Oxidative stress and inflammation in mucopolysaccharidosis type IVA patients treated with enzyme replacement therapy.

Mucopolysaccharidosis type IVA (MPS IVA) is an inborn error of glycosaminoglycan (GAG) catabolism due to the deficient activity of N-acetylgalactosamine-6-sulfate sulfatase that leads to accumulation of the keratan sulfate and chondroitin 6-sulfate in body fluids and in lysosomes. The pathophysiology of this lysosomal storage disorder is not completely understood. The aim of this study was to investigate oxidative stress parameters, pro-inflammatory cytokine and GAG levels in MPS IVA patients. We analyzed urine and blood samples from patients under ERT (n=17) and healthy age-matched controls (n=10-15). Patients presented a reduction of antioxidant defense levels, assessed by a decrease in glutathione content and by an increase in superoxide dismutase activity in erythrocytes. Concerning lipid and protein damage, it was verified increased urine isoprostanes and di-tyrosine levels and decreased plasma sulfhydryl groups in MPS IVA patients compared to controls. MPS IVA patients showed higher DNA damage than control group and this damage had an oxidative origin in both pyrimidine and purine bases. Interleukin 6 was increased in patients and presented an inverse correlation with GSH levels, showing a possible link between inflammation and oxidative stress in MPS IVA disease. The data presented suggest that pro-inflammatory and pro-oxidant states occur in MPS IVA patients even under ERT. Taking these results into account, supplementation of antioxidants in combination with ERT can be a tentative therapeutic approach with the purpose of improving the patient's quality of life. To the best of our knowledge, this is the first study relating MPS IVA patients with oxidative stress.

Experimental evidence for protein oxidative damage and altered antioxidant defense in patients with medium-chain acyl-CoA dehydrogenase deficiency.

The objective of this study was to test whether macromolecule oxidative damage and altered enzymatic antioxidative defenses occur in patients with medium-chain acyl coenzyme A dehydrogenase (MCAD) deficiency. We performed a cross-sectional observational study of in vivo parameters of lipid and protein oxidative damage and antioxidant defenses in asymptomatic, nonstressed, MCAD-deficient patients and healthy controls. Patients were subdivided into three groups based on therapy: patients without prescribed supplementation, patients with carnitine supplementation, and patients with carnitine plus riboflavin supplementation. Compared with healthy controls, nonsupplemented MCAD-deficient patients and patients receiving carnitine supplementation displayed decreased plasma sulfhydryl content (indicating protein oxidative damage). Increased erythrocyte superoxide dismutase (SOD) activity in patients receiving carnitine supplementation probably reflects a compensatory mechanism for scavenging reactive species formation. The combination of carnitine plus riboflavin was not associated with oxidative damage. These are the first indications that MCAD-deficient patients experience protein oxidative damage and that combined supplementation of carnitine and riboflavin may prevent these biochemical alterations. Results suggest involvement of free radicals in the pathophysiology of MCAD deficiency. The underlying mechanisms behind the increased SOD activity upon carnitine supplementation need to be determined. Further studies are necessary to determine the clinical relevance of oxidative stress, including the possibility of antioxidant therapy.

Globotriaosylceramide is correlated with oxidative stress and inflammation in Fabry patients treated with enzyme replacement therapy.

Fabry disease is an X-linked inborn error of glycosphingolipid catabolism due to deficient activity of α-galactosidase A that leads to accumulation of the enzyme substrates, mainly globotriaosylceramide (Gb3), in body fluids and lysosomes of many cell types. Some pathophysiology hypotheses are intimately linked to reactive species production and inflammation, but until this moment there is no in vivo study about it. Hence, the aim of this study was to investigate oxidative stress parameters, pro-inflammatory cytokines and Gb3 levels in Fabry patients under treatment with enzyme replacement therapy (ERT) and finally to establish a possible relation between them. We analyzed urine and blood samples of patients under ERT (n=14) and healthy age-matched controls (n=14). Patients presented decreased levels of antioxidant defenses, assessed by reduced glutathione (GSH), glutathione peroxidase (GPx) activity and increased superoxide dismutase/catalase (SOD/CAT) ratio in erythrocytes. Concerning to the damage to biomolecules (lipids and proteins), we found that plasma levels of malondialdehyde (MDA) and protein carbonyl groups and di-tyrosine (di-Tyr) in urine were increased in patients. The pro-inflammatory cytokines IL-6 and TNF-α were also increased in patients. Urinary Gb3 levels were positively correlated with the plasma levels of IL-6, carbonyl groups and MDA. IL-6 levels were directly correlated with di-Tyr and inversely correlated with GPx activity. This data suggest that pro-inflammatory and pro-oxidant states occur, are correlated and seem to be induced by Gb3 in Fabry patients.

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.

Oxidative stress in patients with mucopolysaccharidosis type II before and during enzyme replacement therapy.

