Inhibition of energy metabolism by 2-methylacetoacetate and 2-methyl-3-hydroxybutyrate in cerebral cortex of developing rats.

Mitochondrial beta-ketothiolase and 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) deficiencies are inherited neurometabolic disorders affecting isoleucine catabolism. Biochemically, beta-ketothiolase deficiency is characterized by intermittent ketoacidosis and urinary excretion of 2-methyl-acetoacetate (MAA), 2-methyl-3-hydroxybutyrate (MHB) and tiglylglycine (TG), whereas in MHBD deficiency only MHB and tiglylglycine accumulate. Lactic acid accumulation and excretion are also observed in these patients, being more pronounced in MHBD-deficient individuals, particularly during acute episodes of decompensation. Patients affected by MHBD deficiency usually manifest severe mental retardation and convulsions, whereas beta-ketothiolase-deficient patients present encephalopathic crises characterized by metabolic acidosis, vomiting and coma. Considering that the pathophysiological mechanisms responsible for the neurological alterations of these disorders are unknown and that lactic acidosis suggests an impairment of energy production, the objective of the present work was to investigate the in vitro effect of MAA and MHB, at concentrations varying from 0.01 to 1.0 mmol/L, on several parameters of energy metabolism in cerebral cortex from young rats. We observed that MAA markedly inhibited CO2 production from glucose, acetate and citrate at concentrations as low as 0.01 mmol/L. In addition, the activities of the respiratory chain complex II and succinate dehydrogenase were mildly inhibited by MAA. MHB, at 0.01 mmol/L and higher concentrations, strongly inhibited CO2 production from all tested substrates, as well as the respiratory chain complex IV activity. The other activities of the respiratory chain were not affected by these metabolites. The data indicate a marked blockage in the Krebs cycle and a mild inhibition of the respiratory chain caused by MAA and MHB. Furthermore, MHB inhibited total and mitochondrial creatine kinase activities, which was prevented by the use of the nitric-oxide synthase inhibitor L-NAME and glutathione (GSH). These data indicate that the effect of MHB on creatine kinase was probably mediated by oxidation or other modification of essential thiol groups of the enzyme by nitric oxide and other by-products derived from this organic acid. In contrast, MAA did not affect creatine kinase activity. Taken together, these observations indicate that aerobic energy metabolism is inhibited by MAA and to a greater extent by MHB, a fact that may be related to lactic acidaemia occurring in patients affected by MHBD and beta-ketothiolase deficiencies. If the in vitro effects detected in the present study also occur in vivo, it is tempting to speculate that they may contribute, at least in part, to the neurological dysfunction found in these disorders.

3-hydroxyglutaric acid enhances glutamate uptake into astrocytes from cerebral cortex of young rats.

A predominantly neurological presentation is common in patients with glutaric acidemia type I (GA-I). 3-hydroxyglutaric acid (3-OHGA), which accumulates in affected patients, has recently been demonstrated to play a central role in the neuropathogenesis of this disease. In the present study, we investigated the in vitro effects of 3-OHGA at concentrations ranging from 10 to 1000 microM on various parameters of the glutamatergic system, such as the basal and potassium-induced release of [3H]glutamate by synaptosomes, as well as on Na+-dependent [3H]glutamate uptake by synaptosomes and astrocytes and Na+-independent [3H]glutamate uptake by synaptic vesicles from cerebral cortex of 30-day-old Wistar rats. First, we observed that exposure of cultured astrocytes to 3-OHGA for 20 h did not reduce their viability. Furthermore, 3-OHGA significantly increased Na+-dependent [3H]glutamate uptake by astrocytes by up to 80% in a dose-dependent manner at doses as low as 30 microM. This effect was not dependent on the presence of the metabolite during the uptake assay, since it occurred even when 3-OHGA was withdrawn from the medium after cultured cells had been exposed to the acid for approximately 1 h. All other parameters investigated were not influenced by this organic acid, indicating a selective action of 3-OHGA on astrocyte transporters. Although the exact mechanisms involved in 3-OHGA-stimulatory effect on astrocyte glutamate uptake are unknown, the present findings contribute to the understanding of the pathophysiology of GA-I, suggesting that astrocytes may protect neurons against excitotoxic damage caused by 3-OHGA by increasing glutamate uptake and therefore reducing the concentration of this excitatory neurotransmitter in the synaptic cleft.

Inhibition of the mitochondrial respiratory chain complex activities in rat cerebral cortex by methylmalonic acid.

Propionic and methylmalonic acidemic patients have severe neurologic symptoms whose etiopathogeny is still obscure. Since increase of lactic acid is detected in the urine of these patients, especially during metabolic decompensation when high concentrations of methylmalonate (MMA) and propionate (PA) are produced, it is possible that cellular respiration may be impaired in these individuals. Therefore, we investigated the effects of MMA and PA (1, 2.5 and 5mM), the principal metabolites which accumulate in these conditions, on the mitochondrial respiratory chain complex activities succinate: 2,6-dichloroindophenol (DCIP) oxireductase (complex II); succinate: cytochrome c oxireductase (complexII+CoQ+III); NADH: cytochrome c oxireductase (complex I+CoQ+complex III); and cytochrome c oxidase (COX) (complex IV) from cerebral cortex homogenates of young rats. The effect of MMA on ubiquinol: cytochrome c oxireductase (complex III) and NADH: ubiquinone oxireductase (complex I) activities was also tested. Control groups did not contain MMA and PA in the incubation medium. MMA significantly inhibited complex I+III (32-46%), complex I (61-72%), and complex II+III (15-26%), without affecting significantly the activities of complexes II, III and IV. However, by using 1mM succinate in the assay instead of the usual 16mM concentration, MMA was able to significantly inhibit complex II activity in the brain homogenates. In contrast, PA did not affect any of these mitochondrial enzyme activities. The effect of MMA and PA on succinate: phenazine oxireductase (soluble succinate dehydrogenase (SDH)) was also measured in mitochondrial preparations. The results showed significant inhibition of the soluble SDH activity by MMA (11-27%) in purified mitochondrial fractions. Thus, if the in vitro inhibition of the oxidative phosphorylation system is also expressed under in vivo conditions, a deficit of brain energy production might explain some of the neurological abnormalities found in patients with methylmalonic acidemia (MMAemia) and be responsible for the lactic acidemia/aciduria identified in some of them.