Tatiane Grazieli Hammerschmidt

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.

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.

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.

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.

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

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‐3β,5α,6β‐triol, both oxidized cholesterol products.

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.