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VOLUME 15 FEBRUARY 1955 NUMBER 2
ORIGINAL ARTICLES
VITAMINS-PAST
AND
PRESENT
By Paul Gy#{246}rgy, M.D.*
I
T is with gratitude and humbleapprecia-tion that I accept the Borden Award for 1954 from the American Academy of Pediatrics. The feeling of gratitude is based on the acknowledgment by pediatric peers and colleagues of research activity con-ducted over a period of many years. This
happens to be the first occasion that a
recipient of the Borden Award given by the Academy of Pediatrics has received previously a Borden Award given by the American Institute of Nutrition for the year 1951. I am inclined to see in this event, a
welcome justification and support of my
professional career with its division of
activities between the clinical-pediatric and the experimental-nutritional fields.
In accordance with the stipulation of the
Award Committee it is proposed to give in
the following, a summary of the research work for which the award was granted. The results achieved were made possible by the
cooperation of a large number of helpful
collaborators.
The problem of qualitative nutrition has been recognized early to be of paramount
From the Department of Pediatrics, School of Medicine, University of Pennsylvania, Philadelphia.
Borden Award Address delivered at the Annual Meeting of the American Academy of Pediatrics, Chicago, October 8, 1954.
#{176}ADDRESS: 3600 Spruce Street, Philadelphia 4, Pa.
importance for the dietary management of
the growing organism: infant and child.
Vitamins are part of this over-all problem. As a young, beginning pediatrician in the early Twenties, deeply immersed in the cinico-chemical study of rickets and tetany, I had been particularly impressed by the use of animals for the solution of nutri-tional problems. At that time, the group
working under McCollum, Park and their
associates at Johns Hopkins, as well as
Alfred Hess and Steinbock had published
their classical studies on experimental rickets in rats. My first attempt to duplicate their observations, including those on the activation of inert oils by ultraviolet
irradi-ation as potent preventives of experimental rickets, had been-for reasons never under-stood-unsuccessful. My first reaction was not to question my own experimental work, but to question animal experiments as a useful tool in clinical nutritional research. Needless to say, future events have amply proven that this judgment was premature and incorrect. Consequently, I fully re-pented, and since that time have upheld
strongly the view that conclusions reached
in animal experiments or in microbiological assays are applicable with great regularity
to conditions in human pathology, and in
particular to nutritional problems.
In the late Twenties, my interest mainly
Moro, was focused on dermatological
condi-tions in infants, with special reference to seborrhoid dermatitis and acrodynia. We tried to approach the problem by feeding various experimental rations to rats. When this work started, approximately 25 years ago, the vitamin B-complex was thought to
consist of 2 separate components: 1) vita-mm B1, the antineuritic factor, and 2) vita-mm B2, the antipellagra factor. The British Committee on Accessory Food Factors in 1927 defined vitamin B2 as “the more
heat-stable, water-soluble dietary factor,recently described and named P-P
(pellagra-preven-tive)factor by Goldberger, Wheeler, Lillie, and Rogers (1926) and found necessary for maintenance of growth and health and pre-vention of characteristic skin lesions in rats, and considered by the latter workers to be
concerned in the prevention of human pellagra.”
Neither B1 nor B2 was at that time avail-able in chemically pure form. We suc-ceeded, in cooperation with Professor Rich-ard Kuhn and Dr. Wagner-Jauregg, in iso-lating from egg-white, milk and liver a yellow fluorescent substance which ap-peared to fit well the definition of vitamin B2. This substance promoted the growth of rats fed a vitamin B-free diet supplemented
with a concentrate of vitamin B1. This sub-stance, which our group originally named “lactofiavin” had been identified chemically -regardless of its original source-as a
derivative of isoalloxazine with a ribose
radical attached. Hence the present term, riboflavin.
