B-1 REPORT OF PROGRESS IN RESEARCH JULY 1, 1944 To JUNE 3O, 1945 NUTRITIONAL BIOCHEMISTRY LABORATORIES DEPARTMENT OF BIOLOGY MASSACHUSETTS INSTITUTE OF TECHNOLOGY B-l (con't. TABLE OF CONTENTS Page Staff of the Laboratories 7 Teaching Activities 8 Research Progress 9 A. Metabolism of Deuteriumated Fatty Acids 9 B. Metabolism of Hydroxy Fatty Acids 10 C. Composition of Mexican Foods 10 D. Radium Metabolism and Experimental Osteogenic Sarcoma 12 E. Nutritional Quality of American Dietaries 12 F. Effect of Large Scale Cooking on the Vitamin Content of Foods 13 Researh Program for 1945 - 46 14 A. Metabolism of Deuteriumated Fatty Acids 14 B. Metabolism of Hydroxy Fatty Acids 14 C. Composition of Ecuadoran Foods 15 D. Composition of Chinese Foods 15 E. Effect of Phytates upon the Absorption and Excretion of Ca, P and Fe 15 F. Effect of Dietary Fat on Requirement of Amino Acids 15 G. Effect of Large Scale Cooking on the Vitamin Content of Foods 16 Publications (1944-45) 16 B-1(con't., FOREWORD For the information of friends and supporters of the laboratories this summary of progress in research by the Nutritional Biochemistry Laboratories of the Massachusetts Institute of Technology during the fiscal year just ended has been prepared. We expect to prepare similar summaries each year in the future. During the past year, the research of the Nutritional Biochemistry Laboratories was generously supported by the Massachusetts Institute of Technology, W.K. Kellogg Foundation, Rockefeller Foundation, Nutrition Foundation, Emma Moore Fund, and Lever Brothers Company. Research during the coming year will be supported by the Massachusetts Institute of Technology , W.K. Kellogg Foundation, Nutrition Foundation, Emma Moore Fund, Lever Brothers Company, Swift and Company, Quaker oaks Company, Hoffman LaRoche Company, and possibly others. We wish to take this opportunity to thank foundation sponsors for their generous support and to thank commercial companies for supporting research in the field of their interest without imposing restrictions which would limit our research activities and interfere with the publication of our results. We hope that our research progress has been commensurate with their confidence in our research ability. Finally, we wish to voice our appreciation for support from Dr. Francis O. Schmitt, In Charge, Department of Biology, to Dr. George R. Harrison, Dean of Science; and to the Administration of the Massachusetts Institute of Technology. B-1 (con't. REPORT OF PROGRESS IN RESEARCH (July 1944 - June 1945) by the NUTRITIONAL BIOCHEMISTRY LABORATORIES DEPARTMENT OF BIOLOGY MASSACHUSETTS INSTITUTE OF TECHNOLOGY STAFF OF THE LABORATORIES The war has not been without its effect upon available research personnel, and we have missed the able services of key personnel. Captain Richard Henderson has been with the Quartermaster Corps of the U.S. Army since 1941, and Pharmacist Mate Albert Nagel has been with the Navy since 1943. We are now hoping for their early return and have continued their names on our masthead. The staff during the period covered by this summary was: Robert S. Harris, S.B., Ph.D. (M.I.T.) Associate Professor, in charge Ernest E. Lockhart, S.B., Ph.D. (M.I.T.) Richard Henderson, S.B. (M.I.T.), absent Henry Sherman, B.A. (Harvard) Elizabeth W. Tapia, A.B. (Wellesley) Helen Lockhart, B.S. (Boston University) Mary Nutter, B.S. (Simmons) Albert H. Nagel, S.B. (M.I.T.), absent Gertrude Nightingale, B.S. (Middlebury) Louise Guild, B.S. (Framingham teachers) Mary Ann Aron, A.B. (Illinois), secretary During the year, Elizabeth W. Tapia and Helen S. Lockhart resigned. Anne F. Knott (B.S., Simmons) and Jane A. Reynolds (B.S. Simmons) were added to the laboratory staff on July 1. On July 1, Dr. Lockhart was promoted to Assistant Professor. Extra-Curricular Activities In addition to teaching and research activities at the Institute, Professor Harris is serving as expert Consultant in Nutrition to the Secretary of War and in this connection has supervised the research program of the Nutrition Laboratory at the Pentagon Building in Washington. he still serves on the Committee on the Nutrition of Industrial Workers of the Food and Nutrition Board of the National Research Council This committee has prepared a monograph on 7 industrial nutrition which will soon be published by the National Research Council. He is serving as Expert Consultant to the Foreign Economic Administration in connection with the Relief and Rehabilitation Division, Office of Food Programs, and in this capacity has recently planned a program for the analysis of the major foods in the Chinese dietary. These analyses will be made in our laboratories during 1945-46. In connection with the nutrition programs in Mexico, he is serving as Honorary Consultant to the Mexican Institute of Nutrition and is supervising the research activities of the Laboratory of Bromatology recently established in Mexico City. Upon request of the Secretary of Health and Assistance of Guatemala, Dr. Harris visited that country in June to make a rapid survey of the nutritional problems of Guatemala, observe present nutrition activities, examine nutrition research facilities and recommend a program for the next few years. He is now preparing a memorandum for the Minister. It is expected that nutritional activities in Guatemala and other Central American countries will play a major role in the development of these programs. Professor Harris has continued to serve as editor of the annual review, "Vitamins and Hormones." Volumes I and II have been published, Volume III will appear in October and Volume IV is in preparation. This book has been very well received by the scientific world. TEACHING ACTIVITIES At the present time the Nutritional Biochemistry Laboratories group teaches the following four courses in the department: 7.22 Chemistry of Nutrition 2 lectures weekly 7.222 Chemistry of Nutrition Laboratory 1 lecture, 4 laboratory hours weekly 7.23 Applied Nutrition 2 lectures, 2 laboratory hours weekly 7.24 Advanced Nutrition 2 lectures, 4 laboratory hours weekly The international implications of nutrition are indicated by the extent to which foreign students have studied with us during the past year. During the period of this report, the following have studied with us: Arturo Vergara Uribe, M.C., Colombia State Department Fellow. After one year with us, he returned in September 1994 to become Chief of the Institute of Nutrition of Colombia. Jose M. Portilla, M.D., Ecuador Commonwealth Fund Fellow. He will return early in September to organize, and to become Chief of, the Institute of Ecuador. Alberto Henriquez, Chile Coordinator's Office Fellow. He returned in July and is expected to become director of the Laboratory of Bromatology of Chile. Aberindo N. Bose, India Sybodh K. Mukherjee, India Syed S. Illusain, India Amy Hun Tsiang, China During the coming year, we will have with us, among others, the following foreign students: Luis Alfredo Gomez Arellano, Dr. of Pharmacology, Ecuador, Commonwealth Fund Fellow Phyllis Kuo-chum Fan, China, Kellogg Foundation Fellow. Chi-hsuen Shou Tsao, China, Kellogg Foundation Fellow. Florence K. C. Wang, China, Kellogg Foundation Fellow. Lenore Yang-Su Loe, China, Kellogg Fellow. Natera Edmundo Rojas, M.D., Mexico, Kellogg Foundation Fellow. RESEARCH PROGRESS In the period covered by this summary, research was conducted on: A. The Metabolism of Deuteriumated Fatty Acids; B. The Metabolism of Hydroxy Fatty Acids; C. The Composition of Mexican Foods; D. Radium Metabolism and Experimental Osteogenic Sarcoma; E. Nutritional Quality of American Dietaries; and F. Effect of Large Scale Cooking on the Vitamin Content of Foods. A. The Metabolism of Deuteriumated Fatty Acids This program has been under way for several years. Its purpose is to study with rates the digestion and metabolism of typical fatty acids; to locate the tissues where these acids are stored, degraded to simpler acids or synthesized into more complex acids; to observe the saturation or desaturation of these acids; to determine the rate of excretion of fatty acid end-products; in effect, to follow the biochemical processes by which food fatty acids are utilized. To do this we "tag" the fatty acids under study with deuterium or heavy hydrogen, synthesize them into triglyceride or fat, and trace their metabolic progress. Involved apparatus has been constructed so that minute quantities of deuterium may be identified in samples of body tissues and in excreta. During the past year, we have demonstrated that the deuterium technique can by very useful in the study of the mechanism of hydrogenation of fats, and that catalytic hydrogenation is not really selective, although the more unsaturated acids become saturated more rapidly. Animal tests have also shown that it takes about two days for an end-product of fat metabolism to appear in measurable amounts in the urine, and seven days pass before the deuterium content of the body [illegible] of rats fed constant amounts of deuteriumated cottonseed oil equilibrium. Iso-oleic acid, the form of oleic acid commonly found in hydrogenated fats, was proven by the deuterium technique to be readily metabolized. The preliminary observation, that dermal tissues contained a disproportionately large amount of "tagged" fat, may be evidence that the skin is more actively involved in the metabolism of food fats than was previously assumed. B. The Metabolism of Hydroxy Fatty Acids We have demonstrated with rats that when the fat in a diet already complete in all known nutrients is replaced in part by a triglyceride containing di-hydroxystearic acid, their growth and development is improved (Archives of Biochemistry, 5, 63, 1944). Furthermore, there is a tendency for these rats to deposit less fat in the dermal tissues. It would appear that hydroxy fatty acids exert a beneficial effect upon rates, but we have not yet found an explanation for this interesting observation. Hydroxy fatty acids occur in food fats, especially those of animal origin. We have also reported that the feeding of larger quantities of these hydroxy acid triglycerides provokes a deficiency syndrome in rats which can be prevented or cured by feeding vitamin K (Science, 96 (2502), 542, 1942). Research of the past year on this subject has shown that the kinds and numbers of bacteria in the intestinal tract are the same when the diet contains hydroxy acids as when it does not. The very low content of vitamin K in the caecum and large intestine indicates that synthesis of this vitamin by bacteria is arrested. Thus, it seems that this syndrome results from a disturbance in the metabolism of the bacteria in the caecum and large intestine. The rat is dependent on the vitamin K synthesized by intestinal bacteria, for when this synthesis is disturbed, vitamin K deficiency results. C. Composition of Mexican Foods For the past several years these laboratories have been collaborating with the Mexican Institute of Nutrition and the Pan American Sanitary Bureau in the training of personnel, in the analysis of typical Mexican foods, in the establishment of a nutritional biochemistry laboratory in Mexico City and in the formulation of a sound school lunch program in Mexico. During the first year of this program, three clinicians were trained in the clinical