Inhibition by propionyl-coenzyme A of N-acetylglutamate synthetase in rat liver mitochondria. A possible explanation for hyperammonemia in propionic and methylmalonic acidemia.

In the search for the mechanism by which hyperammonemia complicates propionic and methylmalonic acidemia the effects of a series of acyl-coenzyme A (CoA) derivatives were studied on the activity of N-acetylglutamate synthetase in rat liver mitochondria using acetyl-CoA as substrate. Propionyl-CoA was found to be a competitive inhibitor. The inhibition constant of 0.71 mM is in the range of concentrations of propionate found in the serum of patients with propionic and methylmalonic acidemia. Propionyl-CoA was also found to be a substrate for N-acetylglutamate synthetase, forming N-propionylglutamate. This compound was a weak activator of rat liver carbamoylphosphate synthetase; the activation constant was 1.1 mM as compared with 0.12 mM for N-acetylglutamate. A decreased level of N-acetylglutamate in liver mitochondria that would follow inhibition of N-acetylglutamate synthetase by propionyl-CoA would be expected to lead to hyperammonemia. Methylmalonyl-CoA, tiglyl-CoA, and isovaleryl-CoA at a concentration of 3 mM caused 30-70% inhibition of N-acetylglutamate synthetase. 3the latter two compounds are readily detoxified by the formation of N-acylglycine conjugates in liver, which may prevent large accumulations and could explain why hyperammonemia is not characteristic of patients with beta-ketothiolase deficiency or isovaleric acidemia in whom these compounds would be expected to be elevated.

Role of N-acetylglutamate and acetyl-CoA in the inhibition of ureagenesis by isovaleric acid in isolated rat hepatocytes.

1 and 10 mmol/l isovalerate strongly inhibited urea synthesis in isolated rat hepatocytes incubated with 10 mmol/l alanine and 3 mmol/l ornithine. Isovalerate also markedly decreased N-acetylglutamate levels, and the decrease correlated with the inhibition of urea synthesis by isovalerate. This compound also lowered cellular levels of acetyl-CoA, a substrate of N-acetylglutamate synthase (EC 2.3.1.1). Isovalerate did not significantly affect the cellular levels of ATP and had no direct effect on N-acetylglutamate synthase activity. These results suggest that the inhibition of urea synthesis by isovalerate is due to decrease in N-acetylglutamate levels.

Effect of overnight constant infusion of human growth hormone (GH)-releasing hormone-(1-44) on 24-hour GH secretion in children with partial GH deficiency.

A continuous infusion (0.5 or 1 microgram/kg X h) of GH-releasing factor-(1-44) [GHRH-(1-44)] was administered from 2000-0800 h to 16 children with GH deficiency, defined as a maximum peak plasma GH less than 11 ng/ml in response to 2 provocative tests [first test; mean, 7.4 +/- 2.6 (+/- SD) ng/ml; second test; mean, 8.4 +/- 2.4 ng/ml]. Eight were boys and 8 girls; their average age was 10 yr, 5 months; and growth was retarded in all [mean, -3 +/- 0.6 (+/- SD)]. Polygraphic monitoring was carried out during the night, and blood samples for plasma GH measurements were drawn every 20 min during the night and the following day. A control study had been carried out in the preceding months with the same children. During GHRH infusion, a significant increase in nocturnal GH secretion occurred; the mean maximum peak increased from 17.5 +/- 3.4 (+/- SD) to 38.7 +/- 3.2 ng/ml, the mean area under the curve from 2243 +/- 459 to 5348 +/- 710 ng/ml, the mean integrated concentration from 4.2 +/- 0.8 to 9.9 +/- 1.3 ng/ml X min, and the mean number of peaks above 5 ng/ml from 2.7 +/- 0.3 to 4.7 +/- 0.4. During GHRH infusion, the 16 children had 2 peaks during the first 4 h of sleep and a third peak at the end of the night. Plasma GH levels the day after the infusions were not significantly increased. We conclude that continuous nocturnal GHRH infusion increases pulsatile sleep GH secretion throughout the night in children with partial GH deficiency.

Results of 1-year growth hormone (GH)-releasing hormone-(1-44) treatment on growth, somatomedin-C, and 24-hour GH secretion in six children with partial GH deficiency.

