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Biomedical subjects

C S Dutra-Filho

Publications and source records attributed to C S Dutra-Filho.

At least 19 recordsLinked to original sources
PubMed2002

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.

Animals↗
PubMed2001

Effects of methylmalonic and propionic acids on glutamate uptake by synaptosomes and synaptic vesicles and on glutamate release by synaptosomes from cerebral cortex of rats.

Neurological dysfunction is common in patients with methylmalonic and propionic acidemias. However, the mechanisms underlying the neuropathology of these disorders are far from understood. In the present study we investigated the in vitro effects of methylmalonic (MMA) and propionic (PA) acids at various concentrations (1 microM-5 mM) on three parameters of the glutamatergic system, namely the basal and potassium-induced release of L-[3H]glutamate by synaptosomes, Na+-dependent L-[3H]glutamate uptake by synaptosomes and Na+-independent L-[3H]glutamate uptake by synaptic vesicles from cerebral cortex of male adult Wistar rats. The results showed that MMA significantly increased potassium-induced but not basal L-[3H]glutamate release from synaptosomes with no alteration in synaptosomal L-[3H]glutamate uptake. A significant reduction of L-[3H]glutamate incorporation into vesicles caused by MMA was also detected. In contrast, PA had no effect on these parameters. These findings indicate that MMA alters the glutamatergic system. Although additional studies are necessary to evaluate the importance of these observations for the neuropathology of methylmalonic acidemia, it is possible that the effects elicited by MMA may lead to excessive glutamate concentrations at the synaptic cleft, a fact that may explain previous in vivo and in vitro findings associating MMA with excitotoxicity.

Animals↗
PubMed2001

Inhibition of glutamate uptake into synaptic vesicles from rat brain by 3-nitropropionic acid in vitro.

The exact mechanisms by which 3-nitropropionic acid (3-NP), a naturally occurring plant and fungal neurotoxin, exerts its neurotoxic effects are not fully understood. However, blockage of ATP synthesis by the irreversible inhibition of succinate dehydrogenase activity, increased production of free radicals, and secondary excitotoxicity have been implicated in its actions. In the present study, synaptic vesicle preparations from brain of adult rats were incubated with 3-NP at final concentrations ranging from 0.01 to 10 mM for the determination of glutamate uptake. The effect of 3-NP on gamma-aminobutyric acid (GABA) and glycine uptake was also studied. Glutamate incorporation into vesicles was inhibited by 3-NP in a dose-dependent manner, whereas doses of up to 10 mM neurotoxin did not affect GABA or glycine uptake. Moreover, 3-NP did not inhibit the ATPase activity of synaptic vesicles. These findings indicate that low concentrations of 3-NP are able to selectively prevent vesicular glutamate storage, and this may represent at least one of the mechanisms responsible for the neurotoxic effects of 3-NP.

Adenosine Triphosphatases↗
PubMed2001

Reduction of large neutral amino acid levels in plasma and brain of hyperleucinemic rats.

Neurological dysfunction is common in patients with maple syrup urine disease (MSUD). However, the mechanisms underlying the neuropathology of this disorder are poorly known. In the present study we investigated the effect of acute hyperleucinemia on plasma and brain concentrations of amino acids. Fifteen-day-old rats were injected subcutaneously with 6 micromol L-leucine per gram body weight. Controls received saline in the same volumes. The animals were sacrificed 30--120 min after injection, blood was collected and their brain rapidly removed and homogenized. The amino acid concentrations were determined by HPLC using orthophtaldialdehyde for derivatization and fluorescence for detection. The results showed significant reductions of the large neutral amino acids (LNAA) L-phenylalanine, L-tyrosine, L-isoleucine, L-valine and L-methionine, as well as L-alanine, L-serine and L-histidine in plasma and of L-phenylalanine, L-isoleucine, L-valine and L-methionine in brain, as compared to controls. In vitro experiments using brain slices to study the influence of leucine on amino acid transport and protein synthesis were also carried out. L-Leucine strongly inhibited [14C]-L-phenylalanine transport into brain, as well as the incorporation of the [14C]-amino acid mixture, [14C]-L-phenylalanine and [14C]-L-lysine into the brain proteins. Although additional studies are necessary to evaluate the importance of these effects for MSUD, considering previous findings of reduced levels of LNAA in plasma and CSF of MSUD patients during crises, it may be speculated that a decrease of essential amino acids in brain may lead to reduction of protein and neurotransmiter synthesis in this disorder.

