anemia vitamin31 and the fact that in
relatively large doses it is able to replace the vitamin in microbiological
tests.32 Other desoxyribosides also function in a similar manner.

HN-C=0

0=C C— CH3

I 1

ch2oh— ch— ch— ch2— ch— n— ch
Ah

Thymidine

The interrelationship between thymidine and the pernicious anemia
vitamin will be discussed in a later section (p. 474).

Strepogenin. This growth principle was originally found in liver and
was effective for certain hemolytic streptococci.33 Subsequently it was
found to be more abundant in certain purified proteins, notably insulin
and trypsinogen, and to be released most effectively by tryptic digestion.34
A convenient test organism which is in current use is L. casei.35 What
appears to be the same substance is also effective in promoting the growth
of mice.36, 37 The available evidence indicates that the substance is of
peptide nature and predominantly acidic, and one synthetic peptide, seryl
glycyl glutamic acid, has been found to have appreciable strepogenin
activity.

If strepogenin is a peptide of known amino acids without any novel
feature in its structure, it could hardly be classified as a B vitamin. The
growth principle as tested for in the usual way has not been obtained
in a form such that a minute amount is effective, so a catalytic function
for strepogenin cannot be taken for granted. It is an important nutritional
principle, however, and it is discussed briefly here because, pending the
complete elucidation of its chemical nature, it is a potential member of
the B vitamin family. Recently, Chattaway et al., using diphtheria or-
ganisms for testing, obtained a highly potent preparation from yeast
which is suggestive of strepogenin.38 It is reported to possess activity by
virtue of the presence of two peptides and a fraction which is stable to
acid hydrolysis, all three of which entities are required for maximum
growth.

16 THE BIOCHEMISTRY OF B VITAMINS

"Vitamin 5i3" represents a growth factor for rats which has been ob-
tained in highly concentrated form by Novak and Hauge.39 The concen-
trate prepared was effective for rats at a level of 2 /^g per day and gave
a maximum response when 10 txg per day were administered. Its ultra-
violet absorption curve showed a maximum at 2820 A, and it exhibited
fluorescence. The material was readily soluble not only in water but also
in acetone, ethanol, ether, chloroform and benzene. If this substance
becomes established as a member of the B family of vitamins, it will be
unique with respect to its solubilities. This fact emphasizes the undesir-
ability of using solubility as a criterion for classifying vitamins.

"Vitamin Bu,'' a crystalline substance having high growth-promoting
activity on reticulocytes and tumor cells in vitro and on the anemia in
rats induced by sulfathiazole administration, has been reported by Norris
and Majnarich.40 It was isolated from urine and contains 19.6 per cent
nitrogen, 4 per cent phosphorus and no cobalt, and is thought to be func-
tionally related to folic acid.41

The literature contains numerous references to additional growth sub-
stances for microorganisms and animals which are potentially members
of the B vitamin family. However, neither their concentration nor char-
acterization has proceeded far enough to justify individual discussion.*
The history of the discovery and identification of individual B vitamins
is replete with nutritional factors, often designated by letter names and
numbers, which are more or less composite in nature and which have
remained poorly defined indefinitely. When a new growth substance is
identified and obtained in crystalline form, usually by the help of micro-
biological tests, confusion with respect to various nutritional factors pre-
viously discovered in animal work tends to be dispelled.

Bibliography

1. Williams, R. J., "A.A.A.S. Research Conference on Cancer," Science Press Print-

ing Co., Lancaster, Pa., 1945, pp. 253-66.

2. Williams, R. J., "Vitamins and Hormones," Vol. I, Academic Press, Inc., New

York, N. Y., 1943, p. 231.

3. Williams, R. R., Ergeb. Vitamin-Hormonjorsch., 1, 213-62 (1938).

4. Rosenberg. H. R., "Chemistry and Physiology of the Vitamins," Interscience

Publishers, New York, N. Y., 1942, pp. 155-70.

* Since the above was written the concentration of three additional potential
members of the B vitamin family has been accomplished: Folinic acid (citrovorium
factor), Bond, T. J., Bardos, T. J., Sibley, M. and Shive, W., J. Am. Chem. Soc. 71,
3852 (1949) and Bardos, T. J , Bond, T. J., Humphreys, J. and Shive, W., ibid.; the
Lactobacillus bulgaricus factor, Williams, W. L., Hoff-Jo'rgensen, E. and Snell, E. E.,
J. Biol. Chem. 177, 933-40 (1949); an acetate factor (pyruvate oxidation factor,
"protogen"), Snell, E. E. and Broquist, H. P.. Arch. Biochem. 23, 326 (1949). Other
bacterial factors are discussed by Snell, E. E., Ann. Rev. Microbiol. Ill, 97 (1949).

B VITAMINS: WHAT THEY ARE 17

5. Williams, R. J., "Advances in Enzymology," Vol. Ill, Interscience Publishers,

New York, N. Y, 1943, pp. 253-87.

6. McNutt, W. S., and Snell, E. E, /. Biol. Chem., 173, 801-2 (1948).

7. Harris, S. H., Heyl, D., and Folkers, K., J. Am. Chem. Soc, 66, 2088-92 (1944).

8. Heyl, D., J. Am. Chem. Soc, 70, 3434-6 (1948).

9. Heyl, D., Harris, S. A., and Folkers, K., /. Am. Chem. Soc, 70, 3429-31 (1948).

10. Heyl, D., et al, J. Am. Chem. Soc, 70, 3669-71 (1948).

11. Hofmann, K., and Winnick, T., J. Biol. Chem., 160, 449-53 (1945).

12. Hofmann, K., "Advances in Enzymology," Vol. Ill, Interscience Publishers,

New York, N. Y., 1943, pp. 289-311.

13. Melville, D. B., "Vitamins and Hormones," Vol. II, Academic Press, Inc., New

York, N. Y, 1944, pp. 29-66.

14. Stokstad, E. L. R., et al, Ann. N. Y. Acad. Sci., 48, 269-72 (1946).

15. Hutchings, B. C, et. al, Ann. N. Y. Acad. Sci., 48, 273-8 (1946)

16. Mowat, J. H, et. al, Ann. N. Y. Acad. Sci, 48, 279-82 (1946).

17. Waller, C. W., et al, Ann. N. Y. Acad. Sci., 48, 283-8 (1946).

18. Pfiffner, J. J., et al, J. Am. Chem. Soc, 69, 1476-87 (1947).

19. Gates, M., Chem. Rev., 41, 63-96 (1947).

20. Woolley, D. W., /. Nutrition, 28, 305-14 (1944).

21. Lane, R. L., and Williams, R. J., Arch. Biochem., 19, 329-35 (1948).
21a. Fischer, E. H., and Bernfeld, P., Helv. Chim. Acta., 32, 1146-50 (1949).

22. McGinnis, J., Norris, L. C, and Heuser, G. F., Proc Soc. Expll. Biol. Med., 56,

197 (1944).

23. Badger, E., J. Biol. Chem., 153, 183-91 (1944).

24. Horowitz, N. H, /. Biol. Chem., 162, 413 (1946).

25. Briggs, G. M., et al, Proc. Soc. Exptl. Biol. Med., 52, 7-10 (1943).

26. Smith, E. L., Nature, 161, 638 (1948).

27. Rickes, E. L., et al, Science, 107, 396 (1948).

28. Ott, W. H., Rickes, E. L., and Wood, T. R., J. Biol. Chem., 174, 1047 (1948).

29. Lillie, J. L., Denton, C. A., and Bird, H. R., ./. Biol. Chem., 176, 1477-8 (1948).

30. Rickes, E. L., et al, Science, 108, 634-5 (1948).

31. Shive, W., Ravel, J. M., and Eakin, R. E., J. Am. Chem. Soc, 70, 2614 (1948).

32. Wright, L. D., Skeggs, H. R., and Huff, J. W., J. Biol Chem., 175, 475-6 (1948).

33. Woolley. D. W., J. Exptl. Med., 73, 487-92 (1941).

34. Wright, L. D., and Skeggs, H. R., /. Bad., 48, 117 (1946).

35. Sprince, H, and Woolley, D. W., J. Exptl. Med., 80, 213-7 (1944).

36. Woolley, D. W., J. Biol. Chem., 159, 753 (1945).

37. Woolley, D. W, J. Biol. Chem., 162, 383-8 (1946).

38. Chattaway, F. W., et al, Biochem. J., 43, lix (1948).

39. Novak, A. F., and Hauge, S. M., J. Biol. Chem., 174, 647 (1948).

40. Norris, E. R., and Majnarich, J. J., Science, 109, 32-3 (1949).

41. Norris, E. R., and Majnarich, J. J., Science, 109, 33-5 (1949).

«$m>

Chapter II A.

DISTRIBUTION OF B VITAMINS

Of the ten members or possible members of the B vitamin family
discussed in Chapter IA, four (inositol, nicotinic acid, choline, and
p-aminobenzoic acid) were well recognized chemicals long before vitamins
were discovered. The earlier information regarding these is therefore
presented briefly first.

Inositol was known during the last century as a compound of wide
distribution having been found in the sprouts, leaves, fruits, seeds and
rhizomes of a considerable number of plants and in the blood, urine and
various organs and tissues of animals such as cattle, guinea pigs and dogs.
It was found also in fowls and cephalopods and in human urine. Early
materials used in the preparation of inositol from natural sources included
walnut leaves, mistletoe berries and beef lung or beef heart. The complete
stereochemical structure of the naturally occurring substance, however,
was not elucidated until 1942. 1- 2

Nicotinic acid has been known chemically for about 80 years as a
product formed by strong oxidation of nicotine. It was first isolated from
natural sources (rice polishings) by Suzuki, Shamimura and Odake3 and
soon after Funk4 isolated it from both yeast and rice polishings in his
attempt to concentrate the anti-beriberi vitamin. Its natural occurrence
was not, however, mentioned in the 4th edition of Beilstein (1935) . It was
only after its coenzymic and vitamin functions were known that its wide-
spread occurrence was recognized.

Choline, first isolated as a hydrolytic product of a phosphatide fraction
in 1865, was originally called "neurin." It was found later to be wide-
spread in ergot, in mushrooms, in the germs of seeds and in other plant
tissues: leaves, fruits, flowers, rhizomes. In these plant sources it was said
to be partly free and partly combined in what are now called phospho-
lipides. It was also found in animal tissues: glandular tissues, brain,
blood, sperm, etc. If, as is commonly thought, lecithins and related
phospholipides are always present in living cells, the universal distribu-
tion of choline in combined form is evident.

p-Aminobenzoic acid has been known chemically since the infancy of
synthetic organic chemistry, but knowledge of its natural occurrence dates
from its isolation from yeast in 1 940-41. 5- G It was administered to dogs

DISTRIBUTION OF B VITAMINS 19

as long ago as 1889 7 and was found to be without physiological effect. It
was found to be excreted unchanged in later experiments.8 Only after its
physiological functioning was suspected 9 has its widespread natural occur-
rence been established.

Evidence for Universal Biological Occurrence of B Vitamins

The universal presence of each B vitamin in living cells is indicated by
microbiological evidence which will be discussed in later paragraphs.
Before this method of study became so generally applicable, it was
realized, for example, that thiamine occurs widely. Williams and Spies*
list in tabular form about 90 different types of products which contain
thiamine. These include animal tissues, fish, dairy products, legumes,
cereals, vegetables, fruits, nuts and yeast. The quantitative information
given by them is based entirely upon animal experiments.

In early investigations involving riboflavin, it was found in yeast, kid-
ney, liver, suprarenals, corpus luteum, egg yolk, egg white, milk, urine,
blood serum and the retinas of fish eyes.10 Advantage was taken in this
case of the fact that riboflavin exhibits characteristic fluorescence. Its
fluorimetric determination will be discussed later. For most of the evidence
bearing on .the universal distribution of the B vitamins in general, how-
ever, we must depend on microbiological evidence.

The first evidence as to the universal occurrence in living matter of
what is now a recognized B vitamin was obtained in connection with
pantothenic acid.11 A systematic study based upon a microbiological test
was presented in 1933, which indicated that the same acid growth deter-
minant was present in all types of living organisms. The materials exam-
ined represented eleven different biological phyla: Chordata, Arthropoda,
Echinodermata, Mollusca, Annulata, Plathylminthes, Mxyomycetes, Bac-
teria, Fungi, Algae and Spermatophytes, and in the case of the more
common phyla several examples were tested. Because of the evidence for
its diverse distribution, this substance was named pantothenic acid (from
the Greek, meaning from everywhere) . It should be noted by contrast
that the seemingly diverse sources of inositol and choline mentioned above
on page 18 represent in each case only three phyla. Likewise, the
evidence for the widespread occurrence of thiamine based upon animal
tests above and the requirements of some insects involved not more
than four phyla: Chordata, Arthropoda, Fungi, and Spermatophytes. It
may be mentioned at this point, though the question will be discussed
later, that ascorbic acid, which is not considered a B vitamin, is widely
present in the tissues of about seven phyla, yet it is not present, to the

* Williams, R. R. and Spies, T. D., "Vitamin Bi (Thiamin) and Its Use in Medi-
cine," The Macmillan Co., New York, N. Y., 1939, 411 pp.

20 THE BIOCHEMISTRY OF B VITAMINS

best of our knowledge, in representative members of the groups repre-
sented by Protozoa, Bacteria and Yeasts. Other examples to be cited later
indicate that widespread occurrence in several phyla is not equivalent to
universal biological occurrence.

It was not apparent at the time, but the root name, pantothen-, could
have been given appropriately to any member of the B vitamin family
because microbiological evidence now reveals that each one is universally
present in the same sense that pantothenic acid is.

The most extensive and systematic study of the distribution of the B
vitamins was undertaken in the writer's laboratories. This series of ex-
plorations was carried with the full realization that data would be subject
to later revision because of improvements in microbiological methods, but
they served to demonstrate, as had not been done before, that thiamine,
riboflavin, nicotinic acid, pantothenic acid, pyridoxine (pyridoxal, see
p. 8), biotin, folic acid and inositol are to be found in any type of
biological material that is examined.

In one study 12 the autolyzates of 50 animal tissues (rat, mouse, beef
and swine) were found to contain substantial amounts of every member
of the group. Embryonic, immature and mature livers, hearts and brains
of rats and chickens were also assayed.13 In other studies enzymatic
digests of seven representative rat tissues,14 seventeen representative
human tissues (three individuals),15 twenty-three human cancers, 18
mouse and rat cancers,16 cell nuclei from heart and cancer tissues 17 were
assayed, always with the same result: substantial amounts of all the
substances in question were found. More comprehensive exploration,18
including thirty-four representative materials from eight different biologi-
cal phyla (Chordata, Arthropoda, Mollusca, Annelida, Protozoa, Bacteria,
Fungi, and Spermatophytes) showed again the universal presence of all
eight substances, as had earlier been shown for pantothenic acid. In an-
other study,19 milk from six species (human, mare, cow, goat, dog, mouse)
was assayed and all eight substances found in every sample.

Numerous scattered studies have contributed information and have
tended to corroborate the universal occurrence of the B vitamins. The
most comprehensive recent compilations of data with respect to their
quantitative distribution in foods are cited in the bibliography.20, 21, 22, 23
Due allowances must be made in every case for the shortcomings of the
methods used.

Contrast Between the Distribution of B Vitamins and that of Other
Vitamins

In view of the possible importance of distribution as a criterion for
determining whether or not a substance should be classified as a B vita-

DISTRIBUTION OF B VITAMINS 21

min, it will be well to examine critically the available information regard-
ing the distribution of those vitamins which are not considered within
this group.

Ascorbic Acid. Of the vitamins not considered in the B group probably
ascorbic acid is most widespread. Certainly, there is no question but that
it is generally, and probably universally, present in the tissues of mature
higher animals.24 Extremely interesting in this connection, however, is
the fact that it is absent from unincubated hen eggs and only appears on
incubation, when the amount increases during the first two weeks.25 Dur-
ing embryonic development, as judged by the acid silver nitrate staining
technique, it does not appear in all of the chick tissues. It disappears from
the liver after the tenth day and does not appear in the adrenals until
the twelfth day.26 These results are in striking contrast to those observed
in connection with the B vitamins. All of these are present in eggs from
the start, even when they are synthesized (e. g., nicotinic acid) by the
chick tissues during incubation.27 Certainly, the B vitamins do not appear
to be absent from any actively growing tissue.

Ascorbic acid likewise is widespread in higher plants. Unlike B vitamins,
however, it is practically absent from typical seeds but is produced during
embryonic development.

It also seems clear that ascorbic acid is present in the tissues of many
lower organisms: earthworms, six molluscs, sea urchins, crustaceans,28
crabs,29 thirteen marine invertebrates, seven marine plants,30 cock-
roaches,31 and mushrooms.32, 33 It is also produced by Aspergillus niger?^
In many of these organisms its distribution in the various organs strongly
suggests that it is of functional importance. The fact that cockroaches
synthesize it and maintain it at about the same level whether or not it
is present in the diet, as do rats, points to the same conclusion.

When we consider the monocellular organisms, bacteria, yeasts and
protozoa, however, we find that the preponderance of evidence indicates
that in these organisms ascorbic acid is generally absent and nonfunc-
tional. Although ascorbic acid has been reported repeatedly to promote
the growth of specific bacteria,35, 36 this effect has always been inter-
pretable as due to the change in the oxidation-reduction potential of the
medium rather than to its action as a specific nutrilite. Though the forma-
tion of vitamin C by bacteria has been reported,36, 37 there is no clear-cut
proof that it is actually ascorbic acid and not some other highly reducing
substance that is produced. There is no doubt that intestinal bacteria
utilize ascorbic acid,38, 39 but in these cases it is probably serving simply
as an energy source. The fact that no one has ever identified ascorbic acid,
either chemically or biologically as a constituent of yeast places it in
striking contrast to the B vitamins, all of which (for which adequate.

22 THE BIOCHEMISTRY OF B VITAMINS

evidence is available) are present in significant amounts in this organism.
The requirement of vitamin C for the growth of certain protozoa (Tricho-
monads) has been reported,40 but not enough information is given to rule
out the effect on the oxidation-reduction potential as an important factor.
The definite absence of ascorbic acid from Paramecium caudatum, P.
bursaria, Stentor coeruleus, Opalina and Nictoterus has also been re-
ported,41 and in the same study it was found that intestinal trypanosomes
in the guinea pig may or may not contain ascorbic acid, depending upon
whether or not the guinea pig diet is deficient.

So far as vitamin C is concerned, it appears to belong definitely in a
different category from the B vitamins in that it is not universally dis-
tributed, being absent from eggs and seeds, from certain embryonic organs
and in general from bacteria, yeast and protozoa.

Vitamin A in its various forms occurs widely in nature, but shows a
marked contrast to the B vitamins inasmuch as it does not appear to be
present in all mammalian tissues,42 and has been found to be absent
during the entire life cycle of cockroaches.43 Its distribution in numerous
lower forms has apparently not been ascertained, but the two facts cited
above are sufficient to show that its distribution shows a marked contrast
to that of the B vitamins. It is interesting that in plants there is some
relationship between the distribution of the carotenoids and ascorbic
acid.44

Vitamins D. Information regarding the distribution of the D vitamins,
except for relatively rich sources, is scanty.45 The fact that they are prac-
tically absent from plant foods and that they appear to be unimportant
for microorganisms indicates that they are not universal. Yeasts, fungi,
and other lower forms contain sterols which may be converted to D vita-
mins by ultraviolet light, but since many of these organisms can live
entirely in the dark, there is no reason to think that D vitamins function
in their physiology. How the D vitamins are distributed in animal organs
and tissues is unknown.

Vitamins E. Comparatively little quantitative information is avail-
able regarding the distribution of tocopherols (E vitamins) in mammalian
tissues,46, 47 though it appears to be generally present. Still less is known
about its presence in lower animals. Its principal known sources are seed
germ oils and certain plants (lettuce, alfalfa) . The mold Phy ' corny ces
appears to contain none at all.48 Yeast evidently contains none because
it is included in vitamin E-deficient diets which are fed to rats. "Royal
jelly," which is fed to the bee larvae that are to become fertile queens, is
devoid of any significant amount of vitamin E.49 In contrast, royal jelly
is a relatively rich source of most of the B vitamins and is even richer in
pantothenic acid than beef liver.50 Sea-urchin eggs are a rich source of

DISTRIBUTION OF B VITAMINS 23

vitamin E 51 ; this is in contrast to hen's eggs which are a poor source of
E vitamins. The occurrence of E vitamins in the germs of seeds (and in
some eggs) makes their distribution resemble that of the B vitamins more
than that of ascorbic acid, but the available evidence indicates that they
are absent and nonfunctional in many lower forms of life.

Vitamins K. The distribution of the K vitamins is particularly inter-
esting in that they are produced by bacteria, are widespread in the
chloroplasts of green plants 52 and are known to function in higher animals.
However, one bacterium out of ten produced no demonstrable amount of
vitamin K, and yeast contains little or none.33 Substances with vitamin K
activity have no effect on the growth, respiration or fermentation of
yeast.54 The studies of Dam and co-workers 54 have shown that vitamin K
is -present in all chlorophyll-bearing plant organs and that it is absent or
present in low amounts in plant organs which normally do not carry
chlorophyll throughout development. Chloroplast preparations were found
to be about 60 times as rich as cytoplasm preparations. Seeds contain very
little. Photosynthesizing algae and bacteria were found to contain vitamin
K.54, 55, 56 Mushrooms were found to contain roughly 1/40 of the amount
in green leaves.

The absence or near absence of K vitamins from seeds, yeast, certain
bacteria, nonchlorophyll-bearing higher plant organs and most animal
tissues makes it appear that their occurrence is not universal. Certainly
their distribution offers a strong contrast to that of the B vitamins.

Quantitative Relationships Pertaining to the Distribution of B Vitamins

Some of the quantitative relationships with respect to the distribution
of the B vitamins are worthy of note, in spite of the fact that available
data are not all dependable. Our present discussion of these relationships
will be limited to six substances: thiamine, riboflavin, nicotinic acid,
pantothenic acid, biotin and inositol. The available data with respect
to pyridoxal, etc., are not sufficiently reliable because of the difficulties
involved in assay and in releasing these forms quantitatively from tissues
without destruction. The available folic acid values are too low by a
variable and unknown amount due to the fact that when most of the
comparable assays were made, enzymes capable of freeing it completely
from tissues were not known.57 Data regarding p-aminobenzoic acid and
choline which might be used for comparative purposes are not available.

From the data compiled in Table 1 it may be noted first that in terms
of the absolute amounts, inositol is always the most abundant and biotin
the least abundant. In 17 out of 24 cases, the following order is main-
tained: (1) inositol, (2) nicotinic acid, (3) pantothenic acid, (4) ribo-

24 THE BIOCHEMISTRY OF B VITAMINS

flavin, (5) thiamine, (6) biotin. Only the following exceptions may be
noted: In five cases (including the 3 seeds) the thiamine content exceeds
that of the riboflavin; in two cases, riboflavin exceeds pantothenic acid;
and in one case pantothenic acid exceeds nicotinic acid. Otherwise the
order of their occurrences falls into the same pattern, and the regularity
observed in the diverse forms is remarkable.

Among the sources given, the values vary least in the case of inositol —
protozoa are 12 times richer than brewers' yeast — and most in the case

Table 1. Relative Abundance of B Vitamins in Whole Organisms (dry wt.).*

Nicotinic

Panto-

Thiamine

Riboflavin

Acid

thenate

Biotin

Inositol

(7/g)

Rat

5.0

10.5

180

0.33

560

Fish

9.5

5.2

0.31

880

Frog

6.4

11.4

0.57

1230

Horned toad

11.

21.

170

0.7

2100

Snake

5.1

45.

142

0.25

1070

Chick embryo

8.3

13.4

405

370

1.75

1180

Red ant

7.3

14.

0.37

2200

Cockroach

16.2

26.

120

0.48

1340

Termite

12.8

26.5

175

0.66

2150

Dros. larvae

24.

47.

210

116

2.05

930

Dros. larvae

23.

43.

195

108

1.95

1320

Oyster

11.

13.

0.53

2700

Earthworm

7.8

25.

0.25

520

Protozoa

38.

17.

105

0.75

3300

A. aerogenes

10.6

43.

240

145

3.9

1360

S. marcescens

27.

35.

235

124

4.1

1160

P. fluorescens

26.

68.

210

7.1

1700

C. butylicum

9.3

55.

250

1.7

860

Mushrooms

8.8

26.

540

138

1.4

1350

Brewers' yeast

8.5

15.2

126

0.07

280

Mold

0.44

4.7

0.10

1280

Wheat seed

5.5

1.8

0.06

1900

Lima bean

5.7

1.4

0.12

1800

Blackeyed peas

8.5

1.5

0.22

2500

* Too much reliance should not be placed upon the exact numerical values, since incomplete extractions
and other limitations are involved in connection with the methods used. The material given represents
the only data on the subject that are available.

of biotin — P. fluorescens is 120 times as rich as wheat seed. Sixty per cent
of the values given in Table 1, however, do not differ from the mean
value for that vitamin by more than a factor of two.

Since microbiological methods are extremely sensitive and can be used
to determine infinitesimally minute amounts, the question may be raised
whether the mere finding of measurable amounts of the various B vitamins
in all organisms is really significant. If we select the lowest values for
each of the vitamins in Table 1 we find that they correspond to the fol-
lowing sources: (1) for thiamine the mold is poorest and the whole rat
a poor second; (2) for riboflavin the three seeds are lowest; (3) for
nicotinic acid again the seeds are lowest; (4) the same is true for panto-
thenic acid; (5) for biotin, wheat and brewers' yeast are the poorest

DISTRIBUTION OF B VITAMINS 25

sources; (6) for inositol, brewers' yeast and rat carcass are the poorest
sources. If the B vitamins are present in any organisms in insignificant
amounts, the ones cited above are likely examples; therefore let us con-
sider these sources individually.

In view of Schopfer's extensive work dealing with the importance of
thiamine for molds, its indispensable role in these organisms can hardly
be questioned. Certainly no one would say that thiamine is insignificant
for rats. The fact that seeds are the poorest sources of riboflavin, nicotinic
acid and pantothenic acid does not indicate a lack of importance of these
vitamins in seed plants, because each increases during germination (at
least in blackeyed peas and lima beans) ,5S and there is abundant indirect
evidence of a diverse nature that they function in the enzyme systems of
seed plants. In view of the importance of biotin as a nutrilite for yeasts,
no one could question that it exists in significant amount in brewers' yeast.
Wheat has about the same amount, and its significance is undoubted,
especially since biotin is relatively abundant in many seed plants. In view
of the importance of inositol as a nutrilite for yeasts, the relatively low
amount in brewers' yeast cannot be taken as an indication of lack of im-
portance. There is no reason to think that inositol is present in rat tissues
in insignificantly low amounts, especially in view of the fact that it is
unevenly distributed in the various tissues in accordance with a definite
pattern, and the total amount present in the carcass of a 200 gram rat is
about 40 mg.

On the basis of these facts we can safely conclude that the B vitamins
are present in all organisms in significant amounts.

The quantitative distribution of the B vitamins in different tissues of
the same species is of interest because of the relative uniformity of the
amounts present and apparent presence of significant amounts in every
tissue and at every stage of development.

The most complete data available are those obtained by the assay of
17 human tissues. In Table 2 is given a summary of the values obtained
from the tissues of three persons, two males and one female. An examina-
tion of these results shows that in 14 out of the 17 tissues, the absolute
amounts of the substances present are in the same order as for the whole
biological kingdom, namely inositol (1), nicotinic acid (2), pantothenic
acid (3) , riboflavin (4) , thiamine (5) , biotin (6) . In the other three tissues
the same order is maintained except that riboflavin slightly exceeds panto-
thenic acid in amount (by from 3-10 per cent).

The variation in content from tissue to tissue is greatest in the case of
biotin (see also Table 1) ; liver is about 50 times as rich as seminal duct.
The variation is least in the case of nicotinic acid; liver is about seven
times as rich as skin. Curiously, when the results in Table 1 are compared
with those in Table 2, a striking resemblance is observed. In the latter

26 THE BIOCHEMISTRY OF B VITAMINS

case, 58 per cent of the values differ by less than a factor of two from the
average value for the vitamin in question, whereas for Table 1, 60 per cent
is the corresponding figure. These data seem to indicate that insofar as
the content of the various B vitamins can be taken as an index of meta-
bolic characteristics, the diversity of these characteristics is just about as
great for the various tissues of a mammal as it is for the various organisms
in the whole biological kingdom.

Unfortunately, data comparable to that given in Tables 1 and 2 are
lacking for each of the vitamins other than those belonging to the B

Table 2. B Vitamins in .

Human T\

Issues, (wet wt.

y/g.)*

Nicotinic

Pantothenic

Thiamine

Riboflavin

Acid

Biotin

Inositol

Heart

3.6

8.3

0.17

500

Liver

2.2

0.74

660

Brain

1.6

2.5

0.58

1510

Lung

1.5

1.9

0.19

400

Kidney

2.8

0.67

1240

Spleen

1.1

3.6

5.4

0.06

1030

Skel. muscle

1.2

2.0

0.035

450

Sm. muscle

1.2

2.3

6.2

0.06

580

Adrenal

1.6

8.2

0.35

690

Stomach

0.56

5.2

6.1

0.19

760

Ileum

0.55

4.2

5.3

0.06

750

Colon

1.0

2.1

0.09

780

Mammary gland

0.43

2.4

3.9

0.04

270

Ovary

0.61

4.3

3.9

0.025

580

Testes

0.8

2.0

0.09

1600

Seminal ducts

0.69

1.0

9.2

2.0

0.015

<100

Skin

0.52

1.2

8.6

3.1

0.022

200

family. From the fragmentary evidence available we can be reasonably
sure that both vitamin A and vitamin C would be found to be absent from
a number of the sources listed in Table 1. Insofar as data are available,
it appears that ascorbic acid is present in all adult human tissues; but
the presence of vitamin A has not been demonstrated in the epithelial
layers of the skin, in ovaries after menopause, in testicles before puberty
or after involution, in the normal duodenal mucosa, etc.42 The complete
absence of vitamin A from the livers of some animals has been reported,59
and tremendous variation in tissue content is common. In this respect,
the quantitative distribution of vitamin A shows a strong contrast to that
of the B vitamins.

Distribution of B Vitamins in Tumors

The quantitative distribution of the B vitamins in cancerous tissues
is interesting both from the standpoint of the B vitamins and because of
the light that it sheds on the cancer problem. In a series of 23 human

DISTRIBUTION OF B VITAMINS 27

tumors of various types almost without exception the same order of
occurrence was observed as in other studies, with inositol the most abun-
dant and biotin the least abundant of the six. Some of the specimens
assayed were estimated to be 70-80 per cent cancer tissue, while a few
contained as little as 20 per cent.16- co- 61- 62> 63

For comparing a group of tissues with each other, on the basis of their
content of B vitamins, a simple mathematical scheme was used by which
to calculate from the assay values the coefficient of uniformity (100 per
cent minus the coefficient of variation) of the group of tissues compared.
On this basis eight diverse normal human tissues showed a uniformity of
only 27 per cent, whereas eight diverse cancers showed a much higher
uniformity, namely, 66 per cent. Three normal human tissues (kidney,
ovary and mammary gland) showed a uniformity of only 11 per cent,
whereas three cancers derived from these same three tissues showed a 60
per cent uniformity. These observations led to the idea that cancer tissue,
regardless of its origin, may represent, from the standpoint of its inherent
metabolic machinery, a specific tissue type.

Further evidence supporting this idea was obtained by comparing vari-
ous groups of tissues as indicated in Table 3.

Table 3. "Vitamin Uniformity" in Human, Rat, and Mouse Normal and
Cancer Tissues.

Coefficient of Uniformity

8 diverse normal rat tissues 29.7
5 rat cancers of diverse origin 62.8

9 heart tissues from mice 76.0
12 diverse mouse cancers 58.0

9 heart tissues, 3 each from human, rat and mouse 61.2

22 cancers (8 human, 5 rat, 9 mouse) 53.3

In all cases when groups of cancers were compared, regardless of whether
they originated in humans, in rats or mice, or in what type of tissue they
originated or whether they were induced or spontaneous, the coefficient
of uniformity was above 50 per cent; however, when diverse tissues, for
example, those in which the cancers originated, were compared, the uni-
formity was far lower than this.

The conclusion derived from these findings is important because it has
been corroborated, largely on the basis of enzyme studies. In this connec-
tion, Greenstein says, "It is possible to speak of cancer tissue in much
the same way as one speaks of hepatic tissue or renal tissue, namely as
a tissue with limited and ascertainable properties." 64

In general, cancer tissue (on a moist basis) tends to have a relatively
low content of B vitamins, perhaps 50 per cent as much as an average of
other mammalian tissues. Part of this difference is due to the relatively
high water content of cancer tissue. However, some of the B vitamins,

28 THE BIOCHEMISTRY OF B VITAMINS

viz., riboflavin, biotin and pyridoxine,* were found to be proportionately
much lower than in other tissues, and inositol and folic acid* were rela-
tively abundant. These findings arc interesting in that both inositol and
folic acid have been found to be effective in causing regression of cancer
when injected intravenously into mice.'1"'

Unfortunately neither the studies of the content of B vitamins nor
extensive enzyme studies have been extended to cover fowl tumors which
are known to be virus-induced. If these tumors should be found to follow
the same pattern, this will constitute further circumstantial evidence that
mammalian tumors are also induced by viruses.

Bibliography

1. Posternak, T., Helv. Chim. Acta, 25, 746-52 (1942).

2. Dangschat, G., and Fischer, H. O. L., Naturwiss., 30, 146-7 (1942).

3. Suzuki, U., Shamimura, T., and Odake, S., Biochem. Z., 43, 89-153 (1912).

4. Funk, C, J. Physiol, 46, 173-9 (1913).

5. Rubbo, S. D., and Gillespie, J. M., Nature, 146, 838-9 (1940).

6. Blanchard, K. C, J. Biol. Chem., 140, 919-26 (1941).

7. Gibbs, W., and Hare, H. A., Am. Chem. J., 11, 435-48 (1889).

8. Hildebrand, H., Beitr. Z. Chem. Physiol. Pathol, 3, 365 (1903).

9. Woods, D. D., Brit. J. Exptl. Path., 21, 74-90 (1940).

10. Euler, H. v., and Alder, E., Z. Physiol Chem., 228, 1-12 (1934).

11. Williams, Roger J., et. al, J. Am. Chem. Soc, 55, 2912-27 (1933).

12. Wright, L. D., et. al, Univ. Texas Pub., 4137, 38-9 (1941).

13. Williams, R. J., Taylor, A., and Cheldelin, V. H., Univ. Texas Pub., 4137, 61-6

(1941).

14. Mitchell, H. K., and Isbell, E. R., Univ. Texas Pub., 4237, 37-40 (1942).

15. Taylor, A., Pollack, M. A., and Williams, R. J., ibid., 41-55.

16. Pollack, M. A., Taylor, A., and Williams, R. J., ibid., 56-71.

17. Isbell, E. R., et al, ibid., 81-3.

18. Woods, A. M., et. al, ibid., 84-6.

19. Williams, R. J., Cheldelin, V. H., and Mitchell, H. K., ibid., 97-104.

20. Adams, G., and Smith, S. L., U. S. Dep. Agr. Misc. Pub., 536, 88 pp. (1944).

21. Cheldelin, V. H, and Williams, R. J., Univ. Texas Pub., 4237, 105-24 (1942).

22. U. S. Dep. Agr. Misc. Pub., 572, (1945).

23. Com. on Food Comp., Natl. Research Council (U. S.) 1944.

24. Giroud, A., Ergeb. Vitamin-H ormonjorsch., 1, 68-113 (1938).

25. Suomalainen, P., Ann. Acad. Sci. Fennicae, Ser. A53, No. 8, 13 pp. (1939).

26. Barnett, S. A., and Bourne, G., Quart. J. Microscop. Sci., 83, 299-316 (1942).

27. Snell, E. E., and Quarles, E., J. Nutrition, 22, 483-9 (1941).

28. Giroud, A., and Ratsimamanga, R., Compt. rend. soc. biol, 120, 763-5 (1935).

29. Ludany, G., Biochem. Z., 284, 108-10 (1936).

30. Eekelen, M. van, Acta. Brevia Neerland. Physiol. Pharmacol. Microbiol, 3,

119-20 (1933).

31. Wollman, E., Giroud, A., and Ratsimamanga, R., Compt. rend. soc. biol, 124,

434-5 (1937).

32. Anderson, E. E., and Fellers, C. R., Proc. Am. Soc. Hort. Sci., 41, 301-4 (1942).

* The values for pyridoxine and folic acid in the publication under discussion16
are of comparative value only, since the release of these vitamins could not be
accomplished in a quantitative manner at the time the investigation was carried out.

DISTRIBUTION OF B VITAMINS 29

33. Kawakami, K., and Miyayosi, EL, Rept. Inst. Sci. Research Manchoukuo, 4,

399-403 (1940).

34. Bernhauer, K., Gorlich, B., and Kocher, E., Biochem. Z., 286, 60-5 (1936).

35. Illenyi, A., Zentr. Bakt. Parasitenk. Abt. I, Orig., 114, (7/8), 502 (1937).

36. Illenyi, A., and Kenessey, S., Zentr. Bakt. Parasitenk. Abt. I, Orig., 146, 204-7

(1940).

37. Berencsi, G., and Illenyi, A., Biochem. Z., 298, 298-300 (1938).

38. Young, R. M., and James, L. H., /. Bad., 44, 75-84 (1942).

39. Kendall, A. I., and Chinn, H., Quart. Bull. Northwestern Univ. Med. School,

15, 205-10 (1941).

40. Cailleau, R., Rept. Proc. 3rd Intern. Congr. Microbiol, 1939, 493 (1940).

41. Roskin, G., and Nastyukova, O., Compt. rend. acad. sci. U.R.S.S., 32, 566-8 (1941).

42. Popper, H., Physiol. Revs., 24, 205-24 (1944).

43. Bowers, R. E., and McCay, C. M., Science, 92, 291 (1940).

44. Giroud, A., et al., Bull. soc. chim. biol, 18, 573-89 (1936).

45. Williams, R. J., "Vitamins and Hormones," Academic Press Inc., New York,

N. Y., 1943, Vol. IV, p. 231.

46. Mason, K. E., J. Nutrition, 23, 71-81 (1942).

47. Hines, L. R., and Mattill, H. A., J. Biol. Chem., 149, 549-54 (1943).

48. Schopfer, W. H., and Blumer, S., Z. Vitaminforsch., 9, 344-9 (1939).

49. Mason, K. E., and Melampy, R. M., Proc. Soc. Exptl. Biol. Med., 35, 459-63

(1936).

50. Pearson, P. B., and Burgin, C. J., Proc. Soc. Exptl. Biol. Med., 48, 415-7 (1941).

51. Lieck, H., and Willstaedt, H., Svensk. Kern. Tid., 57, 134-9 (1945).

52. Gaffron, H., J. Gen. Physiol, 28, 259-68 (1945).

53. Almquist, H. J., Pentler, C. F., and Mecchi, E., Proc. Soc. Exptl. Biol. Med.,

38, 336-8 (1938).

54. Dam, H., Glavind, J., and Nielsen, N., Z. physiol. Chem., 265, 80-7 (1940).

55. Dam, H., Am. J. Botany, 31, 492-3 (1944).

56. Dam, H., Glavind, J., and Svendsen, I., Biochem. J., 32, I, 485-7 (1938).

57. Cheldelin, V. H., et al, Univ. Texas Pub., 4237, 15-36 (1942).

58. Cheldelin, V. H., and Lane, R. L., Proc. Soc. Exptl. Biol. Med., 54, 53-5 (1943).

59. Karrer, P., Euler, H. v. and Schopp, K., Helv. Chim. Acta, 15, 493-5 (1932).

60. Pollack, M. A., et al, Cancer Research, 2, 739-43 (1932).

61. Taylor, A., et al, Cancer Research, 2, 744-7 (1942).

62. Pollack, M. A., et al, Cancer Research, 2, 748-51 (1942).

63. Taylor, A., et al, Cancer Research, 2, 752-4 (1942).

64. Greenstein, J. P., "Biochemistry of Cancer," Academic Press Inc., New York,

N. Y., 1947, p. 370.

65. Lewisohn, R., et al, Proc. Soc. Exptl. Biol Med., 52, 269-72 (1943).

Chapter IMA

COMBINED FORMS— EXTRACTION

It is an experimental fact that in spite of their relatively high water
solubility in the free state, the B vitamins often resist aqueous extraction
from plant and animal materials.

In the case of at least one of the B vitamins (biotin), its distribution
in various tissues was originally determined quantitatively on the assump-
tion that it would be extracted by water, since it exhibited a hydrophilic
character in' the free state. Subsequent investigations showed that a small
and uneven fraction of the vitamin in the various tissues had been ex-
tracted.1 Since the extraction of each of the B vitamins presents special
problems, we must consider them separately.

Thiamine

Historically, yeast and rice polish have been the most important mate-
rials used in attempts to concentrate and purify this vitamin. These two
sources offer considerable contrast, because yeast requires autolysis before
it becomes a favorable source while rice polish can be effectively extracted
directly with acidulated water.2

Since the work of Auhagen 3 and Lohmann and Schuster,4 it has been
recognized that a considerable part of the thiamine in yeast and in animal
tissues is combined in the form of diphosphothiamine (cocarboxylase) ,
and this may undergo cleavage to produce free thiamine. Actually the
diphosphothiamine is itself in combined form in the hoioenzyme, car-
boxylase. Carboxylase has been isolated in pure form and contains 0.46
per cent diphosphothiamine and 0.13 per cent magnesium.5 In the presence
of high salt concentrations the cocarboxylase is firmly bound, but in dilute
salt solutions or in alkaline or acid ammonium sulfate solution it is almost
completely dissociated.6

Various commercial enzyme preparations (taka-diastase, malt diastase,
clarase, polidase, high phosphatase mylase, etc.) catalyze the hydrolysis
of diphosphothiamine, usually at pH 4.5 7- 8 and hence can be used to free
thiamine more or less completely from its natural combination in various
tissues.

Westenbrink 9 postulated the existence of more than one type of com-
bination in undissociated carboxylases, and Sarett and Cheldelin,10 by

COMBINED FORMS—EXTRACTION 31

inhibition studies, found evidence that thiamine itself may form a rela-
tively undissociable linkage with protein, which possesses enzymatic
activity. Tatum and Bell/1 using the same inhibiting agent (pyrithi-
amine) on mutant strains of Neurospora, interpret their somewhat similar
findings to indicate that endogenous thiamine is more effectively utilized
than exogenous thiamine. In a subsequent study of the dissociation of
carboxylases in different yeasts and yeast preparations, Parve and Wes-
tenbrink 12 concluded that the various phenomena could be explained on
the supposition that cocarboxylase combines with proteins other than the
unaltered apoenzyme.

There are a number of other suggestive findings with respect to the
extraction of thiamine from various sources ; the need for further research
is indicated. Of great importance in this connection is the fact that plants
often contain very little of their thiamine in the form of cocarboxylase,
and many unexplained irregularities occur.13- 14 Rice polishings yield free
thiamine without enzymatic treatment. Wheat does not contain cocar-
boxylase, although an enzyme is present which is capable of catalyzing
its hydrolysis.15 Jackbeans and soybeans both contain relatively large
and comparable amounts of thiamine; jackbeans are rich in carboxylase,16
but soybeans are said to have none. Under strongly acid conditions com-
mercial phosphatase preparations were found effective in releasing thia-
mine from wheat embryos, but were wholly ineffective in releasing the
vitamin from brewers' yeast.17 In a wholly different experiment in a
different laboratory, one out of three samples of wheat germ yielded a
slightly increased amount of thiamine upon enzymatic treatment.14 The
availability of the thiamine present in different yeasts varies widely, sug-
gesting differences in the method of combination. In the case of three
brewers' yeast, 93-100 per cent utilization by rats was reported,18 whereas
in the case of some bakers' type yeasts the utilization by human subjects
is said to be as low as 17 per cent (see p. 291 ).19 Utilization by rats in
these cases was also low.20

Unexplained irregularities have also been observed in connection with
animal tissues. In liver, brain and kidney a very large percentage of the
total thiamine is present as cocarboxylase, while in muscle the concentra-
tion of free thiamine may nearly equal to (or in some cases even exceed)
that of cocarboxylase.21, 22 In milk, only 50-60 per cent of the thiamine
is in the free state, and the rest is combined in nondialyzable form, which
is not released by phosphatases but requires a proteolytic agent such as
papain.23- 24 Melnick and co-workers found that thiamine is apparently
destroyed 50-90 per cent when it is incubated with bile, but that subse-
quent treatment with a special enzyme from yeast caused a recovery of
a substantial part of the lost activity. The precursor of this recovered

32 THE BIOCHEMISTRY OF B VITAMINS

activity was not cocarboxylase or its combined form.25 Goodhart and
Sinclair 26 have presented evidence for the existence in serum of thiamine
not in the form of diphosphothiamine bound to protein.

The presence of thiamine-destroying principles in the tissues of fishes,
clams and ferns 27 complicates the extraction of the vitamin from these
sources, as well as the more fundamental problem of whether thiamine
has diverse modes of linkage in the tissues of various organisms. The
inconclusive investigations of Myrback 28 and Shonberg 29 and their respec-
tive associates with respect to the predominant presence or absence of an
oxidized form of thiamine (presumably thiamine disulfide, in which the
thiazole ring is cleaved and the sulfur oxidized) in bakers' yeast, have a
bearing upon the problem of thiamine utilizability mentioned above.
According to Myrback and his associates, bakers' yeast, when highly
aerated, contains no substantial amount of cocarboxylase or thiamine as
such, and this explains the lack of fermentation under these conditions
(Pasteur reaction). Shonberg and his associates deny the existence of the
oxidized form in substantial amounts.

It should be evident that complete information regarding the various
ways in which thiamine is bound in tissues would constitute an important
contribution to understanding the catalytic functions which thiamine
performs.

Riboflavin

The history of riboflavin is closely associated with the flavoproteins of
which it is a part, and from which it may be split by extraction with O.liV
HC1. Warburg and Christian's "old yellow enzyme," 30 which antedated
exact knowledge regarding riboflavin, was the original flavoprotein dis-
covered, and from it riboflavin was subsequently obtained.

Since that time a dozen or more specific flavoproteins have been isolated
in pure form from natural sources, or at least have been concentrated or
identified enzymatically.31-44 Many of these have been obtained from
yeast, others from various animal tissues and from milk, Neurospora and
other molds. In addition, there is the L-amino acid oxidase of snake
venoms on which information is not available as to whether or not it is
a flavoprotein.45 In the great majority of these flavoproteins, the pros-
thetic group is flavin adenine dinucleotide, made up as follows: isoalloxa-
zine-D-ribose-phosphate-phosphate-D-ribose-adenine. However, in three
of the flavoproteins, the "old yellow enzyme," the L-amino acid oxidase
isolated from rat kidney, and cytochrome-c reductase,40 the prosthetic
group is a mononucleotide: isoalloxazine-D-ribose-phosphate. The flavo-
proteins are dissociable into the protein and prosthetic parts which readily
recombine to form the flavoprotein with its original enzymatic activity.

COMBINED FORMS— EXTRACTION 33

By combination of the protein from one flavoprotein with the prosthetic
group from another, new flavoprotein enzymes can be formed.34 Some of
the flavoproteins are thought to have prosthetic groups in addition to the
flavin nucleotides.44

In view of the fact that riboflavin appears always to be attached to
proteins through a phosphate radical, it is not surprising that its extrac-
tion from tissues has not generally been a serious problem. Snell and
Strong 46 recommend for quantitative extraction autoclaving in the pres-
ence of a large amount of water. Extraction with O.liV acid is also men-
tioned by these authors and has been widely used. Neither procedure
effects a quantitative extraction from all materials, even though the
extraction may often be complete or nearly so.8 Recently the use of
0.25Ar HC1 in 25 per cent acetone and 75 per cent water has been recom-
mended.47 The U.S. Pharmacopoeia method involves autoclaving at 15
lbs. pressure for 30 minutes with 0.04./V HC1.

Digestion with a phosphatase preparation to obtain free riboflavin has
theoretical justifications because of the existence of riboflavin bound to
phosphoric acid, and the lack of definite information with respect to the
hydrolysis of this combination under acid conditions. Alkaline hydrolysis
cannot be used because riboflavin is unstable under alkaline conditions.

Recent experimental studies have shown that the cooking (and storage)
of various foods causes a considerable increase in their assayable ribo-
flavin content. This extra amount of riboflavin appears not to be released
by phosphatase preparations, and to be determinable both by fluorometric
and microbiological methods.48, 40

Nicotinic Acid, Nicotinamide

Nicotinic acid occurs principally in the form of its amide, and in tissues
this is for the most part linked directly through the pyridine nitrogen to
a ribose residue which makes up a part of a dinucleotide, such as co-
enzyme I: nicotinamide-ribose-phosphate-phosphate-ribose-adenine. This
coenzyme is in turn linked to various proteins (apoenzymes). Coenzyme
II has a structure similar to coenzyme I except that there is another
phosphate radical the position of which is uncertain. Nicotinamide is
linked in the same manner in both coenzyme I and coenzyme II, either of
which in turn may be combined with a number of proteins. Sumner and
Somers describe about 15 dehydrogenases of which about twice as many
contain coenzyme I as contain coenzyme II.50 The isolation of dinicotinyl
ornithine from the excreta of chicks 51 shows that in natural materials
nicotinic acid may be combined through an amide linkage to other
structures.

34 THE BIOCHEMISTRY OF B VITAMINS

The extraction of nicotinamide or nicotinic acid may be accomplished
in various ways, depending upon the purpose. If one wishes, as is most
often the case, to determine the total amount of the vitamin forms present
and is not concerned with which form is present, he may extract a large
part simply by autoclaving the material with water, as suggested by Snell
and Wright.52 This does not effect quantitative removal in all cases, and
the more common procedure involves autoclaving with N acid or some
similar treatment.53' 54 When applied to animal tissues and milk, this
treatment gives results comparable with those obtained by alkaline diges-
tion or enzyme digestion, but with cereals the values are substantially
higher, due it is thought, to the conversion of some unknown substance
into nicotinic acid.55, 56, 57

Alkaline extraction in the case of cereals, especially if relatively con-
centrated alkali is used, gives values very much higher than by watery
extraction.58 This may be due in part to the conversion of trigonelline
which does not function as a vitamin into nicotinic acid which does.

Enzymatic digestion seems to give satisfactory release so far as the
microbiological determination is concerned,8, 55 because nicotinamide and
the corresponding coenzymes are active in this test; but this method of
extraction has not been generally adopted.

Considerable more research will be required before the extraction of
nicotinic acid and nicotinamide can be completely controlled. This phase
of study has not received adequate study, partly because of the lack of a
satisfactory chemical method which is specific for a single chemical
species.

Pantothenic Acid

Long before the vitamin properties of pantothenic acid were demon-
strated, it was known to exist in a bound form particularly in liver, the
then richest known source.59 From this combination it was freed by
autolysis for purposes of concentration.60

The problem of extraction of pantothenic acid from tissues is different
from that involved in the case of the other vitamins so far discussed
because this vitamin is easily destroyed by hydrolytic cleavage under acid
or alkaline conditions. Autolysis and the use of added enzymes are the
available methods. Using uncooked brain and heart tissues as starting
materials, Cheldelin et al. found that proteolytic enzymes, pepsin, trypsin,
pancreatin, papain were relatively ineffective in the release of pantothenic
acid, and that with hog kidney and spinach, papain was ineffective.
"Takadiastase" in most cases gave the highest yields. Waisman et al.el
found more effective release to be accomplished by pancreatin digestion.
The increased yield due to the use of pancreatin was 2 or 3-fold in the

COMBINED FORMS— EXTRACTION 35

case of some cooked meats. The differences in comparable results from
different laboratories are probably due in part to the fact that crude
enzyme preparations are not uniform. Willerton and Cromwell 62 found
that clarase digestion of yeast and liver preparations caused a several-
fold increase in the available pantothenic acid in some cases, and brought
the assay values for these materials up to the point where they agreed
substantially with chick assay values. In all laboratories phosphatase
preparations are effective in releasing pantothenic acid, and except in the
case of cooked meats, release by this method is at least near the maximum.
Recently Neilands and Strong 63 have made combined use of liver enzyme
and alkaline phosphatase to release pantothenic acid from foodstuffs.
They emphasize the incomplete release of this vitamin by previously used
procedures.

The form (or forms) in which pantothenic acid is bound in natural mate-
rials is largely unknown. On the basis of the fact that pantothenic acid
itself is readily hydrolyzed by acid or alkaline hydrolysis but that
/^-alanine is not readily released from tissues by this means, Williams
postulated that combination, presumably with proteins, takes place
through the /^-alanine portion of the molecule.64 Since esters of panto-
thenic acid are readily hydrolyzed (this can be accomplished without
cleaving the pantothenic acid) and since pantothenic acid has not been
removed from tissues by this means, the amide linkage suggests itself as
most probable. The question of whether pantoic acid is readily split from
the naturally combined forms of pantothenic acid has apparently never
been determined.65

The fact that the coenzyme of Lipmann was found to contain about 10
per cent pantothenic acid has a tremendous bearing upon the problem of
the combined form or forms of pantothenic acid.66 Since the functioning
of pantothenic acid supposedly centers in this coenzyme, it may be pre-
sumed that pantothenic acid occurs naturally combined in this form,
which constitutes the prosthetic group of one or more enzymes. It is inter-
esting that pantothenic acid was freed only very slowly from this co-
enzyme by clarase-papain digestion, according to Cheldelin et al.8 After
/^-alanine had been found as a significant hydrolytic product, a combina-
tion of a liver enzyme and an alkaline phosphatase which together had
previously been found to inactivate the coenzyme was found to release
the pantothenic acid quantitatively.

B6 Group: Pyridoxal, Pyridoxine, and Pyridoxamine

Although the chemistry of vitamin B6 appeared to be cleared up with
the isolation and synthesis of pyridoxine, a biologically active vitamin,
in 1938 and 1939, it was shown conclusivelv vears later 67- 68 that "vitamin

36 THE BIOCHEMISTRY OF B VITAMINS

B6" and pyridoxine are by no means synonymous, and that pyridoxamine
is fundamentally just as important as pyridoxine, and pyridoxal is even
more so.

The question of the combined forms of "vitamin BG" was not answerable
until these findings were made, and all conclusions based upon the sup-
position that vitamin B6 and pyridoxine are one and the same were made
obsolete by these discoveries.

Pyridoxal phosphate, presumably pyridoxal esterified with phosphoric
acid in the 5-position, is now known to be an important coenzyme in-
volved in decarboxylation of amino acids 69 and in transamination.70- 71- 72
This constitutes one of the most important combined forms of vitamin
Bc and is itself the prosthetic group of a number of enzymes, some of
which have been purified.71 Pyridoxal readily forms combinations with
amino acids in vitro, and such combinations may be important in nature.73

Pyridoxamine phosphate is another important bound form of vitamin
BG, and appears to be the principal form occurring in yeast, where it is at
least partly in the free state.74 It is not hydrolyzed by alkaline extraction
and may be extracted by this means from liver and grass preparations.
In these latter materials it exists in combined forms presumably with
proteins, and appears to constitute the predominant form of the vitamin.
Pyridoxamine phosphate is also reported to be present in certain trans-
aminase preparations.75

Pyridoxine, rather than pyridoxal or pyridoxamine, appears to be the
principal form of the vitamin in certain seeds (rice and wheat) , but little
is known regarding its mode of combination.70 These cereals, it will be
noted, are not metabolically active materials, as are those containing
predominantly pyridoxal and pyridoxamine.

The complete extraction of all forms of vitamin Bc in the free con-
dition from tissues in general must take into account each of the forms.
Pyridoxamine phosphate is stable toward alkaline hydrolysis and is
hydrolyzed less readily than pyridoxal phosphate in acid medium.74
Stronger acid {2N) is far less effective in the release of free vitamin from
yeast than is acid of lower concentration (0.055A0 .77, 7S Rabinowitz and
Snell78 found that for many materials autoclaving for 5 hours in 0.055iV
HC1 at 20 lbs. pressure gave maximum yields. A rice bran concentrate,
however, required hydrolysis with 2N acid, and dried green peas and oats
yielded less vitamin B6 with the regular procedure than animal tests
indicated to be present. This discrepancy may be due to intestinal
synthesis.

The problem of extraction of all forms of vitamin Be in the free form
is made less acute by the fact that there appears to be no destruction by

COMBINED FORMS— EXTRACTION 37

acid treatment.78 This makes possible the utilization of one treatment
after another, if necessary.79 The present evidence is against the idea that
other acid labile forms of vitamin BG exist, as once appeared to be the
case.80

Biotin

Though biotin itself is readily water-soluble, only a minute por-
tion of that present in tissues, sometimes as little as 0.1 per cent, is
extracted by hot water.81 Unquestionably biotin exists naturally primarily
in combined forms, but little information is as yet available regarding
these forms.

Avidin,82, 83> 84 a protein constituent of egg white, appears to combine
stoichiometrically with biotin to form a heat-labile complex. Avidin-
biotin constitutes one of the combined forms of biotin, but the distribution
of avidin is limited, so far as is known, to eggs and oviduct tissues,85 and
so from the standpoint of general occurrence this form can be of only
minor importance. The more abundant forms of combined biotin differ
from avidin-biotin in that they are not dissociated in hot water.

Biotin unquestionably is associated with protein enzymes as a coenzyme
or portion thereof, as will be discussed in a later section, but knowledge
regarding these biotin-containing enzymes is almost nonexistent.

Extraction of biotin from various tissues indicates that it may not
always be combined in the same way. For example, exhaustive dialysis
of egg yolk (the original source of biotin used by Kogl and co-workers)
followed by cold-water extraction yields a nondialyzable form which is
active for yeast. Dialysis of liver tissue, on the other hand, does not render
the biotin extractable by cold water, though it could be released by
enzymes.81 Probably the most widely used extraction method is to auto-
clave the material with 6iV sulfuric acid for 2 hours at 15 lbs. pres-
sure si, so, 87. jn most cases this yields the maximum amount of biotin.
Prolonged autoclaving under these conditions causes appreciable destruc-
tion. Autoclaving with 18./V sulfuric acid for 2 hours caused 20-40 per
cent destruction.

This method of extraction cannot be accepted as universally the best
since in some cases, particularly plant materials, there is considerable
destruction.8, 8S This difference in behavior may not be due to differences
in combination but to the presence of substances in certain extracts which
may, under the conditions used, interact with biotin. Incidentally, it may
be remarked that the term "heat lability" as applied to vitamins is a very
uncertain and indefinite one, inasmuch as the substances with which a
vitamin is heated may be fully as important in determining rate of
destruction as is the temperature of heating.

38 THE BIOCHEMISTRY OF B VITAMINS

Inositol

The most widely recognized bound form of inositol is "phytic acid,"
which has been obtained from plant sources in various states of cru-
dity many times, most often in the form of mixed salts. Posternak89
synthesized the hexaphosphoric acid ester of inositol and gave good evi-
dence as to its identity with a natural material, but perusal of the litera-
ture shows that often the term "phytin" (supposedly an acid calcium
magnesium salt of phytic acid) has often been used uncritically for
material of indefinite composition. Other compounds, including lower
phosphoric esters and not agreeing in analysis with the classical formula,
have often been obtained.90- 91 The enzyme phytase,92 which of course is
no more definite than its substrate, is present in plant materials and
catalyzes the hydrolysis of the inositol phosphoric esters.

Inositol is also found in bound form in the phospholipide material.
Anderson 93 first found it in a phosphatide fraction from tubercle bacilli,
and Klenk and Sakai 94 obtained from the cephalin fraction of soybean
phosphatide a material which on hydrolysis yielded inositol and an
inositol monophosphoric ester. Folch and Woolley 95 obtained brain phos-
phatide fractions containing from 6.8 per cent up to 10 per cent inositol,
and Woolley 96 obtained a preparation from soybean, "lipositol," with a
content of 16 per cent inositol. Partial hydrolysis yielded inositol mono-
phosphate. An inositol galactoside linkage was thought to be present.
Complete hydrolysis yielded besides inositol, 15.5 per cent galactose, 8.3
per cent tartaric acid, 23.6 per cent oleic acid, cerebronic, palmitic and
stearic acids totalling about 21 per cent, phosphoric acid 9.8 per cent, and
ethanolamine. The content of the various fat acids and the analysis for
phosphoric acid indicate that the material is not a pure compound, though
the same conclusion holds for many refined phospholipide preparations.

A third, and from the functional standpoint, highly significant form of
bound inositol is pancreatic amylase.97 The purified enzyme not only con-
tains over 0.4 per cent inositol, but inhibition studies show that without
inositol the amylase cannot function.98* The implications of this finding
are far-reaching, especially if it should be found that starch- and
glycogen-splitting enzymes containing inositol are widespread. This might
account for the universal presence of inositol in living cells.

The extraction of the total inositol from tissues in uncombined condi-
tion requires refluxing with 18 per cent HC1 for 6 hours.99 A considerable
but variable portion can be extracted by milder procedures. Autolysis 10°
usually frees considerably less than does enzymatic treatment.8 No criti-
cal study has been found recorded of the way inositol may be released
* See footnote p. 10.

COMBINED FORMS— EXTRACTION 39

from its various combined forms. Piatt and Glock 101 have shown that
when fresh rat tissues are carefully dried in the frozen and finely divided
condition the water extract of the powder contains partly free and partly
combined (requiring acid hydrolysis) inositol.

Choline

The principal bound forms in which choline occurs are the lecithins
and sphingomyelins. Since the chemistry of naturally occurring phos-
pholipides and related compounds is not satisfactory, it being extremely
difficult or impossible to obtain such compounds in the pure state, one
would not dare be dogmatic as to the existence of other lipides in which
choline also is bound.

From the standpoint of physiology, an extremely important bound form
of choline is acetylcholine. From the quantitative standpoint, however,
the amount of natural choline which is bound in this manner is extremely
small.

There are certain close relatives of choline (dimethylaminoethanol,
monomethylaminoethanol, and ethanolamine) all of which are possible
components of lipides. These should be considered in connection with
choline studies.

The extraction of total choline, both combined and uncombined, from
tissues is accomplished by the use of exhaustive absolute methanol ex-
traction.102 Mixtures of ethanol and ether and other solvents have been
used, but the yield in every case is smaller.102- 103

Lecithins which contain most of the bound choline can be precipitated
from an aqueous medium with acetone, leaving free choline in solution.

Acid digestion of tissues has been used to free choline from its combina-
tions.104 Autoclaving the entire tissue with SN HC1 for 2 hours yields a
solution which contains choline in the free form.105

Folic Acid (Pteroylglutamic Acid, P.G.A.)

This vitamin has not been known long enough for its more complex
combined forms to be recognized. There is every reason to suppose that it
acts catalytically as a coenzyme and is therefore bound (perhaps loosely)
to proteins. These protein combinations are unknown.

Of the three combined forms, probably the most revealing is the sim-
plest, i.e., formylfolic acid.100 This has been made synthetically and
evidence for its natural occurrence and biological functioning has accu-
mulated. The next combined form, listed in order of simplicity, has two
extra (three total) glutamic acid residues joined to the glutamic acid
portion by peptide linkage. This was designated "fermentation L. casei
factor," since it was obtained from a fermentation residue.107 A third

40 THE BIOCHEMISTRY OF B VITAMINS

form has a total of seven glutamic acid residues and has been called
"vitamin Bc conjugate." 10S

For the liberation of free folic acid (which is active alike in S. fecaelis,
L. casei and chick tests) from tissues, a special enzyme (or enzymes) is
necessary109-110; drastic acid or alkaline treatment, of course, causes
destruction of the vitamin. Even autoclaving certain bacteria in the
presence of water destroys most of it.111 Treatment with commercial
phosphatase-containing and proteolytic enzymes or autolysis frees only
a fraction of the vitamin present.

Specific enzymatic treatments yield sufficiently divergent results, when
applied to different tissues, to suggest the probability that folic acid
exists in different types of combination.112 The fact that folic acid is
particularly associated with green leaves,113, 114 where it probably func-
tions in a special way, suggests that special combined forms exist in
leaves. The observation of Bird and co-workers 115 that no enzymatic
treatment of plant extracts was found that would cause the microbiolog-
ical assay values to equal those obtained by chick assays, is in line with
this suggestion.

p-Aminobenzoic Acid

Three well defined naturally occurring combined forms of p-amino-
benzoic acid are known: folic acid (including conjugates), rhizopterin 116
and p-aminobenzoylpolyglutamic acid.117 In addition, the acetyl deriva-
tive may occur in blood and urine.118- 119 Other information regarding com-
bined forms is based upon indirect evidence. Most of the p-aminobenzoic
acid of yeast (about 90 per cent) is in the free state; that is, it is extract-
able and utilizable by microorganisms.120, 121 In different tissues tested, the
amounts of "bound" versus total p-aminobenzoic acid varied from 6 per
cent in potatoes to 93 per cent in rat kidney. Eight animal tissues averaged
about 80 per cent bound, whereas miscellaneous materials mostly of plant
origin averaged 44 per cent bound.

The quantitative extraction of p-aminobenzoic acid from tissues offers
difficulties that have not been fully overcome.

Landy and Dicken122 autoclaved the material to be assayed with water
and obtained maximum yields. No data were given as to what other
procedures were used for comparison. Lewis 123 found greater destruction
with acid than with alkali, and autoclaved samples with N NaOH for
30 minutes to obtain assay values. Thompson et al.120 obtained yields
about 3 times as high when the material (beef liver and kidney) was
autoclaved for 1 hour with 6N H2S04, than when alkaline hydrolysis
(mild compared to that used by Lampen and Peterson below) was used.
Under these acid conditions they found about a 15 per cent destruction.

COMBINED FORMS— EXTRACTION 41

Lampen and Peterson 119 made a careful study of hydrolytic conditions
using as the principal material to be assayed a "dry powdered liver
sample." They found liberation by acid hydrolysis to be rapid, but that
it never appeared to be complete. Subsequent digestion of the same soluble
material with alkali caused an increase in the total p-aminobenzoic acid,
indicating that the acid treatment had extracted the compound from the
liver but had not rendered it available to the test organism (CI. aceto-
butylicum) . Freeing of the available form took place more slowly under
alkaline conditions, but proceeded considerably further. The curve for
autoclaving (15 lbs.) with 2N NaOH had not reached a plateau even at
20 hours. Some destruction was observed as a result of long alkaline treat-
ments. The question of whether the p-aminobenzoic acid being formed
during drastic alkaline treatment might be an artifact was investigated,
with negative indications. Preliminary treatment with acid seemed to
cause the subsequent liberation to be more rapid under alkaline con-
ditions.

These four studies cited involved the use of four different assay organ-
isms, and this may be partially responsible for the differences observed.
It is possible that the "aminobenzoicless" Neurospora used in the study
of Thompson et al.120 responds to a conjugate produced by acid treatment,
and that the Clostridium acetobutylicum does not. This would help to
explain the difference between the last two studies cited.

The numerous observations, as well as those relating to folic acid,
suggest that p-aminobenzoic acid may be combined in nature in a number
of ways and that some of these combinations are extraordinarily stable.
Further study is required to clarify the picture.

A comparison of available p-aminobenzoic acid assay values for beef
liver, spinach and egg with corresponding folic acid assay values indicates
that there is 3-10 times as much p-aminobenzoic acid present as could be
derived from the folic acid present. Lampen and Peterson 119 found a
maximum of 8 fxg per gram of p-aminobenzoic acid in liver powder. This
would require 25.6 ixg/gvo. of folic acid if it were all in this form. The
folic acid content of beef liver (no conjugase used) on a dry basis was
2.8 ng/gm (calculated on the basis of 160,000 potency). Even if this
value were to be doubled or tripled by conjugase action, there would
still be more than half of the total p-aminobenzoic acid present in liver
in some conjugated form other than folic acid and its conjugates.

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58. Kodicek, E., Biochem. J., 34, 712-23 (1940).

59. Rohrman, E., Burget, G. E., and Williams, R. J., Proc. Soc. Exptl. Biol. Med.,

32, 473-4 (1934).

60. Williams, R. J., et al, J. Am. Chem. Soc., 60, 2719-23 (1938).

61. Waisman, H. A., et al., J. Nutrition, 23, 239-48 (1942).

62. Willerton, E., and Cromwell, H. W., Ind. Eng. Chem., Anal. Ed., 14, 603-4

(1942).

63. Neilands, J. B., and Strong, F. M., Arch. Biochem., 19, 287-91 (1948).

64. Williams, R. J., "The Biological Action of the Vitamins," Univ. Chicago Press,

Chicago, 111., 1942, p. 122.

65. Sarett, H. P., and Cheldelin, V. H., /. Biol. Chem., 159, 311-9 (1945).

66. Lipmann, F., et al, J. Biol. Chem., 167, 869-70 (1947).

67. Snell, E. E., Guirard, B. M., and Williams, R. J., J. Biol. Chem., 143, 519-30

(1942).

68. Snell, E. E., "Annual Review of Biochemistry," Vol. XV, Stanford Univ. Press,

Stanford University, California, 1946, pp. 383-6.

69. Gunsalus, I. C, Bellamy, W. D., and Umbreit, W. W., /. Biol. Chem., 155,

685-6 (1944).

70. Schlenk, F., and Snell, E. E., /. Biol. Chem., 157, 425-6 (1945).

71. Green, D. E., et al, J. Biol. Chem., 161, 559-82 (1945).

72. Lichstein, H. C, Gunsalus, I. C, and Umbreit, W. W., J. Biol. Chem., 161,

311-20 (1945).

73. Oser, B. L., "Annual Review of Biochemistry," Vol. XVII, Stanford Univ.

Press, Stanford University, Calif., 1948, p. 419.

74. Rabinowitz, J. C, and Snell, E. E., J. Biol. Chem., 169, 643-50 (1947).

75. Schlenk, F., and Fisher, A., Arch. Biochem., 12, 69-78 (1947).

76. Rabinowitz, J. C, and Snell, E. E., J. Biol Chem., 169, 631-42 (1947).

77. Rubin, S. H., Scheiner, J., and Hirschberg, E., J. Biol. Chem., 167, 599-611

(1947).

78. Rabinowitz, J. C, and Snell, E. E., Anal. Chem., 19, 277-80 (1947).

79. Rabinowitz, J. C, and Snell, E. E, J. Biol Chem., 176, 1157-67 (1948).

80. Melnick, D., et al, J. Biol. Chem., 160, 1-14 (1945).

81. Thompson, R. C, Eakin, R. E., and Williams, R. J., Science, 94, 589-90 (1941).

82. Eakin, R. E., Snell, E. E., and Williams, R. J., /. Biol. Chem., 136, 801-2

(1940).

83. Eakin, R. E., Snell, E. E., and Williams, R. J., /. Biol. Chem., 140, 535-43

(1941).

84. Pennington, D. E., Snell, E. E., and Eakin, R. E., J. Am. Chem. Soc, 64, 469

(1942).

85. Hertz, R., and Sebrell, W. H., Science, 96, 257 (1942).

86. Schweigert, B. S., et al, J. Nutrition, 26, 65-71 (1943).

87. Wright, L. D., and Skeggs, H. R., Proc. Soc. Exptl. Biol. Med., 56, 95-8 (1944).

88. Lampen, J. O. Bahler, G. D., and Peterson, W. H., J. Nutrition, 23, 11-21 (1942).

89. Posternak, S., Compt. rend., 169, 138-40 (1919).

90. Anderson, R. J., J. Biol Chem., 18, 441-6 (1914).

91. Boutwell, P. W., J. Am. Chem. Soc, 39, 491-503 (1917).

92. Anderson, R. J., J. Biol. Chem., 20, 483-91 (1915).

93. Anderson, R. J., J. Am. Chem. Soc, 52, 1607-8 (1930).

44 THE BIOCHEMISTRY OF B VITAMINS

94. Klenk, E., and Sakai, R., Z. physiol. Chem.. 258, 33-8 (1939).

95. Folch, J., and Woolley, D. W., J. Biol. Chem., 142, 963-4 (1942).

96. Woolley, G. W., and White, A. G. C, J. Biol. Chem., 147, 581-91 (1943).

97. Williams, R. J., Schlenk, F., and Eppright, M. A., J. Am. Chem. Soc, 66,

896-8 (1944).

98. Lane, R., and Wiliams, R. J., Arch. Biochem., 19, 329-35 (1948).

99. Woolley, D. W., J. Biol. Chem., 140, 453-9 (1941).

100. Williams, R. J., et al, Univ. Texas Pub., 4137, 27-30 (1941).

101. Piatt, B. S., and Glock, G. E., Biochem. J., 37, 709-12 (1943).

102. Engel, R. W., J. Biol. Chem., 144, 701-10 (1942).

103. Rhian, M., Evans, R. J., and St. John, J. L., J. Nutrition, 25, 1-5 (1943).

104. Fletcher, J. D., Best, C. H., and Solandt, O. M., Biochem. J., 29, 2278-84 (1935).

105. Luecke, R. W., and Pearson, P. B., J. Biol. Chem., 155, 507-12 (1944).

106. Gordon, M., et al, J. Am. Chem. Soc, 70, 878 (1948).

107. Angier, R. B., et al, Science, 103, 667-9 (1946).

108. Pfiffner, J. J., et al, J. Am. Chem. Soc, 68, 1392 (1946).

109. Mims, V., Totter, J. R., and Day, P. L., J. Biol. Chem., 155, 401-5 (1944).

110. Bird, O. D., et al, J. Biol. Chem., 163, 649-59 (1946).

111. Stokes, J. L., and Larsen, A., J. Bact., 50, 219-27 (1945).

112. Loo, Y. H., and Williams, R. J., Univ. Texas Pub., 4507, 123-34 (1945).

113. Mitchell, H. K., Snell, E. E., and Williams, R. J., J. Am. Chem. Soc, 63, 2284

(1941).

114. Olson, O. E., Burris, R. H., and Elvehjem, C. A., J. Am. Dietetic Assoc, 23,

200-3 (1947).

115. Bird, O. D., et al, J. Biol. Chem., 159, 631-6 (1945).

116. Rickes, E. L., Chaiet, L., and Keresztesy, J. C., /. Am. Chem. Soc, 69, 2749-51

(1947).

117. Ratner, S., Blanchard, M., and Green, D. E., J. Biol Chem., 164, 691-701

(1946).

118. Harrow, B, Mazur, A., and Sherwin, C. P., J. Biol. Chem., 102, 35-8 (1933).

119. Lampen, J. O. and Peterson, W. H., J. Biol. Chem., 153, 193-202 (1944).

120. Thompson, R. C, Isbell, E. R., and Mitchell, H. K., J. Biol. Chem., 148, 281-7

(1943).

121. Lampen, J. O., et al, Arch. Biochem., 7, 277-86 (1945).

122. Landy, M., and Dicken, D. M., J. Biol. Chem., 146, 109-14 (1942).

123. Lewis, J. C., J. Biol Chem., 146, 441-50 (1942).

Chapter IVA

ASSAY METHODS

There are three important types of assay methods which may, theoret-
ically at least, be developed for each of the B vitamins: (1) chemical
(or physical-chemical) methods; (2) microbiological methods using
bacteria, yeasts and molds; and (3) biological (animal assay) methods.
In addition, it might be desirable, of course, to use combinations of these
methods. Other organisms, such as higher plants or lower animals that
have potentialities for vitamin assay work, may be considered in connec-
tion with gaining the broadest picture. Animal or plant tissues may be
used in some cases. Even human beings can be used for vitamin assay
and the newest (probable) member of the B vitamin family, the anti-
pernicious anemia principle, was assayed for exclusively in this manner
until a useful microbiological test was finally developed.

There are three general purposes which underlie the performance of
vitamin assays: (1) they may be performed for purposes of exploration
with respect to distribution in nature, in foods and in food products; (2)
they may be used as a guide in isolating, purifying and determining the
functions of a principle; (3) they may be used for the assay and control
of commercial concentrates and of synthetic products. The method to be
used must be selected on the basis of its availability and applicability
to the intended purpose. A chemical or physical-chemical method, for
example, might be extremely valuable in testing nearly pure commercial
concentrates or synthetic mixtures, and yet might be of no value what-
ever in connection with the assay of materials where the principle exists
only in traces.

Other things being equal, the chemical and physical-chemical methods,
when applicable, are most advantageous because of their accuracy, speed
and definition. In general, however, such methods become progressively
less applicable as we pass from preparations which are relatively pure
to those in which the substance tested for is present, for example, to the
extent of only a few parts per billion. In the latter cases, chemical or
physical-chemical tests are likely to be far too insensitive. When such
tests are sensitive enough for the assay of a natural mixture, they are
likely to be interfered with by extraneous substances, which, however, it
may be feasible to remove.

46 THE BIOCHEMISTRY OF B VITAMINS

Microbiological tests, in general, rate next to the chemical or physical-
chemical tests with regard to speed and accuracy, and have the tre-
mendous advantage with respect to natural extracts of very great sensi-
tivity and often high specificity. Interference by extraneous substances
is always a possibility in every type of assay, and microbiological tests,
like all others, are more readily applied to concentrates than to trace-
containing mixtures. However, in many cases there is no substantial
difficulty in this regard, and extremely small amounts can be determined
microbiologically.

The importance of microbiological tests in vitamin research may be
gauged by the fact that by their use pantothenic acid, biotin, pyridoxal,
pyridoxamine and folic acid were discovered. They also formed the basis
for the discovery of the vitamin properties of niacin, inositol and p-amino-
benzoic acid, and for the isolation of the anti-pernicious anemia principle.

Biological assays using mammals or fowls constituted the first recog-
nized vitamin tests, and because of the pre-eminent nutritional function
of vitamins, these tests will always remain fundamentally important.
Chemical tests and microbiological tests demand that the vitamin to be
tested shall be in solution. Animal assays do not have this limitation;
and in the sense that vitamins are concerned, by definition, with animal
nutrition, the animal tests are the most direct vitamin tests.

Actually, in animal assays the important factor of availability, as well
as presence, comes in.1 For example, if one were to determine by animal
assay the amount of thiamine in raw bakers' yeast, the relatively low
result would reflect the unavailability of the vitamin present. From the
standpoint of the practical nutrition of animals or human beings the
amounts of the various vitamins present in different foodstuffs are of no
consequence if the vitamins are unavailable, and only a direct test with
animals will give the fundamental information as to how much effective
vitamin is present.

Unfortunately, it is not always safe to carry results obtained using
one species of animals over to other species, because the physiological
availability may not be the same. If one's interest is in human nutrition,
it is necessary to test the materials in question upon human subjects to
gain completely trustworthy information. All nutritional tests on animals
and humans are complicated by the problem of intestinal synthesis of
vitamins, as will be discussed in a later section.

Biological assays for vitamins using experimental animals are costly,
time-consuming and lack accuracy. Nevertheless, for some purposes they
are most necessary because vitamins are fundamentally concerned with
animal and human nutrition.

ASSAY METHODS 47

Thiamine

For physiological research involving thiamine and its functioning,
many different organisms and procedures may profitably be used which
fall outside the scope of our present discussion. We shall be concerned
primarily with methods which are designed to determine thiamine con-
tent, and which neglect all other questions involving availability to any
particular organism.

Thiochrome Method. Since the original publication by Jansen 2 which
forms the basis of this highly important method, dozens of articles deal-
ing with applications, modifications and refinements have been published.
Among the more important of these are those of Hennessy and Cerecedo 3
and Conner and Straub,4 who respectively introduced the use of adequate
base exchange procedures for eliminating interfering substances and
enzyme digestion using commercially available enzyme preparations to
free thiamine from its combination in cocarboxylase.

The experimental essentials of the method as it is now applied include
seven steps:

(1) Quantitative extraction from the material under examination of
all of the free thiamine and cocarboxylase present. This is accomplished
by the use of acidulated water.

(2) Enzymatic digestion of the cocarboxylase present by the use of
clarase or some other suitable commercial preparation rich in phosphatase.

(3) Selective quantitative adsorption of the free thiamine by "Decalso"
or other suitable agent.

(4) Quantitative recovery by elution, yielding a thiamine solution
from which many interfering substances have been discarded.

(5) Oxidation of the thiamine to thiochrome by the use of alkaline
potassium ferricyanide solution.

(6) Extraction of thiochrome by isobutyl alcohol.

(7) Measurement of the fluorescence produced by irradiating the
thiochrome solution with ultraviolet light (fluorophotometer) and evalua-
tion in terms of thiochrome.

The key reaction on which the method is based is the quantitative
production of the fluorescent pigment from thiamine by oxidation.

In the application of this method to specific cases, some of the steps
may become superfluous. For example, in the assay of wheat and wheat
products, Step 2 can be omitted because cocarboxylase is absent, and the
results are the same whether or not this step is performed. If one is deal-
ing with a vitamin concentrate containing free thiamine and not too much
interfering material, steps 1-4 may be dispensed with. If one wishes to
determine free thiamine in the presence of cocarboxylase, Step 2 is

48 THE BIOCHEMISTRY OF B VITAMINS

omitted and advantage is taken of the fact that the pigment produced
by the oxidation of cocarboxylase is not soluble in isobutyl alcohol,
whereas thiochrome is. Experimental details of procedure and a partial
bibliography will be found in the reference cited.5

As ordinarily performed, the thiochrome solution examined (Step 7)
corresponds to the order of 1.0 fxg of thiamine. A micromethod using a
Spekker Fluorometer has been devised6 which is accurate to =•= 20 per cent
when 0.001 /*g of thiamine is determined, and to about ±3 per cent when
the amount of thiamine is 0.05 /*g.

N S

/ \ /\

H3C— C C— NH2HC1 HC C— CH2— CH2OH

II I II II

N C N+ C— CH3

\ y \ / ci-

CH2

Thiamine hydrochloride

N N S

/ \ / \ /\

H3C— C C C C— CH2— CH2OH

I II I II

N C N C— CH3

V \X

H H2

Thiochrome

The thiochrome procedure has been successfully applied to the analysis
of cereals and cereal products,7- s to miscellaneous foods and tissues,9, 10
to urine,11, 12 to blood,13, 14, 14a and to pharmaceuticals.15, 16

Colorimetric Method. Another chemical method of some value involves
the formation of a colored pigment (usually red) when thiamine is
allowed to react in alkaline medium with a diazotized aromatic amine.
The most important amine for this purpose, p-aminoacetophenone, which
yields a red dye insoluble in water but soluble in xylene, was first used
by Prebluda and McCollum.17 The development of quantitative methods,
suitable for laboratory use, involving this reagent is the work of Melnick
and Field.18

The Melnick and Field method, including modifications thereof, has
not been used as extensively as the thiochrome method mainly because
of its lack of sensitivity, though in some laboratories, at least, it has been
found more reliable.15 It requires, in order to be applied, a concentration
of 2-3 fig of thiamine per ml, whereas the thiochrome method is applicable
to solutions containing as little as 0.05 /*g per ml. For the analysis of

ASSAY METHODS 49

relatively potent concentrates or even for clinical examination of urine,19
the colorimetric method is, however, probably superior to any other.

Yeast Fermentation Method. This method, which is probably second
to the thiochrome method with respect to wideness of use, was developed
by Schultz, Atkin and Frey 20-21; it utilizes the fact, discovered by them,
that fermentation by suitable yeast is enhanced by the presence of free
thiamine. In its modified form the method takes advantage of the fact
that thiamine is cleaved quantitatively by sulfite, and the cleavage
products have no effect upon the fermentation. The difference between
the enhancement produced by an untreated solution and a sulfite-treated
one is used as a measure of the thiamine content.

The application of this method requires the use of a special apparatus,
a Fermentometer, which is constructed so that 12 fermentations can be
run simultaneously. One-half gram of yeast is used in each bottle. The
carbon dioxide evolution after 3 hours of fermentation is measured and
the enhancement induced by 1 ng and 2 fig of thiamine are taken as
standards for comparison. Errors in assay under favorable conditions are
not more than a very few per cent.

By the use of a Warburg apparatus thiamine can be assayed on an
ultramicro scale (0.005 fig to 0.025 fig) with errors no greater than ±5
per cent.22

Comparative studies have shown that the yeast fermentation method
gives results generally comparable in accuracy with those obtained by
the thiochrome method.7 16> 23> 24 Details of procedure are described by
the originators.21

Microbiological Growth Methods. The yeast-growth method has not
been used extensively except in the laboratory where it originated, but
for certain types of investigation involving minute amounts of material
it had a distinct advantage. The growth of certain strains of yeast ("Old
Process") in an otherwise complete medium is greatly stimulated by
minute amounts of thiamine.25, 26> 27 Thiamine degradation products are
likely to be effective, however, and these must be ruled out.24 The effect
of thiamine is stimulatory rather than essential for growth, and differ-
ences in growth rates are therefore the basis of the response. By this
means it is possible to determine with satisfactory accuracy in un-
processed materials, amounts of thiamine as little as 0.0001 ^ig, and by
reducing the volume of the cultures, a lower limit can be reached.

The use of the mold Phycomyces Blakesleeanus as a test organism
has been advocated by Schopfer 28 and was used, for example, by
Meiklej ohn 29 for the determination of thiamine in the blood. Sinclair,
however, has emphasized that the response is nonspecific.30 Later this
investigator introduced a correction which was thought to take care of

50 THE BIOCHEMISTRY OF B VITAMINS

extraneous effects.31 Even so, the growth period in the test is 10 days,
and in view of later developments the use of this organism has little to
recommend it.

Niven and Smiley 32 have suggested the use of Streptococcus salivarius
as a test organism. It is extremely sensitive to minute amounts of thia-
mine (0.1 to 2 /i,g per tube) and yields satisfactory assay values. Thiamine
fragments are inactive; cocarboxylase is 40 per cent more active than
thiamine. The method has the disadvantage that the medium has a pH
value of 7.4 and thiamine must be added to it aseptically after auto-
claving.

Sarett and Cheldelin 33 have developed an assay method using Lacto-
bacillus fermenti, which responds in their test to from 0.005 to 0.05 /xg
of thiamine. The recommended growth period is 16 to 18 hours and under
these conditions, cocarboxylase is 30 per cent more active than thiamine.
The method has been applied by the authors to a considerable number
of foods, to animal tissues, and to urine with excellent results.34

Biological Assays. Probably the most widely used biological assay
methods for thiamine involve curative tests on rats. In the U.S.P.
method,35 rats are kept upon a thiamine free diet until they show signs
of acute polyneuritis, whereupon at least eight such rats are given a
standard dose of pure thiamine which "cures" the polyneuritic condition
for from 5 to 15 days, depending upon the dose given and the condition
of the individual rats. As soon as the rats have regressed and reached
the same stage of polyneuritis as before dosing, a single dose of the
unknown material to be tested is administered. If the curative effect of
this preparation lasts as long or longer than that of the standard (based
upon the sum of the cured days for each animal), it is determined to con-
tain as much or more thiamine than the standard.

It is obvious that the assay of unknown mixtures by this method
involves the use of a large number of animals and a large amount of
time, since the depletion period may last as long as 8 or 10 weeks and
the assay period for a single test is from 10 to 30 days longer.

A less time-consuming curative test is one devised by Smith 36 and
modified by Birch and Harris.37 In this modified test animals are placed
upon a thiamine-deficient diet containing, however, a small amount of
thiamine in the form of 0.4—0.5 per cent brewers' yeast. On this diet
they develop polyneuritic symptoms more regularly and do not die of
extreme thiamine deficiency before they become useful test animals.
After depletion, which may require 50 days or more, they are injected
intravenously with the material to be tested and the minimal amount
required to produce a three-day cure is determined. If a larger than
minimal dose is given, the excess can be judged by the longer duration

ASSAY METHODS 51

of the cure, the duration being roughly proportional to the amount of
thiamine administered. The same animal can be used over and over,
5 to 10 times or more, and if laboratory animals are continually kept in
condition for testing, assays can be run with relative speed, and the
results may be as accurate as those obtained by the U.S.P. method.15

Biological tests for thiamine involving rat growth,38 pigeon weight
maintenance,39 prevention of bradycardia in rats involving the use of an
electrocardiograph,37 chick growth,40 and postponement of death from
polyneuritis in chicks41 have all been successfully used; but except for
those interested in the particular species involved, these tests are largely
of historical interest only, since they are not used at present as thiamine
assay methods per se. The "catatorulin" test of Peters and co-workers 42
should also be mentioned in this connection. Oxygen consumption by
brain tissue from avitaminotic pigeons is low, and is increased by minute
amounts of thiamine. The test is sensitive to about 0.2 /xg of thiamine.

Riboflavin

Aside from the rat growth and chick growth methods which are basic,
but belong in a class by themselves because of cumbersomeness and the
time and expense involved, there are two methods that have been widely
used and are recommended for riboflavin assay, namely, the fluorometric
and the microbiological.

Fluorometric Method. This method takes advantage of the fact that
riboflavin fluoresces strongly when exposed to light of wave length 440
to 500 m/x, and the intensity of the fluorescence is proportional to the
concentration of riboflavin in the solution examined. It must be performed
on solutions or extracts containing riboflavin, and the extraction pro-
cedures involve simultaneous hydrolysis of the bound forms (p. 33). If
one were dealing with solutions containing riboflavin as the only fluo-
rescing substance, the application of the method would be relatively
simple. However, in practice, particularly with some types of products,
it is necessary to take elaborate precautions to eliminate the effects of
other substances.

One expedient that is always used to eliminate the effect of interfering
substances involves the use of a sodium hydrosulfite (dithionite) ,
NaoSoO.!, which reduces riboflavin quantitatively to its nonfluorescing
leuco-form, and leaves unaltered some of the other colored fluorescent
substances which may be present. After the fluorometric reading is taken
on the final solution, regardless of previous treatments, the riboflavin is
destroyed with hydrosulfite and a blank fluorometric reading taken. This
reading is subtracted from the riboflavin reading.

52 THE BIOCHEMISTRY OF B VITAMINS

Another means of eliminating the effect of interfering substances is to
reduce with stannous chloride and sodium hydrosulfite all the fluorescent
pigments including riboflavin, and then reoxidize by contact with air;
this procedure brings back riboflavin to its original fluorescent form, but
leaves in reduced form some of the substances which would otherwise
interfere.

Another procedure involves the use of dilute permanganate under con-
trolled conditions to oxidize interfering pigments without affecting the
riboflavin. The excess permanganate is removed with hydrogen peroxide.

Another procedure, regarded as necessary only when the materials
tested are of relatively low potency or highly pigmented, involves adsorb-
ing the riboflavin selectively upon a column of "Florosil" and eluting it
with 20 per cent pyridine in 2 per cent acetic acid. This, of course, leaves
behind many fluorescing substances, but introduces an extra step into
the procedure; hence it is likely to introduce errors as well as expend
time.

Scott and co-workers 43 have recently published a complete procedure
which has been applied with excellent results to many types of materials,
including milk and milk products,44 dried leguminous seeds,45 miscel-
laneous cereal products, fermentation residues, leaf meals, meat and fish
scrap, and yeasts.43 In this method, only two of the expedients discussed
above are used: permanganate oxidation and hydrosulfite reduction at
the end to obtain a blank reading. Details of procedure are to be found
in the original article.43 It is said to require less time than the micro-
biological method.45

A fuller discussion of the various alternate procedures involved in the
fluorometric method will be found elsewhere.46

Microbiological Method. This method, which is essentially that of
Snell and Strong,47 has been widely used and in spite of the natural handi-
cap inherent in the use of an unfamiliar type of technique, it has been
adopted by the U. S. Pharmacopoeia.48

The method is based upon the fact that the growth of Lactobacillus
casei and its ability to produce lactic acid requires riboflavin in the
medium. The riboflavin can be in the free form, but its combined forms,
e.g., flavoprotein, flavin adenine dinucleotide, and presumably riboflavin
phosphate, are equally effective under the experimental conditions pre-
scribed for the assay.49

Theoretically, for this or any other comparable assay, one should
have a basal medium perfect in every other respect except for the lack
of the one item to be assayed for. Actually, it is only necessary in this
case that the basal medium be such that under laboratory conditions other

ASSAY METHODS 53

substances present in the extracts, aside from riboflavin, will have a
negligible effect.

Subsequent investigations dealing with the nutrition of the test organ-
ism have shown that the basal medium recommended by Snell and Strong
is far from ideal in the sense of the previous paragraph. Roberts and
Snell 50 have developed a vastly improved medium in which the organism
responds to riboflavin to a much higher degree. Nevertheless, the original
medium is such that it has given very satisfactory results comparable to
those obtained using the improved medium, and it serves for practical
purposes. If one were assaying materials never assayed before, the
results could be accepted with greater certainty using the more complete
medium of Roberts and Snell.

The most serious extraneous factors that can complicate riboflavin
assay by this method are the fatty substances,51- 52- 53 the effects of which
are sometimes stimulatory and sometimes inhibitory depending upon the
agents, their concentration and upon the presence or absence of other
agents in the extract. These disturbing factors can be eliminated by
preliminary solvent extraction, or more simply by careful filtration of
the extract at pH 4.5 to obtain a clear solution. The latter procedure is
the one used in the U. S. Pharmacopoeia method.

In the application of the method to urine, urea can be present in
sufficient amount to introduce an error due to its inhibitory effect. This
effect can be corrected for if necessary.54

There are two valid methods for evaluating the response of the organism
to riboflavin: turbidimetric measurement of growth after 24 hours or less
(16 hours in case the Roberts-Snell medium is used) and titration of the
acidity developed after 72 hours. Either method gives wholly satisfactory
results; the titration method possibly is a little less exacting, requires no
special apparatus and is specified in the U. S. Pharmacopoeia. The tur-
bidity method, however, gives results overnight which for some purposes
may be a tremendous advantage. It requires a turbidity measuring
device; a suitable photoelectric colorimeter will serve, but even better,
however, is a thermoelectric turbidimeter such as has been in use in the
author's laboratory for twenty years.55

Biological Methods. In view of the satisfactory assay of riboflavin by
the two methods discussed, the animal assay methods are mostly of
historical interest. This does not mean, of course, that animal experiments
involving riboflavin are outmoded.

The rat growth method of Bourquin and Sherman 56 has been used
with minor modifications for a number of years and was the standard
with which the newer methods were initially compared. These investi-
gators were fortunate in preparing an alcoholic extract (80 per cent) of

54 THE BIOCHEMISTRY OF B VITAMINS

whole wheat which contained very little riboflavin but a relatively good
supply of other B vitamins required by rats. Hence, when a riboflavin-
containing material was supplied, its effect was largely due to the ribo-
flavin. The method, of course, was particularly useful in connection with
assaying relatively rich sources. Rats were first depleted for about two
weeks and then fed supplements containing control and unknown
amounts of "vitamin G," and the comparisons evaluated on the basis of
the growth rates over a period of several weeks. Diets of substantially
different character have been used more recently for the assay of ribo-
flavin using rat growth, but we shall not go into detail here.57, 58> 59

Extensive use has not been made of chicks in the assay of riboflavin,
though methods were early proposed.60- 61 An improved method was
devised by Jukes 62 in which the growth response at lower levels was
found to be approximately proportional to the amount of riboflavin fed.
The basal medium contained in addition to yellow corn, wheat middlings,
casein, and supplements, 7 per cent of a "rice bran filtrate," which served
to supply the chicks with the "filtrate factor" (pantothenic acid), and
other unknowns. This was designed to make the test more specific and
was apparently successful.

Nicotinic Acid, Nicotinamide

Two types of methods have been used almost exclusively for the assay
of nicotinic acid or its amide: colorimetric methods and microbiological
methods. Biological methods are relatively unimportant.

Colorimetric Methods. The colorimetric methods which have been
applied widely to nicotinic acid assays all involve the interaction be-
tween it, cyanogen bromide and an aromatic amine. The chemistry of
the reactions is not well known, but rupture of the pyridine ring is thought
to be involved.63 Among the aromatic amines used for this purpose
are p-aminoacetophenone,64 metol 65 (p-methylaminophenol), aniline,66
/3-naphthylarnine,67 and p-phenylenediamine.68

One of the more extensively used amines has been p-aminoaceto-
phenone. This reagent has been investigated by Kodicek, who agrees
with the originators that it is 3 to 5 times as sensitive as aniline or
metol; he has applied it with success to a variety of plant and animal
products.69 It has been applied principally to cereal products by Bina
and co-workers.70

Metol has been used by a number of workers, including Perlzweig and
co-workers,71 Dann and Handler,72 and Steel and collaborators.73 The
color produced is said to be more stable than that obtained with p-amino-
acetophenone,73 and the reaction is reported to be more specific for
nicotinic acid.72 Aniline has been used by Melnick and Field 74, 75 and

ASSAY METHODS 55

Pearson,76 who indicates that it is four times as sensitive as /J-napthyla-
mine.

There have been at least two obstacles in the way of coming to an
agreement upon a chemical method which will be generally recognized
as acceptable. One is the fact that the amount of nicotinic acid appear-
ing in the extract depends upon the method of extraction, and there has
not been common agreement on this point. It is evident that some un-
known substance is converted to nicotinic acid by certain extraction
procedures.69, 77

Another more serious obstacle is the fact that the fundamental reactions
involved in producing the dye or dyes are not understood. The reaction
is not specific for nicotinic acid,75 but with different aromatic reagents
other pyridine derivatives such as nicotinamide, nicotinuric acid and
nicotine as well as pyridine itself react to produce more or less color.

In spite of these obstacles, it is probable that several aromatic amines
can be used successfully for particular types of products with good
success. The colorimetric methods in general are not as sensitive, how-
ever, as the microbiological method which has now become pretty well
standardized and accepted.

Microbiological Method. The principle of this method is the same as
for the riboflavin method, but the microbiological procedure for nico-
tinic acid is in an especially favorable position because of the lack of
any very satisfactory competing chemical method.

As with the microbiological methods generally, the substance to be
assayed must first be extracted and brought into solution. In the Snell
and Wright method,78 which has received wide acceptance,79- so nicotinic
acid, nicotinamide, nicotinuric acid, and cozymase all have the same
biological activity when tested in equimolecular amounts.78 This makes
it unnecessary to bring about complete hydrolysis of the combined
forms. The formation of nicotinic acid by acid and alkali treatment from
precursors which may or may not have vitamin activity,77 presents an
uncertainty which has not been overcome.

The organism used in this assay is Lactobacillus arabinosis 17-5 and
the evaluation of the response involves titration, usually at the end of
72 hours. When this method is used, the turbidity of the extracts does
not interfere. The method is 20 to 100 times as sensitive as the chemical
methods,78 and once the nicotinic acid or its derivative is in solution,
there is no obstacle to its satisfactory determination in amounts down
to 0.05 /xg or less if drop cultures are used.81 The organism used for this
assay is not as sensitive to fat acids as is L. casei. Krehl and co-workers
found that with the possible exception of linoleic acid, they did not
interfere.

56 THE BIOCHEMISTRY OF B VITAMINS

The basal medium as originally proposed has been modified by the
addition of more glucose and sodium acetate, the elimination of half of
the biotin, and the introduction of y>-aminobenzoic acid.82 In the study
referred to earlier in connection with the riboflavin assay, Roberts and
Snell 50 found that the new medium for Lactobacillus casei also supports
heavy growth of Lactobacillus arabinosus in 16 hours, and suggest that
this improved medium may be made the basis of a nicotinic assay. In
spite of improvements which may be made in the basal medium, no
serious errors are introduced by using the medium as originally proposed.

Recently two new microbiological assay methods for nicotinic acid
have been developed, one using a yeast, Torula cremoris,83 the other a
nonpathogenic bacteria, Proteus HX19.8i Both methods are more sensitive
and more rapid than the Snell-Wright method, and it remains to be seen
how widely they are used. One sure advantage, from the standpoint of
research, of having several available methods is the fact that the different
organisms respond characteristically to different natural derivatives.

Biological Tests. Although assays for nicotinic acid have been made
to a limited extent using dogs 85, 8G' 78 and chicks 86 as experimental
animals, the methods have not been standardized. As a result of recently
accumulated knowledge regarding the common transformation of trypto-
phan to nicotinic acid (p. 353) in mammals, it is obvious that no biolog-
ical test which leaves out of consideration this transformation can be
specific for nicotinic acid.

Pantothenic Acid

Although attempts have been made to determine pantothenic acid
chemically by lead tetraacetate oxidation of the lactone derived from it,
no serviceable method was developed,87 and the only available assay
methods are microbiological and biological.

Microbiological Methods. Many microorganisms require pantothenic
acid for growth or are stimulated by it, and all of these are potential test
organisms.

The yeast test served adequately in the discovery and isolation of
this vitamin 88, 89 but later was not used, partly because of the effective-
ness of /^-alanine, a cleavage product, as a yeast growth stimulant, and
also because the pantothenic acid concentration governed the rate of
growth rather than the total growth, as in the case of the lactic acid
bacteria.90, 91 Because of this latter fact, the multiplicity of yeast nutri-
lites,92 and the complicated effects of amino acids,93 it was feared that
the yeast method would not be as specific as would be required in dealing
with all sorts of materials.

Recently the yeast test has been revived: a different strain of yeast

ASSAY METHODS 57

is used, and the ^-alanine effect has been cancelled out by using more
asparagin in the medium; the results appear to be satisfactory.93 It has
the advantage of speed (16 hours) over the titration procedure using
lactic acid bacteria, but has the disadvantage that, as described, the
cultures have to be continuously shaken.

Lactic acid bacteria 94 have been used most extensively for pantothenic
acid assay following the similar methods of Pennington et al.90 and
Strong et al.95 The organism L. casei has been used successfully in the
assay of a great many types of materials.96-106 Fatty substances inter-
fere, as in the case of riboflavin assay,52 but this difficulty can often be
cared for by careful filtration at pH 4.5 or preliminary fat extraction, as
in the case of the riboflavin assay. Inhibiting substances produced by
the clarase digestion of yeasts and other materials are reported to inter-
fere with the determination by causing a downward drift of values with
increasing test dosage.103 Such substances would be most disturbing if
present in low-potency material ; but yeast, which was the worst offender,
is, of course, a relatively rich source. Thompson, Cunningham and Snell 106
found in the assay of canned foods that there was often a downward
drift in the assay values with increasing dosage which, however, tended
to reach a definite level. Presumably in such cases the effect is that of
extraneous stimulating substances which are effective at low dosage levels.
Neal and Strong,104 by suitable supplements to the basal medium, elimi-
nated such drifts which, however, appear not to prevent obtaining satis-
factory assay values, provided the higher dosage values (lower assay
values) are accepted as correct.106

While L. casei has been used extensively as a test organism for pan-
tothenic acid assay, it has the disadvantage that its growth is greatly
affected by fatty substances and by unknowns which must be introduced
into the basal medium in the form of crude extracts. Hoag, Sarett and
Cheldelin 107 and Skeggs and Wright 108 have developed assay methods
using L. arabinosus 17-5. In one laboratory, the Pennington et al.90
medium was modified, principally by using norite-treated autolyzed yeast
and rice bran concentrate in the basal medium in place of the alkali-
treated yeast extract. In the other, the medium of Snell and Wright 7S
for nicotinic acid assay was modified by altering the amounts of some of
the constituents, by introducing xanthine and p-aminobenzoic acid, and
by substituting nicotinic acid for pantothenic acid. For this organism
the effects of fatty substances are less marked, but are enough to intro-
duce substantial errors. Hoag et al. advocate a preliminary ether extrac-
tion for samples of high fat content. Skeggs and Wright introduced oleic
acid, which they found to be stimulative, into the basal medium. If this
is not done, they advocate a preliminary ether extraction. In both

58 THE BIOCHEMISTRY OF B VITAMINS

laboratories it was found that turbidimetric measurement of the response
in 18 hours or less could be used, and in one laboratory 107 titration after
24 to 40 hours was found to be satisfactory.

On the basis of general experience with the two organisms, it seems
likely that L. arabinosus should be a more satisfactory one for pantothenic
acid assays than L. casei. Whether the L. casei methods are too well
entrenched or whether the L. arabinosus methods offer enough advantage
to merit their general adoption remains to be seen.

Among the other microorganisms used for pantothenic acid assay are
Streptococcus lactis,109 Streptobacterium plantarum,110 Proteus mor-
ganii,111' 112, 113 Streptococcus faecalis,114 and Acetobacter suboxidans,
which responds to pantoic lactone and pantoic acid.115, 116

Chick Assay. While it would be feasible to develop an assay method
for pantothenic acid using the growth of any one of a number of animals
as a basis, only chicks have been used with any consistency.

The assay method involves the use of a basal diet the predominant
ingredients of which (yellow corn meal, wheat middlings and commercial
casein) have been heated in the dry state to 120° C for 36 hours. This
heat treatment effectively destroys pantothenic acid. This mixture is
supplemented with minerals, sources of vitamins A, D and K and ribo-
flavin (whey adsorbate) .117> 11S Chicks are kept on a normal diet for four
days and then placed upon the heated diet for 5 days of depletion. After
this they are divided into groups and fed the heated diet supplemented
by standard and "unknown" materials containing pantothenic acid for
comparison. The growth of chicks on the heated diet is very slight, but
in the presence of graded doses of pantothenic acid it is increased in
accordance with the dosage used.

The basal diet as described is not ideal for the purpose of pantothenic
acid assay, but by supplementing it in accordance with newer develop-
ments in the field of chick nutrition it can be made the basis of a test
which grows more and more specific.119

Because of their relative ease and speed, it is probably safe to say that
microbiological assays for pantothenic acid have been run at least ten
times as often as chick assays. The microbiological tests have thus been
more thoroughly explored than has the chick test. In many cases the
microbiological and chick methods give concordant results when direct
comparisons have been made. Yeast and liver preparations have very
often given values by the chick method which are much higher than those
by microbiological methods. According to Willerton and Cromwell 103
this difference largely disappears when the material is properly digested
before the microbiological assay is applied. Other workers have not been
successful in closing this gap.104, 107 In view of the recent findings of

ASSAY METHODS 59

Lipmann et al.120 with respect to the release of pantothenic acid from
coenzyme A, the subject needs to be reinvestigated. Certainly there is
some hazard involved in using crude, non-uniform enzyme preparations,
just as there is in the use of chemicals of low purity.

More recently a rat growth method for the estimation of pantothenic
acid has been proposed in which wheat germ, in addition to thiamine,
riboflavin, pyridoxine, inositol, nicotinic acid and choline, is included in
the basal diet.121 This material is relatively deficient in pantothenic acid
but contains other vitamins essential to rapid growth, and hence is said
to serve excellently as a constituent of the basal diet.

Vitamin B0 — Pyridoxal, Pyridoxamine, Pyridoxine

The available methods for determining these forms of the vitamin
either individually or together are chemical, microbiological and biologi-
cal. Much material which is primarily of historical interest, that ante-
dating the discovery of pyridoxal and pyridoxamine,122 will be omitted
from the present discussion.

Chemical Methods. Since each of the three forms of vitamin BG pos-
sesses a phenolic group in the 3 position and the 6 position is unsubsti-
tuted, it is not surprising that they can be condensed with various
reagents to yield dyes. Among the reagents which have been used in
attempts to develop colorimetric methods for vitamin B6 are diazotized
sulfanilic acid,123 2,6-dichloroquinone chlorimide 124, 125> 126 and diazotized
p-amino acetophenone.127 The earlier attempts were, of course, based
upon the assumption that vitamin BG and pyridoxine are identical.

In natural materials there are numerous phenolic and other reactive
substances, however, and the application of such color reactions to natural
products is a quite different matter from their application to the pure
substances. In developing colorimetric methods it has been necessary to
eliminate or rule out other reacting substances, and in some cases this
has required an elaborate procedure.123 The results obtained using such
precautions have often agreed substantially with animal assays of similar
materials, though actually the number of available animal assay values
on materials which are in any sense duplicable is exceedingly small. The
question of the reliability of the animal assays will be discussed in a later
paragraph.

One of the interesting facts which has a bearing upon the applicability
of this colorimetric procedure is that all three forms of the vitamin give
colors when treated with diazotized sulfanilic acid. Pyridoxal gives a
bright yellow, pyridoxamine yields orange to pink, and pyridoxine gives
an orange color.128 It is easy to see that a natural mixture might give a
a color for which all three components might be partially responsible.

60 THE BIOCHEMISTRY OF B VITAMINS

In view of these facts and because of the existence in nature of other
reacting substances, it seems unlikely that any simple colorimetric pro-
cedure can be worked out for the determination of all forms of vitamin B6.
Spectrophotometry analysis of the colors produced will probably have to
be employed. By the use of borate which under proper conditions forms
an inactive complex with pyridoxine only, it is possible, by difference, to
obtain a color value due to pyridoxine alone.129 This has been applied
using the quinone chloroimide reagent. In the absence of borate under
prescribed conditions both pyridoxal and pyridoxamine are about one-half
as chromogenic as pyridoxine.

Microbiological Methods. Pyridoxine was first shown to be a growth
substance for microorganisms by Moller,130 who found it to be stimulatory
toward lactic acid bacteria and a bottom yeast from sauerkraut. About
this time Schultz, Atkin and Frey 131 and Eakin and Williams 132 inde-
pendently and simultaneously announced its stimulative effect on bakers'
yeast. A yeast growth method {Saccharomyces carlsbergensis) for its
determination as worked out in the Fleischmann Laboratories 133 is at
present the best method for the simultaneous determination of all three
forms of the vitamin. A yeast growth method (Saccharomyces cerevisiae)
was earlier developed and applied by Williams and co-workers,134, 135
which contributed because of contrast with results obtained with Strepto-
coccus lactis (faecalis) to the discovery of "pseudo pyridoxine," later
identified as pyridoxal and pyridoxamine.122, 136

According to Snell and Rannefeld 137 and Melnick and co-workers,129
pyridoxal, pyridoxamine and pyridoxine are approximately equivalent
in their effects on Saccharomyces carlsbergensis; but for Saccharomyces
cerevisiae the newer members of the group are materially less active under
the prescribed conditions.

The effects of pyridoxal, pyridoxamine and pyridoxine on seventeen
test organisms which had been suggested or used, were studied thoroughly
by Snell and Rannefeld,137 and the use of Strep, faecalis as a test organism
for the assay of pyridoxal and pyridoxamine was developed by Rabino-
witz and Snell.114 Pyridoxal has been determined by Rabinowitz et al.131&
It seems feasible by a combination of colorimetric and biological
methods to develop means of determining each of the three members of
the group. Rabinowitz and Snell have determined each of the three com-
ponents by microbiological means.137b The yeast growth method {S.
carlsbergensis) 133 is at present reasonably satisfactory for determining all
three forms together, provided extraction is adequate (p. 36).

Stokes138 has recommended the 5-day mold test using the Neurospora
mutant discovered by Beadle and co-workers. For some studies the 5-day
incubation period would be a serious disadvantage.

ASSAY METHODS 61

Biological Assay. The only biological assay method that has been
used extensively and which has been applied to pyridoxal and pyri-
doxamine is the method of Conger and Elvehjem 139 which has recently
been improved by Sarma, et aL139a This involves the growth response of
rats which had been depleted 4 to 6 weeks on a basal diet containing
B vitamins in the form of thiamine, riboflavin, nicotinic acid, choline and
pantothenic acid in pure form and 1:20 liver concentrate powder. The
growth response is linear with respect to the amount of vitamin B6 added.
As originally designed, this test, like the microbiological tests, was for
the assay of pyridoxine, on the assumption that pyridoxine and vitamin
B6 were identical. Pyridoxamine and pyridoxal are about equally active
in the test, however, so it constitutes a method for determining all three
forms of the vitamin simultaneously. Assay values obtained by this
method for very few duplicable materials are available, and a wider
application of the test, particularly to low-potency materials might easily
show it to possess serious flaws. The biological method of Dimick and
Schreffer,140 which appears to be less specific, has also been employed in
some recent studies.129 Elvehjem has recently suggested an improved basal
diet for the rat growth test.141 This has been investigated in only a pre-
liminary way.

Biotin

The only available methods for the assay of biotin involve the use of
microorganisms or higher animals. The outstanding obstacle to the devel-
opment of a chemical method which would be applicable to natural
material is the fact that biotin occurs naturally in exceedingly low con-
centrations which are beyond the reach of most kinds of chemical tests.

Microbiological Tests:

1. Yeast Growth Method. Yeast was the test organism used ini-
tially in the discovery and isolation of biotin by Kogl and co-workers.142
However, the assay method utilized fresh yeast (heavy seeding) obtained
directly from a brewery and hence was not applicable to laboratories in
general. The most widely used assay method was that developed in the
Texas laboratories, which utilizes a pure culture of bakers' yeast.143

The basal medium for this yeast test contained only known substances,
including inositol, /^-alanine (synthetic calcium pantothenate was not yet
available), thiamine and pyridoxine; 16-hour growth responses were
obtained from the addition to 12-ml cultures of biotin in amounts of 25
to 250 micromicrograms (10 ~12 gram). This method has the advantage of
speed and is also specific to a high degree, since biotin is an extremely
potent nutrilite and the amounts of material (in the form of a tissue

62 THE BIOCHEMISTRY OF B VITAMINS

extract, for example) which need to be added to the cultures are small
enough so that extraneous growth stimulants are not likely to interfere.
This is particularly true if the lower portion of the "growth curve" is
utilized.144 Turbidity and color of extracts for the same reason usually
offer no problem whatever. This method has been used extensively in the
Cornell 145 and Texas laboratories and elsewhere. Hofmann 146 character-
ized it as "simple, accurate and fast," and as "the most satisfactory
technique." In view of the striking effect which amino acids were later
found to have on yeast growth, when the necessary nutrilites are sup-
plied,147 Hertz improved the basal medium by the addition of a casein
digest and by the obvious substitution of calcium pantothenate for
^-alanine.148

The yeast test is not applicable for the determination of biotin in the
presence of desthiobiotin, biotin diaminocarboxylic acid, desthiobiotin
diaminocarboxylic acid,149 oxybiotin,150 biotin sulfoxide methyl ester,151
and a few less active compounds because they too possess biotin activity
for yeast. However, as these are synthetic substances and are not encoun-
tered in many types of investigations, the limitation on the method is
often not serious. Other microbiological procedures are limited to some
extent in the same way, but yeast possesses great synthetic powers and
can utilize some compounds in place of biotin which lactic acid bacteria,
for example, cannot.152

In applying the yeast test to metabolism studies, Oppel 153 found that
only a portion of the total biotin (as measured by the yeast test) of dog,
rabbit, rat and human urines was avidin-combinable, but that "only
minute amounts of the non-avidin-combining biotin were found in diets
and stool specimens." In the case of one subject who consumed 35-55 /xg
of biotin on alternate days but no raw egg white, the "non-combinable
biotin" of the urine was about 35-40 per cent of the total, whereas when
large amounts of egg white and biotin were both consumed, the "non-
combinable" constituted 75-85 per cent of the total urinary excretion.

Burk and Winzler,154 however, reported that uncombinable biotin is
abundant and widespread constituting 90 to 100 per cent of that in the
urine of rats and mice fed avidin and in Squibb urease, 30 to 50 per cent
in "Vitab" (rice bran concentrate) and unhydrolyzed beer, and 1 to 10
per cent in rat lung, spleen, testes, adrenals, lymph nodes, skin, intestinal
tract contents, feces, polished rice, dried yeast and food mixtures. Various
active substances possessing biotin activity were designated by these
investigators as "miotin," "tiotin" and "rhiotin."

Chu and Williams144 corroborated the findings of Oppel in that they
found substantial amounts of "uncombinable biotin" in urine but nowhere
else. They were unable to find evidence for the existence of "uncombinable

ASSAY METHODS 63

biotin" in "Vitab," rat brain, beer or Squibb urease, and warned against
the use of high dosage levels in applying the yeast test. As larger and
larger amounts of extracts are introduced into the yeast medium, the test
becomes less specific, because of the existence of other yeast nutrilites
which may not be in any way related to biotin.155

The existence and significance of the "uncombinable biotin" in urine
has never been clarified. Probably the most reasonable explanation at the
present time is that in urine there exist biotin degradation products which
do not combine with avidin and yet can be utilized by yeast in lieu of
biotin. The existence of such substances has never been completely clari-
fied and their interference with other asasy methods has not been ade-
quately studied.

Hofmann and Winnick 156 have utilized the fact that oxybiotin is active
for yeast in the determination of oxybiotin in the presence of biotin.
Biotin is destroyed by dilute permanganate under conditions that oxybio-
tin is unaffected, so that the response after oxidation is a measure of the
oxybiotin present.

2. Methods Utilizing Lactic Acid Bacteria. Several papers have been
published describing biotin assay methods or modifications thereof, which
utilize L. casei as the test organism.157- 158> 159> 16° Difficulties are encoun-
tered in obtaining a biotin-free basal medium, and the same interferences,
particularly of fatty substances, occur as with other tests involving this
organism. If the titration method is used for evaluating responses, more
time is required to obtain results than in the yeast test.

More recently Wright and Skeggs 161 have developed the use of L.
arabinosus for biotin assay. Because the basal medium needs to contain
only known substances, and for other reasons, this organism appears to
have distinct advantages. A recent survey (1945) is reported to indicate
that seven out of nine prominent laboratories use L. arabinosus for biotin
assays.152 However, the amount of published analytical data accumulated
using this method is small, and any shortcomings that the method may
possess can be discovered only by extensive use. Because of the nature of
the medium and high sensitivity of the organism to biotin, one would not
anticipate any serious difficulties in the general application of the method.
In the test as originally described,161 titration after 72 hours was used to
evaluate responses. Subsequently, Wright 152 has indicated that satisfac-
tory results may be obtained turbidimetrically after about 24 hours of
growth, or by titration after 48 or even 24 hours. Speeding up the
response in this test could doubtless be accomplished by suitable modifica-
tions (if necessary) of the improved medium for L. casei developed by
Roberts and Snell.50

Other biotin assays have utilized as test organisms Rhizobia,154 Clos-

64 THE BIOCHEMISTRY OF B VITAMINS

Iridium butylicum™2 and Neurospora crassa,1G3 among others, but these
have not been used extensively.

Biological Assay. Biological assays of biotin are relatively of little
importance. By introducing raw egg white into the diet of rats they
become deficient in biotin (vitamin H) and develop characteristic lesions.
The daily dose of biotin required to cure this condition was used as a
"unit." 164 Other animals, chicks, guinea pigs, rabbits, monkeys and dogs
are capable of developing egg white injury and may therefore be used
for assay purposes. Ansbacher and Landy 165 produced biotin deficiency
in chicks by feeding a heated diet low in biotin and suggested the use of
this procedure as the basis of an assay method.

Trager 166 has reported the presence in the plasma of various animals,
of fat-soluble material — not oleic acid — which replaces biotin in the tests
involving lactic acid organisms and which is active for biotin-deficient
chicks. Contrary to these findings, however, Axelrod, Mitz and Hof-
mann,1G7 as a result of a thorough study, conclude that the biotin-like
activity present in plasma is explicable on the basis of the content of
known fat acids.

Inositol

For the assay of inositol, chemical and microbiological methods are
available. In addition, some use of experimental animals has been made.

Chemical Method. The only serviceable chemical method for inositol
is that of Piatt and Glock,168 and it is subject to limitations both as to
specificity and to convenience and speed. Earlier chemical methods were
investigated in the author's laboratory 169 and were judged to be almost
valueless. Actual isolation and weighing of the inositol was one of the
expedients used, but this obviously has very serious drawbacks.170

The method of Piatt and Glock involves very briefly: (1) extraction
with water, (2) precipitation of extraneous material with acetone, (3)
removal of glucose by fermentation, (4) removal of other extraneous sub-
stances with base exchange resins, (5) differential oxidation of the resid-
ual glycerol and inositol with periodic acid. The last step takes advan-
tage of the fact that glycerol can be oxidized quantitatively without more
than a very small fraction of the inositol being attacked. As Woolley m
has indicated, however, there are in certain tissues several other sub-
stances closely related to inositol including isomers, and these presumably
would not be eliminated by any of the treatments. On this basis there is
some reason to question the specificity of the method. Furthermore, its
application is relatively exacting and time-consuming.

Microbiological Methods. The microbiological determination of ino-
sitol stems from the finding of Eastcott 172 that inositol serves as a growth

ASSAY METHODS 65

substance for yeast. Woolley 173 and Williams and co-workers m inde-
pendently developed assay methods based upon this observation. The
work of the latter group was in part an outgrowth of earlier work 169 in
the author's laboratory.

The two methods are essentially alike, the main difference being in
some of the constituents of the basal medium. In order to take care of
unknown yeast nutrilites,155 Williams and co-workers introduced into the
basal medium a liver autolyzate which was rich in unknown nutrilites
but relatively poor in inositol. This small amount of inositol caused the
growth in the basal medium to be heavier than would otherwise be the
case; but adequate responses were obtained by the addition of 0.1 /*g to
0.8 fig inositol, and the method was applied extensively 96-101 with success.
Woolley used a dialyzed rice bran extract (which was lacking in free
inositol) as an ingredient of his basal medium because it contained un-
known yeast-stimulating material; he obtained satisfactory results.

It appears that, provided the problem of extraction is cared for (p. 38) ,
either of the yeast methods yields satisfactory results. It is also probable
that either method could be improved if it were reinvestigated in the
light of present knowledge. The extensive application of one of the yeast
methods to the study of the contents of tissues yielded results which in
one case apply to the amount freed by autolysis and in a later study to
the amount freed by enzymatic digestion.96"101 The total inositol values
were generally materially higher than those obtained by autolysis or
enzymatic digestion.85 From the nutritional standpoint the total inositol
values may well be the most important, because bound forms of inositol
are utilizable by mice. On the other hand, the general nutritional impor-
tance of inositol is problematical and the availability of different forms
of inositol to different animals is largely unknown. The fact that phytic
acid interferes with the assimilation of calcium in diets 175 suggests that
inositol is not always freed by digestion and hence is not uniformly avail-
able in this form.

Woolley 176 has made a careful study of the specificity of the yeast test
for inositol, and has found that only meso-inositol is effective. Other
naturally occurring polyalcohols including isomers are inactive, as are
also inositol esters, including phytic acid and soybean lipositol. The bound
inositol in amylase is freed by enzymatic action, since about the same
values were obtained whether enzymatic digestion or acid digestion was
used.177

Another microbiological method for the determination of inositol in-
volves the use of a mutant strain of Neurospora.178 This appears to yield
satisfactory results but has not been extensively applied. The dosage

66 THE BIOCHEMISTRY OF B VITAMINS

levels at which inositol is effective in the test is from 10 to 30 fig, as com-
pared with 0.1 to 0.8 fig in the yeast tests.

Biological Test Using Mice. The ability to cure alopecia in mice which
have been fed inositol-free diets was used as an assay method by Wool-
ley,179 but the results obtained were only roughly quantitative, partly
because relatively few levels were tested. Phytin and mesoinositol, for
example, were found to be "active" when tested at the same level m (0.1
per cent of the diet) . If the active ingredient is inositol, phytin should
be about 1/5 as active. The relative activities of phytin and inositol for
mice would throw some light on the assimilation of phytin and its effects
on calcium assimilation.

Choline

Chemical, microbiological, physiological and animal assay methods for
choline have been described. Each has its usefulness, though the methods
in general are not as satisfactory as many that have been described in
previous sections.

Chemical Assay. A large number of chemical tests have been applied
to the problem of the estimation of choline.180 Among these precipitation
with reineckate is without question the most valuable. The use of this
method has a long history which we shall not take time to trace. Among
more recent papers those by Beattie,181 Jacobi and co-workers,182 Engel,183
Marenzi and Cardini,181 Enteman et al.1S5 Glick,186 and Winzler and
Meserve,187 appear to be most important. The subject has been reviewed
recently by Handler.188

In most cases choline is freed by alkaline hydrolysis, and choline
reineckate precipitated under prescribed conditions is determined colori-
metrically in acetone solution. Marenzi and Cardini,184 however, deter-
mined chromium colorimetrically in the insoluble reineckate, and Winzler
and Meserve 185 used ultraviolet light instead of visible light.

This general method has been applied to tissues and feeds most exten-
sively by Engel 189 and Rhian and co-workers,190 with results which
appear satisfactory. None of the methods is extremely sensitive. For the
direct colorimetric determination of the reineckate in acetone about 200
fig of choline must be in the sample ; when ultraviolet light is used 185 the
minimum determinable amount is about 50 fig. Colorimetric analysis for
chromium 1S4 makes possible a determination of 15 fig. As long as one is
concerned with the total choline content of tissues, which is relatively
high, the methods are serviceable from the standpoint of sensitivity.

Means have been devised whereby most of the interfering substances
commonly found in food materials, including betaine, are eliminated.
Dimethyl aminoethanol may be carried down to some extent along with

ASSAY METHODS 67

choline reineckate, however.191 It is doubtful whether the method could
be used directly, unless by the Winzler and Meserve modification, for the
determination of choline in plants that contain substantial amounts of
alkaloids.

Microbiological Assay. The microbiological method for choline which
has been applied to tissues, blood and urine is that originated by Horowitz
and Beadle,192 and involves the use of a cholineless mutant of Neurospora.
This organism has lost by ultraviolet radiation its ability to synthesize
choline and gives corresponding growth responses when amounts of
choline, 1 fig to 50 ^g, are added to 25 ml of culture medium. The responses
are evaluated after 72 hours' growth by weighing the dried mycelia (ca.
5.0-50 mg). An alternate simplified procedure involves measuring the
diameter of the mold cultures as grown in 16 hours on agar in Petri
dishes.193- 194

The response of this cholineless mutant as well as other cholineless
strains 194 is not quite specific. Betaine and ethanolamine are inactive, but
about 50 per cent of the choline of lecithins 192 is effective in the 72-hour
test; methionine is 0.2 per cent as active as choline; and dimethylamino-
ethanol and monoethylaminoethanol, acetylcholine, phosphorylcholine and
arsenocholine are highly active. Certain other synthetic homologues,
dimethylethylhydroxyethylamine, and diethylmethylhydroxyethylamine
are also highly active.194

One cannot help feeling that these nonspecificities are less important
than they appear. The authors are to be commended for thoroughness in
investigating the question of specificity, and the method should not be
damned because its specificity has been thoroughly investigated. Lecithins
and methionine can be eliminated from test solutions, and the other active
compounds for the most part are not known to occur naturally in quanti-
ties likely to interfere. Dimethylaminoethanol occurs in an alkaloid found
in certain leguminous plants 195 ; but its general distribution, while pos-
sible, and perhaps even probable,196 has not been demonstrated. Leucke
and Pearson have applied the mold method to free choline in plasma and
urine,197 and the free and bound choline of a few animal tissues,198 with
results which appear to be satisfactory and in agreement with those
obtained by the reineckate method. Hodson has applied the method to
milk products.163 A combination of the reineckate and microbiological
methods suggests itself as desirable in crucial cases.

The basis for another microbiological test of limited interest is the
requirement of Type III Pneumococcus for choline.199 A large number of
compounds, mostly synthetic, showed activity, including dimethylethanol-
amine (100 per cent) and ethanolamine (10 per cent). The nonspecificity

68 THE BIOCHEMISTRY OF B VITAMINS

and the pathogenic nature of the organism used are deterrents to its use
as an assay method.

Physiological Assay. An interesting and important method for choline
determination involves acetylating it quantitatively and determining the
acetylcholine by its stimulation of the contraction of isolated muscle from
rabbit intestine. The amount of acetylcholine necessary to elicit a con-
traction about 75 per cent of the maximum may be from 0.01 to 0.03 /xg
per ml, depending upon the muscle preparation and the exact conditions ;
so the method has high sensitivity.200

There are interfering substances and the method possesses many dan-
gerous pitfalls for the chemist,201 but inherently it has great advantages
and it seems likely that cooperative studies by biochemists (who are more
expert in preparing suitable extracts and eliminating by adsorption and
otherwise interfering substances) and physiologists (who are more expert
with respect to dealing with muscle preparations) might evolve an assay
method more sensitive, more specific, and more satisfactory generally
than any yet devised. In its present form it has been applied to many
materials.200 Duplicate assays occasionally differed by almost 30 per cent.

Animal Assays. The development of an animal assay procedure for
choline is complicated by the fact that choline is not an absolute require-
ment; methionine and other naturally occurring substances possess
choline-like activity when fed to animals.202 Engel,1S3 nevertheless, has
developed an assay method for choline and choline-like substances which
depends upon its ability to prevent kidney hemorrhages in rats receiving
a choline-deficient diet. The rats had to be carefully matched and used
in considerable numbers (more often 11 or 12 for each test), but the
results were such that a 10 per cent increase or decrease of the material
tested could be detected. Liver was found to be about 25 per cent more
effective than could be accounted for on the basis of its choline content
as determined chemically by the reineckate method, and this was inter-
preted to mean the presence in it of other substances which are physiologi-
cally like choline. Otherwise, the bioassays agreed with the chemical
method rather closely.

Folic Acid (Pteroyl Glutamic Acid, P.G.A.)

Determination of folic acid may be performed (under delimiting con-
ditions) chemically, microbiologically or by animal assays. None of the
methods has been fully standardized, particularly so as to be readily
applied to tissues of all types.

Chemical Determination. Hutchings, et al.203 have developed a chemi-
cal method for the determination of folic acid and related compounds,
taking advantage of the fact that by reduction in acid solution with zinc

ASSAY METHODS 69

dust, folic acid yields characteristically a pteridine and an aromatic
amine. The latter can be determined colorimetrically after the method of
Bratton and Marshall.204 The presence of aromatic amines in the original
solution before reduction is taken care of by the use of a blank, and the
amount before reduction subtracted from that obtained after reduction
is used as a measure of the folic acid present.

The specificity of the test has not been explored (in spite of the ten
investigators involved) and its sensitivity is not high. It may be used only
for concentrates which contain 5 per cent or more of folic acid.

Microbiological Tests. The two organisms which are most often used
for folic acid assays are L. casei, the organism used initially in observa-
tions dealing with the "riorite eluate factor" 205 and Streptococcus faecalis
R (earlier called Strep, lactis R) which was the principal organism used
in connection with first obtaining folic acid in highly concentrated form 20C
and in determining its distribution in tissue autolyzates and in enzyme-
digested tissues and foods.

The principal differences between the behaviors of the two organisms
in these tests are: (1) L. casei assays are materially affected by the intro-
duction of additional alanine into the medium ; S. faecalis assays are not.207
The same general statement appears to apply also to other amino acids
(leucine, isoleucine, threonine 208> 209) , though direct comparisons appear
not to have been made. (2) L. casei requires for maximum growth un-
known substances such as occur in enzymatically hydrolyzed casein 50
and norite-treated peptone.210 Such addenda have not been required when
S. faecalis is used.210 (3) Thymine in sufficient concentration has stimulat-
ing effect on both organisms as well as certain other lactic acid bac-
teria;211, 212- 213 however, L. casei is considerably more sensitive to small
concentrations.209 On the other hand, thymine in sufficient amounts has
an ultimate effect practically equivalent to folic acid for S. faecalis, but
incomplete fermentation only results when excess thymine is furnished
L. casei.213 This latter observed difference may be eliminated when it is
possible to include all the stimulatory substances affecting L. casei, except
folic acid, in the basal medium. (4) S. faecalis responds to rhizopterin 214
(S.L.R. factor) as it does to folic acid, but this glutamic-acid-free relative
of folic acid is substantially inactive for L. casei. (5) L. casei responds
readily to pteroyl triglutamic acid while for S. faecalis this combined
form is only about 6 per cent as active.215 (Both organisms respond
equally to folic acid and fail to respond to pteroyl heptaglutamic acid.)
(6) L. casei tests have usually involved relatively long growth periods
and titrametric evaluation of the responses. S. faecalis tests, on the other
hand, have usually involved short growth periods and turbidimetric meas-
urement of the response.

70 THE BIOCHEMISTRY OF B VITAMINS

A number of the apparent defects mentioned above are relatively easily
remedied. The basal media can be adjusted to take care of the effects of
alanine and other amino acids, and with less satisfaction the unknown
stimulatory substances.

A drawback so far as L. casei is concerned is the relatively high activity
of thymine — as little as 1 /*g in a sample would be enough to introduce
serious error.209 For S. faecalis, however, folic acid is at least 5000 times as
active as thymine.213 This discrepancy is not as important as might be
supposed, however, since L. casei responds to smaller concentrations of
folic acid than does S. faecalis. Stokstad and Hutchings 216 suggest that
with S. faecalis the total response minus the residual response after selec-
tively destroying the folic acid could be used as a measure of the true
folic acid content. Whether such a procedure is necessary or practical has
not been demonstrated.

The most serious defect of the S. faecalis assay is the response to a
simpler compound, rhizopterin. The degree of importance of this observa-
tion depends upon the distribution of rhizopterin in nature. If it is wide-
spread and relatively abundant, the defect is quite serious. Whether or
not this is so can be determined only by comparative assays on completely
digested samples, using both L. casei and S. faecalis. Because the two
organisms do not respond equally to combined forms of folic acid,215 one
would have to be sure that digestion had been complete before valid con-
clusions could be drawn.

From the foregoing discussion, it is clear that no well standardized and
completely satisfactory microbiological method for folic acid assay has
been devised. Stokstad and Hutchings wisely suggest the use of both L.
casei and S. faecalis.216 Complete digestion of the combined forms without
destruction constitutes one crucial problem (p. 40).

Animal Assay Methods.

Folic acid assays may be performed using chicks, rats or monkeys. The
use of rats involves the feeding of sulfa drugs to prevent intestinal syn-
thesis 217 and involves so many complications and uncertainties that it
does not appear to be a useful assay method at the present time. Use can
be made of rhesus monkeys 21S but the expense and inconvenience could
hardly be justified on the basis of an assay method.

Chick Assay. While O'Dell and Hogan219 and their co-workers used
the cure of anemia in chicks in connection with concentrating "vitamin
Bc," this method of assay has apparently not been applied to the deter-
mination of the vitamin in foods and tissues.

The preferred chick assay method involves the use of a 4-week prophy-
lactic test period. It was applied successfully by Bird et al.220 to natural

ASSAY METHODS 71

materials, but has been only briefly described.221 A number of changes
result from lack of the vitamin,221, 222 including failure to grow, but it is
not apparent how Bird and co-workers 220 calculated their results. In the
assay technic recommended by Day and Totter,223 weights of the chicks
at the end of four weeks are the basis for calculating the results.

The chick assay method as applied by Bird and co-workers yielded
results which in general agreed substantially with microbiological assays
when the materials for the latter were treated enzymatically with prepara-
tions from hog kidney and almonds.220 Plant extracts, however, gave
higher results with chick assays than were obtainable by microbiological
assay. It may be that plants contain combined forms of folic acid which
are not hydrolyzed by any of the treatments used. Chicks respond equally
to folic acid, the tri- and heptaglutamate forms, and presumably to other
combined forms which may occur naturally (p. 31).

/>-Aminobenzoic Acid

While p-aminobenzoic acid can be determined colorimetrically by
diazotizing and coupling with dimethyl-a-napthylamine 224 and by the
use of thiamine as a reagent,225 such methods are neither highly specific
nor are they sensitive in comparison with microbiological methods and
have not been used as general assay procedures.

Animal experiments have demonstrated the physiological effect of feed-
ing p-aminobenzoic acid under prescribed conditions to chicks,226, 227
mice,228, 229 and rats,226, 230 but the effects may be indirect and not always
reproducible,231 and no assay method has resulted from these observations.

Microbiological Assays. Four microbiological assays for p-amino-
benzoic acid have been developed. They utilize respectively, Acetobacter
suboxydans232 Lactobacillus arabinosus 17-5,233 a mutant strain (amino-
benzoicless) of Neurospora23* and CI. acetobutylicum.235 In the table
below is given certain crucial information regarding these tests.

Microbiological Assays for p-Aminobenzoic Acid
Growth

period

Assay range

Method of evaluating

Organism

(hrs.)

(MB)

response

(1) Acetobacter suboxydans

5.0 -50

Turbidity

(2) L. arabinosus

0.15- 0.5

Titration

(3) Neurospora

4.0 -40

Measurement of culture size

(4) CI. acetobutylicum

20-24

0.3 - 1.5

Turbidity

It is probable that each of the four methods is capable of yielding
satisfactory results and for the most part they embody methods which
are now common in microbiological assays. In method 3, the size of the
mold cultures is measured with calipers which is something of an innova-

72 THE BIOCHEMISTRY OF B VITAMINS

tion, and in method 4, the organism has to be grown under anerobic con-
ditions.

Assays for p-aminobenzoic acid are in general exacting partly because
laboratory equipment, glassware, etc., becomes so readily contaminated
with p-aminobenzoic acid that consistent assays are impossible. This is
particularly true when the more sensitive methods are used. Segregation
of the glassware used for this purpose and the use of a separate room are
desirable precautions, in addition to scrupulous care in the cleaning of
glassware.

The problem of extraction of p-aminobenzoic acid, which has already
been discussed (p. 40) , is a crucial one and the four methods above have
never been compared under conditions where the extraction procedures
were the same. The values obtainable for different materials assayed are
therefore usually not comparable. The evidence indicates that the response
in every case is quite specific for p-aminobenzoic acid, in the sense that
no simple known chemicals have more than a slight effect. Information
is lacking as to how the unknown combined forms, which probably exist
naturally, affect the different organisms, and no tests involving pure folic
acid and its conjugates or rhizopterin have been found.

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105. Bauernfeind, J. C, Norris, L. C, and Heuser, G. F., Poultry Sci., 21, 136-41

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ASSAY METHODS 75

106. Thompson, M. L., Cunningham, E., and Snell, E. E., J. Nutrition, 28, 123-9

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107. Hoag, E. H., Sarett, H. P., and Cheldelin, V. H., Ind. Eng. Chem., Anal. Ed.,

17, 60-2 (1945).

108. Skeggs, H. R., and Wright, L. D., J. Biol. Chem., 156, 21-6 (1944).

109. Mitchell, H. K., et al, J. Am. Chem. Soc, 62, 1776-9 (1940).

110. Kuhn, R., and Wreland, T., Ber., 73, 962 (1940).

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113. Pelczar, M. J., and Porter, J. R., Proc. Soc. Exptl. Biol. Med., 47, 3-7 (1941).

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115. Underkofler, L. A., Bantz, A. C, and Peterson, W. H., J. Bact., 45, 183-90 (1943).

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117. Jukes, T. H., /. Biol. Chem., 117, 11-20 (1937).

118. Jukes, T. H., J. Biol. Chem., 129, 225-31 (1939).

119. Jukes, T. H., and McElroy, L. W., Poultry Sci., 22, 438-41 (1943).

120. Lipmann, F., et al, J. Biol. Chem., 167, 869-70 (1947).

121. Bacon, J. S. D, Jenkins, G. N., and Irwin, J. O., Biochem. J., 37, 492-7 (1943).

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123. Swaminathan, M., Nature, 145, 780 (1940).

124. Scudi, J. V., J. Biol. Chem., 139, 707-20 (1941).

125. Bina, A. F., Thomas, J. M., and Brown, E. B., J. Biol. Chem., 148, 111 (1942).

126. Hochberg, M., Melnick, D., and Oser, B. L., /. Biol. Chem., 155, 109-14 (1944).

127. Brown, E. B., Bina, A. F., and Thomas, J. M., J. Biol. Chem., 158, 455-61 (1945).

128. Ormsby, A. A., Fisher, A., and Schlenk, F., Arch. Biochem., 12, 79-81 (1947).

129. Melnick, D., et al, J. Biol. Chem., 160, 1-14 (1945).

130. Moller, E. F., Z. physiol Chem., 254, 285-6 (1938).

131. Schultz, A. S., Atkin, L., and Frey, C. N., /. Am. Chem. Soc, 61, 1931 (1939).

132. Eakin, R. E., and Williams, R. J., /. Am. Chem. Soc, 61, 1932 (1939).

133. Atkin, L., et al, Ind. Eng. Chem., Anal. Ed., 15, 141-4 (1943).

134. Williams, R. J., Eakin, R. E., and McMahan, J. R., Univ. Texas Pub., 4137,

24-6 (1941).

135. Williams, R. J., Univ. Texas Pub., 4237, 9-10 (1942).

136. Snell, E. E., Guirard, B. M., and Williams, R. J., J. Biol Chem., 143, 519-30

(1942).

137. Snell, E. E., and Rannefeld, A. N., J. Biol. Chem., 157, 475-89 (1945).

137a. Rabinowitz, J. C, Mondy, N. I., and Snell, E. E., J. Biol. Chem., 175, 147-53

(1948).
137b. Rabinowitz, J. C, and Snell, E. E., J. Biol Chem., 176, 1157-67 (1948).

138. Stokes, J. L., "Biological Symposia," Vol. 12, Jaques Cattell Press, Lancaster,

Pa., 1947, p. 231.

139. Conger, T. W., and Elvehjem, C. A., J. Biol. Chem., 138, 555-61 (1941).

139a. Sarma, P. S., Snell, E. E., and Elvehjem, C. A., J. Nutrition, 33, 121-8 (1947).

140. Dimick, M. K., and Schreffler, C. B., /. Nutrition, 17, 23-9 (1939).

141. Elvehjem, C. A., "Biological Symposia," Vol. 12, Jaques Cattell Press, Lan-

caster, Pa., 1947, pp. 213-26.

142. Kogl, F., and Tonnis, B., Z. physiol. Chem., 242, 43-73 (1936).

143. Snell, E. E., Eakin, R. E., and Williams, R. J., /. Am. Chem. Soc, 62, 175-8

(1940).

144. Chu, E. J., and Williams, R. J., J. Am. Chem. Soc, 66, 1678-80 (1944).

145. du Vigneaud, V., et al, J. Biol. Chem., 140, 643-51 (1941).

146. Hofmann, K., "Advances in Enzymology and Related Subjects," Vol. 3, Inter-

science Publishers, Inc., New York, N. Y., 1943, pp. 292, 306.

147. Mitchell, H. K, and Williams, R. J., Biochem. J., 34, 1532-6 (1940).

148. Hertz, R., Proc. Soc. Exptl. Biol. Med., 52, 15-7 (1943).

149. Dittmer, K., and du Vigneaud, V., Science, 100, 129-31 (1944).

76 THE BIOCHEMISTRY OF B VITAMINS

150. Pilgrim, F. J., et al, Science, 102, 35-6 (1945).

151. du Vigneaud, V., Chem. Eng. News, 23, 623-5 (1945).

152. Wright, L. D., "Biological Symposia," Vol. 12, Jaques Cattell Press, Lancaster,

Pa., 1947, p. 305.

153. Oppel, T. W., Am. J. Med. Sci., 204, 856-75 (1942).

154. Burk, D., and Winzler, R. J., Science, 97, 57-60 (1943).

155. Williams, R. J., Eakin, R. E., and Snell, E. E., /. Am. Chem. Soc, 62, 1204-7

(1940).

156. Hofmann, K, and Winnick, T., J. Biol. Chem., 160, 449-53 (1945).

157. Shull, G. M., Hutchings, B. L., and Peterson, W. H., J. Biol. Chem., 142, 913-20

(1942).

158. Shull, G. M., and Peterson, W. H., J. Biol. Chem., 151, 201-2 (1943).

159. Landy, M., and Dicken, D. M., J. Lab. Clin. Med., 27, 1086-92 (1942).

160. Tomlinson, F. F., and Peterson, W. H., Arch. Biochem., 5, 221-31 (1944).

161. Wright, L. D., and Skeggs, H. R., Proc. Soc. Exptl Biol. Med., 56, 95-8 (1944).

162. Lampen, J. O., Bahler, G. P., and Peterson, W. H., J. Nutrition, 23, 11-21 (1942).

163. Hodson, A. Z., J. Biol. Chem., 157, 383-5 (1945).

164. Gyorgy, P., J. Biol. Chem., 131, 733-44 (1939).

165. Ansbacher, S., and Landy, M., Proc. Soc. Exptl. Biol. Med., 48, 3-5 (1941).

166. Trager, W., Proc. Soc. Exptl. Biol. Med., 64, 129-34 (1947).

167. Axelrod, A. E., Mitz, M., and Hofmann, K., J. Biol. Chem., 175, 265-74 (1948).

168. Piatt, B. S., and Glock, G. E., Biochem. J., 37, 709-12 (1943).

169. King, Anne, Master's Thesis, Oregon State College, 1938.

170. Winter, L. B., Biochem. J., 34, 249-50 (1940).

171. Woolley, D. W., "Biological Symposia," Vol. 12, Jaques Cattell Press, Lancas-

ter, Pa., 1947, pp. 279-89.

172. Eastcott, E. V., J. Phys. Chem., 32, 1094-11 (1928).

173. Woolley, D. W., J. Biol. Chem., 140, 453-9 (1941).

174. Williams, R. J., et al, Univ. Texas Pub. 4137, 27-30 (1941); see also references

89-94.

175. McCance, R. A., and Widdowson, E. M., Nature, 153, 650 (1944).

176. Woolley, D. W., J. Biol. Chem., 140, 461-6 (1941).

177. Williams, R. J., Schlenk, F., and Eppright, M. A., J. Am. Chem. Soc, 66, 896-8

(1944).

178. Beadle, G. W., J. Biol. Chem., 156, 683-9 (1944).

179. Woolley, D. W., J. Biol. Chem., 139, 29-34 (1941).

180. Best, C. H., and Lucas, C. C, "Vitamins and Hormones," Vol. I, Academic

Press, Inc., New York, N. Y., 1943, pp. 9-16.

181. Beattie, F. J. R., Biochem. J., 30, 1554-9 (1936).

182. Jacobi, H. P., Baumann, C. A., and Meek, W. J., /. Biol. Chem., 138, 571-82

(1941).

183. Engel, R. W., /. Biol. Chem., 144, 701-10 (1942).

184. Marenzi, A. D., and Cardini, C. E., J. Biol. Chem., 147, 363-70 (1943).

185. Entenman, C, Taurog, A., and Chaikoff, I. L., J. Biol. Chem., 155, 13-8 (1944).

186. Glick, D., J. Biol. Chem., 156, 643-51 (1945).

187. Winzler, R. J., and Meserve, E. R., J. Biol. Chem., 159, 395-7 (1945).

188. Handler, P., "Biological Symposia," Vol. 12, Jaques Cattell Press, Lancaster,

Pa., 1947, pp. 361-72.

189. Engel, R. W., J. Nutrition, 25, 441-6 (1943).

190. Rhian, M., Evans, R. J., and St. John, J. L., J. Nutrition, 25, 1-5 (1943).

191. Jukes, T. H, and Dornbush, A. C, Proc. Soc. Exptl. Biol. Med., 58, 142-3 (1945).

192. Horowitz, N. H., and Beadle, G. W., J. Biol. Chem., 150, 325-33 (1943).

193. Thompson, R. C, Isbell, E. R., and Mitchell, H. K., J. Biol. Chem., 148, 281-7

(1943).

194. Horowitz, N. H., Bonner, D., and Houlahan, M. B., J. Biol. Chem., 159, 145-51

(1945).

ASSAY METHODS 77

195. Faltis, F., and Holzinger, L, Bcr., 72B, 1443-50 (1939).

196. Jukes, T. H., and Oleson, J. J., J. Biol. Chem., 157, 419-20 (1945).

197. Lueeke, R. W., and Pearson, P. B., J. Biol. Chem., 153, 259-63 (1944).

198. Lueeke, R. W., and Pearson, P. B., /. Biol. Chem., 155, 507-12 (1944).

199. Badger, E., J. Biol. Chem.., 153, 183-91 (1944).

200. Fletcher, J. D., Best, C. H., and Solandt, O. M., Biochem. J., 29, 2278-84 (1935).

201. Best, C. H., and Lucas, C. C, "Vitamins and Hormones," Vol. I, Academic

Press, Inc., New York, N. Y., 1943, p. 16.

202. Jukes, T. H., "Annual Review of Biochemistry," Vol. 16, Stanford Univ. Press,

Stanford University, Calif., 1947, pp. 193-222.

203. Hutchings, B. L., et al., J. Biol. Chem., 168, 705-10 (1947).

204. Bratton, A. C, and Marshall, E. K., Jr., J. Biol. Chem., 128, 537 (1939).

205. Snell, E. E., and Peterson, W. H., J. Bact., 39, 273-85 (1940).

206. Mitchell, H. K., Snell, E. E., and Williams, R. J., J. Am. Chem. Soc, 63, 2284

(1941).

207. Snell, E. E., Proc. Soc. Exptl Biol. Med., 55, 36-9 (1944).

208. Dolby, D. E., and Waters, J. W., Nature, 153, 139-40 (1944).

209. Krueger, K., and Peterson, W. H., J. Biol. Chem., 158, 145-56 (1945).

210. Tepley, L. J., and Elvehjem, C. A., J. Biol. Chem., 157, 303-9 (1945).

211. Snell, E. E., and Mitchell, H. K., Proc. Natl. Acad. Sci. U.S., 27, 1-7 (1941).

212. Stokstad, E. L. R., J. Biol. Chem., 139, 475-6 (1941).

213. Stokes, J. L., J. Bact., 48, 201-9 (1944).

214. Rickers, E. L., Chaiet, L., and Keresztesy, J. C, J. Am. Chem. Soc, 69, 2749-51

(1947).

215. Hutchings, B. L., et al, Science, 99, 371 (1944).

216. Stokstad, E. L. R., and Hutchings, B. L., "Biological Symposia," Vol. 12.

Jaques Cattell Press, Lancaster, Pa., 1947, pp. 339-60.

217. Day, P. L., and Totter, J. R., ibid., pp. 329-34.

218. Day, P. L., and Totter, J. R., ibid., pp. 316-24.

219. O'Dell, B. L., and Hogan, A. G., J. Biol. Chem., 149, 323-37 (1943).

220. Bird, O. D., et al, J. Biol. Chem., 159, 631-6 (1945).

221. Campbell, C. J., Brown, R. A., and Emmett, A. D., J. Biol. Chem., 152, 483-4

(1944).

222. Campbell, C. J., et al, Am. J. Phxjsiol, 144, 348-54 (1945).

223. Day, P. L., and Totter, J. R., "Biological Symposia," Vol. 12, Jaques Cattell

Press, Lancaster, Pa., 1947, pp. 327-8.

224. Eckert, H. W., J. Biol. Chem., 148, 197-204 (1943).

225. Kirch, E. R., and Bergeim, O., J. Biol. Chem., 148, 445-50 (1943).

226. Ansbacher, S., Science, 93, 164-5 (1941).

227. Briggs, G. M., et al, Proc. Soc. Exptl Biol. Med., 52, 7-10 (1943).

228. Martin, G. J., and Ansbacher, S., J. Biol Chem., 138, 441 (1941).

229. Martin, G. J., and Ansbacher, S., Proc. Soc. Exptl. Biol Med., 48, 118-20 (1941).

230. Martin, G. J., Am. J. Physiol, 136, 124-7 (1942).

231. Henderson, L. M., et al, J. Nutrition, 23, 47-58 (1942).

232. Landy, M., and Dicken, D. M., J. Biol Chem., 146, 109-114 (1942).

233. Lewis, J. C, J. Biol. Chem., 146, 441-50 (1942).

234. Thompson, R. C, Isbell, E. R., and Mitchell, H. K., J. Biol. Chem., 148, 281-7

(1943).

235. Lampen, J. O., and Peterson, W. H., J. Biol Chem., 153, 193-202 (1944).

Chapter VA

BIOGENESIS OF THE B VITAMINS

Some discussion of the biogenesis of the B vitamins in general will be
included below in the section on thiamine. Otherwise we shall treat each
member of the group separately and endeavor to present in the respective
sections the present status of knowledge with regard to where they origi-
nate in nature and in some cases the probable raw materials. Certain
details regarding the biosynthesis of the B vitamins which hinge on plant
and animal metabolism, intestinal bacterial synthesis in relation to sup-
plying the requirements of higher organisms, and upon inhibition studies
will be presented in later chapters.

Thiamine

Several positive answers can be given to the question as to where
thiamine is produced in nature. First, we may say that it is produced
by numerous bacteria, presumably all those which can grow on simple
media, and by many which require other vitamins. Definitive information
on this matter is fragmentary. Peterson and Peterson,1 in a list of 136
organisms which have "growth factor" requirements, indicated 8 which
have been found to synthesize thiamine, 12 others which do not require
thiamine (and presumably synthesize it), 36 which require or at least are
stimulated by it, and 80 for which no information is available. In another
table, "23 organisms reported to synthesize thiamine were listed (p. 299).
Of the bacteria which are stimulated by thiamine, probably a good many
actually synthesize it, as has been demonstrated with certain yeasts.2
Knight 3 has listed 10 bacteria which are capable of synthesizing thiamine,
and Najjar and Barrett4 list 18 such bacteria. The latter list, however,
includes organisms for which synthetic ability was indicated before thi-
amine and other members of the B family of vitamins were clearly dif-
ferentiated. Thompson 5 studied 5 bacteria chosen because of their diverse
characteristics, all of which, however, were capable of being grown on
relatively simple media, and found that they all produced substantial
amounts of thiamine.

The extent to which thiamine synthesis by bacteria in the soil and in
the digestive tracts of animals, particularly in the rumen of cattle, etc.,
figures in the economy of nature is difficult to assess (see Chapter II C),

BIOGENESIS OF THE B VITAMINS 79

but the available evidence indicates that the contribution is substantial.
To think of green plants as the sole substantial producers of thiamine and
other B vitamins is certainly unwarranted.

Second, we can be sure that thiamine is produced in nature by yeasts
and molds. Brewers' yeast was one of the earliest discovered rich sources
of "vitamin B," and that the source of thiamine in yeasts is not entirely
in the culture medium has been amply demonstrated.2,6 While some
strains of the yeast Saccharomyces cerevisiae respond by enhanced growth
to thiamine,7 many do not, and at least some of those which do respond
are capable of thiamine synthesis.2, 8 It is interesting that yeasts, when
furnished with thiazole and pyrimidine intermediates used in the chemical
manufacture of thiamine, may produce relatively large amounts of thi-
amine. This fact has found commercial application.9

It is presumed that thiamine synthesis takes place in many molds
because culture media for them have since Raulin's time been made up
from constituents of known composition excluding all vitamins. More
definite evidence of synthesis has been found, for example, in the case
of Aspergillus oryzae 10 and Aspergillus niger.11 Schopfer and co-workers
have made extensive studies which have revealed that while some fungi
can synthesize thiamine using simple starting materials, others require
either the thiazole or pyrimidine portions of the molecule, or both, before
synthesis and growth are accomplished.12 The vitamin requirements of
fungi have also been investigated to some extent by Williams and Honn 13
and by Robbins and Kavanagh.14

The importance of fungi as producers of thiamine and of other B vita-
mins in nature is indicated. While the number of mold organisms in the
soil is small compared with bacteria, the total mold substance present is
said to be larger. Furthermore, the close association of fungi with the roots
of many green plants is suggestive.

The synthesis of thiamine in green plants during the course of their
natural growth is undoubtedly of prime importance in connection with
producing the thiamine supply of nature. This does not mean, however,
that one can glibly affirm that green plants are the responsible agents in
this synthesis. To demonstrate conclusively the synthetic production of
thiamine by a green plant, one would have to grow the plant aseptically
from seeds rendered aseptic, and show an increase in the thiamine content
of the system. While this is probably possible, it has rarely, if ever, been
done.

In this connection it is necessary to call attention to the most unsatis-
factory state of our knowledge with respect to biochemical facts under-
lying the symbiosis which commonly exists between green plants and
mycorhizal fungi and the bacteria of the soil. Because of this lack of

80 THE BIOCHEMISTRY OF B VITAMINS

knowledge it is impossible to interpret with any certainty the action on
plant growth of animal manures and decaying organic matter — materials
which always contain substantial amounts of B vitamins.

Many experiments have been conducted to ascertain whether or not the
B vitamin content of a crop can be increased by using natural fertilizers.
In general, the effect, if any, has been small.15, 16, 17 In some experiments,
at least, larger crop yields (which means larger total vitamin yields) have
been obtained from plots treated with manure. Of course, it is unsafe to
ascribe the effect of a natural manure to its vitamin content without
further investigation. More often than not, investigations in this general
field have been directed toward practical ends and have not been designed
to answer fundamental biochemical questions.

It should be pointed out in this connection that, in higher organisms
generally, amounts of thiamine beyond an organism's needs are not pro-
duced or stored in quantity, nor are they physiologically valuable or
active. Without an ample supply, however, an organism cannot live.
Hence, the finding that plants grown under different cultural conditions
always contain about the same amount of thiamine throws more light on
the question of how thiamine functions than upon how it originates.

There are several well authenticated facts which have a bearing on the
problem of how thiamine is produced during the growth of green plants
and the extent to which microorganisms participate. (1) Some plant roots
(tomatoes) are tremendously stimulated in growth by very low concen-
trations of thiamine (0.0001 /xg per ml or less)18 such as may occur in the
soil and originate in the bacteria and molds present. (2) Mycorhizal fungi,
which may be either "endotrophic" or "exotrophic," not only play an
indispensable role in the life of such plants as orchids, but are likewise
always associated with many common flowering plants and with many
forest trees (conifers, oaks, beeches, alders, willows, poplars, etc.) . In
many of these latter the mycorhiza are essential to continued life, and
the production of specific B vitamins such as thiamine by the mycorhizal
fungi is not ruled out as an important factor in the symbiotic relationship.
(3) During the early stages of growth of a seedling, especially if the seed
is small and therefore contains little storage food, the seedling is likely
to respond to tissue extracts and vitamin supplements by increased
growth. (4) Pea seedlings grown 8 days in the dark contain one-half to
one-third as much thiamine as those grown in the light, as determined by
the Phyeomyces test.19 (5) Certain plant roots (tomato) are capable of
synthesizing the pyrimidine portion of thiamine and supplying their
thiamine needs if the thiazole portion alone is supplied.20 (6) The bacteria
found in the "rhizosphere" of plants are reported to have more growth
factor requirements than those more distant from the plant roots.21

BIOGENESIS OF THE B VITAMINS 81

It is clear that some of the above facts have a bearing on the problem
of biosynthesis of B vitamins other than thiamine.

An interpretation of the various findings is in line with the idea that
microorganisms — bacteria and molds — often play an important role in
furnishing green plants with thiamine (and probably various other B
vitamins) especially during the initial stages of growth of seedlings.
Synthesis of thiamine by green plants themselves probably can take place,
but in nature a complex symbiosis is the rule rather than the exception.
The nutritional interrelationships between the symbiotic organisms are
mostly obscure, and it would be unscientific to ascribe the effects of the
symbiosis to one organism (the green plant) merely on the basis of its
being more prominent visually.

Little is known about the precise mode of thiamine biosynthesis, beyond
the fact that the last step involves the coupling of the pyrimidine and
thiazole fragments. It has been suggested,22- 23> 24 however, that the thi-
azole fraction may be synthesized by the condensation of methionine,
acetaldehyde and ammonia, since similar condensations are well known:

NH3 CHO— CH3 N- C— CH3

>■

CH3 CH2— CH2— CH— COOH HC C— CH2— CH— COOH

\ / NH2 \ / NH2

S S

The a-amino-/?-(4-methylthiazole-5) -propionic acid so formed may then »
be converted to the thiazole fraction, and it has been demonstrated that
yeast cells are able to perform this latter step in a manner analogous to
that in which they convert most a-amino acids to alcohols:

N C— CH3 N C— CH3

II II HOH || ||

HC C— CH2— CH— COOH > HC C— CH2— CH2OH+C02 + NH3

V k V

Another mode of biosynthesis involving thioformamide and acetopropyl
alcohol has been suggested by the work cited above.

Riboflavin

The bacterial synthesis of riboflavin has been widely observed. Among
the 136 bacteria with "growth factor" requirements listed by Peterson
and Peterson,1 18 synthesize riboflavin, 16 do not require it (and pre-
sumably synthesize it) , while 45 require it for growth, and no information
is available for 57. In another table the authors list 75 bacteria which
have been found to synthesize riboflavin. Molds and fungi are also able
to produce riboflavin ; one fungus Eremothecium ashbyii produces enough
so that it or a derivative crystallizes in the vacuoles.25 The synthesis of

82 THE BIOCHEMISTRY OF B VITAMINS

riboflavin by Aspergillus niger is more effective when the culture medium
is relatively deficient in magnesium.26

The importance of bacteria and fungi as producers of riboflavin in
nature cannot seriously be doubted. The demonstration is particularly
clear in connection with the action of intestinal bacteria and of the or-
ganisms inhabiting the rumen of cattle and sheep.27 Milk is a highly im-
portant nutritional source of riboflavin, and it is clear that the feed which
the cow consumes is not the only source of the vitamin, since the output
of riboflavin in the milk may be ten times as great as the intake in the
food. The riboflavin output in the milk of cows and goats is independent
of the content of the feed, and the rumen content of riboflavin may be
100 times that of the feed. Intestinal synthesis of riboflavin has been
demonstrated in rats,28 fowls 29 and in man,30 and presumably takes place
in animals generally. In ruminants the production takes place higher in
the intestinal tract and the utilization is therefore expedited. Intestinal
synthesis in animals and in man is not sufficient, of course, to insure
against riboflavin deficiency.

The production of riboflavin during the growth of green plants is indi-
cated by the fact that leafy vegetables are generally good food sources,
but little definite information is available as to the locus of the synthesis
or whether symbiotic microorganisms are important. The discussion of
the general topic of B vitamin synthesis in the previous section on
thiamine is applicable also at this point.

Nicotinic Acid

The synthesis of nicotinic acid by bacteria has been observed to be far
less widespread than, for example, the synthesis of riboflavin. In the list
of organisms having growth factor requirements previously mentioned,1
48 bacteria are stimulated by or require nicotinic acid, 9 do not require
it (and presumably carry out its synthesis), for 3 bacteria its synthesis
has been demonstrated, and no information was available concerning 76
others. In the same review, 14 organisms which synthesize nicotinic acid
are listed in another table. Here, as in other cases, some strains of a given
species may have a requirement while other strains do not. Thompson's
study 5 showed that five diverse organisms which are capable of growing
on a relatively simple medium all carry out nicotinic acid synthesis. Very
little attention has been paid to the question of the production of nicotinic
acid by yeasts and fungi, though unquestionably those which can grow
on simple media carry out its synthesis.

While some observations have been made with respect to the stimu-
latory action of nicotinic acid or nicotinamide on plant roots 81- 32 and

BIOGENESIS OF THE B VITAMINS 83

pea embryos,33 and while it is presumed that green plants synthesize
nicotinic acid, little attention has been paid to the problem.

The relatively recent demonstrations that nicotinic acid is nutritionally
replaced by tryptophan in higher animals (p. 279) has, of course, an
important bearing on the problem of its biogenesis. While intestinal syn-
thesis of nicotinic acid in rats has been demonstrated,34 it is not known
how important such synthesis is because nicotinic acid is also reported
to be formed in rat tissues.35 Human beings also excrete more nicotinic
acid degradation products when tryptophan is fed.36 It appears certain
that the production of nicotinic acid from tryptophan in animals is im-
portant in the economy of nature, whether or not bacteria play an impor-
tant role symbiotically in the transformation.

The precise mode in which nicotinic acid is synthesized is at present
under intensive investigation, and our insight into this problem is ham-
pered more by the vast amount of conflicting information than by lack
of data. Present trends tend to emphasize the biosynthetic sequence start-
ing from tryptophan and proceeding as follows:

O
— -.— CH2— CH— COOH ^N— C— CH2— CH— COOH

.NJ NH2 " k^-NH, NH2

Tryptophan Kynurenin

—COOH r |T~ C00H

6-Hydroxy-anthranilic acid Nicotinic acid

This plan has much to support it; it is discussed in greater detail in the
sections that follow (pp. 279, 353). It is, however, well known that both
ornithine and proline are involved in niacin biosynthesis, and these facts
are completely neglected in the above scheme. Proline, ornithine and glu-
tamic acid have all been shown to be effective in increasing the production
of trigonellin by rice plants.

CH,

Trigonellin

THE BIOCHEMISTRY OF B VITAMINS

Indeed, Bovarinck 37 has shown that if glutamic acid and asparagin are
boiled in aerated water containing a trace of manganous sulfate, the solu-
tion will manifest niacin activity. Niacin synthesis by rats is stimulated
by a mixture of glycine and dZ-8-amino-n-valeric acid. It is also known
that guvacine will substitute for nicotinic acid in the nutrition of
Staphylococcus aureus and Proteus vulgaris. A consideration of these facts
has led to the following proposed biosynthetic sequence:

COOH

CH2

Ah-

-NH2
I
COOH

Glutamic acid

H2C CH2

H2C CH— COOH

« Proline

CH2 NH2

/ \ /
CH2 CH

CH2 COOH

NH2

Ornithine

CH2

/ \

CH2 CH2— coon

CH2
NH2

5-Amino-n-valeric acid

CH2

CH— COOH H2O+ glycine

-o

-COOH

NH2

Guvacine

COOH

Nicotinic acid

Pantothenic Acid

Pantothenic acid is required by, or is at least stimulative toward, a
relatively large proportion of the bacteria which have been tested. Thus,
Peterson and Peterson x list 52 bacteria having growth factor require-
ments which respond to pantothenic acid specifically, 2 which synthesize
it and 3 additional ones which do not require it. In another table they
list a total of nine bacteria which are reported to synthesize it. These
include the 5 organisms tested by Thompson,5 all of which grow on rela-
tively simple media and produce other B vitamins, and Rhizobium meli-
lote 38 which grows in the root nodules of legumes.

Despite the relatively small number of bacteria known to synthesize
pantothenic acid, there is good evidence on which to base the opinion that
bacterial synthesis is an important natural source. Its production has
been, demonstrated to take place, for example, in the rumen of sheep and

BIOGENESIS OF THE B VITAMINS

cattle.27, 39 Milk is reported to contain twice as much pantothenic acid
as the total dietary intake of the cow, but the assays on which such
reports are based are not entirely reliable due to incomplete extraction
(p. 34). Synthesis also takes place in rats, particularly in the cecum.
It has been estimated on the basis of balance studies that 10 to 60 per
cent of the requirement of the rat may be furnished in this way.35 The
amount produced is dependent upon the type of diet and the intestinal
flora which is favored. Certain strains of C. diphtheriae, like yeasts, are
able to produce pantothenic acid when ^-alanine alone is supplied, indi-
cating that they possess the metabolic machinery for synthesizing pantoic
acid but not for producing /3-alanine.40 Acetobacter suboxidans, on the
other hand, is stimulated by the pantoic acid portion of the pantothenic
acid molecule and evidently possesses the ability to synthesize /^-alanine
but not pantoic acid.41 Present evidence seems to indicate that the bio-
synthesis of pantothenic acid occurs through the direct coupling of
^-alanine and pantoic acid. A considerable amount of evidence (p. 465)
indicates that the /^-alanine is formed by the decarboxylation of aspartic
acid, while the pantoic acid has been suggested as arising from an amino
acid, pantonine (p. 289), thus:

COOH

CH2

CH— NH2

COOH
Aspartic Acid

CH2OH

CH8— C— CH,

HC— NH2

COOH
"Pantonine"

-co2

CHS

kNH2— CH2— CH2— COOH
^-alanine
CH3

CH2— C CH— COOH

OH CH, OH

Pantoic acid

CH2OH
-C— CH,
CHOH

" NH
CH2
CH2
COOH

Pantothenic
acid

Further discussion bearing on the mechanism of the biosynthesis of pan-
tothenic acid will be given in a later section (p. 464).

The production of pantothenic acid by common strains of bakers' yeast,
in the absence of ^-alanine, is certainly not important, but other yeasts
and fungi which are able to grow on simple media undoubtedly produce
the whole molecule. No worthwhile information regarding the importance
of fungi as producers of pantothenic acid in the soil or elsewhere appears
to be available.

86 THE BIOCHEMISTRY OF B VITAMINS

The relation of pantothenic acid to the growth of green plants has been
studied.38 Aseptically grown alfalfa seedlings were found to respond to
pantothenic acid by increased growth, but when the plants had developed
for several days, they were assayed and found to contain much more
pantothenic acid than could be accounted for other than by synthesis.
These experiments, carried out in the author's laboratory, were performed
at a time when minute amounts of nearly pure pantothenic acid were
available, and there is no reason to doubt the substantial biological purity
of the preparations used. The only criticism of the demonstration of
pantothenic acid synthesis by green plants was the failure to show at the
end of the experiment that molds and other bacteria were entirely absent
from the sand cultures. While there was no macroscopic evidence of mold
or bacterial growth, the resistance of some spores to autoclaving may
introduce a small uncertainty.

Pyridoxal, Pyridoxamine, Pyridoxine

Especially because of relatively recent clarification with respect to the
chemical nature of the "B6 group" of vitamins, the available information
regarding their biosynthesis is relatively unsatisfactory. Studies involving
"vitamin B6" have revealed that of the approximately 40 bacteria about
which information is available, about one-half are stimulated by or re-
quire it, and the other half either are known to synthesize it or have been
shown at least not to require it for growth. The five organisms studied by
Thompson 5 all produced it.

Just how the results of such studies would appear if all three forms
of the vitamin were taken into account is not entirely clear. It is certain,
however, that the three forms are not always interchangeable in nature,
since some organisms are unable to convert pyridoxine into the active
pyridoxal form,42 and the form which predominantly exists differs from
source to source. Pyridoxine appears to be a relatively inactive storage
form which occurs predominantly in metabolically inactive seeds (p. 36) .

Yeasts, molds and fungi which are able to grow on simple media pro-
duce at least one form of the vitamin because assays by a yeast method,
which responds to all forms, indicates the universal presence of these,
as well as the other B vitamins (p. 60). By x-ray induction a mutant
strain of Neurospora crassa was produced early in the investigations of
Beadle and Tatum,43 which is unable to grow unless pyridoxine, pyridoxa-
mine or pyridoxal is added to the medium. For this organism the three
forms are interchangeable, and the x-ray has destroyed a gene which is
essential for the building of any of the forms.

The same uncertainty exists with respect to the mode of production
of the "BG group" during the growth of green plants as exists in connec-

BIOGENESIS OF THE B VITAMINS 87

tion with the other B vitamins. Tomato roots, for example, are stimulated
in growth by pyridoxine,44 and in this respect pyridoxine shows a re-
semblance to some of the other B vitamins.

From recent work it appears that alanine is not, as formerly supposed,45
a direct precursor of vitamin B0 and that D-alanine, which is reported
to be a consistent cell constituent for several organisms, can replace B6
in culture media without giving rise to additional B6.45a

Biotin

The bacterial synthesis of biotin undoubtedly constitutes an im-
portant natural source. While biotin deficiency has often been induced
in animals it is usually by introducing egg white or avidin into the diet,
or administering sulfa drugs, or eliminating bacteria from the intestinal
tract44 that it is accomplished (see p. 428). Usually feeding a biotin-
deficient diet is itself ineffective, though not necessarily so with baby
chicks. Peterson and Peterson * list 21 bacteria which produce biotin,
among them several common intestinal bacteria. The synthesis of biotin
is not limited to the bacteria which fail to respond to it; thus Rhizobia
which are stimulated by biotin (coenzyme R) can be cultured under
conditions in which preformed biotin is excluded.46 As Knight 47 has aptly
pointed out, very limited growth of a microorganism may occur in a
biotin-deficient medium even though the organism is able to synthesize
biotin. The rate of growth may under these conditions be limited by the
rate of synthesis, in which case the addition of biotin to the culture
medium may greatly accelerate growth.

The synthesis of biotin by bacteria is attended often by a large amount
of release into the culture medium. In four organisms studied by Thomp-
son 5 the amount of biotin found in the medium averaged about 8 times
that in the bacterial cells. This release into the medium is not due to
the autolysis of dead cells as it occurs progressively in a rapidly growing
culture.

The bacterial production of biotin in the rumen of cattle has been
demonstrated,48- 49 and rats on a biotin-deficient ration excrete much
more biotin than they take in,50 as do also human beings.51 The presence
of relatively large amounts of biotin in bacterial culture media which
have accidentally been contaminated by organisms from the air bespeaks
the ubiquity of organisms capable of producing this vitamin.

Yeasts and lower fungi as well as bacteria produce biotin. While many
yeasts are stimulated by biotin they often are capable of its synthesis.2, s

Knight 52 lists 12 lower fungi, including Aspergilli and Penicillia, which
do not require biotin, and it is relatively safe to conclude that they
synthesize it. One notable genus of molds that requires biotin, however,

88 THE BIOCHEMISTRY OF B VITAMINS

is the Neurospora, which requires this single vitamin and no other. The
wild strains require biotin; they lack the enzyme (s) necessary for its
synthesis. The mutant strains in addition lack other specific enzymes due
to the destruction of specific individual genes.

Of the work relating biotin to the activities of green plants, one of the
most significant bits is the demonstration that it acts as a growth sub-
stance in stimulating root production on etiolated cuttings of pea roots.53
Stimulative effects of this sort suggest that biotin may not be produced
by green plants, or at least that the synthesis takes place in leaves rather
than roots, and that the roots may depend for at least part of their supply
on soil microorganisms. The stimulative effect of biotin on Rhizobia sug-
gests that the green plants may furnish biotin (synthesis taking place
probably in leaves) to the microorganism as an important factor in the
symbiotic relationship. The ability of some of these organisms to synthe-
size biotin at a slow rate has already been noted. No specific information
appears to be available regarding the relationship of biotin to mycorhizal
growth. In general, we may say that there appears to be no other B
vitamin for which production by microorganisms is as important as it
is in the case of biotin.

Eakin and Eakin 54 have shown that pimelic acid stimulates the
synthesis of biotin by Aspergillus niger, and that this stimulation is
further enhanced by the presence of cysteine or cystine. Pimelic acid
and biotin have been found to be interchangeable as growth stimulants
for some organisms, so that it appears likely that pimelic acid is a pre-
cursor of biotin. There is some evidence to suggest that the synthesis
proceeds through desthiobiotin as a precursor (p. 468).

Folic Acid, Inositol, Choline, p-Aminobenzoic Acid, "Vitamin Bi2"

With respect to the biogenesis of other members of the B vitamin
family, hardly enough information is available to warrant more than a
very brief discussion.

Folic acid is required by a considerable number of microorganisms,
but on the basis of available information it is produced by all those
bacteria which can grow on simple chemically defined media.5 Its produc-
tion in the intestinal tracts of rats has been studied,34 and numerous
demonstrations of deficiency have involved the prevention of intestinal
synthesis by sulfa drugs. Quantitative information in this field is rela-
tively unsatisfactory because of the difficulties of complete release of the
vitamin from its combined forms for assay (p. 40). The fact that deep
green leaves are an unusually rich source of folic acid,55 as well as other
facts regarding its distribution, strongly suggest that this vitamin is
formed in green plants.

BIOGENESIS OF THE B VITAMINS 89

Few studies have dealt with bacterial growth in relation to inositol
synthesis. In the previously mentioned study of Thompson,5 its produc-
tion by five diverse bacteria was demonstrated. One study indicated that
it is not produced to a substantial degree by intestinal organisms in
rats.34 The relative abundance of inositol in plant materials 56> 57 sug-
gests that it is probably synthesized by plants. It is stimulatory to certain
fungi, but in view of the ability of many of these organisms to grow on
simple media, it is presumably synthesized by them.

Choline is probably widely synthesized in nature. Few bacteria have
been found to require it,58 and wild strains of Neurospora evidently
synthesize it.59 In animals, as in Type III Pneumococci,58 methionine and
other compounds containing available methyl groups serve as precursors.
Ethanolamine is probably important as a precursor in the biosynthesis
of choline (p. 353). Choline biosynthesis has been extensively studied
and it is now felt that the exact sequence involved is well known. In brief,
it may be indicated thus:

CH2OH

-CO2
CH2NH2

COOH

Serine

CH2OH

-> CH2

NH2

Ethanolamine

CH2OH

+CH3 I

► CH2

NH— CH3

CH2OH
CH2

■> N— CH3

CH3

+CH3

CH2OH

CH3

CH3 CH3
Choline ion

Certain aspects of this sequence of reactions are considered in greater
detail in a later section (p. 353).

p-Aminobenzoic acid, like choline, is probably produced widely in
nature. Although a considerable number of bacteria are stimulated by
its presence in the culture medium, Peterson and Peterson 1 list 13
bacteria which have been found to synthesize it. Wild strains of Neuro-
spora synthesize this substance as do yeasts. Presumably green plants
do as it enters into the make-up of folic acid, but no direct evidence on
this point has been found.

90 THE BIOCHEMISTRY OF B VITAMINS

Bibliography

1. Peterson, W. H., and Peterson, M. S., Bad. Rev., 9, 49-109 (1945).

2. Williams, R. J., Eakin, R. E., and Snell, E. E., J. Am. Chem. Soc, 62, 1204-7

(1940).

3. Knight, B. C. J. G., "Vitamins and Hormones," Vol. Ill, Academic Press, Inc.,

New York, N. Y., 1945, pp. 105-228b.

4. Najjar, V. A., and Barrett, R., "Vitamins and Hormones," Vol. Ill, Academic

Press, Inc., New York, N. Y., 1945, pp. 27-8.

5. Thompson, R. C, Univ. Texas Pub., 4237, 87-96 (1942).

6. Livshits, M. I., Proc. Sci. hist. Vitamin Research, U.S.S.R., 3, No. 1, 84-8 (1941) ;

CA., 36, 3219 (1942).

7. Williams, R. J., and Roehm, R. R., J. Biol. Chem., 87, 581-90 (1930).

8. Leonian, L. H., and Lilly, V. G., Science, 95, 658 (1942).

9. Standard Brands, Inc., Brit. Patent 532,013, Jan. 15, 1941.

10. Scheunert, A., and Schieblich, M., Biochem. Z., 286, 66-71 (1936).

11. Fawns, H. T., and Jung, A., Biochem. J., 27, 918-33 (1933).

12. Schopfer, W. H., "Plants and Vitamins," Chronica Botanica Co., Waltham,

Mass., 1943, pp. 101-29.

13. Williams, R. J., and Honn, J. M., Plant Physiol, 4, 629-41 (1932).

14. Robbins, W. J., and Kavanagh, V., Botan. Rev., 8, 411-71 (1942).

15. Scheunert, A., Erndhrung, 3, 67-9 (1938).

16. Rowlands, M. J., and Wilkerson, B., Biochem. J., 24, 199-204 (1930).

17. Harris, L. J., J. Agri. Sci., 24, 410-5 (1934).

18. Robbins, W. J., and Bartley, M. A., Science, 85, 246 (1937).

19. Bonner, J., and Green, J., Botan. Gaz., 100, 226 (1934).

20. Robbins, W. J., and Bartley, M. A., Proc. Natl. Acad. Sci. U.S., 23, 358-8 (1937).

21. West, P. M, and Lochhead, A. G, Can. J. Research, 18, C, 129-35 (1940).

22. Harington, C. R., and Moggridge, R. C. G., Biochem. J., 34, 685-9 (1940).

23. Harington, C. R., and Moggridge, R. C. G., J. Chem. Soc, (1939) 443-6.

24. Bonner, J., and Buchman, E. R., Proc. Natl. Acad. Sci. U.S., 24, 431-8 (1938).

25. Guillermond, A., Rev. de My col, 1, 115 (1937).

26. Lavollay, J., and Laborey, F., Compt. rend., 206, 1055-6 (1938).

27. McElroy, L. W., and Goss, H., J. Nutrition, 20, 527-40 (1940).

28. Griffith, W. H., J. Nutrition, 10, 667-74 (1935).

29. Lamoureux, W. F., and Schumacher, R. S., Poultry Sci., 19, 418 (1940).

30. Najjar, V. A., et al, J. Am. Med. Assoc, 126, 357 (1944).

31. Bonner, J., Am. J. Botany, 27, 692 (1940).

32. White, P. R., Am. J. Botany, 27, 811 (1940).

33. Bonner, J., Plant Physiol, 13, 865 (1938).

34. Mitchell, H. K., and Isbell, E. R., Univ. Texas Pub., 4237, 125-34 (1942).

35. Dann, W. J., /. Biol. Chem., 141, 803 (1941).

36. Sarett, H. P., and Goldsmith, G. A., J. Biol. Chem., 167, 293 (1947).

37. Bovarnick, M. R., J. Biol. Chem., 151, 467-75 (1943).

38. McBurney, C. H., Bollen, W. B., and Williams, R. J., Proc Natl. Acad. Sci. U.S.,

21, 301-4 (1935) ; also unpublished observations by the same authors.

39. McElroy, L. W., and Goss, H., J. Biol Chem., 130, 437-8 (1939).

40. Mueller, J. H., and Klotz, A. W., J. Am. Chem. Soc, 60, 3086-7 (1938).

41. Underkofler, L. A., Bantz, A. C, and Peterson, W. H., J. Bad., 45, 183-90 (1943).

42. Snell, E. E., and Rannefeld, A. N, ./. Biol. Chem., 157, 475-89 (1945).

43. Beadle, G. W., and Tatum, E. L., Proc Natl Acad. Sci. U.S., 27, 499-506 (1941).

44. Robbins, W. J., and Schmidt, M. B., Proc Natl. Acad. Sci. U.S., 25, 1-3 (1939).

45. Snell, E. E., and Guirard, B. M., Proc. Natl. Acad. Sci. U.S., 29, 66-73 (1943).
45a. Holden, J. T., and Snell, E. E., /. Biol. Chem., 178, 799-809 (1949).

46. Wilson, J. B., and Wilson, P. W., /. Bad., 43, 329 (1942).

BIOGENESIS OF THE B VITAMINS

Knight, B. C. J. G., "Vitamins and Hormones," Vol. Ill, Academic Press, Inc.,

New York, N. Y., 1945, p. 162.
McElroy, L. W., and Jukes, T. H., Proc. Soc. Exptl. Biol. Med., 45, 296-7 (1940).
Wegner, M. I., et al., Proc. Soc. Exptl. Biol. Med., 45, 769-71 (1940)
Nielsen, E., et al, J. Nutrition, 24, 523-33 (1942).
Oppel, T. W., J. Clin. Invest., 21, 630 (1942).
Knight, B. C. J. G., "Vitamins and Hormones," Vol. HI, Academic Press, Inc.,

New York, N. Y., 1945, p. 167.
Went, F. W., and Thimann, K. V., "Phytohormones," Macmillan Co., New

York, N. Y., 1937, 294 pp.
Eakin, R. E., and Eakin, E. A., Science, 96, 187-8 (1942).
Olson, O. E., Burris, R. H., and Elvehjem, C. A., J. Am. Dietet. Assoc, 23, 200-3

(1947).
Woods, A. M., et al, Univ. Texas Pub., 4237, 84-6 (1942).
Cheldelin, V. H., and Williams, R. J., Univ. Texas Pub., 4237, 105-25 (1942).
Badger, E, J. Biol. Chem., 153, 183-91 (1944).

Section B

THE CATALYTIC FUNCTIONS OF THE
B VITAMINS

Robert E. Eakin

Chapter IB

BIOCHEMICAL REACTIONS AND THEIR CATALYSTS

When a mammal or some lower form of life is deprived of an adequate
amount of one of the B vitamins it requires, serious changes in the
metabolism of the organism occur.* The deviations from the normal
processes induced by the deficiency are reflected in a variety of physio-
logical effects, many of which can be readily detected. In general, how-
ever, the different changes which are observed in even a simple deficiency
of a single vitamin are so diverse in character that it is impossible to
relate the symptoms physiologically or to establish any connection be-
tween the chemical structure of the vitamin and its physiological effects.
An excellent illustration of the complex nature of vitamin function is the
variation in the ways by which a deficiency manifests itself, not only in
different types of life but even among members of the same species (see
Chapter VI C) . Before any plausible explanations for these diverse physi-
ological and clinical effects of the vitamins can be deduced, it is necessary
to explore much deeper and uncover the specific chemical or physical
reactions in which they are participating.

The elucidation of the specific manner in which a vitamin performs its
duties in a biological system usually does not arouse the popular interest
that attends the discovery of a vitamin or the accomplishment of its
synthesis. Nevertheless, for the medical sciences, understanding exactly
how these compounds work, and why, is just as important as knowing
what they are. Until the specific chemical functions of a vitamin are
determined, knowledge about the vitamin is indeed incomplete.

The purpose of this section, then, will be to review those investigations
which have been reported which we feel to be most pertinent in answering
the question, "Into what specific reactions does each B vitamin enter?"
and to correlate these chemical reactions with the observed effects of the
vitamins upon the metabolism of cells and tissues.

During the early investigations on essential dietary factors, it was only
natural that relatively little experimental work was done on the bio-

* A number of topics which will be referred to from a general viewpoint in this
chapter are discussed in more detail in other parts of the book where they are
documented with references to the original publications. The citation of references
in this chapter will be limited for the most part to material not elaborated upon
more thoroughly elsewhere.

96 THE BIOCHEMISTRY OF B VITAMINS

chemical mechanisms in which they functioned. The goal of the pioneers
in the field of vitamin research was to determine the chemical structure
of the vitamins so that their syntheses could be effected and preparations
made available for the treatment of nutritional deficiency diseases. Also,
it was impossible to attack successfully the problem of the mechanisms
of vitamin action prior to the time that the multiple nature of vitamin B
had been clarified, and before potent concentrates of the individual vita-
mins free from other substances having biological activity became
generally available.

Many early investigators of vitamins, particularly those having bio-
chemical training and viewpoints, however, recognized that the vitamins
must act as "catalysts," since only minute amounts were needed to pro-
duce such profound changes in biological systems; but the nature of the
"catalytic activity," of course, at that time could not be explained.

Fortunately, the study of enzymes and enzyme activity was proceeding
simultaneously with the development of the nutritional sciences. It is
interesting to note that 1926 was an epochal year in both fields, for it
marked the isolation for the first time of a vitamin in crystalline form
(thiamine from rice bran)1 and the first isolation of an enzyme (crystal-
line urease from jack bean meal).2 These two accomplishments offered
irrefutable evidence that the activity of both vitamins and enzymes
could, at least in some cases, be attributed to specific chemical compounds.
Previous to this date some scientists had speculated on the relationship
between enzymes and vitamins, and had pointed out that one of the most
likely means by which the vitamins could exert their "catalytic activity"
was to participate in some way in enzymatic reactions. It is probably
difficult for the younger scientists of today, who have always thought of
the vitamins and enzymes as specific chemical entities and who in their
training have always associated vitamin activity with enzyme action, to
realize that twenty-five years ago explaining a vitamin function in terms
of enzymes was only a conjecture, and that even fifteen years ago the
hypothesis that a B vitamin functioned directly in a biochemical reaction
had yet to be demonstrated.

The actual proof that a B vitamin is an integral part of an enzymatic
reaction was not made until 1935, when a component of the "yellow
enzyme" was identified as riboflavin (a substance whose status as a
vitamin at that time was debatable) .3 Two years later an essential part
of the enzyme system required for the biological decarboxylation of
pyruvic acid was shown to be a derivative of thiamine.4 By 1935 nicotina-
mide had been identified as a part of molecules essential for reac-
tions taking place when glucose is utilized by erythrocytes and yeast,5

BIOCHEMICAL REACTIONS AND THEIR CATALYSTS 97

but two years elapsed before the vitamin activity of nicotinamide
(or the corresponding acid) was established.6 It is interesting to note
that, whereas in 1937 a two-year interval had separated the identification
of an enzyme component and its trial in the treatment of the most prev-
alent nutritional disease of this country, today the time that would
elapse between discovery of a new organic molecule essential for some
important enzymatic reaction and its trial in many diseases would be at
most a matter of weeks — an excellent illustration of the change that has
taken place in the last decade in the general acceptance of the enzyme-
vitamin relationship.

A related concept which was early appreciated by some scientists
working with lower types of life, but which was long neglected by most
workers in the field of mammalian nutrition and physiology, was that
of the universal occurrence of the B vitamins and their importance to
all forms of life. The significance of this fact and its effect upon vitamin
research was pointed out in the previous section. Brilliant investigations
early in this century demonstrated that in their metabolism of carbo-
hydrates both mammalian tissues and unicellular organisms (yeast)
utilize almost identical series of enzymatic reactions. Had the intimate
relationship of nutritional factors and enzyme systems been more fully
appreciated, there would have been earlier recognition by all biochemists
of the similarity of the nutritional requirements of lower forms of life
and those of mammals.

