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Orotic aciduria is a severe, hereditary, life-threatening condition, particularly in newborns. An increased orotic acid (OA) content in urine may be a strong indicator of this condition. In this study, we developed a rapid, simple, highly sensitive diagnostic method for use in monitoring the OA levels in urine samples, which were successfully determined using capillary electrophoresis combined with capacitively coupled contactless conductivity detection (CE-C 4 D). A straightforward analysis with an increased sensitivity towards OA and an analysis time of approximately 5 min were realized, and the limit of detection of the developed method was 0.014 mg/L in aqueous solution. The optimized composition of the separation electrolyte was 20 mM 2-(N-morpholino)ethanesulfonic acid/histidine with 0.1 mM cetyltrimethylammonium bromide at a pH of 6.5. The sensitivity of the developed method was significantly increased using a sample stacking approach with a 10% acetonitrile (v/v) plug solution. The method was validated, and satisfactory recoveries of 80.0-92.3% were obtained. The amounts of OA in five urine samples were successfully determined., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Ornithine transcarbamylase deficiency (OTCD) is a monogenic disease of ammonia metabolism in hepatocytes. Severe disease is frequently treated by orthotopic liver transplantation. An attractive approach is the correction of a patient's own cells to regenerate the liver with gene-repaired hepatocytes. This study investigates the efficacy and safety of ex vivo correction of primary human hepatocytes. Hepatocytes isolated from an OTCD patient were genetically corrected ex vivo, through the deletion of a mutant intronic splicing site achieving editing efficiencies >60% and the restoration of the urea cycle in vitro. The corrected hepatocytes were transplanted into the liver of FRGN mice and repopulated to high levels (>80%). Animals transplanted and liver repopulated with genetically edited patient hepatocytes displayed normal ammonia, enhanced clearance of an ammonia challenge and OTC enzyme activity, as well as lower urinary orotic acid when compared to mice repopulated with unedited patient hepatocytes. Gene expression was shown to be similar between mice transplanted with unedited or edited patient hepatocytes. Finally, a genome-wide screening by performing CIRCLE-seq and deep sequencing of >70 potential off-targets revealed no unspecific editing. Overall analysis of disease phenotype, gene expression, and possible off-target editing indicated that the gene editing of a severe genetic liver disease was safe and effective., Competing Interests: Declaration of interests S.C.S. has stock in Yecuris (no Yecuris animals were used in these studies)., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Ornithine transcarbamylase deficiency (OTCD) is a metabolic and genetic disease caused by dysfunction of the hepatocytic urea cycle. To develop new drugs or therapies for OTCD, it is ideal to use models that are more closely related to human metabolism and pathology. Primary human hepatocytes (HHs) isolated from two patients (a 6-month-old boy and a 5-year-old girl) and a healthy donor were transplanted into host mice (hemi-, hetero-OTCD mice, and control mice, respectively). HHs were isolated from these mice and used for serial transplantation into the next host mouse or for in vitro experiments. Histological, biochemical, and enzyme activity analyses were performed. Cultured HHs were treated with ammonium chloride or therapeutic drugs. Replacement rates exceeded 80% after serial transplantation in both OTCD mice. These highly humanized OTCD mice showed characteristics similar to OTCD patients that included increased blood ammonia levels and urine orotic acid levels enhanced by allopurinol. Hemi-OTCD mice showed defects in OTC expression and significantly low enzymatic activities, while hetero-OTCD mice showed residual OTC expression and activities. A reduction in ammonium metabolism was observed in cultured HHs from OTCD mice, and treatment with the therapeutic drug reduced the ammonia levels in the culture medium. In conclusion, we established in vivo OTC mouse models with hemi- and hetero-patient HHs. HHs isolated from the mice were useful as an in vitro model of OTCD. These OTC models could be a source of valuable patient-derived hepatocytes that would enable large scale and reproducible experiments using the same donor., (© 2020 The Authors. Journal of Inherited Metabolic Disease published by John Wiley & Sons Ltd on behalf of SSIEM.)

Background: Aberrant fetal programming in gestational diabetes mellitus seems to increase the risk of obesity, type 2 diabetes, and cardiovascular disease. The inability to accurately identify gestational diabetes mellitus in the first trimester of pregnancy has thwarted ascertaining whether early therapeutic interventions reduce the predisposition to these prevalent medical disorders., Objective: A metabolomics study was conducted to determine whether advanced analytical methods could identify accurate predictors of gestational diabetes mellitus in early pregnancy., Study Design: This nested observational case-control study was composed of 92 gravidas (46 in the gestational diabetes mellitus group and 46 in the control group) in early pregnancy, who were matched by maternal age, body mass index, and gestational age at urine collection. Gestational diabetes mellitus was diagnosed according to community standards. A comprehensive metabolomics platform measured 626 endogenous metabolites in randomly collected urine. Consensus multivariate criteria or the most important by 1 method identified low-molecular weight metabolites independently associated with gestational diabetes mellitus, and a classification tree selected a subset most predictive of gestational diabetes mellitus., Results: Urine for both groups was collected at a mean gestational age of 12 weeks (range, 6-19 weeks' gestation). Consensus multivariate analysis identified 11 metabolites independently linked to gestational diabetes mellitus. Classification tree analysis selected a 7-metabolite subset that predicted gestational diabetes mellitus with an accuracy of 96.7%, independent of maternal age, body mass index, and time of urine collection., Conclusion: Validation of this high-accuracy model by a larger study is now needed to support future studies to determine whether therapeutic interventions in the first trimester of pregnancy for gestational diabetes mellitus reduce short- and long-term morbidity., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Excess urinary orotic acid excretion occurs in patients with some inborn errors of metabolic pathways such as pyrimidine synthesis and urea cycle. Thus, rapid diagnosis of orotic aciduria has a vital importance for patients. In this paper, a novel method for determination of orotic acid in urine samples by ion chromatography with suppressed conductivity detection was investigated. The separation of orotic acid from urine matrix was accomplished by using an anion exchange column with optimized isocratic eluent program which utilized 50 mM NaOH. The other chromatographic conditions were as follows: the suppressor current was 31 mA; the flow rate of mobile phase was 0.25 mL min -1 ; the column temperature was 30 °C; sample loop volume was 10 μL. Under optimized conditions, the limit of detection was 0.2 μmol L‾ 1 . Dramatically elevated orotic acid concentration was observed on pathological urine samples comparing to healthy urines, as expected. There are a good many advantages of the proposed method, but using an environmentally friendly reagent free system, no organic solvent employment and its quick nature, and being a sensitive and reliable method are the most obvious ones. The proposed method, therefore, may be utilized as an alternative technique for clinical laboratories., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objective  Orotic aciduria and deficiency of uridine monophosphate synthetase have been observed in a patient, studied over 10 years, who had no megaloblastic anemia. Excretion of orotic acid and orotidine were 8.24 and 0.52 mmol/mol of creatinine. The ratio of 15.85 differed appreciably from that of 6 patients reported with no megaloblastic anemia. Methods  The analysis of orotidine by gas chromotography mass spectrometry was conducted. Conclusion  Patients with orotic aciduria with and without megaloblastic anemia cannot be distinguished by ratio of orotic acid to orotidine., (Georg Thieme Verlag KG Stuttgart · New York.)

