I’lwl'WM It “H' l > W ‘ W“ ,_l_‘_. Im—x (/3000) EFFECT OF EXCESS DEEYARY NEACiN 3N HiGH AND LOW FAT DIE??- CN SELECTIVE ENZYME AND COENZYME SYSTEM$ Hui THE RAT Times-ts gov Hm Degree 0‘ M. S. MICHIGAN STATE UNIVERSETY Lora. Eiizabeth Long 1962 ABSTRACT EFFECT OF EXCESS DIETARY NIACIN IN HIGH AND LOW FAT DIETS ON SELECTIVE ENZYME AND COENZYME SYSTEMS IN THE RAT by Lora Elizabeth Long Recent reports suggest that excesses of the water-soluble vita- mins, including niacin, may have harmful effects under certain conditions. With the availability of high potency vitamin preparations, widespread enrichment programs, and the indiscriminant use of vitamins by doctors, there is ample justification for the study of excess oral consumption of these vitamins. Moreover, there are available today more sensitive measurements for determining unde- sirable effects, as normal metabolic pathways are defined. The object of this experiment was to study the response of the albino rat to excess dietary niacin. Forty rats were divided into four groups of ten each and fed the following diets: 1) basal, Z) basal + O. 1% of niacin, 3) 40% of fat, and 4) 40% of fat + 0. 1% of niacin. After collecting blood samples for determination of nicotinamide-adenine nucleotides (formerly termed pyridine nucleotides) the animals were sacrificed. Portions of the livers were used for determination of endogenous oxidation, liver nicotinamide-adenine nucleotides, fatty acid oxidase activity, nitrogen, moisture and fat. In both the groups fed 0. 1% of niacin there was a marked increase in blood and liver nicotinamide-adenine nucleotides as compared to the controls. This was evidence that the excess niacin was absorbed and was actually participating in metabolism. When group 3 was compared to group 1, an increase in fatty acid oxidase and nicotinamide nucleotide Lora Elizabeth Long concentrations coupled with a decrease in endogenous respiration was observed, suggesting that fat oxidation was increased in livers from group 3. These changes were not seen in group 4; livers from these animals contained significantly greater amounts of fat. It was postu— lated that the fatty livers resulted from an effective decrease in fat oxidation and that the excess niacin might have affected the synthesis of fatty acid oxidase enzymes. A second explanation for the appearance of fatty livers was that choline was utilized in the methylation of nicotinic acid prior to excretion and not in the synthesis of phospholipid to trans- port fat from the liver. The excess niacin probably resulted in two unrelated effects. The increase in nicotinamide-adenine nucleotide concentrations, which was probably a direct effect of the vitamin excess, occurred in both the high and low fat series. The appearance of fatty livers occurred only in the high fat series. EFFECT OF EXCESS DIETARY NIACIN IN HIGH AND LOW FAT DIETS ON SELECTIVE ENZYME AND COENZYME SYSTEMS IN THE RAT BY Lora Elizabeth Long A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Foods and Nutrition 1962 Approved: .1 . .— J . ’ If a”..- ,‘ , . I ACKNOWLEDGMENT The author wishes to express her sincere gratitude to Dr. Dorothy Arata without whose inspiration and guidance as well as practical assistance the research leading to this thesis would not have been possible. The author would also like to thank Mrs. Norma Booke and Bette Smith for their aid as technicians, and Michigan State University for granting the assistantship which enabled the author to pursue this work. >:< >1: >}: >:< >:< >:< >{z >1: >§< >{< :2: ii TABLE OF CONTENTS I. INTRODUCTION ........ . . . . . .......... II. REVIEW OF LITERATURE ................. A. Isolation of Niacin ................ . . B. Biological Role .......... . . . . . . . . . . C- Nutritional Importance ...... . . . ....... D. Toxicity . . . . . ................... 1. Gross effects ....... . . . ...... 2. Effects on fat metabolism. .......... III. EXPERIMENTAL ............... A. Diets and Experimental Plan . . . .......... B. Chemical Analyses . . . . . . . . ...... . . . . C. Statistical Analysis ........ . . ........ IV.RESULTS ......... ...... A. Effect of Excess Niacin in a Low Fat Diet ...... B. Effect of Excess Niacin in a High Fat Diet C. Effect of Increasing the Fat Content of a Normal Niacin Diet .................. D. Effect of Increasing the Fat Content of a High Niacin Diet ....... . . . . . . ....... V. DISCUSSION ....................... A. Effect of Excess Niacin in the Diet ......... B. Effect of Increasing the Fat Content of the Diet . . . C. Effect of Excess Niacin in the Diet When the Fat Content Has Been Increased ......... VI. SUMMARY AND CONCLUSIONS . . . . . . . . ..... LITERATURE CITED . ............ iii \1U1U1WNN 11 ll 12 13 l4 14 14 15 15 17 17 17 18 21 27 LIST OF TA BLES TABLE Page . Influence of high fat and high niacin diets on growth and foodintakeinalbinorats................. 23 . Influence of high fat and high niacin diets on liver composition in albino rats ...... . . . . . . . . . . 24 . Nicotinamide-adenine nucleotides in rats fed high fat and high niacin diets. . . . . . . ............ 25 . Endogenous oxidation and fatty acid oxidase activity in the rat as affected by high dietary fat and high niacin. . 26 iv I. INTR ODUC TION Pellagra is a deficiency disease characterized by a smooth, red tongue, diarrhea, symmetrical dermatitis and neurological lesions. Although it is not the clear-cut effect of a simple dietary deficiency, the condition can usually be attributed to a lack of niacin or the niacin precursor, tryptophan. Ever since Elvehjem and his associates showed in 1937 that niacin cured the analogous condition, canine black tongue, niacin has been widely used in the treatment of human pellagra. Early studies suggested that therapeutic doses of niacin could be administered with no harmful effects to experimental animals, including man. Now, however, the wide and indiscriminant use of vitamins has made the toxicity of excessive amounts of niacin a potential problem which must be more carefully considered. The present study is an attempt to determine the effects at the cellular level of excess oral consumption of niacin in the albino rat. 11. REVIEW OF LITERATURE A. Isolation of Niacin Niacin, the pellagra