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Isolation and partial characterization of a 5'-nucleotidase specific for orotidine-5'-monophosphate.Proceedings of the National Academy of... Feb 1982A previously unknown 5'nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) (5'-Nase) specific for orotidine 5'-monophosphate (OMP) hs been discovered. This...
A previously unknown 5'nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) (5'-Nase) specific for orotidine 5'-monophosphate (OMP) hs been discovered. This enzyme orotidine 5'-monophosphate phosphohydrolase (OMPase), was isolated from mouse liver microsomes as a separate entity from the nonspecific 5'-Nase. OMPase was partially purified and is shown to cleave OMP to orotidine and inorganic phosphate. The enzyme has negligible activity towards UMP, CMP, dTMP, AMP, IMP, GMP, XMP, 6-azauridine 5'-monophosphate, 1-beta-D-ribofuranosylbarbituric acid 5'-monophosphate (BMF), 2'-UMP, 3'-UMP, 2'-AMP, 3'-AMP, ribose 5-phosphate and beta-glycerophosphate, all of which--with the exception of the 2' or 3' monophosphates, ribose 5'-phosphate, and beta-glycerophosphate--are substrates for 5'-Nase. Both enzymes are inhibited by NaF, but only OMPase is inhibited by SF reagents. OMPase is not inhibited by orotidine, orotate, BMP, concanavalin A, or tetramisole (an alkaline phosphatase inhibitor). OMPase had a Mr 53,000, Km value of 1 mM for OMP, and Vmax value of 49 nmol/min . mg of protein at the present stage of purification. OMPase activity has also been detected in various mammalian tissues including normal human tissues, human tumor xenografts, lymphocytes, and rat liver. OMPase may be responsible, in part, for the low levels of intracellular "free" OMP and for orotidine accumulation in cells treated with 6-azauridine and patients suffering from aortic aciduria.
Topics: 5'-Nucleotidase; Animals; Concanavalin A; Dithionitrobenzoic Acid; Kinetics; Magnesium; Male; Mice; Microsomes; Nucleotidases; Orotate Phosphoribosyltransferase; Pentosyltransferases; Rats; Sodium Fluoride; Substrate Specificity; Tissue Distribution; Uracil Nucleotides; Uridine Monophosphate
PubMed: 6280163
DOI: 10.1073/pnas.79.4.1037 -
The New England Journal of Medicine Jun 1990Ornithine carbamoyltransferase is an X-linked mitochondrial enzyme expressed in hepatocytes and enterocytes. A deficiency of this enzyme results in central nervous...
Ornithine carbamoyltransferase is an X-linked mitochondrial enzyme expressed in hepatocytes and enterocytes. A deficiency of this enzyme results in central nervous system dysfunction, which may be fatal in newborn boys. Milder forms are seen in older boys and girls and in adults. Establishing the carrier status of women at risk for ornithine carbamoyltransferase deficiency is important for determining reproductive and medical risks for affected women. We report a test to establish the carrier status of women at risk for ornithine carbamoyltransferase deficiency. This test relies on the allopurinol-induced accumulation of orotidine, whose synthesis is stimulated by carbamoyl phosphate, a substrate that accumulates in ornithine carbamoyltransferase deficiency. We used anion-exchange, high-performance liquid chromatography to measure urinary orotidine and orotic acid excretion after the administration of a 300-mg oral dose of allopurinol in 25 [corrected] women who were obligate heterozygotes, 13 who were probable heterozygotes, 15 mothers of affected boys from monoplex families (families with only one affected member), 12 mothers of affected girls from monoplex families, and 21 [corrected] normal, unrelated women who were not carriers. Urinary orotidine excretion was increased 3 SD or more above the mean value for the normal women in 95.8 percent of the obligate heterozygotes, 84.6 percent of the probable heterozygotes, 73.3 percent of the mothers of affected boys in monoplex families, and 33.3 percent of the mothers of affected girls in monoplex families, thus establishing that these women were carriers of a mutant ornithine carbamoyltransferase allele. The presence of allopurinol-induced orotic aciduria was not as sensitive or specific an indicator of carrier status as the presence of orotidinuria. We conclude that measurement of urinary orotidine excretion after the administration of allopurinol is a simple and reliable test for the identification of women who are heterozygous for ornithine carbamoyltransferase deficiency.
