Increased peripheral of brain-derived neurotrophic factor levels in phenylketonuric patients treated with l-carnitine.

Phenylketonuria (PKU) is the most common inherited metabolic disorders caused by severe deficiency or absence of phenylalanine hydroxylase activity that converts phenylalanine (Phe) to tyrosine. PKU patients were treated with a Phe restricted diet supplemented with a special formula containing l-carnitine (L-car), well-known antioxidant compound. The lack of treatment can cause neurological and cognitive impairment, as severe mental retardation, neuronal cell loss and synaptic density reduction. Although Phe has been widely demonstrated to be involved in PKU neurotoxicity, the mechanisms responsible for the CNS injury are still not fully known. In this work, we evaluated markers of neurodegeneration, namely BDNF (brain-derived neurotrophic factor), PAI-1 total (Plasminogen activator inhibitor-1 total), Cathepsin D, PDGF AB/BB (platelet-derived growth factor), and NCAM (neuronal adhesion molecule) in plasma of PKU patients at early and late diagnosis and under treatment. We found decreased Phe levels and increased L-car concentrations in PKU patients treated with L-car compared to the other groups, indicating that the proposed treatment was effective. Furthermore, we found increased BDNF levels in the patients under treatment compared to patients at early diagnosis, and a positive correlation between BDNF and L-car and a negative correlation between BDNF and Phe. Our results may indicate that in PKU patients treated with L-car there is an attempt to adjust neuronal plasticity and recover the damage suffered, reflecting a compensatory response to brain injury.

Increased cytokine levels induced by high phenylalanine concentrations in late diagnosis PKU patients compared to early diagnosis: Anti-inflammatory effect of L-carnitine.

Phenylketonuria (PKU) was the first genetic disease to have an effective therapy, which consists of phenylalanine intake restriction. However, there are patients who do not adhere to treatment and/or are not submitted to neonatal screening. PKU patients present L-carnitine (L-car) deficiency, compound that has demonstrated an antioxidant and anti-inflammatory role in metabolic diseases. This study evaluated the effect caused by exposure time to high Phe levels in PKU patients at early and late diagnosis, through pro- and anti-inflammatory cytokines, as well as the L-car effect in patients under treatment. It was observed that there was a decrease in phenylalanine levels in treated patients compared to patients at diagnosis, and an increase in L-car levels in the patients under treatment. Inverse correlation between Phe versus L-car and nitrate plus nitrite versus L-car in PKU patients was also showed. We found increased proinflammatory cytokines levels: interleukin (IL)-1ß, interferons (IFN)-gamma, IL-2, tumor necrosis factor (TNF)-alpha, IL-8 and IL-6 in the patients at late diagnosis compared to controls, and IL-8 in the patients at early diagnosis and treatment compared to controls. Increased IL-2, TNF-alpha, IL-6 levels in the patients at late diagnosis compared to early diagnosis were shown, and reduced IL-6 levels in the treated patients compared to patients at late diagnosis. Moreover, it verified a negative correlation between IFN-gamma and L-car in treated patients. Otherwise, it was observed that there were increased IL-4 levels in the patients at late diagnosis compared to early diagnosis, and reduction in treated patients compared to late diagnosed patients. In urine, there was an increase in 8-isoprostane levels in the patients at diagnosis compared to controls and a decrease in oxidized guanine species in the treated patients compared to the diagnosed patients. Our results demonstrate for the first time in literature that time exposure to high Phe concentrations generates a proinflammatory status, especially in PKU patients with late diagnosis. A pro-oxidant status was verified in not treated PKU patients. Our results demonstrate the importance of early diagnosis and prompt start of treatment, in addition to the importance of L-car supplementation, which can improve cellular defense against inflammation and oxidative damage in PKU patients.

Beneficial in vitro effect of N-acetylcysteine and coenzyme Q10 on DNA damage in neurodegenerative Niemann-Pick type C 1 disease: preliminary results.