Six children with short stature and partial GH deficiency in response to two pharmacological tests received GHRH for 12 months (10 micrograms/kg X day, sc) each evening. Twenty-four-hour GH secretion was studied before and after 3 and 12 months of treatment, and GHRH tests (2 micrograms/kg, iv) were done before and after 6 months of treatment. Plasma somatomedin-C was measured before and after 1.5, 3, 6, 9, and 12 months of treatment. Statural growth was measured at 3-month intervals. Mean growth velocity increased from 4.2 to 8.6 cm/yr, with a good result in five children and no response in the other. The growth response was substantial during the first 3 months. It was maintained during the following 6 months, and then decreased during the last 3 months. The peak plasma GH level in response to GHRH increased from 34.5 +/- 14.2 (+/-SD) ng/mL before treatment to 47.8 +/- 3.4 ng/mL after 6 months of treatment. Twenty-four-hour GH secretion increased in all parameters at 3 months (maximum peak, area under the curve, integrated concentration, and number of peaks) and at 12 months (with the exception of the maximum peak). Nycthemeral secretory profiles became normal, with reappearance of secretory pulses in two children, slight increases in three children, and no change in one child. Plasma somatomedin-C levels rose from 0.8 +/- 0.3 U/mL before treatment to 2.0 +/- 1.0 U/mL at 3 months, then decreased to 1.3 +/- 0.6 U/mL at 12 months. These results indicate that GHRH administered by sc injection for a 1-yr period stimulated growth and GH secretion. However, a decrease in activity was noted during the last 3 months of treatment. Tests for anti-GHRH antibodies were positive in the only child who did not respond to treatment.

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.

Protective effects of SR 57746A in central and peripheral models of neurodegenerative disorders in rodents and primates.

Compounds possessing neurotrophic properties may represent a possible treatment for neurodegenerative disorders such as Alzheimer's disease. SR 57746A, 1-[2-(naphth-2-yl)ethyl]-4-(3-trifluoromethylphenyl)-1,2,5,6- tetrahydropyridine hydrochloride, is a new compound with neurotrophic activity in a number of in vitro preparations. The neurotrophic effects of this compound have been evaluated in vivo using four distinct rat models of neurodegeneration: transient global ischaemia produced by a four-vessel occlusion; septohippocampal lesion produced by injection of vincristine sulphate into the medial septum; sciatic nerve crushing; and acrylamide-induced peripheral neuropathy. Rats were administered vehicle or 2.5-10 mg/kg p.o. SR 57746A, after initiation of the degenerative process, then once daily for 10 days in the first two models, 16 days in the third and 26 days in the fourth model. Median scores for ischaemia-induced neuronal damage were reduced by 30-40% by SR 57746A treatment in hippocampal CA1, CA2, and CA3 regions, and in the dorsal striatum. Twelve days after intraseptal vincristine administration, there was a marked loss of septohippocampal cholinergic neurons, as indicated by reduced choline acetyltransferase activity in both the septum and hippocampus. SR 57746A dose-dependently reversed this reduction in both areas. These results were confirmed by histoenzymological evaluation of hippocampal acetylcholinesterase content. SR 57746A also reversed the loss of hippocampal choline acetyltransferase induced by intraseptal vincristine in marmosets. Behavioral deficits in these models (exploratory behaviour in the former and short-term social memory in the latter) were also significantly reduced by SR 57746A treatment. In the sciatic crush model, sensorimotor function improved more rapidly in rats treated with 10 mg/kg SR 57746A. In this same model, SR 57746A (10 mg/kg/day) also significantly increased the length of regenerated nerve eight days after the crush, as measured using the pinch test. Finally, SR 57746A retarded the onset, reduced the amplitude and accelerated the recovery of acrylamide-induced peripheral neuropathy. Thus, SR 57746A possesses notable neurotrophic activity in a variety of neurodegenerative models in vivo, suggesting that the compound may possess therapeutic potential for the treatment of neurodegenerative diseases.

Growth hormone-releasing factor modifies dopaminergic but not serotonergic activity in the arcuate nucleus of hypothalamus in the rat, as recorded in vivo by differential pulse voltammetry.

