Orotic Acid Excretion Research Articles

Urinary purines and pyrimidines in patients with hyperammonemia of various origins

Excretion patterns of pyrimidines and purines in patients with various types of hyperammonemia have been investigated by 2-dimensional thin-layer chromatography and high pressure liquid chromatography (HPLC). For the quantitative analysis of pseudouridine, uracil and uridine a new procedure has been developed, consisting of pre-fractionation with Dowex 1 × 8, followed by dual column HPLC on a strong anion-exchanger and a reverse phase column. Thymine has also been analyzed in the pre-fractionated urine by a new HPLC method using the reverse phase column in combination with a strong cation-exchange column. Quantitative data for urinary pyrimidines and uric acid in hyperammonemia are given. In patients with a defect in one of the urea cycle enzymes, the level of pyrimidine excretion was found to depend on plasma ammonia concentrations. In other hyperammonemic patients, an increased excretion of orotic acid, uracil and uridine has only been found in one of the two patients with lysinuric protein intolerance, all other patients showing normal excretion patterns. Elevated uric acid excretions have been found frequently in our patients with hyperammonemia, but they did not always coincide with high plasma ammonia levels. A possible explanation for the difference in the excretion levels of the various pyrimidines is discussed.

Bovine Milk Orotic Acid: Variability and Significance for Human Nutrition

Bovine milk, the main source of orotic acid in the human diet, contains approximately 80ng orotic acid per milliliter, .1% of the fat-free solids. Orotic acid is virtually the only component in the acid-soluble nucleotide fraction of bovine milk. Variation is considerable in the concentration of orotic acid from dairy cows. This is primarily attributable to cow-to-cow variation. A secondary factor is stage of lactation; orotic acid is low in colostrum, increases for 10 wk, and remains constant through most of the rest of lactation; upon cessation of milking, orotic acid in lacteal secretions decrease within 6 wk to the low content of colostrum. Orotic acid at 1% in the diet leads to severely fatty livers in rats; more than .1% is necessary to induce the effect. Furthermore, rats appear to be unique in their susceptibility to fatty livers induced by orotic acid. Mice, guinea pigs, hamsters, chicks, dogs, pigs, and monkeys do not respond similarly to diets with 1% orotic acid. The lack of susceptibility in mice is in part due to increased urinary excretion of dietary orotic acid. The basis for the absence of response in other species has not been examined. While research is required, the evidence does not suggest that orotic acid in milk poses a serious problem to human consumers.

Orotic aciduria, an indicator of adequacy of treatment in lysinuric protein intolerance (LPI): 76

Orotic aciduria, an indicator of adequacy of treatment in lysinuric protein intolerance (LPI).The basic defect in LPI is in the transport of the diamino acids in kidney tubuli, jejunum and liver cells. This leads to a deficiency in the liver of the urea cycle intermediates arginine and ornithine, a malfunction of the cycle and an accumulation of ammonia. Like in the other conditions in which hyperammonemia is associated with accumulation of carbamyl phosphate, urinary excretion of orotic acid is excessive. Orotic acid is an intermediate of the synthesis of the pyrimidines from carbamyl phosphate. We measured orotic acid excretion in controls and patients with LPI. The controls in all situations excreted less than 20 μg/kg/hr. The patients excreted normal amounts during fasting (4.6, 2-8, 5 for mean, range, number of subjects). Their excretion was increased in 24-hr urines during a self-chosen low-protein diet (125, 3-366, 7), in 4 to 6 hr urines after a milk load with 0.5 g of protein/kg (488, 251-1747, 3), in 2-hr urines after oral ammonium lactate, 2.5 mmoles/kg (212, 15-1126, 10), and in 6-hr urines after iv alanine, 6.6 mmoles/kg in 90 min (790, 47-1831, 11). If the loads were given with an iv infusion of arginine, ornithine or citrulline, the orotic aciduria did not appear. Given orally, citrulline was the most efficient of the three in preventing orotic aciduria. Orotic aciduria thus is a reliable indicator of the function of the urea cycle in LPI and enables us to monitor the home treatment.