Background: Miller syndrome (post-axial acrofacial dysostosis) arises from gene mutations for the mitochondrial enzyme dihydroorotate dehydrogenase (DHODH). Nonetheless, despite demonstrated loss of enzyme activity dihydroorotate (DHO) has not been shown to accumulate, but paradoxically urine orotate has been reported to be raised, confusing the metabolic diagnosis., Methods: We analysed plasma and urine from a 4-year-old male Miller syndrome patient. DHODH mutations were determined by PCR and Sanger sequencing. Analysis of DHO and orotic acid (OA) in urine, plasma and blood-spot cards was performed using liquid chromatography-tandem mass spectrometry. In vitro stability of DHO in distilled water and control urine was assessed for up to 60h. The patient received a 3-month trial of oral uridine for behavioural problems., Results: The patient had early liver complications that are atypical of Miller syndrome. DHODH genotyping demonstrated compound-heterozygosity for frameshift and missense mutations. DHO was grossly raised in urine and plasma, and was detectable in dried spots of blood and plasma. OA was raised in urine but undetectable in plasma. DHO did not spontaneously degrade to OA. Uridine therapy did not appear to resolve behavioural problems during treatment, but it lowered plasma DHO., Conclusion: This case with grossly raised plasma DHO represents the first biochemical confirmation of functional DHODH deficiency. DHO was also easily detectable in dried plasma and blood spots. We concluded that DHO oxidation to OA must occur enzymatically during renal secretion. This case resolved the biochemical conundrum in previous reports of Miller syndrome patients, and opened the possibility of rapid biochemical screening., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Creatine synthesis and transport disorders, Triple H syndrome and ornithine transcarbamylase deficiency are treatable inborn errors of metabolism. Early screening of patients was found to be beneficial. Mass spectrometry analysis of specific urinary biomarkers might lead to early detection and treatment in the neonatal period. We developed a high-throughput mass spectrometry methodology applicable to newborn screening using dried urine on filter paper for these aforementioned diseases., Methods: A high-throughput methodology was devised for the simultaneous analysis of creatine, guanidineacetic acid, orotic acid, uracil, creatinine and respective internal standards, using both positive and negative electrospray ionization modes, depending on the compound., Results: The precision and accuracy varied by <15%. Stability during storage at different temperatures was confirmed for three weeks. The limits of detection and quantification for each biomarker varied from 0.3 to 6.3 μmol/l and from 1.0 to 20.9 μmol/l, respectively. Analyses of urine specimens from affected patients revealed abnormal results. Targeted biomarkers in urine were detected in the first weeks of life., Conclusions: This rapid, simple and robust liquid chromatography/tandem mass spectrometry methodology is an efficient tool applicable to urine screening for inherited disorders by biochemical laboratories., (Copyright © 2014 Elsevier B.V. All rights reserved.)

An orthogonal signal correction-partial least squares (OSC-PLS) method was developed for the simultaneous spectrophotometric determination of orotic acid (OA), creatinine (CRE), and uric acid (UA) in spiked real samples. By multivariate calibration methods, such as PLS regression, it is possible to obtain a model adjusted to the concentration values of the mixtures used in the calibration range. The effect of OSC used to remove the information unrelated to the target variables is studied. In this study, the calibration model is based on absorption spectra in the 220-320 nm rang for 36 different mixtures of OA, CRE and UA. Calibration matrices contained 1.74-47.00 of OA, 1.13-33.95 of CRE, and 1.68-28.58 of UA in µg/ml. The number of principal component for OA, CRE, and UA with OSC were 3, 4, and 4, and 4, 6, and 5, without OSC, respectively. The evaluation of the prediction errors for the prediction set reveals that the OSC-treated data give substantially lower root mean square error of prediction (RMSEP) values than the original data. The RMSEP for OA, CRE, and UA with OSC were 0.69, 0.20, and 0.53 and 0.80, 0.69, and 0.73 without OSC, respectively. The proposed method was applied for the simultaneous determination of OA, CRE, and UA in spiked biological fluids with satisfactory results., (Copyright © 2012 John Wiley & Sons, Ltd.)