Niemann-Pick type C1 (NP-C1) is a lysosomal storage disease (LSD) caused by mutations in NPC1 gene that lead to defective synthesis of the respective lysosomal transporter protein and cholesterol accumulation in late endosomes/lysosomes (LE/L) compartments, as well as glycosphingolipids GM2 and GM3 in the central nervous system (CNS). Clinical presentation varies according to the age of onset and includes visceral and neurological symptoms, such as hepatosplenomegaly and psychiatric disorders. Studies have been associating the pathophysiology of NP-C1 with oxidative damage to lipids and proteins, as well as evaluating the benefits of adjuvant therapy with antioxidants for this disease. In this work, we evaluated the DNA damage in fibroblasts culture from patients with NP-C1 treated with miglustat, as well as the in vitro effect of the antioxidant compounds N-acetylcysteine (NAC) and Coenzyme Q10 (CoQ10), using the alkaline comet assay. Our preliminary results demonstrate that NP-C1 patients have increased DNA damage compared to healthy individuals and that the treatments with antioxidants can mitigate it. DNA damage may be due to an increase in reactive species since it has been described that NP-C1 patients have increased peripheral markers of damage to other biomolecules. Our study suggests that NP-C1 patients could benefit from the use of adjuvant therapy with NAC and CoQ10, which should be better evaluated in a future clinical trial.

Inflammatory process and oxidative/nitrative stress: in vivo study in mucopolysaccharidosis type IV A patients under long-term enzyme replacement therapy.

Mucopolysaccharidosis type IV A (MPS IVA) is an inborn error of the metabolism (IEM) caused by a deficiency of the enzyme N-acetylgalactosamine 6-sulfate sulfatase (GALNS). Since 2014, enzyme replacement therapy (ERT) is the recommended treatment for these patients. It is known that the inflammatory response is closely related to antioxidant defenses and oxidative stress, and literature shows involvement of oxidative stress in the pathogenesis of IEM. The aim of this study is to investigate the mechanisms of oxidative/nitrative stress and inflammation in patients with MPS IVA under long-term ERT. In the present work we investigate parameters of oxidative/nitrative stress in plasma and urine of MPS IVA patients under long-term ERT and controls, such as plasmatic nitrate/nitrite levels using the LDH Method, urinary di-tyrosine levels by fluorometric method, plasmatic content of sulfhydryl groups, urinary oxidized guanine species by ELISA kit and the plasmatic total antioxidant status. We next evaluated the plasmatic pro and anti-inflammatory cytokines concentration (IL-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, TNF-α) and the expression of factors and enzymes Nrf-2, NF-κß and HO-1, main mediators between inflammation and oxidative stress. In concern to the oxidative/nitrative stress parameters, there was no significant difference between the groups MPS IVA patients under long-term ERT and controls, showing that there is no overproducing of RNS, no protein damage, no DNA/RNA oxidative damage and no modification in the non-enzymatic antioxidant capacity of a tissue to prevent the damage associated to free radical processes in these patients. It was also verified no significant difference between the MPS IVA patients under long-term ERT and controls groups regarding the production of proinflammatory cytokines. About anti-inflammatory cytokines, IL 10 was shown to be elevated in MPS IVA patients under long-term ERT in comparison to the control group. We next evaluated the genic expression of Nrf-2, NF-κß and HO-1and there was no significant difference between the MPS IVA patients under long-term ERT and control groups. In conclusion, MPS IVA patients under long term ERT are not in an inflammatory state and there is no alteration in genic expression in the genes analyzed which are involved in oxidative stress and inflammatory pathways. It is,however, important to consider that absence of imbalance of antioxidant defenses in MPS IVA patients under long term ERT is so far preliminary it is supported by methodologies that are not highly sensitive nor very accurate. Further experiments in future using state-of-the-art methodologies will corroborate these findings. Nevertheless, our results demonstrated the protective effect of the treatment in relation to the parameters studied and the importance of starting treatment in the early stages of the disease.

Molecular profile and peripheral markers of neurodegeneration in patients with Niemann-Pick type C: Decrease in Plasminogen Activator Inhibitor type 1 and Platelet-Derived Growth Factor type AA.