It was in the period immediately following the establishment of the
vitamin-enzyme relationship of riboflavin, thiamine, and nicotinic acid
that the complex tangle of the other B vitamins was unsnarled; the
biological activities of countless factors which had been reported during
the previous twenty years were resolved into pantothenic acid, pyridoxine,
biotin, p-aminobenzoic acid, inositol, and folic acid, or combinations and
derivatives of these substances. Since the three members of the complex
originally recognized were known to function as parts of enzyme systems,
considerable effort in a number of laboratories was directed toward
demonstrating that substances more recently established as B vitamins
were also involved in some type of enzyme reactions as yet uncharacter-
ized, or that these newer vitamins were present in significant amounts in
the purified preparations of known enzymes. Although a number of
suggestive leads were obtained, it was impossible as late as 1943 to
ascribe any definite enzymatic functions to any of these "newer" B
vitamins. The failure to pin these substances down to specific enzymatic
functions led to some speculation that it might be found that not every
B vitamin would exhibit its "catalytic activity" by being an integral part

98 THE BIOCHEMISTRY OF B VITAMINS

of an enzyme system.7 Some weight was given to such interpretations,
since at that time it appeared that there might be almost no end to the
list of growth-promoting substances that would eventually be found;
also, none of the numerous plant and animal hormones had been impli-
cated in specific enzyme reactions.

However, in the five-year period 1943 to 1948, pantothenic acid,
pyridoxal and inositol were found to be parts of definite enzyme systems
involved in fat, protein, and carbohydrate metabolism; and folic acid,
p-aminobenzoic acid, biotin, and a new member of the group which was
isolated during this period, vitamin Bi2, were each shown to function in
one or more fundamental enzymatic reactions which, at the time of this
writing, are in the process of being more fully characterized. During this
same interval it also became increasingly apparent that the number of
unknown B vitamins might not be as great as previously assumed, and
that many of the remaining uncharacterized factors of general biological
significance which had been reported would be found to be mixtures or
derivatives of compounds already known.

Later in this chapter (p. 104) an outline will be given of the funda-
mental types of enzymatic reactions which have been found to constitute
the chemical routes over which the basic processes in carbohydrate,
lipide, and nitrogen metabolism must proceed. A number of these enzyme
systems contain as an indispensable part one of the B vitamins. Of greater
interest is the fact that, except for inositol, each one of the B vitamins is
always required in at least one group of these essential reactions, thus
establishing a definite association between every typical B vitamin and
the processes which form the foundation upon which life is built. If any
one of these types of reactions is lacking, a series of gaps results which
cannot be effectively bridged or by-passed, and the procession of re-
actions necessary for the maintenance of cell activity must cease.* It is
for this reason that these vitamins occur universally and are a pre-
requisite for life.

From the standpoint of organic evolution the thesis that there exist
throughout the entire plant and animal kingdoms a certain number of
essential and fundamental types of enzymatic processes common to all
organisms seems not only reasonable but almost necessary. An apprecia-
tion of this situation has been of invaluable help to those investigators
interested in studying the details of metabolism, for it means that they
can choose as their biological tool any organism or species with which

* See pp. 174, 187, and 202 for a discussion concerning possible exceptions — the
interesting cases where the reactions catalyzed by biotin, pyridoxal, and folic acid
may be by-passed in certain bacteria.

BIOCHEMICAL REACTIONS AND THEIR CATALYSTS 99

they can conveniently work with reasonable assurance that the informa-
tion so obtained will be of general biochemical interest and will be
applicable, with limitations, to other forms of life. As a matter of fact,
the outline of the basic reactions mentioned above was assembled from
information obtained from studying organisms at the extremes on the
scale of biological development.

Now that it is definite that each of the B vitamins can be associated
with an enzymatic reaction, a pertinent question that still must be con-
sidered is: What clear-cut evidence is there that any of the B-vitamins
have fundamental roles which cannot be associated with the enzyme
systems in which they are known to participate? Most of the clinical
symptoms of deficiencies, as well as a number of miscellaneous observa-
tions that have been reported, have not as yet been correlated with the
enzymatic reactions in which the vitamins function; but there is no
clearly defined positive evidence that any of the typical B vitamins has
any indispensable roles other than those associated with the specific type
reactions for which it is required. In view of this it seems reasonable to
offer as a general hypothesis that the only function of the B vitamins
per se is to participate directly in certain specific enzymatic reactions.

In addition to these basic reactions essential for every cell, there are
numerous "specialized" biochemical reactions which are not observed in
all forms of life, but which are characteristic of particular types of
organisms or tissues. These reactions are not necessarily needed by the
individual cells themselves, but they are essential if the complex organiza-
tion of life is to be maintained. Examples of such reactions are photo-
synthesis in green plants, nitrogen fixation in certain types of bacteria,
hormone production by glandular tissue, the reactions involved in trans-
mission of impulses in nerve tissue, etc. Do the B vitamins function in
such reactions? Information that has been obtained on this point seems
scant, especially when one considers the importance of many of these
phenomena and the amount of investigation they have received. It would
seem only natural that in the evolutionary development of the "special-
ized functions" the organism would utilize, wherever possible, the catalysts
already present and functioning in the basic metabolism of the cell. The
little information available on these functions of the vitamins is sum-
marized in a later chapter.

The following sections of this chapter will be devoted to a general
discussion of the fundamental enzyme reactions.

Enzymatic Reactions

Any discussion attempting to explain in detail the mechanisms by which
B vitamins catalyze chemical reactions would necessarily become a

100 THE BIOCHEMISTRY OF B VITAMINS

treatise on enzyme chemistry and hence cannot be undertaken here. How-
ever, before going into the detailed account of the specific reactions in
which the vitamins are involved, it seems advantageous to discuss a few
topics about the general processes and mechanisms of enzyme reactions
which have particular bearing upon points to be treated individually in
the chapters which follow.

A biochemical reaction, like any chemical reaction, involves changes
in the chemical constitution of one or more molecules with an accompany-
ing transformation of energy. These two changes — the material and the
energy — take place simultaneously and cannot be divorced from each
other. There has been an unwarranted tendency on the part of some
workers in the biological sciences to separate biochemical reactions
(including those mediated by derivatives of B vitamins) into energy-
producing reactions and reactions utilized for the synthesis of cellular
components. Such an idea is conveyed in the categorical statements often
made to the effect that "carbohydrates and fats are used for the storage
and production of energy while proteins are used for building cell struc-
ture." In the chemical reactions by which energy is obtained from carbo-
hydrates and lipides, cells are at the same time forming, in addition to
"waste" or excretory products, a large number of compounds which are
either incorporated directly into the structure of cells or are converted
into other compounds which are essential units of cellular constituents.
On the other hand, the energy liberated by the chemical changes occurring
during protein metabolism is utilized in a manner identical to that energy
made available during carbohydrate and lipide metabolism.

Before an enzyme reaction can be considered as well characterized, at
least three things must be known:

(a) the net energy transformations taking place,

(b) the exact chemical changes occurring (i.e., the specific reactants

and products) , and

Energy Transformations in Biological Systems. The role of the bi-
ological catalysts involved in producing energy transformations can be
better appreciated after comparing the conditions involved in cellular
reactions with those employed to effect comparable changes without the
aid of enzymes. In nonbiological systems, the production of utilizable
energy by the oxidation or degradation of organic compounds (wood,
petroleum, alcohol, etc.) almost always involves some kind of combustive
process wherein the chemical energy of the organic compounds is first
converted into heat with an attending production of high temperatures.

BIOCHEMICAL REACTIONS AND THEIR CATALYSTS 101

This thermal energy is then used for work or chemical syntheses by
transforming it into mechanical, electrical, or chemical energy. This
transformation of heat, a "degraded" form of energy, is always inefficient
from the standpoint of thermodynamics, and involves other wasteful
losses due to practical difficulties. In cells, where only small changes in
temperature can be tolerated, heat cannot generally be used to transfer
the energy derived from one metabolic reaction to another process in
which it will be utilized for locomotion, establishment of electrical
potentials, absorption against osmotic pressure, or chemical synthesis.
Energy-producing processes must be carried out in a carefully controlled,
stepwise manner to prevent appreciable rises in temperature and to per-
mit the liberated energy to be stored and utilized as needed.

It has been found that a relatively simple device, though somewhat
unique from the standpoint of classical physics and chemistry, is em-
ployed in most if not all living systems — that of conserving the chemical
energy arising from the degradation and oxidation of organic compounds
(and perhaps from other sources available to some organisms) by con-
verting it into another type of chemical energy, popularly termed "high-
energy phosphate bonds." This conversion may be more accurately
described by saying that the energy-producing metabolic reactions result
in the formation of acid anhydrides of phosphoric acid, compounds which
are most versatile in their reactions and from which the chemical energy
inherent in the acid anhydride linkages can be readily utilized by
biological systems.

Although the B vitamins themselves are not the substances which act
as transporting agents for these high-energy phosphate units, they are
usually involved in the reactions by which these agents are formed and
often in subsequent processes wherein they are utilized. Quantitative
values for the amount of free energy (the energy available for useful
work) liberated or absorbed during a reaction (AF) enable one to predict
which metabolic reactions can be used to create the high-energy units
and which processes will necessarily require expenditure of some of the
cell's reserve of these energy units. This same thermodynamic informa-
tion also enables one to calculate the relative concentrations of the
reactants and products of a reaction at equilibrium. From this it is
possible to determine the direction in which an enzymatic reaction will
proceed under any given set of conditions.

The problem of the "reversibility of a reaction" involves the question
whether a reaction is theoretically capable of proceeding in either direc-
tion. Reversibility implies that a reaction and the reverse transformation
are taking place simultaneously, although the conditions may be such as

102 THE BIOCHEMISTRY OF B VITAMINS

to favor a net change, which is the forward reaction. As a corollary, there
should be some critical set of conditions for a reaction in which there is no
net change, in which case the system will be at equilibrium.

In the past the reversibility of many enzymatic reactions has been
frequently questioned, since attempts to demonstrate enzymatic syn-
thesis, as opposed to hydrolysis and other forms of degradation, in a
direct manner have in some cases proved wholly unsuccessful. Even if
a catalyst is present in excess it may be impossible to achieve the con-
centrations of products required to produce a measurable resynthesis of
the original substances. Today, with isotopes available, it is easy to
demonstrate that in uncomplicated enzymatic reactions the products are
in dynamic equilibrium with the reactants, even though it is sometimes
impossible to achieve the conditions necessary to completely effect the
reverse reaction. This can best be shown by labelling one of the products
with an isotopic atom, adding it to an enzyme system during the course
of a reaction, and stopping the reaction while there is still some of the
reactant left. If the reactant contains some of the isotopic element added
in the product, then it is obvious that the "backward reaction" has been
taking place.

Consequently, today the question should not be, "Is this enzyme re-
action reversible?" but, "Do the conditions required for reversing this
reaction occur or can they be achieved?" To answer the latter question
it is necessary to know the relative concentration of reactants and
products when they are at equilibrium. At equilibrium a reaction can be
forced to go in either direction by making only slight changes in the
concentration of one of the participating substances. In cells a number
of reactions mediated by enzyme systems, including many of these con-
taining B vitamins, are at equilibrium most of the time. The direction in
which these reactions proceed may be constantly alternating because of
the slight changes produced by other reactions in cells. In the reaction,
glycogen + H3P04 +± glucose- 1 -phosphate, only slight changes in the
intracellular concentration of inorganic phosphate are needed to stop
the process in which glycogen is utilized and to initiate its synthesis. This
is an example of one of the important methods by which metabolic proc-
esses are regulated. It is also possible to find reactions at the other
extremes — hydrolytic reactions and some decarboxylations are good ex-
amples. Here, even though the catalyst is present, the ratio of the concen-
trations of products to reactants at equilibrium is so large that it is
impossible to achieve the concentration of the products needed to reverse
the reaction effectively.

A knowledge of relative concentrations at equilibrium is not only of
value in understanding or predicting the mechanisms and chemical routes

BIOCHEMICAL REACTIONS AND THEIR CATALYSTS 103

of metabolic processes, but it also enables the investigator who is studying
an isolated system to have some idea of the concentration conditions that
he must use to demonstrate the activity of an enzyme preparation.

Exact statements of the energy relationships and equilibrium con-
ditions involve the use of thermodynamic concepts and equations. How-
ever, without these it can be stated as an approximation that when the
amount of free energy liberated by a reaction is comparatively small,
then the equilibrium concentrations of the reactants and products can
usually be obtained, such that it is possible for an organism to make use
of either the forward or the reverse reaction. On the other hand, if an
enzymatic reaction liberates a large amount of energy, it would probably
be difficult to establish the concentrations needed to reverse the reaction.
If this reverse process is needed for synthetic purposes, some indirect
route in which high-energy units are utilized will have to be employed
(pp. 218 and 235).

Types of Biochemical Reactions. Next to be considered are the funda-
mental chemical reactions required for the synthesis and interconversion
of constituents essential for cell structure and function. These processes
involve such diverse reactions as the formation and hydrolysis of peptide,
ester, and glucosidic linkages; the oxidation of alcohols, aldehydes, and
amines; the reduction of acids and aldehydes; the hydrogenation and
dehydrogenation of hydrocarbon chains; the formation and cleavage of
carbon-to-carbon bonds; and the synthesis and degradation of hetero-
cyclic compounds. The demands presented by the variety and complexity
of these reactions upon the synthetic abilities of the cell are equalled by
the drastic limitations put upon the conditions under which the reactions
must be carried out. All transformations must take place within a very
narrow temperature range, with extremely low concentrations of reactants,
at an approximately neutral pH and a temperate redox potential. The
powerful but caustic agents which are indispensable to the synthetic
organic chemist must be wholly avoided. Even the simplest types of
biochemical reactions would be impossible under such conditions, if it
were not for the remarkable catalytic abilities of the enzymes.

It is interesting to speculate on the number of enzyme systems re-
quired to account adequately for all these reactions which must be taking
place within cells. The number must indeed be large. If, however, one
tabulates according to type the numerous enzymatic reactions which
have been demonstrated and postulated for the normal metabolism of
carbohydrates and fats, he will find that the reactions can be classified
chemically into a surprisingly small number of groups. By suitable com-
binations of these relatively few types of reactions it is possible to carry

104 THE BIOCHEMISTRY OF B VITAMINS

a carbohydrate or fat through the series of steps by which it is believed
to be normally utilized. These same types of reactions will also ade-
quately account for the synthesis of polysaccharides and fats from the
intermediary compounds arising during metabolism (Chapter IIIB). Un-
fortunately, it is not possible to make this statement general for all
carbohydrates and lipides, since at present one can do nothing more
than speculate about the biosynthesis of sterols.

There still remain to be identified a number of the chemical pathways
by which the individual amino acids, purines and pyrimidines are con-
nected with each other and with the intermediates which they have in
common with carbohydrates and lipides. At the present time this problem
is receiving a great deal of attention and interest, and pertinent findings
appear in almost every current issue of biochemical journals. The
mechanism by which these component units are elaborated into protein
molecules is still an enigma and represents the biggest gap in our knowl-
edge of fundamental biochemical processes.

Below is presented an outline of the general types of enzymatic reactions
necessary to account for the basic processes by which carbohydrates are
known to be utilized and by which fats are presumably synthesized and
degraded. Also included are the fundamental reactions associated with
nitrogen metabolism which appear to be reasonably well characterized
and of general biological importance. A discussion of the specific reactions
and their role in general metabolic processes can be found in the following
chapters.

In this outline, the vitamin or group of vitamins associated with each
particular type of reactions has been indicated. On the basis of present
knowledge, it can be assumed that the specified vitamin or a member of
the specified group will generally be required for the particular type of
enzymatic action irrespective of the specific substrate used or the
biological source furnishing the corresponding enzyme.

Types of Enzymatic Reactions Utilized
in Essential Metabolic Processes

Types oj Reactions Vitamins required

I. Reactions in which acetal, ester, amide, none

and acid anhydride linkages are
formed or cleaved by the addition or
removal of the elements of water or
phosphoric acid.
II. Intramolecular hydrations and dehy- none

drations.
III. Intramolecular isomerizations. none

BIOCHEMICAL REACTIONS AND THEIR CATALYSTS

105

IV. Simple oxidations and reductions in Nicotinic acid

which the following types of double Riboflavin

bonds are created or reduced by the Porphyrins*

removal or addition of hydrogen
atoms (or the addition or loss of elec-
trons) .

A. C=0

C=C

B. C=N-

D. 0=0

Thiamine
(Biotin?)
Pyridoxal

V. Decarboxylations of keto acids and the
reverse reactions.

A. a-Keto acid decarboxylations

B. /?-Keto acid decarboxylations
VI. Reactions of amino acids requiring

activation of the a-position.

A. Transaminations

B. a-Amino acid decarboxylations
VII. Condensations and cleavages creating

or rupturing carbon-to-carbon bonds,
(other than carboxylations) .

A. Aldol type

B. Reactions in which acetyl deriva-
tives are condensed or formed

C. Reactions in which "activated single
carbon unit participates!
cleavedf

VIII. Other reactions in which the single
carbon unit participates!
To place vitamin Bi2 in the classification now would be premature,
since concentrates known to be free from other biological agents have
been available for only a few months. It is possible, however, to indicate
that vitamin Bi2 functions (perhaps indirectly) in the group of reactions
for which p-aminobenzoic acid or folic acid are essential.

* The reasons for including porphyrins with the B vitamins in this discussion will
be taken up later (p. 151).

t This group of reactions is not yet as completely characterized as are the other
types. There is some question concerning the exact compounds which enter into the
reactions and the information on the component parts of the enzymes is still incom-
plete. However, these types of reactions have been included because of the abun-
dance of circumstantial evidence concerning them (see p. 198).

none
Pantothenic acid

p-Aminobenzoic acid
Folic acid

106 THE BIOCHEMISTRY OF B VITAMINS

In the preparation of this outline several types of reactions, which so
far have been demonstrated in only certain forms of life, have been
omitted since their inclusion would necessitate cumbersome qualifying
phrases in the description of the general types of reactions. For example,
the detoxification of amines by amide formation in the liver and the
formation of an acetate ester, acetylcholine, in nerve tissue represent
cases where the reactive acetyl derivative, which usually reacts to form
carbon-to-carbon bonds, has been utilized for specialized functions. From
the standpoint of function, the syntheses of such acetyl amides and
esters (requiring pantothenic acid, p. 195) can be considered entirely
distinct from the amide and ester syntheses necessary to produce fats
and proteins (no vitamin requirement).

It may be that another general type of reaction in which carbon-to-
carbon bonds are formed will eventually be added to this group. Some
microorganisms can effect the condensation of the amino acid serine with
indole to form tryptophan (p. 183). Here it is the hydroxymethyl group
adjacent to the a-carbon atom (rather than the carboxy or amino group)
which reacts after the amino acid is activated by a pyridoxal-containing
enzyme. To date, this reaction is the only instance known in which pyri-
doxal catalyzes the formation of a carbon-to-carbon bond.

Inositol has recently been implicated as a necessary component of
a-amylase, a hydrolytic enzyme. Attempts to associate it with other
hydrolytic enzymes have not yet been reported. The vitamin analyses of
several other purified enzymes catalyzing hydrolysis indicate that
inositol does not function generally in hydrolytic reactions. When poly-
saccharides are synthesized and broken down intracellularly, it is by a
phosphorolytic process rather than a hydrolytic one, i.e., the elements of
phosphoric acid instead of water take part in the formation and cleavage
of glycosidic linkages. It would be interesting to know if inositol is needed
for this type of reaction. If so, then inositol could take its place with the
other members of the B group as a compound which is essential for a
reaction required in the metabolism of practically all cells. The earlier
discovery that inositol is a component of cell lipides (which could
account for its nutritional importance) is probably partly responsible
for so little work being directed toward establishing other possible
functions. The association of inositol with enzymatic activity warrants
a thorough investigation of its catalytic role in metabolism.

In view of the large number of separate reactions involved in the
metabolism of carbohydrates, fats, and proteins and the variety of
chemical compounds produced during these processes, it seems quite
significant that the types of enzymatic reactions are limited; also, that
each vitamin is specific for a given type of reaction, and that all the

BIOCHEMICAL REACTIONS AND THEIR CATALYSTS 107

typical B vitamins appear on this essential list. Cognizance of the relation-
ship between each vitamin and a highly specific type reaction enables
an investigator to predict which B vitamin, if any, will be involved in
a newly discovered enzyme system and which ones would be unlikely to
function. Before it was shown that thiamine functioned only in the
decarboxylation of ot-keto acids, efforts were often made to demonstrate
that thiamine was a component of enzymes which were effective in
decarboxylating other types of acids.

The list of fundamental types of reactions will be incomplete until
the biosyntheses of sterols, certain protein constituents, and some other
essential compounds have been elucidated. On the basis of the informa-
tion now available about the formation and degradation of these com-
pounds it seems reasonable to predict that not too many more types of
reactions will be found necessary to account for their metabolism.

Just as it is possible to produce an almost infinite number of proteins
from combinations of nineteen or so amino acids, so it should be possible,
if the catalysts are available, to produce almost any type of chemical
change which a cell requires by the proper combinations of a limited
number of relatively simple types of reactions. It may be pointed out
here that a limited number of enzymes in the digestive tract are capable
of hydrolyzing an inestimable number of different proteins. Could a
similar number of enzymes accomplish the reverse synthetic processes?
An answer must wait until more knowledge is available concerning the
manner in which the amino acids are placed in order during the synthesis
of proteins. If the amino acids and intermediate peptides are oriented
in some nonenzymatic fashion (by a gene or "organizer"), a small number
of specific enzymes could accomplish the syntheses of most simple pro-
teins.

Such speculation concerning the number of types of enzyme reactions
offers an independent approach on which to base estimates as to the
number of B vitamins yet to be discovered. Undoubtedly several new
types of enzymatic reactions occurring generally throughout the biolog-
ical realms will be found. Some of these will probably require as co-
catalysts specific organic compounds which will be chemically unrelated
to any of the known vitamins or other growth-promoting substances.
When such substances are found, there will be good reasons for grouping
them with the known B vitamins in any classification of biochemical
substances based upon functions.

At one time it seemed necessary to postulate the existence of a large
number of types of enzymatic reactions unrelated to those then known
in order to account for the catalytic activity of the numerous growth-
promoting factors and vitamins which had not yet been associated with

108 THE BIOCHEMISTRY OF B VITAMINS

definite enzymes. Is such an assumption still necessary? This question,
as well as the related question concerning whether or not there are many
undiscovered B vitamins, is certainly debatable. The thesis that there are
only a small number of B vitamins yet to be found may have substantial
foundation.

Components of Enzyme Systems. Once the relationship of a vitamin
to a particular type of chemical reaction has been established, there still
remain many questions to be considered regarding the relationship of
the vitamin to the enzyme system itself.

Although thiamine, riboflavin, and nicotinic acid are as effective agents
as can be found for the treatment of the corresponding nutritional
deficiencies, none of the three is effective as such in the biochemical
reactions for which they are required; each has to be incorporated into
a more complex molecule before it can take part in its reactions. All the
other typical vitamins whose functions have been completely elucidated
have been shown to behave in an analogous manner — they act as cat-
alysts in enzymatic reactions only after they have been built up into
coenzymes of higher molecular weight than the vitamin itself.

Enzyme systems vary considerably in their complexity. The com-
ponent parts of the system acting upon a substrate include:

A. Substance required to produce a suitable environment in which the
reaction can be carried out. This group includes water, and the com-
pounds necessary to establish the proper pH, a suitable redox potential,
and appropriate ionic concentrations.

B. A protein.

C. Co-factors.

(1) Specific inorganic ions.

(2) Specific organic compounds — the coenzymes.

Some of the simpler systems do not require any cofactors; others require
only an activating inorganic ion, or a coenzyme; many enzymes, however,
require both.

There is little specificity about the substances needed to establish a
favorable environment for a reaction, and one has considerable leeway
in his choice of buffering agents, poising agents, and salts which can be
used. These factors should not be confused with or classified as coenzymes.
The concentration of the nonspecific components can usually be varied
over a considerable range. It is impossible to set down a single optimum
value for the pH, redox potential, and salt concentrations, since the most
effective levels will depend upon the other conditions imposed upon the
system. In contrast, the substances listed as cofactors are much more
specific, and during a reaction are combined with the protein component
in stoichiometric amounts. The divalent ions — magnesium, calcium, man-

BIOCHEMICAL REACTIONS AND THEIR CATALYSTS 109

ganese, cobalt, and zinc — are the ionic substances most often encountered.
Sometimes there exists a certain amount of interchangeability among
these metallic ions, but usually it will be found that one particular metal
will be much more effective than any of the others, and probably is the
one associated with the enzyme system in its natural environment.

The organic molecules required as cofactors are highly specific, and in
almost all cases only one specific compound will function; without it
the protein is completely inactive.

Enzyme chemists are always particularly interested in determining
whether a coenzyme is required for a biochemical reaction under in-
vestigation. During the purification of an enzyme, attempts are made
to see if the enzyme molecule can be separated into inactive components
which, upon recombination, will possess the original activity of the
enzyme. If such a dissociation can be achieved, the complete enzyme is
called the holoenzyme; the inactive protein component, the apoenzyme;
and the smaller entities which are split off, the coenzymes or prosthetic
groups. The ease with which the coenzymes may be separated from their
apoenzymes varies considerably. Dialysis has been the method usually
employed for separating these components, since this procedure is less
likely to alter irrevocably the protein molecule than any other means of
separation now known. Extraction of insoluble cellular material with
alkaline or acidic buffers often can effect a resolution of the holoenzyme.
If one is interested in obtaining only the coenzyme, heat will often
liberate the coenzyme but will leave a denatured apoenzyme.

A large number of enzymes exist which cannot be dissociated by
dialysis or any other method yet tried. It may be that this group of
enzymes includes some proteins that contain tightly bound prosthetic
groups which, except for their nondissociability, are analogous to the B
vitamin coenzymes. Indeed, some of the enzymes in which the B vitamins
are implicated have not yet been successfully resolved into their com-
ponent parts. This offers some basis for postulating the existence of
unidentified "factors" which must be incorporated into enzymes catalyz-
ing certain fundamental processes occurring in most cells. From the
standpoint of the nutritional requirements of animals such factors could
be either essential or nonessential.

The classification of organic substances which have been definitely
shown to be prosthetic groups for important enzyme systems is very
simple. They will be found to be one of three types:

(1) adenylic acid or a phosphorylated derivative,

(2) a derivative of one of the B vitamins, or

(3) a metallic complex of a porphyrin.

Very few substances that have been definitely characterized as true

110 THE BIOCHEMISTRY OF B VITAMINS

coenzymes will not fit into this classification. The number of substances
known to function coenzymatically is small, and the B vitamin deriva-
tives constitute a major portion of the list.

Several naturally occurring oxidizing and reducing agents, including
ascorbic acid and glutathione, have been classified as "enzyme activators."
The requirement for these substances, however, is not specific since other
compounds can generally be substituted. It would seem, then, that these
compounds should be considered as poising agents needed to adjust the
redox potential to a level at which the protein will be active. An excep-
tion to this may be the requirement for glutathione, or closely related
compounds, necessary for the functioning of glyoxalase.8 Here gluta-
thione appears to participate chemically in the reaction.

There are a number of important compounds which are essential for
certain cyclic enzymatic processes. Ornithine in the urea cycle and
oxalacetic acid in the tricarboxylic acid cycle (p. 224) are familiar
examples. These substances go through a series of enzymatic transforma-
tions, but are regenerated at the completion of the cycle. For this reason
such a substance is sometimes referred to as a "carrier" for the com-
pound which is produced or consumed during the process. Although it is
possible to show a great deal of similarity between the manner in which
such "carriers" function and the way in which some of the coenzymes
function (p. 137), these "carriers" are commonly thought of as a type of
catalyst distinct from the typical coenzymes.

There are a number of questions concerning the relation of the B vita-
mins to their respective coenzymes and the relation of the coenzymes
to the proteins that will be considered in the following section of this
chapter. The individual coenzymes will be discussed in detail in Chapter
IIB.

The Coenzymes

Intensive research on the structures and functions of the coenzymes
derived from the B vitamins is currently being conducted in a number
of laboratories. It is probable that many important contributions directly
related to the topics to be discussed are about to be reported in the
journal literature. The information available as this book goes to press
will be summarized in the following paragraph.

The coenzymes derived from thiamine, riboflavin, nicotinic acid, and
pyridoxine have all been isolated and their chemical structures deter-
mined. A crystalline preparation of a pantothenic acid-containing
coenzyme has not yet been announced, but the properties and hydrolytic
products of highly active preparations have been reported. Absolute
proof that biotin is a part of a specific coenzyme has not yet been pub-

BIOCHEMICAL REACTIONS AND THEIR CATALYSTS 111

lished. Biotin activity is associated with several molecules more complex
than the simple vitamin itself and it would indeed be unusual if one or
more of these complexes were not a coenzyme for some type of reaction.
Although a great many facts have been learned about the metabolic
processes in which p-aminobenzoic acid, folic acid, and vitamin Bi2
participate, no work has been reported which justifies drawing any con-
clusions concerning the chemical relationship of these vitamins to their
respective coenzymes. Inositol has recently been shown to be an active
constituent of a-amylase. It has not been definitely demonstrated that
this simple molecule is identical with the coenzyme, but it would appear
that this could be so (p. 125) .

Coenzymatic Activity of the Simple Vitamins. Can the B vitamins,
in their simplest chemical forms, ever serve as coenzymes? With the
exception of inositol, just mentioned, there is no evidence that any B
vitamin participates in vivo in a catalytic function until it has been
transformed into its corresponding coenzyme (s).

It is possible in some cases to demonstrate, in vitro, chemical trans-
formations in which a particular vitamin behaves in a manner analogous
to the behavior of its coenzyme, in vivo. An example is the conversion
(transamination) of pyridoxal to pyridoxamine by heating the former
with amino acids. The conditions required for a reasonable yield make
it appear most unlikely that within the cell such nonenzymatic reactions
of the free vitamin could be important. It is also possible to demonstrate
that free riboflavin can take part directly in oxidation and reduction
processes. These reactions are so sluggish in an environment comparable
to that found intracellularly that they would undoubtedly be useless.

Some of the free vitamins produce very distinctive pharmacological
responses (Chapter VC) ; however, these are entirely independent of the
vitamin function of the compound and have no necessary connection
with processes in cellular metabolism for which the vitamin is required.
There are two possible ways in which a free vitamin can conceivably
influence the metabolism of a cell. It is possible for a vitamin to act as
an inhibitor of its own coenzyme (Section D) . Because of the structural
similarity between a vitamin and its coenzyme, it is possible to produce
effects which can best be explained by assuming that the vitamin com-
petes with its coenzyme for the apoenzyme. In in vitro studies with
bacteria and yeast, however, the concentrations of a vitamin required
to produce inhibition are entirely outside the limit which would be found
in natural circumstances. The ability of an organism to detoxify the
inhibitor (the vitamin) by converting it to the coenzyme would also
reduce the probability of achieving concentrations producing inhibition
within the cell.

112 THE BIOCHEMISTRY OF B VITAMINS

High levels of a vitamin might, however, exert an effect which is just
the opposite, i.e., the high concentration might increase the apparent
activity of a coenzyme by slowing down its destruction. Thus, nicotin-
amide has been observed to inhibit the enzymatic destruction of its
coenzyme in minced tissues.9 The investigators making this observation
believed that the extent of inactivation of the coenzyme was decreased
by the increased concentration of one of the products (nicotinamide) of
the inactivating reaction. Thiamine has been shown to be a specific in-
hibitor for certain enzymes which catalyze the hydrolytic destruction of
its coenzyme (p. 156).

Biosynthesis of the Coenzymes. In vivo, the coenzymes are formed by
enzymatic reactions in which the vitamins themselves serve as substrates.
The coenzymes vary in their chemical complexity, and presumably some
vitamin-to-coenzyme transformations require more than one reaction.
The simplest coenzymes are those in which the vitamin is converted to a
phosphoric acid ester. Thiamine, riboflavin, and pyridoxal fall in this
group. Riboflavin, nicotinic acid, pantothenic acid, and perhaps biotin
form more elaborate molecules which contain the nucleotide, adenylic
acid, as a component. The simpler coenzymes have all been synthesized
chemically by direct phosphorylation of the vitamin. Every attempt to
synthesize adenylic acid-containing coenzymes by chemical means has
been unsuccessful.

It is interesting to consider the possible existence of unrecognized vita-
mins required for the reactions converting B vitamins to their coenzymes.
Thus, if some cofactor of a system essential for a reaction which produces
one of the recognized coenzymes could not be synthesized by an animal,
this cofactor could be an essential dietary factor or vitamin. However,
the amount of a catalyst needed to catalyze the formation of a catalyst
would be extremely small; hence the nutritional requirements for such
factors may be extremely minute, and demonstrating their existence would
be difficult. Such a coenzymatic role has been suggested for vitamin Bi2
(in the biosynthesis of the coenzymes of p-aminobenzoic and folic acids,
p. 203). This would account for the clinical activity of vitamin B12 in
one-thousandth the amounts required for other vitamins.

It may often happen that appreciable amounts of a vitamin may be
supplied to an organism in a chemical form which cannot be directly
utilized as the substrate for the synthesis of the necessary coenzyme.
Other reactions must then precede the final transformation. If an or-
ganism is deficient or lacking in enzymes capable of catalyzing the needed
change, then that form of the vitamin will show less activity or be totally
inactive. It appears, for example, that pyridoxine must be oxidized to the
aldehyde before it can be converted into a coenzyme; hence the vitamin

BIOCHEMICAL REACTIONS AND THEIR CATALYSTS 113

B6 activity of pyridoxine for an organism is a measure of that organism's
enzymatic capacity for carrying out the preliminary oxidation (p. 179).

For some cells the coenzyme, or compounds intermediate between the
coenzyme and vitamin in their chemical complexity, must be enzymati-
cally cleaved before absorption can take place, thus necessitating the
complete resynthesis within the cell. An interesting example is cited in a
report in which it was shown that if the monophosphate of thiamine is
supplied as the substrate for the formation of the coenzyme (which is a
di(pyro) -phosphate), the monophosphate must first be hydrolyzed to the
unphosphorylated thiamine.10 In such cases the coenzyme or intermediate
will be useless nutritionally to an organism if it cannot elaborate the
extracellular enzymes needed to degrade the more complex forms of the
vitamin. It is for this reason that the coenzyme of thiamine cannot be
used to supply the nutritional requirements of some thiamine-requiring
yeasts.11

The chemical mechanisms involved in the biosynthesis of a coenzyme
may not always include a reaction involving the vitamin itself. Thus
some bacteria can convert ^-alanine into a pantothenic acid complex
more rapidly than they can produce the complex if pantothenic acid is
the initial substrate.12

Rate of Coenzyme Synthesis. How rapidly is a coenzyme synthesized
from a vitamin? Such conversions can be carried out very quickly by
most cells. This is demonstrated by the rapidity with which one can get
a response when a cell deficient in some coenzyme is supplied the essential
vitamin. There is, to be sure, some lag in time between the addition of the
vitamin to the culture medium and a detectable response due to the
coenzyme formed. It must be remembered that a deficient cell is essen-
tially a dormant cell, since the retardation of vital metabolic processes
forces most of the cell's activities to come to a standstill. It is only natural
that some time should elapse before the cell can reach an active state
again. In cells in which the deficiency is not acute the conversion of a
vitamin to a coenzyme may take place almost immediately. This is
demonstrated in the fermentation assay method for thiamine.13 The yeast
used contains sufficient thiamine coenzyme to carry on some fermentation,
and the cells are in a moderately active phase. The addition of thiamine
to a medium containing such cells results in an immediate increase in the
rate of fermentation.

Extent of Coenzyme Synthesis. There is insufficient information on
which one could base a general statement concerning the relative con-
centrations of all the vitamins and their coenzymes within cells. It ap-
pears, however, that intracellularly the vitamins are predominantly in the
form of their active coenzymes. In blood, for example, it has been shown

114 THE BIOCHEMISTRY OF B VITAMINS

that, whereas the thiamine, nicotinic acid, riboflavin, and pantothenic
acid occur as such in the serum, they are almost entirely in the form of
their respective coenzymes inside the erythrocytes.14, 15> 16> 17 It may be
that part or all of the conversion of a vitamin to a coenzyme takes place
during its absorption into the cell. Many absorptive processes are known
to involve phosphorylation. Intestinal mucosa, a tissue in which absorp-
tive mechanisms are extremely highly developed, has been shown to be
a good source for obtaining preparations which will carry out the en-
zymatic syntheses (phosphorylation) of thiamine and riboflavin co-
enzymes.18, 19

Do all cells possess the enzymes necessary to carry out the syntheses
of their coenzymes from the vitamins? The known cases in which a
vitamin must be furnished in the form of the intact coenzyme are rare.
Two related species of bacteria cannot utilize nicotinic acid or nicotin-
amide (p. 136). Their nutritional requirements are usually supplied in
the form of the intact coenzyme, but actually only a portion of the whole
coenzyme is necessary. Some bacteria do not respond to pantothenic acid,
but must have either the coenzyme or some substance of intermediate
complexity that has been elaborated from pantothenic acid.12 In neither
of these cases can we say that the organisms have a specific requirement
for the coenzyme. Recently some organisms have been found which can-
not utilize unphosphorylated derivatives of pyridoxal.20 The rarity of
cases to date in which the intact coenzymes have been shown to be essen-
tial nutritional factors might lead one to expect that they will seldom
be found to be irreplaceable nutrilites. To draw such a conclusion now
is unwise. The coenzymes of only half of the known vitamins are as yet
chemically identified, and suitable preparations of several of the identified
compounds have not been generally available. At present it is possible
to obtain from commercial sources only two coenzymes — cocarboxylase
(thiamine) and coenzyme I (nicotinic acid). Furthermore, a large number
of organisms have never been successfully grown on media whose chemical
composition is known. Many of the chemically undefined nutritive re-
quirements which can now be furnished only by natural extracts may be
found to be unidentified coenzymes (or their intermediates) of the known
B vitamins.

Number of Coenzymes. Is more than one coenzyme synthesized from
each vitamin? Those coenzymes which function as carriers of specific
groups or atoms naturally will exist in two states. Hydrogen carriers will
exist in the oxidized and reduced forms. A coenzyme which transports an
active group (the active acetyl radical, for example) probably exists part
of the time in chemical combination with the active substance. The situ-
ation in which pyridoxamine phosphate may be formed from pyridoxal

BIOCHEMICAL REACTIONS AND THEIR CATALYSTS 115

phosphate during transaminations is of the same category. A phenomenon
entirely different from this duality in forms of a single coenzyme occurs
when there are synthesized from a single vitamin two specific coenzymes
which cannot be substituted for one another in an enzyme system. Nico-
tinic acid and riboflavin are the only vitamins whose coenzymes are known
at this time to fall definitely in this category.

All the reactions catalyzed by thiamine, pyridoxal, and pantothenic
acid can be explained on the basis of a single coenzyme for each vitamin.

Most of the known enzymatic reactions involving riboflavin require
the more complex coenzyme (called the dinucleotide) . A coenzyme less
complex in structure, riboflavin phosphate (designated as the mono-
nucleotide), however, activates at least three apoenzymes (p. 146). One
of these apoenzymes, that of the old yellow enzyme, can be reactivated
equally well by either the mononucleotide or the dinucleotide. The oxida-
tion mediated by this yellow enzyme proceeds at such a sluggish rate that
it could not be of use in a metabolically active cell; hence it is believed
to be a "derived" enzyme formed during its isolation from a more reactive
flavoprotein. Two other recognized mononucleotide enzymes, cytochrome c
reductase and L-amino acid oxidase, are capable of catalyzing the transfer
of hydrogen atoms at a rate rapid enough to meet adequately the demands
imposed upon them by natural systems. No report has been made con-
cerning the question of whether or not equally active systems could be
formed by using the dinucleotide as the coenzyme.

Most of the reactions catalyzed by nicotinic acid, however, fall into
two distinct classes, each of which utilizes a specific coenzyme. The only
difference in chemical structure between the two nicotinic acid coenzymes
is one phosphate group. There are a few systems which are known to be
activated by either coenzyme. Upon reinvestigation of some systems for
which such claims had originally been made, it was shown that the co-
enzyme containing the extra phosphate group was degraded by traces of
phosphatases present as impurities in the apoenzyme preparation.21 These
phosphatases convert the triphospho-coenzyme (coenzyme II), which
might be inactive itself, into the active diphospho-compound (coenzyme
I), and it would appear that either coenzyme could function.

It may be well, before leaving the topic of vitamin-coenzyme conver-
sion, to speculate as to why a vitamin must be transformed into a co-
enzyme before it is active. In the case of no coenzyme now known does it
appear from the standpoint of theory of how they act that the nonvitamin
portion should be useful or necessary for the catalytic activity observed.
Thus in oxidation and reduction reactions, the addition or donation of
hydrogen atoms during the transfer between substrates takes place in the

116 THE BIOCHEMISTRY OF B VITAMINS

vitamin component of the coenzyme.* In vitro, pyridoxal can participate
in transamination reactions with amino acids without being phos-
phorylated; although the mechanism by which thiamine performs its
catalytic function is not understood, there is no reason to expect that the
phosphate part of the coenzyme molecule is altered during decarboxyla-
tions. It seems in most instances that the chemical changes catalyzed by
a vitamin-containing enzyme are directly mediated by the vitamin com-
ponent of the coenzyme, but the vitamin itself does not readily associate
itself with the apoenzyme; hence, the function of the nonvitamin portion
must in some way be concerned with the mechanism by which the co-
enzyme associates itself with the apoenzyme. Until some better explana-
tion can be offered, it can be postulated that an important reason for the
conversion of a vitamin to its coenzyme is to enable the molecule to con-
tain the chemical groups which are essential for the formation of the
coenzyme-protein bond.

Formation of Holoenzymes. One of the most intriguing properties
exhibited by many proteins is their remarkable ability for combining with
specific compounds of smaller molecular weight. It is impossible to give
any adequate explanation of this phenomenon in terms of the classical
concepts of chemical bonds. The inadequacy of our knowledge is fre-
quently hidden behind the veil of such ambiguous terms as "protein com-
plexes" and "enzyme-substrate union."

The binding of an apoenzyme with its coenzyme should be considered
as a special case of the general phenomenon of combination between
enzymes and substrates. In fact, in a number of reactions the participat-
ing coenzyme can more properly be termed a substrate than a catalyst.
These are the reactions in which coenzymes are chemically altered during
the course of the reaction and must be reconverted to their original form
by a second reaction entirely separate from the first (p. 137). In such
cases the coenzyme is in a strict sense the catalyst for a process, but
not for the individual reactions.

Any attempt to develop a general explanation for the association
between coenzymes and proteins is further complicated by the extreme
variation in the stability of the union. Some systems exist in which the
dissociated apoenzyme and coenzyme are present in greater concentrations
than the holoenzyme; at the other extreme are the enzymes whose pros-
thetic groups are so firmly bound that hydrolysis of the protein is neces-
sary to release the vitamin component. Also, a particular coenzyme does

* In the case of nicotinic acid, the conversion of the tertiary amine (the vitamin)
to the quaternary ammonium base (the coenzyme) is probably essential for estab-
lishing the redox potential appropriate for the reactions catalyzed by the nicotinic
acid-enzyme systems.

BIOCHEMICAL REACTIONS AND THEIR CATALYSTS 117

not necessarily have the same affinity for the different apoenzymes which
it activates.

The most stable type of combination might result from the formation
of an ester or amide from the two components; more labile unions could
resemble those found in compounds which dissociate readily in aqueous
solution — hemiacetals, ammonia-aldehyde types, and hydrates; the bond-
ing in some systems whose degree of dissociation is so sensitive to
electrolyte concentration and pH changes might be explained by ionic
attraction and salt formation.

Until recently no one had ever questioned the assumption that the
union of a substrate and its enzyme was one involving actual physical
contact of the two molecules. However, it has been reported that enzymes
whose surfaces were believed to be completely coated with a polymer
film could still activate their substrates and effectively catalyze re-
actions.22 That this procedure prevented the physical union between
enzyme and substrate has been challenged;23 but if this finding can be
substantiated and shown to be of general application to enzyme systems,
many current concepts concerning enzyme mechanisms, including the
functioning of coenzymes, will have to be radically altered.

Any theory concerning the chemical and physical forces which bind the
components during a reaction should account for the extreme specificity
which the protein may exhibit in its choice of substrates. Often, the
slightest alterations in the structure of a substrate or coenzyme will
affect its capacity for uniting with the enzyme, even though no change
has been made in any of its reactive groups or the isoelectric point. This
is indicative that the spatial configuration is critical.

The similarity in the chemical composition of the nonvitamin moiety
of all the coenzymes is indicative of a general requirement for phosphate
ester and adenylic acid components in the establishment of the coenzyme-
apoenzyme bond. The additional acid and amino groups introduced when
the coenzyme is created increase the number of points where the coenzyme
molecule can become attached to the protein by the formation of a salt.
Perhaps adenylic acid possesses a chemical structure which is particularly
adapted for combining with some configuration common to a number of
proteins. Thus adenylic acid itself serves as a coenzyme for some reactions
p. 134) ; its phosphorylated derivatives are essential dissociable parts of
most enzyme systems in which high-energy phosphate bonds are created
or utilized; and nucleic acids which contain adenylic acid constitute the
prosthetic groups of a number of important types of proteins.

There is a difference in the affinity of apoenzymes for the oxidized and
reduced states of the coenzymes of nicotinic acid. This can be attributed
at least partly to the acid-base changes which accompany the oxidation

118 THE BIOCHEMISTRY OF B VITAMINS

and reduction of the coenzymes. When the coenzyme is reduced, the
quaternary ammonium ion in the pyridine ring is converted to a tertiary
amine. This elimination of a strongly basic group increases the acid prop-
erties of the coenzyme. Since the reduced acidic compound may dissociate
more readily, it leaves the apoenzyme and is free to transport the hydro-
gen atoms to another enzyme system where these atoms will be passed on
to another substrate. Other processes which involve shuttling atoms or
reactive groups from one enzyme system to another may likewise function
because of differences in the degree of dissociation of the two forms of a
coenzyme.

The association of a substrate with its enzyme probably is always
accompanied with a redistribution of intramolecular energies within both
the substrate molecule and the protein. These changes are responsible for
the "activation" of a certain atom or group of the substrate molecule.
When a coenzyme unites with a protein the same thing occurs. The effect
is more than just the tying together of two substances, and even though
the union may be a "loose" one it results in profound changes in some of
the chemical properties and reactivity of both the coenzyme and the
protein.

An interesting example in which a quantitative expression can be
derived for the change that takes place within a coenzyme when it com-
bines with a protein is riboflavin phosphate. The standard redox potential
of the riboflavin coenzyme is considerably different from the potential of
a riboflavin enzyme system.24 This means that in combining with the
apoenzyme some changes take place within the riboflavin moiety which
greatly increase its tendency to accept hydrogen atoms (actually elec-
trons, p. 128) . The magnitude of the changes is such that the system can
catalyze an entirely different group of biological oxidations and reductions
than it could if it retained the potential of the unassociated coenzyme
(p. 146).

The changes in the stability of apoenzymes when dissociated from their
coenzymes can be used to substantiate the fact that the separation of a
coenzyme from its protein may induce critical changes within the protein
molecule. For example, when the coenzyme is removed from a-amylase
by dialysis, the protein rapidly deteriorates and cannot be reactivated
again, even by the addition of the coenzyme. However, it can be shown
that the denatured protein still possesses its original capacity for com-
bining with the coenzyme (p. 125), although the complex formed is
enzymatically inactive. This indicates that in the dissociation of the
holoenzyme an alteration in the molecular structure of the protein occurs
at some position other than the exact site where the coenzyme attaches
itself.

BIOCHEMICAL REACTIONS AND THEIR CATALYSTS 119

The association of a coenzyme with its apoenzyme to form the holo-
enzyme is sometimes an essential step in the biosynthesis of a coenzyme.
Thus, the enzymatic synthesis of cocarboxylase from thiamine requires
the presence of the apoenzyme, and as soon as there has been sufficient
synthesis to saturate the protein the synthesis of the coenzyme ceases.10
A reasonable explanation for this is that, when there is no longer any
apoenzyme present to combine with the coenzyme as it is formed, the
uncombined coenzyme cannot protect itself from the action of phos-
phatases which hydrolyze the coenzyme as rapidly as it is formed.

Specificity of Coenzymes. The inactivity of most hydrolytic and other
degradation products of coenzyme molecules indicates that the structural
specificity required by apoenzymes of their coenzymes usually applies to
the whole coenzyme molecule rather than to one particular type group
or linkage within the molecule. The specificity of some apoenzymes for
their coenzyme cannot be said to be absolute, however. The well sub-
stantiated cases in which an analogue of a vitamin has vitamin-like
activity (Chapter VID) necessitate assuming that coenzymes containing
those vitamin analogues are synthesized enzymatically, and that the re-
sulting analogues of the natural coenzymes are able to combine with the
apoenzymes and catalyze certain reactions. A few analogues of coenzymes
have been prepared synthetically and tested in vitro and found to have
some coenzymatic activity.

Analytical Methods for the Coenzymes. It would be gratifying in a
general discussion of the analytical methods for determining the coen-
zymes (and the enzyme systems in which they participate) to be able to
describe general methods which could be used for all the B vitamin coen-
zymes. Unfortunately, this is not possible at the present time. Studies of
the distribution of the B vitamins in cells and tissues have yielded some
very interesting results (Chapter II A). Such data would have consider-
ably more significance if they indicated how much of the vitamin was
in an active form and how the coenzyme was distributed among the
various systems for which it is required.

It may be that microbiological methods, as general in their applicability
as the vitamin assays, can eventually be developed for the coenzymes.
This will depend upon success in finding organisms which require specif-
ically the intact coenzymes. There are no organisms known which satisfy
this requirement in the strictest sense, the closest case being an organism
whose vitamin B6 requirement can be met only by pyridoxal phosphate
or pyridoxamine phosphate.20 If suitable organisms cannot be found,
inhibitors perhaps will offer the solution to the problem of obtaining
simple general methods for the analyses of the coenzymes. The ideal
inhibitors for these analyses would effectively block the last reaction in

120 THE BIOCHEMISTRY OF B VITAMINS

the conversion of a vitamin to its coenzyme. If such compounds were
available, they could be used to adapt the simple microbiological pro-
cedures used in vitamin determinations to specific assay methods for the
coenzymes.

Today the only specific methods of assay for most coenzymes involve a
direct determination of the effect of the addition of a substance upon an
enzymatic reaction in which the particular coenzyme is the limiting factor.
This entails the preparation of the appropriate apoenzyme, either from
cells or tissues deficient in the coenzyme or from protein preparations in
which the coenzyme has been destroyed or removed. Such enzymatic assay
methods for coenzymes have a number of disadvantages not encountered
in the microbiological methods. They require equipment, chemicals, and
biological preparations which may not be readily available; technically,
more training and skill is usually needed than for a microbiological assay.
In addition, a general standardized procedure for all B vitamin coen-
zymes cannot be used, since the analysis for each coenzyme constitutes
a special method.

Occurrence of Coenzymes. Since one of the reasons for grouping the
B vitamins together is the similarity of their natural distribution, it would
be of interest to know if the enzymes containing them are associated
together physically within cells. Some of the fundamental processes which
involve a number of steps may require at most a single B vitamin coen-
zyme, e.g., the elaborate mechanism by which glycogen is converted to
lactic acid in muscle requires fourteen separate enzymes, but only one
B vitamin coenzyme is needed (p. 219). On the other hand, there are
processes every step of which must be mediated by a different B vitamin.
The conversion of carbohydrate to fat involves a series of reactions in
which each pyruvic acid molecule eventually lengthens a fatty acid by
two carbon atoms. This conversion requires enzymes containing thiamine,
pantothenic acid, nicotinic acid, and probably riboflavin (p. 226). This
series of reactions cannot be demonstrated if the structure of the cell has
been destroyed. It is not known if this process is carried out by a number
of separate enzymes which are physically separated from one another in
the cell. It may be that the transformation requires an enzyme complex
in which the component proteins and coenzymes are actually combined,
and that this system would be inactive if its organization were disturbed.

Recently a process has been described for preparing a protein complex
from mammalian tissue containing all the enzymes necessary for the
aerobic oxidation of pyruvic acid and certain fatty acids through the
tricarboxylic acid cycle. In its isolation, the protein complex containing
all the essential component enzymes separates out as a gel.25 When the
gel loses its ability to carry through the series of reactions there is an

BIOCHEMICAL REACTIONS AND THEIR CATALYSTS 121

alteration in the appearance of the gel. The associated complex of enzymes
is believed to be localized in certain morphological structures occurring
in cytoplasm, the mitochondria.26, 27

Most of the individual reactions utilized in the synthesis of the units
for cell structure and in the production of energy are only component
parts of some process involving a large number of reactions. Usually the
intermediate compounds formed by the reactions are utilized immediately.
In many instances the concentration of these intermediates under normal
conditions is so low that it is difficult to demonstrate their existence ; also
some of the processes in which such intermediates occur are known to
proceed very rapidly. The simplest explanation to account for the rapidity
of a long process, when the concentration of the reactants for a number
of the individual reactions is so low, is that the enzymes are in some way
associated together in the cell.

There probably are also special means by which labile intermediates
and by-products arising in one part of the cell can be transported to
another. If enzyme systems containing cofactors that are easily dissoci-
able are used, it is then possible that a reaction can be carried on in one
part of the cell to furnish chemical intermediates or energy for utilization
elsewhere within the cell. For example, adenosine diphosphate, the co-
enzyme for most reactions in which a high-energy phosphate bond is
generated, is converted in these reactions to adenosine triphosphate. This
form of the coenzyme can dissociate and is free to transport its energy
"unit" to the other parts of the cell where it can be utilized. Hydrogen
atoms arising from the numerous dehydrogenations of cellular reactions
can be transported to other parts of the cell by dissociable coenzymes.
It may be found that several of the highly reactive labile substances like
the activated acetate derivative and the single carbon units are trans-
ported from one part of the cell to another by the coenzymes of panto-
thenic acid and p-aminobenzoic acid. Thus the complete series of enzymes
needed for a particular cell function need not all be located exactly at the
site of the function.

One of the present goals of enzyme chemists is the establishment of
the exact location of the enzyme systems within cells. Some information
has already been obtained by carrying out vitamin and enzyme analyses
on the fractions obtained by separating cells into their gross components.
Any procedure, however, that disrupts cell organization even slightly will
not give all the information needed to explain what actually takes place
within normal cells.

Hence, now that the relationship between most of the vitamins and
their chemical functions has been established, many investigators are
seriously engaged in an equally challenging study — that of associating

122 THE BIOCHEMISTRY OF B VITAMINS

the vitamins and their enzymes with the biological organization of the
cells in which they function.

Bibliography

1. Jansen, B. C. P., and Donath, W. F., Chem. Weekblad, 23, 201 (1926).

2. Sumner, J. B., J. Biol. Chem., 69, 435 (1926).

3. Theorell, H., Biochem. Z., 278, 263 (1935).

4. Lohmann, K., and Schuster, P., Biochem. Z., 294, 188 (1937).

5. Warburg, O., and Christian, W., Biochem. Z., 275, 464 (1935).

6. Elvehjem, C. A., Madden, R. J., Strong, F. M., and Woolley, D. W., J. Am.

Chem. Soc, 59, 1767 (1937).

7. Williams, R. J., Vitamins and Hormones, 1, 229 (1942).

8. Jowett, M., and Quastel, J. H., Biochem. J., 27, 486 (1933).

9. Mann, P. J. G., and Quastel, J. H., Biochem. J., 35, 502 (1941).

10. Weil-Malherbe, H., Biochem. J., 33, 1997 (1939).

11. Williams, R. J., McMahan, J. R.. and Eakin, R. E., University of Texas Pub-

lication, 4137, 34 (1941).

12. King, T. E., Fels, I. G., and Cheldelin, V. H., J. Am. Chem. Soc, 71, 131 (1949).

13. Schultz, A. S., Atkin, L., and Frey, C. N., /. Am. Chem. Soc, 59, 2457 (1937).

14. Goodhart, R. S., and Sinclair, H. M., Biochem. J., 33, 1099 (1939).

15. Kohn, H. I., and Klein, J. R., J. Biol. Chem., 130, 1 (1939).

16. Klein, J. R, and Kohn, H. I., J. Biol. Chem., 136, 177 (1940).

17. Kaplan, N. O., and Lipmann, F., J. Biol. Chem., 174, 37 (1948).

18. Tauber, H., Enzymologia, 2, 171 (1937).

19. Hubner, H., and Verzar, F., Helv. Chim. Acta, 21, 1006 (1938).

20. McNutt, W. S., and Snell, E. E., /. Biol. Chem., 173, 801 (1948).

21. Schlenk, F., Adv. in Enzymology, 5, 212 (1945).

22. Rothen, A., /. Am. Chem. Soc, 70, 2732 (1948).

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Chapter II B

COENZYMES DERIVED FROM B VITAMINS

The problem of determining the specific chemical reactions catalyzed
by a coenzyme derived from a B vitamin is not one which can be attacked
in a straightforward manner. There is no standardized procedure the
utilization of which would assure an investigator of success in determin-
ing the function of a newly discovered vitamin. The relationships between
B vitamins and their enzyme systems have been established through the
correlation of information obtained from many types of investigation of
metabolic processes.

In a critical examination of biochemical processes, there are logical
reasons for studying the enzymatic reactions in their natural environment,
i.e., within intact cells and as an integral part of a series of reactions.
On the other hand, it is impossible to characterize a reaction completely
— to determine the specific compounds reacting and the component parts
of the catalyst — unless an isolated system free from all interfering
phenomena is studied. Obviously, it is not possible to achieve both of
these goals with the same techniques. This difference in purpose has led
to some argument concerning the relative merits of establishing the
existence of biological processes in cells by the use of isotopes, inhibitors,
mutants, etc., and of studying specific isolated enzymatic reactions in
detail. An adequate explanation of metabolism can ultimately be achieved
only by approaching the individual problems from both directions.

The coenzymatic functions of some of the B vitamins were discovered
by investigators whose primary interests were in the enzyme systems they
were isolating; in such cases, specific vitamins have fortuitously been
found to be component parts of particular enzymes — that is, the investi-
gators up to time of finding the presence of a given vitamin, may have
had no particular interest in that or any other vitamin.

In other cases the known functions of B vitamins have been ascertained
as a result of observing the effect of vitamins upon metabolism. In such
direct attempts to study the chemical changes catalyzed by the vitamins,
normal biological systems are compared with those in which the vitamin
has been prevented from functioning. The most direct method of limiting
the reactions catalyzed by a vitamin is to produce a deficiency of that

123

124 THE BIOCHEMISTRY OF B VITAMINS

vitamin in the cells or tissues of an organism requiring the vitamin.
Although this method is naturally limited to organisms which cannot
synthesize adequate amounts of a given vitamin, its applicability has
been greatly extended by inducing mutations in the genes controlling
vitamin syntheses, thus artificially producing organisms with the desired
nutritional requirements.

Reactions requiring a specific vitamin can be blocked even in organisms
which do not normally require the vitamin. This is accomplished by
subjecting the cells to some treatment which inactivates the coenzyme.
Coenzymes essential for some reactions can be destroyed by placing the
organism in an unfavorable environment. A method of general applica-
tion for inactivating specific enzymes within the cell involves the use of
inhibitors. The methods by which inhibitors can be used in investigating
biological reactions is discussed in more detail in the last section of this
monograph.

If the metabolic processes mediated by a coenzyme derived from a
vitamin are blocked by the use of any of the procedures just described,
by-products will accumulate. Chemical identification of the substances
accumulating indicates the nature of the substrate of the transformation
which has been blocked. Finding biochemical substances (chemically un-
related to the vitamin) which will partially or wholly counteract the
changes produced in the deficient organism serves as a means of identi-
fication of some of the products which would normally be formed from
the blocked reactions.

The chemical changes resulting from inactivation of a vitamin-contain-
ing system can be demonstrated by use of classical chemical procedures
only when the substances involved are present in measurable concentra-
tions. In many important instances, however, the intracellular concen-
trations of the reactants and products participating in a series of reactions
are so low that changes from the normal metabolic pattern cannot be
observed by ordinary methods. This obstacle was very effectively sur-
mounted when the isotopes of carbon, hydrogen, nitrogen, sulfur and
phosphorus became available for biological tracer studies. The use of
compounds containing labelled atoms has been responsible for a number
of recent contributions which have established coenzymatic functions of
the vitamins: (1) the unexpected discovery of two extremely important
biochemical intermediates (activated derivatives of formic and acetic
acids) ; (2) the disclosure of a number of fundamental metabolic routes;
and (3) the verification of the existence of postulated reactions previously
undemonstrable.

The purpose of this chapter is to present a discussion of the individual
coenzymes and their enzymes. These discussions will not include those

COENZYMES DERIVED FROM B VITAMINS 125

contributions which have no direct bearing on the functions of the coen-
zymes nor will enzymatic reactions be cited whose existence is not well
substantiated.

Coenzymes Required for the Synthesis and Cleavage of Ester, Acetal, and
Amide Linkages

Most of the high molecular weight compounds — fats, polysaccharides,
proteins, nucleic acids, etc. — must be cleaved into simpler substances
before they can be absorbed and incorporated into essential structures
of cells. This extracellular digestion is catalyzed by a group of enzymes
which directly hydrolyze the ester, acetal, and amide linkages of the
macromolecules. In intracellular syntheses, their hydrolytic products —
simple sugars, fatty acids, amino acids, etc. — are recombined by reactions
which recreate the acetal, ester, and amide bonds. These synthetic reac-
tions within cells cannot be mediated by the same enzymes which cata-
lyzed the hydrolysis, since the intracellular concentrations of amino acids,
free fatty acids-, and monosaccharides are probably never high enough to
reverse the direction of the corresponding hydrolytic reactions. The for-
mation of these larger molecules must proceed by indirect routes by which
are incorporated reactions which introduce the energy necessary for the
coupling of the component units (usually by the formation of phos-
phorylated intermediates) .

With one exception, there is no evidence that the B vitamins function
in either the direct hydrolytic reactions or in the synthetic mechanisms
utilizing phosphorylated intermediates. However, each of the B vitamins
is required for the production of some of the ultimate units from which
the fats, carbohydrates, and proteins are formed, and most of the vitamins
are essential for the energy-producing processes which supply the energy
needed for the synthetic reactions.

Inositol as a Coenzyme. One reaction which is probably an excep-
tion to the statement in the previous paragraph is the hydrolysis of
amylose by a pancreatic enzyme. Inositol appears to function as an
essential component of this enzyme, a-amylase. The coenzymatic activity
of inositol was first suggested when a highly purified preparation of
a-amylase was shown to contain 4.1 mg of inositol per gram.1 Subse-
quently this enzyme was shown to dissociate upon dialysis into a dialyz-
able thermostable component (a coenzyme) and a protein having no
enzymatic activity.2 The dissociation of the enzyme during dialysis pro-
duces a change in the protein component, so that it is no longer enzymati-
cally active even when recombined with the dialyzable fraction. This
change, however, does not affect the capacity of the protein for combining
with the coenzyme. This was demonstrated by showing that the dialyzed

126 THE BIOCHEMISTRY OF B VITAMINS

protein can inactivate an active undialyzed preparation (presumably by
competing with the active enzyme for the coenzyme) ; but a combination
of the dialyzed protein and the dialyzable coenzyme does not exhibit the
inhibitory effect upon an undialyzed preparation. Furthermore, no inacti-
vation of the protein component occurs if the enzyme is dialyzed against
a solution containing the thermostable coenzyme.

In view of these findings a study using inositol inhibitors was carried
out to demonstrate a possible relationship of the coenzyme to inositol.
The y-isomer of 1,2,3,4,5,6-hexachlorohexane, a compound whose inhibi-
tory action on the growth of yeast and molds can be prevented by meso-
inositol, was found to inactivate purified a-amylase preparations.3 The
presence of inositol, however, counteracted this inhibition. To demonstrate
the inactivation of the enzyme, it was necessary that the inhibitor be
incubated with the enzyme for at least fifteen hours. The amount of
inactivation produced by the inhibitor depended on the ratio of inhibitor
to inositol. When the molar ratio of hexachlorohexane to inositol was 10,
only a 10 per cent inactivation occurred. Increasing this ratio to 50 com-
pletely inactivated the enzyme. Thus, the authors concluded that inositol
is an active constituent of a-amylase.

Although the results obtained with the inositol inhibitor are highly
indicative, they do not conclusively answer a pertinent question: Is "free"
inositol the coenzyme of a-amylase? The only logical explanation for the
inhibition by hexachlorohexane and its reversal by inositol in the highly
purified system used is that inositol itself is a dissociable component of
the system, or that it can effectively replace some dissociable component.
It would be interesting to know whether inositol by itself would behave
in a manner identical to the thermostable dialyzable component obtained
by dialysis of the enzyme. If the coenzyme is simply inositol, a-amylase
could be dialyzed against a solution of inositol with no loss in activity;
also, the inactivation of undialyzed preparations by dialyzed protein
fractions could be prevented by the addition of inositol along with the
dialyzed protein.

In considering possible mechanisms by which inositol, which contains
no salt-forming groups, could combine with the apoenzyme, the relation-
ship of the chemical structure of inositol to that of the glucosidic units in
the amylase substrate should not be overlooked.

A crystalline preparation from hog pancreas was used for the dial-
ysis experiments which demonstrated the existence of a coenzyme for
a-amylase. Since that time, a-amylases have been isolated in a crystalline
state from human pancreas,4 human saliva,5 and Bacillus subtilis.G The
hog pancreatic amylase is not the same protein as the enzyme isolated
from human pancreas, but apparently the human salivary amylase is

COENZYMES DERIVED FROM B VITAMINS 127

identical with human pancreatic amylase. Inositol analyses on these
preparations have not been reported, nor is information on the inositol
content of saliva available.

Inositol, according to indications, has two possible biological functions
independent of the one just described. It is a component of certain phos-
pholipides (Section D), and may be an important factor in the regulation
of fat metabolism in mammals. It is impossible to give clear presentation
of the latter function because of disagreements which have not yet been
resolved concerning the manner in which dietary factors influence the pro-
duction of fatty livers. The confusion one encounters in attempting to
follow the literature is due to several facts: different investigators have
used different means of producing fatty livers; the types of lipides de-
posited have not always been well characterized; and it has been neces-
sary to use crude liver extracts rather than pure substances to supply
certain vitamins.7- 8> 9

"Lipocaic" was the name given to an uncharacterized heat-stable sub-
stance (s) present in pancreas which was effective in preventing the fatty
livers induced by diets containing liver extracts of high biotin content.
Inositol, at one time, was claimed to be an active component of lipocaic,
but its activity apparently is not as great as once believed and inositol
per se is not as effective as choline.10

Another lipotropic factor, protein-like in nature, obtained from the
pancreas will prevent the fatty liver which results from pancreatectomy
of dogs. This type of fatty liver can also be treated by adding choline to
the diet. It is believed that the protein-like lipotropic factor functions
by catalyzing during digestion certain hydrolytic reactions which are
necessary to liberate methionine and choline from food, and that these
compounds are the actual agents which prevent the abnormal deposition
of fat in the liver.11 Apparently no effort has been made, however, to
determine if the lipocaic action of inositol may be due to its presence in
the lipotropic protein of the pancreas. If appreciable amounts of inositol
were found in the more active preparations, it would be indicative that
inositol is a component of another hydrolytic enzyme produced by the
pancreas.

Hydrases and Isomerases

A class of reactions distinct from the synthetic and hydrolytic processes
just discussed is the intramolecular hydrations and dehydrations — reac-
tions in which the elements of water are added to or removed from a single
molecule. A number of essential steps in the metabolism of carbohydrates
and fats are of this type: fumaric acid ^± malic acid; phosphogly eerie
acid ^± phosphopyruvic acid ; /3-hydroxybutyric acid ±=> crotonic acid (p.

128 THE BIOCHEMISTRY OF B VITAMINS

222); isocitric acid «=± as-aconitic acid; etc. Each of these reactions
involves the dehydration of a /^-hydroxy acid, a type of reaction often
encountered in organic chemistry. When catalyzed enzymatically, no
vitamin-containing coenzyme has been shown to be required.

The isomerases which act upon phosphohexoses and phosphotrioses
constitute a class of enzymes essential for most, if not all, living organisms
that metabolize carbohydrates. These two enzymes catalyze structural
rearrangements by establishing an equilibrium between:

(1) glucose-6-phosphate and fructose-6-phosphate;

(2) glyceraldehyde-3-phosphate and dihydroxyacetone phosphate.
The conversion of an aldose phosphate to the corresponding ketose phos-
phate may take place as a result of the intermediate formation of an enol
form common to both. A coenzyme requirement for such systems has not
been demonstrated. These reactions produce isomers having the molecular
configuration required for the subsequent aldol condensation or cleavage
by which trioses and hexoses are interconverted (p. 219).

Coenzymes Mediating Biological Oxidations and Reductions

Biological oxidations and reductions constitute a class of enzymatic
processes which has been intensively and thoroughly investigated during
the past half century. Probably no other biochemical phenomenon has
received the amount of attention that has been devoted to the study of
the mechanisms by which organic substances are oxidized and reduced
under the conditions imposed by an environment compatible with life.

It was early recognized that most of the reactions classified as oxida-
tions do not involve molecular oxygen or any of the oxygen-donating
types of compounds commonly employed as oxidizing agents in laboratory
syntheses. The reactions in which metabolic substrates are "oxidized"
can more properly be designated dehydrogenations, for the mechanism is
in most instances one involving the transfer of hydrogen atoms; indeed,
only rarely are oxygen atoms exchanged during the transformation. Even
in aerobic processes, which require molecular oxygen, the particular com-
ponent reaction in which the oxygen is utilized is usually one in which the
oxygen molecule acts as an acceptor for hydrogen atoms which have been
transported from the initial substrate by a series of enzymatic reactions.
The oxygen molecule is, in fact, hydrogenated, forming hydrogen peroxide,
and the oxygen atoms of the oxygen molecule are not incorporated into
any of the organic molecules undergoing oxidation.

A more exact interpretation of the mechanism of hydrogenation-
dehydrogenation reactions can be set forth by explaining the phenomenon
in terms of the donation and acceptance of electrons rather than hydrogen
atoms. Excellent discussions of the current theories regarding the mecha-

COENZYMES DERIVED FROM B VITAMINS 129

nisms of oxidations, particularly those types occurring in biological
systems, have been published recently.12- 13 Since the changes in the struc-
ture of organic molecules can be much more easily indicated in terms of
hydrogen atoms, this convention of depicting "oxidations" will be used
in preference to the more exact one in which the electronic changes are
described.

The enzymatic oxidation of many organic compounds involves no
changes other than the removal of two hydrogen atoms. The catalysts
for this type of reaction are found to contain, as cofactors, derivatives
of either nicotinic acid, riboflavin, or a porphyrin. There are several types
of oxidative processes which are not simple dehydrogenations. Oxidative
decarboxylations and transaminations are illustrative of the more com-
plex types of oxidative reactions. In these cases it will be found that
vitamins other than the ones just mentioned function in the catalytic
processes; these reactions will be considered elsewhere. The dehydrogena-
tions considered below (catalyzed by enzymes containing nicotinic acid,
riboflavin, and porphyrins) are those in which a double bond of one of
the following types is created by the removal of two hydrogen atoms and
an existing double bond in another molecule is reduced by the addition
of these two atoms:

C=0 C=N— C=C 0=0

\ /

£=N—

C=C

/ \

An interpretation of the oxidation and reduction of inorganic substances
can often be most easily made by considering the process in terms of a
galvanic cell. Likewise, the presentation of certain facts pertaining to
biological processes in which there are oxidations and reductions occurring
can be most easily made by drawing an analogy between the biological
systems and a galvanic cell.

The galvanic cell is composed of two half-cells; each half-cell contains
both an oxidized and a reduced form of some substance capable of existing
in the two states (for example, a metal and its ion) ; these oxidized and
reduced components of a half-cell must be in a state of dynamic equilib-
rium; and the two half-cells must be connected by suitable conductors
before chemical changes and energy production take place. When the
circuit between the two half-cells is closed, a reduction will take place at
one electrode, and a compensating oxidation will occur at the other.
Which part (oxidation or reduction) of the overall reaction will occur at
a specific electrode and how much energy will be released by the reaction
are determined by the relative potentials of the two half-cells composing
the cell. At a given temperature and pH this electrode potential of a half
cell is a function of the components of the half-cell and their relative

130 THE BIOCHEMISTRY OF B VITAMINS

concentrations, and this value is a quantitative expression of the capabili-
ties of the component pair as an oxidizing or a reducing agent.

In each of the individual reactions of a biological oxidation there are
two metabolite systems: each system consists of a substance capable of
existing in an oxidized and reduced state ; the oxidized and reduced forms
of a particular system must be in a state of dynamic equilibrium ; and the
two systems must be connected by suitable means before chemical changes
and energy production take place. When this connection between the two
systems is established a reduction will take place in one system and an
equivalent oxidation in the other. Which system will be reduced and
which oxidized and how much energy will be liberated by the reaction are
determined by the relative potentials of the two metabolite systems com-
posing the reaction. In this case, the potential is called the redox poten-
tial; it is a function of the components of the system, their relative
concentrations, the temperature, and, usually, the pH at which the reac-
tion is carried out. This potential is a quantitative expression of the
tendency of the metabolite pair to undergo oxidation or reduction. The
lower the potential (based on the positive and negative notation found
in all biochemical literature) , the greater is the tendency of a system to
accept hydrogen atoms and to exist predominantly in the reduced form.

In many common galvanic cells no catalyst other than water is needed
to establish the equilibrium between the oxidized and reduced states of
the material composing the half-cell. There are instances, however, in
which a catalyst must be introduced to establish this equilibrium. For
example, a platinum electrode must be used to establish the equilibrium
between molecular hydrogen and its oxidized state, the hydrogen ion.
Several systems which often form component parts of biological oxida-
tions (for example, the sulfide-disulfide system, the ascorbic-dehydro-
ascorbic acid system, or certain ferrous-ferric complex systems) require
no catalyst (at least, no enzyme) to establish the equilibrium between
their oxidized and reduced states. When two such systems are coupled, a
nonenzymatic oxidation-reduction reaction occurs. Usually, however, the
situation is more analogous to the hydrogen-hydrogen ion half-cell, and
a catalyst (enzyme) must be introduced to establish the equilibrium
between the oxidized and reduced form of the organic metabolite. The
classical example is the succinic-fumaric acid system. These two acids
cannot be reversibly interconverted by any chemical means yet known,
and they cannot function by themselves as a hydrogen donor or an
acceptor. Yet, in the presence of an appropriate enzyme, these two acids
rapidly reach a state of equilibrium, and this system can act as either an
oxidizing or reducing agent depending upon the potential of the system
with which it becomes linked.

COENZYMES DERIVED FROM B VITAMINS 131

The specific purposes of the enzymes which mediate biological oxida-
tions and reductions are (1) to establish the equilibrium between the
two states of a metabolite system and (2) to link this system with another
of appropriate potential. When these criteria are met a reaction will then
automatically occur.

The relative electrode potential of a half-cell or the relative redox
potential of a metabolite system can be calculated from the standard
potential of the half-cell or the metabolite system. The standard potential
is that potential, referred to an arbitrary standard (the potential of a
|H2 — H+ system), of a half-cell or metabolite system when the oxidized
and reduced states are present in equal concentrations (or, are at equal
activities) . The actual potential of a system having other concentration
ratios differs from the standard potential by a factor which includes the
ratio of the oxidized and reduced forms. Hence the actual potential of a
system and its tendency to act as an oxidizing or reducing agent can be
varied by changing the relative concentration of the two components.

In a galvanic cell it is possible to reverse the direction of the current
and the direction in which the chemical reaction is proceeding by chang-
ing the ratio of the oxidized and reduced forms of the components of the
half-cells, provided the standard potentials of the two half-cells are not
too far apart. If the standard potentials for two metabolite systems
composing a reaction are approximately the same value, it is possible to
cause the reaction to proceed in either direction by establishing appropri-
ate ratios between the oxidized and reduced forms of the two component
systems. Such reactions are those in which the net chemical changes have
been observed to be reversible. If, however, there is appreciable difference
between the standard potentials of the two systems composing the reaction
it is impossible to achieve concentration gradients sufficient to reverse the
usual course of the reaction. In this case the system having the lower
potential will always be observed to be oxidized while the one having the
higher potential will be reduced.

For convenience in studying redox systems, reactions are often set up
in which one of the component systems is an artificial one, i.e., is not
encountered under natural circumstances. The redox dyes, such as
methylene blue, are commonly used for this purpose. Their use often
simplifies the process to be studied, enables the investigator to by-pass
technical difficulties, and through the change in the color of the dyes at
a critical redox potential serves as a convenient indicator in following
the progress of the reaction.

Coenzymes derived from nicotinic acid, riboflavin, and the porphyrins
exist in both oxidized and reduced states and, with their appropriate
apoenzymes, form redox systems. It is not surprising that the enzyme

132 THE BIOCHEMISTRY OF B VITAMINS

systems derived from each of these three cofactors have characteristic,
but widely differing, potentials. Because of this difference, one type of
cofactor is usually much more suitable for a particular reaction than are
the other two. The enzyme systems containing nicotinic acid have the
lowest potential, those containing riboflavin intermediate, and those re-
lated to porphyrins the highest. These vitamin-containing systems either
become linked with one another or with a specific metabolite system, thus
establishing an oxidation-reduction reaction. Such reactions are respon-
sible for most of the biological oxidations which take place. A more
detailed account of individual reactions will be discussed in connection
with their specific coenzymes.

Coenzymes Containing Nicotinic Acid

The first organic coenzyme to be recognized (1904) was a heat-stable
factor which activated preparations of zymase, the complex of enzymes
in yeast which catalyzes the alcoholic fermentation of carbohydrates.14
This cofactor was designated "cozymase." Its chemical constitution, how-
ever, was not established until much later, after a second coenzyme,
coenzyme II (the codehydrogenase of Zwischenferment) , had been dis-
covered, isolated, and shown to be a derivative of nicotinamide (1934) ,15
(Zwischenferment, an enzyme of historical interest, dehydrogenates glu-
coses-phosphate.) A year later nicotinamide was also isolated from
cozymase and the chemical relationship of the two coenzymes was thus
established.16

NH2

CH N

HC C CONH2 /? C N

II I HC || I

HC CH \ C CH

N N N

HC 1 HC 1

HCOH I HCOH
HCOH I HCOH
HC 1 O O HC

111
H2C— O P O P O CH2

O- OH

Diphosphopyridine nucleotide (Cozymase)

Upon hydrolysis the two coenzymes yield identical products; however,
only two mols of phosphoric acid are present in cozymase, while three

COENZYMES DERIVED FROM B VITAMINS 133

mols are liberated from coenzyme II. The results of degradation of the
coenzymes have excluded all but one possible structure for cozymase,17
but have not given sufficient data to establish the exact formula for
coenzyme II. They are both inner salts of ribose dinucleotides in which
the organic bases are nicotinamide and adenine. The problems encoun-
tered in attempting to synthesize such dinucleotides have not yet been
solved, and it has been impossible to verify the proposed structures by
synthesis.

It was originally postulated that the third phosphate group of coenzyme
II formed a linear pyrophosphate "chain" with the two other phosphate
residues.18 However, it is now believed that the additional phosphate is
attached in some position as a "side chain," presumably by forming an
ester with one of the hydroxyl groups of the adenosine component. This
structure would account for the ease with which the triphospho- coenzyme
can be degraded to cozymase, since the reaction would then not entail a
cleavage and resynthesis of the dinucleotide bond during the conversion.

A number of trivial synonyms have been coined for the two coenzymes;
for most of them there are obvious objections. The thiamine coenzyme
is also a cof actor essential for zymase activity; hence, it too is a "co-
zymase" and a "coferment." "Coreductase" and "codehydrogenase" can
logically be applied equally well to coenzymes which contain no nicotin-
amide. Both coenzymes of nicotinamide plus several degradation products
can serve as "Factor V," the growth factor required by Hemophilus
influenzae. Designation of compounds by numerals or by the names of
investigators can be very confusing even to scientists well acquainted
with the historical aspects of biochemistry. Names based upon chemical
structure are the desirable choice. For this reason, diphosphopyridine
nucleotide (DPN) will be used in preference to cozymase, coenzyme I,
codehydrogenase I, coferment of alcoholic fermentation, Harden's cofer-
ment, coreductase, or Factor V; triphosphoptjridine nucleotide (TPN)
will be used to designate the compound which was originally called coen-
zyme II (codehydrogenase II) or Warburg's coferment.

The formula and names given above represent the oxidized states of
the coenzymes. The reducd forms are referred to as the dihydro com-
pounds. In the oxidized state, the nucleus of the nicotinamide exists as
a quaternary pyridinium ion which forms an inner salt with one of the
ionizable acid groups in the pyrophosphate bridge between the two
nucleosides. Upon reduction, the heterocyclic nitrogen atom is converted
to the weakly basic tertiary amine; hence the reduced disphosphopyridine
nucleotide behaves as a dibasic acid. The reduction of either coenzyme is
thus always accompanied by an increase in the number of titratable
hydrogen ions. Which one of the two ortho positions (2 or 6) is hydro-

134 THE BIOCHEMISTRY OF B VITAMINS

genated when the pyridine nucleus is reduced has not yet been established.

The reduction of the coenzymes not only changes the acidic and basic
properties of the compounds, but also causes characteristic alterations in
their absorption spectra, in their stability toward acidic and alkaline
treatment, and in their affinity for their apoenzymes. The reduced coen-
zymes both show in their absorption spectra a very distinct band at
320-360 m^ which is totally absent from the spectra of the oxidized
molecules. The production of this band during reduction of the coenzymes
is so characteristic of these compounds that it can be used as an analytical
method for determining the di- and triphospho nucleotides in purified
preparations.19 Such spectrographs analysis also is an excellent method
for following the progress of enzymatic reductions or other enzymatic
reactions which can be coupled to a reaction involving a nicotinic acid
coenzyme.20 When irradiated with ultraviolet light, only the reduced
coenzymes exhibit a strong whitish fluorescence.21

The coenzymes in their oxidized state are rapidly inactivated by stand-
ing at room temperature in dilute alkaline solution (0.1N), but the rate
of destruction is much slower in acidic solutions (O.liV) ; the reduced
molecules, on the other hand, are inactivated immediately by weakly
acidic conditions, but are unaffected by the alkaline treatment.18

The immediate destruction of the reduced molecules by acid has been
attributed to the formation of an addition compound in which a molecule
of acid adds to one of the double bonds of the dihydropyridine nucleus.22
The stability of the dihydro coenzymes (trivalent nitrogen atom) in
alkaline solution is comparable to the stability of the glucosidic-like link-
age through the nitrogen atom of purine nucleosides (likewise trivalent) .
Hence, the unusual alkaline lability of the oxidized nicotinamide nucleo-
side can probably be attributed to the difference in properties of a glu-
cosidic bond linking a quaternary ammonium nitrogen ion to a pentose.
The initial cleavage occurring when diphosphopyridine nucleotide is sub-
jected to either acidic or basic hydrolysis takes place at the bond linking
the pyridine base to the ribose.

Adenosine diphosphate and adenylic acid can be obtained from the
alkaline hydrolyzates of diphosphopyridine nucleotide,23 the latter pos-
sessing the coenzymatic activity necessary for the activation of certain
apophosphorylases. Attempts to demonstrate (by this enzymatic anal-
ysis) the formation of adenylic acid by a comparable treatment of the
triphosphopyridine nucleotide have been unsuccessful. This is the reason
for believing that the third phosphate group of the latter coenzyme is
attached in the form of a phosphate ester of the adenosine portion of the
molecule rather than as a portion of the pyrimidine nucleoside or the
connecting pyrophosphate bridge. Alkaline cleavage of the former type

COENZYMES DERIVED FROM B VITAMINS 135

compound would yield an esterified derivative of adenylic acid which
would be inactive when tested for "cophosphorylase" activity.24

An enzyme occurring in rabbit tissue has been shown to cleave the pyro-
phosphate linkage in diphosphopyridine nucleotide to produce the two
mononucleotides,248 and an enzyme preparation has been obtained from
almonds which cleaves this coenzyme in a fashion such that the products
are the labile nicotinamide nucleoside, adenosine, and phosphoric acid.25

Since the coenzymes cannot be prepared synthetically, they must be
isolated from natural sources. Yeast is a rich source from which the
diphospho derivative is usually isolated, although it has been suggested
that better preparations can be obtained if muscle is used as the source.
Several investigators have reported alterations in procedure for improv-
ing the older methods of concentration and purification.26' 21 Erythro-
cytes were the source originally used for preparing the triphospho com-
pound;15 it has also been isolated from liver 28 and yeast.29 The diphospho
nucleotide can now be purchased from commercial sources, and prepara-
tions possessing triphospho nucleotide activity can be prepared chem-
ically from the former coenzyme.30 Although in their chemical properties
the di- and triphospho nucleotides are very similar, there are sufficient
differences in their tendencies to be absorbed 29 and in the solubilities of
their salts that their separation from one another is not difficult.29- 31- 32

The dihydro derivatives can be readily prepared by chemical reduction
of the oxidized forms of the coenzymes. Sodium hydrosulfite is a con-
venient reducing agent for this transformation.33

Assay Methods. The quantitative determination of the coenzymes of
nicotinic acid has been carried out by spectographic measurements,
enzymatic analyses, microbiological assays, and chemical methods.

Both the characteristic absorption band at 340 m/x 27 and the fluores-
cence of the dihydrocompounds 21 have been employed for estimating the
coenzyme content of concentrates. These methods do not distinguish
between the di- and triphospho derivatives of nicotinic acid, but they
do serve as a means of establishing the combined amounts of the reduced
forms of the two coenzymes.

The only specific method for determining the concentration of the
individual coenzymes is by use of enzymatic systems. With an appro-
priate apoenzyme an assay for either the diphospho or the triphospho
nucleotide can be accomplished. In this case both the oxidized and reduced
states of the particular coenzyme are equally active and are not dis-
tinguished by the method. In the determination of diphosphopyridine
nucleotide care must be taken to have an apoenzyme preparation free
from phosphatases which would degrade any triphospho coenzyme present
into the compound being measured.

136 THE BIOCHEMISTRY OF B VITAMINS

The activation of a "zymase" system has been the classical method
employed in determining the concentration of diphosphopyridine nucleo-
tide, since the preparation of the apozymase can be readily accom-
plished.34 The repeated extraction of dried brewers' yeast gives a residue
which contains all the components of the alcohol fermentation system
except diphosphopyridine nucleotide. In the presence of the apozymase
preparation the rate of fermentation (measured by following the evolution
of carbon dioxide) gives a direct measure of the amount of coenzyme
introduced into the system. In this process the coenzyme is needed to
accept hydrogen atoms from glyceraldehyde-phosphate and subsequently
to donate them to acetaldehyde. Aerobic processes (such as oxidation of
lactic acid35 and malic acid)27 have also been used for the enzymatic
estimation of diphosphopyridine nucleotide. In addition to the appro-
priate apoenzyme and substrate the system must contain the other
enzymes needed for accepting the hydrogen atoms from the reduced
coenzyme and transporting them to the final hydrogen acceptor. If molec-
ular oxygen is the acceptor, the reaction can be followed manometrically ;
if methylene blue is used, the rate of decolorization is measured.

The oxidation of glucose-6-phosphate (Robinson ester) to 6-phospho-
gluconic acid is catalyzed by a dehydrogenase (Zwischenferment), the
coenzyme of which is triphosphopyridine nucleotide.36 This reaction can
be used as a specific method for the determination of this coenzyme,
since the diphospho nucleotide cannot act here as the hydrogen acceptor.
The reaction can be followed in several ways depending upon the hydro-
gen transporting systems to which it is coupled. The most sensitive and
accurate method is one in which cytochrome-c is the component finally
reduced.37 The rate of reduction of cytochrome-c can be easily followed
spectrometrically. By this procedure quantities as small as 0.02 micro-
gram of the coenzyme can be measured.

Hemophilus influenzae and Hemophilus para-influenzae cannot syn-
thesize the nicotinamide nucleoside from nicotinic acid or nicotinamide;38
consequently, these organisms do not respond to nicotinic acid or nico-
tinamide but must have an exogenous supply of either of the coenzymes
or certain degradation products in which the pyridine-ribose bond is
intact.39 These organisms have been used to assay for the coenzymes by
conventional microbiological procedures. Although this method lacks
specificity because some degradation products are active, the interference
by such substances in most instances probably is negligible.

The chemical reagents employed in the chemical determination of the
free vitamin also react with the coenzymes, and cannot be directly used
to distinguish between the simple vitamin and its more complex deriva-
tives. However, chemical methods can be of use when it is known that

COENZYMES DERIVED FROM B VITAMINS 137

all the nicotinic acid activity of a preparation exists in the form of the
coenzymes or if the various nicotinic acid-containing compounds are
separated by means of paper chromatography, for example.

The use of any of these methods for the analysis of natural substances
depends upon obtaining extracts suitable for analysis. Considerable
variation can often be noted in the analytical results of different in-
vestigators who have tested crude materials. This would indicate that
the accuracy of these methods may be limited by the factors involved
in the preparation of the extracts. The instability of the coenzymes and
the enzymatic destruction which occurs when cells are macerated must
always be taken into account. In making differential assays for the two
coenzymes the possibility of enzymatic interconversion must be con-
sidered. The rapidity with which the original equilibrium of the oxidized
and reduced forms of the two coenzymes can be disturbed can lead to
erroneous conclusions when values for the two states of each of the
coenzyme are sought. The reduced coenzymes, for example, are oxidized
by air during hot-water extraction.18

Occurrence. In view of the number of essential reactions for which
the coenzymes of nicotinic acid are required, it is not surprising that they
have been found in all cells which have been examined for their presence.
It would be most unexpected if some type of cell were found which did
not contain these coenzymes. The quantities of the two individual enzymes
as well as the ratio between the di- and triphospho nucleotides vary over
a wide range for different types of cells.18 Yeast, the richest source of the
diphospho nucleotide yet encountered, contains as high as 0.5 mg per
gram of moist cells. The triphospho nucleotide is always present in
smaller quantities than the simpler coenzyme, which would be expected
inasmuch as it functions in fewer reactions.

The degree of association of these coenzymes with their apoenzymes
is less than that encountered in any of the other B vitamin coenzymes.40
It has been pointed out that the coenzymes really function as substrates
of two independent reactions and that they are, in effect, catalysts for
a complex oxidation-reduction process rather than for simple individual
reactions. The coenzymes must alternate between two separate apo-
enzymes ; the one on which the coenzyme is reduced and the one on which
the reoxidized form is regenerated. Since comparable rates of reaction
are found in both intact cells and in solutions in which there is no organiza-
tion of the enzymes, it is believed that these two apoenzymes between
which the coenzyme shuttles need not be coupled together sterically
within the cell structure.41 These coenzymes have consequently been
described as "mobile coenzymes" in order to contrast their behavior with
that of the fixed coenzymes which remain attached to one apoenzyme

138 THE BIOCHEMISTRY OF B VITAMINS

Because of this mobile function these two coenzymes have been found
to occur in quantities which on an equivalent basis are considerably in
excess of the amounts of apoenzymes. Since the oxidized and reduced
forms of the coenzymes are kept in equilibrium by these apoenzymes,
the ratio of reduced and oxidized molecules will be determined by the
redox potential of the cellular environment. It is not surprising, there-
fore, that of the total diphosphopyridine nucleotide present in a number
of animal tissues, a fairly uniform percentage (35-45 per cent) is in the
reduced state.42 In certain malignant tissues the ratio of reduced form to
oxidized form is increased,43 a change which presumably results from
the lowered redox potential maintained by such cells.

Biosynthesis. The synthesis of the coenzymes from their component
units (those formed upon hydrolysis) can apparently be carried out by
most organisms. The only known instances in which this total synthesis
cannot be accomplished are encountered in the two strains of influenza
bacteria previously mentioned (p. 136) . In higher organisms practically
all the intracellular nicotinic acid is in the form of its coenzymes, but
the extracellular fluids (such as plasma, milk, and urine) contain little,
if any, coenzyme. The enzymatic synthesis of both coenzymes is known
to occur in vitro in most intact cells, and an enzymatic synthesis of
triphosphopyridine nucleotide from nicotinamide, ribose, and adenosine
triphosphate has been reported in which only a cell-free extract was
used.44

Investigations of the specificity of the pyridine component used for
the biosynthesis of the coenzyme have disclosed several interesting varia-
tions among different types of cells in regard to the utilization of nico-
tinic acid and nicotinamide. It appears that the route of synthesis may
differ in different types of cells, and that nicotinamide sometimes cannot
be directly used for the biosynthesis of the coenzymes.45

The synthesis of the triphospho nucleotide from the diphospho com-
pound can be accomplished enzymatically.46 Chemically the conversion
can be made by use of phosphorus oxychloride.30 The phosphorylated
substances formed by the enzymatic and chemical syntheses are assumed
to be identical with the naturally occurring compound, but chemical
proof of their identity has not been established.

When the organization of cells is disrupted the coenzymes of nicotinic
acid are rapidly inactivated by enzymatic hydrolyses. The coenzymes
are vulnerable to attack by hydrolytic enzymes at a number of points.
In animal tissue, most of the enzymes responsible for the inactivation are
believed to hydrolytically cleave the nicotinamide-ribose bond, liberating
free nicotinamide.47

COENZYMES DERIVED FROM B VITAMINS 139

An interesting phenomenon observed in yeast is one in which the
presence of a substrate appears to protect a coenzyme from destruction.
The crude apozymase preparations rapidly inactivate diphosphopyridine
nucleotide when the latter is added alone,48 but if hexose diphosphate is
added simultaneously the destruction does not take place. Likewise, if
the yeast fermentation is inhibited by the presence of the fluoride ion a
rapid decrease in coenzyme content results. Apparently, active fermenta-
tion is necessary to produce the conditions required either for decreasing
the rate of hydrolysis of the coenzyme or for increasing the rate of its
resynthesis from its hydrolytic products. A demonstration of coenzyme
synthesis by these apoenzyme preparations, however, has not as yet beei.
achieved.

Reactions Catalyzed by Nicotinic Acid-containing Coenzymes. Al-
though the reduction of the nicotinic acid coenzymes is often pictured as
the simple addition of two hydrogen atoms to one of the — N=C< bonds
of the pyridine nucleus, the reduction of the coenzyme is probably not
so direct. There is evidence that the reduction is a stepwise procedure
in which the intermediate formation of a stabilized semiquinoid radical
(monohydronucleotides) allows the addition to the coenzyme of a single
hydrogen atom at a time,49 and facilitates the establishment of an equilib-
rium between the oxidized and reduced states.13

A wide degree of variation has been noted in the specificity of various
nicotinic acid dehydrogenases in their requirements for the two coen-
zymes.50 In most of the reactions that have been investigated there is a
distinct preference, if not an absolute requirement, for one or the other
of the two compounds. One enzyme (glutamic acid dehydrogenase from
animal tissues), however, can use either the di- or triphospho nucleotide
equally well.

Approximately forty different enzyme reactions have been reported to
be catalyzed by one or the other of these coenzymes. Although many of
these reactions are probably of limited importance in the normal function-
ing of cells, a number of indispensable reactions taking place during basic
metabolic processes are found in the list.

The redox potentials for most of the important systems with which
pyridine coenzymes are coupled fall in the same range as those of the
coenzymes themselves. Consequently, the direction in which these reac-
tions proceed can be reversed (1) when changes in metabolism alter the
concentration ratio of the metabolite-pair within the cell or (2) when the
coenzyme-reduced coenzyme ratio changes because of variations in the
intracellular redox potential brought about by other processes taking
place. For example, in many instances the direction in which the lactic
acid-pyruvic acid conversion proceeds is constantly alternating because

140 THE BIOCHEMISTRY OF B VITAMINS

of slight changes in the local concentrations of these two substances at
the site of each enzyme molecule or because of variations in the redox
potential during the so-called aerobic-anaerobic phases of cellular activity.
One group of reactions in which these coenzymes participate include
those in which energy-rich phosphate compounds are formed by the
"oxidation" of an aldehyde; the reverse reactions, ones in which energy-
rich bonds are utilized for synthetic purposes, are the "reduction" of acids.
The "oxidation" of phosphoglyceraldehyde is a good example. The alde-
hyde forms a loosely bound addition product with phosphoric acid (a
compound analogous to an aldehyde hydrate) . On dehydrogenation this
inorganic phosphate is converted into a reactive acyl phosphate group
which can be transferred to adenosine diphosphate (ADP) , forming
adenosine triphosphate (ATP) .

o o

H H II o H H ||

ADP + HC— C— C + HO— P— OH + DPN DPN-2H + HC— C— C— OH + ATP

I 1 H I J I

o o o o o

I H H I H

HO— PO HO— PO

i i

H H

11 11

ADP + HC— C— C— O-

i A H

I H H I H H

HO— PO HO— PO

A i

H H

During catabolic phases of activity when glycogen and other organic
substrates are being oxidized for the purpose of supplying energy to cells,
this reaction (and other comparable ones) proceeds in the direction caus-
ing the dehydrogenation of the substrate and the creation of the reactive
phosphate bond. During anabolic phases, when part of the glycogen is
resynthesized by the organism, the reaction proceeds in the reverse direc-
tion and an energy-rich bond of adenosine triphosphate is utilized to
reduce, in effect, an acid to an aldehyde. This reaction represents a general
biological mechanism in which the phosphopyridine nucleotides mediate
the conversion of the latent chemical energy of reduced organic substrates
into the readily utilizable energy of the reactive phosphoric anhydride
compounds, or the reverse process in which available energy is conserved
by the formation of more highly reduced compounds.

The numerous redox systems which have been shown to be enzymati-
cally coupled with the phosphopyridine "coenzymes can be classified

H H

-P— OH

DPN

^ *■

DPN

■211

HC— C— C— O-

-P— OH

ADP

A A

COENZYMES DERIVED FROM B VITAMINS 141

according to the following outline (in which some of the more important
specific systems have been included as examples) : 50, 51

Aldehyde — primary alcohol (acetaldehyde — ethanol)

Ketone — secondary alcohol (pyruvic acid — lactic acid)

(oxalacetic acid — malic acid)
(oxalsuccinic acid — isocitric acid)
(/3-hydroxybutyric acid — acetoacetic
acid)
Acyl phosphate — aldehyde-1-phosphate (1,3-diphosphoglyceric acid — 1,3-diphos-

phoglyceraldehyde)
Acid — -aldehyde (hydrate) (gluconic acid — glucose)

( phosphogluconic acid — glucoses-phos-
phate)
( phosphoglyceric acid — phosphoglycer-
aldehyde)
Imine — amine (iminoglutaric acid — L-glutamic acid)

Both the glucose-6-phosphate and isocitric acid systems, as well as the
glutamic acid dehydrogenase of yeast and bacteria,50 must be coupled
with the triphospho nucleotide; the glutamic dehydrogenase system of
animal tissue requirement is nonspecific; and all the other well charac-
terized enzyme systems react most readily with the diphospho coenzyme.
Several other reactions have been reported to be activated when these
coenzymes are added to specific substrates in the presence of unrefined
preparations of the apoenzymes. These include several interesting chemi-
cal transformations; the reduction of nitrate to nitrite,52 the oxidation of
luciferin (the substrate that on oxidation produces bioluminescence) ,53
and the dehydrogenation of formic acid.54 When crude enzyme prepara-
tions are used, there is always a chance of the observed reaction being a
secondary effect and further characterization should be undertaken before
the reaction is definitely catalogued as to its type.

The Riboflavin-Containing Coenzymes

The role of riboflavin-containing enzymes as hydrogen carriers was
first definitely established in 1932 when a yellow enzyme isolated from
yeast was found to serve as a connecting link in a complex system in
which hexose monophosphate is oxidized and molecular oxygen reduced.55
The specific purpose of the jlavoprotein in this system is to catalyze the
reaction in which the hydrogen atoms accepted by triphosphopyridine
nucleotide from the .substrate are passed on to oxygen. Later, other flavo-
proteins were found to function in a comparable fashion with diphospho-
pyridine nucleotide, and it became evident that one of the fundamental
purposes of the riboflavin enzymes is to serve as "bridges" over which the
hydrogen atoms accumulating on the nicotinic acid coenzymes can be
passed to systems which will carry out the reduction of oxygen. An

142

THE BIOCHEMISTRY OF B VITAMINS

equally important function for the riboflavin coenzymes was also early
appreciated — that of being the initial acceptor for hydrogen atoms in a
number of metabolically important dehydrogenations of aldehydes, amino
acids, and purines.

When the yellow enzyme was discovered, it was realized that the yellow
pigment possessed the characteristics of a "flavin," a class of biological
pigments observed as early as 1879.56 In 1933, riboflavin was isolated in
crystalline form and shown to possess vitamin activity for the rat.57 At
first, it was assumed that riboflavin itself was the chromophoric com-

CH20-
HOCH
HOCH

0
-P— OH

HOCH
CH2

H3C—

c=o

H3C—

Riboflavin phosphate

H2C—
HOCH

-0 — P — 0-

— P-

-0 CH2

HOCH

0 1

1 HOCH

1 Ah

HOCH

CH2

H3C—

/- \

C=0

J— (

H3C—

V
h

sT— <

Flavin adenine dinucleotide

COENZYMES DERIVED FROM B VITAMINS 143

ponent of the yellow enzyme; but the addition of pure riboflavin to the
protein component did not result in reconstitution of the flavoprotein.
Isolation of the pure coenzyme from the enzyme showed the coenzyme to
be a phosphoric acid ester of riboflavin.58

Riboflavin forms two coenzymes. The simpler one is the phosphoric
acid ester just mentioned. This ester is often referred to as a mono-
nucleotide, although in a strict sense this is incorrect since the compound
is derived not from the sugar, D-ribose, but from the corresponding
alcohol, D-ribitol. The more complex coenzyme, called the dinucleotide,
can be described as a molecule in which adenylic acid (muscle) and
riboflavin phosphate are united by the formation of a pyrophosphate bond.

The location of the phosphoric acid at the 5 position on the ribityl unit
has been definitely established. Oxidation of the mononucleotide does not
yield formaldehyde, a product that would be formed were the primary
hydroxyl group unesterified.59 A synthesis of riboflavin-5'-phosphate by
a method establishing the location of the phosphate confirmed the struc-
ture of the mononucleotide, since the synthetic product had the same
coenzymatic activity as the naturally occurring compound.58

The structure of the dinucleotide has not been proved by synthesis.
However, the identity of the two mononucleotides obtained upon hydrol-
ysis is certain.60 The structure indicated in the formula above has been
accepted as the most likely one for the dinucleotide.

Enzymes containing either of the two coenzymes are often referred to
as yellow enzymes or flavoproteins. The coenzymes are sometimes desig-
nated by the method of classification in which the name of the aromatic
nucleus, alloxazine (or more properly, isoalloxazine) , is specified. The
simpler expression, flavin adenine dinucleotide (FAD), is now more
popular than the cumbersome expression isoalloxazine adenine dinucleo-
tide.

The coenzymes of riboflavin resemble the parent vitamin in many of
their physical and chemical characteristics: they exhibit the same charac-
teristic color and fluorescence ; they are decomposed when irradiated ; they
can be reduced by chemical reagents to leuco derivatives; they are auto-
oxidizable {i.e., the reduced leuco compounds can be oxidized by air back
to the original pigment) ; during reduction in strongly acidic solution
there is formed a red intermediate having the properties of a semiquinoid
radical; G1 and the redox potential of the riboflavin phosphate system is
comparable to that of a simple riboflavin system.62

The introduction of the acidic phosphate groups, however, makes the
coenzymes acidic and profoundly affects the capacity of the molecule for
combining with the apoenzyme (p. 145) .

144 THE BIOCHEMISTRY OF B VITAMINS

Assay Methods. A D-amino acid oxidase system is the one commonly
employed for the qualitative and quantitative determination of flavin
adenine dinucleotide.63 No other naturally occurring flavin derivative is
known to exhibit appreciable activity in this assay ; hence, it is used as a
specific test for the dinucleotide coenzyme. The apoenzyme concentrate
used in the assay is obtained by resolving the enzyme present in crude
extracts prepared from hog kidney. As in most flavoproteins, the pigment
dissociates from the protein when a solution of the holoenzyme is acidified,
and the protein component can usually be separated by ammonium sulfate
precipitation. D-alanine is the substrate most often used with this sytsem,
and oxygen is used as the hydrogen acceptor, since methylene blue is
reduced very slowly. The rate of the reaction is followed by measuring
the oxygen uptake or by determining quantitatively either the amount
of ammonia or of the keto acid formed by the oxidation of the amino acid.

No enzymatic method has been developed to determine specifically
riboflavin phosphate. The use of the "old yellow enzyme" — the system
in which riboflavin phosphate was first recognized — will not suffice, since
the system is quite nonspecific in regard to its coenzyme requirements.
The dinucleotide, as well as certain riboflavin derivatives prepared syn-
thetically, can reactivate the apoenzyme (p. 150) . Cytochrome reductase
or L-amino acid oxidase, the only other enzyme systems whose coenzymes
are definitely known to be riboflavin phosphate, may be used. Whether
these systems are specific or not, however, is uncertain since no report has
been made concerning the activity of the dinucleotide when tested with
the apoenzymes of these proteins.

Occurrence. Qualitatively, it is known that the coenzymes of ribo-
flavin have a widespread distribution in cells and tissues. The presence
of flavoenzymes (especially the dinucleotide type) has been demonstrated
in a variety of material, and the coenzymes themselves have been con-
centrated and isolated from microorganisms and several different animal
tissues. Much of the intracellular riboflavin is in a bound form from
which it can be liberated by phosphatases. If riboflavin resembles the
other B vitamins in respect to the distribution of the free vitamin and
its coenzymes, one would expect the intracellular vitamin content to
reflect predominantly the concentration of the coenzymes. However, prac-
tically no quantitative data, based on actual determination of the coen-
zymes, are yet available, nor is it possible to estimate the relative con-
centrations of the two coenzymes.

Biosynthesis. The biosynthesis of riboflavin phosphate from the vita-
min has been carried out in vitro by using phosphorylating enzymes of
the intestinal mucosa of mammals.64 The biosynthesis of the dinucleotide
has never been observed except in intact cells. Since this coenzyme cannot

COENZYMES DERIVED FROM B VITAMINS 145

be prepared synthetically, it can be obtained only by direct isolation from
tissues or yeast, or by separating it from preparations of its flavoproteins.
The ability to synthesize the two coenzymes must be common to most
living organisms, since no instances are known in which either of the
intact coenzymes must be supplied to an organism.

Properties of the Flavoproteins. When the riboflavin coenzymes com-
bine with their apoenzymes to form the flavoproteins, certain changes
can be detected in the properties of the pigment. The flavin no longer
exhibits its characteristic fluorescence, although its yellow color is im-
parted to the enzyme.62

The redox potential of the flavoprotein system is appreciably higher
than for the uncombined flavins, i.e., the flavoprotein has a greater tend-
ency to accept hydrogen atoms and become reduced.62

Several different lines of evidence indicate that riboflavin phosphate
is united to its apoenzyme through both the acid group of the phosphate
ester and the imide nitrogen of the isoalloxazine nucleus, presumably by
the formation of salts with acidic and basic groups of the protein: the
affinity of unphosphorylated riboflavin for the protein is small; the sub-
stitution of groups upon the imide nitrogen destroys all vitamin and
coenzyme activity; the formation of salts of the imide is known to destroy
the fluorescence of isoalloxazines; and the flavin loses its fluorescence
when it combines with apoenzymes to form the flavoproteins.

Unlike the nicotinic acid coenzymes, the flavin coenzymes do not have
to alternate from one apoenzyme to another during the progress of a
reaction. Hence, (in neutral solutions) the riboflavin enzymes are only
slightly dissociated. For this reason, when the "old yellow enzyme" is
resynthesized from its resolved parts, the apoenzyme takes up the added
riboflavin phosphate in stoichiometric fashion and is completely saturated
by the time an equimolecular amount of coenzyme is added. Flavoproteins
are not readily resolved by dialysis in neutral solution, although they are
easily dissociated in acidic solutions in which the pigment dialyzes away
from the protein.65 A simpler procedure is to salt out the protein from
the acid solution leaving the prosthetic group in the supernatant liquid.66
This difference in the stability of the intact enzyme under neutral and
acidic conditions is probably due to ionic and tautomeric changes in the
groups of the coenzyme and the protein which form the salt linkages.

In addition to the differences in motility of their respective coenzymes,
there are a number of other dissimilarities in the enzymatic reactions
catalyzed by flavoproteins and those catalyzed by the nicotinic acid sys-
tems: (1) a considerably smaller number of reactions have been charac-
terized for the flavoproteins; (2) there is much more specificity in the
coupling of the two systems which oxidize and reduce the flavoproteins

146

THE BIOCHEMISTRY OF B VITAMINS

than there is for the nicotinic acid enzyme systems, e.g., a fiavoprotein
which is reduced by a particular substrate can be reoxidized only by cer-
tain specific substances, whereas a pyridine nucleotide reduced by one
metabolic system can be reoxidized by any of a number of other metabolic
systems; (3) many of the reactions catalyzed by the phosphopyridine
coenzymes are those in which the potentials of the reacting systems ap-
proximate those of the coenzymes, and hence the reactions may proceed
in either direction. The redox potential of riboflavin, however, is inter-
mediate between the low values of most organic metabolite systems (and
the nicotinic acid coenzymes) and the high values of the cytochrome
systems or oxygen itself. The differences are of sufficient magnitude that
most reactions catalyzed by flavoproteins may proceed effectively in one
direction only. Consequently, reversal of the direction in which a reaction
catalyzed by a fiavoprotein proceeds is seldom encountered, and it has
become customary to specify one system as the hydrogen donor and the
other as the hydrogen acceptor, rather than to treat them in general
terms of coupled systems.

When the coenzymes of riboflavin are alternately reduced and oxidized
it is believed to be by a process in which the hydrogen atoms are accepted
or donated one at a time. This mechanism is possible since the flavin
nucleus can exist in an intermediate state of reduction in which a stabi-
lized semiquinoid radical is formed — a process which can be observed
experimentally.62 The predominant structures among the various resonance
forms of the coenzymes are indicated as :

H3C-

N N

A/ \/ \

stabilized +H

semiquinoid ^. "*•

radical — H

H3C-

I H

N N

vv v \

H ||
O

Reactions Catalyzed by Flavoproteins. For discussion, the reactions
catalyzed by flavoproteins may be classified in two groups: (1) those in
which the enzyme reacts directly with the primary substrate being metab-
olized, and (2) those in which the pigment is a secondary acceptor of
the hydrogen atoms. The substrates with which the flavoproteins have
been shown to react directly are D-amino acids, L-amino acids, glycine,
L-hydroxy acids, aldehydes, purines, and substances not ordinarily en-
countered under natural conditions, such as quinine and reduced dyes.
In the other group of reactions, the fiavoprotein reacts with an "inter-
mediate carrier," which is either one of the two dihydropyridine nucleo-

COENZYMES DERIVED FROM B VITAMINS 147

tides or a reduced thiamine system which has catalyzed an oxidative
decarboxylation (p. 165).

The reactions of flavoproteins could equally well be classified on the
basis of the substances which have been found to be hydrogen acceptors:
(1) the cytochromes, (2) molecular oxygen, (3) fumaric acid, and (4)
artificial acceptors such as methylene blue.

An interpretation of the primary intracellular functions of flavoproteins
on the basis of results obtained from isolated proteins is hazardous. A
number of instances have been observed in which it appears that the
enzyme after isolation has lost its ability to react with certain substrates.
The rate at which some of the isolated enzymes catalyze reactions in vitro
is too slow to enable them to be of importance in vivo. With some flavo-
proteins which have been isolated it has been impossible to reconstruct
systems using substrates known to occur in living cells. It is still impos-
sible to characterize completely the most important (from a quantitative
standpoint) reaction which flavoproteins mediate in aerobic organisms,
the dehydrogenation of reduced dihydro-diphosphopyridine nucleotide.

The changes which may occur in the enzymatic capacities of the flavo-
proteins during their concentration make it difficult to establish the iden-
tity of enzymes studied by different investigators. On several occasions
enzymes have been obtained from the same source material in separate
laboratories and not all their properties have, checked. Consequently, the
reactions of the flavoproteins will be classified on a general basis accord-
ing to their substrates and no attempt will be made to characterize the
individual enzymes as reported.

Amino acid oxidases. Oxidation of amino acids is a two-step process
in which (1) the a-amino group is dehydrogenated to form an a-imino
acid, and (2) the imino group hydrolyzes spontaneously to yield ammonia
and the corresponding keto acid.

R— C

NH2 NH O

-2H || +H2O ||

COOH — >• R— C— COOH — > R— C— COOH + NH3
H

a-amino acid a-imino acid a-keto acid

Three types of amino acid oxidases which are flavoproteins have been
recognized: (1) D-amino, (2) L-amino, and (3) glycine. The potential
of these systems is such that the reverse process could not be effectively
utilized directly for amino acid synthesis.

D-amino acid oxidase catalyzes the oxidation of all the common
D-amino acids except D-lysine, although there are extreme differences in
the rate of reaction of the individual amino acids.67 The enzyme occurs
in most animal tissues, but its purpose is not understood since its sub-

148 THE BIOCHEMISTRY OF B VITAMINS

strates are not the naturally occurring isomers. Only the dinucleotide can
function as the prosthetic group of this flavoprotein.

L-amino oxidase 68 (L-hydroxy acid oxidase) is an interesting and
unusual enzyme in that it will not only catalyze the oxidation of at least
thirteen amino acids but will also bring about comparable dehydrogena-
tion of L-a-hydroxy acids having structures related to the a-amino acids.69
Its coenzyme is riboflavin monophosphate.70 The enzyme was isolated
from rat kidney. It has been shown that certain bacteria possess a similar
enzyme, some properties of which differ from the animal preparations.71
Molds are the source for a third preparation, the activity of which differs
in minor respects from both the mammalian and bacterial enzymes.72
One of the active principles in snake venom is an L-amino acid oxidase,
and although its relation to the other oxidases just mentioned has not
been ascertained, it is believed to be a flavoprotein.73

Glycine is oxidized by neither of these types of enzymes, but is attacked
by a specific enzyme, glycine oxidase.74 The cofactor is flavin adenine
dinucleotide.

Aldehyde oxidases. It has been shown that there are three flavo-
proteins capable of catalyzing the oxidation of aldehydes. It is believed
that each enzyme of this group contains adenine dinucleotide as its pros-
thetic group. Milk was the first substance to be used as a source of an
"aldehyde oxidizing" flavoprotein, an enzyme which catalyzes the de-
hydrogenation of formaldehyde hydrate and other aliphatic and aromatic
aldehydes.75 Strangely enough, this enzyme, or another flavoprotein which
cannot be separated from it, was later found to catalyze the oxidation
of hypoxanthine and xanthine.76 A second aldehyde oxidase was isolated
from plant tissues,77 but this enzyme was incapable of oxidizing purines.
That it is likewise a flavoprotein has never been demonstrated. Liver has
been found to contain an aldehyde oxidase which differs from liver
xanthine oxidase.78 Penicillium notatum produces a flavoprotein which
was prematurely classified as an antibiotic, notatinP Further investiga-
tion showed this flavoprotein to be a glucose oxidase. The bactericidal
effect of this enzyme is observed only in the presence of glucose and
oxygen in which case hydrogen peroxide accumulates, killing bacteria
which do not contain peroxidases.80 It has been shown that xanthine
oxidase can be made to demonstrate similar action against certain
bacteria.81

Xanthine oxidase. Xanthine oxidase catalyzes the oxidation of hypo-
xanthine and xanthine to uric acid by removing two hydrogen atoms
from the hydrated purine base.

Milk serves as an especially good source of this enzyme, although it is
found widely distributed in animal tissues. Highly purified preparations

COENZYMES DERIVED FROM B VITAMINS 149

of this enzyme show it to be a flavoprotein containing the riboflavin
adenine dinucleotide prosthetic group 66, 82 and perhaps an additional
nonflavin cofactor.83 No naturally occurring purine other than hypo-
xanthine and xanthine is acted upon by this enzyme.84 This high spe-
cificity is interesting when compared with the much lower substrate
specificity exhibited by the aldehyde and amino acid oxidases.

o OH OH

» i S A H

C N C N C N

J C X +H20 N C \ / -2H N C

I CH — *. | || C — >- | II

3 C / HO— C C / \ HO— C C

V/V V/ \ / oh \/ V>

N N N N N N

H H H H

xanthine xanthine hydrate uric acid

Oxygen is the common hydrogen acceptor for the amino acid, aldehyde
and purine oxidases. Methylene blue can be substituted in most of the
reactions if the Thunberg technique (rate of decolorization) is used. In
the case of the D-amino acid oxidase, however, the dye is reduced much
more slowly than is oxygen. In no case is there any evidence that a
cytochrome system is linked with these flavoproteins which catalyze the
direct oxidation of metabolites, i.e., when riboflavin coenzymes directly
accept the hydrogen atoms of organic substrates (rather than through the
intermediation of the pyridine nucleotides) , they transport these atoms
directly to oxygen instead of reducing a cytochrome.

The second group of flavoproteins differs from those just discussed in
at least two important respects: (1) they do not directly catalyze the
dehydrogenation of a metabolite, but have for substrates the reduced
forms of intermediate hydrogen carriers; (2) although they react with
oxygen slowly, cytochrome systems are believed to be the hydrogen
acceptors in vivo.

Flavoproteins can accept hydrogen atoms from three different reduced
coenzyme-enzyme systems: (1) diphosphopyridine nucleotide, (2) tri-
phosphopyridine nucleotide, and (3) probably a reduced thiamine enzyme
system.

The oxidation of reduced diphosphopyridine nucleotide is the specific
function of flavoproteins (designated diaphorases) isolated from yeast,61
heart muscle,85 and milk.86 They possess the following common properties:
(1) their coenzymes are dinucleotides; (2) they can be oxidized by
methylene blue or oxygen, although the latter process is slow; and (3)
there has been no definite demonstration of a system linking these
particular flavoproteins with any specific cytochrome system. Although
it is still believed that there is some means by which these flavin mediators
can pass the hydrogen atoms from reduced diphosphopyridine nucleotide

150 THE BIOCHEMISTRY OF B VITAMINS

to a cytochrome system, and although extensive study has been devoted
to this problem, as yet the proof of such a mechanism is lacking.

The oxidation of the reduced triphosphopyridine nucleotide is carried
out by cytochrome c reductase. This enzyme has been isolated from yeast
and differs from the diaphorases mentioned above in that (1) its coen-
zyme is the riboflavin mononucleotide, (2) its substrate is the triphospho-
pyridine coenzyme, and (3) that the cytochrome system to which it is
linked has been identified.87

Two flavin enzymes: (1) the "old yellow enzyme" 55 which was isolated
from yeast and contains riboflavin phosphate as the prosthetic group,
and (2) the synthetic yellow enzyme 66 in which the dinucleotide is
substituted for riboflavin phosphate upon the same apoenzyme, may act
upon either the di- or triphosphopyridine nucleotides. No cytochrome
system has been shown to function with either of these enzymes; the
flavoproteins in this case pass the hydrogen atoms directly to oxygen
at a sluggish rate and the protein is believed to be a "derived" enzyme
rather than a "native" one.

The other secondary hydrogen donor that is coupled with flavoproteins
is the reduced thiamine system. When thiamine functions in oxidative
decarboxylation reactions involving the removal from a-keto acids of
carbon dioxide and two hydrogen atoms (see p. 165) , either the thiamine
coenzyme or some group of the apoenzyme must be temporarily reduced.
It has been shown, using a crude protein extract from bacteria, that a
flavin dinucleotide must be present when this type of reaction is carried
out aerobically.88 On this basis it has been postulated that a yellow
enzyme is essential for the reoxidation of the reduced thiamine systems.

The mechanism by which energy is conserved in aerobic respiration
is still not clear. Energy balances indicate that there are approximately
three high-energy phosphate bonds formed for every two hydrogen atoms
passed from a substrate via the phosphopyridine and riboflavin nucleo-
tides to oxygen.89

The flavin-containing enzymes which have been enumerated function
primarily as carriers of hydrogen atoms in processes which are believed
to be unidirectional in their natural environment, i.e., the net process is
always one in which organic substrates are the hydrogen donors, and
molecular oxygen (in vivo) is the final acceptor of these atoms. Does
riboflavin function in anaerobic systems? Undoubtedly flavoproteins
catalyze reactions carried out by anaerobic organisms, and in these in-
stances the enzymes must have some organic substrate as a hydrogen
acceptor. To date only one example of this type of reaction has been
disclosed i.e., fumaric dehydrogenase, a flavin adenine dinucleotide

COENZYMES DERIVED FROM B VITAMINS 151

as yet no naturally occurring hydrogen donor has been found and the
catalytic role of the flavoprotein can be demonstrated only when an arti-
ficial donor (a leuco dye) is used.

It is of interest to note that a flavoprotein can catalyze the conversion
of fumaric to succinic acid because it may be indicative of a general
function of flavoproteins which has not yet been established — that of
being a general catalyst for the production or saturation of ethylenic
bonds. A difficultly soluble protein, succinoxidase, catalyzes a similar
reaction (reverse direction) in which succinic acid is dehydrogenated and
a cytochrome is reduced.91 Whether this enzyme is a flavoprotein is ques-
tionable, but it has been shown that the concentration of succinoxidase is
less in the tissues of animals depleted of riboflavin than in the tissues of
animals on adequate diets.92

The Cytochromes

Numerous compounds which are derivatives of porphyrins are essential
for extremely diverse types of biological function ranging from the
oxygen-transporting duties of hemoglobin to the energy transformations
catalyzed by chlorophyll. The porphyrins of particular interest in a dis-
cussion of the functioning of the B vitamins are the ones which act in the
same oxidation systems as do riboflavin and nicotinic acid. These com-
pounds are the cytochromes. It is believed that in the cells of all aerobic
organisms the cytochromes act as the final mediators in most, but not all,
processes in which the utilization of oxygen takes place.

The porphyrins have many properties in common with the B vitamins :
they are found in all forms of life which make use of aerobic processes;
their distribution parallels other B vitamins ; and they constitute a nutri-
tional requirement for some bacteria and protozoa.93 Except for one class
of insects,93a no higher types of life, however, have been encountered
which cannot synthesize their own requirements for the porphyrins. Since
all the known reactions of the porphyrin-containing enzymes are those
in which molecular oxygen is involved, it is not surprising that organisms
which are true anaerobes do not contain detectable amounts of porphyrin
derivatives.

There is still considerable confusion regarding the identity and chemical
function of the cytochromes that have been recognized and characterized
almost entirely on the basis of their absorption spectra — cytochromes a,
ai, a2, a3, b and b2.94 Two cytochrome components, cytochrome c and
cytochrome c oxidase, however, have been well characterized on the basis
of their enzymatic activity, and their function in biological oxidations
has been well established.

152 THE BIOCHEMISTRY OF B VITAMINS

Cytochrome c is a protein (M.W. about 13,000) containing as a pros-
thetic group the same iron-porphyrin unit, hematin, as occurs in hemo-
globin.95 Unlike hemoglobin, though, it functions by changing its valency
state, alternately existing as a ferro and ferric complex. It is not auto-
oxidizable but can react with molecular oxygen only in the presence of a
specific enzyme, cytochrome c oxidase, which is itself a porphyrin deriva-
tive.

The reduction of cytochrome c in vitro at a rate compatible with the
requirements of an actively metabolizing cell can be demonstrated either
with (1) certain dehydrogenases of organic metabolites for which no
other coenzyme requirement is known to exist, or with (2) the reduced
triphosphopyridine nucleotide, provided the neecssary flavin catalyst,
cytochrome c reductase, is present.S7 It seems logical to expect that a
third type system, involving the enzymes by which the majority of
dehydrogenations are initiated, the diphosphopyridine dehydrogenases,
would likewise be associated with the cytochrome c mechanism. In spite
of an intensive search, the enzyme (flavoprotein?) capable of linking the
two systems has yet to be well characterized, although crude preparations
which link the diphosphopyridine nucleotide-cytochrome c systems have
been reported.96, 97

The dehydrogenases having no recognized coenzymes which presumably
pass their hydrogen atoms directly to cytochrome c are: succinic acid
dehydrogenase,91 lactic acid dehydrogenase,98' " oc-glycerophosphate de-
hydrogenase II,100 a formic acid dehydrogenase,101 a fatty acid dehydro-
genase,102 and sarcosine dehydrogenase.103 Most of these enzymes are
intimately associated with the cell structure and cannot be brought into
solution. Since tissues from riboflavin deficient animals have a subnormal
amount of succinic acid dehydrogenase,92 a question can be raised con-
cerning the possibility that not only this enzyme but also other insoluble
cytochrome-linked enzymes may be flavoproteins.

In addition to these natural enzymatic systems, a number of phenols
and amines can directly (nonenzymatically) serve as artificial agents for
reducing cytochrome c.95

As is the case with the other dehydrogenase coenzymes, the reduction
of cytochrome c is brought about by a univalent change in which the
cytochrome c accepts the hydrogen atoms one at a time from the organic
substrates or reduced coenzymes. Each atom so transferred in effect
reduces a ferric-porphyrin group to the ferro complex with the accom-
panying creation of a hydrogen ion:

[H]+Fe+++ complex "^-^ H++Fe++ complex

The reduction causes a very distinctive alteration in the absorption
spectra of the porphyrin complex which can be followed spectrophoto-

COENZYMES DERIVED FROM B VITAMINS 153

metrically.104 An even more sensitive method is a polarigraphic one.105
The reoxidation of reduced cytochrome c is brought about by molecular
oxygen in the presence of cytochrome oxidase, a porphyrin-containing
protein so intimately bound within the structure of cells that it was once
assumed that it could not be extracted and obtained in solution. A soluble
preparation, however, has been recently reported.106 The exact mechanism
of the reaction catalyzed by this enzyme is obscure. Since no hydrogen
peroxide results from the reaction, the oxygen molecule either is reduced
by a process which does not proceed through the reduction state corre-
sponding to hydrogen peroxide, or else this state constitutes an unstable
intermediate which is instantaneously decomposed. In view of the general
peroxidase activity of the iron-porphyrin enzymes, the latter postulate
seems the more reasonable.

When the cytochrome-cytochrome c oxidase systems are blocked by
the heavy metal poisons, CN-, H2S, NaN3, etc., their function in recon-
structed systems or in intact cells can be partially taken over by appro-
priate autooxidizable dyes. When these artificial hydrogen carriers are
used, oxygen is always reduced to hydrogen peroxide.

One more type of iron-porphyrin enzyme, catalase,107 should be men-
tioned, to complete the presentation of the catalysts involved in dehydro-
genations. In all reactions in which the utilization of molecular oxygen
is accomplished by enzymes or catalysts not containing iron or copper,
hydrogen peroxide is formed. If the peroxide were not decomposed at once
it not only would destroy the enzyme system catalyzing its formation but
would inactivate most adjacent proteins as well. Consequently, catalase
is an essential component of all cells having aerobic metabolic processes,
and hence is found widely distributed in nature.

Coenzymes Essential for the Carboxylation and Decarboxylation of Keto
Acids

In the metabolic processes essential for life, a number of keto acids are
continually being formed and utilized. Probably no other type of com-
pound is capable of participating in such a variety of enzymatic reactions.
Because of this, they occupy key positions in most metabolic processes
and are the essential links which interconnect the metabolism of carbo-
hydrates, proteins, and fats. From the standpoint of molecular turnover,
one of the most important mechanisms by which these compounds are
metabolized is decarboxylation, resulting in the cleavage of a carbon-to-
carbon bond and formation of carbon dioxide (or sometimes its reductive
product, formic acid). The reactions can most conveniently be grouped
together as decarboxylase processes. On the basis of the coenzymes
needed as catalysts, this group can be broken down into two definite
classes: those which require thiamine pyrophosphate and those which do

154 THE BIOCHEMISTRY OF B VITAMINS

not. These two classes are also chemically distinct with respect to whether
they involve a- or /?-keto acids.

a-Decarboxylation. Thiamine is essential only when the substrate is
an a-keto acid and only when the reaction results in the rupture of the
bond between the keto carbon atom and the adjacent carboxyl group.

^-Decarboxylation. The other reactions in which carbon dioxide is
formed from a keto acid are those which have been termed ^-decarboxyl-
ations because they result in the direct decarboxylation of acids in which
the keto group is /? to the reacting carboxyl group. Biotin has been asso-
ciated with this type of reaction, although it is questionable if its role
in these reactions is a direct one.

The Coenzyme Derived from Thiamine

Carboxylase, an enzyme which converts pyruvic acid to carbon dioxide
and acetaldehyde, was one of the first components to be recognized in
zymase, the complex of enzymes used by yeast in fermenting sugars.108
Some twenty years after its discovery it was shown (1932) that an
essential thermostable organic component could be removed from the
holoenzyme by washing with weakly alkaline solution.109 This coenzyme
was designated as cocarboxylase. The isolation of the coenzyme in
crystalline form was achieved by using an enzymatic assay method (re-
activation of a carboxylase apoenzyme) to follow the concentration of the
active principle. The chemical structure was established by both degrada-
tion and synthesis and found to correspond to the pyrophosphoric acid
ester of thiamine.110

CH,

O O

=C— CH2— CH20— P— 0— P— OH

OH O-

Thiamine pyrophosphate

The compound has been most often referred to as cocarboxylase, but
other synonyms and abbreviations are frequently encountered — thiamine
pyrophosphate (TPP), diphosphothiamine (DPT), and aneurin pyro-
phosphate (APP) . This coenzyme should not be confused with code-
carboxylase, the name often used to denote pyridoxal phosphate. The
synonym, thiamine pyrophosphate, has been chosen as the one most
suitable for use in the discussions which follow, since it indicates the
vitamin component, describes the chemical nature of the coenzyme, and
eliminates confusion with other types of coenzymes catalyzing decar-

CH3

[3C-

/ \

-c c-

-NH2

c=c

+/ 1

N C-

-CH2-

-N

\ /

V '

c— s

COENZYMES DERIVED FROM B VITAMINS 155

boxylations. Also, there are instances in which the reactions requiring
"cocarboxylase" do not produce or utilize carbon dioxide.

Thiamine pyrophosphate can be prepared synthetically by chemical
phosphorylation of the vitamin in and can be purchased from commercial
firms which stock biochemicals.

The conversion of thiamine to the phosphoric acid ester profoundly
affects its biological reactivity even though, chemically, the change is not
a drastic one. Upon oxidation the coenzyme forms a fluorescent compound,
thiochrome pyrophosphate, by a reaction analogous to that which pro-
duces thiochrome from the vitamin;* the coenzyme can also be cleaved
by sulfurous acid into a pyrimidine and a phosphorylated thiazole;110
the phosphorylation of the vitamin does not alter the susceptibility of
the molecule to cleavage by the "anti-thiamine" enzyme present in raw
fish for the coenzyme is split just as rapidly by this means as is the
vitamin.

The most obvious chemical change in forming the coenzyme is the
creation of a strongly acidic compound from the organic base. This is
accompanied by an increase in the resistance of the thiazole nucleus to
reduction and reoxidation by chemical agents,112 a fact of some impor-
tance when considering possible mechanisms of the functioning of thi-
amine (p. 168).

Assay Methods. The determination of the thiamine pyrophosphate
content of natural materials is usually carried out by a manometric pro-
cedure in which the rate of pyruvic acid decarboxylation is followed.
The crude preparations of the apoenzyme needed for the procedure are
obtained by washing dried yeast cells with an alkaline phosphate buffer.110
The extent of reactivation of the washed cells by extracts (in the presence
of Mg++) is a direct measure of their coenzyme content.

Thiamine itself often shows some activity when it is added to crude
apoenzyme preparations, presumably because of the presence of phos-
phorylating enzymes which convert the vitamin to the coenzyme during
the course of the determination. When brewers' yeast is used as a source
of the apoenzyme, free thiamine is found to exert an "activating" effect
upon the carboxylase enzyme.113 Thiamine itself is completely inactive
when added to the apoenzyme, yet when added to the apoenzyme along
with the coenzyme, it increases the activity of the reconstructed system.
This effect is observed even when the coenzyme is present in excess ; hence
the thiamine effect cannot be attributed to a direct synthesis of additional
coenzyme. This "activation" effect is believed to be due to the ability of

* The acidic pyrophosphoric acid ester of thiochrome (obtained from the coenzyme)
is not extracted from alkaline solutions by butyl alcohol; hence, the coenzyme does
not interfere in the thiochrome assay for the free vitamin when the usual procedures
are used (p. 46).

156 THE BIOCHEMISTRY OF B VITAMINS

thiamine to inhibit certain phosphatases present in the brewers' yeast
which cause the hydrolysis and inactivation of the intact coenzyme.114
Thus thiamine can appear to be active in the system merely because it is
sparing the destruction of its active derivative. The monophosphoric ester
and the pyrimidine moiety of the vitamin behave similarly in "activating"
the carboxylase system from brewers' yeast.

These interfering activities of the vitamin in the manometric assay for
the coenzyme have been eliminated by using bakers' yeast instead of
brewers',115 by adding sodium iodoacetate to inhibit the phosphorylation
of thiamine,116 or by assaying all preparations in the presence of an excess
of thiamine.117 Concise directions for carrying out the manometric deter-
mination of the coenzyme have been published in a standard reference
text.118 An analyst using this procedure on crude materials should be
aware of the many complicating effects which the extraneous matter can
produce.

No organisms are known in which the thiamine requirements can be
met only by its pyrophosphoric ester. Certain atypical strains of gonococ-
cus respond much better to "a cocarboxylase-like substance" than to
thiamine, but their requirement for the intact coenzyme is not absolute.119

Microbiological and chemical methods can be adapted to coenzyme
analysis of tissues and biological products if it is known that all the
"bound" thiamine is the pyrophosphate and if an assay method is used
in which only the free vitamin is active (yeast growth and thiochrome
methods) . If such is the case, the difference in thiamine content of extracts
before and after treatment with phosphatases will represent the amount
of coenzyme. Due to the differences in solubility of the thiochromes result-
ing from the oxidation of thiamine and its phosphate esters, this chemical
method can be adapted for the determination of both the vitamin and
its phosphorylated derivatives.120

The presence of the coenzyme has been directly demonstrated in a
number of different plant and animal tissues and in microorganisms. From
the results obtained, it would appear that intracellularly most of the
thiamine present is in the form of its coenzyme, whereas in plasma and
other extracellular fluids (including urine and cerebrospinal fluid) the
vitamin occurs predominantly in the free state.121 Practically all the
microorganisms so far tested which cannot synthesize their thiamine
requirements can utilize the free vitamin at least as effectively as the
phosphorylated derivatives, whereas many organisms cannot use the co-
enzyme as a nutrient in place of the free vitamin. It is apparent, then,
that the biosynthesis of the coenzyme must take place within the cells
of most organisms and tissues.

Biosynthesis. The presence of enzymes capable of phosphorylating
thiamine has been directly demonstrated in a number of different types

COENZYMES DERIVED FROM B VITAMINS 157

of cells. The results of studies of the biological synthesis indicate that
adenosine triphosphate is probably the usual phosphorylating agent.122* 123
It is needed in only catalytic amounts, however, if other acyl phosphates
are supplied or if the biosynthetic reaction is coupled with enzymatic
processes in which reactive phosphate derivatives are created. A cell free
extract of rat kidney which converts thiamine to its coenzyme has been
reported.124

The pyrophosphate linkage in the coenzyme is readily hydrolyzed by
the phosphatases (distinct from the phosphorylases catalyzing its syn-
thesis) and usually any excess coenzyme present in a cell will be rapidly
hydrolyzed.114 If, however, the coenzyme is combined with apocarboxylase
it is quite resistant to attack by any hydrolytic enzymes present. This
may be one explanation for the fact that the synthesis of the coenzyme
from thiamine in a number of organisms is observed only as long as
uncombined apoenzyme is present.

Mention has already been made of the inhibition of thiamine pyro-
phosphatases by thiamine and its derivatives. This type of inhibition has
been observed only in certain yeast, however. It probably accounts for
the exceptional case in which considerable unbound coenzyme can be
found in yeast cells. If these particular yeasts are cultured in media
containing appreciable amounts of thiamine, they absorb the vitamin
almost quantitatively and convert most of it to the pyrophosphate (in
amounts much greater than could be bound to carboxylase apoen-
zymes).125 The coenzyme synthesized by these cells is not hydrolyzed,
since their specific phosphatases present have been inactivated by the
high thiamine concentrations.

The pyrophosphate linkage between the two phosphate residues is also
readily hydrolyzed by dilute acid. The monophosphoric acid ester formed
by either the acid or enzymatic hydrolysis of the coenzyme is much more
slowly attacked by phosphatases. The presence of appreciable quantities
of the monophosphoric acid ester of thiamine in natural extracts probably
is the result of the breakdown of the coenzyme during the preparation
of the sample.

The mechanism for the formation of holoenzymes from thiamine pyro-
phosphate and its apoenzymes has been the subject of several studies. The
easily prepared apocarboxylase from yeast has been used as the protein
source for the most detailed investigation.126 The presence of the Mg++
or Mn++ ion is essential for the union. Other divalent ions are much less
effective. In earlier reports it had been assumed that the artificially recon-
stituted holoenzyme was different from the original native system, since
the addition of a given amount of thiamine pyrophosphate to washed
yeast cells did not elicit as great a response as was obtained when un-
washed cells containing the equivalent amount of the coenzyme were

158 THE BIOCHEMISTRY OF B VITAMINS

used.127 It is now believed that the difference is due to the presence of
inactivated apocarboxylase and possibly other apoenzymes which com-
bine with some of the added coenzyme and render it unavailable to the
active apocarboxylase. 12<5

That the pyrophosphoric acid group is at least partially responsible
for the association of the apoenzyme with the coenzyme is indicated by
the inhibition of carboxylase activity by other molecules containing
pyrophosphate groups — adenosine triphosphate 123 and thiazole pyro-
phosphate 12s Ca++ ions interfere with the formation of the holoenzyme,
presumably by competing with the Mg++ or Mn++ ions.129 Consequently,
it is desirable to use water free from calcium in the preparation of
apoenzymes. This interference may account for some of the discrepancies
found in the earlier studies on the recombination of the carboxylase
holoenzymes.

Phosphorylated thiamine does not pass through cell membranes
easily.130 This accounts for its relative inactivity as a thiamine source
for certain microorganisms, and explains why it is not as effective as
thiamine in stimulating decarboxylations by tissues from deficient ani-
mals.131 The impermeability of cell membranes to the intact coenzyme
may account for the effects observed when thiamine and its pyrophos-
phate were tested for their relative activities in reversing the inhibition
of bacterial growth induced by pyrithiamine. The vitamin analogue more
effectively inhibits the coenzyme than it does the vitamin— a phenomenon
not ordinarily encountered.132 To account for this, it was postulated that
thiamine is attached to the apoenzyme before it is phosphorylated, and
that the coenzyme so formed is more firmly bound than is preformed
coenzyme. An equally logical explanation is that the coenzyme added to
the medium must be hydrolyzed before absorption can take place, thus
necessitating subsequent resynthesis. The thiamine added in the free form
would be more rapidly absorbed than would the thiamine which had to
be first liberated from the coenzyme; hence the former would produce a
higher intracellular ratio of thiamine to pyrithiamine and would be the
more effective agent for reversing the inhibition.

Reactions Catalyzed by the Thiamine Coenzyme. The enzymatic reac-
tions in which thiamine has been demonstrated to function in vivo are
limited to only two substrates, pyruvic acid and a-ketoglutaric acid, or
their degradation products. Some of these enzyme preparations have been
found to decarboxylate other a-keto acids — for example, a-ketobutyric
and a-ketovaleric acids. The latter compounds, though, have never been
shown to be a part of metabolic processes, and there seems little reason
to believe that these substances normally occur in vivo. Two other a-keto
acids which are important intermediates often formed during metabolism,

COENZYMES DERIVED FROM B VITAMINS 159

oxalacetic acid and oxalsuecinic acid, may be assumed to undergo decar-
boxylase reactions analogous to those found for pyruvic and keto-
glutaric acids. However, these two acids not only are cc-keto, but also
B-keto acids, and they normally undergo /^-decarboxylation rather than
cleaving at the alpha bond.

The reactions of pyruvic acid which are catalyzed by the thiamine
derivative were shown in 1936 not to be limited to simple decarboxyla-
tions. It was found that the coenzyme for carboxylase also is an essential
component of the enzymatic systems by which pyruvic acid is oxidized
in animal tissue, and that the initial reaction is one in which a dehydro-
genation takes place simultaneously with the decarboxylation.133 A num-
ber of different processes are now known which require the thiamine
coenzyme, but all can be explained on the basis of the catalysis of one
of these two types of reactions: (1) simple decarboxylation of either
pyruvic or a-ketoglutaric acid, or (2) oxidative decarboxylation of one
of these two metabolites.

The experimental work which led to the identification of most of the
reactions in which thiamine pyrophosphate participates has been pre-
sented in detail in several reviews.88- 134, 135

The coenzyme of thiamine has been shown to be essential for reac-
tions in which the following substances are produced from pyruvic acid:
acetaldehyde, acetic acid, molecular hydrogen, lactic acid, a reactive
phosphorylated derivative of acetic acid, formic acid, acetoin (acetyl-
methylcarbinol) , acetylethylcarbinol, dicarboxylic acids containing four
carbon atoms, citric acid, a-ketoglutaric acid, acetoacetic acid, and carbon
dioxide and water. On the basis of the diverse chemical nature of the
substances listed it might be inferred that a number of functions would
necessarily have to be ascribed to the single catalyst. However, two
related hypotheses, independently advanced, suggest mechanisms by
which it is possible to explain in terms of a general type reaction most,
if not all, of the reactions, both aerobic and anaerobic, catalyzed by
thiamine pyrophosphate.136' 137 Although the hypothetical intermediates
postulated for these reactions have not been experimentally demonstrated
(and it may be impossible to do so) it is believed that the extension of
these hypotheses offers the most convenient method for presenting the
reactions catalyzed by the thiamine coenzyme and enables one to better
appreciate the chemical relationships between the various products of
these reactions. The individual reactions will therefore be discussed from
the standpoint of the postulated mechanisms.

The diverse reactions of pyruvic acid catalyzed by thiamine-containing
enzymes can be logically explained if it is assumed that the holoenzyme
activates pyruvic acid in a manner such that three fragments are made

160 THE BIOCHEMISTRY OF B VITAMINS

available for recombination in any one of a number of ways to produce
the recognized products of pyruvic acid metabolism.

0 0 r O -,

H || || H ||

HC— C— C— OH — > HC— C + 2H + C02
H L I I J

postulated
pyruvic acid ketenyl

radical

The specific products formed depend upon the manner in which the
enzymes utilized or disposed of the C2 fragment (designated hereafter as
a ketenyl radical) and the atoms of available hydrogen. The ketenyl
radical can react (1) with water to form acetic acid, (2) with phosphoric
acid to yield a phosphorylated derivative of acetic acid, (3) with acetal-
dehyde (or other aldehydes) to form acetoin (or its homologs), (4) with
another ketenyl radical and hydrogen to form the dimer, diacetyl, or (5)
with the available hydrogen- atoms to form acetaldehyde.

The available hydrogen atoms also must be utilized in some fashion if
they are not accepted by the hypothetical ketenyl radical (forming
acetaldehyde) or the dimer, diacetyl (yielding acetoin). They can be
accepted by another molecule of pyruvic acid, forming lactic acid; they
can be disposed of in the form of molecular hydrogen ; they can associate
themselves with the elements of carbon dioxide to produce formic acid;
or under aerobic conditions, the hydrogen atoms can be taken by ribo-
flavin-containing enzymes to be passed on and eventually accepted by
molecular oxygen. The three reactions of a-ketoglutaric acid known to
be catalyzed by thiamine pyrophosphate are analogous to three of the
reactions in which pyruvic acid is the substrate.

Reactions of Pyruvic Acid Catalyzed by Thiamine Pyrophosphate.
Thiamine pyrophosphate and a divalent cation (Mg++ or Mn++) and the
appropriate apoenzyme have been shown to be the coenzymatic factors
for the following eight reactions:

(1) Simple Decarboxylation. The simplest reaction which thiamine
pyrophosphate catalyzes is the direct decarboxylation of pyruvic acid.
When the keto acid molecule is cleaved, the hydrogen atoms become
attached to the C2 fragment and the end products are acetaldehyde and
carbon dioxide:

0 0

H || ||

IC— C— C— OH — >

r on

H ||

HC— C

1 1

H ||

— > HC— C + C02

H H

L CO, -1

acetaldehyde

COENZYMES DERIVED FROM B VITAMINS

161

This reaction is a necessary step in the production of ethanol from sugar
and constitutes the primary method of pyruvate metabolism in yeast
when cultured anaerobically. Carboxylase, the enzyme catalyzing this
reaction, has been shown to occur in yeast, bacteria, fungi, and higher
plants, but it never has been found to constitute a part of the enzymatic
systems by which carbohydrates are utilized in animal metabolism. Free
phosphoric acid is not required for this particular reaction since no
utilizable energy units are produced.

(2) Acetoin Formation. When acetaldehyde is added to preparations
from animal tissues capable of metabolizing pyruvic acid, it is found
that acetoin is formed.138 The reaction is presumed to involve a con-
densation of a reactive ketenyl radical (arising from pyruvic acid) and
a molecule of acetaldehyde forming diacetyl which then acts as the
acceptor for the two hydrogen atoms.

0 0

H || ||
HC— C— C— OH
H

1 1

H ||
HC— C
H

pyruvic acid

+ — >

2H C02

— >

H
HC— C=0
H H

H
HC— (
H I

:— oh

acetaldehyde

acetoin

+ co2

The requirement for inorganic phosphate ion in connection with this
reaction has not been settled. If an energy-containing phosphate inter-
mediate were formed, it would be decomposed and its energy utilized in
the condensation creating the carbon-to-carbon bond (p. 189) . When
propionaldehyde was used as a substrate with pyruvate instead of
acetaldehyde, the homologue of acetoin, acetylethylcarbinol, was the
product of the reaction.

In the absence of acetaldehyde muscle tissues still produce acetoin
from pyruvate but only at one-fourth the rate, and the yield from a given
amount of pyruvate is only half that which would be obtained in the
presence of acetaldehyde. Although the investigators could not detect
free acetaldehyde as an intermediate under these conditions, it is pre-
sumed that the reaction is the result of a two-step process in which
acetaldehyde is a transitory intermediate. The requirement of phosphate
for this reaction has not been determined.

In certain bacteria acetoin is the primary product from the anaerobic
decarboxylation of pyruvic acid.139 The mechanism of acetoin formation
in Aerobacter aerogenes apparently is somewhat different from that in

162

THE BIOCHEMISTRY OF B VITAMINS

animal tissues, since acetaldehyde has not been shown to increase the
yield, i.e., both of the C2 radicals which condense must be formed from
pyruvic acid. It is possible that added acetaldehyde does not unite with
the enzyme system to form the necessary enzyme-substrate complex,
whereas the "acetaldehyde," or its equivalent, produced in situ condenses
instantaneously. Inorganic phosphate was found to be essential for this
reaction, but no phosphorylated intermediates have been directly demon-
strated.

The reverse reaction, in which carbon dioxide is assimilated, has been
reported.140, 141 The enzyme preparation did not require a thiamine
coenzyme for activation, but no evidence was presented to prove that it
was not a bound component of the material used.

(3) Formic Acid Production. Escherichia coli and certain other bac-
terial species have been shown to cleave pyruvic acid in the presence of
inorganic phosphates. Until recently it was assumed that formic acid and
acetyl phosphate were the primary products of this phosphoroclastic
reaction.142 It has recently been shown, however, that the product first
formed from the C2 radical is not acetyl phosphate but is a related com-
pound whose structure is as yet unknown.24s This compound, which in
this chapter is designated as the "phosphoryl-acetyl intermediate" to
distinguish it from acetyl phosphate, is a very reactive acetylating agent
as well as an efficient phosphorylating agent. It is a participant in all
the reactions known to be mediated by the pantothenic acid coenzyme.
(See p. 191 for a detailed discussion of its recognized properties.) The
reaction by which it is formed may be considered to be one in which (1)
the reactive C2 radical is combined in some fashion with phosphoric acid,
perhaps through a common carrier (designated in the formula as X),
and (2) the hydrogen atoms associate themselves with the carbon and
oxygen atoms which usually form carbon dioxide in the other types of
reactions:

0 0

r 0

H || ||

H ||

[C— C— C— OH

HC— C

co2

HC— C 0

I 1

H I ||

+ — >

— >.

X + HC— OH

0 |

HO— P— 0

HO— P— OH

HO— P-

L Ah

phosphoryl

acetyl formic

intermediate acid

An alternate mechanism, advanced when the reaction product was pre-
sumed to be acetyl phosphate, was that phosphoric acid first formed an

COENZYMES DERIVED FROM B VITAMINS

163

acid-carbonyl addition product with the keto acid before the cleavage of
the carbon-to-carbon bond,143 as indicated below:

H 1

HC— C=0 +
H

pyruvic
acid

H O
O— P— OH

C— OH

H 1
HC— C-

)— OH

0— P-

P— OH

keto acid
addition
product

H
HC— C=0

—OH

O— P— OH

acetyl
phosphate

formic
acid

The acid-keto addition product of this earlier hypothesis could very well
be an intermediate in the mechanisms recently postulated.

The "phosphoryl-aeetyl intermediate," if not used immediately for
acetylation, probably reacts with adenosine diphosphate, producing
adenosine triphosphate and acetic acid. In this way most of the energy
resulting from the degradation of pyruvic acid to acetic acid is conserved
in the formation of a high-energy pyrophosphate bond which can be
used by the cell for subsequent energy-requiring processes. The reactions
for the overall process can be summed up in this equation :

Pyruvic acid +H3PO4+ adenosine diphosphate — >•

acetic acid + formic acid +adenosine triphosphate

Attempts to demonstrate the reversibility of this process led to the
experiments which clarified the nature of the acetyl derivative. Synthetic
acetyl phosphate, when added to formic acid in the presence of the enzyme
system, did not yield measurable amounts of pyruvic acid, but biological
preparations of the phosphoryl-aeetyl intermediate (prepared by an
enzymatic synthesis from acetic acid and adenosine triphosphate) were
found to be almost quantitatively converted to pyruvic acid when an
excess of formic acid was used.142a

(4) Production of Molecular Hydrogen by a Phosphoroclastic Splitting
of Pyruvic Acid. Clostridium butylicum possesses an enzyme which car-
ries out a reaction similar to the one just discussed, except that the avail-
able hydrogen atoms are disposed of as molecular hydrogen instead of
combining with the elements of carbon dioxide to form formic acid.
Acetic acid was first thought to be a primary product of the reaction,144
but when phosphate was found to be an essential part of the system it was
postulated, on the basis of substantial evidence, that acetyl phosphate
rather than acetic acid was first formed, and that the acetic acid was a

164 THE BIOCHEMISTRY OF B VITAMINS

product of the decomposition of acetyl phosphate.144 On the basis of the
recent finding discussed above, it is anticipated that the phosphoryl-
acetyl intermediate will be found to be the initial product of the reaction
rather than acetyl phosphate itself, and that the equation representing
the reaction should be:

pyruvic acid +H3PO4 — >- phosphoryl acetyl intermediate +C02+H2

The reaction as indicated might be the result of the summation of two
individual enzymatic reactions — a phosphoroclastic cleavage producing
formic acid, immediately followed by the decomposition by a hydro-
genlyase of the formic acid into carbon dioxide and molecular hydrogen.
This mechanism is excluded, however, since the enzyme preparations do
not decompose formic acid.

(5) The Acetic Acid-Lactic Acid Dismutation of Pyruvic Acid. One
additional means of hydrogen disposal under aerobic conditions has been
observed in bacterial cultures — the case in which the available hydrogen
atoms are accepted by a second molecule of the pyruvic acid substrate.
The products are acetic acid (phosphorylated derivatives?), carbon
dioxide, and lactic acid.

(a) pyruvic acid +H20 >■ (2H)+acetic acid+C02

(or H3PO4?) (phosphorylated?)

(b) pyruvic acid + (2H) >■ lactic acid

Net: 2 pyruvic acid +H20 — >■ lactic acid +acetic acid +C02

This dismutative anaerobic utilization of pyruvic acid has been observed
in a number of animal tissues,145, 14G> 147 and in several species of bac-
teria.88- 147> 148

There is a question which has not yet been conclusively answered:
Is the reaction in which the second molecule of pyruvic acid is reduced
an independent reaction requiring a separate enzyme? If so, is a hydrogen
carrier necessary to transfer the available hydrogen atoms from the
thiamine enzyme to the pyruvic acid reductase? In one instance in which
this reaction was studied in a cell-free system, the evidence favored the
concept of coupled reactions and indicated that hydrogen carriers are
needed to link the two distinct reactions of this dismutation.88

The use of radioactive isotopes has made it possible demonstrate the
assimilation of carbon dioxide by the reverse process.149

(6) Aerobic Production of Acetic Acid. If a hydrogen acceptor (other
than the intermediates or substrate) is available, pyruvic acid can be
metabolized in the fashion indicated below. Under natural conditions, a
riboflavin-containing protein is believed to accept initially the available

COENZYMES DERIVED FROM B VITAMINS 165

hydrogen from the thiamine system, and these atoms are then aerobically
metabolized via a hydrogen transport system.

pyruvic acid+H3P04+riboflavin-containing enzyme >

phosphoryl-acetyl intermediate +C02+reduced flavoprotein
This type of reaction has been thoroughly studied using preparations
from Lactobacillus debruckii as the enzyme source.133 Phosphoric acid is
an essential component of this system and undoubtedly is utilized in the
same manner as in the reactions previously described. If, however, the
phosphoryl-acetyl intermediate is not required for synthetic purposes, it
is degraded, its available energy dissipated as heat, and acetic acid
becomes the end product of the process:

phosphoryl-acetyl intermediate >

acetyl phosphate — >. acetic acid+H3P04

(7) Aerobic Utilization of Pyruvic Acid. Many of the diverse "re-
actions" in pyruvic acid metabolism formerly postulated are now believed
to consist of a series of two or more enzymatic steps. All the processes
have a common initial reaction catalyzed by thiamine pyrophosphate.
This reaction is analogous to the one just described occurring in L.
debruckii, except that in this case a pantothenic acid enzyme picks up
the phosphoryl-acetyl intermediate from the thiamine system:

pyruvic acid+H3P04+riboflavin-containing enzyme + pantothenic acid
coenzyme >■ C02+reduced flavoprotein 4 phosphoryl acetyl inter-
mediate associated with pantothenic acid coenzyme

The many different ways in which the phosphoryl-acetyl intermediate
can be utilized are taken up in the discussion of pantothenic acid function,
but some of the important end products which have been associated with
pyruvic acid and thiamine metabolism will be enumerated here. The
phosphoryl-acetyl intermediate produced from pyruvic acid by the
thiamine-catalyzed reaction is, in the presence of a suitable pantothenic
acid system, used for: acetylating choline; acetylating aromatic amines;
forming acetoacetic acid and its homologues, which are intermediates in
fatty acid synthesis; condensing with oxalacetic acid to form cis-aconitic
acid, which is a precursor of citric acid, a-ketoglutaric acid (and glutamic
acid), the C4 dicarboxylic acids, etc. For this reason thiamine or its
coenzyme has been reported at one time or another as a necessary catalyst
for each of these processes.

The condensation of the reactive intermediate with oxalacetic acid
initiates the tricarboxylic acid cycle by which pyruvic acid is completely
"oxidized" to carbon dioxide and water (p. 224). Consequently, normal
pyruvic acid metabolism in animal tissues can proceed only in the

166

THE BIOCHEMISTRY OF B VITAMINS

presence of pantothenic acid and catalytic amounts of some C4 dicar-
boxylic acid (precursor of oxalacetic acid) in addition to a divalent ion,
inorganic phosphate, thiamine pyrophosphate, a hydrogen transport
system, and oxygen.

(8) Aerobic Oxidation of Pyruvic Acid in the Absence of Phosphate.
Cell-free preparations have been prepared from animal tissue and bacteria
which are capable of oxidizing pyruvic acid in the absence of inorganic
phosphate.150 The reaction observed is:

pyruvic acid + 3^ 02 — >■ acetic acid+C02
Thiamine pyrophosphate is an essential component of this system. Since
a requirement for, or presence of any other cofactors, could not be
demonstrated, the disposition of the hydrogen atoms in this system must
be by a mechanism which has not been previously encountered. When
cells metabolize pyruvic acid in this fashion they presumably cannot
conserve in a chemical form the energy of the oxidation.

In the presence of thiamine pyrophosphate, the enzyme preparation
referred to above can carry out another reaction, not wholly unrelated —
the dismutation of diacetyl.150 It may appear that this dismutation
represents a new type of thiamine function, since it involves neither a
keto acid nor a decarboxylation. However, if only one enzyme in the
preparation is responsible for both pyruvic acid oxidation and diacetyl
dismutation, the latter reaction can be considered as one in which the
enzyme establishes an equilibrium between two different sets of end
products of pyruvic acid metabolism through formation of their common
intermediates, namely, ketenyl radicals and available hydrogen atoms.
The equilibrium is such that the fragments, upon recombination, form
primarily acetic acid rather than diacetyl; but the mechanism for the
formation of acetic acid is the same as if the fragments had been formed
by the decomposition of pyruvic acid. The overall reaction can be repre-
sented in this fashion:

O O

H || || H

HC— C— C— CH

H H

(a) +
2H20

O O
H || l| H

(b) HC— C— C— CH + [2H]

H H

H II

HC— C

1 ' 2H

H OH HO H

II H
C— CH

I I

O OH
H || | H
HC— C— C— CH
H H H

2HC— C + [2H]

H in

o o

II II

Net: 2 H3C— C— C— CH3 + 2 H20
diacetyl

O O OH

II II I

2 H3C— C + H3C— C— CH— CH3

acetic acid acetoin

COENZYMES DERIVED FROM B VITAMINS 167

Reactions of a-Ketoglutarate Catalyzed by Thiamine Pyrophosphate.

ot-Ketoglutaric acid has been shown to be enzymatically decarboxylated
by three different mechanisms.

(1) Simple decarboxylation160

O 0 0 o o

HO— C— CH2— CH«— C— C— OH — >■ HO— C— CH2— CH2— CH + C02
a-ketoglutaric acid succinylsemialdehyde

(2) Aerobic oxidation requiring phosphate151

O 0 0

HO— C— CH2— CH2— C— C— OH + H3P04 — >
a-ketoglutaric acid

O O

II II o

HO— C— CH2-CH2— C— 0-P— OH + C02 + [2H](/7apo-pro^n?)

OH
succinyl phosphate

(3) Aerobic oxidation independent of phosphate150

0 0 0 0 0

HO— C— CH2— CH2— C— C— OH + V202 — > HO— C— CH2— CH2— C— OH + C O
a-ketoglutaric acid succinic acid

Each of these reactions resembles a comparable one in which pyruvic
acid is the substrate; hence they need not be discussed in detail. The
apoenzymes for the two substrates have similar physical properties, but
they are not identical and cannot substitute for one another. The same
ions (Mg++ or Mn++) are required as cofactors. No demonstration has
been made of the existence of a phosphorylated succinyl compound
analogous to the phosphoryl-acetyl intermediate; hence succinyl phos-
phate is shown as the initial product of the phosphorylative oxidation.

A phosphoroclastic cleavage, forming succinyl phosphate and formic
acid, has never been shown to occur in any organism. However, in
muscles perfused with pyruvic acid considerable amounts of succinic
and formic acids accumulate (p. 197). This suggests that there may be
enzymes present to handle the a-ketoglutaric acid (formed from pyruvic
acid) by such an anaerobic cleavage if the oxidative decarboxylation
system is overtaxed or not functioning.

Relationship of the Structure of Thiamine to its Function. Attempts
have naturally been made to correlate the structure of thiamine with the
mechanism by which its coenzyme functions. The initial formation of a

168 THE BIOCHEMISTRY OF B VITAMINS

Schiff's base by the elimination of the elements of water from the amino
group of thiamine and the carbonyl group of the substrate has been
postulated.137 The essentiality of the amino group on the pyrimidine ring
of the vitamin suggests the formation of such an intermediate, but its
existence has not been demonstrated.

Whenever the reaction catalyzed is of an oxidative type the enzyme sys-
tem is momentarily in possession of the equivalent of two available hydro-
gen atoms. The enzyme system must, therefore, exist in both an oxidized
and reduced state. It has been postulated that the coenzyme would be the
most likely component of the enzyme system to undergo a reversible
oxidation and reduction, since such is the case in other enzymes trans-
porting hydrogen atoms. The possibility that some group of the apoenzyme
component (rather than the coenzyme) may be the actual hydrogen
carrier, possibly by constituting a thioldisulfide system, should not be
overlooked. Attempts were first made to demonstrate a reversible reduc-
tion and reoxidation of the thiazole nucleus in a manner analogous to
the pyridine-dihydropyridine interconversions of the nicotinic acid co-
enzymes. This possibility is no longer seriously considered. However, it
has been recently pointed out that the dihydrothiamine pyrophosphate
has never actually been prepared, since all the attempts to reduce the
thiazole nucleus chemically resulted in a cleavage of the molecule at the
methylene bridge connecting the two aromatic nuclei of the vitamin.152

A second mechanism, wherein an oxidized and reduced state of thiamine
would also exist, has been postulated on the basis of the observed thia-
mine activity of "thiamine disulfide," a dimer in which the thiazole
nucleus opens.153, 154 If the suggested equilibrium occurs, the structure
always ascribed to the vitamin represents the reduced, rather than the
oxidized form. Although the disulfide analogues of either thiamine or its
coenzyme are active when tested with intact cells or organisms, these
compounds do not reactivate cell free preparations of apocarboxylase.155
This would indicate that the disulfides are not active oxidized forms of
the vitamin or coenzyme but are instead compounds which, although
inactive per se, can be reduced by cells to form the vitamin or coenzyme
having an intact thiazole nucleus. Since the decarboxylation in which
thiamine disulfide was tested is a nonoxidative one, it can justifiably be
argued that the reaction should not be used for testing the validity of
any hypothesis concerned with oxidized and reduced states of the
coenzyme.

The Function of Thiamine. In making a statement concerning a
general mode of action for the thiamine coenzyme in the decarboxylation
of a-keto acids, one should consider three questions:

COENZYMES DERIVED FROM B VITAMINS

.i-4

9 «

-o— o-
w

-QQ

I
o

o— u

170 THE BIOCHEMISTRY OF B VITAMINS

(1) Are there other cof actors which can catalyze the decarboxylation
reactions of pyruvic and ketoglutaric acids? A cof actor which is essential
for the oxidative decarboxylation of pyruvic acid by certain bacteria has
been demonstrated recently.100 Its structure is not yet known, but on the
basis of stability studies it cannot be related chemically to the usual
coenzyme. However, no report has been made which would justify the
conclusion that the system does not also contain thiamine pyrophosphate.

(2) Is thiamine pyrophosphate necessary for the biological decar-
boxylation of other a-keto acids? One enzyme system in which oxalacetate
is cleaved by an a-decarboxylation (oxidative) rather than a /3-cleavage
has been reported.157 This is an oxidative decarboxylation and produces
malonic acid. The reaction is analogous to the oxidative decarboxylation
of a-ketoglutaric acid in which succinic acid is produced. However, in
this instance, the enzyme has been shown to be a porphyrin-containing
protein and contains no thiamine.158

a-Keto acids are produced by the oxidative deamination of amino
acids. Some of these at least are known to be metabolized by oxidative
decarboxylations (phenylpyruvic acid, for example, is converted to
phenylacetic acid). Thiamine has never been shown to be necessary for
these reactions; but since they have not been studied in well resolved
systems, one cannot make any statement concerning its function in these
reactions.

(3) Is thiamine pyrophosphate a coenzyme for any type of reaction
other than the decarboxylation of a-keto acids? There is no apparent
necessity for postulating any additional type of function for the thiamine
coenzyme, if the reactions of the diacetyl mutase type are regarded as
special cases involving intermediates of a-decarboxylations.

The Coenzymatic Functions of Biotin

The search for the specific enzymatic reactions mediated by biotin has,
at the time of this writing, not been wholly successful. Although consider-
able information is now available concerning metabolic products whose
syntheses depend upon the presence of biotin, the exact reactions in which
the biotin coenzyme participates still cannot be stated with certainty.
The evidence based on all the information reported to date necessitates
the assumption that biotin functions in several processes which seem to
have nothing in common — a situation which, if unexplained, leaves this
one member of the typical B group in a unique category.

Four metabolic processes have been shown to be influenced by the
biotin available to cells or tissues: (1) the ^-decarboxylation of poly-
basic keto acids and the reverse carboxylation ; (2) the biosynthesis of

COENZYMES DERIVED FROM B VITAMINS 171

aspartic acid; (3) the deamination of certain amino acids; and (4) the
biosynthesis of oleic acid. In each instance, however, more than one
mechanism for biotin activity can be justifiably postulated, and in no
case has a specific catalytic function been proved. The questions posed
by the many seemingly unrelated phenomena in biotin metabolism have
stimulated considerable interest, and they are under intensive investi-
gation. The answer to the basic question — does biotin have more than
one type of function? — should be forthcoming soon.

The Role of Biotin in ^-Decarboxylations. The specific enzymatic
systems to which biotin was first tentatively assigned were ^-decar-
boxylases, the enzymes catalyzing the reactions:

oxalacetic acid =^^ pyruvic acid-r-C02
oxalsuccinic acid ^ *" a-ketoglutaric -f C02

The existence of these two reactions had been previously established,
and their importance in metabolism (tricarboxylic acid cycle, p. 224;
carbon dioxide fixation, p. 221) clearly recognized. Both enzymes had
been concentrated and shown to be specific for their respective sub-
strates. They did have comparable equilibrium constants and a require-
ment for the same cofactor, Mn++,159- 16° which is indicative of a common
mechanism. Although no essential organic cofactor can be directly demon-
strated, biotin has been postulated as a component of such systems
because of nutritional relationships between biotin and the metabolites
which participate in those two reactions: (1) oxalacetic acid (or its
amino acid analogue, aspartic acid) effectively replaced the biotin
requirement of microorganisms under certain conditions 161, 162 ; (2)
oxalacetic acid prevented the inhibition of growth effected by a biotin
analogue 161 ; (3) pyruvic acid and biotin in the absence of bicarbonate
were ineffective in meeting the aspartic acid requirements of an organism,
but when the cultures were grown in a bicarbonate-containing medium,
this vitamin could replace the amino acid161; (4) the uptake of carbon
dioxide by a lactobacillus, followed by use of isotopically labelled bicar-
bonate, was not observed until sufficient biotin was added to substitute
for the aspartic acid requirement 164 ; the capacity of biotin-deficient
bacteria to decarboxylate oxalacetate is much less than that of normal
cells,103 and a-ketoglutaric acid prevented the inhibition of a biotin
analogue.161 The most logical explanation for these observations is that
the oxalacetic acid (and aspartic acid) and a-ketoglutaric acid require-
ments of the organisms either can be furnished in the form of the metab-
olites themselves, or can be supplied by the synthetic reactions under
discussion, provided the catalyst — a biotin enzyme — is supplied and is

172 THE BIOCHEMISTRY OF B VITAMINS

not inhibited by biotin analogues. The inability of biotin-deficient tissues
to metabolize pyruvic acid 165, 16C or of biotin-deficient yeast cells to
utilize glucose aerobically 167 can be attributed to the deficit of oxalacetic
acid needed to catalyze the tricarboxylic acid cycle by which these sub-
strates are "oxidized."

However, the following observations which are not in line with this
hypothesis suggest that the function of biotin in /^-decarboxylations may
not be a direct one: (1) aspartic acid, but neither oxalacetic acid nor any
other dicarboxylic acids which can be converted to oxalacetic acid, alters
the biotin requirement of yeast168; (2) the biotin content of oxalacetic
acid decarboxylase preparations from a bacterium decreased during puri-
fication,169 and no biotin at all was found in a purified preparation of
animal origin. However, the biotin might have been in a form which was
inactive in the microbiological assays.170

Biotin Function in Aspartic Acid Synthesis. The sparing effect of
aspartic acid on the biotin requirements of yeast 171, 172 and bac-
teria,161, 1G2- 173> 174 can best be interpreted on the basis of biotin function-
ing either directly or indirectly in the synthesis of this amino acid. Since
aspartic acid is effective when no other C4 dicarboxylic acids are,168 it is
possible that the reaction in aspartic acid synthesis which is catalyzed
by biotin is not one in which oxalacetic acid is directly formed from
pyruvic acid by carboxylation. This would also explain why aspartic
acid is always more effective than oxalacetic acid in substituting for
biotin, and why other C4 dicarboxylic acids, which should be easily con-
verted to oxalacetic acid, are inactive both as substitutes for biotin and
as agents for reversing biotin inhibitors.

The diminished rate of respiration of biotin-deficient yeast 167, 175 is
increased by the addition of either aspartic acid or biotin plus ammonium
salts. Biotin alone is ineffective. This observation suggests that the
reaction catalyzed by biotin in the synthesis of aspartic acid is one in-
volving an amination.

Biotin as a Catalyst for Deaminations. The type of reaction in which
biotin has been most directly implicated is the deamination of certain
amino acids. When bacterial cells are suspended in acid buffers they
rapidly lose their ability to decarboxylate aspartic, malic, and oxalacetic
acids 176 and to deaminate aspartic acid, threonine, and serine.177 Extracts
of dried cells can be reactivated by the addition of yeast extract, or by
biotin plus adenylic acid (muscle) , but not by biotin alone.178 Although
the biotin-adenylic acid mixture is as effective as yeast extract initially,
the combination becomes ineffective after the cell preparations have been
stored; the yeast extract, on the other hand, maintains its ability to
reactivate the stored preparations of the deaminase systems. These

COENZYMES DERIVED FROM B VITAMINS 173

results have been interpreted as showing that a biotin coenzyme is
destroyed by subjecting the cells to an acid environment; that the
coenzyme can be resynthesized from adenylic acid and biotin by fresh
preparations, but that on standing the enzymes bringing about the
synthesis of the coenzymes deteriorate; and that yeast extract contains
the intact coenzyme and, hence, can reactivate the older preparations.

The Role of Biotin in the Synthesis of Oleic Acid. Oleic acid and
related lipides, in the presence of aspartic acid, can effectively replace
biotin in the medium of certain lactobacilli that would otherwise require
this vitamin 179> 18°- 181, 182, 1S3 and can satisfy the biotin requirement of
mosquito larvae.184 This fatty acid can also effectively reverse biotin
inhibitors. The results of such investigations indicate that biotin functions
in the biosynthesis of oleic acid. Efforts to prove that the reverse is true,
i.e., that oleic acid is a precursor of biotin (presumably the aliphatic side
chain attached to the biotin nucleus) , have not been successful.179 Equally
unsuccessful have been the attempts to ascribe the activity of the acid
solely to some physical action (such as alteration of cell permeability) .
The function of biotin in the synthesis of oleic acid is not one in which
carbon dioxide is fixed, for no carbon dioxide (isotopically labelled) is
taken up when biotin is used to satisfy the oleic acid requirement.164

Biotin Coenzymes. The question concerning the number of biotin
coenzymes cannot be satisfactorily answered until the number of "biotins"
is known. There seems to be irrefutable evidence for the existence of two
chemically distinct isomers (Section D), cc-biotin and /?-biotin, having
identical biological properties in all systems in which they have been
compared. There is still some question concerning the exact structure of
the a-isomer, but on the basis of the configuration proposed it is difficult
to see how the two structures could exist in equilibrium or be readily
interconverted.

In addition to these two isomers a number of uncharacterized sub-
stances of diverse complexities have been shown to possess varying degrees
of biotin activity. The existence of these biotin isotels * has been estab-
lished by comparing the biotin activities of naturally occurring substances
with respect to their ability to stimulate growth in different organisms,185
their avidin combinability,185 their rate of migration during chromato-
graphic separations,186 and their effectiveness in counteracting the growth
inhibitions produced by biotin analogues.186 If the composition and dis-
tribution of the biotin coenzyme (s) resembles that of other B vitamin
coenzymes, then it can be assumed that one or more of these naturally

* Chemically distinct compounds which perform the same physiological function.
Williams, R. J., Science, 98, 386 (1943).

174 THE BIOCHEMISTRY OF B VITAMINS

occurring substances, more complex in structure than biotin, will be found
to be a coenzyme.

Biotin and certain of its isotels form very stable complexes with avidin
and other proteins, a combination that cannot be effectively dissociated
by any means yet tried. The release of the biotin component can be
accomplished only by destroying the protein. 1ST A similar nondissociation
of coenzyme-apoenzyme may exist in biotin-containing enzymes.

An insight into the chemical nature of one biotin coenzyme is given
by the investigations on the reactivation of amino acid deaminases
(p. 173). The system which these investigators have developed should
serve as a method which could be adapted for the quantitative determina-
tion of this biotin coenzyme provided the preparations were aged a suffi-
cient length of time so that the component parts of the cleaved (?)
coenzyme in the apoenzyme preparation were no longer active.

Is biotin always an essential cell constituent? In certain "biotin-requir-
ing" bacteria, biotin seems to be essential for only two processes, one
related to the synthesis of aspartic acid and one to oleic acid production.
Any other metabolites produced by biotin-catalyzed reactions are appar-
ently dispensable or else can be obtained by alternate mechanisms. Con-
sequently, if these particular organisms are furnished an exogenous supply
of aspartic and oleic acids, they no longer require detectable amounts of
biotin for growth and reproduction. In this instance the synthesis of
demonstrable quantities of biotin by the organism does not occur. Hence,
this may be one instance in which a B vitamin becomes nonessential for
life. Possibilities which have not been completely eliminated are that
extremely minute quantities of the vitamin are present in the culture
medium as impurities, or else that small amounts are being synthesized
intracellularly. Such undetectable amounts could still be performing
certain duties essential for the survival of the organism. Since the addition
of avidin to the medium does not alter the situation, the presence of an
exogenous source seems unlikely unless the impurity is one of the avidin-
uncombinable isotels.185 Intracellular synthesis, however, cannot yet be
conclusively ruled out, since current assay procedures may not determine
all forms of bound biotin.

The intracellular concentration of both vitamin B6 and folic acid may
likewise be reduced to negligible quantities under similar circumstances
wherein products of their functioning are supplied preformed to bacterial
cells (pp. 187 and 202).

The Coenzyme Activating a-Amino Acids

Most organisms are capable of synthesizing from other metabolic inter-
mediates at least part of their amino acid requirements. Processes of a

COENZYMES DERIVED FROM B VITAMINS 175

reverse type in which amino acids are degraded and metabolized are also
of common biological occurrence. A general method of synthesis or deg-
radation, reductive amination or oxidative deamination, has already
been pointed out in the discussion of reactions catalyzed by the coenzymes
of nicotinic acid and riboflavin (pp. 141 and 147). Several other types
of reactions, however, constitute alternate pathways by which amino
acids may be elaborated or utilized. At least three of these types are
reactions catalyzed by enzymes having a common coenzyme — one derived
from vitamin B6. In several instances, at least, reactions catalyzed by
this vitamin are the only methods by which an organism can adequately
produce particular amino acids and essential metabolites derived from
them.

The Vitamin B6 Coenzyme

A period of almost six years separated the time when pyridoxine was
first synthesized (1939) and the date when the biocatalytic functions of
vitamin B6 were discovered. Then, within a period of a few months, two
distinct metabolic processes involving entirely separate types of chemical
reactions were shown to be dependent upon the presence of vitamin B6
derivatives. Two years later several reactions constituting a third type of
chemical reaction were shown to require the identical pyridoxal coenzyme
as did the two processes earlier recognized.

The decarboxylation of tyrosine and several other amino acids by
bacterial cells had been observed to be catalyzed by an enzyme containing
a dissociable codecarboxylase.lss None of the coenzymes then known,
however, were active in reconstituting purified preparations from which
the coenzyme had been separated, but a concentrate having 15,000 times
the coenzymatic activity of the richest natural source (yeast extract)
had been prepared.189 Concurrently, it was observed that the decarboxyla-
tion of tyrosine by resting cells of a lactic acid bacterium was influenced
by the medium in which the organism had been cultured.190 For the pro-
duction of cells possessing optimum decarboxylase activity the medium
in which the organism was grown had to contain three times the amount
of pyridoxine needed to promote maximum growth. Since the enzymatic
activity of the cells varied according to the amount of pyridoxine fur-
nished them during growth, pyridoxine and its analogues, pyridoxamine
and pyridoxal (which had just become available), were tested with cell
suspensions of bacteria collected from cultures grown on a vitamin B6-free
medium. (The vitamin can be omitted from the medium if high levels
of alanine are used — see p. 187.) The addition of pyridoxal to the "vita-
min-deficient" cells increased the rate of decarboxylation twentyfold;
pyridoxamine and pyridoxine were inactive.191 It was subsequently shown

176 THE BIOCHEMISTRY OF B VITAMINS

that the bacterial cells first phosphorylate the added pyridoxal before
it becomes active,192 and that this phosphorylated pyridoxal is also the
codecarboxylase for at least six of the known amino acid decarboxylases.
The vitamin B6 activity of pyridoxine-supplied bacteria had been
observed in many instances to depend upon an activation of this com-
pound occurring when it was autoclaved with the other components
(particularly the amino acids) of the medium.193 An investigation into
the nature of the chemical changes resulting from the autoclaving led to
the discovery of pyridoxal and pyridoxamine (p. 186) , and to the recog-
nition of a nonbiological reaction (a transamination) by which these two
forms of the vitamin are interconverted.194 Glutamic acid (and most other
common a-amino acids) can serve as an amino donor for the formation
of pyridoxamine from pyridoxal, and a-ketoglutaric acid is an efficient
acceptor for the amino group in the reverse reaction.

NH2 H— C=<

HO— C— CH2— CH2— C— COOH + HO— rT ^— CH2OH
H

-C— COOH + HO— fT^V

KcXJ

glutamic acid pyridoxal

O 0 0

h— c— c-

H— C— NH2

HO— C— CH2— CH2— C— C— OH + HO-^ ^— CH2OH

H3C-

a-ketoglutaric acid pyridoxamine

A similar intermolecular exchange of amino and carbonyl groups, cata-
lyzed by enzymes, had been previously recognized as occurring in the
tissues of animals.195 These enzymes, classified as transaminases, catalyze
the interconversion of certain a-keto and a-amino acids; consequently,
it was suggested that a possible function for vitamin B6 was to serve
as an intermediate in transamination reactions by alternating between
the aldehyde and amine states.194 To test this hypothesis, tissues of
vitamin B6-deficient rats were compared to controls from normal ani-
mals.196 The deficient tissues were found to be definitely inferior in their
ability to catalyze a transamination reaction (the glutamic acid-aspartic
acid system) . The apoenzymes from resolved bacterial decarboxylases
were shown to be reactivated by boiled extracts of transaminase con-
centrates (prepared from animal tissue), indicating the existence of a

COENZYMES DERIVED FROM B VITAMINS 177

common coenzyme for these two systems.197 Subsequently, the reconstruc-
tion of a transaminase system from an inactive apoenzyme and pyridoxal
phosphate was accomplished, thus proving conclusively the identity of
the vitamin B6 coenzyme and cotransaminase.198

Reactions in which tryptophan either is synthesized from indole or is
cleaved to produce it have been demonstrated in bacteria and molds. The
enzymes required can be resolved into inactive components. Synthetic
pyridoxal phosphate can reactivate the apoenzymes 199, 200 and is pre-
sumably identical with naturally occurring "cotryptophanase."

Structure of the Coenzyme. On .the basis of its chemical composition,
pyridoxal phosphate is probably the simplest of the B vitamin coenzymes.
Yet four years have elapsed since the first synthetic preparation of the
coenzyme was made, and its structure still cannot be stated to have been
proved. When pyridoxal is treated with phosphorylating agents, a phos-
phorylated derivative (obtained as a crude barium salt) is formed.192
This "synthetic coenzyme" has the biological properties of the natural
codecarboxylase, cotransaminase, and cotryptophanase.192, 198, 201

The active compound was first prepared synthetically by American
scientists who have now conclusively shown it to be not the 3-phosphate
(the phenolic ester),202 although in their original report properties were
reported for the ester which suggested it was pyridoxal-3-phosphate.203
Meanwhile, Swiss chemists had prepared the acetal of pyridoxal-3-
phosphate by methods which leave no doubt as to the structure of their
product.204 They had claimed codecarboxylase activity for their synthetic
product, but were unable to activate an apotransaminase system with
it.205- 206 The dispute concerning the activity of the 3»phosphate ester has
been resolved by simultaneously testing the products prepared in different
laboratories on the same biological system.202 The activity of the 3-phos-
phate is so low compared to that of the active "synthetic coenzyme" of
the American group that the slight response elicited by the former might
be attribued to traces of an active isomer formed by an intramolecular
transesterification.

The active synthetic derivative when cleaved yields equimolecular
amounts of pyridoxal and inorganic phosphate.203 Since it is not the
phenolic ester, its structure is presumably that of the phosphoric ester of
the hydroxymethyl group (position 5 on the pyridine nucleus). Pyri-
doxamine can be readily produced in quantitative yields by heating
pyridoxal phosphate with an excess of glutamic acid in a neutral solu-
tion,207 and it possesses the specific growth-promoting properties of the
compound prepared by a direct phosphorylation of pyridoxamine.208
The esters are quite stable in alkaline solution (no destruction after
heating for five hours in IN NaOH at 120° C), but they are rapidly

178 THE BIOCHEMISTRY OF B VITAMINS

hydrolyzed when heated with dilute acid (0.05iV) .207 Stronger concentra-
tions of acid are less effective. The ester of the amine is hydrolyzed by
acid more rapidly than is pyridoxal phosphate. Decomposition of the
latter ester was noted when neutral solutions were stored in the refriger-
ator.

H3C

pyridoxal phosphate pyridoxamine phosphate

Assay Methods. The method generally favored for the enzymatic
determination of pyridoxal phosphate is the system in which tyrosine
apodecarboxylase is reactivated. The source of apoenzyme is a dried cell
powder prepared from Streptococcus faecalis R., which has been grown
on an alanine-rich, vitamin B6-deficient medium. A detailed description
of the assay procedure has been published.192 The powder is easily pre-
pared and is stable over long periods of time. When preparations are
assayed with the powder and a substrate, the rate of carbon dioxide
evolution is a measure of the amount of pyridoxal phosphate in the
preparations. More elaborate procedures are needed to prepare the apo-
enzymes of decarboxylases, transaminases, and tryptophanases from
normal cells or tissues; this makes their use as testing agents less con-
venient than the procedure employing deficient cells.

A microorganism which responds only to the phosphorylated deriva-
tives of either pyridoxal or pyridoxamine has been encountered.209 The
amine phosphate is three to five times as active as the aldehyde ester.
The individual determination of each of the unphosphorylated com-
ponents of the B6 group — pyridoxine, pyridoxal, and pyridoxamine —
can be accomplished by a differential method in which samples are
analyzed by use of three organisms which respond differently to the three
compounds.210 If materials are tested by such a procedure both before
and after dilute acid hydrolysis (which cleaves the phosphate ester
linkage) , it is possible to get a reasonably accurate estimate of both the
pyridoxal phosphate and the pyridoxamine phosphate contents of crude
extracts.207 The ease with which the amine and aldehyde forms can be
interconverted, by nonenzymatic as well as enzymatic reactions, should
always be considered when interpreting results obtained by this method.

The formation of the amine phosphate from the aldehyde phosphate
has been followed spectrometrically at an alkaline pH. By this procedure

COENZYMES DERIVED FROM B VITAMINS 179

the two phosphates can be distinguished from each other and from their
nonphosphorylated derivatives.208 This method, of course, is limited to
solutions of the pure substances and cannot be used with extracts of
crude material.

Occurrence. Before the relationship of pyridoxal and codecarboxylase
was recognized, the coenzyme had been shown by enzymatic analyses to
occur in a variety of biological substances.189 Since then, vitamin B6 has
been shown by microbiological assays to occur primarily in bound forms,
presumably the phosphates of pyridoxal and pyridoxamine. On the basis
of distribution studies, using the differential analysis technique previously
mentioned, it appears that pyridoxal phosphate is the predominant form
of vitamin B6 in most animal tissues. Liver appears to be an exception,
for in most samples of this tissue the phosphate of the amine accounts for
the greater part of the vitamin B6 content. Yeast extract is also a rich
source of pyridoxamine phosphate.207

Biosynthesis. It has been observed that the synthesis of pyridoxal
phosphate is rapidly carried out by yeast, molds, and bacteria, but only
from the particular components of the vitamin B6 group that serve to
satisfy the nutritional requirements of each particular organism.212 It
has also been shown that the coenzyme content of rat tissues is directly
related to the dietary intake of pyridoxine. If the bacterial cells are rest-
ing, i.e., suspended in solutions lacking nutrients needed for growth, the
rate of phosphorylation of pyridoxal is decreased to one tenth that
observed in the metabolically active cells ; the conversion of pyridoxamine
to the coenzyme under resting conditions cannot be detected unless a
keto acid (pyruvic acid) is added during the incubation. In this organism
the route of biosynthesis would appear to be limited to the one in which
only pyridoxal can be phosphorylated, i.e., the amine must first undergo
a transamination (enzymatic?) with a keto acid to form the aldehyde.
Studies of this nature have been so limited in number that no general
statement can yet be made concerning the possible utilization by other
organisms of alternate routes of synthesis (via pyridoxamine phosphate,
for example) .

One of the earliest methods of obtaining a "synthetic" coenzyme for
use with preparations of apoenzymes from treated cells was to add
pyridoxal and adenosine triphosphate to the protein preparation before
the substrate for the decarboxylation was introduced.192 Whereas either
the vitamin or phosphorylating agent is inactive when used alone, together
they can effectively replace the missing coenzyme. It is assumed that
the phosphorylation is an enzymatic process catalyzed by a phosphorylat-
ing enzyme present in the crude decarboxylase preparations, although this

180 THE BIOCHEMISTRY OF B VITAMINS

enzymatic biosynthesis of the coenzyme has not been critically studied
and is therefore still poorly characterized.

The phosphorylated esters of the vitamin components are quite stable
and are not as readily inactivated by enzymatic hydrolysis as are the
coenzymes of other vitamins in which pyrophosphoric linkages occur. A
high degree of association between the coenzyme and its recognized
apoenzymes has been demonstrated. This affinity of the apoenzymes for
their coenzymes accounts for the difficulties encountered in trying to
resolve some of the holoenzymes by simple procedures; it may also be
an important factor in preventing the hydrolysis of the coenzyme by
intracellular phosphatases.

The dissociation constant has been measured for a representative
enzyme of each of the three recognized types of pyridoxal-catalyzed
reactions.201, 213 The three values have similar orders of magnitude
and are so small that the amount of uncombined pyridoxal phosphate is
negligible as long as there is even a small excess of an apoenzyme. The
affinity can also be demonstrated by the use of an inhibitor, an analogue
of the coenzyme. The effectiveness of the analogue in inactivating a
decarboxylase system depends upon the order in which the analogue and
the coenzyme are added to the apoenzyme.214 If the coenzyme is added
first the analogue is quite ineffective as an antagonist, whereas if the
order of addition is reversed the analogue is an effective inhibitor. The
association is thus great enough so that the equilibrium between the
inhibitor-apoenzyme and coenzyme-apoenzyme systems is not readily
achieved, and in such an instance considerable time would have to elapse
before the ratio of inhibitor to coenzyme would be the factor determining
the degree of inhibition.

