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.

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.

Rectosigmoid transition perforation by ingá (Inga laurina) seeds in the Brazilian amazon: Case report and surgical treatment.

INTRODUCTION: Foreign body ingestion is a common clinical scenario found in clinical practice. Perforations related to foreign bodies are rare, but they can present as a serious condition in emergency surgery services. The most common sites of perforation are angled areas, such as: ileocecal valve, sigmoid colon, duodenojejunal flexure and small intestine. We are going to describe a rare case of extensive perforation of rectosigmoid transition, without associated obstructive clinical picture, related to voluntary ingestion of foreign body caused by multiple seeds of a typical amazon fruit. PRESENTATION OF CASE: This case report describes the presentation and management of a 46-year-old man who presented signs of acute perforating abdomen, without obstructive condition, after ingestion of foreign body. Imaging examination revealed the presence of foreign bodies with signs of intestinal perforation. Exploratory laparotomy was performed to treat the lesion and remove foreign bodies. DISCUSSION: Bowel perforation by a non-sharp foreign body is a rare complication of object ingestion. Object shape, quantity, narrowing of gastrointestinal tract are factors that can favor perforation. The clinic is not very specific, usually preceded by when obstructive or sub occlusive, and the clinical history is relevant for diagnostic formulation. Regions and cultures with a high intake of food with seeds may constitute an extra risk factor. CONCLUSION: The importance of alerting surgical teams to the possibility of bowel perforation without associated occlusive conditions caused by multiple non-sharp seeds is highlighted, as well as the need for early treatment aiming at favorable clinical outcome.

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.

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.

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.

Protective effect of N-acetyl-L-cysteine and rosuvastatin against oxidative stress in fibroblasts from asymptomatic patients with X-ALD: a preliminary study

Introduction: Several studies in the literature have evaluated the role of oxidative stress and adjuvant therapies for X-linked adrenoleukodystrophy (X-ALD). Here, we investigated whether n-acetyl-L-cysteine (NAC) and rosuvastatin (RSV) could influence the generation of reactive species, redox status and nitrative stress in fibroblasts from asymptomatic patients with X-ALD. Methods: Skin biopsy samples were cultured and treated for 2 hours (37 °C) with NAC and RSV. Results: X-ALD fibroblasts generated high levels of reactive oxygen species. These levels were significantly lower in fibroblasts treated with NAC and RSV relative to untreated samples. The X-ALD fibroblasts from asymptomatic patients also had higher catalase activity, and only NAC was able to increase enzyme activity in the samples. Conclusions: Our results indicated that NAC and RSV were able to improve oxidative stress parameters in fibroblasts from asymptomatic patients with X-ALD, showing that adjuvant antioxidant therapy may be a promising treatment strategy for asymptomatic patients with this disease. (AU)

Oxidative damage in mitochondrial fatty acids oxidation disorders patients and the in vitro effect of l-carnitine on DNA damage induced by the accumulated metabolites.

BACKGROUND: The mitochondrial fatty acids oxidation disorders (FAOD) are inherited metabolic disorders (IMD) characterized by the accumulation of fatty acids of different sizes of chain according to the affected enzyme. METHODS: This study evaluated the lipid peroxidation by the measurement of 8-isoprostanes, nitrosative stress parameters by the measurement of nitrite and nitrate content and DNA and RNA oxidative damage by the measurement of oxidized guanine species in urine samples from long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD), medium-chain acyl-CoA dehydrogenase deficiency (MCADD) and multiple acyl-CoA dehydrogenase deficiency (MADD) patients. Also, we analyzed the in vitro DNA damage by comet assay induced by adipic acid, suberic acid, hexanoylglycine and suberylglycine, separated and in combination, as well as the effect of l-carnitine in human leukocytes. RESULTS: An increase on 8-isoprostanes levels in all groups of patients was observed. The nitrite and nitrate levels were increased in LCHADD patients. DNA and RNA damage evaluation revealed increase on oxidized guanine species levels in LCHADD and MADD patients. The in vitro evaluation revealed an increase on the DNA damage induced by all metabolites, besides a potencialyzed effect. l-carnitine decreased the DNA damage induced by the metabolites. CONCLUSION: These results demonstrate that toxic metabolites accumulated could be related to the increased oxidative and nitrosative stress of FAOD patients and that the metabolites, separated and in combination, cause DNA damage, which was reduced by l-carnitine, demonstrating antioxidant protection. GENERAL SIGNIFICANCE: This work demonstrated oxidative stress in FAOD patients and the genotoxic potential of MCADD metabolites and the protective effect of l-carnitine.