preventing vitamin, was discovered only after a long search. The compound, also called nicotinic acid, was first isolated by Casmir Funk from one of the fractions of the pigeon beri-beri factor in yeast and rice polishings (Funk, '11, '12,, '13). Others (Warburg (_e_t a_._l. , '35 and Vickery, '26) also isolated nicotinic acid in their search for the beri—beri factor but none realized its bio- logical significance. B. Biological Role Not until nicotinamide was shown to be a constituent of Coenzyme II (Warburg and Christian, '34) was its biological function clear. In 1935 Warburg and Christian found Coenzyme II preparation con- tained carbohydrate, phosphoric acid, adenine and a pyridine substance which on hydrolysis yielded nicotinamide. The activity of the prepara- tion paralleled the amount of the pyridine substance present (Warburg _e_t 31. , '35). This active pyridine structure functioned in dehydro- genations by accepting hydrogen ions to form dihydropyridine (Warburg and Christian, '36). Although Coenzyme I had been previously dis- covered by Harden and Young ('05) nicotinamide was not identified as a constituent of the molecule until after the isOlation and characterization of Coenzyme II. Both coenzymes are known to contain nicotinamide and are closely related in structure. Evidence for the interconversion of Coenzyme I and Coenzyme II was provided when an enzyme was isolated from yeast which catalyzed the synthesis of TPN by a direct phosphorylation of DPN by ATP in the presence of magnesium or manganese ion (Kornberg, '50). The mode of linkage of nicotinamide moieties in the coenzyme molecules were studied until the last detail in the proposed structure for DPN was establlshed (Schlenk, '42). It remained only to determine the disposition of the additional (or third) phosphate group on TPN. The three phosphates could either be linked in a chain or the'third phosphate could be esterified to the pentose of the adenosine portion of the molecule. By studying the products of TPN cleavage by the action of nucleotide pyrophosphatase Kornberg and Pricer ('50) were able to establish the structure as that of the esterifi ed pentos e . C. Nutritional Importance Nicotinamide was known to be part of these coenzymes before its nutritional importance was established. In an effort to identify the curative agent for canine black tongue, Elvehjem and his associates initiated a series of studies. Typical symptoms of black tongue were produced in dogs by feeding a modified Goldberg diet, and the effects of feeding certain liver preparations as well as some pure compounds, including niacin, were noted (Koehn and Elvehjem, '36, '37; Frost and Elvehjem, '37; and Elvehjem 3t a_._1. , '38). A phenomenal response was produced by the administration of a single dose of 30 mg of nicotinic acid to a dog showing classical symptoms of black tongue. The dog's appetite improved in a very short time, the mouth lesions disappeared in less than two days, and the growth response was very similar to that obtained with the active liver concentrates (Elvehjem (it 11. , '38). Attempts to isolate the vitamin from the liver concentrates revealed that free nicotinamide made up 40% of the concentrate. The activity of these liver preparations in curing black tongue correlated with the amount of nicotinamide present. Finally, the similarity in activity of the commercially prepared nicotinic acid and nicotinamide to that iso- lated from liver preparations gave rise to the conclusion that the cura- tive factor was nicotinamide itself and not a nicotinamide-containing compound (Elvehjem gt a_._l. , '38). These results were confirmed by Street and Cowgill ('37) who found that 5 mg of commercially prepared nicotinic acid per kilogram body weight per day cured a chronic black tongue condition in two dogs. They observed a prompt resumption of normal appetite and an immediate and sustained increase in body weight following the administration of the compound. The biological significance of nicotinic acid was further supported by studies showing nicotinic acid an essential growth factor for numerous bacteria, such as Staphylococcus aureus (Knight, '37) and Diptheria bacillus (Mueller, '37). Pellagra in human beings and black tongue in dogs were thought to be either analogous or closely related (Goldberg and Wheeler, '28). One of the earliest cases of the successful treatment of pellagra with nicotinic acid was reported by Smith e_t a_._l. ('37). A patient with a history of recurrent pellagra was hospitalized with the typical symptoms of pellagra--glossitis, diarrhea, and dermatitis. The patient was placed on a basic diet low in the pellagra-preventing factor and received 60 mg of nicotinic acid per day by either intravenous or intramuscular injection. The results were dramatic. Normal appetite was restored in 24 hours and the mental confusion had disappeared by 48. There was a great improvement in the appearance of the facial skin after 6 days and the patient appeared completely normal in 12 days. A more complete study on the use of nicotinic acid in the treat- ment of pellagra was made by Spies, e_t a_.l. in 1938. Thirteen non- pellagrous persons were given aqueous solutions of nicotinic acid orally each day in a preliminary study to determine a safe range of dosage. The doses varied in amounts, beginning at a few milligrams and increasing to 200 mg/day. Of these persons, nine eXperienced reactions characterized by flushing, itching and tingling which occurred within 20 minutes after the administration of the nicotinic acid. No reaction was produced when the oral dose was less than 50 mg and when single doses of 30 mg of nicotinic acid were injected intravenously in physio- logical saline solutions. Selection of the eleven pellagrins to be used in the study depended on the presence of glossitis or stomatitis or both. The criterion for effectiveness of the therapeutic agent was to be a disappearance of the typical lesions. All patients who were accepting food were placed on a control diet low in pellagra-preventative factors. They received nicotinic acid either 1) orally, 2) intravenously, or 3) by hypodermoclysis. Again the results were startling. The lesions of the mucous membranes in 11 cases were cured