Mechanistic basis of hypermethioninemia.

Hypermethioninemia is a condition defined as elevated plasma methionine levels and may be a consequence of different conditions that include non-genetic and genetic causes. In severe cases, hypermethioninemia may lead to development of neurological and hepatic impairments, but mechanisms are still not well elucidated. Therefore, this review aims to reunite the knowledge acquired about the methionine-induced brain and liver toxicity focusing on the results obtained by studies from patients, in vitro experiments, and in vivo animal models. In general, some studies have shown that methionine decreases Na+,K+-ATPase activity, induces oxidative stress, increases acetylcholinesterase activity, and leads to dendritic spine downregulation in brain. Concerning to liver, hypermethioninemia seems to provoke changes in cell morphology, lipid accumulation, oxidative stress, inflammation, and ATP depletion. It is possible to infer that oxidative damage is one of the most important mechanisms responsible for methionine toxicity, since different studies showed that this amino acid induces oxidative stress in brain and liver tissues. Besides, reactive oxygen species may mediate other alterations induced by methionine, such as the reduction in brain Na+,K+-ATPase activity, and liver inflammation.

Gestational hypermethioninaemia alters oxidative/nitrative status in skeletal muscle and biomarkers of muscular injury and inflammation in serum of rat offspring.

In this study we evaluated oxidative/nitrative stress parameters (reactive oxygen species production, lipid peroxidation, sulfhydryl content, superoxide dismutase, catalase and nitrite levels), as well as total protein content in the gastrocnemius skeletal muscle of the offspring of rats that had been subjected to gestational hypermethioninaemia. The occurrence of muscular injury and inflammation was also measured by creatine kinase activity, levels of creatinine, urea and C-reactive protein and the presence of cardiac troponin I in serum. Wistar female rats (70-90 days of age) received methionine (2.68 µmol/g body weight) or saline (control) twice a day by subcutaneous injections during the gestational period (21 days). After the rats gave birth, pups were killed at the twenty-first day of life for removal of muscle and serum. Methionine treatment increased reactive oxygen species production and lipid peroxidation and decreased sulfhydryl content, antioxidant enzymes activities and nitrite levels, as well as total protein content in skeletal muscle of the offspring. Creatine kinase activity was reduced and urea and C-reactive protein levels were increased in serum of pups. These results were accompanied by reduced muscle mass. Our findings showed that maternal gestational hypermethioninaemia induced changes in oxidative/nitrative status in gastrocnemius skeletal muscle of the offspring. This may represent a mechanism which can contribute to the myopathies and loss of muscular mass that is found in some hypermethioninaemic patients. In addition, we believe that these results may be relevant as gestational hypermethioninaemia could cause damage to the skeletal muscle during intrauterine life.

Development of an animal model for gestational hypermethioninemia in rat and its effect on brain Na⁺,K⁺-ATPase/Mg²âº-ATPase activity and oxidative status of the offspring.

In the present study we developed a chemically induced experimental model for gestational hypermethioninemia in rats and evaluated in the offspring the activities of Na(+),K(+)-ATPase and Mg(2+)-ATPase, as well as oxidative stress parameters, namely sulfhydryl content, thiobarbituric acid-reactive substances and the antioxidant enzymes superoxide dismutase and catalase in encephalon. Serum and encephalon levels of methionine and total homocysteine were also evaluated in mother rats and in the offspring. Pregnant Wistar rats received two daily subcutaneous injections of methionine throughout the gestational period (21 days). During the treatment, a group of pregnant rats received dose 1 (1.34 µmol methionine/g body weight) and the other one received dose 2 (2.68 µmol methionine/g body weight). Control group received saline. After the rats give birth, a first group of pups was killed at the 7th day of life and the second group at the 21th day of life for removal of serum and encephalon. Mother rats were killed at the 21th day postpartum for removal of serum and encephalon. Both doses 1 and 2 increased methionine levels in encephalon of the mother rats and dose 2 increased methionine levels in encephalon of the offspring. Maternal hypermethioninemia also decreased the activities of Na(+),K(+)-ATPase, Mg(2+)-ATPase and catalase, as well as reduced total sulfhydryl content in the encephalon of the pups. This chemical model seems to be appropriate for studies aiming to investigate the effect of maternal hypermethioninemia on the developing brain during gestation in order to clarify possible neurochemical changes in the offspring.

Mild hyperhomocysteinemia reduces the activity and immunocontent, but does not alter the gene expression, of catalytic α subunits of cerebral Na+,K+-ATPase.

Na(+),K(+)-ATPase is a membrane protein which plays a key role in the maintenance of ion homeostasis that is necessary to neuronal excitability, secondary transport and neurotransmitter uptake. Mild hyperhomocysteinemia leads to several clinical manifestations and particularly cerebral diseases; however, little is known about the mechanisms of homocysteine on cerebral Na(+),K(+)-ATPase. In the present study, we investigated the effect of mild hyperhomocysteinemia on the activity, the immunocontent of catalytic subunits (α1, α2, and α3) and the gene expression of this enzyme. We used the experimental model of mild hyperhomocysteinemia that was induced by homocysteine administration (0.03 µmol/g of body weight) twice a day, from the 30th to the 60th postpartum day. Controls received saline in the same volumes. Results showed that mild hyperhomocysteinemia significantly decreased the activity and the immunocontent of the α 1 and α 2 subunits of the Na(+),K(+)-ATPase in cerebral cortex and hippocampus of adult rats. On the other hand, we did not observe any change in levels of Na(+),K(+)-ATPase mRNA transcripts in such cerebral structures of rats after chronic exposure to homocysteine. The present findings support that the homocysteine modulates the Na(+),K(+)-ATPase and this could be associated, at least in part, with the risk to the development of cerebral diseases in individuals with mild hyperhomocysteinemia.

Maternal Hypermethioninemia Affects Neurons Number, Neurotrophins Levels, Energy Metabolism, and Na+,K+-ATPase Expression/Content in Brain of Rat Offspring.

In the current study, we verified the effects of maternal hypermethioninemia on the number of neurons, apoptosis, nerve growth factor, and brain-derived neurotrophic factor levels, energy metabolism parameters (succinate dehydrogenase, complex II, and cytochrome c oxidase), expression and immunocontent of Na+,K+-ATPase, edema formation, inflammatory markers (tumor necrosis factor-alpha and interleukin-6), and mitochondrial hydrogen peroxide levels in the encephalon from the offspring. Pregnant Wistar rats were divided into two groups: the first one received saline (control) and the second group received 2.68 µmol methionine/g body weight by subcutaneous injections twice a day during gestation (approximately 21 days). After parturition, pups were killed at the 21st day of life for removal of encephalon. Neuronal staining (anti-NeuN) revealed a reduction in number of neurons, which was associated to decreased nerve growth factor and brain-derived neurotrophic factor levels. Maternal hypermethioninemia also reduced succinate dehydrogenase and complex II activities and increased expression and immunocontent of Na+,K+-ATPase alpha subunits. These results indicate that maternal hypermethioninemia may be a predisposing factor for damage to the brain during the intrauterine life.