Amino Acids↗
PubMed2001

Nitric oxide synthase inhibition by L-NAME prevents the decrease of Na+,K+-ATPase activity in midbrain of rats subjected to arginine administration.

In the present study we investigated the effect of acute administration of L-arginine on Na(+),K(+)-ATPase and Mg(2+)-ATPase activities and on some parameters of oxidative stress (chemiluminescence and total radical-trapping antioxidant parameter-TRAP) in midbrain of adult rats. We also tested the effect of L-NAME on the effects produced by arginine. Sixty-day-old rats were treated with an acute intraperitoneal injection of saline (group I, control), arginine (0.8 g/kg) (group II), L-NAME (2 mg/kg) (group III) or arginine (0.8 g/kg) plus L-NAME (2 mg/kg) (group IV). Na(+),K(+)-ATPase activity was significantly reduced in the arginine-treated rats, but was not affected by other treatments. In contrast, Mg(2+)-ATPase activity was not altered by any treatment. Furthermore, chemiluminescence was significantly increased and TRAP was significantly decreased in arginine-treated rats, whereas the simultaneous injection of L-NAME prevented these effects. These results demonstrate that in vivo arginine administration reduces Na(+),K(+)-ATPase activity possibly through free radical generation induced by NO formation.

Animals↗
PubMed2001

L-pyroglutamic acid inhibits energy production and lipid synthesis in cerebral cortex of young rats in vitro.

In the present study we investigated the effects of L-pyroglutamic acid (PGA), which predominantly accumulates in the inherited metabolic diseases glutathione synthetase deficiency (GSD) and gamma-glutamylcysteine synthetase deficiency (GCSD), on some in vitro parameters of energy metabolism and lipid biosynthesis. We evaluated the rates of CO2 production and lipid synthesis from [U-14C]acetate, as well as ATP levels and the activities of creatine kinase and of the respiratory chain complexes I-IV in cerebral cortex of young rats in the presence of PGA at final concentrations ranging from 0.5 to 3 mM. PGA significantly reduced brain CO2 production by 50% at the concentrations of 0.5 to 3 mM, lipid biosynthesis by 20% at concentrations of 0.5 to 3 mM and ATP levels by 52% at the concentration of 3 mM. Regarding the enzyme activities, PGA significantly decreased NADH:cytochrome c oxireductase (complex I plus CoQ plus complex III) by 40% at concentrations of 0.5-3.0 mM and cytochrome c oxidase activity by 22-30% at the concentration of 3.0 mM, without affecting the activities of succinate dehydrogenase, succinate:DCPIP oxireductase (complex II), succinate:cytochrome c oxireductase (complex II plus CoQ plus complex III) or creatine kinase. The results strongly indicate that PGA impairs brain energy production. If these effects also occur in humans, it is possible that they may contribute to the neuropathology of patients affected by these diseases.

Animals↗
PubMed2001

Reduced Na(+), K(+)-ATPase activity in erythrocyte membranes from patients with phenylketonuria.