Niemann-Pick type C1 (NPC1) is a fatal inherited disease, caused by pathogenic variants in NPC1 gene, which leads to intracellular accumulation of non-esterified cholesterol and glycosphingolipids. This accumulation leads to a wide range of clinical manifestations, including neurological and cognitive impairment as well as psychiatric disorders. The pathophysiology of cerebral damage involves loss of Purkinje cells, synaptic disturbance, and demyelination. Miglustat, a reversible inhibitor of glucosylceramide synthase, is an approved treatment for NPC1 and can slow neurological damage. The aim of this study was to assess the levels of peripheric neurodegeneration biomarkers of NPC1 patients, namely brain-derived neurotrophic factor (BDNF), platelet-derived growth factors (PDGF-AA and PDGF-AB/BB), neural cell adhesion molecule (NCAM), PAI-1 Total and Cathepsin-D, as well as the levels of cholestane-3ß,5α,6ß-triol (3ß,5α,6ß-triol), a biomarker for NPC1. Molecular analysis of the NPC1 patients under study was performed by next generation sequencing (NGS) in cultured fibroblasts. We observed that NPC1 patients treated with miglustat have a significant decrease in PAI-1 total and PDGF-AA concentrations, and no alteration in BDNF, NCAM, PDGF-AB/BB and Cathepsin D. We also found that NPC1 patients treated with miglustat have normalized levels of 3ß,5α,6ß-triol. The molecular analysis showed four described mutations, and for two patients was not possible to identify the second mutated allele. Our results indicate that the decrease of PAI-1 and PDGF-AA in NPC1 patients could be involved in the pathophysiology of this disease. This is the first work to analyze those plasmatic markers of neurodegenerative processes in NPC1 patients.

Evaluation of oxidative damage to biomolecules and inflammation in patients with urea cycle disorders.

Urea cycle disorders (UCD) are inborn errors of metabolism that occur due to a loss of function in enzymes and transporters involved in the urea cycle, causing an intoxication by hyperammonemia and accumulation of metabolites. Patients can develop hepatic encephalopathy (HE), severe neurological and motor disabilities, and often death. The mechanisms involved in the pathophysiology of UCD are many and complex, but there are strong indications that oxidative stress and inflammation are present, being responsible for at least part of the cellular damage that occurs in these diseases. The aim of this study was to evaluate oxidative and nitrosative damage and inflammation in UCD, to better understand the pathophysiology mechanisms of these diseases. We evaluated the nitrite and nitrate content, thiobarbituric acid-reactive substances (TBARS), carbonyl protein content and a panel of cytokines in plasma sample of 14 patients. The UCD patients group consisted of individuals affected with ornithine transcarbamylase deficiency (n = 8), carbamoyl phosphate synthetase deficiency (n = 2), argininosuccinate synthetase deficiency (n = 2); arginase 1 deficiency (n = 1) and argininosuccinate lyase deficiency (n = 1). Patients mean age at diagnosis was 5.25 ± 9.86 years-old and mean concentrations were compared with healthy individuals of matched age and gender. We found a significant reduction in nitrogen reactive species in patients when compared to controls. TBARS was increased in patients, indicating lipid peroxidation. To evaluate protein oxidative damage in UCD, the carbonyl content was measured, and the results also demonstrated an increase in this biomarker. Finally, we found that UCD patients have enhanced concentrations of cytokines, with pro-inflammatory interleukins IL-6, IL-8, interferon-γ and TNF-α, and anti-inflammatory IL-10 being increased when compared to the control group. In conclusion, our results demonstrate that oxidative stress and inflammation occurs in UCD and probably contribute to the severe brain damage present in patients.

Evidence of redox imbalance and mitochondrial dysfunction in Niemann-Pick type C 1 patients: the in vitro effect of combined therapy with antioxidants and ß-cyclodextrin nanoparticles.

Niemann-Pick C disease (NPC) is an autosomal recessive genetic disorder resulting from mutation in one of two cholesterol transport genes: NPC1 or NPC2, causing accumulation of unesterified cholesterol, together with glycosphingolipids, within the endosomal/lysosomal compartment of cells. The result is a severe disease in both multiple peripheral organs and the central nervous system, causing neurodegeneration and early death. However, the pathophysiological mechanisms of NPC1 remain poorly understood. Recent studies have shown that the primary lysosomal defect found in fibroblasts from NPC1 patients is accompanied by a deregulation of mitochondrial organization and function. There is currently no cure for NPC1, but recently the potential of ß-cyclodextrin (ß-CD) for the treatment of the disease was discovered, which resulted in the redistribution of cholesterol from subcellular compartments to the circulation and increased longevity in an animal model of NPC1. Considering the above, the present work evaluated the in vitro therapeutic potential of ß-CD to reduce cholesterol in fibroblasts from NPC1 patients. ß-CD was used in its free and nanoparticulate form. We also evaluated the ß-CD potential to restore mitochondrial functions, as well as the beneficial combined effects of treatment with antioxidants N-Acetylcysteine (NAC) and Coenzyme Q10 (CoQ10). Besides, we evaluated oxidative and nitrative stress parameters in NPC1 patients. We showed that oxidative and nitrative stress could contribute to the pathophysiology of NPC1, as the levels of lipoperoxidation and the nitrite and nitrate levels were increased in these patients when compared to healthy individuals, as well as DNA damage. The nanoparticles containing ß-CD reduced the cholesterol accumulated in the NPC1 fibroblasts. This result was potentiated by the concomitant use of the nanoparticles with the antioxidants NAC and CoQ10 compared to those presented by healthy individuals cells ́. In addition, treatments combining ß-CD nanoparticles and antioxidants could reduce mitochondrial oxidative stress, demonstrating advantages compared to free ß-CD. The results obtained are promising regarding the combined use of ß-CD loaded nanoparticles and antioxidants in the treatment of NPC1 disease.

