THE JOURNAL OF NUTRITION
OFFICIAL ORGAN OF THE
AMERICAN INSTITUTE OF NUTRITION
R i c h a r d H . B a r n e s , E d it o r Graduate School of Nutrition Cornell University, Savage Hall Ithaca, New York
H a r o l d H . W i l l i a m s E. N e ig e T o d h u n t e r Associate Editor Biographical Editor
EDITORIAL BOARD
G e o r g e K . D a v i s G e r a l d F. C o m b s R a l p h T . H o l m a n R i c h a r d H . F o l l i s , Jr . R u t h M . L e v e r t o n G e o r g e M . B r ig g s E. L. R. St o k s t a d G e o r g e V . M a n n R . M . F o r b e s Jo h n G. B i e r i M a x O. S c h u l t z e Ju l e s H ir s c h H a r r y P . B r o q u is t A . E. A x e l r o d E. E. H o w e R. G a u r t h H a n s e n
V O L U M E 82
JANUARY - APRIL 1964
PUBLISHED MONTHLY BY THE WISTAR INSTITUTE OF ANATOMY AND BIOLOGY
PHILADELPHIA, PENNSYLVANIA THE JOURNAL OF NUTRITION ® ® Trade mark registered, V. S. Patent Office
Copyright © 1964 by T h e W is t a r I n s t it u t e o f A n a t o m y a n d Bio l o g y
A ll rig h ts reserved Contents
No. 1 JAN U ARY 1964
Sir Jack Cecil Drummond, F.R.S. — A Biographical Sketch. Alice M. Copping ...... 1 Effect of Dietary Restriction of Pregnant Rats on Body Weight Gain of the Offspring. Bacon F. Chow and Chi-Jen Lee ...... 10 Effect of Degree of Fatty Acid Unsaturation in Tocopherol Deficiency- induced Creatinuria. L. A. Witting and M. K. Horwitt...... 19 Phosphorus Requirement of the Baby Pig. E. R. Miller, D. E. Ullrey, C. L. Zutaut, Betty V. Baltzer, D. A. Schmidt, J. A. Hoefer and R. W. Luecke 34 Zinc Deficiency Syndrome in the Young Lamb. E. A. Ott, W. H. Smith, Martin Stob and W. M.B eeson...... 41 Response of the Liver to Prolonged Protein Depletion. IV. Protection of Succinic Oxidase and Succinic Dehydrogenase by Dietary Methionine and Cystine in a Protein-free Ration. J. N. Williams, Jr...... 51 Supplementation of Wheat Gluten Protein. W. L. Banks, Jr., J. B. Allison and R. W. Wannemacher, Jr...... 61 A Determination of the Essential Amino Acid Proportions Needed to Allow Rapid Growth in Chicks. D. C. Dobson, J. O. Anderson and R. E. W a rn ick ...... 67 Relative Potency of Diets Containing Amino Acids and Proteins for Promoting Growth of Rats. W. D. Salmon ...... 76 Fatty Livers Produced in Albino Rats by Excess Niacin in High Fat Diets. I. Alterations in Enzyme and Coenzyme Systems Induced by Sup plementing 40% Fat Diets with 0.1% of Niacin. Lora Long Rikans, Dorothy Arata and Dena C. Cederquist ...... 83 The Amino Acid Composition and Nutritive Value of Proteins. V. Amino Acid Requirements as a Pattern for Protein Evaluation. P. B. Rama Rao, H. W. Norton and B. Connor Johnson ...... 88 Effect of Barbituric Acid, Chlortetracycline and Carbohydrates upon Growth and Gastrointestinal Urease Activity of Chicks. A. P. Alvares, L. H. Harbers and W. J. V is e k ...... 93 Absorption of Preformed Vitamin A from Ligatured Poultry Intestinal Sections. T. E. Shellenberger, D. B. Parrish and P. E. Sanford .... 99 Vanadium Toxicity and Distribution in Chicks and Rats. John N. Hath- cock, Charles H. Hill and Gennard M a tro n e ...... 106 Mineral Balance Studies with the Baby Pig: Effects of Dietary Phosphorus Level upon Calcium and Phosphorus Balance. E. R. Miller, D. E. Ullrey, C. L. Zutaut, J. A. Hoefer and R. W. Luecke...... I l l Adaptation of the Chick to Dietary Energy Source. W. E. Donaldson . . 115
ill IV CONTENTS
Effect of Calcium, Phosphorus and Manganese on the Intestinal Synthesis of Thiamine in Rats. S. K. Meghal and M. C. Nath 121 Effect of the Quality of Dietary Protein on Nitrogen Compounds in the Urine of Rats. Shuhachi Kiriyama and Kiyoshi A sh ida ...... 127 Pantothenic Acid Deficiency in Cats. S. N. Gershoff and L. S. Gottlieb . . 135 Influence of High Protein Diets on Vitamin A Metabolism and Adrenal Hypertrophy in the Chick. G. S. Stoewsand and M. L. S c o tt...... 139 Heat of Combustion Values of the Protein and Fat in the Body and Wool of Sheep. O. L. Paladines, J. T. Reid, A. Bensadoun and B. D. H. van N iek erk ...... 145 Some Biochemical Lesions Associated with Liver Fat Accumulation in Threonine-deficient Rats. Dorothy Arata, Catherine Carroll and Dena C. Cederquist...... 150 Influence of Nicotinic, Picolinic and Pyridine-3-sulfonic Acids on Cho lesterol Metabolism in the Rat. David Kritchevsky and Shirley A. Tepper ...... 157
No. 2 FEBRU ARY 1964
Influences of Dietary Carbohydrate-Fat Combinations on Various Functions Associated with Glycolysis and Lipogenesis in Rats. II. Glucose vs. Sucrose with Corn Oil and Two Hydrogenated Oils. Catherine Carroll ...... 163 Hypertension and Cardiovascular Abnormalities in Starved-Refed Swine. G. S. Smith, Jack L. Smith, M. S. Mameesh, J. Simon and B. Connor Johnson...... 173 Energy Intake and Fertility of Male Chickens. J. E. Parker and G. H. Arscott ...... 183 Effect of Stress from High Protein Diets on Vitamin A Metabolism in Chicks. G. S. Stoewsand and M. L. Scott ...... 188 Studies on the Digestion of Soybeans. H. J. H. de M uelenaere...... 197 Free Amino Acids in Plasma and Tissues of Rats Fed a Vitamin Bs-deficient Diet. Marian E. Swendseid, Juanita Villalobos and Barbara Friedrich ...... 206 Effects of Dietary Supplements on Young Rats Fed High Levels of Zinc. Aden C. Magee and Sandra Spahr ...... 209 Protein and Lysine as Factors in the Cariogenicity of a Cereal Diet. Mary L. D odds...... 217 Protein-Energy Relationship in Adult Rats. I. Effects of Varying Amounts of Dietary Protein and Food Energy on Nitrogen Utilization. Pilar A. Garcia and Charlotte E. Roderuck ...... 224 Protein-Energy Relationship in Adult Rats. II. Qualitative and Quanti tative Variations in Dietary Nitrogen and Effects on Deposition of Hepatic Fat and on Nitrogen Utilization. Pilar A. Garcia and Charlotte E. Roderuck ...... 231 CONTENTS V
Antagonism of Poorly Invertible D-Amino Acids Toward Growth Promotion by Readily Invertible D-Amino Acids. Shrinivas H. Kamath and Clarence P. Berg ...... 237 Antagonism of the D-Forms of the Essential Amino Acids Toward the Promotion of Growth by o-Histidine. Shrinivas H. Kamath and Clarence P. Berg ...... 243
Influence of Various Chelating Agents on the Availability of Zinc. Pran Vohra and F. H. Kratzer ...... 249 Reproductive Performance of Rats Receiving Various Levels of Dietary Protein and Fat. L. R. Richardson, Jackie Godwin, Stella Wilkes and Martha Cannon ...... 257 Effect of Vitamin A and Vitamin A Acid on Cerebrospinal Fluid Pressure and Blood and Liver Vitamin A Concentrations in the Pig. E. C. Nelson, B. A. Dehority, H. S. Teague, A. P. Grifo, Jr. and V. L. Sanger 263 Chemical Pathology of Acute Amino Acid Deficiencies. VI. Influence of Fat Intake on the Morphologic and Biochemical Changes in Young Rats Force-Fed a Threonine-Devoid Diet. Herschel Sidransky and Ethel Verney ...... 269 Exclusion of the Exocrine Pancreatic Secretion: Effect on Digestibility of Soybean and Milk Protein by Baby Pigs at Various Ages. Jerome C. Pekas, Virgil W. Hays and Alan M. Thompson ...... 277 Effect of Zinc Toxicity on Calcium, Phosphorus and Magnesium Metab olism of Young Rats. Alta K. Stewart and Aden C. M a gee...... 287
No. 3 MARCH 1964
Lactose and Calcium Transport in Gut Sacs. Yet-oy Chang and D. M. Hegsted ...... 297 Effects of Dietary Creatine and Related Compounds on Tissue Glycogen Deposition in Rats. W. R. Todd, Marilouise Harris and Lester Laastuen ...... 301 A Mechanism of Interaction between Dietary Protein Levels and Cocci- diosis in Chicks. W. M. Britton, C. H. Hill and C. W. Barber...... 306 Effects of Egg Oil, Cholesterol, Cholic Acid and Choline upon Plasma and Liver Lipids. Jane B. Walker and Gladys A. Em erson...... 311 Importance of Dietary Copper in the Formation of Aortic Elastin. Barry Starcher, Charles H. Hill and Gennard Matrone ...... 318 Dietary Fat and the Structure and Properties of Rat Erythrocytes. II. Stability of the Erythrocyte. Brian L. Walker and F. A. Kummerow 323 Dietary Fat and the Structure and Properties of Rat Erythrocytes III. Response of Erythrocyte Fatty Acids to Various Dietary Fats. Brian L. Walker and F. A. Kum m erow ...... 329 Metabolic Studies of an Amino Acid Imbalance in Cold-exposed Rats. George J. Klain and Robert L. Winders ...... 333 CONTENT
Serum Lipid Levels, Fat, Nitrogen, and Mineral Metabolism of Young Men Associated with Kind of Dietary Carbohydrate. M. Isabel Irwin, Doris D. Taylor and Ruth M. F eeley...... 338 Vitamin A in Serum and Liver of Weanling Pigs Fed a Protein-deficient Diet and Fixed Intakes per Unit of Live Weight of Carotene in Oil. H. D. Eaton, W. B. Boucher and P. C. S h a h ...... 343 Nutrient-to-Calorie Ratios in Applied Nutrition. E. W. Crampton ...... 353 Anti-rachitic Effects of Soybean Preparations for Turkey Poults. C. W. Carlson, J. McGinnis and L. S. Jensen...... 366 Carbohydrate and Fat Metabolism and Response to Insulin in Vitamin Be-deficient Rats. Agnes M. Huber, Stanley N. Gershoff and D. Mark Hegsted ...... 371 Tissue Distribution of a-Aminoisobutyric Acid and Nitrogen Metabolism in the Rat. II. Effects of Environmental Temperature and Dietary Imbalance. L. A. Bavetta and M. E. N im n i...... 379 Amino Acid Composition of Rye Flour and the Influence of Amino Acid Supplementation of Rye Flour and Bread on Growth, Nitrogen Efficiency Ratio and Liver Fat in the Growing Rat. R. Kihlberg and L.-E. Ericson ...... 385 Relationship between Tryptophan — Niacin Metabolism and Changes in Nitrogen Balance. Virginia M. Vivian ...... 395
No. 4 A PR IL 1964
Nutritional Studies with the Guinea Pig. X. Determination of the Linoleic Acid Requirement. M. E. Reid, J. G. Bieri, P. A. Plack and E. L. Andrews...... 401 Response of Digestive Enzymes to Dietary Protein. Jean Twombly Snook and J. H. Meyer ...... 409 Nonspecific Role of Arginine in the Promotion of Muscular Dystrophy in the Chick. K. J. Jenkins, D. C. Hill and Lilian M. Hutcheson . . . . 415 Nutrition and Biochemistry of Survival During Newcastle Disease Virus Infection. II. Relation of Clinical Symptoms and Starvation to Nucleic and Free Amino Acids of Avian Liver. Robert L. Squibb . . . 422 Nutrition and Biochemistry of Survival During Newcastle Disease Virus Infection. III. Relation of Dietary Protein to Nucleic and Free Amino Acids of Avian Liver. Robert L. Squibb ...... 427 Liver Lipid Accumulation in Isoleucine-deficient Rats. R. L. Lyman, C. R. Cook and Mary Ann Williams ...... 432 Effect of Potassium on Juxtaglomerular Cells and the Adrenal Zona Glomerulosa of Rats. Phyllis M. Hartroft and Elizabeth S ow a ...... 439 Adrenal Lipids of Cholesterol-fed Guinea Pigs and Rats. Rosemarie Ostwald, Angela Shannon, P. Miljanich and R. L. L y m a n ...... 443 Studies on the Utilization of Tempeh Protein by Weanling Rats. L. R. Hackler, K. H. Steinkraus, J. P. Van Buren and D. B. H a n d ...... 452 CONTENTS VII
Effect of Thyroxine, Thiouracil and Ambient Temperature on the Utiliza tion of Vitamin A by Vitamin A-deficient Rats. Thomas A. Anderson, Farris Hubbert, Jr. and C. B. Roubicek ...... 457 Influence of Protein and Energy on Growth and Protein Utilization in the Growing Chicken. J. D. Summers, S. J. Slinger, I. R. Sibbald and W. F. P e p p e r...... 463 Effect of Water Intake on Nitrogen Metabolism in Dogs. Ricardo Bressani and J. Edgar B rah am ...... 469 Nutrition of Salmonoid Fishes. XI. Iodide Requirements of Chinook Salmon. A. N. Woodall and Gilles La R och e...... 475 Effect of Pyridoxine Deficiency upon Valine Incorporation into Tissue Proteins of the Rat. Anthony C. Trakatellis and A. E. A xelrod ...... 483 Effect of Zinc Nutrition upon Uptake and Retention of Zinc-65 in the Chick. T. R. Zeigler, R. M. Leach, Jr., M. L. Scott, F. Hnegin, R. K. McEvoy and W. H. Strain ...... 489 Effect of Selenium, Sulfur and Sulfur Amino Acids on Nutritional Mus cular Dystrophy in the Lamb. H. F. Flintz and D. E. H o g u e ...... 495 The Role of Dispensable Amino Acids in the Nutrition of the Rat. L. H. Breuer, Jr., W. G. Pond, R. G. Warner and J. K. Loosli...... 499 Rachitogenic Activity of Soybean Fractions. C. W. Carlson, H. C. Saxena, L. S. Jensen and James M cG in n is...... 507 Index to Volume 82 ...... 513 SIR JACK CECIL DRUMMOND, F.R.S. (1891 — 1952) Published with the permission of the Controller © British Crown of Her Britannic Majesty’s Stationary Office
Sir Jack Cecil Drummond, F.R.S Sir Jack Cecil Drummond, F.R.S. — A Biographical Sketch
( January 12, 1891 — August 4, 1952)
The advance of biochemistry and the Great Britain and other countries remem growth of scientific nutrition from bio ber her kindly grace as hostess in the chemical bases owe much to the work and Drummonds’ home at Chorleywood in Hert personality of Sir Jack Drummond. His fordshire and at functions associated with interests in biochemistry and nutrition Biochemical Society meetings. In the early were extraordinarily diverse and he had a days of the Society, Biochemical dinners great gift for imparting enthusiasm to were exclusively for men and Mrs. Drum others. From this talent, which Drum mond started a delightful custom of enter mond well knew how to develop, the world taining women scientists and wives of the at large has derived great benefit in the biochemists at rival parties. In the course teachers and research workers who were of the years men and women came to dine trained in the Department of Biochemistry together and Mrs. Drummond was always at University College, London. Drummond a happy member of the parties of the early had abounding mental and physical en nineteen-thirties. Overseas students and ergy and was accustomed to work hard visitors were happily received by the Drum and play hard. His vitality was infectious monds in their home. Both took country and he was most successful in training pursuits of gardening, golfing, walking, others for senior posts. Moreover he fre watching birds with great pleasure and quently infused some of his own entertain introduced others to these delights through ing ideas into those around him. His lec their own knowledge. A week-end at Chor tures were never dull and his suggestions leywood was a memorable occasion for on research problems were always illumi many a young student from abroad. nating and sometimes even startling. They Drummond’s first post after graduation certainly aided many a struggling young was as a research assistant at King’s Col research worker to arrive at a satisfactory lege, University of London, where he came development of a difficult problem. into contact with Professor W. D. Halli Early years. Jack Cecil Drummond was burton and Dr. Otto Rosenheim, so that his born on January 12, 1891, at Leicester, initial chemical training was diverted into the only son of Major John Drummond of biochemical channels. Though he moved the Royal Artillery and of Mrs. Drummond. from King’s College to the Research Insti His parents died in India when he was tute of the Cancer Hospital in 1914, Drum very young and he was brought up by an mond’s career was profoundly influenced uncle and aunt, Captain and Mrs. Spinks, by both Halliburton and Rosenheim. At the in a somewhat rigid nonconformist atmos Research Institute he worked under Dr. phere. His early education was at the Casimir Funk who was in charge of bio Roan School, Greenwich, and at the Strand chemical research. In 1912 Funk, then at School of King’s College. From there he the Lister Institute, had published a paper went in 1909 to East London College (now on “The aetiology of the deficiency dis Queen Mary College) and three years later eases” (J. State Med., 1912, 20, 341) in he was graduated with a Bachelor of Sci which he introduced the term “Vitamine” ence degree from the University of London for the first rime. The origin of the term with first class honours in Chemistry. Dur was in Funk’s attempts to isolate from rice ing his student days at East London he polishings an antipolyneuritic principle. met Mabel Straw as a fellow student and The product was a basic nitrogenous sub later married her. Many biochemists in stance, possibly an amine, and on this
J. N u t r i t i o n , 8 2 : ’ 6 4 3 SIR JACK CECIL DRUMMOND, F.R.S. basis Funk suggested that vitamines might University College, 1919-1929. By 1918 be nitrogenous substances essential in the Drummond was already recognised as an diet of birds, man and animals. Drum expert on nutrition and he received the mond himself some years later, in 1920, degree of Doctor of Science. In this year suggested the change to “vitamins,” with also he was appointed to succeed Funk as the omission of the final “e,” as the term Physiological Chemist to the Cancer Hos to cover these essential substances since pital. A year later he moved, on the invita some were not amines. Thus Drummond tion of Professor E. H. Starling, to Univer sity College, London. Here he was made was introduced to the new ideas fertilising Reader in Physiological Chemistry in 1920 the field of nutrition. His second scientific and in 1922 he was appointed to the new publication was written jointly with Funk Chair of Biochemistry at the College. At on the chemical investigation of the phos- this time Katharine Coward, who was later photungstic precipitate from rice polish appointed Head of the Nutrition Depart ings. Studies of amino-acid composition of ment of the Pharmaceutical Society of dietary proteins were in progress at the Great Britain, came to work under Drum same time as the vitamin studies and mond on the fat-soluble accessory food Drummond investigated the possible effects substance, vitamin A. Their joint publica of deficiency of protein and of vitamins on tions over the years 1920-25 form the the growth of tumours in experimental ani framework of much of the fundamental mals. Although by dietary means no in study of vitamin A in foodstuffs and in hibition of tumour growth could be dem pharmaceutical preparations such as cod onstrated without serious general nutrition liver oil. Drummond’s interest in the prac failure in the host animal, such negative tical side of the problem took him into the information was invaluable for planning Norwegian fishing industry to investigate future studies of the relation between nu the processing of cod liver oil and other trition and tumour growth. fish liver oils. His interest in vitamin A With the outbreak of war in August did not preclude attention to vitamin D. 1914, Drummond found to his regret that In collaboration with S. S. Zilva and J. his health did not permit him to serve in Golding, research was carried out on the the Armed Forces. He turned his energies relation of fat-soluble vitamins to rickets to research on food problems carried out at and growth in pigs, and on feeding cod the Cancer Research Institute under Dr. liver oils to cows in winter. The origin of Alexander Paine who taught him the essen fat-soluble vitamins in fish liver oils was tials of methods of feeding diets of con investigated with the aid of marine biolo trolled composition to rats and other gists, and studies were made on the chem experimental animals. War-time studies ical nature of vitamin A. An important included comparative tests on butter and observation made in 1920 with the help of margarine and other butter substitutes O. Rosenheim on the relation of lipo- leading to an interest in fat-soluble as well chrome pigments to vitamin A was the as water-soluble vitamins. Moreover the first indication of the relation between researches on animals and the chemical carotene and vitamin A. Unfortunately the analyses were never purely laboratory ex animal tests in this early study gave nega ercises but led to practical applications as tive results. Some years later it was shown illustrated by a paper in 1918 on “Some that the preparation of the doses of car aspects of infant feeding.” In this publica otene in ethyloleate had permitted oxida tion he pointed out the demand for acces tion of the pigment so that its activity was sory food factors during the period of not demonstrated. Moreover it was not growth and the depletion of the tissues of then appreciated that carotene in solution the mother if the maternal diet was inade was highly susceptible to oxidative destruc quate. He used his experimental observa tion by exposure to light. Drummond him tions to illustrate the point that many self, in a communication at a meeting of substitutes for mother’s milk might be the Biochemical Society, took full responsi seriously deficient in both water-soluble bility for the initial error of observation. and fat-soluble vitamins. This was a gesture characteristic of the BIOGRAPHY O scientific stature and human generosity of tion became the paramount interest in the man which endeared him to those who Drummond’s life. His biochemistry depart were privileged to work in his laboratories ment was running smoothly in the hands and under his guidance. of well-tried colleagues and he was able to Although in the years 1920-30 much of develop practical ideas on the urgent prob Drummond’s attention was given to vita lems of nutrition arising from the eco min A and other matters relating to fats nomic crisis of 1931. He had also had and fat-soluble vitamins D and E, his in some field experience of nutritional defi terest in proteins and in water-soluble vita ciencies beginning with a visit to the fisher mins was maintained. The physiological men of Newfoundland in the course of a role of vitamin B was under investigation study of the cod liver oil industry. There and some of the early work on the complex he observed nutritional deficiencies among nature of vitamin B came from his depart the people caused by the lack of the very ment. At this time the Department of substance present in the products they pre Biochemistry at University College was pared. He surveyed this and related prob small and was part of the Department of lems in the Lane Medical Lectures which Physiology and Biochemistry in the Medi he was invited to give at Stanford Univer cal Sciences Faculty, but the impetus of sity, California, in 1933. He gave a Harvey the research under Drummond soon made Lecture in New York in the same year and the department an important centre which also the Harben Lectures of the Royal exerted an enormous influence on the Society of Arts. In these public lectures he growing subject of biochemistry. At the gave a panoramic view of the modern ideas time of his death in 1952 no fewer than on the functions of the vitamins. His able nine of his pupils were Professors of Bio exposition of such subjects in a way which chemistry in Great Britain or in other coun was intelligible to a lay audience and at tries. By 1925 scientists from many the same time of interest to more scientific countries were anxious to join Drum minds led to extensive demands on his time mond’s research group. They were made for lectures in many countries. This was welcome and the international exchange freely given for he enjoyed travel and com of ideas was one of the most valuable munication with others of like mind. One aspects of research in the Department of of his most extensive journeys was in 1936 Biochemistry. under a short-term Fellowship from the With all the research activity there was Rockefeller Foundation when he visited no neglect of the teaching of medical stu Holland, Germany, Czechoslovakia, Aus dents and of undergraduates who studied tria, Hungary, Switzerland, Poland, Russia, biochemistry as part of a degree in physiol Finland, Sweden, Norway and Denmark in ogy. Drummond’s own lectures were in order to study nutritional conditions. He evitably stimulating and he naturally at had for many years been interested in the tracted a strong team of other teachers. “Peoples’ League of Health,” an organiza He preferred to lecture on the aspects of tion founded in 1917 by Miss Olga Nether- biochemistry which belong to the field of *sole, C.B.E., in Great Britain. This organ nutrition, but his field of vision was wide ization endeavoured to help and to educate and allowed a full participation in the people in the use of food especially in the teaching programme. If all medical stud stringency of unemployment and other sit ents could be inspired by as stimulating a uations leading to poverty and poor nutri teacher as Drummond, their subsequent tion and Drummond was its constant ad attention to problems of nutrition might visor. He became involved in an advisory be more extensive. Much of the distin capacity with many other bodies concerned guished medical research of the past 30 with nutrition including the Ministry of years at University College Hospital and Health, the British Medical Association, other centres derives from men and women and the Health Section of the League of who had premedical biochemistry training Nations. from Drummond. During this time he was beginning to Expanding interests, 1929—1939. In his look into the historical aspects of diet and second decade at University College nutri nutrition. His foreword to ‘The Schoolboy, SIR JACK CECIL DRUMMOND, F.R.S.
His Nutrition and Development,” by G. E. was happily aided by his secretary, Anne Friend (1935) is an admirable example of Wilbraham, who in 1940 became his sec his historical knowledge of his subject. ond wife. Their joint labours were given After a look into the further past he makes first in a delightful series of lectures at the some excellent comments on the nutrition Royal Society of Arts (J. Roy. Soc. Arts, lessons of the 1914-18 war and goes on to 1938, 76, 191; 214; 246). The greatly ex say: panded text was published in 1939 as “The “It is a disheartening task to search the Englishman’s Food. Five Centuries of wreckage of our post-war world in the English Diet.” This enthralling history of hopes of finding some benefit, however diet and dietary practices was the culmina small, which the upheaval brought directly tion of Drummond’s preparatory work for or indirectly to mankind. One there is, al the tremendous part which he was to play though it is seldom recognized. It is the as nutritional adviser in the war years and vast progress and powerful stimulus to fur ther work in the field of nutritional re the years of recovery. search for which the food problems of the The war years. On the outbreak of war war years were to a large extent respon in 1939 Drummond was given leave from sible. Few people adequately realise how the University to be Chief Adviser on Food extensive have been the advances made in Contamination and as a result of his initial nutrition and in the field of practical die activities a manual on “Food and its Pro tetics during the past 20 years. Already as tection from Poison Gas” was published a result of these labours the standard of from the Ministry of Food in 1940. With national physique is noticeably improving his broad vision of nutrition he was by no in this country and the florid forms of nu means fully occupied with the food con tritional disorders, once so common, are tamination work. His own University de fast becoming rarities. partment had temporarily been moved to “But in spite of this progress there is yet Wales, but he kept some academic research a long way to go and in no direction is going at the Courtauld Institute of Bio greater guidance needed than where the chemistry at the Middlesex Hospital, Lon feeding of school children is concerned. don, by courtesy of the Director, Professor How deplorably the subject has been neg E. C. (later Sir Charles) Dodds. On Febru lected we are only just beginning to appre ary 1, 1940, Drummond was officially ap ciate. Almost entirely ignored by the pointed Scientific Adviser to the Ministry of medical profession until recent years, its Food. Immediately prior to this official ap treatment by those responsible for the pro pointment he had submitted to the Ministry vision and preparation of school diets was, of Food a memorandum, “On certain nu with relatively few notable exceptions, de tritional aspects of the food position,” in plorable for a long time. Nor are the par which he reviewed the pre-war nutritional ents free from blame. Many a well-to-do position of Great Britain and the probable father has given far greater attention to effects on it of wartime conditions, par the feeding of his own horses, dogs or farm ticularly in relation to the poorer sections stock than to the diet on which his son of the population. He stressed the need for might be subsisting at a famous public providing bread of high nutritive value, for school.” increasing the consumption of potatoes, This introduction to a most useful oatmeal, cheese and green vegetables, for treatise on adolescent nutrition was written supplying not less than a pint of milk a about the same time as a chapter on “The day to expectant and nursing mothers and Vitamins” contributed to a “Textbook of to all children up to the age of 15, and for Biochemistry,” edited by B. Harrow and fortifying margarine with vitamins A and C. P. Sherwin. In his department, research D. He drew attention to the value of the was in progress on fat-soluble vitamins A, lean meat of the pig as a source of vitamin D and E, on various B vitamins, on carbo B, and at the same time made it clear that hydrate and protein problems. And in there was no special need for large supplies every moment he could find his historical of meat provided adequate supplies of researches were proceeding apace. Here he cheese and fish were available. BIOGRAPHY 7
Within a very short time after his ap out under the guidance of the Accessory pointment, his knowledge and influence Food Factors Committee of the Lister In were clearly reflected in the programme of stitute and the Medical Research Council. food inports for the second year of the war, As a result of the studies the Ministry of to which was appended “A Survey of War Food in 1941 raised the extraction rate of time Nutrition.” This set out in detail flour to 85% and fortified the flour with Drummond’s views on nutritional strategy. calcium to ensure that the daily intake of In the light of the history of nutrition in this essential nutrient should be satisfac the 1914-18 war he made strong recom tory for all groups of the population at a mendations against measures to reduce the time when milk, eggs and meat were in country’s total consumption of food. He short supply. The flour and bread orders was uniquely equipped to see the problems were only one expression of Drummond’s of food supplies and nutritional demands determination that the nutritional value of as a whole and his personal success in col the British diet should not merely be main laborating with statisticians and econo tained but should be improved under war mists produced a food plan for which time conditions. Great Britain must always be profoundly He was fully aware of modern advances grateful. He reviewed the food situation of in food technology and encouraged the im the first world war and endeavoured to porting of dehydrated foods to improve pro plan to avoid the nutritional failures aris tein supplies. Large quantities of dried ing in those years. A comprehensive ra eggs and dried milk were thus obtained tioning system spread supplies evenly over and his appreciation of the difficulties of the population and reviews of home pro the housewife in handling unusual foods duction showed the quota of essential nu led to the information talks and leaflets for trients that could be provided from full use which the Ministry of Food became fam of resources, and the amounts which must ous. Every opportunity was taken to teach be imported in order to balance the dietary simple nutrition in all sections of the com budget. Increased imports were advised of munity. For this purpose the Food Advice cheese, dried and condensed milk, canned Service of the Ministry was established and fatty fish and dried pulses. Concentrates staffed with trained dietitians able to help of vitamins A and D for adding to marga interpret the ideas to the people. rine were considered an import priority. Another valuable contribution to good He recommended that imports of fruit, nutrition and good health with which other than oranges, nuts and eggs in shell Drummond was closely associated was the should be reduced as they were wasteful provision of special foods for mothers and of shipping space. children. He was acutely aware of the spe Recommendations were made also for cial needs of vulnerable groups and as a re satisfying the vitamin C requirement by sult of his efforts schemes were evolved increased consumption of potatoes and for for cheap supplies and priority rationing of increasing the vitamin B< supply by raising fresh milk, for provision of blackcurrant the extraction rate of flour or by fortifying syrup, rose-hip syrup, concentrated orange white flour with synthetic vitamin Bi. The juice and cod liver oil as sources of vita fortification of flour with vitamin Bi was mins. There were also generous allow favoured by many as it left a larger share ances of rationed foods allocated for school of milling products for feeding pigs and meals. It is a measure of Drummond’s poultry. However the Medical Research sense of history that the provision of wel Council Memorandum of August, 1940 fare foods for mothers and children has (Lancet, 1940, 2, 1943) strongly recom been carried on beyond the end of the war mended bread based on a flour of 80 to and beyond the end of rationing as a per 85% extraction. Dr. T. Moran, Director of manent requirement for the health of the the British Flour Millers’ Research Associa population. tion, was closely involved in all the discus Another great enterprise begun by the sions and studies of the bread problem. Ex Ministry of Food under its strenuous Sci perimental work on flours of 72, 80, 821/2, entific Advisor was the Household Dietary 85% and higher extractions was carried Survey. This began in 1941 and is still 8 SIR JACK CECIL DRUMMOND, F.R.S. carried on as a check on the diet of repre Food. He was knighted in 1944 and in the sentative samples of the population. same year he became a Fellow of the The work of the Ministry of Food and Royal Society. In 1946 he received the the Ministry of Health in the distribution U. S. Medal of Freedom with Silver Palms of food supplies was recognized in 1947 by and was elected an Honorary Member of the Lasker Group Award of the American the New York Academy of Sciences. He Public Health Association. The citation was made a Commander (Civil Division) gives at length the details of the work of of the Order of Orange Nassau for his serv the two ministries in maintaining and even ices to Holland. The University of Paris improving health under war time stress conferred on him the Degree of Doctor and recommends awards to the Ministries honoris causa. and to the four leaders concerned: Lord In 1945 he accepted appointment as Woolton, Sir Jack Drummond, Sir Wilson Director of Research to Boots Pure Drug Jameson and Sir John Boyd Orr. Company at Nottingham and resigned his With all his commitments in the Minis chair of Biochemistry at University Col try of Food, Drummond still found time to lege. He did not go to Nottingham until keep in touch with his University Depart 1946 because he was still involved as nu ment which from 1941 was at Leatherhead tritional adviser to the Control Commis in Surrey. From 1941-1944 he was Fuller- sioners in Germany and Austria. ian Professor of Physiology in the Royal Very soon after he had taken up his re Institution and during his tenure of this search post with Boots Pure Drug Company Chair he gave four courses of lectures on he was elected to the Board of Directors of nutritional subjects and two Friday even the Company. Here again his wide experi ing discourses. He was concerned also in ence and his ability to understand and international discussions on nutrition. work with all sorts of people gave him a When the nutritional situation in Malta be full outlet for his energies. He still con came serious in 1942 he flew out to review tinued to travel and to exert his valuable it himself and to advise on relief measures. personal influence for the solution of In 1943 he was delegate to the Hot Springs diverse problems of industry and research. Conference in Virginia so that he was pres ent at the planning sessions which led to He was a powerful catalyst in the process the formation of the Food and Agriculture of lowering barriers of reaction between Organization of the United Nations. scientists in industry and in academic situ Post-war period. In 1943 Drummond ations. became Chairman of the Hospital Catering The bibliography relating to Drum and Diet Committee of the King Edward’s mond’s publications runs to nearly 200 Hospital Fund for London and continued titles yet he found time in life for so very to advise on hôpital food services until many other things than work. He was no 1949. He was the first President of the mean artist and could quickly illustrate a Hospital Caterers’ Association. point of design on paper. He appreciated Towards the end of the war he became art in pictures, in the theater and in music. adviser on nutrition to S.H.A.E.F. and to Moreover he showed those who worked the Allied Post-War Requirements Bureau. with him the way towards appreciation of One of his first tasks was the relief of the the finer things of life. He was good com starving Dutch people before hostilities had pany and enjoyed good food and wine. He finally ceased. A characteristic side line of liked dancing and often organized parties this terrific effort was his visit to his old from his Department to dances and enter friends, Professor and Mrs. Jansen in tainments of the societies to which he be Amsterdam, to assure them of the safety of longed. He was gay and debonair and their son from whom they had not heard seemed enternally young. He made friends for some years since his escape from oc quickly but displayed a tonic critical fac cupied Holland. ulty that had a profound effect on all who Many well deserved honours came to had contact with him in his many spheres Drummond for his work in the Ministry of of influence. BIOGRAPHY 9
He took life with both hands and made perhaps a last tribute to his youthful great use of his opportunities, so that the vitality. news of the murder on August 4, 1952, of Those of us who were his pupils at Uni Sir Jack and Lady Drummond and their versity College or his colleagues at the ten-year-old daughter while they were on Ministry of Food will for years to come camping holiday in France caused infinite continue to be inspired by his achieve dismay to all who had known them. Al ments and to try to carry on the progress of though he was 61 at the time of his death nutrition. he was described as a man of 40 by a A l i c e M. C o p p i n g French newspaper reporter who came to Queen Elizabeth College, the scene of the crime before the identity University of London, of the victims was established. This is London, W.8., England Effect of Dietary Restriction of Pregnant Rats on Body Weight Gain of the Offspring* 1
BACON F. CHOW and CHI-JEN LEE Department of Biochemistry, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, Maryland
ABSTRACT The effect of dietary restriction on various parameters of nutriture has been reported by a number of investigators. In all these studies, the restriction was imposed at various ages after birth. Our present study utilizes the method of dietary restriction but differs from others in that the restriction was imposed on the mother during either the gestation or lactation periods, or both. Its effect on the body weight gain and other characteristics of the offspring are reported. Restriction during gesta tion and lactation of the dietary intake of the rats by as little as 25% of that con sumed by the unrestricted group, resulted in growth stunting of the progenies, anemia and a decreased resistance to hypothermia. Similar effects were observed when the dietary restriction was imposed during the period of gestation only. These adverse effects could be corrected by pituitary extract or growth hormone when the hormone was administered shortly after weaning, but not 3 months after birth.
The effect of dietary restriction on the consumed food plus spillage was measured various parameters of nutriture has been and the food consumption measured by the subject of many studies (1 -6 ). In all difference. Each of the females was then these reports the restriction was imposed returned to the original breeding cage to after birth and at various ages after birth. reconstitute the original groups. On day Our present study utilizes the method of 2, the average amount of food consumed dietary restriction, but differs from others determined for the 9 females maintained in that this stress was imposed on the with the unrestricted diet was calculated mother during the gestation or lactation and quantities corresponding to 0.5 or 0.75 periods, or both. Its effects on body weight that amount were weighed into individual gain ar d other characteristics of the off feed cups and offered to the 15 females in spring are reported here. This communica the 5 restricted groups, after they had been tion contains data obtained in similar stud distributed among individual cages in the ies conducted initially in 1954 and later morning. In most instances, these animals confirmed and expanded in 1962. consumed the entire ration offered. Similarly on day 2, the intake of the un EXPERIMENTAL PROCEDURE restricted group was measured and there Design of experiment. A typical experi after daily. These daily values then con ment involved the use of 8 groups of 3 fe stituted the baseline for setting the intakes males each, chosen at random from a pop for the restricted groups for the balance ulation 3 to 4 months of age. Under our of the experiment. The regimen described standard mating conditions, these together was followed in some experiments during with 8 males, 3 to 4 months of age, com the period of gestation and lactation, and prised 8 breeding groups. Of these groups, in others during gestation only. 3 were assigned for unrestricted intake, After the litters were cast, litter size was the remainder were restricted to levels of arbitrarily adjusted so that no female was either 0.5 or 0.75 of the intake of the un left to nurse more than 8 young, regard restricted females established by the fol less of the previous dietary history. In this lowing technique. way such obvious complications as might The 9 females in the unrestricted groups arise from infection, unequal distribution were transferred to individual cages at of milk and other variables that might re- 9:00 a m on day 1 of the breeding period and each offered a measured amount, usu Received for publication August 5, 1963. 1 This research was supported by U. S. Public Health ally 35 g stock diet. After 8 hours, the un Service grants AM 07210-01 and HD 00475-07.
10 J. N utrition, 8 2 : ’64 MATERNAL DIET AND GROWTH OF THE OFFSPRING 11 suit from excess litter size, were substan TABLE 1 tially avoided. Stock diet Composition of stock diet. In the first kg experiments, begun in 1954, a stock diet Whole wheat 13.5 whose composition is given in table 1 was Maize 9.1 used. In more recent studies, a commercial Rolled oats 8.2 preparation2 was used. Both diets sup Whole milk powder 5.7 Skimmed milk powder 5.7 ported satisfactory reproduction and C asal1 0.5 growth of our stock animals. Fresh kale Liver powder 0.9 was provided in ample amounts 3 times Sodium chloride 0.23 every week. Calcium carbonate 0.12 Ferric citrate 0.1 Hormone preparations used. One hun Copper sulfate 0.02 dred milligrams hog pituitary powder3 were 1A protein preparation of the Crest Foods Com extracted with 100 cm3 0.85% NaCl solu pany, Ashton, Illinois, containing crude casein and tion, adjusted to pH 5.6 and centrifuged. lactalbumin. The clear supernatant was used for subse males are then transferred to individual quent injection. Growth hormone4 with an cages of the same size and kept there with activity of 1 USP unit/mg from bovine their litters until the weanlings are sepa origin was also used. Fifteen milligrams rated at 21 days of age into individual were weighed on a Roller-Smith balance cages of 18 X 18 X 23 cm. and dissolved in 50 cm3 normal saline This mating procedure was used in the solution. preceding experiments. Laboratory animals. In both studies, rats from our colony of the original McCol RESULTS lum strain were used. Our standard mat Effect of dietary restriction during gesta ing procedure is to house one male rat of tion and lactation periods on reproduction established fertility with 3 females in a and body weight changes in pregnant rats. breeding cage (64 X 32 X 50 cm), with Table 2 summarizes the observations of a wood shavings. Under ordinary conditions, all our rats become pregnant within 4 2 Guaranteed analyses: crude protein not less than weeks. When definite physical signs of 23.0%. pregnancy are established by gross observa 3 Courtesy of Armour and Company, Chicago. 4 Courtesy of Merck Sharp and Dohme, Rahway, tion or by increase in body weight, the fe- New Jersey.
TABLE 2 Effect of dietary restriction during gestation and lactation periods on reproduction
Mean A in Treatment1 Pregnancies/ Young/ Avg maternal female litter fetal wt body wt 2
9 9 UR-l 3/3 10 6.0± 0.5 3 40 UR-2 3/3 11 7.0 ±0.7 48 UR-3 3/3 9 7.0±0.7 37
Mean 10 6.7 41.7
R-l 2/3 2 6.0 ±0.0 5 R-2 2/3 6 4 .0± 0.5 1 R-3 2/3 6 4.5 ±0.5 - 5 R-4 2/3 7 3 .0± 1.1 6 R-5 1/3 10 4.5 ± 0.8 - 1 0 R-6 1/3 8 4 .0± 1.1 - 1 0
Mean 6 4.3 -1 .3 1 UR indicates pregnant rats fed ad libitum during the test periods; R indicates restriction of dietary intake of pregnant rats to one-half that of the unrestricted group. • 2 Mean change in maternal body weight indicates weight after delivery minus the initial body weight before mating. 3 se of mean. 12 BACON F. CHOW AND CHI-JEN LEE
typical experiment and shows quite clearly the adverse effects of restriction on repro duction as measured by all parameters re ported. The levels of restriction chosen al lowed the females to go through pregnancy with virtually no weight loss. Weight gain of the offspring from mothers with or without dietary restriction. In studies conducted in 1954 and 1962, the body weight changes of both male and female offspring from both groups of mothers were recorded weekly during the first 4 weeks after weaning, bi-weekly up to 6 months of age and monthly there after. Our routine procedure calls for the removal of feed early in the morning and weighing the animals at least 2 hours later. The results of one of the 1954 studies are shown in figures 1 and 2. Our own stock diet was used. The mean body weights of the offspring in the restricted group were consistently below those of the unrestricted group for both sexes. The lower weight was observed for as long as one year after wards.5 The plateau values where the mean body weights finally level off indicate that the offspring of the restricted groups Fig. 1 Growth rates of male rats born to had a distinctly lower body weight; in the mothers whose dietary intake was unrestricted, case of the male, by as much as 25 to 30% , or restricted to one-half that of the unrestricted group. Data from study conducted in 1954 with 5 Results after 40 weeks not included in figures 1 and 2 due to differences in mortality rates between stock diet prepared in our laboratory are plotted. the 2 groups.
Fig. 2 Growth rates of female rats born to mothers whose dietary intake was unrestricted or restricted to one-half that of the restricted group. Data from study conducted in 1954 with stock diet prepared in our laboratory are plotted. MATERNAL DIET AND GROWTH OF THE OFFSPRING 13 and in the female rat by 15%. In our animals. In another experiment similar 1962 experiment, the commercial labora pituitary extracts were given to young from tory chow diet was used. The results the unrestricted mothers 3 months after plotted in figure 3 are essentially similar to weaning. No corrective effect was ob those just discussed. served. Effect of pituitary hormone on the cor Crude pituitary extract contains a num rection of growth retardation. The gonads ber of factors. To determine whether any of the animals in the restricted group ap growth hormone in the pituitary prepara peared to be underdeveloped. We under tion was responsible for the restoration of took, therefore, to determine whether the growth rate, we injected a highly purified animals from the restricted mothers suf growth hormone preparation 3 times/week fered from pituitary atrophy. To this end, for 3 weeks. This preparation was used in weanlings from the unrestricted group and place of the crude pituitary extract, and from the restricted group were each sub administered to male rats of the restricted divided into 2 groups. One of these re and unrestricted animals. The results of ceived an injection of hog pituitary extract this more recent experiment are given in 3 times/week for 4 weeks, whereas the figure 4. Growth hormone in the dosages other received saline injections as controls. given had essentially the same effect as All animals were given the stock diet ad crude pituitary extract in stimulating the libitum and weighed weekly. The growth growth rates of the restricted animals. It is rate of these animals is recorded in table 3. likely that this, then, is the hormone re The injection of hog pituitary extract sponsible. Data in figure 4 also demon promptly corrected the growth retardation strate that injection of the growth hormone of the restricted animals, but had no ef had no effect on the growth rate of the un fect on the unrestricted animals. This cor restricted animals. rective effect persisted for at least the 8 Anemia. Hematocrit and hemoglobin months during which we observed these determinations were made on blood sam-
Fig. 3 Growth rates of male and female rats fed unrestricted and restricted diets. The reduction in the dietary intake was 25%. The commercial laboratory chow diet was used. 1 4 BACON F. CHOW AND CHI-JEN LEE
TABLE 3 Effect of administration of crude hog pituitary extract on body weight of rats fed, restricted diet1
Body weights
Weeks Male Female in jection Hog Hog Saline Saline pituitary pituitary solution solution extract extract
9 9 9 9 0 28.7± 1.0 2 29.8 ±1.6 22.7 ± 1.0 22.1 ±0.7 1 38.8± 1.1 43.7 ± 1.4 34.0±0.8 42.0 ± 1.0 2 76.8 ±2.0 92.7 ±2.5 62.0 ±1.4 81.0 ± 1.4 3 118.3 ± 3.0 138.0±3.1 78.0 ±1.8 111.0±2.0 6 182.4 ± 3.6 231.0±4.8 117.0± 1.2 159.0 ± 3.0 9 226.0±4.1 301.0±4.7 134.0 ±2.7 196.0 ±5.0
1 Eight rats (21 to 24 days weanling) were used in each group. Hog pituitary extract or a saline solution was administered subcutaneously 3 times/week for 3 weeks only. 2 se o f mean.
Fig. 4 Effect of growth hormone on growth of normal and restricted rats. Purified growth hormone (0.3 m g) was injected subcutaneously to restricted and unrestricted rats at 3 weeks of age every other day for 3 consecutive weeks, after which no injection was given. The growth rates are plotted. pies from rats of both groups at varying were placed in individual plastic boxes ages. Table 4 shows the data for females (perforated for adequate heat transfer). of both groups at 6 and 24 weeks of age. These boxes were small enough to immobil The differences in the hematologic picture ize substantially the animals, yet allowed which were evident at 6 weeks of age were enough room for limited movement. The absent at 24 weeks of age despite the per boxes were then lowered into a large tank sistence of gross effects as indicated by of ice water mixture kept at 0°C. The sur weight difference. vival of both groups of animals is recorded Effects of hypothermia. To determine in table 5. None of the 10 male animals the abilities of rats of the 2 groups to and one out of the 10 females in the un withstand a physical stress such as hypo restricted group died after 15 minutes of thermia, 6-month-old rats of both sexes exposure, whereas 6 out of 10 males and MATERNAL DIET AND GROWTH OF THE OFFSPRING 1 5
TABLE 4 Hematocrit and hemoglobin analyses of female rats of different ages fed restricted and unrestricted diets
Age Treatment1 Six weeks Twenty-four weeks Body wt Ht Hb Body wt Ht Hb
9 % g / 10 0 m l 9 % g / 10 0 m l UR 149 ± 5.0 2 44 ± 1.1 1 3 .2 ± 0 .5 4 250 ± 6 .2 47.0 ±0.68 14.5 ± 0 .2 2 R 7 8 .6 ± 3 .9 38 ± 1 .2 1 1 .6 ± 0 .4 7 215 ± 6 .4 4 7 .1 ± 0 .8 5 14.5 ± 0 .2 4
1 UR indicates pregnant rats fed ad libitum during the test periods; R indicates restriction of dietary intake of pregnant rats to one-half that} of the unrestricted group. 2 All results are expressed as averages of the mean ± se of mean.
TABLE 5 tion (few pregnancies), smaller litters, Mortality rate in hypothermia 1 slightly smaller mean fetal weights and Sex UR a R 2 persistent failure to attain the same weight as controls from mothers fed ad libitum Male 0/10 6/10 during gestation. Female 1/10 7 /1 0 1 Fifteen-minute exposure at 0°C. DISCUSSION 2 UR indicates pregnant rats fed ad libitum during the test periods; R indicates restriction of dietary in Numerous investigations have reported take of pregnant rats to one-half that of the un restricted group. the effects of dietary restriction on rats and other species of animals of all ages. The 7 out of 10 females in the restricted group major observations of these studies, par failed to survive. ticularly as they apply to present investiga Effect of duration of restriction period. tions, are that dietary restriction retards The previously described experiments were the growth and delays the maturation of carried out maintaining restricted intake of animals and that these processes are fre the mothers in the restricted groups up to quently reversible when ad libitum feeding the time of weaning. In table 6, data are is reinstated. This reversibility is well ex reported on an experiment in which dietary emplified in figure 5 which summarizes a restriction was stopped at the end of the study of the effect of intermittent starva period of gestation. Again the effects of tion and ad libitum feeding. Another ma restriction include some evidence of resorp jor observation from studies of this genera
TABLE 6 Effect of dietary restriction during the gestation period only
Body wt of progenies Young/ Avg Treatment1 Pregnancy litter fetal wt Three months Eight months Male Female Male Female
% 9 9 9 9 9 UR 100 ( 3 ) 2 9 6 ± 0 . 2 3 2 1 0 ± 2 0 ( 4 ) 146 ± 7 ( 4 ) 380 ± 2 6 266 ± 2 7 100 (3 ) 10 5 .6 ± 0.4 205± 10 (5) 132± 1 1 ( 3 ) 410 ± 1 1 218 ± 1 4 100 ( 3 ) 9 7 ± 0 .2 196 ± 5 (5) 150± 12 (3) 375 ± 2 2 250 ± 1 2 Mean 100 9.3 6.2 203.7 142.7 388 244.7
R 67 ( 3 ) 5 5 ± 0 .4 135± 18 ( 4 ) 100 ± 6 ( 4 ) 266 ± 1 4 1 5 6 ± 12 67 ( 3 ) 6 4.8 ± 0 .2 1 2 6 ± 10 ( 3 ) 9 6 ± 12 ( 5 ) 284 ± 22 142 ± 1 8 67 ( 3 ) 6 4.6 ± 0 .4 148 ± 21 ( 2 ) 110±8 ( 6 ) 3 1 0 ± 18 176 ± 1 4 50 ( 3 ) 7 4 .2 ± 0 .1 138 ± 6 ( 6 ) 118 ± 17 ( 2 ) 305 ± 3 0 185 ± 2 2 Mean 62.8 6 4.6 136.8 106 291.2 164.8
1 UR indicates pregnant rats fed ad libitum during the test periods; R indicates restriction of dietary in take of pregnant rats to one-half that of the unrestricted group. 2 Numbers in parentheses denote number of animals/ group. 3 se of mean. 16 BACON F. CHOW AND CHI-JEN LEE
Fig. 5 Growth rate of intermittently starved and ad libitum-fed rats. One group of adult rats (6 months old) were starved until their body weights decreased by one-third and were then fed ad libitum until their body weights reached a plateau. They were then starved again for another period, resulting again in a loss of one-third of their original body weight. They were again fed ad libitum. Their growth rates were compared with those of compar able normal rats on whom no starvation had been imposed. is the observation that when moderate re tions and was appropriately conservative striction of dietary intake is imposed and in drawing conclusions from his experi maintained, a very significant prolongation ments. Nevertheless, it was demonstrated of life span is observed. that under these conditions differences in Smith (2 ) has summarized many of body weight of groups characterized by these experiments and has discussed at small and large nursing litter size persisted length the various parameters used to for 12 months or more following inaugu measure the effects of dietary restriction, ration of ad libitum feeding at weaning. although in many instances no precise It has often been stated that in the ear quantitative measurements were reported. lier stages of gestation the fetus receives In virtually all of the studies, dietary re an adequate supply of nutrients from the striction was imposed on the young after mother. It is only in the later stages that birth or at varying times after birth. In the nutritional status of the mother may almost no instance were restrictions im become important to the future develop posed in utero or for that matter, even dur ment of the offspring. Still unproven is the ing lactation, and only recently did Mc- belief that large women tend to give birth Cance (5 ) report studies in which stresses to large babies (7,8) and that poor nutri were imposed during lactation by the de tion of the mother towards the end of vice of controlling the size of the litter be gestation can reduce the size of the new ing nursed. When litter size ranged from born. It has been cited that the weights of as many as 18 to as few as 3, abnormal sit twin lambs can be markedly reduced with uations were induced where limiting fac out decreasing the period of gestation (9 - tors over and above pure nutritional con 11). In addition, it is believed by some siderations entered. Where the litter size obstetricians and veterinarians that the exceeds the number of teats a “survival of size of a baby or an animal at birth is the fittest” situation is induced where the dependent upon the number of young pro weaker offspring are unable to be accom duced with a single pregnancy. However, modated. Moreover, the possibilities of this relationship does not necessarily hold cross infection multiply with larger litter with all species of animals. These general size. McCance recognized these limita izations have been published, but require MATERNAL DIET AND GROWTH OF THE OFFSPRING 17 further experimental support from sys explained satisfactorily by assuming an tematic and controlled studies. insufficient intake of a single limiting nu In the present investigation we were in trient. In the first instance we have ob terested in studying the effect of maternal served similar effects using 2 types of diets diet of rats not only on the birth weights, of entirely different composition. More but also on the weight gain of the progeny over, our stock diet provides at least 2 to over a long period of time, extending to 3 times the known requirements of all 1.5 years or approximately three-fifths of vitamins and other nutrients and, since the life span of our stock animals. In our 1954 studies, we observed that progeny the effects were observed with 25% dietary from mothers fed a restricted diet weighed restriction, this level of reduction of intake less than two-thirds that of the controls could hardly have induced specific nutri even after 1.5 years of ad libitum feeding. ent lack. Actually, in one of our experi It is premature at this time to interpolate ments, additional supplements of known our results to human studies, although it is vitamins were given to the unrestricted possible also, with some bias, to grope for group and the growth stunting was again support from clinical reports. The present observed. Moreover, none of the usual sec studies introduce the stress in utero by re ondary biochemical or clinical signs of stricting the food intake of the pregnant known vitamin deficiencies were observed, rat. The stress, therefore, appears to be although we examined for evidence of fundamentally different and the changes pyridoxine deficiencies by xanthurenic acid following it significantly prolonged. Over excretion or pyridoxal phosphate level in and above the retardation of weight gain, red cells, and for vitamin Eh» deficiency by our observations also include marked determination of vitamin Bi2 activity in se changes in the hematologic picture during rum or liver. the first 6 months of life and lowered re Dwarfism due to zinc deficiency is sistance to a specific external stress such as known and has been studied extensively by artificially induced hypothermia. Whether the altered resistance to hypothermia can Prasad et al. (12). It has been assumed be extrapolated to other stresses, or merely that zinc deficiency is extremely difficult to reflects lower body fat, remains to be achieve experimentally, particularly when elucidated. the experimental animals are housed in Similar but somewhat more marked ef metal cages, as ours were. Moreover, sim fects appear to follow as restriction of die ple calculation from the zinc content of tary intake of the mother is continued dur our diets tends to make zinc deficiency an ing the lactation period but it is not pos unlikely explanation. Perhaps the observed sible from the data in hand to assess the effects arise from deficiency of one or a relative importance of these 2 periods. combination of amino acids or of some Our studies suggest strongly the impor unknown vitamin or other nutritional fac tance of pituitary growth hormone in the t o r s ). Caloric insufficiency alone is not mechanism giving rise to the growth anom likely to explain these results since restora aly. Although Hruza et al. (6) have attempt tion to ad libitum feeding does not achieve ed similarly to incriminate growth hor prompt reversal. Further studies are pro mone, the experiments they reported used jected to explain these alternatives. an excessive amount of preparation and LITERATURE CITED failed to show any responses in less than 6 weeks after administration was started. 1. Osborne, T. B., and L. B. Mendel 1915 The resumption of growth after long continued From general considerations it may well be failure to grow. J. Biol. Chem., 23: 439. that their results are better explained on the 2. Smith, A. H. 1931 Phenomena of retarded basis of spontaneous reversal rather than growth (editorial review). J. Nutrition, 4: specific growth hormone effects. It is al 427. 3. McCay, C. M. 1935 The effect of retarded ways attractive to assume that effects such growth upon the length of life span and upon as we here described can be explained by the ultimate body size. J. Nutrition, 10: 63. invoking the law of the limit. However, it 4. Widdowson, E. M., and R. A. McCance 1960 does not appear that our results can be Some effects of accelerating growth. I. Gen 18 BACON F. CHOW AND CHI-JEN LEE
eral somatic development. Proc. Royal Soc. 9. Wallace, L. R. 1948 The growth of lambs Lond. series B., 152: 188. before and after birth in relation to the 5. McCance, R. A. 1962 Food, growth and level of nutrition, part 1. J. Agr. Sci., 38: 93. time. Lancet, 2: 621. 10. Wallace, L. R. 1948 The growth of lambs before and after birth in relation to the 6. Hruza, Z., P. Fabry, M. Chvapil and E. Holec- level of nutrition, parts 2, 3. J. Agr. Sci., kova 1962 Medical and Clinical Aspects 38: 243. of Aging — Adaptation to undernutrition and 11. Wallace, L. R. 1948 The growth of lambs trauma and influence of over-nutrition on before and after birth in relation to the level biological aging, ed., Herman T. Blumenthal. of nutrition, part 3 (continued). J. Agr. Columbia University Press, New York and Sci., 38: 367. London, p. 42. 12. Prasad, A. S., A. Miale, Jr., Z. Farid, H. H. 7. Widdowson, E. M. 1955 Reproduction and Sandstead and A. S. Schulert 1963 Zn obesity. Voeding, 16: 94. metabolism in patients with the syndrome 8. McKeown, T., R. G. Record 1957 The in of iron deficiency anemia, hepatosplenome- fluence of body weight on reproductive func galy, dwarfism and hypogonadism. J. Lab. tion in women. J. Endocrinol., 15: 410. Clin., 61: 537. Effect of Degree of Fatty Acid Unsaturaiion in Tocopherol Deficiency-induced Creatinuria* 1
L. A. WITTING a n d M. K. HORWITT L. B. Mendel Research Laboratory, Elgin State Hospital, Elgin, Illinois, and Department of Biological Chemistry, University of Illinois College of Medicine, Chicago, Illinois
ABSTRACT The rate of development of creatinuria, in the tocopherol-deficient rat proved to be dependent on the degree of unsaturation of the dietary fatty acids. Data were obtained consistent with relative in vivo rates of fatty acid peroxidation of mono- enoic, dienoic, trienoic, tetraenoic, pentaenoic, and hexaenoic fatty acids in the ratios 0.025:1:2:4:6:8, and tocopherol requirements for constant diminution in the rates of peroxidation in the ratios 0.3:2:3:4:5:6. Muscle phospholipid fatty acid compositions were dependent on the compositions of the dietary fats but were independent of the level of dietary fat between 7.5 and 19%. Within this range :he rates of development of creatinuria and the tocopherol requirements were also independent of the level of dietary fat. One milligram of d-a-tocopheryl acetate per kilogram rat per week delayed the onset of creatinuria by 17 to 20 weeks. When the diet contained suboptimal levels of selenium and sulfur amino acids, creatinuria occurred 30% more rapidly and the tocopherol requirement increased tenfold. Lower levels of the most highly unsaturated fatty acids were noted in the muscle phospholipids of the toccpherol-deficient animals. This is consistent with the concept that susceptibility toward peroxidation increases as the number of double bonds per fatty acid increases.
A variety of pathological conditions arise tion of the various fatty acids rather than in experimental animals fed tocopherol-de to the total amount of unsaturation in the ficient diets. These include encephaloma- dietary fat. That is to say, one mole of lacia, exudative diathesis, and myopathy arachidonate (tetraene) would be expected (nutritional muscular dystrophy) in the to give rise to more than twice as much chick; myopathy, testicular degeneration, peroxidized material as one mole of lino- resorption gestation, and liver necrosis in leate (diene) in the same period of time the rat; steatitis in the cat; and other simi under identical conditions. lar conditions in the rabbit, lamb, monkey, This expectation arises from a consider and calf. In many, if not all of these condi ation of the kinetics of autoxidation in tions, there exists a complex interrelation vitro (10). It is known that free radical ship between dietary concentrations of initiation, RH —> R° and uptake of oxygen sulfur amino acids, biologically available R° + 0 2 —> ROj' are rapid reactions, not de selenium, unsaturated fat, and tocopherol pendent on the nature (degree of unsatu or other non-toxic, fat-soluble antioxidants ration, monoenoic, dienoic, etc.) of the R (1 -4 ). group (where R designates the fatty acid). All 3 of the deficiency states in the chick The overall rate of oxidation must there may be prevented by supplementation with fore be dependent on the rate of the slowest synthetic fat-soluble antioxidants (5 -7 ) reaction (1/10,000 the rate of the preced and the rat has been maintained with a ing reactions) tocopherol-deficient diet for 3 generations k 3 ROi” + RH -» ROOH + R° by supplementation with a synthetic fat- which is extremely dependent on the nature soluble antioxidant (8, 9). Since the major, if not sole, function of a-tocopherol is as a of the R group. Although metal complexes and the presence of protein are expected to biological fat-soluble antioxidant, the pos modify the reaction scheme in vivo, these sibility exists that the onset of deficiency factors should not affect the specific de symptoms can be related to a specific level of lipid peroxidation in the tissue. In this Received for publication August 8, 1963. case, the rate of production of deficiency 1 Supported in part by Illinois Mental Health Fund, The National Vitamin Foundation, Inc., and U.S.P.H.S. should be related to the degree of unsatura Grant A-1126 and Grant NB-03523-02.
