This is the thirteenth in a series of articles recalling scientJlc events in FASEB 50 years ago, including reminiscences of the 1942

Annual Meeting.

1940s Studies of Nicotinic Acid Metabolism and the Relationship of B-Vitamin Utilization to Protein Level in the Diet
By Herbert P Sarett I WENT ro DUKE IN THE FALL OF i938 as a graduate student in biochemistry to work with William Perlzweig. I had spent a year at Cornell getting my M.S. in biochemistry in 1937 with J. B. Sumner, who belatedly received the Nobel Prize in i946 for crystallizing urease in 1926 and showing that enzymes were proteins. It was not easy to find an assistantship to help continue graduate studies in biochemistry Fortunately, while at Cornell I had a stimulating National Youth Administration (NYA) job working with Hans Neurath who was crystallizing proteins and building the equipment to make physicochemical measurements of them. When Neurath went to Duke the following year, he recommended me to Perlzweig for an assistantship that was open; $50 a month was a blessing at the time. There was an enthusiastic and friendly research atmosphere at Duke, which was then a relatively new medical school, and I have many fond memories of warm personal and scientific relationships with staff members in the biochemistry and physiology departments, which were adjacent to each other, as well as those in other departments. The late Phil Handler (who became chairman of the department of Biochemistry after William Perlzweig died in 1949 and then President of the National Academy of Sciences from 1969 to 1981) was a brilliant scientist, a close friend, and most capable in dealing with people socially, scientifically, and politically. He was just starting postgraduate research in the nicotinic acid field with Jack Dann, a bright young Englishman, who unfortunately died of kidney disease in the middle i940s. Hans Neurath, now Professor Emeritus at the University of Washington, was in the early stages of his studies of the physical chemistry of proteins, and Frank Putnam, now Professor Emeritus at the University of Indiana, was starting his research in this area. Fred Bernheim (deceased) was immersed in his manometric studies using the Warburg apparatus, while Jerry Harris (primarily a pediatrician) and Henry Kohn, now Professor Emeritus at Berkeley, were studying the poor oxygen-carrying capacity of sulfhemoglobin, a concern in those days of sulfa drugs. Ed Levy, my first co-worker in the nicotinic acid studies, went on to medical school and is now a retired doctor in Norfolk, Va. Ray Reiser, now Professor Emeritus at Texas A&M, had a postgraduate position measuring fat in the stools of sprue patients for Fred Hanes, Professor of Medicine. Henry Kamin (deceased), a graduate student, stayed at Duke and later served as Chairman of the Food and Nutrition Board Subcommittee on Recommended Dietary Allowances. The biochemistry staff, under Perlzweigs gruff but
en

friendly leadership, was welcomed by all departments to share their clinics, medical meetings, and luncheon tables. A major key to the good relationships between departments was Wilburt C. Davison, the first Dean of Duke Medical School. He was previously head of Pediatrics and Assistant Dean at Johns Hopkins. This most competent man, with his warm and friendly bulk, had organized the entire initial development of the school and recruited promising young men with a future for the faculty (many from Johns Hopkins). In the late 1930s and early i940s the isolation and identification of various B vitamins prompted studies of their roles in metabolism, using both in vivo and in vitro methods. A good deal of the basic knowledge in this area was derived from so-called nutritional biochemical studies in humans and animals. Methods were being developed for assay of the vitamins by colorimetric, fluorometric, and microbiological methods so that they could be measured in foods as well as in blood, tissue, and urine in animal and human studies. In addition, it was discovered that some vitamins, particularly nicotinic acid and pyridoxine, were excreted as metabolites, which had to be identified and measured to get a complete understanding of the utilization of the vitamins. The colonmetric and fluorometric methods were not as specific or sensitive as later methods, and sometimes gave misleading results because of interfering materials in the samples tested or the lack of precision of the colorimeters and spectrophotometers in use at that time. Studies often had to be carried out from several approaches and with different dietary materials in order to make sure of the validity of the findings. As pellagra was widespread in the South, the Department of Medicine at Duke had been one of the groups (but not the Herbert P. Sarett
first) to show in i937 that

this was prevented and cured by nicotinic acid. Perlzweig was starting a group to study the metabolism of nicotinic acid. The first task assigned to Ed Levy and me was to develop a reliable colorimetric method for measuring nicotinic acid compounds in urine. It was found that acid hydrolysis was necessary to convert nicotinamide and nicotinuric acid (glycine conjugate) to nicotinic acid. However, alkaline hydrolysis gave much higher and quite variable values. These and other problems with the assay were discussed by Perlzweig et al. (i, 2). The findings were presented at the i940 Federation meetings in New Oreleans by Perlzweig (1), who attended with most of the senior members of the department, while the graduate students were left behind to teach the biochemistry course. In exploring these puzzling findings, we found that nicotinic acid was formed when trigonelline (N-methylnicotmnic acid), a compound found in coffee and legumes, was heated in alkaline solution in the presence of a source of ammonia

Dr. Sarett is a consultant of nutritional sciences. Correspondence should be addressed to him at 5413 Palm Aire Dr., Sarasota, FL 34243, USA.