Dialysis of fresh preparations of the holoenzyme does not accomplish
its resolution. The most effective means of securing the apoenzyme from
the cellular material of normal organisms, i.e., those supplied adequate
amounts of vitamin B6, is a process of aging. Storing the intact tissue
or extracts for a period of time results in a gradual inactivation of the
preparation and the release of the apoenzymes.198, 199, 215 The process,
however, is not one of simple dissociation but rather one in which destruc-
tion of the coenzyme precedes the dissociation. Dissociation of the com-
plete enzyme is also achieved whenever the source of the enzyme is carried
through the number of fractionating procedures needed to effect a sub-
stantial concentration of the enzyme.216 The extent of destruction of the
coenzyme during such separations has not been indicated.

Reactions Catalyzed by Pyridoxal Phosphate. The chemical changes
catalyzed by pyridoxal phosphate may, on first inspection, appear to
have little in common since they represent extremely divergent types of

COENZYMES DERIVED FROM B VITAMINS 181

reactions: (1) simple decarboxylations, (2) oxidative transfer of amino
groups, and (3) condensations establishing carbon-to-carbon bonds or
the reverse process in which these bonds are cleaved. However, in every
reaction known to be catalyzed by this coenzyme there is a common type
of substrate, an a-amino acid, and in every instance the reaction involves
a group or atom attached to the a- carbon atom.

H COOH COOH H COOH

R— C— C— H R— C— C— H R— C— i— H

H NH2 NH2 H NH2

decarboxylation transamination tryptophan

synthesis and cleavage

I. Amino Acid Decarboxylation. Amino acid decarboxylation is un-
doubtedly an important means by which bacteria can metabolize some
amino acids. This type of reaction also provides these organisms a means
of producing alkaline substances with which they can alter an undesirable
acidic environment.217 In addition, the decarboxylation of amino acids
seems to be the most likely source for certain polyamines which have been
shown to be bacterial metabolites.

Decarboxylation of a-amino acids by mammalian tissues can be demon-
strated, although they never possess activity comparable to that observed
in many bacteria. This type of reaction is not believed to be an important
general mechanism for the catabolism of amino acids, but it may be the
process by which are formed histamine (from histidine), taurine (from
cysteic acid), ethanolamine (from serine), ^-alanine (from aspartic acid),
adrenalin (from 3,4-dihydroxy phenylalanine — "dopa") and putrescine
(from ornithine). Detailed reviews of mammalian and bacterial decar-
boxylases were published in 1945 and 1946.218, 21T

Pyridoxal phosphate has been conclusively shown to be the catalyst
for these six reactions:

H H

R— C— COOH — > C02+R— C— H

NH2 NH2

1. tyrosine >■ C02+tyramine.219

2. dopa — >• C02+3,4-dihydroxyphenylethylamine.219- 197 (precursor of

adrenaline.)

3. lysine >■ C02+cadaverine.220

4. ornithine > C02+putrescine.221

5. arginine >■ C02+argamine.221- 21B

6. glutamic acid >■ C02+7-aminobutyric acid.216

182 THE BIOCHEMISTRY OF B VITAMINS

In every case the apoenzyme used for reconstructing the system was of
bacterial origin, with the exception of dopa decarboxylase.197 No direct
answer has been obtained to the question of whether pyridoxal phosphate
functions in the decarboxylation of other amino acids by mammalian
tissue. Until it has, the lack of a positive demonstration should be at-
tributed to the weakness of such systems in mammalian tissues and the
difficulty of resolving the holoenzyme.

II. Transamination. Pyridoxal phosphate is known to be the coen-
zyme for two transaminase reactions, the so-called glutamic-aspartic
system and the glutamic-alanine system.

O NH2 O 0 0

HO— C— CH2— CH2— C— C— OH + HO— C— CH2— C— OH =^=

glutamic acid oxalacetic

acid

O 0 0 O NH2 O

HO— C— CH2— CH2— C— C— OH + HO— C— CH2— C C— OH

a-ketoglutaric acid aspartic acid

O NH2 O 0 0

2— C (

HO— C— CH2— CH2— C C— OH + H3C— C— C— OH =^=

glutamic acid pyruvic

acid

O 0 0 NH2 O
HO— C— CH2— CH2— C— C— OH + H3C— C C— OH

a-ketoglutaric alanine

acid

A combination of these two systems gives what amounts to an aspartic-
alanine system:

aspartic acid + pyruvic acid ^± oxalacetic acid+alanine.

This reaction was originally believed to be catalyzed by a single distinct
enzyme,222 having another "coenzyme" in addition to pyridoxal phos-
phate. It is now recognized, however, that this reaction is brought about
by a combination of these two enzyme systems and catalytic amounts of
glutamic acid (or a-ketoglutaric acid).223 These latter substances are the
•"coenzyme" of the combined system; they shuttle back and forth between

COENZYMES DERIVED FROM B VITAMINS 183

the two enzymes and thus make possible the coupling of the two reactions.
The individual systems have been found to occur in animal tissue, in
plant material,224 and in bacterial cells, but as yet only those of animal
and bacterial origin have been resolved and tested for their coenzyme
requirements. The existence of a third distinct transaminase is known.
It catalyzes a glutamic-cysteic acid system, but it has not been studied
from the standpoint of its component parts.225 Although the occurrence of
additional transaminases in which other amino acids form part of the
system has been indicated, absolute proof of their existence is still lacking.

III. Reactions in which the Methylene Groups Attached to the a-Car-
bon Atom React. Two instances are known in which pyridoxal phosphate
catalyzes a process in which it is the methylene group bonded to the
a-carbon atom (rather than the amino or carboxyl group) that reacts.
In one tryptophan is the product; in the other, it is the substrate.

A. Synthesis of Tryptophan from Indole and Serine. A mutant of
Neurospora crassa can utilize indole in place of tryptophan.226 An analysis
of the mechanism, using extracts from such cells, demonstrated that the
biosynthesis of tryptophan can be achieved by this reaction.199

H NH2 0

1 II + HO— C— C C— OH —

1 1

1— C— C C— OH

1 1 +H20

k/ H H

V/-

H H

indole serine tryptophan

The equilibrium for this reaction must decidedly favor the synthetic
process, rather than its reverse, since the degradation of tryptophan into
serine and indole cannot be demonstrated with the enzymatic preparation.
B. Non-Oxidative Degradation of Tryptophan. An extract prepared
from Escherichia coli has been found to catalyze the breakdown of trypto-
phan in the following manner201:

H NH2 O 0 0

-c— oh — > rn n + h,c— c— c— oh + nh3

-U-

N N

tryptophan indole pyruvic acid ammonia

After inactivation of the enzyme system by aging and dialysis the
protein can be completely reactivated by pyridoxal phosphate. The pos-
sibility of a two-step reaction in which one enzyme would hydrolytically

184 THE BIOCHEMISTRY OF B VITAMINS

cleave tryptophan to form serine and a second would catalyze the deamin-
ation of serine, yielding the observed products, ammonia and pyruvic
acid, was ruled out when it was shown that the preparation cannot
catalyze the deamination of serine.

An independent investigation has been carried out upon the enzymatic
degradation of tryptophan by cellular extracts from this same organ-
ism.200 In this study, the biological preparations used had apparently not
been adequately resolved into individual enzyme systems, since the pro-
duction of indole and tryptophan was accelerated by each of four sub-
stances: a nicotinic acid coenzyme, free riboflavin, pyridoxal phosphate,
and a porphyrin-containing enzyme (verdoperoxidase) . A maximum rate
of reaction was achieved only when all four of the stimulating factors
were added. By chromatographic analysis it was shown that alanine was
formed during the "reaction." It could, however, have been formed
secondarily from pyruvic acid following the initial cleavage. Were alanine
a primary product of the reaction, it would mean that the cleavage is a
reductive one, and the reaction would of necessity have to be coupled
with a second reaction in which hydrogen atoms are made available. The
effect of the hydrogen-transporting agents upon the rate of indole forma-
tion probably is the result of the oxidative removal of the other products
of the primary reaction.

Relationship of Vitamin BG and Amino Acid Requirements in Bacterial
Metabolism. The determination of the amino acid requirements of sev-
eral bacteria has indicated processes in amino acid syntheses, other than
those completely characterized, for which the vitamin B6 coenzyme is
undoubtedly essential. In most instances, the variations in amino acid
requirements resulting from the addition of pyridoxal or pyridoxamine
can be explained by assuming catalysis of reactions of the types which
have been demonstrated in cell-free systems.

It has been observed that the presence of three amino acids in particular
— lysine, threonine and alanine — radically affects the pyridoxal or pyri-
doxamine requirement of certain bacteria.227, 228, 229 In the absence of
these amino acids the vitamin B6 requirement of these organisms may be
more than ten times that when they are present. It was observed that
these amino acids were no longer essential when pyridoxine was replaced
by pyridoxal or pyridoxamine in the media of certain bacteria. The effect
presumably did not occur as the result of an independent function of
pyridoxine, but rather reflected an inadequate chemical conversion of the
inactive pyridoxine during autoclaving; hence, the organisms were not
supplied sufficient amounts of active precursors of the vitamin B6 coen-
zyme to carry out the synthesis of these three amino acids. Formation
of these acids was independent of the carbon dioxide tension, and it is

COENZYMES DERIVED FROM B VITAMINS 185

presumed that their synthesis probably involves transamination reactions,
either directly or indirectly. Imidazole pyruvic acid has been shown to
replace the histidine requirements of a bacterium provided adequate
vitamin B6 is supplied.230 The amino acid could be formed directly from
the former compound by transamination.

The ability of an organism to use O-amino acids in place of the natural
isomers has been shown to be dependent upon the availability of pyridoxal
or pyridoxamine. Transamination reactions in which optically inactive
keto acids are formed would be a convenient method for making such
compounds utilizable.

The availability of pyridoxal and pyridoxamine also exerts a sparing
action upon the requirements for three additional amino acids — arginine,
phenylalanine and tyrosine.228 In these instances, however, adequate car-
bon dioxide tension must be maintained over the cultures. In a carbonate
free system the vitamin is incapable of altering the requirements for these
particular amino acids. It was at first postulated that a carboxylation
of amines can be effectively used for the biosynthesis of these amino
acids. If such were the case, however, one would have to account for the
origin of these amines by some process other than decarboxylation of
amino acids. In addition, the equilibrium is such that relatively high
concentrations of the rather toxic amines would have to exist for the
reaction to be directed toward amino acid synthesis. When phenylethyl-
amine was supplied the microorganism, only a slight conversion to phenyl-
alanine was observed, even in the presence of high concentrations of
carbon dioxide. It seems probable, therefore, that the carbon dioxide
effect is an indirect one, and that vitamin B6 catalyzes the syntheses of
these three amino acids by some process other than a direct amine car-
boxylation.

The third type of B6 reaction — tryptophan synthesis — has also been
indicated by similar studies of nutritional requirements. Either pyridoxal
or pyridoxamine must be supplied in greater amounts if a bacterium is to
use indole or anthranilic acid (an indole precursor) in place of trypto-
phan.231

In animals, the utilization of tryptophan for nicotinic acid synthesis
is dependent upon an adequate vitamin B6 intake (p. 279) , and low
intakes of vitamin B6 result in abnormal tryptophan catabolism and the
production of urinary products not normally detected (p. 427) .

Vitamin B6 and Fat Metabolism. Several of the earliest observations
upon the physiological results of vitamin B6 deficiencies involved this
vitamin in fat metabolism, particularly the metabolism of the unsaturated
fatty acids.232, 233« 234, 235 No explanation for these observations can be

THE BIOCHEMISTRY OF B VITAMINS

made on the basis of any of the reactions now recognized as being cata-
lyzed by pyridoxal phosphate.

Mechanism of Pyridoxal Phosphate Action. The nonenzymatic trans-
amination reactions in which pyridoxal and pyridoxamine participate can
most logically be explained on the basis of an intermediate formation of
a Schiff's base which can tautomerize and be hydrolytically cleaved in
a manner such as this:

OH OH

H— C— H H— C— H

I H COOH I COOH

N/~\_i=0+H2N-i-H 12! nA-Ln4-H —

HCHOH

HCHOH
pyridoxal

+H20

a-amino
acid

OH OH

H— C— H H— C— H

I COOH +Hi0 I COOH

N<f \— C— N=C ^7^ n/ \— C— NH2 + 0=C

H I -H2° \__/ H I

HCHOH

HCH

imine a-keto

acid

Compounds of the Schiff's base type formed from pyridoxal and amino
acids are believed to exist in biological materials. It has been assumed
that the formation of such an intermediate is the mechanism by which
pyridoxal phosphate activates the amino acid molecule. The energies in
the postulated Schiff's base are localized in such a way that any one of
the groups attached to the cc-carbon atom may undergo a reaction if the
pyridoxal phosphate is attached to the appropriate apoenzyme.

When the substrate for a reaction is an amino acid, the aldehyde
phosphate would be the appropriate state of the coenzyme for the forma-
tion of the postulated intermediate, and pyridoxamine phosphate would
not be expected to be active per se as the coenzyme for decarboxylases or
tryptophanases ; and it is not. However, in transamination reactions
(wherein both amino and keto acids must be present as substrates) it is
postulated that the coenzyme alternates between two states, the aldehyde

COENZYMES DERIVED FROM B VITAMINS 187

and amine, and it would seem logical to expect either the aldehyde or
amine form to be active. Until recently this was thought to be the case.236
Reports of recent experiments in which a highly purified preparation of
glutamic-aspartic transaminase was resolved indicate that in this instance,
at least, only pyridoxal phosphate is active in reconstituting the en-
zyme.208 If this is found to be generally true when refined preparations
are used, the original hypothesis (which led to the discovery of cotrans-
aminase) may have to be modified. It may be that only the aldehyde
phosphate can establish the initial apoenzyme-coenzyme bond.

The phosphorylation of the vitamin is essential for its incorporation
into the enzyme complexes. It has also been demonstrated that an
analogue of the vitamin does not associate to any degree with a decar-
boxylase, and hence is inactive, whereas the phosphorylated analogue is
an effective inhibitor.

The Essentiality of Vitamin BG. The sparing action of alanine,237 and
in particular D-alanine,238 upon the vitamin B6 requirements of some
organisms had been presumed to be due to its utilization in the synthesis
of pyridoxal. Although the decarboxylase and transaminase activities of
cells cultured upon D-alanine in the absence of a vitamin B6 source are
very slight,192^ 198 it has been commonly assumed that the use of D-alanine
results in the synthesis of only the minimum amounts of vitamin B6
needed for growth. It has now been established that D-alanine is not a
precursor of vitamin B6, but is a direct product of its catalytic activity,
or else can indirectly function by sparing the requirement for some metab-
olite produced directly when vitamin BG is available.239, 24°

The phenomenon of the nonessentiality of a B vitamin was discussed
when biotin functions were considered (p. 174). Pyridoxal is another B
vitamin which may be nonessential for the growth and metabolism of
certain organisms. Since its primary function is the synthesis of amino
acids and amines, it may be possible to dispense with its reactions entirely
if the organism is supplied with all the essential products preformed
(L-amino acids, D-alanine, amines, and unidentified products in casein
hydrolyzates) .238

In higher forms of life, where amino acids and hormones like histamine
and adrenalin must be synthesized in situ, and where extensive degrada-
tion of amino acids must precede excretion, the vitamin B6 requirement
could never be completely abolished.

The Coenzymes Involved in Condensations Forming Carbon-to-Carbon
Bonds

In the elaboration of more complex organic compounds from simpler
ones there must be condensation reactions in which carbon-to-carbon

188 THE BIOCHEMISTRY OF B VITAMINS

bonds are established. The condensations carried out by biological sys-
tems resemble the reactions which are commonly employed by organic
chemists, and many of the limitations concerning the types of reactive
compounds and the manner in which they unite are common both to
laboratory syntheses and to those occurring intracellularly.

The earliest enzymatic condensation to be recognized was the one in
which hexoses are formed from trioses by a typical aldol condensation:

HHH OH OH HHHOH

HC— C — C + HC — C— CH =f=^ HC— C — C — C— C— CH

Hh b H i> Hh Ah A A i

HO— P— OH HO— P— OH HO— P— OH HO— P— OH

O OOO

phosphoaldo- phosphoketo- diphosphoketohexose

triose triose

In this reaction, the condensation takes place between a molecule con-
taining a carbonyl group and one containing an active hydrogen atom,
i.e., one bonded to a carbon atom alpha to a carbonyl group. This type of
reaction (aldol condensation) occurs readily in vitro with alkali as a
catalyst; the enzyme (aldolase) which mediates the reaction pictured
above has never been resolved into dissociable components, and is believed
to contain no recognized vitamin. The reaction is one of the essential
steps in the general process by which most organisms metabolize hexoses
or synthesize them from metabolic products. The isomerization of the
phosphate ester of the aldotriose to produce the corresponding ketose is
an essential reaction that must precede the condensation depicted, for the
condensation of two aldotrioses would give a branched chain hexose. Like-
wise, the isomerization of the glucose diphosphate to the corresponding
fructose ester must occur prior to the cleavage, since an aldose cleavage
of glucose could only occur between the a- and /?-carbon atoms, producing
a biose and tetrose.

A second type of condensation often used by organic chemists is the
so-called Claisen type, a condensation which involves (1) the carbonyl
group of an acid anhydride or ester and (2) a carbon and a hydrogen
atom alpha to a carbonyl group. The enzymatic condensations by which
fatty acids, sterols, amino acid precursors, and probably several other
important products are elaborated are of this type.

In Claisen type condensations in biological systems one of the reacting
molecules is usually (if not always) an acyl phosphate, the mixed acid
anhydride of phosphoric acid and an organic acid. In most reactions, it is

COENZYMES DERIVED FROM B VITAMINS 189

an acetic acid derivative. The reaction has been pictured as occurring in
this fashion:

O O

H || O H || O

HC— C— O— P— OH + HC— C— O— P— OH < — >

A H A

H H

O O

H || H || O

HC— C— C— C— O— P— OH + H3PO4
H H ^

Recent investigations have definitely disclosed that the phosphoric acid
derivative of acetic acid is not the simple acetyl phosphate pictured above
(p. 190) , but it probably reacts in a comparable manner. The biologically
active phosphoryl derivative of acetic acid is associated with a panto-
thenic acid coenzyme, and the numerous reactions in which it can be
utilized constitute essential steps in many fundamental processes.

A third type of condensation utilized by living organisms is one which
involves a reactive single carbon unit related to formic acid but not to
carbon dioxide (or the carbonate ion). The only synthetic reaction of
this type now recognized occurs in the conversion of glycine to serine
(p. 201) and is dependent upon a p-aminobenzoic acid-containing enzyme
and possibly vitamin Bi2. The exact mechanism of this type of condensa-
tion is not known, nor is it yet possible to state how important reactions
of this type will ultimately prove to be in the synthetic activities of cells.

A fourth type of condensation which has been observed is one catalyzed
by a thiamine system — the formation of acetoin from the intermediates
formed during the metabolism of pyruvic acid. This reaction has never
been shown to be of value to any cell from the standpoint of synthesis
of cellular components, and it is believed to be a mechanism the only
purpose of which is to dispose of end products of carbohydrate metab-
olism.

Two reactions, catalyzed by pyridoxal phosphate, constitute a fifth
type of condensation: (1) the formation of tryptophan from indole and
serine, and (2) the cleavage of tryptophan into indole, pyruvic acid and
ammonia (p. 183). It should be noted that the a-amino group in the
presence of pyridoxal-containing enzymes is a "potential keto" group
(p. 186) ; hence these reactions of a-amino acids are comparable to those
involving the reactive methylene carbon and hydrogen atoms alpha to
carbonyl groups.

Because the equilibria of the decarboxylation reactions discussed in the

190 THE BIOCHEMISTRY OF B VITAMINS

preceding divisions of this chapter are so decidedly in favor of the cleav-
age of carbon-to-carbon bonds rather than the reverse carboxylation,
these reactions have sometimes been ignored when effective enzymatic
mechanisms for increasing the number of carbon atoms in a molecule are
considered. However, carboxylations, both of the a and B types, catalyzed
by the coenzymes of thiamine and biotin (?), may be utilized to a greater
extent in biological syntheses than was once realized.241

The type of condensation for which the greatest number of reactions
are recognized resembles the Claisen type (second in the listing above) .
The many processes in which it is employed were not understood until
the recent discovery of an essential metabolite — a derivative of acetic
acid. Acetic acid had long been recognized as the principal end product
in certain types of fermentations, but its importance as an intermediate
compound in metabolism was not fully realized until it was shown that
acetic acid (labelled with isotopic atoms), when introduced into organ-
isms, was converted into some activated derivative which participated in
a number of metabolic processes.242 Although the concentration of acetic
acid in normal animal tissues is too low to be measured by conventional
methods, it was shown by the use of isotopes that in a 24-hour period an
adult rat produces a quantity of acetic acid (a phosphorylated derivative)
equal to 1 per cent of its body weight.243 In the meantime, other investi-
gators found that acetic acid was a substrate for (1) the reaction in
which acetylcholine is formed, and (2) the enzymatic acetylation of
aromatic amines. Previously it had been recognized that the acetate ion
which is used for buffering media in which bacteria are grown has func-
tions independent of its buffering capacity,244 and later this metabolic
utilization of acetic acid by bacteria was shown to be related to lipide
synthesis.245, 246

To discuss the reactions in which acetic acid can participate it is nec-
essary to consider the chemical nature of the activated acetyl molecule
which is first formed and with which pantothenic acid is associated. This
reactive compound, which can be formed enzymatically from acetic acid,
is believed actually to be the substance participating in most, if not all
of the acetate reactions. It is a molecule which can act both as a phos-
phorylating and as an acetylating agent247 but it is not identical with
acetyl phosphate.248, 249> 250 Although there were a number of reasons for
assuming the active intermediate to be acetyl phosphate, this compound,
when prepared synthetically, was inactive and could not be substituted
as the substrate replacing the so-called "active acetate" arising from the
oxidative decarboxylation of pyruvate.249 Moreover, the "active acetate"
is not decomposed readily by the specific enzyme, acetyl phosphatase,
whereas the synthetic acetyl phosphate is.248 Partial purification of the

COENZYMES DERIVED FROM B VITAMINS 191

active phosphorylated acetate (prepared enzymatically from acetic acid
and adenosine triphosphate by dried bacterial cells) has been reported.251
It contains equimolecular amounts of reactive acetyl and phosphoryl
groups; hence, it is not acetyl pyrophosphate. In an acidic environment
(pH 1.5) the "active acetate" spontaneously undergoes a transformation
producing a compound which is indistinguishable from synthetic acetyl
phosphate.251 To distinguish the biologically active compound from acetyl
phosphate the former will be referred to as the reactive -phosphoryl- acetyl
intermediate.

Origin of the Reactive Phosphoryl-Acetyl Intermediate. The reactive
acetyl compound is formed during carbohydrate metabolism by the oxida-
tive phosphorylative decarboxylation of pyruvic acid which is catalyzed
by thiamine pyrophosphate (p. 162). It seems logical that this is also
the compound formed during jatty acid (and sterol?) degradation when
the C2 fragments are successively cleaved from the molecule. It probably
is the intermediate formed from ketogenic amino acids when they are
catabolized. Substances metabolically related to fatty acids — acetoacetic
acid and ethanol — undoubtedly are metabolized via metabolic pathway
in which the reactive acetyl intermediate occurs. It has been shown that
the tricarboxylic acids can be cleaved to yield oxalacetic acid and the
reactive acetyl molecule by a reversal of the reactions in which these
acids are formed.241 When an exogenous supply of acetic acid is available,
cells can use a pantothenic acid enzyme, "acetylphosphorylase," to
"activate" the acetic acid molecule; adenosine triphosphate is the phos-
phorylating agent.

The Coenzyme Derived from Pantothenic Acid

Ten years elapsed between the time (1936) that pantothenic acid was
first found to participate in carbohydrate metabolism 253 and the time
that the mechanism of its function was established.

Physiologists interested in nerve metabolism discovered (in 1942) an
enzyme system in brain tissue which was responsible for the synthesis
of acetylcholine from acetic acid, choline, and adenosine triphosphate.254
At about the same time another group of investigators demonstrated the
presence in liver of an enzyme which converts sulfonamides and other
aromatic amines into the less toxic amides by acetylation.249 They showed
that the preparations contained a heat-stable, dissociable component
which would reactivate enzyme systems which had been inactivated by
dialysis. This coenzyme was subsequently shown to be a necessary com-
ponent of the system which acetylated choline.255

Initial attempts to demonstrate the presence of a B vitamin in this
coenzyme were unsuccessful, because pantothenic acid, the essential vita-

192 THE BIOCHEMISTRY OF B VITAMINS

min component, is bound in such a way that it is not released by the
ordinary methods currently used for preparing samples for microbiologi-
cal assay. Acid hydrolysis of the coenzyme did, however, yield appreciable
amounts of /3-alanine.256 The coenzymatic activity of preparations of
various degrees of purity paralleled the yS-alanine content. It was also
shown that pantothenic acid could be liberated in its uncombined form
by use of a combination of a phosphatase and an enzyme obtained from
liver.256, 25T

The presence of pantothenic acid in this coenzyme established at least
one function of this vitamin, and indicated its involvement in reactions
in which acetic acid is utilized. It was realized, however, that neither of
the rather specialized reactions studied could account for the general
importance of the vitamin. This led to the subsequent demonstration of
the essentiality of the pantothenic acid-containing coenzyme for a number
of reactions of general importance in carbohydrate and fat metabolism
where tracer studies and investigations of acetate metabolism in bacteria
had indicated involvement of acetic acid. This coenzyme is now usually
referred to as coenzyme A (A for acetylation) , and will be so designated
in this discussion.

The Chemical Structure of Coenzyme A. The structure of coenzyme
A has not yet been announced. The rate at which it diffuses through
sintered glass membranes indicates that its molecular weight probably
lies between 750 and 850.258 A preparation of the coenzyme containing
11 per cent pantothenic acid, contained 9 per cent phosphorus, 18 per cent
adenine, 22 per cent pentose and some cysteine. On the basis of its panto-
thenic acid content and apparent molecular weight, this preparation was
only 50 per cent pure, but its analysis does indicate the presence of
adenylic acid. Glutamic acid is probably an additional constituent, since
it is essential for the biosynthesis of a pantothenic acid complex,259 which
is believed to be identical with a product obtained during the enzymatic
degradation of the coenzyme.258 Studies of the enzymatic degradation of
the compound indicate that there are at least two linkages which must
be cleaved before pantothenic acid is liberated. Both an intestinal phos-
phatase and an enzyme present in liver extract must be allowed to act
upon the molecule before the coenzyme will be active in the microbiologi-
cal assays usually employed. Either enzyme alone renders the coenzyme
inactive (each producing a different product), but neither enzyme by
itself liberates panthothenic acid.257

Assay Methods. The microorganisms commonly employed in B vita-
min assays cannot utilize coenzyme A.256 Acetobacter suboxydans, which
responds very slowly to pantothenic acid, has been shown to grow rapidly
in the presence of a pantothenic acid conjugate (PAC) concentrated from

COENZYMES DERIVED FROM B VITAMINS 193

liver or heart muscle.259 Intact coenzyme A produces a comparable
response with this organism.258 The cleavage in the coenzyme catalyzed
by the liver enzyme produces a derivative, still phosphorylated, which is
equally active for Acetobacter suboxydans. Incubation of either the intact
coenzyme or its phosphorylated intermediate with phosphatases, however,
destroys the activity for this organism.

The pantothenic acid conjugate (PAC) isolated from heart muscle was
inactive when tested in enzymatic acetylation systems.259 It seems likely,
then, that it is identical with the phosphorylated intermediate derived
from coenzyme A by treatment with the liver enzyme and probably is
formed from coenzyme A by an autolytic process during its concentration
from tissues. A. suboxydans consequently cannot be used for the specific
microbiological assay for the intact coenzyme.

At present the only method of differentiating coenzyme A from some
of its degradation products is by the use of enzyme analyses. By a com-
bination of assays, using Lactobacillus arabinosis, A. suboxydans, and
enzymatic acetylation, it should be possible to work out a differential
assay for coenzyme A, free pantothenic acid, and the two compounds of
intermediate complexity. The activity under various testing conditions
is summarized:

Growth of
Tj. arabinosis

Growth of
A. suboxydans

Enzymatic
Acetylation

+
+

Free pantothenic acid

Coenzyme A

Phosphorylated intermediate (PAC?)

Phosphatase-treated coenzyme =•=

± long incubation or high concentration required.

An enzymatic determination of coenzyme A can be made either by
(1) following the acetylation of choline (using as an indicator a biological
response — muscle contraction) ,260 or (2) determining the rate of acetyla-
tion of aromatic amines.249 As a routine method of analysis, the latter
is preferred because of its greater simplicity and accuracy. A detailed
description of this assay procedure has been published.261 This method
can be conveniently adapted to laboratories equipped for microbiological
analyses. The preparation of a suitable apoenzyme is not a problem, since
crude liver extracts can be used. The coenzyme originally present in these
extracts is completely inactivated if the extract is allowed to stand for
four hours at room temperature.

Occurrence. By use of the rate of acetylation method just described,
the coenzyme A content of a number of animal tissues and various plant
materials has been ascertained.261 The pantothenic acid content of these
sources was simultaneously measured by the conventional microbiological
procedure. A comparison indicates that within cells pantothenic acid exists

194 THE BIOCHEMISTRY OF B VITAMINS

almost exclusively in the form of its coenzyme. As is the case with several
of the other B vitamins, the plasma pantothenic acid is in the form of the
free vitamin, whereas the vitamin within the red blood cells has been
converted quantitatively to the coenzyme.

Biosynthesis. The chemical routes by which pantothenic acid is con-
verted to coenzyme A are not known. Before coenzyme A was discovered,
it had been shown that the addition of pantothenic acid quickly activated
the pyruvate metabolism of bacterial cells which were deficient in panto-
thenic acid.262 Later it was shown that incubation of pantothenic acid-
deficient yeast and bacteria with pantothenic acid resulted in a rapid
synthesis of coenzyme A.263, 264 This indicates that these cells possess
adequate mechanisms for the rapid synthesis of the coenzyme. Since the
tissues of animals receive their pantothenic acid in an uncombined form
from the blood stream, they too must be able to carry out this conversion.
Strangely enough, however, the addition of pantothenic acid to surviving
deficient tissues of ducks and rats in vitro does not result in any demon-
strable synthesis of the coenzyme.265

Incubating resting yeast cells with glutamic acid and pantothenic acid
(or /3-alanine) results in the production of a conjugate which occasionally
shows a thousand times the activity of an unincubated control containing
these same substances.259 That the formation of the conjugate may not
be direct from the vitamin itself was indicated by the fact that the
incubation product of /^-alanine was consistently more active than that
obtained from pantothenic acid.

Reactions Catalyzed by Coenzyme A. Although all the reactions cata-
lyzed by coenzyme A may involve a common substrate, they result in
the formation of a variety of chemical compounds: amides, esters, acid
anhydrides, and compounds produced by the condensation of an acetate
radical with keto acids or acid phosphates.

The equations for enzymatic reactions in which coenzyme A is a cata-
lyst are tabulated on the following page. Following this list is a summary
of the enzyme reactions in which pantothenic acid has been definitely
implicated, but which have not been sufficiently well characterized to en-
able one to say with certainty that coenzyme A is the coenzyme.

It should be noted that the reaction in which the acetyl derivative
condenses with oxalacetic acid to form a tricarboxylic acid is the one
which initiates the cycles by which both carbohydrates and fatty acids
are metabolized aerobically, and by which they are converted to the
dicarboxylic acids, a-ketoglutaric, glutamic, fumaric, tartaric, malic,
oxalacetic, and aspartic acids (p. 223). When carbohydrate metabolism
provides the acetyl molecule, thiamine and pantothenic acid are required;
when fatty acids or ethanol are the source of the reactive phosphoryl-

COENZYMES DERIVED FROM B VITAMINS 195

acetyl intermediate, only pantothenic acid is needed to mediate the initial
condensation.

I. Reactions demonstrated using cell-free preparations in which coenzyme A is the
cofactor.

A. Formation of acid phosphates

acetic acid + ATP — >■ phosphoryl-acetyl intermediate +ADP

B. Formation of esters

choline +acetic acid + ATP — > acetylcholine +ADP+H3P04260

C. Formation of amides

p-aminobenzoic acid (or sulfonamides) + acetic acid -f ATP >■ acetylated

amine+ADP+HsPCV56

D. Condensation reactions*

2 acetic acid+ATP — >■ acetoacetic acid+ADP+H3P04',>D

II. Conversions requiring pantothenic acid which can be demonstrated in vivo by the
use of pantothenic acid antagonists, deficient cells, isotopes,
acetic acid — >■ fatty acids (bacteria)267- 268

acetic acid >■ phloroglucinol-like compound (bacteria)269

acetic acid — > sterols (bacteria)267- 268

acetic acid >■ aromatic amino acids (bacteria)269

acetic acid >■ cz's-aconitate >■ a-ketoglutarate (bacteria)268*

acetic acid — > C02-t-H20 (yeast)264

glucose, or pyruvic acid, or lactic acid — >■ CO2+H2O (rat liver)270- 266 (duck

tissues)266 (bacteria)262- 271
proteins or carbohydrates — >■ fats (rat)272

III. Conversions involving acetic acid for which pantothenic acid has not yet been
shown essential.

acetic acid >■ formic acid (pigeon)273

acetic acid >■ porphyrins (dog)274

ketogenic substances >■ acetone bodies (mammals)

acetic acid+acetic acid — >■ succinic acid (molds)275

The synthesis of "fats from either carbohydrate or protein as far as is
now known must go on through processes in which reactions mediated
by pantothenic acid cause the condensation of reactive acetyl molecules.
The pantothenic acid-requiring reactions in which carbohydrates and
fats are oxidized and in which fatty acids are synthesized are undoubt-
edly essential in many forms of life, while the reactions in which amines
and alcohols are acetylated may be of importance only in specific phyla
having special functions in which an acetylating agent is needed for the
production of acetic acid esters or amides.

No detailed studies of the possible dissociation of coenzyme A from the
apoenzyme while the coenzyme is still associated with the phosphorylated
acetic acid have yet been undertaken; hence, it is not known whether
(1) both the creation and utilization of the "active acetate" must take
place simultaneously in a coupled system, or (2) coenzyme A accepts the
active molecule from one enzymatic reaction, dissociates itself from this

* The reaction in which citric acid is formed by the condensation of an activated
acetate with oxalacetate should be added to the well characterized reactions since
coenzyme A has now been shown to be the coenzyme which reactivates an aged
cell-free preparation capable of accomplishing this synthesis (see footnote p. 223).

196 THE BIOCHEMISTRY OF B VITAMINS

enzyme, and transports the acetyl derivative to another system where it
is utilized.

It is impossible to do more than speculate upon possible linkages that
may exist between coenzyme A and the phosphorylated acetate molecule.
Studies of the specificity of chemical structure for compounds that will
replace or inhibit pantothenic acid have been undertaken (p. 620), but
the information obtained is not sufficient to justify any conclusions con-
cerning the question posed in the previous sentence, or to decide what
groups of the coenzyme are essential for the formation of the enzyme-
coenzyme bond.

Is there more than one pantothenic acid coenzyme? All enzymatic
phenomena in which pantothenic acid has been implicated can be ex-
plained on the basis of a single coenzyme. A fact offering corroborative
evidence for this statement is that all the intracellular pantothenic acid
is in a form which can serve as the active coenzyme for the aromatic
amine acetylation system. If the coenzyme requirement for this enzyme
is specific, then within cells there can be only one pantothenic acid coen-
zyme present in measurable quantities.

Does the pantothenic acid coenzyme mediate reactions other than those
involving acetic acid derivatives? On the basis of information now avail-
able, there seems to be no justification for postulating other functions for
pantothenic acid.

Coenzymes Involved in the Utilization of the Single Carbon Unit

Formic acid is a common by-product of bacterial fermentation. Like
acetic acid, it had long been regarded as a waste product of inefficient
catabolic processes, but its role in essential synthetic reactions had gone
unrecognized. The first indication of its participation in biologically im-
portant synthetic reactions resulted from exploratory tracer studies de-
signed to establish the precursors of purines when they are synthesized
de novo by animal tissues. It was found that neither carbon dioxide nor
the carboxyl group of pyruvic acid could serve as a source of the carbon
atoms in the ureide portion of the purine molecule (positions 2- and 8-),
although it had been anticipated that they would be found to be pre-
cursors of the single carbon units.276 When labelled formic acid was used,
however, the isotopic carbon was incorporated into the purine nucleus
in the 2 and 8 positions.273 This discovery created considerable interest
in possible metabolic roles of formic acid and stimulated further investi-
gations, which have now disclosed other important reactions involving
the single carbon unit derived from formic acid.

No statement can be made at this time as to the exact chemical nature
of this single carbon unit which is normally produced and utilized in

COENZYMES DERIVED FROM B VITAMINS 197

biological systems. It may simply be formic acid; it may be a reactive
derivative similar to the phosphorylated derivatives of acetic acid. An-
other possibility is that the "formyl group" is chemically combined with
a "formate-carrying" coenzyme from the time of its formation until it is
utilized, so that the formic acid or the formate ion would not necessarily
be present as such. Until this question has been settled, it seems unwise
to designate the reactive intermediate as formic acid or formate; hence,
the expression "single carbon unit" has been used.

Origin of the Single Carbon Unit. Exogenous sources of formic acid
itself can be utilized for syntheses requiring the single carbon units (at
least, by bacteria, fowl, and mammals) .277, 273, 278 Consequently, biologi-
cal reactions producing formic acid may serve as sources of the single
carbon unit. Before isotopically labelled compounds were available, free
formic acid had been shown to be formed by:

(1) many species of bacteria, in most cases by a phosphoroclastic
cleavage of pyruvic acid (p. 162) ;

(2) muscle perfused with pyruvic acid (perhaps by a phosphoroclastic
cleavage of oc-ketoglutaric acid) (p. 167) ;

(3) the enzymatic degradation of the imidazole nucleus of histidine
(by histidinase).279

(4) insects (mechanism unexplored).

Other potential sources of the single carbon unit recently disclosed by
the use of tracers are: either carbon atom of glycine,280,281 the /?-carbon
atom of serine,282 the carboxyl carbon atom of acetic acid,276 and the
carbon atoms of the N- and S-methyl groups of choline and methionine.282

Although the carboxyl group of pyruvic acid is the precursor of the
formic acid produced by bacterial fermentation, it has been shown that
this group can not be the primary source of the single carbon unit in the
metabolism of animals.276 The simultaneous accumulation of succinic acid
and formic acid when muscles are perfused with pyruvic acid indicates
that a phosphoroclastic splitting of a-ketoglutaric acid may be an im-
portant source of the single carbon unit in organisms in which pyruvic
acid is metabolized via the tricarboxylic acid cycle (p. 223). Such a reac-
tion could account for the ultimate incorporation of the carboxyl carbon
atom of acetic acid and the carbonyl carbon atom of pyruvic acid into
compounds in the same positions where labelled carbon atoms of formic
acid have been shown to appear. (In aerobic metabolism, these particular
carbon atoms in the acetic and pyruvic acid molecules eventually form
the cc-carboxyl group of a-ketoglutaric acid). Histidine was for a long
time believed to be the precursor of purines in animals since ingestion
of histidine increased the excretion of purines.283 It was naturally assumed
that the histidine was the precursor of the purine's imidazole nucleus.

198 THE BIOCHEMISTRY OF B VITAMINS

Recent studies with isotopic nitrogen have indicated that this concept is
invalid, since the nitrogen atoms of the imidazole ring of histidine are
not used in the formation of purines.284 However, the formic acid or a
derivative arising from the hydrolysis of the N = C — N bonds of histidine
by histidinase could serve as the source for the single carbon units needed
in purine synthesis, and the histidine effect could be entirely attributed
to its activity as a formic-donor. It has not been shown whether or not
the ureide carbon atoms of purines and pyrimidines can be effectively
used as sources for the single carbon intermediate (in reactions which
reverse the processes of their syntheses). Neither serine, glycine, methi-
onine, choline, nor histidine can be considered as primary sources of
the single carbon unit in organisms which do not require an exogenous
supply of these substances.

Vitamins Associated with the Metabolism of the Single Carbon Unit.
Both p-aminobenzoic acid-containing coenzymes and vitamin Bi2
(derivatives?) are catalysts in some stages of the processes by which
formic acid or its derivatives are utilized for synthetic purposes.

Coenzymes Derived from p-Aminobenzoic Acid and Folic Acid

The specific enzyme systems in which folic acid and other p-amino-
benzoic acid derivatives participate are not as yet known. In no instance
can we be certain of the exact structure of the compounds entering into
or produced by the individual reactions, nor do we know the chemical
components of the coenzymes derived from these vitamins. However, a
general hypothesis which explains most of the biochemical reactions in
which these vitamins are implicated can be arrived at from information
acquired during the course of a number of investigations whose primary
objectives were entirely unrelated.

Exploration of the mechanism of sulfonamide inhibition led not only
to the recognition of the vitamin activity of p-aminobenzoic acid, but
also to the establishment of a metabolic relationship between p-amino-
benzoic acid and purines, pyrimidines, and certain amino acids; all these
substances can at least partially overcome the toxic effect of sulfonamides
upon bacterial growth (Chapter II D) . However, the manner in which the
amino acids and nitrogen bases acted as "reversing agents" and their
relationship to p-aminobenzoic acid were at first obscure. It was suggested
that they might be products of reactions involving p-aminobenzoic acid
and that the presence of sulfonamides suppressed their synthesis.285

Subsequent studies on the nutritional requirements of organisms and
mutants requiring an exogenous supply of p-aminobenzoic acid or folic
acid demonstrated that here also the amounts of these vitamins needed

COENZYMES DERIVED FROM B VITAMINS 199

to produce growth responses were definitely determined by the presence
or absence of purines, thymine, and the amino acid, methionine.286, 287

The hypothesis that these essential metabolites were products of
processes, one or more reactions of which involve p-aminobenzoic acid,
was greatly strengthened when quantitative concepts developed for
isolated enzyme systems were shown to be applicable also to the inhibition
of growth in bacteria.288 It was deduced that a simple mathematical
analysis of the manner in which a compound counteracts inhibition of
bacterial growth indicates the role of this substance in the inhibited
system. That is, it is possible to determine whether a substance is (1) the
substrate (or precursor of the substrate) of the reaction, (2) a product
of the reaction (or derived from it), or (3) a part of the catalytic
mechanism of the reaction (Chapter ID).

Whereas the antagonism between p-aminobenzoic acid and the sulfon-
amides is competitive, the concentrations of the pyrimiclines, purines,
and amino acids required to initiate growth in cultures which had been
completely inhibited by sulfonamides were found to be entirely inde-
pendent of the level of the inhibitor, thus substantiating the belief that
these compounds are "products" of p-aminobenzoic acid functioning,289
and that this vitamin is required for their synthesis. What these synthetic
processes have in common, though, was not recognized until (1) tracer
studies had demonstrated the existence of the single carbon intermediate,
and (2) inhibition studies had established the type of reactions catalyzed
by p-aminobenzoic acid.

The link connecting p-aminobenzoic acid with the metabolism of the
single carbon unit was disclosed when the chemical nature of a reactant
in a p-aminobenzoic acid catalyzed process was established.290 A sub-
stance of unknown structure accumulated in the culture medium of
Escherichia coli whenever the growth of the organism was partially in-
hibited by sulfonamides.291 The compound presumably piled up because,
after its formation, sulfonamides blocked the reaction by which it
normally was utilized. Hence it represented a compound related to the
substrate of a p-aminobenzoic acid-catalyzed reaction. The compound
which accumulated was shown to possess a molecular structure which
would make it a logical precursor of purines. It is an imidazole so sub-
stituted that if direct condensation with a single carbon unit were effected,
a naturally occurring purine hypoxanthine would be formed. It was
therefore postulated that one of the functions of p-aminobenzoic acid
was to catalyze a reaction in which the single carbon unit is introduced
into the 2 position of the purine nucleus and that one of the direct effects
of sulfonamide inhibition is the blocking of reactions in which the single
carbon unit participates.290

200

THE BIOCHEMISTRY OF B VITAMINS

-I

H— C

II
H C H

\/ \ I

-N C— N

H C H

\/ \ I
N C— N

C— N

CH + H20

formic 4(5)-arafno-5(4)-

acid imidazolcarboxamide

H— C C— N

hypoxanthine

The possible involvement of folic acid in the metabolism of formic
acid derivatives was indicated a few months later when the structure of
the factor which could be substituted for folic acid in culturing Strepto-
coccus faecalis R. (SLR factor) was shown to be formylpteroic acid
(I).292 The glutamic acid peptide of this substance, formylfolic acid (II),
was synthesized and shown to be more active than folic acid itself in
reversing inhibitors structurally related to folic acid.293

OH
I H

C N 0=C . . O

/ \ / \ I /TA II

N C C— CH2— N— f J— C— OH

H2N— C C CH

I
C N

formylpteroic acid (I)

H
0=C

^C— CH2— N— f 7— O— NH— CH— (CH2)2— COOH

1 II I

H2N— C C CH

^OOH

formyl folic acid (II)

Since p-aminobenzoic acid derivatives occur in both a formylated and
unformylated state, and since both states are biologically active, it is
tempting to postulate that the coenzymes of this vitamin catalyze the
reactions of single carbon units by serving as their carriers in the same
way that the nicotinic acid coenzymes are considered to be hydrogen
carriers. As yet, however, no direct evidence has been offered to prove
or disprove the hypothesis that the coenzymes mediating the single carbon

COENZYMES DERIVED FROM B VITAMINS 201

condensations are alternately formylated in one reaction and then re-
generated by acting as formylating agents in a second.

Reactions Catalyzed by Coenzymes Derived from p-Aminobenzoic Acid
and Folic Acid. Since a direct demonstration of the participation of these
vitamins in any defined enzyme system has not been achieved, no equa-
tions will be written to depict the specific reactions for which they or
their derivatives are required. The following isolated observations, how-
ever, can be explained and integrated if one accepts a general hypothesis
in which it is assumed that: derivatives of p-aminobenzoic acid function
as coenzymes for metabolic reactions in which the single carbon units
(related to formic acid) are utilized for synthetic purposes.

I. Amino Acid Synthesis

A. Serine and Glycine. A p-aminobenzoic acid coenzyme is required
for the reductive condensation by which serine is formed from glycine
and formic derivatives, or for the reverse oxidative cleavage by which
glycine is produced from serine, because: serine increases the sulfon-
amide p-aminobenzoic acid ratio needed to inhibit bacteria294, 295; serine
is a precursor of glycine,296 but in the presence of sulfonamides this con-
version cannot take place 297 ; a synthesis of serine from formic acid and
glycine has been demonstrated280,281; and folic acid has been shown to
be involved in the interconversion of glycine and serine.298

B. Methionine. A p-aminobenzoic acid coenzyme is believed to be an
essential catalyst for some reaction in the reductive process by which the
single carbon unit is converted to an S-methyl group needed for the
synthesis of methionine from homocysteine, because: methionine increases
the sulfonamide p-aminobenzoic acid ratio needed to inhibit bac-
teria,285, 289 and decreases the requirements of organisms (and mutants)
unable to synthesize their own p-aminobenzoic acid 287 ; but homocysteine
is ineffective in either of these situations 2" ; the S-methyl carbon atom
of methionine, isotopically labelled, is found to be incorporated into the
/^-position when serine is formed biosynthetically from glycine.282

II. Purines and Pyrimidines.

Derivatives of folic acid and p-aminobenzoic acid are presumed to be
coenzymes for the reactions in which the single carbon unit is incorporated
into purine and pyrimidine nuclei, because: in the presence of amino
acids the folic acid requirements of some microorganisms can be replaced
by thymine and purines 30° ; in the presence of amino acids, the p-amino-
benzoic acid requirements of other microorganisms can be replaced by
thymine and purines 287 ; in the presence of amino acids the inhibition
produced by folic acid analogues can be prevented by thymine and

202 THE BIOCHEMISTRY OF B VITAMINS

purines (section I) ; in the presence of amino acids the inhibition produced
by p-aminobenzoic acid inhibitors can be prevented by thymine and
purines (section D) ; for some bacteria, 4(5)-amino-5(4)-imidazolcar-
boxamide is partially effective as a purine substitute, and the presence of
formic acid enhances its activity 277 ; this amine accumulates when sul-
fonamides block the synthesis of purines.290 Folic acid can cause a hema-
topoiesis in certain macrocytic anemias; thymine and nucleotides of
purines and pyrimidines also have produced a similar response (p. 414) .

Mention has been previously made of instances in which the nutritional
requirements of certain bacteria for a particular vitamin (biotin, p. 173
or pyridoxal, p. 184) can be completely satisfied by supplying only the
products of the reactions for which the coenzyme of the vitamin is
required, and none of the vitamin itself. This phenomenon was first
demonstrated with folic acid. In a medium containing purines and amino
acids, thymine can substitute for the folic acid required for growth of a
lactobacillus. No synthesis of folic acid could be detected in the cells
grown upon this medium.301

Composition of the Coenzymes. Of a number of questions still un-
answered concerning these vitamins, one of the most perplexing is: Are
p-aminobenzoic acid and folic acid used to form the same coenzymes?
An independent requirement for both p-aminobenzoic acid and folic acid
has never been demonstrated; consequently one cannot, on the basis of
nutritional requirements, imply the existence of separate coenzymes for
these two vitamins. Nothing is known concerning the chemical nature of
their coenzymes; hence, it is impossible to use chemical composition as
a criterion for deciding the question. No coenzymatic activity has yet
been associated with the various combined forms of folic acid and
p-aminobenzoic acid; as a result, a quantitative comparison of the rela-
tive distribution of "combined folic acid" and "combined p-aminobenzoic
acid" cannot provide a solution based upon the distribution of their
actual coenzymes. Nor can a decision be based upon information con-
cerning the biological activities of the two vitamins under various con-
ditions; the relative responses produced by a vitamin, its coenzymes,
and compounds of intermediate complexity depend entirely upon the
type of biological system used; and, except in the case of coenzymatic
activity in cell-free systems, the responses of the various derivatives
have no predictable relationship to their chemical complexity.

Consequently, a definite statement as to the structure of the coenzymes
derived from these two vitamins and their identity or relationship to
each other must be delayed until isolated systems can be employed to
establish the identity of the specific cofactors.

COENZYMES DERIVED FROM B VITAMINS 203

If only coenzymes common to both vitamins exist, there must be two
alternative routes for their syntheses: (1) one in which folic acid is first
synthesized from p-aminobenzoic acid or else must be supplied preformed
to the organism (in which case p-aminobenzoic acid might be ineffective
as a substitute for folic acid) ; and (2) one in which the order of the
assembling of the components is such that folic acid does not constitute
an intermediate (in which case folic acid might be ineffective as a sub-
stitute for p-aminobenzoic acid).*

Biosynthesis of the Coenzymes. Although the chemical nature of the
biosynthetic processes by which coenzymes are formed from folic acid
and p-aminobenzoic acid is unknown, mention should be made of four
observations which may have some bearing on mechanisms of the con-
version of the vitamins to their active forms. (1) The occurrence and
activities of the formyl derivatives of folic and pteroic acids have been
previously pointed out; the formylation of the amino group of a p-amino-
benzoyl moiety may be a reaction occurring at some stage in the forma-
tion of the coenzymes. (2) An enzyme, designated as vitamin Bc conjugase
by its discoverers, catalyzes the hydrolysis of the polyglutamyl deriva-
tives of folic acid which have been found to occur in yeast, plant, and
animal tissues 302 ; this enzyme may catalyze reactions necessary for con-
verting the pteroyl polypeptides into a form which can be used for co-
enzyme synthesis. (3) Vitamin Bi2 has been postulated as functioning
in the utilization of p-aminobenzoic acid, probably by promoting some
reaction which is a necessary step in producing the active cofactors of
p-aminobenzoic and folic acids (p. 207). (4) The amount of folic acid
necessary to cause a remission in pernicious anemia is markedly decreased
if the vitamin is first incubated with a liver preparation of "xanthopterin
oxidase." 303 Folic acid, when incubated either with concentrates pos-
sessing xanthine oxidase activity or with crude extracts of gastric mucosa
is reported to be "activated" in a fashion such that it has vitamin Bu
activity (p. 16), i.e., it can (a) stimulate erythropoiesis in bone marrow
tissue cultures and (b) inhibit proliferation of tumor cells cultured in
vitro. Untreated folic acid is inactive. No further information on the
chemical or functional relationship of vitamin B14 and folic acid has
been disclosed.

* An announcement of the natural occurrence of a group of substances structurally
and functionally related to folic acid which are 100 times as active as folic acid in
preventing the inhibition of a folic acid inhibitor was made in November, 1949
(Bond, T. J., Bardos, T. J., Sibley, M., and Shive, W., J. Am. Chem. Soc, 71, 3852
(1949)). These forms of the folic acid vitamin, designated the folinic acid group,
replace the thymidine requirement of an organism which does not utilize folic acid
itself. Hence, this group may constitute folic acid coenzymes or be compounds more
complex than folic acid itself which are elaborated during the synthesis of the
coenzyme (s).

204 THE BIOCHEMISTRY OF B VITAMINS

Other Catalytic Functions of Folic Acid. The catabolism of tyrosine
is altered in folic acid deficiencies produced in animals,304 in scorbutic
guinea pigs,305 and in humans afflicted with pernicious anemia.306 This
metabolic derangement in the clinical anemias is so consistently observed
that it has been suggested as a test having diagnostic value (p. 416) . In
these three instances the normal oxidation of tyrosine is blocked, causing
the accumulation of phenolic keto acids (probably mono- and dihydroxy-
phenylpyruvic acids). The oral administration of either ascorbic acid,
folic acid, or liver corrects the defective tyrosine metabolism in the
scorbutic guinea pigs307; treating the pernicious anemia patients with
liver extracts (containing vitamin Bi2, but no folic acid) caused a drop
in the concentration of phenolic substances in the blood and urine 308 ;
and addition of folic acid in vitro to liver slices from folic acid-deficient
rats markedly increased their ability to oxidize tyrosine.309 These results
are of considerable interest, inasmuch as both folic acid and ascorbic
acid have been demonstrated to have some metabolic relationship to
vitamins Bi2, both in the nutrition of microorganisms (p. 206) and in
the treatment of pernicious anemia in humans (p. 416) ; however,
the interrelationship in terms of cellular reactions is obscure. It may be
that folic acid is a component of the tyrosine oxidase system and has
functions entirely independent of its role in the metabolism of formic
acid derivatives; or, since only intact tissues have been used, the func-
tion demonstrated for this vitamin in tyrosine oxidation may be an
indirect one. The amounts of folic acid required for correcting the faulty
tyrosine metabolism either in vivo or in vitro are of higher order of magni-
tude than are those required for alleviating other symptoms of folic acid
deficiencies.

Folic acid has been reported to increase the choline esterase content
of blood.310 The concentration of this enzyme, which catalyzes the hy-
drolysis of acetylcholine following its release by neural activity, appar-
ently rises when folic acid is administered to experimental animals or
even when folic acid is added to samples of blood serum in vitro. In an
independent investigation, no evidence was obtained which would sup-
port the original claims of folic acid activity.311 How direct an action
folic acid may have upon choline esterase is not known, but in view of
its other functions it seems unlikely that folic acid would be directly
involved as a coenzyme in this hydrolytic enzyme.

Dopa decarboxylase (extract of rat kidney) has been postulated to
have a coenzyme related to folic acid since pterin analogues inhibit the
activity of the enzyme, and the inhibition can be prevented by folic
acid.312 A pyridoxal analogue, which inhibits tyrosine decarboxylase,
was inactive. The pyridoxal-like activity of folic acid in this particular

COENZYMES DERIVED FROM B VITAMINS 205

instance cannot be easily correlated with any other known function of
folic acid. It may have some relationship to the role of folic acid in the
oxidation of tyrosine.304

Coenzymes Derived From Vitamin Bi2

Vitamin Bi2 and chemically related substances have only recently
become available to investigators interested in studying the chemical
functions of these compounds. As a result, their enzymatic role has not
yet been clearly denned. On the basis of preliminary and incomplete
reports, however, it would appear that the cobalt-containing compounds
effective in the treatment of pernicious anemia should be added to the
list of factors necessary for important reactions of general biological
occurrence. The inclusion of the Bi2 group in the list of B vitamins thus
seems warranted, not only from the standpoint of its distribution and
nutritional importance, but also on the basis of its function.

Until the end of 1948, all available preparations used clinically and
for research were concentrated by procedures which had beeen developed
using clinical responses as the method of assay. Consequently, it is not
surprising that, in addition to the cobalt-containing factors, these crude
concentrates contain appreciable amounts of thymidine and other des-
oxyribosides which possess "vitamin Bi2" activity under some conditions
(p. 206) . The presence of these nucleosides may be desirable from the
standpoint of the effectiveness of the product, but it makes it impossible
to interpret accurately the results of experiments in which "injectable
liver concentrates," "purified antipernicious anemia preparations," "re-
fined liver extracts," etc. were used as sources of vitamin Bi2.

It is impossible as yet to make any statement concerning the chemical
relationship of the substances possessing vitamin Bi2 activity to the
coenzyme (s) derived from them. Two cobalt-containing compounds which
are therapeutically active in the treatment of pernicious anemia have
been reported by English investigators.313 Both these compounds satisfy
the nutritional requirements of a microorganism when tested under con-
ditions where "vitamin Bi2" is essential.314 They can be most easily
characterized by the rate at which they travel during chromatographic
separation. The slower-moving compound, presumably the more complex
of the two, predominates in fresh liver tissue. During autolysis, however,
this form of the vitamin is apparently converted enzymatically to the
faster-moving compound.315 Crystalline substances independently isolated
in the United States to which the names vitamin Bi2 316 and erythrotin 317
have been given are believed to be identical with the faster-moving com-
ponent.

206. THE BIOCHEMISTRY OF B VITAMINS

The ultraviolet absorption spectrum of a crystalline preparation isolated
by a fourth laboratory indicates that the vitamin is "porphyrin-like." 318
One of the cobalt compounds already identified may be an intact co-
enzyme if the constitution of the prosthetic group of the Bi2 enzymes is
no more complex than the porphyrin groups of the iron and copper
porphyrin enzymes.

The reactions taking place during the biosynthesis, digestion, absorp-
tion, and utilization of this vitamin group are still obscure, and its
relationship to the "intrinsic" and "extrinsic" factors has yet to be
explained. A vitamin Bi2 preparation was found to be inactive (when
administered orally to pernicious anemia patients) unless it had been
previously incubated with normal gastric juice.319 Another interesting
fact which further clouds the picture is that the anemias in cattle result-
ing from cobalt deficiency can be corrected only by the oral administra-
tion of cobalt; parenteral injection of the ion is ineffective.320

The type of reactions directly catalyzed by the vitamin Bi2 coenzyme
cannot be stated with certainty, but they definitely are involved in many
of the processes which utilize single carbon units. The vitamin appears
to be intimately associated functionally with folic acid and p-amino-
benzoic acid.

To date, vitamin B12 has been implicated in the following processes:

(1) the syntheses of purines and pyrimidines and their derivatives,

(2) the syntheses of methionine and serine, and

(3) the utilization of p-aminobenzoic acid and folic acid.

The announcement of the isolation of thymidine 321 from antipernicious
anemia preparations resulted in a number of studies designed to determine
what relationship may exist between vitamin B12 and nucleic acid deriva-
tives. When tested on the bacteria which had been found to respond to
vitamin Bi2 {Lactobacillus lactis Dorner and Lactobacillus leishmanii) ,
it was found that either thymidine or the desoxyribosides of other pyrim-
idines and purines could be substituted for this vitamin.322- 323> 324« 325
Also it was shown that, in the presence of vitamin B12 (and folic acid),
purines could be omitted from the medium used to culture L. Lactis.Z2&

In considering possible relationship of vitamin Bi2 to folic acid, it is
important to note that in the cases just mentioned vitamin B12 and
folic acid cannot be effectively substituted for each other. An interesting
demonstration of their independence of function is contained in a report
in which it is implied that the vitamin Bi2 required by L. leishmanii
can be replaced by thymidine but not by thymine, whereas folic acid can
be replaced (somewhat inadequately) by either thymine or thymidine.326
On the basis of the nutritional needs of these two bacteria there has been
some speculation as to the possibility of vitamin Bi2 functioning in the

COENZYMES DERIVED FROM B VITAMINS 207

formation of desoxyribosides, either by promoting the synthesis of a
reactive derivative of desoxyribose 322 or by catalyzing the union of the
sugar with the nitrogen bases.323

Vitamin Bi2 has since been shown to participate in other types of
biosynthetic processes — those by which serine and methionine are formed.
This was first demonstrated by the use of sulfonamide inhibition of
Escherichia coli.29St 317 Two other findings offer collaborative evidence
for the methionine functions: vitamin Bi2 can completely replace the
methionine necessary to promote the growth of a p-aminobenzoic acid
requiring mutant of E. coli, when it is cultured in a medium containing
suboptimal amounts of p-aminobenzoic acid327; and the incidence of
renal damage in rats resulting from a diet deficient in choline and
methionine can be appreciably reduced by the administration of vitamin
B12.328

A very plausible explanation for the manner in which vitamin Bi2
functions has been obtained through the use of inhibitors.295, 317 In E. coli
the biosynthesis of methionine, purines, serine and folic acid (or thymine)
by enzyme systems containing p-aminobenzoic acid can be effectively
blocked by sulfonamide inhibition. But in each of these four distinct
processes the presence of vitamin Bi2 (0.00005 /xgm/ml) reduces by two-
thirds the amount of p-aminobenzoic acid required to counteract the
specific inhibition and restores adequate synthesis of the respective com-
pounds (section D) . The investigators felt that their results could be best
explained by assuming that vitamin B12 is a catalytic factor necessary for
the utilization of p-aminobenzoic acid (and folic acid?). This explanation
could be interpreted to mean that vitamin B12 functions as a catalyst
for the formation of the coenzymes necessary in the reactions involving
single carbon units. Such a hypothesis seems reasonable and would
explain why vitamin Bi2 is involved in the biosynthesis of a variety of
types of chemical compounds. It should be noted that these compounds
include all those in which p-aminobenzoic acid and folic acid are definitely
known to function (p. 201). It would also imply an independent require-
ment of both vitamin Bi2 (catalyst for coenzyme formation) and p-amino-
benzoic acid or folic acid (substrate for coenzyme formation) . Hence
folic acid could not be expected to substitute completely for vitamin Bi2
in the treatment of macrocytic anemias, nor would vitamin Bi2 be ex-
pected to exhibit appreciable activity in the treatment of blood dyscrasias
corrected by folic acid therapy (Chapter VIC).

It has been suggested on the basis of clinical evidence that vitamin Bi2
is necessary for the conversion of folic acid conjugates to other deriva-
tives which are more readily utilized.329, 330, 331 Although the investigators
later retracted their conclusions,332 a direct demonstration of such a

208 THE BIOCHEMISTRY OF B VITAMINS

function for the vitamin appeared in a recent preliminary report.333 The
livers from day-old chicks hatched from eggs laid by hens whose diet
contained no "animal protein" were used as a source of tissue deficient in
vitamin Bi2. These livers were incapable of liberating folic acid from
pteroylheptaglutamic acid. However, the addition of crystalline vitamin
B12 initiated conjugase activity. This would indicate that vitamin B12,
or a derivative of it formed by the liver cells, is a cofactor for some
reaction in the process by which the folic acid conjugate is degraded. The
same reaction, or one related to it chemically, may very well be one of
the essential steps involved in the conversion of folic acid and p-amino-
benzoic acid to their active coenzymes.

The particular reaction (s) catalyzed by Bi2 may be of an oxidative
type if the molecular structure of the vitamin is indicative of its mode
of functioning. All other metallo-porphyrin enzymes mediate oxidative
reactions (p. 151). A similar mechanism for vitamin Bi2 action may be
anticipated if further investigation establishes its structure to be that of
a typical porphyrin.

There is some metabolic relationship, either direct or indirect, between
this vitamin and ascorbic acid (or other reducing compounds). These
agents alter the nutritional requirements of lactobacilli to such an extent
that in their presence the organism can no longer be demonstrated to
require an exogenous supply of vitamin Bi2 or the desoxyribosides.325- 334
The ascorbic acid-vitamin Bi2 effect in microorganisms may be related
to the synergistic effects of liver extracts and vitamin C in the treatment
of pernicious anemia.335

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63. Krebs, H. A., Enzymologia, 7, 53 (1939).

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65. Theorell, H., Biochem. Z., 278, 263 (1935).

66. Warburg, O., and Christian, W., Biochem. Z., 298, 368 (1938).

67. Klein, J. R., and Handler, P., J. Biol. Chem., 139, 103 (1941).

68. Green, D. E., Moore, D. H., Nocito, V., and Ratner, S., J. Biol. Chem., 156,

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69. Blanchard, M. Green, D. E., Nocito, V., and Ratner, S., J. Biol. Chem., 163,

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70. Blanchard, M., Green, D. E., Nocito, V., and Ratner, S., J. Biol. Chem., 161,

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71. Roulet, R., Wydler, H., and Zeller, E. A., Helv. Chim. Acta, 29, 1973 (1946).

72. Knight, S. G., J. Bad., 55, 401 (1948).

73. Singer, T. P., and Kearney, E. B., Fed. Proc, 8, 251 (1949).

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75. Schardinger, F., Z. Untersuch. Nahr. Genussm., 5, 22 (1902).

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78. Lemberg, R., Wyndham, R. A., and Henry, N. P., Australian J. Exptl. Biol.

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80. Schales, O., Arch. Biochem., 2, 487 (1943).

81. Lipmann, F., and Owen, C. R., Science, 98, 246 (1943).

82. Ball, E. G., Science, 88, 131 (1938).

83. Corran, N. S., Dewan, J. G., Gordon, A. H., and Green, D. E., Biochem. J.,

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86. Ball. E. G., J. Biol. Chem., 128, 51 (1939).

87. Haas, E. Horecker, B. L., and Hogness, T. R., J. Biol. Chem., 136, 747 (1940).

88. Lipmann, F., Cold Spring Harbor Symp. Quant. Biol, 7, 248 (1939).

89. Lipmann, F., "Currents in Biochemical Research," Interscience Publishers,

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90. Fischer, F. G., Roedig, A., and Rauch, K., Naturwiss., 27, 197 (1939).

91. Stotz, E., and Hastings, A. B., J. Biol. Chem., 118, 479 (1937).

92. Axelrod, A. E., Potter, V. R., and Elvehjem, C. A., J. Biol. Chem., 142, 85

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93. Lwoff, A., and Lwoff, M., Ann. Inst. Pasteur, 59, 129 (1937).

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94. Stotz, E., "A Symposium on Respiratory Enzymes," The University of Wis-

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98. Stephenson, M., Biochem. J., 22, 605 (1928).

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104. Rosenthal, O., and Drabkin, D. L., J. Biol. Chem., 149, 437 (1943).

105. Carruthers, C, J. Biol. Chem., 171, 64 (1947).

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106. Wainis, W. W., Cooperstein, S. J., Kollen, S., and Eichel, B., J. Biol. Chem.,

173, 145 (1948).

107. Lipton, M. A., Arnold, A., and Berger, J., "Respiratory Enzymes," Burgess

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108. Neuberg, C, and Karczag, L., Biochem. Z., 36, 68 (1911).

109. Auhagen, E., Z. physiol. Chem., 204, 149 (1932).

110. Lohmann, K., and Schuster, P., Biochem. Z., 294, 188 (1937).

111. Weijlard, J., and Tauber, H., J. Am. Chem. Soc., 60, 2263 (1938).

112. Barron, E. S. G., and Lyman, C. M., J. Biol. Chem., 141, 951 (1941).

113. Ochoa, S., and Peters, R. A., Biochem. J., 32, 1501 (1938).

114. Westenbrink, H. G. K., Van Dorp, D. A., Gruder, M., and Veldman, H.,

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115. Lipton, M. A., and Elvehjem, C. A., J. Biol. Chem., 136, 637 (1940).

116. Silverman, M., and Werkman, C. H., Enzymologia, 5, 385 (1938-39).

117. Goodhart, R., /. Biol. Chem., 135, 77 (1940).

118. Sumner, James B., and Somers, G. Fred, "Chemistry and Methods of Enzymes,"

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119. Lankford, C. E., and Skaggs, P. K., Arch. Biochem., 9, 265 (1946).

120. Westenbrink, H. G. K., and Goudsmit, J., Enzymologia, 5, 307 (1938).

121. Ochoa, S., "The Biological Action of the Vitamins," The University of

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122. Lipton, M. A., and Elvehjem, C. A., Cold Spring Harbor Symp. Quant. Biol.,

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123. Weil-Malherbe, H., Biochem. J., 33, 1997 (1939).

124. Cedrangolo, F., and Villano, F., Boll. soc. ital. biol. sper., 17, 558 (1942).

125. Westenbrink, H. G. K., and Veldman, H, Enzymologia, 10, 255 (1942).

126. Parvi, E. P. S., and Westenbrink, H. G. K., Z. Vitaminforschung, 15, 152

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127. Parvi, E. P. S., Chem. Zentr., I, 2000 (1943).

128. Buchman, E. R., Heegaard, E., and Bonner, J., Proc. Nat. Acad. Sci., 26, 561

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129. Grob, E. C, Z. Vitaniinjorschung, 17, 98 (1946).

130. Banga, I., Ochoa, S, and Peters, R. A., Biochem. J., 33, 1109 (1939).

131. Peters, R. A., Biochem. J., 31, 2240 (1937).

132. Saratt, H. P., and Cheldelin, V. H., J. Biol. Chem., 156, 91 (1944).

133. Lipmann, F., Enzymologia, 4, 65 (1937).

134. Ochoa, S., "The Biological Action of the Vitamins," The University of Chicago

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135. Stotz, E., Advances in Enzymology, 5, 145 (1945).

136. Martius, C, Z. physiol. Chem., 279, 96 (1943).

137. Weil-Malherbe, H., Nature, 145, 106 (1940).

138. Green, D. E., Westerfeld, W. W., Vennesland, B., and Knox, W. E., J. Biol.

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139. Silverman, M., and Werkman, C. H., J. Biol. Chem., 138, 35 (1941).

140. Watt, D., and Krampitz, L. 0., Federation Proc, 6, 301 (1947).

141. Gross, N. H., and Werkman, C. H., Arch. Biochem., 15, 125 (1947).

142. Utter, M. R., Werkman, C. H., and Lipmann, F, J. Biol Chem., 154, 723 (1944).

143. Lipmann, F., Advances in Enzymology, 6, 231 (1946).

144. Koepsell, H. J., and Johnson, M. J., J. Biol. Chem., 145, 379 (1942).

145. Krebs, H. A., and Johnson, W. A., Biochem. J., 31, 645 (1937).

146. Weil-Malherbe, H., Biochem. J., 31, 2202 (1937).

147. Barron, E. S. G., and Lyman, C. M., J. Biol. Chem., 127, 143 (1939).

148. Krebs, H. A., Biochem. J., 31, 661 (1937).

149. Wiken, T., Watt, D., White, A. G. C, and Werkman, C. H., Arch. Biochem.,

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150. Green, D. E., Stumpf, P. K., and Zarudnoya, K., J. Biol. Chem., 167, 811 (1947).

212 THE BIOCHEMISTRY OF B VITAMINS

151. Ochoa, S, J. Biol. Chem., 155, 87 (1944).

152. Karrar, P., Graf, W., and Schukri, J., Helv. Chim. Acta, 29, 711 (1946).

153. Zima, 0., and Williams, R. R., Ber. deutsch. chem. Gesellsch., 73, 941 (1940).

154. Zima, 0., Ritsert, K., and Moll, Th., Ztschr. physiol. Chem., 267, 210 (1941).

155. Karrar, P., and Viscontini, M., Helv. Chim. Acta, 29, 711 (1946).

156. O'Kane, D. J., and Gunsalus, I. C, J. Bad., 56, 499 (1948).

157. Vennesland, B., and Evans, E. A., Jr., /. Biol. Chem., 156, 783 (1944).

158. Vennesland, B., Evans, E. A. Jr., and Francis, A. M., J. Biol. Chem., 163, 573

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159. Evans, E. A., Jr., Vennesland, B., and Slotin, L., J. Biol. Chem., 147, 771 (1943).

160. Ochoa, S. and Weisz-Tabori, E., J. Biol. Chem., 159, 245 (1947).

161. Shive, W., and Rogers, L. L., J. Biol. Chem., 169, 453 (1947).

162. Lardy, H. A., Potter, R. L., and Elvehjem, C. A., J. Biol. Chem., 169, 451

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163. Korkes, S. and Ochoa, S., J. Biol. Chem., 176, 463 (1948).

164. Lardy, H. A., Abstract 112th Meeting Am. Chem. Soc, Sept., 1947.

165. Pilgrim, F. J., Axelrod, A. E., and Elvehjem, C. A., /. Biol. Chem., 145, 237

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166. Summerson, W. H., Lee, J. M., and Partridge, C. W. H., Science, 100, 250

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167. Winzler, R. J., Burk, D., and du Vigneaud, V., Arch. Biochem., 5, 25 (1944).

168. Benz, L. G., and Eakin, R. E, Unpublished data.

169. Plaut, G. W. E., and Lardy, H. A., Fed. Proc, 8, 237 (1949).

170. Ochoa, S., et al, J. Biol. Chem., 170, 413 (1949).

171. Koser, S. A., Wright, M. H., and Dorfman, A., Proc. Soc. Exptl. Biol. Med.,

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172. Benz, L. G., Master Thesis, University of Texas, August, 1947.

173. Stokes, J. L., Larsen, A., and Gunness, M., J. Bact., 54, 219 (1947).

174. Potter, R. L., and Elvehjem, C. A., J. Biol. Chem., 172, 531 (1948).

175. Axelrod, A. E., Purvis, S. E., and Hofmann, K., J. Biol. Chem., 176, 695 (1948).

176. Lichstein, H. C., and Umbreit, W. W., J. Biol. Chem., 170, 329 (1947).

177. Lichstein, H. C., and Christman, J. R., J. Biol. Chem., 175, 649 (1948).

178. Lichstein, H. C., J. Biol. Chem., 177, 125 (1949).

179. Williams, V. R., and Fieger, E. A., J. Biol. Chem., 166, 335 (1946).

180. Williams, W. L., Broquist, H. P., and Snell, E. E., J. Biol. Chem., 170, 619

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181. Axelrod, A. E., Hofmann, K., and Daubert, B. F., /. Biol. Chem., 169, 761

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182. Axelrod, A. E., Mitz, M., and Hofmann, K, J. Biol. Chem., 175, 265 (1948).

183. Trager, W., J. Biol. Chem., 176, 133 (1948).

184. Trager, W., /. Biol. Chem., 176, 1211 (1948).

185. Burk, D., and Winzler, R. J., Science, 97, 57 (1943).

186. Jones, M., and Eakin, R. E., unpublished data.

187. Eakin, R. E., Snell, E. E., and Williams, R. J., J. Biol. Chem., 140, 535 (1941).

188. Gale, E. F., Biochem. J., 34, 392 (1940).

189. Gale, E. F., and Epps, H. M. R., Biochem. J., 38, 250 (1944).

190. Bellamy, W. D, and Gunsalus, I. C., /. Bact., 46, 573 (1943).

191. Gunsalus, I. C., and Bellamy, W. D., J. Biol. Chem., 155, 357 (1944).

192.' Umbreit, W. W., Bellamy, W. D., and Gunsalus, I. C., Arch. Biochem., 7, 185
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193. Snell, E. E., Guirard, B. M., and Williams, R. J., J. Biol. Chem., 143, 519 (1942).

194. Snell, E. E, J. Am. Chem. Soc, 67, 194 (1945).

195. Braunstein, A. E., and Kritzmann, M. G., Enzymologia, 2, 129 (1937).

196. Schlenk, F., and Snell, E. E., J. Biol. Chem., 157, 425 (1945).

197. Green, D. E., Leloir, Luis, F., and Nocito, V., J. Biol. Chem., 161, 559 (1945).

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198. Lichstein, H. C, Gunsalus, I. C., and Umbreit, W. W., J. Biol. Chem., 161, 311

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199. Umbreit, W. W., Wood, W. A., and Gunsalus, I. C, J. Biol. Chem., 165, 731

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200. Dawes, E. A., Dawson, J., and Happold, F. C, Nature, 159, 645 (1947).

201. Wood, W. A., Gunsalus, I. C, and Umbreit, W. W., J. Biol. Chem., 170, 313

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202. Umbreit, W. W., and Gunsalus, I. C, J. Biol. Chem., 179, 279 (1949).

203. Gunsalus, I. C, Umbreit, W. W., Bellamy, W. D., and Foust, C. E., J. Biol.

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204. Karrer, P., and Viscontini, M., Helv. Chim. Acta, 30, 52 (1947).

205. Ibid., 30, 524 (1947).

206. Ibid., 30, 528 (1947).

207. Rabinowitz, J. C, and Snell, E. E., /. Biol. Chem., 169, 643 (1947).

208. Umbreit, W. W., O'Kane, D. J., and Gunsalus, I. C, J. Biol. Chem., 176, 629

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209. McNutt, W. S., and Snell, E. E., J. Biol. Chem., 173, 801 (1948).

210. Rabinowitz, J. C, and Snell, E. E., J. Biol. Chem., 176, 1157 (1948).

211. Herbst, E. J., and Snell, E. E., J. Biol. Chem., 176, 989 (1948).

212. Bellamy, W. D., Umbreit, W. W., and Gunsalus, I. C, J. Biol. Chem., 160, 461

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213. O'Kane, D. E., and Gunsalus, I. C, J. Biol. Chem., 170, 425 (1947).

214. Umbreit, W. W., and Waddell, J. G., Proc. Soc. Exptl. Biol. Med., 70, 293

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215. Umbreit, W. W., and Gunsalus, I. C, J. Biol. Chem., 159, 333 (1945).

216. Gale, E. F., and Epps, H. M. R., Biochem. J., 38, 238 (1944).

217. Gale, E. F., Advances in Enzymol, 6, 1 (1946).

218. Blaschko, H., Advances in Enzymol., 5, 67 (1945).

219. Epps, H. M. R., Biochem. J., 38, 242 (1944).

220. Gale, E. F., and Epps, H. M. R., Biochem. J., 38, 232 (1944).

221. Taylor, E. S., and Gale, E. F., Biochem. J., 39, 52 (1945).

222. Braunstein, A. E., and Kritzmann, M. G., Biochimia, 8, 1 (1943).

223. O'Kane, D. E., and Gunsalus, I. C, J. Biol. Chem., 170, 433 (1947).

224. Albaum, H. G., and Cohen, P. P., J. Biol. Chem., 149, 9 (1943).

225. Cohen, P. P., J. Biol. Chem., 136, 565 (1940).

226. Tatum, E. L., and Bonner, D., Proc. Nat. Acad. Sci., 30, 30 (1944).

227. Stokes, J. L., and Gunness, M., Science, 101, 43 (1945).

228. Lyman, C. M., et al., J. Biol. Chem., 167, 177 (1947).

229. Lyman, C. M., and Kiuken, K. A., Federation Proc, 7, 770 (1948).

230. Broquist, H. P., and Snell, E. E., Federation Proc, 8, 188 (1949).

231. Schweigert, B. S., J. Biol. Chem., 168, 283 (1947).

232. Birch, T. W., J. Biol. Chem., 124, 775 (1938).

233. Halliday, N., J. Nutrition, 16, 285 (1938).

234. Quackenbush, F. W., and Steenbock, H., Proc. XVI Intern. Physiol. Congr.

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235. Salmon, W. D., Proc Am. Soc. Biol. Chem., 34, LXXXII (1940). .

236. Schlenk, F., and Fisher, A., Arch. Biochem., 12, 69 (1947).

237. Snell, E. E., and Guirard, B. M., Proc Nat. Acad. Sci., 29, 66 (1943).

238. Snell, E. E, J. Biol. Chem., 158, 497 (1945).

239. Holden, J. T., Furman, C, and Snell, E. E., J. Biol. Chem., 178, 789 (1949).

240. Holden, J. T., and Snell, E. E., J. Biol. Chem., 178, 799 (1949).

241. Ochoa, S., "Currents in Biochemical Research," Interscience Publishers (New

York), 1946, p. 165.

242. Rittenberg, D., and Block. K, J. Biol. Chem., 154, 311 (1944).

243. Block, K., "Currents in Biochemical Research," Interscience Publishers (New

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244. Snell, E. E., Strong, F. M., and Peterson, W. H., Biochem. J., 31, 1789 (1937).

245. Guirard, B. M., Snell, E. E., and Williams, R. J., Arch. Biochem., 9, 361 (1946).

246. Ibid., 9, 381 (1946).

247. Lipmann, F., Advances in Enzymol., 6, 231 (1946).

248. Kaplan, N. 0., and Lipmann, F., Federation Proc, 7, 163 (1948).

249. Lipmann, F., J. Biol. Chew,., 160, 173 (1945).

250. Strecker, H., Krampitz, L. O., and Wood, H. G., Federation Proc, 7, 194 (1948).

251. Kaplan, N. 0., and Lipmann, F., J. Biol. Chem., 176, 459 (1948).

252. Kaplan, N. O, and Soodak, M., Federation Proc, 8, 211 (1949).

253. Williams, R. J, Mosher, W. A., and Rohrman, E., Biochem. J., 30, 2036 (1936).

254. Nachmansohn, D., and Machado, A. L., J. Neurophysiol, 6, 397 (1943).

255. Lipmann, F., and Kaplan, N. O., J. Biol. Chem., 162, 743 (1946).

256. Lipmann, F., et al., J. Biol. Chem., 167, 869 (1947).

257. Novelli, G. D., Kaplan, N. O., and Lipmann, F., J. Biol. Chem., 177, 97 (1949).

258. Novelli, G. D., Flynn, R. M., and Lipmann, F., J. Biol. Chem., 177, 493 (1949).

259. King, T. E., Fels, I. G., and Cheldelin, V. H., J. Am. Chem. Soc, 71, 131 (1949).

260. Nachmansohn, D., and Berman, M., J. Biol. Chem., 165, 55 (1946).

261. Kaplan, N. O., and Lipmann, F., J. Biol. Chem., 174, 37 (1948).

262. Dorfman, A., Berkman, S., and Koser, S. A., J. Biol. Chem., 144, 393 (1942).

263. Novelli, G. D., and Lipmann, F., Arch. Biochem., 14, 23 (1947).

264. Novelli, G. D., and Lipmann, F., J. Biol. Chem., 171, 833 (1947).

265. Olson, R. E., and Kaplan, N. O., J. Biol. Chem., 175, 515 (1948).

266. Soodak, M., and Lipmann, F., J. Biol. Chem., 175, 999 (1948).

267. Guirard, B. M., unpublished observations.

268. Shive, W., Ackermann, W. W., Ravel, J. M., and Sutherland, J. E., J. Am.

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269. Gordon, M., Ph. D. Dissertation, University of Texas, June, 1948.

270. Pilgrim, F. J., Axelrod, A. E., and Elvehjem, C. A., J. Biol. Chem., 145, 237

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271. Hills, G. M., Biochem. J., 37, 418 (1943).

272. McHenry, E. W., and Gavin, G., J. Biol. Chem., 138, 471 (1941).

273. Buchanan, J. M., and Sonne, J. C, J. Biol. Chem., 166, 781 (1946).

274. Block, K., and Rittenberg, D., J. Biol. Chem., 159, 45 (1945).

275. Slade, H. D., and Werkman, C. H., Arch. Biochem., 2, 97 (1943).

276. Buchanan, J. M., Sonne, J. C., and Delluva, A. M., J. Biol. Chem., 166, 395

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277. Shive, W., Lane, A. E., and Eakin, R. E., unpublished observations.

278. Sakami, W., J. Biol. Chem., 176, 995 (1948).

279. Edelbacher, S., Z. physiol. Chem., 157, 106 (1926).

280. Sakami, W., J. Biol. Chem., 178, 519 (1949).

281. Winnick, T., Moring-Claesson, I., and Greenberg, D. M., J. Biol. Chem., 175,

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282. Sakami, W., Federation Proc, 8, 246 (1949).

283. Rose, W. C., and Cook, K. G., J. Biol. Chem., 64, 325 (1925).

284. Shemin, D., and Rittenberg, D., J. Biol. Chem., 167, 875 (1947).

285. Kohn, H. I., Ann. N. Y. Acad. Sci., 44, 503 (1943).

286. Housewright, R. D., and Koser, S. A., J. Infect. Dis., 75, 113 (1944).

287. Lampen, J. 0., Roepke, R. R., and Jones, M. J., J. Biol. Chem., 164, 789 (1946).

288. Shive, W., and Macow, J., J. Biol. Chem., 162, 451 (1946).

289. Shive, W., and Roberts, E. C., J. Biol. Chem., 162, 463 (1946).

290. Shive, W., et al, J. Am. Chem. Soc, 69, 725 (1947).

291. Stetten, M. R., and Fox, C. L., Jr., J. Biol. Chem., 161, 333 (1945).

292. Wolf, D. E., et al., J. Am. Chem.. Soc, 69, 2753 (1947).

293. Gordon, M., Ravel, J. M., Eakin, R. E., and Shive, W., J. Am. Chem. Soc, 70,

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294. Winkler, K. C., and de Haan, P. G., Arch. Biochem., 18, 97 (1948).

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295. Alexander, E. R., Master Thesis, University of Texas, June, 1949.

296. Shemin, D., J. Biol. Chem., 162, 297 (1946).

297. Ravel, J. M., Eakin, R. E., and Shive, W., J. Biol. Chem., 172, 67 (1948).

298. Holland, B. R., and Meinke. W. W., J. Biol Chem., 178, 7 (1949).

299. Shive, W., and Ravel, J. M., unpublished observations.

300. Stokstad, E. L. R., J. Biol. Chem., 139, 476 (1941).

301. Stokes, J. L., J. Bad., 48, 201 (1944).

302. Bird, O. D., el al, J. Biol. Chem., 157, 413 (1945).

303. Jacobson, W., and Good, P. M., Int. Physiol. Cong. Abstracts, Oxford, XVII,

157 (1947).

304. Rodney, G., Swendseid, M. E., and Swanson, A. L., J. Biol. Chem., 168, 395

(1947).

305. Sealock, R. R., and Silberstein, H. E., J. Biol. Chem., 135, 251 (1940).

306. Swendseid, M. E., Burton, I. F., and Bethel, F. H, Proc Soc. Exptl. Biol, and

Med., 52, 202 (1943).

307. Woodruff, W. C., and Darby, W. J., J. Biol. Chem., 172, 851 (1948).

308. Swendseid, M. E., Wandruff, B., and Bethel, F. H., J. Lab. Clin. Med., 32, 1242

(1947).

309. Rodney, G., Swendseid, M. E., and Swanson, A. L., J. Biol. Chem., 179, 19

(1949).

310. Davis, J. E., Proc. Soc. Exptl. Biol, and Med., 63, 287 (1946).

311. Hawkins, R. D., Arch. Biochem., 17, 97 (1948).

312. Martin, G. J., and Beiler, J. M., Arch. Biochem., 15, 195 (1947).

313. Smith, E. L., Nature, 161, 638 (1948).

314. Cuthbertson, W. F. J., and Smith, L. E, Biochem. J., 44, v (1949).

315. Smith, E. L., and Parker, L. F., Biochem. ./., 43, viii (1948).

316. Rickes, E. L., et al, Science, 107, 396 (1948).

317. Shive, W., Ann. N. Y. Acad. Sci., in press.

318. Ellis, B., Petrow, V., and Snook, G., J. Pharm. Pharmacol, 1, 287 (1949).

319. Berk, et al, New England J. Med., 239, 911 (1948).

320. McCance, R. A., Rev. Clin. Espan., 8, 371 (1943).

321. Shive, W. Eakin, R. E., Harding, W. M., Ravel, J. M., and Sutherland, J. E.,

J. Am. Chem. Soc, 70, 2299 (1948).

322. Kitay, E., McNutt, W. S., and Snell, E. E., J. Biol. Chem., 177, 993 (1949).

323. Wright, L. D., Skeggs, H. R., and Huff. J. W., J. Biol Chem., 175, 475 (1948).

324. Kocher, Von v. and Schindler, O., Sonderdruck ans Internationale Zeitschrijt

jiir Vitaminforschung, 20, 441 (1949).

325. Shive, W., Ravel, J. M., and Eakin, R. E., J. Am. Chem. Soc, 70, 2614 (1948).

326. Shive, W., Ravel, J. M, and Harding, W. M., J. Biol. Chem., 176, 991 (1948).

327. Sibley, M., and Shive, W., unpublished observations.

328. Schaefer, A. E., Salmon, W. D., and Strength, D. R., Federation Proc, 8, 395

(1949).

329. Heinle, R. W., and Welch, A. C, Ann. N. Y. Acad. Sci., 48, 343 (1936).

330. Welch, A. D., Heinle, R. W., Nelson, E. M., and Nelson, H. V., J. Biol. Chem.,

164, 787 (1946).

331. Bethell, F. H., et al, Univ. Hosp. Bull, Univ. of Mich., 12, 42 (1946).

332. Welch, A. D., Federation Proc, 6, 471 (1947).

333. Scott, M. L., Hill, C. H, and Norris, L. C., Federation Proc, 8, 249 (1949).

334. Kocher, Von v., Sonderdruck aus Internationale Zeitschrijt jiir Vitaminjor-

schung, 20, 369 (1949).

335. Dyke, S. C., Delia Vida, B. L., and Kelikat, E., Lancet, 2, 278 (1942).

Chapter IIIB

THE FUNCTIONS OF THE B VITAMINS
IN METABOLIC PROCESSES

In Chapter I B there was presented a brief discussion of the funda-
mental reactions common to most organisms. The purpose of the present
chapter is to examine in greater detail those processes the chemical steps
of which have been fairly well established and which are believed to be
generally utilized by many different types of life. There are undoubtedly
some reactions included in this discussion which cannot be demonstrated
to occur in all organisms, and likewise other reactions omitted which
may be universal. The purpose of this chapter would be defeated and
our perspective lost if an attempt was made to be encyclopedic with
respect to all the metabolic reactions which have been demonstrated or
postulated, and to discuss in detail exceptions to the general schemes.
The material presented here is intended (1) to indicate the basic patterns
which are usually followed by most cells, (2) to elucidate the nature of
the chemical reactions involved, and (3) to establish the positions where
the B vitamins are essential.

Organisms show extreme variation in their ability to carry out many
reactions. The numerous end products of glucose metabolism which are
produced by different organisms illustrate this point well. However, the
differences are often one of degree rather than absolute. Although we
often think of one or two specific compounds as being the end products
of a particular fermentation process, actually many substances are pro-
duced. In no fermentation involving intact cells are the principal products
formed exclusively.

Variations in the chemical processes taking place in cells occur (1)
when different organisms are compared, (2) when cells from one tissue
are compared with those of another tissue in the same organism, (3) when
cells at one age are compared with similar cells at a different age, and
(4) when cells and tissues from one individual organism are compared
with those of another individual of the same species. These variations,
taken as a group, are probably more often of a quantitative rather than
a qualitative nature. Many organisms and cells probably have the ability
to carry out fundamental reactions, such as we are considering, even
though such reactions take place slowly and in many cases have not

216

METABOLIC FUNCTIONS OF B VITAMINS 217

been specifically demonstrated. The production of partial genetic blocks
which result, for example, in impaired synthetic abilities on the part of
the cells may cause cells to require for rapid metabolism a substance
which otherwise would never be a limiting factor. In such cases, how-
ever, the cells have not lost their synthetic ability completely, and the
difference between these cells and the unaltered ones is quantitative,
not qualitative. The capacity of cells to adapt themselves to the utiliza-
tion of completely new substances indicates that they possess latent
potentialities with respect to enzymatic reactions which are ordinarily
not observed.

Variations in the fundamental processes in different cells, in different
species, and in different individuals of the same species are of extreme
importance and interest, but their discussion does not belong in a sum-
mary of the chemical processes which appear to be common to cells in
general.

There would be certain advantages in preparing an elaborate diagram-
matic scheme showing all the known and postulated relationships between
the fundamental biochemical compounds. Such a chart could be used to
indicate in a concise manner where the individual reactions discussed in
the previous chapter fit into the general processes of cell metabolism. It
is technically impossible, however, to prepare a diagram that would not
be more confusing than enlightening, since some of the intermediate
compounds, like pyruvic acid, are involved in a multitude of reactions.
The metabolic processes, therefore, will be treated in turn according to
the classical divisions of biochemical substances: carbohydrates, lipides,
and amino acids and proteins. Following this will be a discussion of the
role of the B vitamins in the fundamental physiological processes involv-
ing energy transformations.

The Utilization of Carbohydrates

Most of the chemical steps involved in the utilization of carbohydrates
have been well established, and a number of excellent reviews on this
aspect of metabolism have appeared during the last ten years.1-7 The
general metabolic pathways by which the carbohydrates are stored, de-
graded, or converted to intermediates that can be used for the synthesis
of compounds of other types involve a large number of reactions. These
can be most conveniently considered as components of four different
phases: (1) the synthesis and cleavage of the polysaccharides; (2) the
glycolytic process — glycogen (or starch) ±? pyruvate; (3) the anaerobic
utilization of pyruvate; and (4) the aerobic utilization of pyruvate.

The Synthesis and Cleavage of Polysaccharides. The initial phase of
carbohydrate utilization includes the reactions by which complex sugars

218 THE BIOCHEMISTRY OF B VITAMINS

are enzymatically hydrolyzed extracellularly to yield simpler sugars
which can be absorbed, and the subsequent intracellular processes
by which these absorbed compounds are converted into polysaccharides
in which form they are stored until utilized. A number of enzymes
hydrolyzing carbohydrates have been well characterized. Of these, only
a single specific enzyme, pancreatic amylase, has been shown to contain
a coenzyme (inositol) (p. 125).

The intracellular synthesis of glycogen and starch from simple hexoses
cannot be carried out directly since an input of energy is required for
the formation of the acetal bonds. The energy for the synthesis is intro-
duced by a reaction in which glucose is initially converted to a phosphate
ester8 by adenosine triphosphate, a transformation that uses up an
energy-rich phosphate bond generated previously in some metabolic
reaction. The phosphate ester initially formed, glucose-6-phosphate, is
in equilibrium with its isomer, glucose- 1 -phosphate, due to the presence
of an enzyme which catalyzes this intramolecular transesterification. The
glucose- 1-phosphate molecules polymerize to form the polysaccharide by
a reaction in which the phosphate ester linkage is cleaved (liberating
inorganic phosphate), but an acetal bond is created. The energy trans-
formations involved in this reaction are small and the hexose-phosphate
and polysaccharide are usually in equilibrium.

x glucose-1-phosphate ^ *** polysaccharide +2H3PO4

When an organism expends energy, the phosphoric acid anhydrides, in
which energy has been stored, are hydrolytically cleaved and the in-
organic phosphate concentration increases. This increase in inorganic
phosphate upsets the equilibrium between the hexose phosphate and
glycogen and causes the breakdown of glycogen to glucose- 1-phosphate.
This, in turn, initiates the glycolytic process in which the glycogen is
metabolized. The energy liberated during this process is utilized through
the resynthesis of the phosphoric acid anhydrides from the inorganic
phosphate. The formation of energy-containing phosphate bonds con-
tinues until the cell reaches a state wherein the inorganic phosphate
concentration will have been reduced to such a level that the equilibrium
shifts to favor the formation of glycogen instead of its breakdown. As
far as is now known, the reactions involved in the intracellular formation
of polysaccharides require only adenylic acid and its phosphorylated
derivatives as coenzymes. The B vitamins are involved only indirectly;
they are needed for producing the energy units used in the synthetic
process — the phosphoric acid anhydrides.

Glycolysis. When the second phase of carbohydrate utilization, the
glycolytic process, is initiated by the phosphorolysis of glycogen (or

METABOLIC FUNCTIONS OF B VITAMINS 219

starch), a series of reactions occur which eventually produce pyruvic
acid. Although other ways in which hexoses can be degraded are known,3
the mechanism employed by almost all organisms is the one represented
by the classical scheme of fermentation.1 The individual reactions involve
the formation and cleavage of phosphate esters, isomerizations, the
cleavage of the hexose diphosphate into two triose phosphates (a reaction
which is the reverse of an aldol condensation), the dehydration of a
/^-hydroxy acid, and one dehydrogcnation:

1 . Transesterification :

glucose-1-phosphate ^ "* glucose-6-phosphate

2. Phosphorylation:

glucose-6-phosphate+ATP > glueose-l,6-diphosphate+ADP

3. Isomerization:

glucose-l,6-diphosphate ^ ^ fructose-l,6-diphosphate

4. Aldol cleavage or formation:

fructose-l,6-diphosphate ^ "*• glyceraldehyde-3-phosphate +

dihydroxyacetone phosphate

5. Isomerization:

dihydroxyacetone phosphate ^ *" glyceraldehyde-3-phosphate

6. Nonenzymalic formation and disintegration of a carbonyl-phosphoric acid addition
product (see p. 140):

2(glyceraldehyde-3-phosphate) +2H3P04 =^=^

2 (glyceraldehyde-3-phosphate) -phosphoric acid addition product

7. Hydrogenation-dehydrogenation:

2(glyceraldehyde-3-phosphate) -phosphoric acid addition product +

2DPN =^= 2(3-phosphoglyceroyl phosphate) +2(DPN-2H)

8. Phosphorylation:

2(3-phosphoglvceroyl phosphate) +2ADP -v ^

2(3-phosphoglyceric acid) +2 ATP

9. Transesterification:

2(3-phosphoglyceric acid) =^=^= 2(2-phosphoglyceric acid)

10. Dehydration or hydration (involving /3-hydroxy acid):

2(2-phosphoglyceric acid =^=*= 2(phospho(enol)pyruvic acid)+2H20

1 1 . Phosphorylation:

2(phospho(enol)pyruvic acid)+2ADP =5=^ 2(pyruvic acid)+2ATP

Of the eleven reactions, only one, the dehydrogenation of the diphosphate
derivative of glyceraldehyde, is of the type which requires a B vitamin
coenzyme. The hydrogen atoms from this reaction are accepted by the
diphosphopyridine nucleotide. The net transformation of the organic
molecules brought about by the process and the B vitamin coenzymes
involved may be summed up thus:

hexose unit of polysaccharide
DPN

2 [2H]DPN 2 pyruvic acid

220 THE BIOCHEMISTRY OF B VITAMINS

When this stage of the process is reached, the organism is confronted
with the problem of the disposition of the pyruvic acid and of the hydro-
gen atoms which have been temporarily taken up by the diphosphopyri-
dine nucleotide coenzyme. This coenzyme must be reconverted to the
oxidized form for re-use, for, if no suitable hydrogen acceptor can be
found, the glycolytic process will stop when all the coenzyme is tied up
in its reduced state.

Under anaerobic conditions pyruvic acid itself or other products derived
from it acts as the acceptor for the hydrogen atoms. When oxygen is
available and can be utilized, it can serve as the final acceptor for the
hydrogen atoms and will in addition permit the pyruvic acid to be con-
verted, by reactions involving further dehydrogenations, into compounds
which are in a sense oxidation products; or pyruvic acid may be com-
pletely degraded to carbon dioxide and water.

The reactions into which pyruvic acid can enter are numerous, and
we find a great deal of variation among the various forms of life in the
manner in which they carry forward the carbohydrate metabolism from
this point.5 Probably most often several of the reactions are utilized
simultaneously, although one reaction may predominate to such an extent
that the others are completely overlooked.

Anaerobic Utilization of Pyruvic Acid. When a cell has only a limited
supply of molecular oxygen or when it lacks the porphyrin enzymes
(cytochromes) which catalyze the utilization of molecular oxygen as
the final hydrogen acceptor, it must dispose of the hydrogen atoms
temporarily associated with the reduced nicotinic acid and of the pyruvic
acid by an anaerobic process. The anaerobic processes can be classified
in three groups.

The simplest and most direct process is that in which the pyruvic acid
itself accepts the hydrogen atoms from the coenzyme forming lactic
acid. This type of reaction is the predominant method in a number of
bacteria and in the tissues of vertebrates. The only vitamin involved is
the nicotinic acid which is present in the reduced coenzyme.

hexose unit

DPN

2 [2H]DPN 2 pyruvic acid

DPN

2 lactic acid

A second important type process is one in which the reduction of
pyruvic acid takes place after it has undergone carboxylation.9 Oxalacetic

METABOLIC FUNCTIONS OF B VITAMINS

221

acid is first formed by /?-carboxylation of pyruvic acid and on subsequent
reduction yields malic acid. In biological systems, malic acid is usually
in equilibrium with its dehydration product, fumaric acid. Whether or
not biotin is generally required to mediate the /?-carboxylation step is
still an open question (p. 171), and it may be that this method of carbo-
hydrate utilization is another instance in which the nicotinic acid
coenzyme is the only vitamin coenzyme participating. (Further reduc-
tion of the fumaric acid, utilizing hydrogen atoms from other metabolic
reactions, can occur and results in the production of succinic acid. The
enzyme needed for this reaction, a fumaric reductase, might be expected
to be one of the riboflavin enzymes) (p. 150).

hexose unit
DPN

2 [2H]DPn

2 pyruvic acid

I CO, (BIOTIN COENZYME)
2 oxalacetic acid
— — J.DPN

2 malic acid
|-H20
2 fumaric acid

The third type anaerobic process is one in which pyruvic acid is first
decarboxylated, and the resulting C2 compounds or their condensation
products are then reduced by the hydrogen atoms of the nicotinic acid
coenzyme. The thiamine coenzyme is believed to be absolutely essential
for the production of every one of these fermentation products which are
formed from pyruvic acid by decarboxylation.3 However, they may be
divided into two subgroups depending upon whether or not there is
a pantothenic acid requirement. This requirement appears to be directly
determined by the type of decarboxylation. Those processes in which
thiamine catalyzes a simple decarboxylation to produce acetaldehyde or
its dimer, acetylmethylcarbinol, do not require pantothenic acid. Con-

hexose unit
DPN

2 [2H]dpn

2 pyruvic acid

I THIAMINE PYROPHOSPHATE
2 acetaldehyde + 2 C02

\ DPN |

2 ethanol 2 C02

THE BIOCHEMISTRY OF B VITAMINS

sequently, pantothenate is not required for the synthesis of ethanol or
2,3-butylene glycol (formed by the reduction of acetylmethylcarbinol) .
Many of the thiamine-promoted processes, however, involve an "oxida-
tive" decarboxylation of pyruvic acid which produces the reactive acetyl-
ating intermediate, and hence the subsequent steps in these processes
all require the presence of a pantothenic acid coenzyme (p. 191). The
mechanism of transporting the two additional hydrogen atoms released
by each pyruvic acid molecule during the oxidative decarboxylation is
not known. In aerobic processes they are probably accepted by a ribo-
flavin-containing enzyme.10 In anaerobic processes these hydrogen atoms
are used to reduce the products formed from the acetyl derivatives, pos-
sibly through the intermediation of flavoproteins or some other hydrogen
carrier. A possible mechanism for the formation of butyric acid and butyl
alcohol (based upon demonstrated reactions catalyzed by thiamine,
nicotinic acid, and pantothenic acid) is shown as an example of this
type of process:

hexose unit

DPN

[2H]DPn [2H]di

2 pyruvic acid
— I THIAMINE PYROPHOSPHATE

[2H]? + [2H]? + 2 phosphorylated acetate + 2 C02

I COENZYME A

icetoacetyl phosphate
"4 DPN

hydroxybutyryl phosphate

crotonyl phosphate + H20
-^(FLAVOPROTEIN?) j

butyryl phosphate H20

^JDPN {

butyraldehyde H20

—^(DPN?) j

n-butanol H20

2 CO,

2C02

The synthesis of fatty acids from carbohydrates probably is carried
out by an analogous procedure. Hence, any conversion of carbohydrates
to fats or fat-like substances requires the coenzymes of thiamine, nico-
tinic acid, pantothenic acid, and probably riboflavin. In the synthesis of
fat from proteins, the same vitamins will be essential for the utilization of
those "anti-ketogenic" amino acids which on deamination are metabolized
via processes that involve carbohydrate intermediates.

The Aerobic Utilization of Pyruvate. The first step in the aerobic

METABOLIC FUNCTIONS OF B VITAMINS

223

metabolism of pyruvic acid presumably always involves the thiamine-
requiring oxidative decarboxylation. The hydrogen atoms donated to
riboflavin 10 in this reaction, as well as those donated to the diphospho-
pyridine nucleotide previously during the glycolytic process, are "trans-
ported" by the elaborate dehydrogenase-cytochrome systems described in
Chapter II B and are finally oxidized in reactions which reduce molecular
oxygen. The active phosphorylated acetyl molecule which is formed by
the oxidative decarboxylation of pyruvate can be directly used for energy
by employing it in reactions in which it acts as a phosphorylating agent.11
Acetic acid is then the end product and accumulates. A more common
aerobic mechanism, however, is one in which the activated acetate is
completely oxidized to carbon dioxide and water by a series of reactions
designated as the tricarboxylic acid cycle.2, 4' 12 In this cyclic process,

-2 [2H]DPN

-2 [2H]?

-2 [2H]tpn

hexose unit
| DPN

-2 [2H]Flavin?

2C02

2 pyruvic acid

| THIAMINE PYROPHOSPHATE

2 phosphorylated acetate

-2 H20

COENZYME A

2 ds-aconitic acid
+2 H20

2 isocitric acid
TPN

2 oxalacetic acid

2 oxalsuccinic acid

2C02

-2 [2H]Flavin? 2 C02

i (BIOTIN COENZYME?)
2 ketoglutaric acid

THIAMINE PYROPHOSPHATE

2 succinic acid

(FLAVOPROTEIN?)

2 fumaric acid
+2H20

2 malic acid
DPN

-2 [2H]DPN

dehydrogenase-
cytochrome systems

224 THE BIOCHEMISTRY OF B VITAMINS

the activated acetate is believed to undergo first a condensation with
oxalacetic acid to form as-aconitic acid; then, by a series of dehydro-
genations, hydrations and dehydrations, and decarboxylations the cis-
aconitic acid is degraded to oxalacetic acid, which can then react with
another activated acetate and "carry" it through the same cycle.*

Pantothenic acid is necessary for the initial condensation of the acti-
vated acetate (p. 195) ; thiamine is required for one step in the cycle, the
oxidative decarboxylation of a-ketoglutaric acid; nicotinic acid is neces-
sary for the dehydrogenation of the hydroxy acids, isocitric and malic;
a riboflavin enzyme may be the hydrogen acceptor in the conversion of
succinic acid to fumaric acid (p. 151). It is interesting to note that thia-
mine, riboflavin, nicotinic acid, and pantothenic acid are the vitamins
required for both the aerobic oxidation of carbohydrates and for the
anaerobic processes in which the carbohydrates are converted to fats.

There are a number of compounds of general biological occurrence
and importance which are related chemically to the carbohydrates and
which are undoubtedly produced from them. These include the pentoses,
desoxypentoses, amino sugars, ascorbic acid, inositol, and the hexonic
and hexuronic acids. Nicotinic acid-containing coenzymes have been
shown to catalyze the dehydrogenation of glucose 13 or its phosphate
ester 14 to the corresponding acid, but the involvement of vitamin-con-
taining coenzymes in the biosynthesis of other sugar derivatives has not
yet been established.

The mechanism by which the pentoses and desoxypentoses are generally
formed has not yet been definitely established. This subject is under
active investigation due to the present interest in nucleic acid metabolism.
When the mechanisms for the formation of these compounds, as well as
the other carbohydrate-like substances, are worked out, the vitamin
requirements will probably be obvious; or, conversely, if certain vitamins
are shown to be directly involved in the biosynthesis of any of these
substances, it will give some insight into the mechanisms and the inter-
mediates in their biosyntheses. If, for example, pantothenic acid were
shown to be essential for the synthesis of desoxyribose, a reasonable

* In the past, on the basis of certain tracer experiments, citric acid has been
assigned the role of a metabolic by-product of the tricarboxylic acid cycle rather
than that of a necessary intermediate. In a more critical analysis of these tracer
studies it was pointed out that such an interpretation need not be made, and
subsequent to the preparation of this diagram it was shown experimentally that
citrate, rather than cts-aconitate, is the initial compound formed by the condensation
of the reactive acetyl unit with oxalacetate. (See Stern, J. R., and Ochoa, S., J. Biol.
Chem., 179, 491 (1949) and Potter, V. R., and Heidelberger, C, Nature, 164, 180
(1949)).

METABOLIC FUNCTIONS OF B VITAMINS 225

postulation would be that the precursor of this compound was formed
by a condensation of a triose and an activated acetate unit.

The Metabolism of Lipides

A general scheme of lipide metabolism must account for fatty acid
synthesis and degradation, glycerol formation and utilization, and for
the reactions by which fat molecules are synthesized from their com-
ponent parts, and should also explain the origin of sterols.

Fatty acid metabolism. The mechanism now postulated for the syn-
thesis and degradation of fatty acids 15 is very similar to that shown in
the scheme for the production of butyl alcohol, and the same combination
of vitamins — thiamine, pantothenic acid, nicotinic acid, and riboflavin —
is needed. When proteins are converted to fats a fifth vitamin, pyridoxal
is also undoubtedly required to catalyze the deamination of amino acids.16
A route of synthesis by which butyryl phosphate can be formed from a
hexose unit has been diagrammed (p. 222) . The synthesis of higher fatty
acids presumably is carried out similarly by a series of reactions which
lengthen the carbon chain of a fatty acyl phosphate in some fashion
corresponding to the diagram on the following page. The net result of this
process is an anaerobic utilization of each hexose unit for increasing the
chain of a fatty acid by four carbon atoms.

The discovery of the reactive phosphoryl acetyl compound cleared up
a number of points previously obscure concerning the synthesis and
degradation of fatty acids. This fundamental acetylating agent which
can be formed by a number of metabolic processes furnishes the units
from which fatty acids are constructed, and in turn these same acetyl
units are regenerated when a fat is metabolized. The mechanism of fatty
acid catabolism 13 is believed to be just the reverse of the synthetic proc-
ess; fatty acyl phosphates are degraded by dehydrogenations, hydra-
tions, and phosphoroclastic cleavage of the /3-keto acids to yield the
reactive phosphorylated acetyl units (associated with coenzyme A?) and
the hydrogenated coenzymes of nicotinic acid and riboflavin. If the
aerobic mechanisms for oxidation of the acetyl units (tricarboxylic acid
cycle) are inhibited from functioning or are overloaded by the inter-
mediates of carbohydrate or protein metabolism, then the accumulation
of the acetyl units will result in their condensation, and fats will be
formed. On the other hand, if there is insufficient carbohydrate or pro-
tein degradation to furnish sufficient substrate for the energy-producing
tricarboxylic acid cycle the fats will be degraded to supply the acetyl
units for the oxidative cycle.

THE BIOCHEMISTRY OF B VITAMINS

hexose unit
DPN

fatty acyl phosphate
O
II H2

H203PO— C— C— R

[2H]DPN + [2H]DPN

2 pyruvic acid

[2H]? [2H]? 2 C02 phosphorylated phosphorylated
acetate acetate

COENZYME A
O I O

II H2 || H2
H203PO— C— C— C— C— R

1 DPN

O f OH
II H2 I H2
H203PO— C— C— C— C— R
H

— H20

I H2 H H
H203PO-C— C=C— C— R

(FLAVOPROTEIN?)

|| H2 H2 H2
H203PO— C— C— C— C— R

COENZYME A

O O

II H2 II. H2 H2 H2
H203PO-C— C— C— C— C— C— R

DPN

O OH

|| H2 I H2 H2 H2
H203PO— C— C— C— C— C— C— R
H

-H20

if H2 H H H2 H2
H203PO-C— C=C— C— C— C— R

(FLAVOPROTEIN

o j

H2 H2 H2 H2 H2
H203PO— C— C— C— C— C— C— R

METABOLIC FUNCTIONS OF B VITAMINS 227

It is interesting to note that fatty acids have an appreciable sparing
effect upon the pantothenic acid 17 and riboflavin 1S requirements of lactic
acid bacteria, indicating that a considerable fraction of each of these
vitamins is being used by these organisms in the fat synthesis systems,
but that lipides have only a slight effect upon the nicotinic acid response.19
(A reduction in utilization of an enzyme system is reflected in the lowered
demands of the organism for the cofactors.) The sparing effect of fats
upon the thiamine requirement of mammals (p. 000) can be attributed
to the fact that the body is oxidizing acetyl units primarily derived from
fatty acids rather than those arising from the decarboxylation of pyruvic
acid.

The reverse process, in which a deficiency of an enzyme system alters
the dietary requirements, can also be of physiological importance. For
example, in lactic acid bacteria where the thiamine system is inherently
deficient, the organisms need an exogenous supply of acetate or fatty
acids as a source of acetyl units to supplement the sub-optimal quantities
furnished by the thiamine enzymes.20

An additional problem encountered in the synthesis of fats is that of
the formation of unsaturated fatty acids. The requirement for specific
unsaturated fatty acids in both bacteria and mammals would indicate
that these organisms are deficient in enzymes designed for this purpose.
Enzymes that dehydrogenate fatty acids are known,21 but have never
been shown to be dependent upon any B vitamin coenzymes for activation.
However, the biotin-sparing activity of oleic acid and other unsaturated
fatty acids for certain microorganisms (p. 173) is suggestive of a function
of biotin related in some way to the formation of a double bond by dehy-
drogenation of saturated fatty acids.

Glycerol Formation and Utilization. The glycerol needed for lipide
synthesis is a by-product of carbohydrate metabolism. The phosphoryl-
ated triose, dihydroxy acetone phosphate (formed by the hexose diphos-
phate cleavage), is reduced by the corresponding dehydrogenasi, the
specific hydrogen donor required is the hydrogenated diphosphopyridine
nucleotide. The phosphorylated glycerol is then available for reactions
producing phospholipides — compounds which are assumed to be necessary
intermediates in fat synthesis.

Nicotinic acid, in the form of its coenzyme, is also essential for the
utilization of the glycerol liberated from fat, since the glycerol (after
phosphorylation) must be dehydrogenated by DPN to a triosephosphate
before it can be metabolized in the carbohydrate system.22 After the
initial dehydrogenation, the substrate can be utilized during anabolic
phases of cell activity for synthesis of glycogen or starch; during periods
in which the catabolic activity of the cells predominate, it will be utilized

228 THE BIOCHEMISTRY OF B VITAMINS

in one of the previously described processes in which pyruvic acid is an
intermediate.

Synthesis and Hydrolysis of Fats. A number of lipases from both
plant and animal sources have been thoroughly investigated.23 These
esterases catalyze the hydrolysis of fats, liberating free fatty acids,
glycerol, and the other components found in complex lipides. In no case
has a coenzyme requirement been demonstrated for this hydrolysis.

The synthesis of fats by the reverse reaction can be accomplished in
vitro by these same enzymes if the molar ratio of fatty acids to fat is
greater than two to one.24 Since the intracellular concentration of fatty
acids is very low, the synthesis of fats here must be by a different process. ,
The mechanism probably is similar to that involved in the formation of
polysaccharides from simple sugars and involves phosphorylated inter-
mediates. In the condensations and reductions by which the phosphoryl-
ated acetyl units are elaborated into the fatty acids, it was indicated that
the fatty acid derivatives actually produced are undoubtedly fatty acyl
phosphates. (This supposition is in line with evidence concerning the
reverse process, wherein it has been demonstrated that before a fatty acid
can be degraded enzymatically it must be first converted to the corre-
sponding acyl phosphate.25) The fatty acyl phosphates react directly
with the hydroxyl groups of glycerol phosphate or other alcohols to yield
the esters which constitute the saponifiable lipides. These reactions arc
analogous to those employed for the nonenzymatic synthesis of esters
wherein acyl halides are used. The equilibrium reached in this reaction
is so greatly in favor of ester formation that the reaction can be assumed
for practical purposes to have "gone to completion." Both in the synthesis
of fats and in the utilization of the fatty acids from fats the individual
fatty acids of different carbon chain length appear to be used in a non-
specific and random fashion.20

The vitamin requirements for fat metabolism are summarized in the
diagram on the following page.

Sterol Metabolism. Very little is known about the reactions by which
sterols are formed. Tracer studies using isotopes of carbon first showed
the importance of acetate in the synthesis of sterols.27 An independent
demonstration was provided by the study of the acetate requirements of
organisms which produce lactic acid.20 These bacteria are very limited in
their ability to convert pyruvic acid to the reactive two-carbon unit.
Consequently, they require an exogenous supply of acetate in order to
function normally. Sterols as well as fatty acids were found to have a
significant sparing action upon this acetate requirement of these or-
ganisms.

METABOLIC FUNCTIONS OF B VITAMINS

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230 THE BIOCHEMISTRY OF B VITAMINS

When the function of pantothenic acid was shown to be that of a
mediator of reactions involving active forms of acetate, this vitamin was
naturally implicated in sterol synthesis. It was shown that sterols had a
sparing action upon the pantothenic acid requirement of Lactobacilli,17
and that a sterol could partially reverse the toxicity of pantothenic acid
inhibitors.28 The reversal was noncompetitive, indicating that sterols are
products of a series of reactions, part of which are catalyzed by panto-
thenic acid enzymes.

The mechanism for the formation of both the polynuclear cholane
nucleus and the hydrocarbon side chains is completely unknown, but it
appears impossible to postulate any mechanism involving acetate con-
densations which does not include the hydrogenation of keto groups and
of ethylenic bonds. For these reactions nicotinic acid and riboflavin are
undoubtedly required.

Pantothenic acid, riboflavin, and nicotinic acid may be the only vita-
mins required for sterol synthesis if a cell has a potential source of acetate
in the form of fatty acids or acetate itself. If carbohydrates must be used
as the initial source of carbon and hydrogen, then thiamine, of course,
will be required to form the active acetyl units from pyruvic acid.

Biotin administration at one time was believed to cause the formation
of excessive amounts of cholesterol in the liver.29 Critical reexamination
of this phenomenon has cast doubts upon this role of biotin.30 Biotin.
however, could function in some fashion in the process responsible for
the formation of the unsaturated linkages in the sterol molecules in a
manner comparable to its possible function in the formation of the
ethylenic linkages in oleic acid (p. 227) .

The Metabolism of Nitrogen Compounds

From a chemical standpoint, the metabolism of proteins is more com-
plicated than that of carbohydrates and fats. The presentation of the
basic reactions is likewise more difficult. Unlike the simple sugars or the
fatty acids, the amino acids — the component units of proteins — vary con-
siderably in their chemical structure, and many reactions are necessary to
account for the synthesis of the individual amino acids. Polysaccharides
are usually polymers of a single hexose, and in fats the arrangement of
fatty acids is a random one; but in the formation of the proteins, poly-
merization of the different amino acids must take place according to a
highly specific pattern.

Synthesis and Hydrolysis of Proteins. The proteolytic enzymes cata-
lyzing the direct hydrolysis of the amide bonds require no coenzymes.
Almost nothing is known of the enzymes responsible for the intracellular
synthesis and hydrolysis of proteins. Several enzymatic processes in which

METABOLIC FUNCTIONS OF B VITAMINS 231

simple amide bonds are created have been partially characterized and
shown to require adenosine triphosphate or else a coupling with an aerobic
system in which this phosphorylating agent is presumably generated:

glutamic acid + NH3 >■ glutamine+H2031

benzoic acid+glycine > hippuric acid+H2032

p-aminobenzoic acid+glycine > /j-aminohippuric acid+H2033

The individual steps of these processes and the phosphorylated intermedi-
ates which may be formed have not yet been established. The occurrence
of acyl phosphates derived from the amino acids has never been demon-
strated. Hence, the formation of the peptide bond cannot be explained
on the basis of the utilization of acid anhydrides as was the case in the
formation of the ester and acetal linkages of fats and carbohydrates. No
B vitamin has as yet been directly implicated in the synthetic processes.

Synthesis of Amino Acids. Many of the enzymatic reactions which
are utilized by organisms for the synthesis of their "nonessential" amino
acid requirements have yet to be clearly defined. The disclosure of certain
types of enzymatic reactions of general occurrence has indicated certain
steps which probably take place during most of these syntheses; but the
gaps which still exist in any outline of the total processes indicate how
much remains to be learned before a scheme for amino acid biosynthesis
can be drawn which will in any measure deserve the designation "com-
plete." An effective synthesis of amino acids usually takes place in one
of two ways: (1) by the direct animation of the corresponding keto acid,
or (2) by a reaction in which one a-amino acid is transformed into
another amino acid by a chemical alteration of the molecule which leaves
the original amino and carboxyl groups intact.

The synthesis by amination of a keto acid either (1) utilizes an amino
(or amide) group of some other organic compound, a transamination,
or (2) introduces a molecule of inorganic ammonia into the organic struc-
ture by a reductive amination. In the former instance pyridoxal has been
shown to be required in all cases adequately characterized; in the latter
instance, nicotinic acid. Riboflavin enzymes catalyze most of the recog-
nized oxidative deaminations of amino acids (p. 147) and it may be
assumed that these function in amino acid synthesis by the reversal of
such reactions. The equilibrium established by the flavoprotein enzyme,
however, is so much in favor of deamination that this system has not yet
been shown to be a method by which amino acid synthesis can effectively
occur. However, the glutamic acid dehydrogenases, which are activated
by nicotinic acid coenzymes, catalyze a reaction in which concentrations
can exist which will favor the reverse reaction, amino acid formation.34
It is significant that, of all the amino acids, only glutamic acid has been

232 THE BIOCHEMISTRY OF B VITAMINS

found to participate as a substrate in the reversible nicotinic acid systems.
For this reason, ketoglutaric acid may be an extremely important inter-
mediate for the fixation of ammonia into organic molecules. Equally
significant is the fact that of the known transamination reactions, by which
the a-amino group of one acid can be passed on to other keto acids, all
have as one amino acid component glutamic acid.35 Although only a few
transaminases are known, it seems not unlikely that others exist. If so, a
general scheme for the formation of these amino acids would be the
reductive amination of ketoglutaric acid, followed by a transfer of the
amino group to other keto acids. In this way the ketoglutaric acid-
glutamic acid system could function as a type of ammonia carrier and the
system would be one requiring nicotinic acid and pyricloxal. Since the
amination of ct-ketoglutaric acid is reductive, it entails having the reac-
tion coupled with the dehydrogenation of some organic substrate in order
to supply the hydrogen atoms which will keep regenerating the reduced
form of the nicotinic acid coenzyme. Thus the energy for the conversion
of ammonia to an organic amine is derived indirectly from the "oxidation"
of another substrate. In addition to pyridoxal and nicotinic acid, biotin
may also be another B vitamin directly involved in the biosynthesis of
at least one amino acid, aspartic acid (p. 172).

For many microorganisms the amides of aspartic and glutamic acids,
asparagine and glutamine, are better sources of nitrogen than are inorganic
ammonium salts. It has been shown that glutamine is formed by a reac-
tion in which inorganic ammonia is fixed at the expense of a high energy
phosphate bond in adenosine triphosphate,31 and this conversion of inor-
ganic nitrogen into an intermediate amide nitrogen may often constitute
an essential step in the formation of amino compounds from ammonia.

A number of different observations offer evidence indicating that
the requirements for specific amino acids by organisms reflects a defi-
ciency in their ability to form the carbon skeleton of the essential mole-
cules rather than an incapacity to form the specific amino acid itself.
These observations include (1) the ability of most organisms to utilize
the corresponding keto acids in place of essential amino acids; (2) the
demonstrated equilibrium existing between inorganic ammonia and the
a-amino nitrogen of all amino acids except lysine;36 and (3) the utiliza-
tion of ammonia by mammals in place of the nitrogen usually supplied
by the nonessential amino acid.37

Identified reactions in which one amino acid is formed from another
by changes leaving the original a-amino and carboxyl groups intact
include several transformations which are known to depend upon the
presence of a B vitamin coenzyme:

METABOLIC FUNCTIONS OF B VITAMINS 233

glycine v "* serine (p-aminobenzoic acid, folic acid, vitamin B12)38
homocysteine v "*, methionine (p-aminobenzoic acid, vitamin B12)39
serine >■ tryptophan (pyridoxal)40

Degradation of Amino Acids. The degradation of amino acids can be
conveniently divided into three groups: (1) those in which the amino
acid is first deaminated; (2) those in which the amino acid is converted
into another amino acid; and (3) those in which the amino acid is decar-
boxylated.

The following types of reactions (discussed in detail in the preceding
chapter) which cause the deamination of amino acids have been shown
to be dependent upon the presence of coenzymes of the vitamins indicated :

(1) Oxidative deamination of glycine and most d- and L-amino acids
by riboflavin-containing enzymes.

(2) Oxidative deamination of L-glutamic acid by enzymes whose co-
enzymes contain nicotinic acid.

(3) Transaminations which result in the deamination of glutamic,
aspartic and cysteic acids, alanine, and probably other amino acids by
pyridoxal-containing transaminases.

(4) The simultaneous deamination and degradation of tryptophan by a
system requiring pyridoxal phosphate.

(5) Deamination of aspartic acid, threonine, and serine by biotin-
reactivated systems.

The B-vitamin-catalyzed reactions transforming one amino acid into
another which were previously listed above undoubtedly are important
to the organisms, not only from the standpoint of a means of synthesis,
but also as a means of catabolic utilization of certain amino acids.

The decarboxylation of amino acids results in the formation of mono-
amines, diamines, y-aminobutyric acid, and /^-alanine, and it can be
anticipated that pyridoxal phosphate will be an essential part of most, if
not all, the enzymes carrying out this type of degradation.

A check of the reactions just discussed directly implicates all of the
typical B vitamins, except thiamine and pantothenic acid, in the catalysis
of one or more reactions in which amino acids participate.

Pyrimidines and Purines. The demonstration of a direct involvement
of a B vitamin in reactions utilized for the biosynthesis of purines and
pyrimidines has yet to be accomplished. However, by the use of inhibitors
and isotopically labelled substrates the general routes of synthesis have
been indicated, the important role of the single carbon unit established,
and the essentiality of p-aminobenzoic acid (or folic acid) and vitamin
B12 demonstrated (Chapter IIB). In purine syntheses these two vitamins
may in many instances be required not only for the introduction of the
single carbon unit into the purine nucleus, but also for the biosynthesis

234 THE BIOCHEMISTRY OF B VITAMINS

of glycine (from serine, p. 201) which is needed as the source of the
metabolic unit from which carbon atoms 4 and 5 (and probably nitrogen
atom 7) of the purine nucleus originate.41 In the case of fowls and rep-
tiles which excrete most of their metabolic nitrogen in the form of uric
acid, the extensive purine synthesis which must be accomplished is re-
flected in the unusually high glycine requirement.42

The initial step in the catabolism of purines is the deamination of
adenine and guanine (no vitamin requirement) producing hypoxanthine
and xanthine, which are then oxidized by the flavoprotein, xanthine
oxidase, to yield uric acid. No B vitamin has been implicated in the
further degradations which uric acid has been found to undergo.

Biosynthesis of the B Vitamins. The biological origin of the individual
vitamins has been taken up elsewhere (Chapter VA). However, it is
logical at this point to indicate that the synthesis of one vitamin prob-
ably often depends upon the presence of another B vitamin. In the
intestinal tract the biosynthesis of one B vitamin by bacteria may be
influenced by the dietary level of other vitamins which are essential for
the growth of the intestinal flora accomplishing the synthesis. A more
direct interrelation exists when an enzymatic reaction necessary for the
synthesis of one B vitamin requires another B vitamin as a coenzyme.
No such case has been unequivocally demonstrated, but when the indi-
vidual steps in the biosyntheses of vitamins have been better defined, it
will undoubtedly be found that many of the reactions involved are of the
types which require B vitamin coenzymes. For example, pyridoxal phos-
phate probably catalyzes the decarboxylation of aspartic acid to form
the /^-alanine required for the synthesis of pantothenic acid; and it can
be anticipated that the pyrimidine portion of the pterin (folic acid) and
isoalloxanine (riboflavin) molecules will be formed by processes utilizing
single carbon units in a manner analogous to that observed in purine
synthesis, and will be mediated by a p-aminobenzoic acid coenzyme.

Choline. The methylation of ethanolamine, forming choline, is depend-
ent upon an adequate dietary source of substances containing available
methyl groups 43 (methionine being the most important) or the capacity
of the organism for producing them from other metabolic processes.

In microorganisms it has now been established that the coenzymes in-
volved in the metabolism of the single carbon unit (p-aminobenzoic acid,
folic acid, and vitamin Bi2) likewise function in the conversion of homo-
cysteine to methionine.39 Also, on the basis of studies using isotopically
labelled compounds, it is known that the methyl groups of methionine
and choline can serve as sources of "formate" (p. 197). Hence, it is indi-
cated that the single carbon unit will be found to be one of the precursors
of the available methyl groups. That mammals may possess to a limited

METABOLIC FUNCTIONS OF B VITAMINS 235

extent the enzyme systems capable of the conversion of the single carbon
unit to utilizable methyl groups is indicated in an abstract reporting the
choline-sparing action of vitamin Bi2 in the nutrition of rats and chicks.44
Porphyrins. Lack of either folic acid 45 or vitamin B12 4C (in rats)
results in a decrease in porphyrin synthesis, indicating a possible role of
the single carbon unit in porphyrin metabolism. A recent report indicates
that glycine may be a complete substitute for the folic acid requirement
needed for normal porphyrin synthesis.45 Thus the involvement of the
catalysts of the single carbon unit may be partially or wholly due to their
role in the production of glycine from serine. Glycine had previously been
shown to be one of the metabolic units needed for the biosynthesis of the
pyrrol rings in the porphyrin nucleus.47 Isotopically labelled acetate has
also been shown to be incorporated into the porphyrin structures, impli-
cating a pantothenic acid requirement for porphyrin synthesis.48

Fundamental Physiological Processes Requiring Energy

Knowledge concerning the mechanisms by which the chemical energy
inherent in the organic substrates metabolized by organisms is converted
into other forms of energy must serve as the basis for understanding the
fundamentals of physiological processes. By the degradation of organic
substrates, and in some cases oxidation of inorganic substances, living
organisms transform chemical energy into mechanical energy (including
work against osmotic pressure), thermal energy, electrical energy and
radiant energy. What is known concerning the role that the B vitamins
play in these energy transformations?

Chemical Energy. The general mechanisms by which the energy re-
leased during the oxidation or degradation of organic compounds is made
available to organisms for other purposes which require an energy supply
has been previously indicated. In almost all cases it appears that the
energy is conserved, transported, and eventually utilized through the in-
termediate formation of compounds that are acid anhydrides of phos-
phoric acid, compounds containing the so-called "high energy phosphate
bonds." 49- 50

Three types of reactions for which there is some evidence indicating
the mode of formation of the energy-carrying phosphate bonds have been
discussed in connection with the reactions catalyzed by the coenzymes of
nicotinic acid, thiamine, and pantothenic acid:

(1) The dehydrogenation of an aldehyde-inorganic phosphate addition
product by the coenzymes of nicotinic acid in effect utilizes the energy
derived from the oxidation of an aldehyde to an acid to convert a
molecule of inorganic phosphate to an energy-rich acyl phosphate. (See
p. 140 for the mechanism.)

236 THE BIOCHEMISTRY OF B VITAMINS

(2) The energy liberated in the coupled dehydrogenation and decar-
boxylation of the inorganic phosphate — carbonyl addition products of
pyruvic acid and ot-ketoglutaric acid by thiamine-containing enzymes
produces the energy-rich phosphorylated intermediate (p. 163 and 167).

(3) The degradation of the inorganic phosphate — carbonyl addition
product of /?-ketoacyl phosphates (for example, acetoacetyl phosphate)
results in the cleavage of a carbon-to-carbon bond and the formation of
an additional acyl phosphate (p. 189) .

In addition to these reactions it has been shown that in aerobic proc-
esses additional inorganic phosphate is converted into energy-laden pyro-
phosphates by the reactions in which the hydrogen atoms are transported
to oxygen via the riboflavin and porphyrin-containing enzymes.49 In these
instances the mechanism by which the phosphate transformation is
coupled to the transfer of hydrogen atoms is unknown, but it has been
postulated to take place through the addition of phosphoric acid to
ethylenic bonds.49

Conversely, the energy of the phosphate bonds may be utilized for
synthetic purposes by serving as the sources of energy for the formation
of glucosidic, ester, and probably peptide bonds (reactions requiring no
B vitamins) and for the reductions and condensations catalyzed by nico-
tinic acid, thiamine, and pantothenic acid (reactions which are the reverse
of those tabulated for the formation of the energy-laden bond) .

Mechanical and Thermal Energy. On the basis of the current state
of knowledge it appears that the B vitamins, having catalyzed the proc-
esses by which the high-energy phosphate bonds are formed, have no
further function in transforming this energy into either mechanical work
or thermal energy. Thus, in none of the following instances has a B vita-
min requirement been shown: the contractions of muscle are the result
of the transformation into kinetic energy of the energy liberated during
the hydrolysis of adenosine triphosphate by an enzyme, adenosine triphos-
phatase (a component of muscle myosin)50; the chemical mechanisms
involved in protoplasmic movement (which are responsible for the con-
tractility and mobility of living cells) are not yet understood; the work
against osmotic pressure, performed during absorption, is a process often
involving phosphorylation of the absorbed molecules by adenosine tri-
phosphate; extra thermal energy (over and above that normally resulting
from metabolic processes) can be produced by the hydrolytic action of
pyrophosphatases 23 upon the energy-rich bonds, causing the dissipation
in heat of all the energy of the bonds.

Electrical Energy. Little can be said concerning the manner in which
the energy derived from metabolic reactions is utilized to establish the
electrostatic membrane potentials maintained by viable cells or concern-

METABOLIC FUNCTIONS OF B VITAMINS 237

ing the way the electric currents resulting from localized changes in these
potentials are controlled. Studies on the enzymatic capacities of tissues
in which the electrical potentials are pronounced (nerves and the electric
organs of electric eels and fish) established a correlation between the
acetylcholine metabolism of these tissues and the electrical potentials
which they could develop.51 On the basis of considerable evidence it was
postulated that the electric current responsible for the conduction of
impulses in nerve 51 and muscle fibers 52 was due to the release and
hydrolysis of acetylcholine, a process which was believed to cause a local
change in the permeability of the cell membrane and a resultant flow of
the "action current," and that the energy released during the hydrolysis
of acetylcholine originally was derived from the high-energy phosphate
bonds utilized in the synthesis of acetylcholine. Since this postulate was
advanced, convincing arguments against such a direct involvement of
acetylcholine in conduction have been offered;53 its exact function in
nervous and electric tissue is still open to question.

One of the first functions demonstrated for pantothenic acid was the
requirement for its coenzyme (coenzyme A) in choline acetylase,54 the
enzyme catalyzing the reaction in which choline is acetylated by the
phosphoryl-acetyl intermediate derived either (1) from the oxidative
decarboxylation of pyruvic acid (thiamine pyrophosphate essential) or
(2) from the direct phosphorylation of acetic acid (coenzyme A required) .
Hence, these two coenzymes are of specific importance for the functioning
of cells where conduction takes place.

Radiant Energy. The biological conversion of radiant energy to chem-
ical energy by the reduction of carbon dioxide (photosynthesis) and the
reverse process, the emission of radiant energy during biological oxida-
tions of organic substrates (bioluminescence) , are the result of two proc-
esses whose mechanisms are entirely unrelated. The chemical reactions
responsible for the latter phenomena have been well established, but the
former process, which is indispensable from the standpoint of the economy
of the biological world, cannot yet be described in terms of specific
chemical reactions.

It is surprising, in view of the vast amount of study which has been
devoted to the photosynthetic phenomenon, that nothing has been learned
concerning what roles the B vitamins may play in this process. It can be
anticipated, however, that this question will soon be answered, at least
in part, as a result of current investigations which have already yielded
much information not previously obtained by the classical methods that
have been used in attacking the problem. Two of these recent approachs
which are proving to be especially valuable are the study of photosyn-
thesis from the standpoint of comparative biochemistry 55 and the cor-

238 THE BIOCHEMISTRY OF B VITAMINS

relation of the chemistry of the photosynthetic process with that of
certain chemosynthetic mechanisms which have been well established.56
Chemosynthesis, the assimilation of carbon dioxide by reactions which
utilize energy derived from other metabolic reactions rather than radiant
energy, is the result of the reversal of the processes in which there are
decarboxylation reactions; hence the accomplishment of carbon dioxide
fixation by chemosynthetic means is dependent upon exactly the same
vitamins and enzymes that carry out these carboxylation reactions. It
seems reasonable to expect that many, if not most, of the photosynthetic
"dark reactions" which take place after the initial "light reaction" and
lead to the ultimate formation of carbohydrates will be reactions that are
the reverse of those used for carbohydrate catabolism, and hence will be
catalyzed by the same enzymes and cofactors. When the mechanisms
which the photosynthetic and chemosynthetic processes have in common
are eventually established, then at least some of the functions of the B
vitamins in photosynthesis will have been determined.

Bioluminescence is the result of the action of an enzyme (luciferase)
upon a reduced substrate, dihydroluciferin, in the presence of oxygen.57

luciferase

luciferin-2H+02 > Iuciferin+H202

The release of energy in the form of light is a specific characteristic of
the enzyme, since the oxidation of dihydroluciferin by other agents is not
accompanied by the emission of light. The reduced coenzymes of nicotinic
acid and riboflavin as well as sodium dithionite and hydrogen (Pt cata-
lyst) can be used in place of dihydroluciferin as hydrogen donors for the
luminescent reaction. Luciferin, though once postulated to be a flavin-like
compound, is now known to be chemically related to vitamin K.58

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51. Nachmansohn, D., "Currents in Biochemical Research," Interscience Publishers,

Inc. (New York), 1946, pp. 335-356.

52. Bullock, T. H., Grundfest, H., Nachmansohn, D., and Rothenberg, M. A., /.

N euro physiol., 10, 11 (1947).

53. Grundfest, H., Ann. Rev. Physiol, 9, 477 (1947).

54. Lipmann, F., and Kaplan, N. O., J. Biol. Chem., 162, 743 (1946).

55. Van Niel, C. B., "Photosynthesis in Plants," Iowa State College Press (Ames,

Iowa), 1949, pp. 437-495.

56. Ochoa, S., "Currents in Biochemical Research," Interscience Publishers, Inc.

(New York), 1946, pp. 165-186.

57. Sumner, J. B., and Somers, E. F., op. cit., p. 275.

58. Kluyver, A. J., van der Kirk, G. J. M., and van der Burg, A., Proc Nederl. Akad.

van Wetenschappen, 45, 962 (1942).

Section C

THE ROLE OF THE B VITAMINS IN ANIMAL
AND PLANT ORGANISMS

Ernest Beerstecher, Jr.

PROLOGUE

A logical consideration of the role of the B vitamins in living organisms
might well follow an outline centering around the answers to these four
major questions:

What are the B vitamin requirements of living organisms?

What happens to the B vitamins in living organisms?

How do the B vitamins affect living organisms?

How does B vitamin deprivation affect living organisms?
The first three chapters of this section are an attempt to answer the
first of these questions, while the ensuing chapters are devoted respec-
tively to the last three questions. The answers, however, are by no means
as explicit as are the questions.

Chapter IC

METHODS OF ASSESSING B VITAMIN REQUIREMENTS

General Considerations

After the foregoing consideration of the general nature of the B vitamins
and their role in the chemistry of cells and aggregates of cells, it would
be desirable to proceed by a logical sequence of study to the part played
by the B vitamins in entire organisms. Unfortunately, however, the
present understanding of the facts does not permit so smooth a transition.
In the study of vitamin deficiencies in particular, wherein lie man's most
urgent interests, there is seldom more than a vague similarity between
the biochemical functions of the vitamins as we have considered them
and the clinical characteristics of the avitaminoses. This section of the
monograph is developed largely along a new pathway; it therefore draws
on the more basic biochemical factors previously considered only on those
rare occasions when the circumstances will permit.

Implicit in the study of the B vitamins as a group is the fact that in
nature they always occur together and are essential in the economy of
all living cells. Since the problems involving the requirement of any one
B vitamin are common to all members of the B group, an attempt is made
to present the discussion of these requirements in a general and integrated
way, rather than to stress the consideration of each vitamin individually.

When the supply, whether intracellular or extracellular, of any one of
the B vitamins is cut off, the entire metabolic process rapidly comes to
a standstill. From the standpoint of a single cell, the time required for
this to occur is largely dependent upon how rapidly the various chemi-
cal events progressing within the cell bring about attrition of vitamin-
containing catalyst molecules. In the absence of a renewed supply of
coenzyme, this generally ensues quite rapidly, and the cell becomes func-
tionless in a normal sense when the critical vitamin reaches an inoperably
low level. The quantitative requirement of that cell for any B vitamin
is that amount which it must supply, or which must be supplied to it,
to continue in normal operation. This concept is basically true for cell
aggregates, whether tissues or entire organisms. Cell aggregates for archi-
tectural reasons, however, are able to buffer themselves against deficiency,
and therefore do not respond as rapidly to vitamin privation.

243

244 THE BIOCHEMISTRY OF B VITAMINS

The present discussion will not be primarily concerned with that portion
of the requirement with which living matter is able to supply itself. This
fraction of the requirement has been assessed from a practical standpoint
in the consideration of the sources of vitamins for nutritional purposes,
and from an academic standpoint in the discussion of biosynthetic
processes. Far more expedient is the evaluation of that portion of the
requirement which must be supplied to the cell, tissue, and complete
organism ; and this qualitative and quantitative fraction of the total will
hereafter be referred to as the "requirement," as is the custom. The term
"nutritional requirement" differs critically from this, in that it is that part
of the requirement which must be supplied in the diet, and does not in-
clude that portion which may be supplied by symbiants such as intestinal
bacteria.

The natures of the B vitamin requirements of living organisms are as
diverse as are the forms of life themselves. To a considerable extent the
members of the plant kingdom are able to meet their own B vitamin
requirements, demonstrating thereby a higher degree of synthetic ability
than is found generally in the animal kingdom. This fact is most funda-
mental to the overall economy of life. Green plants have, therefore, been
considered in this discussion primarily as a food source ; and, though they
will be referred to again when considerations arise that seem to warrant
their separate discussion, a more extended consideration of the B vitamins
in green plants does not at present seem practical. Similarly, the role of
the B vitamins in the lower plant forms is of importance from a number
of diverse aspects, which seems to dictate the advisability of their dis-
cussion as the occasion arises rather than by separate treatment. The
B vitamins are therefore presented here in their relationship to the animal
as a whole, while the plant kingdom is considered only as the pattern of
the treatment and as the availability of data permit.

From a purely qualitative standpoint, organisms exist which require
none of the B vitamins, and others exist which require all those now
known and probably still other substances of chemical natures which are
at present unknown. Although many bacteria are able to synthesize all
the B vitamins in sufficient amounts to meet their needs, this cannot be
said at present of any member of the animal kingdom. Indeed, no higher
animal has as yet been found which is able to survive even when all the
presently known B vitamins are supplied; and it must be concluded that
as yet unidentified factors, whether they turn out to be B vitamins or
not, are necessary in animal nutrition. A summary of some of these fac-
tors is given on pp. 12-16.

Qualitative requirements may not, however, be dismissed as a matter
of "required" or "not required," since a variety of factors influence the

METHODS OF ASSESSING B VITAMIN REQUIREMENTS 245

decision. While nutritional requirements of ruminants for B vitamins are
virtually nonexistent, this is only by virtue of an extensive system of
bacterial symbiosis; and ruminants do actually require a supply of these
substances, even though it may be derived from within the confines of the
gastrointestinal tract. Moreover, certain B vitamins may substitute for
others. Thus, as will be shown later, substitutions similar to that of
alanine for vitamin B6 in the nutrition of S. fecaelis R x may occur in the
animal kingdom.

The nature of quantitative B vitamin requirements is by comparison
far more complex and will require extended discussion. It should be
pointed out here, however, that the conditions under which any given
quantitative B vitamin requirement exists are so limited that the fixing
of a practical value for any particular species is possible only within very
broad limits.

Finally, fundamental to the nature of any requirement is the problem
of what criterion shall be taken in judging whether a substance is required
and how much is required.2 Thus, the amount of thiamine necessary to
protect a rat from convulsive seizures might be defined as the require-
ment, even though the animal suffered from other pathological manifesta-
tions. The requirement might also be defined as the amount necessary to
maintain the animal in a state of health, or as the amount required to
promote growth, or optimum growth, or as the amount necessary to pro-
mote longevity. Each of these criteria has found use, and a variety of
similar cases makes it necessary to reach an agreement on, or an under-
standing of, this issue. Prejudicial interpretation of this factor in prison
camps during the recent war has resulted in extensive discussion of defi-
ciency criteria. A more extended discussion of this factor, as it refers to
the prevention of specific pathology, occurs in the following section.
Criteria based on benefits exceeding growth and maintenance are dis-
cussed more fully in later chapters.

Associated with this problem is the one of poor usage and ill-defined
terminology. Expressions such as "health," "physical fitness," "optimal
requirements," and "maximal requirements," are common to the field and
portray clearly the uncertainty and the lack of development of satisfac-
tory scientific criteria and nomenclature.3 One definition of good nutrition,
for instance, is "that condition which permits the development and main-
tenance of the highest state of fitness." 4 "Fitness," however, is a term of
little advantage, since, as Keys points out,5 "everyone knows what it
means but not how to measure it."

In addition to the problem of precise requirements, there is the further
question of recommended intake, i.e., the safety margin believed to be
advisable above the absolute requirement. This consideration is largely

246 THE BIOCHEMISTRY OF B VITAMINS

significant in view of wide individual variations in nutritional require-
ments (p. 273). Unquestionably the most generally accepted recommenda-
tions or "allowances" are those of the Food and Nutrition Board of the
National Research Council (p. 324). Based on broad considerations
and a variety of studies, these are nevertheless frequently challenged,
sometimes because of their interpretation as "requirements" rather than
recommended intakes, but more often on a seemingly valid basis. Indeed,
it is quite apparent that no single recommended level will suffice for all
purposes,6-10 since recommendations for the allowances necessary to ward
off deficiency diseases will obviously differ from those used in planning
a dietary regimen for therapeutic purposes, or those used in planning
broad agricultural or public health programs. These factors will be con-
sidered in greater detail somewhat later.

Methods of Assessing B Vitamin Requirements

Man's first interest has always been with man; but because of the
limitations which have been placed on experimentation involving humans,
great ingenuity has been required in assessing human vitamin require-
ments. A variety of approaches has been employed, all more or less
indirect, and therefore subject to interpretation. It has generally been
possible to determine the nutritional requirements of other species with
far greater precision as a result of the experimental freedom which it is
possible to achieve. This fact led to one of the earlier approaches to
the assessment of human requirements, Cowgill's study of comparative
requirements which is discussed first.

Clear-cut experimental data regarding requirements are rare, and this
is due among other reasons to the difficulty in obtaining animals free from
symbiotic organisms. Some conclusions can be drawn with considerable
certainty in fowls, inasmuch as the interiors of eggs are generally germ-
free. Thus, since the riboflavin content of hens' eggs does not increase
during incubation, we know with certainty that the foetal chicken, at
least, does not synthesize riboflavin.11 Similarly, when hens' eggs are
injected with tryptophan, and then incubated and analyzed, they show
a higher niacin content than uninjected eggs;lla this is among the more
convincing facts that indicate that animal tissues do convert tryptophan
to niacin without the aid of bacterial symbiants. A more adequately
controlled repetition of this work would seem to be desirable. Unfortu-
nately, however, extensive data of this nature are lacking.

Comparative Studies of Requirements. It has long been known that
the food consumption per unit of body weight and the basal metabolic
rate per unit of body weight are much greater for small animals than
for larger ones. It is thus not strange that it was readily apparent from

METHODS OF ASSESSING B VITAMIN REQUIREMENTS 247

the earliest studies that the thiamine requirement per unit body weight
varied from species to species, being greater for smaller animals. Cow-
gill 12 has made an extended study of this relationship and its application
to the determination of human requirements. While the actual data ob-
tained are not generally accepted today, and the relationships obtained
reflect to some extent the impure nature of the crude extracts used at the
time the work was done, the general concept behind the conclusions is
of considerable interest and importance. In studies using the mouse, the
rat, the pigeon, and the dog, and using the maintenance of appetite as a
criterion of satisfaction of the requirement, he found that for any one
species, the thiamine requirement was proportional to the five-thirds
power of the weight of an individual animal, and that the proportionality
constant was characteristic for the species.

Thiamine requirement = KsW i^

When the logarithm of the maxium recorded normal weights ever attained
by an individual of the species is plotted against the logarithms of the
species constants, a straight line is obtained, which may be expressed by
the equation

t,,. ■ • . , ,, v 0.98X W\MxWi
Thiamine requirement (^g/day)= — — ■ —

W max

A study of several species indicated that

' 1.5
and the final equation is thus obtained,

rp,. . . . , ,, s 0.654 XTF.XCali
Thiamine requirement Gug/day) = —

where Wt is the weight of an individual, Wmax is the maximum weight
obtained by the species, and Cal; is the daily food intake in Calories.
Employing the last equation, one concludes that for a 70-kg man {Wmax
is 115 kg) with an intake of 2500 Calories, the daily nutritional thiamine
requirement is about 1000 /xg (1.0 mg).

This general approach has been subjected to considerable criticism, and
is obviously subject to numerous errors.13, 14 Nevertheless, the estimate
so obtained is not greatly different from that obtained by numerous other
means. In view of this fact, it is indeed surprising that this general
approach has not been employed with other members of the B group of
vitamins, where the general principles involved should apply with equal
validity (pp. 319-323) .

Diets controlled to produce a given symptom in a given species. Per-
haps the most direct approach to the assessment of requirements con-

248 THE BIOCHEMISTRY OF B VITAMINS

sists of depleting the nutrition of a factor and then observing the exact
amount of the vitamin required to prevent active clinical manifestations
of deficiency. While this was possibly the earliest technique, only in
recent years has it been directly applied to humans. Even so, such a
direct approach to the problem of vitamin requirements is not entirely
satisfactory for a number of reasons. Primarily, the results obtained will
depend completely upon the symptoms to be prevented. Careful perusal
of the tabulated requirements in Chapter IIIC will demonstrate this
forcibly as it applies to many species, since requirements have been
variously adjudged over wide ranges based on this fact alone. This may
perhaps best be illustrated in the case of the folic acid requirements of
the chick which have been extensively investigated in recent years.15 The
symptoms which are prevented by different levels of folic acid in the
chick diet are indicated in Table 1.

Table 1. Functions Supported in the Chick by Various Levels of

Reference

(15)
(16
(15)
(15)
(15)
(17)

Thus for any vitamin and species a number of criteria may be taken
(and frequently have been taken) as indicating a deficiency or lack of
it. Obviously, the criterion suited to the purpose is that which insures the
"well-being" and "general normal character" of the animal in question,
such terms being about as indefinite as the requirement itself. Since the
full consequences of avitaminoses are seldom realized simultaneously
with the first recognition of the etiology of the disease, the optimal type
of criteria mentioned is not always definable, and realization of this has
done much to foster the study of subclinical deficiencies.

Another difficulty inherent in this approach is that concerned with the
association of specific symptoms with a deficiency. Thus when the list of
vitamins consisted largely of "A," "B," "C," and "D," certain symptoms
were associated with a deficiency of vitamin "B," and only in more recent
times has it been possible to associate certain of these with the precise
chemical factor or factors whose absence was responsible for these symp-
toms. In many cases the exact correlation is still unclear. Green and Brun-
schwig,18 for instance, in assessing the physiological activity of choline,
have only recently come to the conclusion that the factors responsible for
hepatic fatty infiltration and for parenchymal necrosis may not be identi-

Folic Acid

Level of folic acid
(per 100 gm diet) (jig)

Criterion

survival

30
35
45
55

prevention of perosis
normal hemoglobin formation
normal growth
normal feathering

50-110

optimal growth

METHODS OF ASSESSING B VITAMIN REQUIREMENTS 249

cal. Whether or not the finding that thiomalic and thiolactic acids afford
no protection against the latter but seem to promote the fatty infiltration
actually supports this belief remains to be seen.

It is seldom as simple as it might appear to those unfamiliar with the
field to exclude one factor from the diet without simultaneously omitting
another.19 In recent studies using dogs, a diet apparently complete in all
necessary factors but biotin produced paralysis and death. Biotin, more-
over, appeared to prevent these effects.20, 21 Later work 22, 23 showed,
however, that the symptoms were primarily due to a potassium deficiency.
While biotin produced some temporary responses for several hours, the
protective effect of a single adequate dose of potassium lasted for six to
ten weeks. Similar cases are well known throughout the history of the
assessment of B vitamin requirements by depletion methods.

It is only in recent years that it has been possible to apply this type
of study directly to humans. This is due to a considerable extent to the
difficulty in controlling a multitude of variables which must be controlled
in order to obtain significant data. Realizing this, the National Research
Council's Committee on Diagnosis and Pathology of Nutritional Defi-
ciencies has outlined certain of these factors which they feel to be of the
greatest importance.24 While primarily designed for assessing conditions
of deficiency, these factors apply in every sense to the assessment of
requirements in animals as well as humans. Briefly they are: (1) the
adequacy of the criterion for determining the nutritional status; (2) the
nutritional status previous to the experimental period; (3) the diet prior
to and during the experimental period; (4) the conditions which influence
the relationship between the supply and requirements; (5) the indices set
by the observer to measure the criteria; (6) the initial status of the sub-
jects with regard to growth, physical performance, resistance to disease,
etc.; (7) the method of selection of subjects; (8) the number of subjects;
(9) the nature and potency of supplements; and (10) the length of the
experiment.

In addition to these factors, adequate controls are essential. This is
particularly true in human experimentation when psychic factors may be
magnified.25 The supplement must be matched in such control groups with
placebos, indistinguishable from the supplement in taste and appearance.
Not only the subjects, but equally important, the observers, must be
ignorant of which individual receives a placebo and which a supplement.
Even in experimentation designed to determine the essentiality of a nutri-
tional source of some B vitamin, it is necessary to observe the factors
outlined above. In a recent study of the effects of a vitamin B6 deficient
diet in humans,26 almost no definite conclusions could be reached because

250

THE BIOCHEMISTRY OF B VITAMINS

Table 2. Analysis of Beriberi-Producing and Beriberi-Preventing Diets.
(From Williams and Spies)

Designation of Diet

1. Avkroyd No. 3 B

2. Aykroyd No. 13 B

3. Aykroyd No. 2B

4. Strong and Crowell IV

5. Selangor Jail 1892

6. East Indian Navy Natives (fish)

7. Fraser and Stanton I

8. Indian Troops Trincomalee (1900) ....

9. Singapore Prison 1869-75

10. Strong and Crowell II

11. Selangor 1901 Ordinary

12. Kuala Lumpur I

13. East Indian Native Sailors 1874 ,

14. Selangor Jail, Penal, 1902

15. Bilibid Prison 1901-02

16. Selangor Jail, Penal, 1900

17. Negro Laborers Congo I

18. Singapore Prison 1876

19. Philippine Scouts 1908

20. Java Prisons (fish)

21. Java Prisons (dried meat)

22. Madras Native Troops (rice)

23. Dutch E. Ind. Navy, Native 1874 (fish) ,

24. Mediterranean Troops Al

25. PudahGaol

26. Aykroyd No. 1 B

27. Kut-el-Amara British Jan. 22

28. Aykroyd No. 11 B

29. Lascar Seamen

30. Dutch E. Ind. Navy Natives 1878

31. Java Prisons (beef and pork)

32. Aykroyd No. 9 B

33. Aykroyd No. 8 B

34. Selangor 1902 Ordinary

35. Japanese Ryujo Marines

36. Bilibid 1902

37. Aykroyd No. 2 N.B

38. Selangor 1899 Ordinary

39. Aykroyd No. 4 B

40. Selangor 1900 Ordinary

41. Singapore Prison July 1880

42. Akroyd No. 5 B

43. Aykroyd No. 6 B

44. Aykroyd No. 12 B

45. Singapore Prison 1900

46. Mediterranean Troops A2

47. Singapore Prison 1897

48. Dutch E. Ind. Navy, 1874 Europeans .

49. Aykroyd No. 10 B

50. Selangor 1895 Ordinary

Thiamine

per day

Calories

Extent of

(MS)

per day

Cal.

beriberi

225

1974

0.074

300

2480

0.078

308

2520

0.079

268

2310

0.099

XXXX

256

2581

0.099

XXX

420

3810

0.110

XXXX

346

3060

0.113

XXX

446

3922

0.114

XXX

336

2952

0.114

XXX

268

2310

0.115

XXX

402

3150

0.128

XXX

443

3379

0.131

XXX

565

4208

0.134

XXX

284

2084

0.136

XXXX

368

2661

0.138

XXXX

306

2193

0.140

XXXX

475

3295

0.144

XXXX

342

2315

0.148

XXX

578

3908

0.148

XXX

420

2831

0.148

440

2927

0.150

573

3804

0.151

600

3960

0.151

XXX

672

4400

0.153

487

2970

0.164

442

2700

0.164

469

2839

0.165

XXX

934

5530

0.169

670

3956

0.170

948

5534

0.171

550

3211

0.171

818

4720

0.173

676

3870

0.175

482

2690

0.175

521

2875

0.181

XXXX

478

2622

0.182

629

3430

0.183

561

3032

0.185

XXX

495

2660

0.186

468

2454

0.190

XXX

535

2720

0.197

727

3665

0.198

576

2860

0.201

557

2740

0.203

645

3128

0.206

XXX

918

4400

0.208

624

3005

0.208

1091

5189

0.209

632

3020

0.209

618

2942

0.210

METHODS OF ASSESSING B VITAMIN REQUIREMENTS 251

Table 2. Analysis of Beriberi-Producing and Beriberi-Preventing Diets — (Continued).
(From Williams and Spies)

Thiamine T (jug)

Designation of Diet per day Calories Extent of

Gig) per day Cal. beriberi

51. Japan. Ship Ryujo Cadets 651 3072 0.212 XXX

52. Aykroyd No. 7 B 510 2400 0.212 B

53. Megaw and Bhattacharjee Parsibazar . 464 2100 0.220 B

54. Megaw and Bhattacharjee (Campbell) . 506 2200 0.230 B

55. Ryujo Sub-officers 660 2867 0.230 XX

56. Strong and Crowell III 442 1916 0.231 XX

57. Jap. Navy Food Act. 1884 995 4295 0.232 X

58. Ryujo Officers 819 3508 0.233 X

59. Kala Bagan 432 1822 0.237 X

60. Aykroyd No. 7 N.B 751 3160 0.238 No

61. Selangor, Penal, 1895 646 2720 0.238 XXX

62. Selangor, Penal, 1893 710 2982 0.238 X

63. Singapore Prison 1898-99 581 2424 0.240 XXX

64. Bengali Girls 532 2200 0.242 No

65. Singapore Prison Sept.-Dec. 1881 904 3475 0.260 XXX

66. Singapore Prison 1882-85 970 3652 0.266 X

67. Aykroyd No. 1 N.B 703 2590 0.271 No

68. Selangor 1892 1034 3766 0.274 No

69. Mediterranean Troops 1013 3620 0.280 No

70. Megaw and Bhattacharjee Hindus 616 2295 0.281 No

71. Aykroyd No. 3 N.B 764 2710 0 282 No

72. Mediterranean Troops B 1033 3590 0.288 No

73. Aykroyd No. 10 N.B 1035 3690 0.288 No

74. Aykroyd No. 11 N.B 875 2945 0.297 No

75. Aykrovd No. 4 N.B 820 2710 0.300 No

76. Avkroyd No. 9 N.B 1497 4980 0.300 No

77. Selangor 1893-94 Ordinary 965 3142 0.308 No

78. Aykroyd No. 5 N.B 1664 5510 0.302 No

79. Megaw and Bhatt. Anglo-Ind 932 3000 0.310 X

80. Aykroyd No. 12 N.B 1597 4980 0.321 No

81. Bilibid Prison 1912 Manila 941 2911 0.324 No

82. Kut-el-Amara Indians March 11 726 2208 0.328 No

83. Richmond Asylum-Dublin 1897 982 2994 0.328 XXX

84. Kut-el-Amara British March 11 771 2304 0.334 No

85. Garrison, San Juan Jan.-Apr 600 1718 0.350 X

86. Megaw and Bhatt. Mohammedans 778 2180 0.357 No

87. Aykroyd No. 6 N.B 954 2580 0.370 No

88. Indian Native Troops (Improved) 1500 4025 0.373 No

89. Kut-el-Amara British Feb. 10 1063 2835 0.375 No

90. Singapore Prison 1885-1896 585 2514 0.385 No

91. Kut-el-Amara Indians Mar. 4 983 2149 0.457 No

92. Aykroyd No. 8 N.B 2070 5020 0.413 No

93. Kut-el-Amara Indians Jan. 22 1217 2843 0.428 No

94. U. S. Garrison San Juan June 866 1882 0.460 No

95. Kut-el-Amara British Mar. 4 1168 2420 0.481 No

96. Philippine Scouts after 1911 1770 3672 0.482 No

97. Trincomalee Troops 1901 2194 4200 0.522 No

98. Frazer and Stanton 1907-08 1695 3054 0.555 No

99. Kuala Lumpur Asylum 2170 3381 0.624 No

100. Ind. Native Troops Attah 3925 3994 0.985 No

Prevalence of beriberi is indicated by number of X's, except when the number of cases is not recorded

or the group was too small for statistical consideration, in which case the existence of beriberi is indicai
by B.

LI8RAR

,AXi»Aa««>

252 THE BIOCHEMISTRY OF B VITAMINS

a number of these factors mentioned had not been considered in the
experiment.

With rats, mice, chickens, and most other smaller animals, growth has
frequently been taken as a suitable criterion for assessing the fulfillment
of the B vitamin requirement. In humans this is not the case, supposedly
because of the difficulties in the interpretation of such data. Indeed, studies
involving vitamin supplementation of adequate diets have been frequently
subjected to criticism for adopting "growth" as a criterion without any
further consideration of other factors involved. Moreover, from a physi-
ological point of view, "increased" and "improved" growth may be very
different things, there being no guarantee that the most rapid growth rate
is the most desirable.27

Melnick et al.28 have developed B vitamins bioassay methods which
employ human subjects. These methods (p. 283) depend upon the study
of the urinary excretion of the B vitamins, which is presumably a function
of the intake. While not bearing directly upon the problem at hand, the
work of these investigators deserves mention at this time in that it is
subject to the variables previously mentioned, yet illustrates the value
that is attached to carefully controlled experimentation with human
subjects.

Dietary surveys. Somewhat akin to controlled diet studies are those
on healthy and avitaminotic populations. By a careful consideration cf
numerous dietary surveys, it is sometimes possible to estimate the level
of nutrition which will bring about a deficiency of one or more of the
B vitamins. Excellent examples of this approach have resulted from
studies of prison camps and of circumscribed populations during the
recent war, since the diet was frequently rigidly controlled and permitted
unusually accurate assessment of the vitamin intake. One of the classical
studies of this nature, however, is an earlier one dealing with thiamine-
deficient diets in the Orient.

Cowgill 12 studied some 180 human diets in regard to their thiamine and
calorific content and association with beriberi. Williams and Spies 14 later
reassessed these data and arrived at a more accurate estimate of the
minimal human requirement necessary to prevent beriberi. A modified list
of the diets studied in increasing order of the ratio of thiamine to Calories
in the diet is given in Table 2. A summary of the results is given in
Table 3.

The validity of these data and the conclusions derived from them are
borne out adequately by a study of a large number of American dietaries
(Table 4) .29 The thiamine-to-calorie ratio seems generally to be above
that associated with clinical beriberi, other dietary factors (i.e., fat; page

METHODS OF ASSESSING B VITAMIN REQUIREMENTS

253

Table 3. Classification of Diets in Table 2 as to Thiamine Deficiency.
Thiamine Gig /day)

Calories per day

Diets producing
Beriberi

Non-Beriberi

Diets

Total Diets

0.074-0.229
0.230-0.249
0.250-0.279
0.280-

52
9
2
3

Total

100

Table 4. Vitamin Bi Content of Diets of Families of Wage Earners and Low-Salaried
Clerical Workers.

Region, number of families

North Atlantic, 1394 white families

Pacific: 688 white families

East South Central : 426 white families

South: 284 Negro families

Sherman — Average American Diet

(Cowgill p. 186)
American Family on Food Relief

(Cowgill p. 194)
Assumed boderline for clinical beriberi

Weekly

Thiamine

Calories

T(mb)

expenditure per

per day

per
day

per

food constituent

0*e)

Cal.

$1.33-$1.99

600

2550

0.235

2.00- 2.66

735

2960

.248

2.67- 3.32

885

3310

.276

3.33- 3.99

945

3840

.246

4.00- 4.66

1200

4140

.290

1.33- 1.99

780

2570

.303

2.00- 2.66

840

3100

.271

2.67- 3.32

960

3660

.262

3.33- 3.99

1065

4140

.257

4.00- 4.66

1125

4340

.259

.67- 1.32

495

2620

.189

1.33- 1.99

735

3050

.241

2.00- 2.66

855

3470

.246

2.67- 3.32

1095

3980

.275

.67- 1.32

570

2450

.232

1.33- 1.99

840

3460

.243

2.00- 2.66

1180

4470

.242

2.67- 3.32

1260

4880

.259

900

2500

.360
.400

600

2500

.250

276) doubtless contributing a somewhat greater element of safety than is
apparent in these lower values.

Goldberger's classical studies on the nicotinic acid requirement of man
are likewise of a survey nature. Frazier and Friedemann have recently
re-evaluated these studies, including the dietary records of some 1863
human subjects30 and concluded on this basis that the human require-
ment for nicotinic acid, when other factors are present in good supply, is
about 4 mg/day, but may be as high as 7.5 mg/day on a marginal diet
high in corn products. Similarly, Williams 31 studied the B vitamin con-
tent of mixed human diets known to be adequate for human nutrition, of
a highly satisfactory animal ration, and of a rat carcass, and found them
highly similar on an isocalorific basis, as is shown in Table 5. He con-
cluded, therefore, that these values probably indicate "safe" levels for
a daily human intake.

254 THE BIOCHEMISTRY OF B VITAMINS

These values for a recommended intake obviously represent a much
higher level than is arrived at for the requirement by other means. They
are, however, not extravagant as an assessment for practical use in view
of the great variation which may exist in individual needs (p. 273).

Table 5. Vitamin B Content of Various Materials (per 2500 Calories).

Thi-
amine
(mg)

tinic
Acid
(mg)

Ribo-
flavin
(mg)

thenic
Acid
(mg)

Biotin
(mg)

Inosi-
tol
(mg)

Pyrid-
oxine
(mg)

Folic
Acid
(mg)

Mixed diet
Dog food

Rat carcass

3.6
2.8-
4.4
1.86

40.1
24-
40
68

3.67
3.7-
6.2
4.03

11.2
10-
10.9

14.9

0.25
0.114-
1.14
0.124

987

1170-
2909
2.7

1.77

1.13-

1.86

0.93

1.39
0.66-
0.94
1.56

Recommended
daily intake

3.2

3.7

0.14

1000

1.5

1.0

Studies of Vitamin Excretion. A fourth approach to the assessment
of requirements for B vitamins is the evaluation of vitamin excretion
levels in terms of the B vitamin intake. Actually this represents a varia-
tion of a previous approach wherein the excretion is taken as the criterion
of deficiency or sufficiency. Economizing processes in living tissues pre-
sumably work to retain and utilize essential nutrients when the require-
ment is not being met by the nutrition or when a so-called "tissue hunger"
exists for some factor. When, however, the tissues have absorbed as much
of a vitamin as they require, an organism will frequently excrete amounts
of essential food constituents proportional to the intake. Melnick and
co-workers 2S have utilized this latter principle extensively in biological
assay work employing human subjects, and in the study of the biological
availability and inactivation of certain B vitamins. More generally, how-
ever, test doses of a given vitamin are administered to a subject and,
depending upon the portion excreted as compared with known values
from individuals on an adequate diet, an estimate is made of whether
a depletion exists and therefore whether a diet of known vitamin content
meets the requirement. Still simpler but infinitely less satisfactory, one
may attempt to determine whether the level of urinary excretion of a
B vitamin is within the range of values considered to be normal.

This general approach, like the others, has its drawbacks. Unquestion-
ably the greatest errors have been due to the individual variability in the
proportion of an administered test dose which is excreted. Another source
of question has been concerned with the validity of the general principle
that the absorption capacity of the body is a true function of the require-
ment. In general, results obtained by this method agree well with other
evaluations of requirements, although they tend to be somewhat higher.
A third source of error, particularly in earlier studies, is in the measure-
ment of inappropriate excretion products. Increased understanding of

METHODS OF ASSESSING B VITAMIN REQUIREMENTS 255

B vitamin metabolism and of the chemical nature of excretion products
has done much to remedy this situation (p. 365) .

Johnson et al.32 have critically analyzed many of the technical factors
involved in this type of study, devoting special attention to the thiamine,
riboflavin and N'-methylnicotinamide content of urine. A comparison of
fasting specimens, random specimens and samples after an oral loading
test seemed to indicate that fasting urinary excretion studies may be far
more accurate than studies using a loading test. While there is little doubt
that random urine samples are valueless as compared with the fasting
samples, it is apparent (p. 351) that the many factors involved in the
storage and excretion of the B vitamins have caused many workers to
express grave doubt concerning the value of fasting urinary levels of B
vitamins in assessing dietary requirements. This doubt, aggravated by
extensive individual variability, seems well founded, as judged by most
recent studies.33, 34 Dietary interrelationships may operate to make such
errors even greater. Illustrative of this is the demonstration that very
high nicotinamide intakes have been shown to increase thiamine excretion
by as much as 70 per cent.35

In spite of these and other difficulties, much instructive information is
obtainable in this manner. Studies on individuals whose vitamin intakes
have been cut by 28 to 66 per cent from the normal levels show that the
change was generally reflected in the urinary levels within a single week.
In these studies, however, physical changes were not observable through-
out the test period of five weeks.36 There can be little doubt therefore
that when cautiously interpreted, results from urinary excretion studies
may be of extreme value. It is possible to discuss in the limited space
available only a few of the more recent applications of this method, prin-
cipally as illustrative examples of the possibilities of this approach.

In an extended series of studies, Oldham and co-workers, on the basis
of urinary excretion data obtained from urine samples from young
women,37 have decided that the thiamine requirement is somewhat less
than 1.0 mg/day or 20 /^g/kg body weight.

Michelson et al.SH have studied in detail the problems inherent in
assessing the level of thiamine nutrition by this means, and have pointed
out several important considerations. The attainment of excretion equi-
librium at a given intake level requires considerable time, and this was
not realized in much earlier work. They found that a change in daily
intake of thiamine is on an average only half reflected in the excretion
in ten days. These authors studied both thiamine and pyramin * excretion
over prolonged periods, and found large (threefold) variations between
individuals in thiamine excretion at high levels of thiamine intake. They

* 2-methyl-4-amino-5-hydroxymethylpyrimidine.

256 THE BIOCHEMISTRY OF B VITAMINS

report that pyramin excretion increases exponentially with increases in
thiamine intake, approaching linearity at normal levels, as contrasted
with linear increases in thiamine excretion throughout the range studied.
Moreover, pyramin excretion remains measurable at low intakes of thi-
amine, when thiamine excretion falls to essentially zero. For these reasons,
pyramin would seem to be a better end product to study than thiamine.

Comparing the excretion technique with others, Berryman et al.39 find
that urinary excretion of B vitamins falls off rapidly following the change
of men to a deficiency diet, but that fecal levels remain unchanged.
Decline in physical and mental states are more gradual, and these states
improve more slowly when the vitamin supply is increased than do the
urinary excretion levels. The great individual variability in response was
again noted by these workers. Still other studies 40> 41 indicate quite
clearly that a decreased dietary intake is rather rapidly followed by a
decrease in urinary output, followed only gradually by decreases in tissue
content, and only much later by the manifestations of a clinical deficiency.

The most advantageous use of excretion studies in the assessment of
vitamin requirements comes about in those cases in which, unlike thi-
amine, other methods of approach to the requirement are less feasible.
The riboflavin requirement is an example of this situation. Hagedorn,42
in a study of prison inmates, found great variability in riboflavin excre-
tion (0.05-2.4 mg/24 hrs) and in the retention of test doses without
apparent cause, and experienced further difficulty in obtaining useful
results from fasting subjects due to the large errors in measuring such
low riboflavin concentrations as were obtained. Despite the apparent
advantages of dietary control, prison inmates have seldom been a source
of entirely satisfactory data, and other workers have obtained better
results with more satisfactory subject material. Oldham et at. concluded
from a study of the riboflavin excretion of institutionalized children 43
that 1.15-1.6 mg/day of riboflavin met their nutritional requirements.
Other workers, by studying excretion at different levels of intake and the
per cent of a 3-mg test dose of riboflavin excreted in 24 hours at each
level of intake, decided that 1.3-1.5 mg/day is adequate for an intake of
2100-2300 Calories.44 Oldham et al., in their earlier studies, estimated
that the riboflavin nutritional requirement was about 1 mg/day or 0.50
mg per 1000 Calories. A summary of their results is given in Table 6.

All these results on riboflavin agree well with the earlier observations
of R. D. Williams et al.,46 but the requirements arrived at are less than
those proposed by Sebrell et al.i7 It is noteworthy that nearly all workers
are in agreement that fecal riboflavin does not vary with the intake.

Briggs 48 studied two subjects who had been pellagrins, and who were
placed on a "corn-poor" diet containing about 2.4 mg of nicotinic acid

METHODS OF ASSESSING B VITAMIN REQUIREMENTS 257

per day. Although urinary excretion tests suggested a niacin deficiency,
only the mildest of clinical symptoms were observed. Najjar and co-
workers49 in similar experiments maintained subjects on diets containing
1.5-2.0 mg of nicotinic acid per day and observed neither signs of nico-
tinic acid deficiency nor a reduction in N'-methylnicotinamide excretion,
which would suggest depletion of body tissues. These authors attribute
such survival on obviously low levels of nicotinic acid to intestinal syn-
thesis. Cossandi,50 on the basis of loading tests with infants in which
only 1 per cent recovery was obtained as compared with 5-10 per cent
in adults, concluded that up to one year of age there exists a relative
niacin deficiency, more particularly in breast-fed as compared with bottle-
fed infants.

Table 6. Percentage of the Average Total Riboflavin Intake and of the Increase in Average
Intake Excreted at Each Dietary Level.™

Total

Increase in

Total

Increase in

Total Intake

Increase in

eriod

Intake

Excretion

Excreted

Intake
Excreted

(MS)

G«g)

(Mg)

(%)

600

113

1017

417

150

III

1234

217

263

113

7171

5937

4344

4081

1206

325

Normal persons excrete about 2.3 /xg of folic acid per day in urine, and
after oral doses of 5-16 mg, excrete about 28 per cent of the dose according
to a recent study. In patients hospitalized for various causes, recoveries
were much lower, however. There is therefore some reason to believe that
an estimate of human folic acid requirements may be derived from such
studies.51 In the case of some other factors, such a possibility seemingly
does not exist. Thus, since biotin excretion nearly always exceeds the
intake 52 (due to intestinal synthesis) accurate results seem improbable.
In other cases where intestinal synthesis is of a relatively low order, the
error so introduced is not as serious an objection to the method. A more
detailed consideration of many of the factors involved in the validity of
these studies occurs in a later section on B vitamin excretion (p. 364).

Studies of Levels in Various Biological Materials. Closely related to
those studies in which the B vitamin requirement has been assessed on
the basis of excretory levels are those studies of vitamin tissue concen-
trations which contribute to an understanding of B vitamin requirements.
This latter type of study, however, has been very little pursued, to some
extent because of the uncertainty regarding the variable nature of tissue
storage, but in most cases because of the difficulty in obtaining suitable
material.

Studies of fecal vitamin content have proved to be almost totally value-
less, because of both the influence of bacteria directly and the influence

258 THE BIOCHEMISTRY OF B VITAMINS

of other dietary constituents on bacteria. Thus, fecal elimination of most
of the B vitamins in rats is more of a criterion of the dietary protein level
than of any other factor,53 this being particularly true for biotin, panto-
thenic acid, and nicotinic acid. Protein levels likewise influence hepatic
storage of B vitamins, making the liver an uncertain tissue for study. On
diets in which factors other than the B vitamins are held constant, it has
been found 54 that the thiamine content of feces is quite constant and
independent of the intake. Similarly, the biotin in the combined urine
and feces of humans on low, moderate, and high biotin intakes, respec-
tively, is about nine, three, and one to five times the dietary level, or
approximately constant.55

Milk which is generally quite available for study is unfortunately also
influenced in its B vitamin content by a variety of factors other than
dietary vitamin levels55 (p. 347), and has not as yet proved of great
value in this regard. It has been shown, however, that there is some
correlation between the thiamine levels in human blood and in milk, other
factors being constant,57 and that a daily intake of 1.5 mg of thiamine
produces a level of about 20 fig per cent in the milk. Moreover, both the
thiamine and riboflavin levels in milk seem to vary with urinary excre-
tion, indicating some possibility of studies of the requirement during
lactation.58 This type of study has not, however, been extensive as yet.

A variety of other materials has been found somewhat more satis-
factory for assessing tissue vitamin sufficiency. It seems well established
that pork generally reflects the dietary thiamine level of the hog, but
studies have not as yet been reported on the assessment of porcine thia-
mine requirements by such a method.59 This technique of assessing vita-
min requirements, like the urinary excretion method, lends itself best to
those cases where more direct means are not practical.

A typical example of this approach is the work of Czaczkes and Gug-
genheim 60 on the riboflavin requirements of the rat. On diets containing
no riboflavin or 5 fig of riboflavin per day, the riboflavin content of liver,
kidney, muscle, and urine steadily decreases. On a level of 7.5 fig per
day, however, balance is maintained, while on 10 /xg, the tissue content in-
creases. Thus, it seems logical to conclude that for these 50-gm rats, the
riboflavin requirement is about 7.5 fig per day. These workers point out
that the blood content remains constant, while the kidney content does
not show an excess, but readily reflects a deficit. Both liver and muscle,
however, were good indicators of the nutritional status.

Not only must the validity of the particular tissue as an indicator be
established as above, but while employing any tissue, all other dietary
constituents must be kept constant. Thus, it has been found that vitamin
C controls riboflavin storage to a marked degree,61 as does protein. More-

METHODS OF ASSESSING B VITAMIN REQUIREMENTS 259

over, the riboflavin level of the liver is a function not only of dietary
protein, but also of methionine and cystine.02

Interesting results concerning the riboflavin requirements of fowls
have been obtained from studies employing eggs. Jackson and co-
workers 63 have found that the highest level of dietary riboflavin that
would affect the riboflavin concentration in hens' eggs was from 1400 to
1600 tig per pound of feed. This is about 330 /xg per cent and very close
to the requirement of hens as assessed by other means (p. 327) .

In order to maintain a normal blood level of niacin in humans (0.6-0.65
mg per cent) , it has been found that a daily intake of from 12 to 16 mg
is required. Since the blood level of nicotinic acid varies with the dietary
intake, this value may be taken as an estimate (albeit a high one) of the
average human requirement.04 In white rats the liver and spinal fluid
levels of nicotinic acid have also been shown to be a function of niacin
intake,65- 60 but also to be inversely related to the thiamine intake. Old-
ham 39 has concluded that urinary and fecal niacin excretion are inde-
pendent of the intake, each being about 1 mg per day in the young women
studied.

Urinary and fecal pantothenic acid, however, have been found by Old-
ham et al.37 to vary with the dietary intake. Pearson et al. found that in
the chick, both the blood and muscle tissue reflected the dietary supply
of pantothenic acid, although the liver did not.67 Dietary levels of panto-
thenic acid greater than those required for "adequate nutrition," how-
ever, do not further increase the muscle pantothenic acid level in the
chick. Silber 68 similarly finds that in dogs the blood pantothenic acid
level reflects the nutritional supply.

With regard to vitamin B6, studies have shown that the level in rat
liver is independent of the level of intake, for dietary levels greater than
25 /xg per day, thereby suggesting this as the dietary requirement for the
rat. The value so obtained does not vary greatly from other assessments
of this requirement.

Unquestionably, studies of the kind mentioned will be more numerous
in the future. Techniques such as that recently developed for the determi-
nation of riboflavin in very small amounts of serum 68a may eventually
make this approach widespread in nutritional survey work. Moreover,
by similar processes it will be possible eventually to study the nutritional
requirements of individual animal tissues grown in vitro and thus arrive
at a more fundamental understanding of B vitamin requirements. Such
studies, already under way,69, 70 may well be one of the great advances
in this field within the immediate future.

Natural Selection Studies. It has been reported from time to time that
among lower animals at least there exists an instinctive tendency to

260 THE BIOCHEMISTRY OF B VITAMINS

select diets rich in some factor which may be deficient in their nutrition,
and this would seem to provide still another approach to the assessment
of the B vitamin requirements of certain species. However, because of
the imperfect understanding of the phenomena involved and due to the
relatively recent inception of this type of study,71 little progress has been
made with regard to this particular aspect of self-selection diets. Scott
et al.12- 73 have shown clearly, however, that in rats fed appropriately
deficient diets (but not in normal controls) appetites are developed for
thiamine, riboflavin, and the vitamins B6, but not for pantothenic acid.
Much progress in this field will undoubtedly be made in the years im-
mediately ahead as our understanding of the physiological nature of
specific hungers is further developed. (See p. 433.)

The Use of "Anti-Vitamins." Finally, it is worthy of mention that
the new and rapidly developing study of anti-vitamins has contributed
in some degree to our knowledge of B vitamin requirements and seems to
provide an unique approach to their study. Later sections of this mono-
graph will consider in detail how avidin has made possible the elucidation
of the biotin requirement of various species (p. 428), how live yeast
has been used to produce thiamine depletion (p. 291), and how the sulfa
drugs have been similarly employed (p. 298) . It seems possible that the
requirements for as yet unidentified members of the B vitamin group
may await studies of this kind. Challenging in its implications for future
possibilities along these lines is the recent work involving lyco-marasmine.
Plattner and Clausen-Kaas 74 isolated from Fusarium lycospersici Sacc.
a substance, "lyco-marasmine," which is responsible for the wilting of

H2N— CO— CH2 CH3

HOOC— CH— NH— CO— CHo— NH— C— OH

COOH
Lycomarasmine (after Woolley)

plants on which the Fusarium is parasitic. Analysis of this substance
has indicated the probability of a tripeptide nature involving serine,
glycine and aspartic acid,. Strepogenin (p. 15), a possible new member
of the B vitamin group, is considered to be similar in structure, and
because of this it was thought that an anti-vitamin relationship might
exist. This is apparently so, since strepogenin reverses the toxic action of
lycomarasmine, and it therefore seems likely that strepogenin is important
in the higher plants (as it is in bacteria). This conclusion is based on
reasoning by methods analogous to those used in explaining the reversal
of sulfa drug bacterial inhibition with p-aminobenzoic acid (Chapter

IIID) 75,76

METHODS OF ASSESSING B VITAMIN REQUIREMENTS 261

Thus, we find that extensive methodology has accrued about the
assessment of B vitamin requirements. No single method as yet developed
is perfect or free from just criticism. The extent to which more accurate
methods of assessment are required is in itself questionable in view of
the many factors influencing the requirements. Since the very nature of
the nutritional B vitamin requirement is largely a mosaic of these other
factors, it seems quite essential to consider them in some detail in the
following chapter.

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53. Wright, L. D., and Skeggs, H. R., Proc. Soc. Exptl. Biol Med., 63, 327-33 (1946).

54. Alexander, B., and Landwehr, G., J. Clin. Invest., 25, 287-93 (1946).

55. Gardner, J., Parsons, H. T., and Peterson, W. H., Arch. Biochem., 8, 339-48

(1945).

56. Nutrition Revs., 4, 134-7 (1946).

57. Schultz, F. W., Semana med. Buenos Aires, 50, 689-91 (1946).

58. Roderuck, C. E., Williams, H. H., and Macy, I. G., J. Nutrition, 32, 249-65

(1946).

59. Heinemann, W. W., Ensminger, M. E., Cunha, T. J., and McCulloch, E. C,

J. Nutrition, 31, 107-25 (1946).

60. Czaczkes, J. W., and Guggenheim, K., J. Biol. Chem., 162, 267-74 (1946).

61. Cimino, S., Boll. soc. ital. biol. sper., 22, 291-3 (1946).

62. Riesen, H. W., Schweigert, B. S., and Elvehjem, C. A., Arch. Biochem., 10,

387-95 (1946).

METHODS OF ASSESSING B VITAMIN REQUIREMENTS 263

63. Jackson, S. H., Drake, T. G. H., Slinger, S. J., Evans, E. V., and Pocock, R,

J. Nutrition, 32, 567-81 (1946).

64. Gounelle, H., Vallette, A., and Raoul, Y., Compt. rend. soc. biol., 139, 16-17

(1945).

65. Malaguzzi-Valeri, C, and Neri, F., Boll. soc. ital. biol. sper., 17, 644-5 (1942).

66. Malaguzzi-Valeri, C, and Neri, F., Boll. soc. ital. biol. sper., 17, 645-6 (1942).

67. Pearson, P. B., Melass, V. H., and Sherwood, R. M., J. Nutrition, 32, 187-93

(1946).

68. Silber, R. H., J. Nutrition, 27, 425-33 (1944).

68a. Burch, H. B., Bessey, 0. A., and Lowry, H., J. Biol. Chem., 175, 457-70 (1948).

69. Nutrition Revs., 5, 189-90 (1947).

70. Hetherington, D. C, Proc. Soc. Exptl. Biol. Med., 62, 312-15 (1946).

71. Scott, E. M., /. Nutrition, 31, 397-406 (1946).

72. Scott, E. M., and Quint, E., J. Nutrition, 32, 285-91 (1946).

73. Scott, E. M., Smith, S. J., and Verney, E. L., J. Nutrition, 35, 281-6 (1948).

74. Plattner, P. A., and Clauson-Kaas, N., Helv. Chim. Acta, 28, 188-95 (1945).

75. Plattner, P. A., and Clauson-Kaas, N., Experientia, 1, 195-6 (1945).

76. Woolley, D. W., J. Biol. Chem., 176, 1291-1308 (1948).

Chapter II C

FACTORS INFLUENCING B VITAMIN REQUIREMENTS

There are few aspects of an individual, his nutrition, or his environ-
ment that do not to an appreciable extent influence his B vitamin require-
ments. These effects may be exerted by (a) varying the actual physi-
ological requirement, (b) varying the proportion of the total vitamin
intake which may be utilized, or (c) varying the amount of vitamin
supplied by intestinal flora. The ensuing discussion of these effects pro-
ceeds in that order. To consider even a major portion of the factors in-
volved would be neither possible nor practical in this volume. Rather, it
seems expedient to consider briefly those major factors which have proved
to be the most important in influencing B vitamin dietary requirements.
It is impractical to do more than briefly delineate these relationships at
this time, although a more fundamental explanation of their basis in
many cases is provided in the succeeding chapters.

Factors Influencing the Physiological Requirement for the B Vitamins

Species and Strain Variability. Thiamine was the first of the B vitamins
to be discovered and thoroughly studied, and consequently knowledge of
the variability of its requirement among different species is perhaps the
most complete of all. Since many of the more recently discovered
members of the B vitamin family follow the same trends as does thiamine
in this regard, it is advantageous to consider vitamin Bi in greater detail
from this standpoint than would otherwise be the case.

Table 7.

Relationship of the Thiamine Requirement to the Size

of a Species

Thiamine required

Basal

Days

to prevent polyneuritis

Average
weight

required for
polyneuritis

Species

rate

/zg/day /animal

Aig/day/kg

/ug/gm

(gms)

(Cal./kg/day)

to develop

body wt.

food

Tirbolium

confusum

0.5

■

Rice bird

250

9-12

Mouse

160

15-20

0.5

Rat

125

35-60

0.4

Pigeon

400

100

20-30

22.5

0.7

Chick

300

0.7

Dog

8000

0.35

Man

60,000

600

0.60

We have previously mentioned (p. 246) Cowgill's extensive study of
species requirements and his conclusion that smaller species require a
greater amount of thiamine per unit of body weight than do larger ones.