Cytokine profile and cholesterol levels in patients with Niemann-Pick type C disease presenting neurological symptoms: in vivo effect of miglustat and in vitro effect of N-acetylcysteine and coenzyme Q10.

Niemann Pick type C is an inborn error of metabolism (IEM), classified as a lysosomal storage disease (LSD) caused by a dysfunction in NPC transport protein, that leads to intracellular accumulation of non-esterified cholesterol and other lipids. Clinical manifestations are ample, with visceral and neurological symptoms. Miglustat, a molecule that reversibly inhibits glucosylceramide synthase is used as treatment for this disorder. Studies demonstrated the influence of oxidative stress and inflammation in IEM, as well in animal model of NP-C disease. Nonetheless, literature lacks data on patients, so our work aimed to investigate if there is influence of chronic inflammation in the pathophysiology of NP-C disease, and the effect of miglustat, N-acetylcysteine (NAC) and Coenzyme Q10 (CoQ10). We evaluated the plasmatic cytokines in NPC patients at diagnosis and during the treatment with miglustat. Additionally, we performed an in vitro study with antioxidants NAC (1 mM and 2.5 mM) and CoQ10 (5 µM and 10 µM), where we could verify its effect on inflammatory parameters, as well as in cholesterol accumulation. Our results showed that NP-C patients have higher plasmatic levels of pro and anti-inflammatory cytokines (IL-6, IL-8, and IL-10) at diagnosis and the treatment with miglustat was able to restore it. In vitro study showed that treatment with antioxidants in higher concentrations significantly decrease cholesterol accumulation, and NAC at 2.5 mM normalized the level of pro-inflammatory cytokines. Although the mechanism is not completely clear, it can be related to restoration in lipid traffic and decrease in oxidative stress caused by antioxidants.

Nanoparticles containing ß-cyclodextrin potentially useful for the treatment of Niemann-Pick C.

ß-Cyclodextrin (ß-CD) is being considered a promising therapy for Niemann-Pick C (NPC) disease because of its ability to mobilise the entrapped cholesterol from lysosomes, however, a major limitation is its inability to cross the blood-brain barrier (BBB) and address the central nervous system (CNS) manifestations of the disease. Considering this, we aimed to design nanoparticles able to cross the BBB and deliver ß-CD into the CNS lysosomes. The physicochemical characteristics of ß-CD-loaded nanoparticles were evaluated by dynamic light scattering, small-angle X-ray scattering, and cryogenic transmission electron microscopy. The in vitro analyses were performed with NPC dermal fibroblasts and the ß-CD-loaded nanoparticles were tracked in vivo. The nanoparticles showed a mean diameter around 120 nm with a disordered bicontinuous inner structure. The nanoparticles did not cause decrease in cell viability, impairment in the antioxidant enzymes activity, damage to biomolecules or release of reactive species in NPC dermal fibroblasts; also, they did not induce genotoxicity or alter the mitochondrial function in healthy fibroblasts. The ß-CD-loaded nanoparticles were taken up by lysosomes reducing the cholesterol accumulated in NPC fibroblasts and reached the CNS of mice more intensely than other organs, demonstrating advantages compared to the free ß-CD. The results demonstrated the potential of the ß-CD-loaded nanoparticles in reducing the brain impairment of NPC.

Molecular and biochemical biomarkers for diagnosis and therapy monitorization of Niemann-Pick type C patients.

