‘l > I I | \ I L NI‘WIHHII k I ”ll ‘ l s 1 W i ll! THE ENE’LUENCE OF DIETARY EAT ON THE RESPONSE OF THE WEANLING ALBINO RAT TO EXCESSWE ENTAKE OF THIAMBNE AND MACE-N Thesis {*‘or flu Degree 0‘ M. S. MECPLZGAN STATE UNE‘JEESETY EeVerEy Ime- Kioosfzer 1961 LIBRARY Michigan State University ABSTRACT THE INFLUENCE OF DIETARY EAT ON THE RESPONSE OF THE WEANLING.ALBINO RAT TO EXCESSIVE INTAKE OF THIAMINE AND NIACIN by Beverly Jane Klooster Since high potency vitamin preparations are directly avail- able to the consumer, it is possible to consume excess quantities of the water soluble vitamins. This experiment was undertaken to determine if consumption of excess quantities of thiamine or niacin were toxic when added to low or high fat diets. Male albino weanling rats of the Sprague-Dawley strain were distributed among five experimental groups. Each group was fed (ad libitum) one of the following diets during the six-week experi- mental period. Group I 20% casein, 5% fat. Group II 20% casein, 5% fat, 0.1% thiamine. Group III 20% casein, h0% fat. Group IV 20% casein, h0% fat, 0.1% thiamine. Group V 20% casein, h0% fat, 0.1% niacin. At weekly intervals five rats from each group were sacrificed. Livers were removed, weighed, and homogenized with water in a Potter-Elvehjem homogenizer. The homogenate was evaporated to dryness, and ground in a Wiley mill with a ho mesh screen. Pat was determined by ether extractkn1in the Goldfisch apparatus. Nitro- gen was determined by the macro-Kjeldahl method. Beverly Jane Klooster Standard errors were calculated for each mean, Students “t" test was used as a measure of significance. Rats fed the high flat diets (groups III, IV; and V) gained less weight than the rats fed the low fat diets (groups I and II). Rats receiving the high flat-high thiamine diet (group IV) were heavier than those rats on the high fat control diet (group III) throughout the experimental period, but especially from the second through the fourth week. Livers taken from the rats on the high fat diets were signif- icantly smaller than those from rats on the low fat diets regardless of the vitamin composition of the diets. No significant differences in liver moisture or liver nitro- gen were found between any of the groups studied. Increasing the fat content of the diet from 9% to h0% resulted in the accumulation of an excess quantity of fat in the liver. COD? trol rats fed a diet containing b0% fat had a maximum liver fat level of 19% (at four weeks) as compared with.a maximum of 11% (at two weeks) in control rats fed a 5% fat diet. The presence of 0.1% supplementary thiamine in the diet had no sustained effect on liver fat in either the high or low fat diets. However, a.narked increase in liver fat was observed when 0.1% niacin was added to the high fat diet. Rats fed this diet accumulated a maximum of 2h% liver fat in one week. The accumula- tion of fat in the livers of animals fed the high fat-high niacin diet was rapid and sustained. .At the end of six weeks, liver fat levels in these animals were still about 20%. Beverly Jane Klooster Under the conditions of this eXperiment, thiamine was rele- tively non toxic at the 0.1% level regardless of the fat content of the diet. However, a diet containing 10% fat and 0.1% niacin appeared to be toxic to weanling rats, as manifested by the accumu- lation of fat in the livers of the animals in this group. THE INFLUENCE OF DIETARY FAT ON THE RESPONSE OF THE WEANLINGmALBINO RAT TO EXCESSIVE INTAKE OF THIAMINE AND NIACIN By Beverly Jane Klooster .A THESIS Submitted to Michigan.5tate University in partial fulfillment of the requirements for the degree of MASTER OF'SCIENCE Department of Foods and Nutrition 1961 Approved. WWW” ACKNOWLEDGEMENTS The author wishes to express her sincere gratitude to Dr. Dorothy Arata, her major professor, for her guidance and encouragement during the course of this investigation and preparation of this thesis. She also wishes to thank Dr. Dena C. Cederquist, Head of the Department of Foods and Nutritkan, and Dr. Jack J. Stockton of the Department of Microbiology and Public Health for their assistance in planning her course work and for their constructive criticism of this thesis. In addition she wishes to eXpress her appreciation to Dr. Evelyn M. Jones for her kind interest and helpfulness, and to Bette Smith for her cheerfulness and assistance in checking data. ii LIST OF‘IRBLES . . . . . LIST OF FIGURES . . . . INTRODUCTION . . . . . . REVIEW OF LITERATURE . . THIAMINE . . . . Requirement . TABLE OF Fate of dietary thiamine CONTENTS PAGE 0 O O O O O 0 O O C O O O 0 v 0 O I O O O O O O O O O O 0 VI 0 O O O O O C I C O O O O O l o o o o o o o o o o o 0‘ W U) W \A’ chtion 0 O O O O 0 O O O O O O O O C O O O 0 O O 0 Effects of dietary requirement Carbohydrate Fat . . . Protein . Temperature . Metabolism . . Toxicity . . . Vitamin D Vitamin A Thiamine . NIACIN . . . . . Requirement . constituents on thiamine O O O O O O O O O O O O O O O O 0 Relation to amino acids Function . . . . . . . . Metabolism . . . . . . . Toxicity . . . . . . . . iii 0 o o o o o o o o o o o o o \OCDNJ N . . . . . . . . . . . . . . 10 . . . . . . . . . . . . . . 10 . . . . . . . . . . . . . . 12 . . . . . . . . . . . . . . 12 . . . . . . . . . . . . . . 12 . . . . . . . . . . . . . . 13 . . . . . . . . . . . . . . 15 . . . . . . . . . . . . . . 15 . . . . . . . . . . . . . . 15 . . . . . . . . . . . . . . 17 . . . . . . . . . . . . . . 18 . . . . . . . . . . . . . . 18 EXPERIIVENTAL PROCEDURE . RESULTS AND DISCUSSION . SUMNRRY AND CONCLUSIONS TABLES . . . . . . . . . FIGURES ........ LITERATURE CITED . . . . iv PAGE .23 LIST OF TABLES Weight records of animals on experimental Liver weight. ..... . . . . . . . . . . . Liver weight per 100 grams body weight . Per cent moisture in liver . . . . . . . Per cent nitrogen in liver . . . . . . . Per cent fat in liver . . . . . . . . . . PAGE 29 30 31 32 33 3h LIST OF FIGURES FIGURE PAGE 1. GrOWthcurveS00000000000000.0000035 2. Liver fat (per cent dry weight) . . . . . . . . . . . 36 Vi INTRODUCTION INTRODUCTION Early investigators discovered some human and animal diseases were caused by a dietary lack of essential factors we now call vitamins. Since that time the minimum daily recommended allowances have been established and re-evaluated (Dann and Cowgill, 193b, Birch, 1939; Brown and Sturtevant, l9h9). Hypervitaminosis is a recent development as the quantity of vitamins naturally occurring in food is small. To consume an ex- cess of vitamin, great quantities of a specific food high in that vitamin would have to be eaten. Hewever, recent isolation and purification of vitamins with the production of potent vitamin preparations, have made hypervitaminosis a possibility. Vitamins are of two types, fat-soluble and water-soluble. Repeated excess oral doses of the fat-soluble vitamins cause defi- nite deleterious effects in the body (Sebrell and Harris, l95h, Vol. II; Nieman and Klein Obbink, 19514). Excess water soluble vitamins have received little consideration as their solubility resulted in their excretion from the body in the urine. However, high concentrations of these water-soluble vitamins may affect the body in some manner as yet unknown. We are now concerned about excess oral consumption of water-soluble vitamins because of: (l) Initiation of a wideSpread enrichment program. Many cereals and cereal products i.e. flour, bread, and rice, are now enriched with water-soluble vitamins. (2) High potency vitamin preparations. Vitamin prepara- tions containing high concentrations of water-soluble vitamins, eSpecially niacin and thiamine, are directly available to the con- sumer. Usually the most potent preparation is advertised as the best preparation. These vitamin pills contain up to 2000 per cent of (or 20 times) the minimum.dai1y requirement. (3) Indiscriminant use of vitamins by doctors. Stern (1938) stated that intraspinal subarachnoid injection of synthetic vitamin B1 should be tried in the treatment of cancer, and whenever symptoms of obscure origin failed to respond to the usual methods of treatment. Since the possibility of consuming excess quantities of water-soluble vitamins now exists, this experiment was devised to determine if excess quantities of thiamine or niacin were toxic when added to low or high fat diets. The male albino weanling rat was used as the experimental animal. REVIEW OF LITERATURE REVIEW OF LITERATURE THIAMINE Requirement Early investigators using semi-purified diets adequate with respect to protein, fat, carbohydrates, minerals, and water demon- strated the existence of unknown dietary factors essential for normal growth and deve10pment. These factors were subsequentky identified and named "vitamins." Since the discovery of thiamine as a dietary essential, the daily requirement was determined experi- mentally. Animals receiving insufficient quantities of thiamine were polyneuritic and grew at a slower rate than those fed a diet with adequate thiamine. These symptoms of thiamine deficienqy became the criteria used to evaluate the thiamine adequacy of ex- perimental diets. Thiamine requirement was expressed in terms of body weight or food consumption. Dann and Cowgill (193A) found female albino rats weighing fnmm 90-2h0 grams required about 2.1 LU.1 thiamine per 100 grams of body weight. when thiamine was related to food intake, 80-100 micrograms per 100 grams of ration were needed for normal growth (Arnold and Elvehjem, 1938). Earlier, Brodie and MacLeod (1935) related thiamine storage to thiamine intake in an attempt to deve10p another criterion for analyzing the thiamine adequacy of diets. They found livers and 1One International Unit of thiamine is equivalent to 3 micrograms. hearts taken from.animals reared on a normal diet contained ten times as much thiamine per gram as skeletal muscle. Kidney contained about one-half and brain one-third the Quantity of thia- mine present in liver. Only traces of thiamine were found in blood, Spleen, and lungs.2 Therefore, blood thiamine levels were not indicative of thiamine adequacy. Muralt (1911?) stated that thiamine was essential for the normal function of nervous tissue. .As a result, this tissue was one of the last to lose its thiamine content when a state of avita- minosis prevailed. Maximum tissue storage of thiamine occurred in rats when the dietary intake of thiamine was about 30 I.U. per day. .A further increase above this level resulted in no appreciable accumulation of reserves (Leong, 1937a). Leong reported the concentration of thiamine eXpressed as I.U. per gram tissue was five times greater in liver and heart than in muscle. 0f the total quantity of thiamine stored in the body of the "saturated" rat, 50 per cent was found in the muscle while the liver contained 35 per cent. Further study revealed the biologically active form of thiamine was the phosphorylated derivative. Permeability studies using the phOSphorylated and free forms of the vitamin revealed only the free form was readily absorbed by the cell. Therefore, 2Rat bioassay was used to determine the quantity of thia- mine present in the tissue. phosphorylation must have occurred within the cell (Banga, Ochoa, and Peters, 1939). Liver and kidney tissue probably act as storage depots for thiamine because of their high ph03phorylating power. Fate of dietary thiamine Urinary excretion of thiamine in rats receiving 120 I.U. thiamine per day was about 8 per cent of that ingested (Harris and Leong, 1936). The fate of the thiamine retained was possibly (1) storage in the tissues, (2) excretion in the