J. N u t r i t i o n , 8 2 : ’ 64 1 9 2 0 L. A. W ITTING AND M. K. HORWITT
pendence on degree of unsaturation of the of methyl acetate was complete, a stoichio rate-limiting reaction. With a diet suffi metric amount of water was added to de ciently free of tocopherol or other fat-solu stroy the catalyst and the resulting soaps ble antioxidants, fatty acid peroxidation in were removed by filtration. Double distilled the animal tissues may be inevitable, but oleic acid, low in linoleic acid, was ob the rate of peroxidation should be depend tained commercially3 and used for the prep ent on the fatty acid composition of the aration of triolein. Synthetic fats were tissue as influenced by the dietary fat. stored until used in filled mason jars un It is known (1 1) that increasingly der nitrogen at — 5°. greater amounts of a-tocopherol are re The extent of unsaturation was esti quired to prevent the autoxidation of oleic, mated by determining the Hanus iodine linoleic, and linolenic acids in vitro. In the value (1 3 ) and also by calculating a theo rate equation (1 0 ) for the inhibited autoxi retical iodine value from the fatty acid dation reaction composition determined by gas-liquid chro d 0 2/d T = Ki K3 [RH]/K4 [AH], matography (14). The agreement between AH designates tocopherol, methods was excellent. The monoenoic fat, k 4 largely triolein, with an iodine value of 82 K4 arises from R02° + AH —> ROOH + A , supplied 79, 60, 40 and 22 mmoles of and Ki describes the initiation process. unsaturation (RCH = CHR') per 100 g of As the unsaturation of the fatty acid is diet when fed at 25, 19, 12.5 and 7.5% increased, the ratio K3/K4 increases, and levels, respectively. Sufficient saturated fat, must be balanced by the addition of a- essentially palmitoyldistearin (hydrogen tocopherol to decrease the ratio [RH]/[AH]. ated beef fat),4 was included in the re This phenomenon may also occur in vitro. arrangements so that each of the other The present experiment2 was undertaken synthetic fats (table 1) also had an iodine to investigate the quadruple interaction of value of 82. One millimole of R"CH = selenium, sulfur amino acids, tocopherol, CHCH2CH = CHR'" was considered to con and unsaturated fatty acids using an assay stitute 2 mmoles of unsaturation. Accord based on the occurrence of creatinuria, as ingly, a millimole of trienoic, tetraenoic, a symptom of myopathy, in the tocopherol- pentaenoic, and hexaenoic acid would con deficient rat (12). Various levels of 7 dif stitute 3, 4, 5, and 6 mmoles of unsatura ferent synthetic fats were fed with graded tion, respectively, and therefore, each syn levels of tocopherol with and without added thetic fat also supplied 79, 60, 40 and 22 selenium and methionine. mmoles of unsaturation per 100 g of diet Muscle lipids were analyzed and the when fed at the 25, 19, 12.5 and 7.5% fatty acid composition related to the onset levels, respectively. A saturated synthetic of creatinuria and possibly to the occur fat, based on coconut oil rather than hy rence of lipid peroxidation in vivo. drogenated beef fat for reasons of digesti bility, was included in the study. This fat MATERIALS AND METHODS supplied small amounts of linoleic and Preparation of synthetic fats. Naturally oleic acids, and was included to evaluate occurring fats, coconut oil, corn oil, linseed the effect of small amounts of the essen oil, and cod liver oil, were saponified, and tial fatty acid, linoleic, in a saturated fat the nonsaponifiables, including tocopherol, as compared with its effect in the almost extracted into petroleum ether. The fatty completely unsaturated monoenoic fat. acids obtained after acidification were Tocopherol determinations. The syn dried and methylated in the presence of thetic fats contained small amounts of 2,2-dimethoxypropane, methanol, and 2% Emmerie-Engel reactive material (15), but sulfuric acid. After neutralization, extrac 2 Preliminary reports of this work were given at the tion of the product into petroleum ether, 1962 and 1963 m eetings o f the Federation o f A m er ican Societies for Experimental Biology, Atlantic City, and removal of the solvent, the methyl N. J.: L. A. Witting, and M. K. Horwitt 1962 Rat tocopherol needs as a function of n in (CH2CH = CH)„. esters were molecularly distilled and then Federation Proc., 2 1 : 474; L. A. Witting, and M. K. rearranged with glycerol triacetin under Horwitt 1963 The influence of fatty acid structure on relative peroxidizability in vivo . Federation Proc., vacuum at 80 to 120° in the presence of 2 2 : 608. 3 Emery Industries, Cincinnati, Ohio. 0.5% sodium methoxide. When evolution 4 Armour and Company, Chicago, Illinois. TOCOPHEROL-FATTY ACID KINETICS IN VIVO 21
T A B L E 1 Fatty acid composition of synthetic dietary fats
Dietary fat1 Fatty acid type Saturated 2 Monoenoic Dienoic “Mixed “Mixed Trienoic fat” no. 1 fat” no. 2 Polyenoic
Saturated, % 87.4 3 11.7 4 42.5 4 58.5 4 64.8 4 58.8 4 62.4 4 Monoene, % 9.6 81.1 21.2 10.0 12.0 17.3 20.6 Diene, % 3.0 7.2 3 6.0 8.4 4.6 13.1 2.2 Triene, % — — 0.3 23.1 12.1 0.1 1.7 Tetraene, % — — — — 1.4 1.9 2.3 Pentaene, % — — — — 3.2 4.1 5.6 Hexaene, % — — — — 2.9 5.1 5.2 Iodine value 12.4 82.7 8 2.0 82.0 82.0 82.0 82.0 mmole unsaturation/ 100 g fat 47 318 316 316 316 316 316 Estimated peroxidiz ability X 100 3.2 9.2 3 7.1 54.8 77.1 86.5 91.3 Tocopherol, /¿g/g < 0.1 < 0.1 < 0.25 < 0.25 < 0.25 < 0.25 < 0.25
1 For size of groups see tables 2 and 3. 2 Average composition from 0 to 28 weeks. Changed at 18 weeks so that average composition from 0 to 50 weeks was saturated, 90%; monoene, 7.8%; and diene, 2.2%; and estimated peroxidizability (x 10 0), 2.4. 3 Largely lauric and myristic acids. 4 Largely palmitic and stearic acids. after chromatography on zinc ammine car (19), 0.4% DL-methionine and 0.13 ppm bonate impregnated paper (16), it could of selenium as sodium selenite were added be shown that less than 0.25 ug of tocoph to the rations in the B series. In this case erol was present per gram of dienoic, tri- salt mix Illinois 446 (2 0) replaced salts enoic, and polyenoic fat. The saturated USP XIV. The d-a-tocopheryl acetate in and monoenoic fats were either free of coconut oil was given by mouth, by drop tocopherol or contained less than 0.1 ag/g per, 3 times a week. Tocopherol dosage of fat. When small amounts of a-tocoph- (0.2, 0.4, 0.5, 0.6, 1.0, 1.75, and 15 mg erol were added to duplicate samples, re d-a-tocopheryl acetate/kg rat body weight/ coveries were in the 85 to 95% range. week) was calculated for the average ani Although it could not be proven that they mal weight in 100-g brackets (50 to 150 g; were completely free of tocopherol, these 150 to 250 g; 250 to 350 g, etc.). fats probably contained less tocopherol Urinary creatine and creatinine. Twenty- than any others previously reported. four-hour urine samples were collected Animals and diets. Male weanling rats from the rats every 2 to 4 weeks and cre of the Sprague-Dawley strain were used. atine and creatinine concentrations were The basal diet fed was similar to that determined by the method of Bonsnes and described by Century and Horwitt (17) Taussky (21). except that dextrose replaced the corn Fatty acid analyses. The gastrocnemius starch, since the latter material contains and quadriceps muscles were excised tocopherol. Protein content, as casein, was rapidly after death and homogenized in constant at 21.8% of calories and the car methylal-methanol (1 4) containing 0.01% bohydrate content was modified isocalori- a-tocopherol. Neutral lipids and phospho cally to compensate for the various fat lipids were separated by thin layer concentrations (25, 19, 12.5 and 7.5% ). chromatography (2 2) and the fatty acid These diets were fed to the animals in the composition determined by gas-liquid chro A series. Since casein is known to be sub- matography as described previously (14). optimal in sulfur amino acid content (1 8) Tocopherol was present during all stages and also occasionally to be low in selenium of sample preparation and storage. ot ot •2 -S -Si -8 fg. CO to to to 8 CO to to £ to s O s £ s Q) e to to to s s $ 8 to 8 « 8 to f- to ft) « 00 05 to *- to 2 noted (series A: no added selenium or methionine) H «w 'S «H H 'o s P to» rH "in PM « PJ 6 to >* ft to o to 0J M >> X oj § rt oj o o o k. Pi h H 3 O O to to PM PM j i S S 2 to . 4-> PM Pi
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Ci © © © © \ \ © © rH © © © rH © © © © ft © © ft i> r © © © O \ © © \ © N l> © © © © © © © \ rH © Ci © oj rH Ci © © IN © en rH ft Ci ci Ci Ci ft Ci © © © rH Ci ft © ft © r* s? ‘o H-J P3 0 0 w tí S d 0 ÍH 2 'rCJ.iï'oJÎ s ~ ■5«u ä ' o ^ ä * « g tí ‘g*H , S„tío rtglS&W £ fe‘H t 8 § gt-S ! fttí , awS2 ■a|wSS 3 ' -Û ,-2eo Oí Seo 0,,-, ’ ö « tuo 5 ö o > S 2 o, c3 0« S“8 S fi« , ö s c/5H>tí H CU^.H■H ^s ^ d o 1 i co—i p -w n -> 3 ’S S £ S s C i)Jad ) i *CH gS » s « « j ’ ft fi -W Cfl o 2-2 “ 3 3Ê g'S« (tí A ft 'S fi q ■gs ft -S o “ b -0 V oí « a 2 6 L. A. W ITTING AND M. K. HORWITT RESULTS Tep Onset of creatinuria. The creatine-to- 0.1 .2 .3 .4 .5 .6 .7 .8 .9 1.0 creatinine ratios used in the estimation of the time of onset of creatinuria in the animals fed the various synthetic fats in the series A ration are shown in table 2. Data from animals fed the series B ration are presented in table 3. For a description and examples of complete curves on the course of development of creatinuria see Horwitt et al. (12). To avoid the recording of over 2300 creatine-to-creatinine deter minations, only the results of periods just before and just after the onset of signifi cant elevation are included in the tables. From statistical considerations, it was ap parent that a creatine-to-creatinine ratio greater than about 0.4 was indicative of ESTIMATED PEROXIDIZABILITY positive creatinuria since it could be cal Fig. 1 Time in weeks required to produce culated that the probability of such a value creatinuria versus the estimated peroxidizability of the dietary fat (solid line) and time in weeks occurring in a tocopherol-supplemented required to produce creatinuria (reciprocal scale) animal fed these diets was only 0.005. versus the square root of the estimated peroxidiz The higher urinary creatine-to-creatinine ability of the dietary fat (broken line). Data ratios and greater standard deviation of from 19% fat groups in series A are indicated by circles and comparable data from series B by the average ratio found for the tocopherol- triangles. supplemented animals fed the polyenoic fat, is an experimental artifact, later re by 0.025, 1, 2, 4, 6, and 8, respectively. lated to the frequency with which these These numerical values are based on the animals were supplemented with tocoph in vitro maximal rates of oxidation re erol. More frequent supplementation, i.e., ported by Holman (24). The question of daily, instead of 3 times a week, leads to interaction of rates of autoxidation in a normal ratios. mixture has been explored by Gunstone Effect of degree of unsaturation. The and Hilditch (25). A straight line was data in tables 2 and 3 indicate that the obtained when the rate of production of time required for the production of creati creatinuria (1 /T ) was plotted against the nuria as a symptom of nutritional myop square root of the calculated peroxidiz athy in rats fed diets without added vita ability. Addition of selenium and methio min E is dependent on the nature of the nine to the ration increased the time re dietary fat. The fats that produced the quired for the development of creatinuria earliest creatinuria (5 to 7 weeks) were by approximately 40% with any given those (table 1) in which a given amount synthetic fat. of unsaturation was highly concentrated Tocopherol supplementation. When the in a relatively small percentage of the graded levels of tocopherol used in this fatty acid chains, rather than being dis study were administered in series A, little tributed evenly as in the case of the mono- protective effect was noted. One milligram enoic fat, where the production of creati of d-a-tocopheryl acetate per kilogram rat nuria required 17 to 18 weeks. When the per week delayed the onset of creatinuria data from the 19% fat diets were by about 2 weeks. In series B, in which plotted against an estimated peroxidiz- selenium and methionine were added, the ability slightly modified from that onset of creatinuria was delayed by ap previously published (2 3), (fig. 1) two proximately 17 to 20 weeks per milligram hyperbolae resulted. Peroxidizability is of tocopherol when the dienoic, trienoic, calculated as percentages of monoenoic, and polyenoic fats were fed and approxi dienoic, trienoic, tetraenoic, pentaenoic, mately 45 weeks when the monoenoiC fat hexaenoic fatty acids in the diet multiplied was fed (fig. 2). The tocopherol require- TOCOPHEROL-FATTY ACID KINETICS IN VIVO 2 7 crease in hexaenoic acid. Furthermore, an increase in the unsaturation of the neutral lipid occurred. With added dienoic fat, there was an increase of tetraene in the phospholipid and large amounts of diene were noted to “overflow” into the neutral lipid. Although the trienoic fat contributed very little linolenate to the phospholipids, and greatly depressed the arachidonate level, an increase in pentaenes and hex- aene was noted and the neutral fat was high in linolenate. The polyenoic fat led to an extremely high concentration of the docosahexaenoic acid in the phospholipids but did not furnish large amounts of fatty acids suitable for deposition in the neutral lipids. The extreme degree of unsaturation of these fatty acids, however, made their contribution to the estimated peroxidiz- ability of the neutral fat quite important. Effect of muscle fat composition on Fig. 2 Delay in onset of creatinuria versus onset of creatinuria. If the suggestion of tocopherol dosage. Delay in weeks required to Century et al. (2 3 ) as modified herein, produce creatinuria at a given level of tocopherol regarding correlation of muscle fatty acid supplementation minus weeks required to produce creatinuria without tocopherol supplementation. composition to rate of development of cre Data from diets containing polyenoic, dienoic, atinuria is applied to the present data trienoic, and monoenoic synthetic fats are repre (fig. 3) a striking relationship is noted. sented by the symbols: triangle, cross, circle, and From the discontinuous nature of the curve M, respectively. in the region where polyunsaturated fatty ment appeared to be dependent on total acids begin to “overflow” into the neutral unsaturation except in the case of the lipid, it is apparent that peroxidizability of monoenoic unsaturation. the phospholipid fatty acids is of primary Effect of BHA. In an effort to evaluate importance, but that neutral lipid fatty the effects of possible oxidation of dietary acids also affect the rate of development of lipid prior to ingestion, butylated hydroxy- creatinuria. anisole (B H A ) was added to the polyenoic dietary fat at the 0.02% level. Two groups receiving 0.5 and 1.0 mg d-a-tocopheryl acetate/kg rat/week were compared to identical groups receiving the same fat not protected by the addition of this antioxi dant. The BHA had no effect on the rate of development of creatinuria. Effect of dietary fat on muscle fat com position. The fatty acid composition of the rat muscle (gastrocnemius and quad riceps) phospholipids and neutral lipids (table 4) were markedly affected by the different dietary lipids. If the tissue com positions reported for the saturated syn EP0 MUSCLE PHOSPHOLIPID thetic fat diet are taken as a base line, Fig. 3 Time in weeks required to produce cre it will be noted that although the mono atinuria versus the estimated peroxidizability of the muscle phospholipid fatty acids. Data from enoic fat led to similar linoleic (dienoic) 19% fat groups in series A are indicated by cir and arachidonic ( tetraenoic) acid levels in cles and comparable data from series B by tri the phospholipids, there was a marked in angles. 8 2 Effect of level and type of synthetic dietary fat on the fatty acid composition of muscle 1 neutral lipids and phospholipids CO t I gj ^*05 - 3 ' '- H ■H f I s a S!S o S C5/- L"*a O v$> s C5 flic 32 SH 005 M vQ "in n TJ co â a v~/ -' v- V—' CO CO $ q q q q q q o ^ O +1 +1 +1+1C' +1 II II < LO 05 n o fl q i c h 05 rH 05 q 1 CO I> d 6 05 05 q I I rH CO LO i c rH II q co CO rH q rH I I II rH II 0 O > > CD t> t> d CO 00 O q q 05 q d t> i c I I rH I I I I d d o c i-l n i i c rH o c d d cd II 6 N ( d d o c 6 t r 00 rH CO rH q rH rH rH CO i c rH d CO rH q d q i c rH q d rH rH I I d i II II d rH q rH q i c q Cl d I I II H P H H Q rH q q I I II •JH (D fH 0) II II 6 d d i c d d d d d (N q d CO i c CO d q t> rH q LO I I I I II d CO d rH d Cl i c CO d I I rH q q d I I d II II II II A WITTING AND M. . TT IT W R O H K. . M D N A G N I T T I W A. . L CO q d q rH d rH d q rH rH i c q I I cd CO d O! rH q q rH q I I I I II rH rH I I LO I I II q II a a .a | •a LO * S3 * H r II Ü ^ « » » ) ; ) O 0) g;0) ^ O S 2 ¡4 2 § § rH LO d q rH CO 05 rH q Cl I I CO 05 I I Cl id I I d d rH q r> rH rH q rH q q q q rH d rH II q II II +1 i c II II a h 2 x LO i c d i c CO 05 q rH q 05 rH q Cl d i> CM q q rH rH r-1 d i c rH rH I I i c CO rH q I I 1! II q id d q q rH I I q r-i I I II 05 II II d q CO cd 05 i c q r-i id cd L> I I i c q q q q CO I I q LO I I q q II i c q 05 q d II q II 2 i c II CO CO CO 05 CO d 05 LO d 05 d O CO 00 d d +1 05 +1 rH CO 05 +1 +1 +1 CO rH 05 II II X >• 2 O 05 CO o n ü *Si> *Si> •art §$ ’§ -' '- H H * A ^ 0)05 o>w CO q q rH q q CO 05 CO O O d CO co cd q CO t> t> CO id ci 6 05 q rH d LO rH ^ CO CO q 05 q rH d q CO d q CO 05 ( I C O CJ H ID K eu H (U H Q M ^ q rH q rH i c q d q d LO rH d q d q d q rH q d q d q i> q h q q rH t> d I d d d CO r (Nr i c d h q 05 d q rH q d d q d q i co ci q rH q d q d d 00 (M d d d .a 2 > TOs>TO ri (U m g g cd Cl rH q d CM cd q d q q r> q d d rH rH CO O CO CO CO o CO O LO 05 d CO m I I I I 1 Gastrocnemius and quadriceps. 2 Number of samples. 3 SD. 4 Polyunsaturated fatty acids. 3 See page 31 for explanation. TOCOPHEROL-FATTY ACID KINETICS IN VIVO 2 9 As drawn, the lines are parallel and are DISCUSSION of the same slopes on either side of the It seems most practical to discuss the discontinuity. The tissue lipid fatty acid results in terms of 2 distinct but normally compositions determined for the groups fed simultaneous phenomena. The first is the 7.5 and 12.5% fat are comparable to those rate of production of creatinuria with an observed when 19% fat was fed. Data on essentially tocopherol-free diet, and the the rats fed 25% fat are not currently second is the decrease in this rate brought available. Standard errors are not given about by various levels of tocopherol sup for the neutral fat values since the possible plementation. errors in measuring a series of constituents Diets essentially free of tocopherol. at the less than 1% level by gas-liquid Under the specific experimental conditions chromatography are quite large. described herein, it has been possible to Effect of tocopherol deficiency on muscle relate the degree of unsaturation of various fat composition. A group of 62 rats re dietary fatty acids to the rate of production ceiving stripped corn o il5 at the 15% level of creatinuria as a symptom of myopathy in a series B ration was available for mus (nutritional muscular dystrophy) in the cle lipid analysis. Although not a part of rat fed a ration essentially devoid of to the study, as tabulated in tables 1-4, the copherol. Certain experimental procedures comparison of muscle phospholipid fatty must be emphasized since they are either acids of tocopherol-supplemented and to rather unusual or are of such a nature as copherol-deficient rats is of interest, (table to prohibit comparison with various other 5). The lipids from the tocopherol-deficient systems. group are significantly lower in polyunsat The dietary fats used herein were spe urated fatty acids (PU FA ) and the differ cially formulated and do not correspond ence occurs mainly in the most highly to any natural or commercial product. unsaturated fatty acids. Similar data Within the limits of practically available ( table 6) on groups contained in tables 2-4 materials, the basic fat (monoenoic, table show a similar trend but demonstration of 1) was tocopherol-free glycerol triolein statistical significance suffers from the containing enough linoleic acid to meet the smaller group sizes. rat’s essential fatty acid requirements (ap Animals were killed within a few weeks proximately 1% of calories as 18:2) (26). of the first significant increase in urinary The dienoic fat resulted from the substitu creatine-to-creatinine ratio. The decrease tion of one mole of dienoic acid (linoleic) in polyunsaturated fatty acids was of the plus one mole of saturated fatty acid for order of magnitude of 0.1 umole/g wet 5 Distillation Products Industries, Rochester, New weight of tissue. York. TABLE 5 Comparison of fatty acids percentage composition of muscle phospholipids from tocopherol-deficient and tocopherol-supplemented rats fed 15% stripped com o il1 Adequately Tocopherol- Significance Fatty acid supplemented 2 deficient 3 18:2 4 19.9±0.34 5 20.0 ±0.43 NS 6 20:4 18.5 ± 0.31 18.1 ±0.45 NS 22:4 1.7 ± 0.06 1.9 ±0.08 NS 22:5 5.1 ±0.19 3.8 ± 0.21 P < 0.001 22:5 1.1 ± 0.05 0.8 ±0.08 0.005 > P > 0.001 22:6 5.3 ± 0.18 4.6 ±0.27 0.05 > P > 0.025 PU FA 7 53.2 ±0.32 50.6 ±0.64 P < 0.001 1 Prepared by Distillation Products Industries, Rochester, New York, by molecular distillation. 2 Thirty-eight rats/group. 3 Fourteen rats/group. 4 The first figure represents the number of carbon atoms; the second, the number of double bonds. & s e of mean. 6 Not significant. 7 Total polyunsaturated fatty acids. 3 0 L. A. WITTING AND M. K. HORWITT TABLE 6 Comparison of fatty acid percentage composition of muscle phospholipids from tocopherol- deficient and tocopherol-supplemented rats fed various dietary fats Fatty acids, % Dietary fat F a t t y a c i d Significance Adequately Tocopherol- supplemented deficient D i e n o i c ( 9 ) 2 ( 1 8 ) 1 8 ; 2 2 20.5 ± 0.90 3 1 8 . 7 ± 0 . 3 3 0.05 > P > 0.025 2 0 : 4 1 9 . 9 ± 0 . 4 0 2 1 . 0 ± 0 . 5 2 NS 2 2 : 4 1 . 5 ± 0 . 1 3 1 . 6 ± 0 . 1 2 NS 2 2 : 5 3 . 4 ± 0 . 3 0 2 . 8 ± 0 . 2 4 NS 2 2 : 5 1 . 2 ± 0 . 1 0 0 . 9 ± 0 . 0 7 0.05 > P > 0.025 2 2 : 6 4 . 9 ± 0 . 3 7 4 . 0 ± 0 . 2 1 0.05 > P > 0.025 P U F A 4 5 2 . 8 ± 0 . 4 3 4 9 . 5 ± 0 . 6 8 0.005 > P > 0.001 P o l y e n o i c ( 8 ) ( 2 2 ) 1 8 : 2 7 . 8 ± 0 . 8 8 6 . 2 ± 0 . 4 1 NS 2 0 : 4 4 . 0 ± 0 . 5 0 3 . 8 ± 0 . 2 3 NS 2 0 : 5 2 . 6 ± 0 . 3 2 2 . 9 ± 0 . 1 5 NS 2 2 : 5 2 . 6 ± 0 . 3 9 2 . 7 ± 0 . 1 3 NS 2 2 : 6 2 6 . 6 ± 0 . 8 1 2 1 . 7 ± 1 . 0 7 0.02 > P > 0.01 PUFA 4 4 . 6 ± 1 . 0 3 3 8 . 6 ± 1 . 4 3 0.025 > P > 0.020 T r i e n o i c ( 6 ) ( 1 4 ) PUFA 4 9 . 8 ± 1 . 1 0 4 8 . 0 ± 0 . 8 0 NS M o n o e n o i c ( 6 ) ( 7 ) PUFA 42.9 ± 1.10 3 9 . 8 ± 0 . 7 2 0.02 > P > 0.01 S a t u r a t e d ( 3 ) ( 7 ) PUFA 4 2 . 9 ± 1 . 5 0 3 6 . 4 ± 1 . 4 0 0.005 > P > 0.001 1 Num ber of anim als. 2 The first figure represents the num ber of carbon atom s; the second, the num ber of double bonds. 3 s e o f m e a n . 4 Total polyunsaturated fatty acids. 2 moles of monoenoic fatty acid. Similarly, ment, it was necessary that at least one- the trienoic fat was obtained by substitut third of the fatty acids be unsaturated to ing one mole of trienoic acid (hnolenic) facilitate digestion. plus 2 moles of saturated fatty acid for Increasing the number of double bonds 3 moles of monoenoic acid. The polyenoic per fatty acid molecule while keeping the fat contained one mole of tetraenoic acid total number of double bonds in the fat plus 3 moles of saturated fatty acid in constant increased the rate of development place of 4 moles of monoenoic acid, one of creatinuria (tables 2 and 3). If this mole of pentaenoic acid plus 4 moles of were a simple relation, i.e., if diene had saturated fatty acid in place of 5 moles of twice the effect of monoene, and tetraene monoenoic acid, and one mole of hexaenoic had twice the effect of diene, and hexaene acid plus 5 moles of saturated fatty acids had twice the effect of triene, no difference in place of 6 moles of monoenoic acid. would be noticed on the present “constant Mixed fat no. 1 is practically equivalent to unsaturation” diets. a mixture of the synthetic trienoic and Another point requires particular em polyenoic fats, whereas mixed fat no. 2 is phasis. Data were obtained from young, practically equivalent to a mixture of the rapidly growing rats. Tissue lipid fatty synthetic dienoic and polyenoic fats. It acid compositions were markedly influ should be understood that each fat fed at enced by dietary fat (table 4). This ex the same dietary level supplied exactly the perimental condition prohibits direct com same molar quantity of fatty acid unsat parison of the results to data on animals uration, but that potential peroxidizability at a stable weight or those with appreciable increased as the number of double bonds adipose tissue at the start of the experi per molecule increased. Although glyceride ment. At 7.5, 12.5 and 19% levels of any structure was “randomized” by rearrange given dietary fat, the muscle phospholipid TOCOPHEROL-FATTY ACID KINETICS IN VIVO 31 and neutral fat fatty acid compositions amino acids, lowered Ci still further to 2.3. were similar if not practically identical The relation of T0 to the square root of die (table 4). For any given dietary fat at any tary unsaturated fatty acid percentages of these 3 levels, the rate of production of may describe the effective entry of these creatinuria was probably identical (tables materials into tissue lipids under the pres 2 and 3). ent conditions. Tests of the general valid At the 25% level of dietary fat, it may ity of this relationship might be derived be theorized that biosynthesis of saturated from measurements of essential fatty acid and monoenoic fatty acids from carbohy requirements or of the lowering of elevated drate was suppressed to a greater extent serum cholesterol levels (27). than with the diets containing less fat. Effects of tocopherol supplementation. Correspondingly, it would be expected that As might be expected from the tissue lipid low fat diets (less than 7.5% ) would take analyses (table 4) the tocopherol require longer to produce creatinuria since a greater dilution would occur in the tissues ment was found to be independent of die with biosynthetic fatty acids. These points tary fat level from 7.5 to 19% fat. How are under investigation. ever, at lower or higher dietary fat levels The best approximation of the relative as dilution with biosynthesized saturated contribution of the various unsaturated and monoenoic fatty acids increased or de fatty acids to the production of creatinuria creased, respectively, a change in tissue appears to be the ratios of maximal rates lipid fatty acid composition might be ex of oxidation in vitro (2 4 ); 0.025:1:2:4: pected to occur and alter the tocopherol 6:8 for monoenoic, dienoic, trienoic, tetra- requirements accordingly. The protection enoic, pentaenoic, and hexaenoic acids, re afforded by tocopherol, expressed in weeks spectively. Secondary dietary deficiencies required to produce creatinuria with a or nonspecific stresses can be expected to given supplement of tocopherol minus change the rate of production of creatin weeks required to produce creatinuria with uria without changing the dependence on a diet essentially devoid of tocopherol, did dietary fatty acids. The time, in weeks, not show an apparent dependence on the required to produce creatinuria in the A nature of the dietary unsaturated fatty series was approximately 70% of the time, acids at a constant level of unsaturation in weeks, required when the same fat was (fig. 2) except in the case of monoenoic fed in the B series (tables 2, 3). This unsaturation. Relative quantities of to relation prevailed whether a fat was fed copherol required to protect one mole of that produced creatinuria in 7 weeks or if monoenoic, dienoic, trienoic, tetraenoic, one was fed that took 49 weeks to produce pentaenoic and hexaenoic acid, respec creatinuria. tively, are estimated to be approximately In the course of analyzing the data, an in the ratios 0.3:2:3:4:5:6. empirical predictive equation was devel With a diet suboptimal in selenium and oped which may be useful. To, the time in sulfur amino acids, one milligram of d-a- weeks required for the development of tocopheryl acetate per kilogram rat per creatinuria, could be approximated from week delayed the onset of creatinuria by Ci/(EPo)1/2 where EP0, which equals (% approximately 2 weeks, whereas, the same monoenoic acid X 0.025) + (% dienoic amount of tocopherol with a more ade acid X 1 ) + ( % trienoic acid X 2 ) + ( % quate ration gave approximately 17 to 20 tetraenoic acid X 4) + (% pentaenoic weeks protection. A more extensive study acid X 6) + ( % hexaenoic acid X 8 ) , de of the effects of selenium and methionine scribes the dietary fat. With the best diets individually and in combination is in prog used herein, which supplied 7.5 to 19% ress. It is not specifically known that this fat, Ci equaled 7.0. The diets that con is an alteration in rate without effect on tained suboptimal levels of selenium and the fatty acid composition dependence methionine lowered Ci to 5. High (25% ) since neither the high tocopherol supple fat diets lowered Ci to 2.8 and in combina ments necessary to resolve this question, tion these stresses, high fat and subopti nor the key nature of high monoenoic acid mal concentrations of selenium and sulfur fats had been anticipated. 3 2 L. A. WITTING AND M. K. HORWITT Another empirical predictive equation company for generously providing hydro useful under these particular experimental genated beef tallow. conditions may be of interest. The TE, the The assistance of Donald Koehlert in time in weeks to produce creatinuria at a synthesizing the dietary fats, of Ruth Nel given level of tocopherol supplementation son in preparing lipid extracts, of M. Jane (E c), can be approximated by Morton in performing gas-liquid chromato graphic analyses and creatine-to-creatinine determinations, of Emmett Harmon in per where EPT which equals (% monoenoic forming tocopherol analyses, and of Joseph acid X 0.33) + (% dienoic acid X 2) + Wilkins in caring for the animals is grate ( % trienoic acid X 3) + (% trienoic acid fully acknowledged. X 4) + (% pentaenoic acid X 5) + (% LITERATURE CITED hexaenoic acid X 6) describes the percent age composition of the dietary fat. C2 is 1. Harris, R. S., and I. G. Wool, eds. 1962 Symposium on vitamin E and metabolism. about 14 with a good diet but decreases Vitamins and Hormones, 20; 375. rapidly (approximately tenfold) as the ani 2. Schultz, H. W., E. A. Day and R. O. Sinn- mal is stressed by diets suboptimal in se huber, eds. 1962 Lipids and Their Oxida tion. The Avi Publishing Company. Inc., lenium and sulfur amino acids. Given ade Westport, Connecticut. quate data, the equations suggested might 3. Horwitt, M. K. 1961 Vitamin E in Human be used to evaluate the apparent relative Nutrition — An Interpretative Review. Bor rates of fatty acid peroxidation and tocoph den’s Rev. Nutrition Res., 22; 1. 4. Scott, M. L. 1962 Anti-oxidant, selenium erol destruction in vivo. and sulphur amino acids in the vitamin E At an early stage of tocopherol defi nutrition of chicks. Nutrition Abstr. Rev., ciency, lower percentages of the higher 32; 1. 5. Machlin, L. J., R. S. Gordon and K. H. polyunsaturated fatty acids were detected Meisky 1959 The effect of antioxidants on (tables 5 and 6) in the muscle phospho vitamin E deficiency symptoms and produc lipid fatty acids. Similar observations have tion of liver “peroxide” in the chicken. J. been reported by Hove and Seibold (28) Nutrition, 67; 333. 6. Bunnell, R. H., L. D. Matterson, E. P. Sing- in pig liver and adipose tissue, by Bhalerao sen, L. M. Potter, A. Kozeff and E. L. Jung- and Kummerow 6 in chick brain, and by herr 1955 Studies on encephalomalacia Century and Horwitt7 in erythrocytes, liver in the chick. 3. The influence of feeding or injecting various tocopherols and other anti and muscle tissues of the chick. The lower oxidants on the incidence of encephalomala levels of PUFA in the phospholipids of cia. Poultry Sci., 34; 1068. tocopherol-deficient animals are consistent 7. Machlin, L. J., and W. T. Shalkop 1956 with the concept that the lability to peroxi Muscular degeneration in chickens fed diets low in vitamin E and sulfur. J. Nutrition, dation of the PUFA increases as the num 60; 87. ber of double bonds per molecule increases. 8. Crider, Q. E., P. Alaupovic and B. C. Johnson Although the required oxygen, approxi 1960 On the function and metabolism of vitamin E. II. The effects of vitamin E on mately 0.1 nmole 0 2/g wet weight of mus the level of non-vitamin E reducing com cle over a period of 5 to 49 weeks, for lipid pounds present in animal tissues. Biochem. peroxidation could easily have been avail Biophys. Res. Comm., 2; 293. able, there is no direct evidence that this 9. Crider, Q. E., P. Alaupovic and B. C. Johnson 1961 On the function and metabolism of reaction took place. The amount of peroxi- vitamin E. III. Vitamin E and antioxidants dized lipid that may be in the tissue at any in the nutrition of the rat. J. Nutrition, given time is apparently too small for in 73; 64. controvertible direct chemical detection. 10. Lundberg, W. O., ed. 1961 Autoxidation and Antioxidants, vol. 1. Interscience Pub lishers, New York. ACKNOWLEDGMENTS 6 Bhalerao, V. R., M. G. Kokatnur and F. A. Kum We wish to thank Dr. Edward R. merow 1962 The fatty acid composition of lipids extracted from the brain of chicks with symptoms of Perkins, formerly of Armour and Com encephalomalacia. Federation Proc., 22; 290 (ab- pany, for suggestions regarding glyceride 7 Century, B., and M. K. Horwitt 1963 The rela synthesis by the rearrangement reaction tionship of arachidonic acid to nutritional encephalo malacia; implications concerning essential fatty acids and Dr. R. J. Vander Wal of the same in the tissues. Federation Proc., 22; 608 (abstract). TOCOPHEROL-FATTY ACID KINETICS IN VIVO 33 11. Lips, H. J. 1957 Stability of d-a-tocopherol 20. Mameesh, M. S., and B. C. Johnson 1958 alone, in solvents, and in methyl esters of The effect of penicillin on the intestinal fatty acids. J. Am. Oil Chemists’ Soc., 34: synthesis of thiamine in the rat. J. Nutrition, 513. 65: 161. 12. Horwitt, M. K., C. C. Harvey, B. Century and 21. Bonsnes, R. W., and H. H. Taussky 1945 L. A. Witting 1961 Polyunsaturated lipids On the colorimetric determination of creatine and tocopherol requirements. J. Am. Dietet. by the Jaffe reaction. J. Biol. Chem., 158: A., 38: 231. 581. 13. Woodman, A. G. 1941 Food Analysis, ed. 4. 22. Mangold, H. K. 1961 Thin-layer chromatog McGraw Hill Book Co. Inc., New York, p. 185. raphy of lipids. J. Am. Oil Chemists’ Soc., 14. Witting, L. A., C. C. Harvey, B. Century and 38: 708. M. K. Horwitt 1961 Dietary alterations of 23. Century, B., L. A. Witting, C. C. Harvey and fatty acids of erythrocytes and mitochondria M. K. Horwitt 1961 Compositions of skel of brain and liver. J. Lipid Res., 2: 412. etal muscle lipids of rats fed diets containing various oils. J. Nutrition, 75; 341. 15. Emmerie, A., and C. Engel 1938 Colori 24. Holman, R. T. 1954 Progress in the Chem metric determination of a-tocopherol. Rec. istry of Fats and Other Lipids, vol. 2, eds., trav chim. Pays-Bas, 57: 1351. R. T. Holman, W. O. Lundberg and T. Mal 16. Green, J., S. Marcinkiewicz and P. R. Watt kin. Academic Press, Inc., New York, p. 73. 1955 The determination of tocopherols by 25. Gunstone, F. D., and T. P. Hilditch 1946 paper chromatography. J. Sci. Food Agr., The autoxidation of methyl oleate in the 6: 274. presence of small proportions of methyl lin- 17. Century, B., and M. K. Horwitt 1960 The oleate. J. Chem. Soc., 1022. role of diet lipids in the appearance of dys 26. Mohrhauer, H., and R. T. Holman 1963 trophy and creatinuria in the vitamin E- The effect of dose level of essential fatty deficient rat. J. Nutrition, 72: 357. acids upon fatty acid composition of the rat 18. Vars, H. M., and F. N . Gurd 1947 Effect liver. J. Lipid Res., 4: 151. of dietary protein upon the regeneration of 27. Kinsell, L. W. 1963 Some thoughts regard liver protein in the rat. Am. J. Physiol., ing the P:S ratio concept. Am. J. Clin. N u 151: 399. trition, 12: 228. 19. Bonetti, E., and F. Stirpe 1962 Conditions 28. Hove, E. L., and H. R. Seibold 1955 Liver affecting dietary liver necrosis and liver re necrosis and altered fat composition in vita generation in rats. J. Nutrition, 77: 179. min E-deficient swine. J. Nutrition, 56: 173. Phosphorus Requirement of the Baby Pig* 1 E. R. MILLER, D. E. ULLREY, C. L. ZUTAUT, BETTY V. BALTZER, D. A. SCHMIDT, J. A. HOEFER an d R. W. LUECKE Departments of Animal Husbandry, Veterinary Pathology and Biochemistry, Michigan State University, East Lansing, Michigan ABSTRACT Studies were made with 32 baby pigs in 2 trials to determine their dietary phosphorus requirement. Using a synthetic milk diet, the calcium level was maintained at 0.8%, and phosphorus concentration varied from 0.2 to 0.8% of dietary solids with dietary casein consistently supplying 0.2% of phosphorus and USP grade CaHP04-2H20 supplying the additional phosphorus. A dietary phosphorus level of 0.4% appeared adequate to effect normal growth and economy of food utilization. A dietary phosphorus level of 0.5% was adequate to maintain normal concentrations of serum calcium, inorganic phosphorus and alkaline phosphatase and to provide for an adequate rate of skeletal development. To obtain maximal strength of bone and to insure the absence of rachitic lesions it appears necessary to provide the baby pig with 0.6% of dietary phosphorus. Reported studies (1—4) have shown MATERIALS AND METHODS phosphorus requirements of growing-finish Two trials were conducted using 32 York- ing pigs fed natural rations to vary from shire-Hampshire crossbred pigs of either 0.3 to 0.6% of the diet, with the reported sex. Pigs were taken from the sow at one requirement differences largely dependent to three days of age and after environ upon the criteria selected. Combs et al. mental and dietary adjustment with a low (4 ) also studied the phosphorus require phosphorus diet, were assigned to experi ment of pigs between the ages of 2 and 7 mental levels of dietary phosphorus at one weeks of age and concluded that 0.44% of week of age. Bases of treatment assign dietary phosphorus adequately met the ment and environmental conditions were minimal requirement of young pigs fed a similar to those described by Miller et al. fortified corn-soybean meal diet. Zimmer (9 ). The solids of the basal diet consisted man et al. (5 ) obtained optimal perform of 30% of casein,2 10% of lard, 54% of ance with 3- to 7-week-old pigs when their glucose,3 6% of a phosphorus-free mineral high milk-product diet contained 0.6% of mixture (table 1) and vitamins (10). The phosphorus. Freese (6 ) concluded that the diet was made into a 20% of solids, homog phosphorus requirement of young pigs fed enized milk and fed 4 times daily for the liquid milk diets was 1.1% of the dry mat first 3 weeks of trial 1 and the first 2 weeks ter of the diet. Based upon body composi of trial 2. During the final 3 weeks of each tion, Mitchell and McClure (7 ) estimated trial the diet was fed as a dry meal 3 times that the 13.6-kg pig has a phosphorus re daily. When fed as a dry meal 5% of quirement of 0.37% of the dry matter in cellulose4 replaced an equal portion of fat the diet. Lucas and Lodge (8 ) have chosen in the diet. Experimental levels of phos as “preferred estimates” of phosphorus re phorus were 0.2, 0.4 and 0.6% in trial 1 quirement 0.9, 0.65 and 0.45% for the and 0.4, 0.5, 0.6, 0.7 and 0.8% in trial 2, 4.5-, 9-, and 13.6-kg pig, respectively. with 4 pigs per level of phosphorus in In the present study an attempt was each trial. All diets contained 1800 IU of made to assay the phosphorus requirement vitamin D3/kg of solids. Calcium level of of the baby pig reared with a synthetic all diets was maintained at 0.8% and ex- milk diet using as criteria: growth and food economy; level of serum inorganic Received for publication August 5, 1963. 1 Published with the approval of the Director of phosphorus, calcium and alkaline phos the Michigan Agricultural Experiment Station as phatase; concentrations of humeral ash, Journal article no. 3145. 2 Labco, Vitamin-free Casein, The Borden Company, calcium and phosphorus; and measures of New York. 3 Cerelose, Corn Products Company, Argo, illinois. femur density and strength. 4 Solka Floe, Brown Company, Boston. 3 4 J. N u t r i t i o n , 8 2 : ’ 6 4 PHOSPHORUS REQUIREMENT OF THE BABY PIG 3 5 TABLE 1 RESULTS AND DISCUSSION Composition of basal mineral mixture Trial 1. Growth rate and food con % sumption were depressed only in those pigs KC1 (0.002% I) 10.0 receiving less than 0.4% of phosphorus KI 0.002 (table 2) with a significant reduction in FeSO.,-7H,0 1.0 CoCL -6H;0 0.1 rate of growth and feed consumption of CuS04-5H20 0.1 pigs receiving 0.2% of phosphorus becom MnSOj-ILO 0.2 ing apparent after only 2 weeks of the ex ZnSCVHoO 0.4 perimental period. The degree of growth MgS04-7H20 4.0 depression was greater than the degree of NaHCO.i 25.0 CaCOs 33.3 reduction in food consumption in these CaHP04-2H20 0.0 pigs, resulting in a significant reduction in Glucose 1 25.9 the efficiency of food utilization. A level of Total 100.002 0.4% of dietary phosphorus was sufficient 1 Cereiose, Corn Products Company, Argo, Illinois. to support maximal growth rate and economy of food utilization. perimental levels of phosphorus obtained The initial levels of serum inorganic by adjusting the levels of the last 3 in phosphorus were low in all pigs, indicating gredients in table 1, with the casein furn that probably some depletion had occurred ishing 0.2% of phosphorus to each of the during the adjustment period. A level of experimental diets. Studies (11-14) have dietary phosphorus of 0.6% rapidly re indicated excellent usage of dietary phos stored the serum phosphorus to a normal phorus from dicalcium phosphate by pigs, level. Pigs receiving the diet containing and the work of Freese (6 ) and Ludvigson 0.4% of phosphorus did not possess nor and Thorbek (15) indicate that the phos mal levels of serum phosphorus until the phorus of casein is well utilized by the sixth week of the experiment. Serum cal young pig. All diets were analyzed periodi cium levels were normal in all pigs, tend cally by the method of Gomorri (1 6) to ing to be higher in animals receiving lower verify the intended levels of phosphorus. levels of dietary phosphorus. Serum alka Dietary consumption by individual pigs line phosphatase concentration became was accurately measured and recorded. elevated only in the severely phosphorus- All pigs were weighed weekly. Blood deficient pigs. Kidney phosphorus concen was withdrawn bi-weekly from the anterior tration was somewhat increased in phos vena cava for the determination of levels phorus-deficient pigs. No explanation for of serum inorganic phosphorus, calcium this observation is apparent. and alkaline phosphatase by the methods Humeral ash, calcium and phosphorus of Gomorri (16), Mori (17) and Bessey et concentrations were maximal in pigs re al. ( 18), respectively. At the conclusion of ceiving 0.6% of dietary phosphorus. Maxi each trial all pigs were killed and gross mal density of femur and rib were also and microscopic pathological observations exhibited by pigs on this level of phos were made. Various bones, organs and phorus. Furthermore, maximal breaking glands were removed and weighed. The strength, resistance to bending and stress methods of Mori (17) and Gomorri (16) capacity were exhibited by femurs from were adapted to determine calcium and pigs receiving the highest level of phos phosphorus concentration in kidney tissue. phorus supplementation in this trial. Determinations of specific gravity of femur, Strength of femur was largely dependent humerus and eighth rib were made by the upon the thickness of the compact calcified method described by Brown et al. (19). layer of the diaphysis as shown in figure 1. Humeral ash, calcium and phosphorus de Pigs receiving 0.2% of dietary phos terminations were by AOAC (20) methods. phorus exhibited shortened limbs with Strength measurements of the femur were weakened and bent metatarsals and pha made by the methods described by Miller langes (fig. 2). The appearance of the et al. (9 ). Statistical analyses of data were phosphorus-deficient pigs was similar to performed using the multiple range test of that observed in calcium deficiency (9 ) Duncan (21). with the exception that phosphorus-defi- 3 6 MILLER, ULLREY, ZUTAUT, BALTZER, SCHMIDT, HOEFER AND LUECKE TABLE 2 Growth, blood serum and kidney analyses and skeletal development of baby pigs fed different levels of phosphorus (trial 1) Dietary phosphorus level, % of food solids 0.2 0.4 0.6 No. of pigs 4 4 4 Initial weight, kg 3.05 ±0.06 1 3.11 ± 0.16 3.03 ±0.17 Daily gain, kg 0.13 ± 0.01 0.29 ±0.01 ** 0.29 ±0.03 »» Food solids/day, kg 0.24 ±0.01 0.38 ±0.02 »» 0.37± 0.03 »» Food solids/gain 1.85 ± 0.05 1.31 ±0.04 «» 1.28 ±0.07 »» Serum organic P, mg/100 ml Initial 4.1 ±0.2 4.2 ±0.3 3.9 ±0.3 2 Weeks 5.8 ±0.5 5.3 ±0.8 8.8 ±0.8 bb 4 Weeks 2.6 ±0.4 5.2 ± 0 .5 »» 9.2 ± 0.6 bb 6 Weeks 3.2 ±0.3 8.4 ± 0 .9 "« 9.4 ± 0 .4 »» Serum Ca, mg/100 ml Initial 12.1 ±0.4 12.3 ±0.4 12.0 ±0.4 2 Weeks 9.7 ±0.3 10.4 ±0.2 9.3 ±0.5 4 Weeks 12.8 ± 1.1 » 10.7 ±0.8 9.9 ±0.6 6 Weeks 12.8 ± 0 .2 »« 12.2 ± 0 . 3 “» 10.3 ±0.2 Serum alkaline phosphatase, Bessey-Lowry units Initial 11.4 ± 0.8 12.0 ±1.6 12.2 ±0.7 2 Weeks 9.4 ±1.5 14.9 ±1.1 9.8 ±1.7 4 Weeks 8.6 ±1.5 8.4 ±2.0 4.4 ±0.4 6 Weeks 20.5 ±3.5 ^ 8.3 ±0.7 4.5 ±1.4 Kidney analyses P, mg/100 g dry tissue 1550± 10b 1430 ±2 5 1470 ±1 4 Ca, mg/100 g dry tissue 51 ± 4 48 ± 1 40 ± 1 Humeral analyses (dry, fat-free basis) Ash, % 33.4 ±1.8 44.1 ± 1 .0 »» 47.5 ± 0 .7 »» Ca, % 11.6 ±0.7 15.6 ± 0 .4 »» 16.8 ± 0 .3 »“ P, % 5.65 ±0.28 7.91 ±0.07 »» 9.04 ±0.22 bb Ca/P 2.04 ±0.02 »» 1.98 ±0.02 »» 1.87 ± 0.02 Specific gravity Femur 1.11±0.01 1.15± 0.01 »» 1.17 ± 0.01 »» 8th rib 1.13 ± 0.01 1.23 ±0.01 »» 1.27 ± 0.02 »» Weight, g Femur 40.6 ±2.0 65.2 ± 2 .3 » 66.4 ± 3 .2 » 8th rib 3.30 ±0.25 4.49 ±0.39 4.67 ± 0.60 » Femur strength 2 Breaking load, kg 24 ± 1 61 ± 6 »» 81 ± 2 bb Bending moment, kg-cm 48 ± 3 118 ± 8 »» 160 ± 4 bb Moment of inertia, cm4 0.06 ± 0.00 0.12± 0.01 »» 0.14 ±0.01 »" Breaking stress, kg/cm2 470 ±1 6 636 ±6 9 » 785 ±4 0 b 1 s e o f m e a n . a Significantly greater than least value (P < 0.05); aa (P < 0.01). b Significantly greater than both other values (P < 0.05); bb (P < 0.01). 2 For formulae, see Miller et al. (9). cient animals did not exhibit the “humped Examination at necropsy revealed multi back” or “protruding eye” symptoms ex ple rib fractures with calluses in the acutely hibited by calcium-deficient pigs. Perhaps phosphorus-deficient pigs with “beading” failure of the phosphorus-deficient pig to apparent at the costochondral junctions exhibit these symptoms is due to a more (fig. 1). Histopathological findings were uniform cessation of bone and soft tissue similar to those observed in calcium defi growth as compared with the calcium-de ciency (9 ). A single rib fracture was noted ficient pig in which bone growth is prefer in one of the pigs receiving 0.4% of dietary entially interrupted. phosphorus. Histopathological studies re- PHOSPHORUS REQUIREMENT OF THE BABY PIG 3 7 0.2 %P 0.4 %P 0.6 %P 8 th RIBS • P # FEMURS Fig. 1 Eighth rib and cross-section of femur from littermates receiving different dietary levels of phosphorus (trial 1). Note the enlarged costochondral joint (beading) and the cal lus (indicating earlier break) in the rib at left and the lack of compact layer in the corre sponding femur. Breaking loads were 27, 70 and 84 kg for femurs shown from pigs re ceiving 0.2, 0.4 and 0.6% of dietary P, respectively. 38 MILLER, ULLREY, ZUTAUT, BALTZER, SCHMIDT, HOEFER AND LUECKE Fig. 2 Phosphorus-deficient pig exhibiting short limbs, weakened particularly in the metatarsal-phalangeal region. This pig from trial 1 received a synthetic milk diet containing 0.2% P, 0.8% Ca and 1800 IU of vitamin D:l/kg. vealed that the ribs from pigs receiving this level was not significantly affected by die level of phosphorus were fairly normal with tary phosphorus level. Percentages of hu only borderline rachitic lesions. Ribs from meral ash, calcium and phosphorus were pigs receiving 0.6% of dietary phosphorus significantly reduced in pigs receiving the appeared normal in every respect. lowest level of dietary phosphorus. This re Judging from the information obtained duced level of bone ash was further mani from trial 1 it can be concluded that 0.4% fested by the specific gravity of humerus of dietary phosphorus is adequate to pre and rib. Femur strength as measured by vent gross rachitic symptoms and to breaking load, bending moment and break achieve optimal rates of body weight gain ing stress was significantly greater in pigs and economy of food utilization. However, receiving the higher levels of dietary phos it can also be concluded that this level of phorus. dietary phosphorus is inadequate to sup Histopathological studies of rib sections port maximal rate of skeletal development. in this trial revealed moderate rachitic le The second trial was designed to determine sions in all pigs receiving 0.4% of dietary the level of dietary phosphorus that would phosphorus based upon the width and support a maximal rate of skeletal devel regularity of the zone of proliferating car opment as determined by bone composi tilage, the straightness of the line of ossifi tion, density and strength characteristics. cation and the width, straightness and Trial 2. Results of the second trial are regularity of the bony trabeculae. Pigs re presented in table 3. As in the first trial, ceiving 0.5% of dietary phosphorus re 0.4% of dietary phosphorus was adequate vealed ribs varying from fairly normal to also in this trial to support optimal rate of those with borderline lesions of rickets. growth and economy of food utilization. Ribs from pigs receiving the higher levels Serum inorganic phosphorus levels were of dietary phosphorus appeared normal in significantly lower, however, in pigs receiv all respects. ing 0.4% of dietary phosphorus and their The results of trial 2 indicate clearly serum alkaline phosphatase concentration that 0.5% of dietary phosphorus is ade was somewhat elevated. Serum calcium quate to maintain normal concentrations PHOSPHORUS REQUIREMENT OF THE BABY PIG 3 9 TABLE 3 Growth, seruvi analyses and skeletal development of baby pigs (trial 2) Dietary phosphorus level, % of food solids 0.4 0.5 0.6 0.7 0.8 No. of pigs 4 4 4 4 4 Initial weight, kg 1.82 ±0.23 1 1.91 ±0.23 1.82 ±0.12 1.86 ± 0.21 1.91 ±0.18 Daily gain, kg 0.28 ± 0.01 0.28 ± 0.02 0.29 ±0.02 0.29 ±0.02 0.29 ±0.02 Food solids/day, kg 0.85 ±0.02 0.85 ± 0.0 1 0.86 ± 0.02 0.86 ±0.02 0.86 ±0.01 Food solids/gain 1.40±0.04 1.38 ± 0.08 1.35 ±0.07 1.37 ± 0.10 1.37 ±0.06 Serum inorganic P, mg/100 ml Initial 9.4 ±0.1 9.1 ±0.1 10.1 ±0.1 9.1 ±0.6 9.5 ±0.5 3 Weeks 7.7 ±0.3 9.6 ±0.8 10.7 ±0.8 10.2 ±0.2 10.3 ±0.3 5 weeks 8.2 ±0.5 10.4 ± 0 .1 “ 10.0 ±0.1 « 10.4 ± 0 .1 “ 10.3 ±0.1 ‘ Serum Ca, mg/100 ml Initial 10.4 ±0.1 10.4 ±0.1 10.8 ±0.1 11.0 ±0.1 11.0 ±0.1 3 Weeks 11.6 ±0.1 11.0 ±0.1 10.1 ±0.4 10.9 ±0.1 10.5 ±0.1 5 Weeks 11.6 ±0.1 11.0 ±0.1 11.7 ±0.1 10.8 ±0.1 10.9 ±0.1 Serum alkaline phosphatase, Bessey-Lowry units Initial 12.1 ±1.3 16.0 ±2.6 15.4 ±0.8 14.4 ±2.0 14.9 ± 1.4 3 Weeks 14.1 ±1.3 12.4 ±1.2 13.2 ±1.0 11.5 ±0.7 9.3 ±0.8 5 Weeks 11.6 ± 2 . 2 “ 8.5 ±0.7 9.3 ±0.8 8.4 ±0.4 7.6 ±0.4 Humeral analyses (dry, fat-free basis) Ash, % 47.5 ±0.8 52.0 ±0.3 52.1 ± 0.6 ““ 52.6 ± 0 . 6 ““ 53.2 ±0.3 »* Ca, % 16.3 ±0.4 17.8 ±0.1 ““ 17.8 ±0 .2 “* 18.1 ± 0 .3 ““ 18.3 ± 0.2 “” P, % 8.7 ±0.2 9.4 ±0.1 ““ 9.5 ±0.1 *a 9.6 ± 0 . 2 ““ 9.7 ±0.1 aa Ca/P 1.9 ±0.0 1.9 ±0.0 1.9 ±0.0 1.9 ±0.0 1.9 ±0.0 Specific gravity Humerus 1.15 ± 0.02 1.20 ± 0.01 “ 1.20 ± 0.01 “ 1.20 ±0.02 “ 1.21 ±0.01 “ 8th rib 1.24 ±0.01 1.27 ± 0.01 1.30 ±0.01 ““ 1.28 ± 0.01 “ 1.29 ±0.01 “ Femur strength 2 Breaking load, kg 70 ±1 0 93 ±13 99 ± 3 115 ± 16 “ 115 ± 8 * Bending moment, kg-cm 156 ± 21 193 ±2 8 196 ±2 5 236 ± 30 “ 233 ± 1 6 “ Moment of inertia, cm4 0.10 ± 0.02 0.12 ± 0.02 0.12 ±0.01 0.12 ± 0.03 0.12 ± 0.02 Breaking stress, kg/cm2 990 ±9 0 1010 ± 76 1250± 145 1240± 133 1390 ± 1 5 6 “ 1 se o f mean. a Significantly greater than least value (P < 0.05); aa (P < 0.01). 2 For formulae, see Miller et al. (9). of serum calcium, phosphorus and alkaline first 3 weeks of lactation, when sow’s milk phosphatase and to provide for an adequate is usually the sole source of the baby pig’s rate of skeletal development. To obtain nutrition, the concentration of phosphorus maximal strength of bone and to insure in the dry matter of sow’s milk is con the absence of rachitic lesions it appears sistently near this value (0.6% ). necessary to provide the baby pig with 0.6% or more of dietary phosphorus. This LITERATURE CITED is in good agreement with the results ob 1. Aubel, C. E., J. S. Hughes and H. F. Lien- tained by Zimmerman et al. (5 ) and with hardt 1936 Phosphorus requirements in the rations of growing pigs. Kansas Agr. the recommendations of the National Re Exp. Sta. Tech. Bull. 41., Manhattan, Kansas. search Council subcommittee on swine nu 2. Jordan, C. E., J. H. Conrad. M. P. Plumlee trition (2 2) for the 4.5- and 11-kg pig. and W. M. Beeson 1957 Phosphorus and Furthermore, results of the phosphorus protein levels for growing-finishing swine on legume pasture. J. Animal Sci., 16: 1077. analyses of sow’s milk (23-27) show that 3. Chapman, H. L., Jr., J. Kastelic, G. C. Ashton, pos^-colostral phosphorus concentration P. G. Homeyer, C. Y. Roberts, D. V. Catron, increases during lactation but that for the V. W. Hays and V. C. Speer 1962 Calcium 4 0 MILLER, ULLREY, ZUTAUT, BALTZER, SCHMIDT, HOEFER AND LUECKE and phosphorus requirements of growing- for growing-finishing swine. J. Animal Sci., finishing swine. J. Animal Sei., 21: 122. 18; 555. 4. Combs, G. E., J. M. Vandepopuliere, H. D. 15. Ludvigsen, J., and G. Thorbek 1960 Stud- Wallace and M. Roger 1962 Phosphorus ier over pattegrises raekst of ernaering. II. requirement of young pigs. J. Animal Sci., Tilraekst og foderforbrug same kvaelstof-, fos- 21: 3. for- og kalciumomsaetningen hos kunsfigt 5. Zimmerman, D. R., V. C. Speer, V. W. Hays ernaerede pattegrise i alderen fra 5. til 35 and D. V. Catron 1963 Effect of calcium dag. Beretning fra Forsfigslaboratoriet 320, Kpbenhavn. and phosphorus level on baby pig perform 16. Gomorri, G. 1942 A modification of the ance. J. Animal Sci., 22: 658. colorimetric phosporus determination for use 6. Freese, H. H. 1958 Untersuchungen fiber with the photoelectric colorimeter. J. Lab. den Calcium-, Phosphor-, Magnesium-und Clin. Med., 27: 955. Stickstoff-umsatz des Ferkels in Beziehung 17. Mori, K. 1959 Direct microcomplexometric zum Wachstum bei Muttermilch und Mutter- analysis of calcium in biological material. milchersatz. Arch. Tierernahrung, 8: 330. Arch. Biochem. Biophys., 83: 552. 7. Mitchell, H. H.. and F. J. McClure 1937 18. Bessey, O. A., O. H. Lowry and M. J. Brock Mineral nutrition of farm animals. National 1946 A method for the rapid determination Research Council Bull., 99. National Acad of alkaline phosphatase with 5 cubic milli emy of Sciences — National Research Coun meters of serum. J. Biol. Chem., 164; 321. cil, Washington, D. C. 19. Brown, C. J., J. C. Hillier and J. A. Whatley 8. Lucas, I. A. M., and G. A. Lodge 1961 The 1951 Specific gravity as a measure of fat Nutrition of the Young Pig. Commonwealth content of the pork carcass. J. Animal Sci., Agricultural Bureaux, Farnham Royal, 10; 97. Slough, Bucks, England. 20. Association of Official Agricultural Chemists 9. Miller, E. R., D. E. Ullrey, C. L. Zutaut, Betty 1960 Methods of Analysis, ed. 9. Washing V. Baltzer, D. A. Schmidt, J. A. Hoefer and ton, D. C. R. W. Luecke 1962 The calcium require 21. Duncan, D. B. 1955 Multiple range and ment of the baby pig. J. Nutrition, 77; 7. multiple F tests. Biometrics, 11: 1. 10. Miller, E. R., R. L. Johnston, J. A. Hoefer 22. National Research Council, Committee on and R. W. Luecke 1954 The riboflavin re Animal Nutrition 1959 Nutrient require quirement of the baby pig. J. Nutrition, 52: ments of domestic animals. II. Nutrient re 405. quirements of swine, pub. 648. National 11. Chapman, H. L., Jr., J. Kastelic, G. C. Ashton Academy of Sciences — National Research and D. V. Catron 1955 A comparison of Council, Washington, D. C. phosphorus from different sources for grow 23. Hughes, E. H., and H. G. Hart 1935 Pro ing and finishing swine. J. Animal Sci., 14: duction and composition of sow’s milk. J. 1073. Nutrition, 9: 311. 12. Gobble, J. L., R. C. Miller, G. W. Sherritt and 24. Perrin, D. R. 1955 The chemical composi H. W. Dunne 1956 Soft phosphate with tion of the colostrum and milk of the sow. colloidal clay as a source of phosphorus for J. Dairy Res., 22: 103. growing and fattening pigs. Pennsylvania 25. Guegen, L., and E. Salmon-Legagneur 1959 Agr. Exp. Sta. Bull. 609, University Park, La composition du lait de truie: Variations Pennsylvania. des teneurs en quelques elements minéraux 13. Plumlee, M. P., C. E. Jordan, M. H. Kenning- (P, Ca, K, Na, M g). C. R. Acad. Sci., 249: ton and W. M. Beeson 1958 Availability of 784. the phosphorus from various phosphate mate 26. Jylling, B. 1960 Investigations concerning rials for swine. J. Animal Sci., 17: 73. the composition of sow’s milk. Royal Veteri 14. Aldinger, S. M., V. C. Speer, G. C. Ashton, nary Agricultural College Yearbook, Copen V. W. Hays and D. V. Catron 1959 Effect hagen. of feeding different combinations of soft 27. Travnicek, J. 1960 Chemické slozeni mle- phosphate with colloidal clay and dicalcium ziva a rnléka prasnic plemene bilého vslechti- phosphate with and without added fluorine lého. S. Chek. Acad. Med. Ved., 33: 497. Zinc Deficiency Syndrome in the Young Lamb* 1 E. A. OTT, W. H. SMITH, MARTIN STOB a n d W. M. BEESON Purdue University, Lafayette, Indiana ABSTRACT Eighteen lambs averaging 13.5 kg were divided into 6 comparable groups and fed the following diets: basal, basal + 2% polyethylene, basal + 0.61% phytic acid, basal + 0.61% phytic acid + 2% polyethylene, basal -f 100 mg zinc/kg, and basal + 100 mg zinc/kg + 2% polyethylene. The basal diet (2.7 mg zinc/kg) was composed of the following: (in per cent) egg whites, 15.00; cellulose, 18.00; glucose monohydrate, 27.02; starch, 27.02; corn oil, 3.00; minerals and vitamins. The animals fed the diets not supplemented with zinc exhibited the following symptoms: anorexia; depraved appetite (eating wool); reduced growth; reduced feed efficiency; loose wool; swollen hocks; red, wrinkled skin; and open lesions of the skin above the hoof and around the eyes. Tissue changes characteristic of the deficiency were decreased tissue zinc and parakeratotic lesions of the skin. Blood changes typical of the deficient ani mals were decreased serum zinc, decreased serum albumin and increased globulin levels. Excessive salivation, increased rumen propionic acid production and a corre sponding reduction in butyric acid were also associated with the deficiency. The zinc deficiency appeared to be less severe in the lambs fed phytic acid. Feeding 100 mg zinc/kg of the basal diet to deficient lambs for 5 weeks completely alleviated most of the deficiency symptoms. The polyethylene added to the diet had little or no effect on performance but appeared to stimulate rumination. Since Tucker and Salmon (1 ) linked support normal growth and could be ren the parakeratotic condition in swine de dered very low in zinc; 2) to characterize scribed by Kernkamp and Ferrin (2 ) to a the symptoms associated with a zinc de zinc deficiency, a considerable amount of ficiency in the lamb; and 3) to alleviate research has been conducted to determine the deficiency symptoms by feeding zinc. the nutritional requirement of monogas- tric animals for this element. Knowledge METHODS of the ruminant’s requirement for zinc, Eighteen cross-bred native lambs aver however, has not progressed beyond the aging 13.5 kg were divided into 6 com point of recognizing that a need exists. parable groups according to weight, sex This may be due to 2 factors: (a ) only and breeding. The age of the lambs recently have reports indicated that natu ranged from 4 to 8 weeks. Groups were ral deficiencies such as those described by assigned to the following treatments: Legg and Sears (3 ) and Haaranen and basal, basal + 2% polythylene; basal + Hyppola (4 ) may exist; and (b ) the for 0.61% phytic acid; basal + 0.61 phytic mulation of semi-purified diets for rumi acid + 2% polyethylene; basal + 100 mg nants suitable for trace mineral studies zinc/kg; and basal + 100 mg zinc/kg + has been far behind corresponding work 2% polyethylene. The basal diet listed in with monogastrics. table 1, which is a modification of the Miller and Miller (5, 6) described the diet used by Lawlor et al. (10), contains symptoms of zinc deficiency in Holstein 2.7 mg zinc /kg. The polyethylene was calves fed a purified diet containing 3.6 added to the diet of one-half of the lambs mg zinc/kg, but in later work (7 ) could in the form of 3-mm cubes replacing an not produce the deficiency using a natural equal quantity of cellulose to determine diet containing 25 mg zinc/kg. No effect the effect of providing a roughness factor could be found due to high phytic acid to the diet. The phytic acid was added levels as have been described for mono- to 2 of the treatments to test the effect of gastric animals by Plumlee et al. (8 ), and this chelating agent on the zinc fed to Oberleas et al. (9 ). The objectives of this experiment were: Received for publication August 5, 1963. i Contribution from the Department of Animal 1) to develop a ration for lambs that would Sciences, Journal paper no. 2179. J. N u t r it io n , 8 2: ’64 41 4 2 E. A. OTT, W. H. SMITH, MARTIN STOB AND W. M. BEESON TABLE 1 and packed cell volumes were determined Basal d ie t1 and the serum was wet-oxidized and ana lyzed for zinc content by a modification of % the dithizone-bis (2 hydroxyethyl) dithio- Egg whites 2 15.00 carbamate method evaluated by Margerum Cellulose 3 20.00 Glucose monohydrate 4 27.02 and Santacana (11). This method was Starch (corn) 27.02 used for all subsequent zinc analyses. Corn oil 3.00 The lambs were maintained on the first CaHP04'2H20 3.00 phase of the experiment for 10 weeks. At KoCOs-li/aHsO 2.00 MgS04-3H20 0.70 the end of the first phase, one lamb from NaCl 1.00 each of the lots not receiving added zinc Trace mineral mix 5 0.56 and 2 lambs from the lots receiving added Vitamin and antibiotic mix 3 0.56 zinc were killed. Blood samples were taken Choline chloride 0.14 for serum zinc analysis and since Hoefer 1 Diet contained 2.7 mg zinc/kg. 2 Egg white solids, spray, P-19-S (Henningson et al. (12) had found an increase in serum Foods, Inc., New York). globulins and a decrease in albumin in 3 Cellulose Flock, K-856 Pulp (International Filter Corporation), North Tonawanda, New York. zinc-deficient swine, composite samples of 5 Trace mineral mix provided the following in mg/ kg of the diet: iron, 70.40; boron, 14.65; manganese, blood serum were made for each lot and 20.72; copper, 15.86; cobalt, 0.11; molybdenum, 0.33. serum protein fractions determined by 6 Vitamin and antibiotic mix provided the following in mg/kg of the diet: biotin, 0 .2 2 ; naphthoquinone, electrophoresis.2 Total serum proteins were 2.20; pyridoxine, 2.20; folic acid, 4.40; thiamine HC1, 6.60; riboflavin, 8.80; p-aminobenzoic acid, 8.80; determined by the method described by vitamin A (362,000 IU/g), 12.21; Ca pantothenate, 22.00; niacin, 33.00; vitamin E, 33.00; vitamin B12, Lowry (13). Tissue samples were taken 44.00; ascorbic acid, 110.00; inositol, 110.00; vitamin for zinc analysis and histological ex D3, (Delsterol, E. I. du Pont de Nemours & Co., W il mington, Del., 3,000 IU/g), 146.30; and chlortetra- amination. Rumen fluid samples were cycline (Aureomycin, American Cyanamid Co., New York), 22.00. taken from each lamb as it was killed by mixing and sampling the entire reticulo- sheep. The acid was added to the basal rumen contents. The determination of the at the rate of 1.525% of a solution con rumen fluid pH was made within 2 min taining 40% phytic acid. utes after the animal was killed and the The experiment was conducted indoors samples were frozen until volatile fatty and temperatures were maintained be acid (V F A ) analysis could be run. Anal tween 23° and 19°C. When the outside ysis was by a modification3 of a procedure temperature exceeded this, the building described by Erwin (1 4) using an Aero was allowed to equilibrate with the am graph “Hy-Fi” Model 600 gas chromato bient temperature. Due to the early age graph and a 0.3-cm OD by 152-cm stain at which the lambs were weaned, each less steel column packed with 20% LAC group of 3 lambs was placed in a 122- by 296 and 2% phosphoric acid on 60/80 244-cm pen for the first 2 weeks of the acid-washed chromasorb W. The proce experiment to allow them to adjust to the dure readily separates acetic, propionic, new environment. Individual pens were butyric, isovaleric and valeric acids. used for the remainder of the experiment. The 8 lambs remaining from the low Each pen, except those in which the lambs zinc lots were divided into 2 comparable were to receive added zinc, was lined with groups. One group continued to be fed wood to deny the animals access to the the basal + 2% polyethylene and the galvanized pipe frames of the pens. Plas other was given the basal + 100 mg tic feeders and waterers were used. zinc/kg + 2% polyethylene. The 2 lambs Water containing less than 0.1 mg zinc/ remaining from the added zinc lots of liter was supplied from a Zeolite water the first phase continued to be fed the softener. A wooden platform was placed basal + 100 mg zinc/kg + 2% poly in each pen to eliminate the need for ethylene. The repletion phase was con bedding and the pens were washed daily tinued for 5 weeks. At the end of this with soft water. 