and research aspects of nutrition and two chemists were trained in food analysis. A total of 112 samples of Mexican food were collected, frozen with carbon dioxide ice, and shipped by air express. Working with our staff, the Mexican chemists analyzed these samples for moisture, nitrogen, ash, calcium phosphorus, iron, thiamine, riboflavin, niacin, ascorbic acid and vitamin A content. The results of this investigation were recently published (J. Nutrition, 29 (5), 317, 1945). Also the results of a study of the nutritional values of the Mexican tortilla has been published (Science, 102, 91, 1945). Some exceptional foods were revealed by these analyses. A serving of "malva" (an indigenous spinach-like plant was found to contain 40 per cent of the N.R.C. calcium allowance, 90 per cent of the iron, 140 per cent of the vitamin A, 60 percent of the ascorbic acid and significant proportions of the allowances of the other nutrients. "Charales" (an air-dried, small, fresh-water fish) supplied in a 30-gram portion 27 per cent of the protein and 155 per cent of the calcium allowances. "Pulque" (the fermented juice of the maguey cactus) contributed very significant amounts of thiamine, riboflavin, niacin, ascorbic acid and iron. As a whole, the Mexican foods seemed definitely more nutritious than those of the United States. It is clear that with a food pattern so different, any sound Mexican nutrition program should be based on the composition of Mexican foods only. It is interesting to note that our prediction, based on food analysis, that the Mexican diet is weakest in riboflavin and niacin content was confirmed by a clinical study (unpublished) of the people in the Mesquital area by a group from the Rockefeller Foundation. It was observed that the calcium content of the Mexican tortilla is so high that the average Mexican obtains more than 500 mgs. of calcium in the 280 grams of tortilla he eats each day. It is significant that these people would be calcium-deficient were it not for their native practice of using lime in the preparation of corn for tortillas. On the basis of these analyses of Mexican foods, a school lunch program was carried out during an entire school year on approximately 1,000 children. Some seven dietaries were used in rotation, each calculated to supply one-third of the N.R.C. allowance to each child. The lunch cost two to three cents daily. This was less than one-fifth as expensive as comparable lunches formulated on the United States food pattern. The children receiving these lunches lived in the poorest section of Mexico City. The average family size was seven, the family income about 30 cents daily. These children showed no more evidence of malnourishment than a group of 760 middle-class school children studied with the same techniques by our roup in Michigan in 1943 (J. Amer. Diet. Assoc., 19, 182, 1943). Poverty does not necessarily breed malnutrition, nor do riches insure against it. The results in Mexico may be explained as due to a racial difference. But we are convinced that the superior quality and the unrefined nature of Mexican foods, and the firmly established food habits of these propel are important factors also. On the basis of our experience in Mexico, an article has recently been published (Science, 102, 42, 1945) emphasizing the importance of food analysis to the development of a sound agricultural and nutritional program. We can best help other nations by training personnel, providing equipment, and sending experts to help initiate and maintain research programs. We should not impose our foods or food habits upon them; rather we should make available our knowledge and our skills. We have assisted in establishing a food laboratory in Mexico and a trained staff is continuing the food analysis program under our supervision. Plans have been made and funds may soon be granted for a similar program in one or several Latin American countries. Chemists, physicians, agronomists and nutritionists will be trained, foods will be analyzed, a bromatology laboratory will be provided and a filed study will be conducted. After the above nutrition team is trained and started upon a sound program of research and education, it should be successful in establishing nutrition activities in its country. D. Radium Metabolism and Experimental Osteogenic Sarcoma In an effort to determine more accurately the toxicity of radium, we have conducted experiments in which rats have been fed or injected with radium and the effects observed over succeeding months. This work has been summarized in two recent publications (Am. J. Pathology, 20, 1-21, 1944; Am. J. Rosentgenology and Radium Therapy, 52, 353, 1944). Chronic radium poisoning was produced in four series of rats. When 25 to 100 micrograms of radium were given by mouth, primary osteogenic sarcoma resulted in approximately one year. This sarcoma usually developed in the vertebrae and metastases were found in the lung and other organs. These sarcomas have been transplanted to other rats and passed through more than 12 generations. It is our understanding that this is the first successful development of transplantable experimental bone sarcoma. This should greatly assist in bone cancer research. Four to eight per cent of the radium fed by mouth was still retained after four days. At the end of one year, one to seven per cent remained in the animals' bodies. On the other hand, approximately 50 per cent of the radium injected intradermally was retained at the end of a year. A terminal retention of only one microgram of radium was sufficient to produce osteogenic sarcoma. Over 90 per cent of the retained radium was found in the skeleton, the bone showed 100 times the radium concentration of the richest soft tissue (lung). It was hoped that the rat would be useful for estimating the toxic does of radium in man. We have shown, however, that the toxic dose is far greater in the rat than in man. the rat requires 150 times as much radium per kg. body weight and 250 times per kg. skeletal weight. This investigation was made in collaboration with the Department of Physics of M.I.T. and with Dr. Joseph Aub of Harvard Medical School. E. Nutritional Quality of American Dietaries In the past the adequacy of the dietaries of population groups has been measure by one or another type of dietary survey. Though each of these methods has its usefulness, none can accurately predict the actual intake of nutrients by a population. Variation in the nutrient content of each food, in losses during preparation and service, and in the size of servings, combine to make calculation unreliable. It seemed to us that a laboratory measurement of the dietary intake of nutrients was necessary. We analyzed the nutrient content of the [illegible] of a selected group of 71 subjects during the latter part of 1949. Concurrently, the 3,336 returns from a dietary survey, that was conducted on a nation-wide basis, were used to estimate the reliability of the data from the smaller group whose diets were analyzed (J. Am. Diet. Assoc., 20, 742, 1944). The adequacy of the diets was measured by comparison with the Recommended Dietary Allowances of each subject as suggested by the Food and Nutrition Board of the National Research Council (N.R.C. Reprint and Circular Series No. 122, 1945). When so measured, the average daily intake for the group was adequate in ascorbic acid, calcium, and iron, but inadequate in thiamine, riboflavin, and niacin. However, only six per cent of the subjects received the full allowance of all nutrients measured. The percentage of subjects receiving the allowance of each nutrient was as follows: niacin 25 per cent, riboflavin 27 per cent, thiamine 38 per cent, ascorbic acid 52 per cent, calcium 63 per cent and iron 72 per cent. Only 21 per cent of the subjects received as much as three-fourths of the National Research Council Allowance of all nutrients measured. The following percentage received three-fourths of each allowance: niacin 52 per cent, riboflavin 61 per cent, thiamine 68 per cent, ascorbic acid 71 per cent, calcium 87 per cent and iron 91 per cent. Less than 35 percent of the subjects received the more conservative Minimum Daily Requirement as defined by the U.S. Food and Drug Administration. The dietary survey indicated that the quality of the diets studied by chemical analysis was superior to that of the national sample. Thus, it is probable that the dietary intake of the nation at the time of this study was lower than that reported above, and not as high as several government agencies contended. Adequacy of nutrition as measured against the National Research Council allowance reveals that few people in the United States were well fed in 1943. There is no reason to believe that it was significantly better in 1944 or 1945. F. Effect of Large Scale Cooking on the Vitamin Content of Foods There is considerable interest in the losses of certain vitamins which take place during the cooking of foods. An article from these laboratories several years ago (J. Am. Diet. Assoc., 12, 23, 1943) reported that an average of 45 per cent of the ascorbic acid and 35 percent of the thiamine of vegetables was lost during large scale preparation and service. In some instances the losses were as high as 96 per cent. Largely as a result of this publication, Dr. Harris was appointed as Expert Nutrition Consultant to the Secretary of War to set up the Nutrition Laboratory in the Pentagon Building in Washington so that a complete investigation of the losses of nutrients in foods during preparation might be made with the support of the Office of Scientific Research and Development. This laboratory has been in operation for more than one year, and a staff of eight investigators has to date completed studies on potatoes, carrots, cabbage and spinach. The first report of the results of this laboratory is concerned with the effect of cooking upon the vitamin content of potatoes and will be published shortly. In the preparation of mashed potatoes, the only important vitamin loss was in ascorbic acid. During mashing, 18 per cent was lost, and during a subsequent 45 minutes on the steam table, 34 per cent was lost. Potatoes showed losses of 11 per cent ascorbic acid during steaming and 19 per cent during standing for one hour. During boiling, 13 per cent of the ascorbic acid content of potatoes was lost, while 21 per cent more was lost while standing on a steam table. During baking, 12 per cent of the ascorbic acid was lost, while 28 per cent more was lost on standing 90 minutes. There were no losses in thiamine and niacin during the steaming of peeled potatoes, and losses of 17 per cent thiamine and 17 per cent niacin during boiling. From the nutritional standpoint, the steaming of potatoes represents the best method of preparation. It is expected that a series of six or more articles will be published under this laboratory program. RESEARCH PROGRAM FOR 1945-46 During the coming year, the following subjects will be investigated by our laboratories: A. The Metabolism of Deuteriumated Fatty Acids; B. the Metabolism of Hydroxy Fatty Acids; C. The Composition of Ecuadorian Foods; D. The Composition of Chinese Foods; E. The Effect of Phytates upon the Absorption and Excretion of Calcium, Phosphorus and Iron; F. The Effect of Dietary Fat upon Requirement of Amino