Biallelic mutations in the gene encoding DHOdehase [dihydroorotate dehydrogenase (DHODH)], an enzyme required for de novo pyrimidine biosynthesis, have been identified as the cause of Miller (Genée-Weidemann or postaxial acrofacial dysostosis) syndrome (MIM 263750). We report compound heterozygous DHODH mutations in four additional families with typical Miller syndrome. Complementation in auxotrophic yeast demonstrated reduced pyrimidine synthesis and in vitro enzymatic analysis confirmed reduced DHOdehase activity in 11 disease-associated missense mutations, with 7 alleles showing discrepant activity between the assays. These discrepancies are partly explained by the domain structure of DHODH and suggest both assays are useful for interpretation of individual alleles. However, in all affected individuals, the genotype predicts that there should be significant residual DHOdehase activity. Urine samples obtained from two mutation-positive cases showed elevated levels of orotic acid (OA) but not dihydroorotate (DHO), an unexpected finding since these represent the product and the substrate of DHODH enzymatic activity, respectively. Screening of four unrelated cases with overlapping but atypical clinical features showed no mutations in either DHODH or the other de novo pyrimidine biosynthesis genes (CAD, UMPS), with these cases also showing normal levels of urinary OA and DHO. In situ analysis of mouse embryos showed Dhodh, Cad and Umps to be strongly expressed in the pharyngeal arch and limb bud, supporting a site- and stage-specific requirement for de novo pyrimidine synthesis. The developmental sensitivity to reduced pyrimidine synthesis capacity may reflect the requirement for an exceptional mitogenic response to growth factor signalling in the affected tissues.

Ornithine transcarbamylase deficiency (OTCD) is the most common inborn error of urea synthesis. Complete OTCD can result in hyperammonemic coma in the neonatal period, which can rapidly become fatal. Current acute therapy involves dialysis; chronic therapy involves the stimulation of alternate nitrogen clearance pathways; and the only curative approach is liver transplantation. Adeno-associated virus (AAV) vector-based gene therapy would add to current treatment options provided the vector delivers high level and stable transgene expression in liver without dose-limiting toxicity. In this study, we employed an AAV2/8-based self-complementary (sc) vector expressing the murine OTC (mOTC) gene under a liver-specific thyroxine-binding globulin promoter and examined the therapeutic effects in a mouse model of OTCD, the spf (ash) mouse. Seven days after a single intravenous injection of vector, treated mice showed complete normalization of urinary orotic acid, a measure of OTC activity. We further improved vector efficacy by incorporating a Kozak or Kozak-like sequence into mOTC complementary DNA, which increased the OTC activity by five or twofold and achieved sustained correction of orotic aciduria for up to 7 months. Our results demonstrate that vector optimizations can significantly improve the efficacy of gene therapy.

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., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Orotic acid (OA), a marker of hereditary orotic aciduria, is usually used for the differential diagnosis of some hyperammonemic inherited defects of urea cycle and of basic amino acid transporters. This study was aimed to establish age related reference intervals of OA in urine, and for the first time in plasma, and dried blood spot (DBS) from 229 apparently healthy subjects aged from three days to 40 years. The quantification of OA was performed by a previously implemented method, using a stable isotope dilution with 1,3-[(15)N(2)]-orotic acid and hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS). The method has proved to be sensitive and accurate for a quantitative analysis of OA also in DBS and plasma. According to previous studies, urinary OA levels (mmol/mol of creatinine) decrease significantly with age. The upper limits (as 99th %ile) were of 3.44 and 1.30 in groups aged from three days to 1 year (group 1) and from 1 year to 12 years (group 2), respectively; in teenagers (from 13 to 19 years; group 3) and adults (from 20 to 40 years; group 4) urinary levels became more stable and the upper limits were of 0.64 and 1.21, respectively. Furthermore, OA levels in DBS (μM) also resulted significantly higher in subjects of group 1 (upper limit of 0.89) than in subjects of groups 2, 3 and 4 (upper limits of 0.24, 0.21, and 0.29, respectively). OA levels in plasma (μM) were significantly lower in subjects of group 3 (upper limit of 0.30) than in subjects of groups 1, 2, and 4 (upper limits of 0.59, 0.48, and 0.77, respectively). This method was also employed for OA quantification in plasma and DBS of 17 newborns affected by urea cycle defects, resulting sensitive and specific enough to screen these disorders., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Enzyme defects of the urea cycle typically present with significant hyperammonemia and its associated toxicity, in the first few months of life. However, arginase I (ARG1) deficiency, a rare autosomal recessive disorder, has classically been the exception. ARG1 deficiency usually presents later in life with spasticity, seizures, failure to thrive and developmental regression. Neonatal and early infantile presentation of ARG1 deficiency with severe hyperammonemia remains rare and only six such cases have been described. We report a severely affected infant with ARG1 deficiency who presented at 6 weeks of age with lethargy, poor feeding and severe encephalopathy caused by hyperammonemia. The clinical and biochemical features of the proband and six other previously reported cases with neonatal or infantile-onset presentation of ARG1 deficiency with hyperammonemia are reviewed. In addition, the clinical spectrum of seven previously unpublished patients with later onset ARG1 deficiency, who also experienced recurrent hyperammonemia, is presented. Several biochemical abnormalities have been postulated to play a role in the pathogenesis of the neurological changes in ARG1 deficiency including hyperargininemia, elevated guanidino compounds and elevated glutamine levels, as well as the hyperammonemia. The index case demonstrated many of these. The cases reviewed here suggest a genotype/phenotype correlation and advocate for the addition of arginine as a primary target in newborn screening programs., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The measurement of urinary orotic acid excretion is an important test for establishing a diagnosis of hereditary orotic aciduria, a genetic defect of pyrimidine biosynthesis. Measurement of secondary urinary orotic acid elevation is also an important clinical test for the differential diagnosis of hyperammonemia due to some of the primary disorders of the urea cycle including ornithine transcarbamylase (OTC) deficiency, and the hyperornithinemia-hyperammonemia-homocitrullinemia (HHH) syndrome. Low levels of orotic acid are observed in carbamylphosphate synthetase (CPS) defects. This method utilizes a stable-isotope labeled internal standard (1, 3-(15)N-orotic acid), which is added to the standards, controls, and patient samples prior to extraction. Interference from urea is removed by incubation of samples with urease and the orotic acid is derivatized by trimethylsilylation. Quantitation is made against an eight-point standard curve using specific selected ions from both the labeled and unlabeled orotic acid.