feces, (3) destruction in the alimentary tract, or (b) conversion to other compounds dur- ing digestion (Leong, 1937b). Storage of thiamine in the body tissues has been mentioned previously. The second possibility concerned the derivation of‘ fecal thiamine. Fecal excretion of thiamine was measured in adult normal rats on a thiamine-free diet following a single test dose of thiamine given as the International Standard.Acid Clay. Intake was varied from 0-50 I.U., however, the quantity of fecal thiamine remained relatively constant. This indicated the thiamine was readily absorbed, and fecal thiamine was not derived from.food under these conditions. When the thiamine intake was greater than 80 I.U. appreciable amounts were excreted in the feces. These data indicate fecal thiamine was not normally derived from dietary thiamine. The most probable source of fecal thiamine in the normal rat was bacterial synthesis in the lower part of the intestine (Leong,1937b). Function Early investigators attempted to discover the function of thiamine by analyzing its effect upon the body composition of animals. Two groups of rats were pair-fed a fat free ration deficient in thiamine. The average body composition of animals receiving thiamine was: water 6h.9 per cent, fat 9.h per cent, nitrogen 18.5 per cent; whereas those not receiving thiamine was: water 67.8 per cent, fat 3.3 per cent, and nitrogen 21.2 per cent (whipple and Church, 1936). MCHenry and Gavin (1938) also found rats fed a diet containing thiamine stored a greater amount of body fat than control animals pair-fed a fat free dietdeficient in thiamine. They concluded thiamine was active in the synthesis of fat from carbohydrate. However this apparent relationship between thiamine and fat synthesis was considered indirect by Stirn,. Arnold, and Elvehjem (1939). They stated the thiamine deficient rat may preferentially metabolize fat as Opposed to carbohydrate, thus reducing the fat reserves. But, when adequate quantities of thiamine were available, carbohydrate was metabolized normally both for energy and fat syn- thesis. Only recently has the function of thiamine been elucidated. .As stated previously the active form of thiamine is the phOSphoryl- ated form. Insulin was found active in the phOSphorylation of thiamine by increasing the concentration of available adenosine triphOSphate (ATP). Thiamine perphOSphate (TPP) is a constituent of cocarboxylase, a coenzyme capable of removing carboxyl groups from substrates. Many reactions are catalyzed by thiamine pyro- phosphate containing enzyme systems; most are involved in the breakdown of pyruvic acid or other dketo acids (Jansen, l9h9). Therefore, thiamine functioned directly in carbohydrate metabolism. In recent studies Williams and Anderson (1959) found thia- mine deficiency in the rat caused liver neutral lipid (except cholesterol) to fall rapidly to well below the level present in normal control animals. The sudden reintroduction of thiamine by injection caused total lipids to return to a normal level. Williams and Anderson regarded this as an indication that the actual deposi- tion of lipid in the liver was controlled by thiamine, and that thiamine also functioned to allow repletion of liver lipid at a very rapid rate. Therefore, it appeared that the function of thiamine was not simply its action in carbohydrate metabolism. Effects of dietagy constituents on thiamine requirement Carbohydrate .A relationship between dietary carbohydrate and thiamine requirement was suggested by Verhaus, Williams and Waterman in 1935. Vbrhaus stated, the larger the carbohydrate intake the greater the demand for thiamine in the human and lower animals. Consequently a depletion of thiamine would take place more rapidly on a high car- bohydrate than on a low carbohydrate diet. The type of carbohydrate in the diet also influenced the dietary requirement for thiamine, but only when coprOphagy was permitted (Guerrant and Butcher, 1935). Thus the effect of different types of dietary carbohydrate was indirect, being mediated thnaugh its acceleration or depression of bacterial thiamine synthe- sis. The metabolic thiamine requirement of the rat depended upon the quantity of carbohydrate and was independent of the type of dietary carbohydrate. Thus when c0pr0phagy was permitted, animals consuming a carbohydrate that stimulated thiamine production in the gut actually had two sources of thiamine: thiamine in the diet and thiamine in the consumed feces. Morgan and Yudkin (1959) believed the thiamine Sparing action of sorbitol was due to the immediate absorption of the thia- mine produced in greater quantities by the intestinal bacteria when sorbitol was included in the diet. They later discovered pre- vention of c0pr0phagy caused the rats to lose weight and die with thiamine deficiency symptoms. These observations supported the theory that thiamine synthesized by intestinal flora was not readily absorbed from the gut, if at all. Therefore, the quantity of carbohydrate in the diet directly influenced the dietary thiamine requirement, whereas the type of carbohydrate was only indirectly related. Fat .Adult male rats consuming a low thiamine-high fat diet lost less weight than those consuming a low thiamine-low fat diet (Kemmerer and Steenbock, 1933). This seemed to indicate that fat was thiamine sparing. However, analysis of liver and muscle from both groups of animals indicated the concentration of thiamine was the same in both groups. In addition, the liver cocarboxylase level of rats fed a low thiamine-high fat diet was similar to liver tissue from thiamine deficient polyneuritic rats (Stirrr, Arnold, and Elvehjem, 1939). An eXplanation for these data was given by Stirii, Arnold and Elvehjem (1939). The thiamine deficient rat may preferentially metabolize