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Essays on Paths of Discovery in the Biomedical Sciences
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CHAPTER 3
BERIBERI AND THE COENZYME FUNCTION OF VITAMIN B1

The bacteriological triumphs of Louis Pasteur, Rudolph Koch, and their followers so caught the imagination of scientists and doctors in the last two decades of the nineteenth century and early years of the twentieth century that it was almost impossible to imagine a disease caused by anything but microbes or poisons. Medical research was primarily directed at the difficult but immensely satisfying tasks of tracking down the micro-organisms responsible for a variety of infectious diseases, isolating microbial toxins, and preparing vaccines and antitoxins. In 1890, when the Dutch government commissioned a team of physicians to investigate a disease which had begun to devastate the Dutch East Indian colonies about twenty years earlier, everyone fully expected to find a bacterium or parasite or, just possibly, some kind* of poison. The slow realization that the disease, beriberi, was not caused by some tiny organism or a potent toxin, but rather by the lack of minute quantities of an unknown and indispensable nutrient in the diet, required a major shift in medical and biological thinking. Over the first four decades of this century, research on beriberi and on vitamin B1 (thiamine)(1), both exemplified and helped to bring about remarkable changes in our assumptions about disease, nutrition, and the intimate workings of the living cell.

The number of vitamins, the variety of their physiological effects, and their confusing propensity for appearing in combinations make the history of their discovery a long and complicated story. R. R. Williams, who synthesized thiamine in 1936 and who then wrote the first book devoted to a single vitamin, used as a frontispiece to Vitamin B1 (Thiamin) and Its Use in Medicine a thick cable: each separate strand represented one of the various components of the vitamin B1 complex as they had been laboriously unraveled by medical, physiological, and chemical research between 1897 and 1938.

In this chapte rwe will trace only one of these strands, that involved with the discovery and identification of vitamin B1 and then with the elucidation of its biochemical role, as a coenzyme which functions in the metabolic breakdown of sugars.


Disease, Biochemistry, and Nutrition: 1900

Many physicians reacted with scorn when Pasteur promulgated the germ theory of infectious disease in the 1860's and 70's, but by the last decade of the nineteenth century the medical profession had largely adopted the new ideas. By 1900, when microbiology was a wellestablished discipline, biochemistry was just beginning to emerge as a field. The word itself had hardly been invented, and the first journals, societies, university departments, and classes in biological chemistry or biochemistry were founded only in the first decade of the twentieth century.

Biochemistry's most obvious progenitor was physiological chemistry, but the problems that the new science addressed were in some ways more general than those of physiological chemistry - as the bio prefix implied and ir, other ways much more specific. In particular, the mixed crew of physiological and organic chemists, medical researchers, pathologists, bacteriologists, and physiologists who came to call themselves biochemists paid special attention to the chemical processes that occurred within the living cell. The development of the new field was given impetus by several discoveries in the 1890's which suggested that cells produce catalysts which perform the chemical reactions of oxidation, fermentation, respiration, and synthesis inside the cell. Catalysts, or enzymes, which could start or accelerate the breakdown of compounds outside the cell had long been known, but in 1897 scientists first learned that the enzyme of fermentation, zymase, could be extracted from ground-up cells and used to ferment sugars. This discovery, which earned Edward Buchner one of the earliest Nobel Prizes in Chemistry in 1907, showed that the chemical components of the cell could be studied piece-meal, that the cell was not a single structural unit which would lose all its physiological properties the moment its membrane was broken (Kohler 1970, pp. 162-178; Kohler 1971, pp, 35-61; Kohler 1973, pp. 181-196).

The two major problems that the new science of biochemistry set out to solve were the chemical structure and role of enzymes, and the chemical properties [END OF PAGE 27] of protoplasm. The cell was commonly viewed as a bag of protoplasm and protoplasm was believed to be a colloidal, jelly-like chemical compound of immense size and complexity, and of such fragility that it had to be studied intact. This mysterious substance somehow absorbed and incorporated nutrients, synthesized all manner of products, respired and fermented, grew and reproduced. Biologists and chemists were daunted by protoplasm, and any attempt to analyze it was considered foolhardy by many.

However, Buchner's extraction of zymase as well as the work being done on toxins and antitoxins suggested to biochemists that at least some of the cell's chemical components did have what was then understood to be molecular structure. These compounds and the reactions in which they participated could be studied in cell extracts in the test tube. However, the difficulties that early biochemists faced were tremendous. Their extracts were crude; methods of extracting cell consitutents were harsh; techniques for analyzing the extracts were arduous and time-consuming. Even the basic chemical nature of enzymes was a matter of great debate, for many biologists and chemists did not believe that enzymes were proteins even after James Sumner in 1926 purified and crystallized the enzyme urease, and showed that it was a protein (Fruton 1972, p. 156 ff).

The first generation of biochemists spent little time studying nutrition on the cellular and intracellular level, although for at least a quarter of a century nutrition had been one field where physiology and organic chemistry had come together. In the late nineteenth century, research in nutrition had followed two main lines: determining the energy values of foodstuffs, and trying to devise the physiologically and economically optimal balance of "the nutritional trinity" of protein, carbohydrate, and fat (Armsby 1906, quoted in McCollum 1957, P. 153; lhde and Becker 1971, pp. 20-26). To early twentieth-century nutrition researchers, the measurements of human energy needs and of the caloric content of foods were extremely impressive, because they provided strong quantitative data that seemed to demonstrate a "real measure of nutritional needs, independent of, and apparently superior to, considerations based upon chemical details (Hopkins 1929, pp. 211, 216). In contrast, analysis of the proportions of the elements in different foodstuffs to obtain a "proximate analysis" of their protein, carbohydrate, fat, mineral, and fiber content seemed to be leading nowhere, although over 55; lhde and Becker 1971, pp. 20-26).

Both kinds of nutritional research ignored a point which was becoming more and more obvious in practical animal feeding experiments: the theoretical categories of nutrients were too poorly defined to be useful. Even research on minerals, which were obviously chemically distinct and comparatively easy to study, was hampered by assumptions about the nature of life and protoplasm. Bunge, whose students turned out so many papers on mineral nutrients, firmly believed that animals could only use the more complicated organic mineral compounds produced by plants. Bunge's view followed from the general feeling that animal protoplasm was chemically so complicated and unique a substance that it could only make use of organic substances like itself. This assumption made Bunge ignore the implications of work done by two of his students, Lunin and Socin, who came close to proving the existence of unknown nutrients later named vitamins -.simply because the synthetic diets Lunin and Socin had tried on mice included inorganic salts rather than the vital organic compounds (Hopkins 1929, pp. 213-214).


Beriberi, Chickens, and Rice

The disease beriberi has long been known in the Far East, for there is a Chinese record of a beriberi epidemic dating from 2697 BC (Williams and Spies 1938, p. 17). In the early seventeenth century, a Dutch physician, Jacobus Bontius, described the symptoms of beriberi which he had observed in himself and his patients in Java, and a similar disease named ship beriberi had been known to attack Western sailors on long voyages (Bontius 1642, pp. xxi-xxiii, 106, 111). In the latter half of the nineteenth century, beriberi became a major public health problem in Asia. It became endemic in many countries, and was, especially apt to devastate groups of people who were compelled to live, sleep, eat, and work together, such as prisoners, sailors, hospital and lunatic asylum patients, and mothers and their infants. In any of the forms it took, beriberi was a ravaging disease. Some victims suffered from acute burning sensations on the skins of their feet and from grave weakness or paralysis in the legs as they wasted away to mere skeletons. For others, the emaciation muscular atrophy was disguised by the swellings of edema. Still others, babies especially, died suddenly of [END OF PAGE 28] acute heart failure. Many patients suffered all the various manifestations in succession, and all too often the disease was fatal.

The military were particularly alarmed by the ravages of beriberi in army camps and on navy vessels. A young medical officer in the Japanese navy, K. Takaki, heard from his father how beriberi had killed many men of the Imperial Palace Guard in 1862, and in his own work at the Naval Hospital, Takaki sometimes found as many as three-quarters of the patients ill with beriberi. "Such conditions," Takaki wrote in 1906 when he explained why he had devoted so much of his life to the study and eradication of beriberi, "used to strike my heart cold when I came to think of the future of our Empire, because if such a state of health went on without discovering the cause and treatment of beriberi, our navy would be of no use in time of need" (Takaki 1906, p. 1369; Williams 1961, pp. 19-25; Harris 1938, pp. 52-56). Takaki's fears were shared by the Dutch colonial authorities in Indonesia - who were losing so many soldiers to beriberi that they could not win a guerilla war against Surnatran rebels - and by the American colonial authorities in the Phillipines - who watched helplessly as a tenth of the native police force came down with beriberi every year (Jansen 1956, p. 260; Williams 1961, p. 4). For such reasons, governments thought it important to find out what caused and what might cure beriberi.

Since the disease obviously seemed to be contagious, its cause was assumed to be a micro-organism. The first task thus was to isolate the bacterium responsible, and Takaki spent five years (1875-1880) in England learning bacteriology for this purpose alone. The starting point for the ultimately successful Dutch investigation also was bacteriological. In 1886 the Dutch government appointed a pathologist, Pekelharing, and a neurologist, Winkler, as a commission to study the destruction of the peripheral nervous system by beriberi and to search for the infectious agent. Before going to Java, Pekelharing and Winkler stopped in Berlin to visit Koch's lab and learn about the latest developments in bacteriology. There they met Christian Eijkman, a young Dutch doctor on leave from the Dutch East Indies colonial army, who asked to join the commission as an assistant. In the colonies, the commission soon believed they had found a coccus in beriberi patients which could not be found in healthy people. Although they published this conclusion, Pekelharing still had doubts as to whether they really had isolated the bacterial cause of beriberi. A young medical officer, J. van Eecke, who worked closely with the commission and did bacteriological research on his own, put it very bluntly: "Whether the micro-organism we found is the pathogen or not is a question that must be settled unequivocally. The short history of bacteriology is already over-rich in examples of premature conclusions" (van Eecke 1887, p. 85).

To decide the question according to the rigorous canons of bacteriology he had learned from Koch, Eijkman remained in Djarkarta as a civilian medical researcher after Pekelharing and Winkler went home to the Netherlands in 1888. Eijkman began by trying to give animals beriberi, inoculating them with infectious matter from beriberi patients in the hospital. For his experimental animals, he made a somewhat unorthodox but fortunate choice. He used chickens, probably because they were cheap, easy to obtain, and easy to care for, but also perhaps because he had heard that, during an outbreak of beriberi in the Moluccas, large numbers of the birds and hens had also suffered from the disease (Jansen 1959, pp. 70, 74). The bewildering variety of forms that beriberi took presented Eijkman with a problem: how would he know if his hens had caught beriberi? He decided to look for evidence of degeneration of the peripheral nerves (polyneuritis), a prominent and painful symptom of human beriberi. But for several months the hens showed no signs of anything resembling beriberi (Jansen 1956, pp. 260-65).

