- Email: [email protected]
Absence of Fatty Livers in Rhesus Monkeys Fed Orotic Acid1
A b s e n c e o f F a t t y Livers in Rhesus M o n k e y s Fed Orotic Acid 1 M. KORYCKA-DAHL, T. RICHARDSON, and C. H. AMUNDSON Department of Food Science University of Wisconsin Madison 53706 J. R. ALLEN Department of Pathology University of Wisconsin Madison 53706 ABSTRACT
milk (12) consumed by human subjects exerts a definite hypocholesterolemia. Consequently orotic acid, skim milk, or fermented milk in the diet of rhesus monkeys may be hypocholesterolemic, but at the same time 1% orotic acid in the diet may induce a fatty liver. This paper reports a preliminary study of effects of orotic acid and milk powders in the diet on blood and liver lipids of rhesus monkeys.
Pairs of rhesus monkeys were fed for 10 wk a basal diet containing 1% orotic acid or 10% nonfat milk powders. Amounts of total lipids in the liver and hepatic morphology were normal after 10 wk indicating that orotic acid in the diet did not induce fatty livers in rhesus monkeys. INTRODUCTION
Fatty livers are induced in rats fed orotic acid in the diet (6, 14, 19, 20 22). The formation of fatty livers in rats induced by orotic acid is believed due to impaired hepatic assembly and/or secretion of very low density lipoproteins (VLDL) (19) resulting in hepatic triglyceride accumulation with concomitant depression of serum lipids (including cholesterol) (14, 22). Although orotic acid and skim milk powders which contain large amounts of orotic acid cause fatty livers in rats (16), orotic acid does not appear to do so in chickens, mice, dogs, hamsters, pigs, guinea pigs, and monkeys (2, 3, 21). It is not known what effect, if any, orotic acid has on lipids of human liver. However, Portman (17) has suggested that rhesus monkeys are similar to man in their response to changes in diet with resultant changes in blood lipids, indicating rhesus monkeys would be ideal primates for studying effects of orotic acid on blood and liver lipids. Also, orotic acid given to humans at relatively high doses has a slight but significant hypocholesterolemic effect (9). Two liters per day of skim milk (8) or fermented
Received April 11, 1979. iThis research was supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison. 1979 J Dairy Sci 62:1801--1803
MATERIALS AND METHODS
Six rhesus monkeys (four males and two females), approximately 3 yr old, were fed a semipurified basal diet for monkeys for 2 wk. The basal diet was (% by weight): ground wheat (30.0), alfalfa meal (2.0), yellow corn (36.1), soybean meal (12.0), sucrose (10.0), corn gluten meal (5.0), animal fat (1.0), brewers dried grain (1.0), calcium carbonate (1.5), calcium phosphate dibasic (1.0), trace mineral salts (.3), and fat and water soluble vitamins (.1). After 2~wk adaptation to the basal diet, monkeys were divided into three pairs and were fed the following experimental diets for 10 wk: pair 1, basal diet; pair 2, 1% orotic acid in basal diet; pair 3, 10% nonfat milk powders in basal diet. When the diet contained 10% milk powders, the yellow corn and the soybean meal components each were decreased by 5%. Pair 3 received the diet containing 10% nonfat dry milk for the first 6 wk. The next 4 wk they were fed the diet containing 10% nonfat dry yogurt to which lactose was added to compensate for that amount utilized during fermentation. Since skim milk was reported to be hypocholesterolemic in humans (8), nonfat dry milk was fed at 10% to observe any hypocholesterolemic effect that it might have in monkeys. However, no hypocholesterolemic effect over that of the basal diet was observed after
1801
1802
KORYCKA-DAHL ET AL.