Three subtypes of hereditary orotic aciduria are described in the literature, all related to deficiencies in uridine monophosphate synthase, the multifunctional enzyme that contains both orotate: pyrophosphoryl transferase and orotidine monophosphate decarboxylase activities. The type of enzyme defect present in the subtypes has been re-examined by steady-state modelling of the relative outputs of the three enzymic products, uridine monophosphate, urinary orotic acid and urinary orotidine. It is shown that the ratio of urinary outputs of orotidine to orotate provides a means of testing for particular forms of enzyme defect. It is confirmed that the type I defect is caused by loss of uridine monophosphate synthase activity. Cells and tissue of type I cases have a residual amount of activity that is qualitatively unchanged: the relative rates of the transferase and decarboxylase do not differ from those of wild-type enzyme. The single claimed case of type II, thought to be due to specific inactivation of orotidine monophosphate decarboxylase, is shown to have a product spectrum inconsistent with that claim. It is proposed that this type II form does not differ sufficiently to be accepted as separate from type I. The third subtype, hereditary orotic aciduria without megaloblastic anaemia, occurs in two cases. It has the product spectrum expected of a defect in orotidine monophosphate decarboxylase. This form is the only one that appears to have a qualitatively different uridine monophosphate synthase. The possibility that orotidine monophosphate may control flux through the pyrimidine biosynthesis pathway in hereditary orotic aciduria is discussed.

Ornithine transcarbamylase (OTC) deficiency, the most common urea cycle disorder, is associated with severe hyperammonemia accompanied by a high risk of neurological damage and death in patients presenting with the neonatal-onset form. Contemporary therapies, including liver transplantation, remain inadequate with considerable morbidity, justifying vigorous investigation of alternate therapies. Clinical evidence suggests that as little as 3% normal enzyme activity is sufficient to ameliorate the severe neonatal phenotype, making OTC deficiency an ideal model for the development of liver-targeted gene therapy. In this study, we investigated metabolic correction in neonatal and adult male OTC-deficient Spf(ash) mice following adeno-associated virus (AAV)2/8-mediated delivery of the murine OTC complementary DNA under the transcriptional control of a liver-specific promoter. Substantially supraphysiological levels of OTC enzymatic activity were readily achieved in both adult and neonatal mice following a single intraperitoneal (i.p.) injection, with metabolic correction in adults being robust and life-long. In the neonates, however, full metabolic correction was transient, although modest levels of OTC expression persisted into adulthood. Although not directly testable in Spf(ash) mice, these levels were theoretically sufficient to prevent hyperammonemia in a null phenotype. This loss of expression in the neonatal liver is the consequence of hepatocellular proliferation and presents an added challenge to human therapy.

Mutant proteins have the potential to exert dominant-negative effects that might limit the therapeutic efficacy of their wild-type counterparts after gene transfer. For ornithine transcarbamylase (OTC) deficiency, in vitro studies have suggested the presence of dominant-negative effects, however, supporting in vivo studies have not been conducted. In this study, we exploited the capacity of recombinant adeno-associated virus (rAAV) 2/8 vectors to deliver transgenes to the mouse liver with high efficiency to determine whether expression of selected OTC mutant proteins exert inhibitory effects on endogenous wild-type OTC enzymatic activity. Using site-directed mutagenesis we constructed three OTC mutants with a theoretical or reported in vitro capacity to exert dominant-negative effects, and delivered these to the liver using rAAV2/8. Each mutation had been earlier identified in patients with OTC deficiency. Treated mice showed no increase in urinary orotic acid levels or reduction in OTC activity despite supra-physiological expression of the mutant proteins, consistent with an absence of dominant-negative effects. These data have important implications for the development of gene therapy strategies for OTC deficiency and validate a model system in which potential dominant-negative effects of specific mutations in prospective patients can be examined empirically before gene therapy.

Modern instrumentation allows the measurement of organic acids in urine in their physiological concentration ranges. Eight of the compounds that are reported can serve as markers for specific toxicant exposure or detoxification challenges. Xylene exposure causes elevation of 2-methylhippurate, and orotic acid elevation reveals ammonia challenge that exceeds the capacity of the urea cycle. General hepatic detoxification stimulation by natural compounds, drugs, or xenobiotic compounds causes elevated levels of glucaric acid. Abnormalities of alpha-hydroxybutyrate, pyroglutamate, and sulfate can indicate up-regulated glutathione biosynthesis, impaired reformation of glutathione in the gamma-glutamyl cycle, and depleted total body glutathione status, respectively. Patterns of these compounds measured in a simple overnight urine specimen help to identify focal areas of clinical concern and monitor patient responses to detoxification interventions.