fat as opposed to carbohydrate. This allowed the ani- mals on a low thiamine-high fat diet to lose less weight than those on a low thiamine-low fat diet. However, since the dietary intake of thiamine was low, the animal's thiamine stores were depleted. Salmon and Goodman (1937) analyzed the vitamin B Sparing action of various natural fats and synthetic esters. The effec- tiveness of esters of single fatty acids in alleviating the thiamine deficiency symptoms in rats depended upon the length of the fatty acid carbon chain. Maximum effectiveness was found at the 8 carbon fatty acid with longer or shorter chains showing less effectiveness. The beneficial effect of fat in a low thiamine diet was an alleviation of metabolic stress on the animal by decreasing the carbohydrate intake. Protein The effect of protein on the dietary thiamine content was similar to the effect of fat. Increasing the protein content at the eXpense of carbohydrate reduced the thiamine requirement. On a high protein diet (6h per cent casein) the rat re- quired 20 micrograms of thiamine per day, whereas on a high carbohydrate diet (6h per cent sucrose), 33 micrograms per day were required for normal growth (Wainio, 19h2). 10 Likewise, the concentration of thiamine in the carcass and liver was not affected by the quantity of protein in the diet. emerging Diets containing adequate vitamins for the maintenance of rats adapted to temperate coolness contained insufficient thiamine for rats maintained at tr0pical warmth. This was true because the rats did not consume enough ration at the high temperature to meet the dietary thiamine requirement (Mills, Cottingham, and Taylor, 19h8). Using growth.after partial depletion as the criterion of adequacy, Hegsted.and McPhee (1950) found rats maintained at a low temperature (55° F) required 50 per cent more thiamine per day than rats maintained at a high temperature (780 F). Animals maintained at the lower temperature were consuming 25 per cent more calories which accounted for only part of the increased requirement for thiamine. At 78° F rats required 0.161; - 0.168 milligrams thiamine per 1000 non-fat calories compared with 0.191 - 0.203 milligrams per 1000 non-fat calories at 55° F. Metabolism The use of thiamine labeled with S35 allowed Khmelevskii (1959) to trace thiamine metabolism in liver and kidneys of rats. Tests were made at various times after thiamine S3S administration to identify thiamine decomposition.products. During the entire period of study the concentration of labeled thiamine decomposi— tion.products remained at a low level. Khmelevskii regarded this as an indication that organ tissues did not store the decomposition products. The ratio between the concentration of free thiamine 11 535 and thiamine s35 phosphoesters in liver and kidney showed no fluctuation, indicating an active balance between phoSphoryLated and free thiamine. Urine contained large quantities of 535 labeled decomposition products, while the feces contained small quantities of free thiamine, ph03phoesters, and thiamine decomposition products. .Analysis of rabbit urinary and fecal excretions for h to 6 2h-hour periods following administration of radiothiamine, gave an average total recovery of 77 per cent of the 535 after administration by stomach tube, 86 per cent after intramuscular injection, and 5k per cent after intravenous injection. The neutral sulfur fraction of urine contained more than 50 per cent of the recovered 53;. The greatest portion of this was recovered in the first 2h-hour period following oral administration.0kzrett and Cerecedo, 1958). Isola- tion of the main metabolites during the first 2h hours following oral administration indicated that unchanged thiamine s35 and the thiazole S3S moiety accounted for approximately 95 per cent of the 535 fraction of the urine. Thiamine $35 and thiazole S35 were excreted in a ratio of 2:1. Grebennik and Zakharova (1959) found after subcutaneous injection of thiamine 535, 81.73 per cent was in the urine and 7.12 per cent in the feces. Only 0.69_per cent of the 535 was excreted in the oxidized form. These experiments indicated that thiamine in the blood and tissue fluids was easily filtered out in the kidney and appeared in the urine. In.addition, a very small portion of the thiamine re- entered the digestive tract to be excreted with the feces. 12 Toxicity Many substances required by the body in small quantities become toxic when administered in large quantities. .Administration of excess quantities of the fat soluble vitamins results in toxicity. Vitamin D Excessive intake of vitamin D was characterized by the development of specific symptoms of hypervitaminosis. The histo- logical changes were similar in children and adults, although fatalities seemed o) occur more frequently in the young (Sebrell and Harris, l95h, Vol. II). There was a diffuse calcinosis in the joints, synovial membranes, kidneys, myocardium, pulmonary alveoli, parathyroid glands, large and medium sized arteries, con- junctivae, cornea, and the acid secreting portion of the stomach. The abnormal calcification could be seen grossly as a whitish, chalky material. In the early stages of hypervitaminosis the bones may show accelerated calcification of the provisional zone of cal- cification with thickening of the periosteum in more advanced cases. In later stages diffuse demineralization of the bones and inter- ference with cartilage growth can be observed. Vitamin.A Nieman and Klein Obbink (l95h) reported excess vitamin A suppressed normal keratinization. Continued excessive intakes caused Spontaneous fracture of the tibia and femur. The fractures resulted from accleration of longitudinal bone growth. Reduced formation of dentine was found as well as hemmorrhage and inflamma- tion of mucous