Urinary Orotic Excretion Research Articles

Carbamoylphosphate synthetase 1 (CPS1) deficiency: clinical, biochemical, and molecular characterization in Malaysian patients.

Carbamoyl phosphate synthetase 1 (CPS1) deficiency is a rare autosomal recessive disorder of ureagenesis presenting as life-threatening hyperammonemia. In this study, we present the main clinical features and biochemical and molecular data of six Malaysian patients with CPS1 deficiency. All the patients have neonatal-onset symptoms, initially diagnosed as infections before hyperammonemia was recognized. They have typical biochemical findings of hyperglutaminemia, hypocitrullinemia, and low to normal urinary excretion of orotate. One neonate succumbed to the first hyperammonemic decompensation. Five neonatal survivors received long-term treatment consisting of dietary protein restriction and ammonia-scavenging drugs. They have delayed neurocognitive development of varying severity. Genetic analysis revealed eight mutations in CPS1 gene, five of which were not previously reported. Five mutations were missense changes while another three were predicted to create premature stop codons. In silico analyses showed that these new mutations affected different CPS1 enzyme domains and were predicted to interrupt interactions at enzyme active sites, disturb local enzyme conformation, and destabilize assembly of intact enzyme complex. All mutations are private except one mutation; p.Ile1254Phe was found in three unrelated families. Identification of a recurrent p.Ile1254Phe mutation suggests the presence of a common and unique mutation in our population. Our study also expands the mutational spectrum of the CPS1 gene.

Effects of arginine treatment on nutrition, growth and urea cycle function in seven Japanese boys with late-onset ornithine transcarbamylase deficiency

The aim of this study was to investigate the effects of arginine on nutrition, growth and urea cycle function in boys with late-onset ornithine transcarbamylase deficiency (OTCD). Seven Japanese boys with late-onset OTCD enrolled in this study resumed arginine treatment after the cessation of this therapy for a few years. Clinical presentations such as vomiting and unconsciousness, plasma amino acids and urinary orotate excretion were followed chronologically to evaluate urea cycle function and protein synthesis with and without this therapy. In addition to height and body weight, blood levels of proteins, lipids, growth hormone (GH), insulin-like growth factor-I (IGF-I) and IGF-binding protein -3 (IGFBP-3) were monitored. The frequency of hyperammonemic attacks and urinary orotate excretion decreased significantly following the resumption of arginine treatment. Despite showing no marked change in body weight, height increased gradually. Extremely low plasma arginine increased to normal levels, while plasma glutamine and alanine levels decreased considerably. Except for a slight increase in high-density lipoprotein cholesterol level, blood levels of markers for nutrition did not change. In contrast, low serum IGF-I and IGFBP-3 levels increased to age-matched control levels, and normal urinary GH secretion became greater than the level observed in the controls. Arginine treatment is able to reduces attacks of hyperammonemia in boys with late-onset OTCD and to increase their growth.

Extremely Short Small Bowel Induces Focal Tubulointerstitial Fibrosis

ABSTRACTBackgroundArginine becomes an essential amino acid after massive resection of the small bowel as a result of decreased biosynthesis of citrulline in the remaining small bowel. It is also reported that nitric oxide (NO) is synthesized from l ‐arginine by NO synthase (NOS), and NO is involved in the regulation of blood flow in the kidney. The authors observed a patient with an extremely short small bowel, showing focal tubulointerstitial fibrosis. The experiment was designed to clarify whether massive small bowel resection (SBR) produces focal tubulointerstitial fibrosis in the kidney.MethodsAn experimental study was performed using 4‐week‐old rats with 90% proximal SBR either with or without arginine supplementation for 6 weeks after surgery.ResultsIn rats without arginine supplementation, low plasma levels of citrulline and arginine increased urinary excretion of orotate, and focal tubulointerstitial fibrosis was observed 6 weeks after 90% SBR. The data from plasma amino acid chromatography and increased excretion of urinary orotate suggested the presence of arginine deficiency. The kidney pathology was similar to that of our patient. Rats with arginine supplementation after 90% SBR and pair‐fed control rats without 90% SBR showed almost normal glomeruli and tubulointerstitium.ConclusionsExperimental study strongly suggests that arginine deficiency causes focal tubulointerstitial fibrosis in the kidney after massive SBR.