264

FACTORS INFLUENCING B VITAMIN REQUIREMENTS 265

To a large extent this is probably due to the increased relative food
intake of smaller animals, which is in turn related to the greater body
surface and basal metabolic rate of small animals per unit of body
weight. The data in Table 7 1 are presented only to illustrate these
relationships, and are not proposed as absolute values in any case.

Beyond doubt, similar tabulations might be made for the other B
vitamins (p. 319) .

Strain differences may be as marked as the differences between species
and a large number have been reported. For instance, Light and Cracas 2> 3
compared the thiamine requirements of three different strains of rats and
found indications of considerable variation in the requirement. Their
data are summarized in Table 8.

Table 8. Thiamine Requirements of Three Strains of Rats.
Average growth in grams per rat for a five-week test period

On a level of On a level of On a level of

Strain 2 pg Thiamine/rat/day 4 /ig Thiamine/rat/day 8 /<g Thiamine/rat/day

1 33.3±1.9 52.8±2.3

2 29.8±3.38

3 14.0±1.64 27.0±1.04 52.9±2.8

Lamoureux and Hutt 4 developed strains of white leghorn chickens
which gained 50 per cent more in weight than other strains on a given
thiamine intake. Engel 5 similarly obtained strains of rats varying
broadly in their choline requirements. On a given dietary intake one
strain suffered 1.5 per cent deaths as contrasted with 42.4 per cent for
the other group. The females of the former strain had an incidence of
kidney hemorrhages of 19.7 per cent as compared with 93.9 per cent for
the latter group. Lucas, Heuser, and Norris,6 in their studies of chick
nutrition, found that Red Rock cross chicks require as much as 20 times
the vitamin B6 levels in their diet as is required by other strains such
as White Leghorns in order to prevent a severe vitamin Bc deficiency.
Similarly, Ershoff 7 has found that while most rats do not require nico-
tinic acid, one strain did not nurse their young while on a niacin deficient
diet, although they appeared otherwise normal. The report of Rhoads
et al.8 that there is no difference in the response of colored and white
children to normal and vitamin supplemented diets should not be inter-
preted as meaning that differences in vitamin requirements do not exist
between human races, although there is little information available bear-
ing upon this nutritionally important question.

Variations with Age and Weight. Mention has previously been made
(p. 247) of Cowgill's conclusion that the thiamine requirement varies
within a species according to the five-thirds power of the body weight.
This has been disputed,1 and it is at present felt that there is no general
relationship between the thiamine requirement and the age or weight of

266 THE BIOCHEMISTRY OF B VITAMINS

an individual, except as these factors influence the calorific intake. Thus,
for a given species, the requirement expressed as the vitamin-to-calorie
ratio is independent of the weight of the individual. General considera-
tions would indicate that this is similarly true for the other B vitamins,
and it is possible that this constitutes a distinguishing characteristic of
the B group.

Unfortunately, little work has as yet been done on the effects of age,
as distinguished from weight, on B vitamin requirements. Rafsky and
Newman 9> 10 have studied the niacin requirement of the aged, and more
recently their thiamine excretion.11 A study was made of 31 persons, 14
men and 17 women, ranging from 65 to 81 years of age and on adequate
thiamine intakes (0.51-1.11 mg/day). Forty-five per cent of the subjects
were found to excrete less than 50 y per day of thiamine, which is con-
siderably below the generally accepted normal value. Despite the fact
that a similar percentage of the group had a low gastric hydrochloric
acid secretion and six had total achlorhydria, this was not correlatable
with the low thiamine excretion. Mills et al.12 have recently shown that
the thiamine requirement of adult rats per gram of food increases greatly
with old age and increasing weight. Since in the aged the caloric require-
ments may be much less than in younger individuals, these would seem
to be findings of great significance. The presumption that in old age the
efficiency of vitamin utilization is greatly curtailed would seem to be of
considerable theoretical interest to the growing field of geriatrics.123

In the extremely young, the situation is somewhat more lucid. In
children from one to ten years there is a gradual increase in caloric
requirement with age, and the B vitamin requirements parallel this.13
This is not to say that extraneous factors may not influence the require-
ment in young individuals and then become insignificant later. Thus,
Schweigert et al.14 found that young mice were much more sensitive to
the influence of protein levels on the niacin requirement than were older
ones. A variety of similar observations will be discussed at later points
in our studies. Suffice it to say that much of our knowledge of B vitamin
requirements has been derived from experiments upon adults, and much
remains to be learned before this can safely be extrapolated to the very
young and the very old.

Influence of Sex. The influence of sex on the B vitamin requirements,
so far as is now known, is exerted by virtue of the known differences in
basal metabolism and caloric intake between the sexes, and the increased
requirement during reproduction. Up to five years, the average caloric
intakes of boys and girls are the same, and this appears true of the B
vitamin requirements also. Subsequently, however, boys have a higher
caloric intake and therefore a higher requirement for B vitamins.13 Here

FACTORS INFLUENCING B VITAMIN REQUIREMENTS 267

again, however, the complete picture is not available. For example, it is
not entirely clear why the administration of higher levels of choline pro-
duces a considerable growth increase in female turkeys at 24 weeks, but
not in females at 10 weeks, nor in male turkeys at all.15 Morris, Palmer,
and Kennedy, in studying the relationship of efficiency of food utilization
to inheritance in rats,16 found that there were appreciable sex differences,
the average female efficiency being 70 per cent greater than that for the
male during the six-week study period. It seems likely, however, that
present advances in our understanding of the effect of the sex hormones
on metabolism in general, and in particular upon protein metabolism,
may ultimately serve to clarify certain of these relationships.

Influence of Occupation. While it is commonly stated that persons
engaged in hard physical labor require an increased amount of vitamins
in their nutrition, there is little to support so sweeping a statement.17
Nevertheless, the effect of work in increasing the caloric requirement
does undoubtedly bring about increased B vitamin requirements (but
probably not increased requirements for other vitamins), although this
increase may not be as great as for the caloric requirement. As con-
trasted with such hard physical labor, Forbes 18 has pointed out that
emotionally tiring work performed by the average industrial worker is
completely unstudied in regard to its effect on B vitamin requirements.
During recent years, as our understanding of nutritional values has
improved, there has been in the United States a tendency for the calorific
value of diets to rise.13 Concurrently there has therefore been an increase
in B vitamin requirements, but there is little to indicate to what extent
the increased pace of life in these same years has influenced our nutri-
tional vitamin requirements.

In occupations involving such extreme physical exertion that perspi-
ration is excessive, there is some possibility that increased vitamin excre-
tion affects the B vitamin requirement. This possibility is considered at
greater length in the paragraphs that follow.

Effects of Climate. Throughout the war years there appeared a variety
of conflicting reports with regard to the effects of climate on vitamin
requirements. Many of these were particularly concerned with the thia-
mine requirement, which probably serves as an excellent criterion for
the problem in question. Since in tropical climates there is a decreased
caloric requirement,19 it might be anticipated that there would be de-
creased B vitamin requirements. Despite this, Mills et at.20' 21 found that
rats have increased thiamine requirements at higher temperatures, and
Sarett and Perlzweig 22 later extended these studies by measuring the
tissue thiamine in rats at different temperatures. Edison, however, found
that rats at 90° F and 70 per cent relative humidity required no more

268 THE BIOCHEMISTR Y OF B VITAMINS

thiamine than those at 72° F and 50 per cent relative humidity (and
perhaps less) for normal growth.23

A solution to this dilemma came with the study of Kline, Friedman
and Nelson,24 wherein the basal diets were thiamine-free and the thiamine
dosage was administered separately. Therefore, thiamine intake was not
a function of food intake, as it had been in the previous studies. The
rats were allowed to develop polyneuritis and then a dose of thiamine
hydrochloride was given and an observation made of the length of time
that protection was afforded. It was found by this method that 6 ^g of
thiamine hydrochloride protected a rat for an average of 8.7 days of 78° F
and 12.4 days at 85° F. In a second experiment a series of ten rats under-
went four successive depletion and treatment periods. The results are
summarized in abbreviated form in Table 9.

Table 9. Effect of Temperature on the Thiamine Requirement of Rats.

Dose of thiamine

Temperature
(°F)

Days of

:riment

Period

hydrochloride (7)

protection

—

8.7
12.4

9.1

11.6

9.9

0-4

It was found, moreover, that with a given daily dietary intake of
thiamine, there was more rapid growth of rats at 90° F than at 78° F.
Earlier workers were unable to arrive at this conclusion because the rats
used were receiving optimal amounts of thiamine, under which conditions
the food intake, and therefore the growth response, were controlled by
the environmental temperature and could not therefore be related to the
thiamine intake or requirement.

Mills et al.25 point out nevertheless that since vitamin intake is nor-
mally a function of the amount of food eaten, their conclusions are still
significant. Extending their data to chicks, they have found the poly-
neuritis threshold level is 1 mg/kg diet at 70° F and 3 mg/kg at 90° F.
At these temperatures no differences were found in the chick require-
ments for folic acid, niacin, pyridoxine, or choline. (The choline require-
ment apparently varies with the temperature for rats.) It is thus
apparent that the previously conflicting evidence regarding the tempera-
ture effect is in reality in no conflict at all, and that the question depends
upon whether the thiamine requirement or the amount actually consumed
is under consideration.

Despite the obvious and acknowledged importance of these findings, it
has been pointed out that man differs from the rat to a considerable
degree in his mechanism for temperature regulation, and that data per-

FACTORS INFLUENCING B VITAMIN REQUIREMENTS 269

fectly valid for fur-bearing animals are not always as significant when
applied to man for this reason. Extensive studies have therefore been
made upon the increased excretion of B vitamins in sweat.

Spector, Hamilton, and Mitchell 2G have found that an increase in
relative humidity from 65 to 92 per cent at 32.2° C produces an increase
in the average net loss of body weight of 176 per cent, but an increase
of 221 per cent in the average dermal excretion of pantothenic acid by
humans. When the temperature was increased from 28.9 to 38.3° C, the
hourly dermal excretion similarly increased from 5.1 to 27.7 tig, and the
total urinary and dermal excretion was increased 11.6 per cent. Johnson,
Mitchell and Hamilton 27 made similar studies on inositol excretion in
sweat, and found that it increased from 27 Atg/hour under comfortable
conditions to 118 itg/hour under hot, moist conditions. The general opinion
at present resulting from these studies is that B vitamin losses in sweat
under tropical conditions are quite small as compared to the normal
urinary output, and that there is thus considerable support for the belief
that tropical climates do not increase B vitamin requirements in humans.

With regard to the other extreme of climate, there has been less actual
study, perhaps because no one has as yet seriously questioned the
obviously increased requirement necessary in cold climates to support
the increased caloric requirement.2711 Johnson and Kark 2S have presented
data showing a linear relation between voluntary caloric intake in men
(American soldiers), and the environmental temperature, the range being
from 92° F and 3100 Calories to -30° F and 4900 Calories! Studies have
shown 29 that a nutritional vitamin intake greater than that normally
required is without effect in enhancing the ability of men to withstand
the harmful effects of repeated exposure to cold climates.

Effects of Pregnancy and Lactation. Extensive studies have been made
of the effects of pregnancy and lactation on vitamin requirements, and
there is no doubt that under these circumstances there is a considerable
increase in the required dietary level of B vitamins. Thus, Williams 30
points out that in Manila the majority of women of the poorer classes
show signs of deficiency during the child-bearing age. In many of these
same regions, moreover, infantile beriberi is widespread. To a large extent
at least this is due to the increased metabolic rate in pregnancy, and to
lactation. Since the total metabolism has been shown to be equal to the
metabolism of the mother plus the metabolism of the fetus, it might be
anticipated that the thiamine requirement under such conditions could
be calculated. There is not at present sufficient evidence to assess the
validity of such a process, however.

Siddell and Mull 31 have made a study of urinary excretion in a group
of 42 pregnant women. Their results are shown in Table 10.

270 THE BIOCHEMISTRY OF B VITAMINS

All the patients in this study seemed quite normal and gave birth to
normal and well-nourished children. Since normal adults on well-balanced
diets containing about 0.86 mg. of thiamine per day excrete more than
200 fig of thiamine per day in the urine,32 it would seem from this study
that there is little justification for the widespread vitamin supple-
mentation of the diets of pregnant women. Beyond this, however, there
is little information on the problem at hand, since the women were all
on ample diets and it is not possible to say how much their food intake
varied during the course of pregnancy.

Table 10. Average 24-hr. Thiamine Excretion (^g) During Pregnancy.

Group I Group II Group III

Trimester Well-balanced Same diet + 0.75 mg Same diet + 1.50 mg

diet only thiamine/day thiamine/day

1 286 428

2 263 620 932

3 249 483 1131

Kennedy and Palmer,33 from studies on sows and on rats on egg-white
diets, have concluded that biotin is needed early in the life of the fetus,
and also later for normal lactation. The existence of a high biotin require-
ment in rapidly growing tissue is not entirely unexpected in view of its
biochemical function (p. 170).

During lactation the B vitamin requirement remains high, since it still
represents the requirement of two or more individuals. When lactation
is possible, but for some reason does not occur, any increased vitamin
requirement is imperceptible. Apparently, the extent of the increased
requirement during pregnancy and lactation is not the same for each
vitamin. Thus, rats normally require about three times as much panto-
thenic acid as pyridoxine, but during lactation the ratio increases to six
times or higher.34

A number of factors work to raise the requirements during lactation,
chief among these being the fact that the physiological requirement of
the mother is now logically an amount such as will provide a sufficient
level in the milk to nourish the infant. It is quite clear that the thiamine
level in the milk is largely dependent upon the amount in the diet (p. 347) .
Therefore, a large increase in dietary intake might conceivably be neces-
sary to provide a sufficiently high level in milk. Rats are stated to require
five times the maintenance amount of thiamine to nurse a litter success-
fully.35, 36 For these reasons the study of increased B vitamin require-
ments during lactation received quite early attention,37 although little
scientific elucidation occurred until recent years.

Roderuck et aL3S have recently made excellent studies of the thiamine
and riboflavin requirements of humans during lactation. Fourteen women
were studied for an extended period during which analyses were made

FACTORS INFLUENCING B VITAMIN REQUIREMENTS 271

of duplicate meals, of 24-hour collections of urine, and of fasting one-
hour samples of urine. During the period of lactation the thiamine intake
varied from 0.73 to 1.59 mg/day and the riboflavin intake from 2.2 to
3.6 mg/day. Sample data from one individual in which the average
thiamine intake was 1.11 mg/day and the average daily riboflavin intake
was 2.95 mg are given in Table 11.

Table 11. Average Daily Secretion of Thiamine and Riboflavin in Hvn.an Milk.

Days

Thiamine

Total thiamine

Riboflavin

Total riboflavin

post partum

in milk

secretion in milk

in milk

secretion in

(nig %)

(mg)

(mg %)

milk (mg)

0.002

0.014

0.033

0.26

0.005

0.086

0.040

0.72

0.015

0.126

0.048

0.41

162

0.012

0.112

0.042

0.38

239

0.013

0.089

0.043

0.29

302

0.016

0.062

0.046

0.18

Total thiamine secretion in milk was from 2 to 12 per cent of the
intake, and total riboflavin, 6 to 32 per cent. For all subjects, the maxi-
mum daily thiamine secreted in milk never exceeded 15 per cent of the
intake and varied in urine from 2 to 57 per cent of the intake. Assuming
that there is no waste of thiamine in the production of milk by mammary
tissue, and since there was no indication of an avitaminosis in the sub-
jects, the authors conclude that a 15 per cent increase in the dietary
supply of the requirements of normal women supplied any increased
requirements due to lactation. On this basis the National Research
Council's recommendations of 3 mg of riboflavin per day and 2 mg of
thiamine per day on a 3000-Calorie basis seem adequate, and the absolute
requirement is obviously much lower. The National Research Council
also suggests 20 mg per day as a suitable nicotinic acid intake under
similar conditions. Their recommendations for lactation all involve an
increase of from one-half to one-third of the level (on a 3000-Calorie
basis) for a very active normal woman.

Pathological States of the Body. It is readily apparent that a large
variety of pathological situations might arise which could hamper the
efficient utilization by the organism of an otherwise adequate B vitamin
supply. Some of these which occur with sufficient frequency to permit
their recognition as distinct clinical entities are discussed in later chapters
as deficiency conditions (Chap. VI C) . Many other clinical and acute
pathological conditions, however, appear only from time to time, and yet
effectively cause a distinct rise in B vitamin requirements. While such
conditions are of major importance in hospital dietetics, there is not at
present sufficient information to permit rational treatment in these cases.
Largely because of this, "shot-gun vitamin therapy" has become a rather
standardized procedure.

272 THE BIOCHEMISTRY OF B VITAMINS

Perhaps of the most striking interest in recent years has been the
study of B vitamin requirements in severe injury. Most of such studies
have shown, however, only that increased vitamin intakes produce some
type of beneficial result. Thus, Govier and Greer 39 found that in dogs
with hemorrhage-induced shock, although all eventually succumbed, the
average survival time of thiamine-treated controls was 2.4 times that
of an untreated group. They found that the elevated pyruvic acid (4.5
mg per cent),* blood sugar and blood lactic acid levels occurring in
shock were all returned to normal by thiamine administration. Moreover,
a high plasma thiamine level seems to add to the resistance to the onset
of shock.40 Finally, cocarboxylase is dephosphorylated in shock, but is
apparently resynthesized as a result of thiamine administration.41, 42 In
these cases, as Govier points out, a thiamine deficiency in a sense may
exist even though there is an ample thiamine supply, since the thiamine
is converted to a metabolically useless form. Greig 43 has shown that this
same situation prevails in anoxia, and that similar breakdowns of co-
enzyme I and flavin adenine dinucleotide may occur in these cases. Thus,
the requirements under such acute pathological conditions are obviously
considerably elevated at least for thiamine, riboflavin and niocin.

These observations have been confirmed to some extent in humans.
Andreae, Schenker and Browne 43 studied riboflavin excretion in 23 cases
of burns and injuries. Among healthy controls about one-half of a 5-mg
oral dose of riboflavin was retained, whereas patients with acute injuries
showed a much higher retention for 3 to 5 days immediately following
the injury. Subsequently there was abnormally high riboflavin excretion,
followed by a return to normal at about ten days following the injury.
One might suppose that the vitamin retained during the first period (or
that formed from the coenzyme decomposition) was not further used,
but stored in some unusual manner for a period and then excreted. Leven-
son et al.45 studied six patients with severe, acute surgical conditions and
found similar abnormalities in thiamine, riboflavin and nicotinic acid
metabolism. All this work seems to accord well with the known facts
regarding coenzyme breakdown 46 (p. 352) .

There has been a variety of reports regarding the effects of gastro-
intestinal disturbances and surgery upon B vitamin requirements. In a
typical case, signs of pellagra were observed some four weeks after
stomach resection. These disappeared on thiamine and nicotinic acid
therapy. In another case, polyneuritis attendant upon adhesions of the
omentum was cured by relaparotomy followed by thiamine therapy.47
Little definite information is available regarding the basis for such effects.

The effects of endemic disease on the B vitamin requirements are vir-

* Normal, 1-2 mg per cent.

FACTORS INFLUENCING B VITAMIN REQUIREMENTS 273

tually unstudied and present a field of real interest for obvious reasons.
Other aspects of disease and its interrelationships with B vitamins are
more fully presented in Chapter VI C. At present it is possible to do little
more than recognize the existence of a vast and unstudied group of so-
called "conditioned nutritional deficiencies," wherein pathological con-
ditions have raised individual B vitamin requirements.

It has previously been mentioned (p. 247) that the thiamine require-
ment varies with the metabolic rate of an individual. Under conditions
in which there is an augmented metabolism (hyperthyroidism, prolonged
fever) the requirement is therefore notably increased. Details as to the
extent of this increase are lacking, however.

Inherent Individual Variations in B Vitamin Requirements. When
finally we consider the existence of the many factors which go to delineate
the vitamin requirements of a species and a strain under highly defined
conditions, we are still faced with the indisputable fact that individual
animals, and indeed litter mates, differ from each other in many ways,
among which are their B vitamin requirements. R. J. Williams, in a more
extended discussion of the aspects of individual variability in metabolic
patterns, has pointed out the extreme importance of such variation. He
states:48

"It would be presumed on the basis of what we know about the inherit-
ance of enzyme catalysts and the heritability of vitamin requirements in
animals that the requirements for each vitamin would be inherited as a
separate unit. A requirement for one vitamin might be very high, for
another it might be low, and for a third it might be about average, and
so on. The inheritance of individual vitamin requirements, which is closely
akin to the inheritance of enzyme catalysts, does not rule out the fact
that environmental conditions such as infectious disease may alter re-
quirements and make for variation, though information on this point is
largely lacking. . . ."

"On the basis of what we know about the requirements of animals it is
safe to assume that individual human beings differ widely from one
another in the amounts of different vitamins that they require. It is not
at all improbable that specific individuals may have requirements for
certain vitamins which are several times those of their associates. These
differences may be due to relative failure to digest or assimilate, in-
creased tendency to excrete, a failure in the ability to build the vitamins
into the tissues, or to other reasons."

"As I have said, information on variation in vitamin requirements is
largely lacking; those who have been investigating vitamins in nutrition
have not been interested in possible individual differences but have been
pleased if they could get information about the average man, and have

274

THE BIOCHEMISTRY OF B VITAMINS

been content to neglect the exceptional individual whose performances
are out of line. Even information regarding the average man has been
difficult to obtain."

"Casual information suggesting individual variability in vitamin re-
quirements is readily available. Probably every doctor who deals in his
practice with vitamin requirements could cite cases of unusual benefits
from vitamins, or cases in which administration of a vitamin was effective
in one case and wholly ineffective in another."

Figure 1.

.6 .8 1.

REQUIREMENT

1.6

1.2 1.4

IN MC/OAY

Distribution of the daily thiamine requirement among fifteen individuals.

Actual cases of individual variations might be enumerated in great
length. In some, the variability may be extreme. For instance, even when
all presently recognized vitamins are added to the diets of chicks it is
known that a certain small percentage of them (about 2 per cent) still
develop perosis.49 Bloomfield 50 found great variation in the weight losses
among rats on deficiency diets, and found that after recovery, the same
individuals again lose the most weight on a second test on the deficiency
diet.

Berryman et al.51 have come across the same problem in B vitamin
excretion studies, finding that different human individuals, even under
the best controlled conditions, may still excrete widely varying amounts

FACTORS INFLUENCING B VITAMIN REQUIREMENTS 275

of vitamins. In the study of Denko et alP on the B vitamin excretion of a
number of individuals, individual differences are readily apparent, and
the similarities in excretion levels between a pair of brothers are most
noteworthy in suggesting the genetic basis which must exist to explain
individual variations (p. 369).

Pett has considered this aspect of the vitamin requirements at great
length and has emphasized the great danger of taking the average, mean,
or modal value for a dietary standard.53 He points out that when such a
standard is taken, a distribution curve for this figure should be available.
In line with this suggestion he has presented such a curve for thiamine
prepared from data in the literature.54 These data are indicated in Fig-
ure 1. Unfortunately, however, no one has apparently as yet endeavored
to prepare more accurate data of this kind for thiamine or for the other
members of the B vitamin group.

Factors Involving the Nature of the B Vitamin Nutritional Supply

For a given individual the amount of a B vitamin which may be re-
quired nutritionally varies considerably according to the efficiency with
which the vitamin actually present in the food may be utilized. Thus,
other components of the diet or circumstances attendant upon dietary
habit may influence the requirement. It has also been found that when
more than one form of a given vitamin exists in nature, the relative
potencies of these forms for various species may not be the same, and
some forms may be completely unavailable due to their occurrence in
"bound" states which cannot be utilized by the animal in question. Fur-
ther specific inhibitions and inactivations exist which in some cases pre-
vent utilization of the vitamin. All these things effectively influence the
amount of the vitamin which must be present in the diet to meet the needs
of the organism, and they must therefore be considered in some detail.
Still other factors conditioning the requirement, such as the effect of the
state of nutrition on the proportion of vitamin assimilated, are at present
too poorly understood to provide more than passing recognition of their
existence.

Effect of Other Nutritional Components. The ways in which other
components of the diet may influence the B vitamin requirement are
varied and only a few interrelationships are well understood. In some
cases it is quite clear that a given B vitamin is involved in the metabolism
of some particular nutritional component and that the amount of that
component consumed will directly influence the vitamin requirement. In
other cases, certain species are able to utilize biosynthetic precursors of
the vitamin so as to increase their supply. In many instances it is not
clear whether the animal itself or symbiotic organisms bring about the

276 THE BIOCHEMISTRY OF B VITAMINS

particular effect. Conflicting reports exist in many cases, making a thor-
ough understanding of many relationships impossible, at least for the
present. The discussion which follows then is of necessity a brief one,
intended merely to point out the major associations of this type which are
recognized at present.

Effect of Carbohydrates and the Calorific Intake. Thiamine functions
in the form of thiamine pyrophosphate (cocarboxylase) in the metabolism
of carbohydrates (the decarboxylation of pyruvate and ketoglutarate)
(p. 158) . For this reason the thiamine requirement is determined almost
completely by the carbohydrate intake. It is apparently immaterial to
the requirement whether the carbohydrate is used immediately for energy
or converted to fat.55 Because of this, the thiamine requirement is fre-
quently expressed in terms of thiamine per Calorie, or even better per
"non-fat Calorie." Fat, and to a lesser extent protein, by displacing car-
bohydrate from the diet, exert a "sparing action" on the thiamine re-
quirement.

Dann 56 has maintained rats for a period of a year on a substantially
thiamine-free diet containing 80 per cent protein and no carbohydrate.
At the end of this period, the animals appeared to be in excellent condi-
tion although they had not grown rapidly. In this case, it would appear
that intestinal synthesis by symbionts was ample to meet most require-
ments on the carbohydrate-free diet.

Thiamine is not required in the immediate metabolism of ethyl alcohol,
although it was long believed otherwise; and thiamine deficiencies in
alcoholics are believed to be due to low intakes and possibly poor assimi-
lation. Lowry et al.57 found in fact that alcohol delayed the symptoms of
an acute thiamine deficiency in rats on a deficiency diet, when it either
supplemented or replaced part of the carbohydrate of the diet. Westerfeld
and Doisy,58 in similar studies with pigeons, found that either alcohol or
fat had a thiamine-sparing action, 16 grams of fat being equivalent in
this regard to 9 grams of alcohol. It therefore seems well established that
the isocaloric substitution of alcohol or fat for carbohydrate decreases
the thiamine requirement. Recent studies have also shown that replace-
ment of the dietary carbohydrate on an isocaloric basis with ethyl alcohol
results in an increased excretion of thiamine, N'-methylnicotinamide, and
pyridoxic acid, but not pantothenic or folic acid.59

High caloric diets also apparently create an increased folic acid require-
ment, and this adds to the many other difficulties inherent in assessing
the foli'c acid requirements of the chick (p. 248) . Luckey and co-workers
further found the nature of the diet to be a major factor.60 Diets high
in fat or in which the only carbohydrate was glucose, sucrose or starch
necessitated a much higher level of folic acid than did cornmeal and

FACTORS INFLUENCING B VITAMIN REQUIREMENTS 277

dextrin diets or high protein -low fat diets. In experiments with rats it has
also been found that diets containing dextrin as the carbohydrate require
less pyridoxine than diets in which sucrose is the carbohydrate com-
ponent.01

Effect of Proteins and Amino Acids. Much attention has been drawn
to the effects of protein levels on the riboflavin requirement. Reference
has previously been made to the studies of Czaczkes and Guggenheim 62
on the correlation of the dietary requirements of rats for riboflavin with
tissue and urinary riboflavin levels. In this study it was found that the
fat or protein content of the diet profoundly affected the requirement.
These results are summarized in Table 12. It is apparent that at least
part of this variation is due to variation in bacterial synthesis in the
gut, and it seems doubtful whether increased protein increases the ribo-
flavin requirement beyond this. Trufanov 63 has found that in rats on a
low protein diet the riboflavin content of the urine increases, while that
of liver and muscle decreases. He claims moreover that there is no further
synthesis of flavin-adenine-dinucleotide in the liver and tissues after fifty
days on this diet. There is little apparent relationship between these data
and the observation that thiamine and riboflavin tend to counteract the
degenerative changes wrought upon the liver and spleen of experimental
animals by protein-free diets.64

Table 12. The Effect of Protein on the Riboflavin Requirement of the Rat.

% of Calories as Daily Riboflavin Riboflavin

Diet ; — Riboflavin _ Level Level Remarks

Fat Protein Excretion in Organs in Feces

Standard 20% 20%

High-fat 40% 20% diminished diminished diminished Requires two times

amount of ribofla-
vin required to
maintain normal
level in organs.

Low-fat 2% 20% increased increased increased Requires half of

normal amount.

High-protein 20% 34% diminished diminished diminished Requires twice nor-
mal amount.

Low-protein 20% 11% no change no change no change Lack ability to store

riboflavin.

Our present understanding of the function of vitamin B6 in amino acid
metabolism * would lead one to predict that an increased dietary protein
level would increase the vitamin B6 requirement and this is found to be
the case. Thus, in mice on a vitamin B6-deficient 50 per cent casein diet,
the liver vitamin Bc after 3 weeks was 0.97 /ug/gm, whereas in a similar
series on 10 per cent casein, the level was 3.7 /^g/gm and reached the low

* Lyman's studies of bacteria are particularly interesting in this regard.65 For
instance, with pyridoxine present, L. arabinosus loses its nutritional requirements for
threonine, lysine, and alanine.

278 THE BIOCHEMISTRY OF B VITAMINS

level previously mentioned only after 12 weeks.14 Still more direct evi-
dence was produced by Morgan et al.,6G who were able to produce a
vitamin B6 deficiency in dogs on a 45.8 per cent casein diet in 79 to 123
days, but no deficiency on an 18 per cent casein diet for 169 to 190 days.

Pantothenic acid is believed to function in the metabolism of carbo-
hydrates and more particularly in acetylation processes, and one might
expect that it might be less required in diets in which the main energy
component was protein. This expectation may be further strengthened by
the fact that amino acids function as precursors for both the /^-alanine
and pantoic acid moieties of pantothenic acid, and may thus encourage
synthesis in the intestine. Nelson and Evans 67 have recently shown that
rats raised on a pantothenic acid-deficient diet fare much better on a
high-protein diet (64 per cent casein) than on a lower one (24 per cent
casein) .

The nutritional requirement for choline is greatly affected by the
protein intake of the diet, largely by virtue of the interrelationship
between choline and the amino acids serine and methionine. The precursor
function of serine for choline is discussed elsewhere (p. 89) in relation-
ship to the general cellular metabolism. The reciprocal relationships of
choline and methionine for growth and lipotropism 68 are most frequently
encountered in animal nutrition, however. Choline functions in the trans-
port of fats, and in the absence of an adequate supply a variety of symp-
toms, including a neutral fat type fatty liver, may develop. Methionine
may replace choline in the diet inasmuch as it serves as a source of methyl
groups for choline synthesis in vivo. Thus, in the presence of ample
serine, rats may grow at a normal rate (41.8 gms/21 days) and have
normal liver lipides (8.9 mg per cent) if the diet contains 1200 mg per
cent of methionine.68 In the presence of 500 mg per cent of methionine,
however, the growth rate is only 23.8 gms/21 days and liver lipides are
24.7 mg per cent. Addition to this diet of 100 mg per cent of choline
restores liver lipides to normal but does not improve the growth rate.
These data taken from Treadwell's study are summarized in Table 13,
and are interpreted as meaning that on a choline-free diet, the methionine
requirement is 1200 mg per cent, of which about half is required for
lipotropism (i.e., choline synthesis) and half for growth.

However, it should also be noted that starch, or some impurity therein
other than choline, has an appreciable effect in preventing the develop-
ment of the hemorrhagic kidneys found in young white rats on a choline-
deficient diet.69- 70

For chick growth, a very similar relationship holds, as it does in a
variety of other animals. McKittrick 71 has found that the essential
choline (the limit beyond which reduction of choline cannot proceed with-

FACTORS INFLUENCING B VITAMIN REQUIREMENTS 279

out reducing growth below optimum levels, regardless of the methionine
present) was about 0.1 per cent of the diet (containing 0.55 per cent
L-cystine) and the essential level of methionine was 0.5 per cent of the
diet.

Thus, it seems generally true that the choline requirement is quite
dependent upon methionine and serine. A further discussion of these inter-
relationships is found in a later chapter.

Table 13.

Effect of Cystine and Methionine

on the Choline Requirement.

Choline

Methionine

Cystine

% Gain in

Liver Lipides

g/100 gm

mg/100 gm

weight in

gm/100 gm

diet

21 days

moist tissue

500

100

' 23.8

24.7

600

100

37.0

20.5

700

100

38.3

18.8

1000

100

43.3

14.9

1200

100

41.8

8.9

500

100

23.8

24.7

500

200

26.4

24.3

500

300

29.3

26.1

500

400

30.6

21.9

500

600

26.5

16.1

500

100

23.8

24.7

100

500

100

20.2

7.0

200

500

100

27.2

6.6

Effect of Tryptophan on the Nicotinic Acid Requirement. While in
reality a subject belonging to the discussion of the previous section, the
effect of tryptophan on the nicotinic acid requirement is of such far-
reaching importance that it merits entirely separate treatment. For the
sake of clarity of discussion, however, many of the aspects of the closely
associated effect of corn on the nicotinic acid requirement will be treated
in greater detail in a later section dealing specifically with inhibitory
effects.

Despite the fact that by 1938 the identity of nicotinic acid as the
pellagra-preventive vitamin was well established, it was still apparent
that other factors were involved in the etiology of this condition. Aykroyd
and Swaminathan 72 observed that in Moldavia the staple corn diet sup-
plied 15 mg of nicotinic acid daily and that there was endemic pellagra,
whereas in southern India the 5 mg/day of nicotinic acid derived from
rice diets produced only rare cases. A variety of similar observations led
to the conclusion that corn in some manner antagonized the utilization
of nicotinic acid. Krehl et al.73 showed, moreover, that in the rat, which
normally is able to synthesize its own supply of nicotinic acid, corn grits
almost completely prevented growth, but that this effect was entirely
reversed by the addition of 1 mg per cent of nicotinic acid to the diet.
Moreover, the nicotinic acid requirement of dogs on purified diets con-

280 THE BIOCHEMISTRY OF B VITAMINS

taining corn grits was tripled.74 Wintrobe 75 found a similar relationship
in young pigs.

In Krehl's study it was found that -raising the casein in the basal diet
from a level of 15 per cent to a level of 20 per cent also prevented the
antagonism due to corn. Both lysine and tryptophan occur in low concen-
trations in corn but in higher levels in casein; and Krehl et a/.7G found
that whereas lysine was not effective, 0.05 per cent of L-tryptophan was
as effective in reversing the effect of corn as was nicotinic acid itself. At
the time, this was believed to be due to intestinal synthesis of nicotinic
acid by bacteria, which were believed unable to grow on corn diets due
to inadequate tryptophan.. It was also found 77 that in diets in which
wheat gluten or gelatin (which are lower in tryptophan) were used as the
protein supplement, poor growth ensued which was cured by either trypto-
phan or nicotinic acid. When egg albumin, fibrin or soybean globulin were
used as the protein components 7S it was found that no inhibition occurred
when corn grits were added to the diet, and that this could be explained
on the basis of the high tryptophan content of these protein materials.
Thus, while an antagonist effect of corn was not ruled out, its low trypto-
phan content was at least in part concerned with the nicotinic acid-
deficiencies observed.

Singal and co-workers 79 found that there was an increased urinary
excretion of niacin and its metabolic products in rats when tryptophan
was added to the corn grit diet. Rosen, Huff, and Perlzweig 80 similarly
observed that 50 mg of dl- or L-tryptophan either given orally or sub-
cutaneously produced a five- to tenfold increase in nicotinic acid excretion
over the level excreted on 15 per cent casein diets. Moreover, there was
a relative constancy of fecal nicotinic acid whether or not tryptophan
was administered. From these observations it became readily apparent
that the tryptophan effect was not due to intestinal synthesis, and that
tryptophan must therefore be involved in nicotinic acid metabolism, prob-
ably as a precursor. This, moreover, seemed more in line with the observa-
tion that, when 4 mg/day of 3-pyridylmethylketone, a structural
analogue of nicotinic acid, was fed to mice on niacin-free diets, 11 out of
12 died within 10 days, but that this toxic action was prevented by both
nicotinic acid and tryptophan.81 This work was based on the well estab-
lished theory of the interference by structural analogues with the opera-
tion of a metabolite through saturation of enzymes involved in the
metabolism of the latter substance, and reversal of such toxic action by
precursors or products of the metabolite. It led naturally to the re-
emphasis of a "pellagragenic" agent in corn, which acts in a manner
similar to that of 3-pyridylmethylketone (Chap. VI D). More recently,
work with Neurospora mutants 82 makes it seem certain that the effect

FACTORS INFLUENCING B VITAMIN REQUIREMENTS 281

of tryptophan is as a metabolic precursor of nicotinic acid. The mode of
conversion is by no means known at this time, however, nor is there
unequivocal reason to select the indole-nitrogen over the a-amino-nitrogen
as the precursor of that nitrogen atom which occurs in niacin (see p. 356) .

Recent studies have indicated that the tryptophan-nicotinic acid rela-
tionship holds with a variety of other species. The lack of growth of
guinea pigs on a corn-soybean oil meal-alfalfa ration is reversed by
nicotinic acid or tryptophan.83 Chicks on a 10 per cent gelatin diet exhibit
depressed growth rates which are corrected by 5 mg of nicotinic acid or
200 mg of DL-tryptophan per 100 gm of diet.84 Krehl has found 77 that
for rats 1 to 1.5 mg of nicotinic acid are equivalent to 50 mg of tryptophan
per 100 gm of diet. Monkeys, however, develop a deficiency on such diets,
which is corrected by neither nicotinic acid nor tryptophan, nor both, but
by liver powder or by lyophilized liver.85 Various other workers have also
recently produced a variety of evidence to show that man does actually
convert tryptophan into nicotinic acid8687 (see p. 354). Present indica-
tions are that neither indole, anthranilic acid, nor indoleacetic acid may
serve in lieu of tryptophan as a nicotinic acid precursor.88 Additional
data bearing on the problem of the conversion of tryptophan to nicotinic
acid will be found in the discussion of nicotinic acid metabolism in Chap-
ters V A and IV C. Without further consideration it seems fair to assume
that, for most higher animals at least, the nicotinic acid requirement can
be stated only in the light of some estimate of the tryptophan intake.
Frazier and Friedemann 89 have shown that on corn-free diets the human
requirement may be as low as 4 mg/day of nicotinic acid, but that this
is increased to about 5 mg/day by corn when there is a high vitamin and
protein level, and to 7.5 mg/day when vitamin and protein levels are
lower.

Of considerable interest is the recent report 90 that bacteriologically
sterile Drosophila melanogaster require both tryptophan and niacin, and
that the niacin requirement is increased by higher tryptophan levels in
the diet. This indicates that there may be wide variation in the animal
kingdom with regard to this interrelationship, and to some extent tends to
suggest the theory that there is at least contributory intestinal synthesis
of nicotinic acid by bacteria. Other recent work showing that sulfonamides
decrease the excretion of nicotinic acid metabolites 91 has further brought
this earlier hypothesis into prominence again. Nutritional studies with
"germ-free animals" (p. 300) may do much to resolve this dilemma, and
it seems probable that both factors may eventually be shown to be
involved.

Vitamin Interrelationships. Strangely enough, little is as yet known
about the effects of vitamins on the requirements of other vitamins. As

282 THE BIOCHEMISTRY OF B VITAMINS

concerns the fat-soluble group, it has been reported that the thiamine
requirement of the rat is considerably increased (as indicated by loading
tests) in vitamin A deficiency, and that large doses of thiamine delay the
appearance of symptoms of avitaminosis A.92

While rats are normally not susceptible to nicotinic acid deficiency, it
has been reported that a multiple deficiency of nicotinic acid, pantothenic
acid, and p-aminobenzoic acid produces a syndrome similar to that of
pellagra. While all these factors are required for its cure,93 nicotinic acid
alone can delay the symptoms. There is at present a rapidly increasing
body of evidence which indicates that nicotinic acid and folic acid are
intimately associated in some manner in the cure of the symptoms of
nicotinic acid deficiency.94 This problem is more appropriately discussed
in later chapters (pp. 408 and 412) .

There is at present little explanation for the observation that thiamine
and cholic acid substitute for pantothenic acid in the Hall strain of
Clostridium botulinum Type A.95

Nutritional Customs, Habits, and Taboos. Of great practical concern
to the nutritionist is the problem of dietary habits and their effect on
nutritional requirements. Generally the effect of such habits is exerted
through obvious means (excessive carbohydrate intake, high protein diets,
high raw egg intake, etc.), but subtle factors may also be at work which
may influence dietary calculations to a considerable degree. Thus even
drinking water may contain appreciable quantities of B vitamins, and the
consumption of excessive quantities of coffee with its relatively high
trigonellin content might certainly influence the nicotinic acid requirement
(p. 288) . The habit of the Mexican natives of consuming beans with their
tortillas undoubtedly prevents widespread pellagra in Mexico. Such fac-
tors should then influence the calculation of adequate dietary require-
ments as much as they influence the adequacy of the diet itself. While it
is necessary to abbreviate what might well be an extended discussion on
this point, it is none the less important to emphasize the fundamental
nature of sociological aspects in assessing nutritional requirements.

The Processing of Foods. Whereas the vitamin requirements of an
individual might well be met on the basis of the native vitamin content
of the diet, the situation may well be otherwise by the time the dietary
components have been marketed, stored, canned, cooked, baked, or other-
wise modified by circumstances of time, temperature, and chemical treat-
ment. Losses of B vitamins in food preparation vary widely with the
food and the precise methods involved; there have been a large number
of studies and publications on this subject. The topic, however, does not
fall sufficiently within the realm of this monograph to merit a detailed
discussion. Moreover, the ready availability of extensive data on this

FACTORS INFLUENCING B VITAMIN REQUIREMENTS 283

topic further lessens the expediency of a consideration of it here. It should
be emphasized in passing, however, that the topic is of extreme impor-
tance in estimating the B vitamin requirements of any population, and
must be weighed carefully in evaluating the adequacy of diets and of new
processes in food technology.

Biological Potency and Availability. It frequently occurs among the
B vitamins that one vitamin may have a number of structural forms, the
biological activities of which differ for a given species, and between dif-
ferent species. The requirement for a B vitamin in a given animal cannot
be met by a form that is without activity for that animal, regardless of
how high its activity may be for another. It is necessary to insure that in
the diet there is an adequate amount of B vitamin analogues which are
active for the species in question, if the requirement is to be met. For this
reason, the nutritional requirement cannot be stated accurately in terms
of, say, vitamin B6, since vitamin B(i has a number of active forms with
different biological potencies. It is therefore necessary to consider, as one
aspect of the problem of assessing the nutritional requirements, what the
various naturally occurring forms of the B vitamins are, and how they
compare with each other as regards their biological activity.

Beyond this, it has been found in recent years that certain forms of
some B vitamins are not available to animals for use; indeed the
problem of the availability of vitamins, even when they are present in
otherwise adequate amounts, is one worthy of considerable attention.

Human Bioassay Techniques. While it is generally possible to deter-
mine the vitamin requirements of lower animals and the potencies of
various diets or vitamin derivatives for any given species by a suitable
direct approach, it is seldom possible to employ such direct methods with
man himself. Laboratory depletion studies upon members of the human
race are relatively rare,06 and indirect methods must generally be used.
These have largely employed studies of vitamin excretion.

Melnick et al.97 have applied such studies to a number of problems.
Generally the assay involves suitably sized groups of about five individ-
uals on diets containing ample vitamins for their requirements. Twenty-
four-hour samples of urine are collected and the basal level of vitamins
determined. Immediately thereafter a known dose of vitamin is admin-
istered, and the percentage of this dose recovered in the urine is observed.
When the test dose is then administered, it is assumed that the percentage
recovery in the urine is similar, and the potency of the test material can
presumably thus be calculated. Despite the fact that there are large
individual variations in basal levels, control experiments are reported to
indicate an accuracy generally as good as that obtained in other animal
assays. In view of the various factors discussed previously in regard to

284 THE BIOCHEMISTRY OF B VITAMINS

excretion studies, it is apparent that such an approach must involve sev-
eral assumptions {e.g., that the excretion is a function of the intake) and
techniques {e.g., eventual testing of the excreted products by nonhuman
means) which leave much to be desired. Nevertheless, when the conditions
are adequately controlled, considerable valuable information may be so
obtained, and extended further studies of this nature seem to be urgently
required. The application of such human bioassay techniques to the study
of the availability of thiamine in yeast98-99 (p. 291) and its enzymatic
destruction 10° (p. 292) , have indeed opened a valuable new approach in
the science of nutrition.

The Relative Potencies of the Various Naturally Occurring Forms of
the B Vitamins. It seems most probable that much remains to be dis-
covered concerning the variety of naturally occurring substances possess-
ing B vitamin activity. This is largely due to the great difficulties inherent
in the separation of minute quantities of structurally similar compounds.
Recently techniques employing a combination of paper partition chroma-
tography 101 and plate growth of assay organisms 102 have been developed,
and these show promise of adding to our knowledge of nutrilite derivatives
in general. For the present, however, the discussion which follows must
be considered in terms of the probability of the existence in nature of a
far greater number of B vitamin isotels.103

The discussion of these various vitamin forms might well be undertaken
in a pedagogic fashion from the standpoint of their relationship to the
generally accepted form(s) of the vitamin, but it is certainly worthwhile
to point out that other rather obvious categories of isotels do exist. Thus
we have the immediate vitamin structures such as the B6 "triad" and the
folic acid "triad." Secondly, bound forms, available and unavailable, are
known for most of the B vitamins. Vitamin precursors frequently show
vitamin activity as in the case of the precursors of choline and nicotinic
acid, and the functional forms, frequently coenzymes, are generally active.
Catabolic products of vitamin metabolism also show activity for some
species. Finally, there is a large group of biologically active substances
which seem to have little apparent structural relationship to the B vita-
mins themselves, although one might theorize extensively on their activity.
For practical purposes, however, it has been deemed advisable to consider
the various known analogues in relation to each of the individual vita-
mins, and thus in a more systematic, though less erudite fashion.

(1) Thiamine

Thiamine may occur in free and combined forms (p. 30), and little
is known of the nature of bound thiamine. It is apparent however that
various animal species may utilize some, but not all, of these bound

FACTORS INFLUENCING B VITAMIN REQUIREMENTS 285

forms. Thiamine also occurs in the form of the pyrophosphate (cocar-
boxylase), but the relative potency of this form for living things other
than bacteria is apparently unknown. (Certain strains of Neisseria gonor-
rheae even require this form for growth, and thiamine itself is not only
inactive, but inhibits growth, being competitively reversed in its action
by the pyrophosphate.) 104 Certain protozoa at least, and perhaps a few
higher animals, are able to use either the thiazole or the pyrimidine frac-
tion of thiamine for growth,105 so that in a general sense, the presence
of these substances and their activity must be considered in the nutrition
of members of phyla below the chordates.

A thiol form of thiamine, and its oxidized disulfide form (and their
pyrophosphates), are believed to exist,106 and both thiamine and cocar-
boxylase disulfide have been shown to be active in the catatorulin test
with deficient pigeon brain (p. 51). The presence of these forms must
be considered, if they are found to have biological activity for any species
under consideration.

Finally, Polonovski et al.107 recently reported an interesting series of
studies in which a number of natural and synthetic pterins were found
to substitute for thiamine (or riboflavin) in pigeons and rats. Fluores-
cyanine (a fluorescent pigment from the scales of certain fish) was able
to eliminate the symptoms of deficiency in thiamine-deficient rats and
pigeons, and to increase the oxygen uptake and carbon dioxide evolution
from thiamine deficient rat brain. Injection of 50 to 100 /xg/day of a
number of synthetic pterins produced similar effects, and the fact that
oral dosage was similar in effect showed that the result was not due to
intestinal synthesis by microorganisms. Indeed the pterins were found to
be incapable of replacing thiamine for microorganisms. It was also re-
ported that in cecectomized rats 10S xanthopterin, isoxanthopterin, folic
acid, and lumazine could be substituted for thiamine, isoxanthopterin
being the most active. Such results, if verified, suggest the possibility
that the biological and structural specificity of many of the B vitamins
(and other nutritionally active substances) may not be as great as has
been generally presumed heretofore, and that the specificity may rest
rather in the chemical structure and groupings involved in the precise
functions of the vitamin.

(2) Riboflavin

Riboflavin is known to exist in free and combined forms and as the
5-phosphate, in the form of the flavin-adenine-dinucleotide, and in both
oxidized and reduced states (p. 32). The relative potencies and avail-
ability of these forms, however, for various species, are not known, though
it is generally assumed that riboflavin is seldom present in forms in which

286 THE BIOCHEMISTRY OF B VITAMINS

it cannot be used. In the previously mentioned work of Polonovski et al.107
it was found that certain pterins could substitute for riboflavin in pigeons
and rats. Ten jxg per day of fluorescyanine produced a growth rate in
riboflavin deficient rats of 2 gm/day, similar to that produced by ribo-
flavin. A large variety of synthetic riboflavin derivatives have been pre-
pared, and much has been learned in this manner regarding the structural
specificity associated with the riboflavin molecule, but these substances
per se are of little interest in the present discussion.

(3) Nicotinic Acid

There are probably more known nutritionally active substances isotelic
with nicotinic acid than with any of the other B vitamins. Many of these
analogues are known to function in nature either as precursors or products
in nicotinic acid metabolism, and there seems to be considerable variation
in the abilities of various species to utilize these analogues. In general the
activity of nicotinic acid is believed due to its conversion to nicotinic
acid amide and thence to Coenzymes I and II, and consequently sub-
stances that may be readily converted to nicotinic acid or nicotinic acid
amide may be expected to show nicotinic acid activity. Mueller's finding
that nicotinic acid is more potent than nicotinic acid amide for diphtheria
organisms,109 and the discovery by Dorfman et al.110 that nicotinic acid
amide is more potent than Coenzymes I or II for dysentery organisms,
while in discord with this view, do not prove unequivocally that it is
untenable, but rather indicate the fastidious nature of certain cells with
regard to their nutritional source of building blocks for intracellular
coenzymes.

In order to obviate an unduly lengthy discussion regarding the activity
of these derivatives for various species, the available information is sum-
marized in Table 14. The effects of a large number of synthetic pyridine
derivatives which do not occur in nature have been reviewed adequately
in the literature m and in Chapter VI D of this monograph. Much of the
tryptophan-nicotinic acid interrelationship has been worked out with
Neurospora mutants, and for that reason data are included to show the
metabolites which have been found active for some Neurospora mutants.

Few quantitative interrelationships are known. It is generally true that
nicotinic acid and nicotinic acid amide are about equally active, though
exceptions exist, particularly among bacteria. The drug coramine is not
nearly as active as nicotinic acid, but seems to be sufficiently active to be
an effective therapeutic agent in man. /3-Picoline has a low activity, but
the /?-methyl group can apparently be oxidized to niacin. For rats on a
corn diet, Krehl 7T found 1.0-1.5 mg nicotinic acid to be approximately

FACTORS INFLUENCING B VITAMIN REQUIREMENTS 287

equivalent to 50 mg of tryptophan. For chicks, 5 mg of nicotinic acid are
equivalent to about 200 mg of DL-tryptophan,84 and for dogs, 1 gm of
L-tryptophan is equivalent to 5.6-10 mg of nicotinic acid.lllb There are
considerable individual variations in this regard, but in general the overall
efficiency of the conversion seems to be of the order of 1 or 2 per cent.
Only L-tryptophan is effective. Fifty to 100 gm of protein per day would
be required to meet the niacin requirement of man solely through synthesis
from tryptophan on this basis. Since many animals excrete N'-methyl-
nicotinamide (F2), it was thought that some might be able to demethylate
this product to nicotinamide, but to date only divergent and generally
questionable results have been obtained (p. 359).

Table 14. Nicotinic Acid Activity of Some M etabolically Related Compounds.

Compound Man Dog Chick Neurospora

Nicotinic acid

Nicotinamide

Coenzyme I

Coenzyme II

Nicotinuric acid

-(?)

Trigonellin

N'-methylnicotinamide (F2)

±(?)

/3-Picoline

/3-Aminopyridine

Tryptophan

Kynurenine

Anthranilic acid

3-Hydroxyanthranilic acid

Coramine (synthetic drug)
(N,N-diethylnicotinamide)

N-methylnicotinamide

Quinolinic acid1110*

* Active for the rat in increasing F2 excretion and growth. llta

It has been stated as a generalization that animal tissues contain a
preponderance of nicotinamide, and that plant tissues contain a smaller
and more variable amount in this form.112 There is also evidence indicat-
ing that cereals contain a nicotinic acid precursor that is liberated only
upon alkaline extraction. This precursor is apparently unavailable to dogs
or chicks unless liberated by hydrolysis prior to feeding.113-115

The recent isolation of dinicotinylornithine from natural sources pro-

288 THE BIOCHEMISTRY OF B VITAMINS

vides still another niacin derivative which may figure in meeting the
nicotinic acid requirements of man.110

It has been previously mentioned that cooking may modify the nutri-
tional value of a vitamin source. An interesting example of improved
nutrition resulting in this manner is in the case of coffee. Raw coffee con-
tains a preponderance of trigonellin over nicotinic acid, but in the roast-
ing process the trigonellin is largely converted to nicotinic acid, so that a
cup of coffee generally contains from 1 to 2 mg of nicotinic acid.117

(4) The Vitamins B6

The three major forms of vitamin B6 are pyridoxine, pyridoxal, and
pyridoxamine, to which may be added the functional vitamin form, code-
carboxylase, or pyridoxal phosphate, and pyridoxamine phosphate, which
is now known to be a nutritional requirement for certain lactic acid
bacteria. In addition, "bound" forms exist, and there is increasing evi-
dence to suggest the possibility of a variety of vitamin B6-amino acid
complexes (Schiff bases) with biological activity. Finally the ability of
some lactic acid bacteria to employ certain amino acids interchangeably
with vitamin BG may eventually be found to have broader biological
significance.118

In vitamin BG-deficient mice,119 using xanthurenic acid excretion as a
criterion of response, Miller and Bowman found that pyridoxine hydro-
chloride returned the excretion to normal within four days, while pyridoxal
and pyridoxamine required seventeen and twenty-four days respectively.
Growth rates on the three analogues and survival times on lower levels
similarly indicated the same order of activities.

For the rat, it has been shown that the three forms are about equally
active 120 in promoting growth and in restoring the ability to convert
tryptophan to niacin. 120a It has also been reported that for the rat and
chick, all three forms and pyridoxal phosphate are equally active when
fed by eye dropper or injected intraperitoneally,121 although when added
to the ration, pyridoxine appears somewhat more active. The three forms
are also known to be equally active in promoting growth and blood
regeneration in vitamin B6-deficient dogs.122

Although certain studies would suggest the existence of further ana-
logues of vitamin B6 in nature,123 the work of Rabinowitz and Snell 123- 124
indicates strongly that the B6 trilogy, their phosphates, and their protein-
bound forms account for the vitamin B6 content of tissues. Little is as yet
known of the biological activity of the various products of vitamin B6
metabolism in mammals, although 4-pyridoxic acid is known to be inac-
tive for rats, chicks, and all the bacteria so far tested.

FACTORS INFLUENCING B VITAMIN REQUIREMENTS 289

(5) Pantothenic Acid

Pantothenic acid is known to occur in the form of the free acid, as
coenzyme A, in one or more bound forms, and possibly in the form of
conjugates with one or more amino acids (glutamic acid particularly has
been suggested). In addition, certain lower organisms at least can utilize
either the /^-alanine or the pantoyl fraction of the molecule in lieu of
pantothenic acid120,1-7; and an amino acid which has recently been
indicated as occurring naturally has been suggested as a precursor of
pantoic acid, and may have pantothenic acid activity.128 Coenzyme A is
apparently available to animals, but /^-alanine is inactive for at least
rats and chicks, and probably all higher animals. Pantothenyl alcohol, a
synthetic compound presumably not occurring in nature, is reported to be
utilized by humans as readily as pantothenic acid, and may in some cases
be even more effective, perhaps due to its apparently greater stability to
acid.129-132 It cannot be oxidized by bacteria to the acid, however, and so
is inactive in supporting bacterial growth.

(6) Biotin

Whereas biotin may have a large number of stereoisomers, there is little
evidence to indicate that they occur naturally. There are reputed to be,
however, as yet unelucidated avidin uncombinable forms, and bound
forms. The bound forms which occur naturally seem to be readily utilized
by most animals. There is also some evidence for the existence of a biotin
coenzyme which is more active than biotin. 132a O-Heterobiotin (oxybiotin)
which is a synthetic analogue, and desthiobiotin, which may possibly
occur naturally, have however been studied quite extensively and com-
pared to biotin carefully as regards their potency. Other substances with
structures as yet unknown, and some with structures unlike that of biotin
(oleic acid) substitute for biotin in some lower forms of life.133

McCoy et al.134 have shown that DL-oxybiotin is about 17 per cent as
effective as biotin for the chick. It is 25 per cent as active as D-biotin for
Saccharomyces cerevisiae and Lactobacillus casei, and 50 per cent for
L. arabinosus.1*5 It is inactivated by avidin, and its effect for L. casei is
inhibited by desthiobiotin. DL-Oxy biotin prevents chick dermatitis at
levels of about 20 ^g per 100 g diet.130 Using growth and the disappearance
of skin lesions as criteria, DL-oxybiotin is found to be only 4 per cent as
active as D-biotin in curing egg-white injury in the rat.137

Tatum has suggested that desthiobiotin functions in the biosynthesis
of biotin.138 Desthiobiotin is one-twentieth as active as D-biotin in curing
egg-white injury in rats.135 Wright et al. have recently isolated a crystal-
line biotin-protein complex.191

290 THE BIOCHEMISTRY OF B VITAMINS

(7) Folic Acid

The elucidation of the structure and functions of the folic acid group
of compounds has come in recent years, so that comparatively little is
known as to the relative merits of its various analogues. Folic acid is
known to occur in free and bound forms, as the tri- and heptaglutamate,
and also in formylated derivatives. Methods for assessing responses gen-
erally depend upon hematopoietic response, and a variety of substances
have been found to influence such responses (e.g., pyridoxic acids, p. 421).
Even 5-methyl uracil (thymine) in doses of 4.5 gm or more produced
hematological responses in six patients in relapse stages of Addisonian
pernicious anemia,139 which is in line with the suspected function of folic
acid in purine and pyrimidine formation. Indeed, for bacteria at least, the
replacement of the pteridin moiety of folic acid by a quinazoline or a
benzimidazole ring gives compounds with some biological activity.140- 141
Petering et al.14'2 found that a combination of 4-pyridoxic acid and folic
acid was better than folic acid alone in stimulating growth and hemo-
globin formation in deficient chicks, although other workers have been
unable to confirm this. Folic acid and the tri- and heptaglutamate are
all active in curing sprue,143 and are active for monkeys. The mono-
and triglutamate at least are active in nutritional macrocytic anemia,
and folic acid at least is active in treating pernicious anemia. (The hepta-
glutamate has up till now given highly divergent results.144* 145 The
recently isolated vitamin B]2 should also be mentioned at this point as
a substance which in minute quantities is capable of alleviating pernicious
anemia. Both pteroic acid and formylpteroic acid are inactive for L. casei
or humans,140"148 while pteroylheptaglutamate is inactive for bacteria,
but is twice as active as the monoglutamate for Tetrahymena gelii.1*9
Various other possibilities exist for the presence of folic acid activity
in natural sources 149a which are more conveniently discussed in a later
chapter (pp. 413 to 422).

(8) Choline

Because choline is a structural part of such a wide variety of naturally
occurring compounds, its analogues in a sense may be considered to be
numberless. In the phospholipides, however, when digestive hydrolysis
occurs, free choline is doubtless liberated, so that the efficiency of lipide
choline is quite high. This variety of choline derivatives, however, makes
the assessment of choline requirements difficult, since it involves an esti-
mate of the ability of an organism to liberate the choline.150 In addition
to its esters, the various substances involved in choline synthesis are also
active in many cases. Mono- and dimethylethanolamine are active in

FACTORS INFLUENCING B VITAMIN REQUIREMENTS 291

preventing perosis in chicks, for instance, but the mono- derivative will
not promote growth, and the dimethyl compound only does so to the
extent that methionine is present.151 These relationships are discussed at
greater length elsewhere (p. 353).

(9) p-Aminobe?izoic Acid

p-Aminobenzoic acid occurs in both free and combined forms in nature
and as part of the folic acid group of compounds. Little is known beyond
this concerning the variety of its occurrence, or the relative potency of
its forms (p. Ill D).

(10) Inositol

Inositol occurs in nature in the free form, as the hexaphosphoric ester
and its salts, and in certain "cephalins." 152 In addition it occurs in bound
forms that have been as yet unelucidated.153 The majority of these forms
are apparently available to most higher animals, except those cases where
the insoluble calcium or magnesium salts of the phosphate make solution
and liberation of the inositol impossible.

Availability of Thiamine from Yeast. Mention has been made of the
existence of bound and/or unavailable forms of a number of the B vita-
mins. In no case, however, has this fact been so vitally important and so
vividly demonstrated as in the case of the thiamine of yeast. Particularly
is this so because of the extensive use of yeast as a thiamine source.

It has been known for some time that dried yeast is superior to live
yeast as a thiamine source for rats,154 but it was not until recent years
that this was shown to be true for man." In some cases indeed, as little
as 17 per cent of the total thiamine was found to be available for nutri-
tional purposes in humans.97, 9S Recently, it has been shown that live
yeast, when added to a diet containing adequate thiamine,155 decreases
the available thiamine in the diet, and the technique has even been em-
ployed to produce thiamine deficiencies in man. In one experiment, for
instance, five women on a diet containing 1.6 mg per day of thiamine were
fed 15 gm of live baker's yeast, which decreased the average urinary
thiamine level from 374 to 101 fig per day. As little as 150 gm of yeast
was found to depress the excretion to 40 ^g per day.

A variety of studies on this phenomenon have resulted in the conclusion
that the effect is due to the inability of the intestine to absorb the thi-
amine, for mechanical reasons. An inverse relationship exists in such
cases between the fecal and urinary thiamine, and it appears that a large
proportion of the living yeast cells ingested pass through the intestinal
tract without rupturing. These cells apparently have an active thiamine
uptake, and the yeast thiamine, whether free or phosphorylated, does not

292 THE BIOCHEMISTRY OF B VITAMINS

readily diffuse out of the intact cell. In this manner a mechanical barrier
materially influences the availability of the thiamine. Boiling, or other
measures which fracture the yeast cells effectively, destroy the anti-
thiamine effect. In general, the other B vitamins of live yeast appear to
be similarly unavailable for nutritional purposes by virtue of the same
effect. Effects such as these are by no means limited to yeast, and empha-
size the desirability of obtaining information regarding the availability
of the B vitamins in any particular food source for any species dependent
upon that source.155*

Natural Inhibitors and Inactivators

It has been discovered in recent years that there exists in nature a
group of substances which are able to exercise a pronounced effect upon
the nutritional value of B vitamins, either by destroying them, by irre-
versibly binding them, or by competing with them for some enzyme
system involved in the function of the vitamin. Typical of these effects
are the thiaminases, which enzymatically destroy thiamine; avidin, which
tightly binds biotin; and a possible pellagragenic factor in corn, which
competes with nicotinic acid. In addition to these rather clear-cut cases,
a large number of less thoroughly studied examples are known where the
nature of the effect is not as yet clear. The consideration of these various
effects is here undertaken in the order mentioned, to emphasize the type
of mechanism involved, rather than the details of any specific case.

The effects of a large group of synthetic inhibitors are considered in a
later section; the application of the use of inhibitors and inactivators to
the assessment of B vitamin requirements has already received passing
mention (p. 260).

Enzymatic Inactivation of B Vitamins. It was in 1932 on the farm
of J. S. Chastek that a fatal disease of domestic foxes was first observed,
although it was not until some years later, when the affliction had reached
major economic proportions, that it was realized that the fatal symptoms
followed closely upon the incorporation of raw fish into the animal stock
diet. The careful studies of Green et al.156 indicated clearly that the afflic-
tion, which generally terminated fatally in from two to three months,
was probably due to a thiamine-splitting enzyme in the raw fish. This
was soon shown to be so,15T and the thiaminase was found in most un-
cooked fresh water fish and molluscs, and in a few salt-water forms. It
was also found that the enzyme produced similar effects in the diet of
cats 158 and could potentially act in this manner in man. It was also active
in suppressing the growth-promoting effects of thiamine on yeast (En-
domyces vernalis) .159 The enzyme consists of a heat-labile, nondialyzable

FACTORS INFLUENCING B VITAMIN REQUIREMENTS

293

and a heat-stable, dialyzable fraction (possibly manganese), and catalyzes
the overall reaction:

CH3
N=C— NH2 C=C— CH2CH2OH

CH3— C C CH2 ±N

N— CH

ci- c— s

N=C— NH2

-i L

CH3

C=C— CH2— CH2OH

that is,

CH3— C C CH2OH + N

R'CH2N+R3 + H20 =± R'CH2OH + NR3 + HJ

+ H-

The reaction is somewhat unique in that a hydrogen ion is formed in the
process. Studies of the reaction employing enzyme extracts yield unidenti-
fied pyrimidine derivatives, while the pyrimidylmethyl alcohol is only
obtained by using whole tissue suspensions. Whereas a number of o-amino
derivatives of aromatic compounds are able to inhibit this reaction, m-
substituted compounds generally activate it. This activation by substances
such as ?n-nitroaniline and m-aminobenzoic acid is believed to occur by
virtue of a combination between the ra-amino group of the accelerator and
the 5-methylene group of the pyrimidine moiety as it is split off. When
ra-nitroaniline is used as the accelerator (or acceptor) for instance, it is
possible to isolate from the reaction mixture N-(2-methyl-6-amino-
pyrimidyl-5-methyl) -m-nitroaniline. Presumably the enzyme itself cata-
lyzes only the first reaction, a sort of transmethylation in which the
methyl group is substituted with a pyrimidine derivative, and in which
the substituted methyl group is transferred from a quaternary amine to
a primary amine. Subsequent reactions in vivo then hydrolyze the sec-
ondary amine formed to the pyrimidylmethyl alcohol.159* The enzyme is
of additional interest in that it rapidly destroys the thiamine in dead
carp, unless the enzyme is inactivated by immediate heating after death.
Employing known bioassay techniques (p. 283), it has been found that
42 per cent of a test dose of 7.5 mg of thiamine (and 50 per cent of the
thiamine in the basal diet) is destroyed by the consumption of 100 gm.of
raw clams.100 The anti-thiamine activity of a large variety of plant mate-
rials has been shown to be due to nonenzymatic factors, and the "Chastek
factor" remains the principal example of enzymatic B vitamin destruction.
Inactivation of B Vitamins by Binding Agents. In 1916 1G0 Bateman

294 THE BIOCHEMISTRY OF B VITAMINS

made the observation that raw egg white has a toxic effect when incor-
porated into the diet, and in 1927 Boas 161 observed the presence in certain
foods of an organic substance that would protect against this effect. As
the result of extended subsequent researches Eakin et al. found that the
symptoms of egg-white toxicity were due to the presence in egg white of
a protein, avidin,162'164 which is able to bind biotin in a firm complex which
is not readily broken by the usual digestive processes. An enzyme is
present in the blood, however, which can break the combination. Subse-
quently, the feeding of raw egg white has become a standard procedure
in producing the symptoms of biotin deficiency in a wide variety of
animals.165

It has recently been suggested that the retardation of sexual develop-
ment in chickens, which occurs on diets containing otherwise optimal
amounts of vitamins and minerals, but large amounts of whole milk
powder, may be due to a similar avidin-like effect.166

Inhibition of B Vitamin Activity by Competitive Action. For three
centuries preceding the discovery that nicotinic acid deficiency was in-
volved in the etiology of pellagra, it was realized that the affliction was
associated with the use of corn as a major portion of the diet, and it was
suggested from time to time that pellagra was due to some toxic agent
in the corn. This theory completely disappeared for a time when it was
shown that pellagra primarily indicates a nicotinic acid deficiency. It
was soon realized, however, that in consideration of the amounts of
nicotinic acid and tryptophan present in various corn diets, there remained
far too high an incidence of pellagra. Thus on the rice diets of India,
which provided about 5 rag of nicotinic acid per day, pellagra was rare,
while it was endemic in Moldavia, where the staple corn diets provided
15 mg per day.72 Similar paradoxes were encountered broadly in the
United States.167 It thus appears that corn does, in fact, contain a pella-
gragenic factor.

"Woolley 168 has suggested that the toxic action may be due to some
structural analogue of nicotinic acid, or its associated metabolites, which
competes with the nutrilite for some enzyme (Chap. VI D), thus prevent-
ing the full nicotinic acid activity. A one hundred thousandfold concentra-
tion of the factor was achieved, using mice as assay animals, and the toxic
effect was found to be reversed by nicotinamide. Since it was reported
that 3-indoleacetic acid produced a corn-like pellagragenic action in rats,
and this substance is known to be present in corn in considerable amounts,
heteroauxin was believed to be the toxic agent.169 Subsequent experiments,
however, have indicated this to be untrue, and the chemical nature of
the pellagragenic agent in corn, if it exists, remains as yet unknown.170-172

Subsequent to the study of thiaminase, it was found that a wide variety

FACTORS INFLUENCING B VITAMIN REQUIREMENTS 295

of plant materials have anti-thiamine activity, and it was at first thought
that a similar enzymatic principle was involved in these plants. Later,
however, it was found that these antithiamine effects were due to ther-
mostable principles, so that it now appears, but not unequivocally, that
an antimetabolite principle is involved.

Bhagvat and Devi m found that chloroform-water extracts of rice
polishings, ragi {Eleasine coracana) , green grain {Phaseolus radiatus) ,
mustard seed {Brassica juncea) , yellow cotton seed (Gossipum sp.), and
linseed (Linum usitatissi?num) have an inactivating effect on both free
and combined thiamine. The active factor contains a dialyzable and a
nondialyzable portion. Incubation for short periods of the crude material
or organic extract with thiamine or thiamine-containing products indi-
cated a loss of thiamine activity, as shown by thiochrome tests and rat
and pigeon assays. The product was, however, active for mosquito larvae,
suggesting the possibility of a chemical cleavage analogous to the bisulfite
cleavage, in which the fission products have activity for some species.
This would not involve inhibitory effects in the sense that we are con-
sidering them, and chemical nonenzymatic inactivation would be more
analogous to enzymatic destruction if this mode of action is proved to
exist. Unfortunately, further information upon this point is lacking. "Fern
poisoning," which afflicts horses and cattle that consume considerable
amounts of the fern Pteris aquilina, has also been shown to exert its toxic
effect by producing a thiamine deficiency,174 and has indeed been proposed
as another way of bringing about such deficiencies experimentally. In
this case the highly thermostable material seems to differ from the active
substance in Bhagvat and Devi's work in some characteristics, and
although the mode of action is unknown, an antimetabolite effect seems
unlikely.

A naturally occurring vitamin B6 inhibitor has been shown to exist in
linseed oil meal,174a and vitamin B6 deficiency is rapidly induced in
chicks when their diet contains 30 per cent of this meal. Usual dietary
levels of vitamin B6 do not affect the inhibitor, but synthetic pyridoxine
added in higher levels completely prevents the anti-pyridoxine activity.
Water pretreatment of the meal also abolishes the activity, but it does
not change the vitamin B(i content.

Rat growth on a low casein diet is inhibited by threonine, and this
inhibition is prevented by either tryptophan or niacin. 174b Both threonine
and phenylalanine are said to intensify the symptoms of niacin-trypto-
phan deficiency in the rat.174c

The possible presence in synthetic vitamin preparations of impurities
which have inhibitory effects should be given some consideration. A
recent example of this was the report that folic acid inhibited the activity

296 THE BIOCHEMISTRY OF B VITAMINS

of milk and liver xanthopterin oxidase and xanthine oxidase. 174d- e A later
report 174f indicated that the effect was due to an impurity, probably a
pteridyl aldehyde photofission product, which also had the ability to
inhibit rabbit liver quinine oxidase. It is at present uncertain whether
or not this impurity could account for the undesirable neurological effects
observed in the treatment of macrocytic anemias with synthetic folic
acid. (p. 416)

It has recently been reported that the feeding of 300 to 500 mg of
adenine per day to dogs produces a pellagra-like condition, and it seems
possible that the large excess of this substance might well interfere by
competition with the function or synthesis of some other metabolite.175

Mention has previously been made (p. 260) of the toxic effect of lyco-
marasmine upon certain species, and the reversal of this effect by strepo-
genin. In species that require nutritional strepogenin, it seems apparent
that the ingestion of quantities of the tomato wilt might readily influence
nutritional requirements for strepogenin.

It is also worthy of passing mention that such synthetic products as
araboflavin and dulcitoflavin inhibit riboflavin activity in rats;176 and
naturally occurring analogues of a similar nature, should they exist, might
be expected to be similarly effective in causing increased requirements for
riboflavin in the diet. These and other synthetic inhibitors are considered
in a later section.

Miscellaneous Antivitamin Effects. Various other instances of anti-
vitamin effects have been reported from time to time in the literature,
but have not appeared to be of sufficient importance to merit further
study. A review of these reports would be tedious and uninformative.
Mention should be made, however, of several recent reports of this general
nature.

With the advent of rapid transportation and refrigeration, a variety
of new and strange food plants may appear in the markets of the civilized
world. It should be anticipated that from some of these, unique dietary
problems may arise. The fact that a plant has been utilized as a food
by some remote native population is not in itself a guarantee of the de-
sirability of its incorporation into a diet. Indeed, B vitamin deficiencies
are common among primitive tribes (contrary to the general opinion),
and have probably existed from prehistoric times. While this is due to
a wide variety of factors, antivitamin effects play at least some part.
An interesting example of these points is the case of manioc.177 A prepara-
tion of this plant, called "gari," is one of the staple foods in Nigeria,
although its incorporation into the diet produces a syndrome (in native
school children), which is improved considerably by riboflavin (but not
niacin) and cured by the administration of marmite.

FACTORS INFLUENCING B VITAMIN REQUIREMENTS 297

It is unnecessary, however, to consider strange food sources to find
such effects. Gross dietary distortions in a normal dietary regime may
bring such quantities of an apparently innocuous substance into the diet
that effects which are usually too slight to measure become important.
Thus the addition of various quantities of roasted coffee to the diet of
rats and dogs produced in the dogs diuresis, catharsis, weight loss, flaccid
paralysis, greying of the hair, an eye condition, and convulsions; and
produced alopecia, weight loss, edema, and death in the rats.178 In dogs,
inositol appeared to cure the paralysis, and biotin cured the "weepy
eye" symptoms. The curative properties of inositol and whole liver were
not, however, complete in the rat. The alopecia in the rat appears to be
due to the caffeine. The similarity of caffeine to various other purines
involved in metabolism (and the known presence in coffee of a wide
variety of other analogues of B vitamins and other metabolites) might
suggest the existence of such toxic effects.

Finally, new processing methods in food technology may result in
antivitamin effects by the production of synthetic antivitamins. Thus
the agenizing of flour (bleaching with NC13) forms products which have
been shown to be responsible for canine distemper, and it has been sug-
gested 179 that this is due to chlorination of the aromatic groups in the
gluten. Such chloro-amino acids are known to be inhibitors of amino acid
metabolism, and similarly modified B vitamins would be capable of a
similar effect. To date, fortunately, no such case has been reported.

Influence of the Intestinal Flora upon the B Vitamin Requirement

It has long been known that the microorganisms of the digestive tract
are in many cases able to synthesize many of the B vitamins, and that
these may well have a significant role in supplementing the nutritional
supply in order to meet the B vitamin requirement. Studies of this aspect
of vitamin nutrition have been numerous and extensive, but because of
the equivocal nature of the techniques available for such studies, the
results obtained have been at best qualitative, and frequently difficult
to interpret. Because of the uncertain nature of the results, the vast
amount of data bearing on the subject, and the presence in the literature
of excellent reviews on this topic,180"181 it does not seem practical or
expeditious in this volume to consider the problem in detail. For this
reason, only a brief summary of the major points of interest is here
presented.

Numerous studies have dealt at great length with the analysis of in-
testinal and rumenal flora, and the abilities of microorganisms to
synthesize the various B vitamins. Some organisms which are known to
synthesize specific B vitamins are listed in Table 15. Analyses have shown

298 THE BIOCHEMISTRY OF B VITAMINS

a varied and extensive list of organisms which inhabit the intestinal tract,
frequently predominated by the coliform group, lactobacilli, enterococci,
and yeasts.182 Intestinal bacteria which require nutritional sources of
B vitamins also exist, e.g., lactobacilli (p. 307), most frequently in rela-
tively small numbers although this fortuitous fact is seldom considered
and has been little investigated. The nature of the factors influencing the
intestinal flora, and its variation among species has been studied at
length, but the valid information derived from such studies is limited for
the most part to the influence of diet on the nature of the organisms
present.182a

The nature of the carbohydrate, protein, fat, and vitamin content of
the diet has a considerable effect upon the relative numbers of the various
types of organisms to be found in the intestinal lumen, depending upon
the selective effects of these dietary constituents in promoting or depress-
ing the growth of the various organisms. The phenomenon of "refec-
tion," 183 in which high starch diets protect experimental rats on a
B-deficient diet, has long been known, and is generally believed to be due
to the stimulation of thiamine-producing organisms in the rat intestine.
Various drugs also have pronounced effects, and antibiotic substances
may depress intestinal vitamin synthesis to a very low level.

It is generally recognized that the entire B vitamin nutritional require-
ment of ruminants may be met by the microflora of the rumen, so that
B-avitaminoses are rare in this group of animals. In young ruminants,
before the microflora is well established, some deficiencies may occur,
however.184 In most other higher animals, a lesser and more variable
part of the requirement is so met. Limited evidence suggests that the
diet of the pig is more subject to fluctuations in intestinal synthesis than
most other species,185 although the reasons for this are unknown. Rela-
tively little is known at all concerning the effects or nature of intestinal
flora in the nonmammalian vertebrates, or in the invertebrates, although
there is ample evidence to suggest that bacterial vitamin synthesis may
be a critical factor in fulfilling nutritional requirements in the entire
plant and animal kingdoms.

The study of the extent to which intestinal synthesis may affect the
nutritional requirement in any given case has been approached in several
ways. Most frequently sulfonamides and antibiotics have been fed, and
their effect on the nutritional requirement as judged by the various tech-
niques previously discussed (Chap. I C) has been observed.186 Such
studies applied to man and domestic and laboratory animals have given
us most of the existing data regarding the intestinal synthesis of the B
vitamins in general, and biotin and folic acid in particular. Other special
diets have also been used from time to time with some success, when the

FACTORS INFLUENCING B VITAMIN REQUIREMENTS 299

Table 15. Some Common Bacteria which Synthesize

B Vitamins*

Organism

Thia-
mine

Ribo- Niacin Vitamin
flavin B6

Panto-
thenic
acid

Biotin Folic
acid

Inositol

Pfeiffer's bacillus

B. vulgatus

+ +

B. proteus

B. subtilis

B. adhoerans

B. lactis aerogenes

B. alcaligenes
fecalis

+ +

Dysentery bac.
strain

Corynebacterium
diphtheriae

B. aerogenes

+ +

B. mesentericus

+ +

E. coli

+ +

Ps. fluorescens

+ + +

+ +

Prot. vulgaris

+ + +

+ +

CI. butylicum

+ + +

+ +

B. bifidus

Lactic acid bact.
strain

B. vulgaris

Aerobacter
aerogenes

+ +

Staph, flavis

Prop. bact. strain

Serratia mares

Ps. aeruginosa

E. typhosa

* Neither are the data in this table complete, nor is the table a complete list. Rather is it meant to
indicate representative data, and includes only those cases where study has definitely shown synthesis
by the indicated organism.

+ = synthesize the indicated B vitamin.

diet could be suitably balanced so as to affect adversely the growth of
the bacteria without influencing the dietary requirement of the host.187
Cecectomy has given some valuable data, but is technically difficult, and
therefore has been rarely used.188 In addition, this technique creates a
different animal from the normal one — a factor worth weighing in both

300 THE BIOCHEMISTRY OF B VITAMINS

this technique and in those methods where drugs are involved, which may
likewise modify other factors influencing the requirement. In many cases,
moreover, cecectomy merely serves to shift the area of bacterial synthe-
sis to another portion of the intestinal tract.

Some idea of the extent of bacterial synthesis in humans may be
gained from the excellent study of Denko et al.,52 in which the urinary
and fecal excretion of eight B vitamins was measured over a period of
time in seven individuals on controlled and analyzed diets. They found
that the urinary and fecal folic acid averaged 5.5 times the intake,
p-aminobenzoic acid 2.3 times, biotin 3.8 times, and pantothenic acid 1.1
times. Riboflavin was slightly less than the intake, and thiamine, niacin,
and vitamin B6 considerably less. In this latter regard, however, certain
known metabolic products of vitamin BG, niacin, thiamine and riboflavin
were not measured ; hence it is likely that the excretion of these vitamins,
too, may exceed the intake.

It was also found in this same study that the fecal excretion of ribo-
flavin and p-aminobenzoic acid was 1.5 times the urinary excretion; fecal
thiamine and niacin were double the urinary excretion; fecal biotin was
four times as much; and fecal folic acid 75 times as much. Only about
60 per cent of the pantothenic acid and pyridoxine appeared in the urine,
however. It would thus appear that in these well nourished individuals,
bacterial synthesis of the B vitamins may well exceed the normal intake.

Most nearly approaching the ideal from the scientific standpoint would
be the study of the B vitamin requirements of bacteriologically sterile
animals. While it has been possible to obtain such animals for over fifty
years by aseptic delivery on Cesarean section, it has been only in recent
years that the techniques have been so improved as to be practical by
the efforts of Glimmer 189 and Reyniers.190 Reyniers et al. have been able
to obtain and raise to maturity a variety of "germ-free" animals in this
fashion; and the study of the nutritional requirements of these animals
promises to be one of the great advances in the science of nutrition in the
years ahead. Unfortunately even in this case, however, the animals must
be considered as very different from their symbiotic "cousins," and results
so obtained will be viewed with caution because of this fact. Life as it
exists is a highly symbiotic process, and the knowledge that is so obtained
with germ-free animals will not apply with certainty to animals living
in natural surroundings. No one of the approaches mentioned can be
expected to supply the desired information ; but over an extended period,
the synthesis of data obtained by the various techniques will result in an
increase in knowledge regarding the functioning of intestinal symbionts.

FACTORS INFLUENCING B VITAMIN REQUIREMENTS 301

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160. Bateman, W. G., J. Biol. Chem., 26, 263-91 (1916).

161. Boas, M. A., Biochem. J., 21, 712-24 (1927).

162. Eakin, R. E., Snell, E. E., and Williams, R. J., J. Biol. Chem., 140, 535-43

(1941).

163. Eakin, R. E., McKinley, W. A., and Williams, R. J., Science, 92, 224-5 (1940).

164. Eakin, R. E., Snell, E. E., and Williams, R. J., J. Biol. Chem., 136, 801-2 (1940)

165. Hertz, R., Physiol. Revs., 26, 479-94 (1946).

166. Alibrandi, A., Arch, fisiol, 44, 163-79 (1945).

167. Dann, W. J., Federation Proc, 3, 159-61 (1944).

168. Woolley, D. W., J. Biol. Chem., 162, 179-80 (1946).

169. Kodicek, E., Carpenter, K. J., and Harris, L. J., Lancet, 251, 491-2 (1946).

170. Rosen, F., and Perlzweig, W. A., Arch. Biochem., 15, 111-14 (1947).

171. Krehl, W. A., Carvalho, A, and Cowgill, G. R., Federation Proc, 6, 413 (1947).

172. Sarett, H. P., and Goldsmith, G. A., Federation Proc, 6, 288 (1947).

173. Bhagvat, K., and Devi, P., Indian J. Med. Research, 32, 131-7 (1944).

174. Weswig, P. H., Freed, A. M., and Haag, J. R., J. Biol. Chem., 165, 737-8 (1946).

175. Raska, S. B., Science, 105, 126-7 (1947).

176. Emerson, G. A., Wurtz, E., and Johnson, O. H., J. Biol. Chem., 160, 165-7 (1945).

177. Moore, D. F., J. Trop. Med. Hyg., 45, 129-31 (1942).

178. Briggs, G. M., Jr., Mills, R. C, Elvehjem, C. A., and Hart, E. B., Proc. Soc

Exptl. Biol. Med., 51, 59-61 (1942).

179. Mellanby, E., Brit. Med. J., 2, 885 (1946).

180. Najjar, V. A., and Barrett, R., Vitamins and Hormones, 3, 23-48 (1945).

181. Nutrition Revs., 4, 76-8 (1946).

182. Evenson, A., McCoy, E., Geyer, B. R, and Elvehjem, C. A., /. Bad., 51, 513-21

(1945).
182a. Gall, L. S., Illingworth, B. A., Cowgill, G. R., and Fenton, P. F., J. Nutrition,
35, 27-38 (1948).

183. Roscoe, M. H., Hyg., 27, 103-7 (1927).

184. Johnson, B. C, Wiese, A. C, Mitchell, H. H., and Nevens, W. B, J. Biol.

Chem., 167, 729-36 (1947).

185. Nutrition Revs., 3, 81-2 (1945).

186. Daft, F. S., and Sebrell, W. H., Vitamins and Hormones, 3, 49-72 (1945).

187. Friedman, H., J. Nutrition, 12, 165-72 (1936).

188. Taylor, A., Pennington, D., and Thacker, J., Univ. Texas Pub., 4237, pp. 135-44

(1942).

189. Glimstedt, G., Skand. Arch. Physiol, 73, 48-62 (1936).

190. Reyniers, J. A., Trexler, P. C, and Ervin, R. F., Lobund Rept., 1, 1-120 (1946).

191. Wright, L. D., Cresson, E. L., Skeggs, H. R., Wood, T. R., Peck, R. L., Wolf,

D. E, and Folkers, K., J. Am. Chem. Soc, 72, 1048 (1950).

Chapter IMC

THE B VITAMIN REQUIREMENTS OF ANIMALS
AND PLANTS

Requirements of Invertebrates

Biochemistry in the broadest sense is concerned with the chemistry of
all living species. Generally, however, biochemical investigations have
been most intense in those fields that seem the most likely to bring the
accomplishment of immediate results. Coupled with this, the ever urgent
need for deeper insight into the factors involved in human nutrition, and
to a lesser extent in the nutrition of domestic animals, has resulted in a
neglect in the study of the nutritional requirements of invertebrates.

In very recent years, however, a number of factors have brought about
some change for the better in this regard. In the first place, commercial
interests, increasingly mindful of the economic benefits of research, have
diverted investigations in several cases into this field of inquiry. The
manufacturers of insecticides and those in the sea-food industries have thus
taken an active interest in what quite recently was purely an academic
subject. Secondarily, the problems of the recent war and life in tropical
places have brought about a renaissance in parasitology, which has re-
sulted in considerable new knowledge of the vitamin requirements of a
number of parasitic and vector invertebrates.

Aside from the purely academic, commercial, and medical reasons for
inquiry into invertebrate nutrition, an increasing awareness of the benefits
to be derived from a study of comparative biochemistry seems likely to
bring about even greater interest in this field. We have already con-
sidered one such topic — Cowgill's application of comparative requirements
in assessing human vitamin requirements (p. 246). The challenge for
investigation in comparative biochemistry is very great. In many common
phyla (porifera, coelenterata, echinodermata, annelida) literally nothing
is known about vitamin requirements (or indeed nutritional require-
ments) . Still greater is the challenge, because up to the time of this
writing, no animal had as yet been reared and bred on a chemically
defined diet. In view of these considerations there are many limitations to
anything resembling a thorough review of invertebrate B vitamin require-
ments, and as to these limitations we are helpless.

THE B VITAMIN REQUIREMENTS OF ANIMALS AND PLANTS 307

One might for comparative purposes wish to start a review with a
consideration of the B vitamin requirements of green plants. Most exist-
ing evidence indicates that green plants are able to meet their own vitamin
requirements by synthesis. Such plants, however, are seldom studied under
sterile conditions, and plant embryos and roots are known to require in
many cases a nutritional source of B vitamins ; hence this field of investi-
gation offers limitless opportunities for study. A brief consideration of
green plant requirements, therefore, occurs later in this chapter.

Passing briefly to the bacteria whose B vitamin requirements have
been studied extensively, we find a broad spectrum of requirements.
While many bacteria are able to meet all their nutritional requirements
by synthesis, others require one or many of the known B vitamins, and
some are even more fastidious.1, 2 The B vitamin requirements of bac-
teria have been extensively reviewed several times in recent years,3 and
various aspects of bacterial and plant nutrition are considered in greater
detail elsewhere (p. 336) . As concerns the most "simple" living forms,
the viruses, we know so little of their metabolism and nutrition that
almost nothing can be said.4

Like the bacteria, the protozoa constitute a heterogeneous group nu-
tritionally as well as taxonomically. Many of the more plant-like forms
grow well in inorganic salts media, and have the ability to synthesize all
the B vitamins, while the so-called "higher" protozoa require most if
not all of them. Still others require as yet unidentified growth factors.
The nutrition of the protozoa has been intensively studied in recent
years, and excellent reviews are available.5, 6

Thus in passing from the higher plants to the higher animals, we pass,
in general, from cells requiring none to cells requiring most of the B
vitamins. At the juncture of the two kingdoms, we find single-celled
organisms that run the gamut of requirements, in general resembling nu-
tritionally the kingdom that they most resemble on the basis of other
conventional taxonomical considerations. If the line of demarcation be-
tween the plant and animal kingdoms is thus sharply drawn nutritionally
within the single-celled forms, we may expect that as we ascend the
evolutionary scale in the animal kingdom, no perceptible trend in B vita-
min requirements will occur, i.e., the lowest metazoan forms would re-
quire many if not most of the B vitamins. As shown in the discussion
which follows, the limited information available suggests that this is
essentially so. The recent development of methods for the study of the
biosynthetic abilities of developing embryos should even further assist
in examining this generalization in the years to come.7

The Lower Invertebrates. In considering the B vitamin requirements
of the lower invertebrates the absolute lack of evidence concerning the

308 THE BIOCHEMISTRY OF B VITAMINS

nutrition of the Porifera and Coelenterata creates a great gap. The
economic importance of these phyla may soon, however, induce sufficient
effort into the study of this group of animals to overcome the experimental
difficulties inherent in the investigation of their nutrition.

Regarding the Plathelminthes, studies on the nutrition of Planaria
maculata have shown that a heat-labile, ether-soluble fraction of liver
is required for growth,s and that a similar and possibly identical factor
is inactivated by egg white. Despite the conflicting nature of the descrip-
tions, there is some reason for believing the factor to be biotin. It has
been shown 9 that the number of tapeworms (Hymenolepis diminutia)
in rats on various vitamin-deficient diets varies broadly. Thiamine-
deficient rats have worm populations similar to those of normal rats,
whereas rats deficient in the "B complex" vitamins have reduced numbers
of worms. We shall see shortly that, by contrast, studies of parasitic
round worm infections in humans and rats show increased populations
of worms in malnutrition.

The fish tapeworm, Diphyllobothrium latum, infects humans and causes
a macrocytic anemia differing from Addisonian pernicious anemia in that
spinal cord involvement is rare, gastric acid is seldom decreased, the
anemia is cured by removal of the worm, etc. Extracts or suspensions of
the worm are inactive, and considerable evidence suggests that the effect
produced may possibly be due to absorption by the worm of the anti-
pernicious anemia factor (vitamin Bi2) in the host, in a manner similar
to that in which live yeast may deplete the intestinal tract of thiamine
(p. 291 ),10 and produce the corresponding avitaminosis. One is caused
to wonder in passing whether a metabolic factor is involved in the pro-
duction of rat liver sarcoma by the flat worm Cysticercus.11

The nutrition of a number of round worms (Anguilla oxophilla,1'2
Neoplectana glaseri,13 and Ancylostoma caninum14) has been studied,
but never in such a manner as to indicate much with regard to their
B vitamin requirements. All require complex media (yeast autolysate-
peptone, dextrose-veal infusion-toe yeast, live bacteria) and in many
cases, the diet is still inadequate for reproduction without supplementa-
tion with other materials of a complex nature.

It is generally believed that hookworm infection in man is commonest
in areas where malnutrition is prevalent, and that the improvement of
the general dietary status of an individual may frequently purify the
body of the parasite. Moreover, in experiments using vitamin-deficient
rats and the round worm Nippostrongylus muris,15 it was found that
thiamine- and riboflavin-deficient rats contained appreciably more para-
sites than did normal controls. Mention of this work is made to empha-
size the unreliability of data obtained by deducing the nutritional require-

THE B VITAMIN REQUIREMENTS OF ANIMALS AND PLANTS 309

ments of the parasite from the nutritional status of the host, since it is
extremely improbable that the diminished amounts of thiamine and
riboflavin per se improved the nutrition of the parasite. Unfortunately,
much of our knowledge of the nutrition of parasitic organisms stems
from data of this nature.

If the information so far presented seems sparse, it is even more strik-
ing that data are virtually nonexistent regarding the B vitamin require-
ments of the Echinodermata and Annelida. This is particularly strange
in view of the relative ease in the handling of such animals as the earth-
worms and leeches. Similarly, but more obviously because of experi-
mental difficulties, nothing at all is known of the B vitamin requirements
of Mollusca, although some indirect data 1G suggest that the requirement
is a complex one.

The Arthropoda. We have seen that the requirement for no single
B vitamin has been definitely established for any metazoan invertebrate
below the Arthropoda, and a similar lack of information exists with
regard to the economically important Crustacea. The Insecta, then, repre-
sent the only class of metazoan invertebrates concerning which we have
reliable information as to B vitamin requirements, and for some insects
relatively complete data are available. For this reason alone, the discus-
sion of this class is more extensive than that of the other phyla.

Aside from the need for information regarding the nutritional require-
ments of insects, there are many factors to encourage their study. In
general, they are readily bred in a small space and with little time and
effort, and require a minimum of food and equipment for their study.
Various species are herbivorous, carnivorous, and omnivorous, and hence
vary widely in their natural nutrition. Factors such as weight, food and
liquid intake, and environment may be readily controlled, and for these
reasons insects are generally ideal experimental animals. Insects have
the further advantage in nutritional experimentation in that they possess
symbiotic microorganisms which may function to meet their requirements,
in a manner similar to that of the vertebrates. Thus of five beetles studied
by Fraenkel and Blewett,17 Tribolium confusum and Ptinus Tectus
required thiamine, riboflavin, nicotinic acid, pyridoxine, pantothenic acid,
biotin and choline, while Lasioderma serricorne, Sitodrepa panicea, and
Silvanus surinamesis did not require all of these because of the presence
of intraocellular symbionts. It is possible, on the other hand, to raise many
insects in a germ-free condition, a feat involving great difficulties with
higher animals. Under such sterile conditions the nutritional require-
ments are generally found to be more fastidious, reflecting extra needs,
for metamorphosis, etc.

310 THE BIOCHEMISTRY OF B VITAMINS

I + * M ++

S S -

1 J

0 -H

S o

i 1

■■§ &

cq g .a
III

+ +

* 41 ++ +

fill + + + + + + + +

++++++ ++ +

++++++ ++

++++++ ++ +

.1 ?! Hi I ill

s "^ -2 §> g -S s -2 -g "2 e
S 8 ^ 3 ~ s g a-g H -S *=

?5 H I ! ^ » « & e •« ^| a .e

13 §■$ s-l I Si isl Htl!

THE B VITAMIN REQUIREMENTS OF ANIMALS AND PLANTS 311

To an extent exceeding that in the vertebrates, insect B vitamin re-
quirements vary with the phases of the animal's life. Larval requirements
are much higher (and B vitamin content apparently lower) than in the
adult, and the requirements for successful pupation, exuviation, and
reproduction may be even more extreme. Thus Aedes aegypti larvae from
bacteriologically sterile eggs complete metamorphosis on a sterile diet

^40

«0
til

o
<30

I-
I
o

2 10

500 fG

:&=^

■OQ ^G

10 20 30

AGE OF NYMPHS (DAYS)

Figure 2. Growth of cockroach nymphs on various levels of choline.

only when it contains adequate amounts of biotin,18 and the creation of
abnormally high B vitamin levels in serum causes premature shedding
of the old skin of Tristoma infestans larvae.19 The high level of panto-
thenic acid in the royal jelly of the bee is also suggestive in this regard.20
While a relatively large number of insects has been studied, the number
is actually small in view of the great variety of known species. This
variety provides the broadest possible latitude for experimental work, as
previously stated, and with relatively few difficulties. Illustrative of this
fact are the studies of Sarma et al. with the rice moth larva (Corey ra
cephalonica St.), which have demonstrated in a variety of ways to be
discussed later the similarity that exists between vertebrate and arthropod
vitamin metabolism. In a manner similar to that in which higher experi-

312

THE BIOCHEMISTRY OF B VITAMINS

mental animals are kept, the larvae are reared in a 30° C incubator on
a whole wheat stock diet. Ten day-old larvae are then placed upon appro-
priate experimental diets, and growth measured by weekly weighing. In
this manner, the experimental results obtained accord beautifully with
those obtained in nutritional research with higher animals.

Table 17. Quantitative B Vitamin Requirements of Several Insect Species
(ng/unit of diet)

Species
Aedes aegypti

Drosophila melanogaster

Galleria mellonella

Tribolium confusum

Blatella germanica

0.4

xg/ml

4.0

Mg/gm

1.0

Mg/ml
0.05-0.10

Mg/gm

Folic
acid

0.2

Mg/gm

Biotin

0.05

Mg/ml

0.10

Mg/gm

2000.-4000.

Mg/gm

Qualitatively, the B vitamin requirements of insects appear to resemble
closely those of the vertebrates. A summary of much of the existing data
in this regard is given in Table 16. The limited amount of quantitative
data as yet available is insufficient to make valid comparisons, but is
summarized in Table 17. Consideration of these data in terms of their
relationship to insect vitamin composition 34, 35 shows that, as in higher

Table

18. Results of B Vitamin Deprivation j

or Several Insect Speciei

Vitamin
missing from

Tribolium

Ptinus

Silvanus

Number

Diet

Growth

sur-

Indext

Growth

sur-

Index

Growth

sur-

Index

rate*

vivingt

rate

viving

rate

viving

Thiamine

714

140

1552

Riboflavin

846

Niacin

Vitamin B6

1120

349

1456

Pantothenic

Acid

140

103

Choline

1672

1036

1424

Inositol

1649

1666

1365

PABA

1548

1764

1316

Yeast-fed

controls

100

1800

100

1800

100

1600

* Growth Rate: Reciprocal of the period in days in which 50% of the insects were completely developed,
expressed as the percentage of the period required by a yeast fed control group.

t Number surviving: The number of the original group of twenty insects that completed development.
% Index: Product of the growth rate and number surviving.

animals, there is a rough correlation between the amounts of the various
vitamins required and the amounts found in the animal. The nicety of
response obtained in determining requirements is indicated by Figure 2,
which shows the growth of cockroach nymphs on various levels of

THE B VITAMIN REQUIREMENTS OF ANIMALS AND PLANTS 313

choline.21 Such responses, however, must be assessed in terms not only
of weight, but also of the time required to reach the adult stage, and the
number of nymphs surviving. Indeed some workers use the product of
the number of insects reaching a certain stage of development and the
reciprocal of the average time required to reach that stage as a criterion

Day*.

Figure 3. Increase in pyruvic acid in Corcyra cephalonica St. larvae on a thiamine

deficient diet. Ten larvae were used in each test group. Pyruvic Acid is in mg per

100 gm of larvae.

of response.18 Certain interesting conclusions are apparent when such a
criterion is applied to some of Fraenkel and Blewett's data,17 as shown
in Table 18. Species differences are readily apparent, as witness the
critical nature of riboflavin for Ptinus. Certainly riboflavin, niacin, and
pantothenic acid stand out for these three species as the B vitamins most
critically required in their nutrition.

314

THE BIOCHEMISTRY OF B VITAMINS

Because of the nicety of quantitative response obtainable, Sarma and
co-workers have employed Corcyra as an assay animal for thiamine,
riboflavin, and pyridoxine, and obtained results which agree well with
those obtained by other methods. Strain differences, however, are suf-
ficiently great that "pure" strains would be desirable for routine insect
assay work.30- 31- 36

Figure 4. Growth of Corcyra Cephnlonicn St. larvae with various amounts of
biotin concentrate added to an egg-white diet. Ten larvae per test group.

Of even greater interest, however, is the work done by this group on
vitamin metabolism in insects.37 In a manner entirely analogous to that
of higher animals, thiamine-deficient Corcyra larvae show fatty tissue
and nerve degeneration, and the pyruvic acid content of the deficient
larvae increases to eight times that of the normal animals (p. 403), and
decreases again on a high thiamine diet. Again, vitamin B6 deficient larvae
on high tryptophan diets excrete a yellow pigment which disappears on
supplementation of the diet with pyridoxine, in a manner remarkably
suggestive of the abnormal xanthurenic acid (yellow) excretion in vita-
min B6 deficient mammals (p. 428). As in mammals, tryptophan alone

THE B VITAMIN REQUIREMENTS OF ANIMALS AND PLANTS 315

is able to increase excretion of the pigment. Finally, raw egg white or
avidin concentrate, when added to the diet, causes death of the larvae in
about four weeks, while pretreatment of the concentrate or egg white by
means which destroy avidin permits normal larval growth. When larvae
are changed from egg white diets to diets containing various levels of
biotin, the growth is renewed at rates in accordance with the biotin
present.37 The nicety of these responses is illustrated in Figures 3 and 4.
Thus in many ways at least the rice moth larva resembles the vertebrates
in its metabolism of thiamine, pyridoxine, and biotin.171-80 The extension
of these studies of the Coonoor Nutrition Research Laboratories will be
anticipated with great interest.

The insects mentioned above represent a number of the more important
orders of the class Insecta. Many others have not been investigated. The
order Thysanura (silverfish), because of its primitive nature, ubiquitous
distribution, and economic importance, certainly challenges the investi-
gator. Indeed the entire class Arachnida, for many reasons important,
remains virtually unstudied with regard to its B vitamin requirements.
Perhaps the single exception to this statement is the work of de Meillon
and co-workers on the blood-sucking tick, Ornithodorus moubata. These
workers found that this tick (as well as the common bedbug, Cimex
lactularis) , when feeding on thiamine-deficient rats, requires an average
of 79.7 days to develop as compared with 41.1 days on normal rats.38
They further found that definite toxic effects were observable in blood-
sucking arthropods that fed on rabbits injected with y-hexachlorocyclo-
hexane, suggesting an interference with inositol metabolism.39 It is truly
remarkable that the vast effort put into the study of tick-borne diseases
has not produced similar more extended studies of the nutrition of the
insect vectors. Indeed future studies of insect-borne disease may well
start with a study of the nutrition of the host, as an approach to insecti-
cides that might be organized on logical lines of reasoning, based upon
present knowledge of the inhibition of vitamin metabolism.

In summary, our knowledge of invertebrate nutrition is practically
nonexistent, and, at best, is based upon presumptive evidence, in the
phyla below Arthropoda. This scant evidence, however, suggests that
these lower phyla have extensive B vitamin requirements.40 Among the
Arthropoda, information approaching adequacy exists only in the class
Insecta, where the available data indicate that B vitamin requirements
are as extensive as those of the vertebrates. The problems involved in
invertebrate nutrition are such as to offer great inducement to the in-
vestigator, and are further significant for physiological, medical, economic,
and taxonomic reasons.

316 THE BIOCHEMISTRY OF B VITAMINS

The B Vitamin Requirements of Green Plants

Because the major source of the B vitamins for the animal kingdom
ultimately comes from green plants, and because green plants are so
frequently referred to as autotrophic, and even further because of the
widespread culture of plants in mineral salts-water solutions, it is cus-
tomary to think of them as having no B vitamin requirements. It is true
that the green plant as a whole will grow without an apparent exogenous
supply of B vitamins or other organic nutrients. It is also true, however,
that in many cases the plant may be deriving considerable nourishment
from symbiotic microorganisms. It also seems reasonably well established
that many green plants may not be able to synthesize sufficient B vita-
mins for optimum growth. It has been frequently observed (and also
denied) that thiamine, at least, frequently causes more luxuriant growth
of some plants, when added to the nutriment. Added riboflavin has been
reported to be of benefit to eggplant cultured in a synthetic medium,41
and it seems likely that other specific cases may arise involving others
of the B vitamins.

Green plant embryos, young roots, cuttings, and pollen grains are,
however, heterotrophic, and all are dependent upon supplies of at least
some of the B vitamins from stored foods or other portions of the plant.
While only a limited amount of information is available, it seems that
thiamine and riboflavin 42> 43 have pronounced effects on the germination
of pollen grains. Biotin is highly active in stimulating the growth of roots
on cuttings,44, 182 but in this case its activity has been likened to that of a
hormone. Indeed biotin functions in an auxin-like fashion in roots, in
which it is concentrated in the tips (and also in the tips of coleoptiles) .45
Biotin is synthesized by all roots, and even excreted in considerable
amounts.40 Its effect on root formation in peas, for instance, is quite
marked, inducing a 100 per cent increase in root formation on pea cut-
tings.44 The pea embryo, however, cannot synthesize biotin, and in this
case the function of biotin is more likely associated with the metabolic
activities of the embryo than with its differentiation. Biotin shares with
auxin a and b the structural feature of a five-membered ring joined to
a valeric acid derivative, and may well owe its auxin-like activity to this
resemblance.

The roots and embryos of green plants have been more extensively
studied, but plant tissue culture techniques are generally so recent in
origin as to have supplied little information. Excised roots have been
grown in apparently sterile media, and show a wide variety of organic nu-
tritional requirements. White has found some nine amino acids to be re-
quired by excised tomato roots — histidine, phenylalanine, lysine, leucine,

THE B VITAMIN REQUIREMENTS OF ANIMALS AND PLANTS 317

isoleucine, valine, glutamic acid, proline, and serine.47- 48 Thiamine is
apparently not synthesized by any roots,49 but is necessary for their
growth, and is active in dilutions as low as 1 :4x 1013. 50 There is also some
evidence to indicate that it is a stimulating factor for root formation.51
Roots are apparently able to utilize the thiazole and pyrimidine fractions
of thiamine about as well as thiamine itself.52 Some, such as the tomato
root, require only the thiazole portion, being able to synthesize the
pyrimidine part; but others, such as the pea root, can synthesize neither
part, requiring a nutritional source of both.

Work with plant embryos requires that the embryo be removed from
the seed and the cotyledons from the embryo as early as possible. When
these precautions are taken, the resulting embryo is found to be a
heterotrophic organism, requiring a nutritional source of at least some
of the B vitamins. Thiamine and nicotinic acid have been found to be
required by most roots and embryos, while pyridoxine is required by a
lesser number. The requirement for riboflavin and pantothenic acid is
less clearly known, due to less thorough study. The requirements for the
more recently discovered members of the B group of vitamins have not
been reported to date. A summary of much of the known data is shown
in Table 19.

Table 19. Known B Vitamin Requirements of the Roots and Embryos of Some Green Plants

Nicotinic

Pantothenic

Species

Thiamine

Riboflavin

Pyridoxine

acid

Biotin

Tomato roots

Pea roots

—

Pea embryos

Cosmos roots

—

Radish roots1

—

Alfalfa roots

—

Flax roots

—

Clover roots

—

Cotton roots

—

Carrot roots

—

Datura roots

Sunflower roots

+ = require (for thiamine, require both parts of molecule).
S = stimulates growth.
— = not required.

1 = thiamine and niacin alone as effective as yeast extract.

2 = requires thiazole portion of thiamine only.

In summary, we find that the ability of green plants to synthesize B
vitamins is very unevenly distributed, and is concentrated generally in
those parts of the plant reserved specifically for general synthetic activity.
To what extent the activities of the various tissues of animals vary in
respect to B vitamin synthesis is largely unknown. While green plants as
a whole survive readily without an exogenous source of B vitamins, it
seems apparent that parts of the plant (and phases in its life) exist that

318 THE BIOCHEMISTRY OF B VITAMINS

need some, and may in the future be shown to need most, of the B vitamins
required by other organisms.

The B Vitamin Requirement of the Vertebrates

Since we have considered earlier the many factors that may so pro-
foundly influence B vitamin requirements, it would indeed be strange to
set down absolute specifications for the vitamin intake of man or any other
species. A casual survey of the literature reveals data pertaining to the re-
quirements of many species, and in some cases, excellent reviews of the
requirements of a particular species are available (man,53 mouse,54 birds,56
pigs,57 ruminants,58 other animals59,181). For the sake of convenience
alone some of the useful data are reproduced here in tabular form. Any
discussion of their validity, however, would be redundant, and is de-
liberately avoided. In addition, the current National Research Council
table of recommended daily dietary allowances for humans is here repro-
duced for the convenience of those who are constrained to settle upon
some figure for dietary calculations.00 The amounts cited in this table,
as in any other, might well be subjected to extended discussion, but a
critical appraisal of human B vitamin requirements would require a
volume in itself.61 It may be said, however, that it would be difficult for
practical reasons to construct a diet meeting the stated allowances that
was still inadequate in B vitamin content. It is thus hoped that the salient
fact emerging from our discussion of B vitamin requirements is that even
in a monograph devoted to the subject of the B vitamins, the authors
cannot conscientiously make a statement as to the precise requirements
of any species for any vitamin.

Reference was made earlier to Cowgill's comparison of the thiamine
requirements of different species (p. 246). It would be expected that a
similar general relationship would hold for the other B vitamins, and
this is apparently so, judging from the limited data available. Despite
the many errors, qualifications, and interpretations inherent in any presen-
tation of such data, Figures 5 to 14 are presented, to emphasize the
general trend that exists for smaller species to have a higher relative B
vitamin requirement than larger ones.183

When one considers at length the conflicting evidence regarding whether
or not various animal species require certain B vitamins, the conclusion
is inevitably reached on the basis of the existing evidence that all the
species studied require an exogenous source of all the B vitamins, with
the probable exception of nicotinic acid (and choline, although this has
"nonvitamin" functions and the requirement should be considered in a
different light because of this) . In those cases where there is no nutritional
requirement, intestinal flora account adequately for the discrepancy. In

THE B VITAMIN REQUIREMENTS OF ANIMALS AND PLANTS 319

i 10

BODY WEIGHT (KG)

100

Figure 5. Relationship of the size of a species to its thiamine requirement.
10

1 10

BODY WEIGHT (KG)

Figure 6. Relationship of the size of a species to its riboflavin requirement.

THE BIOCHEMISTRY OF B VITAMINS

.1 1 10

BODY WEIGHT (KG)

Figure 7. Relationship of the size of a species to its nicotinic acid requirement.

BODY WEIGHT (KG)

Figure 8. Relationship of the size of a species to its pyridoxine requirement.

THE B VITAMIN REQUIREMENTS OF ANIMALS AND PLANTS 321

1 10

BODY WEIGHT (KG)

Figure 9. Relationship of the size of a species to its pantothenic acid requirement.

BODY WEIGHT (KG)

Figure 10. Relationship of the size of a species to its biotin requirement.

1 10

BODY WEIGHT (KG)

Figure 11. Relationship of the size of a species to its Folic Acid requirement.

1 10

BODY WEIGHT (KG)

Figure 12. Relationship of the size of a species to its Choline requirement.

BODY WEIGHT (KG)
Figure 13. Relationship of the size of a species to its inositol requirement.

1 10 100

BODY WEIGHT (KG)

Figure 14. Relationship of the size of a species to its p-aminobenzoic acid

requirement.

324

THE BIOCHEMISTRY OF B VITAMINS

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THE BIOCHEMISTRY OF B VITAMINS

Table 21. Some Reported Requirements of Humans for B Vitamins

Thiamine

Riboflavin
Nicotinic acid

1.0 - 1.8 mg per day

3.2 mg per day

1.06 mg per day
0.44 mg per 1000 Cal.

1. 5- 1.8 mg per day

3.7 mg per day
1.3 - 1.4 mg per day

10.0 -18.0 mg per day

40.0 mg per day

15.0 -20.0 mg per day

Remarks Referenc

Recommended intake and

not requirement (60)

Based on good diets (62)

Minimum (63)
Minimum for "normal"

male

Recommended intake (60)

Recommended intake (62)

Based on excretion studies (64)

Recommended intake (60)

Recommended intake (62)
Based on blood level

studies (65)

1.5 - 2.0 mg per day

Based on excretion studies

(66)

4.0 - 7.5 mg per day

Minimum requirement

(67)

Pantothenic acid

9.0 -12.0 mg per day

or per 2500 Cal.

Recommended

(62, 68)

Vitamin B6

1.5 mg per day

Recommended

(62)

Biotin

0.15- 0.3 mg per day

Recommended

(69)

0.14 mg per day

Recommended

(62)

Folic acid

0.20 mg per day

Recommended

(62)

Inositol

1.0 gm per day

Recommended

(62)

Under remarks, "recommended" means that the authors cited in the reference infer that this level is
suitable for the normal diet of individuals in order to maintain a state of good health. It will be noted
that in some cases where there may be no nutritional requirement under normal circumstances, it is never-
theless suggested that a level of the vitamin be provided in the diet as a precaution in view of our lack of
knowledge regarding the subject.

Table 22,

Some Reported Requirements of Rats for B Vitamins

Vitamin

Amount

Remarks

Reference

Thiamine

0.0125 mg per day

Minimum

(70)

0.02% solution ad lib

Doubles life span

(71)

Riboflavin

0.0075 mg per day

Based on tissue levels

(72)

0.010 mg per day

Minimum

(70)

0.018 mg per day

Minimum

(73)

0.036-0.090 mg per day

For pregnant rats based on

tissue level studies

(74)

Pantothenic acid

0.01 mg per day

For optimal growth

(75)

0.10 mg per day

For reproduction

(76)

Vitamin Be

0.15 mg per 100 gm diet

Maximum growth. Growth

is linear to 0.075 mg

(77)

0.01 mg per day

Minimum

(78)

Biotin

0.001-0.003 mg per day

Recommended. Avidin re-

quired to deplete.

(79)

Inositol

20.0 mg per day

Cures deficiency symptoms.
Required in diet only by

cotton rats.

(80)

p-Aminobenzoic

acid

0.75 mg per day

Cures nutritional achromo-

trichia

(81)

Choline

1.5-5.0 mg per day

To maintain lipid tissues

(82)

120-200 mg/kg per day

Prevents kidney degenera-
tion and liver lipotropism

(83)

THE B VITAMIN REQUIREMENTS OF ANIMALS AND PLANTS 327

Table 23. Some Reported Requirements of Mice for B Vitamins
Vitamin Amount Remarks

Thiamine

Riboflavin

Pantothenic acid
Vitamin B6

Biotin
Inositol

p-Aminobenzoic
acid

0.005 mg per day

0.008

0.010

0.005

0.0015 mg per gm food

0.004 mg per day

0.030 mg per day

0.0005 mg per gm food

0.001

0.008 mg per 100 gm diet

10.0 mg per 100 gm diet

0.25 mg per day

Maintenance of body weight of

weanlings
Suboptimal growth
Normal growth
Maintenance in adults
Minimum requirement for adults
Minimum requirement for adults
Minimum requirement
Half normal growth of weanlings
Good growth of weanlings

Cures deficiency symptoms

Prevents depigmentation of fur

(85)
(85)
(85)
(86)
(87)
(88)
(89)
(54)
(90)
(91)
(84)

(84)

Table 24. Some Reported Requirements of Chicks for B Vitamins
Vitamin Amount Remarks Reference

Thiamine

Riboflavin

Nicotinic acid
Pantothenic acid

Vitamin B6

Biotin

Folic acid
Vitamin BJ2

p-Aminobenzoic

acid
Choline

Strepogenin

0.080 mg per 100 gm diet

0.150 mg per 100 gm diet
0.170 mg per 100 gm diet
0.29-0.36 mg per 100 gm diet
0.23-0.25 mg per 100 gm diet

1.5 mg per 100 gm diet
1.8 mg per 100 gm diet
0.5 mg per 100 gm diet
0.6 mg per 100 gm diet
1.4 mg per 100 gm diet
2.0 mg per 100 gm diet

0.66-1.0 mg per 100 gm diet
1.4 mg per 100 gm diet

0.30-0.40 mg per 100 gm diet

0.030 mg per day

4.0 mg per 100 gm diet

0.2 mg per 100 gm diet

0.01 mg per 100 gm diet
0.0025-.010 mg per day
0.020 mg per 100 gm diet

0.050 mg per 100 gm diet

6.0 mg per 1000 gm diet

30.0 mg per 1000 gm diet

7.5 mg per 100 gm diet
100-200 mg per 100 gm diet

12,000 mg units per 100 gm
diet

Prevents deficiency and

suppressed growth (92)

Optimal growth (93)

Recommended level (94)

Recommended (95-98)
To maintain hatchability

of eggs in adults (99-102)

Recommended (94)

On 10% gelatin diet (103)

To prevent dermatitis (104)

For optimal growth (105)

Optimal growth (106)
Increases rapidity of

growth (107)
Minimum (108)
Minimum. Done with de-
pleted chicks (104)
Minimum (109, 110)
Minimum (111)
Minimum to prevent de-
ficiency (112)
To prevent anorexia,
weight loss, and de-

creased hatchability

(113)

Minimum

(94, 114)

Minimum

(73)

DL-O-Heterobiotin mini-

mum

(115)

Recommended (see Table

p. 248)

(116-121)

Stimulates chick growth

on animal protein fac-

tor deficient diet

(123)

Above optimum require-

ment

(123)

Recommended

(122)

Recommended to prevent

perosis

(124-126)

Optimum growth

(127)

328 THE BIOCHEMISTRY OF B VITAMINS

view of this fact, the nutritional requirement is a rather flexible quantity,
depending as it does upon the difference between the physiological require-
ment and the bacterial synthesis; and it is easy to understand the large
number of conflicting reports in the literature on this topic.

In an extended discussion of human nutrition, it would be appropriate
to consider at this point what the actual average consumption of B
vitamins was for the many population groups in the world that have
been studied in this regard. This formidable task is not here undertaken
because the quantity of information is so great, the quality so poor, and
the space available so limited. Suffice it to say that for the research
statistician a wealth of material lies buried in the literature awaiting
analyses from which may come a better understanding of the B vitamins
in nutrition. Since the actual consumption of the B vitamins varies among
individuals, groups, and geographical areas even more than does the
requirement, no simple analysis of this subject is now possible.

Table 25. Some Reported Requirements of Other Domestic Birds for B Vitamins
(mg per 100 gm diet)

Vitamin and species Amount

Thiamine

Pigeon 0.125

Riboflavin

Poult 0.3-0.4

Duck 0.3

Nicotinic acid

Poult 5.0

2-5 mg
Pantothenic acid

Ducklings 1.1

Vitamin B6

Duck 0.25

Folic acid

Poult 0.08

Choline

Poult 170.0

Inositol

Poult 1000.0

Philosophic Considerations

In retrospect, the members of the plant kingdom seem able to synthesize
the B vitamins while the members of the animal kingdom, as far as we
know, have completely lost this ability, except for nicotinic acid. General-
izations are always dangerous, but it is remarkable how consistently this
one holds true. While parts of plants may be heterotrophic, there is no
certainty that small vital cell groups in animal tissue do not have the
ability to synthesize B vitamins. Further, whereas certain Thallophytes
are heterotrophic and certain protozoa autotrophic, the general nutritional

Remarks

Reference

Minimum requirement

(128)

Minimum
Required for growth

(94, 112, 129)
(130, 131)

On 10% gelatin diet
For good growth, etc.

(132)
(129, 133)

Minimum

(131)

Minimum

(134)

Required to prevent deficiency

(129, 135, 136)

Required to prevent perosis

(94)

Maximum growth

(129)

THE B VITAMIN REQUIREMENTS OF ANIMALS AND PLANTS 329

trend in even these intermediate species follows closely trends in their
morphological classification. If one were compelled to state a single dis-
tinctive difference between the two kingdoms, it would be difficult to find
a better one than that of the ability to synthesize the B vitamins.

Table 26. Some Reported Requirements of Miscellaneous Mammals for B Vitamins

Vitamin and species

Amount

Remarks

Reference

Thiamine

Monkey

0.04 rag per day

To maintain body weight

(137-139)

0.05 mg per day

For growth response

0.075 mg per day

Optimum growth

Dog

0.0275-0.075 mg per 100

gm diet

Requirement

(140)

Swine

0.037 mg/kg/day

Requirement

(141)

Cat

less than 0.05 mg per day

Requirement

(142)

Riboflavin

Dog

0.01-0.02 mg/100 gm

body weight

Requirement

(143)

Swine

0.002 0.006 mg/100 gm

body weight

Requirement

(144)

Fox

0. 12-40 mg per 100 gm

diet

Requirement

(145)

Monkey (young)

0.025-0.030 mg/kg/body

weight

Requirement

(146)

Nicotinic acid

Monkey

5.0 mg per day

Requirement

(147)

Dog

0.5-1.5 mg/kg/day

Requirement

(148-151)

Pig

6.5-10 mg per day

Normal growth

(152-154)

Rabbit

0.2 mg per kg

Minimum for maintenance

(155-157)

0.5-1 mg per kg

Slight growth

5.-10 mg per kg

Maximum growth

Fox

0.39-2 mg per kg body

weight

Requirement

(145)

Pantothenic acid

Dog (young)

0. 1 mg/kg body wt/day

Requirement (adults less)

(158)

Pig (growing)

7.8-11.8 mg/100 lbs

animal/day

Requirement

(159)

Fox

0.25-1.5 mg/100 gm food

Requirement

(145)

Biotin

Pig

0.1 mg per day

To prevent alopoecia, etc.

(160)

Folic acid

Monkey

0.1 mg per day

Requirement

(161)

0.2-0.3 mg per day Bc

Requirement

(162)

Fox

0.5 mg per day

Adequate diet

(163)

Mink

less than 0.05 mg/day

(164)

Choline

Dog

50 mg/kg body weight

Requirement

(165-167)

Why this nutritional dichotomy developed in the evolutionary process
is a matter for considerable speculation. It is apparent that both plants
and animals may survive upon the earth in a biological balance when
only the one group has the higher synthetic ability, and a philosophy
might readily be worked out in terms of a conserving economy in the
distribution of metabolic abilities. More practically, since on a global
scale the plant and animal kingdoms are interdependent, this difference

330 THE BIOCHEMISTRY OF B VITAMINS

may provide one means whereby the animal kingdom cannot exceed the
balance that exists, just as the plant kingdom is held in check by the
carbon dioxide content of the air, the supply of which is influenced by
the animal population. It may be surmised that the dichotomy may have
developed by a stepwise genetic process, leaving in the primitive forms
of both kingdoms {T hallo phytes and protozoa) members having the
ability to synthesize intermediate numbers of B vitamins; and on this
basis the fact that the protozoa and fungi vary in their abilities would
be expected. Arriving thus at a common primitive progenitor with fully
developed synthetic abilities does not seem as improbable as arriving at
one with none, according to present concepts 168 of evolution.

Despite the un verifiable nature of these suggestions, one is tempted to
wonder why nicotinic acid stands out as an exception to the fact that
animals cannot synthesize B vitamins. (There is some evidence that
Tetrohymena gelii and Drosphila melanogaster may be exceptions to
this). If one were forced into the ludicrous discussion as to which one of
the B vitamins is the most vital, he would probably select niacin. Ulti-
mately, life is characterized by activities; these require energy, and
energy is generally derived in animals from high-energy phosphate bonds,
which are most frequently created by a reduction involving Cozymase
and thus nicotinic acid. An extremely primitive system might conceivably
subsist with this B vitamin, whereas the other B vitamins could not
serve this prime purpose of energy production. (In plants, the utilization
of radiant energy presumably involves a reduction of fixed carbon dioxide,
and here again it may well be that nicotinic acid is the first vitamin
involved in the linear process.) It may be considered providential, at
least, that more flexibility is permitted animals in niacin synthesis, allow-
ing the conversion from tryptophan. It may also be because of the par-
ticularly crucial place assigned to niacin in metabolism, that animals,
which are still limited by the tryptophan nutrition and the efficiency of
the process, tend to store nicotinic acid in as nearly finished a product
as possible, nicotinamide,169 whereas the less limited plants store it
primarily as nicotinic acid. Indeed, our existing scanty knowledge sug-
gests that the routes of synthesis of niacin may differ in the animals and
higher plants, since asparagin, glutamic acid, proline and ornithine
seem to be intimately involved in plant synthesis,170 but not in animals,
while tryptophan generally seems to be the major precursor in animals,
but not in plants. This point in the discussion at least should receive
elucidation in the near future.

THE B VITAMIN REQUIREMENTS OF ANIMALS AND PLANTS 331

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77. Sarma, P. S., Snell, E. E., and Elvehjem, C. A., J. Biol. Chem., 165, 55-63 (1946).

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79. Ringrose, A. T., Norris, L. C, and Heuser, G. F., Poultry Sci., 10, 166-77 (1931).

80. Pavcek, P. L., and Baum, H. M., Science, 93, 502 (1941).

81. Martin, G. J., and Ansbacher, S., J. Biol Chem., 138, 441 (1941).

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91. Nielsen, E., and Block, A., J. Nutrition, 28, 203-8 (1944).

92. Arnold, A., and Elvehjem, C. A., J. Nutrition, 15, 403-10 (1938).

93. Jukes, T. H., and Heitman, H„ Jr., J. Nutrition, 19, 21-30 (1940).

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25, 47-51 (1946).

96. Bethke, R. M., and Record, P. R., Poultry Sci., 21, 147-54 (1942).

97. Bolton, W., J. Agr. Sci., 34, 198-206 (1944).

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99. Davis, H. J., Norris, L. C, and Heuser, G. F., Poultry Sci., 17, 81-6 (1938).

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25, 47-51 (1946).

102. Clandinin, D. R., Poultry Sci., 25, 223-31 (1946).

103. Briggs, G. M., Jr., Mills, R. C, Elvehjem, C. A., and Hart, E. B, Proc. Soc.

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104. Jukes, T. H., J. Biol. Chem., 129, 225-31 (1939).

105. Bauernfeind, J. C, and Norris, L. C, Science, 89, 416-17 (1939).

106. Jukes, T. H., J. Biol. Chem., 117, 11-20 (1937).

107. Bird, H. R., and Rubin, M., Poultry Sci., 25, 87-9 (1946).

108. Richardson, L. R., Hogan, A. G., and Kempster, H. L., Missouri Agr. Exp.

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109. Briggs, G. M., Jr., Mills, R. C, Hegsted, D. M., Elvehjem, C. A., and Hart,

E. B., Poultry Sci., 21, 379-83 (1942).

110. Hogan, A. G., Richardson, L. R., Patrick, H., O'Dell, B. L., and Kempster,

H. L, Poultry Sci., 20, 180-3 (1941).

111. Hegsted, D. M., Oleson, J. J., Elvehjem, C. A., and Hart, E. B., J. Biol. Chem.,

130, 423-4 (1939).

112. Lucas, H. L., Heuser, G. F., and Norris, L. C, Poultry Sci., 25, 137-42 (1946).

113. Cravens, W. W., Sebesta, E. E., Halpin, J. G., and Hart, E. B., Poultry Sci.,

25, 80-2 (1946).

114. Hegsted, D. M., Niells. R. C, Briggs, G. M. Jr., Elvehjem, C. A., and Hart,

E. B., J. Nutrition, 23, 175-9 (1942).

115. McCoy, R. H., Felton, J. R., and Hofmann, K., Arch. Biochem., 9, 141-7 (1946).

116. Robertson, E. I., Daniel, L. J., Farmer, F. A., Norris, L. C, and Heuser, G. F.,

Proc. Soc. Exptl. Biol. Med., 62, 97-101 (1946).

117. Oleson, J. J., Hutchings, B. L., and Sloane, N. H., J. Biol. Chem., 165, 371-5

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118. Daniel, L. J., Farmer, F. A., and Norris, L. C, J. Biol. Chem., 163, 349-50

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119. Hutchings, B. L., Oleson, J. J., and Stoksted, E. L. R., J. Biol. Chem., 163,

447-53 (1946).

120. Luckey, T. D., Moore, P. R., Elvehjem, C. A., and Hart, E. B., Science, 103,

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121. Petering, H. G., Marvel, J. P., Glausier, C. E., Jr., and Waddell, J., J. Biol.

Chem., 162, 477-89 (1946).

122. Briggs, G. M, Jr., Luckey, T. D., Mills, R. C, Elvehjem, C. A., and Hart,

E. B., Proc. Soc. Exptl. Biol. Med., 52, 7-10 (1943).

123. Ott, W. H, Rickes, E. L., and Wood, T. R., J. Biol. Chem., 174, 1047-8 (1948).

124. Jukes. T. H., Oleson, J. J., and Dornbush, A. C, J. Nutrition, 30, 219-23 (1945).

125. Record, P. R., and Bethke, R. M., Poultry Sci., 21, 271-6 (1942).

126. Abbott, O. D., and DeMasters, C. U., J. Nutrition, 19, 47-55 (1940).

334 THE BIOCHEMISTRY OF B VITAMINS

127. Scott, M. L., Norris, L. C, and Heuser, G. F, J. Biol. Chem., 167, 261-72 (1947).

128. Swank, R. L., and Bessey, 0. A., /. Nutrition, 22, 77-89 (1941).

129. Jukes, T. H., Stokstad, E. L. R., and Belt, M., J. Nutrition, 33, 1-12 (1947).

130. Fritz, J. C, Archer, W., and Barker, D., Poultry Sci., 18, 449-54 (1939).

131. Hegsted, D. M., and Perry, R. L., J. Nutrition, 35, 411-17 (1948).

132. Briggs, G. M., Jr., J. Nutrition, 31, 79-84 (1946).

133. Briggs, G. M., Jr., J. Biol. Chem., 161, 749-50 (1945).

134. Hegsted, D. M., and Rao, M. N., J. Nutrition, 30, 367-74 (1945).

135. Richardson, L. R., Hogan, A. G., and Kempster, H. L., J. Nutrition 30, 151-7

(1945).

136. Lance, B. G., and Hogan, A. G., J. Nutrition, 36, 369-79, (1948).

137. Waisman, H. A., and McCall, K. B., Arch. Biochem., 4, 265-79 (1944).

138. Leblond, C. P., and Chaulin-Serviniere, J., Am. J. Med. Sci., 203, 100-9 (1942).

139. McCarrison, R., Brit. Med. J., I, 249-53 (1920).

140. Arnold, A., and Elvehjem, C. A., Am. J. Physiol, 126, 289-98 (1939).

141. Van Etten, C., Ellis, N. R., and Madsen, L. L., J. Nutrition, 20, 607-25 (1940).

142. Everett, G. M., Am. J. Physiol, 141, 439-48 (1944).

143. Street, H. R., and Cowgill, G. R., Am. J. Physiol, 125, 323-34 (1939).

144. Hughes, E. H., J. Nutrition, 20, 233-8 (1940).

145. Schaefer, A. E., Whitehair, C. K., and Elvehjem, C. A., J. Nutrition, 34, 131-9

(1947).

146. Cooperman, J. M., Waisman, H. A., McCall, K. B., and Elvehjem, C. A.,

J. Nutrition, 30, 45-57 (1945).

147. Harris, L. J., Biochem. J., 32, 1479-81 (1938).

148. Margolis, G., Margolis, L. H., and Smith, S. G., J. Nutrition, 16, 541-8 (1938).

149. Sebrell, W. H., Onstott, R. H., Fraser, H. F., and Daft, F. S., J. Nutrition, 16,

355-62 (1938).

150. Elvehjem, C. A., Madden. R. J., Strong, F. M., and Woolley, D. W., J. Biol

Chem., 123, 137-49 (1938).

151. Birch, T. W., J. Nutrition, 17, 281-92 (1939).

152. Hughes, E. H., Hilgardia, 11, 595-612 (1939).

153. Wintrobe, M. M., Am. J. Physiol, 126, 375-87 (1939).

154. Braude, R., Kon, S. K., and White, E. G., Biochem. J., 40, 843-55 (1947).

155. Hogan, A. G., and Hamilton, J. W., J. Nutrition, 23, 533-43 (1942).

156. Wooley, J. G., and Sebrell, W. H., J. Nutrition, 29, 191-9 (1944).

157. Olcese, O., Pearson, P. B., and Schweigert, B. S., J. Nutrition, 35, 577-90 (1948).

158. Schaefer, A. E., McKibbin, J. M, and Elvehjem, C. A., J. Biol. Chem., 143,

321-30 (1942).

159. Hughes, E. H., and Ittner, N. R., J. Animal Sci., 1, 116-19 (1942).

160. Cunha, T. J., Lindley, D. C., and Ensminger, M. E., J. Animal Sci., 5, 219-25

(1946).

161. Cooperman, J. M., Elvehjem, C. A., McCall, K. B., and Ruegamer, W. R.,

Proc. Soc. Exptl. Biol. Med., 61, 92-7 (1946).

162. Cooperman, J. M., McCall, K. B., Ruegamer, W. R., and Elvehjem, C. A.,

J. Nutrition, 32, 37-45 (1945).

163. Schaefer, A. E., Whitehair, C. K., and Elvehjem, C. A., Arch. Biochem., 12,

349-57 (1947).

164. Schaefer, A. E., Whitehair C. K., and Elvehjem, C. A., Proc. Soc. Exptl. Biol.

Med., 62, 169-74 (1946).

165. McKibbin, J. M., Thayer, S., and Stare, F. J., J. Lab. Clin. Med., 29, 1109-22

(1944).

166. Ruegamer, W. R., Michaud, L., Elvehjem, C. A., and Hart, E. B., Am. J.

Physiol, 145, 23-7 (1945).

167. Enterman, C, and Chaikoff, I. L., J. Biol. Chem., 138, 477-85 (1941).

168. Horowitz, N. H., Proc. Natl. Acad. Sci. U. S., 31, 153-7 (1945).

THE B VITAMIN REQUIREMENTS OF ANIMALS AND PLANTS 335

169. Krehl, W. A., de la Huerga, J., Elvehjem, C. A., and Hart, E. B., J. Biol. Chem.,

166, 53-7 (1946).

170. Ellinger, P., and Kader, M. M. A., Biochem. J., 42, ix (1948).

171. Sarma, P. S., and Sreenevasaya, M., Current Sci., 8, 551 (1939).

172. Sarma, P. S., and Sreenevasaya, M., Current Sci., 10, 525-6 (1941).

173. Ramaswami, S., Sarma, P. S., and Sreenevasaya, M., Current Sci., 11, 53-4

(1942).

174. Swamy, B. G. L., and Sreenevasaya, M., Current Sci., 11, 147-8 (1942).

175. Sarma, P. S., and Bhaguat, K, Current Sci., 11, 331-2 (1942).

176. Swamy, B. G. L., and Sreenevasaya, M., Current Sci., 9, 493 (1940).

177. Sarma, P. S., and Bhaguat, K., Current Sci., 11, 394 (1942).

178. Sarma, P. S., Indian J. Med. Research, 31, 161-3 (1943).

179. Sarma, P. S., Indian J. Med. Research, 31, 165-71 (1943).

180. Bhaguat, K., and Sarma, P. S., Indian J. Med. Research, 31, 173-81 (1943).

181. Schweigert, B. S., Vitamins and Hormones, 6, 55-67, (1948).

182. Bonner, J., and Bonner, H., Vitamins and Hormones, 6, 225-275, (1948).

183. Beerstecher, E., Jr., Science, 111, 300-312, (1950).

Chapter IVC

METABOLISM OF THE B VITAMINS

It is proposed to outline in this chapter the essential facts concerning
the processes which the B vitamins undergo from the time they are
synthesized or ingested by the organism until they are excreted. As in
other chapters in this section, the information in most cases is empirical ;
a true understanding of the subject does not exist. In both plants and
animals, however, there are certain broad categories which may be referred
to as "stages" in the metabolism. For convenience, these may be referred
to as digestion, absorption, distribution, anabolism and catabolism, and
excretion, and the following discussion proceeds in this order. Since plant
metabolism involves many differences in detail, and its consideration is
limited by the meager amount of data bearing upon the subject, it is con-
sidered independently.

Metabolism in Plants

Origin in the Plant. The precise pathways involved in B vitamin
biosynthesis have been considered briefly in an earlier section. The cyto-
logical location of this synthesis in the single-celled plants is not known,
but it seems relatively certain that the synthesis occurs in the leaves of
the higher green plants, which are generally considered to be the major
focus of synthetic activity in the plant. Since many alkaloids are also
synthesized in this highly active metabolic area,224 it is no wonder that
some B vitamin moieties may be recognized in certain alkaloids (arecoline,
guvacine, nicotine, ergot alkaloids) . B Vitamins are also supplied to the
higher plants from the soil and from symbiotic microorganisms, although
the extent to which this source is important to the higher plants is
unknown. The B vitamins reach the heterotrophic portions of the plant
largely through the translocation stream by which other leaf synthetic
products are transported, but also by diffusion from neighboring cells and
by absorption from the exterior environment.

Digestion and Cellular Absorption in Plants. Since the B vitamins are
frequently found in plants in bound forms, it is apparent that the plants
must first release the stored vitamins in order to utilize them for their
own purposes, and annual cyclic variations in the relative amounts of
free and bound vitamins are known to occur in some cases. Since plants

336

METABOLISM OF THE B VITAMINS 337

are capable of binding the B vitamins, either to create structural enzyme-
coenzyme forms or insoluble storage forms, our knowledge of the mech-
anism of catalysis would suggest that they also would be able to liberate
these forms. If, as suggested (p. 316), biotin functions as a hormone in
plants, flowing downward from the tip, then its solubilizing release from
the bound form in an auxin-like fashion (p. 37) is of major importance
in plant differentiation.1- 225 In the case of many Thallophytes, digestion
as we know it may frequently occur outside the plant. Although the bac-
teria particularly are well known for their ability to liberate vitamins
from combination, in some cases, as in the avidin-biotin complex, even
bacteria may be incapable of digesting the bound form. There is a more
detailed discussion of enzymatic liberation of B vitamins earlier in this
volume (Chap. Ill A).

It is generally assumed that water-soluble forms of the B vitamins
may diffuse readily in and out of plant cells, but that bound forms, par-
ticularly protein-bound forms, do not. These assumptions are frequently
unjustified, and are based largely on analogy with questionable data in
the animal kingdom. Since there is little evidence bearing directly on the
subject in plant cells and tissues, it seems pertinent at least to point out
that the diffusion across a cell membrane or tissue barrier may involve
intermediate formation, and is most frequently a selective process not
involving simple diffusion in the strictly physical sense. The active
absorption of thiamine by live yeast in the animal intestine is particularly
significant in this regard. Further, some bound forms, even proteinaceous
ones, may be quite capable of diffusion across "semipermeable" mem-
branes. Further extended studies will therefore be required to determine
the nature of this process in plants.

Distribution, Catabolism, and Excretion in Plants. The salient facts
with regard to the distribution of the B vitamins in plants have been
considered in an earlier section (Chap. II A). Thiamine, vitamin B,3,
niacin, pantothenic acid, and biotin seem largely concentrated in the seeds,
while riboflavin, inositol, and folic acid are most concentrated in the
leaves. Without dwelling unduly upon this latter fact, and without ques-
tioning the importance of all B vitamins to the photosynthetic process,
it seems of sufficient interest to note that inositol occurs in leaves largely
as the hexaphosphate,2 and may function in this case as a phosphate
storage form; that riboflavin has been implicated in phototropism in
plants,3 a phenomenon that is closely integrated with photosynthesis; and
that folic acid may well have a very special role in the photosynthetic
process.

Strangely, little is known concerning the role of the B vitamins in
photosynthesis, and in one recent monograph on photosynthesis 4 no
B vitamin is even so much as mentioned in the index. It is known that

338 THE BIOCHEMISTRY OF B VITAMINS

pantothenic acid is not synthesized in the leaves of green plants until the
photosynthetic process commences, but whether this is a cause or an
effect is uncertain. Beyond this, little is known of annual cyclic variations
in those plants that maintain their photosynthetic ability throughout
the year.

In the Thallophyta, and within single cells in general, very little is as
yet known with regard to the possible localization of B vitamins. It is not
possible to report any reliable information as to distribution between the
nucleus, cytoplasm, and cell wall, or as to distribution in different parts
of the cytoplasm, or as to distinct changes during mitosis. There will un-
doubtedly be much intensive study of this subject during the next few
years as a result of the intensive research efforts now in effect in the field
of cytochemistry, and of the rapidly increasing number of techniques now
being reported which should facilitate such study.220

Practically nothing is known with regard to the breakdown products
of B vitamins in green plants. A fertile and relatively simple field for
exploration awaits the investigator of this point, with the information
found in animal catabolism well developed to serve as a guide. In the
case of the Thallophyta, furthermore, the breakdown products of B vita-
mins have been but little more studied, despite the vast amount of infor-
mation available on the bacterial catabolism of other metabolites.

Plants excrete B vitamins into the surrounding medium, and vitamin
production by certain bacteria has already been noted (p. 299). Indeed
in certain molds {Eremothecium ashbyae, Ashbya gossypii) riboflavin
excretion is so pronounced as to form riboflavin crystals about the myce-
lium.5 Higher plants secrete vitamins into the soil from their roots, and in
several instances (i.e., thiamine, inositol) extended studies have been made
of the subject; these have indicated that the high localized vitamin con-
tent of the soil is a factor in increasing the bacterial population in these
areas.6 The exact process involved in excretion, as in absorption, however,
is unknown, and such factors as threshold values are as yet unavailable.

Digestion in the Animal Organism

The many complex activities that proceed in the animal gastrointestinal
tract may produce three major changes in the nutritional vitamin forms:
liberation, activation, and destruction. Most frequently, the bound unab-
sorbable vitamin complexes may be broken down and the vitamin liber-
ated and made available for absorption. Associated with this process, the
cellular structure of the food may (or may not) be destroyed. Less often,
the vitamin may be so modified in the intestinal tract as to provide a
more active fprm than that ingested, for example, a form more closely
related to the functional form. Finally, destruction or inactivation of

METABOLISM OF THE B VITAMINS 339

B vitamins may occur. These effects may be produced as the result of
enzymes indigenous to the animal, or as the result of intestinal microflora;
but in most cases it is not now possible to distinguish whether one or
both factors are involved. The establishment of whether the digestive
effect is due to animal or microfloral action is important, since the assess-
ment of factors which may affect the digestive process must be made in
terms of which member of this digestive partnership is involved. When
large amounts of a nutritional component are processed in the digestive
tract, bacterial effects upon the component may be negligible; but in the
case of small amounts of catalytic materials, bacteria may move the
digestive process into a radically different channel.

The part of the B vitamin nutritional intake which is supplied by
bacteria is (most logically) largely present in the intestine in free form,
and is not further materially affected by the digestion. A large fraction
of the exogenous B vitamin nutrition is in bound form, however. In some
cases cooking of the food may suffice to break the complex. This is reported
to be markedly true in the case of riboflavin, and to a variable and lesser
extent in the case of the other vitamins; it is, of course, dependent upon
the conditions of temperature, pH, and concentrations of other ingredients.
Cooking does not produce sufficient liberation of available forms to be
considered as a major factor in the process. While some liberation may
occur in the stored uncooked food as the result of ripening processes or
autolysis, this factor seems not to be of any considerable importance in
the overall liberation. Thus, water-soluble choline compounds (choline
glycerophosphoric esters) appear quite rapidly when rat intestine and
stomach are allowed to autolyze, but there is only very slow liberation
in lung and kidney autolysates, and scarcely any in brain, liver, and
heart.7

The B vitamin-protein complexes of the food are to a great extent
broken down in the gastrointestinal tract, and a limited amount of
evidence suggests that this process occurs largely in the duodenum. This
process may not be as efficient in some cases as in others, and in the case
of pantothenic acid complexes particularly, there is some evidence to
suggest that the liberation is not as complete as in the case of the other
B vitamins.8 Since it seems quite likely that the functional form of
pantothenic acid, unlike most of the other B vitamins, involves a union
of the vitamin with an amino acid (glutamic acid), it seems pertinent
to suggest that the binding of pantothenic acid may be stronger for this
reason. Similarly the apparent unavailability of some of the more complex
forms of folic acid to some bacteria and to pernicious anemia patients
suggests that a vitamin connected in its functional form (coenzyme) to

340 THE BIOCHEMISTRY OF B VITAMINS

an amino acid {e.g., glutamic acid) may not be as readily released as
vitamins not normally so linked in the functional form.

A brief consideration of the structures of the B vitamins shows that a
diversity of active groups is present which may participate in binding to
proteins. Carboxylic acid groups are present in five of the B vitamins,
hydroxyl groups in six, aliphatic or aromatic primary amino groups in
four, and phenolic groups in three, with a scattering of other active struc-
tural groupings. Generally phenolic groups and aromatic amino groups
remain intact in metabolism so as to function in redox reactions; and
hydroxyl groups are frequently phosphorylated in functional forms. It
would, therefore, seem most likely that the B vitamins are generally
bound via their acid or aliphatic amino groups or via the acid groups of
their phosphates to suitable active groups in proteins, and that the
amido and salt linkages so formed should be quite readily hydrolyzed
by pH extremes or certain phosphatases and digestive enzymes. Thus
thiamine, riboflavin, choline, and inositol are the B vitamins which have
well known and widely occurring phosphates, but few other good groups
for protein binding, and so may logically be bound via phosphate mole-
cules to protein ; whereas nicotinic acid, pantothenic acid, p-aminobenzoic
acid, biotin, and folic acid do not have widely distributed (or well
known) phosphates, but do have carboxylic acid groups capable of combin-
ing with free protein amino groupings. Nicotinamide may possibly be
bound through its amide grouping ; indeed either it or nicotinic acid might
be produced upon liberation, depending upon the course of the action.
The B6 vitamins most logically would be bound via their 5-hydroxy-
methyl group (phosphorylated?) since other active groupings are
involved in the vitamin function and must necessarily remain intact.
Such reasoning supposes that each vitamin contains separate groupings
for performing its primary function and for attaching it to its protein
enzyme — an hypothesis which fits well the existing information on this
subject. In vitro studies with pure enzyme preparations should do much
to elucidate the nature of the binding involved in each case, and the
enzymes capable of vitamin liberation. The fact that the avidin-biotin
complex is not broken by the intestinal processing indicates that the
nature of the binding may not be deduced with complete accuracy on
the basis of structural considerations alone, although the — CO — NH —
grouping common to both biotin and protein linkages may be suggestive
of secondary binding effects.

Individual differences in digestive ability, whether within physiological
or pathological limits, are doubtless among the important factors which
influence the broad individual differences in B vitamin requirements.
This fact is extremely evident in the case of the folic acid conjugases,

METABOLISM OF THE B VITAMINS 341

which may be well utilized by normal persons, but are apparently un-
available to patients with pernicious anemia, although folic acid itself
is effective. In this instance there is some reason to believe that "vitamin
B12" functions in the enzyme which hydrolyzes vitamin Bc conjugate to
folic acid.9 A variety of other disturbances which involve the gastro-
intestinal tract have similarly been shown to produce B vitamin de-
ficiencies, and may involve drastically reduced abilities to liberate bound
forms of the B vitamins. Among healthy persons, the differences may not
be so manifest, but they undoubtedly do exist and may markedly pre-
dispose certain individuals and groups to avitaminoses. The factors
influencing intestinal liberation of the B vitamins are at least as manifold
as those affecting the digestive process in the broader sense. They lack
experimental elucidation at present, but may be surmised generally on
the basis of our overall knowledge of gastrointestinal digestion. Because
of the lack of data, a more extended consideration of these factors is
not now possible.

Mention has previously been made of the unavailability of the B
vitamins in live yeast (p. 291) , and this must be considered in the broader
sense as a digestive limitation. It seems equally certain that other cellular
forms that are not disrupted during digestion may similarly withhold
their vitamins, so that cellular disintegration is a critical factor in the
digestive process. While the cells of most food material are apparently
not as resistant to fracture as are yeast cells, further investigation of
this point is merited. The possibility of irreversible adsorption of the B
vitamins upon other nutritional components in the intestine seems also
worthy of consideration in this regard. Fuller's earth adsorbates of rice
polishing extract were early used as a thiamine standard, but it has been
subsequently shown that only about half of the thiamine present could
be eluted in the animal digestive tract.10 Similar adsorbents are now
broadly used as medicants, and undoubtedly they similarly limit the
available thiamine in some cases. Cellulose may exert a similar effect,
although the evidence now available seems to disprove this belief.11

The free vitamin may in some instances be converted by the digestive
process to an even more active form, although this is not apparently a
general process. Phosphorylation and bacterial conversion to functional
forms probably account for the cases in which this is so, although it is
by no means certain that other vital changes in vitamin structure do not
occur in the intestinal tract of some species. The conversion of the higher
homologues of folic acid to folic acid in the animal digestive tract must,
moreover, be considered as one example of an increase in vitamin activity
in the light of our present understanding of this group of substances. In
addition, it is probable that many other such apparent effects are actually

342 THE BIOCHEMISTRY Ut' B VITAMINS

due to the favorable influence of the exogenous vitamin on endogenous
bacterial synthesis in general, rather than to actual interconversion of an
exogenous molecule by a bacterium to a more active derivative of the
same molecule.

Finally, the chemical processes encountered in the digestive tract un-
doubtedly result in the destruction of some percentage of ingested B
vitamins; this factor is a major one in certain of the cases where paren-
teral vitamin administration is markedly more effective than feeding
per os. Biotin, for instance, is said to be five times more active parenter-
al^,12 and it might be expected that the — CH— NH— CO— NH— CH—
structure of its ring would receive some destruction by enzymes active
upon peptide linkages. For the same reason, folic and pantothenic acids
may be hydrolyzed to some extent in the intestine, and there may be
some cleavage of carbon — nitrogen bonds, such as those on the ribitol in
riboflavin and on the thiazole moiety in thiamine. Pyridoxal and pyridox-
amine are also less active and may be partially decomposed when fed
orally, as might be anticipated from their general chemical reactivity
in vitro. Vitamin B12 is readily destroyed by the digestive processes, and
it is essential that this vitamin be protected by conversion into a bound
form. Ternberg and Eakin 12a have recently shown that "intrinsic factor"
(p. 415), a protein material present in the gastric juices, has this ability
of combining with vitamin Bi2 and protecting it from digestive destruc-
tion. In pernicious anemia, intrinsic factor is absent from the gastric
juice, and a vitamin Bi2 deficiency results due to the digestive destruction
of the unprotected vitamin.

Since our nutrition is oral, and the B vitamin nutritional requirement
is a summation of all these effects, digestive destruction is not generally
a matter for extreme concern. In experimentation or medication where
parenteral administration is used, it is important, however, to take
cognizance of the fact that a much larger amount of vitamin may reach
the animal via this route than by supplying a similar amount orally,
from the standpoint both of the physiological effects that may result, and
of exceeding the limits below which the vitamin is not toxic. As will be
shown later in some detail, parenteral administration is most commonly
practiced with the two vitamins that appear to be most toxic, thiamine
and nicotinic acid. In other cases, as in these, extrapolation of the oral
therapeutic dose to the parenteral one is dangerous, largely because of our
limited knowledge of the degree of destruction of the oral dose in the
intestine.

Finally, it should be pointed out that despite the fact that the B vita-
mins are markedly soluble in water and generally just as insoluble in
organic solvents, they may in some cases nevertheless have to undergo

METABOLISM OF THE B VITAMINS 343

transformations, like the fat-soluble vitamins, to render them absorbable.
This is most certainly so in the case of some of the absorbed forms of
choline, and may be true of other vitamins, such as inositol. For some
of the lower forms of life, for which the nutrition must reach the absorp-
tive membrane in particulate form, and liquid media will not suffice, a
similar consideration may well be involved. In any case, the water
solubility of a substance cannot be considered a -priori to be the final
end to be achieved in the digestive process.

The Absorption of B Vitamins

The state of knowledge of the processes involved in the absorption of
the individual B vitamins is to a large extent a function of the time that
the B vitamin in question has been well recognized. The more explicit
information which is available concerning thiamine and riboflavin absorp-
tion indicates clearly that the absorption of the B vitamins cannot be
regarded as a simple process, even though it is frequently assumed to be
for the more recently discovered vitamins. Passage of a metabolite across
a living membrane seldom is a matter of passive transfer or simple
diffusion. This, is certainly a good generalization for the B vitamins, even
in view of the lack of much experimental data to verify the assertion. It
should also be reemphasized at this point that vitamin-protein complexes
may be absorbed in some cases, just as undigested proteins in general
may be absorbed to a limited degree.

In the case of both thiamine and riboflavin, it is believed that phos-
phorylation occurs in the intestinal mucosa prior to absorption.13 Through-
out the gamut of physiological processes, phosphorylation is a frequent
adjunct to the passage of metabolites of many kinds across membranes,
so that in the cases of thiamine and riboflavin the process is by no means
unique. Both thiamine and its phosphate are readily absorbed in the
small gut;14 and although thiamine occurs in the blood plasma in the
free form15 (part of which may be again secreted in the gastric juice),
duodenal phosphorylase readily phosphorylates thiamine in vitro,1Q> 17
so that phosphorylation and dephosphorylation apparently are involved
in passage across the intestinal wall into the circulation, as in the case
of transfer of many other metabolites. Riboflavin, its 5'-phosphate, and
its adenine dinucleotide are all available to the higher animals, but it is
possible that the coenzymes are broken down prior to absorption. Free
riboflavin is phosphorylated prior to absorption,13, 18 and the phosphoryla-
tion may be clone in vitro with mucosal extracts. Interference with the
process by iodoacetate or by adrenalectomy in rats causes a prompt lack
of free riboflavin absorption and ensuing cessation of growth, which may

344 THE BIOCHEMISTRY OF B VITAMINS

however be prevented by nutritional riboflavin-5'-phosphate or the
dinucleotide coenzyme form.19

Only scant knowledge exists in other cases. Free B vitamins are
generally believed to be readily absorbed in the intestine, but probably
not as such. It is not certain whether cozymase and inositol phosphate
are absorbed directly or first split to simpler products, but both nicotinic
acid and its amide are apparently absorbed. Recent evidence indicates
that coenzyme A is probably hydrolyzed prior to pantothenate absorp-
tion.20 By analogy with thiamine and riboflavin, it seems possible that
many of these vitamins undergo phosphorylation prior to absorption;
this point merits further investigation. Since absorption processes and
"thresholds" are apparently major factors in influencing the individual
differences in efficiency of B vitamin utilization and requirements, it is
indeed surprising that more extended study of this subject has not been
undertaken to date.

It is readily apparent that a large number of factors may influence
the ability to absorb the B vitamins. Diets high in fats may mechanically
prevent ready access of the vitamin to the absorptive membrane.
Pharmaceutical derivatives of riboflavin have been prepared with such
low solubility as to be poorly absorbable. In the case of renal resorption
of amino acids, competition may exist between amino acids that are
resorbed by a similar mechanism for the limited metabolic activity of
the membrane,21 and it seems quite logical that an excess of metabolites
that are transported across the intestinal wall by a mechanism (e.g.,
phosphorylation) similar to that involved with a vitamin may diminish
the absorbability of that vitamin. This factor may explain the observa-
tion that the presence of food in the gastrointestinal tract diminishes the
absorption of calcium pantothenate in dogs.22 Certainly any factor that
influenced the phosphorylation activity of the intestine would have this
effect, and this consideration may well be advanced as one other that
favors a balanced diet containing a variety of nutrients. At present, how-
ever, it is entirely impossible to estimate what portion of vitamin mal-
nutrition is due to absorption difficulties. It is known that wide variations
do exist in the ability to use dietary components, and further extended
studies are necessary to show what effect the state and nature of the
nutrition have on the ability of an individual to utilize B vitamins. We
have already noted (p. 300) that large amounts of B vitamins occur in
the feces, and it is most uncertain what portion of this material is of
exogenous origin and whether its presence is due to its inavailability to
the animal or to its not being required. Over 60 per cent of the B vitamins
of feces are said to be water-soluble, and should be available.23 Perhaps
by a better understanding of the absorptive process, we may some day

METABOLISM OF THE B VITAMINS 345

hope to understand this, as well as the innumerable clinical reports deal-
ing with the marked beneficial effects of large doses of some one vitamin
in an individual where the pathology bears no apparent relation to the
medication.

Distribution of the B Vitamins

Much specific information has been included in an earlier chapter on
the occurrence and distribution of the B vitamins that might be recon-
sidered at this point. It would seem more appropriate, however, to con-
sider in more general terms and from a somewhat dynamic standpoint
the various general relationships that exist in the distribution process,
leaving the interpretation of the voluminous data found in the literature
on B vitamin occurrence for those whose needs justify the labor that must
necessarily be involved in so arduous a task. The allocation by the cir-
culation of the various B vitamins to the tissues is a complex process, and
we can at best do little more than guess at the principles involved.

State and Levels in the Circulation. Once the B vitamins are intro-
duced into the circulation, they are rapidly distributed between the
cellular elements and the plasma, and almost as rapidly between the
blood and the tissues, so that an equilibrium is usually maintained. It
is generally true that the plasma does not in itself modify the B vitamins,
although in some cases "binding" may occur. The blood is generally in-
capable of any vitamin liberation, being unable to break, for instance,
the avidin-biotin complex.24 Parenteral administration of the complex,
however, results in its destruction by tissue oxidative processes.

The thiamine in blood plasma is largely in the free state, but not
entirely so, whereas that in the cellular elements of the blood is mostly,
if not entirely in the form of cocarboxylase.25 Apparently all nucleated
cells are capable of performing the phosphorylation, and adult red blood
cells are believed to have acquired their cocarboxylase content prior to
loss of the nucleus while in the bone marrow.26 The total blood thiamine
in normal humans generally ranges between 8 and 9 fig per cent, although
some workers have reported values as high as 14.5 fig per cent.27 Of this,
apparently 70-90 per cent is esterified.28"30 Pig blood apparently contains
about 20 fig per cent;31 oxblood, 5.7 fig per cent;25 pigeon blood 20.2
fig per cent;25 rat blood 7 ^g per cent;32-33 and rabbit blood about 28.3
fig per cent of total thiamine.34 Various reported figures relating to the
distribution of free and esterified thiamine between plasma and cells,
and in pregnancy, and placental and amniotic fluid,36 and infant blood 37
may be found in the literature, but are not at present sufficiently sub-
stantiated to merit general acceptance.

346 THE BIOCHEMISTRY OF B VITAMINS

Riboflavin is converted by the red blood cells (probably only nucleate
erythrocytes) and most other cells of the body into flavin adeninedinucleo-
tide, but this reaction cannot be performed by the plasma.35- 38 There
is apparently a rather constant equilibrium between the coenzyme levels
in cells and plasma; and the level in the whole blood remains quite steady
under most conditions. Human blood is said to contain the equivalent of
about 21.2 ixg per cent, varying somewhat annually in places where there
are marked seasonal dietary changes.39 The blood plasmas of several
Brazilian snakes contain levels ranging from 180 to 300 /^g per cent.40

CHICK^

^^^■"OChick

^c£-^"

HJ 30-

Q
O

°MAN

v-^IC

10-

rf'MAN

.01 .03 .10 .30 1.0

REQUIREMENT (MG/kg/dAY)

Figure 15. Relationship of the requirements by various species for thiamine and
pantothenic acid to the blood levels of these vitamins.

Nicotinic acid and nicotinamide enter the blood, as far as is now known,
as such, although it is possible that small amounts of cozymase may
escape digestion and be directly absorbed. Blood plasma contains both
the acid and the amide, and these are converted to cozymase41 in
nucleate blood cells (for the most part white cells, which contain very
little of the free vitamin) . In humans, the blood level normally ranges
from 400 to 700 fig per cent, of which about one-third is free and the
balance is combined as coenzyme.42-45 The blood level of nicotinic acid
is not generally lowered appreciably in pellagra.46 Horse blood contains
about 180 fig per cent free and 140 ^g per cent combined nicotinic acid,
while in the cow the corresponding levels are about 268 and 332 fig per
cent, respectively.

Pantothenic acid is found in the blood of man in concentrations of
about 30 fig per cent,47 and in chicks in levels of about 40 fig per cent
(50 fig per cent in the plasma),48 but little is known of its state or dis-

METABOLISM OF THE B VITAMINS 347

tribution, and even less of the states of the remainder of the B vitamins
in the circulation. On the basis of thiamine, riboflavin, and nicotinic acid,
it would appear that free B vitamins entering the plasma are unchanged
there, but largely enter the nucleate blood and tissue cells where they are
converted to coenzymes, leaving only a low residual free vitamin content
in the blood. How true this is of the other vitamins remains to be deter-
mined.

With regard to the relationship of the blood levels of B vitamins to the
nutritional requirement for these vitamins, three general relationships are
apparent. Except those vitamins that may be supplied to a large extent
by endogenous sources such as niacin and biotin, the blood levels of any
given vitamin for a number of species tend to increase as the nutritional
requirements on a unit body weight basis increase, i.e., as the sizes tend
to decrease (p. 246) .227 This is in line with Williams' observation that there
is a "tendency for the vitamin content to be lower in the tissues of larger
animals." 49 With the same exceptions, for any given species, the blood
levels of various B vitamins tend to vary with the vitamin requirements.
These relationships are shown in Fig. 15. Finally, as indicated in an
earlier section, at levels of vitamin intake below the nutritional require-
ment, the blood level tends to reflect the intake, whereas at higher dietary
levels it does not. The urinary levels, conversely, reflect only the higher
intake levels, for obvious reasons. With regard to this last generalization,
so many qualifications and apparent exceptions exist, and, as previously
mentioned, so many other dietary factors influence the balance, that it
must be taken only as a rather self-evident and frequently demonstrable
trend, and not relied upon quantitatively.

Levels in Milk. For a number of readily apparent reasons, there has
been extensive study of the levels of the various B vitamins in the milk
of a number of species. Much of the existing knowledge as to actual levels
is summarized in Table 27. Present limited information suggests that
thiamine, biotin, and inositol occur in milk in bound forms (or in more
firmly bound forms than the other B vitamins) ; but little is actually
known about the precise vitamin forms in milk. Within certain limits,
the vitamin content of the milk reflects that of the diet. Among a number
of species, it is known that the B vitamin content of the milk generally
increases as the requirement increases, and in any one species, the relative
amounts of B vitamin in milk tend to vary with the requirements, as
shown in Fig. 16.

The colostrum in ruminants seems to be higher in vitamins (Bi and
B2) than is the adult milk,50 whereas the inverse seems to be true in the
human. Macy et al.51' r>2 have shown that the following variations occur
in human milk:

348

THE BIOCHEMISTRY OF B VITAMINS

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METABOLISM OF THE B VITAMINS

349

(a) Thiamine: 0.9-2.4 fig per cent during first few days after parturi-
tion, rising to 8.1 fig per cent on tenth day and about 148 fig per cent in
mature milk. Free thiamine starts at 1 fig per cent, rising to 5-7 fig per
cent, and averaging 37 per cent of the total.

r 10r100

-I
2

L.01

A MOUSE
O MAN

OPYRIDOXINE

10 MOUSE (>*C/DAY) 100

1000

1 MAN (MG/DAY) 10

100

VITAMIN REQUIREMENT

Figure 16. Relationship of the B vitamin requirements of man and the mouse to
the levels of B vitamins found in the milk of these species.

(b) Riboflavin: 19.6 jug per cent on first day of lactation, rising to
39.2 fig per cent on the tenth day, with free riboflavin rising more slowly.
Mature milk contained a total of about 35-49 fig per cent, with free ribo-
flavin composing 43-86 per cent of the total.

(c) Nicotinic acid: 100 fig per cent on the first day, decreasing slightly
from second to fourth day and then rising rapidly to about 250 fig per
cent on tenth day. Mature milk contained 175-200 fig per cent.

350 THE BIOCHEMISTRY OF B VITAMINS

(d) Pantothenic acid: 48 /tg per cent on first day to 245 tig per cent
on the fourth day and rising to 304 tig per cent on the tenth day, with
mature milk containing about 250 tig per cent.

(e) Biotin: Very low first five days, rising to about 0.38 tig per cent
on ninth day and 0.80 tig per cent in mature milk.

Pearson's data on cows and ewes show, however, that these trends are
not necessarily general. Thus:50

(a) Cow colostrum contains 62 tig per cent thiamine and 610 /xg per
cent riboflavin, while cow milk contains 38 tig per cent thiamine and
177 tig per cent riboflavin.

(b) Ewe colostrum contains 180 tig per cent thiamine and 2008 tig per
cent riboflavin, while ewe milk contains 60 tig per cent thiamine and
436 tig per cent riboflavin.

(d) Pantothenic acid is higher in the milk of both species.

All workers in this field seem generally agreed that variations between
individuals are great in regard to milk vitamin content. Since the level
in the milks of individuals receiving similar diets must involve the intes-
tinal-circulatory absorption threshold, the excretory thresholds, and the
circulatory-acinus threshold, there is ample reason to anticipate such
marked individual differences. In consideration of the low content of
human and cows' milk in relationship to infant requirements, particularly
in regard to thiamine and niacin, at a time when there is only limited
intestinal synthesis in the young, and of the broad individual differences
in human milk, the concept of milk as a perfect nutrient for the young
should not be accepted uncritically.

Distribution and Storage in Tissues and Body Fluids. The B vitamins
are circulated and pass readily from the blood into the various tissues and
fluids of the body, in each case establishing an equilibrium between the
content of the tissue and the environment. A discussion of tissue profiles
and various interrelationships that exist in this regard are to be found
elsewhere in this volume. Ultimately, studies of distribution constants
may make possible the calculation of tissue contents from vitamin intakes,
but this is not now generally possible. In the tissues, the vitamins are
frequently fixed into firmly bound coenzyme-enzyme complexes or vita-
min-protein storage forms. It has been shown that practically all the
pantothenic acid of the body tissues is in coenzyme form,53, 54 and it
seems apparent that in the cases of most other B vitamins it will eventu-
ally be shown that the coenzyme form accounts for the majority of the
vitamin present. It has previously been mentioned that nicotinamide is
the predominating form of that vitamin in animal tissues (p. 330), while

METABOLISM OF THE B VITAMINS 351

nicotinic acid predominates in plant tissues. In animal tissues and yeast
pyridoxal and pyridoxamine predominate, while pyridoxine is present in
larger amounts in plants.55 Of theoretical interest in this regard is the
fact that oxybiotin functions per se in animal tissues and is not converted
to biotin.56 Its storage in tissues parallels closely that of biotin.57 Vitamin
storage in animals is a limited process, being influenced by many factors,
most frequently by the protein intake, as previously mentioned (p. 277) .
Whereas storage doubtless occurs throughout the body, the liver seems to
be particularly effective as a storage depot in many cases. As previously
noted, rats on a low-protein diet are not able to store riboflavin, regard-
less of the intake, but excrete unused excesses promptly, whereas high
riboflavin intakes on a high-protein diet bring about prompt liver storage
of riboflavin.58 Similar relationships have been observed for other vita-
mins,59 although storage abilities vary among species and for each
vitamin. Thus the depletion of some vitamin in one species {e.g., thiamine
in the rat) may cover a long period of progressive pathology, while in
others (e. g., thiamine in the mouse), the depletion is so rapid that death
is one of the first observable symptoms.60 For this reason, the general
nature of and trends in B vitamin storage are so erratic as to make
generalization impossible for practical purposes. At best, the possibilities
of building up extensive B vitamin reserves are limited, unlike the fat-
soluble vitamins, and current trends in nutritional thought along these
lines should be considered critically in regard to both humans and farm
animals.61 The desirability of increasing the vitamin level in meats in
certain areas is great, and it has been shown that it is possible to increase
the thiamine level in hog tissues by feeding higher levels of thiamine in
the diet.62 It should be noted that the hearts and livers of hogs on lower
thiamine levels, however, had more riboflavin than did those tissues from
animals receiving adequate thiamine. In the chicken, increases in muscle
pantothenic acid cannot be achieved by dietary supplementation with
pantothenic acid levels above those required for adequate nutrition, how-
ever. A considerable amount of data has recently shown that within limits
the riboflavin content of hens' eggs may be increased by increasing the
dietary riboflavin, and in such cases when the efficiency and limits of
effectiveness are known, higher feeding levels are obviously desirable
in fowls.63-66

Synthesis and Catabolism in Animals

The biogenesis of the B vitamins has been discussed at some length m
an earlier chapter, but certain considerations make it seem appropriate
to review some aspects of this subject as it applies to the higher animals.
Whereas most animals have the ability to convert the commonly recog-

352 THE BIOCHEMISTRY OF B VITAMINS

nized forms of the B vitamins to their respective functional forms, only
in the cases of nicotinic acid and choline are the animals apparently able
to synthesize the vitamin from some precursors. Special consideration
must be given to the synthesis of these vitamins, therefore, and also to
the breakdown of nicotinic acid, concerning which we have considerable
knowledge. The transformation of the other B vitamins to their excretory
products has been little studied and few data are available in their regard.
Coenzyme Synthesis. Thiamine, riboflavin, pyridoxal, nicotinamide,
and pantothenic acid are known to function in the specific coenzymes
cocarboxylase, flavin mononucleotide and dinucleotide, codecarboxylase,
coenzymes I and II, and coenzyme A, respectively, all of which contain
phosphate. In the case of cocarboxylase, flavin mononucleotide, and
codecarboxylase, the vitamin phosphate apparently constitutes the entire
coenzyme, and the synthesis of the coenzymes by phosphorylases is ap-
parently performed within the various cells of the animal in a rather
direct manner. This is certainly so in the case of thiamine, since prepara-
tions from liver and kidney, and to a lesser extent muscle and brain
actually convert it to cocarboxylase.67 Rat kidney extracts in phosphate
buffer at pH 8.4 and containing a trace of arsenite are particularly
effective in this respect,68 and it has been shown that some energy-
yielding system is necessary to perform the phosphorylation,69 as would
be expected. It is unclear, however, just how the vitamin B6 triad func-
tions in maintaining the codecarboxylase concentration, although it seem.s
likely that pyridoxine is first oxidized and then phosphorylated to
pyridoxal phosphate; this then functions in a reversible transamination
system with pyridoxamine phosphate in those processes which activate
the a carbon atom of a-amino acids, such as transamination, decarboxyla-
tion, and trytophan decomposition. Present evidence indicates at least
that pyridoxamine requires the presence of a keto acid for its conversion
to pyridoxal 69 (p. 176) . It further seems likely, but is not proved, that
riboflavin is first phosphorylated and then coupled with adenylic acid,
since adenylic acid and riboflavin-5'-phosphoric acid are formed on the
enzymatic hydrolysis of the "fiavin-adenine-dinucleotide." 70 In this
regard, riboflavin must be considered to be a step more evolved toward
the coenzyme than nicotinic acid, animals apparently not being able
to couple ribitol to the 6,7-dimethyl-isoalloxazine nucleus, as they are
able to couple ribose to nicotinamide. The steps involved in the con-
version of nicotinamide to coenzymes I and II are not known, but by
analogy with riboflavin one might expect the process to involve com-
binations with ribose, phosphorylation, and coupling with adenylic acid
successively. It has been shown that rabbit brain contains a DPN

METABOLISM OF THE B VITAMINS 353

nucleosidase which splits only nicotinamide from DPN, whereas rab-
bit kidney contains a pyrophosphatase which forms adenylic acid and
nicotinamide mononucleotide from DPN; n and it may be that the
coenzyme synthesis similarly has more than one route. The synthesis
of the coenzyme does not directly involve pyridoxine or its derivatives.72
The DPN of chick embryos increases with the niacin content, suggesting
that the ability to synthesize coenzymes is present in the animal from
the earliest stages of its development.73 Apparently only leucocytes and
other nucleated cells are able to perform the synthesis.41, 74 The tissues
of pantothenic acid-deficient rats and ducks exhibit a deficiency of coen-
zyme A in a manner analogous to many of the other B vitamins. Nitrogen,
arsenite, and glucose are reported to interfere with coenzyme A synthesis
in vitro.75 With regard to biotin, there is some evidence to indicate that
adenylic acid is involved in the synthesis of its coenzyme,228 which is re-
ported to be more active biologically than biotin itself.76 Steps involved
in the formation of active forms of the other B vitamins are unknown
and must await further elucidation of the structure of the functional
forms.

Choline Synthesis. Any discussion of choline synthesis must be con-
sidered in the light of the fact that in many ways choline is not a typical
B vitamin, and appears to occur and function in relatively large amounts
in the living organism. In an earlier chapter it has been shown that in
Neurospora and other organisms, choline is synthesized via the proc-

CH2OH CH2OH CH2OH CH2OH CH2OH

CH— NH2 — > CH2 — > CH2 — > CH2 — > CH2

I I I I I

COOH NH2 NH— CH3 N— CH3 N"

CH3

CH3 CH3 CH3

and that the necessary methyl groups may be derived from a source such
as methionine or betaine. Apparently the same series of reactions occurs
in all the higher animals if sufficient serine and methionine are available,79
and in practice it is necessary to limit the methionine content of the diet
in order to produce choline deficiencies. Isotopic studies in the rat have
done much to show that the pathway indicated above does in fact exist
in animals.so

Nicotinic Acid Synthesis. It has previously been mentioned that ani-
mals have the ability to convert tryptophan to nicotinic acid (p. 83),
and that in Neurospora the pathway presumably involves kynurenine,
3-hydroxy-kynurenine, and 3-hydroxy-anthranilic acid (p. 280) , but that

354 THE BIOCHEMISTRY OF B VITAMINS

even in Neurospora the exact sequence is not as yet well understood.
While there is much reason to believe that the process is similar in the
higher animals, there is not at present sufficient evidence to indicate that
this is unequivocably so.

The primary indications that tryptophan is converted to nicotinic acid
in animals are the facts that the administration of tryptophan to the
rat,sl horse,83 pig,84 dog,85 calf,86 and humans,87 results in the excretion
of increased amounts of nicotinic acid metabolites in the urine,88 and
that tryptophan supplants niacin in preventing a niacin deficiency in the
rat,89 chick,90 mouse,91 dog,85 pig,84 guinea pig,82 rabbit 92 and humans.93
While many authorities have attempted to explain these facts on the
basis of intestinal bacterial synthesis, a number of ingenious experiments
have largely eliminated this possibility. Thus the injection of chick eggs
with tryptophan causes an increase in niacin content.95 In addition, a
variety of measures designed to lower or minimize the effects of intestinal
symbiants have been without effect upon the conversion.96 Perhaps the
only known exceptions to this fact in the animal kingdom are the cases of
germ-free Drosophila — which require both tryptophan and niacin, and in
which tryptophan, when increased, causes a higher niacin requirement 97
and Tetrahymena.98 It is also known that 3-pyridylmethylketone, a
structural analogue of niacin, is toxic to mice.99 Since nicotinic acid pre-
vents its toxicity, the analogue presumably interferes with niacin forma-
tion, and since tryptophan similarly reverses the toxicity, it presumably
functions as a niacin precursor.

The balance of the data bearing on the problem center around the fact
that in vitamin B(i deficiency there is a decrease in urinary kynurenic acid
and an increase in urinary xanthurenic acid, and that simultaneously
there is a decrease in nicotinic acid metabolites in the urine.100 Since
kynurenic acid is known to be derived from tryptophan,101 and kynurenine
is a known intermediate in niacin synthesis from tryptophan in Neuros-
pora, it seems logical to suppose that vitamin B6 is involved somewhere
in the intermediate process. Actually, however, kynurenine does not lead
to increased nicotinic acid synthesis in the rat, as indicated by the
N'-methylnicotinamide excretion studies, and kynurenine does not pro-
duce growth in the rat in the absence of tryptophan and niacin.102-104
3-Hydroxy-anthranilic acid does bring about increased growth and in-
creased niacin and F2 excretion in rats, however.105 Since, however, the
experimental basis for these reports was limited, and the work is not as
yet confirmed, it seems best at present to leave the subject of the involve-
ment of kynurenine in animal metabolism open while considering the
known facts regarding this general metabolic pathway.

METABOLISM OF THE B VITAMINS 355

In tracer studies with DL-tryptophan-/3-C 14, the labelled atom may be
found in kynurenic acid, but not in niacin.

-CH2CH(NH2)COOH r^N-CO— CH2CH(NH2)COOH

"* L J-NH2
H

This indicates clearly that the conversion does not involve the pyridine
ring of kynurenic acid as a ring precursor of niacin.100 When, however,
DL-tryptophan-3-C 14 is employed, the tracer atom is found in the car-
boxylic acid group of niacin,107 strongly suggesting that it is the benzene
ring of tryptophan which eventually becomes the pyridine ring of nicotinic
acid.

CH2CH(NH2)COOH ^ VCOCH2CH(NH2)COOH

V ^^-NH2

-COOH f VCOOH

-NH2 ~^ l^N

The ring rearrangement presumably involved in the final step of the
process is of a type not entirely unknown to organic chemistry. Very
recently Henderson and co-workers 107a-c have shown that a probable
intermediate in this step is quinolinic acid. Rats on a 9% casein diet
containing little niacin but 2.5% added DL-tryptophan excrete 6-11.
mg of this acid per clay. The injection of 1. mM of tryptophan or 3-
hydroxyanthranilic acid into such rats increases their niacin excretion
tenfold, their F2 excretion twenty-five to one hundredfold, and their
quinolinic acid excretion one hundred to three hundredfold. Quinolinic
acid is slightly active for rats in replacing dietary niacin, is active in
some Neurospora strains, and accumulates in the culture medium of
other strains. Rat liver slices or homogenates are able to convert 3-
hydroxyanthranilic acid to quinolinic acid, and quinolinic acid is con-
verted by weak acid to a substance having niacin activity for Lactobacil-
lus arabinosus.

356 THE BIOCHEMISTRY OF B VITAMINS

Kynurenic acid (4-hydroxyquinaldic acid) was the earliest known
product of tryptophan metabolism in the higher animals,108 and it is
known that the liver is one of the principal sites of its formation. Only
the L-form of tryptophan is active in its formation, and 3-indole-pyruvic
acid and kynurenine are equally as active as tryptophan in kynurenic
acid formation.109 In the rabbit, which normally excretes only kynurenic
acid, thiamine deficiency produces a simultaneous excretion of kynurenine,
so that thiamine presumably catalyzes the conversion of kynurenine to
kynurenic acid.110 Kynurenic acid will not substitute for tryptophan in
the diet. Work with methylkynurenic acid and methyltryptophan deriva-
tives in the rabbit further indicates that it is the a-amino nitrogen atom
of the tryptophan that ends up in the kynurenic acid structure, rather
than the pyrrole nitrogen.111 The presence in urine of two other products,
xanthurenine (3-hydroxykynurenine) and xanthurenic acid (4,8-di-
hydroxyquinaldic acid)112, 113 has been recognized more recently. As pre-
viously stated, the occurrence of the yellow xanthurenic acid in the urine of
various species (rice moth larvae,114 mice,115 rats,116- 117 rabbits,118
dogs,118 swine,119) made vitamin B6 deficient by depletion diets or by an
inhibitor, 4-desoxypyridoxine,120 is closely associated in some manner
with the pathway of niacin synthesis, and is the only certain intermediate
point so far found that can be studied in animals. The general interpre-
tation of this fact is that vitamin B6 normally mediates tryptophan
metabolism so as to prevent (or limit)121 its oxidation to xanthurenic
acid. A quinine oxidase, recently isolated, has the property of oxidizing
the carbon atom next to the nitrogen atom in a large variety of hetero-
cyclic compounds.122 Although this enzyme is involved in niacin break-
down, as we shall see shortly, it does not affect tryptophan, xanthurenic
acid, or kynurenic acid, and is thus probably not involved in niacin
synthesis. It does oxidize indole, but neither indole, 3-indoleacetic acid,
nor anthranilic acid is effective in niacin synthesis.123 Thus, in summary,
whereas there is considerable direct and indirect evidence concerning the
general pathway of niacin synthesis in animals, there is little certainty
as to the actual steps involved, and the problem remains as one of the
outstanding ones to be worked out in the future.

Products of Nicotinic Acid. There is probably more evidence bearing
upon the nature of the products formed from nicotinic acid in animals
than upon the metabolism of all of the other B vitamins combined. This
is largely because these products are diverse in nature and occur in rela-
tively large amounts. Further, because of their diversity, they have com-
plicated the study of nicotinic acid requirements by means of the urinary
excretion methods, which have been so successful in other cases. It is

METABOLISM OF THE B VITAMINS

357

therefore now possible to account for a fair portion of the nicotinic acid
metabolized in the animal body (see p. 365).

There are at present some eight known derivates of nicotinic acid which
are excreted by various animal species. These are:

-COOH

Nicotinic acid

-COOH

CH3
Trigonellin
-CO— NH— CH2COOH

-CONH2
Nicotinamide

CH3

N'-Methylnicotinamide (F2)

■CO— NH2

Nicotinuric acid

COOH
COOH

Quinolinic acid

CH3
N'-Methyl-6-pyridone-3-carboxylamide

,-CO— NH— (CH2)3— CH— NH— CO-
kNJ ioOH

Dinicotinylornithine

In addition to these compounds, evidence has been presented to show
that many other nicotinic acid metabolites are formed in the body.124-126
It seems well established that coenzymes I or II are not excreted, and
there is little evidence to suggest that riboside derivatives of niacin are
found in the excreta, so that these coenzymes are apparently hydrolyzed
at the nicotinamide bond as a general procedure in their metabolism
(see p. 352).

Our knowledge of niacin metabolism in various species must be inter-
preted carefully, in view of the fact that much earlier work was done
with analytical procedures that were not specific or sensitive, and that
certain compounds closely related to those measured were not known
until recent years. Most species probably excrete at least some nicotinic
acid and nicotinamide as such, although there is some conflicting evidence

358 THE BIOCHEMISTRY OF B VITAMINS

on this point. Thus, in the dog earlier reports stated that there was no
free nicotinic acid or nicotinamide in the urine, 12T whereas later workers
report its presence.128 It has similarly been stated that birds excrete
only nicotinic acid,129 although this seems unlikely, and indeed they
have been shown to excrete dinicotinylornithine in their droppings
(chicks) ,130

It has long been known that when pyridine is administered to animals,
it is excreted in the form of N'-methylpyridinium ions,131 and hetero-
cyclic nitrogen compounds in general seem to be largely methylated
prior to excretion by both plants and animals. Even before the role of
nicotinic acid as a vitamin was known, it had been shown that this sub-
stance, when administered to dogs, was excreted as trigonellin (and
nicotinic acid),128 and it might be anticipated that trigonellin would be
a major metabolic product of niacin. Despite the fact that the dog ex-
cretes nearly all of a 100-mg dose of nicotinic acid as trigonellin and
nicotinic acid, rabbits do not excrete trigonellin,128 and apparently can-
not methylate niacin. While humans excrete some trigonellin, it is now
believed that this is all exogenous, and the result only of the trigonellin
ingested. Oral administration of trigonellin to humans does not result in
significantly increased excretion of other nicotinic acid metabolites, but
does result in almost complete excretion of the entire dose as trigonel-
lin.132-136 Although trigonellin is apparently ineffective in curing niacin
deficiency in at least some species, generalizations are dangerous since,
as in the case of other metabolites to be considered, results may vary
depending upon the magnitude of the dosage and the nutritional state
of the animal. At present it would seem best to believe that whereas in
some species (e.g., the dog) trigonellin may be a major end product of
niacin metabolism, it is probably found in only very small amounts in
many others. While there is apparently little tendency to amidate trig-
onellin in animals, since ingested trigonellin is largely excreted as such,
trigonellin in the dog may result from deamidation of N'-methylnico-
tinamide. This may well be the case in view of the ability of all animals
studied to methylate nicotinamide, and the relatively lower levels of free
nicotinic acid available to the dog for conversion directly to trigonellin.
We may well assume for the present, therefore, that trigonellin arises
as a product of methylated nicotinamide, and that animals that do not
excrete measurable trigonellin lack the tendency to deamidate F2.

N'-methylnicotinamide (F2) is apparently one of the major metabolic
products of nicotinic acid in all the animals so far studied in this regard
(man,87 rat,81 horse,83 calf ,8G pig 84) . It is said to account for 94 per cent
of the total nicotinic acid and its metabolites found in the urine.23 When
rats are fed large amounts of niacin in which the acid group is labelled

METABOLISM OF THE B VITAMINS 359

with C13, 95.7 per cent of the original concentration of C13 is recovered as
F2.143 Tracer work with C14 indicates, however, that both nicotinic acid
and its amide are to some extent decarboxylated,144 although the products
are as yet unknown. F2 is distinguished in urine by its formation of a
bluish white fluorescent compound when examined under ultraviolet
light in the presence of alkali and butanol,137, 138 and its part in nicotinic
acid metabolism has been realized only during the past few years. It
does not promote growth in Lactobacillus arabinosus (which can utilize
both nicotinamide and nicotinuric acid) nor in Leuconostoc mesenteroides
(which cannot utilize low levels of nicotinamide or any levels of nico-
tinuric acid, but requires free niacin).139 Liver slices in vitro, but not
kidney or muscle tissue, convert nicotinamide to F2, and in vivo studies
have shown that the intact liver is capable of performing this methylation
at a rapid rate, whereas the intact kidney is not.140 The liver is thus be-
lieved to be the sole site of F2 formation; and F2 formation from nicotina-
mide has been shown to be a valuable test of hepatic function, employing
as the test dose a physiological compound within physiological levels, as
contrasted with the usual tests employing substances foreign to the ani-
mal body.141, 142 The reaction has been of some value in the study of
transmethylation in healthy persons, and it appears that in studies with
rat liver slices, the methylation is frequently enhanced by methionine.140
Bearing upon the nature of the methylation process, as well as the utiliza-
tion of F2 as a nicotinamide source, is the demonstration that the incor-
poration of 2 per cent of F2 into the Griffith and Wade fatty liver diet of
four rats for three months resulted in liver fats of 24.5, 8.2, 10.6 and
23.5 per cent, as compared to rats on the unsupplemented diet with liver
fats of 36.1, 39.1, 39.7 and 34.7 per cent. It would thus appear that the
methyl group of F2 is available for choline formation (p. 353) ,145

As a corollary of this latter study,146 it was found that 1 per cent F2
in the diets of four rats on this diet produced after three months an
average daily excretion of 11.8 fig per day of nicotinamide, as compared
with 4.5 fig per day in four controls on this diet. While these data have
been criticized on the basis of the smallness of the groups and the pos-
sibility of nicotinamide contamination in the large amounts of F2 em-
ployed, general metabolic considerations would make it seem likely that
F2 was at least to some extent available as a nicotinamide source. Tests
have to date strongly indicated that F2 is ineffective in the treatment of
pellagra 146 and blacktongue,147 although in the latter case seriously con-
flicting reports do exist.148, 149

That F2 is derived rather directly from nicotinamide is shown by the
facts (1) that nicotinamide is more effective than nicotinic acid in pro-
ducing urinary F2,150 (2) that oral doses of equivalent amounts of nico-

360 THE BIOCHEMISTRY OF B VITAMINS

tinamide and F2 produce similar amounts of urinary F2,151 and (3) that,
as previously mentioned, rats fed nicotinic acid in which the carbon
atom of the carboxylic acid group was labelled with C13 excrete amounts
of labelled F2 in the urine which indicate that all of the F2 is derived
from nicotinic acid. F2 is absent from the urine of pellagrins and its ex-
cretion is in general a function of the nicotinic acid and tryptophan in-
take of animals.152 Wide individual variations exist, however, and some
individuals with an apparently adequate niacin intake do not excrete
any F2.153 There are, moreover, apparently significant variations among
species in their ability to form F2. Thus in the horse on a low-niacin diet
tryptophan stimulates only slightly higher nicotinic acid excretion, and
no increase in F2 excretion,154 whereas in the cotton rat tryptophan
stimulates an increase in both,155 but principally in the F2.156 In humans,
tryptophan apparently does not affect niacin excretion, but does increase
the excretion of F2 markedly.157 About half the niacin metabolites ex-
creted by calves are active for Lactobacillus arabinosus, the remainder
being largely F2.158 In vitamin B6 deficient rats, it has previously been
mentioned that there is no F2 excretion. When pyridoxine is administered
to these animals, there is a prompt disappearance of xanthurenic acid
from the urine, but only a very gradual reappearance of F2.159 Therefore,
although F2 may be formed rather directly from nicotinamide, it would
seem that such a wide variety of factors influence its formation that ex-
tended study will yet be required to assess its exact place in niacin
metabolism.

