Introduction

Diagnostic value of urinary orotic acid Introduction Orotic acid (1,2,3,6-tetrahydro-2,6-dioxo-4-pyrimidinecarboxylic acid; uracil-6-carboxylic acid) is a minor component of the diet. It is found in whey and root vegetables, such as carrots and beets. In contrast to sheep’s and goat’s milk, cow’s milk contains a relatively large amount of orotic acid, while human milk lacks this compound. The nutritional importance of orotic acid lies in its role as a growth factor in feed deficiencies and as a protective agent for liver. Orotic acid improves the symptoms due to folate or cobalamin deficiency and increases intracellular levels of nucleotides and nucleic acids. Large doses of magnesium orotate (3 g/day) markedly improve left ventricular function and exercise tolerance in patients with coronary heart disease probably by correcting a relative deficiency of nucleotide precursors or by increasing myocardial energy supply. Orotic acid has been used therapeutically also in the treatment of neonatal jaundice, hyperlipoproteinemia, degenerative retinal disease and gout. Orotic acid is still referred to as vitamin B13, but it is not really recognised as a vitamin because it is manufactured by the human body and intestinal flora. Prolonged exposure to elevated orotic acid concentrations may cause noxious effects. Addition of orotic acid to rat diet causes hepatic steatosis. Orotic acid is a promoter of hepatic carcinogenesis in rat treated with 1,2 dimethylhydrazine, enhances preneoplastic and neoplastic lesions in hamsters and stimulates proliferation of K 562 leukemic cells in vitro. The estimated LD50 of orotic acid in mice is 2000 mg/kg, when administered per os, and 841 mg/kg, when administered via intraperitoneal injection.  [1]

Metabolic disorders

Because renal excretion of orotic acid is very efficient and urinary values integrate changes over time, orotic acid measurements are more relevant in urine than in plasma. The distribution of the urinary orotic acid values for healthy subjects shows an asymmetrical pattern with a mean value of 1.13 μmol/mmol creatinine and a mode of 0.62 μmol/mmol creatinine. A study on single urine samples from 168 healthy adults in Nagoya General Hospital gave a reference range of 0.26–3.20 μmol/mmol creatinine by using HPLC method. Values less than 10 μmol/mmol creatinine have been reported in other studies in healthy adults. Females have higher levels of orotic acid (0.36–3.20 μmol/mmol creatinine) than males do (0.26–1.91 μmol/mmol creatinine). Levels are higher in infants of 1–12 months (0.76–4.10 μmol/mmol creatinine) than in newborns, older children or adults. Orotic acid excretion is about half normal during starvation because of a lowered rate of production and utilisation. The starvation adaptation of orotic acid excretion occurs more rapidly than does the decrease in urinary nitrogen loss. The response to refeeding of acutely malnourished normal male is an increase in orotic acid excretion with a decrease in whole body protein catabolism. Slight increases in urinary orotic acid have been reported in pregnancy and premature birth. Slight to moderate increases have been observed in patients with heart failure, hypertension, diabetes, malignancy, benign tumors, cerebral infection, and trauma. Adult alcoholics show elevated urinary orotic acid-to-creatinine ratios early after drinking episodes, suggesting that liver damage may cause increased orotic acid excretion. An unusually high urinary excretion of orotic acid was found in a patient with hepatocellular carcinoma without cirrhosis, as a result of partial tumor occlusion of the hepatic veins, tumor breakdown and portosystemic shunting.[2]

