Determination of urinary orotic acid and uracil by capillary zone electrophoresis

We describe a simple method for measuring orotic acid and uracil concentration in urine by capillary zone electrophoresis in 20 mM Na-borate buffer, pH 9.2. The method was applied for studying a patient with HHH (hyperornithinemia, hyperammonemia and homocitrullinuria) syndrome. A high value of uracil excretion was found during periods of relatively low orotic acid excretion and normal ammonemia. The orotic acid level in urine was increased by increasing protein intake.     

Über die Gewöhnung von Ziegen an eiskaltes Tränkwasser

90 urine samples obtained in three lamb trials and one experiment using adult wethers were analyzed for their contents of orotic acid and creatinine. The average daily excretion of orotic acid accounted for 0.5 mg to 1.5 mg (35 μg to 130 μg/W^0.75) with a high individual variation. Correlation coefficients between orotic acid and other urinary constituents were low indicating an entirely different response to metabolic variations. There was only a weak relationship to live weight, protein retention and rumen fluid traits. Defaunation reduced the orotic acid excretion (significant in the adult wethers) whereas the addition of rumen‐protected lysine as well as the use of different dietary carbohydrate sources were without effect. The urinary excretion of creatinine increased with live weight and age from 0.4 g/d in the 20 kg lambs to 1.7 g/d in the adult 53 kg wethers. The correlations with live weight were close whereas the apparently negative correlation with protein retention was not real as could be evaluated by calculation of the partial correlations. There was a close correlation of creatinine with total N, urea and allantoin. Neither defaunation nor rumen‐protected lysine and the kind of carbohydrate source had significant effects on creatinine. The use of orotic acid and creatinine as indicators of metabolic disorders were discussed. Easy application in practical diagnosis without quantitative urine collection might be possible by the determination of orotic acid in the milk of cows and of the creatinine/N ratio in urine.     

Radioassay of orotic acid phosphoribosyltransferase and orotidylate decarboxylase utilizing a high-voltage paper electrophoresis technique or an improved 14CO2- release method.

Orotic acid phosphoribosyltransferase (EC 2.4.2.10) and orotidylate decarboxylase (EC 4.1.1.23) can be assayed independently of one another by the high voltage paper electrophoresis method described here, which separates orotic acid, OMP, and UMP, the substrates and/or products of these enzymes, from each other. The relative migration of other compounds, mainly other nucleotides, their bases, or other intermediates of the UMP biosynthetic pathway, has also been recorded. The method has allowed us to observe that OMP is not released to any significant degree from the enzyme complex of these two enzymes that occurs in Ehrlich ascites cells; rather orotic acid is converted stoichiometrically by the enzyme complex to UMP. For purification of the enzyme complex, we have found the release of ¹⁴CO₂ from [¹⁴C]carboxyl-labeled orotic acid (when phosphoribosyl pyrophosphate and Mg²⁺ are present) preferable to the HVPE method as a routine assay procedure. The most economical CO₂-absorbant for the assay of the enzyme complex or for orotidylate decarboxylase (and possibly for other enzymes which release CO₂) is an NaOH-soaked paper strip. As detailed here, its use allows one to repeatedly reuse the scintillation vials and fluid.     

Diagnostic value of urinary orotic acid levels: applicable separation methods

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. This review provides an overview of metabolic aberrations that may be related to increased orotic acid levels in urine, and summarises published methods for separation, identification and quantitative determination of orotic acid in urine samples. Applications of high-performance liquid chromatography, gas chromatography, and capillary electrophoresis to the analysis of urinary specimens are described. The advantages and limitations of these separation and identification methodologies as well as other less frequently employed techniques are assessed and discussed.     

Automated determination of orotic acid, uracil and pseudouridine in urine by high-performance liquid chromatography with column switching

A column-switching high-performance liquid chromatographic method, requiring no sample preparation apart from filtration, is described for quantification of urinary orotic acid, uracil and pseudouridine. The analyses were carried out using a reversed-phase octadecylsilane-bonded column for sample clean-up and a cation-exchange column for separation; 5–20 ]sml samples of urine were directly analysed, and more than 100 samples could be analysed consecutively. Each sample required only 30 min. Detection limits of these compounds were 5 pmol. Creatinine-related urinary uracil excretion was lowest in the newborn period (17.3 ± 14.4 μmol/g of creatinine). A patient with partial ornithine transcarbamylase deficiency and his mother usually excreted a high level of uracil during the period of normal orotic acid excretion and normal serum ammonia level.     

