Effect of genistein and coenzyme Q10 in oxidative damage and mitochondrial membrane potential in an attenuated type II mucopolysaccharidosis cellular model.

Mucopolysaccharidosis type II (MPS II) is an inborn error of the metabolism resulting from several possible mutations in the gene coding for iduronate-2-sulfatase (IDS), which leads to a great clinical heterogeneity presented by these patients. Many studies demonstrate the involvement of oxidative stress in the pathogenesis of inborn errors of metabolism, and mitochondrial dysfunction and oxidative stress can be related since most of reactive oxygen species come from mitochondria. Cellular models have been used to study different diseases and are useful in biochemical research to investigate them in a new promising way. The aim of this study is to develop a heterozygous cellular model for MPS II and analyze parameters of oxidative stress and mitochondrial dysfunction and investigate the in vitro effect of genistein and coenzyme Q10 on these parameters for a better understanding of the pathophysiology of this disease. The HP18 cells (heterozygous c.261_266del6/c.259_261del3) showed almost null results in the activity of the IDS enzyme and presented accumulation of glycosaminoglycans (GAGs), allowing the characterization of this knockout cellular model by MPS II gene editing. An increase in the production of reactive species was demonstrated (p < .05 compared with WT vehicle group) and genistein at concentrations of 25 and 50 µm decreased in vitro its production (p < .05 compared with HP18 vehicle group), but there was no effect of coenzyme Q10 in this parameter. There was a tendency for lysosomal pH change in HP18 cells in comparison to WT group and none of the antioxidants tested demonstrated any effect on this parameter. There was no increase in the activity of the antioxidant enzymes superoxide dismutase and catalase and oxidative damage to DNA in HP18 cells in comparison to WT group and neither genistein nor coenzyme q10 had any effect on these parameters. Regarding mitochondrial membrane potential, genistein induced mitochondrial depolarization in both concentrations tested (p < .05 compared with HP18 vehicle group and compared with WT vehicle group) and incubation with coenzyme Q10 demonstrated no effect on this parameter. In conclusion, it is hypothesized that our cellular model could be compared with a milder MPS II phenotype, given that the accumulation of GAGs in lysosomes is not as expressive as another cellular model for MPS II presented in the literature. Therefore, it is reasonable to expect that there is no mitochondrial depolarization and no DNA damage, since there is less lysosomal impairment, as well as less redox imbalance.

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.

Alterations of Plasmatic Biomarkers of Neurodegeneration in Mucopolysaccharidosis Type II Patients Under Enzyme Replacement Therapy.

Mucopolysaccharidosis type II (MPS II) is a disorder caused by a deficient activity of iduronate-2-sulfatase, a lysosomal enzyme responsible for degrading glycosaminoglycans (GAGs). The abnormal storage of GAGs within lysosomes disrupts cellular homeostasis and leads to a severe symptomatology. Patients present neuropsychiatric impairment characterized by mental retardation and impaired cognition. The aim of this study was to quantify four neurodegeneration biomarkers in plasma: brain-derived neurotrophic factor (BDNF), platelet-derived growth factor (PDGF-AA), neural cell adhesion molecule (NCAM) and cathepsin-D, as well as to identify possible correlations with urinary GAGs in seven patients undergoing treatment with ERT (Elaprase® 0.5 mg/kg of body weight). Patients with both severe and attenuated forms of MPS II showed signs of neurodegeneration in neuroimaging exams. Patients have a decrease in BDNF and PDGF-AA concentrations, and an increase in NCAM level compared to controls. No alterations in cathepsin-D concentration were seen. GAGs levels were higher in patients than in controls, but no significant correlations between GAGs and biomarkers were observed. These results evidence that patients have neurodegeneration and that monitoring these biomarkers might be useful for assessing this process. To this date, this is the first work to analyze these plasmatic markers of neurodegeneration in patients.

Treatment of maple syrup urine disease: Benefits, risks, and challenges of liver transplantation.