Hyperlysinemia with hyperammonemia and homocitrullinuria: 51

We studied a male patient (K.L.) aged 5 years, who had had feeding difficulties during infancy after weaning, and was later found to be slightly mentally and motorically retarded. The presenting symptom was speech difficulty with marked dysarthria and dysphasia. The liver and spleen were normal in size, leukocyte and platelet counts have always been normal, as have the liver enzymes and function tests. There was a clear aversion against berries and sweet food, but not against proteins. Plasma lysine was continuously 10-20 times higher than normal, and urinary excretion of lysine and homocitrulline were elevated. Blood ammonia was normal during fast, but moderately increased after meals. In an i.v. L-alanine load, 6.6 mmoles/kg of body weight given in 5 or 90 min, the patient developed marked hyperammonemia (peak values 465 uM and 760 uM, respectively) and vomited. Serum urea rose slowly and less than in the controls. Urinary orotic acid excretion was increased during the hyperammonemia but not on a self-chosen diet. Oral load with L-lysine-HC1, 0.5 mmoles/kg of body weight, caused a normal molar increase in plasma lysine and blood ammonia remained normal. The patient apparently has a new possibly inherited hyperammonemic disease, linked to constant hyperlysinemia and homocitrullinuria.

Effect of allopurinol on pyrimidine metabolism in human white blood cells. Role of the salvage pathway.

The administration of allopurinol leads to an increase of urinary excretion of orotic acid and orotidine (Fox et al. 197o) due to an inhibition of orotidine-5-phosphate decarboxylase (ODC).

Ornithine transcarbamylase deficiency in mutant mice I. Studies on the characterization of enzyme defect and suitability as animal model of human disease.

Summary: Eighty-four young mice born from matings of four sparse-fur (spf)/+ females and two 22A +/Y males, were classified according to spf phenotype for males and orotic acid excretion and ornithine transcarbamylase (OTC) activity for females. The OTC activity of 15.2 ± 1.32 μmole citrulline/mg protein/hr shown by hemizygous affected males was 13% of that of normal males. Heterozygous females showed a much wider variation with a mean activity of 56% of normals. Apparent Km and Vmax of the liver OTC in respect of carbamyl phosphate (CP) and ornithine (ORN) were measured for the hemizygous affected males, heterozygous females, and normal males and females. Km and Vmax (CP) for hemizygous affected males were significantly lower than normal males, normal females, and heterozygous females. Mean Km and Vmax (CP) values of heterozygous females were also significantly different from normal groups and hemizygous affected males. There were no significant differences for the values of Km (ORN) among various groups. However, the Vmax (ORN) of hemizygous males was significantly lower than the other three groups. Vmax (ORN) of the heterozygous females was also lower than the normal groups. Mean value of 18.38 ± 5.13 μmole orotic acid/mg creatinine, excreted by spf/Y males, was significantly higher than all other groups. The average excretion of 7.38 ± 5.63 μmole by heterozygous females was also significantly higher than normal males (0.72 ± 0.23 μmole) and females (0.54 ± 0.27 μmole). The high orotic acid excretion by mutant mice underlines the basic similarity of these animals to the human counterpart. There was no significant difference in the excretion of urinary urea between normal males and affected hemizygous males, or between normal females and heterozygous females. Speculation: Lower Km for carbamyl phosphate might be a characteristic of OTC from mutant mice with spf gene, which could be detected by urinary orotic acid measurement. These mice would prove to be useful animal models for studying nutritional and therapeutic measures to alleviate the consequences of OTC deficiency in children. Characterization of the defect of a mutant enzyme in an animal model should lead to greater understanding of the human disease counterpart. These studies would also be helpful in investigation of other inherited hyperammonemic syndromes.