2 Electrophoresis was as described for procedure B Blood samples were taken at the begin in the Beckman Instruction Manual on the Spinco ning of the experiment and at the seventh, Model R Paper Electrophoresis system. 3 Rumsey, T. S. 1963 Corn silage in rations for tenth, and fifteenth week. Hemoglobin lactating dairy cows. M.S. Thesis, Purdue University. ZINC DEFICIENCY SYNDROME IN THE YOUNG LAMB 4 3 phase all lambs were killed and samples loosening, rubbing and eating of the fleece. comparable to those taken at 10 weeks These symptoms were noted during the were taken. fourth week in 2 lambs and were followed The data were analyzed by analysis of by swelling of the area between the hocks variance techniques as outlined by Snede- and the hoof and lesions of the skin just cor (15). The Duncan multiple range test above the hoof and around the eyes. The (16) was used to test for significant differ skin areas that were devoid of wool be ences among the treatment means. came slightly red and wrinkled. Encrusted areas developed on the ventral surfaces RESULTS AND DISCUSSION of the body and the skin of the animals The lambs consumed the ration readily showing the most severe conditions was but found some difficulty in adjusting to it. easily damaged. Similar external symp This was evidenced by the variations in toms have been described by Miller and the consistency of the feces during the Miller (5,6) as characteristic of zinc-de first 3 to 4 weeks; however, the problem ficient calves and by Kernkamp and Ferrin did not persist. During the third week, a (2 ) as swine parakeratosis. By the eighth difference in feed consumption, rate of week, all the lambs except one were show gain and feed-to-gain ratio became evi ing some of the symptoms. The symptoms dent between the lots receiving added zinc appeared in the remaining lamb very and the other treatments. These differ rapidly during the tenth week. Additional ences became progressively more pro symptoms present in the most acute cases nounced throughout the remainder of the include excessive salivation when eating experiment. Visible symptoms of the de and ruminating, shivering, swollen joints, ficiency (fig. 1) were first manifest by the and a characteristic stance which included Fig. 1 Left, zinc-deficient lamb after fed basal diet for 15 weeks. Right, lamb of the same age which was fed the basal + 100 mg zinc/kg diet for the same period. 44 E. A. OTT, W. H. SMITH, MARTIN STOB AND W. M. BEESON placing the feet very close together and value for each lot was called the “gross arching the back. The sequence of ap symptom score.” The subjectivity of the pearance for the symptoms was not con score necessitated the exclusion of some stant between lambs. symptoms from the evaluation because of Due to the difficulty inherent in de the difficulty in assessing their severity. scribing the progression of a syndrome in At the end of the first phase the differ a group of animals, a numerical evalua ence in the gross symptom score (table 2) tion was made of each lamb’s severity of between the lots with and without added 4 symptoms: fleece condition, general ap zinc was significant (P < 0.01). A signifi pearance of the skin, lesions around the cantly (P < 0.05) more severe condition feet and lesions around the eyes. The score was also found in the lambs fed the basal was established by giving each area a zero diet than in those receiving added phytic if normal, and up to a 5 for extreme ab acid. normality. Thus, each animal could have Average daily gain and feed consump a maximal score of 20 points. The mean tion (table 3) were significantly (P < 0.01) TABLE 2 Gross symptoms score1 for lambs fed basal, basal + phytic acid and basal + 100-mg zinc/kg diets Week of experiment Treatment 8 9 10 Phase 1(1 0 weeks) Basal 9.2 11.3 14.3 2 Basal + phytic acid 5.8 8.0 10.0 Basal + zinc 3 0 0 0 Phase 2 (repletion for 5 weeks) Week of experiment 10 11 12 13 14 15 Basal 10.8 3 14.2 4 15.5 5 15.8 5 15.8 5 15.8 5 Basal + zinc (repletion) 11.2 10.2 8.2 5.0 2.5 0.2 Basal + zinc 6 0 0 0 0 0 0 1 Gross symptoms score was calculated by giving each lamb a score of 0 if normal, and up to 5 if extremely abnormal for 4 different symptoms. Mean values were taken for each lot. 2 Difference between lambs fed the basal and basal + phytic acid was significant (P < 0.05). 3 Value based on lambs remaining after severely affected lambs were killed at the end of phase 1. 4 Difference between lambs fed basal and basal + zinc repletion diet was significant (P < 0.05). 3 Difference between lambs fed basal and basal + zinc (repletion) diet was significant (P < 0.01). 6 Since the lambs that received added zinc for the entire period did not exhibit any of the symptoms evaluated, they were considered significantly different from the deficient lambs and were not included in the analyses. TABLE 3 Performance data of lambs fed basal, basal + phytic acid and basal + 100-mg zinc/kg diets No. of Avg Daily Feed-to- Treatment animals daily gain gain feed ratio kg kg Phase 1(10 weeks) Basal 6 0.03 0.45 16.85 Basal + phytic acid 6 0.05 0.51 9.44 1 Basal-1-zinc 6 0.25 2 0.97 2 3.99 2 Phase 2 (repletion for 5 weeks) Basal 4 0.02 0.75 31.62 Basal-(-zinc (repletion) 4 0.29 3 1.15 3.99 Basal-fzinc 2 0.32 3 1.94 6.03 1 Value significantly different from that for the basal diet (P < 0.05). 2 Value significantly different from the other 2 treatments (P < 0.01). 3 Values significantly different from that for the basal diet (P < 0.01). ZINC DEFICIENCY SYNDROME IN THE YOUNG LAMB 4 5 greater for the zinc-supplemented lambs. Blood analyses showed very little differ Lambs receiving added zinc and added ence in hemoglobin and packed cell vol phytic acid were significantly (P < 0 .0 1 ) umes at the end of the first phase. Serum more efficient than the lambs fed the basal zinc levels (fig. 3) decreased to a level of diet, and those receiving the added zinc about 20 ng/100 ml. Lambs with low zinc were more efficient (P < 0.05) than those levels at the start of the experiment tended receiving the phytic acid. to exhibit the external symptoms first. Response to the repletion diet was dra During the second phase of the experi matic. Feed consumption increased imme ment there was a tendency for the afore diately and the external symptoms started mentioned factors in the repleted lambs to regress (fig. 2). Lesions healed; skin to approach the values for the lambs main lost its red, wrinkled appearance; and ex tained with the added zinc diets for 15 cessive salivation ceased. The lambs did weeks. Serum zinc levels in the lambs not lose their appetite for wool for about that had continued to be fed the low zinc 2 weeks, but by this time the old wool had diet for 15 weeks remained the same as tightened and new growth was evident in the 10-week value. Results of serum pro the denuded areas. Growth response was tein analysis (table 4) indicate that sig also immediate. Rate of gain increased nificant (P < 0.05) increases in the serum each week until it was comparable to that globulin levels occur in the deficient of the lambs receiving the added zinc diet lambs. Fractionation of cn- and a2- por for the entire experiment. Lambs fed the tions of the globulins was incomplete and basal diet gained significantly (P < 0.01) since their relative proportion to the 13- more slowly than either of the other globulins appeared to be constant the 3 groups. portions were combined. As a group this L X p . 1 a. R e p le te d July 10. I H 2 Lamb 2 2 Fig‘. 2 Left, zinc-deficient lamb after fed basal diet for 10 weeks. Right, same lamb after 5 weeks on the basal 100 mg zinc/kg diet. 4 6 E. A. OTT, W. H. SMITH, MARTIN STOB AND W. M. BEESON CO E o CT> O iC c a> W eek s Fig. 3 Growth rate and serum zinc levels of lambs fed deficient and adequate zinc diets. ------, indicates basal diet; — □ — , basal plus phytic acid; — O— , basal plus zinc; and — • — , basal plus zinc (repletion). ZINC DEFICIENCY SYNDROME IN THE YOUNG LAMB 47 TABLE 4 Concentrations of albumin and globulins in lamb serum 1 Total Treatment protein Albumin Total globulins a- and ß-globulins 7 -globulins g/100 m l g/100 n il % g / 1 0 0 m l % g/100 m l % g/100 m l % Phase 1 (10 weeks) Basal 10.28 3.92 38.1 6.34 61.8 3.14 30.6 3.20 31.2 Basal + phytic acid 9.88 3.70 37.5 6.17 62.4 3.35 34.0 2.82 28.5 Basal + zinc 7.01 4.33 61.8 2 2.68 2 38.1 2 1.95 27.8 0.72 3 10.4 3 Phase 2 (5 weeks) Basal 11.05 4.80 43.5 6.24 56.5 2.83 25.6 3.41 30.9 Basal-}- zinc (repletion) 8.18 4.62 56.5 3.56 43.5 2.05 25.1 1.50 18.4 Basal zinc 7.65 4.72 61.8 2.92 38.2 1.97 25.8 0.95 12.4 1 The relative percentages of the various fractions were analyzed by electrophoresis, the total protein concentrations were by the Folin-Lowry method. 2 Values significantly different from the other 2 treatments (P < 0.01). 3 Values significantly different from the other 2 treatments (P < 0.05). fraction showed a tendency to increase reductions were noted in the heart, mus in the deficient lambs but not significantly. cle, pancreas and wool. Similar reductions The y- fraction, however, showed a sig in tissue zinc were observed in deficient nificant (P < 0.05) increase in the de calves by Miller and Miller (6 ). Samples ficient lambs. Albumin levels tended to taken at the end of the second phase decrease in the deficient lambs but the showed a general increase in zinc content differences were nonsignificant. This de of the repleted lambs when compared with crease in the serum albumin may possibly the deficient lambs. Differences in the be a first-order effect of the deficiency; kidney, liver, pancreas and spleen were however, the increased globulin level is significant. Exceptions to this were the probably a response to secondary infection brain and skin. Zinc content of the brain resulting from the open lesions of the skin. was higher in the lambs not receiving The zinc analyses of the tissues, as added zinc. The zinc level of the skin did listed in table 5, indicate some reduction not show a decrease in the deficient ani in all tissues taken from the deficient ani mals. This is contrary to what might be mals at the end of phase 1. Significant expected in observing the other symptoms TABLE 5 Zinc content of tissue Phase 1 Phase 2 Tissue Basal + Basal + Basal phytic Basal + Basal zinc Basal + acid zinc (repletion) zinc fig Zn / g fresh tissue fig Zn/g fresh tissue Brain 10.8 10.2 12.7 11.2 10.4 8.5 Heart 15.6 1 15.2 1 17.5 18.5 21.2 21.2 Kidney 15.6 17.0 17.7 12.1 2 15.6 15.8 Liver 24.4 28.4 35.1 27.2 2 43.4 39.6 3 Muscle 25.7 4 21.6 1 33.9 -- 5 -- 5 ___ 5 Pancreas 11.2 4 11.2 4 17.4 11.63 16.2 18.0 Skin 5.3 4.0 5.5 7.1 8.8 6.5 Spleen 21.6 19.6 22.3 20.0 6 21.6 22.2 Wool 98.8 1 101.4 1 120.0 -- 7 104.6 121.5 1 Values significantly different from the basal + zinc diet (P < 0.05). 2 Values significantly different from other 2 treatments (P < 0.01). 3 Value significantly different from the repleted group (P < 0.05). 4 Values significantly different from the basal + zinc diet (P < 0.01). s Samples of muscle tissue for lambs on phase 2 were lost during analysis. 6 Value significantly different from other 2 treatments (P < 0.05). 7 Lambs on the basal diet did not have enough wool for analysis at the end of phase 2. 4 8 E. A. OTT, W. H. SMITH, MARTIN STOB AND W. M. BEESON of the deficiency. A possible explanation ceiving phytic acid appeared to be less may be the variations in the samples ana severely affected. The lambs that were lyzed due to varying amounts of hair and repleted for 5 weeks lost all excessive wool, which are high in zinc, and varying stratum corneum; however, the 5-week amounts of subcutaneous fat. These vari period appeared to be insufficient for the ations in sample uniformity were not a stratum germinativum to return to nor problem with respect to the analysis of mal. Examination of the adrenals, pitui other tissues but may have had consider tary, thyroid, spleen, pancreas, liver, ru able influence on accuracy of the analysis men, reticulum, omasum, abomasum, of the skin. small intestine, kidney and brain did not Histopathological examination of the reveal any changes which could be asso skin revealed a typical parakeratotic con ciated with the deficiency. dition. Skin samples taken from the hock The differences shown by the gross and flank areas (fig. 4) showed thickening symptoms score, feed efficiency and histo of the stratum corneum with retention of logical examination appear to indicate the nuclei, accumulation of debris, thick that the phytic acid may reduce the sev ening of the stratum germinativum, elon erity of the zinc deficiency in sheep. The gation of the rete pegs and acanthosis in mechanism of this action is not clear, but the deficient lambs. Skin from lambs re may relate to the phosphorus supplied by Fig. 4 Photomicrographs of skin sections from lambs receiving different levels of zinc. Upper left, lamb fed basal for 10 weeks, deficient. Upper right, lamb fed basal + 100 mg zinc/kg, normal. Lower left, lamb repleted for 5 weeks. Lower right, lamb fed basal + 1.525% phytic acid solution for 10 weeks. ZINC DEFICIENCY SYNDROME IN THE YOUNG LAMB 4 9 TABLE 6 Molar percentage of volatile fatty acids and pH of rumen fluid from lambs fed the basal, basal + phytic acid and basal + 100-mg zinc/kg diets with and without polyethylene Treatment pH Acetic Propionic Butyric Isovaleric Valeric acid acid acid acid acid Phase 1(10 weeks ) Basal 7.13 47.2 30.6 10.3 5.4 6.3 Basal + phytic acid 6.42 60.6 27.2 8.2 1.3 2.6 Basal + zinc 6.30 57.0 24.8 10.9 2.3 6.1 Polyethylene 6.17 49.3 27.6 13.6 1 1.4 5.4 1 No polyethylene 6.90 61.6 1 26.1 6.6 3.2 2.4 Phase 2 (repletion for 5 weeks) Basal 7.05 51.3 37.5 5.7 2.3 3.2 Basal-f zinc (repletion) 6.45 58.6 21.6 2 14.1 2 0.7 5.0 Basal + zinc 6.55 54.8 18.7 2 19.2 2 1.0 6.2 1 Differences between treatments are significant (P < 0.05). 2 Values significantly different from the basal (P < 0.01). the phytic acid. Tillman (1 7) showed ACKNOWLEDGMENTS that the phosphorus in phytic acid is avail Our appreciation to R. H. Tetzloff for able to the ruminant and Lewis et al. (18) care of the animals; Mrs. L. Reitz for her demonstrated that under certain condi assistance and advice on laboratory pro tions the addition of phosphorus to a low cedures; Dr. W. R. Featherston for serum zinc diet for swine reduced the severity of protein analysis; and Dr. A. L. Delez for his the resulting parakeratosis. Similar con advice on the histopathological changes ditions may exist in this diet because it in the deficient animals. supplied calcium and phosphorus at a level of 0.70 and 0.48% respectively, but LITERATURE CITED the added phytic acid resulted in a final phosphorus content of 0.65% or nearly a 1. Tucker, H. F., and W. D. Salmon 1955 Parakeratosis or zinc deficiency in the pig. 1:1 ratio with the calcium. Proc. Soc. Biol. Med., 88: 613. Differences in the rumen fluid pH were 2. Kernkamp, H. C. H., and E. F. Ferrin 1953 not significant; however, there was a trend Parakeratosis in swine. J. Am. Vet. Med. A., toward higher pH values in the deficient 123: 217. lambs (table 6). This may be attributed 3. Legg, S. P., L. Sears 1960 Zinc sulphate treatment of parakeratosis in cattle. Nature, to the increased salivation which they ex 186: 1061. hibited. At the end of the first phase no 4. Haaranen, S., and K. Hyppola 1961 Cure, significant differences in VFA proportions prevention of dairy cattle itch and hair slick could be attributed to the high or low zinc ing with zinc. Feedstuffs, 33: 46: 38. diets. Polyethylene additions resulted in 5. Miller, J. K., and W. J. Miller 1960 Devel opment of zinc deficiency in Holstein calves a significant increase (P < 0.05) in the fed a purified diet. J. Dairy Sci., 43: 1854. percentage of butyric and valeric acids 6. Miller, J. K., and W. J. Miller 1962 Experi with a corresponding decrease in acetic mental zinc deficiency and recovery of calves. acid. This variaiton is probably due to the J. Nutrition, 76; 467. differences in rumination. Since all the 7. Miller, J. K., W. J. Miller and C. M. Clifton 1962 Calf response to starters of varying lambs were given polyethylene during the zinc contents. J. Dairy Sci., 45; 1536. second phase, a further check on the poly 8. Plumlee, M. P , D. R. Whitaker, J. H. Conrad, ethylene’s effect could not be made. The W. H. Smith, H. E. Parker and W. M. Beeson additional 5 weeks of feeding the basal diet 1960 The effect of phytic acid and other organic factors on zinc utilization by the brought about a significant reduction (P < growing pig. Feedstuffs, 32; 34: 10. 0.01) in butyric acid production and a 9. Oberleas, D., M. E. Muhrer and B. L. O’Dell corresponding increase of propionic acid. 1962 Effects of phytic acid on zinc avail The repleted lambs corresponded well with ability and parakeratosis in swine. J. Animal Sci., 57. the lambs which had been fed the basal 21: 10. Lawlor, M. J., W. H. Smith and W. M. Beeson + 100 mg zinc/kg diet for the entire 15- 1963 Semi-purified diets for sheep. J. Ani week period. mal Sci., 22: 481. 50 E. A. OTT, W. H. SMITH, MARTIN STOB AND W. M. BEESON 11. Margerum, D. W., and G. Santacana 1960 and rumen fluid by gas chromatography. J. Evaluation of methods for trace zinc deter Dairy Sci., 44: 1768. mination. Anal. Chem., 32: 1157. 15. Snedecor, G. W. 1956 Statistical Methods, 12. Hoefer, J. A., E. R. Müler, D. E. Ullrey, H. D. ed. 5. Iowa State University Press, Ames. Ritche and R. W. Luecke 1960 Interrela 16. Duncan, D. B. 1955 Multiple range and tionships between calcium, zinc, iron and multiple F tests. Biometrics, 11: 1. copper in swine feeding. J. Animal Sei., 19: 17. Tillman, A. D., and J. R. Brethour 1958 249. Utilization of phytin phosphorus by sheep. 13. Lowry, O. H., N. J. Rosebrough, A. L. Farr J. Animal Sci., 17: 104. and R. J. Randall 1951 Protein measure 18. Lewis, P. K., Jr., W. G. Hoekstra, R. H. Grum- ment with the Folin phenol reagent. J. Biol. mer and P. H. Phillips 1956 The effect of Chem., 193: 265. certain nutritional factors including calcium, 14. Erwin, E. S., G. J. Marco and E. M. Emery phosphorus and zinc on parakeratosis in 1961 Volatile fatty acid analysis of blood swine. J. Animal Sci., 15: 741. Response of the Liver to Prolonged Protein Depletion IV. PROTECTION OF SUCCINIC OXIDASE AND SUCCINIC DEHYDROGENASE BY DIETARY METHIONINE AND CYSTINE IN A PROTEIN-FREE RATION J. N. WILLIAMS, JR. Laboratory of Nutrition and Endocrinology, National Institute of Arthritis and Metabolic Diseases, U. S. Department of Health, Education and Welfare, Public Health Service, National Institutes of Health, Bethesda, Maryland ABSTRACT The influence of including 0.3% DL-methionine in a protein-free ra tion upon liver succinic oxidase, succinic dehydrogenase, and protein was studied in the adult male rat. The changes were followed as a function of time during protein depletion followed by repletion. Enzyme assay systems were used in which the mito chondria were “damaged” by including calcium ions in the media, thus allowing free access of substrates and dye acceptor to the enzymes. The results with succinic oxi dase and succinic dehydrogenase were almost identical, clearly indicating that the rate- limiting step in the succinic oxidase system under these conditions is at the initial dehydrogenase. In the group receiving the protein-free ration without methionine, the activities of the enzymes decreased to 40% of normal after 8 weeks with a slight in crease toward normal after about 15 weeks. In the group receiving methionine in the protein-free ration, after an initial decrease to 60% of normal after 3 weeks (which was identical to the response in the group receiving no protein and no methionine), a rapid increase toward normal occurred with normal values for these enzymes being reached after 15 weeks. No significant change occurred in the pair-fed controls throughout the study. Protection of liver protein by methionine in a protein-free ration was also observed. Results from further studies with all other essential amino acids as well as cystine, arginine, and glutamic acid, added individually to the protein-free ration at levels equivalent to 0.3% methionine, indicated that cystine as well as methionine significantly protected the liver cells against loss of succinic oxidase and succinic dehydrogenase. No other amino acid tested showed this effect. Both cystine and methionine also protected liver cells from loss of other protein but to a less ex tent than succinic oxidase and succinic dehydrogenase. The initial results of an investigation of low level in protein-free rations, offers cellular changes in the liver of the rat as a some as yet unelucidated protection of cer function of protein depletion have been tain liver components (3, 4). In addition presented in previous reports of this series it has been found that methionine or cys (1 -3 ). In the investigation an attempt tine in a protein-free ration causes a sev was made to define, biochemically and eral-fold increase in liver lipids.* 1 A de physiologically, the adaptation of liver cells tailed report of this work will be given to prolonged protein depletion followed by separately. Recently Wannemacher and repletion. In particular, emphasis was Allison2 have reported that addition of me placed on those components in the cell thionine to a protein-free diet prevented most closely related to the maintenance of loss of liver ribonucleic acid (R N A ) and energy metabolism. To this end a detailed protein and tended to increase serum al study of the adaptive response of the suc bumin levels to above normal levels. In cinic oxidase system — the initial dehy the first 2 publications of the present series drogenase and the individual components of the associated electron transport sys Received for publication July 5, 1963. 1 Williams, J. N., Jr., and A. D. Jasik 1963 Pro tem — as a model mitochondrial system, duction of fatty livers by methionine and cystine. Abstracts, Sixth International Congress of Biochem was carried out. istry, Edinburgh. A few brief reports have appeared in the 2 Wannemacher, R. W., and J. B. Allison 1963 The effect of methionine upon serum and liver pro literature that methionine, when fed at a tein metabolism. Federation Proc., 22: 202 (abstract). J. N u t r i t i o n , 8 2 : ’ 6 4 5 1 5 2 J. N. WILLIAMS, JR. (1, 2 ), 0.3% DL-methionine, which had Phase 1. Adult male rats of the originally been added to supplement the Sprague-Dawley strain were fed a com 20% casein ration, was retained in the plete purified diet (diet R l ( 1 ) ) for a 3- protein-free ration. Since this level of me week adjustment period. The complete diet thionine, although quite low, might have consisted of: (in per cent) casein, 20; influenced the results, it was important to DL-methionine, 0.3; corn oil, 5; salts N re-examine some of the variables studied plus molybdate (6 ), 6.5; glucose monohy earlier both with and without methionine drate, 63.5; choline chloride, 0.2; ¿-inositol, included in the protein-free ration. Pre 0.02; and water soluble-vitamin mix in liminary results indicated that methionine glucose monohydrate (7 ), 4.5. Fat-soluble did influence the results with succinic vitamins (1 ) were given weekly to each oxidase and dehydrogenase. Therefore, a rat in 2 drops of corn oil. The rats, which study of the effects not only of methionine weighed 290 g after the initial adjustment but also of cystine, arginine, glutamic acid period, were separated into 4 groups. (as control) and each of the other essen Group 1 received diet R l ad libitum. tial amino acids was undertaken. The re Group 2 received diet R l but was pair-fed sults of these studies are presented here with group 4 ( average daily food consump with. tion). Group 3 received ad libitum diet R l In the present paper succinic dehydro from which the casein was omitted. Group genase has also been re-studied using a 4 received ad libitum diet R l from which system in which the mitochondrial mem both casein and methionine were omitted. brane permeability barrier to the electron The food consumption of groups 3 and 4 acceptor in the assay system was elimi was almost identical. nated. In the second communication of At zero time and after various time in this series (2 ), it was pointed out that the tervals, rats from each group were killed method then used for estimating succinic and the livers removed, rapidly weighed, dehydrogenase, which utilized intact mito and chilled. Succinic oxidase was deter chondria, probably did not give a true mined by the procedure of Potter (8 ). In measure of the enzyme activity. Singer this method the mitochondria are damaged and Lusty (5 ) had reported while the by adding calcium to the assay medium. paper was in press that the use of phena- Succinic dehydrogenase was assayed using zine methosulfate as electron acceptor for the same system as for succinic oxidase ex assaying succinic dehydrogenase in intact cept for the following changes: 0.1 ml of rat liver mitochondria gave erroneously low 3% phenazine methosulfate was included values since the membrane of undamaged in a side-arm of the flask, and 0.3 ml of mitochondria exhibits a permeability bar 0.01 m KCN was added to the main com rier toward the dye. If the membrane was partment, both at the expense of water. damaged, for example by including cal After temperature equilibration, the phen cium ions in the medium, the barrier could azine methosulfate and substrate were be eliminated. added to the main compartment and oxy gen uptake recorded after 0.5 minute and EXPERIMENTAL again after 5.5 minutes. The difference be The study was divided into 2 phases. tween the 2 readings gave the rate of ini In the first phase the changes in succinic tial oxidation of succinate via the initial oxidase and succinic dehydrogenase associ dehydrogenase. ated with prolonged protein depletion were Nitrogen was determined in the first followed as a function of time of protein phase of the study by a semi-micro Nessler- depletion followed by repletion. In this ization procedure as described previously phase the effect of including methionine in (1 ). Nitrogen X 6.25 was taken as the the protein-free ration was studied. In the value for protein. In the second phase pro second phase the effects of adding individ tein was determined directly by the method ually all other essential amino acids as well of Lowry et al. (9 ). Liver DNA was esti as cystine, glutamic acid, and arginine at mated by using the extraction procedure of equimolar levels in a protein-free ration Schneider (1 0) followed by color develop were studied. ment with diphenylamine (11). AMINO ACID PROTECTION OF MITOCHONDRIAL ENZYMES 53 Since the enzyme assay for only 4 rats Student’s t test to determine whether the could be handled satisfactorily on any one differences between the means of groups 2- day, the points on the figures in the Results 4 versus 1, groups 3 and 4 versus 2, and section represent the averages for the ani group 4 versus 3 at any one time are sig mals killed from about the fifth day before nificant. The method of noting the signifi to the fifth day after the indicated day. cant differences is shown in the legend of One rat from each of the 4 groups was usu the figures. The results in phase 2 are ally killed on a single day so that rats from presented as bar graphs for the 14 differ the control groups were run simultaneously ent groups. The results have been ana with experimental rats whenever possible. lyzed statistically and the range of 2 stand The animals surviving after 102 days of ard errors is drawn for each bar to denote depletion were re-fed the complete diet ad libitum until nearly normal body weight significance of difference among the had been reached. Group 2 was still pair- means. fed with group 4 during the repletion. The Phase 1. In Figure 1 are presented the numbers of animals killed for each point results for the response of liver succinic were as follows: zero time, 8; 24, 56, and oxidase to protein depletion, with and with 102 days, 7 to 10 of each group; 3 days out added 0.3% DL-methionine. Results post-repletion, 4 to 8 of groups 2-4; 6 days for the ad libitum- fed ( group 1) and pair- post-repletion, 4 of groups 2-4; 15 days fed controls (group 2) have been separated post-repletion, 4 of groups 2-4; 25 days from the 2 protein-deficient groups ( groups post-repletion, 8 of group 1; 56 and 86 days 3 and 4) for clarity. The values for the post-repletion, 6 to 12 of each group. ad libitum-fed controls are presented as Phase 2. When it had been ascertained microliters of oxygen uptake per hour per from the first phase of the study that me milligram of DNA. The results for the thionine had an effect on the enzymes be other 3 groups are presented as percentage ing studied, another group of 112 rats was of the ad libitum-fed control values. This set up for an initial adjustment period was done to negate the influence of the fluc with the complete diet. After the adjust tuations in the points for group 1 upon the ment period, the rats were separated into other groups. Thus a clearer picture of the 14 groups of 8 rats each (groups 1A-14A) effects of the diets per se on the variables and fed the individual diets for 8 weeks. is obtained. The diet fed to each group is shown in the Three-tenths per cent methionine in the legend of figure 4. The purpose of study protein-free diet markedly spares succinic ing all of these amino acids was to ascer oxidase. After the initial loss in both tain whether the effects observed in phase groups 3 and 4 to 60% of normal, the 1 were specific for methionine or whether values for group 4 continued to decrease other essential amino acids as well as cys until at 56 days after depletion, the value tine, arginine, or glutamic acid (as con was only 40% of normal. The presence of trol) also showed specific effects. methionine, however, enabled group 3 to Enzyme assays and other analyses were maintain a significantly higher level of suc carried out as described above in phase 1. cinic oxidase than that of group 4 after the initial loss. In fact the activity per cell of RESULTS AND DISCUSSION group 3 returned almost to normal by the The results are presented as activity or one hundred second day of depletion. It concentration per milligram DNA. Since cannot be decided at present whether this the concentration of DNA per average liver return toward normal of group 2 represents cell nucleus has been found to be constant an actual resynthesis of the enzyme system during protein deprivation in adult rats from body protein, whether it is a type of (12), activity or concentration of liver enzyme induction, or whether it is a reflec cellular components expressed per unit tion of less loss of the enzyme system with weight of DNA should be directly propor respect to other cellular components. When tional to the activity or concentration of a protein was replaced in the diet of group 4 component per average liver cell. The re the succinic oxidase activity rebounded to sults in phase 1 have been analyzed using above normal and then declined slowly 5 4 J. N. WILLIAMS, JR. Fig. 1 The response of liver succinic oxidase to prolonged feeding of a protein-free ration with and without 0.30% Dr-methionine, followed by repletion. (I, A LC ), • . indicates ad libitum-fed controls; (II, PF C ), A, indicates pair-fed controls; (III, N P + M ), ■ , indicates protein deficient rats supplemented with 0.30% DL-methionine; (IV , N P ), O (small open circle), indicates protein-deficient rats. Repletion was begun at the arrows. The results for groups II-IV are expressed as percentage of the ad libitum-fed controls (I ). Tests for significance of difference between means Group versus Group Notation for P < 0.01 II, III, IV i O (large circles) III, IV i i □ IV h i \ / ' v The numbers of rats at each point are given in the text. AMINO ACID PROTECTION OF MITOCHONDRIAL ENZYMES 5 5 cates ad libitum-fed controls; (II, PF C ), A, indicates pair-fed controls; (III, NP + M ), g , indicates protein-deficient rats supplemented with 0.30% DL-methionine; (IV , N P ), O (small open circle), indicates protein-deficient rats. Repletion was begun at the arrows. The results for groups II-IV are expressed as percentage of the ad libitum-controls (I ). Tests for significance of difference between means Group versus Notation for P < 0.05 Group ( see exception below ) II, III, IV I O (large circles) III, IV ii □ IV in \ / ',X P was < 0.01 in all cases except for IV versus I at 24 days, and III versus II at 56 days. 5 6 J. N. WILLIAMS, JR. Fig. 3 The response of liver protein (N x 6.25) to prolonged feeding of a protein-free ration with and without 0.30% DL-methionine, followed by repletion. (I, A LC ), • _ indicates ad libitum-fed controls; (II, PF C ), ▲, indicates pair-fed controls; (III, N P + M ), ■ , indicates protein-deficient rats supplemented with 0.30% DL-methionine; (IV , N P ), O (small open circle), indicates protein-deficient rats. Repletion was begun at the arrows. The results for groups II-IV are expressed as percentage of the ad libitum-fed controls (I ). Tests for significance of difference between means Group versus Group Notation for P < 0.01 II, III, IV i O (large circles) III, IV ii □ IV h i \ ✓ The numbers of rats at each point are given in the text. AMINO ACID PROTECTION OF MITOCHONDRIAL ENZYMES 5 7 toward normal. The values for the pair-fed methosulfate (the dye acceptor) to the en controls did not change significantly zyme. throughout the study. Thus the effects of A similar protective effect of methionine 0.3% methionine in the protein-free ration was observed in the initial study of this and of the protein-free ration itself on suc series (2 ). However, the magnitude of the cinic oxidase are specific effects of the absolute values was considerably less in diets and not of changes in food intake. the earlier study since the mitochondria The results for succinic dehydrogenase were kept intact. (fig. 2) were almost identical to those for The protection of succinic oxidase by succinic oxidase. This indicates clearly methionine as indicated in figure 1 can that in the succinic oxidase system the thus probably be explained by methionine rate-limiting step lies in the initial dehy protection of the initial dehydrogenase. drogenase. In the present studies, the Since succinic dehydrogenase is the rate- measurement of succinic dehydrogenase limiting step in the system, any changes was carried out with mitochondria dam observed in the succinic oxidase system aged by calcium addition, thus allowing would be a reflection of changes in suc free access of substrate and phenazine cinic dehydrogenase. JEA MA ETA YA VIA HA KA XA XIA m i a SMA NP + + + + + + + + + + + arg cyst girt hist isol leuc lys meth $ala threo trypt GROUP Fig. 4 Succinic oxidase activity per milligram DNA in rats fed a normal control diet (I A ), a protein- and amino acid-free diet (IIA ) and protein-free diets containing various amino acids equimolar with 0.30% L-methionine (IIIA-XIVA) for 56 days. The meanings of the abbreviations in the figure are as follows: arg, 0.42% L-arginine-HCl; cyst, 0.24% L-cystine; glut, 0.30% L-glutamic acid; hist, 0.42% L-histidine-HCl-ftO; isol, 0.20% L-isoleucine; leuc, 0.26% L-leucine; lys, 0.37% L-lysine-HCl; meth, 0.30% L-methionine; For comparing the enzyme responses whereas the levels of the enzymes were un with changes in other liver protein, liver changed in that group. protein data from the same animals from Phase 2. In figures 4-6 are presented which the enzyme results were obtained the results of studies with groups of rats are presented in figure 3. Although pro fed the protein-free diets containing other tein was lost from the liver cells in both amino acids added individually to the pro groups 3 and 4, the specific pattern of loss tein-free ration. In addition to methionine, was different from that for the enzymes. cystine protected succinic oxidase and de Protein was continuously lost from the liver hydrogenase (figs. 4, 5). None of the other cells of both groups of rats throughout the amino acids tested had any significant ef depletion in contrast with an elevation of fect on these enzymes. The effects of cys succinic oxidase and dehydrogenase per tine and methionine appear to be equiva cell in group 3 after 24 days and in group lent. The effect of these 2 amino acids on 4 after 56 days. Nevertheless methionine liver protein is shown in figure 6. Although protected the liver cells to some extent both amino acids prevented the loss of pro against loss of protein. Also the animals tein from the liver cells to some extent, the in the pair-fed control group [2] lost sig degree of protection was not as great as nificant protein from their liver cells, with succinic oxidase and dehydrogenase. GROUP Fig. 5 Succinic dehydrogenase actvity per milligram DNA in rats fed a normal control diet (I A ), a protein- and amino acid-free diet (IIA ) and protein-free diets containing various amino acids equi molar with 0.30% L-methionine (IIIA —XIV A ) for 56 days. The meanings of the abbreviations are given in the legend of figure 4. Twice the standard error of each group is represented by the vertical line through the mean. Each mean is obtained from 7 to 8 rats. AMINO ACID PROTECTION OF MITOCHONDRIAL ENZYMES 59 Fig. 6 Liver protein per milligram DNA in rats fed a normal control diet (I A ), a protein- and amino acid-free diet (IIA ) and protein-free diets containing various amino acids equimolar with 0.30% L-methionine (I I I A -X I V A ) for 56 days. The meanings of the abbreviations are given in the legend of figure 4. Twice the standard error of each group is represented by the vertical line through the mean. Each mean is obtained from 7 to 8 rats. The results with groups 1A, 2A, and 10A Since succinic dehydrogenase contains corroborate the results in figures 1-3 al sulfhydryl groups that are necessary for its most exactly at the 56-day point. Histidine activity, it is inviting to suggest that me appears to have had a slight effect in pre thionine and cystine protect the enzyme by venting loss of liver protein. protecting the sulfhydryl groups. However, The absolute values for succinic oxidase if the only effect of methionine and cystine and dehydrogenase activities in phase 1 are is to protect activity in this manner, why somewhat higher on the average than in should they also prevent loss of liver pro phase 2 (compare ALC, figs. 1 and 4, and 2 tein? Until more can be learned about and 5). It is difficult to explain why this amino acid conservation mechanisms and occurred. However, it is the author’s ex interrelationships among amino acids with perience that different groups of rats, al respect to protein re-synthesis in protein though all of the same strain and treated deficient animals, it would be premature to as identically as possible, often show state with certainty a mechanism for their slightly different average enzyme activities actions in the present paper. A possible from one year to the next. In the present lead for further work is given by the results study, phase 1 was carried out approxi of Aschkenasy (13), who observed that if mately a year before phase 2. However, low protein diets are supplemented with in each phase appropriate controls were methionine, the livers of male rats become included simultaneously with the experi hypertrophied. If the animals are first mental groups to negate this effect. adrenalectomized, this effect of methionine 60 J. N. WILLIAMS, JR. is not obtained. The answers to some of 6. Fox, M. R. S., and G. M. Briggs 1960 Salt these questions are being sought at present. m ixtures for purified-type diets. III. An im proved salt m ixture for chicks. J. Nutrition, 7 2 : 2 4 3 . ACKNOWLEDGMENTS 7. Fox, M. R. S., L. O. Ortiz and G. M. Briggs The author wishes to thank Alice Jasik 1955 Toxicity of ethionine in the young for her competent technical assistance in chick. Agr. Food Chem ., 3: 436. these studies and Mrs. Esther Hurley and 8. Um breit, W . W ., R. H. Burris and J. F. Stauf Woodrow Duvall for feeding and care of fer 1959 M anom etric Techniques. Burgess the animals and for preparation of the Publishing Com pany, M inneapolis, p. 173. diets used. 9. Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall 1951 Protein measure m ent w ith the Folin phenol reagent. J. Biol. LITERATURE CITED C h e m . , 193: 2 6 5 . 1. W illiam s, Jr., J. N. 1961 Response of the 10. Schneider, W . C. 1945 Phosphorus com liver to prolonged protein depletion. I. Liver pounds in anim al tissue. I. Extraction and weight, nitrogen, and deoxyribonucleic acid. estim ation of desoxypentose nucleic acid and J. Nutrition, 73: 199. of pentose nucleic acid. J. Biol. Chem ., 161: 2. W illiam s, Jr., J. N. 1961 Response of the 2 9 3 . liver to prolonged protein depletion. II. The 11. Davidson, J. N., and C. Waym outh 1944 succinic oxidase system and its component Tissue nucleic acids. 3. The nucleic acid enzym es. J. Nutrition, 73: 210. 3. W illiam s, Jr., J. N. 1963 Response of the and nucleotide content of liver tissue. Bio liver to prolonged protein depletion. III. Co chem . J., 38: 379. enzyme Q. Arch. Biochem . Biophysics, 101: 12. Thom son, R. Y., F. C. Heagy, W . C. Hutchin 5 1 2 . son and J. N. Davidson 1953 The deoxyri 4. Boissier, J., and A. Aschkenasy 1950 Ef bonucleic acid content of the rat cell nu fects comparés de l’adm inistration de gly- cleus and its use in expressing the results of cocolle, de thiam ine et de m éthionine sur les tissue analysis, w ith particular reference to organs de rats privés protéines alim entaires. the com position of liver tissue. Biochem . J., Bull. Soc. Chem ie Biologique, 32: 942. 5 3 : 4 6 0 . 5. Singer, T. P., and C. J. Lusty 1960 Per 13. Aschkenasy, A. 1955 Suppression par la m eability factors in the assay of m itochon surrénalectom ie de la protection des foie par drial dehydrogenases. Biochem . Biophys. Res. m éthionine contre les effects du régim e hypo- Com m ., 2; 276. protidique. C. R. Soc. Biologique, 149: 6 6 3 . Supplementation of Wheat Gluten Protein' W . L. BAN KS, JR .,2'3 J. B. A LLISO N a n d R. W. W ANNEM ACHER. JR. Bureau of Biological Research, Rutgers — The State University, New Brunswick, New Jersey ABSTRACT Male weanling W istar rats were fed diets containing wheat gluten (group 1), wheat gluten supplemented with lysine monohydrochloride (group 2). wheat gluten plus casein (group 3), casein (group 4 ), and egg album in (group 5) as the dietary protein sources, as w ell as a diet free of protein (group 6). The nitrogen growth index of 8.6 for the group 1 anim als was im proved by either m ethod of sup plem entation to 16.7, values less than the 20.2 and 27.0 for the group 4 and 5 ani m als, respectively. Liver protein D N A and RN A /D N A ratios increased w ith both nitro gen intake and nutritive value of the dietary protein except in those anim als in group 2. The values for these ratios in anim als in groups 1 and 2 were the same. In the m uscle, these ratios increased w ith both type and quantity of dietary protein without exception. In the brain, these param eters were unaltered by quantity or quality of dietary protein. W hen these cellular param eters were com pared at nitrogen intakes producing anim als that gained 50 g over the experim ental period of 28 days, con siderable difference was noted in these quantities as a result of feeding the various proteins even though the anim als would have gained the same weight. Wheat gluten is an example of an “un promoting qualities of unsupplemented balanced” protein, deficient in the amino wheat gluten, casein and egg albumin acid, lysine (1 ). Supplementation of protein. wheat gluten with an optimal quantity of this amino acid4 resulted in an increased METHODS rate of growth in young rats, a rate that Groups of 10 male weanling rats of the was almost equal to that associated with Wistar strain were fed diets containing rats fed casein. Gain in body weight is wheat gluten, wheat gluten supplemented only one parameter that may be used to with lysine monohydrochloride (4.7 g/ assess the nutritive value of a dietary pro 100 g protein), wheat gluten plus casein tein. Recently, more attention has been (2.1:1), casein, or egg albumin protein. given to responses of individual cells and These diets were prepared in an agar gel tissues to dietary protein changes. Allison base (table 1) (3 ) and fed ad libitum at et al. (2 ), for example, examined the re several protein levels. Wheat gluten was sponse of protein/DNA and RNA/DNA in supplemented with lysine and casein at several tissues to variation in quantity and levels that would produce an equivalent quality of dietary protein. These investi growth response over the experimental gators found that protein/DNA increased period. Daily food consumption and with nitrogen intake in liver, muscle and weekly body weight changes were recorded. kidney, but this ratio remained essentially After 28 days, the animals were killed constant in the brain. The ratio increased and samples were taken of the brain, also in liver and muscle as the nutritive gastrocnemious muscle, and liver. A 2.0-g value of the dietary protein was increased. sample of the liver was homogenized in Nutritive value had less effect upon pro 10.0 ml of a 0.35 m sucrose solution buff tein/DNA in the kidney and little or no ered with bicarbonate to a pff of 7.4. effect upon the ratio in the brain. Weighed samples of the brain and muscle The object of the present paper was to were homogenized in 5.0 ml of the sucrose study the changes in some of the param Received for publication July 22, 1963. eters associated with protein biosynthesis 1 This work was supported in part by a United States Public Health Service Grant AM-04341. in various tissues which result from sup 2 A Johnson and Johnson pre-doctoral Fellow. plementation of wheat gluten protein with 3 Present address: School of Aerospace Medicine, Brooks AFB, Texas. lysine or casein. These 2 supplemented 4 Middleton, E. J. 1959 A study on the lysine supplementation of wheat gluten. Ph.D. Thesis, Rut proteins were compared with the growth gers University, New Brunswick, New Jersey. J. N u t r itio n , 82: ’64 6 1 62 W. L. BANKS, JR., J. B. ALLISON AND R. W. WANNEMACHER, JR. TABLE 1 Basal agar gel d iet1 m g / 1000 g 9 d ry d ie t P r o t e i n 1 8 0 T h i a m i n e 1 0 S u c r o s e 1 3 4 R i b o f l a v i n 2 0 D e x t r o s e 2 1 2 P y r i d o x i n e 1 0 D e x t r i n 1 4 0 M e n a d i o n e 1 5 L a r d 2 3 1 N i a c i n 8 0 S a l t 4 0 Pantothenic acid 8 0 A g a r 3 3 I n o s i t o l 2 0 0 Cod liver oil 2 0 F o l i c a c i d 0 . 5 a-Tocopherol 2 1 0 B i o t i n 0 . 5 1 0 0 0 p-Am inobenzoic acid 8 0 . 0 2 0 0 0 . 0 W a t e r 1 4 0 0 C h o l i n e Ascorbic acid 2 . 0 2 4 0 0 units/1000 g d ry d ie t V i t a m i n A 4 0 , 0 0 0 V i t a m i n D 4 , 0 0 0 1 The protein content of the diet can be varied by isocaloric replacement of the protein with sucrose and dextrose (3). 2 100 mg in 10 g hydrogenated cottonseed oil (Crisco, Procter and Gamble, Cincinnati). buffer. Ribonucleic acid (RNA) and deoxy tein/DNA, and protein/RNA in the liver. ribonucleic acid (D N A ) were determined The RNA/DNA ratio increased with nitro using a modification5 of the ultra-violet gen intake, the greatest increase being as absorption techniques suggested by Hutch sociated with the feeding of egg albumin ison and Munro (4 ). Total protein was and the least with the feeding of wheat estimated by the Biuret method of Layne gluten. Supplementing wheat gluten with (5 ) on an alkaline hydrolyzate of an acid- lysine did not result in a greater increase insoluble precipitate of the homogenate in RNA/DNA ratio than was noted in ani from which the lipid had been extracted. mals fed unsupplemented wheat gluten. Standard errors were determined for all Supplementing with casein, however, im values and a probability value (P ) of less proved the RNA/DNA ratio but not to the than 0.01 was taken as the level for statis value observed for casein alone. The pro- tical significance. tein/DNA ratio followed the same pattern illustrated for RNA/DNA, the most protein RESULTS being formed in animals fed egg, less in Nitrogen growth index, a measure of the animals fed casein or wheat gluten supple growth promoting quality of a dietary pro mented with casein, and least in those fed tein (6 ), was determined for the various wheat gluten or wheat gluten supple diets. The indexes determined for differ mented with lysine. These data suggest ent proteins were wheat gluten, 8.6; casein, that there is a good correlation between 20.2; and egg albumin, 27.0. Supplement protein in the liver and total RNA, which ing wheat gluten with either the free amino is confirmed by the constant ratio, protein/ acid, lysine, or casein protein improved the RNA, illustrated in figure 1. nitrogen growth index to a value of 16.7. These results demonstrate that the maxi Thus, the value of wheat gluten as a die mal protein/DNA, resulting from feeding tary protein source for body weight gain egg albumin, was not obtained by feeding was improved equally by either type of large intakes of proteins with low nutritive supplementation although the amino acid values. Supplementing wheat gluten with patterns were not the same. The signifi lysine did not improve protein biosynthesis cance of these differences in patterns will in the liver above the results obtained while be discussed later (table 2). feeding unsupplemented wheat gluten. It The data plotted in figure 1 illustrate 5 Banks, W. L., Jr. 1963 Studies on the supple the effects of nitrogen intake and quality mentation of wheat gluten protein in the growing rat. Ph.D. Thesis, Rutgers University, New Brunswick, of dietary protein upon RNA/DNA, pro New Jersey. WHEAT GLUTEN SUPPLEMENTATION 63 TABLE 2 Amino acid intakes producing a body weight gain of 50 g with various dietary proteins W h e a t W h e a t E g g W h e a t M i n i m a l A m i n o a c i d C a s e i n g l u t e n g l u t e n a l b u m i n g l u t e n v a l u e s 1 + c a s e i n + l y s i n e 9 9 9 9 9 9 T o t a l N 1 .9 2 . 5 3 . 0 3 . 0 5 . 8 1 .7 A r g i n i n e 0 . 7 1 0 . 6 1 0 . 7 1 0 . 6 9 1 .3 3 0 . 4 1 H i s t i d i n e 0 . 2 8 0 . 4 8 0 . 4 5 0 . 3 7 0 . 7 2 0 . 2 5 I s o l e u c i n e 0 . 7 2 0 . 9 9 0 . 9 8 0 . 8 5 1 .6 5 0 . 6 8 L e u c i n e 1 . 0 4 1 . 5 6 1 .5 3 1 .3 5 2 . 6 1 0 . 9 3 L y s i n e 0 . 8 6 1 . 2 7 0 . 8 0 1 .2 6 0 . 7 3 0 . 8 0 Phenylalanine 0 . 7 1 0 . 8 5 0 . 9 7 0 . 9 4 1 . 8 2 0 . 5 9 T y r o s i n e 0 . 4 0 0 . 8 1 0 . 6 9 0 . 5 2 1 .0 1 0 . 3 7 Total arom atic 1 .1 1 1 . 6 6 1 . 6 6 1 . 4 6 2 . 8 3 0 . 9 6 C y s t i n e 0 . 3 5 0 . 0 6 0 . 3 1 0 . 4 4 0 . 8 6 _ M e t h i o n i n e 0 . 5 0 0 . 5 1 0 . 4 2 0 . 3 1 0 . 6 0 ___ T o t a l S 0 . 8 5 0 . 5 7 0 . 7 3 0 . 7 5 1 . 4 6 0 . 6 5 T h r e o n i n e 0 . 5 8 0 . 7 0 0 . 6 4 0 . 5 3 1 . 0 2 0 . 5 1 T r y p t o p h a n 0 . 1 3 0 . 1 5 0 . 1 6 0 . 1 7 0 . 2 8 0 . 1 1 V a l i n e 0 . 8 9 1 . 1 5 1 .0 1 0 . 8 0 1 . 5 5 0 . 8 1 P r o l i n e 0 . 5 1 2 . 0 9 2 . 6 6 2 . 6 5 5 . 1 2 ___ Glutam ic acid 1 . 5 3 3 . 5 0 5 . 7 0 6 . 5 6 1 2 . 7 5 ___ Aspartic acid 1 .3 1 1 . 1 4 0 . 9 5 0 . 7 1 1 . 3 6 ___ G l y c i n e 0 . 4 5 0 . 3 1 0 . 5 3 0 . 6 2 1 . 2 0 — 1 Allison et al. (7). is possible that the free lysine added to the considered the limiting essential amino wheat gluten was absorbed and transported acid in wheat gluten. When lysine was in such a way that the improved growth of added to wheat gluten, however, the essen the rats when fed the supplemented pro tial amino acids were all in excess of tein was associated primarily with growth minimal values, suggested by Allison et al. of tissue other than the liver, tissue such (7 ), except for threonine and valine. Sup as muscle. Figure 2 illustrates the increase plementing wheat gluten with casein re in protein/DNA in the muscle with nitro sulted in lysine as the limiting amino acid, gen intake, the greatest increase being as whereas sulfur amino acids were limiting sociated with the feeding of casein and for casein. Thus, each dietary protein wheat gluten supplemented with casein. presented a different problem in meeting Adding lysine to wheat gluten, however, the amino acid requirements for protein increased the protein/DNA ratio above biosynthesis. that of those fed unsupplemented wheat The data in figure 3 summarize the re gluten at the higher intakes. sults obtained during this gain of 50 g. An One way to compare the effect of feeding objection to a comparison at such a low various dietary proteins upon the variables gain in weight is the restriction put upon discussed in this paper would be to make the proteins of high nutritive value which the comparison at some fixed gain in body would, with higher intake and greater weight. The amino acid intakes that pro growth, produce a cell with more protein/ duced a gain in body weight of 50 g in DNA than would be possible with the pro rats fed various dietary proteins are re tein of lower nutritive value. Nevertheless, corded in table 2. These data suggest that the difference in nitrogen growth index is the gain of 50 g in rats fed wheat gluten well established at this gain of 50 g as illus was associated with excess essential amino trated by the data plotted in figure 1. The acid when compared with minimal values, RNA/DNA and protein/DNA ratios ob except for lysine. Thus, lysine could be served in the liver illustrate the poor re- 64 W. L. BANKS, JR., J. B. ALLISON AND R. W. WANNEMACHER, JR. Fig. 1 RNA/DN A, protein/DNA and protein/RNA ratios vs. nitrogen intake for 28 days from the livers of anim als fed wheat gluten (solid triangles), wheat gluten supplemented with lysine m onohydrochloride (solid circles), wheat gluten plus casein (solid squares), casein (open circles), egg album in protein (open triangles), and a protein-free diet (P FD ) (open squares). A ll curves were drawn as an approxim ate fit of the experim ental points. Plus signs on curves indicate 50-g gain in body weight. sponse of the liver-to-lysine supplementa in the muscle of animals fed wheat gluten tion of wheat gluten. The unusually large may reflect a compensating mechanism RNA-to-DNA ratios in the muscles of the whereby the muscle protein is catabolized animals fed wheat gluten may reflect the to supply amino acids which could correct relatively large intake of nitrogen in these the amino acid pattern of wheat gluten. animals which was necessary to obtain a There was little difference in the protein./ 50-g weight gain. The low protein/RNA DNA ratio in the muscles of animals fed WHEAT GLUTEN SUPPLEMENTATION 6 5 NITROGEN INTAKE - g. Fig. 2 Protein/DNA vs. nitrogen intake for 28 days of the muscles of anim als fed wheat gluten (solid triangles), wheat gluten supplem ented with lysine m onohydrochloride (solid circles), w heat gluten plus casein (solid squares), casein (open circles), and a protein- free diet (PFD ) (open squares). NITROGEN LIVER LIVER LIVER GROWTH WEIGHT RNA/DNA PROTEIN/DNA INDEX 9 7 / 1 / \ / \ / \ 7; / s / \ 1 7 BRAIN RNA/DNA Fig. 3 A com parison of cellular constituents at nitrogen intakes that produced a gain in weight of 50 g. The black bars record data obtained w hile feeding wheat gluten (W G ); the other bars beginning w ith the stippled bar illustrate data obtained w hile feeding wheat gluten supplemented with lysine (W G + L), wheat gluten plus casein (W G + C A S), casein (C A S), and egg album in (E A ), respectively. 6 6 W. L. BANKS, JR., J. B. ALLISON AND R. W. WANNEMACHEE, JR. either wheat gluten or wheat gluten sup LITERATURE CITED plemented with lysine. Supplementation of 1. Grau, C. R., and M. Kamei 1950 Amino wheat gluten with casein, however, re acid im balance and growth requirem ents for sulted in a higher protein/DNA ratio in the lysine and m ethionine. J. Nutrition, 41: 8 9 . muscle, a ratio that was equivalent to that 2. Allison, J. B., R. W . W annem acher, Jr., W . L. Banks, Jr., W . H. W unner and R. A. Gomez- obtained in animals fed casein. A greater Brenes 1962 Dietary proteins correlated gain in body weight than 50 g in animals w ith ribonuclease, ribonucleic acid and tis fed wheat gluten supplemented with lysine sue proteins. J. Nutrition, 78: 333. resulted in an improved protein/DNA ratio 3 Allison, J. B., R. W . W annem acher, Jr., R. Hilf, J. F. Migliarese and M. L. Crossley in the muscle (fig. 2). Thus, nutritive 1954 Dietary protein and tumor-host rela value often can be expressed best at more tionship in the rat. J. N utrition, 54: 593. adequate nitrogen intakes. These data 4. Hutchison, W . C., and H. N. Munro 1961 demonstrate also that a nitrogen intake The determ ination of nucleic acid in biolog ical m aterials. Analyst, 86; 786. from various dietary protein sources to 5. Layne, E. 1957 Spectrophoretic and turbi- produce the same gain in weight may re dim etric m ethods of m easuring proteins. III. sult in different biochemical responses in Biuret method. In Methods in Enzym ology, various tissues. A constant response, how Vol. 3. Academ ic Press, Inc., New York, p. ever, independent of quality of protein is 4 5 0 . 6. Allison, J. B., W . H. Fitzpatrick 1960 illustrated for the brain in figure 3. The Dietary Proteins in Health and Disease. complete significance of a nitrogen growth Charles C Thom as, Springfield, Illinois. index, therefore, requires a detailed study 7. Allison, J. B., R. W . W annem acher, Jr., E. of each tissue as it exists in the dynamic Middleton and T. Spoerlein 1959 Diet pro tein requirem ents and problems of supple metabolic state of the body as a whole. m entation. Food Technol., 13: 5 9 7 . A Determination of the Essential Amino Acid Proportions Needed to Allow Rapid Growth in Chicks’ D. C. DOBSON, J. O. ANDERSO N a n d R. E. W ARNICK Poultry Department, Utah State University, Logan, Utah A BS T R A C T A series of experim ents was conducted to determ ine how the essential am ino acids in a ration should be proportioned to allow the chick to grow at a rapid rate. In the approach used, the level of each essential am ino acid in a ration was adjusted until the growth rate was decreased about the same am ount when a con stant fraction of any essential am ino acid was removed. A purified-type ration was fed which contained approxim ately 9.0% essential am ino acids and 2.88% nitrogen provided by a m ixture of proteins and am ino acids. The ration considered to have the best essential am ino acid balance was calculated to contain 1.28% arginine, 0.43% histidine, 1.15% lysine, 1.30% leucine, 0.80% isoleucine, 0.95% valine, 1.33%' phenylalanine and tyrosine, 0.20% tryptophan, 0.73% m ethionine and cystine, and 0.78% threonine. Chicks fed a ration w ith the natural isom er of these am ino acids at these levels gained 25% more weight and their gain/feed ratio was 15% higher than that of chicks fed a ration adjusted to the N ational Research Council m inim um requirem ent levels. These am ino acid levels are not m inim um requirem ents; replacing sim ultaneously 12.3% of all 12 w ith an equal level of nitrogen as glutam ic acid did not decrease growth rate. Experiments have been conducted to The chick’s requirement for arginine is determine the chick’s minimum require higher when casein provides most of the ment for each of the essential amino acids. protein than when commercial-type ra In a typical experiment conducted to de tions are fed. Anderson and Dobson (2 ) fine a requirement, chicks were fed graded observed that the high arginine require levels of the amino acid in a ration that ment associated with rations based on contained the proper level of protein and casein could be reduced by replacing part energy, and adequate levels of other re of the casein with any of the several mix quired nutrients. The lowest level of the tures of nonessential amino acids. No par amino acid that would allow the chick to ticular one of the nonessential amino grow at the maximum rate obtained and acids was required in the mixtures. Klain with the highest feed efficiency, was con et al. (3 ) reported that chicks fed rations sidered to be the minimum requirement. containing amino acids in the proportions Members of the National Research Coun occurring in a casein ration, also required cil (1 ) have reviewed reports of research a relatively high level of arginine. Casein conducted to define requirements and se is an unsually rich source of most of the lected a set of requirements which has essential amino acids except arginine. The been used extensively as a guide in formu high arginine requirement associated with lating commercial broiler rations. the casein rations is apparently related The requirement for each essential to the relatively high level of one or more amino acid varies with the protein and of the essential amino acids in the ration. energy content of the ration and cannot Experiments have been conducted, us be expressed as a constant percentage of ing a ration whose protein mixture did not a ration. In stating the amino acid re include casein, to study what effect in quirements of the chick, the protein and creasing the level of each essential amino energy levels of the ration are usually spe acid would have on the arginine require- cified. The requirements given by the NRC are for a ration with 20% protein Received for publication July 29, 1963. 