Acids; and G. The Effect of Large Scale Cooking on the Vitamin Content of Foods. Other subjects may be added as the year progresses. A. The Metabolism of Deuteriumated Fatty Acids The study of the metabolism of "tagged" fatty acids will continue, with major emphasis upon the metabolism of oleic acid. B. The Metabolism of Hydroxy Fatty Acids This investigation will consist of two parts. In one, an in vitro study will be made of the effect of di-hydroxy stearic acid upon the synthesis of vitamin K by intestinal bacteria (coli- aerogenous types). We wish to determine the manner in which hydroxy stearic acid interferes with this biochemical process. Possibly it blocks an important enzyme system. In the second part of this program, we will use white rats and investigate the effect o hydroxy fatty acids upon the gastro-intestinal synthesis of vitamins other than vitamin K, notably thiamine, riboflavin and niacin. There is no reason to suppose that the [illegible] on these vitamins will be similar. D. The Composition of Ecuadorian Foods In collaboration with Dr. Gomez, we plan to analyze food samples for Ecuador. It will be interesting to compare the composition of these foods with the of similar foods from Mexico, for we have already noted that differences in strain may result in marked differences in nutritional quality. D. The Composition of Chinese Foods A project has been set up in collaboration with the Foreign Economic Administration, the War Department and the Chinese Government whereby data on the composition of Chinese foods will be obtained. Under this program, Dr. Walker of the Foreign Economic Administration will collect and stabilize samples of more than two hundred foods, taken at several seasons of the year and from various part of China. The Army Air Forces Command will transport these sample to Cambridge where they will be analyzed by four Chinese in collaboration with our laboratory personnel. These Chinese have been awarded fellowships by the W. K. Kellogg Foundation for a twelve-month period. They will be trained in the methods of food analysis and in nutrition science, with the expectation that they will return later to China and enter the employ of the Chinese Institute of Nutrition. In many respects this program will resemble that which we carried out in collaboration with the Mexican Institute of Nutrition. E. Effect of Phytates upon the Absorption and Excretion of Calcium, Phosphorus and Iron For many years certain cereal foods have been accused of interfering with the absorption of minerals. The phytates of foods are held to be responsible for this interference. The literature on this subject is confusing. We propose to study first the effect of relatively pure phytates upon the absorption and excretion of iron, calcium and phosphorus. These minerals will be made radioactive, by cyclotron bombardment, incorporated into inorganic salts and mixed in the dietary. The metabolism will be traced by radioactivity-measuring techniques. After this series is completed, we will study the effect of phytates in cereals (oats, corn, rye, etc.) upon the absorption and excretion of these mineral salts. Finally, if we find that phytates do interfere with mineral metabolism, we propose to devise techniques which may be used in the treatment of cereals to minimize the interference. It is possible that the effect of phytates upon the metabolism of minerals in the average dietary is insignificant, but this should be proven. F. Effect of Dietary Fat upon the Requirement of Amino Acids Others have shown that a diet poorly balanced with respect to essential amino acid content caused a less satisfactory metabolism of fat (ketosis) than a diet well balanced in amino acid content. We have hypothesized that the content of fat in the diet may have an influence upon the amounts of essential amino acids required for the growth and development of the rat. We propose to determine whether this "sparing action" exists by using a series of diets, each deficient in a different essential amino acid but adequate in all others, and containing different proportions of fat. G. Effect of Large Scale Cooking on the Vitamin Content of Foods The program at the Nutrition Laboratory of the Pentagon is continuing under the sponsorship of the War Department, supported by grants from the Office of Scientific Research and Development. During the coming year, the losses in vitamin content of vegetables and meats during preparation and service on a large scale will be measured. It is expected that this program will be completed within the next year. PUBLICATIONS Reprints of publications by members of the staff of the Nutritional Biochemistry Laboratories are available to anyone requesting them. The publications of the past year have been: 1. Harris, R.S. and K. Thimann: Vitamins and Hormones, Vol. II. Academic Press, Inc. 1944. 2. Harris, R.S., H. Sherman and E.E. Lockhart: "Effect of glycerides of hydroxy fatty acids upon growth and development," Archives of Biochem., 5, 63, (Sept.) 1944. 3. Evans, R.D., R.S. Harris and J.W.M. Bunker: "Radium metabolism in rats, and the production of osteogenic sarcoma by experimental radium poisoning," Amer. J. Roent. Rad. There, 52, 353, (Oct.) 1944. 4. Lockhart, E.E., R.S. Harris, E.W. Tapia, H.S. Lockhart, M.K. Nutter, V. Tiffany and A.H. Nagel: "Study of the nutritional quality of dietaries by chemical analysis," J. Amer. Diet. Assoc., 20, 742, (Dec.) 1944) 5. Cravioto, R., E.E. Lockhart, R.K. Anderson, F. de P. Miranda and R.S. Harris: "Composition of typical Mexican foods," J. Nutrition, 29, 317, (May) 1945. 6. Harris, R.S.: "An approach to the nutrition problems of other nations," Science, 102, 42 (July) 1945. 7. Cravioto, R., R.K. Anderson, E.E. Lockhard, F. de P. Miranda and R.S. Harris: "Nutritive value of the Mexican tortilla," Science, 102, 91, (July) 1945. 8. Harris, Robert S.: "Reexamining our national nutrition program," New York Legislative Document, 73, 1944. B-6 REPORT OF PROGRESS IN RESEARCH REPORT II July 1, 1945 to June 30, 1946 NUTRITIONAL BIOCHEMISTRY LABORATORIES DEPARTMENT OF FOOD TECHNOLOGY MASSACHUSETTS INSTITUTE OF TECHNOLOGY B-6 (con't.) TABLE OF CONTENTS Page Staff of the Laboratories.......................................7 Teaching Activities.............................................8 Research Progress...............................................9 A. Metabolism of Deuteriumated Oleic Acid................9 B. Effect of Dihydroxystearic Acid upon Intestinal Synthesis of Vitamins................................10 C. Effect of Fat upon Nitrogen Retention................10 D. Composition of Chinese Foods.........................10 E. Composition of Ecuadorian Foods......................11 F. Effect of Phytates in Oats on Absorption of Radioactive Iron ....................................11 G. Effect of Large-Scale Cooking on the Vitamin Content od Food..............................................12 Research Program Projected for 1946-47.........................13 A. Metabolism of Deuteriumated Oleic Acid...............13 B. Synthesis of Essential Fatty Acids by the Rat........13 C. Effect of Dihydroxystearic Acid upon Intestinal Synthesis of Vitamins................................13 D. Effect of Fat upon Nitrogen Retention................13 E. Composition of Central American Foods................13 F. Effect of Phytates in Oats in Absorption of Radioactive Iron.....................................14 G. Effect of Timing upon the Metabolism of Essential Amino Acids..........................................14 H. Browning of Ascorbic Acid when Treated by Heat.......14 I. Vitamin Losses in Foods Cooked by the Chinese Method.15 B-6 (con't.) FOREWORD One year ago the Nutritional Biochemistry Laboratories of the Massachusetts Institute of Technology published its first "Report of Progress in Research" covering the year ending June 30, 1945. This report contained an account of the research and teaching activities of the staff, briefly summarized the year's research activities, and outlined the program for the year ahead. This report was prepare so that those interested in the work of our laboratories might have a periodic summary of research progress. It was so well received that we have prepared this second report, covering the period July 1, 1945 to June 30, 1946. During the past year the research of the Nutritional Biochemistry Laboratories has been generously supported by the Nutrition Foundation, Inc the W. K. Kellogg Foundation, the Central American Nutrition Foundation (United Fruit Company), Lever Brothers Company, Quaker Oats Company, Swift and Company as well as the Massachusetts Institute of Technology. Research during the year 1946-47 will be supported by the Nutrition Foundation, the Central American Nutrition Foundation, the Chicago Livestock and Meat Board, Quaker Oats Company, Swift and Company, Hoffman LaRoche, Inc., the Charles H. Hood Dairy Foundation and possibly others. We grasp this opportunity to thank the foundations and commercial companies who support our research program without imposing restrictions which would limit our activities or the dissemination of our research results by publication in scientific journals. We trust that our progress has justified their investment in our research program. We have deeply appreciated the support and encouragement which Prof. William L. Campbell, head of the Department of Food Technology and which the administration of the Massachusetts Institute of Technology has given our program. THE STAFF OF THE NUTRITIONAL BIOCHEMISTRY LABORATORIES B-6 (con't.) REPORT OF PROGRESS IN RESEARCH (July 1, 1945 -- June 30, 1946) by the NUTRITIONAL BIOCHEMISTRY LABORATORIES DEPARTMENT OF FOOD TECHNOLOGY MASSACHUSETTS INSTITUTE OF TECHNOLOGY Staff of the Laboratories The staff during the period covered by this summary was: Robert S. Harris, S.B., Ph.D. Professor in Charge Ernest E. Lockhart, S.B., Ph.D. Hazel E. Munsell, B.A., Ph.D. Richard Henderson, S.B. Henry Sherman, B.A., Ph.D. Louis O. Williams, B.A., Ph.D. Mary Nutter, S.B. Gertrude Nightingale, B.S. Louise Guild, B.S. Anne F. Knott, S.B. Samuel A. Goldblith, S.B. Isabel MacClellan, Secretary During this period Mr. Sherman received his Ph.D. degree and Professor Harris was advanced to a full professorship. The Laboratories were transferred from the Department of Biology to the Department of Food Technology on March 1, 1946. Dr. Hazel E. Munsell joined the staff on May 1. Mr. Henderson was honorably discharged from the Army in March with a rank of Major in the Quartermaster Corps after four years with three years' service in Australia. Mr. Goldblith was honorably discharged in February with the rank of Captain. Three and a half years of his four years of service were spent as a prisoner of war in Bataan and in Japan. He received a citation for gallantry in action and was awarded the Silver Star medal. Mesrs. Richard Henderson, Merton P. Lamden and Leon M. Sharpe are conducting research for use in their theses to be submitted for credit toward degrees of Doctor of Philosophy. EXTRA-CURRICULAR ACTIVITIES Professor Harris continued to serve until October 1, 1945, as Expert Consultant to the Foreign Economic Administration and as 7 Expert Consultant in Nutrition to the Secretary of War through June [illegible] 1946. He is still serving as a member