Extremely short small bowel induces focal tubulointerstitial fibrosis.

Arginine becomes an essential amino acid after massive resection of the small bowel as a result of decreased biosynthesis of citrulline in the remaining small bowel. It is also reported that nitric oxide (NO) is synthesized from l-arginine by NO synthase (NOS), and NO is involved in the regulation of blood flow in the kidney. The authors observed a patient with an extremely short small bowel, showing focal tubulointerstitial fibrosis. The experiment was designed to clarify whether massive small bowel resection (SBR) produces focal tubulointerstitial fibrosis in the kidney. An experimental study was performed using 4-week-old rats with 90% proximal SBR either with or without arginine supplementation for 6 weeks after surgery. In rats without arginine supplementation, low plasma levels of citrulline and arginine increased urinary excretion of orotate, and focal tubulointerstitial fibrosis was observed 6 weeks after 90% SBR. The data from plasma amino acid chromatography and increased excretion of urinary orotate suggested the presence of arginine deficiency. The kidney pathology was similar to that of our patient. Rats with arginine supplementation after 90% SBR and pair-fed control rats without 90% SBR showed almost normal glomeruli and tubulointerstitium. Experimental study strongly suggests that arginine deficiency causes focal tubulointerstitial fibrosis in the kidney after massive SBR.

The effects of various inhibitors on the regulation of orotic acid excretion in sparse-fur mutant mice (spf/Y) deficient in ornithine transcarbamylase

Experiments were conducted to determine whether the excessive orotic aciduria, induced in sparse-fur male mice (spf/Y) deficient in ornithine transcarbamylase (OTC), may be regulated by some inhibitors, such as acivicin (0.014 mmol/100 g body weight, i.p.), N- (phosphonoacetyl)- l-aspartate (PALA, 2.5 mg/100 g body weight, i.p.), adenine (3 g/kg diet) and cycloheximide (0.35 mmol/kg body weight, i.p.). We also administered ornithine (1 mmol/100 g body weight, i.p.), a substrate of the urea cycle, to alleviate the metabolic deficiency of arginine in spf/Y mice which may also be responsible for excessive orotic aciduria. The orotic aciduria remained insensitive to acivicin, indicating mitochondria as the source of carbamyl phosphate. However, orotate excretion was significantly decreased by PALA ( P < 0.01), due to its effect on the aspartate transcarbamylase activity. The ingestion of adenine resulted in an increase ( P < 0.05) of urinary orotate, suggesting the blockage of the utilization of orotate for nucleotide biosynthesis. Ornithine administration led to a reduction ( P < 0.01) of the excretion of orotate induced by the OTC deficiency in these mice, indicating that one of the regulatory steps in its synthesis may be the availability of ornithine. There were no changes in urinary orotate excretion in spf/Y mice when treated with cycloheximide. On the other hand, pretreatment with cycloheximide in an artificial model of OTC deficiency (Swiss-ICR normal mice on an arginine-deficient diet treated thereafter with norvaline, an inhibitor of OTC), caused a significant decrease in urinary orotate. These results suggest that spf/Y mice are unique in that the increased synthesis of orotate is not sensitive to cycloheximide. Perhaps this may reflect an adaptive phenomenon developed by the mutant mice to handle excess mitochondrial carbamyl phosphate and orotic acid.

The metabolism and urinary excretion of orotic acid in hyperargininaemic sheep.