Background: Helper-dependent adenoviral vectors (HDAd) can mediate long-term phenotypic correction in the ornithine transacarbamylase (OTC)-deficient mice model with negligible chronic toxicity. However, the high doses required for metabolic correction will result in systemic inflammatory response syndrome in humans. This acute toxicity represents the major obstacle for clinical applications of HDAd vectors for the treatment of OTC deficiency. Strategies for reducing the dose necessary for disease correction are highly desirable because HDAd acute toxicity is clearly dose-dependent., Methods: We analysed a potent expression cassette and the hydrodynamic injection for the ability to reduce the HDAd dose necessary for phenotypic correction in OTC-deficient spf-ash mice., Results: We have developed a vector containing a potent expression cassette expressing the OTC transgene, which allowed phenotypic correction at lower doses. Our results suggest that vector expressing greater OTC levels allows correction of orotic acid overproduction at lower doses that make clinical translation more relevant. We were able to further reduce the minimal effective dose by delivering the vector through the hydrodynamic injection technique., Conclusions: Vectors containing the expression cassette used in the present study, combined with other strategies for improving HDAd therapeutic index, will likely permit application of these vectors for the treatment of OTC deficiency as well as other urea cycle disorders., ((c) 2008 John Wiley & Sons, Ltd.)

A young patient with hepatocellular carcinoma receiving chemotherapy presented with encephalopathy. Evaluation of the patient revealed a metabolic profile consistent with ornithine transcarbamoylase (OTC) deficiency, an inherited disorder of the urea cycle. The evaluation yielded a plasma amino acid analysis consistent with OTC deficiency. However, genetic analysis did not reveal a somatic mutation of the OTC gene in this patient. The hyperammonemic encephalopathy was reversed by the infusion of arginine, a common treatment for hereditary OTC deficiency. This case may represent a distinct syndrome of reversible hyperammonemia in patients with hepatocellular carcinoma.

High-resolution (1)H NMR spectroscopy of body fluids has proved to be very useful in diagnostics of inherited metabolic diseases, whereas (13)C NMR remains almost unexploited. In this paper the application of (13)C NMR spectroscopy of fivefold concentrated urine samples for diagnosis of selected metabolic diseases is reported. Various marker metabolites were identified in test urine samples from 33 patients suffering from 10 different diseases, providing information which could be crucial for their diagnoses. Spectra were accumulated for 2 h or overnight when using spectrometers operating at 9.4 or 4.7 T magnetic fields, respectively. Interpretation of the measurement results was based on a comparison of the peak positions in the measured spectrum with reference data. The paper contains a table with (13)C NMR chemical shifts of 73 standard compounds. The method can be applied individually or as an auxiliary technique to (1)H NMR or any other analytical method.

The urinary excretion of orotic acid, an intermediate in the pyrimidine biosynthetic pathway, is markedly increased in many inborn errors of the urea cycle and in a number of other disorders involving arginine metabolism. Carbamoyl phosphate, which accumulates within hepatic mitochondria in patients with ornithine transcarbamoylase deficiency, can diffuse to the cytosol and enter the pyrimidine pathway, resulting in greatly increased orotic acid production and excretion. This orotic aciduria also occurs in inborn errors of the mitochondrial ornithine/citrulline transporter, arginase, argininosuccinate synthetase, and argininosuccinate lyase. Increased orotic acid excretion is also found in a number of hypoargininemic states, such as lysinuric protein intolerance. However, orotic aciduria should not be used uncritically as an index of arginine deficiency because it is found in patients with arginase deficiency who exhibit hyperargininemia. Increased orotic acid excretion can also arise as a result of impairments of pyrimidine synthesis, whether brought about by a genetic defect (e.g., in UMP synthase) or by drugs that inhibit the terminal part of the pathway (e.g., allopurinol or 6-azauridine). When used appropriately, measurement of urinary orotic acid is a valuable tool for the study of many derangements of arginine metabolism, including arginine depletion, and to assess the efficacy of therapies used to replete this amino acid.

A 4-months-old calf of Japanese black cattle was diagnosed with orotic aciduria by gas-chromatography/mass-spectrometry (GC/MS). Until now orotic aciduria had not been reported in Japanese black cattle. The animal showed repeated diarrhea. The hematocrit was low, and microcytes and acanthocytes were observed in blood smears. The calf had lower serum total protein concentrations with a higher blood ammonia concentration. Needle-shaped crystals of orotic acid were observed in urinary sediments. Sequence homologous analysis with cattle uridine monophosphate synthase DNA indicated silent mutation in the affected calf.

Background: 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., Results: 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., Conclusion: Arginine treatment is able to reduces attacks of hyperammonemia in boys with late-onset OTCD and to increase their growth.

The purpose of this study was to determine the efficacy of novel recombinant adeno-associated viral (AAV) vector constructs in correcting metabolic defects in the liver in two strains of ornithine transcarbamylase (OTC)-deficient mice (spf and spf-ash). AAV vectors expressing mouse OTC were produced with capsids from AAV2 and the novel serotypes AAV7, 8, and 9. OTC-deficient mice were infused with these vectors as well as a control AAV2/8 vector expressing LacZ. In vivo activity of OTC was assessed by measuring a surrogate marker, urine orotate. The novel vectors restored orotate levels to virtually normal 15 days after infusion, and each persisted to 1 year posttreatment. Liver OTC enzyme activity in spf mice was substantially higher in animals receiving novel vectors compared to those receiving AAV2 vectors. Animals receiving novel OTC-expressing vectors lived longer than those treated with AAV2 OTC or untreated controls, and they were tolerant to a challenge with NH3 at 21 days and beyond, which caused severe morbidity in control OTC-deficient animals. Numerous mice, representative of all treatment groups followed for +250 days, were observed to have either nodules or discrete tumors in the liver, the etiology of which is the subject of a companion paper.