promptly by means of nicotinic acid. The pellagrous glossitis, stomatitis, ptyalism, vaginitis and proctitis did not reappear while the patients received the treatment, although glossitis and stomatitis did recur in one patient when nicotinic acid treatment was stopped. The authors concluded that a dose of O. 5 gm of nicotinic acid administered orally appeared safe and sufficient as a curative agent. Fifty to 80 mg/day by intravenous injection and 100 mg of nicotinic acid in one liter of physiological saline by hypo- dermoclysis were optimum when nicotinic acid was administered by these means (Spies e_t a}. , '38). D. Toxicity 1. Gross effects . Although early workers noted no gross undesirable effects of niacin in doses necessary to cure pellagra or canine black tongue, it was necessary to study the toxicity of the vitamin in larger doses. It was also necessary to establish whether or not a chronic toxicity would result after prolonged administration of niacin. The toxicity of this compound was established by experiments designed to determine the lethal doses of niacin and several other compounds. For the rat the minimal lethal dose of nicotinic acid injected as a neutralized solution was 3 to 7 mg/kg body weight (Chen e_t a_._1. , '38; Unna, '39, and Brazda and Coulson, '46). At least one of these groups (Brazda and Coulson, '46) felt that the nonspecific toxicity observed was the effect of large amounts of hypertonic solutions. However, acute toxicity was also observed when nicotinic acid was administered orally. When 2 gm of nicotinic acid/day was fed to a small dog along with his ration a definite toxicity occurred after 5 days. The dog suffered spasmodic vomiting and failure to eat but returned to normal upon discontinuation of the nicotinic acid (Elvehjem (_e_t a_._l. , '38). Other workers found similar results when two dogs received 2 gm nicotinic acid daily in capsule form. The dog which had received a total of 40 gm of nicotinic acid over a period of 20 days had convulsions, loss of appetite, and excretion of bloody feces. Post mortem examina- tion revealed ulceration of the gastrointestinal tract and fatty meta- morphosis of the liver (Chen e_t 341.. , '38). Because he felt the symptoms of toxicity observed in Chen's and Elvehjem's dogs were due to the acidity of the compound administered, Unna ('39) repeated these experi- ments using a neutralized solution of nicotinic acid. Two grams of nicotinic acid/kg/day had no adverse effects on the adult dogs in his study. There were no indications in any of these studies that sublethal doses of nicotinic acid administered over longer periods of time caused any chronic effects. Five mice injected with 500 mg nicotinic acid/kg/day for four weeks were reported to have survived with an increase in body weight (no comparison to normal weight gain was cited) (Chen _e_t 321' , '38). Dogs given oral doses of 1.0, 0. 5, O. 2, and 0.06 gm nicotinic acid/day also showed no gross effects, had gained in weight and appeared normal after 8 weeks (Chen e_t all. , '38). In an effort to study gross and micro- scopic effects of prolonged administration, ten 6-week-old rats were fed 1 gm nicotinic acid/kg/day for 40 days. No toxic symptoms were observed and the weight increase was normal as compared to control animals. There were no microscopic changes in the heart, lungs, spleen, kidney, intestinal tract, bone marrow, and genital organs (Unna, '39). However, an injection of a 0. 5% solution of nicotinic acid (5 to 10 mg) frequently raised the blood pressure in cats and rabbits by 10 to 20 mm (Unna, '39 and Hunt and Renshaw, '29).) This increase was also observed with an injection of hydrochloric acid adjusted to the same pH as the nicotinic acid solution, but not with an injection of neutralized nicotinic acid. Respiration and electrocardiographs of heart action in these animals remained unaltered after injection (Unna, '39). A 1% solution of nicotinic acid was also reported to cause no hyperemia or ulceration in guinea pigs or any effects on autonomic ganglia in rabbit intestine (Chen, '38). The studies on the toxicity of niacin were abandoned because they lacked practical application, There was evidence that niacin had a therapeutic range as wide as 1:1000, that excesses were not stored but were either destroyed or excreted (Unna, '39), and that sublethal doses did not appear to have any effects on growth, respiration, heart action, the nervous system, or blood pressure (Chen, e_t a_._l. , '38, Unna, '39, Hunt and Renshaw, '29, and Brazda and Coulson, '46). 2. Effects on fat metabolism. Later however, Handler and Dann ('42) observed the appearance of fatty livers in rats when nicotinic acid was introduced into purified diets. Animals fed a 10% casein diet unsupplemented with choline had 17. 2% of fatty acids (per cent of wet weight) in their livers. When 1% of nicotinic acid was incorporated into the same diet liver fatty acids were increased to 24. 1%. Addition of choline or betaine (0. 15%) to either diet reduced liver fatty acids to 4. 9% and 4. 1% respectively, comparable to per cent fatty acids present in livers from control. animals. The authors then repeated the experiment using a 20% casein basal diet unsupplemented with choline, to which was added 2% of nicotinic acid. The livers of the animals fed the nicotinic acid were decidedly fatty (11. 8% fatty acids) when compared to those of the con- trols (4. 2% fatty acids). Addition of choline to the nicotinic acid diet completely prevented the formation of fatty livers (livers with increased fatty acid content). The authors concluded that nicotinic acid appeared to increase the fatty acid content of livers in rats fed either the low protein diet (10% casein) or the diet adequate in protein (20% casein) as compared with the respective control groups. In both cases the addition of betaine or choline prevented the increase in liver fatty acids. Gaylor El: 11' ('60) studied the effect of moderately high levels of niacin on serum cholesterol in chicks and rats. The livers of rats fed diets containing 1% of nicotinic acid and O. 10% choline appeared to contain slightly more fat than those from the controls, although the level of significance was not reported. Because of the use of niacin in reducing serum cholesterol levels in human beings (Parsons and Flinn, '57 and Altschul and Hoffer, '58) there have been several studies in this decade on niacin and sterol metabolism. In one study (Parsons and Flinn, '59) 3 gm of nicotinic acid per day administered to patients on unrestricted diets lowered the serum cholesterol levels, particularly the beta-lipoprotein fractions. The only side reaction observed was flushing. Otherwise, 17 patients receiving from 3 to 7. 