Symposium: Experiments That Changed Nutritional Thinking

Symposium: Experiments That Changed Nutritional Thinking

Experiments That Changed Nutritional Thinking1

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 usesmade 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 experimentationstrength 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 recog-tific method begins, not with the accumulation of facts, but nized as being inadequate, and a new and radically differentwith recognition of an unsolved problem. This leads to conjec- hypothesis is proposed as the result of unusual insight, usuallyture about a solution, i.e., formulation of a hypothesis. The coupled with new methods. This leads to a new paradigm, andessence of the process is to subject hypotheses to critical exami- a period during which it is consolidated follows. An examplenation and experimental tests that have the potential to refute given by Kuhn of a scientific revolution is the discovery bythem. It is basically a process for detecting error; its strength Copernicus, at a time when essentially all astronomers believedlies in its self-correcting nature. If a hypothesis fails to with- that the earth was the center of our solar system, that in factstand 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 varioushypotheses vigorously to ensure that they are not rejected sciences, Kuhn proposes, is characterized by a series of para-without being tested thoroughly. Although the ability of a digms interspersed with periods of ‘‘normal science’’ duringhypothesis to withstand such tests does not establish unequivo- which problems falling within the limits of the prevailing para-cally that it is valid, as assumptions that are false are eliminated digm are explored. This view is complementary to that ofby repeated testing, we achieve an increasingly better approxi- Popper, who emphasized the need for constant hypothesis test-mation 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 isprotein 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 med-basis of feeding studies with dogs. Subsequently the German icine are the discoveries of Harvey, Lavoisier and Darwin, eachschool 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 arterieshypothesis was challenged by Fick and Wislicenus in 1866 in passed to the veins and circulated back to the heart. This wasan elegant nitrogen balance study performed during a moun- the demise of the hypothesis, postulated by Galen in the 2ndtain climbing expedition. Their results, interpreted by century, that the blood oscillated back and forth within theFrankland, proved conclusively that the hypothesis was errone- arterial system. Lavoisier’s discovery, in 1777, that combustionous (Paper 2). Nonetheless, the assumption that a high protein was a chemical process in which oxygen combined with otherintake was uniquely important in stimulating vigor of mind elements with the release of energy made untenable the cen-and body was accepted for another 40 years until it was chal- tury-old hypothesis that combustion represented loss of ‘‘phlo-lenged in 1904 by Chittenden, who demonstrated that healthy giston.’’ Charles Darwin in his classic study The Origin of Spe-young men remained vigorous with a protein intake about half cies, published in 1859, assembled evidence that new speciesthat recommended by Voit (Paper 5).

had evolved continuously over millions of years. His theoryThomas Kuhn, another philosopher of science, has con-of evolution demonstrated that biblical creationism, the beliefthat species arose intact through supernatural intervention,and which was almost universally accepted in countries that1 Presented as part of the minisymposium ‘‘Experiments That Changed Nutri-

tional Thinking’’ given in first part at Experimental Biology 95 on April 11, 1995 had adopted Western religions, was incompatible with scien-in Atlanta, GA, and in second part at Experimental Biology 96 on April 16, 1996, tific observations.in Washington, DC. This symposium was sponsored by the American Society for

All of the symposium presentations that follow discuss ex-Nutritional Sciences. Guest editors for the symposium publication were KennethJ. Carpenter, University of California, Berkeley, CA, Alfred E. Harper, University periments that influenced nutritional thinking. Some describeof Wisconsin, Madison, WI and Robert E. Olson, University of South Florida, experiments that challenged accepted concepts and resultedTampa, FL.

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

0022-3166/97 $3.00 q 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 withinthe 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 compo-field. 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 understandingessentiality of organic and inorganic micronutrients. Despite a the role of minerals in nutrition. The demonstration by Stock-number of observations during the 19th century that diets man in 1893 that inorganic iron was used efficiently for hemo-composed of purified food constituents did not support growth globin synthesis corrected this erroneous assumption (Paperor even life, this shift did not occur suddenly as the result of 3). Thirty-five years later, Hart and associates discovered thata single discovery; it occurred over a period of more than 60 copper was essential for the utilization of iron in hemoglobinyears. The lag was attributable in large measure to resistance formation. It is now known that copper promotes uptake ofto the new paradigm by many scientists who were influenced iron by transferrin and increases the utilization of iron byby 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 andminerals were the sole principles of a nutritionally adequate colorless oils both had vitamin A activity, a conflict betweendiet. Only after the inadequacy of Liebig’s hypothesis had been competing hypotheses about the nature of vitamin A precur-demonstrated in many experiments that should have changed sors was resolved by Thomas Moore, who in 1930 showed thatnutritional thinking, but did not, was the new paradigm gener- the yellow b-carotene was converted to colorless vitamin Aally accepted. Four of the papers describe experiments that in the animal body (Paper 11). Thiamin was shown by Loh-contributed 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 metabo-in 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 protein-disease was prevented by including rice polishings or beans or free extracts of yeast could cure pellagra posed a problem thatwater extracts of them in the diet. He concluded that chickens was resolved when Krehl and colleagues discovered in 1945needed an organic complex provided in adequate quantities that the amino acid tryptophan was a precursor of niacin inby rice polishings and beans but not by polished rice. His the body (Paper 15). That complex interactions and antago-observations had little immediate effect on orthodox nutri- nisms can occur among trace minerals was discovered by Dicktional views, even though they ultimately contributed to the and associates, who observed that copper deficiency occurs inbasis for the new paradigm (Paper 4). animals with a normally adequate intake of copper if theirLiebig’s concept that the nutritional value of foods and intake of molybdenum and/or sulfate is high (Paper 16). Infeeds 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 Also during the 1970s, through the work of Kodicek inthat calves from cows fed an all-wheat ration survived only a

Cambridge and Deluca in Wisconsin, the prevailing view thatshort time even though the wheat ration was balanced forvitamin D acted directly to promote intestinal absorption ofmajor nutrients to match an all-corn ration that proved to becalcium and regulation of bone metabolism was shown to be infully adequate. This was a clear demonstration of the inade-error. They discovered that vitamin D, through the combinedquacy of Liebig’s concept (Paper 6).actions of the liver and kidney, was converted to a hormoneSubsequently, McCollum found that rats fed a simplifiedthat mediated the actions attributed to vitamin D. This repre-diet of casein, carbohydrate and minerals stopped growing un-sented a new concept: the action of a vitamin depending onless supplied with a fat-soluble factor present in butter but notits conversion to a hormone (Paper 19).in olive oil. Rats fed a polished rice diet were found to need

Another major paradigm shift in nutrition resulted froma water-soluble factor B, as Grijns had shown, as well as thediscoveries about the ability of the body to synthesize andfat-soluble factor A (Paper 7). During this period, Holst anddegrade nutrients and tissue constituents. The shift occurredFroelich in Norway induced a scurvy-like disease in guineain phases, two of which are discussed in the symposium.pigs by feeding them diets resembling those of Grijns. This

Claude Bernard, the great French physiologist, conjectureddisease was prevented by providing the guinea pigs with lemonabout the source of glucose in the blood of dogs consuming ajuice or cabbage.diet that contained neither sugar nor starch. By a series ofAlso, between 1909 and 1914, Osborne and Mendel atcarefully conducted experiments during the 1850s, he discov-Yale, following on an earlier observation by Hopkins in Cam-ered liver glycogen and the process of gluconeogenesis bybridge that tryptophan was essential for the survival of mice,which glucose and glycogen could be synthesized in the liverdiscovered that some plant proteins did not support growth offrom non-glucose precursors, enabling this organ to supplyrats unless the rats were supplemented with other amino acidsglucose to the blood (Paper 1).(Paper 8). Hopkins, and Funk in London, both postulated in

The use of isotopically labeled compounds by Schoen-1912 that diseases such as scurvy, beriberi and rickets wereheimer in the 1930s to follow the metabolic fate of fatty acidsdietary deficiency diseases. Only between 1910 and 1915, afterand amino acids administered orally revealed for the first timethese and other demonstrations of the inadequacy of Liebig’sthat these nutrients were incorporated rapidly into depot fatconcept, was the new paradigm of the essentiality of minorand body proteins, respectively, and that their metabolitesconstituents of foods widely accepted.continued to be excreted over many days. Through his work,Acceptance of the new paradigm was followed by a periodthe concept of distinct exogenous (dietary) and endogenousof unparalleled discovery in nutritional science from about(tissue) metabolism was replaced with the concept of the ‘‘dy-1915 to the 1950s, during which some 40 essential nutrientsnamic state of metabolism,’’ the continuous breakdown of tis-were identified and characterized and their functions explored.

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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common pool from which new tissue components were synthe- We believe that these proceedings illustrate, on the onehand, the tremendous advances resulting from the scientificsized (Paper 14).

The demonstration by Becker and colleagues that sucrose approach to nutrition and, on the other, the importance ofcontinually maintaining a critical approach to even well-ac-and fructose are toxic to young pigs and calves represents

an extension of this paradigm, one of many, illustrating that cepted hypotheses and concepts.metabolic pathways for some nutrients may not be functionalat birth and undergo development during the early stages of Paper 1: The Liver Forms, Stores andgrowth (Paper 18).

Secretes Glucose (Claude Bernard,With the successive discoveries of essential nutrients be-tween 1915 and 1950 and the virtual disappearance of dietary 1860)deficiency diseases, emphasis in nutrition was on ensuring that

Presented by Patricia B. Swan, Department of Food Science anddiets would provide adequate quantities of all essential nutri-Human Nutrition, Iowa State University, Ames, IA 50011 asents to prevent impairment of growth and development. Al-part of the minisymposium ‘‘Experiments That Changed Nutritionalthough it was recognized that requirements declined with in-Thinking’’ given at Experimental Biology 95, April 11, 1995, increasing age, little attention was given to the long-term effectsAtlanta, GA.of total food intake. One of the first challenges to the paradigm

that if essential nutrient intake was adequate throughout life, In 1834, the 21-year-old Claude Bernard left the hills ofother dietary factors would be of little consequence, came the Rhone Valley and went to Paris to seek his fortune as afrom Clive McCay. He argued that short-term trials with the playwright. A professor of literature at the Sorbonne read oneemphasis on rapid growth did not provide an adequate test of of his plays, Arthur of Brittany, and counseled Bernard to enrollthe most desirable nutritional state throughout life. He found in medical school instead. Heeding this advice, Bernard en-that, although rats allowed to freely eat a nutritionally ade- tered the College de France in the fall (Bernard 1979).quate diet grew most rapidly, those allowed only restricted There he became intrigued by lectures in physiology givenamounts of food could survive much longer (Paper 13). Com- by Francois Magendie. Most chemists and physiologists of thepeting hypotheses about the basis for these effects remain unre- time believed that only plants synthesized lipids, carbohydratessolved, but they have opened new directions in nutritional and proteins, whereas animals merely degraded them. Thethinking, especially in relation to appropriate body weight and macromolecules within the body were therefore assumed toenergy 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 sometimesdistracted attention from investigations of the nonnutritional seemed to be more fat in an animal’s body than could havecomponents of foods and from the ancient paradigm that foods come from its diet. Bernard was captivated by Magendie’s dem-contain nutriment, medicines and poisons. The finding that onstrations of the intricacies of animal physiology, and frombroccoli in the diet increased resistance of guinea pigs to X- 1841 to 1844 he served as his laboratory assistant, gainingirradiation and that this effect was not related to its contribu- knowledge of techniques in animal experimentation (Holmestion of known nutrients shifted attention back to the nonnutri- 1974).ent components of foods (Paper 17). There is now evidencethat substances in cruciferous plants and some other foods may

Studies on Glucoseincrease resistance to cancers. These observations have led toacceptance of a scientifically based form of the paradigm that Soon Bernard began his own experiments, studying diges-foods can affect health by their contributions of chemicals tion and certain functions of the nervous system. He extendedother than essential nutrients through their influence on sus- the digestion studies to examine the fate of sugars within theceptibility 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 tractlearned through the use of animal models. It also illustrates (Grmek 1968). Cane sugar, injected directly into a vein, wasthat caution must be exercised in extrapolating findings in one excreted unchanged in the urine; injected grape sugar, how-species to another. For example, rats were an excellent choice ever, disappeared. Thus, glucose seemed to be the major formfor 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 inspecies led a leader in the field to conclude that these diseases the dog’s blood. Thus, glucose was a normal constituent ofin humans were not, after all, due to dietary deficiencies. These blood, at least after starch consumption, not just a sign of theexperiments also illustrate the need for caution in assuming diabetic condition as had been thought previously (Grmekthat 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’sconcepts do not necessarily make earlier ones obsolete; several idea, he conducted experiments with dogs that he thoughtmay exist together and overlap, with all being valid frameworks would show glucose was burned in the lungs; however, thesefor investigation. What is the outlook for new paradigms and experiments yielded contradictory or uninterpretable resultsconcepts in nutritional science? Application of techniques (Grmek 1968, Holmes 1974). In the early experiments, he hadfrom genetics and molecular biology to nutritional problems only insensitive methods for detecting and quantifying glucosehas led in recent years to advances in understanding the roles and needed to use large quantities. He was assuming that theseof 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 hehormones, and responses of the immune system. Undoubtedly sometimes fed the glucose and sometimes injected it. Improve-other new paradigms and concepts, unanticipated now, will ment of a method for the detection of glucose based on its

ability to reduce copper in an alkaline potassium tartrate solu-follow.



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tion significantly improved his results. Gradually Bernard im- its chemical similarity to starch in plants and reported that itwas present in opalescent extracts of the liver and formed aproved his experimental techniques, and the early work set

the stage for later, more successful, experiments. white precipitate when alcohol was added. It gave a red-winecolor with iodine and was hydrolyzed by diastase, from saliva orplants, to produce glucose (Bernard 1857a, 1857b, and 1857c).The Source of Blood Glucose

In July 1848, Bernard conducted an experiment with a A Productive Decadefemale that had been nursing a litter of pups. He did not feed

Within 10 years, Claude Bernard had made three majorher for one day and, as expected, found no glucose in herdiscoveries: 1) Glucose is a normal constituent of liver. 2)gastrointestinal tract, but to his surprise, he did find glucoseLiver is the source of blood glucose. 3) Liver forms glucosein her blood (Grmek 1967). ‘‘What was the source of thisand stores it as glycogen, which, upon degradation, yieldsglucose?’’ After this experiment, he altered the direction ofglucose.his research to find the answer to this question (Grmek 1968,

Bernard’s experiments and the theories he derived fromHolmes 1974).them were major contributions to the science of nutritionalIn August, Bernard used a dog that had been fed only meatphysiology. His exceptional skill in the surgery required for(no carbohydrates) for eight days and found a large amountthese studies, and the understanding that he developed re-of glucose in the portal vein, smaller amounts in the heartgarding the use of intact animals in experimentation, earnedand the neck, but none in the chyle, stomach, intestine orhim recognition as the ‘‘father of experimental medicine.’’urine. He exclaimed that the source of this glucose was ‘‘in-His major textbook (Bernard 1865) became a classic in thecomprehensible’’ (Grmek 1968).field, and he later received many honors, including member-A few days later, using a dog that had been fed only lardship in L’Academie Francaise (Bernard 1979, Olmstedand tripe, he found no glucose in the mesentery (before the1938).portal vein), but ‘‘enormous’’ quantities of glucose in the liver

(Grmek 1968). Within the next four days, Bernard measuredthe glucose content of the liver of many different species, Literature Citedfinding significant amounts of glucose in most. He concluded

Bernard, C. (1849) De l’origine du sucre dans l’economie 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 anim-

aux. C. R. Hebd. Acad. Sci. 31: 571–574.Barreswil 1848).Bernard, C. (1853) Recherches sur une nouvelle fonction du foie. These pre-Subsequent experiments provided evidence that the liver

sentee a la Faculte des Sciences de Paris. Imprimerie de L. Martinet, Paris,was the source of glucose in the body. By placing a tie France.

Bernard, C. (1855) Sur le mecanisme de la formation du sucre dans le foie.between the liver and the portal vein, Bernard was able toC. R. Hebd. Acad. Sci. 41: 461–469.show that the source of glucose previously found in the

Bernard, C. (1857a) Les phenomenes glycogeniques du foie. Soc. Biol. 2eportal vein was the back flow of blood from the liver, not Serie 4: 3–7.Bernard, C. (1857b) Sur le mecanisme physiologique de la formation du sucrean alternative source prior to the liver (Bernard 1849 and

dans le foie. C. R. hebd. Acad. Sci. 44: 578–586.1850). For this work he received the Prize for Physiology inBernard, C. (1857c) Remarques sur la formation de la matiere glycogene du1851 (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 a l’Etude de la Medecine Experimentale. J. B.Bailliere et fils, Paris, France.concept of the body’s ability to regulate its internal environ-

Bernard, C. (1878) Lecons sur les Phenomenes de la Vie Communs aux Anim-ment (Bernard 1878). aux et aux Vegetaux. 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: ClaudeBernard and the Internal Environment, pp. 17–27. Marcel Dekker, New York,

In 1855 Magendie died, and Bernard was named to the NY.Grmek, M. D. (1967) Catalogue des Manuscrits de Claude Bernard. MassonChair in Physiology at the College de France (Olmsted 1938).

et Cie, Editeurs, Paris, France.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 glyco-to digestion, diabetes, toxins and the nervous system. During genic function of the liver. J. Hist. Biol. 1: 141–154.

Holmes, F. L. (1974) Claude Bernard and Animal Chemistry. Harvard Universitythis time, he typically measured glucose in duplicate in severalPress, 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 notmake the duplicate measurement until the following day, afterthe liver had been allowed to stand at room temperature over- Paper 2: Protein Cannot Be the Solenight. To his surprise, the content of glucose had increased Source of Muscular Energy (Fick,markedly (Bernard 1855, Grmek 1967). Wislicenus and Frankland, 1866)Bernard next decided to perfuse an isolated liver with coldwater until the perfusate was free of glucose. He then allowed Presented by Kenneth J. Carpenter, University of California,the liver to stand at room temperature for some hours. Upon Berkeley, CA 94720-3104 as part of the minisymposium ‘‘Experi-resuming perfusion, he again found significant amounts of glu- ments That Changed Nutritional Thinking’’ given at Experimentalcose in the perfusate. It appeared, therefore, that something Biology 96, April 16, 1996, in Washington, D.C.within the liver was giving rise to glucose and it clearly wasnot making glucose from other elements in the blood. He took By 1865 it had been the general ‘‘textbook’’ view for over

20 years that the energy needed for muscular contraction camethis as proof that there was a source of glucose within the liver(Bernard 1855). from the destruction of a portion of the muscle’s own sub-

stance, i.e., protein. This had been stated by the organic chem-Bernard then began the tedious job of isolating the gluco-genic material present in the liver. He eventually recognized ist Justus Liebig in his influential Animal Chemistry. On page




233, he added that the protein broke up during the release of TABLE 2energy and that the nitrogenous fraction was converted to ureaand excreted by the kidney, so that the total amount of work The results from the climbing trialperformed (i.e., both internally, as in the heart muscles, and

Fick Wislicenusexternally) was proportional to the nitrogen excreted in theurine (Liebig 1840).

Body weight (/ objects carried), kg 66 76Liebig’s second point was essentially disproved by theUrinary N (0500–1900 h),1 g 5.74 5.55finding 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 proteinthey had worked a treadmill than on days when they had (at 4.37 kcal/g)2 kcal 157 151.6

Work equivalent (at 423 kg-m/kcal),rested (Smith 1862). However, it was still possible that pro-kg-m 66,400 64,100tein had been the sole muscle fuel and that more had broken

Net work in ascending 1956 mdown on rest days by some alternative mechanism. It cer-against gravity, kg-m 129,000 149,000tainly seemed that nitrogen intake was the main determi-

nant 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 considerableamount 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 morningprotein to urea and carbon dioxide were known, and also they set out, carrying urine collection equipment, andthe relation of heat energy to mechanical work, it should be walked steadily up a steep path until, at 1320 h, they reachedpossible to determine whether the amount of body protein another hotel at the summit. They were in a cold mistmetabolized 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 dayFrankland, was at work in England developing a method for until 1900 h on their exercise day, their only food was cakesmeasuring the heat of oxidation of organic materials. High made from starch paste fried in fat; they also drank stronglypressure ‘‘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 themanganese dioxide with the test material in a little ‘‘diving subsequent calculations, with slight ‘‘rounding off ’’ of theirbell’’ immersed in an insulated water tank. Using a series values, are set out in Table 2. It is seen that even withoutof controls to adjust his results for the rise in temperature making any allowance for the internal work of breathingof 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 efficient, the quantity of protein metabolized was insuffi-1866). cient to have provided the energy needed for their climb;The most directly relevant results are set out in Table 1. in fact it was 51% for one subject and 43% for the other.He assumed that metabolized protein yielded one-third of

The climbers concluded that ‘‘the burning of protein can-its own weight of urea, and he therefore subtracted thenot be the only source of muscular power’’ (Fick and Wisli-residual gross energy of this quantity of urea when estimatingcenus 1866). And Frankland, on page 684 of his review ofthe energy released from the metabolism of 1 g of proteinthese and other results, added: ‘‘Like every other part of thein the body.body the muscles are constantly being renewed; but thisBy this time it also seemed well established that the me-renewal is not perceptibly more rapid during great muscularchanical equivalent of heat was such that the energy neededactivity than during comparative quiescence. After the sup-to raise 1 kg a distance of 423 m was at least approximatelyply of sufficient albuminized matter [protein] in the food toequivalent to 1 kilocalorie (Joule 1843).provide for the necessary renewal of the tissues, the bestNow the human trial was needed. Fick and his universitymaterials for the production, both of internal and externalcolleague Johannes Wislicenus passed a night at a hotel nearwork, are non-nitrogenous material. . .’’ (Frankland 1866).

