Severe Hyperhomocysteinemia Decreases Respiratory Enzyme and Na(+)-K(+) ATPase Activities, and Leads to Mitochondrial Alterations in Rat Amygdala.

Severe hyperhomocysteinemia is caused by increased plasma levels of homocysteine (Hcy), a methionine derivative, and is associated with cerebral disorders. Creatine supplementation has emerged as an adjuvant to protect against neurodegenerative diseases, due to its potential antioxidant role. Here, we examined the effects of severe hyperhomocysteinemia on brain metabolism, and evaluated a possible neuroprotective role of creatine in hyperhomocysteinemia, by concomitant treatment with Hcy and creatine (50 mg/Kg body weight). Hyperhomocysteinemia was induced in young rats (6-day-old) by treatment with homocysteine (0.3-0.6 µmol/g body weight) for 23 days, and then the following parameters of rat amygdala were evaluated: (1) the activity of the respiratory chain complexes succinate dehydrogenase, complex II and cytochrome c oxidase; (2) mitochondrial mass and membrane potential; (3) the levels of necrosis and apoptosis; and (4) the activity and immunocontent of Na(+),K(+)-ATPase. Hcy treatment decreased the activities of succinate dehydrogenase and cytochrome c oxidase, but did not alter complex II activity. Hcy treatment also increased the number of cells with high mitochondrial mass, high mitochondrial membrane potential, and in late apoptosis. Importantly, creatine administration prevented some of the key effects of Hcy administration on the amygdala. We also observed a decrease in the activity and immunocontent of the α1 subunit of the Na(+),K(+)-ATPase in amygdala after Hcy- treatment. Our findings support the notion that Hcy modulates mitochondrial function and bioenergetics in the brain, as well as Na(+),K(+)-ATPase activity, and suggest that creatine might represent an effective adjuvant to protect against the effects of high Hcy plasma levels.

Lipid, Oxidative and Inflammatory Profile and Alterations in the Enzymes Paraoxonase and Butyrylcholinesterase in Plasma of Patients with Homocystinuria Due CBS Deficiency: The Vitamin B12 and Folic Acid Importance.

Cystathionine-ß-synthase (CBS) deficiency is the main cause of homocystinuria. Homocysteine (Hcy), methionine, and other metabolites of Hcy accumulate in the body of affected patients. Despite the fact that thromboembolism represents the major cause of morbidity in CBS-deficient patients, the mechanisms of cardiovascular alterations found in homocystinuria remain unclear. In this work, we evaluated the lipid and inflammatory profile, oxidative protein damage, and the activities of the enzymes paraoxonase (PON1) and butyrylcholinesterase (BuChE) in plasma of CBS-deficient patients at diagnosis and during the treatment (protein-restricted diet supplemented with pyridoxine, folic acid, betaine, and vitamin B12). We also investigated the effect of folic acid and vitamin B12 on these parameters. We found a significant decrease in HDL cholesterol and apolipoprotein A1 (ApoA-1) levels, as well as in PON1 activity in both untreated and treated CBS-deficient patients when compared to controls. BuChE activity and IL-6 levels were significantly increased in not treated patients. Furthermore, significant positive correlations between PON1 activity and sulphydryl groups and between IL-6 levels and carbonyl content were verified. Moreover, vitamin B12 was positively correlated with PON1 and ApoA-1 levels, while folic acid was inversely correlated with total Hcy concentration, demonstrating the importance of this treatment. Our results also demonstrated that CBS-deficient patients presented important alterations in biochemical parameters, possibly caused by the metabolites of Hcy, as well as by oxidative stress, and that the adequate adherence to the treatment is essential to revert or prevent these alterations.

Hyperprolinemia induces DNA, protein and lipid damage in blood of rats: antioxidant protection.

The present study investigated the effects of hyperprolinemia on oxidative damage to biomolecules (protein, lipids and DNA) and the antioxidant status in blood of rats. The influence of the antioxidants on the effects elicited by proline was also examined. Wistar rats received two daily injections of proline and/or vitamin E plus C (6th-28th day of life) and were killed 12h after the last injection. Results showed that hyperprolinemia induced a significant oxidative damage to proteins, lipids and DNA demonstrated by increased carbonyl content, malondialdehyde levels and a greater damage index in comet assay, respectively. The concomitant antioxidants administration to proline treatment completely prevented oxidative damage to proteins, but partially prevented lipids and DNA damage. We also observed that the non-enzymatic antioxidant potential was decreased by proline treatment and partially prevented by antioxidant supplementation. The plasma levels of vitamins E and C significantly increased in rats treated exogenously with these vitamins but, interestingly, when proline was administered concomitantly with vitamin E plus C, the levels of these vitamins were similar to those found in plasma of control and proline rats. Our findings suggest that hyperprolinemia promotes oxidative damage to the three major classes of macromolecules in blood of rats. These effects were accomplished by decrease in non-enzymatic antioxidant potential and decrease in vitamins administered exogenously, which significantly decreased oxidative damage to biomolecules studied. These data suggest that antioxidants may be an effective adjuvant therapeutic to limit oxidative damage caused by proline.

Reduction of butyrylcholinesterase activity in plasma from patients with disorders of propionate metabolism is prevented by treatment with L-carnitine and protein restriction.