The synthesis, blood plasma turnover and urinary excretion of the orotic acid in normo- and hyperargininaemic sheep was investigated. The whole-body orotate formation was evaluated indirectly by the measurement of urinary orotate excretion after blockage of pyrimidine pathway with 6-azauridine (4 hour i.v. infusion of 0.2 mg.kg-1.min-1). Simultaneous infusion of L-arginine (2.5 mumols.kg-1.min-1) significantly elevated the blood plasma arginine, ornithine and urea level, however, it did not significantly influence urinary orotate excretion. In normoargininaemia blood plasma turnover of exogenous orotic acid amounted to 4.9 min and 67% of this compound was eliminated through the kidneys. The renal clearance of orotic acid amounted to 21.7 ml.min-1.kg-0.75. Hyperargininaemia elevated blood plasma turnover to 8.2 min, and diminished the renal clearance of this metabolite to 13.7 ml.min-1.kg-0.75. These results indicate that hyperargininaemia and hyperornithinaemia do not change the whole body synthesis of orotic acid in sheep but they can affect renal excretion of this metabolite, particularly at the rate of tubular secretion close to saturation.

Arginine-Deficient Diets Alter Plasma and Tissue Amino Acids in Young and Aged Rats

Blood and urine metabolites were measured in two experiments for young (2-mo-old) and aged (20-mo-old) male Sprague-Dawley rats fed arginine-devoid diets made isonitrogenous to a control 1.12% arginine diet by adding alanine or glycine. Diet, fed for 7 or 13 d, had little effect on urinary or plasma ammonia and urea. Urinary orotate excretion was more than 40-fold higher in rats fed the arginine-deficient diets (P less than 0.01) in both experiments. Source of nonessential N (alanine or glycine) in the arginine-deficient diets did not alter orotic acid excretion or plasma or urine ammonia or urea. Changes in plasma arginine, alanine and glycine concentrations reflected the levels of these amino acids in the diet. Tissue ornithine levels reflected dietary arginine level, but tissue citrulline was unaffected by dietary arginine. Glutamate and glutamine were greater in the plasma and liver of rats fed arginine-deficient diets. Plasma concentrations of glutamate and glutamine were positively correlated with urinary orotic acid excretion (P less than 0.05) and ornithine and arginine were negatively correlated with orotic acid excretion (P less than 0.01). Increased tissue glutamine may be related to the greater orotate excretion in rats fed arginine-devoid diets. The metabolic responses to dietary arginine deficiency were similar in young and aged rats. In general, concentrations of amino acids in plasma, liver and spleen were higher in aged rats.

Nitrogen Metabolism in Sickle Cell Anemia: Free Amino Acids in Plasma and Urine

Twenty-four hour urinary levels and fasting plasma concentrations of free amino acids (AA) were evaluated in adult sickle cell anemia (HbSS) and age-matched HbAA subjects with comparable daily energy (E) and protein (N) intake. HbSS elicited significant reduction in the sum of plasma indispensable (EAA) with no change in the dispensable (NAA) amino acids, resulting in a prominent (P less than 0.01) reduction in the EAA/NAA ratio from 71% to 55%. Arg (-38%), Leu (-32%), Val (-28%) and His (-32%) were among the AA most severely affected. Despite a twofold increase in 24-hour urine volume in HbSS compared with HbAA subjects, total urinary losses of EAA and NAA were markedly reduced in the former group, with Arg (-53%) and Gly (-56%) levels most prominently affected. Compared with HbAA controls, HbSS subjects showed a three-fourfold increase in 24-hour urinary orotate excretion that had no relationship to amount of N intake. The results indicated that adult subjects with HbSS, who consumed adequate N and E as per the RDA for healthy individuals, behaved like normal HbAA controls on a low protein diet. There was evidence that the HbSS subject might be in a precarious state with respect to sufficiency of several amino acids, particularly L-Arg, which is now classified as conditionally indispensable for the human.

Late-onset ornithine transcarbamylase deficiency in male patients

We report on 21 male patients who presented after 28 days of age with ornithine transcarbamylase (OTC) deficiency, which we define as late-onset OTC deficiency. These patients appeared normal at birth, but irritability, vomiting, and lethargy, which were often episodic, later developed. The age at presentation ranged from 2 months to 44 years. Biochemical testing revealed hyperammonemia, hyperglutaminemia, hypocitrullinemia, increased urinary orotate excretion, and decreased liver OTC activity measured in vitro, which ranged from 0% to 15% of normal. Male patients who were older at presentation had a somewhat different pattern of presenting symptoms and were more likely to die. These data illustrate the phenotypic variability of OTC deficiency. Unexplained episodes of repetitive or protracted vomiting in association with progressive alterations in behavior or neurologic findings should suggest the diagnosis of a urea cycle defect (or another symptomatic inborn error of metabolism), regardless of the age or medical history of the patient.