5 gm of niacin/day for one year exhibited no abnormalities in complete blood count, blood glucose, non-protein nitro- gen, serum bilirubin, cephalin flocculation, thymol turbidity, trans- aminase, alkaline phosphatase, total protein, or electrophoretic patterns of serum proteins. Needle biopsies of the liver showed no signs of fatty metamorphosis or other abnormalities (Parsons and Flinn, '59). Apparently the relationship of niacin to sterol metabolism is some- what different in the rat than in the human. While the administration of relatively high levels of niacin causes a decrease in serum cholesterol in the human, the reverse seems to be the case in the rat (Hardy St 341. , '60). Male albino rats fed a diet containing 1% of nicotinic acid for four weeks exhibited an increase in total cholesterol content of the blood. Liver pyridine nucleotides were also increased by about 100% in niacin treated rats as compared with controls. Since nicotinamide also in- creased the concentration of the coenzymes but did not increase serum cholesterol, the increased concentration of the coenzymes was believed to be unrelated to the increase in serum cholesterol. Studies with carbon labeled compounds showed that rats fed high levels of niacin incorporated more of an injected dose of acetate-l-CM into liver sterols and less into fatty acids than did the control animals (Merril, '58 and Hardy ‘1" e_tl. , '60). The addition of nicotinic acid in vitro to liver slices from unsupplemented rats produced the same effect, suggesting a control mechanism for partitioning off acetate into synthesis of sterols or fatty acids which could be altered by the amount of nicotinic acid present. These data suggest that the elevated levels of fat observed in rats fed a diet containing high niacin (Handler and Dann, '42 and Gaylor e_t a_1_1. , '60) was probably not due to an increased synthesis of fatty acids in the liver. Fatty livers induced by excess niacin must reflect a decreased rate of oxidation or a decreased transport rather than an increased synthesis . 10 Recent reports suggest that excesses of the water-soluble vita- mins, including niacin, may have harmful effects under certain con- ditions. With the availability of high potency vitamin preparations, widespread enrichment programs, and the indiscriminant use of vitamins by doctors, there is ample justification for the study of excess oral consumption of these vitamins. Moreover, there are available today more sensitive measurements for measuring any undesirable effects, as normal metabolic pathways are defined. A pilot experiment1 was conducted in this laboratory to determine if excess quantities of thiamin or niacin would be toxic when incorporated into high or low fat diets andfed to male albino rats. In a 40% fat diet 0. 1% of niacin significantly increased the fat content of the liver above the 40% rat control. The weight gain, percentage moisture and nitrogen of the livers were comparable to that of the control rats. The same diets were used in the present experiment. Metabolic effects of excess niacin were studied in male albino rats through the determination of blood and liver nicotinamide-adenine nucleotides, 7‘ endogenous oxidation and activity of the fatty acid oxidase system in liver homogenates . lBeverly Jane Klooster, "Influence of dietary fat on the response of the weanling albino rat to excessive intake of thiamin and niacin. " (Unpublished Master's thesis, Department of Nutrition, Michigan State University, 1961), p. 26. 2The coenzymes involved were formerly termed diphospho- and triphosphopyridine nucleotides (DPN and TPN, respectively). By recom- mendation of the Council of the International Union of Biochemistry a new nomenclature was adopted in 1961. The coenzymes are now referred to as nicotinamide-adenine dinucleotide (NAD) and nicotinamide-adenine dinucleotide phosphate (NADP). This new nomenclature will be used in this paper. III. EXPERIMENTAL A. Diets and Experimental Plan Forty male weanling albino rats of the Sprague-Dawley strain were divided into four groups for the experiment. Group 1 received the basal diet providing the following in grams per 100 gm diet: casein, 20; salts W,1 4'; choline, 0.15; corn oil, 7‘ 5; and vitamin mix, 0. 25. The vitamin mix contained the following in milligrams/100 gm diet: vitamin A powder (20,000 IU/gm), 2.5; calciferol, 0.1; thiamin, 0.4; riboflavin, O. 8; pyridoxine, O. 25; calcium pantothenate, 2. 0; inositol, l. 0; folic acid, 0.02; vitamin Blz,0.0023; biotin, 0.01; para-aminobenzoic acid, 0. 2; menadione, 0.4; and sucrose to make 0. 25 gm. Groups 2, 3 and 4 received diets as follows: Group 2--basal diet + 0.1% of niacin Group 3--basal diet containing 40% of fat Group 4--basal diet containing 40% of fat + 0. 1% of niacin. Sucrose was adjusted in the diets to make up the weight differences. Each group was composed of 10 animals with the average initial 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 and records were kept of the animals' food intake and weight gain. lWes son modification of Osborne and Mendel salt mixture. Science, 752339.1932. ZContaining 7. 5mg of a-tocopherol acetate. 31510.1(7otrituration of vitamin B12 with mannitol. ll 12 After 21 and 42 days blood samples were taken from the tails for determination of nicotinamide-adenine nucleotides. After 44 days the animals were stunned by a sharp blow on the head and decapitated. The livers were removed and portions were taken for the chemical analyses to be described below. B . Chemical Analys es Blood nicotinamide- adenine nucleotides . Blood nicotinamide-adenine nucleotides (NAD, NADP and NMNI) were determined by the Kring and Williams ('54) method with two modifi- cations. The sample volume was increased to 0. 