the monkeys had consumed the nonfat dry milk diet for 6 wk so they were changed to a nonfat dried yogurt diet to determine if fermented milk had any hypocholesterolemic effect as reported for humans (7, 12, 13, 18). Orotic acid was purchased from Aldrich Chemical Co., Milwaukee, WI; nonfat dry milk was from Carnation Co., Los Angeles, CA; nonfat dry yogurt was prepared in the University of Wisconsin dairy plant. Lactose was assayed by the method o f Lawrence (11), and orotic acid was determined by the procedure of Okonkwo and Kinsella (15). F o o d intake was constant throughout the experiment (200 g/monkey per day). Body weight, cholesterol, and triglycerides of serum of each animal were measured weekly. There were no obvious differences in b o d y weight gains. All blood samples were from animals fasted overnight. At the termination of the experiment, livers were biopsied for chemical and morphological evaluation. Cholesterol and triglycerides in blood serum were determined by the Bio-Dynamics Unitest System Kit (Bio-Dynamics/bmc, Indianapolis, IN); total liver lipid was determined by the method of Folch et al. as given by Christie (1). RESULTS AND DISCUSSION
Because of a scarcity in the supply of rhesus monkeys for these experiments, there were insufficient numbers of animals in the experiment to allow statistical analyses of the results. However, there was adecline in serum cholesterol in all three pairs of monkeys over the 10 wk.
The average decreases as percent of initial serum cholesterol were 20, 16, and 22 for pairs 1, 2, and 3. Thus, skim milk, yogurt, and orotic acid in the diet did not possess hypocholesterolemic effect above that of the basal diet. This may reflect the hypocholesterolemic effect of the soya proteins (4) in the diet. However, there was definitely no hypercholesterolemic response to the caseins in the milk-based diets as reported by Hamilton and Carroll (5). There were no definitive trends in serum triglycerides, with those samples fromanimals on the basal diet being slightly less at the end of the experiment. In contrast to the results for rats (2, 6, 14, 19, 20, 22), orotic acid at 1% of the diet did not cause fatty livers in rhesus monkeys after 10 wk on the diet as judged b y assay of the total liver lipid (Table 1) and microscopic examination of liver biopsy slices. Hepatic morphology was normal for all animals. Total lipid content o f the liver varied from 5.1 to 7.6% of wet weight. Kerr et al. (10) have reported that total lipids comprise 8.13 + .17% of the wet weight of liver in adult rhesus monkeys; however, juvenile monkeys, as in this experiment, accumulate less lipid in the liver than adults. Preliminary results by Valti et al. (21) reported that orotic acid at 4% in the diet did not induce fatty livers in an unspecified strain of monkey after 20 days of feeding. There is a possibility that our semipurified diet contained enough adenine to reverse any orotic acid-induced accumulation of hepatic tri-
TABLE 1. Effects of dietary orotic acid on the lipid content of rhesus monkey livers. Pair no.
Diet
Orotic acid in the diet
1
Basal
--(mg/kg)-0
2
Basal + orotic acid
10,000
3
Milk powdersb
61-84
Animals
Total lipids in wet livera
Male Female
5.06 7.55
Male Female
6.12 7.56
Male Male
6.24
(%)
7.07
aAssayed after 10 wk on indicated diets. bMonkeys consumed the diet containing 10% nonfat dry milk during weeks 0 to 6 and 10% nonfat dry yogurt during weeks 6 to 10. Journal of Dairy Science Vol. 62, No. 11, 1979
TECHNICAL NOTE glycerides. A d e n i n e sulfate, as low as .25% o f t h e diet, reversed o r o t i c a c i d - i n d u c e d f a t t y liver in rats (6, 22). Milk c o n t a i n s virtually no adenine, b u t w h o l e grain w h i c h was used t o prepare t h e basal diet m i g h t c o n t a i n m o d e r a t e quantities o f purines. F u r t h e r e x t e n s i o n o f this work should exclude adenine containing c o m p o n e n t s f r o m the basal diet, or it s h o u l d be d e m o n s t r a t e d t h a t this diet induces f a t t y livers in rats.
ACKNOWLEDGMENTS The a u t h o r s gratefully a c k n o w l e d g e M. L. S u n d e for t h e d i e t a r y r e g i m e n s in t h e s e studies and H. Gierke for preparing t h e dried y o g u r t .