When rats were fed a diet supplemented with crystalline vitamin B1 and with ribo-flavin added, they showed only slight and temporary weight gain. For continued growth and normal development, further supplements to the diet were required such as, extracts from yeast, liver, rice bran, etc. Consequently, it was concluded that ribo-flavin, or vitamin B2 as it is still called, was
only one member of the more comprehen-sive vitamin B2 complex. It is different from the specific pellagra-preventive factor (P-P)
of Goldberger and his associates, as first shown by us in cooperation with Harris and Birch, and independently by Elvenhjem and his associates, who then succeeded in identifying nicotinic acid (amide) with the antipellagra factor. Skin manifestations seen in rats kept on a riboflavin-free diet had not been found very striking or specific and, except for very definite seborrheic qualities, were of a more trivial nature. They were certainly not characteristic of a pellagra-like disease.
The discovery of riboflavin opened the
way to the unravelling of the vitamin B2
complex. In biochemical research it
repre-sented a special milestone. Riboflavin was the first vitamin recognized as part of an enzyme system. Therefore, it was not only a vitamin but also a dietary pro-enzyme. It bridged the gap between an essential nu-trient and cell enzymes and cellular metab-olism. Today, with the general acceptance of this idea, it is not surprising that water-soluble vitamins represent essential parts of enzyme systems.
In just 5 years, from 1933 to 1938, Sebrell and Butler clearly proved riboflavin as an essential exogenous food constituent for the human organism. Ariboflavinosis is a
generally accepted clinical entity, the
spe-cificity of which is perhaps less well
delineated than that of several other vita-min-deficiency diseases.
The history of the next member of the vitmain B2 complex, studied and isolated
by us, vitamin B6, was less straightforward,
at least with regard to its recognition as
an essential food constituent for man. Its discovery and identification as a member of the vitamin B2 complex posed no diffi-culties. Rats kept on a vitamin B-free diet,
supplemented with crystalline vitamin B1
and ribroflavin, soon developed character-istic signs of a specific deficiency.
ORIGINAL ARTICLES 121 B6 manifested itself in rats by edema,
red-ness, scaliness of the paws, snout, nose and ears, thus of the most distal parts of the body. Correspondingly we called the condi-tion rat acrodynia without any prejudice whether this condition is in its etiology and pathogenesis analogous to human acrodynia. In collaboration with Birch, we further observed that fat with a high percentage of unsaturated fatty acids, at that time also called vitamin F, prevented and beneficially influenced these cutaneous manifestations
of vitamin B6 deficiency. In young animals,
such as rats, mice and pigs, convulsive seiz-ures appeared as signs of a deficiency of vitamin B6. In dogs lacking vitamin B6,
microcytic anemia developed. These ab-normal conditions responded specifically to vitamin B6 medication. In many instances vitamin B6 deficiency in animals became apparent only by nonspecific retardation of
growth.
Vitamin B6, as originally isolated, was an alcohol. Following our suggestion it is now called pyridoxine. But B6 also occurs as aldehyde, pyridoxal and as amine, pyridox-amine, the latter also in phosphorylated
form. In natural food products, such as
milk or yeast, it is present chiefly as pyri-doxal and pyridoxamine. The credit for the recognition of these chemically slightly dif-ferent and also biologically by no means identical forms of vitamin B6 belongs chiefly to the excellent investigative work of
Snell and his associates.Today we speak of vitamin B6 more or less as a complex, and
of pyridoxine, pyridoxal and pyridoxamine
as members of the vitamin B6 group.
During the past 2 years, almost 20 years after the discovery of vitamin B6, clinical evidence has accumulated to reveal, beyond any doubt, that B6 has a very real
place in human nutrition. Holt and his
associates were the first group to illustrate that deprivation of vitamin B6 may cause
convulsions in young infants. Hunt, Stokes
and their associates have reported convul-sions in a young infant with apparent con-genitally increased vitamin B6 requirement
or dependency. Finally, scores of infants
were seen lately suffering from irritability
and convulsive seizures. These infants were fed a liquid, autoclaved commercial milk formula, which when analyzed showed a
very low content of vitamin B6. This same formula, when not autoclaved but just sprayed, contained a greater amount of vitmain B6 content and in addition caused no deleterious effects on infants.