These conclusions were not immediately accepted, butthey stimulated further long-term trials that were confirma-TABLE 1 tory, although Liebig himself never admitted in so manyword that he had been wrong (Carpenter 1994).Frankland’s presentation of his results for the energy values

of protein and urea1

Name of substance dried at Heat units Kg-m Literature Cited1007C (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.

Fick, A. & Wislicenus, J. (1866) On the origin of muscular power. Phil. Mag.substance:Lond. (4th ser.) 31: 485–503.When burnt in oxygen

Frankland, E. (1866) On the source of muscular power. R. Institution Proc. 4:Beef muscle purified by ether 5.10 2161661–685.Purified albumen 5.00 2117

Joule, J. P. (1843) On the calorific effects of magneto-electricity and on theUrea 2.21 934mechanical 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 toBeef 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.Soc. Lond. 151: 747–834.1 From Frankland (1866).



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Paper 3: Inorganic Iron Can Be Used toBuild Hemoglobin (Stockman, 1893)

Presented by Richard A. Ahrens, Department of Nutrition andFood Science, College of Agriculture and Natural Resources, Uni-versity of Maryland, College Park, MD 20742-7521 as part ofthe minisymposium ‘‘Experiments That Changed NutritionalThinking’’ given at Experimental Biology 96, April 16, 1996, inWashington, DC.

The condition of anemia was originally named morbus virgi-neus by Johannes Lange (Lange 1554). Lange was a physicianof Lemberg and Rector of Leipzig University. He consideredthis disease to be peculiar to virgins and to be due to a retentionof menstrual blood. His therapy involved instructing virginsafflicted with this disease to marry as soon as possible. He cited FIGURE 1 Hemoglobin responses of chlorotic patients to ferrousno less an authority than Hippocrates, in his treatise De Morbis sulfide (in keratin capsules, 550 mg/d), iron citrate (subcutaneous, 32

mg/d) and bismuth oxide (9.6 g). The response times between the initialVirginum, as also recommending marriage to cure this disease.and final observations were 12 d for ferrous sulfide, 10 d for iron citrateJ. Varandal renamed this disease ‘‘chlorosis’’ (Varandaland 9 d for bismuth oxide. The percentages given on the y-axis are1615). The popular English term was the ‘‘green sickness,’’based on the clinical standard for hemoglobin in use in 1893. Generatedreferring to the greenish hue assumed by Caucasians whenfrom the data of Stockman (1893).their blood is low in hemoglobin. Chlorosis soon became a

central feature in medical textbooks describing the diseases ofwomen. Because chlorosis was a sign of virginity, European During the 1880s, Gustav von Bunge wrote two influential

papers in which he concluded that only organic sources ofartists often painted young women during this era with a green-ish hue. In art, if not in fact, chlorosis was a widespread condi- iron were of value in treating chlorosis (Bunge 1885 and 1889).

Von Bunge’s interest in iron dated back to 1874 when hetion.By the mid 19th century, the disease of chlorosis was ac- analyzed both blood and milk and recognized that blood was

rich in iron and milk had very little. He developed a philoso-cepted by many physicians as being associated with neuroticand hysterical manifestations (Bullough and Voght 1973). phy that people are always best served when they get essential

nutrients from foods. That philosophy also applied to iron. ToChlorosis became a form of neurosis. This view of chlorosiswas an impediment to the acceptance of dietary therapy for quote von Bunge, ‘‘Why should a patient buy his iron in the

pharmacy and not on the market with the usual foodstuffs?’’its treatment. It was a refinement of the view that anemia wasdue to virginity in women, but it continued to perpetuate a This is a philosophy with many adherents today. As von Bunge

implemented what he believed, however, it soon became asex bias. Bullough and Voght (1973) pointed out that sex biasflourished during the latter half of the 19th century as a male personal crusade in which he claimed that ‘‘the iron which

the doctors give to chlorotics to form hemoglobin is not ab-‘‘backlash’’ against women’s demands for more education,greater political equality and the elimination of male stereo- sorbed at all.’’

As Gustav von Bunge got into his crusade he was soontypes about woman’s place. Medical practitioners were almostall men, and many of them were hostile to any change in the claiming that iron therapy was successful because of the power

of suggestion. It was well known, after all, that most chloroticsstatus quo in male-female relationships. Medical schools thathad admitted a few women early in the 19th century began were women and often exhibited nervous or psychic distur-

bances. He felt that this made them ‘‘highly suggestible.’’ Theto reject female applicants purely on the basis of their sex.Nursing schools were established in growing numbers to pro- true villains, according to von Bunge, were those who advised

young women to practice vegetarianism. He spent much ofvide a alternative for females. By the latter part of the 19thcentury, chlorosis became an extremely common diagnosis what remained of his life arguing against vegetarianism and

was enthusiastic about the nutritional value of meat in the(Clark 1887). It is necessary to appreciate this historical con-text to understand some of the resistance to accepting a nutri- human diet. He died in 1920, just as Prohibition was beginning

as the ‘‘noble experiment’’ in the United States. He was anent deficiency as the cause of this disease.Pierre Blaud in France recommended the use of pills con- implacable foe of alcohol consumption all his life and looked

forward to the results of this experiment with U.S. citizens astaining ferrous sulfate for the treatment of chlorosis (Blaud 1832).The average dose amounted to approximately 150 mg/d, and the guinea pigs. He anticipated that a model U.S. society

would result from Prohibition and that Europe would thenconsiderable success was achieved. Despite this success, however,there was considerable resistance to the acceptance of chlorosis soon follow this great example (McCay 1953). It is undoubt-

edly fortunate that he did not live to see the result of thisas a simple dietary iron deficiency. One of the obstacles to beovercome was the just-discussed sex bias that tended to associate particular experiment.

When he wasn’t blaming the power of suggestion for thechlorosis with the neuroses of women. Another obstacle to beovercome, however, was the inability of investigators using the beneficial effects of inorganic dietary iron on chlorosis, von

Bunge had a second explanation. Bullough and Voght (1973)balance method to demonstrate that inorganic iron could beabsorbed from the gastrointestinal tract. V. Kletzinsky conducted noted that such contradictions were common among research-

ers studying ‘‘women’s diseases’’ during the late 19th century.a series of experiments (Kletzinsky 1854). In all of his studies,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-equal to the amount of inorganic iron ingested. A third obstacleto be overcome was the toxic effect of intravenous injections of teria of hydrogen sulfide, which then reacted with organic iron

compounds in the ingesta to produce insoluble ferrous sulfide.ferrous sulfate in dogs.




more commonly between the advent of menstruation and the consummationIf inorganic salts of metals having insoluble sulfides were givenof 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.Gellschaft. Aerzte Wien 2: 281–289.iron compounds free for absorption.

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.

Stockman, R. (1893) The treatment of chlorosis by iron and some other drugs.the test (Stockman 1893). He did tests on chlorotic patients toBr. Med. J. I: 881–885, 942–944.determine if inorganic iron worked directly or by the indirect

Stockman, R. (1895) On the amount of iron in ordinary dietaries and in somemechanism of binding with hydrogen sulfide. His results are articles of food. J. Physiol. 18: 484–489.

Varandal, J. (1615) De Morbis et Affectibus Mulierum Libri Tres. Lyons, France.summarized in Figure 1. He gave subcutaneous daily injectionsof ferrous citrate providing 32 mg of iron to three chloroticyoung women and found an increase from 44% to 52% of Paper 4: A Micronutrient Deficiency innormal hemoglobin concentration in 10 d. After 24 d the

Chickens (Grijns, 1896–1901).women had blood hemoglobin concentrations that were 72%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 partenclosed in keratin capsules that released the iron salt in the of the minisymposium ‘‘Experiments That Changed Nutritionalsmall intestine. Iron in this form could not be expected to Thinking’’ given at Experimental Biology 95, April 11, 1995, inbind any additional hydrogen sulfide. Nevertheless he found Atlanta, GA.an increase from 48% to 60% of normal hemoglobin concen-tration in 12 d. After 33 d the women had blood hemoglobin Recently we reported on Christiaan Eijkman’s work onconcentrations that were 84% of normal. He also gave 9.6 g/ polyneuritis in chickens performed during the 1890s in Indo-d of bismuth dioxide to chlorotic women having blood hemo- nesia (Carpenter and Sutherland 1995). The timeline showsglobin concentrations that were 55% of normal and found how early it was when this work was being performed: atthese hemoglobin levels to be only 54% of normal 9 d later. the same time as Atwater’s calorimetry work, and before theManganese dioxide gave a similar result. These latter two salts ‘‘vitamine’’ concept of Funk and McCollum’s studies of fat-were quite capable of removing hydrogen sulfide from the gut, soluble factors (Table 1). This was a time when the infectiousbut 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 inBunge’s hypothesis by Ralph Stockman in 1893 should have Indonesia at that time, that he recognized a similarity withmade it apparent that inorganic iron had great value as a polyneuritis seen in chickens (Eijkman 1990). He found thatnutrient. In another paper two years later, Stockman (1895) polyneuritis appeared in fowls when they were fed a diet ofshowed that chlorosis in young women was explained by their polished rice, but that by adding the silver skin, which hadlow overall intake of food, particularly of meat, which resulted been removed during polishing, the polyneuritis could be pre-necessarily 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 nerveneed. He showed, through the use of a more specific analytical poison produced during the fermentation of starch in theprocedure for iron in foods that avoided interference from chicken’s crop and that the silver skin contained an antidotestarch, 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 helittle, 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 hadfar 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 Germanyto question the therapeutic value of inorganic iron in anemia (Grijns 1901). In 1892 he was sent to Indonesia to assist inthrough the 1920s. Gustav von Bunge died in 1920. The old another of Eijkman’s studies, that of the physiological adaptionconcept of ‘‘chlorosis’’ is also long gone (Fowler 1936). How- of Europeans to tropical conditions. But Grijns was shortlyever, precautions are still needed to ensure an adequate intake recalled to military service, and when he was able to returnof iron. In the United States, white bread and many breakfast to Batavia (modern-day Jakarta), Eijkman had already left forcereals are routinely fortified with inorganic iron, and pregnant Holland. Grijns was then appointed to carry on the investiga-women 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 physiologi-drain on people’s blood supply, iron deficiency anemia remainsa serious problem.

TABLE 1Literature Cited

Dates of some significant papers in nutritional scienceBlaud, 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.

1901 Grijns (need for unidentified micronutrients)Bunge, G. (1885) Ueber die Assimilation des Eisens. Hoppe-Seyler Z. Physiol.1907 Holst and Frohlich (guinea pig scurvy)Chem. 9: 49–59.1912 Funk (‘‘vitamine’’ concept)Bunge, G. (1889) Uber die Aufnahme des Eisens in den Organismus des Sau-

glings. Z. Physiol. Chem. 13: 399–406. 1914 McCollum (fat-soluble factors)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



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Grijns was also looking for a food material that when givenTABLE 2in small amounts with polished rice, would prevent an out-break of polyneuritis. He tested the mung bean (which he hadGrijns’ key experiments1

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 composi-

of white rice cause not cured. tion of these two legumes, he saw that soybean was the richerpolyneuritis?in protein, fat and minerals but less effective as an anti-neuritic2. Was the removal of fat with No, adding oil to the diet didsubstance (Table 3). This supported his belief that polyneuritisthe silver skin the cause of the not prevent the disease.

disease? was not caused by a lack of these three nutrients. In later3. 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 thereforedeveloped polyneuritis. had the same experience with Phaseolus radiatus (mung bean)4. Could the protective No, the extracts did not

as with the seed coat of the rice . . . at every attempt tosubstance be extracted from prevent or cure the diseaseisolate the active constituents, they perished . . . in differentrice silver skin and mung because they decomposed

bean? during extraction. conditions they apparently became decomposed’’ (Grijns5. 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 tosago, tapioca and arenga starch diets did not prevent polyneuri-1 Based on the paper by Grijns (1901). tis. However, removing starch and feeding meat alone did curethe condition. From these results, Eijkman had concluded thatstarch was a significant harmful factor in the etiology of poly-cal and pharmacological properties of the tannin contained inneuritis, but this explanation did not satisfy Grijns. He felt itred rice’’ and to determine if the pigment found in red riceimportant to determine whether polyneuritis could developcould be considered as a curative or preventive remedy forindependently of starch consumption. He therefore fed fourberiberi.birds meat that had been extracted with water for 2 d, and allGrijns was aware that it was not just red rice that preventeddied with signs of polyneuritis. He then fed eight birds meatpolyneuritis but all unpolished rice, and he decided to continuethat had been autoclaved, and six of these also developedto study the whole silver skin and not just to focus on thepolyneuritis. Thus Grijns concluded that the development ofpigment. His first feeding experiments confirmed Eijkman’spolyneuritis was not connected with starch and was evenconclusions that polyneuritis was not caused by a lack of fat,wholly independent of the presence of carbohydrate. Theseprotein or mineral (Table 2). In his 1901 report, Grijns re-experiments also confirmed that the nerve degeneration wasmarked: ‘‘In judging the suitability of a food, we have notnot caused by a lack of protein (Table 2).finished when we have determined the quantity of albumen

In a discussion of polyneuritis and beriberi, Grijns put forth. . . fat, carbohydrates and salts, even when we have appliedtwo explanations for the symptoms that occurred: ‘‘either wethe corrections for digestibility. We can indeed calculate frompresume a deficiency, a partial starvation, . . . or . . . therethis whether a balance of nitrogen will be possible with it andis a microorganism which exercises a degenerative influencewhether the work which must be performed bother internallyon the nerves’’ (Grijns 1901). Concerning the possibility of aand externally, can be obtained from it, but not whether per-deficiency or partial starvation, Grijns stated that very littlemanent health is possible.’’was known about the metabolism of the peripheral nervousGrijns believed that a number of substances existed, whosesystem and that ‘‘if for the maintenance of the peripheralactions were not explained, but which played an importantnervous system, a certain substance or group of substances ispart in the prevention of disease. He illustrated this idea withindispensable, which are immaterial for the metabolism of thetwo examples: ‘‘how very difficult it is, in spite of all themuscles, then it may be assumed that very little of them ischemical analyses of mother’s milk, to find a good substitutenecessary. When therefore in certain foods the substances in-for it and how frequently we find that, when we think onedispensable for the nervous system are lacking or are presenthas been found, we are again disappointed’’ and ‘‘the peculiarin insufficient quantity, in the first place any reserve supply,fact that scurvy, which usually develops from lack of freshwhich is present either in the nerve itself or in the blood orfood, which sometimes occurs on long sea voyages, is usuallyin some other organ, will be used up . . . (and) disturbancescured when the patients can again obtain fresh meat and freshwill develop.’’greens.’’ He concluded that still-unknown substances may be

He explained that polyneuritis did not develop with totalresponsible.Grijns used two approaches for investigating these ‘‘un-

known substances.’’ One was to prepare different fractions fromTABLE 3the silver skin, and the other was by comparative assay (Grijns

1901). He first boiled rice bran in a large quantity of waterComposition of mung bean (P. radiatus java) and soybeanfor 24 h and then strained, filtered and evaporated the liquor

(S. hispida tumida java)1to give a dried extract. He used fowls that were already con-suming a polished rice diet and gave them the extract via a

Mung bean Soybeanstomach tube. All the birds died with symptoms of polyneuritis.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

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




starvation, because in this situation the muscles were drawn TABLE 1upon to provide the needed protein and that this process re-leased 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 healthGrijns used the notion of individual differences to account for and strength’’)1why some birds did not develop polyneuritis: ‘‘one personneeds a much larger quantity of food than another to maintain Authority Protein Energyhis physical equilibrium, while doing the same work . . . If

g kcaltherefore the total metabolism shows important differences,there is no reason why, separate tissues which together furnish

Ranke 100 2324the total metabolism, should not have individual differences.Munk 105 3022Therefore a food which contains just enough of the still un-Voit (1881) 118 3055known nerve nutritive substances for one fowl contains too Rubner 127 3092

little for another.’’ Moleschott 130 3160In regard to the concept of a microorganism causing nerve Atwater (1894) 125 3315

degeneration, Grijns believed that this depended on the nour-1 From McCay (1912), based on dietary intake studies.ishment of the tissue to resist infectious organisms. He con-

cluded 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 partbe absent without serious injury to the peripheral nervous through the expanded use of the nitrogen balance approachsystem . . . The distribution of these substances in the differ- (developed initially by Boussingault for his studies in Alsaceent food stuffs is very unequal . . . Attempts to separate them on the utilization of foodstuffs by milch cows), others beganhave resulted in their disintegration . . . (showing) they are to question whether intakes lower than those shown in Tablevery complex’’ (Grijns 1901). 1 would not only be adequate but possibly offer benefits for

improvements in health. Arguably, the most significant ofthese others was Russell Henry Chittenden. Thus, BenedictLiterature Cited (1906) says: ‘‘Of all the experiments heretofore made in whichthe low protein diet was used, none can compare with theCarpenter, K. J. & Sutherland, B. (1995) Eijkman’s contribution to the discov-

ery of vitamins. J. Nutr. 125: 155–163. exhaustive series of experiments recently completed by Profes-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.-Indie 30: 295–334

and after the ‘‘dust had really settled’’ they were destined to(1890); 32: 353–362 (1893); 36: 214–269 (1896), van der Heij, D. G., transl.).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-Indie 41: 1–110. [Reprinted in English translation in Grijns, G. (1935).

In 1882, Chittenden was appointed Professor of Physiologi-Researches on Vitamins 1901–1911. J. Noorduyn en Zoon, Gorinchem, Hol-land. cal Chemistry at the Sheffield Scientific School at Yale Uni-

versity, two years after he had completed his Ph.D. degree inphysiological chemistry, the first such degree awarded by a

Paper 5: Dietary Protein Standards Can university in the United States (Vickery, 1945).Chittenden (1904) presented a detailed account of his seriesBe Halved (Chittenden, 1904)

of experiments in a monograph entitled Physiological Econ-omy of Nutrition: With Special Reference to the MinimalPresented by Vernon R. Young and Yong-Ming Yu, School ofProteid Requirement of the Healthy Man. An ExperimentalScience and Clinical Research Center, Massachusetts Institute ofStudy. This is remarkable considering that his experimentsTechnology, Cambridge, MA 02139 as part of the minisymposiumbegan only in 1902 and continued well into 1904 and that‘‘Experiments That Changed Nutritional Thinking’’ given at Exper-this occurred well before the convenience afforded by com-imental Biology 96, April 16, 1996, in Washington, DC.puter-based data retrieval and summary techniques, not tomention desktop publishing. In this publication, he indicatesThe essentiality of a dietary substance, which was later

named ‘‘protein’’ by the brilliant Swedish chemist Jac Berzelius that he had first questioned the premise that the dietetic stan-dards adopted by mankind represented the real needs or re-(Korpes 1970), had been recognized by the middle of the 18th

century by Beccari and by Haller (Munro 1969 and 1985). quirements of the body (p. 3, Chittenden 1904): ‘‘We mayeven question whether simple observation of the kinds andHowever, it was not until about a century later that definitive

pronouncements were made about the dietary needs for pro- amounts of foods consumed by different classes of people underdifferent conditions of life have any very important bearingteins in human subjects. Thus, surveys of diets in the United

Kingdom by Lyon Playfair, in Germany by Carl Voit, in the upon this question.’’ He was the sort of mentor that any stu-dent would have been privileged to serve under: willing toUnited States by Wilbur Atwater, as well as by others in other

countries, revealed, in relation to protein intake and the total challenge dogma and chart an entirely new experimental ap-proach.fuel value of the diet, that ‘‘all over the world people who can

obtain such food as they desire use liberal rather than small His experiments began with an opportunity to observe forseveral months the dietary habits of Horace Fletcher, an Amer-quantities . . .’’ (Benedict 1906). It was from these kinds of

data that conclusions were drawn about the necessary intakes ican of independent means. Chittenden noted that Fletcher’snitrogen intake averaged 7.19 g, and in the words of Dr. An-of protein, and Voit, who commanded considerable attention

and scientific respect, concluded that—based on his assess- derson, the director of the Yale Gymnasium, ‘‘Mr. Fletcher ofVenice performs this work with greater ease and with fewerment of his work in Munich—the protein intake of the aver-

age working man should be 118 g daily and that higher intakes noticeable bad results than any man of his age and conditionI have worked with’’ (p. 14, Chittenden 1904).would be necessary for heavy workers. Atwater, a pupil of Voit,



1026S SUPPLEMENT

the body, certainly under ordinary conditions of life’’ (p. 475,TABLE 2Chittenden 1904).

