Experiments That Changed Nutritional Thinking - TUUM EST

Symposium: Experiments That Changed Nutritional Thinking 

Experiments That Changed Nutritional Thinking 1 

Kenneth J. Carpenter, 2 Alfred E. Harper* and Robert E. Olson † 

Department of Nutritional Sciences, University of California, Berkeley, CA; *University of Wisconsin, Madison, 

WI; and † University of South Florida, Tampa, FL 

The objective of this two-part symposium, begun in 1995 cluded that major advances in science do not occur gradually, 

and continued in 1996, was to describe some of the discoveries but suddenly, and constitute ‘‘scientific revolutions.’’ He uses 

made during the past 150 years that changed the direction of the term ‘‘paradigm’’ to describe the theoretical assumptions, 

thinking in nutrition. These discoveries all illustrate the laws and techniques that dominate scientific experimentation 

strength of the scientific method as a process for gaining reli- by a particular community of scientists during a given period. 

able knowledge of the natural world. 

Eventually, however, observations that are at variance with 

Philosopher of science Karl Popper proposed that the scien- the current paradigm are encountered. The paradigm is recogtific 

method begins, not with the accumulation of facts, but nized as being inadequate, and a new and radically different 

with recognition of an unsolved problem. This leads to conjec- hypothesis is proposed as the result of unusual insight, usually 

ture about a solution, i.e., formulation of a hypothesis. The coupled with new methods. This leads to a new paradigm, and 

essence of the process is to subject hypotheses to critical exami- a period during which it is consolidated follows. An example 

nation and experimental tests that have the potential to refute given by Kuhn of a scientific revolution is the discovery by 

them. It is basically a process for detecting error; its strength Copernicus, at a time when essentially all astronomers believed 

lies in its self-correcting nature. If a hypothesis fails to with- that the earth was the center of our solar system, that in fact 

stand a test with the potential to refute it, it must be discarded the sun was the center of the solar system and the planets, 

or modified. It is equally important, nonetheless, to defend including Earth, revolved around it. The history of the various 

hypotheses vigorously to ensure that they are not rejected sciences, Kuhn proposes, is characterized by a series of parawithout 

being tested thoroughly. Although the ability of a digms interspersed with periods of ‘‘normal science’’ during 

hypothesis to withstand such tests does not establish unequivo- which problems falling within the limits of the prevailing paracally 

that it is valid, as assumptions that are false are eliminated digm are explored. This view is complementary to that of 

by repeated testing, we achieve an increasingly better approxi- Popper, who emphasized the need for constant hypothesis testmation 

of reality. 

ing and modification to ferret out error. Scientific revolutions 

The process is illustrated by two articles on early studies of have occurred in biology and medicine, of which nutrition is 

protein that were included in the symposium. During the a part, since the time of Hippocrates. 

1820s, protein was accepted as an essential nutrient on the Some examples of scientific revolutions in biology and medbasis 

of feeding studies with dogs. Subsequently the German icine are the discoveries of Harvey, Lavoisier and Darwin, each 

school of Liebig and Voit postulated on theoretical grounds of which made existing paradigms obsolete. Harvey, in 1628, 

that protein was the source of energy for muscular work. This 

discovered that blood pumped by the heart through the arteries 

hypothesis was challenged by Fick and Wislicenus in 1866 in 

passed to the veins and circulated back to the heart. This was 

an elegant nitrogen balance study performed during a mounthe 

demise of the hypothesis, postulated by Galen in the 2nd 

tain climbing expedition. Their results, interpreted by 

century, that the blood oscillated back and forth within the 

Frankland, proved conclusively that the hypothesis was erronearterial 

system. Lavoisier’s discovery, in 1777, that combustion 

ous (Paper 2). Nonetheless, the assumption that a high protein 

was a chemical process in which oxygen combined with other 

intake was uniquely important in stimulating vigor of mind 

elements with the release of energy made untenable the cenand 

body was accepted for another 40 years until it was chaltury-old 

hypothesis that combustion represented loss of ‘‘phlolenged 

in 1904 by Chittenden, who demonstrated that healthy 

young men remained vigorous with a protein intake about half 

giston.’’ Charles Darwin in his classic study The Origin of Spe- 

that recommended by Voit (Paper 5). 

cies, published in 1859, assembled evidence that new species 

Thomas Kuhn, another philosopher of science, has conof 

evolution demonstrated that biblical creationism, the belief 

had evolved continuously over millions of years. His theory 

that species arose intact through supernatural intervention, 

1 Presented as part of the minisymposium ‘‘Experiments That Changed Nutritional 

Thinking’’ given in first part at Experimental Biology 95 on April 11, 1995 had adopted Western religions, was incompatible with scien- 

and which was almost universally accepted in countries that 

in Atlanta, GA, and in second part at Experimental Biology 96 on April 16, 1996, 

in Washington, DC. This symposium was sponsored by the American Society for 

tific observations. 

Nutritional Sciences. Guest editors for the symposium publication were Kenneth All of the symposium presentations that follow discuss ex- 

J. Carpenter, University of California, Berkeley, CA, Alfred E. Harper, University periments that influenced nutritional thinking. Some describe 

of Wisconsin, Madison, WI and Robert E. Olson, University of South Florida, 

Tampa, FL. 

experiments that challenged accepted concepts and resulted 

2 To whom correspondence should be addressed. in their displacement with new ones; others are reports of 

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0022-3166/97 $3.00 1997 American Society for Nutritional Sciences. J. Nutr. 127: 127: 1017S–1053S, 1997. 

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discoveries that arose from exploration of specific aspects of sentative experiments of this expansion of knowledge within 

the new concepts. Several fit Kuhn’s concept of scientific revo- the new paradigm. 

lutions that bring about a rapid change in the paradigm of a Iron was known early in the 19th century to be a compofield. 

nent of hemoglobin, but the belief that only organically bound 

A major paradigm shift in nutrition was the discovery of the iron was available to the body was an obstacle to understanding 

essentiality of organic and inorganic micronutrients. Despite a the role of minerals in nutrition. The demonstration by Stocknumber 

of observations during the 19th century that diets man in 1893 that inorganic iron was used efficiently for hemocomposed 

of purified food constituents did not support growth globin synthesis corrected this erroneous assumption (Paper 

or even life, this shift did not occur suddenly as the result of 3). Thirty-five years later, Hart and associates discovered that 

a single discovery; it occurred over a period of more than 60 copper was essential for the utilization of iron in hemoglobin 

years. The lag was attributable in large measure to resistance formation. It is now known that copper promotes uptake of 

to the new paradigm by many scientists who were influenced iron by transferrin and increases the utilization of iron by 

by the great prestige of Liebig and who accepted, almost as erythroblasts for hemopoiesis (Paper 10). 

dogma, his concept that energy sources, protein and a few After the discovery that yellow carotenoid pigments and 

minerals were the sole principles of a nutritionally adequate colorless oils both had vitamin A activity, a conflict between 

diet. Only after the inadequacy of Liebig’s hypothesis had been competing hypotheses about the nature of vitamin A precurdemonstrated 

in many experiments that should have changed sors was resolved by Thomas Moore, who in 1930 showed that 

nutritional thinking, but did not, was the new paradigm gener- the yellow b-carotene was converted to colorless vitamin A 

ally accepted. Four of the papers describe experiments that in the animal body (Paper 11). Thiamin was shown by Lohcontributed 

to the shift in paradigm. 

mann and Schuster in 1937 to be a component of the coen- 

Gerrit Grijns, in the 1890s, extended the work of Eijkmann zyme thiaminpyrophosphate, and its role in pyruvate metaboin 

Java (Indonesia) showing that chickens fed a diet of white lism in the animal body was elaborated by Peters (Paper 12). 

rice developed polyneuritis, a disease resembling beriberi. The Observations by Goldberger that protein as well as proteindisease 

was prevented by including rice polishings or beans or free extracts of yeast could cure pellagra posed a problem that 

water extracts of them in the diet. He concluded that chickens was resolved when Krehl and colleagues discovered in 1945 

needed an organic complex provided in adequate quantities 

that the amino acid tryptophan was a precursor of niacin in 

by rice polishings and beans but not by polished rice. His 

the body (Paper 15). That complex interactions and antagoobservations 

had little immediate effect on orthodox nutrinisms 

can occur among trace minerals was discovered by Dick 

tional views, even though they ultimately contributed to the 

and associates, who observed that copper deficiency occurs in 

basis for the new paradigm (Paper 4). 

animals with a normally adequate intake of copper if their 

Liebig’s concept that the nutritional value of foods and 

intake of molybdenum and/or sulfate is high (Paper 16). In 

feeds could be predicted from their proximate composition 

1972, selenium was shown by Rotruck and co-workers to be 

(nitrogen, ether extract, ash, and carbohydrate by difference) 

essential for the action of glutathione peroxidase (Paper 20). 

was tested directly by Hart and colleagues in 1907. They found 

that calves from cows fed an all-wheat ration survived only a 

Also during the 1970s, through the work of Kodicek in 

short time even though the wheat ration was balanced for 

Cambridge and Deluca in Wisconsin, the prevailing view that 

major nutrients to match an all-corn ration that proved to be 

vitamin D acted directly to promote intestinal absorption of 

fully adequate. This was a clear demonstration of the inadeerror. 

They discovered that vitamin D, through the combined 

calcium and regulation of bone metabolism was shown to be in 

quacy of Liebig’s concept (Paper 6). 

Subsequently, McCollum found that rats fed a simplified actions of the liver and kidney, was converted to a hormone 

diet of casein, carbohydrate and minerals stopped growing unsented 

a new concept: the action of a vitamin depending on 

that mediated the actions attributed to vitamin D. This repre- 

less supplied with a fat-soluble factor present in butter but not 

in olive oil. Rats fed a polished rice diet were found to need its conversion to a hormone (Paper 19). 

a water-soluble factor B, as Grijns had shown, as well as the Another major paradigm shift in nutrition resulted from 

fat-soluble factor A (Paper 7). During this period, Holst and discoveries about the ability of the body to synthesize and 

Froelich in Norway induced a scurvy-like disease in guinea degrade nutrients and tissue constituents. The shift occurred 

pigs by feeding them diets resembling those of Grijns. This in phases, two of which are discussed in the symposium. 

disease was prevented by providing the guinea pigs with lemon Claude Bernard, the great French physiologist, conjectured 

juice or cabbage. 

about the source of glucose in the blood of dogs consuming a 

Also, between 1909 and 1914, Osborne and Mendel at diet that contained neither sugar nor starch. By a series of 

Yale, following on an earlier observation by Hopkins in Camered 

liver glycogen and the process of gluconeogenesis by 

carefully conducted experiments during the 1850s, he discovbridge 

that tryptophan was essential for the survival of mice, 

discovered that some plant proteins did not support growth of which glucose and glycogen could be synthesized in the liver 

rats unless the rats were supplemented with other amino acids from non-glucose precursors, enabling this organ to supply 

(Paper 8). Hopkins, and Funk in London, both postulated in glucose to the blood (Paper 1). 

1912 that diseases such as scurvy, beriberi and rickets were The use of isotopically labeled compounds by Schoendietary 

deficiency diseases. Only between 1910 and 1915, after heimer in the 1930s to follow the metabolic fate of fatty acids 

these and other demonstrations of the inadequacy of Liebig’s and amino acids administered orally revealed for the first time 

concept, was the new paradigm of the essentiality of minor that these nutrients were incorporated rapidly into depot fat 

constituents of foods widely accepted. 

and body proteins, respectively, and that their metabolites 

Acceptance of the new paradigm was followed by a period continued to be excreted over many days. Through his work, 

of unparalleled discovery in nutritional science from about the concept of distinct exogenous (dietary) and endogenous 

1915 to the 1950s, during which some 40 essential nutrients (tissue) metabolism was replaced with the concept of the ‘‘dywere 

identified and characterized and their functions explored. namic state of metabolism,’’ the continuous breakdown of tis- 

Several of the articles included in the symposium discuss repre- sues with the constituents of both food and tissues entering a 

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HISTORY OF NUTRITION 

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common pool from which new tissue components were synthe- We believe that these proceedings illustrate, on the one 

sized (Paper 14). 

hand, the tremendous advances resulting from the scientific 

The demonstration by Becker and colleagues that sucrose approach to nutrition and, on the other, the importance of 

and fructose are toxic to young pigs and calves represents continually maintaining a critical approach to even well-ac- 

an extension of this paradigm, one of many, illustrating that cepted hypotheses and concepts. 

metabolic pathways for some nutrients may not be functional 

at birth and undergo development during the early stages of 

growth (Paper 18). 

Paper 1: The Liver Forms, Stores and 

With the successive discoveries of essential nutrients between 

1915 and 1950 and the virtual disappearance of dietary 1860) 

Secretes Glucose (Claude Bernard, 

deficiency diseases, emphasis in nutrition was on ensuring that 

diets would provide adequate quantities of all essential nutri- Presented by Patricia B. Swan, Department of Food Science and 

ents to prevent impairment of growth and development. Alpart 

of the minisymposium ‘‘Experiments That Changed Nutritional 

Human Nutrition, Iowa State University, Ames, IA 50011 as 

though it was recognized that requirements declined with increasing 

age, little attention was given to the long-term effects Thinking’’ given at Experimental Biology 95, April 11, 1995, in 

of total food intake. One of the first challenges to the paradigm Atlanta, GA. 

that if essential nutrient intake was adequate throughout life, 

In 1834, the 21-year-old Claude Bernard left the hills of 

other dietary factors would be of little consequence, came the Rhône Valley and went to Paris to seek his fortune as a 

from Clive McCay. He argued that short-term trials with the 

playwright. A professor of literature at the Sorbonne read one 

emphasis on rapid growth did not provide an adequate test of 

of his plays, Arthur of Brittany, and counseled Bernard to enroll 

the most desirable nutritional state throughout life. He found in medical school instead. Heeding this advice, Bernard enthat, 

although rats allowed to freely eat a nutritionally adetered 

the Collège de France in the fall (Bernard 1979). 

quate diet grew most rapidly, those allowed only restricted There he became intrigued by lectures in physiology given 

amounts of food could survive much longer (Paper 13). Com- by François Magendie. Most chemists and physiologists of the 

peting hypotheses about the basis for these effects remain unre- time believed that only plants synthesized lipids, carbohydrates 

solved, but they have opened new directions in nutritional 

and proteins, whereas animals merely degraded them. The 

thinking, especially in relation to appropriate body weight and macromolecules within the body were therefore assumed to 

energy intake for adults. 

come largely preformed from the diet (Holmes 1974). A few 

Emphasis on the paradigm of nutritional essentiality also 

skeptics questioned these ideas, because there sometimes 

distracted attention from investigations of the nonnutritional seemed to be more fat in an animal’s body than could have 

components of foods and from the ancient paradigm that foods 

come from its diet. Bernard was captivated by Magendie’s demcontain 

nutriment, medicines and poisons. The finding that onstrations of the intricacies of animal physiology, and from 

broccoli in the diet increased resistance of guinea pigs to X- 1841 to 1844 he served as his laboratory assistant, gaining 

irradiation and that this effect was not related to its contribu- knowledge of techniques in animal experimentation (Holmes 

tion of known nutrients shifted attention back to the nonnutri- 1974). 

ent components of foods (Paper 17). There is now evidence 

that substances in cruciferous plants and some other foods may 

increase resistance to cancers. These observations have led to 

Studies on Glucose 

acceptance of a scientifically based form of the paradigm that Soon Bernard began his own experiments, studying digesfoods 

can affect health by their contributions of chemicals tion and certain functions of the nervous system. He extended 

other than essential nutrients through their influence on sus- the digestion studies to examine the fate of sugars within the 

ceptibility to certain diseases. 

body and demonstrated that cane sugar (sucrose) was con- 

The work reviewed here illustrates how much has been verted to grape sugar (glucose) in the gastrointestinal tract 

learned through the use of animal models. It also illustrates (Grmek 1968). Cane sugar, injected directly into a vein, was 

that caution must be exercised in extrapolating findings in one excreted unchanged in the urine; injected grape sugar, howspecies 

to another. For example, rats were an excellent choice ever, disappeared. Thus, glucose seemed to be the major form 

for studies of vitamin A and thiamin deficiencies, but failure of sugar used within the animal body, and when Magendie, 

to produce the equivalent of either pellagra or scurvy in this assisted by Bernard, fed starch to a dog, glucose was found in 

species led a leader in the field to conclude that these diseases the dog’s blood. Thus, glucose was a normal constituent of 

in humans were not, after all, due to dietary deficiencies. These blood, at least after starch consumption, not just a sign of the 

experiments also illustrate the need for caution in assuming diabetic condition as had been thought previously (Grmek 

that observations made at one stage of life apply throughout 1968). 

life. 

Early in 1848, Bernard began a systematic study to learn 

The history of nutrition illustrates that new paradigms and where glucose is used within the body. Following Lavoisier’s 

concepts do not necessarily make earlier ones obsolete; several idea, he conducted experiments with dogs that he thought 

may exist together and overlap, with all being valid frameworks would show glucose was burned in the lungs; however, these 

for investigation. What is the outlook for new paradigms and experiments yielded contradictory or uninterpretable results 

concepts in nutritional science? Application of techniques (Grmek 1968, Holmes 1974). In the early experiments, he had 

from genetics and molecular biology to nutritional problems only insensitive methods for detecting and quantifying glucose 

has led in recent years to advances in understanding the roles and needed to use large quantities. He was assuming that these 

of nutrients and their metabolites in the regulation of gene large quantities would be used almost instantly. Moreover, 

expression with respect to metabolic adaptations, the action of he used animals of various physiological conditions, and he 

hormones, and responses of the immune system. Undoubtedly sometimes fed the glucose and sometimes injected it. Improveother 

new paradigms and concepts, unanticipated now, will 

follow. 

ment of a method for the detection of glucose based on its 

ability to reduce copper in an alkaline potassium tartrate solu- 



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tion significantly improved his results. Gradually Bernard improved 

his experimental techniques, and the early work set 

the stage for later, more successful, experiments. 

The Source of Blood Glucose 

its chemical similarity to starch in plants and reported that it 

was present in opalescent extracts of the liver and formed a 

white precipitate when alcohol was added. It gave a red-wine 

color with iodine and was hydrolyzed by diastase, from saliva or 

plants, to produce glucose (Bernard 1857a, 1857b, and 1857c). 

In July 1848, Bernard conducted an experiment with a A Productive Decade 

female that had been nursing a litter of pups. He did not feed 

her for one day and, as expected, found no glucose in her Within 10 years, Claude Bernard had made three major 

gastrointestinal tract, but to his surprise, he did find glucose discoveries: 1) Glucose is a normal constituent of liver. 2) 

in her blood (Grmek 1967). ‘‘What was the source of this Liver is the source of blood glucose. 3) Liver forms glucose 

glucose?’’ After this experiment, he altered the direction of and stores it as glycogen, which, upon degradation, yields 

his research to find the answer to this question (Grmek 1968, glucose. 

Holmes 1974). 

Bernard’s experiments and the theories he derived from 

In August, Bernard used a dog that had been fed only meat them were major contributions to the science of nutritional 

(no carbohydrates) for eight days and found a large amount physiology. His exceptional skill in the surgery required for 

of glucose in the portal vein, smaller amounts in the heart these studies, and the understanding that he developed re- 

and the neck, but none in the chyle, stomach, intestine or garding the use of intact animals in experimentation, earned 

urine. He exclaimed that the source of this glucose was ‘‘in- him recognition as the ‘‘father of experimental medicine.’’ 

comprehensible’’ (Grmek 1968). 

His major textbook (Bernard 1865) became a classic in the 

A few days later, using a dog that had been fed only lard field, and he later received many honors, including memberand 

tripe, he found no glucose in the mesentery (before the ship in L’Académie Française (Bernard 1979, Olmsted 

portal vein), but ‘‘enormous’’ quantities of glucose in the liver 1938). 

(Grmek 1968). Within the next four days, Bernard measured 

the glucose content of the liver of many different species, Literature Cited 

finding significant amounts of glucose in most. He concluded 

Bernard, C. (1849) De l’origine du sucre dans l’économie animale. Arch. Gen. 

that liver of healthy animals contains glucose independent of 

de Med. 18: 303–319. 

a source of glucose in the diet (Bernard 1850, Bernard and Bernard, C. (1850) Sur une nouvelle fonction du foie chez l’homme et les animaux. 

C. R. Hebd. Acad. Sci. 31: 571–574. 

Barreswil 1848). 

Bernard, C. (1853) Recherches sur une nouvelle fonction du foie. Thèse pre- 

Subsequent experiments provided evidence that the liver 

sentée a la Faculté des Sciences de Paris. Imprimerie de L. Martinet, Paris, 

was the source of glucose in the body. By placing a tie 

France. 

between the liver and the portal vein, Bernard was able to Bernard, C. (1855) Sur le mécanisme de la formation du sucre dans le foie. 

show that the source of glucose previously found in the 

C. R. Hebd. Acad. Sci. 41: 461–469. 

Bernard, C. (1857a) Les phénomènes glycogéniques du foie. Soc. Biol. 2e 

portal vein was the back flow of blood from the liver, not Serie 4: 3–7. 

an alternative source prior to the liver (Bernard 1849 and Bernard, C. (1857b) Sur le mécanisme physiologique de la formation du sucre 

1850). For this work he received the Prize for Physiology in 

dans le foie. C. R. hebd. Acad. Sci. 44: 578–586. 

Bernard, C. (1857c) Remarques sur la formation de la matière glycogène du 

1851 (Olmsted 1938) and the doctorate of science (Bernard 

foie. C. R. hebd. Acad. Sci. 44: 1325–1331. 

1853). It was also the beginning of Bernard’s important Bernard, C. (1865) Introduction à l’Etude de la Médecine Experimentale. J. B. 

concept of the body’s ability to regulate its internal environ- 

Baillière et fils, Paris, France. 

Bernard, C. (1878) Leçons sur les Phénomènes de la Vie Communs aux Animment 

(Bernard 1878). 

aux et aux Végétaux. vol. 1, Paris, France. 

Bernard, C. & Barreswil, C. (1848) De la presence du sucre dans le foie. C. R. 

Hebd. Acad. Sci. 27: 514–515. 

Search for the Source of Glucose in the Liver Bernard, J. (1979) The life and scientific milieu of Claude Bernard. In: Claude 

Bernard and the Internal Environment, pp. 17–27. Marcel Dekker, New York, 

NY. 

Grmek, M. D. (1967) Catalogue des Manuscrits de Claude Bernard. Masson 

et Cie, Editeurs, Paris, France. 

In 1855 Magendie died, and Bernard was named to the 

Chair in Physiology at the Collège de France (Olmsted 1938). 

In this role he continued to pursue a variety of studies related 

Grmek, M. D. (1968) First steps in Claude Bernard’s discovery of the glycoto 

digestion, diabetes, toxins and the nervous system. During genic function of the liver. J. Hist. Biol. 1: 141–154. 

this time, he typically measured glucose in duplicate in several Holmes, F. L. (1974) Claude Bernard and Animal Chemistry. Harvard University 

Press, Cambridge, MA. 

tissues of the animals he was studying. On one occasion he 

Olmsted, J.M.D. (1938) Claude Bernard, Physiologist. Harper, New York, NY. 

made the first measurement on a liver on one day, but did not 

make the duplicate measurement until the following day, after 

Presented by Kenneth J. Carpenter, University of California, 

Berkeley, CA 94720-3104 as part of the minisymposium ‘‘Experi- 

ments That Changed Nutritional Thinking’’ given at Experimental 

Biology 96, April 16, 1996, in Washington, D.C. 

the liver had been allowed to stand at room temperature overnight. 

To his surprise, the content of glucose had increased 

markedly (Bernard 1855, Grmek 1967). 

Bernard next decided to perfuse an isolated liver with cold 

water until the perfusate was free of glucose. He then allowed 

the liver to stand at room temperature for some hours. Upon 

resuming perfusion, he again found significant amounts of glucose 

in the perfusate. It appeared, therefore, that something 

within the liver was giving rise to glucose and it clearly was 

not making glucose from other elements in the blood. He took 

this as proof that there was a source of glucose within the liver 

(Bernard 1855). 

Bernard then began the tedious job of isolating the glucogenic 

material present in the liver. He eventually recognized 

Paper 2: Protein Cannot Be the Sole 

Source of Muscular Energy (Fick, 

Wislicenus and Frankland, 1866) 

By 1865 it had been the general ‘‘textbook’’ view for over 

20 years that the energy needed for muscular contraction came 

from the destruction of a portion of the muscle’s own substance, 

i.e., protein. This had been stated by the organic chemist 

Justus Liebig in his influential Animal Chemistry. On page 




1021S 

233, he added that the protein broke up during the release of 

energy and that the nitrogenous fraction was converted to urea 

TABLE 2 

and excreted by the kidney, so that the total amount of work 

The results from the climbing trial 

performed (i.e., both internally, as in the heart muscles, and 

externally) was proportional to the nitrogen excreted in the 

urine (Liebig 1840). 

Fick Wislicenus 

Liebig’s second point was essentially disproved by the Body weight (/ objects carried), kg 66 76 

Urinary N (0500–1900 h),1 g 5.74 5.55 

finding that prisoners receiving a constant daily ration of Protein metabolized (N 1 6.25), g 35.9 34.7 

food excreted no more urinary nitrogen during 24 h in which Caloric equivalent of protein 

they had worked a treadmill than on days when they had (at 4.37 kcal/g)2 kcal 157 151.6 

rested (Smith 1862). However, it was still possible that prokg-m 

Work equivalent (at 423 kg-m/kcal), 

tein had been the sole muscle fuel and that more had broken 

66,400 64,100 

down on rest days by some alternative mechanism. It ceragainst 

gravity, kg-m 

Net work in ascending 1956 m 

tainly seemed that nitrogen intake was the main determinant 

129,000 149,000 

of its output. 1 Fick and Wislicenus (1866). 

A Swiss physiologist, Adolf Fick, saw that the best condi- 2 Frankland (1866). 

tions for a critical experiment would be to do a considerable 

amount of measurable work while eating a protein-free diet. 

Then if the heat energy obtained from the oxidation of the foot of a convenient mountain. At 0500 h next morning 

protein to urea and carbon dioxide were known, and also 

they set out, carrying urine collection equipment, and 

the relation of heat energy to mechanical work, it should be 

walked steadily up a steep path until, at 1320 h, they reached 

possible to determine whether the amount of body protein 

another hotel at the summit. They were in a cold mist 

metabolized was sufficient to have powered the work done. 

throughout the climb and did not believe that they had had 

Fortunately, Fick’s brother-in-law, the chemist Edward 

significant losses from sweating. From noon the previous day 

Frankland, was at work in England developing a method for 

until 1900 h on their exercise day, their only food was cakes 

measuring the heat of oxidation of organic materials. High 

made from starch paste fried in fat; they also drank strongly 

pressure ‘‘bomb’’ calorimeters had not yet been developed, 

sweetened tea and some beer and wine over the period. 

but he was able to ignite a mix of potassium chlorate and 

Their results for their urinary nitrogen excretion, and the 

manganese dioxide with the test material in a little ‘‘diving 

subsequent calculations, with slight ‘‘rounding off’’ of their 

bell’’ immersed in an insulated water tank. Using a series 

values, are set out in Table 2. It is seen that even without 

of controls to adjust his results for the rise in temperature 

making any allowance for the internal work of breathing 

of the bath, he was able to obtain an impressive set of results 

and respiration, and even if the muscular system were 100% 

for a long series of food materials and for urea (Frankland 

1866). 

efficient, the quantity of protein metabolized was insuffi- 

The most directly relevant results are set out in Table 1. 

cient to have provided the energy needed for their climb; 

He assumed that metabolized protein yielded one-third of 

in fact it was 51% for one subject and 43% for the other. 

its own weight of urea, and he therefore subtracted the 

The climbers concluded that ‘‘the burning of protein canresidual 

gross energy of this quantity of urea when estimating 

not be the only source of muscular power’’ (Fick and Wisli- 

the energy released from the metabolism of 1 g of protein 

cenus 1866). And Frankland, on page 684 of his review of 

in the body. 

these and other results, added: ‘‘Like every other part of the 

By this time it also seemed well established that the merenewal 

is not perceptibly more rapid during great muscular 

body the muscles are constantly being renewed; but this 

chanical equivalent of heat was such that the energy needed 

to raise 1 kg a distance of 423 m was at least approximately activity than during comparative quiescence. After the sup- 

equivalent to 1 kilocalorie (Joule 1843). 

ply of sufficient albuminized matter [protein] in the food to 

Now the human trial was needed. Fick and his university provide for the necessary renewal of the tissues, the best 

colleague Johannes Wislicenus passed a night at a hotel near materials for the production, both of internal and external 

work, are non-nitrogenous material. . .’’ (Frankland 1866). 

