Carlos Alberto Yasin Wayhs

Reduction of lipid and protein damage in patients with disorders of propionate metabolism under treatment: a possible protective role of L-carnitine supplementation.

Disorders of propionate metabolism are autosomal recessive diseases clinically characterized by acute metabolic crises in the neonatal period and long‐term neurological deficits whose pathophysiology is not completely established. There are increasing evidences demonstrating antioxidant properties for l‐carnitine, which is used in the treatment of propionic and methylmalonic acidemias to increase the excretion of organic acids accumulated in tissues and biological fluids of the affected patients. In this work we aimed to evaluate lipid (malondialdehyde content) and protein (carbonyl formation and sulfhydryl oxidation) oxidative damage in plasma from patients with propionic and methylmalonic acidemias at the moment of diagnosis and during treatment with l‐carnitine. We also correlated the parameters of oxidative damage with plasma total, free and esterified l‐carnitine levels. We found a significant increase of malondialdehyde and carbonyl groups, as well as a reduction of sulfhydryl groups in plasma of these patients at diagnosis compared to controls. Furthermore, patients under treatment presented a marked reduction of the content of protein carbonyl groups, similar to controls, and malondialdehyde content in relation to patients at diagnosis. In addition, plasma total and free l‐carnitine concentrations were negatively correlated with malondialdehyde levels. Taken together, the present data indicate that treatment significantly reduces oxidative damage in patients affected by disorders of propionate metabolism and that l‐carnitine supplementation may be involved in this protection.

Prevention by L-carnitine of DNA damage induced by propionic and L-methylmalonic acids in human peripheral leukocytes in vitro.

Propionic acidemia (PAemia) and methylmalonic acidemia (MMAemia) are inborn errors of propionate metabolism characterized by the accumulation of, respectively, propionic and l-methylmalonic acids (and their metabolites) in the blood and tissues of affected patients. The conditions lead to severe metabolic complications in the neonatal period and to long-term neurological manifestations. Treatment for these disorders consists of a protein-restricted diet, supplemented with synthetic formulas of amino acids, but excluding isoleucine, threonine, valine and methionine; and l-carnitine, to promote detoxication. In vitro and in vivo studies have demonstrated that lipid and protein oxidative damage may be involved in the pathophysiology of these diseases, but DNA damage has not been fully investigated. In this work, we evaluated in vitro the effects of PA and MMA, in the presence or absence of l-carnitine, on DNA damage in peripheral leukocytes, as determined by the alkaline comet assay, using silver staining and visual scoring. PA and MMA induced a DNA damage index (DI) significantly higher than that of the control group. l-Carnitine significantly reduced PA- and MMA-induced DNA damage, in a concentration-dependent manner. Our findings indicate that PA and MMA induce DNA damage and l-carnitine is able to prevent this damage.

Experimental evidence of oxidative stress in plasma of homocystinuric patients: A possible role for homocysteine

Homocystinuria is an inherited disorder biochemically characterized by high urinary excretion of homocystine and increased levels of homocysteine (Hcy) and methionine in biological fluids. Affected patients usually have a variety of clinical and pathologic manifestations. Previous experimental data have shown a relationship between Hcy and oxidative stress, although very little was reported on this process in patients with homocystinuria. Therefore, in the present study we evaluated parameters of oxidative stress, namely carbonyl formation, malondialdehyde (MDA) levels, sulfhydryl content and total antioxidant status (TAS) in patients with homocystinuria at diagnosis and under treatment with a protein restricted diet supplemented by pyridoxine, folate, betaine, and vitamin B(12). We also correlated plasma Hcy and methionine concentrations with the oxidative stress parameters examined. We found a significant increase of MDA levels and carbonyl formation, as well as a reduction of sulfhydryl groups and TAS in plasma of homocystinuric patients at diagnosis relatively to healthy individuals (controls). We also verified that Hcy levels were negatively correlated with sulfhydryl content and positively with MDA levels. Furthermore, patients under treatment presented a significant reduction of the content of MDA, Hcy and methionine concentrations relatively to patients at diagnosis. Taken together, the present data indicate that lipid and protein oxidative damages are increased and the antioxidant defenses diminished in plasma of homocystinuric patients, probably due to increased reactive species elicited by Hcy. It is therefore presumed that oxidative stress participates at least in part in the pathogenesis of homocystinuria.