REFERENCES 1 Christie, W. W. 1973. Lipid analysis. Pergamon Press, Oxford. 2 Durschlag, R. P., and J. L. Robinson. 1977. Metabolic consequences of orotic acid consumption in several mammalian species. Fed. Proc. 36:1127 (Abstr.) 3 Durschlag, R. P., and J. L. Robinson. 1978. Metabolic fate of orotic acid in relation to hepatic fat accumulation. Fed. Proc. 37:328 (Abstr.) 4 Fumagalli, R., R. Paoletti, and A. N. Howard. 1978. Hypocholesterolaemic effect of soya. Life Sci. 22:947. 5 Hamilton, R.M.G., and K. K. Carroll. 1976. Plasma cholesterol levels in rabbits fed low fat, low cholesterol diet. Effects of dietary proteins, carbohydrates and fibre from different sources. Atberosclerosis 24:47. 6 Handschumacher, R. E., W. A. Creasey, J. J. Jaffe, C. A Pasternak, and L. Hankin. 1960. Biochemical and nutritional studies on the induction of fatty livers by dietary orotic acid. Proc. Nat. Acad. Sci. 46:178. 7 Hepner, G., R. Fried, S. St. Jeor, L. Fusetti, and R. Morin. 1979. Hypocholesterolemic effect of yogurt and milk. Amer. J. Clin. Nutr. 32:19.
1803
8 Howard, A. N., and J. Marks. 1977. Hypocholesterolaemic effect of milk. Lancet 2:255. 9 Kelley, W. N., M. L. Greene, 1. H. Fox, F. M. Rosenbloom, R. 1. Levy, and J. E. Seegmiller. 1970. Effects of orotic acid on purine and lipoprotein metabolism in man. Metabolism 19:1025. 10 Kerr, G. R., J. A. Campbell, A. C. Helmuth, and H. A. Waisman. 1971. Growth and development of the fetal rhesus monkey (Macaca mulatta). I1. Total nitrogen, protein, lipid, glycogen, and water composition of major organs. Pedlar. Res. 5:151. 11 Lawrence, A. J. 1968. The determination of lactose in milk products. Australian J. Dairy Technol. 21:103. 12 Mann, G. V. 1977. A factor in yogurt which lowers cholesterol in man. Atherosclerosis 26: 335. 13 Mann, G. V., and A. Spoerry. 1974. Studies of a surfactant and cholesteremia in the Maasai. Amer. J. Clin. Nutr. 27:464. 14 Novikoff, P. M., and D. Edelstein. 1977. Reversal of orotic acid-induced fatty liver in rats by doffbrate. Lab. Invest. 36:215. 15 Okonkwo, P. O., and J. E. Kinsella. 1969. Orotic acid in food milk powders. Amer. J. Clin. Nutr. 22:532. 16 Okonkwo, P. O., and J. E. Kinsella. 1974. Fatty livers induction by orotic acid contained in skim milk powder. Experientia 30:993. 17 Portman, O. W. 1970. Nutritional requirements (NRC) of nonhuman primates. Page 87 in Feeding and nutrition of nonhuman primates. R. S. Harris, ed. Academic Press, New York. 18 Richardson, T. 1978. The hypocholesteremic effect of milk - a review. J. Food Prot. 41:226. 19 Sabesin, S. M., S. Frase, and J. B. Ragland. 1977. Accumulation of nascent lipoproteins in rat hepatic Golgi during induction of fatty liver by orotic acid. Lab. invest. 37:127. 20 Standerfer, S. B., and P. Handler. 1955. Fatty liver induced by orotic acid feeding. Proc. Soc. Exp. Biol. Med. 90:270. 21 Valli, E.A., D.S.R. Sarma, and P. S. Sarma. 1968. Species specificity in orotic acid-induced fatty liver. India. J. Biochem. 5:120. 22 Windmueller, H. G. 1964. An orotic acid-induced adenine-reversed inhibition of hepatic lipoprotein secretion in the rat. J. Biol. Chem. 239:530.