These conclusions were not immediately accepted, but 

TABLE 1 

they stimulated further long-term trials that were confirmatory, 

although Liebig himself never admitted in so many 

Frankland’s presentation of his results for the energy values word that he had been wrong (Carpenter 1994). 

of protein and urea1 


Name of substance dried at Heat units Kg-m Literature Cited 

100C (kcal) of force 

Carpenter, K. J. (1994) Protein and Energy: A Study of Changing Ideas in Nutri- 

Energy developed by 1 of each 

tion. Cambridge University Press, New York, NY. 

substance: 

Fick, A. & Wislicenus, J. (1866) On the origin of muscular power. Phil. Mag. 

When burnt in oxygen 

Lond. (4th ser.) 31: 485–503. 

Beef muscle purified by ether 5.10 2161 Frankland, E. (1866) On the source of muscular power. R. Institution Proc. 4: 

Purified albumen 5.00 2117 

661–685. 

Urea 2.21 934 

Joule, J. P. (1843) On the calorific effects of magneto-electricity and on the 

mechanical value of heat. Phil. Mag. Lond. (3rd ser.) 23: 435–443. 

When consumed in the body 

Liebig, J. (1840) Animal Chemistry or Organic Chemistry in its Application to 

Beef muscle purified by ether 4.37 1848 

Physiology and Pathology (W. Gregory, trans.). Owen, Cambridge, MA. (Re- 

Purified albumen 4.26 1803 printed 1964 in facsimile by Johnson Reprint Corp., New York, NY.) 

Smith, E. (1862) On the elimination of urea and urinary water. Phil. Trans. R. 

1 From Frankland (1866). 

Soc. Lond. 151: 747–834. 


1022S 

SUPPLEMENT 

Paper 3: Inorganic Iron Can Be Used to 

Build Hemoglobin (Stockman, 1893) 

Presented by Richard A. Ahrens, Department of Nutrition and 

Food Science, College of Agriculture and Natural Resources, University 

of Maryland, College Park, MD 20742-7521 as part of 

the minisymposium ‘‘Experiments That Changed Nutritional 

Thinking’’ given at Experimental Biology 96, April 16, 1996, in 

Washington, DC. 

The condition of anemia was originally named morbus virgineus 

by Johannes Lange (Lange 1554). Lange was a physician 

of Lemberg and Rector of Leipzig University. He considered 

this disease to be peculiar to virgins and to be due to a retention 

of menstrual blood. His therapy involved instructing virgins 

afflicted with this disease to marry as soon as possible. He cited FIGURE 1 Hemoglobin responses of chlorotic patients to ferrous 

no less an authority than Hippocrates, in his treatise De Morbis sulfide (in keratin capsules, 550 mg/d), iron citrate (subcutaneous, 32 

Virginum, as also recommending marriage to cure this disease. mg/d) and bismuth oxide (9.6 g). The response times between the initial 

J. Varandal renamed this disease ‘‘chlorosis’’ (Varandal and final observations were 12 d for ferrous sulfide, 10 d for iron citrate 

1615). The popular English term was the ‘‘green sickness,’’ and 9 d for bismuth oxide. The percentages given on the y-axis are 

referring to the greenish hue assumed by Caucasians when based on the clinical standard for hemoglobin in use in 1893. Generated 

from the data of Stockman (1893). 

their blood is low in hemoglobin. Chlorosis soon became a 

central feature in medical textbooks describing the diseases of 

women. Because chlorosis was a sign of virginity, European During the 1880s, Gustav von Bunge wrote two influential 

artists often painted young women during this era with a greenish 

papers in which he concluded that only organic sources of 

hue. In art, if not in fact, chlorosis was a widespread condi- iron were of value in treating chlorosis (Bunge 1885 and 1889). 

tion. 

Von Bunge’s interest in iron dated back to 1874 when he 

By the mid 19th century, the disease of chlorosis was ac- analyzed both blood and milk and recognized that blood was 

cepted by many physicians as being associated with neurotic rich in iron and milk had very little. He developed a philoso- 

and hysterical manifestations (Bullough and Voght 1973). phy that people are always best served when they get essential 

Chlorosis became a form of neurosis. This view of chlorosis nutrients from foods. That philosophy also applied to iron. To 

was an impediment to the acceptance of dietary therapy for quote von Bunge, ‘‘Why should a patient buy his iron in the 

its treatment. It was a refinement of the view that anemia was pharmacy and not on the market with the usual foodstuffs?’’ 

due to virginity in women, but it continued to perpetuate a This is a philosophy with many adherents today. As von Bunge 

sex bias. Bullough and Voght (1973) pointed out that sex bias implemented what he believed, however, it soon became a 

flourished during the latter half of the 19th century as a male personal crusade in which he claimed that ‘‘the iron which 

‘‘backlash’’ against women’s demands for more education, the doctors give to chlorotics to form hemoglobin is not abgreater 

political equality and the elimination of male stereo- sorbed at all.’’ 

types about woman’s place. Medical practitioners were almost As Gustav von Bunge got into his crusade he was soon 

all men, and many of them were hostile to any change in the claiming that iron therapy was successful because of the power 

status quo in male-female relationships. Medical schools that of suggestion. It was well known, after all, that most chlorotics 

had admitted a few women early in the 19th century began were women and often exhibited nervous or psychic disturbances. 

to reject female applicants purely on the basis of their sex. 

He felt that this made them ‘‘highly suggestible.’’ The 

Nursing schools were established in growing numbers to pro- true villains, according to von Bunge, were those who advised 

vide a alternative for females. By the latter part of the 19th young women to practice vegetarianism. He spent much of 

century, chlorosis became an extremely common diagnosis what remained of his life arguing against vegetarianism and 

(Clark 1887). It is necessary to appreciate this historical context 

was enthusiastic about the nutritional value of meat in the 

to understand some of the resistance to accepting a nutri- human diet. He died in 1920, just as Prohibition was beginning 

ent deficiency as the cause of this disease. 

as the ‘‘noble experiment’’ in the United States. He was an 

Pierre Blaud in France recommended the use of pills con- implacable foe of alcohol consumption all his life and looked 

taining ferrous sulfate for the treatment of chlorosis (Blaud 1832). forward to the results of this experiment with U.S. citizens as 

The average dose amounted to approximately 150 mg/d, and the guinea pigs. He anticipated that a model U.S. society 

considerable success was achieved. Despite this success, however, would result from Prohibition and that Europe would then 

there was considerable resistance to the acceptance of chlorosis soon follow this great example (McCay 1953). It is undoubtedly 

as a simple dietary iron deficiency. One of the obstacles to be 

fortunate that he did not live to see the result of this 

overcome was the just-discussed sex bias that tended to associate particular experiment. 

chlorosis with the neuroses of women. Another obstacle to be When he wasn’t blaming the power of suggestion for the 

overcome, however, was the inability of investigators using the beneficial effects of inorganic dietary iron on chlorosis, von 

balance method to demonstrate that inorganic iron could be Bunge had a second explanation. Bullough and Voght (1973) 

absorbed from the gastrointestinal tract. V. Kletzinsky conducted noted that such contradictions were common among researchers 

a series of experiments (Kletzinsky 1854). In all of his studies, 

studying ‘‘women’s diseases’’ during the late 19th century. 

the amount of iron recovered in the feces was almost exactly Von Bunge adopted Kletzinsky’s theory (1854) that susceptible 

patients became chlorotic through the production by gut bac- 

teria of hydrogen sulfide, which then reacted with organic iron 

compounds in the ingesta to produce insoluble ferrous sulfide. 

equal to the amount of inorganic iron ingested. A third obstacle 

to be overcome was the toxic effect of intravenous injections of 

ferrous sulfate in dogs. 




1023S 

If inorganic salts of metals having insoluble sulfides were given 

more commonly between the advent of menstruation and the consummation 

of womanhood. Lancet 2: 1003–1005. 

as dietary supplements in large quantities, these should take 

Fowler, W. M. (1936) Chlorosis—an obituary. Ann. Med. Hist. 8: 168–177. 

up most of the hydrogen sulfide, leaving more of the organic Kletzinsky, V. (1854) Ein Kritischer Beitrag des Chemiatrie des Eisens. Z. 

iron compounds free for absorption. 

Gellschaft. Aerzte Wien 2: 281–289. 

Lange, J. (1554) Medicinalium Epistolarum Miscellanea, pp. 74–77. Basel, 

Ralph Stockman of the Edinburgh University School of 

Switzerland. 

Medicine put Kletzinsky’s, and thereby von Bunge’s, theory to McCay, C. M. (1953) Gustav B. Von Bunge. J. Nutr. 49: 2–19. 

the test (Stockman 1893). He did tests on chlorotic patients to Stockman, R. (1893) The treatment of chlorosis by iron and some other drugs. 

determine if inorganic iron worked directly or by the indirect 

Br. Med. J. I: 881–885, 942–944. 

Stockman, R. (1895) On the amount of iron in ordinary dietaries and in some 

mechanism of binding with hydrogen sulfide. His results are articles of food. J. Physiol. 18: 484–489. 

summarized in Figure 1. He gave subcutaneous daily injections Varandal, J. (1615) De Morbis et Affectibus Mulierum Libri Tres. Lyons, France. 

of ferrous citrate providing 32 mg of iron to three chlorotic 

young women and found an increase from 44% to 52% of 

normal hemoglobin concentration in 10 d. After 24 d the 

Paper 4: A Micronutrient Deficiency in 

women had blood hemoglobin concentrations that were 72% Chickens (Grijns, 1896–1901). 

of normal. Stockman then tried giving another four subjects 

Presented by Barbara Sutherland, Department of Nutritional Sci- 

550 mg/d of iron by mouth in the form of ferrous sulfide and 

ences, University of California, Berkeley, CA 94720-3104 as part 

enclosed in keratin capsules that released the iron salt in the 


small intestine. Iron in this form could not be expected to 


bind any additional hydrogen sulfide. Nevertheless he found 

Atlanta, GA. 

an increase from 48% to 60% of normal hemoglobin concentration 

in 12 d. After 33 d the women had blood hemoglobin Recently we reported on Christiaan Eijkman’s work on 

concentrations that were 84% of normal. He also gave 9.6 g/ polyneuritis in chickens performed during the 1890s in Indod 

of bismuth dioxide to chlorotic women having blood hemo- nesia (Carpenter and Sutherland 1995). The timeline shows 

globin concentrations that were 55% of normal and found how early it was when this work was being performed: at 

these hemoglobin levels to be only 54% of normal 9 d later. the same time as Atwater’s calorimetry work, and before the 

Manganese dioxide gave a similar result. These latter two salts ‘‘vitamine’’ concept of Funk and McCollum’s studies of fatwere 

quite capable of removing hydrogen sulfide from the gut, soluble factors (Table 1). This was a time when the infectious 

but they had no value in treating chlorosis. 

theory of disease was dominant, and it was while Eijkman was 

It would seem that the elegant refutation of Gustav von looking for an infectious cause of beriberi, a serious disease in 

Bunge’s hypothesis by Ralph Stockman in 1893 should have Indonesia at that time, that he recognized a similarity with 

made it apparent that inorganic iron had great value as a polyneuritis seen in chickens (Eijkman 1990). He found that 

nutrient. In another paper two years later, Stockman (1895) polyneuritis appeared in fowls when they were fed a diet of 

showed that chlorosis in young women was explained by their polished rice, but that by adding the silver skin, which had 

low overall intake of food, particularly of meat, which resulted been removed during polishing, the polyneuritis could be prenecessarily 

in low iron intake, at a time when the combined vented or cured. From the results of many feeding experiments, 

burdens of growth and menstrual blood loss increased their Eijkman concluded that the polyneuritis was due to a nerve 

need. He showed, through the use of a more specific analytical poison produced during the fermentation of starch in the 

procedure for iron in foods that avoided interference from chicken’s crop and that the silver skin contained an antidote 

starch, that the diet of anemic young women was particularly to this poison. 

low in iron, partly because these young women were eating so Eijkman’s work was cut short by ill health, and in 1896 he 

little, and most of that was bread. 

had to return to Holland. The research was continued by 

The reputation of Gustav von Bunge at that time, however, another young Dutch military surgeon, Gerrit Grijns. He had 

far exceeded the reputation of Ralph Stockman. As Carpenter obtained his medical education in Holland and then studied 

(1990) has pointed out, poorly conducted research continued physiology in the laboratory of Carl Ludwig in Germany 

to question the therapeutic value of inorganic iron in anemia (Grijns 1901). In 1892 he was sent to Indonesia to assist in 

through the 1920s. Gustav von Bunge died in 1920. The old another of Eijkman’s studies, that of the physiological adaption 

concept of ‘‘chlorosis’’ is also long gone (Fowler 1936). How- of Europeans to tropical conditions. But Grijns was shortly 

ever, precautions are still needed to ensure an adequate intake recalled to military service, and when he was able to return 

of iron. In the United States, white bread and many breakfast to Batavia (modern-day Jakarta), Eijkman had already left for 

cereals are routinely fortified with inorganic iron, and pregnant Holland. Grijns was then appointed to carry on the investigawomen 

are advised to take iron supplements. In the Third tions into the cause of polyneuritis in chickens. 

World, particularly where hookworm infestation is a chronic His official commission was to ‘‘investigate the physiologidrain 

on people’s blood supply, iron deficiency anemia remains 

a serious problem. 

TABLE 1 

Literature Cited 

Dates of some significant papers in nutritional science 

Blaud, P. (1832) Sur les maladies chlorotiques et sur un mode de traitment 

specifique dans ces affections. Rev. Med. Franc. Etrang. 1: 337–367. 

1889 Atwater (chemistry of U.S. foods) 

Bullough, V. & Voght, M. (1973) Women, menstruation and nineteenth-century 

1896 Eijkman (polyneuritis from polished rice) 

medicine. Bull. Hist. Med. 47: 66–82. 

Bunge, G. (1885) Ueber die Assimilation des Eisens. Hoppe-Seyler Z. Physiol. 

1901 Grijns (need for unidentified micronutrients) 

Chem. 9: 49–59. 

1907 Holst and Fröhlich (guinea pig scurvy) 

Bunge, G. (1889) Uber die Aufnahme des Eisens in den Organismus des Sauglings. 

Z. Physiol. Chem. 13: 399–406. 

1914 McCollum (fat-soluble factors) 

1912 Funk (‘‘vitamine’’ concept) 

Carpenter, K. J. (1990) The history of a controversy over the role of inorganic 1926 Jansen and Donath (thiamin isolated) 

iron in the treatment of anemia. J. Nutr. 120: 141–147. 

1936 Williams and Cline (thiamin synthesized) 

Clark, A. (1887) Observations on the anaemia or chlorosis of girls, occurring 



1024S 

SUPPLEMENT 

TABLE 2 

Grijns was also looking for a food material that when given 

in small amounts with polished rice, would prevent an outbreak 

Grijns’ key experiments1 

of polyneuritis. He tested the mung bean (which he had 

noticed was often included in chicken feed) and the soybean. 

The question asked The answer The results of his feeding experiments showed that both the 

skin and kernel of the mung bean prevented polyneuritis; how- 

1. Did a lack of minerals in a diet No, diseased chickens were ever, the soybean was less effective. Comparing the composiof 

white rice cause 

not cured. 

polyneuritis? 

tion of these two legumes, he saw that soybean was the richer 

2. Was the removal of fat with No, adding oil to the diet did in protein, fat and minerals but less effective as an anti-neuritic 

the silver skin the cause of the not prevent the disease. substance (Table 3). This supported his belief that polyneuritis 

disease? 

was not caused by a lack of these three nutrients. In later 

3. Was a lack of protein causing No, birds fed a supplement of experiments he found that extracts of mung bean were just as 

the disease? high protein soybean still labile as those from silver skin. He stated that ‘‘we therefore 

developed polyneuritis. 

4. Could the protective No, the extracts did not 

had the same experience with Phaseolus radiatus (mung bean) 

substance be extracted from prevent or cure the disease as with the seed coat of the rice . . . at every attempt to 

rice silver skin and mung 

because they decomposed isolate the active constituents, they perished . . . in different 

bean? during extraction. conditions they apparently became decomposed’’ (Grijns 

5. Was starch required to No, it appeared in chickens fed 1901). 

produce polyneuritis? just autoclaved meat. Eijkman had reported that the addition of some meat to 

1 Based on the paper by Grijns (1901). 

sago, tapioca and arenga starch diets did not prevent polyneuritis. 

However, removing starch and feeding meat alone did cure 

the condition. From these results, Eijkman had concluded that 

cal and pharmacological properties of the tannin contained in starch was a significant harmful factor in the etiology of poly- 

red rice’’ and to determine if the pigment found in red rice neuritis, but this explanation did not satisfy Grijns. He felt it 

could be considered as a curative or preventive remedy for important to determine whether polyneuritis could develop 

beriberi. 

independently of starch consumption. He therefore fed four 

Grijns was aware that it was not just red rice that prevented birds meat that had been extracted with water for 2 d, and all 

polyneuritis but all unpolished rice, and he decided to continue died with signs of polyneuritis. He then fed eight birds meat 

to study the whole silver skin and not just to focus on the that had been autoclaved, and six of these also developed 

pigment. His first feeding experiments confirmed Eijkman’s polyneuritis. Thus Grijns concluded that the development of 

conclusions that polyneuritis was not caused by a lack of fat, polyneuritis was not connected with starch and was even 

protein or mineral (Table 2). In his 1901 report, Grijns reexperiments 

also confirmed that the nerve degeneration was 

wholly independent of the presence of carbohydrate. These 

marked: ‘‘In judging the suitability of a food, we have not 

finished when we have determined the quantity of albumen not caused by a lack of protein (Table 2). 

. . . fat, carbohydrates and salts, even when we have applied In a discussion of polyneuritis and beriberi, Grijns put forth 

the corrections for digestibility. We can indeed calculate from two explanations for the symptoms that occurred: ‘‘either we 

this whether a balance of nitrogen will be possible with it and presume a deficiency, a partial starvation, . . . or . . . there 

whether the work which must be performed bother internally is a microorganism which exercises a degenerative influence 

and externally, can be obtained from it, but not whether perdeficiency 

or partial starvation, Grijns stated that very little 

on the nerves’’ (Grijns 1901). Concerning the possibility of a 

manent health is possible.’’ 

Grijns believed that a number of substances existed, whose was known about the metabolism of the peripheral nervous 

actions were not explained, but which played an important system and that ‘‘if for the maintenance of the peripheral 

part in the prevention of disease. He illustrated this idea with nervous system, a certain substance or group of substances is 

two examples: ‘‘how very difficult it is, in spite of all the indispensable, which are immaterial for the metabolism of the 

chemical analyses of mother’s milk, to find a good substitute muscles, then it may be assumed that very little of them is 

for it and how frequently we find that, when we think one necessary. When therefore in certain foods the substances in- 

has been found, we are again disappointed’’ and ‘‘the peculiar dispensable for the nervous system are lacking or are present 

fact that scurvy, which usually develops from lack of fresh in insufficient quantity, in the first place any reserve supply, 

food, which sometimes occurs on long sea voyages, is usually which is present either in the nerve itself or in the blood or 

cured when the patients can again obtain fresh meat and fresh in some other organ, will be used up . . . (and) disturbances 

greens.’’ He concluded that still-unknown substances may be will develop.’’ 

responsible. 

He explained that polyneuritis did not develop with total 

Grijns used two approaches for investigating these ‘‘unknown 

substances.’’ One was to prepare different fractions from 

the silver skin, and the other was by comparative assay (Grijns 

TABLE 3 

1901). He first boiled rice bran in a large quantity of water 

for 24 h and then strained, filtered and evaporated the liquor Composition of mung bean (P. radiatus java) and soybean 

to give a dried extract. He used fowls that were already consuming 

(S. hispida tumida java)1 

a polished rice diet and gave them the extract via a 

stomach tube. All the birds died with symptoms of polyneuritis. 

Mung bean 

Soybean 

Increasing the dose of the extract further had no effect; neither 

Albumin 21% 42% 

did feeding the residue from the extracted bran. Grijns con- Fat 4.1% 28% 

cluded that the ‘‘protective substances of the silver skin were Ash 3.6% 5.7% 

for the most part lost through the methods of preparation 

used.’’ 

1 Modified from table of analyses published by Grijns (1901). 




1025S 

starvation, because in this situation the muscles were drawn 

upon to provide the needed protein and that this process released 

TABLE 1 

the ‘‘protective substance,’’ which therefore became Some early dietary standards (‘‘minimum for average man, 

available to the nerves so that degeneration was prevented. under average conditions, doing moderate work, in health 

Grijns used the notion of individual differences to account for 

and strength’’)1 

why some birds did not develop polyneuritis: ‘‘one person 

needs a much larger quantity of food than another to maintain 

his physical equilibrium, while doing the same work . . . If 

Authority Protein Energy 

therefore the total metabolism shows important differences, 

there is no reason why, separate tissues which together furnish 

g 

kcal 

the total metabolism, should not have individual differences. Ranke 100 2324 

Munk 105 3022 

Therefore a food which contains just enough of the still un- 

Voit (1881) 118 3055 

known nerve nutritive substances for one fowl contains too Rubner 127 3092 

little for another.’’ Moleschott 130 3160 

In regard to the concept of a microorganism causing nerve Atwater (1894) 125 3315 

degeneration, Grijns believed that this depended on the nourishment 

of the tissue to resist infectious organisms. He concluded 

1 From McCay (1912), based on dietary intake studies. 

that, irrespective of the causal factor of polyneuritis, 

‘‘there occur in various natural foods substances which cannot supported this conclusion (Table 1). However, and in part 

be absent without serious injury to the peripheral nervous through the expanded use of the nitrogen balance approach 

system . . . The distribution of these substances in the differ- (developed initially by Boussingault for his studies in Alsace 

ent food stuffs is very unequal . . . Attempts to separate them on the utilization of foodstuffs by milch cows), others began 

have resulted in their disintegration . . . (showing) they are to question whether intakes lower than those shown in Table 

very complex’’ (Grijns 1901). 

1 would not only be adequate but possibly offer benefits for 

improvements in health. Arguably, the most significant of 


these others was Russell Henry Chittenden. Thus, Benedict 

(1906) says: ‘‘Of all the experiments heretofore made in which 

Carpenter, K. J. & Sutherland, B. (1995) Eijkman’s contribution to the discovery 

of vitamins. J. Nutr. 125: 155–163. 

exhaustive series of experiments recently completed by Profesthe 

low protein diet was used, none can compare with the 

Eijkman, C. (1990) Polyneuritis in chickens, or the origin of vitamin research. 

sor Chittenden of New Haven.’’ Indeed, they were impressive 

(Papers originally published in Geneeskd. Tijdschr. Ned.-Indië 30: 295–334 

(1890); 32: 353–362 (1893); 36: 214–269 (1896), van der Heij, D. G., transl.). and after the ‘‘dust had really settled’’ they were destined to 

Hoffman-la Roche, Basel, Switzerland. 

have an enduring and profound effect on the course of research 

Grijns, G. (1901) Over polyneuritis Gallinarum. Geneeskundig Tijdschrift v. 

in, and thinking about, human nutritional requirements. 

Ned-Indië 41: 1–110. [Reprinted in English translation in Grijns, G. (1935). 

Researches on Vitamins 1901–1911. J. Noorduyn en Zoon, Gorinchem, Holland. 

cal Chemistry at the Sheffield Scientific School at Yale Uni- 

In 1882, Chittenden was appointed Professor of Physiologiversity, 

two years after he had completed his Ph.D. degree in 

physiological chemistry, the first such degree awarded by a 

Paper 5: Dietary Protein Standards Can university in the United States (Vickery, 1945). 

Be Halved (Chittenden, 1904) 

The essentiality of a dietary substance, which was later 

named ‘‘protein’’ by the brilliant Swedish chemist Jac Berzelius 

(Korpes 1970), had been recognized by the middle of the 18th 

century by Beccari and by Haller (Munro 1969 and 1985). 

However, it was not until about a century later that definitive 

pronouncements were made about the dietary needs for proteins 

in human subjects. Thus, surveys of diets in the United 

Kingdom by Lyon Playfair, in Germany by Carl Voit, in the 

United States by Wilbur Atwater, as well as by others in other 

countries, revealed, in relation to protein intake and the total 

fuel value of the diet, that ‘‘all over the world people who can 

obtain such food as they desire use liberal rather than small 

quantities . . .’’ (Benedict 1906). It was from these kinds of 

data that conclusions were drawn about the necessary intakes 

of protein, and Voit, who commanded considerable attention 

and scientific respect, concluded that—based on his assessment 

of his work in Munich—the protein intake of the average 

working man should be 118 g daily and that higher intakes 

would be necessary for heavy workers. Atwater, a pupil of Voit, 

Presented by Vernon R. Young and Yong-Ming Yu, School of 

Science and Clinical Research Center, Massachusetts Institute of 

Technology, Cambridge, MA 02139 as part of the minisymposium 

‘‘Experiments That Changed Nutritional Thinking’’ given at Experimental 

Biology 96, April 16, 1996, in Washington, DC. 

Chittenden (1904) presented a detailed account of his series 

of experiments in a monograph entitled Physiological Economy 

of Nutrition: With Special Reference to the Minimal 

Proteid Requirement of the Healthy Man. An Experimental 

Study. This is remarkable considering that his experiments 

began only in 1902 and continued well into 1904 and that 

this occurred well before the convenience afforded by com- 

puter-based data retrieval and summary techniques, not to 

mention desktop publishing. In this publication, he indicates 

that he had first questioned the premise that the dietetic standards 

adopted by mankind represented the real needs or requirements 

of the body (p. 3, Chittenden 1904): ‘‘We may 

even question whether simple observation of the kinds and 

amounts of foods consumed by different classes of people under 

different conditions of life have any very important bearing 

upon this question.’’ He was the sort of mentor that any student 

would have been privileged to serve under: willing to 

challenge dogma and chart an entirely new experimental approach. 

His experiments began with an opportunity to observe for 

several months the dietary habits of Horace Fletcher, an Amer- 

ican of independent means. Chittenden noted that Fletcher’s 

nitrogen intake averaged 7.19 g, and in the words of Dr. An- 

derson, the director of the Yale Gymnasium, ‘‘Mr. Fletcher of 

Venice performs this work with greater ease and with fewer 

noticeable bad results than any man of his age and condition 

I have worked with’’ (p. 14, Chittenden 1904). 



1026S 

SUPPLEMENT 

TABLE 2 

the body, certainly under ordinary conditions of life’’ (p. 475, 

Chittenden 1904). 

Perhaps it ought to be noted that these experiments did 

not actually establish an average, minimum physiological re- 

quirement for dietary protein in these groups of subjects because 

1) the low protein diets were freely chosen by the professional 

group, 2) the athletes were instructed to diminish the 

intake of protein but without imposition of a specific diet, 

and 3) the soldiers were given meals that contained lowered 

amounts of protein than provided by ordinary army rations 

and apparently with some reduction in the total ‘‘fuel value’’ 

of the food. The food given to each soldier was weighed, and 

at the close of every meal the uneaten food was determined 

and subtracted from the initial weight. 

Although there has been some concern about the precision 

and accuracy of the nitrogen balance data generated from 

Chittenden’s experiments (McCay 1912, Carpenter 1994), including 

the reliability of the urine and fecal collections and 

determination of nitrogen intake, the results of these series of 

experiments were coherent and dramatic. They presented a 

strong case that the physiological needs for protein were much 

lower than values represented by free-choice intakes of dietary 

protein. 