Parenteral (i.v.) injection of growth hormone-releasing factor (GRF) increases the height of the 3,4-dihydroxyphenylacetic acid oxidation peak (peak 2) but does not change 5-hydroxyindole extracellular content (peak 3) in the arcuate nucleus of the hypothalamus, both peaks being recorded by the differential pulse voltammetry technique using a single specifically pretreated monopyrolytic carbon fibre electrode. Conversely, no significant changes are observed in the peak 2 and peak 3 heights recorded in the medial or in the lateral nucleus of the hypothalamus. These data suggest a specific interaction between GRF and the dopaminergic system.

Potentiation by piridoxilate of the synthesis of hippurate from benzoate in isolated rat hepatocytes. An approach to the determination of new pathways of nitrogen excretion in inborn errors of urea synthesis.

The synthesis of hippurate from benzoate as compared to ureagenesis has been investigated in isolated rat hepatocytes. Half-maximal synthesis of hippurate was observed at 0.3 mmol/l benzoate. In the presence of 1 mmol/l benzoate, hippurate synthesis proceeded linearly with time at a rate of 40 +/- 10 mumol X h-1 X g-1 dry weight. This provided less than 10% of nitrogen epuration supported by concomitant urea synthesis (350 +/- 82 mumol X h-1 X g-1 dry weight). The incorporation of benzoate to hippurate was markedly limited by the availability of glycine. Half-maximal hippurate synthesis was observed at 2 mmol/l glycine. In the absence of glycine, piridoxilate, a glyoxylate derivative, markedly potentiated hippurate synthesis. Half-maximal stimulation was observed at 10 mmol/l piridoxilate. In the presence of 10 mmol/l piridoxilate, hippurate synthesis (420 +/- 35 mumol X h-1 X g-1 dry weight) provided more than 50% of nitrogen epuration supported by urea synthesis. It is concluded that supplementation with nitrogen-free analogues of glycine such as piridoxilate are required to potentiate hippurate synthesis in an attempt to replace ureagenesis as an alternative pathway of waste nitrogen excretion in inborn errors of urea synthesis.

Biotin dependent carboxylase activities in normal human and multicarboxylase deficient patient fibroblasts: relationship to the biotin content of the culture medium.

Three mitochondrial carboxylase activities can be depleted in skin fibroblasts from patients with the neonatal form of multiple carboxylase deficiency when the biotin content of the culture medium is lowered to 25 nmol/l. On the other hand this depletion can be achieved in control fibroblasts or in fibroblasts from patients with the late onset form of the deficiency when avidin (50 U/l) is added to the culture medium. The kinetics of the carboxylase activity decrease are nevertheless identical for control and for both types of fibroblasts. After depletion, control and late onset form fibroblasts recover their carboxylase activities at the same rate, whereas fibroblasts with the neonatal form of deficiency need longer times or higher concentrations of biotin to restore their carboxylase activities. These results are consistent with previous hypotheses concerning the origin of both forms of the deficiency. In addition, this technique provides a convenient access to human apocarboxylases, i.e. substrates for in vitro holocarboxylase synthetase investigation.

Inhibition of urea synthesis by pent-4-enoic acid: potentiation by ammonia.

Pent-4-enoic acid inhibited ureagenesis approximatively 90% in rat hepatocytes incubated with pyruvate, ammonia and ornithine. Inhibition of ureagenesis was much less with alanine as substrate (approximatively 10%). The addition of ammonia led to a drastic dose-dependent inhibition of ureagenesis by pent-4-enoate. Half-maximum effect of ammonia was observed at 0.2 mM concentration. Concomitant cellular concentrations of N-acetylglutamate were also drastically modified by the addition of ammonia as was the accumulation of citrulline. These data suggest that ammonia may seriously interfere with the metabolism of pent-4-enoic acid and leads to a dramatic potentiation of its toxicity.

Action of the antiepileptic drug, valproic acid, on fatty acid oxidation in isolated rat hepatocytes.

Valproate at 0.1 to 5 mM strongly inhibited oxidation of 1-(14C)-palmitate in isolated rat hepatocytes. Valproate at the same concentrations markedly decreased ketogenesis from 1 mM oleate. Valproate in a dose up to 5 mM did not significantly affect cellular concentration of ATP but lowered beta-hydroxybutyrate/acetoacetate and lactate/pyruvate ratios which paralleled its effect on ketogenesis. Moreover concomitant acetyl-CoA levels were drastically decreased by valproate. From this it may be concluded that inhibition of fatty acid oxidation by valproate results in reduced production of two carbons units and a drop of NADH/NAD+ ratio in rat hepatocyte. This suggests that valproate seriously interferes with beta-oxidation of physiological long-chain fatty acids.