Perhaps it ought to be noted that these experiments didA typical day’s record of R. H. Chittenden’s diet and nitrogennot actually establish an average, minimum physiological re-balance after 18 mo on his self-imposed experiment1quirement for dietary protein in these groups of subjects be-

Sunday, June 26, 1904 cause 1) the low protein diets were freely chosen by the profes-Diet sional group, 2) the athletes were instructed to diminish the

Breakfast: Coffee 122 g, cream 31 g, sugar 8 g. intake of protein but without imposition of a specific diet,Dinner: Roast lamb 50 g, baked potato 52 g, peas 64 g, biscuit and 3) the soldiers were given meals that contained lowered82 g, butter 12 g, lettuce salad 43 g, cream cheese 21 g,

amounts of protein than provided by ordinary army rationstoasted crackers 23 g, blanc mange 164 g.and apparently with some reduction in the total ‘‘fuel value’’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. of the food. The food given to each soldier was weighed, andBody weight Å 57.4 kg (initial weight in November 1902 Å 65 kg. at the close of every meal the uneaten food was determined

Age 47 y) and subtracted from the initial weight.Protein intake Å 0.64 g/kg Although there has been some concern about the precisionN balance Å 00.07 g N

and accuracy of the nitrogen balance data generated fromEnergy intake Å 1549 kcalChittenden’s experiments (McCay 1912, Carpenter 1994), in-cluding the reliability of the urine and fecal collections and1 Data from Chittenden (1904).determination of nitrogen intake, the results of these series ofexperiments were coherent and dramatic. They presented aBack then was no different than today, in that Chittendenstrong case that the physiological needs for protein were muchneeded financial support for the conduct of his investigations.lower than values represented by free-choice intakes of dietaryHe secured funds from the Carnegie Institution of Washingtonprotein.and the Bache Fund of the National Academy of Sciences,

Chittenden’s conclusions were neither quickly nor univer-and he also received large donations from Fletcher and Johnsally accepted. For example, McCay (1912) referred to theH. Patterson of Dayton, Ohio.onslaught to Chittenden’s findings and ideas, but he used hisThe overall investigation consisted of three major experi-own data from dietary surveys of Bengalis, as well as the data ofments, each characterized by a long-term period of dietaryothers, to reach a conclusion that ‘‘Voit stands today absolutelyprotein restriction combined with nitrogen excretion measure-vindicated.’’ Although Cathcart (1911) was in ‘‘completements and supplemented in some studies with assessments ofagreement with Professor Chittenden’s statement that life canphysical and mental well-being. Chittenden (1904) statesbe maintained and frequently maintained at a high level on‘‘The writer, fully impressed with his responsibility in the con-relatively low protein intake,’’ he was not sure if it was desir-duct of an experiment of this kind, began with himself inable to keep a low intake as a general rule, and he expressedNovember 1902.’’ He therefore served as one of the subjectsconcern about the quality of protein. Later, he (Cathcart 1921)in his study of five university professors and instructors, includ-voiced reservations that were related to the lowered resistanceing his former student Lafayette Mendel, who by that timeto disease in persons consuming low protein diets. However,had become professor.Chittenden (1911) had already argued that the problem withOn the basis of his own experience (Table 2), including theMcCay’s studies was that the diets of the populations studieddisappearance of the rheumatic problem he had been havingin India lacked unidentified trace nutrients. This was probablyin his knee joint and the findings with the other four Yaletrue, in retrospect, given the public health problems of iron,professionals, Chittenden concluded that the minimum ‘‘pro-vitamin A and iodine deficiencies that are prevalent in south-teid’’ requirement was 93–103 mg N/kg body wt (about 0.6–east Asia today.0.64 g proteinrkg01

rd), which anticipates, by 80 years, theThe protein standards for healthy individuals continuedmean requirement figure of 0.6 g proteinrkg01

rd01 propos-to be set by the opinions of individuals until national anded by FAO/WHO/UNU (1985)!international committees were convened to establish dietaryThe next two series of studies confirmed and strengthenedrecommendations. An early international committee was setthese initial findings; one of these was with 13 members of aup by the League of Nations, and in 1936 the recommendationdetachment from the U.S. Army Hospital Corps, who werewas that ‘‘the protein intake for all adults should not fall belowhoused in Vanderbilt Square at Yale for 6 mo. The study1 gramme of protein per kilogramme of body weight . . .’’included measures of physical and mental condition and of(League of Nations 1936). No scientific justification was pre-blood composition in addition to nitrogen excretion and bal-sented in support of the recommendation. In 1943 the U.S.ance over the 6-mo period. The conclusion was that 50 g ofFood and Nutrition Board of the National Academy of Sci-protein daily can establish nitrogen equilibrium and that thereences issued its first Recommended Dietary Allowances, andis, at this approximate intake, a maintenance of physicalin this report 66 g of protein daily was recommended. Thesestrength and vigor and an ability to respond to sensory stimuli.early figures proposed by expert groups were somewhat higherIn a follow-up letter written to Chittenden by one of thethan those found to be sufficient by Chittenden, but they wereparticipants, Private First Class J. Steltz, and on behalf of thefar below the liberal standards that had been widely adoptedother men, he stated: ‘‘The men are all in first-class condition.during the middle 19th and on into the early part of the. . . We eat very little meat now as a rule, and would willingly20th century. It seems clear, however, that by the 1950s thego on another test.’’ The extensive findings in a third seriesmetabolic approach used by Chittenden and others for estab-involving eight Yale University athletes merely served to repli-lishing protein requirements had been well embraced, to thecate all of these data and the interpretations that had beenexclusion of the dietary intake approach followed by Voit,drawn from them.Atwater and others. For example, at the Princeton ConferenceThus, Chittenden concluded that one-half of the 118 g ofin 1955, W. R. Aykroyd, the director of the nutrition divisionprotein called for daily by the ordinary dietary standards is

quite sufficient to meet all the real physiological needs of of the Food and Agriculture Organization, stated: ‘‘We need




Vickery, H. B. (1945) Russell Henry Chittenden. 1856–1943. National Acad-not linger on Carl Voit and his recommendations on proteinemy of Sciences USA, Biographical Memoirs, Volume 24 (Second Memoir),requirements, which were over influenced by what was ob- pp. 59–103. National Academy of Sciences, Washington, DC.

served among the population of Munich in 1880’’ (FAO1957a). Indeed, the first FAO report specifically concernedwith protein requirements (FAO 1957b) depended heavily Paper 6: Liebig’s Concept of Nutritionalupon the review by Sherman et al. (1920) of the literature Adequacy Challenged (Hart et al., 1911)on nitrogen balance and adult requirements, which includedextensive 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 Changedday 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 dailysafe practical allowance of 0.66 g/kg. Although the more recent Imagine that we have fallen back 90 years through time. Itrecommendations (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, vita-it served as a vindication of the data obtained and conclusions mins or fatty acids. Liebig’s concept from the 1850s that pro-drawn from his visionary studies, commenced 50 years earlier tein, a few minerals, and sources of energy (fat and carbohy-in 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 bybutions in the areas of digestive physiology and the action of leaders in the field, including Voit in Germany and Atwaterproteolytic enzymes, an activity that had been enhanced by and Langworthy at the U.S. Department of Agriculturehis year-long sojourn with Kuhne in Heidelberg, and in toxi- (Harper 1993). Although several investigators have ques-cology, including heavy metal poisoning and disorders created tioned the validity of Liebig’s concept, their reports lie buriedby 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 Agriculturalto 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 cowsmember 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 Hartthat he is skeptical of Liebig’s claim that the ‘‘physiologicalLiterature Citedvalue’’ 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 to16: 409–437. test Liebig’s hypothesis.Carpenter, K. J. (1994) Protein and Energy. A Study of Changing Ideas in Nutri-

In 1907, in collaboration with G. C. Humphrey of the Ani-tion. Cambridge University Press, New York, NY.Cathcart, E. P. (1911) Discussion. Br. Med. J. 11: 664. mal Husbandry Department, Hart plans what will come to beCathcart, E. P. (1921) The Physiology of Protein Metabolism. Longmans known as the ‘Wisconsin single grain experiment’. He hiresGreen, London, U.K.

E. V. McCollum to conduct the chemical analyses and HarryChittenden, R. H. (1904) Physiological Economy in Nutrition: With Special Ref-erence to the Minimal Proteid Requirement of the Healthy Man. An Experimen- Steenbock as a student assistant. The objective of the experi-tal Study. Frederick A. Stokes Co., New York, NY. ment is to compare the performance of four groups of heifersChittenden, R. H. (1911) Discussion on the merits of a low protein diet. Open-

fed rations composed entirely of the corn, wheat or oat planting 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

The animals, 16 Shorthorn heifers about 6 mo of age andOrganization of the United Nations, Rome, Italy.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 fourFAO/WHO/UNU (1985) Energy and Protein Requirements. Report of a Joint rations, designed to provide 14 lb dry matter/d, consist of theFAO/WHO/UNU Expert Consultation. World Health Organization Technical

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 wheat straw 7; and a mixed ration consisting of equal parts ofSciences, Washington, DC.the other three.Korpes, J. E. (1970) Jac Berzelius. His Life and Work. Almquist & Wiksell,

Stockholm, Sweden. The average amounts consumed by the four groups duringLeague of Nations (1936) The Problem of Nutrition. Report on the Physiological the course of the experiment (14.5 to 15.2 lb/d) did not differBases of Nutrition, Vol. II. Technical Commission of the Health Committee,

appreciably. Values for crude digestibility of the different ra-official no. A.12(a).IIB, League of Nations Publications Department, Geneva,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.Weight gains of the groups after 1 and 3 y are shown inMunro, H. N. (1969) Historical introduction: the origin and growth of our pres-

ent concepts of protein metabolism. In: Mammalian Protein Metabolism (Mu- Table 1. Although the corn-fed group gained considerablynro, H. N. & Allison, J. B., eds.), Vol. 1, pp. 1–29. Academic Press, New York, more weight than the wheat-fed group, variability withinNY.

groups was such that, as can be seen from the large SD, theMunro, H. N. (1985) Historical perspective on protein requirements: objectivesfor the future. In: Nutritional Adaptation in Man (Blaxter, K. & Waterlow, J. C., results did not provide convincing evidence that the growtheds.), pp. 155–167. John Libbey, London, U.K. responses were different.

Sherman, H. C., Gillett, L. H. & Osterberg, E. (1920) Protein requirement of The first clear evidence of differences in the responses ofmaintenance in man and the minimum efficiency of bread protein. J. Biol.Chem. 41: 97–109. the groups was from observations on the appearance of the



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ration could not be predicted reliably from measurements ofTABLE 1its total digestible nutrients and energy content, 2) the differ-ences in performance were unlikely to be due to differencesWeight gain of heifers fed single grain rations1

in the protein component because animals fed the diet thatGroup 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 inadequaciespounds were unlikely because the mineral supplement did not improve

Corn 471 { 64 726 { 55 the performance of cows consuming the wheat ration. TheyOat 408 { 55 824 { 89did not claim to have eliminated these possible explanationsMixture 410 { 37 724 { 149conclusively.Wheat 353 { 67 665 { 86

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 essentialnutrient. They did, however, propose that the physiologicalanimals after the first year. Cows consuming the corn rationvalue of a food or feed could be determined by measuringhad smooth coats, were full through the chest, and appearedgrowth or other responses of animals fed diets in which ahealthy. Those consuming the wheat ration had rough coats,portion of a basic ration was replaced by the product to bewere of smaller girth, and appeared gaunt. The other twotested.groups were intermediate between the corn- and wheat-fed

Is it possible, with hindsight, to identify specific nutritionalgroups.deficits that can account for the inferior performance of theThere were major differences in reproductive performancecows fed the wheat ration? Levels of calcium and magnesium(Table 2). Calves born to cows consuming the corn rationwere low in the wheat ration. A level of 0.16% of magnesiumwere strong and vigorous in both years and all lived. The cowsis adequate for dairy cattle, but the level of 0.16% for calciumconsuming the wheat ration all delivered 3–4 wk prematurely.is marginal (Shepherd and Converse 1939) and 0.3% is nowTheir calves were weak both years, and none lived beyond 12recommended (Scott 1986). Nonetheless, the reproductived. Again, results for the other two groups were intermediate.performance of cows consuming the wheat ration, as Hart andIn 1909, the first year of calving, cows consuming the oat andcolleagues noted, was not improved when they were givenmixture rations produced weak calves, with only two from thesupplements of calcium and magnesium. Also, the fat contentoat group and one from the mixture group living beyond a fewof the wheat ration was low. Dairy cattle require at least 2%days. In 1910, the performance of these two groups was better.of fat (Shepherd and Converse 1939). Low milk production,The calves were carried to term, and all of the calves fromas was observed in the wheat-fed group, is an early sign ofthe oat-fed group and two from the mixture-fed group livedfatty acid deficiency in lactating dairy cows.but were weaker than those from the corn-fed group. Average

In addition, the carotenoid content of forage crops declinesweights of the calves, were 78, 72, 62 and 49 lb for groups fedduring storage, and vitamin A depletion occurs commonly incorn, oat, mixture and wheat, respectively.cows maintained during the winter on forage that has beenMilk production of each group was measured for 30 d eachstored for several months. A predominant sign of vitamin Ayear following cessation of colostrum secretion. The differencedeficiency in dairy cows is premature calving, with the calvesbetween the amounts of milk produced by the wheat- andoften born dead or surviving for only a short time. Reproduc-corn-fed groups was large (Table 1). Average values for thetive performance of cows receiving the corn ration, whichgroups (lb/d { SD, number of observations in parentheses)would be expected to provide a high level of carotenoids, waswere as follows: corn-fed group, 26 { 3.5 (8); oat-fed group,excellent. That of the cows fed the wheat ration was poor.22.9 { 6.5 (6); mixture-fed group, 20.4 { 1.5 (5); wheat-fedMcCollum (1964) attributed this to loss of most of the leavesgroup, 14.1 { 2.6 (4). (In calculating the average for theof the wheat plant during threshing. However, reproductivewheat-fed group I deleted two values, one for a cow that diedperformance of the oat-fed group was poor the first year butof illness, and one abnormally low value). No differences weremuch better the second, suggesting that the carotenoid con-observed in milk composition (total solids, total protein, ca-tent of feedstuffs varies from year to year. It would thus seemsein, ash or fat) or in the characteristics of the milk fat.

The wheat ration was known from chemical analyses toprovide less calcium, magnesium and potassium than the other TABLE 2rations. Two cows in the wheat-fed group were therefore givena supplement of these minerals during 1 y of the study to raise Condition and survival of calves1

their levels of intake to those provided in the diets of theGroup 1909 1910other groups. The condition of the cows was not noticeably

improved. The calf produced by one of them was small andCorn Strong and vigorous both years; all livedweak and lived only a few hours.

6d premature None prematureAfter the experiment was completed, some animals wereOat All weak 3 fairly strong, 1 weakswitched 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 prematureto corn improved rapidly. It produced a calf weighing 81 lb, Mixture 1 fair, 1 weak 1 weak, 1 fairly strong

2 died, 1 lived 1 born dead, 2 livedcompared with 47 and 48 lb for calves produced the previous1 aborted2 y. One cow that was switched from the corn diet to the12 d premature 4 d prematurewheat ration supplemented with extra calcium, magnesium

Wheat 3 weak, 1 stillborn All weak. 2 cows diedand potassium deteriorated. Its calf was stillborn 18 d prema- None lived ú12 d None livedturely and weighed only 36 lb, compared with 93 and 85 lb 26 d premature 24 d prematurefor those born the previous 2 y.

1 Adapted from Hart et al (1911).The authors concluded that 1) the nutritive value of a




highly probable that differences in reproductive performance At Wisconsin, McCollum’s initial major responsibility wasof the groups were due to differences in vitamin A status, that of analytical chemist in a single-grain feeding trial withowing to differences in the carotenoid content of the rations, heifers, started in early 1907, at the suggestion of Professorpossibly complicated in the case of the wheat-fed group by an Emeritus S. M. Babcock. The trial, directed by E. B. Hart toinadequate 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 ofdence from research in the United States that the nutritive four groups fed different rations. Three of the groups were fedvalue 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 fromdeathknell for Liebig and Voit’s concept of a nutritionally all three plant sources. As judged by the proximate method ofadequate diet. It contributed, together with observations by chemical analysis for estimating nutritional adequacy, all fourEuropean investigators on the inadequacies of purified or re- rations should have been of similar value. However, the calvesstricted 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 thatthinking about the nutritional essentiality of constituents of he and others connected with the project had overlooked thefoods. Maynard (1962) stated that the findings of Hart and fact that, in harvesting the crops used to prepare the experi-his associates ‘‘stimulated much of the work which resulted in mental rations, the leaves of the corn and oat crops largelylater 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 referencesorganic substances in animals. These continued at Wisconsin to this noted experiment the fortuitous flaw has been over-for 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 concurrentals, especially of iodine and copper; the discoveries of McCol- and diligent searching of the available literature—especiallylum and associates of the nutritional essentiality of fat-soluble the abstracts in Maly’s yearbooks—persuaded McCollum as toA and water-soluble B, and that there were two essential fat- the real possibility of some indispensable nutrients remainingsoluble factors, A and D; and the contributions of Steenbock unrecognized. This, plus his discussions with Dr. Babcock, con-and associates to knowledge of vitamin D and their discovery vinced him that the heifer feeding project would not alonethat vitamin A activity was associated with the yellow carot- yield really significant new knowledge, but it stimulated himenoid 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 nutritionalLiterature Citedrequirements 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: Nutri- Small animals were desirable because they eat little, and wetional Essentiality: A Changing Paradigm (Roche, A. F., ed.), pp. 3–11. Report

could do the extensive chemical work necessary for purifyingof the Twelfth Ross Conference on Medical Research, Ross Products Division,the dietary ingredients. Small animals grow rapidly to maturity,Abbott Laboratories, Columbus, OH.

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) Physiologi-They opposed the use of rats in an agricultural experimentcal effect on growth and reproduction of rations balanced from restricted

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. pressing work. He procured ‘‘a dozen young albino rats from aMaynard, L. A. (1962) Early days of nutrition research in the United States ofAmerica. 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 limitedMcCollum, E. V. (1964) From Kansas Farm Boy to Scientist. University of Kan-

knowledge in dealing with small experimental animals, hesas 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 PavlovianShepherd, J. B. & Converse, H. T. (1939) Practical feeding and nutritional re-

ideas on relationships between the taste of food and its digest-quirements 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 resultswere obtained. However, this and other experiences advancedPaper 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 womanUniversity, Bloomington, IN 47405-6203 as part of the minisymp- resident of Madison, Marguerite Davis, who had recently grad-sosium ‘‘Experiments That Changed Nutritional Thinking’’ given uated in chemistry at the University of California at Berkeley,at Experimental Biology 95, April 11, 1995, in Atlanta, GA. and asked if she might take care of the rats.There soon began the experiments that were in time identi-In July 1907, Elmer Verner McCollum began his academic

fied as McCollum’s biological method for the analysis of food.career as instructor in agricultural chemistry at the UniversityThe first definitive publication of research in which Miss Davisof Wisconsin, after completing A.B. and M.S. degrees in chem-had a significant role was in establishing the ‘‘necessity ofistry at the University of Kansas and a Ph.D. degree in organiccertain lipins in the diet’’ (McCollum and Davis 1913). Thischemistry plus a year of postdoctoral work in physiological

chemistry at Yale University. was an unexpected outcome in the early phases of their exten-



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Two years later, McCollum and his graduate student Corne-lia Kennedy suggested the provisional use of alphabetical termsfor this lipin and other essential ‘‘organic complex(s),’’ usinga prefix designating characteristic solubility. Thus for this es-sential factor(s) they proposed the term ‘‘fat-soluble A’’(McCollum and Kennedy 1916). Soon this gave way to theterm ‘‘vitamin A.’’

McCollum and Davis also focused on discovering the natureof the nutritional deficiencies of the cereal grains. Particularattention was given to the nature of the dietary deficienciesof rice. In their article were 42 growth charts (McCollum andDavis 1915). Each chart portrayed the growth for each rat inan experimental lot, the composition of the ration used, thekind of test substance (accessory) added, if any, etc. Some ofthe results are summarized in Table 1. In their introductionthey stated in part:

‘‘In the present communication we present experimental datashowing the specific properties of polished and of unpolishedrice as a food, and show the supplementary relationship be-tween these and certain purified and naturally occurring food-stuffs . . . . These studies, in addition to extending our knowl-edge concerning the dietary position of rice, have contributedto our understanding of the factors involved in normal nutri-tion, especially as regards the unknown accessory constituentsof the diet which have received so much attention in recentyears in connection with the ‘deficiency diseases’, scurvy andberi-beri.’’

FIGURE 1 Prepared from McCollum’s growth charts. This male A substantial proportion of the research was on ‘‘the supple-rat received 3% cottonseed oil in its diet for the 7 wk of period 1 mentary relationship between certain extracts of naturally oc-and then received 3% olive oil that had been shaken with soaps from curring foodstuffs and polished rice’’ in which most of thesaponified butter fat. The dotted line represents the normal growth rate

extractants employed were water, ethanol and acetoneof these rats (from McCollum and Davis 1914, chart 2, rat B).(McCollum and Davis 1915).