Na(+), K(+)-ATPase activity was determined in erythrocyte membranes from 12 phenylketonuric patients of both sexes, aged 8.8 +/- 5.0 y, with plasma phenylalanine levels of 0.64 +/- 0.31 mM. The in vitro effects of phenylalanine and alanine on the enzyme activity in erythrocyte membranes from healthy individuals were also investigated. We observed that Na(+), K(+)-ATPase activity was decreased by 31% in erythrocytes from phenylketonuric patients compared with normal age-matched individuals (p < 0.01). We also observed a significant negative correlation between erythrocyte Na(+), K(+)-ATPase activity and plasma phenylalanine levels (r = -0.65; p < 0.05). All PKU patients with plasma phenylalanine levels higher than 0.3 mM had erythrocyte Na(+), K(+)-ATPase activity below the normal range. Phenylalanine inhibited in vitro erythrocyte Na(+), K(+)-ATPase activity by 22 to 34%, whereas alanine had no effect on this activity. However, when combined with phenylalanine, alanine prevented Na(+) K(+)-ATPase inhibition. Considering that reduction of Na(+), K(+)-ATPase activity occurs in various neurodegenerative disorders leading to neuronal loss, our previous observations showing a significant reduction of Na(+), K(+)-ATPase activity in brain cortex of rats subjected to experimental phenylketonuria and the present results, it is proposed that determination of Na(+), K(+)-ATPase activity in erythrocytes may be a useful peripheral marker for the neurotoxic effect of phenylalanine in phenylketonuria.

Adolescent↗
PubMed2001

Chronic postnatal administration of methylmalonic acid provokes a decrease of myelin content and ganglioside N-acetylneuraminic acid concentration in cerebrum of young rats.

Levels of methylmalonic acid (MMA) comparable to those of human methylmalonic acidemia were achieved in blood (2-2.5 mmol/l) and brain (1.35 umol/g) of rats by administering buffered MMA, pH 7.4, subcutaneously twice a day from the 5th to the 28th day of life. MMA doses ranged from 0.76 to 1.67 umol/g as a function of animal age. Control rats were treated with saline in the same volumes. The animals were sacrificed by decapitation on the 28th day of age. Blood was taken and the brain was rapidly removed. Medulla, pons, the olfactory lobes and cerebellum were discarded and the rest of the brain ("cerebrum") was isolated. Body and "cerebrum" weight were measured, as well as the cholesterol and triglyceride concentrations in blood and the content of myelin, total lipids, and the concentrations of the lipid fractions (cholesterol, glycerolipids, phospholipids and ganglioside N-acetylneuraminic acid (ganglioside-NANA)) in the "cerebrum". Chronic MMA administration had no effect on body or "cerebrum" weight, suggesting that the metabolites per se neither affect the appetite of the rats nor cause malnutrition. In contrast, MMA caused a significant reduction of plasma triglycerides, but not of plasma cholesterol levels. A significant diminution of myelin content and of ganglioside-NANA concentration was also observed in the "cerebrum". We propose that the reduction of myelin content and ganglioside-NANA caused by MMA may be related to the delayed myelination/cerebral atrophy and neurological dysfunction found in methylmalonic acidemic children.

Animals↗
PubMed2001

Inhibition of in vitro CO2 production and lipid synthesis by 2-hydroxybutyric acid in rat brain.

2-Hydroxybutyric acid appears at high concentrations in situations related to deficient energy metabolism (e.g., birth asphyxia) and also in inherited metabolic diseases affecting the central nervous system during neonatal development, such as "cerebral" lactic acidosis, glutaric aciduria type II, dihydrolipoyl dehydrogenase (E3) deficiency, and propionic acidemia. The present study was carried out to determine the effect of 2-hydroxybutyric acid at various concentrations (1-10 mM) on CO2 production and lipid synthesis from labeled substrates in cerebral cortex of 30-day-old Wistar rats in vitro. CO2 production was significantly inhibited (30-70%) by 2-hydroxybutyric acid in cerebral cortex prisms, in total homogenates and in the mitochondrial fraction. We also demonstrated a significant inhibition of lipid synthesis (20-45%) in cerebral cortex prisms and total homogenates in the presence of 2-hydroxybutyric acid. However, no inhibition of lipid synthesis occurred in homogenates free of nuclei and mitochondria. The results indicate an impairment of mitochondrial energy metabolism caused by 2-hydroxybutyric acid, a fact that may secondarily lead to reduction of lipid synthesis. It is possible that these findings may be associated with the neuropathophysiology of the situations where 2-hydroxybutyric acid is accumulated.