of the Committee on Nutrition of the Pan American Sanitary Bureau and in that connection is taking a leading part in organizing the Institute of Nutrition of Central America and Panama and in planning the first four years of its program. In connection with this program, a chemist, a physician, an agronomist and a nutritionist from each of the participating countries will be granted a one year fellowship for study in the United States. A large laboratory will be established in Guatemala City for the analysis of native foods as well as sample diets taken in the various countries. This laboratory will be directed by a chemist from the United States. The physicians and nutritionists will collaborate in a field study of the nutritional and clinical status of the population in one areas so that they may develop their skills in survey work under the leadership of an American physician. The INCAP is to be a permanent organization concerned with food and nutrition problems of Central America. It will be directed by an executive board consisting of one member from each country, one from the Pan American Sanitary Bureau and one from the Kellogg Foundation which is providing funds for the fellowship program and for the equipment of the laboratory. This is an interesting opportunity for international collaboration on an important problem, for nutrition and malnutrition are not limited by political boundaries or racial inheritance. The Charles H. Hood Dairy Foundation has appointed Dr. Harris to its Advisory Committee for a period of five years. TEACHING ACTIVITIES The following four courses were taught by members of the staff of the Nutritional Biochemistry Laboratories: 20.04 Chemistry of Food 2 lectures, 4 laboratory hours weekly 20.32 Chemistry of Nutrition 2 lectures weekly 20.32 2 Chemistry of Nutrition Laboratory 1 lecture, 4 laboratory hours weekly 20.34 Applied Nutrition 2 lectures, 2 laboratory hours weekly 20.36 Advanced Nutrition 2 lectures, 4 laboratory hours weekly The following foreign students have studied with us and worked in our laboratories during this period: Leonel Fierro del Rio, M.D. A professor of bromotology at the Institute of Tropical Diseases, Mexico. Luis Alfredo Gomez Arelani, [illegible] Ecuador, Commonwealth Fund Fellow Dr. Gomez will return home to direct the Laboratory [illegible] of the Institute of Nutrition of Ecuador. Subodh K. Mukherjee, India Fellow. Lenore Yang-Su Loe, China, W. K. Kellogg Foundation Fellow. Chi-hsuen Shou Taso, china, W. K. Kellogg Foundation Fellow. Florence K. C. Wang, China, W. K. Kellogg Foundation Fellow. Ying Hsueh Wu, China, W. K. Kellogg Foundation Fellow. When the training of the above four Chinese students is completed, they are to join the staff of the food laboratory of the National Institute of Health in Peiping, China, and continue the program of analysis of Chinese foods which was initiated by our laboratories. The following distinguished persons visited our laboratories during the year: Mr. Dinor Walter Invernizzi, Monteviodeo, Uruguay. Dr. Salvador Zubiran, Rector de la Universidad National Autonoma de Mexico, Mexico. Dr. B.C.F. Jansen, Holland Dr. Hsien, Wu, Director of the Chinese Institute of Nutrition. Dr. F. W. Clements, Australian Institute of Anatomy. Dr. Eleanor D. Mason, Department of Physiology and Nutrition, Women's Christian College, Madras, India. RESEARCH PROGRESS During this period research was conducted on: A. The Metabolism of Deuteriumated Oleic Acid; B. Effect of Hydroxystearic Acid upon the Intestinal Synthesis of Vitamins; C. Effect of Fat upon Nitrogen Retention; D. Composition of Chinese Foods; E. Composition of Central American Foods; F. Composition of Ecuadorian Foods; G. Effect of Large Scale Cooking on the Vitamin Content of Foods. A. Metabolism of Deuteriumated Iso-Oleic Acid Deuteriumated iso-oleic acid was fed in the diets of rats. There was an increase in the iodine numbers of the fatty acids in the carcasses and hides as well as in their deuterium content, indicating that deuterium content, the liver fatty acids were more rapidly replaced than the fatty acids of other tissues confirming previous reports that the liver plays an important role in fatty acid metabolism. The appearance of large amounts of fatty acid containing deuterium in the kidneys indicated that the composition of kidney fat may be rapidly affected by dietary fat. The low deuterium content of the brain fatty acids demonstrated that the rate of change of the character of the fat in this organ is relatively slow. 9 In terms of total fat metabolism the liver may not be the most important metabolic tissue and dermal fat and carcass fat may be much more important in fat metabolism than was once thought. There were 40 grams of fat in the carcasses to every gram in the liver. The presence of deuterium in the saturated fatty acids in the carcass demonstrates that the rat can hydrogenate iso-oleic acid. The presence of deuterium in the urine and body fluids proves that the organism can metabolize iso-oleic acid. Spectrophotometric analyses of the carcass and hide fatty acids demonstrated that arachidonic acid had been synthesized. It has been thought that this "essential" fatty acid cannot be synthesized by the rat and must be supplied in the diet. Our data suggests that the rat can synthesize this acid if certain non-essential fatty acids are supplied in the diet. The octabromide of arachidonic acid contained deuterium, further proof of the synthesis of this fatty acid. The demonstration of "essential" fatty acid synthesis is of considerable importance. B. Effect of Dihydroxysteric Acid upon Intestinal Synthesis of Vitamins The manuscript on the "Effect of Eihydroxystearic Acid on Vitamin K Synthesis by Rats" has been completed and will be presented before the meeting of the American Chemical Society in September and published shortly afterward. In this paper it was reported that vitamin K deficiency developed more rapidly in male than in female rats receiving the hydroxy fatty acid; that no vitamin K was present in the intestines of these rats, indicating an arrest in vitamin K synthesis; that neither the kinds nor numbers of intestinal bacteria were affected by the fatty acid feeding, indicating that the deficiency syndrome did not result from a bactericidal or bacteriostatic action upon the intestinal flora but possibly from a blocking of the biochemical system involved in the synthesis of vitamin K; and that the physiological requirement of the male rat for vitamin K is higher than that of the female rat. C. The Effect of Fat upon Nitrogen Retention We are studying the effect of the level of fat in the diet upon the metabolism of a protein (gelatin) deficient in several amino acids, when fed in diets otherwise adequate to rats. the results of the nitrogen balance measurements in the first trial indicated that the fat in the diet had caused a sparing action and that this sparing action increased as the fat content was increased toward 20 per cent of the diet. The sparing action decreased again when the diet contained 30 per cent fat. 10 When the experiment was repeated, using [illegible] place of cornstarch, no sparing action was noted; in fact, the nitrogen balance became more negative as the fat content of the diet was increased. This may indicate that the sparing [illegible] of fats is affected by the nature of the carbohydrate component of the diet. At the moment it is difficult to interpret the series of experiments that have been run in this study. It is interesting that the groups of rats consumed iso-coloric quantities of diet each day in spite of the fact that they were fed ad libitum and that they were fed several varieties of diet. D. Composition of Chinese Foods In collaboration with the Foreign Economic Administration, the U. S. Ware Department and the Chinese Institute of Nutrition, samples of food have been collected in China and shipped to the United States by air for analysis in our laboratories. Four Chinese chemists, provided with fellowships by the W. K. Kellogg Foundation, were trained in our laboratories in the methods of food analysis and assigned to analyze the foods. The program had started when hostilities ceased and the program immediately lost its importance to our armed forces. However, the project was allowed to run for the full year because the data would be useful to relief agencies and to the Chinese Government. Fifty-six foods were analyzed for moisture, ash, nitrogen, calcium, phosphorus, iron, carotene, thiamine, riboflavin, and niacin content. Several of the foods were found to be especially nutritious. Mu-erh (Anricularie anricula-judae-Schrot) was the most notable, as this fungus contained considerable amounts of protein, iron, calcium and niacin. The iron content (185 mgs. per 100 gms.) is the highest of any food know to us. The blossom of the day lily (Hemerocallis flava Z.) is important especially because of its high calcium and iron content. Soybeans, peas, horse beans, kidney beans, apricot kernels and peanuts are also high in nutrient content. Analyses of several soybean products revealed that procedures used in their preparation of often increased the nutrient content. The results of this study of Chinese foods are included in a manuscript now in preparation for publication. E. Composition of Ecuadorian Foods A number of samples of staple foods was received from Ecuador and analyzed for nutrient content by Dr.Jose Portilla and Dr. Alfredo Gomez, working under our direction. Chochos were rich in nitrogen (6.4 per cent) and calcium (135 mgs. per cent), lentils were rich in iron (15.3 mgs per cent) and cocoa was rich in iron (14.8 mgs. per cent). It is expected that this study will be continued in the laboratory to be established in Ecuador. F. Effect of Phytates upon Absorption of Radioactive Iron This investigation was designed to demonstrate the extent to which the phytates contained in cereal (oats) interferes with the 11 absorption of iron from the intestinal tract of children. It is the current belief that phytates (hexa-phosphoric acid salts of inositol) react with minerals in foods to form insoluble mineral salts which are unabsorbable. By determining the radioactivity of the haemoglobin taken subsequent to feeding radioactive iron with phytate-containing food, it is possible to determine the rate and extent of the uptake of iron from the intestinal tract. If these data are compared with similar date from experiments in which no phytates are given, it is possible to estimate the extent to which these phytates interfere. In our experiment we are using a group of 18 children, 10 to 14 years old. In five successive periods equal amounts of iron were fed in (1) a breakfast, (2) a glass of milk, (3) a serving of oatmeal, (4) a glass of milk to which sodium phytate was added, and (5) a glass of water. The feedings in periods (1), (3) and (4) contained equal amounts of phytates an the remaining two contained none. This investigation is still continuing. Preliminary results indicate that phytates impair the effectiveness of iron absorption and that this interference is much greater when phytates are administered in solutions than when they are fed with food. It was noted that foods, per se, markedly interfere with iron absorption. For example, only two-thirds as much iron was absorbed when administered with milk as with water. G. Effect of Large-Scale Cooking on the Vitamin Content of Food This study was conducted in the Nutrition Laboratory of the War Department in the Pentagon, Washington, D.C., under the supervision of Dr. harris in his capacity as expert Consultant to the Secretary of War. Dr. Munsell was a prominent member of the staff of the Nutrition Laboratory and directed its research for part of the year, before coming to Cambridge. A marked destruction of ascorbic acid was noted when carrots were steamed and still greater losses were noted when carrots were boiled. Further destruction of this vitamin was noted when cooked carrots stood on the steam table for one hour. Steamed carrots lost about 18 per cent, and boiled carrots lost 20 per cent to 50 per cent of their vitamin, riboflavin, niacin, pantothenic acid and biotin content. Most of the vitamins "lost" were found in the cooking water. Only slight losses of the B-vitamins and carotene were noted when carrots were held for one hour on a steam table. Carotene losses were less than seven per cent on boiling or steaming. From the standpoint of flavor, aroma, consistency and appearance as well as retention of vitamins, steaming of carrots is recommended as superior to boiling. Furthermore, carrots should be cooked without peeling. Similar studies on spinach, cabbage, corn and various cuts of meat have also been studied and manuscripts are being prepared for publication. 