This was in regard to answering the question of the presenceof ‘‘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 acetonestating that: extract in supplementing a diet based on polished rice. McCol-‘‘During the past year we have been engaged in a study of the lum and Davis (1915) concluded that ‘‘such knowledge, wheninfluence of the composition and quantity of the inorganicavailable for a wide variety of foodstuffs must, we believe, becontent of the ration on the growth of the rat. In this workof great value in the formulation of human dietaries whichwe have employed rations compounded of pure casein, carbo-will promote health.’’ Thus this study contributed to the illu-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 workby lard, with a considerable degree of success. . . .’’

In their commentary on these germinal experiences with ratsTABLE 1fed such rations they wrote:

‘‘The fact that a rat of 40 to 50 grams in weight can growGrowth of young rats fed polished rice withnormally during three months or more on such rations, then

cease to grow but maintain its weight and a well nourished different supplements1appearance for weeks and then resume growth on a rationcontaining 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 sub-stances the animals run out of some organic complex which is 308 316 317 383 395indispensable 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.)

Approx. initialIn this article they had shown that butter fat and eggsweight g 100 40 60 60 40contain the ‘‘lipins’’ and that none exists in lard or olive oil.

Also, they showed that the organic complex was extractable Dietwith ether. In 1914 they reported that the new fat-soluble Polished rice 96 91 91 — 82

Unpolished rice — — — 88 —factor was transferred to olive oil by the following procedure:Rice polishings — — — — 10‘‘Butter fat was saponified with potassium hydroxide in alcohol.Salt mix 4 4 4 2 3The resulting soap was dissolved in water and olive oil wasEgg albumen — 5 — — —thoroughly emulsified in the soap solution. The olive oil wasCasein — — — 5 —of the same sample which had been tested on rats and foundButter fat — — 5 5 5to be of no value in protecting them against decline on the

basal diet. The emulsion was then broken with ether, and the Approx. weightolive oil was recovered in that solvent. After evaporating the gain in 8 wk, g 0 or less 0–15 loss 65 50ether, the olive oil was found by a feeding test to have acquiredthe nutritive quality of the butter fat.’’ (McCollum and Davis 1 Based on data in five different growth charts selected from 42

published by McCollum and Davis (1915).1914)




on rice was in progress they learned more about the findings TABLE 1of E. B. Vedder and others on beriberi, and they ‘‘identifiedthe 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’’ as their protein source1(McCollum 1957).

Diet 1 Diet 2 (17.64%McCollum presented these findings in a lecture before the(18% gliadin) gliadin, 0.36% lysine)prestigious Harvey Society in New York in 1917 and concluded

by stating: ‘‘These [supplements] have peculiar [not yet under-No. rats 4 2stood] dietary properties which the best chemical talent ofFood eaten, g 99 149today fails to recognize, but which are readily demonstrable Lysine, from gliadin,2 mg 160 240

by biologic tests’’ (McCollum 1917). Lysine, from supplement, mg — 535Five 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 viewEst. protein gain,2 g /0.99 /5.63to discovering the nature of the deficiencies of individual natu- Est. Lysine gain,2 mg /79 /450ral 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, andprotein, 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 whichenhance its value. The method is applicable in another modi- 12 years the younger, was the son of Jewish immigrants runningfication, 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 cameportant foods with respect to any one dietary constituent.’’ back on to the faculty, loved teaching and, unusually for hisThe 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 differentLiterature Cited vegetable proteins and demonstrating their chemical individu-

ality and, in particular, how different they were in amino acidDay, H. G. (1974) Elmer Verner McCollum 1879–1967, A Biographical Memoir.composition from animal proteins. Did that mean that theyBiographical Memoirs 45: 263–335. National Academy of Sciences, Washing-

ton, DC. were necessarily nutritionally inferior? This was, obviously, ofMcCollum, E. V. (1917) The supplementary dietary relationships among our interest to Mendel, too, because he had been part of the bignatural foodstuffs. Harvey Society Lecture Series 12: 151–180; also in J. Am.

Yale study a few years earlier that used human subjects andMed. Assoc. 68: 1379–1386.McCollum, E. V. (1922) The Newer Knowledge of Nutrition: The Use of Food ended with the recommendation that Atwater’s dietary stan-

for 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 forMcCollum, E. V. (1957) A History of Nutrition. The Sequence of Ideas in Nutri-the 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 en-phy 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 dietadult rats and dogs, but that acid hydrolysates could not do soduring growth. J. Biol. Chem. 15: 167–175.

McCollum, E. V. & Davis, M. (1914) Observations on the isolation of the sub- (Henriques and Hansen 1905). This was attributed to thestance 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. esized that, although animals could manufacture linear aminoMcCollum, E. V. & Davis, M. (1915) The nature of the dietary deficiencies ofrice. 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 recoveringall their urinary nitrogen and thus getting spurious positivePaper 8: Some Amino Acids Arebalances. They thought that the only really satisfactory wayIndispensable for Growth (Osborne andto test the adequacy of dietary proteins with or without the

Mendel, 1914–1916) addition of amino acids was to have young animals grow sub-stantially in size, using purified diet components.Presented by Kenneth J. Carpenter, Department of Nutritional

They soon found difficulties in maintaining growth in ratsSciences, University of California, Berkeley, CA 94720-3104 asusing starch, lard and mineral mixes even with casein as thepart of the minisymposium ‘‘Experiments That Changed Nutritionalprotein source. They obtained improved growth by addingThinking’’ given at Experimental Biology 96, April 16, 1996, indried ‘‘protein-free milk,’’ i.e., separated milk from which theAtlanta, GA.proteins had been precipitated by acidification and then boil-ing (Osborne and Mendel 1911). After the further inclusion‘‘Osborne and Mendel’’—one of the most important and

long-lasting collaborations in the history of nutritional sci- of butter fat, growth continued to the mature weight of theiranimals (Osborne and Mendel 1914). With gliadin as theirence—and between two very different characters. Thomas

Osborne was from a well-established New England family of protein source in place of casein, rats hardly changed weightfor 3 mo. But with added lysine they grew well.lawyers and bankers and the fifth generation to go to Yale. He

lived all his life in New Haven, worked in the Agricultural At this time, with only crude gravimetric methods of analy-sis available, they believed that gliadin (isolated from wheat)Experiment Station with no students and did not like to face

an audience (Fruton 1995, Vickery 1932). Lafayette Mendel, had no lysine. Therefore, because rats would maintain weight



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Literature CitedTABLE 2Abderhalden, E. (1909) Weiterer Beitrag zur Frage nach der Verwertung von

Mean 3-wk weight changes of young rats receiving 18% zein tief abgebautem Eiweiss in Tierischen Organismus. Z. Physiol. Chem. 61:194–199.as their protein source1

Becker, S. (1968) The Emergence of a Trace Nutrient Concept through AnimalFeeding 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.Sci. Biogr. Mem. 18: 123–155.g

Fruton, J. S. (1995) Thomas Burr Osborne and chemistry. Bull. Hist. Chem.17: 1–8.22 None 020 ({ 5) Henriques, V. & Hansen, C. (1905) Uber Eiwessynthese im Tierkorper. 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.Osborne, T. & Mendel, L. B. (1914) Amino-acids in nutrition and growth. J.[Normal weight gain on a control diet] [/35]

Biol. Chem. 17: 325–349.Osborne, T. & Mendel, L. B. (1916) The amino-acid minimum for maintenance

and growth, as exemplified by further experiments with lysine and trypto-1 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. Bi-

ogr. Mem. 14: 261–304.Willcock, E. G. & Hopkins, F. G. (1906) The importance of individual aminowith gliadin as the sole protein, lysine did not seem to be

acids in metabolism. J. Physiol. 35: 88–102.required for maintenance. However, by 1915, with an im-proved analytical procedure for lysine, they had found thatgliadin 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 thatpart of the lysine in the rats’ new tissues had to have come Presented by Leslie M. Klevay, U.S. Department of Agriculture,from the free amino acid supplement. They did not report any Grand Forks Human Nutrition Research Center, Grand Forks,actual calculations to support this point, but Table 1 repro- ND 58202 and Robert E. Olson, College of Medicine, Universityduces the relevant estimates that they could, and perhaps did, of South Florida, Tampa, FL 33606 as part of the minisymposiummake. ‘‘Experiments That Changed Nutritional Thinking’’ given at Exper-

Lysine could not apparently by synthesized by rats, even imental Biology 96, April 16, 1996, in Washington DC.though it had no cyclic group. This was an important pointbecause it had been thought it was the amino acids containing Microbiology was transforming medicine at the end of the

Victorian era. By the time Funk coined the term ‘‘vitamine’’cyclic groups that animals would be found to be unable tosynthesize. On the other hand, rats could apparently maintain in 1912, the causative organism for tuberculosis had been

known for 30 years. It is not surprising that infection wasthemselves with dietary protein of very different compositionfrom that of their own tissues. considered more likely than dietary deficiency to be the cause

of pellagra. Both toxicity and heredity, two other causes ofThis idea was confirmed by their parallel results with zein(from corn), which is completely lacking in lysine and also disease known at the turn of the century, had also been sug-

gested.both tryptophan and glycine (Osborne and Mendel 1916).They showed most of their results with zein as growth curves Dementia, dermatitis, diarrhea and finally death associated

with a diet of meat, maize and molasses described the pellagrafor individual rats, because they changed their supplementsfrom time to time, but Table 2 summarizes their results from syndrome. Unfortunately, the ‘‘meat’’ consumed by poor peo-

ple was high in fat and low in protein. The dermatitis is photo-just the first few weeks of feeding.It is clear that without tryptophan the rats did worst. With sensitive and confined to the areas of skin exposed to sunlight;

Casal’s (1691–1759) necklace is the eponym attached to ‘‘theit, but without lysine, they did better, though on average stilllosing a little weight. With both amino acids added, they grew area of erythema and pigmentation around the neck in pella-

gra’’ (Terris 1964). The dementia was usually of the manic-fairly rapidly, at about two thirds of the optimal rate. Theyconcluded, as Willcock and Hopkins (1906) had earlier, that depressive type and severe enough to justify admission to a

mental institution.perhaps the more rapid loss in the absence of tryptophan meantthat this amino acid was needed for some special priority func- Joseph Goldberger, who contributed extensively to our un-

derstanding of the causes of pellagra, was born in Austria intion, requiring tissue loss to provide it, and that in its presencethere was at any rate less breakdown of protein. 1874 and immigrated with his parents to the United States in

the 1880s. He grew up in New York City and entered the CityThe work clarified the importance of amino acid composi-tion as a determinant of protein quality and demonstrated College of New York as a high school graduate in 1890 to

study engineering but changed his field to medicine two yearsexamples of three types of amino acids: lysine that could notbe synthesized and was needed almost entirely for growth, later by enrolling at the Bellevue Hospital Medical College.

He obtained his MD degree in 1895 and after interning fortryptophan needed for maintenance as well as growth, andglycine that could be synthesized so that its supply was not one year and practicing medicine in New York and Pennsylva-

nia for three additional years, he joined the U.S. Public Healthlimiting.Osborne and Mendel’s persistence with long-term growth Service in 1899. He served as a quarantine officer in various

ports including New Orleans, Tampico, Veracruz and Havanaas a measure of nutritional adequacy, in parallel with the workof McCollum’s group in Wisconsin, opened up a new approach and studied yellow fever and typhus transmission by mosquitos

in those areas. In 1909, he solved the cause of Schamberg’sto the search for vitamins as well as the discovery of furtheressential amino acids (Becker 1968, Carpenter 1994). disease, a pigmented dermatitis, prevalent in crew members




on private yachts and in men living in private dwellings and berger and Wheeler themselves were the first subjects, eachreceiving 5 mL of the defibrinated blood by intramuscularboarding houses in the Philadelphia area. Goldberger and

Schamberg (1909) observed that these men slept on straw injection and also secretions. Three days later, Goldberger atefeces from an acutely ill patient together with the urine andmattresses, and they finally identified a mite (Pediculoides ven-

tricosus) as the vector for the disease. Thus, Goldberger had dermatitis scales from two other patients. As a result, Gold-berger developed diarrhea that lasted for about a week, butconsiderable experience in epidemiology and knowledge of

infectious diseases when he was assigned by the Surgeon Gen- despite this both he and Wheeler joined three other volunteersfor a similar round of tests with both injections of defibrinatederal in 1913 to undertake a study of the causation of pellagra.

Pellagra was not recognized as a problem in the United blood from three patients and the oral consumption of scalesand excreta. To maximize the chance of catching any infectionStates until early in the 20th century. In 1912, Lavinder of

the U.S. Public Health Service estimated that more than from stools, they used fresh fecal material from the rectum ofpellagrins by using an enema and then blended material from25,000 cases of pellagra had occurred in the United States in

the previous five years and that the case fatality rate was 40%. five subjects into pills that were consumed by the volunteers.The volunteers also took sodium bicarbonate before and afterThe dominant thinking in the United States at the time Gold-

berger began his investigations was that pellagra was an infec- consuming these materials to reduce the acidity of the stomachto prevent a possible bacteriocidal action of gastric juice. Mrs.tious disease. As a result of studies in South Carolina, the

Thompson-McFadden Pellagra Commission concluded in Goldberger received one injection of blood. Both Goldbergerand Wheeler felt stiffness after the intramuscular injections,1913 that ‘‘1) The supposition that the ingestion of good or

spoiled maize is the essential cause of pellagra is not supported and several volunteers felt nauseous after ingestion of feces.Nonetheless, after five to seven months none showed any signby our study; and 2) Pellagra is in all probability a specific

infectious disease communicable from person to person by of pellagra. Because Goldberger’s group had failed to demon-strate any transmissibility of an infectious agent to themselvesmeans at present unknown.’’ These conclusions were elabo-

rated by Siler et al. (1914). from pellagrins and had demonstrated both the preventativeand curative action in pellagrins of diets rich in animal foods,In less than three months after beginning his investigation,

Goldberger (1914) published his first paper on pellagra. In a they felt secure in their conclusion that the disease was dietaryand not infectious. Nonetheless, they conducted another epi-document of a little over three pages, Goldberger summarized

the epidemiology of the disease as follows. Pellagra cannot be demiologic study of seven cotton mill villages in South Caro-lina beginning in 1916, which showed that the disease wascommunicable. The cause is dietary. Prevention should result

from a ‘‘reduction in cereals and vegetables and canned foods not high in villages with poor sanitation but was high invillages with poor diets.that enter to a large extent in the dietary of many of the

people in the South and an increase in fresh animal food In 1920, the question remaining was: What was the agentin animal foods that prevented pellagra? Because the biologicalcomponent such as fresh meats, eggs and milk.’’ In support of

these views Goldberger pointed out that 1) in institutions value of animal protein was in general better than the valuesfor proteins in cereals and vegetables, Goldberger and Tannerwhere pellagra was prevalent, no case had ever occurred in

nurses or attendants; 2) the disease was essentially rural; and (1922) proposed that an amino acid might be the pellagrapreventative factor. Tanner actually conducted a trial of tryp-3) it was associated with poverty, which in turn was associated

with a diet deficient in animal foods. tophan in one pellagrous patient, which caused marked im-provement of the dermatitis but little change in the diarrhea.These conclusions, however, were reached by epidemiologic

methods involving the association of variables and did not He reported the finding in a progress report to Goldberger,but the result was not followed up (Tanner 1921, quoted byconstitute proof of the etiology of the disease. Goldberger and

his colleagues then proceeded to attempt 1) to cure pellagra Hundley 1954). Goldberger also tested various foods in anattempt to cure black tongue, the pellagrous analogue in dogs.by changing the diet of pellagrins to one rich in animal foods

and 2) to demonstrate by direct studies the possible infectivity Goldberger died prematurely at age 55 in 1929. He thusdidn’t live to see the cure of black tongue with niacin asof secretions, scales and excreta from pellagrins. A year after

publishing his first paper on pellagra, Goldberger and his co- reported by Elvehjem et al. in 1937, although Goldberger andSebrell (1930) did induce remission of this canine disease withworkers demonstrated in back-to-back papers (Goldberger etliver extract. His idea that amino acids were critical in theal. 1915, Willets 1915) that pellagra could be prevented inpathogenesis of pellagra was confirmed by a finding by Krehlinstitutionalized patients by a diet that included generouset al. (1945), who proved that nicotinic acid could be formedamounts of milk, eggs, meat, beans and peas and that pellagrafrom tryptophan. Subsequently Vilter et al. (1949) showedcould be successfully treated by the same regimen.that tryptophan would cure pellagra in humans.The second part of Goldberger’s plan was to demonstrate

In summary, Goldberger was a well-trained physician, athe nontransmissibility of pellagra by contact with nasopha-brilliant epidemiologist and an imaginative clinical investiga-ryngeal secretions, blood and excreta from pellagrins (Gold-tor. He studied a variety of infectious diseases and pellagra,berger 1916). In a heroic study on themselves conducted bywhich was not infectious, with a multidisciplinary approachGoldberger, Sydenstricker, Tanner, Wheeler, Willets, Gold-that included epidemiology. He is still lauded as a exemplarberger’s wife and an additional 10 volunteers, defibrinatedof clinical epidemiology (Elmore and Feinstein 1994).blood, nasopharyngeal secretions, feces, urine and dermatitic

scales were administered enterally and parenterally in an at-tempt to cause pellagra. It must be noted that physicians in

Literature Citedthe public health service at that time had learned to acceptsuch exposures as the risk of dealing with infectious diseases, Elmore, J. G. & Feinstein, A. R. (1994) Joseph Goldberger; an unsung hero of

American clinical epidemiology. Ann. Intern. Med. 121: 372–375.and in fact Goldberger himself had contracted both yellowElvehjem, C. A., Madden, R. J., Strong, F. M. & Wooley, D. W. (1937) Relationfever and typhus from his previous work. Various tissues, nasal

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.Goldberger, J. (1914) The etiology of pellagra: the significance of certain epi-of various grades of severity, including three fatal cases. Gold-



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demiological observations with respect thereto. Public Health Rep. 29: 1683– anemic animals by dietary methods, however. In the preceding1686. paper in their series, data were presented showing that ironGoldberger, J. (1916) The transmissibility of pellagra: experimental attempts

salts of great purity were ineffective in correcting an anemiaat transmission to the human subject. Public Health Rep. 31: 3159–3173.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 (anPub. Health Rep. 24: 973–975.acid extract of ash) was very potent in restoring normal hemo-Goldberger, J. & Sebrell, W. H. (1930) The black tongue preventive value of

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 estab-

logic factor in pellagra. Public Health Rep. 37: 462–486.lished copper as an essential nutrient. Although the authorsGoldberger, J., Waring, C. H. & Willets, D. G. (1915) A test of diet in the preven-

tion of pellagra. South. Med. J. 8: 1043–1044. were modest, concluding only that the experiments ‘‘point toGoldberger, J., Wheeler, G. A. & Sydenstricker, E. (1918) A study of the diet the need for a more intensive study of the role of small amounts

on nonpellagrous and of pellagrous households in textile mill communities in of inorganic substances in the diet,’’ McCollum (1957) suggestsSouth Carolina in 1916. J. Am. Med. Assoc. 71: 944–949.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, biochem-tryptophan. Science 101: 489–491. ists, and pathologists.’’Siler, J. F., Garrison, P. E. & MacNeal, W. J. (1914) Pellagra, a summary of the

Figure 1 contains charts on six rats of some 50 presented.first progress report of the Thompson-McFadden Commission. J. Am. Med.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 against time. Intakes of 0.3 g of Cohn’s liver preparation (ap-Hundley in Sebrell, W. H. & Harris, R. S. (1954) The Vitamins: Chemistry,proximately equivalent to 1.7 g of dried beef liver) producedPhysiology, Pathology, Volume II, page 553. Academic Press, New York, NY.