Topics: Adult; Allopurinol; Chromatography, High Pressure Liquid; Female; Genetic Carrier Screening; Heterozygote; Humans; Middle Aged; Ornithine Carbamoyltransferase; Ornithine Carbamoyltransferase Deficiency Disease; Orotic Acid; Uridine
PubMed: 2342523
DOI: 10.1056/NEJM199006073222305 -
PloS One 2015Ornithine transcarbamylase deficiency (OTCD, OMIM# 311250) is an inherited X-linked urea cycle disorder that is characterized by hyperammonemia and orotic aciduria. In...
Ornithine transcarbamylase deficiency (OTCD, OMIM# 311250) is an inherited X-linked urea cycle disorder that is characterized by hyperammonemia and orotic aciduria. In this report, we describe a new animal model of OTCD caused by a spontaneous mutation in the mouse Otc gene (c.240T>A, p.K80N). This transversion in exon 3 of ornithine transcarbamylase leads to normal levels of mRNA with low levels of mature protein and is homologous to a mutation that has also been described in a single patient affected with late-onset OTCD. With higher residual enzyme activity, spf-J were found to have normal plasma ammonia and orotate. Baseline plasma amino acid profiles were consistent with mild OTCD: elevated glutamine, and lower citrulline and arginine. In contrast to WT, spf-J displayed baseline elevations in cerebral amino acids with depletion following immune challenge with polyinosinic:polycytidylic acid. Our results indicate that the mild spf-J mutation constitutes a new mouse model that is suitable for mechanistic studies of mild OTCD and the exploration of cerebral pathophysiology during acute decompensation that characterizes proximal urea cycle dysfunction in humans.
Topics: Amino Acid Sequence; Amino Acids; Animals; Biological Transport; Body Weight; Brain; Disease Models, Animal; Humans; Mice; Molecular Sequence Data; Mutation, Missense; Ornithine Carbamoyltransferase; Ornithine Carbamoyltransferase Deficiency Disease; Orotic Acid; Phenotype; Poly I-C; Protein Structure, Tertiary; Rats; Survival Analysis
PubMed: 25647322
DOI: 10.1371/journal.pone.0116594 -
Molecular Genetics and Metabolism Feb 2012Ornithine transcarbamylase deficiency (OTCD), the most common and severe urea cycle disorder, is an excellent model for developing liver-directed gene therapy. No...
Ornithine transcarbamylase deficiency (OTCD), the most common and severe urea cycle disorder, is an excellent model for developing liver-directed gene therapy. No curative therapy exists except for liver transplantation which is limited by available donors and carries significant risk of mortality and morbidity. Adeno-associated virus 8 (AAV8) has been shown to be the most efficient vector for liver-directed gene transfer and is currently being evaluated in a clinical trial for treating hemophilia B. In this study, we generated a clinical candidate vector for a proposed OTC gene therapy trial in humans based on a self-complementary AAV8 vector expressing codon-optimized human OTC (hOTCco) under the control of a liver-specific promoter. Codon-optimization dramatically improved the efficacy of OTC gene therapy. Supraphysiological expression levels and activity of hOTC were achieved in adult spf(ash) mice following a single intravenous injection of hOTCco vector. Vector doses as low as 1×10(10) genome copies (GC) achieved robust and sustained correction of the OTCD biomarker orotic aciduria and clinical protection against an ammonia challenge. Functional expression of hOTC in 40% of liver areas was found in mice treated with a low vector dose of 1×10(9) GC. We suggest that the clinical candidate vector we have developed has the potential to achieve therapeutic effects in OTCD patients.
Topics: Adult; Animals; Dependovirus; Gene Expression; Genetic Therapy; Genetic Vectors; Humans; Liver; Mice; Ornithine Carbamoyltransferase; Ornithine Carbamoyltransferase Deficiency Disease; Orotic Acid
PubMed: 22133298
DOI: 10.1016/j.ymgme.2011.10.020 -
Canadian Medical Association Journal Sep 1972Recurrent vomiting without apparent cause should alert the physician to the possibility of a disorder of ammonia metabolism. Crystalluria in a three-month-old male...
Recurrent vomiting without apparent cause should alert the physician to the possibility of a disorder of ammonia metabolism. Crystalluria in a three-month-old male infant with a history of intermittent vomiting since birth and incipient coma led to the discovery of orotic aciduria. A diagnosis of ornithine carbamyl transferase (OCT) deficiency was derived from study of the liver after the infant had died; residual activity was about 5% of normal. Ammonia intoxication was the presumed cause of death. Overproduction of orotic acid and other pyrimidines reflects the deficiency of OCT. The possibility of genetic heterogeneity for the hereditary trait under observation must be considered because it may influence prognosis and counselling.