Journal of Dairy Science Vol. 62, No. 11, 1979
milk (12) consumed by human subjects exerts a definite hypocholesterolemia. Consequently orotic acid, skim milk, or fermented milk in the diet of rhesus monkeys may be hypocholesterolemic, but at the same time 1% orotic acid in the diet may induce a fatty liver. This paper reports a preliminary study of effects of orotic acid and milk powders in the diet on blood and liver lipids of rhesus monkeys.
Pairs of rhesus monkeys were fed for 10 wk a basal diet containing 1% orotic acid or 10% nonfat milk powders. Amounts of total lipids in the liver and hepatic morphology were normal after 10 wk indicating that orotic acid in the diet did not induce fatty livers in rhesus monkeys. INTRODUCTION
Fatty livers are induced in rats fed orotic acid in the diet (6, 14, 19, 20 22). The formation of fatty livers in rats induced by orotic acid is believed due to impaired hepatic assembly and/or secretion of very low density lipoproteins (VLDL) (19) resulting in hepatic triglyceride accumulation with concomitant depression of serum lipids (including cholesterol) (14, 22). Although orotic acid and skim milk powders which contain large amounts of orotic acid cause fatty livers in rats (16), orotic acid does not appear to do so in chickens, mice, dogs, hamsters, pigs, guinea pigs, and monkeys (2, 3, 21). It is not known what effect, if any, orotic acid has on lipids of human liver. However, Portman (17) has suggested that rhesus monkeys are similar to man in their response to changes in diet with resultant changes in blood lipids, indicating rhesus monkeys would be ideal primates for studying effects of orotic acid on blood and liver lipids. Also, orotic acid given to humans at relatively high doses has a slight but significant hypocholesterolemic effect (9). Two liters per day of skim milk (8) or fermented
Received April 11, 1979. iThis research was supported by the College of Agricultural and Life Sciences, University of Wisconsin, Madison. 1979 J Dairy Sci 62:1801--1803
MATERIALS AND METHODS
Six rhesus monkeys (four males and two females), approximately 3 yr old, were fed a semipurified basal diet for monkeys for 2 wk. The basal diet was (% by weight): ground wheat (30.0), alfalfa meal (2.0), yellow corn (36.1), soybean meal (12.0), sucrose (10.0), corn gluten meal (5.0), animal fat (1.0), brewers dried grain (1.0), calcium carbonate (1.5), calcium phosphate dibasic (1.0), trace mineral salts (.3), and fat and water soluble vitamins (.1). After 2~wk adaptation to the basal diet, monkeys were divided into three pairs and were fed the following experimental diets for 10 wk: pair 1, basal diet; pair 2, 1% orotic acid in basal diet; pair 3, 10% nonfat milk powders in basal diet. When the diet contained 10% milk powders, the yellow corn and the soybean meal components each were decreased by 5%. Pair 3 received the diet containing 10% nonfat dry milk for the first 6 wk. The next 4 wk they were fed the diet containing 10% nonfat dry yogurt to which lactose was added to compensate for that amount utilized during fermentation. Since skim milk was reported to be hypocholesterolemic in humans (8), nonfat dry milk was fed at 10% to observe any hypocholesterolemic effect that it might have in monkeys. However, no hypocholesterolemic effect over that of the basal diet was observed after
1801
1802
KORYCKA-DAHL ET AL.