Orotic acid overproduction

Abnormally high urinary levels of orotic acid have been found in conditions that evoke hyperammonemia and accumulation of intramitochondrial carbamoyl phosphate, which may diffuse into the cytosol and stimulate de novo pyrimidine synthesis. With the exception of arginine and ornithine, amino acids as well as ammonia salts induce orotic aciduria in rat. Arginine-deficient diet increases liver carbamoyl phosphate and urinary orotic acid level in many species, while arginine infusion reduces orotic acid excretion in sheep and rat when a high nitrogen diet is given. However, it has been reported that an arginine-deficient diet does not cause hyperammonemia or orotic aciduria in adult humans since the de novo arginine synthesis is sufficient for the maintenance of normal cellular metabolism. In most cases, orotic aciduria in humans arises from inherited defects of enzymes involved in the urea cycle after the synthesis of intramitochondrial carbamoyl phosphate. An estimate of the overall incidence of these inborn errors gives a value of 1 per 9400. Ornithine transcarbamylase deficiency constitutes 70% of the cases, argininosuccinate synthetase deficiency 16%, arginonosuccinate lyase deficiency 12%, and arginase deficiency 2%. Clinical presentation of the patients is very similar and related to hyperammonemia, which is common to all these diseases. The variability of the symptoms presumably depends on genomic factors and on the metabolic consequences of the various enzyme deficiencies. For instance, variability in expression of ornithine transcarbamylase deficiency in heterozygous females is related to the proportion of hepatocytes in which the normal or mutant allele is on the active X chromosome. Arginonosuccinate lyase deficiency of a degree similar to ornithine transcarbamylase deficiency may not be as severe a disease because newly synthesised argininosuccinate may serve as a waste nitrogen product. Acute change in consciousness and hyperammonemia in ornithine transcarbamylase deficiency with late onset can simulate Reye’s syndrome. However, in Reye’s syndrome without inherited transcarbamylase deficiency normal excretion of pyrimidines has been reported.[3]

Biological relevance of the analytical results

Urinary orotic acid determination is a useful tool for screening hereditary orotic aciduria and for differentiating the hyperammonemia disorders which cannot be readily diagnosed by amino acid chromatography, thus reducing the need for enzyme determination in tissue biopsies. Hyperammonemia is caused by congenital disorders of the urea cycle and it is frequently found in organic aciduria and several aminoacidopathies. The amino acid analysis in plasma and urine allows the diagnosis of citrullinemia, argininosuccinic aciduria, argininemia, gyrate atrophy, HHH syndrome, lysinuric protein intolerance, and non-ketonic hyperglycinemia. However, the amino acid pattern is non specific in N-acetylglutamate synthetase deficiency, carbamoyl phosphate synthetase deficiency, ornithine transcarbamylase deficiency, and organic acidurias. Among this latter group of diseases, the elevation of orotic acid may be useful for identifying ornithine transcarbamylase deficiency. If orotic acid is not elevated, organic acidurias should be searched for by determining short fatty acids and organic acids in plasma and urine. Diagnosis of N-acetylglutamate synthetase deficiency and carbamoyl phosphate synthetase deficiency can be made by assaying the enzymes in liver biopsy and in rectal or duodenal tissue, respectively. It has been reported that orotic acid excretion associated with ornithine transcarbamylase deficiency increases in the morning, whereas those in healthy subjects remain at baseline. 

It has been suggested that the increase in orotic acid excretion in the patients is due to the protein intake at breakfast after starvation throughout the whole night. Because orotic acid may go undetected in patients with ornithine transcarbamylase deficiency in the afternoon of a non-hyperammonemic period, urinary orotic acid concentration does not appear to be an ideal diagnostic index for the inherited disease. Urinary uracil has been recently proposed, instead of orotic acid, for a more useful indicator of ornithine transcarbamylase deficiency. Orotic acid determination in the allopurinol loading test (300-mg oral dose) may be a useful diagnostic tool to establish the carrier status of women at risk for ornithine carbamoyltransferase deficiency. Although the predictive value of the test is good, both orotic acid and orotidine should be measured to reduce the risk of misclassification. Moreover, orotic aciduria is not as sensitive or specific an indicator of heterozygosis as the presence of orotidinuria. Finally, an increased excretion of orotic acid in sick children is relatively frequent and a positive allopurinol load test may not indicate a specific urea cycle defect.

References

[1] G.K. Hinkel, H.W. Kintzel, R. Schwarze. "Prevention of hyperbilirubinemia in premature and newborn infants using orotic acid". Dtsch. Gesundheitsw., 27 (1972), p. 2414.

[2] G. Muller. "Metabolic effects of orotic acid". Z. Gesamte Inn. Med., 39 (1984), p. 269.

[3] P. Collipp. "Orotic acid, inosine and nucleosides in the treatment of degenerative retinal diseases: a double blind study". Curr. Ther. Res., 42 (1987), p. 235.