Analytical derivatization-a tool for determination of orotic acid

Derivatization of orotic acid (OA) into various forms (trimethylsilylderivate, alkyl ester and per-methylated derivate) and their evaluation by GC/MS is described. The tested approach includes ion-exchange SPE clean-up, evaporation and chemical reaction with different types of derivatization agents (N,O-bis-(trimethylsilyl)trifluoroacetamide with trimethylchlorosilane, butanol with acetylchloride and ethereal solution of diazomethane). Derivate originated in the reaction with diazomethane was used for determination of urinary orotic acid by GC/MS. Detection limit of 0.28 micromol l(-1) was reached using the ion 82 m/z in single ion monitoring (SIM) mode. Linearity of the method was tested within the range of 3.4-2503.4 micromol l(-1) covering physiological and pathological levels of orotic acid in urine sample. Recoveries were within the range 93.7-110.6%. Application of the method on the patient with defect of ornithine transcarbamylase (OTC) was demonstrated as well.     

Orotic acid biosynthesis in rat liver: studies on the source of carbamoylphosphate.

Measurements of the incorporation of radiolabeled precursors into orotic acid in tissue slices and minces provided evidence of the participation of the intramitochondrial carbamoylphosphate synthetase (CPSase-I) in the de novo biosynthesis of pyrimidines in rat liver. Ammonia, the only nitrogen source utilized by CPSase-I, markedly stimulated the incorporation of NaH¹⁴CO₃ into orotic acid in liver slices, and ornithine, which enhances the intramitochondrial consumption of carbamoylphosphate (CP) in citrulline synthesis, antagonized the stimulation by ammonia. Sensitivity of the incorporation of NaH¹⁴CO₃ into orotic acid to stimulation by ammonia was found to increase with age in concert with the emergence of CPSase-I in the liver during late fetal and neonatal development. Tissues lacking in CPSase-I activity did not exhibit the responses to ammonia and ornithine observed with the adult rat liver. While the occurrence of CPSase-I in the liver contributes extensively toward the exceptionally high capacity of that tissue for the de novo biosynthesis of orotic acid, our results also indicate that the physiological rate of orotic acid biosynthesis in rat liver is approximately one-third of capacity; the incorporation of NaH¹⁴CO₃ into orotic acid averaged 488 nmol/g of tissue in 3 h in the presence of toxic levels of ammonia, but declined to 160 nmol/g of tissue in 3 h when physiological levels of both ammonia and ornithine were provided. However, the rate of orotic acid biosynthesis observed with physiological concentrations of ammonia and ornithine could be reduced further, to about one-quarter of the physiological rate, by providing additional ornithine; thus, physiological levels of ornithine do not prevent the escape of intramitochondrial CP into the cytoplasm. Finally, over 80% of the incorporation of NaH¹⁴CO₃ into orotic acid at physiological levels of ammonia and ornithine was found to be ammonia dependent, and all but a small fraction of the ammonia-dependent incorporation could be blocked by providing ornithine in amounts in excess of physiological. These results indicate that CPSase-I is the major source of CP in the biosynthesis of hepatic pyrimidines under normal (physiological) conditions as well as in ammonia toxicity.     

Pyrimidine biosynthesis and its regulation in the developing rat brain.

—Measurements of the incorporation of [¹⁴C]NaHCO₃ into orotic acid, uridine nucleotides and RNA in tissue minces establish the occurrence of the complete orotate pathway for the de novo biosynthesis of pyrimidines in rat brain. Selective inhibition of the incorporation of various radiolabelled precursors into orotic acid by uridine demonstrates the operation of a feedback control mechanism in brain minces and indicates carbamoylphosphate synthetase to be the site of inhibition; purine nucleosides were similarly found to inhibit the de novo biosynthesis of pyrimidines. The activity of the orotate pathway, as assessed by the rate of incorporation of [¹⁴C]NaHCO₃ into orotic acid, was found to be very high in fetal brain and to decline rapidly with neurological development; the mature rat brain exhibits less than 1% of the activity of the fetal brain at 18 days of gestation. Comparative studies on the ability of minces of the brain and several extraneural tissues to utilize [¹⁴C]NaHCO₃ and [¹⁴C]aspartate as precursors of orotic acid lead us to speculate that variations in the ability of tissues to synthesize orotic acid de novo are determined by similar variations in their ability to synthesize carbamoylphosphate.     