20 ml and the hydrogen peroxide was added to the alcohol solution before the blood sample was introduced. Fluorescence was developed by the method used by Carpenter and Kodicek ('50) and read on a fluorometer using B-1 and PC-l filters. Hematocrits were determined for each sample and the results were reported as micrograms nicotinamide-adenine nucleotides per milliliter red blood cells. Hematocrits. Fresh blood samples were drawn into heparinized capillary tubes which were then sealed off at one end and centrifuged at 4000 rpm for 10 minutes in a Serval refrigerated centrifuge. Hematocrits were calcu- lated by the following formula: packed cell volume (mm) divided by total volume (mm). Live r nicotinamide-adenine nucleotides . Immediately after the removal of the liver a 0. 50 gm piece was excised from one lobe and immersed in a 2% nicotinamide solution for analysis of nicotinamide-adenine nucleotides by the method of lNicotinamide mononucleotide. 13 Robinson e_zt a_._l. , ('47). Following homogenization in a Potter-Elvehjem homogenizer, the extracts were made to 100 ml with the nicotinamide solution and 0. 5 ml aliquots used for the development of fluorescence by the method described for blood nicotinamide-adenine nucleotides (Carpenter and Kodicek, '50). Endogenous oxidation and fatty acid oxidase. Endogenous oxidation and activity of the fatty acid oxidase system were measured by manometric procedures (Lehninger, '55) using the Warburg apparatus. A 2. 50 gm portion of chilled liver was homogenized in a chilled Potter-Elvehjem homogenizer with 5.0 ml of 0. 25 M sucrose. A 1. 0 ml aliquot of the homogenate was pipetted into chilled Warburg flasks. All flasks were allowed to equilibrate for five minutes at 250C. in the Warburg bath, and readings were taken at 5-minute intervals. Calculations were based upon the 10- or lS-minute interval where activity was highest. Moisture, lipid, and nitrogen analyses. The remaining portions of the liver, which had been stored frozen, were allowed to thaw at room temperature and homogenized in water in a Potter-Elvehjem homogenizer. The homogenates were quantitatively transferred to weighed evaporating dishes and dried at 900C. for twelve hours. Per cent fat based on the dry weight of the livers was determined by extracting 1. 000 gm of the dried ground livers with ether on the Goldfisch apparatus for three hours. Nitrogen was determined by the Macro Kjeldahl method on 0. 250 gm samples of the fat extracted livers and expressed as per cent fresh weight of the liver. C . Statistical Analysis Standard errors of the means were calculated on all data and student's "t" test used as a measure of significance. IV. RESULTS A. Effect of Excess Niacin in a Low Fat Diet The addition of 0. 1% of niacin to the basal (5% fat) diet did not affect growth, food consumption, or efficiency of food ultilization in the experimental rats when compared to the control animals (groups 1 and 2, Table 1). Per cent fat, per cent moisture and per cent nitro— gen of the liver were likewise unaffected by this treatment (Table 2). However, a significant (p < 0. 01) increase in nicotinamide-adenine nucleotide levels in both blood and liver tissues (Table 3) was observed in rats fed the high niacin diet. This increase was apparent at three weeks and had not changed appreciably by six weeks. There were no significant differences between groups 1 and 2 in endogenous oxida- tion or in the activity of the fatty acid oxidase system (Table 4). B. Effect of Excess Niacin in a High Fat Diet When the fat content of the basal diet was increased to 40%, the addition of excess niacin caused significant changes in liver composition (p < O. 01). Livers of rats fed high niacin (group 4) contained more fat and less moisture than livers from the normal niacin group (3, Table 2). An increase in the nicotinamide-adenine nucleotide concentration of both blood and liver was observed in the high niacin group (Table 3). The significant (p < 0. 01) increase. observed in blood nicotinamide- adenine nucleotides at three weeks was sustained throughout the 6-week period. There were no changes in growth, food intake, efficiency of food utilization (Table l) or per cent nitrogen of the liver (Table 2). _ Endogenous oxidation was significantly higher (p < 0. 05) in livers from 14 15 group 4 as compared to those from group 3, but there were no differences in the activity of the fatty acid oxidase system between the two groups (Table 4). C. Effect of Increasing the Fat Content of a Normal Niacin Diet When the high and low fat control groups are compared with each other (groups 1 and 3), some interesting phenomenon may be noted. Rats fed a diet containing 40% of fat (group 3) demonstrated a significantly lower rate of endogenous oxidation (p < 0.05) and a higher activity of the fatty acid oxidase system (p < 0.02) in their livers as compared to the controls (group 1, Table 4). Moreover, the concentration of nicotinamide- adenine nucleotides in livers was higher (p < 0.01) in the animals from group 3 (Table 3). No effect on per cent fat, per cent moisture, or per cent nitrogen of the liver was noted when the fat content was increased in the normal niacin diet; yet, there was a marked effect on growth and food intake. A significant (p < 0. 01) depression in growth and food intake was observed in the animals fed the 40% fat diet as compared to the controls (Table 1). Efficiency of utilization, measured as grams of weight gain/100 calories did not differ among any of the four experimental groups (Table 1) . D. Effect of Increasing the Fat Content of a High Niacin Diet When excess (0. 1%) niacin was added to both high and low fat con... trol groups (groups 2 and 4), a different series of responses was obtained. There were no differences between these groups in the concentration of liver nicotinamide-adenine nucleotides, activity of the fatty oxidase system, or endogenous oxidation. In the presence of excess niacin the most specific effect of increasing the dietary fat from 5 to 40% was on the 16 concentration of fat in the liver. Liver fat levels increased from 11% (group 2) to 19% (group 4) as a result of the elevated dietary fat. Moisture and nitrogen levels from both groups were not significantly different. The effect on growth and food intake of increasing dietary fat in the presence of excess niacin was identical with the effect of increasing the fat content in the diets containing normal amounts of niacin. The high fat diet significantly inhibited growth and decreased food intake (p < 0. 