Like Pasteur, however, Eijkman was prepared to take advantage of accidents and "chance" observations. One day in 1889 he found his chickens sick with something that closely resembled beriberi. The signs of polyneuritis were especially plain: the unhappy birds staggered and collapsed, their wings drooped in partial paralysis, their heads and necks were pulled sharply backwards by peculiar muscular spasms. But it seemed unlikely that the inoculations were responsible, for the inoculated chickens and the uninoculated controls were equally sick and nearly three-fourths of each flock died. When Eijkman examined the dead birds, he found that both groups showed the distinctive signs of nerve degeneration that he knew so well from his microscopic studies of nerves from beriberi victims. Six months later, the few birds who survived what Eijkman cautiously called polyneuritis gallinarum (polyneuritis of chickens) recovered quite suddenly and did not relapse.

On investigating the sudden outbreak of polyneuritis and its equally sudden and mysterious disappearance, Eijkman discovered that the chickens' diet had been [END OF PAGE 29] changed twice. When he began his unsuccessful inoculations, the birds were fed crude unmilled rice. Then the servant in charge of feeding them begged table scraps of polished white rice from the hospital; after a few weeks of eating this rice, the birds fell ill. Later, when a new hospital cook "refused to allow military rice to be taken for civilian chickens," their feed again consisted of the crude, unpolished rice (Eijkman 1929, p. 203). Soon afterwards the chickens got well and stayed healthy. Moreover, sick birds recovered when they were fed the rice polishings and rice germ. Clearly, something about the white rice caused polyneuritis gallinarum, and something in the red skin and germ of the unmilled rice prevented and cured the disease.

Eljkman's "lucky accident" and his simple test of his observation gave him a new freedom in his work on beriberi, for he could now produce the disease at will in his experimental animals. This still left him open, however, to the criticism that chicken polyneuritis was not the same disease as human beriberi, a problem that was met when Eijkman took advantage of a "natural experiment" with Javanese prisoners. He asked the medical inspector of the prison system to collect information about the incidence of beriberi in the island's jails and about the kinds of rice their 300,000 prisoners ate. The inquiry revealed that in jails where prisoners milled their own rice by hand according to their local custom, the inmates came down with beriberi at only 1/300th the rate found in jails where prisoners were given industrially milled polished white rice. Indeed, all but one of the 37 jails where prisoners prepared their own rice were completely free from beriberi (Eijkman 1929, pp. 205-206). To Eijkman, although not to the prison authorities or other experts in tropical medicine, the results seemed unequivocal.

When Eijkman tried to explain why polished rice should be so dangerous and unmilled rice with its germ and skin intact should be so beneficial, he fell back on the prevailing assumptions of bacteriology. If the cause of a disease was not a micro-organism, he reasoned, then there must be a toxin which was neutralized by something in the red skin and germ. Eijkman continued to believe for many years that the starchy white grain contained an active toxic substance, a belief strengthened by his showing in 1896 that beriberi was not caused by a dietary protein deficiency.(2)

In 1896 Gerrit Grijns, a young surgeon who had served periodically as Eijkman's assistant since 1893, succeeded Eijkman as director of the Djarkarta laboratory. The Dutch East Indies government asked Grijns to "investigate the physiological and pharmacological properties of the tannin contained in red rice, and possibly other constituents of this kind of rice which might require consideration in relation particularly to beriberi" (Grijns 1935, p. 1). He was assured that he should not restrict his research to this problem, but instead should extend Eijkman's results to discover more about the connection between diet and polyneuritis and about the anti-beriberi properties of red rice.

Grijins continued the work on chicken polyneuritis, first by finishing Eijkman's incomplete experiments on "salt-starvation" as a possible explanation for the harmful white rice diet. Over the next four years, he improved Eijkman's experimental techniques, and he explored many ideas. Did the origin of the rice make a difference? Was the loss of fats contained in the rice skin significant? Was the starch of rice, sago, and tapioca really positively harmful, as Eijkman had concluded in his 1895 and 1896 reports? What was the active substance in rice skin? Could other foods cure beriberi as effectively as rice skin and rice germ?

In casting about for other foods that prevented or cured beriberi, Grijns happened on a kind of pea, Phaseolus radiatus (Katjang idjoe in Malayan), which was often used in Java to supplement chicken-feed. The results of tests with this kind of pea in birds who were so ill with beriberi that they had to be fed by hand were striking; within three or four days they could feed themselves, and within ten days they could walk easily again. Both the skin and kernel of the pea were equally efficacious, but the peas lost their curative and preventive powers when cooked in steam at 120'C. The discovery of the peas' effectiveness led to a series of experiments which neatly disproved Eijkman's theory of toxins produced by starches, by showing that polyneuritis could be produced in pigeons and fowl with a diet that contained no starch or carbohydrate at all.

Grijns' report in 1901 on his five years of work with nearly two hundred fifty birds began by recapitulating and arguing against theories of a bacterial or toxic cause of beriberi. At the same time, he stressed that his work strengthened Eijkman's important finding that chicken polyneuritis and human beriberi were the same. He concluded with the first clear statement of the existenc: of previously unknown "protective substances" whose absence from the diet led to "partial starvation." [END OF PAGE 30]

There occur in various natural foods, substances, which cannot be absent without serious injury to the peripheral nervous system. The distribution of these substances in the different food-stuffs is very unequal. Of those examined, phaseolus radiatus and cajanus indicus were the richest, and polished rice the poorest, in these substances. The separation of these substances meets with the difficulty that they are so easily disintegrated. This disintegration, which takes place in a damp, warm place, shows that they are very complex substances. They cannot be replaced by simple chemical compounds (Grijns 1935, p. 38).

To judge by the dates of Grijns' various experiments, he had had serious doubts about Eijkman's toxin theory Within a year of starting his research, and he probably hypothesized the existence and distribution of the iprotective substance early in 1898. His experiments between 1898 and 1901, designed to clear up details about the chemical constituents of substitues for rice skin and the rice skin itself, were founded on Grijns' belief that a new vital dietary substance was involved.

Grijns' first paper had little effect when it was published in 1901. Both Eijkman and he published their results in the medical journal of the Dutch East Indies and summarized them in the leading medical journal of the Netherlands; but their articles were in Dutch, which severely limited their audience. Moreover, as late as 1906 Eijkman was still hesitant about accepting Grijns' "partial starvation" theory, for he would not discard his own toxin hypothesis until he had repeated all the experiments on potato starch and peas (Eijkman 1906, pp. 156-58). (it thus is interesting to see how carefully Eijkman avoided discussing his long-held toxin and antidote theory in his 1929 Nobel Prize lecture; he only spoke about the "anti-neuritic principle" of the rice skin, not at all about the poisons in the starchy grain! ) Only two Norwegian researchers, Holst and Frolich, who were studying ship beriberi, tried to follow up Eijkman's and Grijns' research immediately. Believing that tests on mammals would be more instructive than tests on birds for explaining human pathology, they chose the guinea pig as their experimental animal. Their results were unexpected; they were able to produce scurvy, but not beriberi, and switched their research to take advantage of this finding (Holst 1907, pp. 619-633).

Although little attention was paid to Eijkman's and Grijns' work at the time, the problem of beriberi was not being ignored. During the first decade of the 20th century, several other physicians in Asia arrived more or less independently at the connection between a diet of polished rice and beriberi, and provided valuable new evidence for what would become the vitamin theory. Braddon, an English doctor, observed striking differences among four cultures living in Malaya in regard to their use of rice and their incidence of beriberi. The Malays who milled their rice at home by hand, the Tamils who parboiled their rice before removing the rice skin, and the Europeans who did not eat rice were all free from beriberi, but the Chinese who ate imported rice suffered severely from the disease. Braddon believed that a toxin, perhaps from a saprophyte, formed in white rice as it grew stale. His theory was tested by Fletcher on patients in a lunatic asylum, where nearly half the patients had beriberi and a quarter of those died of it. He confirmed the relationship between the rice diet and the disease, but left the explanation open.

In 1907, Fraser and Stanton tried different kinds of rice diets on Javanese laborers who were building roads in isolated areas of Malaya. After finding that a white rice diet did indeed produce beriberi, they tried something that, rather surprisingly, Eijkman and Grijns had not done: they tried to extract the hypothetical toxins from white rice. In addition, they showed that if an alcoholic extract were made of parboiled rice, the rice after extraction was no longer any better than milled white rice. But the extract could be fed along with polished rice and prevent the beriberi that the polished rice diet would usually cause. Fraser and Stanton concluded that the polished white rice lacked "some substance or substances essential for the normal metabolism of nerve tissues" - a clear statement of the deficiency theory (Williams 1961, pp. 43-49). Fraser and Stanton presented their work at the first meeting of the Far Eastern Association of Tropical Medicine in 1910, at which time the association resolved that: "sufficient evidence has been produced in support of the view that beriberi is associated with the continuous consumption of white (polished) rice as the staple article of diet, and the Association accordingly desires to bring this matter to the notice of the various governments concerned" (Williams 1961, pp. 48-49).

Fraser and Stanton's report was heard by an American, Vedder, who also knew of Eijkman's and Grijns' [END OF PAGE 31] work. Vedder enthusiastically adopted Fraser and Stanton's deficiency theory because he had already seen that Grijns' results implied a kind of nutritional deficiency that was simpler and more fundamental than that implied by Griins' phrase "protective substances." To help him find out exactly what the essential substances in extracts of rice skin or parboiled rice were, Vedder recruited a young chemist at the Manila Bureau of Science, R. R. Williams. Vedder's enthusiasm was contagious, and for years Williams worked in his spare time until he at last succeeded in isolating and synthesizing the active anti-beriberi principle (Williams 1961, pp. 1,48-50,95 ff).


Rats, Milk, and Minimal Diets

While physicians in Asia were proving a strong connection between disease and diet and formulating the new theory of nutritional deficiencies of unknown substances, a different kind of research in the United States and England yielded other important evidence of the need for accessory food factors. For these workers, the impetus for undertaking such research did not come out of a desire to understand a particuiar disease, although several of them did have practical applications in view. They hoped to determine the simplest diet for farm animals and experimental animals and to use that knowledge as the basis for further nutritional experimentation. By 1906, there were already about a dozen papers scattered throughout the literature and published in at least four different languages, which described chemically simplified or synthetic diets. As a rule, the authors of these papers knew little, if anything, about one another's work, and very often they did not follow up on their own observations. Looking back, we can see that they were on the right track, but they had no way of being sure at the time.