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 liversVilter, R. W., Mueller, J. F. & Bean, W. B. (1949) The therapeutic effect of tryp-

to produce extracts more efficacious than whole liver in curingtophan 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 liverpreparation was low in iron as shown by chemical analysis.The response of rat 615 was similar to that of rat 596 whenPaper 10: Copper as a Supplement toash 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 potentPresented 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. RatChanged 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

soluble in hydrochloric acid. Deciding to make their ash frac-Although Hopkins (1906) and Funk (1912) shrewdly pre-tionation ‘‘more dramatic,’’ they treated this acid extract withdicted early in the 20th century that some diseases occurredhydrogen sulfide. The hemoglobin of rat 690 was restored bybecause of dietary deficiency of accessory or essential foodthe ‘‘small but distinct precipitate of sulfides.’’ After this pre-factors, the observations they cited were ‘‘unknown to medicalcipitate was ‘‘filtered off,’’ the filtrate was treated with ammo-men and chemists of that period’’ (McCollum 1957). Physiolo-nia and ammonium sulfide; rat 688 died without improvementgists, biochemists and even the public (Allen 1931) caughtof hemoglobin when given this second precipitate. Thus, itup by 1928. Many important discoveries on the role of organicwas found that the active element had an acid-insoluble sul-nutrients were made in the 1920s because Hopkins and Funkfide; other elements besides copper were possible (Sorumhad opened their eyes (McCollum 1957).1949). Acid ‘‘extracts of the ash of lettuce . . . could notThe 1926 edition of Osler’s influential textbook (Osler andbe separated sharply into an active and inactive fraction byMcCrae 1926) listed two classes of anemia. The secondary orprecipitation with ammonia.’’symptomatic class included those due to blood loss, infection

The chronology of these experiments is not obvious. Perhapsor intoxication. The primary or essential class included onlythe rat number can be a guide. ‘‘Shortly before the time’’ thatchlorosis, pernicious anemia and sickle cell anemia. Of thesethe fractions of the Cohn preparation were studied, a trial ofthree, only chlorosis had an effective treatment. Osler referredcopper was made. Figure 2 shows the response of rat 621, theto the treatment of chlorosis by iron therapy as ‘‘one of therat from which the essentiality of copper can be inferred. Whenmost brilliant instances—of which we have but three orsupplemental iron with copper was begun, growth had ceasedfour—of the specific action of a remedy’’ and stated ‘‘It is aand hemoglobin was 2.68 g/dL. Growth resumed and hemoglobinminor matter how iron cures chlorosis.’’ Osler did not infer theincreased to 9.35 in 2 wk, 10.9 after 6 wk and then reached 13.3existence of iron deficiency anemia (chlorosis was of unknowng/dL. ‘‘Without the copper addition, the rise in hemoglobin wouldcause). Anemias from deficiency of ascorbic acid, cobalt, folate,not have occurred.’’ ‘‘This preliminary experiment was with butniacin, pantothenic acid, pyridoxine, riboflavin, thiamine, vi-a single animal but the effect was so convincing and helpful’’tamin E, etc., were unrecognized (Wintrobe 1967).that the chart is recorded ‘‘if for no other reason than its historicalHart, Steenbock, Waddell and Elvehjem certainly were wellinterest.’’ A dozen other rats were studied at three doses of copper.informed about nutritional opportunities and contemporaryResponse to 0.01 mg of copper was less rapid than the responsenutritional knowledge; they may have been somewhat aheadto 0.05 or 0.1 mg. The intermediate dose was similar to thatof physicians who read Osler. Their experimental design re-found in the ash of the liver preparation.sembled that of Whipple et al. (1920), who used phlebotomy

The authors knew that copper occurs in plant and animalto make dogs anemic and then measured blood regeneration toassay dietary components for nutritional value. They produced tissues, but ‘‘no definite function has been assigned to it except




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 theresponse 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, War- the authors suggest that copper is an essential nutrient. Surpris-ingly little has been written on the definition of nutritionalburg and Krebs had reported copper in human blood and in

serum of ‘‘dog, cat, rat, guinea pig, frog, chicken and goose.’’ essentiality, although concepts from the trace element era havebeen collected and reviewed (Klevay 1987). Although ratsIn cattle, liver was found to be highest in copper among the

organs, with lean meat being considerably lower. grow reasonably well when deficient in copper, they cannotcomplete their life cycle without it.‘‘Recently the use of liver and liver extracts has come into

prominence . . . in the treatment of pernicious anemia.’’ ‘‘The The experiments of Hart et al. (1928) leading up to thisfact that this preparation was found effective in the treatment of discovery about copper were done without the use of statistics.anemia in the rat exactly as it has been found effective in the Hill (1965) recalls some of his own work done in this era intreatment of pernicious anemia in man appeared to us rather which results were so clearcut that it had not occurred to himsignificant.’’ That the preparation contained copper and that to use a test of significance. He discusses more recent situationscopper alone was effective in rats suggested ‘‘more than a casual when to ‘‘decline to draw conclusions without standard errorsincidental connection.’’ It was realized, however, that the ‘‘treat- can . . . be silly.’’ I’m told that replication by repetition andment of the two anemias need not necessarily be alike.’’ confirmation in more than one species prevented false conclu-

sions in this earlier era. Thus, this experiment is all the moreThoughts from the 1990s remarkable, although it is not quite fair to say that it was done

with only a single rat.Instead of being the seventh paper in a series on iron, thiscould have been the first in a series on copper. Nowhere did Hart et al. (1928) cited seven references. The method for



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important, because this assay validated the other assays. Nodata were available on the ‘‘form in which Cu occurs in liver’’or on ‘‘various materials’’ in which chemical form may havea ‘‘modifying effect upon the action of Cu.’’ These commentsare valid in regard to much recent work on the biology ofcopper, as well.

Literature CitedAllen, 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 ab-

normalities 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 perni-cious 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 role of copper in carbohydrate metabo-

lism. 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 (Bratter, P. &

liver preparation was measured by the xanthate method with- Schramel, P., eds.), pp. 43–60. Walter de Gruyter & Co, Berlin, Germany.Lei, K. Y. & Carr, T. P. (1990) Role of Copper in Lipid Metabolism, CRC Press,out citation. Inspection of papers I, V, IV and VI in their

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

ed. D. Appleton and Company, New York, NY.binometers, methods not likely to be as reliable as the cyan-Percival, S. S. (1995) Neutropenia caused by copper deficiency: possiblemethemoglobin 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 thecytochrome oxidase of rat tissues. J. Biol. Chem. 129: 729–737.pernicious anemia; another contained a ‘‘substance capable of

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 cop-

cages were made of galvanized iron. per deficient swine. Am. J. Pathol. 41: 603–621.Sorum, C. H. (1949) Introduction to Semimicro Qualitative Analysis, p. 5. Pren-Nutritional scientists became preoccupied with the hema-

tice-Hall, New York, NY.tology of copper for more than a half century after this work.Whipple, G. H., Robscheit, F. S. & Hooper, C. W. (1920) Blood regenerationThe reason for this preoccupation is not clear, but it may have following simple anemia. Am. J. Physiol. 53–54: 236–262.Wintrobe, M. M. (1967) Clinical Hematology, 6th ed. Lea & Febiger, Philadel-been created by the vigor of Cartwright and Wintrobe. Perhaps

phia, PA.just as the Buchners’ concepts of cell-free fermentation wereresisted by many because of Pasteur’s experiments on spontane-ous generation, many in nutrition began to think that hematol- Paper 11: The Conversion of Caroteneogy was everything in relation to copper. to Vitamin A (Thomas Moore, 1930)A few were more flexible, however, and searched for othercharacteristics of copper deficiency. Keil and Nelson (1934) Presented by James Allen Olson, Department of Biochemistry anddiscovered what now is called glucose intolerance. Bennetts et Biophysics, Iowa State University, Ames, IA 50011-3260 as partal. (1942) first noticed cardiac catastrophes. The hematologists of the minisymposium ‘‘Experiments That Changed Nutritionalwere not totally inactive in this latter field (Shields et al. Thinking’’ given at Experimental Biology 96, April 16, 1996, in1962). Surely if the hypercholesterolemia of copper deficiency Washington, DC.were not nearly invisible in plasma or serum (in contrast tohypertriglyceridemia, which is mild in deficiency), copper and In the early 1900s, Frederick Gowland Hopkins, Casimir

Funk and others realized that minor organic components oflipid metabolism would have been associated long before 1973(Klevay). Now (Lei and Carr 1990) there is much more re- foods played essential roles in growth and nutritional well-

being. These minor essential constituents of the diet weresearch being published on cardiovascular effects of copper de-ficiency than on hematology, although interest in the latter is termed ‘‘vitamines,’’ primarily because the first one discovered,

thiamine, clearly possessed an amine function. That these mys-reviving with a shift toward leukocytes (Percival 1995). It maybe interesting to know what pathology might have been found terious vitamines were present not only in water-soluble ex-

tracts of living materials but also in fat-soluble extracts wasby Hart et al. (1928) with even limited necroscopy. Sometimespathology is obvious (Allen and Klevay 1978). soon noted by several investigators both in the United States

and in Europe. McCollum and Davis (1913) at the UniversityHart et al. (1928) commented that ‘‘Only when some im-portant function . . . lends itself . . . to quantitative measure- of Wisconsin and Osborne and Mendel (1913) at Yale Univer-

sity first identified foods containing this fat-soluble growthment are conditions suitable for making progress . . . .’’ Theyhad adequate methods for measuring copper, hemoglobin, etc., factor. Active foods included butter, egg yolk, whole milk

powder, cod liver oil and many pigmented fruits and vegeta-but the bioassay using rat 621 and others perhaps was most




TABLE 1

Comparison of ingested and stored carotene and vitamin A1

Total liver units

Daily dose Total ingested BlueTreatment of carotene n units (yellow) (610–630 nm) Yellow

Depleted 0 10 0 0 1–10Carotene 10–50 mg 4 770–4400 0 7–16Carotene 750 mg 4 24,000–39,000 2000–3700 40–110Red palm oil 1.55 g 2 83,000–130,000 4500–5000 280–400Fresh 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 late1920s. The two key objectives of his study were 1) to provepigmented fruits and vegetables.

Between 1913 and 1919 two different types of foods clearly that purified carotene preparations did not contain vitamin Aand 2) to show unambiguously that carotene was convertedshowed activity: 1) animal foods that were colorless or showed

only a slight yellow color and 2) yellow-colored foods, largely to vitamin A in vivo.In the mid-1920s, it had become clear that carotene andof plant origin. The question then arose of the nutritional

relationship between these two types of foods. There were vitamin A were quite different substances, based on their color,absorption spectra, solubility, and reactivity with antimonyseveral possibilities: 1) that the two different types of com-

pounds were independently active on some physiologic system trichloride. In this latter reaction, vitamin A gave a vivid bluecolor at a wavelength maximum of 610–630 nm, whereasessential for growth; 2) that the pigmented compounds were

active as such, or possibly were converted to active, so-called carotene gave a green-blue color of lesser intensity at a peakwavelength of 590 nm. The absorption maximum of b-caro-‘‘leuko’’ forms; and 3) that only one of the types of compounds

was active, but that the other type was contaminated with the tene was approximately 450 nm, whereas liver oils containingvitamin A absorbed very weakly, if at all, at that wavelengthactive ingredient. In 1919, Harry Steenbock wrote: ‘‘It appears

reasonably safe, at least as a working hypothesis, to assume but showed a maximum absorption at 328 nm. The main in-strument used in these studies was the Lovibond tintometer.that the fat-soluble vitamin is a yellow plant pigment or a

closely related compound.’’ By its use, two types of units were defined: yellow units, whichwere high for b-carotene and very low for vitamin A, and blueThe following year, Palmer and Kempster (1920) tested this

hypothesis. They clearly showed that chicks grew well and units, based on the antimony trichloride reaction, which werevery strong for vitamin A and weak for b-carotene. Thus, thelaid eggs when fed a carotenoid-free diet supplemented with

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 to100 for vitamin A.into turmoil. If the yellow plant pigments (carotenoids) were

not the active growth-promoting compounds, why indeed, in Moore’s full paper was published in the Biochemical Journalin 1930. Moore first determined whether or not carotenoidthe absence of animal products, did they stimulate growth and

development? preparations contained vitamin A. He crystallized carotene 12times from a concentrated extract of carrots. He then com-A variety of factors confounded a clear analysis of the prob-

lem at that time. First of all, the chemical structures neither pared the growth-promoting ability of his crystalline carotenewith that of a cod liver oil concentrate. Approximately equalof vitamin A nor of carotenoids were known. Second, all lipid

preparations, whether from animal or plant sources, contained amounts of carotene and of the oil concentrate stimulated thegrowth of vitamin A–deficient rats. The oil concentrate gavemany impurities. Third, some plant carotenoids stimulated

growth, but other equally colored plant extracts did not. Four, the expected strong blue color at 610–630 nm, characteristicof vitamin A. If the carotene preparation had contained vita-other nutritional inadequacies often plagued the interpretation

of results. For example, vitamin E was discovered to be an min A as a contaminant, then it also should have given astrong blue color. But it did not. Thus, Moore concluded thatessential fat-soluble vitamin only in the early 1920s. Finally,

the methodology for the measurement both of vitamin A from the carotene preparation did not contain vitamin A.Moore then turned to studies on the conversion of caroteneanimal sources and of carotenoids from plant sources was rudi-

mentary. to vitamin A in vivo. He fed 22 rats a vitamin A and carotene–free diet for 28 to 77 d. Ten of the depleted rats were killed,Following Palmer and Kempster’s work, the generally ac-

cepted view was that carotenoid preparations were contami- and their livers were analyzed for yellow and blue units. There-after, the remaining rats were supplemented with variousnated with the colorless vitamin, which was found in pork

liver and other animal tissues. Thus, carotenoids per se were sources of carotene for 16 to 55 d. These rats were then killed,and their livers were analyzed. The data presented in Moore’sthought to be inactive constituents of these extracts.

Thomas Moore, born in 1900, started his research career paper are summarized in Table 1. The 10 depleted rats showeda few yellow units but no blue units in their liver. Small dosesin the mid-1920s at the Dunn Nutrition Laboratory associated

with Cambridge University in England. Following studies on of carotene stimulated the growth of animals but did not muchchange the presence of blue or yellow units in the liver. Onthe importance of light exposure in the formation of biologi-

cally active carotenoids in plants, he focused his attention on the other hand, when large doses of carotene or red palm oil,which predominantly contains a- and b-carotene in roughlythe nutritional relationship between carotene and vitamin A.

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 TABLE 1increased. Fresh carrots, given ad libitum to the animals,showed a similar effect. Bisulfite-binding substances from normal and

Moore then made the following calculation. Because the vitamin B1–deficient pigeons and rats1ratio of yellow to blue units for b-carotene is 11 to 1, the

Animal Normal Deficient Curednumber of yellow units of carotene corresponding to the blueunits found in the liver (2000–3700) when large doses of

mg/100 mL bloodcarotene were given would be expected to be 22,000–41,000.The amount of yellow units actually present, however, was

Pigeon 3.96 11.31 5.29only 40–110 (Table 1), a very small fraction of the amountRat 4.22 9.39expected to support an active role of carotene per se. Thus,

Moore (1930) concluded: ‘‘It is impossible that the colour 1 Data from Thompson and Johnson (1935). Approximately 2.5 mgreaction 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 wouldaccount for its physiological activity. The conclusion must bereached that carotene, or some part thereof, if it should later Peters 1929 and 1930) also concluded that there was moreprove to be heterogeneous, behaves in vivo as a precursor of lactic acid present in the brains of pigeons showing acutethe 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 musclecal 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 thia-conclusions. Thus, the dilemma of the relationship between min (in concentrated form) to brain tissue from deficient pi-the colored compounds largely found in plant foods and the geons, with added lactate, increased the in vitro rate of oxygennearly colorless compounds of liver had been resolved. Need- consumption to that of tissue from normal birds (Meiklejohn etless 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 (Pe-ters and Thompson 1934). This made an immediate metabolicLiterature Citedconnection to carbohydrate metabolism whereby glucose had

Karrer, P., Morf, R. & Schoepp, K. (1931) Zur kenntnis des vitamins A in fisch- recently been shown by Embden and Meyerhof to be degradedtranen. Helv. Chim. Acta 14: 1431–1436.

in animals to pyruvate. However, the lack of a significantMcCollum, E. V. & Davis, M. (1913) The necessity of certain lipins duringgrowth. 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 wasoil vitamin A from carotene. VI. The conversion of carotene to vitamin A in puzzling.vivo. Biochem. J. 24: 692–702.

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, that the abnormally large amount of pyruvate formed in thefecundity and reproduction of fowls. J. Biol. Chem. 39: 299–313.deficient brain in vivo was not detectable because the metabo-Steenbock, H. (1919) White corn vs. yellow corn and a probable relationship

between the fat-soluble vitamine and yellow plant pigments. Science 50: 352– lite largely diffuses out into the blood stream. Using pigeons353. and rats, they measured pyruvate indirectly with other keto

compounds that can form bisulfite complexes, and they securedthe fractional amount of pyruvate in the bisulfite-binding com-Paper 12: The Co-enzyme Function ofpounds by direct isolation of the pyruvate 2,4-dinitrophenylhy-Thiamin (Peters et al., 1929–1937) drazone and estimating it colorimetrically. Their findings aresummarized in Table 1. It should be noted that Platt and LuPresented by Donald B. McCormick, Department of Biochemistry,(1935) bridged from the experimental animals to humans byEmory University School of Medicine, Atlanta, GA 30322 as partconcordantly reporting the presence of pyruvate in the bloodof the minisymposium ‘‘Experiments That Changed Nutritionaland cerebrospinal fluid of beriberi patients in the Orient, whereThinking’’ given at Experimental Biology 95, April 11, 1995, inthe story began.Atlanta, GA.

The significance of findings importantly focused by the in-vestigators at Oxford, and even the broader value of basicDescriptions of a particular human illness that were re-

corded over a thousand years ago in the Far East reflect what research with its use of animals, was well reviewed by R. A.Peters (1936) in his lecture delivered at the National Hospital,later was termed beriberi (Gubler 1991, McCormick 1988).

Eijkman and then Grijns, working in Batavia, used chickens Queen-Square. In summarizing ‘‘What has been learnt,’’ Petersstates: ‘‘We may now take stock of the position. A purely in-as a model for the disease, and they found in the 1890s that

chickens developed polyneuritis when fed white rice, but that vitro research with brain tissue of the bird was started in thefirst instance to improve the test for vitamin B-1 and laterrice bran acted as a preventive. Further studies in the 1920s

at the Eijkman Institute in the Dutch West Indies elaborated extended to elucidate the enzyme with which the vitamincooperated. It has not only helped to settle these problemssome general characteristics of the necessary micronutrient in

rice bran. The bird model was extended by R. A. Peters and his but it has proved the existence of pyruvate in normal metabo-lism. It has also shown that an in-vitro research upon braincolleagues, who used pigeons at Oxford University in England.

The connection of a role for the anti-beriberi factor in tissue which takes advantage of the in-vitro labours of bio-chemists, can be applied to in-vivo events. This is an im-carbohydrate metabolism became apparent with the report of

Japanese workers (Inawashiro and Hayasaka 1928) that lactic portant step in this field. It is further encouraging that thework has led to the detection of pyruvate in the blood ofacid disappeared more slowly from the blood of beriberi pa-

tients after exercise. Investigators at Oxford (Kinnersley and beriberi patients, which may well prove diagnostic. Surely we




Fisher, R. B. (1931) CLIII. Carbohydrate metabolism in birds. III. The effects ofTABLE 2 rest and exercise upon the lactic acid content of the organs of normal andrice-fed pigeons. Biochem. J. 25: 1410–1418.

Elucidation of cocarboxylase as thiamin pyrophosphate1 Gubler, C. J. (1991) Thiamin. In: Handbook of Vitamins (Machlin, L. J., ed.),2nd ed., pp. 233–281. Marcel Dekker, New York, NY.

Horecker, B. L., Smyrniotis, P. Z. & Klenow, H. (1953) The formation of sedo-Isolation of cocarboxylase (100 kg yeast r 750 mg HCl salt)heptulose phosphate from pentose phosphate. J. Biol. Chem. 205: 661–682.1. Preparation of alcoholic heat extract.

Inawashiro, R. & Hayasaka, E. (1928) Studies on effect of muscular exercise2. 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 beri-

reprecipitation 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 me-

tabolism in birds; relation between lactic acid content of brain and symptomspyridine.of opisthotonus in rice-fed pigeons. Biochem. J. 23: 1126–1136.5. Fractional precipitation with methanol-ether.

Kinnersley, H. W. & Peters, R. A. (1930) Carbohydrate metabolism in birds;6. Precipitation of poorly soluble picrolonates.brain localisation of lactic acidosis in avitaminosis B1 and its relation to origin7. Precipitation of poorly soluble Ba and Ag salts.of symptoms. Biochem. J. 24: 711–722.8. Precipitation with phosphotungstic acid and crystallization Lohmann, K. & Schuster, Ph. (1937) Untersuchungen uber 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 C12H21O8N4P2SCl (Shils, M. E. & Young, V. R., eds.), 7th ed., pp. 355–361. Lea & Febiger,Philadelphia, PA.Acid hydrolyses pyrophosphate ester

Meiklejohn, A. P., Passmore, R. & Peters, R. A. (1932) The independence ofSulfite splitting pyrimidine and thiazole partsvitamin B1 deficiency and inanition. Proc. R. Soc. B. 111: 391–395.UV absorption same as thiamin monophosphate

Peters, R. A. (1936) The biochemical lesion in vitamin B1 deficiency. Applica-Biological researchtion 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.

Platt, B. S. & Lu, G. D. (1935) Proc. Physiol. Soc., 3rd Gen. Congr., Chinese1 Outlined from Lohmann and Schuster (1937).Med. Assoc.

Thompson, R.H.S. & Johnson, R. E. (1935) LXXX. Blood pyruvate in vitamincould not have a better instance of the ultimately practical B1 deficiency. Biochem. J. 29: 694–700.value of a purely academic research.’’

