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.     

The use of a triton X-100-based scintillant for counting fractions from density gradients

The properties of an aqueous scintillation mixture containing butyl-PBD as the sole scintillant and using Triton X-100 as emulsifier are described. This counting mixture, which is considerably cheaper than other published mixtures for aqueous samples, is shown to perform extremely satisfactorily with polysomes and RNA labeled by prior injection of [¹⁴C]orotic acid. When, however, this counting mixture is used with ³H-labeled samples, the density gradient solutes sucrose and cesium chloride are shown to quench the counting of RNA and polysomes but not of toluene or orotic acid.     

Radiation-induced conversion of 5,6-dihydropyrimidines to the corresponding pyrimidines in N2O-saturated aqueous solutions

The effect of transition metal salts on the radiation-induced conversion of 5,6-dihydropyrimidines to the corresponding parent pyrimidines was studied in N2O-saturated aqueous solution at pH 7.0. The yield of the pyrimidines increased in sigmoidal forms with the increased one-electron reduction potential of the transition metal salts. The radiolysis of 5,6-dihydroorotic acid suggested that the 6-yl radical of the acid undergoes oxidation by transition metal salts to give orotic acid, whereas the corresponding 5-yl radical readily liberates CO2 to give uracil radical anion.     

Relation between intracellular Na ion activity and tension of sheep cardiac Purkinje fibers exposed to dihydro-ouabain.

The intracellular Na ion activity (aiNa) and the contractile tension (T) of sheep cardiac Purkinje fibers were simultaneously measured employing recessed-tip Na+-selective glass microelectrodes and a mechano-electric transducer. The aiNa of 6.4 +/- 1.6 mM (mean +/- SD, n = 56) was obtained in fibers perfused with normal Tyrode's solution. The changes in aiNa and T were measured during and after the exposure of fibers to a cardiac glycoside, dihydro-ouabain (DHO) in concentrations between 5 X 10(-8) M and 10(-5) M. The exposure time to DHO was 15 min. Both aiNa and T did not change in fibers exposed to 5 X 10(-8) M DHO, and the threshold concentration for the effect of DHO appeared to be around 10(-7) M. In DHO concentrations greater than the threshold, the increases in aiNa and T strongly correlated during the onset of DHO effects. The recoveries of aiNa and T were variable and slow, being dependent on the DHO concentration. In those fibers which recovered from the effects of DHO, the time-course of aiNa recovery was similar to that of T recovery. In fibers exposed to DHO of 5 X 10(-6) M or greater, the apparent toxic effects were observed in both action potential and contraction after an initial increase in T. The fibers manifesting the apparent toxic effects has a aiNa of approximately 30 mM or greater. The results of this study indicate that the increase in aiNa is associated with the positive inotropic action of the cardiac glycoside.     

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.     

Early effects of dietary orotic acid upon liver lipid synthesis and bile cholesterol secretion in rats

Dietary orotic acid is known to cause impaired fatty acid synthesis and increased cholesterol synthesis in rats. We found that the impaired fatty acid synthesis occurs during the first day of orotic acid feeding and, in studies with albumin-bound [1-14C]palmitic acid, an associated decrease in the rate of esterification of this fatty acid into triacylglycerol, phospholipid, and cholesteryl ester was observed. These changes may result from the known decreases in liver levels of adenine nucleotides or, as reported here, from decreased liver CoASH levels in orotic acid-fed rats. The increase in hepatic cholesterol synthesis occurred during the second day of orotic acid feeding. It was detected by increased incorporation of [1,2-14C]acetate into cholesterol by liver slices and by a 7-fold increase in HMG-CoA reductase activity. At the same time the biliary output of cholesterol was increased 2-fold and studies using 3H2O revealed that the output of newly synthesized cholesterol in bile was increased 5-fold. The content of cholesteryl ester in hepatic microsomes decreased during orotic acid feeding but free cholesterol was unchanged. The findings are interpreted to suggest that the increased bile cholesterol secretion caused by orotic acid is a result of impaired hepatic cholesterol esterification and that the increase in HMG-CoA reductase activity is a result of diminished negative feedback due to the depleted content of cholesteryl ester in the hepatic microsomes.     

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.     

Abnormal hepatic nucleotide pools in sparse fur (spf) mutant mice deficient in ornithine transcarbamylase.