Maple syrup urine disease (MSUD) is caused by a deficiency in the activity of the branched-chain α-ketoacid dehydrogenase (BCKD) complex, promoting the accumulation of the branched-chain amino acids (BCAA) leucine, isoleucine, and valine, as well as their respective α-keto acids. MSUD is an autosomal recessive hereditary metabolic disorder characterized by ketoacidosis, ataxia, coma, and mental and psychomotor retardation. The mechanisms involved in the brain damage caused by MSUD are not fully understood. Early diagnosis and treatment, as well as proper control of metabolic decompensation crises, are crucial for patients' survival and for a better prognosis. The recommended treatment consists of a high-calorie diet with restricted protein intake and specific formulas containing essential amino acids, except those accumulated in MSUD. This treatment will be maintained throughout life, being adjusted according to the patients' nutritional needs and BCAA concentration. Because dietary treatment may not be sufficient to prevent neurological damage in MSUD patients, other therapeutic strategies have been studied, including liver transplantation. With transplantation, it is possible to obtain an increase of about 10% of the normal BCKD in the body, an amount sufficient to maintain amino acid homeostasis and reduce metabolic decompensation crises. However, the experience related to this practice is very limited when considering the shortage of liver for transplantation and the risks related to the surgical procedure and immunosuppression. Thus, the purpose of this review is to survey the benefits, risks, and challenges of liver transplantation in the treatment of MSUD.

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.

Evaluation of biochemical profile and oxidative damage to lipids and proteins in patients with lysosomal acid lipase deficiency.

Lysosomal acid lipase deficiency (LALD) is an inborn error of metabolism that lacks satisfactory treatment, which leads to the development of severe hepatic and cardiac complications and may even lead to death. In this sense, knowledge of the mechanisms involved in the pathophysiology of this disorder becomes essential to allow the search for new therapeutic strategies. There are no studies in the literature investigating the role of reactive species and inflammatory processes in the pathophysiology of this disorder. Therefore, the aim of this work was to investigate parameters of oxidative and inflammatory stress in LALD patients. In this work, we obtained results that demonstrate that LALD patients are susceptible to oxidative stress caused by an increase in the production of free radicals, observed by the increase of 2-7-dihydrodichlorofluorescein. The decrease in sulfhydryl content reflects oxidative damage to proteins, as well as a decrease in antioxidant defenses. Likewise, the increase in urinary levels of di-tyrosine observed also demonstrates oxidative damage to proteins. Furthermore, the determination of chitotriosidase activity in the plasma of patients with LALD was significantly higher, suggesting a pro-inflammatory state. An increase in plasma oxysterol levels was observed in patients with LALD, indicating an important relationship between this disease and cholesterol metabolism and oxidative stress. Also, we observed in LALD patients increased levels of nitrate production. The positive correlation found between oxysterol levels and activity of chitotriosidase in these patients indicates a possible link between the production of reactive species and inflammation. In addition, an increase in lipid profile biomarkers such as total and low-density lipoprotein cholesterol were demonstrated in the patients, which reinforces the involvement of cholesterol metabolism. Thus, we can assume that, in LALD, oxidative and nitrosative damage, in addition to inflammatory process, play an important role in its evolution and future clinical manifestations. In this way, we can suggest that the study of the potential benefit of the use of antioxidant and anti-inflammatory substances as an adjuvant tool in the treatment will be important, which should be associated with the already recommended therapy.

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.

Evaluation of oxidative stress and mitochondrial function in a type II mucopolysaccharidosis cellular model: in vitro effects of genistein and coenzyme Q10.

Mucopolysaccharidosis type II (MPS II or Hunter Syndrome) is a lysosomal disease caused by deficient degradation of glycosaminoglycans (GAGs) heparan sulfate and dermatan sulfate due to the deficiency of the enzyme iduronate-2-sulfatase. The main treatment for MPS II is the administration of the recombinant form of the enzyme, in a process known as enzyme replacement therapy (ERT). Oxidative damage can contribute to the pathophysiology of MPS II and treatment with ERT can reduce the effects of oxidative stress. For a better understanding of pathophysiology of MPS II, we evaluated biomarkers of mitochondrial dysfunction, DNA (Deoxyribonucleic acid) damage, antioxidant defenses, reactive species production and lysosomal size in IDS-deficient HEK 293 cells and investigate the in vitro effect of genistein and coenzyme Q10 (CoQ) on these biomarkers. An increase in the production of reactive species was demonstrated, as well as an increase in the activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT). Also, an increase in lysosomal volume and oxidative damage to DNA were verified. There was no evidence of a change in mitochondrial function in this cell model. In the HEK 293 (human embryonic kidney 293) knockout (KO) HP10 cell model we found that genistein at concentrations of 25 and 50 µm decreased in vitro the production of reactive species and the activity of the SOD enzyme, showing an antioxidant protective effect. Still, in these cells we verified that the coenzyme Q10 in the concentrations of 5 and 10 µm decreased in vitro the activity of the SOD enzyme and in the concentration of 10 µm decreased in vitro the DNA damage, also demonstrating antioxidant protection. In conclusion, MPS II knockout cells demonstrated oxidative stress and DNA damage and genistein, as well as coenzyme Q10, have been shown to have an important protective effect in vitro against these oxidative damages.