During the period when the metabolic connections of vita- Paper 13: To Live Longer, Eat Less!min B1 to carbohydrate utilization at the level of pyruvate (McCay, 1934–1939)were being established, other work was leading to elucidation

Presented by Patricia B. Swan, Department of Food Science andof the structure of the vitamin (Gubler 1991, McCormickHuman Nutrition, Iowa State University, Ames, IA 50011 as1988). The correct empirical formula of vitamin B1 deter-part of the minisymposium ‘‘Experiments That Changed Nutritionalmined by A. Windaus in 1932 recognized the inclusion ofThinking’’ given at Experimental Biology 96, April 16, 1996 insulfur, and by 1936–1937 R. R. Williams and his coworkersWashington, DC.determined the full structure and accomplished synthesis of

what we today call thiamin. In 1925 Clive McCay took a postdoctoral position at YaleWith 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 ofwork 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 anthis functional coenzyme form of vitamin B1 was elucidated by instructor in chemistry at Texas A&M for a year. He wasLohmann and Schuster (1937), who isolated and characterized awarded a M.S. in biochemistry by Iowa State College inthiamin pyrophosphate starting with 100 kg of yeast. The steps 1923 and a Ph.D. with C.L.A. Schmidt at the University ofinvolved 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 totions within a-keto acid decarboxylase subunits of three gen- turn his attention to questions more biological in natureeral 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 Men-chain a-keto acids, operating in our bodies. A second im- del’s laboratory (Osborne et al. 1917) demonstrating that fe-portant role for the coenzyme of thiamin in the direct metabo- male rats whose food intake was restricted were able to repro-lism of carbohydrates is within transketolase, where thiamin duce at more advanced ages than usual. McCay asked Mendelpyrophosphate mediates interconversions with pentose phos- why they had not carried their experiments longer to deter-phates (Horecker et al. 1953). A better index of thiamin status mine the extent to which the life span of these rats had beenin humans evolved with development of erythrocyte transke- increased. Mendel replied that McCay, as a young man, wastolase assays. However, the classic work of R. A. Peters and in a better position to undertake such a long-term experimenthis 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. May-liams working on thiamin structure and of Lohmann and nard, the head of the animal husbandry department at CornellSchuster 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-yearstanding. career at Cornell, retiring in 1962 (Loosli 1974).

Nutrition and LongevityLiterature CitedShortly after he went to Cornell, McCay began his firstAuhagen, E. (1932) Co-Carboxylase, ein neues Co-Enzyme der alkoholischen

Garung. Hoppe-Seyler’s Z. Physiol. Chem. 204: 149–167. study of the effects of food restriction on the life span of rats,



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TABLE 2TABLE 1

Effects of food restriction on the weight and density of theEffects of food restriction on life span of rats1

femur of rats1Median age

Group Ave. age at death Group Mean weight Density

d I UnrestrictedMales (n Å 11) 0.741 1.22

I Unrestricted Females (n Å 18) 0.540 1.21Males (n Å 14) 483 522 II Restricted at weaningFemales (n Å 22) 801 820 Males (n Å 7) 0.486 1.15

II Restricted at weaning Females (n Å 13) 0.398 1.13Males (n Å 13) 820 797 III Restricted 2 wk after weaningFemales (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 1.14Males (n Å 15) 894 919Females2 (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 Longevitypublishing the first report from this work in 1934 while some In a second experiment, McCay and his group fed all ratsof the rats were yet alive (McCay and Crowell 1934). McCay identical amounts of a nutrient-dense diet, feeding thatand his co-author remarked on the long-held viewpoint that amount required to just maintain the weight of the restrictednutrition and life span were related (Darby 1990). Yet, they rats. This allowed them to determine whether the unlimitednoted, the science of nutrition had focused on the young, consumption of a nutrient-dense diet had shortened the lifegrowing animal to the exclusion of the study of the adult or of the control group from the first experiment, and they con-aging animal. cluded it had not. The growth of the rats was controlled byThey wrote: ‘‘In this day when both children and animals varying the amount of a mixture of sucrose, cooked starch andare being fed to attain a maximum growth rate, it seems a lard (38:57:5) that was fed, with the control rats being allowedlittle short of heresy to present data in favor of the ancient to eat as much as they chose. In addition, the control grouptheory that slow growth favors longevity.’’ They cited the ear- was divided, with half the rats being fed cod liver oil and halflier work of Osborne et al. (1917) and their own previous (as well as all rats with restricted intakes) being fed irradiatedobservations that trout with very low protein intake ‘‘failed to yeast and carotene as sources of fat-soluble vitamins. Supple-grow [but remained alive and] lived twice as long as those mentation with these different sources of fat-soluble nutrientsthat grew.’’ They postulated that ‘‘something was consumedin growth that is essential for the maintenance of life’’ (McCayand Crowell 1934). TABLE 3

Accordingly, McCay and Crowell selected from their col-ony 106 rats, both male and female, and divided them into Effects of various lengths of food restriction onthree groups. They fed them all the same nutrient-dense diet the life span of rats1from the time of weaning. However, Group I was fed unlimitedamounts of the diet, whereas the others were fed restricted Age at deathamounts of the diet, either from the time of weaning (Group

Group Ave. RangeII) or after 2 wk (Group III). Restricted rats were maintainedat a constant weight until once about every 100 d when they

dwere allowed to grow about 10 g because of the feeding ofsucrose or beef liver to all rats.

I Unrestricted2A 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) 643 404–965data concerning the length of life of these rats. The female rats II Restricted until d 300

Males (n Å 4) 865 805–1018that were fed unrestricted amounts of diet lived significantlyFemales (n Å 5) 811 555–1183longer than the comparable males (median age at death 820 vs

Restricted until d 500522 d). Male rats that were fed restricted amounts of diet, how-Males (n Å 5) 806 366–1103ever, 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 700lengthened by food restriction and the slower rate of growth, Males (n Å 4) 874 772–1025

Females (n Å 6) 912 406–1320whereas the life of the slower-growing females was not lengthenedRestricted until d 1000greatly, if at all, by food restriction.

Males (n Å 5) 882 336–1127McCay et al. (1935) also observed the effects of food restric-Females (n Å 4) 1033 815–1320tion 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

liver oil, were combined.some of the bones ‘‘crumbled in the course of dissection.’’




these data for guidance of human beings he might summarizeTABLE 4by stating: ‘Eat what you should, after that eat what you willbut not too much.’ ’’Effects of food restriction on weight and density

McCay’s scientific work was interrupted by his military ser-of the femur of rats1vice 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 spanof rats and other species. He is best remembered by his students

g for the course he taught for several years on the history ofnutrition. A collection of some of these lectures was published

I Unrestricted posthumously (McCay 1972). McCay died of a heart attackMales 0.70 1.10 in 1967.Females 0.57 1.21

II Restricted until d 300Males 0.63 1.18 Literature CitedFemales 0.45 1.13

Darby, W. J. (1990) Early concepts on the role of nutrition, diet and longevity.Restricted until d 500In: Nutrition and Aging (Prinsley, D. M. & Sandstead, H. H., eds.). Alan R. Liss,Males 0.54 1.18New York, NY.Females 0.47 1.13

McCay, C. M. (1939) Chemical aspects of aging. In: Problems of AgingRestricted until d 700(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 uponFemales 0.39 1.11 aging. In: Cowdry’s Problem of Aging (Lansing, A. I., ed.), chapter 6. Williams &

Restricted until d 1000 Wilkins, Baltimore, MD.Males 0.38 1.06 McCay, C. M. (1973) Notes on the History of Nutrition Research (Verzar, F.,

ed.). Hans Huber Publishers, Berne, Switzerland.Females 0.37 1.03McCay, C. M. & Crowell, M. F. (1934) Prolonging the life span. Sci. Monthly

39: 405–414.1 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) Retardedgrowth, life span, ultimate body size and age changes in the albino rat after3 and 4 represent the combination of these two treatmentsfeeding 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 45:294–295.the results of the first, began with 106 rats. Thirty-three were

fed unlimited amount of energy, and 73 were fed restrictedamounts. Thirty-five of the restricted rats, however, died due Paper 14: The Dynamic State of Bodyto two failures in the heating system. On d 300, the remaining

Constituents (Schoenheimer, 1939)38 rats from the restricted group were divided into four sub-groups and fed unlimited energy starting on d 300, 500, 700 Presented by Robert E. Olson, Department of Pediatrics, Collegeor 1000 (McCay et al. 1939). of Medicine, University of South Florida, Tampa, FL 33606 asAll groups of rats with restricted intakes contained longer- part of the minisymposium ‘‘Experiments That Changed Nutritionallived individuals than did the control group. At the time that Thinking’’ given at Experimental Biology 95, April 11, 1995, inthe longest-lived control rat died (d 965), 18 out of the 38 Atlanta, GA.rats whose intakes were restricted were still alive. When therestricted rats were allowed to eat normally, they resumed The discoveries of Rudolf Schoenheimer and his colleaguesgrowth with few exceptions. Those exceptions were certain at Columbia University in the period from 1933 to 1941 revo-individuals whose food intake had been restricted for 1000 d. lutionized our understanding of the metabolism of fatty acidsRestricted 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 re-rats in the second experiment. Again, both measures tended ceived his medical degree from the University of Berlin into decrease with increasing length of time of food restriction. 1922. Following a year of clinical training he did postdoctoralBone growth (length) was also measured, and the authors con- work in Leipzig with Karl Thomas from 1923 to 1926 andcluded: ‘‘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. Inmale 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, wherehe came in contact with Harold Urey, who had discoveredConclusionsdeuterium in 1932, and David Rittenburg, one of Urey’s stu-dents who had also joined the biochemistry faculty at Colum-McCay summarized the conclusions he drew from these

experiments in a chapter he wrote (McCay 1939) and later bia. Together they planned experiments with various isotopiccompounds, including heavy water, and substrates labeled withrevised (McCay 1952) as a contribution to a book on aging.

‘‘This indicated that the life span was flexible and that the deuterium and 15N; these experiments were to revolutionizeconcepts of fat and protein metabolism.possibility of its extension was unknown as well as that the

retarded animals tended to outlive those that matured nor- At the time that Schoenheimer initiated his isotopic studiesof intermediary metabolism it was generally believed that themally’’ (McCay 1952). ‘‘The second experiment gave essen-

tially the same results as the first and indicated clearly that major components of the body, including body fat and protein,were chemically stable. It was assumed that there was verythe retarded growth was the essential feature’’ (McCay 1952).

He went on to say: ‘‘If one were to draw conclusions from 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 rateas the synthesis in the first experiment (Fig. 1). Schoenheimeralso demonstrated that [2H]-palmitic acid was not only depos-ited in the fat of rats but also converted into other fatty acids,including stearic and oleic acids but not linoleic acid, whichhad been shown earlier to be essential.

Studies of Protein Metabolism

Schoenheimer and his associates next turned to the study ofprotein metabolism. They prepared doubly labeled L(–)leucinecontaining 3.60 atoms % excess deuterium in its hydrogenatoms and 6.54 atoms % excess 15N in its nitrogen atom.This concentration of both isotopes was high enough to allowadmixture with several hundred parts of ordinary leucine andstill permit detection by mass spectrometry. Adult rats werefed a stock diet containing the marked leucine and observedfor 3 d, during which time there was no change in weight. Atthe end of the 3-d period the excreta and body tissues wereanalyzed. About 30% of the 15N administered was found inthe excreta (2.2% of the isotope in the feces and 27% in theurine). The body protein retained the remaining 65% of theactivity, indicating that the isotopic N had been incorporatedmainly into tissue protein. This finding was totally inconsistentwith Folin’s view of exogenous protein metabolism.

It was found that different organs were not equally effectivein the fixation of dietary nitrogen. As shown in Table 1, theFIGURE 1 The synthesis and destruction of fatty acids in mice

(Schoenheimer and Rittenberg 1936). Used with permission of the Jour- proteins of the internal organs, serum and intestinal tract werenal of Biological Chemistry, Bethesda, MD. the most active. The proteins of muscle showed less activity,

but, because they constitute by far the largest part of the ani-mal, the low concentration of 15N actually represented a highvarious organs in the body except for replacement of tissue absolute amount of isotope. In fact, 66% of the administeredcaused by ‘‘wear and tear.’’ Fat depots in the body were consid- isotope was recovered in muscle and only 33% in the combinedered to be a reserve source of energy that were called upon internal organs.only when the body was faced with serious food restriction. Table 2 shows that not only was there an active incorpora-Otherwise, fat metabolism occurred only for purposes of oxidiz- tion of leucine into various organs and tissues, but the 15Ning dietary fat to produce energy. also appeared in all other tissue amino acids studied exceptSimilar views were held regarding protein metabolism, lysine. Particularly prominent in this exchange were glutamicmostly as a result of studies by Rubner in Germany and Folin and aspartic acids. This transamination observed by Schoen-in the United States. In 1905, Folin concluded on the basis heimer in these studies was explained enzymatically byof studies of the excretion of nitrogenous substances in urine Braunstein and Kretzmann in 1937. The 15N in arginine wasthat nitrogen metabolism could be divided into endogenous more enriched in liver than in kidney, mostly because of the(tissue) and exogenous (dietary) phases (Folin 1905). E. V. ornithine-urea cycle in liver, discovered by Krebs and Hensel-McCollum et al. (1939) complimented Folin in the fifth edi- eit in 1932.tion of the Newer Knowledge of Nutrition by saying, ‘‘Folin When the deuterium and 15N contents of the administeredpossessed the genius to solve the problem (of protein metabo- doubly labeled leucine were both measured, it was found thatlism) in its main outlines, and his interpretation of the mecha- the D/15N ratio in leucine was increased from 100:182 in thenism is almost universally excepted.’’ Folin’s view became the administered compound to 100:108 in the carcass, indicatingparadigm for protein metabolism that persisted to the middle

1930s when Schoenheimer began his work.

TABLE 1Studies of Fat Metabolism

15N Content of protein nitrogen in rat organs afterIn Schoenheimer’s first study of the turnover of body con- consumption of L(–)leucine for 3 d1

stituents, he used deuterated water to track the biosynthesisof body fat. A proton from water is taken up in fatty acid Organ 15N excessbiosynthesis in the reductive steps catalyzed by NADPH. Fig-ure 1 shows the enrichment of fatty acids in the depot fat of Serum 1.67

Liver 0.94mice over a period of 19 d. The isotope content of total fattyIntestine 1.49acids of the mice reached a maximum at d 6 with a 1

2 time ofKidney 1.382.5 d. The total enrichment indicated that 14% of all fattyHeart 0.89acids (mostly in adipose tissue) was replaced in this experi- Muscle 0.31

ment. To demonstrate the destruction of fatty acids in micefed a carbohydrate-rich diet, mice of the same weight were fed 1 Data from Schoenheimer (1942). Values are calculated for 100

atom percent 15N in leucine.for 5 d a diet consisting of 20% fat enriched with deuterium




Folin, O. (1905) A theory of protein metabolism. Am. J. Physiol. 13: 117–138.TABLE 2 Krebs, H. A. & Henseleit, K. (1932) Untersuchungen uber die Harnstoffbildungim Tierkorper. 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 Knowledgeof Nutrition, 5th ed., pp. 80–82. MacMillan, New York, NY.by rats for 3 d1

Schoenheimer, R. (1942) The Dynamic State of Body Constituents. HafnerPublishing, New York, NY.

Amino Schoenheimer, R., Ratner, S. & Rittenberg, D. (1939) Studies in protein metab-acid Liver Muscle olism. X. The metabolic activity of body proteins investigated with L(–)leucine

containing two isotopes. J. Biol. Chem. 130: 703–732.Schoenheimer, R. & Rittenberg, D. (1936) Deuterium as an indicator in theLeucine 7.95 1.90

study of intermediary metabolism, VI. Synthesis and destruction of fatty acidsGlutamate 1.85 0.89in the organism. J. Biol. Chem. 114: 381–396.Aspartate 1.16 0.70

Arginine 0.89 0.25Tyrosine 0.50 0.20Lysine 0.06 Paper 15: Tryptophan’s Role as aAmide N 0.78 0.51 Vitamin Precursor (Krehl et al., 1945)

1 Data from Schoenheimer (1942). Calculated for 100 atom per centPresented by LaVell M. Henderson, Professor Emeritus, Depart-15N in leucine administered.ment of Biochemistry, University of Minnesota, St. Paul, MN55108 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 oftransamination as a major reaction of amino acids. When canine black tongue was recognized as the counter-

Schoenheimer and coworkers then gave [15N]ammonium part of pellagra, about 1925, experimental nutritionistscitrate 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 andmate. 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 containedportant enzymes for ammonia fixation into amino acids. Most enough niacin that it should prevent black tongue and pella-of the isotope in arginine was in the amidine group and was gra. Krehl and co-workers confirmed this, but they found thatremoved by arginase, leaving a very small amount in ornithine the niacin requirement of dogs was three times greater whenin accord with the ornithine-urea cycle. corn diets rather than sucrose-casein diets were consumed.

In these experiments, [15N]ammonia was also incorporated Rats fed sucrose-casein diets had been found not to require ainto creatine, as expected from the work of Borsook and Dub- dietary source of niacin, but the same group now tested ratsnoff, who in 1941 demonstrating arginine to be a precursor with corn diets. They reported (Krehl et al. 1945a) that re-of creatine. What Schoenheimer then showed was that the placement of 40% of their 15% casein diet with corn gritsformation of creatinine from [15N]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 ratsof the curve indicated that about 2% of total body creatine did require niacin. Corn is unusual in having a particularlywas being converted to urinary creatinine (and replaced by low level of the essential amino acid tryptophan in its mixednew synthesis) per day. This also explained the constancy of proteins. Two months later, Krehl et al. (1945b) reported thatcreatinine excretion in the urine, which Folin had interpreted tryptophan added at 40 mg/100 g diet replaced niacin in re-to 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 hy-pothesis discussed earlier as follows: ‘‘It is scarcely possibleto reconcile our findings with any theory which requires a TABLE 1distinction between two types of nitrogen. . . . The excreted

Effect of corn grits (CG), cystine (Cys), threonine (Thr),nitrogen may be considered as a part of the metabolic pooltryptophan (Trp) and niacin on the growth of weanling ratsoriginating from interaction of dietary nitrogen with the rela-

tively large quantities of reactive tissue nitrogen.’’ fed 9% casein dietsIn summary, Schoenheimer introduced a new and dynamic

Dietary groupparadigm for fat and protein metabolism, replacing the staticview of Folin and others. In fact, the idea of the major body

Basal diet Basal Basal / niacinconstituents as a dynamic biochemical system has been ex-tended more recently to include all aspects of metabolism,

g/wkincluding systems involved in transport, hemopoiesis, endo-crine and cytokine activity and even the genome. 15% Casein 29 29

9% Casein / 40% CG 7 279% Casein / 40% CG / Trp 31Literature Cited9% Casein 109% Casein / 0.2% Cys 12 17Borsook, H. & Dubnoff, J. W. (1941) The formation of glycocyamine in animal

tissues. J. Biol. Chem. 138: 389–394. 9% Casein / 0.2% Cys /Braunstein, A. E. & Kritzmann, M. G. (1937) Uber den Ab- und Aufbau von 0.078 Thr 3 19

Aminosauren durch Umaminierung. Enzymologia 2: 129–140.



1044S SUPPLEMENT

diet is elevated, protein synthesis increases, tryptophan isdrawn into this function at the expense of the alternate path-way to niacin, and the vitamin deficiency results. That thisexplanation for the effect of threonine is valid is supported bythe results of similar imbalance studies that showed that lysine,valine, leucine and isoleucine also produce growth inhibitionthat is reversed by tryptophan or niacin (Koeppe and Hender-son 1955).FIGURE 1 Intermediates in the conversion of tryptophan to niacin

Many isotopic labeling studies with rats have confirmedin N. crassa and rats.the conversion of tryptophan to niacin. The first evidenceregarding the reactions involved came from experiments with

The most likely explanation for the interchangeability of mutants of the mold Neurospora crassa (Fig. 1). The first fourniacin, required at 1 mg% of the diet, and tryptophan, required compounds in this scheme were verified in animal systems,at 40 times that molar concentration, was the conversion of and quinolinic acid was added when it was identified as antryptophan to niacin. The fact that 2% acid-hydrolyzed casein excretory product of tryptophan in mammals and a rather(tryptophan destroyed) could replace 40% corn grits in causing ineffective niacin substitute in rats and a niacin-less mutantthe deficiency suggested that an increase in the levels of other of N. crassa (Henderson 1949).amino acids was inducing the deficiency. This effect was Quinolinic and picolinic acids are formed by the incubationtermed ‘‘amino acid imbalance.’’ The amino acids most effec- of 3-hydroxyanthranilate with mammalian liver (Fig. 2). Nei-tive in producing the deficiency were threonine and lysine ther is degraded to carbon dioxide even in vivo. That they(Hankes et al. 1948), but additional sulfur amino acids were arise from intermediates in the degradation of tryptophan toneeded to maximize the effect. Therefore, in subsequent studies glutarate was established by the report of Gholson et al.0.2% L-cystine was added to the basal diet (Table 1). The (1962). It is evident that picolinate and quinolinate arise byexplanation offered for the effect of other amino acids on the formation of cyclic Shiff’s bases from intermediates in thegrowth of rats fed the 9% casein / 0.2% L-cystine diets is degradation of tryptophan, and they can be considered sideillustrated as follows: reaction products of the degradative pathway.