Monitoring of Phenylalanine Levels in Patients with Phenylketonuria Using Dried Blood Spots: a Comparison of Two Methods

Abstract Phenylketonuria (PKU) is caused by deficient activity of phenylalanine hydroxylase (PAH), responsible for the conversion of phenylalanine (Phe) to tyrosine (Tyr). Monitoring of patients with PKU requires the measurement of Phe in plasma using high-performance liquid chromatography (HPLC) or in dried blood spots (DBS) using different techniques to adjust treatment strategy. The objective of this study was to evaluate Phe levels in DBS measured by two different methods and compare them with Phe levels measured in plasma by HPLC. We analyzed 89 blood samples from 47 PKU patients by two different methods: fluorometric method developed in-house (method A) and the commercially available PerkinElmer® Neonatal Phenylalanine Kit (method B) and in plasma by HPLC. The mean Phe levels by method A, method B, and HPLC were 430.4±39.9μmol/L, 439.3±35.4μmol/L, and 442.2±41.6μmol/L, respectively. The correlation values between HPLC and methods A and B were 0.990 and 0.974, respectively (p < 0.001 for both). Our data suggest that methods A and B are useful alternatives for monitoring Phe levels in patients with PKU, with method A being in closer agreement with the reference standard (HPLC).

The value of fasting in the diagnosis of medium-chain acyl-CoA dehydrogenase deficiency / O valor do jejum no diagnóstico da deficiência de acil-CoA desidrogenase de cadeia média

ABSTRACT Female patient carrier of medium-chain acyl-CoA dehydrogenase deficiency (MCADD) with recurrent clinical episodes of hypoglycemia and altered level of consciousness, presented changes in blood acylcarnitine profile by tandem mass spectrometry and in the urinary organic acid analysis by gas chromatography/mass spectrometry (GC/MS). This case demonstrates the importance of fasting prior biological sample collection (when possible) when MCADD is suspected, and emphasizes that the time/momentum of biological sample collection is crucial to diagnosis, considering the possibility that MCADD is underdiagnosed in Brazil.

RESUMEN Paciente portadora de deficiencia de acil-CoA deshidrogenasa de cadena media (MCADD) con episodios clínicos recurrentes de hipoglucemia y alteración de consciencia presentó mudanzas en el perfil de acilcarnitinas en la sangre con técnicas de espectrometría de masas en tándem y en el análisis de ácidos orgánicos urinarios mediante cromatografía de gases acoplada a espectrometría de masas. Este caso demuestra la importancia de la toma de muestras biológicas en ayunas (se posible) cuando se sospecha de MCADD y destaca que el tiempo/momento de extracción de la muestra biológica es valioso para el diagnóstico, considerando la posibilidad de que la MCADD es subdiagnosticada en Brasil.

RESUMO Paciente portadora de deficiência de acil-CoA desidrogenase de cadeia média (MCADD), com episódios clínicos recorrentes de hipoglicemia e alteração de consciência, apresentou alterações no perfil de acilcarnitinas em sangue por espectrometria de massas em tandem e na análise de ácidos orgânicos urinários por cromatografia gasosa acoplada à espectrometria de massa. Este caso demonstra a importância da coleta de amostra biológica em jejum (se possível) quando há suspeita de MCADD e ressalta que o tempo/momento de coleta da amostra biológica é importante para o diagnóstico, considerando a possibilidade de a MCADD ser subdiagnosticada no Brasil.

Increased levels of hexacosanoic acid in the brain of Winstar rats: a behavioral study

Introduction: X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal metabolic disorder associated with mutations in the ATP-binding cassette sub-family D member1 (ABCD1) gene. Practically all male patients with X-ALD develop adrenocortical insufficiency during childhood and progressive myelopathy and peripheral neuropathy in adulthood. However, some male patients develop a fatal cerebral demyelinating disease named cerebral adrenoleukodystrophy. Although the exact mechanisms underlying brain damage in X-ALD are still poorly elucidated, it is known that hexacosanoic acid (C26:0) accumulation represents a hallmark in the pathogenesis of this disease. In this study, we examined whether an overload of C26:0 injected in Wistar rats was capable of causing behavioral changes in these animals. Methods: Egg lecithin in ethanol was dried under a nitrogen stream and mixed with C26:0 methyl ester. Male Wistar rats at 2-3 weeks of age were obtained from Universidade Federal do Rio Grande do Sul (UFRGS), divided into 8 groups, and submitted to an open field test. We then analyzed line crossings (locomotion and exploration), rearing (orienting and investigatory responses), grooming (anxiety manifestation), and latency to move for each animal. Results: Animals subjected to C26:0 administration presented fewer crossings and rearing episodes and a higher latency to move 45 minutes after C26:0 injection. The present work yields experimental evidence that C26:0, the main accumulated metabolite in X-ALD, can cause behavioral alterations in rats such as the impairment of locomotion and exploratory capabilities, as well as a reduction in orienting and investigatory responses. Conclusion: Although our results are preliminary, they are extremely important for future studies that investigate C26:0 accumulation and locomotor impairment in patients with X-ALD. (AU)