The great French chemist, Dumas, for example, attempted to invent a substitute for mother's milk during the siege of Paris in 1870. The disastrous effects of his mixtures of emulsified fat and sweetened protein - in theory a complete food - led him to conclude that a vitally important ingredient was lacking, but Dumas did not try to identify the differences between real milk and his artificial concoction. The work by Lunin in 1880 and Socin in 1891 on inorganic minerals and salts in the diet also suggested that milk and egg yolks contain more than just the basic protein, fat, carbohydrate, and minerals. They used very small numbers of experimental animals, so their results were not conclusive; and their professor, Bunge, as we have mentioned, discouraged them on theoretical grounds from continuing (Ihde and Becker 1971, pp. 11-12). Ironically, the one scientist who did take Lunin and Socin's work seriously was C. A. Pekelharing, the Dutch pathologist who had sought a bacterial cause of beriberi in 1888. In 1905 he published the results of several years of research which demonstrated the existence of -an unknown substance in milk which even in very small quantities is of paramount importance to nourishment. If this substance is absent, the organism loses its power to assimilate the well-known principal parts of food. . Undoubtedly this substance not only occurs in milk, but in all sorts of food stuffs both of vegetable and animal origin" (Pekelharing 1905, p. 122).

In Great Britain the most important work on simplified diets at the beginning of the twentieth century was done by Frederick Gowland Hopkins, one of the most influential figures in the history of vitamin research and biochemistry. As a young man, Hopkins trained for a career as an "analyst," which then meant a professional analytic chemist. At the age of 28, he decided to study medicine and as a medical student began doing valuable research in physiological chemistry and pathology. As he recalled in his 1929 Nobel Prize address, however, his clinical work excited an interest in nutrition:

Early in my career I became convinced that current teaching concerning nutrition was inadequate, and while still a student in hospital in the earlier eighteen nineties I made up my mind that the part played by nutritional errors in the causation of disease was underrated. The current treatment of scurvy and rickets seemed to me to ignore the significance of the old recorded observations. I had then a great ambition to study those diseases from a nutritional standpoint (Hopkins 1929, p. 217).

Rather than going into clinical medicine, Hopkins took up an unexpected offer in 1898 to "develop ... teaching and research in the chemical side of physiology" at Cambridge (Needham and Baldwin 1949, p. 20). Sixteen years later, the university founded a chair of biochemistry, appointed Hopkins the first professor, and put him in charge of the new department of biochemistry

.

Hopkins' research in his first years at Cambridge concentrated on proteins and amino acids. "I realized," he said in 1929, "as did many others at the last century's close, that for a full understanding of so many other [END OF PAGE 32] aspects of biochemistry, further knowledge of proteins was then a prerequisite; and . . . I did my best to contribute to that knowledge" (Hopkins 1929, p. 217). By capitalizing on his discovery of an impurity in acetic acid while teaching a laboratory session on proteins, Hopkins was able to isolate and identify and identify tryptophane, an amino acid whose existence had only been postulated before then. Hopkins followed this achievement by showing that the protein of maize, zein, lacked this ismino acid. More important still, he found that an artificial diet which contained adequate amounts of fats, carbohydrates, and minerals with zein as the only protein, could not keep mice alive; as soon as tryptophane was added to supplement the zein, the mice survived much longer.

Hopkins realized clearly and quite early in his work on proteins that a variety of nutrients were required for health and life. In an interesting speech to the Society of Public Analysts in 1906, "The Analyst and the Medical Man," Hopkins stressed that:

No animal can live upon a mixture of pure protein, fat, and carbohydrate, and even when the necessary inorganic material is carefully supplied the animal still cannot flourish. The animal body s adjusted to live either upon plant tissues or on the tissues of other animals, and these contain countless substances other than the proteins, carbohydrates, and fats. Physiological evolution, I believe has made some of these well-nigh as essential a; are the basal constituents of diet.

He cited lecithin as one such substance that was already known, and pointed to rickets and scurvy as diseases in which empirical dietary cures had been found long ago. These illnesses and other less obvious or severe "nutritive errors" were, Hopkins insisted, certainly due to "minimal quantitative factors" in the diet. He asked the analytic chemists to help the doctors by identifying and isolating these "unknown substances with unknown properties, present in complex mixtures." His audience's response to this appeal was disappointing; only one chemist addressed himself to the question of diet and disease, and then simply to point how impossible difficult, expensive, and time-consuming such analysis of foodstuffs would be (Hopkins 1906, pp. 394-96, 401).

At the time of this speech, Hopkins had been using simplified diets to test the qualitative effects of different proteins and amino acids on the growth of young rats. Although the addition of tryptophane to zein lengthened survival time, the rats still did not grow properly. By substituting casein (milk protein) for zein, by adding a small quantity of milk to the simplified diet, or by using yeast extracts to make the dull, tasteless diet more appetizing, he could bring the young rats' growth patterns back to normal. From 1906 to 1912 Hopkins tried to understand how these small changes in the diet could make so great a difference in the rats' growth. For a number of reasons, however, he published nothing and only gave a couple of talks on his experiments before 1912 (Hopkins 1929, p. 218; Dale 1948, pp. 130-131 ).(3)

While Hopkins moved from studies of the nutritive values of different proteins to the inadequacies of synthetic diets, American researchers at the Wisconsin and Connecticut agricultural stations undertook similar research. Like Hopkins, they began with the assumption that all proteins do not possess equal food value. At the Connecticut Agricultural Experiment Station in New Haven, Thomas Osborne began preparing pure vegetable proteins and analyzying their amino acid content from the 1890's on. A young chemist from Kansas, Elmer V. McCollum, came to Yale in 1904 to do graduate work in organic chemistry. He specialized in physiological chemistry, took courses on nutrition from Lafayette Mendel and Russell Chittenden, the leading American authorities, and worked on amino acid determinations at Osborne's laboratory. McCollum recalled hearing Mendel lecture on Hopkins' work on supplementing zein with tryptophane in 1906-07; he also remembered that none of his teachers ever mentioned beriberi, scurvy, rickets, or pellagra (McCollum 1953, p. 301).

In 1907, while Osborne and Mendel proceeded with tests of the nutritive values of their purified proteins, McCollum went to the University of Wisconsin's College of Agriculture and Research Station to perform chemical analysis of cattle feeds. McCollum did a thorough search of the literature on animal nutrition and collected accounts of other attempts to restrict diets in order to determine the requirements for particular substances. He was "astonished to find that every effort which had been made to feed animals on such mixtures (of isolated proteins, carbohydrates, fats, and mineral-salt mixtures) had resulted in prompt failure of their health," and he realized then "that the most important discovery to be made in nutrition would be the elucidation of the cause or causes of these failures" (McCollum 1953, pp. 304-05).

For a number of reasons, the cattle-feeding experi-[END OF PAGE 33]ments that McCollum was to conduct proved impractical. And so, in 1907, he set up the first rat colony in America for nutritional studies. McCollum kept in touch with his Yale professors and in 1909 Mendel and Osborne also began using rats as experimental animals for their protein tests.

Like Hopkins, McCollum worried about the palatability of the insipid simplified diets he used on his rats, and tried to add flavor and variety to their diet. In so doing, he unwittingly saved his rats from vitamin deficiencies by allowing them to eat whey -contaminated lactose and their own feces - both sources of vitamins not otherwise provided by the simplified diet. In a 1913 paper, McCollum and his co-worker, Margaret Davis, showed that fats, or something associated with fats, extracted by ether from butter or egg yolks, contained something necessary to the growth of young rats. As long as the diet consisted of casein, carbohydrates, salt, and lard, they were able to keep the rats alive and growing for a few weeks, after which the rats did not gain any more weight. But the addition of ether-extracts of butter or yolk to the diet at this point would set the rats growing rapidly (McCollum and Davis 1913).

Meanwhile, at the Connecticut research station, Osborne and Mendel had been working along similar lines; establishing a basic diet of "protein-free (skim) milk," carbohydrates, salts, and lard, and then testing the effects of different proteins. But until 1912 they were unable to get rats to grow full size on their basic diet (Osborne and Mendel 1911, pp. 618-698). Then, in desperation, they reconsidered every constituent of milk, realized that all their milk substitutes and diets lacked the cream of milk, and tried replacing lard with butterfat. The results were so immediately successful that they began to write up the new experiments for publication (Osborne and Mendel 1913a, b). To their chagrin, however, their rivals McCollum and Davis won what had been a conscious race between the Connecticut and Wisconsin researchers. In the summer of 1913 Osborne wrote to Mendel, "I have just received the July issue of the Journal of Biological Chemistry and notice McCollum's article in it, which I assume you have seen. If not, I might say that he could just as well have taken his data from our notebooks as from his" (Becker 1970, p. 158-59).


The "Vitamine" Theory: 1911-1920

Hopkins, McCollum and Davis, and Osborne and Mendel gave a new direction to nutritional studies by their agreement that complete growth required the food factor in milk, by their demonstration of the value of rats as experimental animals, and by their use of growth rate as the measure of dietary deficiency. But much of the force of their papers came from their explicit recognition that their work and the research on beriberi and scurvy were closely allied. For this idea, they were indebted in some measure to Casimir Funk, a young Polish chemist working in England, who had begun to study the beriberi-preventing substance in rice skin and yeast in 1911. Funk's review of the literature, his bold speculations about the nature of deficiency diseases, and his invention of the word "vitamine" as a name for the mysterious food factors implicated in deficiency diseases, all argued that there was an underlying unity to the diverse studies of beriberi, scurvy and the growth promoting factor in milk. In effect, Funk did what Hopkins had failed to so with the inssights so cleasly expressed in the 1906 speech to the analysts: he provided a theory of nutrition and disease that was as dramatic and as amenable to scientific verification as Pasteur's germ theory of infectious disease. From 1912, when the new word first appeared in print, to the 1940's, vitamin research was as exciting a field as bacteriology had been in the late ninteenth and early twentieth century.

After receiving his Ph.D. in organic chemistry in 1904, and then studying under the noted biochemist, Gabriel Bertrand, at the Institut Pasteur in Paris, Funk did research on amino acids and proteins in synthetic diets for dogs with the physiological chemist, Abderhalden. In 1910, an English friend in Abderhalden's laboratory found Funk a job at the Lister Institute in London, where his attention was soon directed to the problem of beriberi (Harrow 1955, pp. 35-39).