Chittenden’s conclusions were neither quickly nor univer- 

sally accepted. For example, McCay (1912) referred to the 

onslaught to Chittenden’s findings and ideas, but he used his 

own data from dietary surveys of Bengalis, as well as the data of 

others, to reach a conclusion that ‘‘Voit stands today absolutely 

vindicated.’’ Although Cathcart (1911) was in ‘‘complete 

agreement with Professor Chittenden’s statement that life can 

be maintained and frequently maintained at a high level on 

relatively low protein intake,’’ he was not sure if it was desir- 

able to keep a low intake as a general rule, and he expressed 

concern about the quality of protein. Later, he (Cathcart 1921) 

voiced reservations that were related to the lowered resistance 

to disease in persons consuming low protein diets. However, 

Chittenden (1911) had already argued that the problem with 

McCay’s studies was that the diets of the populations studied 

in India lacked unidentified trace nutrients. This was probably 

true, in retrospect, given the public health problems of iron, 

vitamin A and iodine deficiencies that are prevalent in south- 

east Asia today. 

The protein standards for healthy individuals continued 

to be set by the opinions of individuals until national and 

international committees were convened to establish dietary 

recommendations. An early international committee was set 

up by the League of Nations, and in 1936 the recommendation 

was that ‘‘the protein intake for all adults should not fall below 

1 gramme of protein per kilogramme of body weight . . .’’ 

(League of Nations 1936). No scientific justification was presented 

in support of the recommendation. In 1943 the U.S. 

Food and Nutrition Board of the National Academy of Sci- 

ences issued its first Recommended Dietary Allowances, and 

in this report 66 g of protein daily was recommended. These 

early figures proposed by expert groups were somewhat higher 

than those found to be sufficient by Chittenden, but they were 

far below the liberal standards that had been widely adopted 

during the middle 19th and on into the early part of the 

20th century. It seems clear, however, that by the 1950s the 

metabolic approach used by Chittenden and others for establishing 

protein requirements had been well embraced, to the 

exclusion of the dietary intake approach followed by Voit, 

Atwater and others. For example, at the Princeton Conference 

in 1955, W. R. Aykroyd, the director of the nutrition division 

of the Food and Agriculture Organization, stated: ‘‘We need 

A typical day’s record of R. H. Chittenden’s diet and nitrogen 

balance after 18 mo on his self-imposed experiment1 

Sunday, June 26, 1904 

Diet 

Breakfast: Coffee 122 g, cream 31 g, sugar 8 g. 

Dinner: Roast lamb 50 g, baked potato 52 g, peas 64 g, biscuit 

82 g, butter 12 g, lettuce salad 43 g, cream cheese 21 g, 

toasted crackers 23 g, blanc mange 164 g. 

Supper: Iced tea 225 g, sugar 29 g, lettuce sandwich 51 g, 

strawberries 130 g, sugar 22 g, cream 40 g, sponge cake 31 g. 

Body weight Å 57.4 kg (initial weight in November 1902 Å 65 kg. 

Age 47 y) 

Protein intake Å 0.64 g/kg 

N balance Å00.07 g N 

Energy intake Å 1549 kcal 

1 Data from Chittenden (1904). 

Back then was no different than today, in that Chittenden 

needed financial support for the conduct of his investigations. 

He secured funds from the Carnegie Institution of Washington 

and the Bache Fund of the National Academy of Sciences, 

and he also received large donations from Fletcher and John 

H. Patterson of Dayton, Ohio. 

The overall investigation consisted of three major experi- 

ments, each characterized by a long-term period of dietary 

protein restriction combined with nitrogen excretion measure- 

ments and supplemented in some studies with assessments of 

physical and mental well-being. Chittenden (1904) states 

‘‘The writer, fully impressed with his responsibility in the conduct 

of an experiment of this kind, began with himself in 

November 1902.’’ He therefore served as one of the subjects 

in his study of five university professors and instructors, includ- 

ing his former student Lafayette Mendel, who by that time 

had become professor. 

On the basis of his own experience (Table 2), including the 

disappearance of the rheumatic problem he had been having 

in his knee joint and the findings with the other four Yale 

professionals, Chittenden concluded that the minimum ‘‘proteid’’ 

requirement was 93–103 mg N/kg body wt (about 0.6– 

0.64 g proteinrkg 01 rd), which anticipates, by 80 years, the 

mean requirement figure of 0.6 g proteinrkg 01 rd 01 propos- 

ed by FAO/WHO/UNU (1985)! 

The next two series of studies confirmed and strengthened 

these initial findings; one of these was with 13 members of a 

detachment from the U.S. Army Hospital Corps, who were 

housed in Vanderbilt Square at Yale for 6 mo. The study 

included measures of physical and mental condition and of 

blood composition in addition to nitrogen excretion and bal- 

ance over the 6-mo period. The conclusion was that 50 g of 

protein daily can establish nitrogen equilibrium and that there 

is, at this approximate intake, a maintenance of physical 

strength and vigor and an ability to respond to sensory stimuli. 

In a follow-up letter written to Chittenden by one of the 

participants, Private First Class J. Steltz, and on behalf of the 

other men, he stated: ‘‘The men are all in first-class condition. 

. . . We eat very little meat now as a rule, and would willingly 

go on another test.’’ The extensive findings in a third series 

involving eight Yale University athletes merely served to repli- 

cate all of these data and the interpretations that had been 

drawn from them. 

Thus, Chittenden concluded that one-half of the 118 g of 

protein called for daily by the ordinary dietary standards is 

quite sufficient to meet all the real physiological needs of 




1027S 

not linger on Carl Voit and his recommendations on protein Vickery, H. B. (1945) Russell Henry Chittenden. 1856–1943. National Acad- 

emy of Sciences USA, Biographical Memoirs, Volume 24 (Second Memoir), 

requirements, which were over influenced by what was obpp. 

59–103. National Academy of Sciences, Washington, DC. 

served among the population of Munich in 1880’’ (FAO 

1957a). Indeed, the first FAO report specifically concerned 

with protein requirements (FAO 1957b) depended heavily Paper 6: Liebig’s Concept of Nutritional 

upon the review by Sherman et al. (1920) of the literature 

on nitrogen balance and adult requirements, which included 

Adequacy Challenged (Hart et al., 1911) 

extensive reference to Chittenden’s work. Parenthetically, Presented by Alfred E. Harper, University of Wisconsin, Madison, 

Sherman’s paper might be viewed as a forerunner of modern- WI, as part of the minisymposium ‘‘Experiments That Changed 

day meta-analysis! In any event, this 1955 FAO committee Nutritional Thinking’’ given at Experimental Biology 96, April 16, 

suggested that the average minimum requirement of adults for 

1996, in Washington, DC. 

reference protein was 0.35 g/kg body wt and proposed a daily 

safe practical allowance of 0.66 g/kg. Although the more recent Imagine that we have fallen back 90 years through time. It 

recommendations (FAO/WHO/UNU 1985) differ from those is 1906. We know that protein and a few minerals (sodium, 

given in the 1957 report of FAO, this latter assessment un- potassium, calcium, phosphorus, iron) are essential nutrients, 

doubtedly would have given Chittenden great satisfaction, and but we are unaware of the essentiality of trace elements, vitait 

served as a vindication of the data obtained and conclusions mins or fatty acids. Liebig’s concept from the 1850s that prodrawn 

from his visionary studies, commenced 50 years earlier tein, a few minerals, and sources of energy (fat and carbohyin 

the former New Haven residence of Joseph E. Sheffield. drates) are the sole principles of a nutritionally adequate diet 

Although Chittenden explored and made important contri- still dominates nutritional thinking and is widely accepted by 

butions in the areas of digestive physiology and the action of leaders in the field, including Voit in Germany and Atwater 

proteolytic enzymes, an activity that had been enhanced by and Langworthy at the U.S. Department of Agriculture 

his year-long sojourn with Kühne in Heidelberg, and in toxi- (Harper 1993). Although several investigators have quescology, 

including heavy metal poisoning and disorders created tioned the validity of Liebig’s concept, their reports lie buried 

by alcohol, it was his studies of the protein requirements of in the scientific literature (McCollum 1957, p. 201). 

humans that may well be regarded as his greatest contribution E. B. Hart has just been appointed Professor of Agricultural 

to the advancement of nutritional science. When Chittenden Chemistry at the University of Wisconsin to succeed S. M. 

died on Boxing Day (December 26) 1943, he had been a Babcock. Babcock has observed that milk production by cows 

member of the National Academy of Sciences for more than consuming rations composed of different feedstuffs differs con- 

53 years! siderably even when the rations are formulated to have the 

same gross composition (proximate analysis). He tells Hart 


that he is skeptical of Liebig’s claim that the ‘‘physiological 

value’’ of a ration can be predicted from knowledge of its gross 

Benedict, F. G. (1906) The nutritive requirements of the body. Am. J. Physiol. chemical composition (Hart 1932) and encourages Hart to 

16: 409–437. 

Carpenter, K. J. (1994) Protein and Energy. A Study of Changing Ideas in Nutritest 

Liebig’s hypothesis. 

tion. Cambridge University Press, New York, NY. 

In 1907, in collaboration with G. C. Humphrey of the Ani- 

Cathcart, E. P. (1911) Discussion. Br. Med. J. 11: 664. mal Husbandry Department, Hart plans what will come to be 

Cathcart, E. P. (1921) The Physiology of Protein Metabolism. Longmans 

Green, London, U.K. 

known as the ‘Wisconsin single grain experiment’. He hires 

Chittenden, R. H. (1904) Physiological Economy in Nutrition: With Special Reference 

to the Minimal Proteid Requirement of the Healthy Man. An Experimen- Steenbock as a student assistant. The objective of the experi- 

E. V. McCollum to conduct the chemical analyses and Harry 

tal Study. Frederick A. Stokes Co., New York, NY. 

ment is to compare the performance of four groups of heifers 

Chittenden, R. H. (1911) Discussion on the merits of a low protein diet. Openfed 

rations composed entirely of the corn, wheat or oat plant 

ing paper. Br. Med. J. 11: 656–662. 

FAO (1957a) Human Protein Requirements and Their Fulfillment in Practice. or a mixture of the three, all formulated to be closely similar 

Proc. Conf. in Princeton, U.S. (1955) (Waterlow, J. C. & Stephen, J.M.L., eds.), 

in gross composition and energy content (Hart et al. 1911). 

p. 2. FAO Nutrition Meetings Report Series no. 12, Food and Agriculture 

Organization of the United Nations, Rome, Italy. 

The animals, 16 Shorthorn heifers about 6 mo of age and 

FAO (1957b) Protein Requirements. FAO Nutritional Studies, no. 16. Food and weighing 300–400 pounds (lb), are to be carried to maturity 

Agriculture Organization of the United Nations, Rome, Italy. 

and through two consecutive reproductive periods. The four 

FAO/WHO/UNU (1985) Energy and Protein Requirements. Report of a Joint 

FAO/WHO/UNU Expert Consultation. World Health Organization Technical rations, designed to provide 14 lb dry matter/d, consist of the 

Report Series 724, World Health Organization, Geneva, Switzerland. following (lb/d): corn meal 5, corn gluten 2, corn stover 7; 

Food and Nutrition Board (1943) Recommended Dietary Allowances. National oat meal 7, oat straw 7; ground wheat 6.7, wheat gluten 0.3, 

Research Council Reprint and Circular Series, no. 115. National Academy of 

Sciences, Washington, DC. 

wheat straw 7; and a mixed ration consisting of equal parts of 

Korpes, J. E. (1970) Jac Berzelius. His Life and Work. Almquist & Wiksell, the other three. 

Stockholm, Sweden. 

The average amounts consumed by the four groups during 

League of Nations (1936) The Problem of Nutrition. Report on the Physiological 

Bases of Nutrition, Vol. II. Technical Commission of the Health Committee, 

the course of the experiment (14.5 to 15.2 lb/d) did not differ 

official no. A.12(a).IIB, League of Nations Publications Department, Geneva, appreciably. Values for crude digestibility of the different ra- 

Switzerland. 

tions averaged 65 { 3% for both dry matter and nitrogen and 

McCay, D. (1912) The Protein Element in Nutrition. Edward Arnold, London, 

were not significantly different. 

U.K. 

Munro, H. N. (1969) Historical introduction: the origin and growth of our present 

concepts of protein metabolism. In: Mammalian Protein Metabolism (Mu- Table 1. Although the corn-fed group gained considerably 

Weight gains of the groups after 1 and 3 y are shown in 

nro, H. N. & Allison, J. B., eds.), Vol. 1, pp. 1–29. Academic Press, New York, 

more weight than the wheat-fed group, variability within 

NY. 

Munro, H. N. (1985) Historical perspective on protein requirements: objectives groups was such that, as can be seen from the large SD, the 

for the future. In: Nutritional Adaptation in Man (Blaxter, K. & Waterlow, J. C., results did not provide convincing evidence that the growth 

eds.), pp. 155–167. John Libbey, London, U.K. 

responses were different. 

Sherman, H. C., Gillett, L. H. & Osterberg, E. (1920) Protein requirement of 

maintenance in man and the minimum efficiency of bread protein. J. Biol. The first clear evidence of differences in the responses of 

Chem. 41: 97–109. 

the groups was from observations on the appearance of the 



1028S 

SUPPLEMENT 

TABLE 1 

ration could not be predicted reliably from measurements of 

its total digestible nutrients and energy content, 2) the differences 

Weight gain of heifers fed single grain rations1 

in performance were unlikely to be due to differences 

in the protein component because animals fed the diet that 

Group Year 1 Year 3 provided a mixture of proteins did not grow as well as some 

of those fed the single grain diets, and 3) mineral inadequacies 

pounds 

were unlikely because the mineral supplement did not improve 

Corn 471 { 64 726 { 55 

Oat 408 { 55 824 { 89 the performance of cows consuming the wheat ration. They 

Mixture 410 { 37 724 { 149 did not claim to have eliminated these possible explanations 

Wheat 353 { 67 665 { 86 conclusively. 

They stated ‘‘we have no adequate explanation of our re- 

1 Values are means { SD. Adapted from Hart et al (1911). sults.’’ They did not attribute the inferior performance of the 

wheat-fed group to a deficit of some unidentified essential 

animals after the first year. Cows consuming the corn ration nutrient. They did, however, propose that the physiological 

had smooth coats, were full through the chest, and appeared value of a food or feed could be determined by measuring 

healthy. Those consuming the wheat ration had rough coats, growth or other responses of animals fed diets in which a 

were of smaller girth, and appeared gaunt. The other two portion of a basic ration was replaced by the product to be 

groups were intermediate between the corn- and wheat-fed tested. 

groups. 

Is it possible, with hindsight, to identify specific nutritional 

There were major differences in reproductive performance deficits that can account for the inferior performance of the 

(Table 2). Calves born to cows consuming the corn ration cows fed the wheat ration? Levels of calcium and magnesium 

were strong and vigorous in both years and all lived. The cows were low in the wheat ration. A level of 0.16% of magnesium 

consuming the wheat ration all delivered 3–4 wk prematurely. is adequate for dairy cattle, but the level of 0.16% for calcium 

Their calves were weak both years, and none lived beyond 12 is marginal (Shepherd and Converse 1939) and 0.3% is now 

d. Again, results for the other two groups were intermediate. recommended (Scott 1986). Nonetheless, the reproductive 

In 1909, the first year of calving, cows consuming the oat and performance of cows consuming the wheat ration, as Hart and 

mixture rations produced weak calves, with only two from the colleagues noted, was not improved when they were given 

oat group and one from the mixture group living beyond a few supplements of calcium and magnesium. Also, the fat content 

days. In 1910, the performance of these two groups was better. of the wheat ration was low. Dairy cattle require at least 2% 

The calves were carried to term, and all of the calves from of fat (Shepherd and Converse 1939). Low milk production, 

the oat-fed group and two from the mixture-fed group lived as was observed in the wheat-fed group, is an early sign of 

but were weaker than those from the corn-fed group. Average fatty acid deficiency in lactating dairy cows. 

weights of the calves, were 78, 72, 62 and 49 lb for groups fed In addition, the carotenoid content of forage crops declines 

corn, oat, mixture and wheat, respectively. 

during storage, and vitamin A depletion occurs commonly in 

Milk production of each group was measured for 30 d each cows maintained during the winter on forage that has been 

year following cessation of colostrum secretion. The difference stored for several months. A predominant sign of vitamin A 

between the amounts of milk produced by the wheat- and deficiency in dairy cows is premature calving, with the calves 

corn-fed groups was large (Table 1). Average values for the often born dead or surviving for only a short time. Reproduc- 

groups (lb/d { SD, number of observations in parentheses) tive performance of cows receiving the corn ration, which 

were as follows: corn-fed group, 26 { 3.5 (8); oat-fed group, would be expected to provide a high level of carotenoids, was 

22.9 { 6.5 (6); mixture-fed group, 20.4 { 1.5 (5); wheat-fed excellent. That of the cows fed the wheat ration was poor. 

group, 14.1 { 2.6 (4). (In calculating the average for the McCollum (1964) attributed this to loss of most of the leaves 

wheat-fed group I deleted two values, one for a cow that died of the wheat plant during threshing. However, reproductive 

of illness, and one abnormally low value). No differences were performance of the oat-fed group was poor the first year but 

observed in milk composition (total solids, total protein, catent 

of feedstuffs varies from year to year. It would thus seem 

much better the second, suggesting that the carotenoid con- 

sein, ash or fat) or in the characteristics of the milk fat. 

The wheat ration was known from chemical analyses to 

provide less calcium, magnesium and potassium than the other 

rations. Two cows in the wheat-fed group were therefore given 

TABLE 2 

a supplement of these minerals during 1 y of the study to raise 

their levels of intake to those provided in the diets of the 

Condition and survival of calves1 

other groups. The condition of the cows was not noticeably 

improved. The calf produced by one of them was small and 

Group 1909 1910 

weak and lived only a few hours. 

Corn 

Strong and vigorous both years; all lived 

After the experiment was completed, some animals were 

6d premature 

None premature 

Oat All weak 3 fairly strong, 1 weak 

switched to other rations for an additional year (1910–1911). 2 died, 2 lived All lived 2.5 d premature 

The vigor and health of one cow that was switched from wheat 

12.5 d premature 

to corn improved rapidly. It produced a calf weighing 81 lb, Mixture 1 fair, 1 weak 1 weak, 1 fairly strong 

compared with 47 and 48 lb for calves produced the previous 

2 died, 1 lived 1 born dead, 2 lived 

2 y. One cow that was switched from the corn diet to the 

1 aborted 

wheat ration supplemented with extra calcium, magnesium 

12 d premature 4 d premature 

Wheat 3 weak, 1 stillborn All weak. 2 cows died 

and potassium deteriorated. Its calf was stillborn 18 d prema- None lived ú12 d None lived 

turely and weighed only 36 lb, compared with 93 and 85 lb 26 d premature 24 d premature 

for those born the previous 2 y. 

The authors concluded that 1) the nutritive value of a 

1 Adapted from Hart et al (1911). 




1029S 

highly probable that differences in reproductive performance At Wisconsin, McCollum’s initial major responsibility was 

of the groups were due to differences in vitamin A status, that of analytical chemist in a single-grain feeding trial with 

owing to differences in the carotenoid content of the rations, heifers, started in early 1907, at the suggestion of Professor 

possibly complicated in the case of the wheat-fed group by an Emeritus S. M. Babcock. The trial, directed by E. B. Hart to 

inadequate intake of fat and a marginal intake of calcium. whom McCollum was directly responsible throughout his 10 

The results of this experiment provided the first clear evi- years at Wisconsin, consisted of comparing the responses of 

dence from research in the United States that the nutritive four groups fed different rations. Three of the groups were fed 

value of a diet depended on factors other than its content of exclusively rations prepared from a single cereal grain plant: 

protein, a few minerals, and energy sources. It sounded the corn, oats or wheat. The fourth group received a mixture from 

deathknell for Liebig and Voit’s concept of a nutritionally all three plant sources. As judged by the proximate method of 

adequate diet. It contributed, together with observations by chemical analysis for estimating nutritional adequacy, all four 

European investigators on the inadequacies of purified or re- rations should have been of similar value. However, the calves 

stricted diets for chickens, rats and guinea pigs (see introduc- from the wheat-fed group all died. 

tion to this symposium), to a sharp change in the direction of In his autobiography, McCollum (1957) pointed out that 

thinking about the nutritional essentiality of constituents of he and others connected with the project had overlooked the 

foods. Maynard (1962) stated that the findings of Hart and fact that, in harvesting the crops used to prepare the experihis 

associates ‘‘stimulated much of the work which resulted in mental rations, the leaves of the corn and oat crops largely 

later discoveries on vitamins, amino acids, and trace minerals.’’ remained intact. However, the wheat leaves were so fragile 

During the course of the experiment, Professor Hart initi- that virtually all were lost. Thus, ‘‘We actually fed [some of] 

ated projects to investigate metabolism of minerals and of the cows wheat grain and straw.’’ In the many other references 

organic substances in animals. These continued at Wisconsin to this noted experiment the fortuitous flaw has been overfor 

over 50 years (Elvehjem 1953). From them came the contri- looked. 

butions of Hart and associates to the knowledge of trace miner- The failure of the wheat diet, together with his concurrent 

als, especially of iodine and copper; the discoveries of McCol- and diligent searching of the available literature—especially 

lum and associates of the nutritional essentiality of fat-soluble the abstracts in Maly’s yearbooks—persuaded McCollum as to 

A and water-soluble B, and that there were two essential fat- the real possibility of some indispensable nutrients remaining 

soluble factors, A and D; and the contributions of Steenbock unrecognized. This, plus his discussions with Dr. Babcock, conand 

associates to knowledge of vitamin D and their discovery vinced him that the heifer feeding project would not alone 

that vitamin A activity was associated with the yellow carot- yield really significant new knowledge, but it stimulated him 

enoid pigments of plants (Ihde 1965). 

to think and move in a new and fertile direction. After some 

four months he suggested to Dean Henry and Dr. Hart that 


‘‘the most promising approach to the study of the nutritional 

requirements of animals was through experiments with small 

Elvehjem, C. A. (1953) Edwin Bret Hart. J. Nutr. 51: 1–14. animals fed simplified diets composed of purified nutrients. 

Harper, A. E. (1993) Nutritional essentiality: historical perspective. In: Nutritional 

Essentiality: A Changing Paradigm (Roche, A. F., ed.), pp. 3–11. Report Small animals were desirable because they eat little, and we 

of the Twelfth Ross Conference on Medical Research, Ross Products Division, could do the extensive chemical work necessary for purifying 

Abbott Laboratories, Columbus, OH. 

the dietary ingredients. Small animals grow rapidly to maturity, 

Hart, E. B. (1932) Obituary of Stephen Moulton Babcock. J. Assoc. Off. Agric. reproduce at an early age, and have a short span of life. . . . 

Chem. (Feb.): iii–v. I recommended using rats . . .’’ (McCollum 1964). 

Hart,E. B.,McCollum,E. V.,Steenbock,H.&Humphrey,G. C. (1911) Physiological 

effect on growth and reproduction of rations balanced from restricted They opposed the use of rats in an agricultural experiment 

sources. Univ. Wis. Agric. Exp. Stn. Res. Bull. 17: 131–205. 

station, but Babcock encouraged him and McCollum pro- 

Ihde, A. I. (1965) The basic sciences in Wisconsin. Wis. Acad. Trans. 54 (part 

ceeded on his own, without discontinuing any of his other 

A): 33–41. 

Maynard, L. A. (1962) Early days of nutrition research in the United States of pressing work. He procured ‘‘a dozen young albino rats from a 

America. Nutr. Abs. Rev. 32: 345–355. 

pet-stock dealer in Chicago and paid for them myself’’ 

McCollum, E. V. (1957) A History of Nutrition. Houghton-Mifflin, Boston, MA. 

(McCollum 1964). Owing to his meager resources and limited 

McCollum, E. V. (1964) From Kansas Farm Boy to Scientist. University of Kanknowledge 

in dealing with small experimental animals, he 

sas Press, Laurence, KS. 

Scott, M. L. (1986) Nutrition of Humans and Selected Animal Species. Wiley, began to learn through trial and error. His extensive reading 

New York, NY. 

program had given him some acquaintance with the Pavlovian 

Shepherd, J. B. & Converse, H. T. (1939) Practical feeding and nutritional reideas 

on relationships between the taste of food and its digestquirements 

of young dairy stock. In: Food and Life. Yearbook of Agriculture, 

pp. 597–638. U.S. Department of Agriculture, Washington, DC. 

ibility. Thus he focused for a while on adding to the purified 

diets different flavors pleasant to people. No promising results 

were obtained. However, this and other experiences advanced 

Paper 7: Young Rats Need Unknown 

his wisdom in the selection of research hypotheses and the 

Growth Factors (McCollum, 1913–1917) planning of experiments. 

Most fortunately, two years after he had gone to Wisconsin, 

Presented by Harry G. Day, Department of Chemistry, Indiana 

he was voluntarily and unexpectedly joined by a young woman 

University, Bloomington, IN 47405-6203 as part of the minisympresident 

of Madison, Marguerite Davis, who had recently gradsosium 

‘‘Experiments That Changed Nutritional Thinking’’ given 

at Experimental Biology 95, April 11, 1995, in Atlanta, GA. 

uated in chemistry at the University of California at Berkeley, 

and asked if she might take care of the rats. 

In July 1907, Elmer Verner McCollum began his academic There soon began the experiments that were in time identicareer 

as instructor in agricultural chemistry at the University fied as McCollum’s biological method for the analysis of food. 

of Wisconsin, after completing A.B. and M.S. degrees in chem- 

The first definitive publication of research in which Miss Davis 

istry at the University of Kansas and a Ph.D. degree in organic 

chemistry plus a year of postdoctoral work in physiological 

chemistry at Yale University. 

had a significant role was in establishing the ‘‘necessity of 

certain lipins in the diet’’ (McCollum and Davis 1913). This 

was an unexpected outcome in the early phases of their exten- 



1030S 

SUPPLEMENT 

Two years later, McCollum and his graduate student Cornelia 

Kennedy suggested the provisional use of alphabetical terms 

for this lipin and other essential ‘‘organic complex(s),’’ using 

a prefix designating characteristic solubility. Thus for this essential 

factor(s) they proposed the term ‘‘fat-soluble A’’ 

(McCollum and Kennedy 1916). Soon this gave way to the 

term ‘‘vitamin A.’’ 

McCollum and Davis also focused on discovering the nature 

of the nutritional deficiencies of the cereal grains. Particular 

attention was given to the nature of the dietary deficiencies 

of rice. In their article were 42 growth charts (McCollum and 

Davis 1915). Each chart portrayed the growth for each rat in 

an experimental lot, the composition of the ration used, the 

kind of test substance (accessory) added, if any, etc. Some of 

the results are summarized in Table 1. In their introduction 

they stated in part: 

‘‘In the present communication we present experimental data 

showing the specific properties of polished and of unpolished 

rice as a food, and show the supplementary relationship between 

these and certain purified and naturally occurring foodstuffs 

. . . . These studies, in addition to extending our knowledge 

concerning the dietary position of rice, have contributed 

to our understanding of the factors involved in normal nutrition, 

especially as regards the unknown accessory constituents 

of the diet which have received so much attention in recent 

years in connection with the ‘deficiency diseases’, scurvy and 

beri-beri.’’ 

FIGURE 1 Prepared from McCollum’s growth charts. This male A substantial proportion of the research was on ‘‘the supplerat 

received 3% cottonseed oil in its diet for the 7 wk of period 1 mentary relationship between certain extracts of naturally ocand 

then received 3% olive oil that had been shaken with soaps from 

curring foodstuffs and polished rice’’ in which most of the 

saponified butter fat. The dotted line represents the normal growth rate 

of these rats (from McCollum and Davis 1914, chart 2, rat B). 

extractants employed were water, ethanol and acetone 

(McCollum and Davis 1915). 