Prevention by L-carnitine of DNA damage induced by 3-hydroxy-3-methylglutaric and 3-methylglutaric acids and experimental evidence of lipid and DNA damage in patients with 3-hydroxy-3-methylglutaric aciduria.

3-hydroxy-3-methylglutaric aciduria (HMGA) is an inherited disorder of the leucine catabolic pathway in which occurs a deficiency of the 3-hydroxy-3-methylglutaryl-CoA lyase enzyme. Therefore, the organic acids 3-hydroxy-3-methylglutaric (HMG) and 3-methylglutaric (MGA), mainly, accumulate in tissues of affected patients. Lately, much attention has been focused on free radicals as mediators of tissue damage in human diseases, causing lipid peroxidation, protein oxidation and DNA damage. The treatment of this disease is based in a restricted protein ingest and supplementation with l-carnitine (LC), an antioxidant and detoxifying agent. In the present work, we investigated the in vitro oxidative damage to DNA induced by the accumulation of organic acids and oxidative stress parameters in vivo of patients with 3-HMG, as well as the effect of the recommended therapy. The in vitro DNA damage was analyzed by the alkaline comet assay in leukocytes incubated with HMG and MGA (1 mM, 2.5 mM and 5 mM) and co-incubated with LC (90 µM and 150 µM). The in vivo urinary 15-F2t-isoprostane levels and urinary oxidized guanine species were measured by ELISA kits in patient's urine before and after the treatment with LC. HMG and MGA induced a DNA damage index (DI) significantly higher than that of the control group. The DI was significantly reduced in the presence of LC. It was also verified a significant increase of oxidized guanine species and urinary isoprostane levels, biomarker of oxidative DNA damage and lipid peroxidation respectively, in patients before treatment. After the treatment and supplementation with LC, patients presented significantly lower levels of those biomarkers. Analyzing the data together, we can conclude that HMGA patients present oxidative lipid and DNA damage, which is induced by HMG and MGA, and the antioxidant therapy with LC can prevent that kind of injuries.

Screening for organic acidurias and aminoacidopathies in high-risk Brazilian patients: Eleven-year experience of a reference center.

Organic acidurias and aminoacidopathies are groups of frequent inborn errors of metabolism (IEMs), which are caused by mutations in specific genes that lead to loss of protein/enzyme or transport function with important deleterious effects to cell metabolism. Since a considerable number of such disorders are potentially treatable when diagnosed at an early stage of life, diagnosis is crucial for the patients. In the present report, we describe symptomatic individuals referred to our service that were diagnosed with these disorders from 2006 to 2016. We used blood and urine samples from 21,800 patients suspected of aminoacidopathies or organic acidemias that were processed by the analytical techniques reverse phase high-performance liquid chromatography for amino acid quantification and gas chromatography coupled to mass spectrometry for organic acid detection. Analysis of dried blood spots by liquid chromatography-tandem mass spectrometry was used in some cases. We detected 258 cases of organic acidurias, and 117 patients with aminoacidopathies were diagnosed. Once diagnosis was performed, patients were promptly submitted to the available treatments with clear reduction of mortality and morbidity. The obtained data may help pediatricians and metabolic geneticists to become aware of these diseases and possibly expand newborn screening programs in the future.

DNA damage induced by alloisoleucine and other metabolites in maple syrup urine disease and protective effect of l-carnitine.

Maple syrup urine disease (MSUD) is an inherited deficiency of the branched-chain α-keto dehydrogenase complex, characterized by accumulation of the branched-chain amino acids (BCAAs) and their respective branched chain α-keto-acids (BCKAs), as well as by the presence of alloisoleucine (Allo). Studies have shown that oxidative stress is involved in the pathophysiology of MSUD. In this work, we investigated using the comet assay whether Allo, BCAAs and BCKAs could induce in vitro DNA damage, as well as the influence of l-Carnitine (L-Car) upon DNA damage. We also evaluated urinary 8-hydroxydeoguanosine (8-OHdG) levels, an oxidative DNA damage biomarker, in MSUD patients submitted to a restricted diet supplemented or not with L-Car. All tested concentrations of metabolites (separated or incubated together) induced in vitro DNA damage, and the co-treatment with L-Car reduced these effects. We found that Allo induced the higher DNA damage class and verified a potentiation of DNA damage induced by synergistic action between metabolites. In vivo, it was observed a significant increase in 8-OHdG levels, which was reversed by L-Car. We demonstrated for the first time that oxidative DNA damage is induced not only by BCAAs and BCKAs but also by Allo and we reinforce the protective effect of L-Car.