C. J. Martin, the first director of the Lister Institute of Preventive Medicine, was interested in tropical diseases and encouraged his friend Braddon's work on beriberi in Malaya. Martin also was a close friend of Hopkins, and knowing Hopkins' research on tryptophane deficiency in zein, Martin guessed that the cause of beriberi might be some other sort of amino acid deficiency in polished rice (Chick 1956, pp. 134-39, 197-98). Since Funk had experience with amino acid determinations, Martin suggested that he analyze the amino acid content of the rice and rice skins Braddon had sent to the Institute (Harrow 1955, p. 39). At the time, Martin's amino acid hypothesis was as plausible as any other theory about beriberi - and there were a good many. Although there was considerable agreement [END OF PAGE 34] by 1911 that eating too much polished rice caused beriberi (witness the resolution of the Far East Association of Tropical Medicine), the reason for the ill effects of polished rice and the good effects of the rice skin was by no means settled. In addition to the toxinantidote theory held by Eijkman, Braddon, and others, and the protective substance-deficiency theory held by Grijns and Fraser and Stanton, there were other reasonable suggestions: a deficiency of fat, a deficiency of phosphorus, a deficiency of nitrogen, an excess of carbohydrate, a lack of a particular phosphorus-containing substance called phytin (Williams 1961, pp. 14-15).

When Funk and the first of several collaborators, E. A. Cooper, set to work analyzing extracts from rice skins in 1911, they had very little idea what to look for. The extant literature, they remarked, gave very few clues to the chemical nature of the protective substance from rice polishings, except that it seemed to be neither a salt or a protein.

Cooper and Funk's first experiments sought to learn whether the substance could still cure beriberi after it was subjected to extensive chemical manipulations, whether it had a simple or complicated chemical structure, and whether it belonged to a known class of chemical compounds. They quickly showed that "it is very improbable that polyneuritis is the result of a deficiency in phosphorus compounds." The active extract they obtained also lacked carbohydrate or protein groups, although it did include a significant amount of nitrogen. And, as others had found, very small amounts of the extract sufficed to cure beriberi in pigeons (Cooper and Funk 1911, pp. 1266-67; Funk 1911, p. 400; Funk 1912a, p. 149).

Funk coined the word "vitamine"(4) for his 1911 paper in the Journal of Physiology to describe the nitrogen-containing substance he and Cooper had extracted from yeast and rice skin. But the staff at the Lister Institute, to whom he was required to submit drafts of his research papers, and the editors of the Journal of Physiology disliked the new word. Their opposition was quite sensible: the substance had not been proved to be an amine or amino acid - as the word implied - and, in general, the indiscriminate coining of words in science led to confusion. Funk was not obliged, however, to show drafts of review articles to his colleagues at the Institute, and at the suggestion of a fellow Polish scientist, he wrote an article on "The Etiology of Deficiency Disease" for the widely read Journal of State Medicine in 1912 (Harrow 1955, pp. 41-43). Here he was free to introduce and explain what he meant by his catchword, "vitamine."

It is now known that all of these diseases (beriberi, polyneuritis, epidemic dropsy, scurvy, experimental scurvy in animals, infantile scurvy, ship beriberi, pellagra), with the exception of pellagra, can be prevented and cured by the addition of certain preventive substances; the deficient substances, which are of the nature of organic bases, we will call "vitamines;" and we will speak of a beriberi or scurvy vitamine, which means a substance preventing the particular disease (Funk 1912a, p. 164).

Although Funk did not give the etymology of "vitamine" in this paper, it clearly was a compound of vita, "life" in Latin, and amine, a nitrogenous base. Thus, a "vitamine" meant a nitrogenous amine which is necessary to life: a much larger claim than "a substance preventing the particular disease," and a claim that made Funk's colleagues at the Institute and on the Journal of Physiology uneasy. In later years Funk protested that he did not mean that all vitamines had to be amines, but this point was not clear in his early papers and caused considerable confusion (Funk 1922, p. 169, n. 1).

In his essay of 1912, Funk also surveyed the evidence for grouping human and experimental beriberi, scurvy, and ship beriberi under the general category of "deficiency diseases," and perspicaciously argued that pellagra and possibly rickets also belonged to this class although the vitamines involved were probably different from those of beriberi and scurvy. He told of the attempts by himself and other investigators to extract the active substance, the beriberi vitamine, from rice skins, yeast, and Katjang idjoe beans. Speculating on the possible chemical and metabolic relationships between the beriberi vitamine and the scurvy vitamine, it seemed likely to him that the animal body could transform the scurvy vitamine into the beriberi vitamine, but not vice-versa. And, based on what he knew of the experiments by Osborne and Mendel and by Hopkins with simplified diets supplemented by milk, Funk supposed "that the substance facilitating growth found in milk is similar, if not identical, with the vitamines described by me" (Funk 1912a, p. 169).

The reactions to Funk's work were mixed. In general, his attempts to isolate the active substance in rice skin were greeted with enthusiasm. Only a few weeks after [END OF PAGE 35] Cooper and Funk's preiiminary 1911 communication on rice-skin extracts in Lancet, a column in Lancet described how a German explorer in New Guinea had successfully prevented beriberi among the members of his expedition by eating a pottage of red rice and Katjang idjoe beans cooked together every day. The anonymous Lancet writer remarked on the new progress of knowledge about the disease:

In future we are to expect that, thanks to the work of Mr. E. A. Cooper and Dr. Casimir Funk (The Lancet, Nov. 4, p. 1266), the leader of an expedition will be able to take all the special substance required to keep beriberi away from his men in a one-ounce bottle in his pocket. Of such are the triumphs of medicine. (Anonymous 1911c)

But the proposal of the new word, vitamine, and its theoretical implications aroused much controversy. During the next ten years Funk campaigned vigorously on behalf of his idea. In 1914 he published a long German monograph, Die Vitamine, which was revised and translated into English in 1922. Although he moved several times - from England to Canada, from Canada to the United States, then to Poland, to Paris, and back to the United States in 1939 - Funk continued to do research on vitamines and to publish papers on his work in English, German, and Polish (Harrow 1955, pp. 5598). Less than five years after Funk coined the word, "vitamine" became a widely used and accepted term in scientific and popular writing. In September 1915, for example, Scientific Amerkan published a condensation of an article on "Vitamines and Their Importance for the Maintenance of Health." A year later an editorial in Science, "A New Phase in the Science of Nutrition," described the progress of vitamine research and showed how well Funk's term caught on:

The word "vitamine" has come into our vocabulary since the latest dictionaries were published. Etymologically it means an amine that is essential to life, and it was coined by C. Funk as a generic name for a group of substances, of unknown chemical composition, small quantities of which appear to be a necessary constituent of a wholesome human diet . . . An absence or insufficiency of vitamines in the diet brings on diseases now known as "avitaminoses" or "deficiency diseases," of which scurvy and beriberi are the principal representatives. Science already recognizes two vitamines - viz., antiscorbutic vitamine, which prevents scurvy, and antineuritic vitamine, which prevents beriberi in man and polyneuritis in birds. There may be others.

The investigation of the vitamines has made great strides in the past two years. The subject is beginning to crop up in the newspapers and in general literature, not to mention the small talk of the dinner table, where everything on the menu invites classification from the point view of the "vitaminologist." (Science 1916 p. 453)

By the early 1920's writers were using the the term metaphorically: "A book . . . . so full of the vitamines of literature," "The vitamines of the spirit and. . . true religion" (Oxford English Dictionary Supplement, 1933). But, although widely used, the word and the concepts it stood for still upset many scientists. As late as 1948, the eminent physiologist and pharmacogist, Sir Henry Dale, regretted that Hopkins had not used "the right, which would have been generally accorded to him (in view of his contribution to vitami research)" to suppress so inappropriate a term (Dal. 1948, p. 131). Osborne and Mendel, in their second 1913 paper, "The Influence of Butter-fat on Growth," wrote that butterfat probably did contain "something analogous to the so-called vitamines which Funk considers to be necessary for life," but they saw some important objections to Funk's generalization.

Without minimizing the importance of the new field of research and the new viewpoints in nutrition which are presented by these recent findings, we may nevertheless hesitate to accept the extreme generalizations which have already been proposed on the basis of the evidence obtained largely from the investigation of pathological conditions. . . It is still rather early to generalize on the role of accessory "vitamines" when the ideal conditions in respect to the familiar fundamental nutrients and inorganic salts adequate for prolonged maintenance are not completely solved (Osborne and Mendel 1913b, pp. 429-30).

They argued further that a substance which maintained health, like Funk's beriberi and scurvy vitamines, might be a very different kind of thing from their butterfat accessory factor which promoted normal growth. In any case, their butterfat growth -promot i ng substance was not nitrogenous - the one chemical characteristic Funk seemed to insist upon by his use of the suffix amine. [END OF PAGE 36]

McCollum made similar criticisms in his popular lectures on nutrition in 1918. He disliked the vita prefix because it implied that vitamines were more essential to life than, for example, the indispensable amino acids. And, McCollum felt, Funk had exaggerated the number of deficiency diseases, erred in his chemical description of the curative substances, and foolishly denied the significance of the fat-soluble substance. The popularity of Funk's word, McCollum said bluntly, was deplorable: "There has become fixed in the minds of students of nutrition and of the reading public an altogether extravagant idea regarding the importance of the substances to which Funk gave the name 'vitamines' " (McCollum 1918, pp. 84, 113-14). Hopkins and Abderhalden preferred other terms to express the idea of indispensable dietary constituents of undetermined chemical composition: "accessory food factors," "food hormones," "nutramines" (Needham and Baldwin i1949, p. 166; Abderhalden 1919, p. 39).

Despite (or perhaps because of) these disagreements about words and the things they stood for, more and more scientists became interested in the nature of vitamins and in the practical applications of what McCollum in 1918 called "the newer knowledge of nutrition." Funk's former colleagues at the Lister Institute (he had taken an appointment at the London Cancer Hospital in 1913) made vitamin research their main contribution to the war effort: they studied the quantitative distribution of vitamins in different foods; and, in response to an epidemic of beriberi among Australian soldiers stationed on Lemnos, they prepared vitamin-concentrates from yeast and eggs to supplement army rations. Funk's assistant, Jack C. Drummond, performed some early experiments on the effects of vitamins on tumor growth rates and worked with Hopkins on the wartime Food Committee of the Royal Society, making recommendations about food rationing and vitamin enrichment of margarine. In the United States, McCollum, with various collaborators, continued his research on rat diets, trying to distinguish among the various indispensable food complexes by their different physiological effects. R. R. Williams was given unofficial permission by his biochemist-chief at the Food and Drug Service to work part-time with Atherton Seidell on the isolation and synthesis of the beriberi vitamin, even though "at that time, of course, vitamins were not recognized as having a legitimate place in food chemistry" (McCollum 1918, passim; Williams 196 1, p. 107).

In the Netherlands and Java, Eijkman and his coworkers continued their studies of beriberi and the active substance in rice skins; similar research was underway in Japan. Pellagra and rickets, which Funk and Hopkins had guessed might be due to vitamin deficiencies, received new attention from physicians, scientists, and public health services in American and Britain.