High-protein nutrition during pregnancy increases neuroinflammation and homocysteine levels and impairs behavior in male adolescent rats offspring.

AIMS: Throughout gestation, proteins in the diet are a source of essential amino acids that are crucial for proper healthy fetal growth and development. The present study was proposed to investigate the effect of high-protein diet consumption throughout pregnancy on redox homeostasis, neuroinflammatory status and amino acid levels, including homocysteine, in the male adolescent rats offspring's cerebral cortex. We also performed a battery of behavioral tests to evaluate maternal care, olfactory preference, exploratory capacity, habituation, memory, anxiety- and depression-like behavior motor activity in the offspring. MAIN METHODS: After pregnancy confirmation, the pregnant rats were randomly divided into two groups, according to the diet: group 1, (control) standard diet containing 20 % protein, and group 2, the high-protein diet containing 50 % protein. Throughout the gestational period, the pregnant rats received experimental diets. KEY FINDINGS: Results showed an increase in homocysteine levels and neuroinflammatory mediators in the offspring's cerebral cortex from pregnant rats supplemented with a high-protein diet throughout pregnancy. Besides decreasing histidine levels in offspring's serum. The results also revealed an impairment in memory and motricity and an increase in anxiety-like behavior in the offspring supplemented with a high-protein diet throughout pregnancy. Our findings showed a significant effect of high-protein diet consumption throughout pregnancy on offspring's neurobiochemistry, which can negatively impact behavioral performance. SIGNIFICANCE: Our results reinforce the importance of consuming a balanced diet during the gestational period, especially macronutrients such as proteins since the fetus is sensitive to the mother's diet during pregnancy which may impact the development of the offspring.

Clinical findings of patients with hyperammonemia affected by urea cycle disorders with hepatic encephalopathy.

Urea cycle disorders (UCD) are a group of genetic diseases caused by deficiencies in the enzymes and transporters involved in the urea cycle. The impairment of the cycle results in ammonia accumulation, leading to neurological dysfunctions and poor outcomes to affected patients. The aim of this study is to investigate and describe UCD patients' principal clinical and biochemical presentations to support professionals on urgent diagnosis and quick management, aiming better outcomes for patients. We explored medical records of 30 patients diagnosed in a referral center from Brazil to delineate UCD clinical and biochemical profile. Patients demonstrated a range of signs and symptoms, such as altered levels of consciousness, acute encephalopathy, seizures, progressive loss of appetite, vomiting, coma, and respiratory distress, in most cases combined with high levels of ammonia, which is an immediate biomarker, leading to a UCD suspicion. The most prevalent UCD detected were ornithine transcarbamylase deficiency, followed by citrullinemia type 1, hyperargininemia, carbamoyl phosphate synthase 1 deficiency, and argininosuccinic aciduria. Clinical symptoms were highly severe, being the majority developmental and neurological disabilities, with 20% of death rate. Laboratory analysis revealed high levels of ammonia (mean ± SD: 860 ± 470 µmol/L; reference value: ≤80 µmol/L), hypoglycemia, metabolic acidosis, and high excretion of orotic acid in the urine (except in carbamoyl phosphate synthetase 1 [CPS1] deficiency). We emphasize the need of urgent identification of UCD clinical and biochemical conditions, and immediate measurement of ammonia, to enable the correct diagnosis and increase the chances of patients' survival, minimizing neurological and psychomotor damage caused by hepatic encephalopathy.