This was in regard to answering the question of the presence 

of ‘‘water-soluble accessory’’ in polished and unpolished rice. 

sive biological analysis of foods. In this paper they began by 

Water extract of wheat embryo was far superior to an acetone 

stating that: 

extract in supplementing a diet based on polished rice. McCol- 

‘‘During the past year we have been engaged in a study of the 

influence of the composition and quantity of the inorganic lum and Davis (1915) concluded that ‘‘such knowledge, when 

content of the ration on the growth of the rat. In this work available for a wide variety of foodstuffs must, we believe, be 

we have employed rations compounded of pure casein, carbowill 

promote health.’’ Thus this study contributed to the illu- 

of great value in the formulation of human dietaries which 

hydrates, and salt mixtures made up of pure reagents, and the 

same rations in which a part of the carbohydrate was replaced mination of all their studies on cereal grains. While their work 

by lard, with a considerable degree of success. . . .’’ 

In their commentary on these germinal experiences with rats 

fed such rations they wrote: 

TABLE 1 

‘‘The fact that a rat of 40 to 50 grams in weight can grow 

normally during three months or more on such rations, then 

Growth of young rats fed polished rice with 

cease to grow but maintain its weight and a well nourished 

different supplements1 

appearance for weeks and then resume growth on a ration 

containing certain naturally occurring food-stuffs would lead 

Lot (no. of male and female rats) 

one to the belief that on these mixtures of purified food substances 

the animals run out of some organic complex which is 308 316 317 383 395 

indispensable for further growth but without which mainte- (5 M, 1 F) (6 M) (6 M, 1 F) (3 M, 2 F) (4 F) 

nance in a fairly good nutritive state is possible.’’ (See Fig. 1.) 

In this article they had shown that butter fat and eggs Approx. initial 

contain the ‘‘lipins’’ and that none exists in lard or olive oil. 

weight g 100 40 60 60 40 

Also, they showed that the organic complex was extractable Diet 

with ether. In 1914 they reported that the new fat-soluble Polished rice 96 91 91 — 82 

factor was transferred to olive oil by the following procedure: Unpolished rice — — — 88 — 

‘‘Butter fat was saponified with potassium hydroxide in alcohol. 

Rice polishings — — — — 10 

The resulting soap was dissolved in water and olive oil was 

Salt mix 4 4 4 2 3 

thoroughly emulsified in the soap solution. The olive oil was 

Egg albumen — 5 — — — 

of the same sample which had been tested on rats and found 

Casein — — — 5 — 

to be of no value in protecting them against decline on the 

Butter fat — — 5 5 5 

basal diet. The emulsion was then broken with ether, and the Approx. weight 

olive oil was recovered in that solvent. After evaporating the gain in 8 wk, g 0 or less 0–15 loss 65 50 

ether, the olive oil was found by a feeding test to have acquired 

the nutritive quality of the butter fat.’’ (McCollum and Davis 1 Based on data in five different growth charts selected from 42 

1914) 

published by McCollum and Davis (1915). 




1031S 

on rice was in progress they learned more about the findings 

of E. B. Vedder and others on beriberi, and they ‘‘identified 

TABLE 1 

the nutritive value of the substance in their water extracts Mean 3-wk response of young rats receiving gliadin ({lysine) 

of wheat germ and egg yolk with the anti-beriberi factor’’ 

(McCollum 1957). 

as their protein source1 

McCollum presented these findings in a lecture before the 

Diet 1 Diet 2 (17.64% 

prestigious Harvey Society in New York in 1917 and concluded 

by stating: ‘‘These [supplements] have peculiar [not yet under- 

(18% gliadin) gliadin, 0.36% lysine) 

stood] dietary properties which the best chemical talent of No. rats 4 2 

Food eaten, g 99 149 

today fails to recognize, but which are readily demonstrable Lysine, from gliadin,2 mg 160 240 

by biologic tests’’ (McCollum 1917). Lysine, from supplement, mg — 535 

Five years later, in the second edition of his book The Newer Total lysine intake, mg 160 775 

Knowledge of Nutrition (McCollum 1922) he wrote: 

Weight gain, g /6.2 /35.2 

‘‘The biological method . . . was first developed with a view 

Est. protein gain,2 g /0.99 /5.63 

to discovering the nature of the deficiencies of individual natu- 

Est. Lysine gain,2 mg /79 /450 

ral food-stuffs. For this purpose the food under investigation is 

the principal component of the diet and is supplemented with 1 Data from Osborne and Mendel (1916) 

small additions of one or more purified food substances (e.g., 

2 Assuming 0.92% lysine in gliadin, 8.0% in rat body proteins, and 

protein, inorganic salts, vitamins) [but even in 1922 no such 16% protein in the weight gain by the rats. 

isolated and chemically identified nutrient had been reported], 

in order to bring to light the nature of the additions which 

enhance its value. The method is applicable in another modi- 12 years the younger, was the son of Jewish immigrants running 

fication, however, which has yielded much valuable informa- a clothing store in New Haven and a brilliant student at Yale. 

tion concerning the relative values of many of our more im- After two years of postdoctoral work in Germany, he came 

portant foods with respect to any one dietary constituent.’’ back on to the faculty, loved teaching and, unusually for his 

The final and most comprehensive presentation on the bio- time, encouraged young women as well as men to do graduate 

logical method for the analysis of foods is in his history of work. His field was metabolism (Chittenden 1938). 

nutrition (McCollum 1957). In 1909, when they began to collaborate, Osborne was 50 

years old and had already spent over 20 years isolating different 


vegetable proteins and demonstrating their chemical individuality 

and, in particular, how different they were in amino acid 

Day, H. G. (1974) Elmer Verner McCollum 1879–1967, A Biographical Memoir. 

Biographical Memoirs 45: 263–335. National Academy of Sciences, Washington, 

composition from animal proteins. Did that mean that they 

DC. 

were necessarily nutritionally inferior? This was, obviously, of 

McCollum, E. V. (1917) The supplementary dietary relationships among our 

natural foodstuffs. Harvey Society Lecture Series 12: 151–180; also in J. Am. 

interest to Mendel, too, because he had been part of the big 

Med. Assoc. 68: 1379–1386. 

Yale study a few years earlier that used human subjects and 

McCollum, E. V. (1922) The Newer Knowledge of Nutrition: The Use of Food ended with the recommendation that Atwater’s dietary stanfor 

the Preservation of Vitality and Health, 2d ed. Macmillan, New York, NY. 

dards for protein could be halved from 120 g to about 60 g for 

McCollum, E. V. (1957) A History of Nutrition. The Sequence of Ideas in Nutrithe 

average man, with no qualification as to the type of protein. 

tion Investigations. Houghton Mifflin, Boston, MA. 

McCollum, E. V. (1964) From Kansas Farm Boy to Scientist. The Autobiogra- By 1909, several European workers had reported that enphy 

of Elmer Verner McCollum. University of Kansas Press, Lawrence, KS. 

zyme digests of proteins could support nitrogen balance in 

McCollum, E. V. & Davis, M. (1913) The necessity of certain lipins in the diet 

during growth. J. Biol. Chem. 15: 167–175. 

adult rats and dogs, but that acid hydrolysates could not do so 

McCollum, E. V. & Davis, M. (1914) Observations on the isolation of the sub- (Henriques and Hansen 1905). This was attributed to the 

stance in butter fat which exerts a stimulating effect on growth. J. Biol. Chem. 

destruction of tryptophan by acid digestion, and it was hypoth- 

19: 245–250. 

McCollum, E. V. & Davis, M. (1915) The nature of the dietary deficiencies of esized that, although animals could manufacture linear amino 

rice. J. Biol. Chem. 23: 181–230. 

acids, only the plant kingdom could synthesize cyclic ones 

McCollum, E. V. & Kennedy, C. (1916) The dietary factors operating in the such as tryptophan (Abderhalden 1909). 

production of polyneuritis. J. Biol. Chem. 24: 491–502. 

Osborne and Mendel were skeptical of short-term nitrogen 

balance trials with rats, because of the problem of recovering 

Paper 8: Some Amino Acids Are 

all their urinary nitrogen and thus getting spurious positive 

Indispensable for Growth (Osborne and 

balances. They thought that the only really satisfactory way 

to test the adequacy of dietary proteins with or without the 

Mendel, 1914–1916) 

addition of amino acids was to have young animals grow substantially 

in size, using purified diet components. 

Presented by Kenneth J. Carpenter, Department of Nutritional 

Sciences, University of California, Berkeley, CA 94720-3104 as They soon found difficulties in maintaining growth in rats 

part of the minisymposium ‘‘Experiments That Changed Nutritional 

using starch, lard and mineral mixes even with casein as the 

Thinking’’ given at Experimental Biology 96, April 16, 1996, in protein source. They obtained improved growth by adding 

Atlanta, GA. 

dried ‘‘protein-free milk,’’ i.e., separated milk from which the 

proteins had been precipitated by acidification and then boiling 

‘‘Osborne and Mendel’’—one of the most important and 

(Osborne and Mendel 1911). After the further inclusion 

long-lasting collaborations in the history of nutritional sci- of butter fat, growth continued to the mature weight of their 

ence—and between two very different characters. Thomas animals (Osborne and Mendel 1914). With gliadin as their 

Osborne was from a well-established New England family of protein source in place of casein, rats hardly changed weight 

lawyers and bankers and the fifth generation to go to Yale. He for 3 mo. But with added lysine they grew well. 

lived all his life in New Haven, worked in the Agricultural At this time, with only crude gravimetric methods of analysis 

Experiment Station with no students and did not like to face 

available, they believed that gliadin (isolated from wheat) 

an audience (Fruton 1995, Vickery 1932). Lafayette Mendel, had no lysine. Therefore, because rats would maintain weight 



1032S 

SUPPLEMENT 

TABLE 2 


Mean 3-wk weight changes of young rats receiving 18% zein 

Abderhalden, E. (1909) Weiterer Beitrag zur Frage nach der Verwertung von 

tief abgebautem Eiweiss in Tierischen Organismus. Z. Physiol. Chem. 61: 

as their protein source1 

194–199. 

Becker, S. (1968) The Emergence of a Trace Nutrient Concept through Animal 

Feeding Experiments. Ph.D. thesis, University of Wisconsin. University Micro- 

Amino acid 

films Inc., Ann Arbor, MI. 

supplement to diet Mean weight Carpenter, K. J. (1994) Protein and Energy: A Study of Changing Ideas in Nutri- 

No. rats (g/100 g) change ({SD) tion. Cambridge University Press, New York, NY. 

Chittenden, R. H. (1938) Lafayette Benedict Mendel, 1872–1935. Natl. Acad. 

g 

Sci. Biogr. Mem. 18: 123–155. 

Fruton, J. S. (1995) Thomas Burr Osborne and chemistry. Bull. Hist. Chem. 

17: 1–8. 

22 None 020 ({ 5) 

Henriques, V. & Hansen, C. (1905) Über Eiwessynthese im Tierkörper. Hoppe- 

6 0.54% Tryptophan 07 ({ 6) Seyler’s Z. Physiol. Chem. 43: 417–446. 

7 0.54% Tryptophan /22 ({ 5) Osborne, T. & Mendel, L. B. (1911) Feeding Experiments with Isolated Food 

/ 0.54% lysine Substances. Carnegie Inst. Washington. Publ. 156. 

[Normal weight gain on a control diet] [/35] 

Osborne, T. & Mendel, L. B. (1914) Amino-acids in nutrition and growth. J. 

Biol. Chem. 17: 325–349. 

Osborne, T. & Mendel, L. B. (1916) The amino-acid minimum for maintenance 

1 

and growth, as exemplified by further experiments with lysine and trypto- 

Data from Osborne and Mendel (1914). 

phane. J. Biol. Chem. 25: 1–12. 

Vickery, H. B. (1932) Thomas Burr Osborne, 1859–1929. Natl. Acad. Sci. Biogr. 

with gliadin as the sole protein, lysine did not seem to be Mem. 14: 261–304. 

Willcock, E. G. & Hopkins, F. G. (1906) The importance of individual amino 

acids in metabolism. J. Physiol. 35: 88–102. 

required for maintenance. However, by 1915, with an improved 

analytical procedure for lysine, they had found that 

gliadin did contain about 0.9% lysine, but they assumed that 

Paper 9: Pellagra Is Not Infectious! 

the rat’s muscle proteins, like those of animals’ muscles that 

(Goldberger, 1916) 

had been analyzed, contained approximately 8% lysine, so that 

part of the lysine in the rats’ new tissues had to have come 

from the free amino acid supplement. They did not report any 

actual calculations to support this point, but Table 1 reproduces 

the relevant estimates that they could, and perhaps did, 

make. 

Lysine could not apparently by synthesized by rats, even 

though it had no cyclic group. This was an important point 

because it had been thought it was the amino acids containing 

cyclic groups that animals would be found to be unable to 

synthesize. On the other hand, rats could apparently maintain 

themselves with dietary protein of very different composition 

from that of their own tissues. 

This idea was confirmed by their parallel results with zein 

(from corn), which is completely lacking in lysine and also 

both tryptophan and glycine (Osborne and Mendel 1916). 

They showed most of their results with zein as growth curves 

for individual rats, because they changed their supplements 

from time to time, but Table 2 summarizes their results from 

just the first few weeks of feeding. 

It is clear that without tryptophan the rats did worst. With 

it, but without lysine, they did better, though on average still 

losing a little weight. With both amino acids added, they grew 

fairly rapidly, at about two thirds of the optimal rate. They 

concluded, as Willcock and Hopkins (1906) had earlier, that 

perhaps the more rapid loss in the absence of tryptophan meant 

that this amino acid was needed for some special priority function, 

requiring tissue loss to provide it, and that in its presence 

there was at any rate less breakdown of protein. 

The work clarified the importance of amino acid composition 

as a determinant of protein quality and demonstrated 

examples of three types of amino acids: lysine that could not 

be synthesized and was needed almost entirely for growth, 

tryptophan needed for maintenance as well as growth, and 

glycine that could be synthesized so that its supply was not 

limiting. 

Osborne and Mendel’s persistence with long-term growth 

as a measure of nutritional adequacy, in parallel with the work 

of McCollum’s group in Wisconsin, opened up a new approach 

to the search for vitamins as well as the discovery of further 

essential amino acids (Becker 1968, Carpenter 1994). 

Presented by Leslie M. Klevay, U.S. Department of Agriculture, 

Grand Forks Human Nutrition Research Center, Grand Forks, 

ND 58202 and Robert E. Olson, College of Medicine, University 

of South Florida, Tampa, FL 33606 as part of the minisymposium 


Biology 96, April 16, 1996, in Washington DC. 

Microbiology was transforming medicine at the end of the 

Victorian era. By the time Funk coined the term ‘‘vitamine’’ 

in 1912, the causative organism for tuberculosis had been 

known for 30 years. It is not surprising that infection was 

considered more likely than dietary deficiency to be the cause 

of pellagra. Both toxicity and heredity, two other causes of 

disease known at the turn of the century, had also been suggested. 

Dementia, dermatitis, diarrhea and finally death associated 

with a diet of meat, maize and molasses described the pellagra 

syndrome. Unfortunately, the ‘‘meat’’ consumed by poor people 

was high in fat and low in protein. The dermatitis is photo- 

sensitive and confined to the areas of skin exposed to sunlight; 

Cásal’s (1691–1759) necklace is the eponym attached to ‘‘the 

area of erythema and pigmentation around the neck in pellagra’’ 

(Terris 1964). The dementia was usually of the manic- 

depressive type and severe enough to justify admission to a 

mental institution. 

Joseph Goldberger, who contributed extensively to our understanding 

of the causes of pellagra, was born in Austria in 

1874 and immigrated with his parents to the United States in 

the 1880s. He grew up in New York City and entered the City 

College of New York as a high school graduate in 1890 to 

study engineering but changed his field to medicine two years 

later by enrolling at the Bellevue Hospital Medical College. 

He obtained his MD degree in 1895 and after interning for 

one year and practicing medicine in New York and Pennsylvania 

for three additional years, he joined the U.S. Public Health 

Service in 1899. He served as a quarantine officer in various 

ports including New Orleans, Tampico, Veracruz and Havana 

and studied yellow fever and typhus transmission by mosquitos 

in those areas. In 1909, he solved the cause of Schamberg’s 

disease, a pigmented dermatitis, prevalent in crew members 




1033S 

on private yachts and in men living in private dwellings and berger and Wheeler themselves were the first subjects, each 

boarding houses in the Philadelphia area. Goldberger and receiving 5 mL of the defibrinated blood by intramuscular 

Schamberg (1909) observed that these men slept on straw injection and also secretions. Three days later, Goldberger ate 

mattresses, and they finally identified a mite (Pediculoides ventricosus) 

feces from an acutely ill patient together with the urine and 

as the vector for the disease. Thus, Goldberger had dermatitis scales from two other patients. As a result, Gold- 

considerable experience in epidemiology and knowledge of berger developed diarrhea that lasted for about a week, but 

infectious diseases when he was assigned by the Surgeon Gen- despite this both he and Wheeler joined three other volunteers 

eral in 1913 to undertake a study of the causation of pellagra. for a similar round of tests with both injections of defibrinated 

Pellagra was not recognized as a problem in the United blood from three patients and the oral consumption of scales 

States until early in the 20th century. In 1912, Lavinder of and excreta. To maximize the chance of catching any infection 

the U.S. Public Health Service estimated that more than from stools, they used fresh fecal material from the rectum of 

25,000 cases of pellagra had occurred in the United States in pellagrins by using an enema and then blended material from 

the previous five years and that the case fatality rate was 40%. five subjects into pills that were consumed by the volunteers. 

The dominant thinking in the United States at the time Goldberger 

The volunteers also took sodium bicarbonate before and after 

began his investigations was that pellagra was an infec- consuming these materials to reduce the acidity of the stomach 

tious disease. As a result of studies in South Carolina, the to prevent a possible bacteriocidal action of gastric juice. Mrs. 

Thompson-McFadden Pellagra Commission concluded in Goldberger received one injection of blood. Both Goldberger 

1913 that ‘‘1) The supposition that the ingestion of good or and Wheeler felt stiffness after the intramuscular injections, 

spoiled maize is the essential cause of pellagra is not supported and several volunteers felt nauseous after ingestion of feces. 

by our study; and 2) Pellagra is in all probability a specific Nonetheless, after five to seven months none showed any sign 

infectious disease communicable from person to person by of pellagra. Because Goldberger’s group had failed to demonstrate 

means at present unknown.’’ These conclusions were elaborated 

any transmissibility of an infectious agent to themselves 

by Siler et al. (1914). 

from pellagrins and had demonstrated both the preventative 

In less than three months after beginning his investigation, and curative action in pellagrins of diets rich in animal foods, 

Goldberger (1914) published his first paper on pellagra. In a they felt secure in their conclusion that the disease was dietary 

document of a little over three pages, Goldberger summarized and not infectious. Nonetheless, they conducted another epi- 

the epidemiology of the disease as follows. Pellagra cannot be demiologic study of seven cotton mill villages in South Carolina 

communicable. The cause is dietary. Prevention should result 

beginning in 1916, which showed that the disease was 

from a ‘‘reduction in cereals and vegetables and canned foods not high in villages with poor sanitation but was high in 

that enter to a large extent in the dietary of many of the villages with poor diets. 

people in the South and an increase in fresh animal food In 1920, the question remaining was: What was the agent 

component such as fresh meats, eggs and milk.’’ In support of in animal foods that prevented pellagra? Because the biological 

these views Goldberger pointed out that 1) in institutions value of animal protein was in general better than the values 

where pellagra was prevalent, no case had ever occurred in for proteins in cereals and vegetables, Goldberger and Tanner 

nurses or attendants; 2) the disease was essentially rural; and (1922) proposed that an amino acid might be the pellagra 

3) it was associated with poverty, which in turn was associated preventative factor. Tanner actually conducted a trial of trypwith 

a diet deficient in animal foods. 

tophan in one pellagrous patient, which caused marked improvement 

These conclusions, however, were reached by epidemiologic 

of the dermatitis but little change in the diarrhea. 

methods involving the association of variables and did not He reported the finding in a progress report to Goldberger, 

constitute proof of the etiology of the disease. Goldberger and but the result was not followed up (Tanner 1921, quoted by 

his colleagues then proceeded to attempt 1) to cure pellagra Hundley 1954). Goldberger also tested various foods in an 

by changing the diet of pellagrins to one rich in animal foods attempt to cure black tongue, the pellagrous analogue in dogs. 

and 2) to demonstrate by direct studies the possible infectivity Goldberger died prematurely at age 55 in 1929. He thus 

of secretions, scales and excreta from pellagrins. A year after didn’t live to see the cure of black tongue with niacin as 

publishing his first paper on pellagra, Goldberger and his co- reported by Elvehjem et al. in 1937, although Goldberger and 

workers demonstrated in back-to-back papers (Goldberger et Sebrell (1930) did induce remission of this canine disease with 

al. 1915, Willets 1915) that pellagra could be prevented in liver extract. His idea that amino acids were critical in the 

institutionalized patients by a diet that included generous pathogenesis of pellagra was confirmed by a finding by Krehl 

amounts of milk, eggs, meat, beans and peas and that pellagra et al. (1945), who proved that nicotinic acid could be formed 

could be successfully treated by the same regimen. 

from tryptophan. Subsequently Vilter et al. (1949) showed 

The second part of Goldberger’s plan was to demonstrate that tryptophan would cure pellagra in humans. 

the nontransmissibility of pellagra by contact with nasophabrilliant 

In summary, Goldberger was a well-trained physician, a 

ryngeal secretions, blood and excreta from pellagrins (Goldtor. 

epidemiologist and an imaginative clinical investiga- 

berger 1916). In a heroic study on themselves conducted by 

He studied a variety of infectious diseases and pellagra, 

Goldberger, Sydenstricker, Tanner, Wheeler, Willets, Goldthat 

which was not infectious, with a multidisciplinary approach 

berger’s wife and an additional 10 volunteers, defibrinated 

included epidemiology. He is still lauded as a exemplar 

blood, nasopharyngeal secretions, feces, urine and dermatitic of clinical epidemiology (Elmore and Feinstein 1994). 

scales were administered enterally and parenterally in an attempt 

to cause pellagra. It must be noted that physicians in 

the public health service at that time had learned to accept 


such exposures as the risk of dealing with infectious diseases, Elmore, J. G. & Feinstein, A. R. (1994) Joseph Goldberger; an unsung hero of 

and in fact Goldberger himself had contracted both yellow 

American clinical epidemiology. Ann. Intern. Med. 121: 372–375. 

fever and typhus from his previous work. Various tissues, nasal Elvehjem, C. A., Madden, R. J., Strong, F. M. & Wooley, D. W. (1937) Relation 

of nicotinic acid and nicotinic amide to canine black tongue. J. Am. Chem. 

secretions and excreta were obtained from 17 cases of pellagra Soc. 59: 1767–1768. 

of various grades of severity, including three fatal cases. Gold- Goldberger, J. (1914) The etiology of pellagra: the significance of certain epi- 



1034S 

SUPPLEMENT 

demiological observations with respect thereto. Public Health Rep. 29: 1683– anemic animals by dietary methods, however. In the preceding 

1686. 

Goldberger, J. (1916) The transmissibility of pellagra: experimental attempts 

paper in their series, data were presented showing that iron 

at transmission to the human subject. Public Health Rep. 31: 3159–3173. salts of great purity were ineffective in correcting an anemia 

Goldberger, J. & Schamberg, J. F. (1909) Epidemic of an urticarioid dermatitis of rats confined to a diet of cow’s milk. However, an equal 

due to a small mite (Pediculoides ventricosus) in the straw of mattresses. 

amount of iron fed as ash of lettuce, corn or beef liver (an 

Pub. Health Rep. 24: 973–975. 

Goldberger, J. & Sebrell, W. H. (1930) The black tongue preventive value of acid extract of ash) was very potent in restoring normal hemo- 

Minot’s liver extract. Public Health Rep. 45: 3064–3070. 

globin. 

Goldberger, J. & Tanner, W. F. (1922) Amino acid deficiency probably the etio- 

The seventh paper (Hart et al. 1928) in their series establogic 

factor in pellagra. Public Health Rep. 37: 462–486. 

Goldberger, J., Waring, C. H. & Willets, D. G. (1915) A test of diet in the prevention 

of pellagra. South. Med. J. 8: 1043–1044. 

were modest, concluding only that the experiments ‘‘point to 

lished copper as an essential nutrient. Although the authors 

Goldberger, J., Wheeler, G. A. & Sydenstricker, E. (1918) A study of the diet 

the need for a more intensive study of the rôle of small amounts 

on nonpellagrous and of pellagrous households in textile mill communities in 

South Carolina in 1916. J. Am. Med. Assoc. 71: 944–949. 

of inorganic substances in the diet,’’ McCollum (1957) suggests 

Krehl, W. A., Tepley, L. J., Sarma, P. S. & Elvehjem, C. A. (1945) Growth re- that this experiment and other similar ones on trace elements 

tarding effects of corn in nicotinic acid–low rations and its counteraction by ‘‘broadened immensely the outlook of physiologists, biochemtryptophan. 

Science 101: 489–491. 

Siler, J. F., Garrison, P. E. & MacNeal, W. J. (1914) Pellagra, a summary of the 

ists, and pathologists.’’ 

first progress report of the Thompson-McFadden Commission. J. Am. Med. Figure 1 contains charts on six rats of some 50 presented. 

Assn. 62: 8–12. 

Each chart showed both rat weight and hemoglobin plotted 

Tanner, W. F. (1921) Progress report to Goldberger (unpublished and cited by 

Hundley in Sebrell, W. H. & Harris, R. S. (1954) The Vitamins: Chemistry, 

against time. Intakes of 0.3 g of Cohn’s liver preparation (ap- 

Physiology, Pathology, Volume II, page 553. Academic Press, New York, NY. proximately equivalent to 1.7 g of dried beef liver) produced 

Terris, M. (1964) Goldberger on Pellagra. Louisiana State University Press, a ‘‘marked growth response’’ in rat 597 without much improve- 

Baton Rouge, LA. 

ment in hemoglobin. Cohn et al. (1927) fractionated livers 

Vilter, R. W., Mueller, J. F. & Bean, W. B. (1949) The therapeutic effect of trypto 

produce extracts more efficacious than whole liver in curing 

tophan in human pellagra. J. Lab. Clin. Med. 34: 409–413. 

Willets, D. G. (1915) The treatment of pellagra by diet. South. Med. J. 8: 1044– anemia. Rat 596 responded with improved hemoglobin when 

1047. 

given 0.5 mg of iron. Thus bioassay confirmed that the liver 

preparation was low in iron as shown by chemical analysis. 

Paper 10: Copper as a Supplement to The response of rat 615 was similar to that of rat 596 when 

ash of the liver preparation was fed with iron, thereby exclud- 

Iron for Hemoglobin Building in the Rat 

ing ‘‘the existence of an organic factor’’ being of benefit in 

(Hart et al., 1928) 

these experiments. 

Because several types of ash had been found ‘‘very potent 

Presented by Leslie M. Klevay, U.S. Department of Agriculture, 

in restoring to normal the hemoglobin,’’ an attempt to frac- 

Grand Forks Human Nutrition Research Center, Grand Forks, 

tionate the ash was made ‘‘quite early in our experiments.’’ 