In 1920 Jack Drummond helped lessen the remaining disagreement in the new field by proposing a reform of nomenclature. He acknowledged the great convenience of having a single word to name all of the "so-called accessory food factors" and noted the confusion that accompanied the proliferation of synonyms for Funk's vitamines. But the word vitamine, despite its wide adoption by 1920, was still unfortunate because "the termination '-ine' is one strictly employed in chemical nomenclature to denote substances of a basic character, whereas there is no evidence which supports his (Funk's) idea that these indispensable dietary constituents are amines" (Drummond 1920, p. 660). Drummond suggested that the final e in "vitamine" should be dropped. The result, "vitamin," would fit under the Chemical Society's nomenclature rule, which allowed "a neutral substance of undefined composition to bear a name ending in '-in'." The various individual vitamins could then be called by the letters of the alphabet which had already been used to differentiate among them: vitamin A, vitamin B, vitamin C. Funk opposed the change because, he said in 1922, "I still believe in the nitrogenous nature of these substances," and he continued to call them by the name he had invented until 1937 (Funk 1922, p. 39 n. 2; Harrow 1955, p. 200). Otherwise, the new spelling was adopted rapidly, although, as we shall see, the designations of individual vitamins presented other difficulties.

If the "young science of vitamins" needed further legitimation it was provided in 1929 by the award of the Nobel Prize in Medicine and Physiology to Christian Eijkman and Sir Frederick Gowland Hopkins for their "discoveries of the antineuritic and the growth-promoting vitamins .... which .... are foundation stones of the science of vitamins" (Liljestrand 1929, p. 198). Hopkins used the occasion to recount the early history of vitamin studies, paying tribute to the foreshadowings of Lunin, Socin, and Pekelharing, noting Grijns' correct interpretation of Eijkman,s experiments on chicken beriberi, and assessing the work and writings of Casimir Funk.

The award to Hopkins must have made Funk bitterly angry. Three years before, Funk had protested in a letter [END OF PAGE 37] to Science that Hopkins did not deserve to be called " the discoverer of vitamines," that Hopkins' 1912 paper came "so late that it exerted a relatively small influence on the development of the whole subject," and that Hopkins' earlier work, described in the 1906 speech, had been unknown to other workers until 1912 (Funk 1926, pp. 455-56). Hopkins' conclusion in the Nobel Prize lecture that Funk had "not received too much, but too little credit for his vitamin research as a whole" was hardly consoling, since Hopkins still claimed priority for his own experiments and ideas on the physiological functions of vitamins (Hopkins 1929). As in all priority disputes, both sides had justification for their rival claims to recognition. It is idle for us to try to apportion credit or judge the relative significance of their various contributions, not to mention those of their predecessors like Pekelharing and Eijkman or their contemporaries like McCollum and Osborne. But we should underscore one point: the rapid acceptance of the idea of vitamins was probably as much due to Hopkins' and Funk's enthusiastic, skillful evangelizing as to the two biochemists' actual scientific work on the chemistry and physiology of accessory food factors (Becker 1970, pp. 159-161).


The Race for Vitamin B1

No doubt scientists would have tried to isolate, analyze, and synthesize the anti-beriberi factor in rice skins and the growth-facilitating factor in butterfat even if there had been no theory linking the two, for it was clear that each of these substances was of physiological and medical importance. But the vitamin hypothesis made it all the more imperative to obtain the substances in pure form. Only then could one know whether the crude extracts contained more than one active substance, whether vitamins did constitute a new class of chemical compounds (as Funk seemed to assert), and whether the various vitamins all performed similar physiological functions. At least four different research teams were engaged in the effort to isolate the beriberi vitamin, a task of very great difficulty. Then, once the pure vitamin had been isolated, it was possible to work out its correct structure. Once the structure was known, the synthesis of vitamin B1 in turn was a comparatively simple albeit also lengthy task.

R. R. Williams, who was deeply involved with every stage of this endeavor, estimated in 1938 that no other substance in the history of biochemistry had cost so much to isolate and identify as vitamin B1: "The first gram of pure vitamin must have cost an aggregate of several hundred thousand dollars" (Williams and Spies 1938, P. 138). That cost is only one measure of the importance, both theoretical and practical, that scientists attached to gaining knowledge of the anti-beriberi substance. The quest for vitamin B1 served as a school for biochemists from 1912 to 1940. "To mention all the names of those who have participated in some phase of the project," Williams wrote, "is to call the roll of half the mature biochemists in England and the United States. The project bulked equally large upon the horizon of Dutch, Japanese . . . French (and, somewhat later, German) biochemistry. . . (Williams and Spies 1938, p. 138).

The high cost in labor, time, and materials was largely due to the nature of the vitamin itself. To those who worked on its isolation, the vitamin's ability to act effectively in very small doses raised two immediate problems: the anti-beriberi factor was to be found in nature only in very small quantities, and its presence in crude extracts was hard to assay.(5) The enormous quantities of rice skin or yeast that it took to yield even a tiny bit of the vitamin astonished everyone who worked on the problem. For example, Jansen and Donath, who carried on the long tradition of Dutch beriberi research in Eijkman's laboratory in Djarkarta, began with 100 kilograms of rice bran to obtain 100 milligrams of pure vitamin (Jansen 1956, pp. 274-277).

The problems involved both in extracting a tiny bit of vitamin from tons of rice bran or from yeast and in measuring its ability to cure beriberi were complicated by a confusion that was as much a dispute over theory as it was a problem with chemical techniques. During the first years of vitamin research, the leading researchers could not agree how many vitamins there were. Funk, for instance, believed for several years that only the beriberi vitamin and the scurvy vitamin fit his criteria for a vitamin. Accordingly, he at first rejected McCollum's fat-soluble growth factor because it did not cure beriberi (or any other known deficiency disease) and because it was not an amine (Funk 1922, p. 117; Funk 1925, pp. 157-58). McCollum, in turn, insisted that only his fat-soluble growth factor, "fat-soluble A", which did cure an eye disease, and Funk's anti-beriberi factor, "water-soluble B", were true vitamins. His early experiments led him to assert that scurvy, rickets, and pellagra were not vitamin deficiency diseases (McCollum 1918, pp. 30 ff). But the nature of vitamin B - as Drummond had renamed the beriberi vitamin and [END OF PAGE 38] "water-soluble B" - was itself in question. McCollum showed in 1918 that "water-soluble B" was as necessary to the growth of rats as "fat-soluble A". Was vitamin B then a single substance with several distinct physiological functions, or was vitamin B a mixture of different active substances?

Early in the 1920's Eijkman and Jansen, among others, declared their belief that the water-soluble growth factor which could be extracted from yeast was not identical to the beriberi-preventing factor (Williams and Spies 1939, p. 130). Jansen and Donath clearly made this a working assumption in their choice of an assay for vitamin B - only the cure or prevention of beriberi could serve as a valid test for the presence of the beriberi vitamin. Their decision was vindicated in 1926 by Smith and Hendrick, who demonstrated that the anti-beriberi factor in yeast could not survive pro- longed heat, while the rat-growth factor in yeast did retain its activity after heating (Williams and Spies 1938, pp. 130-31). Thus, vitamin B was not a single substance but rather a complex of at least two vitamins: the thermolabile, anti-neuritic factor (that would be called vitamin B1, aneurin, or Thiamine), and the thermostabile, growth-promoting factor (Dutcher 1928, 6 pp. 206-209).(6)

Despite the many problems of technique and theory, Jansen and Donath succeeded in isolating almost pure crystals of the beriberi vitamin in 1926. Williams, who also was working intensively to isolate the vitamin, regarded this feat as a "landmark . . . . example of a systematically planned and executed pursuit of a chemical objective" (Williams and Spies 1938, p. 139). After eight years of labor, the Dutch chemists obtained 100 milligrams (1/280 of an ounce) of the almost pure substance. They sent "some scores of milligrams" to Eijkman, who had the pleasure of confirming their claim that this substance was the vitamin and that astonishingly small amounts of it could cure or prevent beriberi in birds (Jansen 1956, p. 274-77).

Jansen and Donath's long and tedious work had shown that the anti-beriberi vitamin could be isolated from natural sources, and, they believed, they also had determined its correct empirical formula. Other groups immediately set out to repeat and modify the isolation process, and to check Jansen and Donath's analysis of thiamine's chemical components. In 1931, a team of chemists at Gottingen led by A. Windaus found that Jansen and Donath had made a critical error in their analysis: they had overlooked the presence of sulphur in the thiamine molecule. As Williams commented, "What a shock it must have been to Jansen and Donath to learn of this mistake after all the years of grueling work they had expended on the isolation!" (Williams 1961, p. 117).

Two years later, in 1933, successful isolation was also reported by R. A. Peters and his colleagues in England, and by Williams' group in the United States. Williams' group confirmed Windaus' empirical formula, and, by several innovations in the isolation process, succeeded in improving the yield of pure crystalline thiamine by four or five fold.

Now an intense race began to determine the structure of thiamine and then to synthesize the vitamin (see Williams 1961; Wuest 1962). The race was triggered not only by the desire of chemists to understand how thiamine was made and to synthesize it, gaining undeniable prestige in the process. The pharmaceutical industry knew that the commercial rewards, too, would be high for the company that acquired patent rights to manufacture the anti-beriberi vitamin. Thus, impelled by a variety of motives, three major groups of researchers and pharmaceutical companies pursued the structure and synthesis of thiamine: R. R. Williams et al. in the U. S., at first independently, but later supported by Merck and Co.; A. R. Todd and F. Berjel in England, sponsored by the Swiss company, Hoffman-La Roche, Ltd; and, in Germany, H. Andersag and K. Westphal in the Elderfeld Laboratories of the giant chemical firm, I. G. Farben.

Scientific priority in the race fell to R. R. Williams and his colleagues in the U. S., who first published the complete synthesis procedure in August 1936. Not having realized that his work on thiamine had significant commercial interest, Williams was surprised to find that he was engaged in a highly competitive race, and even more surprised to find himself embroiled in a legal dispute over patent rights with I. G. Farben. Williams and his colleagues had assigned the patent to a nonprofit Research Corporation, to insure that the profits from the manufacture of vitamins would be used to support scientific research and to eradicate dietary diseases. The courts ultimately awarded the patent rights in the United States and Canada - which soon became the largest market for vitamin B1 in the world to Williams et al., Research Corporati6n, and its two licensees, Merck and Co. and Hoffman-La Roche. After eight years of effort, we recall, Jansen and Donath in 1926 had finally extracted 100 milligrams of crystalline [END OF PAGE 39] thiamine from natural sousrces. Could they, one wonders, have envisaged that by 1950 the U. S. manufacturers of synthetic thiamine would be producing 100 metric tons a year!