Analysis of Variance↗
PubMed2000

Inhibition of glutamate uptake into synaptic vesicles of rat brain by the metabolites accumulating in maple syrup urine disease.

Maple syrup urine disease is an inherited metabolic disorder characterized by tissue accumulation of branched-chain amino acids and their corresponding keto acids in the affected children. Although this disorder is predominantly characterized by neurological symptoms, only few studies were carried out to investigate its neuropathology. In this study we investigated the effect of the metabolites accumulating in maple syrup urine disease on the in vitro uptake of [3H]glutamate by synaptic vesicles of rat brain. Synaptic vesicle preparations from whole brain of male adult Wistar rats (200-250 g) were incubated with the branched-chain amino acids and their corresponding keto acids at final concentrations ranging from 0.25 to 10 mM for the determination of glutamate uptake. Glutamate uptake was significantly inhibited by L-leucine, L-isoleucine, L-2-ketoisocaproic acid and L-2-keto-3-methylvaleric acid by approximately 60%, whereas L-valine and L-2-ketoisovaleric acid showed no effect. We also verified that the metabolites probably act by competitive inhibition. Therefore, it is possible that extracellular glutamate levels may be increased in maple syrup urine disease and that excitotoxicity may be involved in the neuropathology of this disorder.

Animals↗
PubMed2000

Propionic and L-methylmalonic acids induce oxidative stress in brain of young rats.

The in vitro effects of propionic and L-methylmalonic acids on some parameters of oxidative stress were investigated in the cerebral cortex of 21-day-old rats. Chemiluminescence, thiobarbituric acid-reactive substances (TBA-RS) and total radical-trapping antioxidant capacity (TRAP) were measured in brain tissue homogenates in the presence of propionic or L-methylmalonic acids at concentrations ranging from 1 to 10mM. Both acids significantly increased chemiluminescence and TBA-RS and decreased TRAP, indicating a simulation of lipid peroxidation and a reduction of tissue antioxidant potential. Other organic acids tested which accumulate in some organic acidemias (suberic, sebacic, adipic, 3-methylglutaric and 4-hydroxybutyric acids) did not affect these parameters. This study provides evidence that free radical generation may participate in the neurological dysfunction of propionic and methylmalonic acidemias.

Animals↗
PubMed2000

Quinolinic acid inhibits glutamate uptake into synaptic vesicles from rat brain.

Quinolinic acid (QA) is an endogenous and potent neurotoxin associated with the neurotoxicity of various common diseases. The uptake of neurotransmitters into synaptic vesicles is an important event involved in the storage and release of neurotransmitters by vesicles. The influence of QA on the uptake of glutamate, GABA and glycine into rat brain synaptic vesicles was investigated. QA (0.3-10 mM) significantly inhibited (>50%) the uptake of glutamate into synaptic vesicles, whereas QA at concentrations up to 10 mM had no significant effect on GABA or glycine uptake. Such results indicate that QA is able to selectively inhibit the vesicular uptake of glutamate, without interfering with the uptake of the inhibitory neurotransmitters GABA and glycine. These findings might be related to the neurotoxic effects of QA in the brain.

Animals↗
PubMed2000

Effect of phenylalanine and p-chlorophenylalanine on Na+, K+-ATPase activity in the synaptic plasma membrane from the cerebral cortex of rats.

Na+, K+-ATPase activity was measured in synaptic plasma membrane from cerebral cortex of Wistar rats subjected to experimental phenylketonuria, i.e., chemical hyperphenylalaninemia induced by subcutaneous administration of 5.2 micromol phenylalanine / g body weight (twice a day) plus 0.9 micromol p-chlorophenylalanine / g body weight (once a day). The treatment was performed from the 6th to the 14th postpartum day and rats were killed 12 h after the last injection. Synaptic plasma membrane from cerebral cortex was prepared by a discontinuous density sucrose gradient for Na+, K+-ATPase activity determination. The results showed that the enzyme activity was decreased by 30% in animals subjected to experimental phenylketonuria when compared to control. The in vitro effects of the drugs on Na+, K+-ATPase activity were also investigated. Phenylalanine and p-chlorophenylalanine inhibited the enzyme activity and this inhibition was reversed by alanine. In addition, competition between phenylalanine and p-chlorophenylalanine for binding to the enzyme was observed, suggesting a common binding site for these substances. Our results suggest that reduction of Na+, K+-ATPase activity may be one of the mechanisms related to the brain dysfunction observed in human PKU.