Inhibition of ureagenesis by valproate in rat hepatocytes. Role of N-acetylglutamate and acetyl-CoA.

Valproate (0.5-5 mM) strongly inhibited urea synthesis in isolated rat hepatocytes incubated with 10 mM-alanine and 3 mM-ornithine. Valproate at the same concentrations markedly decreased concentrations of N-acetylglutamate, an essential activator of carbamoyl-phosphate synthetase I (EC 6.3.4.16), in parallel with the inhibition of urea synthesis by valproate. This compound also lowered the cellular concentration of acetyl-CoA, a substrate of N-acetylglutamate synthase (EC 2.3.1.1); glutamate, aspartate and citrulline were similarly decreased. Valproate in a dose up to 2 mM did not significantly affect the cellular concentration of ATP and had no direct effect on N-acetylglutamate synthesis, carbamoyl-phosphate synthetase I and ornithine transcarbamoylase (EC 2.1.3.3) activities.

Potentiation by ammonia of the metabolic effects of pent-4-enoate in isolated rat hepatocytes.

The metabolic effects of pent-4-enoate were studied in isolated rat hepatocytes; 1 mM-pent-4-enoate did not significantly inhibit gluconeogenesis from lactate, alanine and glycerol, but significantly decreased glucose synthesis from pyruvate. The addition of 1 mM-NH4Cl led to a drastic inhibition of glucose synthesis from all these substrates. In hepatocytes incubated with 10 mM-alanine and 1 mM-oleate, pent-4-enoate at 0.05-1 mM slightly inhibited glucose synthesis and ketogenesis. The addition of ammonia resulted in a dramatic potentiation of the metabolic effects of pent-4-enoate. Half-maximum effect of ammonia was observed at 0.2 mM concentration. Concomitant cellular concentrations of ATP and acetyl-CoA were also decreased by the addition of ammonia, as were lactate/pyruvate ratio and beta-hydroxybutyrate/acetoacetate ratio. These data suggest that ammonia seriously interferes with the cellular metabolism of pent-4-enoate and leads to a dramatic potentiation of its effects.

A new family affected by the syndrome of hyperornithinaemia, hyperammonaemia and homocitrullinuria.

We have reported three cases of hyperornithinaemia associated with hyperammonaemia and homocitrullinuria (HHH). They deal with two brothers and a sister from a family where the parents and four other children are healthy on clinical and biochemical examination. The biochemical findings in our patients indicate the existence of a defect in the transport of ornithine into the mitochondria. Cultured skin fibroblasts from two of these patients incorporated six times less [14C]ornithine into protein as compared to control cells. The most characteristic sign of the clinical picture is the progressive spastic paraparesis found in one of the cases. Ornithine supplementation and restricted protein intake may be useful in the treatment of this syndrome since after three years of treatment the clinical response was favourable and the patients showed no adverse clinical effects.

Characterization of enzymatic deficiencies of branched chain amino-acid catabolism in human fibroblasts by genetic complementation.

Leucine and Isoleucine metabolism in cultured skin fibroblasts from patients with leucinosis, beta-Ketothiolase deficiency, propionic, methylmalonic and isovaleric acidemia, was compared with that in normal fibroblasts. A simple assay was developed using (U14C) Isoleucine and (U14C) Leucine as substrates. Radioactive incorporation into protein aminoacids were measured. The (U14C) branched chain aminoacid incorporation into proteins provides an estimation of the protein synthesis and the incorporation ratio (14C) Aspartate + (14C) Glutamate/(14C) branched chain aminoacid, measures the integrity of the metabolic pathway. Complementation tests permits to characterize the genetic defect. The incorporation ratio was significantly decreased in blocked pathways, namely in leucinosis and isovaleric acidemia in the presence of (U14C) Leucine and in Leucinosis, beta-Ketothiolase deficiency, propionic and methylmalonic acidemia in the presence of (U14C) Isoleucine. There was a significant restoration of activity in mutant strains with distinct genetic defects after polyethylene-glycol fusion. This assay provides a valuable tool to screen for enzymatic deficiencies of branched chain aminoacid catabolism.