1 Approved as Utah Agricultural Experiment Station and 900 kcal of productive energy 454 g. Journal Paper no. 359. J. N u t r it io n , 82: ’ 64 67 6 8 D. C. DOBSON, J. O. ANDERSON AND R. E. WARNICK ment of chicks (2 ). Increasing the lysine mum growth rate obtained. Thus it ap level produced a reduction in growth rate, peared that even when the protein and which was partly overcome by adding energy content of a ration were specified, more arginine. This indicated that the the essential amino acid content of the high lysine level in casein is the factor mixture that would allow the most rapid most responsible for the high arginine re growth rate could not be defined on the quirement of chicks fed rations based on basis of minimum requirements alone. casein. When both lysine and arginine Apparently a balance among the essential were added, growth rate was further in amino acids was also required. creased by adding a mixture of methio The present paper reports the results of nine, threonine, and histidine. The lysine a series of experiments conducted to deter and arginine addition evidently increased mine how the essential amino acids should the requirement for one or more of the be proportioned in a ration to allow the other 3 amino acids. chick to grow at a rapid rate. In the ap Anderson and Dobson (2 ) also reported proach used in these experiments, a ration results of other experiments which indi was said to contain “balanced proportions” cated that the amount of lysine required of the amino acids when the growth rate to allow the chick to grow at the maximum obtained with the ration decreased by the rate also was increased when the levels same amount when a given fraction of any of the other 9 essential amino acids in a one of the essential amino acids was re ration were increased. Under these cir moved. cumstances the lysine requirement did not EXPERIMENTAL appear to be increased as much as the Determining the balanced proportions. arginine requirement. A series of 36 experiments was conducted A limited number of experiments have to adjust the level of each essential amino been conducted to determine whether the acid in a ration until the growth rate ob overall essential amino acid level in the tained with the ration was reduced by the ration affects the requirements for in same amount when a given fraction of dividual amino acids other than lysine and any one of the essential amino acids was arginine.2 When a ration was fed in which removed. The amino acids considered the total essential amino acid/protein ratio essential in these experiments are listed was relatively high, comparable to that in in the lower section of table 1. a ration based on casein, the amount of In the first experiment, a basic ration leucine recommended by the NRC was was formulated which was judged to have adequate. The listed requirements for his a reasonable balance among the essential tidine and threonine proved inadequate in amino acids. This ration and 10 modifica a test of a similar ration. The results in tions were fed to chicks in an effort to dicated that a ration including the essen determine which of the essential amino tial amino acids at the NRC requirement levels would not constitute the best bal 2 The results of these experiments have not been published in detail. They were conducted primarily anced mixture that might be produced. A to determine whether it would be worthwhile to con duct the work reported in this paper. Our previous variety of diets were fed to chicks to de research indicated that the lysine or arginine require termine the adequacy of requirements ments increased as the level of the other essential amino acids in the ration increased. The unpublished adopted by the NRC. These diets varied in results suggested that the same was true of the re quirements for the other essential amino acids. It their total essential amino acid content appeared unlikely, however, that any arbitrarv in and in their balance among the essential crease in the total level of essential amino acids in a ration would produce an increase in the requirement amino acids. Requirements determined in for a particular amino acid. Our ability to demon strate these increases in requirements probably de this way conceivably would not be the best pended on starting with certain proportions among balanced set that might be developed. the essential amino acids. After these unpublished experiments were conducted, our ability to judge The increases in individual amino acid amino acid balance in these rations appeared some what questionable. Further experiments of the same requirements, as produced by increasing type were not considered worthwhile at that time. The results of the unpublished experiments were the levels of the other essential amino presented at the 1959 meeting of the Poultry Science acids (2 ),3 were not associated with sig Association. Dobson, D. C., and J. O. Anderson 1959 Effect of essential amino acids in the diet on the nificant changes in the gross energy or chick s requirement for specific amino acids. Poultry Sci., 38: 1199 (abstract). r protein levels of the ration, or in the maxi 3 Unpublished results. AMINO AGID BALANCE 6 9 TABLE 1 Composition of the protein and amino acid portion of several rations and their calculated essential amino acid content1 1 2 3 4 5 6 7 g/kg of ration Blood fibrin 33.6 31.6 50.0 50.0 50.0 50.0 50.0 Casein 27.2 25.6 56.5 56.5 56.5 56.5 56.5 Isolated soybean protein 69.9 65.8 0 0 0 0 0 Gelatin 39.3 36.9 32.0 32.0 32.0 32.0 32.0 L-Arginine-HCl 2.95 3.69 5.2 6.17 5.04 4.26 3.50 1,-Histidine-HC1 1.05 1.30 0 1.60 1.23 0.94 0.68 1,-Lysine-HC1 2.26 2.97 0.99 2.86 1.81 1.09 0.38 L-Leucine 1.94 2.60 4.61 3.61 2.66 2.01 1.36 DL-Isoleucine 3.64 4.36 1.36 5.36 4.20 3.40 2.60 dl-V aline 4.36 5.22 2.24 5.24 3.86 2.90 1.96 DL-Phenylalanine 1.14 1.86 4.04 3.44 2.50 1.80 1.12 L-Tyrosine 1.66 1.95 1.93 1.53 1.03 0.72 0.39 L-Tryptophan 0.36 0.46 0.20 0.20 0.05 0 0 DL-Methionine 1.77 2.30 2.01 1.51 1.22 1.02 0.82 L-Cystine 2.11 2.28 2.40 2.20 1.97 1.79 1.63 DL-Threonine 3.44 4.16 0.48 4.08 2.94 2.16 1.38 Glycine 1.92 2.80 4.73 4.73 4.73 4.73 4.73 L-Glutamic acid 10.37 13.76 46.59 27.21 40.42 49.6 58.5 Calculated essential amino acid levels, % of ration Arginine 1.28 1.28 1.20 1.28 1.19 1.12 1.06 Histidine 0.43 0.43 0.30 0.43 0.40 0.38 0.36 Lysine 1.15 1.15 1.00 1.15 1.07 1.01 0.95 Leucine 1.30 1.30 1.40 1.30 1.21 1.14 1.08 Isoleucine 0.80 0.80 0.60 0.80 0.74 0.70 0.66 Valine 0.95 0.95 0.80 0.95 0.88 0.83 0.79 Phenylalanine 0.67 0.67 0.70 0.67 0.62 0.59 0.55 Tyrosine 0.66 0.66 0.70 0.66 0.61 0.58 0.55 Tryptophan 0.20 0.20 0.20 0.20 0.19 0.18 0.18 Methionine 0.40 0.40 0.45 0.40 0.37 0.35 0.33 Cystine 0.33 0.33 0.35 0.33 0.31 0.29 0.27 Threonine 0.78 0.78 0.60 0.78 0.72 0.68 0.65 Total essential 8.95 8.95 8.30 8.95 8.31 7.85 7.43 1 The composition of the remainder of these rations is given in the text of this paper. 2 Most of the amino acids used in all rations were purchased from Nutritional Biochemicals Corporation, Cleveland. acids were provided by the basic ration at was removed from a basic ration to pro relatively high levels and which at rela duce one modified ration. The methionine tively low levels in relation to the needs level equaled 50 to 65% of the total level of the chick. Using this information a of these 2 amino acids in all rations. A new, and presumably better-balanced basic composite fraction of phenylalanine and ration was formulated. The new basic tyrosine was removed from a basic ration ration was then fed in a similar ex to produce another of the modified rations. periment. This procedure was followed The phenylalanine level equaled 50 to throughout the entire series. 55% of the total level of these amino acids A fraction of each essential amino acid in all rations. was removed in turn from each basic If removing a fraction of an amino acid ration to produce its 10 modifications. The from the basic ration decreased growth fraction removed was constant for all rate more than the average decrease ob essential amino acids in any one experi tained, it was assumed that the basic ment, but it varied from 10 to 20% in the ration contained a relatively low level of different experiments conducted. A com that amino acid. If the decrease in weight posite fraction of methionine and cystine gained was less than average, it was as 7 0 D. C. DOBSON, J. O. ANDERSON AND R. E. WARNICK sumed that the basic ration contained a but considered only one-half as effective relatively high level of that amino acid. as the natural isomer. The basic ration fed in each experiment The individual amounts of 16 or 17 contained a total of 2.88% nitrogen and amino acids in the proteins used in these approximately 9.0% of the essential amino rations were determined by ion exchange acids provided by a mixture of proteins chromatography after hydrolysis with 6 n and amino acids. The 9.0% essential HC1, by a procedure adapted from those amino acid level was arbitrarily fixed at given by Moore and Stein (4 ), Moore et al. the beginning of the series of experiments. (5 ), and Spackman et al. (6 ). Details on This level is less than that in rations fed the procedure are given elsewhere.8 These to broilers in commercial production, but values, which were used in calculating the more than the sum of the NRC mini- amino acid content of the rations fed, are mium requirements for these amino acids shown in table 2. Since the tryptophan (8.3% ). Tables giving the amino acid and some of the cystine were destroyed composition of ingredients used in com during the acid hydrolysis, the values for mercial broiler production indicated that these amino acids were taken from the the ratio between the protein content and literature, as indicated in table 2. the total level of essential amino acids in The amino acid content of each ration commercial rations would be about two. was calculated to the nearest 0.01%, but Therefore, the protein level (N X 6.25) it is realized that the errors involved in in the experimental rations was 18%. determining the balanced proportions do Glycine, glutamic acid, and proline, not justify expressing them with this ac sometimes listed as essential amino acids curacy. The essential amino acid levels in for the chick, were not considered essen the basic rations were adjusted in incre tial in these experiments. It was recog ments of 0.025%. nized that chicks grow more rapidly when All rations fed in these experiments these amino acids are included in their contained the mineral mixture of Fox and diet than when they are not, but their Briggs (8 ) at a level of 6%, unless they levels in a ration were not deemed as criti contained amino acid hydrochlorides. If cal as those of the amino acids classed as the rations included amino acid hydro essential in these experiments. All rations chlorides, the chloride level of the ration fed in these experiments contained these was adjusted by replacing part of the 3 amino acids, but no attempt was made sodium and potassium chlorides of the to determine what proportions of each mineral mixture with the corresponding were needed to make a well balanced ra bicarbonates. Each kilogram of ration con tion. Limiting these studies to the amino tained 100 g of a vitamin mixture which acids considered essential simplified the provided the following: (in m g) thiamine- experiments. HC1, 8; riboflavin, 10; pyridoxine, 8; nia Mixtures of isolated soybean protein,4 cin, 100; folic acid, 2; biotin, 0.3; Ca-D- casein,5 blood fibrin,6 and gelatin,7 pro pantothenate, 20; choline chloride, 2000; vided most of the amino acids in the vitamin Bi2, 0.01; menadione sodium bi rations used to define the amino acid bal sulfite, 5.4; and df-a-tocopheryl acetate, 40. ance. The defining rations also contained The mixture also provided 10,000 IU of enough of an amino acid mixture to adjust vitamin A and 1500 ICU of vitamin D2/kg the level of each essential amino acid to of ration. Glucose monohydrate9 was used the desired level and allow for the desired as the vitamin carrier. All rations con reductions. L-Glutamic acid was used to tained 3% refined corn or cottonseed oil. adjust the nitrogen level to 2.88%. Kjel- 4 ADM C-l Assay Protein, Archer-Daniels-Midland dahl determinations were made of the Company, Cincinnati, Ohio. 5 Obtained from Nutritional Biochemicals Corpora nitrogen content of all protein supple tion, Cleveland. ments. DL-Methionine was used and was 6 Obtained from Armour Livestock Bureau, Chicago. 7 Swift’s Superclear Gelatin, Swift and Company, considered effective as the natural isomer. Chicago. 8 Dobson, D. C. 1961 Essential amino acid compo The racemic form of isoleucine, valine, sition of a balanced chick diet. Ph. D. Thesis. Utah State University. phenylalanine, and threonine was used 9 Cerelose, Corn Products Company, New York. AMINO ACID BALANCE 71 TABLE 2 Values used in calculating the a m in o acid content of rations Isolated Amino acid soybean Blood Casein Gelatin protein fibrin g/16 g N Arginine 7.3 6.8 4.2 8.2 Histidine 2.7 2.5 3.2 1.1 Lysine 6.4 8.4 8.7 3.9 Leucine 9.1 7.7 10.3 3.1 Isoleucine 5.2 4.7 5.9 1.2 Valine 5.1 5.4 7.4 2.8 Phenylalanine 5.0 4.3 4.9 2.2 Tyrosine 3.4 5.1 5.4 0.6 Tryptophan 1.0 1 2.4 2 1.5 2 0 2 Methionine 1.3 2.1 2.9 0.5 Cystine 0.6 1 1.9 0.4 2 0.2 2 Threonine 4.1 6.6 4.6 1.8 Glycine 4.3 5.4 2.3 24.2 Glutamic acid 18.5 13.2 22 1 11.0 Proline 4.4 12.8 8 2 6.0 Serine 5.2 6.0 6.4 3.2 Aspartic acid 12.5 3.8 3.6 9.1 Alanine 4.9 4.2 8 9 11.8 1 Value given by Archer-Daniels-Midland, Cincinnati. 2 Values taken from Block and Weiss (7). Glucose monohydrate was used to adjust of chicks fed a ration with the essential the rations to 100% . amino acids at the NRC requirement levels Vantress X Nichol’s-108 chicks of both was compared with that of chicks fed a sexes were fed a commercial-type ration ration with these amino acids at the levels for 5 to 7 days. They were then distributed which the preceding series of experiments to experimental groups of 6 to 8 chicks had indicated were well balanced. Results each on the basis of their weights. The of the last 2 of these experiments are re experimental rations were fed for 9 to 10 ported in this paper. In the first of these days. These periods were the same for experiments this type of comparison was all groups in any one experiment, but they made by feeding 2 rations in which a mix varied with the different experiments. The ture of the natural isomer of each of 16 chicks were housed in electrically heated amino acids provided all of the amino batteries with wire floors. Feed and water acids; another comparison was made in were offered ad libitum. the same experiment using rations that Only the results of the last 4 experi contained proteins and amino acids. ments of the series conducted to determine One ration without protein contained the balanced proportions are presented in the established NRC requirement level of this paper. Rations 1 and 2 (table 1) each essential amino acid, although NRC were the basic rations fed in the 4 experi levels are stated to be for a 20% protein ments reported here. The level of each ration and the ration fed contained only essential amino acid was reduced by 15% the equivalent of 18% protein. These and by 20% of the level in the basic ra levels are the same as calculated for ration tions in these 4 experiments. The 15% 3 as given in table 1. The second ration and some of the 20% reductions were contained the essential amino acids at made by replacing the proper amount of the calculated levels of rations 1 or 4 each essential amino acid in ration 1 with ( table 1). Both amino acid rations con glutamic acid. Where a 20% reduction tained a mixture of essential amino acids, could not be made by using ration 1, the 2% glycine, 0.6% proline, 0.75% aspara reduction was made using ration 2. gine, and enough glutamic acid to adjust Comparison of balanced and NRC re the nitrogen content to 2.88% . They also quirement proportions. The performance contained 0.6% magnesium trisilicate and 72 D. C. DOBSON, J. O. ANDERSON AND R. E. WARNICK 0.3% aluminum hydroxide. The amino determine the dry matter. Nitrogen was acid mixture, the antacids, and the sodium determined in the dried residue using a and potassium bicarbonates for a ration Kjeldahl apparatus. Ether extract was also were thoroughly mixed in a blender with determined in the residue. enough ethanol and water mixture (1 :3 ) When data were analyzed statistically, to make a slurry. The rations contained the analysis of variance was used (9 ). 2% cellulose;10 starch was the carbohy drate except for the glucose monohydrate RESULTS AND DISCUSSION used as a carrier for the vitamin mixture. Table 3 shows the results of the last 4 The amino acid slurry was dried on the experiments of the series conducted to starch and cellulose. These rations con determine how the essential amino acids tained 3% corn oil and the same vitamin should be proportioned to make a balanced mixture as the rations fed in defining the mixture. The decreases in growth rate, balanced proportions. The mineral mix produced by decreasing the level of each ture included also provided an identical essential amino acid by 15 or 20% of the level of each mineral except for that added ration 1 level, were not the same for all by the antacids. amino acids at either level of reduction. Another comparison of the balanced If ration 1 had balanced proportions of and the NRC requirement proportions was the essential amino acids, equal decreases made by feeding rations 3 through 7 in growth rate would not be expected in (table 1). Ration 3 was formulated to an experiment of this type because of contain the NRC requirement level of each the random variations in growth rate of essential amino acid, whereas rations 4 chicks. These random variations limited through 7 contained different total quan our ability to define the balanced propor tities of these amino acids in the propor tions. Also, it was realized that this ap tions used in ration 1. A single mixture of proach might not define the proportions 3 proteins provided most of the amino that would allow the fastest growth rate. acids in these rations. Different amino The series of experiments was termi acid mixtures were added to the protein nated after the 4 experiments reported mixture to produce rations with the de because further experiments using this sired amino acid levels. Rations 4 through approach did not then appear to be justi 7 contained progressively smaller amounts fied. The decreases in growth rate pro of each essential amino acid except that duced by removing the same fraction of rations 6 and 7 contained identical trypto each essential amino acid in turn were phan levels. All of the tryptophan in ra sufficiently similar to negate plans for fur tion 6 was provided by the protein mixture ther experiments. Results presented in and could not be reduced without chang table 3 and those of preceding experi ing the mixture. Enough glutamic acid ments were evaluated in making this de was added to each ration to adjust the cision. The essential amino acid propor nitrogen level provided by proteins and tions in ration 1 (table 1) are considered amino acids to 2.88%. Glucose mono well balanced, although it may be possible hydrate was used to adjust these rations to achieve a mixture that would allow to 100%. They contained 0.3% mag faster gains. It must be realized that under nesium trisilicate, 0.15% aluminium hy conditions which deviate from those used droxide, 3% corn oil, and 2% cellulose. in defining these proportions, another set The mineral and vitamin mixtures were of proportions might be better balanced. the same as those added to the amino acid The calculated amino acid levels shown rations. for ration 1 in table 1 reflect errors in the Three average chicks fed each of the amino acid content assumed for the pro rations in experiment 2 were killed with teins in the ration. The cystine level for chloroform after they had fasted for 2 ration 1 in table 1 is 0.01% higher than hours. The entire carcass of each bird, that calculated for the ration by using the including the contents of the gastrointes values given in table 2. Cystine in the pro- tinal tract, was ground and dried in an 10 Alphacel, Nutritional Biochemicals Corporation, oven at 100°C to a constant weight to Cleveland. AMINO ACID BALANCE 7 3 TABLE 3 Results obtained when the level of each essential amino acid in rations 1 or 2 was decreased by 15 or 20% Avg Amino acid Rations Decreased Gain/feed daily ratio 1 decreased 1, 2 levels gain 1 % % 9 None (ration 1 or 2) — — 13.0 0.63 Arginine 1.28 1.09 12.4 0.61 1.02 12.3 0.60 Histidine 0.43 0.37 12.4 0.62 0.34 11.9 0.62 Lysine 1.15 0.98 12.0 0.62 0.92 11.1 0.60 Leucine 1.30 1.11 12.2 0.64 1.04 11.1 0.59 Isoleucine 0.80 0.68 12.3 0.62 0.64 10.6 0.59 Valine 0.95 0.81 12.1 0.63 0.76 11.6 0.61 0.67/ 0.57/ Phenylalanine/ 12.5 0.63 Tyrosine / 0.66/ 0.56/ 0.54/ 11.8 0.62 0.52/ Tryptophan 0.20 0.17 11.9 0.60 0.16 13.0 0.62 0.40/ 0.34/ Methionine/ 12.5 0.62 Cystine / 0.33/ 0.28/ 0.32/ 12.1 0.61 0.26/ Threonine 0.78 0.66 12.7 0.63 0.62 11.9 0.62 1 The average gain and gain/feed ratio of chicks fed rations 1 or 2 are based on 12 groups of 6 chicks each; all of the other values are based on 4 groups of 6 chicks each. Approximate significant difference in rate of gain (P < 0.05) is 1.1 g/day; in gain/feed ratio, 0.025. tein mixture was determined after oxida 8.95% total level of the essential amino tion to cysteic acid by the procedure of acids must be higher than a chick needs Bidmead and Ley (10). The cystine level for rapid growth when a ration with 18% found in the mixture was higher than that protein and the energy levels of these calculated by using the values given in rations is fed. table 2. While the NRC set of minimum require When the level of each essential amino ments and the essential amino acid levels acid was reduced by 20% of the ration 1 in ration 1 show a high correlation, some level, weight gain was reduced about 10% important differences may be noted. The on the average, and the gain/feed ratio 2 sets of values can be compared in table was reduced about 4%. A 15% reduction 1. Ration 1 contains a total of 8.95% of in the level of each essential amino acid these amino acids, whereas ration 5 has reduced weight gain about 5% and the the essential amino acids in the same pro gain/feed ratio about 1.5% . When 10% portions as ration 1 but with a total of reductions in the amino acid levels were 8.3%. Ration 3 has these amino acids in made in other experiments, the weight the NRC requirement proportions, also gains were decreased about 1 %. The with a total of 8.3%. The balanced pro 74 D. C. DOBSON, J. O. ANDERSON AND R. E. WARNICK portions defined by these experiments have gain was 26% more and the gain/feed relatively more histidine, isoleucine, threo ratio 15% more when the ration contained nine, and valine, and relatively less phe the essential amino acids in the balanced nylalanine and tyrosine, leucine, and proportions, than when the ration con methionine and cystine. tained the NRC minimum requirement The balanced proportions defined by levels. Both differences were statistically our experiments may not be the best bal significant (P < 0.005). The performance anced values that might be defined. How of the chicks fed either ration was as good ever, chicks fed rations with the essential as that obtained when rations were fed amino acids in these newly defined pro that contained intact protein and the same portions have gained weight faster and levels of the essential amino acids (com utilized their feed more efficiently than pare results obtained with amino acid chicks fed similar rations with the essen rations with those obtained when rations tial amino acids in the proportions given 3 and 4 were fed in experiment 1). Gen as minimum requirements by the NRC. erally, it has been assumed that a high fat Results of the last experiment con level was needed in a diet to allow chicks ducted to compare the 2 sets of values, to grow rapidly when fed rations in which using rations in which mixtures of the all of the amino acids are provided in the natural isomer of each of 16 amino acids free form. This was not the case with provided all amino acids, are presented either of the rations fed in these experi in the upper part of table 4. The rate of ments. TABLE 4 Results obtained when 18% protein rations were fed with the essential amino acids in the NRC (1) requirement proportions or in balanced proportions with different total levels 1 Carcass content 2 Essential Total Avg amino acid essential Exp. Gain/feed amino no. daily ratio proportions acids gain matterDry Proteinprotein extract Ether % of of live weight ration 9 % Rations containing the natural isomer of 16 amino acids NRC requirement 8.30 1 11.9 0.52 Balanced 8.95 1 15.0* 0.60* Rations containing proteins and amino acids NRC requirement, ration 3 8.30 1 12.0 0.54 8.30 2 10.9 0.52 29.4 17.1 2.73 avg 11.4 0.53 Balanced, ration 4 8.95 1 14.3 0.58 8.95 2 14.0 0.60 30.6 16.4 2.62 avg 14.1* 0.59* Balanced, ration 5 8.30 1 12.9 0.59 8.30 2 13.6 0.59 29.3 16.9 2.70 avg 13.2* 0.59* Balanced, ration 6 7.85 1 14.0 0.60 7.85 2 13.9 0.60 29.9 16.7 2.67 avg 13.9* 0.60* Balanced, ration 7 7.43 1 13.4 0.60 7.43 2 13.2 0.58 31.0 16.8 2.69 avg 13.3* 0.59* 1. Duplicate groups of 5 chicks/treatment in experiment 1, and triplicate groups of 6 in experiment 2. Chicks were 7 days old at the time experiments were started. Their average starting weights were 104 and 99 g in experiments 1 and 2, respectively. The experiments lasted 8 and 9 days, respectively. 2 Results from analysis of 3 chicks fed each ration in experiment 2. * Significantly more than gain or gain/feed ratio with the ration containing the essential amino acids at the NRC requirement levels, P < 0.005. AMINO ACID BALANCE 75 Results obtained when the balanced and The results presented in table 4 are typi the NRC requirement proportions were cal of results obtained in other similar compared by feeding rations 3 through 7 experiments conducted near the end of the are presented in table 4. Chicks fed the series of experiments that determined the 4 rations with the essential amino acids balanced proportions. The level of each in the balanced proportions gained signifi essential amino acid in ration 1 is not a cantly more and utilized feed more effi minimum requirement, but the essential ciently than chicks fed the ration adjusted amino acid proportions in ration 1 are considered better-balanced than the pro to the NRC requirement levels. The higher portions given as minimum requirements total level of essential amino acids in ra of the chick by the NRC. tion 4 evidently was not responsible for the higher growth rate and feed efficiency LITERATURE CITED obtained, as compared with the results 1. National Research Council, Committee on with ration 3. The 9.0% total essential Anim al Nutrition 1960 Nutrient require amino acid level, which was set somewhat ments of domestic animals, no. 1. Nutrient requirements of poultry. Pub. 827, National arbitrarily in the series of experiments con Academy of Sciences — National Research ducted to determine the balanced propor Council, Washington, D.C. tions, was apparently higher than neces 2. Anderson, J. O., and D. C. Dobson 1959 sary for chicks to grow rapidly when their Amino acid requirements of the chick. 2. Effect of total essential amino acid level in ration provided the energy and protein the diet on the arginine and lysine require levels fed in these experiments. The level ments. Poultry Sci., 38: 1140. of each essential amino acid in ration 1 3. Klain, G. J., H. M. Scott and B. C. Johnson or ration 4 is not to be considered as a 1959 Arginine requirement of chicks fed a crystalline amino acid diet simulating the minimum requiment for chicks fed a ra composition of casein. Poultry Sci., 38: 488. tion with 18% protein and the energy 4. Moore, S., and W . H. Stein 1954 Proce level of the rations fed in these experi dures for the chromatographic determination ments. All essential amino acid levels in of amino acids on four per cent cross-linked sulfonated polystyrene resins. J. Biol. Chem., ration 4 were decreased 12.3% to produce 211: 893. ration 6, but these decreases did not de 5. Moore, S., D. H. Spackman and W. H. Stein crease the growth rate or the feed effi 1958 Chromatography of amino acids on ciency. Even a simultaneous 17.3% re sulfonated polystyrene resins. Anal. Chem., 30: 1185. duction (ration 7) in the level of essential 6. Spackman, D. H., W. H. Stein and S. Moore amino acids except tryptophan did not 1958 Automatic recording apparatus for produce a statistically significant reduc use in chromatography of amino acids. Anal. tion in either growth rate or feed efficiency. Chem., 30: 1190. 7. Block, R. J., and K. W. Weiss 1956 Amino At the end of the last experiment, the Acid Handbook. Charles C Thomas, Spring- percentage of dry matter, protein, and field, Illinois. ether extract was determined in the car 8. Fox, M. R. S., and G. M. Briggs 1960 Salt casses of 3 chicks fed each ration. The mixtures for purified-type diets. III. An im proved salt mixture for chicks. J. Nutrition, averages are shown in table 4. Although 72: 243. small differences in composition were 9. Snedecor, G. W . 1956 Statistical Methods. noted, none were statistically significant. Iowa State College Press, Ames. Thus, the evidence did not indicate that 10. Bidmead, D. S., and F. J. Ley 1958 Quan titative amino acid analysis of food proteins differences in body composition would ex on single ion-exchange column. Biochem. plain the differences in weight gain. Bioph. Acta., 29: 562. Relative Potency of Diets Containing Amino Acids and Proteins for Promoting Growth of Rats* 1'2 W. D. SALMON Auburn University Agricultural Experiment Station, Department of Animal Science, Auburn, Alabama ABSTRACT The necessity for inclusion of certain nonessential ammo acids and of the high levels of essential amino acids sometimes used in diets for rats was investi gated. The rate of weight gain of rats produced by the best amino acid diets was com pared with that produced by diets containing intact protein or combinations of intact protein and amino acids. Data are presented on weight gains of young rats subjected to 23 different dietary treatments for 8 weeks. Accumulated weight gains for the first 4-weeks appeared to be somewhat more dependable than for the first 2-week period; there was no advantage in extending the test over an 8-week period. A mean weight gain of 44+ g/rat/week for a 4-week period was supported by a protein-free diet con taining 14 amino acids and 2.16% total nitrogen; of this 0.66% was supplied by L-glutamic acid. A diet containing 19 amino acids and 3.03% total nitrogen was less effective. Similar diets containing 20% of casein, lactalbumen or protein equivalent of dried lean beef supported mean weight gains of 50+ g/rat/week over the same period. This was a significantly higher (P < 0.005) rate of gain than was produced by the best amino acid diet. When adequately supplemented with amino acids, 25% of peanut meal, 20% of wheat gluten or 10% of casein in the diet likewise produced mean weight gains of 50+ g/rat/week over a 4-week period. Thus, it was shown that there was a growth-stimulating effect of the intact proteins not observed in any of the amino acid combinations tested. Beginning with the classic investiga teins for relative efficacy in supporting tions of Rose and associates in 1931, nu weight gains in young rats. Data at sig merous experiments have been conducted nificant variance with the above conclu on the growth rate of rats or other species sions of Greenstein and Winitz and of fed diets containing amino acids as the Sauberlich are presented in the present sole source of dietary nitrogen. In some paper. experiments with rats the growth rate has been good; generally it has not equaled MATERIALS AND METHODS that produced by a similar diet containing Weanling male rats of the Charles River an adequate level of intact proteins of CD strain were used in all experiments. good quality. In a comprehensive review They were fed commercial rat breeder of the literature related to this problem diet for 2 days after they were received in through 1957 by Greenstein and Winitz the laboratory. They were then weighed ( 1 ) , they stated that “at the present time and placed in individual, screen-bottom diets of chemically defined components cages suspended on roll-paper type metal can equal the nutritive capacity of diets racks. Temperature of the animal room composed of whole protein.” Data pre was maintained between 22° and 24°C. sented from this laboratory by Sauberlich Each treatment group in an experiment (2 ) led to a similar conclusion, but this consisted of 5 rats. They were fed the re was not supported by the recent report of spective experimental diets in glass jars Breuer et al. (3 ). immediately after being placed in individ Additional studies were made to explore ual cages and daily thereafter. Tap water the necessity for the high levels of essen was supplied ad libitum. The rats were tial amino acids used by Sauberlich and Received for publication August 27, 1963. others and the need for inclusion of cer 1 Supported in part by PHS Research Grant AM tain nonessential amino acids in the diet. 01175 from National Institute of Arthritis and Me tabolic Diseases, Public Health Service. Diets containing amino acids have been 2 Some of the data in this paper were presented at the Federation of American Societies for Experimental compared wth those containing intact pro Biology Meetings in Atlantic City, New Jersey, 1962. 76 J. N u t r it io n , 82: ’ 64 AMINO ACIDS VERSUS PROTEINS IN DIETS FOR RATS 7 7 TABLE 1 tractor with steam-jacketed extraction ves Amounts of basal components in the test diets sels in which the casein was kept hot 9/100 g while the solvent percolated through. The Am ino acids or proteins l peanut meal was a high-grade solvent- S u c r o s e 1 0 . 0 process product made from shelled pea L a r d 2 0 . 0 nuts. It was extracted by the same method Cornstarch 2 q . s . V itam in prem ix no. 6 3 2 . 0 as the casein. The lactalbumen7 was ob Salts no. 8 3 5 . 0 tained commercially, and used as received. A g a r 1 .0 The wheat gluten was used as received Choline chloride 0 . 3 from the manufacturer.8 A scorbic acid 4 5 0 .1 Penicillin “G” sodium s 0 . 0 1 RESULTS mg/100 g F o l a c i n 0 . 2 Mean weight gains of rats by treatments B i o t i n 0 . 0 5 during periods of 2, 4 and 8 weeks are shown in tables 2 and 3. With the excep M l 100 g tion of treatments A, B, H and N, the only V i t a m i n D 2 4 Vitam in B !26 5 differences in treatments were in the amino acid or protein constituents of the 1 Shown for each treatment in tables 2 and 3. 2 In amounts to make 100 g. diets and in the levels of corn starch 3 For amounts of vitamins and minerals supplied see Materials and Methods in text. necessitated by such changes. In the for 4 Ascorbic acid was omitted from treatment H. mulation of these diets, their adaptability 5 Penicillin was omitted from treatments A and N. 6 Vitamin B 12 was omitted from treatments A, B, for use in studies of choline deficiency was and N. considered. For this reason cystine was weighed at approximately the same time arbitrarily included at a fixed level to of day each week, at which time the feed keep the level of methionine as low as jars were removed and replaced with clean consistent with maximal growth rate at ones. The diets were mixed in quantities tainable with such diets. of 1 kg and stored at 1° to 2°C until fed. The mean weight gains for the various The basal component composition of the diet treatments were analyzed by Duncan’s test diets is shown in table 1, except for multiple range test (4 ) separately for the the individual amino acids and proteins 2-, 4-, and 8-week periods. For the 2-week which varied as listed for each treatment period, a significant decrease in weight in tables 2 and 3. Vitamin premix no. 6 gain resulted from omitting vitamin BJ2 with a sucrose base, supplied the following (treatment B ) or vitamin Bi2 and peni amounts of vitamins: (in mg/kg of diet)3 cillin (treatment A ) from the diet contain menadione, 50; riboflavin, 20; thiamine- ing 8 nonessential amino acids (treat HC1, 20; pyridoxine-HCl, 24; Ca panto ment C ) (P < 0.05) or from the diet con thenate, 50; niacin, 100; inositol, 200; taining 20% of extracted casein (treat D-a-tocopheryl acetate, 55; and vitamin A ment N vs. treatment O) (P < 0 .0 1 ). At palmitate, 100,000 USP units. Folacin, both the 4- and 8-week periods, the dele vitamin B[2, biotin and vitamin D2 were tion of vitamin Bi2 and penicillin from the not included in the premix but were added amino acid diet or from the 20% extracted separately in solution form to each diet. casein diet decreased weight gains signifi The amounts are shown in table 1. Salts cantly (P < 0.01). The results from treat- no. 8 supplied the following quantities 3 Appreciation is expressed to Distillation Products Industries Inc., Rochester, New York, for the dry of minerals: (% of diet) CaHPO.,-2 H20, vitamin A (PG B-250 and the vitamin E supplement 3.15; KC1, 0.6; MgSO.,. 0.4; NaHCOs, 0.7; (type F-50) and to Merck Sharp and Dohme Research Laboratories, Rahway, New Jersey, for the other vita Fe citrate, 0.123; MnSOrH.O, 0.010; mins used. . . , 4 Obtained from Nutritional Biochemicals Corpora ZnC03, 0.010; CuS04, 0.004; KI, 0.003. tion, Cleveland, or Mann Research Laboratories, New The amino acids were obtained commer York, except for DD-methionine, N.F. grade, which was provided by the Dow Chemical Company, Midland, cially.4,5 The casein (no. 1 nutritional Michigan. , _ 5 Appreciation is expressed to Dow Chemical Com grade)6 was extracted for 48 hours with pany, Midland, Michigan, for the methionine and to General Mills, Minneapolis, for the wheat gluten. 82% (vol) methanol and for 48 additional 6 Obtained from A. Adler and Son, Philadelphia. hours with 100% methanol. The extrac 7 Obtained from Nutritional Biochemicals Corpora tion, Cleveland. tion was made in a continuous-type ex 3 Obtained from General Mills, Minneapolis. TABLE 2 Percentages of test components in diets and mean weight gains of rats by treatments doÒHHHO H H 'i^ H q ic ^ ddiooÒ ^ o n q c o ì r- rH i r O Ò t> O O oqroq^i>ioq^^ co q q q q ^ q q q c ^ o rid n H i H ^ q dcodd d n o c d i r i n o ó c c q i q - q r q d ^ ^ d q d o c q c d i r i n d c i - r d i j r d d d i o - r c i n q d c o i - q r d q ^ i d - d q q o c d i - r i n d c i - r co ò q o c o q d ò q ^ ^ q q c ^ in in q q ^ o q q cocooNNinc^uoTf ddr-i r-id ridcidciddddodrii i r ’fi d ' o o c d n i d n t d n i d o i c c o c d q i q u c q d q i q q r d i - q ir c n d ic q - r ’d d o q d q n d o o c o c ÒÒ0ÓÒr-ÌnÌr-Ìr-iÒ iqi; coco'i ^qqin d H H i c i r CO d d d cqqq^qqinin^ ÒÒCOÒr-ÌcNr-ir-ÌO Ò Ò C O O O r H r - i r H o <*J O x fi f cobfl tu u P § I ’§ s h s - Ì ' 3 l l ' ¿ o hiOhi aj u £ fitì S g Q fi ^ d5 CD-fi o fifi S * cj M * fi rfi ' S . , 'S .5 +■» (D Qfi fi fi CD _c rH © rH O rH coqiococococoiqioiocoH q r - Ì Ocoq r - q q q i O r H rH Oq co »H q q q O r-i rHrHO rH O q q q q n i o c i r d d d d d d i o d i c d i r qiococococoioiqiqcoH qco o o c i r d d o o d d i c d i c d i r C O C O O io O D - I | IC ni o ni c O o o ri o i q C o c q O C D I O I O| eoo q co io co q | - | | qcqiotNo N C 6 r-i O r-i COCO oni oòòòò I ri co ò ò in ò ò io n iò n io r i n d i r d i r I I I I N ( O r-5 O r-i CO CO CO hiannojjjQUZHZS a| H ! 2 ? H fi 2 § 5 A -fi ; j j j j fi rfi 03 0) I I .O 'So.O 2 g » « n 3 « 5 o3 3 CD O i Pif Pi O-3 « H d 3 M fi fi ! l i M oj fi O Ci I I j I I 1^1 I ) « H o e 0) h CO'fiKStj, 'OS s. -'PO'S a o a m H(NO ClrH TfCD h n in m q q q rH q ^ O O CO i r ) o m r-i ni ni r-i (M O 05 l> cn < q > o3 coO W> h in i n o -*-> o3. ni b D -, -, D b i CO £ S ^ M TT co ^ ^ CO ■ TT S M £ 1 Vitamin B12 and penicillin were omitted from diet in treatment A. 2 Vitamin B12 was omitted from diet in treatment B. :f Ascorbic acid was omitted from diet in treatment H. 4 S D . Percentages of test components in diets and mean weight gains of rats by treatments > £ C/5 H P & O P H § £ M t d 0 00 —I 1 rH 11 1 1 9 °. 0 0 t rH 0 d in — I I 1 r I I 1 1 O 1 1 tOrHO»HOrH d 0 CO — O T O T—I O I T- © T—I O w (N co O q ^ 1 1 1 O O ^ T—1 rH CO d CM d d CM q CM in CD q CD d 1 CO 1 0 d CO d CO 0 CO °. ° 1 O 1 d q CO d q CO CM d q CO 0 1 Vitamin B 12 and penicillin were omitted from diet for treatment N. 8 0 W. D. SALMON ments A and B show that the deletion of vised formulations were generally higher vitamin Bn depressed growth as much as than those supported by treatments D and deletion of both vitamin Bn and penicillin. E, the differences were statistically signifi Thus, there was no vitamin Bn-sparing cant only for treatments L and M. As com effect by penicillin in this diet. pared with treatment D, there was a de When ascorbic acid was omitted from crease in the level of 10 amino acids in the diet, there was a decrease in mean treatment L. This was exclusive of the weight gains (treatment G vs. treatment 5 nonessential amino acids that were H ). The decrease was significant (P < omitted from treatment L and for which 0.05) at the 2- and 4-week periods and there was some compensatory increase in approached significance for the entire 8- glutamic acid. Of the 10 amino acids week period. The inclusion of 0.50% of that were decreased, 5 were decreased creatine in the diet containing the full 50% or more; in addition, threonine was complement of nonessential amino acids, decreased 40% and valine 32% . Thus the had no effect on weight gains at any period revised formulations represent a rather (treatment C vs. D). substantial reduction in dietary levels of When the essential amino acids were essential amino acids as well as of total maintained at the high level of treatment nitrogen. Several tests were made to as D, mean weight gains were not depressed certain whether the omission of glycine by omission of alanine, aspartic acid, and tyrosine from the diet would affect asparagine, proline and serine from the the rate of gain. When glycine alone or diet, provided that glutamic acid was in both glycine and tyrosine were omitted, creased to maintain the same nitrogen there was a decrease in mean weight gains. level (treatment E). In one experiment, Pending further study, these 2 amino acids the results of which are not included in were routinely included in all amino acid the tables, mean weight gains were like diets reported in this paper; the level of wise not depressed by the omission of tyrosine was substantially reduced, how these 5 amino acids, when the glutamic ever, for treatments I, J, K, L and M. acid level was held at 4.0% and arginine- The best amino acid diets (treatments HC1 was increased to 1.52 or 2.40% . The L and M ) were compared with various nitrogen levels of the diets representing diets that contained intact protein. These these changes were 2.80 and 3.04%, re were 20% of extracted casein (treatment spectively. It was likewise found in this O), 15% of extracted casein supplemented experiment that mean weight gains were with amino acids (treatment P ), 10% of not affected by reducing the glutamic acid extracted casein supplemented with amino from 8.4 to 6.0% without increasing acids (treatment Q) 5% of extracted arginine; the nitrogen content of this diet casein superimposed upon the amino acid was 2.80%. Later tests on levels of glu levels in treatment J (treatment R ), 20% tamic acid in diets containing lower levels of wheat gluten supplemented with amino of total nitrogen, showed no statistically acids (treatments S and T ), 25% of ex significant differences in weight gains pro tracted peanut meal supplemented with duced by diets containing 6.0, 7.0, or 8.0% amino acids (treatment U ), 20% of lactal- of glutamic acid and 0.80% of arginine- bumen (treatment V ) and 21.4% of dried HC1; gains were slightly larger with 7.0 lean beef (treatment W ). Analysis of vari or 8.0% than with 6.0% and significantly ance was made on the data by periods. larger (P < 0.05) than with 5.0% of glu At the 2-week period, mean weight gains tamic acid in the diet. produced by treatments P, Q, S, U, V, W Numerous tests in which the major were significantly greater (P < 0.005) than essential amino acids were individually those produced by treatments L and M, decreased stepwise were conducted. The but were not significantly greater for treat data from these tests are not included in ments O, R and T. The accumulated mean the tables, but data from revised formula weight gains for the 4-week period were tions based on these tests are included significantly greater than those produced (treatments F, G, I, L and M ). Although by treatments L and M for treatments O, mean weight gains supported by the re Q, S, U, V, W (P < 0.005) P (P < 0.01) AMINO ACIDS VERSUS PROTEINS IN DIETS FOR RATS 8 1 and T (P < 0 .1 0 ). The accumulated mean a test will include the period of maximal weight gains for the 8-week period exhibit gain. There appears to be no advantage a pattern similar to that for the 4-week in extending the test to 8 weeks. Increas period with minor shifts in rank of some ing the number of animals in a 4-week treatments. Significant increases over test would be preferable. gains produced by treatments L and M were shown by treatments O, P, Q, S, T, DISCUSSION V, W ( P < 0.005) and U (P < 0.05). The data presented in this paper are in Treatment R produced no significant in agreement with reports of other investi crease over treatments L and M at any gators that an excellent rate of growth period. The results show that the intact in rats can result from feeding protein- proteins tested had a growth-stimulating free diets containing crystalline amino effect greater than that of the best protein- acids. They afford conclusive proof, how free amino acid diets. The diet containing ever, that such diets are not as potent as 10% of casein supplemented with amino protein-containing diets for promotion of acids supported a rate of weight gain com growth in this species. Diets containing parable to that supported by the diet con adequate levels of good quality protein pro taining 20% of casein or equivalent levels duced mean weight gains of 6 to 8 g/rat/ of lactalbumen or dried lean beef. The week more during a 4-week period than addition of 5% of casein to an amino acid the best amino acid diet tested. The differ diet (treatment R ), however, did not pro ence was significant (P < 0.005). Diets duce a statistically significant increase containing incomplete proteins or 10% of over the weight gain produced by the casein, when they were adequately supple amino acid treatments L and M. mented with amino acids produced weight The length of time required to obtain gains comparable to those produced by the definitive data from experiments of this diets containing adequate levels of good nature is of major importance. A break quality protein. Apparently there was a down of the mean weight gain for the growth-stimulating substance in the pro 23 treatments by periods shows that the teins tested that was not present in the maximal rate of gain generally occurred amino acid diets. Several sources of un in the 3- to 4-week period and the lowest identified growth factors were tested as rate of gain during the 5- to 8-week period. supplements to the amino acid diets. These During the first 2-week period the mean included the dietary levels: (on a % solids weight gain in grams per rat per week was basis) fish solubles, 5; distiller’s solubles, 40, during the second 2-week period 47 5; dried whey, 5; beef extract, 5; liver ex and during the final 4-week period only tract 2 or pepsin, 1. None of these pro 33. When recorded as accumulated mean duced any appreciable increase in weight weight gains, the differences were masked gain. Supplementation with 5% of pan to some extent but were still obvious. In creas powder, whole fiver powder or thy this case the mean weight gain for the mus powder produced slight increases but 23 treatments in grams per rat per week no more than 5% of casein, which was for the 2-week period was 40, for the 4- shown in treatment R not to produce a week period 43.5 and for the 8-week period significant increase. 38; the coefficient of variation was higher Treatment L represents a simplification for the 2-week period (0.15869075) than of the amino acid mixture and a substan for the 4-week period (0.10958502) or the tial decrease in the levels of essential 8-week period (0.10329268). Moreover a amino acids as compared with treatment treatment, as for example treatment O vs. C or D. It is a less expensive diet without L and M, occasionally failed to show a any decrease in effectiveness in supporting difference from another treatment at the weight gains. It may not represent mini 2-week period, but showed a significant mal levels of essential amino acids re difference at the 4- and 8-week periods. quired for maximal weight gains. In most It appears that a 2-week test period may be cases the levels are somewhat above the useful for preliminary screening tests, but standards proposed by Rose et al. (5, 6). a 4-week test is more dependable. Such The diets used by those workers, however, 8 2 W. D. SALMON contained only 2 to 4% fat, whereas the LITERATURE CITED diets used in the experiments reported 1. Greenstein, J. P., and W . W initz 1961 here contained 20% fat. The latter diets Chem istry of the Am ino Acids, vol. 1. John represented a considerably higher energy W iley and Sons, Inc., New York, pp. 245- 4 3 2 . level and supported a substantially higher 2. Säuberlich, H. E. 1961 Growth of rats fed rate of weight gain. It is questionable protein-free diets supplem ented w ith purified whether minimal requirements of essen am ino acids. J. Nutrition, 74: 2 9 8 . tial amino acids or of total nitrogen re 3. Breuer, L. H., Jr., W . G. Pond, R. G. W arner quired for maximal growth of rats can be and J. K. Loosli 1963 A comparison of several am ino acid and casein diets for the accurately ascertained until it is possible growing rat. J. N utrition, 80: 2 4 3 . to include in the diet the growth-stimu 4. Duncan, D. B. 1955 Multiple range and lating substance that is present in intact m ultiple F tests. Biom etrics, 11: 1 . proteins. 5. Rose, W . C., L. C. Sm ith, M. W om ack and M. Shane 1949 The utilization of the nitrogen of ammonium salts and certain ACKNOWLEDGMENTS other compounds in the synthesis of non- Appreciation is expressed to A. E. Drake essential am ino acids in vivo. J. Biol. Chem .. and W. H. Hearn of the Research Data 181: 3 0 7 . Analysis Laboratory of Auburn University 6. Wretlind, K. A., and W. C. Rose 1950 Methionine requirement for growth and for the statistical analysis of the data pre utilization of its optical isomers. J. Biol. sented. C h e m . , 198: 6 9 7 . Fatty Livers Produced in Albino Rats by Excess Niacin in High Fat Diets I. ALTERATIONS IN ENZYME AND COENZYME SYSTEMS INDUCED BY SUPPLEMENTING 40% FAT DIETS WITH 0.1% OF NIACIN* 1'2 LORA LONG RIKANS, DOROTHY ARATA a n d DENA C. CEDERQUIST Department of Foods and Nutrition, College of Home Economics, Michigan State University, East Lansing, Michigan ABSTRACT Forty m ale weanling albino rats were divided into 4 groups and fed 20% casein diets of high fat (4 0 % ) or low fat (5 % ) content w ith adequate or exces sive quantities of niacin. The niacin content of both the high and low fat diets was increased by supplem enting the diets with 0.1% of niacin. Two control groups, one fed high fat and the other low fat, containing adequate (0.5 m g/100 g) quantities of niacin, were fed the diet sim ultaneously. Blood sam ples were collected for pyridine nucleotide determ inations on the twenty-first and the forty-second day of the experi ment. The anim als were killed after 44 days and the livers analyzed for pyridine nucleotide concentration, fatty acid oxidase activity, and proxim ate com position. Re sults from this study indicate that excess niacin enters m etabolic pathw ays to produce at least 2 unrelated effects, increased concentrations of pyridine nucleotides in blood and liver and increased levels of fat in the liver. The first effect occurs regardless of the level of fat in the diet, but the second occurs only in conjunction w ith a high level of dietary fat. The possibility that an increased consum ption of niacin and fat increases the anim als’ requirem ent for choline is discussed. Only a few studies have been conducted the development of more sensitive meas to determine whether excessive dietary urements of normal metabolic pathways levels of the water-soluble vitamins have provide ample justification for a study of any effects on the growth and develop the oral consumption of large quantities of ment of experimental animals. In partic niacin. ular, there is little mention in the litera Recent observations suggest that excess ture of hypervitaminosis niacin. The lethal niacin may have some harmful effects in dose of this water-soluble vitamin (2 to 7 g animals. The introduction of 2% of niacin niacin/kg body weight) was determined in a 10% fat diet not supplemented with for the rat by Chen et al. (1 ), Unna (2 ), choline, significantly increased the level and Brazda and Coulson (3 ), and for the of fat in the livers of rats. The addition of dog by Elvehjem et al. (4 ). Sublethal choline or betaine to the same diet reduced doses were tolerated without gross signs of the liver fat level to that of the control toxicity or inhibition of growth, and the group (7 ). In addition, Gaylor et al. (8 ) studies were abandoned because they reported that livers from rats fed 10% fat lacked practical applications. diets containing 1% of niacin (and 0.1% Now, however, the widespread enrich of choline) contained slightly more fat ment programs and the availability of high than those from controls, although the potency vitamin preparations have made level of significance was not reported. possible the indiscriminate ingestion of Pilot experiments conducted in this labora large quantities of niacin. One recognized tory corroborated this observation. Livers treatment of hypercholesterolemia involves from rats fed 0.1% of niacin in a 40% large oral doses of niacin (3 to 7.5 g Received for publication June 12, 1963. niacin/day) which often produce a flush 1 Journal Article no. 3167 from the Michigan Agri ing reaction (5 ) and, possibly, liver dys cultural Experiment Station. 2 This work was supported in part by a grant from function (6 ). These factors, along with the National Institutes of Health, Project no. A-6093. J. N u t r itio n , 82: ’64 83 8 4 LORA LONG RIKANS, DOROTHY ARATA AND DENA C. CEDERQUIST corn oil diet -contained significantly more termined for each blood sample and re fat than those from control animals. The sults were reported as micrograms pyri present experiment was undertaken to dine nucleotides per milliliter red blood study the effect produced by feeding male cells. rats rations containing high levels of nia Liver pyridine nucleotides. Immedi cin and fat. An attempt was made to ately after the removal of the liver a 0.50-g determine the metabolic fate of the excess sample was excised from one lobe for niacin and the nature of the biochemical determination of pyridine nucleotides by lesion(s) associated with fat accumula the method of Robinson et al. (11). Fluo tion in the liver. rescence was developed by the method of Carpenter and Kodicek (10). METHODS Endogenous oxidation and fatty acid Forty male weanling albino rats of the oxidase. Endogenous oxidation and activ Sprague-Dawley strain were divided into ity of the fatty acid oxidase system were 4 groups. Group 1 received a basal diet pro measured by a modification of the mano- viding the following in g/100 g diet: case metric procedures described by Lehninger in, 20; salts W,3 4; choline, 0.15; vitamin (1 2) using the Warburg apparatus. A mix, 0.25; corn oil,4 5; and sucrose, 70.6. 2.50-g portion of chilled liver was homog The vitamin mix contained the following enized with 5.0 ml of 0.25 m sucrose. A in mg/100 g diet: vitamin A powder 1.0-ml aliquot of crude liver homogenate (20,000 IU/g), 2.5;5 calciferol, 0.1; thia was pipetted into chilled Warburg flasks. mine, 0.4; riboflavin, 0.8; niacin, 0.5; vita All flasks were allowed to equilibrate for min Bi2, 0.002;6 pyridoxine, 0.25; Ca pan 5 minutes at 25.0°C in the Warburg bath, tothenate, 2.0; menadione, 0.4; inositol, and manometer readings were taken at 1.0; folic acid, 0.2; biotin, 0.01; p-amino- 5-minute intervals. Calculations of en benzoic acid, 0.2; and sucrose to make zyme activity were based upon the 10- or 0.25 g. The diet for group 2 was identical 15-minute interval where activity was with that for group 1 except that 0.1% highest. of niacin was added at the expense of Moisture, lipid, and nitrogen analyses. sucrose. Animals in groups 3 and 4 re The remaining portions of the livers that ceived diets identical with diets 1 and 2, had been stored frozen, were allowed to respectively, except that 35 additional thaw at room temperature and homoge grams of corn oil replaced 35 g of sucrose. nized with water in a Potter-Elvehjem Food and water were provided ad libitum homogenizer. The homogenates were quan and records were kept of food intake and titatively transferred to weighed evapo weight gain throughout the experimental rating dishes and dried at 90 °C for 12 period. hours. Fat was determined by subjecting On the twenty-first and forty-second a 1.000-g sample of the dried ground liver days of the experiment blood samples were to continuous ether extraction for 3 hours taken from the tails of all animals for on the Goldfisch apparatus and weighing determination of pyridine nucleotides. On the ether-soluble lipoidal material. Nitro the forty-fourth day the animals were gen was determined by the macro-Kjeldahl stunned by a sharp blow on the head and method on 0.250-g samples of the fat-free decapitated. The livers were removed for livers and calculated as percentage of the the chemical analyses described below. fresh weight of liver. Blood pyridine nucleotides. Blood pyri 3 Wesson, L. G. 1932 A modification of the Os- dine nucleotides (DPN,7 TPN,8 and NMN 9) bome-Mendel salt mixture containing only inorganic constituents. Science, 75: 339. Obtained as Salt were determined by the Kring and W il Mixture-W from Nutritional Biochemicals Corpora liams (9 ) method with 2 modifications. tion, Cleveland. 4 Containing 7.5 mg of a-tocopheryl acetate. The sample volume was increased to 0.20 5 Containing 50 IU vitamin A, slightly exceeding the nutritional requirement for the rat. Brown, R. A. ml and hydrogen peroxide was added to and M. Sturtevant 1949 The vitamin requirements the alcohol solution before the blood sam of the growing rat. Vitamins and Hormones, 7: 171. 6 Two milligrams of a 0.1% trituration of vitamin ple was introduced. Fluorescence was de B12 with mannitol. veloped by the method of Carpenter and 7 Diphosphopyridine nucleotide. 8 Triphosphopyridine nucleotide. Kodicek (10). Red cell volumes were de 9 Nicontinamide mononucleotide. EXCESS NIACIN IN HIGH FAT DIETS 85 Standard errors of the means were cal by 6 weeks. Livers from animals fed the culated for all data and Student’s t test 40% corn oil diet (group 3) contained used to measure significance. higher levels (P < 0 .0 1 ) of pyridine nu cleotides than did those from animals fed RESULTS the 5% corn oil diet (group 1), although Growth and food intake data are pre these levels were not as high as in the sented in table 1. There were no differ groups fed excess niacin (2 and 4). Con ences in weight gain or food intake be centrations of blood pyridine nucleotides tween groups fed excess niacin and their were comparable in groups 1 and 3. respective controls (groups 1 vs. 2 and 3 Endogenous oxidation and the activity vs. 4). However, a significant reduction of the fatty acid oxidase system were af (P < 0.01) in both weight and food intake fected by increasing the fat content of was observed in groups fed 40% corn oil the diet (table 3). The endogenous oxida diets (3 and 4) when compared with tion was significantly lower (P < 0.05) groups fed 5% com oil diets whether or and fatty acid oxidase activity higher not excess niacin was present in the diets (P<0.01) in livers from animals fed (groups 3 vs. 1 and groups 4 vs. 2). Effi 40% fat diets (group 3) when compared ciency of food utilization, expressed as g with those fed 5% fat diets (group 1). weight gain/100 kcal consumed, did not These differences were not noted when differ among the 4 groups. excess niacin was added to these diets The concentration of pyridine nucleo (groups 2 and 4). tides in livers was markedly different be Data from analyses of the liver for tween groups fed adequate and excessive moisture, fat, and nitrogen are sum amounts of niacin (table 2). The addition marized in table 4. The percentage of of 0.1% of niacin to a diet containing nitrogen based on wet weight of the liver either 5 or 40% of fat10 (groups 2 vs. 1 was not changed by alterations in the diet and 4 vs. 3) resulted in a significant in (table 4). However, differences appeared crease (P<0.01) in pyridine nucleotide 10 Fat was supplied as corn oil in all the diets used concentrations in both blood and liver in this experiment. A totally different set of results tissues. This increase was apparent at could be obtained with a more saturated fat, a pos sibility presently under investigation in this labora 3 weeks and had not changed appreciably tory. TABLE 1 Growth and food intake of albino rats fed diets high in fat or high in niacin, or both G r o u p 1 D i e t W eight gain Food intake Efficiency 2 g/week kcal /week g/100 kcal i 5% Corn oil 3 5 ± 1 3 3 2 6 ± 8 3 1 1 ± 2 3 2 5% Corn oil+ 0.1% niacin 3 6 ± 1 3 5 5 ± 8 1 0 ± 2 3 40% Corn oil 2 3 ± 1 2 7 8 ± 1 0 8 ± 2 4 40% Cornoil + 0.1% niacin 2 4 ± 1 2 8 8 ± 1 0 8 ± 2 1 Each group consisted of 10 anim als. 2 W e i g : ht gain/100 kcal consum ed. 3 SE Of m e a n . T A B L E 2 Pyridine nucleotides in the livers of rats fed diets high in fat or high in niacin, or both B l o o d 3 W e e k s 2 6 W e e k s 3 L i v e r fig/ml RBC fig/ml RBC ng/g liver 1 5 % C o m o i l 2 3 0 ± 2 5 3 2 8 0 ± 2 5 3 1 2 6 0 ± 6 9 3 2 5% Com oil + 0.1% niacin 4 0 8 ± 4 5 4 3 4 ± 4 1 2 5 5 7 ± 2 2 0 3 40% Corn oil 1 7 8 ± 1 6 2 3 5 ± 4 2 1 8 6 6 ± 6 0 4 40% Cornoil + 0.1% niacin 4 1 2 ± 7 0 3 9 1 ± 3 0 2 3 7 9 ± 1 5 2 1 From 6 to 10 animals used for each analysis. 2 Length of time fed experimental diet. 3 se or mean. 8 6 LORA LONG RIKANS, DOROTHY ARATA AND DENA C. CEDERQUIST TABLE 3 Endogenous oxidation and fatty acid oxidase activity of rats fed diets high in fat or high in niacin, or both E n d o g e n o u s Fatty acid G r o u p 1 D i e t o x i d a t i o n o x i d a s e nl Oz/hr/g liver /il Oz/hr/g liver i 5% Corn oil 1 7 7 5 ± 7 3 2 3 3 1 ± 3 4 2 2 5% Corn oil+ 0.1% niacin 1 7 9 0 ± 5 6 4 0 5 ± 3 7 3 40% Corn oil 1 5 5 5 ± 4 5 5 0 0 ± 2 8 4 4 0 % C o r n o i l + 0 . 1 % n i a c i n 1 7 5 0 ± 6 1 4 6 5 ± 4 1 1 From 8 to 10 animals used for each analysis. 2 s e o f m e a n . TABLE 4 Liver composition of rats fed diets high in fat or high in niacin, or both F a t N i t r o g e n G r o u p 1 D i e t M o i s t u r e w e t w t 2 W e t w t 2 D r y w t 3 % % % % i 5% Corn oil 71.0± 0.4 4 3.07± 0.08 4 3 . 2 ± 0 . 1 4 10.9 ± 0.5 4 2 5% Cornoil + 0.1% niacin 7 0 . 1 ± 0 . 3 3 . 1 2 ± 0 . 0 6 3 . 4 ± 0 . 1 1 1 . 3 ± 0 . 3 3 40% Corn oil 7 1 . 2 ± 0 . 2 3 . 2 1 ± 0 . 0 6 3 . 8 ± 0 . 3 1 3 . 1 ± 1 .1 4 40% Cornoil + 0.1% niacin 6 9 . 3 ± 0 . 3 3 . 2 4 ± 0 . 0 4 5 . 8 ± 0 . 4 1 9 . 0 ± 1 .1 1 Each group consisted of 10 animals. 2 Calculated on the basis of fresh weight of the liver. 3 Calculated on the basis of dry weight of the liver. 