12 RESEARCH PROGRAM PROJECTED FOR 1946-47 During the coming year research on the following subjects will be conducted by our laboratories: A. The Metabolism of Deuteriumated Oleic Acid; B. The Synthesis of Essential Fatty acids by the Rat; C. Effect of Fat upon Nitrogen Retention; E. Composition of Central American Foods; F. Effect of Phytates in Oats on Absorption of Radioactive Iron; G. Effect of Timing upon the Metabolism of Essential Amino Acids; H. Browning of Ascorbic Acid when Treated by Heat; I. Vitamin Losses in Foods Cooked by Procedure Common in China. Other subjects may be added from time to time. A. Metabolism of Deuteriumated Oleic Acid This study will continue with the expectation that the research on deuteriumated oleic acid will be completed and research on deuteriumated stearic acid initiated. B. Synthesis of Essential Fatty Acids We propose to investigate further the biological synthesis of arachidonic acid hoping to determine its precursor. First we will compare the role of oleic acid with that of claidic acid in effecting this synthesis. C. Effect of Dihyroxystearic Acid upon Intestinal Synthesis The investigation of the effort of this acid upon the intestinal synthesis of thiamine, riboflavin, and niacin will be completed during the coming year. Concurrently we will observe the effects of a deficiency of each of these B-vitamins upon the intestinal synthesis of the other two. D. Effect of Fat upon Nitrogen Retention We are studying the effect of the nature of the carbohydrate component of the diet upon the sparing action of fat on nitrogen retention. These results will aid in interpreting the data of the previous experiments and in planning future work on the sparing action of fat on protein. E. Composition of Central American Foods We have received a generous grant from the Untied Fruit Company which will enable us to make a three year study of the composition of all foods grown in central America. One of our staff is now in that region, observing the foods eaten by the people, collecting specimens for botanical identification, stabilizing samples so that they may be shipped to Cambridge by air, and making valuable observations of the food habits of the people. he will cover all parts of Central America and collect samples of all foods in each region. it is hoped that the program will be completed within three years and that reliable data on several samples of each important food will be obtained. The data will be published in scientific journals from time 13 to time. At the termination of this program we wish to assemble all the [illegible] data on the composition of Central American foods so that this information may be available in one reference source. We feel that results of this food analysis program will have an important effect upon the food and nutrition problems of these countries. A knowledge of the composition of foods should help the farmer to decide the crops he should plant and the genetic strains he should select so that the harvest may be most nutritious as well as most abundant, should help the nutritionist in determining dietaries which will give the best nutrition for the lowest cost, should help agricultural economists in formulating national policies, and should assist all people toward a more efficient use of available plant and animal products. F. Effect of Phytates in Oats on the Absorption of Radioactive Iron This investigation should be concluded within the next year. We are hoping for additional support so that we may compare the availability of phytates in various cereals and cereal products and availability of minerals in cereals by a destruction or inactivation of the phytate component. G. Effect of Timing upon the Metabolism of Essential Amino Acids A preliminary experiment with rats has indicated that it is important that the protein of each meal be balanced in its essential amino acid content. Zein was the sole protein in these diets. It was supplemented with the tow essential amino acids (lysine and tryptophane) in which it is most deficient. Tryptophane was given in equal quantities in the control and test diets. The control group of rats received the lysine supplement in the diet every day while the test group received lysine in the diet every third day and on that day three times as much was given. Thus over a period of weeks, both groups consumed the same diets and only the time at which the lysine was fed was varied. the group fed the test diet (lysine every third day). Thus the timing of amino acid feeding has an effect upon the efficiency of metabolism of food protein. This result may have great significance for it may indicate that not only the food for the week but the food of each meal should be balanced in amino acid content. Experiments using a variety of amino acids and several proteins have been planned so that this important observation may be well substantiated. H. Browning of Ascorbic Acid when Treated by Heat The non-enzymatic darkening of citrus products subjected to heat and the question of the relationship of this darkening to loss in ascorbic acid content has led us to investigate the mechanism of color formation in heated aqueous solutions of ascorbic acid. It has been reported in the literature that iso-ascorbic acid 14 produces color less readily than ascorbic acid and also is oxidized in preference to it. Thus, iso-ascorbic acid may [illegible] be useful as an anti-oxidant to protect ascorbic acid in foods. At present it is impossible to differentiate iso-ascorbic acid from ascorbic acid except by biological assay. Since iso-ascorbic acid is only five per cent as active biologically as ascorbic acid it is important that a convenient method of analysis be developed. We will work on this problem during the coming year. I. Vitamin Losses in Foods Cooked by Procedures Common in China In China foods are commonly cooked by "stir-frying," in which vegetables and meats are cut into thin slices or small cubes and added to a pan containing a shallow layer of hot fat. Since the cooking times of the different components are different, the ingredients are added separately so that the cooking of all will be completed together. Generally, stir-frying requires two to three minutes and the dishes are very flavorous. This method of cooking may be advantageous from several view points for the cooking time is extremely short and immersion of the food in oil reduces vitamin destruction and the leaching out of vitamins and minerals. We are studying the vitamin losses where foods are cooked according to authentic recipes. Our Chinese follows will cook the foods and conduct the analyses. PUBLICATIONS Reprints of publications by members of the staff of the Nutritional Biochemistry Laboratories are available to anyone requesting them. The publications during the past year have been: 1. Harris, R.S. and K. V. Thimann; Vitamins and Hormones, Vol. III Academic Press, Inc., 1945. 2. Sherman, H., M.K. Nutter, E.E. Lockhart, and R. S. Harris; "Studies on fatty acid metabolism by the deuterium technique, Oleic acid." Abstracts of papers, 100th meeting of American Chemical Society, p. 14A April 1940. 3. Harris, R. S.: "Nutrient loss through large-scale cooking,: American Cookery, Vol. 51, No. 3, p. 20, November, 1945. 4. Cravioto, R.B., E. E. Lockhart, F. de Miranda and R. S. Harris: "Contenido nutritivo de cietos tipicos alimentos mexicanos," Boletin de la Oficina Santiaria Panamericana, Agosto, 1945. 5. Harris, R.S.: "Essentials of a post-war nutrition program," New York Legislative Document No. 76, 1940. 6. Harris, R.S., et al.: "The nutrition of industrial workers," National Research Council, Reprint and Circular Series No. 123 (Sept.) 1945. 7. Munsell, H.E. "Ascorbic acid content of the mango in relation to variety," Food Research 10, 95, 1945. 8. Streightoff, F., H.E. Munsell, B.A. Ben-Dor, M.L. Orr, M.H. Leonard S.R. Ezekiel and F. G. Koch, "Effect of large-scale methods of preparation on the vitamin content of food: II Carrots," J. Amer. Diet. Assoc. 2:(6), 511, 1946. 9. Goldblith, S.A.: "Sanitation in Bataan and its aftermath," The Military Engineer, 1946. 15 B-7 REPORT OF PROGRESS IN RESEARCH REPORT III July 1, 1946 to June 30, 1948 NUTRITIONAL BIOCHEMISTRY LABORATORIES DEPARTMENT OF FOOD TECHNOLOGY MASSACHUSETTS INSTITUTE OF TECHNOLOGY TABLE OF CONTENTS Page Foreword........................................................3 Staff of the Nutritional Biochemistry Laboratories..............4 Public Service Activities of the Staff..........................4 Papers Presented at Scientific Meetings.........................5 Teaching Program................................................5 Special Foreign Students........................................6 Distinguished Visitors..........................................6 Research Progress...............................................7 A. Metabolism of Deuteriumated Iso-Oleic Acid............7 B. Synthesis of Essential Fatty Acids (Arachidonic Acid) by the Rat............................................7 C. Interrelation between Pyridoxine and Arachidonic Acid.8 D. Nutritional Value of Elaidic Acid.....................8 E. Effect of Fat Content of Diet upon Nitrogen Retention.9 F. Effect of Dihydroxystearic Acid on Thiamine Synthesis and Storage...........................................9 G. Effect of Dihyroxystearic Acid on Riboflavin Synthesis and Storage...........................................9 H. Composition of the Edible Plants of Central America...9 I. Nutritional Advantage of the Concurrent Feeding of Essential Amino Acids................................10 J. Effect of Phytates upon the Absorption of Radioactive Iron.................................................11 K. Vitamin Losses in Foods Cooked by Chinese Procedures.11 L. Stability of Solutions of Pure Ascorbic and Dehydroascorbic