Topics: Ammonia; Chromatography, Gas; Coma; Humans; Infant; Infusions, Parenteral; Liver; Male; Metabolism, Inborn Errors; Ornithine Carbamoyltransferase; Orotic Acid; Spectrophotometry, Ultraviolet; Urea; Vomiting
PubMed: 5074751
DOI: No ID Found -
Nucleic Acids Research Mar 1988The sparse fur with abnormal skin and hair (Spf-ash) mouse is a model for the human X-linked hereditary disorder, ornithine transcarbamylase (OTC) deficiency. In Spf-ash...
The sparse fur with abnormal skin and hair (Spf-ash) mouse is a model for the human X-linked hereditary disorder, ornithine transcarbamylase (OTC) deficiency. In Spf-ash mice, both OTC mRNA and enzyme activity are 5% of control values resulting in hyperammonemia, pronounced orotic aciduria and an abnormal phenotype characterized by growth retardation and sparse fur. Using microinjection, we introduced a construction containing rat OTC cDNA linked to the SV40 early promoter into fertilized eggs of Spf-ash mice. The expression of the transgene resulted in the development of a transgenic mouse whose phenotype and orotic acid excretion are fully normalized. Thus, the possibility of correcting hereditary enzymatic defect by gene transfer of heterologous cDNA coding for the normal enzyme has been demonstrated.
Topics: Amino Acid Metabolism, Inborn Errors; Animals; Cloning, Molecular; Gene Expression Regulation; Genetic Engineering; Liver; Mice; Mice, Mutant Strains; Mice, Transgenic; Ornithine Carbamoyltransferase; Ornithine Carbamoyltransferase Deficiency Disease; Pedigree; Phenotype
PubMed: 3162766
DOI: 10.1093/nar/16.5.2099 -
World Journal of Hepatology May 2015The clinical manifestations of hyperammonemia are usually easily identifiable to the clinician when associated with liver disease and lead to prompt diagnosis and...
The clinical manifestations of hyperammonemia are usually easily identifiable to the clinician when associated with liver disease and lead to prompt diagnosis and treatment. However, hyperammonemia-induced encephalopathy is rare in adults in the absence of overt liver disease, thus diagnosis is often delayed or missed leading to potentially life threatening complications. Without proper treatment, such patients can decompensate rapidly with poor outcomes including seizures, coma, and death. Early assessment of plasma ammonia levels in patients with normal hepatic function and characteristic symptoms of encephalopathy can lead to early intervention while investigating the underlying etiology. We describe a patient who presented with a 2-year progression of waxing and waning acute mental status changes after a Roux-en-Y gastric bypass surgery. He was found to have elevated ammonia level as well as orotic aciduria; results consistent with a urea cycle disorder. After consulting neurology as well as toxicology, he ultimately improved after dietary protein restriction, sodium benzoate and lactulose therapy. While rare, clinicians should have a high index of suspicion for late onset urea cycle disorders in symptomatic patients presenting with encephalopathy secondary to hyperammonemia.
PubMed: 25954483
DOI: 10.4254/wjh.v7.i7.1007 -
Molecular Therapy : the Journal of the... Sep 2004The urea cycle disorders (UCDs) are important models for developing gene replacement therapy for liver diseases. Long-term correction of the most common UCD, ornithine...
The urea cycle disorders (UCDs) are important models for developing gene replacement therapy for liver diseases. Long-term correction of the most common UCD, ornithine transcarbamylase (OTC) deficiency, has yet to be achieved in clinical or preclinical settings. The single human clinical trial using early-generation adenovirus (Ad) failed to show any biochemical correction. In adult OTC-deficient mice, an E1/E2-deleted Ad vector expressing the mouse OTC gene, but not the human, was only transiently therapeutic. By using post-transcriptional overexpression in the context of the less immunogenic helper-dependent adenoviral vector, we achieved metabolic correction of adult OTC-deficient mice for >6 months. Demonstrating this result were normalized orotic aciduria, normal hepatic enzyme activity, and elevated OTC RNA and protein levels in the absence of chronic hepatotoxicity. Overexpressing the human protein may have overcome two potential mechanisms accounting for poor cross-species complementation: a kinetic block at the level of mitochondrial import or a dominant negative effect by the mutant polypeptide. These data represent an important approach for treating human inborn errors of hepatocyte metabolism like the UCDs that require high-level transduction and gene expression for clinical correction.