The urea cycle in the rett syndrome

The in vivo functioning of the urea cycle in the Rett syndrome (RS) was investigated using alanine load test. The test was carried out in girls with RS and their mothers. These results were compared with those on normal females, males and obligate carriers for ornithine carbamoyltransferase deficiency. Post load hyperammonaemia was not seen in any of the RS girls who were not on medication (valproate). Increased orotate excretion was found in some of the RS girls and mothers. The pattern of urinary excretion of orotate in RS girls and their mothers after a standardised alanine load was similar to that found in female carriers of OCT deficiency. This finding can be explained by a mutant OCT, subject to the Lyon effect, and linked to the gene abnormality in RS or by the presence of an abnormal X-linked mitochondrial protein synthesised in cytoplasma and positioned in close proximity to OCT. Such a protein abnormality could also affect mitochondrial functions in other cells, e.g. in the developing brain.

Effect on lipid in liver and serum, and some urinary components of dietary supplement of excess lysine given to previously starved or non-starved rats.

In order to obtain further information on the changes in liver lipids, either a basal or a lysineexcess diet was refed to previously starved rats or fed to previously non-starved rats. Liver lipid accumulation was observed in previously starved rats refed the lysine-excess diet for 7 days, but not in rats without previous starvation. The liver lipid did not accumulate with another 8 days' feeding (15 days' refeeding). The addition of methionine alone or in combination with threonine to the lysineexcess diet had no effect on the liver lipid level. The decrease in serum triacylglycerol in rats refed the lysine-excess diet was preceded by lipid accumulation in the liver. Urinary potassium during the initial two days increased with refeeding and feeding. Marked excretion of orotate was observed for 2 days from the initiation of refeeding of the lysine-excess diet and it then decreased. Thus, such a marked increase in the urinary excretion of orotate might be associated with the stimulation of orotate biosynthesis and with lipid accumulation in the liver.

Determination of urinary orotate excretion by high-performance liquid chromatography

A new reversed-phase high-performance liquid chromatography procedure for the determination of urinary orotate excretion is described. It is a selective, sensitive and rapid method, suitable for the differentiation of inherited metabolic diseases with abnormal orotate metabolism.

Effect of partial ornithine carbamoyltransferase deficiency on urea synthesis and related biochemical events.

The biochemical response to an intravenous alanine load of 0.25 g/kg was studied in nine adult female relatives of children with ornithine carbamoyltransferase deficiency. Six were classified as affected by partial deficiency and three as unaffected. The plasma ammonium concentration showed no change after the alanine load in the unaffected group, but marked increases occurred in all but one of the affected groups. The maximum rate of urea synthesis after the alanine load was decreased by 37% (P = 0.02) and delayed by 43% (P = 0.02) in the affected group. In the affected group a low rate of urea synthesis was associated with high urinary orotate excretion, high maximum plasma ammonium concentration and delay in the time taken to reach the maximum rate of urea synthesis (Kendall concordance W = 0.55, P less than 0.05). The effects of a higher dose of alanine and of oral protein were compared. The alanine load of 0.25 g of alanine/kg body weight was shown to provide an adequate stimulus to urea synthesis with a more rapid return of ammonium concentration to the pre-load level than with the protein load. The implication of these results in determining the distribution of flux control of urea synthesis, the discrepancy between them and predicted results and the necessary modifications to quantitative simulations are discussed.