It has been realized for some time that the level of the excretion of
F2 is the result of two major factors: its synthesis from nicotinamide
and its conversion to still other metabolic products.160 It has recently
been discovered that liver contains an enzyme capable of oxidizing
quinine and many other nitrogen heterocycles, the enzymatic oxidation
characteristically involving the conversion of the carbon atom adjacent
to the nitrogen into a keto group.122, 123 This oxidase readily converts F2
into N'-methyl-6-pyridone-3-carboxylamide in vitro, and it has been
found that the administration of 600-900 mg of nicotinamide to humans
results in the urinary excretion of about 100 mg of this same compound.
It thus appears that this substance is a major product of niacin metab-
olism in at least some species. To date, however, it is uncertain as to what
extent its presence may have influenced earlier determination of other
niacin metabolites. In any case it seems likely that further analogous
pyridones may eventually be discovered among the metabolic products
of niacin.

Nicotinuric acid (nicotinylglycine) was found to be a major product
of niacin metabolism in the dog long before niacin was implicated as a

METABOLISM OF THE B VITAMINS 361

vitamin.161 Rabbits similarly excrete nicotinuric acid, but humans ap-
parently do not.102 In vitro studies with hog intestinal hippurase have
shown that it cleaves nicotinuric and hippuric acids at roughly equal
rates, and it has been suggested therefore that nicotinuric acid is prob-
ably formed by hippurase in vivo.16* Although this point lacks further
verification, it seems likely that the liver and kidneys may well perform
this function along with their more extensive hippuric acid-synthesizing
functions.

Dinicotinylornithine has been isolated from dried chicken droppings,130
but nothing is known as yet concerning its metabolism or significance.
The structure is of particular interest, however, because of the relationship
of ornithine to nicotinic acid synthesis in the higher plants (p. 84) . The
recent isolation of quinolinic acid from rat urine is mentioned on page
355, but little more is yet known concerning the significance of this
substance in the urine. 107a-c

A considerable portion of ingested nicotinic acid remains still to be
accounted for, and several probable, but as yet undetected, products have
been suggested in the preceding discussion. In the studies previously
mentioned employing nicotinic acid in which the acid group was labelled
with C14, it was shown that 15 per cent of the C14 of the acid group
disappears from mice as exhaled carbon dioxide, so that considerable
decarboxylation, possibly preceded by ring rupture, occurs. These studies
further showed that the gross metabolism of niacin and its amide is
identical in the mouse.144 It thus appears likely that in addition to the
3-carboxylic acid derivatives of pyridine now known, other pyridine
derivatives may also occur and eventually be detected. In view of this
likelihood, a reinvestigation of the numerous pyridine derivatives already
discovered in nature with regard to their urinary occurrence would seem
to be in order.124- 125- 164

Metabolic Products of the Other B Vitamins. As previously stated,
little is actually known of the metabolic products of the other B vitamins,
although one might hazard extensive guesses as to the probable hydro-
lytic, oxidative, and conjugative changes that they may logically undergo
by analogy with the metabolism of other structurally similar compounds.
Rather than venture so far, therefore, it seems better to outline what
little is known in this regard, leaving the speculation for a time when
it might be better strengthened by the discovery of new and suggestive
data in this largely unstudied realm of knowledge.

On normal levels of dietary intake, only a small portion of the B vita-
mins consumed can be accounted for on the basis of their excretion in
urine. This may be due to their conversion to as yet unidentified but
closely related decomposition products,165- 166 or to their conversion to

362 THE BIOCHEMISTRY OF B VITAMINS

very dissimilar products. In this connection it should be pointed out that
they may be completely metabolized to carbon dioxide, water, and am-
monia. Tracer studies have indicated that the nutritionally active isomer
of inositol is at least partially converted to glucose in the rat,167 and the
probable existence of similar conversions of some other vitamins may
make it impossible ever to obtain completely balanced data on intake and
excretion. This seems even more likely since the urinary and fecal excre-
tion so frequently exceeds the intake (pp. 300 and 368) , and it is uncertain
what portion of the B vitamins present in both urine and feces originated
in the diet.

A variety of types of metabolic conversions break down the B vitamins
to their known excretory products. Urine apparently contains no cocar-
boxylase, so that the thiamine present in urine must largely result from
the action of phosphatases on the pyrophosphate.168 Thiamine is split
in the human body in apparently much the same way as by the thiaminase
of aquatic animals (p. 292), and the resulting pyrimidine moiety, called
pyramin (2-methyl-4-amino-5-hydroxymethylpyrimicline) is found in the
urine in considerable quantities.169 When an individual is saturated with
thiamine, practically all excess thiamine given can be recovered either as
thiamine or pyramin,170 so that it would appear that there are not nor-
mally other major end products of thiamine metabolism. However, al-
though the amount of thiamine excreted is highly characteristic for the
individual (p. 255) , pyramin excretion seems to be a relatively constant
process.171 Since on the same diets one individual may consistently ex-
crete three times as much thiamine as another, while excreting an essen-
tially identical level of pyramin, it is apparent that still another pathway
of thiamine metabolism must exist.

Riboflavin is principally excreted as such, but varying amounts up to
half of the total may be excreted as the phosphate.172 Aquaflavin, or
uroflavin, a degradation product of riboflavin, is also found in most urine
samples.173 In cow, goat, and sheep milk and urine there is a considerable
discrepancy between riboflavin determinations done fluorometrically and
microbiologically, and it appears that an as yet structurally unidentified
degradation product of riboflavin is present which exhibits marked fluores-
cence but no microbiological activity.174 As this metabolic product is not
present in the urine of humans or rats, it is possible that the product is
formed in the rumen of the animals mentioned. Certain microorganisms
are well known for their ability to oxidize riboflavin to lumichrome,175
and such a reaction may be responsible for the presence of this fluores-
cent pigment in the urine and milk of ruminants.

Pantothenic acid is partially excreted as such, but a large part of that
ingested has an unknown fate.176, 177 It is well established that there is

METABOLISM OF THE B VITAMINS 363

no pantoyl lactone in the urine, and that the lactone administered intra-
venously or orally is recovered in the urine quantitatively and unchanged ;
hence hydrolysis of the vitamin molecule probably does not normally
occur to any appreciable extent.178 Since the tissue vitamin is almost
solely in the form of the coenzyme, it would seem that the search for
traces of coenzyme fragments in the urine may well lead to clues as to
the exact structure of this coenzyme. Synthetic pantothenyl alcohol is
converted by the body to pantothenic acid,179 but since this former com-
pound apparently does not appear in nature, the reaction has no great
significance.

T
o

Lumichrome

Little is known of the breakdown of folic acid in the body, but it is
known that "conjugase" of the liver, kidney and pancreas converts the
triglutamate and heptaglutamate to folic acid.180 Strangely enough, in
vitro studies with chicken pancreas conjugase have shown that only the
terminal glutamic acid molecule is removed from the triglutamate, so
that there is a considerable lack of understanding of how folic acid is
ultimately produced in vivo.181 Even in sprue, administered pteroyl
triglutamate is converted to the monoglutamate, as indicated by urinary
excretion of the latter.182 The fact that folic acid-free liver extracts
further increase the excretion of folic acid in this case suggests that the
metabolism of the conjugates is in some manner mediated by the ery-
throcyte maturation factor.

Although little is known of the normal metabolism of p-aminobenzoic
acid, larger amounts when administered are excreted as the acetylated
product.183 Normal rats acetylate about 70 per cent of the excreted por-
tion of a 1 to 2.5-mg dose of p-aminobenzoic acid, and this ability is
decreased in pantothenic acid-deficient rats.184 Sulfanilamide is similarly
acetylated in the animal body,185 and if, as seems likely, the mechanism
is the same, the process involves adenosine triphosphate, acetate, and
coenzyme A.186 The activity of liver preparations in this regard suggests
that this organ is at least one major site of the reaction. Since however
all these considerations refer to levels of p-aminobenzoic acid that are
somewhat above the physiological range, there can be no absolute cer-

364 THE BIOCHEMISTRY OF B VITAMINS

tainty that a similar mechanism is involved normally in PABA metab-
olism.

Regarding vitamin B6, apparently small amounts of all three forms
may be excreted in the urine. The major metabolic product of pyridoxine,
however, is 4-pyridoxic acid, which accounts for about five-sixths of the
known vitamin B6 metabolites in the urine.

COOH
I
HO-^\-CH2OH

CH:

4-Pyridoxic acid

Urinary choline appears to be entirely in the free state, although there
does not seem as yet to be ample evidence to substantiate the absence
of other derivatives. Practically nothing is known of the breakdown
products of biotin, or "vitamin Bi2," although the latter is said to be
stored by both normal and pernicious anemia patients, even when it is
administered in high dosage.187 Whereas a variety of biotin metabolic
products have been suggested, the recent demonstration of the biotin-
like activity for microorganisms of a number of structurally unrelated
compounds (p. 173) seems to cast serious doubts on the existing data
concerning the presence of biotin degradation products in urine.

Thus at present there are many gaps in our knowledge of the break-
down products of the B vitamins. These gaps may reasonably be ex-
pected to be filled in the years immediately ahead, largely by the use of
isotopic tracer techniques, which have already been so preeminently suc-
cessful in problems of this kind, and also by means of the rapidly devel-
oping technique of paper chromatography, which is so well adapted to the
separation and identification of small amounts of structurally similar
compounds. As in many other cases discussed in this monograph, this
field is one in which true progress has barely commenced.

Excretion

A great many of the aspects of B vitamin excretion have been discussed
previously in regard to the assessment of B vitamin requirements (p.
254) , the effects of climate and other factors upon the requirements
(p. 269), the concentration in milk (p. 347) which may be regarded as
an excretory product, and with regard to the breakdown products of the
B vitamins (p. 361) . Still other aspects are more appropriately discussed
in the consideration of deciency states. Consequently our present discus-

METABOLISM OF THE B VITAMINS 365

sion will be in the nature of a summary of the general picture at present
available of the levels of excretion themselves.

Levels in Urine. Table 28 summarizes the major known end products
of vitamin metabolism found in the urine. In cases where a variety of
products exist, variations occur in the relative amounts of the products,
and in some cases at least these variations have been extensively studied.
For the most part, however, little is known of the significance of the
various forms and their relative amounts.

Table 28. Known Urinary Products of Vitamin Metabolism

B Vitamin Urinary form

Thiamine Thiamine (but not pyrophosphate)

Pyramin
Riboflavin Riboflavin (and phosphate)

Uroflavin
Nicotinic acid Nicotinic acid (and amide)

N'-methylnicotinamide

N'methyl-6-pyridone-3-carboxylamide

Nicotinuric acid

Quinolinic acid
Vitamin B6 Vitamin B6

4-Pyridoxic acid
Pantothenic acid Pantothenic acid

Biotin Biotin

Folic acid (group) Folic acid

p-Aminobenzoic acid Acetyl PABA

Choline Choline

Inositol Inositol

The excretion of the B vitamins and their derivatives via the kidney
or skin involves processes considerably more complex than passive diffu-
sion. While few data are available on the topic, it appears that the renal
and dermal thresholds are maintained, in some cases at least, by inter-
mediate formation similar to that which occurs upon the passage of a
vast number of other metabolites across biological membranes. Whereas
there have been some studies of the renal clearance and glomerular filtra-
tion of the B vitamins,186, 187 this field of endeavor is relatively unex-
ploited at present.

In considering the levels of B vitamin excretion in humans, and their
relation to the dietary intake, one could do little better than to consider
the data shown in Tables 29 and 30, which are taken from the excellent
study of Denko et ai.23 The subjects were seven healthy men from 23 to
28 years of age, who were maintained on a balanced diet for a twelve-
week period. Various aspects of these data have been previously discussed
(pp. 275 and 300) , and they are sufficiently complete and lucid that an
extended discussion of their various aspects at this time would seem un-
warranted. The data are in excellent agreement with the great mass of
published material on this topic, and have the advantage of being far

THE BIOCHEMISTRY OF B VITAMINS

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368 THE BIOCHEMISTR Y OF B VITAMINS

more complete than most similar studies. Those interested in specific
aspects of the excretion problem may readily find a vast current literature
dealing with the individual vitamins: thiamine,188-196 riboflavin,197"204
niacin,205-211 pantothenic acid,212"213 biotin,214' 215 folic acid,216-218 vita-
min B6,219 p-aminobenzoic acid and inositol,220 and the antipernicious
anemia factor.221 Problems dealing with obstetrics and pediatrics are
similarly well covered in the current literature,188- 199- 20°- 207> 210> 222 and
ready access to the vast earlier literature on the topic may be gained
through these references. Unfortunately, there is not at present sufficient
information to draw any conclusion as to the variations in levels among
various species, however.

Fecal and Dermal Excretion. As shown in Tables 29 and 30, except
for pantothenic acid and pyridoxine, fecal excretion of the B vitamins far
exceeds urinary excretion, but there is little information to indicate what
part of the fecal excretion represents dietary intake and what part in-
testinal synthesis. Undoubtedly the greater part of the fecal vitamin
content is of bacterial origin, since the combined fecal and urinary ex-
cretion of biotin, pantothenic acid, PABA, and folic acid exceeds the
intake, and since the remainder of the B vitamins have other major
products of metabolism which were not considered in this study. More-
over, the consideration of dermal excretion was not made in assessing these
data. Furthermore, there is no apparent correlation between the com-
bined urinary and fecal excretion and the dietary intake.

It seems quite certain that fecal thiamine is almost entirely in the form
of cocarboxylase, in contrast with urinary thiamine.194 Little is known
of the precise form of the other vitamins in the feces, although from 60
to 100 per cent of the fecal B vitamin content is said to be "water-sol-
uble." 27 Similarly, little is known as to the precise forms of the B vita-
mins excreted in sweat. Vitamin concentrations in sweat appear to be
quite similar to those in urine, and it is consequently generally felt that
under normal environmental conditions the dermal excretion is negligible
by comparison with urinary excretion. Pantothenic acid normally occurs
in sweat in concentrations of about 3.8 fig per cent,212 and the fact that
this level cannot be increased by higher dosage with pantothenic acid
suggests that the excretory ability of the skin is more limited than that
of the kidney. Inositol and PABA occur in sweat in concentrations of
about 21. fig per cent and 0.24 fxg per cent, respectively.220

Recent studies on urinary and dermal excretion of- pyridoxine and its
derivatives are of interest in that they strongly suggest that sweat con-
tains metabolites of B vitamins quite similar to those in urine. Table 31,
taken from Johnson, Hamilton and Mitchell's paper,223 indicates that
sweat normally contains pyridoxal, pyridoxamine and 4-pyridoxic acid.

METABOLISM OF THE B VITAMINS 369

When higher levels of pyridoxine are administered, dermal excretion of
pyridoxine also occurs.

Table 31. Average Dermal and Urinary Excretion of Pyridoxine and its Metabolites

. Sweat (mg/8 hours) *

pyridoxal + 4-pyridoxic
pyridoxine pyridoxamine* acid

Subject

2
3

— ■ — Urine (mg/24 hours)

pyridoxal + 4-pyridoxic
pyridoxinef pyridoxamine* acid

0.114
0.187
0.118

0.192
0.375
0.236

2.83
3.02
3.29

0.030
0.030
0.032

0.159
0.193
0.257

0.131

0.181

3.36

0.035

0.189

Average

0.138

0.246

3.13

0.031

0.198

% of total

3.9

6.8

89.3

13.6

86.4

* Measured in terms of pyridoxal.

t The data cannot be accepted in a quantitative sense, because the assay methods used are now known
to give results that do not represent the specific analogues indicated. The data do illustrate the points
cited in the text, however.

Individual Variations. Despite the fact that there may be large daily
variations in the excretion of most of the B vitamins, marked individual
differences do exist of such a nature that for some of the B vitamins,
two individuals on the same dietary intake may excrete widely divergent
amounts. This is to be expected in consideration of the many genetically
controlled enzyme systems that mediate vitamin metabolism. Fecal B
vitamin excretion varies more than does the urinary excretion, since the
bulk of fecal vitamins is controlled by the bacteria present rather than
by the host; but even in this case individuals do differ by virtue of these
differences in intestinal tracts, etc. This may be noted, for instance, by
considering the values in Table 29 for subjects 4 and 6. These subjects
were brothers, and a distinct resemblance occurs in the urinary and fecal
excretion of both thiamine and riboflavin.

An excellent example of urinary individual differences is that of thia-

Table 32. Urinary and Fecal Excretion of Thiamine and Pyramin by Six Men on

Three Days*

Thiamine (/ig/day)

Pyramin 0ig/day)t

Subject

Urine

Feces

Urine

Feces

II III mean

I III mean

2
3
4
5
6

118
36
60
85
55
62

75 79 91
21 39 32
48 53 54
83 86 85
46 49 50
52 60 58

540
510
800
550
720
666

187
162
148
178
188
159

169 157 171

170 142 158
178 178 168
198 179 185
173 146 169
169 150 159

383

208
37

532
61
94

Mean

54 61 62

631

170

176 159 168

219

* In the above table, the measurements of thiamine were not made on the same three days as were
the measurements of pyramin.

t Expressed as 2-methyl-4-amino-5-ethoxymethylpyrimidine.

370 THE BIOCHEMISTRY OF B VITAMINS

mine excretion, as shown in Table 32. The subjects in this study of
Mickelson et al.171 were on a thiamine intake of approximately 1.5 mg per
day, and under these conditions distinct differences are shown in thia-
mine excretion among the subjects. Pyramin excretion, however, was
most remarkably constant from day to day and individual to individual,
and has been suggested for this reason as a more accurate indication of
the status of thiamine nutrition as judged by excretion studies (p. 256).
It will be noted that fecal thiamine does not vary with either the thia-
mine or pyramin excretion in the urine. Certainly the investigation of
the source and high variability of fecal pyramin would seem to be a
worthwhile undertaking, together with the extension of this study to a
larger number of individuals.

Relationship to Dietary and Body Levels. The levels of the various B
vitamins found in the urine do not correlate with the requirements for
these vitamins because of the variety of ways in which the various mem-
bers of the group are metabolized. Above a certain minimal level, the
amount of a vitamin in the diet is generally correlated with its content in
the urine. In a general manner, the levels in the urine resemble those in
milk and in sweat. As yet there are insufficient data to tell whether this
is also true of saliva. The addition of some one vitamin in a single high
dose to the diet of an individual "saturated" with a B vitamin causes an
increase in the blood level rather rapidly, but an almost equally rapid
fall to normal and corresponding increase in the urine. A full understand-
ing of these relationships, however, must await the elucidation of the
variations which occur in the various B vitamin derivatives, the fate
of the unaccounted for fraction of the intake, and the source and qualita-
tive nature of the fecal fraction of the vitamins eliminated. These prob-
lems at present are all under intensive investigation, and may well be to
a large extent resolved in the near future.

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173. Koschara, W., Z. physiol. Chem., 232, 101-16 (1935).

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35, 499-509 (1948).

180. Daniel, L. J., Scott, M. L., Norris, L. C, and Hauser, G. F., J. Biol. Chem.,

160, 265-71 (1945).

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183. Bernhard, K., Z. physiol. Chem., 267, 91-8 (1940).

184. Riggs, T. R., and Hegsted, D. M., J. Biol. Chem., 172, 539-45 (1948).

185. Lipmann, F., Federation Proc, 4, 97 (1945).

186. Nachmansohn, D., and John, H. M., J. Biol. Chem., 158, 157-71 (1945).

187. Blanchaer, M. C, and Cameron, E., J. Nutrition, 35, 391-7 (1948).

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194. Alexander, B., and Landwehr, G., J. Clin. Invest., 25, 287-93 (1946).

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200. Dubrauzky, V., and Blazso, S., Z. Vitaminj orsch., 14, 2-13 (1943).

201. Hathaway, M. L., and Lobb, D. E., J. Nutrition, 32, 9-18 (1946).

202. Davis, M. V., Oldham, H. G., and Roberts, L. J., J. Nutrition, 32, 143-61 (1946).

203. Hou, H. C, and Dju, M. Y., Chinese Med. J., 61, 121-30 (1942).

204. Truvanov, A. V., Biokhimiya, 11, 33-43 (1946).

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376 THE BIOCHEMISTRY OF B VITAMINS

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and Folkers, K., J. Am. Chem. Soc, 72, 1048 (1950).

Chapter VC

PHYSIOLOGICAL, PHARMACOLOGICAL, AND
TOXICOLOGICAL EFFECTS

The four preceding chapters have presented the available evidence
bearing upon the problems of what the B vitamin requirements of living
things are, and the metabolic fate of the vitamins themselves, and the
next chapter is to deal with the effects of B vitamin deprivation. In ac-
cordance with the outline proposed for this section, it is necessary
to consider here certain overall effects of the B vitamins on living or-
ganisms.

It is immediately apparent that the major effect of the B vitamins is
to maintain the animal and all its parts in an efficient functional state,
and regarding this aspect there would seem to be little cause for extended
discussion. The exact manner in which the B vitamins function in the
maintenance of cellular and tissue metabolism has been considered at
length in earlier sections. Moreover, the possible role of the B vitamins
in preventing and curing various pathological conditions, primarily
avitaminotic and otherwise, is more conveniently discussed in the next
chapter, dealing with deficiency states. There are however certain special
relationships which exist between the various B vitamins and specific
physiological systems and functions, and these require some brief con-
sideration at this point.

In considering the biological effects of any substance it is important
to realize that different concentrations of a substance frequently mani-
fest markedly different activities. What may be an innocuous and even
required substance at one level of administration may become a danger-
ous drug at another. There is frequently a distinct intermediate level at
which the substance becomes effective in producing in the activity of
the organism certain changes which are neither particularly hazardous
in the usual sense nor of a nature similar to its physiological function.
These three levels of activity — -the physiological, the pharmacological,
and the toxicological — are quite distinct for a number of the B vitamins.
The cause of the varying activities exhibited by different concentrations
of these and other substances is obscure, but it may be surmised that
B vitamins in increased concentrations may have two major effects: they
may increase the normal physiological reaction to a point where it be-

377

378 THE BIOCHEMISTRY OF B VITAMINS

comes so disproportionate as to upset the usual functions and become
manifest, or they may at higher concentrations react in enzyme systems
in which they are normally alien, in the manner of analogue inhibitors
(Sect. D) . These considerations have not as yet been intensively investi-
gated, and there is little real knowledge in this regard.

The study of the pharmacology and toxicology of the B vitamins is of
interest largely in that it throws light on two major problems: what the
effects of large doses or prolonged treatment with a given vitamin will
be, and to what extent the special physiological relationships to be con-
sidered here (and their derangement, considered in the next chapter) may
be related to the pharmacological activity of the vitamin. Most author-
ities now feel that there is little real relationship between the often ap-
parent similarity in physiological and pharmacological activities of the
B vitamins, being so decided largely by virtue of the thousandfold differ-
ence that generally exists between the two levels. Whether or not such
an opinion is justified, it is certainly true that the three levels of activity
of the B vitamins can be most adequately expressed in terms of micro-
grams, milligrams, and grams, respectively. In this regard, one of the
most distinctive and remarkable things about the B vitamin group is
certainly the high physiological activity per unit weight, coupled with the
low toxicity exhibited by these substances which, on the basis of struc-
tural considerations alone, might be predicted to be highly toxic. A
further reason for the consideration of certain pharmacological aspects
of the B vitamins lies in the aspect that the precise mode of action of
most drugs is as yet unknown. Present knowledge indicates, however,
that it will eventually be shown that most drugs, at least, exert their
action through specific enzyme systems. In view of the fact that so many
of these systems involve B vitamins as coenzymes, it may be surmised
that a growing field of interest will develop in B vitamin-drug interrela-
tionships.

Physiological Relationships

Many of the special physiological interrelationships of the B vitamins
are only discernible in the light of their derangement, and are therefore
almost of necessity discussed in the next chapter. Most of the others
seem to be associated with certain categories that have been established
by tradition in the field of physiology — metabolism, endocrine function
and reproduction, nervous function, and mental activity. It is apparent
that these terms have little real meaning from the standpoint of being
distinct functional entities: they are so interdependent as to be in prac-
tice inseparable. Nevertheless they do convey sufficient indications of
various aspects of physiology for the present purpose, and are therefore

PHYSIOLOGY, PHARMACOLOGY, AND TOXICOLOGY 379

used here as reference points rather than to designate functional boun-
daries.

Metabolic Interrelationships. Many of the relationships between vita-
mins and metabolism primarily involve endocrine function and are dis-
cussed in a following section, while other relationships that involve bio-
chemically specific metabolic reactions are discussed at other appropriate
places. Drawing the many facets of metabolism together results in the
concept of "total metabolism" or the overall metabolic rate. The met-
abolic rate is a function of all the factors that go into a myriad of
metabolic processes, and may be limited by any factor that is deficient,
so that when any B vitamin is present in inadequate amounts, the metab-
olism may be slowed down, and addition of that B vitamin may increase
the metabolic rate to the point where some other factor becomes limiting.
Beyond this, the B vitamins have little effect upon the metabolic rate in
the sense that many other physiologically active substances do. When
for some reason the metabolic rate is increased, the heightened cellular
activity of the body results in an increased vitamin attrition and there-
fore an increased requirement for the B vitamins, as it does other nutri-
tional elements.

As far as is now known, these same considerations apply to the inter-
relationships between the B vitamins and growth. Supplementation of
the diet with increased amounts of B vitamins will undoubtedly increase
the growth rate in many cases,117 but this effect is primarily due to the
fact that in these cases there are originally present insufficient supplies of
vitamin to meet the requirements for the -maximum rate of growth per-
mitted by other metabolic factors. Whereas there is some evidence to
indicate that certain B vitamins may function specifically to increase
the mitotic rate of certain plant tissues, it seems unlikely that such an
effect occurs in the higher animals. When B vitamins are added to the
diet to a point that they no longer stimulate growth, it is apparent that
some other factor then becomes limiting in the process. This factor may
well be one whose limiting nature is unimportant, but it may also be
one that will result in serious consequences. Thus in an overly rapid rate
of growth various other nutritional substances associated with protein
metabolism or bone formation may become limiting; and as previously
stated, there is no assurance that the fastest rate of growth is the best.
There would seem to be some justification, therefore, in permitting growth
to proceed at what experience has taught is a reasonable rate, allowing
the limiting catalytic activity of the B vitamin, when it exists, to remain
the controlling factor. Because of the high rate of turnover of raw mate-
rials in the growing animal, and the consequent high metabolic rate,
there is an increased B vitamin requirement on a weight basis. In well

380 THE BIOCHEMISTRY OF B VITAMINS

nourished populations, however, this factor seems adequately compen-
sated for by the increased appetite and consumption of other nutritional
materials. Except when specific factors make it seem expedient, there
consequently seems little justification for the B vitamin supplementation
of the diets of growing children. When however the practice is instituted,
supplementation of the diet with a variety of other important "nutritional
factors would also seem to be highly desirable.

Endocrine Function and Reproduction. * The known interrelationships
between the B vitamins and endocrine function are numerous;1*1 at our
present state of knowledge many are nebulous, but a few cases at least
have received considerable attention. The endocrine glands represent
areas of high metabolic activity, and it is logical to assume that they
would demonstrate unusual sensitivity to changes in vitamin balance for
that reason. They function, moreover, in a catalytic role frequently asso-
ciated with the control of specific metabolic processes, and the effects
produced by the superimposition of two catalytic factors would for
kinetic reasons be extremely pronounced.

As mentioned earlier, B vitamin deficiency results in a pathological
condition of all the body tissues, and depleted glandular tissue will gen-
erally manifest such a condition by cytological changes and deranged
function. While such a condition is not always observed in all the en-
docrines, there seems ample evidence to indicate that it does occur in
those cases where other factors do not produce death before the effect
is observable. It is remarkable that relatively little is known about the
hypophysis in this regard, and this fact may be a reflection of special
preservative powers possessed by this key gland. In other cases, little is
known regarding vitamin interrelationships aside from the natural de-
generative changes occurring in deficiency. Certain special relationships
are known, however, that seem to involve more than this, and it is with
these that our concern here rests. They involve primarily the thyroids,
the adrenal cortex, and the gonads. Were our knowledge adequate, it
would of course be apparent that these relationships extend through these
endocrines to the others.

Thyroid relationships. The thyroid gland exerts its effects by virtue
of its relationship to the metabolic rate, which in turn has a profound
influence on B vitamin requirements. There is seldom any pronounced
thyroid change in B vitamin deficiency states. Experimental hyper-
thyroidism in animals produces a number of characteristic changes — loss
of weight, interrupted sexual cycles, hepatic damage, cardiovascular dis-
orders, and a drop in liver glycogen. These symptoms can for the most

* The function of the B vitamins as plant hormones has been discussed earlier
(p. 316), and has recently been thoroughly reviewed in the literature (1).

PHYSIOLOGY, PHARMACOLOGY, AND TOXICOLOGY 381

part be prevented or cured by B vitamin, liver or yeast administra-
tion.2, 3- 115 The thiamine content of rat tissues decreases when the animal
is rendered hyperthyroid, and there is increased thiamine excretion.4, 5
Thiamine administration in some cases of Graves' disease has been shown
to be beneficial, and it seems apparent that many of the symptoms of
hyperthyroidism are due to the conditioned malnutrition caused by in-
creased vitamin requirements. There is known to be a real possibility that
the thyroid and other glands have a marked influence on vitamin metab-
olism, particularly at points involving absorption and excretion, but the
nature of such effects is at present too obscure to permit any detailed
consideration. The involvement of the so-called "digestive hormones" in
this regard would seem to be of special interest, but has been little
studied.

Adrenal Cortical Relationships. Pantothenic acid-deficient animals
develop a characteristic lesion of the adrenal cortex,6 and adrenalectomy
in such animals prevents the typical gray hair syndrome,7 although
desoxycorticosterone administration permits hair graying to occur.8 This
relationship has been extensively studied, and is discussed in greater
detail in the next chapter. Despite the fact that there is still considerable
disagreement as to whether apantothenosis produces adrenal hypofunc-
tion or hyperfunction, it is at least clear that there is involved here an
intimate relationship between pantothenic acid, desoxycorticosterone, and
melanin formation, and that this bids fair to be the first direct vitamin-
hormone interrelationship to be understood in any detail. Many of the
other symptoms of pantothenic acid deficiency, such as the deranged
water and salt balance, are undoubtedly manifest through adrenal mal-
function.9-12

Interrelationships with the Gonads and Reproduction. Whereas cer-
tain of the fat-soluble vitamins have been linked with sexual and repro-
ductive functions by tradition, there is no fundamental reason to attribute
such a role to them and not the B vitamins. B Vitamin deficiency most
certainly results in sterility, and this point will be mentioned again later.
There are rather specific manners, however, in which the androgenic and
estrogenic substances are related to the B vitamins, and these are con-
sidered here.

Certain steroid hormones, largely androgenic in nature, have now been
found to have an important role in nitrogen, inorganic phosphorus, and
potassium retention, in castrate animals, eunuchs, and normal animals
including man.13 The administration of testosterone, for instance, pro-
duces an abrupt drop in urea excretion but also a hypoproteinemia, and
it is felt that nitrogen retention is involved with the laying down of
tissue protein. Whether nitrogen retention is a cause or result of this

382 THE BIOCHEMISTRY OF B VITAMINS

is unclear, but testosterone simultaneously produces a marked rise in renal
arginase and e?-amino acid oxidase, so that it is felt that the kidney is in
some manner involved in these overall retention effects. It has previously
been pointed out that the role of the B vitamins is closely associated with
nitrogen metabolism in a variety of ways including absorption, storage,
and metabolic function. It is therefore apparent that the B vitamins must
be considered from a number of standpoints in interpreting the overall
metabolic effects of the androgens.14

Considerably more concise is the relationship between the B vitamins
and estrogen inactivation.15-19 A great variety of ingenious experimental
work has shown that the liver inactivates estrogen and androgens both
in vivo and in vitro. In the case of estrogens, but not androgens, this
inactivation does not occur when the experimental animals are on thi-
amine or riboflavin deficient diets, and it has been suggested that the
liver inactivation normally involves oxidative steps which are impaired
in deficiency. Indeed, it seems well established that cozymase is critical
in this conversion.20' 21 Chick liver converts testosterone to 17-keto-
steroids, while rat liver carries the process beyond this, further metab-
olizing the latter substances.22 It has been pointed out moreover, that
the differences in action on estrogens and androgens during deficiency
works to produce a severe imbalance, and working more or less from this
viewpoint, Biskind et al.23 have reported the successful use of B vitamin
therapy in the treatment of cases of menorrhagia, metrorrhagia, cystic
mastitis, and premenstrual tension. Administration of large doses of
thiamine or riboflavin rapidly restores the ability of livers in deficient
animals to detoxify estrone or alpha-estradiol, but the restoration of
diethylstilbestrol inactivation is less rapid.24 There is no apparent rela-
tionship between liver damage and function in this regard. Inanition and
malnutrition, particularly protein deficiency, in general exert a similar
effect, and it has been claimed that the effects of thiamine and riboflavin
in this regard are not specific, the effect being due to the inanition pro-
duced.25 While this may be true, it seems clear that livers from animals
made deficient in pyridoxine, pantothenic acid, biotin and vitamin A do
not lose their inactivating ability.26 It has been suggested, moreover, that
the pronounced gynecomastia seen in many male prisoners-of-war in the
Orient during the last war may have been due to this effect brought about
by the accompanying malnutrition — quite frequently accompanied by
ariboflavinosis and mild beriberi.27 Excess estrogen, moreover, is known
to cause adrenal hypertrophy, and it seems likely that the adrenal hyper-
trophy associated with certain avitaminoses in rats may be a result of
this same impaired liver function.28

A second relationship of considerable interest involves the fact that the

PHYSIOLOGY, PHARMACOLOGY, AND TOXICOLOGY 383

marked (fortyfold) hypertrophy induced in the chick oviduct by stil-
bestrol does not occur in folic acid-deficient chicks and to only a moderate
degree in pantothenic acid-deficient chicks.20 Thus in experiments in
which chicks were maintained on appropriate deficient diets for fifteen
to nineteen days following which they received daily subcutaneous in-
jections for six days of 0.5 mg of stilbestrol, autopsy revealed folic
acid-deficient chicks to have an average oviduct weight of 62 mg;
pantothenic-deficient chicks had average oviduct weights of 281 mg, with
weights for controls receiving 20 jxg of folic acid daily from birth of 450
mg. It has been shown that there is a direct quantitative relationship
between the oviduct response to stilbestrol and the dietary level of folic
acid. Riboflavin and pyridoxine at least do not behave in a similar man-
ner.30 Later work has indicated strongly that a similar relationship is
true in the folic acid-deficient monkey on estradiol treatment, and that
it is probably a general phenomenon.31 It has been suggested 32 that all
rapid cell proliferation requires special amounts of folic acid for the
necessarily rapid synthesis of nuclear thymine and purine bases. This
suggestion is of some particular interest in view of the role of folic acid
in erythropoiesis and the observed effects of folic acid analogues on tumor
growth.33

Reference to this response of the chick oviduct calls for at least passing
mention of the fact that it is in the oviduct that biotin-binding avidin
is laid down in the hen's egg.34 An extended discussion of the genesis and
significance of this naturally occurring antivitamin is not expedient at
this time, although it should be noted that there is as yet no acceptable
explanation for the functional occurrence of this substance. Despite earlier
hopes, little of significance from the standpoint of an understanding of
its fundamental role in metabolism has been discovered.

A consideration of various aspects of the reproduction process — game-
togenesis, mating behavior, the estrus cycle, embryonic development,
lactation, and maternal instinct — shows at once that adequate B vitamin
nutrition is essential to the process for many reasons that may be con-
sidered in terms of general health.35 Thus, the production of viable sperm
and ova, development of the embryo, and the maintenance of satisfactory
lactation are linked to B vitamin activity, largely because normally func-
tioning cellular and tissue elements are essential to these processes. No
specific B vitamin relationship is known to be directly involved beyond
this, although special "lactation factors" have been reported in experi-
mental work from time to time.30-39 Indirectly, B vitamin-endocrine
relationships may influence these processes as well as mating and maternal
behavior. The estrus cycle, and all the phases of reproduction directly
influenced by estrogens, are directly concerned with the B vitamins in

384 THE BIOCHEMISTRY OF B VITAMINS

the manner previously mentioned, however. It would thus appear that
the role of the B vitamins in the reproductive process per se is no more
than a composite of the various facets previously discussed: the increased
requirements (p. 269), the general metabolism, and the endocrine rela-
tionships. The clinical synthesis of these items is a problem beyond the
realm of this monograph.

Nerve Function. The "special" functions of the B vitamins considered
in this section may be considered as "special" largely because they occur
in the higher animals and may not be considered in terms of the funda-
mental metabolism of a single cell. Thus endocrine relationships and the
metabolism of large aggregates of cells are considerations peculiar to
higher animals as contrasted to single cells. Nervous function is similarly
a specialized activity, and one in which several vitamins are particularly
involved in addition to their fundamental role in the life of the neuron.
From the standpoint of maintaining the lipoid myelin sheath and the
transfer of the nerve impulse along the nerve cell, special relationships
may well exist which can as yet only be surmised. For nerve impulse
transmission, in which acetylcholine is critically involved, thiamine and
pantothenic acid at least must play particularly important roles.
Coenzyme A is known to function in the acetylation of choline, and the
vital nature of this reaction would dictate a critical role in the metabolic
interchange in the neurone.

Thiamine, by virtue of its role in the conversion of pyruvate to acetate,
is obviously important in the supplying of a constant source of acetyl
groups for the pantothenate-mediated coupling, thus:

thiamine

pyruvate >■ acetate") coenzyme A

+ > — > acetylcholine

choline/

It has been observed that diffusible thiamine is present in stimulated
nerves in four to eight times the concentration that it exists in resting
nerves, as measured by bradycardia tests and Phycomyces tests, but that
equal amounts are indicated by yeast fermentation tests.40, 41 This excess
of diffusible thiamine disappears rapidly after stimulation, however. In
nerve poisoned with iodoacetate, by contrast, there is an apparent pre-
ponderance of diffusible thiamine in resting nerve.42 Thiamine (but not
cocarboxylase) is said to block acetylcholine action upon the heart, more-
over.43 The significance of these observations on the basis of the role of
thiamine proposed above is not too clear, and indeed Muralt has proposed
that thiamine must be considered not only as a catalyst, but also as a
metabolic substance in nerve biochemistry.44 The justification for such

PHYSIOLOGY, PHARMACOLOGY, AND TOXICOLOGY 385

a proposal, however, does not at present seem sufficient to warrant its
general acceptance.

Mental Activity. A further field of interest characteristic of animals
involves reflex and mental activity, and because of the pronounced defects
that occur in these functions in vitamin deprivation, various groups have
sought to establish some special relationship between them and the B
vitamins. To a large extent such attempts have been unsuccessful. There
is no doubt that mental activity as measured by sensory acuity and per-
formance tests of various kinds, reflex activity, and general personality
are all influenced greatly in B vitamin deficiency of even a mild nature.
Thus dogs that have developed conditioned reflexes to auditory stimuli
such that they showed a 100 per cent differentiation between two tones
for some months, lose their ability to differentiate in from four to fifteen
days on B vitamin-deficient diets.45 While this loss became worse over
a two-month deficiency period, other deficiency signs were lacking in the
experiment. Vitamin supplementation rapidly restored the ability to dif-
ferentiate, and similar results were obtainable by repeating the sequence
on the same animals.

A number of studies have been made to determine whether thiamine
supplementation improves the mental response of children,46- 47 and the
results have indicated that such is not the case when the individuals
concerned have an adequate thiamine intake. Similar results from adults
on adequate and restricted diets 48-50 seem to indicate clearly that inade-
quate B vitamin nutrition results in a frequently severe and general
breakdown in mental, personality and reflex processes, but that supple-
mentation with B vitamins above the level required for adequate nutrition
as assessed by other means has little or no beneficial effect.

Pharmacological and Toxicological Effects 50a

Previous mention has been made of the several important reasons for
a brief consideration of the pharmacodynamic action of the B vitamins.
Such a discussion might well be undertaken either from the standpoint
of the pharmacological effects observed in particular physiological sys-
tems, or in terms of the known effects of structurally similar compounds.
The latter alternative, i.e., the consideration of each B vitamin separately,
is here adopted because of the desirability of considering the picture so
obtained in the light of deficiency symptoms. The impetus of current
research in this field is such that what is at present a sparsely studied
topic may be expected to expand in the immediate future to a major field
of endeavor. Certainly such an event will be essential before a thorough
understanding of the biochemical processes involved in drug action can
be elucidated and drug design can become a science.

386 THE BIOCHEMISTRY OF B VITAMINS

Thiamine. The pharmacodynamic action of thiamine may best be
described as "curare-like." 51 In frogs, rats, and dogs, except for the
dosage, c?e:c£ro-tubocurarine and thiamine have been demonstrated to have
almost identical effects. Rapid intravenous injection of 50 mg/kg of
thiamine hydrochloride into dogs causes respiratory paralysis, hypoten-
sion, bradycardia, and vasodilation, but if respiration is maintained these
effects are transient. Toe contractions resulting from stimulation of the
isolated sciatic nerve are prevented by both thiamine and dextro-tubo-
curarine, although the muscle still responds to direct stimulation. Similar
effects may be demonstrated on the exposed sciatic nerve and gastro-
cnemius muscle of frogs. These results seem closely related to the
symptoms following administration of lethal doses to experimental
animals — weakness, tetany, labored breathing, and death from respiratory
failure. Thiamine has found some use in the treatment of labor pains52
in intramuscular doses of 60 mg.

The lethal doses of thiamine by intravenous administration are 125,
250, 300 and 350 mg/kg for the mouse, rat, rabbit and dog respectively,
and the toxic subcutaneous and oraT doses are about six and forty times
as high respectively. There is no apparent cumulative effect. The thera-
peutic index (ratio of. therapeutic dose to minimum lethal dose) is there-
fore about 600 for mice, 5000 for rats, and 70,000 for dogs. Haley 54 has
carefully determined thiamine toxicity for the mouse and rabbit and finds
that for the mouse the intravenous LD 50 * is 84.24 mg/kg, with a stand-
ard error of ±1.14 for the mononitrate salt. Intraperitoneally it is 387.3
mg/kg ±1.65; and 329.8 ±3.93 for the hydrochloride. For the rabbit the
average intravenous lethal dose of the mononitrate is 112.58 mg/kg and
for the hydrochloric 117.45 mg/kg. He quotes Molitor as having found
the intravenous hydrochloride toxicity for the mouse to be 85 mg/kg.
The rapidity of the onset of thiamine pharmacological activity is such
as to largely preclude any possibility of action other than interference
in some enzyme system. Death seems to result generally from paralysis
of the respiratory center of the medulla,55, 56 and the administration of
thiamine by cisternal puncture 57 or direct application to the cerebral
cortex 5S produces particularly marked effects.

Thiamine toxicity in man is well known and presents a number of
special problems.59 It appears reasonably certain that the untoward
effects reported as resulting from thiamine administration in man result
either from sensitization and the resulting allergic type of response or in
a very few cases from some inherent susceptibility to this compound. All
the cases reported in this regard, including two deaths 60, 61 and several
near deaths 62 had histories of extensive previous thiamine therapy, and
* The dose that produces death in 50 per cent of the test animals given that dose.

PHYSIOLOGY, PHARMACOLOGY, AND TOXICOLOGY 387

the doses involved have never exceeded 100 mg or so, and were generally
less (5-10 mg) . They were generally intravenous. These levels are far
below the lethal dose that might be anticipated from toxicity data in
lower animals. Unfortunately, however, little accurate toxicity data
exist for lower animals, and much of the data mentioned earlier is subject
to extreme question as regards its accuracy. The symptoms described in
acute cases involve those classically associated with anaphylaxis,63-67
and in some cases at least hypersensitivity has been demonstrated by
passive transfer.68 In sensitive persons, subcutaneous injection of small
amounts of thiamine generally causes large wheals,68 although the validity
of such a skin test has been challenged.69 Other symptoms often asso-
ciated with thiamine toxicity include herpes zoster,70- 71 headache, trem-
bling, and a rapid pulse. The symptoms have been likened to those
occurring from an overdose of thyroid extract,72 and in general bear little
resemblance to the curare-like action seen in lower animals.73-82 Attempts
to sensitize rabbits, dogs, and guinea pigs to thiamine have been unsuc-
cessful.83

Thiamine toxicity is a major problem from the practical standpoint
because large doses of thiamine are so frequently administered. It is for
this reason as much as any that a large number of toxic effects have been
reported for this B vitamin and not for the others. It seems quite clear
that the effects observed in man have little or no relationship to those
seen in lower animals — that in man the result is an allergic one in prob-
ably every case reported.84, 85 That similar results occur from a large
variety of other popular pharmaceuticals — the sulfa drugs are a classic
example — would indicate that such an effect might well be anticipated.
Some workers have strongly advocated the complete abandonment of high
thiamine therapy, particularly by intravenous administration, for this
reason. In any case, it is readily apparent that two things are badly
needed at present: a thorough evaluation of individual responses to
thiamine, and accurate data on the toxicity levels of thiamine for lower
animals other than the mouse. Such information should certainly precede
any acceleration in the present trends toward massive vitamin therapy.

Nicotinic Acid. Nicotinic acid and its amide like many pyridine
derivatives, have marked pharmacological activity. One of the most
important manifestations of this in man is the nitroid reaction, which is
similar to the effects of histamine and caused by nicotinic acid but not
the amide.86-92 Shortly after a suitable dose of nicotinic acid is adminis-
tered, there is a marked flushing of the face, neck, and arms. The reaction
is due to a transient vasodilation which lasts for an hour or so and may
be accompanied by itching or burning. This flushing action is said to be
prevented by a previous oral dose of 30-60 gm of glycine.93 There is an

388 THE BIOCHEMISTRY OF B VITAMINS

increased peripheral blood flow 94 and a rise in cutaneous temperature.
The reaction is not considered to be a "frank toxic manifestation" 95 in
view of the low dosage that evokes it and its absence on nicotinamide
administration, the latter being about twice as toxic as the free acid.
Approximately 30 mg of niacin per day orally may be sufficient to arouse
the reaction, while 10 mg intravenously or 60 mg intramuscularly are
frequently effective. At present, 16-18 mg of nicotinic acid are generally
added to each pound of flour in this country, and there has been at least
one report of a flushing reaction from this source. In general it seems
that there is a great individual variation in response, that all individuals
react to some dosage, and that the manifestation is a harmless one. It
does however make the amide the vitamin isotel of choice, since oral
doses as high as 500 mg are without a nitroid effect.

The pharmacological effects of niacin and many other closely related
pyridine derivatives have been intensively studied, and it should be men-
tioned that the diethylamide of nicotinic acid, coramine or nikethamide,
is a widely used respiratory stimulant.96, 97 Nicotinic acid, as regards its
flushing action, has been likened to histamine, acting in a manner
antagonistic to epinephrine. Nicotinic acid has little or no effect on the
blood pressure or pulse rate however, its effects on peripheral blood flow
being apparently local ones. It is without effect on the autonomic ganglia,
isolated rabbit intestine, or isolated guinea pig uterus. Nicotinic acid has
been compared with its amide, the mono- and diethyl amides, and pyridine
as regards their effects on curare, prostigmine, and acetylcholine action
on the quadriceps and soleus muscles of cats anaesthetized with nem-
butal.98 These results are indicated in Table 33.

Table 33. Some Pharmacological Effects of Niacin and Similar Compounds

. Effect on ■-

Derivative Curare Effect Prostigmine Effect Acetylcholine Effect

Pyridine antagonistic reinforcing enhancing

Nicotinic acid antagonistic antagonistic enhancing

Nicotinamide antagonistic reinforcing enhancing

Ethylnicotinamide reinforcing reinforcing enhancing

Diethylnicotinamide reinforcing reinforcing

It has similarly been convenient to compare the toxicity of niacin with
that of many structurally similar compounds. As regards chronic toxicity,
it seems that doses as high as 2 gm/kg daily for several months do not
affect dogs, rats, or chicks.99 Brazda and Coulson have determined the
subcutaneous LD 50's of a number of pyridine derivatives for rats,100
and their results are summarized in Table 34. Unna 101 has similarly
found the LD 50 of niacin for mice and rats to be from 4-5 gm/kg
subcutaneously and 5-7 gm/kg orally, and the toxicity of nicotinamide

PHYSIOLOGY, PHARMACOLOGY, AND TOXICOLOGY 389

Table 34. LD 50 of Several Pyridine Derivatives for 50-100 gm
Rats by Subcutaneous Injection

LD 50

Derivative

(gm/kg)

Order of Toxicity

Pyridine

1.0

5.00

Pyridine methochloride

0.28

17.9

Niacin

5.0

1.0

Niacin methochloride (Trigonellin)

5.0

1.0

Nicotinamide

1.68

3.0

Nicotinamide methochloride (F2)

2.40

2.08

Coramine

0.24

20.8

Coramine methochloride

1.90

2.63

to be about double this. By contrast the closely related alkaloid, arecoline,
has a subcutaneous lethal dose for mice of 0.065 gm/kg.102

^ ^N^

■N
I
CH3

Arecoline Nicotine

Minimum lethal doses have also been determined for niacin intravenously
for a number of species and nicotine was similarly assessed in parallel
experiments,103 with the results shown in Table 35.

Table 35. Intravenous Toxicity of Sodium Nicotinate and Nicotine

Lethal Dose (mg/kg)-

Species Nicotine Sodium nicotinate

White mice 0.8 4500

Rats 1.0 3500

Guinea pigs 4.5 3500

Death from niacin, as from nicotine, follows severe clonic convulsions.
Other symptoms associated with niacin toxicity include generalized
paralysis, depressed respiration, and cyanosis, death generally occurring
in from 12 to 36 hours.

Attention should also be called to the fact that large but sublethal
doses of pyridine derivatives may have a growth depressing effect by
virtue of the fact that they are generally excreted as INT-methyl deriva-
tives, and they may thus draw critically on the available methyl group
supply of the body.

Choline. The pharmacological effects of choline have been well known
for some time 104 and choline chloride has found some use as a therapeutic
agent. It exerts a muscarine effect peripherally, stimulating parasym-
pathetic endings, and also a nicotine effect, stimulating and paralyzing

390 THE BIOCHEMISTRY OF B VITAMINS

autonomic ganglia. By vagal stimulation it slows and weakens the heart
and causes a drop in blood pressure. Choline increases salivary, lacrimal,
and other secretions and both gastroc and intestinal peristalsis. Doses of
600 mg in 240 ml physiological saline may be slowly administered intra-
venously (15-20 minutes) without great danger.

Levels of 1, 2, and 4 per cent choline in chick diets produce retarded
growth but no other apparent ill effects. In rat diets, 2.7, 5, and 10 per
cent choline decreases rat growth by 20, 45, and 100 per cent, respec-
tively. When 1 per cent choline, corresponding to 500 mg/kg/day is
used, it is not effective. The 2.7 per cent level corresponds to 1350
mg/kg/day. Drinking water containing 1 per cent choline decreases
growth, the dose being 750 mg/kg/day.105 The LD 50 of choline given
intraperitoneally to mice is 320 mg/kg, and the oral LD 50 for rats is 6.7
gm/kg. Other studies have reported the oral LD 50 for rats to vary from
3.4 to 6.1 gm/kg depending upon the solution concentration used.106, 109
It has been pointed out that the same toxicity applied to man would mean
that minimum effects would occur at 15-70 gm/day and the LD 50 dose
would be about 200-400 gm.107

Pyridoxine. The lethal dose (LD 50) of pyridoxine for rats by sub-
cutaneous injection is about 3.1 gm/kg.108 The oral LD 50 is 4. gm/kg.
Toxic symptoms involve tonic convulsions some 24 hours after dosing,
the hind limbs being stretched away from the body. These convulsive
attacks continue for from several days to three weeks unless death inter-
venes. At lethal doses death occurs in from 36 to 72 hours. The closeness
of the subcutaneous and oral toxic doses is of course a reflection of the
delayed nature of the symptoms. Autopsy of these animals reveals adrenal
enlargement with occasional cortical hemorrhage. In man pyridoxine is
said to have a sedative effect. Daily feeding of 10 mg/kg to rats, dogs,
and monkeys for a period of three months produces no significant change,
and 20 mg/kg given to cats intravenously is ineffective, as are single
doses up to one gram.110

Riboflavin. There is little or no information available regarding the
toxicology and pharmacology of riboflavin. Five thousand times the
therapeutic dose for mice (i.e.: 340 mg/kg) is not effective in producing
any observable action and it has been estimated that on this basis 20 gm
per day for a 70-kg man should be harmless.111-113

Folic Acid. Folic acid is relatively nontoxic, judging by the limited
available data. The intravenous LD 50 in mg/kg is 600 for the mouse,
500 for the rat, 410 for the rabbit, and 120 for the guinea pig. Death is
apparently caused by the obstruction of the renal tubules as the result
of precipitation of folic acid. Taylor and Carmichael 116 have recently
found a high sex differential in the toxicity of folic acid for dba mice,

PHYSIOLOGY, PHARMACOLOGY, AND TOXICOLOGY 391

the males being much more resistant. Thus, whereas 600. mg/kg. pro-
duced a 100 per cent mortality in females, 1600 mg/kg. did not kill any-
male mice. Five mg/kg intraperitoneal^ does not affect rats or rabbits
when administered daily over long periods, but doses of 50 to 75 mg/kg
do have some slight effects. All efforts to demonstrate any pharmacologi-
cal activity have been unsuccessful.114

Pantothenic Acid, Biotin, p-Aminobenzoic Acid, and Inositol. Little
is known concerning the pharmacology of pantothenic acid. Intravenous
doses as high as 100 mg have been given to man without apparent
effects" (p. 422). Monkeys have been fed as high as one gram per day
and rats have also been given one gram per rat per day without apparent
harmful effects. Biotin, p-aminobenzoic acid and inositol are also known
to be relatively nontoxic but have been little studied in this regard. A
number of esters of p-aminobenzoic acid (the ethyl ester, "Benzocaine,"
and the butyl ester, "Butesin") have however been widely used as local
anaesthetics.

Summary of Relationships

It is apparent that the B vitamins play certain physiological roles in
the higher animals that differ from their maintenance functions in cellular
metabolism, and these roles occur by virtue of the existence of differen-
tiated physiological functions in the higher animals. In these cases the
vitamins undoubtedly act in the same type of reaction, but the process in
which the reaction occurs is not one characteristic of all living cells. The
fact that only a few such relationships can be demonstrated lucidly at
present is probably the result of the state of our learning rather than an
indication of a limitation to the number of "special cases." It seems
entirely reasonable to expect that in the evolutionary scale, increased
morphological complexity would demand an increased complexity in
catalyst functions, and this view should receive considerable elucidation
in the years ahead.

The symptoms of vitamin deprivation, and physiological and phar-
macological levels of B vitamin administration, apparently bear little
relationship to one another. To some extent, this may be a reflection upon
the present status of knowledge. It is also apparent however that closely
similar structural analogues may have vastly different pharmacodynamic
properties. Of considerable theoretical and practical interest in this regard
is the allergic reaction to thiamine, since the symptoms in hypersensitivity
bear little or no relationship to the structure of the causative reagent. The
implications of this situation in regard to future plans for high vitamin
therapy are of the greatest significance, and it may be anticipated that
similar sensitivities will appear for other B vitamins. One cannot but

392 THE BIOCHEMISTRY OF B VITAMINS

wonder whether modern man will be forced by a trick of his genius to
desensitize himself against these substances so critical to his existence.

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49. Henderson, C. R., Wheeler, N. C., Johnson, H. C., Cogswell, R. C., Jr., Berry-

man, G. H., Ivy, A. C., Friedemann, T. E., and Youmans, J. B., Am. J. Med.
Sci., 213, 488-93 (1947).

50. Graves, P. R., Brit. Med. J., 1, 253 (1947).

50a. Molitor, H., and Emerson, G., Vitamins and Hormones, 6, 69-100 (1948).

51. Smith, J. A., Foa, P. P., and Weinstein, H. R., Science, 108, 412 (1948).

52. Shub, P., Am. Rev. Soviet Med., 4, 476 (1947).

53. Molitor, H., Sampson, W. L., E. Merck's Jahresber., 50, 51-64 (1936).

54. Haley, T. J., and Flesher, A. M., Science, 104, 567-8 (1946).

55. Smith, J. A., Foa, P. P., Weinstein, H. R., Ludwig, A. S., and Wertheim, J. M.,

J. Pharmacol. Exptl. Therap., 93, 294-304 (1948).

56. Hecht, G., and Weese, H., Klin. Wochschr., 16, 414-5 (1936).

57. Stern, E. L., Am. J. Surg., 39, 495 (1938).

58. Dias, M. V., Science, 105, 211-3 (1947).

59. Lancet, 252, 223-4 (1947).

60. Reingold, I. M., and Webb, F. R., J. Am. Med. Assoc, 130, 491-2 (1946).

61. Mills, C. A., /. Am. Med. Assoc, 117, 1500-1 (1941).

62. Schiff, L., J. Am. Med. Assoc, 117, 609 (1941).

63. Stiles, M. H., J. Allergy, 12, 507 (1941).

64. Stiles, M. H., J. Am. Med. Assoc, 117, 954 (1941).

65. Eisenstadt, W. S., Minnesota Med., 25, 861 (1942).

66. Leitner, Z. A., Lancet, 245, 474-5 (1943).

67. Stein, W., and Morgenstein, M., Ann. Internal Med., 20, 826 (1944).

68. Laws, C. L., J. Am. Med. Assoc, 117, 176 (1941).

69. Kulz, F. J., Investigative Dermatol, 5, 135 (1942).

70. Sternberg, C. L., Am. J. Digestive Diseases, 5, 680-1 (1938).

71. Sternberg, C. L., J. Am. Med. Assoc, 116, 2713 (1941).

72. Mills, C. A., J. Am. Med. Assoc, 116, 2101 (1941).

73. Demole, V., Kongressber., XVI Int. Physiol. Kongr., Zurich, 1938, p. 19.

74. Pick, E. P., and Unna, K., J. Pharmacol. Exptl. Therap., 83, 59-70 (1945).

75. Smith, J. A., Science, 106, 622 (1947).

76. Smith, J. A., Foa, P. P., and Weinstein, H. R., Federation Proc, 6, 204 (1948).

77. Smith, J. A., Foa, P. P., Weinstein, H. R., Ludwig, A. S., and Wertheim, J. M.,

Federation Proc, 7, 116-7 (1948).

78. Smith, J. A., Foa, P. P., Weinstein, H. R., Ludwig, A. S., and Wertheim, J. M.,

J. Pharmacol. Exptl. Therap., 93, 294-304 (1948).

79. Haley, T. J, Proc. Soc Exptl. Biol. Med., 68, 153-7 (1948).

80. Perla, D., Proc Soc. Exptl. Biol. Med., 37, 169-72 (1937).

81. Zaidi, S. H., Indian Med. Gaz., 82, 181 (1947).

82. Hamovici, H., and Pick, E. P., Proc Soc. Exptl Biol Med., 62, 234-7 (1946).

83. Molitor, H., Federation Proc, 1, 309-15 (1942).

84. Leitner, Z. A., Lancet, 252, 345-6 (1947).

85. Haley, T. J., Lancet, 252, 500 (1947).

86. Smith, D. T., Ruffen, J. M., and Smith, S. G., /. Am. Med. Assoc, 109, 2054-5

(1937).

394 THE BIOCHEMISTRY OF B VITAMINS

87. Sebrell, W. H., and Butler, R. E., J. Am. Med. Assoc, 111, 2286-7 (1938).

88. Spies, T. D., Bean, W. B., and Stone, R. E, J. Am. Med Assoc, ill, 584-92

(1938).

89. Popkin, R. J., Am. Heart J., 18, 697 (1939).

90. Field, H., Jr., and Robinson, W. D., Am. J. Med. Sci., 199, 275-6 (1940).

91. Alport, A. C, and Ghalioungui, P., Arch. Schiffs- u. Tropen-Hyg., 43, 34 (1939).

92. Robertson, D. S., Brit. Med. J., 1, 339 (1941).

93. Bean, W. B., and Spies, T. D., J. Am. Med. Assoc, 114, 439 (1940).

94. Abramson, D. I., and Katzenstein, K. H., Am. J. Med. Sci., 200, 96-102 (1940).

95. Nutrition Revs., 4, 65-7 (1946).

96. Goodman, L., and Gilman, A., "The Pharmacological Basis of Therapeutics,"

Macmillan, New York, 1941, p. 270.

97. Huidobro, F., and Jordon, J., J. Pharmacol. Exptl. Therap., 86, 49-59 (1946).

98. Valenzuela, F., and Huidobro, F., J. Pharmacol. Exptl. Therap., 92, 1-14 (1948).

99. Spies, T. D., Hightower, P. P., and Hubbard, L. H., /. Am. Med. Assoc, 115,

292-7 (1940).

100. Brazda, F. G., and Coulson, R. A., Proc Soc Exptl. Biol. Med., 62, 19-21

(1946).

101. Unna, K., J. Pharmacol. Exptl. Therap., 65, 95-103 (1939).

102. Hunt, R., and Renshaw, R. R., J. Pharmacol. Exptl. Therap., 35, 75-98 (1929).

103. Chen, K. K., Rose, C. L., and Robbins, E. B., Proc. Soc. Exptl. Biol. Med., 38,

241-5 (1938).

104. "The Merck Index," 5th Ed., Merck and Co., Inc., Rahway, N. J., 1940, p. 142.

105. Melass, V. H., Pearson, P. B., and Sherwood, R. M., Proc. Soc Exptl. Biol.

Med., 62, 174-6 (1946).

106. Neuman, M. W., and Hodge, H. C, Proc. Soc. Exptl. Biol. Med., 58, 87-8

(1945).

107. Nutrition Revs., 5, 116-18 (1947).

108. Unna, K., and Antopol, W., Proc. Soc Exptl. Biol. Med., 43, 116-18 (1940).

109. Hodge, H. C, Proc. Soc. Exptl. Biol. Med., 58, 212-15 (1945).

110. Unna, K., and Molitor, H., Am. J. Physiol, 129, 483-4 (1940).

110a. Weigand, C. G., Eckler, C. R., and Chen, K. K., Proc. Soc. Exptl. Biol. Med.,
44, 147-51 (1940).

111. Kuhn, R, and Boulanger, P., Z. physiol. Chem., 241, 233-8 (1936).

112. Kuhn, R., Klin. Wochschr., 17, 222-3 (1938).

113. Demole, V., Z. Vitaminforsch., 7, 138-43 (1938).

114. Harned, B. K., Cunningham, R. W., Smith, H. D., and Clark, M. C, Ann. N. Y.

Acad. Sci., 48, 289-98 (1946).

115. Ershoff, B. H., Proc. Soc. Exptl. Biol. Med., 70, 398-401 (1949).

116. Taylor, A., and Carmichael, N., Proc. Soc. Exptl. Biol. Med., 71, 544-5 (1949).

117. Wetzel, N. C. Fargo, W. C, Smith, I. H., and Helikson. J., Science, 110, 651-3

(1949).

Chapter VIC

B VITAMIN DEFICIENCY STATES

General Considerations

When for some reason the supply of one of the B vitamins to an animal
organism is diminished to a level below the nutritional requirement, the
B vitamin content of the organism slowly diminishes, as a result of both
molecular attrition and excretion. The time required for the shortage to
become manifest varies with both of these factors and with the rate at
which the nutritional deficit is incurred. Eventually, however, the short-
age of vitamin will be felt in the individual metabolizing cells of the
body and the cellular function becomes retarded or deranged in some
other manner. The various organs and tissues of the body vary in their
ability to fend for themselves under such adverse conditions, as evidenced
by the fact that deficiencies do not primarily affect all portions of the
animal with equal severity, and that death from B vitamin deficiency
generally results from the loss of function of some one most susceptible
organ or tissue, and seldom from general inanition.

Since all the B vitamins are required by every living cell, and except
for nicotinic acid and choline, animal tissues are apparently incapable
of B vitamin synthesis, it would appear that all B vitamin deficiencies
might result in largely the same symptoms in any given species of animal.
This is generally true, except for those instances where a particular
tissue has an unusually high requirement for some one vitamin;1 in this
case the cells of that tissue may suffer disproportionately from the deficit,
and the tissue will develop a specific pathology in response. It is there-
fore believed that the symptoms of the avitaminoses are generally not
due to any particular biochemical effect but rather to a diminished func-
tion of the cells of the various body tissues, resulting in a pathological
condition of the tissue concerned.

From the overall standpoint, the progress of an avitaminosis generally
follows a rather set pattern, in which the course of the disease is first
manifest in general feelings of poor health, accompanied by a decreased
fasting urinary vitamin excretion level. As the symptoms progress there
is a gradual decrease in tissue vitamin levels until the point is reached
where clinical deficiency signs and symptoms occur and macroscopically
and microscopically demonstrable pathology results2 (see Figure 17).

395

396 THE BIOCHEMISTRY OF B VITAMINS

The symptoms associated with B vitamin deficiencies are consequently
of a broad and general nature,3 affecting all the organs and systems of
the animal. Neurological symptoms are manifest psychiatrically and by
both central and peripheral neurological malfunction and degeneration.4
Cardiovascular and gastrointestinal symptoms are almost invariably
found. Epithelial degeneration is characteristic, and hematological and
endocrinological involvements are pronounced in deficiencies of at least
some of the B vitamins. A more detailed consideration of specific defi-
ciencies follows in a later section, but it is important to emphasize at
this point the general nature of the symptomatology, which frequently
makes clinical diagnoses of avitaminoses extremely difficult. A conse-
quence of this fact is that much of our knowledge concerning deficiencies
has come from "epidemiological" studies, where the disease was deduced
from the nature of the diet. Largely as a result of this development, a
diagnosis of an avitaminosis in an individual living in a generally well
nourished population area is seldom made, and our understanding of
individual differences in requirements and of subclinical vitamin defi-
ciencies is a recent but only slowly progressing effort to remedy this.2, 5- 6

B Vitamin deficiencies generally occur as the result of an unbalanced
diet, and when a dietary intake is so poor as to bring the level of one
B vitamin below a critical level, it rather frequently happens that
more than one vitamin is lacking in the diet in adequate amounts. Con-
sequently, many of the classical pictures of deficiency are actually com-
pound deficiencies, and clear-cut clinical cases involving a deficiency of
one and only one nutritional factor are seldom seen.7-12 This fact is
exemplified by the many difficulties experienced by most earlier workers,
and in some cases by workers even today, in producing diets for animal
experimentation which are lacking in only the factor under study.13 As a
result of the compound nature of most clinical avitaminoses, there is a
great wealth of practical information available on the pathology of
naturally occurring deficiency diseases, but much less information on the
picture involved in any particular B vitamin deficiency. In considering
clinical data bearing upon the medical aspects of deficiency, it is there-
fore most important to keep in mind the probable complex nature of the
deficiency. Moreover, many case reports from nonendemic populations
involve deficiencies secondary to some other affliction, in which case the
symptomatology is similarly a complex one in which the distribution of
symptoms between a number of causative factors is difficult if not im-
possible to analyze.14, 15

Finally, there is a growing realization at present that the incipient
early stages of deficiency may manifest themselves over extended periods
in individuals upon a slightly submarginal B vitamin intake.16 The

B VITAMIN DEFICIENCY STATES 397

extent to which such subclinical deficiencies occur is at present unknown,
but a growing body of evidence seems to suggest that even in populations
having a high standard of living there may be a high incidence.17 The
early rapid commercial overexploitation of vitamins which resulted in a
universal "vitamin consciousness" has now been followed by a reactionary
era of public resistance to the general topic of vitamins, with the result
that progress in the study and treatment of subclinical deficiencies may
well be extremely difficult for some years.

An extended discussion of B vitamin deficiencies has not been under-
taken in this volume for a number of reasons. Primarily this monograph
is concerned with the more biochemical aspects of B vitamins, and a very
extensive discussion of clinical material would be out of place. The clini-
cal picture, moreover, is more appropriately one for clinicians and pathol-
ogists, and is not within the scope of interest of either the authors or most
of the readers of this book. The references found in this section, therefore,
are generally the recent publications which add something to the long
known overall picture of deficiency. Finally, a great number of excellent
clinical treatises exist, which may be turned to by those whose interests in
this aspect go beyond the coverage given here. In keeping with the pur-
poses set forth in the preface, therefore, this chapter presents a broad gen-
eral survey of the essential facts concerning the B avitaminoses, omitting
a voluminous mass of supplementary data of interest only to those who
would pursue the clinical problems in an extended manner. Only by this
course may the vast majority of those for whom this treatise is chiefly
intended hope to obtain some picture of the general field of B vitamin
deficiencies.

B Vitamin Deficiencies in the Lower Forms of Life

Very little is known regarding the natural occurrence of B vitamin
deficiencies in organisms other than the vertebrates. In single-celled
organisms, the lack of some required B vitamin results in a cessation of
growth, although some portions of the metabolic machinery may func-
tion for short periods under these conditions. For this reason there is
little to be gained from a consideration of the known facts regarding this
condition in bacteria and protozoa, aside from certain metabolic de-
rangements that might be discussed, as they shed light upon similar
situations in the higher animals. Perhaps the nearest approach to such
a state among the green plants occurs when some foreign toxic substance
inhibits the activity of a growth factor, as in the case of the lycomaras-
mine inhibition of a strepogenin activity in the tomato (p. 260). Labora-
tory-induced deficiencies have been produced in at least one lower animal,
the rice moth as previously mentioned (p. 314), it having been rendered

398 THE BIOCHEMISTRY OF B VITAMINS

deficient in each of several B vitamins by means of techniques similar
to those used with higher animals. Certain insecticides such as "Gam-
mexane" (hexachlorocyclohexane) may function to produce conditioned
deficiencies in some other insects by antagonizing vitamin activity,20 and
it is interesting to reflect on the possible action of certain "weed killers"
by a similar mechanism. In general there seems to be little present inter-
est in this general field of endeavor, however, despite the many intriguing
possibilities that exist for improving the growth of both plants and
economically important lower animals, and the destruction of undesirable
members of both kingdoms by vitamin analogues.

B Vitamin Deficiencies in the Higher Animals

Introduction. While the roles of thiamine and nicotinic acid in the
etiology of beriberi and pellagra have been recognized for only a rela-
tively few years, these two important B avitaminoses have been recog-
nized as human afflictions for centuries. Beriberi was well known and is
clearly described in the literature of the 7th century in China, although its
spread to Japan was not apparent until a thousand years later, possibly
as a result of the introduction of polished rice into the Japanese diet at
that time. Descriptions of pellagra apparently do not occur before the
early part of the 18th century, although the affliction probably occurred
before this time. It is difficult to estimate the full effect of these two
diseases during the course of history. In the Philippine Islands as late
as 1947 the mortality rate due to beriberi was 132 per 100,000 popula-
tion, being second only to pulmonary tuberculosis.18 In the Japanese
navy prior to Takaki's work the incidence was 23 per cent.19 Just as beri-
beri is endemic in the Orient, so pellagra has been in many other areas
of the world, including the southern United States. In 1941 in this country
there were 1868 deaths from this disease reported.20 All these figures
constitute only that small part of the total cases that terminated fatally,
and reflect only those deaths reported to the authorities and known to be
due to this cause. Moreover, the indirect effects of this malnutrition un-
doubtedly resulted in a far higher mortality than the avitaminosis itself.
At present, with the technical ability available to eradicate these and
other avitaminoses, the overall picture is changing only slowly, because
of the enormous problems involved in practical implementation of the
prophylaxis and therapy in most of the populations where these diseases
occur.21

The discussion of the avitaminoses which follows must of necessity be
from the standpoint of a consideration of undesirable afflictions that have
no place in a well regulated society. It is indeed difficult to picture disease
as serving any good purpose, and yet in some instances we find reports of

B VITAMIN DEFICIENCY STATES 399

increased resistance to some infectious agents in deficiency states.22-24
Of even greater interest, however, is a consideration of the increased
muscular activity which results in some cases of nutritional deprivation.
It has been shown repeatedly in rats deprived of thiamine or riboflavin
(but not carotenol or mineral salts) that in the period before the usual
signs of deficiency occur, the running activity of these animals is mark-
edly increased. Wald and Jackson 25 have pointed out that in a free en-
vironment this activity would increase the probability of the animal
encountering what it lacks. "In effect it represents a gamble in which
the animals' metabolic reserves are staked against the chance of finding
its necessities. The possibility of a successful outcome for the individual,
however, is not the only point of the reaction. It probably represents also
the behavioral basis of mammalian emigration." These authors feel that
emigration serves more to relieve nutritional pressures on the home
population than to rescue the individual emigre from starvation.

Thiamine Deficiency. There are three known major causes of thiamine
deficiency in the higher animals. Of primary importance, the lack of an
adequate nutritional source of thiamine results in a number of conditions
typified in man by beriberi. Vying with this for importance, the second
cause is an increased or inherently high requirement for thiamine in spe-
cific individuals, due (1) to physiological conditions previously men-
tioned in the discussion of vitamin metabolism, (2) to the action of toxic
agents or pathological conditions, or (3) to circumstances involving an
abnormal dietary. Finally, mention has previously been made of the
production of deficiencies of thiamine in nature by the action of thia-
minases and other anti-thiamine compounds. Since the last two etiological
factors have already been discussed in some detail, and their result is
much the same as in the case of primary nutritional deficiency, the dis-
cussion will be principally concerned with this latter situation.

Beriberi is endemic in regions where polished rice is a major item in
the nutrition, polished rice containing about 6 per cent (0.02 mg. per
cent) of the thiamine of brown rice (0.3-0.4 mg. per cent) . White wheat
flour and other high carbohydrates staples in the diet have also been
responsible for beriberi from time to time.27, 28 Certain other factors,
essentially those which increase the thiamine requirement,29 tend to pre-
dispose toward the condition. The incidence is higher in warm months
and under condition of high humidity, much commoner in men than
women, most frequent between the ages of 15 and 30 years, and greater
in individuals having other forms of disease.

Infant beriberi is also common in many children breast-fed by mothers
with beriberi, and is most common in the second month of life. A recently
reported acute case in the United States was found in an infant at birth

400 THE BIOCHEMISTRY OF B VITAMINS

that was delivered by a mother with only mild symptoms, however.30
In the Philippines in 1947 about sixty-six per cent of the deaths from
beriberi were infants,18 and it may be that infantile beriberi is commoner
in the United States than is generally appreciated.31, 32

Thiamine deficiency in the Western world is generally a more subtle
affliction, for although outbreaks of beriberi have been known here, it is
not common. Generally the result of inadequate thiamine in this case
is manifest in one of two ways: by cardiac manifestations 2S or by neuro-
logical symptoms.33-35 The latter type is commonly referred to as nutri-
tional polyneuritis, and is a frequent result of chronic alcoholism. Similar
to this form, but frequently of less severity, is an almost general incidence
of very mild beriberi in the Orient, and below this in severity is the wide-
spread existence of so-called "subclinical" thiamine deficiency,6 which, as
medical experience progresses, becomes more and more clearly defined and
less and less "sub"-clinical. The epidemic dropsy seen in India and Africa
may well be an atypical form of beriberi.36 Another frequently referred
to but relatively rare form of thiamine deficiency is the encephalopathy
of Wernicke, or Wernicke's disease.37 Naturally occurring thiamine
deficiency in vertebrates other than man is apparently rare, the "Chastek"
paralysis of foxes (p. 292) being perhaps the best known example. In
general, the symptoms of thiamine deficiency in most animals follow
closely those in human beriberi.

Beriberi is generally referred to as acute or chronic, and "wet" or "dry,"
the latter terminology being dependent upon the presence or absence of
severe edema. The symptoms of the disease are conveniently considered
in three categories: cardiovascular, neurological, and edematous. It
appears that palpitation of the heart and dyspnea (difficult or labored
breathing) are among the earliest symptoms ; there is hypertrophy of the
heart, and its action is increased. Ellis found that in 125 cases of beriberi
that came to autopsy the average heart weight was 379 gm as compared
with 255 gm for 204 patients dying from other causes.38 The pulse be-
comes rapid (120-130), and subject to change upon the slightest exertion.
Diastolic pressure alone is low. Despite these pronounced changes, there
is often little change in the electrocardiogram. Nervous symptoms are
both motor and sensory but there is seldom any sensory disturbance
apparent that is not located peripherally. The sensory symptoms involve
hyperaesthesia (increased sensitivity) commencing in the lower extrem-
ities and frequently the finger tips, followed by pain and frequently
cramps. In severe cases other areas of the body may be involved, but the
motor disturbances are generally limited to the lower extremities.

It is of interest that the sensory disturbances do not reflect the distri-
bution of particular nerves, and that both motor and sensory symptoms

B VITAMIN DEFICIENCY STATES 401

in many cases are more pronounced on one side of the body. Motor
disturbances may vary from a general sense of weakness to absolute
paralysis. In severe cases there is extensive edema which is present only
to an incipient degree in the milder ones. Fever and vomiting also occur
in the advanced stages; there is no respiratory pulsation of the diaphragm,
and severe pulmonary edema aggravates the generally terminal cardiac
failure. While the disease may run a protracted course throughout most
of which dietotherapy provides rapid recovery, a much feared variation
which occasionally develops — Shoshin — brings about rapid aggravation
of the symptoms and death within a few days. Findings on autopsy are
generally in line with those to be expected from the symptoms: edematous
effusions, typical cardiac changes, and frequent peripheral nervous degen-
eration. Histologically demonstrable nerve degeneration has never, how-
ever, been proved to be the result of thiamine deficiency.39

In the less severe deficiencies more frequently encountered in the West-
ern world, loss of appetite is one of the first symptoms.40 In many cases
there is an accompanying disturbance in gastric motility and in experi-
mental animals, at least, a more severe gastric disturbance frequently
occurs. It has been shown that in thiamine-deficient rats there is a much
greater volume of gastric secretion, but there is no change in acidity,
peptic power, or total chloride concentration.41 There is a high incidence
of gastric ulcer in protein-deficient rats on a restricted calorific intake
which is independent of thiamine intake; but thiamine-deficient rats
have a greater incidence, number, and severity of lesions than rats having
adequate thiamine.41, 42

In the type of deficiency manifest largely by cardiac symptoms, the
most frequent disturbances are dyspnea, tachycardia (increased heart
rate), and palpitation, and the general picture with its less frequently
occurring auxiliary symptoms of edema, pulmonary congestion, and
systolic and diastolic murmurs is such as to show little difference from the
similar phase in beriberi itself. Weiss and Wilkins state that thiamine
deficiency in their experience is a more frequent cause of heart disease
than either subacute bacterial endocarditis or congenital heart disease.43
In the neuritic type of disturbance, mild cases frequently manifest only
absence of knee jerks, plantar dysesthesia (impaired sensitivity of the
sole of the foot), and a tenderness of the calf muscles; prompt recovery
generally results from thiamine administration. In more severe cases these
symptoms become progressively worse, resembling those of beriberi,
although in this case there may be a more frequent involvement of mental
difficulties (Korsakoff's syndrome?). The course of the disease may be as
short as a few weeks, but is frequently much longer, and when acute,
seldom responds completely to thiamine therapy.

402 THE BIOCHEMISTRY OF B VITAMINS

Wernicke's disease is marked by rather different symptoms, largely of
a cerebral nature.37 A typical hemorrhagic lesion of the third and fourth
ventricles of the brain frequently associated with polyneuritis and
alcoholism (and found in thiamine-deficient pigeons) is the principal
anatomical sign, while symptoms involve lethargy and excitability, coma,
nystagmus (a rapid involuntary oscillation of the eyeball) , vomiting, and
cardiac and respiratory irregularities. The reasons for the manifestation
of thiamine deficiency in different individuals in such a variety of ways
are unknown. It has been variously proposed that the critical factor is a
matter of degree of deficiency, of compound deficiencies or pathologies, or
of varying sensitivities of the several affected systems in different indi-
viduals. A large number of other animal species have been rendered
thiamine-deficient and their symptomatology studied; to a large extent,
the principal symptoms are those found in beriberi.

In the complete absence of thiamine mice die so rapidly that typical
symptoms do not develop; but in a more gradual depletion they cease to
grow, lose weight, undergo convulsions (especially when spun by the tail),
and exhibit brain and muscular lesions and testicular degeneration.44

Rats follow a similar course, exhibiting a failure to grow and a loss of
weight, followed by typical convulsions and polyneuritis. Assay methods
exist for thiamine which depend on either growth,45 or the prevention or
cure of polyneuritis in rats.46 The normal heart rate of rats is from 500 to
530 beats per minute, and on a thiamine-free diet this drops to 250 to 300
per minute after about two weeks. The remission of this bradycardia has
also found considerable use as a thiamine assay technique, because of its
rapidity, economy of animals, and relative ease and accuracy.47

Thiamine deficiency in the fox, ferret, and mink is generally referred
to as Chastek paralysis (p. 292) . In young foxes, death occurs before
other typical symptoms occur; but in older ones, a period of anorexia is
followed by general weakness, ataxia (lack of ability to coordinate
muscular movements), and spastic paralysis. The paralysis is character-
ized by extreme board-like stiffness with the heads drawn back, but by
no signs of mental effects. Death may occur before the paralysis is com-
plete, or may not ensue until the complete paralysis has been developed
for some time. Autopsy reveals cardiac edema and degeneration, and
hepatic congestion, hemorrhage, and necrosis. Typical Wernicke lesions
are found in the brain of foxes after about 40 days' depletion, whereas
they occur in dogs only after prolonged chronic deficiency.

Thiamine deficiency has been studied in a wide variety of other animals,
important among which are cats,48 monkeys,49 cattle,50 and birds. Eijkman
and Grijn's original observations on thiamine deficiency and the curative
effects of rice polishings were made on chickens. Pigeons were extensively

B VITAMIN DEFICIENCY STATES 403

used as assay animals in earlier work, both the cure of polyneuritis and
the maintenance of weight having been used as the measured criteria in
the assays.51 Thiamine has also been measured by its effect on the oxygen
uptake of avitaminotic pigeon brain tissue, once referred to as the
"catatorulin effect." 52 It is of interest in this regard that differences
apparently occur in the ability of tissues from different species to take
up oxygen.53 Deficient pigeons first exhibit a general lack of activity and
cease to eat normally. One of the most characteristic symptoms of
thiamine deficiency in the pigeon is head retraction, and this symptom
has been utilized as a criterion in assay work. The rapid cure by thiamine
administration of this grotesque, rumed-feather, drawn back neck with
upside down head appearance (opisthotonus) is one of the most sensa-
tional experimental pictures conceivable, particularly in view of the
convulsions and death which normally follow rapidly upon this stage. As
in rats, pigeons that are thiamine-deficient exhibit a marked bradycardia.

The principal biochemical features involved in thiamine deficiency are
an increased blood and urinary pyruvate and lactate and decreased
thiamine and cocarboxylase. As previously discussed (p. 255), thiamine
retention in loading tests is increased. The pyruvism is obviously due to
the lack of ability to convert pyruvate to acetate, a reaction involving
thiamine. Since the oxidative metabolism of pyruvate is further blocked
in the cyclophorase system at the thiamine-mediated conversion of
ketoglutarate to succinate, the decreased oxygen uptake of thiamine-
deficient pigeon brain would certainly be expected. Studies on the pyru-
vate exchange in the heart of thiamine-deficient dogs indicate that the
heart normally oxidizes completely the products regularly formed within
it from carbohydrate metabolism, and in addition some of the products
derived from other organs via the blood, and that in thiamine deficiency
this function is so impaired that the increased blood pyruvate may to a
large extent originate from the heart.54 Since many factors influence blood
pyruvate and lactate levels, it seems that pyruvate and lactate determi-
nations are not more sensitive and accurate as diagnostic tests for
thiamine deficiency than thiamine levels themselves.55

There is some evidence to indicate that episthotonos in the pigeon and
some other symptoms in other species are a consequence of lactate accu-
mulation, which may be demonstrated in the liver, heart, muscles, and
brain. There is similarly an accumulation of tissue pyruvate but the
actual amounts of either acid are so small as to make doubtful their
contribution to the toxic effect. Other evidences of a deranged carbo-
hydrate metabolism follow as a sequence to the blocked pyruvate oxida-
tion. Pigeons exhibit a hyperglycemia and depletion of liver glycogen,
and rats show abnormally high glucose tolerance curves (reduced glucose

404 THE BIOCHEMISTRY OF B VITAMINS

tolerance). In human beriberi, too, some degree of hyperglycemia is
common. It is noteworthy in this regard that both the islets of Langerhans
and the adrenal medulla are frequently hypertrophied in beriberi. It has
also been observed that there is a decrease in gastric acidity in beriberi,
frequently progressing to achlorhydria, and Goodhart and Sinclair 56
have demonstrated a definite correlation between gastric acidity and blood
cocarboxylase. Mention should be made of the evidence that milk from
women suffering from beriberi contains a toxic factor, as do the blood,
urine and tissues of thiamine-deficient experimental animals, and that
such toxic factors may be in part responsible for some of the deficiency
symptoms.57 Methylglyoxal has been suggested in this regard, although
there is insufficient evidence available to evaluate properly the numerous
papers dealing with the presence of this substance in thiamine-deficient
animals.

Horwitt 58 has recently given considerable attention to the study of
blood lactate and pyruvate in mild thiamine deficiency and has concluded
that the basal levels are of little diagnostic value, since at this stage the
organism can still retain a blood equilibrium. Only after pronounced
clinical signs of deficiency become apparent do the blood levels change,
and even then exceptions occur. During mild deficiency, lactate and
pyruvate determination following glucose administration are significant
if correlated with the blood glucose. It was further found in this study
that mild exercise after glucose administration made it readily possible
to detect distinct characteristics in mild thiamine deficiency at an early
stage in its development. A formula termed the "Index of Carbohydrate
Metabolism" or "(CI)" was developed to relate the amounts of blood
lactate (L), pyruvate (P), and glucose (G) in milligrams per cent, and
a change in this index seemed to be highly indicative of thiamine restric-
tion.

(CI)

0-3+(i5p-3

Patients receiving 200 /xg of thiamine per day showed increases of
this index to a pathological level within 10 weeks or less, and clinical
signs of deficiency followed within one to four months after the first
significant rise. This study merits particular attention from the student
of thiamine nutrition because of its extended nature (three years), its
coordinated approach involving biochemical, clinical, neurological, and
psychological investigations, its carefully controlled nature, and the
tremendous emphasis placed on the study and reporting of individual data
rather than average data. While it was primarily concerned with the

B VITAMIN DEFICIENCY STATES

405

problems of thiamine and riboflavin nutrition in gerontology and mental
disease, it is one of the major contributions to the literature on the subject
of vitamin deficiency, and may well serve as a model for further studies
of this nature, much needed with regard to nonclinical individuals and
cases involving the remainder of the B vitamin group.

10 15 20 25 0 5 10 15 20

Days on Thiamine Deficient Diet

Figure 17. Decline in urinary and tissue levels of thiamine during the course of
thiamine depletion in the rat.

Previous mention has been made of the fact that in thiamine restriction,
the rapid drop in urinary thiamine is followed by a much more gradual
decline in tissue levels. Salsedo et al.59 have studied these changes in
thiamine-deficient rats, and their results are summarized in Figure 17.
These workers have suggested that the drop in tissue levels coinciding

406 THE BIOCHEMISTRY OF B VITAMINS

with the time at which urinary thiamine disappears may provide a means
of assessing the thiamine requirement from urinary studies. It is further
apparent that brain tissue seems particularly able to preserve its thiamine
content in periods of thiamine deficiency.

Riboflavin Deficiency. Riboflavin deficiency was first recognized in
man about 1935, although experimental deficiencies in a number of
animals had been produced considerably before this time. The vast
majority of cases of ariboflavinosis seem to be associated with other
deficiencies, characteristically pellagra, but a sufficient number of cases
of noncomplicated deficiency have now been observed to indicate that
the affliction is a rather common and distinct clinical entity.00 Whereas
there is little to indicate its extent, it seems apparent that ariboflavinosis
is relatively common in the southeastern United States, in the Orient,
and among native populations in the West Indies. The major cause of
riboflavin deficiency so far as is known is an inadequate intake, and corn
and polished rice are low enough in riboflavin to insure a deficiency if
some other high riboflavin source is not incorporated into the diet.

The symptoms of ariboflavinosis in man principally involve the mouth,
tongue, nose, and eyes, although general body weakness and dermatitis
may also occur.61 Primarily there is an inflammation of the tip and
margin of the tongue (glossitis), and lesions at the muco-cutaneous
juncture of the mouth with the development of painful fissures.02 There
is an increased redness of the lips, but a pallor to the mucosa, and
a scaly, greasy desquamation about the nose and ears. A nasolabial
seborrhea and seborrhoic and follicular keratosis of the face is common.
Undoubtedly, however, the glossitis is the most prominent symptom, the
papillae being large and flattened, and the tongue itself being a reddish
purple color, appearing clean, but frequently manifesting fissures. Of
equal diagnostic importance, however, is the marked corneal vasculariza-
tion and attendant ocular symptoms involving circum-corneal injection,
photophobia, burning of the eyes, corneal opacity, and pigmentation of
the iris.63- 64 There is little doubt that the eye is one of the most sensitive
organs of the body to riboflavin deficiency. Out of 47 cases of ariboflavin-
osis studied by Sydenstricker et al.05 six had cataract.

Ariboflavinosis is identical with an affliction of children long known in
the southern United States as "perleche." The symptoms of ariboflavinosis
generally respond promptly to riboflavin administration, but only in
patients with teeth does the cheilosis disappear. Ariboflavinosis in
edentulous patients presents certain unique problems, therefore, with
regard to successful therapy.66 Ariboflavinosis is common in many parts
of Africa, and in Nigeria it is said to be present in 16 per cent of the
adult population. A syndrome also seen in Africa in which about 50 per

B VITAMIN DEFICIENCY STATES 407

cent of the cases are under two years of age is known as Kwashiorkor,
or "infantile pellagra," and is thought to be a compound deficiency in
which ariboflavinosis is predominant.07- 68 A recent case of ariboflavinosis
in India was characterized by vulval pathology, a symptom not generally
seen in cases reported from other areas,09 and still other atypical forms
may arise among different population groups.

Riboflavin deficiency was first studied in the rat (in 1926) and termed
"rat pellagra," and the curative agent was termed the "pellagra-preven-
tive factor," although it is now known that the symptoms studied were
those of ariboflavinosis, and that the factor involved was riboflavin and
not the main curative agent for human pellagra. The first symptoms in
the rat are a cessation of growth, and this fact has long been used as the
basis for a riboflavin assay technique. Shortly thereafter there is a general
loss of fur and a characteristic symmetrical dermatitis of the ears, upper
chest, and extremities. The tail becomes dry and scabby, the eyeballs
sunken and lids swollen; corneal vascularization occurs as in man, and
is among the most striking characteristics. Cataract has been reported by
some workers, but there is much disagreement as to its general occur-
rence.70 Granulocytopenia and anemia also occur in severe cases 71 of
ariboflavinosis in rats, dogs, swine, and monkeys, although in some cases
folic acid deficiency seems to be involved in an unclear manner with this
symptom. Swine are said to develop cataracts when suffering from aribo-
flavinosis, but there is apparently no corneal vascularization.

In young chicks on a prolonged mild deficiency there is a highly
characteristic "curled toe paralysis." On a completely deficient diet chicks
exhibit an acute paralysis and dystonia, rapidly followed by death.
Dermatitis is rare but does occur in turkeys. Eggs from hens on a low
riboflavin diet fail to hatch, although injection of riboflavin into the egg
on the first day of incubation remedies this defect.72

Riboflavin deficiency has been extensively studied in dogs. These
animals rapidly become weak, reluctant to walk, and exhibit an ataxia.
Vomiting and occasional convulsion also occur. After some time in this
unhealthy stage the animals develop a diarrhea, followed suddenly by
collapse, coma, and death within a few hours. This rather dramatic rapid
terminal stage is not seen in rats, and only in chicks on a completely
deficient diet.73

The biochemical changes occurring in ariboflavinosis have not been
well studied, although a number of pertinent observations have been
made. There is of course a diminished level of riboflavin and its adenine
dinucleotide in the body and urine under deficiency conditions. The
xanthine ovidase (p. 148) activity of deficient rat liver, as measured by
oxygen consumption rates, is very low.74 Liver slices from deficient rats

408 THE BIOCHEMISTRY OF B VITAMINS

have a decreased D-amino acid oxidase activity, which is increaed by
the addition of riboflavin dinucleotide, indicating that the apoenzyme is
present and only the coenzyme deficient.75 In ariboflavinosis there are,
moreover, some signs of a water metabolism derangement similar to those
that have been observed in pantothenic acid deficiency (p. 424) ,76 In
hens and dogs that have died of ariboflavinosis there is an increased liver
fat content, and a high fat diet seems to aggravate the symptoms of
riboflavin deficiency in rats.

It has been suggested that the oxidative role played by riboflavin in the
normally avascular cornea is counteracted in deficiency by supply of the
needed oxygenation by vascularization. It has been reported that in
riboflavin deficiency there is an increased magnesium requirement.77
Since riboflavin and thiamine have been reported to have limited sparing
actions on each other and since magnesium is involved as a cofactor with
thiamine, it is possible that the magnesium effect is due to increased
thiamine activity. B vitamins have been reported to reverse atabrine
toxicity for rats,78 and riboflavin may be active in this regard, as it is
in reversing atabrine inhibition in the tryptophanase reaction.79 Generally,
however, it is apparent that there is little true understanding of the
biochemical picture in ariboflavinosis.

Nicotinic Acid. Nicotinic acid deficiency occurs most frequently in
the form of pellagra, although classical pellagra is not purely a nicotinic
acid deficiency. This disease is common throughout the world, frequently
compounded with beriberi and other avitaminoses, and at one time or
another has been either endemic or epidemic in nearly every area except-
ing northern Europe. It is endemic in the southeastern United States,80
in South Africa,9- 81 and in some other tropical and semitropical areas of
the world. It is considered in many respects to be more dangerous than
beriberi, and is certainly the major clinical avitaminosis encountered in
the United States. It is considered to be an economic disease, as Drum-
mond says, a matter of pounds, shillings, and pence.82

Most generally pellagra is associated with populations in which corn
is a staple in the diet, just as polished rice is in beriberi. As previously
mentioned (p. 279), this appears to be due to the low nicotinic acid and
tryptophan content of the corn, coupled with the possible presence of a
pellagragenic factor in the corn. Pellagra and other somewhat atypical
forms of niacin deficiency also occur, however, under other circumstances
when the dietary levels of niacin and tryptophan are low.83

As with thiamine, nicotinic acid deficiency is a frequent result of
chronic alcoholism and has been observed secondary to drug addiction.84
It is readily apparent, however, that there are many factors other than
nutritional ones which are secondarily involved in the etiology of pel-

B VITAMIN DEFICIENCY STATES 409

lagra. Sunlight in some manner and to an unknown extent plays an
important role in the disease.85 With the exception of the characteristic
vaginal and scrotal lesions, the early skin lesions generally follow exposed
parts of the body, and it has been suggested that some photodynamic
action at the site of the chromatophores may promote this pathological
characteristic. Strong sunlight is also known to cause relapse in patients
in remission. It has further been suggested that the characteristic out-
break of the disease in the spring may involve sunlight, although it seems
more likely that this is due to the accumulated effects of the nutritional
deficiency acquired during the winter months.

A number of factors combine to make pellagra particularly perni-
cious. Pellagragenic diets are generally deficient in thiamine, riboflavin,
and pantothenic acid, and perhaps still other factors, and the mul-
tiple nature of the avitaminosis has a weakening effect on the body
which leaves it poorly able to withstand the most pronounced deficiency
of niacin. Disturbed gastrointestinal function also plays a major role in
the etiology of niacin deficiency. Dysentery, colitis, intestinal parasitism,
and surgery are all known to be strong predisposing factors toward
pellagra, as they are toward beriberi. It would seem that in these
deficiencies there is a strong probability of the creation of a vicious cycle,
the deficiency leading to further gastrointestinal disturbances which in
turn encourage the avitaminosis. The exact manner in which such organic
afflictions promote deficiency is unknown, although faulty digestion and
absorption seem likely. It is of considerable interest that gastric prepara-
tions have been successfully used for many years in the treatment of
pellagra, and there is at least some evidence to indicate that their efficacy
may not be due to the niacin content.86 (Certain liver fractions which
have a low niacin content are similarly quite active) .

In its essence, as Goldberger pointed out, the major causes of pellagra
seem to involve first dietary factors, and secondly factors that prevent
the normal utilization of the diet by the individual.87 The disease is com-
mon in children and persons of all ages and racial stocks, being most
pronounced in adult married women. Finally, when all the known etio-
logical factors are taken into consideration, it is still difficult to explain
the fact that in both man and experimental animals and without any
dietary change, there is a frequent spontaneous remission of niacin
deficiency which, however, is seldom long-lasting.

As previously stated, pellagra characteristically develops in the early
months of the year. In some acute cases the symptoms become progres-
sively worse and death ensues. In the majority, however, the symptoms
develop for only a few months and then disappear for the most part and
in some cases never return. Generally there is an annual recurrence

410 THE BIOCHEMISTRY OF B VITAMINS

which continues until the progressively weakened condition of the pel-
lagrin results in death, most often in about five years. Nicotinic acid
therapy is highly and rapidly effective for all the symptoms that are
clearly due to the niacin deficiency, but since pellagra is a multiple
deficiency, complete cure is obtained only by multiple vitamin therapy.
In advanced cases where irreversible pathological changes have occurred,
of course, vitamin therapy has only limited efficacy.

The cardinal symptoms of pellagra are often referred to as the three
"D's," dermatitis, diarrhea, and dementia. Gastric disturbances, anorexia,
headache, and loss of weight and strength are among the earliest symp-
toms. Diarrhea may occur quite early, and frequently becomes one of
the severest symptoms in later stages. Para-sprue, a condition that is
widespread in India and similar in many respects to sprue (p. 417) , is
undoubtedly a compound deficiency, principally involving niacin de-
ficiency, and distinguishable from true sprue by the nonfatty nature of
the stools.8s In pellagra, redness of the tip and margin of the tongue 89
and lining of the mouth, and a characteristic gingivitis and gastric
irritation are almost invariable; at the height of the disease the tongue
becomes swollen and cracked, and peels and appears cyanotic in many
cases.90 Nausea is frequent, and along with this and the diarrhea, 40 per
cent of the cases develop an achlorhydria. The dermatitis is bilaterally
symmetrical in most but not all cases,85 clearly defined, and generally
restricted to a necklace or gauntlet pattern about the neck, and to the
dorsal surfaces of the hands and forearms, although it may occur on
other areas exposed to sunlight (the face and lower legs) and on areas
subject to chafing. Frequent characteristic scrotal lesions are also believed
to be due to niacin deficiency, although this point seems uncertain.91- 92
The dermatitis generally originates in the form of deep red areas that
gradually become brown and coalesce and later become thickened and
scaly.

Unlike the symptoms in beriberi or ariboflavinosis, there is a high
incidence of mental symptoms in pellagra, and these may range from
mild psychoneurosis and insomnia to stupor or mania. The existence of
acute mental disorders in this case, and their rapid cure by niacin therapy
merits special consideration from the standpoint of its bearing upon the
nature of mental disease in general. The longstanding and widespread
concept of a purely psychological etiology in the insanities has resulted in
little general advance in the cure of these diseases. Very recently a few
workers have attempted to discover physiological bases for mental dis-
turbances, but in the face of widespread opposition from groups that hold
the "psychic" hypothesis. A thorough consideration of the dramatic cure
of the mania of pellagra by niacin should do much to promote further in-

B VITAMIN DEFICIENCY STATES 411

vcstigations of mental disease from the nutritional and metabolic stand-
points, and to remove the laissez faire policy now applying to the natural
sciences in the study of the mental processes.

A variety of nervous symptoms also occur in pellagra, but it is uncer-
tain to what extent these arc due to an accompanying thiamine defi-
ciency.93- 94 While there are apparently no marked cardiovascular changes
in pellagra, a macrocytic hyperchromic anemia is frequent.05 Atypical
forms of pellagra are frequently encountered as a result of compound
deficiencies. In addition, most workers feel that subclinical pellagra is
common in the United States, and to some considerable degree even in
the North. Finally, recent years have brought forth an increasing number
of reports of cases of nervous and mental disease that have responded
markedly to niacin therapy. To a lesser extent there have been obtained
similar remissions of other of the characteristic symptoms of pellagra
that occasionally appear singly in individuals not exhibiting the general
pattern of the avitaminosis.

Post-mortem examination of pellagrins shows little to indicate the
cause of the disease other than the skin lesions. There is seldom any
change in the stomach, but the colon is thickened, red and typically
stippled with cystic lesions. Nervous lesions are common, but generally
believed to be due to deficiencies of other factors. The heart appears quite
normal, but the liver, while normal in size, is yellowish gray and mottled,
and characterized by fatty degeneration and fibrosis. From the overall
standpoint, it is apparent that the marked differences between the symp-
tomatology of niacin deficiency and that of thiamine and riboflavin defi-
ciency do not in any way reflect obvious relationships of these vitamins
to their coenzyme functions. Indeed, the mental symptoms associated
with pellagra might far more logically result from beriberi, since
thiamine occupies a special place in nerve function (p. 384) ; and since
niacin and riboflavin function metabolically in an intricate fashion, it is
strange indeed that there is not a greater similarity between the symptoms
of their deficiencies.

Until recent years, the mortality from pellagra varied, but was gener-
ally above 66 per cent of the cases. Recently, however, due to improved
dietotherapy and hospital methods, and the specific use of niacin, this
has dropped to below 5 per cent. Niacin, in doses of from 50 to 1,000 mg
daily when administered orally (or intravenously in acute cases) , gener-
ally achieves a rapid remission of the symptoms of niacin deficiency, the
redness of the tongue disappearing in a day and the lingual ulcers in
several days, nausea and vomiting ceasing immediately, the mental
symptoms vanishing within a week, and the dermatitis regardless of its
severity eventually disappearing completely.

412 THE BIOCHEMISTRY OF B VITAMINS

In areas where pellagra is endemic, dogs frequently develop a canine
counterpart of the disease known as "blacktongue." In general the symp-
toms follow closely those found in humans. The mouth is typically dark
red due to necrosis, which in turn causes a drooling appearance and a
fetid odor. There is a generalized gastrointestinal disturbance, and the
scrotal lesions seen in human pellagra are generally found in black-
tongue in dogs. A macrocytic anemia is frequently found,96 and almost
invariably nervous degeneration occurs. The same disease has been pro-
duced in dogs in experimental studies by the use of niacin-deficient diets.
Because rats do not readily develop a niacin deficiency, dogs have
been of great value in the experimental study of niacin deficiency. Recent
work has indicated, however, that the frequent anemia seen in dogs with
blacktongue may primarily be a simultaneous folic acid deficiency.97, 98
That the etiology of pellagra and blacktongue is not fully understood is
readily apparent from the fact that the injection of saline alone is fre-
quently curative for blacktongue.99 In any case it is apparent that many
of the symptoms of blacktongue induced in dogs on certain diets are
those of folic acid deficiency, and that this condition in the dog is
frequently more similar to sprue than pellagra, the balance of the symp-
toms in blacktongue depending largely on the diet by which the deficiency
is induced.100

Pig pellagra also occurs naturally, although with much less frequency,
and pigs have been used somewhat in the study of niacin deficiency 101
because of the possibility of producing this condition experimentally. In
this case there is no glossitis or stomatitis ; the symptoms involve anorexia,
slowed growth, a scurfy skin, colitis, and a diarrhea which is generally
followed within a month by death. The condition responds promptly to
50 mg/day of niacin. Monkeys fed on a niacin-deficient diet similarly
become ill and develop anorexia, diarrhea, and dermatitis, and are cured
by doses of 5 to 25 mg of niacin. As previously mentioned, rats do not
normally develop a niacin deficiency on niacin deficient diets, nor do
lambs.

Biochemical changes in niacin deficiency are not well studied. A change
in the urine that frequently occurs in pellagra and has been used in
laboratory diagnosis involves an increase in pigments which have been
variously identified as coproporphyria I and III or urorosein and indi-
rubin.102 It is generally presumed that these pigments are formed as a
result of impaired liver function, and may be responsible when deposited
in the skin for the characteristic photosensitive effects in pellagra. It
seems well established now, however, that this diagnostic criterion is of
little or no value, since a similar result occurs in a number of unrelated
conditions, and since many pellagrins do not give a positive reaction.103

B VITAMIN DEFICIENCY STATES 413

Significantly, however, the original work upon which this test was based
was done upon a group of pellagrins most of whom suffered the disease
as a consequence of alcoholism, and pellagra secondary to alcoholism may
differ in some respects from other forms. As a result of the extreme nausea
and diarrhea, a hyperproteinemia may develop in advanced cases and a
disturbance of acid-base equilibrium may also occur. The decrease in
urinary levels (and blood and tissue levels in extreme cases) of niacin
and its metabolites has already been mentioned in earlier chapters; this
constitutes one of the most marked biochemical changes occurring during
early or mild deficiency.104 It has also been reported that pellagrins are
extremely sensitive to insulin and are refractory to adrenalin, but these
factors have not as yet received adequate study.105, 106

Folic Acid and Vitamin B]2 Deficiency.107-111 A large number of different
clinical conditions result in the production of anemias, and in recent years
it has become readily apparent that many of these have a definite nutri-
tional deficiency involved in their etiology. Of these a rather significant
number of anemias have been found to respond to a greater or lesser
degree to the administration of the two most recently identified members
of the B group of vitamins, folic acid and vitamin Bi2. While it is by
no means certain that these anemias are caused by a nutritional deficiency,
it is apparent that for one reason or another the bodies of patients so
afflicted do not receive an adequate supply of these factors. It is undoubt-
edly true that a deficiency of any of the B vitamins would ultimately
result in anemia,112- 113 and anemias are associated with deficiencies of a
number of them; but the acute macrocytic anemias that respond to the
two vitamins here discussed are so marked and well recognized as to
leave little doubt that these factors have a particularly important func-
tion in normal erythrocyte physiology. The conditions that respond to
these factors are almost without exception marked by severe anemia, but
this is generally only one of a group of symptoms associated with each
clinical entity to be considered here. Thus there is no doubt that deficien-
cies of these vitamins are manifest throughout the body, and the weight
given the hematological aspect should not distract attention from the
other symptoms of these avitaminoses.

A unique situation exists with regard to these avitaminoses: a number
of factors other than low dietary levels contribute to the majority of the
recognized cases, and these factors bring about a number of different,
prevalent, clinically identifiable forms of the deficiency. The situation is
even more unusual in that two different members of the B group of
vitamins are generally effective in curing most of the deficiency condi-
tions, and that a naturally occurring pterin and pyrimidine are also
known which are effective, though to a lesser degree. This multiplicity of

414 THE BIOCHEMISTRY OF B VITAMINS

curative factors is undoubtedly due to the fact that the substances in-
volved are closely related to one another metabolically, and various con-
siderations may eventually cause the folic acid group and vitamin Bi2
to be considered as a functional unity.

It is important to realize that the afflictions considered here have been
recognized for a great many years; they are widespread, and in terms
of their overall effects they compete with beriberi and pellagra as avita-
minoses: it is for that reason that their discussion occurs at this point.
As a consequence, the consideration of these conditions upon which numer-
ous volumes have been written must be extremely abbreviated. Further,
the discoveries of the folic acid group of substances and vitamin Bi2 and
their remarkable clinical efficacy have been so recent that there is at the
time of writing a considerable degree of uncertainty and even confusion
regarding the true picture of their function. The metabolic function of
these vitamins, discussed elsewhere (p. 198), is at present only poorly
understood. 113a For these reasons it seems best to present in barest outline
form the facts as they now stand, realizing full well that in these para-
graphs more than any other, the overall concept may change drastically
within a few months.

The following clinical conditions have been found to respond in some
degree to one or both of the vitamins here discussed:

(a) Addisonian pernicious anemia.

(b) sprue

(d) macrocytic anemias of pregnancy

(e) macrocytic anemias (megaloblastic) of infancy

(f) macrocytic anemias following surgery, alcoholism, cancer, and
infectious agents.

In addition, thymine (5-methyluracil) in massive doses has been found
to be effective in a number of these conditions.

CH3

HN I
H

thymine

It seems most convenient to discuss the effects of these vitamins and
thymine in relation to each of the conditions mentioned above, and in
the approximate order given.

B VITAMIN DEFICIENCY STATES 415

Addisonian pernicious anemia is a spontaneously occurring macrocytic
anemia further characterized by a permanent, histamine-refractory
achlorhydria, hyperplastic megaloblastic bone marrow, glossitis, and
frequent neurological manifestations ranging from peripheral neuritis to
degenerative lesions of the spinal cord. It is frequently accompanied by
depigmentation of the hair, increased urinary urobilin, and increased
plasma iron and bilirubin. There are generally high blood and urinary
levels of phenolic compounds.114 Tyrosine seems to increase the efficacy
of some liver extracts, and it is readily apparent that there is a close
interrelationship between folic acid, ascorbic acid, and tyrosine metab-
olism;115,116 but the precise manner in which folic acid influences the
ascorbic acid-controlled oxidation of tyrosine is at present obscure. Im-
paired absorption of tyrosine has been made the basis of a "tolerance
test" in which, after a 4-gm oral dose, the blood levels reach a peak only
after three hours, as contrasted with one hour in healthy persons. Per-
nicious anemia patients differ from those with cirrhosis in that the blood
tyrosive level is markedly elevated.117' 118 During remissions there is occa-
sionally a transitory edema of unexplained etiology,119 and a few perni-
cious anemia patients exhibit cardiac symptoms.120

The cause of pernicious anemia is unknown, but it has generally been
considered to be an acquired metabolic defect that results in an impair-
ment of erythrocyte maturation. Liver has long been known to sustain
patients with the disease, but its continued consumption is necessary, and
highly potent concentrates of the antipernicious anemia factor have been
used parenterally for some time. As previously mentioned, achlorhydria
is an invariable accompaniment of this disease, and it has been found
that the gastric juice from normal persons when incubated with beef
steak produces a curative substance, but the gastric juice from patients
does not. The active principle is produced by the mucosa of the pyloric
and cardiac regions of the stomach, and the commencement of the
duodenum, and desiccated defatted hog stomach may be used thera-
peutically as an alternative to liver. Ternberg and Eakin have recently
shown 120a that the active principle in gastric juice, long designated as
the "intrinsic factor," is in reality a protein which combines with vitamin
B12 (extrinsic factor) to form a complex which is resistant to the destruc-
tive changes wrought upon vitamin Bi2 itself by the digestive processes
(p. 342) . In pernicious anemia, the intrinsic factor is absent, and conse-
quently a "conditioned" vitamin Bi2 deficiency results even in the pres-
ence of an adequate nutritional supply, since the vitamin consumed is
destroyed before it can be absorbed.121"123 It has been suggested that
these interrelationships favor the substitution of the term erythrotin for
vitamin B12, apoerythein for intrinsic factor, and erythein for the complex.

416

THE BIOCHEMISTRY OF B VITAMINS

Thymine, folic acid, and vitamin B12 are all effective in bringing about
a remission of the symptoms of pernicious anemia, but the relative effec-
tive doses of these substances varies a millionfold. From 5 to 10 grams
of thymine daily, or approximately 10-mg daily of folic acid are required,
while an initial intramuscular injection of 15 fig of vitamin Bi2 is effective
for a number of days. Spies et aL124 have compared the effects of these
three substances in a case of pernicious anemia that was admitted to the
hospital for treatment three successive times. Their graphs are reproduced

•2 20

10 -

THYMINt

1 1 1 1 1 1 1 1 1 1 1 1 1 1

.!...■«.«

FOLIC AdO

. i ........... i

VTTAMW fta

ii i ii 1 1 1 n 1 1 1

......... .^ »■ i

Figure 18. Hematological response in a case of pernicious anemia.

in Figure 18 and are of particular value because of the analogous data
obtained from cases of tropical sprue and nutritional macrocytic anemia
shown in Figures 19 and 20.

The evidence now available suggests that pteroyl heptaglutamate is
not appreciably active in pernicious anemia,125- 126 and that none of the
folic acid vitamins are effective in curing all the symptoms. Indeed,
neither folic acid nor thymine prevents the development of subacute
combined degeneration of the spinal cord, nor do they retard it once it
is initiated.127 Folic acid in some cases seems to aggravate the neurologi-
cal symptoms, although this effect may possibly be due to impurities in

B VITAMIN DEFICIENCY STATES

417

the preparations used 128 (p. 295) . In this regard, however, there is good
evidence to indicate that folic acid increases cholinesterase formation in
the body, and folic acid is apparently quite effective in counteracting the
hyperchromic anemia produced by choline injection. It therefore seems
well to reserve judgment in this regard until the functions of folic acid
in neural physiology are more clearly elucidated.129, 13° Recent indications
are that vitamin Bi2 does improve the neurological status of at least most
patients with subacute combined degeneration of the spinal cord.131* 132

THYMINE

10,000,000 M9.*&%
bm mouth

. 1 1 1 1 1 1 1 1 1 1 1 1

FOUCACIO

10,000 a* doily

by mouth

ZS*.

""■"■"""

1 1 1 1 1 1 1 1 ii i ii 1 1

3 io

S 8

6
5

Figure 19. Hematological response in a case of tropical sprue.

The achlorhydria of pernicious anemia does not respond to treatment by

any known means.

Sprue is a clinical condition that has been well recognized for some two
centuries and is characterized by a macrocytic anemia with moderate
leucopenia and bone marrow hyperplasia, steatorrhea 133 (high fat in the
feces), glossitis, diarrhea, skin pigmentation, loss of body weight, and
impaired absorption from the gastrointestinal tract. It differs from para-
sprue, which is more basically a niacin deficiency and in which the
diarrhea is of the nonfatty type.88 It is generally felt to be a deficiency
disease resulting from impaired absorption of the antipernicious anemia

VITAMIN Bn
,23/ia.l.M.

418

THE BIOCHEMISTRY OF B VITAMINS

factor. There is generally an accompanying deficiency of the fat-soluble
vitamins due to faulty absorption,134-135 together with nightblindness,
hypocalcemia and hypoprothrombinemia.136 It differs from pernicious
anemia in the skin pigmentation, frequent presence of the intrinsic factor,
frequent occurrence before puberty, general presence of gastric acid,
absorption defects, and frequent association with poor diets. There is a
typically flat oral glucose curve, but a normal intravenous one, and no rise
in blood phosphate after oral glucose. Impaired glucose absorption in this
case has been interpreted as due to an impaired phosphorylation mecha-
nism in the intestinal wall, fructose absorption being apparently normal.137

FOUC ACIO
10,000 /tf. daily

XS:

i 1 1 i i i I 1 1 1 1 1 1 i

i i i 1 1 i i i

VITAMIN B„
15/tf.lM.

v/*

I I 1 1 1 1 1 1 1 1 1

I 1 1 1 1 II 1 1 1 1 1 1 I

3579 II 13 13579 II 13 13579 It 13
DAYS

Figure 20. Hematological response in a case of nutritional macrocytic anemia.

Frequent classification into tropical and nontropical varieties seems justi-
fied only on the basis of its more frequent occurrence and severer forms
in the tropics. Sprue must be regarded as a multiple deficiency, initiated
perhaps by infectious conditions or general malnutrition, and propelled
by the diarrhea and other factors that make absorption inefficient and
create a vicious cycle. The symptoms generally respond well to liver
therapy and improved diet. The response of sprue to thymine, folic acid,
and vitamin Bi2, is almost identical with that of pernicious anemia, and
similar data from the Spies et al. study previously mentioned 124 is shown
for a case of tropical sprue in Figure 19. In separate studies,138 it has
been shown that massive doses of thymine are followed by at least a
year of freedom from sprue and its symptoms. Pteroyldiglutamic and

B VITAMIN DEFICIENCY STATES 419

triglutamic acids are also both effective in the treatment of sprue.139, 140
Some cases of sprue, like those of pernicious anemia, are apparently
refractory to folic acid however.141

Nutritional macrocytic anemia is an ill-defined condition that resembles
both sprue and pernicious anemia, and in many cases may actually repre-
sent stages of one or the other of these diseases. It frequently differs
from sprue in that there is a normal oral glucose curve, and from perni-
cious anemia in the frequent presence of gastric acid and intrinsic factor.
It is most generally a compound deficiency and is often associated with
more marked manifestations of other avitaminoses, such as pellagra.
Spies et aL124 have presented evidence to show a similar efficacy in this
case of thymine, folic acid, and vitamin Bi2 to that in sprue and pernici-
ous anemia, as shown in Figure 20.

The macrocytic anemia of pregnancy is also an ill-defined condition
similar to those already discussed, but characterized by its temporary
nature. It is generally thought to be a manifestation of the heavy demands
upon the mother made by the foetus, since recovery generally follows
delivery, and continuous therapy is seldom necessary. This condition
responds to folic acid,142 thymine,127 or liver therapy, vitamin B12 being
as yet unreported upon. It has recently been reported, however, that this
condition, which is cured by crude liver preparations, does not respond
to some highly purified preparations that are very active against per-
nicious anemia, and there is therefore considerable reason to suspect a
basic difference between the two types of anemia.143, 144

The megaloblastic macrocytic anemias of infancy and childhood con-
stitute a number of clinical entities: celiac disease or steatorrhea, gen-
erally characterized as infantile sprue; true pernicious anemia of childhood
which is extremely rare; a condition characterized as "temporary
pernicious anemia"; and "goat's milk" anemia. The first three of these
conditions are known to respond to folic acid therapy, the last being as
yet unreported upon. The effects of thymine and vitamin Bi2 in these
conditions are also unknown.145

Finally, a variety of miscellaneous conditions, such as diarrhea, gastric
cancer, gastrectomy, and alcoholism, have resulted in conditions of
macrocytic anemia which resemble more or less the syndrome noted above,
and have as their basis the absence of extrinsic factor or an inadequate
supply of folic acid or vitamin Bx2. Some of these, such as gastrectomy
and the so-called "chronic diarrheas," have already been shown to respond
well to folic acid, but further studies are as yet lacking. A macrocytic
anemia caused by infestation with the fish tapeworm, Diphyllobothrium
latum, resembles pernicious anemia in many respects and is noteworthy
because of the extreme nature of the causative agent in this case. This

420 THE BIOCHEMISTRY OF B VITAMINS

latter condition responds to removal of the parasite, to anti-pernicious
anemia liver extract, and stomach preparations, no report of vitamin
therapy being as yet available.146

It is thus apparent that a variety of common long recognized severe
afflictions must now be considered as avitaminoses, and that the symptoms
of these diseases generally respond well to both folic acid and vitamin
Bi2. It is equally apparent, however, that much work remains to be done
in elucidating the relationships of these B vitamins to the full measure
of the antipernicious effect of liver, and to erythrocyte maturation. There
are present indications that vitamin Bi2 may function in the synthesis
of folic acid, and that folic acid functions in purine synthesis.147 The
relationship of these observations to the etiology of the macrocytic
anemias remains for the present obscure, as do the relations of the other
B vitamins to these hematological symptoms.148

Deficiencies in Vertebrates other than Man. Folic acid deficiencies
have been produced in a number of lower animals, most notably
chicks,149, 164 and monkeys.150 In chicks the symptoms involve slow growth
and deficient feathering and a macrocytic anemia, and in monkeys,
diarrhea, gingivitis, and a macrocytic anemia. Folic acid is effective in
causing a remission in both cases. Folic acid deficiencies have been pro-
duced in mice by the use of the inhibitors, 1-methylfolic acid and
4-aminofolic acid (Chap. VD).151 Typical deficiency has also been pro-
duced in rats. Both humans and rats develop severe blood dyscrasias
when treated with certain chemotherapeutic agents. In rats the agranulo-
cytosis and bone marrow hypoplasia produced in this manner (sulfa-
guanidine, sulfasuxidine) respond rapidly to folic acid administration.
Folic acid deficiency also apparently occurs in rats made deficient in
pantothenic acid (p. 423) ,152 and in some cases of blacktongue in
dogs96,98 (p. 412), in which case the syndrome is more like canine sprue
than pellagra. Similar unclear relationships exist between folic acid and
the anemias resulting from riboflavin deficiency in rats, dogs, swine, and
monkeys.153- 154 Rats also have impaired lactation when folic acid-defi-
cient.155 Folic acid is only partially effective in treating the blood changes
induced in rats by gastrectomy,156 and thymine is ineffective in replacing
folic acid in the rat for either hematopoiesis or lactation, or in the chick
at all.157

N^S,XN^OH

HN=LN.

xanthopterin

B VITAMIN DEFICIENCY STATES 421

Salmon and trout both develop anemias that are cured or prevented
by xanthopterin158 and by folic acid; and xanthopterin apparently159
has at least some activity in the monkey and possibly the goat's milk
anemia of rats, but not in the chick.100 The incubation of xanthopterin
with rat liver gives results that indicate, though not unequivocally, that
this substance is converted to folic acid or enhances the liberation of
folic acid from bound forms. Studies with regard to Bi2 deficiency in
lower animals are as yet fragmentary, although the apparent identity
of this vitamin with the "animal protein factor" 161 and the "cow manure
factor" 162 provides some evidence of the necessity of this factor in the
diets of rats and chicks and of the symptoms resulting from some degree
of nutritional deprivation. Anemia has been produced in a pig, however,
by using vitamin-free casein and 2 per cent sulfasuxidine in the diet,
and a remission obtained by the use of purified liver extract; thus vitamin
BX2 deficiency may be attainable in a number of species when the con-
ditions are properly selected. Prior to the isolation of vitamin Bi2, how-
ever, a satisfactory biological response to the antipernicious anemia
factor in animals other than man had been long and ardently sought for
in vain as an assay device. The relationship which may exist between
vitamin B12 and cobalt metabolism should now be reviewed in the light
of the cobalt content of vitamin Bi2 and the known lowering of vitamin
B6 blood levels in cobalt-deficient animals.

Mention should be made of the apparent stimulatory effect of a- and
/?-pyracins upon the pteroyltriglutamate activity on hemoglobin forma-
tion and growth in anemic chicks. Either of the pyracins alone is ineffec-
tive. However, either pyracin with the triglutamate stimulates its effective-
ness (/?-pyracin being somewhat more active) in improving growth and

O — CH2

-CH

a-pyracin fi-pyracin

preventing anemia. /?-pyracin does not further augment the activity of
the monoglutamate in the anemic chick. All three forms of folic acid are
apparently active to some degree for both chicks and monkeys, and the
pyracins seem to influence favorably the conversion of the triglutamate
to the monoglutamate in the chick. Whether this implies an involvement
of pyridoxine metabolism in pteroylmonoglutamate formation from its
higher conjugates remains unknown. Indeed, recent reports have chal-

422 THE BIOCHEMISTRY OF B VITAMINS

lenged the fact that the pyracins have any biological activity whatsoever
in this regard.

Formylpteroic acid (rhizopterin, SLR factor) is inactive in curing rat
leukopenia, and its magnesium salt and the magnesium salt of formylfolic
acid, presumably a functional form of folic acid, are both apparently
inactive in hematopoiesis in man,163 as is pteroic acid itself. Indeed,
formylpteroic acid is inactive in curing folic acid deficiency in rats or
chicks in concentrations 50 times the curative dose of folic acid. The
present limited evidence regarding the inefficacy of formylfolic acid in
man should be viewed with caution, however. These observations are of
interest in view of the inactivity of pteroylpolyglutamates for bacteria,
unlike man, and the high activity of formylpteroic acid in some bacteria.

Pantothenic Acid Deficiency. While there is no acute widespread disease
of man now known to be associated with a lack of pantothenic acid, there
is great danger in assuming that widespread and dangerous deficiencies
of this vitamin do not exist. The function of riboflavin as a vitamin had
been well established for a considerable period before it was realized
that its lack was responsible for any special difficulty in man — far less
the serious consequences that we now associate with ariboflavinosis. The
efficacy of pantothenic acid in curing an increasing number of individual
cases of various acute symptoms would seem to suggest, therefore, that
as diagnostic experience is acquired, pantothenic acid deficiency may be
discovered to be a relatively common affliction in man. There is little
doubt that pantothenic acid deficiency is commonly present in many con-
ditions of malnutrition, and is a frequent complication in beriberi, aribo-
flavinosis, and pellagra; and a low blood pantothenic acid level is fre-
quently observed in these three conditions. A number of cases of peripheral
neuritis and delirium tremens and one of Korsakoff's syndrome that did
not respond to other vitamins have apparently responded promptly to
pantothenic acid.

Clear-cut pantothenic acid deficiencies have been produced in a variety
of other animals. Indeed before its isolation and identification, this sub-
stance unknowingly had been studied by various groups as a factor for
weight maintenance in pigeons (vitamin B3) , the chick "anti-dermatitis"
factor, and a liver filtrate factor required by rats.165

Pantothenic acid deficiency in the chick results in extensive spinal cord
lesions, thymus involution, a fatty liver, keratitis, dermatitis, and re-
tarded feathering. In black chicks there is a feather depigmentation, and
force-feeding chicks on the deficiency diet results rapidly in death. Hens
are apparently more resistant to deficiency, but do eventually develop a
mild dermatitis of the lower shanks and feet when on a pantothenic acid-
free diet. A deficiency of any proportions, however, drastically curtails

B VITAMIN DEFICIENCY STATES 423

reproduction and hatchability of eggs, although its effect on egg produc-
tion is only slight.

In rats, deficiency of this vitamin results in a wide variety of symp-
toms, including graying of the hair in dark rats, "blood-caked whiskers"
due to porphyrin deposition from the Harderian gland, dermatitis, sore
mouth and nose, subcuticular hemorrhage, kidney and heart damage,
marked and highly characteristic adrenal damage, and sudden death.
In highly deficient rats there is also a severe anemia, granulocytopenia,
and bone marrow hypoplasia. The anemia responds, but only slowly, to
pantothenic acid, whereas the granulocytopenia is apparently a folic acid
deficiency. It seems, therefore, that the one deficiency is able to create a
deficiency of another factor (or factors) in this case. In this regard it
has been shown that feeding rats a purified diet containing sulfasuxidine
causes a reduction in hepatic stores of folic acid, pantothenic acid, and
biotin, but not of other B vitamins. Under these circumstances a typical
pantothenic acid deficiency develops on a diet which normally would
contain an adequate level of the vitamin, and administration of folic acid
and biotin (but not pantothenic acid) causes recovery from the avita-
minosis.166

The course of pantothenic acid deficiency in dogs is quite different from
that in the foregoing cases, being marked by the rapidity that the symp-
toms progress. Sudden prostration or coma, convulsions, violent gastro-
intestinal disturbances, and an accelerated respiration and heart rate all
are prominent and terminate rapidly in death. The suddenness of the
onset of these symptoms makes treatment quite difficult and frequently
ineffectual. Autopsy reveals a generally severe gastroenteritis, hemorrhagic
kidneys, a mottled fatty liver, and a mottled thymus, but little adrenal
damage.

Whereas pantothenic acid deficiency has beeen studied a number of
times in mice, the results seem generally to be complicated by the uncer-
tain role played by other nutritional factors in the symptoms observed.
Although graying of hair is caused in mice on a pantothenic acid-
deficient diet and can be cured by administration of the vitamin, biotin
administration seems necessary for the indefinite maintenance of a nor-
mal pelt. Alopecia is also caused in mice by abiotinosis; but it has been
claimed that the curative effect of pantothenic acid in this case is by
stimulation of intestinal inositol synthesis, inositol deficiency being
responsible for the alopecia. Adult mice lose weight on a pantothenic
acid-deficfent diet; and in deficient mice, while the adrenals remain
normal, there are desquamative dermatosis, myelin degeneration in the
sciatic nerve and spinal cord with accompanying paralysis of the hind
quarters, spinal curvature, and a serous exudate from the eyes.

424 THE BIOCHEMISTRY OF B VITAMINS

Deficient pigs show poor growth and become emaciated, develop a
rough, dry coat, and later lose their hair, have a prominent gastro-
enteritis, and an uncoordinated gait, described as goose-stepping with
the hind legs.167- 168

Pantothenic acid is now known to function as part of coenzyme A in
acetylation processes. These processes are at present believed to include
the acetylation of choline and aromatic amines, and the condensation of
oxalacetate to form cis-aconitate, although they may be much more
extensive (p. 195). While a large number of facts of biochemical interest
are known concerning pantothenic acid deficiency, these do not as yet
fit neatly together to provide a clear-cut explanation of the "biochemical
lesions" that occur in this condition. An almost certain factor, however,
lies in the adrenal atrophy that is so pronounced in rats, since many of
the symptoms of the avitaminosis are characteristic of adrenal pathology.
While the biogenesis of the adrenocortical hormones is unknown, it is at
least tempting to suggest that it proceeds through an acetylation at the
17 position which may be pantothenate-mediated, followed by oxidation
of the 21 -methyl group, in somewhat the following manner:

J + CH3CO~ C°enZyme-^>
CH,

[0]

CH2OH

progesterone-like I

intermediate 9^

desoxycorticosterone, etc.

In this manner failure of the acetylation process might well result in
adrenal insufficiency. Desoxycorticosterone itself is not effective in treat-
ing the hair graying in deficient animals, but other closely related keto-
steroids are well known for their efficacy in restoring hair pigmentation
in man under many other conditions. It has been shown, moreover, that
deficient animals do actually have an adrenal cortical insufficiency, which
produces such sequelae as the loss of abdominal fat, decreased testicular
function, deranged water metabolism and reduced growth.169-171

B VITAMIN DEFICIENCY STATES 425

Studies on adrenalectomized rats have done much to elucidate this
relationship.172 Adrenalectomy has been shown to accelerate the growth
of new hair in both normal and pantothenic acid-deficient rats, and in
the avitaminotic animals it restores the color of the hair and the skin.
Finally, it accelerates recovery when pantothenic acid is administered.
A bluish pigmentation of the skin develops in the adrenalectomized rat;
it reaches a maximum after about two weeks and then fades. This color-
ation is due to widespread accumulation of melanin in hair bulbs and
follicles, and hair graying in pantothenate deficiency results from atrophy
of the hair apparatus and a cessation of melanin deposition. The skin
pigmentation occurs in all the operated animals, but lasts for a shorter
time in the deficient animals. Finally, in adrenalectomized deficient rats,
desoxycorticosterone acetate prevents the effects of the operation on hair
growth and color. It is thus clear that any adrenal insufficiency in panto-
thenic acid deficiency is not primarily concerned with the absence of
desoxycorticosterone, since the latter substance is necessary to produce
the gray hair symptom.173 This vitamin-hormone interrelationship is one
that merits further study in the immediate future.

The bronzing of the skin in Addison's disease is in some ways sug-
gestive of the fundamental role of the adrenals in pigmentation. The
structural relationships between the medullary adrenaline and the known
precursors of melanin, and between adrenaline and tyramine, which is an
endocrine substance controlling pigmentation in certain molluscs, further
suggest that both portions of the suprarenal gland function in pigmenta-
tion. The avitaminosis-induced hair graying and porphyrin deposition
might conceivably both be linked to the adrenal atrophy. It is well known
that the adrenals are intimately associated with the mineral balance,
and a number of interesting factors in apantothenosis are undoubtedly
associated with this fact. Low salt diets seem to favor the hair-graying
process, and deficient animals have an increased salt appetite. The in-
volvement of the adrenals in water balance recalls the blood-soaked
whiskers appearance of rats due to water deprivation — a condition char-
acteristic of this avitaminosis but in this case not cured by pantothenic
acid.

While nervous lesions are common to most B vitamin deficiencies, the
role of pantothenic acid in acetylcholine synthesis would cause one to
predict particularly severe symptoms in this case. If pantothenate func-
tions in the multiple acetate condensation that occurs in fat synthesis
and oxidation, then the fatty liver symptom found in the avitaminosis
might be explained.

Finally, other symptoms of pantothenate deficiency are doubtless
associated with the role of pantothenate in carbohydrate metabolism.

426 THE BIOCHEMISTRY OF B VITAMINS

It is believed that the hypoglycemia (and presumed low liver glycogen)
seen in dogs in advanced stages of depletion is responsible for the coma
that occurs in these animals. The fact that thyroid administration in-
creases the pantothenate requirement is apparently associated with the
overall metabolic stimulation, since the pyridoxine and thiamine require-
ments are similarly increased.

Pantothenic acid is apparently associated with the mobilization of
liver riboflavin, but the mechanism is obscure.174 Vitamin B6 deficient
animals are said to be more resistant to hair graying in pantothenate
deficiency, and cystine is believed to speed the remission of this symptom
on pantothenate treatment; but the reasons for these relationships are
unknown.175 Finally, and perhaps most peculiar, is the evidence that
chronic zinc chloride poisoning causes a syndrome extremely similar to
pantothenate deficiency, that is said to respond to administration of the
vitamin.176

Vitamin B0 Deficiency. As in the case of pantothenic acid, no well
recognized syndrome in man is known to be due to vitamin B6 deficiency,
although a variety of conditions have been reported to respond to vitamin
B6 administration. There seems little doubt that vitamin B6 deficiency
is a factor in some compound deficiencies, such as beriberi, pellagra, and
sprue, but attempts to deplete humans of this vitamin specifically have
been generally quite unsuccessful (p. 249).

It has been reported that many of the nervous symptoms that remain
in pellagrins after thiamine, riboflavin, and niacin therapy respond rapidly
to pyridoxine administration.177 There is also some evidence that the
characteristic cheilosis of ariboflavinosis may also frequently be due to
vitamin B6 deficiency, this vitamin having been effective in the cure of
this symptom in a number of cases.178 Other reports suggesting the
efficacy of pyridoxine in epilepsy, Parkinson's disease, pseudohyper-
trophic muscular dystrophy, and macrocytic anemia are scattered and
lack general confirmation, although it is entirely certain that some
benefit would result in any one of these conditions should the nature
of the affliction in some manner interfere with the normal degree of
efficiency in vitamin B6 metabolism. Generally, therefore, it seems that
vitamin BG plays an important role in human nutrition, but the circum-
stances attending vitamin Bc deficiency in man are as yet obscure.179

Vitamin B6 deficiency has been induced in a number of animals by the
use of depletion diets, and the rat has been much studied in this regard.
Most characteristic of the vitamin B6-depleted rat is the dermatitis or
acrodynia, which is manifest on the peripheral portions of the animal
such as the tail, ears, nose, mouth and paws. There is an accompanying
scaliness and edema, an ulcerated tongue, and cessation of growth with

B VITAMIN DEFICIENCY STATES 427

accompanying reduction of the accessory organs of reproduction and
decreased sexual behavior. In adult mice, vitamin B6 deficiency results
in failure to maintain body weight, and death inside of two months. In
young mice there is a paralysis of the hind legs, but no dermatitis which is
frequently seen in the rat. In adult mice, however, the acute stages of
deficiency do show pathological skin manifestations and frequently
necrotic tails.

There is no dermatitis in the apyridoxic chick, but growth is slow and
accompanied by anorexia and general signs of debility. Pigs and dogs
have also been studied with regard to vitamin B6 deficiency and both
develop anemias.lso' 1S1 In the pig the anemia is microcytic, the bone
marrow hyperplastic, and both it and the spleen and liver are siderotic
(contain excessive iron deposits). One of the most significant changes
seen in dogs, rats, chicks, and pigs during vitamin B6 deficiency is the
typical epileptiform seizures.182 The attack appears suddenly, the animal
running about wildly in great excitement, then falling and undergoing
both clonic and tonic convulsions, followed by coma and collapse. Slow,
confused recovery follows upon the rather brief period during which the
attack occurs. Nervous degeneration occurs in acute vitamin B6 defi-
ciency, as in the case of the other B vitamins, and in the dog at least
there is pronounced cardiac hypertrophy.

The epileptiform seizures seen in lower animals have been responsible
for the study of pyridoxine efficacy in human epilepsy. The results in
this regard, however, have not been promising. Davenport and Daven-
port 183 have shown that pyridoxine increases the electroshock threshold
of mildly apyridoxic rats, but not of normal animals. Glutamic acid
similarly increases the threshold, and tryptophan, which intensifies
vitamin B6 deficiency, lowers it. In severe deficiency, pyridoxine causes
only a slow rise in the electroshock threshold unless there has been
previous loading with glutamic acid. These facts all tend to suggest that
maintenance of transaminase activity is critical for a high electroshock
threshold — a suggestion with many implications in the field of brain
metabolism and mental disease.

Dairy cattle apparently develop a natural vitamin B6 deficiency — a
fact that is remarkable both because of the resistance of ruminants to
avitaminoses, and the apparently rare occurrence of natural apyridoxosis.
In cattle the affliction is manifest by anorexia, thinness, a poor hair coat,
retarded growth, and most characteristic of all by a poikilocytosis. This
condition has been found to respond rapidly to pyridoxine administration,
and it will be of interest to see whether the corresponding poikilocytosis
in man (sickle cell anemia) bears any relationship to vitamin B6 defi-
ciency.184 In cobalt deficiency in sheep there is apparently a lowering of

428 THE BIOCHEMISTRY OF B VITAMINS

blood vitamin B6 levels — a fact further involving vitamin B6 with vitamin
B12 185 in the overall picture of normal hematopoiesis. The relationship
of the pdracins to folic acid has already been discussed (p. 421).

Previous mention has been made of many of the biochemical aspects
of vitamin B6 deficiency. There is a lowering of blood, tissue and body
levels of this vitamin in most cases, and loading tests have been employed
in the diagnosis of apyridoxosis. Studies of the anemia produced in swine
have resulted in the conclusion that hemoglobin synthesis is decreased
in this deficiency. Thus during vitamin B6 deficiency in swine, erythrocyte
protoporphyrin is reduced by over 50 per cent, plasma iron is more than
doubled and plasma copper is decreased by 20 per cent, while urinary
coproporphyrin remains constant. It is thus felt that the fundamental
disturbance involves impaired protoporphyrin synthesis.186

One of the most striking changes that occurs in apyridoxosis is the
interference in tryptophan metabolism which results in increased urinary
excretion of xanthurenic acid and decreased excretion of kynurenic acid
by deficient animals. Various aspects of this derangement have been dis-
cussed in relation to the earlier study of vitamin metabolism (p. 354) .
Transaminase activity is distinctly decreased in vitamin B6-deficient
rat tissue,187-190 and can be increased by the addition of pyridoxal and
adenosine triphosphate. Pyridoxine is said to alleviate the toxic symptoms
of DL-serine in rats.191

Biotin Deficiency. So far as is now known, biotin deficiencies are not
naturally occurring, probably largely because of the considerable intes-
tinal synthesis of this vitamin. Deficiencies induced in a number of species
by the feeding of avidin-containing raw egg white are well known, how-
ever. Sydenstricker et al. fed a group of human volunteers a diet con-
taining 30 per cent of the total calories in the form of desiccated egg
white and observed a fine scaly dermatitis ; a variety of mental symptoms,
including depression, lassitude, hallucinations and panic; pallor; and a
drop in urinary excretion of biotin from 29-62 fig per day to 3.5-7.5 /xg
per day after ten weeks on the diet. All these symptoms disappeared after
administration of biotin.192 There have been a number of reports of suc-
cessful biotin therapy of various skin disturbances (acne vulgaris, rosacea,
furunculosis, baldness due to seborrhea), but there is at present insuffi-
cient evidence to support such claims.

Egg-white injury has been extensively studied in the rat, the symptoms
being highly typical. There is a cessation of growth, a spectacle-eyed
appearance, a desquamous dermatitis of the neck and groin which becomes
generalized, and stiffened joints, which result in an awkward movement
of the animal. Progressive emaciation terminates in death. Such rats are
said to be unusually susceptible to infection with various organisms,

B VITAMIN DEFICIENCY STATES 429

Trypanosoma lewisi infection and pneumonia having been particularly
studied.193 A deep brown pigmentation has also been reported on the back
of deficient rats, particularly males (and deficient human infants) . Male
rats are said to be more sensitive than females.

Biotin-deficient chicks also develop a characteristic dermatitis: the feet
become calloused and cracked, the corners of the mouth and the area
about the beak develop severe lesions, and the eyelids become swollen
and stick together. Similar symptoms of no distinctive interest occur as
the result of egg-white feeding to a wide variety of other animals —
rabbits, monkeys,194, 195 mice, guinea pigs, and swine. Little is known of
the biochemical changes that occur in abiotinosis. Studies with liver slices
from biotin-deficient rats have shown that added biotin increases the
efficacy of this material in utilizing lactate, and it may be that the con-
version of pyruvate to oxalacetate will be shown to be seriously impaired
in this deficiency (p. 171 ).196 Impaired ovalacetate formation might cause
a collateral impairment of pyruvate oxidation, which may explain the
observed depression in oxygen consumption of biotin-deficient duck
heart.197

p-Aminobenzoic Acid Deficiency. Relatively little is known about
p-aminobenzoic acid (PABA) deficiency and its incidence in man, or
about the relationship of this vitamin to another in which it is contained,
folic acid. There is good evidence to indicate, however, that it is effective
in the treatment of certain types of hair graying in man. Folic and
pantothenic acids have been reported to have similar effects, and it has
been suggested that p-aminobenzoic acid may act through folic acid
synthesis. There is some reason to believe that pantothenic acid functions
with PABA in hair pigmentation. The effect of PABA ultimately is in-
volved in melanin formation however, and the oxidation of tyrosine to
melanin may in some manner be related to the apparent relationship
between tyrosine and folic acid (p. 415).

PABA stimulates the growth of rats and chicks on a deficiency diet,
and is a growth requirement for certain microorganisms. In the black or
piebald rat, deficiency results in graying of the hair (nutritional achromo-
trichia) — a condition which, like hydroquinone-induced achromotrichia,
can be cured by PABA administration. Female albino rats have been
reported to have lactation disturbances when fed a PABA-deficient diet.
The high efficacy of the sulfonamides in inhibiting bacterial growth indi-
cates that PABA plays an unusually critical role in cellular metabolism,
particularly since analogues of many other metabolites have not proved
effective therapeutic agents. It would seem, therefore, that PABA occu-
pies some key metabolic position, which when blocked causes the break-
down of a number of metabolic sequences. The reversal of sulfanilamide

430 THE BIOCHEMISTRY OF B VITAMINS

by folic acid and vitamin B12 indicates indeed that PABA is intimately
related to these substances (see Chapter HID). In view of these con-
siderations it is remarkable that the physiological effects of PABA and
its deficiency in the higher animals are not more pronounced.

Inositol Deficiency. Inositol deficiency is little understood, probably
because of the paucity of evidence regarding this avitaminosis in man.
Possibly the only effect known in man is its prevention of the fatty liver
induced by cholesterol feeding. Mice and rats apparently require inositol
for normal growth, and mice on deficiency diets become hairless and
develop a severe dermatitis. Rats develop a spectacle-eyed appearance
and a high-cholesterol fatty liver. The alopoecia and other symptoms
undergo remission on inositol administration. The fatty livers induced
by biotin or liver extract are similarly cured by inositol, but not choline.198
Inositol does not affect "fat type" fatty livers as does choline, however.
Along with the association of inositol as one of the lipocaic factors of
the pancreas, other relationships appear. Inositol has recently been
reported to be highly efficacious in the treatment of diabetes mellitus,
and despite the caution that must be used in viewing such reports, it
seems probable that as yet unknown dietary considerations will even-
tually be shown to play an integral role in the etiology of this disease.
The relationship is more interesting in view of the possible role of
inositol as a coenzyme in amylase (p. 125) .199- 20°

One other aspect of inositol deserves mention at this point. Beyond its
wide occurrence in the free state and as its esters and their salts, inositol
is found in certain cephalins called "lipositols," which occur in brain
tissue, soybean oil, etc. Regardless of what other roles they play, these
cephalins undoubtedly function in the mobilization and transport of
fat, and the lipotropic activity of inositol may be due to its occurrence
in lipositols. Beyond this, the antagonistic activities between streptomycin
and lipositol in bacteria suggest that lipositol may play an integral role
in cellular function.201

Choline Deficiency. Choline deficiency occurs naturally among domes-
tic fowls and it is thought to occur on rare occasion in other animals, but
it may be readily induced in most species. An important function of
choline is as a supply of methyl groups for transmethylation,202 and
although this function is vital, it is not a vitamin function. For this
reason, and because the topic is a large and important one in its own
right,203 the function of choline as a raw material for methionine synthesis
cannot be considered at length in this monograph. It is recognized, how-
ever, that choline may function as a vitamin by acting catalytically as
a carrier of methyl groups, or as a donor in the methylation of some other
catalyst. It is difficult to attribute a vitamin function to choline by virtue

B VITAMIN DEFICIENCY STATES 431

of its fat transport functions which involve large amounts of the sub-
stance that are not compatible with our present concept of B vitamin
activity. One might associate a vitamin function with choline for its role
in acetylcholine synthesis, were it not so difficult to extend this to micro-
organisms, some of which require a nutritional choline source. Because
of the multiplicity of symptoms associated with its deficiency and despite
the several possible reasons for these, it is difficult to discuss choline
deficiency in the light of an avitaminosis, although it may well be true
that choline functions as a vitamin, and that some of the manifestations
of its deficiency are due to the breakdown of this function.

The usual symptoms of choline deficiency are retarded growth, fatty
livers with interlobular cirrhosis, renal tubular degeneration, enlarged
spleen, and a generalized hemorrhagic condition.204- 205 In chicks and
turkeys, perosis is also characteristic and egg production subsides. In
albino rats normal lactation ceases, and young rats raised from mothers
approaching such a state develop a flaccid paralysis of the hind quarters.
There is a high nonprotein nitrogen excretion in choline deficiency, and
the general indications of impaired renal and hepatic function. Choline
is said to depress the polycythemia induced by cobalt 206 and to prevent
the necrosis, cirrhosis, and cancer of liver induced by "butter yellow"
(dimethylaminoazobenzene) .207 Neoplasms are frequently formed, more-
over, in the livers of choline-deficient rats.208 There is little in this overall
picture to suggest the consequences that result from an impaired function
of choline in cellular metabolism, and further studies of choline function
in cellular and enzyme systems where fat mobilization and transmethyla-
tion effects can be eliminated are needed to elucidate the vitamin role of
choline. Choline deficiency has been studied in a variety of other animals,
dogs being particularly suitable for such studies.209

The Relationship of the B Vitamins to Various "Nondeficient" States.

A number of pathological conditions are known which, while they bear
no apparent relationship to any of the avitaminoses studied, yet respond
to B vitamin, liver, or yeast therapy. Whether this is due to insufficient
study of the therapeutic effects of the various members of the B group
in each case, or the presence of as yet unidentified factors is at present
uncertain. Also worthy of consideration are a wide variety of conditions
of presumably known etiology which bear no apparent relationship to
avitaminoses, but respond favorably to B vitamin therapy. At the present
time, when the impetus for study of most of the classical nutritional
deficiencies has subsided, the possibilities for spectacular advances in this
new field seem unlimited. Only a few suggestions are as yet available,
however, to indicate the directions in which such advance may proceed.

432 THE BIOCHEMISTRY OF B VITAMINS

A list of topics that have been suggested in this regard is provided in
Table 36. Only two are of sufficient promise to merit any greater discus-
sion at present.

p-Aminobenzoic acid has recently been shown to be an highly effective
and specific chemotherapeutic agent against various rickettsial diseases.

Table 36. Various Pathological Conditions Associated with or
Reported as Responding to B Vitamin Therapy
Condition Reference

Infectious Diseases
Bacterial

Salmonella infection

210,211

Tuberculosis

212

Tabes dorsalis

213

Pneumonia

214

General

215-218

Protozoa

Trypanosomes

193

Malaria

219, 220

Rickettsiae and Viruses

Rocky Mountain Spotted Fever and Typhus

221

Equine encephalomyelitis

222

Poliomyelitis

223

Immunity

Antibody production

224, 225

Invasion

226

Phagocytosis

227

Resistance

228, 229

Organic Diseases

Eye, ear, nose, and throat disorders

230-232

Mental and neurological disorders

233-238

Genetotrophic diseases, alcoholism

239

Diabetes

240-242

Inanition and anestrous

243

Arthritis

244

Cancer

245

Disorders of pregnancy

246

Anorexia nervosa

247

Intestinal obstruction, etc.

248, 249

Seborrhea

250

Injuries

Burns

251

Shock

252-254

Anoxia

253, 255

Wounds

256

General

257

In mice infected with murine typhus, incorporation of 3 per cent of PABA
into the diet results in a 100 per cent survival, compared with zero sur-
vival for untreated controls. Similar results have been obtained with
Rocky Mountain spotted fever in guinea pigs,221 and the use of PABA
in humans infected with these diseases has been shown to be highly
effective. The precise reasons for these effects are obscure, however. The

B VITAMIN DEFICIENCY STATES 433

results so obtained are of particular interest in that analogues of this
vitamin have met with such spectacular success in combatting bacterial
infections.

Of interest from both theoretical and practical standpoints is the work
of Williams et al. on genetotrophic diseases.239 Whereas the realization
that there may exist genetically induced high vitamin requirements is
not entirely new, the recent coupling of such high requirements with
nutritional states to produce a hitherto unconsidered type of deficiency
disease merits attention. Experimental evidence has as yet been produced
bearing on only one — compulsive drinking. Thus, whereas rats from an
ordinary colony normally show a wide range in individual appetites for
alcohol as measured by self-selection of water or 10 per cent alcohol,
animals on deficient diets tend eventually to drink large quantities of
alcohol. The alcohol consumption so induced may be cured by nutritional
means by correcting the nutritional deficiency to which the particular
animal is subject. The extension of these studies to clinical trials with
humans, and to other diseases,259 should certainly be one of the most
fascinating directions in which results are to be expected in the near
future in this field.

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206. Davis, J. E., Am. J. Physiol, 127, 322-7 (1939).

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221. Anigstein, L., and Bader, M. N., Science, 101, 591-2 (1945).

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B VITAMIN DEFICIENCY STATES 439

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Section D

THE COMPARATIVE BIOLOGICAL ACTIVITIES OF THE
B VITAMINS AND RELATED COMPOUNDS

By William Shive

Chapter ID

INTRODUCTION AND THEORETICAL CONSIDERATIONS

The preparation and testing of analogues of biologically active com-
pounds has long been an accepted approach in the search for more active
principles. This approach was very successful in the field of chemotherapy
and pharmacology but has yielded meager results with compounds
analogous to the B vitamins as far as nutrition is concerned.

A few compounds have been found to be partially, but almost never
fully, active in replacing their analogous B vitamins in the nutrition of
various organisms. Other analogues have been observed to replace the
corresponding vitamin in carrying out some, but not all, of its biological
functions. In most instances, analogues have been found to be essentially
inert biologically; however, in some cases, certain analogues of the B
vitamins and of related metabolites have been found to be toxic for some
organisms. The action of substances which are toxic for an organism has
been presumed to involve a combination of the substance with an essen-
tial cell constituent. This type of action is usually represented by a
combination of the toxic agent with an enzyme in such a fashion that the
enzyme can no longer effectively serve as a catalyst.

Much of our knowledge concerning enzymes has been obtained by the
use of toxic substances capable of reducing the velocity of enzymatic
reactions. According to the modes of action of these inhibitory substances,
the inhibitions can usually be classified into one of two general types.
One type is termed competitive inhibition, in which case the effectiveness
of the inhibitor in preventing an enzymatic reaction depends upon the
concentration of the substrate, and a direct relationship exists between
the rate of the reaction and the relative concentrations of inhibitor and
substrate. The inhibitor appears to compete with the substrate for the
same reactive groups of the enzyme. In the other type, noncompetitive
inhibition, the effective enzyme concentration is diminished by the in-
hibitor irrespective of the concentration of the substrate.

Noncompetitive inhibitors such as fluoride,1- 2- 3 which prevents the
functioning of enolase and allows the accumulation of 2-phosphoglycerate,
have been utilized successfully as specific enzyme "poisons" permitting
the substrate to accumulate in a biological system. In the fluoride inhibi-
tion of enolase, which converts 2-phosphoglycerate to 2-phosphopyruvic

443

444 THE BIOCHEMISTRY OF B VITAMINS

acid, the fluoride forms a fluorophosphate with the magnesium of enolase.4
Iodoacetate appears to inhibit noncompetitively enzymes which contain
sulfhydryl groups essential for their activity. It has been postulated that
hydrocyanic acid, mercuric salts, sodium azide, hydroxylamine and many
other noncompetitive inhibitors likewise react with specific groups or ions
essential for the activity of the enzymes. These inactivations are not pre-
vented by excess substrate, as is the case in the inactivation of an enzyme
by a competitive type of inhibitor.

The earliest reports of inhibitions which appear to be of a competitive
nature concerned inhibitions of enzymatic reactions by products of the
reactions.5 Maltose and glucose inhibit maltose production from starch
by malt amylase.6 In this and many other instances, such inhibitions
induced by the products of a reaction cannot be ascribed totally to a
reversal of the reaction by a mass action effect. Thus, fructose or /?-glu-
cose prevents the hydrolysis of sucrose by yeast saccharase, and a-glucose
competitively prevents the action of Aspergillus saccharase;7 glycine as
well as alanine prevents the hydrolysis of glycylglycine by intestinal
peptidases;8 pyruvic acid inhibits competitively the dehydrogenation of
lactic acid by lactic dehydrogenase;9 and ornithine prevents the hydrol-
ysis of arginine by arginase.10 In all these cases the products of the
enzymatic reactions appear to be able to combine with the enzyme in
competition with the substrate. In a number of instances compounds
somewhat similar to the substrate were known to prevent an enzymatic
reaction. Thus, 3-methylxanthine as well as guanine, uric acid or 1- or
7-methylguanine prevents the oxidation of xanthine by xanthine oxidase.11
Some of the inhibitions were shown to be competitive; e.g., physostigmine
competitively prevents the hydrolysis of acetylcholine by choline es-
terase.12

The realization that substances structurally related to the substrate
may inhibit the action of an enzyme came with the report of Quastel and
Wooldridge 9 that the dehydrogenation of succinic acid by succinic de-
hydrogenase was competitively prevented by malonic acid and a number
of structurally related compounds.

Although in this and in many other cases the competitive inhibitors
structurally resembled the substrate, and although the fundamental
theories for both competitive and noncompetitive inhibitions had been
previously developed, especially for isolated enzyme systems, the impor-
tance of inhibition of enzymatic action by an analogue of the substrate
as related to chemotherapy and growth inhibition was not fully realized
until the report of Woods and Fildes in 1940 13- 14 that the bacteriostatic
action of sulfanilamide was competitively prevented by p-aminobenzoic
acid, a compound previously not known to have a biological function.

INTRODUCTION AND THEORETICAL CONSIDERATIONS 445

This report prompted a widespread search for chemotherapeutic agents
among compounds which were structurally related to catalytic metab-
olites, but which inhibited the biological functioning of the metabolite.
Many investigators directed their efforts toward the discovery of new
and effective chemotherapeutic agents. Others prepared and utilized
analogues of metabolites in the study of biochemical transformations.
Out of these efforts a new field and new tools for the study of biochemistry
have been developed.

Inhibition of Enzymatic Action

Inhibition of the functioning of an enzyme by an inhibitor results in a
decrease in the rate of the enzymatic reaction and of the biological process
in which the enzyme is involved. The mode of action of substances in-
hibiting biological processes has in general been determined either by a
study of the relative concentrations of the inhibitor and substrate (metab-
olite) which are necessary to obtain a defined degree of inhibition after
the lapse of a specified time, or by a study of the effect of the substrate
on the rate of the process at a specified concentration of inhibitor. For a
competitive inhibition, the ratio of the concentration of inhibitor to the
concentration of substrate necessary for a defined degree of inhibition of
a biological process in a specified time is constant over a range of con-
centrations and is termed the inhibition index. The effect of the substrate
on the rate of the biological process inhibited by a competitive analogue
is such that at high concentrations of substrate the rate approaches that
of the normal process in the absence of the inhibitor. Such data are usually
presented graphically, as subsequently indicated.

The Inhibition Index. Competition of an inhibitor I (analogue) with
a substrate S (metabolite) for an enzyme is frequently represented by
the following equations analogous to those first developed by Michaelis
and Menten,15 where P represents the product and ES and EI represent
the enzyme-substrate complex and enzyme-inhibitor complex, respec-
tively :

E+S =^= ES — > E+P
E+I =f=*= EI
By the Law of Mass Action:

[E][S] K

lESj = Ks (1)

where K8 is the dissociation constant of the enzyme-substrate complex,
and

[E] [I]
[EI] '

KI (2)

446 THE BIOCHEMISTRY OF B VITAMINS

where Ki is the dissociation constant of the enzyme-inhibitor complex.
By dividing equation 2 by equation 1, one obtains

m ki[ei) (

[S] KS[ES] K J

If [Et] represents the total enzyme concentration, both free and com-
bined, by definition

[Et] = [E]+[EI)+[ES] (4)

In the application of the above equations to biological systems, certain
experimental conditions designed to limit some of the variables greatly
simplify the problem. In order to study the effect of an inhibitor on an
entire biological system, conditions must be such that an observable effect
on the rate of a biological process will result from the interaction of the
inhibitor with an enzyme; this specific enzymatic reaction then becomes
the limiting reaction of the biological process. In an isolated enzyme
system, the observable effect may be a decrease in the rate of formation
of a product. The observable effect on bacterial cells or any isolated
culture of cells such as tissue cultures may be a decreased growth rate or
complete inhibition of growth; or the effect observed in an animal or
embryo may be on the rate of growth, time of survival or time necessary
for the development of certain deficiency symptoms. Hence, any observ-
able effect resulting from a decreased rate of reaction of an inhibited
enzyme system can be used to determine the effect of an inhibitor on a
specific enzyme system. One method of studying the mode of action of
an inhibitor is to determine the relationship between the concentration
of inhibitor and the concentration of substrate necessary to obtain a
defined observable effect within a constant period of time. Other experi-
mental conditions of the biological system are not allowed to vary. For
example, in bacterial studies, the concentrations of inhibitor just neces-
sary to attain a defined inhibition of growth, e.g., maximum or half-
maximum inhibition, are determined with variable concentrations of
substrate under conditions of a defined medium, constant size of inoculum,
and a defined time and temperature of incubation.

The quantitative response of biological systems under such conditions
is dependent upon the rate, r, of the limiting reaction, which can be
expressed as follows:

r = k[ES] (5)

where k is the rate constant for the reaction. Under the defined experi-
mental conditions, the variables which may affect [ES] and in turn the
rate of the reaction are the concentrations of the inhibitor and substrate
as well as the total enzyme concentration, [Et]. The inhibitor and sub-

INTRODUCTION AND THEORETICAL CONSIDERATIONS 447

strate are usually employed at concentrations sufficiently high that their
utilization by the biological system does not appreciably alter their con-
centrations during the experimental period. If the total enzyme concen-
tration of the biological system does not vaiy during the course of the
experiment, the variables of equation 3 and 4 are not a function of time.
Even in a system in which cells are multiplying, the mother and daughter
cells have similar composition, and the concentrations of their intra-
cellular enzymes would not be expected to vary appreciably. Conse-
quently, for both static and growing systems, the enzyme concentration
in all its various forms can be assumed to be constant during the course
of the experiment and not a function of time.

Varied concentrations of inhibitor and substrate are established at the
outset of the experiment in such a manner that one can determine the
critical ratio which will reduce the response of the biological system to a
denned quantity after the lapse of a constant period of time. The total
response of the organism, then, is a function of the rate of the limiting
inhibited enzymatic reaction and of time. In order to obtain the defined
response after a constant period of time, the rate of the limiting reaction,
r, must be reduced to a value which is specific for this particular selected
response. The rate of the inhibited reaction is proportional to the con-
centration of the enzyme-substrate complex; hence, to achieve the critical
rate which will produce the chosen biological response in a specified time,
the concentration of the active complex [ES] must be reduced to a specific
value, CEs, at the onset of the process.

For increasing concentrations of substrate and inhibitor, particularly
those approaching enzyme saturation, the value of [E] approaches zero
and becomes negligible in comparison with [EI]. If the total enzyme
concentration, [Et], is assumed to be constant for the reasons previously
indicated, it is apparent from equation 4 that the concentration of the
enzyme-inhibitor complex, [EI], must be essentially a constant value,
CEi, since [Et] and [ES] are constant, and [E] can be neglected.

By substitution of these constant values, CEi and CEs for [EI] and
[ES], respectively, in the general equation 3, one obtains an equation
for the molar ratio of analogue to metabolite which must be established
to produce the necessary inhibition that will result in the defined response
of the biological system under the conditions outlined above. This ratio
is a constant, K, the inhibition index.

[7] KiCei Tr ^v

wr^cTs=K (6)

The assumption that the intracellular concentration of all the forms of
a particular enzyme in a biological system does not vary with changes in
the substrate and inhibitor concentrations may not always be valid. The

448 THE BIOCHEMISTRY OF B VITAMINS

decrease in concentration of enzyme-substrate complex induced by the
inhibitor may possibly affect the biosynthesis of the enzyme by a system
of multiplying cells. Any such change in concentration of enzyme under
these conditions would be expected to be a function of [ES] and of time.
It has been shown that even if such changes in the intracellular concen-
tration of the enzyme do occur during the course of an experiment, there
still is a specific unique value of [/]/[S] which must be initially estab-
lished in order to obtain the specified response of the system at the end
of a constant experimental period.16

It can likewise be demonstrated that even when a growing system
develops a resistance to an inhibitor during the course of an experiment,
there is still a definite initial ratio of inhibitor to substrate concentration
for the particular system which will produce the specific response for the
constant experimental period.

For experimental conditions in which the concentrations of inhibitor
and substrate must be regulated in a medium outside the biological sys-
tem wherein the reaction takes place, i.e., bacterial growth experiments,
etc., the ratio of the concentrations of the inhibitor to the substrate
within the cell is a function of this ratio of the concentrations existing
outside the cell. Consequently, this latter ratio is also constant for a
defined inhibition after a specific period of time and is termed the inhibi-
tion index for such systems, even though it does not represent the actual
intracellular ratio.

For biological systems in which high concentrations of inhibitor and
substrate cannot be employed, e.g., embryonated eggs, the amount of
the substrate synthesized by or present in the system must be considered.
The inhibition index would be [/]/([Si] + [S2]), where [Si] represents
the contribution of the biological system and [S2] represents the con-
tribution of the exogenous supply to the total concentration of substrate.

If an inhibitor reacts with the enzyme-substrate complex, a constant
inhibitor-substrate ratio for a defined inhibition is not obtained, and the
inhibition is of the noncompetitive type.

Even though a vitamin is initially utilized as substrate in the forma-
tion of a coenzyme, an analogue may prevent the combination of this
coenzyme with an apoenzyme. This can be illustrated by the following:

Ea+Co =*=* EaCo

Ea+I ^^ EaI

EaCo+S ^± EaCoS

P+EaCo

where Ea, Co and / represent the apoenzyme, coenzyme and inhibitor,
respectively. EaCo, and EaI represent the complete enzyme and the
apoenzyme inhibitor complex, respectively. S, EaCoS and P represent the

INTRODUCTION AND THEORETICAL CONSIDERATIONS 449

substrate, enzyme-substrate complex and the product of the enzyme
system. The following equation can be derived in a manner analogous
to equation (3) :

[7] Kj[EJ]

[Co] KC[E a Co] K)

where Kr and Kc are the dissociation constants for the apoenzyme-in-
hibitor complex and for the complete enzyme, respectively. By the Law
of Mass Action:

[S] [Ea Co] ..

[EaCoS]=Ks (8)

where Kg is the dissociation constant of the enzyme-substrate complex.
The total apoenzyme concentration, [Ea] becomes

[Ea J = [Ea] + [EJ] + [Ea Co] + [Ea Co S] (9)

In the application of these equations to the determination of the effect
of the concentrations of inhibitor and coenzyme on the degree of inhibi-
tion of a biological system, the rate of the reaction in which the final
product, P, is formed would be expected to govern the rate of the biologi-
cal process. The effect of any change in substrate concentrations, resulting
from the lack of the complete enzyme in optimal concentration, would
be a function of time under the experimental conditions. For a defined
degree of inhibition after a constant experimental period, the rate of the
reaction is limited in the manner previously indicated for substrate
inhibition. Consequently, the concentration of the enzyme-substrate com-
plex, [EaCoS] , must be reduced to a defined amount at the outset of the
biological process in order to attain a defined inhibition. Since [S] is not
a variable at the outset of the experiment, it is apparent from equation
(8) that the concentration of EaCo must be reduced initially to a defined
amount. Since the concentration of [Ea] becomes negligible, in comparison
with [EJ], with increasing concentrations of inhibitor and coenzyme, and
since the total enzyme concentration is considered to be constant, it fol-
lows from equation (9) that EJ must then become a defined concentra-
tion initially in order for a defined inhibition to be obtained.

Since both EnC0 and EJ must initially be defined quantities for a defined
inhibition, it follows from equation (7) that the ratio of inhibitor to
coenzyme necessary for a defined inhibition is constant. Consequently,
the inhibition index may be applied to such a system.

The problem of the reverse rate of reaction being so slow that equilib-
rium conditions are not attained becomes a reality for reactions of many
apoenzymes with their coenzymes or analogues of the coenzymes. In such
cases, it is essential that both the inhibitor and coenzyme be added

450 THE BIOCHEMISTRY OF B VITAMINS

simultaneously in order to study the enzyme system; otherwise, one of
the factors may combine with the enzyme completely before the other
has a chance to combine with the apoenzyme. This problem exists particu-
larly with isolated and nongrowing systems.

Effect of Substrate on Velocity of Inhibited Enzymatic Reactions.17- 18
In some instances, determination of the rate of a biological process involv-
ing an inhibited enzyme system has advantages over the inhibition index
method both in ease of obtaining experimental data and in its interpreta-
tion. This is true particularly for biological systems in which concentra-
tions of inhibitor and substrate sufficiently high to approach enzyme
saturation cannot be employed. Such rate studies are frequently employed
in the elucidation of the mechanisms of inhibition of isolated enzyme
systems. The equations which may be applied to a general system of this
type are analogous to those previously indicated in the derivation of the
inhibition index. Under these conditions of suboptimal substrate concen-
trations the possibility of the combination of the inhibitor with the
enzyme-substrate complex as well as with the free enzyme must be
considered. Thus,

E +Szi=±ES — >P+E
E + I ^=± EI

ES + I =^= ESI

where the symbols represent the quantities previously indicated in the
inhibition index method, and ESI represents the enzyme-substrate-
inhibitor complex. By the Law of Mass Action, equations (1) and (2)
apply to the dissociation of the enzyme-substrate complex and to the
dissociation of the enzyme-inhibitor complex. Similarly, the dissociation
constant, KSj, of the enzyme-substrate-inhibitor complex can be obtained.

[ES][I]

-[ESiY= SI (10)

The total enzyme concentration, [Et], may be represented as follows:

[Et] = [E]+[EI} + [ES} + [ESI] (11)

Solving equation (1) for [E], equation (2) for [EI], and equation (10)
for [ESI] and substituting these values in equation (11), one obtains:

_, Ks[ES].Ks[ES)[I).wq] .[ES][I] f .

Et=~TsT+ k,[S] +[ES]+~IGr (12)

The velocity of the enzymatic reaction is proportional to f ES] , so that
r=k[ES], where r is the rate and k is the rate constant of the reaction.
The maximum rate, R, of the enzymatic reaction is similarly proportional
to the total enzyme concentration since [ES] becomes equal to [Et]

INTRODUCTION AND THEORETICAL CONSIDERATIONS 451

under such conditions. Thus, the ratio, R/r is equal [Et]/[ES] for which
an expression can be obtained from equation (12) by dividing by [ES] :

[Et]_R_Ks .Ks[I] M , [/] m,

[ES]~ r ~ [SVKAS^^Ks! KX*}

These equations can be applied with slight modification to (a) en-
zymatic reaction in the absence of an inhibitor, (b) competitive inhibition
in which the inhibitor combines only with the free enzyme and not with
the enzyme-substrate complex and (c) noncompetitive inhibition in
which the inhibitor combines with the enzyme-substrate complex and
may or may not combine with the free enzyme.

In the absence of an inhibitor, the [EI] and [ESI] terms> of equation
(11) are zero, and the corresponding terms of equation (13) can be
omitted to obtain an expression for the velocity of an enzymatic reaction
in terms of the concentration of the substrate.

Hbj+s <14)

In the case of competitive inhibition, the inhibitor does not combine
with the enzyme-substrate complex, so that the [ESI] term of equation
(11) and the corresponding term of equation (13) can be omitted.

1 [Ks Kb[I]1 1
S]\_R "1~ KiRyR

r [S]\_R ' KjRJ ' R (15)

For strictly noncompetitive inhibition, the inhibitor combines with both
the enzyme and the enzyme-substrate complex; so equation (13) rear-
ranged applies:

1 i[ks Ks[I}~] 1
r [S}\_R^ KjRyR

+mk (16)

Since the dissociation constants, Kj and KSi may not be identical, the
extreme can be represented by a competitive inhibition in which the
inhibitor does not combine with the enzyme-substrate complex and by a
noncompetitive enzyme-substrate inhibition in which inhibitor does not
combine with the free enzyme but combines only with the enzyme-
substrate complex. In the latter case, which has been termed "uncompeti-
tive" inhibition,18 the [El] term of equation (11) and the corresponding
term of equation (13) can be omitted:

i=**-+I+_M- (17)

r R[SVR^RKsi K }

In order to determine the type of inhibition, the rate of an enzymatic
reaction is determined over a wide range of substrate concentrations in
the absence of an inhibitor and at two or more concentrations of inhibitor.

452

THE BIOCHEMISTRY OF B VITAMINS

By plotting the reciprocal of the rate, 1/r, against the reciprocal of the
concentration of the substrate, 1/[S], a linear relationship should result
at a constant concentration of inhibitor. The characteristics of slope and
extrapolated intercept at 1/[S]=0 are as follows for various types of
inhibitions.

1/[S]

Figure 1. Relationship of reciprocal of the rate of an enzymatic reaction to the
reciprocal of substrate concentration as a method of determining type of inhibition.

Competitive Inhibition

Noncompetitive Inhibition

_._._._. "Uncompetitive" Inhibition
-x-x-x-x In absence of Inhibitor

For the uninhibited enzyme reaction, the extrapolated intercept should
represent 1/R, where R is the maximum rate of the enzymatic reaction
at enzyme saturation, and the slope should be Ks/R. The ratio of slope
to intercept is the dissociation constant of the enzyme-substrate complex.

For competitive inhibition, the intercept for various concentrations of
the inhibitor is constant, 1/R. However, the slope of the linear relation-
ship is Ks/R + Ksm/KjR, and is dependent upon the concentration of
the inhibitor.

For noncompetitive inhibition, both the slope and intercepts are
changed. The usual case is such that the combination of the inhibitor
with either enzyme or enzyme-substrate complex occurs with approxi-
mately equal affinity, so that Ki and KSi are approximately equal. Under

INTRODUCTION AND THEORETICAL CONSIDERATIONS

453

such circumstances the slope and the intercept are increased by a factor
of 1 + [I]/KI} as compared to the linear relationship in the absence of the
inhibitor.

For the specific case of noncompetitive inhibition ("uncompetitive in-

«r /■

V ■ x

/ 1 *

'5b

!-/„

/ • ^ ""

♦ ' ^* ""

* ^^ i

4^ !

V^ i ^

AT *

S^ S* X»«»«««K««»»»

<+H

* ' ^ •- '" ^

y ^ ■ — i

^ •*** i

■*

/0*

/ / . **

— » «— » .— . V—> ,— » «— I «— J »— . ^ .-* W^ W^ *-» V

1/[S]

Figure 2. Relationship of the reciprocal of the rate of a biological process to the

reciprocal of the substrate concentration.
_._._._. Relationship in the absence of an inhibitor.
Relationship in the presence of a competitive inhibitor at a defined

concentration.
mmwwmhp- Maximum rate of the biological process at optimal substrate con-
centrations.
Extrapolated theoretical values for rate exceeding the maximum rate

of the biological process which is then limited by other enzymatic

processes.
Maximum value of 1/[S] for biological systems either synthesizing or

containing unknown amounts of substrate.
x x x x x x x Maximum value of the reciprocal of rate of the biological process for

systems synthesizing or containing unknown amounts of substrate

under experimental conditions.
-x-x-x-x Extension of theoretical relationships for biological systems incapable

of synthesis of substrate or not containing unknown amounts of

substrate,
hibition") in which the inhibitor combines only with the enzyme-substrate
complex, addition of the inhibitor does not change the slope as compared
to the linear relationship in the absence of inhibitor; but the intercept
increases by a factor of 1 + [I]/K8I.

These changes are indicated in Figure 1, which illustrates the varia-

454 THE BIOCHEMISTRY OF B VITAMINS

tions of slope and intercept which distinguish competitive inhibition from
noncompetitive inhibitions.

A more specialized type of inhibition, "quadratic" inhibition, has been
described, in which the plots for different concentrations of inhibitor have
different intercepts and begin with near zero slopes which change to a
common slope identical with that in the absence of the inhibitor.18 This
is easily differentiated from competitive inhibition.

In a consideration of the rates of biological processes rather than of
isolated enzymes, the application of the methods of differentiation of
competitive inhibition from other types by rate studies is somewhat com-
plicated by the interrelationship of the rate of the biological process and
the rate of the specific inhibited enzymatic reaction. In the presence of
the inhibitor at effective concentrations, the rate of the specific enzymatic
reaction is the limiting process in eliciting an observable response from
the biological system. Under such conditions, the reciprocal of the rate
of the biological process plotted against the reciprocal of the substrate
concentration would give a linear function over a defined region in the
plot. The extrapolated intercept would not represent the maximum rate
of the biological process, but rather the theoretical maximum rate of the
biological process when the inhibited enzyme is saturated with substrate
and is still the limiting reaction. This is a purely hypothetical condition,
since many enzyme systems other than the specific one concerned would
most likely be somewhat less efficient under these conditions. As indicated
in Figure 2, the rate can increase only to the maximum rate of the
complete biological process and not to the maximum theoretical rate of
the specific enzyme.

Another complication is the fact that many biological systems have the
ability to synthesize the substrate. Hence, while the concentration of the
substrate supplied to the system can be diminished to zero, the concentra-
tion synthesized by the biological system limits the lowest concentration
of substrate attainable. Thus, the value of 1/[S] can be increased only
to a definite value, and further decreases in exogenous supply of the
substrate do not alter the rate of the biological process. This is indicated
in Figure 2. With increasing 1/[S] values the transition from the normal
linear relationship of 1/[S] versus 1/r to the constant minimum rate of
the biological process for a defined concentration of inhibitor may be
either an abrupt change or a gradual one depending upon the system.
For example, there may be a very abrupt change in systems in which the
concentration of substrate outside the biological system is not appreciably
altered by the biological system, and the concentration of substrate
within and without the system attain equilibrium. Concentrations of
exogenous substrate below that synthesized by the system would not

INTRODUCTION AND THEORETICAL CONSIDERATIONS 455

alter the internal concentration of the factor, while concentrations of
the externally supplemented substrate higher than that synthesized by the
system would attain an equilibrium with the concentration within the
system. Since the amount of substrate synthesized would merely be addi-
tive to total external and internal concentrations, the total external and
resulting internal concentration would not be materially affected by such
synthesis in many systems, e.g., inhibition of growth of a small inoculum
in bacterial growth medium.

On the other hand, the transition may be gradual in some systems, e.g.,
embryonated eggs, in which the amount of substrate synthesized or
initially present is appreciable in comparison with the total amount of
substrate supplied. The transition indicated in Figure 2 is minimized
to indicate the intermediate effect between two possible extremes.

These effects have not seriously limited the application of this method
in determining the type of inhibition in certain biological systems, but
may have altered some of the quantitative aspects of data derived by the
method.

This method was applied by Wyss 19 to the effect of p-aminobenzoic
acid on the growth inhibition of Escherichia coli resulting from the action
of sulfanilamide. The results indicate that sulfanilamide competitively
inhibits the functioning of p-aminobenzoic acid. The rate of growth of
the organism inhibited by sulfanilamide was considered to be proportional
to the rate of the inhibited enzymatic reaction, and the total enzyme
concentration of the bacterial cell was assumed to be constant, since
mother and daughter cells have the same enzyme concentration. Essen-
tially, it is assumed that no resistance to sulfonamide develops under the
testing conditions. Although some resistance may develop during the
determination of growth rate, it does not seriously affect the qualitative
results, but may affect conclusions which are based on quantitative cal-
culation. This method has also been applied to the reversal by p-amino-
benzoic acid of the toxicity of other sulfonamides for Escherichia coli.20

By a method similar to that used in the derivation of the above rate
equations, an expression can be derived for the competitive inhibition of
the combination of a coenzyme with an apoenzyme. Thus

where the symbols are identical with those previously indicated. In the
graphical presentation of the data with 1/r plotted against l/[Co], the
intercept of 1/r at l/[Co]=0 does not change with changes in inhibitor
concentration, but the slope of the linear relationship increases with
increasing concentrations of inhibitor.

456 THE BIOCHEMISTRY OF B VITAMINS

Biological Action of Analogues of B Vitamins. Although some vitamin
analogues are converted in vivo to the corresponding vitamin, such is not
always the case. An analogue may combine with the enzyme involved in
the conversion of the vitamin to its coenzyme. The enzyme-analogue
complex either may be nonfunctional, resulting in competitive inhibition,
or it may function in a manner identical with that of the vitamin. In the
latter case an analogue of the normal coenzyme will be formed. If the
modified coenzyme cannot be utilized by the proper apoenzyme, the result
is still a competitive inhibition of coenzyme formation. However, if
modified coenzymes resulting from the analogue can be utilized by the
apoenzymes, the analogue is capable of replacing the vitamin in the
biological system. Some modified coenzymes apparently can be utilized
by only some of the apoenzymes which normally combine with the natu-
ral coenzyme. In such cases, supplements of the products of the enzyme
systems which cannot utilize the modified form of the coenzyme are
essential for the vitamin-like activity of an analogue which can replace
all other coenzyme functions of the vitamin. Similar considerations apply
in case more than one coenzyme is derived from the vitamin. Only one
of the coenzymes might be replaced by a similar product derived biologi-
cally from the analogue.

The analogue of a vitamin may prevent the utilization of a vitamin at
any stage in the biosynthesis and functioning of the coenzyme; however,
if two successive stages in the utilization of a vitamin are inhibited by
a single analogue, the vitamin would be capable of preventing the inhibi-
tion only over a small range of concentrations.

Structural Modifications Producing Vitamin Analogues with Biological
Activity. In order for a compound to inhibit the functioning of or replace
a natural metabolite in a biological system, the substance must possess
the ability to combine with a particular enzyme utilizing the metabolite.
The ability to combine with a particular enzyme at the specific point at
which the metabolite associates with the enzyme is determined not only
by the presence of certain functional groups which are instrumental in
effecting such a combination, but also upon the shape, size and configura-
tion of the molecule. Consequently, most of the compounds which either
replace or inhibit a vitamin in its functioning in a biological system are
related structurally to the vitamin.

The types of modifications which produce compounds with such biologi-
cal activities include replacement of groups occurring in the metabolite
as follows: -S- by -CH = CH- _CH2-CH2- or -S02-; benzene
nucleus by aromatic heterocyclic nuclei such as thiophene, pyrimidine,
pyridine, etc. nuclei ; -COOH by -S02-NH2, -S03H, -S02-R, -SO-R,
-P03H2, -P02H, -As03H2, -CH2OH, -COR, etc.; -CH3 by -CI,

INTRODUCTION AND THEORETICAL CONSIDERATIONS 457

-C2H5, etc.; -NH2 by -OH, -N02, etc.; -H by -CI, -CH3, -OH,
etc., and these and other groups by such similar groups.

A number of excellent reviews by Woolley,21 24 by Welch,2"' and by
Roblin 2G have indicated the developments in the general field of competi-
tive inhibitors of the biochemical functioning of essential metabolites and
have indicated more extensively the changes in structure most likely to
produce metabolic antagonists.

The biological activities of the B vitamins and related compounds in-
cluding modifications such as these indicated above are listed in subse-
quent chapters. The biological activities in replacing the vitamin are
indicated in terms of per cent activity on a molar basis relative to the
particular B vitamin. The inhibitory activities are indicated in terms of
the inhibition index which, unless otherwise indicated, is the lowest ratio
of the concentration of the inhibitor to the concentration of the corre-
sponding B vitamin at which maximum inhibition of the biological system
occurs.

Bibliography

1. Embden, G., and Haymann, C, Z. physiol. Chem., 137, 154 (1924).

2. Lohmann, K., Biochem. Z., 222, 324 (1930).

3. Embden, G., and Deuticke, H. J., Z. physiol. Chem., 230, 50 (1934).

4. Warburg, O., and Christian, W., Naturwiss., 29, 589 (1941); Biochem. Z., 310,

384 (1941).

5. Tammann, G., Z. physiol. Chem., 16, 271 (1892); Armstrong, E. F., Proc Soc.

Roy. Soc. (London), 73, 516 (1904).

6. Wohl, A., and Glimm, E., Biochem. Z., 27, 349 (1910).

7. Kuhn, R., Z. physiol. Chem., 129, 57 (1923).

8. Euler, H. v., and Josephson, K., Z. physiol. Chem., 157, 122 (1926).

9. Quastel, J. H., and Woolridge, W. R., Biochem. J., 22, 689 (1928).

10. Gross, R. E., Z. physiol. Chem., 112, 236 (1920).

11. Coombs, H. L, Biochem. J., 21, 1259 (1927).

12. Loewi, O., and Navratil, E., Arch. ges. Physiol. (Pfluger's), 214, 678 (1926).

13. Woods, D. D., and Fildes, P., Chem. Ind., 59, 133 (1940).

14. Woods, D. D., Brit. J. Exptl. Path., 21, 74 (1940).

15. Michaelis, L., and Menten, M. L., Biochem. Z., 49, 333 (1913).

16. Shive, W., "Conference on Development and Uses of Antimetabolites," New

York Acad. Sci., Feb., 1949 (in press).

17. Lineweaver, H., and Burk, D., J. Am. Chem. Soc, 56, 658 (1934).

18. Ebersole, E. R., Guttentag, C, and Wilson, P. W., Arch. Biochem., 3, 399 (1944).

19. Wyss, O., Proc. Soc. Exptl. Biol. Med., 48, 122 (1941).

20. Klotz, I. M., and Gutmann, H. R., J. Am. Chem. Soc, 67, 558 (1945).

21. Woolley, D. W., Science, 100, 579 (1944).

22. Woolley, D. W., Harvey Lectures, Ser. 41, 189 (1946).

23. Woolley, D. W., Advances in Enzymol, 6, 129 (1946).

24. Woolley, D. W., Physiol. Revs., 27, 308 (1947).

25. Welch, A. D., Physiol. Revs., 25, 687 (1945).

26. Roblin, R. O., Chem. Revs., 38, 255 (1946).

Chapter II D

UTILIZATION OF COMPETITIVE ANALOGUE-METABOLITE

INHIBITION IN THE ELUCIDATION OF BIOCHEMICAL

PROCESSES INVOLVING VITAMINS

Rapid methods of developing relatively specific assays for metabolites
of biochemical systems and of elucidating the biochemical processes in-
volving these substances are desirable in the study of biochemistry. One
of the recently developed methods whereby this can be accomplished
involves a study of the effects of known metabolites and of biological
extracts on a specific competitive analogue-metabolite inhibition of a
biological system. These effects can be studied by a variety of testing
techniques to determine the interrelationship of the metabolite to the
substances affecting the inhibition. The ability of unknown substances in
natural extracts to exert an influence on the system can be used as a basis
for assay of unknown naturally occurring substances directly related to
the metabolite. Consequently, this method, which has been termed inhibi-
tion analysis, offers a direct approach to specific problems of biochemistry
which may otherwise be difficult to solve.

Theoretical Considerations

From a theoretical standpoint,1-5 exogenous substances other than the
metabolite which are capable of preventing the inhibitory effect of the
analogue in competitive analogue-metabolite inhibitions of biological sys-
tems include (1) substances which increase the effective concentration
of the metabolite, e.g., precursors of the metabolite; (2) the product, or
its equivalent, of the inhibited enzymatic reaction; (3) substances which
decrease the quantity of the product necessary for normal functioning of
the biological system, i.e., substances exerting a "sparing action" on the
product of the inhibited enzyme system; (4) agents increasing the effec-
tive concentration of the inhibited enzyme; and (5) substances which
assist in the destruction of the inhibitory analogue.

(1) Precursor Effect. The effective concentration of the metabolite
may be increased in a biological system by supplementation of the system
with several types of substances other than the metabolite. The most
common is a limiting precursor, an additional amount of which allows
the organism to synthesize an increased concentration of the metabolite.

458

COMPETITIVE ANALOGUE-METABOLITE INHIBITION 459

However, the addition of a catalytic factor normally limiting the biosyn-
thesis of the metabolite may also cause an increased synthesis, resulting
in a higher concentration of the metabolite in the biological system.
Further, if the metabolite is utilized in reactions which are not essential
for the response of the biological system, substances which prevent the
loss of the metabolite by such pathways may also increase the effective
concentration of the metabolite; but the magnitude of such an effect
would usually be relatively small in comparison with other precursor
effects.

A relatively simple testing technique has been developed which charac-
terizes the precursor type of effect on a competitive analogue-metabolite
inhibition of a biological system. The increased metabolite concentration
resulting from enhanced synthesis by the biological system results in a
corresponding increase in the amount of analogue (inhibitor) necessary
for a defined inhibition. However, if the metabolite is supplied to the
system in concentrations in excess of that from the enhanced synthesis
by the biological system, substances which exert such a precursor effect
do not appreciably influence the amount of inhibitor necessary for the
defined inhibition in the presence of the metabolite at such a concentra-
tion. Thus, the minimum inhibitory concentration of the analogue is
increased by such substances, but the inhibition index determined over
the remainder of a range of concentrations of the metabolite is not altered.

In many instances increasing concentrations of a limiting precursor of
a metabolite may prevent the inhibition caused by the analogue over a
rather wide range of concentrations. However, the conversion of the pre-
cursor to the metabolite usually becomes less efficient at higher concen-
trations, and the concentration of precursor relative to the inhibitory
analogue of the metabolite for a defined inhibition increases markedly
with increasing concentrations.

(2) Product Effect. If the product of the inhibited enzymatic reaction
is of such a nature that it can be supplied to the biological system from
an external source, the inhibited enzyme system becomes nonessential for
the biological process as a whole when this product is made available to
the system in adequate quantities. If the analogue does not prevent the
functioning of any other enzymes utilizing the metabolite, it is no longer
inhibitory to the biological system.

However, the metabolite (substrate) may be utilized by several differ-
ent enzymes involved in the synthesis of several products, Pi, P2, P3, etc.,
and the specific analogue, 7, may prevent the conversion of the metabo-
lite, S, to one or more of these products. If more than one system is
inhibited by the analogue, one of these enzyme systems, e.g., Eu would
be expected to become the limiting reaction of the biological process

460 THE BIOCHEMISTRY OF B VITAMINS

before the others. The equations relating the inhibition index to equilib-
rium constants and other constants (p. 447) would apply to the particular
enzyme system, E1} and the inhibition index, K1} would be related to this
particular enzyme system.

Although the analogue may not prevent the formation of P2, it may at
higher concentrations prevent the conversion of S to P3 sufficiently to
inhibit the biological system. Consequently, an exogenous supply of Pi
would not completely prevent the toxicity of the analogue, since another
enzyme system, Es, becomes the limiting process of the biological system,
and the particular equilibrium, rate, and other constants of this enzyme
determine the inhibition index, K3. Thus, in the presence of adequate
amounts of Pi, the inhibition index is increased from the K\ value cor-
responding to the inhibition of the biosynthesis of Pi, to the value Ks,
corresponding to the inhibition of the biosynthesis of P3.