Lysinuric protein intolerance (LPI) is an autosomal recessive transport disorder of the dibasic amino acids. The defect leads to deficiency of lysine, arginine, and ornithine and, secondarily, to a functional disorder of the urea cycle. Transient postprandial hyperammonemia and subsequent persistent protein aversion, linked with several other biochemical and clinical characteristics of the disease, suggest an increased risk for maternal and fetal complications during pregnancy and delivery. Our unique material on the outcomes of 18 pregnancies of 9 Finnish mothers with LPI and the follow-up of their 19 children shows that maternal LPI is truly associated with increased risk of anemia, toxemia, and intrauterine growth retardation during pregnancy and bleeding complications during delivery. Successful pregnancies and deliveries can still be achieved with careful follow-up of blood pressure and laboratory values. The children of the mothers with LPI generally develop normally. Special care of maternal protein nutrition and control of ammonemia, anemia, and toxemia during pregnancy are essential. We propose centralization of deliveries to obstetric units with capability to deal with bleeding complications and rare inborn errors of metabolism.

We report a male patient with a history of recurrent idiopathic vomiting, normal plasma ammonia and glutamine concentrations in acute phase, who died at 3 years of age. Ornithine transcarbamylase deficiency was diagnosed after detecting elevated urinary orotate concentrations in a sample collected just before death, and the diagnosis was confirmed by DNA analysis.

Background: Female patients with ornithine transcarbamylase deficiency (OTCD) show a wide range of clinical severity, from asymptomatic to lethal hyperammonemia. It is important to establish a simple method to distinguish symptomatic from asymptomatic patients., Methods: Uracil and orotic acid concentrations were analyzed in three female patients with OTCD at both the hyperammonemia-attack and interval stages. These concentrations were compared with those in asymptomatic female patients reported previously., Results: Uracil concentrations in symptomatic female patients were uniformly higher than those in asymptomatic female patients at both the hyperammonemia-attack and interval stages., Conclusion: Uracil may present a useful index for detecting OTCD female patients who are destined to suffer from hyperammonemia attack. Further data on uracil concentrations are necessary to establish the threshold for distinguishing symptomatic from asymptomatic subjects.

Background: The small bowel is believed to play a crucial role in endogenous arginine synthesis. Therefore, an insufficient arginine supply in the situation of massive intestinal resection might impede normal arginine metabolism. This study sought to determine the clinical and metabolic effects of an arginine-free diet in stable short-bowel patients., Methods: Four patients, mean age 49 years (range: 26-67), mean time from intestinal resection 46 m (range: 15-97), and remnant small bowel of 30 to 100 cm consumed an L-amino acid arginine-free diet (egg pattern) for 5 days (0.9 g protein equivalent/kg/d plus malabsorption adjustments). Fasting plasma amino acids, ammonium, and blood chemistries were assessed at days 0, 3, and 5. Urinary orotate, orotidine, uric acid, urea, creatinine, and total nitrogen were evaluated daily., Results: Significant decreases in plasma levels of arginine, ornithine, and hydroxyproline occurred at day 5. A decreasing trend in plasma citrulline and a significant plasma glutamine increase were also observed in the same period. Conversely, ammonium concentrations remained normal. Regarding urine compounds, striking orotic aciduria with a peak at day 4 (14-fold vs baseline) and significant decreases in uric acid and urea excretion were found. There were no relevant clinical events., Conclusions: Despite the limited number of patients in our work and their relative heterogeneity, our results support the idea of the indispensability of an exogenous arginine supply in humans under short bowel syndrome conditions. Studies in larger series are needed to further investigate these findings.

An 18 month-old, intact female American Shorthair cat was presented for evaluation of stunted growth and postprandial depression. Fasting serum ammonia and serum bile acid concentrations were above reference ranges at 396 microg/dl and 6.5 micromol/ l and their postprandial concentrations were 785 microg/dl and 9.5 micromol/l, respectively. The initial tentative diagnosis of a portosystemic shunt was excluded by mesenteric portography and histopathology of the liver. The cat was then suspected of a urea cycle enzyme deficiency and its urine was analyzed by gas chromatography-mass spectrometry. A presumptive diagnosis of ornithine transcarbamylase deficiency was made on the basis of the detection of orotic acid and uracil.

We have studied the influence of dietary arginine on tissue arginine content, and arginine metabolism in CD1 mice. Dietary arginine restriction produced by feeding mice with a low arginine diet (0.06%) produced a marked decrease in arginine concentrations in the plasma, skeletal muscle and kidney of female mice (72%, 67% and 54%, respectively) while in male mice the decreases were smaller (58% in blood and 18% in the skeletal muscle). This diet abolished not only the sexual dimorphism in arginine content observed in mice fed with the diet containing 1% arginine, but also reduced renal activities of arginase and nitric oxide synthase in the female mice and ornithine decarboxylase and the decarboxylation of arginine in the male mice. Urinary putrescine excretion was dramatically reduced by arginine restriction in the male mice whereas orotic acid excretion increased about 30 fold in both sexes; urea and creatinine excretion did not change. Taken together our results indicate that dietary arginine plays a relevant role in the maintenance of the sexual dimorphism in arginine content and arginine metabolism in CD1 mice, and that this may have physiological significance because of the important effects that arginine-derived products exert on a variety of cellular processes.

Analytical potentialities of capillary zone electrophoresis in the separation system with tandem-coupled columns to the spectral identification and determination of orotic acid (OA) in urine by diode array detection (DAD), coupled to the separation system via optical fibers, were investigated. A very significant "in-column" clean-up of OA from urine matrix was reached in the separation stage of the tandem by combining a low pH (2.8) with complexing effects of electroneutral agents [alpha- and beta-cyclodextrins, poly(vinylpyrrolidone) and 3-(N,N-dimethyldodecylammonio)propanesulfonate]. Due to this, its DAD spectral data could be acquired in the detection stage of the tandem with almost no disturbances by matrix co-migrants. The concentration limits of detection obtained under such working conditions for a 200-nl sample load of OA and 320 microm I.D. capillary tubes were 3.5 micromol/l (218 nm) and 0.4 micromol/l (280 nm). Using chemometry procedures (target transformation factor analysis, fixed size moving window-evolving factor analysis, orthogonal projection approach and fixed size moving window-target transformation factor analysis) in processing of the acquired spectral data, the presence of OA in the loaded urine matrix could be confirmed with confidence when its concentration was 10 micromol/l or slightly less.