ND 58202 as part of the minisympsosium ‘‘Experiments That 

All experiments were done with the same iron intake. Rat 

Changed Nutritional Thinking’’ given at Experimental Biology 95, 

620 had improved hemoglobin with an acid extract of ash, 

April 11, 1995, in Atlanta, GA. 

indicating that the effective ‘‘substance (or substances)’’ were 

Although Hopkins (1906) and Funk (1912) shrewdly pretionation 

‘‘more dramatic,’’ they treated this acid extract with 

soluble in hydrochloric acid. Deciding to make their ash fracdicted 

early in the 20th century that some diseases occurred 

because of dietary deficiency of accessory or essential food hydrogen sulfide. The hemoglobin of rat 690 was restored by 

factors, the observations they cited were ‘‘unknown to medical the ‘‘small but distinct precipitate of sulfides.’’ After this premen 

and chemists of that period’’ (McCollum 1957). Physiolonia 

and ammonium sulfide; rat 688 died without improvement 

cipitate was ‘‘filtered off,’’ the filtrate was treated with ammo- 

gists, biochemists and even the public (Allen 1931) caught 

up by 1928. Many important discoveries on the role of organic of hemoglobin when given this second precipitate. Thus, it 

nutrients were made in the 1920s because Hopkins and Funk was found that the active element had an acid-insoluble sul- 

had opened their eyes (McCollum 1957). 

fide; other elements besides copper were possible (Sorum 

The 1926 edition of Osler’s influential textbook (Osler and 1949). Acid ‘‘extracts of the ash of lettuce . . . could not 

McCrae 1926) listed two classes of anemia. The secondary or be separated sharply into an active and inactive fraction by 

symptomatic class included those due to blood loss, infection precipitation with ammonia.’’ 

or intoxication. The primary or essential class included only The chronology of these experiments is not obvious. Perhaps 

chlorosis, pernicious anemia and sickle cell anemia. Of these the rat number can be a guide. ‘‘Shortly before the time’’ that 

three, only chlorosis had an effective treatment. Osler referred the fractions of the Cohn preparation were studied, a trial of 

to the treatment of chlorosis by iron therapy as ‘‘one of the copper was made. Figure 2 shows the response of rat 621, the 

most brilliant instances—of which we have but three or rat from which the essentiality of copper can be inferred. When 

four—of the specific action of a remedy’’ and stated ‘‘It is a supplemental iron with copper was begun, growth had ceased 

minor matter how iron cures chlorosis.’’ Osler did not infer the and hemoglobin was 2.68 g/dL. Growth resumed and hemoglobin 

existence of iron deficiency anemia (chlorosis was of unknown increased to 9.35 in 2 wk, 10.9 after 6 wk and then reached 13.3 

cause). Anemias from deficiency of ascorbic acid, cobalt, folate, g/dL. ‘‘Without the copper addition, the rise in hemoglobin would 

niacin, pantothenic acid, pyridoxine, riboflavin, thiamine, via 

single animal but the effect was so convincing and helpful’’ 

not have occurred.’’ ‘‘This preliminary experiment was with but 

tamin E, etc., were unrecognized (Wintrobe 1967). 

Hart, Steenbock, Waddell and Elvehjem certainly were well that the chart is recorded ‘‘if for no other reason than its historical 

informed about nutritional opportunities and contemporary interest.’’ A dozen other rats were studied at three doses of copper. 

nutritional knowledge; they may have been somewhat ahead Response to 0.01 mg of copper was less rapid than the response 

of physicians who read Osler. Their experimental design refound 

in the ash of the liver preparation. 

to 0.05 or 0.1 mg. The intermediate dose was similar to that 

sembled that of Whipple et al. (1920), who used phlebotomy 

to make dogs anemic and then measured blood regeneration to The authors knew that copper occurs in plant and animal 

assay dietary components for nutritional value. They produced tissues, but ‘‘no definite function has been assigned to it except 





1035S 

FIGURE 1 Each square is 4 wk wide. The diagonal line on the growth line shows when additions to the diet of cow’s whole milk were begun. 

These additions were made 6 d per week. The top panel illustrates the response of rats to the liver preparation; the bottom panel shows the 

response to acid extract of liver preparation ash. Rat responses modified from charts of Hart et al. (1928). 

in the case of some molluscs and crustacea.’’ McHargue, Warburg 

and Krebs had reported copper in human blood and in 

serum of ‘‘dog, cat, rat, guinea pig, frog, chicken and goose.’’ 

In cattle, liver was found to be highest in copper among the 

organs, with lean meat being considerably lower. 

‘‘Recently the use of liver and liver extracts has come into 

prominence . . . in the treatment of pernicious anemia.’’ ‘‘The 

fact that this preparation was found effective in the treatment of 

anemia in the rat exactly as it has been found effective in the 

treatment of pernicious anemia in man appeared to us rather 

significant.’’ That the preparation contained copper and that 

copper alone was effective in rats suggested ‘‘more than a casual 

incidental connection.’’ It was realized, however, that the ‘‘treatment 

of the two anemias need not necessarily be alike.’’ 

Thoughts from the 1990s 

Instead of being the seventh paper in a series on iron, this 

could have been the first in a series on copper. Nowhere did 

the authors suggest that copper is an essential nutrient. Surprisingly 

little has been written on the definition of nutritional 

essentiality, although concepts from the trace element era have 

been collected and reviewed (Klevay 1987). Although rats 

grow reasonably well when deficient in copper, they cannot 

complete their life cycle without it. 

The experiments of Hart et al. (1928) leading up to this 

discovery about copper were done without the use of statistics. 

Hill (1965) recalls some of his own work done in this era in 

which results were so clearcut that it had not occurred to him 

to use a test of significance. He discusses more recent situations 

when to ‘‘decline to draw conclusions without standard errors 

can . . . be silly.’’ I’m told that replication by repetition and 

confirmation in more than one species prevented false conclusions 

in this earlier era. Thus, this experiment is all the more 

remarkable, although it is not quite fair to say that it was done 

with only a single rat. 

Hart et al. (1928) cited seven references. The method for 


1036S 

SUPPLEMENT 

important, because this assay validated the other assays. No 

data were available on the ‘‘form in which Cu occurs in liver’’ 

or on ‘‘various materials’’ in which chemical form may have 

a ‘‘modifying effect upon the action of Cu.’’ These comments 

are valid in regard to much recent work on the biology of 

copper, as well. 


Allen, F. L. (1931) Only Yesterday, p. 160. Harper and Brothers Publishers, 

New York, NY. 

Allen, K.G.D. & Klevay, L. M. (1978) Cholesterolemia and cardiovascular abnormalities 

in rats caused by copper deficiency. Atherosclerosis 29: 81–93. 

Bennetts, H. W., Harley, R. & Evans, S. T. (1942) Studies on copper deficiency 

of cattle: the fatal termination (‘‘falling disease’’). Aust. Vet. J. 18: 50–63. 

Cohn, E. J., Minot, G. R., Fulton, J. F., Ulrichs, H. F., Sargent, F. C., Weare, J. H. & 

Murphy, W. P. (1927) The nature of the material in liver effective in pernicious 

anemia. I. J. Biol. Chem. 74: 1xix–1xxii. 

Funk, C. (1912) The etiology of the deficiency diseases. J. State. Med. 20: 

341–368. 

Hart, E. B., Steenbock, H., Waddell, J. & Elvehjem, C. A. (1928) Iron in nutrition. 

VII. Copper as a supplement to iron for hemoglobin building in the rat. J. Biol. 

Chem. 77: 797–812. 

Hill, A. B. (1965) The environment and disease: association or causation? Proc. 

R. Soc. Med. 58: 295–300. 

Hopkins, F. G. (1906) The analyst and the medical man. Analyst 31: 385–404. 

Keil, H. L. & Nelson, V. E. (1934) The rôle of copper in carbohydrate metabolism. 

J. Biol. Chem. 106: 343–349. 

FIGURE 2 Modified from chart V (Hart et al. 1928). Klevay, L. M. (1973) Hypercholesterolemia in rats produced by an increase in 

the ratio of zinc to copper ingested. Am. J. Clin. Nutr. 26: 1060–1068. 

Klevay, L. M. (1987) Dietary requirements for trace elements in humans. In: 

measuring hemoglobin was not among them. Copper in the 

Trace Element-Analytical Chemistry in Medicine and Biology (Brätter, P. & 

liver preparation was measured by the xanthate method with- 

Schramel, P., eds.), pp. 43–60. Walter de Gruyter & Co, Berlin, Germany. 

out citation. Inspection of papers I, V, IV and VI in their Lei, K. Y. & Carr, T. P. (1990) Role of Copper in Lipid Metabolism, CRC Press, 

Boca Raton, FL. 

series revealed no method of iron analysis. Hemoglobin was 

McCollum, E. V. (1957) A History of Nutrition. Houghton Mifflin, Boston, MA. 

measured with either Feischl-Miescher or Newcomer hemoglo- Osler, W. & McCrae, T. (1926) The Principles and Practice of Medicine, 10th 

binometers, methods not likely to be as reliable as the cyan- 

ed. D. Appleton and Company, New York, NY. 

Percival, S. S. (1995) Neutropenia caused by copper deficiency: possible 

methemoglobin method. Similarly, it is not clear which of the 

mechanisms of action. Nutr. Rev. 53: 59–66. 

Cohn fractions was used. Two were active in treatment of Schultze, M. O. (1939) The effect of deficiencies in copper and iron on the 

pernicious anemia; another contained a ‘‘substance capable of 

cytochrome oxidase of rat tissues. J. Biol. Chem. 129: 729–737. 

Shields, G. S., Coulson, W. F., Kimball, D. A., Cartwright, G. E. & Winthrobe, M. 

reducing blood pressure.’’ One can infer (Schultze 1939) that 

(1962) Studies on copper metabolism XXXII. Cardiovascular lesions in copcages 

were made of galvanized iron. per deficient swine. Am. J. Pathol. 41: 603–621. 

Nutritional scientists became preoccupied with the hematice-Hall, 

Sorum, C. H. (1949) Introduction to Semimicro Qualitative Analysis, p. 5. Prentology 

New York, NY. 

of copper for more than a half century after this work. 

Whipple, G. H., Robscheit, F. S. & Hooper, C. W. (1920) Blood regeneration 

The reason for this preoccupation is not clear, but it may have following simple anemia. Am. J. Physiol. 53–54: 236–262. 

been created by the vigor of Cartwright and Wintrobe. Perhaps 

just as the Buchners’ concepts of cell-free fermentation were 

resisted by many because of Pasteur’s experiments on spontaneous 

generation, many in nutrition began to think that hematol- 

Wintrobe, M. M. (1967) Clinical Hematology, 6th ed. Lea & Febiger, Philadel- 

phia, PA. 

Paper 11: The Conversion of Carotene 

to Vitamin A (Thomas Moore, 1930) 

ogy was everything in relation to copper. 

A few were more flexible, however, and searched for other 

characteristics of copper deficiency. Keil and Nelson (1934) Presented by James Allen Olson, Department of Biochemistry and 

discovered what now is called glucose intolerance. Bennetts et Biophysics, Iowa State University, Ames, IA 50011-3260 as part 

al. (1942) first noticed cardiac catastrophes. The hematologists of the minisymposium ‘‘Experiments That Changed Nutritional 

were not totally inactive in this latter field (Shields et al. Thinking’’ given at Experimental Biology 96, April 16, 1996, in 

1962). Surely if the hypercholesterolemia of copper deficiency Washington, DC. 

were not nearly invisible in plasma or serum (in contrast to 

hypertriglyceridemia, which is mild in deficiency), copper and In the early 1900s, Frederick Gowland Hopkins, Casimir 

lipid metabolism would have been associated long before 1973 Funk and others realized that minor organic components of 

(Klevay). Now (Lei and Carr 1990) there is much more re- foods played essential roles in growth and nutritional wellbeing. 

search being published on cardiovascular effects of copper deficiency 

These minor essential constituents of the diet were 

than on hematology, although interest in the latter is termed ‘‘vitamines,’’ primarily because the first one discovered, 

reviving with a shift toward leukocytes (Percival 1995). It may thiamine, clearly possessed an amine function. That these mys- 

be interesting to know what pathology might have been found terious vitamines were present not only in water-soluble extracts 

by Hart et al. (1928) with even limited necroscopy. Sometimes 

of living materials but also in fat-soluble extracts was 

pathology is obvious (Allen and Klevay 1978). 

soon noted by several investigators both in the United States 

Hart et al. (1928) commented that ‘‘Only when some important 

and in Europe. McCollum and Davis (1913) at the University 

function . . . lends itself . . . to quantitative measure- of Wisconsin and Osborne and Mendel (1913) at Yale Univer- 

ment are conditions suitable for making progress . . . .’’ They sity first identified foods containing this fat-soluble growth 

had adequate methods for measuring copper, hemoglobin, etc., factor. Active foods included butter, egg yolk, whole milk 

but the bioassay using rat 621 and others perhaps was most powder, cod liver oil and many pigmented fruits and vegeta- 




1037S 

TABLE 1 

Comparison of ingested and stored carotene and vitamin A1 

Total liver units 

Daily dose Total ingested Blue 

Treatment of carotene n units (yellow) (610–630 nm) Yellow 

Depleted 0 10 0 0 1–10 

Carotene 10–50 mg 4 770–4400 0 7–16 

Carotene 750 mg 4 24,000–39,000 2000–3700 40–110 

Red palm oil 1.55 g 2 83,000–130,000 4500–5000 280–400 

Fresh carrots Ad libitum 2 ND 5300–16,000 250–600 

1 Data from Moore 1930. ND Å not determined. 

bles, whereas inactive foods included lard, olive oil and non- studies on the conversion of carotene to vitamin A in the late 

pigmented fruits and vegetables. 

1920s. The two key objectives of his study were 1) to prove 

Between 1913 and 1919 two different types of foods clearly that purified carotene preparations did not contain vitamin A 

showed activity: 1) animal foods that were colorless or showed and 2) to show unambiguously that carotene was converted 

only a slight yellow color and 2) yellow-colored foods, largely to vitamin A in vivo. 

of plant origin. The question then arose of the nutritional In the mid-1920s, it had become clear that carotene and 

relationship between these two types of foods. There were vitamin A were quite different substances, based on their color, 

several possibilities: 1) that the two different types of compounds 

absorption spectra, solubility, and reactivity with antimony 

were independently active on some physiologic system trichloride. In this latter reaction, vitamin A gave a vivid blue 

essential for growth; 2) that the pigmented compounds were color at a wavelength maximum of 610–630 nm, whereas 

active as such, or possibly were converted to active, so-called carotene gave a green-blue color of lesser intensity at a peak 

‘‘leuko’’ forms; and 3) that only one of the types of compounds wavelength of 590 nm. The absorption maximum of b-carowas 

active, but that the other type was contaminated with the tene was approximately 450 nm, whereas liver oils containing 

active ingredient. In 1919, Harry Steenbock wrote: ‘‘It appears vitamin A absorbed very weakly, if at all, at that wavelength 

reasonably safe, at least as a working hypothesis, to assume but showed a maximum absorption at 328 nm. The main instrument 

that the fat-soluble vitamin is a yellow plant pigment or a 

used in these studies was the Lovibond tintometer. 

closely related compound.’’ 

By its use, two types of units were defined: yellow units, which 

The following year, Palmer and Kempster (1920) tested this were high for b-carotene and very low for vitamin A, and blue 

hypothesis. They clearly showed that chicks grew well and units, based on the antimony trichloride reaction, which were 

laid eggs when fed a carotenoid-free diet supplemented with very strong for vitamin A and weak for b-carotene. Thus, the 

a colorless extract of pork liver. The field was suddenly thrust ratio of yellow to blue units was 11 to 1 for carotene but 1 to 

into turmoil. If the yellow plant pigments (carotenoids) were 100 for vitamin A. 

not the active growth-promoting compounds, why indeed, in Moore’s full paper was published in the Biochemical Journal 

the absence of animal products, did they stimulate growth and in 1930. Moore first determined whether or not carotenoid 

development? preparations contained vitamin A. He crystallized carotene 12 

A variety of factors confounded a clear analysis of the problem 

times from a concentrated extract of carrots. He then comof 

at that time. First of all, the chemical structures neither pared the growth-promoting ability of his crystalline carotene 

vitamin A nor of carotenoids were known. Second, all lipid with that of a cod liver oil concentrate. Approximately equal 

preparations, whether from animal or plant sources, contained amounts of carotene and of the oil concentrate stimulated the 

many impurities. Third, some plant carotenoids stimulated growth of vitamin A–deficient rats. The oil concentrate gave 

growth, but other equally colored plant extracts did not. Four, the expected strong blue color at 610–630 nm, characteristic 

other nutritional inadequacies often plagued the interpretation of vitamin A. If the carotene preparation had contained vita- 

of results. For example, vitamin E was discovered to be an min A as a contaminant, then it also should have given a 

essential fat-soluble vitamin only in the early 1920s. Finally, strong blue color. But it did not. Thus, Moore concluded that 

the methodology for the measurement both of vitamin A from the carotene preparation did not contain vitamin A. 

animal sources and of carotenoids from plant sources was rudimentary. 

Moore then turned to studies on the conversion of carotene 

to vitamin A in vivo. He fed 22 rats a vitamin A and carotene– 

Following Palmer and Kempster’s work, the generally accepted 

free diet for 28 to 77 d. Ten of the depleted rats were killed, 

view was that carotenoid preparations were contami- and their livers were analyzed for yellow and blue units. There- 

nated with the colorless vitamin, which was found in pork after, the remaining rats were supplemented with various 

liver and other animal tissues. Thus, carotenoids per se were sources of carotene for 16 to 55 d. These rats were then killed, 

thought to be inactive constituents of these extracts. 

and their livers were analyzed. The data presented in Moore’s 

Thomas Moore, born in 1900, started his research career paper are summarized in Table 1. The 10 depleted rats showed 

in the mid-1920s at the Dunn Nutrition Laboratory associated a few yellow units but no blue units in their liver. Small doses 

with Cambridge University in England. Following studies on of carotene stimulated the growth of animals but did not much 

the importance of light exposure in the formation of biologically 

change the presence of blue or yellow units in the liver. On 

active carotenoids in plants, he focused his attention on the other hand, when large doses of carotene or red palm oil, 

the nutritional relationship between carotene and vitamin A. which predominantly contains a- and b-carotene in roughly 

Unconvinced by the ‘‘contaminant’’ hypothesis, he initiated equivalent amounts, were administered, the blue units in the 



1038S 

SUPPLEMENT 

liver rose dramatically but the yellow units were only modestly 

increased. Fresh carrots, given ad libitum to the animals, 

TABLE 1 

showed a similar effect. 

Bisulfite-binding substances from normal and 

Moore then made the following calculation. Because the 

ratio of yellow to blue units for b-carotene is 11 to 1, the 

vitamin B1–deficient pigeons and rats1 

number of yellow units of carotene corresponding to the blue 

units found in the liver (2000–3700) when large doses of 

carotene were given would be expected to be 22,000–41,000. 

Animal Normal Deficient 

mg/100 mL blood 

Cured 

The amount of yellow units actually present, however, was 

Pigeon 3.96 11.31 

only 40–110 (Table 1), a very small fraction of the amount 

Rat 4.22 9.39 

expected to support an active role of carotene per se. Thus, 

5.29 

Moore (1930) concluded: ‘‘It is impossible that the colour 

1 Data from Thompson and Johnson (1935). Approximately 2.5 mg 

reaction of carotene could conceal any underlying colour reac- of each averaged number is not pyruvate. 

tion due to the liver oil vitamin A in such amounts as would 

account for its physiological activity. The conclusion must be 

reached that carotene, or some part thereof, if it should later Peters 1929 and 1930) also concluded that there was more 

prove to be heterogeneous, behaves in vivo as a precursor of lactic acid present in the brains of pigeons showing acute 

the vitamin.’’ 

symptoms of vitamin B1 deficiency. R. B Fisher (1931) further 

Soon thereafter, Karrer et al. (1931) determined the chemi- noted the slower loss of lactate from heart and skeletal muscle 

cal structures both of carotene and of vitamin A. Karrer’s of deficient pigeons after exercise. 

structural determinations were in full accord with Moore’s Peters and associates then demonstrated that adding thiaconclusions. 

Thus, the dilemma of the relationship between min (in concentrated form) to brain tissue from deficient pithe 

colored compounds largely found in plant foods and the geons, with added lactate, increased the in vitro rate of oxygen 

nearly colorless compounds of liver had been resolved. Need- consumption to that of tissue from normal birds (Meiklejohn et 

less to say, Moore’s findings have stood the test of time. al. 1932). Two years later it was shown that adding crystalline 

thiamin also increased the metabolism of added pyruvate (Peters 

and Thompson 1934). This made an immediate metabolic 


connection to carbohydrate metabolism whereby glucose had 

Karrer, P., Morf, R. & Schoepp, K. (1931) Zur kenntnis des vitamins A in fischrecently 

been shown by Embden and Meyerhof to be degraded 

tranen. Helv. Chim. Acta 14: 1431–1436. 

McCollum, E. V. & Davis, M. (1913) The necessity of certain lipins during in animals to pyruvate. However, the lack of a significant 

growth. J. Biol. Chem. 15: 167–175. 

difference between pyruvate content of normal and vitamin 

Moore, T. (1930) LXXIX. Vitamin A and carotene. V. The absence of the liver 

B1–deficient brains before incubation with added lactate was 

oil vitamin A from carotene. VI. The conversion of carotene to vitamin A in 

vivo. Biochem. J. 24: 692–702. 

puzzling. 

Osborne, E. V. & Mendel, L. B. (1913) The relation of growth to the chemical The explanation was provided by the work of R.H.S. 

constituents of the diet. J. Biol. Chem. 15: 311–326. 

Thompson and R. E. Johnson (1935), who correctly supposed 

Palmer, L. S. & Kempster, H. L. (1920) Relation of plant carotenoids to growth, 

fecundity and reproduction of fowls. J. Biol. Chem. 39: 299–313. 

that the abnormally large amount of pyruvate formed in the 

Steenbock, H. (1919) White corn vs. yellow corn and a probable relationship deficient brain in vivo was not detectable because the metabobetween 

the fat-soluble vitamine and yellow plant pigments. Science 50: 352– lite largely diffuses out into the blood stream. Using pigeons 

353. 

and rats, they measured pyruvate indirectly with other keto 

compounds that can form bisulfite complexes, and they secured 

Paper 12: The Co-enzyme Function of the fractional amount of pyruvate in the bisulfite-binding compounds 

by direct isolation of the pyruvate 2,4-dinitrophenylhy- 

Thiamin (Peters et al., 1929–1937) 

drazone and estimating it colorimetrically. Their findings are 

Presented by Donald B. McCormick, Department of Biochemistry, summarized in Table 1. It should be noted that Platt and Lu 

Emory University School of Medicine, Atlanta, GA 30322 as part (1935) bridged from the experimental animals to humans by 

of the minisymposium ‘‘Experiments That Changed Nutritional concordantly reporting the presence of pyruvate in the blood 

Thinking’’ given at Experimental Biology 95, April 11, 1995, in and cerebrospinal fluid of beriberi patients in the Orient, where 

Atlanta, GA. 

the story began. 

The significance of findings importantly focused by the investigators 

Descriptions of a particular human illness that were recorded 

at Oxford, and even the broader value of basic 

over a thousand years ago in the Far East reflect what research with its use of animals, was well reviewed by R. A. 

later was termed beriberi (Gubler 1991, McCormick 1988). Peters (1936) in his lecture delivered at the National Hospital, 

Eijkman and then Grijns, working in Batavia, used chickens Queen-Square. In summarizing ‘‘What has been learnt,’’ Peters 

as a model for the disease, and they found in the 1890s that states: ‘‘We may now take stock of the position. A purely in- 

chickens developed polyneuritis when fed white rice, but that vitro research with brain tissue of the bird was started in the 

rice bran acted as a preventive. Further studies in the 1920s first instance to improve the test for vitamin B-1 and later 

at the Eijkman Institute in the Dutch West Indies elaborated extended to elucidate the enzyme with which the vitamin 

some general characteristics of the necessary micronutrient in cooperated. It has not only helped to settle these problems 

rice bran. The bird model was extended by R. A. Peters and his but it has proved the existence of pyruvate in normal metabolism. 

colleagues, who used pigeons at Oxford University in England. 

It has also shown that an in-vitro research upon brain 

The connection of a role for the anti-beriberi factor in tissue which takes advantage of the in-vitro labours of biochemists, 

carbohydrate metabolism became apparent with the report of 

can be applied to in-vivo events. This is an imcarbohydrate 

Japanese workers (Inawashiro and Hayasaka 1928) that lactic portant step in this field. It is further encouraging that the 

acid disappeared more slowly from the blood of beriberi patients 

work has led to the detection of pyruvate in the blood of 

after exercise. Investigators at Oxford (Kinnersley and beriberi patients, which may well prove diagnostic. Surely 

we 




1039S 

Fisher, R. B. (1931) CLIII. Carbohydrate metabolism in birds. III. The effects of 

TABLE 2 

rest and exercise upon the lactic acid content of the organs of normal and 

Elucidation of cocarboxylase as thiamin pyrophosphate1 

rice-fed pigeons. Biochem. J. 25: 1410–1418. 

Gubler, C. J. (1991) Thiamin. In: Handbook of Vitamins (Machlin, L. J., ed.), 

2nd ed., pp. 233–281. Marcel Dekker, New York, NY. 

Isolation of cocarboxylase (100 kg yeast r 750 mg HCl salt) 

Horecker, B. L., Smyrniotis, P. Z. & Klenow, H. (1953) The formation of sedo- 

1. Preparation of alcoholic heat extract. 

heptulose phosphate from pentose phosphate. J. Biol. Chem. 205: 661–682. 

Inawashiro, R. & Hayasaka, E. (1928) Studies on effect of muscular exercise 

2. Barium precipitation and elution. 

in beri-beri; influences of muscular exercise upon gas and carbohydrate me- 

3. Precipitation from an acid solution with ethanol and tabolism. (Resynthesis of lactic acid, acidosis and entity of fatigue in berireprecipitation 

with methanol. beri.) Tohoku J. Exp. Med. 12: 1–28. 

4. Absorption on Frankonit KL and elution with hot dilute Kinnersley, H. W. & Peters, R. A. (1929) Observations upon carbohydrate metabolism 

pyridine. 

in birds; relation between lactic acid content of brain and symptoms 

5. Fractional precipitation with methanol-ether. 

of opisthotonus in rice-fed pigeons. Biochem. J. 23: 1126–1136. 

6. Precipitation of poorly soluble picrolonates. 

Kinnersley, H. W. & Peters, R. A. (1930) Carbohydrate metabolism in birds; 

7. Precipitation of poorly soluble Ba and Ag salts. 

brain localisation of lactic acidosis in avitaminosis B 1 and its relation to origin 

of symptoms. Biochem. J. 24: 711–722. 

8. Precipitation with phosphotungstic acid and crystallization 

Lohmann, K. & Schuster, Ph. (1937) Untersuchungen über die Cocarboxylase. 

as the hydrochloride salt. Biochem. Z. 294: 188–214. 

Chemical research McCormick, D. B. (1988) Thiamin. In: Modern Nutrition in Health and Disease 

Empirical analyses C 12 H 21 O 8 N 4 P 2 SCl (Shils, M. E. & Young, V. R., eds.), 7th ed., pp. 355–361. Lea & Febiger, 

Acid hydrolyses pyrophosphate ester 

Philadelphia, PA. 

Sulfite splitting pyrimidine and thiazole parts 

Meiklejohn, A. P., Passmore, R. & Peters, R. A. (1932) The independence of 

UV absorption same as thiamin monophosphate 

vitamin B 1 deficiency and inanition. Proc. R. Soc. B. 111: 391–395. 

Biological research 

Peters, R. A. (1936) The biochemical lesion in vitamin B 1 deficiency. Application 

of modern biochemical analysis in its diagnosis. Lancet 1: 1161–1165. 

Reconstitution of pyruvate carboxylase activity from beer 

Peters, R. A. & Thompson, R.H.S. (1934) CXXI. Pyruvic acid as an intermediary 

and baker’s yeasts. 

metabolite in the brain tissue of avitaminous and normal pigeons. Biochem. 

J. 28: 916–925. 

1 Outlined from Lohmann and Schuster (1937). 

Platt, B. S. & Lu, G. D. (1935) Proc. Physiol. Soc., 3rd Gen. Congr., Chinese 

Med. Assoc. 

Thompson, R.H.S. & Johnson, R. E. (1935) LXXX. Blood pyruvate in vitamin 

could not have a better instance of the ultimately practical B 1 deficiency. Biochem. J. 29: 694–700. 

value of a purely academic research.’’ 

Paper 13: To Live Longer, Eat Less! 

During the period when the metabolic connections of vitamin 

B1 to carbohydrate utilization at the level of pyruvate 

were being established, other work was leading to elucidation 

(McCay, 1934–1939) 

of the structure of the vitamin (Gubler 1991, McCormick Presented by Patricia B. Swan, Department of Food Science and 

1988). The correct empirical formula of vitamin B1 deterpart 


Human Nutrition, Iowa State University, Ames, IA 50011 as 

mined by A. Windaus in 1932 recognized the inclusion of 

sulfur, and by 1936–1937 R. R. Williams and his coworkers Thinking’’ given at Experimental Biology 96, April 16, 1996 in 

determined the full structure and accomplished synthesis of Washington, DC. 

what we today call thiamin. 