Regulation of pyrimidine biosynthesis by purine and pyrimidine nucleosides in slices of rat tissue

Evidence of the primary sites for the regulation of de novo pyrimidine biosynthesis by purine and pyrimidine nucleosides has been obtained in tissue slices through measurements of the incorporation of radiolabeled precursors into an intermediate and end product of the pathway. Both purine and pyrimidine nucleosides inhibited the incorporation of [¹⁴C]-NaHCO₃ into orotic acid and uridine nucleotides, and the inhibition was found to be reversible upon transferring the tissue slices to a medium lacking nucleoside. The ammonia-stimulated incorporation of [¹⁴C]NaHCO₃ into orotic acid, which is unique to liver slices, was sensitive to inhibition by pyrimidine nucleosides at physiological levels of ammonia, but this regulatory mechanism was lost at toxic levels of ammonia. Adenosine, but not uridine, was found to have the additional effects of inhibiting the conversion of [¹⁴C]orotic acid to UMP and depleting the tissue slices of PRPP. Since PRPP is required as an activator of the first enzyme of the de novo pathway, CPSase II, and a substrate of the fifth enzyme, OPRTase, these results indicate that adenosine inhibits the incorporation of [¹⁴C]NaHCO₃ into orotic acid and the incorporation of [¹⁴C]orotic acid into UMP by depriving CPSase II and OPRTase, respectively, of PRPP. Uridine or its metabolites, on the other hand, appear to control the de novo biosynthesis of pyrimidines through end product inhibition of an early enzyme, most likely CPSase II. We found no evidence of end product inhibition of the conversion of orotic acid to UMP in tissue slices.     

Production of uridine 5′-monophosphate by Corynebacterium ammoniagenes ATCC 6872 using a statistically improved biocatalytic process

Attempts were made with success to develop a two-step biocatalytic process for uridine 5′-monophosphate (UMP) production from orotic acid by Corynebacterium ammoniagenes ATCC 6872: the strain was first cultivated in a high salt mineral medium, and then cells were harvested and used as the catalyst in the UMP production reaction. Effects of cultivation and reaction conditions on UMP production were investigated. The cells exhibited the highest biocatalytic ability when cultivated in a medium containing corn steep liquor at pH 7.0 for 15 h in the exponential phase of growth. To optimize the reaction, both “one-factor-at-a-time” method and statistical method were performed. By “one-factor-at-a-time” optimization, orotic acid, glucose, phosphate ion (equimolar KH₂PO₄ and K₂HPO₄), MgCl₂, Triton X-100 were shown to be the optimum components for the biocatalytic reaction. Phosphate ion and C. ammoniagenes cell were furthermore demonstrated as the most important main effects on UMP production by Plackett–Burman design, indicating that 5-phosphoribosyl-1-pyrophosphate (PRPP) synthesis was the rate-limiting step for pyrimidine nucleotides production. Optimization by a central composition design (CCD) was then performed, and up to 32 mM (10.4 g l⁻¹) UMP was accumulated in 24 h from 38.5 mM (6 g l⁻¹) orotic acid. The yield was threefold higher than the original UMP yield before optimization.     

Orotic acid biosynthesis in arginine-deficient rats.

Arginine deficiency is associated with a mild orotic aciduria. Liver slices from rats fed a purified l-amino acid diet with (control) and without arginine supplementation were used for studies of [¹⁴C]bicarbonate incorporation into orotic acid. The nanomoles of orotic acid synthesized in isolated liver slices from both control and arginine-deficient animals increased linearly with time. Orotic acid biosynthesis was significantly greater in liver slices than slices of heart, muscle, kidney, and minced spleen. The order of orotate biosynthesis from [¹⁴C]bicarbonate was liver > spleen = kidney > muscle > heart. Arginine deficiency resulted in a significant stimulation of liver orotic acid biosynthesis. This stimulation in pyrimidine biosynthesis can account for a major portion of the orotic aciduria. Orotic acid synthesis from spleens isolated from arginine-deficient rats was also enhanced compared with controls. Although the rate of orotic acid biosynthesis is small relative to liver production, the spleen may contribute slightly to increased orotic aciduria in the arginine-deficient rat. Arginine supplementation in vitro to livers from rats fed either the control of arginine-deficient diet resulted in a significant reduction in synthesis of orotic acid. Dietary arginine may play a key role in regulating mitochondrial carbamoyl phosphate utilization into both pyrimidine and urea biosynthesis.     