01) but there was no difference in efficiency of food utilization. V. DISCUSSION A. Effect of Excess Niacin in the Diet Rats fed the diets containing a supplement of 0. 1% of niacin showed marked increases (almost two-fold) in the concentrations of nicotinamide-adenine nucleotides in both blood and liver tissues regardless of the fat content of the diet. Thus, this study provides strong supportive evidence that the excess niacin present in the diet was absorbed and participated in metabolic reactions, refuting the theory of "inert excretion. " These findings are in agreement with others (Williams e_t ail. , '50, '51 and Burch it :11. , '55) who reported elevated nicotinamide-adenine nucleotide concentrations when tryptophan and excessive amounts of niacin (5 to 20 mg niacin/day) were supple- mented to niacin deficient rats. The same effect was observed in studies on the effect of niacin on sterol metabolism (Gaylor.e_t a_._l. , '60 ). B. Effect of Increasing the Fat Content of the Diet Increasing the corn oil content of the diet from 5 to 40% reduced the growth rate of the rats whether or not excess niacin was present. This is in agreement with the findings of Barboriak _e_t a_l. ('58) who reported that rats receiving liquid vegetable oils in diets showed sig- nificantly smaller weight gains than did rats fed solid fats. The decrease in growth was undoubtedly a direct result of the lowered food intake observed in the groups fed high fat diets. This is supported by the lack of any significant differences in efficiency of utilization of food between any of the groups. In a study by Deuel (_e_t a1. ('46) the per cent fat in the diet was varied from 0 to 50%. The differences in growth depended on 17 l8 variations in the amounts rats consumed, although efficiency of food utilization varied only slightly, as in this experiment. In the groups fed normal amounts of niacin, increasing the level of dietary fat produced three changes: 1) the activity of the fatty acid oxidase system was increased, 2) the level of nicotinamide-adenine nucleotides was increased, and 3) the rate of endogenous oxidation was decreased. The increased activity of the fatty acid oxidase system and the increased concentrations of nicotinamide-adenine nucleotides probably reflected an adaptive response on the part of the animal. In order to metabolize the extra quantity of fat ingested, an increased synthesis of the enzymes comprising the fatty acid oxidase system apparently was accomplished. Since the nicotinamide-adenine dinucleo- tides are necessary cofactors in the oxidation of fat (Lehninger, '45), the requirement for these coenzymes would also be elevated. Endogenous respiration may have been decreased due to an increased economy of utilization of food energy. This result has been observed in rats when the per cent of fat in the diet was increased from 2 to 30% (Forbes e_t a_._l. , '46). It would appear that the rats in this study adjusted to a higher level of fat in the diet by increasing the rate of fat oxidation in the liver, thus increasing the economy of calorie utilization. C. Effect of Excess Niacin in the Diet When The Fat Content Has Been Increased The livers of rats fed diets containing 40% of fat and 0. 1% of niacin contained significantly greater amounts of fat than those from the control animals on a 40% fat diet. Since fatty livers were not pro- duced by excess niacin in the low fat diets, we may assume that the extra fat in the liver was dietary in origin. Furthermore, we can assume that excess niacin in the high fat diets must have affected the l9 pathways involved in the metabolism of dietary fat because of the abnormally high accumulation of fat in the livers of these rats. The increase in fatty acid oxidation observed when the fat content was increased in the basal diet to 40% (group 3) did not occur when excess niacin was added to this ration (group 4). This might suggest that the excess quantity of niacin in group 4 interferred with the animals' ability to increase the synthesis of enzymes involved in fatty acid oxi- dation in response to elevated concentration of substrate. If excess niacin does interfere with the synthesis of the fatty acid oxidase system in the presence of high fat, then fat oxidation would be reduced and animals fed excess niacin in a high fat diet could develop fatty livers. A second explanation for the appearance of fatty livers in these animals is that addition of niacin induced a state of choline deficiency. This suggests the action of excess niacin in causing fatty livers is indirect. The theory of an induced choline deficiency is supported by the evidence of Handler and Dann ('42) discussed previously. Nicotinic acid or its amide is detoxified in the liver by methylation to form N'-methylnicotinamide. The methylation involves the donation of a methyl group by methione (Cantoni, '51) which can in turn be derived from choline by oxidation to betaine and the subsequent transfer of a methyl group to homocysteine (Muntz, '50). However, in a study with a soluble enzyme system from rat liver, nicotinamide was methylated by methione but choline could not serve as the methyl donor either in the presence or absence of homocysteine (Cantoni, '51). The possibility that some enzymatic capacity was lost in the preparation of this system must be considered, since the synthesis of methionine should have proceeded in the presence of choline and homocysteine. The methyl group could have been supplied by the newly synthesized methionine. If one proceeds with the assumption that choline is used to methylate niacin, or nicotinamide which is the active compound, then increasing the amount of niacin in the diet would increase the requirement 20 for choline. This would decrease the amount of choline available for fat metabolism via phospholipid formation and could cause an increase of fat in the liver by decreasing fat transport (Perlmanand Chaikoff '39). Fatty livers observed under