4 s e o f m e a n . in percentage fat“ and percentage mois niacin. Thus, this study provides strong ture in livers from the high fat groups supportive evidence that at least a portion (3 and 4). Livers from animals in group of the excess niacin present in the diet was 4 (excess niacin) contained significantly absorbed and participated in metabolic re greater amounts of fat (P<0.01) and actions, refuting the theory of “inert excre less moisture (P < 0.01) than those from tion.” These observations are in agree group 3 (adequate niacin). When livers ment with those of others (8, 13-15) who from animals in the low fat series were reported elevated pyridine nucleotide con compared (groups 1 and 2) no differences centrations when excessive amounts of were observed in fat or moisture as a re niacin were administered to rats. sult of including excess niacin in the diet. A second effect of consuming excess quantities of niacin was observed in some DISCUSSION oxidative processes. Livers from animals Growth data from this experiment cor fed a 40% fat diet demonstrated an in roborate earlier observations (2, 7) that creased activity of the fatty acid oxidase excessive niacin has no growth inhibiting system and a decreased rate of endogenous effect on the rat. The growth depression oxidation when compared with their con noted in this study resulting from increas trols fed 5% fat diets. These changes, ing the fat content of the diet from 5 to coupled with an increase in liver pyridine 40% probably reflected the lowered food nucleotides, probably reflect an adaptive intakes observed in the groups fed high response on the part of the rat to the in corn oil diets. This is supported by the creased fat content of the diet. The ani lack of any significant differences in effi mals appeared to have adjusted to the ciency of utilization of food between any higher level of dietary fat by increasing of the 4 groups. the rate of fat oxidation in the liver, thus The effect of excess niacin was shown increasing the economy of calorie utiliza most dramatically in pyridine nucleotide tion in the liver. These changes did not concentrations which were increased mark 11 A ll liver fat data cited in text are based on dry edly (almost twofold) in rats fed excess weight of liver. EXCESS NIACIN IN HIGH FAT DIETS 8 7 occur when the diet contained excess nia 5. Parsons, W . B., and J. H. Flinn 1959 Re cin, suggesting that the excess niacin in duction of serum cholesterol levels and beta- lipoprotein cholesterol levels by nicotinic terfered in some manner with the adap acid. Arch. Int. Med., 103; 123. tive response of the animals to high levels 6. Rausen, A. R., and D. Adlersburg 1961 of dietary fat. Idiopathic (hereditary) hyperlipem ia and The extent to which the metabolism of hypercholesterem ia in children. Pediatrics, methyl groups may be involved in the de 2 8 : 2 7 6 . 7. Handler, P., and W . J. Dann 1942 The velopment of fatty livers under these condi inhibition of rat growth by nicotinam ide. tions cannot be ignored (7 ). Prior to its J. Biol. Chem ., 146: 357. excretion, nicotinamide is methylated by 8. Gaylor, J. L., R. W . F. Hardy and C. A. methione (1 6) which can, in turn, be de Baum ann 1960 Effect of nicotinic acid rived from choline (17). Thus choline is in and related compounds on sterol metabo lism in the chick and rat. J. Nutrition, 70: volved in the detoxication of niacin. More 2 9 3 . over, Griffith (1 8 ) observed an increased 9. Kring, J. P., and J. N. W illiam s, Jr. 1954 severity of a choline deficiency in young Differential determ ination of pyridine nu rats by increasing the fat content of the cleotides and N '-m ethylnicotinam ide in blood. diet. J. Biol. Chem ., 207: 851. 10. Carpenter, K. J., and E. Kodicek 1950 The In the study reported here, increasing fluorim etric estim ation of N'-m ethylnicotina- the concentration of both niacin and fat m ide and its differentiation from coenzyme in the diet (group 4) would serve to in I. Biochem . J., 46: 421. crease the animals’ requirement for cho 11. Robinson, J., N. Levitas, F. Rosen and W . A. Perlzweig 1947 The fluorescent condensa line. Thus the fatty livers observed in this tion product of N'-m ethylnicotinam ide and group could reflect a mild choline de acetone. IV. A rapid method for determi ficiency. nation of pyridine nucleotides in anim al However, the fatty livers observed under tissues. The coenzyme content of rat tis sues. J. Biol. Chem ., 170: 653. conditions reported here did not demon 12. Lehninger, A. L. 1955 Fatty acid oxida strate the classical biochemcial symptoms tion in m itochondria: Methods in Enzymol- of choline deficiency. The repression in ogy, vol. 1, eds., S. P. Colowick and N. O. fatty acid oxidation which reportedly oc Kaplan. Academ ic Press, Inc., p. 545. curs in choline-deficient liver slices (19, 13. W illiam s, J. J., Jr., P. Feigelson and C. A. Elvehjem 1950 Relation of tryptophan and 20) was not observed in this experiment. niacin to pyridine nucleotides of tissue. J. That is, oxidation of octanoate was com Biol. Chem ., 187: 597. parable in livers from groups fed high fat 14. W illiam s, J. N., Jr., P. Feigelson, S. S. diets with normal or excessive amounts of Shaninian and C. A. Elvehjem 1951 Fur ther studies on tryptophan-niacin-pyridine niacin. It is possible that the excess niacin nucleotide relationships. J. Biol. Chem ., 189: in the biological system may have com 6 5 9 . plicated the classical symptoms of choline 15. Burch, H. B., C. A. Storvick, R. L. Bicknell, deficiency. H. C. Kung, L. G. Alejo, W . A. Everhart, The extent to which choline is involved O. H. Lowry, C. G. King and O. A. Bessey 1955 Metabolic studies of precursors of in the effects induced by excess niacin is pyridine nucleotides. J. Biol. Chem ., 212: currently under investigation. 8 9 7 . 16. Cantoni, G. L. 1951 Methylation of nico tinam ide w ith a soluble enzym e system from LITERATURE CITED rat liver. J. Biol. Chem ., 189: 203. 1. Chen, K. K., C. L. Rose and E. B. Robbins 17. M untz, J. A. 1950 The inability of choline 1938 Toxicity of nicotinic acid. Proc. Soc. to transfer a m ethyl group directly to hom o Exp. Biol. M ed., 38: 241. cysteine for m ethionine form ation. J. Biol. 2. Unna, K. 1939 Studies on the toxicity and Chem ., 182: 489. pharmacology of nicotinic acid. J. Phar 18. Griffith, W . H. 1940 Choline metabolism. m acol. Exp. Therap., 65: 95. III. The effect of cystine fat and cholesterol 3. Brazda, F. G., and R. A. Coulson 1946 on hem orrhagic degeneration in young rats. Toxicity of nicotinic acid and some of its J. Biol. Chem ., 132: 639. derivatives. Proc. Soc. Exp. Biol. M ed., 62: 19. Artom , C. 1953 Role of choline in the oxi 1 9 . dation of fatty acids by the liver. J. Biol. 4. Elvehjem , C. A., R. J. M adden, F. M. Strong Chem ., 205: 100. and D. W . Wooley 1938 The isolation and 20. Rees, D. E., and K. L. Kline 1957 Metabo identification of the anti-black tongue factor. lism of octanoate by fatty liver slices. Am . J. J. Biol. Chem ., 12: 137. Physiol., 190: 446. The Amino Acid Composition and Nutritive Value of Proteins V. AMINO ACID REQUIREMENTS AS A PATTERN FOR PROTEIN EVALUATION* 1 P. B. RAM A RAO,2 H. W . NORTON a n d B. CONNOR JOHNSON Division of Animal Nutrition, University of Illinois, Urbana, Illinois ABSTRACT The pattern of essential amino acid requirements of the growing rat has been used as a reference “protein” for estim ating the nutritive value of various proteins. A “requirem ent index” is proposed as a chem ical estim ate of the nutritive value of proteins. Its correlation w ith the biological value of 11 proteins is highly significant (r = 0.9654). Several attempts have been made to TABLE 1 evaluate the nutritive value of proteins on Pattern of amino acids required the basis of their amino acid compositions. by the growing rat Mitchell and Block (1 ) used the amino acid composition of whole egg protein as % of diet the standard for computing a “chemical L-Histidine 0 . 2 5 score” of protein, based on the most limit L - L y s i n e 0 . 9 0 L-Tryptophan 0 . 1 1 ing amino acid. Later, Mitchell (2 ) com L-Isoleucine 0 . 5 5 puted a modified essential amino acid in l -V a l i n e 0 . 5 5 dex, modifying that of Kiihnau (3 ), who L - L e u c i n e 0 . 7 0 proposed human milk as the standard, and L-Threonine 0 . 5 0 L-M ethionine °-16l0 3 4 j 0 0 . bso U that of Oser (4 ), but retaining whole egg L - C y s t i n e protein as the standard. The essential L-Phenylalanine 0 - 4 2 1 Q 7 2 amino acid requirements should serve bet L - T y r o s i n e 0 . 3 0 r ter as a standard for calculating such Plus nonessential am ino acid m ixture 1 to m ake amino acid indexes. For this purpose, we a total conventional protein content of 10% redetermined the minimal requirements ( 1 . 6 g N ) . for the essential amino acids and the opti 1 100 g of the nonessential amino acid mixture con mal protein level for maximal growth and tains: 12.3 g DL-serine; 41.6 g n-glutamic acid; 12.7 g body nitrogen retention of the growing rat L-aspartic acid; 5.9 g DL-alanine; 23.7 g n-proline, and 3.8 g glycine. (5 -7 ). In the present paper a “requirement index” is proposed which is the geometrical Rat growth with the minimal amino acid mean of the several amino acids, each ex diet. The results of a study of growth pressed as a percentage of its standard with this amino acid mixture and with a value, except that values exceeding 100% 10% protein, whole egg protein diet, are are reduced to 100%. The calculated re presented in table 2. Growth with the quirement indexes for a number of pro minimal amino acid diet and the whole teins are compared with published biologi egg protein diet was almost equal at about cal values. 5 g/day, and the efficiency of protein utili The reference pattern of the minimal zation (protein efficiency ratio = 3.9) was essential amino acid requirements of the the same in the 2 groups. growing rat is shown in table 1. The opti mal protein level (N X 6.25) for maximal Received for publication August 19, 1963. growth and nitrogen retention was found 1 This work was supported in part by a contract with the Surgeon General’s Office, Department of the to be 10% of the diet, with a mixture of Army, contract no. DA-49-007-MD-544. The opinions expressed in this publication are those of the authors nonessential amino acids as in casein pro and not necessarily those of the Department of the viding the nonessential amino acid nitro Army. 2 Present address: Department of Biochemistry, gen (6 ). Indian Institute of Science, Bangalore 12, India. 88 J. N u t r it io n , 8 2 : ’64 PROTEIN EVALUATION 8 9 TABLE 2 Rat growth ivith the minimal amino acid diet A v g A v g D i e t w e i g h t f o o d P E R 2 g a i n 1 i n t a k e 9 9 W hole egg protein 3 62.6 ± 2.4 1 1 6 2 3 . 9 ± 0 . 1 9 1 Am ino acid diet 5 9 . 5 ± 2 . 4 1 5 2 3 . 9 ± 0 . 2 6 1 Average of 6 male rats in each group; average initial weight 56 g; fed ad libitum for 12 days. 2 Protein efficiency ratio: gram weight gain per gram conventional protein (N x 6.25) intake. 3 Twelve grams of laboratory-prepared whole egg protein replaced the essential amino acids and the nonessential amino acid mixture in the basal diet to provide a protein level of 10% (N x 6.25); see table 1. 4 s e o f m e a n . Correlation between biological values ample, in the case of whole egg protein, and calculated indexes. To calculate the lysine, threonine, and histidine are limit correlation coefficients between the chemi ing, whereas in soybean meal methionine cal and biological methods of evaluation of + cystine, lysine, threonine, and valine are the nutritive value of proteins, the amino limiting in that order. acid composition of 15 proteins was taken The calculated nutritive values of 15 dif from the summary tables of Block and ferent proteins and their biological values Bolling (8 ). The biological values of these are shown in table 3. The “chemical score” proteins (table 3) were taken from litera is the percentage of the most limiting ture (2 ), except for casein for which our amino acid as compared with the standard. own data were used.3 The calculated per The requirement index is calculated essen centage of limiting amino acids in these tially according to Mitchell’s (2 ) method proteins as compared with the amino acid for the calculation of the modified essen requirement pattern (table 1) are sum tial amino acid index. For both values the marized in table 4 in order from the most requirement pattern was used as the stand limiting to the marginally limiting amino ard reference “protein.” acid. The number of limiting amino acids Figure 1 illustrates the correlation be varies from protein to protein. For ex- tween the chemical scores, calculated ac- TABLE 3 Biological values of proteins, and scores based on the requirement pattern B i o C h e m R e q u i r e P r o t e i n l o g i c a l i c a l m e n t v a l u e s c o r e 1 i n d e x 2 1 W hole egg protein 9 7 7 8 9 5 2 Egg album in 9 3 7 2 9 4 3 G e l a t i n 2 2 0 0 4 C a s e i n 7 9 7 8 9 6 5 Lactalbum in 8 4 9 2 9 9 6 W hole m ilk 9 2 8 4 9 7 7 Beef liver 7 7 7 8 9 5 8 W hole wheat 6 7 3 0 7 6 9 W heat flour 5 2 2 1 6 8 10 W hole corn 6 2 2 6 7 7 1 1 O a t s 6 5 4 0 8 2 1 2 R i c e 7 0 3 6 8 1 13 Peanut m eal 5 7 3 2 6 7 14 Cottonseed m eal 6 4 3 9 7 7 15 Soybean m eal 7 5 7 2 9 1 1 Percentage of the most limiting amino acid in Fig. 1 Relation of biological value to chem ical protein as compared with the requirement pattern (table 1). s c o r e . 2 Calculated according to Mitchell (4 ) but using the amino acid requirements as standard instead of 3 V. C. Metta and B. C. Johnson. Army Report, egg protein. unpublished. 9 0 P. B. RAMA RAO, H. W. NORTON AND B. CONNOR JOHNSON T A B L E 4 Essential amino acids limiting in several proteins as compared with the requirement pattern Lim iting essential am ino acid P r o t e i n ( % of requirem ent levels) 1 W hole egg protein Lysine (78), threonine (86), histidine (96) 2 Egg album in Lysine (72), threonine (8 4 ), histidine (96) 3 G e l a t i n Tryptophan (0 ), m ethionine + cystine (18), isoleucine (31), histidine (3 6 ), threonine (3 8 ), phenylalanine + tyrosine (39), leucine (50), valine (51), lysine (55) 4 C a s e i n M ethionine-f cystine (78), threonine (90), lysine (94) 5 Lactalbum in Histidine (92) 6 W h o l e m i l k M ethionine + cystine (8 4 ), threonine (94), lysine (97) 7 B e e f l i v e r Lysine (7 8 ), isoleucine (8 7 ), m ethionine + cystine (92) 8 W hole wheat Lysine (30), threonine (66), isoleucine (73), valine (78), histidine (8 4 ), m ethionine + cystine (8 6 ) 9 W heat fiour Lysine (21), threonine (54), tryptophan (72), valine (75), isoleucine (7 7 ), m ethionine + cystine (7 8 ), histidine (8 8 ) 1 0 W h o l e c o r n Lysine (26), tryptophan (55), threonine (74), m ethionine+ cystine (92), valine (97) 1 1 O a t s Lysine (40), threonine (72), m ethionine 4-cystine (76), iso leucine (90), histidine (92), valine (98) 1 2 R i c e Lysine (36), histidine (68), threonine (76), m ethionine+ cystine (86), isoleucine (95) 1 3 Peanut m eal Threonine (32), lysine (33), methionine + cystine (52), valine (80), isoleucine (84), histidine (84), tryptophan (91), leucine (96) 1 4 Cottonseed m eal Lysine (39), threonine (60), isoleucine (73), m ethionine-f cystine (78), leucine (8 5 ), valine (88) 1 5 Soybean m eal Methionine + cystine (72), lysine (76), threonine (78), valine (97) cording to Mitchell and Block (1 ) using the requirement pattern as the standard, and the biological values of 15 proteins. The correlation coefficient (0.89) is highly significant and compares well with that (0.846) reported by Mitchell (2 ) using egg protein as the standard. Figure 2 illustrates similarly the corre lation of the biological value with the re quirement index. Obviously 11 samples fit one fine and 3 (whole egg, egg albumin, and whole milk) do not. Their departures from the regression line fitting the 11 points are statistically significant, the least exceeding 8 standard deviations. The gela tin point has been omitted from the regres sion calculation because its requirement index is so extremely uncertain. This un certainty is traceable mainly to the trypto phan content, reported to be 0% , of which Fig. 2 Relation of biological value to require the relative error is extremely large. If the m ent index (w hole egg protein, egg album in, and whole m ilk protein, designated by A , and gelatin, tryptophan content were 1%, the require designated by O, not included in fitting regres ment index would be 25, moving from far sion lin e). PROTEIN EVALUATION 91 above to somewhat below the line fitted to few units, nowhere near enough to bring 11 other proteins. An examination of chart the points into reasonable agreement with 4 of Mitchell (2 ), with egg protein used as the 0 to 100 line. 3) The relative digesti the standard, shows that most of the points bilities of the different proteins are not are also on a line of similar position to that considered in the total amino acid analysis. plotted here where the pattern of amino However, it is difficult to believe that low acid requirements served as standard. ered digestibilities would release for ab sorption amino acid mixtures from such If no other factor were involved, biologi divergent proteins as to put them so satis cal value (which is by definition that per factorily on one line 17% below theoreti centage of the protein absorbed which is cal. Sheffner et al. (1 0 ) have suggested used by the animal) and the degree of an assay to include digestibility, and their correspondence of the amino acid pattern data do not indicate that the pattern of of a protein to the required amino acid amino acids released by digestion is of this pattern should follow a line from (0, 0) lower biological value. 4) Another possible to (100, 100). Egg and whole milk proteins explanation involves the so-called non- appear to fit such a line. All other proteins essential amino acids. If, in fact, one or (except perhaps gelatin) fit very well the more of these are essential, as are glutamic straight line Y = 0.8276X with standard acid, arginine, and glycine for the chick error of estimate only 2.5 units of biologi (11-13), such requirements have not been cal value. This slope is significantly (P < used in calculating the requirement index 0.01) less than unity, the biological values and possibly could account for the excesses falling about 17% below the 0 to 100 fine. over observed biological values. There are 4 obvious possible explana That the requirement index is a good tions for this observation, and for egg and beginning towards calculating biological milk falling on the theoretical line: 1) If value from amino acid composition is indi cated by the correlation of 0.9654 for 11 a protein has a deficiency of one amino samples. The possibility of multiple re acid it must have an excess of another. gression of biological value on amino acid Perhaps this excess depresses the deter percentages was considered, but the multi mined biological value. This can apply at ple correlation was only 0.9823 with 9 in most to histidine, lysine, threonine, and dependent variables, not reaching the 1% methionine plus cystine, because of the significance level. excesses of other amino acids in egg and milk, and it is not easy to see that a recal LITERATURE CITED culation of the requirement index allowing 1. M itchell, H. H., and R. J. Block 1946 Some somehow for excesses could account for relationships between the am ino acid content much of the discrepancy. 2 ) An error in of proteins and their nutritive values for the the determination of the amino acid re rat. J. Biol. Chem ., 163: 5 9 9 . quirements giving too low a value for some 2. Mitchell, H. H. 1954 The dependence of biological value of food proteins upon their one or more amino acid requirements content of essential am ino acids. W issensch. would give erroneously high calculated in Abhandl. deutsch. Akad. Landw. Berlin, 2: dexes. However, a regression line calcu 2 7 9 . lated using whole egg as the standard 3. Kühnau, J. 1949 Biochem ie des Nahrungs- (which is significantly higher in several eiweisses. Angew . Chem ie, 61 .■ 357. 4. Oser, B. L. 1951 Method for integrating amino acids, but lower in lysine) is not essential am ino acid content in the nutri very different in slope. These 2 nitrogen tional evaluation of protein. J. Am . Dietet. sources give very similar growth rates and A., 27: 396. nitrogen retentions. The N requirement 5. Ram a Rao, P. B., V. C. M etta and B. C. John son 1959 The am ino acid composition and determinations using the 2 N sources the nutritive value of proteins. I. Essential gave 10% protein requirement for the am ino acid requirem ents of the growing rat. amino acid mix (6 ) and 10 to 12% for J. Nutrition, 69: 3 8 7 . whole egg (9 ), the egg being limiting in 6. Ram a Rao, P. B , V. C. M etta and B. C. John lysine. Assuming a higher value for the son 1960 The am ino acid composition and nutritive value of proteins. III. The total pro requirement for any one amino acid can tein and the nonessential nitrogen require decrease the requirement index by but a m ent. J. Nutrition, 71: 3 6 1 . 9 2 P. B. RAMA RAO, H. W. NORTON AND B. CONNOR JOHNSON 7. Rama Rao, P. B., H. W. Norton and B. C. 10. Sheffner, A. L., R. Adachi and H. Spector Johnson 1961 The amino acid composition 1956 Measurement of the net utilization of and nutritive value of proteins. IV. Phenyl heat-processed proteins by means of the pep alanine, tyrosine, methionine, and cystine re sin digest-residue (PDR) amino acid index. quirements of the growing rat. J. Nutrition, J. Nutrition, 60: 507. 73: 38. 11. Klain, G. J., H. M. Scott and B. C. Johnson 8. Block, R. J., and D. Bolling 1951 The 1960 The amino acid requirement of the amino acid composition of foods and pro growing chick fed a crystalline amino acid teins, ed. 2. Charles C Thomas, Springfield, diet. Poultry Sci., 39: 39. Illinois. 12. Greene, D. E., H. M. Scott and B. C. Johnson 9. Mitchell, H. H., and J. R. Beadles 1952 1960 A need for glycine in crystalline The determination of the protein require amino acid diets. Poultry Sci., 3 9 : 512. ment of the rat for maximum growth under 13. Almquist, H. J., and C. R. Grau 1944 The conditions of restricted consumption of food. amino acid requirements of the chick. J. J. Nutrition, 47: 133. Nutrition, 28: 325. Effect of Barbituric Acid, Chlortetracycline and Carbohydrates upon Growth and Gastro intestinal Urease Activity of Chicks* 1 2 A. P. ALVARES, L. H. HARBERS - a n d W J. VISEK Department of Pharmacology, The University of Chicago, Chicago, Illinois ABSTRACT Results are presented from 100 lots of 15 chicks/lot fed basal diets containing sucrose, dextrose or starch and supplemented with barbituric acid (1.28g/kg) or chlortetracycline (0.1 g/kg) alone or in combination. These supplements when added alone produced the greatest increase in growth with the sucrose diet and the smallest with the starch diet. The 2 supplements in combination did not show an additive effect. The efficiency of feed utilization followed that of growth. Gastrointes tinal ammonia production and ureolytic activity were suppressed only when there was an observed increase in growth with the additives. Ureolytic activity in the small intes tine was inversely related to the growth response of the various basal carbohydrate diets; however, lowest activity did not reflect greatest gain for all treatments. The response to barbituric acid, a cyclic urea compound, appears to be dependent on the carbohydrate sources, an observation similar to that noted with antibiotics. Earlier studies have demonstrated that have been difficult to reproduce consist urea breakdown in the gastrointestinal ently (15). Others have reported differ tract may be altered by dietary anti-micro ences in utilization of various nutrients bial agents (1). One of the products, in animals fed antibiotics (16). Stokstad ammonia, has been suggested to be a toxin et al. (1 7 ) have shown that the growth re whose production is suppressed by these sponse to antibiotics is dependent upon the agents (2 -6 ). Animals immunized to crys type of carbohydrate in diets of chicks. talline jackbean urease have also demon In the studies reported herein 100 lots of strated increased growth and efficiency of 15 chicks/lot were used to determine their feed utilization concurrently with de response to barbituric acid and chlortetra pressed urease activity in their gastrointes cycline when either or both were added to tinal tracts (7, 8). Urease produced by soil purified diets containing various carbo bacteria m ay be inhibited by various cyclic hydrates, and to study the effects of these urea compounds (9, 10). Barbituric acid, additives on gastrointestinal ammonia con a suggested anti-metabolite of uracil (1 1 ), centration and urease activity. An attempt which is similar in chemical structure to was also made to determine the relation the above compounds, has also been shown ship between growth response and ureo to depress urease activity in vitro.3 More lytic activity in the small and large intes recent work with chicks fed a sucrose- tin e. casein diet indicates that barbituric acid EXPERIMENTAL will enhance growth concurrently with a One-day-old Van tress-Arbor acre cock reduction in ammonia concentration and erels having an initial weight of 35 to 45 g urease activity in the gastrointestinal tract were used in all experiments. A 28-day ( 1 2 ). growth period was used as described previ It has been shown that different carbo ously (12). In any set of experiments 3 hydrates may alter the requirements for carbohydrates, sucrose, dextrose or starch, other nutrients (1 3 ) and that these differ ences in requirements are accompanied by Received for publication July 29, 1963. 1 Supported in part by Atomic Energy Commission changes in the number and type of flora Contract AT (11-1) 670, and the Herman Frasch inhabiting the gastrointestinal tract (14). Foundation, New York. 2 Public Health Service Postdoctoral Fellow. Changes in gastrointestinal flora have also 3 Visek, W. J., A. I. Jacobson, M. E. Iwert and been reported following addition of anti A. P. Alvares 1961 Depression of urease activity in the gastrointestinal tract by chemical agents. Fed biotics to the diet although these changes eration Proc., 20: 370 (abstract). J. N utrition, 82; ’64 93 94 A. P. ALVARES, L. H. HARBERS AND W. J. VISEK were studied simultaneously. In all studies, of chicks fed diets containing sucrose, dex experimental diets had the same composi trose or starch with and without barbi tion as the basal diet ( diet 1, table 1 ), ex turic acid (1.28 g/kg of diet). These were cept for additions in grams per kilogram followed by 2 successive experiments of as follows: diet 2, barbituric acid 1.28; 24 lots each (table 2). diet 3, Chlortetracycline 0.1; and diet 4, In the experiments using 24 lots, the barbituric acid 1.28 plus Chlortetracycline chicks were fasted for 4 hours prior to kill 0.1. There were several preliminary ex ing at the end of the 28-day experimental periments ( table 1 ) with a total of 52 lots period. Ten randomly selected chicks from TABLE 1 Summary of preliminary investigations with cockerels fed various carbohydrates and supplemented with barbituric acid for 4 weeks Weight gain Diet 1 0-2 Weeks 0-4 Weeks 0-4 Weeks 9 9 g/ g feed 1 Sucrose (12 ) 2 75 ± 2 3 239 ± 7 0.51 ±0.02 2 Sucrose + barbituric acid (1 2 ) 82 ± 2 250 ± 7 0.56 ±0.01 1 Dextrose (8) 86 ± 5 236 ± 9 0.47 ±0.03 2 Dextrose-(-barbituric acid (8 ) 88 ± 5 240 ± 7 0.48 ±0.03 1 Starch (7 ) 85 ± 5 257 ± 13 0.54 ±0.01 2 Starch + barbituric acid (5 ) 8 9 ± 4 257 ± 1 8 0.56 ±0.04 1 Diet 1, basal: casein (Nutritional Biochemicals Corp., Cleveland), 20; carbohydrate, 61.6; gelatin (Wilson and Co., Chicago), 8; bone ash (Wilson and Co., Chicago), 2; calcium gluconate (Gazzolo Drug and Chemical Co., Chicago), 5; salt mixture, 1.4; choline mixture in casein, 1; and vitamin mixture (17) in casein, 1. Diet 2: basal plus barbituric acid, 1.28 g/kg. The salt mixture contained: (mg/100 g diet) NaCl, 420; K2HPO4, 420; KH2PO4, 315; MgSC>4, 175; MnS0 4 -H2 0 , 28; ferric citrate, 35; CUSO4 5 H2O, 1.4; KI, 0.42; Zn acetate-2H20, 1.1725; Al2 (S 0 4 ) 3 - I 8 H2O, 1.12; C0 CI2 6 H2O, 0.2675; NiS04*6H20, 0.31; KBr, 0.56; sodium molybdate, 0.035. 2 Figures in parentheses indicate number of lots, with 15 birds/lot. 3 Mean + se. TABLE 2 W eight gain of cockerels fed basal diets containing various carbohydrates and supplemented w ith either barbituric acid, Chlortetracycline, or both1>2 Weight gain Diet 3 0—2 Weeks 2—4 Weeks 0-4 Weeks 9 9 9 Sucrose 1 Basal 9 5 ± 7 4 170± 19 265 ± 1 4 2 Barbituric acid 116± 14 5 181 ± 9 297 ± 9 3 Chlortetracycline 1 2 0 ± 5 6 1 9 9±3 7 319 ± 8 6 4 Barbituric acid + Chlortetracycline 123 ± 5 5*6 193 ± 6 7 3 1 6 ± 9 6 Dextrose 1 Basal 123 ± 11 190 ± 7 313 ± 18 2 Barbituric acid 123 ± 9 199 ± 4 322 ± 11 3 Chlortetracycline 131 ± 1 3 7 211 ± 20 342 ±3 3 4 Barbituric acid + Chlortetracycline 133 ± 7 7 201 ± 1 2 334 ±1 9 Starch 1 Basal 117± 2 213 ± 6 330 ± 7 2 Barbituric acid 1 1 8±4 208 ± 5 326 ± 5 3 Chlortetracycline 121 ± 3 7 214 ± 10 335 ± 8 4 Barbituric acid + Chlortetracycline 130 ± 3 7 203 ± 8 333 ± 8 1 Data are averages of 2 successive experiments with 24 lots. 2 Four lots/treatment, with 15 birds/lot. 3 Diet 2, basal plus barbituric acid (Eastman Organic Chemicals, Rochester, N.Y.), 1.28 g/kg. Diet 3, basal plus Chlortetracycline (Lederle Laboratories, Pearl River, N.Y.), 0.1 g/kg. Diet 4, basal plus barbituric acid, 1.28 g/kg, plus Chlortetracycline, 0.1 g/kg. 4 Mean ± se. 5 Barbituric acid vs. no barbituric acid significant at P < 0.05. 6 Chlortetracycline vs. no Chlortetracycline significant at P < 0.01. 7 Chlortetracycline vs. no Chlortetracycline significant at P < 0.05. CARBOHYDRATES AND INTESTINAL UREASE ACTIVITY 95 each lot were killed. The small and large acid enhanced weight gain and feed effi intestines plus contents were rapidly re ciency with a sucrose diet, whereas little moved, immediately frozen in dry ice and or no effect was observed with either dex stored at — 10°C. Prior to analyses, the trose or starch (table 1). intestines from each lot were thawed at Combined growth data from the subse room temperature. The small and large in quent experiments are shown in table 2. testines were separated and pooled into During the first 2 weeks the addition of respective groups and weighed. The speci barbituric acid to the basal diet containing mens were homogenized with 0.9% NaCl sucrose increased weight gains by 11% at 4°C in a W aring Blendor for 3 minutes. (P<0.05), whereas no significant in The total homogenates of the small and creases were noted with this agent when large intestines were diluted to 400 and added to diets containing dextrose or 300 ml, respectively. Am monia production starch. During this same period chlortetra- and urease activity were estimated as de cycline added to the sucrose diet increased scribed previously (12). Modifications em growth by 15% (P < 0.01), whereas when ployed were the use of 3-ml homogenate added to the dextrose or starch diets it in aliquots and determination of liberated creased growth by 7% (P < 0.05). Weight ammonia with a Van Slyke-Cullen urea gains during the 2- to 4-week period fol apparatus using 5 m l saturated K2C03 solu lowed those of the first 2-week period but tion with an aeration time of 30 minutes. the effects of the additives were less appar All of the incubations for ammonia pro ent. For the overall experimental period of duction and ureolytic activity were com 4 weeks the difference in growth was great pleted within 0.5 hour after the intestines est with the diet containing sucrose and were thawed. The data from each carbo least with that containing starch. The hydrate experiment were subjected to 2- main effect of barbituric acid with the way analysis of variance using orthogonal sucrose diet was an increase in growth of comparisons as described by Snedecor (18). 4% , whereas that of chlortetracycline was 13% (P<0.01). When barbituric acid RESULTS was the only additive (diet 2), growth was Data from the preliminary experiments improved by an average of 12% over the involving 52 lots suggested that barbituric basal diet (diet 1) but this difference was TABLE 3 Efficiency of feed utilization of cockerels fed diets containing various carbohydrates and supplemented w ith either barbituric acid or chlortetracycline , o r b o t h Weight gain Diet 0—2 Weeks 2-1 Weeks 0—4 Weeks g/ g feed g !g feed g/ g feed Sucrose i Basal 0.51 ±0 .0 2 1 0.51 ±0.05 0.52± 0.04 2 Barbituric acid 0.63 ±0 .0 3 2 0.52 ±0.03 0.55 ±0.01 4 3 Chlortetracycline 0.61 ±0 .0 2 3 0.53 ±0.02 0.55 ±0 .0 2 5 4 Barbituric acid + chlortetracycline 0.67 ± 0.01 2-3 0.55 ± 0.01 0.59 ±0 .0 2 4'5 Dextrose 1 Basal 0.67 ± 0.01 0.54 ±0.01 0.58 ± 0.01 2 Barbituric acid 0.66 ±0 .0 2 0.55 ±0.01 0.59 ± 0.01 3 Chlortetracycline 0.65 ±0.03 0.56 ±0.02 0.60 ±0 .0 2 4 Barbituric acid + chlortetracycline 0.67 ± 0.01 0.54 ±0.01 0.59 ±0.01 Starch 1 Basal 0.63±0.01 0.57 ± 0.01 0.58 ±0.01 2 Barbituric acid 0.63 ±0.01 0.56 ±0.01 0.58±0.01 3 Chlortetracycline 0.65±0.01 3 0.57 ± 0.01 0.60 ±0.01 4 Barbituric acid + Chlortetracycline 0.65 ±0.01 3 0.55 ±0.01 0.59 ±0.01 1 Mean ± se. 2 Barbituric acid vs. no barbituric acid significant at P < 0.01. 3 Chlortetracycline vs. no chlortetracycline significant at P < 0.01. 4 Barbituric acid vs. no barbituric acid significant at P < 0.06. 5 Chlortetracycline vs. no chlortetracycline significant at P < 0.06. 9 6 A. P. ALVARES, L. H. HARBERS AND W. J. VISEK not statistically significant. Chlortetra grams per gram wet weight, are shown in cycline alone (diet 3) showed an increase table 4. The ammonia concentration and of 20% (P < 0 .0 2 ). The combined effects the urease activity were consistently higher of the agents on growth were not additive, in the large intestine than in the small there being only 19% more growth than intestine. In the group of chicks fed the control with the two in combination. In sucrose diet, the addition of barbituric acid the dextrose group, Chlortetracycline in alone resulted in a depression of ammonia creased body weight gain by 6% which concentration in the small and large intes was not statistically significant, whereas tine by 18 and 13%, respectively, when barbituric acid treatment showed no effect. compared with the basal group. There was For the starch group, neither barbituric a decrease of 10% in the small intestines acid nor Chlortetracycline affected gains in of chicks fed chlortetracycline. In the dex body weight. trose group, addition of barbituric acid re The data on efficiency of feed utilization, sulted in a significant (P < 0.01) depres obtained by dividing the total weight gain sion of ammonia in the large intestine. per lot by the total feed consumed, axe pre Barbituric acid alone depressed ammonia sented in table 3. In general, efficiency of concentration in chicks fed the starch basal feed utilization paralleled that of growth. diet, in both the small and large intestine. Chicks fed starch diets showed the highest The differences in urease activity of the efficiency and those fed sucrose the lowest. gastrointestinal tracts plus contents of the Both additives influenced utilization of su chicks fed various carbohydrate basal diets crose throughout the experiment, whereas were greatest in the small intestine. This the effects with starch were manifested activity was highest with the sucrose diet during the first 2 weeks. No effect was and lowest with the starch diet. Urease noted with dextrose. activity in the small intestine was lowered Gastrointestinal ammonia concentration by barbituric acid or chlortetracycline and ureolytic activity, expressed in micro either alone or in combination with the TABLE 4 Am monia concentration and hydrolysis of urea by gastrointestinal contents of 4-week-old cockerels fed diets containing various carbohydrates and supplemented with barbituric acid or chlortetracycline, or both 1 n h 3 Urea split Diet Small Large Small Large intestine intestine intestine intestine ng/g wet wt M l9 wet let tig/g wet wt ¡xg/g w e t w t Sucrose 1 Basal 179 + 1 6 2 450 ±9 0 121 ± 18 844 ± 7 1 2 Barbituric acid 152 ± 16 398 ± 72 77 ± 12 3 769+137 3 Chlortetracycline 162 ± 13 445 ± 4 7 87 ± 25 684 ± 93 4 4 Barbituric acid-t- chlortetracycline 188 ± 9 465 ± 26 6 2 ± 20 3 557 ±2 0 4 Dextrose 1 Basal 171 ± 13 446 ± 23 107 ± 18 568 + 88 2 Barbituric acid 175± 13 316 ± 2 1 3 94 ±1 8 709 + 78 3 3 Chlortetracycline 187 ± 7 537 ± 9 66 ± 8 542 ±5 6 4 Barbituric acid + chlortetracycline 172± 12 373 ± 3 7 5 8 5 ±4 0 762 ± 86 3 Starch 1 Basal 205 ± 20 497± 77 59 ± 7 737+132 2 Barbituric acid 186± 16 409 ± 76 66 ±2 3 495 + 84 3 Chlortetracycline 193 ± 2 6 452 ±6 8 67 ± 16 601 ±1 38 4 Barbituric acid + chlortetracycline 195 ± 2 5 524 ±6 7 85 ±2 0 669 ±1 35 1 Ten birds/lot; 4 lots/treatment. 2 Mean ± s e . 2 Barbituric acid vs. no barbituric acid significant at P < 0.05. 4 Chlortetracycline vs. no chlortetracycline significant at P < 0.05. 5 Barbituric acid vs. no barbituric acid significant at P < 0.01. CARBOHYDRATES AND INTESTINAL UREASE ACTIVITY 9 7 TABLE 5 Correlation coefficients of growth upon ureolytic activity in the large and small intestines of chicks Between Within Source Simple treatments treatments Overall Small intestine -0 .3 4 5 1 -0 .7 2 0 2 + 0.325 Large intestine -0 .2 8 2 1 -0 .5 2 4 -0 .1 9 3 Sucrose Small intestine -0 .4 8 1 2 — 0.85C -0 .1 9 2 Large intestine -0 .4 0 4 -0 .90 3 -0 .1 8 5 Dextrose Small intestine -0 .40 1 -0 .9 5 0 1 + 0.460 Large intestine -0 .3 0 8 + 0.116 -0 .4 5 2 Starch Small intestine + 0.143 + 0.260 + 0.136 Large intestine + 0.054 + 0.190 + 0.045 1 Significant at P < 0.05. 2 Significant at P < 0.06. sucrose and dextrose diets. Addition of as that containing sucrose produced the these agents to the starch diet, however, least. The sucrose diet supplemented with resulted in a slight increase in ureolytic Chlortetracycline led to greater differences activity. The urease activity of the large in growth and efficiency of feed utilization intestine of the chicks fed sucrose was de in chicks than when it was added to a creased 1 5 % relative to control with the starch diet. These results parallel those addition of barbituric acid and 3 0 % with obtained by Stokstad et al. (17). Addition Chlortetracycline (P < 0.05). In the dex of barbituric acid to a sucrose diet resulted trose group, addition of barbituric acid in growth differences which were greatest resulted in elevation of urease activity, during the first 2 weeks of feeding. Al whereas Chlortetracycline showed no ef though barbituric acid improved growth fects. With the starch diet, addition of bar concurrently with suppression of ureolytic bituric acid suppressed the ureolytic ac activity, there was no additive effect with tivity by 1 5 % , whereas Chlortetracycline Chlortetracycline, suggesting that these failed to show any effects. exert their effect on the bacterial flora Correlation coefficients for 4 - week through a common pathway. Barbituric growth and gastrointestinal ureolytic activ acid, when added to the sucrose diet, im ity at the time of death were determined proved efficiency of feed utilization with overall and with respect to carbohydrate depression of ammonia concentration and source (table 5). Simple correlations of ureolytic activity of the gastrointestinal gain with ureolytic activity in the small tract. These results are similar to those and large intestines were negative except obtained previously in this laboratory (12). for the starch group. Significant negative Chlortetracycline and barbituric acid correlations were found between gain and were not as effective when added to the ureolytic activity in both segments of the dextrose diet as when added to a sucrose gastrointestinal tract over all treatments diet, although the increased gains with the (P < 0.05) and in the small intestine (P < additives were accompanied by a depres 0.06) for the sucrose group. Between treat sion of the ureolytic activity in the small ment analyses followed that of the simple intestine. W ith the starch diet, the addi correlations, whereas the within treat tives did not increase the gain in weight ments correlations gave no definite pat of the chicks and there was no significant tern. depression of ureolytic activity in the gas trointestinal tract. DISCUSSION The correlation analyses support the The basal diet containing starch pro hypothesis that when the addition of Chlor duced the greatest growth in chicks, where tetracycline or barbituric acid will enhance 98 A. P. ALVARES, L. H. HARBERS AND W. J. VISEK growth, there is a concurrent depression of National Academy Sciences — National Re search Council, Washington, D. C., p. 84. ureolytic activity. The data show that the 4. Dintzis, R. Z., and A. B. Hastings 1953 type of carbohydrate in the diet influences The effect of antibiotics on urea breakdown the response of chicks to barbituric acid. in mice. Proc. Nat. Acad. Sci., 3 9 : 571. Various carbohydrates have been shown to 5. Sherlock, S. 1958 Pathogenesis and man agement of hepatic coma. Am. J. Med., 24: alter the intestinal flora (1 4 ) and the pos 805. sibility exists that urease-producing bac 6. Silen, W., H. A. Harper, D. L. Mawdsley and teria may be suppressed leading to a W. L. Weirich 1955 Effect of antibacterial decrease in production of various entero- agents on ammonia production within the toxins including ammonia. Analysis of intestine. Proc. Soc. Exp. Biol. Med., 8 8 : 138. 7. Dang, H. C., and W. J. Visek 1960 Effect data within treatment groups, however, of urease injection on body weights of grow does not suggest that lowest ureolytic activ ing rats and chicks. Proc. Soc. Exp. Biol. ity is associated with greatest weight gain. Med., 105: 164. Although the amount of growth varies 8. Harbers, L. H., R. F. Hendrickson, H. N. Bass and W. J. Visek 1963 Effect of urease im with chlortetracycline and barbituric acid, munity upon rats with controlled vitamin A their overall effects on growth, ammonia intakes. Proc. Soc. Exp. Biol. Med., 1 1 3 : 420. concentration and urease activity in the 9. Gray, C. T., J. C. Gerhart and M. S. Brooke gastrointestinal tract are similar. The evi 1959 Inhibition of urease by alloxan and alloxanic acid. Nature (London), 1 8 4 : 1936. dence brought forth in this paper demon 10. Gray, C. T., M. S. Brooke and J. C. Gerhart strates that the mode of action of chlor- 1961 Metabolism of alloxanic acid in a soil tetracycline, an antimicrobial agent, and microorganism. J. Bacteriol., 8 1 : 755. of barbituric acid, a cyclic urea derivative, 11. Woods, D. D. 1942 The mode o f action of chemotherapeutic agents. Biochem. J., 36: 3. when fed to growing chicks, is dependent 12. Harbers, L. H., A. P. Alvares, A. I. Jacobson on the carbohydrate source. The data also and W. J. Visek 1963 Effect of barbituric suggest that under certain dietary regi acid and chlortetracycline upon growth, am mens increased growth of chicks may be monia concentration and urease activity in associated with depressed urease activity the gastrointestinal tract of chicks. J. Nutri tion, 80: 75. of the gastrointestinal tract. 13. Harper, A. E., and C. A. Elvehjem 1957 A review of the effects of different carbohy ACKNOWLEDGMENTS drates on vitamin and amino acid require The authors acknowledge the assistance ments. J. Agr. Food Chem., 5: 754. of the American Meat Institute Founda 14. Gyorgy, P. 1961 Recent advances in hu man nutrition. Little, Brown and Company, tion, Chicago, Illinois, for use of facilities. Cambridge, Massachusetts, chap. 22. 15. Jukes, T. H. 1957 Antibiotics in nutrition. LITERATURE CITED Medical Encyclopedia Inc., New York, p. 43. 1. Visek, W. J., J. M. Baron and D. M. Switz 16. Barnes, R. H., E. Kwong, K. Delany and G. 1959 Urea metabolism and intestinal ureo Fiala 1960 The mechanism of the thia lytic activity of rats fed antimicrobial agents. mine-sparing effect of penicillin in rats. J. J. Pharm. Therap., 1 2 6 : 359. Nutrition, 71: 149. 2. Francois, A. C., and M. Michel 1955 Ac 17. Stokstad, E. L. R „ T. H. Jukes and W. L. tion de la penicillin et de Taureomycine sur Williams 1953 The growth promoting ef les propriétés de la flore intestinale du porc. fects o f aureomycin on various types of diets. C. R. Acad. Sci., 2 4 0 : 1 2 4. Poult. Sci., 32: 1054. 3. Francois, A. C. 1956 First International 18. Snedecor, G. W. 1956 Statistical Methods Conference on Antibiotics in Agriculture, Iowa State College Press, Ames, p. 237. Absorption of Preformed Vitamin A from Ligatured Poultry Intestinal Sections* 1'2 T. E. SHELLENBERGER,3 D. B. PARRISH a n d P. E. SANFORD Kansas State University, Manhattan, Kansas ABSTRACT Absorption of aqueous dispersions of vitamin A was studied, using ligatured intestinal sections as modified in vivo systems. After injecting vitamin A acetate into duodenum, posterior small intestine, or cecum, the vitamin A content of duodenal and intestinal tissues was found to be similar, but that in blood serum was 50% higher after duodenal injections; little vitamin A was absorbed from the cecum. Absorption was better in younger and egg-strain birds than in older and broiler-strain birds. Vitamin A acetate was absorbed faster from the duodenum than from the small intestine, and also resulted in vitamin A increasing to higher levels in blood serum and liver. Vitamin A acetate was absorbed faster than the palmitate and led to higher blood serum and liver vitamin A levels. About 90% of the vitamin A in intestinal tissues was in the ester form, mainly higher fatty acid types, and about 65% of that in blood serum was in the alcohol form, whether vitamin A acetate, palmitate, or alcohol was injected. When serum vitamin A levels reached 11 ,ug/100 ml from single injections of vitamin A, small, but measurable, quantities of vitamin A were observed in liver. Results of simultaneous injections of ethoxyquin and vita- min A varied. Mechanisms of absorption are discussed. Vitamin A is more readily absorbed from and vitamin and manganese sulfate pre the intestinal tract when administered as mixes in wheat shorts, 2.35. Except for aqueous dispersions or in milk emulsions vitamin A, this diet was adequate for nor than as solutions in oil (1 -4). There is, mal growth and health. Chicks were raised however, apparently little published infor in electrically heated, wire-floor batteries mation on absorption of different forms of in rooms at 24 to 28°. Feed and water vitamin A by poultry (5 ), and none on were given ad libitum. absorption from ligatured intestinal sec When 5 to 10 weeks of age, chicks were tions. fed the basal diet, free of vitamin A, for 7 This study was made to compare absorp to 10 days to deplete the small pre-experi- tion of aqueous dispersions of vitamin A mental reserves. Preliminary results indi alcohol, acetate, and palmitate from dif cated that blood serum vitam in A was less ferent ligatured intestinal sections of poul than 5 pg/100 ml and liver vitam in A less try and to compare their effects on intes than 3 ug/liver after depletion. tinal and blood serum vitamin A alcohol Isolation of intestinal sections. V ita m in and ester levels and ratios. The effect of A-depleted chicks were anesthetized lightly an antioxidant, ethoxyquin, on the absorp with ether. A lateral incision was made in tion of vitamin A acetate also was investi the abdominal wall so that sections of in gated. Ligatured intestinal sections were testine could be manipulated. Ligatures used as modified in vivo systems. were applied to duodenal sections, poste rior to the ventriculus and anterior to the EXPERIMENTAL entrance of the bile and pancreatic ducts, Experimental animals. Chicks used being careful to avoid damage to the pan- were obtained from local hatcheries, or Received for publication August 1, 1963. were hatched from eggs laid by hens re 1 Contribution no. 41, Department of Biochemistry, ceiving a carotenoid-free diet.4 Chicks were and no. 279, Department of Poultry Science, Kansas Agricultural Experiment Station, Manhattan, Kansas. fed a diet containing 880 IU vitam in A/kg, 2 Portion of a dissertation presented by the senior composed of carotenoid-free ingredients as author as partial fulfillment of requirements for the Ph.D. degree. For a more extensive treatment and follows: (in per cent) white corn, 58.5; data not presented see University Microfilms, Inc., Ann Arbor, Michigan, no. 61-1002. soybean oil meal (44% ), 31; nonfat dry 3 Present address: Stanford Research Institute, milk, 2; brewer’s yeast, 2; bone meal, 2; Menlo Park, California. 4 Poultry Science Department, Kansas State Univer CaCOs, 1; NaCl, 0.5; cottonseed oil, 0.65; sity. J. N u t r it io n , 82: ’64 99 100 T. E. SHELLENBERGER, D. B. PARRISH AND P. E. SANFORD créas. Posterior small intestine segments5 RESULTS were isolated by applying one ligature pos Values for vitamin A content of intes terior to the yolk stalk and another ante tine, blood serum, and liver — 4 hours rior to the blind end of the ceca, being after injection of vitamin A acetate into careful to avoid rupture of blood vessels. intestinal sections — are summarized in The cecum was ligatured as far from the table 1. Although vitamin A levels of the blind end as possible. Preparations of duodenal and small intestine tissues were vitamin A were injected into the ligatured similar, average blood serum and liver sections using a hypodermic syringe. Liga vitamin A content was more than 50% tured sections were replaced in the abdom larger after vitam in A acetate was injected inal cavity and the incision sutured. Birds into the duodenal sections. After the ace were killed at times after injection indi tate was injected into ligatured cecal sec cated in tables 1-4, and samples were tions, only a small quantity of vitamin A taken for vitamin A and carotenoid deter was recovered from the tissues (table 1), minations. Sections were washed free of blood serum vitamin A was much lower lumen contents at collection time. than following duodenal or intestinal in Preparation of vitam in A dispersions. jections, and there was no increase of vita Aqueous dispersions of vitam in A acetate, min A in livers. Thus, ligatured duodenal, palmitate, and alcohol6 were prepared prior posterior small intestinal, and cecal sec to each trial by dissolving the vitam in A in tions differed markedly in absorption of 10 ml of ether, adding 25 ml of 20% poly aqueous dispersions of vitamin A acetate. oxyethylene sorbitan monooleate (Tween Studies were undertaken to test effect of 40)7 containing a few drops of 95% eth age and type of chick on absorption of anol, and removing the volatile solvents vitamin A acetate from duodenal and pos under vacuum at 50°. terior small intestinal sections. Compari Analytical m ethods. Analyses for total sons were made at 5, 7 and 9 weeks of age, vitamin A in tissue, liver, and intestine with both broiler strain and egg strain were made by a modification of the pro chicks given 740 to 760 units vitamin A/ cedure of Neff et al. (6 ),8 utilizing the bird. Blood serum vitamin A content was Carr-Price procedure with reagents pre higher in younger and in egg-strain birds,9 pared as described by the AOAC (7). mainly, no doubt, reflecting size of bird in Blood serum vitamin A was determined by 5 The segment between yolk stock and ceca, approxi the Kimble method ( 8 ). Vitam in A alcohol mately the anterior one-half of the ileum. and esters were separated by a modifica 6 Vitamin A acetate was USP vitamin A reference solution. Vitamin A palmitate was 1 million IU/g tion of the chromatographic procedure of synthetic product in com oil, Chas. Pfizer and Com- any. Vitamin A alcohol was a crystalline product, Parrish et al. (9 ). Vitamin A esters were Sistillation Products Industries, Inc., Rochester, New resolved into low and high molecular York. 7 Atlas Powder Company, Wilmington, Delaware. weight fatty acid components by the pro 8 Peroxide-free ether, and redistilled petroleum sol vents were used in extractions. cedure of Kaiser and Kagan (10). 9 See footnote 2. TABLE 1 Absorption of vitam in A acetate from ligatured duodenal and posterior small intestine sections Vitamin A content Site of No. of injection experimental Intestinal tissue Blood serum Liver birds Avg Range Avg Range Avg Range 1'ig/tissue fig / 100 m l fig / liv e r None 1 4 i 2 2 2 » Duodenum 7 * 44 27-55 22 12-29 14 5-17 Posterior small intestine 7 * 48 35-57 14 10-16 8 5-11 Cecum 12 ‘ 5 6 2 1 Received 0.5 ml carrier only. 2 Cecum tissue. 3 Only 2 values in this average. received 600 or 620 USP units preformed Yitamin A acetate in 1 ml 20% Tween 40. AH birds killed 4 hours after injection of vitamin A. 5 385 or 500 USP units vitamin A acetate in 0.5 ml 20% Tween 40; liver data only from birds injected with higher level vitamin A. ABSORPTION OF VITAMIN A 101 relation to quantity of vitamin A injected. samples. Thus aqueous dispersions of Differences in duodenal and posterior small vitamin A acetate injected into ligatured intestinal absorptions were, in general, as duodenal sections were absorbed into the illustrated by table 1. blood and deposited in the liver faster, and To determine the time required for vita reached higher levels within 2 to 3 hours min A to appear in intestinal tissue, blood than that injected into ligatured posterior serum, and liver, samples were taken for small intestine. analyses at definite intervals following in Comparisons were made also of the rela jection of vitamin A acetate into ligatured tive duodenal absorptions of vitamin A duodenal sections (table 2). Considerable palmitate and acetate as dispersions with vitam in A was observed in duodenal tissue Tween 40. After acetate was injected, within 0.25 hour, increased to about 70 duodenal vitamin A content was more ug/tissue at 0.5 hour, then decreased ir than twice that after palmitate was in regularly. Blood serum vitamin A was jected (table 3). Blood serum vitamin A 9 ng/100 ml within 0.5 hour after duo was 3 times higher in the acetate-treated denal injection, and increased further dur than in the palmitate-treated birds, and ing the next 3 to 4 hours. Liver vitamin A liver vitamin A content was 5 times increases lagged only slightly, being 8 ng greater in the former. 0.75 hour after injection, and increasing To study the role of esterification and substantially in the next 1 to 3 hours. hydrolysis during absorption and the effect Vitam in A was observed in ligatured pos on the state of vitam in A in blood, analyses terior small intestinal tissue also within were made of the vitamin A ester and 0.25 hour after vitamin A acetate was in alcohol content and ratios in intestinal jected, and it increased further in 2 hours tissues and blood serum following injec (table 2). Blood serum vitamin A con tions of aqueous dispersions of vitamin A tent increased slowly, but within 2 hours acetate, palmitate, and alcohol into liga it was some 10 times that of those in the tured duodenal sections. Four hours after initial samples. Vitamin A deposition in injections, birds were killed and samples creased even more slowly in the livers; 2 saved for chromatographic resolution of hours after injection, liver vitamin A was vitam in A alcohcl and esters. Blood sera of only 4 times greater than that in the initial the 6 chicks in each lot were pooled to give TABLE 2 Absorption and deposition of vitam in A in tissues at various time intervals following injection of vitam in A acetate into ligatured duodenal and posterior small intestinal (P.S.I.) s e c tio n s Average vitamin A content Time Duodenal Blood Liver P.S.I. Blood tissue serum tissue serum Liver h o u r 8 fig / tissu e fig/100 m l jig / liv e r jig /tissue jig / 100 m l jig / liv e r 0 2 ( 2 )> 0 2 (2 ) 1 (2 ) 2 ( 6 ) 1 (5 ) 1 (6 ) 0.25 58 (2 ) i (2 ) 1 (2 ) 25 (2 ) 1 (1 ) 1 (2 ) 0.5 73 (3 ) 9 (3 ) 3 ( 3 ) 4 2 (5 ) 2 ( 4 ) 2 ( 5 ) 0.75 71 (2 ) 13 (2 ) 8 (2 ) 52 (2 ) 3 (2 ) 2 ( 2 ) 1 71 (3 ) 21 (3 ) 13 (3 ) 54 (5 ) 4 (4 ) 2 ( 5 ) 1.25 58 (2 ) 22 (2 ) 16 (2 ) 43 (4 ) 7 (3 ) 2 ( 4 ) 1.5 74 (3 ) 28 (3 ) 18 (3 ) 64 (6 ) 9 (5 ) 2 (6 ) 1.75 52 (2 ) 28 (2 ) 18 (2 ) 51 (4 ) 1 0 (3 ) 2 ( 4 ) 2 74 (1 ) 37 (1 ) 1 7 (1 ) 58 (4 ) 11 (3 ) 4 (4 ) 2.25 72 (2 ) 14 (2 ) 5 (2 ) 2.5 42 (1 ) 39 (1 ) 2 0 (1 ) 70 (3 ) 16 (2 ) 9 (3 ) 2.75 47 (1 ) 17 (1 ) 14 (1 ) 3 60 (1 ) 43 (1 ) 25 (1 ) 69 (2 ) 23 (1 ) 9 ( 2 ) 3.5 45 (1 ) 49 (1 ) 20 (1 ) 3 7 (1 ) 1 4 (1 ) 4 51 (1 ) 47 (1 ) 20 (1 ) 'Number of birds from which samples were taken. In duodenal studies, each received 700 to 710 USP units vitamin A acetate in 1 ml 20% Tween 40, and in posterior small intestinal studies, 700 to 740 USP units. 3 No vitamin A detected. 102 T. E. SHELLENBERGER, D. B. PARRISH AND P. E. SANFORD TABLE 3 Absorption of preformed vitamin A palmitate and acetate from ligatured duodenal sections 1 Vitamin A levels No. of Vitamin A birds form Duodenal Blood administered tissue serum Liver fig /tissue fig/100 m l fig / liv e r 4 2 None i 3 i 6 3 Palmitate 25 18 4 6 4 Acetate 58 55 20 1 Birds killed for samples 4 hours after vitamin A injections. 2 Negative controls. Received carrier only. 3 Received 680 or 700 USP units vitamin A palmitate in 1 ml 20% Tween 40. 4 Received 680 or 700 USP units vitamin A acetate in 1 ml 20% Tween 40. sufficient vitamin A for chromatography. lower. Most of the vitamin A ester in both Results (table 4) indicated that about tissue and serum was recovered as higher 90% 10 of the vitam in A injected as acetate molecular weight types. was recovered from the duodenal tissue as Regardless of the form of vitamin A ester. Both acetate and higher fatty acid injected, 85 to 90% of the vitamin A re esters of vitamin A were detected by chro covered in the doudenal mucosa and walls matography, indicating at least partial hy was the ester. In blood serum, some 30 to drolysis and re-esterification. Blood serum 35% of the vitamin A recovered after in vitamin A was about 65% alcohol and jecting vitamin A acetate or alcohol was 35% ester; only higher molecular weight the ester (mostly of higher fatty acid fatty acid esters were detected. types), whereas only vitam in A alcohol was Although even more vitam in A palmitate detected in serum after injecting vitamin than acetate was injected, there was less A palmitate. vitamin A in duodenal tissue (table 4 ), in Different quantities of vitam in A acetate dicating that the palmitate was not so were injected into ligatured duodenal sec readily absorbed. About 85% of the duo tions to study the relation of blood serum denal vitamin A was the ester and repre vitamin A levels to liver storage. Blood se sented by higher molecular weight types, rum and liver samples were analyzed 4 similar to trials with acetate. Vitamin A hours after the injections. When birds alcohol, but no ester, was detected in the were injected with 355 USP units vitamin blood serum. 10 Vitamin A ester and alcohol content was based Results with injections of vitamin A on quantities recovered by chromatography. Total alcohol generally agreed with those ob recoveries of vitamin A by column chromatography ranged from 83 to 108% of that determined prior to tained by injecting acetate (table 4 ); how chromatography. Percentage recoveries are m foot notes to table 4 when recoveries were not practically ever, total duodenal vitam in A content was 100%. TABLE 4 Tissue vitamin A ester and alcohol levels 4 to 4.5 hours after injection of various forms vitam in A into ligatured duodenal sections Duodenal tissue Blood serum 1 Form No. birds vitamin A injected injected Alcohol Ester Total % Alcohol Ester % ester Total ester /ig /tissue fig/100 m l Vitamin A acetate 2 6 5 49 54 91 24 14 38 37 Vitamin A palmitate 3 6 2 12 14 4 86 12 0 s 12« 0 Vitamin A alcohol7 6 3 21 24 8 88 26 10 36 9 28 1 Pooled serum sample. 2 Birds received 760 USP umts vitamin A in 1 ml 20% Tween 40. 3 Birds received 1025 USP units vitamin A in 1 ml 20% Tween 40. 4 Recovery of 108% , based on values before chromatography, s No apparent vitamin A ester recovered. 6 Recovery of 83%, based on values before chromatography. 7 Birds received 700 IU vitamin A in 1 ml 20% Tween 40. 8 Recovery of 103% , based on values before chromatography. 9 Recovery of 95% , based on values before chromatography. ABSORPTION OF VITAMIN A 103 A, serum vitamin A content was 11 ug/ ence is explainable, in part, in terms of 100 ml and liver vitam in A was 5 ug/liver. absorptive surfaces in the 2 sections. Al Substantial liver vitam in A storage (11 ug) though the posterior small intestinal sec was observed in birds injected with 670 tion was almost twice as long as the duo USP units vitam in A, where a blood serum denal section, villi are longer and more vitamin A content of 20 ug/100 ml was numerous per area in the duodenum (15). attained.11 The vitamin A content of duodenal tis In similar studies of injections into liga sue was 2 to 4 times higher after injection tured posterior small intestine sections, of vitam in A acetate than after injection of birds given 412 USP units vitamin A aver the palmitate ( tables 3 and 4). Most of aged 11 ug/vitamin A /100 ml of blood se the vitamin A recovered was in the higher rum, but liver vitamin A content was not molecular ester form; the remainder was substantially higher than that of controls. largely vitam in A alcohol. Eden and Sellers With 615 USP units vitamins A, serum (16) maintained that vitamin A esters vitamin A levels averaged 14 ug/100 ml were hydrolyzed and re-esterified during and liver, 10 ug/liver. absorption instead of being absorbed in Data on relation of blood serum vitamin the unaltered form. Similar conclusions A levels and liver vitamin A storage also were reached by Murthy et al. (1 7 ). Krish- are shown in table 2. Birds had a serum namurthy et al. (1 8 ) reported some hydrol vitamin A content of 9, 13 and 21 ug/ ysis of natural vitamin A esters by small 100 ml at 0.5, 0.75, and 1 hour, respec intestinal tissue. Mahadevan et al. (19) tively, after vitamin A was injected into observed that in vitro hydrolysis of vitam in ligatured duodenal sections, at which times A palmitate was activated by sodium tau- the liver vitam in A content was 3, 8 and 13 rocholate but inhibited by the presence of ug/liver, respectively. In general, moderate large amounts of Tween 20 and Tween 80. liver vitamin A storage occurred only after This effect alone, if also true in vivo, could serum vitamin A levels of some 12 to 15 account for the decreased absorption noted ug/100 ml were attained. with the vitamin A palmitate. Hydrolysis Effects of low concentrations of dietary of vitamin A esters by liver homogenates, antioxidants on vitamin A and carotene however, varies inversely with chain length utilization and on growth have been re of the fatty acid portion (20, 21 );13 this ported (11-14), but these effects probably may be true also in the intestine. are complicated by degree of protection Although during active absorption a pro given vitamin A during feed storage and portion of the free vitamin is present in while in the upper gastrointestinal tract mucosae and muscles of the small intes after ingestion. tine and in mesenteric lymph fluid (22), In a series of trials 335 or 715 USP units it is transported from the intestine prin vitamin A acetate dispersed in 20% Tween cipally as long-chain fatty acid esters and 40, with or without 0.03% ethoxyquin,12 by the lymphatics. Moore (2 ) concluded was injected into ligatured duodenal sec that the lymphatics are more important tions to determine the effect of the antioxi than the portal vein for absorption of vita dant, ethoxyquin, on absorption. Duodenal min A. However, Imbesi (4 ) suggested tissue, serum, and liver were analyzed for that vitamin A acetate was absorbed vitamin A 1.25 or 4 hours later. Although through both lymph and portal systems, there was an indication of decreased ab and Murray and Grice (23) observed that sorption at 1.25 hours, definite effects blocking the lymphatic system of rats at could not be discovered because of vari the thoracic duct did not interfere with ability within treatments. vitamin A absorption and suggested the portal system as an important alternative DISCUSSION route for absorption. Vitam in A acetate injected into duodenal 1 1 See footnote 2. sections appeared in the blood earlier, was 12 Santoquin, Monsanto Chemical Company, St. deposited in the liver faster, and reached Louis. 13 High, E. G., J. R. Powell and H. B. Bright 1957 higher levels than when injected into pos I n v iv o and in v itr o hydrolysis of vitamin A esters and esterification of vitamin A alcohol. Federation terior small intestine sections. This differ Proc., 16: 388 (abstract). 104 T. E. SHELLENBERGER, D. B. PARRISH AND P. E. SANFORD Normally, as confirmed in this study 4. Imbesi, A. 1958 Vitam in A absorption through the blood and lymph. Atti. Accad. with chicks, most of the vitamin A in Peloritana Pericolanti, Classe Sci. Med.