Oxidative Imbalance, Nitrative Stress, and Inflammation in C6 Glial Cells Exposed to Hexacosanoic Acid: Protective Effect of N-acetyl-L-cysteine, Trolox, and Rosuvastatin.

X-linked adrenoleukodystrophy (X-ALD) is an inherited neurometabolic disorder caused by disfunction of the ABCD1 gene, which encodes a peroxisomal protein responsible for the transport of the very long-chain fatty acids from the cytosol into the peroxisome, to undergo ß-oxidation. The mainly accumulated saturated fatty acids are hexacosanoic acid (C26:0) and tetracosanoic acid (C24:0) in tissues and body fluids. This peroxisomal disorder occurs in at least 1 out of 20,000 births. Considering that pathophysiology of this disease is not well characterized yet, and glial cells are widely used in studies of protective mechanisms against neuronal oxidative stress, we investigated oxidative damages and inflammatory effects of vesicles containing lecithin and C26:0, as well as the protection conferred by N-acetyl-L-cysteine (NAC), trolox (TRO), and rosuvastatin (RSV) was assessed. It was verified that glial cells exposed to C26:0 presented oxidative DNA damage (measured by comet assay and endonuclease III repair enzyme), enzymatic oxidative imbalance (high catalase activity), nitrative stress [increased nitric oxide (NO) levels], inflammation [high Interleukin-1beta (IL-1ß) levels], and induced lipid peroxidation (increased isoprostane levels) compared to native glial cells without C26:0 exposure. Furthermore, NAC, TRO, and RSV were capable to mitigate some damages caused by the C26:0 in glial cells. The present work yields experimental evidence that inflammation, oxidative, and nitrative stress may be induced by hexacosanoic acid, the main accumulated metabolite in X-ALD, and that antioxidants might be considered as an adjuvant therapy for this severe neurometabolic disease.

Oxidative damage in glutaric aciduria type I patients and the protective effects of l-carnitine treatment.

The deficiency of the enzyme glutaryl-CoA dehydrogenase, known as glutaric acidemia type I (GA-I), leads to the accumulation of glutaric acid (GA) and glutarilcarnitine (C5DC) in the tissues and body fluids, unleashing important neurotoxic effects. l-carnitine (l-car) is recommended for the treatment of GA-I, aiming to induce the excretion of toxic metabolites. l-car has also demonstrated an important role as antioxidant and anti-inflammatory in some neurometabolic diseases. This study evaluated GA-I patients at diagnosis moment and treated the oxidative damage to lipids, proteins, and the inflammatory profile, as well as in vivo and in vitro DNA damage, reactive nitrogen species (RNS), and antioxidant capacity, verifying if the actual treatment with l-car (100 mg kg-1 day-1 ) is able to protect the organism against these processes. Significant increases of GA and C5DC were observed in GA-I patients. A deficiency of carnitine in patients before the supplementation was found. GA-I patients presented significantly increased levels of isoprostanes, di-tyrosine, urinary oxidized guanine species, and the RNS, as well as a reduced antioxidant capacity. The l-car supplementation induced beneficial effects reducing these biomarkers levels and increasing the antioxidant capacity. GA, in three different concentrations, significantly induced DNA damage in vitro, and the l-car was able to prevent this damage. Significant increases of pro-inflammatory cytokines IL-6, IL-8, GM-CSF, and TNF-α were shown in patients. Thus, the beneficial effects of l-car presented in the treatment of GA-I are due not only by increasing the excretion of accumulated toxic metabolites, but also by preventing oxidative damage.

Selective Screening of Fatty Acids Oxidation Defects and Organic Acidemias by Liquid Chromatography/tandem Mass Spectrometry Acylcarnitine Analysis in Brazilian Patients.