Topics: Adenoviridae; Animals; Genetic Therapy; Hepatitis B Virus, Woodchuck; Hepatocytes; Humans; Male; Mice; Mice, Transgenic; Ornithine Carbamoyltransferase; Ornithine Carbamoyltransferase Deficiency Disease; RNA, Messenger; Regulatory Sequences, Nucleic Acid
PubMed: 15336649
DOI: 10.1016/j.ymthe.2004.05.036 -
JIMD Reports Jan 2021The combination of neonatal hyperammonemia, lactic acidosis, ketonuria, and hypoglycemia is pathognomonic for carbonic anhydrase VA (CA-VA) deficiency. We present two...
Two cases of carbonic anhydrase VA deficiency-An ultrarare metabolic decompensation syndrome presenting with hyperammonemia, lactic acidosis, ketonuria, and good clinical outcome.
The combination of neonatal hyperammonemia, lactic acidosis, ketonuria, and hypoglycemia is pathognomonic for carbonic anhydrase VA (CA-VA) deficiency. We present two cases of this rare inborn error of metabolism. Both newborns with South Asian ancestry presented with a metabolic decompensation characterized by hyperammonemia, lactic acidosis and ketonuria; one also had hypoglycemia. Standard metabolic investigations (plasma amino acids, acylcarnitine profile, and urine organic acids) were not indicative of a specific organic aciduria or fatty acid oxidation defect but had some overlapping features with a urea cycle disorder (elevated glutamine, orotic acid, and low arginine). Hyperammonemia was treated initially with nitrogen scavenger therapy and carglumic acid. One patient required hemodialysis. Both have had a favorable long-term prognosis after their initial metabolic decompensation. Genetic testing confirmed the diagnosis of carbonic anhydrase VA (CA-VA) deficiency due to biallelic pathogenic variants in . These cases are in line with 15 cases previously described in the literature, making the phenotypic presentation pathognomonic for this ultrarare (potentially underdiagnosed) inborn error of metabolism with a good prognosis.
PubMed: 33473334
DOI: 10.1002/jmd2.12171 -
Molecular Therapy : the Journal of the... May 2011Urea cycle defects presenting early in life with hyperammonemia remain difficult to treat and commonly necessitate liver transplantation. Gene therapy has the potential...
Urea cycle defects presenting early in life with hyperammonemia remain difficult to treat and commonly necessitate liver transplantation. Gene therapy has the potential to prevent hyperammonemic episodes while awaiting liver transplantation, and possibly also to avert the need for transplantation altogether. Ornithine transcarbamylase (OTC) deficiency, the most prevalent urea cycle disorder, provides an ideal model for the development of liver-targeted gene therapy. While we and others have successfully cured the spf(ash) mouse model of OTC deficiency using adeno-associated virus (AAV) vectors, a major limitation of this model is the presence of residual OTC enzymatic activity which confers a mild phenotype without clinically significant hyperammonemia. To better model severe disease we devised a strategy involving AAV2/8-mediated delivery of a short hairpin RNA (shRNA) to specifically knockdown residual endogenous OTC messenger RNA (mRNA). This strategy proved highly successful with vector-treated mice developing severe hyperammonemia and associated neurological impairment. Using this system, we showed that the dose of an AAV rescue construct encoding the murine OTC (mOTC) cDNA required to prevent hyperammonemia is fivefold lower than that required to control orotic aciduria. This result is favorable for clinical translation as it indicates that the threshold for therapeutic benefit is likely to be lower than indicated by earlier studies.
Topics: 3' Untranslated Regions; Animals; Dependovirus; Disease Models, Animal; Gene Knockdown Techniques; Gene Transfer Techniques; Genetic Therapy; Genetic Vectors; HEK293 Cells; Humans; Hyperammonemia; Liver; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Transgenic; Ornithine Carbamoyltransferase; Ornithine Carbamoyltransferase Deficiency Disease; Orotic Acid; RNA, Messenger; RNA, Small Interfering
PubMed: 21386824
DOI: 10.1038/mt.2011.32