Nomenclature continued to be a problem for several more years after thiamine had been analyzed and synthesized. Vitamin B1 was a widely accepted term, but seemed to belong too much to the era of confusion over the heterogeneity of vitamin B. Jansen proposed "aneurin" as an abbreviation for "anti-neuritic vitamin," a word still used in the British Pharmacopeia. But the American Council of Chemistry and Pharmacy of the American Medical Association rejected this name because it made a therapeutic claim for the substance, and because it could be confused with "aneurism." The Japanese vitamin researchers tended to use the name "oryzanin," while R.A. Peters in England preferred "torulin" - words that referred to the source of the vitamin in rice and yeast. In 1937 R.R. Williams suggested that, since "aneurin" had met with such objections in the United States, it might be convenient and acceptable to use a name which reflected the vitamin's chemical peculiarities: "thiamin," where the thiaprefix referred to the sulphur atom and thiazole ring (Williams and Spies 1938, pp. 134-35). Later, the American Chemical Society noted the presence of an amino group in the molecule and changed the name to "thiamine" - a small indirect victory for Casimir Funk's original nomenclature.


Thiamine, Coenzymes, and Carbohydrate Metabolism

While organic chemists were hard at work isolating, analyzing, and synthesizing pure vitamin B1, physiologists and biochemists were busy trying to understand the vitamin's role in metabolism. Unfortunately, the organic chemists and the biochemists and physiologists of the 1920s and 30s could not help each other with their tasks, for the chemical structure of the vitamin offered no clues to its functions, and vice versa. There was no chemical test for the presence of vitamin B1, for instance, that would determine its distribution in the tissues, and the low yields of the early isolation procedures forestalled any systematic physiological experimentation with the pure crystals. As a result, the concurrent investigations of the chemistry and physiology of vitamin B1 did not converge until 1937, when the newly synthesized vitamin was used to confirm the identity of thiamine with cocarboxylase, a coenzyme which had just been shown to play a major role in carbohydrate metabolism.

Almost as soon as an accessory food factor was postulated as the cause of berioen, scientists suggesting a variety of ways that such a factor might act in metabolism. Eijkman's original hypothesis was that it neutralized toxins produced by starches. The edema suffered by many beriberi patients made some observers think that the vitamin was involved in water metabolism, while others felt that the vitamin was involved in the body's ability to use phosphorus. Funk's discovery that the vitamin contained a pyrimidine ring suggested to him that the vitamin might have something to do with nucleic acids, although the functions of the latter were completely obscure at the time. The rapidly accumulating knowledge of hormones and their importance in metabolism made other early researchers, such as Hopkins, Schaumann, and Funk think that vitamins might work as "exogenous hormones," that is, hormones that had to be supplied by nutriments rather than by biosynthesis within the body. Somewhat akin to this idea was Hopkins' early view of "nutritive errors": the effects of the missing food factors were comparable to the "inborn errors of metabolism" described by his close friend, Sir Archibald Garrod (see Chapter 5). Thus, the accessory food factors might be necessary at particular stages of metabolic pathways, although Hopkins could not specify whether the factors served as catalysts or substrates (Hopkins 1906, p. 396). In 1911 Funk suggested that vitamins might serve as "mother-substances," precursors to other essential metabolites (Funk 1914, p. 6). Because such small amounts of vitamins had such striking effects, Seidell among others speculated that they might be related to enzymes (Seidell 1924, p. 440).

Funk and others considered yet another possibility, which proved to be the best guess about the function of the beriberi vitamin. In 1914 Funk observed that if pigeons on a vitamin-free diet were fed extra carbohydrates, they developed polyneuritis more rapidly. Experiments on pigeons fed with various combinations of carbohydrates and vitamin extracts suggested that vitamin-free artificial diets with a high proportion of carbohydrates cause a "marked disturbance of the carbohydrate metabolism" which could be quickly reversed by the vitamin. Braddon and Cooper came to similar conclusions at the same time (Funk and Schbnborn 1914, pp. 328-33 1).

Although the idea that vitamin B1 was somehow involved in carbohydrate metabolism gave a focus to research, it was not as helpful as the vitamin researchers must have hoped, for carbohydrate metabolism was [END OF PAGE 40] in itself one of the central problems of biochemistry. A common refrain in historical accounts of developments in biochemistry is, "the process was turning out to be much more complex than had been imagined ten years before" (Fruton 1972, p. 343; Peters 1939, 1071, note). In the case of carbohydrate metabolism and thiamine, it took several decades before the varied rimental observations of fermentation, glycolysis, respiration, and vitamin activity could be sorted into a coherent scheme for the breakdown of sugar and the tapping of its energy.

Two quite different lines of biochemical work led to the conclusion in 1936-37 that vitamin B1 acted as a coenzyme in the reactions of pyruvic acid, an important intermediate in both the anaerobic fermentation of sugar to alcohol and carbon dioxide, and the aerobic breakdown of sugar in respiration to carbon dioxide and water. The research by Karl Lohmann, which culminated in the isolation of cocarboxylase and the recognition of this coenzyme as the phosphoric ester of thiamine, was part of a long, intensive investigation into what is now called the Embden-Meyerhof pathway: the anaerobic degradation of glucose to pyruvic acid, and thence to ethanol and carbon dioxide in yeast (fermentation), or to lactic acid in muscle (glycolysis). The second line of research, that by R.A. Peters and his collaborators, was specifically aimed at understanding the cell's need for thiamine: they wanted to pinpoint the lesion caused by the vitamin deficiency, but they also realized that their work ought to cast some light on the details of carbohydrate metabolism.

Karl Lohmann worked as a senior staff member in Otto Meyerhof's research laboratory from the mid-1920s to 1936, where his ability as a superb organic chemist beautifully complemented Meyerhof's keen scientific imagination in their studies of glycolysis, muscle contraction, and fermentation (Proc. Conf. Hist. Devel. Bioenerg. 1973, pp. 70-73, 169). Among his many accomplishments, the achievement for which Lohmann is best known was his isolation of adenine triphosphate (ATP) in 1929. Later, ATP was shown to be the compound which trapped the free energy to drive other reactions in the cell. Although Lohmann recognized some of the implications for energy transport by ATP, he was chiefly interested in the compound as a coenzyme required for the transfer of phosphate to and from intermediates in the pathway of glycolysis and fermentation (Cori 1973, pp. 163, 166; Fruton 1972, pp. 366-69).

The question Lohmann wanted to answer once he had isolated ATP was: what does the coenzyme ATP have to do with the coferment of zymase discovered by Harden and Young in 1906?(7) It was through the pursuit of this question, in a series of complex biochemical studies, that Lohmann and Schuster in 1937 announced the purification of a new coenzyme, cocarboxylase, and proved that "the organic 'ground substance' of cocarboxylase, is diphosphorylated aneurin (vitamin B1;" that is, "cocarboxylase is diphosphorylated aneurin (vitamin B1)" (Lohmann and Schuster 1937, p. 300).

Unfortunately, it is not clear from Lohmann's account of his investigations when or how he first suspected the identity of the coenzyme as thiamine diphosphate. The publicity that had been given to R. R. Williams' recent analysis of thiamine's structure, and the subsequent race to synthesize the vitamin may well have alerted Lohmann to compare the two compounds. Obviously, only the availability of pure crystals of thiamine from natural sources and from Williams' synthesis made a definitive identification possible.

There was another reason, though, for Lohmann to think of his coenzyme in terms of vitamins. Between 1932 and 1935 Warburg, Kuhn, and Theorell had shown that riboflavin (vitamin B2 in one nomenclature) was the coenzyme for the so-called "yellow enzyme" which, according to Warburg, shuttled hydrogen from the oxidation of sugar to the respiratory chain to react with oxygen (Ball 1973, pp. 98-99). Lohmann mentioned the work on riboflavin in the discussion part of his cocarboxylase paper and drew attention to another similarity between the two vitamins: not only were they both the "ground-substances" of coenzymes, but they also needed phosphate groups to be added onto the vitamin molecule before they could act as coenzymes. It was a point that the discoverer of ATP was bound to notice. It is also proof that Lohmann did not yet know that the nicotinic acid amide in Warburg's other coenzyme, DPN, was the anti-pellagra vitamin although Warburg himself had suggested this identity. When he first crystallized nicotinic acid amide crystals from DPN in 1934, Warburg declared, "I am quite sure this will turn out to be a vitamin." But despite his research on the coenzyme function of riboflavin, Warburg was not interested enough in vitamins to follow up his hunch about nicotinic acid amide (Theorell 1962, p. 2). Thus, it was not until late 1937, after Lohmann and Schuster's paper on cocarboxylase, that Conrad Elvehiern published his identification of niacin as yet [END OF PAGE 41] another vitamin which acts as a phosphorylated structure in a coenzyme. Not surprisingly, the identification within so short a span of time of three members of the vitamin B complex as parts of coenzymes involved in major metabolic pathways suggested that all vitamins have coenzyme functions, setting in motion a great deal of new biochemical research.

The second major line of research leading to the identification of vitamin B1's coenzyme function was that pursued by R.A. Peters and his colleagues at Oxford. While Peters and Lohmann arrived, independently, at the same conclusions about thiamine's role in intermediary metabolism, their work was strikingly different in both techniques and goals. Karl Lohmann, as we have noted, was an organic chemist, whose work relating to vitamin B, was part of a complex series of basic biochemical analysis being conducted by Meyerhof's laboratory group. R.A. Peters, on the other hand, had been trained as a physician, and for personal and professional reasons "could never forget the hospital and the wish to improve care for people" (Peters, personal communication). Peters saw his research on thiamine as an attempt to explain the pathology of vitamin deficiency in biochemical rather than histological or anatomical terms. He introduced the striking phrase "biochemical lesion," to "crystallize the idea that pathological disturbances in tissues were initiated by changes in their biochemistry" (Peters 1963, p. 1). Although the ultimate aim of Peters' research was to pinpoint a derangement in a biochemical pathway, his use of pigeons as an assay forced him to keep in mind the gross consequences of the biochemical lesion upon the whole organism.

After completing his clinical studies, Peters served as a medical officer in France until he was recalled to England at the request of the eminent physiologist, Sir Joseph Bancroft, "to work on antidotes to gas poisoning" (Peters, personal communication). It was in this particular context of war-impelled research that Peters' attention was first directed to vitamin B1. In 1920, working in Hopkins' laboratory at Cambridge on the effects of poisons on protozoa, Peters found that his protozoa would not grow on their artificial medium unless an alcoholic extract of yeast was added, and thus his interest was directed to vitamin B1. Beginning in 1922, and continuing after his appointment as professor of biochemistry at Oxford in 1923, Peters began the effort to isolate vitamin B1 from bakers' yeast.

