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.     

鸟氨酸氨基甲酰转移酶速率法测定的初步探讨

目的建立鸟氨酸氨基甲酰转移酶(OCT)自动化分析的方法。方法在磷酸盐缓冲液(pH值7.2)条件下,以瓜氨酸为底物,经一系列酶偶联反应,尼克酰胺腺嘌呤二核苷酸(NADH+H+)被氧化为氧化型辅酶Ⅰ(NAD+),340 nm处测定吸光度(A)变化值,A值下降的速率与待测样本中的OCT含量成正比关系,间接求出OCT的活性。结果本法最适pH值为7.2,最适底物浓度为2.0 mmol/L。批内、批间平均变异系数(CV)分别为3.80%、4.08%。米氏常数(Km)值为0.16 mmol/L。回收率达91.56%。在320 U/L内线性良好。参考范围为0~18 U/L。结论本方法能够快速、简便、准确的测定OCT活性,适用于各类全自动生化分析仪。     

鸟氨酸氨基甲酰转移酶速率法测定的初步探讨

目的 建立鸟氨酸氨基甲酰转移酶(OCT)自动化分析的方法.方法 在磷酸盐缓冲液(pH值7.2)条件下,以瓜氨酸为底物,经一系列酶偶联反应,尼克酰胺腺嘌呤二核苷酸(NADH+H+)被氧化为氧化型辅酶Ⅰ(NAD+),340 nm处测定吸光度(A)变化值,A值下降的速率与待测样本中的OCT含量成正比关系,间接求出OCT的活性.结果 本法最适pH值为7.2,最适底物浓度为2.0 mmol/L.批内、批间平均变异系数(CV)分别为3.80%、4.08%.米氏常数(Km)值为0.16 mmol/L.回收率达91.56%.在320 U/L内线性良好.参考范围为0～18 U/L.结论本方法能够快速、简便、准确的测定OCT活性,适用于各类全自动生化分析仪.     

鸟氨酸氨甲酰转移酶缺乏症患儿合并吞咽障碍的护理

总结1例鸟氨酸氨甲酰转移酶缺乏症患儿合并吞咽障碍的护理经验。护理要点包括：缓解期给予低蛋白饮食护理、加强疾病知识宣教、吞咽障碍护理、患儿及家长的心理护理等,经过2个月的治疗及护理,患儿病情被控制、吞咽功能改善。     

女性新生儿期起病型鸟氨酸氨甲酰基转移酶缺乏症1例并文献复习

回顾性分析1例女性新生儿期起病型鸟氨酸氨甲酰基转移酶缺乏症患儿的临床资料及基因检测结果。患儿女性,3天,主要表现为反应差、抽搐、昏迷及高氨血症。血瓜氨酸水平(4.12μmol/L)降低及尿乳清酸水平显著升高(166.3μmol/L),二代测序及QPCR验证发现OTC基因外显子1-10的杂合缺失,母亲携带外显子2和外显子4的杂合缺失,父亲、姐姐及双胞胎弟弟未携带该基因外显子缺失。系国内首次报道女性新生儿期起病外显子1-10全部缺失的鸟氨酸氨甲酰基转移酶缺乏症。     

Assay of ornithine carbamoyltransferase activity in human liver using carbon-labeled ornithine and thin-layer chromatography

Ornithine carbamoyltransferase (EC 2.1.3.3) activity in human liver homogenates has been measured using 14C-labeled ornithine and unlabeled carbamoyl phosphate. A thin-layer chromatographic (TLC) procedure is used to separate the radioactive substrate and product, ornithine and citrulline, respectively, and the regions of the chromatogram corresponding to ornithine and citrulline are cut out and counted in a liquid scintillation spectrophotometer. The method has the following advantages: (1) the radioactive substrate ornithine is more stable in solution than carbamoyl phosphate, (2) 14C-labeled ornithine is available in higher specific activity than carbamoyl phosphate, (3) all radioactivity may be accounted for by using the TLC system, (4) the developed thin-layer chromatogram is stable indefinitely, (5) in contrast to colorimetric assays, other compounds in the raction mixture do not interfere with the citrulline determination, and (6) most importantly, the rate of the enzyme reaction at various time intervals can be determined by taking aliquots from the same incubation tube.     