Species Comparison of the Influence of Ammonia on Orotic Acid and Urea Biosynthesis in Liver

Although orotic aciduria occurs in both male and female rats fed an arginine-deficient diet, only hepatocytes from male rats exhibited an enhanced rate of orotate biosynthesis to increasing ammonium compared to controls. Inhibition of incorporation [14C]NaHCO3 into urea by norvaline in the rat and mouse was accompanied by a 20 and 25% increase in orotate biosynthesis, respectively. Injection of ammonium chloride (2 mmol/kg) or consumption of a diet devoid of arginine resulted in an increased urinary orotate excretion in the rat. Injection of a similar quantity of ammonium chloride in mice did not result in an orotic aciduria. The influence of ammonia and arginine on the biosynthesis of orotic acid and urea in isolated liver slices from various mammals was also examined. In rat, mouse and pig liver, increasing quantities of ammonia stimulated the incorporation of [14C]NaHCO3 into orotic acid. In porcine, bovine and ovine liver the incorporation of [14C]NaHCO3 into orotic acid was low and was minimally effected by supplemental ammonia. Addition of arginine to the incubation medium diminished the incorporation of [14C]NaHCO3 into orotic acid in the liver of all species examined except the cow and sheep. This decrease was accompanied by a stimulation of urea biosynthesis in the rat, but a depression in the pig. Addition of ammonium (5.0 mM) markedly increased the incorporation of [14C]NaHCO3 into urea in all mammalian liver slices examined. These studies show that the liver of various mammals can respond to increasing ammonia by increasing orotic acid biosynthesis. However, species differences in the response to ammonia are evident.

New defects of pyrimidine metabolism.

The number of inherited defects of the pyrimidine metabolism described so far is small, compared to that of the purine metabolism. Combined deficiency of orotate phosphoribosyltransferase (OPRT) (EC 2.4.2.10) and orotidine 5′-monophosphate decarboxylase (ODC) (EC 4.1.1.23), designated as type I hereditary orotic aciduria, presents with characteristic clinical features such as hypo-chromic anemia with a megaloblastic bone marrow and crystalluria. Only six patients have been described and, as far as we know, new cases have not been discovered recently. ODC deficiency with similar clinical phenomena and leading to increased urinary excretion of orotate and orotidine has been detected in only one patient (1). A third defect, a deficiency of pyrimidine 5′-nucleotidase (Py-5N). (EC 3.1.3.5.) in erythrocytes, is associated with chronic hemolytic anemia and prominent basophylic stippling of the erythrocytes due to accumulated pyrimidine nucleotides. An increasing number of patients have been reported, their detection being facilitated by the typical phenomena. We do not know whether the urinary pyrimidine profile in this condition is abnormal.

Effects of orotic acid ingestion on urinary and blood parameters in humans

Pertinent urinary and blood parameters were examined in 29 normal adults ingesting orotic acid by capsule once a week. Upon ingestion of 6 g orotic acid, urinary excretion of orotate and urate was increased in all subjects but creatinine and urea were unaffected. Mean orotate excretion was 624 mg or 10.5% of dose ingested, although individual variability was large (1–26%). In five persons studied more extensively, excretion of excess orotate was proportional to the amount ingested and was generally complete within 8 hours. Orotic acid ingestion led to uricosuria and the pattern for urate excretion was similar to that for orotate. Blood parameters before participation in the study and 24 hours after ingesting 6 g orotic acid were within normal ranges. However, serum urate and cholesterol were decreased significantly. The decrease in blood urate reflects the uricosuric effect of orotic acid. The hypocholesteremic response to orotic acid ingestion in humans warrants further investigation.

Spontaneous animal models of ornithine transcarbamylase deficiency: studies on serum and urinary nitrogenous metabolites.

Various groups of spf (sparse fur) and spf ash (allele with abnormal skin and hair) mice, were compared in respect of their liver ornithine transcarbamylase (OTC) activity, urinary orotate and concentrations of serum NH3 and glutamine. While liver OTC activity was comparable in both the strains, the excretion of urinary orotate was lower in spfash mice. In contrast to spf females, the spf ash/+ heterozygotes cannot be distinguished from normal +/+ females, as urinary orotate excretion has no significant correlation with liver OTC activity. However, serum glutamine is significantly correlated with liver OTC deficiency in both the strains. Administration of 1% sodium benzoate to spf/Y males resulted in a pattern of excretion of hippurate and urea N similar to clinical trials in humans. Urinary orotate was significantly reduced, while ad lib dietary intake increased almost 3 fold. Both the spontaneous animal models have considerable potential to be used in the investigation of the etiology, specific pathology and nutritional therapy of congenital hyperammonemias.