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(ammonium salts or urea) (3). We standardized the conditions for this conversion and used it to measure total Nmethylnicotinic acid compounds in urine in our studies of nicotinic acid metabolism in humans and dogs (4, 5). In the nicotinic acid metabolism studies, diets free of coffee and legumes had to be used in order to minimize exogenous sources of trigonelline. In addition, smoking was curtailed because any nicotine or pyridine compounds in urine gave some color with the CNBr reagent used to measure nicotinic acid. In humans, N-methylnicotinamide was later shown to be the main metabolite in urine; this could be measured fluorometrically (6) or by the colorimetric method used for N-methylnicotinamide or trigonelline (7). These methods were also used in later studies to show that tryptophan was an important precursor of nicotinic acid in humans (8). We also became interested in determining whether the level of protein in the diet affected the utilization of various B vitamins. Dogs and rats were fed diets with adequate levels of vitamins that differed only in protein level (very low, moderate, and high), and urinary excretion of thiamine, riboflavin, and nicotinic acid compounds was measured (9, 10). On the low-protein diet, riboflavin excretion was fivefold higher than on the high-protein diets. Excretion of nicotinic acid compounds was also higher on the low-protein diet, but thiamine excretion was not affected by the level of protein in the diet. Urinary excretion of vitamins and metabolites after injected test doses of riboflavin, nicotinic acid, and pyridoxme in dogs on low- and high-protein diets also showed much greater retention of these vitamins on the high-protein diet. These and other studies suggested that B vitamins such as riboflavin, nicotinic acid, and pyridoxine were required in anabolism of protein, whereas thiamine was not. This was confirmed in a 26-day balance study in four groups of young rats fed high- or low-protein diets with high or low levels of B complex vitamins (11). Good growth and nitrogen retention were found only when there was adquate protein and B vitamins in the diet; riboflavin and nicotinic acid levels in the carcasse and livers were low in all animals fed low protein diets regardless of vitamin levels in the diet. Thiamine levels in the animals were related to thiamine levels in the diet and not to protein levels. These findings from the early I940s helped provide a good foundation for a basic understanding of the metabolism and utilization of several B vitamins.

Federation meetings have been most stimulating and productive, and I rarely missed any since I first attended at Chicago in 194i. There was a large delegation from Duke at Boston in 1942; the department had a big party, which gave the younger staff a chance to meet many prominent biochemists and nutritionists of that era.

REFERENCES
W. A., Levy, E. D., and Sarett, H. P. (1940) On the quantitative determination of nicotinic acid and its derivatives in urine.] Riot. Ghem. 133, lxxiv-lxxv 2. Perlzweig, W. A., Levy, E. D., and Sarett, H. P. (1940) Nicotinic acid derivatives in human urine and their determination. ] Riot. Ghem. 136, 729-745 3. Sarett, H. P., Perlzweig, W. A., and Levy, E. D. (1940) Synthesis and excretion of trigonelline. j Riot. Ghem. 135, 483-485 4. Sarett, H.P., Hugg, J. W., and Perlzweig, W. A. (1942) Studies 1. Perlzweig, in nicotinic acid metabolism. I. The fate of nicotinic acid in

man.] Nutr 23, 23-34 5. Sarett, H. P. (1942) Studies in nicotinic acid metabolism. II. The fate of nicotinic acid in normal and blacktongue dogs. ] Nutr. 23, 35-45 6. Huff, J. W., and Perlzweig, W. A. (1943)] Biol. Ghem. 150, 395-400
7. Sarett, H. P. (1943) A direct method methyl derivatives of nicotinic acid 159-164 for the determination of Nin urine.] Riot. Ghem. 150,

8. Sarett, H. P., and Goldsmith, G. A. (1947) The effect of tryptophan on the excretion of nicotinic acid derivatives in humans.
Riot. Chem. 167, 293-294 9. Sarett, H. P., Klein, J. R., and Perlzweig, W. A. (1942) The effect of variations in protein intake on the urinary excretion of riboflavin, nicotinic acid, pyridoxin and thiamin in dogs. Federation Proc. 1, 132-133 (abstr.) 10. Sarett, H. P., Klein, J. R., and Perlzweig, W. A. (1942) The effect of the level of protein intake upon the urinary excretion of riboflavin and nivotinic acid in dogs and rats.] Nutr. 24, 295-306

11. Sarett, H. P., and Perlzweig, W. A. (1943) The effect of protein and B-vitamin levels of the diet upon the tissue content and balance of riboflavin and nicotinic acid in rats. ] NuIr. 25,
173-183

2488

50 YEARS AGO

Vol. 6

Anril

1992