Study of ammonia metabolism in a patient with ornithine transcarbamylase deficiency using an 15N tracer

Ammonia metabolism was studied in an 8-year-old girl with ornithine transcarbamylase (OTC) deficiency, using 15N-tracer. Changes in the incorporation of 15N into amino acids and urea were examined after 15NH4Cl administration. The recovery of total 15N in the urine of the patient in 3 days was 28.5% of the administered 15N whereas that of a control was 69.3%. They were mostly urea. The recovery of 15N-urea in the patient was 28.8% of the control in 1 day, 32% in 2 days and 33.3% in 3 days after the administration of 15NH4Cl. A larger amount of 15N was incorporated into glutamine (alpha-amino N) and glutamate and 15N was incorporated more rapidly into alanine, asparagine and serine in the patient than in the control. The incorporation into ornithine was less in the patient than in the control.     

Serum level of ornithine carbamoyltransferase is influenced by the state of Kupffer cells

BACKGROUND: The ratio of ornithine carbamoyltransferase (OCT) to alanine aminotransferase (ALT) or glutamate dehydrogenase (GDH) in serum has been suggested as an indicator for the diagnosis of hepatocellular carcinoma and alcoholic liver disease, respectively. However, the mechanisms responsible for the increase in these ratios are still unclear. METHODS: Wistar rats were pretreated with lipopolysaccharide (LPS) or gadolinium chloride (GD) before being administered with thioacetamide (TAA, 200 mg/kg, ip). Serum OCT and ALT levels were compared with control values. Half-lives of the enzymes in circulation were evaluated after the intravenous injection of the purified enzymes into rats with or without the pretreatment. RESULTS: The serum level of OCT at 24 h after the administration of TAA was significantly lower in the LPS-treated group, and not influenced by pretreatment with GD. The half-life of OCT was prolonged from 1.06+/-0.14 to 2.07+/-0.29 h (p<0.05) by the pretreatment with GD, but not influenced by the administration of LPS. No change was observed in the clearance of GDH or ALT among the pretreatments. CONCLUSIONS: Leakage into and clearance from the circulation of OCT are influenced by whether Kupffer cells are activated or not. OCT alone or in combination with other markers may be a useful indicator for Kupffer cell activation as well as mitochondrial damage in hepatic cells.     

Some kinetic properties of liver ornithine carbamoyl transferase (oct) in a patient with oct deficiency

Some kinetic properties of liver OCT from a patient with OCT deficiency were studied. Contrary to controls, in which two pH optima were observed (pH 7.7 and pH 8.5), only the pH optimum of 8.5 could be demonstrated in our patient. From K_M studies at pH 7.7 and pH 8.5, the most striking abnormalities in comparison with human controls were (a) a strongly increased K_M (ornithine) at pH 7.7, but less pronounced at pH 8.5, (b) a higher V_max at pH 8.5 compared with the V_max at pH 7.7 and (c) the absence of substrate inhibition at pH 8.5 of ornithine was elevated up to a concentration above approximately 1.5 mM.     

Restimulation-induced apoptosis of T cells is impaired in patients with X-linked lymphoproliferative disease caused by SAP deficiency