Since Px becomes the first limiting product, an external supply of P3
would not be expected to exert any effect on the inhibition in the absence
of Pi. However, if both Px and P3 are supplied to the biological system,
the analogue either becomes ineffective as an inhibitor of the system, or
at a still higher inhibition index it prevents an additional function of the
metabolite.

Hence, regardless of the metabolite concentration, the addition of the
product of an inhibited enzyme system will result either in a complete
reversal of the toxicity of the analogue, or in a higher analogue-metabolite
ratio corresponding to the inhibition index of another enzyme system
utilizing the metabolite. If an analogue prevents the formation of a series
of products from the metabolite, there is a definite order in which the
products must be added if the effect of each substance is to be demon-
strated. In the absence of a product the biosynthesis of which is inhibited
at a lower inhibition index, all other products the biosyntheses of which
are inhibited at higher inhibition indices do not affect the inhibition index.

The general shape of the graph obtained by plotting the growth response
of the biological system against increasing concentration of an inhibitor
at a constant concentration of substrate is related to a specific enzyme
system and is dependent upon the dissociation constants, etc., of the
particular system (p. 447) . Since two separate enzyme systems involving
the same metabolite and inhibitory analogue would not be expected to
have similar dissociation constants, etc., the general shape of such a graph
or any type of data which depends upon such constants can be employed
to show that different enzyme systems are involved. The ratio of the
index for maximum inhibition to that for half-maximum inhibition can
be useful in such cases.

COMPETITIVE ANALOGUE-METABOLITE INHIBITION 461

(3) "Sparing Effect" on the Product. The following equation can be
obtained by solving equation (4) (p. 446) for [EI\ and substituting the
value in equation (3) (p. 446).

[I]_Kj([Et]-[ES]-[E])

[S] KS[ES] Uy;

If exogenous substances act in such a manner as to decrease the amount
of product, P, necessary for a constant response of the biological system,
the concentration of the enzyme-substrate complex must be decreased
accordingly, to maintain a defined response of the system in the presence
of such substances. This can be accomplished by increasing the ratio of
inhibitor to substrate. Since [E] is relatively small and can be neglected,
and since [Et] is relatively constant and is large with respect to \ES]
under the testing conditions, it is apparent from equation (19) that the
inhibition index varies approximately inversely with [EJS], which is
defined by the amount of product necessary for the defined response of
the biological system. Consequently, the decrease in the amount of product
required by the biological system for a constant response as a result of
the addition of substances exerting a "sparing action" is reflected by a
proportional decrease in [ES] , and a practically proportional increase in
the inhibition index.

If the product, P, of the inhibited enzyme system is involved in the
biosynthesis of secondary products, Pa, Pb, Pc, etc., these secondary
products can exert an effect on the inhibited biological system. If a sec-
ondary product is formed by a reversible reaction and if the reverse
reaction can take place under the testing conditions, the secondary product
will affect the inhibition in a manner analogous to the primary product.
However, in many instances the secondary products cannot form the
primary product by a reversible reaction. In such cases, one particular
secondary product would be expected to become the substance limiting
the biological response, and would be capable of exerting a "sparing
action" on the primary product. A definite order in which the secondary
products exert their effects would be expected.

While analogues of vitamins may in some cases inhibit at the coenzyme
stage, analogues often prevent coenzyme formation from the vitamin, as
illustrated in Figure 3. The coenzyme in turn is required by several
apoenzymes to form a series of enzymes, Ea> Eb, Ec, etc. These apoenzymes
vary in their relative concentrations and in their affinity for the coenzyme,
and the enzymes are required by the biological system in different con-
centrations. Consequently, one particular enzyme system, e.g., Ea, requir-
ing the coenzyme would be expected to become the limiting factor of
the biological system. If the product, Pa, of such an enzyme is supplied

462 THE BIOCHEMISTRY OF B VITAMINS

to the biological system, a decreased rate of coenzyme formation would
be essential to maintain the defined response. To decrease the rate of
coenzyme formation, an increase in the inhibition index is required such
that another enzyme system, e.g., EC) requiring the coenzyme becomes
the limiting system of the biological process. There is a definite order
in which the secondary products exert their effects. If a biological system
is somewhat deficient in a secondary substrate, e.g., Sa, when the conver-
sion of this substrate to product, Pa, is the limiting reaction of the system,
supplements of this substrate will allow the reaction to proceed more
efficiently. To maintain the defined inhibition of the system, the concen-
tration of the enzyme, Ea, must be decreased by decreasing the rate of
coenzyme formation. This necessitates an increase in the ratio of analogue
to vitamin. The increase in the inhibition index resulting from such addi-
tions will seldom approach that obtained with the product, Pa.

Figure 3. Relationship of Enzymatic Reactions to Inhibition of Coenzyme Formation
by Analogues of Vitamins

Substances which prevent the product of the inhibited enzyme reaction
from being utilized in a manner not essential for the biological response
would be expected to exert a "sparing action" on the product. Undoubtedly
still other types of substances exerting such an action will be discovered.

(4) Changes in Total Effective Enzyme Concentration. From equa-
tion (19) it is apparent that exogenous substances which increase the
total effective enzyme concentration, Et, will affect an inhibition in a
manner such that an increased ratio of analogue to metabolite is required
in order to obtain the same degree of inhibition as in the absence of such
substances. Since [ES] is small in comparison with [Ef] and is main-
tained at a defined value under these conditions where only [Et], [I] and

COMPETITIVE ANALOGUE-METABOLITE INHIBITION 463

[S] are varied, the inhibition index varies approximately directly with
the total effective enzyme concentration.

The substances which can produce such an increase in the effective
enzyme concentration include (a) a limiting coenzyme or a precursor of
a limiting coenzyme when excess apoenzyme is synthesized by the biologi-
cal system; and (b) a limiting second substrate which is involved in the
utilization of the inhibited substrate in the enzymatic reaction. Since the
biological system normally synthesizes these substances in near optimal
amounts, these effects usually are relatively moderate in comparison with
the other types.

(5) Destruction of the Inhibitor. Substances which allow rapid destruc-
tion of an inhibitory analogue by the biological system will render the
analogue ineffective as an inhibitor; however, if destruction occurs more
slowly, other effects, e.g., one similar to a precursor effect, may be ob-
served.

Other Theoretical Considerations. Reversing agents of type 1 are
easily distinguished from those of types 2, 3, and 4 by determination of
the inhibition index over a wide range of concentrations. Since substances
of the latter three types exert their action by increasing the inhibition
index, methods whereby these groups can be further differentiated are
desirable. A number of substances exerting such an effect on competitive
inhibition can usually be classified into the various groups. Since reversing
agents of type 2 but not of types 3 and 4 involve more than one enzyme,
data which involve the dissociation and other constants will permit classi-
fication of substances of type 2. Hence, the ratio of the inhibition index
for maximum to that for half-maximum inhibition would be expected to
change with the addition of an agent of type 2, but not of types 3 and 4.
Reversing agents of type 3 and type 4 exert their effects independently
of each other and are synergistic; but neither exerts an effect on the in-
hibition in the presence of the product of type 2.

When two analogues prevent enzymatic reactions at two successive
stages in a biosynthetic sequence, a synergistic action of the inhibitions
is usually obtained. When the first biosynthetic transformation is inhib-
ited to such an extent that the biosynthesis of the immediate product is
reduced to half the normal rate, the amount of an analogue of the product
necessary to inhibit completely the response of the biological system is
reduced to half. However, the amount of the first analogue necessary to
reduce the rate of the reaction to half the normal rate is often only a small
fraction of that necessary to inhibit completely the response of the bio-
logical system. Consequently, the effects of the inhibitors are not additive
but synergistic. Demonstration of such synergistic effects can often be
useful in indicating such a biosynthetic sequence.

464 THE BIOCHEMISTRY OF B VITAMINS

Application of Inhibition Analysis to Elucidation of Biochemical
Transformations

Biosynthesis of Pantothenic Acid. The specificity of the yeast assay
for pantothenic acid which resulted in the discovery of the vitamin is
dependent upon the presence of asparagine in the medium.6 Asparagine
prevents the response of the organism to /^-alanine, which in the absence
of asparagine replaces pantothenic acid in stimulating the growth of
yeast. Similar results have been obtained with a number of a- and /?-amino
acids,7 and with propionic acid 8 (p. 645) .

The involvement of aspartic acid in the biosynthesis of /^-alanine and
of pantothenic acid in Escherichia coli has been demonstrated with two
analogues of aspartic acid. Cysteic acid ° and hydroxyaspartic acid 1
competitively prevent the functioning of aspartic acid in Escherichia coli
cultured in a salts-glucose medium. The inhibition indices are 30-100 and
3-16, respectively. If a supplement of either /^-alanine or pantothenic
acid is added to the growth medium, cysteic acid does not affect the
growth of the organism even at relatively high concentrations; but
hydroxyaspartic acid at high concentrations still prevents it. The inhibi-
tion index in the latter case is increased to 20-30. Thus, cysteic acid
apparently prevents only the conversion of aspartic acid to ^-alanine,
whereas hydroxyaspartic acid prevents that reaction and at least one
additional transformation involving aspartic acid.

Inhibitions of the biosynthesis of pantothenic acid by several analogues
of pantoic acid have been reported. Salicylic acid has been considered in
this category10 (p. 646). Either cc-hydroxy-/?,/?-dimethylbutyric acid or
/?,y-dihydroxy-/?-methylbutyric acid prevents growth of Saccharomyces
cerevisiae G. M. stimulated by /3-alanine, but does not affect growth
stimulated by pantothenic acid u (p. 646) ; hence, these compounds
apparently prevent the biosynthesis of pantothenic acid by preventing
the combination of pantoic acid with /^-alanine.

Biochemical Functions of Pantothenic Acid. A study of the effect of
known metabolites on the inhibition of pantothenic acid synthesis by
cysteic acid in Escherichia coli has been utilized in an effort to determine
the metabolic function of pantothenic acid.12 As the organism becomes
deficient in synthesis of the vitamin and the corresponding coenzyme, one
particular secondary enzyme system would be expected to become the
first limiting reaction for growth, since the various apoenzymes requiring
the coenzyme would be expected to differ in their affinity for the coenzyme
and in the quantity of the coenzyme required for normal enzymatic
activity. Addition of the product of this secondary enzyme system would
allow growth until the rate of synthesis of pantothenic acid was decreased

COMPETITIVE ANALOGUE-METABOLITE INHIBITION 465

to an extent such that another secondary enzyme system requiring the
factor became the limiting reaction of the biological system. Since the
rate of synthesis of pantothenic acid under the testing conditions is
determined by the ratio of cysteic acid to aspartic acid, the addition of
such a product of a secondary enzyme system would be expected to result
in an increase in the inhibition index — a type 3 effect.

Such an effect is obtained with citric acid, as-aconitic acid, or a-keto-
glutaric acid. The inhibition index determined over a range of aspartic
acid concentrations is increased approximately tenfold when one of these
substances is added to the medium of Escherichia coli. Although inactive
alone, supplements of oxalacetic and pyruvic acids together increased the
inhibition index slightly. Acetate had a similar slight effect. These effects
were not comparable to those of the tricarboxylic acids or of a-ketoglu-
taric acid. Pantoic acid was inactive. Since the sparing action of citric
acid, ra's-aconitic acid, or oc-ketoglutaric acid cannot be duplicated by
the precursors of the tricarboxylic acids, it appears that in Escherichia
coli pantothenic acid functions in the biosynthesis of the tricarboxylic
acids, citric and as-aconitic acids, from oxalacetic acid and pyruvate or
acetate. It has since been shown that coenzyme A, which was found to
contain pantothenic acid while the work just mentioned was being com-
pleted,13 functions in the oxidation of acetate in yeast 14 and in the forma-
tion of citric, acid by pigeon liver from oxalacetic acid and acetate.15
Phenylpantothenone has been reported to prevent the conversion of
pantothenic acid to coenzyme A in yeast.16

The "sparing action" of a-ketoglutaric acid on pantothenic acid and
the precursor effect of glutamic acid in the biosynthesis of aspartic acid
result in an unusual effect of glutamic acid on the toxicity of cysteic acid
for Escherichia coli. Apparently a very rapid transamination with
oxalacetic acid results in the formation of essentially equivalent amounts
of aspartic acid from supplementary glutamic acid. The presence of a-keto-
glutaric acid results in a "sparing action" on the product of the system
such that glutamic acid is 3 to 10 times as effective as aspartic acid in
preventing the toxicity of cysteic acid.9 This puzzling situation of an
apparent precursor of a metabolite being more effective than the metabo-
lite itself led to the elucidation of the complete cycle in which glutamic
acid simultaneously acts as a limiting precursor and as the product of a
secondary enzyme system utilizing the product of the blocked enzymatic
reaction as indicated in Figure 4.

At an inhibition index of approximately 3,000, N-pantoyl-n-butylamine
inhibits the utilization of pantothenic acid by Lactobacillus arabinosus
17-5. A supplement of either oleic acid or "Tween 80" increases the inhibi-
tion index approximately tenfold to a value of 30,000. Since either acetate

466

THE BIOCHEMISTRY OF B VITAMINS

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COMPETITIVE ANALOGUE-METABOLITE INHIBITION 467

or oleic acid is required by Lactobacillus arabinosus for growth, it appears
that pantothenic acid functions in the conversion of acetate to oleic acid
or its equivalent.12

A strain of Leuconostoc mensenteroides which requires either acetate
or an aromatic amino acid (phenylalanine, tryptophan or tyrosine) for
growth is inhibited by N-pantoyl-n-butylamine.5 The inhibition is pre-
vented competitively by pantothenic acid, resulting in an inhibition index
of 300 in the presence of either aromatic amino acids or acetate; but in
the presence of both an aromatic amino acid and acetate the inhibition
index is increased to 3,000. Phloroglucinol, particularly in the presence of
increased phosphate, was just as effective as the aromatic amino acids,
which are interchangeable in exerting this effect. Although ineffective
in replacing the aromatic compounds, sterols such as cholesterol and
coprosterone exert a sparing effect on the amount of phloroglucinol or
aromatic amino acids necessary to prevent the toxicity of the analogue
of pantothenic acid. The data suggest that pantothenic acid functions in
the biosynthesis of an intermediate common to the biosynthesis of all
these aromatic amino acids, and possibly to the biosynthesis of sterols
which are not readily reconverted to the intermediate. Phloroglucinol,
which can be considered a condensation product of three acetate radicals,
either is such an intermediate or is converted to it by the organism.5

In studies with Proteus morganii, the relative effects of glutamic acid
and cis- aconitic acid suggested that glutamic acid might be involved in
pantothenic acid metabolism in still an additional manner. Accordingly,
a number of synthetic conjugates of pantothenic acid, including those
with all the naturally occurring amino acids and with certain peptides,
have been prepared.5 The conjugates containing glutamic acid were some-
what more active than pantothenic acid in preventing the toxicity of
analogues of pantothenic acid for certain lactic acid bacteria.5 Concen-
trates of coenzyme A contain glutamic acid and glycine,17 as well as
cysteine or cystine.18 A naturally occurring conjugate of pantothenic
acid is also reported to contain glutamic acid.19

Biochemical Interrelationships Involving Biotin. The inhibitory effect
of y-(3,4-ureylenecyclohexyl) butyric acid on the growth of Lactobacillus
arabinosus is prevented competitively by biotin, resulting in an inhibition
index of approximately 30,000 in a medium from which aspartic acid is
omitted.20 Either aspartic acid or oxalacetic acid affects the inhibition
in such a manner that the inhibition index is increased to approximately
300,000. Although sodium bicarbonate, particularly in the presence of
pyruvate, exerts an effect on the inhibition index, the two substances
together are not as effective as either aspartic acid or oxalacetic acid.
Oleic acid or "Tween 80" has no effect on the inhibitory action of the

468 THE BIOCHEMISTRY OF B VITAMINS

analogue in the absence of an exogenous aspartic acid supplement; but the
inhibition index determined in the presence of both aspartic and oleic
acids varies from 1,000,000 to 10,000,000. Often when the inhibition index
is relatively low (1,000,000), cfs-aconitic acid and related tricarboxylic
acids increase the inhibition index several fold. However, the effect of
the tricarboxylic acids is not always obtained; consequently, even if the
tricarboxylic acids are essential products, presumably derived from
cc-ketoglutaric acid, additional enzymatic reactions involving biotin are
essential for growth of Lactobacillus arabinosus. The implications of
these data are indicated in Figure 5, which shows that the biotin
analogue presumably prevents the formation of a coenzyme which is
either directly or indirectly concerned with the carboxylation of pyruvic
acid to form aspartic acid, the biosynthesis of oleic acid or an analogous
product, and at least one additional product.5, 20

The carboxylation of pyruvic acid as a function of biotin was inde-
pendently and almost simultaneously discovered in three different
laboratories.21, 20, 22, 2:i An interrelationship of aspartic acid and biotin
had been established for yeast much earlier,24 and was extended to
bacteria.25 The ability of oleic acid to replace the nutritional requirement
of some microorganisms for biotin had been previously reported.26

Homobiotin, which inhibits the utilization of biotin by Lactobacillus
casei at a relatively low inhibition index, does not inhibit the growth of
that organism in a medium containing oleic acid,27 even at relatively
high concentrations. Since analogues of biotin do not usually inhibit the
utilization of biotin synthesized by an organism, it is possible that oleic
acid as a limiting product reduces the biotin requirement of Lactobacillus
casei to such a point that the organism is capable of synthesis of the small
amount of the vitamin needed for other enzyme systems. The possibility
of complete replacement of a vitamin by all the products of the enzyme
systems in which it functions must be considered in this case. In view
of the results with Lactobacillus arabinosus, it appears that synthesis
of the small biotin requirement by the organism may be the most logical
explanation.

Desthiobiotin competitively prevents the toxicity of 2-oxo-4-imidazol-
idinecaproic acid for Escherichia coli.28 The inhibition index is approxi-
mately 100. Since biotin, at slightly greater concentration than the lowest
giving any response, prevents the toxicity of even relatively high con-
centrations of the desthiobiotin analogue, it appears that the analogue
prevents the formation of biotin from a metabolite identical with or
similar to desthiobiotin. A supplement of either glutamic acid or a-keto-
glutaric acid prevents the toxicity of the inhibitor in such a manner as
to increase the inhibition index to 300. Either glutamic acid or cc-keto-

COMPETITIVE ANALOGUE-METABOLITE INHIBITION

glutaric acid exerts a "sparing effect" on biotin which results in an in-
crease in the ratio of analogue to desthiobiotin necessary for the defined
inhibition of growth of Escherichia coli.20 Since neither cis-aconitic acid
nor citric acid exerted such an effect, one would suppose by analogy with
the carboxylation of pyruvic acid that biotin functions in the decar-
boxylation of oxalsuccinicacid.20

Inhibition
index

C02+CH3— C— COOH

7-(3,4-Ureylenecyclohexyl)-
butyric acid

30,000

HO— C— CH2— C— COOH

O NH2

II I

HO— C— CH2— CH— COOH

Biotin

Precursor

Biotin coenzyme

Inhibition
index

Precursor *- Oleic acid

300,000

Inhibition
index

*• Unknown product

1,000,000 to

10,000,000

Figure 5.

Interrelationships of Biotin Indicated by Inhibition Analysis with
7-(3,4-Ureylenecyclohexyl)butyric acid

Biochemical Functions of p-Aminobenzoic Acid and Folic Acid. Sub-
stances other than p-aminobenzoic acid which prevent the toxicity of
sulfonamides have been known to occur in natural extracts for some
time.29- 30 Methionine 30_33 has some ability — which is enhanced by
purines 32, 33 — to prevent the toxicity of sulfanilamide for Escherichia
coli. Adenine or hypoxanthine is reported to be as active on a weight
basis as p-aminobenzoic acid in preventing the protective action of sul-
fanilamide against infections of Streptococcus hemolyticus in mice.34
Purines under specific conditions also prevent the toxicity of sulfonamides
for lactic acid bacteria 35 and for Eremothecium ashbyii.3Q

Such effects of structurally unrelated compounds were early considered
to be evidence against the competitive analogue-metabolite theory of
sulfonamide action. However, adequate explanations for such action are

470

THE BIOCHEMISTRY OF B VITAMINS

offered by inhibition analysis, which allows some insight into the meta-
bolic functions of this and related vitamins.

Sulfanilamide, in preventing the functioning of p-aminobenzoic acid
in Escherichia coli, inhibits a series of biochemical transformations, as
indicated in Figure 6.5, 37, 3S The inhibition index determined in a salts-

HS— CH2

NH2
-CH2— CH— COOH

Inhibition NH2
Index
> CH3— S— CH2— CH2— CH— COOH

Homocysteine (or related
precursor)

3000

Methionine

//
NH2— C

I
C— N

II /'
NH2— C— NH

5(4)-Amino-4(5)-imidazolecarbox-
amide (or derivative)

NH2— CH2— C— OH

Glycine

10,000

30,000

I
HC

N— C-

>CH
-NH

Hypoxanthine

Xanthine

Adenine

Guanine

or derivatives

NH2

HOCH2— CH— COOH

Serine

CH3

OH NH2
-CH— CH— COOH

Threonine

Precursor

100,000

NH— C
NH— CH

-CH3

Thymine
(or derivative,
e.g., thymidine)

Figure 6, Interrelationships Involving p-Aminobenzoic Acid Determined by
Inhibition Analysis with Sulfanilamide

glucose medium is approximately 3000; but in the presence of supple-
ments of methionine a higher ratio, approximately 10,000, is required for
the same degree of inhibition of growth of the organism.37 The inhibition
is further affected by certain purines, which are ineffective in the absence
of supplementary methionine.37 The inhibition index is approximately
30,000 when determined in a medium containing both methionine and
purines. The purines and derivatives effective in replacing the purine

COMPETITIVE ANALOGUE-METABOLITE INHIBITION 471

requirement under these conditions include adenosine, xanthine, guanine
and inosine. Adenine not only is ineffective, but is usually synergistic
with sulfonamides in preventing growth of Escherichia coli under these
conditions.37 Thus, the first limiting enzymatic reaction involves the
biosynthesis of methionine, probably from homocysteine, since the latter,
a precursor, does not affect the inhibition.5, 3S The next limiting transfor-
mation, which becomes apparent when exogenous methionine is supplied
to the organism, is the biosynthesis of purines, presumably from 5(4)-
amino-4(5)-imidazolecarboxamide or a derivative of this amine, since
the amine accumulates in the medium under these conditions of sul-
fonamide inhibition.39, 40 Although the amine stimulates growth of
Lactobacillus arabinosus in a manner similar to purines and disappears
slowly from the medium during the process,5 many organisms including
Escherichia coli cannot utilize the amine, and it remains unchanged in
the medium. It appears likely that the free amine is derived from a con-
jugated form, e.g., riboside or desoxyriboside, which is the normal pre-
cursor of purines.

Glycine, which increases the production of the aminoimidazolecarboxa-
mide in Escherichia coli, is apparently a precursor of the amine.41 Al-
though less effective than glycine, threonine similarly increases the pro-
duction of the amine and is apparently converted into glycine by the
organism. The inability of serine to replace glycine, particularly in view
of reported mutants of Escherichia coli requiring either serine or glycine
for growth, indicates that the conversion of serine to glycine is blocked
by sulfanilamide.41

The involvement of p-aminobenzoic acid in the biosynthesis of serine
is further indicated by the effect of serine on the inhibition of growth of
Escherichia coli by sulfanilamide in a medium containing both methionine
and purines.38 The inhibition index is increased from 30,000 to 50,000 —
100,000 by supplements of serine under these conditions.5- 3S

Thymine and folic acid are somewhat interchangeable in affecting the
inhibition index determined in a medium containing methionine, purines
and serine.38 The inhibition index is usually increased two- or threefold
to approximately 200,000 to 300,000.5, 3S High concentrations of sul-
fanilamide which are necessary for determination of these high inhibition
indices often do not affect solely enzymes utilizing p-aminobenzoic acid;
consequently, the results are sometimes variable.5' 38

For each of these products to exert its effect on the inhibition of growth
of Escherichia coli by sulfanilamide, the products must be added in a
definite sequence as indicated in Figure 6. All products whose biosyntheses
are related to lower inhibition indices must be available to the organism
from an exogenous supply in order that a product whose biosynthesis is

472 THE BIOCHEMISTRY OF B VITAMINS

related to a higher inhibition index may exert its effect on the inhibition.
However, the order in which the products exert their effect may vary to
some extent under different testing conditions and with inocula treated
differently. For example, it is occasionally possible in Escherichia coli to
obtain an effect with folic acid in the absence of serine.

The requirement of certain mutant strains of Escherichia coli for
p-aminobenzoic acid can be replaced by a mixture of amino acids, purines
and thymine, indicating further the involvement of p-aminobenzoic acid
in the biosynthesis of these substances.42

The index for inhibition of growth of Lactobacillus arabinosus by
sulfanilamide is increased from 100 to 1000 by purines.37 For this organism
and Streptobacterium plantarum 10S, thymine at high concentrations in
the presence of purines is reported to prevent the toxicity of sulfonamides
over a wide range of concentrations.43 Both purines and thymine are
necessary to replace the requirement of p-aminobenzoic acid for growth
of Lactobacillus arabinosus.43 Thymine exerts a similar effect on the
toxicity of sulfanilamides for certain strains of Streptococcus jaecalis
and Streptococcus zymogenes.44

One common feature in the metabolic reactions in which p-aminobenzoic
acid participates is the involvement of a single carbon unit in the final
product. Hence, it appears that p-aminobenzoic acid functions directly or
indirectly in the introduction of single carbon units into purines, pyrimi-
dines, serine (from glycine), and methionine (from homocysteine or a
related precursor) . A close association of all these single carbon units with
the possibility of a common precursor is suggested by these results. The
incorporation of isotopically labelled formate in the 2 and 8 positions of
uric acid previously indicated a single carbon unit precursor for the
purines.45 The involvement of a common precursor was further indicated
recently by results indicating the incorporation of isotopically labelled
formate into the /3-carbon atom of serine,46 and the incorporation of
isotopically labelled methyl groups of choline into the /3-carbon atom of
serine.47

On the basis of published research the interrelationship of p-amino-
benzoic acid and folic acid cannot be completely described. Folic acid
has little or no activity in replacing the nutritional requirement of a
number of organisms for p-aminobenzoic acid. However, sulfonamides
prevent the biosynthesis of a microbiologically active form(s) of folic
acid in a wide variety of organisms which do not require folic acid for
growth (p. 490). Furthermore, the biological functions of folic acid closely
parallel those of p-aminobenzoic acid. Thus, thymine and purines replace
or partially replace the folic acid requirement of certain organisms,48-50
and with Lactobacillus casei, inhibition analysis indicates that folic acid

COMPETITIVE ANALOGUE-METABOLITE INHIBITION 473

functions in the biosynthesis of these substances.51 The inhibition index
obtained with methylfolic acid, a competitive analogue of folic acid, is
30 in the absence of purines, 100 in their presence, and 1000 in the pres-
ence of both purines and thymine.51 Thymine is inactive alone. For Strep-
tococcus faecalis R, the toxicity of methylfolic acid is completely pre-
vented by both purines and thymine.52 Purines alone exert a very slight
effect on the inhibition index, and thymine alone exerts no effect. An
involvement of folic acid in the biosynthesis of serine in Streptococcus
faecalis R 53 appears to be similar to the involvement of p-aminobenzoic
acid in the same biosynthesis in Escherichia coli.

The involvement of p-aminobenzoic acid in biosyntheses involving a
single carbon unit led to a search for folic acid derivatives capable of
serving as a formate carrier. Pteroylhistidine which was prepared syn-
thetically did not exert any pronounced activity. However, the structure
of rhizopterin, N10-formylpteroic acid, gave a clue as to how formate
could be carried by a functional derivative of folic acid. Accordingly,
formylfolic acid was prepared and found to be approximately 30 times
as active as folic acid in preventing the toxicity of methylfolic acid for
Streptococcus faecalis R. The derivative was just as active as folic acid
in promoting growth of this and a number of other organisms requiring
folic acid.54

While it is not possible as yet on the basis of published results to con-
clude whether p-aminobenzoic acid and folic acid are converted to an
identical coenzyme, or whether two or more different coenzymes are in-
volved, inhibition analysis applied to natural extracts has been useful in
the discovery of a form of folic acid which is more widely active, and of
other related factors from which a solution to the perplexing problem
appears possible in the near future.

Utilization of Inhibition Analysis in the Development of Assays for
Naturally Occurring Unknown Factors

The techniques of inhibition analysis can be used to develop micro-
biological assays for unknown factors which may be difficult to detect by
other means. This offers a specific, direct approach to the discovery and
isolation of factors related to a specific metabolite. Such a problem pre-
sented itself recently when folic acid was found to be effective in the
treatment of pernicious anemia, but was not the anti-pernicious anemia
principle (s) of refined liver extracts. It was highly desirable at that time
to develop specific assays for unknown substances related to folic acid
or p-aminobenzoic acid and occurring in refined liver extracts. It has been
reported that through the use of inhibition analysis and related ap-
proaches more than twenty different assays have been developed for

474 THE BIOCHEMISTRY OF B VITAMINS

unidentified substances related to p-aminobenzoic acid, folic acid or re-
lated factors.5

Thymidine. One particular factor was especially interesting since it
prevented the toxicity of methylfolic acid for Leuconostoc mesenteroides
8293 and the toxicity of either sulfanilamide or 6,7-diphenyl-2,4-diami-
nopteridine for Lactobacillus arabinosus 17-5.5, 55 Since an inhibition anal-
ysis indicated that the factor was a product of the biological functioning
of folic acid and p-aminobenzoic acid, the high activity of refined liver
extracts suggested the possibility that the factor might be a conjugated
form of folic acid. However, on isolation of the factor from hog liver, a
colorless crystalline compound was obtained and identified as thymidine
which was present in some refined liver extracts to the extent of 1 per
cent of the solids.55

The inhibition index for Leuconostoc mesenteroides inhibited by meth-
ylfolic acid is increased from 3,000 to 30,000 by the addition of thymi-
dine to the medium. Although thymine and thymidine are interchangeable
for many organisms, thymine is inactive in the above tests. Consequently,
the biosynthesis of thymidine does not appear to take place through the
intermediate formation of thymine.55

The Vitamin B^. Group. Of the many tests developed for factors
occurring in refined liver extracts, five were found to involve a func-
tionally related group of factors involved in the biochemical functioning
of p-aminobenzoic acid in Escherichia coli.5 One testing method utilized
sulfanilamide in a concentration sufficient to prevent the biosynthesis of
methionine in Escherichia coli grown in a salts-glucose medium supple-
mented with known vitamins, xanthine, thymine, serine and glutamic
acid. Under these conditions, the organism responded to very small
amounts of refined liver extract; and by use of this assay technique, a
crystalline red compound was isolated from refined liver extracts after
a 20,000-fold concentration.5 Because of its distinctive color and biological
properties, the factor was termed "erythrotin." This factor is apparently
identical with or closely related to vitamin B12.5, 56 Still another factor,
which moves more slowly in organic solvents used as eluants during
various chromatographic separations, is also active in the Escherichia
coli assay; this has been tentatively named "erythrotide." 5 The root
prefix erythro- has been suggested as a basis for naming the individual
members of this group of factors which have the same biological function
but slightly different chemical structures.

Although methionine replaces erythrotin for Escherichia coli under the
testing conditions, the latter compound is from 100,000 to 300,000 times
as active. Consequently, no interference by methionine was encountered

COMPETITIVE ANALOGUE-METABOLITE INHIBITION 475

in the isolation of the factors from liver or in the assay of relatively
good sources of this group of factors.5

Whether the biosynthesis of methionine, purines, serine or thymine (or
folic acid) is the limiting reaction in the growth of Escherichia coli
inhibited by sulfanilamide, the inhibition index is increased about three-
fold by the addition of erythrotin at a concentration of 0.0005 y per
10 cc as indicated in Table l.5 This effect is not enhanced by increasing
the concentrations of erythrotin, even to 0.1 y per 10 cc. From these
results it is apparent that erythrotin plays a catalytic role in the bio-
synthesis of methionine, purines, serine, thymine and additional products.
The catalytic role of erythrotin involves either the conversion of
p-aminobenzoic acid to the coenzyme form involved in these syntheses or
a separate catalytic role in combining the single carbon unit into these
metabolites. Thus, the close association of the functioning of the vitamin
Bi2 group and p-aminobenzoic acid is further indicated. The biological
action of other unknown factors which are currently being isolated by
similar techniques will further clarify the exact role of this new vitamin.

Table 1. Effect of Erythrotin on Sulfanilamide Inhibition of E. coli

-Inhibition Index-

With added
Without added Erythrotin

Supplement Erythrotin 0.00057 per 10 cc

None 3,000 10,000

Methionine, 100 y per 10 cc 10,000 30,000

Methionine, 100 y per 10 cc \ m nnn , nn nnn

Xanthine, 100 y per 10 cc / 30'000 100,000

Methionine, 100 y per 10 cc "i

Xanthine, 100 y per 10 cc J 50,000-100,000 200,000-300,000

Serine, 100 y per 10 cc J

Methionine, 100 y per 10 cc l

Xanthine, 100 t per 10 cc I 100,000-200,000 300,000-500,000

serine, 100 y per 10 cc ' ' ' '

Folic acid, 0.03 y per 10 cc >

It is interesting that neither thymidine nor other desoxyribosides which
replace the vitamin Bi2 group for both Lactobacillus lactis and Lacto-
bacillus leichmanni is active in replacing erythrotin for Escherichia
coli.5 Similarly ascorbic acid, glutathione and related compounds at con-
centrations which can replace vitamin Bi2 under certain conditions for
these lactic acid bacteria do not affect appreciably the response of
Escherichia coli in the sulfonamide assay.5

Mechanisms of Resistance to Competitive Inhibitors

If an organism is allowed to grow in the presence of sub-inhibitory con-
centrations of a vitamin analogue or drug, the concentration of the in-

476 THE BIOCHEMISTRY OF B VITAMINS

hibitor can usually be increased gradually until the organism becomes
resistant to relatively high concentrations of the inhibitory compound.
The resistance acquired by the organism in the early stages of the de-
velopment of resistant strains is usually lost after the organism is cul-
tured in the absence of the inhibitor. The initial resistance may be highly
specific for the inhibitory analogue, or the organism may be sensitive to
analogues of similar structure. However, the resistance gained after
prolonged culturing in the presence of the inhibitor usually is relatively
permanent, and may be specific not for the individual inhibitor but for
the majority, if not all, of the inhibitory analogues of the vitamin, the
functioning of which is prevented by the inhibitor against which the
resistance is developed.

The development of resistant strains of an organism would be expected
to result either from a selection of a naturally occurring mutant strain,
or from an induced mutation, or a combination of both. Obviously, in
the presence of the drug, the environment favors the selection of natural
resistant strains, and no convincing data indicating that an inhibitor
specifically induces the mutation to a strain which is resistant have been
presented. While the process of development of resistance is gradual in
most instances, suggesting that the final mutant strain which possesses
the resistance is not present in the initial population, the possibility
exists that continual selection of progeny more and more resistant to the
inhibitor allows the isolation of a mutant strain which occurs normally,
but only so infrequently that an ordinary culture would have little chance
initially of containing a single such resistant cell.

Many types of biochemical differences between normal strains of an
organism sensitive to an inhibitory analogue of a metabolite and strains
resistant to the inhibitor could account for the development of resistant
strains. Biochemical differences between resistant and parent strains
include: (1) increase in the biosynthesis and concentration of the metab-
olite in the cells; (2) increase in the effective concentration of the in-
hibited enzyme; (3) increase in the synthesis of other factors limiting
the utilization of the product of the inhibited enzyme system, i.e., factors
which exert a "sparing action" on the product; (4) more extensive de-
velopment of other mechanisms by which the product of the inhibited
enzyme system is synthesized; (5) presence of an enzyme of slightly
different structure with normal affinity for the metabolite, but with less
or no affinity for the analogue, i.e., different cells of the same organism
may not necessarily produce structurally identical molecules of an en-
zyme catalyzing the same reaction in all the cells, and such variations
could conceivably occur within a single cell; (6) presence of an enzyme
system which destroys the inhibitor; (7) decrease in cell permeability
specifically to the inhibitor.

COMPETITIVE ANALOGUE-METABOLITE INHIBITION 477

Many organisms resistant to an inhibitory analogue do produce in-
creased amounts of the metabolite. This can be demonstrated by assay of
the cultures for the metabolite. Although in such a case considerably
more of the analogue is required for inhibition of growth in the absence
of an exogenous supply of the metabolite, the inhibition index appearing
with increased supplemental concentrations of the metabolite does not
differ from that of the normal strain.

For resistant strains of organisms showing increased inhibition indices,
classification in the above groups is difficult, since resistant organisms of
all the types 2-5 would be expected to have increased inhibition indices.
If the resistant organisms of type 4 are able to dispense completely with
the normal route of biosynthesis, such organisms would be expected to be
completely resistant to the analogue.

Destruction of the inhibitor by the organism is known to account for
the resistance in some strains which tolerate larger amounts of the in-
hibitor. If the rate of destruction of an inhibitory analogue is not too
rapid, the inhibition index determined at high concentrations of metab-
olite and inhibitor would not be expected to be appreciably altered.

Specific phases of acquired resistance to the inhibitory action of ana-
logues are discussed in subsequent sections.

Competitive Metabolite Antagonists and Biochemical Genetics

When Escherichia coli is sub-cultured in an inorganic salts-glucose
medium containing sulfanilamide and methionine as well as glycine,
serine and xanthine, a strain develops which requires methionine for
growth 58 and cannot utilize homocystine.59 The mutant strain does not
develop in the absence of either sulfanilamide or methionine. Though
evidence has been presented which indicates that the mutant strains of
this type have a greater growth rate than the parent strain,00 the mech-
anism by which the mutant strain is obtained appears to be more com-
plex than that of spontaneous mutation and selection. This mutant strain
is particularly interesting, since sulfanilamide prevents the biosynthesis
of methionine at the stage corresponding to the deficiency of the mutant
strain.59 Similarly, a strain of Escherichia coli requiring both methionine
and purines has been obtained from serial sub-cultures in the presence
of sulfanilamide in a medium containing both methionine and purines.61
The purine requirements of the mutant strain are analogous to the re-
quirements of the parent strain cultured in the presence of sulfonamides.01
From these results, it appears possible that competitive inhibitors of
metabolites may play a role in the elucidation of the biochemical rela-
tionship of enzyme to gene.

From crosses of a sulfanilamide-resistant strain to wild type, a strain
of Neurospora which appeared to require sulfanilamide for growth was

478 THE BIOCHEMISTRY OF B VITAMINS

obtained.62 p-Aminobenzoic acid inhibited the growth-promoting action
of sulfanilamide in a competitive manner. The ratio of p-aminobenzoic
acid to sulfanilamide giving growth inhibition was approximately 0.001.
Sulfanilamide was not specific, since sulfapyridine and sulfathiazole also
promoted growth. The latter was the most effective. A double mutant
obtained from crosses of the sulfonamide-requiring strain and a p-amino-
benzoic acid-requiring strain required both p-aminobenzoic acid and
sulfanilamide for growth, a maximum being obtained at a ratio of
sulfanilamide to p-aminobenzoic acid of approximately 1000.63 Investiga-
tion of this double mutant revealed that low concentrations of p-amino-
benzoic acid stimulated the growth of the organism in the absence of
sulfanilamide, but that higher concentrations of p-aminobenzoic acid
were toxic to the organism.64 The toxicity of p-aminobenzoic acid at these
higher concentrations could be prevented by sulfanilamide. Thus, the
sulfonamide-requiring mutant produced more than the tolerated amount
of p-aminobenzoic acid.64 The detrimental enzymatic transformations
involved in the utilization of p-aminobenzoic acid are inhibited by sul-
fanilamide, which thereby prevents the toxicity of excess p-aminobenzoic
acid. A similar situation exists with yeasts for which thiamine is toxic,
and the toxicity is prevented by pyrithiamine.65

More and more it is being realized that the basis for many of the
genetic blocks of enzymatic reactions involves inhibitions by normal
metabolic products of the organism. These inhibitions are not unrelated in
character to those obtained with synthetic analogues of metabolites.

Bibliography

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COMPETITIVE ANALOGUE-METABOLITE INHIBITION 479

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480 THE BIOCHEMISTRY OF B VITAMINS

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Chapter MID

p-AMINOBENZOIC ACID

The discovery by Woods 1 in 1940 that p-aminobenzoic acid, first
synthesized by Fischer2 in 1863, is an essential metabolite was not the
result of nutritional studies, but was made on the basis of the ability of
the factor to prevent the bacteriostasis of sulfanilamide and related com-
pounds. Even the evolvement of the sulfonamides and related compounds
as chemotherapeutic agents was almost as novel, particularly since sul-
fanilamide had been synthesized by Gelmo 3 in 1908, approximately a
quarter of a century before the chemotherapeutic action of this type of
compound was discovered.

The inhibitory activity of many dyes against bacteria in vitro led to
the preparation of a number of azo compounds including those in which
diazotized sulfanilamide was coupled with a number of aromatic amines
and related compounds.4, 5 The activity of one of these compounds, pron-
tosil,6 attracted widespread attention because of its effectiveness in vivo,

NH2
ICl-HjN— f ?— N=N— <f ?— £

prontosil

particularly against staphylococcal and /^-hemolytic streptococcal sep-
ticemia.7 However, the compound was inactive in preventing the growth
of these bacteria in vitro. Also, it was found that a number of active
compounds could be prepared by coupling diazotized sulfanilamide with
a variety of aromatic amines and phenols; but the sulfanilamide portion
of the molecule was highly specific, since a wide variety of other diazo-
tized amines did not form active compounds on coupling with these same
aromatic amines and phenols.8 The specificity of sulfanilamide in the
synthesis of active azo derivatives and the fact that the azo compounds
were not effective in vitro led to the discovery that sulfanilamide was
fully active in replacing prontosil.8 Reduction of the azo group in vivo
apparently allows the formation of the active principle, which is also
active in vitro.

Studies of the mechanism of the bacteriostasis produced by sulfanila-

481

482 THE BIOCHEMISTRY OF B VITAMINS

mide led to indirect evidence suggesting that inactivation of essential
enzymes was involved.9, 10 Extracts of streptococci,11 Brucella abortus 12
and yeast x were effective in preventing the toxicity of sulfanilamide for
streptococci ; and the active substance from yeast extract reversed inhibi-
tion in a manner reminiscent of the competitive inhibition of enzyme
reactions by substances structurally related to the substrate.1 By testing
substances structurally related to sulfanilamide, Woods x discovered that
p-aminobenzoic acid was extremely effective in preventing competitively
the toxicity of sulfanilamide, and that the compound exhibited a high
degree of specificity.

S02NH2

NH2 NH2

p-aminobenzoic acid sulfanilamide

It was proposed that p-aminobenzoic acid is an essential metabolite
synthesized in adequate amounts by many organisms but that it might be
an essential growth factor for some organisms.1

Within a short time the nutritional importance of the compound was
demonstrated by Rubbo and Gillespie,13 who isolated p-aminobenzoic
acid as the N-benzoyl derivative from yeast, and demonstrated that
p-aminobenzoic acid is an essential growth factor for Clostridium aceto-
butylicum. p-Aminobenzoic acid was subsequently isolated from yeast
in the free form and as the N-acetyl derivative by Blanehard,14 and as the
methyl ester by Kuhn and Schwartz.15 Many other organisms were sub-
sequently shown to require this factor.

Specificity

The specificity of the nutritional requirements of various organisms
for p-aminobenzoic acid, as well as the specificity of this substance in
preventing the toxicity of sulfonamides for various organisms, is indicated
in Table 2, in which the activities of various compounds related to
p-aminobenzoic acid are listed.

A majority of the compounds which possess activity in replacing
p-aminobenzoic acid are substances which presumably can be easily con-
verted to p-aminobenzoic acid by the various organisms. These include
4-aminocyclohexanecarboxylic acid, p-nitrobenzoic acid, p-hydroxylami-
nobenzoic acid, N-glycosido-p-aminobenzoic acids, esters and certain
amide derivatives of p-aminobenzoic acid — all of which can be converted

p-AMINOBENZOIC ACID

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484 THE BIOCHEMISTRY OF B VITAMINS

easily to p-aminobenzoic acid by oxidation, reduction or hydrolytic reac-
tions. However, there is no such explanation for the activity of several
compounds which apparently may be utilized as such in carrying out the
biochemical functions of the growth factor. Among these are 2-fluoro-4-
aminobenzoic acid 21 and the corresponding 2-bromocompound,21 as well
as 2-aminopyrimidine-5-carboxylic acid.31

COOH

NH2 NH2

2-fluoro-Jf.-aminobenzoic acid 2-aminopyrimidine-5-carboxylic acid

2-Fluoro-4-aminobenzoic acid is reported by Wyss, Rubin and Strands-
kov 21 to be approximately 38 per cent as effective as p-aminobenzoic
acid in promoting growth of either Clostridium acetobutylicum or a
mutant strain of Neurospora crassa requiring p-aminobenzoic acid for
growth, and has approximately this same relative activity /38 per cent)
in preventing the toxicity of sulfanilamide for Escherichia coli. Since the
ratio of sulfanilamide to 2-fluoro-4-aminobenzoic acid is indicated to be
constant over a range of concentrations, the utilization of the analogue as
such in building the appropriate coenzyme is indicated. If conversion to
p-aminobenzoic acid were the mechanism of action, the per cent conver-
sion would be expected to decrease with increased concentration of the
analogue.

2-Hydroxy-4-aminobenzoic acid (4-aminosalicylic acid) is particularly
interesting. Though this compound appears to inhibit effectively the
utilization of p-aminobenzoic acid in Mycobacterium tubercidosis*1'42
it does not have such an action in a wide variety of other organisms
(p. 525) . On the contrary, for two mutant strains of Escherichia coli
which require p-aminobenzoic acid, the hydroxy derivative is 4 to 16
per cent as effective as p-aminobenzoic acid in promoting growth.28 Thus,
the analogue replaces p-aminobenzoic acid in promoting growth of one
organism and inhibits the functioning of this factor in another organism.

The activity of p-nitrobenzoic acid varies considerably. It is almost
inactive in preventing the toxicity of sulfanilamide for Streptococcus
hemolyticus, but is almost as effective as p-aminobenzoic acid in promot-
ing growth of some strains of Clostridium acetobutylicum. The nitro
compound is only slightly active for a "p-aminobenzoicless" strain of
Neurospora crassa,24 and inhibits the growth of Streptococcus viridans 43
(p. 522). The growth inhibition is prevented by p-aminobenzoic acid or

p-AMINOBENZOIC ACID 485

by long incubation, particularly with higher concentrations of nitro-
benzoic acid, indicating that the organism actually converts the p-nitro-
benzoic acid slowly to p-aminobenzoic acid. With growth, the nitro
compound is converted into p-aminobenzoic acid in sufficient quantity
that the conversion can be demonstrated by quantitative determination
of diazotizable amine and by the ability of the product to reverse the
toxicity of sulfathiazole for the organism.44

The activity of p-hydroxylaminobenzoic acid in preventing the toxicity
of sulfanilamide for Streptococcus hemolyticus approaches that of
p-aminobenzoic acid.1 Although it is possible that samples of the compound
contain traces of p-aminobenzoic acid, such highly active substances
would not be expected to contain sufficient p-aminobenzoic acid as a
contaminant to account for the high activity.1 The difference in activity
of p-nitro- and p-hydroxylaminobenzoic acids for Streptococcus hemoly-
ticus is interesting.

For Staphylococcus aureus, 4-aminocyclohexanecarboxylic acid is ap-
proximately 77 per cent as effective as p-aminobenzoic acid in preventing
the toxicity of sulfanilic acid. This type of dehydrogenation in vivo also
occurs with nicotinic acid derivatives (p. 607). p-Aminobenzaldehyde is
apparently as active as p-aminobenzoic acid for Clostridium acetobuty-
licum.

While the N-alkyl derivatives of p-aminobenzoic acid are almost de-
void of activity,30 N-glycosido-p-aminobenzoic acids retain the activity
of p-aminobenzoic acid. N-D-Ribosido- and N-L-arabinosido-p-amino-
benzoic acid are as active as p-aminobenzoic acid for Clostridium aceto-
butylicum.10

Since p-aminobenzoylglycine as well as p-nitrobenzoylglycine is just
as active as p-aminobenzoic acid for Clostridium acetobutylicum,18 the
cleavage of peptide groups appears to occur. However, p-aminobenzamide
is utilized inefficiently, if at all, by most of the organisms and inhibits
the functioning of p-aminobenzoic acid in some strains of Escherichia
coft45-46 (p. 522). p-Acetamidobenzoic acid is utilized only with diffi-
culty by many organisms. Clostridium acetobutylicum appears to be able
to utilize p-benzamidobenzoic acid about one-tenth as effectively as
p-aminobenzoic acid,17 but the activity for what is presumably another
strain of this organism is reported to be considerably less.19

p-Aminophenylacetic acid has been reported to be ten times as effective
as p-aminobenzoic acid for Clostridium acetobutylicum,17 but other re-
ports indicate that the compound is only 0.002 to 0.01 per cent as effec-
tive as p-aminobenzoic acid as a growth factor,18"20 and it is ineffective
in preventing the toxicity of sulfanilamide for this organism.47 For other

486 THE BIOCHEMISTRY OF B VITAMINS

organisms the analogue is not particularly effective in replacing p-amino-
benzoic acid (Table 2).

Alkyl esters of p-aminobenzoic acid are not readily hydrolyzed by
many organisms. Also, considerable variation in activities has been re-
ported in some cases. Thus, ethyl p-aminobenzoate is reported to be
from less than 0.1 per cent 19 to almost 100 per cent as effective as
p-aminobenzoic acid for Clostridium acetobutylicum.11

An ester such as diethylaminoethyl p-aminobenzoate (procaine) ap-
pears to be more readily hydrolyzed by various organisms than either
the methyl or ethyl esters. Since this and related compounds are widely
used as local anesthetics, the ability of these compounds to prevent
sulfonamide-induced bacteriostasis has received widespread atten-
tion.30- 40' 48<54 As indicated in Table 2, procaine is almost one-fourth as
active as p-aminobenzoic acid for Streptobacteriwn plantarum 26 and
Streptococcus hemolyticus. It is also utilized by Acetobacter sub oxy dans,22
Clostridium acetobutylicum,13, 17~20 Escherichia coli,M Staphylococcus
aureus,110 Eberthella typhosa,Gi and pneumococci.48' 63 The inhibitory
effect of sulfanilamide on sprouting wheat is decreased by a number of
local anesthetics derived from p-aminobenzoic acid.59

Different sulfonamides are similarly affected by procaine. The latter
compound has been reported to prevent the bacteriostasis resulting from
sulfanilamide,50- 53> 61- 63 sulfapyridine,40'48 sulfathiazole,40- 49- 60 bis(p-
aminophenyl) sulfoxide,49 bis (p-aminophenyl ) sulf one,49 p-aminophenyl
p-hydroxyphenyl sulfone,49 p-aminophenyl p-nitrophenyl sulfoxide,49 and
prontosil.62

Other anesthetics containing p-aminobenzoic acid are also active.
y - Dimethy lamino - a, 8 - dimethylpropyl, y - diethy lamino - B, B - dimethyl-
propyl, /?-diethylaminoisohexyl and y-di-n-butylpropyl p-aminobenzoates
are 1-10, 0.1-1.0, 1.0 ca. and 0.1 ca. per cent, respectively, as effective as
p-aminobenzoic acid in preventing the toxicity of sulfapyridine for
Escherichia coli.40 For Staphylococcus aureus, the first two compounds
are somewhat more than 1 per cent, and procaine approximately 10 per
cent, as effective as p-aminobenzoic acid in preventing the toxicity of
sulfathiazole.40 y-Di-n-butylpropyl p-aminobenzoate is considerably less
active than procaine in preventing sulfanilamide bacteriostasis.61

Procaine is reported to be hydrolyzed by an esterase in human and
mouse blood.50 The mortality of mice infected with streptococci and
treated with sulfanilamide is slightly greater when procaine is injected
with the sulfanilamide.50, 53 The effect is significant only when maximum
nontoxic doses of procaine are administered.53 The pneumococcal thera-
peutic action of sulfapyridine in mice is decreased by administration of
local anesthetics derived from p-aminobenzoic acid.51 Procaine counter-

p-AMINOBENZOIC ACID 487

acts the therapeutic effect of locally applied sulfathiazole against gas-
gangrene,60 and antagonizes the action of sulfanilamide on pneumococci
in vivo as well as in vitro.™

From the in vitro activity of procaine for Streptococcus hemolyticus 1
(Table 2) it has been calculated that the average amount of procaine
used in minor surgery should be sufficient to inhibit all the sulfanilamide
in the human body, even during intensive treatment.53' 58 The concentra-
tion in the pleural fluid of patients under procaine anesthesia has been
reported to be 0.0002 per cent, which is sufficient to prevent completely
the bacteriostasis resulting from 0.05 per cent sulfapyridine for Pneumo-
coccus III (T 3-1) in vitro.48

However, procaine and its metabolic products are rapidly excreted
within 10 to 12 hours by the rabbit,52 and presumably by other organisms.
This tends to minimize the deleterious effect of such anesthetics on the
chemotherapeutic action of sulfonamides. Three clinical cases, one strep-
tococcal infection treated with sulfanilamide and one streptococcal and
one pneumococcal infection on sulfapyridine therapy, have been pre-
sented in which procaine was used as a local anesthetic without more
than transient ill effect on therapy.53 However, the use of local anesthetics
derived from p-aminobenzoic acid is contraindicated during sulfonamide
therapy.

Since /?-dimethylaminoethyl p-n-butylaminobenzoate (Table 2) is rela-
tively inactive in preventing sulfonamide bacteriostasis, a group of esters
and amides of p-alkylaminobenzoic acids have been prepared as possible
local anesthetics with diminished ability to prevent sulfonamide bac-
teriostasis. These compounds in general were ineffective in preventing
sulfonamide bacteriostasis for Escherichia coli and Streptococcus hemo-
lyticus?0- 56> 57

N-(p-Aminobenzoyl)-L-glutamic Acid. The first indication of the in-
volvement of glutamic acid in the metabolism of p-aminobenzoic acid
was the report of Auhagen,05 who found that N- (p-aminobenzoyl) -l-
glutamic acid is eight to ten times as effective as p-aminobenzoic acid in
preventing the toxicity of sulfanilamide for Streptobacterium plantarum
10 S.

H2N— / \— CO— NH— CH— CH2— CH2— COOH
\=S COOH

N-{p-aminobenzoyl)-i^-glutamic acid

The corresponding p-aminobenzoyl derivatives of D-glutamic acid,
L-aspartic acid, d- or L-leucine, glycine or glycylglycine were compara-

THE BIOCHEMISTRY OF B VITAMINS

o M-3

6 aft

ill

1 a 11

I • -

e - 2 m'5

^ | g S-|

1 5 fill

8 -a

^ a a-S

g K

S « o . 2

"3 § c f S

Is"

©'--;

CO "tf ■

oo<

o o<

&\-v co

O O <N

o o ooooo

oo o o o

i COCO O^h

ooo oo

o-h'o
o
co

o
oco

ooo oo

O i

•;3 03

o3 —

.5°

■^3 0

E'S

O >

2
-6

QJ 03

Otf

0' Cj

p-AMINOBENZOIC ACID 489

tively inactive in preventing the bacteriostasis induced by sulfanilamide
for this organism.65

These tests have been repeated at various concentrations of sulfanila-
mide or sulfathiazole to obtain data which indicate that N-(p-amino-
benzoyl)-L-glutamic acid competitively prevents the toxicity of the
sulfonamides and presumably is not the metabolic product of the enzyme
system inhibited by sulfonamides.66

The pronounced activity of p-aminobenzoylglutamic acid over p-amino-
benzoic acid has been observed only with two organisms under specific
conditions. Even with Streptobacterium plantarum, differences in strains
or testing conditions have resulted in failures 67> 68> 69 to duplicate the
results of Auhagen.65 One such result is indicated in Table 3. However,
p-aminobenzoylglutamic acid is indicated in the same table to be con-
siderably more effective than p-aminobenzoic acid for Lactobacillus
arabinosus, but only in preventing the toxicity of low concentrations of
sulfapyridine.69 However, adaptation to grow without p-aminobenzoic
acid occurs under such conditions and may account for the unusual ac-
tivity. At higher concentrations of inhibitor, p-aminobenzoylglutamic
acid becomes relatively ineffective.69

In promoting growth of Lactobacillus arabinosus, p-aminobenzoyl-
glutamic acid is only about 25 per cent as effective as p-aminobenzoic
acid after 20-24 hours' incubation, but approaches the activity of
p-aminobenzoic acid after 64 hours' incubation (77 per cent).69, 70

In preventing the bacteriostasis resulting from the action of sulfanila-
mide on Escherichia coli, Clostridium acetobutylicwm, Streptococcus
pyogenes, Diplococcus pneumoniae Type I, and Lactobacillus arabinosus,
N-(p-aminobenzoyl)-L-glutamic acid is only 0.017, 0.012, 0.25, 0.1 and
5.0 per cent, respectively, as effective as p-aminobenzoic acid.68 The glu-
tamate is only 0.067 and 0.13 per cent as effective as p-aminobenzoic acid
in promoting the growth of Clostridium acetobutylicum and Acetobacter
suboxydans, respectively.68 N-(p-Aminobenzoyl)-L-glutamic acid is also
less active than p-aminobenzoic acid in preventing the toxicity of sulfa-
pyridine for Diplococcus pneumoniae Type III and Streptococcus hemo-
lytics Group A,16 and the toxicity of sulfadiazine for Streptococcus
faecalis Ralston and Streptococcus zymogenes 26 CI.

While the enhanced activity of N-(p-aminobenzoyl)-L-glutamic acid
over p-aminobenzoic acid is not widespread, the results with a single
organism gave an early indication of the involvement of glutamic acid
in the metabolism of p-aminobenzoic acid. With the discovery of p-amino-
benzoic acid as a constituent of folic acid, the presence of glutamic
acid in the folic acid molecule became an obvious possibility.

490 THE BIOCHEMISTRY OF B VITAMINS

p-Aminobenzoylpolyglutamyl Peptide from Yeast. A conjugate of
p-aminobenzoic acid with 10 or 11 L-glutamic acid residues and one un-
known amino acid residue presumably acidic in nature has been isolated
from yeast.75- 76 This conjugate accounts for 20 to 30 per cent of the total
amount of p-aminobenzoic acid occurring in yeast. Approximately 400
mg of the purified peptide was obtained from 50 kg of dried yeast. As
the amino group of p-aminobenzoyl peptide is diazotizable, it appears that
the aromatic acid is conjugated by means of the carboxyl group to a
peptide chain of 10 or 11 L-glutamic acids with presumably a terminal
unidentified amino acid. By analogy with the folic acid group, it seems
probable that the conjugate may have y-glutamyl units in the pep-
tide chain. The ability of this conjugate to prevent the hydrolysis of a
conjugate of folic acid (p. 571) further emphasizes this similarity.

As indicated in Table 3, the conjugate of p-aminobenzoic acid is rela-
tively inert in promoting growth of organisms requiring p-aminobenzoic
acid or in preventing the toxicity of sulfonamides.75

Folic Acid and Related Compounds. The first conclusive indication of
an interrelationship of p-aminobenzoic acid and folic acid was the report
of Miller,77 who indicated that for both normal and resistant strains of
Escherichia coli, sulfonamides at concentrations which do not affect
growth decrease markedly the production of microbiologically active
forms of folic acid. The biosynthesis of biotin is not affected under these
conditions. Earlier work had indicated that p-aminobenzoic acid stimu-
lated growth and increased approximately threefold the formation of
microbiologically active forms of folic acid in mixed culture of bacteria
from the fowl intestine.78 p-Aminobenzoic acid was also reported to en-
hance the synthesis by Mycobacterium tuberculosis of factors with ac-
tivities of vitamin Bi0 and Bn, which are chick factors replaceable by
folic acid.79

N C C— CH2— NH— (/ N>— CO— NH— CH— CH2— CH2— COOH

H2N— C C CH COO

\ / \ s

N N

folic acid

The elucidation of the structure of folic acid (p. 565) made the relation-
ship apparent, since folic acid contains p-aminobenzoic acid in combina-
tion with a pteridine group and glutamic acid. Initially the possibility
that p-aminobenzoic acid functioned solely in the biosynthesis of folic
acid presented itself. This possibility was also indicated by the resistance
to sulfonamide-inhibition of organisms requiring folic acid for growth.80

p-AMINOBENZOIC ACID 491

However, it was observed even before the elucidation of the structure of
folic acid that concentrations of this vitamin corresponding to those
ordinarily required for organisms incapable of folic acid synthesis do
not prevent the toxicity of sulfonamide for a number of organisms.66, 80, 81
Even high concentrations of folic acid do not affect the toxicity of sulfon-
amides for Escherichia coli 66 and many other organisms.66, 80, 81, 82 Fur-
thermore, mutant strains of Escherichia coli 83 and of Neurospora crassa,
which require p-aminobenzoic acid for growth, do not respond to folic
acid. For many organisms which require p-aminobenzoic acid, folic
acid is either not utilized at any concentration or is utilized less effectively
than p-aminobenzoic acid.66 However, Lampen and Jones 71, 72 have
shown that folic acid is more active than p-aminobenzoic acid in support-
ing the growth of Streptococcus faecalis (Ralston), and that it prevents
noncompetitive^ the toxicity of sulfonamides for this organism as indi-
cated in Table 3. The amount of folic acid necessary for reversal of
sulfonamides approximates that necessary for growth of the organisms
in the absence of sulfonamides. The toxicity of sulfonamides for Strep-
tococcus faecalis G1Y2, Streptococcus zymogenes 26 CI, Streptococcus
durans S10, or Streptococcus liquefaciens 815 is similarly reversed by low
concentrations of folic acid.71, 72 For a number of organisms, such as
Lactobacillus arabinosus and Streptobacterium plantarum, the concentra-
tion of folic acid necessary to prevent the toxicity of sulfonamides is
considerably higher than the concentration of p-aminobenzoic acid neces-
sary to stimulate growth of the organisms.69 However, approximately the
same amount of folic acid is required for reversal of any concentration
of sulfonamides.66, 69 The noncompetitive reversal suggests that folic acid
can supply the normal metabolic intermediate involved in the biosyn-
thesis of the appropriate coenzyme, but is considerably less effectively
utilized than p-aminobenzoic acid. The utilization of folic acid for these
organisms cannot be ascribed to its conversion to p-aminobenzoic acid.
p-Aminobenzoic acid, however, is converted by Lactobacillus arabinosus
to microbiologically active forms of folic acid.70 The reversal by folic
acid of the sulfadiazine inhibition of psittacosis virus (Strain 6 BC) in
embryonated eggs (Table 3) is somewhat analogous to the results with
Lactobacillus arabinosus and Streptobacterium plantarum.73, 74 Either
p-aminobenzoic acid or folic acid prevents the chemotherapeutic effect of
sulfathiazole in mice infected with Toxoplasma (RH strain).84

The activity of folic acid under certain conditions in preventing the
toxicity of sulfonamides (p. 471) for Escherichia coli indicates that the
organism has some slight ability to utilize folic acid in the biosynthesis
of the coenzyme involved in production of thymine. However, folic acid
cannot substitute for p-aminobenzoic acid in the biosynthesis of meth-
ionine, purines and serine in Escherichia coli (p. 470) .

492 THE BIOCHEMISTRY OF B VITAMINS

Two different explanations for the various published data interrelating
p-aminobenzoic acid and folic acid are possible. Folic acid can be con-
sidered as differing from the normal metabolic intermediate in the biosyn-
thesis of the coenzyme derived from p-aminobenzoic acid; and organisms
would be expected to have varying abilities to utilize folic acid in forming
the coenzyme. In Escherichia coli, the addition of a number of products
(methionine, purines and serine) might be expected to exert a sparing
action such that the small amount of folic acid utilized by the organism
would be sufficient for growth. This explanation would conform to the
theory that a single coenzyme is derived from both p-aminobenzoic acid
and folic acid.

The other explanation would require that more than one coenzyme is
formed from folic acid and p-aminobenzoic acid. Thus, folic acid would
perform only part of the biochemical functions of p-aminobenzoic acid.
The data which are difficult to explain by such a theory include the ability
of folic acid to prevent completely the toxicity of sulfonamides and to
fulfill the growth requirements in place of p-aminobenzoic acid for cer-
tain organisms. If this second explanation is valid, such data would indi-
cate that a rapid conversion of folic acid to the other coenzymes
occurs by a process resistant to sulfonamides, or that the products of the
functioning of the coenzyme derived from p-aminobenzoic acid but not
folic acid are supplied in the medium of such organisms.

Although published reports do not indicate conclusively whether or not
two or more coenzymes are derived from p-aminobenzoic acid and folic
acid, the low activity of folic acid for many organisms suggests that
other active forms of the vitamin will be found.

The exact biochemical step in the conversion of p-aminobenzoic acid
to folic acid which is prevented by sulfonamides has not been elucidated.
It is interesting that pteroic acid and p-aminobenzoylglutamic acid
reverse the toxicity of sulfonamides competitively, so that neither repre-
sents the product of the inhibited enzyme system. Since both p-aminoben-
zoylglutamic acid and pteroic acid are less effective than p-aminobenzoic
acid over a range of concentrations (Table 3), it appears that the bio-
synthesis of folic acid does not proceed through these compounds as
intermediates. It has been suggested that sulfonamides may prevent a
combination of p-aminobenzoic acid with reductone — a, /3-dihydroxy-
acrolein. If so, this may represent a stage in the biosynthesis of folic acid,
but convincing biochemical evidence has not been presented.85 2-Amino-
4-hydroxypteridine-6-carboxaldehyde has been suggested as a possible
intermediate which is prevented by sulfonamide from combining with
p-aminobenzoic acid.86 Only glucose and p-aminobenzoic acid are essen-
tial, and glutamic acid is stimulatory in the biosynthesis of microbiologi-

p-AMINOBENZOIC ACID 493

cally active forms of folic acid by Streptobacterium plantarum.87 Synthesis
of this factor is inhibited by either sulfanilamide or sulfathiazole, and
the inhibition is prevented competitively by p-aminobenzoic acid.87 The
synthesis of the folic acid group by yeast is also inhibited by sulfona-
mides.ss

The utilization of pteroic acid by Streptococcus faecalis R, which
requires folic acid for growth, is not inhibited by sulfonamides.71

Pteroyl-di-y-glutamylglutamic acid has an action similar to that of
folic acid for organisms which utilize either p-aminobenzoic acid or folic
acid (Table 3). The action of sulfonamides on such organisms is non-
competitively reversed by the triglutamate at a concentration somewhat
higher than that of folic acid essential for a similar response.

Inhibitory Analogues of p-Aminobenzoic Acid

After the discovery of the therapeutic activity of sulfanilamide,8 a
tremendous number of analogous compounds were prepared and tested
as possible chemotherapeutic agents. Actually, many of the more efficient
sulfonamides known today were in common use before the discovery by
Woods1 of the interrelationship of these compounds with p-aminobenzoic
acid. Although not among the first analogues synthesized and tested,
sulfapyridine was the first of the ^-substituted sulfonamides found to

ON— i
H2N-/ \-S02-NH-C

S — CH
sulfapyridine sulfathiazole

be superior to sulfanilamide. Sulfapyridine was particularly outstanding
at the time for its curative effect in pneumonia,89 and was apparently
first prepared by Ewins and Phillips 90 but was prepared by several
others.91 One of the most potent of the sulfonamides, sulfathiazole, was
first reported by Fosbinder and Walter.92 The synthesis of two more very
effective sulfonamides, sulfadiazine and sulfamethyldiazine (sulfamer-
azine), was first reported by Roblin, et aL93

HC CH

32N-f \-S02-NH-C CH H2N-<f V*

sulfadiazine sulfacetamide

Table 4. Sulfanilamide, Sulfathiazole, and Sulfapyridine as Inhibitory Analogues of
p-Aminobenzoic Acid

■ Inhibition Index—

Sulfanil- Sulfa-

Sulfa-

- — Efficiency Ratios"—^
Sulfa- Sulfa-

Supple-

Organism

amide

pyridine

thiazole

pyridine

thiazole

mentary
References

Aerobacter aerogenes

3220b 10°

456 100

72100

Clostridium

acetobulylicum

23000c 13

Corynebacteriu m

diphtheriae

200-1600101

Diplococcus

pneumoniae

1600

ca.102

280
ca.102

110

ca.102

5.7102

15102

36, 101-104

Escherichia coli

33306 10°

450& 100

41& 100

7 4100

Slioo

1, 49, 101,
102, 105-113

Lactobacillus

acidophilus

80006 10°

1336 100

60100

Lactobacillus

arabinosus 17-5

100<* 80

Lactobacillus

pentosus 124-2

200 d 80

Mycobacterium

tuberculosis

16600 h lli
1000c 115

2800 b 1U

1066H4
20c 11S

5.9114

157114
50115

116-118

Neisseria gonorrhoeae

100101

119-121

Neisseria

meningococcus

5-1000* 101

Pasteurella pestis

4.5cll3

45c 113

Proteus vulgaris

4000 b 10°

556 100

73100

110, 122

Pseudomonas

aeruginosa

133306ino

184& 100

73100

Salmonella enteritidis

100123

Salmonella

typhimurium

66506 10°

926 100

72100

Staphylococcus aureus

46606 10°

416moo

536 100

II2100

ggioo

36. 109, m>.

Streptobacterium

plantarum

15Q6 26

636 26

356 26

2.426

4.326

128, 129

Streptococcus hemo-

lytics (Richards)

5000c >

1000c »

36, 49, 119,
130, 131

Aspergillus niger

10^ / 105

132

Neurospora crassa"

15006 24

Saccharomyces

cerevisiae 139

159QM33

1270° 133

540 5 133

1.3133

2 9133 134-136

Polytomella caeca

3.7105

Strigomonas oncopelti

210,000c 137

Trichophyton

purpureum

400c 138

Rice seedings

4 ca.139

Tomato roots

35-506
ca.140

27-36 b
ca.140

17*
ca.140

1-2"°

2-3140

Wheat seedings

8 ca.14

Other organisms for which the toxicity of sulfonamides in vitro is prevented by p-aminobenzoic acid
include: Bacillus subtilis;1*2 Brucella paramelitensis;im Eberthella typhosa;nl Proteus friedlanderi;110 Pseu-
domonas pyocyanea;li2 Salmonella paratyphi;111 Salmonella schottmuelleri;'" Shigella dysenteria;m Botrytis allii
Munn;ul Fusarium caeruleum (Lib.) Sacc.;141 Penicillium diaitatum Sae<\;'» t'hlordla (pigmented and non-
pigmented)143, l44; Nitzschia polea var. debilis (fresh water diatom)145, 146' »7; Flax seed;148 Onion rootlets;149
Pea roots160' 151 and shoots.151

0 Ratio of the activity of the sulfonamide to sulfanilamide.100

b For half-maximum growth.

e Molecular equivalents of sulfonamide neutralized by one molecular equivalent of p-aminobenzoic acid.

d In the absence of purines.

• Varies with different strains.
/ AtpH 7.1, 2000 at pH 3.7.

9 Either p-aminobenzoicless strains 1663 or 5359 or parent strain.

* Inhibition index depends on strain. Mitis, intermedia*, and gravis strains are susceptible.

p-AMINOBENZOIC ACID

495

Sulfacetamide (albucid) ,94 because of low toxicity, is useful in urinary-
infections. Sulfaguanidine,95, °6 succinylsulfathiazole,97, 9S and phthalyl-
sulfathiazole °7, 9S have been synthesized and, being sparingly absorbed
from the bowel, are useful in treatment of intestinal infections.

Table 5. Sulfonamides, Sulfones, Sulfoxides and Related Inhibitory Analogues of
p-Aminobenzoic Acid

Supplemen-
Inhibition Efficiency tary

Index Ratio0 References

Compound

Sulfadiazine

Sulfacetamide

Sulfaguanidine

Sulfanilic acid

bis(4-Amino-
phenyl)sulfone

Organism

Escherichia coli
Staphylococcus aureus
Mycobacterium tuberculosis
Pasteurella pestis
Proteus vulgaris
Escherichia coli
Staphylococcus aureus
Streptococcus hemolyticus B
Neisseria gonorrhoeae
Escherichia coli
Staphylococcus aureus
Streptococcus hemolyticus B
Saccharomyces cerevisiae 139
Streptococcus hemolyticus
Escherichia coli

Streptobacterium plantarum 10 S
Staphylococcus aureiis

Escherichia coli
Mycobacterium tuberculosis
Streptobacterium plantarum 10 S
Streptococcus hemolyticus B

Other sulfonamides, sulfones, sulfoxides and related compounds the toxicity of which is prevented by
p-aminobenzoic acid include: Nl-arylsulfanilamides: N'-phenyl-,130 N'-o-tolyl-,130 N'-o-chlorophenyl-,130
N^o-hydroxyphenyl-,130 N^p-hydroxyphenyl-,130 N'-p-aminophenyl-,130 N'-p-nitrophenyl-,127 and Nl-m-
carboxyphenylsulfanilamides,130 and N'.N'-dimethyl-l-sulfanilamidobenzenesulfonamide.127, 129' 13° N1-
Heterocyclicsulfanilamides: 2-sulfanilamido derivatives of pyrimidine,151 4-methylpyrimidine,130 5-chloro-
pyrimidine,d 156 6-bromopyrimidine,<i 156 4-amino-5-bromopyrimidine,156 5-bromo-4-methylpyrimidine,156
5-bromo-4,6-dimethylpyrimidine,156 5-(2,3-dibromopropyl)-4, 6-dimethylpyrimidine,156 4-methylthia-
zole,127' 130 4-phenyl-5-methylthiazole,127, 13° 5-bromothiazole,159 5-chlorothiazole,156 5-bromo-4-methyl-
thiazole,156 5-chloro-4-methylthiazole,156 5-pyridinesulfonamide,127' l3° 4-methyldiazine,110 4,6-dimethyl-
diazine,110 5-chloropyridine,156 thiazoline,127, 130 5-methylthiadiazole,121, m 5-ethylthiadiazole,121' 131
5-7i-propylthiadiazole,121 5-isopropylthiadiazole,121 and 5-isobutylthiadiazole121 and 2-(2-chlorosulfanila-
mido)pyrimidine156 and 3,4-dimethyl-5-sulfanilamidoiso-oxazole.157 Nl-Acylsulfanilamides and miscellaneous
sulfonamides: N4-sulfoxymethylsulfanilamide (sodium salt),130 N'-3,4-dimethylbenzoylsulfanilamide,158
sulfanilamidoacetic acid,130 p-hydroxylaminobenzenesulfonamide.101 Sulfones and sulfoxides: 2-amino-
phenyl 4-aminophenyl sulfone,154 bis(2-aminophenyl)sulfone,164 bis(3-aminophenyl) sulfone,154 4-ace-
tylaminophenyl 4-nitrophenyl sulfone,154 4-aminophenyl 4-hydroxyphenyl sulfone,49 4-aminophenyl
4-benzylidineaminophenyl sulfone,154 4-aminophenyl 5-amino-2-pyridyl sulfone,'59 promin,155 bis(4-amino-
phenyi) sulfoxide,149, I54 bis(4-acetamidophenyl)sulfoxide,154 4-aminophenyl phenyl sulfoxide,154 4-amino-
phenyl 4-nitrophenyl sulfoxide,49' 154 4-acetamidophenyl 4-nitrophenyl sulfone49, 154 4-aminophenyl 4-chlo-
rophenyl sulfone,154 4-aminophenyl 4-iodophenyl sulfone,154 4-iodophenyl 4-nitrophenyl sulfone.154
Miscellaneous Analogues: p-aminophenyldimethylsulfonium /3-naphthalenesulfonate,160 bis(4-aminophenyl)
disulfide,109 bis(4-aminophenyl)diselenide.d109

° Ratio of the activity of the sulfonamide to that of sulfanilamide.100

6 For half-maximum growth.

0 Molecular equivalents of sulfonamide neutralized by one molecular equivalent of p-aminobenzoic acid.

d Toxicity of these compounds only partially reversed by p-aminobenzoic acid.

43b 100

78ioo

106, 108, 110

92 b 100

51ioo

124, 125

1436114

1161U

4.5«ui

50110

5346 100

6ioo

5346 100

9100

800c 130

2.9130

121

39606 10°

0.84100

108

457O6 100

1.0100

2500c 13°

0.59130

12806 133

1.2»

15,000c *

0.331

130

l,000e112

0.1112

5,000626

0.0326

129

10 ca.162

153

1390c 108

2.0108

3326H4

50114

154, 155

286 26

5.326

129

249c 130

6.6130

The remarkable chemotherapeutic activity of this group of compounds
has stimulated the preparation of thousands of compounds somewhat
related in structure to sulfanilamide. An excellent monograph by
Northey " on sulfonamides and related compounds includes comprehen-

496 THE BIOCHEMISTRY OF B VITAMINS

sive lists of these compounds together with their activities. Consequently,
only these compounds which have been studied with respect to their rela-
tionship to p-aminobenzoic acid are included in this monograph.

Sulfonamides, Sulfones and Related Analogues: Activity and Reversals
with p-Aminobenzoic Acid in Vitro

After the appearance of the report of Woods 1 indicating the competi-
tive interrelationship of p-aminobenzoic acid and the sulfonamide drugs,
this effect was confirmed by many others, and the effect of p-aminoben-
zoic acid on the toxicity of related inhibitory analogues was determined
for a wide variety of organisms. The organisms for which the toxicity of
sulfanilamide, sulfathiazole or sulfapyridine in vitro is prevented by
p-aminobenzoic acid are indicated in Table 4. It is apparent that numer-
ous species of bacteria, fungi, higher plants, diatoms, yeast and flagellates
are inhibited by these sulfonamides, and the inhibition is prevented
competitively by p-aminobenzoic acid. Similar results have been obtained
with a large number of N1-substituted sulfanilamides, sulfones, sulfoxides
and similar compounds structurally related to p-aminobenzoic acid
(Table 5).

As indicated in these tables, the inhibition indices of an individual
inhibitor vary considerably for different organisms. Also, organisms
which require folic acid for growth are highly resistant to inhibition by
any of the analogues of p-aminobenzoic acid. However, the relative
efficiencies of the different sulfonamides in inhibiting the growth of various
organisms are rather consistent, and it has been suggested that there is
no specificity in the ability of sulfonamides to inhibit specific organ-
isms.100 While this is true for most organisms, there are many exceptions.
For example, sulfathiazole and sulfapyridine are only slightly more
effective for Streptobacterium plantarum than sulfanilamide, and bis-
(4-aminophenyl)sulfone, which is only slightly more effective for
Escherichia coli than sulfanilamide, is fifty times as efficient as sulfanila-
mide for Mycobacterium tuberculosis. The sulfone is more active than

NHr-/ Y-SO2-/ ^>-NH2

bis (4-aminophenyl) sulfone

sulfathiazole for Streptobacterium plantarum, while sulfathiazole is con-
siderably more effective than the sulfone against Escherichia coli. Even
though the order of the relative activities of sulfanilamide, sulfapyridine
and sulfathiazole is the same for essentially all organisms, there is little
difference in their inhibitory ability against organisms such as Saccha-

p-AMINOBENZOIC ACID 497

romyces cerevisiae, tomato roots and Streptobacterium plantarum. The
order of effectiveness in some cases depends upon the experimental
method, since the dose-response curves for the inhibitory action of the
various sulfonamides are not identical under different testing conditions.
For example, the growth of the fresh-water diatom, Nitzschia palea var.
debilis, is inhibited by sulfanilamide, sulfapyridine and sulfathiazole,
with decreasing effectiveness in the order named; however, p-aminoben-
zoic acid increases in effectiveness in preventing the toxicities of the
sulfonamides in the reverse of the order named.145-147 A similar situation
exists with Saccharomyces cerevisiae 133 and with the nonpigmented alga,
chlorella.143 At low concentrations of p-aminobenzoic acid, sulfapyridine
is less effective for Saccharomyces cerevisiae than sulfanilamide, but at
higher concentrations of p-aminobenzoic acid such that considerable
growth occurs, sulfapyridine is more inhibitory to the organism than is
sulfanilamide.133 Differences in the shape of dose-response curves appear
to account for these unusual effects.

The sulfonamides, sulfones, sulfoxides, etc., which inhibit the utilization
of p-aminobenzoic acid usually possess a free amino group in the position
para to the sulfur-containing substituent. In a few cases, inhibitory activ-
ity has been reported for compounds in which the amino group is replaced
by groups, such as nitro, acetylamino, alkylamino, glycosidoamino, etc.,
which presumably may be converted into a free amino group in the in-
hibited biological system. Usually these compounds are less active than
the analogous compound with the free amino group. Also substitution of
the aromatic ring of this series of sulfur analogues of p-aminobenzoic acid
usually results in a decrease or complete loss of inhibitory activity.

The most effective modifications of sulfanilamide involve N^sub-
stituents. In general the inhibitory activity is decreased if the substituent
is an alkyl or cycloalkyl group, but is usually increased if the substituent
is an aromatic heterocyclic group. Similarly, the most effective sulfones
and sulfoxides contain an aromatic group in conjunction with a p-amino-
phenyl substituent.

In contrast to the unusual activity of N-(p-aminobenzoyl)-L-glutamic
acid compared with p-aminobenzoic acid in preventing the toxicity of
sulfonamides for certain organisms under specific conditions (p. 487),
the corresponding sulfonamide, N-sulfanilyl-L-glutamic acid not only
does not have increased inhibitory power but is relatively inactive.67, 1C1

Reversals with p-Aminobenzoic Acid in Vivo

Shortly after the preliminary report of Woods and Fildes1 concerning
the ability of p-aminobenzoic acid to prevent the inhibitory action of
sulfonamides, Selbie 1G2 found that the therapeutic activity of sulfanila-

498

THE BIOCHEMISTRY OF B VITAMINS

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p-AMINOBENZOIC ACID 499

mide against infections of Streptococcus hemolyticus in mice was also
antagonized by p-aminobenzoic acid. These findings were rapidly con-
firmed with this and other organisms for a wide variety of sulfonamides,
sulfones and sulfoxides as indicated in Table 6. This in vivo testing
allowed extensions of the interrelationship of p-aminobenzoic acid and
the sulfonamide drugs to pathogenic protozoa such as Toxoplasma, Plas-
modium lophurae and Plasmodium gallinaceum, and to such viruses as <
psittacosis and the virus of lymphogranuloma venereum.

Because p-aminobenzoic acid is rapidly converted into inactive forms,
e.g., conjugates in animals, and is rapidly excreted in comparison to
sulfonamides and related compounds, the amount of the factor necessary
to prevent the therapeutic action of the sulfonamide drugs is often high
in comparison with amounts required for in vitro testing.167, 1T4 However,
if the concentration of p-aminobenzoic acid at the site of the infection
is compared with the corresponding concentration of sulfonamide, results
comparable with those for in vitro testing are obtained.174 Failure to
make such comparisons has resulted in conflicting reports in some cases.

The specificity of particular sulfonamide drugs for certain organisms
appears to be greater in vivo than in vitro. Of a group of 33 sulfanilamide
derivatives, including the N1 -heterocyclic sulfanilamides, none was found
to have significantly greater therapeutic activity than sulfanilamide
against Streptococcus hemolyticus C-203 infections in mice. However, the
^-heterocyclic sulfanilamides were more effective than sulfanilamide
against infections of Diplococcus pneumoniae.175 Of a group of N1-acyl-
sulfanilamides, a few were found to be effective against Streptococcus
hemolyticus, Diplococcus pneumoniae and Escherichia coli infections in
mice ; however, some of the compounds were effective only against Strep-
tococcus hemolyticus and Escherichia coli, while another was effective
only against Escherichia coli and not against the other two.176 Conse-
quently, there appears to be a higher degree of specificity of chemothera-
peutic activity in vivo than in vitro.

Sulfonamides, sulfones and related analogues of p-aminobenzoic acid
are reported to prevent the effects of endotoxins of microorganisms.177
Although some attempts to verify the early work failed,178, 179 the effects
of sulfonamides and related compounds on the action of endotoxins of
certain bacteria have been verified. 1S0"183 Sulfanilamide allows a signifi-
cant increase in the number of mice surviving injection of the endotoxins
of Salmonella typhimurium without affecting the process of immuniza-
tion.182- 183 The therapeutic effect of sulfanilamide was prevented by ad-
ministration of p-aminobenzoic acid.183 The effect of 4-aminophenyl
4-nitrophenyl sulfoxide in protecting 40 to 80 per cent of the mice from
a lethal dose of typhoid endotoxin is antagonized by injected p-amino-

500 THE BIOCHEMISTRY OF B VITAMINS

benzoic acid.184 The treatment with the sulfoxide does not affect the
immunization of the animal against the endotoxin or the organism.184
Similar results were obtained with endotoxin from a particularly virulent
strain of Escherichia coli.18*

Inhibitions by Sulfonamides Unaffected by p-Aminobenzoic Acid

Sulfapyrazine, sulfadiazine and sulfathiazole inhibit completely the
growth of Bacterium tularense, but p-aminobenzoic acid does not affect
the inhibition.113 Although such cases are unusual for these analogues,
there are a number of sulfonamide derivatives related structurally to
p-aminobenzoic acid which are inhibitory, but the inhibition is not re-
versed by p-aminobenzoic acid. The toxicity of 2-, 3-, 5- and 7-sulfanila-
midoindazoles for Brucella melitensis is only slightly reversed by
p-aminobenzoic acid.1S5 3',5'-Dibromosulfanilanilide is strongly inhibitory
to numerous strains of pneumococci, hemolytic streptococci and staphy-
lococci, but is only slightly inhibitory to Friedlanders' bacillus, Escher-
ichia coli, Pseudomonas aeruginosa and various types of dysentery bacilli.
However, the strong inhibition observed with the gram-positive cocci is
not prevented by p-aminobenzoic acid, but this vitamin reverses the
slight inhibition obtained with the gram-negative bacilli.186 Similarly,
the toxicity of a series of 3',4'- and 3',5'-halogenosulfanilanilides is also
unaffected by p-aminobenzoic acid for certain organisms.187, 188 The tox-
icity of p-aminomethylbenzenesulfonamide is not affected by p-amino-
benzoic acid or by p-aminomethylbenzoic acid for a wide variety of
organisms.189, 190

For some organisms the toxicity of sulfanilamide is only partially
counteracted by p-aminobenzoic acid. This is true of onion rootlets, for
which p-aminobenzoic acid is toxic at higher concentrations.149 Although
very effective at low concentrations in preventing the toxicity of sul-
fanilamide, p-aminobenzoic acid is toxic at higher concentrations for pea
roots.150 A similar situation exists with flax seed, which germinates slowly
or not at all in the presence of relatively high concentrations of sulfanil-
amide, and the growth of the seedlings is retarded by lower concentra-
tions of the drug. Although p-aminobenzoic acid counteracts the inhib-
itory action, root development is not quite restored to normal. Higher
concentrations of p-aminobenzoic acid are toxic.148 For Lupinus albus
seedlings 191 p-aminobenzoic acid enhances the toxicity of sulfanilamide.

In systems in which the toxicity of sulfonamides or related compounds
is not affected by p-aminobenzoic acid, it appears that in most instances
enzymatic reactions other than those concerned with the utilization of
p-aminobenzoic acid are involved. This does not preclude the possibility
that in some instances a sequence of two reactions is prevented by the

p-AMINOBENZOIC ACID 501

analogue or that the analogue combines irreversibly with the enzyme,
or that some other such phenomenon prevents a competitive reversal by
p-aminobenzoic acid.

Mechanism of Action of Sulfonamides and Correlation of Activities with
Physical Properties and Structure

Attempts to correlate the inhibitory activities of the N1-substituted
sulfonamides and related compounds with structure or some physical
property of the compounds have resulted in several different theories as
to the mode of action of these substances. However, the theory of com-
petitive inhibition of the utilization of p-aminobenzoic acid as an essen-
tial metabolite1 explains more effectively the data which have accumu-
lated.

Application of the Michaelis-Menten equations adapted to the rate of
an inhibited reaction was made by Wyss 192 to demonstrate the com-
petitive nature of the relationship between p-aminobenzoic acid and
sulfanilamide for Escherichia coli (p. 455) . A similar treatment was ap-
plied to the inhibition index by Wood, who suggested that the variations
in the bacteriostatic activity of the different sulfonamides might be the
result of differences in affinities for the enzyme involved in the function-
ing of p-aminobenzoic acid.108

Sulfanilamide does not displace any appreciable amount of p-amino-
benzoic acid from cells of Streptococcus hemolyticus,193 and there is no
appreciable binding of sulfanilamide labelled with radioactive sulfur in
the cells of Escherichia coli.ld4 Thus, p-aminobenzoic acid appears to be
converted into a coenzyme form, and very little of the total p-amino-
benzoic acid of cells exists in a combination from which it is displaced by
sulfonamide. This is further indicated by the observation that bacteria
are capable of undergoing a definite, limited number (six or seven) of
cell divisions in the presence of any effective drug concentration, re-
gardless of the inoculum employed.195, 19C After inhibition is obtained,
p-aminobenzoic acid is reported to exert its effect immediately under
certain conditions.158* m

Correlation of Activities with Ionization of Sulfonamides. The influence
of pH on the inhibitory activity of sulfonamides and on the ability of
p-aminobenzoic acid to prevent the toxicity of these drugs was first indi-
cated by Lwoff and co-workers.198 The amount of sulfanilamide necessary
to prevent the reproduction of the flagellate, Polytomella caeca, was five
times greater at pH values below 3.1 than at values above 5.5. The
change in inhibitory activity of sulfanilamide appeared to occur only
between these two points. The ratio of sulfanilamide to p-aminobenzoic
acid at which complete reversal of the inhibition is obtained increases

502 THE BIOCHEMISTRY OF B VITAMINS

from 1 at a pH range of 9.2-7.55 to 1,200 at pH 3.65, but then decreases
to 380 at pH 2.25. These values corrected for the relative activity of
sulfanilamide give 1, 245 and 76, respectively, for the relative activities
of p-aminobenzoic acid. Since the point of maximum activity corresponds
closely to the isoelectric point of p-aminobenzoic acid, it was proposed
that the undissociated molecules penetrate into the cell at a greater rate
than the ions.

For Escherichia coli and Aspergillus niger, sulfanilamide was less
effective in acidic than in neutral media; however, the variation with
Escherichia coli was very slight and only fivefold for Aspergillus niger.198
Consequently, any theory must account for such small variations.

Some degree of correlation of the inhibitory activity of sulfonamides
with their ability to dissociate to sulfonamide ions was subsequently
noted.199- 200 The ratios of the concentration of sulfanilamide, sulfa-
pyridine, sulfathiazole or sulfadiazine to that of p-aminobenzoic acid at
which inhibition of growth of Escherichia coli becomes apparent are ap-
proximately 500, 40, 8 and 8, respectively, at pH 7. However, these ratios
calculated on the basis of the ratio of sulfonamide anion to p-amino-
benzoate, are 1.4, 1.4, 4.9 and 6.4, respectively.199 Approximately eight
times as much sulfanilamide is required to inhibit the growth of Esche-
richia coli at pH 6.8 than at pH 7.8. 199 At pH 9, there is comparatively
little difference in the activity of sulfathiazole and sulfanilamide.200 Since
an increase in the pH of the culture medium favors ionization of the
sulfonamides, the increased effectiveness of sulfonamides with increased
pH of the medium and the similar activities of certain sulfonamide ions
led to the consideration that the anion is the active form of the ionizable
sulfonamides.199' 200

Subsequently, it was observed that the activity of the sulfonamides
increased with pH of the culture medium only up to the point where
ionization is approximately half complete.124- 201 Actual decreases in
bacteriostatic activity are observed with highly ionized sulfonamides
such as sulfadiazine, when the pH is increased above the point of 50 per
cent ionization. Thus, when the pH of the medium exceeds the pKa of
the sulfonamide, the activity begins to decline. For sulfadiazine, an
increase of approximately 8.8 fold in concentration is required for bac-
teriostasis when the pH is increased from 6.5, where the sulfonamide is
approximately 50 per cent ionized, to 8.9 where the sulfadiazine is ap-
proximately 99.6 per cent ionized.

These results led to the suggestion that only the molecular form pene-
trated the cell wall of bacteria while only the ionic form combines with
the enzyme which is inhibited.201

The decreasing ability of p-aminobenzoic acid (pKa4.68) to prevent

p-AMINOBENZOIC ACID

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504 THE BIOCHEMISTRY OF B VITAMINS

the toxicity of sulfonamide with increasing pH of the medium 124> 198 is
parallelled by growth studies with a mutant strain of Neurospora crassa
which requires considerably less p-aminobenzoic acid in media at pH 4
than at higher pH levels.202

The effects of pH on the activities of several sulfonamides, including
some which do not ionize, are indicated in Table 7. These data were
derived by North ey " from original data supplied by Wyss. Escherichia
coli was used for the data below pH 7 and Streptococcus faecalis in tests
above pH 7. At pH 7 the results with both organisms agreed so closely
that only an average of the results was indicated. The inhibition indices
are expressed in terms of the concentration of the sulfonamide to
p-aminobenzoic acid, the ratio of the molecular forms of the substances in
the medium, and the ratio of the ionic forms in the medium.

If only the molecular forms penetrate to the site of action, and if the
pH within the cell is assumed to be constant over the pH range indicated,
the ratio of molecular forms of sulfonamide to p-aminobenzoic acid in
the medium would be proportional to the corresponding ratio of ionic
forms within the cell. Since this ratio is not constant for the inhibition,
it does not appear that the relative activity of sulfonamides can be ex-
plained solely on the basis of greater permeability of the molecular form
and greater activity of the ionic form. However, the pH inside the cell
may be affected more by the pH of the medium than is generally realized.

From the data presented in Table 7, it is apparent that the sulfonamides
are usually most effective at a pH almost equal to their pKa, that at any
given pH the most active sulfonamide is usually the one with a pKa ap-
proximating that pH, and that non-ionic sulfonamides increase in ac-
tivity with increases in pH over the range indicated. Similar results for
changes in the activity of sulfonamides with changing pH of the medium
have been reported for Mycobacterium tuberculosis.203

On the assumptions that (1) only the molecular form of sulfonamides
penetrate the cell wall, (2) only the ionic form of sulfonamides acting
within the cell inhibit the biological system, (3) that all sulfonamide
ions have equal inhibitory activity within the cell, and (4) that the pH
within the cells of Escherichia coli is 6, Northey " derived an empirical
equation relating activity of a sulfonamide to the fraction ionized in the
medium and within the cell. Thus,

log l/CR = \og Xi (1-XO +7.2573

where CR is the minimum molar concentration producing the inhibition
in medium at pH 7, Xi is the fraction of drug ionized within the cell, X0
is the fraction ionized in the medium, and the last figure is — log k, where

p-AMINOBENZOIC ACID

505

k is a proportionality constant derived empirically from the experimental
maximum activity of sulfonamides at pH 7, as indicated in Figure 7.

The curve for this expression when plotted as indicated in Figure 7
agrees closely with the experimental curve for Escherichia coli obtained
from the data given in Table 8. Similar experimental data are obtained
with other organisms, as indicated in Table 8. This theory, of course,

+• /of

°\\
\°

°/ /
/•/

\ \n

//
//

\
\
\

/
//

\
\

pKa

Figure 7. Experimental and theoretical relationships of activity of sulfonamides to
acid dissociation constants.

Experimental curve.204

_._._._. Theoretical (Negativity of S02 group).204
Theoretical (Ionization).99

does not account for the high activity of certain sulfonamides which do
not ionize.

Correlation with Negativity of the S02 Group. Bell and Roblin 204
have presented a theory correlating the negativity of the S02 group of
ISP-substituted sulfonamides with their ability to prevent the growth of
Escherichia coli. According to the theory, the more negative the S02 group
of an N^substituted sulfanilamide derivative, the greater its bacterio-
static power. Since p-aminobenzoic acid is more than 99 per cent ionized
in a medium buffered at pH 7, the first formula of Figure 8 indicates the
main form in which the vitamin exists in the medium and also within

506

THE BIOCHEMISTRY OF B VITAMINS

Table 8. Dissociation Constants and Bacteriostatic Activity of N-Substituted Sulfon-
amides and Related Compounds

Compound

p-Aminobenzoic acid

NMSulfanilylsulf anilamide

Sulfanilylcyanamide

N'-Ethylsulfonylsulfanilamide

Sulfanilylglycine

N'-Chloroacetylsulfanilamide

3-Sulfanilamido-4-methylfurazan

5-Sulfanilamido-3-methylisooxazole

3-Sulfanilamido-5-methyloxadiazole

IN^-Benzoylsulf anilamide

4-Sulfanilamido-l,2,4-triazole

2-Sulfanilamido-l,3,4-thiadiazole

N^p-Aminobenzoylsulfanilamide

N^-Acetylsulfanilamide

Sulfanilylurea

2-Sulfanilamido-5-methylthiadiazole

5-Sulfanilamido-2-chloropyrimidine

2-Sulfanilamidopyrazine

4-Sulfanilamidopyrimidine

Sulfadiazine

2-Sulfanilamidooxazole

5-Sulfanilamidopyrimidine

3-Sulfanilamidopyridazine

2-Sulfanilamido-4-methylpyrimidine

5-Sulfanilamido-2-bromopyridine

Sulfathiazole

2-Sulfanilamido-5-bromopyridine

2-Sulfanilamido-4,6-dimethylpyrimidine

2-Sulfanilamido- 4-methylthiazole

NMSulfanilylsulfanilamide

3-Sulfanilamidopyridine

4-Sulfanilamidopyridine

N3-Sulfanilylmetanilamide

Sulfapyridine

2-Sulfanilamido-5-aminopyridine

5-Sulfanilamido-2-aminopyridine

2-Sulfamilamido-4-aminopyrimidine

N^-Phenylsulfanilamide

2-Sulfanilamidoimidazole

N^m-Tolylsulfanilamide

N'-p-Tolylsulfanilamide

N^o-Tolylsulf anilamide

N^-p-Aminophenylsulfanilamide

Sulfanilamide

N^Methylsulfanilamide

N '-Furf urylsulf anilamide

N^Hydroxyethylsulf anilamide

N \ N LD imethylsulf anilamide

Sulfanilylguanidine

Sulfanilylaminoguanidine

4,4'-Diaminodiphenylsulfone

Minimum

Inhibitory Concentration X 105

Mycobacterium

tuberculosis

Streptococcus

Escherichia

var. hominis 607 hemolyticus B

pKa2M

coli™

114

130

4.68

2.89

60.0

2.92

100

3.10

1000

3.52

>90.0

3.79

10.0

4.10

1.0

4.2

0.6

4.40

2.0

6.0

4.57

0.3

4.66

>80.0

4.77

0.6

5.20

0.5

5.38

0.7

1.56

5.42

10.0

5.45

0.2

0.28

5.80

0.1

6.04

0.08

0.15

6.17

0.1

6.48

0.08

0.15

1.03

6.5

0.08

6.62

0.2

7.06

0.08

7.06

0.2

7.12

0.2

7.12

0.08

0.15

7.15

0.5

7.37

0.3

7.79

0.2

0.34

7.85

0.5

7.89

0.2

8.00

2.5

8.23

2.0

8.43

0.6

1.2

1.22

8.47

0.6

4.7

8.82

2.0

9.44

20.0

9.60

3.0

2.22

9.72

40.0

9.74

5.0

9.82

5.0

9.96

10.0

3.57

10.22

5.0

1.72

10.43

20.0

4.54

10.77

30.0

10.88

20.0

10.92

50.0

30.0

10.0

7.69

0.9

2.0

0.62

0.69

p-AMINOBENZOIC ACID

507

the cell of the organism which has a buffering capacity presumably in
the same pH range. The molecular and ionized forms of ^-substituted
sulfonamides are also indicated in Figure 8. From the standpoint of geo-
metrical considerations, the p-aminobenzenesulfonyl group and the
p-aminobenzoate ion are very similar, the bond distances differing only
slightly, as indicated in Figure 8. Sulfones likewise are similar to the
p-aminobenzoate ion.

6.7 A.

H H

\ /

N
1

()

|<-2.3 A-»|
y-Aminobenzoate ion
Figure 8

/ \l

t S

/ \

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I-*— 2.4 A-»|

Sulfonamide

Sulfonamide ion

The Structures of the p-Aminobenzoate Ion and Molecular and
Ionic Forms of Sulfonamides.

If the substituent group, R, of the ^-substituted sulfonamide func-
tions solely in affecting the combining power of the p-aminobenzenesul-
fonyl group with the enzyme and the substituent within itself does not
possess groups which aid in this combining power, the activity of the
sulfonamide will depend upon the effect of the substituent group on the
combining power of the reactive groups, presumably the basic amino
group and the S02 group. Since only slight variations occur in the ioniza-
tion constants for the basic amino group of the ^-substituted sulfon-
amides, differences in combining power of these sulfonamides with the
inhibited enzyme cannot be attributed to the reactivity of the basic
amino group, and no relationship between the bacteriostatic activity and
these constants has been noted for these analogues. However, this may
become an important consideration for other analogues.

The acid dissociation constants of ^-substituted sulfonamides vary
over a wide range, indicating that the properties of the sulfonamide
group are influenced greatly by the N1 substituent.

Since the p-aminobenzoate ion possesses an electronic charge which
greatly increases the negative character of the C02" group, Bell and
Roblin postulate that this negative character of the C02" increases the
affinity with which the molecule combines with the enzyme, and that the
more negative the S02 group in the sulfonamides, the greater the ability

508 THE BIOCHEMISTRY OF B VITAMINS

of the sulfonamide to compete with the p-aminobenzoate ion for the
enzyme.

The ionic form of a sulfonamide has an electronic charge on the amide
nitrogen. Since the S02 group is electron-attracting, the charge on the
adjacent atom is shared. This results in a more negative S02 group in
the ionized sulfonamide as compared with the corresponding group in the
un-ionized form. Consequently, the ionized form of the sulfonamide
would be expected by this theory to be considerably more active than the
molecular form.

Sulfanilamide (pKa3.7x 10-11) at pH 7 is only slightly ionized. Sub-
stitution of an electron-attracting group at the N1 position allows the
hydrogen on this nitrogen to escape as a proton much more easily, since
the electron density of the nitrogen atom is decreased by the electron-
attracting group. As the electron-attracting power of the N1 substituent
increases, the degree of ionization of the sulfonamide increases.

However, as the electron-attracting power of the N1 substituent in-
creases, the S02 group becomes less negative, since the two groups com-
pete for the electrons surrounding the nitrogen. Conversely, as the electron
donating power of the N1 substituent increases, the S02 group becomes
more negative; however, simultaneously the degree of ionization de-
creases. These two opposing effects on activity of the sulfonamide would
be expected to result in a maximum activity at a definite pKa value,
according to the theory.

With the assumption that the bacteriostatic activity of ^-substituted
sulfonamides is proportional to the potential of the S02 group and that
the potential of the S02 group is influenced by the inductive effect of
the N1 substituent, Bell and Roblin derived equations relating the bio-
logical activity of sulfonamide ion with the inductive constant of the
N1 substituent. Thus,

log (k/xC R) = a(12.3- / Ra)- 1 Ra2 = 4.39 -0.255/ R

Similarly, the biological activity of the molecular form was related to the
inductive constant. Thus,

\og(k/a-x)CR) = a(-1.3-IRa)-IRa2= -0.464-0.255 IR

where k is a proportionality constant, determined experimentally to be
0.001, x is the fraction of the compound in the ionized state, CR is the
minimum molar concentration of the compound required for bacterio-
stasis, IB is the inductive constant of the N1 substituent, and a is the
fraction of the inductive effect transmitted across each bond and is taken
as 1/2.8, the value of Branch and Calvin for a covalent bond.
From the above equations, the logarithm of the ratio of the activity of

p-AMINOBENZOIC ACID 509

the molecular form to the ionized form is 4.85, which means that the ion
is approximately 104-85 times as active as the corresponding molecular
form. This seems to be somewhat high when the activities of nonionizing
analogues are compared to similar ionizing forms.

The inductive constants of Branch and Calvin for various radicals are,
according to Bell and Roblin,204 a linear function of the pKa values of
the corresponding sulfanilamides with the radicals substituted in the N1
position.

J* = -1.33 p#„+ 13.88

With these three equations, the activity of a sulfonamide could be
determined from the ionization constant. In Figure 7, theoretical activi-
ties for the ionic and molecular forms at pH 7 are plotted against pKa
and compared with the experimental activities. The point of maximum
activity occurs at a pKa value of 6.7, which agrees very well with the
experimentally determined maximum activity. The pKa of the most active
sulfonamide varies with the pH, since the fraction ionized changes for
the various sulfonamides with changes in pH.

The theoretical considerations applicable to the data with one organism,
Escherichia coli in this instance, are not necessarily applicable to other
organisms, since the combining power of the sulfonamide group may not
in all cases be the limiting factor for interaction with the enzyme, and
the enzyme may in itself differ to some extent from organism to organism.

Correlation With Ionization Constant. With the assumptions (1) that
only the sulfonamide ion combines with the enzyme; (2) that a constant
quantity of enzyme must be combined with the sulfonamide for inhibi-
tion of growth of Escherichia coli to occur; (3) that the dissociation
constant for the enzyme-sulfonamide ion complex is a function of the
ionization constant of the sulfonamide; and (4) that a maximum exists
for the activity of sulfonamides correlated with ionization constant, an
equation derived from the equilibrium constants by Klotz 205 indicates
that at this maximum, the logarithm of the dissociation constant of the
enzyme-sulfonamide ion complex is a linear function of the logarithm of
the ionization constant of the sulfonamide. Thus,

dlnKp [H+]
dlnKa K°a + [H+] J

where Kp represents the dissociation constant of the enzyme-sulfonamide
complex, K is the ionization constant of the sulfonamide, and K° is the

r ' a 'a

ionization constant of the most effective sulfonamide at the hydrogen
ion concentration, [H+]. Since the ratio of d In Kp to d In Ka is con-

510 THE BIOCHEMISTRY OF B VITAMINS

stant under the assumed conditions over a range of Ka values at a con-
stant[H+] , K = kKf , where k is an integration constant. Substitution of
kKf for K in an expression relating the ionization constants of sulfona-
mide and dissociation constants of enzyme-drug complexes resulted in an
equation relating the activity of the sulfonamides to their ionization
constants. However, the evaluation. of k and / is dependent upon experi-
mental data. Since the assumptions used in obtaining the equation are
the theoretical considerations for which proof is desirable in advance of
data, the equation has little to offer other than an empirical expression
of the interrelationship of pKa and sulfonamide activity. The final ex-
pression is very similar to the one derived by Bell and Roblin.204

The assumption that a maximum exists in the activity for variable pKa
values is based on the theory that the basic ionic form of the sulfonamide
combines with the enzyme which acts as an acid.206 With increasing
ionization constants, the sulfonamides would be expected to increase in
activity; however, the increase in acidity of the sulfonamides would re-
sult in a decrease in basicity of the ion and decrease the combining power
of the ion with the enzyme. Consequently, a maximum in activity may
be expected from these theoretical considerations. These considerations
do not, however, account for the relatively high activity of certain non-
ionizing analogues of p-aminobenzoic acid.

Effect of Resonance. It has been suggested that the activity of sulfa-
nilamides and related compounds is associated with the contribution of
the resonating form in which there is a separation of charge, such that a
positively charged coplanar amino group is the fundamental factor and
the negative character of the S02 group is a concomitant factor associated
with the resonating form.207- 208 The interpretation of some experimental
data on physical properties of sulfanilamide supporting this conclusion 209
has been questioned.210 Compounds such as a vinylog of sulfanilamide 211
and the p-aminophenyldimethylsulfonium ion,160 which might be expected
to have appreciable activity according to this theory, have been found to
be relatively inactive. Other related theories have been proposed.212

Miscellaneous Factors Influencing Sulfonamide Activity

Inhibitory analogues of two metabolites of a biosynthetic sequence
usually exert synergistic inhibitory effects. Either ethionine 213 or meth-
oxinine,214 both of which are inhibitory analogues of methionine for
Escherichia coli, displays synergistic effects with sulfonamides. Similarly
5-amino-7-hydroxy-l-v-triazolo[d]pyrimidine, an inhibitory analogue of
guanine, and sulfonamides are synergistic in inhibiting the growth of
Staphylococcus aureus and Escherichia coli.

Many compounds have been reported to act synergistically with sul-

p-AMINOBENZOIC ACID 511

dine,225- 226 thiourea,225- 226 urethane,215- 21C- 223 asparagine,217 hexyl carba-
mate,227 6-benzylthiouracil,228 5,6-tetramethylene thiouracil,228 O-ethyl-
isourea,224 dicyandiamide, N-methyl thiourea,224 penicillin,229 and
n-propyl, isopropyl, n-butyl and isobutyl carbamates.230

Although the synergism of sulfonamides with urea has been con-
firmed,231- 232 failures to confirm the synergism have also been reported.233
Actually either additive or synergistic activity may be observed depend-
ing upon the experimental conditions employed.

Ethyl carbamate is reported to exert an antisulfonamide effect on
luminous bacteria,234 and a slight effect on the toxicity of sulfanamide
for Streptococcus hemolyticus 235 and Escherichia coli.23r>

The bacteriostatic activity of sulfathiazole is reported to increase with
temperature above 37° C for Escherichia coli and for Streptococcus
pyogenes.236 p-Aminobenzoic acid becomes less effective in preventing
the toxicity of sulfathiazole and becomes a more potent inhibitor itself
at higher concentrations under these temperature conditions.236

Biological Effects of Sulfonamides and Related Compounds

Effect of Biochemical Transformation. The effect of methionine,
purines (or derivatives), serine or thymine (or derivatives, e.g., thymi-
dine) in preventing the toxicity of sulfonamides are discussed separately
(pp. 469 and 473). The involvement of p-aminobenzoic acid and related
catalytic factors in the biosyntheses of these factors is indicated by the
results of such inhibition studies.

The possibility that p-aminobenzoic acid has a role in the biosynthesis
of other metabolites has been indicated by the ability of certain metab-
olites to exert an effect on the toxicity of sulfonamides. For example,
arginine, histidine, lysine, methionine, glutamic acid and aspartic acid
are reported to have some ability to prevent the toxicity of sulfonamides
for Proteus vulgaris.287 Valine, and to a lesser extent lysine and isoleucine,
prevent the toxicity of sulfanilamide for Escherichia coli in a medium
containing methionine, purines, serine and either thymine or folic acid.238
Tryptophan has some ability to prevent the toxicity of sulfathiazole for
Staphylococcus aureus.239' 240

Various sulfonamides inhibit the growth of Eremothecium ashbyii, and
the inhibition is paralleled by a decrease in formation of flavin, pre-
sumably riboflavin.241

The phosphorus content of yeast is increased by growth in the presence
of sulfanilamide (200 y per cc) from 1.9 to 2.6 mg per g of dry cells. A
slight increase in the nitrogen content is also noted under similar condi-
tions; p-aminobenzoic acid (1 y per cc) counteracts these effects.134-242

512 THE BIOCHEMISTRY OF B VITAMINS

Effect on Respiration. Since the early indication 243> 244 that sulfon-
amides inhibit the respiration of certain bacteria and other microorgan-
isms, numerous investigators have attempted to correlate inhibition of
respiration with inhibition of growth by sulfonamides. While both inhibi-
tions occur simultaneously in some organisms,244-246 the inhibition of
growth of most organisms by sulfonamides appears to involve essential
metabolic reactions not directly associated with respiration.247 Respira-
tion is inhibited by certain sulfonamides which are without chemothera-
peutic activity.248

The inhibition of growth of Staphylococcus aureus by sulfapyridine
is reported to be prevented partially by coenzymes I or II, but not by nico-
tinic acid.249 The ability of these coenzymes to prevent the toxicity of
sulfapyridine has been questioned on the basis of failures to confirm this
effect with both Staphylococcus aureus and a strain of Escherichia coli
requiring nicotinic acid for growth.250 However, it has been shown that
the ability of coenzyme I to exert such an effect on Staphylococcus aureus
is dependent on the use of a small inoculum, and apparently is related
to the growth-stimulating action of the coenzyme.251, 252 High concentra-
tions of nicotinic acid (100 y per cc) are reported to prevent the toxicity
of low concentrations of sulfapyridine for Lactobacillus arabinosus.253
Nicotinamide, cozymase, and nicotinamide-riboside exert a similar effect
at somewhat lower concentrations (1 to 5 y per cc) in preventing the
toxicity of sulfapyridine (2 y per cc).253

Some attempts were unsuccessful in demonstrating interference of
sulfanilamide, sulfapyridine or sulfathiazole in the functioning of
cozymase in yeast fermentation and in several systems in rat liver.254

However, sulfapyridine appears to inhibit competitively the stimula-
tion by nicotinamide of the respiration of nicotinamide-deficient cells of
dysentery bacilli utilizing glucose.255-257 Greater inhibitory activity was
observed if the sulfapyridine was added prior to the vitamin. Similar
results were obtained with cozymase; but since p-aminobenzoic acid did
not affect the inhibition, the inhibitory action does not appear to be
related to growth inhibitions which are prevented by p-aminobenzoic
acid. Other sulfonamides showed no definite inhibition of nicotinamide-
stimulated respiration.255-257

The sulfonamides, particularly sulfanilamide, prevent the combination
of coenzyme II with the apoenzyme from yeast which oxidizes glucose-
6-phosphate to phosphohexonic acid.258 The sulfonamides react irrevers-
ibly with the apoenzyme and compete with the prosthetic group for the
apoenzyme. Coenzyme II counteracts approximately fifty times its con-
centration of sulfanilamide. Glucose-6-phosphate also counteracts the
inhibition to some extent, but p-aminobenzoic acid is ineffective in pre-

p-AMINOBENZOIC ACID 513

venting the inhibition. The effect of the sulfonamides on cytochrome c,
cytochrome c reductase, and lactic dehydrogenase is much less pro-
nounced, and cytochrome oxidase is not affected.258 p-Aminobenzoic acid
and a number of other aromatic acids are reported to inhibit lactic acid
dehydrogenase.259

Sulfathiazole is reported to inhibit to a greater extent than other sulfon-
amides the anaerobic decarboxylation of pyruvic acid by Staphylococcus
aureus, Escherichia coli and yeast.260 The partial inhibition of carboxylase
of yeast or Staphylococcus aureus is prevented to some extent by cocar-
boxylase 261 and to a greater extent by p-aminobenzoic acid.262 p- Amino-
benzoic acid at higher concentrations was inhibitory to carboxy-
lase.261, 262, 263 It was considered that these results provided additional
evidence for the hypothesis that the mode of action of sulfonamides
involves the respiratory enzymes.

Although sulfonamides have been reported to inhibit the oxidation of
glucose,246- 255' 256- 263- 264> 265 glycerol, lactate and pyruvate,266"267 inhibi-
tions of respiration by sulfonamides appear to be either unrelated or at
most indirectly related to the inhibition of the catalytic role of p-amino-
benzoic acid.

Effects on Nutrition of Animals. Certain sulfonamides fed to rats in
a highly purified diet which alone supports normal growth and develop-
ment cause the appearance of typical signs of dietary deficiencies.268-272
These symptoms often do not develop in animals receiving the sulfona-
mide in stock or natural diet.272-273 Although the mechanism by which
sulfonamides exert such an effect is not completely understood, experi-
mental evidence has usually been interpreted as indicating that the defi-
ciencies develop as a result of the bacteriostatic action of the sulfonamides
on the intestinal bacteria which synthesize certain factors required by
the animals. The intestinal flora is usually markedly affected in animals
receiving sulfonamide.

The symptoms which develop on administration of sulfaguanidine or
succinylsulfathiazole in highly purified diets to rats include alopecia,
achromotrichia, porphyrin-stained whiskers, anemia, leukopenia, agranu-
locytosis and hypocellularity of the bone marrow. These symptoms, as
well as the retardation in growth and increase in prothrombin time which
result from the toxic action of the sulfonamides, are overcome by supple-
ments of folic acid and biotin.271, 273-279 Vitamin K counteracts only the
prothrombin effect.274- 275 p-Aminobenzoic acid prevents the effect of
sulfaguanidine,268-274 but is reported not to counteract the effect of suc-
cinylsulfathiazole.272

The hepatic storage of folic acid and biotin decreases in rats on a
highly purified diet as compared with stock or natural diets; however,

514 THE BIOCHEMISTRY OF B VITAMINS

inclusion of succinylsulfathiazole further decreases hepatic storage of
these vitamins.280 Administration of folic acid and biotin returns the
hepatic storage of these vitamins to normal. A marked decrease in hepatic
storage of pantothenic acid which occurs on administration of succinyl-
sulfathiazole is not corrected by administration of large amounts of
pantothenic acid, whether administered orally or parenterally ; however,
supplements of folic acid and biotin allow normal storage of pantothenic
acid in the liver, besides alleviating the symptoms normally attributed
to pantothenic acid deficiency.280

Rats on stock diets containing succinylsulfathiazole are normal with
respect to hepatic vitamin storage, with the exception of folic acid. The
amount of folic acid in the liver is significantly decreased, but is still
many times that found in deficient animals.281

Succinylsulfathiazole accentuates the folic acid deficiency induced by
strain of lactation in rats. The leucopenia and granulocytopenia produced
under these conditions are especially severe.282

The leukopenia, granulocytopenia and anemia produced in rats by
sulfanilamide, sulfathiazole and sulfadiazine are prevented by either folic
acid or p-aminobenzoic acid.283

Sulfapyridine fed at a level of 1 per cent of the diet produces symptoms
of pantothenic acid deficiency, i.e., roughening of the fur, coproporphyrin
deposits on the noses, wrists and whiskers, hemorrhagic necrosis of the
adrenal glands, retardation of growth, and in black rats achromotrichia.
All these symptoms except retardation of growth are relieved by rela-
tively large supplements of pantothenic acid.284- 285 The ability of panto-
thenic acid to counteract these effects is in contrast to the deficiency
obtained with succinylsulfathiazole.

Xanthopterin has been reported to alleviate the leukopenia of rats fed
a purified diet containing succinylsulfathiazole. 285a However, several at-
tempts to confirm this effect were not successful.278- 279, 286> 287 More re-
cently, xanthopterin has been reported to produce an immediate response
in alleviating the anemia in rats fed a synthetic diet containing 1 per
cent sulfathiazole.28S Folic acid was effective only after a delay of three
to five days.288 Subsequent results indicated that the optimal dose of
xanthopterin was 1 mg per kg of body weight and that 10 mg not only
was ineffective but intensified the anemia.289 Similar results were obtained
with xanthopterin and folic acid in stimulating cell proliferation of iso-
lated bone marrow.290

Ascorbic acid is reported to be effective in treatment of the leucopenia
occurring in rats fed a purified diet containing either succinylsulfathiazole
or phthalylsulfathiazole.291

Sulfonamides are reported to have a delayed carcinogenic action,
though less than that of dibenzanthracene in albino rats and mice.292

p-AMINOBENZOIC ACID 515

Increased excretion of urobilins has been observed in rats treated with
sulfonamides.293

The addition of succinylsulfathiazole to a highly purified diet deficient
in inositol inhibited the growth of rats and caused alopecia, which was
cured with inositol.294

Phthalylsulfathiazole administered to the pig on a diet deficient in
inositol and biotin caused a syndrome which could be prevented by the
addition of biotin, but was also largely alleviated by inositol.295 It has
been suggested that the sulfa drugs inhibit intestinal microorganisms
which synthesize inositol and thereby cause a deficiency.296-298 Sulfa-
guanidine in combination with inositol reduces the fertility of female
albino rats, whereas either compound alone has no effect.299

Succinylsulfathiazole fed to pigs on a purified diet produces an
anemia 300, 301 which responds to treatment with either folic acid, or less
effectively with purified liver extracts. Earlier failures 302, 303 to accom-
plish this result have been explained on the basis of the relatively long
period of treatment necessary to obtain this effect. Both folic acid and
biotin are necessary to prevent the effects of the sulfonamide on the pig;
however, folic acid administered without biotin accelerates the appearance
of biotin deficiency symptoms.301 Thus, the interrelationships of these
factors in the pig appear to parallel those in the rat.

The nutritional requirements of rabbits 304 and chicks 305, 306 are re-
ported to be affected by sulfaguanidine. On the other hand, succinylsulfa-
thiazole does not appear to increase the nutritional requirements of
chicks.307

Succinylsulfathiazole added to purified diets fed to mice retards the
growth of the animals.308 Supplements of folic acid and biotin together
do not counteract this effect ; but these factors, together with liver extract,
prevent the toxic action of the sulfonamide on growth of the animals.
Concentrates of vitamin Bi2 almost completely replace the liver extract.308

Sulfanilamide, 125 mg injected daily, prolonged nerve chronaxia and
shortened muscle chronaxia in rats.308a These effects, which are similar to
those in chronic alkalosis, are prevented by daily oral administration of
ethyl p-aminobenzoate (50 y), nicotinic acid (400 y), riboflavin (20 y),
or ascorbic acid (5 mg) . No effects were observed with thiamine, adenine,
pantothenic acid or pyridoxine.

3-Hydroxysulfanilamide has been identified in human urine as a
metabolic product of sulfanilamide.309

It has been reported that the toxicity for rats of bis(4-a-aminovaleryl-
phenyl)sulfone or related sulfones is diminished by administration of
either p-aminobenzoic acid or ascorbic acid.310

Miscellaneous Effects. p-Aminobenzoic acid is reported to counteract
the depressant effect of sulfanilamide on isolated frog hearts.311 While

516 THE BIOCHEMISTRY OF B VITAMINS

p-aminobenzoic acid alone at low concentrations exerts no effect on
rhythm or amplitude, sulfanilamide at low concentrations increases
amplitude, but does not accelerate rhythm, and at high concentrations
stops the frog heart.311 p-Aminobenzoic acid may under certain conditions
inhibit the contractions of isolated frog heart.312 The incidence of con-
vulsions after intramuscular injection of procaine in guinea pigs is
Teduced by previous administration of either diethylaminoethanol or
p-aminobenzoic acid, or to a greater extent by both compounds.313 Other
compounds structurally related to p-aminobenzoic acid and diethylamino-
ethanol exert a similar effect.313 No inhibition of the peripheral local
anesthetic action of procaine occurs with these compounds.313 The effects
are presumably unrelated to the catalytic roles of p-aminobenzoic acid.

The effect on intestinal contractions of procaine in physiologically
active concentrations is suppressed by sulfanilamide in concentrations
eight times that of procaine.314

Either p-aminobenzoic acid or diethylaminoethanol prevents competi-
tively the typical convulsive action of procaine in guinea pigs.315

The formation of a yellow pigment in cultures of Mycobacterium
tuberculosis in the presence of p-aminobenzoic acid or procaine is inhib-
ited by sulfanilamide.316- 317 The oxidation of p-aminobenzoic acid to a
red-colored substance by peroxidase from horseradish is also prevented
by sulfanilamide.317

Sulfanilamide, sulfathiazole or sulfapyridine inhibits the peroxidase
reaction, but not phenol oxidase with p-aminobenzoic acid as a sub-
strate.317

Carbonic anhydrase is inhibited by sulfanilamide,318 but the inhibition
is only partially counteracted by p-aminobenzoic acid.319

An interesting adsorption effect has been reported in which sulfanila-
mide (0.1 per cent) decreases by 44 per cent the amount of methylene
blue adsorbed by charcoal. p-Aminobenzoic acid (0.028 per cent) com-
pletely prevented the effect of sulfanilamide on the adsorption.320 The
possibility that sulfonamides act bacteriostatically by reducing cellular
and colloidal adsorption was suggested.320

Resistance to Sulfonamides

Natural Resistance. Many organisms, either isolated from patients or
tested at random, have been found to possess a "natural" resistance to
the sulfonamides.321-333 For example, two strains of Staphylococcus
aureus isolated from patients with severe infections differed in their
susceptibility to sodium sulfathiazole before treatment was adminis-
tered;321 and from 168 patients with pneumonia, meningitis or endo-
carditis, moderately resistant organisms were isolated from six cases.322

p-AMINOBENZOIC ACID 517

Different types of pneumococci 323, 324, 327> 328, 332 vary in their suscepti-
bility to various sulfonamides. Similar results are obtained with different
strains of Clostridium,320 gonococcus 329 and /^-hemolytic streptococci.331, 333
Marked disparity in the inhibitory ability of a specific sulfonamide for
10 strains of Shigella sonnet has been noted.325

Although clinical and in vitro observations usually correspond,329- 334, 335
in vitro resistance does not always indicate in vivo resistance, and vice
versa.528, 329, 332, 336-339 Sulfathiazole resistance was induced in vitro in
Shigella paradysenteriae Flexner, but this resistance was not exhibited in
vivo in white mice.337 By isolating pneumococci from patients and fol-
lowing the changes in their resistance during the course of therapy, it
was found that the in vitro resistance of an organism was decreased.332
Of organisms isolated from patients who have shown resistance in sul-
fonamide therapy, some are not resistant in vitro. Although variation in
resistance of isolated gonococci corresponded to the clinical reaction of
the patient in most cases, an in inYro-susceptible strain has been isolated
from a patient resistant to sulfathiazole therapy, and an in tnYro-resistant
strain has been isolated from a patient who had responded to this treat-
ment.329 These and similar observations in clinical studies with gono-
cocci 339 suggest that this particular type of resistance is dependent
entirely upon the environment. It appears that in vivo resistance may
sometimes be due to a host factor (and in vitro resistance, to a constituent
of the culture medium) , which counteracts the action of the sulfonamide.

Acquired Resistance. Organisms serially transferred in vitro in in-
creasing concentrations of the sulfonamides may be made resistant to the
action of these drugs. Resistance may also be developed in vivo during
sulfonamide administration to the host. Many organisms have been
shown capable of developing resistance to the sulfonamides: Escheri-
chia coli,11' 25°- 34°-344 hemolytic streptococci,331, 345 349 Streptococcus pyo-
genes,350 Neisseria gonorrheae,334' 335- 350a-357 Neisseria meningitidis ,35G
Neisseria catarrhalis35* and Neisseria sicca356 Brucella abortus,12 Bru-
cella paramelitensis342 pneumococci,18, 26(5, 322, 324, 330, 332, 336, 338, 358"369
Staphylococcus aureus,1' 18, 321, 342, 37°-37G Staphylococcus pyogenes,350
Shigella paradysenteriae337' 377-379 Shigella sonnei18' 325, 337, 377, 37S Myco-
bacterium ranae,380 Friedlander's bacillus,343 Acetobacter sub oxy dans,381
Polytomella caeca,104 and Endamoeba histolytica.382 Between strains of
one species there is often a wide variation in the ease with which resist-
ance may be developed,351' 359> 302 but the number of resistant organisms
developing from a single strain often does not vary significantly.351

Development of resistance in vivo may be rather difficult or may occur
readily, depending upon the organism. In a study of pneumococci from
72 infected patients, a strain with striking resistance developed in only

518 THE BIOCHEMISTRY OF B VITAMINS

one case;361 however, resistance of organisms isolated from patients after
prolonged treatment is usually increased. Gonococci isolated from infected
patients not cured after sulfathiazole had been administered for 6 days
(6 gm/day) were resistant to 0.5 mg per cent of sulfathiazole in vitro,
and some grew in 25-50 mg per cent, while strains from cured patients
were susceptible to this concentration.355

A wide variation in the extent to which resistance may be developed
in different strains has also been observed.321, 359, 377

Although resistance to a certain bacteriostatic concentration of one
sulfonamide is usually accompanied by an equal resistance to similar
bacteriostatic concentrations of the other sulfonamides,327- 341, 351, 353, 356,
357, 362, 363, 365 th}s js not a m\e wjthout exceptions.345, 350, 377 For example,
strains of Shigella paradysenteriae Flexner, Shigella sonnei (Ch) and
Shigella sonnei (Ma) , made resistant to sulfathiazole by transfer in in-
creasing concentrations, were resistant also to sulfapyridine, sulfadiazine
and sulfanilamide, but not to sulfapyrazine. Their resistance to sulfa-
cetamide varied.377- 378

Cross-resistance between sulfonamides and penicillin does not appear
to take place,330- 344, 350' 359 and it has been shown that development of
resistance to sulfanilamide does not influence susceptibility to strepto-
mycin, atebrin, sodium salicylate, 3,5-dibromosalicylic acid and synthelin
(decamethylenediguanidine) .344

A high degree of resistance acquired after a great many transfers on
sulfonamide-containing media is usually permanent.330, 338, 350, 356, 360, 363,
383, 384 However, partial resistance acquired through short contact with
the drug is usually temporary, and is lost after repeated transfer on sul-
fonamide-free media.338, 360, 367, 383

Several factors affecting the degree of resistance acquired have been
observed. It has been indicated that, unless the drug is present in suffi-
cient quantity to inhibit growth of Escherichia coli, very little resistance
is developed, and that the degree of resistance developed varies with the
concentration used.341 However, it is also reported that by repeated trans-
fer of cultures of Escherichia coli on low concentrations of the drug,
resistant strains can be developed.250 With Brucella abortus an increase
in the time of incubation increases the degree of resistance developed.12
In the presence of p-aminobenzoic acid, no sulfonamide resistance could
be produced with Escherichia coli, and the presence of methionine delayed
its acquisition.250 Organisms made resistant in a plain infusion broth con-
taining peptone were not resistant when tested in a synthetic medium.250

Because pneumococci surviving the first exposure to sulfapyridine were
significantly more resistant than any organisms of the original parent

p-AMINOBENZOIC ACID 519

strain,385 it was suggested that the acquisition of sulfonamide-resistance
is a sudden, spontaneous "mutation" occurring continuously, but becom-
ing evident only when conditions are suitable for selective propagation
of resistant cells.351 The gradual increase in resistance of Staphylococcus
aureus to sulfonamides occurring at random time during serial transfer
in sodium sulfathiazole 37C was interpreted to indicate mutation and
selection. The slow and apparently multiple process in obtaining resistant
strains of Bacterium lactis aerogenes and early stage reversal of resistance
in the absence of the inhibitor, indicating a slower growth rate for the
resistant strain, have been presented as evidence in favor of an adaptive
mechanism.3S3

From the standpoint of the permanence of resistance in organisms as
related to the clinical use of sulfonamides, it is of interest that the cultiva-
tion of a mixed population of sulfanilamide-resistant and susceptible
cells of Escherichia coli for 10 passages in synthetic media free of sulfa-
nilamide resulted in a "weeding out" of the resistant strain, as shown by
subsequent exposure of the culture to the drug.344

The converse of resistance is obtained by culturing in hemolyzed horse
blood staphylococci and some streptococci which are relatively insensitive
to sulfonamide. The organisms develop marked sensitivity to the sul-
fonamides.386

Mechanism of Resistance. A significant increase in production of
p-aminobenzoic acid or a related anti-sulfonamide compound over that in
parent or nonresistant strains has been observed in sulfonamicle-resistant
strains of Staphylococcus aureus,18- 353- 371, 373> 387, 389 gonococci,334 strains
of Clostridium,326 Brucella paramelitensis,342 Escherichia coli,340 strepto-
cocci,340 Polytomella caeca,104 Diplococcus pneumoniae,340' 388, 390 and

Table 9. The Inhibition Index and Minimum Inhibitory Concentration of 2-Sulfanil-

amido-4-methylthiazole for Resistant and Original Strains of

Staphylococcus aureus127

Minimum

Effective

Inhibitor

Relative

Concentration

Antisulfonamide

Strain

X105' *

Inhibition Index*

Produced'

B (original)

0.31

<20

B (resistant)

10.0

220

<20

Nr. VI (original)

0.18

<20

Nr. VI (resistant)

6.67

133

28.5

Nr. IX (original)

0.21

<20

Nr. IX (resistant)

4.67

100 <*

Nr. X (original)

1.67

Nr. X (resistant)

12.0

Nr. VII (original)

1.25

° Concentration necessary to reduce growth to one-third of controls.

b Ratio of concentration of sulfamethylthiazole (0.001 M) to p-aminobenzoic acid.

c Relative antisulfonamide activity against strain B of extracts prepared from the various strains.

d Arbitrary value.

520 THE BIOCHEMISTRY OF B VITAMINS

Shigella sonnei.388 The synthesis of anti-sulfonamides occurs in both the
presence and the absence of sulfonamides.371, 373 The sulfonamide in-
hibitors may be restricted to the cells or released into the medium.340
Although the inhibitor in most cases noted is p-aminobenzoic acid, some
inhibitors exhibit different properties and presumably are not p-amino-
benzoic acid.340

Conversely, in experiments with certain resistant strains of Shigella
paradysenteriae Sonne,18 Diplococcus pneumoniae Type I,18 and Sta-
phylococcus aureus3^1 there was no demonstrable increase in synthesis
of p-aminobenzoic acid,18 and for certain resistant strains of Neisseria
gonorrheae increased synthesis of p-aminobenzoic acid was insufficient to
account entirely for such resistance.355 A sulfathiazole-resistant strain of
staphylococci with no increased production of an anti-sulfonamide was
found to require smaller amounts of p-aminobenzoic acid for reversal of
sulfonamides than the parent strain.389

Ivanovics 127 compared the minimum inhibitory concentrations of sul-
famethylthiazole and the inhibition indices obtained with resistant and
parent strains of Staphylococcus aureus. The data listed in Table 9
indicate that the resistance of this organism can be accounted for by
increased production of p-aminobenzoic acid or a related sulfonamide
antagonist, or by more efficient utilization of p-aminobenzoic acid, or by
a combination of the two mechanisms. The former mechanism is char-
acterized by an increased minimum inhibitory concentration for the
resistant organisms, but does not involve a change in the inhibition index.
More efficient utilization of p-aminobenzoic acid results in an increased
inhibition index.

Differences Between Resistant and Parent Strains. Although the virul-
ence of an organism is usually unchanged in the acquisition of sulfonamide
resistance,206- 331, 35S- 389> 392 in some cases contrary observations have been
made.336- 337> 348- 393 Certain strains of Shigella sonnei 337 and /^-hemolytic
streptococci 348 became nonvirulent on becoming resistant to sulfonamides.
Meningococci which became resistant to sulfanilamide lost their virulence,
while those resistant to sulfapyridine remained virulent.393 A sulfonamide-
resistant strain of gonococcus lost its pathogenicity, and it could not be
restored by treatment with p-aminobenzoic acid.120

More often than not, the morphology of the resistant strain remains the
same as that of the parent strain;266, 338- 350' 363, 367 however, changes have
been noticed in some cases. The more resistant strains of staphylococci
studied produced a nonfat-soluble yellow pigment in the presence of
sulfonamides.372 However, strains which were not as highly resistant did
not produce this pigment. It is suggested that this pigment may be derived
from p-aminobenzoic acid.

p-AMINOBENZOIC ACID 521

Resistant strains of pneumococci did not lose type-specific characteris-
tics, and were still susceptible to anti-serum.303

Sulfonamide-resistant strains of pneumococci 26e produce less hydrogen
peroxide than the parent strain, and the ability to form hydrogen peroxide
is lost by strains of a-streptococci which become resistant to sulfathi-
azole;349 but these strains no longer require riboflavin for growth and are
more effective than the parent strain in oxidizing a number of sub-
strates.349 This contrasts to resistant strains of pneumococci, which are
less effective in oxidizing glycerol, lactate and pyruvate but not glucose.266
Strains of Shigella with the greatest fermentation activity are more
resistant to sulfonamides.325 Resistant strains of Mycobacterium ranae 38°
produce diazotizable arylamines. Development of resistance and increased
arylamine formation in Staphylococcus aureus is reported not to be asso-
ciated, but the arylamine in this case is apparently derived from trypto-
phan, and is not p-aminobenzoic acid.375

Other Inhibitory Analogues of p-Aminobenzoic Acid

Shortly after the discovery of the competitive relationship of p-amino-
benzoic acid and the sulfonamides, many compounds differing from the
sulfonamides but related in structure to p-aminobenzoic acid were pre-
pared and tested. The compounds of this group which inhibit the utiliza-
tion of p-aminobenzoic acid by various organisms are indicated in Table
10. It is apparent that neither a free amino nor an acidic radical is essen-
tial for the inhibitory action of an analogue of p-aminobenzoic acid.
Nitro and acetamido groups may replace the amino group for certain
inhibitory analogues, while the modification of the carboxyl group can
be extended to a variety of changes. Utilization of carboxamide, ketone
and alcohol groups, as well as arsonic, phosphonic and phosphonous acid
groups in place of the carboxyl group of p-aminobenzoic acid and related
analogues results in some instances in inhibitory analogues. Some in-
hibitory analogues contain an isosteric, heterocyclic ring in place of the
aromatic ring structure of p-aminobenzoic acid, while other inhibitory
analogues are substituted p-aminobenzoic acids. Of a large number of
analogues of p-aminobenzoic acid with substituents in the aromatic
nucleus, only a few inhibit the utilization of this vitamin.16, 21 Usually
disubstituted p-aminobenzoic acids are inactive, indicating the possibility
that one side of the ring structure of the vitamin must be intact for com-
bination with the appropriate enzymes.

Halogeno-4-aminobenzoic Acid. Although 2-fluoro-4-aminobenzoic acid
is approximately one-third as effective as p-aminobenzoic acid in promot-
ing growth of certain organisms and in preventing the toxicity of sulfanil-
amide (p. 484), 3-fluoro-4-aminobenzoic acid is almost as effective as

THE BIOCHEMISTRY OF B VITAMINS

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524 THE BIOCHEMISTRY OF B VITAMINS

sulfanilamide in preventing the growth of Escherichia coli, and 2-chloro-
4-aminobenzoic acid is more active than sulfanilamide against Escherichia

COOH

NH2 NH2

2-ch!oro-J+-aminobenzoic acid 3-fluoro-^-aminobenzoic acid

coli.21 However, in the presence of methionine, 2-chloro-4-aminobenzoic
acid not only does not exert an inhibitory effect on Escherichia coli but
has a slight ability to prevent the toxicity of sulfanilamide for that
organism. The 2-chloro derivative also has some ability to prevent the
toxicity of sulfonamides for Diplococcus pneumoniae, Streptococcus
pyogenes and Streptococcus hemolyticus.16' 32, 33 Strains of Escherichia
coli which are resistant to inhibition by 2-chloro-4-aminobenzoic acid are
obtained in a single transfer in the presence of the analogue; however,
these strains resistant to 2-chloro-4-aminobenzoic acid are not resistant
to sulfonamides, but sulfonamide-resistant strains are resistant to inhi-
bition by the 2-chloro derivative.400

Pantothenic acid is reported to be as effective as p-aminobenzoic acid
in preventing the toxicity of 2-chloro-4-aminobenzoic acid;401 however,
since this effect is not observed after short incubation periods 402 and
since resistance 400 to the inhibition occurs after a longer incubation
period in the absence of pantothenic acid, it appears that this effect may
be associated with the resistance phenomenon. Either thiamine or glu-
tamic acid exerts effects analogous to pantothenic acid on this inhibi-
tion.402

In contrast to slight growth-promoting activity of 2-bromo-4-amino-
benzoic acid,21' 33 which may possibly be attributed to contamination
with p-aminobenzoic acid, 3-bromo-4-aminobenzoic acid has some in-
hibitory action against Escherichia coli and Streptococcus hemolyticus
Group A.16 3-Chloro-4-aminobenzoic acid is somewhat more inhibitory
than the corresponding 3-bromo derivative for Escherichia coli. The 3-
chloro derivative is also effective against Streptococcus pyogenes.

4-Amino-2-hydroxybenzoic Acid (^-Aminosalicylic Acid) and Related
Analogues. Since benzoic and salicylic acids were reported to stimulate
the oxygen consumption and carbon dioxide production of tubercle ba-
cilli,403 Lehmann,404, 405 in attempting to find an inhibitor of the stimula-
tory action, tested 60 substances of similar structure and found that the

p-AMINOBENZOIC ACID 525

most effective compound, p-aminosalicylic acicl, inhibited growth of the
tubercle bacillus at concentrations as low as 1.5 y per cc. At this concen-

NH2

-p-amino salicylic acid

tration the inhibitor was bacteriostatic rather than bactericidal. The
compound fed at a concentration of 5 per cent of the diet was not toxic
for rats, mice or rabbits, but guinea pigs became emaciated, lost hair
and died within two weeks on such a diet. In guinea pigs infected with
Mycobacterium tuberculosis, the analogue tended to retard the disease,
but only at concentrations at which some of the animals died. However,
no toxic manifestations on administration of 10 to 15 g daily were ob-
served in patients with tuberculosis. Patients responded with a prompt
fall in temperature, improvement in general condition, gains in weight
and appetite, and increases in red cells and hemoglobin.

p-Aminosalicylic acid in vitro is a very effective bacteriostatic agent
at concentrations of 0.1-1.5 y per cc for tubercle bacillus,404-411 but does
not have appreciable inhibitory activity against other microorgan-
isms.408- 412- 413

In the presence of 1 y per cc of p-aminobenzoic acid, the amount of
p-aminosalicylic acid necessary for inhibition of growth of Mycobac-
terium tuberculosis H37Rv is increased approximately 16 fold.42 Salicylic
acid also prevents the toxicity of ^-aminosalicylic acid in a somewhat
competitive manner, but is only 2-6 per cent as effective as p-amino-
benzoic acid.395

Contrasting with the inhibitory action on Mycobacterium tuberculosis,
^-aminosalicylic acid replaces p-aminobenzoic acid in stimulating the
growth of two mutant strains of Escherichia coli which require p-amino-
benzoic acid for growth.28 The analogue depending upon the testing con-
ditions is from 4 to 16 per cent as effective as p-aminobenzoic acid.28 It
appears that ^-aminosalicylic acid inhibits the utilization of p-amino-
benzoic acid for tubercle bacilli, but is utilized in place of the vitamin
by Escherichia coli. Such activity, if general among microorganisms,
would account for the unusual specificity of p-aminosalicylic acid as an
inhibitory analogue.

Strains of tubercle bacilli which are resistant to streptomycin are
sensitive to ^-aminosalicylic acid,42, 407 and the two compounds have been

526 THE BIOCHEMISTRY OF B VITAMINS

reported to act synergistically in inhibiting the growth of some strains
of the organism.411 With other strains, only additive effects are ob-
served.42, 414 Nevertheless, the utilization of ^-aminosalicylic acid con-
current with streptomycin to prevent the selection of resistant strains is
indicated.

Although p-aminosalicylic acid is reported to have little therapeutic
activity in rabbits or guinea pigs infected with certain strains of tubercle
bacilli,405, 408, 414 the analogue appears to suppress experimental tubercu-
losis in mice,42 as determined by histopathologic examination.41"' The
effect of streptomycin on the analogue appears to be additive in vivo.

^-Aminosalicylic acid is rapidly adsorbed and excreted by human sub-
jects as well as by laboratory animals.414, 416419 Although blood levels of
the compound can be maintained in the animals for several hours, ap-
proximately 85 per cent of the administered dose is recoverable from the
urine within ten hours.416 Recovery as conjugated amines was highest
(60 per cent of total dose) in man and apparently negligible in the dog.410
Both the free ^-aminosalicylic acid and the N-acetyl derivative can be iso-
lated from the urine of rabbits after administration of p-aminosalicylic
acid.417 In the urine of one human subject, three compounds containing
a free amino group and two conjugated amines have been detected subse-
quent to the administration of ^-aminosalicylic acid.416 Among these,
unchanged p-aminosalicylic acid, p-aminosalicyluric acid and N-acetyl-
p-aminobenzoic acid have been identified.416

4-Amino-3-hydroxybenzoic acid is about one-third as effective as sul-
fanilamide against Streptococcus pyogenes, and has a slight chemothera-
peutic action in mice infected with that organism.32 The corresponding
3-methyoxy derivative is somewhat less inhibitory to Streptococcus
hemolyticus Group A.16

Other Substituted p-Aminobenzoic Acids. Both 2, 4-diamino- and
3,4-diaminobenzoic acids are antagonists of p-aminobenzoic acid for
Diplococcus pneumoniae,33 and compare favorably with sulfanilamide in
inhibiting the growth of Escherichia coli.21 Considerable loss in activity
is observed 16 with acetylation of the 2-amino group of the former com-
pound.16

3-Methyl-4-aminobenzoic acid has some inhibitory activity against
Escherichia coli and Streptococcus hemolyticus. The corresponding
2-methyl derivative is less effective against Escherichia coli, and is inac-
tive against Streptococcus hemolyticus. The amide of the 3-methyl
derivative is less active than the parent compound.

Isosteres. A number of compounds which are isosteric with p-amino-
benzoic acid are rather potent inhibitory analogues of the vitamin.
5-Nitrothiophene-2-carboxylic acid and the corresponding amide are more

p-AMINOBENZOIC ACID 527

active than sulfanilamide for several organisms (Table 9). The latter
compound is approximately ten times as active as sulfanilamide for

HC CH HC CH

N02— C C— CO— NH2 CH3— CO— NH— C C— CO— CH3

5-nitrothiophene~2-carboxamide meihyl-2-(5-acetamidothienyl) ketone

Escherichia coli. While the heterocyclic amide is approximately ten times
as active as the free acid for Escherichia coli, the acid is approximately
twice as effective as the amide against both Streptococcus hemolyticus
Group A and Diplococcus pneumoniae Type III. The corresponding amino
compound, 5-aminothiophene-2-carboxamide, is inactive as an inhibitor
for these organisms.16 Methyl 2-(5-acetamiclothienyl) ketone is more
active than sulfanilamide in inhibiting growth of Escherichia coli, but is
somewhat less effective against Streptococcus hemolyticus and Diplococ-
cus pneumoniae. A similar situation exists for 6-aminonicotinic acid,
which is several times as active as sulfanilamide against Escherichia
coli and is essentially as active against Streptococcus hemolyticus; but
the analogue does not inhibit the growth of Diplococcus pneumoniae
Type III. It is interesting that this analogue is reported to be an antag-
onist of nicotinic acid rather than of p-aminobenzoic acid in Staphylococ-

-COOH
NH2

6-aminonicotinic acid

cus aureus.420 The pyrimidine derivative corresponding to this pyridine
analogue, 2-amino-5-pyrimidinecarboxylic acid, is reported to have slight
activity in preventing the toxicity of sulfonamides (p. 484) .

Ketone Analogues. As indicated in Table 9, p-aminophenyl ketones
are very effective inhibitory analogues of p-aminobenzoic acid. p,p'-
Diaminobenzil is several times as active as sulfanilamide in inhibiting
the growth of either Streptobacterium plantarum or Staphylococcus
pyogenes. This combination of two carbonyl groups results in a more
effective inhibitor than a single carbonyl. Thus, the diketone is approx-
imately 20 to 60 times as effective as p,p'-diaminobenzophenone. Slight
chemotherapeutic activity was observed with p,p'-diaminobenzophenone

H2N-/ \_ CO— CO— / \-NH2 H2N-/ \_CO— / VnH,

p ,p '-diaminobenzil p,p'-diaminobenzophenone

528 THE BIOCHEMISTRY OF B VITAMINS

in mice infected with streptococci, gonococci and meningococci. The
compound had only a slight effect against staphylococci and pneumococci
in vivo.396 Although the inhibitory activity of p,p'-diaminobenzoin against
Streptobacterium plantarum approached that of p,p'-diaminobenzil for
short periods of incubation, the benzoin derivative decreased markedly
in activity with increased incubation periods to only a small fraction of
the activity of the benzil derivative.26 It was suggested that the organism

■— f \-CO— CHOH— / \— NH2 H2N-/ \— i

p,p'-diaminobenzoin p-aminoacetophenone

oxidized the benzoin derivative to p-aminobenzoic acid, thereby effecting
a reversal of the inhibition. p-Aminobenzophenone is about 10 to 25 per
cent as effective as sulfanilamide in preventing growth of Streptobac-
terium plantarum, and p;p'-diaminodesoxybenzoin and p-aminobenzophe-
none are only slightly inhibitory to that organism. p-Nitroacetophenone
appears to be more effective than p-aminoacetophenone against Strepto-
bacterium plantarum.

Miscellaneous Analogues. p-Nitrobenzoic acid at low concentrations
inhibits the growth of Streptococcus viridans, and the growth inhibition
is prevented by p-aminobenzoic acid; at higher concentrations the
nitro compound does not inhibit growth of the organism. Under these con-
ditions, the organism produces a diazotizable amine in the medium which
has anti-sulfonamide properties.44 Consequently, it appears that reversals
noted at high concentrations are the result of the action of p-amino-
benzoic acid accumulating from reduction of the inhibitor by the organ-
ism.

p-Aminobenzamide is an inhibitory analogue of p-aminobenzoic acid
for Escherichia coli, but is not particularly effective. Similar results have
been obtained with 2- (4-aminobenzamido) pyridine against Streptobac-
terium plantarum.

Ethyl p-aminobenzoate is an inhibitory analogue of p-aminobenzoic
acid for Streptobacterium plantarum.421

o-Aminobenzoic acid at a very high inhibition index is toxic for Stri-
gomonas oncopelti137 and m-aminobenzoic acid has some inhibitory ac-
tivity against Streptobacterium plantarum.421 For the latter organism a
high concentration of benzoic acid is toxic, and the inhibition is prevented
by p-aminobenzoic acid.421

Even p-aminobenzyl alcohol has some ability to inhibit the utilization
of p-aminobenzoic acid by some organisms.4203 6-Amino-2-naphthoic acid
is slightly inhibitory to Streptobacterium plantarum.396

p-AMINOBENZOIC ACID 529

Analogues of p-Aminobenzoic Acid Containing Arsenic, Antimony or
Phosphorus. The sodium salt of arsanilic acid (p-aminobenzenearsonic
acid), which has been termed atoxyl, was one of the first compounds
found to have trypanosomacidal activity. The discovery of the activity of
p-aminobenzoic acid in preventing the toxicity of the sulfonamides for
bacteria prompted analogous research on arsanilic acid. The effect of
arsanilic acid in retarding the growth of Escherichia coli as measured by
oxygen consumption is counteracted by p-aminobenzoic acid.45 With
some strains of the organism, complete inhibition of growth occurs, and
in one case the inhibition index for half-maximum growth has been found
to be approximately 15,000. 107 The analogue is only about 3 per cent as
effective as sulfanilamide. Methionine also prevents the toxicity of
arsanilic acid, as well as sulfonamides, for Escherichia coli.107

OH OH

HO— PO

NH2 NH2

arsanilic acid phosphanilic acid

The trypanosomacidal activity of arsanilic acid, which is attributed to
the formation of the arsenoxide, is not affected by p-aminobenzoic acid in
a test which renders Trypanosoma equiperdum noninfective for young
rats.107 However, the chemotherapeutic action of arsanilic acid in Try-
panosoma equiperdum infections in mice is reported to be counteracted
by certain doses of p-aminobenzoic acid and sulfanilamide.42121 These
results suggest the possibility that substances of analogous structure pre-
vent the conversion of arsanilic acid to the active form. Further evidence
for such possibilities is indicated by reports that relatively large amounts
of p-aminobenzoic acid prevent the toxicity for rats of otherwise lethal
doses of arsanilic acid and related arsonic acids.422-425 However, p-amino-
benzoic acid apparently does not prevent the therapeutic effects of these
arsenicals against Trypanosoma equiperdum in rats. If p-aminobenzoic
acid is not administered within a few hours after the arsenical, it is not
effective in preventing the toxic manifestations of the drugs.425 p-Amino-
benzoic acid is not particularly effective in preventing the toxic effects of
trivalent arsenicals, especially that of ra-amino-p-hydroxyphenylarsen-
oxide.425 The effect of an otherwise lethal dose of neoarsphenamine is
counteracted by p-aminobenzoic acid in dogs.426 The trypanocidal ac-
tivity of y-(p-arsenosophenyl) butyric acid both in vivo and in vitro is

530 THE BIOCHEMISTRY OF B VITAMINS

also counteracted by p-aminobenzoic acid.427 The question of specificity
of p-aminobenzoic acid in eliciting some of these responses has not been
adequately determined.

The toxicity of salvarsan, neosalvarsan, or neostibosan for Streptobac-
terium plantarum is prevented by p-aminobenzoic acid.129

Similarly, the toxic manifestations caused by administration of sodium
??i-chloro-p-acetylaminobenzenestibonate to rats are counteracted by
p-aminobenzoic acid, which does not appreciably affect the therapeutic
activity of the drug against Trypanosoma equiperdum*28

Phosphanilic acid inhibits the growth of Streptobacterium plantarum,
and p-aminobenzoic acid prevents the inhibition. The inhibition index for
half-maximum growth is approximately 12,000.26 Both phosphanilic acid
and phosphanilamide are reported to inhibit with varying degrees of
activity the growth of Escherichia coli, Staphylococcus aureus and Sal-
monella typhimurium.*29 Phosphanilic acid also inhibits the growth of
Mycobacterium tuberculosis in vitro.430, 431 Administered three times
daily in doses of 15 mg, it is not toxic to mice, but only low concentra-
tions in the blood are attained.431

p-Aminobenzenephosphorous acid is slightly less inhibitory than sulfa-
nilamide to the growth of Escherichia coli. p-Aminobenzoic acid prevents
the inhibitory effect of the analogue.432

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Chapter IVD

BIOTIN

The stimulating effect of small amounts of natural extracts on the
growth of yeast was first described in 1901 by Wildiers,1 who gave the
name "bios" to this growth-promoting substance. Bios was subsequently
shown to be a group of factors, one of which was isolated from dried
Chinese chick egg yolk in 1935 by Kogl,2 who characterized the compound
as the methyl ester, determined its empirical formula and named it
"biotin."3 du Vigneaud and co-workers isolated biotin from liver4 and
from a milk concentrate 5 and completed the proof of structure.6 The
biotin molecule is indicated by the following formula:

H2C CH— CH2— CH2— CH2— CH2— COOH

biotin

(cis-hexahydr o-2-oxo-l H-thieno [3,4] imidazole-4-valeric acid)

The synthesis of the vitamin first reported by Harris et at? has since been
accomplished in a number of laboratories by different methods.

Specificity*

There are four diastereoisomers or eight optically active modifications
corresponding to the structure of biotin. All the racemic diastereoisomers,
DL-biotin,7- 8 DL-epi-biotin,9 DL-allobiotin 7> 8 and DL-e?w"-allobiotm,8

* A naturally-occurring complex of biotin, biocytin, has recently been isolated
in crystalline form from yeast extract (Wright, L. D., el al., J. Am. Chem. Soc, 72,
1048 (1950). Biocytin, m. p. 230-240° (dec.) is on a molar basis just as active as
biotin for Lactobacillus casei but is inactive for Lactobacillus arabinosus. On acid
hydrolysis of biocytin, an equivalent of 40=t= 4 per cent of biotin determined micro-
biologically is produced. Biocytin also replaces biotin in the nutrition of Lactobacillus
delbmckii LD5, Lactobacillus acidophilus, Streptococcus faecalis R, Neurospora
crassa and Saccharomyces carlsbergensis, but the complex is inactive for Lacto-
bacillus pentosus and Leuconostoc mesenteroides P-60 as well as Lactobacillus
arabinosus.

542

BIOTIN 543

have been synthesized. Elucidation of the structure of the four racemic
modifications has been accomplished to some extent by the conversion of
the biotin stereoisomers to the corresponding desthio derivatives. This
conversion is effected by the removal of the sulfur of biotin by reduction
with Raney's nickel.10 The structure of the resulting product, desthiobiotin,
allows only two racemic forms, since the asymmetry of carbon atom 2 in
biotin is destroyed by the reduction. DL-Biotin and Dh-epi-biotin are

HN NH

HC CH

CH3 CH2— (CH2)4— COOH
desthiobiotin

reduced to the same DL-desthiobiotin,9 while both DL-allobiotin and
DL-epi-allobiotin give rise to the same DL-allodesthiobiotin.11 Thus,
biotin and epi-biotin differ by being epimeric at carbon 2 where the side
chain is attached. Allobiotin is similarly epimeric with epi-allobiotin.
Although the exact configuration of these compounds is unknown, the
relationship between the epimeric biotins and epimeric allobiotins has
been resolved by the relative ease of hydrolysis of the ureylene groups
of allobiotin and epi-allobiotin as compared with biotin. The resulting
diamino compounds derived from hydrolysis of the allobiotins are also
less easily reconverted by the action of phosgene into the original com-
pounds than are epimeric biotins. These results suggest that nitrogens
of the ureylene group of the epimeric biotins have a as-configuration,
whereas the nitrogens of this group in the epimeric allobiotins have a
trans configuration as illustrated below:

CO
/ \

NH H NH H

•s

NH H

H NH

\ /

CH(CH2)4(

H2C CH(CH2)4COOH H2C CH(CH2)4COOH

biotin, ep'\-biotin allobiotin, epi-allobiotin

Of the four racemic diastereoisomers, all except DL-biotin have been
reported to be essentially inactive biologically as indicated in Table 11.

544

THE BIOCHEMISTRY OF B VITAMINS

Table 11. Specificity of the Configuration of Biotin.

Compound

Organism

Activity

Reference

(+)-Biotin

100

(-)-Biotin

Rat
Chick

Lactobacillus casei
Lactobacillus arabinosus

0(<13)

0.023

0.006-0.019

12
13

DL-Biotin

Rat
Chick

Lactobacillus casei
Lactobacillus arabinosus
Saccharomyces cerevisiae

50 ca
49

51, 50
50

14. 15

DL-epi-Biotin

Saccharomyces cerevisiae

DL-Allobiotin

Rat
Chick

Lactobacillus casei
Lactobacillus arabinosus

0
0

0.0029
0.002

12
13
13
14

DL-epf-Allobiotin

Lactobacillus arabinosus

However, the racemic biotin is only half as active as the natural biotin
which is dextrorotatory. Synthetic ( + ) biotin is just as active as natural
biotin, whereas the synthetic ( — ) biotin is essentially inactive. The small
amount of activity which has been found is usually attributed to con-
tamination of the sample with traces of the dextrorotatory form.

Kogl and co-workers 16~19 have concluded that two isomeric forms of
biotin exist in nature, a-biotin isolated from egg yolk and /3-biotin
isolated from liver by the procedure of du Vigneaud et al.4 A mixture of
a-biotin (m. p. 220° C) and ^8-biotin (m. p. 232-3° C) melted at 197-
202° C; and a mixture of the methyl ester of a-biotin (m. p. 161-2° C)
and the methyl ester of /?-biotin (m. p. 163-4° C) showed a melting point
depression of 20-30° C. Specific rotations of the two biotins and their
methyl esters differed markedly. More significant was the isolation of a
sulfocaproic acid by hydrolysis of a-biotin to a diaminocarboxylic acid,
oxidation of the latter with lead tetracetate to an aldehyde, and finally
oxidation of the aldehyde with potassium permanganate in a series of
steps. This sulfocaproic acid, which was converted by alkali fusion to a
product identified as a, /?-dimethylbutyric acid, was found to be identical
with the synthetic ( — ) form of a-isopropyl-/?-sulfopropionic acid when
the melting points and mixed melting points of the anilides and m-tolu-
idine salts of each were studied. Since there is no branching in the carbon
skeleton of biotin, such a degradation product would indicate that another
structural form exists.

Early work on the biological activity of the two biotins indicated that
/3-biotin was approximately twice as active as a-biotin for yeast, Rhizo-

BIOTIN 545

bium trifolii and Clostridium butylicum}' 20 However, more recent experi-
ments 21 on a sample of a-biotin isolated by Kogl indicate that with
corrections for impurity of the natural biotin and corrections for the
inactivity of ( — ) biotin in DL-/?-biotin, a-biotin possesses 90-96 per cent
of the activity of synthetic DL-/?-biotin for Lactobacillus casei 7469,
Lactobacillus pentosus 124-2, Saccharomyces cerevisiae Y-30, Clostridium
acetobutylicum S-9 and Neurospora crassa 1-A wild. If two such isomeric
forms of biotin exist, it is indeed remarkable that for a wide variety of
organisms the isomers have essentially identical biological activities.

Oxybiotin (O-Heterobiotin). The synthesis of DL-hexahydro-2-oxo-lH-
furo[3,4]imidazole-4-valeric acid, the biologically active oxygen analogue
of biotin, was reported almost simultaneously by Hofmann 22 and by
Duschinsky et al.23 Both O-heterobiotin 23 and oxybiotin 24 have been
suggested as trivial names for the analogue which is indicated by the fol-
lowing formula:

CO
HN NH

HC CH

H2C CH— (CH2)4— COOH

oxybiotin (O-heterobiotin)

Since this analogue might exist in stereoisomeric modifications similar
to those of biotin, all four theoretically possible racemic forms of 3,4-
diamino-2-tetrahydrofuranvaleric acids were prepared.25 Attempts to con-
vert these to the corresponding hexahydro-2-oxo-lH-furo[3,4]imidazole-
4-valeric acids were successful with only two of the four diastereoisomers.
Since only the two c{s-3,4-diamino-2-tetrahydrofuranvaleric acids formed
bicyclic ureylene derivatives, it appears that the tetrahydrofuran ring
is more planar in its configuration than the tetrahydrothiophene ring,
which apparently allows the formation of ircms-ureylene derivatives. As
in the case of biotin, the spatial arrangement of the side chain with
respect to the ureylene group of the two diastereoisomers is still unknown.

Of the two racemic diastereoisomers of the oxygen analogue of biotin,
only one, DL-oxybiotin (which is identical with DL-O-heterobiotin) ,26
possesses appreciable biological activity. The other, DL-epi-oxybiotin,25
is reported to have slight activity (0.1 per cent that of oxybiotin) for
Lactobacillus arabinosus; however, it is suggested that it is likely that
the activity is caused by DL-oxybiotin as a contaminant. The racemic
modification of the biologically active form has not as yet been resolved;

546 THE BIOCHEMISTRY OF B VITAMINS

however, if it is analogous to biotin, only one of the optically active
forms would be expected to be biologically active.

As indicated in Table 12, this oxygen analogue of biotin is capable of
replacing the vitamin in the nutrition of a wide variety of organisms.
For Lactobacillus arabinosus and Lactobacillus pentosus 124-2, DL-oxy-
biotin is as active as DL-biotin, giving growth responses at various
concentrations identical with those obtained with biotin. However, the

Table 12. The Biological Activity of Dh-Oxijbiotin (O-Helerobiotin).

Activity
per cent
Organism of (+) biotin Reference

Rat 6.0-2.9^e 27- 28- 31

Chicks 17* 27'29

20-2'. d 30

Lactobacillus arabinosus 50* 24' 41

Lactobacillus pentosus 124-2 50 33

Lactobacillus casei 40 2i- 34

22-25 23' 31

Streptococcus faecalis R 0(pH 6.6)' 32

7.5(pH 7.3)' 32

Rhizobium trifolii 12-1.3" 34

Saccharomyces cerevisiae 25-10c 23' 24, 31- 34

Saccharomyces carlsbergensis 209 31

° If the biological activity resides in only one of the optically active forms of the oxygen analogue, these
values should be doubled.

4 Biotin deficiency induced by feeding raw egg white with biotin deficient diet; DL-oxybiotin injected
intramuscularly or subcutaneously.

• The activity of the analogue relative to ( + ) biotin varies at different concentrations of the analogue;
i.e., the dose-response plots differ in shape for the two compounds. The relative activity of the analogue
tends to decrease with increases in concentrations.

d Animals on biotin deficient diet only.

« Activities of 29-44 per cent dependent upon pH of the medium have been reported.32

/ Activity reported to be dependent upon pH of medium.

» Slight variations at different concentrations.

activity of this analogue is not so pronounced for other organisms, and
relative to biotin, this analogue is often less effective in obtaining the
maximum response of the organism than in eliciting a suboptimal re-
sponse. This is particularly true of Rhizobium trifolii and Saccharomyces
cerevisiae. For Saccharomyces cerevisiae, oxybiotin is approximately 25
per cent as effective as ( + ) biotin in stimulating the fermentation rate
of biotin-deficient cells.40

It is interesting to note that the activity of oxybiotin for Streptococcus
faecalis R has been reported to be dependent upon the pH of the medium.
The analogue is approximately 7.5 per cent as active as ( + ) biotin at
pH 7.3, but it is essentially inactive at pH 6.6.

In the rat oxybiotin administered by daily subcutaneous injections
completely cured the skin lesions resulting from biotin deficiency caused
by feeding raw egg white, and spastic paralysis of the hind legs observed
in a few animals was also completely cured with the oxygen analogue.28
In stimulating growth, DL-oxybiotin was from 2.9 to 6.0 per cent as

BIOTIN 547

effective as ( + ) biotin. The lower figure was obtained for near maximal
response of the organism, whereas the higher value represents the relative
efficiency in eliciting a suboptimal response. Hence, in plots of dose-
response for the rat, the general shape of the curves for oxybiotin and
biotin differ.

As little as 8 y of DL-oxy biotin injected intramuscularly in chicks
(White Leghorn cockerels) rendered deficient in biotin by supplementing
a biotin-deficient diet with raw egg white caused the disappearance of
the mandibular lesions within a week, and some healing of the feet.
After three weeks, the hard, scaly, cracked skin of the bottom of the
feet sloughed off, leaving normal tissue; but edema was still evident in
some segments of the toes of the chicks. No deficiency symptoms of
chicks on a biotin-deficient diet were obtained if a supplement of oxy-
biotin was provided; chicks becoming deficient on the diet were cured
by oxybiotin. Injected intramuscularly, DL-oxybiotin was approximately
17 per cent as active as ( + ) biotin in eliciting growth response of day-
old chicks maintained for one week on a biotin-deficient diet containing
raw egg white. The plots of dose-response for both compounds are similar
in shape.29

These results contrast sharply with those of another group,30 who
report that the oxygen analogue, when administered as a supplement in
the diet instead of being injected intramuscularly, fails to replace biotin
completely for growth of the chick (White Leghorn cockerels) on a
biotin-deficient diet. It is reported that the racemic analogue at 20 y per
100 g of diet is approximately 20 per cent as active as ( + ) biotin in
promoting growth, but at high concentrations of the analogue in the diet
it is only 0.5-3 per cent as effective as biotin. The relative activities are
reported to vary inversely with the amount of analogue fed. Since optimal
growth as obtained with biotin was not attained with any concentration
of the analogue up to 1000 y per 100 g of diet, it was suggested that the
analogue fulfilled only a part of the function of biotin in the chick. The
oxygen analogue is reported to have about one-third the activity of
biotin in curing the dermatitis which develops in the deficient animals.
It will be interesting indeed if the activity of oxybiotin (O-heterobiotin)
can be demonstrated to be dependent upon the method of administration
to the chick.

Although several analogues which were very active in replacing the
corresponding vitamins had previously been prepared and tested, the
oxygen analogue of biotin afforded the first opportunity for development
of specific assays useful in determining whether the analogue was con-
verted into the vitamin or was utilized as such by organisms in which
it had vitamin activity. Two direct assays for oxybiotin have been

548 THE BIOCHEMISTRY OF B VITAMINS

developed; they depend upon the destruction of biotin by Raney's
nickel 35 and by oxidation with permanganate.30 The oxygen analogue is
not appreciably affected by either of the reagents, but Raney's nickel
quantitatively converts biotin to desthiobiotin and permanganate oxidizes
biotin to the corresponding sulfone. Since neither desthiobiotin nor the
sulfone of biotin possesses any appreciable activity for Lactobacillus
arabinosus, as well as for several other organisms, such organisms which
respond to oxybiotin may then be utilized for a direct assay for the
oxygen analogue.

Indirect assays for oxybiotin by assaying directly for biotin in the
presence of oxybiotin have been developed. The response of Lactobacillus
arabinosus to moderate amounts of oxybiotin is prevented by either 800
my per 10 cc of biotin sulfone or 70 y per 10 cc of y- (3,4-ureylenecyclo-
hexyl) butyric acid.37 Under these conditions, neither of the inhibitors
appreciably affects the utilization of biotin by the organism. The biotin
is thus determined directly, and from the response of the organism to
the assay sample in the absence of the inhibitors a differential assay for
oxybiotin is obtained. Of course, large amounts of oxybiotin overcome
the toxicity of the inhibitors and would prevent the determination of
relatively small amounts of biotin.

Another indirect assay depends upon the inability of Streptococcus
jaecalis R to utilize the oxygen analogue effectively. This permits the
estimation of biotin in an extract with only slight interference from the
oxygen analogue. By simultaneous assays with Lactobacillus arabinosus
and Streptococcus jaecalis R, a direct assay for biotin and a differential
assay for the oxygen analogue of biotin have been developed.32

With these assays, oxybiotin has been demonstrated not to occur
naturally in any of the organisms tested, and it has been possible to show
that the analogue is utilized as such without prior conversion to biotin.
Thus, permanganate destroys the biotin activity of hydrolysates of cells
of either Saccharomyces cerevisiae or Rhizobium trifolii grown in a biotin-
containing medium, but does not destroy the biotin-like activity of such
hydrolysates from cells of the organism grown in the presence of oxy-
biotin instead of biotin.36 In balance experiments 38 with Saccharomyces
cerevisiae 139 grown in a medium containing 10 my of DL-oxybiotin
per 250 cc, essentially all (94-97 per cent of the compound) was recovered
from the medium and cells. At higher concentrations (100 my per 250 cc),
the recovery was lower (84 per cent) . By three different methods, the
Raney's nickel method, the permanganate method, and the differential
growth inhibitor method, it was demonstrated that oxybiotin alone
(with the exception of the small amount of biotin added with the in-
oculum) accounted for all the biotin-like activity in the cells.38

BIOTIN 549

With the permanganate method, oxybiotin has been demonstrated as
the substance accounting for the biotin-like activity of hydrolysates of
cells of Lactobacillus pentosus 124-2 grown in an oxybiotin medium.33
No biotin could be demonstrated in the hydrolysates of the cells by the
differential growth inhibitor method. There was no significant difference
in the growth rate of the organism when oxybiotin replaced biotin in
stimulating growth. Either oxybiotin or biotin was absorbed from the
medium into the cell in larger amounts than necessary for growth, and
balance studies indicated that destruction of either compound became
apparent only at relatively high concentrations, where recoveries were
as low as 15 per cent.

After several weeks of intramuscular injections of oxybiotin, the liver,
heart, spleen, lung and a sample of leg muscle were removed from chicks
which had been maintained on a biotin-deficient diet containing dry raw
egg white.39 All these tissues assayed for oxybiotin by both the perman-
ganate method and the Raney's nickel method had a high content of
oxybiotin. The actual biotin content of the tissues was essentially iden-
tical with that of tissues from biotin-deficient chicks. Injections of chicks
with microbiologically equivalent amounts for Lactobacillus arabinosus
of either oxybiotin or biotin result in storage of these compounds in
similarly equivalent concentrations in the various tissues. Oxybiotin was
found to be bound in the tissues in a manner similar to that of biotin.
It was only partially liberated by hot water, but was readily freed by
acid hydrolysis.

In contrast to this, it has been reported that administration of the
oxygen analogue orally to biotin-deficient chicks does not result in
appreciable accumulation of the analogue in the liver or leg muscle, even
though the analogue gives a growth response and cures the dermatitis of
the chicks.30

However, in total balance studies,39 analysis of acid hydrolysates of
whole chicks grown on a biotin-deficient diet and of their combined
excreta by both the permanganate method and the Raney's nickel method
has demonstrated that the biotin content of chicks injected with oxy-
biotin was identical with that of control chicks and not significantly
different from that of newly hatched chicks. The amount of biotin
excreted by chicks injected with oxybiotin was identical with that ex-
creted by control chicks on the same biotin-deficient diet. Also, of a
total dosage of 32 or 64 y of DL-oxy biotin injected into chicks over a
period of two weeks, essentially all (75 per cent) of the oxybiotin was
recovered and was distributed almost equally between the chick and
excreta. Injected biotin was similarly recovered.

These results with the chick and other organisms demonstrate that the

550

THE BIOCHEMISTRY OF B VITAMINS

biological action of oxybiotin is a property of the molecule. Since it is
not converted by the organisms into biotin, it must be utilized per se in
the formation of the necessary coenzyme (s), which in turn can function

Table 13. Growth-promoting Activities of Analogues of Biotin and Oxybiotin.

Compound

(+)-Desthiobiotin°'8

DL-Desthiobiotin" •'

DL-Desthioallobiotin
Biotin methyl ester'

DL-Oxybiotin methyl ester'
Biotin sulfoxide methyl ester8

Biotin sulfone*

«s-3,4-Diamino-2-tetra-
hydrothiophenevaleric acid'

Organism

Saccharomyces cerevisiae

Lactobacillus casei

Saccharomyces cerevisiae

Rats*

Saccharomyces cerevisiae

Lactobacillus casei

Saccharomyces cerevisiae

Lactobacillus casei

Saccharomyces cerevisiae

Ratsc

Lactobacillus casei

Saccharomyces cerevisiae

Saccharomyces cerevisiae F.B.

Lactobacillus casei

Rats

Lactobacillus arabinosus

Lactobacillus casei

Rats

Lactobacillus arabinosus

Lactobacillus casei

Lactobacillus arabinosus
Lactobacillus casei

Saccharomyces cerevisiae
Lactobacillus arabinosus
Lactobacillus casei
Saccharomyces cerevisiae
Lactobacillus casei
Saccharomyces cerevisiae
Lactobacillus casei

DL-ct's-3,4-Diamino-2-

tetrahydrothiophenevaleric

acid'
DL-as-3,4-Diamino-2-

tetrahydrofuranvaleric

acid'
DL-Hexahy dro-2-oxo-l H-

furo[3,4]imidazole-4-

pentanol'
DL-Hexahydro-2-oxo-4-

methyl-lH-furo[3,4]imid-

azole
( + )-f ,77-Diaminopelargonic

acid*
DL-5-Methyl-2-thiono-4-

imidazolidinecaproic acid

■Active for Saccharomyces cerevisiae (25 strains), Saccharomyces chodati, Saccharomyces macedoniensis,
Endomycopsisfibuliger,Debaryomyces matruchoti v subglobosus, Mycoderma valida, Mycotorula lactis, Schizo-
saccharomyces pombe, Torula lactosa, Zygosaccharomyces lactis, Zygosaccharomyces marxianus, Neuro-
spora crassa, Neurospora sitophila, Ceratostomella ips 438, Ceratostomella montium, Leuconostoc mesenteroides,
Penicillium notatum 21464, Escherichia coli 58, Ceratostomella reukanfi, Schwanniomyces occidentalis 116.14,51
54. 55 Inactive for Ceratostomella pini 416, Sordaria fimicola, Lactobacillus arabinosus, amd Rhizobium trifolii
205," Penicillium chrysogenum 62078."- "

• Compound competitively inhibits utilization of biotin for this organism.

• Biotin deficiency induced with raw egg white.

d Only 35-50 per cent of optimal growth obtained with biotin is attained; inactive in the absence of
aspartic acid.

• Configuration analogous to (+) biotin.
/ Configuration analogous to Di^-biotin.

Activity

per cent

(+) biotin

References

100

60-50

43, 44

0.1-0.01

12, 44

100

16-10

100 ca.

0.1<*

47, 48, 63

0.1

49, 53

0(<0.01)

0«1)

12, 47

1.8

3.6

0(0.4)

0.01

0.35-0.5

0.13

0.07-0.03

0.0001

10, 48

0.06

rather efficiently in place of the natural coenzyme. It appears that the
sulfur atom of biotin is not essential for its biological action.

Desthiobiotin. In the course of structural studies,10 natural biotin was
treated with Raney's nickel to remove the sulfur atom of the tetrahydro-

BIOTIN 551

thiophene ring. The resulting product, ( + ) desthiobiotin, gave an un-
usual result in the biotin assay with Saccharomyces cerevisiae.*1 ( + )
Desthiobiotin was just as active as ( + ) biotin, and the dose-response
curves of the assays were essentially identical. However, the derivative
not only was inactive for Lactobacillus casei but inhibited competitively
the response of the organism to biotin.42, 51

The activity of desthiobiotin is limited to only one of the four optically
active forms of the compound. Synthetic DL-desthioallobiotin is inactive
for Saccharomyces cerevisiaef synthetic DL-desthiobiotin is approx-
imately half as active as ( + ) desthiobiotin.43,44

As indicated in Table 13, the utilization of desthiobiotin in place of
biotin is rather widespread. Organisms which utilize desthiobiotin have
been found to convert the compound into substances which possess
biotin-like activity for organisms which require biotin, but do not utilize
desthiobiotin.14, 42, 63 The biotin content of cells of Saccharomyces cerevi-
siae grown on desthiobiotin was determined by differential assay with
Lactobacillus casei and the yeast. When present in low concentration, the
desthio compound was apparently converted quantitatively into biotin;
however, at higher concentrations it was less effectively converted into
biotin, and resting cells did not convert any measurable amounts of the
compound to biotin or biotin-like substances.14, 42 Further evidence for
the conversion of desthiobiotin to biotin is afforded by the observation
that for Lactobacillus casei, Lactobacillus arabinosus and Rhizobium
trifolii, Raney's nickel destroys the biotin activity of cells of Saccha-
romyces cerevisiae grown on either biotin or desthiobiotin.14 Perman-
ganate also destroys the activity.36 This contrasts with the results obtained
with oxybiotin.

The natural occurrence of desthiobiotin is suggested by the accumula-
tion in an x-ray induced biotinless mutant of Penicillium chrysogenum,
strain 62078, of a substance which has the biological properties of
desthiobiotin. The substance is inactive for Lactobacillus casei, but active
for Neurospora crassa and Escherichia coli 58, a mutant strain requiring
biotin.54 The last two organisms utilize either desthiobiotin or biotin
while the Penicillium mutant requires biotin and is unable to utilize
desthiobiotin. Escherichia coli accumulates a biotin precursor which may
be desthiobiotin.62

From these observations it appears that desthiobiotin is a normal
precursor, or is converted to a normal precursor of biotin by a number of
organisms. These results are further substantiated by results with a
desthiobiotin inhibitor (p. 468).

Pimelic acid, which was shown to be an essential growth factor for
certain strains of diphtheria bacillus,56 can be replaced by biotin in the

552 THE BIOCHEMISTRY OF B VITAMINS

nutrition of the Allen strain of the organism.57 Pimelic acid, however,
does not replace biotin or desthiobiotin for a majority of organisms requir-
ing biotin.54' 58 Pimelic acid, and also suberic and azelaic acids, enhanced
effectively the biosynthesis of biotin in Aspergillus niger, an organism
which requires neither biotin nor pimelic acid.59 The biosynthesis is fur-
ther enhanced by certain sulfur compounds, such as cystine and cysteine.
An increase in the accumulation of a substance similar to desthiobiotin in
the biotinless mutant of Penicillium chrysogenum, strain 62078, is ob-
tained on supplementing the medium with pimelic acid.54 Hence, the
effects of pimelic acid and desthiobiotin appear to be those of precursors
of biotin as indicated below:

HOOC— (CH2)6— COOH — ^

CO CO

/ \ / \

HN NH HN NH

HC CH — > HC CH

CH3 CH2— (CH2)4— COOH H2C CH— (CH2)4— COOH

\ /

Other Stimulatory Biotin Analogues. As indicated above, the oxygen
analogue of biotin and precursors of biotin replace the vitamin in the
nutrition of a wide variety of organisms. A considerable number of
analogues and derivatives of biotin and oxybiotin possess considerable
activity. The activities of these compounds are indicated in Table 13.
In many instances, biotin is formed from the derivatives, but in several
cases the activities appear to be inherent in the analogue.

It is interesting to note that the methyl ester of biotin is inactive for
Lactobacillus casei but fully active for Saccharomyces cerevisiae. The
methyl ester of oxybiotin is slightly less active than the free acid for the
latter organism.

The sulfoxide of biotin is as active as biotin for Saccharomyces cere-
visiae, but the sulfone which is an antagonist of biotin for Lactobacillus
casei has only slight growth-promoting activity for the yeast. This slight
activity is dependent upon the presence of aspartic acid in the medium.
Hence, it appears that the sulfone cannot replace all the functions of
biotin for this organism. Growth obtained with the sulfone at any con-
centration never attained more than 35 to 50 per cent of the maximum
growth obtained with cultures grown on biotin. The results indicate that
the sulfone may be utilized as such without prior conversion to biotin.

The diamino acids obtained on hydrolysis of biotin, desthiobiotin and

BIOTIN • 553

oxybiotin have activities up to 10 per cent of that of biotin for a number
of organisms.

NH2 NH2 NH2 NH2

H— C C— H H— C C— H

H2C CH— (CH2)4— COOH

I I

CH3 CH2— (CH2)<— COOH

cis-S ,4-diamino-2-tetrahydro- f ,t]-diaminopelargonic acid

thiophenevaleric acid

H-i

NH2 NH2
I
C— H

H2C CH— (CH2)4— COOH

cis-3,4-diamino-2-tetrahydrofuranvaleric acid

On the basis of the activity of the diaminocarboxylic acid derived from
biotin, it has been proposed that biotin may function as a coenzyme
involved in utilization of carbon dioxide.60

Slight activity has been noted for an oxybiotin analogue in which the
valeric acid side chain has been replaced by a methyl group. The activity
of the 4-pentanol derivative corresponding to oxybiotin may be the
result of oxidation of the compound to oxybiotin. Analogous to this
result, it has been reported that replacement of the alcohol group to form
a number of sulfur analogues results in compounds possessing slight
activity. Thus, DL-hexahydro-2-oxo-lH-furo [3,4] imidazole-4- (5-pen-
tanesulfonic acid) has slight growth-promoting activity for both Lacto-
bacillus arabinosus and Saccharo?nyces cerevisiae; and both DL-hexa-
hydro-2-oxo-4- (5-benzylthiopentyl) -lH-furo [3,4] imidazole and DL-hex-
ahydro-2-oxo-4-(5-mercaptopentyl)-lH-furo [3,4] imidazole are slightly
active in replacing biotin for Lactobacillus arabinosus.61

Inhibitory Analogues of Biotin

Desthiobiotin and Related Compounds. Desthiobiotin, which was
obtained by du Vigneaud and associates 10 during structural studies on
cleavage of biotin with Raney's nickel, was found to possess growth-
promoting activity comparable to biotin for Saccharo?nyces cerevisiae.*1
On the other hand, it was found to prevent competitively the utilization
of biotin by Lactobacillus casei.i2< 51 The activities of this derivative
stimulated the preparation and testing of numerous analogues. These
compounds are listed in Table 14. The inhibitory activities, unless indi-

554

THE BIOCHEMISTRY OF B VITAMINS

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cated otherwise, are listed in terms of the molar inhibition ratio,48 which
has been defined as the number of molecules of the inhibitor necessary
to prevent the biological effect of one molecule of biotin. The molar in-
hibition ratio 4S of inhibitor to biotin is obtained experimentally by
determining the amount of the inhibitor required to reduce the growth
obtained with 0.0002 y of biotin to the level of growth obtained with
0.0001 y of biotin. The molar inhibition ratio is approximately twice the
inhibition index for half-maximum inhibition of growth, and is usually
considerably less than the inhibition index just necessary for complete
inhibition of growth. In replacing biotin with oxybiotin (O-heterobiotin)
or desthiobiotin, amounts of the compounds biologically equivalent to
the indicated amounts of biotin are used for determining the molar
inhibition ratios.

Since ( + ) desthiobiotin is approximately twice as active as dl-
desthiobiotin in preventing the utilization of biotin by Lactobacillus
casei, the inhibitory action appears to be the result of the action of only
the dextrorotatory form, which is also the only form which appears to
exert a growth-promoting activity for Saccharomyces cerevisiae. The
oxygen analogue of biotin is also capable of preventing the toxicity of
desthiobiotin for Lactobacillus casei, but is much less effective. The
results indicate that the affinity of oxybiotin for the enzyme involved is
considerably less than that of biotin.

The more effective inhibitory analogues of desthiobiotin, which are
indicated by the following formulas, represent modifications of desthio-
biotin in which the length of the side chain containing the carboxyl group
CO CO

/ \ / \

HN NH HN NH

HC CH H2C CH

CH, CH2— (CH2)7— COOH CH2— (CH2)4— COOH

VL-5-methyl-2-oxo-4~ mj-2-oxo-It-imidazolidine-

imidazolidinepelargonic acid caproic acid

CO
/ \

HN NH

HC CH

CH3

CH2-(CH2)4— S03H

T>ii-5-methyl-2-oxo-Jr-iinidazolidine-
pentanesidfonic acid

is varied, the 5-methyl group is omitted, or the carboxyl group is replaced
by a sulfonic acid group. These compounds have not as yet been reported
to be particularly effective antagonists of biotin.

556 THE BIOCHEMISTRY OF B VITAMINS

Nevertheless, a few of these analogues appear to be very potent in
preventing the utilization of desthiobiotin for certain organisms (Table
14). This is especially true of DL-2-oxo-4-imidazolidinecaproic acid for
Escherichia coli. Since biotin prevents the inhibition noncompetitively,
the analogue appears to prevent the conversion of desthiobiotin to biotin
(p. 468). Whether a similar situation exists with other analogues has
not been determined. In some cases both the conversion of desthiobiotin
to biotin and the utilization of biotin may be prevented by the inhibitor.

Table 15. Sulfide and Sidfone Analogues of Biotin.

Molar Inhibition Ratio

Compound

Organism

Biotin

Desthio-
biotin

References

DL-Norbiotin

Saccharomyces cerevisiae
Lactobacillus casei

1,000
13,000

71
71

dl-Ho mobiotin

Saccharomyces cerevisiae
Lactobacillus casei
Lactobacillus arabinosus

700
130

43,000

71
71
71

DL-Bishomobiotin

Saccharomyces cerevisiae
Lactobacillus casei

30,000
7,000

DL-Trishomobiotin

Saccharomyces cerevisiae
Lactobacillus casei

50,000
3,000

Biotin sulfone0

Lactobacillus casei
Neurospora crassa
Escherichia coli 58 b

280
1,000 ca
1,000 ca

1,000 ca
1,000 ca

48
54
64

DL-Homobiotin sulfone

Saccharomyces cerevisiae
Lactobacillus casei

40,000
400

71
71

DL-Bishomobiotin
sulfone

Saccharomyces cerevisiae
Lactobacillus casei

60,000
8,000

71
71

DL-Trishomobiotin
sulfone

Saccharomyces cerevisiae
Lactobacillus casei

60,000
6,000

71
71

" Also inhibitory to Lactobacillus arabinosus :ui<l Staphylococcus aureus.™
b A mutant strain requiring biotin or desthiobiotin for growth.

Desthiobiotin is reported to inhibit the functioning of biotin in Sordari
fimicola and Ceratostomella pini 416. Desthiobiotin is also reported to
have an inhibitory effect on the growth of tumors.70 The response of
Lactobacillus arabinosus to biotin is increased by the presence of desthio-
biotin, even though the desthio compound is inactive alone.51

Neither 5-methyl-2-oxo-4-imidazolidinebutyric acid 64 nor 5-methyl-4-
hexyl-2-imidazolidone 66 has appreciable inhibitory activity for either
Lactobacillus casei or Saccharomyces cerevisiae.

Sulfide and Sulfone Analogues of Biotin. The most potent inhibitors
of the utilization of biotin which have been reported are the homologues
of biotin, and the sulfone of biotin and its homologues.48, 54, n Of these,
biotin sulfone and DL-homobiotin are the most effective antagonists of
biotin, as indicated in Table 15. When comparing the potencies of vitamin

BIOTIN

557

analogues, the organism must be specified. While biotin sulfone can, in
the presence of aspartic acid, replace biotin in stimulating the growth of

HN
Hi-

h2c

CH— (CH2)4— COOH
"SC-2
biotin sulfone

CO
HN NH

HC CH

H2C CH— (CH2)5— COOH

homobiotin

Saccharomyces cerevisiae, it is an effective antagonist of biotin for
Lactobacillus casei. Even homobiotin is not a particularly effective an-
tagonist of biotin for Lactobacillus arabinosus.