conditions reported here could be explained on this basis. However, the animals in this study did not demonstrate the classical biochemical symptoms of a choline deficiency. Artom ('53) has shown that the oxidation of long chain fatty acids is depressed in liver preparations from choline deficient animals. This observation was confirmed by Rees and Kline ('57) who demonstrated a decreased utilization of octanoate by livers taken from choline deficient animals. Further, Rees and Kline ('57) observed a decreased rate of endogenous oxidation in these livers. In the study reported here, no depression of the liver fatty acid oxidase system nor of endogenous oxidation was seen in rats fed high fat, high niacin diets, when compared with high fat control rats. Therefore, while it is possible that the fatty livers seen in group 4 were caused by a choline deficiency, the excess niacin in the biological system may have complicated the classical bio- chemical symptoms of such a deficiency. It is obvious from the results of this study that excess niacin enters metabolic pathways to produce at least two unrelated effects; increased concentration of nicotinamide-adenine nucleotides in blood and liver tissues and increased level of fat in livers. That coenzyme concentrations are increased by feeding excess niacin in both the low and the high fat series but fat content of the liver is increased by excess niacin only in the high fat series is evidence that the effects are unrelated. The elevation of the coenzyme concentrations is probably a direct effect of the vitamin excess; the production of fatty livers may be either a direct or an indirect effect of the excess niacin. Further work is in progress in this laboratory to determine the effect of increasing the level of choline in a diet containing both high fat and high niacin. VI. SUMMARY AND CONCLUSIONS The object of this study was to determine the reSponse of the albino rat to excess dietary niacin. Forty rats were divided into four groups of ten each and fed diets as follows: group 1, basal; group 2, basal +0.1% niacin; group 3, 40% of fat; and group 4, 40% of fat + 0.1% of niacin. Blood nicotinamide—adenine nucleotide concentrations were determined at 21 and 42 days. The animals were sacrificed after 44 days a'nd samples of the liver were taken for manometric determination of fatty acid oxidase activity and endogenous respiration and chemical analyses of liver nicotinamide-adenine nucleotides, nitrogen, fat, and moisture. In both groups fed 0. 1% of niacin (groups 2 and 4) there was a marked increase in blood and liver nicotinamide-adenine nucleotides when compared to the controls regardless of dietary fat levels. This was evidence that the excess niacin was absorbed and was actively participating in metabolism. When group 3 was compared to group 1 an increase in fatty acid oxidase activity, an increase in liver nicotinamide— adenine nucleotides, and a decrease in endogenous respiration were observed which led the author to conclude that fat oxidation was increased in the livers of animals fed a diet containing 40% of fat. These changes were not seen in group 4 (high fat and high niacin); livers from these animals contained significantly greater amounts of fat than did those from rats fed the high fat control diet (group 3). It was postulated that the fatty livers resulted from an effective decrease in fat oxidation and that the excess niacin might have affected the synthesis of fatty acid oxidase enzymes. A second explanation for the appearance of fatty livers in this group was that choline was being utilized in the 21 22 detoxication of nicotinic acid and not in the synthesis of phospholipids to transport fat from the liver. It seemed probable that the excess niacin had two separate and distinct effects. The elevation of nicotin- amide-adenine nucleotides levels which occurred when excess niacin was introduced into either high or low fat diets was probably a direct effect of the excess vitamin. The production of fatty livers, however, occurred only when excess niacin was incorporated in a high fat diet and could be either a direct effect of the vitamin excess or a secondary effect of a choline deficiency. 23 Table 1. Influence of high fat and high niacin diets on growth and food intake in albino rats. Weight Gain Food Intake Efficiencyl Group Diet gm/week cal/week gm/lOO cal 1’- basal 35 :1: 13 326 :t 83 10.9 i 23 2 0.1% niacin 36il 355:1:8 10.042 3 40% corn oil 23 :1: 1 278 :1: 10 8.2 :l: 2 4 40% corn oil 24 *1 288 i 10 8.5 :1: 2 + 0.1% niacin 1Weight gain per 100 calories consumed. zEach‘ group consisted of ten animals. 3Standard error of the mean. 24 Table 2. Influence of high fat and high niacin diets on liver composition in albino rats. Per cent Per cent Per cent Groupl Diet Fatz Moisture Nitrogen3 1 basal 10.9:1:0.53 71.0:0.43 3.07:1:0.08‘ 2 0.1%niacin ll.3:1:0.3 70110.3 3.123:O.06 3 40% corn oil 13.131.1 71.23.02 3.21:0.06 4 40% corn oil 19031.1 69310.3 3.24:1:0.04 + 0.1% niacin 1Each group consisted of ten animals. ZCalculated on the basis of dry weight of liver. 3‘Calculated on the basis of fresh weight of liver. 4Standard error of the mean. 25 Table 3. Nicotinamide-adenine nucleotides in rats fed high fat and high niacin diets. Blood Liver H /m1 RBC Hg /gm Group Diet 3 weeks 6 weeks1 liver 1 basal 230 4 252 280 4 252 1260 4. 692 2 0.1% niacin 408 4 45 434 4 41 2557 4 220 3 40% corn oil 178 :1: 16 235 d: 42 1866 :1: 60 4 40% corn oil 412 4 70 3914 30 2379 4152 + 0.1% niacin 1Length of time on experimental diet. zStandard error of the mean. 26 Table 4. Endogenous oxidation and fatty acid oxidase activity in the rat as affected by high dietary fat and high niacin. Endogenous Fatty Acid Oxidation Oxidase Group Diet (.11 02/ hr/ gm liver 1 basal 1775 4 731 3314 341 2 0.1% niacin 1790 4 56 405 4 37 3 40% corn oil 1555 4 45 500 4 28 4 40% corn oil 1750 4 61 465 4 41 + 0.1% niacin 1Standard error of the mean. LITERATURE CITED Altschul, R. and A. Hoffer 1958 Effect of nicotinic acid upon serum cholesterol and upon basal metabolism rate of young normal adults. Arch. Biochem. 