In 1925 Clive McCay took a postdoctoral position at Yale 

With the recognition that decarboxylation of pyruvate was University, with Lafayette B. Mendel, an experience that 

a biochemical step somehow involving vitamin B1, the earlier would lead directly to his classical studies of the effects of 

work of E. Auhagen (1932), who separated the alkaline labile nutrition on the longevity of rats. He had been born (1898) 

coenzyme called ‘‘co-carboxylase’’ from the yeast pyruvate in Indiana, received an A.B. degree in physics and chemistry 

‘‘carboxylase,’’ was given new importance. The structure of from the University of Illinois in 1920, and had served as an 

this functional coenzyme form of vitamin B1 was elucidated by instructor in chemistry at Texas A&M for a year. He was 

Lohmann and Schuster (1937), who isolated and characterized awarded a M.S. in biochemistry by Iowa State College in 

thiamin pyrophosphate starting with 100 kg of yeast. The steps 1923 and a Ph.D. with C.L.A. Schmidt at the University of 

involved are shown in Table 2. 

California, Berkeley in 1925. With his strong background in 

It can now be appreciated that thiamin pyrophosphate func- the chemical aspects of biochemistry, McCay was ready to 

tions within a-keto acid decarboxylase subunits of three gen- turn his attention to questions more biological in nature 

eral types of multi-enzymic a-keto acid dehydrogenases, (Loosli 1974). 

namely those for pyruvate, a-ketoglutarate and branched- At Yale, McCay learned of the earlier experiments in Menchain 

a-keto acids, operating in our bodies. A second im- del’s laboratory (Osborne et al. 1917) demonstrating that feportant 

role for the coenzyme of thiamin in the direct metabo- male rats whose food intake was restricted were able to reprolism 

of carbohydrates is within transketolase, where thiamin duce at more advanced ages than usual. McCay asked Mendel 

pyrophosphate mediates interconversions with pentose phos- why they had not carried their experiments longer to deterphates 

(Horecker et al. 1953). A better index of thiamin status mine the extent to which the life span of these rats had been 

in humans evolved with development of erythrocyte transke- increased. Mendel replied that McCay, as a young man, was 

tolase assays. However, the classic work of R. A. Peters and in a better position to undertake such a long-term experiment 

his associates remains a seminal example of the prelude to (Loosli 1974). 

thiamin coenzyme functions, whereas the efforts of R. R. Wil- While at Yale, McCay became acquainted with L. A. Mayliams 

working on thiamin structure and of Lohmann and nard, the head of the animal husbandry department at Cornell 

Schuster ascertaining the coenzymic nature of its pyrophos- University, who was on leave to work with Mendel. Maynard, 

phate lead us toward the modern era of biochemical under- impressed with McCay, hired him. Thus, he began his 35-year 

standing. career at Cornell, retiring in 1962 (Loosli 1974). 



Auhagen, E. (1932) Co-Carboxylase, ein neues Co-Enzyme der alkoholischen 

Gärung. Hoppe-Seyler’s Z. Physiol. Chem. 204: 149–167. 

Nutrition and Longevity 

Shortly after he went to Cornell, McCay began his first 

study of the effects of food restriction on the life span of rats, 


1040S 

SUPPLEMENT 

TABLE 1 

TABLE 2 

Effects of food restriction on life span of rats1 

Effects of food restriction on the weight and density of the 

femur of rats1 

Median age 

Group Ave. age at death Group Mean weight Density 

d 

I Unrestricted 

Males (n Å 11) 0.741 1.22 

I Unrestricted Females (n Å 18) 0.540 1.21 

Males (n Å 14) 483 522 II Restricted at weaning 

Females (n Å 22) 801 820 Males (n Å 7) 0.486 1.15 

II Restricted at weaning Females (n Å 13) 0.398 1.13 

Males (n Å 13) 820 797 III Restricted 2 wk after weaning 

Females (n Å 23) 775 904 Males (n Å 9) 0.484 1.09 

III Restricted 2 wk after weaning Females (n Å 10) 0.432 1.14 

Males (n Å 15) 894 919 

Females2 (n Å 19) 826 894 1 Data taken from McCay et al. (1935). 

1 Data taken from McCay et al. (1935). 

2 Two female rats in this group died at a young age due to high Table 2 provides data regarding the weight and density of the 

temperatures in the animal room. They were not included in the calcula- femur at the time of death. 

tion of results. 

Confirmation of the Relationship of Diet to Longevity 

publishing the first report from this work in 1934 while some 

In a second experiment, McCay and his group fed all rats 

of the rats were yet alive (McCay and Crowell 1934). McCay 

and his co-author remarked on the long-held viewpoint that 

identical amounts of a nutrient-dense diet, feeding that 

nutrition and life span were related (Darby 1990). Yet, they 

amount required to just maintain the weight of the restricted 

rats. This allowed them to determine whether the unlimited 

noted, the science of nutrition had focused on the young, 

consumption of a nutrient-dense diet had shortened the life 

growing animal to the exclusion of the study of the adult or 

of the control group from the first experiment, and they conaging 

animal. 

cluded it had not. The growth of the rats was controlled by 

They wrote: ‘‘In this day when both children and animals 

varying the amount of a mixture of sucrose, cooked starch and 

are being fed to attain a maximum growth rate, it seems a 

lard (38:57:5) that was fed, with the control rats being allowed 

little short of heresy to present data in favor of the ancient 

to eat as much as they chose. In addition, the control group 

theory that slow growth favors longevity.’’ They cited the earwas 

divided, with half the rats being fed cod liver oil and half 

lier work of Osborne et al. (1917) and their own previous 

(as well as all rats with restricted intakes) being fed irradiated 

observations that trout with very low protein intake ‘‘failed to 

grow [but remained alive and] lived twice as long as those 

yeast and carotene as sources of fat-soluble vitamins. Supple- 

mentation with these different sources of fat-soluble nutrients 

that grew.’’ They postulated that ‘‘something was consumed 

in growth that is essential for the maintenance of life’’ (McCay 

and Crowell 1934). 

Accordingly, McCay and Crowell selected from their colony 

TABLE 3 

106 rats, both male and female, and divided them into 

Effects of various lengths of food restriction on 

three groups. They fed them all the same nutrient-dense diet 

the life span of rats1 

from the time of weaning. However, Group I was fed unlimited 

amounts of the diet, whereas the others were fed restricted 

Age at death 

amounts of the diet, either from the time of weaning (Group 

II) or after 2 wk (Group III). Restricted rats were maintained 

at a constant weight until once about every 100 d when they 

Group Ave. Range 

were allowed to grow about 10 g because of the feeding of 

d 

sucrose or beef liver to all rats. 

I Unrestricted2 

A full report was published on this initial experiment a year Males (n Å 17) 670 308–896 

after the preliminary report (McCay et al. 1935). Table 1 presents Females (n Å 16) 643 404–965 

data concerning the length of life of these rats. The female rats II Restricted until d 300 

that were fed unrestricted amounts of diet lived significantly Males (n Å 4) 865 805–1018 

longer than the comparable males (median age at death 820 vs Females (n Å 5) 811 555–1183 

522 d). Male rats that were fed restricted amounts of diet, how- 

Restricted until d 500 

Males (n Å 5) 806 366–1103 

ever, lived as long, and in some cases longer, than did the females Females (n Å 5) 990 793–1078 

whose intake was restricted. The life of the male rats was clearly Restricted until d 700 

lengthened by food restriction and the slower rate of growth, Males (n Å 4) 874 772–1025 

whereas the life of the slower-growing females was not lengthened Females (n Å 6) 912 406–1320 

greatly, if at all, by food restriction. 


McCay et al. (1935) also observed the effects of food restric- 

Males (n Å 5) 882 336–1127 

Females (n Å 4) 1033 815–1320 

tion on the growth and composition of several tissues. Their 

most striking observation was of the fragility of the bones taken 1 Data taken from McCay et al. (1939). 

from the rats fed restricted amounts of diet. They reported that 

2 Data for controls, fed either carotene / irradiated yeast or cod 

some of the bones ‘‘crumbled in the course of dissection.’’ liver oil, were combined. 




1041S 

TABLE 4 

these data for guidance of human beings he might summarize 

by stating: ‘Eat what you should, after that eat what you will 

Effects of food restriction on weight and density 

but not too much.’ ’’ 

of the femur of rats1 

McCay’s scientific work was interrupted by his military ser- 

vice during World War II, but after the war he continued his 

Group Weight Density studies of the effects of nutrition on aging and the life span 

of rats and other species. He is best remembered by his students 

g 

for the course he taught for several years on the history of 

nutrition. A collection of some of these lectures was published 

I Unrestricted 

posthumously (McCay 1972). McCay died of a heart attack 

Males 0.70 1.10 

Females 0.57 1.21 in 1967. 

II Restricted until d 300 

Males 0.63 1.18 

Females 0.45 1.13 



Darby, W. J. (1990) Early concepts on the role of nutrition, diet and longevity. 

Males 0.54 1.18 

In: Nutrition and Aging (Prinsley, D. M. & Sandstead, H. H., eds.). Alan R. Liss, 

Females 0.47 1.13 

New York, NY. 

McCay, C. M. (1939) Chemical aspects of aging. In: Problems of Aging 


(Cowdry, E. V., ed.), chapter 21. Williams & Wilkins, Baltimore, MD. 

Males 0.55 1.15 

McCay, C. M. (1952) Chemical aspects of aging and the effect of diet upon 

Females 0.39 1.11 aging. In: Cowdry’s Problem of Aging (Lansing, A. I., ed.), chapter 6. Williams & 


Wilkins, Baltimore, MD. 

Males 0.38 1.06 McCay, C. M. (1973) Notes on the History of Nutrition Research (Verzar, F., 

Females 0.37 1.03 

ed.). Hans Huber Publishers, Berne, Switzerland. 

McCay, C. M. & Crowell, M. F. (1934) Prolonging the life span. Sci. Monthly 

1 39: 405–414. 

Data taken from McCay et al. (1939). 

McCay, C. M., Crowell, M. F. & Maynard, L. A. (1935) The effect of retarded 

growth upon the length of life span and upon the ultimate body size. J. Nutr. 

10: 63–79. 

did not significantly alter the results, and the data in Tables McCay, C. M., Maynard, L. A., Sperling, G. & Barnes, L. L. (1939) Retarded 

3 and 4 represent the combination of these two treatments 

growth, life span, ultimate body size and age changes in the albino rat after 

feeding diets restricted in calories. J. Nutr. 18: 1–13. 

(McCay et al. 1939). 

Osborne, T. B., Mendel, L. B. & Ferry, E. L. (1917) The effect of retardation of 

The second experiment, designed to confirm and extend growth upon the breeding period and duration of life of rats. Science 45: 

the results of the first, began with 106 rats. Thirty-three were 

294–295. 

fed unlimited amount of energy, and 73 were fed restricted 

amounts. Thirty-five of the restricted rats, however, died due 

Paper 14: The Dynamic State of Body 

Constituents (Schoenheimer, 1939) 

to two failures in the heating system. On d 300, the remaining 

38 rats from the restricted group were divided into four subgroups 

and fed unlimited energy starting on d 300, 500, 700 

Presented by Robert E. Olson, Department of Pediatrics, College 

or 1000 (McCay et al. 1939). 

of Medicine, University of South Florida, Tampa, FL 33606 as 

All groups of rats with restricted intakes contained longerpart 


lived individuals than did the control group. At the time that 


the longest-lived control rat died (d 965), 18 out of the 38 

Atlanta, GA. 

rats whose intakes were restricted were still alive. When the 

restricted rats were allowed to eat normally, they resumed The discoveries of Rudolf Schoenheimer and his colleagues 

growth with few exceptions. Those exceptions were certain at Columbia University in the period from 1933 to 1941 revoindividuals 

whose food intake had been restricted for 1000 d. lutionized our understanding of the metabolism of fatty acids 

Restricted rats did not attain the full size of the control rats. and amino acids and led to the concept of metabolic turnover. 

Table 4 presents the weight and density of the femur of Rudolf Schoenheimer was born in Berlin in 1898 and rerats 

in the second experiment. Again, both measures tended ceived his medical degree from the University of Berlin in 

to decrease with increasing length of time of food restriction. 1922. Following a year of clinical training he did postdoctoral 

Bone growth (length) was also measured, and the authors con- work in Leipzig with Karl Thomas from 1923 to 1926 and 

cluded: ‘‘The bones of the males respond to the realimentation then in Freiburg with Ludwig Aschoff from 1926 to 1933, 

more promptly than those of the female. The bones of the where he began work on the metabolism of cholesterol. In 

male retain the power to grow larger than those of the female’’ 1933, following the political upheaval in Germany, he emi- 

(McCay et al. 1939). 

grated to the United States to take up an appointment in the 

Department of Biochemistry at Columbia University, where 

he came in contact with Harold Urey, who had discovered 

Conclusions 

McCay summarized the conclusions he drew from these 

experiments in a chapter he wrote (McCay 1939) and later 

revised (McCay 1952) as a contribution to a book on aging. 

‘‘This indicated that the life span was flexible and that the 

possibility of its extension was unknown as well as that the 

retarded animals tended to outlive those that matured normally’’ 

(McCay 1952). ‘‘The second experiment gave essentially 

the same results as the first and indicated clearly that 

the retarded growth was the essential feature’’ (McCay 1952). 

He went on to say: ‘‘If one were to draw conclusions from 

deuterium in 1932, and David Rittenburg, one of Urey’s students 

who had also joined the biochemistry faculty at Columbia. 

Together they planned experiments with various isotopic 

compounds, including heavy water, and substrates labeled with 

deuterium and 15 N; these experiments were to revolutionize 

concepts of fat and protein metabolism. 

At the time that Schoenheimer initiated his isotopic studies 

of intermediary metabolism it was generally believed that the 

major components of the body, including body fat and protein, 

were chemically stable. It was assumed that there was very 

little exchange of nutrient molecules between the diet and the 



1042S 

SUPPLEMENT 

and 80% whole wheat bread. Animals were killed at intervals. 

The destruction of the labeled fat proceeded at the same rate 

as the synthesis in the first experiment (Fig. 1). Schoenheimer 

also demonstrated that [ 2 H]-palmitic acid was not only deposited 

in the fat of rats but also converted into other fatty acids, 

including stearic and oleic acids but not linoleic acid, which 

had been shown earlier to be essential. 

FIGURE 1 The synthesis and destruction of fatty acids in mice 

(Schoenheimer and Rittenberg 1936). Used with permission of the Journal 

of Biological Chemistry, Bethesda, MD. 

Studies of Protein Metabolism 

various organs in the body except for replacement of tissue 

caused by ‘‘wear and tear.’’ Fat depots in the body were consid- 

ered to be a reserve source of energy that were called upon 

only when the body was faced with serious food restriction. 

Otherwise, fat metabolism occurred only for purposes of oxidiz- 

ing dietary fat to produce energy. 

Similar views were held regarding protein metabolism, 

mostly as a result of studies by Rubner in Germany and Folin 

in the United States. In 1905, Folin concluded on the basis 

of studies of the excretion of nitrogenous substances in urine 

that nitrogen metabolism could be divided into endogenous 

(tissue) and exogenous (dietary) phases (Folin 1905). E. V. 

McCollum et al. (1939) complimented Folin in the fifth edi- 

tion of the Newer Knowledge of Nutrition by saying, ‘‘Folin 

possessed the genius to solve the problem (of protein metabo- 

lism) in its main outlines, and his interpretation of the mecha- 

nism is almost universally excepted.’’ Folin’s view became the 

paradigm for protein metabolism that persisted to the middle 

1930s when Schoenheimer began his work. 

Schoenheimer and his associates next turned to the study of 

protein metabolism. They prepared doubly labeled L(–)leucine 

containing 3.60 atoms % excess deuterium in its hydrogen 

atoms and 6.54 atoms % excess 15 N in its nitrogen atom. 

This concentration of both isotopes was high enough to allow 

admixture with several hundred parts of ordinary leucine and 

still permit detection by mass spectrometry. Adult rats were 

fed a stock diet containing the marked leucine and observed 

for 3 d, during which time there was no change in weight. At 

the end of the 3-d period the excreta and body tissues were 

analyzed. About 30% of the 15 N administered was found in 

the excreta (2.2% of the isotope in the feces and 27% in the 

urine). The body protein retained the remaining 65% of the 

activity, indicating that the isotopic N had been incorporated 

mainly into tissue protein. This finding was totally inconsistent 

with Folin’s view of exogenous protein metabolism. 

It was found that different organs were not equally effective 

in the fixation of dietary nitrogen. As shown in Table 1, the 

proteins of the internal organs, serum and intestinal tract were 

the most active. The proteins of muscle showed less activity, 

but, because they constitute by far the largest part of the animal, 

the low concentration of 15 N actually represented a high 

absolute amount of isotope. In fact, 66% of the administered 

isotope was recovered in muscle and only 33% in the combined 

internal organs. 

Table 2 shows that not only was there an active incorporation 

of leucine into various organs and tissues, but the 15 N 

also appeared in all other tissue amino acids studied except 

lysine. Particularly prominent in this exchange were glutamic 

and aspartic acids. This transamination observed by Schoen- 

heimer in these studies was explained enzymatically by 

Braunstein and Kretzmann in 1937. The 15 N in arginine was 

more enriched in liver than in kidney, mostly because of the 

ornithine-urea cycle in liver, discovered by Krebs and Henseleit 

in 1932. 

When the deuterium and 15 N contents of the administered 

doubly labeled leucine were both measured, it was found that 

the D/ 15 N ratio in leucine was increased from 100:182 in the 

administered compound to 100:108 in the carcass, indicating 

TABLE 1 

Studies of Fat Metabolism 

15N Content of protein nitrogen in rat organs after 

In Schoenheimer’s first study of the turnover of body conconsumption 

of L(–)leucine for 3 d1 

stituents, he used deuterated water to track the biosynthesis 

of body fat. A proton from water is taken up in fatty acid Organ 15N excess 

biosynthesis in the reductive steps catalyzed by NADPH. Figure 

1 shows the enrichment of fatty acids in the depot fat of Serum 1.67 

mice over a period of 19 d. The isotope content of total fatty 

Liver 0.94 

acids of the mice reached a maximum at d 6 with a 1 Intestine 1.49 

2 time of 

2.5 d. The total enrichment indicated that 14% of all fatty 

Kidney 1.38 

Heart 0.89 

acids (mostly in adipose tissue) was replaced in this experi- Muscle 0.31 

ment. To demonstrate the destruction of fatty acids in mice 

fed a carbohydrate-rich diet, mice of the same weight were fed 1 Data from Schoenheimer (1942). Values are calculated for 100 

for 5 d a diet consisting of 20% fat enriched with deuterium atom percent 15N in leucine. 




1043S 

Folin, O. (1905) A theory of protein metabolism. Am. J. Physiol. 13: 117–138. 

TABLE 2 Krebs, H. A. & Henseleit, K. (1932) Untersuchungen über die Harnstoffbildung 

im Tierkörper. Z. Physiol. Chem. 210: 33–45. 

15N Content of amino acids after consumption of L(–)leucine McCollum, E. V., Orent-Keiles, E. & Day, H. G. (1939) The Newer Knowledge 

by rats for 3 d1 

of Nutrition, 5th ed., pp. 80–82. MacMillan, New York, NY. 

Schoenheimer, R. (1942) The Dynamic State of Body Constituents. Hafner 

Publishing, New York, NY. 

Amino Schoenheimer, R., Ratner, S. & Rittenberg, D. (1939) Studies in protein metabacid 

Liver Muscle olism. X. The metabolic activity of body proteins investigated with L(–)leucine 

containing two isotopes. J. Biol. Chem. 130: 703–732. 

Leucine 7.95 1.90 Schoenheimer, R. & Rittenberg, D. (1936) Deuterium as an indicator in the 

study of intermediary metabolism, VI. Synthesis and destruction of fatty acids 

Glutamate 1.85 0.89 

Aspartate 1.16 0.70 

Arginine 0.89 0.25 

Tyrosine 0.50 0.20 

in the organism. J. Biol. Chem. 114: 381–396. 

Lysine 0.06 Paper 15: Tryptophan’s Role as a 

Amide N 0.78 0.51 

Vitamin Precursor (Krehl et al., 1945) 

1 Data from Schoenheimer (1942). Calculated for 100 atom per cent 

15N in leucine administered. 

Presented by LaVell M. Henderson, Professor Emeritus, Department 

of Biochemistry, University of Minnesota, St. Paul, MN 

55108 as part of the minisymposium ‘‘Experiments That Changed 

that a chemical reaction had occurred by which more than Nutritional Thinking’’ given at Experimental Biology 95, April 11, 

one third of the labeled nitrogen in the original leucine was 1995, in Atlanta, GA. 

replaced by ordinary nitrogen. This was further validation of 

transamination as a major reaction of amino acids. 

When canine black tongue was recognized as the counter- 

Schoenheimer and coworkers then gave [ 15 N]ammonium part of pellagra, about 1925, experimental nutritionists 

citrate to immature rats fed a low protein diet. On this unnatu- adopted the dog for their studies of the causation of pellagra. 

ral diet the animals lost weight, but despite the rapid disappear- Two hundred years after Casal’s description of this syndrome, 

ance of body proteins the rats synthesized new amino acids it was established as a deficiency of a new B-vitamin, niacin, 

from dietary ammonia. The amide N in glutamine and aspara- by the experiments of Elvehjem et al. (1938). 

gine was highly enriched, as was the a-amino group of gluta- L. J. Teply used the microbiological method of Snell and 

mate. Glutamine synthetase, glutamate dehydrogenase and Wright to assay a variety of foods for their niacin activity. 

carbamoyl phosphate synthetase are now known to be im- Contrary to expectations, he found that corn contained 

portant enzymes for ammonia fixation into amino acids. Most enough niacin that it should prevent black tongue and pellaof 

the isotope in arginine was in the amidine group and was gra. Krehl and co-workers confirmed this, but they found that 

removed by arginase, leaving a very small amount in ornithine the niacin requirement of dogs was three times greater when 

in accord with the ornithine-urea cycle. 

corn diets rather than sucrose-casein diets were consumed. 

In these experiments, [ 15 N]ammonia was also incorporated Rats fed sucrose-casein diets had been found not to require a 

into creatine, as expected from the work of Borsook and Dub- dietary source of niacin, but the same group now tested rats 

noff, who in 1941 demonstrating arginine to be a precursor with corn diets. They reported (Krehl et al. 1945a) that reof 

creatine. What Schoenheimer then showed was that the placement of 40% of their 15% casein diet with corn grits 

formation of creatinine from [ 15 N]creatine was a spontaneous reduced growth from 25 to 4 g/wk. When niacin was added, 

reaction without isotope dilution over a 29-d period. The slope growth was restored so that under these conditions the rats 

of the curve indicated that about 2% of total body creatine did require niacin. Corn is unusual in having a particularly 

was being converted to urinary creatinine (and replaced by low level of the essential amino acid tryptophan in its mixed 

new synthesis) per day. This also explained the constancy of proteins. Two months later, Krehl et al. (1945b) reported that 

creatinine excretion in the urine, which Folin had interpreted tryptophan added at 40 mg/100 g diet replaced niacin in reto 

indicate a separate ‘‘endogenous metabolism’’ of protein. versing the growth suppression caused by corn grits (Table 1). 

Schoenheimer et al. (1939) took issue with the Folin hypothesis 

discussed earlier as follows: ‘‘It is scarcely possible 

to reconcile our findings with any theory which requires a 

distinction between two types of nitrogen. . . . The excreted 

TABLE 1 

nitrogen may be considered as a part of the metabolic pool Effect of corn grits (CG), cystine (Cys), threonine (Thr), 

originating from interaction of dietary nitrogen with the relatively 

tryptophan (Trp) and niacin on the growth of weanling rats 

large quantities of reactive tissue nitrogen.’’ 

In summary, Schoenheimer introduced a new and dynamic 

fed 9% casein diets 

paradigm for fat and protein metabolism, replacing the static 

Dietary group 

view of Folin and others. In fact, the idea of the major body 

constituents as a dynamic biochemical system has been extended 

Basal diet Basal Basal / niacin 

more recently to include all aspects of metabolism, 

including systems involved in transport, hemopoiesis, endocrine 

g/wk 

and cytokine activity and even the genome. 15% Casein 29 29 

9% Casein / 40% CG 7 27 


9% Casein / 40% CG / Trp 31 

9% Casein 10 

Borsook, H. & Dubnoff, J. W. (1941) The formation of glycocyamine in animal 9% Casein / 0.2% Cys 12 17 

tissues. J. Biol. Chem. 138: 389–394. 9% Casein / 0.2% Cys / 

Braunstein, A. E. & Kritzmann, M. G. (1937) Über den Ab- und Aufbau von 

Aminosäuren durch Umaminierung. Enzymologia 2: 129–140. 

0.078 Thr 3 19 



1044S 

SUPPLEMENT 

FIGURE 1 Intermediates in the conversion of tryptophan to niacin 

in N. crassa and rats. 

Trp / AA (Thr or Lys) r Protein 

f 

Niacin 

When an essential amino acid, such as threonine, is supplied 

at just below the optimum level, moderately good growth oc- 

curs and the marginal tryptophan level meets the needs for 

both protein and niacin synthesis. When the threonine in the 

The most likely explanation for the interchangeability of 

niacin, required at 1 mg% of the diet, and tryptophan, required 

at 40 times that molar concentration, was the conversion of 

tryptophan to niacin. The fact that 2% acid-hydrolyzed casein 

(tryptophan destroyed) could replace 40% corn grits in causing 

the deficiency suggested that an increase in the levels of other 

amino acids was inducing the deficiency. This effect was 

termed ‘‘amino acid imbalance.’’ The amino acids most effective 

in producing the deficiency were threonine and lysine 

(Hankes et al. 1948), but additional sulfur amino acids were 

needed to maximize the effect. Therefore, in subsequent studies 

0.2% L-cystine was added to the basal diet (Table 1). The 

explanation offered for the effect of other amino acids on the 

growth of rats fed the 9% casein / 0.2% L-cystine diets is 

illustrated as follows: 

diet is elevated, protein synthesis increases, tryptophan is 

drawn into this function at the expense of the alternate pathway 

to niacin, and the vitamin deficiency results. That this 

explanation for the effect of threonine is valid is supported by 

the results of similar imbalance studies that showed that lysine, 

valine, leucine and isoleucine also produce growth inhibition 

that is reversed by tryptophan or niacin (Koeppe and Henderson 

1955). 

Many isotopic labeling studies with rats have confirmed 

the conversion of tryptophan to niacin. The first evidence 

regarding the reactions involved came from experiments with 

mutants of the mold Neurospora crassa (Fig. 1). The first four 

compounds in this scheme were verified in animal systems, 

and quinolinic acid was added when it was identified as an 

excretory product of tryptophan in mammals and a rather 

ineffective niacin substitute in rats and a niacin-less mutant 

of N. crassa (Henderson 1949). 

Quinolinic and picolinic acids are formed by the incubation 

of 3-hydroxyanthranilate with mammalian liver (Fig. 2). Nei- 

ther is degraded to carbon dioxide even in vivo. That they 

arise from intermediates in the degradation of tryptophan to 

glutarate was established by the report of Gholson et al. 

(1962). It is evident that picolinate and quinolinate arise by 

formation of cyclic Shiff’s bases from intermediates in the 

degradation of tryptophan, and they can be considered side 

reaction products of the degradative pathway. 

The limited activity of exogenous quinolinate as a dietary 

replacement for niacin cast doubt on its intermediary role in 

the formation of niacin. This limited activity probably results 

from the failure of the salts of quinolinic acid to penetrate the 

cells in which it normally arises. It is a strong acid and because 

it is obliged to enter cells in the undissociated form, a proper 

pH for its penetration is much below physiological pH values. 

The role of quinolinate in the formation of niacin was clarified 


FIGURE 2 

Simplified scheme for the total metabolism of tryptophan and its conversion to pyridinium compounds, including niacin. 



1045S 

by the observations of Nishizuka and Hayaishi (1963), who origin of the two-way interaction concept but extends the 

showed that it is converted to nicotinic acid mononucleotide concept to that of a three-way interaction with copper, molyb- 

in the presence of 5-phosphoribosyl-1-pyrophosphate by a sol- denum and sulfur acting on each other individually and collectively. 

uble enzyme system from rat liver (Fig. 2). 