Background and Aims: The small intestine contains several enzymes involved in arginine synthesis and converts glutamine to citrulline, the major compound for endogenous arginine synthesis. This study was conducted to assess the plasma status of urea-cycle intermediates and orotic urinary excretion in short-bowel patients., Methods: Thirteen stable short-bowel syndrome patients (7 men; 60.2+/-15.2 years) were studied. Patients were divided into moderately resected (Group A; n=6) and severely resected (Group B; n=7) according to their remnant bowel length (Group A: 61-150 cm; Group B: < or =60 cm). All subjects were consuming an oral diet plus dietetic supplements. Plasma urea-cycle amino acids, ammonium and urinary orotic acid were determined., Results: Plasma glutamine levels were significantly higher in both patient groups than in the control group (P<0.001). Regarding citrulline, Group B levels were significantly lower vs. controls (P<0.001). Comparisons between patient groups showed higher arginine in Group A (P<0.05) and non-statistically lower citrulline in Group B. Blood ammonium and orotic urinary excretion were normal., Conclusions: Although plasma citrulline and glutamine alterations were found, patients showed no hyperammonemia or orotic aciduria, which suggests a certain degree of adaptation in arginine and related amino acid metabolism, when an adequate dietary supply of arginine is provided.

Quantification of orotic acid (uracil-6-carboxylic acid) in urine is an important tool to diagnose some inherited diseases, such as urea cycle disorder (OTCD) and hereditary orotic aciduria. New rapid analytical methods are necessary to provide high-throughput orotic acid analyses. A new analytical method has been developed for the rapid analysis of orotic acid in urine by liquid chromatography coupled with ion spray tandem mass spectrometry (LC/MS/MS). After a sample dilution 1:20, the analysis was performed in the selected reaction monitoring mode in which orotic acid was detected through the transition m/z 155 to 111. The retention time was 3.9 min in a 4.5-min analysis. Daily calibration between 0.5-5.0 micromol/L of orotic acid, corresponding to 10-100 micromol/L in urine before the 1:20 dilution, offered consistent linearity and reproducibility. Interassay coefficient of variance (c.v.) was 4.97% at a mean concentration of 10.99 micromol/L. The sensitivity and specificity of tandem mass spectrometry permitted a high volume of analyses of orotic acid. The sample preparation is simple, inexpensive and not time demanding., (Copyright 2003 John Wiley & Sons, Ltd.)

Background: Hyperammonemia, hypoglycemia, hepatopathy, and ventricular tachycardia are common presenting features of carnitine-acylcarnitine translocase deficiency (Mendelian Inheritance in Man database: *212138), a mitochondrial fatty acid oxidation disorder with a lethal prognosis. These features have not been identified as the presenting features of mitochondrial cytopathy in the neonatal period., Case Presentation: We describe an atypical presentation of mitochondrial cytopathy in a 2 day-old neonate. She presented with a Reye-like syndrome episode, premature ventricular contractions and ventricular tachycardia. Initial laboratory evaluation exhibited a large amount of 3-methylglutaconic acid on urine organic acid analysis, mild orotic aciduria and a nonspecific abnormal acylcarnitine profile. The evaluation for carnitine-acylcarnitine translocase deficiency and other fatty acid oxidation disorders was negative. The patient later developed a hypertrophic cardiomyopathy and continued to be affected by recurrent Reye-like syndrome episodes triggered by infections. A muscle biopsy exhibited signs of a mitochondrial cytopathy. During the course of her disease, her Reye-like syndrome episodes have subsided; however, cardiomyopathy has persisted along with fatigue and exercise intolerance., Conclusions: This case illustrates that, in the neonatal period, hyperammonemia and ventricular tachycardia may be the presenting features of a lethal carnitine-acylcarnitine translocase deficiency or of a mitochondrial cytopathy, associated with a milder clinical course. This association broadens the spectrum of presenting phenotypes observed in patients with disturbed mitochondrial energy metabolism. Also, the presence of 3-methylglutaconic aciduria suggests mitochondrial dysfunction and mild orotic aciduria could potentially be used as a marker of mitochondrial disease.

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.

Objectives: To determine the proportion of urine amino and organic acids screening tests (UMS) undertaken for patients referred with developmental delay or intellectual disability (DD/ID), and within the group with DD/ID, to determine the diagnostic yield, the proportion of diagnoses with a therapy and the associated recurrence risks., Methods: A retrospective review of request forms and results of UMS, in individuals older than 28 days, referred to the Women's and Children's Hospital, North Adelaide, between 1 January 1992 and 31 December 1998 was carried out. Urine was analysed by ion exchange chromatography (amino acids), gas chromatography/mass spectrometry (organic acids), colorimetric assay (orotic acid) and stable isotope-dilution mass spectrometry (trimethylamine)., Results: A total of 3316 samples were received, 1447 being from patients with DD/ID. A diagnosis was determined for 1.8% of all referrals. For patients with DD/ID, the diagnostic yield was 1.1%, with a similar yield for isolated DD/ID and DD/ID with other features (9/828 vs 7/619; chi2 = 0.006; P = 0.93). Specific therapies were available for 69% of diagnoses associated with DD/ID and 87.5% had known Mendelian or mitochondrial inheritance., Conclusion: Urine metabolic screening is an important part of the evaluation of children with DD/ID as it can enable families to make reproductive decisions and children to receive appropriate therapy early.