Hyperammonemia in Inherited Metabolic Diseases.

Ammonia is a neurotoxic compound which is detoxified through liver enzymes from urea cycle. Several inherited or acquired conditions can elevate ammonia concentrations in blood, causing severe damage to the central nervous system due to the toxic effects exerted by ammonia on the astrocytes. Therefore, hyperammonemic patients present potentially life-threatening neuropsychiatric symptoms, whose severity is related with the hyperammonemia magnitude and duration, as well as the brain maturation stage. Inherited metabolic diseases caused by enzymatic defects that compromise directly or indirectly the urea cycle activity are the main cause of hyperammonemia in the neonatal period. These diseases are mainly represented by the congenital defects of urea cycle, classical organic acidurias, and the defects of mitochondrial fatty acids oxidation, with hyperammonemia being more severe and frequent in the first two groups mentioned. An effective and rapid treatment of hyperammonemia is crucial to prevent irreversible neurological damage and it depends on the understanding of the pathophysiology of the diseases, as well as of the available therapeutic approaches. In this review, the mechanisms underlying the hyperammonemia and neurological dysfunction in urea cycle disorders, organic acidurias, and fatty acids oxidation defects, as well as the therapeutic strategies for the ammonia control will be discussed.

Evidence that Oxidative Disbalance and Mitochondrial Dysfunction are Involved in the Pathophysiology of Fatty Acid Oxidation Disorders.

Mitochondrial fatty acid ß-oxidation disorders (FAODs) are a group of about 20 diseases which are caused by specific mutations in genes that codify proteins or enzymes involved in the fatty acid transport and mitochondrial ß-oxidation. As a consequence of these inherited metabolic defects, fatty acids can not be used as an appropriate energetic source during special conditions, such as prolonged fasting, exercise or other catabolic states. Therefore, patients usually present hepatopathy, cardiomyopathy, severe skeletal myopathy and neuropathy, besides biochemical features like hypoketotic hypoglycemia, metabolic acidosis, hypotony and hyperammonemia. This set of symptoms seems to be related not only with the energy deficiency, but also with toxic effects provoked by fatty acids and carnitine derivatives accumulated in the tissues of the patients. The understanding of the mechanisms by which these metabolites provoke tissue injury in FAODs is crucial for the developmental of novel therapeutic strategies that promote increased life expectancy, as well as improved life quality for patients. In this sense, the objective of this review is to present evidence from the scientific literature on the role of oxidative damage and mitochondrial dysfunction in the pathogenesis of the most prevalent FAODs: medium-chain acyl-CoA dehydrogenase (MCAD), long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) and very long-chain acyl-CoA dehydrogenase (VLCAD) deficiencies. It is expected that the findings presented in this review, obtained from both animal model and patients studies, may contribute to a better comprehension of the pathophysiology of these diseases.

L-carnitine protects DNA oxidative damage induced by phenylalanine and its keto acid derivatives in neural cells: a possible pathomechanism and adjuvant therapy for brain injury in phenylketonuria.

Although phenylalanine (Phe) is known to be neurotoxic in phenylketonuria (PKU), its exact pathogenetic mechanisms of brain damage are still poorly known. Furthermore, much less is known about the role of the Phe derivatives phenylacetic (PAA), phenyllactic (PLA) and phenylpyruvic (PPA) acids that also accumulate in this this disorder on PKU neuropathology. Previous in vitro and in vivo studies have shown that Phe elicits oxidative stress in brain of rodents and that this deleterious process also occurs in peripheral tissues of phenylketonuric patients. In the present study, we investigated whether Phe and its derivatives PAA, PLA and PPA separately or in combination could induce reactive oxygen species (ROS) formation and provoke DNA damage in C6 glial cells. We also tested the role of L-carnitine (L-car), which has been recently considered an antioxidant agent and easily cross the blood brain barrier on the alterations of C6 redox status provoked by Phe and its metabolites. We first observed that cell viability was not changed by Phe and its metabolites. Furthermore, Phe, PAA, PLA and PPA, at concentrations found in plasma of PKU patients, provoked marked DNA damage in the glial cells separately and when combined. Of note, these effects were totally prevented (Phe, PAA and PPA) or attenuated (PLA) by L-car pre-treatment. In addition, a potent ROS formation also induced by Phe and PAA, whereas only moderate increases of ROS were caused by PPA and PLA. Pre-treatment with L-car also prevented Phe- and PAA-induced ROS generation, but not that provoked by PLA and PPA. Thus, our data show that Phe and its major metabolites accumulated in PKU provoke extensive DNA damage in glial cells probably by ROS formation and that L-car may potentially represent an adjuvant therapeutic agent in PKU treatment.