membranes. Hypothrombenemia and degenerative effects 13 in heart, kidney, and liver were noted as well as reduced erythrodyte countsdue to a hyperplastic bone marrow. In general, lowering the vitamin intake reversed the toxic effects. Continued administration of toxic doses of vitamin.A to rats during a period of several days caused symptoms of chronic intoxication: weight loss, muscular weakness, loss of hair, sore- ness and bleeding in the skin, swelling of the palpebrae, ex0phthala- mos, stiffness of the limbs, limping, spontaneous fractures, internal hemorrhages, and eventually death (Rodahl, 1950). Thiamine To obtain.any toxic effects from thiamine in eXperimental animals maintained on adequate diets, parenteral doses of several thousand times the daily requirement must be given (Sebrell and Harris, 195R, Vo1. III). Since thiamine preparations used for in- jection contained a preservative, it was necessary to determine which component (thiamine or preservative) was responsible for the toxic effects. Haley and Flasher (19h6) found the toxic effects observed in rabbits were due to the thiamine in the preparation. _ They added that this was not an anaphylactic response caused by hypersensitivity to thiamine. Excess thiamine in.the blood following injection repressed the respiratory center in the medulla, decreasing the oxygen content of the blood causing aSphyxial convulsions and cardiac arrhythemia. Death followed due to respiratory arrest. Experimentation with dogs revealed if artificial respiration was maintained after in- jection of thiamine until the concentration of thiamine in the 11: blood fell to a tolerable level, spontaneous respiration was resumed (Smith, _e_t_a_l_., 19118). Parenteral doses of thiamine in man also resulted in toxic reactions. Laws (l9hl) and Schiff (l9hl) both reported almost fatal responses in man following repeated injection with thiamine hydro- chloride. Both patients had received repeated thiamine injections previously without any untoward effects. A hypersensitivity to thiamine may have deve10ped causing an anaphylactic shock when the thiamine injection was given. An instance of sudden death following intravenous injection of thiamine hydrochloride in man was reported due to anaphylactic shock by Rinegold and Webb (19h6). Stiles (19h1) suggested a solution of 5 milligrams thiamine hydrochloride per milliliter be used for a cutaneous sensitivity test prior to injection. However, because of the critical nature of the concentration.emplqyed in the intradermal test, a positive test was not conclusive proof of sensitivity to thiamine (Kalz, 19b2). In man, no toxic effects have been reported following oral administration of thiamine. The addition of high levels of thiamine (50 times the ade- quate level) to a diet deficient in riboflavin, pyridoxine, and pantothenate had no significant influence on weight or food effic- iency of rats when compared to a diet with adequate thiamine and deficient in riboflavin, pyridoxine, and panthothenate (Morrison and Sarett, 1959). In another study, four groups of rats were fed for six months on diets containing 0, ho, 200, or 1000 parts thiamine 15 ehydroxyethyl disulfite respectively per one million parts of basal diet. Even at the highest level of intake, weight gain, food intake, food efficiency, and organ weights were not affected. Anatomical and histological examination revealed no differences between groups (Ishikawa‘et‘al., 1959). The toxicity of thiamine thdroxyethyl disulfite was negligible in rats at a dose over 2500 tines the usual intake of human beings in relation to body weight. Niacin Requirement Niacin is also one of the water soluble B vitamins. Niacin requirements first were studied in the same manner as thiamine using growth as a measurement of adequacy. Birch (1939) found that comp plete absence of niacin from a 20 per cent casein diet for over 150 days was not accompanied by any specific physiological dysfunction in the rat such as was seen in dogs and swine. This did not, however, exclude the possibility that niacin was necessary for nor- mal growth, but indicated dietary niacin was not required. Evidence for synthesis of niacin by the rat was given by Dann and Kohn (l9hO) and by Dann (l9hl). Brown and Sturtevant (19h?) questioned whether niacin should be considered an essential dietary nutrient for the rat since it can by synthesized by the rat. glation to amino acids Since it was proven that the rat was capable of synthesizing niacin, experimentation was conducted to determine the dietary precursors of niacin. Early experiments relating protein to 16 niacin requirement led Huff and Perlzweig (l9b2) to suggest, "the tissues of the rat are capable of synthesizing nicotinic acid from the simplest of ammonium salts, amines, and amino acids, and that any contribution of the intestinal bacteria to this synthesis is of small order of magnitude." Niacin synthesis was actually per- formed by the tissues of the rat, whereas, thiamine synthesis was a function of intestinal bacteria. When large quantities of corn were included in a low niacin diet, pellagra resulted, whereas casein in the diet failed to pro- duce pellagra. This action of casein could not be explained by the presence of niacin in casein. Conversion of tryptOphan to niacin was responsible for the action of casein. Growth retarda— tion caused by inclusion of ho per cent corn grits in a 9 per cent casein diet could be counteracted by addition of either 50 milli- grams of tryptOphan or 1.0 milligramcfniacin per 100 grams of diet (Krehl gt‘gl., 19h5).3 Growing rats maintained on a ration in which tryptophan was the limiting amino acid showed a marked de- crease in niacin synthesis. When tqyptophan was added to the diet, niacin synthesis increased (Hundley, 19h7). Niacin synthesis from dietary tryptOphan followed this scheme as cited by Lushbough and Schweigert (1958} in their review article: TryptOphan -— kynurenine —- 3-hydroxy kynurem’ne -—- 3-hydroxy anthranilic acid -—- l-amino-hpformyl-l, 3-butadiene -—- 1, 2-dicarboxy1ic acid -—- quinolinic acid -- nicotinic acid. .4; 31h man.55.8 mg tryptophan is; equivalent to 1 mg niacin. (Goldsmith, Miller, and Unglaub, 1961). 