Nitrogen-Stimulated Orotic Acid Synthesis and Nucleotide Imbalance1

Home , Carbamoyl phosphate, Dihydroorotase, Orotic acid, Orotic aciduria, Uric acid

[CANCER RESEARCH (SUPPL.) 52. 2082s-2084s. April I. 1992] Nitrogen-stimulated Orotic Acid Synthesis and Nucleotide Imbalance1

Willard J. Visek2

University of Illinois, College of Medicine, Urbana, Illinois 61801

Abstract bound to the inner mitochondria! membrane. The cytoplasmic enzymes reside in two separate multifunctional complexes. One Orotic acid, first discovered in ruminant milk, is an intermediate in contains carbamoyl phosphate synthetase II, aspartate trans- the pyrimidine biosynthesis pathway of animal cells. Its synthesis is carbamylase, and dihydroorotase, whereas the other includes initiated by the formation of carbamoyl phosphate (CP) in the cytoplasm, orotate phosphoribosyl transferase and orotodine-5"-phosphate with ammonia derived from glutamine. Ureotelic species also form CP in the first step of urea synthesis in liver mitochondria. For that, ammonia decarboxylase (2, 3). A deficiency of the latter two enzyme is derived from tissue fluid. When there is insufficient capacity for activities results in accumulation of orotate and a profound rise detoxifying the load of ammonia presented for urea synthesis, CP leaves in its excretion in the urine, a condition known as hereditary the mitochondria and enters the pyrimidine pathway, where orotic acid orotic aciduria (4). This bifunctional protein complex with its biosynthesis is stimulated, orotic acid excretion in urine then increases. two enzyme activities is also referred to as UMP synthase. Orotic acid synthesis is abnormally high with hereditary deficiencies of A severe deficiency of UMP synthase elevates urinary orotic urea-cycle enzymes or uridine monophosphate synthase. It is also ele acid excretion in humans to 1500 mg/day, compared with the vated by ammonia intoxication and during feeding of diets high in protein, usual 2.5 mg/day. Megaloblastic anemia and growth retarda high in lysine with respect to arginine, or deficient in arginine, ornithine, and di rulline. Rats fed 1% orotic acid or diets deficient in urea-cycle tion are accompanying conditions (4). Robinson et al. (5) dis amino acids develop fatty livers, which has not been demonstrated in covered an analogous condition in Holstein cows, which show other species. Humans consuming 6 g of orotic acid daily have not shown a partial deficiency of UMP synthase in their RBC. This hered adverse effects. Rats fed 1% orotic acid or arginine-deficient diets also itary condition exhibits an autosomal recessive mode of inher showed more and larger foci positive for -y-glutamyl transpeptidase and itance, with hÃ©tÃ©rozygotesshowinghigh concentrations of or- more liver tumors after administration of carcinogens and partial hepa- otate in their milk and urine and no other known detrimental tectomy. Orotic acid feeding was also associated with the tendency for consequences. The homozygous recessive genotypes die in utero development of larger mammary tumors induced by chemical carcinogens near the 40th day after conception (6). in rats and with development of urinary bladder calculi containing high concentrations of orotic acid in mice. Conditions that raise tissue orotic acid change purine-pyrimidine ratios. It is unknown whether tissue oro- Ammonia-induced Orotic Aciduria tate concentrations play a role in the recently observed enhanced prolif eration of cells in the colon of rats fed high-protein, high-fat diets or in Kesner (7) first reported that injections of ammonium salts the promotion of chemically induced colon cancer by intrarectal admin raised urinary orotic acid excretion by rats. Ureotelic species istration of ammonium acetate. also show elevated orotic acid excretion with ammonia intoxi cation from hereditary urea cycle deficiency (8); injections of Introduction toxic doses of urease (9) or amino acids (10); diets lacking the three urea cycle amino acids ornithine, citrulline, and arginine Orotic acid, an intermediate in pyrimidine biosynthesis, (11); or diets containing excess lysine, which is an antagonist which is its only known biological source (Fig. 1), was discov of arginine (12). Elevation of orotic acid excretion and fatty ered in bovine milk by Biscaro and Belloni in 1905 (1). Pyrim liver caused by excess lysine can be overcome by feeding adenine idine biosynthesis starts with the formation of CP,1 which is or allopurinol (13). Recent studies also show that there are 2- also an intermediate in the initial steps of urea biosynthesis. 3-fold elevations in urinary orotate in concert with high intakes The formation of CP, which is essential for pyrimidine synthe of dietary protein in otherwise complete diets consumed by sis, occurs in the cytosol of all tissues in a reaction catalyzed by other species (14, 15). Increasing the load of tissue ammonia carbamoyl phosphate synthetase II, whereas CP destined for for detoxification by the urea cycle increases the formation of urea synthesis is formed in the liver mitochondria of ureotelic CP by liver mitochondria of ureotelic species. When CP syn species in a reaction catalyzed by carbamoyl phosphate synthe thesis exceeds the capacity for its use in subsequent steps of tase I. Tissue fluid is the source of ammonia for CP synthesis urea synthesis, CP escapes into the cytosol and the synthesis of in mitochondria and glutamine is the source in cytosol. The orotate is increased in the cytosolic pyrimidine pathway (14, committed step in the synthesis of pyrimidines is the formation 16). Because it is water soluble and readily excreted by the of carbamoyl aspartate, catalyzed by aspartate carbamoylase. kidney, levels of orotate rise rapidly in the urine as its synthesis This is followed by cyclization to form dihydroorotate and increases. Its level of excretion has been suggested as an indi dehydrogenation to yield orotate. Orotate, a precursor of nu cator of ammonia intoxication and thereby an indirect measure cleic acid pyrimidines, reacts with 5-phosphoribosyl pyrophos- of the balance between tissue ammonia concentrations and the phate to form orotidine-5-phosphate, which then undergoes capacity for urea synthesis. The quantities of orotate excreted decarboxylation to yield UMP, the direct precursor of all other are not sufficient to play a significant role as a form of excreted pyrimidine nucleotides. All enzymes of this pathway are cyto- nitrogen (17). plasmic except for dihydroorotic acid dehydrogenase, which is 1Presented at "Nutrition and Cancer," the first conference of the International Effects of Orotic Acid Consumption Conference Series on Nutrition and Health Promotion, April 17-19, 1991, Atlanta, GA. Milk of ruminants contains higher concentrations of orotic ! To whom requests for reprints should be addressed, at University of Illinois, acid than does milk from other species, where it normally ranges College of Medicine, 190 Medical Sciences Building, 506 South Mathews Avenue. Urbana, IL 61801. from 0 to 5 Mg/ml and approximates the concentrations in other 3The abbreviation used is: CP, carbamoyl phosphate. tissues and fluids. Orotic acid in milk of normal cows is elevated 2082s

