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.

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).

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.

In vivo intracerebral administration of L-2-hydroxyglutaric acid provokes oxidative stress and histopathological alterations in striatum and cerebellum of adolescent rats.

Patients affected by L-2-hydroxyglutaric aciduria (L-2-HGA) are biochemically characterized by elevated L-2-hydroxyglutaric acid (L-2-HG) concentrations in cerebrospinal fluid, plasma, and urine due to a blockage in the conversion of L-2-HG to α-ketoglutaric acid. Neurological symptoms associated with basal ganglia and cerebelar abnormalities whose pathophysiology is still unknown are typical of this neurometabolic disorder. In the present study we evaluated the early effects (30min after injection) of an acute in vivo intrastriatal and intracerebellar L-2-HG administration on redox homeostasis in rat striatum and cerebellum, respectively. Histological analyses of these brain structures were also carried out 7 days after L-2-HG treatment (long-term effects). L-2-HG significantly decreased the concentrations of reduced (GSH) and total glutathione (tGS), as well as of glutathione peroxidase (GPx) and reductase (GR) activities, but did not change the activities of superoxide dismutase and catalase in striatum. Furthermore, the concentrations of oxidized glutathione (GSSG) and malondialdehyde (MDA), as well as 2',7'-dichlorofluorescein (DCFH) oxidation and hydrogen peroxide (H2O2) production, were increased, whereas carbonyl formation and nitrate plus nitrite concentrations were not altered by L-2-HG injection. It was also found that the melatonin, ascorbic acid plus α-tocopherol, and creatine totally prevented most of these effects, whereas N-acetylcysteine, the noncompetitive glutamate NMDA antagonist MK-801, and the nitric oxide synthase inhibitor L-NAME were not able to normalize the redox alterations elicited by L-2-HG in striatum. L-2-HG intracerebellar injection similarly provoked a decrease of antioxidant defenses (GSH, tGS, GPx, and GR) and an increase of the concentrations of GSSG, MDA, and H2O2 in cerebellum. These results strongly indicate that the major accumulating metabolite in L-2-HGA induce oxidative stress by decreasing the antioxidant defenses and enhancing reactive oxygen species in striatum and cerebellum of adolescent rats. Regarding the histopathological findings, L-2-HG caused intense vacuolation, lymphocyte and macrophage infiltrates, eosinophilic granular bodies, and necrosis in striatum. Immunohistochemistry revealed that L-2-HG treatment provoked an increase of GFAP and a decrease of NeuN immunostaining, indicating reactive astroglyosis and reduction of neuronal population, respectively, in striatum. Similar macrophage infiltrates, associated with less intense vacuolation and lymphocytic infiltration, were observed in cerebellum. However, we did not observe necrosis, eosinophilic granular bodies, and alteration of GFAP and NeuN content in L-2-HG-teated cerebellum. From the biochemical and histological findings, it is presumed that L-2-HG provokes striatal and cerebellar damage in vivo possibly through oxidative stress induction. Therefore, we postulate that antioxidants may serve as adjuvant therapy allied to the current treatment based on a protein-restricted diet and riboflavin and L-carnitine supplementation in patients affected by L-2-HGA.

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

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

Selective screening for organic acidemias by urine organic acid GC-MS analysis in Brazil: fifteen-year experience.

BACKGROUND: The gas chromatography/mass spectrometry (GC/MS) method for organic acid analysis was established in developed countries since 1980s, but due to the small number of experienced clinical biochemists in this field and also the short availability of mass spectrometers scarce reports exist on the prevalence of organic acidemias (OAs) in developing countries like Brazil. METHODS: During January 1994 to July 2008, we analyzed organic acids by GC/MS in urine specimens obtained from Brazilian children with clinical suspicion of metabolic diseases. RESULTS: Two hundred and thirty four cases of disorders of organic acid metabolism, including 218 OAs (3.17%), were diagnosed among 6866 patients investigated. The most frequent disorders were primary lactic acidemia (57), methylmalonic acidemia (34), glutaric acidemia type I (33), propionic acidemia (18), 3-hydroxy-3-methylglutaric aciduria (17), L-2-hydroxyglutaric aciduria (9) and multiple carboxylase deficiency (9). Fourteen cases of mitochondrial fatty acid oxidation disorders, as well as 12 aminoacidopathies and 4 cases of vitamin B12 deficiency were also detected. Prompt treatment following diagnosis led to a better outcome in a considerable number of patients. CONCLUSION: Detection of OAs in loco in developing countries is important despite the implied extra costs, since it allows rapid therapy in many cases with a significant reduction of morbidity and mortality and makes the physicians more aware of these pathologies.

Effect of in vivo administration of ethylmalonic acid on energy metabolism in rat tissues.

High concentrations of ethylmalonic acid (EMA) occur in tissues and biological fluids of patients affected by deficiency of short-chain acyl-CoA dehydrogenase activity, as well as in other illnesses characterized by neurological and muscular symptoms. Considering that the pathophysiological mechanisms responsible for the clinical manifestations of these diseases are virtually unknown, in the present work we developed a chemical in vivo model of ethylmalonic acidemia in young Wistar rats for neurochemical and behavioral studies through subcutaneous administration of EMA to young rats. The doses of EMA administered subcutaneously varied according to the age of the animals, being injected 3, 4, and 6 micromol g(-1) of body weight in rats of 7, 14, and 21 days, respectively. The concentrations of the acid were measured in blood and brain at regular intervals after a single injection (30-120 min) and reached the highest concentrations (3.0 mM and 0.5 micromol g(-1), approximately 0.5 mM), respectively, after 30 and 60 min of EMA injection. Next, we investigated the effects of acute EMA administration on the activities of complexes I-III, II, II-III, and IV of the respiratory chain in cerebral cortex and skeletal muscle, as well as on the activity of creatine kinase in cerebral cortex, striatum, skeletal muscle, and cardiac muscle of rats of 14 days of life. Control rats were treated with saline in the same volumes. We verified EMA administration did not change these enzymatic activities in all tissues studied. Although transient high concentrations of EMA did not alter important parameters of energy metabolism, it cannot be ruled out that chronic administration of this organic acid would disrupt energy metabolism.