Sparse fur hemizygous male mice are over 90% deficient in ornithine transcarbamylase and exhibit increased synthesis of orotic acid. Because our earlier studies have demonstrated that orotic acid is a liver tumor promoter in the rat, it was of interest to determine whether this genetic disorder also increases the risk of tumor promotion. The results revealed that the livers of mutant mice showed a fourfold increase in uridine nucleotides and a 50% decrease in adenosine nucleotides compared to corresponding controls, a pattern of nucleotide pool imbalance similar to that seen in the livers of rats exposed to orotic acid under promoting conditions. Creation of such an imbalance appears to be important for orotic acid to exert its promotional effects. Sparse fur mutant mouse may, therefore, be an ideal animal model to study the tumor-promoting effects of orotate.     

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.     

Acetate-mediated production of orotic acid by ura3 mutants of Candida albicans

Candida albicans ura3 mutants were found to produce large amounts of orotic acid when the growth medium was supplemented with sodium acetate. Experiments with 13C-labeled acetate showed that the acetate served as a precursor of orotic acid. This system of acetate-mediated production of orotic acid is similar to other documented microbial producers in yield but unique for its acetate requirement.     

Utilization of labelled uridine, cytidine and orotic acid for determination of ribonucleic acid synthesis in mouse liver

[3H]uridine and [3H]orotic acid were equally utilized for labelling of RNA in mouse liver. Incorporation of [3H]cytidine was 2-3 times as high as that of [3H]-labelled uridine or orotic acid. These results differ from findings in rat liver, where both cytidine and orotic acid are better utilized for RNA labelling than is uridine. The ratio between liver RNA [3H]-activity and volatile [3H]-activity was 2, 3 and 13, respectively, at 300 min after injection of labelled uridine, orotic acid and cytidine, indicating an efficient chanelling of cytidine into liver anabolic pathways.     

Dietary orotic acid affects antioxidant enzyme mRNA levels and oxidative damage to lipids and proteins in rat liver

We investigated the effects of the dietary addition of orotic acid on liver antioxidant enzymes, mRNA levels of these enzymes, and peroxidative products by comparing casein with soy protein as the source of dietary protein. Rats fed the casein diet accumulated more liver lipids than those fed the soy protein diet when orotic acid was added. The addition of orotic acid lowered both the activity of liver Cu, Zn-superoxide dismutase and the level of Cu, Zn-superoxide dismutase mRNA. The addition of orotic acid led to a significant increase in the contents of conjugated dienes and protein carbonyls in the liver. In addition, dietary soy protein protected the increase in the levels of lipids and proteins peroxide induced by orotic acid. The addition of orotic acid to the casein diet increased the activities of both serum ornithine carbamoyltransferase and alanine aminotransferase. Thus, liver damage might result from the increased superoxide anion due to the decrease in the activity of hepatic superoxide dismutase, as well as increase in the production of hepatic peroxidative products in rats fed the casein diet with orotic acid.     

Relationship between the activity of the orotic acid pathway and glucose content of roots of Cucurbita pepo

Abstract. Apical 3-cm root segments excised from 2-d-old squash seedlings ( Cucurbita pepo L. cv. Early Prolific Straightneck) that were germinated and grown between sheets of paper towelling moistened with H₂O₂ incorporated 144±10 nmol (± SE. n = 15) NaH¹⁴CO₃ into uridine nucleotides (∑UMP) per gram intact roots during the 3-h incubation period ( Lovatt, Albert & Tremblay, 1979 ). Continued culture of squash seedlings in this manner for an additional 24 or 48 h had no effect or slightly increased (30%) the activity of the orotic acid pathway. However, transfer of 2-d-old seedlings to Shive's nutrient solution reduced the activity of the orotic acid pathway within 15 h to 2.3 nmol NaH¹⁴CO₃ incorporated into ∑UMP per gram apical 3-cm root segments during the 3-h incubation period. The observed decrease in capacity to synthesize pyrimidine nuclotides de novo paralleled the reduction in glucose content of the roots and was reversed by supplying glucose during hydroponic culture in Shive's nutrient solution. Root elongation was not affected by the reduced activity of the orotic acid pathway nor by the decreased level of available glucose.     

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.     

STUDIES ON THE ORIGIN OF PYRIMIDINES FOR BIOSYNTHESIS OF NEURAL RNA IN THE RAT

Uridine was far superior to orotic acid in labelling the RNA in incubated slices of rat brain. On the other hand, uridine and orotic acid were equally effective in labelling the RNA of hepatic or renal slices In rats in vivo, uridine, but not orotic acid, labelled brain RNA, and the cerebellar RNA contained the most label. In contrast, both uridine and orotic acid labelled hepatic RNA. Only when surgical intervention prevented peripheral metabolism of orotic acid, thereby raising its concentration in the plasma, did neural tissue utilize this precursor for limited biosynthesis of RNA. However, among the tissues studied, the preference for uridine over orotic acid for RNA synthesis was unique to neural tissue.     