Urinary excretion of orotic acid, orotidine and other pyrimidines in a patient with purine nucleoside phosphorylase deficiency

Urinary orotidine and orotic acid have been determined in a patient with purine nucleoside phosphorylase (PNP) deficiency under various dietary and therapeutic conditions. For this purpose a new procedure for the analysis of both compounds has been developed, consisting of prefractionation with Dowex 1X8, followed by two HPLC steps on a μ Bondapak NH 2 and a μ. Bondapak C 18 column. With this method normal as well as slightly elevated excretions of orotic acid have been found in our patient. No evidence was obtained for inhibition of OPRT by purine (deoxy)nucleosides as a cause of pyrimidine starvation. A significant increase of urinary orotidine was found after loading with allopurinol. For comparison excretory values in a patient with ornithine transcarbamylase deficiency and also in a patient with orotic aciduria type I are shown. The possible cause of the slight increase in urinary orotic acid in our patient has been discussed.

Mechanism for Fatty Liver Induction in Rats Fed Arginine Deficient Diets

Arginine deficiency is known to lead to marked alterations in pyrimidine biosynthesis and the excessive loss of urinary orotic acid. Orotic acid feeding is known to lead to hepatic steatosis. These studies show that arginine deficiency also results in a marked increase in liver lipids in the rat. The majority of the increased liver lipid can be accounted for by triglyceride accumulation. Increased liver lipid infiltration was found to be independent of the sex of the rat. Accompanying this increase was a decrease in serum triglycerides and cholesterol concentrations. Fatty infiltrations induced by arginine deficiency could be reversed by refeeding an arginine enriched diet. Adenine supplementation (0.30%) to the arginine deficient diet also completely prevented the induction of fatty livers. Adenine supplementation resulted in a dramatic increase in urinary orotic acid excretion in the arginine deficient rat. Guanine supplementation (0.5%) to an arginine deficient diet reduced but did not prevent the induction of fatty livers. The similarities of fatty livers induced by arginine deficiency and orotic acid feeding are discussed.

Cytosine and orotic acid in urine of immunodeficient children.

We describe procedures for determining cytosine and orotic acid in urine. We determine cytosine by cation-exchange analysis with either HCl or pH 5.2 buffer as eluent. Orotic acid is first separated by an anion-exchange separative procedure; after lyophilization, the product is subjected to "high-pressure" liquid chromatography for further separation and detection. We analyzed urine from normal subjects and from immunodeficient children. Three children with severe combined immunodeficiency had increased levels of cytosine in urine (23-160 mmol/mol creatinine); one child with severe combined immunodeficiency and two children with other immunodeficiencies had normal urinary levels (less than 2 mmol/mol creatinine). Orotic acid excretion in urine was normal (1-5 mmol/mol creatinine) in all of th immunodeficient children. We discuss the possible significance of the increased cytosine excretion in the three children with severe combined immunodeficiency.

Lysinuric protein intolerance ( LPI ): treatment with citrulline and lysine

LPI is an autosomal recessive defect of diamino acid transport in renal tubular and intestinal epithelium, and hepatocytes. This leads to deficiency of ornithine and other urea cycle intermediates, and inadequate function of the cycle which results in hyperammonemia after protein intake, and protein aversion. Lysine shortage may contribute to the growth failure of the patients. Our patients have earlier been treated with an arginine supplement. We have now shown that, while the intestinal absorption is very poor for arginine, it is intact for citrulline, another urea cycle intermediate. Since autumn 1976 our patients have been given 1.5 g/kg of dietary protein daily supplemented with 2-3 g citrulline and 1-2 g lysine. The protein aversion decreased in 13/21 patients, growth accelerated in 12/21, and urinary orotic acid excretion rate normalized in 9/11 as a sign of improved nitrogen tolerance. 4 patients had very fragile and sparse hair; this normalized during the therapy. Gastrointestinal complaints appeared during the treatment in 6/21; these disappeared when lysine supplementation was stopped. Hepato- and splenomegalia, increased plasma lactate dehydrogenase, leukocytopenia and trombocytopenia, and fasting plasma and urinary amino acids remained unchanged. They may depend on lysine deficiency. The severe intestinal transport defect of lysine seems to invalidate the oral route for lysine supplementation. We suggest that citrulline, possibly with lysine, should replace arginine in the treatment of LPI.