Protective effects of L-carnitine on behavioral alterations and neuroinflammation in striatum of glutaryl-COA dehydrogenase deficient mice.

Glutaric acidemia type 1 (GA1) is caused by glutaryl-CoA dehydrogenase deficiency that leads to a blockage in the metabolic route of the amino acids lysine and tryptophan and subsequent accumulation of glutaric acid (GA), 3-hydroxyglutaric acids and glutarylcarnitine (C5DC). Patients predominantly manifest neurological symptoms, associated with acute striatal degeneration, as well as progressive cortical and striatum injury whose pathogenesis is not yet fully established. Current treatment includes protein/lysine restriction and l-carnitine supplementation of (L-car). The aim of this work was to evaluate behavior parameters and pro-inflammatory factors (cytokines IL-1ß, TNF-α and cathepsin-D levels), as well as the anti-inflammatory cytokine IL10 in striatum of knockout mice (Gcdh-/-) and wild type (WT) mice submitted to a normal or a high Lys diet. The potential protective effects of L-car treatment on these parameters were also evaluated. Gcdh-/- mice showed behavioral changes, including lower motor activity (decreased number of crossings) and exploratory activity (reduced number of rearings). Also, Gcdh-/- mice had significantly higher concentrations of glutarylcarnitine (C5DC) in blood and cathepsin-D (CATD), interleukin IL-1ß and tumor factor necrosis alpha (TNF-α) in striatum than WT mice. Noteworthy, L-car treatment prevented most behavioral alterations, normalized CATD levels and attenuated IL-1ß levels in striatum of Gcdh-/- mice. Finally, IL-1ß was positively correlated with CATD and C5DC levels and L-car was negatively correlated with CATD. Our results demonstrate behavioral changes and a pro-inflammatory status in striatum of the animal model of GA1 and, most importantly, L-car showed important protective effects on these alterations.

Preliminary results of PBA-loaded nanoparticles development and the effect on oxidative stress and neuroinflammation in rats submitted to a chemically induced chronic model of MSUD.

Maple syrup urine disease (MSUD) is a genetic disorder that leads the accumulation of branched-chain amino acids (BCAA) leucine (Leu), isoleucine, valine and metabolites. The symptomatology includes psychomotor delay and mental retardation. MSUD therapy comprises a lifelong protein strict diet with low BCAA levels and is well established that high concentrations of Leu and/or its ketoacid are associated with neurological symptoms. Recently, it was demonstrated that the phenylbutyrate (PBA) have the ability to decrease BCAA concentrations. This work aimed the development of lipid-based nanoparticles loaded with PBA, capable of targeting to the central nervous system in order to verify its action mechanisms on oxidative stress and cell death in brain of rats subjected to a MSUD chronic model. PBA-loaded nanoparticles treatment was effective in significantly decreasing BCAA concentration in plasma and Leu in the cerebral cortex of MSUD animals. Furthermore, PBA modulate the activity of catalase, superoxide dismutase, glutathione peroxidase and glutathione reductase enzymes, as well as preventing the oxidative damage to lipid membranes and proteins. PBA was also able to decrease the glial fibrillary acidic protein concentrations and partially decreased the reactive species production and caspase-3 activity in MSUD rats. Taken together, the data indicate that the PBA-loaded nanoparticles could be an efficient adjuvant in the MSUD therapy, protecting against oxidative brain damage and neuroinflammation.

Parameters of Oxidative Stress and Behavior in Animals Treated with Dexametasone and Submitted to Pentylenetetrazol Kindling.