The wide variation among animal species with regard to In 1938 Ferguson and co-workers reported that the forage 

the effectiveness of tryptophan as a source of niacin seems to grown in the ‘‘teart’’ area of southern England contained 

result from the degree to which 2-acroleyl-3-aminofumarate is higher concentrations of molybdenum than that of non-teart 

decarboxylated (Fig. 2). In cat liver, 86% is decarboxylated herbage. The teart area is an area of approximately 20,000 

and only 9% is converted to quinolinate (Suhadolnik et al. acres on which grazing cattle and sheep developed severe 

1957). At the other extreme, the rat liver decarboxylates only scouring and exhibited poor performance. Horses were not 

12%, leaving 80% to form quinolinate. Livers from eight other affected. The authors observed that addition of soluble molybdate 

species, including dogs and humans, fall between these two 

to normal forage, in amounts equivalent to that consumed 

extremes. Cats cannot substitute tryptophan for niacin, in the teart forage, produced the same signs of diarrhea in 

whereas rats do not require niacin at the usual levels of tryptophan 

dairy cows. Although this paper did not show interaction of 

intake. Humans and dogs have a limited capacity to molybdenum with another specific nutrient, it did show the 

substitute tryptophan for niacin, so the clinical deficiency appears 

detrimental effect of trace quantities of molybdenum in the 

even with moderate intakes of tryptophan. diet of ruminants (Ferguson et al. 1938). 

The prevalence of pellagra in poor, corn-eating populations Evidence of an antagonistic copper-molybdenum interacin 

southern Europe, but not in natives of the western hemi- tion came from Australia. The original observation in Australia 

sphere, has been explained by the fact that the natives of the 

was made during the investigation of an entirely different 

Americas used hot lime or other alkaline solutions in the problem, enzootic jaundice, a disease in sheep that results from 

preparation of their tortillas from corn meal. The observations chronic copper toxicity. While seeking an explanation for a 

of Krehl et al. (1945b), 50 years ago, have provided an explana- chronic copper toxicity that occurred in specific areas of the 

tion for the variation in niacin requirements among species country, Dick and Bull (1945) observed incidentally that mo- 

and a partial explanation for the association between corn lybdate supplementation of cows and sheep decreased the liver 

consumption and pellagra, based upon the very limited tryptophan 

storage of copper. Similar observations were made in New 

content of corn. A full understanding of this association Zealand by Cunningham (1950), who studied the interaction 

will depend upon the identification of the postulated substances 

of copper and molybdenum in relation to ‘‘peat scours.’’ In the 

in corn that form the vitamin on heating with alkali. United States, Comar et al. (1949) showed that molybdenum 

supplementation lowered copper storage in livers of cattle, 


and the effect occurred with intakes that gave rise to copper 

deficiency in areas of Florida. This series of studies established 

Elvehjem, C. A., Madden, R. J., Strong, F. M. & Woolley, D. W. (1938) The isothe 

concept of Cu-Mo antagonism and suggested that excess 

lation and identification of the anti-black tongue factor. J. Biol. Chem. 123: 

137–149. 

dietary molybdenum might induce copper deficiency. It also 

Gholson, R. K., Nishazuka, Y., Ichiyama, A., Kawai, H., Nakamura, S. & Hayaishi, suggested that a low molybdenum concentration in forage of 

O. (1962) New intermediates in the catabolism of tryptophan in mammalian 

normal copper content might predispose sheep to copper toxliver. 

J. Biol. Chem. 237: PC2043–2045. 

Hankes, L. V., Henderson, L. M. Brickson, W. L. & Evlehjem, C. A. (1948) Effect icity. 

of amino acids on the growth of rats on niacin-tryptophan deficient rations. Although these experiments demonstrated Cu-Mo antago- 

J. Biol. Chem. 174: 873–881. 

nism, it soon became apparent that another dietary factor 

Henderson, L. M. (1949) Quinolinic acid metabolism II. Replacement of nicotinic 

acid for the growth of the rat and Neurospora. J. Biol. Chem. 181: 677– 

influenced the interaction. The concept of a three-way interac- 

685. tion among Cu-Mo-S emerged from a series of papers (Dick 

Koeppe, O. J. & Henderson, L. M. (1955) Niacin-tryptophan deficiency re- 

1952, 1953a, 1953b and 1954) that constitute the basis of this 

sulting from imbalances in amino acid diets. J. Nutr. 55: 23–33. 

present article. The first of the series (Dick 1952) showed that 

Krehl, W. A., Teply, L. J. & Elvehjem, C. A. (1945a) Corn as an etiological factor 

in the production of nicotinic acid deficiency in the rat. Science 101: 283. 

an unknown dietary constituent besides molybdenum had an 

Krehl, W. A., Teply, L. J., Sarma, P. S. & Elvehjem, C. A. (1945b) Growth-re- effect on copper storage in livers of sheep. The salient data 

tarding effect of corn in nicotinic acid–low rations and its counteraction by 

tryptophane. Science 101: 489–490. 

are shown in Table 1. Clearly the amount of copper stored 

Nishizuka, Y. & Hayaishi, O. (1963) Enzymic synthesis of niacin nucleotides increased as the proportion of oat to alfalfa (lucerne) hay in 

from 3-hydroxyanthranilic acid in mammalian liver. J. Biol. Chem. 238: the diet increased with supplemental copper and molybdenum 

PC483–485. 

Suhadolnik, R. J., Stevens, C. O., Decker, R. H., Henderson, L. M. & Hankes, L. V. 

each remaining constant at 10 mg/d. With equal proportions 

(1957) Species variation in metabolism of 3-hydroyanthranilate to pyridinecarboxylic 

acids. J. Biol. Chem. 228: 973–982. 

6-mo period. This value compares with an earlier observed 

of the two forages, total liver copper increased 35 mg over the 

increase of approximately 135 mg that occurred under similar 

conditions without added molybdenum. When oat hay was 

Paper 16: The Concept of Trace 

Element Antagonism: The Cu-Mo-S 

Triangle (Dick, 1952–1954) 

The precise origin of the concept of antagonistic interaction 

of two or more essential trace elements is not entirely clear, 

but this essay reviews some of the first documented evidence 

for such a physiological antagonism. It not only describes the 

Presented by Boyd L. O’Dell, Department of Biochemistry, University 

of Missouri, Columbia, MO 65211 as part of the minisym- 

posium ‘‘Experiments That Changed Nutritional Thinking’’ given 

at Experimental Biology 96, April 16, 1996, in Washington, DC. 

the sole source of forage, the increase was 113 mg even in the 

presence of additional molybdenum. It is also notable that the 

molybdenum concentration in the blood was 10-fold higher 

in sheep that consumed oat compared with alfalfa hay. This 

experiment showed clearly that a naturally occurring component 

of alfalfa that is largely absent in oat hay affected the 

physiological interaction of copper and molybdenum. 

The next paper in the series (Dick 1953a) described experiments 

that identified inorganic sulfate as the component of 

alfalfa that lowered the blood molybdenum. Dick (1953a) 

found that an aqueous extract of alfalfa hay was rich in sulfate 

ion and that the extract had the same molybdenum-lowering 



1046S 

SUPPLEMENT 

TABLE 1 

pairs liver copper storage. Pertinent data from two experiments 

are presented in Table 2. All sheep consumed approximately 

Liver copper and blood molybdenum concentrations of sheep 10 mg of copper and some were supplemented with 10 mg of 

fed different sources of forage supplemented with 10 mg molybdenum per day. When the animals consumed oat hay, 

each of copper and molybdenum per day for 6 mo1 the addition of molybdenum had no effect on liver copper 

storage, but when they consumed alfalfa it markedly decreased 

Dietary forage Increase in Blood Mo copper storage. When the oat hay diet was supplemented with 

(chaffed hay) liver Cu2 conc.3 2.2 or 4.4 g of sulfate per day as well as with molybdenum, 

copper storage was reduced to the same degree as in sheep fed 

mg mcg/100 mL alfalfa supplemented with molybdenum. The lower level of 

sulfate (2.2 g) was just as effective as the higher level in de- 

Lucerne 9 48 

creasing liver copper in sheep consuming oat or alfalfa hay. 

Lucerne 3:oat 1 19 96 

Lucerne 1:oat 1 35 180 These experiments clearly established the existence of a three- 

Lucerne 1:oat 3 70 378 way interaction among copper, molybdenum and sulfate as 

Oat 113 447 regards liver copper storage. 

In spite of the decrease in liver copper storage observed 

1 Adapted from Dick (1952); n Å 6 animals per group. when the diet was supplemented with both molybdenum and 

2 The increase in total liver copper is the difference between the sulfate, the concentration of blood copper was increased, as 

estimated initial content and that at slaughter; the initial value was 

shown by the data summarized in Table 3. The blood copper 

approximately 27 mg. 

3 The values are the means of weekly blood samples taken over the concentrations rose with increasing levels of both molybdeperiod. 

num and sulfate, and there appeared to be an interaction. 

Even though the blood copper concentration increased, the 

effect as the hay itself. The results summarized in Figure 1 

copper was not available for biological function. Sheep fed 

show that the administration of potassium sulfate lowered 

high levels of molybdenum and sulfate and a nominally ade- 

blood molybdenum in a sheep fed oat hay supplemented with 

quate level of copper developed signs of copper deficiency even 

10 mg of molybdenum per day. The lower blood molybdenum 

with blood copper concentrations that were normal or greater 

resulted from increased excretion of molybdenum via the kidliver 

copper concentration. Clearly, under these conditions of 

than normal. There was loss of crimp in the wool and decreased 

ney, an effect that was independent of urine volume. This and 

related experiments established the Mo-S interaction. 

high molybdenum and sulfate intake, blood copper was not a 

Subsequently, Dick (1953b) showed that sulfate is also the 

valid measure of copper status because there was evidence of 

factor in alfalfa that, in combination with molybdenum, imconcentrations. 

copper deficiency in spite of normal or elevated blood copper 

An explanation of the Cu-Mo-S interaction must account 

for the fact that S renders both copper and molybdenum biologically 

unavailable and that molybdenum lowers copper 

availability only in the presence of dietary S. Suttle (1974) 

pointed out that the formation of cupric tetrathiomolybdate, 

a highly insoluble complex, could occur in the sulfide-rich 

environment of the rumen. This would account for low copper 

absorption but not for the high blood copper concentration. 

Normally all of the copper in blood is solubilized by trichloroacetic 

acid (TCA), but this was not true in cases of high 

molybdenum intake. To explain this phenomenon, Dick et al. 

(1975) prepared di, tri- and tetrathiomolybdates and observed 

that their addition to blood in vitro resulted in a TCA-insoluble 

fraction that contained most of the copper. When tetrathiomolybdate 

was administered to sheep intravenously, there 

was an immediate rise in the TCA-insoluble copper in the 

blood. Thus, formation of copper thiomolybdates, particularly 

CuMoS 4 , in the rumen accounts for the poor absorption of 

copper when the intake of molybdenum is high. The absorption 

of thiomolybdate and subsequent formation of CuMoS 4 

in the blood accounts for the high concentration of blood 

copper that is not bioavailable. 

The experiments described here revealed a three-way interaction 

of copper, molybdenum and sulfate in ruminant animals, 

in which bacterial fermentation plays a major role in 

digestion. The interaction in monogastric animals is much less 

dramatic, because there is less sulfide available to form the 

thiomolybdate complexes. Ruminal microflora normally play 

FIGURE 1 Effect of a single oral dose of potassium sulfate on 

a major role in this three-way interaction, but the interaction 

the blood concentration (mcg/100 mL) and urinary excretion (mg/d) of 

molybdenum. The sheep was fed oat hay and drenched daily with 10 can be demonstrated in monogastric animals by administration 

mg of Mo as ammonium molybdate. Collections were made over 7 d; of thiomolybdates. Interestingly, tetrathiomolybdate has been 

the time of sulfate administration is indicated by arrow. Figure 4 from used to treat Wilson’s disease in human patients (Brewer et 

Dick (1953a). 

al. 1994). This genetic disease results in accumulation of cop- 




1047S 

TABLE 2 

Change in total liver copper in sheep fed forages supplemented with copper, molybdenum and sulfate for periods of 81–114 d1 

Dry forage Cu intake Mo intake Sulfate intake D Liver Cu2 

mg/d g/d mg 

Oat hay 10.0 0.3 — /54 

Oat hay 10.0 10.3 — /40 

Lucerne 9.8 0.5 — /20 

Lucerne 9.8 10.5 — 012 

Oat hay 9.9 0.5 2.2 & 4.43 /15 

Oat hay 9.9 10.7 2.2 & 4.4 020 

Lucerne 10.1 0.7 2.2 & 4.4 /39 

Lucerne 10.1 11.1 2.2 & 4.4 018 

1 Adapted from Tables 3 and 5 of Dick (1953b). 

2 Calculated change in total liver copper during periods of 82 and 114 d; the mean d 0 values, determined by biopsy, were 52 and 84 mg, 

respectively. 

3 The effects of adding 2.2 and 4.4 g of sulfate, as the potassium salt, were not different, and the data were combined. 

Dick, A. T. (1956) Molybdenum and copper relationships in animal nutrition. In: 

Inorganic Nitrogen Metabolism (McElroy, W. D. & Glass, B., eds.), pp. 445– 

473. The Johns Hopkins Press, Baltimore, MD. 

Dick, A. T. & Bull, L. B. (1945) Some preliminary observations on the effect of 

molybdenum on copper metabolism in herbivorous animals. Aust. Vet. J. 21: 

70–72. 

Dick, A. T., Dewey, D. W. & Gawthorne, J. M. (1975) Thiomolybdates and copper- 

per in tissues, and thiomolybdate counteracts copper toxicity 

by complexation of the cupric ion and prevention of its absorption. 

What lesson derives from this work? In the first place, one 

cannot easily predict the outcome of good science regardless of 

its origin. In this case a project that was designed to determine 

the toxicology of a disease in sheep led to the elucidation of molybdenum-sulphur interaction in ruminant nutrition. J. Agric. Sci. 85: 567– 

a complex interaction related to copper deficiency and eventu- 

568. 

ally to a compound used in the treatment of a human disease. 

Ferguson, W. W., Lewis, A. H. & Watson, S. J. (1938) Action of molybdenum in 


Comar, C. L., Singer, L. & Davis, G. K. (1949) J. Biol. Chem. 180: 913–922. 

Cunningham, I. J. (1950) Copper and molybdenum in relation to diseases of cattle 

and sheep in New Zealand. In: Copper Metabolism (McElroy, W. D. & Glass, 

B., eds.), pp. 246–273. Johns Hopkins Press, Baltimore, MD. 

Brewer, G. J., Dick, R. D., Johnson, V., Wang, Y., Yuzbasiyan-Gurkan, V., Kluin, 

K. & Aisen, A. (1994) Treatment of Wilson’s disease with ammonium tetrathio- Plants 

molybdate. I. Initial therapy in 17 neurologically affected patients. Arch. Neurol. 

51: 545–554. 

Dick, A. T. (1952) The effect of diet and of molybdenum on copper metabolism 

in sheep. Aust. Vet. J. 28: 30–33. 

Dick, A. T. (1953a) The effect of inorganic sulphate on the excretion of molybdenum 

in the sheep. Aust. Vet. J. 29: 18–24. 

Dick, A. T. (1953b) The control of copper storage in the liver of sheep by inorganic 

sulphate and molybdenum. Aust. Vet. J. 29: 233–238. 

Dick, A. T. (1954) Preliminary observations on the effects of high intakes of molybdenum 

and of inorganic sulphate on blood copper and on fleece character 

in crossbred sheep. Aust. Vet. J. 30: 196–202. 

nutrition of milking cows. Nature (Lond.) 141: 553. 

Suttle, N. F. (1974) Recent studies of the copper-molybdenum antagonism. Proc. 

Nutr. Soc. 33: 299–305. 

Paper 17: Reduced Radiation Damage 

from Ingestion of Cabbage Family 

Presented by Mindy S. Kurzer, Department of Food Science and 

Nutrition, University of Minnesota, St. Paul, MN 55108 as part 



Washington, DC. 

By 1950, it had been noted that experiments on the effects 

of radiation in guinea pigs showed great variability in the 

results. Mme. M. Lourau and O. Lartigue (Lourau and Lartigue 

1950) observed that although each series of experiments was 

TABLE 3 

fairly homogeneous, when one compared experiments, the lethal 

radiation dose varied by a factor of two or more. The 

Changes in blood copper concentrations in sheep fed a 

cause of this variability in response was unknown. They stated 

forage diet supplemented with graded levels of sulfate and that it was necessary to make a systematic study of the factors 

given variable daily doses of molybdenum for 3 d1 

responsible for these differences in radiosensitivity. 

Lourau and Lartigue were the first to show that diet compo- 

Increase in blood copper with indicated Mo dose2 

sition was one of these factors. In the experiment reported in 

Dietary sulfate 

1950, they studied 114 male guinea pigs. Test diets consisted 

intake 15 mg Mo 30 mg Mo 60 mg Mo 90 mg Mo 

of oat and bran supplemented with either cabbage at 50 g/d 

g/d % 

or beets at 75 g/d. The animals received a single whole-body 

radiation dose. Table 1 summarizes the results. For a given 

1.5 1.4 5.2 15.3 25.3 radiation dose, mortality was far greater in the animals fed 

1.8 1.1 9.0 13.5 30.3 beets: the first death was at 100 roentgen (R) with 100% 

3.1 9.7 15.3 26.1 46.1 mortality at 200 R. In the animals fed cabbage, the first death 

5.7 15.6 16.7 67.1 74.1 

was at 250 R, with 100% mortality at 500 R. 

In addition to their finding that guinea pigs fed beets died 

1 Data from Table 1 of Dick (1954). 

2 Change in the blood copper concentration of individual sheep over 

a 4-d period; the mean initial concentration was 81 mcg/100 mL. The 

daily intake of copper was 10 mg. 

at lower radiation doses than those fed cabbage, Lourau and 

Lartigue commented that only certain lesions differed between 

the two groups. Although bone marrow changes were identical 



1048S 

SUPPLEMENT 

TABLE 1 

The first experiment on the effect of diet on radiation mortality in guinea pigs1 

Radiation dose (R) 

100 150 200 250 300 350 400 500 

mortality, % 

Cabbage — — 0 10 25 50 75 100 

Beets 2.5 — 100 — 100 — 100 — 

1 Data from Lourau and Lartigue (1950). 

in the two groups, at equal radiation doses, animals fed beets control group would have allowed them to distinguish between 

had more severe and widespread hemorrhages than those fed their two explanations (toxic substances in beets vs. protective 

cabbage. 

substances in cabbage). 

The authors proposed two explanations for the dietary effect Spector and Calloway used an oat and bran control diet and 

on radiosensitivity. They suggested that 1) cabbage may con- the control diet supplemented with beets, cabbage or broccoli. 

tain substances that protect against radiation damage, such as They irradiated their four groups of guinea pigs with a radiation 

vitamins P and C; or 2) beets may contain substances that dose of 400 R. Their results are shown in Table 3. Although 

become toxic after irradiation. They preferred the second explanation, 

beet consumption did not affect mortality, consumption of 

that beets were toxic to irradiated guinea pigs. This either cabbage or broccoli significantly reduced mortality from 

was supported by the observation that the LD 50 for the animals irradiation. Thus, they proved that Lourau and Lartigue were 

consuming beets was about 150 R, considerably lower than incorrect in their conclusion that beets contained a toxic substance 

the usually reported LD 50 of 250 R. 

and that Duplan was correct as to the protective effects 

A few years later, M. Jean-Francois Duplan showed that in of cabbage. 

fact, Lourau and Lartigue’s first explanation was correct, that Calloway and colleagues went on to investigate the sub- 

cabbage did offer protection against radiation damage (Duplan stance conferring the protective effects (Calloway et al. 1963). 

1953). Duplan studied 70 male guinea pigs fed oat and bran Because the basal diet was devoid of vitamin A, and animals 

diets. The test diets were supplemented with either cabbage consuming this diet were known to become vitamin A deficient, 

or carrots in this study, and the animals received a single 

they suggested that sources of vitamin A might be able 

radiation dose. 

to decrease the radiosensitivity of guinea pigs. Their results 

The results of Duplan are shown in Table 2. For a given are shown in Table 3. In this experiment, they confirmed 

radiation dose, the animals consuming cabbage had much previous findings that both cabbage and broccoli lowered mortality 

lower mortality than those consuming carrots. He saw no differences 

in irradiated animals. They found that a number of other 

in lesions, but he did note that the animals consuming b-carotene–containing foods also exerted some beneficial effects. 

carrots lost much more weight than those consuming cabbage. 

Mortality after 20 d was significantly lowered by concarrots 

Duplan’s results suggested that Lourau and Lartigue may sumption of the b-carotene–containing vegetables that they 

have been incorrect in concluding that beets contained a toxic tested. Beets, apples and white potatoes had no effect. Supple- 

substance. Rather than concluding that carrots contained a mentation with all essential vitamins reduced mortality somewhat, 

toxic substance, Duplan concluded that cabbage lowered the 

but not to the same degree as supplementation with 

radiosensitivity of guinea pigs. He speculated that the radioprotective 

substances may have been antioxidant goitrogens present 

in the cabbage. 

Spector and Calloway (1959) continued this investigation 

TABLE 3 

of the dietary factors that protect against radiation damage. Further experiments on the effect of diet on radiation 

They very importantly noted that Lourau and Lartigue did not 

mortality in guinea pigs 

have a control group in their experiment. The addition of a 

Mortality at 20 d after irradiation 

with 400 R 

TABLE 2 

The second experiment on the effect of diet on radiation 

mortality in guinea pigs1 

Radiation dose (R) 

300 500 1000 

mortality, % 

Cabbage 0 6.5 87.5 

Carrots 50 86 — 

1 Data from Duplan (1953). 

Spector and 

Calloway et al. 

Supplement Calloway (1959) (1963) 

mortality, % 

None 100 97 

Beets 90 — 

Cabbage 50 54 

Broccoli 35 42 

Alfalfa 0 

Mustard greens 12 

Green beans 31 

Lettuce 44 

All vitamins 72 




1049S 

vegetables. Supplementation with pure vitamin A or b-caro- stract contained few details, and the work in question was 

tene alone had little effect on mortality. 

apparently never published as a full-length paper. 

Calloway and co-workers found that when an adequate pu- Physiologists interested in reproduction had demonstrated 

rified diet was fed, the animals were somewhat resistant to in the late 1940s that fetal blood of sheep contained a high 

radiation damage, suggesting that part of the beneficial effects concentration of fructose (Bacon and Bell 1949, Barklay et al. 

of broccoli may have been due to improved nutritional status. 1949, Hitchcock 1949), and Goodwin (1957) later showed 

On the other hand, the beneficial effects of broccoli could that fetal blood of pigs was also rich in fructose, although the 

not be duplicated by feeding a mixture of 48 chemically pure level in the blood of pigs 24 h after birth was very low. Alexan- 

ingredients patterned on the known composition of broccoli. der et al. (1955) identified glucose as the precursor of fetal 

The experiments of the three groups considered together fructose, with the site of conversion being the placenta. Newsuggested 

that a nonnutrient substance present in cabbage ton and Sampson (1951), though not mentioning it in their 

and broccoli protected the guinea pigs from radiation damage. paper, must have assumed that fructose was an important en- 

Experiments performed by Calloway to isolate the substance ergy source for fetal pigs, and perhaps newborn pigs as well. 

from alfalfa found it to be present in the water-soluble fraction, They obtained pigs at birth and deprived them of food for 

although the protective substance itself was not isolated. periods of 24 to 48 h, or until blood glucose had fallen to 20 

These early experiments laid the foundation for subsequent mg/100 mL or less. These hypoglycemic comatose pigs were 

work showing the beneficial effects of fruit and vegetable con- then given intravenous injections of various sugar solutions. 

sumption in humans. Plants are now known to contain, in Glucose injection produced a dramatic resuscitative response 

addition to vitamins and minerals, many nonnutrient com- within a few minutes, and galactose gave a positive response 

pounds that have important biological effects such as antioxidant, 

also, although less immediate and less dramatic. Fructose injec- 

anticarcinogenic, antimicrobial, antiinflammatory, anti- tion was without benefit, and none of the six pigs given fruc- 

viral and antimutagenic effects. These nonnutrient compounds tose were resuscitated. Taken together, the work of Johnson 

include lignans, indoles, coumarins and flavonoids, which in (1949) and Newton and Sampson (1951) suggested that baby 

1994 were reported to protect mice from the effects of radiation pigs could not effectively utilize either sucrose or fructose. 

(Shimoi et al. 1994). Perhaps these were the elusive substances 

responsible for the radioprotective effects first observed over The Key Feeding Experiments 

45 years ago. 

In the early 1950s there was little interest in early weaning 


of pigs, and it was common practice to wean pigs at 8 wk of 

age. Also, this period was marked by keen interest in the 

Calloway, D. H., Newell, G. W., Calhoun, W. K. & Munson, A. H. (1963) Further 

newly discovered role of antibiotics and vitamin B-12 in pig 

studies of the influence of diet on radiosensitivity of guinea pigs, with special nutrition. Today, there is great interest in artificial rearing of 

reference to broccoli and alfalfa. J. Nutr. 79: 340–348. 

newborn pigs, so that effective carbohydrate sources in syn- 

Duplan, J.-F. (1953) Influence of dietary regimen on the radiosensitivity of the 

thetic milk diets are very important. 

guinea pig. C. R. Acad. Sci. 236: 424–426. 

Lourau, M. & Lartigue, O. (1950) The influence of diet on the biological effects D. E. Becker at the University of Illinois published three 

produced by whole body X-irradiation. Experientia 6: 25–26. 

papers in 1954 that involved extensive testing of carbohydrate 

Shimoi, K., Masuda, S., Furugori, M., Esaki, S. & Kinae, N. (1994) Radioprotecsources 

for pigs ranging in age from 1 d to 16 wk (Becker and 

tive effect of antioxidative flavonoids in g-ray irradiated mice. Carcinogenesis 

15: 2669–2672. 

Terrill 1954, Becker et al. 1954a and 1954b). With pigs during 

Spector, H. & Calloway, D. H. (1959) Reduction of X-irradiation mortality by the period 1 to 10 d of age, liquid diets containing casein and 

cabbage and broccoli. Proc. Soc. Exp. Biol. Med. 100: 405–407. 

various sugars were fed with ad libitum access, with the sugar 

component representing 56.6 g per 100 g of dry ingredients 

(Becker et al. 1954b). Of the seven pigs fed each diet, six fed 

the sucrose diet died, five fed the fructose diet died, and only 

one fed the dextrose diet died. Among surviving pigs, those 

fed dextrose gained weight, whereas those fed sucrose or fruc- 

tose lost weight. Pigs fed either sucrose or fructose also exhibited 

severe diarrhea. 

With pigs fed various carbohydrate sources (56.6 g/100 g 

dry solids) during the period 7 to 35 d of age (Becker et al. 

1954a), three of the eight pigs fed sucrose died, whereas mortal- 

ity was minimal in pigs fed lactose, dextrose, dextrin or cornstarch. 

Although the surviving pigs fed sucrose gained body 

weight as effectively as those fed the other carbohydrate 

sources, the three pigs that died had severe diarrhea. This 

experiment suggested that at least some pigs by 7 d of age can 

effectively hydrolyze sucrose in the gut and can also utilize 

both glucose and fructose for energy. Subsequently, Becker 

and Terrill (1954) demonstrated that 12-wk-old pigs could 

effectively utilize sucrose (50% of dry diet), but depressed 

growth and moderate diarrhea resulted when the semipurified 

soybean meal diet contained 50% lactose. 

Paper 18: Toxicity of Sucrose and 

Fructose for Neonatal Pigs (Becker et 

al. 1954) 

Presented by David H. Baker, Department of Animal Sciences and 

Division of Nutritional Sciences, University of Illinois at Urbana- 

Champaign, Urbana, IL 61801 as part of the minisymposium 


Biology 96, April 16, 1996, in Washington, DC. 