The urinary excretion of orotic acid and orotidine, measured by an isotope dilution assay

Unknown concentrations of orotic acid can be measured by competition with a known amount of [ carboxyl- 14C] orotic acid for reaction with a limiting amount of phosphoribosylpyrophosphate in the presence of orotate phosphoribosyltransferase and orotidine monophosphate decarboxylase. The dilution of the specific radioactivity in the product 14CO 2 is a sensitive and accurate measure of the amount of orotic acid present in the sample. Orotidine can also be determined after hydrolytic cleavage to orotic acid. The method was used to measure orotic acid and orotidine in urine samples from newborns, healthy controls and patients with gout or deficiency of hypoxanthine-guanine phosphoribosyltransferase receiving allopurinol. Urinary excretion of orotic acid and orotidine in newborns was similar whether the infants were breast-fed or received milk powder. The excretion of orotidine was increased in all patients receiving allopurinol. After allopurinol administration orotic acid excretion was increased in gouty patients but close to normal values in patients with deficiency of hypoxanthine-guanine phosphoribosyltransferase. The results are discussed in relation to the mechanism by which allopurinol inhibits pyrimidine metabolism.

Orotic acid excretion in some wild-type strains of Escherichia coli K-12

During rapid growth, the excretion of pyrimidines, predominantly uracil, is a common phenomenon in procaryotes and eucaryotes. In Escherichia coli, some K-12 strains excrete orotic acid and not uracil. This is caused by a mutation in the pyrF gene.

Orotic Aciduria in the Female Rat and Its Relation to Dietary Arginine

Metabolic abnormalities in arginine deficient female rats are similar to those previously observed in males. Increased excretion of orotic acid and citric acid was observed in both immature and mature arginine deficient female rats. Although urea excretion was unchanged in the immature females, it was depressed in mature females fed the arginine deficient diet. Arginine deficiency depressed urinary ammonia indepen dently of age, weight and feed consumption. Nonpregnant rats showed significantly reduced quantities of citrate in their urine if their purified 12% casein diet was supplemented with 1.0% arginine. Urinary urea, citric acid, and orotic acid increased during gestation when a 12% casein basal diet was fed. Addition of 1.0% arginine abolished the increase in urinary excretion of citrate. Arginine supplementation also reduced orotic acid excretion during late gestation (days 16 to 18) and early lactation. Mean body weight gains exclusive of fetuses and placental tissue during gestation were 37 g for rats fed 1% arginine and 2 g for those fed the unsupplemented casein basal diet. Nonpregnant rats fed the casein diet showed considerable daily variation in urinary metabolites with arginine supplementation causing only a reduction in urinary citrate. Urine ob tained without respect to age, parity or nutritional history contained higher concentrations of orotic acid from pregnant women than from nonpregnant women and the concentrations increased with duration of pregnancy. J. Nutr. 108: 1281-1288, 1978.

Purine metabolism and immunodeficiency: urinary purine excretion as a diagnostic screening test in adenosine deaminase and purine nucleoside phosphorylase deficiency.

1. We have compared urinary purine excretion by two different methods in three separate paediatric disorders of purine metabolism: purine nucleoside phosphorylase deficiency, adenosine deaminase deficiency and adenine phosphoribosyltransferase deficiency. 2. The abnormal purines identified in each case were specific for the defect and directly related to it: adenine in adenine phophoribosyltransferase deficiency; the abnormal nucleosides inosine, guanosine and their corresponding deoxyribosides in purine nucleoside phosphorylase deficiency; deoxyadenosine in adenosine deaminase deficiency, the latter having previously been identified erroneously as adenine after degradation in the acidic conditions used. 3. Deoxyriboside excretion was specific for the two defects associated with immunodeficiency; adenine for adenine phosphoribosyltransferase deficiency and 2,8-dihydroxyadenine urolithiasis. The results obtained by a quantitative method were reflected in a simple rapid qualitative technique, isotachophoresis of the urine. 4. Purine overexcretion (principally inosine) was evident only in purine nucleoside phosphorylase deficiency, which emphasizes the importance of hypoxanthine salvage for the overall control of purine production in man. In none of these disorders was any inhibition of pyrimidine biosynthesis as reflected by oroticaciduria noted. Orotic acid excretion was within normal limits in all cases. 5. From these results it is suggested that the associated immunodeficiency in adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency relates directly to the intracellular accumulation of 2′-deoxyribosides and not indirectly to an inhibitory effect on pyrimidine metabolism.

