Ana Maria Brusque

Methylmalonate administration decreases Na+,K+-ATPase activity in cerebral cortex of rats.

Buffered methylmalonate (MMA) was injected s.c. into rats twice a day at 8 h intervals from 5 to 25 days of age (chronic treatment), or into 10-day-old rats three times a day at 1 h intervals (acute treatment). Control rats received saline in the same volumes. Na+, K+-ATPase and Mg2+-ATPase activities were determined in the synaptic plasma membranes from cerebral cortex of rats. Na+, K+-ATPase activity was reduced by 30–40% in MMA-treated rats, whereas Mg2+-ATPase activity was not. In contrast, MMA at final concentrations ranging from 0.1 to 2.0 mM had no in vitro effect on these enzyme activities. However, when brain homogenates were incubated with 2 mM MMA before membrane preparation, Na+, K+-ATPase activity was decreased by 44%. Furthermore, this reduction was totally prevented by the simultaneous addition of glutathione and MMA, suggesting that oxidation of thiol groups or other oxidative damage to the enzyme could be responsible for this effect.

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.

Effect of chemically induced propionic acidemia on neurobehavioral development of rats.

High levels of propionic acid (PPA) comparable to those of human propionic acidemia were achieved in blood (1-5 mmol/l) and brain (1 micromol/g) of rats by administering saline-buffered propionate (pH 7.4) subcutaneously twice a day from the 6th to the 28th day of life. PPA doses ranged from 1.44 to 1.92 micromol/g body weight as a function of animal age. Control rats were treated with saline in the same volumes. Growth and development of physical landmarks were assessed by monitoring the following parameters daily: body weight, upper incisor eruption, eye opening, and hair coat. Development of some reflexes was also monitored, and a specific subset of motor skills was evaluated at days 14 and 21 of life by the free-fall righting test and the spontaneous alternation test. Chronic PPA administration had no effect on body weight, cerebral cortex weight, or cerebellum weight, but caused slight but significant delays in the day of appearance of hair coat and eye opening, indicating an effect of PPA on the development of physical parameters. Free-fall righting was impaired in PPA-treated animals. On the other hand, PPA administration had no effect on the performance of the animals in the spontaneous alternation tests. Long-term effects of early PPA administration were investigated by assessing animal performance in an aversive task (two-way shuttle avoidance task) and in a nonaversive (open-field task) behavioral task at 60 days of age. PPA-treated rats did not habituate to the open field, and presented a lack of retention of the shuttle-avoidance task. Our results suggest that early postnatal PPA administration to rats alters normal development and induces long-term behavioral deficits in aversive and nonaversive tasks.

Inhibition of Na+, K+-ATPase from rat brain cortex by propionic acid

BUFFERED propionic acid was injected s.c. into rats twice a day at 8 h intervals from the 6 to 21 days of age. Control rats received saline in the same volumes. The animals were weighed and killed by decapitation at 23 days. Whole brain and cerebral cortex were weighed and synaptic plasma membranes were prepared from cortex for the determination of Na+,K+-ATPase and Mg2+-ATPase activities. Body, whole brain and cortical weights were similar in the two groups, suggesting that propionic acid does not cause malnutrition in rats. Na+,K+-ATPase activity was significantly reduced by 30% in membranes from the propionate-treated group, whereas Mg2+-ATPase activity was not. In another set of experiments, synaptic plasma membranes were prepared from cerebral cortex of 23-day-old rats and incubated with propionic acid at final concentrations ranging from 0.1 to 2.0 mM. Na+,K+-ATPase activity, but not Mg2+-ATPase activity, was inhibited by 22–32%. Since propionic acid concentrations in plasma of chronically treated rats and of propionic acidaemic children are of the same order of magnitude as those tested in vitro, the results suggest that the inhibition of Na+,K+-ATPase activity may be related to the neurological dysfunction of patients affected by propionic acidaemia.

Serum and liver lipids in rats and chicks fed with diets containing different oils.

OBJECTIVES Because dietary fat composition is determinant for serum cholesterol level, which is related to cardiovascular disease, we evaluated the effects of diets containing saturated (coconut oil) or polyunsaturated fatty acids (soybean oil) supplemented or not with dietary cholesterol on serum and liver lipid composition in two animal species. METHODS Male Wistar rats (21 d old) were assigned to one of seven groups and fed with commercial diet or diets containing 5% or 20% soybean oil or 20% coconut oil with or without 1% cholesterol. Chicks were assigned to one of four groups and fed with diets containing 15% soybean oil or 15% coconut oil with or without 1% cholesterol. RESULTS In rats, the accumulations of hepatic cholesterol and triacylglycerols were higher in the group fed 20% soybean oil and 1% cholesterol than in the group fed 20% coconut fat and 1% cholesterol. The highest serum levels of cholesterol and triacylglycerols were observed in the group fed coconut oil and cholesterol, compared with the group fed soybean oil and cholesterol. Triacylglycerol, high-density lipoprotein, and total cholesterol serum levels increased with diet containing coconut oil and cholesterol. In chicks, the highest hepatic cholesterol accumulation occurred in the group fed 15% coconut fat and 1% cholesterol. Total and high-density lipoprotein cholesterol levels increased with diet containing coconut oil and cholesterol, although none of these diets modified serum triacylglycerol levels. CONCLUSIONS The type of experimental animal model and the diet composition influence lipid metabolism.