BACKGROUND AND PURPOSE: Oxidative stress (OS) is defined as an excessive production of reactive oxygen species that cannot be neutralized by the action of antioxidants, but also as an alteration of the cellular redox balance. The relationship between OS and epilepsy is not yet fully understood. The objective of this study was to evaluate the effect of dexamethasone on OS levels and memory in the kindling model induced by pentylenetetrazole. METHODS: The animals were divided in six groups: control group that received no treatment, vehicle group treated with vehicle, diazepam group, and groups treated with dexamethasone (1, 2 and 4 mg/kg). Treated animals received pentylenetetrazole in alternated days for 15 days. Inhibitory avoidance test was conducted in 2 hours and OS was evaluated after animal sacrifice. RESULTS: Regarding the treatment with dexamethasone, there was no significant difference when compared to the control groups in relation to the inhibitory avoidance test. On OS levels, there was a decrease in catalase activity levels in the hippocampus and an increase in thiobarbituric acid reactive substances and glutathione peroxidase levels in the hippocampus. CONCLUSIONS: The anticonvulsant effect of dexametasone remains uncertain. Immunological mechanisms, with the release of cytokines and inflammatory mediators, seem to be the key to this process. The mechanisms that generate OS are probably related to the anticonvulsant effects found.

Clinical, biochemical and molecular findings of 24 Brazilian patients with glutaric acidemia type 1: 4 novel mutations in the GCDH gene.

Glutaric aciduria type 1 (GA-1) is a rare but treatable inherited disease caused by deficiency of glutaryl-CoA dehydrogenase activity due to GCDH gene mutations. In this study, we report 24 symptomatic GA-1 Brazilian patients, and present their clinical, biochemical, and molecular findings. Patients were diagnosed by high levels of glutaric and/or 3-hydroxyglutaric and glutarylcarnitine. Diagnosis was confirmed by genetic analysis. Most patients had the early-onset severe form of the disease and the main features were neurological deterioration, seizures and dystonia, usually following an episode of metabolic decompensation. Despite the early symptomatology, diagnosis took a long time for most patients. We identified 13 variants in the GCDH gene, four of them were novel: c.91 + 5G > A, c.167T > G, c.257C > T, and c.10A > T. The most common mutation was c.1204C > T (p.R402W). Surprisingly, the second most frequent mutation was the new mutation c.91 + 5G > A (IVS1 ds G-A + 5). Our results allowed a complete characterization of the GA-1 Brazilian patients. Besides, they expand the mutational spectrum of GA-1, with the description of four new mutations. This work reinforces the importance of awareness of GA-1 among doctors in order to allow early diagnosis and treatment in countries like Brazil where the disease has not been included in newborn screening programs.

Disruption of mitochondrial functions and oxidative stress contribute to neurologic dysfunction in organic acidurias.

Organic acidurias (OADs) are inherited disorders of amino acid metabolism biochemically characterized by accumulation of short-chain carboxylic acids in tissues and biological fluids of the affected patients and clinically by predominant neurological manifestations. Some of these disorders are amenable to treatment, which significantly decreases mortality and morbidity, but it is still ineffective to prevent long-term neurologic and systemic complications. Although pathogenesis of OADs is still poorly established, recent human and animal data, such as lactic acidosis, mitochondrial morphological alterations, decreased activities of respiratory chain complexes and altered parameters of oxidative stress, found in tissues from patients and from genetic mice models with these diseases indicate that disruption of critical mitochondrial functions and oxidative stress play an important role in their pathophysiology. Furthermore, organic acids that accumulate in the most prevalent OADs were shown to compromise bioenergetics, by decreasing ATP synthesis, mitochondrial membrane potential, reducing equivalent content and calcium retention capacity, besides inducing mitochondrial swelling, reactive oxygen and nitrogen species generation and apoptosis. It is therefore presumed that secondary mitochondrial dysfunction and oxidative stress caused by major metabolites accumulating in OADs contribute to tissue damage in these pathologies.

Free carnitine and branched chain amino acids are not good biomarkers in Huntington's disease.