-Biol., serum is the alcohol (2, 22, 24). The 5 6 : 203 (cited in Chem. Abs., 53: 20350d). alcohol of serum may arise from 2 pos 5. Week, E. F., and F. J. Sevigne 1949 Vita sible sources : absorption of vitamin A hy min A utilization studies. I. The utilization of vitamin A alcohol, vitamin A acetate, and drolyzed in the intestinal lumen, or hy vitamin A natural esters by the chick. J. drolysis of serum vitamin A esters by Nutrition, 3 9 : 233. tissues other than the small intestine. A 6. Neff, A. W., D. B. Parrish, J. S. Hughes and small quantity (10 to 15% ) of intestinal L. F. Payne 1949 The state of vitamin A vitamin A was found as free alcohol; this in eggs. Arch. Biochem., 2 1 : 315. 7. Association of Official Agricultural Chemists may be significant, since it could be the 1960 Methods o f Analysis, ed. 9. W ashing source of blood serum vitamin A alcohol, ton, D. C., p. 652. via the portal system at a rate preventing 8. Kimble, M. S. 1939 The photocolorimetric build-up in intestinal tissue. Another pos determination of vitamin A and carotene in human plasma. J. Lab. Clin. Med., 24: 1055. sible source of serum vitamin A alcohol is 9. Parrish, D. B., G. H. Wise and J. S. Hughes hydrolysis of blood vitamin A esters by 1947 The state of vitam in A in colostrum other tissues. Krishnamurthy et al. (18) and in milk. J. Biol. Chem., 167: 673. reported that pancreatic tissue actively 10. Kaiser, E., and B. M. Kagan 1956 Separa hydrolyzed natural vitamin A esters, liver tion of vitamin A alcohol and vitamin A esters by paper chromatography. Arch. Bio tissue was inactive, whereas splenic tissue chem. Biophys., 63: 118. hydrolyzed a small quantity. Slow hydrol 11. Matterson, L. D., R. H. Bunnell, L. D. Stin ysis of the palmitate in intestinal lumen son, E. P. Singsen and L. M. Potter 1955 and thus lower concentrations of vitamin The effect of incorporating diphenyl-p-phen- ylenediamine (DPPD) into a ration contain A in duodenal tissue, plus other tissue ing various levels of vitamin A from fish oils. hydrolysis may account for lack of vitamin Poult. Sci., 34: 1080. A esters in post-absorptive blood serum 12. Shellenberger, T. E., D. B. Parrish and P. E. after the palmitate was injected. Further Sanford 1957 Effects o f antioxidants, DPPD and BHT, on health, production and work is needed to define the mechanism reproduction o f laying hens. Poult. Sci., 36: controlling vitamin A alcohol levels of 1313. b lood . 13. Tiews, J. 1957 The influence o f antioxi Transport of vitamin A across the in dants and antibiotics on the utilization of testinal wall apparently is energy-depend vitamin A by chicken and rat. Landwirtsch. Forsch., Sonderh., no. 9: 148. ent (22), for it was accelerated by extra 14. Ascarelli, I. 1957 The use of vitamin A glucose or ATP, or both, whereas glycolytic protected by DPPD ( diphenyl-p-phenylene- inhibitors and uncoupling agents inhibited diamine) for the growth of chickens. Poult. or reduced absorption. If ethoxyquin, an Sci., 36: 549. 15. Calhoun, M. L. 1954 Microscopic Anatomy antioxidant, decreases absorption, as our of the Digestive Systems of the Chicken. study indicated, it might be through inter Iowa State College Press, Ames, Iowa. ference in energy dependent processes. 16. Eden, E., and K. C. Sellers 1950 Hydrolysis and esterification of vitamin A during ab sorption. Biochem. J., 46: 261. LITERATURE CITED 17. Murthy, S. K., S. Mahadevan, P. S. Sastry 1. Jacobson, N. L., R. S. Allen, J. T. Blake and and J. Ganguly 1961 Fatty acid specificity P. G. Homeyer 1954 The effect of method for the esterification of vitamin A and cho of administration on the absorption and lesterol by intestinal and pancreatic enzymes storage of vitamin A by dairy calves. J. in rats. Nature, 1 8 9 : 482. Nutrition, 54: 143. 18. Krishnamurthy, S., P. S. Sastry and J. Gan guly 1957 Studies on vitam in A esterase. 2. Moore, T. 1957 Vitamin A. Elsevier Pub II. The hydrolysis of natural vitamin A ester lishing Company, New York. within the living animal. Indian J. Med. 3. Wise, G. W., N. L. Jacobson, R. S. Allen and Res., 45: 391. S. P. Yang 1958 Rate of absorption of 19. Mahadevan, S., S. K. Murthy, S. Krishna carotene and of vitamin A from the alimen murthy and J. Ganguly 1961 Studies on tary tract of dairy calves. II. Effects of vitam in A esterase. 4. The hydrolysis and methods of dispersion and of administration. synthesis of vitamin A esters by rat intestinal J. Dairy Sci., 4 1 : 143. mucosae. Biochem. J., 79: 416. ABSORPTION OF VITAMIN A 105 20. Krause, R. F., and L. T. Powell 1953 The 23. Murray, T. K., and H. C. Grice 1961 Ab i n v i t r o hydrolysis and esterification of vita sorption of vitamin A after thoracic duct min A. Arch. Biochem. Biophys., 44: 57. ligation in the rat. Canad. J. Biochem. 21. Laughland, D. H. 1958 The hydrolysis of Physiol., 3 9 : 1103. vitamin A esters by rat liver homogenates. 24. Parrish, D. B , G. H. Wise and J. S. Hughes Arch. Biochem. Biophys., 7 3 : 95. 1948 Effect of vitamin A supplements upon 22. Ganguly, J. 1960 Absorption, transport, and the state of vitamin A in blood serum of the storage of vitamin A. Vitamins and Hor dairy cow and in blood serum and liver of mones, 1 8 : 387. its neonatal calf. J. Biol. Chem., 1 7 2 : 355. Vanadium Toxicity and Distribution in Chicks and Rats* 1'2 JOHN N. HATHCOCK, CHARLES H. HILL a n d GENNARD MATRONE North Carolina State College, of the University of North Carolina, Raleigh, North Carolina ABSTRACT Studies of the effect of dietary vanadium revealed that 25 ppm vana dium added to a dry skim milk-glucose monohydrate diet was toxic to chicks as indi cated by both depression of growth and mortality. Ammonium metavanadate and vanadyl sulfate were equally toxic. The addition of the essential trace elements, iron, copper, molybdenum, cobalt, zinc, and manganese to the basal diet did not affect the toxicity of vanadium. Scandium, titanium, and niobium, elements which resemble vanadium in many of their chemical and physical parameters, were not toxic when fed at 200 ppm nor did they influence the toxicity of vanadium. Radioisotope studies revealed that scandium and niobium were poorly absorbed or poorly retained compared to vanadium. EDTA completely prevented the toxicity of vanadium apparently by pre venting its absorption from the intestinal tract. Ingested V48 was found to be mainly concentrated in the hone and kidney of young chicks and in the kidney of adult rats. Dietary vanadium in low concentration TABLE 1 has been found to be toxic to rats and B a s a l d ie t chicks (1 -4 ). Although considerable work % has been carried out, the nature of vana Dried skim milk 60.0 Glucose dium toxicity remains unknown. 32.5 Cottonseed oil 1 5.0 The work presented in this report was DL-Methionine 0.3 undertaken to: 1) further study vanadium L-Arginine 0.5 toxicity in chicks and rats; 2) determine Glycine 0.5 NaCl 0.5 target organs by use of radioactive vana MgSO„-7H20 0.5 dium; 3) compare effects of vanadium 9/kg with effects of elements similar to it; and M nS04-H20 0.22 4) determine the effects of a dietary che FeS04-7H20 0.53 lating agent on vanadium toxicity. Cu S04-5HoO 0.10 Vitamin mix 2 1.98 MATERIALS AND METHODS 1 Wesson Oil, The Wesson Oil Company, New Orleans, La. The chicks used in the non-radioisotope 2 Supplies per kilogram of diet: Vitamin A, 11,000 USP units; vitamin D, 1980 IC units; thiamine, 3.52 studies, straight-run White Plymouth mg; riboflavin, 5.72 mg; folic acid, 1.10 mg; a-tocoph- Rocks from a commercial hatchery, were eryl acetate, 2 2 . 0 mg; menadione sodium bisulfite, 0.792 mg; biotin, 0.176 mg; pantothenic acid, 18.3 mg; weighed individually and distributed at niacin, 52.3 mg; pyridoxine, 5.7 mg; and cyano- random into experimental lots at one day cobalamin, 8 .8 ¡ig. of age. They were housed in electrically dium. Two lots of 20 chicks each were fed heated battery brooders with raised wire e a c h diet. floors. Water and feed were supplied ad In the third experiment. 100 ppm iron, libitum. The basal diet used in all these 25 ppm copper, 70 ppm manganese, 100 studies is presented in table 1. Individual ppm zinc, 50 ppm cobalt, and 50 ppm body weights of the chicks were recorded molybdenum were added to the basal diet weekly and mortality was recorded daily. in both the presence and absence of 25 In the first experiment, ammonium metavanadate was fed to chicks at levels Received for publication August 5, 1963. 1 Published with the approval of the Director of the of 10 and 25 ppm vanadium in the diet, Experiment Station as Paper no. 1678 of the Journal and in the second experiment, both am Series. 2 Supported in part by grants from the National monium metavanadate and vanadyl sul Institutes of Health ( AM05651-02), the Herman Frasch Foundation, and the Moorman Manufacturing Com fate were fed at a level of 25 ppm vana- pany. 106 J. N u t r i t i o n , 82. ’64 VANADIUM TOXICITY 107 ppm vanadium as ammonium metavana In the fourth study, 4 rats were each date. Each of these diets was fed to 2 given 5 uc of scandium-46, as scandium lots of 20 chicks each. trichloride, and 4 others were each given In the fourth study, scandium, titanium, 5 uc of niobium-95, as niobium oxalate. and niobium were each fed at 200 ppm After 4 days, certain organs and the ex alone and in combination with 50 ppm creta were analyzed for content of these vanadium to 2 lots of 10 chicks each. The radioactive elements. compounds used were scandium trichlo The results were analyzed for statistical ride, titanium tetrachloride, potassium significance by Duncan’s multiple range niobate, and ammonium metavanadate. test ( 5 ) . In the fifth experiment, vanadium at 25 ppm was fed in the absence and presence RESULTS AND DISCUSSION of disodium ethylenediaminetetraacetate The first study was conducted to deter (EDTA) at 0.93 and 2.78 mmole/kg. mine the level of vanadium which would These levels of EDTA are 2 and 6 times have toxic effects when included in the the molar concentration of vanadium. Two basal diet. The results, presented in table lots of 20 chicks each were fed each diet. 2, experiment 1, show that the addition of In the radioisotope experiments, 6-week- 10 ppm vanadium to the basal diet failed old chicks or mature rats were each orally to affect either the growth or mortality of administered a quantity of the radioiso the chicks at 2 weeks of age. However, tope in 0.5 ml of solution. After allowing 25 ppm vanadium significantly depressed time for the adsorption and assimilation of growth and increased mortality. The con the radioisotopes, the animals were killed trol and 10 ppm vanadium groups were and samples of the tissues and excreta carried to 6 weeks of age and no signifi were taken for both counting and weigh cant differences were observed in either ing. The radioactivities of the counting weight gains or mortality. samples were determined in a deep-well scintillation counter and compared on the TABLE 2 basis of net counts per minute per gram Effects of dietary vanadium on chicks of dry weight of the tissue. In the first of these experiments, 5.6 uc 2-Week 2-Week Vanadium of vanadium-48, in the form of vanadyl mortality 1>2 gain dichloride, were administered orally to % 9 each of 6 chicks. Forty-eight hours later, Experiment 1 15.0“ 75.7“ the radioactive vanadium content of vari 0 ppm 10 ppm 3 15.0“ 69.9 “ ous organs was determined. 25 ppm 3 95.0 b 10.0 b In the second radioisotope experiment, 3 chicks fed the basal diet and 3 others Experiment 2 5.0 “ 77.1 * fed that diet supplemented with EDTA at None NH 4VO3 4 82.5 b 16.9 b 0.93 mmole/kg were each given 5.1 uc VOSOP 85.0 b 23.0 b of vanadium-48. Forty-eight hours later, the radioactive vanadium contents of liver, 1 Mean of 2 replicates of 20 chicks each. 2 Numbers with same superscripts are not signifi kidney, bone, and bone marrow were de cantly different (P — 0.05). 3 As NH4VO3 . te rm in e d . 4 At 25 ppm vanadium. For the third experiment, 3 rats were fed the basal diet and 3 others were fed The second siudy was conducted to de that diet with EDTA added at 0.93 mmole/ termine whether 2 different vanadium kg. Three days after beginning to feed the compounds with vanadium of different animals these diets, they were orally given valences would have different effects on 10 uc of vanadium-48 each. The urine and chicks when included in the diet. The re feces were collected separately. Forty- sults, shown in table 2, experiment 2, in eight hours later, the radioactive vana dicate that, in the diet, vanadyl sulfate dium content of the liver, kidney, and with the vanadium in 4+ valence, and bone was determined as well as that of the ammonium metavanadate with the vana urine and feces. dium in 5+ valence are equally toxic. 108 JOHN N. HATHCOCK, CHARLES H. HILL AND GENNARD MATRONE This toxicity manifested with a low die TABLE 4 tary concentration indicates some spe Effects of elements similar to vanadium cificity in the action of vanadium. That 2-Week 2-Week Treatment1 gain 2>3 is, with this level of vanadium the deleter mortality 2>3 ious effects observed could hardly be due % 9 0.0* 100.1 * to such a nonspecific property of metals None V 90.0» 23.5» as the binding of sulfhydryl groups of pro Sc 0.0 * 104.2 » teins in general. Ti 5.0 * 101.1 * 5.0 * 101.2 * It was possible that vanadium was Nb V + S c 100.0» — antagonistic to an essential trace metal V + Ti 100.0» — analogous to the antagonism of zinc for V + Nb 100.0» — copper (6 ) or tungsten for molybdenum 1 Vanadium at 50 ppm as NH4VO3 , scandium at (7). To determine whether this was so, 200 ppm as ScCl3 , titanium at 200 ppm as TiCLj, and niobium at 200 ppm as (KsO)«'(NasOs)®*I6 H2O. in the third experiment, vanadium was fed 2 Mean of 2 replicates of 10 chicks each. 3 Numbers with same superscripts are not signifi at a concentration of 25 ppm in the basal cantly different (P — 0.05). diet and in that diet supplemented with Studies with other elements have indi essential trace minerals. The results, pre cated that chelating agents can reverse sented in table 3, show that the additional m etal toxicity by chelation and subsequent amounts of the trace elements had no excretion (8 ) or by prevention of absorp effect on the toxicity of vanadium as meas tion (9). In the fifth experiment, EDTA ured by either growth inhibition or mor was added at 2 and 6 times the molar ta lity. concentration of vanadium in the diet to TABLE 3 determine whether this chelating agent Effect of vanadium on basal and fortified diets would affect the toxicity produced by 25 ppm dietary vanadium. The results, pre 2-Week 2-Week Supplement1 mortality 2>3 gain 2'3 sented in table 5, show that both levels of EDTA protected the chicks against the % 9 vanadium toxicity. None 0.0 » 82.2* Vanadium 65.0 b 21.3 b TABLE 5 Trace minerals 2.5 » 77.6* V + trace minerals 60.0 » 26.4 b Effects of EDTA1 on vanadium toxicity 1 4 3 2-Week 3-Week Vanadium at 25 ppm as NH VO ; trace mineral Dietary supplement supplement includes 1 0 0 ppm iron as FeS0 4 -7 H2 0 , 1 0 0 mortality 2»3 gain *-3 ppm zinc as ZnCl2, 70 ppm manganese as MnSO^HfoO, 50 ppm cobalt as CoCl2 -6 H2 0 , 50 ppm molybdenum % 9 as Na2Mo0 4 -2 H2 0 , and 25 ppm copper as CuSCV None 5.0* 90.4* 5H20. Vanadium 4 62.5» 30.5» 2 Mean of 2 replicates of 20 chicks each. 3 Numbers with same superscripts are not signifi V 4+ 0.93 cantly different (P^O .05). mmole/kg EDTA 12.5* 78.3 * V 4 + 2.78 The 3 elements, scandium, titanium, mmole/kg EDTA 5.0* 89.4* and niobium, are very similar to vana 1 Added as disodium ethylenediaminetetraacetate. dium: the ions of these elements, Sc3+, 2 Mean of 2 replicates of 20 chicks each. 3 Numbers with same superscripts are not signifi Ti4+, and Nb5+ are isoelectronic in their cantly different (P — 0.05). 4 Vanadium at 25 ppm which equals 0.46 mmole/ valence shells with the V5+ ion and all kg, as NH4VO3. have a favored coordination number of The results of the studies thus far re six. The similarities of these elements to ported raised several questions: 1) What vanadium suggested that they, too, might are the target organs of dietary vanadium? be toxic or might influence the toxicity of 2) How does EDTA protect the chick from vanadium. However, the results of the vanadium toxicity? 3) Are scandium, fourth experiment, presented in table 4, titanium, and niobium absorbed in ap indicate that, at 200 ppm in the diet, scan preciable amounts from the intestinal dium, titanium, and niobium are not toxic tract? The radioisotope studies presented as measured by growth or mortality, nor in this report were undertaken to answer do they affect the toxicity of vanadium. these questions. VANADIUM TOXICITY 109 The results of the first of these experi TABLE 7 ments, presented in table 6, show the dis Effect of EDTA 1 on radiovanadium distribution tribution of the radioactivity in the tissues of the chicks 48 hours after administra Tissue Control EDTA i tion of the radioactive vanadium. The con mean net count/ m in/g dry zut centrations were found to be greatest in Experiment 1 2 the bone and kidney. Liver 4,843 1,028 Kidney 21,210 1,760 TABLE 6 Bone 3 69,843 19,888 Marrow 4 5,075 375 Radiovanadium distribution in chick tissues Experiment 2 5 Tissue Liver 1,805 333 mean net count/ Kidney 8,942 2,127 m in/g dry wt Bone 6 155 131 Bone 1 13,346 Urine 25,879 9,130 Kidney 10,931 Feces 168,998 737,139 Liver 2,292 1 Concentration in the diet was 0.93 mmole/kg as Spleen 1,693 disodium ethylenediaminetetraacetate. # Gonads 2 1,491 2 Each value is the mean of 3 chicks. Duodenum 1,200 3 From the central section of the right femur with the marrow removed. Lung 981 4 Marrow from the femur section mentioned above. Heart 882 5 Each value is the mean of 3 rats. Proven triculus 861 6 From the central section of the right femur includ ing marrow. Skin 700 Brain 527 Muscle 3 505 the rats, as it had in the chicks, the pres Pancreas 231 ence of EDTA in the diet produced lower mean net count/ concentrations of vanadium-48 in the tis m in / m l sues. The urine of the EDTA-fed rats, also, Whole blood 275 contained less radioactive vanadium than Serum 201 Blood cells 4 444 that of the control rats. The feces of the EDTA-fed rats, however, contained more 1 From the central section of the right femur; in cludes marrow. radioactivity than that of the control rats. 2 Mean includes 3 males and 3 females; mean for These results indicate that in the presence ovaries was 4% less than for testes. 3 From the right thigh. of dietary EDTA less vanadium is absorbed 4 Calculated from the activities of the whole blood and serum. from the intestinal tract. Thus, EDTA probably prevented vanadium toxicity by The next experiment was conducted to preventing its absorption from the intes determine the distribution of radioactive tin e. vanadium in certain tissues of the chick The low activity of the bone of all the both in the presence and absence of die rats may be explained by the fact that they tary EDTA. The results, presented in were mature adults, whereas the chicks table 7, experiment 1, show that dietary in the previous experiments were young EDTA decreased the concentration of and growing rapidly. radioactive vanadium in all the tissues There are no data in the literature studied. Whether this decrease was due which provide an estimate of the absorp to increased excretion or decreased absorp tion of scandium, titanium, and niobium. tion could not be determined from this It was possible that these elements, unlike study with chicks. vanadium, were not absorbed to any ap To obtain some knowledge of the ex preciable extent from the intestinal tract. cretion and absorption patterns of vana To estimate the absorption of these ele dium in the presence and absence of die ments from the diet, radioisotopes of scan tary EDTA, rats were used in the next dium and niobium were used to determine experiment. The distributions of radio the absorption and distribution patterns active vanadium in the tissues and ex of these elements when orally adminis creta of rats fed the basal diet and rats tered. None of the known radioisotopes fed the EDTA-containing diet are pre of titanium have a half-life long enough sented in table 7, experiment 2. Again in for use in this type of experiment. The 110 JOHN N. HATHCOCK, CHARLES H. HILL AND GENNARD MATRONE TABLE 8 Distribution of scandium-46 and niobium-95 1 Tissue Scandium-46 2 Niobium-95 3 mean net count! % o f fe c a l mean net count/ % o f fe c a l m in/g dry wt co u n ts 4 m in/g dry wt co u n ts 4 Liver 69.0 0.0025 207.2 0.0121 Spleen 178.0 0.00045 414.5 0.0024 Kidney 78.8 0.00048 943.8 0.0085 Femur 3 10.2 0.00028 356.5 0.0016 Muscle 5 142.2 0.0034 152.2 0.0016 Urine 320.0 0.0071 5,609.5 0.2300 1 All numbers given are means for 4 rats. 2 As ScCl3. 3 As Nb(HC 2 04 )5. 4 ^ 1 i 4. a Count/min whole organ 4 Calculated as: —=-----— —;—. . . > — X 100. Count/min total feces 5 Values for these tissues given on wet weight basis. results of this experiment, presented in 4. Romoser, G. L., W. A. Dudley, L. J. Machlin table 8, are shown as mean net counts and L. Loveless 1961 Toxicity of vana dium and chromium for the growing chick. per minute per gram of dry weight, and Poult. Sci., 4 0 : 1171. as a percentage of fecal counts.3 The re 5. Duncan, D. B. 1955 Multiple range and sults indicate that scandium and niobium multiple F tests. Biometrics, 1 1 : 1. are poorly absorbed from the intestinal 6. Smith, S. E., and E. J. Larson 1946 Zinc toxicity in rats. J. Biol. Chem., 1 6 3 : 2 9 . tract. 7. Leach, R. M., and L. C. Norris 1957 Stud LITERATURE CITED ies on factors affecting the response of chicks to molybdenum. Poult. Sci., 3 6 : 1131. 1. Berg, L. R. 1963 Evidence of vanadium 8. Sivjakov, K. I., and H. A. Braun, Jr. 1959 toxicity resulting from the use of certain The treatment of acute selenium, cadmium, commercial phosphorus supplements in chick and tungsten intoxication in rats with cal rations. Poult. Sci., 42: 766. cium disodium ethylenediaminetetraacetate. 2. Franke, K. W., and A. L. Moxon 1937 Toxicol. Appl. Pharmacol., 1 : 602. The toxicity of orally ingested arsenic, selen 9. Longcope, W. T., and J. A. Luetscher, Jr. ium, tellurium, vanadium, and molybdenum. 1946 Treatment of acute mercury poison J. Pharmacol. Exp. Therap., 6 1 : 89. ing by BAL. J. Clin. Invest., 25: 557. 3. Nelson, T. S., M. B. Gillis and H. T. Peeler 1962 Studies of the effect of vanadium on Count/min whole organ 3 Calculated as: chick growth. Poult. Sci., 4 1 : 519. Count/min totaf feces Mineral Balance Studies with the Baby Pig: Effects of Dietary Phosphorus Level upon Calcium and Phosphorus Balance* 1'2 E. R. M ILLER, D. E. ULLREY, C. L. ZUTAUT, J. A. HOEFER a n d R. W. LUECKE Departments of Anim al Husbandry and Biochemistry, Michigan State University, East Lansing, M ichigan ABSTRACT Calcium and phosphorus balance studies were conducted on 29 baby pigs receiving a synthetic milk diet containing phosphorus levels o f 0.2, 0.4, 0.5, 0.6, 0.7 and 0.8% with 0.8% calcium. Growth rate, food intake and mineral retention were greatly depressed in pigs receiving 0.2% of phosphorus. Increasing dietary phos phorus levels to 0.5% resulted in increased total phosphorus retention (g ) and per centage phosphorus retention. Increasing dietary phosphorus levels beyond 0.5% did not increase phosphorus retention (g ) but decreased percentage phosphorus retention. Dietary phosphorus levels below 0.5% resulted in reduced calcium retention (g) whereas increasing dietary phosphorus levels above 0.5% did not affect calcium bal ance. The dietary phosphorus requirement for optimal utilization of calcium and phosphorus is therefore about 0.5%. Mineral balance studies with animals of phosphorus in each of the diets with provide information concerning mineral USP grade CaHP04-2H20 supplying the absorption and retention and offer a means additional phosphorus for the higher die of assessing the effects of dietary nutrient tary levels. Dietary calcium level for all levels and other factors upon mineral utili pigs was maintained at 0.8% by appropri zation. Few mineral balance studies with ate reduction of CaC03 when C aH P04-2H20 baby pigs have been conducted. Danish was increased. Thus the sources of dietary (1), German (2, 3), Canadian (4 ) and calcium were CaC03 and CaHP0r2H20 American (5, 6) balance trials concerned and the sources of dietary phosphorus were with calcium and phosphorus utilization of casein and CaHP04-2H20. All diets con young pigs receiving sow’s milk, sow’s milk tained 1800 IU of vitam in D3/kg of solids. substitutes or synthetic milk diets have Pigs used in the balance studies were been reported. placed in metal metabolism cages3 for a 3- The present study was undertaken to de to 5-day adjustment period prior to the col termine the effects of dietary phosphorus lection period. Pigs were removed from the level upon calcium and phosphorus utiliza metabolism cage to other cages to be fed tion by the baby pig as determined by (4 times daily while fed the liquid diet or mineral balance trials and to provide sup 3 times daily with dry diet) an amount of plementary information toward a more ac food and water which they would consume curate determination of the dietary phos within a 5- to 10-minute period. Their phorus requirement of the baby pig (7 ). mouths were wiped clean and then they were returned to the collection cages. Con MATERIALS AND METHODS stant daily feed intakes were maintained Baby pigs used in the study were from 2 throughout the remainder of the adjust trials conducted to determine their phos ment period and the 3-day collection phorus requirement (7). Calcium and Received for publication August 5, 1963. phosphorus balance studies were conducted 1 Published with the approval of the Director of the Michigan Agricultural Experiment Station as with pigs receiving 0.2, 0.4 or 0.6% of die Journal article no. 3179. 2 Presented in part before the meeting of the tary phosphorus in the first trial, and 0.4, American Institute of Nutrition, April, 1962 (Federa 0.5, 0.6, 0.7 or 0.8% of dietary phosphorus tion Proc., 21 : 308, 1962, abstract). 3 LC-108/A, Geo. H. Wahman Manufacturing Com in the second trial. Casein furnished 0.2% pany, Baltimore, Maryland. J. N u t r itio n , 82: ’64 111 112 MILLER, ULLREY, ZUTAUT, HOEFER AND LUECKE period. Feces were collected separately from urine by means of a fine screen placed above the collection tray. Urine contamina tion was minimal because of the firm na ture of the feces. In the first trial, collec tions were made at 2, 4 and 6 weeks of age with pigs receiving the synthetic diet as a liquid during the first 2 collections and as a dry meal in the final collection. In the second trial collections were made during the final 2 weeks ( 5 and 6 weeks of age) with all pigs receiving the dry meal diet. Rate of dietary consumption was de pressed only in pigs receiving 0.2% of die tary phosphorus. Total dietary intakes of pigs fed other levels of dietary phosphorus were equated during simultaneous collec tion periods. Total fecal collections were dried in a low temperature (70°C ) convection oven, weighed, finely ground and stored in air tight containers. Total urine collections Fig. 1 Phosphorus retention of baby pigs as affected by age and phosphorus level (synthetic were acidified with 6 M HC1 to a pH of be milk diet). tween 1 and 2, the volume accurately measured and 100-ml samples stored in In the second trial littermates fed the air-tight bottles. Analyses of fecal and different levels of dietary phosphorus re urine calcium and phosphorus concentra ceived equal food intakes during simultane tions were by modifications of the methods ous collections. The effects of dietary phos of Varley (8 ). Statistical analyses of data phorus level upon calcium and phosphorus were performed using the multiple range utilization are shown in data presented in test of Duncan (9 ). table 1. Pigs receiving 0.4 or 0.5% of die RESULTS AND DISCUSSION tary phosphorus excreted similar amounts Results of balance studies conducted of fecal and urinary phosphorus. Animals during the first trial are presented graphi receiving the higher concentrations of die cally in figures 1 and 2. Dietary intake was tary phosphorus excreted proportionately depressed very early in the trial in pigs greater amounts of phosphorus, particu receiving 0.2% of dietary phosphorus. larly in the urine, than animals receiving Animals receiving the 2 higher levels of 0.5% of phosphorus. This resulted in dietary phosphorus received equal food in similar phosphorus retention by pigs re takes. Level of dietary phosphorus had a ceiving dietary phosphorus levels of 0.5 to positive effect upon calcium and phos 0.8% which was significantly greater than phorus retention at each age. Daily cal that of pigs receiving 0.4% of phosphorus. cium and phosphorus retention increased Pigs receiving the lower levels of dietary with age at each level of dietary phos phosphorus retained a higher percentage phorus. A great increase in calcium and of the phosphorus intake than animals re phosphorus retention occurred from 4 to 6 ceiving 0.7 or 0.8% of phosphorus. weeks of age in pigs receiving 0.4 or 0.6% Fecal calcium excretion was significantly of dietary phosphorus. This increased re greater by pigs receiving 0.4% of phos tention was prim arily due to the greater in phorus than by animals receiving the takes of calcium and phosphorus that oc higher levels of dietary phosphorus. This curred when the dietary form was changed appears to be an effect of the low level of from a liquid (20% of solids) to a dry dietary phosphorus per se, but it is also m ea l. possible that this reduced apparent calcium DIETARY PHOSPHORUS AND MINERAL BALANCE 113 5.56 absorption may be due to dietary source of calcium since more of the calcium in the low phosphorus diet is being supplied in the less soluble carbonate form. The latter possibility does not, however, appear to be operative since no stepwise decrease in fecal calcium excretion occurs with other alterations of CaC03 and CaHP04-2H20 as dietary phosphorus level is increased be yond 0.5%. Furthermore, calcium digesti bility studies with rats (1 0 ) and with cat tle (1 1 ) have shown no significant differ ences in calcium utilization when added to the diet in the carbonate or phosphate forms. Urinary calcium excretion was un affected by dietary phosphorus level, con tributing 5 to 10% of the total calcium ex creted. This relationship between urinary and fecal calcium excretion has been shown to be quite consistent (1, 5, 6). In creased fecal calcium excretion by pigs re ceiving 0.4% of phosphorus resulted in a significantly reduced calcium retention. Since average dietary calcium intakes were equated the percentage calcium retention of pigs receiving 0.4% phosphorus was also significantly less than that of pigs re ceiving higher levels of dietary phosphorus. The effects of dietary phosphorus level upon calcium and phosphorus utilization Fig. 2 Calcium retention of baby pigs as af fected by age and phosphorus level (synthetic by the baby pig are best illustrated by the milk diet). graphs in figures 3 and 4. These indicate TABLE 1 Calcium and 'phosphorus excretion and retention as affected by dietary phosphorus level Dietary phosphorus level, % 0.4 0.5 0.6 0.7 0.8 No. of collections 4 4 4 4 4 Daily food intake, g 1 700 700 700 700 700 P balance Daily P intake, g 1 2.8 3.5 4.2 4.9 5.6 Daily fecal P, g 0.3 ±0.03 * 0.3 ± 0.03 0 .5 ± 0.04 0.8 ±0.03 bb 0.8 ±0.14 b‘ Daily urinary P, g 0.1 ±0.07 0.1 ± 0.03 0.5 ±0.03 1 .0 ± 0 .1 5 bb 1.5 ± 0.13 bb Daily P retention, g 2.4 ±0 .2 3 .1 ± 0.3 « 3.2 ±0 .4 * 3.1 ± 0 .2 » 3.3 ± 0 .3 » P retention, % 84 ± 2 .5 bb 88 ± 2 .3 bb 75 ± 2 .8 b 63 ±1 .5 59 ±5 .6 Ca balance Daily Ca intake, g 1 5.6 5.6 5.6 5.6 5.6 Daily fecal Ca, g 2 .0 ± 0.2 1.1 ± 0.1 “ 1.2±0.1 *> 1.3 ± 0.1 *» 1 .2± 0.2 *» Daily urinary Ca, g 0.1 ±0.08 0.1 ± 0 .0 2 0.1±0.01 0.1 ±0.01 0.1 ±0.02 Daily Ca retention, g 3.4 ±0 .3 4.4 ± 0.5 » 4.3 ± 0 .6 » 4 .2 ± 0.4 * 4.3 ± 0.6 » Ca retention, % 60 ±2 .9 78 ± 3.3 * 78 ± 4 .3 » 75 ±0 .8 » 77 ±5 .4 * 1 Coefficient of variation ( standard deviation -±mean) of all intake means is 0.14. 2 SE. * Significantly different from 0.4% P mean value (P < 0.05); aa P < 0.01. i» Significantly greater than least 2 values (P < 0.05); bb P < 0.01. 114 MILLER, ULLREY, ZUTAUT, HOEFER AND LUECKE that the dietary phosphorus level for opti mal utilization of calcium and phosphorus is about 0.5%. LITERATURE CITED 1. Ludvigsen, J., and G. Thorbek 1960 Stud- ier over pattegrises raekst of ernaering. II. Tilraekst og foderforbrug samt kvaelstof-, fosfor- og kalciumomsaetningen hos kunsfigt ernaerda pattergrise i aideren fra 5. til 35. dag. Beretning fra Forspgslaboratoriet 320, Kpbenhaven. 2. Freese, H. H. 1958 Undersuchungen liber den Calcium-, Phosphor-, Magnesium- und Stickstoff-umsatz des Ferkels in Beziehung zum Wachstum bei Muttermilch und Mutter- milchersatz. Arch. Tierernahrung, 8 : 330. 3. Lenkeit, W., and H. H. Freese 1958 Zum Wachstum bei Muttermilch und Muttermil- chersatz. Ztschr. Tierernahrung u. Futter- mittelk, 1 3 : 243. 4. W hiting, F., and L. M. Bezeau 1958 The calcium, phosphorus and zinc balance in pigs DIETARY P (%) as influenced by the weight of pig and the Fig. 3 Phosphorus balance as affected by die level of calcium, zinc and vitamin D in the tary phosphorus level. ration. Canad. J. Animal Sci., 3 8 : 109. 5. Hansard, S. L., W. A. Lyke and H. M. Crowder 1961 Absorption, excretion and utilization of calcium by swine. J. Animal Sci., 20: 292. 6. Miller, E. R., D. E. Ullrey, C. L. Zutaut, B. V. Baltzer, D. A. Schmidt, J. A. Hoefer and R. W. Luecke 1962 Calcium requirement o f the baby pig. J. Nutrition, 77: 7. 7. MiUer, E. R., D. E. Ullrey, C. L. Zutaut, B. V. Baltzer, D. A. Schmidt, J. A. Hoefer and R. W. Luecke 1963 Phosphorus requirement of the baby pig. J. Nutrition, 82: 34. 8. Varley, H. 1960 Practical Clinical Chem istry. Interscience Publishers, Inc., New York. 9. Duncan, D. B. 1955 Multiple range and multiple F tests. Biometrics, 1 1 : 1. 10. Hayami, H., Y. Matsuno, K. Mori, M. Suzki and S. Saito 1955 The utilization of cal cium from various sources. V III. A com parison of calcium sulfate, carbonate and phosphate. National Institute of Nutrition Annual Report (Tokyo), 1 9 5 5 : 24. (Cited in Chem. Abstr., 5 1 : 16753.) 11. Hansard, S. L., H. M. Crowder and W. A. Lyke 1957 Biological avaUability of cal Fig. 4 Calcium balance as affected by dietary cium in feeds for cattle. J. Animal Sci., 1 6 : phosphorus level. 437. Adaptation of the Chick to Dietary Energy Source' W. E. DONALDSON Department of Poultry Science, North Carolina Agricultural Experiment Station, Raleigh, North Carolina ABSTRACT One-day-old chicks were maintained from 0 to 14 days with high- carbohydrate (CHO) or high-fat (fat) diets. The chicks were then given fatty acid or glucose labeled with C14 and placed in metabolism cages. Excreta and respiratory CO. were collected for 24 hours to determine C14 dose retention and C14 Oz production. Production of C14 02 was used as an index of fatty acid or glucose oxidation. Fat-fed chicks oxidized fatty acid at a maximal rate regardless of age, dietary choline level or dietary fatty acid level. The CHO-fed chicks oxidized fatty acid maximally at one day of age, but the rate decreased with age. Diet had no effect on the oxidation of glucose-C14. Feeding the fat diet to CHO-adapted chicks at the time of fatty acid-C14 administration resulted in an increase of fatty acid oxidation to levels comparable to fat adapted chicks. When glucose-C14 was administered, tissues of chicks fed the CHO diet contained approximately 3 times as much radioactivity in the fat-soluble fraction as did the tissues of chicks fed fat. The results are discussed in terms of growth response to dietary fat. Rand et al. (1 ) obtained consistent of the consistent growth responses ob growth responses with chicks by the eqi- tained by the addition of fat to chick diets caloric substitution of corn oil for glucose. when the ratio of metabolizable energy to The growth responses to corn oil were ob other nutrients is held constant. tained under both ad libitum and equalized The experiments reported here were de feeding techniques. Carcasses of chicks signed to test the above hypothesis. fed either corn oil or glucose had a similar protein content despite the differences in PROCEDURE body weight. These workers suggested that R earing ■procedures. Female White Rock the corn oil effect could have been exerted chicks ranging in age from one-day-old to by increased efficiency of metabolizable 14 days were used. The chicks from each energy utilization or protein utilization. experiment were maintained in electrically Tepperman et al.2 observed that liver heated batteries with raised-wire floors. slices from fat-fed rats oxidize palmitic-1- One-half of the chicks were fed a high- C14 acid more rapidly than do slices from carbohydrate diet and the remaining half carbohydrate-fed rats. This result coupled were fed a high-fat diet (table 1). In ex with other observations (2 ) suggested a periment 2, choline was inadvertently metabolic adaptation dependent upon die omitted from both diets. tary energy source. Shifts in fatty acid- At appropriate ages, 3 chicks fed each oxidizing enzymes were postulated as a diet, selected according to body weight,3 possible explanation of the observed dif were administered a single oral dose of fe re n c e s . palm itic-l-CJ! acid, linoleic-l-C14 acid, or Boell (3 ) stated that the avian embryo uniform ly labeled glucose-C14 (size, specific derives over 90% of its total caloric re activity, and type of dose are listed under quirement from fatty acid oxidation. Received for publication July 1, 1963. The above results are compatible with 1 Contribution from the Poultry Science Department, the hypothesis that at hatching, the chick North Carolina Agricultural Experiment Station, Raleigh, North Carolina. Published with the approval is adapted to a fatty acid-oxidizing type of the Director of Research as paper no. 1653 of the metabolism and that inclusion of large Journal Series. 2 Tepperman, J., H. M. Tepperman and M. P. Schul- amounts of carbohydrates in the diet re man 1955 Oxidation of palmitic acid-l-C14 by liver slices of fat and carbohydrate diet-adapted rats. sults in adaptation to a carbohydrate-oxi Federation Proc., 14: 152 (abstract). 3 The corn oil diet consistently gave approximately dizing type metabolism and loss of fatty a 10% growth response over the glucose diet. Chicks acid oxidizing ability. Such a hypothesis of similar body weight were chosen from each dietary treatment so that differences in metabolic rate due to becomes attractive as a partial explanation differences in body weight would not result. J. N u t r itio n , 82 : ’ 64 115 116 W. E. DONALDSON TABLE 1 Total C14 0 2 activity of samples collected Composition of test diets 1 from each chick was assayed by the method of Harlan.5 High- Excreta samples from each age group in Calbdiltdrate tat diet each experiment were pooled according to % % diet. The excreta were diluted to 250 ml Glucose 2 51.70 — with distilled water and mixed in a W aring Com oil 3 — 28.05 Blendor for 5 minutes. Duplicate 0.2-ml Soybean oil meal, samples were withdrawn and placed on 37.94 47.24 50% protein 20-ml counting vials containing scintil Cellulose 4 4.75 17.72 Trace mineralized salt 0.47 0.59 lator. The vials were prepared by filling Dicalcium phosphate 3.70 4.61 with thixotropic gel powder6 * and adding Ground limestone 0.95 1.18 15 ml of scintillator solution. Scintillator 0.02 0.03 Manganese sulfate solution was prepared by mixing 378 ml DL-Methionine 0.28 0.35 Vitamin mixture of absolute ethanol with 600 ml of toluene (glucose carrier)5 0.19 0.23 containing 0.4% PPO (2,5-diphenyloxa- zole) and 0.0015% dimethyl POPOP (1, i The calculated ratios of metabolizable energy to protein were constant for both diets. 4-bis - (2,4-m ethyl-5-phenyloxazolyl) - ben 2 Cerelose, Corn Products Company, New York. zene). The samples were counted and re 3 Fatty acid composition was determined by gas- liquid chromatography as follows: ( % ) myristic, counted after addition of toluene-C14 inter 1.08; palmitic, 9.18; stearic, 2.56; oleic, 29.38; lino- leic, 55.90; and linolenic, 1.90. Mazola, Corn Products nal standard. This method gave similar Company, New York. results to a procedure in which the excreta 4 Solka Floe, Brown Company, Boston. s Each 100 g of the vitamin mixture supplied: were extracted with diethyl ether-ethanol- (milligrams) thiamine• HC1, 176; Ca pantothenate, HC1 solvent and the extract counted in 924; niacin, 2640; riboflavin, 286; pyridoxine-HCl, 286; biotin, 8 .8 ; folic acid, 55; vitamin K, 39.6; vita toluene containing 0.4% PPO and 0.01% min E acetate, 1100; choline chloride, 6400; and POPOP. vitamin B12 , 440 /¿g; vitamin A 550,000 IU; and vitamin D3, 99,000 ICU. In experiment 3, the 14-day old chicks were killed after C 02 collection by dislocat results). The proper doses were placed in ing the neck. The carcasses from the gelatin capsules and inserted into the crops 3 chicks on each treatment were pooled of the chicks. Glucose was in water solu and passed through a meat grinder. The tion. Fatty acids were dissolved in benzene ground tissues were thoroughly mixed and solution and were pipetted into capsules 10-g aliquots were blended with 10 ml dis and the benzene removed under dry nitro tilled water for 5 minutes in a Servall gen. These chicks were then placed in Blendor. Duplicate 500-mg samples were glass metabolism cages. Respiratory C02 made up to 30 ml with distilled water and was collected for 24 hours by means of were extracted by the addition of 1 ml drawing air through the cages with a vac concentrated HC1 and 20 ml diethyl ether. uum pump and bubbling it through 200 ml The samples were shaken for 5 minutes, o f 2 .5 n NaOH for 1- and 7-day-old chicks 20 ml of 95% ethanol were added and and 200 ml of 5.0 n NaOH for 14-day-old shaking continued for another 5 minutes. chicks. Excreta were collected for 24 The ether layer was withdrawn by pipette hours. Feed and water were available ad and the samples were washed with three libitum during the experiments. The chicks 10-ml portions of ether. Twenty milliliters were fed the same diets while in the of ether were added and the samples again metabolism cages as previously except that were shaken for 5 minutes. The ether was in experiment 4 the chicks were starved withdrawn and the samples washed as for 2 hours before being given the C14 dose previously. The ether extracts and wash after which the diets were reversed. ings were pooled and evaporated under Chem ical procedures. All radioassays were made using a liquid scintillation spec 4 Tri-Carb, Packard Instrument Company, La Grange, Illinois. trometer.4 All samples were assayed suffi 5 Harlan, J. W. 1961 Liquid scintillation counting ciently long to reduce counting error to of aqueous carbonate solutions. Atomlight, no. 18: 8-11. Published by New England Nuclear Corpora < 1.25% . Background ranged between 55 tion, Boston. 6 Cab-O-Sil, Packard Instrument Company, La and 65 count/min. Grange, Illinois. DIET ADAPTATION IN CHICKS 117 dry nitrogen. The residues were weighed total amount of linoleic acid oxidized was and taken up in 15 ml of scintillator solu greater than the total palmitic acid oxida tion and radioassayed by the same method tion in the fat-fed chicks (assuming that as the excreta samples. The water-ethanol the proportions of labeled to unlabeled layer was sampled and reduced to < 0.5 ml fatty acids oxidized were constant) since under dry nitrogen. These samples were the com oil contained more linoleic than radioassayed as above. Moisture determi palmitic (55.9% vs. 9.2% ). The data sug nations were made on the tissues by drying gest that the chick oxidizes fat per se and for 24 hours at 90°C. that the proportion of a fatty acid that is oxidized is not affected by the level of that RESULTS AND DISCUSSION fatty acid in the dietary fat. The omission The first experiment was designed to of added choline chloride from both diets measure the influence of dietary energy in experiment 2 did not result in markedly source on fatty acid oxidation. From the decreased C14 0 2 production from the la working hypothesis, a relatively high rate beled fatty acid as compared to experiment of fatty acid oxidation was assumed in 1. The calculated levels of choline (1100 newly hatched chicks. Furthermore, the and 1320 mg/kg in the carbohydrate and oxidation rate presumably would be main fat diets, respectively) appeared to be ade tained with fat feeding and decrease with quate for normal fatty acid oxidation. carbohydrate feeding. The results, shown Experiment 3 was designed to determine in table 2, support the hypothesis. Al whether dietary energy source affected glu though the standard errors of the mean cose oxidation. The working hypothesis C14 CL. production were high, at 14 days of suggested that glucose oxidation would be age, the differences in oxidation rate be m inim al in a newly hatched chick and that tween carbohydrate- and fat-fed chicks was the m inim al oxidation level would be m ain statistically significant (P = 0.01). tained relatively constant with age when Table 3 shows the recovery of C14 0 2 fat was fed. Conversely, carbohydrate from one-day-old chicks in experiment 1 as feeding would result in increased glucose a function of time. These data indicate oxidation with age. The results are shown that the differences in C14 0 2 recovery are in table 5. In contrast to expectation, a reflection of differing oxidation rates and C14 0 2 recovery was relatively constant re not of a time lag in absorption of the gardless of diet. labeled fatty acid. The calculated percentage of the total The second experiment was designed to calories contributed by protein, carbohy determine whether the oxidation rate of a drate and fat to the experimental diets are fatty acid is related to the level of that shown in table 6. Comparison of these fatty acid in the diet. The results are calculations with the observed oxidation shown in table 4. The pattern of C14 0 2 rates of labeled fatty acid or glucose shows recovery was similar when either labeled that the total quantities of fatty acid oxi palmitic or linoleic acids were fed. The dized were greater with the fat diet, and TABLE 2 Effect of diet on palm itic-l-C14 acid oxidation by chicks (exp. 1) C14 Avg Apparent C14 C14 o2 Diet Age 1 dosage 2 body wt retention v produced 3 days fic / c h ic k 9 % % CHO i 4 43 93.6 42.2 ±6.60 Fat i 4 43 92.9 51.7 ±8.32 CHO 7 3 72 95.0 32.8 ±9.05 Fat 7 3 72 96.7 48.8 ±4.80 CHO 14 4 135 83.1 24.3 ±5.93 Fat 14 4 135 97.5 54.5 ±1.58 1 Three chicks per treatment. 2 Palmitic-l-C14 acid, specific activity 10 mc/mmole. 3 Mean values of % absorbed dose recovered ± s e . 118 W. E. DONALDSON conversely the total quantities of glucose et al. (1 ) observed a 5% difference in oxidized were greater on the carbohydrate nitrogen retention between glucose- and diet. Total calories oxidized were calcu corn oil-fed chicks. This 5% difference lated by applying oxidation rates for 14- would result in a negligible difference be day-old chicks from experiments 1 and 3 tween diets in protein calories oxidized. to the calorie values for the diets. These These calculations suggested that if carbo calculations are also shown in table 6. It hydrate-fed chicks were suddenly shifted was assumed that the protein calories oxi to the fat diet, the level of fatty acid oxida dized were similar for both diets. Rand tion would have to increase or else meta bolic rate would decrease. TABLE 3 In experiment 4 chicks maintained with E ffe c t of diet on palmitic-l-C14 acid oxidation by a high-carbohydrate diet were shifted to a c h ic k s at various time intervals after high-fat diet immediately after labeled C14 administration (exp. 1) fatty acid administration at 14 days of age. The data in table 7 show clearly that diet Hours after C14 C44 o 2 Diet administration 1 produced 2 reversal results in reversal of C14 0 2 produc tion (viz., level of C14 0 2 production from CHO 2 12.6 4 22.0 labeled fatty acid is a function of diet dur 6 27.2 ing C02 collection and not prior dietary 24 42.2 experience). It is concluded that 1) the Fat 2 12.1 chick is able to adjust its metabolism to 4 26.9 accommodate radical changes in dietary 6 35.8 energy source in relatively short periods of 24 51.7 time, and 2) any shift in glucose or fatty 1 Three day-old chicks per treatment. See table 2 for acid oxidizing enzyme levels due to feeding details of treatment. is not great enough to appreciably affect 2 Cumulative mean values of % absorbed dose recovered. oxidation rate. TABLE 4 E ffe c t o f diet on palm itic-l-C11 a cid and linoleic-l-C 14 a cid oxidation by chicks (exp. 2) C14 Diet Label Age i Avg Apparent C14 C44 02 dosage 2 body wt retention produced 3 days fie/ch icli 9 % % CHO palmitic 13 4 127 87.5 24.4±4.70 Fat palmitic 13 4 127 90.7 50.0 ± 5.76 CHO linoleic 14 4 129 90.9 17.6 ±3.22 Fat linoleic 14 4 133 93.9 44.2 ±0.57 1 Three chicks per treatment. 2 Palmitic-l-C14 acid and linoleic-l-C14 acid, specific activities 5 mc/mmole. 3 Mean values of % absorbed dose recovered ± s e . TABLE 5 E f f e c t of diet on uniformly labeled d-glucose-C14 oxidation by chicks (exp. 3) C1-» Avg Diet Age 1 Apparent C14 C14 o2 dosage 2 body wt retention produced 3 days n c /chicli 9 % % CHO i i 39 95.4 46.9 ± 0.57 Fat i i 39 94.3 45.6 ± 5.89 CHO 7 2 73 97.3 45.7 ± 1.47 Fat 7 2 73 94.1 49.5 ± 1.78 CHO 14 2 138 93.5 54.1 ±1.73 Fat 14 2 137 87.5 56.9 ±3.22 1 Three chicks per treatment. 2 Uniformly labeled D-glucose*C14, specific activity 3.2 mc/mmole. 3 Mean values of % absorbed dose recovered + s e . DIET ADAPTATION IN CHICKS 119 The data on glucose and fatty acid oxi fed birds. Based on calculated diet anal dation do not establish a metabolic basis ysis, total carbohydrate-to-fat conversion for growth response of chicks to dietary was approximately 12 times higher per fat. A possible metabolic basis is provided 100 g of feed consumed with the carbo by carcass analysis data from experiment hydrate diet. Total energy available for 3 (glucose-C14 administration). N o statis tissue synthesis should be comparable for tical analysis was possible since pooled chicks fed both diets if we assume that en samples were used. Carcass moisture and ergy requirements for maintenance and fat content were slightly lower in the fat- body activity are similar. This implies that fed chicks. The data show that the carbo since carbohydrate-fed chicks require a hydrate-fed chicks converted considerably measureable amount of energy to synthe more carbohydrate to fat than did the fat- size tissue fat (whereas fat-fed chicks de posit dietary fatty acids per se), energy TABLE 6 available for tissue protein synthesis is re Calculated caloric composition of and energy duced, and growth is reduced. If body production from the experimental diets weight gain decreases in comparison with fat-fed chicks, total energy requirements CHO diet Fat diet also decrease. But, at any given body % of total calories from : weight, total energy available for tissue Protein 21.9 22.1 protein synthesis could be reduced because CHO 77.6 18.2 of the requirement for tissue fat synthesis. Fat 0.5 59.7 For the above hypothesis to be true, nitro Kilocalories oxidized/100 kcal consumed: 1 gen retention must be poorer with carbo Protein Constant (K )2 Constant (K )2 hydrate feeding. Biely and March (4 ) ob CHO 41.9 10.3 Fat 0.1 32.5 served that dietary fat had no consistent effect on nitrogen retention in the chick. Total 42.0+ K 42.8 + K These workers used a balance technique in 1 Based on C14 O2 recovery from 14-day-old chicks which they measured feed intake and col from experiments 1 and 3. lected feces and urine during successive 2 It was assumed that amino acid oxidation was similar on both diets. 24-hour periods, but no marker was incor- TABLE 7 Effect of diet reversal during C14 O 2 collection on palm itic-l-C11 acid oxidation by chicks (exp. 4)1 Diet C14 Avg Apparent C14 C14 O2 Diet during C14 O2 Age 2 collection dosage 3 body wt retention produced 4 days yc/chick 9 % % CHO Fat 14 2 139 7 5 . 1 52.8 ±1 .8 2 Fat CHO 14 2 140 90.6 40.0 ± 1.00 _,_.1 Chicks were starved for 2 hours, administered the C14 dose and fed the opposite diet during CO2 collection. 2 Three chicks per treatment. 3 Palmitic-l-C14 acid, specific activity 10 me/mmole. 4 Mean values of % absorbed dose recovered ± s e . TABLE 8 Effect of diet on carcass composition and deposition of uniformly labeled D-glucose-C14 in chicks (exp. 3)1 Absorbed dose in tissues Diet Age Avg Carcass Carcass body wt moisture fat H20- Fat- soluble soluble days 9 % % % % CHO 14 138 71.3 11.5 19.6 14.4 Fat 14 137 70.0 9.7 31.0 5.0 1 All values based on pooled samples of 3 chicks. 120 W. E. DONALDSON porated in the feed. Rand et al. (1) showed by a failure of the reaction acetyl coen that dietary fat gave consistent improve zyme A to malonyl coenzyme A and not by ments in nitrogen retention. In these stud reduced TPN H supply or availability. ies, carcass protein analyses were used to Although there is question as to how calculate nitrogen retention. The experi feeding fat inhibits lipogenesis, there is no ments cited are typical of numerous con question that inhibition occurs. The re flicting reports in the literature concerning sults presented herein support this concept. dietary fat and nitrogen retention. Tepperman and Tepperman (5) reviewed LITERATURE CITED extensive data on carbohydrate oxidation 1. Rand, N. T., H. M. Scott and F. A. Kum- and lipogenesis. The data suggest that merow 1958 Dietary fat in the nutrition when the liver is exposed to increased con of the growing chick. Poultry Sci., 37; 1075. 2. Tepperman, H. M., and J. Tepperman 1955 centrations of glucose (such as with car Ketogenesis in rats on high carbohydrate and bohydrate feeding) hexose monophosphate high fat diets. Am. J. Physiol., 180: 511. shunt (H M P) activity is increased. In 3. Boell, E. J. 1955 Energy exchange and en creased HMP activity results in an in zyme development during embryogenesis. In Analysis of Development, eds., B. H. W illier, creased supply of reduced triphosphopyri- P.A. Weiss and V. Hamburger. W. B. Saun dine nucleotide (TPN H ). Since TPNH is ders Company, Philadelphia. required for reductive fatty acid synthesis, 4 Biely, J., and B. March 1957 Fat studies it is postulated that control of lipogenesis in poultry. 7. Fat and nitrogen retention in chicks fed diets containing different levels is regulated by TPN H availability. Glucose of fat and protein. Poultry Sci., 36; 1235. oxidation under conditions of fat feeding 5. Tepperman, J., and H. M. Tepperman 1960 follows the gycolytic pathway, and HMP Some effects of hormones on cells and cell activity is reduced. constituents. Pharmacol. Rev., 12; 301. 6. Bortz, W., S. Abraham and I. L. Chaikoff More recently, Bortz et al. (6 ) presented 1963 Localization of the block in lipogenesis convincing evidence that the block in lipo resulting from feeding fat. J. Biol. Chem., genesis resulting from feeding fat is caused 238; 1266. Effect of Calcium, Phosphorus and Manganese on the Intestinal Synthesis of Thiamine in Rats S. K. M EGHAL a n d M. C. N A T H University Department of Biochemistry, Amravati Road, Nagpur, India ABSTRACT When rats were fed thiamine-inadequate diets containing different levels of calcium and phosphorus with or without manganese, the urinary and fecal excretion of thiamine increased in rats receiving a low level of calcium and phos phorus and supplementation of manganese to this diet resulted in more urinary and fecal thiamine excretion. Higher levels of calcium and phosphorus in the diets, with or without manganese, and diets without an added amount of these minerals decreased the urinary and fecal excretion of thiamine. The presence of manganese in the diets reduced the lactobacilli count in the cecum and increased the coliform count. Rats receiving low calcium and phosphorus diets with or without manganese stored more thiamine in the liver and cecum than did other groups, whereas no increase was noted in the thiamine content of the heart. This observation indicates that low dietary cal cium and phosphorus, with or without manganese, enhanced thiamine synthesis by the intestinal flora. This thiamine might be ultimately available to rats after coprophagy. A great deal of research has been car ferent amounts of calcium and phosphorus ried out on the effect of carbohydrates, with or without manganese on the intesti fats, proteins and antibiotics, etc., on the nal flora and biosynthesis of thiamine. intestinal synthesis of thiamine, but little attention has been directed toward the EXPERIMENTAL influence of salts and minerals. The in Weanling albino rats were used in this testinal bacteria present in animals are study. They were grouped uniformly with coliforms, anaerobes and lactics. O f these respect to sex, weight and litter, and were 3 microbes, the coliform organisms have caged individually, with food and water been shown to synthesize thiamine to the given ad libitum. They were fed the thia greatest extent (1 ). Recently we have re mine-inadequate diet for 5 days. The thia ported that the intestinal flora and thia mine-inadequate basal diet contained: (in mine synthesis depend upon the dietary per cent) vitamin-free casein (acid alco constituents (2, 3). Friedman (4) reported hol-extracted), 25; dextrin, 20; sucrose, that when rats became rachitic, the num 37; groundnut oil, 15; and salt mixture ber of acid-producing organisms dimin (10), 1.5. The following vitamins were ished, whereas non-acid-producing organ added per kilogram of diet: (in m illigram s) isms increased, and also that rachitic rats thiamine-HCl, 0.4; riboflavin, 7; pyri- had alkaline feces. Eppright et al. (5 ) doxine-HCl, 5; Ca pantothenate, 40; nia studied the influence of mineral salts on cin, 40; inositol, 200; biotin, 0.2; folic the intestinal flora and postulated from acid, 2; vitamin B>2, 0.1; 2-methyl naph their experiment that a diet low in inor thoquinone, 10; vitam in A acetate, 3; vita ganic salts favored coliform flora. min D, 0.04; and a-tocopheryl acetate, 50. The essentiality of dietary manganese The salt mixture recommended by for rats was demonstrated by Orent and Krieger and Steenbock (10) was used be McCollum (6 ) and Kemmerer et al. (7). cause it is free from calcium and phos Hill and Holtkamp (8 ) have recently re phorus. For this experiment manganese ported that storage of thiamine in liver sulfate was also removed from this salt increased with supplementation of man mixture. The required amount of calcium ganese and thiamine. Balakrishnan and was added to each diet as calcium carbo De (9 ) have reported that a diet low in nate, and phosphorus as an equimolar phosphorus and high in calcium enhanced mixture of monobasic and dibasic phos- thiamine synthesis in rats. The present paper presents the results of feeding dif Received for publication September 9, 1963. J. N u t r it io n , 82: ’ 64 121 122 S. K. MEGHAL AND M. C. NATH phates of potassium (11). The details of Each liver and heart was weighed and the amounts of calcium and phosphorus frozen and the thiamine content of each in the diets are given in table 1. When sample was determined by the method of manganese was incorporated in the diet it Kratzing and Slater (15). The ceca of was fed at a level of 0 . 0 1 4 % manganese 3 rats from each group were removed and as manganese chloride. their contents were collected immediately Rats were divided into 10 groups, with under aseptic conditions. The cecal con 6 rats per group. The first 5 groups re tents of each rat were refrigerated at ceived diets A, B, C, D, and E without 4° C before the next rat was killed. The manganese, respectively, and the remain cecal contents obtained from 3 rats in ing groups received the same diets as each group were pooled, mixed and 0.1 g described above, respectively, but supple of the mixed sample was weighed asep mented with manganese. Since rats were tically. The weighed samples were sus kept in wire-bottom cages, they consumed pended in 10 ml of sterile saline (0.9% ) their feces along with the diets and no and serial dilutions were made in sterile care was taken to prevent coprophagy dur saline. Plate counts of the numbers of ing the experiment. The experiment was organisms in the cecal contents were made carried out for 4 weeks. Urine and feces with Mackonkey’s agar media for coli were collected the last 3 days of the week. form bacteria, with nutrient agar media Urinary thiamine was determined by the for “total number of organisms,” and the thiochrome procedure as modified by Maw- lactobacillus selection medium (16) for son and Thompson (12), and fecal thia lactobacilli. All plates were incubated at mine as described by the Association of 37° C for 72 hours before counts were Vitamin Chemists (13). made of the colonies. During the 4-week experiment, the The cecal contents of the remaining rats weekly fecal coliform count was taken of 3 from each group were weighed and di rats from each group. The feces of each rat gested in a flask with a sufficient amount were collected aseptically the last 3 days of o f 0.1 n HC1; samples were then prepared the week on sterile filter paper sheets and in the recommended manner and total the sheets were pressed together to remove thiamine was estimated by the method of as much moisture as possible and then Hennessey and Cerecedo (17). the feces were transferred to weighed RESULTS sterile test tubes. A uniform suspension of about 0.1 g of feces with 10 ml of saline The effect of different levels of calcium (0.9% NaCl solution) was made under and phosphorus with or without manga aseptic conditions. Further dilutions were nese on the urinary and fecal excretion of made with the saline, and the coliform thiamine is presented in table 2. The daily colonies in the feces were counted by the urinary and fecal excretion of thiamine plate method, using Mackonkey’s agar increased in the group receiving low cal medium (14). cium and low phosphorus (diet D ) with At the end of the fourth week, all ani out manganese. Manganese supplementa mals were killed by a sharp blow on the tion of the above diet also increased daily head and the livers and hearts excised. urinary and fecal excretion of thiamine TABLE 1 Calchivi and phosphorus composition of diets 1 Diets A B C D E Ca (CaC03), % o f diet 0.42 0 . 1 0 0.42 0 . 1 0 __ P (KH..PO4 ) 0.22 0 . 2 2 0.05 0.05 — P (K 5HPO 4 ) 0.10 0 . 1 0 0.025 0.025 — P from casein 2 0.22 0 . 2 2 0.22 0.22 0 . 2 2 Total P, % of diet 0.54 0 . 5 4 0.295 0.295 0 . 2 2 1 These minerals were given along with the basal diet. 2 About 0.86% P. Effect of different levels of calcium and phosphorus with and without supplementation of manganese on the urinary and fecal excretion of thiamine P 0) 0) X o 00 CO c 6 6 o 6 o o O O 6 6 6 6 6 6 6 6 6 CM CO LO CO rf 6 o 6 o 6 o 6 6 o CO o CO CO rP CM 6 6 6 10 O CO ' CD CM CO 6 6 O CO co o o i m c co co o c io io m c m c O m c 3 A PH A .S? A V s o .