Chronic early leucine administration induces behavioral deficits in rats

Sustained levels of leucine comparable to those of human Maple Syrup Urine Disease (MSUD) were achieved in blood and brain of rats by subcutaneous leucine administration twice a day from the 6th to the 28th day of life. Control rats were treated with saline in the same volumes. Behavioral studies using aversive and nonaversive tasks were performed during adult age. Chronic early leucine treatment impaired acquisition of a two-way shuttle avoidance task and altered habituation to an open field. Our results suggest that early postnatal leucine administration induces long-lasting behavioral deficits.

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.

Dehydroepiandrosterone increases synaptosomal glutamate release and improves the performance in inhibitory avoidance task

Dehydroepiandrosterone (DHEA) exerts multiple effects in the rodent central nervous system (CNS), mediated through its nongenomic actions on several neurotransmitter systems, increasing neuronal excitability, modulating neuronal plasticity and presenting neuroprotective properties. It has been demonstrated that DHEA is a potent modulator of GABA(A), NMDA and Sigma receptors. In the present study, we investigated the effect of DHEA on (i) basal- and K(+)-stimulated l-[(3)H]glutamate release from synaptosomes (both in vitro and ex vivo), (ii) synaptosomal l-[(3)H]glutamate uptake (in vitro), and (iii) an inhibitory avoidance task (in vivo). The results indicated that DHEA in vitro increased glutamate release by 57%, and its intracerebroventricular infusion increased the basal-[(3)H]glutamate release by 15%. After 30 min of intraperitoneal administration, DHEA levels in the serum or CSF increased 33 and 21 times, respectively. Additionally, DHEA, intraperitoneally administrated 30 min before training, improved memory for inhibitory avoidance task. Concluding, DHEA could improve memory on an inhibitory avoidance task, perhaps due to its ability to physiologically strength the glutamatergic tonus by increasing glutamate release.

In vitro effects of selenite and mercuric chloride on liver thiobarbituric acid–reactive substances and non-protein thiols from rats: Influences of dietary cholesterol and polyunsaturated and saturated fatty acids☆

OBJECTIVE We measured the in vitro effects of mercuric chloride (Hg2+) and selenite (Se4+) on hepatic 2-thiobarbituric acid-reactive substances (TBARS) and non-protein sulfhydryl (NPSH) levels of rats fed diets enriched with polyunsaturated or saturated fatty acids with and without cholesterol. METHODS Male Wistar rats (21 d old) were assigned to one of four groups and fed diets containing 20% soybean oil, 20% soybean oil plus 1% cholesterol, 20% coconut oil, or coconut oil plus 1% cholesterol. After the feeding period (6 wk), body weight gain was equal in all groups. TBARS levels and NPSH content were measured after in vitro exposure to mercuric chloride (100 microM) and sodium selenite (25 microM) for 1 h. RESULTS The lipid peroxidation, measured as TBARS levels in the control group, were statistically higher in hepatic homogenates of rats fed diets containing soybean oil than in groups fed coconut oil (P = 0.009). However, cholesterol supplementation did not change TBARS levels. Selenite alone did not modify TBARS production, whereas mercury alone significantly increased TBARS levels. Moreover, Se4+ protected against mercury-induced lipid peroxidation only in rats fed diets containing coconut oil. In the control group, dietary fat acids did not change NPSH levels. Selenite produced higher oxidative effects toward NPSH content, whereas Hg2+ decreased NPSH levels only in liver from rats fed diets containing soybean oil. NPSH levels were higher after concomitant exposure to Se4+ and Hg2+ chloride that after exposure to Se4+ alone, suggesting an interaction between Hg2+ and Se4+. Catalase activity was higher in animals fed diets containing soybean oil. Dietary cholesterol decreased glutathione peroxidase activity. CONCLUSION Together these results indicated that the protective effect of Se4+ against mercury-induced lipid peroxidation depends on dietary fat saturation.

Ganglioside alterations in the central nervous system of rats chronically injected with methylmalonic and propionic acids.

Neurological dysfunction and structural cerebral abnormalities are commonly found in patients with methylmalonic and propionic acidemia. However, the mechanisms underlying the neuropathology of these disorders are poorly understood. We have previously demonstrated that methylmalonic and propionic acids induce a significant reduction of ganglioside N-acetylneuraminic acid in the brain of rats subjected to chronic administration of these metabolites. In the present study, we investigated the in vivo effects of chronic administration of methylmalonic (MMA) and propionic (PA) acids (from the 6th to the 28th day of life) on the distribution and composition of gangliosides in the cerebellum and cerebral cortex of rats. Control rats were treated with the same volumes of saline. It was first verified that MMA and PA treatment did not modify body, cerebellum, or cortical weight, nor the ganglioside concentration in the cerebral cortex of the animals. In contrast, a significant reduction in total ganglioside content in the cerebellum of approximately 20–30% and 50% of control levels occurred in rats injected with MMA and PA, respectively. Moreover, chronic MMA and PA administration did not interfere with the ganglioside pattern in the cerebral cortex, whereas the distribution of individual gangliosides was altered in the cerebellum of MMA- and PA-treated animals. Rats injected with MMA demonstrated a marked decrease in GM1 and GD3, whereas chronic PA treatment provoked a significant reduction of all ganglioside species, with the exception of an increase in GM2. Since gangliosides are closely related to the dendritic surface and other neural membranes, indirectly reflecting synaptogenesis, these ganglioside abnormalities may be associated with the brain damage found in methylmalonic and propionic acidemias.