Protein and lipid damage in maple syrup urine disease patients: l-carnitine effect

Maple syrup urine disease (MSUD) is an inborn error of metabolism biochemically characterized by elevated levels of the branched chain amino acids (BCAA) leucine, isoleucine, valine and the corresponding branched‐chain α‐keto acids. This disorder is clinically characterized by ketoacidosis, seizures, coma, psychomotor delay and mental retardation whose pathophysiology is not completely understood. Recent studies have shown that oxidative stress may be involved in neuropathology of MSUD. l‐Carnitine (l‐Car) plays a central role in the cellular energy metabolism because it transports long‐chain fatty acids for oxidation and ATP generation. In recent years many studies have demonstrated the antioxidant role of this compound. In this work, we investigated the effect of BCAA‐restricted diet supplemented or not with l‐Car on lipid peroxidation and in protein oxidation in MSUD patients. We found a significant increase of malondialdehyde and of carbonyl content in plasma of MSUD patients under BCAA‐restricted diet compared to controls. Furthermore, patients under BCAA‐restricted diet plus l‐Car supplementation presented a marked reduction of malondialdehyde content in relation to controls, reducing the lipid peroxidation. In addition, free l‐Car concentrations were negatively correlated with malondialdehyde levels. Our data show that l‐Car may have an antioxidant effect, protecting against the lipid peroxidation and this could represent an additional therapeutic approach to the patients affected by MSUD.

Oxidative stress in Niemann-Pick type C patients: a protective role of N-butyl-deoxynojirimycin therapy.

Niemann‐Pick type C (NPC) is a rare neurodegenerative disorder biochemically characterized by the accumulation of cholesterol and glycosphingolipids in late endosomes and lysosomes of the affected patients. N‐butyl‐deoxynojirimycin is the only approved drug for patients with NPC disease. It inhibits glycosphingolipid synthesis, therefore reducing intracellular lipid storage. Although the mechanisms underlying the neurologic damage in the NPC disease are not yet well established, in vitro and in vivo studies suggest an involvement of reactive species in the pathophysiology of this disease. In this work we aimed to evaluate parameters of lipid and protein oxidation, measured by thiobarbituric acid‐reactive species (TBA‐RS) and protein carbonyl formation, respectively, as well as the enzymatic and non‐enzymatic antioxidant defenses in plasma, erythrocytes and fibroblasts from NPC1 patients, at diagnosis and during treatment with N‐butyl‐deoxynojirimycin. We found a significant increase of TBA‐RS in plasma and fibroblasts, as well as increased protein carbonyl formation and decreased total antioxidant status (TAS) in plasma of untreated NPC1 patients as compared to the control group. In addition, erythrocyte glutathione peroxidase (GSH‐Px) activity was increased, whereas CAT and SOD activities were normal in these patients. We also observed that patients treated with N‐butyl‐deoxynojirimycin normalized plasma TBA‐RS and TAS, as well as erythrocyte GSH‐Px activity. Taken together, the present data indicate that oxidative stress is increased in patients with NPC1 disease and that treatment with N‐butyl‐deoxynojirimycin is able to confer protection against this pathological process.

Protein and lipid oxidative damage in streptozotocin-induced diabetic rats submitted to forced swimming test: the insulin and clonazepam effect

Diabetes may modify central nervous system functions and is associated with moderate cognitive deficits and changes in the brain, a condition that may be referred to as diabetic encephalopathy. The prevalence of depression in diabetic patients is higher than in the general population, and clonazepam is being used to treat this complication. Oxidative stress may play a role in the development of diabetes complications. We investigated oxidative stress parameters in streptozotocin-induced diabetic rats submitted to forced swimming test (STZ) and evaluated the effect of insulin (STZ-INS) and/or clonazepam (STZ-CNZ and STZ-INS-CNZ) acute treatment on these animal model. Oxidative damage to proteins measured as carbonyl content in plasma was significantly increased in STZ group compared to STZ treated groups. Malondialdehyde plasma levels were significantly reduced in STZ-INS and STZ-INS-CNZ groups when compared to STZ rats, being significantly reduced in STZ-INS-CNZ than STZ-INS rats. The activities of the antioxidant enzymes catalase, superoxide dismutase and glutathione peroxidase showed no significant differences among all groups of animals. These findings showed that protein and lipid damage occurs in this diabetes/depression animal model and that the associated treatment of insulin and clonazepam is capable to protect against oxidative damage in this experimental model.

Oxidative Stress Parameters in Urine from Patients with Disorders of Propionate Metabolism: a Beneficial Effect of l -Carnitine Supplementation

Propionic (PA) and methylmalonic (MMA) acidurias are inherited disorders caused by deficiency of propionyl-CoA carboxylase and methylmalonyl-CoA mutase, respectively. Affected patients present acute metabolic crises in the neonatal period and long-term neurological deficits. Treatments of these diseases include a protein restricted diet and l-carnitine supplementation. l-Carnitine is widely used in the therapy of these diseases to prevent secondary l-carnitine deficiency and promote detoxification, and several recent in vitro and in vivo studies have reported antioxidant and antiperoxidative effects of this compound. In this study, we evaluated the oxidative stress parameters, isoprostane and di-tyrosine levels, and the antioxidant capacity, in urine from patients with PA and MMA at the diagnosis, and during treatment with l-carnitine and protein-restricted diet. We verified a significant increase of isoprostanes and di-tyrosine, as well as a significant reduction of the antioxidant capacity in urine from these patients at diagnosis, as compared to controls. Furthermore, treated patients presented a marked reduction of isoprostanes and di-tyrosine levels in relation to untreated patients. In addition, patients with higher levels of protein and lipid oxidative damage, determined by di-tyrosine and isoprostanes levels, also presented lower urinary concentrations of total and free l-carnitine. In conclusion, the present results indicate that treatment with low protein diet and l-carnitine significantly reduces urinary biomarkers of protein and lipid oxidative damage in patients with disorders of propionate metabolism and that l-carnitine supplementation may be specially involved in this protection.