lation of nascent lipoproteins in the Golgi apparatus and an H,N-C-COO" -- --Ã•Y 2 | Aspanate 'I accompanying depression in blood lipids. Paired-feeding studies CH, iranscaibamoylase O = CV HC-COOH by Durschlag and Robinson (25) showed that the depression I H was related to orotate intake and not to other dietary conditions. {N-Carbamoylaspartate CHj Adding uracil plus cytosine, guanine, hypoxanthine, or xanthine XC.-COO- for 7 days did not prevent the condition (26). Valli et al. (27) fed diets with 1% orotic acid to mice, chicks, and monkeys, and [pihydfoorotate | Bloomfield et al. (28) fed 2% orotic acid to chicks for 4 weeks. DlhydroorolateIf Neither group of investigators reported accumulation of hepatic DehydrogenaseL â€ž¿+ fat in their experimental animals. Durschlag and Robinson (25) examined the consequences of feeding 1% orotic acid, for up to 30 days, in a basal diet common to mice, guinea pigs, hamsters, pigs, and rats, under conditions of paired feeding. Only pigs and rats showed increased liver weight and only rats showed hepatic steatosis. None of the other species fed the same basal diet, nor monkeys fed a different basal diet containing orotic I Unaylale (UMP) [ | Orolidylale IOMP)| acid, showed changes in liver fat. Rats and mice showed a Fig. 1. Pathway of pyrimidine nucleotide biosynthesis. depression in adenosine-uridine nucleotide ratios, presumably because of competition for 5-phosphoribosyl-pyrophosphate, in early lactation and then stabilizes around 80 pg/m\, or about an intermediate required for orotate metabolism and purine 0.1-0.2% of the nonfat total solids (18). Bovine milk and other nucleotide synthesis. The depression was greater in rats than in dairy products are the only human foods known to contain mice. Kelley et al. (21) reported similar results in human substantial quantities. In 1955 Standerfer and Handler (19) subjects, with depletion of 5-phosphoribosyl-pyrophosphate reported that feeding orotic acid caused fatty livers. This re concentrations in the RBCs of the subjects after they had sponse, which appears specific for rats, has served as a stimulus consumed 6 g of orotic acid. for research on the biological effects of orotic acid, because of the importance of cow milk in the diet of infants and other Orotic Acid in Tumor Promotion humans. On the basis of present evidence, the risks to humans from usual levels of orotate consumption appear to be minimal, Original observations of the promotion of chemically induced and the mechanism underlying the development of fatty livers liver cancers by orotic acid feeding were described by Sarma in rats is unknown. FallÃ³n et al. (20) found that i.v. injection and associates (29, 30). Male Fischer 344 and Sprague Dawley of orotic acid into humans caused prompt urinary excretion of rats have shown more and larger foci positive for 7-glutamyl orotate and urate, and they suggested that the two metabolites transpeptidase, which ultimately develop into hepatic nodules compete for renal reabsorption. Orally administered orotic acid and hepatocellular carcinoma. These have occurred after ad did not increase excretion of either compound. Kelley et al. ministration of 1,2-dimethylhydrazine or benzo(a)pyrene and (21) showed increased urinary uric acid after orotic acid inges partial hepatectomy (31). In studies at another laboratory, rats tion by humans, but they did not determine orotic acid excre fed 1% orotic acid also tended to show larger chemically in tion. They found that orotic acid taken orally decreased serum duced mammary tumors (32). The precancerous liver lesions uric acid in their patients whether serum uric acid was normal advance to histologically defined carcinomas and appear not to or elevated. They also found that 6 g of orotic acid taken daily be strain