% + w> w $ q is m s w e r e re d u c e d in ra ts r e c e iv in g th e e th g in iv e c e r ts ra in d e c u d re e v e a r e h w to s r ru a s o e h p m p sp is a o h p e s e d n n a a g n a m m iu s t lc ow u a o c sh f ith o w e c or n ls e ia v r ith a le w v w lo f o e h T is s ly a n a e h T rats. f o hi di showed a i i ease. s is h T to . e s a e s re e c n in a g n t a h m lig s f a o d e n w o h itio s d d a t ie m d r n h g a lifo u g o is o r c o th th m l r a c n lifo e fe o v e c e e h th T n o rats. e th t c f o ffe e s m is n le a g b r a o r o v are fa t n a u o c m r lifo o c l a c n fe a f m o ta in a te d a u t q n ese e ie d th a ffic t -in u a s e th in m r o ia th t n ie fic e e d th r e ith e in t ie d s ru o h sp o h p nt i for Di A ( mal ci m iu lc a c l a im t p (o A t ie D . ra flo l a tin s te in orm or sms; a i efect s a w t c ffe e t h lig s a t u b ; s m is n a rg o m r fo t ie d t. c is th ffe e le to b a r o e v s e fa n a y g n n a a m e v a h f o t o n n id d tio ta n e m le p ow phosphorus). s u r o h p s o h p w lo hi ne-nadequat et t cal um m iu lc a c t u o ith w t ie d te a u q e d a -in e in m ia th . ) 8 1 ( l e v le % 5 at t n a ific n t n ig u s o c lly m a r tic lifo tis o c ta s l a c fe ly k e e w e th n o e s e n a g m iu lc a c f o ls e e v v o le b a r e e th h ig to h d e c ia ed n , d r u th d e o a v n e ro f w s p o a o w e H r o is e s m e s e th s n a a yn w s g iet. n t d a c l m a ffe e tin n s e is h te th w in d n a e th e in m n o t c ffe e i or he at vi ow w lo e g h in T iv e c e r s. p ts grou ra ll e a th f in o e e g m a ra s to s e th tic a s p e a h w h ic h w e th n o t c ffe e le b a r o v fa e m so d a h e s e n a g e in m ia th f o n tio e r c x e l a c fe d n a y r a in r u f on t f coli o c l a c fe e th n o t c ffe e o n d a h e s e n a g n a m th e e ffe c t o f d iffe r e n t le v e ls o f c a lc iu m an d d an m iu lc a c f o ls e v le t n e r iffe d f o t c ffe e e th g in c n a h n e w an lo d t a a h th t s ie d te a ic d in te a u q e is d h a T -in e in m . n tio e r c x e l a c e f p ro v e d th e th ia m in e c o n te n t o f liv e r an d d an r e liv f o t im n te e s n e o n c a g n e a in m m ia ith th w t e ie th d is d th e v f o ro p n tio ta n e m n a m t a th n io s lu c n o c e th to s d a le ly ite fin e d e th d e s a re c e d ) A t ie (d s ru o h sp o h p d n a a n d o p tim a l p h o s p h o r u s ) w ith o r w ith o u t t u o ith w r o m iu lc a ith c w ) w s o u l r ( o h p B s o t h ie p d l d a n a tim p ) o s u r d o n h a p s o h p d n a p u s n e v e d n a t n e im r e p x e e th t u o h g u ro th s h o w n w ith d ie t C (o p t im a l c a lc iu m and and m iu lc a c l a im t p (o C t ie d ith w n w o h s ci ow phos us i he hi ia th e th in s ru o h sp o h p w lo d n a m iu lc a c f o t hi ne e in m ia th rt a e h on t c ffe le e p p o u n s d a h ; ) t u b 4 m u le c e b c a (t e g ra to s e in m ia th e tissu gnii l e n t s recei ng g in iv e c e r s a w ose th it ). d 1 an in .0 0 A < d t e ie c d P u ( red g E in h iv t it e ie w c tly d e n r r a o ific ose n ith th ig w s s f ru o o h t sp a o h th p w lo d n a m iu lc a c cal um and manganese ( et E) had d a h ) E t ie (d e s e n a g n a m d n a m iu lc a c t u o ae ncreased i hoe at recei ng g in iv e c e r ts ra ose th in d e s a e r c in are t n u o c n i biory efect he nary a d an y r a in r u e th n o t c ith ffe w e t ie d y r e h ito T ib h in t. n e an rim e p x e e th t u o h g u ro th et t wihout Our u O . e s e n a g n a m t u o ith w r o ith w D t ie d utmanganese was gr er ( < 01) han n a th ) 1 .0 0 < (P r te a re g s a w e s e n a g n a m t ou n t e 3 a ae esent ng g in w o h s d te n e s re p are ta a d 3 le b ta In cont s a he iorm m r lifo o c e th d an ts n te n o c l a c e c e h T the d e s a e r c in ) P w lo + a C w o l ( D t ie D 123 124 S. K. MEGHAL AND M. C. NATH TABLE 3 Effect of different levels of calcium and phosphorus with or without manganese on the weekly fecal coliform count Coliform counts in weeks D iet1 Without Mn With Mn 1 2 3 4 1 2 3 4 A 4.91 2 4.94 4.93 4.85 4.94 5.01 5.03 5.03 B 4.89 4.88 4.87 4.94 4.99 4.91 5.03 4.97 C 5.00 5.07 5.08 5.10 5.12 5.14 5.17 5.16 D 5.00 5.11 5.19 5.23 5.25 5.26 5.26 5.27 E 4.68 4.50 4.44 4.44 4.66 4.57 4.51 4.44 1 Diet A, high Ca + high P; diet B, low Ca + high P; diet C, high Ca + low P; diet D, low Ca + low P; and diet E, very low P without Ca. 2 Average logio number of bacteria per gram of wet feces. TABLE 4 Liver, heart and cecal thiamine and cecal flora as influenced by calcium, phosphorus and manganese Logio number of bacteria/g Liver Heart Cecal Total wet cecal contents D iet1 thiamine 2 thiamine 2 cecal thiamine 2 contents n ^ Total Lacto- Coliform count bacilli M / g g-g/g v-g/g 9 Without supplementation of Mn A 1.32 ±0.08 3 1.20 ±0.02 1.25 ±0.04 2.02 4.65 5.07 4.91 B 1.08 ±0.03 1.08 ±0.04 1.01 ±0.03 1.66 4.34 4.90 4.78 C 1.60± 0.06 1.09±0.07 1.32 ±0.04 2.10 4.92 5.23 4.91 D 2.28 ±0.08 4 1.22± 0.06 1.89 ±0.06 3.12 5.14 5.39 4.76 E 0.88 ±0 .0 2 5 0.98 ±0.03 0.98 ±0.02 1.40 4.07 4.69 5.45 With supplementation of Mn A 1.65±0.05 1.21 ±0.06 1.58 ±0.03 2.16 4.71 5.14 3.93 B 1.05 ±0.03 1.12± 0.03 1.51 ±0.02 1.74 4.36 4.95 4.04 C 2.07 ±0 .0 7 1.18±0.02 1.52 ±0.03 2.80 4.96 5.30 4.50 D 3.29 ±0.06 * 1.28 ±0.03 2.37 ±0.09 3.95 5.44 5.44 4.50 E 0.92 ±0.03 5 1.19±0.05 0.93 ±0.03 1.52 4.07 4.71 4.35 1 Diet A, high Ca + high P; diet B, low Ca + high P; diet C, high Ca + low P; diet D, low Ca + low P; diet E, very low P without Ca. 2 On wet-weight basis. 3 se of mean. 4 Significantly increased from diet A at P < 0.01. 5 Significantly reduced from diet A atP < 0.01. results also indicate that those fed diets DISCUSSION without manganese showed higher lacto- Inclusion of the Osborne and Mendel bacillus counts than those receiving man salt mixture by Eppright et al. (5 ) in the ganese. This indicates that manganese diet of rats produced an intestinal flora might have an inhibitory effect on lacto- predominating lactobacilli and it has been bacilli and might be favorable for coliform further reported that, when calcium and organisms. phosphorus were added to a low salt diet, The growth data (figs. 1, 2) show that the flora was high in lactobacilli. Our diet D without manganese increased the results confirm that low calcium and low body weight of rats and supplementation phosphorus increased the coliform count of manganese to this diet had more pro and decreased the lactobacilli. Recent nounced effect on the body weight of rats. work of Balakrishnan and De (9 ) shows However, the diet with low phosphorus that high calcium and low phosphorus in resulted in a decrease in body weight. The creased the urinary and fecal excretion statistical analysis shows that these growth of thiamine in the presence of dietary data are significant at 5% level. manganese. Our results, however, indicate MINERALS AND THIAMINE BIOSYNTHESIS 125 A, high Ca+ high P B, low Ca + high P C, high Ca + low P D, low Ca + low P E, very low P without Ca WEEKS Fig. 1 Effect of different levels of calcium and phosphorus without manganese in thia mine-inadequate diet on weekly growth of rats. § O A, high Ca+ high P 05 H B, low Ca + high P X C O 3 A C, high Ca + low P £ D, low Ca + low P !* Q 8 O E, very low P without Ca M E Z WEEKS Fig. 2 Effect of different levels of calcium and phosphorus with manganese in thiamine- inadequate diet on weekly growth of rats. that more thiamine was excreted in urine min content (niacin, riboflavin and panto and feces with a diet low in calcium and thenic acid) of livers of duck. Our results, phosphorus even though manganese was with rats, however, indicate that the man absent from the diet. Supplementation of ganese increases the thiamine storage in manganese to the above diet increased the the liver of rats and reduces cecal lacto- rate of thiamine excretion in urine and bacilli. Hill and Holtkamp (8 ) have shown feces. Reen and Pearson (1 9) reported that supplementation of the diet to an in that during manganese deficiency there take of 0.17 mg of manganese/day, in was no significant alteration in the vita creased the average thiamine content of 126 S. K. MEGHAL AND M. C. NATH liver and, furthermore, an increase of the 7. Kemmerer, A. R., C. A. Elvehjem and E. B manganese intake did not affect the thia Hart 1931 Studies on the relation of man ganese to the nutrition of mouse. J. Biol. mine storage. The metabolic interrelation Chem., 92; 623. ship between thiamine and manganese 8. Hill, R. M., and D. E. Holtkamp 1954 was first reported by Perla et al. (20), but Storage of dietary manganese and thiamine from our results it is possible that this in the rat. J. Nutrition, 53; 73. 9. Balakrishnan, S., and S. S. De 1951 Effect effect of manganese may be attributed to of calcium and phosphorus on the intestinal the change in the intestinal flora brought synthesis of vitamin Bi. Indian J. Physiol. about by its presence in the diet. The same Allied Sci., 5; 123. reasoning applies to the effect of calcium 10. Krieger, C. H., and H. Steenbock 1940 and phosphorus which have been reported Cereals and rickets. XII. The effect of cal cium and vitamin D on the availability of to affect the intestinal flora. It is also well phosphorus. J. Nutrition, 20; 125. established that the intestinal flora is 11. Fuhr, I., and H. Steenbock 1943 Effect known to synthesize thiamine. of dietary calcium, phosphorus and vitamin The results of our present investigations D on the utilization of iron. II. Effect of vitamin D on body iron and hemoglobin pro indicate that low calcium and low phos duction. J. Biol. Chem., 147: 65. phorus with or without manganese in the 12. Mawson, E. H., and S. Y. Thompson 1948 thiamine-inadequate diet enhances more A note on the estimation of vitamin Bi in thiamine synthesis than high calcium and urine. Biochem. J., 43; 2. low phosphorus in the diet as reported by 13. Association of Vitamin Chemists 1951 Methods of Vitamin Assay, ed. 2. Interscience Balakrishnan and De (9 ). It has also been Publishers, Inc., New York. observed in this laboratory that coproph- 14. Balakrishnan, S., and R. Rajagopalan 1952 agy is essential in order to make available Studies on the influence of feeding milk and flora-synthesized thiamine and to promote curd on the intestinal synthesis of vitamin Bi. Indian J. Physiol. Allied Sci., 6; 143. synthesis of thiamine in rats (21). 15. Kratzing, C. C., and E. C. Slater 1950 Effect of sulfonamides on the aneurine econ LITERATURE CITED omy in animals. Biochem. J., 47; 24. 1. Najjar, V. A., and R. Barrett 1945 The 16. Rogosa, M., A. J. Mitchell and R. F. W ise synthesis of B vitamins by intestinal bac man 1951 A selective medium for the teria. Vitamins and Hormones, 3: 23. isolation and enumeration of oral and fecal 2. Nath, M. C., and S. K. Meghal 1961 Effect lactobacilli. J. Bact., 62; 132. of carbohydrates on the intestinal synthesis 17. Hennessey, D. J., and L. R. Cerecedo 1939 of thiamine in rats. Biochem. J., 81: 220. The determination of free and phosphor- 3. Meghal, S. K., and M. C. Nath 1962 In ylated thiamine by a modified thiochrome hibitory effect of sulfaguanidine on the bio assay. J. Am. Chem. Soc., 61: 179. synthesis of thiamine in rats and the bene 18. Snedecor, G. W. 1956 Statistical Methods, ficial role of penicillin and hydrolyzed glu ed. 5. Iowa State College Press, Ames. cose cycloacetoacetate. J. Nutrition, 76: 265. 19. Reen, R. V., and B. Pearson 1955 Man 4. Friedman, H. 1936 Study of the fecal ganese deficiency in the duck. J. Nutrition, flora and lime test of normal rats, rachitic 55; 225. rats and healing rachitic rats. J. Nutrition, 20. Perla, D., M. Sandberg and O. M. Holly 12: 165. 1939 Interdependence of vitamin Bi and 5. Eppright, E. S., G. Valley and A. H. Smith manganese. III. Manganese, copper and iron 1937 Influence of the salt in the diet on the metabolism in normal rats. Proc. Soc. Exp. intestinal flora of the albino rat. J. Bact., Biol. Med., 42; 371. 34; 81. 21. Meghal, S. K., and M. C. Nath 1963 Effect 6. Orent, E., and E. V. McCollum 1931 Effect of prevention of coprophagy on the thiamine of deprivation of manganese in the rat. J. sparing action of mustard. Nature (Lond.). Biol. Chem., 92: 651. 198: 89. Effect of the Quality of Dietary Protein on Nitrogen Compounds in the Urine of Rats SHUHACHI KIRIYAMA* 1 a n d KIYOSHI ASHIDA Laboratory of Food and Nutrition, Department of Agricultural Chemistry, Nagoya University, Anjo, Aichi, Japan ABSTRACT To learn in more detail the effect of dietary protein quality on the ratio of urinary N compounds of rats, the excretion rate of urea, aliantoin, creatinine and their partition were determined when the rats of 4 ages (21-, 27-, 61- and 158- day-old) were fed each diet containing casein and wheat gluten at the 14% level. The nutritive value of both proteins was simultaneously compared using the usual methods for biological value, net protein utilization, net protein ratio and the ratio of creatinine N to total urinary N. In general, urea excretion increased with the de crease of protein quality and increased with protein intake or age, or both. Although aliantoin excretion was more complex and less variable than urea excretion, that of the casein groups was consistently higher than that of gluten groups except in the case of the 158-day-old rats. In using the ratios between urinary N compounds to describe nutritive value of dietary protein, the values for (allantoin/urea) X protein intake were comparable to that of other criteria: biological value or net protein ratio, al though the effect of age and dietary protein quality on aliantoin excretion needs fur ther investigation. It is well known that Folin (1 ) investi which is one of the “lowest” as protein gated indirectly the metabolism of body source. This study showed great variations protein by analyzing the urinary N com in urea and aliantoin excretion. pounds. Relatively few studies on the rela The present paper deals with a survey tionship between urinary N constituents of the change of N compounds in urine of and dietary protein quality have been re rats fed casein and gluten as the protein ported. Two of these are the pioneering source at 4 defined stages of age, and with investigations of Murlin et al. (2, 3) in a comparison between prevailing methods which protein quality was expressed by the to evaluate protein quality biologically. percentage of creatinine N in total uri nary N. METHODS AND MATERIALS The change in either the quantity or Sixty Wistar-strain male rats were used quality, or of both, of the ingested protein throughout this study. Four separate ex causes alteration of body protein metabo periments were carried out using rats of lism which influences the relative excre 4 ages, 21. 27, 61. and 158 days old, re tion rates of catabolic end products of spectively. protein and of other materials related to In each experiment, 15 rats were se protein metabolism (mainly N compounds). lected from standardized animals that had If some regularity is found in the inter been fed a “standard” 25% casein diet for relationship between protein quality and one week. They were then divided into urinary N compounds, it will become pos 3 subgroups, and started to be fed the test sible by the analysis of urinary N com diets A, B, and C as shown in table 1. All pounds to determine the quality of dietary diets were fed ad libitum. Immediately protein. Murlin’s investigations were also before daily feeding, the synthetic test along this line. diets were mixed with water and made In a preceding report (4 ), the authors into a soft ball. The animals older than determined urinary urea, aliantoin, and 27 days of age had been maintained be- creatinine, etc., when rats were fed a diet containing at various levels, egg albumin which is one of the proteins with the Received for publication August 1, 1963. 1 Present address: The National Institute of Nutri “highest” biological value, and gelatin tion, 1 Toyamacho, Shinjukuku, Tokyo, Japan. J. N u t r it io n , 82: ’ 64 127 128 SHUHACHI KIRI Y AM A AND KIYOSHI ASHIDA TABLE 1 Composition of “standard” and test diets “Standard” Test diets 1 Constituents diet A B C % % % % a-Starch 2 65 89 75 75 Casein 25 — 14 — Gluten — — — 14 Sesame oil 5 5 5 5 Salts-B 3 4 4 4 4 Vitamins 4 1 1 1 1 Feces’ marker (C r203 or Si02) — 1 1 1 Total 100 100 100 100 Average protein content ( N X 6.25) 21.0 0 11.5 11.9 1 The test diets for 21- and 158-day-old rats contained one per cent more of a-starch, substituting feces’ marker. 2 This is an instant starch or pregelatinized starch made from potato-^-starch. 3 Harper (15). 4 This mixture was identical with Harper’s (15) except for omission of ascorbic acid and a-tocopherol. fore standardization with the stock diet These experiments were performed dur used in our laboratory. ing the period extending from October, Urine was collected from the animals 1959, to March, 1960. while they were maintained in individual Environmental temperature ranged from metabolic cages. The first urine collection 20 to 25°C under air conditioning. period was for 3 days after 3 days’ feeding The biological value was determined by of test diets, and the second collection the method of Njaa (8 ), and the net pro period, for another 3 days after one day’s tein utilization (N P U ) was calculated by interval. As a preservative for the urine, multiplying digestibility by the biological 10 ml of n sulfuric acid were placed in value. each collection flask at the beginning of Net protein ratio (N P R ), which is a the collection period. modification of protein efficiency ratio pro At the end of both urine collection posed by Bender and Doell (9 ), was cal periods the rats were removed from meta culated from the difference between the bolic cages. The cages, screens, and the gains in weight of the test and non-protein funnels which were fitted with filter paper, groups divided by the weight of ingested were washed down with hot distilled protein during 10 days of feeding. water, the washings being combined with the collected urine. RESULTS AND DISCUSSION The pooled 3-day urine samples ob The daily N intake of the 61-day-old rats tained from each rat were diluted up to was the highest and was approximately 250 ml with distilled water and a portion 1.5 times higher than that of the 21- and of this diluted sample was preserved with 27-day-old rats. In each experiment, nitro a small amount of chloroform and stored gen intakes of the casein and gluten in deep-freeze ready for analysis. Chro groups were about the same except at the mium oxide, Cr203 was used to mark feces. second period for the 21- and 158-day-old The urine samples were analyzed in dupli rats, as shown in figure 1. cate for allantoin, urea and creatinine by The digestibility of the test proteins did colorimetry using Kotaki’s electrophotom not differ significantly among ages and eter at 500, 430, and 530 mu, and the individuals (table 2). methods described by Young and Conway Each group showed its own character (5 ), Engel and Engel (6 ) and Clark and istic growth curves (figs. 2-5). As ex Thompson (7 ), respectively. pected, the casein groups grew more Nitrogen determination was carried out rapidly than the gluten groups, although by the Kjeldahl method. in the case of the 158-day-old rats the URINARY N COMPOUNDS AND DIETARY PROTEIN QUALITY 129 21 27 61 158 days old Fig. 1 Average nitrogen intake per day per rat at each stage of age, when rats were fed the casein (C ) and wheat gluten (G ) diet. White and shaded columns represent the value at the first and second periods. Vertical bars represent standard error of the mean. TABLE 2 True digestibility 1 of casein and wheat gluten of rats at the various stages of age in each period Age in days Diet Period 21 27 61 158 Casein p-i 98.6±0.37 2 93.1 ±1.59 94.1 ±0.49 96.5 ±0.93 p-ii 97.1 ±0.38 93.4 ±0.21 98.0 ±0.87 95.5 ± 0.67 Gluten p-i 95.1 ±0.86 94.1 ±0.91 97.2 ±0.61 98.5 ±0.54 p -ii 96.1 ±1.14 92.9 ±0.78 96.1 ±1.09 92.7 ±0.86 1 N intake — (Fecal N in protein-fed rat — fecal N in nonprotein-fed rat) N intake x 1 0 0 . 2 se of mean. Fig. 2 Growth rate of 21-day-old rats fed test Fig. 3 Growth rate of 27-day-old rats fed test diets. O------O, casein; A ------A, wheat gluten; diets. O------O, casein; A ------A, wheat gluten; X------X, protein-free groups. Vertical bars rep X------X, protein-free groups. Vertical bars rep resent standard error of the mean. resent standard error of the mean. 130 SHUHACHI KIRIYAMA AND KIYOSHI ASHIDA Urea excretion of the gluten groups was consistently higher than that of the casein groups (table 3), even though N intakes were almost the same (fig. 1). In growing animals, urea excretion increased in the second period except for that of the 21- day-old rats fed the gluten diet, whereas no difference was noted in the 158-day-old animals. Hats fed gluten consistently ex creted a greater percentage of the urinary Fig. 4 Growth rate of 61-day-old rats fed test diets. O------O, casein; A ------A, wheat gluten; ------X, protein-free groups. Vertical bars rep resent standard error of the mean. growth curve of the former became sta tionary approaching the latter. These re sults are reasonable considering that the amino acid composition of casein is better Fig. 5 Growth rate of 158-day-old rats fed test diets. O------O, casein; A ------A, wheat gluten; balanced with respect to the requirement X------X, protein-free groups. Vertical bars rep pattern of rats than that of gluten. resent standard error of the mean. TABLE 3 Average urea excretion and percentage of urea N in total urinary N at the various stages of age and periods zvhen rats zuere fed casein, gluten and nonprotein diets Period P-I Period P-II Age Diet Urea N Urea N Urea in total in total urinary Urea urinary N N d a y s m g / r a t / d a y % m g / r a t / d a y 9o 21 Nonprotein 8.4 ± 1.3 1 33.8 7.1 ± 1.1 35.2 Casein 93.4 ± 8.3 60.3 108 ± 3.2 62.6 Gluten 180 ±12.1 77.0 155 ± 8.9 76.3 27 Nonprotein 23.8 ± 3.7 53.9 16.4 ± 1.2 54.2 Casein 118 ± 2.7 62.5 134 ± 5.4 73.0 Gluten 150 ±12.6 70.0 223 ± 3.4 77.6 61 Nonprotein 45.6 ± 4.0 47.7 29.5 ± 3.4 44.2 Casein 202 ±12.1 60.9 226 ± 13.8 68.2 Gluten 342 ±24.9 70.2 413 ±17.9 77.9 158 Nonprotein 44.0 ± 7.0 45.3 46.7± 5.8 48.1 Casein 217 ±21.1 69.2 205 ±21.6 68.3 Gluten 313 ±22.6 74.5 320 ±24.0 75.7 1 se of mean. URINARY N COMPOUNDS AND DIETARY PROTEIN QUALITY 131 N as urea than rats receiving casein irre spective of age or period of collection (table 3). In the nonprotein group, the percentage of urea N in total urinary N was significantly lower than that of both protein groups. This value was in a state of equilibrium in first period. This may have occurred because urea excretion ceases more rapidly than excretion of other N compounds immediately after the protein intake is suspended. Allantoin excretion of the casein groups was higher than that of the gluten groups except in the 158-day-old rats (table 4), but the difference in allantoin excretion between the 2 groups was not as marked as the differences in urea excretion. Allan toin excretion of the casein groups in creased along with growth up to the sec ond period, but in the gluten groups it was almost the same in both periods except in the 27-day-old group. This might be con sidered to be due to the extremely slow growth rate of the gluten groups as com pared with that of casein groups. The in crements of allantoin excretion appear to Fig. 6 The difference of allantoin excretion be affected not only by absolute body rate in various ages. The values are plotted in weight, but also by growth rate of rats. micrograms of allantoin per gram of body weight The larger the animal the greater is, in per day against each age. O and A : represent casein and gluten groups, respectively. general, its total allantoin excretion; but when allantoin excretion is computed per Actually, as reported in the previous stud unit of body weight, the smaller the ani ies (4 ), when rats were fed extremely low mal, the higher the value is (fig. 6). quality protein such as gelatin, allantoin excretion decreased. However, as shown TABLE 4 above, allantoin excretion was affected Average allantoin excretion of rats at the various rather by age than by dietary protein qual stages of age and periods when fed casein, ity. Thus, allantoin excretion is complex, gluten and nonprotein diets and the effect of age and dietary protein Groups quality on allantoin excretion should be Period Nonprotein Casein Gluten investigated further. mg/daylrat vig/day/rat mg/day/rat It was apparent that the changes of uri nary N compounds obtained above were 21-day-old caused by the quality of the ingested pro p -i 12.4 ± 0.5 1 20.0 ± 1.3 15.5 ± 0.6 p -i i 7.9±0.3 23.3 ±1.3 13.5 ±0.8 tein; when the quality or quantity of die tary protein, or both, are unbalanced or 27-day-old in excess of the requirements of rats, it p i 7.6± 1.5 14.7 ±1.8 9.4 ±1.4 is considered that the portion of dietary p -i i 8.4 ±0.6 22.8 ±1.9 19.9 ± 0.9 protein utilized for body protein synthesis 61-day-old would be small, and urea excretion would p i 25.8 ±1.4 37.9 ± 0.8 34.9 ±0.7 become relatively high. On the other hand, p -i i 20.8 ±0.8 43.5 ±1.2 33.0 ± 1.1 in view of present knowledge of the inti 158-day-old mate correlation of ribonucleic acid metab p i 31.4 ±1.7 32.2 ± 1.7 34.8 ±1.2 olism with protein synthesis, it would be p -ii 28.7 ±1.0 36.5 ± 1.9 34.4 ±1.2 expected that allantoin excretion would 1 se of mean. be increased with the increase of nutritive 132 SHUHACHI KIRIYAMA AND KIYOSHI ASHIDA value of ingested protein, because it ap o f ( A / U ) X Ip and NPR of 21-day-old rats pears probable that when protein synthesis were appreciably lower than those of older actively increases, the increased excretion rats, but the relative values of biological of allantoin as the catabolic end product value and NPU of 21-day-old rats were of purine bases derived from nucleic acids slightly higher than the values of both might be attributed to the increased turn 27- and 61-day-old rats, especially in the over rate of ribonucleic acids. second period. The values of NPR of both Indirect evidence for the above assump the casein and gluten groups successively tions has been reported by Muramatsu and approached each other as age progressed Ashida (10, 11), who showed that urea or physiological development proceeded. excretion increased almost linearly and The ratio of creatinine N to total N allantoin excretion increased curvilinearly excreted is an expression developed by against the increment of dietary level or Murlin et al. (2, 3). Their studies are ingested amounts of casein. Also, Ashida based on creatinine excretion not being and Harper (12), and Schimke (13) have affected by exogenous proteins or by other reported a similar response of urea ex nutrients. The values obtained by this cretion in their studies on the biological method are shown in table 5. The values regulation of protein metabolism. for the casein are always higher than for W e have attempted to determine the nu gluten. And, there are general trends simi tritive value of dietary protein by pursuing lar to biological value in the relative such variations as described above. Part values. Because complete urine collection of this research has been reported previ is not required in this method, C/Nt is ously (4, 14). In these reports, the values simple and useful in the assessment of of allantoin (A)/urea (U ) ratio were protein quality when the protein intake greatly lowered with the increase of pro of various protein groups is equal because tein consumption, because urea excretion this value would greatly fluctuate by the was primarily dominated by protein intake change of IP. (Ip). Hence, to eliminate the effect due In case of the casein diet, the absolute to variation of protein consumption within ( A / U ) X Ip values for 27-day-old rats were a group, (A /U ) was multiplied by Ip. Sub lower than those of the 21-day-old group. sequently, obtained values of (A /U ) X I, This might be explained either by the fell into a certain constant point within rapidly decreased allantoin excretion with a group, and they were unbalanced only age (cf. fig. 6) or by the increased urea by protein quality. The values of (A/U ) excretion per unit of IP in 27-day-old rats. X Ip and also the values obtained by apply Decreased utilization of absorbed protein ing some of prevailing methods to esti mate the nutritive value of protein are TABLE 5 summarized in table 6. Ratios of creatinine excreted (mg) to total urinary At first glance, it appears that within a nitrogen (mg), (C/Nt) X 100, and relative values when rats were fed casein defined stage of age the (A /U ) X I p values or wheat gluten diets of the casein group are constantly higher than that of the gluten group. (C/Nt) x 100 G/C Age Period Only the biological value in the 27-day- Casein Gluten x 100 2 old rats of the gluten group in the first days period was significantly higher than the 21 p i 2.89 ±0.08 1 1.72 ± 0.08 59.5 general trend. The biological value and p -i i 3.02 ±0.06 2.02 ±0.08 66.8 net protein utilization are expressed on a 27 p i 2.45 ±0.08 2.00± 0.12 81.7 percentage scale, whereas (A /U ) X I p and p -i i 2.59± 0.16 1.73 ±0.05 66.8 net protein ratio are expressed as a rela tive value. Therefore, direct comparison 61 p i 4.02 ±0.07 2.69 ±0.20 66.9 p -i i 4.41 ±0.16 2.56±0.15 58.1 between these methods of measurement is impossible. To compare them, the rela 158 p i 7.20 ±0.48 5.71 ±0.33 79.3 tive expression (the value of gluten/the p -i i 7.91 ±0.74 5.69 ±0.39 71.9 value of casein X 100) is given in the 1 se of mean. 2 The ratio of the value for gluten to the value lower part of table 6. The relative values for casein (% ). Comparison of methods evaluating protein quality when rats were fed casein and wheat gluten diets at each stage of age and period QUALITY PROTEIN DIETARY AND COMPOUNDS N URINARY PP * i—* ’5) £ o o O a > C\J 3 i—I © CO © © 00 'S q d © +1 +1 © d 00 rH q © CD + 1 q d +1 rH CD G d q d q rH co q d q +1 +1 CO d O rH © + 1 P 1u ,C0 oj !> J3 0) CO h Ü s CN U rH © CD © © q © © CN q © © © © d q d O Ü \ U (N t- co d © rH d c: © © o q © q © d q d O © © o s. O rH © d © q © © d © q © rH q © q © © q o N O C O rH © © © q ■d rH © CD © (N q q d q © d 3 The ratio of th« value for gluten to the value for casein (% ). 133 134 SHUHACHI KIRIYAMA AND KIYOSHI ASHIDA may be assumed from the growth curves. 2. Murlin, J. R., T. A. Szymanski and E. C. At this age, however, the relative values Nasset 1948 Creatinine nitrogen percent age as a check on the biological values of between the casein and gluten groups are proteins. J. Nutrition, 36: 171. comparable to the others. Higher absolute 3. Murlin, J. R., A. D. Hayes and K. Johnson values in 61-day-old rats might be also ex 1953 Correlation between the biological plained by the higher growth rate of this value of protein and the percentage of group caused by higher food intake. In creatinine N in the urine. J. Nutrition, 51: 149. this case, both urea and allantoin excre 4. Kiriyama, S., and K. Ashida 1960 The tion increased, but the increased urea ex relation between quantity and quality of cretion was not large enough to decrease dietary protein and nitrogen compounds in the (A /U ) X Ip value. The fact that rel urine of rat. I. The ratio of the amount of allantoin to that of urea in urine of the rats X atively low absolute values of (A /U ) fed egg albumin or gelatin as protein source. Ip were obtained in the 158-day-old rats Seikagaku (J. Japanese Biochem. Soc.), 32: (although they had the highest weight) 185. might be due to extremely slow gain. 5. Young, E. G., and C. F. Conway 1942 Esti Similar explanations also appear possible mation of allantoin by the Rimini-Schryver reaction. J. Biol. Chem., 142: 839. in the case of the gluten diet for 61-day-old 6. Engel, M. G., and F. L. Engel 1947 The rats although the situation is reversed. colorimetric microdetermination of urea ni We have not been able to decide whether trogen by the xanthydrol method. J. Biol. a consideration of allantoin excretion was Chem., 167: 535. essential in the calculation of nutritive 7. Clark, L. C. Jr., and H. L. Thompson 1949 Determination of creatine and creatinine in value. However, surveying the partition urine. Anal. Chem., 21: 1218. of nitrogenous constituents in urine would 8. Njaa, L. R. 1959 A shortened version of be pertinent and of great significance the Mitchell method for determination of from the following point of view. All or the biological value of protein. Growth rate ganisms take up their nutrients from their as a source of error. Brit. J. Nutrition, 13: 137. environments and excrete their wastes 9. Bender, A. E., and B. H. Doell 1957 Bio continually, and by these basal processes logical evaluation of proteins: a new aspect. they can support life. As long as life is Brit. J. Nutrition, 11: 140. maintained by a balance of the above 2 10. Muramatsu, K., and K. Ashida 1955 Com basal processes, organisms acquire a fixed position of nutrients and metabolism of tis sue protein. II. The effect of varying levels metabolic pattern corresponding to the of dietary protein upon the enzyme activities nutrient intake. This “corresponding meta and nitrogen metabolites. J. Agr. Chem. Soc. bolic pattern” can be secured only by Japan, 29: 725. changing the ratios among the velocities 11. Muramatsu, K., and K. Ashida 1957 Com position of nutrients and metabolism of tis of numerous reaction processes in the sue protein. IV. Relation between rat liver body. Therefore, the degree of inconsist xanthine oxidase, arginase activity and uri ence, in both quantity and quality of the nary nitrogen metabolites. J. Agr. Chem. nutrients ingested, between the nutrient Soc. Japan, 31: 607. pattern of food and requirement pattern of 12. Ashida, K., and A. E. Harper 1961 Meta bolic adaptations in higher animals. VI. animals would be reflected in the velocity Liver arginase activity during adaptation to ratios of reaction processes in the body, high protein diet. Proc. Soc. Exp. Biol. Med., and consequently, these fluctuations, espe 107: 151. cially of protein metabolism, would be 13. Schimke, R. T. 1962 Adaptive character istics of urea cycle enzymes in the rat. J. come detectable in urine with a certain Biol. Chem., 237: 459. ratio among various urinary nitrogenous 14. Kiriyama, S., and K. Ashida 1962 The re end products. lation between quantity and quality of die Studies on the responses of urinary N tary protein and nitrogen compounds in partition to the graded incorporation of a urine of rat. II. The values of (allantoin/ urea) X protein intake of adult rats fed the single essential amino acid into amino diets incorporating egg albumin, casein or acid mixtures are now in progress. wheat gluten as protein source. Japanese J. Nutrition, 20: 121. LITERATURE CITED 15. Harper, A. E. 1959 Amino acid balance 1. Folin, O. 1905 Laws governing the chemi and imbalance. I. Dietary level of protein cal composition of urine. Am. J. Physiol., and amino acid imbalance. J. Nutrition, 68: 13: 66. 405. Pantothenic Acid Deficiency in Cats* 1 S. N. GERSHOFF a n d L. S. GOTTLIEB Department of Nutrition, Harvard School of Public Health, the Mallory Institute of Pathology, Boston City Hospital, and Tufts University School of Medicine, Boston, Massachusetts ABSTRACT Cats were fed purified diets containing varying levels of calcium pantothenate. In pantothenic acid-deficient cats the most prominent changes were growth failure and histologic changes in the small intestines and the liver. The re sults of gross and histologic examinations, measurements of acetylation of urinary p-aminobenzoic and the ratio of urinary pantothenic acid to creatinine indicate that in this study, cats required approximately 5 mg of calcium pantothenate/kg of diet. During the past 10 years, a program de Throughout the study, hematological ex signed to determine the nutrient require aminations were made and when the cats ments of cats has been pursued in this died or were killed, they were autopsied laboratory. In the present paper, panto and their tissues fixed in 10% buffered thenic acid deficiency in cats will be de formalin. Sections of heart, lung, trachea, scribed and the minimal requirements for gastrointestinal tract, liver, spleen, kidney, pantothenic acid of cats fed purified diets adrenal, lymph node, skin and bone mar will be reported. row were stained routinely with hema toxylin and eosin and sections of gastro MATERIALS AND METHODS intestinal tract were stained by the periodic In this work, kittens approximately 3 acid-Schiff reaction. months old of mixed breed and sex were used. Before being admitted to the labora RESULTS tory, the cats were dewormed, dusted Eight cats were fed diets without added against external parasites, and vaccinated calcium pantothenate. The terminal stages against feline distemper. The animals were of acute deficiency were observed in 2 of housed in individual cages and maintained these after 2 months, and after 4 to 4.5 with food and water ad libitum. The puri months in the other six. The deficiency fied diet used consisted of: (in per cent) state was characterized chiefly by emacia casein, 32.1; sucrose, 37.6; corn oil, 12.5; tion. Loss or greying of hair and blood hydrogenated fat, 12.5; cod liver oil, 1.0; dyscrasias were not observed. Four cats salts IV (1 ), 4; and choline, 0.3 and (in receiving 1 mg of calcium pantothenate/kg mg/kg diet) thiamine, 4; riboflavin, 8; of diet died after being fed the diet for pyridoxine, 4; niacin, 40; folic acid, 1; approximately 3.5 to 4.5 months. Of the biotin, 0.2; menadione, 1; and vitamin Bi2, 4 cats fed 3 mg of calcium pantothenate/ 0.1. The levels of calcium pantothenate kg of diet, three died after 6 months, two fed were 0, 1, 3, 5, 10 and 20 mg/kg of of massive respiratory infections and one diet. lived 9.5 months. Five milligrams of cal During the third experimental month, cium pantothenate per kilogram of diet the cats were injected intraperitoneally appeared to be sufficient to support good with 6 mg of p-aminobenzoic acid/kg of growth and health in 4 cats. Two groups body weight and acetylated and total uri of 4 cats were fed the diets containing 5 nary p-aminobenzoic acid were determined with the reagents and in the manner de Received for publication July 11, 1963. 1 Supported in part by grants-in-aid from the Nutri scribed for sulfanilamide by Bratton and tion Foundation and the Fund for Research and Marshall (2 ). In the fourth experimental Teaching, Department of Nutrition, Harvard School of Public Health, Boston, Massachusetts, and by month, urinary creatinine (3 ) and panto Public Health Service Research Grant AM-04165 (National Institute of Arthritis and Metabolic Dis thenic acid (4 ) estimates were made. eases). J. N u t r it io n , 82: ’ 64 135 136 S. N. GERSHOFF AND L. S. GOTTLIEB TABLE 1 W eight changes of cats fed diets with varying levels o f calcium ■pantothenate 1 Month Ca No. of pantothenate cats 0 1 2 3 4 m g/kg of diet 9 9 9 9 9 0 8 1019 1141 1351 1363 (6 )2 1162(6) 1 4 1272 1452 1520 1412 1495(3) 3 4 1273 1460 1788 1808 1628 5 4 1108 1564 2128 2348 2555 10 4 873 988 1493 1802 2073 20 4 966 1419 1781 2041 2285 1 Each group contained equal numbers of male and female kittens. Mean weights of each group are presented in grams. 2 Figures in parentheses represent number of cats in group alive. and 10 mg of calcium pantothenate/kg for the number of round cells in the tunica almost 2 years and cats have been main propria. Some animals showed a loss of tained with diets containing 20 mg of cal polarity of the nuclei of the columnar cells. cium pantothenate/kg for up to 3 years The glands were occasionally dilated and before being killed. contained protein material and degenerat Table 1 presents the growth records of ing polymorphonuclear lymphocytes. In the 6 groups of cats during the first 4 some animals, the epithelial columnar cells months of this study. These data indicate were enlarged in size with vesicular nuclei that following an initial growth response, at the bases of the crypts with many gland the cats receiving 0, 1 or 3 mg of calcium like forms containing inspissated protein pantothenate/kg of diet started to lose material. The tops of the villi in some weight during the third or fourth month, animals appeared to show infarct necrosis whereas the mean weights of the cats in and one animal showed focal areas of re the other groups continued to increase. pair of a previous ulcer with loss of villi Lesions attributable to pantothenic acid and increase of fibrous tissue in the tunica deficiency were observed in cats receiving with tract glands showing inflammatory 0, 1 and 3 mg of calcium pantothenate/kg cells. Sections of the colon showed no of diet. The livers and intestinal tracts of changes or slight dilatation of the glands these animals were particularly affected. and inspissated protein and polymorphonu The livers showed moderate to marked clear lymphocytes. fatty metamorphosis and both fine and Histologic changes related to the panto coarse vacuolar formation. Lipid was thenic acid content of the diets were not evenly deposited and showed no zonal pref observed in the other tissues examined. erence. There was no increase in portal Histologic changes associated with panto fibrous tissue and no evidence of cirrhosis. thenic acid deficiency were not observed in A few of the livers showed moderate cats receiving 5, 10 or 20 mg of calcium amounts of lipofuschin pigment in hepatic pantothenate/kg of diet except for a slight cells and in portal areas. Lesions of the fatty metamorphosis observed in the liver gastrointestinal tract were confined pri of one of the animals fed 5 mg of calcium pantothenate/kg of diet. marily to the small bowel. Sections of the In figure 1, the results of the studies of esophagus and stomach were normal ex acetylation of p-aminobenzoic acid and cept for evidence of superficial acute ero urinary excretion of pantothenic acid are sions in 2 stomachs. In the small intes presented. These data show that as the tines, some animals showed giant blunted content of the diets increased from 3 to villi in the areas of the jejunum and upper 5 mg of pantothenic acid/kg, there were ileum as well as a brown-yellow lipofuschin marked increases in the excretion of acet- present at the tips in fibrocytes and macro ylated p-aminobenzoic acid and the panto phages. In some, there was an increase in thenic acid, creatinine ratio. PANTOTHENIC ACID DEFICIENCY IN CATS 137 MG CALCIUM PANTOTHENATE PER KILO OF DIET Fig. 1 Effect of dietary levels of calcium pantothenate on the urinary excretion of acetylated p- aminobenzoic acid and pantothenic acid. DISCUSSION acetylation reactions, is well established. The effects of pantothenic acid defi In vitamin requirement studies, determina ciency vary widely from species to species. tions of urinary levels of the vitamin being In the cat, the most prominent changes studied and metabolites whose excretion is were growth failure and histologic changes related to dietary intake of the vitamin are in the small intestines and the liver. Many often used as measures of the degree of of the deficient animals showed evidence tissue saturation with the vitamin. Higgs of bronchopneumonia or lobar pneumonia. and Hegsted (7, 8) have reported that fol Dermatitis, achromotrichia, adrenal and lowing the injection of 6 mg of p-amino- kidney necrosis and blood dyscrasias, benzoic acid/kg in normal rats and rabbits, which have been often reported in defi approximately 70% of the p-aminobenzoic cient animals of other species, were not acid excreted in the next 24 hours was observed in this work. The histologic acetylated. Pantothenic acid-deficient rats changes of the gastrointestinal tract are acetylated 50% of a 1-mg dose and 37% similar but not identical to those described of a 2.5-mg dose. In this work cats receiv in the pig (5 ). The spinal cord was not ing the highest level of pantothenic acid examined in this study, but ganglion cells, acetylated approximately 40% of a 6 mg/ present in the periadrenal fat showed no kg p-aminobenzoic acid load test and de evidence of chromatolysis. There was no ficient cats about 17%. As the calcium evidence of necrosis of the adrenal cortex pantothenate content of the diets increased as has been previously noted in the rat (6 ) from 3 to 5 mg/kg there was a sharp in and no evidence of cortical hemorrhage. crease in acetylation of p-aminobenzoic However, cats that died while being fed the acid from 20 to 37%. There was also a experimental diet showed marked loss of sharp increase in the urinary ratio of pan cortical lipid. tothenic acid to creatinine. However, this A dietary source of pantothenic acid is ratio did not reach a maximum until 10 mg required for all species of animals that of calcium pantothenate/kg of diet were have been studied. The role of this vitamin fed. It appears from these data and the re in intermediary metabolism, particularly sults of gross and histological examinations 138 S. N. GERSHOFF AND L. S. GOTTLIEB that the minimal requirement of cats fed Pantothenic acid deficiency in swine with the purified diets used in this work was ap particular reference to the effects on growth and on the alimentary tract. Bull. Johns proximately 5 mg of calcium pantothe- Hopkins Hosp., 73; 313. nate/kg of diet. This is considerably less 6. Deane, H. W., and J. M. McKibbin 1946 than the recommended minimal require The chemical cytology of the rat adrenal cor ments of other laboratory animals (9-11), tex in pantothenic acid deficiency. Endo most of which require 8 mg or more of cal crinology, 38: 385. 7. Riggs, T. R., and D. M. Hegsted 1948 The cium pantothenate/kg of diet for growth effect of pantothenic acid deficiency on acet when fed diets of less caloric density than ylation in rats. J. Biol. Chem., 172: 539. those used in this study. 8. Riggs, T. R., and D. M. Hegsted 1951 Size of dose and acetylation of aromatic amines LITERATURE CITED in the rat. J. Biol. Chem., 193: 669. 1. Hegsted, D. M., R. C. Mills, C. A. Elvehjem 9. National Research Council, Committee on and E. B. Hart 1941 Choline in the nutri Animal Nutrition 1953 Nutrient Require tion of chicks. J. Biol. Chem., 138; 459. ments for Dogs, pub. 300. National Academy 2. Bratton, A. C., and E. K. Marshall, Jr. 1939 of Sciences — National Research Council, A new coupling component for sulfanilamide Washington, D. C. determination. J. Biol. Chem., 128: 537. 10. National Research Council, Comittee on Ani 3. Clark, L. C., and H. L. Thompson 1949 De mal Nutrition 1960 Nutrient Requirements termination of creatine and creatinine in of Poultry, pub. 827. National Academy of urine. Anal. Chem., 21: 1218. Sciences — National Research Council, W ash 4. Hoag, E. H., H. P. Sarett and V. H. Cheldelin ington, D. C. 1945 Use of Lactobacillus arabinosus 17-5 11. National Research Council, Committe on for microassay of pantothenic acid. Ind. Animal Nutrition 1962 Nutrient Require Eng. Chem. (Anal, ed.), 17; 60. ments of Laboratory Animals, pub. 990. N a 5. Wintrobe, M. M., R. H. Follis, Jr., R. Alcay- tional Academy of Sciences — National Re aga, M. Paulson and S. Humphreys 1943 search Council, Washington, D. C. Influence of High Protein Diets on Vitamin A Metabolism and Adrenal Hypertrophy in the Chick G. S. STOEWSAND a n d M. L. SCOTT Department of Poultry Husbandry and Graduate School of Nutrition, Cornell University, Ithaca, New York ABSTRACT High dietary levels of isolated soybean protein, purified isolated soy bean protein, casein-gelatin, vitamin-free casein, or amino acid mixtures formulated to simulate isolated soybean protein were administered to chicks. When adequate dietary intake of the high protein diets was obtained, a significantly decreased liver vitamin A storage was observed as compared with that of pair-fed chicks consuming the same protein at moderate dietary levels. Isolated soybean protein diets had the most marked effects in reducing vitamin A liver stores. The depletion of vitamin A ester in the liver was more complete than the depletion of vitamin A alcohol. En largement of the adrenal glands occurred in chicks consuming the high isolated soy bean protein diets. This enlargement appeared to be due primarily to hypertrophy of the adrenal cortical tissue. Studies with a number of species have levels in liver and blood were lowered. shown that high protein diets reduce vita Erwin et al. (6 ) reported steers fed iso min A in the liver and decrease survival caloric diets showed decreased liver vita time of animals fed diets low in vitamin A. min A reserves when higher amounts of In an earlier report from this laboratory protein were fed. (1 ) it was shown that chicks consuming High levels of dietary protein also have high amounts of isolated soybean protein been observed at times to produce adrenal had a decreased amount of vitamin A in gland hypertrophy in mammals. Tepper- liver and blood serum as compared with man et al. (7 ) and Leathern (8 ) reported vitamin A levels in pair-fed chicks receiv that high dietary protein levels increased ing a normal amount of this protein. Olsen rat adrenal weights. Ingle et al. (9 ), how et al. (2 ) observed that vitamin A liver ever, did not observe significantly higher stores of chicks decreased when protein- adrenal gland weights in rats fed high deficient diets were made adequate by amounts of protein, and in one experiment supplementation with additional protein. by Benua and Howard (1 0) mice fed high However, in this study the increased levels of protein did not have hyper growth obtained from the higher protein trophied adrenals. diets may have been at least partially re The results presented in the present sponsible for the reduced liver vitamin A paper provide additional evidence that high stores, since the vitamin A requirement is levels of dietary protein from various directly related to body weight (3 ). Fer- sources influence the vitamin A status of rando and Mainguy * 1 observed decreased the chick and that high dietary protein liver vitamin A in chicks when the protein levels also cause hypertrophy of the ad content of the diet was gradually increased renals in young chicks. from 16 to 36%. Mayer and Krehl (4 ) observed that the average survival time of METHODS AND MATERIALS rats fed a 60% casein-vitamin A-deficient Experiment 1. Twelve groups of 14 diet was markedly decreased compared one-day-old White Plymouth Rock cockerel with that of rats fed vitamin A-deficient diets containing normal protein levels. Received for publication September 3, 1963. 1 Ferrando, R., and P. Mainguy 1962 Influence du Bohman et al. (5 ) observed that when taux de protides et de lipides totaux de la ration sur extra protein was fed to wintering beef la reserve hepatique de vitamine A chez le poulet. Twelfth World’s Poultry Congress, Proceedings. Syd calves on range, vitamin A and carotene ney, Australia, pp. 168-171. J. N u t r it io n , 82: ’64 139 140 G. S. STOEWSAND AND M. L. SCOTT chicks obtained from a commercial hatch fed chicks was determined by Duncan’s ery were randomly distributed into 12 pens new multiple range test according to Steel within an electrically heated chick battery, and Torrie (12). and 6 treatments were randomly assigned Experiment 2. The second experiment to the pens, 2 lots per treatment. At the was conducted with chicks of the same end of the first week, 10 chicks were breed and from the same source as those selected on the basis of similar body used in experiment 1. At one day of age, weights to be maintained with the respec 11 chicks per lot were randomly allotted tive diets for 3 additional weeks. The com to each of 15 experimental pens. At the position of the diets is presented in table 1. end of one week 8 chicks were selected to Either isolated soybean protein or casein be maintained with the experimental treat and gelatin were used as the source of ments for 3 additional weeks. Each treat protein in this experiment. Chicks eating ment was fed to triplicate lots of chicks. the moderate (normal) protein diets (2 The diets were similar to the isolated soy and 4) were either pair-fed to the chicks bean protein diets used in experiment 1 consuming the corresponding high protein (diets 1 and 2, table 1). All chicks were diets (1 and 3) or were fed ad libitum fed the same amount of feed consumed by (diets 5 and 6). After 4 weeks of age, the chicks receiving the high protein diet 5 livers of chicks from each pen were col which showed the lowest feed consump lected, immersed immediately in liquid ni tion. trogen, and stored at — 10°C until liver To determine whether impurities in the vitamin A analyses were made. The livers crude, commercial isolated soybean protein were analyzed individually for vitamin A exerted any effect upon vitamin A liver according to the method of Ames et al. storage, 2 experimental groups received (11). Statistical significance of the differ “purified” isolated soybean protein2 sub ences in liver vitamin A content of pair- stituted for “crude” soy protein in both diet TABLE 1 Composition of experimental diets Diet no. 1 2 3 4 5 6 % % % % % % Isolated soybean protein 1 84.35 28.00 ___ — — — Casein — — 63.15 21.00 — — Vitamin-free casein 2 — — — — 77.605 25.00 Gelatin — — 22.00 7.00 — — Glucose monohydrate — 58.05 — 57.15 — 58.845 Cellulose 3.00 3.00 3.00 3.00 3.00 3.00 Stripped lard 3 3.00 3.00 3.00 3.00 3.00 3.00 Mineral mixture 5.63 4 5.63 4 5.63 4 5.63 4 6.21 5 6.21 5 Vitamin mixture 6 1.00 1.00 1.00 1.00 1.00 1.00 DL-Methionine 1.20 0.70 0.80 0.80 0.90 0.30 L-Cystine 1.00 — — — — — L-Arginine-HCl — — 0.80 0.80 4.50 1.50 Glycine 0.50 0.30 — — 2.40 0.80 Choline chloride (70% ) 0.22 0.22 0.22 0.22 0.22 0.22 N aH C 03 — — — — 1.00 — Antioxidant7 0.0125 0.0125 0.0125 0.0125 0.0125 0.0125 % Protein ( N X 6.25) 74.9 25.0 80.6 26.6 83.3 27.4 1 Archer-Daniels-Midland, Minneapolis. 2 Nutritional Biochemicals Corporation, Cleveland. 3 Distillation Products Industries; Rochester, New York. 4 Mineral mixture supplies per kg of diet: (in g) CaHP04, 20.70; CaC03, 14.80; KH2P04, 10.00; KC1, 1.00; NaCl, 6.00; NaHCOs, 0.80; MgS04, 3.00; FeS04-7H20, 0.333; MnS04 H20, 0.333; KI, 0.0026; ZnO, 0.062; CuS04- 5H20, 0.0167; CoCl2-6H20, 0.0017; NaMo04-2H20, 0.0093; Na2Se03, 0.0001. 5 Chloride low mineral mixture contains: (g/kg) CaHP04, 17.00; CaC03, 18.30; KH2P04, 13.77; NaC2H302, 8.42; NaHCOs, 0.80; MgS04, 3.00; FeS04-7H20, 0.333; MnS04 H20, 0.333; KI, 0.026; ZnO, 0.125; CuS04-5H20, 0.0167; CoC12-6H20, 0.0017; NaMo04-2H20, 0.0083; Na2Se03, 0.0001. 6 Vitamin mixture supplies per kg of diet: (in IU ) Vitamin A, 5,000; vitamin D3, 975; (in m g) a-tocopheryl acetate, 600; niacin, 50; Ca pantothenate, 20; riboflavin, 10; thiamine-HC1, 10; pyridoxine-HCl, 10; folic acid, 4.0; menadione sodium bisulfite, 1.52; biotin, 0.5; (in /¿g) vitamin Bi2, 20 (diets 2, 4, 6 ); 50 (diets 1, 3); 60 (diet 5); and 9.9 g of added glucose monohydrate. 7 Santoquin, Monsanto Chemical Company, St. Louis. PROTEINS, VITAMIN A AND ADRENALS 141 TABLE 2 Effect of washing isolated soybean protein on body weight and liver vitamin A of chicks fed approximately the same amount of diet Vitamin A in the liver 2 Lot Treatment Protein 4-Week no. in diet1 weights Total Liver retention 3 Alcohol Ester % 9 IU / liv e r % i High purified 80.5 236 4 78 4.7 + 0 2 High crude 77.6 2 2 0 0 0 0 0 3 Moderate purified 24.6 267 429 22.5 + + 4 Moderate crude 23.5 255 354 18.6 + + 5 Moderate crude + washing 27.0 241 471 26.3 + + 1 N x 6.25. 2 Diet contained 5,000 IU vitamin A/kg of diet. 3______Total liver vitamin A/chick x 100 Total amount of vitamin A consumed/chick 4 All diets pair-fed equivalent to the feed consumed ad libitum by the chicks receiving the “high crude” protein diet. TABLE 3 1 and diet 2. A further treatment group Amino acid diets that simulate isolated soybean (lot no. 5 of table 2) received diet 2 with protein diets in experiment 3 the additional washings from the “purifica tion” of the soy protein to make the total High i Moderate “soy protein impurities” equivalent to those protein protein in the high “crude” soy diet. Livers were collected, pooled (4 per lot) % % and analyzed for vitamin A. In addition, L-ArginineHCl 6.71 2 . 0 2 vitamin A alcohol and vitamin A acetate L-Aspartic acid 1.29 0.40 L-Cystine 1.49 0.13 were separated by thin layer chromatog L-Glutamic acid 12.35 3.74 raphy3 from aliquots of the ether solutions Glycine 4.21 1.30 of liver vitamin A. These were determined L-Histidine-HCl 2 . 1 1 0.64 qualitatively. DL-Isoleucine 9.69 2.93 L-Leucine 6.07 1.82 Experiment 3. The third experiment L-Lysine-HCl 5.49 1.67 was conducted to determine whether the DL-Methionine 2 . 0 0 0.94 depressing effect of the high soybean pro DL-Phenylalanine 8.07 2.44 tein upon liver vitamin A storage was a L-Proline 2 . 0 2 0.60 DL-Serine 2.82 0.84 peculiarity of soybean protein or whether DL-Threonine 6.31 1.92 it also occurred when casein diets or pure DL-Tryptophan 1.62 0.49 amino acid diets were fed. L-Tyrosine 2.76 0.82 White Plymouth Rock X Van tress cock DL-Valine 8.89 2.69 erels produced by a controlled flock of Glucose monohydrate — 59.545 Stripped lard 2 3.00 3.00 hens were used in this experiment. Ten Cellulose 3.00 3.00 chicks were randomly placed in each of 8 Mineral mixture 3 6 . 2 1 6 . 2 1 Vitamin mixture 2 1 . 0 0 1 . 0 0 2 The isolated soybean protein was washed with Choline chloride (70% ) 0 . 2 2 0 . 2 2 41 °C tap water at a pH of 4.6, pressed in a small cheese press, dried in a forced-air draft oven at 65°C NaHCOu 1.50 0.50 and finely ground. Analysis for total sulfur by the Antacid 4 1 . 0 0 1 . 0 0 sodium peroxide method (17) showed the sulfur was Antioxidant 2 reduced from 0.55% in the “ crude” protein to 0.15% 0.0125 0.0125 in the “purified” isolated soybean protein. 3 Glass plates, 20x20 cm, were spread with 27% silica gel G. Aliquots (50 to 100 /¿liters) of the di % Protein (N X 6.25) 64.2 20.5 ethyl ether solution from the total vitamin A analysis were pipetted onto the base line of the plate which 1 At the end of the first week of the experiment an was then placed into a covered glass jar containing a additional 2.25% NaHC03 and 2.25% KHCO3 was mixed solvent of 75% petroleum ether (bp 30° to 60°) mixed into this diet in order to neutralize the HC1 and 25% diethyl ether. After the solvent had risen radical of the basic amino acids. 10 cm above the base line, the plates were either quickly placed under a UV light and the separated 2 See table 1; vitamin B12 ( f i g/kg of diet) high, 60; vitamin A alcohol and vitamin A acetate spots out moderate, 20. lined with a pencil before fading ensued, or a 25% 3 See table 1, footnote 5. solution (w /v) of antimony trichloride in chloroform 4 Gelusil ( aluminum hydroxide — magnesium tri was applied by dropper and the blue color of the silicate), Warner-Chilcott Laboratories, Morris Plains, Carr-Price reaction with vitamin A compounds was New Jersey. observed in the separated spots. 142 G. S. STOEWSAND AND M. L. SCOTT duplicated treatments; 6 chicks were se nificance by the method described in ex lected at one week of age and continued to periment 1. be fed the experimental diets for the sub Both adrenal glands were dissected from sequent 3 weeks as in experiments 1 and 6 chicks per dietary treatment, trimmed of 2. Two treatments received the same excess tissue and weighed. Since the body “high” and “moderate” crude or purified weights of the chicks varied widely, an ad isolated soybean protein diets used in ex justment of the paired adrenal weights was periment 2. The other treatments were made by an analysis of covariance (12). high and moderate amino acid diets for The results are presented in table 4.4 mulated to simulate isolated soybean pro tein (table 3) and the high and moderate RESULTS AND DISCUSSION vitamin-free casein diets (table 1, diets 5 The results of experiment 1 are shown and 6). Chicks consuming the moderate in table 5. Chicks consuming the high iso or normal protein diets were pair-fed to lated soybean protein diet showed a sig the chicks fed the respective high protein nificant decrease in total liver vitamin A as diets. Vitamin A was fed at 100,000 IU/kg compared with chicks fed the other diets. of diet to measure fecal vitamin A which Chicks fed the high casein-gelatin diet had was done by the same method as the liver significantly lower levels of total liver vita vitamin A analyses used in experiments 1 min A than the chicks pair-fed the moder and 2. Vitamin A absorption was deter ate casein-gelatin diet, but these were much mined during the last week of the experi higher than total liver vitamin A levels of ment. Feed intake and total excreta were the chicks fed the high isolated soybean carefully measured for 4 days, and aver 4 The standard error of the ^difference between the aged on an individual chick basis. The adjusted adrenal pair means (sd) of chicks consuming vitamin A liver retention values presented the same high and moderate protein diets were used in determining significance of the differences (i.e., in table 4 were analyzed for statistical sig t~ d/sd where d is the difference of the two means). TABLE 4 Effect of high levels of dietary protein and amino acids on 4-week body weights, utilization of vitamin A, and chick adrenal pair weights Liver vitamin A 2 Mean Treatment Protein 4-Week in diet1 wt Apparent Retained adrenal absorption Total in liver 3 pair wt 4 % 9 % % mg 1 High crude isolated soy protein 76.2 209 (12/12)5 73 11,000 44 6 56.5 7 2 Moderate crude isolated soy protein 24.0 262 (12/12) 83 24,106 75 35.7 3 High purified isolated soy protein 80.5 220 (12/12) 73 15,325 61 50.9 7 4 Moderate purified isolated soy protein 24.8 254 (12/12) 74 21,312 74 35.2 5 High casein 83.3 99 (9/12) 93 11,544 53« 35.8 6 Moderate casein 27.4 269 »(12/12) 83 27,018 76 27.4 7 High amino acids 64.2 110 (6/12) 82 16,167 72 39.9 8 Moderate amino acids 20.5 165 (12/12) 82 20,223 82 32.5 1 N x 6.25. 2 Diet contained 100,000 IU vitamin A/kg diet. 3 ______Total liver vitamin A/chick Total vitamin A apparently absorbed/chick X 4 Adjusted to a common body weight by analysis of covariance. 5 Number of chicks survived/number of chicks started. 6 Significantly (P < 0.05) lower than the respective moderate protein pair-fed controls. 7 Significantly higher (P < 0.01) than the respective moderate protein pair-fed controls. 8 Pair-fed to high isolated soy protein diets. PROTEINS, VITAMIN A AND ADRENALS 143 TABLE 5 Effect of high levels of isolated soybean protein and casein-gelatin on 4-week weight, feed conversion, and vitamin A liver storage Total Protein 4-Week G feed/ mean Treatment in diet wt g gain liver vitamin A 2 % 9 IU /liver 1 High isolated soy protein 74.9 270 2.04 60 “ 3 2 Moderate isolated soy protein (pair-fed to lot no. 1) 24.9 278 1.88 807 1)0 3 High casein-gelatin 80.5 269 1.72 646 » 4 Moderate casein-gelatin (pair-fed to lot no. 3) 26.3 263 1.74 916 « 5 Moderate isolated soy protein (ad libitum) 24.9 541 1.64 1387 d 6 Moderate isolated casein-gelatin (ad libitum) 26.3 468 1.60 1348 d 1 N x 6.25. 2 Diet contained 5,000 IU vitamin A/kg of diet. 3 Values that are significantly different (P < 0.05) are followed by different letters. protein diet. Analyses showed no vitamin mental period, perhaps because of certain A or carotene in the casein used in this excess amino acid toxicities (13). experiment. Liver vitamin A, expressed as the The results of experiment 2 are shown amount of vitamin A retained as a per in table 2. In this experiment no vitamin centage of the total amount of vitamin A A was detected in livers of chicks fed the apparently absorbed, again showed a sig high crude soybean protein diet. A very nificant decrease in the chicks consuming low level of liver vitamin A was observed the high crude isolated soybean protein in the chicks consuming the high purified diet and in chicks consuming the high protein diets. The results with the moder casein diet. However, the high casein-fed ate protein diets were approximately equiv chicks had livers that were very small alent and there was no decrease in liver (average weight < 3 g ) at 4 weeks of age, retention of vitamin A when the washings and the amount of vitamin A/g of liver from the “purification” of the crude iso was not decreased compared with that of lated protein were added to the moderate the larger chicks fed the moderate protein soybean diet. The small amount of liver diets. The chicks fed the high purified iso vitamin A in the chicks consuming the lated soy protein diet had lower vitamin A high purified protein diet was shown to be liver stores than the pair-fed chicks con vitamin A alcohol; there was no detectable suming the moderate purified isolated soy vitamin A ester in the livers of these protein diet, but this difference was not chicks. Both vitamin A ester and alcohol quite significant (P < 0.05). were observed in the livers of all chicks re The 2 high isolated soybean protein diets ceiving the moderate protein diets. produced chicks with significantly heavier The results of experiment 3 are pre adrenal glands as compared with those of sented in table 4. Chicks receiving the chicks receiving the moderate protein diets. high casein diet could not consume a nor Histological examination (hematoxylin and mal amount of food because the casein be eosin stain) of the adrenals showed no came impacted in the beaks of the chicks, changes in these enlarged glands except for which interfered markedly with food con some hypertrophy of the cortical tissue, sumption. Chicks consuming the high especially of the periphery of the organ. amino acid diet grew poorly and had 50% Further studies in this laboratory (1 4 ) in mortality at the end of the 4 week experi- dicate that chicks fed high isolated soybean 144 G. S. STOEWSAND AND M. L. SCOTT protein diets have an increased production 5. Bohman, V. R., M. A. Wade and C. Torrell of corticosterone. 