For us pediatricians, 2 important con-clusions may be emphasized from these observations. 1) Young infants are especially sensitive to vitamin B6 deficiency and re-spond to this deficiency with a range ex-tending from hyperirritability to severe epi-leptiform convulsive fits. 2) In milk and perhaps in other natural products as well, autoclaving may incur severe deterioration. The naturally occurring forms of B6, pyri-doxal and pyridoxamine, are much more heat labile than pyridoxine.
There is a need for intensive investiga-tion on the effect of autoclaving or of any other form of preservation on the biologi-cal nutritional value of food products. The newly created Committee on Nutrition of the American Academy of Pediatrics will find a wide-open field for exploration and improvement.
Attempts to produce cutaneous lesions by dietary means led to the study of the con-dition which developed in rats when they were fed a diet containing a large propor-tion of unheated egg white. In confirmation of previous observations by Boas we found this specific scaly-seborrheic experimental dermatosis in rats to respond to a protec-tive dietary factor which we called vitamin H. The best sources for this protective
fac-tor were the kidney, liver, yeast and
egg-yolk. Incorporation of raw egg-white in a diet was essential in rats for the production of the corresponding pathological condi-tion. It was observed also in chickens fed rations not containing egg-white, but in-stead supplemented with pure or purified
fractions of the known-at least at that
complex. In collaboration with du Vigneaud we were able to show that vitamin H was identical with biotin. Biotin is a microbio-logical growth factor originally recognized and isolated by Kogl.
In further associations with Roger R.
Williams and his co-workers we were able to demonstrate that the injurious effect of raw egg-white was due to the formation of
a complex between biotin and a particular
albumin-like constituent of egg-white,
called avidin. Avidin exerted its harmful
effect only after ingestion by preventing the
absorption of biotin from the intestine and
consequently producing a conditioned,
sec-ondary biotin deficiency. When adminis-tered parenterally, we found avidin not only harmless, but that its admixture of biotin became liberated and acted then in
a curative capacity.
Today, biotin is recognized as another
member of the vitamin B2 complex. It is
one of the most powerful vitamins known.
It is manufactured in the intestinal flora of rats and apparently of man in amounts which may fully cover the normal require-ment. Although the clinical picture of ex-perimental biotin deficiency resembles that
of seborrheic dermatis and similar
desqua-mative cutaneous conditions, the proper
role of biotin in human pathology has not yet been determined. Our own relevant studies furnished no positive convincing results.
A deficiency of pantothenic acid in rats may also be associated with the production of cutaneous lesions. Depigmentation of the fur, for example the grayng of black or piebald coated rats, has been found by us to be a characteristic sequela of deficiency of pantothenic acid or-to a lesser extent-of biotin.
Throughout the next chapter of our stud-ies on the vitamin B2 complex we observed more or less fortuitously, various mani-festations of hepatic injury. These manifes-tations were produced apparently by purely dietary means. During the next 15 years, up
to the present, we concentrated mainly on
the analysis and further ramification of these initial findings. We were able to re-produce in rats through dietary hepatic
injury, both its acute form, necrosis as well as its subacute and chronic manifestation, diffuse fibrosis and cirrhosis. In both forms, protein deficiency plays an important part together with choline deficiency in the sub-acute, chronic form of fatty cirrhosis. It is our contention that when rats are fed a ration deficient in choline and in its pre-cursors, such as methionine, then diffuse fibrosis as it develops is not secondary to the initial fatty infiltration of the liver, but is due to a general hepatic cell damage; the fatty infiltration being only one coin-cidental manifestation of this injury.