or carcinogen specific.4 Possible mechanisms under for 4-7 days led to modest decreases in plasma lipids and lying the cancer-promoting actions of orotic acid are discussed cholesterol. in the contribution by Manjeshwar and associates in this sym Robinson and Dombrowski (22) studied the pharmacology posium (33). of orotic acid in 29 healthy adult volunteers who had consumed The considerable variation in the rate of excretion of ingested 0-6 g at weekly intervals for 4 consecutive weeks. After inges orotate between species may be a determinant of the kinds of tion of 6 g, their excretion of orotate and urate increased pathological changes that ensue. For example, the relatively significantly, but creatinine and urea excretion remained unaf low excretion rate, which is <8% in 24 h for rats, may enhance fected. Excretion of orotate was proportional to the dose and tumor promotion in the liver, which is associated with greater was generally complete within 8 hours, and the excretion pat depression in purine-pyrimidine ratios in rats than in mice. tern for urate was similar. Mean excretion of orotate was 10.5% Mice excreted 27% of their labeled orotic acid in the urine after of the dose, varied from 1% to 26%, and was unrelated to p.o. administration (25). Albino mice developed a high inci gender. As reported by Kelley et al. (21), blood cholesterol dence of urinary bladder stones of high orotate content after decreased after orotic acid ingestion. Orotate excretion after consuming a semipurified diet containing 1% orotic acid (34). the ingestion of placebos averaged 2.52 Â±0.11 mg/day, which Orotic acid synthesis is enhanced by increasing protein in is between the values of 1.5 and 2.8 mg/day reported by Tax et take, and high protein intake is correlated with enhanced cancer al. (23) and Visek et al. (24), respectively. As reported by others, incidence. All amino acids are potential sources of ammonia, excretion was similar whether expressed as mg/24 h or mg/g and more ammonia is released by metabolic processes when creatinine. Durschlag and Robinson (25) found that rats ex creted an average of 7.6% of an ingested tracer dose of [I4C] protein intakes rise. Ammonia shortens cell life span and in creases the protein and RNA content of the intestinal mucosa orotate, and mice 27.4%, in 24 h. (17). High-protein, high-fat diets increase ammonia concentra TriglycÃ©ridesare the form of fat deposited in the liver of rats tions in the lumen and cell proliferation in the mucosa of the fed 0.5% or more orotic acid in their diet. This has been attributed to decreased secretion of lipoproteins, with accumu 4T. S. Elliott et al., unpublished observations. 2083s

chondrial carbamoy Iphosphate for utilization in extramitochondrial reactions colon of rats (35). It was also shown that ammonia causes in rat liver. Arch. Biochem. Biophys., Â¡62:357-368, 1974. damage of the colonie mucosa (36) and that intrarectal infusions 17. Visek, W. J. Ammonia metabolism, urea cycle capacity and their biochemical of ammonium acetate increase the incidence and dysplasia of assessment. Nutr. Rev., 37: 273-282, 1979. 18. Robinson, J. L. Bovine milk orotic acid: variability and significance for chemically induced carcinoma in rats (37). Whether ammonia human nutrition. J. Dairy Sci., 63: 865-871, 1980. causes these changes by direct toxic action on cells or whether 19. Standerfer, S. B., and Handler, P. Fatty liver induced by orotic acid feeding. the changes are secondary to changes in nucleotide synthesis or Proc. Soc. Exp. Biol. Med.. 90: 270-271, 1955. 20. FallÃ³n, H. J., Free, E., Ill, Block, J., and Seegmiller, J. E. 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2084s

Willard J. Visek

Cancer Res 1992;52:2082s-2084s.

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