Acid.................................12 M. Estimation of Ascorbic Acid in Food Preparations.....12 N. Retention of Vitamins During Cooking of Enriched Farina...............................................12 O. Protein Quality of Certain Foodstuffs................13 Research Program Projected for 1948-49.........................13 A. Metabolism of Deuteriumated Lauric Acid..............13 B. Nutritional Importance of Petroselinic and Petroselaidic Acids..................................14 C. Metabolic Interrelationships between the B Complex Vitamins and Arachidonic Acid........................14 D. Composition of the Edible Plants of Central America..14 E. Effect of Phytates upon the Absorption of Radioactive Calcium..................................14 F. Influence of Liver Extract upon Rats on "Complete" Synthetic Diets......................................14 G. Effect of Iso-Oleic Acid Content of the Diet upon Growth...............................................15 H. Nutritive Value of "Hard" Fats.......................15 I. Dermal Absorption of Deuteriumated Soaps and Fatty Acids................................................15 J. Synthesis of Glycogen from Fats......................15 Publications from the Laboratories.............................15 FOREWORD The previous two "Reports of Progress in Research" of the Nutritional Biochemistry Laboratories of the Massachusetts Institute of Technology have been received so well by our friends that we have been encouraged to prepare this brief summary of the research accomplishments of the past two years, and an outline of the program planned for the year ahead. During the period beginning July I, 1940 and ending June 30, 1948, the research program of the Nutritional Biochemistry Laboratories has been generously supported by the Nutrition Foundation, The Central American Nutrition Foundation (United Fruit Company), the Charles H. Hood Dairy Foundation, the National Vitamin Foundation, Hoffman LaRoche, Inc., The National Live Stock and Meat Board, Swift and Company, Quaker Oats Company, The Procter and Gamble Company, and the Massachusetts Institute of Technology. Our research program during the academic year beginning July I, 1948 will be supported by The Nutrition Foundation, The National Vitamin Foundation, The Central American Nutrition Foundation, Quaker Oats Company. The Procter and Gamble Company and possibly others. We welcome this opportunity to express our thanks to the foundations and commercial companies who give financial support to our research program without imposing restrictions which limit either our activities or the publication of the results of our research. We earnestly hope that our progress has justified their investment. We wish also to voice our appreciation to Professor William L. Campbell. Head of the Department of Food Technology, for his sympathetic cooperation; our thanks to the Administration of the Massachusetts Institute of Technology for its continued support; and to extend greetings to former students, former colleagues and present friends now scattered over the face of the earth. The Staff of the Nutritional Biochemistry Laboratories REPORT OF PROGRESS IN RESEARCH (July 1, 1946-June 30, 1948) by the NUTRITIONAL BIOCHEMISTRY LABORATORIES DEPARTMENT OF FOOD TECHNOLOGY MASSACHUSETTS INSTITUTE OF TECHNOLOGY STAFF OF THE LABORATORIES The staff during the period covered by this report included the following: ROBERT S. HARRIS, S.B., Ph.D. Professor in Charge HAZEL E. MUNSELL, B.A., M.A., Ph.D. HENRY SHERMAN, B.A., Ph.D. Louis O. WILLIAMS, B.A., Ph.D. *RICHARD HENDERSON, S.B. *,GERTRUDE NIGHTINGALE, B.S. LOUIS P. GUILD, B.S. *ANNE F. KNOTT, S.B. *SAMUEL A. GOLDBLITH, S.B. *LEON M. SHARPE, S.B. CYNTHIA B. TROESCHER, S.B. *MERTON p. LAMDEN, S.B. *LAURA M. CAMPLING, B.A. ANDREW C. PEACOCK, S.B. *RUTH E. EMERY, B.S. RITA E. BRENZ, B.S. JUNE WISKIND, B.S. BOBBE P. FAULDERS, B.S. ISABEL W. MacCLELLAN, Secretary Those marked with an asterisk left the staff during the perIod of this report. Leon M. Sharpe, Richard Henderson and Merton P. Lamden received the Ph.D. degree, and Samuel A. Goldblith the M.S. degree. Dr. Sharpe has joined the staff of Brookhaven, Laboratories, Long Island, New York; Dr. Henderson is now Assistant Professor of Bacteriology at Syracuse University, and Dr. Lamden is Assistant Professor of Biochemistry at the University of Vermont Medical School. PUBLIC SERVICE ACTIVITIES OF THE STAFF Professor Harris continues to serve as, member of the Committee of Nutrition of the Pan-American Sanitary Bureau, and Scientific 4 Director of the research and training program of the [illegible] Nutrition of Central America and Panama. He is chairman of the Cambridge Nutrition Council; a member of the nutrition committee of the Cambridge Red Cross, and the Massachusetts Central Health Council. PAPERS PRESENTED AT SCIENTIFIC MEETINGS During the period covered by this report the following papers were presented at scientific meetings: "Effect of Dihydroxystearic Acid on Vitamin K Synthesis by Rats," presented by G. Nightingale. American Chemical Society, September, 1946. "The Nutrition Problem of Central America," presented by R.S. Harris, New York State Dietetic Association, May, 1947; also presented before Eastern Massachusetts Dietetic Association, February, 1948. "Biological Synthesis of Arachidonic Acid," presented by H. Sherman, American Oil Chemical Society, Chicago, October, 1947. "Pyridoxine and Fat Metabolism," presented by H. Sherman, Nutrition Symposium, Columbia University, December, 1947. "Program of the Central American Nutrition Foundation," presented by R.S. Harris at Santa Tecla, El Salvador, January, 1948; also presented at Escuela Agriola Pan-americans, Zamarano, Honduras, February, 1948. "Estimation of Ascorbic Acid in Food Preparations," presented by S.A. Goldblith, American Chemical Society, New York, September, 1947. "Composition of Central American Foods. I. Honduras," presented by H.E. Munsell, American Institute of Nutrition, Atlantic City, March 1948. "Effect of Phytate on the Absorption of Radioactive Iron," presented by R.S. Harris, American Institute of Nutrition Atlantic City, March 1948. "Use of Isotopes in Biochemical Research," presented by A.C. Peacock, Wellesley College, April, 1948. TEACHING PROGRAM The following six courses are taught by members of the Staff of the Nutritional Biochemistry Laboratories: 20.04 Chemistry of Food 20.32 Chemistry of Nutrition 20.322 Chemistry of Nutrition Laboratories 20.34 Applied Nutrition 20.36 Advanced Nutrition 20.38 Nutritional Evaluation of Food Processes 5 SPECIAL FOREIGN STUDENTS The following foreign students studied with us and worked in our laboratories during the past two years: Luis Alfredo Gomez Arellano, D. Pharm., Laboratory of Bromotology, Institute of Nutrition, Ecuador. Stubodh K. Mukherjee, India. Isabel A. Escobar, Hospital de Enfermedades de la Nutricion, Mexico. Dr. Sarah Baberly, Hebrew University, Palestine. Dr. Walter A. Lindberg, Institute of Hygiene, University of Oslo, Norway. Karin M. Wold, Control Laboratory, Pharmaceutical Chemistry, Norway. Darsban S. Bhatia, India. Nripendra L. Lahiry, India. Alfonso C. Parra, Colombia, State Department Fellow. Guillermo Arroyave Borjes, Guatemala, Institute of Nutrition of Central America and Panama. Salvador A. Pizzati, Honduras, Institute of Nutrition of Central America and Panama. Andre A. Campos, El Salvador, Institute of Nutrition of Central America and Panama. DISTINGUISHED VISITORS The following were among the distinguished persons who visited our laboratories during this period: Prof. Harry Lundin, Institute of Technology, Sweden. Dr. A. G. van Veen, Chief of Eijkman Institute, Batavia, Java. Dr. M. van Eckelen, Chief of Central Institute for Nutrition Research, Holland. Dr. William R. Aykroyd, Food and Agricui Lure Organization, United Nations. Dr. H. E. Magee, Ministry of Health, England. Dr. Werner G. Jaffee, Ministry of Agriculture, Venezuela. Dr. Maria Szezgiel, Warsaw, State Institute of Hygiene, Poland. Dr. Chium-Tong Ling, National Institute of Health, China. Dr. G. Sankitran, Professor of Physiology, All Indian Institute of Hygiene, Calcutta. Dr. Guillermo Garcia Lopez, Instituto Coordinador de Nutricion de Cuba, Havana. Dr. A. Szezygiel, State Institute of Hygiene, Department of Nutrition, Warsaw, Poland. Dr. Tulio Ganduglia, Buenos Aires, Argentina. Dr. Santiago Nadelman, Buenos Aires, Argentina. Dr. Karl Wuhrmann, Swiss Federal Institute of Technology. Sir Christopher W. M. Coa, Advisor to British Colonial Secretary. Dr. F. N. Woodward, United Kingdom Scientific, Mission. Dr. G. D. Bhavnani, Sind College, Karachi, India. Dr. C. K. Chu, Director, National Institute of Health, Chia. 6 Dr. Cheng-fa Wong, Director Institute of Nutrition, China. Dr. M. V. Radhakrishna Rao, Haffkino Institute, Bombay, India Dr. Hunt Khumart, Officers in Charge of [illegible] Military Government, Germany. Prof. Josue de Castro, Rio de Janeiro, Brazil. Dr. S. S. De. Indian Institute of Science, India. Dr. S. Wan, National Defense Medical Center, China. Dr. K. Cheung, Dean, Army Medical College, China. Dr. Sverre Stene, Norwegian Institute of Public Health, Norway Dr. Conrado Piscual, Manila, Philippines. Dr. Kaj Dessan, Copenliagen, Denmark. Dr. A. Ulhoa Cintra, Hospital Clinices, Brazil. Dr. H. C. Hou, Director, Institute of Nutrition, China. Dr. P. R. Peacock, Glasgow, Scotland. Dr. Edmundo Rojas, Hospital de Enfermedades de Ia Nutricio Mexico City. RESEARCH PROGRESS A. Metabolism of Deuteriumated Iso-Oleic Acid The use of deuterium to "tag" fatty acids has made it possible to study their metabolic rate. A group of rats was fed a synthetic diet containing fourteen per cent triolein for fourteen days, [illegible] given the same synthetic diet but containing seven per cent triolein and seven per cent tri-di-deuterio-iso-olein for forty-two days. Representative groups were killed periodically during the experimental period and the fatty acids of the carcasses, hides, livers, kidney and brains were analyzed for a deuterium content. The results indicated a rapid introduction of the diet fat into the fatty acids of carcasses, hides, and kidneys, and a slow introduction into the fat acids of the brain. This confirms data in Report II. The fatty acid in the carcasses and hides became longer and more unsaturated the experimental, period, continued. These studies with deteriorated iso-oleic acids indicate that the rat can metabolize iso-acid and that the dermal tissues are actively involve in fat metabolism. RESEARCH PROGRESS B. Metabolism of Deuteriumated Iso-Oleic Acid In the studies with tri-di-deuterio-iso-olein mentioned under Section A above, the fatty acids from the carcasses and hides were analyzed spectrophotometrically and it was found that the arachidonic acid content of the carcass fat increased as the experiment period continued. Thus, it appears that arachidonic acid was synthesized by the rats. This conclusion was greatly strengthened [illegible] our finding that deuterium was present in the arachidonic acid molecule for it indicated that the arachidonic acid was synthesized from a deuteriumated iso-oleic acid in the diet. This evidence that the rat can synthesize an "essential" fatty acid is of considerable scientific interest. Dr. Sherman has prepared a review of the chemistry