X-linked lymphoproliferative disease (XLP) is a rare congenital immunodeficiency that leads to an extreme, usually fatal increase in the number of lymphocytes upon infection with EBV. It is most commonly defined molecularly by loss of expression of SLAM-associated protein (SAP). Despite this, there is little understanding of how SAP deficiency causes lymphocytosis following EBV infection. Here we show that T cells from individuals with XLP are specifically resistant to apoptosis mediated by TCR restimulation, a process that normally constrains T cell expansion during immune responses. Expression of SAP and the SLAM family receptor NK, T, and B cell antigen (NTB-A) were required for TCR-induced upregulation of key pro-apoptotic molecules and subsequent apoptosis. Further, SAP/NTB-A signaling augmented the strength of the proximal TCR signal to achieve the threshold required for restimulation-induced cell death (RICD). Strikingly, TCR ligation in activated T cells triggered increased recruitment of SAP to NTB-A, dissociation of the phosphatase SHP-1, and colocalization of NTB-A with CD3 aggregates. In contrast, NTB-A and SHP-1 contributed to RICD resistance in XLP T cells. Our results reveal what we believe to be novel roles for NTB-A and SAP in regulating T cell homeostasis through apoptosis and provide mechanistic insight into the pathogenesis of lymphoproliferative disease in XLP.     

Molecular genetic studies of mitochondrial ornithine transporter deficiency (HHH syndrome)]

Mitochondrial ornithine transporter deficiency has been called HHH syndrome, because this disorder is characterized by three biochemical abnormalities; hyperornithinemia, hyperammonemia, and homocitrullinuria, and presents with various neurological symptoms; mental retardation, spastic paraparesis with pyramidal signs, cerebellar ataxia and episodic disturbance of consciousness or coma due to hyperammonemia. We identified four mutations in the mitochondrial ornithine transporter gene (ORNT1) of Japanese patients with HHH syndrome. These include a nonsense mutation (R179X), associated with exon skipping, missense mutations (G27E, P126R), and an insertion of AAC between codons 228 and 229, leading to an insertion of amino acid Asn. Especially, R179X was detected 4 of 7 Japanese patients (8 of 14 alleles), implying that this is a common mutation in Japanese population.     

Ratio of serum ornithine carbamoyltransferase to alanine aminotransferase as a potent indicator for hepatocellular carcinoma

OBJECTIVES: To evaluate the clinical advantage of the ratio of serum ornithine carbamoyltransferase (OCT) to alanine aminotransferase (ALT) in the diagnosis of hepatocellular carcinoma (HCC). DESIGN AND METHODS: Serum levels of hepatic enzyme markers and their combinations were evaluated and compared with those of two other markers for HCC. RESULTS: OCT/ALT was significantly higher in case of HCC than chronic hepatitis or liver cirrhosis. Its sensitivity (64.3%) was higher than those of alpha-fetoprotein and PIVKA-II (21.4% and 42.9%, respectively). Fluctuations of OCT/ALT before and after treatment were similar to those of alpha-fetoprotein. CONCLUSIONS: OCT/ALT is a potent indicator for the diagnosis and the prognosis of HCC.     

A direct method for the estimation of ornithine carbamoyltransferase activity in serum.

Ornithine carbamoyltransferase (EC 2.1.3.3) activity was estimated by determining the amount of citrulline produced. The citrulline was colorimetrically measured by using a diacetylmonoxime-thiosemicarbazide reaction without deproteinization. The color complex which had the maximum absorbance at 515 nm was stable at room temperature in daylight for at least several hours. The proposed method was superior to other methods already reported in sensitivity, stability and simplicity.     

Direct micromethod for colorimetry of serum ornithine carbamoyltransferase activity, with use of a linear standard curve.

Determination of serum ornithine carbamoyltransferase (EC 2.1.3.3) activity can be a valuable diagnostic tool in the detection of liver diseases involving cytolytic processes. I describe a micromethod for measuring this activity in serum, in which the reaction product, citrulline, is measured colorimetrically in the incubation mixture without prior deproteinization. To eliminate the interference of serum protein precipitation, the concentration of sulfuric acid in the color reagent has been decreased, without substantial loss of sensitivity. Optimizing the conditions of citrulline determination, in which antipyrine and 2,3-butanedione monoxime are used, has resulted in a linear standard curve. The color formed by citrulline is found to be stable in room lighting and sensitive only to direct sunlight. The precision of the method is inversely correlated to serum enzyme activity, the CV varying between 4.6 and 21.1%.     

A pilot study of in vivo liver-directed gene transfer with an adenoviral vector in partial ornithine transcarbamylase deficiency.

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.