The limited activity of exogenous quinolinate as a dietaryreplacement for niacin cast doubt on its intermediary role in

Trp / AA (Thr or Lys) r Proteinf

Niacin the formation of niacin. This limited activity probably resultsfrom the failure of the salts of quinolinic acid to penetrate thecells in which it normally arises. It is a strong acid and becauseWhen an essential amino acid, such as threonine, is suppliedit is obliged to enter cells in the undissociated form, a properat just below the optimum level, moderately good growth oc-pH for its penetration is much below physiological pH values.curs and the marginal tryptophan level meets the needs for

both protein and niacin synthesis. When the threonine in the 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.




by the observations of Nishizuka and Hayaishi (1963), who origin of the two-way interaction concept but extends theconcept to that of a three-way interaction with copper, molyb-showed that it is converted to nicotinic acid mononucleotide

in the presence of 5-phosphoribosyl-1-pyrophosphate by a sol- denum and sulfur acting on each other individually and collec-tively.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 foragegrown in the ‘‘teart’’ area of southern England containedthe effectiveness of tryptophan as a source of niacin seems to

result from the degree to which 2-acroleyl-3-aminofumarate is higher concentrations of molybdenum than that of non-teartherbage. The teart area is an area of approximately 20,000decarboxylated (Fig. 2). In cat liver, 86% is decarboxylated

and only 9% is converted to quinolinate (Suhadolnik et al. acres on which grazing cattle and sheep developed severescouring and exhibited poor performance. Horses were not1957). At the other extreme, the rat liver decarboxylates only

12%, leaving 80% to form quinolinate. Livers from eight other affected. The authors observed that addition of soluble molyb-date to normal forage, in amounts equivalent to that consumedspecies, including dogs and humans, fall between these two

extremes. Cats cannot substitute tryptophan for niacin, in the teart forage, produced the same signs of diarrhea indairy cows. Although this paper did not show interaction ofwhereas rats do not require niacin at the usual levels of trypto-

phan intake. Humans and dogs have a limited capacity to molybdenum with another specific nutrient, it did show thedetrimental effect of trace quantities of molybdenum in thesubstitute tryptophan for niacin, so the clinical deficiency ap-

pears even with moderate intakes of tryptophan. diet of ruminants (Ferguson et al. 1938).Evidence of an antagonistic copper-molybdenum interac-The prevalence of pellagra in poor, corn-eating populations

in southern Europe, but not in natives of the western hemi- tion came from Australia. The original observation in Austra-lia was made during the investigation of an entirely differentsphere, has been explained by the fact that the natives of the

Americas used hot lime or other alkaline solutions in the problem, enzootic jaundice, a disease in sheep that results fromchronic copper toxicity. While seeking an explanation for apreparation of their tortillas from corn meal. The observations

of Krehl et al. (1945b), 50 years ago, have provided an explana- chronic copper toxicity that occurred in specific areas of thecountry, Dick and Bull (1945) observed incidentally that mo-tion for the variation in niacin requirements among species

and a partial explanation for the association between corn lybdate supplementation of cows and sheep decreased the liverstorage of copper. Similar observations were made in Newconsumption and pellagra, based upon the very limited trypto-

phan content of corn. A full understanding of this association Zealand by Cunningham (1950), who studied the interactionof copper and molybdenum in relation to ‘‘peat scours.’’ In thewill depend upon the identification of the postulated sub-

stances in corn that form the vitamin on heating with alkali. United States, Comar et al. (1949) showed that molybdenumsupplementation lowered copper storage in livers of cattle,and the effect occurred with intakes that gave rise to copperLiterature Citeddeficiency in areas of Florida. This series of studies established

Elvehjem, C. A., Madden, R. J., Strong, F. M. & Woolley, D. W. (1938) The iso- the concept of Cu-Mo antagonism and suggested that excesslation and identification of the anti-black tongue factor. J. Biol. Chem. 123:dietary molybdenum might induce copper deficiency. It also137–149.

Gholson, R. K., Nishazuka, Y., Ichiyama, A., Kawai, H., Nakamura, S. & Hayaishi, suggested that a low molybdenum concentration in forage ofO. (1962) New intermediates in the catabolism of tryptophan in mammalian normal copper content might predispose sheep to copper tox-liver. J. Biol. Chem. 237: PC2043–2045.

icity.Hankes, L. V., Henderson, L. M. Brickson, W. L. & Evlehjem, C. A. (1948) Effectof 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 nico-influenced the interaction. The concept of a three-way interac-tinic acid for the growth of the rat and Neurospora. J. Biol. Chem. 181: 677–

685. tion among Cu-Mo-S emerged from a series of papers (DickKoeppe, 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 thatKrehl, W. A., Teply, L. J. & Elvehjem, C. A. (1945a) Corn as an etiological factorin 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 datatarding effect of corn in nicotinic acid–low rations and its counteraction by are shown in Table 1. Clearly the amount of copper storedtryptophane. Science 101: 489–490.

increased as the proportion of oat to alfalfa (lucerne) hay inNishizuka, Y. & Hayaishi, O. (1963) Enzymic synthesis of niacin nucleotidesfrom 3-hydroxyanthranilic acid in mammalian liver. J. Biol. Chem. 238: the diet increased with supplemental copper and molybdenumPC483–485. each remaining constant at 10 mg/d. With equal proportionsSuhadolnik, R. J., Stevens, C. O., Decker, R. H., Henderson, L. M. & Hankes, L. V.

of the two forages, total liver copper increased 35 mg over the(1957) Species variation in metabolism of 3-hydroyanthranilate to pyridinec-arboxylic acids. J. Biol. Chem. 228: 973–982. 6-mo period. This value compares with an earlier observed

increase of approximately 135 mg that occurred under similarconditions without added molybdenum. When oat hay wasPaper 16: The Concept of Tracethe sole source of forage, the increase was 113 mg even in theElement Antagonism: The Cu-Mo-S presence of additional molybdenum. It is also notable that the

Triangle (Dick, 1952–1954) molybdenum concentration in the blood was 10-fold higherin sheep that consumed oat compared with alfalfa hay. ThisPresented by Boyd L. O’Dell, Department of Biochemistry, Uni- experiment showed clearly that a naturally occurring compo-versity of Missouri, Columbia, MO 65211 as part of the minisym- nent of alfalfa that is largely absent in oat hay affected theposium ‘‘Experiments That Changed Nutritional Thinking’’ given physiological interaction of copper and molybdenum.at Experimental Biology 96, April 16, 1996, in Washington, DC.

The next paper in the series (Dick 1953a) described experi-ments that identified inorganic sulfate as the component ofThe precise origin of the concept of antagonistic interactionalfalfa that lowered the blood molybdenum. Dick (1953a)of two or more essential trace elements is not entirely clear,found that an aqueous extract of alfalfa hay was rich in sulfatebut this essay reviews some of the first documented evidence

for such a physiological antagonism. It not only describes the ion and that the extract had the same molybdenum-lowering



1046S SUPPLEMENT

pairs liver copper storage. Pertinent data from two experimentsTABLE 1are presented in Table 2. All sheep consumed approximately10 mg of copper and some were supplemented with 10 mg ofLiver copper and blood molybdenum concentrations of sheepmolybdenum per day. When the animals consumed oat hay,fed different sources of forage supplemented with 10 mgthe addition of molybdenum had no effect on liver coppereach of copper and molybdenum per day for 6 mo1storage, but when they consumed alfalfa it markedly decreasedcopper storage. When the oat hay diet was supplemented withDietary forage Increase in Blood Mo

(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 ofsulfate (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 These experiments clearly established the existence of a three-Lucerne 1:oat 1 35 180

way interaction among copper, molybdenum and sulfate asLucerne 1:oat 3 70 378regards liver copper storage.Oat 113 447

In spite of the decrease in liver copper storage observed1 Adapted from Dick (1952); n Å 6 animals per group. when the diet was supplemented with both molybdenum and2 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 copperapproximately 27 mg.concentrations rose with increasing levels of both molybde-3 The values are the means of weekly blood samples taken over thenum and sulfate, and there appeared to be an interaction.period.Even though the blood copper concentration increased, thecopper was not available for biological function. Sheep fedeffect as the hay itself. The results summarized in Figure 1high levels of molybdenum and sulfate and a nominally ade-show that the administration of potassium sulfate loweredquate level of copper developed signs of copper deficiency evenblood molybdenum in a sheep fed oat hay supplemented withwith blood copper concentrations that were normal or greater10 mg of molybdenum per day. The lower blood molybdenumthan normal. There was loss of crimp in the wool and decreasedresulted from increased excretion of molybdenum via the kid-liver copper concentration. Clearly, under these conditions ofney, an effect that was independent of urine volume. This andhigh molybdenum and sulfate intake, blood copper was not arelated experiments established the Mo-S interaction.valid measure of copper status because there was evidence ofSubsequently, Dick (1953b) showed that sulfate is also thecopper deficiency in spite of normal or elevated blood copperfactor in alfalfa that, in combination with molybdenum, im-concentrations.

An explanation of the Cu-Mo-S interaction must accountfor the fact that S renders both copper and molybdenum bio-logically unavailable and that molybdenum lowers copperavailability 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-richenvironment of the rumen. This would account for low copperabsorption but not for the high blood copper concentration.Normally all of the copper in blood is solubilized by trichloro-acetic acid (TCA), but this was not true in cases of highmolybdenum intake. To explain this phenomenon, Dick et al.(1975) prepared di, tri- and tetrathiomolybdates and observedthat their addition to blood in vitro resulted in a TCA-insolu-ble fraction that contained most of the copper. When tetrathi-omolybdate was administered to sheep intravenously, therewas an immediate rise in the TCA-insoluble copper in theblood. Thus, formation of copper thiomolybdates, particularlyCuMoS4, in the rumen accounts for the poor absorption ofcopper when the intake of molybdenum is high. The absorp-tion of thiomolybdate and subsequent formation of CuMoS4

in the blood accounts for the high concentration of bloodcopper that is not bioavailable.

The experiments described here revealed a three-way inter-action of copper, molybdenum and sulfate in ruminant ani-mals, in which bacterial fermentation plays a major role indigestion. The interaction in monogastric animals is much lessdramatic, because there is less sulfide available to form thethiomolybdate 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 interactionthe blood concentration (mcg/100 mL) and urinary excretion (mg/d) ofcan be demonstrated in monogastric animals by administrationmolybdenum. The sheep was fed oat hay and drenched daily with 10of thiomolybdates. Interestingly, tetrathiomolybdate has beenmg of Mo as ammonium molybdate. Collections were made over 7 d;used to treat Wilson’s disease in human patients (Brewer etthe time of sulfate administration is indicated by arrow. Figure 4 from

Dick (1953a). al. 1994). This genetic disease results in accumulation of cop-




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 — /54Oat hay 10.0 10.3 — /40Lucerne 9.8 0.5 — /20Lucerne 9.8 10.5 — 012Oat hay 9.9 0.5 2.2 & 4.43 /15Oat hay 9.9 10.7 2.2 & 4.4 020Lucerne 10.1 0.7 2.2 & 4.4 /39Lucerne 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:per in tissues, and thiomolybdate counteracts copper toxicityInorganic Nitrogen Metabolism (McElroy, W. D. & Glass, B., eds.), pp. 445–by complexation of the cupric ion and prevention of its absorp- 473. The Johns Hopkins Press, Baltimore, MD.

tion. What lesson derives from this work? In the first place, one Dick, A. T. & Bull, L. B. (1945) Some preliminary observations on the effect ofmolybdenum on copper metabolism in herbivorous animals. Aust. Vet. J. 21:cannot easily predict the outcome of good science regardless of70–72.its origin. In this case a project that was designed to determine Dick, A. T., Dewey, D. W. & Gawthorne, J. M. (1975) Thiomolybdates and copper-

the toxicology of a disease in sheep led to the elucidation of molybdenum-sulphur interaction in ruminant nutrition. J. Agric. Sci. 85: 567–568.a complex interaction related to copper deficiency and eventu-

Ferguson, W. W., Lewis, A. H. & Watson, S. J. (1938) Action of molybdenum inally to a compound used in the treatment of a human disease.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.Literature Cited

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 Paper 17: Reduced Radiation Damageand sheep in New Zealand. In: Copper Metabolism (McElroy, W. D. & Glass,

B., eds.), pp. 246–273. Johns Hopkins Press, Baltimore, MD. from Ingestion of Cabbage FamilyBrewer, 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- Plantsmolybdate. I. Initial therapy in 17 neurologically affected patients. Arch. Neu-rol. 51: 545–554. Presented by Mindy S. Kurzer, Department of Food Science and

Dick, A. T. (1952) The effect of diet and of molybdenum on copper metabolism Nutrition, University of Minnesota, St. Paul, MN 55108 as partin sheep. Aust. Vet. J. 28: 30–33.of the minisymposium ‘‘Experiments That Changed NutritionalDick, A. T. (1953a) The effect of inorganic sulphate on the excretion of molybde-

num in the sheep. Aust. Vet. J. 29: 18–24. Thinking’’ given at Experimental Biology 96, April 16, 1996, inDick, A. T. (1953b) The control of copper storage in the liver of sheep by inorganic Washington, DC.sulphate and molybdenum. Aust. Vet. J. 29: 233–238.Dick, A. T. (1954) Preliminary observations on the effects of high intakes of molyb-

By 1950, it had been noted that experiments on the effectsdenum and of inorganic sulphate on blood copper and on fleece characterin crossbred sheep. Aust. Vet. J. 30: 196–202. of radiation in guinea pigs showed great variability in the

results. Mme. M. Lourau and O. Lartigue (Lourau and Lartigue1950) observed that although each series of experiments was

TABLE 3 fairly homogeneous, when one compared experiments, the le-thal 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 statedforage 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 inDietary sulfate 1950, they studied 114 male guinea pigs. Test diets consistedintake 15 mg Mo 30 mg Mo 60 mg Mo 90 mg Mo

of oat and bran supplemented with either cabbage at 50 g/dor beets at 75 g/d. The animals received a single whole-bodyg/d %radiation dose. Table 1 summarizes the results. For a givenradiation dose, mortality was far greater in the animals fed1.5 1.4 5.2 15.3 25.3

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 death5.7 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 died1 Data from Table 1 of Dick (1954).at lower radiation doses than those fed cabbage, Lourau and2 Change in the blood copper concentration of individual sheep overLartigue commented that only certain lesions differed betweena 4-d period; the mean initial concentration was 81 mcg/100 mL. The

daily intake of copper was 10 mg. 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 100Beets 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 betweentheir two explanations (toxic substances in beets vs. protectivehad more severe and widespread hemorrhages than those fed

cabbage. substances in cabbage).Spector and Calloway used an oat and bran control diet andThe authors proposed two explanations for the dietary effect

on radiosensitivity. They suggested that 1) cabbage may con- the control diet supplemented with beets, cabbage or broccoli.They irradiated their four groups of guinea pigs with a radiationtain substances that protect against radiation damage, such as

vitamins P and C; or 2) beets may contain substances that dose of 400 R. Their results are shown in Table 3. Althoughbeet consumption did not affect mortality, consumption ofbecome toxic after irradiation. They preferred the second ex-

planation, that beets were toxic to irradiated guinea pigs. This either cabbage or broccoli significantly reduced mortality fromirradiation. Thus, they proved that Lourau and Lartigue werewas supported by the observation that the LD50 for the animals

consuming beets was about 150 R, considerably lower than incorrect in their conclusion that beets contained a toxic sub-stance and that Duplan was correct as to the protective effectsthe usually reported LD50 of 250 R.

A few years later, M. Jean-Francois Duplan showed that in of cabbage.Calloway and colleagues went on to investigate the sub-fact, Lourau and Lartigue’s first explanation was correct, that

cabbage did offer protection against radiation damage (Duplan stance conferring the protective effects (Calloway et al. 1963).Because the basal diet was devoid of vitamin A, and animals1953). Duplan studied 70 male guinea pigs fed oat and bran

diets. The test diets were supplemented with either cabbage consuming this diet were known to become vitamin A defi-cient, they suggested that sources of vitamin A might be ableor carrots in this study, and the animals received a single

radiation dose. to decrease the radiosensitivity of guinea pigs. Their resultsare shown in Table 3. In this experiment, they confirmedThe results of Duplan are shown in Table 2. For a given

radiation dose, the animals consuming cabbage had much previous findings that both cabbage and broccoli lowered mor-tality in irradiated animals. They found that a number of otherlower mortality than those consuming carrots. He saw no dif-

ferences in lesions, but he did note that the animals consuming b-carotene–containing foods also exerted some beneficial ef-fects. Mortality after 20 d was significantly lowered by con-carrots lost much more weight than those consuming cabbage.

Duplan’s results suggested that Lourau and Lartigue may sumption of the b-carotene–containing vegetables that theytested. Beets, apples and white potatoes had no effect. Supple-have been incorrect in concluding that beets contained a toxic

substance. Rather than concluding that carrots contained a mentation with all essential vitamins reduced mortality some-what, but not to the same degree as supplementation withtoxic substance, Duplan concluded that cabbage lowered the

radiosensitivity of guinea pigs. He speculated that the radiopro-tective substances may have been antioxidant goitrogens pres-ent in the cabbage. TABLE 3

Spector and Calloway (1959) continued this investigationof the dietary factors that protect against radiation damage. Further experiments on the effect of diet on radiationThey very importantly noted that Lourau and Lartigue did not mortality in guinea pigshave a control group in their experiment. The addition of a

Mortality at 20 d after irradiationwith 400 R

TABLE 2Spector and Calloway et al.

Supplement Calloway (1959) (1963)The second experiment on the effect of diet on radiationmortality in guinea pigs1

mortality, %

Radiation dose (R)None 100 97Beets 90 —300 500 1000Cabbage 50 54Broccoli 35 42mortality, %Alfalfa 0Mustard greens 12

Cabbage 0 6.5 87.5 Green beans 31Carrots 50 86 — Lettuce 44

All vitamins 721 Data from Duplan (1953).




vegetables. Supplementation with pure vitamin A or b-caro- stract contained few details, and the work in question wasapparently never published as a full-length paper.tene alone had little effect on mortality.

Calloway and co-workers found that when an adequate pu- Physiologists interested in reproduction had demonstratedin the late 1940s that fetal blood of sheep contained a highrified diet was fed, the animals were somewhat resistant to

radiation damage, suggesting that part of the beneficial effects concentration of fructose (Bacon and Bell 1949, Barklay et al.1949, Hitchcock 1949), and Goodwin (1957) later showedof broccoli may have been due to improved nutritional status.

On the other hand, the beneficial effects of broccoli could that fetal blood of pigs was also rich in fructose, although thelevel in the blood of pigs 24 h after birth was very low. Alexan-not be duplicated by feeding a mixture of 48 chemically pure

ingredients patterned on the known composition of broccoli. der et al. (1955) identified glucose as the precursor of fetalfructose, with the site of conversion being the placenta. New-The experiments of the three groups considered together

suggested that a nonnutrient substance present in cabbage ton and Sampson (1951), though not mentioning it in theirpaper, must have assumed that fructose was an important en-and broccoli protected the guinea pigs from radiation damage.

Experiments performed by Calloway to isolate the substance ergy source for fetal pigs, and perhaps newborn pigs as well.They obtained pigs at birth and deprived them of food forfrom alfalfa found it to be present in the water-soluble fraction,

although the protective substance itself was not isolated. periods of 24 to 48 h, or until blood glucose had fallen to 20mg/100 mL or less. These hypoglycemic comatose pigs wereThese early experiments laid the foundation for subsequent

work showing the beneficial effects of fruit and vegetable con- then given intravenous injections of various sugar solutions.Glucose injection produced a dramatic resuscitative responsesumption in humans. Plants are now known to contain, in

addition to vitamins and minerals, many nonnutrient com- within a few minutes, and galactose gave a positive responsealso, although less immediate and less dramatic. Fructose injec-pounds that have important biological effects such as antioxi-

dant, anticarcinogenic, antimicrobial, antiinflammatory, anti- tion was without benefit, and none of the six pigs given fruc-tose were resuscitated. Taken together, the work of Johnsonviral and antimutagenic effects. These nonnutrient compounds

include lignans, indoles, coumarins and flavonoids, which in (1949) and Newton and Sampson (1951) suggested that babypigs could not effectively utilize either sucrose or fructose.1994 were reported to protect mice from the effects of radiation

(Shimoi et al. 1994). Perhaps these were the elusive substancesresponsible for the radioprotective effects first observed over The Key Feeding Experiments45 years ago.

In the early 1950s there was little interest in early weaningof pigs, and it was common practice to wean pigs at 8 wk of

Literature Cited age. Also, this period was marked by keen interest in thenewly discovered role of antibiotics and vitamin B-12 in pigCalloway, D. H., Newell, G. W., Calhoun, W. K. & Munson, A. H. (1963) Furthernutrition. Today, there is great interest in artificial rearing ofstudies of the influence of diet on radiosensitivity of guinea pigs, with special

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.