and biochemistry of arachidonic acid which will be submitted for publication 7 C. Interrelation between Pyridoxine and Arachidonic Acid [illegible] a fed a pyridoxine-deficient diet, rats develop a scaliness of the paws and tails which is hardly distinguishable from the syndrome which develops from a deficiency in "essential" fatty acids. Others have demonstrated that pyridoxine is necessary for the formation of fat from protein. From this we have reasoned that there may be an interrelationship between pyridoxine and "essential" fatty acids. We have demonstrated that this deficiency condition can be cured by feeding pyridoxine but that it is not affected by feeding linoleic acid. The effects of pyridoxine have been confirmed in a repeat experiment. The evidence indicates that pyridoxine deficiency not only decreases the appetite of rats but also the efficiency of food utilization. It appears that rats store only small amounts of pyridoxine, for their growth rate was retarded within five days after they were placed on a pyridoxic-free ration. The rats fed no pyridoxine consumed nearly twice as much food per gram gain in weight as those fed pyridoxic in fat-free diets or in diets containing olive oil. More fat was deposited in their tissues when pyridoxine and/or fat, was present in the diet. The dermatitis which developed after two to four weeks on a fat-free pyridoxine-free diet was partially cured by feeding pyridoxine, and completely cured when both pyridoxine and linoleic acid were added. The percentage of arachidonic acid in the carcass fatty acids was lowest when olive oil and pyridoxine were present in the diet of the rats. The total amount of arachidonic acid was greatest in those rats fed pyridoxine in the diet. It is obvious that pyridoxine and arachidonic acid are interrelated in metabolism. D. Nutritional Value of Elaidic Acid In the experiments mentioned under "Section B of this report both iso-oleic acids and normal oleic acid were present in the diet. The question arises whether the iso-oleic fatty acids were used in the synthesis of arachidonic acid. In an attempt to answer this question one group of rats, was given a fat-free synthetic diet; a (80-85 per cent normal oleic acid) added; and a third group was given this diet with 14 per cent elaidic acid (an iso-oleic acid) added. After 42 days the animals were destroyed, the fatty acids extracted from the carcasses and livers, and these acids analyzed for arachidonic acid content. The animals on the fat-free diet developed a very mild scaliness of the feet and tails. Since, in a later experiment, this condition was prevented by feeding additional amounts of the various vitamins in the supplement, it is considered possible that olive oil or elaidin in the diet exerts a vitamin-sparing action. The carcasses of the rats fed the fat-free diet contained definitely less fat, and the carcass fatty acids, were bore saturated, than those from the rats fed olive oil or elaidic acid. The total arachidonic acid content of these fat samples was determined by both the poly- 8 100 mg., 170 mg. and 220 mg. of arachidonic acid per rat as a result of feeding the claidin, fat-free and olive oil diets, respectively. On a gram basis, the arachidonic acid [illegible] the carcass fatty acids from the groups fed (1) a fat-free diet, (2) this diet with elaidin or (3) with olive oil was 1.4 per cent, 1.3 per cent and 0.6 per cent, respectively. Further, research may show that these differences in the arachidonic acid content of the carcass fats were due to differences in rate of utilization of this acid in metabolism. A series of diets containing 15 per cent or 25 per cent of gelatine and 0 per cent, 10 per cent, 20 per cent, 30 percent or 40 per cent of hydrogenated fats, and various carbohydrates (sucrose, dextrose and cornstarch) were fed to rats. The effect of the amount of fat and the type of carbohydrate upon nitrogen retention, growth and food utilization was determined. The rates tended to eat isocaloric amounts of diets when fed ad libitum, even when these diets varied widely (0 per cent to 40 per cent) in fat content. Nitrogen retention increased as the fat content of the diet decreased, and the carbohydrate content of the diet increased. Thus, carbohydrates exert a protein-sparing actions of sugar, cornstarch and dextrose are similar. Tryptophan (0.2 per cent) added to gelatine diets caused an increased in food and caloric intake, a reduction in weight loss, an increase in fat deposition in the liver and carcass, and an increase in nitrogen retention. Diets containing gelatine (25 per cent) as the source of amino acids caused an abnormally low deposition of fat in addition of 0.2 per cent tryptophan. Tryptophan seems to be involved in the biochemical process whereby fat is deposited in the tissues of rats. F. Effect of Dihydroxystearic Acid on Thiamine Synthesis and Storage In a previous study (Arch. Biochem. 12, 381, 1947) it was shown that the synthesis of vitamin K in the intestinal tracts of rats was stopped when dihydroxystearic acid was fed in the diet. The effect of this hydroxy fatty acid on thiamine synthesis has been studied, and no adverse effect was noted. In fact, there was inconclusive evidence that the thiamine content of the colon and cecal contents, and of the liver and gastrocnemius muscle, was increased as a result of feeding dihydroxystearic acid. G. Effect of Dihydroxystearic Acid on Riboflavin Synthesis and Storage A study similar to that mentioned in Section F showed that dihydroxystearic acid in the diet had no effect upon the intestinal synthesis of riboflavin. II. Composition of the Edible Plants of Central America On July 1, 1946 a three-year study was begun to determine the composition of the edible plants of Central America. One member 9 of the staff is resident there and is responsible for the collection and identification of specimens from all parts of that area in all seasons, and for the preparation and shipment of samples. Procedures have been developed for the stabilization of samples so that they may be air-expressed to Cambridge and analyzed here by four of our staff. Determinations are made of moisture, ether (or vitamin A), thiamine riboflavin, niacin, and ascorbic acid. As of June 30, 1948, a total of 507 samples had been analyzed and 6032 analyses had been completed. A number of highly nutritious plant food has been "discovered" under this program. The nutrient content of many varieties had not previously been determined by any laboratory. I. Nutritional Advantage of the Concurrent Feeding of Essential Amino Acids In the past it has been assumed that the quality of the protein is of no importance in one meal, if it is satisfactory during a week, a month or even longer. According to this reasoning, a deficient intake of certain amino acids in one meal may be compensated by an excess intake of these amino acids in subsequent meals. We have long questioned the soundness of this reasoning and have now put it to a test. Groups of weanling rats were fed a basal diet containing casein (30), cornstarch (55.4), hydrogenated fat (190), tryptophan (0.17), histidine (0.13), salt-mixture (4) and vitamin mixture (0.36). Control groups received the basal diet + 1 per cent lysine. Test groups received the basal diet and the basal diet + 2 per cent lysine at alternate feedings. The lysine replaced equal weights of cornstarch in the diet. Groups were fed (1) one hour and fasted seven hours, (2) one hour and fasted five hours, and (3) one hour and fasted three hours throughout successive experiments lasting 27 days. A time-interval feeder was devised for the automatic opening and closing of the feeding jars. Significant differences were noted in nitrogen retention and in body weight gain, weight gain per gram of diet eaten, weight gain per gram of lysine eaten. These differences were greater as the feeding intervals lengthened. Thus a delay of three hours or longer in the feeding of lysine as a supplement to a lysine-low diet interfered with metabolism. Lysine must be fed concurrently with other amino acids in the diet for most efficient utilization by the rat. The growth and development of the groups receiving the complete diet at every meal was always superior, even when the feeding interval was only three hours. This may be interpreted to mean that the effects of a poor protein in one meal cannot be counteracted by a complementary protein in the next meal; but that a balanced mixture must be consumed at every meal if the protein is to be utilized most efficiently. These results suggest that breakfast foods compounded of several complementary foodstuffs (cereals, legumes, etc.) while superior from the nutrition standpoint, would also be superior dietetically. 10 J. [illegible] In this project 17 adolescent boys were used to study the relative absorption of radioactive iron from five [illegible]: (a) water, (b) milk, (c) milk and rolled oats, (d) milk and sodium phytate, and (e) milk, rolled oats, tomato juice, bread and omelet. The iron content of all meals, and the amounts of milk in the last four meals, were the same. Meals (c), (d) and (e) contained the same quantity of phytate; the others were phytate-free. It was found that the following percentages of the radioactive iron were absorbed, respectively, from the five different test meals: (a) 12.4, (b) 8.3, (c) 4.8, (d) 0.7 and (c) 2.5. These results indicate that milk interferes with the absorption of iron from the gastrointestinal tract. Since the drop in iron absorption when milk and rolled oats was fed was less than twice the reduction produced by milk alone, it is evident that much of the phytate naturally present in rolled oats is inert. The observation that the absorption of iron from atypical breakfast (c) was only one-fifth that from the aqueous solution (a) suggests that medicinal iron would be much more effectively absorbed if taken between meals with water. This study was undertaken to determine whether the phytates in a typical cereal (oats) interfere with iron absorption. The evidence indicates that phytates interfere but little, and that other factors (such as probably the mass of the food itself) probably are responsible for the relatively poor utilization of food iron. K. Vitamin Lones in Foods Cooked by Chinese Procedures When vegetables and meats are "stir-fried" by the Chinese method, a shallow layer of fat is heated in a pan to about 280 degrees C and the diced ingredients are added in such an order that the cooking of all ingredients is completed at the same time. This method of cooking may be advantageous because the cooking time is short (generally 2-3 minutes), little of the vitamins and minerals is leached out and discarded, and the vitamins are protected against oxidation by the oil. A series of 19 authentic Chinese recipes was used to study the vitamin losses during "stir-frying." Competent Chinese women chemists prepared the dishes and analyzed the foods before and after cooking. Each recipe was prepared in duplicate. One portion was treated with five per cent oxalic acid to preserve it until analyzed, while the second was "stir-fryed." In each case the entire recipe (raw or stir-fried) was slurried and the carotene, thiamine, riboflavin, niacin and ascorbic acid content determined by chemical procedures. Results were compared on an anhydrous basis. The losses varied considerably in the various recipes. The losses in carotene were very low, and in 13 recipes there was an apparent increase. In general, the losses were less than 10 per cent for niacin, 15 percent for riboflavin, 20 per cent for thiamine and 35 per cent for ascorbic acid. These losses compare very favorably with those reported on foods cooked by Western methods. 