Animals↗
PubMed2000

Platelet Na+, K+-ATPase activity as a possible peripheral marker for the neurotoxic effects of phenylalanine in phenylketonuria.

The in vitro effects of phenylalanine or alanine alone or combined on Na+, K+-ATPase activity in membranes from human platelets were investigated. The enzyme activity was assayed in membranes prepared from platelet-rich plasma of healthy donors. Phenylalanine or alanine were added to the assay to final concentrations of 0.3 to 1.2 mM, similar to those found in plasma of phenylketonuric patients. Phenylalanine inhibited Na+, K+-ATPase activity by 20-50% [F(4,25)=11.47 ; p<0.001]. Alanine had no effect on Na+, K+-ATPase activity but when combined with phenylalanine prevented the enzyme inhibition. These results, allied to others previously reported on brain Na+, K+-ATPase activity, may reflect a general inhibitory effect of phenylalanine on this important enzyme activity. Therefore, it is possible that measurement of Na+, K+-ATPase activity in platelets from PKU patients may be a useful peripheral marker for the neurotoxic effects of phenylalanine.

Alanine↗
PubMed2000

Inhibition of energy production in vitro by glutaric acid in cerebral cortex of young rats.

The present study investigated the effects of glutaric acid (GA), which predominantly accumulates in glutaric acidemia type I (GA-I), on some in vitro parameters of energy metabolism in cerebral cortex of rats. We first evaluated CO2 production from [U-14C] acetate, as well as ATP levels in brain of young Wistar rats. The effect of the acid on the activities of the respiratory chain complexes were also investigated. GA was tested at final concentrations ranging from 0.5 to 5.0 mM. GA significantly reduced brain CO2 production by 50% at the concentrations of 0.5 to 3.0 mM, ATP levels by 25% at the concentration of 3.0 mM, succinate:cytochrome C oxireductase (complex II plus CoQ plus complex III) by 25% at 5 mM concentration, and NADH:cytochrome C oxireductase (complex I plus CoQ plus complex Ill) by 25% at 2.5 and 5 mM concentrations. The results strongly indicate that GA impairs brain energy production. If these effects also occur in humans, it is possible that they may contribute to the neuropathology of patients affected by GA-I.

Acetates↗
PubMed1999

Proline administration decreases Na+,K+-ATPase activity in the synaptic plasma membrane from cerebral cortex of rats.

Buffered proline was injected subcutaneously into rats twice a day at 8 h intervals from the 6th to the 28th day of age. Control rats received saline in the same volumes. The animals were weighed and killed by decapitation 12 h after the last injection. Cerebral cortex was used for the determination of Na+,K+-ATPase and Mg2+-ATPase activities. Body, whole brain and cortical weights were similar in the two groups. Na+,K+-ATPase activity was significantly reduced (by 20%) in membranes from the proline-treated group compared to the controls, whereas Mg2+-ATPase activity was not affected by proline. In another set of experiments, synaptic plasma membranes were prepared from cerebral cortex of 29-day-old rats and incubated with proline at final concentrations ranging from 0.1 to 2.0 mM. Na+,K+-ATPase activity, but not Mg2+-ATPase activity, was inhibited by 20-30%. Since proline concentrations in plasma of chronically treated rats and of type 11 hyperprolinemic children are of the same order of magnitude as those tested in vitro, the results suggest that reduction of Na+,K+-ATPase activity may contribute to the neurological dysfunction found in some patients affected by type II hyperprolinemia.

Amino Acid Metabolism, Inborn Errors↗