Kinetic analysis of a new human ornithine carbamoyltransferase variant.

A new human enzymatic variant was found in a patient with ornithine carbamoyltransferase (carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) deficiency. This mutant enzyme has decreased affinity, with an abnormal Km value for ornithine (3-5-times greater than control at all pH values). The maximal velocity (V) varied with pH as a normal enzyme but the sigmoid curve obtained (V vs. pH) is shifted towards alkaline pH values. The pK of the functional catalytic group is 8.3 instead of 6.65 of a control enzyme. At its optimum pH the V of the mutant enzyme is greater than the V of the normal enzyme. Other mutant enzyme proteins with abnormal affinity for ornithine have already been described. They all are different from the reported here.

Superoxide-induced deimination of arginine in hematopoietic cells.

Murine bone marrow cells can produce citrulline directly from L-arginine without intermediate ornithine. An L-arginine-dependent biochemical pathway synthesizing L-citrulline and nitrate, coupled to an effector mechanism has also been recently demonstrated in murine cytotoxic activated macrophages. We show herein that L-citrulline synthesis in murine bone marrow cells can be induced by the generation of superoxide. It can take place in an arginine-free medium, suggesting the implication of a superoxide-dependent peptidyl arginine deiminase.

Efficiency of substitution of 2-ketoisocaproic acid and 2-ketoisovaleric acid in the diet of normal and uremic growing rats.

Effects of various intakes of the ketoanalogues of leucine (KICA) and valine (KIVA) on growth, nitrogen, and urea excretion were examined and compared to those of an optimal intake (A) of the corresponding amino acids. Diet KICA and KIVA contents varied from 1 to 4 times A. In controls, growth was significantly reduced with equimolar substitution, corrected with twice A, and unchanged at higher levels. Doubling KICA corrected growth except with substantial anorexia. In uremic rats fed KIVA, growth was corrected at twice A. Low-KICA diets reduced plasma-leucine level; higher KICA diets normalized plasma leucine and revealed branched-chain amino acid (BCAA) antagonism. Changes in 2-ketoacids were unrelated to those of BCAA. In uremia, KICA decreased plasma and urinary urea without changing nitrogen retention. Ketoacid substitution for amino acids was 50% efficient in normal rats and not altered by uremia. BCKAs, specifically KICA, could modify urea metabolism.

Developmental changes of citrullinogenesis, mitochondrial N-acetylglutamate content and N-acetylglutamate synthetase in fetal and neonatal rats.

A low citrullinogenesis (less than 60 per cent of the adult value) was observed throughout the suckling period when mitochondria isolated from newborn rat liver were incubated in vitro with L-glutamate or succinate as oxidizable substrates. The adult value was reached after weaning. From birth to weaning, intact mitochondria synthesized more citrulline when supplemented with L-glutamate than with succinate. The low citrullinogenesis could not be explained by low carbamoylphosphate synthetase-I and ornithine transcarbamoylase activities that reached adult values at birth. The decreased citrullinogenesis seen for the first three days of life seemed to be related to the low intramitochondrial concentration of N-acetylglutamate, an activator of the carbamoylphosphate synthetase-I. The concentration of this activator did not differ from that reported for adult rat liver mitochondria after the fourth day of life. The discrepancy between the normal value of N-acetylglutamate concentration and the low activity of the N-acetylglutamate synthetase (15 to 30 per cent of the adult activity) is discussed on the basis of acetyl-CoA or L-glutamate availability in mitochondria isolated from newborn or young rats.

Localization of copper-zinc superoxide dismutase mRNA in human hippocampus by in situ hybridization.

The cellular localization of copper-zinc superoxide dismutase (CuZn SOD) mRNA was determined in the human hippocampus by in situ hybridization with a 35S-labelled DNA probe complementary to human CuZn SOD mRNA. A positive hybridization signal was detected in pyramidal cell layers CA1-CA4 of Ammon's horn (CA), pyramidal cells of subiculum and in the granule cells of the dentate gyrus. The fact that CuZn SOD gene expression is important in neurones which are preferentially vulnerable in neurodegenerative processes such as Alzheimer's disease, suggests a role played by oxygen free radicals in the mechanism of nerve cell death.

Transglutaminase in erythrocytes of cystic fibrosis patients.

Transglutaminase activity was determined in erythrocytes from patients with cystic fibrosis and control subjects. No differences were observed between the two groups either for enzymatic activity or for the activating effect of calcium ions. The previously described abnormal metabolism of polyamines in cystic fibrosis cannot be related to an altered function of transglutaminases.

Ion-exchange chromatography and clinical criteria in the screening of the aminoacidopathies.

By a retrospective study on a two-year period (1494 patients) we have obtained evidence that the best method of screening aminoacidopathies is given by the ion-exchange chromatography of amino acids. This method gave the highest rates of diagnosis and allowed diagnosis of aminoacidopathies that other techniques such as thin-layer chromatography would fail to detect because the concentrations of some amino acids, although pathological, were too low. The diagnostic rate was markedly improved by a skilled clinical selection of the population studied.

Blood and urinary amino acids in children receiving total parenteral nutrition.

Plasma concentrations and urinary outputs of amino acids were estimated in nineteen patients receiving intravenous hyperalimentation to evaluate the adequacy of dosage and composition of the infusates for the maintenance of normal blood concentrations of essential amino acids. The use of high concentrations of branched chain amino acids seems to be appropriate for valine and isoleucine but not for leucine. The high concentration of cysteine in the infusates used induces a very high urinary excretion of cysteine and cystine and are ineffective to bring the decreased plasma cystine levels back to normal.