73: 420. Artom, C. 1953 Role of choline in the oxidation of fatty acids by the liver. J. Biol. Chem. 205: 100. Barboriak, J. J., W. A. Krehl, G. R. Cowgill, and A. A. Whedon 1958 Influence of high fat diets on growth and development of obesity in the albino rat. J. Nutr. 64: 241. Brazda, F. G. and R. A. Coulson 1946 Toxicity of nicotinic acid and some of its derivatives. Proc. Soc. Exp. Biol. 81 Med. 62: 19. Burch, H. B., C. A. Storvick, R. L. Bicknell, H. C. Kung, L. G. Alejo, W. A. Everhart, O. H. Lowry, C. G. King, and O. A. Bessey 1955 Metabolic studies of precursors of pyridine nucleotides. J. Biol. Chem. 212: 897. Cantoni, G. L. 1951 Methylation of nicotinamide with a soluble enzyme system from rat liver. Ibid. 189: 203. Carpenter, K. J. and E. Kodicek 1950 The fluorimetric estimation of N'-methylnicotinamide and its differentiation from coenzyme I. Biochem. J. 46: 421. Chen, K. K., C. L. Rose, and E. B. Robbins 1938 Toxicity of nicotinic acid. Proc. Soc. Exp. Biol. 81 Med. 38: 241. Deuel, H. J., E. R. Meserve, E.Str.aub, C. Hendrick, and B. T. Scheer 1946 The effect of fat level of the diet on general nutrition: I. Growth, reproduction, and physical capacity of rats receiving diets containing various levels of cottonseed oil or margarin fat ad libitum. . J. Nutr. 33: 569. Elvehjem, C. A., R. J. Madden, F. M. Strong, and D. W. Wooley 1938 The isolation and identification of the anti-black tongue factor. J. Biol. Chem. 12: 137. 27 28 Forbes, E. B., R. W. Swift, R. F. Elliot, and W. H. Janes 1946 Relation of fat to economy of food utilization. J. Nutr. 31: 113. Frost, D. V. and C. A. Elvehjem 1937 Further studies on factor W. J. Biol. Chem. 121: 255. Funk, C. 1911 On the chemical nature of the substance which cures polyneuritis in birds induced by a diet of polished rice. J. Physiol. 43: 395. 1912 Further experimental studies on beri-beri. The action of certain purine and pyrimidine derivatives. Ibid. 45: 489. 1913 Studies on beri-beri. VII. Chemistry of the vitamin- fraction from yeast and rice polishings. Ibid. 46: 173. Gaylor, J. L., R. W. F. Hardy, and C. A. Baumann 1960 Effect of nicotinic acid and related compounds on sterol metabolism in the chick and rat. J. Nutr. 70: 293. Goldberg, J. and G. A. Wheeler 1928 Experimental black tongue in dogs and its relation to pellagra. Pub. Health Rep. 43: 172. Handler, P. and W. J. Dann 1942 The inhibition of rat growth by nicotinamide. J. Biol. Chem. 146: 357. Harden, A. and W. J. Young 1905 The alcoholic ferment of yeast- juice. J. Physiol. 32: i. Hardy, R. W. F., J. L. Gaylor, and C. A. Baumann 1960 Biosynthesis of sterols and fatty acids as affected by nicotinic acid and related compounds. J. Nutr. 71: 159. Hunt, R. and R. R. Renshaw 1929 Action of certain heterocyclic compounds on the autonomic nervous system. J. Pharmacol. Exp. Therap. 35: 75. Knight, B. C. J. G. 1937 XCVII. The nutrition of Staphylococcus aureus; nicotinic acid and vitamin B1. Biochem. J. 31: 731. Koehn, C. J. and C. A. Elvehjem 1936 Studies on vitamin G (B2) and its relation to canine black tongue. J. Nutr. 11: 67. 1937 Further studies on the concentration of the antipellagra factor. J. Biol. Chem. 118: 693. - 29 Kornberg, A. 1950 Enzymatic synthesis of triphosphopyridine nucleotide. Ibid. , 182: 805. Kornberg, A. and W. E. Pricer, Jr. 1950 On the structure of triphos- phopyridine nucleotide. Ibid. 186: 557. Kring, J. P. and J. N. Williams, Jr. 1954 Differential determination of pyridine nucleotides and N'-methylnicotinamide in blood. Ibid. 207: 851. Lehninger, A. L. 1945 On the activation of fatty acid oxidation. Ibid. 161: 437. 1955 Fatty acid oxidation in mitochondria: Methods of Enzymology Vol. I-Ed. by S. P. Colowick and M. 0. Kaplan, p. 545. Merril, J. M. 1958 Effect of nicotinic acid on the incorporation of radiocarbon into cholesterol circulation. Res. 6: 482. Mueller, J. H. 1937 Nicotinic acid as a growth accessory for the Diphtheria bacillus. J. Biol. Chem. 120: 219. Muntz, J. A. 1950 The inability of choline to transfer a methyl group directly to homocystein for methione formation. Ibid. 182: 489. Parsons, W. B., Jr. and J. H. Flinn 1957 Reduction in elevated blood cholesterol levels by large doses of nicotinic acid: Preliminary report. J. A. M. A. 165:234. Parsons, W. B. and J- H- Flinn 1959 Reduction of serum cholesterol levels and beta-lipoprotein cholesterol level by nicotinic acid. A.M.A. Arch. Int. Med. 103: 123. Perlman, I. and I. L. Chaikoff 1939 Radioactive phosphorus as an indicator of phospholipid metabolism: V. On the mechanism of the action of choline upon the liver of the fat-fed rat. J. Biol. Chem. 127: 211. Rees, D. E. and D. L. Kline 1957 Metabolism of octanoate by liver slices. Am. J. Physiol. 190: 446. Robinson, J., N. Levitas, F. Rosen, and W. A. Perlzweig 1947 The fluorescent condensation product of N'-methylnicotinamide and acetone: IV. .A rapid method for determination of pyridine nucleotides in animal tissues. The coenzyme content of rat tissues. J. Biol- Chem. 170: 653. 30 Schlenk, F. 1942 Identification of the carbohydrate group in the nicotinamide nucleotides. J. Biol. Chem. 146: 619. Smith, D. T., J. M. Ruffin and S. G. Smith 1937 Pellagra success- fully treated with nicotinic acid: a case report. J.A. M.A. 109: 2054. Spies, T. D., C. Cooper and M. A. Blanhehop 1938 The use of nicotinic acid in the treatment of pellagra. Ibid. 110: 622. Street, H. R. and G. R. Cowgill 1937 The cure of canine black tongue with nicotinic acid. Proc. Soc. Exp. Biol. Med. 37: 547. Unna, K. 1939 Studies on the toxicity and pharmacology of nicotinic acid. J. Pharmacol. Exp. Therap. 65: 95. Vickery, H. B. 1926 Simple nitrogeneous constituents of yeast. J. Biol. Chem. 68: 585. Warburg, O. and W. Christian 1933 The yellow enzyme and its functions. Biochem. Z. 266: 377. Chem. Abs. 28: 794 (1934). 1936 Pyridine as the active group of dehydrogenating enzymes. Biochem. Z. 286: 147. Chem. Abs. 30: 8262b (1936). and A. Griese 1935 The active group of the coenzyme from red blood cells. Biochem. Z. 279: 143 (1935). Chem. Abs. 29:8022. Williams, J. N., Jr., P. Feigelson and C. A. Elvehjem 1950 Relation of tryptophan and niacin to pyridine nucleotides of tissue. J. Biol. Chem. 187: 597. and S. S. Shaninian, and C. A. Elvehjem 1951 Further studies on tryptophan-niacin-pyridine nucleotide relationships. Ibid. 189:659. 3‘ y "I ‘ ’1 AAAAAAAAA ”'71 111111121 111 11111111111111.1111) 93