Analogues of Oxybiotin (O-Heterobiotin). The activities of the hom-
ologues of oxybiotin and related compounds are indicated in Table 16.
Oxybiotin analogues are either inactive or relatively ineffective in pre-
venting the functioning of biotin, but they may prevent the utilization
of oxybiotin. It appears that oxybiotin and its analogues do not combine

Table 16.

Analogues of Oxybiotin (O-Heterobiotin).

Molar Inhibition Ratio

Compound

Organism

Biotin

Oxybiotin

(O-Hetero

biotin)

References

DL-Bisnoroxybiotin

Saccharomyces cerevisiae
Lactobacillus arabinosus

> 500,000

72
72

DL-Noroxybiotin

Saccharomyces cerevisiae
Lactobacillus arabinosus

> 500,000

143,000

72
72

DL-Homooxybiotin

Saccharomyces cerevisiae
Lactobacillus arabinosus

445,000
> 500,000

7,400
225,000

40, 72

DL-Bishomodxybiotin

Saccharomyces cerevisiae
Lactobacillus arabinosus

> 500,000

30,000

DL-Hexahydro-2-oxo-l H-

furo[3,4]imidazole-4-
(4-butanesulfonic acid)
(Oxybiotin sulfonic acid)

Saccharomyces cerevisiae

1,460,000

16,600

40, 61

DL-Hexahydro-2-oxo-4-
(4-benzylthiobutyl)-lH-
furo[3,4]imidazole

Saccharomyces cerevisiae

740,000

9,300

40, 61

" No significant inhibition.

as strongly with the enzyme involved as does biotin and its corresponding-
analogues. The preparation of the lower and higher homologues of oxy-
biotin has allowed some comparison of their inhibitory activities in the
presence of oxybiotin with the activities of the corresponding analogues
of biotin in the presence of biotin. For Saccharomyces cerevisiae, dl-

558 THE BIOCHEMISTRY OF B VITAMINS

bishomooxybiotin prevents the utilization of oxybiotin at approximately
the same molar ratio at which DL-bishomobiotin prevents the utilization
of biotin. However, this does not hold true for inhibitions with homooxy-
biotin and homobiotin. The molar ratios determined with the correspond-
ing growth factors differ by a factor of ten, and the discrepancy increases
to over 100 fold for the next lower homologues of oxybiotin and biotin.
In the presence of ammonium sulfate, biotin or oxybiotin stimulates
the fermentation rate of biotin-deficient yeast. If either DL-homooxy-
biotin or the sulfonic acid corresponding to oxybiotin is added before

CO CO

HN NH HN NH

HC CH HC CH

H2C CH— (CH2)6— COOH H,C CH— (CH2)4— S03H

o o

homooxybiotin hexahydro-2-oxo-l H-furo[8,4]imidazole-

4~(4~butanesulfonic acid)
(oxybiotin sulfonic acid)

oxybiotin or biotin, the analogues prevent the fermentation at inhibition
ratios of 3,750 and 5,000 respectively, for oxybiotin, and 375,000 and
600,000 for biotin. If the inhibitors are added after the addition of biotin,
no inhibitory effect is observed, indicating that the inhibitors prevent
the conversion of biotin to a functional form which is not antagonized by
the analogues. The effect of aspartic acid on fermentation is not altered
by the inhibitors.40

DL-Hexahydro-2-oxo-4- (5-benzylthiopentyl) -lH-furo [3,4] imidazole,
DL-hexahydro-2-oxo-4- (4-mercaptobutyl) -lH-furo [3,4] imidazole, and
DL-hexahydro-2-oxo-4- (5-mercaptopentyl) -lH-furo [3,4] imidazole are re-
ported to have an inhibitory effect on Saccharomyces cerevisiae.

Ureylenephenyl and Ureylenecyclohexylbutyric and Valeric Acids. A
group of 2,3- and 3,4-ureylenephenyl and ureylenecyclohexylbutyric
and valeric acids have been synthesized and found to inhibit the utiliza-
tion of biotin in a number of organisms. The activities of these compounds
against Lactobacillus casei and Saccharomyces cerevisiae are indicated
in Table 17. y-(2,3-Ure3^1enecyclohexyl) butyric acid and S-(2,3-urey-
lenecyclohexyl) valeric acid are the most effective compounds against
yeast, whereas y- (3,4-ureylenecyclohexyl) butyric acid was the most effec-
tive against Lactobacillus casei. Two diastereoisomers distinguished by
different melting points were obtained in the case of each of the 2,3-
ureylenecyclohexyl-derivatives; however, in contrast to other biotin

BIOTIN

559

HN NH

\ /

HC — CH

H2C CH— (CH2)3— COOH

H2C — OH2

y-{2,3-ureylenecyclohexyl)butyric acid

HN NH

HC— CH
/ \

H2C CH— (CH2)4— COOH

H2C — CH2

b-{2 ,3-ureylenecyclohexyl)
valeric acid

HC— CH
/ \

H2C CH2

H2C — CH— (CH2)3— COOH
y-(8,4-ureylenecyclohexyl)butyric acid

analogues and stereoisomers, the biological activities of the diastereoiso-
mers were almost identical.

Table 17. Ureylenebenzene and Cyclohexane Derivatives as Inhibitory Biotin Analogues

Molar

Lactobacillus

Inhibition Ratio *

Saccharomyces

Analogue

casei

cerevisiae

7-(2,3-Ureylenephenyl)butyric acid

25,000,000

310,000

■y-(2,3-Ureylenecyclohexyl)butyric acid,"

12,500,000

1,500

m. p. 218-220°

7-(2,3-Ureylenecyclohexyl)butyric acid,"

6,250,000

1,500

m. p. 192-194°

5-(2,3-Ureylenephenyl)valeric acid

6,250,000

2,500,000

5-(2,3-Ureylenecyclohexyl)valeric acid,"

31,000

3,000

m. p. 222-226°

5-(2,3,-Ureylenecyclohexyl)valeric acid,°

31,000

3,000

m. p. 183-184°

7-(3,4-Ureylenephenyl)butyric acid6

1,500,000

6,250,000

7-(3,4-Ureylenecyclohexyl)butyric acid6

4,000

156,000

S-(3,4-Ureylenephenyl)valeric acid

750,000

1,560,000

8-(3,4-Ureylenecyclohexyl)valeric acid

31,000

156,000

0 Stereoisomeric modifications distinguished only by melting point.
6 Inhibits growth of Lactobacillus arahinosus.

Avidin

On a well balanced diet to which relatively large quantities of dried
egg white have been added, rats lose their hair and develop a severe
dermatitis and skin hemorrhages; these symptoms are accompanied by

560 THE BIOCHEMISTRY OF B VITAMINS

nervous disorders and loss of weight. A spasticity develops which in the
later stages of the deficiency causes the rats to assume a typical kangaroo-
like posture, and unless the condition is remedied, death ensues.74

The principle in egg white which is responsible for the detrimental effect
has the properties characteristic of a protein; that is, it is destroyed by
heat or mild hydrolysis with dilute acid and is precipitated by such
agents as ammonium sulfate. A naturally occurring substance which
exerts a protective action against this egg white injury has been termed
vitamin H by Gyorgy.75 The identity of vitamin H with biotin was
finally established 76> 77, 78 by testing a sample of biotin methyl ester
isolated by Kogl.2

The protein in raw egg white which renders biotin unavailable to
animals was found also to prevent the utilization of biotin by Saccharo-
myces cerevisiae 79 and other microorganisms requiring biotin for growth.
Thus, a protein constituent of the egg white forms a stable, nondialyzable
complex with biotin. The combination between the protein and biotin
was found to occur in stoichiometric amounts. With the microbiological
test for the egg white factor, the protein which combines with biotin has
been isolated by Eakin and associates 79, 80 in crystalline form and named
avidin. Purified avidin produced effects in rats similar to those caused
by dried egg white.81 With the assumption that one molecule of biotin
combines with one molecule of protein, the molecular weight of avidin
has been calculated to be 43,500. The isoelectric point occurs at approxi-
mately pH 10.82 Some loss of activity was inherent in crystallization, since
the potency of a crystalline sample of avidin was approximately 4,000
units per gram as compared with 7,000 units per gram for amorphous
preparations. By definition one unit of avidin combines with 1 y of
biotin.80

It is interesting to note that avidin passes through the alimentary tract
of animals unchanged and can be demonstrated in the feces.83 Even liver,
kidney and proteolytic enzymes are inactive in liberating biotin combined
with avidin.84 However, destruction of avidin by heat treatment liberated
material with biotin-like activity.

The ability of various analogues of biotin to combine with avidin has
been determined. For example, it has been reported that sufficient avidin
completely inhibits the growth promoting activity of biotin sulfone for
Saccharomyces cerevisiae.47 Since limited amounts of avidin added to a
medium containing excess biotin sulfone produce responses in the yeast
characteristic of biotin but not of biotin sulfone, it appears that biotin
sulfone liberates biotin from the complex.

A method for determining the avidin-combinability of biotin analogues
has been developed by Wright and Skeggs,85,86 in which the "relative

BIOTIN 561

affinity" for avidin is determined by varying the analogue concentration
when biotin and avidin are present in stoichiometric amounts. The "rela-
tive affinity" is expressed as a ratio of concentration of analogue to biotin
at which one-half of the biotin remains free and available for growth of
a test organism. With this technique, ( — ) biotin and DL-allobiotin were
found to have no significant affinity for avidin, but DL-epi-allobiotin has
an affinity ratio of approximately 6. The affinity ratios for DL-desthiobio-
tin and 8- (2,3-ureylenecyclohexyl) valeric acid are 10 and 14, respectively.
y-(3,4-Ureylenecyclohexyl) butyric acid and 8-(3,4-ureylenecyclohexyl)-
valeric acid have a definite ability to combine with avidin, but the ratios
are too high to be determined.

Other than those mentioned above, the following have been found to
combine with avidin: DL-oxy biotin,34 DL-oxybiotin methyl ester,34 dl-
hexahydro-2-oxo-lH-furo[3,4]imidazole-4-pentanol 34 and 2-oxo-4-imid-
azolidinevaleric, caproic, enanthic and caprylic acids.48

The inability of «s-3,4-diamino-2-tetrahydrothiophenevaleric acid
(the diaminocarboxylic acid corresponding to biotin) to combine with
avidin was the basis for the first suggestion that avidin combinability
was a function of the imidazolidone ring.48> 49 This is further substantiated
by the inability of £,??-diaminopelargonic acid,48 an analogous derivative
of desthiobiotin, and DL-c{s-3,4-diamino-2-tetrahydrofuranvaleric acid,34
corresponding to oxybiotin, to combine with avidin.

Since desthiobiotin48 and other derivatives not containing the sulfur
atom combine with avidin, the sulfur atom does not appear essential for
the bonding. Also, the methyl ester of oxybiotin 34 and the alcohol
analogue of oxybiotin 34 in which the carbinol group replaces the carboxyl
group combine with avidin, indicating that the carboxyl group is not
essential for this activity. However, there appears to be structural
specificity with regard to the configuration of the molecule necessary for
avidin combinability.

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562 THE BIOCHEMISTRY OF B VITAMINS

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BIOTIN 563

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62. Auhagen, E., Naturwiss., 33, 221 (1946).

63. Leonian, L. H., and Lilly, V. G, J. Bad., 49, 291 (1945).

64. Duschinsky, R., and Dolan, L. A., /. Am. Chem. Soc, 67, 2079 (1945).

65. Dittmer, K., and du Vigneaud, V., J. Biol. Chem., 169, 63 (1947).

66. Duschinsky, R., and Dolan, L. A., /. Am. Chem. Soc, 68, 2350 (1946).

67. Rogers, L. L., and Shive, W., /. Biol. Chem., 169, 57 (1947).

68. McKennis, H., Jr., and du Vigneaud, V., J. Am. Chem. Soc, 68, 832 (1946).

69. Duschinsky, R., and Rubin, S. H., /. Am. Chem. Soc, 70, 2546 (1948).

70. Keresztesy, J. C., Laszlo, D., and Leuchtenberger, C, Cancer Research, 6, 128

(1946).

71. Goldberg, M. W., Sternbach, L. H., Kaiser, S., Heineman, S. D., Scheiner, J.,

and Rubin, S. H., Arch. Biochem., 14, 480 (1947).

72. Hofmann, K., Chen, C., Bridgwater, A., and Axelrod, A. E., /. Am. Chem. Soc,

69, 191 (1947).

73. English, J. P., Clapp, R. C., Cole, Q. P., Halverstadt, I. F., Lampen, J. O., and

Roblin, R. O., Jr., /. Am. Chem. Soc, 67, 295 (1945).

74. Boas, M. A., Biochem. J., 21, 712 (1927).

75. Gyorgy, P., J. Biol. Chem., 131, 733 (1939).

76. Gyorgy, P., Melville, D. B., Burk, D., and du Vigneaud, V., Science, 91, 243

(1940).

77. du Vigneaud, V., Melville, D. B., Gyorgy, P., and Rose, C. S., Science, 92, 62

(1940).

78. Gyorgy, P., Rose, C. S., Hofmann, K., Melville, D. B., and du Vigneaud, V.,

Science, 92, 609 (1940).

79. Eakin, R. E., Snell, E. E., and Williams, R. J., J. Biol. Chem., 140, 535 (1941).

80. Pennington, D., Snell, E. E., and Eakin, R. E., J. Am. Chem. Soc, 64, 469 (1942).

81. Gyorgy, P., Rose, C. S., Eakin, R. E., Snell, E. E., and Williams, R. J., Science,

93, 477 (1941).

82. Woolley, D. W., and Longsworth, L. G., J. Biol. Chem., 142, 285 (1942).

564 THE BIOCHEMISTRY OF B VITAMINS

83. Gyorgy, P., and Rose, C. S, Science, 94, 261 (1941)

85.

gyorgy, P., and Rose, C. S., Proc. Soc. Exptl. Biol. Med., 53, 55 (1943)
Wright, L. D., and Skeggs, H. R., Arch. Biochem., 12, 27 (1947)

It 160L(lM7fkegg8' H' R" and CleSSOn' E- L>' Pr°C- SoC' ^ BioL Med->

Chapter VD

THE FOLIC ACID GROUP

The isolation of members of the folic acid group brought together a
number of divergent studies. The earliest report of a biological activity
which can now be attributed to the folic acid group is the striking effect
of yeast extract in relieving a nutritional anemia occurring particularly
in pregnant women.1 The nutritional deficiency was reproduced in
monkeys,2, 3 and the factor necessary to prevent the deficiency was later
termed vitamin M.4, 5 The folic acid group also accounts for the active
principles which were essential for growth (Factor U)6 and prevention
of nutritional anemia (vitamin Bc) in chicks,7, 8 and essential for the
growth of Lactobacillus casei (Norit Eluate Factor)9,10 and of Strepto-
coccus faecalis R.11

The isolation of the first crystalline member of the folic acid group
was reported by Pfiffner et al.,12 who obtained folic acid from liver.
Shortly afterward, Stokstad 13 also reported the isolation of the same
compound from liver. An enzymatic digest of a yeast concentrate was
used as another source material for the isolation of the crystalline mate-
rial.14 Folic acid has also been isolated in essentially pure form from
spinach.15 Other members of this group of factors were obtained in crystal-
line form within a short period of time.16, 17, 18 Structural studies on folic
acid indicated a xanthopterin-like unit,1923 p-aminobenzoic acid,21-23
and glutamic acid,21-23 combined as indicated by the following formula:

A N
N C C— CH2— NH— (/ J— CO— NH— CH— CH2— CH2— COOH

H,N— C C CH ^=/ COOH

\y \ /

N N

folic acid (N-pteroyl-h-glutamic acid)

The complete structure and synthesis 24, 25 were announced simul-
taneously with the structure of another member of this complex, a factor
derived from the cultural broth of an unidentified organism belonging
to the genus Cory neb acterium.11 This factor was identified as a pteroyldi-

565

566 THE BIOCHEMISTRY OF B VITAMINS

glutamylglutamic acid 21_23 and later synthesized.26 By synthesis, the
structure was established as N-pteroyldi-y-glutamylglutamic acid.

C N r. * O

N C C-CHVNH-^ VcO-CNH-CH-CH^CHo-Oo-NH-CH-CHj-CHj-COOH

H2N-C C CH \=/ COOH COOH

N-pteroyldi-^-glutamylglutamic acid

Another crystalline compound which prevents anemia in chicks, but is
not appreciably active for either Lactobacillus casei or Streptococcus
faecalis R, was isolated from yeast.18 This compound was termed vitamin
Bc conjugate, and later was demonstrated to be a pteroylhexaglutamyl-
glutamic acid.27 An enzyme widely distributed in animal tissues 28~32
hydrolyzes the conjugate to a form which is active for Streptococcus
faecalis R and Lactobacillus casei. Folic acid was isolated after enzymatic
digestion of a concentrate of the conjugate from yeast.14

A fourth crystalline member 16 of the folic complex was isolated from
the fermentation liquors of Rhizopus nigricans and given the trivial name
rhizopterin.33 This compound was subsequently shown to be N10-formyl-
pteroic acid and synthesized 34, 35 from pteroic acid which was previously
prepared synthetically in the course of studies on folic acid.24

OH O

I II

C N H— C

N C C— CH2— N— f

H2N— C C CH

TJ N

Nl0-formylpteroic acid (rhizopterin)

The relative biological activities of these naturally occurring members
of the folic acid complex as well as a number of synthetic, related com-
pounds are indicated in Table 18. All activities, except those for rat and
man, represent the ability of derivatives to replace folic acid as an
essential nutritional factor under controlled experimental conditions.

Supplementary folic acid is not ordinarily required by rats on a puri-
fied diet, but rats fed sulfaguanidine or succinylsulfathiazole in such a
purified diet grow slowly 6G and develop an anemia, an agranulocytosis,
a leucopenia, and a hypocellularity of bone marrow,67, 68 which are cor-
rected by administration of folic acid.55, 69 The effect of sulfonamides,

THE FOLIC ACID GROUP

567

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568 THE BIOCHEMISTRY OF B VITAMINS

which has been attributed to decreased synthesis by intestinal bacteria,
affords a deficiency with which the efficacy of folic acid and its derivatives
can be evaluated for the rat. The effectiveness of folic acid in treatment
of pernicious anemia,70-72 sprue,73 and nutritional macrocytic anemia 70-72
has made available a means of demonstrating in human subjects the
activities of compounds related to folic acid. Increased excretion in the
urine of microbiologically active forms of folic acid on administration of
microbiologically inactive forms of the vitamin has also been used as a
criterion for the utilization of the compounds in human subjects. Although
only small amounts, less than 1 per cent of the normal total intake of
folic acid in human subjects, are regularly excreted in the urine in micro-
biologically active forms,74,75 the administration of relatively large
amounts of folic acid (1-15 mg) often results in urinary excretion of 15
to 75 per cent of the administered dose within 24 hours ;42, 76 however,
the amount excreted depends somewhat on the individual.

Of the two optically active stereoisomers with the structure of folic
acid, only the naturally occurring L-modification appears to be biologi-
cally active, since the racemic modification is approximately half as effec-
tive as the naturally occurring folic acid for Lactobacillus casei and
Streptococcus faecalis R.22 Also, D-folic acid (N-pteroyl-D-glutamic
acid) does not replace L-folic acid in stimulating the production of the
Rous sarcoma (p. 595) in folic acid-deficient chicks.77

Although N-pteroyl-a-glutamylglutamic acid is only about 1 per cent
as effective as folic acid in the nutrition of Lactobacillus casei and Strep-
tococcus faecalis R,26, 36 this folic acid derivative is completely effective
for the chick,36 and also replaces folic acid in stimulation of the Rous
sarcoma virus in folic acid-deficient chicks.77 Administration of N-pteroyl-
a-glutamylglutamic acid (6.5 mg) either orally or intravenously to normal
male human subjects results in an increased excretion of folic acid
amounting to 50 to 75 per cent of that excreted when an equivalent
amount of folic acid (5 mg) is administered.38 Although there is a lag
in the initial rate of excretion, particularly following intravenous admin-
istration, a major portion of the excretion takes place within the first
six hours.38 Pteroyl-a-glutamylglutamic acid has also been reported to
be effective in treatment of pernicious anemia and macrocytic nutritional
anemia, but does not appear to be as active as folic acid.39, 40

Contrasting markedly to the corresponding a-glutamyl derivative,
N-pteroyl-y-glutamylglutamic acid is almost as active as folic acid for
Lactobacillus casei and Streptococcus faecalis R.26

With the exception of Streptococcus faecalis R, N-pteroyldi-y-glutamyl-
glutamic acid is essentially as effective as folic acid for all the organisms
listed in Table 18. With Lactobacillus casei and particularly with Strepto-

THE FOLIC ACID GROUP 569

coccus faecalis R, a sigmoid curve is obtained on plotting the growth
response against concentration of the triglutamate.78, 79

Pteroyldi-y-glutamylglutamic acid, administered either orally or in-
travenously, is just as effective as folic acid in effecting an increase in
the urinary excretion of forms of folic acid active for Streptococcus
faecalis R; however, a very slight lag in the rate of excretion is noted.38
Even so, most of the active substances derived from the triglutamate are
excreted during the first six hours.38

The earliest indication of a beneficial effect of the triglutamate in
human subjects was a report of slight activity in a patient with nutri-
tional macrocytic anemia.41 Administration of 5 mg of the triglutamate
per day gave a reticulocyte response of 12 per cent on the ninth day,
with a subsequent increase in red cell count.41 Subsequently, parenteral
administration of the triglutamate (daily dose equivalent to 3.1 mg of
folic acid) was shown to increase markedly the excretion of folic acid,
and to have pronounced beneficial effects in relieving the clinical and
hematologic manifestations of sprue in a patient previously treated with
liver extracts.42 Treatment of a sprue patient with intramuscular injec-
tions of a crystalline sample of the pteroyldi-y-glutamylglutamic acid
(5 mg twice daily) alone resulted in a rise in erythrocyte count and
hemoglobin, clinical improvement, and maximum reticulocytosis of 38
per cent on the fourth day of therapy.43 Administration of 3 mg daily of
the triglutamate by intramuscular injection to a patient with pernicious
anemia in relapse resulted in a submaximal hemopoietic response, accom-
panied by subjective improvement.44 This is in contrast to an earlier
report indicating that a concentrate of the triglutamate was inactive
when administered to two patients in doses equivalent to 3.6 and 2.3 mg
of folic acid daily.45 However, from more recent work, it appears that
the triglutamate is utilized by patients with pernicious anemia.39

Monkeys rendered anemic and leucopenic by vitamin M-deficient
diets respond to an intramuscular injection of 3 mg of pteroyldi-y-gluta-
mylglutamic acid. Administered in divided doses over a period of three
days, it produces prompt and complete remission lasting for ten to
thirty days.54

The deficiency of folic acid resulting from administration of succinyl-
sulfathiazole or sulfaguanidine in the diet of rats is corrected by pteroyldi-
y-glutamylglutamic acid as well as by folic acid.55 The triglutamyl
derivative also has a preventive and corrective effect on anemia induced
by bleeding rats fed a purified diet containing succinylsulfathiazole.56
The leucopenia which develops in rats given thyroid powder orally or
thyroxine injections and fed thiourea in a purified diet is relieved by the
triglutamate.57 Granulocytopenia, which develops in rats on a purified

570 THE BIOCHEMISTRY OF B VITAMINS

diet, deficient in riboflavin, is also corrected by pteroyldi-y-glutamyl-
glutamic acid.57a

Pteroyldi-y-glutamylglutamic acid is just as effective on a molar basis
as folic acid in promoting growth and hemoglobin formation in
chicks.17- 59, 60 Early work indicated that, while folic acid was effective
alone, chicks required a supplementary factor for the utilization of the
pteroyltriglutamic acid. This factor could be replaced by 5- or 4-pyri-
doxic acid (p. 657) or their lactones (a- or /?-pyracin) .8°-82 4-Pyridoxic
acid was the more effective of the two isomers. Attempts 59, 60 to confirm
this supplementary effect have not been successful, with the exception
of a single experiment involving a comparison between two groups of
six chicks each.83 In hemorrhagic anemia in hens, either pteroyltriglutamic
acid or 4-pyridoxic acid, or more effectively, a combination of the two
compounds, was reported to exert a beneficial action in hastening the
regeneration of hemoglobin.84 The yield of folic acid, determined with
Streptococcus faecalis R from an incubation mixture of pteroyltriglu-
tamic acid and chicken liver, increased twofold on supplementing the
mixture with 4- or 5-pyridoxic acid.85

N-Pteroyldi-y-glutamylglutamic acid is also effective in replacing
folic acid in stimulating the Rous sarcoma virus in folic acid-deficient
chicks.77

This pteroyltriglutamic acid on a molar basis is just as effective as folic
acid in maintaining the concentration of microbiologically active forms of
folic acid in the blood of turkey poults on a folic acid-deficient diet.61
The triglutamate, however, is more effective than folic acid in increasing
the blood concentrations of conjugates of folic acid which are hydrolyzed
by chicken pancreas.61

A conjugase preparation from chicken pancreas86 hydrolyzes N-pteroyl-
di-y-glutamylglutamic acid as well as N-(p-aminobenzoyl)di-y-glutamyl-
glutamic acid and produces one molecule of glutamic acid for each
carboxyl group liberated.87 Assay with S. faecalis R of the reaction mix-
ture resulting from the action of the enzyme preparation on the pteroyl-
triglutamic acid indicated the liberation of activity equivalent to one folic
acid for each glutamic acid. These results suggest that the triglutamate
is hydrolyzed only to the diglutamate, which is approximately as active
as folic acid for S. faecalis R. Since one equivalent of glutamic acid shows
an inhibitory effect on the enzymatic reaction, it is suggested that the
glutamic acid formed may prevent the reaction from going to completion.87

The pteroylhexaglutamylglutamic acid, vitamin Bc conjugate, possesses
on a molar basis only about one per cent of the activity of folic acid for
the two bacteria, Lactobacillus casei and Streptococcus faecalis R, which
are commonly used for the assay of folic acid.12 On the other hand, both

THE FOLIC ACID GROUP 571

this conjugate and the triglutamate, on a molar basis, are as active as folic
acid for Tetrahymena gelii W.62 Earlier work on a deficient medium indi-
cated the possibility that these conjugates may be more effective than
folic acid.63

Oral or parenteral administration of pteroylhexaglutamylglutamic acid
to normal human subjects produces a prompt excretion of microbiolog-
ically active forms of folic acid. On daily administration of equivalent
amounts of the conjugate or folic acid (4 mg), comparable amounts of
microbiologically active forms of folic acid (approximately 30-35 per cent
of the administered dose) are excreted daily.46 A normal subject given
2.8 mg of the conjugate daily by intramuscular injection excreted 8.3
per cent of microbiologically active forms equivalent to folic acid, whereas
administration of an equivalent amount of folic acid resulted in the
urinary excretion of 16 per cent of microbiologically active forms.47 Oral
administration of pteroylhexaglutamylglutamic acid (equivalent to 8.4
mg of folic acid daily) caused a rapid clinical improvement in a patient
with sprue; however, only small amounts of microbiologically active
forms of the vitamin were excreted in the urine.42 The conjugate appears
to be similarly active in nutritional macrocytic anemia.39,51

However, the ability of pernicious anemia patients to utilize the conju-
gate appears to vary.48 Many patients in relapse respond hematologically
and excrete increased amounts of folic acid following the administration
of pteroylhexaglutamylglutamic acid.39, 49_51 Yet there are reports of
failure by pernicious anemia patients in relapse to respond either hema-
tologically or with increased excretion of microbiologically active forms
of the vitamin.47-50, 52 Quantities as high as 54 mg of the conjugate admin-
istered daily in exceptional cases of pernicious anemia have failed to
increase the urinary excretion of microbiologically active forms of folic
acid.50 It has been demonstrated that even in normal individuals the
ability of the conjugate to cause increased urinary excretion of micro-
biologically active forms of folic acid can be almost completely inhibited
by administration of a conjugate preparation containing conjugase in-
hibitor or by administration of yeast extract 46 which contains consider-
able amounts of conjugase inhibitor.88 No evidence for the excretion of the
conjugate as such has been found.46

Nucleic acid,89 proteins,90 and a p-aminobenzoylpolyglutamyl derivative
of an unidentified amino acid,91 which has been isolated from yeast,92
inhibit the conjugase enzyme. The inhibition with the polypeptide is com-
petitive.91 Conjugase inhibitors appear to be widely distributed in nature
and are known to occur in liver and spinach as well as in yeast.46 It has
been suggested that the conjugase inhibitors may play an important role
in the utilization of the conjugate and may account for at least some of

572 THE BIOCHEMISTRY OF B VITAMINS

the variations in the response of pernicious anemia patients to the
conjugate.46, 49

Pteroylhexaglutamylglutamic acid is just as active as folic acid in cor-
recting the succinylsulfathiazole-induced leucopenia in rats if the factors
are administered orally.58 Injected parenterally, the conjugate is not quite
so effective as folic acid. Simultaneous oral administration of a conjugase
inhibitor and the heptaglutamate cause a 50 per cent decrease in the
urinary excretion of microbiologically active forms of folic acid as com-
pared with controls on the conjugate alone; however, the hematologic
response is not decreased by the conjugase inhibitor.58

From these results, it appears that the hematopoietic activity of folic
acid derivatives may not necessarily be reflected in urinary excretion of
microbiologically available forms of folic acid on administration of the
derivatives, and may not be dependent upon preliminary formation of
folic acid before conversion to the active coenzyme.

On the basis of growth and production of hemoglobin, vitamin Bc con-
jugate (pteroylheptaglutamic acid) administered in the diet is approxi-
mately 60-65 per cent as active on a molar basis as folic acid for chicks.12
It has been reported that livers of day-old chicks from eggs of hens main-
tained on a diet containing no animal protein are almost devoid of
pteroylheptaglutamic acid conjugase; however, vitamin Bi2 and 4-pyri-
doxic acid are reported to be synergistic in producing a marked increase
in the conjugase activity of preparations from such livers.93

Also of interest is the demonstration of pteroylheptaglutamic acid
conjugase in the blood of turkey poults as well as other animals, including
human beings.94

No hematologic responses have been noted after administration of
either pteroic acid or N10-formylpteroic acid (rhizopterin) to patients
with pernicious anemia or nutritional macrocytic anemia.53, 95 These
compounds are also inactive in replacing folic acid for other animals
as indicated in Table 18. A slight response has been reported for
N10-formylpteroic acid in replacing folic acid for Tetrahymena gelii W.64
Both the formyl derivative and pteroic acid have only very slight growth-
promoting effect on Lactobacillus casei; 16, 24 however, the formyl deriva-
tive is just as effective as folic acid in the nutrition of Streptococcus
faecalis R.16 Depending upon the time of incubation, the activity of
pteroic acid approaches that of folic acid for this organism.65 Streptococ-
cus faecalis 732, Streptococcus faecalis F24, Streptococcus zymogenes
5C1 and Streptococcus durans 98A are also able to utilize either folic acid
or formylpteroic acid (rhizopterin).96 Suspensions of resting cells of these
organisms as well as Streptococcus faecalis R convert formylpteroic acid
to folic acid or an analogous substance.96 Streptococcus faecalis S108 A,

THE FOLIC ACID GROUP 573

Lactobacillus bulgaricus 05, and Lactobacillus delbruckii LD50 require
folic acid for growth, but cannot utilize formylpteroic acid.96

Formylfolic acid, which is utilized as effectively as folic acid by Strep-
tococcus faecalis R and Lactobacillus casei37 produces reticulocytosis and
increases the hemoglobin and the number of red and white blood cells and
platelets in pernicious anemia patients, but is reported to be less active
than folic acid when administered orally.53

N-[p-(4-Quinazolyl) benzoyl] glutamic acid is reported to be approxi-
mately 1 to 10 per cent as active as folic acid in stimulating the growth
of Streptococcus faecalis R. However, the results indicate that consider-
able growth was obtained in the absence of exogenous folic acid, at least
one-half that which was obtained by addition of either folic acid or the
analogue.97 The possibility of a sparing action of the analogue on folic
acid cannot be excluded on the basis of the data presented; however,
analogues of vitamins can, in many instances, carry out the metabolic
functions of the vitamins. This quinazolyl analogue does not produce a
hematologic response in patients with pernicious anemia.53

N10-Methylpteroylglutamic acid, which is inhibitory to some organisms
(p. 580), replaces folic acid in stimulating the virus causing the Rous
sarcoma in folic acid-deficient chicks.77

An x-methylfolic acid (p. 575) , 2-desamino-2-hydroxypteroic acid,
2-desamino-2-hydroxypteroylglutamic acid, and pteroylaspartic acid are
all inactive clinically in treatment of the anemias responding to folic
acid.53

The diamide of folic acid neither increases the urinary folic acid con-
centrations in human subjects nor appreciably stimulates the growth of
Lactobacillus casei or Streptococcus faecalis R.38 However, the methyl
ester of folic acid is approximately 10 per cent as active as the free acid
for Lactobacillus casei,13 but probably somewhat less active for Strepto-
coccus faecalis R.19

Xanthopterin and Related Pterins. The hemopoietic effect of xanthop-
terin in alleviating the anemia resulting from the maintenance of rats
on a diet of goat's milk was reported almost a decade before the structure
of folic acid was known.98 Xanthopterin has also been reported to relieve
the anemia produced by feeding a high-protein diet to fingerling Chinook
salmon.99 Treatment of cytopenic monkeys maintained on a vitamin
M-deficient diet with synthetic xanthopterin gave a reticulocyte response
and increased the number of red and white blood cells.100 The growth
inhibition and leucopenia in rats maintained on a purified diet deficient
in folic acid and containing succinylsulfathiazole has been reported to
respond partially to xanthopterin.101 Attempts to reproduce these results
have been only partially successful; however, incubation of rat livers

574 THE BIOCHEMISTRY OF B VITAMINS

with xanthopterin increases the production of microbiologically active
forms of folic acid.75

The leucopenia and anemia in rats resulting from the action of sulfa-
thiazole is reported to be alleviated by xanthopterin, folic acid, or a sub-
stance termed vitamin Bi4, which increases cell proliferation in a beef
bone marrow suspension but inhibits proliferation of a suspension of
Brown Pearce rabbit tumor cells.102 Vitamin Bi4, isolated from human
urine, is much more effective than xanthopterin ; yet the activity of both
xanthopterin and folic acid is reportedly increased by incubation with
xanthine oxidase from milk, or with gastric mucosa of rats. 2-Amino-4-
hydroxy-7-methylpteridine counteracts the effects of both xanthopterin
and vitamin B14.102> 103

Since xanthopterin is inactive in treatment of pernicious anemia 95
and does not replace folic acid for chicks,104, 105 the relationship between
xanthopterin and folic acid in the rat is somewhat obscure. The possibility
that this and related pterins may prevent the metabolism and loss of folic
acid in enzymatic reactions not involved in hemapoiesis and growth must
also be considered. This is further emphasized by the ability of a number
of pterins and related substances to exert a beneficial action in chicks on
a folic acid-deficient diet. Thus, fed at 20 mg per 100 g diet over a four
week period the following compounds gave on occasion what appeared to
be significant growth stimulation of chicks: 105 2,4-dihydroxy-6,7-dicar-
boxypteridine, 2-mercapto-4-hydroxy-7-carboxypteridine, 2-amino-4-
hydroxy-7-carboxypteridine, 2-amino-4,6-dihydroxy-7-carboxypteridine,
2,4-dihydroxy-6-(or 7)-hydroxy-7(or 6) -carboxymethylpteridine, 2-ami-
no-4-hydroxy-6 (or 7)-hydroxy-7(or 6) -methylpteridine, 2,4-dihydroxy-6
(or 7)-hydroxy-7(or 6) -methylpteridine, bisalloxazine, 6-amino-2,4,8-tri-
hydroxypyrimido(4,5-e)pteridine, 2,6-dihydroxy-4,5-diaminopyrimidine,
and alloxan under conditions of suboptimal concentrations of folic acid.
Of these compounds, only 2-amino-4-hydroxy-7-carboxypteridine and
2-mercapto-4-hydroxy-6,7-dicarboxypteridine under similar conditions
stimulated hemoglobin formation. 2,4-Diamino-6,7-dicarboxypteridine,
2,4-diamino-7-carboxypteridine, 2,4-dihydroxy-7-carboxypteridine, and
2-mercapto-4-hydroxy-7-carboxypteridine were also found to stimulate,
under certain conditions, hemoglobin formation in chicks on suboptimal
concentrations of folic acid.105 The presence of folic acid in suboptimal
amounts is essential for the response of all these factors.105 With 2-amino-
4-hydroxypteridine, 2,4-diaminopteridine, 2-amino-4-hydroxy-6,7-dicar-
boxypteridine, 2,4-diamino-6,7-dimethylpteridine, 2-amino-4-hydroxy-7-
methylpteridine, 2,4-diamino-7-methylpteridine, or 2,4-diamino-6,7-
diphenylpteridine, no stimulatory effects were noted on either growth or
hemoglobin formation.105

THE FOLIC ACID GROUP 575

Inhibitory Analogues of Folic Acid

The first synthetic inhibitory analogue of folic acid was reported by
Martin, Tolman and Moss,106 who found that a D-methylfolic acid, pre-
pared from 2,4,5-triamino-6-hydroxypyrimidine, cc,/?-dibromobutyralde-
hyde and N-(p-aminobenzoyl)-D-glutamic acid, competitively prevented
the utilization of folic acid by Streptococcus faecalis R. Subsequently,
numerous synthetic analogues of folic acid, which prevent the biological
functions of the vitamin, have been reported. These inhibitory analogues
represent a variety of modifications of the vitamin; some of the more
active inhibitors are modifications in which a methyl group has been
placed in the pteroyl radical, an amino group has replaced the 4-hydroxyl
group of the pteridine ring, a substituent group has been placed at the
N10-position, or the glutamic acid moiety has been replaced or, in case of
modified pteroic acids, omitted. A group of pterins — particularly 2,4-
diaminopteridines, with only slight structural resemblance to folic acid
in contrast to other folic acid analogues — have been reported to be effec-
tive antagonists of folic acid for a number of organisms, including some
which synthesize folic acid.

Substituted Folic, Pteroic and Pteroylaspartic Acids

Inhibitory analogues of folic acid which can be classed as substituted
folic, pteroic, or pteroylaspartic acids are listed with inhibition indices
in Table 19.

x-Methylfolic Acids. The condensation product from a,/3-dibromo-
butyraldehyde, 2,4,5-triamino-6-hydroxypyrimidine, and N- (p-amino-
benzoyl)-D-giutamic acid inhibits the growth of Streptococcus faecalis
R, and the toxicity is competitively prevented by folic acid. The inhibi-
tion index is approximately 150.10G Although no evidence has been pre-
sented on the exact structure of this antagonist, it has been designated as
7-methylfolic acid.106

A similar product was reported which was derived from N-(p-amino-
benzoyl)-L-glutamic acid and is considerably more inhibitory for Strep-
tococcus faecalis R.107, 108 The inhibition index for this L-z-methylfolic
acid is approximately 20 to 30.107 The product also inhibits the utiliza-
tion of folic acid by Lactobacillus casei at an inhibition index of 100 to

1000 107, 109

Either L-z-methylfolic acid or sulfathiazole (1 to 10 mg) prevents the
growth of Staphylococcus aureus 209 in a bouillon medium. The toxicity
of the methylfolic acid is reported to be prevented by relatively high
concentrations (1-10 mg) of p-aminobenzoic acid, folic acid, pteroic acid
or sulfathiazole, whereas the toxicity of sulfathiazole is prevented only
by p-aminobenzoic acid and pteroic acid.109b

Administration of this analogue to a wide variety of organisms results

576

THE BIOCHEMISTRY OF B VITAMINS

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578 THE BIOCHEMISTRY OF B VITAMINS

in the appearance of symptoms characteristic of folic acid deficiency (p.
413). Thus, L-z-methylfolic acid inhibits competitively the utilization of
folic acid in rats fed a purified diet supplemented with succinylsulfa-
thiazole.107 The analogue-folic acid ratio at which the deficiency symp-
toms appear is approximately 3000. The deficiency symptoms produced at
this ratio and more acutely at higher ratios of analogue to vitamin include
reduced rate of growth, lowered hemoglobin values, and pronounced re-
duction of the white cell count, with a greater reduction in granulocytes
than in lymphocytes. Inanition and severe diarrhea develop, the fur
becoming rough and unkempt in appearance. A red pigment accumulates
about the vibrissae, and necrotic and ulcerative changes are produced in
the oral cavity, particularly in animals which die. The oral lesions pro-
duced with the antagonist are usually not characteristic of a folic acid
deficiency in rats, but are produced by folic acid deficiency in the
monkey.110 In such animals, a general necrotic condition within the
mouth and inflamed lungs with considerable congestion are noted. The
gastrointestinal tracts of most animals are empty and atonic; the livers,
spleens, hearts and other organs except the genital system are normal in
size and appearance, but uteri are always small and atrophic. In the
bone marrow, the maturation of cells of the erythroid series and of granu-
locytes are seriously impaired. If animals are treated with sufficient folic
acid just prior to the expected appearance of the terminal moribund
state, which usually occurs within one to two weeks after the onset of
the syndrome, the rats recover rapidly and appear normal within four
weeks. After recovery, the only abnormal effect is an enlarged spleen,
which in some cases is four times the normal size.107

Although female mice on a purified basal diet containing succinylsulfa-
thiazole do not develop deficiency symptoms within six weeks, supple-
mentation of the diet with crude L-z-methylfolic acid (10 g per kg)
results in death of a majority of the animals within six weeks, and the
surviving animals are left in a moribund condition.111 These surviving
animals are emaciated but do not have the chromodacryorrhea, ruffled
fur, and other characteristics of rats similarly treated. The livers are
yellow, though normal in size and texture, but the uteri are atrophic. In
contrast to the rat, in which the reduction of the granulocyte count is
more pronounced, the cells of both the myeloid and lymphoid series are
inhibited to almost the same extent by methylfolic acid in the mouse.
Since complete protection against the analogue is afforded by 0.1 g of
folic acid for each 10 g of crude inhibitor, the inhibition index is greater
than 100.111

Chicks on a folic acid-deficient diet develop symptoms which include
slow growth, poor feathering and low hemoglobin content of the blood.

THE FOLIC ACID GROUP 579

These symptoms are prevented by folic acid, but are aggravated by l-z-
methylfolic acid, which prevents the utilization of folic acid.111 The
inhibition index is somewhat lower than 10,000. The symptoms of defi-
ciency resulting from 1 g of the inhibitor per kg of diet are completely
prevented by 10 mg of folic acid per kg of diet.111

L-z-Methylfolic acid (1 g per kg of diet) administered to pigs on a
purified diet deficient in folic acid and containing succinylsulfathiazole
causes the development of deficiency symptoms characterized by severe
anemia, profuse diarrhea, diminished appetite, decreased growth rate,
some loss of hair and unkempt appearance.112 The formation of erythro-
cytes and granulocytes is inhibited. Adequate amounts of folic acid
prevent toxicity of the analogue. Administration daily of the extrinsic
factor for pernicious anemia (p. 415) derived from 100 g of crude casein
together with 80 to 150 cc of fresh neutralized human gastric juice re-
sulted in improved appetite in one pig, and both growth and hemato-
poiesis were initiated and continued for many weeks after cessation of
therapy.112 Liver extracts corresponding to those used in treatment of
pernicious anemia also allow remissions,113 but the pig apparently cannot
be maintained indefinitely without supplementation of folic acid.113, 114
While the inhibitory effect of the antagonist can be modified to some
extent by these extracts, pigs receiving 2 g of the analogue per kg of a
diet which contained adequate quantities of extrinsic factor respond only
partially, if at all, to liver extract; however, administration of folic acid
to such animals allows rapid relief of the anemia.114 The antipernicious
anemia principle of liver does not appear to be present in normal amounts
in the liver of pigs with remissions induced by folic acid, even with simul-
taneous feeding of extrinsic factor.113

Although dogs appear normal on a purified diet with folic acid omitted,
the animals on the diet supplemented with L-z-methylfolic acid develop
deficiency symptoms characterized by slow growth, or loss in weight,
emaciation, alopecia, anemia and ulceration of the skin. Liver extract
gives only a slight hemopoietic response, but administration of sufficient
folic acid prevents the toxic effects of the analogue and causes marked
responses in the deficient animals.115 Rhesus monkeys appear to be re-
sistant to the L-x-methylfolic acid.116

The development of the larvae of Drosophila melanogaster in a syn-
thetic medium containing growing yeast is arrested by supplementing the
medium with L-ar-methylfolic acid.117' 118 In medium containing 0.25
per cent of the analogue, only 0.53 per cent of the larvae survived, and at
a concentration of 1 per cent, all the larvae died. Supplementation of the
medium containing 1 per cent of antagonist with an adequate quantity

580 THE BIOCHEMISTRY OF B VITAMINS

of folic acid increases the adult emergence to 64 per cent. Controls in the
absence of the analogue showed a survival value of 74 per cent.117, 118

Estrogen-induced growth responses of the genital tract of the chick is
prevented by L-rc-methylfolic acid at a concentration of 1 per cent in the
diet.119 The inhibitory effect is completely prevented by injection of 4 mg
of folic acid four times daily.119 It is interesting that such estrogen-in-
duced growth is not obtained in folic acid-deficient chicks and monkeys.120

Addition of L-z-methylfolic acid (50 y per cc) to human blood cell
cultures in a medium containing 30 to 35 per cent human umbilical cord
serum in a balanced salt solution results in marked erythrophagocytosis
by granulocytes, as compared with blood cell cultures in the absence of
the analogue.121 Addition of folic acid (5 y per cc) prevents the effect
of the analogue. A maturation arrest in the erythroid series does not
develop under these conditions.121

Other Methylfolic Acids. Effects similar to those of z-methylfolic
acid have been noted recently with 9-methylfolic acid, which competi-
tively inhibits the utilization of folic acid for rats, mice and chicks.116

C N CH3

N C C— CH— NH— f V- CO— NH— CH— CH2— CH2— COOH

I II I \=/ I

H2N— C C CH COOH

V/ \ /

N N

9-methylfolic acid [N-{9-methylpteroyl)-h-glutamic acid]

However, N10-methylfolic acid is almost a thousand times as effective as
9-methylfolic acid in competitively preventing the functions of folic acid
in Streptococcus faecalis R (Table 19). The N10-methylfolic acid, in
contrast to other analogues of folic acid, is capable of stimulating the
growth of the Rous sarcoma in folic acid-deficient chicks.77 The N10-
methyl analogue also has an effect analogous to L-z-methylfolic acid,
causing marked erythrophagocytosis by granulocytes in human blood cell
cultures.121

H2N— C

C N CH3

N C C— CH2— N— (' V- CO— NH— CH— CH,— CH2— COOH

II I w/ I

C CH — COOH

N N

Nl0-methylfolic acid [N-(N10-methylpteroyl)-L,-glutamic acid]

THE FOLIC ACID GROUP 581

The analogue with a combination of two methyl groups at the 9- and
N10-positions (9,N10-dimethylfolic acid) is considerably more effective
than 9-methylfolic acid in inhibiting the growth of Streptococcus faecalis
R, but is less effective than N10-methylfolic acid.

Substituted Pteroic Acids and Related Compounds. A larger group,
such as phenacyl, in the N10-position appears to be detrimental to the
inhibitory activity of an analogue. This is indicated more clearly in the
pteroic acid series of N10-derivatives, which become progressively less
effective as antagonists of folic acid for Streptococcus faecalis R as the
size of the substituent group increases from the N10-methyl (Table 19).

C N CH3

N C C— CH2— N— (' \— COOH

i ii i x^/

H2N— C C CH

X / \ /

N N

Ni0-methylpteroic acid

The pteroic acid analogues are as a general rule less effective than the
corresponding folic acid analogues. Although the data in Table 19 indi-
cate that z-methylpteroic acid approaches the activity of z-methylfolic
acid, it is only 10 to 20 per cent as effective as x-methylfolic acid in pre-
venting the utilization of folic acid by Streptococcus faecalis R when
the two analogues are tested under identical conditions.108, 126 The chloro-
methylpteroic acid prepared from 4-amino-2-chlorobenzoic acid, cc,/?-
dibromobutyraldehyde and 2,4,5-triamino-6-hydroxypyrimidine is ap-
proximately as inhibitory as .r-methylfolic acid. It is interesting that
4-amino-2-chlorobenzoic acid can replace p-aminobenzoic acid in this
folic acid analogue without appreciably altering its inhibitory action, and
can also inhibit the utilization of p-aminobenzoic acid by Escherichia
coli (p. 524) .

. I

C N

N C C— CO— NH— V \— CO— NH— CH— CH2— CH2— COOH

I II I \_/ I

H2N— C C C— OH COOH

7-hydroxy-9-oxofolic acid [N-(N-(2-amino-4,7-dihydroxypteridine-6-car-
boxyhjl)-p-aminobenzoyl)~'L-glutamic acid]

582 THE BIOCHEMISTRY OF B VITAMINS

7-Hydroxy-9-oxofolic acid (2-amino-4,7-dihydroxypteridine-6-carbox-
ylyl-p-aminobenzoylglutamic acid) , which is more effective as an inhibitor
of growth of Lactobacillus casei than of Streptococcus faecalis R, con-
trasts with a number of the other analogues listed in Table 19 which are
more effective in preventing growth of Streptococcus faecalis R. Whereas
1 mg per day of this compound injected intraperitoneal^ is tolerated by
rats weighing approximately 125 g, a single dose of 10 mg is fatal within
24 hours. Simultaneous administration of 10 y of folic acid prevents the
toxicity of the analogue.

An isomer of the 7-hydroxy-9-oxofolic acid with the 6- and 7-substitu-
ents exchanged (2-amino-4,6-dihydroxypteridine-7-carboxylyl-p-amino-
benzoylglutamic acid) is less than one-third as active as the isomer with
the general structure corresponding to folic acid.

Pteroylaspartic Acids. N-Pteroyl-L-aspartic acid inhibits the growth
of both Lactobacillus casei and Streptococcus faecalis R, as indicated in
Table 19. If pteroic acid, pteroyl-y-glutamylglutamic acid, or pteroyl-
di-y-glutamylglutamic acid are employed in place of folic acid for Strep-

I
C N i k

N C C— CH2— NH— ([ V-CO— NH— CH— CH2— COOH

i ii i X=J i

H2N— C C CH COOH

N N

N-pteroyl-Jj-aspartic acid

tococcus faecalis R, the inhibition indices for half maximum growth are
2-3, 4.2-17.2 and 0.21-0.32, respectively. Folic acid with an index of
37-55 prevents the toxicity of the inhibitor most effectively ; the trigluta-
mate is least effective.

A marked decrease in growth rate and decreased hemoglobin levels
are observed in chicks receiving the inhibitor, which causes these defi-
ciency symptoms to appear when administered at 500 times the concen-
tration of folic acid.

Administration of the inhibitor to rats at concentrations up to 4.5 mg
per day did not cause any significant decrease in growth rate or the ap-
pearance of deficiency symptoms even with animals receiving carboxy-
sulfathiazole.

N-(x-Methylpteroyl)-L-aspartic acid is somewhat less effective than
pteroylaspartic acid in preventing the utilization of folic acid by Strep-
tococcus faecalis R.

THE FOLIC ACID GROUP 583

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584 THE BIOCHEMISTRY OF B VITAMINS

4-Amino-4-desoxyfolic Acid and Related Analogues

A series of synthetic analogues of folic acid which have an amino group
in place of the 4-hydroxyl of the pteridyl moiety of folic acid are char-
acterized by very potent inhibitory activities; with few exceptions these
either are not prevented or are prevented only to a limited extent by
folic acid. The analogues are extremely toxic for a majority of the organ-
isms which require folic acid, and cause symptoms which are character-
istic of folic acid deficiency even though folic acid does not in many
instances show appreciable ability to prevent the toxic manifestations.
The biological activities of analogues of this type are indicated in Table
20.

4-Amino-4-desoxyfolic Acid [N- (4- Amino -4- desoxypteroyl-L- glutamic
Acid]. From Table 20, it is apparent that 2-amino-4-desoxyfolic acid
inhibits markedly the growth of Streptococcus faecalis R; however, the
various members of the folic acid group have very little effect on the
amount of inhibitor necessary to prevent growth of the organism.129 Only

NH2

f \

N C C— CH2— NH— ^ J— CO— NH— CH— CH2— CH2— COOH

H2N— C C CH ^==/^ COOH

N N

Jf.-amino-4-desoxy folic acid [N-(Jt.-amino-Jt.-desoxy'pteroyl)-ij-glutamic acid]

about a three-fold increase in the concentration of the analogue is neces-
sary to obtain the same degree of growth inhibition when the folic acid
concentration is increased one hundred-fold. Growth stimulated by
pteroic acid is most susceptible to the inhibitor, and no effect on the
toxicity is noted by increasing one hundred-fold the concentration of
pteroic acid.129

The toxicity of the analogue becomes apparent in chicks at a concen-
tration of 1 mg per kg of diet containing 0.5 mg of folic acid. At 5 mg
per kg of the diet, the 4-amino analogue is lethal for all the chicks.129
The onset of the symptoms is rapid in comparison with dietary depletions.
To prevent the lethal effects of 4 mg of analogue per kg of diet, approxi-
mately 96 mg of folic acid per kg of diet are required. Intermediate
concentrations of folic acid permit partial survivals, but there is little
inhibition of growth of chicks surviving sub-lethal concentrations of the
analogue.129

A similar situation exists with rats. As little as 10 y per day or 1 mg

THE FOLIC ACID GROUP 585

per kg of diet is fatal to all the animals. The symptoms of the toxicity
include severe watery diarrhea, resulting in extreme dehydration, loss
of weight, porphyrin-stained whiskers and hemoconcentration. Signs of
nervous involvement and anoxia develop prior to death, and extensive
tissue changes, including intestinal lesions and a very hypoplastic bone
marrow, are observed.129 In the peripheral blood, marked granulocyto-
penia and reticulocytopenia and a moderate lymphopenia develop simul-
taneously.132 Very high concentrations of pteroylglutamic acid (20-30
mg per kg of diet) are required to prevent the effects of even the mini-
mum lethal concentration. The folic acid content of the liver appears to
be decreased as indicated by microbiological assay.133 The LD50 of the
analogue for rats receiving single doses is 4.5 ±1.4 mg per kg.132 Regard-
less of the size of the dose above the minimum lethal dose, the course of
the fatal intoxication is not altered. The animals are unaffected for ap-
proximately one day, but all fatalities usually occur on the third or fourth
day. Oral administration is as effective as parenteral injection, and frac-
tional doses are cumulative and are possibly more effective than a single
dose. Animals surviving the minimum lethal dose have a transient re-
tardation in growth with subsequent rapid recovery.

At a concentration of 0.3 mg per kg of diet, 4-amino-4-desoxyfolic
acid tends to lower slightly the hemoglobin and white cell count of
mice.134 The effect is prevented by high concentrations of folic acid, but
the high mortality of mice fed 1 mg of the analogue per kg of diet is not
affected by folic acid even at high concentrations (100 mg per kg of diet).
Gross examination at autopsy revealed no lesions.138 The LD50 for mice
receiving a single dose of the analogue is 1.9 ±0.3 mg. per kg.132 Frac-
tional doses were cumulative and almost as toxic as the single dose. The
LD50 can be increased several fold by repeated administration of folic
acid (47 mg per kg daily) or pteroyltriglutamic acid (500 mg per kg
daily) before and after administration of the 4-amino analogue. Neither
thymine nor refined liver extract has such an effect on the toxicity of the
analogue.132

Guinea pigs given daily subcutaneous injections of 0.5 to 5.0 mg of
4-amino-4-desoxyfolic acid lose weight and with few exceptions die within
11 to 28 days.135 The symptoms of the toxicity noted include normocytic
anemia, leucopenia, agranulocytosis, thrombocytopenia and hypoplasia
of the bone marrow. Although refined liver extract does not prevent the
anemia or leucopenia, folic acid in concentrations 25 to 100 times that
of the analogue appears to prevent the development of leucopenia or
thrombocytopenia, but not the anemia.135

The 4-amino analogue administered to dogs in daily doses of 0.05 to
0.1 mg per kg of body weight is fatal to approximately half the animals

586 THE BIOCHEMISTRY OF B VITAMINS

within ten days.136 The effects of the analogues are similar to those in
other animals and include: hemorrhagic diarrhea, weight loss, leucopenia,
hemoconcentration and degeneration of the bone marrow. Erythropoiesis
and myelopoiesis are inhibited. Lymphopoiesis is less affected.130

Monkeys are susceptible to the inhibitory action of 4-amino-4-desoxy-
folic acid, in contrast to the effect of L-x-methylfolic acid.116

The characteristic growth response to estrogens of the genital tracts
of female chicks 137 and rats 137 and newly metamorphosed frogs 138
(Rana clamitans) is inhibited by 4-amino-4-desoxyfolic acid. The de-
crease in the response of the female frog oviducts to estradiol resulting
from administration of the amino analogue is not affected by supple-
mental folic acid even at 100 times (5 mg) the concentration of the
analogue (0.05 mg) .138 High concentrations of folic acid in both the chick
and rat tend to prevent the effect of the analogue.137

4-Amino-4-desoxyfolic acid also prevents the development of larvae of
Drosophila melanogaster in a synthetic medium containing growing yeast.
Folic acid does not appear to prevent this inhibition.117, 118

The analogue also causes a marked erythrophagocytosis by granu-
locytes in human blood cell cultures.121

Marked effects of the 4-amino analogue on the blood islets in 6- to
8-day old chick embryos have been reported to be altered by folic acid,
but not by either refined liver extract or vitamin Bi2.139

Liver tissue from rats depleted of folic acid on a succinylsulfathiazole
diet has a decreased ability to oxidize tyrosine as compared with liver
tissue from normal animals.139a The addition of folic acid in vitro par-
tially restores the ability to oxidize tyrosine, but neither liver extract
nor pteroylheptaglutamate has this effect. Liver tissue from rats fed
4-amino-4-desoxyfolic acid is similarly deficient in its ability to oxidize
tyrosine, but no effect on the system is obtained in vitro with folic acid,
vitamin Bi2, or liver extract. The administration of folic acid or refined
liver extract to the rat prevents the effects of the analogue on the ability
of the liver to oxidize tyrosine. The analogue does not inhibit the oxida-
tion of tyrosine by liver slices in vitro.139*

4-Amino-4-desoxy-N10-methylfolic Acid [N-(4-Amino-4-desoxy-N10- meth-
ylpteroyl)-L-glutamic Acid]. In contrast to the effects of 4-amino-4-
desoxyfolic acid, the toxicity of 4-amino-4-desoxy-N10-methylfolic acid
for Streptococcus faecalis R is prevented competitively by folic acid, as
indicated in Table 20.131 However, folic acid has little ability to prevent
the toxicity of the analogue for rats.131 Animals receiving sub-lethal
amounts of 4-amino-4-desoxy-N10-methylfolic acid have normal growth;
and except for a little alopecia and occasional but never severe chromo-
dacryorrhea, anemia and leucopenia, the animals do not show symptoms

THE FOLIC ACID GROUP 587

characteristically caused by other analogues. At 3 mg per kg of diet, the
analogue is fatally toxic to all the rats, moderate anemia, leucopenia and
granulocytopenia developing shortly before death. This fatal effect of the
minimum lethal concentration is prevented only by high concentrations
of folic acid (100 mg per kg of diet). Gross examination at autopsy does
not reveal the usual lesions and pathology observed with other folic acid
analogues. The viscera are normal in appearance, with no lesions or

NH2

C N CH3

N C C— CH2— N— (' \>— CO— NH— CH— CH2— CH2— COOH

I
COOH

. / \ •

N N

4-amino-4-desoxy-N10-methylfolic acid

pathological changes other than a slight enteritis. Animals administered
lethal amounts of the analogue seldom died before the fifth or sixth day,
but all the animals susceptible to that concentration died within the
second week. Animals surviving beyond the second week after admin-
istration of the analogue were not subsequently affected.131

It is interesting that different patterns of symptoms are obtained with
various analogues of folic acid.

A slight growth-retarding effect of 4-amino-4-desoxy-N10-methylfolic
acid (3 mg per kg of diet containing 0.1 mg of folic acid) on chicks is
prevented by folic acid (10 mg per kg of diet).131

The analogue also causes marked cytological changes in the blood
islets of 6- to 8-day old chick embryos. Folic acid, but neither liver ex-
tracts nor vitamin B12, alters this effect.139

Other 2,4-Diaminopteridyl Analogues of Folic Acid. Both 4-amino-4-
desoxypteroic acid and 4-amino-9,N10-dimethyl-4-desoxyfolic acid are
effective inhibitory analogues of folic acid for Streptococcus faecalis R,
as indicated in Table 20.122, 124- 130 4-Amino-4-desoxypteroyl-L-aspartic
acid has some activity in inhibiting neoplastic growth (p. 593). A prelim-
inary report indicates that the aspartic analogue is toxic for mice, rats
and dogs, and produces pathologic changes similar to those caused by
the other 4-amino-4-desoxypteroyl derivatives.140a

Substituted Pteridines and Pyrimidines

2,4-Diamino- and Related Pteridines. The first indication of the
great affinity of certain 4-aminopteridines for enzymes related to the
utilization of folic acid in biological systems was reported by Daniel,
et al.140 They found that a group of 2,4-diaminopteridines synthesized by

THE BIOCHEMISTRY OF B VITAMINS

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THE FOLIC ACID GROUP 589

Mallette, Taylor and Cain 141 was extremely effective in inhibiting the
growth of Streptococcus faecalis R. The effects of these pteridines and
related compounds on a number of organisms are indicated in Table 21.
The inhibitory effects of these pteridines are prevented in a competitive
manner by folic acid, particularly for Streptococcus faecalis R. The most
effective inhibitors of these pteridines are those with aromatic substitu-
ents in the 6,7-positions, e.g., 2,4-diamino-6,7-diphenylpteridine, 2,4-
diaminophenanthro(9,10-e)pteridine and 2,4-diaminoacenaphtho(l,2-e)-
pteridine.

NH2

A n jTx

H2N— C C C

N C C

N N

2 ,4-diamino-6 ,7-diphenylpteridine

2 ,4,-diamino-6 ,7-phenanthro(9 ,10-e)pteridine

A large number of the pteridines indicated in Table 21 were prepared
in an effort to find more soluble derivatives with the same potent in-
hibitory properties as the 2,4-diaminopteridines with 6,7-aromatic sub-
stituents. However, substitution of the aromatic groups lowered the
activity, regardless of the nature of the substituent group. Acetylation of
the 2- or 4-amino groups did not appreciably alter the activity, but
methylation of these amino groups resulted in considerable loss of in-
hibitory power.143

The 2,4-diaminopteridines contrast markedly with the 2-amino-4-
hydroxypteridines which, though more closely related structurally to folic
acid, do not possess antibacterial activity; on the contrary, some have
very slight growth-stimulating effects.140

2,4-Diamino-6,7-diphenylpteridine inhibits the growth of Lactobacillus
arabinosus, which does not require exogenous folic acid.140 Both p-amino-

590 THE BIOCHEMISTRY OF B VITAMINS

benzoic acid and folic acid prevent the toxicity to a certain extent, but do
not prevent the toxicity of concentrations of 3 y per cc or greater. This
type of phenomenon is characteristic of the corresponding 4-amino
analogues of folic acid for a number of organisms. However, it is interest-
ing that the substituted pteridine inhibits the system of enzymes related
to folic acid in an organism which synthesizes the vitamin. Most folic
acid analogues are not able to prevent effectively the utilization of this
vitamin group in organisms which synthesize the vitamin.

Some of the 2,4-diaminopteridines act synergistically with sulfonamides
in preventing growth of Escherichia coli, Staphylococcus aureus and Lacto-
bacillus casei.li4i For example, a minimum of either 1500 y per 10 cc of
2,4-diamino-6,7-dimethylpteridine or 1000 y per 10 cc of sulfathiazole is
necessary to prevent the growth of Staphylococcus aureus, but a combina-
tion of 20 y per 10 cc of each of the two compounds allows approximately
the same degree of inhibition. Similarly, a minimum of either 2000 y per
10 cc of the same pteridine or 50 y per 10 cc of sulfathiazole prevents
the growth of Escherichia coli, but a mixture of 100 y per 10 cc of the
pteridine and 20 y per 10 cc of sulfathiazole prevents growth of the
organism. 2,4-Diamino-6,7-diphenylpteridine, at a concentration which is
ineffective alone, will reduce more than ten-fold the amount of sulfa-
thiazole necessary for inhibition of growth of Lactobacillus arabinosus.
These results indicate that two biological processes in sequence are
inhibited.144

Although 2,4-diamino-6,7-diphenylpteridine does not affect growth or
hemoglobin formation in chicks,105 it is reported to affect blood formation,
particularly leucocytes in rats.133 The pteridine appears to cause a decrease
of folic acid in the liver of chicks,105 but no decrease in the folic acid
content of the liver is noted for rats.133 Other pterins tested with rats
included 2,4-diamino-6,7-dimethylpteridine, 2,4-diamino-6,7-di (4-amino-
phenyl) pteridine and 2-amino-4-hydroxy-6,7-diphenylpteridine.133

In chicks, both 2-amino-4-hydroxy-6,7-dimethylpteridine and 2-amino-
4-hydroxy-6,7-diphenylpteridine (20 mg per 100 g of diet containing 15 y
of folic acid) inhibit growth of chicks and decrease hemoglobin formation.
The inhibitory effect is prevented by folic acid at 60 y per 100 g of diet.105
2-Amino-4-hydroxy-6(or 7)-hydroxy-7(or 6) -methylpteridine appears to
inhibit hemoglobin formation, but stimulates growth of chicks on a
diet containing suboptimal concentrations of folic acid. 2,4-Dihydroxy-
benzo(e) pteridine was slightly inhibitory to both growth and hemoglobin
formation, whereas 2,4,6-trihydroxy-7-carboxypteridine was inhibitory
only to hemoglobin formation.105 It is interesting that 2-amino-4-
hydroxypteridine-7-carboxylic acid and related pteridines prevent the
oxidation of either xanthopterin or xanthine by a xanthine oxidase prepa-

THE FOLIC ACID GROUP 591

ration from whey.145 2-Amino-4-hydroxy-6-formylpteridine is also an
effective inhibitor of xanthine oxidase and related enzymes.146

Pyrimidine Derivatives. The toxicity of either 2,4-diamino-6,7-di-
methylpteridine or 2,6-diaminopurine for Lactobacillus casei is reported
to be prevented in a competitive manner by either folic acid or purines
under highly specific conditions, particularly with concentrations of folic
acid which limit growth.147 The reversal of the toxicity of the pteridine
occurs over a relatively restricted range with purines, whereas the reversal
by folic acid can be accomplished over a wide range of concentration of
inhibitor.147 This indicates that the effect of the purines on the inhibited
system is probably indirect. The reversal of the toxicity of 2,6-diamino-
purine differs from the usual purine effect in that adenine prevents the
toxicity over a much greater range of concentrations than does folic
acid.147 This suggests the possibility that the folic acid effect is indirect,
presumably in eliciting synthesis of adenine, which in turn competes with
the diaminopurine. The possibility exists, however, that a single com-
pound, such as 2,6-diaminopurine, may effectively inhibit the utilization
of two different metabolites. 2-Amino-4-hydroxypteridine derivatives,
2-aminopurine, 2-amino- and 2,4-diamino-6,7-dihydroxypteridines simi-
larly produce inhibitions of Lactobacillus casei stimulated by folic acid;
these inhibitions are prevented by purines. These compounds, however,
do not inhibit growth of Lactobacillus casei in the presence of thymine
and purines, 'this contrasts to 2,4-diamino-6,7-dimethylpteridine, which
inhibits growth of the organism under either condition.147 2,4-Diamino-
5-methyl- and 5,6-dimethylpyrimidines are intermediate in their behavior
toward reversal with purines and folic acid.147 Growth of Lactobacillus
casei stimulated by either thymine or folic acid is inhibited by 5-nitro-
uracil;148' 149 however, the toxicity of 5-nitrouracil is prevented by uracil
under conditions in which folic acid exerts no effect.150

Miscellaneous

Miscellaneous analogues of folic acid which have been found to exert
inhibitory effects on the utilization of folic acid by some organisms are
indicated in Table 22.

4-Desoxy folic Acid is reported to decrease the apparent folic acid con-
tent of the liver and produce both anemia and leucopenia in rats on a
diet supplemented with succinylsulfathiazole.133

N-\N -( 2-Amino-/+,6-dihydroxypteridine-7 ' -carboxylyl) -p-aminoben-
zoyl]--L-glutamic acid is isomeric with 7-hydroxy-9-oxofolic acid (Table
19) and is less than half as effective in inhibiting the growth of Lacto-
bacillus casei as is the isomer resembling folic acid.125

Quinoxaline has a slight inhibitory effect on the growth of Streptococcus

THE BIOCHEMISTRY OF B VITAMINS

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THE FOLIC ACID GROUP 593

faecalis R, which is prevented by folic acid.151 Quinoxaline derivatives
more closely related to folic acid are considerably more effective than the
parent compound;125 however, the compounds with modified ring struc-
tures in place of the pteridine ring of folic acid have not as yet been
found to be particularly effective as inhibitory analogues of folic acid
(Table 22).

N[N '-{2- Amino- 4- hydroxy -6 -pteridylmethyl) -p-aminobenzenesulfo-
nyl]-h-glutamic acid has recently been synthesized, but no biological data
were reported.154

A number of analogues of folic acid including N-(2-amino-4-hy-
droxy-6-pteridylmethyl) -p-aminophenol, N- (2-amino-4-hydroxy-6-pter-
idylmethyl) -p-aminohippuric acid, N- (2-amino-4-hydroxy-6-pteridyl-
methyl) -4-amino-2-chlorobenzoic acid, 2-hydroxy-2-desaminopteroic
acid, 2-hydroxy-2-desaminofolic acid, N-(2-amino-4-hydroxy-6-pteridyl-
methyl) -p-aminobenzenesulfonic acid, N- [N- (4- (6-aminoquinazoline) )
p-aminobenzoyl] glutamic acid, N- [N- (4-quinazoline) -p-aminobenzoyl] -
glutamic acid, N- (4-aminoquinazoline) -p-aminobenzenesulfonamide,
N- [N- (4- (6-nitroquinazoline) ) -p-aminobenzoyl] glutamic acid, N[N- (4-
(6-chloroquinazoline) ) -p-aminobenzoyl] glutamic acid, N- [4- (6-chloro-
quinazoline) ] -p-aminobenzoic acid, N-(2,4-dihydroxy-6-pteridylmethyl)-
p-aminobenzenesulfonic acid, and N-[N-(2,4-dihydroxy-#-methyl-6-pter-
idylmethyl)-p-aminobenzoyl] glutamic acid have been prepared, and
tested for their effects on the blood pressure of the dog, inhibitory effects
on 3,4-dihydroxyphenylalanine decarboxylase, and their inhibitory action
on Streptococcus faecalis R.126

Previous observations indicated that L-.T-methylfolic acid inhibited to
some extent the action of the decarboxylase of 3,4-dihydroxyphenyl-
alanine 155 and lowered the blood pressure of the dog.156 Many of the
above compounds, as well as other related inhibitory analogues at con-
centrations of 30 to 300 y per cc, show some inhibitory action on the
decarboxylase enzyme.130, 157 The inhibition is reported to be prevented
by folic acid in extremely high concentrations, ten to a hundred times the
concentration of the inhibitors. Also, some of these analogues affect the
blood pressure of the dog. These effects are reported to be nullified by the
injection of 250 mg of folic acid per kg of body weight. The pteroic acid
analogues are more effective than the folic acid analogues for the hypo-
tensive effect.126

The Effect of Compounds Related to Folic Acid on Cancer

An extensive study of B vitamins in normal and cancer tissue has indi-
cated that folic acid is relatively the most abundant of the vitamins in
cancer tissue. Since there is similarity in the content of the B vitamins

594 THE BIOCHEMISTRY OF B VITAMINS

in cancer tissues regardless of the host organism or site of appearance or
means of induction, the general patterns of the metabolism of cancer
tissues appear to be related.158

The folic acid from livers of rats bearing Walker tumor transplants is
liberated by autolysis either in phosphate-sodium chloride or acetate
buffer, whereas maximal release of folic acid from livers of normal rats
requires both Clarase and phosphate-sodium chloride buffer. The acetate
buffer does not replace the phosphate buffer.

Pteroyldi-y-glutamylglutamic Acid. The first direct effect on growth of
cancer by a member of the folic acid group was reported by Leuchten-
berger, et al.,100 who found that the growth of Sarcoma 180 in female
Rockland mice was reduced to approximately 35 per cent of the controls
by intravenous injection of 5 y of a concentrate containing members of
the folic acid group, or by the same number of injections of 0.4 y of
fermentation Lactobacillus casei factor (pteroyldi-y-glutamylglutamic
acid) . Complete regressions of spontaneous breast cancers in mice were
observed in 38 among 89 (43 per cent) animals treated with daily injec-
tions of 5 y of the pteroyltriglutamate. The incidence of the development
of new tumors was decreased from 14 out of 60 mice in the controls to 1
out of 89 mice treated with the triglutamate.161 The liver Lactobacillus
casei factor (pteroylglutamic acid) is inactive in effecting this inhibitory
action on the tumors.161 Additional data on the inhibition of transplanted
tumors and of spontaneous tumors have been reported by this group.162
However, failures in attempts to confirm these results using sarcoma 180
or spontaneous breast cancer of mice have been reported.163-166

Preliminary clinical reports of the use of pteroyldi-y-glutamylglutamic
acid (teropterin) in the treatment of cancer have indicated that it is
nontoxic and in 500-mg doses relieves pain in most cases, if not all,
thereby allowing a reduction in amount of sedation or analgesia required
and in some cases obviating the necessity for use of opiates. The patients,
after treatment with the compound, are cheerful, exhibit a sense of well-
being and are more mentally alert. Although some patients with advanced
malignancy have shown considerable improvement on treatment with
the compound, the results in most instances cannot be evaluated objec-
tively because other treatments known to have desirable effects preceded
or were concurrent with the administration of the triglutamate.167-169

Effect of Folic Acid and Related Compounds on Rous Chicken Sarcoma.
Folic acid and related compounds have a profound effect on the avian
neoplasm first described by Rous.170 The Rous chicken sarcoma, which
is transmitted by a filtrable agent, can be controlled either by regulation
of the amount of folic acid in the diet of the chicks or by administration
of certain folic acid antagonists.77, 170-172 Thus, injection of a sterile,

THE FOLIC ACID GROUP 595

aqueous suspension of infected tissue into the breast of baby chicks on
an ordinary diet resulted in development of tumors in approximately 95
per cent of the chicks; but out of 40 similarly injected chicks on a folic
acid-deficient diet, no tumors developed. However, the frequency of tumor
development with chicks on the same deficient diet but injected daily with
folic acid (100 y), pteroyl-a-glutamylglutamic acid (500 y), pteroyltri-
glutamic acid (500 y), or N10-methylfolic acid (100 y) was 90, 90, 100,
and 80 per cent, respectively. No such action was noted with similar
injections of pteroyl-D-glutamic acid, pteroic acid, pteroylaspartic acid,
N10-methylpteroic acid or p-aminobenzoyltriglutamic acid.

On a normal diet on which 95 per cent of the chicks developed tumors
by the twentieth day, folic acid antagonists tended to prolong or com-
pletely inhibit the development of the tumor, but were in many instances
rather toxic to the host. Thus, daily intraperitoneal injections, begin-
ning at the time of inoculation with tumor of 5-6 day old chicks, of
4-amino-4-desoxypteroyl-L-aspartic acid (400 y), 4-amino-4-desoxy-
pteroyl-D-glutamic acid (400 y), 4-amino-N10-methyl-4-desoxypteroyl-
glutamic acid (100 y), and 4-amino-N10-methyl-4-desoxypteroic acid
(100 y) decreased the percentage of chicks developing tumors by the
twentieth day to 40, 0, 70, and 60 per cent, respectively. Pteroylaspartic
acid and pteroyl-D-glutamic acid did not exhibit any appreciable in-
hibitory activity on the tumor. Concentrations of the compounds which
were inhibitory to tumor growth resulted in impairment of health and
eventual loss of life, particularly when the compounds were injected. The
therapeutic index was slightly more favorable in older animals and least
favorable with one-day old chicks. The method of administration was
also important, and the best results were obtained on administering the
antagonists in the diet. Thus, 80 mg per kg of diet of 4-amino-4-desoxy-
pteroylaspartic acid or a similar amount of 4-amino-4-desoxypteroyl-D-
glutamic acid was relatively nontoxic to one-day old chicks, and prevented
for 17 days the development of tumors in 75 and 55 per cent of the chicks,
respectively. All the untreated controls developed tumors.

4-Amino-4-desoxyfolic acid is suitable only for treatment of adult birds
since it is extremely toxic for growing chicks. Any concentration affecting
tumor growth was lethal to the chick. However, 1 mg of 4-amino-4-
desoxyfolic acid, administered daily by intraperitoneal injection, pre-
vented the development of the tumor in approximately 60 per cent of
adult birds as compared with 20 per cent obtained with 4-amino-4-desoxy-
pteroylaspartic acid administered similarly. Even at this dosage for adult
birds, 4-amino-4-desoxyfolic acid produces noticeable emaciation in adult
birds, and a few injections of 10 y are lethal to baby chicks. Doses of
0.25 mg of folic acid, pteroyltriglutamic acid or pteroyldiglutamic acid

596 THE BIOCHEMISTRY OF B VITAMINS

protect approximately 60, 50 and 10 per cent of baby chicks against the
toxic effect of 10 y doses of 4-amino-4-desoxyfolic acid which are other-
wise fatal. The antagonist under these conditions did not prevent tumor
growth in baby chicks.

Leukemia. The greater sensitivity to folic acid deficiency of leuco-
poiesis in the myeloid series 107 led to the suggestion that folic acid
analogues might be used as chemotherapeutic agents in myelogenous
leukemia.

In a preliminary report on the use of folic acid derivatives in the
treatment of human leukemia m it is indicated that pteroyldi-y-glutamyl-
glutamic acid, pteroyldiglutamic acid, pteroylaspartic acid and N-methyl-
pteroic acid do not alter the blood picture or bone marrow in cases of
chronic leukemia and multiple myeloma; however, in cases of acute
leukemia, administration of the folic acid antagonists resulted in a tem-
porary reduction of the total white cells.173

A preliminary report of the effect of inhibitory folic acid analogues on
acute leukemia in children indicated that temporary remissions can be
induced with 4-amino-4-desoxyfolic acid.174 The effect of N10-methyl-
pteroic acid and pteroylaspartic acid is questionable. Upon treatment with
4-amino-4-desoxyfolic acid, the white cell count tended to return to a
normal level both in patients with an initially high count and in those
with marked leukopenia at the outset of the therapy. A decline was
observed in the percentage of immature cells with a marked decrease in
blast forms, which disappeared in some cases from the peripheral blood.
The relative percentages of mature leucocytes approached normal values
in the peripheral blood. In the bone marrow a decrease or disappearance
of leukemic cells and variation from hypoplasia to almost normal pattern
was observed. However, the analogue is toxic, giving effects which include
stomatitis with early ulceration. Indications are that the remissions are
only temporary. Similar temporary favorable results have been reported
by others.175

Only slight and irregular effects in increasing the survival time of mice
injected with transmitted leukemia Ak 1394 and Akm 9417 are obtained
on administration of 4-amino-4-desoxyfolic acid which was, however,
somewhat more effective against the Ak 4 and C 1498 strains of leu-
kemia.176 4-Amino-N10-methyl-4-desoxyfolic acid allowed a longer sur-
vival time than did nitrogen mustards in mice injected with Akm 9417,
Ak 4 or C 1498 strains of leukemia, but was not effective with the Ak 1394
strain. Nitrogen mustards allow a slight increase in survival time of mice
with the C 1498 strain, but 4-amino-N10-methyl-4-desoxyfolic acid in-
creases the survival time slightly more than 200 per cent. By the cytocidal
method, no activity could be shown against the cells of leukemia Akm

THE FOLIC ACID GROUP 597

9417, which are completely inactivated by methylbis(chloroethyl) amine
at four times the LD50.176

The average survival time of mice with Ak 4 strain of leukemia is
prolonged significantly by 2,6-diaminopurine as well as 4-amino-4-des-
oxyfolic acid.177

Sarcoma 180. 4-Amino-4-desoxyfolic acid at 0.19 to 0.42 mg per kg
retards the rate of growth of sarcoma 180 in mice. Inhibitions of tumor
growth to the extent of more than 50 per cent and on occasions as high
as 94 per cent of the controls are obtained, but the mice lose weight up to
20 per cent under these conditions.178 Pteroyl-a-glutamylglutamic acid
administered intraperitoneally or intravenously does not affect growth of
the tumor.176

4-Amino-4-desoxyfolic acid is fatal to many of the animals under con-
ditions giving marked inhibition of tumor growth.179

A partial reversal of the inhibition of tumor growth is obtained by
administration of folic acid with the 4-amino-4-desoxyfolic acid. Folic
acid alone exerts some inhibition of tumor growth when administered at
high concentration.180

4-Amino-4-desoxy-N10-methylfolic acid has a more favorable thera-
peutic index than 4-amino-4-desoxyfolic acid.181 At 1.5 mg per kg per day,
the amino methyl analogue shows marked inhibition of growth of sarcoma
180, the tumors being only 3-10 per cent of the size of controls after one
week, and causes very little loss in weight of the animals, with only 7 per
cent fatalities during the injection period. Slightly higher concentrations
(2.5 mg per kg per day) of the analogue are lethal to almost half the
animals. Male mice appear to tolerate the analogue better than female
mice.181

4-Amino-4-desoxypteroylaspartic acid also has a more favorable thera-
peutic index than 4-amino-4-desoxyfolic acid in inhibition of sarcoma 180

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THE FOLIC ACID GROUP 599

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600 THE BIOCHEMISTRY OF B VITAMINS

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THE FOLIC ACID GROUP 601

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128. Seeger, D. R., Smith, J. M., Jr., and Hultquist, M. E., J. Am. Chem. Soc, 69,

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129. Oleson, J. J., Hutchings, B. L., and SubbaRow, Y., J. Biol. Chem., 175, 359

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602 THE BIOCHEMISTRY OF B VITAMINS

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132. Philips, F. S., and Thiersch, J. B., J. Pharmacol. Exptl. Therap., 95, 303 (1949).

133. Swendseid, M. E., Wittle, E. L., Moersch, G. W., Bird, 0. D., and Brown, R.

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134. Franklin, A. L., Stokstad, E. L. R., and Jukes, T. H., Proc. Soc. Exptl. Biol.

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135. Minnich, V., and Moore, C. V., Fed. Proc, 7, 276 (1948).

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137. Hertz, R., Proc. 39th Annual Meeting Am. Assn. Cancer Research, March,

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154. Viscontini, M., and Meier, J., Helv. Chim. Acta, 32, 877 (1949).

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THE FOLIC ACID GROUP 603

160. Leuchtenberger, C, Lewisohn, R., Laszlo, D., and Leuchtenberger, R., Proc.

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Biol. Med., 70, 396 (1949).

Chapter VI D

THE NICOTINIC ACID GROUP

Specificity

Although nicotinic acid was prepared synthetically in 1867 by the
oxidation of nicotine,1, 2- 3 it was not isolated from natural products until
1912.4, 5, c jn 1934^ nicotinamide was isolated from coenzyme II by War-
burg and Christian,7 who thereby demonstrated the first biological role
of the factor since coenzyme II was recognized as a hydrogen-transporting
coenzyme. Euler, Albers, and Schlenk 8 shortly afterward obtained nico-
tinamide from coenzyme I. The structure of coenzyme I originally pro-
posed by Schlenk and Euler 9 is indicated as follows:

N=C— NH2

r^N-CO-NH2 H(L i_N

II J II II >CH

xNr N— C— N'

H— 6 H— i-

H— C— OH | H— C— <

-OH

H— C— OH I H— C— (

H-i-

CH2 — O-

coenzyme I; cozymase

Coenzyme II contains an additional phosphate group and can be converted
into coenzyme I.10 The exact location of this phosphate group is still
questionable.

The first indication of the importance of this group of factors in nutri-
tion was presented by A. Lwoff and M. Lwoff,11 who demonstrated that
a factor essential for growth of certain bacteria of the Hemophilus
group,12- 13 the "V" factor, was replaced by either coenzyme I or II, both
of which possess properties analogous to those of the "V" factor. Shortly
thereafter, the role of nicotinic acid as an essential growth factor was
demonstrated for Staphylococcus aureus by Knight14 and for Coryne-
bacterium diphtheriae by Mueller.15 The activity of nicotinic acid and
nicotinamide in preventing blacktongue in dogs was subsequently demon-

604

THE NICOTINIC ACID GROUP 605

strated by Elvehjem and co-workers.16 These reports, particularly with
the extension of the biological role of nicotinic acid to treatment of human
pellagra,17- 1S stimulated an intense search among analogues of nicotinic
acid for those which possessed biological activity.

The specificity of the nicotinic acid group for various organisms is
indicated in Table 23. It is interesting to note that nicotinic acid cannot
replace nicotinamide in the nutrition of certain strains of Pasteurella,
and that nicotinamide is essentially inactive in replacing nicotinic acid for
Leuconostoc mesenteroides 9135 and 8293 and Leuconostoc dextranicum
8086. Quantitative variations in ability to utilize these compounds are
common among various organisms. For example, nicotinamide is approxi-
mately ten times as active as nicotinic acid for certain dysentery bacilli,
but nicotinic acid is ten times as effective as nicotinamide in stimulating
the growth of Cory meb acterium diphtheriae.15 Most organisms, however,
use nicotinic acid and its amide with about the same efficiency. The
Hemophilus group which requires the "V" factor cannot utilize either
nicotinic acid or its amide. For Hemophilus parainfluenzae and Hemo-
philus influenzae, the "V" factor requirement is satisfied most effectively
by coenzyme I or by the equally active dihydrocoenzyme I.50, 51 Desamino-
coenzyme I is only 60 per cent as active as coenzyme I. As indicated in
Table 23, nicotinamide riboside replaces the coenzyme, but is considerably
less active on a molar basis. Growth response of the organism to increas-
ing concentrations of the riboside is not proportional to that obtained
with corresponding concentrations of coenzyme I. Coenzyme II is also
less active than nicotinamide riboside, but the growth response closely
parallels that of the riboside, indicating the possibility that it is utilized
by prior conversion to the riboside before coenzyme I synthesis.50, 51 The
amount of nicotinamide riboside just necessary for detectable growth of
Hemophilus influenzae or Hemophilus parainfluenzae is less than that of
coenzyme I for the same, response. However, for appreciable growth con-
siderably more of the riboside than of coenzyme I is essential.50- 51

As indicated in Table 23, organisms which require nicotinic acid or
nicotinamide for growth can usually utilize the coenzymes. However,
Leuconostoc mesenteroides 8293 and Leuconostoc dextranicum 8086 can-
not utilize effectively either coenzyme I or II.39a Furthermore, coenzyme
I injected intravenously is reported to have no therapeutic effect on
canine blacktongue,25 but administered orally in rats, it is reported to be
more active than nicotinamide.54 In many instances, coenzymes I and II
are less effective as microbial growth factors than either nicotinic acid
or nicotinamide.38, 46 Although neither Leuconostoc mesenteroides 8293
nor Leuconostoc dextranicum 8086 can utilize exogenous coenzyme I or
II, both organisms synthesize from nicotinic acid a factor which replaces

606

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608 THE BIOCHEMISTRY OF B VITAMINS

these coenzymes in the nutrition of Hemophilus parainfluenzae.3da Similar
results are obtained with Leuconostoc mesenteroides 9135 30a and with
Lactobacillus arabinosus 17-5, in which nicotinic acid largely exists as
coenzyme I.55

Most metabolites of nicotinic acid which are excreted in the urine of
animals are not effectively utilized by many organisms which require
nicotinic acid or nicotinamide for growth. Thus, trigonelline appears to be
inactive for most organisms; however, for Torula cremoris 2512 it is
almost as active as nicotinic acid. ISP-Methyl nicotinamide chloride has
only very slight ability to replace the nicotinic acid group for a few
organisms. Nicotinuric acid is utilized only with difficulty by many
organisms, but for some, e.g., Lactobacillus arabinosus, it is approximately
as active as nicotinic acid.

Although nicotinuric acid was initially reported on the basis of a single
test to have a relatively high curative action in canine blacktongue,24 it
has been proposed 26 on the basis of additional tests that the compound
probably should be grouped with pyrazinecarboxylic acid,30' 31 pyrazine-
2,3-dicarboxylic acid,30' 31 quinolinic acid,30 3-aminopyridine,56 2-amino-
nicotinic acid,26 and pyrimidine-4-carboxylic acid.20 These compounds do
not act regularly as blacktongue-preventives, but are sometimes able to
replace nicotinic acid, at least partially, in the diet of dogs for extended
periods. Nicotinuric acid administered to dogs maintained on a low
nicotinic acid diet is excreted almost quantitatively without metabolic
change.57 This offers further evidence of the inability of the dog to utilize
this compound. It is also interesting that nicotinuric acid administered by
intravenous injection to human subjects was almost quantitatively ex-
creted unchanged in the urine.58

The majority of the biologically active analogues of nicotinic acid are
compounds which can be converted to the vitamin by the organism. How-
ever, the activities, even though of a low order, of thiazole-5-carboxylic
acid or its amide, thiazole-5-sulfonic acid, N-(2-pyridyl)-3-pyridinesul-
fonamide, pyrazinecarboxylic acid, and 2,3-pyrazinedicarboxylic acid can-
not be explained on this basis. It has been suggested that some of these
may actually be utilized as such without prior conversion to nicotinic acid.

Administration of pyrazinecarboxylic acid and quinolinic acid was re-
ported to produce an increase in "V" factor activity in the blood and
urine of human subjects;22-23 but this has not been verified by subse-
quent work,30 in which a slightly different assay method for the "V"
factor was employed. Quinolinic acid, pyrazine monocarboxylic acid, or
pyrazine-2,3-dicarboxylic acid administered orally to male subjects did
not cause a rise in the "V" factor content of blood cells. Neither was
synthesis of "V" factor obtained on incubation of the three acids under

THE NICOTINIC ACID GROUP 609

sterile condition with defibrinated human blood. However, these results
do not necessarily contradict the reported activity of these compounds
in treatment of pellagra, since a change of the "V" factor content of the
blood is not a reliable method of determining utilization of analogues
of nicotinic acid. This is particularly true in view of the fact that oral
administration of nicotinamide to human subjects does not cause a
change in the "V" factor concentration of the blood; but oral administra-
tion of nicotinic acid is followed by a prompt elevation of the concentra-
tion of this factor, as determined by Hemophilus influenzae.59' 60 The
increase in concentration of the factor is paralleled by changes in the
rate at which erythrocytes oxidize lactate and malate.

The activity of the esters of nicotinic acid for chicks increases with
increasing chain length from ethyl to n-butyl.33 n-Butyl nicotinate is
almost as active as nicotinic acid. For bacteria, however, the activity
appears to decrease with increasing chain length. Animals appear in most
instances to utilize both the esters and N-substituted amides more effec-
tively than most bacteria. The search for utilizable but water-insoluble
forms of nicotinic acid has largely been centered around the esters and
N-substituted amides.29, 52, G1- 62 These forms of the vitamin are needed
for enrichment of certain foods, such as corn grits and white rice, which
are customarily subject to rinsing before cooking.

Methyl nicotinate has been reported to be the most active form of the
vitamin for certain organisms. For example, with glucose as a substrate,
the respiration of dysentery bacilli grown on a medium deficient in nico-
tinamide can be stimulated by coenzyme I, coenzyme II, nicotinamide,
nicotinic acid, methyl nicotinate or other derivatives.63 Methyl nicotinate
is reported to be more active than nicotinamide, which in turn is more
active than coenzyme I or II or nicotinic acid. It was suggested that,
since these variations cannot be explained by differences in rate of diffu-
sion, the results are incompatible with the theory that nicotinamide
serves simply as a precursor of coenzyme I or II or both.03

A similar high activity of methyl nicotinate has been reported 38 for
Proteus vulgaris 3056. The activity of nicotinamide and methyl nicotinate
varies with the pH of the medium. At pH 7.2, these activities are 78
and 92 per cent, respectively, of nicotinic acid; however, at pH 7.8-8.1,
the activities are 215 and 636 per cent, respectively. Thus, methyl nico-
tinate is significantly more active than either nicotinic acid or nicotin-
amide under these conditions. For other strains of Proteus, nicotinic acid,
nicotinamide, and coenzyme I have been reported 41 to be equally ef-
fective.

The growth requirements of 189 strains of Proteus were investi-
gated, and strains of P. vulgaris, P. anindolo genes, P. par a-americ anus.

610 THE BIOCHEMISTRY OF B VITAMINS

P. americanus, P. ammonia, P. mirabilis, P. asiaticus and P. nocutarnum
were found to have essentially the specificity as indicated in Table 23 for
Proteus, with the exception of quinolinic acid, which was inactive at the
concentration employed.42

After injection into rats of compounds related to nicotinic acid, the
increase in urinary excretion of nicotinamide methochloride has been re-
ported in terms of per cent of theoretically possible increase 34 as follows:
nicotinic acid, 11.8; nicotinamide, 22.7; N-ethylnicotinamide, 17.2; N,N-
diethylnicotinamide, 16.8; N-phenylnicotinamide, 6.5; N- (4'-methyoxy-
phenyl) nicotinamide, 6.2; N-benzylnicotinamide, 5.8; nicotinamide
methochloride, 62.7; quinolinic acid, 5.2; and 3-picoline, 41.3. No increase
in urinary excretion of the metabolite was reported for nicotinonitrile,
trigonelline, or N-cyclohexylnicotinamide or related methylcyclohexyl
derivative. The extent to which 3-picoline is metabolized is interesting.

Even compounds closely related to nicotinic acid are not capable of
replacing the vitamin. Thus, isonicotinic acid,24- 35, 36, 39a> 42, 43, 44 picolinic
acidj2o, 24, 35, 36, 38, 39a, 42, 43, 44 dinicotinic acid,35- 45 cinchomeronic acid,45
6-methylnicotinic acid,24, 36, 39a- 45 2,4-dimethylpyridine-3-carboxylic
acid,43 2,4,6-trimethylpyridine-3,5-dicarboxylic acid,40- 43 2-aminonico-
tinic acid,42, 49 or pyridine betaine-3-carboxylic acid 42, 49 cannot replace
nicotinic acid or its amide in the nutrition of a wide variety of organisms.
With the exception of Staphylococcus aureus, the hexahydro derivative
of nicotinic acid, nipecotic acid 24, 36, 45 is not dehydrogenated. Ethyl
/?-oxo-3-pyridinepropionate (ethyl nicotinoacetate)35, 45 is not cleaved by
some organisms, and nicotine 36, 43 is not oxidized by many organisms.
N-Cyclohexylnicotinamide as well as N-(2-methyl-cyclohexyl) nicotin-
amide cannot replace nicotinic acid in the nutrition of larvae of Tribolium
confusum, but the corresponding N-phenyl derivative is active.34 Other
compounds which are inactive in replacing the vitamin for some organisms
include arecoline,35- 45 ^-aminopyridine,20- 35> 45 N,N-diethylthionicotin-
amide,38 thiopicolinamide,38 quinoline-2-carboxylic acid,38 quinoline-3-
carboxylic acid,38 pyridine-3-sulfonic acid,24- 36> 43' 45 pyridines-sulfon-
amide,38 /3-acetylpyridine,24- 45 benzoic acid,35- 49 pyridine,24- 40- 45 2-pico-
line,20- 36- 38- 44 and 4-picoline.44

Of the last group, 3-acetylpyridine and pyridine-3-sulfonic acid exerted
a lethal toxic effect for dogs deficient in nicotinic acid, but did not exert
such an action on normal animals.24 An occasional report has listed some
members of this group as exerting a slight stimulating effect,38 or as
being capable of replacing nicotinic acid: e.g., 2,6-dimethylpyridine-3,5-
dicarboxylic acid and dinicotinic acid were reported to give some im-
provement in pellagra;20 pyridine-3-sulfonic acid and nicotine were re-
ported to be active for Proteus 38- 40 and Streptobacterium plantarum.38

THE NICOTINIC ACID GROUP 611

The order of activity reported is such that contamination with the vita-
min cannot be ruled out, particularly since other reports indicate
inactivity.

Inhibitory Analogues of the Nicotinic Acid Group

Although a large number of analogues of nicotinic acid and related
compounds have been tested as inhibitors, only a few have been reported
to inhibit competitively the functioning of nicotinic acid in biological
systems.

3-Pyridinesulfonamide. The first specific reversal of the toxicity of
an analogue by nicotinamide was reported by Mcllwain,04 who found
that the inhibition of growth of Staphylococcus aureus caused by 3-

-S02NH2

3-pyridinesulfonamide

pyridinesulfonamide was competitively prevented by the vitamin. The
inhibition indices were 50,000, 250,000 and 1,250,000 for incubation
periods of 19, 26 and 43-96 hours, respectively. Growth promoted by
nicotinic acid is affected much less by the analogue, and the inhibition
under these conditions was essentially negligible, even though nicotinic
acid is somewhat less effective than nicotinamide in promoting growth
of the organism. Inhibition of growth promoted by a preparation of co-
enzyme I was not only more intense, but also was not prevented by addi-
tional amounts of coenzyme I. It was concluded that with Staphylococ-
cus aureus, nicotinic acid is not used solely for synthesis of this coenzyme.

With Proteus vulgaris,64 growth promoted by nicotinamide was pre-
vented by 3-pyridinesulfonamide, but the analogue was less inhibitory
to Proteus vulgaris than to Staphylococcus aureus. The inhibitory effect
of the compound on growth changed with time and usually disappeared
within a few days. The analogue was less effective in preventing growth
stimulated by cozymase and was ineffective in preventing growth stimu-
lated by nicotinic acid.

The toxicity of pyridine-3-sulfonamide for Streptobacterium plantarum
is prevented over a narrow range of concentrations by nicotinic acid,
nicotinamide, coenzyme I, 5-thiazolecarboxylic acid, and heavy metal
salts, particularly iron salts.65

3-Pyridinesulfonamide does not inhibit the growth of Escherichia
coli,64- 66 some strains of Proteus vulgaris, or the flagellate, Polytomella
caeca66

612 THE BIOCHEMISTRY OF B VITAMINS

3-Pyridinesulfonic Acid. The growth of Proteus vulgaris has been re-
ported to be effectively inhibited by 3-pyridinesulfonic acid (M/100).64

S02OH

3-pyridinesulfonic acid

Although the toxicity of the inhibitor was not competitively prevented,
increased concentrations of nicotinic acid reversed the inhibition after
three to four days. When growth of the organism was promoted by an
equivalent concentration of nicotinamide, 3-pyridinesulfonic acid did not
inhibit it. Growth promoted by a preparation of coenzyme I was more
strongly inhibited than that obtained with nicotinic acid as a growth
stimulant. These indications that nicotinamide may not be used by
Proteus vulgaris solely for the synthesis of coenzyme I have been pointed
out by Mcllwain.64

With Staphylococcus aureus, the inhibition of growth by 3-pyridinesul-
fonic acid was prevented to some extent by nicotinic acid in a somewhat
competitive manner.64 The inhibition index was 10,000 and 250,000 for
incubation periods of 23 hours and 5-6 days, respectively.

The toxicity of pyridine-3-sulfonic acid (4xlO"3M) for Proteus vul-
garis is reported67 to be prevented by either nicotinic acid (lXl0_3M)
or by thiazole-5-carboxamide (lxlO_3M). The thiazole-5-carboxamide
without the inhibitor shows slight inhibition of growth of the organism.
The possibility that thiazole-5-carboxamide may actually function in the
organism in place of nicotinic acid was suggested.

For Lactobacillus acidophilus (Hadley), 3-pyridinesulfonic acid caused
half-maximum inhibition of growth at an index of 1700.68 However, no
data indicating reversal of the inhibition were indicated.

Some inhibition of growth of rats was obtained by administration of
3-pyridinesulfonic acid in a diet containing a low amount of protein.69
The inhibition of growth was prevented by either nicotinic acid or higher
amounts of protein in the diet. However, 3-pyridinesulfonic acid did not
appreciably affect the onset and reversibility of the effects of nicotinic
acid deficiency in dogs.70 The sulfonic acid does not produce symptoms of
nicotinic acid deficiency in mice.71

Another interesting effect is stimulation of growth of Staphylococcus
aureus by low concentrations of 3-pyridinesulfonic acid (M/5000 to
M/1000) , which has been reported to occur in the presence of suboptimal
concentrations of nicotinic acid; however, no stimulation was obtained
even at high concentrations (M/100) in the absence of nicotinic acid.53

THE NICOTINIC ACID GROUP 613

Methyl 3-Pyridyl Ketone (3-Acetylpyridine). For Streptobacterium
plantarum, 3-acetylpyridine is toxic only at high concentrations, and the

-COCH3

methyl 3-pyridyl ketone (3-acetylpyridine)

inhibition is not reversed by nicotinic acid.72 However, the analogue is
toxic for nicotinic acid-deficient dogs but not for normal dogs.24 This
suggested the possibility that it might be used to produce symptoms of
nicotinic acid deficiency in mice.73 With doses of 2 to 4 mg per day, the
animals began to breathe rapidly very soon after administration of the
analogue. In a few hours, difficulties in control of the hind legs developed.
Within two days, complete paralysis of the hind legs resulted. The mice
appeared emaciated, extremely wet and unkempt. The skin became very
red and inflamed, and, after four to seven days, fiery red tongues devel-
oped in about half the animals. Supplementing the ration with nicotinic
acid for three or four days prior to administration of the analogue pre-
vented the disease; however, only partial success was reported for at-
tempts to cure animals ill with the deficiency disease.

Tryptophan also prevents the toxicity of 3-acetylpyridine, which causes
the pellagra-like manifestations.74 The amino acid in amounts as little as
0.1 per cent of the diet was sufficient to protect the animals, and was as
active as nicotinic acid in exerting the protective action.

3-Acetylpyridine injected into the yolk-sac is toxic for a 4-day old
developing chick embryo.75 Sublethal concentrations cause certain mal-
developments of the chick, such as undersized, deformed legs and a general
edema-like condition over the body. However, approximately 600 y of
3-acetylpyridine per egg was lethal within 24 hours. The toxicity of the
analogue was prevented entirely when sufficient nicotinamide was injected
simultaneously. The inhibition index required for the lethal effect in all
the eggs was 15.4-16.7. The lowest ratio of analogue to metabolite just
necessary not to exert any lethal effects was 13.6-14.5. However, in order
to prevent maldevelopment of the chick, a still lower ratio of analogue
to metabolite was essential. As compared with nicotinamide, nicotinic acid
and tryptophan exerted much weaker effects in preventing the toxicity
of the analogue for the chick embryo, but there appears to be some slight
ability of the embryo to utilize nicotinic acid and tryptophan in place of
nicotinamide at this stage of development.

Marked electrocardiographic abnormalities of an isolated rabbit's heart
occurred on perfusion with 3-acetylpyridine.76 Administration of nicotin-

614 THE BIOCHEMISTRY OF B VITAMINS

amide prevented these abnormalities.76 A beneficial effect of nicotinic
acid on the isolated heart had been previously observed in the absence
of an inhibitory analogue,77 and in clinical studies marked alterations in
the electrocardiogram, which disappear promptly after nicotinic acid
therapy, have been noted in patients with pellagra.78

Other Analogues of Nicotinic Acid and Nicotinamide. 6-Aminonico-
tinic acid which inhibits the utilization of p-aminobenzoic acid for a

-COOH r^N— CO— CH2— CO-

NH2-^N '

6-aminonicotinic acid dibenzoylmeihane

{1 ,3-diphenyl-l ,3-propanedione)

number of organisms (p. 527) prevents the growth of Staphylococcus
aureus in a synthetic medium at a concentration of 1 y per cc.79 The
growth inhibition is reported to be prevented by either nicotinic acid or
nicotinamide at concentration 0.1 to 0.01 that of the inhibitor. p-Amino-
benzoic acid does not affect the inhibition.79

l,3-Diphenyl-l,3-propanedione (dibenzoylmethane) inhibits the growth
of Proteus vulgaris. The inhibition of growth is prevented by sufficient
nicotinamide. The inhibition index is reported to be approximately 100.42a

Thiazole-5-carboxamide exerts a slight toxic effect for Staphylococcus
aureus at relatively high concentrations.53 This effect contrasts with the
ability of the compound to prevent the toxicity of 3-pyridinesulfonic acid
for Proteus vulgaris.

2- (5'-Thiazolecarboxamido) pyridine neither inhibits nor promotes
growth of Staphylococcus aureus.53

N-2-Pyridyl-3-pyridinesulfonamide is reported to be less effective as
an inhibitor than the corresponding sulfonamide or sulfonic acid.80 It has
been indicated (Table 23) that it replaces the requirement of Staphylo-
coccus aureus for nicotinic acid.53

The toxicities of coramine, picolinic acid, pyridine-3-sulfonic acid,
thionicotinamide, thiopicolinamide, quinoline-3-carboxylic acid, quinoline-
2-carboxylic acid (quinaldinic acid) , and N,N-diethylpyridine-3-sulfon-
amide were all found to be of a low order for Proteus vulgaris and
Streptobacterium plantarum, and were not prevented by nicotinic acid or
nicotinamide. The latter two were toxic themselves at a concentration
approaching that of some of these analogues.65 5-Thiazolecarboxylic acid
was toxic for Streptobacterium plantarum only at high concentrations at
which its inhibitory effects were not prevented by nicotinic acid or
amide.65

THE NICOTINIC ACID GROUP 615

A number of analogues of nicotinic acid have been reported to be inac-
tive as inhibitory analogues of the vitamin. These include; 1,2-dinicotinyl-
hydrazine,81 nicotinamide,82 nicotinhydrazide, 2-fluoronicotinic acid,83
6-fluoronicotinic acid,83 5-bromonicotinic acid, and 5-bromonicotinamide.

The synthesis of 5-fluoronicotinic acid and 5-fluoronicotinamide has

-COOH F-f^,-CONH2

5-fluoronicotinic acid 5-fluoronicotinamide

been recently reported, but the biological action of the analogues are not
as yet available.84 Preliminary tests have indicated that these compounds
are the most effective nicotinic acid antagonists as yet reported. The
analogues, particularly the acid, inhibit a large number of bacteria at
relatively low inhibition indices.85

Natural Antagonisms Related to Nicotinic Acid. Pellagra has long been
associated with the eating of corn, and evidence has been presented which
indicates that nicotinic acid is required in higher amounts if corn is
included in the diet of animals.81, 86, 87 In fact, rats which ordinarily do
not require nicotinic acid need either a supplement of nicotinic acid or
additional tryptophan in the diet to overcome the retardations of growth
resulting from the inclusion of corn grits in a low-protein diet.88 Since
supplementary tryptophan administered either orally or subcutaneously
to the rat causes increased excretion of metabolites of the nicotinic acid
group, this animal appears to synthesize nicotinic acid from tryptophan.89
Administered to patients with pellagra, tryptophan (6-g oral doses)
causes a remission of typical acute pellagrous lesions with increased
urinary excretion of N1-methylnicotinamide.90 Tryptophan does not, how-
ever, affect the nicotinic acid requirement of many organisms, e.g., Tetra-
hymena geleii W 91 and most lactobacilli. Evidence has been presented
indicating that the conversion of tryptophan to nicotinic acid in the rat
is impaired by certain amino acids, such as glycine and threonine, by
tryptophan-free proteins, or by corn grits.92, 93 However, the mode of
action of the responsible substances is largely unknown. In mice fed a
low-protein diet a weakly basic water-soluble substance which has been
concentrated about 100,000 times from corn causes a disease similar to
pellagra which is preventable or curable with nicotinamide.94 Indoleacetic
acid has been reported to retard growth of rats in a manner similar to
corn, and its effect on growth is prevented by either nicotinic acid or
tryptophan.95 Although other reports fail to confirm this effect of indole-
acetic acid,69, 96 there appear to be naturally occurring materials which

616 THE BIOCHEMISTRY OF B VITAMINS

exert an inhibition either directly or indirectly on nicotinic acid synthesis
or functioning in animals. This type of action is probably more wide-
spread than has been generally recognized.

The synthesis of nicotinamide from ornithine or ammonium lactate by
Escherichia coli or by mixed cultures of rat caecum contents is inhibited
by either 2-,4-,5-, or 7-methyltryptophan.97 The involvement of ornithine
and 8-amino-n-valeric acid in the biosynthesis of guvacin (1,2,5,6-
tetrahydronicotinic acid) and nicotinic acid had been suggested before
the interrelationship of tryptophan and nicotinic acid was discovered.98
The effect of these substances on nicotinic acid synthesis and its relation-
ship to the tryptophan process is still obscure.

Analogues of 3-Hydroxyanthranilic Acid. A study of the requirements
of a number of mutant strains of Neurospora revealed that one strain
could utilize either tryptophan, kynurenine, 3-hydroxyanthranilic acid, or
nicotinic acid for growth.99 Another strain could utilize only hydroxy an -
thranilic acid or nicotinic acid,100, 101 and still another required nicotinic
acid and could not utilize hydroxyanthranilic acid, which accumulated in
the medium.100- 101 These results indicate that kynurenine and hydroxyan-
thranilic acid represent successive steps in the conversion of tryptophan
to nicotinic acid or nicotinamide.

It has been reported that 3-methoxyanthranilic acid is significantly
inhibitory to the conversion of tryptophan to nicotinic acid by Neuro-
spora.102 Since methylation of 3-hydroxyanthranilic acid may occur in
nature, and since methyl-2-methylamino-3-methoxybenzoate has been
isolated from the seeds of two species of Nigella, these or similar naturally
occurring substances as dietary constituents may affect the transformation
of tryptophan to nicotinic acid.

Inhibitions Involving Coenzyme I. The functioning of coenzyme I in
glucose dehydrogenase and lactic acid dehydrogenase is prevented compet-
itively by 3-pyridinesulfonic acid.103 The ratios of inhibitor to coenzyme
for half-maximum inhibition are approximately 730 and 780, respectively.
Nicotinic acid and nicotinamide also effectively inhibit these enzymes,
but nicotinamide methiodide is inactive. 3-Pyridinesulfonamide or its
methiodide is somewhat less active than the corresponding sulfonic acid.
Although a number of other compounds were inhibitory for these enzymes,
the inhibitory effects of many of the substances, e.g., salicylic acid and
adenosine, were not prevented by coenzyme I.

Salicylic acid prevents the function of coenzyme I in glucose fermenta-
tion by a zymase preparation (yeast).104 The inhibition is prevented in
a competitive manner by coenzyme I over a range of concentrations. The
ratio of concentration of salicylic acid to coenzyme I necessary for half-
maximum inhibition of the fermentation is 662-692. At high concentra-

THE NICOTINIC ACID GROUP 617

tions, nicotinic acid, nicotinamide, and trigonelline were found to inhibit
glucose fermentation in this system.

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618 THE BIOCHEMISTRY OF B VITAMINS

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THE NICOTINIC ACID GROUP 619

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Chapter VII D

PANTOTHENIC ACID

Specificity

Even before the complete structure of pantothenic acid was determined
to be D-N-(a,y-dihydroxy-/?,/?-dimethylbutyryl)-/?-alanine, preliminary
data indicating that the factor was an hydroxy acid conjugated with
^-alanine by an amide linkage l led to the preparation of a number of
analogous compounds. Some of these possessed partial activity.2-4 Subse-
quent to the announcement of the structure of pantothenic acid, a number
of more closely related analogues of the vitamin were prepared and tested
for activity in replacing pantothenic acid in the nutrition of organisms
requiring the vitamin.

In Table 24, the specificity of pantothenic acid is indicated by com-
parison of its activity with that of its analogues. The activity of panto-
thenic acid resides only in the dextrorotatory form, which has been
indicated to be the D-configuration by application of Hudson's amide
rule.8, 21- 22 l-( — )-Panthothenic acid appears to be inactive for organisms
requiring the intact vitamin.

The methyl and ethyl esters of pantothenic acid are comparable to the
vitamin in promoting the growth of rats,6"9 but ethyl pantothenate is only
6.8 per cent as effective as pantothenic acid for Lactobacillus casei.G' 7

Acetylation is reported to destroy the activity of pantothenic acid in
natural extracts for chicks 23 and bacteria.24 However, synthetic ethyl
monoacetyl D-pantothenate (Table 24) is approximately as active as
pantothenic acid for both rats and chicks, but is only 0.7 per cent as
effective as the vitamin for Lactobacillus casein 7 It is necessary to
hydrolyze methyl monoacetyl pantothenate to obtain maximal activity
with Streptococcus faecalis R.2 Mono-p-nitrobenzoyl D-pantothenic acid
(N-a-p-nitrobenzoxy-y-hydroxy-/?,/?-dimethylbutyryl-/?-alanine) is inac-
tive for Lactobacillus casei.7

The analogues which replace pantothenic acid are usually only partial
substitutes for the vitamin in the nutrition of most organisms; however,
some analogues appear to be capable of completely replacing the vitamin
in the nutrition of some organisms. Of the analogues which appear to act
without prior conversion to pantothenic acid, N- (a-hydroxy-£,/?-di-
methylolbutyryl) -^-alanine ("hydroxypantothenic acid") and N-(a-

620

PANTOTHENIC ACID 621

hydroxy-/? -methyl -/?-methylolvaleryl) -^-alanine ("methylpantothenic
acid") are the most effective in replacing the nutritional requirement of
pantothenic acid for a wide variety of organisms. The "methylpantothenic
acid" appears to be somewhat mqre active than "hydroxypantothenic
acid" for several organisms, but neither of the substituted pantothenic
acids exerts an action comparable with that of the vitamin on a wide
variety of organisms. In many instances, the activity of the analogue
compared with that of the vitamin is greater at low than at high dosage.

Preparation of two of the four diastereoisomers of "methylpantothenic
acid" has recently been reported.25 DL-a-Keto-/3-methyl-/?-methylolvalero-
lactone was resolved into the two optically active forms which were
reduced to the corresponding a-hydroxy lactones by yeast. Since yeast
reduces cc-keto-/3,/?-dimethylbutyrolactone to ( — ) -pantolactone,26 the two
optically active homologues of pantolactone presumably have the same
D-configuration on the a-carbon, and the two disastereoisomeric "methyl-
pantothenic acids" prepared from these lactones differ only in configura-
tion on the /3-carbon and have a configuration analogous to pantothenic
acid on the a-carbon. The "methylpantothenic acid" A with a configura-
tion on the /3-carbon presumably similar to alloisoleucine determined by
analogy in melting points is 27.8 per cent as active as pantothenic acid.
The "methylpantothenic acid" B, presumably with configuration similar
to isoleucine, is 62.5 per cent as active as pantothenic acid. Tested at a con-
centration of 14 y per cc of cinchonidine salt, neither of these diastere-
oisomeric "methylpantothenic acids" allowed a maximum growth response
of Streptobacterium plantarum 10 S in the absence of pantothenic acid.25

Since DL-N-(a-hydroxy-^-ethyl-^-methylolvaleryl) -^-alanine is in-
active,12 apparently only one of the ^-methyl groups of the butyryl por-
tion of pantothenic acid can be modified without complete loss of the
biological activity of the vitamin.

All the analogues capable of replacing pantothenic acid to any extent
retain at least one of the two hydroxyl groups. Modifications in vivo
may account for the activity of a number of the less effective analogues.

Some analogues of pantothenic acid which are inhibitory have been
found to replace pantothenic acid partially. These will be discussed
separately.

The alcohol corresponding to pantothenic acid (pantothenyl alcohol)
has been found to be as effective as pantothenic acid in preventing
achromotrichia in rats.16 However, this activity is the result of conver-
sion of the alcohol in vivo to pantothenic acid.17 Other warm-blooded
animals have been found to carry out this oxidation.17 After administra-
tion of pantothenyl alcohol, pantothenic acid was excreted in the urine
of male human subjects in amounts equal to or slightly greater than after
administration of an equivalent amount of calcium pantothenate (100

622 THE BIOCHEMISTRY OF B VITAMINS

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624 THE BIOCHEMISTRY OF B VITAMINS

mg). When larger amounts were administered, the urinary excretion of
pantothenic acid was decidedly greater after administration of panto-
thenyl alcohol than after administration of an equivalent amount of
either sodium or calcium pantothenate.17, 27, 28 Pantothenyl alcohol can-
not replace pantothenic acid in the nutrition of lactic acid bacteria. On
the contrary, it inhibits the utilization of pantothenic acid by these
organisms,19 as subsequently discussed.

While /^-alanine but not pantoic acid can replace pantothenic acid in
the nutrition of some organisms such as yeast 29, 30 and some strains of
Corynebacterium diphtheriae,3U3S pantoic acid, but not /^-alanine, is
effective in replacing the vitamin for other organisms such as Acetobacter
subdoxydans 621, 34 Streptococcus hemolyticus H 69 D,35 and one strain of
Clostridium septicum36 Either pantoic acid or /^-alanine alone accelerates
the rate of growth of certain strains of Brucella suis ;37 the two combined
are more effective, but still are not as effective as the intact vitamin.
When capable of replacing pantothenic acid, pantoic acid usually is con-
siderably more active than pantolactone.38 The results suggest that
pantolactone must be hydrolyzed before utilization in the synthesis of
pantothenic acid.

Coenzyme A,39 the coenzyme which accounts for the major portion of
the bound pantothenic acid, is inactive in replacing pantothenic acid in
the nutrition of numerous organisms. These include Lactobacillus ara-
binosus 17-5, Saccharomyces cerevisiae, and Lactobacillus casei. However,
coenzyme A administered intraperitoneal^ is fully active in the chick
assay, but on oral administration only 61 per cent of the activity was
observed.40 Both coenzyme A 41 and a naturally occurring conjugate of
pantothenic acid 42 are appreciably more effective than pantothenic acid
in promoting the growth of Acetobacter suboxydans 621. A product ob-
tained from coenzyme A by enzymatic action of liver extracts has growth-
promoting properties for Acetobacter suboxydans similar to that of the
conjugate of pantothenic acid.41 Intestinal phosphatase acts on coenzyme
A to form a still different product, which does not have enhanced ac-
tivity for Acetobacter suboxydans 41 and is not effective for other micro-
organisms.43 Both intestinal phosphatase and liver enzymes are required
for the conversion of coenzyme A to a form which is utilized by Lacto-
bacillus arabinosus and most other microorganisms, and is presumably
free pantothenic acid.43

Inhibitory Analogues of Pantothenic Acid

Pantoyltaurine. The first growth inhibition specifically and competi-
tively reversed by pantothenic acid was reported by Snell,44 who pre-
pared and tested N-(cc,y-dihydroxy-/?,/?-dimethylbutyryl) taurine as an

PANTOTHENIC ACID 625

inhibitory analogue of pantothenic acid for lactic acid bacteria. Kuhn,
Wieland, and Moller 45 independently and almost simultaneously re-
ported similar findings. Barnett and Robinson 15 prepared, and Mc-
Ilwain 46 tested the same analogue independently, but published their
results at a later date with the suggestion of the name "pantoyltaurine"
for the analogue. The term "pantoyl" for the a,y-dihydroxy-/?,/3-dimethyl-
butyryl radical, as suggested by Mcllwain,46 has since been widely used.
The two optically active forms of pantoyltaurine have been prepared
from d- ( — ) - and l- ( + ) -pantolactone by fusion with the sodium salt
of taurine at 120° C for five hours. The resulting product from the l-
pantolactone was only about one-tenth as active as that from the lactone
of D-configuration.44 If milder conditions were employed for the con-
densation and the products carefully purified by chromatography and
conversion to the quinine salt, the differential in activity was even
greater, about 32-fold.45 Since the lactone intermediates were not op-
tically pure and since the reactions are such that some racemization takes
place, it seems probable that only the d-( + ) form of pantoyltaurine
corresponding to the configuration of the active form of pantothenic acid
exerts a bacteriostatic activity. This specificity of configuration has sub-
sequently been confirmed with other analogues of pantothenic acid.

Table 25. Pantoyltaurine.

Test Organism

Inhibition Index Reference

Streptococcus hemolyticus

500 47

Corynebacterium diphtheriae (Gl)

500 "

Lactobacillus arabinosus 17-5

1,000 44

Diplococcus pneumoniae

1,000 47

Streptobacterium plantarum

2,000 «

Propionibacterium pentosaceum P-ll

5,000 44

Saccharomyces cerevisiae GM

8,000 44

Streptococcus faecalis R

10,000 44

Lactobacillus pentosus 124-2

150,000 44

Leuconostoc mesenteroides P-60

150,000 44

Other lactobacilli

Table 33

As indicated in Table 25, pantoyltaurine inhibits the growth of a wide
variety of microorganisms which require pantothenic acid for growth.
The growth inhibition is counteracted specifically by pantothenic acid,
and becomes apparent only when the ratio of analogue to pantothenic
acid surpasses a critical value. The minimum ratio necessary for max-
imum inhibition of growth, the inhibition index, is indicated in this table.
This competitive relationship exists for each of the organisms over wide
ranges in concentration. Inhibition indices vary with time of incubation,
composition of the medium, size of inoculum, strain of the organism, etc.
Consequently, the values indicated in Table 25 are only approximate,
since the results from different laboratories do not agree exactly.

626 THE BIOCHEMISTRY OF B VITAMINS

Organisms which do not require pantothenic acid in their nutrition are
usually not affected by pantoyltaurine. This phenomenon is widespread
among the analogues of pantothenic acid and will be discussed separately.
The toxicity of pantoyltaurine for Streptobacterium plant arum was pre-
vented to some extent by large concentrations of /^-alanine, and to a
larger extent by mixtures of pantolactone and /3-alanine. Since the mix-
tures of pantolactone and /^-alanine do not promote growth in the ab-
sence of pantothenic acid, it appears that either a chemical or enzymatic
conversion of the pantoyl radical of the analogue to pantothenic acid
takes place during the testing.

Early reports 48 indicated that pantothenic acid deficiency in mice
could be produced by long-continued oral administration of pantoyl-
taurine, but this has not been substantiated. On the contrary, no toxic
symptoms have been observed on the administration, either orally or
subcutaneously, of pantoyltaurine to mice 49~51 or rats.51

Insulin-treated, depancreatized dogs on a diet deficient in pantothenic
acid were fed 1 g of pantoyltaurine daily. After three days, complete
refusal of food occurred, but during the first three days with constant
food intake, the urinary nitrogen rose, whereas hemoglobin and cell vol-
ume fell sharply.52 It has not been indicated whether pantothenic acid
has a beneficial effect in preventing these effects.

Pantoyltaurine, however, constitutes the first case of an effective
chemotherapeutic agent being designed in accordance with the concept
of competitive analogue-metabolite growth inhibition. Mcllwain and
Hawking 51 reported that rats were protected from 10,000 lethal doses of
a virulent strain of streptococcus and less completely from 1,000,000
lethal doses by frequent subcutaneous doses of pantoyltaurine. Although
pantoyltaurine is rapidly excreted by rats, the ratio of pantoyltaurine to
pantothenic acid in the blood could be maintained above the range neces-
sary for in vitro inhibition. Administration of pantothenic acid with a
subsequent increase in its concentration in the blood resulted in reversal
of the therapeutic effect of pantoyltaurine, indicating that the mode of
action in vivo was analogous to that in vitro. Because of a higher con-
centration of pantothenic acid in the blood of mice, pantoyltaurine did
not exert such a protective action for these animals. Sulfonamide-resist-
ant streptococci were just as sensitive to pantoyltaurine as the nonresist-
ant strains.

In a series of studies on the mode of action of pantoyltaurine, Mc-
llwain 53 found that low concentrations of pantoyltaurine inhibited the
initiation of growth of ^-hemolytic streptococci, but, when the analogue
was added to growing cultures, the inhibitory effect on growth was not
apparent until after a latent period of an hour or more. The action of

PANTOTHENIC ACID 627

pantothenate in preventing growth inhibition was similarly delayed. The
disappearance of pantothenic acid from the culture medium of either
streptococci or Corynebacterium diphtheriae was quickly inhibited by
pantoyltaurine and promptly recurred after removal of pantoyltaurine.
By quantitative experiments, it was shown that the amount of panto-
thenic acid consumed in such a process by streptococci and other
organisms during normal growth was in considerable excess of their
ordinary needs. No correlation was apparent between the rate of destruc-
tion of pantothenic acid by an organism and the sensitivity of the
organism to pantoyltaurine. The process of pantothenic acid destruction 54
has been shown to be independent of growth and oxygen consumption,
but was associated with the presence of glucose and a casein hydrolyzate
(or related materials) and perhaps magnesium ions. Pantoyltaurine in-
hibited both growth and the destruction of pantothenic acid, but did
not prevent glycolysis appreciably. However, inhibition of glycolysis
prevented the inactivation of pantothenic acid. The concentrations of
pantoyltaurine (or other analogues) necessary to prevent the disappear-
ance of pantothenic acid from the medium varied over a 300-fold range
with strains of streptococci, of Corynebacterium diphtheriae, and of
Proteus morganii, but were correlated with the concentrations required
for inhibition of growth of the respective organisms.

Pantothenic acid was found to be present in a bound form in two
strains of /^-hemolytic streptococci. Autolysis or enzymatic digestion
liberated free pantothenic acid, but even high concentrations of pantoyl-
taurine did not displace the pantothenic acid from the combined form.
Pantothenic acid which was loosely bound was released into saline solu-
tions, but pantoyltaurine did not decrease the quantity remaining with
the organism. Thus, no gross displacement of pantothenic acid occurred.55

From these results, it was concluded that pantoyltaurine acts as a
bacteriostatic agent by preventing the conversion of pantothenic acid
to a functional derivative in susceptible bacteria.

In pantothenic acid-deficient yeast, pantoyltaurine does not inhibit
fermentation processes stimulated by pantothenic acid.56 In Streptococcus
hemolyticus,57 pantoyltaurine inhibits the rate of glycolysis slightly
(10-25 per cent), and the effect was prevented by pantothenic acid which
alone accelerates glycolysis to a slight extent.57 The concentrations of
pantoyltaurine affecting glycolysis had no effect on growth.

Pantoyltaurine depressed the growth of Streptococcus hemolyticus by
increasing the lag period and decreasing the rate of growth.57 The
logarithmic period of growth involved two phases — an initial phase dur-
ing which the rate of growth was considerably decreased, as compared
with normal growth, and a later phase characterized by an almost normal

628 THE BIOCHEMISTRY OF B VITAMINS

growth rate which was less susceptible to increasing concentrations of
the inhibitor.

Mcllwain 33 obtained resistant strains of Streptococcus hemolyticus and
Corynebacterium diphtheriae by serially subculturing the parent strains
in increasing concentration of pantoyltaurine. The strains of streptococci
which were resistant to pantoyltaurine were just as sensitive to sulfanila-
mide as the parent strain. Also, sulfanilamide-resistant strains were
found to be as susceptible to pantoyltaurine as the parent strain. The
resistant strains of streptococci retained their requirement for pantothenic
acid and did not appear to destroy pantoyltaurine; but the resistant
strains of diphtheriae bacilli were capable of utilizing /^-alanine instead
of pantothenic acid, whereas /3-alanine did not replace pantothenic acid
in the nutrition of the parent strain. Selection for strains of diphtheria
bacilli which could utilize ^-alanine was accomplished in the absence of
pantoyltaurine. These strains were resistant to bacteriostasis by pantoyl-
taurine. Proteus morganii, which normally is not sensitive to inhibition
by pantoyltaurine, was found to be inhibited by a mixture of salicylic
acid and pantoyltaurine under conditions in which the single components
of the mixture were inactive. A similar action was observed in both
normal and resistant streptococci. Pantothenic acid was effective in pre-
venting the toxic effects of the mixtures. Strains of Proteus morganii
have since been found which are susceptible to inhibition by pantoyl-
taurine with very high inhibition indices, approximately 200,000.33

Analogues Related to Pantoyltaurine. Pantoyltauramide and some of
its substituted derivatives, as well as sulfones, sulfoxides, sulfides, a
disulfide, and a mercaptan related to pantoyltaurine have been prepared.
The formulas and names are listed in Table 26.

DL-N-Pantoyltauramide was found to be active against Streptococcus
hemolyticus, Diplococcus pneumoniae, and Corynebacterium diphtheriae,
the inhibition indices being 2000, 10,000-50,000, and 2000-10,000, respec-
tively.46 Although it was less active than pantoyltaurine in vitro, and
against streptococcal infections in rats, D-N-pantoyltauramide admin-
istered to chicks intravenously in enormous doses (2 g or more per kg
per day) was found to exert a marked suppressive action upon the growth
of Plasmodium gallinaceum in chicks, but not in ducks. The antimalarial
activity in terms of quinine equivalents was 0.03. The inactivity of panto-
thenic acid analogues in ducks is caused by a difference in the host rather
than susceptibility of the organism, but this difference was not the result
of variations in the pantothenic acid content of the blood of the hosts.
The study of the antimalarial activity of analogues of pantothenic acid
was begun subsequent to the discovery that the addition of pantothenic
acid to an appropriate medium containing duck erythrocytes parasitized

PANTOTHENIC ACID 629

with Plasmodium lophurae lengthened the survival period of the para-
sites.63

The condensation product of DL-pantolactone and DL-a-phenyltaurine
does not appreciably affect the growth of Lactobacillus arabinosus.G0

DL-N-Pantoyl-/?-mercaptoethylamine and the corresponding disulfide
have been found to be approximately as active as pantoyltaurine in pre-
venting the utilization of pantothenic acid by Lactobacillus arabinosus.
Di(N-pantoyl-/?-aminoethyl) sulfide and the corresponding sulfoxide

Table 26. Pantoyltaurine and Related Inhibitory Analogues of Pantothenic Acid.

Formula Name References

R°-NH-CH2-CH2-S02-OH DL-N-Pantoyltaurine 15^ «. «• 46

D-N-Pantoyltaurine 44, 45

L-N-Pantoyltaurine **• **

R-NH-CH2-CH2-S02-NH2 DL-N-Pantoyltauramide 1B- 46

D-N-Pantoyltauramide 88' 89
R-NH-CH2-CH(C6H6)-S02-OHc N-Pantoyl-^-amino-a-phenylethane-

sulfonic acid 60

R-NH-CH2-CH2-S02-N(Ri)(R2) Substituted Pantoyltauramides Table 27

R-NH-CH2-CH2-SH DL-N-Pantoyl-/3-mercaptoethylamine 59

(R-NH-CH2-CH2)2-S2 Di(N-Pantoyl-jS-aminoethyl) disulfide6 "

(R-NH-CH2-CH2)2S Di(N-Pantoyl-/3-aminoethyl) sulfide11 59

(R-NH-CH2-CH2)2SO Di(N-Pantoyl-/3-aminoethyl) sulfoxide" B9

(R-NH-CH2-CH2)2S02 Di(N-Pantoyl-/3-aminoethyl) sulfone* 69

R-NH-CH2-CH2-S-C6H6 D-(N-Pantoyl-/3-aminoethyl) phenyl

sulfide 61
R-NH-CH2-CH2-SO-C6H6 D-(N-Pantoyl-/3-aminoethyl) phenyl

sulfoxide 61
R-NH-CH2-CH2-S02-C6HB D~(N-Pantoyl-/3-aminoethyl) phenyl

sulfone 61

R-NH-CH2-CH2-S-C6H4C1 D-(N-Pantoyl-/3-aminoethyl) p-chloro- 61

phenyl sulfide
R-NH-CH2-CH2-S02-C6H4-CH3 DL-(N-Pantoyl-/3-aminoethyl) p-tolyl

sulfone 62
R-NH-CH2-CH2-S02-C6H4-NH2 Di^(N-Pantoyl-/3-aminoethyl) p-

aminophenyl sulfone 62
R-NH-CH2-CH2-S02-C6H4-OCH3 DL-(N-Pantoyl-/3-aminoethyl) p-

methoxyphenyl sulfone 62

« R represents the pantoyl group, H0CH2-C(CH3)2-CH0H-C0-.
*> A crystalline compound, one of the two diastereoisomers.
c Presumably a mixture of diastereoisomers.

and sulfone were reported to be less effective than pantoyltaurine
in inhibiting the growth of Lactobacillus arabinosus. In vivo tests indi-
cated that these compounds were less effective than pantoyltaurine in
preventing streptococcal infections in rats.

D(N-Pantoyl-/?-aminoethyl) phenyl sulfide, the corresponding sulfoxide
and sulfone, and the corresponding p-chlorophenyl sulfide have been pre-
pared 64 and tested for antimalarial activity G1 in chicks infected with
Plasmodium gallinaceum. The quinine equivalents obtained for the sup-
pressive action were 0.8, 1.5, 1.0 and 1.0, respectively.

The phenyl sulfide and sulfoxide have also been found to be effective
against Trichomonas vaginalis with inhibition indices of 3.1 and 140r

630 THE BIOCHEMISTRY OF B VITAMINS

respectively, for 9 days' incubation in vitro.G5 The phenyl sulfide is also
effective in vitro against Trichomonas foetus and Trichomonas gallinae.
However, even at high concentrations, it did not affect Trichomonas
vaginalis infection in monkeys and human beings.

The phenyl sulfide has been reported to have an extremely low acute
and chronic oral toxicity in several animal species,06 and local irritation
effects are not obtained even upon repeated administration. An anemia
developed in monkeys which gradually disappeared upon discontinuing
the administration of the compound. Smooth muscle was generally stimu-
lated by the analogue.

DL(N-Pantoyl-/?-aminoethyl) p-tolyl sulfone and the corresponding
p-aminophenyl and p-methoxyphenyl sulfones inhibited competitively
the functioning of pantothenic acid in Lactobacillus casei with inhibition
indices of approximately 6400, 6400 and 1600, respectively. Streptococcus
pyogenes was also inhibited by these compounds, but only a slight chemo-
therapeutic effect was obtained in experimental streptococcal infections
in rats.

Substituted Pantoyltauramides. Since pantoyltaurine and its amide
had been found to inhibit competitively the utilization of pantothenic
acid in a relatively large number of bacteria, many substituted amides
of pantoyltaurine were prepared as possible chemotherapeutic agents,
particularly against the malarial parasites and streptococci. The activities
of these compounds against Streptococcus hemolyticus C203 in vitro and
in infected mice and as suppressives against the malarial parasites in
chicks are shown in Table 27. Streptococcus viridans, Streptococcus
agalactiae, and pneumococci were also susceptible to this group of in-
hibitors. Sulfonamide-resistant streptococci were just as susceptible to
the substituted pantoyltauramides as the normal strains.

It is interesting to note that the substituted amides are extremely effec-
tive inhibitors of the utilization of pantothenic acid in Streptococcus
hemolyticus C203.70 The more active of the compounds are approximately
10 to 20 times as effective as pantoyltaurine in vitro. Also, the protective
activity of the substituted pantoyltauramides in experimental strepto-
coccal infections in mice is in contrast to the inability of pantoyltaurine
to exert any protective action for mice. Even the chemotherapeutic activity
of pantoyltaurine which was observed in rats was obtained only with
large doses administered frequently.51

All the substituted pantoyltauramides were relatively nontoxic to mice
and rats, and with most of the analogues both the chemotherapeutic
activity and in vitro inhibition of growth were prevented by higher con-
centrations of pantothenic acid. Of the compounds listed, the two most
effective in maintaining blood levels and exerting a protective action on

PANTOTHENIC ACID

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632 THE BIOCHEMISTRY OF B VITAMINS

mice infected with Streptococcus hemolyticus C203 were D-pantoyltaur-
amido-4-chlorobenzene and D-pantoyltauramido-3,5-dibromobenzene.
However, only limited concentrations of these two compounds were found
to be reversed by pantothenic acid. This afforded an interesting compari-
son of the relative activities of analogues of d- and L-configuration. The
concentration of L-pantoyltauramido-3,5-dibromobenzene necessary for
inhibition of growth was found to be essentially equal to the lowest con-
centration of the D-form which pantothenic acid did not competitively
reverse. Also, L-2-(pantoyltauramido) pyridine was inactive at a ratio of
inhibitor to pantothenic acid of 6400, while the inhibition index of the
compound of D-configuration was 100. Hence, the analogues of L-con-
figuration appear to be inactive as competitive antagonists of pantothenic
acid.

These pantoyltauramide derivatives also have antiplasmodial activity
in blood, but not in sporozoite-induced infections of Plasmodium gal-
linaceum in chickens. This suggests that the blood phases of this infection
require pantothenic acid. The tissue phases either may not require the
vitamin or may obtain sufficient supplies of pantothenic acid or other
agents in the tissues to prevent the inhibitory action of the pantothenic
acid analogues. The most active analogue is D-pantoyltauramido-4-chloro-
benzene, which is four times as active as quinine in a standard four-day
trophozoite-induced infection (in brackets in Table 27), or sixteen times
as active in a more sensitive test where the peak parasitemias were not
reached until seven days after infection. The suppressive action of the
analogues was completely prevented by addition of adequate quantities
of pantothenic acid to the diet.

The pantoyltauramides were found to be toxic for chicks, but only at
concentrations well above those required for therapeutic doses.69 At a
concentration of 0.5 per cent in the diet, D-pantoyltauramido-4-chloro-
benzene reduced food intakes markedly and caused death of birds after
the twelfth day. At concentrations of 0.1 per cent, the analogue caused
only slightly reduced weight gains in a ten day period, and 0.025 per cent
(40 mg/kg/day) of the analogue did not affect growth of the chicks. The
toxicity of even 0.5 per cent of the analogue in the diet was prevented by
supplementation of 0.025 per cent of calcium D-pantothenate. An increase
was obtained in immature erythrocytes which was directly related to the
size of the dose of the analogue and length of treatment, although there
was no effect on the total erythrocyte count. Concentrations in the diet
as low as 0.0067 per cent gave significant increases in immature erythro-
cytes in ten days. However, rats receiving 0.25 g/kg/day of the analogue
maintained a normal blood picture even after 30 days of treatment.
Pantoyltauramidobenzene, pantoyltauramido-5-bromobenzene, pantoyl-

PANTOTHENIC ACID 633

tauramido-4-methoxybenzene, pantoyltauramido-4-methylbenzene and
pantoyltauramido-4-nitrobenzene caused similar alterations in the blood
picture of chicks. The toxicities were roughly proportional to the anti-
malarial activities.

N-Pantoyl Amino Acids. Investigations concerning the specificity of
pantothenic acid have resulted in the preparation of a number of
N-pantoyl derivatives of amino acids or their esters. DL-Pantolactone
was condensed with the esters of DL-alanine, DL-/?-aminobutyric acid,
L-aspartic acid and DL-lysine, and the resulting products were found to
be inactive in replacing pantothenic acid as a growth factor.71 In the
isolation of pantothenic acid from livers of tunny fish, an impurity which
was present in higher concentrations than pantothenic acid was found
to be a homologue of pantothenic acid, composed of L-leucine and a
pantoic acid homologue containing seven carbon atoms. This suggested
the preparation of the pantoyl derivative of L-leucine, which was found
to be inactive in replacing pantothenic acid for Streptobacterium plan-
tarum.12 Pantoyl-y-aminobutyric acid has been found to be unable to
replace pantothenic acid in preventing achromotrichia in rats.16 Also, the
pantoyl derivatives of lysine, leucine, and valine have no marked activity
in replacing the requirement of Proteus morganii for pantothenic acid.15

Condensation products of DL-pantolactone with glycine, DL-a-amino-
butyric acid, DL-a-aminoisobutyric acid, DL-a-amino-a-ethylbutyric
acid, and DL-norvaline are essentially inactive for Lactobacillus ara-
binosus 17-5.73 Corresponding condensation products of phenylalanine,
2-aminocyclohexanecarboxylic acid, and nipecotic acid are also essentially
inactive for Lactobacillus casei.62

Prepared as a possible substitute for pantothenic acid, the cc-methyl
analogue of pantothenic acid was found to have slight growth-promoting
activity, particularly for Saccharomyces cerevisiae G. M.; however, the
compound was found to prevent the utilization of pantothenic acid by
Lactobacillus casei.74' Several N-pantoyl amino acids, as indicated in
Table 28, have since been found to be competitive antagonists of panto-
thenic acid. With the possible exception of N-pantoyl-e-aminocaproic
acid, N-pantoyl-/?-aminobutyric acid appears to be the most effective of
this group as an antagonist of pantothenic acid. Since the product tested
is a mixture of two racemic diastereoisomers, the inhibitory activity may
result from the action of only one of the four forms, and certainly results
from the action of no more than the two forms containing the pantoyl
group of D-configuration. For comparison with the other analogues, the
inhibition indices for N-pantoyl-/?-aminoisobutyric acid presumably
should be doubled, since it alone was prepared from D-pantolactone.

In some cases with the a-substituted pantothenic acids, N-pantoyl-

634

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PANTOTHENIC ACID 635

isoserine and N-pantoyl-/?-aminoisobutyric acid, complete inhibition of
growth was not obtained regardless of the amount of inhibitor added.75
For such organisms not completely inhibited, these compounds alone
partially and ineffectively replaced pantothenic acid in stimulating
growth; however, growth induced by pantothenic acid was readily in-
hibited by these compounds to a level corresponding to their own stimu-
latory effect, but no further. In cases of this type, almost complete
inhibition of growth was obtained before stimulation by the antimetab-
olite became apparent. The growth stimulated by any concentration of
these analogues was only a fraction of that obtained with maximal con-
centrations of pantothenic acid.75

The relative concentrations of these antimetabolites required to pro-
duce half-maximum and maximum inhibition varied considerably with
the various test organisms. The variation was from slightly more than
twofold for Leuconostoc mesenteroides P-60 to more than tenfold for
Lactobacillus arabinosus 17-5 and Lactobacillus casei.75

The condensation products of DL-pantolactone and the sodium salts
of DL-alanine, DL-a-aminoisobutyric acid, DL-norvaline, DL-valine,
DL-phenylalanine, DL-serine, L-asparagine, p-aminobenzoic acid and
sulfanilamide were inactive as inhibitory analogues of pantothenic acid
for Streptobacterium plantarum. However, the pantoyl derivatives of
norvaline and alanine were somewhat inhibitory against yeast, whether
the growth was stimulated by /^-alanine or by pantothenic acid.77

Pantothenones. Replacement of the carboxyl group of p-aminobenzoic
acid by various ketone groups resulted in compounds which competitively
inhibited the corresponding metabolite.78 In determining whether or not
this type of alteration could be used extensively in obtaining antimetab-
olites, Woolley and Collyer79 prepared phenyl-D-pantothenone and
found it to be somewhat effective in preventing competitively the utiliza-
tion of pantothenic acid for several organisms, as indicated in Table 29.
It should be noted that the inhibition indices are for half-maximum
inhibition. These values are usually only a fraction of those for maximum
inhibition of growth. For Escherichia coli, Saccharomyces cerevisiae, and
Endomyces vernalis, phenyl-D-pantothenone reduced the growth to half-
maximum at concentrations of 60, 33 and 39 y per cc, respectively, in the
presence of 0.04 y per cc of pantothenic acid. However, the inhibitions
were not reversed by supplementary pantothenic acid for these organisms,
which synthesize pantothenic acid. Saccharomyces cerevisiae requires,
of course, the /^-alanine portion in order to carry out this synthesis. It is
interesting to note in Table 29 that the toxicity of phenyl pantothenone
is reversed by pantothenic acid for a strain of Staphylococcus aureus,
which was found to grow wrell without exogenous pantothenic acid but

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PANTOTHENIC ACID 637

was stimulated appreciably in its growth in the presence of the vitamin.
Usually organisms which synthesize pantothenic acid are not affected
by most of the analogues of that vitamin.

The activity of pantoyltauramide as an antimalarial led to the testing
of other analogues of pantothenic acid including phenyl pantothenone,
which was found to be effective as a suppressive agent in blood-induced
infections of Plasmodium gallinaceum in chicks.61 As a result of this
activity, other analogues related to phenyl pantothenone were prepared.80
The activities of these ketones are indicated in Table 29. Whereas p-tolyl
D-pantothenone is the most effective inhibitor against Lactobacillus
casei, p-chlorophenyl D-pantothenone is the most effective antimalarial
agent.

An impure preparation of methyl pantothenone was found to inhibit
the growth of Saccharomyces cerevisiae and Lactobacillus casei.79 This
inhibitory effect was not reversed by pantothenic acid, and the prepara-
tion was at lower concentrations 1 per cent as active as the vitamin in
stimulating growth of these two organisms. The possibility of contamina-
tion of the analogue with pantothenic acid appears possible.

The toxicity of phenyl pantothenone for Saccharomyces cerevisiae is
not reversed by pantothenic acid, but certain amino acids at relatively
high concentrations prevent the toxicity of the inhibitor.81 L-Histidine
(0.26 mg per cc) is the most effective amino acid in preventing the
toxicity of 160 y per cc of the inhibitor. L-Glutamic acid is somewhat
less effective, and L-proline, L-aspartic acid, L-asparagine and glycine
were about half as effective as L-glutamic acid, which also has a slight
effect on the toxicity of phenyl pantothenone for Lactobacillus casei.
Very slight effects were noted with serine, threonine, alanine and lysine,
but other amino acids were inactive.

Pantothenyl Alcohol and Related Compounds. As previously indicated,
pantothenyl alcohol is converted by warm-blooded animals into panto-
thenic acid and serves as an available source of that vitamin for these
organisms; however, as indicated in Table 30, the alcohol analogue not
only is not utilized in place of the vitamin by lactic acid bacteria, but
on the contrary it prevents competitively the utilization of pantothenic
acid by these organisms.19 Pantothenyl alcohol prevents the utiliza-
tion of pantothenic acid in Leuconostoc mesenteroides P-60 at an in-
hibition index of 300, whereas pantoyltaurine is inactive at a ratio of
200,000. On the other hand, pantoyltaurine is approximately 5 times as
active as pantothenyl alcohol in preventing the functioning of pantothenic
acid in Lactobacillus arabinosus 17-5. Such variations in sensitivity to
pantothenic acid analogues may result solely from differences in perme-
ability of various microbial cells, but more likely the variations are the

THE BIOCHEMISTRY OF B VITAMINS

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640 THE BIOCHEMISTRY OF B VITAMINS

result of differences in cellular interactions. The structures of the enzymes
utilizing pantothenic acid may be slightly different, the analogues may
prevent different reactions in the utilization of pantothenic acid, or dif-
ferences in the ability of the organisms to convert the analogues to other
forms may exist.

As indicated in Table 30, alteration of the structure of pantothenyl
alcohol tends to lower the inhibitory activity of the analogue. D-p-Tolyl-
pantothenol is more active than the corresponding methylpantothenol,
but is only 0.1 as active as the corresponding pantothenone for Lacto-
bacillus casei. However, in chicks with blood-induced infections of
Plasmodium gallinaceum, both p-tolylpantothenol and p-tolylpanto-
thenone possess approximately the same activity as quinine.61

N-Pantoylalkylamines and Related Compounds. A series of N-pantoyl
amines 19, 82 have been found to be relatively effective competitive
antagonists of pantothenic acid for Leuconostoc mesenteroides P-60, but
somewhat less effective as antimetabolites for Lactobacillus casei and
Lactobacillus arabinosus 17-5, as indicated in Table 31.

For Leuconostoc mesenteroides, the effectiveness of the different pantoyl
alkyl amines as inhibitory analogues of pantothenic acid increases con-
siderably as the length of the alkyl chain is increased to four carbons,
and then slowly decreases with increasing chain length. The derivatives
containing a branched alkyl are less active than the corresponding n-alkyl
derivatives. For Lactobacillus arabinosus 17-5 and Lactobacillus casei,
no definite trend in effectiveness with increasing chain length was
apparent; both the n-propyl- and the w-heptylamides were more effective
than the intervening members of the series.

D-N-Pantoyl-/?-phenylethylamine 80 also has a very slight activity
(quinine equivalent, 0.03) against blood-induced Plasmodium gallinaceum
infections in chicks.61

N-Pantoyl-n-butylamine and, less effectively, pantoyltaurine are re-
ported to inhibit competitively the utilization of pantothenic acid as a
carbon source for a strain of Pseudomonas.83

Miscellaneous Pantoyl Derivatives. DL-Pantamide and DL-panthy-
drazide have been synthesized and found to be antagonists of pantothenic
acid functioning in Lactobacillus casei.®2 At concentrations of 0.025 and
2.5 y per cc of pantothenic acid, 1,250 and 10,000 y per cc, respectively,
of pantamide, and 6 and 5,000 y per cc, respectively, of panthydrazide
inhibited growth of the organism.62

For Leuconostoc mesenteroides P-60, DL-pantothenonitrile, DL-panto-
thenyl amine, and DL-N-pantoyl-/?-methoxyethylamine prevent com-
petitively the functioning of pantothenic acid.82 The inhibition indices
are 10,000, 40,000 and 15,000, respectively. It is interesting to note

PANTOTHENIC ACID 641

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642 THE BIOCHEMISTRY OF B VITAMINS

that the last compound is not so effective as either the w-butylamine
or ethanolamine derivative.82 DL-N-Pantoyl-/3-diethylaminoethylamine,
DL-N-pantoyl-5-diethylamino-2-pentylamine, and DL-N-pantoyl-p-anisi-
dine are essentially inactive as pantothenic acid antagonists for Lacto-
bacillus casei.62

N-Substituted /3-Alanines and Related Compounds. Modification of the
pantoyl group of pantothenic acid has resulted with few exceptions in
compounds possessing slight activity in replacing the vitamin or in com-
pounds with neither stimulatory nor inhibitory properties. Almost one-
third of the reported N-substituted /^-alanine analogues of pantothenic
acid are active at least partially in replacing the vitamin in the nutrition
of some organisms (cf. Table 24).

The N-substituted derivatives of /3-alanine which have been found to
be inactive in replacing pantothenic acid are listed in Table 32. The
ester of a-amino-y-hydroxy-/?,/?-dimethylbutyryl-/?-alanine, although in-
active alone in replacing pantothenic acid, supplements suboptimal con-
centrations of pantothenic acid in stimulating growth of Lactobacillus
arabinosus. As indicated in Table 32, nearly half of these compounds
were not tested as possible antimetabolites of pantothenic acid. Although
many of the compounds were found to be slightly inhibitory to the test or-
ganisms, the inhibitions were with few exceptions not prevented by panto-
thenic acid. Thus, y-hydroxyvaleryl- and y-hydroxybutyryl-/3-alanine
prevent the growth of several organisms, but the toxicity is not abolished
by pantothenic acid.

Salicylyl-/3-alanine, mandelyl-/?-alanine, and acetyl mandelyl-/3-alanine
have been reported to be moderately active pantothenic acid antagonists ;
however, the data presented for mandelyl-/?-alanine do not indicate a
competitive inhibition.

Drell and Dunn 73, 84 have shown that N-a,y-dihydroxy-/?,/?-dimethyl-
valeryl-/3-alanine (w-methylpantothenic acid) competitively prevents the
utilization of pantothenic acid by a large number of lactic acid bacteria
and by Streptococcus hemolyticus. This compound is the first N-substi-
tuted /^-alanine to be reported as an effective antimetabolite of panto-
thenic acid. The analogue exists in two racemic diastereoisomeric forms,
the relative activities of which are unknown.

As indicated in Table 33, w-methylpantothenic acid inhibits the growth
of a wide variety of organisms for which pantothenic acid is essential;
however, it does not affect the growth of organisms not requiring the
vitamin for growth. As is common with a number of inhibitors, stimulation
of growth was obtained in many instances at concentrations of the an-
alogue just lower than that necessary for inhibition of growth. At rela-
tively high concentrations of pantothenic acid, the inhibition index was

PANTOTHENIC ACID 643

significantly increased, particularly for Lactobacillus casei and Lacto-
bacillus fermenti. After complete inhibition of growth of these organisms
was attained, further increases in the concentration of the analogue stimu-
lated growth of the organisms. Thus, at high concentrations of pantothenic
acid, complete inhibition of growth is never attained, even though the
analogue alone is incapable of stimulating growth of the organisms.

w-Methylpantothenic acid is effective against streptococci both in vitro
and in vivo. Mice were protected from an 80 per cent fatal infection of

Table 33. Comparison of Activity of u-Methylpantoyl Derivatives with Pantoyltaurine.™

Organism

Leuconostoc citrovorum 8082°
Lactobacillus fermentatus 4006
Lactobacilhis pentoaceticus 367
Lactobacillus brevis 8257
Leuconostoc citrovorum 797
Leuconostoc citrovorum 7013
Streptococcus faecalis R 8043
Lactobacillus casei 7469
Lactobacillus helveticus 335
Lactobacillus helveticus 6345
Lactobacillus lycopersici 4005
Leuconostoc dextranicum 8358
Leuconostoc dextranicum 8086
Leuconostoc mesenteroides 9135
Leuconostoc mesenteroides 8293
Lactobacillus fermenti 36-9338
Lactobacillus gayoni 8289
Leuconostoc dextranicum 8359
Leuconostoc mesenteroides P-60 (8042)
Lactobacillus pentosus 124-2
Lactobacillus arabinosus 17-5 (8014)
Lactobacillus brassicae 8041
Lactobacillus mannitopoeus

° American Type Culture Collection number.

b Stimulation of growth obtained at concentrations just below inhibitory levels.
c Half-maximum inhibition; complete inhibition of growth not obtained at index of 280,000.
d Stimulation of growth, no inhibition obtained.

• Stimulation of growth obtained at concentrations of analogue higher than that just necessary for
maximum inhibition. Effect enhanced by higher concentrations of pantothenic acid.

a /^-hemolytic streptococcus (Group A, type 23, No. 1072) when the
analogue was administered in the diet for four days prior to infection at
a concentration 200 times that of pantothenic acid.

Production of a pantothenic acid deficiency in weanling mice has been
accomplished by supplementation of the diet with w-methylpantothenic
acid.90 On a pantothenic acid-deficient diet, the survival time decreased
from 8 to 9 weeks to 5, 4, 2.5 and 1.5 weeks by supplementation with 0.06,
0.2, 0.5 and 1.5 per cent, respectively, of the analogue in the diet. With
diets containing 2 and 6 mg per cent of pantothenic acid and a 100-fold
excess of the analogue, the survival time was about the same as on the

Inhibition Index *

d-Methylpanto- to-Methylpantoyl-

Pantoyl-

thenic acid

taurine

2400

4200 b

150

5 1,000 b

113,000

270

175,000

85,000*

270

75,000

140,000

330

7,300

8,500 b

330

6,000 h

5,1006

330

26,000

35,000

450 d

16,500

15,000

500 b

51,0006

42,5006

5506

44,000 b

57,000b

800

> 333,000*

51,000

900

2,200

850

900

5,000

1,350

900

> 333,000*

1,350

1,100

4,400

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2,200 d

> 1,000,000*

130,000

2,200 b

> 333,000*

225.0006

2,7006

3,500

1,400

3,300

> 1,000,000*

250,000

4,000

> 333,000

5,200

22,000

4,700

7,500

> 333,000

13,0006

> 333,000*

225,000*

644 THE BIOCHEMISTRY OF B VITAMINS

pantothenic acid-deficient diet. Administration of an adequate amount of
pantothenic acid prevented or cured the deficiency disease caused by the
analogue. Strain differences in mice were observed, and graying of fur was
noted in only one of five experimental groups.

Analogues of Pantothenic Acid Modified in Both the Pantoyl and
/?-Alanyl Groups. A homologue of pantoyltaurine, DL-N-/3,8-dihydroxy-
7,y-dimethylvaleryltaurine (homopantoyltaurine)15 is an antagonist of
pantothenic acid for Streptococcus hemolyticus with an inhibition index
of 20,000.47 The inhibitory activity of this analogue is in contrast to that
of the corresponding ^-alanine derivative (homopantothenic acid). The
^-alanine derivative exerts only a slight toxic effect, which is not reversed
by pantothenic acid. On the other hand, homopantoyltaurine is much less
effective than the corresponding pantoyl derivative (pantoyltaurine),
which inhibits growth at an index of 500.

Another homologue of pantoyltaurine, N-ot,y-dihydroxy-/?,/?-dimethyl-
valeryltaurine (w-methylpantoyltaurine) , also has properties intermediate
between the corresponding y3-alanine and pantoyl derivatives.73 w-Methyl-
pantoyltaurine inhibits competitively the utilization of pantothenic acid
in a large number of lactic acid bacteria as indicated in Table 33; how-
ever, it is much less effective than w-methylpantothenic acid, but is more
effective in some cases than pantoyltaurine.

An unusual result was obtained with N-cc,y-dihydroxy-/?,/?-dimethyl-
valeryl-/?-aminobutyric acid, which prevents the growth of Escherichia
coli at a concentration of 100 y per cc.91 The toxicity is prevented by
pantothenic acid. Usually analogues of pantothenic acid either do not
prevent the growth of organisms which synthesize the vitamin, or, if
growth is prevented at high concentrations, the toxicity is not prevented
by supplements of the vitamin.

The activity of DL-N-a-hydroxy-/?,/?-dimethylbutyryltaurine,14 which
prevents the utilization of pantothenic acid (inhibition index, 2000 for
half-maximum) or pantoic acid (inhibition index, 800 for half-maxi-
mum) by Acetobacter sub oxy dans, is in contrast with the corresponding
^-alanine derivative, which possesses activity in replacing pantothenic
acid as a growth factor for this organism (cf. Table 24).

N - ( y - Hydroxybutyryl) taurine and N4 - (diacetylpantoyl) sulfanil-
amide have been reported to be antagonists of pantothenic acid for
Lactobacillus caseif® however, the former was found to have little
growth-inhibitory action with other organisms.6S N-a,y-Dihydroxy-/3,/3-
dimethylvaleryl-L-leucine,73 N- (/3-benzoylethyl) -a-hydroxycaproamide,80
N- (£-benzoylethyl ) caproamide,80 DL-N-a,y-dihydroxy-/?,/?-diphenyl-

butyryltaurine,60 DL-N-a-tosyl-y-hydroxy-y8,y3-dimethylbutyryltaurine,60

PANTOTHENIC ACID 645

N-butyryltaurine 68 and DL-N-a,y-dihydroxybutyryltaurine 68 are also
relatively inactive as inhibitors of pantothenic acid functioning.

The two optically active forms of N-a-keto-/?-methyl-/3-methylol-
valeryltaurine are reported to have slight activity in inhibiting the growth
of Streptobacterium plantarum 105.25 However, N-ct-keto-/3,/?-dimethyl-y-
hydroxybutyryltaurine does not show such an inhibitory effect.87

Analogues of ^-Alanine. The unintentional use of an inhibitor of the
utilization of /^-alanine led to the discovery of pantothenic acid as a
growth factor for yeast by Williams and co-workers.29, 92 Asparagine
added to the medium prevented the response of yeast to /^-alanine and
allowed a very specific microbiological assay for the vitamin.

Not only asparagine, which resembles /3-alanine structurally, but also
other a-amino acids in general prevent the utilization of /^-alanine by
yeast;93,94 thus L-glutamic acid, L-aspartic acid, L-glutamine, glycine,
DL-alanine, DL-serine, DL-a-aminobutyric acid, DL-threonine, DL-norvaline,
DL-norleucine, dl- valine, DL-leucine, DL-methionine, and DL-cysteine show
an inhibitory effect on yeast growth stimulated by ^-alanine. However,
no such effect is obtained when growth is stimulated by pantothenic
acid, except for somewhat less specific inhibitions obtained only with
methionine and cysteine.76 Proline, D-leucine, arginine, glycylglycine,
and a-aminoisobutyric acid do not inhibit growth stimulated by /?-
alanine.

The more effective inhibitors of the utilization of /3-alanine have been
/?-amino acids. These include /3-aminobutyric acid,95 isoserine,96 and
phenyl-/?-alanine.7G /3-Aminobutyric acid competitively inhibits the in-
crease in respiration of yeast brought about by /?-alanine when present
in the ratio 1000:1, but does not have any effect on the increase brought
about by the minimum effective concentration of pantothenic acid.97 The
inhibitory effect on respiration which is paralleled by a similar effect on
growth is less if the analogue is added several hours after the /3-alanine.
Taurine,95 /?-alanylglycine,95 and N-methyl-/?-alanine 9G are ineffective
for this strain of yeast. Both taurine and pantoyltaurine have been found
to inhibit only one strain of yeast out of seventeen when growth was
stimulated by ^-alanine.30 Taurine had no effect on growth stimulated by
pantothenic acid with this strain of yeast. However, pantoyltaurine in-
hibited the utilization of pantothenic acid in this and all other strains of
yeast.

a,/?-Diaminopropionic acid is reported to have a marked growth-inhibit-
ing effect on Corynebacterium diphtheriae, both mitis and gravis strains,
presumably by competing with the essential growth factor, ^-alanine.98

a-Methyl-/?-alanine has been found to be slightly active in replacing
/^-alanine for the growth of Saccharomyces cerevisiae G. M.74

646 THE BIOCHEMISTRY OF B VITAMINS

L-Carnosine is approximately one-fourth as active as /3-alanine for a
strain of diphtheria bacillus " and for Saccharomyces cerevisiae, Fleisch-
mann's strain 139,100 but is not active for Saccharomyces cerevisiae
G. M.30 D-Carnosine was inactive for the diphtheria bacillus.

The inhibitory effect of propionic acid for Escherichia coli,101 for Sac-
charomyces cerevisiae,102 and to some extent for Acetobacter suboxy-
dans,102 is prevented by ^-alanine. For Saccharomyces cerevisiae the
inhibition index is approximately 10,000. Pantothenic acid completely
reverses the toxicity at its minimum effective concentration so that it
appears that propionic acid also prevents the conversion of ,/3-alanine to
pantothenic acid in these organisms. Acetic acid acts in an analogous
manner with this strain of yeast, but is much less effective.

Analogues of Pantoic Acid. Compounds structurally similar to pantoic
acid and possessing growth inhibiting properties are listed in Table 34.
However, these compounds are not necessarily competitive antagonists
of pantoic acid. On the contrary, most of them cannot be considered
solely as competitive inhibitors of the functioning of pantoic acid.

Thus, a group of pantoic acid analogues reported by Cheldelin and
Schink14 prevent the growth of Acetobacter suboxydans either in the
presence of pantothenic acid or pantoic acid as indicated in Table 34.
However, growth of Saccharomyces cerevisiae G. M. stimulated by panto-
thenic acid was not prevented by either a-hydroxy-/3,/?-dimethylbutyric
acid or /?,y-dihydroxy-/3-methylbutyric acid, but the growth stimulated
by /^-alanine was prevented by the analogues. This suggests that the two
analogues prevent the biosynthesis of pantothenic acid in the yeast under
these conditions, presumably by competition with pantoic acid or a related
metabolite. With Acetobacter suboxydans the analogues appear to prevent
the utilization of pantothenic acid rather than its synthesis.

Ivanovics 103 has reported that salicylic acid at low concentrations
appears to prevent the synthesis of pantothenic acid in Escherichia coli.
This effect is exerted to a lesser extent by acetylsalicylic acid or phenyl-
salicylate but not by thiosalicylic acid or salicylamide. The inhibitory
action of low concentrations of salicylic acid was prevented in a somewhat
noncompetitive manner by pantothenic acid. Also, larger amounts of
pantoic acid as well as some amino acids were effective in preventing the
toxicity. Although valine alone was singly the most active of the amino
acids, mixtures of methionine with valine, leucine, isoleucine, or lysine
were more effective. These mixtures were almost equivalent to a casein
hydrolysate which caused an eight fold increase in pantothenic acid syn-
thesis. Thiamine and vitamin K have also been reported to prevent par-
tially the inhibitory effect of salicylic acid.104

Pantoic acid is approximately nine times more active than pantolactone

PANTOTHENIC ACID

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648 THE BIOCHEMISTRY OF B VITAMINS

in antagonizing the inhibitory action of salicylic acid on Escherichia
coli.105 Pantoyltaurine and certain pantoyltauramides have been found
to prevent the toxicity of salicylate but are only about 3 per cent as active
as pantoic acid.106 It is suggested that the organism effects a partial
hydrolysis of the analogues forming pantoic acid which prevents the
toxicity of salicylic acid.

DL-a-Amino-/?,/?-dimethyl-y-hydroxybutyric acid, which has been
termed pantonine, has been found to be as effective as DL-pantoic acid
in preventing the toxicity of salicylic acid for Escherichia coli, but was
inactive in replacing the pantoic acid or pantothenic acid requirement of
Acetobacter sub oxy dans.107

With Staphylococcus aureus, pantothenic acid prevented the toxicity
of low concentrations of salicylic acid in almost a competitive manner;
the anti-bacterial index varied only from 50,000 to 26,000 over a 16-fold
range of concentrations of pantothenic acid.103 The vitamin was approxi-
mately 20,000 times as active as pantolactone. Ascorbic acid, pimelic acid,
and purine bases are reported to enhance the anti-salicylate action of
pantothenic acid and to permit ^-alanine, which is not otherwise effective,
to prevent the toxicity of the inhibitor.108 The purine bases alone, particu-
larly guanine, prevent to some extent the bacteriostatic action of salicylic
acid.

The toxicity of salicylic acid for a strain of Clostridium septicum is
reported to be related to the utilization of pantoic acid.36

A strain of Proteus morganii which required pantothenic acid or pantoic
acid for growth was unaffected by salicylic acid except at high concentra-
tions.103 The toxicity of such high concentrations of the inhibitor was not
affected by pantothenic acid.

The reproductive phase of Trypanosoma lewisi infection is prolonged
and the number of organisms is increased in rats by restricting panto-
thenic acid in the diet. Repeated administration of salicylic acid results
similarly in prolonging the reproduction phase and in an exalted para-
sitemia in the rats.109

Mandelic acid has an inhibitory action which is largely prevented by
pantothenic acid in some strains of Escherichia coli;110 however, inhibi-
tion of growth of many strains is not obtained with mandelic acid except
at high concentrations, the effects of which are not prevented by panto-
thenic acid.111

Bibliography

1. Williams, R. J., Truesdail, J. H.. Weinstock. H. H.. Jr., Rohrmann. E., Lyman,

C. M., and McBurney, C. H., J. Am. Chem. Soc, 60, 2719 (1938).

2. Mitchell, H. K., Weinstock, H. H., Jr., Snell, E. E., Stanberry, S. R., and Wil-

liams, R. J., /. Am. Chem. Soc, 62, 1776 (1940).

PANTOTHENIC ACID

3. Woolley, D. W., and Hutchings, B. L., J. Bad., 38, 285 (1

4. SubbaRow, Y., and Rane, L., J. Am. Chem. Soc, 61, 1616 (1939).

5. Stiller, E. T., Harris, S. A., Finkelstein, J., Keresztesy, J. C, and Folkers, K.,

J. Am. Chem. Soc, 62, 1785 (1940).

6. Unna, K., and Muschett, C. W., Am. J. Physiol, 135, 267 (1942).

7. Harris, S. A., Boyack, G. A., and Folkers, K., J. Am. Chem. Soc., 63, 2662 (1941).

8. Griissner, A., Gatzi-Fichter, M., and Reichstein, T., Helv. Chim. Acta, 23, 1276

(1940).

9. Reichstein, T., and Griissner, A., Helv. Chim. Acta, 23, 650 (1940).

10. Mitchell, H. K., Snell, E. E., and Williams, R. J., J. Am. Chem. Soc, 62, 1791

(1940).

11. Zschiesche, E., and Mitchell, H. K., Proc. Soc. Exptl. Biol. Med., 45, 565 (1940).

12. Henze, H. R., and Nease, A. H., (unpublished work) ; Nease, A. H., Disserta-

tion, University of Texas (1943).

13. Woolley, D. W., and Hutchings, B. L., /. Bad., 39, 287 (1940).

14. Cheldelin, V. H., and Schink, C. A., J. Am. Chem. Soc, 69, 2625 (1947).

15. Barnett, J. W., and Robinson, F. A., Biochem. J., 36, 357, 364 (1942).

16. Pfaltz, Z., Z. Vitaminforsch., 13, 236 (1943); Chem. Abstr., 38, 6339 (1944).

17. Burlet, E., Jubilee Volume, Emil Barell, Reinhardt, Ltd. Co., Basle, Switzer-

land, 1946, p. 92; Burlet, E., Z. Vitaminjorsch., 14, 318 (1944); Chem. Abstr.,
41, 2782 (1947).

18. Schnider, O., Jubilee Volume, Emil Barell, Reinhardt, Ltd. Co., Basle, Switzer-

land, 1946, p. 85.

19. Snell, E. E., and Shive, W., J. Biol. Chem., 158, 551 (1945).

20. Hegsted, D. M., Proc. Soc. Exptl. Biol. Med., 69, 571 (1948).

21. Parke, H. C, and Lawson, E. J., J. Am. Chem. Soc, 63, 2869 (1941).

22. Hudson, C. S., J. Am. Chem. Soc, 39, 462 (1917).

23. Woolley, D. W., Waisman, H. A., Michelsen, O., and Elvehjem, C. A., /. Biol.

Chem., 125, 715 (1938).

24. Snell, E. E., Strong, F. M., and Peterson, N. H., Biochem. J., 31, 1789 (1937).

25. Wieland, T., and Moller, E. F, Chem. Abstr., 43, 4225 (1949); Ber., 81, 316

(1948).

26. Kuhn, R., and Wieland, T., Ber., 75, 121 (1942).

27. Schmidt, V., Acta Pharmacol. Toxicol. (Copenhagen), 1, 120 (1945); Chem.

Abstr., 40, 6127 (1946).

28. Rubin, S. H., Cooperman, J. M., Moore, M. E., and Scheiner, J., J. Nutrition,

35, 499 (1948).

29. Williams, R. J., and Rohrmann, E., J. Am. Chem. Soc, 58, 695 (1936).

30. Sarett, H. P., and Cheldelin, V. H., J. Bad., 49, 31 (1945).

31. Mueller, J. H., Proc. Soc. Exptl. Biol. Med., 36, 706 (1937) ; Mueller, J. H., and

Cohen, S., J. Bad., 34, 381 (1937); Mueller, J. H., and Klotz, A. W., J. Am.
Chem. Soc, 60, 3086 (1938).

32. Evans, W. C, Handley, W. R. C, and Happold, F. C, Brit. J. Exptl. Path.,

20, 823 (1939); Evans, W. C, Happold, F. C, and Handley, W. R. C, Brit.
J. Exptl. Path., 20, 41 (1939).

33. Mcllwain, H., Brit. J. Exptl. Path., 24, 203, 212 (1943).

34. Underkofler, L. A., Bantz, A. C, and Peterson, W. H., J. Bad., 45, 113 (1943).

35. Woolley, D. W., J. Biol. Chem., 130, 417 (1939).

36. Ryan, F. J., Schneider, L. K., and Ballentine, R., J. Bad., 53, 417 (1947).

37. Koser, S. A., Breslove, B. B., and Dorfman, A., J. Infectious Diseases, 69, 114

(1941); J. Bad., 41, 37 (1941).

38. Sarett, H. P., and Cheldelin, V. H., J. Biol. Chem., 159, 311 (1945).

39. Lipmann, F., Kaplan, N. O., Novelli, G. D., Little L. C, and Guirard, B. M.,

J. Biol. Chem., 167, 869 (1947).

40. Hegsted, D. M., and Lipmann, F., J. Biol. Chem., 174, 89 (1948).

41. Novelli, G. D., Flynn, R. M., and Lipmann, F., J. Biol. Chem.. 177. 493 (1949)

650 THE BIOCHEMISTRY OF B VITAMINS

42. King, T. E., Locher, L. M., and Cheldelin, V. H., Arch. Biochem., 17, 483

(1948); Abstracts, Am. Chem. Soc, 114th Meeting 59c (1948); King, T. E.,
Fels, I. G., and Cheldelin, V. H., J. Am. Chem. Soc, 71, 131 (1949).

43. Novelli, G. D., Kaplan, N. 0., and Lipmann, F., J. Biol. Chem., 177, 97 (1949) ;

Lipmann, F., Kaplan, N. O., and Novelli, G. D., Fed. Proc, 6, 272 (1947).

44. Snell, E. E., J. Biol. Chem., 139, 975 (1941); 141, 121 (1941).

45. Knhn, R., Wieland, W., and Moller, E. F., Ber., 74, 1605 (1941).

46. Mcllwain, H., Brit. J. Exptl. Path., 23, 95 (1942).

47. Mcllwain, H., Biochem. J., 36, 417 (1942); Mcllwain, H., Barrett, J., and

Robinson, F. A., Chem. and Ind., 61, 96 (1942).

48. Snell, E. E., Chan, L., Spiridanoff, S., Way, E. L., and Leake, C. D., Science,

97, 168 (1943).

49. Woolley, D. W., and White, A. G. C, Proc. Soc. Exptl. Biol. Med., 52, 106

(1943).

50. Unna, K., Proc. Soc. Exptl. Biol. Med., 54, 55 (1943).

51. Mcllwain, H., and Hawking, F., Lancet, 1943, I, 449.

52. Gaebler, O. H., and Herman, E. V., Fed. Proc, 6, 254 (1947).

53. Mcllwain, H., Biochem. J., 39, 279 (1945) ; Proceeding, 38, XXVIII (1944).

54. Mcllwain, H., and Hughes, D. E., Biochem. J., 38, 187 (1944); 39, 133 (1945).

55. Mcllwain, H., Biochem. J., 39, 329 (1945).

56. Teague, P. C, and Williams, R. J., J. Gen. Physiol, 25, 777 (1942).

57. Mcllwain, H., Biochem. J., 38, 97 (1944).

58. Mead, J. F., Rapport, M. M., Senear, A. E., Maynard, J. T., and Koepfli, J. B.,

J. Biol. Chem., 163, 465 (1946).

59. Barnett, J., J. Chem. Soc, 1944, 5.

60. Barnett, J., Dupre, D. J., Holloway, B. J., and Robinson, F. A., J . Am. Chem.

Soc, 1944, 94.

61. Wiselogle, F. Y., "A Survey of Antimalarial Drugs 1941-1945," Vol. I, p. 138,

174, J. W. Edwards, Ann Arbor, Michigan, 1946.

62. Madinaveitia, J., Martin, A. R., Rose, F. L., and Swain, G., Biochem. J., 3J,

85 (1945).

63. Trager, W., J. Exptl. Med., 77, 411 (1943).

64. Koepfli, J. B., et al. (unpublished work) California Institute of Technology.

65. Johnson, G., and Kupferberg, A. B., Proc. Soc Exptl. Biol. Med., 67, 390 (1948).

66. Singher, H. O., Millman, N., and Bosworth, M. R., Proc Soc Exptl. Biol. Med.,

67, 388 (1948).

67. Mead, J. F., and Koepfli, J. B., J. Org. Chem., 12, 295 (1947).

68. Winterbottom, R., Clapp, J. W., Miller, W. H., English, J. P., and Roblin,

R. 0., Jr., J. Am. Chem. Soc, 69, 1393 (1947).

69. Brackett, S., Waletzky, E., and Baker, M., J. Parasitol, 32, 453 (1946).

70. White, H. J., Lee, M. E., Jackson, E. R., Himes, A. T., and Alverson, C, Fed.

Proc, Part II, 5, 214 (1946).

71. Weinstock, H. H, Jr., May, E. L., Arnold, A, and Price, D., J. Biol. Chem.,

135, 343 (1940).

72. Kuhn, R., and Wieland, T., Ber., 73, 962 (1940).

73. Drell, W., and Dunn, M. S., J. Am. Chem. Soc, 70, 2057 (1948).

74. Pollack, M. A., J. Am. Chem. Soc, 65, 1335 (1943).

75. Shive, W., and Snell, E. E., Science, 102, 401 (1945).

76. Nielsen, N., Hartelium, V., and Johansen, G., Naturwiss., 32, 294 (1944) ; Chem.

Abstr., 40, 3154 (1946) ; Compt. rend. trav. lab. Carlsberg, Sir. physiol, 24,
39 (1944); Chem. Abstr., 40, 5796 (1946).

77. Nielsen, N., and Roholt, K., Acta Pharmacol. Toxicol. (Cophenhagen), 1, 207

(1945).

78. Auhagen, E., Z. physiol. Chem., 27 A, 48 (1942).

79. Woolley, D. W., and Collyer, M. L., J. Biol. Chem., 159, 263 (1945).

PANTOTHENIC ACID 651

80. Lutz, R. E, Wilson, J. W., Ill, Deinet, A. J., Harncst, G. H., Martin, T. A.,

and Freek, J. A., J. Org. Chem., 12, 96 (1947).

81. Woolley, D. W., J. Biol. Chem.; 163, 481 (1946).

82. Shive, W., and Snell, E. E., J. Biol. Chem., 160, 287 (1945).

83. Metzger, W. I., /. Bad., 54, 135 (1947).

84. Drell, W., and Dunn, M. S., Abstracts of Papers, 112th Meeting, Am. Chem.

Soc, 5c (1947).
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85. Drell, W., and Dunn, M. S., J. Am. Chem. Soc., 68, 1868 (1946).

86. Holly, F. W., Barnes, R. A., Koniuszy, F. R., and Folkers, K., J. Am. Chem.

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87. Wieland, T., Chem. Ber., 81, 323 (1948); Chem. Abstr., 43, 4223 (1949).

88. Woolley, D. W., /. Biol. Chem., 134, 461 (1940).

89. Moss, J. N., Urist, H., and Martin, G. J., Arch. Biochem., 19, 213 (1948).

90. Drell, W., and Dunn, M. S., Abstracts of Papers, 115th Meeting, Am. Chem.

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Chem. Soc, 69, 2567 (1947).

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Chem. Soc, 61, 1421 (1939).

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Chem. Abslr., 41, 2773 (1947).

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96. Nielsen, N., and Johansen, G., Naturwiss., 31, 235 (1943).

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Chapter VIM D

THE VITAMIN B6 GROUP

Although a dermatitis, termed acrodynia, which is characteristic of
vitamin B6 deficiency, was observed as early as 1926 in rats fed a deficient
diet,1 it was not until 1934 that Gyorgy 2 established that the "rat pel-
lagra preventitive" factor was a new B vitamin (vitamin B6) ; it was
subsequently isolated in crystalline form independently in five different
laboratories.3-7 However, in 1932 Ohdake 8 isolated from rice polishings
a compound apparently identical with vitamin B6, but did not recognize
it as a vitamin.

The structure of vitamin B6 indicated below was first elucidated by
Kuhn and co-workers 9< 10, 11 and subsequently was confirmed by inde-
pendent work of other laboratories.12-15

CH2OH

vitamin B6 (pyridoxine, adermin)

The synthesis of vitamin B6 was accomplished shortly afterward by
Harris and Folkers,16 by Kuhn and co-workers,17 and by Morii and
Makino.18

Specificity

During the proof of structure and synthesis of vitamin B6, a number of
structurally related compounds were prepared and tested for vitamin B6
activity. The activities of these compounds in replacing vitamin B6 for
various organisms are indicated in Table 35.

Although many of the organisms respond similarly to the various ana-
logues of pyridoxine, there are a few instances which contrast markedly.
Thus, 2-ethyl-3-hydroxy-4,5-bis(hydroxymethyl) pyridine, which is as
active as pyridoxine for stimulation of growth of excised tomato roots, in-
hibits the utilization of pyridoxine by Ceratostomella ulmi; but 2-methyl-
3-hydroxy-4-ethoxymethyl-5-hydroxymethylpyridine, which is 1 to 5 per
cent as active as pyridoxine for Ceratostomella ulmi, is quite injurious

THE VITAMIN B« GROUP

653

6! ■zzUlTUVIUVjd o =

rnu3)3Vqo)d3J)g 2

Sn33030jd3J,fg 2 28 CD ^ iO

_ O TO CD

8 «

LZ3VIS103J.33 oio (N

S33ftW04T>i{33Vg 22 rt

wS?OOJ O^BUJOl o o o ^

g§0ssgg5 55 55

■2 s g-??.

«-' ofc " ^

I -.all

£&-§!
rtja^ o j>

mil

ilfli
Iflll

bog >,oj2

654 THE BIOCHEMISTRY OF B VITAMINS

to tomato roots. Desoxypyridoxine (2,4-dimethyl-3-hydroxy-5-hydroxy-
methylpyridine) possesses growth-promoting activity for several micro-
organisms, but is an antagonist of pyridoxine for the rat and certain other
animals (p. 601). 2-Methyl-3-hydroxy-4-methoxymethyl-5-hydroxy-
methylpyricline and 2-methyl-3-hydroxy-4,5-epoxydimethylpyridine are
utilized effectively by Lactobacillus casei, but are inactive for Sac-
charomyces carlsbergensis. The relatively high activity of 2-methyl-3-
hydroxy-4,5-bis(bromomethyl) pyridine for both Lactobacillus casei and
Saccharomyces carlsbergensis is interesting.

For many organisms, the triacetate of pyridoxine is as active as
pyridoxine, but this compound is not utilized by Lactobacillus casei. On
the other hand, both pyridoxine triacetate and tribenzoate are more
effective for Streptococcus faecalis R than is pyridoxine. Pyridoxine
diacetate is almost ten times as effective as pyridoxine for Streptococcus
faecalis R. These as well as some prior results indicated that compounds
exist which are considerably more active than pyridoxine for Streptococ-
cus faecalis R.

Pyridoxamine and Pyridoxal. A naturally occurring form of vitamin
B6 which was more active than pyridoxine was discovered by Snell,
Guirard and Williams,33 who found that assays for the pyridoxine content
of natural extracts with Streptococcus faecalis R gave values for the
pyridoxine content several hundred to several thousand times as great as
could be accounted for on the basis of pyridoxine actually present. This
"pseudopyridoxine" was found to have properties similar to pyridoxine,
and tissues of animals deficient in vitamin B6 also contained less "pseudo-
pyridoxine."

It was demonstrated by Snell 34 that pyridoxine was almost inactive
unless autoclaved with the medium or with certain amino acids. Carpenter
and Strong ~>6 independently found that mild oxidation of pyridoxine
yielded a substance with increased activity for Lactobacillus casei.
Snell 27, 35 had indicated that mixtures resulting from the reaction of
pyridoxine with animating agents or mild oxidizing agents were more
active for Streptococcus faecalis R than the original vitamin. The proper-
ties of the active substance indicated that both an amine and an aldehyde
derived chemically from pyridoxine were the active principles. With this
and additional evidence, the structures of the two substances were limited
essentially to four possibilities, which were synthesized in pure form by
Harris, Heyl and Folkers 36 by methods analogous to those used by Snell.
Biological tests of the synthetic compounds revealed that 2-methyl-3-
hydroxy-4-aminomethyl-5-hydroxymethylpyridine and 2-methyl-3-hy-
droxy-4-formyl-5-hydroxymethylpyridine were the active amine and

THE VITAMIN Be GROUP 655

aldehyde which have been given the trivial names, pyridoxamine and
pyridoxal, respectively.

CH2NH2 CHO

— CH2OH HO-< ^— CH2OH

pyridoxamine pyridoxal

On the basis of their structures, Snell 35 proposed that pyridoxal and
pyridoxamine might have a role in biological transamination, and showed
that reversible interconversion of pyridoxal and pyridoxamine occurred
by transamination reactions with amino acids.37 Vitamin B6-deficient
rats were found to have a marked decrease in transaminase activity com-
pared to normal rats.38

Pyridoxal, but not pyridoxamine, added to cells of Streptococcus
faecalis R stimulated tyrosine decarboxylase,39 which was previously
known to be influenced by the concentration of pyridoxine added to the
growth medium.40 Gunsalus, Bellamy and Umbreit 41 subsequently found
that adenosine triphosphate was essential for maximum activity of
pyridoxal in the system, and prepared a phosphorylated derivative of
pyridoxal which was active in the absence of the phosphorylating agent.
The enzyme was later isolated in a cell-free state and resolved into an
apoenzyme and a coenzyme. The coenzyme was replaced by the phos-
phorylated pyridoxal.42 One of the methods of preparation 41 indicates
that the 5-hydroxymethyl is the point of phosphorylation, but definite
proof of the structure is still lacking.41* This pyridoxal phosphate has
been found to be a prosthetic group of transaminase and other enzymes
(p. 177) , and can be converted by heating with glutamic acid to pyridox-
amine phosphate, which functions as a cotransaminase but not as a
codecarboxylase.43 The discovery of this group of factors, which have
been termed the vitamin B6 group, stimulated studies of comparative
activities which are indicated in Table 36.

Injected pyridoxine, pyridoxal and pyridoxamine are equally active
for dogs, rats and chicks; however, either pyridoxal or pyridoxamine
is less active than pyridoxine for rats, mice and chicks when mixed with
the diet. This loss of activity is prevented in the case of rats and
chicks when these substances are fed by dropper. A possible explanation
has been suggested 45 that pyridoxal and pyridoxamine are more suscept-
ible to destruction or utilization by intestinal bacteria than is pyridoxine.
This conclusion is supported by the fact that Streptococcus faecalis R and
presumably many other bacteria do not remove appreciable amounts of

656

THE BIOCHEMISTRY OF B VITAMINS

pyridoxine from the medium.32 Pyridoxal phosphate fed with the diet is
similarly less active than pyridoxine, but when injected the two sub-
stances are equally active for rats.45

Table 36. Biological Activities of the Vitamin B6 Group.

, Activity, per cent of most active form .

•a

o
>>

-a
•c

— 01

Cm a

■eg

References

100°

100*

100 *>

100h

100*

31, 45

100'

50cac

65 cac

45, 46

100c

75 cac

' 55 CELcd

100 b

1006

100c

50cac

80"

0.16

100

100 ca

20-33

100

16 ca

100

0.085

100

0.70

6ca

0.7

31, 60, 61

0.015

68.8

100

7 ca

66-200e

31, 50, 51

0.102

100

27.5

0.001'

100/

0.013"

31.8"

100»

100

10 ca

0.13

62.3

100

0.16

100

39.5

0.19

100

4.76

0.16

100

0.31

100

87.5

0.26

100

3.96

0.01-0.03

30 ca

100

0.08

35 ca

100

86.3

100

93.6

12 ca

31, 60, 61

100

33-98

13-52

100

51-98

18-61

51.1

100

87.5

100

Organism

Rats

Mice
Chicks

Tetrahymena geleii
Lactobacillus helveticus
Lactobacillus acidophilus
Lactobacillus casei
Streptococcus faecalis R
Streptococcus lactis L 101
Lactobacillus arabinosus

Leuconostoc mesenteroides
Streptococcus lactis UT
Streptococcus lactis 374
Streptococcus mastitidis G-2
Streptococcus mastititis 97-B
Streptococcus zymogenes H69D5
Bacillus lactis acidi Bl-1
Clostridium perfringens
Clostridium welchi (B P6K)
Saccharomyces carlsbergensis
Saccharomyces cerevisiae GM
Saccharomyces oviformis
Ceratostomella ulrni
Neurospora sitophila 299

° Injected in radial vein. Activities indicated are also the same for ability to promote blood regimenta-
tion in anemic dogs.

b Injected intraperitoneally or fed by dropper; pyridoxal, pyridoxal phosphate or pyridoxamine are less
active than pyridoxine if fed in the diet.

« Fed in diet.

d Ability to utilize pyridoxal increased after first week. Xanthurenic acid excretion data support the
growth data.

• Dose-response curves for pyridoxamine phosphate and pyridoxamine differ. Figures express extreme
variation in relative activity which increases as pyridoxamine is increased.

/ Activity in stimulating growth, vitamin B6 not essential for growth.

"Activity in allowing growth to occur on indole in place of tryptophan.

It is interesting to note that two organisms — a strain of Lactobacillus
helveticus and a strain of Lactobacillus acidophilus — require the phos-
phorylated forms of pyridoxamine or pyridoxal and cannot utilize pyri-
doxal, pyridoxamine or pyridoxine. Pyridoxamine phosphate is from 3 to
6 times as active as pyridoxal phosphate for these organisms.

Most bacteria utilize either pyridoxal or pyridoxamine more effectively
than pyridoxine. The normal habitat of most of these organisms is of

THE VITAMIN B* GROUP 657

animal origin where pyridoxal and pyridoxamine predominate.55 Pyri-
doxine occurs in as large or larger amounts than pyridoxal and pyri-
doxamine in plants.55

Yeast and molds utilized pyridoxine very effectively and in some
instances more effectively than either pyridoxal or pyridoxamine.