Is type 1b glycogenosis related to an anomeric preference for glucose-6-phosphate uptake by hepatic microsomes?

The contradictory data obtained in studies of type 1b glycogenosis would be explained by an anomeric preference for the capture of glucose-6-phosphate by hepatic microsomes. The alpha anomers of hexoses would be utilized preferentially by glycogenolysis and gluconeogenesis, while the beta anomers of hexoses would be preferentially utilized for glycolysis.

[Metabolism of amino acids and urea cycle: animal models of enzymopathies]. / Métabolisme des acides aminés et cycle de l'urée: modèles animaux des enzymopathies.

This article is a review of the animal models which have been described until now for the inborn errors of amino acid metabolism and urea cycle. Two approaches have been explored to obtain such animal models: firstly oral or parenteral administration of the amino acid(s) accumulated in some enzymatic defects of the amino acid catabolism or urea cycle--specific enzymatic inhibitors have sometimes been used; secondly, the genetic approach, which is often the best method. Systematic screening of rat or mouse mutants obtained by experimental mutagenesis was performed in some cases. The relationships between the metabolic disturbances observed either in the inborn errors of metabolism or in the experimental models are discussed as well as the origin of the discrepancies often observed between human and animal syndromes.

Serotonin in experimental histidinemia.

An experimental histidinemia was obtained in rats by in vivo administration of nitromethane, a histidase inhibitor. The magnitude of increase in plasma histidine in the nitromethane-treated rats was in the same range as that in the histidinemic subjects. No modifications were observed in the serotonin concentrations in blood or in various areas of brain between the nitromethane-treated rats and the control rats. No dramatic modifications of serotonin metabolism seem to be implicated in histidinemia, unlike phenylketonuria.

Eleventh week amniocentesis for prenatal diagnosis of some metabolic diseases.

We report the results of 23 prenatal diagnoses performed at the 11th or 12th week of gestation by the simultaneous analysis of chorionic villi (for direct or indirect enzymatic analysis) and cell-free amniotic fluid (for search of accumulated catabolites). For six cases of citrullinaemia, four cases of argininosuccinic aciduria, seven cases of propionic acidaemia, and six cases of methylmalonic acidaemia, three discrepancies were observed between the two methods used. The amniotic fluid analysis for accumulated catabolites seems to be a safe method and should always be used in conjunction with the enzymatic assays performed for the prenatal diagnosis of these diseases.

Renal Histidinuria.

One patient with abnormal histidinuria and a normal plasma histidine concentration is presented. This patient had myoclonic seizures like another patient previously reported with histidinuria. The effect of a peroral histidine loading test was studied. The relationship between renal histidinuria and neurological abnormalities is discussed.

A new enzymatic pathway of citrullinogenesis in murine hemopoietic cells.

Citrullinogenesis is demonstrated when murine bone marrow cells are incubated with dialyzed secondary mixed leukocyte culture supernatant. The identity of citrulline in bone marrow cell supernatants has been established by gas chromatographic mass spectrometric analysis. It is shown that, in our model, citrulline synthesis proceeds directly from arginine without intermediate ornithine production, ruling out the involvement of ornithine transcarbamylase (EC 2.1.3.3.). Moreover, none of the other enzymatic activities described for catalyzing citrullinogenesis, i.e. arginine deiminase or peptidyl arginine deiminase can be demonstrated. The generation of oxygen radicals is necessary for this enzymatic reaction. It is induced by a thermolabile protein produced during the antiallograft immune response with a molecular weight of about 150,000.

Inborn errors of amino acid metabolism. The best strategy for their diagnosis.

We performed a cost-effectiveness analysis to evaluate whether a pediatrician who suspects an inherited disease of amino acid metabolism should refer the child to a specialist in inborn errors of amino acid metabolism or should prescribe the usual screening test, chromatography of amino acids. Actual hospital costs were used to value the referral, the tests, and the complications that occur when the diagnosis is not recognized. The percent of confirmed diagnoses was chosen as a measure of effectiveness. We conclude that it is more cost-effective for a pediatrician to refer the child to a specialist, that the best strategy in the absence of a referral is to prescribe thin-layer chromatography, and that the least cost-effective strategy is to perform ion-exchange chromatography immediately.

Age-related reference values for free amino acids in first morning urine specimens.

We determined age-related reference values for urinary free amino acids (in mmol/mol creatinine) in first morning urine specimens from 360 control subjects who were divided into nine age groups: birth to 1 month, 1-6 months, 6-12 months, 1-2 years, 2-4 years, 4-7 years, 7-10 years, 10-13 years, and older than 13 years. Except for taurine and 3-methylhistidine, the concentration of all the amino acids decreased with increasing age. The use of these results to detect aminoacidopathies and tubulopathies is discussed.

Ascorbate-cyanide test on red blood cells in uremia: effect of guanidinopropionic acid.

Red blood cells (RBC) of uremic or control subjects have been exposed to an oxidative stress by ascorbate and cyanide. The spectrophotometric determination of the sulfhemoglobin production has been used as a reliable method for the measurement of the hexose monophosphate shunt. The sulfhemoglobin production by RBC was significantly higher in uremic than in control subjects. Uremic plasma increased this production in uremic as well as in control RBC while control plasma was ineffective under the same conditions. In vitro the guanidinopropionic acid had the same toxic effect on normal RBC. These results suggest that the abnormal sulfhemoglobin production by uremic RBC might be due to an increased plasma concentration of guanidinopropionic acid in uremia.