McRoberts and Hogan (1944) fed a liquid synthetic milk 

diet (30 g sucrose and 30 g casein per 100 g dry solids) to 

newborn pigs and reported poor performance and diarrhea, 

despite addition of ‘‘unrecognized factors’’ (vitamins) to the 

diet in the form of brewer’s yeast or beef liver extract. Subse- 

quently, Bustad et al. (1948) tried a similar synthetic milk 

formulation for newborn pigs, and even when lactose was sub- 

stituted for sucrose, the pigs had severe diarrhea and failed to 

survive longer than 22 d. In 1949, S. R. Johnson presented an 

abstract at the FASEB meeting in Detroit wherein it was reported 

that 2-d-old pigs would thrive on a synthetic milk 

diet containing lactose or glucose but would experience severe 

diarrhea and death when the diet contained sucrose as the 

carbohydrate source (Johnson 1949). Unfortunately, this ab- 

Enzymatic Development in Pigs 

The work of Bailey et al. (1956) and Walker (1959) with 

small intestinal and pancreatic extracts from pigs at various 



1050S 

SUPPLEMENT 

ages showed that intestinal sucrase activity was extremely low Bacon, J.S.D. & Bell, D. J. (1948) Fructose and glucose in the blood of fetal 

sheep. Biochem. J. 42: 397–405. 

in newborn pigs and increased 10-fold at 1 wk, 60-fold at 2 

Bailey, C. B., Kitts, W. D. & Wood, A. J. (1956) The development of the digeswk 

and 200-fold at 5 wk of age. Intestinal lactase activity, on tive enzyme system of the pig during its preweaning phase of growth. B. 

the other hand, was high at birth but declined thereafter. 

Intestinal lactase, sucrase and maltase. Can. J. Agric. Sci. 36: 51–58. 

Barklay, H., Haas, P., Huggett, A.S.G., King, G. & Rowley, D. (1949) The sugar 

Aherne et al. (1969a and 1969b) repeated some of Becker’s 

of foetal blood, the amniotic and allantoic fluids. J. Physiol. 109: 98–102. 

earlier studies and found that 2- and 4-d-old pigs could not Becker, D. E. & Terrill, S. W. (1954) Various carbohydrates in a semipurified 

utilize either sucrose or fructose, whereas 7-d-old pigs survived 

diet for the growing pig. Arch. Biochem. Biophys. 50: 399–403. 

when fed these sugars, although weight gains were somewhat 

Becker, D. E., Ullrey, D. E. & Terrill, S. W. (1954a) A comparison of carbohy- 

drates in a synthetic milk diet for the baby pig. Arch. Biochem. Biophys. 48: 

lower in pigs fed sucrose or fructose than in those fed glucose 178–183. 

or lactose. An intestinal loop in situ procedure together with Becker, D. E., Ullrey, D. E., Terrill, S. W. & Notzold, R. A. (1954b) Failure of the 

assessment of blood sugar concentration established that frucnewborn 

pig to utilize sucrose. Science 120: 345–346. 

Bustad, L. K., Ham, W. E. & Cunha, T. J. (1948) Preliminary observations on 

tose was absorbed from the gut intact, with little, if any, con- using a synthetic milk for raising pigs from birth. Arch. Biochem. 17: 249– 

version to glucose in the gut mucosa. This result was confirmed 

260. 

by stomach tubing experiments wherein 3-, 6- and 9-d-old pigs Cori, G. T., Ochoa, S., Slein, M. W. & Cori, C. F. (1951) The metabolism of 

fructose in liver: isolation of fructose-1-phosphate and inorganic pyrophosshowed 

marked elevations in blood fructose but not glucose 

phate. Biochem. Biophys. Acta 7: 304–317. 

when fructose was intubated. Urinary fructose excretion ac- Goodwin, R.F.W. (1957) The relationship between concentration of blood 

counted for a sizable portion of the fructose administered, par- 

sugar and some vital body functions in the newborn pig. J. Physiol. 136: 208– 

217. 

ticularly in 3- and 6-d-old pigs. Intestinal fructokinase activity 

Hitchcock, M.W.J. (1949) Fructose in the sheep foetus. J. Physiol. 108: 117– 

was very low in pigs of all ages, and hepatic fructokinase activ- 126. 

ity was also low in 3-d-old pigs but tended to increase with Huber, J. T., Hartman, P. A., Jacobson, N. L. & Allen, R. S. (1958) Digestive 

enzyme activity in the young calf. J. Dairy Sci. 41: 743 (abs.). 

age or fructose feeding or both. 

Johnson, S. R. (1949) Comparison of sugars in the purified diet of baby pigs. 

It seems clear that the toxicity of sucrose for neonatal pigs 

Fed. Proc. 8: 387 (abs.). 

is caused by a low activity of intestinal sucrase. Also, pigs McRoberts, V. F. & Hogan, A. G. (1944) Adequacy of semipurified diets for the 

pig. J. Nutr. 28: 165–174. 

appear to absorb fructose intact, and during the first week of 

Newton, W. C. & Sampson, J. (1951) Studies on baby pig mortality. VII. The 

life they are poorly equipped to phosphorylate fructose in the 

effectiveness of certain sugars other than glucose in alleviating hypoglycemic 

liver (Cori et al. 1951) to facilitate its metabolism to triose 

coma in fasting newborn pigs. Cornell Vet. 41: 377–381. 

units, hence energy. That 2-d-old pigs experience diarrhea 

Pettigrew, J. E., Zimmerman, D. R. & Ewan, R. C. (1971) Plasma carbohydrate 

levels in the neonatal pig. J. Anim. Sci. 32: 895–899. 

when fed fructose suggests that not all of the ingested fructose Velu, J. G., Kendall, K. A. & Gardner, K. E. (1960) Utilization of various sugars 

is absorbed. Some thus may pass down to the lower gut where it 

by the young dairy calf. J. Dairy Sci. 43: 546–552. 

probably causes a fermentative type of diarrhea. Interestingly, Walker, D. M. (1959) The development of the digestive system of the young 

animal. II. Carbohydrase enzyme development in the young pig. J. Agric. Sci. 

Pettigrew et al. (1971) found that the magnitude of blood 

52: 357–363. 

fructose concentration at birth was negatively correlated with White, C. E., Piper, E. L., Noland, P. R. & Daniels, L. B. (1982) Fructose utilization 

for nucleic acid synthesis in the fetal pig. J. Anim. Sci. 55: 73–76. 

pig performance at 32 h of age. 

Young dairy calves seem to be similar to young pigs with 

regard to sucrose and fructose utilization (Velu et al. 1960). Paper 19: Discovery of the Vitamin D 

Huber et al. (1958) showed that young calves had very low 

activities of intestinal sucrase. 

Endocrine System 

Presented by Hector F. DeLuca, Department of Biochemistry, 

The Remaining Mystery 

University of Wisconsin-Madison as part of the minisymposium 

Why is fructose virtually absent in maternal blood, whereas ‘‘Experiments That Changed Nutritional Thinking’’ given at Experits 

level in fetal blood is higher than that of all nonfructose imental Biology 95, April 11, 1995, in Atlanta, GA. 

reducing sugars combined, at all stages of gestation? The sheep 

In the early part of this century, during the discovery of 

work of Andrews et al. (1960) showed that perfused liver from 

the vitamins by McCollum and coworkers (McCollum and 

fetal and newborn lambs cannot metabolize fructose to carbon 

Davis 1913, McCollum et al. 1916) and by Osborne and Mendioxide. 

If, as appears to be the case, fructose is not functioning 

del (1917), came the idea that rickets (a disease rampant in 

as an energy source for the fetus, what is its role and why is 

northern Europe and northern United States) might be a diit 

so plentiful in fetal blood, amniotic fluid and allantoic fluid? 

etary deficiency. The truth of this idea was readily demon- 

Is it possible that fructose may be required for synthesis of 

strated by Sir Edward Mellanby (1919), who raised the quesa 

cellular constituent required in only microquantities? The 

tion whether the antirachitic activity might be due to the fatwork 

of White et al. (1982) with fetal pigs indicated significant 

soluble vitamin A, discovered by McCollum et al. (1916). 

recovery of [U- 14 C]fructose in muscle and liver RNA and 

However, McCollum and coworkers clearly demonstrated in 

DNA. This suggests that fetal fructose may serve as an im- 

1922 that a separate fat-soluble substance was responsible for 

portant precursor of ribose-5-phosphate that is needed for nuhealing 

rickets (McCollum et al. 1922). 

cleic acid biosynthesis in the fetus. 

In the subsequent two decades came the discovery of the 

irradiation process for producing vitamin D (Steenbock and 


Black 1924), the isolation and identification of the structures 

Aherne, F. X., Hays, V. W., Ewan, R. C. & Speer, V. C. (1969a) Absorption and of vitamins D 2 (Askew et al. 1931) and D 3 (Windaus et al. 

utilization of sugars by the baby pig. J. Anim. Sci. 29: 444–450. 

1936) and a general understanding of the physiologic function 

Aherne, F. X., Hays, V. W., Ewan, R. C. & Speer, V. C. (1969b) Glucose and 

of vitamin D in calcium absorption in the small intestine and 

fructose in the fetal and newborn pig. J. Anim. Sci. 29: 906–911. 

Alexander, D. P., Andrews, R. D., Huggett, A.S.G., Nixon, D. A. & Widdas, W. F. the mineralization of the skeleton (Nicolaysen and Eeg-Larsen 

(1955) The placental transfer of sugars in the sheep: studies with radioactive 1953). Kodicek and his group were pioneers in vitamin D 

sugar. J. Physiol. 129: 352–366. 

metabolism research, using first bioassay and then very weakly 

Andrews, W.H.H., Britton, H. G., Huggett, A.S.G. & Nixon, D. A. (1960) Fruclabeled 

vitamin D 2 (1956). After a decade of investigation, 

tose metabolism in the isolated perfused liver of the foetal and newborn 

sheep. J. Physiol. 153: 199–208. 

this group concluded that vitamin D functions directly without 




1051S 

further metabolism (Kodicek 1956). As illustrated by a paper 1972, Holick et al. 1972), whereas its precursor (25-OH-D 3 ) 

stating that vitamin D itself is responsible for its biological had little biological activity in the nephrectomized rat. This 

activity in intestine (Haussler and Norman 1967), this idea provided clear evidence that the two-step process is required 

persisted throughout most of the 1960s. 

for vitamin D activation for function. Another important proof 

The underlying key to success in this area must be attributed resulted when Fraser et al. (1973) demonstrated that vitamin 

to the chemical synthesis of radiolabeled vitamin D of high D dependency rickets type I (an autosomal recessive genetic 

enough specific radioactivity to permit experiments with truly disorder causing severe rickets despite normal intakes of vitamin 

physiologic amounts of vitamin D (Neville and DeLuca 1966). 

D) could be healed with the provision of physiologic 

Biologically active polar metabolites of vitamin D were discov- amounts of synthetic 1,25-(OH) 2 D 3 , leaving no doubt of the 

ered (Lund and DeLuca 1966, Norman et al. 1964), and further two-step activation process in humans. 

research demonstrated that the more polar metabolite not only While the identification of peak 5 proceeded, Boyle et al. 

had higher biological activity but also acted more rapidly (Morii 

(1971) found that the production of peak 5 metabolite in vivo 

et al. 1967). This provided the impetus for the isolation is strongly dependent upon the dietary calcium levels. Low 

and chemical identification of 25-hydroxyvitamin D 3 (25-OH- calcium diets resulted in massive conversion to 1,25-(OH) 2 D 3 , 

D 3 ) in 1968 (Blunt et al. 1968) and its proof of structure by whereas high calcium diets and vitamin D adequacy resulted 

chemical synthesis that same year (Blunt and DeLuca 1969). in the production of another metabolite that proved to be 

The chemical synthesis of this compound allowed for the intro- 24,25-(OH) 2 D 3 . Pursuit of this led to the demonstration that 

duction of a high specific activity tritium label in the side chain the parathyroid gland mediated this regulation (Garabedian et 

of 25-OH-D 3 . This permitted experiments demonstrating that al. 1972). Thus, in response to low blood calcium, parathyroid 

25-OH-D 3 rapidly disappears and is converted to more polar hormone is secreted that in turn stimulates 1a-hydroxylase in 

metabolites (Cousins et al. 1970): Lawson et al. (1969) found the kidney to produce the vitamin D hormone. The vitamin 

that the intestine contained a major vitamin D metabolite D hormone together with parathyroid hormone then provides 

called ‘‘peak P,’’ which seemed to have lost some of its label for the mobilization of calcium from bone and renal reabsorption 

that was in the 1-position and was thus called a tritium-deficient 

of calcium, and 1,25-(OH) 2 D 3 by itself provides for the 

metabolite. This loss of tritium was not confirmed else- absorption of calcium and phosphorus (DeLuca 1974). Thus, 

where, but the existence of the intestinal polar metabolite the basic tenets of the vitamin D endocrine system were dis- 

called ‘‘4B’’ was also reported (Haussler et al. 1968). 

covered from 1968 to 1973, as summarized in the Federation 

Continuing isolation and identification of metabolites in Proceedings lecture delivered in 1974 (DeLuca 1974). 

the DeLuca group resulted in the finding of at least three polar 

metabolites, one of which proved to be 25,26-dihydroxyvitamin 

D 3 (Suda et al. 1970b). Another was believed to be 21,25- 


Askew, R. A., Bourdillon, R. B., Bruce, H. M., Jenkins, R.G.C. & Webster, T. A. 

dihydroxyvitamin D 3 (Suda et al. 1970a), and a third was 

(1931) Proc. R. Soc. B 107: 76–90. 

present in such small amounts in blood that its identification Blunt, J. W. & DeLuca, H. F. (1969) Biochemistry 8: 671–675. 

was not possible. It soon became clear that the functional Blunt, J. W., DeLuca, H. F. & Schnoes, H. K. (1968) Biochemistry 7: 3317– 

metabolite in a target tissue could only be isolated with cer- 

3322. 

Boyle, I. T., Gray, R. W. & DeLuca, H. F. 

tainty from that target tissue. Therefore, from the intestines 68: 2131–2134. 

(1971) Proc. Natl. Acad. Sci. U.S.A. 

of 1600 vitamin D–deficient chickens given 3 H-labeled vitanology 

Boyle,I. T.,Miravet,L.,Gray, R. W.,Holick,M. F.&DeLuca,H. F. (1972) Endocri- 

90: 605–608. 

3 came the isolation of what was termed ‘‘peak 5’’ by 

min D 

Cousins, R. J., DeLuca, H. F. & Gray, R. W. (1970) Biochemistry 9: 3649–3652. 

the DeLuca group. After several chromatographic steps, 8 mg of 

Fraser, D. R. & Kodicek, E. (1970) Nature (Lond.) 228: 764–766. 

what still seemed to be slightly impure metabolite was obtained Fraser, D., Kooh, S. W., Kind, H. P., Holick, M. F., Tanaka, Y. & DeLuca, H. F. 

(Holick et al. 1971). From the fact that this metabolite was 

(1973) N. Engl. J. Med. 289: 817–822. 

Garabedian, M., Holick, M. F., DeLuca, H. F. & Boyle, I. T. (1972) Proc. Natl. 

known to have a tertiary hydroxyl came the idea that this 

Acad. Sci. U.S.A. 69: 1673–1676. 

metabolite could be specifically modified and rechromato- Gray, R., Boyle, I. & DeLuca, H. F. (1971) Science 172: 1232–1234. 

graphed. Thus, the trimethylsilyl (TMS) derivative of the me- Haussler, M. R., Myrtle, J. F. & Norman, A. W. (1968) J. Biol. Chem. 243: 4055– 

4064. 

tabolite was made and treated so as to remove the silyl groups 

Haussler, M. R. & Norman, A. W. (1967) Arch. Biochem. Biophys. 118: 145– 

from the secondary alcohol functions but not from the tertiary 153. 

alcohol on the 25-position (Holick et al. 1971). This was Holick, M. F., Garabedian, M. & DeLuca, H. F. (1972) Science 176: 1146–1147. 

Holick, M. F., Schnoes, H. K., DeLuca, H. F., Suda, T. & Cousins, R. J. (1971) 

followed by chromatographic purification, providing 2 mg of 

Biochemistry 10: 2799–2804. 

pure 25 TMS metabolite. Mass spectrometry and specific Kodicek, E. (1956) In: Ciba Foundation Symposium on Bone Structure and 

chemical reactions were used to identify the structure as 1,25- 

Metabolism (Wolstenhome, G.W.E. & O’Connor, C. M., eds.), pp. 161–174. 

Little, Brown and Co., Boston, MA. 

dihydroxyvitamin D 3 [1,25-(OH) 2 D 3 ] (Holick et al. 1971). 

Lawson, D.E.M., Wilson, P. W. & Kodicek, E. (1969) Biochem. J. 115: 269– 

At the same time, Fraser and Kodicek (1970) made the 277. 

important discovery that their ‘‘peak P’’ metabolite (‘‘peak 5’’ Lund, J. & DeLuca, H. F. (1966) J. Lipid Res. 7: 739–744. 

metabolite from the DeLuca laboratory and ‘‘peak 4B’’ from 

McCollum, E. V. & Davis, M. (1913) J. Biol. Chem. 25: 167–175. 

McCollum, E. V., Simmonds, N., Becker, J. E. & Shipley, P. G. (1922) J. Biol. 

the Norman group) is produced exclusively in the kidney. This Chem. 53: 293–312. 

important discovery was readily confirmed (Gray et al. 1971, McCollum, E. V., Simmonds, N. & Pitz, W. (1916) J. Biol. Chem. 27: 33–43. 

Norman et al. 1971). The Kodicek group generated large 

Mellanby, E. (1919) Lancet 1: 407–412. 

Morii, H., Lund, J., Neville, P. F. & DeLuca, H. F. (1967) Arch. Biochem. Bioamounts 

of the metabolite in vitro, but it was of insufficient phys. 120: 508–512. 

purity to allow identification. Proof of the 1,25-(OH) 2 D 3 struc- Neville, P. F. & DeLuca, H. F. (1966) Biochemistry 5: 2201–2207. 

ture came finally through direct chemical synthesis by a long 

Nicolaysen, R. & Eeg-Larsen, N. (1953) Vitam. Horm. 11: 29–60. 

Norman, A. W., Lund, J. & DeLuca, H. F. (1964) Arch. Biochem. Biophys. 108: 

procedure demonstrating that the configuration of the hy- 12–21. 

droxyl on the 1-position is indeed 1a (Semmler et al. 1972). Norman, A. W., Midgett, R. J., Myrtle, J. F. & Nowicki, H. G. (1971) Biochem. 

Thus, the active metabolite of vitamin D proved to be 1a,25- 

Biophys. Res. Commun. 42: 1082–1087. 

Osborne, T. B. & Mendel, L. B. (1917) J. Biol. Chem. 31: 149–163. 

(OH) 2 D 3 . Furthermore, this metabolite proved to be equally Semmler, E. J., Holick, M. F., Schnoes, H. K. & DeLuca, H. F. (1972) Tetrahedron 

Lett. 40: active in nephrectomized or sham-operated rats (Boyle et al. 

4147–4150. 



1052S 

SUPPLEMENT 

Steenbock, H. & Black, A. (1924) J. Biol. Chem. 61: 405–422. In August 1969, John Rotruck, perusing the Biochemistry 

Suda, T., DeLuca, H. F., Schnoes, H. K., Ponchon, G., Tanaka, Y. & Holick, M. F. 

(1970a) Biochemistry 9: 2917–2922. 

Journal, came across a paper by R. E. Pinto and W. Bartley 

Suda, T., DeLuca, H. F., Schnoes, H. K., Tanaka, Y. & Holick, M. F. (1970b) (1969). The paper was on the effect of age and sex on glutathi- 

Biochemistry 9: 4776–4780. 

one peroxidase and glutathione reductase activities in rat liver 

Windaus, A., Schenck, F. & von Werder, F. (1936) Hoppe-Seyler’s Z. Physiol. 

homogenates. This paper presented a pathway of glutathione 

Chem. 241: 100–103. 

metabolism that connected glutathione with the enzyme glutathione 

peroxidase. The reduced form of glutathione converts 

Paper 20: Glutathione Peroxidase: hydrogen peroxide to water most efficiently when it is catalyzed 

A Role for Selenium (Rotruck 1972) by the enzyme glutathione peroxidase. A figure in this paper 

illustrated the proposed relationship of glutathione, glutathi- 

Presented by Richard A. Ahrens, Department of Nutrition and one reductase, glutathione peroxidase and NADPH. It was 

Food Science, College of Agriculture and Natural Resources, Uni- also noted that glucose metabolism is the main source of 

versity of Maryland, College Park, MD 20742 as part of the NADPH. With this figure in front of him, John began to 

minisymposium ‘‘Experiments That Changed Nutritional Think- develop that same afternoon a hypothesis that selenium played 

ing’’ given at Experimental Biology 95, April 11, 1995, in Atlanta, a role in glutathione peroxidase. He generated a one-page 

GA. 

research proposal that was presented to Hoekstra, complete 

The first practical biologic interest in selenium occurred in with misspelled words and typographical errors, that same afthe 

1930s, when selenium was associated with alkali disease. ternoon. Hoekstra said that, as far as he knew, this was the 

This disease was called more graphically the ‘‘blind staggers’’ first research proposal of an involvement of selenium with 

in certain northern range areas and resulted from selenium glutathione peroxidase, and he reacted with enthusiasm. John, 

poisoning from forage grown on those shale soils. The standard however, had another year of heavy course work ahead of him 

treatment for such poisoning was to provide very small before he began to fully implement his proposal in the summer 

amounts of certain arsenic compounds in the feed or water of of 1970. Over the next several months the actual experimenta- 

livestock. 

tion progressed rapidly, culminating in studies demonstrating 

However, K. Schwarz and C. M. Foltz (1957) reported that incorporation of 75 Se into glutathione peroxidase. This portion 

traces of selenium prevented liver necrosis in certain vitamin of John’s Ph.D. dissertation took about 12 months to complete. 

E–deficient rats. Because vitamin E was known to be an antithis 

research reported on a glucose-dependent protection by 

The first publication (Rotruck et al. 1971) to come from 

oxidant nutrient, a role for selenium in repairing or preventing 

oxidative damage was soon being sought. The first paper de- dietary selenium against hemolysis of rat erythrocytes in vitro. 

scribing the enzyme glutathione peroxidase was also published That was also the year that John Rotruck left the University 

in the same year by G. C. Mills (1957). Glutathione peroxidase of Wisconsin and joined Procter and Gamble, where he had 

was reported to be an erythrocyte enzyme that protects hemothat 

research was first reported at the 1972 FASEB meetings 

a long career from which he retired in 1995. An abstract from 

globin from oxidative breakdown. Nobody in 1957 linked 

these two discoveries! 

(Rotruck et al. 1972a), and a subsequent publication on the 

In 1968 John T. Rotruck was assigned to the research laboselenium 

appeared later that year in the Journal of Nutrition 

prevention of oxidative damage to rat erythrocytes by dietary 

ratory of Professor W. G. Hoekstra to complete his Ph.D. studies 

at the University of Wisconsin. As these things often hapactually 

used the methodology from the original paper by Mills 

(Rotruck et al. 1972b). For this portion of the research, John 

pen, John Rotruck was initially assigned to work with another 

faculty member who promptly went off on a sabbatical leave. (1957) on glutathione peroxidase to establish that glutathione 

Hoekstra worked largely with the metabolic effects of zinc. He peroxidase was virtually nonexistent in selenium-deficient rats. 

was no particular expert in selenium or glutathione peroxidase, These papers from Madison did generate some controversy, 

but he was familiar with the whole field of trace mineral rephotometric 

method to study bovine erythrocyte glutathione 

because L. Flohe’ (1971), working in Germany, used a spectro- 

search. Although attempts to understand selenium’s mode of 

action had focused on its potential role as an antioxidant, peroxidase and reported that it contained ‘‘no non-protein 

these theories had not been uniformly accepted. Indeed, John prosthetic group.’’ He responded negatively to the 1972 reports 

Rotruck recalls that some faculty members at the University by Rotruck, but he did decide to recheck his earlier concluof 

Wisconsin did not believe that selenium could be part of sions. In the next year, the last part of John Rotruck’s disserta- 

an enzyme. In 1968 and 1969, he was engaged largely in taking tion research was published in Science and demonstrated the 

classes prior to the heavy research engagement that was to uptake of 75 Se by glutathione peroxidase and the fact that 

characterize his last two years in the Ph.D. program in bio- selenium was tightly bound to the enzyme (Rotruck et al. 

chemistry. 

1973). The controversy ended when Flohe’ et al. (1973) pub- 

Being an enthusiastic graduate student, John Rotruck read lished a letter reporting that glutathione peroxidase was, inthe 

literature on glutathione biochemistry because of the simiworkers 

deed, a selenoenzyme. The 1973 paper by Rotruck and colarities 

between selenium and sulfur chemistry and biochemisviews 

was identified as a Nutrition Classic in Nutrition Re- 

try. Glutathione is the common name of g-glutamylcysteinylin 

in 1980 and as a ‘‘Citation Classic’’ in Current Contents 

glycine. It is therefore a tripeptide composed of glutamic acid, 

1988. A RDA for selenium was established in 1989, and in 

cysteine and glycine. The sufhydryl group of cysteine is reactive 1992 John Rotruck and Bill Hoekstra received the Klaus 

and tends to form disulfide bonds with other glutathione mole- Schwarz Commemorative Medal for this work. 

cules when oxidized. Glutathione functions as a reducing agent John Rotruck recalls that it was the research environment 

to maintain other molecules in the reduced form and can at his time in the Biochemistry Department at the University 

convert hydrogen peroxide in the body to water. When it is of Wisconsin that made such discoveries possible. Just across 

oxidized to its double form linked by disulfide bonds, it can the hall from him, Hector DeLuca’s students were explaining 

be reduced to the sulfhydryl form. The enzyme that does that how vitamin D worked. Down the hall from them was John 

is called glutathione reductase, and it uses NADPH as a source Suttie’s laboratory where the role of vitamin K was being 

of hydrogen to achieve this reduction. 

explained. Nobel Prize winners came to speak on their research 





1053S 

on a frequent basis. It just felt normal to him that a graduate Pinto, R. E. & Bartley, W. (1969) The effect of age and sex on glutathione 

peroxidase activities and on aerobic glutathione oxidation in rat liver homogestudent 

should make important discoveries. If you didn’t do 

nate. Biochem. J. 112: 109–115. 

it, you felt almost inferior. At the time he didn’t realize that Rotruck, J. T., Hoekstra, W. G. & Pope, A. L. (1971) Glucose-dependent protection 

by dietary selenium against haemolysis of rat erythrocytes in vitro. 

he had made a ‘‘once in a lifetime’’ discovery. Now he does! 

Nature New Biol. 231: 223–224. 

Rotruck, J. T. et al. (1972a) Relationship of selenium to GSH peroxidase. Fed. 

Proc. 31: 691 (abs. 2684). 

Literature Cited Rotruck, J. T., Pope, A. L., Ganther, H. E. & Hoekstra, W. G. (1972b) Prevention 

of oxidative damage to rat erythrocytes by dietary selenium. J. Nutr. 102: 

Flohe’, L. (1971) Die Glutathioneperoxidase: Enzymologie und biologische As- 689–696. 

pekte. Klin. Wochenschr. 49: 669–683. 

Rotruck, J. T., Pope, A. L., Ganther, H. E., Swanson, A. B., Hafeman, D. G. & 

Flohe’, L., Gunzler, W. A. & Schock, H. H. (1973) Glutathione peroxidase: a Hoekstra, W. G. (1973) Selenium: biochemical role as a component of gluselenoenzyme. 

FEBS Lett. 32: 132–138. tathione peroxidase. Science 179: 588–590. 

Mills, G. C. (1957) Hemoglobin catabolism. I. Glutathione peroxidase, an eryth- Schwarz, K. & Foltz, C. M. (1957) Selenium as an integral part of factor 3 

rocyte enzyme which protects hemoglobin from oxidative breakdown in the against dietary necrotic liver degeneration. J. Am. Chem. Soc. 79: 3292– 

intact erythrocyte. J. Biol. Chem. 229: 189–197. 

3293.

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