11 L. Stability of Solutions of Pure Ascorbic and Dehydroascorbic Acid [illegible] two most acceptable procedures for the analysis of ascorbic acid in foods are the indophenol titration method of Bessey and the phenythydraxien method of Roe. Frequently, data obtained by these methods do not agree. We have measured the ascorbic and dehydroascorbic acid content of freshly prepared and aged aqueous solutions of pure ascorbic acid, and have compared the stabilizing effects of oxalic acid and metaphosphoric acid on these solutions. The results of these experiments have been published (Science 107, 226, 1948). It was found that ascorbic acid is stable in 0.5 per cent oxalic-10 per cent acetic acid solutions for at least 12 days at 4 degrees C. In five percent metaphosphoric acid-10 per cent acetic acid solutions it is stable for at least eight days at 4 degrees C. Oxalic acid seems preferable as a preservative for ascorbic acid not only because it is more effective than metaphosphoric acid but also because it is more stable, less expensive and more convenient to use. Though both metaphosphoric acid and oxalic acid exert a stabilizing effect on ascorbic acid, neither acid prevents the transformation of dehydroascorbic acid into derivatives which are not convertible to ascorbic acid by hydrogen sulfide treatment. The Roe method (using 2,4-dinitrophenylhydraxine) probably estimates the ascorbic acid originally present in a foodstuff when it was garden-fresh, while the Bessey method (using 2,6-dichlorophenolindophenol) measures only what is biologically active at the time of analysis. This may resolve the controversy on this subject in the literature. M. Estimation of Ascorbic Acid in Food Preparations Continuing the research mentioned in the previous section, we have demonstrated that the Roe method and Bessey method are equally valid for measuring the ascorbic acid content in garden-fresh materials studied was in the reduced form. The results of this study have been published (Anal. Chem. 20, 649, 1948). Samples slurried with four parts of 0.5 per cent oxalic acid and stored for as long as two week at room temperature (70-77 degrees F) may be assayed by the Bessey method to determine the ascorbic acid at the time of harvesting. Thus, by using a stabilized slurries of garden-fresh edible plants their ascorbic acid content at the time of harvest may be measured by the Roe method in a laboratory remote from the harvest area. We are applying this method in our Central American food analysis program. These methods may also be used to establish the freshness of vegetable foods. N. Retention of Vitamins During Cooking of Enriched Farina Several laboratories have studied the retention of vitamins during the cooking of enriched farina, with discordant results. This question is of considerable importance, since there is little value in 12 enriching farina with vitamins [illegible] during cooking. Three preparations of enriched farina were [illegible] in our study. These were prepared individually so that each batch contained known amounts of the enrichment ingredient. The first contained added thiamine, riboflavin, and niacin, the second added thiamine, added thiamine, riboflavin, and niacin, the second added thiamine, riboflavin, niacin and a mineral phosphate mixture. Each batch was cooked in a saucepan on an electric stove according to home procedures with care to have this procedure the same for all batches. Each entire batch was slurried for sampling. The average retentions for the three batches were as follows: thiamine, 98.9, 97.0, 96.6 per cent; riboflavin, 94.9, 98.4, 94.7 per cent; and niacin, 95.2, 100.0, 94.6 per cent. It is evident that less than five per cent of the thiamine, riboflavin or niacin content is destroyed during the cooking of enriched farina by home procedures. The results of this study have been published (J. Amer. Diet. Assoc. 24, 314, 1948). O. Protein Quality of Certain Foodstuffs Feeding experiments on 260 male rats showed that (1) the protein of egg albumen and of corn germ is of excellent biological value, (2) the protein of rolled oats is superior to that of soya flour and of Mexican rice, (3) the protein of white corn is inferior to that of yellow corn, (4) the preparation of the Mexican tortilla impairs the nutritional value of the corn protein, and (5) the protein of peanut flour is inferior to that of soya flour. Rats fed uncooked beans died a toxic agent, or that they are indigestible. These results were published in a Mexican scientific journal (Ciencia 7, 203, 1947). RESEARCH PROGRAM PROJECTED FOR 1948-49 During the coming year research on the following subjects will be conducted in our laboratories: A. The Metabolism of Deuteriumated Lauric Acid; B. Nutritional Importance of the Petroselinic and Petroselaidic Acids; C. Metabolic Interrelationships Between the B-Complex Vitamins and Arachidonic Acid; D. Composition of the Edible Plants of Central America; E. Effect of Phytates upon the Absorption of Radioactive Calcium; F. Influence of Liver Extract upon Rats on "Complete" Synthetic Diets; G. Effect of the Iso-oleic Acid Content of the Diet upon Growth; H. Nutritive Values of "Hard" Fats; I. Dermal Absorption of Deuteriumated Soaps and Fatty Acids; and J. Synthesis of Glycogen. A. Metabolism of Deuteriumated Lauric Acid Having completed a study of deuteriumated oleic acid we now wish to study other fatty acids which are commonly found in our food fats. Cocoanut oil is rich in lauric acid. A deuteriumated form of lauric acid will be prepared, and its metabolism will be 13 studies [illegible] the same procedures as were employed in the deuterium [illegible] oleic acid study. B. The Nutritional Importance of the Pedtroselinic and Petroselaidic Acids Our results with elaidic acid, and the controversial results of others who have worked with vaccenic acid, indicate that the various iso-oleic acids may differ in their nutritional values. We plan to study the nutritional values of oleic acids. Next we will study tripetroselinin and triptroselaidin, the triglycerides of the cis- and trans-isomers, respectively, of [illegible] oleic acid. C. Metabolic Interrelationship between the B-Complex Vitamins and Arachidonic Acid We have already accumulated considerable evidence that pyridoxine and arachidonic acid are interrelated in metabolism and possibly are synergistic. In searching for an explanation of this relationship we plan to test vitamins other than pyridoxine. Each of the B-complex vitamins will be omitted from the diets of rats to determine the effect of each omission upon the arachidonic acid content of tissue fats. D. Composition of Central American Foods This fascinating and fundamental survey of the nutritional resources of Central America will continue for another year. The we plan to write a monograph which will be a discussion of the food plants of Central America from the viewpoint of the botanist, the food chemist and the nutritionist. There remains the problem of stimulating an agricultural and food program in these countries which will lead to a better use of indigenous plant resources. E. Effect of Phytates upon the Absorption of Radioactive Calcium Most of the scientific literature on the effect of phytates in foods upon the utilization of minerals is concerned with studies of calcium absorption rather than that of iron, but in no case has the radioactive tracer technique been used to study calcium in relation to this problem. Now that radioactive calcium has been made available from the atomic pile, it is possible to conduct these experiments, but before preceding with such a study, it must be proven that radioactive calcium is not toxic for human beings. As a preliminary to this program, the absorption and excretion of radioactive calcium will be studied in rats. F. Influence of Liver Extract upon Rats on "Complete" Synthetic Diets In a preliminary experiment it has been observed that when a nutritionally "complete" experimental diet, containing liberal quantities of 14 vitamins, was supplemented with liver extract, the male animals were stimulated to grow more rapidly. The female rats seemed unaffected by the liver supplement. The factor in liver which is responsible for the growth of male rats must be investigated. 14 G. Effect of the Iso-oleic Acid Content of the Diet upon Growth When oils are hydrogenated in the production of commercial hydrogenated fats, considerable quantities of iso-oleic acids are formed which are different from normal oleic acid in chemical and physical properties and may also differ in biological value. An experiment in which the nutritional response to fats containing various percentages of iso-oleic acids is measured would be useful. H. Nutritive Value of "Hard" Fats There is still a question whether the absorption of fat from the intestine is dependent upon the "stearic acid" content of the fat, the saturated fatty acid content and/or the arrangement of the fatty acids i the triglyceride molecule. Also, it is not known whether the growth of rats is influenced by the arrangement of the fatty acids on the glycerol molecule. An investigation has been planned as an attack on this problem. I. Dermal Absorption of Deuteriumated Soaps and Fatty Acids Little is known of the ability of soaps or fatty acids to penetrate the dermal tissues of human beings. This subject has considerable theoretical and practical importance, and a study of the excretion of deuterium by subjects to whose skin deuteriumated compounds have been applied, would be valuable. J. Synthesis of Glycogen from Fats To our knowledge, no convincing experiment has yet been performed which proves the synthesis of glycogen from fats. We plan to isolate glycogen from the livers and carcasses of rats which have received deuteriumated lauric acid, and examine the glycogen for deuterium content. A "tagged" glycogen will be presumptive evidence of its synthesis from fat. PUBLICATIONS FROM THE LABORATORIES Reprints of publications by members of the staff of the Nutritional Biochemistry Laboratories are available to anyone requesting them. The following scientific papers were published during the period July 1, 1946 to June 30, 1948: 1. Sherman, H., M.K. Nutter, E.E. Lockhart, and R.S. Harris, "Studies on Farry Acid Metabolism by the Deuterium Technique I. Oleic Acid." Proc. Amer. Chem. Soc. p. 14A, 1946. 2. Tapia, M.A., F. deP. Miranda, and R.S. Harris, "Calidad en Proteinas de Alimentos Seleccionadas," Ciencia (Mexico) 7, 203, 1946. 3. Harris, R.S. and K.V. Thimann, "Vitamins and Hormones," Vol. IV, 400 pp., Academic Press, Inc. 1946. 4. Harris, R.S., "The Nutrition Problem of Mexico," J. Am. Dietet. A., 22, 974, 1946. 5. Harris, R.S., "Essentials of a Post-War Nutrition Program," N.Y. Legis. Doc. 76, 48, 1946. 6. Harris, R.S., "El complejo vitaminico B en la nutricion humans." Archives Urguayes de Medicina Cirugia y Especialidades 30, 1, 1947. 15