the monkeys had consumed the nonfat dry milk diet for 6 wk so they were changed to a nonfat dried yogurt diet to determine if fermented milk had any hypocholesterolemic effect as reported for humans (7, 12, 13, 18). Orotic acid was purchased from Aldrich Chemical Co., Milwaukee, WI; nonfat dry milk was from Carnation Co., Los Angeles, CA; nonfat dry yogurt was prepared in the University of Wisconsin dairy plant. Lactose was assayed by the method o f Lawrence (11), and orotic acid was determined by the procedure of Okonkwo and Kinsella (15). F o o d intake was constant throughout the experiment (200 g/monkey per day). Body weight, cholesterol, and triglycerides of serum of each animal were measured weekly. There were no obvious differences in b o d y weight gains. All blood samples were from animals fasted overnight. At the termination of the experiment, livers were biopsied for chemical and morphological evaluation. Cholesterol and triglycerides in blood serum were determined by the Bio-Dynamics Unitest System Kit (Bio-Dynamics/bmc, Indianapolis, IN); total liver lipid was determined by the method of Folch et al. as given by Christie (1). RESULTS AND DISCUSSION
Because of a scarcity in the supply of rhesus monkeys for these experiments, there were insufficient numbers of animals in the experiment to allow statistical analyses of the results. However, there was adecline in serum cholesterol in all three pairs of monkeys over the 10 wk.
The average decreases as percent of initial serum cholesterol were 20, 16, and 22 for pairs 1, 2, and 3. Thus, skim milk, yogurt, and orotic acid in the diet did not possess hypocholesterolemic effect above that of the basal diet. This may reflect the hypocholesterolemic effect of the soya proteins (4) in the diet. However, there was definitely no hypercholesterolemic response to the caseins in the milk-based diets as reported by Hamilton and Carroll (5). There were no definitive trends in serum triglycerides, with those samples fromanimals on the basal diet being slightly less at the end of the experiment. In contrast to the results for rats (2, 6, 14, 19, 20, 22), orotic acid at 1% of the diet did not cause fatty livers in rhesus monkeys after 10 wk on the diet as judged b y assay of the total liver lipid (Table 1) and microscopic examination of liver biopsy slices. Hepatic morphology was normal for all animals. Total lipid content o f the liver varied from 5.1 to 7.6% of wet weight. Kerr et al. (10) have reported that total lipids comprise 8.13 + .17% of the wet weight of liver in adult rhesus monkeys; however, juvenile monkeys, as in this experiment, accumulate less lipid in the liver than adults. Preliminary results by Valti et al. (21) reported that orotic acid at 4% in the diet did not induce fatty livers in an unspecified strain of monkey after 20 days of feeding. There is a possibility that our semipurified diet contained enough adenine to reverse any orotic acid-induced accumulation of hepatic tri-
TABLE 1. Effects of dietary orotic acid on the lipid content of rhesus monkey livers. Pair no.
Diet
Orotic acid in the diet
1
Basal
--(mg/kg)-0
2
Basal + orotic acid
10,000
3
Milk powdersb
61-84
Animals
Total lipids in wet livera
Male Female
5.06 7.55
Male Female
6.12 7.56
Male Male
6.24
(%)
7.07
aAssayed after 10 wk on indicated diets. bMonkeys consumed the diet containing 10% nonfat dry milk during weeks 0 to 6 and 10% nonfat dry yogurt during weeks 6 to 10. Journal of Dairy Science Vol. 62, No. 11, 1979
TECHNICAL NOTE glycerides. A d e n i n e sulfate, as low as .25% o f t h e diet, reversed o r o t i c a c i d - i n d u c e d f a t t y liver in rats (6, 22). Milk c o n t a i n s virtually no adenine, b u t w h o l e grain w h i c h was used t o prepare t h e basal diet m i g h t c o n t a i n m o d e r a t e quantities o f purines. F u r t h e r e x t e n s i o n o f this work should exclude adenine containing c o m p o n e n t s f r o m the basal diet, or it s h o u l d be d e m o n s t r a t e d t h a t this diet induces f a t t y livers in rats.
ACKNOWLEDGMENTS The a u t h o r s gratefully a c k n o w l e d g e M. L. S u n d e for t h e d i e t a r y r e g i m e n s in t h e s e studies and H. Gierke for preparing t h e dried y o g u r t .