For the few organisms tested, with the exception of the two requiring
the phosphorylated form of the vitamin, pyridoxal phosphate is only 3
to 10 per cent as effective as pyridoxal, but pyridoxamine phosphate under
certain conditions is more active than any other member of the vitamin
B6 group for Streptococcus faecalis R.

Pyridoxic Acid. A metabolite of pyridoxine occurring in human urine56
has been isolated, identified as 2-methyl-3-hydroxy-4-carboxy-5-hydroxy-
methylpyridine, synthesized and given the trivial name pyridoxic acid by
Huff and Perlzweig.57- 5S

COOH

HO— i^S-CHjOH

ch34v

pyridoxic acid

Pyridoxic acid is the chief metabolic product of either pyridoxine,
pyridoxal or pyridoxamine.59 Oral administration of pyridoxal to human
subjects results in excretion of significantly higher amounts of pyridoxic
acid than does administration of pyridoxine or pyridoxamine. After ad-
ministration of pyridoxamine, almost equivalent amounts of pyridoxal
and pyridoxamine are excreted; but when pyridoxal or pyridoxine is
ingested, the form fed is the chief form of the vitamin in the urine.
Although injection of pyridoxine increased the pyridoxamine and pyri-
doxal content of the urine, no evidence could be obtained for the conver-
sion of either pyridoxal or pyridoxamine to pyridoxine. The recoveries in
these four forms from ingested pyridoxal, pyridoxine or pyridoxamine
were 70, 45 and 31 per cent, respectively.

It is interesting to note that pyridoxic acid is inactive in replacing the
vitamin B6 group in the nutrition of dogs,44 Streptococcus faecalis R,31
Lactobacillus casei,31 and Saccharomyces carlsbergensis.31

Both the lactone of pyridoxic acid and the lactone of 2-methyl-3-
hydroxy-4-hydroxymethyl-5-carboxypyridine have been reported to en-
hance the effect of pteroyldi-y-glutamylglutamic acid in promoting growth
and preventing anemia in chicks on a purified diet containing adequate
amounts of pyridoxine (p. 570) .60, 61 Attempts to confirm these effects
have been unsuccessful.61*1 The substances, designated as fi- and a-pyracin,
respectively, have been reported not to have any appreciable vitamin B6

658 THE BIOCHEMISTRY OF B VITAMINS

activity for certain microorganisms.31 a-Pyracin, as judged by survival
of the animals on a vitamin B6-cleficient diet, appears to have slight
pyridoxine activity in mice.46 The possibility of contamination with
pyridoxine was suggested, since a-pyracin is inactive for chicks.47

Analogues of Pyridoxal and Pyridoxamine. 2-Methyl-3-hydroxy-4-
hydroxymethyl-5-aminomethylpyridine, an isomer of pyridoxamine, is
only 0.002, 1.4, 0.22 and 0.5 per cent as active as pyridoxamine for
Streptococcus faecalis R, Lactobacillus casei, Saccharomyces carls-
bergensis, and rats, respectively. The activity for Lactobacillus casei is
slightly exaggerated, since it is compared with pyridoxamine, which is
relatively inactive as compared with pyridoxal for this organism.31
2-Methyl-3-hydroxy-4-hydroxymethyl-5-formylpyridine, the correspond-
ing isomer of pyridoxal, is 0.005-0.01, 0.03, and 29-73 per cent as effective
as pyridoxal for Streptococcus faecalis R, Lactobacillus casei and Sac-
charomyces carlsbergensis, respectively. The 5-formyl derivative appar-
ently can be utilized effectively by yeast, which presumably reduces the
formyl group. Rats cannot effectively convert this compound to the
vitamin.31

The ethyl acetal derived from the hemiacetal of pyridoxal is 50 to 75
per cent as active as pyridoxal for Streptococcus faecalis R and Lacto-
bacillus casei and just as effective as the vitamin for Saccharomyces
carlsbergensis. This activity, however, is attributed to the hydrolysis of
the acetal.31

Since vitamin BG functions in transamination reactions, the biological
activity of Schiff bases and analogous compounds of the amino acids and
pyridoxal are of interest. Eighteen pyridoxylamino acids corresponding
to the formula indicated below have been prepared 62 and tested 63 for

R
CH2— NH— CH— COOH

HO— ^S— CH2OH

their ability to replace the vitamin B6 group for a variety of organisms.
These compounds were prepared by reductive condensation of pyridoxal
with the following amino acids: DL-alanine, DL-aspartic acid, L-aspar-
agine, DL-glutamic acid, L-glutamic acid, glycine, DL-isoleucine, dl-
leucine, L-leucine, L-lysine, DL-methionine, DL-norleucine, DL-phenyl-
alanine, DL-serine, DL-threonine, DL-tryptophan, L-tyrosine and dl-
valine. None of these compounds is more than 0.5 per cent as active as
pyridoxal hydrochloride on a weight basis for Saccharomyces carlsber-

THE VITAMIN Be GROUP 659

gensis, Streptococcus faccalis and Lactobacillus casei. For a strain of
Neurospora sitophila, the activities were for several of the compounds
between 0.1 and 1 per cent that of pyridoxal hydrochloride. For the rat,
definite but limited activity has been noted for some of the pyridoxal-
amino acids.

After pyridoxylamino acids are autoclaved in dilute aqueous solution,
the resulting solution possesses high activity.63 Since the liberation of
active substances could be prevented by antioxidants, such as ascorbic
acid, cysteine or the complete basal medium, it was proposed that an
oxidative and hydrolytic cleavage — presumably by intermediate for-
mation of Schiff bases — occurred, with the formation of pyridoxal and
pyridoxamine. This is substantiated by the fact that pyridoxylideneani-
line and other Schiff bases tested were found to be as active as pyridoxal.
The thiazolidinecarboxylic acid formed from L-cysteine and pyridoxal
[2-(2-methyl-3-hydroxy-5-hydroxymethyl-4-pyridyl) -4 -thiazolidinecar-
boxylic acid] was also as active as pyridoxal for all organisms. The
activity is attributed to pyridoxal formed by cleavage of the product
in aqueous solutions, since the products of cysteine with other aldehydes
act similarly.03

Pyridoxyl-/?-alanine and the condensation product formed between
histidine and pyridoxal [4- (2-methyl-3-hydroxyl-5-hydroxymethyl-4-
pyridyl)-l-imidazo(c)tetrahydropyridine-6-carboxylic acid] were in-
active.63

No significant antivitamin effect was noted for any of the pyridoxyl
or pyridoxylidene compounds tested.63

By condensation of a number of amines, including some pressor amines,
with pyridoxal and reduction of the pyridoxylidene derivative, a number
of pyridoxyl amines have been prepared. These include pyridoxyltrypta-
mine, pyridoxyl-/?-phenylethylamine, pyridoxyltyramine and pyridoxyl-
benzylamine, which have activities between 50 and 100 per cent that
of pyridoxine for rats. These compounds are considerably more effective
than the corresponding pyridoxylamino acid in replacing the vitamin BG

Inhibitory Analogues of the Vitamin B6 Group

Demonstration of a growth inhibition prevented by pyridoxine was
first reported by Robbins and Ma,23 who showed that the toxicity of
certain pyridoxine analogues for Ceratostomella ulmi was prevented by
sufficient pyridoxine. These analogues were 2-ethyl-3-hydroxy-4,5-bis-
(hydroxymethyl) pyridine, 2-methyl-3-amino-4-hydroxymethyl-5-amino-
methylpyridine, 2-methyl-3-amino-4-ethoxymethyl-5-aminomethylpyri-
dine, and 2,4,5-trimethyl-3-hydroxypyridine. Robbins 24 previously

THE BIOCHEMISTRY OF B VITAMINS

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THE VITAMIN Ba GROUP

661

reported that 2,4,5-trimethyl-3-hydroxypyridine and 2-methyl-3-hy-
droxy-4-ethoxymethyl-5-hydroxymethylpyridine inhibited the growth
of excised tomato roots, but did not show that the toxicity was prevented
by pyridoxine. These and other inhibitory analogues of the vitamin Bc
group are listed in Table 37. The analogues, with one exception, contain
one or more of the following variations from the structure of pyridoxine:
2-ethyl group replacing the 2-methyl group, a methyl group replacing an
hydroxymethyl group, or either an amino or alkyoxyl group replacing
an hydroxyl group. These types are illustrated by the following inhibitory
analogues:

CH2OH

H0:A

-CH2OH

2-ethyl-8-hydroxy-4,5-bis
(hydroxymethyl)pyridine

CH3

I
HO— if^'S— CH2OH

desoxypyridoxine

CH2OCH3

CH2OH

-CH2OH

£-methyl-3-hydroxy-4-inethoxymethyl-
5-hydroxymethylpyridine

-CH2NH2

2-m ethyl-3-amino-Jf-hydroxy-
methyl-5-aminomethylpyridine

2-Ethyl-3-hydroxy-4,5-bis(hydroxymethyl)pyridine. The index at which
the ethyl homologue of pyridoxine inhibits the growth of Ceratostomella
ulmi is approximately 50.25 At that ratio of analogue to vitamin, no
growth occurs, but at a ratio of 10, approximately half-maximal growth
is obtained. This analogue is as effective as pyridoxine in stimulating the
growth of excised tomato roots (Table 35). 24 This high activity in replac-
ing the vitamin for one organism and the very potent inhibiting action
on another organism illustrate that a line of demarkation cannot be
drawn between stimulatory and inhibitory properties of analogues of
metabolites.

Desoxypyridoxines. Investigation of the vitamin activity of analogues
of pyridoxine led to the discovery that 2,4-dimethyl-3-hydroxy-5-hy-
droxymethylpyridine (a desoxypyridoxine) was very toxic for pyridoxine-
deficient chicks.65 Day-old female single comb White Leghorn chicks,
after being maintained on an adequate diet for three days, were placed
on a purified diet deficient in pyridoxine for a period of six days before
administration of the desoxypyridoxine. Administration of as little as

662 THE BIOCHEMISTRY OF B VITAMINS

two doses of desoxypyridoxine (100 y on alternate days) at approximately
six times the concentration of pyridoxine intake (16 y) resulted in the
death of all the chicks. Without simultaneous administration of pyri-
doxine, as little as 16 y doses of desoxypyridoxine gave similar results.
From the growth response of the chick to pyridoxine, the apparent
pyridoxine activity resulting from administration of a mixture of the
vitamin and analogue was determined. The difference between the amount
of the vitamin administered and the apparent activity of the mixture
was considered the amount of vitamin activity counteracted by the
analogue. On this basis, two molecules of the inhibitor were necessary
to counteract the vitamin activity of one molecule of pyridoxine at both
suboptimal and optimal concentrations of the vitamin for the deficient
chicks.65

Normal chicks on a diet containing adequate amounts of pyridoxine
were able to tolerate a total dosage of at least 600 y of the analogue, a
level six times the lethal concentration for more than half the pyridoxine-
deficient chicks on an analogous diet.65

Almost 100 per cent mortality of the chick embryos resulted from
the injection of 1 mg of desoxypyridoxine into eggs at the outset of
incubation.66 The inhibitory effects were prevented by simultaneous
injection of one of the vitamin BG group. The ratios of analogue to the
vitamin at which only 50 per cent of the embryos survived were: 20 for
pyridoxal hydrochloride, 50 for pyridoxamine dihydrochloride, and 100
for pyridoxine hydrochloride. However, desoxypyridoxine was not toxic
to the embryo when injected after six days of incubation, except at
high concentrations (2.5-5 mg per egg) at which the toxicity was not
prevented by any of the three forms of vitamin B6.66

If desoxypyridoxine is administered to weanling rats in a purified
diet deficient in pyridoxine, the rate of production of acrodynia was
increased and the symptoms of vitamin B6 depletion were aggravated.67
Pyridoxine, as well as pyridoxal and pyridoxamine, prevents the toxicity
of the analogue. The ratio of desoxypyridoxine to pyridoxine at which
the dermatitis appears is approximately 50. With stock rations, the ratio
was 175. Since the other members of the vitamin B6 group act in a manner
analogous to pyridoxine, the increased ratio with the stock ratios cannot
be accounted for on the basis of their presence in the stock diet. Adult
rats previously maintained on a stock diet were not affected appreciably
during a test period on a purified diet deficient in pyridoxine; however,
on the deficient diet supplemented with 0.5 mg per cent of desoxypyri-
doxine, rats showed deficiency symptoms, acrodynia and loss of weight,
at an average of 55 days.67

Marked reproductive upsets occur in normal female rats on a pyri-

THE VITAMIN Be GROUP 663

doxine-deficient diet containing 0.5 mg per cent of desoxypyridoxine.73
If a change to the deficient diet containing the analogue is made on the
day of breeding, the effects noted, such as 10 per cent resorption, are not
as drastic as those resulting from placing the animals on the diet prior
to breeding. If the animals are placed on the diet 22 days before breed-
ing, resorption occurs in all cases and failure of implantation occurred
to a significant extent (29 per cent). The percentage of resorptions and
number of young born dead increased, whereas the average number of
young per litter and average weight of the young decreased with the
increase in number of days the animals were maintained on the deficient
diet before breeding. Supplementation with pyridoxine on the day of
breeding counteracted the adverse effects of the desoxypyridoxine.73

Administration of desoxypyridoxine to rats receiving tryptophan
causes small increases in the excretion of xanthurenic acid and ky-
nurenine, products which are known to be excreted as a result of vitamin
B6 deficiency (p. 428). The increase produced by the analogue in excre-
tion of xanthurenic acid and kynurenine was significantly greater in rats
partially depleted of vitamin B6. Desoxypyridoxine produced this met-
abolic dysfunction almost immediately, in contrast with the period of
time necessary for occurrence of deficiency symptoms. Supplements of
pyridoxine prevented these effects of desoxypyridoxine.

With mice on a pyridoxine-deficient diet, desoxypyridoxine produces
dermatitis, "ring tail" condition, unstable gait and other symptoms com-
parable to those produced in the rat.68

Desoxypyridoxine fed to an insulin-treated, depancreatized dog on a
diet deficient in pyridoxine increased the fasting blood sugar but did not
cause glycosuria or affect the hemoglobin, cell volume or serum
chlorides.75

Administration of desoxypyridoxine to mice and rats causes atrophy
of both normal and neoplastic lymphoid tissue.76 Lymphosarcoma trans-
plants showed marked regression following administration of desoxy-
pyridoxine. The regression was associated with extensive pyknosis and
caryorrhexis of tumor lymphocytes and transformation of tumor cells
into apparent multinucleated giant cells.76 When pyridoxine is given
simultaneously with the analogue, the latter has no effect. Similarly,
atrophy of the spleen, thymus and lymph nodes has been reported for
puppies, chicks and monkeys following the administration of desoxy-
pyridoxine.69 Impairment of the immune response in rats has also been
noted, and the anamnestic reaction is abolished in acute pyridoxine
deficiency.76 There is not, however, an increased rate of antibody destruc-
tion, as the disappearance of antibodies following passive immunization
is not accelerated by a pyridoxine deficiency.76 A progressive decrease

664 THE BIOCHEMISTRY OF B VITAMINS

in erythrocyte count, hemoglobin, and hematocrit with a microcytic and
hypochromic anemia was observed in puppies receiving desoxypyri-
doxine.69 The animals lost weight and died within two months. Monkeys
receiving desoxypyridoxine similarly developed microcytic anemia,
leucopenia and lymphopenia.69

Desoxypyridoxine inhibits the multiplication of T2r+ Escherichia coli
bacteriophage without affecting growth of the bacteria. The inhibition
of virus production is prevented by sufficient pyridoxine, as well as by
formic, acetic, butyric, valeric and pyruvic acids, glucose-6-phosphate,
and less effectively by lactic, malic, fumaric and succinic acids.76a

Desoxypyridoxine is reported to inhibit the stimulatory action of
pyridoxine or pyridoxal for Saccharomyces cerevisiae. Pyridoxal appears
to be more effective in preventing the inhibitory action of low concentra-
tions of the analogue.70 At low concentrations the analogue alone is
reported to stimulate the growth of a strain of Saccharomyces cerevisiae.27
It is interesting to note that desoxypyridoxine has slight growth-promot-
ing activities for a number of microorganisms (Table 35).

Since the effect of desoxypyridoxine is more pronounced in animals
with restricted vitamin B6 intake, the mechanism of action of the in-
hibitor did not appear to be solely that of strict competition with the
vitamin. Desoxypyridoxine, even at concentration of 1 mg per cc, does
not affect tyrosine decarboxylase from Streptococcus faecalis R,72' 77
aspartic-glutamic transaminase from heart muscle of the horse,72 or the
tryptophanase system of Escherichia coli.72 Once pyridoxal phosphate is
associated with these enzymes, desoxypyridoxine does not displace it.
Furthermore, desoxypyridoxine does not prevent the combination of
pyridoxal phosphate with apoenzymes of the decarboxylase or the tran-
saminase.72 Desoxypyridoxine has no effect on the formation of pyridoxal
phosphate from pyridoxal and suboptimal amounts of adenosine tri-
phosphate in Streptococcus faecalis R.72 However, when adenosine tri-
phosphate is present in excess and pyridoxal is limiting, desoxypyridoxine
exerts some effect when the mixture is assayed with the apoenzyme of
tyrosine decarboxylase from Streptococcus faecalis R.72 This inhibition
is attributed to the formation of desoxypyridoxine phosphate.

Desoxypyridoxine phosphate exerts no effect on 3,4-dihydroxyphenyl-
alanine decarboxylase 77a and only a slight effect on tyrosine decar-
boxylase.72, 77 However, if it is allowed to compete with pyridoxal phos-
phate for the apoenzyme, either by addition prior to or simultaneous
with the natural coenzyme, complete inhibition of the tyrosine decar-
boxylase occurs at a ratio of inhibitor to coenzyme of 1000.72 If the
apoenzyme is allowed to combine with the coenzyme first, desoxypyri-
doxine phosphate at the same relative concentration after a short incuba-

THE VITAMIN B, GROUP 665

tion inhibits only to the extent of 12 per cent.72 This indicates a very
slow rate of dissociation of the enzyme and that considerable time would
be required for attainment of equilibrium. This slow rate of attainment
of equilibrium has been advanced as the explanation for the more pro-
nounced effect of desoxypyridoxine in animals on a vitamin B6-deficient
diet.72

2,4,5-Trimethyl-3-hydroxypyridme, a bisdesoxy pyridoxine, is toxic
for Ceratostomclla ulmi with an inhibition index of 830-250,25 and is also
toxic for excised tomato roots.24

2-Methyl-3-hydroxy-4-alkoxy-5-hydroxymethylpyridines. Tests with chicks
by methods analogous to those used for desoxypyridoxine indicate that
2-methyl-3-hydroxy-4-methoxymethyl-5-hydroxymethylpyridine is al-
most as effective as desoxypyridoxine in preventing the utilization of
pyridoxine. Approximately 4 molecules of the methoxy analogue
counteracts the response of 1 molecule of pyridoxine. Although both
desoxypyridoxine and the methoxy analogue act similarly in many
respects, the effects of otherwise lethal doses of desoxypyridoxine are
easily counteracted, even after a considerable period of time, by admin-
istration of pyridoxine; however, pyridoxine administered subsequent to
the methoxy analogue was generally ineffective in preventing death of
chicks. Thus, if the modes of action of the two compounds are similar
to the extent that the phosphorylated derivatives are the active inhibitory
forms, the rate of dissociation of the complex of the phosphate of the
methoxy analogue with appropriate apoenzymes would be expected to
be similar to the slow rate observed with pyridoxal phosphate.

Hypoplasia, or failure of development of lymphoid elements, was the
outstanding feature in the spleens of chicks receiving the methoxy
analogue.69 Daily feeding of 1 mg per kg of the methoxy compound to
puppies on a vitamin BG-deficient diet resulted in death after 1 to 4
weeks. One pup, however, on this dosage remained alive for a month
and maintained blood values only slightly lower than those of the animals
receiving pyridoxine, indicating some activity of the analogue in replac-
ing pyridoxine for this animal.00

When the 4-methoxy analogue of pyridoxine and tryptophan was
administered to rats deficient in vitamin Br„ the animals excreted less
xanthurenic acid and kynurenine than the animals receiving tryptophan
alone.74 However, in normal animals, the analogue tended to increase
the amounts of these products excreted. This indicates that the analogue
may inhibit the action of pyridoxine to some extent; however, the in-
creased excretion of pyridoxic acid on administration of the analogue
indicated that it is cleaved and presumably utilized to some extent.74

666 THE BIOCHEMISTRY OF B VITAMINS

This is also indicated by the activity of the analogue in replacing the
vitamin in the nutrition of rats (Table 35).

2-Methyl-3-hydroxy-4-ethoxymethyl-5-hydroxymethylpyridine is re-
ported to inhibit the growth of excised tomato roots.24

Other Analogues of Pyridoxine. Both 2-methyl-3-amino-4-hydroxy-
methyl-5-aminomethylpyridine and the corresponding 4-ethoxymethyl
analogue are inhibitory for Ceratostomella ulmi, and the toxicity is pre-
vented by sufficient pyridoxine. The inhibition indices are 250 and 2500,
respectively.25 A homologue of the last compound, 2-ethyl-3-amino-4-
ethoxymethyl-5-aminomethylpyridine, prevents the stimulatory action of
pyridoxine for Saccharomyces cerevisiae.70 For relatively high concentra-
tions the inhibitor appears to prevent the stimulatory action of an
equivalent amount of pyridoxine. Growth, however, is never completely
prevented by the analogue.70

Another inhibitory analogue of pyridoxine has been reported 70 to be
2-methyl-3-hydroxy-4-hydroxymethylpyridine ; however, this compound
is actually derived from 2-picoline by sulfonation, fusion of the sulfonate
with alkali, conversion to a dialkylaminomethyl derivative by the Man-
nich reaction and hydrolysis to a (hydroxymethyl)picolinol. The inter-
mediate hydroxypicoline has been shown to be 6-methyl-3-hydroxy-
pyridine.78, 79 Consequently, this analogue probably is 6-methyl-3-
hydroxy-2(or 4) -hydroxymethylpyridine. The analogue prevents the
stimulatory action of pyridoxine on Saccharomyces cerevisiae at a molar
ratio of 250.70 A similar revision of structure is necessary for a group of
(dialkylaminomethyl) picolinols prepared even earlier by analogous re-
actions.80

2-Methyl-3-hydroxy-5-hydroxymethylpyridine has been reported to
be slightly inhibitory.81- 82 Numerous other analogues have been pre-
pared81, 82 including some pyrimidine analogues 83, 84 of pyridoxine, but
neither growth-promoting nor growth-inhibiting properties have been
reported for these compounds.

Irradiation of pyridoxamine under aerobic or anaerobic conditions
produces a mixture with antibacterial properties.85 The active principle
inhibits the growth of a wide variety of gram-negative organisms. The
effect of the vitamin B6 group on the inhibition has not been determined.85

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72. Umbreit, W. W., and Waddell, J. G., Proc Soc. Exptl. Biol. Med., 70, 293 (1949).

73. Nelson, M. M., and Evans, H. M., Proc. Soc Extpl. Biol. Med., 68, 274 (1948).

74. Porter, C. C, Clark, I., and Silber. R. H., J. Biol. Chem., 167, 573 (1947).

75. Gaebler, O. H., and Herman, E. V., Fed. Proc, 6, 254 (1947).

76. Stoerck, H. C, Fed. Proc. 7, 281 (1948) ; ./. Biol. Chem. 171, 437 (1948).
76a. Wooley, J. G., and Murphy, M. K, J. Biol. Chem., 178, 869 (1949).

77. Beiler, J. M., and Martin, G. J., J. Biol. Chem., 169, 345 (1947).
77a. Martin, G. J., and Beiler, J. M., Arch. Biochem., 15, 201 (1947).

78. Parker, E. D., and Shive, W., J. Am. Chem. Soc, 69, 63 (1947).

79. McElvain, S. M., and Goese, M. A., J. Am. Chem. Soc, 65, 2233 (1943).

80. Brown, R. F, and Miller, S. J., J. Org. Chem. 11, 388 (1946).

81. Perez-Medina, L. A., Mariella, R. P., and McElvain, S. M., /. Am. Chem. Soc,

69, 2574 (1947).

82. Mariella, R. P., and Leech, J. L., J. Am. Chem. Soc, 71, 331 (1949).

83. McCasland, G. E., Tarbell, D. S., Carlin, R. B., and Shakespeare, N., J. Am.

Chem. Soc, 68, 2390 (1946).

84. McCasland, G. E., and Tarbell, D. S., /. Am. Chem. Soc, 68, 2393 (1946).

85. Shwartzman, G., and Fisher, H., J. Biol. Chem., 167, 345 (1947).

Chapter IXD

RIBOFLAVIN

In 1933, one of the members of the vitamin B2 complex was identified
as lactochrome, a naturally occurring yellow pigment,1 first concentrated
from milk in 1879,2 and obtained in crude form from the same source
in 1925.3 This yellow pigment, which has a characteristic green fluores-
cence, was obtained in crystalline form from both egg white (ovoflavin)1
and milk (lactoflavin).1- 4 Subsequent chemical studies by Karrer and
Kuhn and their co-workers limited the structural possibilities for the
vitamin to the isomeric 6,7-dimethyl-9-(tetrahydroxyamyl)-isoalloxa-
zines. By preparation of the possible isomers, the structure was finally
established by Karrer and co-workers 3> 6 and Kuhn and co-workers,7* 8
who independently synthesized the vitamin in 1935. The vitamin which
has since been termed riboflavin has the following structure:

CH2OH

I
HO— C— H

HO— C— H

I
HO— C— H

H-i-

CH,-^ Y C C=0

N C

riboflavin
[6, 7-dimethyl-9- (d,1 '-ribityiyisoalloxazine]

While the purification and proof of structure of riboflavin were being
accomplished, simultaneous investigations showed the vitamin to be
associated with certain enzymes. In 1932, Warburg and Christian 9
isolated a coenzyme essential for the functioning of the yellow enzyme.
This coenzyme was found to be related to vitamin B2,10' u and in 1936
its identity with synthetic riboflavin-5'-phosphoric acid was demon-
strated.12 In vitro, riboflavin can replace the coenzyme, but only at

670

THE BIOCHEMISTRY OF B VITAMINS

relatively high concentrations, which suggests that the phosphate group
facilitates combination of the coenzyme with the apoenzyme.

Since a large number of compounds analogous to riboflavin were pre-
pared in the final synthetic approach to the structure of the vitamin
and were tested for activity in replacing the vitamin in the nutrition of

Table 38.

Specificity of Riboflavin.

-l-KyHlW ivy } pci V.C110

Bacillus

Lactobacillus

lactis

Old Yellow

Compound

Rat

casei

acidi

Enzyme"

6,7-Dimethyl-9-(D,l'-ribityl)-

100

isoalloxazine (riboflavin)

6-Ethyl-7-methy]-9- (d, 1 '-ribityl)-

>5013

100 ca

75-100

isoalloxazine

(90-100)e- 3°

(70-100)*. 30

6-Methyl-9-(D, 1 '-ribity 1)-

>25, <50

4-10

6-15

87-9216

isoalloxazine

13, 14, 15

(45-65)*- 30

(45-65)*. 30

7-Methyl-9-(D,l'-ribityl)-

50 ca

12-20

11-16

isoalloxazine

13, 16, 17

(75-100)*- 30

(70-90)*. 30

6,7-Dimethyl-9-(D, 1 '-arabityl)-

0/. 30

0/.30

0*. M

isoalloxazine

6,7-Dimethyl-9-(L,l'-arabityl)-

30 ca*

0/. 30

0/.30

76-8132

isoalloxazine

6, 18, 23, 26

6-Methyl-9- (l, 1 '-arabityl)-

< 10<M5

35 ca15

isoalloxazine

6,7-Trimethj'lene-9-(L, 1 '-arabityl)-

<10<i, 15

46 ca15

isoalloxazine

6,7-Tetramethylene-9-(L,l'-

<10d-15

30-3315

arabityl) isoalloxazine

Riboflavin tetraacetate

100 ca"

Q30

0*. 32

Riboflavin-5'-phosphate

10028, 29

10031

150'. 32

Flavin-adenine-dinucleotide

10031

105'. 33

a The activities relative to that of riboflavin are calculated from data of each reference. Inactivity under
the testing conditions is indicated by 0. Upper limits and in one instance the lower limit of activity are
indicated where data are not available for accurate estimate.

b Inhibitory to growth.22 This compound was reported in earlier work to have slight activity13' I7-21 but
an Amadori rearrangement appears to have occurred during synthesis resulting in contamination of the
sample with riboflavin.

c Maximum growth not obtained at any concentration.

d Slightly active. 20 y per day kept the animals alive without much growth, but animals receiving 10 y
per day died in 2 to 5 weeks.

• Maximum growth obtained with the analogue at any concentration reaches only the per cent indicated
in brackets of the maximum response to riboflavin.

t Analogue inactive alone but enhances the response of the organism to riboflavin.

o Data from separate experiments not strictly comparable on a quantitative basis. Per cent indicated
represents the relative oxygen consumption in the presence of comparable quantities of analogue unless
otherwise indicated.

* Considered inactive; however, slight activities of enzyme preparations in absence of added analogue
do not permit accurate estimates of low activities.

' Relative oxygen consumption obtained with 30 y riboflavin-5'-phosphate compared with 150 y ribo-
flavin. As little as 2.5 y riboflavin-5'-phosphate is essentially as active as 30 y in this system.
i Calculated on basis of activity (70%) in comparison to riboflavin-5'-phosphate.33

rats, the specificity of the structure for vitamin B2 activity has been
extensively studied by the time proof of structure of the vitamin was
achieved. The activities of certain analogues in replacing riboflavin in
the nutrition of rats and certain microorganisms are indicated in Table
38.

Many compounds analogous to riboflavin were also tested for activity
as a coenzyme of the yellow coenzyme. Oxygen uptake and methylene
blue decolorization were determined for the test substances when glucose-

RIBOFLAVIN 671

6-phosphate was oxidized to phosphogluconic acid by yeast hexosemono-
phosphate dehydrogenase. The coenzyme II required for the oxidation
was supplied from horse blood, and the yellow enzyme necessary to com-
plete the system was provided as the apoenzyme. The activities of the
analogues in replacing riboflavin as a coenzyme for the yellow enzyme
are shown in Table 38.

Another coenzyme containing riboflavin was discovered in 1936 by
Das 34 as a dialyzable coenzyme of an amino acid oxidase, and was later
isolated by Straub 35 and by Warburg and Christian.30' 37 The latter
investigators demonstrated that the coenzyme contained a flavin and
adenine in the form of a dinucleotide. This coenzyme, which is considered
a combination of adenylic acid and riboflavin-5'-phosphate by a pyro-
phosphate bond, is more versatile in its action than riboflavin-5'-phos-
phate, which cannot replace the dinucleotide for many apoenzymes.38
The dinucleotide can replace riboflavin phosphate in the yellow enzyme,
and Lactobacillus casei utilizes either of these coenzymes as efficiently as
riboflavin.

Of the eight stereoisomers corresponding to the structure of riboflavin,
seven of these have been synthesized. These include the stereoisomers
containing in place of the 9-D,l'-ribityl group of riboflavin the following
groups: L,l'-ribityl,17 D,l'-arabityl,12- 17"22 L,l'-arabityl,6- 18> 23"26 d,1'-
lyxityl,17 D,l'-xylityl,5- «• 23- 27 or 9-L,l'-lyxityl. The 9-L,l'-lyxityl
stereoisomer (L-lyxoflavin) has recently been isolated from human heart
muscle and synthesized,39 but no biological tests were reported. Of the
other stereoisomers, only the D,l'-xylityl and L,l'-arabityl derivatives
are reported to have activity in replacing the vitamin in the nutrition of
rats. These two compounds are effective sometimes for only a few weeks
with an average growth of the animals of about 30 g.18- 27 The standard
weight gain for rats of 40 g in 30 days with 8 y per day of riboflavin was
never obtained, even with as large amounts as 150 y per day of either
of the two stereoisomers.27

Only slight alterations of the riboflavin structure can be made if the
biological activity is retained. The analogues most effective in replacing
the vitamin are those containing modifications of the benzene ring of
riboflavin (Table 38). The analogue with an ethyl group in place of the
6-methyl group appears to be almost as active as riboflavin for rats,
Lactobacillus casei and Bacillus lactis acidi. Modifications involving the
elimination of either the 6- or 7-methyl group possess appreciable
biological activity of the vitamin; however, at least one of the methyl
groups is essential for this activity, since, in contrast to these compounds
and the corresponding 6,7-dimethyl derivatives, 9-(D,l-ribityl)isoalloxa-
zine 40 and 9-(d or L,l'-arabityl)isoalloxazine 6- 26> 41 are inactive in

672 THE BIOCHEMISTRY OF B VITAMINS

replacing riboflavin for rats and as a coenzyme in the yellow enzyme.32
Other known modifications of riboflavin involving the benzene ring have
only slight stimulatory effects, or are inactive in replacing the vitamin.
The 6,7-dimethyl groups of the L-arabityl stereoisomer of riboflavin can
be replaced by either a 6,7-trimethylene or 6,7-tetramethylene group
without complete loss of the biological activity for rats and the yellow
enzyme (Table 38). 7-Ethyl-9(D,r-ribityl)isoalloxazine has a growth-
promoting effect for rats, but the response is not constant.13 Essentially
inactive alone, 5,6-benzo-9-(D,r-ribityl)isoalloxazine 13, 30 and 6-ethyl-
7-methyl-9-(L,l'-arabityl)isoalloxazine 13, 30 enhance the response of
Lactobacillus casei and Bacillus lactis acidi to riboflavin.30 No growth-
promoting activity for rats has been reported for either 5,6-dimethyl-9-
(L,l'-arabityl)- or 6,8-dimethyl-9-(D,r-ribityl)isoalloxazine.42 6,8, Di-
methyl-9-(D or L,l'-arabityl(isoalloxazine 43 and 5,7-dimethyl-9-(D or
L,r-arabityl)isoalloxazine43 are inactive for rats and as a coenzyme for
the yellow enzyme. 5,6-Benzo-9-(L,r-arabityl)isoalloxazine is inactive
for rats.13

Replacement of the D-ribityl-group in riboflavin by glycosido-group-
ings results in total loss of biological activity. Thus, 6,7-dimethyl-9-(D-
or L,l'-arabinosido) and 6,7-dimethyl-9-(D-ribosido)isoalloxazine are
inactive in replacing riboflavin in the nutrition of the rat 44 or in function-
ing as a coenzyme for the yellow enzyme.32

With exception of the D-xylityl and L-arabityl stereoisomers of ribo-
flavin, substitution of similar groups for the 9-D,l'-ribityl group in ribo-
flavin produces inactive substances. These are exemplified by compounds
containing the following substituents in the 9-position: D-l'-desoxy-
ribityl,34 L,l'-rhamnityl,6 or n-amyl.24, 27, 45 Similarly, a complete loss
of activity is obtained with analogues of 7-methyl-9-(D,r-ribityl)-
isoalloxazine in which the ribityl group is replaced by the L,l'-ara-
bityl,0-26 D,r-xylityl,'''- 2e D,l'-sorbityl,6- 26 D-l'-dulcityl,5- c or D,l'-man-
nityl 5- 6 group.

Substitution of a methyl group in the 3 position of riboflavin results
in complete loss of vitamin activity for the rat.8 The 3,6,7-trimethyl-
9-(D,l/-ribityl)isoalloxazine is also inactive in the yellow enzyme test.
Since this analogue does not combine with the protein, and since neither
it nor the yellow enzyme shows the fluorescence characteristic of ribo-
flavin and the free coenzyme, it has been proposed that the 3-position
is one point of attachment of coenzyme to the apoenzyme.46 Because the
riboflavin-5'-phosphate combines more readily than the free vitamin
with the protein, the phosphate group has been considered as another
point of combination with the apoenzyme.46

When riboflavin is esterified, the resulting derivatives vary consider-
ably in their ability to replace riboflavin for biological systems. Activity

RIBOFLAVIN 673

appears to depend on whether or not the test organism is able to effect
hydrolysis of a given ester. The tetra-acetyl derivative is almost as active
as riboflavin for rats,27 but is inactive in the nutrition of Lactobacillus
casei and Bacillus lactis acidi30 and as a coenzyme for the yellow
enzyme.32 The triacetate derivative of riboflavin-5'-phosphate is also
inactive in the yellow enzyme test.32 Riboflavin-5'-phosphate admin-
istered either orally or parenterally in the rat is fully as active as ribo-
flavin.29 The sulfate of riboflavin shows some activity as a coenzyme for
the yellow enzyme.32 Riboflavin mono-, di-, tri- and tetrasuccinates have
been prepared in the search for more soluble forms of the vitamin.47 For
Lactobacillus casei, the latter two are essentially inert, whereas the first
two are respectively 60 and 18 per cent as active as riboflavin. For the
rat, the activities on a molar basis are 100, 65, 21 and 0 per cent, respec-
tively, of riboflavin. The inactivity of the tetrasuccinate contrasts with
the high activity reported for the tetracetate in replacing riboflavin for
the rat.47

Both the mono- and diacetone derivatives of riboflavin are active in
the nutrition of rats.25- 27 However, the condensation of riboflavin with
chloral and with levulinic acid produced acetals which are inactive for
both Lactobacillus casei and the rat.47

The reaction of riboflavin with formaldehyde produces methylol deriva-
tives which are more soluble in water than the vitamin. The mono-
methylol derivative retains approximately 55 per cent of the activity of
the vitamin but polymethylol derivatives are much less active.48

2-Amino-4,5-dimethyl-r-D-ribitylaminobenzene increases the response
of Lactobacillus casei to suboptimal concentrations of riboflavin, but
alone at concentrations of 20 to 40 y per cc, it is 0.003 per cent as effective
as riboflavin.31 In the presence of alloxan, the activity is increased to as
high as 0.35 per cent that of riboflavin. Alloxan alone is inactive, and
neither riboflavin nor flavin-adenine-dinucleotide affects this transforma-
tion. It is suggested that the organism has some slight ability to com-
bine these two components to form riboflavin.31

It has been reported that isoxanthopterincarboxylic acid, 2-thio-6-
hydroxypteridine, or lumazine can replace riboflavin or thiamine, or both,
in preventing changes in chronaxia in rats.49 In similar experiments with
pigeons, isoxanthopterin, 6-hydroxypteridine, 2,6-diaminopteridine,
leucopterin, or lumazine are reportedly active in replacing riboflavin or
thiamine.49

Inhibitory Analogues of Riboflavin

Although a number of analogues of riboflavin have been prepared, only
a few appear to inhibit specifically the functioning of riboflavin in
biological systems. These and related inhibitors are indicated in Table

674

THE BIOCHEMISTRY OF B VITAMINS

tOOO
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3 S 3 S S

ss: sacs

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S S u u p G «

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CO"o"<M~

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lis

1-2.2

!« .

=*s*

RIBOFLAVIN 675

39. Of the substances listed, quinine and atebrin do not appear to be
specific in their action since riboflavin, though affecting the inhibition,
apparently does not act in a competitive manner. However, quinine,
atebrin, and other antimalarials do affect certain isolated enzyme systems
in which the riboflavin coenzymes function.

6,7-Dichloro-9(D,r-ribityl)isoalloxazine [Dichloroflavin] . Noting that re-
placement of methyl by chloro substituents on the benzene nucleus of
aromatic compounds changes the crystallographic properties so little that
uninterrupted series of mixed crystals often result, Kuhn, Weygand, and
Moller50 prepared 6,7-dichloro-9(D,l'-ribityl)isoalloxazine as a possible
competitive inhibitor of riboflavin in biological systems. The analogue,
which was the first antagonist of riboflavin to be reported, inhibits the
growth of Staphylococcus aureus and Streptobacterium plantarum, but
does not restrict the growth of yeast or Bacillus lactis acidi. Riboflavin
competitively prevents the inhibitory action of the analogue, and the
inhibition indices for half-maximum growth are 50, 70, and 280 for

OH OH OH
CH2— C— C C CH2OH

A k k

ci— k^^Jl //C\

N C

dichloroflavin[6,7-dichloro-9(D,l'-ribityl)isoalloxazine]

tests incubated 3, 4, and 6 days, respectively, with Staphylococcus aureus.
With Streptobacterium plantarum, similar inhibition indices are 25, 60,
130, and 165 for incubation periods of 2, 3, 4, and 6 days, respectively.
The inhibitory behavior of dichloroflavin has been explained on the basis
of the difference in the redox potentials of dichloroflavin, E0= —0.095 V
(pH 7), and riboflavin, E0=— 0.185 V (pH 7). The vitamin analogue
probably cannot participate in the oxidation-reduction reactions medi-
ated by the riboflavin coenzymes.50

Neither dichloroflavin nor its 5'-phosphate affect the activity in vitro
of D-amino acid oxidase from liver or xanthine oxidase from milk even
at concentrations 1000-fold greater than that of the riboflavin coen-
zyme.56

Isoriboflavin [5,6-Dimethyl-9(D,l'-ribityl)isoalloxazine]. An isomer of
riboflavin, 5,6-dimethyl-9-(D,r-ribityl)isoalloxazine, at levels of 2 mg

676

THE BIOCHEMISTRY OF B VITAMINS

per day, restricts the growth of riboflavin-deficient rats to a much greater
extent than does the deficiency of the vitamin alone.51 This intake of
isoriboflavin almost completely inhibits the growth-promoting effect of
10 y per day of riboflavin, but the inhibitory effect can be prevented
entirely by the daily administration of 40 y of the vitamin.51

Isoriboflavin even at concentrations 100,000 times that of riboflavin
does not inhibit the growth of Lactobacillus casei; and negligible activity

OH OH OH

I I I
CH2 C C C CH2OH

I I I

H H H

CH3-J

isoriboflavin [5,6-dimethyl-9-(D,l'-ribityl)isoalloxazine]

(less than 0.5 per cent) is obtained in attempts to replace the requirement
for riboflavin, with either isoriboflavin or its tetra-acetyl derivative.57
However, isoriboflavin markedly stimulates the acid production and
growth of Lactobacillus casei in the presence of suboptimal levels of
riboflavin or flavin-adenine-dinucleotide.31

D-Araboflavin [6,7-Dimethyl-9-(D,l'-arabityl)isoalloxazine]. Administra-
tion of D-araboflavin (200 y per day) to rats on a riboflavin-deficient
diet retards growth and increases the mortality rate beyond that which

CH2-

H OH OH

I I !

-C C C CH2OH

I I I

OH H H

CH3
CH3

C=0

I
,NH

D-araboflavin [6 ,7-dimethyl-9- (D,l '-arabityl) isoalloxazine]

could be attributed to deficiency of the vitamin alone.22 Only one out of
ten rats survives by the third week. The analogue (200 y per day) also
decreases the rate of growth of rats receiving low amounts of riboflavin
(10 y per day) to such an extent that no growth takes place by the third

RIBOFLAVIN 677

week. L-Araboflavin (200 y per day), which possesses some growth-
promoting properties itself, appears to reduce very slightly the growth
of rats receiving low amounts of riboflavin (10 y per day).22 D-Arabo-
flavin, at a concentration of 25 y per cc, also inhibits the growth of an
unidentified strain of lactic acid bacteria.

Galactoflavin [6,7-Dimethyl-9-(D,l'-dulcityl)isoalloxazine]. The admin-
istration of galactoflavin (1.0 to 2.16 mg) daily by stomach tube increases
the mortality rate and decreases the rate of growth of rats on a ribo-
flavin-free diet. The analogue (2.16 mg per day) completely inhibits the

-CH2OH

galactoflavin [6 ,7-dimethyl-9-{D ,1' -dulcityl)isoalloxazine\

response of the animals to 10 y daily of riboflavin and markedly inhibits
the response to 40 y daily of the vitamin. The inhibitory effect of 2.16 mg
of the analogue is almost, but not completely, prevented by 200 y per day
of riboflavin. The index at which growth inhibition is noted is approxi-
mately 10.

Lumichrome and Lumiflavin. The growth of a mutant strain (51602)
of Neurospora which requires riboflavin when incubated at 31-34° C, but
not when incubated at 25° C, is inhibited by both lumichrome and lumi-
flavin.53 The inhibition resulting from either compound is competitively
prevented by riboflavin. In tests incubated for 84 hours at 31° C, the
inhibition index for lumichrome was 2.2-2.5 for half-maximum and 6-8
for complete inhibition of growth. Lumiflavin was only about one-
twentieth as effective as lumichrome.53

CH3

N NH N N

CH3-L^>X ^Cx Jm CH3-l^Jx A .NH

N C N C

lumichrome lumiflavin

(6 ,7-dimethylalloxazine) (6,7,9-trimethylisoalloxaziiie)

678 THE BIOCHEMISTRY OF B VITAMINS

Natural extracts also contain a substance (s) which inhibits growth of
the mutant. The toxicity is competitively prevented by riboflavin, but
the properties of the toxic material suggest that neither lumichrome nor
lumiflavin is responsible for the inhibitory activity of natural extracts.53

The growth of Lactobacillus casei stimulated by suboptimal amounts
of riboflavin is inhibited by high concentrations of lumiflavin; however,
in the presence of increased amounts of riboflavin, a stimulatory action
is exerted by the analogue.31 Lumiflavin inhibits the utilization of flavin-
adenine-dinucleotide more effectively than the utilization of riboflavin
by the organism.31

2,4 - Diamino - 7,8 - dimethyl- 10(d,1' - ribityl) 5,10 - dihydrophenazine. The
growth of Lactobacillus casei in the presence of 0.03 y per cc of riboflavin
is completely inhibited by 200 y per cc of 2,4-diamino-7,8-dimethyl-
10(D,r-ribityl)5,10-dihydrophenazine.54 The toxicity is prevented by

OH OH

CH2 C C—

-C —

-CH2OH

i i
H H

CH3-

■T

/Vy™'

CH3-

■VT

]

I NH2

2,4-diamino-7,8-dimethyl-l 0- (Z>, 1 '-ribityl) -5, 1 0-dihydrophenazine

increasing the concentration of riboflavin to 100 y per cc. The inhibition
index appears to be approximately 6600. Half-maximum inhibition of
growth of Hemolytic streptococcus H69D and Lactobacillus arabinosus
resulted from addition of 330 y per cc of the diaminophenazine to a
medium containing 0.03 y per cc of riboflavin. Higher concentrations of
riboflavin prevent this inhibition of growth. Staphylococcus aureus, Strep-
tococcus faecalis R, and Escherichia coli are not affected in their growth
by the analogue. Because of the instability of the diaminophenazine,
reduction of the corresponding 2,4-dinitrophenazine with finely divided
iron in the culture medium is a desirable procedure. However, correspond-
ing results are obtained with purified diaminophenazine previously pre-
pared by reduction of the dinitrophenazine with tin.54

The dinitrophenazine produces in mice a very mild riboflavin deficiency
characterized by greasy, unkempt fur, by hyperirritability, and by a
slightly reduced rate of growth. Sufficient amounts of riboflavin prevent
the appearance of these changes.

RIBOFLAVIN

679

Flavin-adenine-dinucleotide prevents the toxicity of the diamino-
phenazine for Lactobacillus casei in a manner analogous to riboflavin,
and is equally effective.31

Atebrin, Quinine and Related Antimalarials. The discovery that ate-
brin inhibits the oxygen consumption of various organisms resulted in
investigations of possible relationships to the riboflavin coenzymes.58, 59, 60
It was demonstrated that atebrin inhibits D-amino acid oxidase 60 and
prevents the combination of the apoenzyme of cytochrome reductase
with riboflavin-5'-phosphate.61 Although riboflavin-5'-phosphate at a

CH30

CH2 CH2 CH— CH=CH2

I I I
HOCH— CH CH2 CH2

quinine

CH30

CH3

C2Hg

NH— CH— CH2— CH2— CH2— N
I \

I C2H6

atebrin

ratio of 1 to 500 prevents the inhibitory action of atebrin, the inhibition
resulting when atebrin is added to the apoenzyme of cytochrome reduc-
tase prior to the coenzyme is not affected by the coenzyme.61

A study of the effect of a number of compounds on the D-amino acid
oxidase system has revealed that a large number of compounds related to
quinine and atebrin inhibit the enzyme.02 Since increased concentrations
of flavin-adenine-dinucleotide prevent the inhibition resulting from a
number of these compounds, competitive inhibition is indicated. The rela-
tive activities of these compounds as compared with quinine at two dif-
ferent temperatures are indicated in Table 40. Atebrin, auramine, and
novalauramine are somewhat more effective than quinine, while plas-
mochin and a number of quinoline derivatives are approximately as

680 THE BIOCHEMISTRY OF B VITAMINS

Table 40. Competitive Inhibitors of Flavin-Adenine-Dinucleotide for
D- Amino Acid Oxidase62

. Quinine Equivalent*

At 37° C At 30° C

Auramine 7

Atebrin 2.5 2

Novalauramine 2

Quinine 1 1

Quinine methochloride 1 1

6-Methoxyquinoline 1 1

Plasmochin 1

7-Chloro-4-(4-diethylamino-l-methylbutyl-

amino)-2-methylquinoline 1

7-Chloro-4- (4-diethylamino- 1-methylbutyl-

amino)-3-methylquinoline 0.4 0.5

7-Chloro-4- (4-diethylamino- 1-methylbutyl-

amino)quinoline 0.5 0.5

a-(Diamylaminomethyl)-l,2,3,4-tetrahydro-

9-phenanthrenemethanol
Sulfathiazole
Sulfapyridine
Sulfadiazine
Sulfanilamide
Benzenesulfonamide
N- (4-Diethylamino- 1 -methylbutyl)-/3-

(p-dimethylaminophenyl)alanine
Aniline
Pyridine
0L-a-(p-Dimethylaminophenyl)glycine

* The ratio of concentrations of quinine and inhibitor required to give the same amount of inhibition at
any concentration of flavin-adenine-dinueleotide.

active as quinine. The sulfonamides are considerably less effective. The
dissociation of the coenzyme from D-amino acid oxidase does not occur
readily at 30° C, but dissociation is readily detectable at 37° C. At the
latter temperature, the inhibitions resulting from quinine appear to be
reversible and competitive with the coenzyme. The dissociation constants
for two different enzyme preparations are 4.6 X 10 7 and 6.9 X 10 7 for the
enzyme complex and 4.6 X 1(H and 8.9 X 1(H for the quinine-apoenzyme
complex. With atebrin, two types of inhibition appear to exist. One is

(CH3)2— N+=< \=C— <f \-N(CH3)2

ci: 7X=/ I \=J

NH2
auramine

C2H5

0.07

0.2

0.04

0.015

0.07

0.015

0.04

0.01

0.02

0.007

0.03

0.007

NH— CH— CH2— CH,— CH2— N

I \

CH3 C2H6

plasmochin

RIBOFLAVIN 681

rapid, reversible, and competitive, but the other is slower and apparently
irreversible.02

Quinine, atebrin, and a number of antimalarials prevent the growth of
Lactobacillus casein The inhibition is overcome to some extent, but
apparently not strictly competitively in most instances, by increasing
the riboflavin content of the medium. Thus, in media containing 0.25 y
and 2.5 y per cc of riboflavin, the maximum concentrations, respectively,
in mg per cc at which visible growth of Lactobacillus casei is observed
are 0.6 and 1.75 mg of quinine, 0.06 and 0.25 mg of atebrin, 0.02 and 0.1
mg of propamidine, 0.0034 and 0.01 mg of methylene blue, 0.29 and 1.22
mg of 2-p-chloroanilino-4-/3-diethylaminoethylamino-6-methylpyrimi-
dine,63 0.42 and 2.5 mg of 2-p-chlorophenylguanidino-4-/?-diethylamino-
ethylamino-6-methylpyrimidine,64 and 0.02 and 0.06 mg of 2-(6'-bromo-
B- naphthy lamino ) - 4 - diethylaminoethylamino - 6 - methy lpyrimidine.55, 65
Other inhibitory substances not related to these compounds structurally
were not affected by additional riboflavin.55

NH— CH2— CH2— N(C2H6)2

I
C

°t)

N CH

I II

C C— CH3

N N

2-p-chloroanilmo-4-^-diethyla7ni?ioethyla7nino-6-niethylpyrimidine

CI— <r

NH— CH2— CH2— N (C2H5)2
I

C
,/ \
NH N C

H I II

C C C— CH3

./ \ / \ /

N N N

H H

S-p-chlorophenylguanidino-4-^-diethylaminoethylamino-6-methylpyrimidine

Atebrin has an inhibitory action on the tryptophanase activity of viable
cells of Escherichia coli but has little effect on the cell-free enzyme. The
inhibitory action of atebrin on the cells is reduced by addition of supple-
mentary riboflavin, and the effect has been attributed to the influence of
accumulated pyruvate on the system.

Miscellaneous Analogues of Riboflavin. At concentrations of 25 y per
cc, 9-(D,r-sorbityl)isoalloxazine inhibits the growth of Bacillus lactis

682 THE BIOCHEMISTRY OF B VITAMINS

acidi, and either 6,7-dimethyl-9-(D,l'-xylityl)isoalloxazine or 9-(d,1'
arabityl)isoalloxazine inhibits the growth of an unidentified strain of
lactic acid bacteria.50

The following isoalloxazines have been reported to be inactive as
inhibitory analogues of riboflavin for Bacillus lactis acidi and the uniden-
tified strain of lactic acid bacteria: 9-(L,l'-arabityl)-, 9-hydroxyethyl-,
3-methyl-9- (D,l'-sorbityl) -, 5,6-dimethyl-9- (L,l'-arabityl) -, 6,7-di-
methyl-9- (D,l'-sorbityl) -, 3,6,7-trimethyl-9- (D,l'-sorbityl) -, 6,7-tetra-
methylene-9-(L,l'-arabityl)-, 5,6-benzo-9-methyl-, 9-phenyl-, 6-methyl-
9-(D,r-ribityl)isoalloxazine, 6,7-dimethyl-9-isoalloxazineacetic acid, and
9-isoalloxazineacetic acid. l,2-Dimethyl-4-amino-5-D,r-ribitylamino-
benzene and the corresponding 4-nitro derivative are also inactive.50

Although 2-amino-4,5-dimethyl-l,D-ribitylaminobenzene inhibits the
oxidation of riboflavin by Pseudomonas riboflavina, the inhibition does
not appear to be competitive.57 No growth-inhibiting effect of 2-amino-
4,5-dimethyl-l-ribitylaminobenzene, even at relatively high concentra-
tions, is obtained with Lactobacillus casei.57

p-Monomethylaminoazobenzene and p-dimethylaminoazobenzene,
which are hepatic carcinogens, inhibit the growth of both Lactobacillus
casei and Saccharomyces cerevisiae.67 Riboflavin tends to prevent the
toxicity of these compounds for each organism.67

Bibliography

1. Kuhn. R., Gyorgy, P., and Wagner^Tauregg, T., Ber., 66, 317, 576, 1034 (1933).

2. Blyth, A. W., /. Chem. Soc, 35, 530 (1879).

3. Bleyer, B., and Kallmann, 0., Biochem. Z., 155, 54 (1925).

4. Ellinger, P, and Koschara, W., Ber., 66, 315, 808, 1411 (1933).

5. Karrer, P., Schopp, K., and Benz, F., Helv. Chim. Acta, 18, 426 (1935).

6. Euler, H. v.. Karrer, P., Malmberg, M., Schopp, K., Benz, F., Becker, B., and

Frei, P., Helv. Chim. Acta, 18, 522 (1935).

7. Kuhn, R., Reinemund. K., Kaltschmitt, H., Strobele, K., and Trischmann, H.,

Naturwiss., 23, 260 (1935).

8. Kuhn, R., Reinemund, K., Weygand, F., and Strobele, K., Ber., 68, 1765 (1935).

9. Warburg, 0., and Christian, W., Naturwiss., 20, 688, 980 (1932) ; Biochem. Z., 258,

496 (1933); 263, 228 (1933); 242, 206 (1931); 254, 438 (1932); 257, 492 (1933);
266, 377 (1933).

10. Banga, I., and Szent-Gyorgyi, A., Biochem. Z., 246, 203 (1932) ; Banga, I., Szent-

Gyorgyi, A., and Vargha, L., Z. physiol. Chem., 210, 228 (1932).

11. Theorell, H., Biochem. Z., 275, 37 (1934); Kuhn, R., and Rudy, H., Ber., 68,

383 (1935).

12. Kuhn, R., Rudy, H., and Weygand, F., Ber., 69, 1543 (1936); Kuhn, R., and

Rudy, H., Ber., 69, 1974 (1936).

13. Karrer, P., and Quibell, T. H, Helv. Chim. Acta, 19, 1034 (1936).

14. Karrer, P., and Strong, F. M, Helv. Chim. Acta, 18, 1343 (1935).

15. Kuhn, R., Vetter, H., and Rzeppa, H. W., Ber., 70, 1302 (1937).

16. Karrer, P., Euler, H. v., Malmberg, M., and Schopp, K., Svensk. Kem. Tid.,

47, 153 (1935).

17. Karrer, P., Salomon, H., Schopp, K., Benz, F., and Becker, B., Helv. Chim.

Acta, 18, 908 (1935).

RIBOFLAVIN 683

18. Euler, H. v., Karrer, P., and Malmberg, M., Helv. Chim. Acta, 18, 1336 (1935).

19. Kuhn, R., and Weygand, F., Ber., 68, 1282 (1935).

20. Kuhn, R., and Weygand, F., Ber., 70, 769 (1937); Karrer, P., Ber., 70, 2565

(1937).

21. Weygand, F., Ber., 73, 1259 (1940).

22. Euler, H. v., and Karrer, P., Helv. Chim. Acta, 29, 353 (1946).

23. Kuhn, R., and Weygand, F., Ber., 67, 1939, 2084 (1934).

24. Kuhn, R., and Weygand, F., Ber., 68, 166 (1935).

25. Kuhn, R., Rudy, H., and Weygand, F., Ber., 68, 625 (1935).

26. Karrer, P., Schopp, K., Bonz, F., and Pfachler, K., Helv. Chim. Acta, 18, 69

(1935).

27. Kuhn, R., Angew. Chem., 49, 6 (1936).

28. Kuhn, R., Rudy, H., and Weygand, F., Ber., 69, 1543 (1936); Kuhn, R., and

Rudy, H., Ber., 68, 383 (1935) ; Z. physiol. Chem., 239, 47 (1936).

29. Gyorgy, P., Proc. Soc. Exptl. Biol. Med., 35, 207 (1936).

30. Snell, E. E., and Strong, F. M., Enzymologia, 6, 186 (1939).

31. Sarett, H. P., J. Biol. Chem., 162, 87 (1946).

32. Kuhn, R., and Rudy, H., Ber., 69, 2557 (1936).

33. Warburg, 0., and Christian, W., Biochem. Z., 298, 368 (1938).

34. Das, N. B., Biochem. J., 30, 1080, 1617 (1936).

35. Straub, F. B., Nature, 141, 603 (1938).

36. Warburg, O., and Christian, W., Naturwiss., 26, 201, 235 (1938).

37. Warburg, O., and Christian, W., Biochem. Z., 296, 294 (1938); 298, 150, 368

(1938).

38. Haas, E., Biochem. Z., 298, 378 (1938).

39. Pallares, E. S., and Garza, H. M., Arch. Biochem., 22, 63 (1949).

40. Karrer, P., Salomon, H., Schopp, K, and Benz, F., Helv. Chim. Acta, 18,

1143 (1935).

41. Kuhn, R., and Weygand, F., Ber., 68, 1001 (1935).

42. Karrer, P., and Strong, F. M.. Helv. Chim. Acta, 19, 483 (1936).

43. Kuhn, R., Desnuelle, P., and Weygand, F., Ber., 70, 1293 (1937).

44. Kuhn, R., and Strobele, K., Ber., 70, 747 (1937).

45. Kuhn, R., and Weygand, F., Ber., 67, 1941 (1934).

46. Kuhn, R., and Boulanger, P., Ber., 69, 1557 (1936).

47. Furter, M. F., Haas, G. J., and Rubin, S. H., J. Biol. Chem., 160, 293 (1945).

48. Schoen, K., and Gordon, S. M., Arch. Biochem., 22, 149 (1949).

49. Busnel, R. G., Chauchard, P., Mazoue, H., and Polonovski, M., Compt. rend.

soc. biol., 140, 50 (1946).

50. Kuhn, R., Weygand, F., and Moller, E. F., Ber., 76, 1044 (1943).

51. Emerson, G. A., and Tishler, M., Proc. Soc. Exptl. Biol. Med., 55, 184 (1944).

52. Emerson, G. A., Wurtz, E., and Johnson, O. H, /. Biol. Chem., 160, 165 (1945).

53. Mitchell, H. K., and Houlahan, M. B., Am. J. Botany, 33, 31 (1946).

54. Woolley, D. W., /. Biol. Chem., 154, 31 (1944).

55. Madinaveitia, J., Biochem. J., 40, 373 (1946); Biochem. J., 38, xxvii (1944).

56. Karrer, P., and Ruckstuhl, H., Bl. Schweiz. Akad. Med. Wiss., 1, 236 (1945).

57. Foster, J. W., J. Bact., 48, 97 (1944).

58. Fulton, J. D., and Christophers, S. R., Ann. Trop. Med., 32, 77 (1938).

59. Martin, S. J., Cominole, B., and Clark, B. B., /. Pharmacol, 65, 156 (1939).

60. Wright, C. I., and Sabine, J. C, J. Biol. Chem., 155, 315 (1944).

61. Haas, E., /. Biol. Chem., 155, 321 (1944).

62. Hellerman, L., Lindsay, A., and Bovarnick, M. R., J. Biol. Chem., 163, 553 (1946).

63. Curd, F. H. S., and Rose, F. L., J. Chem. Soc, 1946, 343.

64. Curd, F. H. S., and Rose, F. L., J. Chem. Soc, 1946, 362.

65. Curd, F. H. S., Raison, C. G, and Rose, F. L., J. Chem. Soc, 1946, 366.

66. Dawson, J., Biochem. J., 40, xli (1946); Dawes, E. A., and Happold, F. C,

Biochem. J., 44, 349 (1949).

67. Miller, E. C, Kingsley, H. N., and Miller, J. A., Cancer Research, 7, 730 (1947).

Chapter XD

THIAMINE*

Introduction

Between 1884 and 1912, it was established that beriberi in man 1> 2- 3
and polyneuritis (beriberi) in fowls 4 and in rats 5 are deficiency diseases
caused by the lack of some substance which is present only in certain
foods, and it was shown that rice bran is a relatively rich source of this
necessary substance. The isolation of this substance (which in the Euro-
pean literature is known as aneurine and in the American literature is
called thiamine) presented many difficulties; it was not until 1926 to 1934
that crystalline preparations approaching purity were obtained 6~10 and
the empirical formula was established beyond reasonable doubt. In 1936,
Williams u and, independently, Grewe 12 showed that thiamine has the
structure represented by the following formula:

ci-

CH2 N+ C— CH3

CH3— '^ ^J— NH2 HC C— CH2CH2OH

* \/

thiamine

Synthesis of this compound was achieved in the same year by Williams
and Cline 13 and by Andersag and Westphal.14

Specificity

The results of tests on pigeons and on rats indicate that on a molar
basis, the hydrochloride, hydrobromide,13 hydroiodide,15 the sulfate and
nitrate salts as well as the acetate, benzoate, chaulmoograte and phos-
phate esters 17 of thiamine possess substantially the same antineuritic
activity.

Thiamine in the form of the pyrophosphate ester (cocarboxylase) is
involved in tissue oxidation of carbohydrates, particularly in reactions
involving decarboxylation of pyruvic acid;18 consequently, it might be
expected that thiamine would be capable of reversible oxidation and

* By A. D. Barton and Lorene L. Rogers.

684

THIAMINE 685

reduction. Early investigation of this possibility showed that thiamine,
under suitable conditions, took up one mole of hydrogen when reduced
catalytically or by means of sodium hydrosulfite,19 but the reduction
product was biologically inactive. On the other hand dihydrothiamine
pyrophosphate, prepared by catalytic hydrogenation, was found to be as
active as thiamine pyrophosphate.19 However, none of these reduction
products was autoxidizable.

Later investigations showed that under conditions as mild as those
prevailing in growing tissues, thiamine 20 and thiamine pyrophosphate 21
(cocarboxylase) can be oxidized either by dilute hydrogen peroxide at
pH 7.5 or by iodine in alkaline solution to form the corresponding di-
sulfide derivative without loss of vitamin Bi activity; this conversion
involves the opening of the thiazole ring, and the disulfide may be repre-
sented by the following formula:

CH2 CH3 CH3 CH2

CH3

-C C N

J-NH2 CH0 C-S-S-C CHOHtNJ^

CH2 GH2

I I

CH2OH CH2OH

-CH3

The disulfide can be reduced by cysteine or glutathione.22 More re-
cently it has been shown that thiamine pyrophosphate disulfide is inactive
in the yeast carboxylase enzyme system,23 and although fermenting yeast
is able to reduce the cocarboxylase disulfide, it appears that the disulfide
form may not be involved in the biological functioning of thiamine.

More vigorous oxidation of thiamine yields thiochrome,24 a yellow
compound with intense blue fluorescence; this compound is biologically
inactive, except for a few microorganisms.25

H2
fVV-C-CH3

! II

nvt C C— CH2CH2OH

thiochrome

In general, it appears that animals require the complete thiamine
molecule, but plants and many microorganisms can utilize a mixture of
the pyrimidine and thiazole components for the synthesis of vitamin Bi.
Some organisms require only one of the components, and others are cap-

686 THE BIOCHEMISTRY OF B VITAMINS

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THIAMINE 687

able of growth without an external supply of either of these components or
thiamine. These requirements are summarized in Table 41. Some strains
of Neisseria gonorrhoeae, when first isolated from a human host, cannot
utilize thiamine but require cocarboxylase.25a

Organisms which require only one or both of the components of thia-
mine rather than the intact molecule or do not require an external supply
of thiamine or its components apparently achieve partial or in the latter
case complete synthesis of thiamine. For example, Bacillus subtilis can
be grown in a thiamine-free medium, and the resulting broth will sup-
port the growth of Staphylococcus aureus, which requires thiamine or its
two components.26 Moreover, although Mucor ramannianus requires the
thiazole component and Rhodotorula rubra requires the pyrimidine com-
ponent of thiamine, these two organisms can grow together in thiamine-
free media.27 Katznelson 27a reported that Bacillus paraalvei requires
thiamine or a mixture of its components, or at least the thiazole com-
ponent, for growth in a medium devoid of cystine, phenylalanine, valine
and leucine. Given the three last-named amino acids with either cysteine,
glutathione, or cystine (in a reducing medium) or even sodium thiogly-
colate or sodium thiosulfate or ascorbic acid, this organism can grow
without added thiamine or its components. These results suggest that
this organism requires thiamine in its metabolism but in adequate media
is able to synthesize the thiamine it requires.

The biological activity of a large number of analogues of thiamine and
its pyrimidine and thiazole components has been determined, as indicated
in Tables 42, 43, and 44. As a result of these tests, it is evident that the
thiamine molecule can undergo very little modification without extensive
loss of vitamin Bx activity. Substitution of an ethyl, propyl or iso-
propyl group for the 2'-methyl group or an ethyl group for the 4-methyl
group are the only modifications which did not produce drastic reduction
in the vitamin Bx activity. In addition to the skeletal structure of thi-
amine, the 4'-amino group, the 5-/3-hydroxyethyl group and the absence
of substitution in the 2-position appear to be essential for significant
vitamin Bx activity.

A number of thiamine analogues containing a pyridine ring instead of
the thiazole ring 58~63 have been found to be inactive in growth tests on
the pigeon, the rat, and several microorganisms. The fact that two of
these analogues are active in stimulating the production of carbon dioxide
by yeast 63 is due to the ability of the yeast to cleave these analogues and
use them as a source of the pyrimidine component of the thiamine mole-
cule.

Although many microorganisms can synthesize thiamine when supplied
with suitable intermediates and thus do not require an external supply

THE BIOCHEMISTRY OF B VITAMINS

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692 THE BIOCHEMISTRY OF B VITAMINS

of thiamine, nevertheless it appears that vitamin Bi is required in their
metabolism, and the results of the experiments with analogues of the
pyrimidine and thiazole components of thiamine (summarized in Tables
43 and 44) indicate that for many microorganisms the structural spec-
ificity of vitamin Bi is substantially the same as for animals.

Recently it was reported that the administration of lumazine, xanthop-
terin, isoxanthopterin or folic acid to rats and pigeons on a low thiamine
diet not only restores chronaxia to normal, but also cures polyneuritis
and permits normal growth. This effect is observed when these compounds
are injected into normal rats and when they are administered to cae-
cumectomized rats, which indicates that their activity is not due to the
synthesis of extra thiamine by the intestinal flora. However, these com-
pounds do not replace thiamine as growth factors for Glaucoma or for
Polytomella caeca. These results suggest that these compounds may exert
a sparing action on thiamine but are not able to replace it entirely.

Bonner 67 has shown that pea roots require both the pyrimidine and
thiazole components of thiamine for growth. Growth tests with analogues
(Tables 43 and 44) indicate that their structural requirements for vita-
min Bx activity are not quite so stringent as those of many other or-
ganisms. Recently it was reported that 4-methyl-5-thiazoleacetamide
stimulates plant germination.69b

The possibility that the thiazole portion of thiamine may be derived
from an amino acid precursor, /?-(4-methylthiazolyl-5) -alanine, was early
considered.68, 69 While neither Staphylococcus aureus nor Phycomyces
blakesleeanus utilized the amino acid derivative, the compound replaced
the thiazole moiety in stimulating pea roots.

By assay with Phycomyces blakesleeanus, the formation by pea roots
of the thiazole portion of thiamine was demonstrated with this amino acid
as well as with 4-methylthiazole derivatives with the following substitu-
ents in the 5-position: -CH2-CH2C1, -CH = CH2, -CH2-CH2NH2,
and — CH2— CH2OH. However, with 4-methylthiazole derivatives con-
taining either -CHOH-CH3 or -CH.-CHOH-CH-, at position 5, and
with — CH2 — CH2OH and — CH3 at positions 5 and 2, respectively, syn-
thesis of the thiazole moiety by pea roots could not be demonstrated.67*1
Consequently, since the last compounds are also effective in promoting
growth of pea roots, it appears that these compounds possess activity
without prior conversion to the normal thiazole moiety, while the activity
of the other compounds may be ascribed at least in part to their conver-
sion to the normal metabolite. The mode of action of these compounds
which are not converted to the thiazole moiety is still obscure since
analogues of thiamine possessing some of these modifications are inactive
for pea roots (Table 42).

THIAMINE

693

Table 45. The Activity of Antagonists Related to Thiamine.

Analogue

l-(2'-Methyl-4'-amino-5'-pyrimidylmethyl)-
2-methyl-3-(/3-hydroxyethyl)pyridinium
chloride (pyrithiamine)

3-(2'-Methyl-4'-hydroxy-5'-pyrimidyl-
methyl)-4-methyl-5-(S-hydroxyethyl)
thiazolium chloride (oxythiamine)

3-(2'-Butyl-4'-amino-5'-pyrimidylmethyl)-
4-methyl-5-(/3-hydroxyethyl)thiazolium
chloride

2-Methyl-4-amino-5-bromoethylpyrimidine

2-Methyl-4-amino-5-bromomethylpyrimidine

2-Methyl-4-amino-5-chloromethylpyrimidine
2-Methyl-4-amino-5-hydroxymethylpyrimidine
2-Methyl-4-amino-5-aminomethylpyrimidine
2- Methyl-4-aminopy rimidine

2-Methyl-4-amino-5-ethoxymethylpyrimidine

4-Methyl-5-(/3-hydroxyethyl)thiazole

pyrophosphate
Sulfathiazole

Organism

Mouse
Rat

Ceratostomella

fimbriata
Ceratostomella

pennicillata
Phytophthora

cinnamoni
Chalaropsis

thielavioides
Lactobacillus

fermenti
Endomyces vernalis
Penicillium

digitatum
Mucor ramannianus
Saccharomyces

cerevisiae
Staphylococcus

aureus
Staphylococcus

aureus
Salmonella

gallinarum
Lactobacillus

acidophilus
Neurospora crassa
Lactobacillus

arabinosus
Escherichia coli
Escherichia coli
Mouse

Inhibition
Index"

ca206
ca 20 b

7, 19

11
50
10c

130
160
80°

800

800
2000

700

1000

1900
400,000

Rat

Mouse

Rat, mouse

Lactobacillus

fermentum
Lactobacillus

fermentum
Yeast

carboxylase
Yeast carboxylase

40,000
2,000,000 d
20,000
Not
greater
than 25
ca 20 b

f
i
f
> 20,000
3000c

20,000"

10c
ca 160c' *

° For half-maximum growth.

b Approximately 40 moles of the inhibitor nullified the effect of one mole of thiamine.

c Using thiamine pyrophosphate as the growth factor.

d For Clostridium butylicum, Lactobacillus casei, Lactobacillus delbruckii, Lactobacillus pcntoaceticus.
Streptococcus lactis R, Propionibacterium pentosaceum and hemolytic streptococcus H69D, the pyrithiamine
inhibition index is greater than 2X106.

e Quantitative data not available.

I Subcutaneous administration of the inhibitor (7-9 mg to rats or 1-1.5 mg to mice) to animals main-
tained on a diet which appeared adequate for growth caused severe cramps and often death.

a If thiamine pyrophosphate is used as the growth factor, 90% inhibition occurs when the inhibitor/
growth factor ratio is 5000.

* One mole of cocarboxylase was reported to prevent the union of 322 moles of sulfathiazole with the
carboxylase protein of yeast washed with alkaline phosphate buffer solution.

694 THE BIOCHEMISTRY OF B VITAMINS

The interaction of thioformamide with 3-chloro-5-hydroxy-2-pentanone,
which forms the thiazole portion of the thiamine molecule in vitro, also
occurs in pea root cells. 5-Hydroxy-2-pentanone (CH3— CO— CH2— CH2
— CH2OH) is also utilized by the root cells for the thiazole synthesis, but
4-hydroxy-2-pentanone did not allow formation of 4-methyl-5- (a-hy-
droxyethyl) thiazole which has appreciable activity for pea roots.67a

Inhibitory Analogues of Thiamine

The first report of an analogue of thiamine which may interfere with
the utilization of this vitamin is that of Abderhalden,70 who observed in
1939 that the administration of 2-methyl-4-amino-5-bromomethylpyrimi-
dine (as well as the 5-bromoethyl homologue) to rats maintained on a diet
which appeared to be adequate for growth led to acute cramps and death
of the animals. It was found that this effect could be prevented by the
addition of corn sprouts or more dried yeast to the diet. These results
were confirmed by Morii,30 who showed that the 5-hydroxy, 5-bromo,
and 5-chloro analogues, but not the 5-amino analogue, also produced
spasms in mice as well as in rats.

In 1940, Buchman, et al.71 showed that the thiazole pyrophosphate
portion of cocarboxylase (thiamine pyrophosphate) inhibited the car-

N C— CH3

HC C— CH2CH2— O— PO— O— PO— OH

\/ II

S OH OH

Jf.-methyl-5-^-hydroxyeihylthiazole pyrophosphate

boxylase system of yeast, in which cocarboxylase is the coenzyme. Later,
Sevag, et al.12 reported that sulfathiazole specifically inhibits the cocar-
boxylase system of yeast. This effect can be overcome by the addition
of cocarboxylase, one mole of cocarboxylase being able to prevent the
union of 322 moles of sulfathiazole with the carboxylase protein. Subse-
quently, it was shown that p-aminobenzoic acid, although itself slightly
inhibitory, largely overcame the inhibitory effect of sulfathiazole on the
carboxylase system of Staphylococcus aureus and Escherichia coli. More-
over, these carboxylase systems are also inhibited (although to a lesser
extent) by sulanilamide, sulfapyridine, sulfadiazine, 2-sulfanilamido-
5-ethyl-4-thiazolone, 2-aminopyridine, 2-aminothiazole and 2-amino-
pyrimidine,72 some of which possess very little structural similarity to
thiamine. These results suggest that the relationship between thiamine
and sulfathiazole may be more obscure than it appeared initially.

Robbins 60 reported that pyrithiamine (heteroaneurine), l-(2'-methyl-
4'- amino - 5'-pyrimidylmethyl) - 2-methyl- 3 - (/?- hydroxyethyl) pyridinium

TH I AM INF 695

bromide, (the analogue of thiamine having a pyridine ring instead of the
thiazole ring) , slightly inhibited the growth of Phy corny ces blakesleeanus,
which requires thiamine or its pyrimidine and thiazole components for
growth. However, the inhibition was reversed by the addition of the
thiazole component, which indicated that this organism is able to cleave
the pyrithiamine molecule and use the pyrimidine component. This in-
terpretation was supported by the fact that in low concentration, this
analogue stimulated the growth of Pythiomorpha gonapodioides, which
requires only the pyrimidine component of thiamine. Large amounts of
the analogue were toxic, but this effect could be overcome by the addition
of the pyrimidine or the thiazole component of thiamine.

Woolley and White 32a found that pyrithiamine competitively inhibited
the growth of a number of organisms which require an external supply
of thiamine or its components, whereas it was without effect on organisms
which did not require thiamine. Those species which required intact
thiamine were about ten times as sensitive as those which needed only
the pyrimidine portion of thiamine, and about one hundred times as
sensitive as those stimulated by the pyrimidine and thiazole components
of thiamine. The pyrithiamine inhibition indices for Endomyces vernalis
and Mucor ra?nannianus were 130 and 800, respectively, but these or-
ganisms were not inhibited by 2-methyl-3-(/?-hydroxyethyl) pyridine,
even in concentrations as high as 100 y per cc. The resistance of the
organisms which do not require thiamine could not be attributed to their
synthesis of abnormally large amounts of thiamine since there was no
significant increase in the synthesis of this vitamin when the organisms
were grown in the presence of pyrithiamine. Subsequently, Woolley76
reported that a new strain of Endomyces vernalis was obtained by grow-
ing a culture in media containing pyrithiamine; it was not inhibited by
twenty-five times the concentration of pyrithiamine which produced 50
per cent inhibition in the parent strain. It still required thiamine or its
pyrimidine portion as a growth factor, but it was able to utilize small
amounts of the pyrithiamine also. It appears that resistance to inhibition
by pyrithiamine may depend in part on the ability of the organism to
cleave the molecule into its pyrimidine and pyridine components, since
the latter does not interfere with the growth of organisms which are
inhibited by pyrithiamine.

Wyss 77 reported that the pyrithiamine inhibition indices for Staphy-
lococcus aureus and Escherichia coll were 700 and 20,000, respectively.
When injected into mice in concentrations which were not toxic to the
animals, pyrithiamine was not anti-bacterial in the blood. Dreiser,
Scholtz and Spies 7S reported that pyrithiamine is inhibitory to the growth

696 THE BIOCHEMISTRY OF B VITAMINS

of Lactobacillus acidophilus; the inhibition index was found to be ap-
proximately 1900.

Sarett and Cheldelin 79 observed that pyrithiamine, 2-methyl-4-amino-
pyrimidine and 2-methyl-4-amino-5-ethoxymethylpyrimidine inhibit the
utilization of either thiamine pyrophosphate or thiamine phosphate more
effectively than the utilization of thiamine for growth of Lactobacillus
fermentum and Penicillium digitatum. The possibility that dephospho-
rylation of the pyrophosphate was inhibited by the analogues and that
some thiamine in the free form was essential for growth was considered,
but small amounts of thiamine did not affect the inhibition by the ana-
logues of the utilization of larger amounts of the pyrophosphate. On the
basis of these results, it was suggested that thiamine is attached to the
apoenzyme before phosphorylation.

In 1943, Woolley and White 82 reported that the feeding of pyrithia-
mine to mice induced characteristic polyneuritic symptoms of thiamine
deficiency, whereas the animals merely lost weight and died without
polyneuritic symptoms on a low-thiamine diet. The effect could be pre-
vented or reversed by the administration of thiamine; about forty moles
of the pyrithiamine nullified one mole of thiamine. Emerson 83 obtained
similar results after administering pyrithiamine to rats. In this case also,
the inhibition index was approximately 20.

Wilson and Harris 83a have recently pointed out that repeated analyses
of pyrithiamine hydrobromide, which had been assigned the formula
Ci4H2oN4OBr2, gave values which did not correspond closely to this
formula. Furthermore, the nitrogen values on different samples were
inconsistent. These authors report the preparation of a compound whose
constants do correspond to this formula, and the new compound has been
named neopyrithiamine. In rats its activity in inhibiting thiamine hydro-

CH,-^ ^-NH2HBr

CH3CH2CH2OH
neopyrithiamine hydrobromide

chloride is four times as great as that of pyrithiamine. Rabinowitz and
Snell 83b have shown that neopyrithiamine alleviates the growth inhibi-
tion observed in yeast grown in the absence of vitamin B6, but in the
presence of thiamine. It is concluded that neopyrithiamine acts as a com-
petitive inhibitor to overcome the toxic effect of the added thiamine.

Although oxythiamine, the 4'-hydroxy analogue of thiamine, is reported
to have 0.5 per cent of the antineuritic activity of thiamine for pigeons,50

THIAMINE 697

it has no vitamin action on rats,49 and in doses of 25 to 50 y per day it is
fatally toxic to young mice maintained on a low vitamin Bi diet supple-

ci-

n— CH2 N+ C— CH3

CH3— L JLOH HC C— CH2CH2OH

oxythiamine

mented with 1 y of thiamine per day.74 The 5-/?-chloroethyl and 5-/3-
bromoethyl analogues of oxythiamine were not toxic, even when admin-
istered in doses as high as 100 y per day. More recently, it was reported
that a low-thiamine diet provided some protection for mice against
infection with the Lansing strain of poliomyelitis virus. Similar protec-
tion, though less marked, was afforded by the administration of oxythia-
mine.75

In 1945, Emerson and Southwick 84 demonstrated that the administra-
tion of the 2'-butyl homologue of thiamine to rats maintained on a sub-
optimal intake of thiamine produced polyneuritis and subnormal growth,
both characteristic of thiamine deficiency. This effect was counteracted

N^^— CH2 N+ C— CH3

C4H,JL. J-NH2 HC C-CH2CH2OH

N \/

butylthiamine

by increasing the thiamine intake; the inhibition index was approx-
imately 20.

Ochoa and Peters 80 reported that thiamine, as well as a number of
pyrimidine analogues, stimulated the carboxylase system of yeast which
had been washed with an alkaline phosphate buffer solution. Westen-
brink, et al.sl showed that this apparent "stimulation" was actually due
to the fact that these compounds inhibited the dephosphorylation of the
cocarboxylase by a phosphatase present in the yeast. From the results
which are summarized in Table 46, it is evident that the inhibitory effect
depends on the presence of the 4-aminopyrimidine group. Weil-Malherbe
demonstrated that the presence of excess thiamine also slightly inhibits
the synthesis of cocarboxylase by yeast. 81a

Weswig, Freed and Haag 85 reported that rats placed on diets contain-
ing bracken fern which had been air-dried and ground developed symp-

698 THE BIOCHEMISTRY OF B VITAMINS

toms suggestive of thiamine deficiency after about ten days, and death
ensued about twenty days later. If treated orally with 0.5 mg of thiamine
per day, animals showing severe anorexia, emaciation and polyneuritis
promptly recovered. The toxicity was not decreased when the air-dried
fern was heated at 105° C in air for 18 hours. The causative agent is
essentially insoluble in ethyl ether and in acetone, but appears to be
slightly soluble in 92 per cent ethyl alcohol.

Table 46. The Inhibition of the De phosphorylation of Cocarboxylase in Yeast.

Analogue Inhibition Index Reference

Thiamine hydrochloride 15° 80

between 0.2 and 20 81

3-(2'-Methyl-4'-amino-5'-pyrimidylmethyl)- 75° 80

4-methyl-5-(/3-hydroxypropyl)thiazolium

chloride
2-Methyl-4-amino-5-aminomethylpyrimidine 100-150" 80

hydrochloride 50-150 81

2-Methyl-4-amino-5-thioformamidomethyl- 400" 80

pyrimidine hydrochloride
2-Methyl-4-hydroxy-5-thioformamidomethyl- b 80

pyrimidine hydrochloride

° Ochoa and Peters80 reported that these compounds stimulated the carboxylase system of yeast which
had been washed with alkaline phosphate buffer solution. Westenbrink81, et al. showed that the apparent
' stimulation" was due to inhibition of the dephosphorylation of the cocarboxylase. These inhibition indices
are calculated from the data of Ochoa and Peters on the assumption that 50% "stimulation" corresponded
to 50% inhibition of dephosphorylation of the cocarboxylase present and they represent the ratio
moles of inhibitor , . , . „ .. .. . ,. „

= ; 1 which gives .)(.)' r "stimulation.

moles of cocarboxylase

b Ochoa and Peters reported that this compound did not "stimulate" the carboxylase system.

The Chastek paralysis of foxes was shown by Green, Carlson and
Evans 86 to be caused by diets containing large amounts of raw fish,
which apparently contained a substance capable of inactivating the
thiamine in the feed and thus caused a thiamine deficiency. Later in-
vestigations demonstrated that a similar effect was produced in chicks 87
and in cats.88 In each case, the toxicity could be overcome by the admin-
istration of large amounts of thiamine. The toxic substance was found to
be an enzyme,89 and it was shown by Krampitz, Woolley and White 90
to cleave the thiamine molecule at the methylene bridge, liberating ulti-
mately 2-methyl-4-amino-5-hydroxymethylpyrimidine and 4-methyl-5-
(/?-hydroxyethyl)thiazole. The thiazole component was liberated directly,
but the pyrimidine portion apparently was first converted to an inter-
mediate and liberated in a subsequent reaction. This was confirmed by
Hennessy, Warner, Falk and Truhlar,91 who isolated a crystalline inter-
mediate after the destruction of thiamine by clam tissue. Analysis indi-
cated that the molecular formula was CsH1eN.iO3S.2HCl but the structure
of the compound was not reported. Sealock and Davis 92 found that
m-nitroaniline and m-aminobenzoic acid stimulated the Chastek paralysis
thiaminase in vitro, and they concluded that this effect was due to the

THIAMINE 699

formation of a secondary amine by combination of the amino group of
the "activator" with the 5-methylenc group of the pyrimidine component
of the thiamine. They succeeded in isolating N-(2-methyl-4-amino-5-
pyrimidylmethyl)-m-nitroanilinc from the reaction mixture produced
when thiamine was enzymatically destroyed in the presence of ?>i-nitro-
aniline.

Sealock and Goodland 93 found that the cleavage of thiamine by the
Chastek paralysis enzyme is inhibited by a number of thiazole deriva-
tives. 3-o-Aminobenzyl-4-methylthiazolium chloride at a concentration

ci-

aCH2— N+ C— CH3
NH2 Hi! L

\ /

3-o-aminobenzyl-4-methyUhiazolium chloride

of 5 X 10~4 mole/liter was found to produce 100 per cent inhibition of the
enzymatic destruction of thiamine at the same molar concentration, and
the extent of the inhibition proved to be dependent upon the inhibitor-
thiamine ratio. A like concentration of 3-/?-aminoethyl-4-methylthia-
zolium chloride caused 56 per cent inhibition of the destruction of the
vitamin. The other compounds tested (3-o-nitrobenzyl-, 3-/3-phthalimido-
ethyl-, 3-ethyl-, 3-phenyl-, 3-ethyl-2-methyl-, and 3-methyl-5-/3-
hydroxyethyl-4-methylthiazolium chlorides as well as several 6-amino-
pyrimidine compounds) were either only slightly effective or completely
without activity as inhibitors. The importance for inhibitory activity of
the amino group in the position analogous to that of the 4'-amino group
of thiamine is indicated by the high activity of the o-aminobenzyl deriva-
tive and the low activity of the corresponding compounds which do not
possess this group. Further evidence for the importance of the position
of the amino group in the benzyl portion of the thiazole derivative was
obtained by Sealock and Livermore,94 who showed that whereas the
o-aminobenzyl derivative was highly inhibitory, the corresponding
p-aminobenzyl analogue was almost without inhibitory action and the
m-aminobenzyl compound was markedly stimulatory. It was also demon-
strated that the 4-methyl group is not an essential feature of the inhibitor
molecule, for the corresponding 2-methyl derivative had equal or slightly
greater inhibitory action. However, placing a methyl group in both the
2 and 4 positions of the thiazole ring resulted in a compound with less
than 50 per cent of the inhibitory activity of the corresponding analogues
in which only one of those positions was substituted.

700 THE BIOCHEMISTRY OF B VITAMINS

Soodak and Cerecedo 74 reported that oxythiamine inhibits the Chastek
paralysis thiaminase, but quantitative data are not available. Bhagvat
and Devi 95 found that certain cereals, legumes and oil seeds contain a
factor capable of destroying thiamine. Apparently, the factor is not an
enzyme, since it is extracted by chloroform-water mixtures and is stable
to heat, including autoclaving. The end products are believed to be
pyrimidine and thiazole derivatives, since mosquito larvae were able to
utilize for growth the breakdown products produced when thiamine was
destroyed by extracts from ragi or carp tissue, but were unable to utilize
the breakdown products produced by autoclaving or treating thiamine
with sulfite or sodium hydroxide. The extraction from the flesh of
Corbicula strata of a thiaminase which deaminized the pyrimidine ring
but did not open the thiazole ring was announced by Murata.96

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THIAMINE 701

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48a. Schopfer, W. H., Ann. inst. Pasteur, 61, 781 (1938).

49. Bergel, F., and Todd. A. R, J. Chem. Soc, 1504 (1937).

50. Slobodin, Y. M., and Zigel, M. S., /. Gen. Chem. U.S.S.R., 11, 1019 (1941);

C. A., 36, 6542 (1942).

51. Price, C. C, Leonard, N. J., and Reitsema, R. H., /. Am. Chem. Soc, 68, 766

(1946).

52. Bowman, A., /. Chem. Soc, 494 (1937).

53. Pesina, A. G., and Zigel, M. S., Proc Sci. Inst. Vitamin Research, U.S.S.R.,

3, No. 1, 94 (1941); C. A., 36, 3008 (1942).

54. Buchman, E. R., and Richardson, E. M., J. Am. Chem. Soc, 67, 395 (1945).

55. Price, D., and Pickel, F. D., /. Am. Chem. Soc, 63, 1067 (1941).

56. Dornow, A., Ber., 73, 156 (1940).

57. Dornow, A., and Schacht, W., Chem. Ber., 80, 505 (1947).

58. Baumgarten, P., and Dornow, A., Ber., 73, 44 (1940).

59. Abderhalden, E., and Abderhalden, R., Arch. ges. Physiol. (Pfliiger's), 244, 142

(1940).

60. Robbins, W. J., Proc. Natl Acad. Sci. U. S., 27, 419 (1941).

61. Schopfer, W. H., Compt. rend. soc. phys. hist. nat. Geneve, 58, 64 (1941); (in

Arch. sci. phys. nat., 23, suppl. 64 (1941)).

62. Dornow, A., and Machens, H., Chem. Ber., 80, 502 (1947).

63. Finkelstein, J., and Elderfield, R. C., J. Org. Chem., 4, 365 (1939).

64. Robbins, W. J., Proc. Natl. Acad. Sci., U. S., 28, 352 (1942).

65. Abderhalden, E., and Abderhalden, R., Arch. ges. Physiol. (Pfliiger's), 240, 746

(1938).

66. Robbins, W. J., and Kavanagh, F., Proc. Natl Acad. Sci. U. S., 24, 229 (1938).

67. Bonner, J., Am. J. Botany, 25, 543 (1938).

67a. Bonner, J., and Buchman, E. R., Proc. Natl. Acad. Sci. U. S., 24, 431 (1938).

68. Buchman, E. R., and Richardson, E. M., J. Am. Chem. Soc, 61, 891 (1939).

69. Harington, C. R., and Moggridge, R. C. G., J. Chem. Soc, 443 (1939).

702 THE BIOCHEMISTRY OF B VITAMINS

69a. Fox, S. N., Sargent, H., and Buchman, E. R., /. Am. Chem. Soc, 67, 496

(1945).
69b. Sahashi, Y., Nakayama, A., Uchida, M., Ionoue, J., and Mizuno, H., /. Sci.

Research Inst. Tokyo, 43, No. 1191, 56 (1948).

70. Abderhalden, R., Klin. Wochschr., 18, 171 (1939).

71. Buchman, E. R., Heegard, E., and Bonner, J., Proc Natl. Acad. Sci. U. S., 26,

561 (1940).

72. Sevag, M. G., Shelburne, M., and Mudd, S., J. BacL, 49, 65 (1945); J. Gen.

Physiol, 25, 805 (1942).

73. Sevag, M. G., Henry, J., and Richardson, R. A., J. Bad., 49, 71 (1945).

74. Soodak, M., and Cerecedo, L. R., J. Am. Chem. Soc, 66, 1988 (1944); Fed.

Proc, 6, 293 (1947).

75. Jones, J. H., Foster, C., and Henle, W., Proc. Soc, Exptl. Biol. Med., 69, 454

(1948).

76. Woolley, D. W., Proc Soc. Exptl. Biol. Med., 55, 179 (1944).

77. Wyss, 0., J. BacL, 46, 483 (1943).

78. Dreizen, S., Scholz, E., and Spies, T. D., Proc Soc. Exptl. Biol. Med., 68, 620

(1948).

79. Sarett, H. P., and Cheldelin, V. H, J. Biol. Chem., 156, 91 (1944).

80. Ochoa, S., and Peters, R. A., Biochem. J., 32, 1501 (1938).

81. Westenbrink, H. G. K, Van Dorp, D. A., Gruber, M, and Veldman, H,

Enzymologia, 9, 73 (1940)
81a. Weil-Malherbe, H., Biochem. J., 33, 1997 (1939).

82. Woolley, D. W., and White, A. G. C, J. Biol. Chem., 149, 285 (1943).

83. Emerson, G. A., Abstract of Papers, ACS, April 14-18, 1947, p. 42b.
83a. Wilson, A. N., and Harris, S. A., /. Am. Chem. Soc, 71, 2231 (1949).
83b. Rabinowitz, J. C, and Snell, E. E., Federation Proc, 8, 240 (1949).

84. Emerson, G. A., and Southwick, P. L., J. Biol. Chem., 160, 169 (1945).

85. Weswig, P. H., Freed, A. M., and Haag, J. R., J. Biol. Chem., 165, 737 (1946).

86. Green, R. G., Carlson, W. E., and Evans, C. A., J. Nutrition, 21, 243 (1941).

87. Spitzer, E. H., Coombes, A. I., Elvehjem, C. A., and Wisnicky, W., Proc. Soc.

Exptl. Biol. Med., 48, 376 (1941).

88. Smith, D. C, and Proutt, L. M., Proc. Soc. Exptl. Biol. Med., 56, 1 (1944).

89. Sealock, R. R., Livermore, A. H., and Evans, C. A., J. Am. Chem. Soc, 65, 935

(1943).

90. Krampitz, L. O., and Woolley, D. W., J. Biol. Chem., 152, 9 (1944).

91. Hennessy, D. J., Warner, S., Falk, K. G., and Truhlar, J., Abstract of Papers,

ACS, April 8-12, 1946 (p. 20B).

92. Sealock, R. R., and Davis, N. C, J. Biol. Chem., 177, 987 (1949).

93. Sealock, R. R., and Goodland, R. L., J. Am. Chem. Soc, 66, 507 (1944).

94. Sealock, R. R., and Livermore, A. H., /. Biol. Chem., 177, 553 (1949).

95. Bhagvat, K., and Devi, P., Indian J. Med. Research, 32, 131 (1944).

96. Murata, K., Rept. Osaka Municipal Research Inst. Domestic Sci., 17, No. 1,

137 (1946).

97. Bonner, J., and Bonner, H., Vitamins and Hormones, 6, 225 (1948).

Chapter XI D

BIOLOGICAL ACTIVITIES OF OTHER NUTRITIONAL
FACTORS OF DOUBTFUL STATUS*

Choline
Introduction

The presence of choline as an integral structural unit as phospholipides
was recognized soon after the middle of the last century; lecithins and
sphingomyelins from both plant and animal sources were found to con-
tain the substance. The nutritional importance of choline was not demon-
strated until relatively recently.

In a series of investigations which followed the discovery of insulin by
Banting and Best in 1922, it was found that the administration of
lecithin prevents the abnormal accumulation of fat in the livers of de-
pancreatized dogs injected with insulin.1- 2- 3 Subsequently it was shown
that the development of fatty livers in rats maintained on a high-fat,
low-protein ration can be prevented or cured by the inclusion of choline
in the diet.4 The lipotropic action of lecithin was therefore attributed to
the presence of choline in its structure.

It is now known that a dietary deficiency of choline may cause a
variety of metabolic disturbances. These effects are discussed in detail
in several excellent review papers.5, 6- 7 For rats, a lack of choline results
in cessation of growth, infiltration of fat in the livers, and hemorrhagic
degeneration of the kidneys. For chicks and turkey poults, a choline
deficiency causes cessation of growth and perosis or "slipped tendon
disease."

In addition to its involvement in animal and avian nutrition, choline
is required as a growth factor by a number of microorganisms including
certain strains of pneumococci (Types I, II, III, V, VIII) s- 9 and "choline-
less" mutants of Neurospora crassa.10' n

Studies dealing with the choline molecule suggest that the metabolic
effects of choline may be divided into two groups: (a) those which de-
pend on the effectiveness of choline as a source of the "transferable
methyl group," and (b) those which depend on the presence of the intact
choline molecule.

* By Thomas J. Bardos, Lorene L. Rogers, and A. D. Barton.

703

704

THE BIOCHEMISTRY OF B VITAMINS

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NUTRITIONAL FACTORS OF DOUBTFUL STATUS 705

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706 THE BIOCHEMISTRY OF B VITAMINS

Efforts to determine what specific actions of choline are responsible
for the gross physiological changes observed in choline deficiency led to
the discovery of a functional relationship between choline and methionine.
In 1938 Tucker and Eckstein 12 reported that methionine exerts an effect
on liver fat similar to that shown by choline, du Vigneaud and co-work-
ers 13 later observed that methionine can be replaced in the diet of rats
by homocysteine, provided choline or betaine is simultaneously supplied.
They suggested that this effect is due to the conversion of homocysteine
to methionine by the transfer of methyl groups from choline.14 As a
result of these and subsequent investigations it is now apparent that
choline, betaine and methionine can serve as metabolic sources of the
"transferable methyl group," which may be an essential dietary con-
stituent.

Choline apparently functions as the intact molecule in the synthesis of
some of the phospholipides which play an important role in the regula-
tion of fat metabolism as well as in the synthesis of acetylcholine. The
latter compound has attracted much attention as a "chemical transmitter"
of the nerve impulse.

Specificity

A considerable number of compounds have been tested for their ability
to alleviate the various symptoms produced by choline deficiencies in
rats and in chicks and turkey poults. The results of these tests are sum-
marized in Table 47.

The ability of various compounds to permit growth of the rat or the
chick on a choline-methionine-free diet supplemented with homocysteine
has been interpreted to mean that these compounds are able to transfer
methyl groups to homocysteine to form methionine. This conversion has
been demonstrated following the administration of deuteriocholine 45 and
deuteriobetaine.46 On this basis the following compounds have been found
to be methyl donors: choline, choline derivatives such as lecithin and
phosphorylcholine, monoethylcholine, betaine, dimethylthetin (sulfobe-
taine), methylethylthetin and dimethylpropiothetin. The minimal re-
quirement for methyl donor activity appears to be the presence of at least
one methyl group attached directly to an onium pole. That such activity
may also be conditioned by enzyme specificity is suggested by the fact
that transmethylations utilizing choline, betaine and dimethylthetin are
catalyzed by three different enzymes.47 This view is further supported by
the observation that sulfobetaine is an active methyl donor, while sulfo-
choline is inactive in this respect. Moreover, dimethylpropiothetin ex-
hibits a marked growth-promoting activity, whereas its nitrogen analogue,

NUTRITIONAL FACTORS OF DOUBTFUL STATUS

707

/3-alanine betaine, is toxic and apparently does not serve as a methyl
donor.

The effects of choline in preventing fatty livers and hemorrhagic
kidneys in rats and mice as well as the antiperosis effect in chicks and
turkeys and the growth promoting effects on mutants No. 34486 and

Table 48. Activity of Choline Derivatives on Growth of a Strain of
Pneumococcus (Type III).9

Compound

Structural Formula

Activity

Choline chloride

(CH,),N(Cl)-CH,-CH,-OH

100

Dimethylethanolamine

(CH3)2N-CH2-CH2OH

100

Methyldiethanolamine

(CH3)N(CH2-CH2-OH)2

100

Triethylcholine chloride

(C2H6)3-N(C1)-CH2-CH2-0H

100

Diethylethanolamine

(C2H6)2-N-CH2-CH2-OH

100

Dimethy lethy 1 hydroxyethyl-
ammonium chloride

(CH3)2(C2H6)N(C1) • CH2 • CH2 • OH

100

Ethanolamine

H2NCH2CH2OH

10(80)

Diethanolamine

HN(CH2-CH3-OH)2

50(95)

Triethanolamine

N(CH2CH2OH)3

100(95)

10.

Tetraethanolammonium
hydroxide

(HO)N(CH2-CH2-OH)4

25(85)

11.

N-Acetylethanolamine

CH3CONHCH2CH2OH

10(23)

12.

a-Ethylethanolamine

H2N-CH(C2H5)-CH2OH

67(80)

13.

a-Ethyl-a-hydroxymethyl-
ethanolamine

H2N-C(C2H6)(CH2-OH)CH2-OH

25(100)

14.

a-Methyl-a-hydroxymethyl-

ethanolamine
a.a-Dimethylethanolamine

H2N-C(CH2-OH)(CH3)CH2OH

12(90)

15.

H2N-C(CH3)2-CH2-OH

10(31)

16.

a,a-Dimethylcholine chloride

(CH3)3N(C1)-C(CH3)2-CH2-0H

100(20)

17.

a,a-Dihydroxymethylethanol-

H2N-C(CH2OH)2-CH2OH

10(5)

18.

amine
7-Diethylaminopropanol

(C2H6)2N ■ CH2 • CH2 • CH2 • OH

83(100)

19.

/3,7-Propanediol-a-diethylamine (C2H5)2N • CH2 • CH(OH) • CH2 ■ OH

50(100)

20.

Diethylmethyl-/3,7-dihydroxy-
propylammonium chloride

(C2H5)2(CH3)N(C1) • CH2 • CHOH • CH2OH

100(20)

21.

Acetylcholine chloride

(CH3)3N(Cl)-CH2-CH2-0-CO-CH3

100(7)

22.

Acetyl-/3-methylcholine
chloride

(CH3)3N(Cl)-CH2-CH(CH3)-0-CO-CH3

100(7)

Even when tested at concentrations up to 50 7 /ml. of medium the following com-
pounds were inactive: N-phenylethanolamine, 2-nitro-l-butanol; serine; ethylamine;
ethylenediamine; glycine; sarcosine; betaine; j3-methoxyethylamine; carnitine; calcium
phosphorylcholine; urethane of /3-methylcholine chloride; carbamylcholine chloride.

a Percent activity on molar basis relative to choline chloride necessary for maximal response of the or-
ganism to the compound. Bracketed figures indicate percent of maximal growth attained with the most
effective concentration of the compounds which do not give growth equivalent to that obtained with 5 y
per cc. of choline chloride.

No. 47904 of Neurospora crassa appear to depend on a function of choline
which involves the utilization of the intact molecule. The ability of
choline to form phospholipides may be a critical factor, since it has been
shown that analogues possessing choline activity, e.g., triethylcholine 48, 49
and sulfocholine,20 can be incorporated into the body phospholipides of
rats. Inasmuch as these two compounds are not methyl donors, it appears
that the utilization of choline as an intact molecule is independent of its
ability to furnish methyl groups. Additional support for this hypothesis

708 THE BIOCHEMISTRY OF B VITAMINS

is provided by the fact that compounds which fail to function as methyl
donors such as arsenocholine, diethylmethylhydroxyethylammonium
chloride and homocholine, are active in preventing fatty livers in the
rat or perosis in the chick, whereas such methyl donors as betaine and
methionine do not prevent perosis in the chick.

Choline has been found to be essential for the growth of certain strains
of pneumococci (Types I, II, V, VIII) .8 A number of choline analogues
have been tested as growth factors for a Type III strain;9 the results of
these tests are summarized in Table 48. Of the known functions of
choline in animal nutrition, its utilization in phospholipide formation
appears to be its most likely role in the metabolism of the pneumococcus,
since ethanolamine, diethanolamine and triethanolamine are also effective.
The activity of these compounds, together with the inactivity of methio-
nine, betaine, phosphorylcholine, etc., suggest that for this organism
choline does not have a significant role in transmethylation.

Two mutant strains, No. 34486 and No. 47904, which arose from the
ultraviolet irradiation of a culture of wild type Neurospora crassa were
found 10- n to require choline for growth in a medium which supported
the growth of the wild type organism. Both mutants also responded to
acetylcholine, arsenocholine, phosphorylcholine, dimethylaminoethanol,
dimethylethylhydroxyethylammonium chloride, diethylmethylhydroxy-
ethylammonium chloride, triethylcholine and methionine. The following
compounds were inactive for both mutants: betaine, creatine, sarcosine,
ethanolamine, neurine, diethylaminoethanol, dimethylamine, trimethyl-
amine and tetramethylammonium chloride. Of considerable interest is
the fact that mutant No. 34486 can utilize monomethylaminoethanol but
No. 47904 cannot; under suitable conditions, this compound accumulates
in the latter organism.48 These results were considered to indicate that
methylaminoethanol is an intermediate in the synthesis of choline by
Neurospora and that the block in mutant No. 34486 precedes the forma-
tion of methylaminoethanol, whereas the block in mutant No. 47904 fol-
lows it. The structural specificity exhibited suggests that in Neurospora
the predominant function of choline depends on its acting as an intact
molecule rather than as a methyl donor.

Inhibitors Related to Choline

Triethylcholine. Keston and Wortis 50 reported in 1946 that even
though triethylcholine is lipotropic when fed in small quantities to rats,
the compound is acutely toxic when injected into mice. The toxicity is
completely prevented by the simultaneous injection of an equal weight
of choline chloride. It was further observed that the action of choline in
the contraction of isolated frog muscle is blocked by triethylcholine while

NUTRITIONAL FACTORS OF DOUBTFUL STATUS 709

C2H5
5— N— CH2— CH2— OH

ci-

C2H6

triethylcholine chloride

the action of acetylcholine is unaffected. These data indicate that the
choline analogue may interfere with the synthesis of acetylcholine from
choline.

L-Penicillamine (/3,,5-Dimethylcysteine). Wilson and du Vigneaud 51
found that the growth of young albino rats was inhibited when L-penicil-

CH3 NH2
HS C CH COOH

CH3

L-penicillamine

lamine hydrochloride hydrate was added to the diet, and that normal
growth was restored by feeding choline chloride. However, this inhibitor
appears not to be competitive with choline, for aminoethanol was found
to be even more effective than choline in overcoming the growth inhibi-
tion. Dimethylaminoethanol and monomethylaminoethanol were also
effective in restoring the normal rate of growth, but methionine, cysteine,
cystine, homocysteine and homocystine were without activity. These in-
vestigators suggested that "penicillamine may exert its toxic action by
blocking either the synthesis or the utilization of aminoethanol."

Coramine (N,N-Diethylnicotinamide). A type of inhibition involving
choline was observed by Wilson and Leduc.52 Weanling mice were fed on
a low protein, choline-deficient diet to which coramine was added in
varying amounts. The compound at a concentration of 0.25 per cent per-

-C— N(C2H6)2

coramine

mitted very slight growth; 0.5 per cent allowed maintenance without
growth; and 1.0 per cent caused a loss in weight. The inhibitory effects of
coramine are reversed by the addition of choline to the diet. Even though
coramine is structurally unrelated to choline, its detoxication apparently
takes place by transmethylation and its presence in the diet increases
the requirement for a methyl donor. Choline must function here only as

710 THE BIOCHEMISTRY OF B VITAMINS

a methyl donor and not as a lipotropic agent, since fatty livers do not
develop on a 1 per cent coramine diet.

Ethionine (S-Ethylhomocysteine). It was early shown 53 that ethionine
is toxic to rats on a low methionine diet and that the toxicity is prevented
by the simultaneous administration of methionine. Methionine also re-

NH2

C2H6— S— CH2— CH2— CH— COOH
ethionine

verses the growth inhibition produced in Escherichia coli by ethionine.54
The latter compound was used by Stekol and Weiss 55 in an effort to
determine whether the inhibition of growth in rats is the result of a block
in the utilization of methionine per se, or whether there is interference
with the utilization of some of the metabolites which normally originate
from methionine. They found that choline alone can alleviate the growth
inhibition caused by ethionine just as methionine can. The other sub-
stances tested, cystine, homocystine and cystathionine, were without
effect on the inhibition. It is suggested that the increased need for choline
which is created by the administration of ethionine may result in a
diversion of the labile methyl group for greater synthesis of choline, thus
decreasing the amount of methionine available for growth. The possibility
of the incorporation of ethionine into the tissue protein of the rat was
also considered, but it was stressed that further experimentation must
be done before a rigorous interpretation of the data will be possible.

In this discussion no attempt has been made to treat the many in-
hibitors of enzyme systems in which choline is involved, but only those
which may have a bearing on the status of choline as a doubtful member
of the B vitamin complex.

Inositol

The role of inositol as a growth factor for yeast was discovered in
1928 by Eastcott,56 who isolated it from tea and recognized it as the
active constituent of Lukas' "Bios I." Subsequent work has shown that
it is a complementary growth factor for some strains of yeast; i.e., it is
relatively ineffective alone, but in combination with other B vitamins, it
often causes a striking increase in growth.57, 5S The amount of inositol
necessary to produce these growth effects is from 100 to 1000 times larger
than the effective concentrations of the other B vitamins, and the effects
obtained are largely dependent on the particular strain of yeast under
investigation. Inositol is also a complementary growth factor for various

NUTRITIONAL FACTORS OF DOUBTFUL STATUS

711

Meso-inositol

L 2, 3t 5

4, 6

Epi-inositol

l,2t?f4,,5
6

Scyllitol

It l> 5
2, A, 6

OH OH

OH H

1,-inositol
1. 2« A
3, 5, 6

d.-inositol
1. 2. 5
3, A, 6

jjjjco-inosltol
3^

Allo-lnosltol

1. 2t > !i
5. 6

oj/h

H\OH

1,2,1

,*,5.6

1. E. 2

». 5. 6

Figure 9.

712 THE BIOCHEMISTRY OF B VITAMINS

fungi, including Nematospora gossypii,5d> 60 Lophodermium pinastri 60 and
Eremothecium ashbyii.®1 The compound has been shown to be an absolute
essential for only a few organisms, such as Rhizopus suinus 62 and
"inositolless" mutant strains of Neurospora crassa.63

Woolley in 1940 isolated the "mouse anti-alopecia factor" from liver
concentrate and identified it as inositol.64, 65 He showed that a loss of hair
and a severe dermatitis developed in mice on a purified diet and that the
administration of inositol cured these symptoms.

Vitamin-like effects of inositol have also been reported for rats,66, 67, 68, 69
guinea pigs,70 hamsters,71, 72 chicks,73, 74 and pigs.75

Specificity

Inositol, in contrast to other B vitamins, has a number of naturally
occurring, closely related analogues. Several of its geometrical isomers and
their derivatives are present in many natural products.

Nine geometrical isomers of inositol are theoretically possible if the
six carbon atoms of the cyclohexane ring are considered to be coplanar.
These nine forms76 are schematically represented below (Figure 9).
Four of these isomers (?^eso-inositol, ( + )- and ( — ) -inositol, and
scyllitol) are known to occur naturally either in the free state or in the
form of their esters or ethers. Their configurations as they are repre-
sented in Figure 9 have been established by the work of Posternak.79, 80, 81
Three others isomers (epwnositol, aUo-inositol and muco-inositol) have
been synthesized and characterized. 79, 82, 83

The possibility of additional isomers of compounds structurally related
to inositol has been indicated by x-ray studies on the /^-isomer of 1,2,3,4,-
5,6-hexachlorocyclohexane and the corresponding bromo derivative.
These studies 77, 78 indicate that the six carbon atoms do not lie in one
plane but in two parallel planes. This so-called "puckered ring" (chair
form) would make 16 isomers theoretically possible including six optically
active forms. While such structures may be stable in crystalline form,
it is possible that in solution such isomers resulting solely because of the
"puckered ring" or chair form of the cyclohexane ring may become inter-
convertible.

The biological activity of the four naturally occurring isomers and
some of their most common derivatives 76 are listed in Table 49. Of all
these substances only meso-inositol was found to have vitamin activity
for both yeast and mice.84 Indeed, in most instances only meso-inositol
or compounds which can readily form meso-inositol appear to be effective.
Esters of meso-inositol are active for mice but inactive for yeast. For
yeast only mytilitol (the methyl homologue of scyllitol) and hydroxy-
mytilitol have some activity in addition to meso-inositol itself.

NUTRITIONAL FACTORS OF DOUBTFUL STATUS 713

Table 49. Specificity of meso-Inositol.

.cuvity, y0 inos
g

IlIOl

1
O

Compound

§ 1

§.

"f-sg

O 03

■si

meso-Inositol

100

107

100

108

100

106

meso-Inositol monophosphate

106

50-100

109

meso-Inositol tetraphosphate

106

meso-Inositol hexaphosphate

(Phytin)
meso-Inositol hexaacetate

106

+
106

ept-Inositol

2.9

109

(— )-Inositol

106

r i

108

4.5

109

106

(— )-Inositol monomethyl ether

(Quebrachitol)
(+)-Inositol

0, <1

106, 107

106

U0.4109J
22

109

108

20.7

109

106
106

(+)-Inositol monomethylether

(Pinitol)
Scyllitol

Mytilitol*

106

107

106

<10

109

/o 61\

\2.9109j
8.1

109

106

+
106

Isomytilitol*

19.7

109

0.87

109

Hydroxymytilitol

7.5

109

Hydroxyisomytilitol

<10

2.9

109

0.15

109

Quercitol (pentahydroxy-

cyclohexane)
5-Desoxy-(+ )-inositol

0, <1

106, 107

1
109

2.9

109

108

106

Scyllo-ms-inosose

("biochemical inosose")
epi-ms-Inosose

("chemical inosose")
Soybean cephalin

106

93.6

109

17.4

109

13.9

109

Arabitol, sorbitol, dulcitol, mannitol and other sugar alcohols are inactive for yeast.107

* According to Posternak,81 mytilitol is methylscyllitol, and isomytilitol is methyl-?neso-inositol.

Schopfer 62> 85- 86 has tested the specificity of inositol for Rhizopus
suinus and the "inositolless" mutant of Neurospora crassa. Meso-inositol
is highly specific for both these organisms. (Table 49)

An interesting stereochemical specificity has been observed in the rate
of the enzymatic oxidation of the inositols to ketones and diketones by

714 THE BIOCHEMISTRY OF B VITAMINS

Acetobacter suboxydans.80' 87, 88, 89 According to Magasanik and Char-
gaff,89 "only those hydroxyls are oxidized that are situated in a polar
plane." This would mean that in the case of meso-inositol only the cis-
hydroxyl in the 2 position is attacked. Schopfer 62 believed this hydroxyl
to be necessary for vitamin activity. If this is true, scyllo-ms-inosose,
which is active for Eremothecium ashbyii (Table 49), must be reduced
to the corresponding meso-inositol before it is utilized by the organism.

Inhibitory Analogues of Inositol

The theory was advanced by Slade 90- 91 in 1945 that the powerful
insecticidal action of y-hexachlorocyclohexane ("Gammexane") could be
explained on the basis of its structural similarity to meso-inositol. He
assigned to the y-isomer the configuration corresponding to that of meso-
inositol and pointed out that this compound has a much higher toxicity
for insects than do the a-, B-, and S-isomers. It was proposed that the
y-isomer exerts its inhibitory action by blocking the functioning of
inositol in some important enzyme system.

Recent x-ray studies 92 have indicated that the configuration of the
y-isomer does not correspond to that of meso-inositol; nevertheless,
Slade's theory concerning the antagonism between "Gammexane" and
meso-inositol stimulated several studies, and it was demonstrated that
in some cases the toxic action of y-hexachlorocyclohexane could be af-
fected by meso-inositol.

Kirkwood and Philips 93 found that the Gebruder Mayer strain of
yeast, which normally requires 1 y of inositol per ml of medium for
maximal growth, was strongly inhibited by 60 y per ml of y-hexachloro-
cyclohexane. This inhibition was "progressively but not completely"
reversed by the addition of 1 to 6 y of meso-inositol per ml of medium.
This would correspond to a molar inhibition ratio of about 30. The a-,
B-, and 8-isomers of hexachlorocyclohexane also had a slight inhibitory
effect, but only the inhibition caused by the y-isomer could be reversed
by meso-inositol.

The y-isomer of hexachlorocyclohexane completely inhibits the growth
of Nematospora gossypii, whereas the /3-isomer is inactive and the
a-isomer is only slightly inhibitory.94 The inhibition caused by 10-60 y
per ml of the y-isomer was reduced to 50 per cent by the addition of 60 y
per ml of meso-inositol and to 6 per cent by 100 y per ml of the vitamin.

Schopfer et al.95 found that flavinogenesis is inhibited in Eremothecium
ashbyii var gossypii by y-hexachlorocyclohexane and that the growth
medium is completely decolorized. The effects of 800 y of the inhibitor
are prevented by the addition of 5 y of meso-inositol. Scyllo-ms-inosose

NUTRITIONAL FACTORS OF DOUBTFUL STATUS 715

is also effective in preventing the inhibition, but ( + ) -inositol, scyllitol
and other related compounds are inactive.

Chargaff et al.96 found that meso-inositol is able to prevent the meta-
phase arrest and tumor formation induced in Allium Cepa by either
colchicine or y-hexachlorocyclohexane, first observed by Nybom and
Knutsson.97 Meso-inositol appears to be specific in producing this effect;
other related compounds were inactive.

It was recently reported 98 that y-hexachlorocyclohexane, after an in-
cubation period of 16 hours, completely inhibited the enzymatic activity
of a sample of purified pancreatic a-amylase which had been shown to
have a relatively high inositol content. The inhibition was competitively
prevented by the addition of meso-inositol. Fischer and Bernfeld " were
not able to repeat this work, and suggested that the inositol-containing
amylase was only partially purified.

Meillon 10° observed that the blood of rabbits injected with y-hexa-
chlorocyclohexane was toxic for certain blood-sucking anthropods and
that this toxicity was not reversed by injections of inositol. Schopfer95
found that either the toxicity of the inhibitor or the reversing action of
meso-inositol was negligible or inconsistent in many fungi. He suggested
that these inconsistencies may be due to the biosynthesis of inositol by
these organisms.

Other studies have indicated that S-hexachlorocyclohexane is more
toxic than the y-isomer for the ciliate Glaucoma piriformis,101 for the
eggs of sea urchins,102 and for many bacteria.103 In none of these cases
was meso-inositol effective in preventing the toxicity.

In view of all the above experiments, it seems reasonable to conclude
that the toxicity of y-hexachlorocyclohexane for various organisms is not
dependent on a single mechanism. Even though the y-isomer may in some
cases interfere with enzymatic reactions involving meso-inositol,
there are apparently interrelationships other than these which are
involved.

Carter and his co-workers 104 found an interesting relationship between
streptomycin and lipositol, a phospholipide containing 16 per cent inositol
in a combined form. Soy bean lipositol, as well as preparations from
brain infusion, prevented the antibacterial action of streptomycin on
Eberthella typhosa and Staphylococcus aureus. Since there is present in
lipositol an inositol-galactose structure which bears some resemblance to
the streptomycin molecule, the authors suggested the possibility of a
metabolite-antimetabolite relationship. It has also been observed 105 that
lipositol is slightly active in replacing streptomycin for streptomycin-
requiring mutants of Escherichia coli.

716

THE BIOCHEMISTRY OF B VITAMINS

Iron Porphyrins (Hemes) As Growth Factors and Inhibitors
The same porphyrin nucleus which is present in heme, the nonprotein
component of hemoglobin, occurs in the prosthetic group of various im-

H3C-

HC-

-CH=CH2

-CH

H3C-
HOOCCH2CH2-

HC-

N Fe N

v v

HOOCCH2CH2—

=CH

-CH3

iron protoporphyrin IX
(heme or protoheme)

-CH3

-CH=CH2

portant enzymes, including catalase, peroxidase and the cytochromes.
Compounds whose structures are identical with or closely related to that
of iron protoporphyrin IX have been found to be present in all anima1
and plant cells that have been examined with the exception of some
anaerobic bacteria.

Among the organisms which have been found unable to synthesize
protoporphyrin IX, the precursor of heme, are bacteria (Hemophilus
influenzae), protozoa (certain trypanosomidae), and one insect species
(Triatoma infestans). Consequently, protoporphyrin IX is an essential
growth factor for these organisms. Although it has never been classified
as a B vitamin, its mode of action, its role in respiratory enzyme systems,
and its probable (but still unknown) relationships to some of the B vita-
mins may justify its inclusion here.

The role, mechanism of action and chemical structure of the iron por-
phyrins have been discussed in two excellent review papers by Granick
and Gilder no and by Lwoff .ni

It was discovered as early as 1892 that Hemophilus influenzae did not
grow unless a small amount of blood or hemoglobin was added to the
culture medium.112 It was shown by Davis113 and by Thjotta and
Avery 114 that this organism required two growth factors, a heat-stable
substance X which is found in hemoglobin and a heat-labile factor V
which is present in yeast and in fresh animal and vegetable tissues.

Lwoff and Lwoff 115 found that the V factor could be replaced by coen-

NUTRITIONAL FACTORS OF DOUBTFUL STATUS 717

zymes I or II. Olsen 116 showed that the growth-stimulating properties
of the X factor could be produced by heme (iron protoporphyrin), as well
as by hemoglobin, but that hematoporphyrin, hemocyanin, bilirubin,
chlorophyll, and pyrrole were inactive. He concluded that the growth-
promoting activity of heme was connected with its function in the per-
oxidase enzyme system. This conclusion was later shown to be erroneous
by Lwoff, who demonstrated that heme is also used for the synthesis of
cytochrome c m and that the growth-promoting activity and the per-
oxidase activity are not necessarily related.111

Granick and Gilder 118> 119- 12°- m have investigated thoroughly the
specificity of heme as the growth factor X for Hemophilus influenzae.
They found that protoporphyrin IX could replace heme in all cases; in
fact, as is shown in Table 50, the iron-free compound has even higher
activity in some instances than heme itself. Evidence was obtained that
protoporphyrin IX was converted into heme by these organisms, showing
that Hemophilus influenzae is capable of inserting iron into the proto-
porphyrin ring. It was also demonstrated that peroxidase and catalase
enzyme systems were formed from the protoporphyrin added to the heme-
free culture medium. The observation that the direct addition of heme
to the medium was often less effective than the addition of protoporphyrin
was attributed to the fact that heme is quite readily destroyed by even
traces of hydrogen peroxide. This view was supported by the fact that
substances which are able to destroy H202 enhance the growth of the
organism in the presence of the various iron porphyrins whereas they
do not affect the activity of the iron-free protoporphyrin.119 Neither
cytochrome c nor crystalline beef catalase replaced factor X in stimulat-
ing the growth of these organisms.

The iron-free porphyrins which do not contain vinyl groups, including
deutero-, hemato-, meso-, and coproporphyrins, do not replace proto-
porphyrin in promoting growth of Hemophilus influenzae. Mesoporphyrin
in small concentration did support growth of the "rough" Turner strain
but larger concentrations of the compound were inhibitory. However,
when these porphyrins lacking vinyl groups were converted into the
corresponding iron porphyrins and then supplied to the organisms, they
were found to support growth in seven of the ten strains tested (See
Table 50). These data suggest that the vinyl group is essential for the
insertion of iron into the porphyrin ring, but not for the growth-promoting
activity of the iron porphyrins. It appears that the nonvinyl-containing
iron porphyrins cannot carry out all the functions of iron protoporphyrin,
since the cultures grown on the former compounds do not possess the
ability to reduce nitrates to nitrites (Table 50). Also, maximum growth
is not always obtained in the presence of such iron porphyrins. Theorell

718

THE BIOCHEMISTRY OF B VITAMINS

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NUTRITIONAL FACTORS OF DOUBTFUL STATUS 719

and his co-workers 122 have reported that the protein moiety of horse-
radish peroxidase combines with protoheme, mesoheme, and deuteroheme
to form substances with 100, 53, and 63 per cent, respectively, of the
original enzyme activity.

Since heme is known to be a constituent of oxygen-activating enzyme
systems, it has been generally accepted that it was indispensable only
for aerobic life processes. It was thought that Hemophilus influenzae, a
facultative anaerobe, would not require heme when grown under anaerobic
conditions. This view was supported by experiments of Kopp 123 and
Eirund,124 but it was later shown by Gilder and Granick 119 that a small
amount of heme was required even for anaerobic growth. According to
Lwoff,111 heme possibly functions also in enzyme systems other than
those concerned with the activation of oxygen.

The nutritional requirements of some trypanosomidae were studied by
Lwoff and Lwoff.111- 115- 117- 125- 126- 127> 128- 129> 13° These parasites, which
live in the digestive tubes of certain flies, could be grown on artificial
medium only if it contained blood. Lwoff showed that blood can be
replaced by either heme or the iron-free protoporphyrin in its role of
stimulating the multiplication and respiration of Strigomonas fasciculata,
but that all the nonvinyl-containing porphyrins and hemes, as well as
cytochrome c, peroxidase and the "active iron" of Bandisch, were in-
active. Hence, Strigomonas fasciculata like Hemophilus influenzae is able
to insert iron into the protoporphyrin molecule.

The only insect which has been found to require heme for growth is
the assassin bug, Triatoma infestans.lzx' 132 The artificially fed larvae
of this insect require either blood or heme in their diet in order for
normal growth to occur.

In the course of their investigations Granick and Gilder 118 discovered
that when iron-free porphyrins were added to a medium containing either
protoporphyrin or iron protoporphyrin, the former substances inhibited
the growth of Hemophilus influenzae. The inhibition was of the competi-
tive type, the molecular ratio of the nonvinyl-containing porphyrin to
protoporphyrin at almost complete inhibition being nearly constant. The
molecular ratio of hemato-, deutero-, and coproporphyrin fco protopor-
phyrin for almost complete inhibition was approximately 10 to 1. A
similar competition was also observed between iron protoporphyrin and
other iron porphyrins. All these compounds support growth of the or-
ganism, but only iron protoporphyrin forms enzymes which reduce nitrate
to nitrite. When iron mesoporphyrin was added to a medium containing
a suboptimal concentration of heme, the growth of Hemophilus influenzae
was enhanced but its ability to reduce nitrates was decreased. These
observations led Granick and Gilder to conclude that a competition exists

720 THE BIOCHEMISTRY OF B VITAMINS

between the vinyl-containing and nonvinyl-containing porphyrins for
certain apoenzymes.

The free ionizable propionic acid side chains seem to be essential for
biological activity of the various porphyrins. When these propionic groups
were esterified, none of the compounds tested either supported or inhibited
the growth of Hemophilus influenzae.118 Apparently the free carboxyl
groups are necessary for the attachment of the iron porphyrins to the
basic groups of the apoenzyme.

High concentrations of heme inhibit the growth of some bacteria or
even cause lysis. Cultures of Bacillus subtilis disappear 24 hours after the
addition of blood to the medium and their growth is completely inhibited
by heme in a concentration of 1 : 125,000.133 C orynebacterium diphtheriae
is inhibited by even low concentrations of heme, especially under aerobic
conditions.134 Lwoff m attributes these effects to the inhibition in vivo .of
the succinic acid dehydrogenase enzyme system by heme, a phenomenon
observed in vitro by Keilen and Hartree.135 Since the biosynthesis of
heme occurs in these same bacteria, this may be considered an antibiotic
effect. Iron mesoporphyrin has also been reported to be an inhibitor for
many bacteria.136

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112. Pfeiffer, R., Deut. med. Wochschr., 18, 28 (1892).

113. Davis, D. J., J. Injections Diseases, 21, 392 (1917).

NUTRITIONAL FACTORS OF DOUBTFUL STATUS 723

114. Thjotta, T., and Avery, 0. T., J. Exptl. Med., 34, 97 (1921).

115. Lwoff, A., and Lwoff, M., Ann. inst. Pasteur, 59, 129 (1937).

116. Olsen, O., Centr. Bakt. Parasitenk. I Abt., 85, 12 (1920).

117. Lwoff, A., Compt. rend soc. biol, 122, 1041 (1936).

118. Granick, S., and Gilder, H., J. Gen. Physiol, 30, 1 (1946).

119. Gilder, H., and Granick, S., J. Gen. Physiol, 31, 103 (1947).

120. Granick, S., and Gilder, H., Science, 101, 540 (1945).

121. Granick, S., Ann. N. Y. Acad. Sci., 48, 657 (1947).

122. Theorell, H., Bergstrom, S., and Akeson, A., Arkiv. Kemi., Mineral. GeoL,

Ser. A16, No. 13, 1 (1943).

123. Kopp, H., Centr. Bakt. Parasitenk., I Abt. 105, 54 (1927).

124. Eirund, A., Centr. Bakt. Parasitenk, I Abt., Ill, 195 (1929).

125. Lwoff, M., Ann. inst. Pasteur, 51, 55 and 707 (1933).

126. Lwoff, A., Compt. rend. soc. biol, 113, 231 (1933).

127. Lwoff, A., Centr. Bakt. Parasitenk., I Abt., 130, 498 (1934).

128. Lwoff, A., Proc. Roy. Soc. (London), B124, 5 (1937).

129. Lwoff, M., Compt. rend. soc. biol, 121, 419 (1936).

130. Lwoff, M., "Recherches sur le pouvoir de synthese des Flagelles Trypanosom-

ides," Monographies del' Institut Pasteur, Paris, 1940.

131. Lwoff, M., and Nicolle, P., Compt. rend. soc. biol, 138, 164, 205, 341, (1944).

132. Lwoff, M., and Nicolle, P., Compt. rend. soc. biol, 139, 879 (1945).

133. Van Heyningen, W. E., Nature, 162, 114 (1948).

134. Glass, V., J. Path. Bad., 49, 548 (1939).

135. Keilin, D., and Hartree, E. F., Biochem. J., 41, 503 (1947).

136. Kammerer, H., Verhandl. deut. Ges. inn. Med., 31, 704 (1914).

INDEX

Absorption of B vitamins,

factors influencing, 344

in animals, 343-345

in plants, 336-337
Abundance of B vitamins, in whole or-
ganisms, 24
Acetaldehyde, formation of, 221
Acetate factor, 16 ff
Acetic acid,

aerobic production of, 164

as metabolic intermediate, 190
Acetic acid-lactic acid dismutation, 164

hydrogen carriers for, 164
Acetoin, from pyruvate, 161
Acetylcholine, 39

formation in nerve tissue, 106

role in production of electrical energy,
237
Acetylmethylcarbinol, formation of, 221
Acetylphosphorylase, 191
Achlorhydria, 266
Acid phosphates, formation of, 195
Acid labile forms of pyridoxine, 37
cis-Aconitate, formation of, 195
Activated acetyl molecule, 190
"Active acetate," 190
Active forms of B vitamins (see coen-
zymes of individual vitamins)
Addisonian pernicious anemia, 415

therapy, 415
Addison's disease, pigmentation in, 425
Adenine, toxicity of, 296
Adenosine diphosphate,

as phosphorylating agent, 191

from DPN, 134
Adenosine triphosphate,

in biosynthesis of thiamine, 157

in muscular contraction, 236
Adenylic acid, role in coenzyme forma-
tion, 117
Adermin, 652

Adrenal cortical relationships, 381
Adrenal cortex, effect of pantothenate

deficiency on, 380, 424
Adrenal hypertrophy, 382
Adrenalectomy, effects of, 425
Adrenaline, from DOPA, 181
Aerobic respiration, energy conservation

in, 150
Age, effect on vitamin requirements, 265-

266
Aged, vitamin requirements of, 266
Agenizing of flour, 297

Alanine, relation to bacterial pyridoxine

requirement, 184
/3-Alanine, 35

from aspartic acid, 85, 181

relation to pantothenic acid, 466

role in biosynthesis of pantothenic
acid, 85
Alcohol, effect on B vitamin require-
ments, 276
Alcoholism, chronic,

as genetotrophic disease, 433

with nutritional polyneuritis, 400
Aldehyde oxidases, 148
Aldol cleavage, in glycolysis, 219
Aldolase, 188
Alkaloids, in plants, 336
Alloxazine, 143
Amides, formation of, 195
Amines, detoxification of, 106
Amino acid decarboxylation, pyridoxal

phosphate in, 181
a-Amino acids,

coenzyme activating, 174

effect on choline requirement, 278

effect on riboflavin requirement, 277

folic acid in synthesis of, 201

PABA in synthesis of, 201

relation to bacterial pyridoxine re-
quirements, 184

requirements for plants of, 316-317

role of a-ketoglutarate in synthesis of,
232

synthesis of aromatic, 195

vitamins required for degradation of,
233

vitamins required for synthesis of, 231
D-Amino acid oxidase, 147
L-Amino acid oxidase, 32, 147, 148
;>Aminobenzoic acid

analogues, 291, 484-486

anti-pernicious anemia vitamin in uti-
lization of, 203, 207

assay methods, 71-72

biochemical function, 11-12, 469, 488

biochemical interrelationships, 470

biosynthesis, 89

combined forms, 40-41

deficiency, 429-430

distribution, 18-19

effect of species size on requirement
of, 323

excretion of, 365-370

725

726

INDEX

p-Aminobenzoic acid — Continued

glutamic acid in metabolism of, 487-
489

hydrolytic conditions, 41

inhibitory analogues, 493-500, 521-530

isosteres, 527-528

metabolic relations to purines, pyri-
midines, 198

metabolism of, 363-364

relation to folic acid, 202, 472, 490-493

relation to nicotinic acid, 282

relation to pantothenic acid, 282

relation to thymidine, 474

requirements, 326, 327

role in amino acid synthesis, 201

role in carbon to carbon bond forma-
tion, 189

role in purine and pyrimidine syn-
thesis, 201

role in single carbon unit metabolism,
472

specificity, 482-483

structure, 11

sulfonamide antagonism to, 199

sulfonamides in inhibition analysis of
function, 470-471

toxicity, 391
p-Aminobenzoylglutamyl peptide, 490
7-Aminobutyric acid, formation of, 181
Amino sugars, 223
a-Amylase,

dissociation of, 125

inositol in, 10, 38, 106, 125

occurrence, 126
Analogues of B vitamins, 443, 456-460
(See also individual vitamins)

effect on coenzyme formation, 461

pharmacological properties, 391
Androgens,

inactivation of, 382

role in nitrogen, phosphorus and potas-
sium retention, 381-382
Anemia, pernicious, 204, 415-417

excretion of folic acid in, 571

thymine in therapy of, 414
Anemias,

associated with folic acid, vitamin Bi2
deficiency, 413

due to cobalt deficiency, 206

"goat's milk," 419

megaloblastic macrocytic, 419

nutritional macrocytic, 419

pregnancy macrocytic, 419

tapeworm in macrocytic, 419-420

"temporary pernicious," 419
Aneurin, 6

Aneurin pyrophosphate, 164
Animal-protein factor, 14
Animals,

absorption of B vitamins in, 343-345

catabolism of B vitamins in, 351-370

digestion in, 338-343

Animals — Continued

germ-free, 300

synthesis of B vitamins in, 351-356
Annelida, B vitamin requirements of, 309
Anoxia,

coenzyme breakdown in, 272

effect on B vitamin requirements, 272
Antagonists, biochemical genetics in

competitive, 477-478
Anthranilic acid, 287
Antibiotics, use in intestinal studies, 298
"Anti-dermatitis factor," 422
Anti-malarials, relation to riboflavin co-
enzymes, 679-681
Antipernicious anemia vitamin,

absorption spectra, 206

activity of nucleosides, 205

activity of thymidine, 205

as folic acid analogue, 290

characterization of active compounds,
205

coenzymatic role of, 112

coenzymes, 206

description, 14

dosage, 14

function, 14, 207, 474-475

inhibition studies, 207

interrelation to ascorbic acid, 204

interrelation to folic acid, 206-207

reactions catalyzed by, 206

role in carbon to carbon bond forma-
tion, 189

role in formation of serine and methi-
onine, 207

role in utilization of PABA, 203, 207
Antipernicious anemia vitamin, defi-
ciency of,

anemias associated with, 413

pernicious anemia, 415-416

sprue, 417-418
Anti-thiamine effects, 295
"Anti-thiamine enzyme," in raw fish, 155
"Anti-vitamins," use of, 260-261
Apocarboxylase, 157
Apoenzymes, 31, 109

differential affinity for nicotinic acid
coenzymes, 118
Apoerythein, 415
Apophosphorylases, 134
APP, 154
Araboflavin, 296
Arecoline, 336, 389
Arginine, formation of, 181
Arthropoda, B vitamin requirements of,

309
Ascorbic acid,

distribution, 21-22

effect on riboflavin storage, 258

from carbohydrates, 223

interrelation to carotenoids, 22

interrelation to vitamin Bi2, 204

oxidation-reduction potential effect, 21

INDEX

727

Aspartic acid,

/3-alanine from, 85, 181

function of biotin in synthesis of, 172

interrelation to pantothenic acid, 466
Aspartic-alanine system, 182
Assay methods, (See also individual vita-
mins)

biological, advantages of, 46

chemical and physical-chemical, advan-
tages of, 45

microbiological, advantages of, 46

purposes, 45
Assays of urinary B vitamins, in humans,

252, 254, 283
Atebrin, relation to riboflavin coenzymes,

679-681
Auramine, 680

Autoclaving, effect on pyridoxine, 176
Auximers, 316

Auxin activity of vitamins, 316
Availability of B vitamins, 46, 283
Avidin, 12, 37, 559-561

as egg white constituent, 294

production in oviduct, 383

properties of, 560

Bacteria,

B vitamin requirements of, 307

intestinal, effect on B vitamin require-
ments, 297

synthesis of B vitamins by, 299-300
Beriberi,

analysis of diets, in 250-251

historical, 398

incidence, 398, 399

infant, 399

symptomology, 400-401
Betaine, 10

Biochemical genetics, competitive antag-
onists and, 477-478
Biochemical reactions, 95 et seq.

types of, 103 et seq.
Biocytin, 542 ff
Biological effects of B vitamins, 377 et

seq.
Biological materials, B vitamin levels in,

257-259
Biological oxidations and reductions (See
dehydrogenations)

coenzymes mediating, 128

mechanisms, 128
Biological potency of B vitamins, 283
Biological systems, energy transforma-
tions in, 100
Bioluminescence, 237-238
"Bios I," 710

Biosynthesis of coenzymes, 112
Biotin,

analogues, 289, 553-559

as essential cell constituent, 174

assay methods, 61-64

bacterial production of, 87-88

Biotin — Continued

biochemical interrelationships, 467-468

biosynthesis, 87-88

coenzymes, 173

combined forms, 37

distribution, 23-24

effect of potassium on deficiency, 249

effect of species size on requirements
of, 321

effect on pantothenate requirements,
278

effect on root production, 88

excretion, 87, 365-370

extraction, 37

functions, 170, 172

hormone activity of, 337

in deaminations, 172-173

in synthesis of aspartic acid, 172

in synthesis of oleic acid, 173

inhibitory analogues, 553-559

requirements, 326, 327, 329

role in ^-decarboxylations, 154, 171

sparing action of oleic acid on, 227

specificity, 542-545

stereoisomers, 542-544

stimulatory analogues, 545-553

structure, 8

toxicity, 391

"uncombinable," 62
Biotin deficiency,

biochemical aspects of, 429

in dogs, 249

symptomology, 428
a-Biotin, 173
^-Biotin, 173
Blacktongue, 412
Blood,

B vitamin levels in, 345-347

nicotinic acid content of, 258
Bound forms of B vitamins, 284
Bradycardia tests, 384
2,3-Butylene glycol, 222

Cadaverine, formation of, 181
Caffeine, toxicity of, 297
Caloric intake, effect on B vitamin re-
quirements, 276
Cancer tissue, (See also tumors)

B vitamin content, 27

vitamin uniformity in, 27

water content, 27
Carbohydrates,

effect on B vitamin requirements, 276

related compounds from, 223

synthesis of fatty acids from, 222

utilization of, 217 et seq.
Carbon to carbon bond formation, 106,
181 et seq.

role of PABA in, 189

role of thiamine in, 189

role of vitamin Bi2 in, 189

728

INDEX

Carbonyl-phosphoric acid addition prod-
uct, 219
Carboxylase, 30, 154
Carboxylation of keto acids, coenzymes

for, 153
Carotenoids, 22
Carriers, 110
Catabolism, of B vitamins in animals,

351-370
Catalase, 153
"Catalins," 5
Catatorulin effect, 403
Cecectomized rats, 285
Cecectomy, in study of intestinal vita-
min synthesis, 299
Cell aggregates, 243
Chastek paralysis, 292, 402

of foxes, 292, 400
Chemical energy, conservation of, 101
Chemosynthesis, 238
Chicks,

B vitamin requirements of, 327

DPN content of embryos, 352

strain differences, 265
Children, B vitamin requirements of, 266
Chloroamino acids, 297
Cholamine (see ethanolamine)
Cholic acid, relation to thiamine, 282
Choline, 10

analogues, 290, 704-705

as source of "formate," 234

assay methods, 66 et seq.

biological activity, 706-708

biosynthesis, 88-89, 234-235, 353

combined forms, 39

deficiency, 430-431, 703

distribution, 18

effect of amino acids on requirements
of, 278

effect of species size on requirements
of, 322

essential, 278-279

ethanolamine as precursor, 89

excretion, 365-370

extraction, 39

functions, 430

inhibitory analogues, 708-710

in phospholipids, 11, 39

methyl groups from, 10-11

pharmacological action, 389-390

relation to ethanolamine, 89

requirements, 326, 327, 328, 329

specificity, 706-708

structure, 10

toxicity, 390
Choline acetjdase, 237
Choline esterase, increase of associated

with folic acid, 204
CI, 404

Citrovorum factor, 16 ff
Claisen-type condensation, 188

Climate, effect on B vitamin require-
ments, 267
Cobalt deficiency, anemia in cattle from,

206
Cocarboxylase, 154
Codecarboxylase, 177
"Codehydrogenase," 133
Codehydrogenase I, 133
Coefficient of uniformity, 27
Coelenterata, nutrition of, 307
Coenzyme A,

assay methods, 192

biosynthesis, 194

in acetylation of choline, 237

occurrence, 193-194

reactions catalyzed by, 194 et seq.

structural studies on, 192
Coenzyme I, 33, 133, 286, 604

inhibitions involving, 616-617
Coenzyme II, 33, 132, 286
Coenzyme of Lipmann, 35
Coenzymes,

analytical methods for, 119 et seq.

biosynthesis of, 112

classification of, 109

from B vitamins, 110-111

meaning of, 108

"mobile," 137

number of, 114

occurrence of, 120 et seq.

role of adenylic acid in formation of,
114

separation of, 109

specificity of, 115, 119
Coenzyme synthesis, 343-344, 352-353

extent of, 113

rate of, 113
Coenzvmatic activity of simple vitamins,

111
Cofactors, 108
Coffee,

toxicity of, 297

trigonellin in, 288
"Coferment," 133

Coferment of alcoholic fermentation, 133
Cold, effect on B vitamin requirements,

268
Colostrum, B vitamins in, 347
Combined forms of B vitamins, (See
also individual vitamins) 30 et seq.
Competitive inhibition, 295, 443
"Concentrates" of B vitamins, meaning,

13
Condensations, carbon to carbon bonds,

187 et seq. 195
Conservation of chemical energy, 101
Controlled diets, 247 et seq.
Cooking,

effect on B vitamins, 339

effect on riboflavin, 33
"Cophosphorylase" activity, 135
Coramine, 287

INDEX

729

"Coreductase," 133

Corn, pellagragenic agent in, 280, 292
Cotransaminase, 177
"Cotryptophanase," 177
Cow-manure factor, 14, 421
"Cozymase," 132, 133

in sex hormone inactivation, 382
Crustacea, B vitamin requirements of,

309
Customs, effect on B vitamin require-
ments, 282
Cuttings, B vitamin requirements of

plant, 316
Cysteic acid, taurine from, 181
Cystic mastitis, B vitamin treatment of,

382
Cytochrome-C, 152

reduction mechanism, 152-153

reoxidation, 153
Cytochrome-C reductase, 32

oxidation of reduced TPN by, 150
Cytochromes (see also porphyrins),

absorption spectra, 151

Daily dietary allowances of B vitamins

for humans, table of, 324
"Dark reactions," 238
Deamination, biotin as catalyst for, 172-

173
Decarboxylation,

of amino acids, 181

of keto acids, enzymes for, 153 et seq.
/3-decarboxylation, biotin in, 154, 171
Decarboxylase processes, 153, et seq.
Deficiency states of B vitamins, 395 et
seq. (See also individual vitamins)

beriberi, 398-406

compound, 396

criteria, 248

in higher animals, 398 et seq.

in insects, 312-313

in lower forms of life, 397-398

in primitive tribes, 296

in young ruminants, 298

muscular activity in, 399

pathology from, 395

pellagra, 398, 408-412

subclinical, 396-397

symptomology, 248 et seq., 396
Definition of B vitamins, 5
Dehydration-hydration, in glycolysis, 219
Dehydrogenation,

enzymes in, 129

in glycolysis, 219
Deprivation of B vitamins, effect on in-
sects, 312
Dermal excretion of B vitamins, 368-369
Desthiobiotin, 468, 543, 550-552
Desoxycorticosterone, relation to panto-
thenic acid, 381
Desoxypentoses,

from carbohydrates, 223

Desoxypentoses — Continued

mechanism of formation, 224
Desoxyribosides, Vitamin Bi« activity of,

205
Destruction of B vitamins in intestine,

337-338
Detoxification of amines, 106
Diacetyl, dismutation of, 166
Diaphorases, 149
Dietary surveys, 252-254
Diets,

controlled, 247 et seq.

special, use of in study of intestinal
syntheses, 298

vitamin B content of mixed, 254
Digestion,

in animals, 338-343

in plants, 336-338
Dihydroluciferin, 238
3,4-dihydroxyphenylalanine, 181
3,4-dihydroxyphenylethylamine, forma-
tion of, 181
Dimethylethanolamine, 290
Dinicotinyl ornithine, 287, 357
Diphosphopyridine nucleotide,

structure of, 132

as hydrogen acceptor in glycolysis, 219
Diphosphothiamine, 154
Disease, effect on B vitamin require-
ments, 273
Dismutation,

acetic acid-lactic acid, 164

diacetyl, 166
Distribution of B vitamins, 18 et seq.,
345 et seq. (See also individual vita-
mins)

in body fluids, 350-351

in body tissues, 350-351

in circulating blood, 345-347

in human tissues, 26

in milks, 347-350

in tumors, 27

quantitative relationships, 23

significance of, 24-25

uniformity of, 25
Dogfood, vitamin B content of, 254
Domestic birds, Vitamin B requirements

of, 328
DOPA, 181
DOPA decarboxylase, 182

relation of coenzyme of, to folic acid,
204
DPN, 133

oxidation of reduced form by dia-
phorases, 149

ratio of reduced to oxidized form in
malignancy, 138
DPT, 154

Dropsy, epidemic, 400
Dulcitoflavin, 296

730

INDEX

Echinodermata, B vitamin requirements

of, 309
Effects of B vitamins, 377 et seq. (See
also specific vitamins)

pharmacological, 385-391

toxicological, 385-391
Eggs,

experiments with, 246

riboflavin content of, 351
Egg white diets, effect on insects, 314
Embryonic development, B vitamins

and, 383
Endocrine function, effect of B vitamins

on, 380
Endocrine glands, metabolic activity of,

380-384
Energy,

acetylcholine metabolism in produc-
tion of, 237

chemical, 235

conservation of chemical, 101

conservation of, in aerobic respiration,
150

electrical, 236-237

from pyrophosphate bonds, 163

mechanical, 236

radiant, 237-238

thermal, 236
Energy transformations,

in biological systems, 100, 235 et seq.

nicotinic acid coenzyme in, 140
Environment, effect on B vitamin re-
quirement, 264
Enzymatic action, inhibition of, 445
Enzymatic reactions, 99 et seq.

general processes of, 100

inactivation of B vitamins, 292

outline of types, 104-105

requirements for characterization, 100

vitamins required, 104-105
Enzyme activators, 110
Enzyme systems, components of, 108
"Enzyme-substrate union," 116
Enzymes, 108

changes in total effect of concentration
of, 462

coenzymes, 108

cofactors, 108

environmental conditions, 108

hydrolytic, 125

"poisons," 443
Ergot alkaloids, 336
Erythein, 14-15, 415
Erythropoiesis,

stimulation of, 203

folic acid and, 383
Erythro-, 475
Erythrotide, 475
Erythrotin, 14-15, 415, 475
Essential choline, 278-279
Esters, formation of, 195
a-Estradiol, detoxification of, 382

Estrogens,

inactivation by liver, 382

effect of B vitamins on inactivation,
382
Estrone, detoxification of, 382
Estrus cycle, B vitamins and, 383
Ethanol, formation of, 222
Ethanolamine, 10, 181, 290
Excretion of B vitamins

dermal, 368-369

fecal levels, 366-369

individual variations, 369-370

in plants, 337-338

studies on, 254-257

urinary levels, 364-368
Excretion of pyramine, 255
Extraction of B vitamins, 30 et seq.
"Extrinsic" factors, 206
Exuviation, B vitamin requirements in,
311

Factors, unidentified, 244
"Factor V," 133
FAD, 143

Fasting urine specimens, 255
Fat metabolism, relation of vitamin B8
to, 185-186

vitamin requirements for, 229
Fats,

effect on B vitamin requirements, 276

formation of, 195, 228

hydrolysis of, 228
Fat soluble compounds, 4
Fat soluble vitamins, relation to repro-
duction, 381
Fatty acid dehydrogenase, 152
Fatty acids,

metabolism of, 225 et seq.

role of Coenzyme A in formation, 195

role of phosphoryl-acetyl compound in
metabolism, 225

sparing effect on vitamins, 227

synthesis from carbohydrates, 222

unsaturated, formation of, 227

vitamins required in metabolism, 225
Fatty liver, prevention of, 127
Feces,

B vitamin excretion in, 256, 257, 300,
344, 366-367, 368-369

pantothenate content, 259

daily excretion of B vitamins in, 366-
367
Fermentation L. casei factor, 39
"Fern poisoning," 295
Fertilizers, effect of on B vitamins in

crops, 80
Fever, effect on B vitamin requirements,

273
"Filtrate factor," 54
"Fitness," 245

Flavinadeninedinucleotide, 142, 143
Flavoproteins, 7, 32, 141

INDEX

731

Flavoproteins — Continued
L-amino acid oxidase, 32
comparison of reactions catalyzed by
to those of nicotinic acid system, 145
cytochrome-C reductase, 32

diaphorases, 149

in production of ethylenic bonds, 151

intracellular forms, 147

"old yellow enzyme," 32

properties of, 145

reaction types catalyzed by, 146

redox properties of, 145

types of, 147

union with apoenzyme, 145
Flour, agenizing of, 297
Fluids, distribution and storage of B

vitamins in, 350-351
Fluorescyanine, 285-286
Folic acid,

p-aminobenzoic acid in activity of
xanthopterin, 421

analogues, 290

assay methods, 68-71

biological activity of, 568-574

biosynthesis, 88

choline esterase, increase associated
with, 204

combined forms, 39-40

defective metabolism of tyrosine and,
204

distribution, 40

effect of related compounds on cancer,
593-597

effect of species size on requirements
of, 322

excretion, 365

formyl derivative, 39

functions, 488

hematopoieses from, 202

inhibitory analogues, 575-593

in single carbon unit metabolism, 200

isolation, 565

liberation by enzymes, 40

metabolism, 363

oviduct response to stilbestrol and, 383

pyridoxal-like activity of, 204-205

relation to PABA, 202, 472, 490-493

relation to pernicious anemia, 204

relation to vitamin Bi2, 206

relation to thymidine, 424

requirements, 326, 327, 328, 329

role in amino acid synthesis, 201

role in erythropoiesis, 383

role in purine and pyrimidine syn-
thesis, 202

specificity, 566-568

structure, 9, 493

therapeutic use in pernicious anemia,
416

toxicity, 390-391
Folic acid coenzymes, biosynthesis, 203
Folic acid deficiency, 413-422

Folic acid deficiency — Continued

anemias associated with, 413

in vertebrates, 420

in chicks, 248

pernicious anemia and, 415, 416

sprue, 417
Folinic acid, 16 ff, 203 ff
Foods, processing of, effect on B vitamin

content, 282
"Formate carrying," coenzyme, 197
Formic acid,

formation of, 197

incorporation into purines, 196

production from pyruvate, 162
Formic acid dehydrogenase, 152
Formylfolic acid, 39

structure, 200
"Formyl group," 197
Formylpteroic acid, structure, 200, 422
Fowls, B vitamin requirements of, 259
Foxes,

thiamin deficiency in, 292, 400

Chastek paralysis, 292, 400
Free energy, prediction of reactions from,

101
Fumaric acid,

conversion to succinic acid, 151, 221

formation, 221
Fumaric dehydrogenase, 150
Fumaric reductase, 221
Function of B vitamins in nutrition, 5
Function of B vitamins in metabolism,

216 et seq.
Functional forms of B vitamins, 284

Galvanic cell, action of, 129
Gametogenesis, B vitamins in, 383
Genetic blocks, partial, 217
Genetotrophic diseases, 433
Geriatrics, 266

Germ-free animals, studies with, 300
Germination, B vitamin requirements

for, 316
Glucose oxidase, 148
Glucose-6-phosphate, oxidation of, 136
Glutamic acid,

as amino donor, 176

in metabolism of PABA, 487-489
Glutamic-alanine system, 182
Glutamic-aspartic system, 182
Glutamic-aspartic transaminase, 187
Glutamic-cysteic acid system, 183
Glutathione, in function of glyoxalase,

110
Glycerol, formation and utilization, 227-

228
D-Glycerophosphate dehydrogenase, 152
Glycine, conversion to serine, 233
Glycine oxidase, 148
Glycogen, phosphorolysis of, 218-219
Glycolytic process,

energy considerations, 218

732

INDEX

Glycolytic process — Continued

mechanism, 218
Grave's disease, thiamine administration

in, 381
Gray hair syndrome, 381
Green plants,

as food source, 244

B vitamin deficiencies in, 397

B vitamin requirements of, 316-318
Growth,

as criterion for vitamin sufficiency, 250

effect of B vitamins on, 379

"improvement" in, 250

"increase" in, 250
Guvacine, 336
Gynecomastia, in malnutrition, 382

Habits, effect on B vitamin requirements,

282
Harden's coferment, 133
Heart action, effect of thiamine on, 384
Heat lability as applied to vitamins, 37
Heavy metal poisons, 153
Hematopoiesis, 202
Hemes, 716-720 (See porphyrins)
Hepatic fatty infiltration, 248
Heteroauxin, 294

Hexonic acid, from carbohydrates, 223
Hexuronic acid, from carbohydrates, 223
High energy phosphate bonds, 101, 163
Higher animals, B vitamin deficiencies

in, 398 et seq.
Histamine, formation of, 181
Histidine,
as single carbon unit donor, 198
histamine from, 181
Holoenzymes, 30, 109
formation of, 116
stability of, 117
Homobiotin, 468
Homocysteine, conversion to methionine,

233, 234
Hormone-vitamin interrelationships, 380-

384
Human experimentation,
bioassay methods for vitamins in, 252,

254-257, 283
variables in, 249
Human tissue, distribution of B vitamins

in, 26
Humans,
B vitamin requirements of, 326
table of daily dietary allowances for,
324
Humidity, effect on B vitamin require-
ments, 267
Hungers, specific, 260
Hydrases, 127 et seq.
Hydrogen, production of from pyruvate,

163
Hydrogen acceptor, DPN as, 219

Hydrogen carriers, for acetate-lactate
dismutation, 164

Hydrogenation-dehydrogenation, in gly-
colysis, 219

/3-hydroxy acids, dehydration of, 128

L-Hydroxyacid oxidase, 148

3-Hydroxyanthranilic acid, 287

Hyperthyroidism,
symptomology of, 380
B vitamins in treatment of, 381

Hypophysis, 380

Hypoxanthine, formation of, 199

Illness, effect on B vitamin requirements,

271
Inactivation of B vitamins,

enzymatic, 292

in intestine, 337-338
Inactivators, natural, 292
Index of Carbohydrate Metabolism, 404
Indole, tryptophan from, 183
"Infantile pellagra," 407
Inhibition,

competitive, 443

determination of type, 452-453

index, 445-449

mass action effect in, 444

non-competitive, 443

of enzymatic action, 445

"quadratic," 454

reaction rates in, 450-451

synergistic action of, 463
Inhibition analysis,

application of, 464

in assay development for unknown
factors, 473
Inhibitors,

destruction of, 463

mechanism of resistance to competi-
tive, 475-477

natural, 292
Injury, effect on B vitamin requirements,

272
Inositol,

analogues, 291

assay methods, 64-66

biological activity, 712-714

biosynthesis, 89

combined forms, 38

deficiency, 430

distribution, 18

effect of species size on requirement of,
323

excretion of, 365

extraction, 38-39

formation from carbohydrates, 223

in amylase, 10, 38, 106, 125, 218

in phospholipides, 38, 106, 127

inhibition studies on, 125

inhibitory analogues, 714-715

requirements, 326, 327, 328

specificity, 712-714

INDEX

733

Inosital — -Continued

stereoisomers, 711-712

structure, 10, 711-712

therapeutic use, 430

toxicity, 391
Insects,

B vitamin requirements of, 309-315

effect of egg-white diets on, 314

pyruvism in, 313

symbiosis in, 309

vitamin metabolism in, 314-315
Intake of B vitamins, recommended, 245
Intermediates, transport of labile, 121
"Intermediate carrier," 146
Interrelationships of B vitamins, 281-282,
379-380

thiamine-vitamin A, 282
Intestinal flora, 264

effect on B vitamin requirements, 297
Intestinal synthesis of B vitamins, 298
"Intrinsic factors," 206
Invertebrates, B vitamin requirements

of, 306-315
Iodoacetate inhibition of thiamine phos-
phorylations, 155
Isoalloxazine, 143

Isoalloxazine adenine dinucleotide, 143
Isomerases, 127 et seq.
Isomerization, in glycolysis, 219
Isotels, 173
Isotopic labeling, 102
Isoxanthopterin, 285

Jackbean mean, crystalline urease from,
96

Ketenyl radical, reactions of, 160

a-Ketoglutaric acid,
as amino acceptor, 176
as source of single carbon unit, 197
decarboxylation by thiamine coen-
zyme, 158
reactions catalyzed by TPP, 167
role in synthesis of amino acids, 232
role of coenzyme in synthesis, 195

Korsakoff's syndrome, 401

Kwashiorkor, 407

Kynurenin, 287
in synthesis of nicotinic acid, 83

Labile intermediates, transport of, 121
Labor, effect on B vitamin requirements,

267
Labor pains, thiamine in treatment of,

356
Lactation,

B vitamins and, 383

effect on B vitamin requirements, 269
"Lactation factors," 383
Lactic acid dehydrogenase, 152
Lactobacillus bulgaricus factor, 16 ff
Lactochrome, 669

Lactoflavin, 669

Larvae, B vitamin requirements of, 311

Lecithins, 39

Leukemia, effect of folic acid analogues,
596

Liberation of B vitamins, in intestinal
tract, 338

"Light reaction," 238

Linseed meal, anti-pyridoxine effect of,
295

Lipides, metabolism of, 225, et seq.

"Lipocaic," 127

Lipositol, 38, 430

Liver, inactivation of sex hormones by,
382

Loading test, 255

Lower forms of life, B vitamin deficien-
cies in, 397

Luciferase, 238

Luciferin, 238

Lumazine, 285

■Lumichrome, 363

Lycomarasmine, 260, 296

Lysine, relation to bacterial vitamin Ba
requirements, 184

Macrocytic anemias, 419-420
Magnesium ions, as cofactors for thi-
amine coenzyme, 158
Malic acid, formation of, 221
Malignant tissues, ratio of oxidized to

reduced form of DPN in, 138
Mammals, B vitamin requirements of,

329
Manioc, 296
Manganese ions,

as cofactors for thiamine coenzyme,
158

as cofactors in biotin enzymes, 171
Mass action effect in inhibition, 444
Maternal instinct, B vitamins and, 383
Mating behavior, B vitamins and, 383
Melanin production, 381
Menorrhagia, B vitamin treatment of,

382
Mental activity,

effect of B vitamins on, 385

effect on B vitamin requirements, 267
Mental response, effect of thiamine on,

385
Metabolic interrelationships of B vita-
mins, 379-380
Metabolic products,

of nicotinic acid, 356-361

of other B vitamins, 361-364
Metabolic rate, 264, 379

effect of B vitamins on, 379

effect on B vitamin requirements, 273
Metabolism, "total," 379
Metamorphosis, nutrition in, 309
Methionine, 10

essential level, 279

734

INDEX

Methionine — Continued

from homocysteine, 233, 234

methyl groups from, 10, 234

role of vitamin Bi» in formation, 207
Methyl groups,

from choline, 10-11

from methionine, 234
N'-methyl nicotinamide, 357
N'- methyl - 6 - py ridone -3- carboxy lamide,

357
Metorrhagia, B vitamin treatment of,

382
Mice, B vitamin requirements of, 327
Microbiological tests in discovery of B

vitamins, 15
Milks,

aldehyde oxidases in, 148

B vitamins in, 20, 258, 347-350

diaphorases from, 149

pantothenate in, 85

riboflavin in, 82, 271

thiamine in, 271

xanthine oxidase in, 148
"Mobile coenzymes," 137
Modification of B vitamins in intestinal

tract, 337-338
Molds, riboflavin excretion in, 338
Monomethylethanolamine, 290
"Mouse anti-alopecia factor," 712
Muscle,

diaphorases from, 149

DPN from, 135
Muscular activity in B vitamin defi-
ciency, 399
Mycorrhizial fungi, 80-81
Myelin sheath, 384

National Research Council, table of rec-
ommended dietary allowances for
humans, 324
Natural selection studies, 259-260
Nerve function, effect of B vitamins on,

384
Nerve metabolism, role of vitamins in,

384
Neuron, 384
Neurospora, 10
Niacin (see nicotinic acid)
Nicotinamide (see nicotinic acid)
Nicotine, 336, 389

Nicotinic acid, (see also nicotinic acid-
type compounds)
activity of glutamic acid and aspara-

gine, 84
analogues, 286
assay methods, 54-56
biological activity of, 604-611
biosynthesis, 82-83, 330, 353-356
combined forms, 33
content in blood, 259
distribution, 18

Nicotinic acid — Continued

effect of species size on requirement of,
320

effect of tryptophan on requirement
of, 279

excretion, 365

extraction, 34

inhibitory analogues, 280, 611-617

interrelation to PABA, 282

interrelation to pantothenic acid, 282

lethal dosage, 389

liberation by enzymes, 34

natural antagonisms related to, 615-616

pharmacological action, 387-388

products of, 356-361

replaceability by tryptophan, 279

requirements, 317, 324, 326, 327, 328,
329

specificity, 604, 611

stimulatory action on plant roots, 82-
83

structure, 7

therapeutic use, 411

toxicity, 387-388
Nicotinic acid coenzymes, (see also
DPN, TPN)

absorption spectra, 134

assay methods, 135

biosynthesis, 138

extraction, 137

inactivation of, 134
Nicotinic acid deficiency, 408-412

biochemical changes in, 412

blacktongue, 412

para-sprue, 410

pellagra, 408-412

symptomology, 409
Nicotinic acid type compounds,

pharmacological action of, 388

occurrence, 137

oxidized and reduced forms, 133-134

reactions catalyzed by, 139 et seq.

redox systems coupled with, 140

role in energy transformations, 140

sources, 135

specificity, 139
Nicotinuric acid, 287, 357
Nitrogen compounds, metabolism of, 230

et seq.
Nitrogen retention, effect of androgens

on, 381-382
Nitroid reaction, 387
"Norite-eluate factor," 69
Nucleosides, vitamin Bi2 activity of, 205
Nucleotides, 7

Nutrition, effect of sulfonamides on, 512
Nutritional customs, effect on B vitamin

requirements, 202
Nutritional function of B vitamins, 5
"Nutritional requirement," 244
Nutritional status, 249

INDEX

735

Nutritional viewpoint of meaning of B

vitamins, 13
Nutritional polyneuritis, 400
with chronic alcoholism, 400

Occupation, effect on B vitamin require-
ments, 267

Occurrence of B vitamins, reason for
universal, 98

"Old yellow enzyme," 32, 144, 150

Oleic acid,
biotin in synthesis of, 173
sparing action on biotin, 227

Ornithine, putrescine from, 181

Oviduct hypertrophy, effect of stilbestrol
on, 383

Ovoflavin, 669

Oxalacetic acid, formation of, 221

Oxidases, amino acid, 147-148

Oxidation-reduction potential, effect of
ascorbic acid, 21

Oxybiotin, 8, 545-550

PAC, 192-193

Pancreatic amylase, 38, 218

Pantoic acid, 35

from pantonine, 85
"Pantonine," 85
Pantothen-, 20
Pantothenic acid,
^ analogues, 289
/ assay methods, 56-59

biological activity, 620-624

biosynthesis, 84-86

coenzyme (see coenzyme A)

combined forms, 34

conjugate, 192-193
„ distribution, 19-20

effect of biotin on requirement of, 278

effect of species size on requirement of,
321

excretion, 365

extraction, 34

fecal content of, 259

functions, 465-466

inhibition of, 464

inhibitory analogues, 624-648

interrelation to /3-alanine, 466

interrelation to aspartic acid, 466

interrelation to nicotinic acid, 282

interrelation to PABA, 282

interrelation to desoxycorticosterone,
melanin, 381
, liberation by enzymes, 34-35

metabolism, 362-363

oviduct response to stilbestrol and, 383

relation to plant growth, 86

role in sterol synthesis, 230

requirements, 326, 327, 328, 329

sparing effect of fatty acids on, 227

specificity, 620-624
., structure, 7

Pantothenic acid — Continued

toxicity, 391
v universal occurrence, 19

urinary content of, 259

Pantothenic acid deficiency,

adrenal hypertrophy in, 424

biochemical nature of, 424

symptomology, 423
Partial genetic block, 217
Pasteur reaction, 32
Pathological states,

effect on B vitamin requirements, 271

relation of B vitamins to, 431-433
Pellagra,

causation, 409

distribution, 408

effect of tryptophan in, 615

historical, 398

in pigs, 412

incidence, 398

mental symptoms, 410

pathology, 410-411

role of sunlight in, 409

symptomology, 409
Pellagragenic agent, in corn, 280, 294
"Pellagra-preventive factor," 407
Pentoses,

from carbohydrates, 223

mechanism of formation, 224
"Perleche," 406
Pernicious anemia,

Addisonian, 415

excretion of folic acid in, 571

hematological response in, 416

relation of folic acid to, 204

symptomology, 415

therapy, 415

thymine, folic acid, vitamin B]2 in
therapy, 416
Perspiration, B vitamins in, 269, 368-369
PGA, 39
pH, effect on sulfonamide activity, 502-

505
Pharmacological action of B vitamins

(see individual vitamins)
Pharmacological level, 377
Phenylethylamine, formation of, 181
Phlorglucinol-like compounds, formation

of, 195
Phosphatases, 157

Phosphate bonds, high energy, 101, 236
Phospholipides,

choline in, 11, 39

inositol in, 38
Phosphoroclastic reaction, 162, 335
Phosphorus retention, effect of androgens

on, 381-382
"Phosphoryl-acetyl intermediate," 162

end products of, associated with thia-
mine metabolism, 165

736

INDEX

"Phosphoryl-acetyl intermediate — Cont'd

origin of, 191

role in fatty acid metabolism, 225
Phosphorylases, 157
Phosphorylation,

in glycolysis, 219

of B vitamins, 343-344
Photosynthesis, 237-238, 337
Phototrophism, 337
Phycomyces test, 80, 384
Phyla, pantothenate in various, 19-20
"Physical fitness," 245
Physiological interrelationships of B vita-
mins, 378-379
Physiological level, 377
Physiological requirements, 264
Phytic acid, 38
Phytin, 38
/3-Picoline, 286-287
Pigs, pellagra in, 412
Pimelic acid,

as biotin precursor, 88

effect on biotin production, 88
Pituitary gland, 380
Placebos, use of, 249
Plant cuttings, B vitamin requirements

of, 316
Plant embryos, B vitamin requirements

of, 307, 316
Plant roots, B vitamin requirements of,

307, 316
Plants, green,

absorption in, 336-337

B vitamin deficiencies in, 397

digestion in, 336-337

distribution of B vitamins in, 337

excretion of B vitamins in, 336-338

metabolism of B vitamins in, 336-338

origin of B vitamins in, 337
Plant tissues,

aldehyde-oxidases in, 148

amino acid requirements of, 316-317

B vitamin requirements of, 316-318

mitotic rates of, 379
Plasmochin, 680
Platyhelminthes, B vitamin requirements

of, 308
Poising agents, enzyme activators as, 110
Poisons, heavy-metal, 153
Pollen grains, B vitamin requirements of,

316
Polyneuritis, nutritional, 400

time required for development, 264

with chronic alcoholism, 400
Polysaccharides, synthesis and cleavage

of, 217 et seq.
Porifera, nutrition of, 308
Porphyrins,

as growth factors, 716

as inhibitors, 716

B vitamin-like properties of, 151

biological activity, 716-720

Porphyrins, — Continued

role of single carbon unit in biosyn-
thesis of, 235
Potassium,

effect of androgens on retention of, 381-
382

relation to biotin deficiency, 249
Potency of B vitamins, 283
Potentials,

actual, 131

redox, 130

standard, 131
Precursors of B vitamins, 284
Pregnancy,

anemia in, 419

effect on B vitamin requirements, 268
Premenstrual tension, B vitamin treat-
ment of, 382
Primitive tribes, deficiencies in, 296
Prison camps, dietary surveys in, 252
Procaine, effect on sulfonamide therapy,

486
Processing of food, effect on B vitamin

content of, 282
Products of B vitamins, 284
Prontosil, 481

Prosthetic groups, classification of, 109
Protein-complex, preparation of, 120-121
"Protein-complexes," 116
Proteins,

effect on B vitamin requirements-, 276

effect on pyridoxine requirements, 278

effect on riboflavin requirements, 277

synthesis and hydrolysis of, 230 et seq.
Protogen, 16

Protozoa, B vitamin requirements of, 307
"Pseudopyridoxine," 654
Pterins, effects in anemia, 573-574
Pteroylglutamic acid, 9, 39-40
Pteroylheptaglutamate, 290
Pupation, B vitamin requirements for, 311
Purines, catabolism of, 234

folic acid in synthesis of, 201

incorporation of single carbon unit
in, 196

PABA in synthesis of, 201

vitamins required in synthesis of, 233-
234
Putrescine, 181
a-Pyracin, 421
0-Pyraein, 421

Pyramin, excretion of, 255, 369
Pyridine derivatives, excretion of, 389
Pyridoxal (see also pyridoxine, Vitamin
B8 coenzyme)

analogues, 658-659

assay methods, 59-61

biological activity, 655, 656

dissociation constants of enzymes of,
180

isolation, 654

phosphate, 7, 36, 178

INDEX

737

Pyridoxal — Continued

structure, 8

sparing action on amino acids, 185
Pyridoxamine (see also pyridoxine, Vita-
min B6 coenzyme)

analogues, 658-659

assay methods, 59-61

biological activity, 655-656

formation from pyridoxal phosphate,
177

isolation, 654

phosphate, 7, 36, 178

structure, 8

sparing action on amino acids, 184
4-Pyridoxic acid, 288, 290, 364, 657-658
Pyridoxine,

acid-labile forms, 37

analogues, 288

antagonistic effects, 295

as storage form, 86

assay methods, 59-61

biological activity, 652-654, 656

biosynthesis, 86-87

coenzymes (see vitamin B6 coenzymes)

combined forms, 36

distribution, 36

effect of autoclaving on, 176

effect of protein on requirements of,
278

effect of species size on requirements
of, 320

essentiality of, 187

excretion of, 365

extraction, 36

inhibitory analogues, 659-666

metabolism, 364

pharmacological action, 390

relation to fat metabolism, 185-186

relation to growth of plants, 86-87

requirements, 317, 318

specificity, 652-654

structure, 8

toxicity, 390
Pyridoxine deficiency,

biochemical aspects, 428

symptomology, 426-427
Pyrimidines,

folic acid in synthesis of, 202

PABA in synthesis of, 201

vitamins required in synthesis of, 233-
234
Pyrophosphate bond, energy from, 163
Pyruvate oxidation factor, 16 ff
Pyruvic acid,

acetate-lactate dismutation of, 164

acetoin from, 161

aerobic oxidation, without phosphate,
166

aerobic utilization of, 165, 220

anaerobic utilization of, 220

as hydrogen acceptor, 220

/3-decarboxylation of, 221

Pyruvic acid — Continued
decarboxylation of, 158, 160, 221
formic acid production from, 162
phosphoroclastic cleavage of, 163
reduction after carboxylation, 220-221

Pyruvism, in insects, 312

"Quadratic" inhibition, 454

Quinine, relation to riboflavin coenzymes,

679-681
Quinine oxidase, 296
Quinolinic acid, formation of, 287, 355,

357

Random urine specimens, 255

Rat carcass, vitamin B content of, 254

Rats, requirement for B vitamins, 326

requirement for vitamin Ba, 299

strain differences, 265
Reaction rate,

effect of substrate in inhibition of, 450

in absence of inhibition, 451

in competitive inhibition, 451

in noncompetitive inhibition, 451
Reactions,

reversibility of, 101

types of biochemical, 103
Recommended daily intake of B vita-
mins, 254
Redox dyes, 131
Redox potential, 130
Redox systems, coupled with nicotinic

acid coenzyme, 141
"Refection," 298
Relaparotomy, B vitamin deficiency

after, 272
Reproduction,

B vitamin deficiency and, 381-382

essentiality of B vitamins in, 383
Requirements for B vitamins (see also
individual vitamins)

bioassay methods, in humans, 252

comparative studies of, 246 et seq.

criteria in fixing of, 245

effect of age on, 265-266

effect of anoxia on, 272

effect of caloric intake on, 276

effect of carbohydrates on, 276

effect of climate on, 267

effect of customs on, 282

effect of disease on, 272

effect of fats on, 276

effect of fever on, 273

effect of illness on, 271

effect of injury on, 272

effect of intestinal flora on, 297

effect of lactation on, 269

affect of metabolic rate on, 273

affect of other nutritional components
on, 275-276

effect of pregnancy on, 269

effect of proteins on, 276

738

INDEX

Requirements for B vitamins — Cont'd

effect of sex on, 266-267

effect of shock on, 272

effect of size on, 264

effect of species size on, 319-323

effect of surgery on, 272

effect of thyroid on, 380-381

effect of weight on, 266

effect of work on, 267

environment as factor, 264

factors influencing, 264, et seq.

growth as criterion for, 252

maximal, 245

methods of assessing, 243

of aged, 266

of arthropoda, 309-315

of bacteria, 307

of chicks, 327

of children, 266

of domestic birds, 328

of fetuses, 270

of fowls, 258

of humans, 326

of insects, 309-315

of invertebrates, 306-315

of mammals, 329

of mice, 327

of plant roots and embryos, 307

of protozoa, 307

of rats, 326

of tissues, 259

of vertebrates, 318-330

of viruses, 307

of worms, 308

optimal, 245

qualitative requirements, 244

quantitative requirements, 245

recommended intake, 245

specie variability and, 264

strain variability and, 265
Resection, effect on B vitamin require-
ments, 272
Resistance, mechanism in competitive in-
hibition, 475-477

mechanism of sulfonamide, 519-520

to sulfonamides, 516-521
Reversibility of a reaction, 101
Rhizopterin, 422, 566

in folic acid assay, 70

PABA in, 40
Riboflavin,

analogues, 285-286

assay methods, 51-56

biological activity of, 670-673

biosynthesis, 81-82

combined forms, 32-33

cooking and storage effect on, 33

determination of, in serum, 259

distribution of, 19

effect of ascorbic acid on storage, 258

effect of protein on requirement, 277

Riboflavin — Continued

effect of species size on requirement of,
319

excretion, 271, 338, 365

extraction, 33

flavoproteins, 32

function, in anaerobic systems, 150

inhibitory analogues, 673-682

in milk, 82

metabolism of, 362

"old yellow enzyme," 32

requirements, 317, 324, 326, 327, 328,
329

sparing effect of fatty acids on, 227

specificity, 670-673

stereoisomers, 671

structure, 6

toxicity, 390
Riboflavin coenzymes (see also flavopro-
teins)

as hydrogen carriers, 141

assay methods, 144

biosynthesis, 144

mechanism of oxidation and reduction,
146

occurrence, 144
Riboflavin deficiency,

biochemical changes in, 407-408

distribution, 406

symptomology, 406
Rice bran, crystalline thiamine from, 96
Rice moth larva, studies on, 311-312
Robinson ester, 136
Rous chicken sarcoma, 594-596
"Royal jelly," 22
Rumen,

synthesis of pantothenic acid in, 85

synthesis of riboflavin in, 82

synthesis of thiamine in, 78-79
Ruminants, deficiency in young of, 298

Sarcoma, 180, 597

effect of folic acid on, 594-596
Sarcosine dehydrogenases, 152
"Saturation" with B vitamins, 370
Schiff's base formation, 186
Secondary products, effect in inhibition,

461
Self-selection studies, 259-260
Serine,

ethanolamine from, 181

from glycine, 233

role of vitamin Bis in formation of, 207

trypotophan from, 183, 233
Sex, influence on B vitamin requirements,

266-267
Sexual function, B vitamin deficiency

and, 381-382
Shock,

coenzyme breakdown in, 272

effect on B vitamin requirements, 272

INDEX

739

Sickness, effect on B vitamin require-
ments, 271
Significance of distribution of B vita-
mins, 24-25
Silverfish, 315
Single carbon unit,
blocking of reactions by sulfonamides,

200
coenzymes in utilization of, 196 et seq.
folic acid in, 200
in purines, 196
origin, 197
sources, 197

vitamins associated with metabolism
of, 198 et seq.
Size, effect on B vitamin requirements of

species, 319, 323
SLR factor, 200, 422
Sodium iodoacetate, inhibition of phos-
phorylation of thiamine, 156
Snake venom, 148
Solubility as criterion for classification of

vitamins, 16
Solubility of B vitamins in water, 30
Sources of B vitamins, 30 et seq.
"Sparing effect" in inhibition studies,

461
Specificity of B vitamins (see specific B

vitamins)
Specificity of coenzymes, 115
Species, variability of, 264
Species size, relation of B vitamin re-
quirements to, 264, 319-323
Sphingomyelins, 39
Sprue, 417-418
symptomology, 417
therapy, 418
thymine in, 418
Standard potential, 131
Sterile animals,
studies with, 300
metabolism of, 228, 230
Sterility, B vitamin deficiency and, 381-

382
Sterols,
role of Coenzyme A in synthesis of,

195, 230
vitamins required in synthesis of, 230
Stilbestrol, effect on oviduct, 383
Storage of B vitamins, 255, 258-259

in tissues and fluid, 350-351
Storage of foods, effect on riboflavin

content, 33
Strain, variability of, 264
Strepogenin, 15, 260, 296, 397
Substrate, effect on rate of inhibited

enzymatic reactions, 450
Succinic acid dehydrogenase, 152
Succinoxidase, 151
Sulfonamides,
activity in reversal with PABA, 496-499

Sulfonamides — C ontimied
antagonism to chemotherapeutic ac-
tion, 498
antagonism to PABA of, 199, 494, 500
biological effects of, 511-516
blocking of single carbon unit metab-
olism by, 200
effect of ionization on activity, 501-

502
effect of mass action on activity, 501
effect of pH on activity, 502-505
effect of physical properties on activity,

501
effect of procaine on therapy with, 486
effect of resonance on activity, 510
effect of structure on activity, 501
effect of sulfonyl-negativity on activ-
ity, 505-509
effect on nutrition, 513
effect on respiration, 512
in inhibition analysis of PABA func-
tion, 470-471
inhibitions unaffected by PABA, 500
miscellaneous effects of, 515, 516
resistance to, 516-521

Su If ones,
activity in reversal with PABA, 496-

499
antagonism to PABA, 495

Sulfonyl group, effect of negativity on
sulfonamides, 505-509

Symbiosis,
in green plants, 79
in insects, 309

Symbiotic microorganisms, 79

Synergism, in inhibition, 463

Synthesis of B vitamins in animals, 351

Synthesis of coenzymes,
extent of, 113
rate of, 113

Synthetic ability, impairment of, 217

Taboos, effect on B vitamin requirement,

282
Tapeworm, in anemia, 419-420
Temperature, effect on B vitamin re-
quirements, 267-268
Testosterone, effect of on excretion of
nitrogen, phosphorus, potassium, 381-
382
Thiaminase, 292-293
Thiamine,

activating effect on carboxylase, 155

analogues, 284-285, 688- 690

antagonistic effects, 295

as cofactor, 133

assay methods, 47-51

availability, from yeast, 291

biological activity of, 687-694

biosynthesis, 78-81

cocarboxylase, 30

combined forms, 30-31

740

INDEX

Thiamine — Continued

content of diets of, 253

curare-like action of, 386

decline in urinary and tissue levels, 405

distribution, 19

-disulfide, 32

effect of species size on requirement,
264, 319

effect of yeast on dietary, 291

effect on heart action, 384

effect on mental activity, 385

effect on nervous impulse, 384

effect on plant and seedling growth, 80

excretion, 271, 365

extraction, 31

functions, 168, 170

inhibition of phosphorylation of, 155

inhibitory analogues, 693-700

interrelation to cholic acid, 282

interrelation to vitamin A, 282

lethal dosage, 386

pharmacological action, 386

-pyrophosphate, 154

relation of structure to function, 167-
168

requirements, 264, 317, 324, 326, 327,
328, 329

sources, 30

sparing effect, 276

specificity, 684-694

structure, 4

therapeutic index, 386

therapeutic use, 356, 381, 386

toxicity of, 386-387

variability in requirements of, 274
Thiamine coenzymes,

assay methods, 155

biosynthesis, 157

formation of holoenzyme from, 157

impermeability of cell membranes to,
158

inhibition by thiamine, 157

reactions catalyzed by, 158 et seq.

role in carbon to carbon bond metab-
olism, 189
Thiamine deficiency,

beriberi, 399-400

biochemical features of, 403

causes of, 399

in foxes, 292, 402

mild, 404

nutritional polyneuritis, 400

requirements to prevent, 264
Thiamine disulfide, thiamine activity of,

168-169
Thiochrome, 685
Thiochrome method, 47-48
Thiochrome pyrophosphate, 155
Threonine, relation to pyridoxine require-
ment, 184
Thunberg technique, 149

Thymidine, 15

interrelation to antipernicious anemia
vitamin, 205

interrelation to PABA and folic acid,
474

structure, 15
Thymine, 290, 414

in anemia therapy, 414, 416

in sprue, 418

structure, 414
Thyroid gland,

effect on B vitamin metabolism, 381

effect on B vitamin requirements, 380
Thysanura, 315
Ticks, 315

"Tissue hunger," 254
Tissues,

B vitamins in, 20, 258, 350-351

B vitamins in human tissue, 26

cancer, 27

requirements for B vitamins of, 259

"vitamin uniformity" in, 27
Tocopherols, distribution, 22
Toxicological level, 377
TPN, 133

oxidation of reduced forms by cyto-
chrome-C-reductase, 150
TPP, synthetic preparation of, 154-155
Transaminases, 176
Transaminations, 176
Transesterifications, in glycolysis, 219
Transport of labile intermediates, 121
Tricarboxylic acid cycle, 223
Trigonellin, 83, 357
Triphosphopyridine nucleotide, 133
Tropical climate, effect on B vitamin re-
quirements, 267
Tryptophan, 83

as substitute for nicotinic acid, 83,
279

effect on excretion of niacin products,
83

effect on niacin requirement, 279

non-oxidative degradation, 183

synthesis, 183, 233
Tumors, 26-27

B vitamin content of, 28

inhibition of proliferation of cells of,
203

virus-induced, 28
Tyramine, formation of, 181
Tyrosine, defective metabolism of, 204
Tyrosine apodecarboxylase, 178
Tyrosine decarboxylase, 204

Uncombinable biotin, 62-63
Unidentified factors in animal nutrition,

244
Uniformity, coefficient of, 27
Uniformity of distribution of B vitamins,

INDEX

741

"Uniformity, vitamin," in tissues, 27
Universal distribution of B vitamins, 19-
20

reason for, 98
Unknown factors, inhibition analysis in

assay development for, 473
Unsaturated fatty acids, formation of,

227
Urease, from jack-bean meal, 96
Urinary specimens,

B vitamins in, 252, 254-257

pantothenate in, 259
Urine,

excretion of B vitamins in, 364-368

nicotinic acid products in, 356-361

other vitamin products in, 365

"V" factor activity, 608-609
Variability,

individual, 255, 369-370

species, 264

strain, 264
Variables in human experiments, 249
Variation, individual in thiamine require-
ments, 273-274
Venom, snake, 148
Verdoperoxidase, 184
Vertebrates, B vitamin requirements of,

318-330
Viruses, B vitamin requirements of, 307
Virus-induced tumors, 28
Vitamin A,

distribution, 22

interrelation to thiamine, 282
Vitamin assays (see assay methods, in-
dividual vitamins)
Vitamin B

biosynthesis, 78 et seq. 234-235

content of various materials, 254, 257-
259

end products of, 256

levels in feces, 256-257

studies in excretion of, 254-257

Vitamin Bi (see thiamine)

Vitamin B2 (see riboflavin)

Vitamin B3 (see pantothenic acid)

Vitamin Be (see pyridoxine, pyridox-
amine, and pyridoxal)

Vitamin Bu> (see antipernicious anemia
vitamin)

Vitamin B13, 16

Vitamin Bi4, 16 ff, 203

Vitamin Bc conjugase, 203

Vitamin Bc conjugate, 40

Vitamin Be coenzyme,

assay methods, 178

biosynthesis, 179-180

coenzyme for glutamic-aspartic system,
182

coenzyme for glutamic-alanine system,
182

condensations with, 189

in reactions of methylene groups, 183

in transaminations, 176

inhibition studies, 180

mechanism of action, 186

occurrence, 179

reactions catalyzed by, 180 et seq.

relation to bacterial amino acid re-
quirements, 184

sources, 179

structure, 177
Vitamin C (see ascorbic acid)
Vitamin D, 22

Vitamin E (see tocopherols)
Vitamin G (see riboflavin)
Vitamin H (see biotin)
Vitamin K, 23

Vitamins required in enzymatic reac-
tions, 104-105
"Vitamin-uniformity" in tissues, 27

Warburg's coferment, 133

Water content of cancer tissues, 27

Water solubility of B vitamins, 30

Weed killers, action of, 398

Weight, effect on B vitamin require-
ments, 266

Wernicke's disease, 400, 402

Work, effect on B vitamin requirements,
267

Worms, B vitamin requirements of, 308

Xanthine, uric acid from, 149
Xanthine oxidase, 148, 296
Xanthopterin, 285, 288

folic acid activity of, 421

effect in anemias, 573-574
"Xanthopterin oxidase," 203, 296
Xanthurenic acid excretion, 314

Yeast,

availability of thiamine in, 291

diaphorases from, 149

DPN from, 135

effect on dietary thiamine, 291
Yeast fermentation tests, 384
Yellow enzyme, 96

Zwischen ferment, 132
Zymase, 154

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