It has been shown by Klaus Schwarz
dur-ing the last war in Germany and independ-ently by us following the war that vitamin E is a very essential and determining factor in the prevention of acute massive hemor-rhagic necrosis of the liver as it develops
regularly in those rats which are fed a
diet containing yeast as the sole source of
protein. We have further shown in
collab-oration, and in independent “side-line” studies with Catharine S. Rose that the red cells of rats deficient in vitamin E are read-ily hemolyzed in vivo by alloxan or in vitro
by dialuric acid, the reduction product of alloxan. This test proved to be a convenient method to determine the extent of deple-tion of vitamin E in rats.
We as well as Gordon with our mutual collaborators, were able to demonstrate with the hemolysis test, using hydrogen peroxide in lieu of dialuric acid as the
hemolyzing agent, the presence of a
physi-ological vitamin E deficiency in newborn human infants.
Red blood cells of rats kept on a
necro-genic diet as they become more depleted in
vitamin E gradually become more
ORIGINAL ARTICLES 123 cells become resistant to in vitro hemolysis
by dialuric acid. Thus it appears that yeast, which is free from vitamin E, must contain some other substance which might be re-sponsible for this double protective effect of high doses of yeast, i.e., prevention of hepatic necrosis and that of hemolysis by dialuric acid.
Vitamin E is a powerful biological anti-oxidant. Over 10 years ago we made the observation that yeast, liver, rice bran and wheatgerm, all excellent sources of the
vitamin B complex, were able to delay
rancidity of fat. This indicated a high anti-oxidant activity which-at least in yeast and in liver as well as in aqueous extracts of rice bran and wheatgerm-must have been due to cellular constituents unlike vitamin E. We had to assume that the effect of yeast in reversing hemolysis by dialuric acid and -where given in large amounts-in prevent-ing liver necrosis in rats must have been based on an antioxidant effect, in the wider sense of the underlying physico-chemical
mechanism.
Investigations in the isolation and
chemi-cal characterization of the antioxidant con-stituents of yeast and their possible role in experimental dietary hepatic necrosis are
in progress.
Antibiotics, in particular aureomycin, ter-ramycin, and penicillin have been found effective in delaying the development of experimental hepatic necrosis and fibrosis in rats. This effect is probably mediated by
the intestinal flora either by sparing
essen-tial nutrient or by elimination of harmful bacterial metabolites.
In the foregoing, vitamins and their re-lated substances were discussed. They are of great importance for the growing organ-ism, animal as well as infant and child, but of no really specific interest to the pedi-atrician. Our present most intensive investi-gative preoccupation centers more directly on the particular nutritional significance and value of human milk for the human infant.
In cooperation with a number of
associ-ates we were able to establish the presence of hitherto unrecognized N-containing car-bohydrates in human milk which exert a powerful growth-promoting effect on a special strain of
Lactobacillus
bifidus
calledthe
Penn
variant. In contrast, the compara-ble activity of cow’s milk is only about 1/40 of human milk. This study is at present branching out in many directions of bio-chemical as well as of nutritional and clini-cal interest. We were able to show that human colostrum, human milk, meconium and gastric mucin are especially rich in this microbiological growth factor. Thisso-called Bifidus Factor occurs in dialyzable
and undialyzable, in other words in small
and in large, molecular form. It exerts a
growth-promoting effect in rats, and-at
least in the form of crude gastric
mucin-it prevents fatty infiltration and cirrhosis of
the liver in rats fed a diet low in protein
and choline. Purified fractions of the Bifidus
Factor have demonstrable antiviral activity
on influenza and mumps virus tested in
em-bryonated eggs or chorio-allantoic mem-brane. Seifter and Baeder have shown powerful lipemia clearing effect by high molecular fractions obtained from human milk, even when given by mouth. In 2 pa-tients suffering from pernicious anemia, colostrum appeared to act as a good substi-tute for the Intrinsic Factor.
It is too early to draw any definite
con-elusion from this small number of observa-tions. However, the hope seems to be justi-fied that this new approach to an old prob-lem may yield results of lasting significance.
Accomplishments cannot be achieved
without trial, tribulations and multiple re-current disappointments. The most helpful single factor, apart from the timeliness of
the line of research, is the deliberate