Brain effect of insulin and clonazepam in diabetic rats under depressive-like behavior

Diabetes mellitus is characterized by hyperglycemia resulting from defects on insulin secretion, insulin action, or both. It has recently become clear that the central nervous system is not spared from the deleterious effects of diabetes, since diabetic encephalopathy was recognized as a complication of this heterogeneous metabolic disorder. There is a well recognized association between depression and diabetes, once prevalence of depression in diabetic patients is higher than in general population, and clonazepam is being used to treat this complication. Oxidative stress is widely accepted as playing a key mediatory role in the development and progression of diabetes and its complications. In this work we analyzed DNA damage by comet assay and lipid damage in prefrontal cortex, hippocampus and striatum of streptozotocin-induced diabetic rats submitted to the forced swimming test. It was verified that the diabetic group presented DNA and lipid damage in the brain areas evaluated, when compared to the control groups. Additionally, a significant reduction of the DNA and lipid damage in animals treated with insulin and/or clonazepam was observed. These data suggest that the association of these two drugs could protect against DNA and lipid damage in diabetic rats submitted to the forced swimming test, an animal model of depression.

DNA damage in leukocytes from pretreatment mucopolysaccharidosis type II patients; protective effect of enzyme replacement therapy.

Mucopolysaccharidosis type II (MPS II) is an X-linked recessive disease caused by deficiency of the lysosomal enzyme iduronate-2-sulfatase, leading to progressive accumulation of glycosaminoglycans in nearly all cell types, tissues and organs. Enzyme replacement therapy reduces the storage of these substances in the lysosomes. Oxidative stress is related to the pathophysiology of many disorders, including inborn errors of metabolism. Oxidative damage to protein and lipid has been described in MPS types I and III. The aim of this study was to analyze DNA damage, as determined by the alkaline comet assay using silver staining, in peripheral leukocytes from MPS II patients before treatment and during the first six months of enzyme replacement therapy. We also correlated DNA damage with lipid and protein oxidative damages, analyzed by plasma malondialdehyde levels and carbonyl group content, respectively. We found a significant increase in lipid and protein damage in MPS II patients before treatment when compared to controls. Also, our results showed greater DNA damage in terms of damage index (DI) in pretreatment MPS II patients (DI=18.0 ± 2.4) when compared to controls (DI=66.0 ± 2.0). Enzyme replacement therapy led to a significant decrease in levels of malondialdehyde and DNA damage when compared to pretreatment, but did not reach control values. Significant positive correlations between DNA damage and malondialdehyde levels, as well as carbonyl group content, were observed. Our findings indicate that MPS II patients are subject to DNA damage and that enzyme replacement therapy is able to protect against this process.

L-carnitine supplementation decreases dna damage in treated MSUD patients

Maple syrup urine disease (MSUD) is an inherited disorder caused by severe deficient activity of the branched-chain α-keto acid dehydrogenase complex involved in the degradation pathway of branched-chain amino acids (BCAAs) and their α-ketoacid derivatives. MSUD patients generally present ketoacidosis, poor feeding, ataxia, coma, psychomotor delay, mental retardation and brain abnormalites. Treatment consists of dietary restriction of the BCAA (low protein intake) supplemented by a BCAA-free amino acid mixture. Although the mechanisms of brain damage in MSUD are poorly known, previous studies have shown that oxidative stress may be involved in the neuropathology of this disorder. In this regard, it was recently reported that MSUD patients have deficiency of l-carnitine (l-car), a compound with antioxidant properties that is used as adjuvant therapy in various inborn errors of metabolism. In this work, we investigated DNA damage determined by the alkaline comet assay in peripheral whole blood leukocytes of MSUD patients submitted to a BCAA-restricted diet supplemented or not with l-car. We observed a significant increase of DNA damage index (DI) in leukocytes from MSUD patients under BCAA-restricted diet as compared to controls and that l-car supplementation significantly decreased DNA DI levels. It was also found a positive correlation between DI and MDA content, a marker of lipid peroxidation, and an inverse correlation between DI and l-car levels. Taken together, our present results suggest a role for reactive species and the involvement of oxidative stress in DNA damage in this disorder. Since l-car reduced DNA damage, it is presumed that dietary supplementation of this compound may serve as an adjuvant therapeutic strategy for MSUD patients in addition to other therapies.