"When Jansen and Donath published their fascinating isolation from rice polishings," Peters recall, "I decided to continue our work with yeast, because it would become necessary ultimately to be sure that the vitamin B1 in yeast was the same compound as that in rice polishings. And, we realized early that the Jansen and Donath crystals were not quite pure" (Peters, personal communication). Thus, along with the Winclaus group in Germany and Williams and Seidell in the United States, Peters' group pursued and eventually accomplished the goal of obtaining pure vitamin B1 extract from yeast.

From 1922/23 on, Peters states, his work on vitamin B1 "was part of a larger plan," one inspired both by his clinical training and his work as a biochemist. "I thought that by isolating one factor dealing with a specific clinical condition, I could begin to clear up the muddle as to how many B factors existed" (Peters, personal communication). Within this framework, the problem of testing the vitamin activity of the yeast extract fractionations led Peters to the question: how did the vitamin deficiency produce the dramatic symptoms of beriberi? He and his colleagues felt that the cure of polyneuritic symptoms in pigeons was a more certain sign of vitamin activity than either the prevention of symptoms or the progress of normal growth. But, as Williams and others had objected, it was all too easy to produce false cures or remissions by injecting the sick birds with glucose and water. So, during the mid-1920s, Peters sought a reliable test of a cure. Opisthotonus, the convulsive neck spasm which pulls the head of a pigeon sharply back, seemed to be the most obvious and important clinical sign of a genuine vitamin deficiency. "Hence," Peters recalls, "I sat down to make the pigeon opisthotonus test quantitative and reliable. This I succeeded in doing by standardizing conditions of feeding, and by never using birds which took over a month to develop the head retraction, when other deficiencies seemed to intervene" (Peters, personal communication)' If the opisthotonus appeared after a pigeon had lived for a couple of weeks on a polised rice diet, Peters found, it could often be relieved for a day or two by glucose and water. But, if the neck spasm re-appeared within a month or less on the deficient diet and could no longer be relieved by glucose, then the only cure was the vitamin (Peters 1963, pp. 6-8).

Study of the head retraction symptom, and their observations of pseudo-cures with glucose, led Peters [END OF PAGE 42] the next stage of research:

the convulsive state induced by the deficiency drew my attention to the central nervous system and provided the stimulus to examine this for possibly enzymic changes; until then attention had been focused upon the peripheral nervous system, which had become emphasized wrongly through the extensive use of the word "Polyneuritic." (Peters 1963, pp. 8-9).

The temporary "cures" produced by glucose suggested at the glucose might counteract a low blood sugar el in the sick birds. Comparison of the blood sugar level in sick and normal pigeons did not give any real answer, so Peters tried to lower the blood sugar in healthy birds by insulin injections. As others had also discovered, Peters reported in 1929, he found

that ordinary doses of insulin had no effect upon the birds. Large doses generally gave convulsions .... to my surprise the convulsions exactly resembled those of avitaminosis . . . . So far as I know this has not been previously pointed out. In view of this fact, Mr. Kinnersley and I were led to examine systematically the various features of the carbohydrate "cycle" in the brains of avitaminous birds. (Peters 1929, p. 272).

By 1936, the study of the "carbohydrate cycle" undertaken by Peters and his associates had yielded important findings about the function of thiamine. He and his co-workers had shown as well that biochemistry made a "new approach to pathological analysis" possible. Working with rather large amounts of wet tissue rather than with the cell-thick sections used by histologists, they could detect "changes too subtle to be revealed upon the histological specimen, changes in the behavior of essential enzyme systems present." They had, by this approach, first located the site of the biochemical lesion of vitamin B1 deficiency in the lower parts of the brain, and then pinpointed the lesion in the oxidation system of the 3-carbon stage (lactic and pyruvic acid) of sugar metabolism. While Embden and Meyerhof's experiment had left open the possibility that the presence of pyruvic acid was an artifact of their techniques, Peters' techniques proved that pyruvic acid actually was a normal intermediary in carbohydrate metabolism. They also had shown that the acute symptom of the neck spasm in thiamine-deficient pigeons was probably not due to a toxic build-up of pyruvic acid, but to the deficiency of the energy which the oxidation of pyruvic acid would normally produce. In short, Peters and his colleagues had shown "that an in-vitro research. . . which takes advantage of the in-vitro labours of biochemists can be applied to in-vivo events" (Peters 1936). Even in its unfinished state, Peters' work was a convincing demonstration of the power of biochemical research to explain normal and pathological phenomena at a new level of detail.

Lohmann's isolation of cocarboxylase in 1937 was a triumphant confirmation of Peters' conclusions that vitamin B1 was the coenzyme directly concerned with the metabolism of pyruvic acid. But Lohmann's results raised new questions. The first and easiest to answer was: was there any important difference in the activity of the free vitamin B1 which Peters had used and the phosphorylated vitamin B1 which Lohmann had found? Between 1937 and 1939 Peters' group included the Spanish biochemist, Severo Ochoa, who had just come from Meyerhof's laboratory at Heidelberg and thus knew about Lohmann's work first hand (Proc. Conf. Hist. Devel Bioenerg., 1973 pp. 169-170, 184). Ochoa showed that both in brain and yeast, the free vitamin had to be phosphorylated to become cocarboxylase before it could act on pyruvic acid; the in vitro brain tissue preparations Peters had been using luckily contained the enzymes and phosphates necessary for this step (Peters 1963, pp. 18-19).

A much more puzzling problem remained: what was the aerobic oxidative reaction of pyruvic acid and cocarboxylase in carbohydrate metabolism in the brain? Lohmann had shown how pyruvic acid was broken down to acetaldehyde by a simple removal of carbon dioxide (i. e. decarboxylation) in the anaerobic fermentation reaction of yeast. But Peters was dealing with a system which included oxygen and in which pyruvic acid was somehow completely broken down by both oxidation (i.e. dehydrogenation) and decarboxylation to yield carbon dioxide, water, and energy. The most satisfactory path for the oxidation of pyruvic acid would have been the citric acid cycle which Hans Krebs and W.A. Johnson proposed in 1937. Unfortunately, when Banga, Ochoa, and Peters tried to test this pathway, they found that only a few of the intermediates in the citric acid cycle would help speed the oxidation of pyruvic acid (in the presence of cocarboxylase). So in 1939 Peter's group was forced to conclude that, in the brain at least, cocarboxylase set pyruvic acid on some other important pathway which also required [END OF PAGE 43] the uptake of oxygen. They suggested that the pathway might be the one outlined by Szent-Gyorgl in 1937: some of the compounds found in the Krebs cycle served to transport hydrogen from pyruvic acid to the respiratory chain and ultimately to oxygen (Fruton 1972, pp. 380-381; Banga, Ochoa, and Peters 1939a, b). With this proposal, they claimed that "we may . . . . consider that we know now the main facts about the biochemistry of vitamin B1" (Banga, Ochoa, and Peters 1939a, P. 1109).


Epilogue

Time would prove that Banga, Ochoa, and Peters were in fact premature with their claim to know the "main facts of the biochemistry of vitamin B1.- Over the next decade, Fritz Lipmann - with important contributions from Ochoa and Feoclor Lynen - clarified the role of pyruvic acid as the "crossroads" or "hub" of carbohydrate metabolism. In working out the steps which link pyruvic acid to Kreb's citric acid cycle, Lipmann showed that cocarboxylase did indeed mediate both the simple decarboxylation of pyruvic acid in alcoholic fermentation and the oxidative decarboxylation of pyruvic acid in the first step of the aerobic pathway (Lipmann 1971, pp. 27-54, 119-127).

Lipmann's work marked the end of an era of vitamin B1 research. For those who had participated in the discovery of vitamin-deficiency diseases, the identification of vitamins, their chemical isolation and synthesis, and finally the elucidation of their function in intermediary metabolism, the pioneering excitement was over and it was time to turn to other fields. But there were still many questions about vitamin B1 which needed answers, many practical implications to work out, and many new avenues of research that would be opened up.

One major line of research, the practical application of knowledge about the vitamin's chemistry and function has, in effect, continued the original thrust of Eijkman's beriberi research. His original hope in studying beriberi was to find the cause of the disease and then to find a cure, but, as we have seen, Eijkman was more successful at finding a cure for beriberi than at finding out the primary cause.(8) It is impressive to see how, ever since Eijkman, many of the scientists who worked hardest at basic research arising out of his observations of beriberi's cure also became leaders in the application of their results to medicine and human nutrition. After isolating vitamin B1, for example, Jansen immediately used the process to prepare vitamin pill for beriberi patients in the Dutch East Indies. R.R. Williams, in turn, assigned the profits from his patent of thiamine synthesis to the nonprofit Research Corporation, the American Friends Service Committee, and Williams-Waterman Fund for the Combat of Dietary Diseases, in order to sponsor research in human nutrition and dietary diseases (Williams 1961, pp. 168-189; Williams 1956). And, during World War II, R. A. Peters turned his knowledge of the pyruvate oxiclase system back to the problem of poison gases, which had indirectly led him to the study of thiamine. He and his laboratory were able to show how the arsenical gas, levvisite, interfered with pyruvic acid metabolism, and they subsequently developed an effective antidote.

Medical researchers and physiologists also have investigated a variety of factors which affect the thiamine requirements of micro-organisms, experimental animals, and man: the proportions of fat, protein, and total calories in the diet, the use of antibiotics or sulfa drugs, the intestinal bacterial synthesis of thiamine or its inhibitors, and interactions with hormones, minerals, and other vitamins. Such work has suggested other metabolic pathways in which thiamine could be involved, and it has become clear that thiamine in its phosphorylated form takes part in at least two dozen biochemical reactions - more than any other coenzyme known (Bhuvaneswaran and Screenivasan 1962, pp. 580-585; Williams 1961, pp. 140 ff; Brin 1962; Breslow 1962; Gunsalus 1956).

From the vantage point of historical retrospect, we can see that the scientific research inspired by the study of beriberi was done at two different levels of complexity and detail, which correspond roughly to the concerns of the science of nutrition and of the science of biochemistry. For both sciences, the beriberi and vitamin B1 research posed important problems and contributed to the theoretical foundations of the field. When Eijkman began his investigation of the relationship between diet and beriberi, nutritional science was at a standstill. The results of the beriberi research, coupled with the work on simplified diets, gave the study of nutrition an entirely new entity to deal with. What were these mysterious 'vitamines'? How many were there? How did their absence cause disease? What was their chemical nature? The glory and intellectual satisfaction that shortly before had gone to the discoverer of a new microbial pathogen now went to the scientist who discerned the deficiency of a [END OF PAGE 44] vitamin in a disease or who isolated or synthesized a vitamin. The attempt to understand the function of vitamins at a deeper level paralleled the development of biochemistry as a discipline, and a remarkable number of eminent biochemists cut their scientific teeth on the problem of vitamin B1. The successful elucidation of thiamine's precise role in carbohydrate metabolism was seen as a triumphant vindication of the primary program of biochemical research, the dissection of intermediary metabolism.