Ornithine transcarbamylase deficiency (OTCD) is an inborn error of urea synthesis that has been considered as a model for liver-directed gene therapy. Current treatment has failed to avert a high mortality or morbidity from hyperammonemic coma. Restoration of enzyme activity in the liver should suffice to normalize metabolism. An E1- and E4-deleted vector based on adenovirus type 5 and containing human OTC cDNA was infused into the right hepatic artery in adults with partial OTCD. Six cohorts of three or four subjects received 1/2 log-increasing doses of vector from 2 x 10(9) to 6 x 10(11) particles/kg. This paper describes the experience in all but the last subject, who experienced lethal complications. Adverse effects included a flu-like episode and a transient rise in temperature, hepatic transaminases, thrombocytopenia, and hypophosphatemia. Humoral responses to the vector were seen in all research subjects and a proliferative cellular response to the vector developed in apparently naive subjects. In situ hybridization studies showed transgene expression in hepatocytes of 7 of 17 subjects. Three of 11 subjects with symptoms related to OTCD showed modest increases in urea cycle metabolic activity that were not statistically significant. The low levels of gene transfer detected in this trial suggest that at the doses tested, significant metabolic correction did not occur.

Potentialities of capillary zone electrophoresis with on-line isotachophoresis sample pretreatment and diode array detection (ITP-CZE-DAD) to the separation, detection and identification of trace analytes present in biological matrices were investigated. Urine represented a multicomponent, variable and high ionic strength matrix while orotic acid was chosen as a model analyte of a practical clinical relevance in this investigation. Using the ITP-CZE combination in the column-coupling configuration of the separation system ITP provided an enhanced sample load capacity to the separation system (a 30 microl sample injection volume), concentrated the analyte and served as an on-line sample clean up technique. On the other hand, CZE performed a final separation of the analyte from matrix constituents present in the ITP pretreated sample and provided favorable conditions for its detection and identification by DAD. Using current correction and smoothing procedures analytically relevant DAD spectra of orotic acid could be obtained also in instances when this was injected in a model sample at a 2 x 10(-7) mol/l concentration (an estimated limit of determination of orotic acid at a 218 nm detection wavelength). ITP-CZE separations of urine samples (based on differences in acid-base properties and host-guest complexations of the analyte and matrix anionic constituents) led to significant sample clean ups. Consequently, DAD spectra of orotic acid matching its reference spectrum, could be acquired also in instances when the acid was present in urine matrices (loaded in 30 microl injection volumes of 20-fold diluted urine samples) at 4-6 x 10(-7) mol/l concentrations. Here, residual trace matrix interferents prevented a closer approach to the above value attainable for model samples. Although this work was focused only on one analyte and urine matrix it implies very promising potentialities of the ITP-CZE-DAD combination in the identification and quantitation of trace analytes present in biological matrices, in general.

Background: 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., Methods: 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., Results: 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., Conclusions: Experimental study strongly suggests that arginine deficiency causes focal tubulointerstitial fibrosis in the kidney after massive SBR.

A liver transplantation from an asymptomatic mother, who was a carrier of ornithine transcarbamylase deficiency, to her daughter, who had severe manifestation, was successfully performed. One-year monitoring of plasma amino acid and urinary orotate/orotidine levels revealed no abnormality in the urea cycle in either subject.

The most direct test of functional capacity of the liver in nitrogen disposal is to stress the urea cycle with a high protein load. This has been used in the diagnosis of heterozygosity for ornithine carbamoyltransferase deficiency for many years by measuring the subsequent excretion of orotic acid in urine. Reports have shown some ambiguity in both this and the more recent allopurinol test. We investigated the effects of different foods as the protein load and of different analytical methods. A standardized protocol was developed, giving 35 g protein per m2 surface area as steamed fat-free chicken breast to be eaten within 30 min. Urine was collected at zero time and over 0-2, 2-4 and 4-6 h. Compliance was checked by assessing excretion of amino acids. Diagnostic sensitivity was improved by reference to the change in excretion, i.e. the ratio of excretions 2-4 h/0-2 h. Extension of the test to 6 h gave no diagnostic advantage over a 4 h test. Comparison of the analysis of total orotic acids by the photometric method of Harris and Oberholtzer, the reference method for this study, with that by the method of Goldstein and colleagues showed that the latter gave erratic results with some false positives. However, comparison of the method of Harris and Oberholtzer with specific orotic acid analysis by a modification of the stable-isotope internal standard method of Rimoldi and colleagues yielded the same diagnoses. The improved protein load test gave a clearly positive result in all 16 obligate heterozygotes and 2 possible heterozygotes tested from 14 kindred, and a clearly negative result in all 18 control subjects and all 6 of the possible heterozygotes who were later shown by DNA studies not to carry the family mutation. The test appears at least as sensitive and specific as the allopurinol test, and is more convenient because of the short period of sample collection.

The urea cycle disorders (UCDs) represent a group of inherited metabolic diseases with hyperammonemia as the primary laboratory abnormality. Affected individuals may become comatose or die if not treated rapidly. Diagnosis of a UCD requires a high index of suspicion and judicious use of the laboratory. It is important to rule out other conditions causing hyperammonemia that may require different treatment. The astute clinician may suspect a specific UCD in the appropriate clinical setting, but only laboratory results can confirm a specific diagnosis. The importance of the laboratory in helping the clinician to differentiate among various causes of hyperammonemia, in confirming a specific UCD, in carrier testing, and in prenatal diagnostic testing is highlighted in this review.