BACKGROUND: Inborn errors of metabolism (IEM) are diseases which can lead to accumulation of toxic metabolites in the organism. AIM OF THE STUDY: To investigate, by selective screening, mitochondrial fatty acid oxidation defects (FAOD) and organic acidemias in Brazilian individuals with clinical suspicion of IEM. METHODS: A total of 7,268 individuals, from different regions of Brazil, had whole blood samples impregnated on filter paper which were submitted to the acylcarnitines analysis by liquid chromatography/tandem mass spectrometry (LC/MS/MS) at the Medical Genetics Service of Hospital de Clínicas de Porto Alegre, Brazil, during July 2008-July 2016. RESULTS: Our results showed that 68 patients (0.93%) were diagnosed with FAOD (19 cases) and organic acidemias (49 cases). The most prevalent FAOD was multiple acyl CoA dehydrogenase deficiency (MADD), whereas glutaric type I and 3-OH-3-methylglutaric acidemias were the most frequent disorders of organic acid metabolism. Neurologic symptoms and metabolic acidosis were the most common clinical and laboratory features, whereas the average age of the patients at diagnosis was 2.3 years. CONCLUSIONS: Results demonstrated a high incidence of glutaric acidemia type I and 3-OH-3- methylglutaric acidemia in Brazil and an unexpectedly low incidence of FAOD, particularly medium-chain acyl-CoA dehydrogenase deficiency (MCADD).

Inflammatory profile in X-linked adrenoleukodystrophy patients: Understanding disease progression.

X-linked adrenoleukodystrophy (X-ALD) is an inherited disease characterized by progressive inflammatory demyelization in the brain, adrenal insufficiency, and an abnormal accumulation of very long chain fatty acids (VLCFA) in tissue and body fluids. Considering that inflammation might be involved in pathophysiology of X-ALD, we aimed to investigate pro- and anti-inflammatory cytokines in plasma from three different male phenotypes (CCER, AMN, and asymptomatic individuals). Our results showed that asymptomatic patients presented increased levels of pro-inflammatory cytokines IL-1ß, IL-2, IL-8, and TNF-α and the last one was also higher in AMN phenotype. Besides, asymptomatic patients presented higher levels of anti-inflammatory cytokines IL-4 and IL-10. AMN patients presented higher levels of IL-2, IL-5, and IL-4. We might hypothesize that inflammation in X-ALD is related to plasmatic VLCFA concentration, since there were positive correlations between C26:0 plasmatic levels and pro-inflammatory cytokines in asymptomatic and AMN patients and negative correlation between anti-inflammatory cytokine and C24:0/C22:0 ratio in AMN patients. The present work yields experimental evidence that there is an inflammatory imbalance associated Th1, (IL-2, IL-6, and IFN-γ), Th2 (IL-4 and IL-10), and macrophages response (TNF-α and IL-1ß) in the periphery of asymptomatic and AMN patients, and there is correlation between VLCFA plasmatic levels and inflammatory mediators in X-ALD. Furthermore, we might also speculate that the increase of plasmatic cytokines in asymptomatic patients could be considered an early biomarker of brain damage and maybe also a predictor of disease progression.

DNA damage in homocystinuria: 8-oxo-, 8-dihydro-2'-deoxyguanosine levels in cystathionine-ß-synthase deficient patients and the in vitro protective effect of N-acetyl­L­cysteine

Introduction: Homocysteine (Hcy) tissue accumulation occurs in a metabolic disease characterized biochemically by cystathionine ß-synthase (CBS) deficiency and clinically by mental retardation, vascular problems, and skeletal abnormalities. Previous studies indicate the occurrence of DNA damage secondary to hyperhomocysteinemia and it was observed that DNA damage occurs in leukocytes from CBS-deficient patients. This study aimed to investigate whether an oxidative mechanism could be involved in DNA damage previously found and investigated the in vitro effect of N-acety-L-cysteine (NAC) on DNA damage caused by high Hcy levels. Methods: We evaluated a biomarker of oxidative DNA damage in the urine of CBS­deficient patients, as well as the in vitro effect of NAC on DNA damage caused by high levels of Hcy. Moreover, a biomarker of lipid oxidative damage was also measured in urine of CBS deficient patients. Results: There was an increase in parameters of DNA (8-oxo-7,8-dihydro-2'- deoxyguanosine) and lipid (15-F2t-isoprostanes levels) oxidative damage in CBS-deficient patients when compared to controls. In addition, a significant positive correlation was found between 15-F2t-isoprostanes levels and total Hcy concentrations. Besides, an in vitro protective effect of NAC at concentrations of 1 and 5 mM was observed on DNA damage caused by Hcy 50 µM and 200 µM. Additionally, we showed a decrease in sulfhydryl content in plasma from CBS-deficient patients when compared to controls. Discussion: These results demonstrated that DNA damage occurs by an oxidative mechanism in CBS deficiency together with lipid oxidative damage, highlighting the NAC beneficial action upon DNA oxidative process, contributing with a new treatment perspective of the patients affected by classic homocystinuria.