D. E. Becker at the University of Illinois published threeLourau, M. & Lartigue, O. (1950) The influence of diet on the biological effectsproduced 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) Radioprotec- sources for pigs ranging in age from 1 d to 16 wk (Becker andtive effect of antioxidative flavonoids in g-ray irradiated mice. Carcinogenesis

Terrill 1954, Becker et al. 1954a and 1954b). With pigs during15: 2669–2672.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 sugarcomponent representing 56.6 g per 100 g of dry ingredients(Becker et al. 1954b). Of the seven pigs fed each diet, six fedPaper 18: Toxicity of Sucrose and the sucrose diet died, five fed the fructose diet died, and only

Fructose for Neonatal Pigs (Becker et one fed the dextrose diet died. Among surviving pigs, thosefed dextrose gained weight, whereas those fed sucrose or fruc-al. 1954)tose lost weight. Pigs fed either sucrose or fructose also exhib-

Presented by David H. Baker, Department of Animal Sciences and ited severe diarrhea.Division of Nutritional Sciences, University of Illinois at Urbana- With pigs fed various carbohydrate sources (56.6 g/100 gChampaign, Urbana, IL 61801 as part of the minisymposium dry solids) during the period 7 to 35 d of age (Becker et al.‘‘Experiments That Changed Nutritional Thinking’’ given at Exper- 1954a), three of the eight pigs fed sucrose died, whereas mortal-imental Biology 96, April 16, 1996, in Washington, DC. ity was minimal in pigs fed lactose, dextrose, dextrin or corn-

starch. Although the surviving pigs fed sucrose gained bodyMcRoberts and Hogan (1944) fed a liquid synthetic milk weight as effectively as those fed the other carbohydrate

diet (30 g sucrose and 30 g casein per 100 g dry solids) to sources, the three pigs that died had severe diarrhea. Thisnewborn pigs and reported poor performance and diarrhea, experiment suggested that at least some pigs by 7 d of age candespite addition of ‘‘unrecognized factors’’ (vitamins) to the effectively hydrolyze sucrose in the gut and can also utilizediet in the form of brewer’s yeast or beef liver extract. Subse- both glucose and fructose for energy. Subsequently, Beckerquently, Bustad et al. (1948) tried a similar synthetic milk and Terrill (1954) demonstrated that 12-wk-old pigs couldformulation for newborn pigs, and even when lactose was sub- effectively utilize sucrose (50% of dry diet), but depressedstituted for sucrose, the pigs had severe diarrhea and failed to growth and moderate diarrhea resulted when the semipurifiedsurvive longer than 22 d. In 1949, S. R. Johnson presented an soybean meal diet contained 50% lactose.abstract at the FASEB meeting in Detroit wherein it was re-ported that 2-d-old pigs would thrive on a synthetic milk Enzymatic Development in Pigsdiet containing lactose or glucose but would experience severediarrhea and death when the diet contained sucrose as the The work of Bailey et al. (1956) and Walker (1959) with

small intestinal and pancreatic extracts from pigs at variouscarbohydrate source (Johnson 1949). Unfortunately, this ab-



1050S SUPPLEMENT

Bacon, J.S.D. & Bell, D. J. (1948) Fructose and glucose in the blood of fetalages showed that intestinal sucrase activity was extremely lowsheep. 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 diges-

wk 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.Intestinal lactase, sucrase and maltase. Can. J. Agric. Sci. 36: 51–58.the other hand, was high at birth but declined thereafter.

Barklay, H., Haas, P., Huggett, A.S.G., King, G. & Rowley, D. (1949) The sugarAherne 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

diet for the growing pig. Arch. Biochem. Biophys. 50: 399–403.utilize either sucrose or fructose, whereas 7-d-old pigs survivedBecker, D. E., Ullrey, D. E. & Terrill, S. W. (1954a) A comparison of carbohy-when fed these sugars, although weight gains were somewhat

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.

Becker, D. E., Ullrey, D. E., Terrill, S. W. & Notzold, R. A. (1954b) Failure of theor lactose. An intestinal loop in situ procedure together withnewborn pig to utilize sucrose. Science 120: 345–346.assessment of blood sugar concentration established that fruc-

Bustad, L. K., Ham, W. E. & Cunha, T. J. (1948) Preliminary observations ontose was absorbed from the gut intact, with little, if any, con- using a synthetic milk for raising pigs from birth. Arch. Biochem. 17: 249–260.version to glucose in the gut mucosa. This result was confirmed

Cori, G. T., Ochoa, S., Slein, M. W. & Cori, C. F. (1951) The metabolism ofby stomach tubing experiments wherein 3-, 6- and 9-d-old pigsfructose in liver: isolation of fructose-1-phosphate and inorganic pyrophos-showed 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 bloodsugar and some vital body functions in the newborn pig. J. Physiol. 136: 208–counted for a sizable portion of the fructose administered, par-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.Huber, J. T., Hartman, P. A., Jacobson, N. L. & Allen, R. S. (1958) Digestiveity was also low in 3-d-old pigs but tended to increase with

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.).

McRoberts, V. F. & Hogan, A. G. (1944) Adequacy of semipurified diets for theis caused by a low activity of intestinal sucrase. Also, pigspig. 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. Thelife they are poorly equipped to phosphorylate fructose in the effectiveness of certain sugars other than glucose in alleviating hypoglycemic

coma in fasting newborn pigs. Cornell Vet. 41: 377–381.liver (Cori et al. 1951) to facilitate its metabolism to triosePettigrew, J. E., Zimmerman, D. R. & Ewan, R. C. (1971) Plasma carbohydrateunits, hence energy. That 2-d-old pigs experience diarrhea

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 sugarsby the young dairy calf. J. Dairy Sci. 43: 546–552.is absorbed. Some thus may pass down to the lower gut where it

Walker, D. M. (1959) The development of the digestive system of the youngprobably causes a fermentative type of diarrhea. Interestingly,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 utiliza-tion 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 withregard to sucrose and fructose utilization (Velu et al. 1960). Paper 19: Discovery of the Vitamin DHuber et al. (1958) showed that young calves had very low Endocrine Systemactivities of intestinal sucrase.

Presented by Hector F. DeLuca, Department of Biochemistry,The Remaining Mystery University of Wisconsin-Madison as part of the minisymposium

‘‘Experiments That Changed Nutritional Thinking’’ given at Exper-Why is fructose virtually absent in maternal blood, whereasimental Biology 95, April 11, 1995, in Atlanta, GA.its level in fetal blood is higher than that of all nonfructose

reducing sugars combined, at all stages of gestation? The sheep In the early part of this century, during the discovery ofwork of Andrews et al. (1960) showed that perfused liver from the vitamins by McCollum and coworkers (McCollum andfetal and newborn lambs cannot metabolize fructose to carbon Davis 1913, McCollum et al. 1916) and by Osborne and Men-dioxide. If, as appears to be the case, fructose is not functioning del (1917), came the idea that rickets (a disease rampant inas an energy source for the fetus, what is its role and why is northern Europe and northern United States) might be a di-it 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 ques-a cellular constituent required in only microquantities? The tion whether the antirachitic activity might be due to the fat-work of White et al. (1982) with fetal pigs indicated significant soluble vitamin A, discovered by McCollum et al. (1916).recovery of [U-14C]fructose in muscle and liver RNA and However, McCollum and coworkers clearly demonstrated inDNA. This suggests that fetal fructose may serve as an im- 1922 that a separate fat-soluble substance was responsible forportant precursor of ribose-5-phosphate that is needed for nu- healing 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 andLiterature Cited Black 1924), the isolation and identification of the structures

of vitamins D2 (Askew et al. 1931) and D3 (Windaus et al.Aherne, F. X., Hays, V. W., Ewan, R. C. & Speer, V. C. (1969a) Absorption andutilization 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 andfructose in the fetal and newborn pig. J. Anim. Sci. 29: 906–911.

the mineralization of the skeleton (Nicolaysen and Eeg-LarsenAlexander, D. P., Andrews, R. D., Huggett, A.S.G., Nixon, D. A. & Widdas, W. F.(1955) The placental transfer of sugars in the sheep: studies with radioactive 1953). Kodicek and his group were pioneers in vitamin Dsugar. 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) Fruc- labeled vitamin D2 (1956). After a decade of investigation,tose metabolism in the isolated perfused liver of the foetal and newbornsheep. J. Physiol. 153: 199–208. this group concluded that vitamin D functions directly without




further metabolism (Kodicek 1956). As illustrated by a paper 1972, Holick et al. 1972), whereas its precursor (25-OH-D3)had little biological activity in the nephrectomized rat. Thisstating that vitamin D itself is responsible for its biological

activity in intestine (Haussler and Norman 1967), this idea provided clear evidence that the two-step process is requiredfor vitamin D activation for function. Another important proofpersisted throughout most of the 1960s.

The underlying key to success in this area must be attributed resulted when Fraser et al. (1973) demonstrated that vitaminD dependency rickets type I (an autosomal recessive geneticto the chemical synthesis of radiolabeled vitamin D of high

enough specific radioactivity to permit experiments with truly disorder causing severe rickets despite normal intakes of vita-min D) could be healed with the provision of physiologicphysiologic amounts of vitamin D (Neville and DeLuca 1966).

Biologically active polar metabolites of vitamin D were discov- amounts of synthetic 1,25-(OH)2D3, leaving no doubt of thetwo-step activation process in humans.ered (Lund and DeLuca 1966, Norman et al. 1964), and further

research demonstrated that the more polar metabolite not only While the identification of peak 5 proceeded, Boyle et al.(1971) found that the production of peak 5 metabolite in vivohad higher biological activity but also acted more rapidly (Mo-

rii et al. 1967). This provided the impetus for the isolation is strongly dependent upon the dietary calcium levels. Lowcalcium diets resulted in massive conversion to 1,25-(OH)2D3,and chemical identification of 25-hydroxyvitamin D3 (25-OH-

D3) in 1968 (Blunt et al. 1968) and its proof of structure by whereas high calcium diets and vitamin D adequacy resultedin the production of another metabolite that proved to bechemical synthesis that same year (Blunt and DeLuca 1969).

The chemical synthesis of this compound allowed for the intro- 24,25-(OH)2D3. Pursuit of this led to the demonstration thatthe parathyroid gland mediated this regulation (Garabedian etduction of a high specific activity tritium label in the side chain

of 25-OH-D3. This permitted experiments demonstrating that al. 1972). Thus, in response to low blood calcium, parathyroidhormone is secreted that in turn stimulates 1a-hydroxylase in25-OH-D3 rapidly disappears and is converted to more polar

metabolites (Cousins et al. 1970): Lawson et al. (1969) found the kidney to produce the vitamin D hormone. The vitaminD hormone together with parathyroid hormone then providesthat the intestine contained a major vitamin D metabolite

called ‘‘peak P,’’ which seemed to have lost some of its label for the mobilization of calcium from bone and renal reabsorp-tion of calcium, and 1,25-(OH)2D3 by itself provides for thethat was in the 1-position and was thus called a tritium-defi-

cient metabolite. This loss of tritium was not confirmed else- absorption of calcium and phosphorus (DeLuca 1974). Thus,the basic tenets of the vitamin D endocrine system were dis-where, but the existence of the intestinal polar metabolite

called ‘‘4B’’ was also reported (Haussler et al. 1968). covered from 1968 to 1973, as summarized in the FederationProceedings lecture delivered in 1974 (DeLuca 1974).Continuing isolation and identification of metabolites in

the DeLuca group resulted in the finding of at least three polarmetabolites, one of which proved to be 25,26-dihydroxyvita- Literature Citedmin D3 (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 D3 (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.Blunt, J. W., DeLuca, H. F. & Schnoes, H. K. (1968) Biochemistry 7: 3317–was not possible. It soon became clear that the functional

3322.metabolite in a target tissue could only be isolated with cer-Boyle, I. T., Gray, R. W. & DeLuca, H. F. (1971) Proc. Natl. Acad. Sci. U.S.A.tainty from that target tissue. Therefore, from the intestines 68: 2131–2134.Boyle, I. T.,Miravet,L.,Gray,R. W.,Holick,M. F.&DeLuca,H. F. (1972) Endocri-of 1600 vitamin D–deficient chickens given 3H-labeled vita-

nology 90: 605–608.min D3 came the isolation of what was termed ‘‘peak 5’’ byCousins, 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.(1973) N. Engl. J. Med. 289: 817–822.(Holick et al. 1971). From the fact that this metabolite was

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.

Haussler, M. R., Myrtle, J. F. & Norman, A. W. (1968) J. Biol. Chem. 243: 4055–graphed. Thus, the trimethylsilyl (TMS) derivative of the me-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.Holick, M. F., Garabedian, M. & DeLuca, H. F. (1972) Science 176: 1146–1147.alcohol on the 25-position (Holick et al. 1971). This wasHolick, 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

Metabolism (Wolstenhome, G.W.E. & O’Connor, C. M., eds.), pp. 161–174.chemical reactions were used to identify the structure as 1,25-Little, Brown and Co., Boston, MA.dihydroxyvitamin D3 [1,25-(OH)2D3] (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.

Lund, J. & DeLuca, H. F. (1966) J. Lipid Res. 7: 739–744.important discovery that their ‘‘peak P’’ metabolite (‘‘peak 5’’McCollum, E. V. & Davis, M. (1913) J. Biol. Chem. 25: 167–175.metabolite from the DeLuca laboratory and ‘‘peak 4B’’ fromMcCollum, 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.McCollum, E. V., Simmonds, N. & Pitz, W. (1916) J. Biol. Chem. 27: 33–43.important discovery was readily confirmed (Gray et al. 1971,Mellanby, E. (1919) Lancet 1: 407–412.Norman et al. 1971). The Kodicek group generated largeMorii, H., Lund, J., Neville, P. F. & DeLuca, H. F. (1967) Arch. Biochem. Bio-amounts of the metabolite in vitro, but it was of insufficient phys. 120: 508–512.Neville, P. F. & DeLuca, H. F. (1966) Biochemistry 5: 2201–2207.purity to allow identification. Proof of the 1,25-(OH)2D3 struc-Nicolaysen, R. & Eeg-Larsen, N. (1953) Vitam. Horm. 11: 29–60.ture came finally through direct chemical synthesis by a longNorman, A. W., Lund, J. & DeLuca, H. F. (1964) Arch. Biochem. Biophys. 108:procedure demonstrating that the configuration of the hy- 12–21.Norman, A. W., Midgett, R. J., Myrtle, J. F. & Nowicki, H. G. (1971) Biochem.droxyl on the 1-position is indeed 1a (Semmler et al. 1972).

Biophys. Res. Commun. 42: 1082–1087.Thus, the active metabolite of vitamin D proved to be 1a,25-Osborne, T. B. & Mendel, L. B. (1917) J. Biol. Chem. 31: 149–163.(OH)2D3. Furthermore, this metabolite proved to be equally Semmler, E. J., Holick, M. F., Schnoes, H. K. & DeLuca, H. F. (1972) Tetrahe-

dron Lett. 40: 4147–4150.active in nephrectomized or sham-operated rats (Boyle et al.



1052S SUPPLEMENT

Steenbock, H. & Black, A. (1924) J. Biol. Chem. 61: 405–422. In August 1969, John Rotruck, perusing the BiochemistrySuda, T., DeLuca, H. F., Schnoes, H. K., Ponchon, G., Tanaka, Y. & Holick, M. F. Journal, came across a paper by R. E. Pinto and W. Bartley(1970a) Biochemistry 9: 2917–2922.

(1969). The paper was on the effect of age and sex on glutathi-Suda, T., DeLuca, H. F., Schnoes, H. K., Tanaka, Y. & Holick, M. F. (1970b)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 glutathioneChem. 241: 100–103.metabolism that connected glutathione with the enzyme gluta-thione peroxidase. The reduced form of glutathione convertsPaper 20: Glutathione Peroxidase: hydrogen peroxide to water most efficiently when it is catalyzedby the enzyme glutathione peroxidase. A figure in this paperA Role for Selenium (Rotruck 1972)illustrated the proposed relationship of glutathione, glutathi-

Presented by Richard A. Ahrens, Department of Nutrition and one reductase, glutathione peroxidase and NADPH. It wasFood Science, College of Agriculture and Natural Resources, Uni- also noted that glucose metabolism is the main source ofversity of Maryland, College Park, MD 20742 as part of the NADPH. With this figure in front of him, John began tominisymposium ‘‘Experiments That Changed Nutritional Think- develop that same afternoon a hypothesis that selenium playeding’’ given at Experimental Biology 95, April 11, 1995, in Atlanta, a role in glutathione peroxidase. He generated a one-pageGA. research proposal that was presented to Hoekstra, complete

with misspelled words and typographical errors, that same af-The first practical biologic interest in selenium occurred internoon. Hoekstra said that, as far as he knew, this was thethe 1930s, when selenium was associated with alkali disease.first research proposal of an involvement of selenium withThis disease was called more graphically the ‘‘blind staggers’’glutathione peroxidase, and he reacted with enthusiasm. John,in certain northern range areas and resulted from seleniumhowever, had another year of heavy course work ahead of himpoisoning from forage grown on those shale soils. The standardbefore he began to fully implement his proposal in the summertreatment for such poisoning was to provide very smallof 1970. Over the next several months the actual experimenta-amounts of certain arsenic compounds in the feed or water oftion progressed rapidly, culminating in studies demonstratinglivestock.incorporation of 75Se into glutathione peroxidase. This portionHowever, K. Schwarz and C. M. Foltz (1957) reported thatof John’s Ph.D. dissertation took about 12 months to complete.traces of selenium prevented liver necrosis in certain vitamin

The first publication (Rotruck et al. 1971) to come fromE–deficient rats. Because vitamin E was known to be an anti-this research reported on a glucose-dependent protection byoxidant nutrient, a role for selenium in repairing or preventingdietary selenium against hemolysis of rat erythrocytes in vitro.oxidative damage was soon being sought. The first paper de-That was also the year that John Rotruck left the Universityscribing the enzyme glutathione peroxidase was also publishedof Wisconsin and joined Procter and Gamble, where he hadin the same year by G. C. Mills (1957). Glutathione peroxidasea long career from which he retired in 1995. An abstract fromwas reported to be an erythrocyte enzyme that protects hemo-that research was first reported at the 1972 FASEB meetingsglobin from oxidative breakdown. Nobody in 1957 linked(Rotruck et al. 1972a), and a subsequent publication on thethese two discoveries!prevention of oxidative damage to rat erythrocytes by dietaryIn 1968 John T. Rotruck was assigned to the research labo-selenium appeared later that year in the Journal of Nutritionratory of Professor W. G. Hoekstra to complete his Ph.D. stud-(Rotruck et al. 1972b). For this portion of the research, Johnies at the University of Wisconsin. As these things often hap-actually used the methodology from the original paper by Millspen, John Rotruck was initially assigned to work with another(1957) on glutathione peroxidase to establish that glutathionefaculty member who promptly went off on a sabbatical leave.peroxidase was virtually nonexistent in selenium-deficient rats.Hoekstra worked largely with the metabolic effects of zinc. He

These papers from Madison did generate some controversy,was no particular expert in selenium or glutathione peroxidase,because L. Flohe’ (1971), working in Germany, used a spectro-but he was familiar with the whole field of trace mineral re-photometric method to study bovine erythrocyte glutathionesearch. Although attempts to understand selenium’s mode ofperoxidase and reported that it contained ‘‘no non-proteinaction had focused on its potential role as an antioxidant,prosthetic group.’’ He responded negatively to the 1972 reportsthese theories had not been uniformly accepted. Indeed, Johnby Rotruck, but he did decide to recheck his earlier conclu-Rotruck recalls that some faculty members at the Universitysions. In the next year, the last part of John Rotruck’s disserta-of Wisconsin did not believe that selenium could be part oftion research was published in Science and demonstrated thean enzyme. In 1968 and 1969, he was engaged largely in takinguptake of 75Se by glutathione peroxidase and the fact thatclasses prior to the heavy research engagement that was toselenium was tightly bound to the enzyme (Rotruck et al.characterize his last two years in the Ph.D. program in bio-1973). The controversy ended when Flohe’ et al. (1973) pub-chemistry.lished a letter reporting that glutathione peroxidase was, in-Being an enthusiastic graduate student, John Rotruck readdeed, a selenoenzyme. The 1973 paper by Rotruck and co-the literature on glutathione biochemistry because of the simi-workers was identified as a Nutrition Classic in Nutrition Re-larities between selenium and sulfur chemistry and biochemis-views in 1980 and as a ‘‘Citation Classic’’ in Current Contentstry. Glutathione is the common name of g-glutamylcysteinyl-in 1988. A RDA for selenium was established in 1989, and inglycine. It is therefore a tripeptide composed of glutamic acid,1992 John Rotruck and Bill Hoekstra received the Klauscysteine and glycine. The sufhydryl group of cysteine is reactiveSchwarz Commemorative Medal for this work.and tends to form disulfide bonds with other glutathione mole-

John Rotruck recalls that it was the research environmentcules when oxidized. Glutathione functions as a reducing agentat his time in the Biochemistry Department at the Universityto maintain other molecules in the reduced form and canof Wisconsin that made such discoveries possible. Just acrossconvert hydrogen peroxide in the body to water. When it isthe hall from him, Hector DeLuca’s students were explainingoxidized to its double form linked by disulfide bonds, it canhow vitamin D worked. Down the hall from them was Johnbe reduced to the sulfhydryl form. The enzyme that does thatSuttie’s laboratory where the role of vitamin K was beingis called glutathione reductase, and it uses NADPH as a source

of hydrogen to achieve this reduction. explained. Nobel Prize winners came to speak on their research




Pinto, R. E. & Bartley, W. (1969) The effect of age and sex on glutathioneon a frequent basis. It just felt normal to him that a graduateperoxidase activities and on aerobic glutathione oxidation in rat liver homoge-student 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 pro-tection 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) Preven-tion 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 glu-selenoenzyme. 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 3rocyte enzyme which protects hemoglobin from oxidative breakdown in the against dietary necrotic liver degeneration. J. Am. Chem. Soc. 79: 3292–

3293.intact erythrocyte. J. Biol. Chem. 229: 189–197.



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Symposium: Experiments That Changed Nutritional Thinking