REFERENCES 1 Christie, W. W. 1973. Lipid analysis. Pergamon Press, Oxford. 2 Durschlag, R. P., and J. L. Robinson. 1977. Metabolic consequences of orotic acid consumption in several mammalian species. Fed. Proc. 36:1127 (Abstr.) 3 Durschlag, R. P., and J. L. Robinson. 1978. Metabolic fate of orotic acid in relation to hepatic fat accumulation. Fed. Proc. 37:328 (Abstr.) 4 Fumagalli, R., R. Paoletti, and A. N. Howard. 1978. Hypocholesterolaemic effect of soya. Life Sci. 22:947. 5 Hamilton, R.M.G., and K. K. Carroll. 1976. Plasma cholesterol levels in rabbits fed low fat, low cholesterol diet. Effects of dietary proteins, carbohydrates and fibre from different sources. Atberosclerosis 24:47. 6 Handschumacher, R. E., W. A. Creasey, J. J. Jaffe, C. A Pasternak, and L. Hankin. 1960. Biochemical and nutritional studies on the induction of fatty livers by dietary orotic acid. Proc. Nat. Acad. Sci. 46:178. 7 Hepner, G., R. Fried, S. St. Jeor, L. Fusetti, and R. Morin. 1979. Hypocholesterolemic effect of yogurt and milk. Amer. J. Clin. Nutr. 32:19.
1803
8 Howard, A. N., and J. Marks. 1977. Hypocholesterolaemic effect of milk. Lancet 2:255. 9 Kelley, W. N., M. L. Greene, 1. H. Fox, F. M. Rosenbloom, R. 1. Levy, and J. E. Seegmiller. 1970. Effects of orotic acid on purine and lipoprotein metabolism in man. Metabolism 19:1025. 10 Kerr, G. R., J. A. Campbell, A. C. Helmuth, and H. A. Waisman. 1971. Growth and development of the fetal rhesus monkey (Macaca mulatta). I1. Total nitrogen, protein, lipid, glycogen, and water composition of major organs. Pedlar. Res. 5:151. 11 Lawrence, A. J. 1968. The determination of lactose in milk products. Australian J. Dairy Technol. 21:103. 12 Mann, G. V. 1977. A factor in yogurt which lowers cholesterol in man. Atherosclerosis 26: 335. 13 Mann, G. V., and A. Spoerry. 1974. Studies of a surfactant and cholesteremia in the Maasai. Amer. J. Clin. Nutr. 27:464. 14 Novikoff, P. M., and D. Edelstein. 1977. Reversal of orotic acid-induced fatty liver in rats by doffbrate. Lab. Invest. 36:215. 15 Okonkwo, P. O., and J. E. Kinsella. 1969. Orotic acid in food milk powders. Amer. J. Clin. Nutr. 22:532. 16 Okonkwo, P. O., and J. E. Kinsella. 1974. Fatty livers induction by orotic acid contained in skim milk powder. Experientia 30:993. 17 Portman, O. W. 1970. Nutritional requirements (NRC) of nonhuman primates. Page 87 in Feeding and nutrition of nonhuman primates. R. S. Harris, ed. Academic Press, New York. 18 Richardson, T. 1978. The hypocholesteremic effect of milk - a review. J. Food Prot. 41:226. 19 Sabesin, S. M., S. Frase, and J. B. Ragland. 1977. Accumulation of nascent lipoproteins in rat hepatic Golgi during induction of fatty liver by orotic acid. Lab. invest. 37:127. 20 Standerfer, S. B., and P. Handler. 1955. Fatty liver induced by orotic acid feeding. Proc. Soc. Exp. Biol. Med. 90:270. 21 Valli, E.A., D.S.R. Sarma, and P. S. Sarma. 1968. Species specificity in orotic acid-induced fatty liver. India. J. Biochem. 5:120. 22 Windmueller, H. G. 1964. An orotic acid-induced adenine-reversed inhibition of hepatic lipoprotein secretion in the rat. J. Biol. Chem. 239:530.
Journal of Dairy Science Vol. 62, No. 11, 1979