Amino Acid Induced Orotic Aciduria

Twenty amino acids were examined for their effects on urinary orotic acid excretion. Except for arginine and ornithine, all of the remaining amino acids tested induced a mild orotic aciduria in rats 2 hours post feeding. Two ammonium salts, and urease also acted, as inducers of orotic aciduria. The ammoneogenic properties of the amino acids tested could not solely explain the induced excretion of orotic acid. Only serine, glutamine, NH4Cl, (NH4)2CO3, and urease increased orotic acid excretion in the 24 hour fasted rat. Administration of 0.5 mmoles of arginine or ornithine ameliorated the mild orotic aciduria induced by either glycine or lysine. Arginine was shown to be more efficacious in preventing glycine induced orotic aciduria than was ornithine. Amino acid induced orotic aciduria is dependent upon the physiological state of the animal, varying with the state of digestion and the supply of arginine.

Dietary Protein Intake and Arginine Requirements in the Rat

Experiments were conducted with immature rats fed L-amino acid purified diets varying in total N and arginine. The experiments demonstrated that total N intake was the factor responsible for increased orotic acid excretion during arginine deficiency. Increased orotic acid excretion was accompanied by increased liver transaminase activities and increased liver concentrations of NH4-N and glutamine. Arginine requirements for growth and normal metabolite excretion increased as dietary N was increased. Accompanying elevated urinary citrate during N deprivation and arginine deficiency was a depression of liver isocitrate dehydrogenase activity. Citrate excretion was lower if arginine was fed as the HCl compared to the free base. During a partial or total arginine deficiency citrate excretion was elevated at varying dietary N concentrations. Urinary pH was not significantly changed by level of dietary N or arginine.

A VARIANT OF CARNOSINEMIA WITH NORMAL SERUM CARNOSINASE ACTIVITY IN AN INFANT

The six week old son of a first cousin marriage presented with hyperammonemia, transient acidosts, hepatosplenomegaly, failure to thrive, and seizures. Quantitative plasma and urine amino acids, including lysine clearance, were normal, orotic acid excretion was not increased, and no organic acid abnormalities were evident by GLC-mass spectroscopic analysis. Notably, during periods when the patient was placed on a diet containing 1 gm/kg/day protein intake, arginine supplementation (100 mgm/kg/day) consistently and significantly lowered plasma ammonia (on arginine, 82 μgm% ± 3, S.E.; off arginine, 113 ± 8; p < .01). The mean value for plasma ammonia during periods on a 2gm/kg/day protein diet without arginine (80 ± 17) was lower than on 1 gm/kg/day protein (p=0.1). Liver biopsy was examined by light and electron microscopy. Enzymes of the urea cycle were assayed by Dr. P. J. Snodgrass, revealing a partial deficiency of CPS (0.49 μmol/mg prot/hr; normal neonatal mean 1.13, range 0.81-1.41), assayed in the presence of excess N-acetylglutamate. Since arginine activates glutamate N-acetylase and N-acetylglutamate produced by this reaction is a cofactor for CPS, we postulate that arginine, supplied as such or through moderate amounts of dietary protein, lowered plasma ammonia in our patient by stimulating residual CPS activity.