17 It ix;‘well established that tryptOphan and niacin iare interconvertible. Function Niacin occurs in animals mainly as the amide which is generally found in the form of diphosphopyridine nucleotide (DPN) and triphosphOpyridine nucleotide (TPN). DPN was formerly called Coenzyme I and TPN Coenzyme II. These compounds act as hydrogen carriers, undergoing reversible oxidation reduction reactions. In the presence of a dehydrogenase DPN accepts hydrogen forming reduced DPN. Reduced DPN is oxidized by an apprOpriate flavin nucleotide in the initial reaction of the electron transport system. Rat liver pyridine nucleotide was below normal when the diet of the adult contained 1.5 milligrams per cent niacin but no tryptophan. However, if tryptOphan was fed with 20 milligrams per cent niacin, liver pyridine nucleotide increased to the same level as that found in rats fed tryptOphan and 1.5 milligrams per cent niacin. This indicated high levels of niacin had little effect upon liver pyridine nucleotide content (Williams, Feigelson, and Elvehjem, 1950). waever, in young rats, dietary niacin had no effect upon liver pyridine nucleotide either in the presence or absence of dietary tryptOphan. In young rats dietary tryptOphan appeared more important than niacin in maintaining liver pyridine nucleotide levels. Dietary tryptophan added to the non-protein ration increased liver pyridine nucleotides almost to normal in both young and adult rats. In adult rats, however, niacin had no effect on the liver 18 pyridine nucleotides, even when fed at very high levels (650 mg per cent). In young rats fed the nonpprotein rations, high dietary niacin appeared to Spare liver pyridine nucleotides. The effect was not as marked as with equivalent levels of dietary tryptOphan. (Williams, Feigelson, Shahinian, and Elvehjem, 1951). Even during severe niacin deficiency in the dog, the DPN level of the blood, kidney cortex, and brain remained constant, whereas the DPN content of liver and muscle was lower (Axelrod, Madden,and Elvehjem, 1939). The blood DPN level in man may have been increased by in- gestion of large amounts of niacin. HOwever, the results were variable depending upon the quantity of niacin ingested. There- fore, in borderline cases of deficiency disease diagnosis could not be made flomblood DPN levels. Metabolism Six urinary metabolites were demonstrated chromatographically after injection of radioactive nicotinic acid and nicotinamide: N—methylnicotinamide, nicotinuric acid, nicatinic acid, N-methyl-é- pyridone-3-carboxylamide and nicotinamide. .Another unknown compound was separated. This compound has not been identified; it was not trigonelline, nicotinic acid N-methyl betaine (Leifer gt‘gl., 1951). ToXicity Niacin toxicity was studied in rats and dogs by Chen, Rose, and Robbins (1938). Two dogs were fed daily 235 23 two grams of nicotinic acid in a capsule. One dog died after 19 days though the medication was stopped on the twelfth day. Fatty metamorphosis 19 of the liver was apparent in both animals. Symptoms of toxicity noted prior to death were bloody feces, anorexia,and convulsions. Hewever, Unna (1939) found none of these toxic effects in dogs re- ceiving excess oral doses of niacin which had been neutralized prior to administration. Unna suggested the acidity of the niacin may lave been responsible for the toxic reaction observed by Chen st 31. A group of ten six-week old rats was fed one gram of sodium nicotinate per kilogram daily over a period of to days. The weight of the experimental animals increased as regularly as the controls. When the rats were sacrificed, gross and miscrosc0pic xamination showed no pathological changes in heart, lungs, spleen, kidneys, intestinal tnact, bone marrow, and genital organs. No symptoms of toxicity were observed. Acute toxicity was of minor concern to practical vitamin therapy, however, chronic toxicity deserved more attention since vitamins were likely to be taken over a prolonged period and with- out supervision.by a physician. Daily feeding of several hundred times the maintenance doses of niacin over the entire life Span of rats, failed to produce gross toxic effects (Mblitor, l9h2). Nicotinamide was demonstrated to be several hundred times more toxic than nicotinic acid. Inclusion of l per cent nicotin- amide in a 10 per cent casein diet almost completely inhibited the growth of rats of both sexes. One per cent nicotinic acid had no effect upon growth.but did induce fatty livers. Even 2 per cent nicotinic acid had only a slight effect upon growth (Handler and Dann, l9h2). The explanation given for fatty liver induction was a deprivation of methyl groups because of trigonelline synthesis. 