1959 Effect on animal fat and protein sup plement in range beef cattle. J. Animal Sci., The results of these experiments show, 18: 567. therefore, that high protein diets, especially 6. Erwin, E. S., R. S. Gordon and J. W . Algeo high soybean protein diets, cause a marked 1963 Effect of antioxidant, protein and reduction in total vitamin A liver stores, energy on vitamin A and feed utilization in steers. J. Animal Sci., 22: 341. and also that this reduction is accompan 7. Tepperman, J., F. L. Engel and C. N. H. Long ied by a complete depletion of vitamin A 1943 Effect of high protein diets on size esters in the liver and at the same time a and activity of the adrenal cortex in the marked hypertrophy of the adrenal cortex. albino rat. Endocrinology, 32: 403. 8. Leathern, J. H. 1951 Adrenal and thyroid According to evidence presented in a re weights in rats following high protein diets view on vitamin A storage and transport by and testosterone propionate. Exp. Med. Surg., Ganguly (1 5 ) it appears that the animal 9: 138. maintains the vitamin A alcohol content of 9. Ingle, D. J., G. B. Gunther and J. Nezamis 1943 Effect of diet in rats on adrenal its tissues at the expense of the liver ester, weights and on survival following adrenalec whereas the liver vitamin A alcohol content tomy. Endocrinology, 32: 410. is usually quite constant. Selye (1 6 ) and 10. Benua, R. S., and E. Howard 1945 The Constantinides5 have shown that high pro effect of dietary protein on adrenal weights and on growth after unilateral adrenalectomy. tein diets augment various other stresses Endocrinology, 36: 170. in animals. Since, in the present studies, 11. Ames, S. R., H. A. Risley and P. L. Harris the high crude isolated soybean protein 1954 Simplified procedure for extraction diet has been shown to decrease vitamin and determination of vitamin A in liver. Anal. Chem., 26; 1378. A liver reserves and at the same time pro 12. Steel, R. G. D., and J. H. Torrie 1960 Prin duces increases in adrenal cortical size and ciples and Procedures of Statistics. McGraw corticosterone production, it appears pos Hill Book Company, New York. sible that perhaps vitamin A utilization is 13. Snetsinger, D. C., and H. M. Scott 1961 mediated either directly or indirectly via The relative toxicity of intraperitoneally in jected amino acids and the effect of glycine adreno-cortical mechanisms. and arginine thereon. Poultry Sci., 40: 1681. 14. Stoewsand, G. S., and M. L. Scott 1964 LITERATURE CITED Effect of stress from high protein diets on 1. Stoewsand, G. S., and M. L. Scott 1961 E f vitamin A metabolism in chicks. J. Nutrition, fect of protein on utilization of vitamin A in in press. the chick. Proc. Soc. Exp. Biol. Med., 106: 15. Ganguly, J. 1960 Absorption, transport and 635. storage of vitamin A. Vitamins and Hor 2. Olsen, E. M., J. D. Harvey and H. D. Branion mones, 18: 387. 1959 Effect of dietary protein and energy 16. Selye, H. 1946 The general adaptation syn levels on the utilization of vitamin A and drome and the diseases of adaptation. J. carotene. Poultry Sci., 38: 942. Clin. Endocrinol., 6: 117. 3. Guilbert, H. R., and G. H. Hart 1935 Mini 17. Association of Official Agricultural Chemists mum vitamin A requirements with particular 1960 Methods of Analysis. Washington, reference to cattle. J. Nutrition, 10: 409. D. C. 4. Mayer, J., and W. A. Krehl 1948 The rela 5 Constantinides, P. 1950 Influence of diet com tion of diet composition and vitamin C to position on alarm reaction to acute stress. Federation vitamin A deficiency. J. Nutrition, 35: 523. Proc., 9: 25 (abstract). Heat of Combustion Values of the Protein and Fat in the Body and W ool of Sheep* 1'2 O. L. PALADINES,3 J. T. REID, A. BENSADOUN and B. D. H. V A N NIEK E R K 4 Department of Animal Husbandry, Cornell University, Ithaca, New York ABSTRACT The heat of combustion values of protein and fat were determined in the ash-free, dry matter of the bodies of 63 sheep, and of the wool of 45 sheep. As a consequence of the ranges in age at the time of slaughtering and the dietary treat ments imposed, the animals represented a wide range in body composition. After each animal was shorn and slaughtered, the ingesta-free body was separated into 4 parts, consisting mainly of the blood, viscera, hide, and carcass. The mean calorific values (kcal/g) of the protein and fat, respectively, in the various body parts were as follows: blood, 5.854 ± 0.052 and 7.435 ± 0.052; viscera, 5.428 ± 0.057 and 9.312 ± 0.057; hide, 5.458 ± 0.086 and 9.305 ± 0.086; and carcass, 5.327 ± 0.068 and 9.424 ± 0.068. For the total, ingesta-free body, the calorific values were 5.379 ± 0.051 kcal/g of protein and 9.405 ± 0.051 kcal/g of fat. As the result of the manner by which they were derived, these values represent the calorific value of the composite body proteins and lipids. As a consequence, these values are more accurate for use in studies involving the metabolism and storage of energy than are the older values which are based chiefly on the analysis of body (generally muscle) proteins and body fats purified to various degrees. Although the calorific value of fat (9.405 kcal/g) representing the total body was only slightly lower than the commonly used value (9.5 kcal/g), the calorific value of protein representing the total body (5.379 kcal/g) was considerably lower than that (5.7 kcal/g) which is generally used. The calorific value of wool protein was found to be 5.609 ± 0.0023 kcal/g and that of wool fat was 9.741 ± 0.0035 kcal/g. Since wool protein contained 16.85% of nitrogen, the ob ligatory factor for converting the percentage of nitrogen to the percentage of protein is 5.933. The use of body composition estimated of various species. However, the values by indirect methods as the basis of in do not necessarily represent the body as direct calorimetry requires the use of cal a whole, and they do not take into account orific values of the composite proteins and substances other than pure protein and fats of the animal body. Also, in the use triglycerides deposited in the ash-free, dry of the slaughter-analysis method as an matter of the animal body. Since the indirect calorimetric method, the applica chemical composition of the nitrogen- tion of calorific values for the directly containing substances and lipids deter determined body protein and fat would mines their energy value, the analysis of obviate the need to determine heat of purified preparations isolated from a few combustion values on the body tissues. tissues could yield erroneous values with Although the average values of 5.7 respect to application to the total body. kcal/g of protein and 9.5 kcal/g of fat as Received for publication August 5, 1963. 1 This investigation was supported by a research recorded by Armsby (1 ) have been used grant (A-2889) from the National Institute of Arthritis extensively, their accuracy when applied and Metabolic Diseases, U. S. Public Health Service. 2 The data presented here are a part of those pre to the composite proteins and fats of the sented in the Ph.D. degree thesis by O. L. Paladines to the Graduate School, Cornell University, 1963. body has been questioned by Blaxter and 3 Recipient of Organization of American States Rook (2 ) and Stroud.5 Armsby’s values, scholarship (1960-62) and Rockefeller Foundation fellowship (1962-63); present address: Inter-American derived from the analytical data of chiefly Institute of Agricultural Sciences, Turrialba, Costa Rica. late-nineteenth century German workers, 4 Present address: Grootfontein College of Agricul ture, Middelburg, South Africa. represent the calorific values of protein 5 Stroud, J. W. 1961 The development of indirect and fat isolated principally from muscles methods for the estimation of the chemical composi tion and energy value of living cattle. Ph.D. Thesis, and fat depots, respectively, of animals Cornell University, Ithaca, New York. J . N utrition, 8 2 : ’64 145 146 O. L. PALADINES, J. T. REID, A. BENSADOUN AND B. D. H. VAN NIEKERK The purpose of the study to be reported currently with each of the chemical anal here was to determine the calorific values yses listed below, the dry matter content of the protein and fat representing the was determined by freeze-drying. total body and the wool of sheep. Analytical methods. Ash was deter mined by the common AO AC method (3 ) EXPERIMENTAL PROCEDURE and nitrogen was determined by the Animals, dietary history and slaughter macro-Kjeldahl method using the boric- and tissue-sampling procedures. The acid modification proposed by Scales and present study was conducted in connec Harrison (4 ). However, for the purposes tion with a nutritional experiment in of this study, the protein content of the which 63 sheep were slaughtered and body-tissue groups was computed as the analyzed. Just prior to the beginning of difference between the total weight and the experimental feeding period, 18 sheep, the sum of the moisture, ether extract, of which 9 were 8 months old and 9 were and ash contents. This scheme was con 20 months old, were slaughtered and an sidered to be more accurate than multiply alyzed. Each of the remaining 45 sheep ing the nitrogen content by an arbitrary was fed a diet composed of either chopped protein factor since the nitrogen content hay; pelleted, finely ground hay; or a pel of the protein is different in the various leted mixture of 55% hay and 45% corn tissues. On the basis of the data obtained meal for 196 days (at which time their in the present studies, it is indicated that age was either 15 or 27 months) at one of the nitrogen content of the composite pro 3 levels of intake: low (slightly above teins of the ingesta-free, wool-free body maintenance); medium (intermediate be of sheep is of the order of 15.58%, and tween low and high); and high (ad libi that the factor for converting nitrogen to tum). Just prior to slaughter, the sheep protein is 6.42. were shorn and the wool produced since Fat was determined as the difference in the beginning of the feeding period was weight between the freeze-dried sample analyzed separately from the remainder and the freeze-dried residue remaining of the body. after extraction with ether. Four to The body was separated into 4 analyt 8 samples weighing approximately 10 g ical groups: 1) blood, obtained by sever were extracted continuously for 7 days. ing the main vessels in the neck; 2) vis Thus, the calorific values derived for so- cera, which included the empty digestive called “fat” in the present study apply tract, mesenteric fat, lungs, heart, liver, strictly to the ether-soluble fraction in the pancreas, spleen, and urinary bladder; 3) water-free body parts. A complete extrac hide, including remnants of wool not re tion of lipids would require the use of a moved by a close shearing, ears and the mixture of polar and non-polar solvents hooves; and 4) one-half of the skinned, (e.g., methanol and chloroform). How eviscerated carcass, with superficial fat, ever, for the purposes of indirect calor one kidney with its fat and including one- imetry in which the lipid fraction is half of the skinned head. The carcass was determined most conveniently by ether divided into halves along the median line extraction, the values obtained in the pres and the left side was used in the chemical ent work are strictly valid and applicable. analysis. Each analytical group, except The heat of combustion value of the blood which was freeze-dried in toto, was tissue samples was determined with an ground and mixed 5 or 6 times by a high- oxygen bomb calorimeter. capacity grinder and samples weighing 1 The wool samples obtained from the to 2.3 kg were freeze-dried. The freeze- 45 sheep used in the feeding experiment dried samples were mixed with crushed were studied. The wool fiber and fat were dry ice and ground in a Wiley mill separated by successive extractions of the equipped with a screen having holes 1.59 wool with ether and washings with warm mm in diameter. During the grinding water. Moisture, ash, protein and gross process the samples gained some moisture energy in the clean wool fiber and mois and in the final form for analysis con ture and gross energy in the ether extract tained about 95% of dry matter. Con of the wool, were determined directly. CALORIFIC VALUE OF BODY PROTEIN AND FAT 147 The heat of combustion of the ash-free, Calorific value of body protein and fat. dry matter of the 4 body-tissue groups and In table 2 are summarized the statistics of the total, ingesta-free body was parti concerned with the relationships between tioned between the protein and fat. This the heat of combustion value and the per was effected by regressing the calorific centages of protein and fat in the various value on the percentage of protein or fat tissue groups and the reconstituted, in in the ash-free, dry matter of the tissue gesta-free body. Both the regression and groups. The intercept values of the re correlation coefficients are highly signifi spective equations represent the calorific cant. (Since protein and fat constitute value of fat and protein. The calorific 100% of the ash-free, dry matter, the cor value of the protein and fat of wool was relation between the calorific value and computed on the moisture-free, ash-free fat concentration is identical to that be basis. tween the calorific value and protein con A more detailed description of animals, centration.) dietary treatments and the slaughter and Although the correlation coefficient be analytical procedures used is given in tween the concentration of fat or protein the report of Van Niekerk et al. (5 ). and the calorific value of blood is highly significant, it is nevertheless too low for RESULTS AND DISCUSSION the relationship to have prediction value. Chemical composition and gross energy The low correlation between these vari value of ash-free, dry matter. The com ables for blood is associated with the nar position of the ash-free, dry matter of the row range in the variables. As shown in bodies of the 63 sheep studied is shown table 1, the protein content of blood in table 1. The wide range in these data ranged only from 94.20 to 100.00%. The is the result of the range in the dietary few values observed below 98% probably treatments imposed and in the age of the reflect dilution by small quantities of animals. superficial body fat which, during the TABLE 1 Chemical coviposition of the ash-free, dry bodies of 63 sheep Protein Calorific value Body Fat part Mean Range Mean Range Mean Range % % % % kcal/g kcal/g Blood 98.80 94.20-100.00 1.20 0.00- 5.80 5.873 5.723-5.991 Viscera 33.50 12.88- 73.38 66.50 26.62-87.12 8.011 6.535-8.877 Hide 74.05 61.28- 81.63 25.95 18.37-38.72 6.473 6.076-7.038 Carcass 40.18 18.31- 66.68 59.82 33.32-81.69 7.778 6.758-8.667 Ingesta-free body 43.33 20.57- 71.20 56.67 28.80-79.43 7.661 6.607-8.581 TABLE 2 Relation between the concentration of protein or fat and the calorific value of the ash-free, dry matter of the sheep body Regression equations 1 Body Correlation coefficient 2 S,-.*3 St4 part Protein Fat Blood Y = 7.435-0.01583X Y = 5.854+ 0.01583X 0.341 0.052 0.00564 Viscera Y = 9.312 —0.03881X Y = 5.428+ 0.03881X 0.995 0.057 0.00051 Hide Y = 9.305-0.03847X Y = 5.458+ 0.03847X 0.893 0.086 0.00248 Carcass Y = 9.424 — 0.04097X Y = 5.327+ 0.04097X 0.989 0.068 0.00079 Total, ingesta free body Y = 9.405-0.04025X Y = 5.379+ 0.04025X 0.994 0.051 0.00057 1 In equations, Y = calorific value (kcal/g), and X = protein or fat (% of ash-free, dry matter). 2 Correlation coefficient between calorific value (kcal/g of ash-free, dry matter) and the concentration of protein or fat (% of ash-free, dry matter); all coefficients highly significant (P < 0.01). 3 se of estimate; highly significant (P < 0.01). 4 sd of regression coefficient. 148 O. L. PALADINES, J. T. REID, A. BENSADOUN AND B. D. H. VAN NIEKERK slaughtering process, inadvertently fell and whole, ingesta-free body. However, into the blood-drip pan and were analyzed the calorific value of blood protein was as a normal component of the blood. significantly higher than that of the protein As the correlation coefficients, standard of the hide (P < 0.05), and the viscera, deviations from regression, and standard carcass, and the ingesta-free body (P < deviations of the regression coefficients re 0.01). The calorific value of the fat of corded in table 2 indicate, the relation blood was significantly lower than that of ships between the concentration of fat or the fat of the other body parts at the same protein and the calorific value of the other levels of probability as indicated above for tissue groups have a high degree of pre protein. diction value. Of these, the relationship The calorific value per gram of nitrogen for the hide was the lowest. This is prob or crude protein (nitrogen X 6.25) can be ably the result of variable amounts of computed from the data in table 3. These wool remaining on the hide even though values for the total, ingesta-free, wool- the animals were shorn closely. free body are 0.838 kcal/g of nitrogen and From the regression equations shown 5.238 kcal/g of nitrogen X 6.25. in table 2, the heat of combustion value Comparison of results of present study of protein and of fat can be computed with those of older investigations. A com individually. Since the ash-free, dry mat parison of the data in table 3 with those ter is composed wholly of protein and fat, of other investigators reveals that the cal the regression equation for either protein orific values of protein determined in the or fat may be used. For example, substi present study for the carcass (5.327 kcal tution of the values, 0.0 and 100.0 for X /g) and the whole, ingesta-free body (5.379 in the protein regression will resolve the kcal/g) are considerably lower than the calorific value of fat and protein, respec average values of 5.656 and 5.7 kcal/g tively, for a given tissue group or the total proposed by Rubner (6 ) and Armsby (1 ), body. Conversely, substitution of the same respectively. On the other hand, the values values in the fat regression will allow the derived in the present study are in good calorific value of protein and fat, respec agreement with that (5.322 kcal/g) of the tively, to be computed for a given tissue tissue proteins (containing 16% nitrogen) group or the whole body. Obviously, the of cattle determined by Blaxter and Rook intercept value of the protein-regression (2 ) and the calorific value of 5.447 kcal/g equation represents the calorific value of of protein representing the total body of fat and the intercept value of the fat- cattle as reported by Stroud.6 The differ regression equation is the calorific value ence between the various values is trace of protein. The calorific values so com able to the method by which the values puted and their 95% confidence intervals were derived. The early workers were con are presented in table 3. Statistical treat cerned with determining the calorific value ment of the mean calorific values of the of isolated muscle proteins in varying protein and fat revealed no significant dif ferences among the viscera, hide, carcass 6 See footnote 5. TABLE 3 Calorific value of the protein and fat in the sheep body Calorific value Body Protein Fat part 95% Confidence 95% Confidence Mean interval Mean interval hcal/g hcal/g Blood 5.854 5.748-5.960 7.435 6.299-8.571 Viscera 5.428 5.296-5.560 9.312 9.190-9.434 Hide 5.458 5.242-5.674 9.305 8.899-9.711 Carcass 5.327 5.161-5.493 9.424 9.274-9.574 Total, ingesta-free body 5.379 5.257-5.501 9.405 9.290-9.520 CALORIFIC VALUE OF BODY PROTEIN AND FAT 149 states of purity and did not necessarily The calorific value of both the protein intend that their values be applied to the and fat of wool is strikingly higher than total proteins of the body. Nevertheless, that of the corresponding components of their values have been used for this pur the body proper. These differences are re pose, even though they do not correspond flected in the great differences in the chem to the protein of the total body. The ical composition between wool and other method used by Stroud7 is the same as tissues. Block (7 ) reported that wool pro that used in the present study. The method tein is a keratin containing 14.3% of cys employed by Blaxter and Rook (2 ), al tine, 3.9% of sulfur, and 16.8% of nitro though similar to that of the present study, gen. Other tissues such as the hair, hoof, involved the regression of the calorific and horn also contain keratins. In the value of various tissues on their nitrogen present study it was found that wool pro concentration. It is noteworthy that the tein contains 16.85% of nitrogen, a value calorific value (5.322 kcal/g) of protein almost identical to that (16.8% ) reported (% nitrogen X 6.25) in the body tissues by Block (7 ). As a consequence, it is sug of cattle as determined by Blaxter and gested that the factor, 5.933 (having the Rook (2 ) is almost identical to the value 95% confidence interval of 5.930 to 5.936), (5.327 kcal/g) derived for the carcass of should be used to compute the protein sheep in the present study. Although concentration from the percentage of nitro Stroud’s value (5.447 kcal/g) for protein gen in wool. of the total body of cattle is slightly higher than that (5.379 kcal/g) obtained for ACKNOWLEDGMENTS sheep in the present study, the 2 values The authors wish to thank L. D. Van are essentially the same considering the Vleck for his suggestions concerning sta errors attached to the estimates. tistical analyses, Mrs. Thelma Zablan for Although in better agreement with re help with chemical analyses, and R. E. cent observations than was the case for Allyn, J. Mauboussin, and R. P. Gerbasi the calorific value of protein, the average for help in the care and slaughtering of calorific value of fat (9.5 kcal/g) sug experimental animals. gested by Armsby (1 ), is somewhat higher than recent studies have revealed. The LITERATURE CITED following values (kcal/g) have been ob 1. Armsby, H. P. 1917 The Nutrition of Farm tained: 9.367, for cattle tissues (2 ); 9.499, Animals. The Macmillan Company, New for cattle tissues (Stroud);8 and 9.405 for York. sheep tissues (present study). It is prob 2. Blaxter, K. L., and J. A. F. Rook 1953 The able that these values are not significantly heat of combustion of the tissues of cattle in relation to their chemical composition. Brit. different from 9.5 kcal/g. J. Nutrition, 7: 83. Calorific value of wool protein and fat. 3. Association of Official Agricultural Chemists The mean calorific value of the protein of 1955 Official Methods of Analysis, ed. 8. wool was 5.609 ± 0.0023 kcal/g, and the Washington, D. C. 95% confidence interval was 5.607 to 4. Scales, F. M., and H. E. Harrison 1920 5.611 kcal/g. The mean calorific value of Boric acid modification of the Kjeldahl method for crop and soil analysis. J. Ind. wool fat was 9.741 ± 0.0035 kcal/g, with Eng. Chem., 12; 350. the 95% confidence interval of 9.738 and 5. Van Niekerk, B. D. H., J. T. Reid, A. Ben- 9.744 kcal/g. Since the degree of varia sadoun and O. L. Paladines 1963 Urinary tion about the means is so small, a high creatinine as an index of body composition. degree of accuracy is attached to the use J. Nutrition, 79: 463. 6. Rubner, M. 1885 Calorimetrische untersu- of these values for the prediction of the chungen. Ztschr. Biol., 21: 250. calorific values of wool fat and protein. 7. Block, R. J., and D. Bolling 1948 The The use of these values in future experi Determination of Amino Acids, rev. ed. Bur ments would obviate the need to determine gess Publishing Company, Minneapolis. the heat of combustion value of wool fiber 7 See footnote 5. and fat, which is difficult and laborious. 8 See footnote 5. Some Biochemical Lesions Associated with Liver Fat Accumulation in Threonine-deficient Rats’ DOROTHY ARATA, CATHERINE CARROLL2 a n d DENA C. CEDERQUIST Department of Foods and Nutrition, College of Home Economics, Michigan State University, East Lansing, Michigan ABSTRACT The metabolism of 2 groups of co-factors, the pyridine nucleotides and the adenosine polyphosphates, was altered in fatty livers produced in weanling rats by feeding a low protein diet deficient in threonine. Both total pyridine nucleo tide levels and the PN/PNH ratio were significantly decreased by the twenty-fifth day in livers from threonine-deficient animals. These values returned to control levels upon subsequent supplementation with threonine. The amount of labile phosphorus de rived from ADP and ATP from the fatty livers was significantly less than that from livers of control rats. The activity of the DPN-cytochrome c reductase enzyme system was significantly lower in livers from threonine-deficient rats than in livers from threonine-supplemented rats throughout the experimental period. The activity of the fatty acid oxidase system declined sharply during the early stages of fat accumulation in the liver. The subsequent, complete recovery of this enzyme system was followed closely by a return of liver fat concentrations to near-control levels. The ultimate dependence of all the above functions on active electron transport is noted, and the suggestion made that a threonine deficiency might affect some phase of this system. A threonine imbalance, characterized animals. In addition, the activity of an by accumulation of excess fat in the liver, enzyme system involved in reoxidation of can be produced in weanling rats by feed DPNH during electron transport, DPN- ing a 9% casein diet supplemented with cytochrome c reductase, was measured in methionine and tryptophan.3 This study livers from threonine-deficient and from was undertaken to investigate further the control rats. To assess the extent of in effects of a threonine deficiency on the volvement of ATP in this fatty liver syn metabolic functions of liver tissue. drome, determinations were made of labile Evidence has accumulated which sug phosphorus from the adenosine polyphos gests a faulty metabolism of the adenosine phates, and of endogenous inorganic phos polyphosphates and pyridine nucleotides phorus. Finally, livers were assayed for in livers infiltrated with fat under different fatty acid oxidase activity, inasmuch as experimental conditions. Dianzani (1 ) both DPN and ATP are obligatory co noted a disruption in the metabolism of factors in the series of reactions involved both of these groups of co-factors in fatty in the oxidation of fatty acids (4, 5). livers resulting from carbon tetrachloride poisoning, white phosphorus posioning, EXPERIMENTAL or choline deficiency. Total pyridine nu A total of 90 male, weanling rats of the cleotide levels, as well as the ratio of the Sprague-Dawley strain was used in this oxidized to the reduced forms (P N /PNH), study. For each series, the animals were were lower in the fatty livers than in divided into 2 groups. The rats in group 1 those from control rats (1 ). Furthermore, were fed a basal ration consisting of the ATP concentration in liver tissues was Received for publication August 5, 1963. decreased by fatty infiltration induced by 1 Supported in part by United States Air Force con tract no. AF 49(657-276). Journal Article no. 3201 any of the above means (2 ). In fatty from the Michigan Agricultural Experiment Station. livers associated with threonine deficiency, 2 Present address: Department of Home Economics, University of Arkanasas, Fayetteville, Arkansas. Arata et al. (3 ) observed a similar reduc 3 Singal, S. A., V. P. Sydenstricker and J. M. Little john 1949 The lipotropic action of threonine. Fed tion in pyridine nucleotides. An exten eration Proc., 8: 251 (abstract). 4 The PN/PNH ratios were determined by one of sion of the latter study is reported here. the authors (D.A.) in the Department of Biochemistry, The PN/PNH ratios 4 were determined University of Wisconsin, under the direction of Dr. J. N. Williams, Jr. His helpful guidance is hereby for fatty livers and for livers from control gratefully acknowledged. 150 J. N u t r i t i o n , 8 2 : ’64 THREONINE IMBALANCE IN WEANLING RATS 151 following: (in grams) casein, 9.0; choline used in place of 0.5 ml of mitochondrial chloride, 0.15; salts,5 4.0; vitamin mix,6 suspension. The DPN-cytochrome c re 0.25; corn oil,7 5.0; DL-methionine, 0.3; ductase activity was determined by the DL-tryptophan, 0.1; and sucrose to 100. method of Potter (9 ). Crude malic de The diet fed to rats in group 2 was iden hydrogenase preparations required for this tical with that above, except that 0.36 g system were isolated from fresh beef DL-threonine was added at the expense of hearts as described by Potter (9 ). Fat was sucrose. determined in dried, ground liver homog Pyridine nucleotide content of liver tis enates by continuous ether extraction in sues was determined after rats had been a Goldfisch apparatus. fed the experimental diets for 25 days. Livers were assayed for labile phosphorus RESULTS (from ADP and A TP), endogenous in Results from the differential assay for organic phosphorus, fat and for activities pyridine nucleotide content of the livers of the DPN-cytochrome c reductase and are summarized in table 1. The data for fatty acid oxidase enzyme systems, after total pyridine nucleotide values are in 2, 3, and 4 weeks of feeding. A 4-week agreement with those reported previously experimental period was judged to be of (3 ). Livers from threonine-deficient rats insufficient length for assay of the fatty (group 1) contained more than twice as acid oxidase system. A second experiment much PNH as the livers from control rats, was devised wherein fatty acid oxidase whereas the concentration of the oxidized activity was measured over a period of form of these coenzymes (P N ) was sig 6 weeks. The data from the 2 experiments nificantly decreased. The decrease in PN were combined. together with the increase in PNH in Total pyridine nucleotide, oxidized pyri livers from threonine-deficient rats re dine nucleotide (P N ), and reduced pyri sulted in a greatly reduced PN/PNH ratio, dine nucleotide (P N H ) were determined equal to only about one-quarter of the according to a procedure developed by ratio in livers from control rats. In view Feigelson et al. (6 ). Total labile phos of these results, it was considered perti phorus from ADP and ATP was deter nent to determine whether a short interval mined by charcoal adsorption of the adenosine phosphates from aliquots of s Wesson, L. G. 1932 A modification of the Os- borne-Mendel salt mixture containing only inorganic liver homogenates, followed by hydrolysis constituents. Science, 75: 339. Obtained as Salt in HC1 to release the labile phosphorus Mixture-W from Nutritional Biochemicals Corporation, Cleveland. from the polyphosphates (7 ). Fatty acid 6 The vitamin mix provided the following in mg/ 100 g ration: thiamine HC1, 0.5; riboflavin, 0.5; niacin, oxidation was measured manometrically, 1.0; pvridoxine, 0.25; Ca pantothenate, 2.0; inositol, using the Warburg apparatus. The method 10.0; folic acid, 0.02; vitamin B12, 0.002; biotin, 0.01; vitamin A powder, 10.0 (200 USP units); and cal used was essentially that of Lehninger for ciferol, 0.18 (150 USP units). mitochondrial preparations (8 ), except 7 Mazola, Corn Products Company, Argo, Illinois, containing 7.5 mg a-tocopheryl acetate and 0.375 mg that 1 ml whole homogenate (33% ) was menadione/5 g. TABLE 1 Liver pyridine nucleotide 1 and DPN-cytochrome c reductase 2 data from rats fed 9% casein diets with and without supplementary threonine' for 2 zveeks DPN-cyto Total chrome c Group No. PNH "> PN/PNH no.3 rats pyridine PN 4 reductase nucleotide activity 1 9 7 7 3 ± 9 6 674 ±2 1 99 ±1 4 6.9 139± 11 2 9 1 0 2 1 ± 1 0 986 ± 1 4 35 ± 8 28.2 182 ±1 3 1 Expressed as /¿g/g fresh tissue. 2 Expressed as/filter 0 2 /hr/g fresh tissue. ^ . 3 Group 1, 9% of casein, no supplementary threonine; group 2, 9% of casein 4- 0 .3 b % ot D L - threonine. 4 Oxidized pyridine nucleotide. 5 Reduced pyridine nucleotide. 6 se of mean. 152 DOROTHY ARATA, CATHERINE CARROLL AND DENA C. CEDERQUIST of feeding the threonine-supplemented liver fat levels had reached maxima in diet to the remaining rats in group 1 2 to 3 weeks. In this experiment the peak would cause the PN/PNH ratio to return in liver fat deposition appeared more rap to a normal level. Six rats from group 1 idly than in previous runs. As a conse and 3 rats from group 2 were maintained quence, the sampling procedures were with their respective diets for a period of improperly spaced and a clear cut peak 39 days. At that time, 3 of the 6 rats in was not observed. An additional experi group 1 were fed diet 2 (9% of casein + ment was devised as an adjunct to the one threonine). All animals were fed for an reported here. Data were collected during additional 5 days. At the close of this the period of fat accumulation in liver 5-day period, the rats were killed and their tissues and through the diminishing phase livers analyzed for PN and PNH. Rats of the liver fat curve. Data from the 2 that had been maintained with the threo experiments were combined, and are pre nine-deficient diet for a total of 44 days sented in table 2. Liver fat levels in the had an average liver PN/PNH ratio of control group (group 2) did not increase 4.5. Those rats maintained with the con significantly above the levels at zero time. trol diet for 44 days had a ratio of 16.0. Fatty acid oxidase activity per gram of The rats fed the deficient diet for 39 days liver from rats fed the threonine-deficient and the control diet for 5 days had an diet was significantly lower than the ac average PN/PNH ratio of 19.9, essentially tivity in livers from control rats during identical with that of the control group. the phase of fat deposition in the liver. The activity of the DPN-cytochrome c During fat mobilization out of the fiver, reductase system in liver was depressed activity of the fatty acid oxidase system in throughout the experimental period in threonine-deficient rats was significantly threonine-deficient rats as compared with greater than the activity of the same sys threonine-supplemented rats (table 1). tem during fat deposition (fig. 1). This difference was significant at the 5% When fatty acid oxidase activities were level. calculated on the basis of the total weight A progressive accumulation of fat in of fiver tissues, a similar curve was ob livers of threonine-deficient rats was again tained. In fact, the depression in the total observed. In previous studies (3, 10) activity of this enzyme system was more TABLE 2 Liver fat and fatty acid oxidase data from rats fed 9% casein diets with and without supplementary threonine for 6 weeks 1 Group 1 2 Group 2 3 No. Fatty acid rats Liver fat Fatty acid oxidase activity Liver fat oxidase activity % of dry wt fil Oz/hr/g liver % of control % of dry wt fil Oz/hr/g liver 12 8.2 ± 1.0 4 552 ±4 3 4 100 8 .2 ± 1.0 4 552 ±4 3 4 8 15.4 ±1 .5 449 ± 4 0 84 7.0 ±0 .8 532 ± 39 4 20.2±2.1 318 ±2 4 41 8.4 ±1 .2 782 ± 83 4 23.2 ±6 .6 267 ± 7 6 43 10.3± 1.3 621 ± 6 0 5. 31.7±2.6 4 5 0 ± 51 108 13.5±4.7 4 1 5±4 5 5 17.4 ±2 .9 565 ±4 5 128 7.8 ±2 .4 441 ± 6 6 5 11.2±3.7 6 1 4±4 7 146 6.4 ±0 .5 442 ±5 4 4 17.1 ± 2 .2 735 ± 1 3 137 8.3 ±0 .4 538 ± 3 5 5 11.9 ± 0.7 470 ± 8 0 81 5.6 ±0 .4 581 ± 7 2 1 These figures represent a compilation of 2 separate experiments. The time factor varied somewhat between the experiments; therefore, time notations would be approximate and thus are omitted. The complete experiment ran for 6 weeks and the peak in liver fat deposition occurred at approxi mately 2 weeks. 2 Fed 9% of casein, no supplementary threonine. 3 Fed 9% of casein + 0.36% of DL-threonine. 4 se of mean. THREONINE IMBALANCE IN WEANLING RATS 153 WEEKS Fig. 1 Changes in fatty acid oxidase activity in livers from threonine-deficient rats dur ing accumulation and removal of excess fat. severe during the phase of fat deposition, during the fourth week (fig. 2). Amounts falling to 26% of the control. These re of labile phosphorus from the threonine- sults were spot-checked in another group deficient group were significantly lower of rats, and the same relationship was (P < 0 .0 1 ) than those from the control observed. group throughout the experimental period The amount of labile phosphorus de (table 3). The concentration of endoge rived from ADP and ATP in livers from nous inorganic phosphorus in livers from threonine-deficient rats (group 1) varied both groups of rats increased at compar somewhat over the 4-week period, but able rates for 3 weeks. However, during showed no substantial fluctuations from the fourth week, when labile phosphorus the level established at zero time. In livers had leveled off in the control group, there from the control group, on the other hand, was a much sharper increase in inorganic labile phosphorus values increased sharply phosphorus in this group than in the de in the first 2 weeks, continued to increase ficient group (table 3). The significance during the third week, then leveled off of this observation is undetermined. 154 DOROTHY ARATA, CATHERINE CARROLL AND DENA C. CEDERQUIST TABLE 3 Liver phosphorus data from rats fed 9% casein diets with and without supplementary threonine Weeks No. Group 1 1 Group 2 2 fed diet rats fig/ total liver % of control fig/ total liver Labile phosphorus from ADP and ATP 0 4 307 100 307 2 8 343 ±3 3 3 61 560 ±5 3 3 3 4 289 ±4 8 46 627 ±1 5 4 4 359 ±4 7 56 639 ±6 6 Endogenous inorganic phosphorus 0 4 735 100 735 2 8 723 ±4 4 82 880 ±6 9 3 4 865 ± 62 86 1008 ±91 4 4 1079 ±145 59 1829 ±183 1 Fed 9% of casein, no supplementary threonine. 2 Fed 9% of casein + 0.36% of DL-tbreonine. 3 se o f mean. decreased rapidly to near-control levels. It was not a true inverse relationship, since enzyme activity changes preceded liver fat changes. This suggests that the fatty acid oxidase system is an important instrument in regulating liver fat levels. The activity curve for fatty acid oxidase presented here is strikingly similar to ac tivity curves for xanthine oxidase and malic dehydrogenase in threonine-defi cient rats (10). These enzymes, too, began to recover before the accumulated fat was mobilized out of the liver. A similar ob servation was made by Arata et al. (3 ), namely, maximal stimulation of endoge nous oxidation by added DPN in livers from threonine-deficient rats occurred be fore the peak in liver fat deposition. Using histological techniques for the determina tion of liver fat induced by carbon tetra chloride, Recknagle and Anthony (1 1) suggested that biochemical lesions and liver fat accumulation are not interde pendent. However, the observations above Fig. 2 Labile phosphorus from ADP and ATP in livers from threonine-deficient and threonine- strongly suggest a definite temporal rela supplemented rats. tionship between changes in the activities of some enzyme systems and movement DISCUSSION of fat into and out of the liver. Concurrent changes in activity of the The temporal changes in the activities fatty acid oxidase system and in concen of the oxidative enzymes mentioned above tration of fat in livers from threonine- appear to be nonspecific responses to the deficient rats appeared to follow a pattern deficiency of threonine. It is unlikely that (fig. 1). As the activity of the enzyme any of these systems reflects a primary system decreased, fat began to accumulate biochemical lesion. The appearance of in the livers; then, after the enzyme sys fatty livers in threonine-deficient animals tem had fully recovered, fat concentration may, in fact, be a compensatory mecha THREONINE IMBALANCE IN WEANLING RATS 155 nism to allow recovery of the enzyme tem, and is one of the chief sites of con systems in a manner similar to the one version of DPNH to DPN; ATP is required suggested by Wilgram et al. (1 2 ) for for synthesis of DPN (18, 20); the rate of choline deficiency. fatty acid oxidation is dependent on avail The disturbance observed in fatty acid ability of DPN; and the oxidation of fatty oxidation could have been secondary to acids is a major route for removal of fat changes in concentration of pyridine nu from the liver. Also, the activity of the cleotides, since DPN acts as a coenzyme cytochrome oxidase enzyme system, which in the fatty acid oxidase system (5, 13). accepts electrons derived from DPNH, is The reduced quantity of total PN observed depressed in livers of threonine-deficient in the livers of threonine-deficient rats in rats.8 A primary attack on one of the links this experiment would limit the supply of in electron transport would probably affect DPN available for fatty acid oxidation. the normal functioning of other compo At the same time, the pile-up of PNH nents of the system, through deficiency of (including DPNH and TPN H ) would tend substrates or pile-up of products, or both, to favor fatty acid synthesis (14-16). In and could conceivably lead to all the le fact, Yoshida and Harper (1 7) demon sions noted above. strated an increased rate of fatty acid The fact that the activity of the fatty synthesis from C14-labeled acetate in threo acid oxidase system returned to normal or nine-deficient rats. above, whereas the PN/PNH ratio and the The major factor contributing to the activity of the DPN-cyctochrome c reduc depression in total pyridine nucleotide con tase system remained at suboptimal levels, tent of the fatty livers could well have suggests that some biochemical adaptation been a defect in ATP metabolism. The took place. It is possible that the limited decrease in labile phosphorus in livers amount of DPN available was used pref from threonine-deficient rats probably indi erentially for fatty acid oxidation, that cates a restricted quantity of ATP. Dian- adjustments occurred in PN/PNH transhy- zani (2 ) reported the concentration of drogenase activities, that alternate path ATP to be markedly reduced and that of ways became more active, or that some ADP somewhat increased in fatty livers, other adaptive change occurred. The data resulting in a net decrease in total ADP presented here do not provide sufficient evi plus ATP. When Stekol et al. (18) induced dence to identify a specific mechanism by a decrease in liver ATP levels by feeding which fatty acid oxidation and liver fat ethionine, they noted a parallel decrease levels approached control levels with time, in DPN synthesis. In the present study, The temporal behavior of these systems the decrease in labile phosphorus values has important implications with respect in threonine-deficient rats (expressed as to the original lesion. The complete re percentage of control) tended to parallel covery of the PN/PNH ratio in depleted the decrease in fatty acid oxidase activity rats after only 5 days’ supplementation in the same rats during the period of fat with threonine, together with the failure accumulation in the liver. to recover with time of those systems most The metabolic lesions observed in this closely associated with transfer of electrons study (decreased supply of ATP, depressed from DPNH to cytochrome c, strongly sug activity of the DPN-cytochrome c reduc gest that a threonine imbalance directly tase system, greatly reduced PN/PNH influences a phase of electron transport ratio, lower levels of total pyridine nucleo essential for maximal reoxidation of tides, and depressed rate of fatty acid DPNH. oxidation, with accumulation of fat in the LITERATURE CITED liver) all appear to point to some portion of the electron transport system as the 1. Dianzani, M. U. 1955 Content and distribu tion of pyridine nucleotides in fatty livers. possible primary target of a threonine de Biochem. Biophys. Acta, 17: 391. ficiency. The major mechanisms for ATP 2. Dianzani, M. U. 1957 The content of adeno generation are coupled with electron trans sine polyphosphates in fatty livers. Biochem. port (1 9); DPN-cytochrome c reductase is J., 65: 116. a component of the electron transport sys 8 Unpublished observations. 156 DOROTHY ARATA, CATHERINE CARROLL AND DENA C. CEDERQUIST 3. Arata, D., G. Svenneby, J. N. Williams, Jr. mitochondrial degeneration. J. Biol. Chem., and C. A. Elvehjem 1956 Metabolic factors 234: 1052. and the development of fatty livers in par 12. W ilgram , G. F., C. F. Holloway and E. P. tial threonine deficiency. J. Biol. Chem., Kennedy 1960 The content of cytidine di 219: 327. phosphate choline in the livers of normal 4. Lehninger, A. L. 1945 The relationship of and choline-deficient rats. J. Biol. Chem., the adenosine polyphosphates to fatty acid 235: 37. oxidation in homogenized liver preparations. 13. Green, D. E. 1954 Fatty acid oxidation in J. Biol. Chem., 157: 363. soluble systems of anmal tissues. Biol. Rev., 5. Lehninger, A. L. 1945 On the activation of Cambridge Phil. Soc., 29: 330. fatty acid oxidation. J. Biol. Chem., 161: 14. Van Baalan, J., and S. Gurin 1953 Co 437. factor requirements for lipogenesis. J. Biol. 6. Feigelson, P., J. N. Williams, Jr. and C. A. Chem., 205: 303. Elvehjem 1950 Spectrophotometric estima 15. Langdon, R. G. 1957 The biosynthesis of tion of pyridine nucleotides in animal tissue. fatty acids in rat liver. J. Biol. Chem., J. Biol. Chem., 185: 741. 226; 615. 7. Seitz, I. F. 1956 Determination of adeno 16. Wakil, S. J. 1961 Mechanisms of fatty sine di- and tri-phosphates. Bull. Exp. Biol. acid synthesis. J. Lipid Res., 2: 1. Med. (U .S.S.R.), 22: 235. 17. Yoshida, A., and A. E. Harper 1960 Effect 8. Lehninger, A. L. 1955 Fatty acid oxidation of threonine and choline deficiencies on the in mitochondria. Methods in Enzymology, metabolism of C14-labeled acetate and pal- vol. 1, eds., S. P. Colowick and M. O. Kaplan. mitate in the intact rat. J. Biol. Chem., Academic Press, Inc., New York, p. 545. 235: 2586. 9. Potter, V. R. 1946 The assay of animal 18. Stekol, J. A., E. Bedrak, U. Mody, N. Burnette tissues for respiratory enzymes. V. The malic and C. Sommerville 1963 Inhibition of the dehydrogenase system. J. Biol. Chem., 165: synthesis of diphosphopyridine nucleotide by 311. ethionine administration in the liver of rats 10. Carroll, C., D. Arata and D. C. Cederquist and mice. J. Biol. Chem., 238: 469. 1960 Effect of threonine deficiency on 19. Lehninger, A. L., C. L. Wadkins, C. Cooper, changes in enzyme activity and liver fat T. M. Devlin and J. L. Gamble, Jr. 1958 deposition with time. J. Nutrition, 70; 502. Oxidative phosphorylation. Science, 128: 450. 11. Recknagel, R. O., and D. D. Anthony 1959 20. Preiss, J., and P. Handler 1958 Biosyn Biochemical changes in carbon tetrachloride thesis of diphosphopyridine nucleotide. II. fatty liver: separation of fatty changes from Enzymatic aspect. J. Biol. Chem., 233: 493. Influence of Nicotinic, Picolinic and Pyridine-3-sulfonic Acids on Cholesterol Metabolism in the Rat1 DAVID KRITCHEVSKY a n d SHIRLEY A. TEPPER The Wistar Institute, Philadelphia, Pennsylvania ABSTRACT The addition of 1% of nicotinic, picolinic or pyridine-3-sulfonic acid to a cholesterol-free rat diet for 3 weeks did not affect serum cholesterol levels. Liver cholesterol levels of the nicotinic acid-fed rats were lower than those of the controls. When these compounds were fed at 1% of the diet to rats also receiving 2% cholesterol and 0.5% cholic acid, all test groups displayed elevated serum cholesterol levels and the nicotinic acid group also had a higher liver cholesterol content than did the controls. Hepatic cholesterogenesis from sodium acetate-l-C14 in rats fed 1% nicotinic acid or pyridine-3-sulfonic acid was significantly reduced, whereas that of the group fed picolinic acid was below that of the control group but not significantly so. The mechanism by which nicotinic acid in vivo effects of these compounds upon exerts its hypocholesterolemic effect is not cholesterol metabolism in the rat and their yet clear (1). Neither common deriva comparison with nicotinic acid. tives, such as nicotinamide (2), nor metab olites, such as nicotinuric acid (3), display METHODS any hypocholesterolemic properties. The All studies were carried out in male authors of the initial report on the action Wistar rats weighing 150-160 g. The basal of nicotinic acid (4 ) suggested that it test diet contained, per 100 g: (in grams) might act by increasing the in vivo oxida cereal mixture, 70; wheat germ, 6.25; skim tion of cholesterol. Studies of the influ milk powder, 20; vitamin mixture, 2.75; ence of nicotinic acid on the oxidation of and test compound, 1.0.3 Sufficient water cholesterol by rat liver mitochondria (5 ) was added to make a thick dough. This showed that mitochondrial preparations diet provides 20% protein, 11% fat and from livers of rats fed nicotinic acid oxi 62% carbohydrate and was well received dized more cholesterol-26-C14 to Cl10 2 than by the animals. The control diet contained did similar preparations from untreated 70 g of cereal mixture. In the hypercho- rats. It was further observed that addition lesterolemic diet, cholesterol (2 g) and of nicotinic acid (6 to 15 m g) to control cholic acid (0.5 g) were added at the ex mitochondrial preparations also enhanced pense of the cereal mixture. Rats were cholesterol oxidation. A nicotinic acid maintained with the respective diets for homologue, 3-pyridylacetic acid has been 3 weeks at which time they were killed shown to lower serum cholesterol levels in and the serum and liver analyzed for cho man (6 ) and to inhibit TritonMnduced lesterol by the Mann (10) modification of hypercholesterolemia in rats (7). In ex the method of Zlatkis et al. (11). Aliquots periments with a series of nicotinic acid of the liver were dissolved in 15% alco homologues, only 3-pyridylacetic acid was holic KOH and the color reaction carried *123 active in vitro in enhancing cholesterol Received for publication August 19, 1963. oxidation by rat liver mitochondria (8). 1 This work was supported, in part, by USPHS Re In a subsequent series of experiments (9 ) search Grant HE-03299 and Career Award 5-K6-HE-734 from the National Heart Institute. it was observed that although neither 2 Rohm and Haas, Philadelphia. 3 Cereal mixture, Pablum, Mead Johnson and Com nicotinamide nor its homologues affected pany, Evansville, Indiana; wheat germ, Kretschmer mitochondrial oxidation of cholesterol, 2 Corporation, Carrollton, Michigan; skim milk powder, Carnation Company, Los Angeles; vitamin mixture, compounds related to nicotinic acid (pico Vionate, E. R. Squibb and Sons, New York; nicotinic acid, Eastman Organic Chemicals, Rochester, New linic acid, pyridine-2-carboxylic acid, and York; picolinic acid-3-pyridinesulfonic acid, and cholic pyridine-3-sulfonic acid) increased oxida acid, Calbiochem, Inc., Los Angeles; cholesterol, W il son and Company, Chicago; sodium acetate-l-C14, tion. The present report deals with the New England Nuclear Corporation, Boston. J. N u t r i t i o n , 8 2 : ’ 6 4 157 158 DAVID KRITCHEVSKY AND SHIRLEY A. TEPPER out on the petroleum ether extract of the effects of 1% of nicotinic acid, nicotina saponification mixture. In the biosynthesis mide and isonicotinic acid (pyridine-4- experiment each animal was given an carboxylic acid) upon the serum and fiver intraperitonial injection of sodium acetate- cholesterol levels of rats fed a diet con 1-C14 (1 uc/100 g ) and killed 6 hours later. taining 10% lard for 4 weeks. The nico One-gram aliquots of the livers of these tinic acid group showed somewhat higher animals were taken for cholesterol deter serum cholesterol levels and lower fiver mination. The remaining fiver was also cholesterol levels than did the controls. saponified and the nonsaponifiable mate The picolinic acid group showed lower rial extracted with petroleum ether. The serum and fiver cholesterol levels than the digitonide complex of the cholesterol pres control group and nicotinamide had no ent in the dried extract was prepared. The effect. Our data are presented in table 1. digitonides were dissolved in 1 ml of In 3 weeks none of the test compounds Hyamine4 and assayed for radioactivity (nicotinic acid, picolinic acid, pyridine-3- using a Packard Tri-Carb Liquid Scintilla sulfonic acid) had a signficant effect upon tion Spectrometer (12). serum cholesterol levels and the fiver cho lesterol levels of the nicotinic acid-fed RESULTS AND DISCUSSION group were lower than those of the 3 other In our earlier experiments (5 ) we found groups. No anorexia (as evidenced by that the addition of 0.75% nicotinic acid reduced weight gain) was observed in any to a cholesterol-free diet over a 3-week group. period had no significant effect on the Since the clinical use of nicotinic acid serum cholesterol levels of either male or involves its administration to hypercho- female rats. Duncan and Best (13) made lesterolemic subjects, of greater interest a similar observation in rats fed 1% nico was the influence of nicotinic acid and tinic acid for either 8 or 42 days. After the other test compounds upon the serum 8 days they noted no differences in the and fiver cholesterol levels of rats being fiver cholesterol levels of the fed or con tested with a hypercholesterolemic regi trol rats, but at 42 days the fiver choles men. Nath et al. (16) observed that when terol levels of the nicotinic acid-fed rats 1% nicotinic acid was fed to rats in a were somewhat higher than those of the diet containing 1% cholesterol and 0.5% control group. This observation was true cholic acid there was an increase in both in pair-fed rats as well as in rats fed ad serum and fiver cholesterol levels com libitum. Friedman and Byers (14) re pared with those of controls. Gaylor et al. ported that the addition of 1% nicotinic (15) added 1% nicotinic acid to the diets acid to the diet lowered cholesterol levels of rats pre-fed cholesterol (1% ) and cholic in rats examined at either 12 or 14 days acid (0.5% ) and reported a retardation of and attributed this to an anorexic effect cholesterolemia. Friedman and Byers (14) of the drug since the nicotinic acid-fed carried out a study in which 1% nicotinic groups gained less weight than did the con acid was fed to rats with a modified Hart- trols. Duncan and Best (1 3 ) also observed roft (17) hypolipotropic diet and observed smaller weight gains in nicotinic acid fed a retardation of hypercholesterolemia in rats. Gaylor et al. (15) compared the 4 Packard Instrument Company, La Grange, Illinois. TABLE 1 Autopsy data — rats fed normal diets plus 1 % test compound for 21 days Group Liver Serum Survival Wt gain Liver wt cholesterol cholesterol 9 9 m g / g m g / 1 0 0 m l Nicotinic acid 4/5 1 06± 3.9 1 8.9 ± 0 .8 1 1.71 ±0.09 1 78.4± 4.5 1 Picolinic acid 4/5 89 ± 6.8 9.8 ±0 .9 1.86±0.11 89.3 ± 11.6 Pyridine-3-sulfonic acid 5/5 87 ± 12.4 9.3 ± 0 .7 2.23 ±0.22 2 84.9 ± 11.2 Control 5/5 9 4 ± 13.3 9.7 ±1 .2 1.96±0.05 2 75.2 ± 7.1 1 se of mean. 2 Test for significance: nicotinic acid vs. pyridine-3-sulfonic acid or control 0.05 > P > 0.01. PYRIDINE ACIDS AND CHOLESTEROL METABOLISM 159 ad libitum fed rats but none in pair-fed lesterol pools were also similar for the 2 groups. Schon (18) reported that 1% groups. We have calculated serum-liver nicotinic acid had only a slight effect on cholesterol pools (1 9 ) for all the groups of the liver cholesterol levels of rats fed a rats used in both experiments and these special hypolipotropic diet. Our own ob are presented in table 3. The serum-liver servations upon the serum and liver cho cholesterol pools of all the test groups were lesterol concentrations in rats fed 1% somewhat lower than that of the control in nicotinic, picolinic or pyridine-3-sulfonic the series in which cholesterol was present acid, 2% cholesterol and 0.5% cholic acid in the diet. With the control diet only the for 3 weeks (table 2) show that all 3 test nicotinic acid-fed group had a strikingly groups had higher cholesterol levels than smaller serum-liver cholesterol pool. These did the controls, and the nicotinic acid data show the relatively small contribution group also had significantly higher liver of the serum cholesterol to this pool in cholesterol levels. The serum cholesterol either diet. data are in agreement with those of Nath The question of how nicotinic acid af et al. (16) whose experimental design was fects synthesis of cholesterol by rat liver substantially similar to ours. The control has been studied by a number of investi group exhibited a greater weight gain than gators with conflicting results. In table 4 did any of the test group. Liver weights of we have attempted to summarize the pre the nicotinic and picolinic acid-fed rats vious results. Duncan and Best (13) ob were significantly lower than those of the served a slightly decreased incorporation controls. of acetate-l-C14 into the liver cholesterol Duncan and Best (13) observed that of rats fed 1% nicotinic acid for 42 days the total carcass cholesterol content of rats but the difference was not significant. fed 1% nicotinic acid was the same as Hardy et al. (22) reported the duration of that of the controls. The serum-liver cho feeding affected cholesterogenesis. After TABLE 2 Autopsy data — rats fed cholesterol (2% ) cholic acid (0.5% ) diet plus 1% of test compound for 21 days Liver Serum Group Survival Wt gain Liver wt cholesterol cholesterol 9 9 m g / g v ig / 1 0 0 m l Nicotinic acid 8/8 39 ± 4.5 '■* 9.1 ± 0 .4 Lb 2.73 ± 0.21 >■« 229.6 ±2 6.6 1 Picolinic acid 3/8 58 ±7 .2 9.6± 0.7 c 1.89 ±0.44 222.3 ±87.0 Pyridine-3- sulfonic acid 7/8 48 ± 7 .0 11.8±0.5 d 1.64 ±0.25 227.0 ±37.4 Control 7/8 6 9 ±8 .5 12.8 ±0 .5 2.18 ±0.15 176.3 ± 9.8 1 se of mean. a Significance, control vs. nicotinic acid, 0.01 > P > 0.001. b Significance, control vs. nicotinic acid, P < 0.001. c Significance, control vs. picolinic acid, 0.01 > P > 0.001. d Significance, pyridine-3-sulfonic acid vs. nicotinic acid, 0.01 > P > 0.001; pyridine-3-sulfonic acid vs. picolinic acid, P < 0.05. e Significance, control vs. nicotinic acid, P = 0.05. TABLE 3 Serum-liver cholesterol pools for rats fed 1% nicotinic, picolinic and pyridine-3-sidfonic acid with normal and hypercholesterolemic diets for 21 days Normal diet Cholesterol diet Group Serum Liver Total Serum Liver Total m g m g m g m g m g rug Nicotinic acid 6.1 152.2 158.3 13.0 248.4 261.4 Picolinic acid 6.5 182.3 188.8 13.6 181.4 195.0 Pyridine-3-sulfonic acid 6.3 207.4 213.7 13.5 192.3 205.8 Control 5.6 190.1 195.7 11.4 279.0 290.4 160 DAVID KKITCHEVSKY AND SHIRLEY A. TEPPER TABLE 4 Effect of nicotinic acid (N A ) on hepatic cholesterol synthesis in rats Rat strain Type of experiment Indicator Results Reference Sprague-Dawley intact, NA-fed acetate-C14 increase (2 0 ) Sprague-Dawley liver slices, NA added acetate-C14 increase (2 0 ) Long Evans intact, NA-fed biliary excretion no change (1 4 ) Holtzman intact, NA-fed acetate-C14 no change (1 3 ) Hooded liver slices, NA added acetate-C14 decrease (2 1 ) Holtzman liver slices, N A pre-fed acetate-C14 increase (2 2 ) Holtzman liver slices, N A added acetate-C14 no change (2 2 ) Long Evans liver slices, N A added acetate-C14 decrease (7 ) Long Evans liver slices, N A added mevalonate-C14 decrease (7 ) 3 days of nicotinic acid administration acid and 231% for nicotinic acid (22). cholesterol synthesis was below that of the Pyridine-3-acetic acid (7 ) was active to controls as it was after 7 days but at 14, the same extent as nicotinic acid in in 21 or 28 days there was an increase in hibiting biosynthesis of cholesterol from cholesterogenesis. acetate or mevalonate. The experiments of Hardy et al. (22) We administered sodium acetate-l-C14 were carried out using livers from rats fed intraperitoneally (1 uc/100 g) to each of a diet containing 10% lard which may in the rats fed the cholesterol-free diet after itself affect cholesterol biosynthesis (23, 3 weeks, killed them 6 hours later and 24). Addition of nicotinic acid directly to assayed the liver cholesterol-C14 as the digi- the incubation mixture generally exerted tonide. Our observations are detailed in little effect. Bizzi and Grossi (7 ) observed table 5. The biosynthesis of cholesterol that nicotinic acid added at a level of 2 was significantly reduced in the groups nmole increased cholesterol synthesis from fed nicotinic or pyridine-3-sulfonic acid. acetate but not from mevalonate; but at The hepatic synthesis of cholesterol is 20 nmole nicotinic acid inhibited biosyn affected by the cholesterol content of the thesis from either precursor. liver (25, 26). In our series of experiments Of the experiments in which compounds the average fiver cholesterol content of related to nicotinic acid were used Perry the nicotinic acid-fed group was lower (21) and Hardy et al. (22) observed that than that of the controls. If the percent nicotinamide had no effect upon choles age of C14 incorporation is multiplied by terol biosynthesis when added to the incu the total fiver cholesterol content some bation mixture. In rats pre-fed the test idea of how fiver cholesterol affects bio compound for 3 weeks neither nicotina synthesis might be obtained. mide nor isonicotinic acid enhanced hepa The results of such a calculation in the tic cholesterol synthesis to the extent that groups described in table 5 would be: 1% nicotinic acid did. The increases ob nicotinic acid, 73.1; picolinic acid, 133.1; served with these compounds were 127% pyridine-3-sulfonic acid, 77.8 and control, for nicotinamide, 146% for isonicotinic 195.0. TABLE 5 Biosynthesis of cholesterol from acetate by rats maintained with 1% nicotinic, picolinic or pyridine-3-sulfonic acid for 21 days Group No. Incorporated C14 P 2 % Nicotinic acid 4 0.480±0.071 1 0.05 > P > 0.01 Picolinic acid 4 0.730 ±0.192 N.S.3 Pyridine-3-sulfonic acid 5 0.375 ±0.019 0.05 > P > 0.01 Control 5 1.026 ±0.204 — 1 se of mean. 2 Significance compared with control. 3 Not significant. PYRIDINE ACIDS AND CHOLESTEROL METABOLISM 161 In the other experiments carried out in to nicotinic acid and nicotinamide. Arch. which cholesterol biosynthesis was deter Int. Pharmacodyn., in press. 10. Mann, G. V. 1961 A method for choles mined in intact rats fed nicotinic acid, terol in serum. Clin. Chem., 7: 275. Merrill (20) fed 0.8% nicotinic acid and 11. Zlatkis, A., B. Zak and A. J. Boyle 1953 A assayed biosynthesis after 8 days and new method for the direct determination of Friedman and Byers (14) fed 1% of this serum cholesterol. J. Lab. Clin. Med., 4 1 : 486. compound and measured biosynthesis at 12. Shapiro, I. L., and D. Kritchevsky 1963 2 weeks. Duncan and Best (13) noted a Radioassay of cholesterol-C14 digitonide. slight, but not significant, increase in Anal. Biochem., 5: 88. hepatic cholesterogenesis in rats fed 1% 13. Duncan, C. H., and M. M. Best 1960 Lack nicotinic acid for 8 days and a non-signifi of nicotinic acid effect on cholesterol metab olism in the rat. J. Lipid Res., 1: 159. cant decrease in rats fed 1% nicotinic acid 14. Friedman, M., and S. O. Byers 1959 for 42 days. In view of the observations Evaluation of nicotinic acid as an hypocho- of Hardy et al. (22) on the time effect of lesteremic and anti-atherogenic substance. nicotinic acid on cholesterol biosynthesis, J. Clin. Invest., 38: 1328. 15. Gaylor, J. L., R. W. F. Hardy and C. A. this factor should be taken into consider Baumann 1960 Effects of nicotinic acid ation in comparing data. 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