HYPERAMMONEMIA WITH PARTIAL CARBAMYL PHOSPHATE SYNTHASE (CPS) DEFICIENCY RESPONSIVE TO ARGININE THERAPY

The six week old son of a first cousin marriage presented with hyperammonemia, transient acidosis, hepatosplenomegaly, failure to thrive, and seizures. Quantitative plasma and urine amino acids, including lysine clearance, were normal, orotic acid excretion was not increased, and no organic acid abnormalities were evident by GLC-mass spectroscopic analyaia. Notably, during periods when the patient was placed on a diet containing 1 gm/kg/day protein intake, arginine supplementation (100 mgm/kg/day) consistently and significantly lowered plasma ammonia (on arginine, 82 μgm% ± 3, S.E.; off arginine, 113 ± 8; p<.01). The mean value for plasma ammonia during periods on a 2gm/kg/day protein diet without arginine (80 ± 17) was lower than on 1 gm/kg/day protein (p=0.1). Liver biopsy was examined by light and electron microscopy. Enzymes of the urea cycle were assayed by Dr. P. J. Snodgrass, revealing a partial deficiency of CPS (0.49 μmol/mg prot/hr; normal neonatal mean 1.13, range 0.81-1.41), assayed in the presence of excess N-acetylglutamate. Since arginine activates glutamate N-acetylase and N-acetylglutamate produced by this reaction is a cofactor for CPS, we postulate that arginine, supplied as such or through moderate amounts of dietary protein, lowered plasma ammonia in our patient by stimulating residual CPS activity.

Nitrogen retention in rats fed on diets enriched with arginine and glycine. 1. Improved N retention after trauma.

1. Nitrogen retention was measured in adult rats (250-350 g) subjected to the trauma of hind-leg fracture and given diets with or without arginine plus glycine supplementation. Observations were also recorded on excretion of creatine, creatinine, allantoin, and orotic acid. Liver and skeletal muscle transaminase activities were also determined. 2. When traumatized rats weighing approximately 250 g were given a diet with 200 g casein/kg, supplemented with 20 g arginine and 10 g glycine/kg (EC diet) or a casein diet made isonitrogenous with the EC diet by addition of aspartic acid (C diet), a 60-70% increase in N retention was observed for the first 5 d post-injury for animals consuming the EC diet. A soya-bean (S) diet, isonitrogenous to the diet containing 20% casein, supplemented with arginine and glycine was as effective as the EC diet in promoting significantly better N retention of traumatized rats (350 g) in comparison to rats given the C diet. 3. When the dietary casein content was reduced to 100 g/kg, supplements of 10 g arginine and 5 g glycine or 20 g arginine and 10 g glycine/kg did not improve N retention. It is suggested that both protein quality and protein quantity are important following injury. 4. An increased excretion of creatine was observed in traumatized rats given the high-protein diets supplemented with arginine and glycine. No consistent changes were noted for urine creatinine. 5. 5. Urine allantoin levels remained stable after leg-fracture in rats consuming either the C or EC diets. Differences in the levels of urine orotic acid were found during both the pre- and post-injury periods in rats given the C, EC or S diets. 6. The mechanisms responsible for the improved N retention of traumatized rats consuming the high-protein diets with supplements of arginine and glycine may be related to the role of arginine both as a constituent of muscle tissue and as an intermediate in the urea cycle. 7. In traumatized rats fed the C or EC diets, liver transaminase activity increased whereas the transaminase activity in skeletal muscle decreased. These results support the recent concept that the increased excretion of N following injury arises from diminished reutilization of amino acids by muscle tissue without an acute increase in the rate of muscle catabolism.

Enzymes of Ammonia Detoxication after Portacaval Shunt in the Rat

At high systemic blood concentrations ammonia may be partially deviated into the pathway of pyrimidine synthesis, as has been observed in different genetic defects of the urea cycle. The portacaval shunt (PCS) rat presents an animal model to study ammonia detoxication without an underlying enzyme defect in the urea cycle. Since ammonia may induce a deviation into the pyrimidine pathway by influencing enzymatic reactions involved in this pathway, the activity of carbamylphosphate synthetase and aspartate transcarbamylase in liver as well as the excretion of orotic acid in the urine were measured in rats 10, 20 and 30 days after PCS. The results suggest that in this experimental model ammonia may be channeled into the pyrimidine pathway leading to a stimulation of the first enzymatic step and to an increased excretion of orotic acid.