BACKGROUND: Huntington's disease (HD), caused by an expanded CAG repeat at HTT, has no treatment, and biomarkers are needed for future clinical trials. OBJECTIVE: The objective of this study was to verify if free carnitine and branched chain amino acids levels behave as potential biomarkers in HD. METHODS: Symptomatic and asymptomatic HD carriers and controls were recruited. Age, sex, body mass index (BMI), age of onset, disease duration, UHDRS scores, and expanded CAG tract were obtained; valine, leucine, isoleucine, and free carnitine were measured. Baseline and longitudinal analysis were performed. RESULTS: Seventy-four symptomatic carriers, 20 asymptomatic carriers, and 22 non-carriers were included. At baseline, valine levels were reduced in symptomatic and asymptomatic HD carriers when compared to non-carriers. No difference in free carnitine or isoleucine+leucine levels were observed between groups. BMI of symptomatic individuals was lower than those of non-carriers. Valine levels correlated with BMI. Follow-up evaluation was performed in 43 symptomatic individuals. UHDRS total motor score increased 4.8 points/year on average. No significant reductions in BMI or valine were observed, whereas free carnitine and isoleucine+leucine levels increased. CONCLUSIONS: Although valine levels were lower in HD carriers and were related to BMI losses observed in pre-symptomatic individuals, none of these metabolites seem to be biomarkers for HD.

Free carnitine and branched chain amino acids are not good biomarkers in Huntington's disease / Carnitina livre e aminoácidos de cadeia ramificada não são bons biomarcadores na doença de Huntington

ABSTRACT Background: Huntington's disease (HD), caused by an expanded CAG repeat at HTT, has no treatment, and biomarkers are needed for future clinical trials. Objective: The objective of this study was to verify if free carnitine and branched chain amino acids levels behave as potential biomarkers in HD. Methods: Symptomatic and asymptomatic HD carriers and controls were recruited. Age, sex, body mass index (BMI), age of onset, disease duration, UHDRS scores, and expanded CAG tract were obtained; valine, leucine, isoleucine, and free carnitine were measured. Baseline and longitudinal analysis were performed. Results: Seventy-four symptomatic carriers, 20 asymptomatic carriers, and 22 non-carriers were included. At baseline, valine levels were reduced in symptomatic and asymptomatic HD carriers when compared to non-carriers. No difference in free carnitine or isoleucine+leucine levels were observed between groups. BMI of symptomatic individuals was lower than those of non-carriers. Valine levels correlated with BMI. Follow-up evaluation was performed in 43 symptomatic individuals. UHDRS total motor score increased 4.8 points/year on average. No significant reductions in BMI or valine were observed, whereas free carnitine and isoleucine+leucine levels increased. Conclusions: Although valine levels were lower in HD carriers and were related to BMI losses observed in pre-symptomatic individuals, none of these metabolites seem to be biomarkers for HD.

RESUMO Introdução: A doença de Huntington (DH), causada por uma repetição CAG expandida no HTT, não possui tratamento e biomarcadores são necessários para futuros ensaios clínicos. Objetivo: Nosso objetivo foi verificar se os níveis de carnitina livre e aminoácidos de cadeia ramificada se comportam como potenciais biomarcadores na DH. Métodos: Portadores sintomáticos e assintomáticos e controles foram recrutados. Idade, sexo, índice de massa corporal (IMC), idade de início, duração da doença, escores UHDRS e trato CAG expandido foram obtidos; valina, leucina, isoleucina e carnitina livre foram medidas. Foram realizadas análises basal e longitudinal. Resultados: Setenta e quatro portadores sintomáticos, 20 portadores assintomáticos e 22 não portadores foram incluídos. No início do estudo, os níveis de valina estavam reduzidos em portadores de DH sintomáticos e assintomáticos quando comparados aos não portadores. Não foram observadas diferenças nos níveis de carnitina livre ou isoleucina + leucina entre os grupos. O IMC dos indivíduos sintomáticos foi menor que o dos não portadores. Níveis de valina correlacionaram-se com o IMC. Avaliação de acompanhamento foi realizada em 43 indivíduos sintomáticos. A pontuação do escore motor total da UHDRS aumentou 4,8 pontos/ano em média. Não foram observadas reduções significativas no IMC ou na valina, enquanto os níveis de carnitina livre e isoleucina+leucina aumentaram. Conclusões: Embora os níveis de valina tenham sido menores nos portadores de DH e estivessem relacionados às perdas de IMC observadas em indivíduos pré-sintomáticos, nenhum desses metabólitos parece ser biomarcador para a DH.