BACKGROUND: Niemann-Pick type C (NP-C), one of 50 inherited lysosomal storage disorders, is caused by NPC protein impairment that leads to unesterified cholesterol accumulation in late endosomal/lysosomal compartments. The clinical manifestations of NP-C include hepatosplenomegaly, neurological and psychiatric symptoms. Current diagnosis for NP-C is based on observation of the accumulated cholesterol in fibroblasts of affected individuals, using an invasive and time expensive test, called Filipin staining. Lately, two metabolites that are markedly increased in NP-C patients are arising as biomarkers for this disease screening: 7-ketocholesterol and cholestane-3ß,5α,6ß-triol, both oxidized cholesterol products. OBJECTIVE: In this work, we aimed to evaluate the performance of cholestane-3ß,5α,6ß-triol analysis for the screening and monitoring of NPC patients, correlating it with chitotriosidase levels, Filipin staining and molecular analysis. It was investigated 76 non-treated individuals with NP-C suspicion and also 7 patients with previous NP-C diagnosis under treatment with miglustat, in order to verify the cholestane-3ß,5α,6ß-triol value as a tool for therapy monitoring. RESULTS: Considering molecular assay as golden standard, it was verified that cholestane-3ß,5α,6ß-triol analysis presented 88% of sensitivity, 96.08% of specificity, a positive and negative predictive value calculated in 91.67% and 94.23%, respectively, for the diagnosis of NP-C. Chitotriosidase levels were increased in patients with positive molecular analysis for NP-C. For Filipin staining, it was found 1 false positive, 7 false negative and 24 inconclusive cases, showing that this assay has important limitations for NP-C diagnosis. Besides, we found a significant decrease in cholestane-3ß,5α,6ß-triol concentrations in NP-C patients under therapy with miglustat when compared to non-treated patients. CONCLUSION: Taken together, the present data show that cholestane-3ß,5α,6ß-triol analysis has a high potential to be an important NP-C screening assay, and also can be used for therapy monitorization with miglustat in NP-C patients.

Investigation of correlation of urinary globotriaosylceramide (Gb3) levels with markers of renal function in patients with Fabry disease.

Fabry disease (FD) is a disorder that results from mutations of hydrolase α-galactosidase A. The enzymatic defect leads to accumulation of globotriaosylceramide (Gb3) in the kidney. Substrate deposition is related to tissue damage in FD, but the relation of urinary Gb3 levels in patients and the renal function markers remain not completely understood. Once nephropathy is one of the main features of FD and is marked by an insidious development, we investigated a possible correlation of Gb3 with biochemical markers of nephropathy including albuminuria, estimated glomerular filtration rate (eGFR), serum creatinine and urea, and proteinuria in male and female patients under or not enzyme replacement therapy (ERT).Gb3, proteinuria and albuminuria were increased in male and female FD patients. We found no correlation between urinary Gb3 levels and all renal function parameters evaluated in Fabry patients (in both sexes and using or not ERT). On the other hand, albuminuria showed negative correlation with eGFR only in male under or not ERT, demonstrating that albuminuria seems to be an early marker of renal function alteration. In conclusion, the results suggest that urinary Gb3 level does not reflect the renal function and that albuminuria is an important biomarker in male FD patients.

Oxidative Stress in Homocystinuria Due to Cystathionine ß-Synthase Deficiency: Findings in Patients and in Animal Models.

Homocystinuria is an inborn error of amino acid metabolism caused by deficiency of cystathionine ß-synthase (CBS) activity, biochemically characterized by homocysteine (Hcy) and methionine (Met) accumulation in biological fluids and high urinary excretion of homocystine. Clinical manifestations include thinning and lengthening of long bones, osteoporosis, dislocation of the ocular lens, thromboembolism, and mental retardation. Although the pathophysiology of this disease is poorly known, the present review summarizes the available experimental findings obtained from patients and animal models indicating that oxidative stress may contribute to the pathogenesis of homocystinuria. In this scenario, several studies have shown that enzymatic and non-enzymatic antioxidant defenses are decreased in individuals affected by this disease. Furthermore, markers of lipid, protein, and DNA oxidative damage have been reported to be increased in blood, brain, liver, and skeletal muscle in animal models studied and in homocystinuric patients, probably as a result of increased free radical generation. On the other hand, in vitro and in vivo studies have shown that Hcy induces reactive species formation in brain, so that this major accumulating metabolite may underlie the oxidative damage observed in the animal model and human condition. Taken together, it may be presumed that the disruption of redox homeostasis may contribute to the tissue damage found in homocystinuria. Therefore, it is proposed that the use of appropriate antioxidants may represent a novel adjuvant therapy for patients affected by this disease.

Biomolecules damage and redox status abnormalities in Fabry patients before and during enzyme replacement therapy.