Species Specificity of Arginine Deficiency-Induced Hepatic Steatosis

Hepatic steatosis resulting from the consumption of an arginine-deficient diet in the rat was found to occur independent of age or size. The rate of lipid biosynthesis as indicated by in vitro incorporation of 14C-acetate was significantly increased in rats fed an arginine-deficient diet when expressed per milligram liver. Supplementation of the arginine-deficient diet with 1% ribose, 1% hypoxanthine or 0.2% adenine depressed the fatty infiltration caused by arginine deficiency. Inosine, xanthine or uracil supplementation did not significantly alter the fatty infiltration, liver orotic acid biosynthesis or urinary orotic excretion induced by the arginine deficiency. Increased orotic acid excretion was also observed in the mouse, hamster and rabbit fed a diet devoid of arginine. However, consumption of the arginine-deficient diet for 21 days did not significantly alter the liver lipid content of mice, hamsters or rabbits. Although the fatty infiltration appears to be limited to the rat, altered liver nucleotides were observed in rats, hamsters and rabbits fed an arginine-deficient diet. Similarities of arginine deficiency and orotic acid feeding in various species are discussed.arginine deficiency steatosis orotic acid

Asymptomatic hyperammonemia in low birthweight infants.

At 0-3 days of age the plasma ammonium concentration in full term appropriate for gestational age (AGA) infants was (mean +/- SEM) 27.5 +/- 0.5 micron; a value similar to that reported in adults. Ammonium levels in low birthweight AGA and SGA groups were 47.0 +/- 2.0 micron and 45.1 +/- 3.3 micron respectively; significantly elevated (P less than 0.001) as compared to the full term group. These increased ammonium levels persisted at 3-5 weeks of age. Associated with the hyperammonemia was a significant (P less than 0.01) decrease in plasma alpha-ketoglutarate concentration: 11.8 +/- 1.0 micron, in the low birthweight AGA as compared to 20.7 +/- 0.6 micron in the full term AGA infants. There was an inverse linear correlation between plasma concentrations of ammonium and alpha-ketoglutarate r = -0.86, P less than 0.001. Urinary orotate excretion was significantly elevated (P less than 0.05) in low birthweight AGA infants. There was no difference in the plasma concentrations of glutamine, glutamate, or alanine among the various groups. Hyperammonemia was not associated with neurologic dysfunction.

HYPERAHMONEMIA IN PREMATURE INFANTS

Plasma levels of ammonium, glutamine, glutamate, alanine, α-ketoglutarate and urinary excretion of orotate were measured in full-term, n=41, premature small for gestational age (SGA), n=9, and premature appropriate for gestation age (AGA), n=27, infants. At 0-3 days of Hfe there was a significant difference (p<.001) in plasma ammonium concentrations in the premature SGA and AGA infants (mean±SEM) 45±3. 47±2 μM as compared to the full-term infants, 27±1 μM (adult normal <30 μM). When the two premature groups reacned term by weight or gestational age they were still hyperammonemic (39±3, 47±3 μM). It was not until 6-8 weeks of postnatal age that plasma ammonium levels became normal in all the prematures (29±1 μM). Associated with the hyperammonemia was a significant (p<.01) decrease of plasma α-keto-glutarate concentration, 12±1 μM, in premature AGA (n=13), compared to 22±1 μM in full-term (n=17) infants and an increase (p<.05) in urinary orotate excretion, 9±2 as compared to 4±1 μg/mg creatinine. There was a significant, p<.001, relationship between birth weight and plasma ammonium concentration (r=-0.65) and between α-keto-glutarate and plasma ammonium levels (r=-0.86). There were no differences in the plasma concentrations of glutamine, glutamate or alanine. Both premature groups were α-symptomatic. Possible mechanisms responsible for these abnormalities include development delay of one or more of the enzymes related to the urea cycle or urea cycle substrate deficiency.