At the first level, it is easy to see how closely basic nutritional research was tied to the clinical study of disease. This connection is somewhat less direct, less obvious in the biochemical stage of thiamine research, yet even here the disease of beriberi continued to serve as the inspiration for much of the work. It is probably true that Lohmann's work on cocarboxylase was aimed at understanding one more piece of a complex metabolic process, and as far as we know, he and Meyerhof had no special medical question in mind. For them, the basic research puzzle of carbohydrate metabolism was quite difficult and interesting enough in itself. But for R. R. Williams (and probably B. C. P. Jansen) the motives for studying vitamin B1 were certainly mixed. Williams has testified how strongly his first-hand experience of the ravages of beriberi in the Phillipines and the dramatic cures with Vedder's rice-polish moved him to pursue the problem to the end, despite the many obstacles he faced. Part of the urgency he felt in the race to synthesize thiamine arose from his certainty that his competitors, all organic chemists in pharmaceutical firms, would claim exclusive rights over thiamine manufacture for their own firms and thus raise the price of thiamine beyond anything that beriberi victims in Asia could afford. And, Williams also found thiamine an absorbing problem in organic chemistry.

When I began my work with Vedder in Manila, and for twenty years thereafter, I never thought of the antineuritic vitamin as something having monetary value. It was merely a baffling scientific problem, the solution of which would interest the rice eaters of Asia. For several years I did not visualize any probability that the knowledge of its structure would be highly pertinent to the basic science of nutrition, nor that its availability in pure form would become a significant factor in the economy or welfare of any people in the West. I worked at the job of isolating it, partly to justify my curiosity as to why or how it worked, and partly as a humanitarian contribution to the very poor and ignorant of Asia. (Williams 1961,p.164)

For R. A. Peters, the motives are still harder to untangle. In addition to his clinical interests, he gladly acknowledged the great influence that Hopkins and Hopkins' outlook had on his choice of problems and methods. His success in finding the biochemical lesion of vitamin B1 deficiency proved, Peters has often said, how right Hopkins had been to urge the unraveling of metabolic pathways as the chief problem of "the dynamic side of biochemistry" (Peters 1963, p. 15; Peters 1929, p. 216; Peters 1957, pp. 371 ff). But Peters' initial decision to study vitamin B1 was as a side issue which needed to be settled before the research on the pharmacological effects of poison gases could proceed. Then, we have seen, he became absorbed by the challenge of isolating vitamin B1 as part of the larger puzzle of how many vitamin B factors there were. Through this research, in turn, Peters was led to search for the primary "lesion" of thiamine deficiency, a biochemical quest in part founded on the bizarre character of the neck spasm in vitamin-deficient pigeons and the astonishing speed of the cure. These two phenomena caught his imagination in a way that the tediously slow rat-growth test simply could not match:

I always felt that at least for myself a very important aspect of vitamin B studies was the dramatic change in the polyneuritic pigeon, so-called, on dosing with thiamine, i.e., the change from convulsive opisthotonus to normality. The impact which these facts made upon myself was no more remarkable than that upon students .... it was easy to realize that something fundamental would be found out if these events could be understood and this is what induced me to study much further this and the biochemical conditions of the brain. (Peters 1963, pp. 8, 13; 1957, p. 373)

Although the biochemical puzzle quickly became the major focus of Peters' research, his use of the polyneuritic pigeons kept him from ever losing sight of the in vivo effects of the biochemical phenomena he saw in vitro. And it was this clearcut connection between in vitro and in vivo events that made Peters' explanation of thiamine's role in pyruvic acid oxidation in the brain so convincing to both physicians and biochemists.

In the course of half a century, research on beriberi and thiamine has directly contributed to two major [END OF PAGE 45] discoveries in basic science: the discovery of the existence of vitamins, and then the discovery of the role of vitamins as coenzymes which catalyze crucial steps in intermediary metabolism. Is there any prospect that the study of beriberi can still lead to new fundamental ideas in biology? Or has the study of vitamins at all levels been so successful that, as some vitamin researchers have mournfully argued in recent years, there is nothing left to do, that the field is complete and therefore dead? (Wuest 1962, p. 400; Schneider 1963, p, 157; Zbinden 1962, p. 550.

History has often showed that it is unwise to speak eulogies over a dead science, for too often the corpse has proved to be a phoenix. At least two major questions about beriberi and vitamin B1 were not answered during the golden age of vitamin research. How exactly do all the terrible symptoms of beriberi - the burning of the nerves, paralysis, edema, emaciation, heart failure, convulsive spasms - follow from the initial biochemical lesion, whether that lesion be the cocarboxylase deficiency in pyruvic acid metabolism or the lack of thiamine in yet another process? And why is there such variation among individuals and among species in their susceptibility to thiamine deficiency? The first question hints at the possibilities of new levels of complexity in neurophysiology. The second suggests a new approach to the interaction of environment and heredity (Schneider 1963, pp. 162-169. It may still be premature to claim that we know the main facts about beriberi and vitamin B1. [END OF PAGE 46]


Notes
Chapter 3

(1) "Thiamin" was later changed to "Thiamine" by the American Chemical Society to reflect the presence of an amino group in the molecule.

(2) Between 1882 and 1906 Takaki succeeded in virtually eliminating beriberi from the Japanese navy by adding European foods to the Japanese rice-based diet; his theory was that the ratio of nitrogen to carbon (i.e., protein to carbohydrate) in tne Japanese sailors' diet was too small. A Dutchman, Van Leent, had developed a similar theory in 1879 after observing the differences in diet and the rate of beriberi between the Europeans and the East Indians in the Dutch East Indian navy. Eijkman, who knew Van Leent's work, but not Takaki's, was able to show in 1896 that the amount of nitrogen in the rice skin and germ was negligible, and that replacing such a small amount of protein with protein from other foods would not cure beriberi. Therefore, he concluded, beriberi was not caused by a dietary deficiency of protein. He also initiated research into the possibility that the rice skin might contain necessary mineral salts. (Eijkman 1929, pp. 200-201).

(3) Because Hopkins did not publish, it is hard to trace the lines of his research except through his recollections and those of his friends. He apparently began by performing experiments like those of Lunin and Socin (although he was ignorant of their work and Pekelharing's) to convince himself that the diet of purified basic constituents really was inadequate for survival, let alone growth. He noticed that the mice grew fairly well when fed some batches of commercial casein, while on others they died quickly. The mysterious growth factor could be extracted by alcohol from the growth-supporting casein, leaving that casein incapable of helping the mice grow. Later, Hopkins discovered that the yeast extract he had been using to make the purified diets more tasty also contained this factor and indeed was more effective than the casain extracts. (it was at this point that he made his comments to the public analysts in 1906.) Most of his research up to 1912 dealt with yeast extracts and fractionations - time-consuming work because each test had to follow the growth of rats over 4 to 9 weeks (Dale 1948, p. 131; Hopkins 1912, pp. 454-460).

(4) "Vitamine" was the accepted spelling until 1920. Because Funk's spelling had definite chemical and theoretical implications, we will use this spelling in discussing this early phase of vitamin research.

(5) The work that went into the development of reliable, sensitive biological or chemical tests of thiamine's presence could be the subject of a separate chapter, one that would illustrate well the importance of technique in both clinical and basic research. Significant assays developed for thiamine after the time period covered in this chapter include that discovered in 1936-37 by Bergel and Todd: an oxidation reaction of thiamine to thiochrome, a compound which emits a strong blue fluorescence that can be easily and accurately measured. Then, in 1938, M. Lwoff devised a sensitive microbiological assay for thiamine which used protozoa to test for the release of thiamine by stimulated nerves (Sebrell and Harris 1972, p. 147). These and other assays have been widely used to find out how much thiamine is needed for the normal growth of microorganisms, plants, animals, and people.

(6) Over the next few years, the heat-stable factor was itself shown to be a mixture of several physiologically and chemically distinct substances, Ironically, Funk and a team of researchers had identified one of these substances, nicotinic acid (niacin) as early as 1911-12 in their attempts to isolate the beriberi vitamin (McCollum 1957, pp. 310-311). But it was not until 1937 that niacin was proven to be the vitamin lacking in the diets of pellagra victims, a deficiency disease fully as devastating as beriberi (Etheridge 1972, p. 205 ff). While the intricate details of the unraveling of the vitamin B complex do not concern us here, it is important to realize how perplexing the multiple properties of vitamin B were to the people who had to use these properties as their only means of determining the presence of the vitamin they hoped to isolate.

(7) The discovery of coenzymes began in 1906, when Arthur Harden and William John Young at the Lister Institute showed that zymase from yeast could not ferment glucose unless two other factors were present: phosphate and "a dialysable substance which is not destroyed by heat" (Harden and Young 1906, p. 25; Fruton 1972, pp. 297, 344). In 1918 this second factor - called coferment by Harden and Young, and cozymase by Euler and Myrback in the 1920's - was found in animal tissues by Otto Meyerhof. Then, in 1921, he and Gustav Embden showed that coferment was as necessary to glycolysis in muscle tissue as it was to alcoholic fermentation in yeast. Meyerhof, Embden, and several other biochemists saw that this common requirement for coferment implied a fundamental similarity between the pathways of alcoholic fermentation and glycolysis and, even more important, it implied a "unity of biochemistry" among all kinds of living organisms (Fruton 1972, p. 346). However, little was done to determine the chemical identity of this necessary companion to zymase until the 1930's.

(8) Beriberi is still the most important manifestation of vitamin B1 deficiency, and although much research has been done on its pathology, cure, and prevention, it has remained a significant public health problem in southeast Asia. Its modern victims, though, are now more likely to be rural women and babies than the soldiers, sailors, prisoners, and hospital patients who suffered so often from beriberi at the turn of the century. In the course of this century, more and more people have taken to eating factory-polished white rice rather than milling their own rice. the polished tastes and cooks better, it keeps better, and it has a certain 'social prestige.' To enrich the ric)f-adds to its cost; laws requiring enrichment are often hard to enforce; and so beriberi persists (Salcedo 1962, p. 573; Sebrell 1962, pp. 566567). In the U.S. beriberi has never been a great problem, save in conjunction with other dietary deficiencies like pellagra (now rare, thanks to the general enrichment of staple cereal products) or the general malnutrition of acute alcoholism. [END OF PAGE 47]


Chapter 3
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