Effects of dietary protein type on the response of lipid metabolism to orotic acid in rats

The effects of orotic acid supplementation to casein, egg protein, soy protein and wheat gluten diets on the lipids of liver and serum were compared. When orotic acid was added, the contents of total lipids and triacylglycerol in the liver of the casein group were significantly higher or tended to be higher than those of the other three dietary groups. Dietary orotic acid had no effect on the food intake. The liver weight, and liver total lipids, triacylglycerol, cholesterol and phospholipids were increased or tended to be increased by the addition of orotic acid. The serum triacylglycerol level was decreased by the addition of orotic acid to either the casein or soy protein diet. Thus, the response to liver lipid accumulation induced by orotic acid feeding depended on the dietary protein type.     

Studies on the Accumulation of Orotic Acid by Escherichia coli K12

More than 300 mg/liter of orotic acid was found to accumulate in the supernatants of the cultures of wild type strains of E. coli K12. The pyrimidine precursor was accumulated in a synthetic medium such as glucose-ammonium sulfate medium. The substance was isolated from the culture, crystallized, and identified as orotic acid. Orotic acid was excreted mainly during logarithmic phase of the bacterial growth. Yeast extract or nutrient broth stimulated bacterial growth, but suppressed orotic acid accumulation. E. coli strains other than K12 failed to accumulate orotic acid.

The results suggest that the accumulation of orotic acid is specific to E. coli K12.     

Supplementation of orotic acid to the casein, but not to egg protein, soy protein, or wheat gluten diets, increases serum ornithine carbamoyltransferase activity

Effects of dietary supplementation of orotic acid to a diet containing the casein protein were compared with diets containing egg protein, soy protein, or wheat gluten on lipid levels in the liver and serum and activities of ornithine carbamoyltransferase (OCT) and alanine aminotransferase in the serum of rats. We found that supplementation of orotic acid to each diet increased the contents of the liver total lipids, triacylglycerol, and phospholipids compared with those not supplemented. The contents of liver total lipids, triacylglycerol, cholesterol, and phospholipids in rats fed the casein diet were significantly higher than those of rats fed the other three diets when orotic acid was supplemented. The levels of triacylglycerol, cholesterol, and phospholipids in the serum of rats fed the casein diet were markedly decreased by addition of orotic acid. The supplementation of orotic acid significantly increased the activities of both serum OCT and alanine aminotransferase in rats fed the casein diet, but not in rats fed the other diets. In conclusion, liver lipid accumulation induced by dietary orotic acid depends on the type of dietary protein. The enhancement of serum OCT activity may result from liver lipid accumulation in rats fed the casein diet supplemented with orotic acid, demonstrating hepatic damage.     

Multiple inhibitor analysis of the brequinar and leflunomide binding sites on human dihydroorotate dehydrogenase

Brequinar and the active metabolite of leflunomide, A77 1726, have been clearly shown to inhibit human dihydroorotate dehydrogenase (DHODH), but conflicting mechanisms for their inhibition have been reported. DHODH catalyses the conversion of dihydroorotate (DHO) to orotate concurrent with the reduction of ubiquinone. This study presents data that indicates brequinar is a competitive inhibitor versus ubiquinone; A77 1726 is noncompetitive versus ubiquinone and both are uncompetitive versus DHO. 2-Phenyl 5-quinolinecarboxylic acid (PQC), the core moiety of brequinar also shows competitive inhibition versus ubiquinone. Multiple inhibition experiments indicate that PQC (and thus brequinar) and A77 1726 have overlapping binding sites. Both PQC and A77 1726 are also mutually exclusive with barbituric acid (a competitive inhibitor versus DHO). In addition, we failed to observe brequinar binding to E.orotate by isothermal titration calorimetry (ITC). These results indicate that the E.DHO.inhibitor and E.orotate.inhibitor ternary complexes do not form. The absence of these complexes is consistent with the two-site ping-pong mechanism reported for DHODH. This kinetic data suggests that recent crystal structures of human DHODH complexed with orotate and A77 1726 or brequinar may not represent the relevant physiological binding sites for these inhibitors [Liu, S., Neidhardt, E. A., Grossman, T. H., Ocain, T., and Clardy J. (2000) Structure 8, 25-33].