Marion Deon

Evidence that oxidative stress is increased in plasma from patients with maple syrup urine disease

Maple syrup urine disease (MSUD) or branched-chain α-keto aciduria (BCKA) is an inherited disorder caused by a deficiency of the branched-chain α-keto acid dehydrogenase complex (BCKAD) activity. The blockage of this pathway leads to tissue accumulation of the branched-chain amino acids (BCAA) leucine, isoleucine and valine and their respective keto-acids. The clinical features presented by MSUD patients include ketoacidosis, convulsions, coma, psychomotor delay and mental retardation. The mechanism of brain damage in this disease is still poorly understood. However, an increase in lipid peroxidation in vitro in cerebral cortex of young rats as well as a decrease in the antioxidant defenses has been previously observed. In the present work we evaluated different oxidative stress parameters, named reactive species of thiobarbituric acid (TBARS), total antioxidant reactivity (TAR) and total antioxidant status (TAS) in plasma of MSUD patients in order to evaluate whether oxidative stress is involved in this disorder. We verified a marked increase of plasma TBARS measurements, which is indicative of increased lipid peroxidation, as well as a decrease on plasma TAR reflecting a deficient capacity to efficiently modulate the damage associated with an increased production of reactive species. In contrast, TAS was not changed indicating that the total content of antioxidants in plasma of patients affected by MSUD was not altered. These results suggest that free radical generation is elicited in MSUD and is possibly involved in the pathophysiology of the tissue damage found in this disorder.

Investigation of oxidative stress parameters in treated phenylketonuric patients

Phenylketonuria (PKU) is the most frequent disturbance of amino acid metabolism being caused by severe deficiency of phenylalanine hydroxylase activity. Untreated PKU patients present severe mental retardation whose pathophysiology is not completely estabilished. Despite the low-Phe diet, a considerable number of phenylketonuric patients present a mild to moderate psychomotor delay and decreased cognitive functions. In the present study we evaluated various parameters of oxidative stress namely thiobarbituric acid-reactive species (TBA-RS), total antioxidant reactivity (TAR) and activities of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) in two groups of treated PKU patients, one with well controlled and the other with high Phe blood levels in order to investigate whether blood Phe concentrations could be correlated with the extend of oxidative stress. We initially verified a marked increase of TBA-RS, and a decrease of TAR in plasma, as well as a reduction of erythrocyte GSH-Px activity which were similar in both groups of PKU patients, when compared to controls of similar ages. In contrast, CAT and SOD activities were not altered in PKU patients. These results show that oxidative stress occurs in PKU patients and that this pathogenic process is probably not directly correlated to Phe blood levels.

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.

l-Carnitine Blood Levels and Oxidative Stress in Treated Phenylketonuric Patients

Aimsl-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.

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.

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.

Oxidative stress in plasma from maple syrup urine disease patients during treatment

Maple Syrup Urine Disease (MSUD) is an autossomal recessive metabolic disorder caused by a deficiency of branched-chain α-keto acid dehydrogenase complex activity leading to accumulation of the branched-chain amino acids leucine, isoleucine and valine and their corresponding branched-chain α-keto acids. Affected patients usually present hypoglycemia, ketoacidosis, convulsions, poor feeding, coma, psychomotor delay and mental retardation. Considering that the pathophysiology of MSUD is still poorly understood, in this study we evaluated some parameters of oxidative stress, namely thiobarbituric acid-reactive substances (TBARS), total antioxidant reactivity (TAR) and total antioxidant status (TAS) in plasma from treated MSUD patients presenting high and low plasma leucine levels. We verified a significant increase of TBARS (lipid peroxidation) and a decrease of TAR (capacity to rapidly react with free radicals) in plasma from treated MSUD patients with low and with high plasma levels of leucine compared to the control group. It was also verified that TAS (quantity of tissue antioxidants) was not altered in plasma from treated MSUD patients with low and high blood leucine levels. Finally, we found no correlation between leucine, valine and isoleucine levels with the various parameters of oxidative stress. These results are indicative that increased lipid oxidative damage and decreased antioxidant defenses occur in plasma of MSUD patients and that the accumulating branched-chain amino acids are probably not directly associated to oxidative stress in this disorder.

Induction of lipid peroxidation and decrease of antioxidant defenses in symptomatic and asymptomatic patients with X-linked adrenoleukodystrophy.

Patients affected by X‐linked adrenoleukodystrophy (X‐ALD) present a progressive brain and peripheral demyelination and adrenal cortex insufficiency, associated with accumulation of the very long chain fatty acids (VLCFA) hexacosanoic acid (C26:0) and tetracosanoic acid (C24:0) in different tissues and biological fluids. X‐ALD is characterized by heterogeneous clinical phenotypes. Seven clinical variants have been described for this genetic disorder, being the childhood cerebral form (CCER), adrenomyeloneuropathy (AMN) and asymptomatic the most common clinical forms. In a previous work, we showed evidence that oxidative stress is involved in the pathophysiology of X‐ALD symptomatic patients. In the present study, we compared oxidative stress parameters, namely thiobarbituric acid reactive substances (TBA‐RS) and total antioxidant status (TAS), in plasma from patients with CCER, AMN and in asymptomatic X‐ALD patients. It was observed that symptomatic and asymptomatic X‐ALD patients presented a significant increase of plasma TBA‐RS measurement, indicating a stimulation of lipid peroxidation. Furthermore, lipid peroxidation was higher in AMN, as compared to CCER and asymptomatic patients. We also observed that the total antioxidant defenses (TAS) were decreased in symptomatic but not in asymptomatic X‐ALD patients. Therefore, it may be presumed that asymptomatic patients seem to be protected against oxidative stress because of their normal antioxidant defenses and that other factors besides oxidative damage may be responsible for the severity of the symptoms in X‐ALD and need to be investigated.

Oxidative stress parameters in diabetic rats submitted to forced swimming test: the clonazepam effect

Diabetes-associated depression may occur due to changes in the quality of life imposed by treatment, or may be a consequence of the biochemical changes accompanying the disease. We evaluated the oxidative stress from diabetic animals submitted to an experimental model of depression and the effects of clonazepam. Male Wistar rats were induced to diabetes with streptozotocin and submitted to forced swimming test. Clonazepam was administered 24, 5 and 1 h before test. The animals were sacrificed by decapitation, and plasma and erythrocytes were separated, as well as hippocampus, cortex and striatum. Reactive species of thiobarbituric acid (TBARS) and total antioxidant reactivity (TAR) as well as antioxidant enzyme activities catalase (CAT) and superoxide dismutase (SOD) were evaluated. Results showed a significant increase of TBARS and a significant decrease of TAR in plasma from diabetic animals, which was altered by clonazepam. There were no effects of CAT and SOD activities in erythrocytes from tested animals. The results observed in hippocampus showed a significant increase of TBARS from diabetic rats, altered by clonazepam, and no one alteration was verified in TAR. The significant increase of TBARS and the significant decrease of TAR in cortex from diabetic rats were not altered by clonazepam administration. There were no alterations of TBARS and TAR in striatum from tested animals. Besides, clonazepam reverses the immobility in diabetic rats. Considering the action of clonazepam, it is suggested that it could be an alternative therapeutic for depression to diabetic patients, once it could give a protection against free radicals.

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.