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.

Inhibition of mitochondrial creatine kinase activity from rat cerebral cortex by methylmalonic acid.

Accumulation of methylmalonic acid (MMA) in tissues and biological fluids is the biochemical hallmark of patients affected by the neurometabolic disorder known as methylmalonic acidemia (MMAemia). Although this disease is predominantly characterized by severe neurological findings, the underlying mechanisms of brain injury are not totally established. In the present study, we investigated the effect of MMA, as well as propionic (PA) and tiglic (TA) acids, whose concentrations are also increased but to a lesser extend in MMAemia, on total (tCK), cytosolic (Cy-CK) and mitochondrial (Mi-CK) creatine kinase (CK) activities from cerebral cortex of 30-day-old Wistar rats. Total CK activity (tCK) was measured in whole cell homogenates, whereas Cy-CK and Mi-CK were determined, respectively, in cytosolic and mitochondrial preparations from rat cerebral cortex. We verified that tCK and Mi-CK activities were significantly inhibited by MMA at concentrations as low as 1 mM, in contrast to Cy-CK which was not affected by the presence of the acid in the incubation medium. Furthermore, PA and TA, at concentrations as high as 5 mM, did not alter CK activity. We also observed that the inhibitions provoked by MMA were fully prevented by pre-incubation of the homogenates with reduced glutathione, suggesting that the inhibitory effect of MMA was possibly mediated by oxidation of essential thiol groups of the enzyme. Considering the importance of CK for brain metabolism homeostasis, our results suggest that inhibition of this enzyme by increased levels of MMA may contribute to the neurodegeneration of patients affected by MMAemia and explain previous reports showing an impairment of brain energy metabolism and a reduction of brain phosphocreatine levels caused by MMA.

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.

[Nurses' concepts on their work in the clinical model of health care]. / Concepções de enfermeiras sobre seu trabalho no modelo clínico de atenção à saúde.

The aim of this study was to know the nurses' conception about the emphasis of the activities that they develop in the work process, in the clinic model assistance. This study is a qualitative research, with dialetic approach. To form the sample one nurse was chosen in each of the 17 units of a university hospital in Porto Alegre/RS. The data were collected through semi-structured interview. To analyze the data the material was classified in relevant structures, from which nine categories were established. The results showed that, although there is a prevalence of management activities of the care concerning the assistance activities, the nurses considered that these dimensions are indissociable in their work. It was concluded that the relation between assisting and managing are not excluding and that the management is one of the dimensions of nursing care.