Fabry disease (FD) is caused by deficient activity of the lysosomal enzyme α-galactosidase A. Its substrates, mainly globotriaosylceramide (Gb3), accumulate and seem to induce other pathophysiological findings of FD. Once enzyme replacement therapy (ERT) is not completely efficient on preventing disease progress in FD patients, elucidating the underlying mechanisms in FD pathophysiology is essential to the development of additional therapeutic strategies. We investigated 58 Fabry patients (23 male and 35 female) subdivided into two groups (at diagnosis and during long-term ERT) and compared them to healthy individuals. Fabry patients at diagnosis presented altered glutathione (GSH) metabolism (higher GSH levels, lower glutathione peroxidase - GPx - and normal glutathione reductase - GR - activities), higher lipid peroxidation levels (thiobarbituric acid reactive species - TBARS - and malondialdehyde - MDA), nitric oxide (NO(.)) equivalents and urinary Gb3. Fabry patients on ERT presented GSH metabolism similar to controls, although lipid peroxidation and urinary levels of NO(.) equivalents remained higher whereas Gb3 levels were lower than at diagnosis but still higher than controls. These data demonstrated that redox impairment occurs in Fabry patients before and after ERT, probably as a consequence of Gb3 accumulation, providing targets to future therapy approaches using antioxidants in combination with ERT in FD.

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.

Urinary biomarkers of oxidative damage in Maple syrup urine disease: the L-carnitine role.

Maple syrup urine disease (MSUD) is a disorder of branched-chain amino acids (BCAA). The defect in the branched-chain α-keto acid dehydrogenase complex activity leads to an accumulation of these compounds and their corresponding α-keto-acids and α-hydroxy-acids. Studies have shown that oxidative stress may be involved in neuropathology of MSUD. L-carnitine (L-car), which has demonstrated an important role as antioxidant by reducing and scavenging free radicals formation and by enhancing the activity of antioxidant enzymes, have been used in the treatment of some metabolic rare disorders. This study evaluated the oxidative stress parameters, di-tyrosine, isoprostanes and antioxidant capacity, in urine of MSUD patients under protein-restricted diet supplemented or not with L-car capsules at a dose of 50 mg kg(-1) day(-1). It was also determined urinary α-keto isocaproic acid levels as well as blood free L-car concentrations in blood. It was found a deficiency of carnitine in patients before the L-car supplementation. Significant increases of di-tyrosine and isoprostanes, as well as reduced antioxidant capacity, were observed before the treatment with L-car. The L-car supplementation induced beneficial effects on these parameters reducing the di-tyrosine and isoprostanes levels and increasing the antioxidant capacity. It was also showed a significant increase in urinary of α-ketoisocaproic acid after 2 months of L-car treatment, compared to control group. In conclusion, our results suggest that L-car may have beneficial effects in the treatment of MSUD by preventing oxidative damage to the cells and that urine can be used to monitorize oxidative damage in patients affected by this disease.

Increased oxidative stress in patients with 3-hydroxy-3-methylglutaric aciduria.

3-hydroxy-3-methylglutaric aciduria (HMGA; OMIM 246450) is a rare autosomal recessive disorder, caused by the deficiency of 3-hydroxy-3-methylglutaryl-CoA lyase (4.1.3.4), which results in the accumulation of 3-hydroxy-3-methylglutaric (HMG) and 3-methylglutaric (MGA) acids in tissues and biological fluids of affected individuals. Recent in vivo and in vitro animal studies have demonstrated that the accumulation of these metabolites can disturb the cellular redox homeostasis, which can contribute to the neurological manifestations presented by the patients. So, in the present work, we investigated oxidative stress parameters in plasma and urine samples from HMGA patients, obtained at the moment of diagnosis of this disorder and during therapy with low-protein diet and L-carnitine supplementation. It was verified that untreated HMGA patients presented higher levels of urinary di-tyrosine and plasma thiobarbituric acid-reactive substances (TBA-RS), which are markers of protein and lipid oxidative damage, respectively, as well as a reduction of the urinary antioxidant capacity. Treated HMGA patients also presented an increased protein oxidative damage, as demonstrated by their higher concentrations of plasma protein carbonyl groups and urinary di-tyrosine, as well as by the reduction of total sulfhydryl groups in plasma, in relation to controls. On the other hand, HMGA patients under therapy presented normal levels of TBA-RS and urinary antioxidant capacity, which can be related, at least in part, to the antioxidant and antiperoxidative effects exerted by L-carnitine. The results of this work are the first report showing that a redox imbalance occurs in patients with HMGA what reinforces the importance of the antioxidant therapy in this disorder.