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.     

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.     

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.     

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.     

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.     

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.     

Effects of Acetaldehyde on Growth and Biosynthesis in an Algal Flagellate Polytomella caeca*

SYNOPSIS. Eight mM acetaldehyde prevented growth of Polytomella caeca in acetate medium and differentially changed the labeling by acetate-2-¹⁴C of chromatographically separated RNA hydrolysate products. Four mM acetaldehyde also prevented growth in acetate medium unless uridine, thymidine, guanosine, uracil, thymine or quanine were present; then growth was delayed by 2 or 4 days. Orotidine, orotic acid, dihydroortic acid, cytosine, cytidine, adenosine and adenine had no effect on growth in acetate medium containing 4 mM acetaldehyde. One mM acetaldehyde promoted growth in acetate medium and also could serve as a sole carbon source. One mM propionaldehyde, but not butyraldehyde, was also an adequate carbon source. Four mM acetaldehyde, as a sole carbon source, supported growth only when uridine was present.     

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.     

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.     

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.     

Ghrelin and orotic acid increased in subclinical mastitis

Hormone ghrelin and orotic acid accelerate wound healing as well as controlling inflammation and immunity. We have, therefore, investigated the serum and milk levels of ghrelin and orotic acid in dairy cows with (n = 21) or without (n = 21) subclinical mastitis. Acylated and des-acylated ghrelin as well as orotic acid concentration were detected by using high performance liquid chromatography (HPLC). The results revealed that ghrelin level in milk and serum was significantly higher in dairy cows with subclinical mastitis than that of dairy cows without subclinical mastitis. This was also the case when the orotic acid concentrations in dairy cows with subclinical mastitis were compared with those dairy cows without subclinical mastitis. In conclusion, ghrelin and orotic acid occur in particularly high concentrations in subclinical mastitis, and might, therefore, be required in greater amounts for tissue repair and may be also used as a indicator for subclinical mastitis.     

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.     

Prebiotic synthesis of orotic acid parallel to the biosynthetic pathway

By heating an aqueous solution of aspartic acid and urea, carbamylaspartic acid is first formed and then the molecule is cyclized to dihydroorotic acid (DHO) with loss of water. Irradiation of an aqueous solution of DHO with a tungsten lamp yields orotic acid by photo-dehydrogenation of the molecule. This pathway of orotic acid formation is quite similar to that of biosynthesis of the molecule.     

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.     

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.     

DNA and RNA Syntheses by Intraerythrocytic Stages of Plasmodium knowlesi*

Incorporation of the nucleic acid precursors, orotic acid, adenosine, thymidine, and uridine, was studied in various stages of intraerythrocytic Plasmodium knowlesi from infected rhesus monkeys. Incubation of the parasitized erythrocytes with the precursors was for 3 hr periods using a plasma-free culture medium. The samples containing primarily rings, early trophozoites, or late trophozoites incorporated orotic acid, adenosine, and uridine into RNA; however, these stages exhibited negligible or very low levels of incorporation of any of the precursors into DNA. The sample containing late trophozoite and schizont stages incorporated orotic acid, adenosine, and uridine into RNA, and orotic acid, adenosine, and very low levels of thymidine into DNA. These results indicate that DNA synthesis (the S phase of the cell cycle) occurs very close to the time of nuclear division, and that either the G1 or G2 phase is very short in P. knowlesi. It was also observed that adenosine and orotic acid, 2 precursors which are incorporated into both DNA and RNA, are utilized differently by the intraerythrocytic parasites. Incorporation of orotic acid into RNA and DNA and adenosine incorporation into DNA were continuous for the entire incubation period, whereas incorporation of adenosine into RNA was very low during the last 2 hr of each period. It was further demonstrated that the parasites utilized exogenous uridine for synthesis of RNA, and that the older parasite stages incorporated thymidine into DNA.     

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.     

Dietary orotic acid accentuates the hepatic response to phenobarbital in rats.

In rats treated with phenobarbital for 3 days and simultaneously fed a semisynthetic diet containing 1.0% orotic acid, the extent of the increases in liver microsomal phosphatidylcholine, phosphatidylethanolamine, total RNA, total protein, and cytochrome P-450 were significantly greater than they were in rats treated identically with phenobarbital but without dietary orotic acid. This is attributed primarily to the stimulation of hepatic phosphatidylcholine synthesis by dietary orotic acid. In the absence of phenobarbital, orotic acid was shown to cause some increase in liver smooth endoplasmic reticulum components, but not cytochrome P-450. Orotic acid also decreased the activity of microsomal phosphatidylethanolamine N-methyltransferase, which may have contributed to the increase in the microsomal content of phosphatidylethanolamine. The hypothesis is advanced that phospholipid availability is a limiting factor in the hepatic response to phenobarbital. When more phospholipid is available to provide the structural framework for biogenesis of endoplasmic reticulum, all of the hepatic actions of phenobarbital, including induction of cytochrome P-450, are amplified.     

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.     

Orotic acid added to casein, but not to egg protein, soy protein, or wheat gluten diets increases 1,2-diacylglycerol levels and lowers superoxide dismutase activities in rat liver.

Effects of the dietary addition of orotic acid to a diet containing casein as a sole protein source on lipid levels in the liver and serum, activities of antioxidant enzymes in the liver, and some enzyme activities in serum, were compared with other diets containing egg protein, soy protein, or wheat gluten, respectively. 1. The contents in the liver of each lipid were increased by the addition of orotic acid as compared with those values without it. The orotic acid added to the casein diet caused accumulation of more liver total lipids, triacylglycerol, 1,2-diacylglycerol, and phospholipids than those fed three other diets. 2. The addition of orotic acid to the casein, but not to the other three diets, lowered the activities of liver superoxide dismutase and increased the activities of both serum ornithine carbamoyltransferase and alanine aminotransferase. Thus, the significant increase in serum ornithine carbamoyltransferase activities as the marker of liver lesions may result from the marked accumulation of liver lipids, decreased activities of hepatic superoxide dismutase, and the increased level of hepatic 1,2-diacylglycerol, followed by possibly the increased level of superoxide anion and increased activity of protein kinase C in rats fed the casein diet with orotic acid added.