OBJECTIVE: We investigated the relationship between butyrylcholinesterase (BuChE) activity and lipid oxidative damage in patients with disorders of propionate metabolism, before and after treatment with protein restriction and L-carnitine. DESIGN AND METHODS: BuChE activity and malondialdehyde (MDA) were measured in plasma from eight untreated patients (at diagnosis) and from seven patients under treatment with protein restriction and L-carnitne supplementation (100mg/kg/day). RESULTS: We verified a significant reduction of butyrylcholinesterase activity, as well as an increased MDA formation in plasma from untreated patients. However, treated patients presented MDA and BuChE activity similar to controls. Furthermore, butyrylcholinesterase activity was negatively correlated with MDA concentrations in these patients. CONCLUSION: The results suggest that an increased free radicals formation may be involved in the decrease of butyrylcholinesterase activity, possibly contributing to the neurological damage of these disorders, and that treatment with L-carnitine and low-protein diet possibly is able to prevent this damage.

Homocysteine induces oxidative-nitrative stress in heart of rats: prevention by folic acid.

Hyperhomocysteinemia is a risk factor for cardiovascular disease, stroke, and thrombosis; however, the mechanisms by which homocysteine triggers these dysfunctions are not fully understood. In the present study, we investigated the effect of chronic hyperhomocysteinemia on some parameters of oxidative stress, namely thiobarbituric acid reactive substances, an index of lipid peroxidation, 2',7'-dichlorofluorescein (H(2)DCF) oxidation, activities of antioxidant enzymes named superoxide dismutase and catalase, as well as nitrite levels in heart of young rats. We also evaluated the effect of folic acid on biochemical alterations elicited by hyperhomocysteinemia. Wistar rats received daily subcutaneous injection of homocysteine (0.3-0.6 µmol/g body weight) and/or folic acid (0.011 µmol/g body weight) from their 6th to the 28th day of life. Controls and treated rats were killed 1 h and/or 12 h after the last injection. Results showed that chronic homocysteine administration increases lipid peroxidation and reactive species production and decreases enzymatic antioxidant defenses and nitrite levels in the heart of young rats killed 1 h, but not 12 h after the last injection of homocysteine. Folic acid concurrent administration prevented homocysteine effects probable by its antioxidant properties. Our data indicate that oxidative stress is elicited by chronic hyperhomocystenemia, a mechanism that may contribute, at least in part, to the cardiovascular alterations characteristic of hyperhomocysteinemic patients. If confirmed in human beings, our results could propose that the supplementation of folic acid can be used as an adjuvant therapy in cardiovascular alterations caused by homocysteine.

Chronic hyperhomocysteinemia alters antioxidant defenses and increases DNA damage in brain and blood of rats: protective effect of folic acid.

We have previously demonstrated that acute hyperhomocysteinemia induces oxidative stress in rat brain. In the present study, we initially investigated the effect of chronic hyperhomocysteinemia on some parameters of oxidative damage, namely total radical-trapping antioxidant potential and activities of antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase), as well as on DNA damage in parietal cortex and blood of rats. We also evaluated the effect of folic acid on biochemical alterations elicited by hyperhomocysteinemia. Wistar rats received daily subcutaneous injection of Hcy (0.3-0.6 micromol/g body weight), and/or folic acid (0.011 micromol/g body weight) from their 6th to their 28th day of life. Twelve hours after the last injection the rats were sacrificed, parietal cortex and total blood was collected. Results showed that chronic homocysteine administration increased DNA damage, evaluated by comet assay, and disrupted antioxidant defenses (enzymatic and non-enzymatic) in parietal cortex and blood/plasma. Folic acid concurrent administration prevented homocysteine effects, possibly by its antioxidant and DNA stability maintenance properties. If confirmed in human beings, our results could propose that the supplementation of folic acid can be used as an adjuvant therapy in disorders that accumulate homocysteine.

Supplementation with vitamins E plus C or soy isoflavones in ovariectomized rats: effect on the activities of Na(+), K (+)-ATPase and cholinesterases.

Since a previous study demonstrated that ovariectomized rats present an activation of Na(+), K(+)-ATPase and acetylcholinesterase (AChE) activities, in the present study we investigated the influence of vitamins E plus C or soy isoflavones on the effects elicited by ovariectomy on the activities of these enzyme in hippocampus of ovariectomized rats. We also determined the effect of the same compounds on the reduction of serum butyrylcholinesterase (BuChE) activity caused by ovariectomy. Female adult Wistar rats were assigned to one of the following groups: sham (submitted to surgery without removal of the ovaries) and ovariectomized. Seven days after surgery, animals were treated for 30 days with a single daily intraperitoneous injection of vitamins E (40 mg/kg) plus C (100 mg/kg) or saline (control). In another set of experiments, the rats were fed for 30 days on a special diet with soy protein or a standard diet with casein (control). Rats were sacrificed after treatments and the hippocampus was dissected and serum was separated. Data demonstrate that vitamins E plus C reversed the activation of Na(+), K(+)-ATPase and AChE in hippocampus of ovariectomized rats. Conversely, soy protein supplementation reversed the increase of AChE activity, but not of Na(+), K(+)-ATPase activity, caused by ovariectomized group. Neither treatment was able to reverse the reduction of serum BuChE activity. Furthermore, treatments with vitamins E plus C or soy were unable to reverse the decrease in estradiol levels caused by ovariectomy. Our findings show that the treatment with vitamins E plus C significantly reversed the effect of ovariectomy on hippocampal Na(+), K(+)-ATPase and AChE activities. However, a soy diet that was rich in isoflavones was able to reverse just the increase of AChE. Neither treatment altered the reduction in serum BuChE activity. Taken together, these vitamins and soy may have a protective role against the possible brain dysfunction observed in some menopause women. Vitamins E plus C and soy isoflavones may be a good alternative as a novel therapeutic strategy.