20 HOwever, Leifer 3t 31. (1951) found trigonelline was not a meta- bolite of niacin metabolism. Sarett (191a) stated N—methyl nicotinamide is closely related to trigonelline and may comprise a large part of what has been measured as trigonelline in earlier work, thus Handler and Dann may have had the correct idea even though they may have incorrectly identified the end product. The growth inhibition due to nicotinamide was prevented by the administration of methionine and choline plus homocystine, but not by choline, betaine, homocystine or cystine alone. Fatty liver formation induced by nicotinic acid and nicotinamide was prevented by feeding methionine, choline and betaine, but increased when cystine or homocystine were fed (Handler and Dann, l9h2). Brazida and Coulson (l9h6) found methylation decreased the toxicity of nicotinamide but had little or no apparent influence on the toxicity of nicotinic acid. Therefore, methylation was not the only factor concerned in the toxicity of these compounds. They stated the toxicity of non-methylated compounds appeared to be directly related to structure, rather than a depletion of the body stores of methyl groups in the process of detoxication. EXPERIMENTAL PROCEDURE EXPERIMENTAL PROCEDURE The percentage composition of the basal diet (1) fed the control group (I) was as follows: sucrose, 71; vitamin-free casein, 20; corn oil, 5; salts wt-u; vitamin mix, 0.25; and choline chloride, 0.15. The vitamin mixture contained in milligrams per kilogram of ration: Vitamin.A, 25.0; calciferol, 1.0; thiamine hydrochloride, h.0; riboflavin, 8.0; niacin, 5.0; pyridoxine, 2.5; calcium.panto- thante, 20.0; inositol, 10.0; folic acid, 0.2; menadione, h.0; vitamin 812, 0.02; biotin, 0.1; p-aminobenzoic acid, 2.0; -toc0pher- 01, 75.0. Diet 2 was identical with diet 1 except that diet 2 cons tained an additional 0.1 gram thiamine per 100 grams of ration. Diets 3, h, and 5 were prepared by increasing the corn oil of the basal diet from 5 per cent to hO per cent at the eXpense of sucrose. Diet 3 served as the control diet in the high fat series, with a vitamin mixture identical with that in the basal diet. Diets h and 5 contained an additional 0.1 per cent thiamine and 0.1 per cent niacin, respectively. Male albino weanling rats of the Sprague-Dawley strain were distributed by weight among five eXperimental groups. Each group was composed of 30 animals with the average weight of any one group not exceeding that of any other by more than one gram. The animals were housed individually in cages with one-half inch raised wire-mesh bottoms. Food and water were provided ad libitum during the six week experimental period. The room was air conditioned and maintained between 7b and 76° F. The animals were hWesson modification of Osborne and Mendel salt mixture. Science 752339, 1932. 21 22 weighed weekly. At weekly intervals five rats from each experimental group were sacrificed by decapitation. Livers were removed, rinsed in water, blotted free of excess moisture and weighed. The livers were then homogenized with water in a Potter-Elvehjem homogenizer, and stored in the frozen state. Prior to analysis, the frozen homogenates were allowed to thaw for one hqur at naom temperature, transferred quantitatively to an evaporating dish and evaporated to dryness (12 hours) in a drying oven at 95° C. The dried residues were weighed and ground in a Wiley mill with a ho mesh screen. One gram samples were weighed for fat extraction in the Goldfisch apparatus. About 0.3 gram of the fat extracted liver was weighed for nitrogen determina- tion by the nacro-Kjeldahl metlnd. Standard errors were calculated for each mean, Student's "t" test was used as a measure of significance. RESULTS AND DISCUSSION RESULTS.AND DISCUSSION No effect of high quantities of thiamine upon growth was observed in the low fat series (Table 1). Body weights of rats in group II (high thiamine) were not significantby different from trose of rats in group I (control). In the high fat series, no significant difference in growth was observed between group III (control) and group V (high niacin). Throughout the experiment these groups did not vary by more than 3 grams. chever, the rats in group IV (high thiamine) were consistently heavier than group III throughout the entire experiment (Figure 1). The two groups were significantly different from the second through the fourth week (P 0.01). .After the fourth week, although the animals in group IV were consistently heavier than those in group III, the difference between these two groups was not statistically signifi- cant. waever, this same trend in weight between groups III and IV was observed in a pilot study. The failure to demonstrate significant differences between these groups after the fourth week of this experiment was probably due to two factors. Each week the size of the papulation decreased,1 with a resulting decrease in the number of degrees of freedom with which the two groups could be compared. .At the same time, as the animals increased in size, individual variations between animals within a group increased. 1Five animals were sacrificed from each group at weekly intervals. 23 21: Thus, the decreased size of the sample and the increased individual variation resulted in higher standard errors and lower degrees of freedom. Both factors served to reduce the level of significance between means after the fourth week. Increasing the fat content of the diet from.5 per cent to to per cent did.decreasethe growth rate. Rats fed the high fat diets grew at a significantly slower rate than did rats fed the corresponding low fat diets (compare group 1‘33 III and group II.X§ IV in Figure 1). This reduced rate of growth in rats fed a high fat diet is in agreement with the findings of Barboriak $3.3l‘ (1958) and Harrill 33 3;. (1959). Liver weight data are recorded in Table 2. The livers fiom.rats fed the high fat diets (groups III, IV, and V) were significantly smaller than those from.rats fed the low fat diets (groups I and II). The addition of excess thiamine to either the low or the high fat diet and the addition of excess niacin to the high fat diet did not alter this observation. Since the animals on the high fat diets were smaller (Table 1), liver weight was calculated per 100 grams of body weight (Table 3) to determine whether or not the smaller livers observed in the high fat series were just a function of total body weight. Liver weight per 100 grams body weight of animals in the high fat groups (III, IV, and V) was significantky lower than the low flat groups (I and II) after the second week of the experiment. This suggested that the presence of a large quantity of fat in the diet inhibited growth of liver tissue to a greater extent than it retarded body growth. .Addition of excess thiamine to either the high fat or low fat series, or the 25 addition of excess niacin to the high fat series caused no signifi- cant change in liver weight expressed in terms of body weight. The animals on the high fat diets ate a smaller quantity of ration than those on the low fat diets. Food consumption records kept during a two week pilot study indicated that rats on the high fat diets ate an average of 100 grams during the two weeks, whereas, rats on the low fat diets consumed an average of 151 grams. Liver moisture and liver nitrogen data are presented in Tables h and 5 respectively. In the low fat series, addition of 0.1 per cent thiamine to the basal diet did not alter either the moishxeror nitrogen content of the livers. Likewise, in the high fat series, excess thiamine or niacin had no effect upon liver moisture or nitrogen. Liver fat in both control group (I and III) increased dur- ing the first two weeks post-weaning (Table 6). The rate at which liver fat was deposited in the low fat control group (I) was vir- tualby identical with the rate of liver fat deposition in the high fat control group (III) for the first three weeks (Figure 2). However, at the fourth week a significant rise (P 0.01) in liver fat was observed in group III. The reason or reasons for this observation are not clear. The addition of 0.1 per cent thiamine to the basal diet containing either low fat or high fat had no significant effect on liver fat levels. The liver fat curves from animals fed an excess quantity of thiamine (Groups II and IV) roughly followed the liver fat curves of the respective control animals (Figure 2). HOwever, when rats were fed the high fat basal diet supple- mented with 0.1 per cent niacin, the increase in liver fat above the 26 control animals was significant at the l per cent level. The deposi- tion of liver fat in the high niacin-high fat group (V) was partic- ularly rapid during the first week post-weaning. The liver flat level declined slightby during the second week and remained relatively cons tant thereafter. Since the composition of livers taken from rats in group V did not differ from.control rats with respect to (a) weight of liver, (b) liver moisture, or (0) liver nitrogen, the constituent which was replaced by fat in group V is unknown. When 0.1 per cent niacin was added to the high fat diet, the balance between fat synthesis, transport, storage,and degrada- tion was in some way disturbed. Which one of these factors, or combination of factors, was responsible for the increased deposition of liver fat in this group has not been determined. SUT’ITJ’ARY AND CONCLIE IONS SUNNY AND CONCLUSIONS Five groups of 30 male albino weanling rats per group were fed five experimental diets: Group I 20% casein, 5% fat. Group II 20% casein, % fat, 0.1% thiamine. Group III 20% casein, h0% fat. Group IV 20% casein, b0% fat, 0.1%thiamine. Group V 20% casein, h0% fat, 0.1% niacin. Five animals from.each group were sacrificed weekly during the six- week experimental period. Rats fed the high fat diets (group III, IV, and V) gained less weight than rats fed the low fat diets (groups I and II). Growth of rats on the high flat-high thiamine diet (group IV) was greater than that of rats on the high rat control diet (group III) throughout the experimental period, but especially from the second through the fourth week. Livers from rats on the high fat diets were significantly smaller than those on the low fat diets regardless of the vitamin composition of the diets. No significant differences in liver moisture and liver nitro- gen were found between any of the groups stadied. Increasing the fat content of thediet from 5 per cent to to per cent resulted in the develOpment of moderately fatty livers. Control rats fed a to per cent fat diet had a maximum liver fat level of 19 per cent at h weeks, as compared with a maximum of 11 per cent at two weeks in control rats fed a 5 per cent fat diet. 27 28 The presence of 0.1 per cent supplementary thiamine in either the high fat or low fat diet had no sustained effect on liver fat. Hewever, a marked reSponse was observed when 0.1 per cent niacin was added to the high fat diet. Rats fed this diet deposited a maximum liver fat of 2b per cent in one week. The response of the animal to excess quantities of niacin was rapid and sustained. .At the end of six weeks, liver fat levels in these animals were still about 20 per cent. 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FIGURES GRAMS WEIGHT IN 260 240 220 200 I80 I60 I40 I20 l00 80 60 40 35 FIGURE l GROWTH CURVES /, .\ V V \ 0 \\.. -v-v- snow I -V-V- GROUP 11 -o—o— GROUP 111 -o-o- snoop I! -a—u- saoup I l L 3 4 TIME IN WEE KS or- at mxum3 2. m1; m n v n N _ O J A J 4 0 J 0/ . /DflFVA» 1 b H 0000010101 0 /> 0 NH 05010 .IOIOI D 1 O 000 0:00.00 :01... 1 00 .5000 uplpn a 1 H 0.5010 IDIDI L 0 0 J [.000 o/ oIIIIIIIIo/ 1 /m o .0 0 L D D l D 1 .0 D l 0 00.1053 >mo kzwomwn: P4...— mm>_n_ 1 N mmeC O. N. Q. 0. ON «N #N ON 1” 1.9030836 LITERATURE CITED LITERATURE CITED .Arnold, A. and C. A. Elvehjem 1938 Studies on the Vitamin Bl Requirements of Growing Rats. J. Nutr. 15:h29—hh3. .Axelrod, A. E., R. J. Madden, and C. A. Elvehjem 1939 The Effect of a Nicotinic Acid Deficiency Upon the Coenzyme I Conten‘ of Animal Tissues. J. Biol. Chem. 131:85-93. Banga, I., S. Ochoa, and R. A. Peters 1939 CXXXV. Pyruvate Oxidation in Brain. VI The.Active Form of Vitamin B1 and the Role of Ch Dicarboxylic Acids. Biochem. 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