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder caused by deficiency of the enzyme iduronate-2-sulfatase, responsible for the degradation of glycosaminoglycans dermatan and heparan sulfate. Once the generation of free radicals is involved in the pathogenesis of many diseases, including some inborn errors of metabolism, the aim of this study was to evaluate blood oxidative stress parameters in MPS II patients, before and during 6 months of enzyme replacement therapy. We found significantly increased levels of malondialdehyde and carbonyl groups in plasma as well as erythrocyte catalase activity in patients before treatment compared to the control group. Plasma sulfhydryl group content and total antioxidant status were significantly reduced before treatment, while superoxide dismutase enzyme was not altered at this time when compared to controls. During enzyme replacement therapy, there was a significant reduction in levels of malondialdehyde when compared to pretreatment. Sulfhydryl groups were significantly increased until three months of treatment in MPS II patients in comparison to pretreatment. There were no significant alterations in plasma total antioxidant status and carbonyl groups as well as in catalase and superoxide dismutase activities during treatment in relation to pretreatment. The results indicate that MPS II patients are subject to lipid and protein oxidative damage and present reduction in non-enzymatic antioxidants, suggesting a possible involvement of free radicals in the pathophysiology of this disease. Also, the results may suggest that enzyme replacement therapy seems to protect against lipid peroxidation and protein damage in these patients.

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.

Effects of insulin and clonazepam on DNA damage in diabetic rats submitted to the forced swimming test.

Diabetes mellitus (DM) is a chronic hyperglycemic state. DM may be associated with moderate cognitive deficits and neurophysiologic/structural changes in the brain (diabetic encephalopathy). Psychiatric manifestations seem to accompany this encephalopathy, since the prevalence of depression in diabetic patients is much higher than in the general population, and clonazepam is being used to treat this complication. The excessive production of oxygen free radicals that may occur in diabetes induces a variety of lesions in macromolecules, including DNA. In this work, we analyzed DNA damage in leukocytes from streptozotocin-induced diabetic rats submitted to the forced swimming test. The DNA damage index was significantly elevated (DI=61.00 ± 4.95) in the diabetic group compared to the control group (34.00 ± 1.26). Significant reductions of the damage index were observed in diabetic animals treated with insulin (45.00 ± 1.82), clonazepam (52.00 ± 1.22), or both agents (39.00 ± 5.83, not significantly different from control levels). Insulin plus clonazepam can protect against DNA damage in stressed diabetic rats.

L-carnitine blood levels and oxidative stress in treated phenylketonuric patients.

AIMS: L-carnitine exerts an important role by facilitating the mitochondrial transport of fatty acids, but is also a scavenger of free radicals, protecting cells from oxidative damage. Phenylketonuria (PKU), an inborn error of phenylalanine (Phe) metabolism, is currently treated with a special diet consisting of severe restriction of protein-enriched foods, therefore potentially leading to L-carnitine depletion. The aim of this study was to determine L-carnitine levels and oxidative stress parameters in blood of two groups of PKU patients, with good and poor adherence to treatment. METHODS: Treatment of patients consisted of a low protein diet supplemented with a synthetic amino acids formula not containing Phe, L-carnitine, and selenium. L-carnitine concentrations and the oxidative stress parameters thiobarbituric acid reactive species (TBARS) and total antioxidant reactivity (TAR) were measured in blood of the two groups of treated PKU patients and controls. RESULTS: We verified a significant decrease of serum L-carnitine levels in patients who strictly adhered to the diet, as compared to controls and patients who did not comply with the diet. Furthermore, TBARS measurement was significantly increased and TAR was significantly reduced in both groups of phenylketonuric patients relatively to controls. We also found a significant negative correlation between TBARS and L-carnitine levels and a significant positive correlation between TAR and L-carnitine levels in well-treated PKU patients. CONCLUSIONS: Our results suggest that L-carnitine should be measured in plasma of treated PKU patients, and when a decrease of this endogenous component is detected in plasma, supplementation should be considered as an adjuvant therapy.

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.

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.

Effect of short- and long-term exposition to high phenylalanine blood levels on oxidative damage in phenylketonuric patients.

Phenylketonuria is the most frequent disturbance of amino acid metabolism. Treatment for phenylketonuric patients consists of phenylalanine intake restriction. However, there are patients who do not adhere to treatment and/or are not submitted to neonatal screening. These individuals are more prone to develop brain damage due to long-lasting toxic effects of high levels of phenylalanine and/or its metabolites. Oxidative stress occurs in late-diagnosed phenylketonuric patients, probably contributing to the neurological damage in this disorder. In this work, we aimed to compare the influence of time exposition to high phenylalanine levels on oxidative stress parameters in phenylketonuric patients who did not adhere to protein restricted diet. We evaluated a large spectrum of oxidative stress parameters in plasma and erythrocytes from phenylketonuric patients with early and late diagnosis and of age-matched healthy controls. Erythrocyte glutathione peroxidase activity and glutathione levels, as well as plasma total antioxidant reactivity were significantly reduced in both groups of patients when compared to the control group. Furthermore, protein oxidative damage, measured by carbonyl formation and sulfhydryl oxidation, and lipid peroxidation, determined by malondialdehyde levels, were significantly increased only in patients exposed for a long time to high phenylalanine concentrations, compared to early diagnosed patients and controls. In conclusion, exposition to high phenylalanine concentrations for a short or long time results in a reduction of non-enzymatic and enzymatic antioxidant defenses, whereas protein and lipid oxidative damage only occurs in patients with late diagnosis.