A sensitive, nonradiometric assay for dihydroorotic acid dehydrogenase using anion-exchange high-performance liquid chromatography

A new method to assay the mitochondrial pyrimidine de novo enzyme, dihydroorotate (DHO) dehydrogenase, which catalyzes the dehydrogenation of DHO, with orotic acid as the product was developed. The assay was optimized using a rat liver mitochondrial preparation. Orotic acid was quantified with high-performance liquid chromatography using an anion-exchange column (Partisil-SAX) with uv detection at 280 nm. Isocratic elution with low phosphate buffer at pH 4.0 was used. The detection limit was 20 pmol per injection, which is comparable to previously described radiometric assays. The HPLC assay was compared with a spectrophotometric assay measuring orotic acid formation in a deproteinized reaction mixture. Absorbance was measured at the optimal wavelength for orotic acid, 278.5 nm. This assay is less sensitive and less specific than the HPLC assay, which can also detect UMP which might be formed from orotic acid in whole homogenates. With both assays kinetic parameters of the enzyme were determined. In the high concentration range (80-1000 microM) both Km and Vmax values were comparable. With the HPLC assay the concentration range was extended down to 12 microM and initial rates could be determined. The apparent Km was about 12 microM. The HPLC assay is also suitable for use in the study of inhibition of DHO dehydrogenase.     

Studies on the Utilization of Hydrocarbon by Microorganisms

An uracil-requiring mutant (KY7122) of Arthrobacter paraffineus KY4303 (ATCC15591) was found to accumulate orotic acid and orotidine on n-paraffine as a sole carbon source.

Both substances were definitely indentified as orotic acid and orotidine, from the results on column and paper chromatography, UV and IR absorption spectra, elementary analysis and analyses of hydrolysate.

Cultural conditions for orotic acid and orotidine fermentation were then investigated. As the carbon source n-paraffines from C₁₄ to C₁₆ were the most suitable for the fermentation, and sorbitol, fructose and mannitol were best utilized for the growth, and orotidine produced from them were twice as much as those from hydrocarbon. The addition of 200 mg of uracil and 2 g of C. S. L to 1 liter of medium was most optimal for orotic acid and orotidine fermentation.

Orotic acid and orotidine accumulations were enhanced by the addition of either l-tyrosine, l-leusine, l-threonine, gluconate or meat extract.     

Measurement of urinary pyrimidine bases and nucleosides by high-performance liquid chromatography

A rapid procedure for the isolation, separation, identification and measurement of urinary pyrimidine bases and nucleosides by high-performance liquid chromatography (HPLC) is presented. The initial isolation of these compounds from urine was accomplished with small disposable ion-exchange columns. HPLC was performed on a silica gel column with a mobile phase composed of methylene chloride, methanol and 1 M aqueous ammonium formate buffer. Peaks were recorded at both 254 nm and 280 nm and the response ratio was used in conjunction with the elution volume for compound identification. The minimum detectable amount (signal-to-noise ratio = 2) ranged from 0.2 ng for uracil to 2.2 ng for cytidine. Linearity and recovery for thymine, uracil, uridine, pseudouridine, orotic acid and orotidine added to urine was demonstrated over almost a 10³ concentration range. The potential application of this method for the study of inborn errors in the urea cycle is discussed.     

Improvement of the energy supply and contractile function in normal and ischemic rat hearts by dietary orotic acid

AimsAs cardiac performance is closely related to its energy supply, our study investigated the effect of the orotic acid cardioprotective agent on the pathways of energy supply, in both conditions of normal flow and ischemia.Main methodsMale Wistar rats were fed during nine days with a balanced diet only or supplemented with 1% orotic acid.Key findingsDietary administration of orotic acid increased the cardiac utilization of fatty acids, activity of the lipoprotein lipase, expression of the gene of peroxisome proliferator-activated receptor α and its target enzymes. In addition, orotic acid increased the myocardial uptake and incorporation of glucose, glycogen content and level of GLUT4, concentration of glycolytic metabolites and lactate production in both experimental conditions, baseline and after regional ischemia.SignificanceThus, in orotic acid hearts there was a simultaneous stimulus of fatty acid oxidation and glycolytic pathway, reflected in increased energetic content even in pre-ischemia. The analysis of the cardiac contractility index showed a positive inotropic effect of orotic acid due, at least in part, to the increased availability of energy. The result allows us to suggest that the metabolic changes induced by orotic acid result in appreciable alterations on myocardial contractile function.     

Regulation of Pyrimidine Biosynthesis in Intact Cells of Cucurbita pepo

The occurrence of the complete orotic acid pathway for the biosynthesis de novo of pyrimidine nucleotides was demonstrated in the intact cells of roots excised from summer squash (Cucurbita pepo L. cv. Early Prolific Straightneck). Evidence that the biosynthesis of pyrimidine nucleotides proceeds via the orotate pathway in C. pepo included: (a) demonstration of the incorporation of [¹⁴C]NaHCO₃, [¹⁴C]carbamylaspartate, and [¹⁴C]orotic acid into uridine nucleotides; (b) the isolation of [¹⁴C]orotic acid when [¹⁴C]NaHCO₃ and [¹⁴C]carbamylaspartate were used as precursors; (c) the observation that 6-azauridine, a known inhibitor of the pathway, blocked the incorporation of early precursors into uridine nucleotides while causing a concomitant accumulation of orotic acid; and (d) demonstration of the activities of the component enzymes of the orotate pathway in assays employing cell-free extracts.     

Pteridine-requiring dihydroorotate hydroxylase from Crithidiafasciculata

Dihydroorotic acid is converted to orotic acid in $\text{Crithidia}$ by hydroxylation and subsequent dehydration. The hydroxylase is soluble, stable to acid (pH 4.0), destroyed by alkali (pH 11) and by heat (55° for 3 min.). Activity is rapidly lost upon standing at 4° and upon freezing. Its activity is optimum at pH 7.4. Its isoelectric point is 6.2. It has an absolute dependence on O₂ and a reduced pteridine. Pteridine reductases are present in cell extracts which, in the presence of NADH, permits the efficient use of biopterin (the oxidized form of the naturally occurring pteridine in this organism) as a cofactor for the hydroxylase.     

Elevated urinary excretion of orotic acid in sheep caused by intraruminal infusion of sodium propionate.

1. The effect of sodium propionate on urinary excretion of orotic acid was investigated. 2. Solutions containing sodium propionate or NaCl, 750 mM/day each, were continuously infused into the rumen for 10 days. 3. During NaCl infusion, an urinary orotic acid excretion of 290 +/- 80 micrograms/day was noted. The intraruminal infusion of sodium propionate raised the concentration of propionic acid in the rumen fluid from 14.0 +/- 0.9 to 26.9 +/- 1.9 mM. 4. During this experimental period the excretion of orotic acid via urine significantly increased to 492 +/- 30 micrograms/day. Parameters of nitrogen balance were not altered by propionate. 5. It is suggested that the site of propionate action in intact sheep is in the pyrimidine synthesis pathway.     

The capacity of orotic acid production in pyrimidinedeficient mutants ofBrevibacterium ammoniagenes

Eight uracil-dependent mutants ofBrevibacterium ammoniagenes CCEB 364 and three mutants ofCorynebacterium sp. 9366 were checked for the production of precursors of nucleic acids. Four of the strains liberated into the medium a substantial amount of orotic acid. The production of orotic acid by a mutant ofBrevibacterium ammoniagenes (1043) was examined on mineral media containing varying amounts of glucose in the presence of uracil. The optimum concentration of glucose for the production of orotic acid was found to be 5–8%. On media to which natural substrates were added the orotic acid production increased substantially. The maximum production (6.5 g orotic acid/liter) was reached in a medium containing 0.5% yeast extract and 5% glucose; addition of uracil to this medium had no effect on the production. The maximum rate of production occurred between 24 and 72 h of fermentation. After this period the concentration of orotic acid in the medium decreases.     

Studies on the Accumulation of Orotic Acid by Escherichia coli K12

The mechanism whereby Escherichia coli K12 accumulates orotic acid in culture fluid was studied. Pyrimidine compounds were incorporated effectively into cells of E. coli K12, stimulated the growth, and depressed the accumulation; while purine compounds were not so much consumed by the microorganism for its growth, and affected the accumulation to a lesser extent. On the other hand, E. coli B unable to accumulate orotic acid utilized less effectively pyrimidine compounds for its growth than strain K12.

It is supposed, therefore, that in the de novo pathway for pyrimidine synthesis in E. coli K12 the step from orotic acid to 5′-UMP is genetically depressed so that orotic acid is accumulated when pyrimidine compounds, that would cause a feedback inhibition of orotic acid synthesis upon incorporation, are not supplemented.