Daniella de Moura Coelho

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.

Hexacosanoic and docosanoic acids plasma levels in patients with cerebral childhood and asymptomatic X-linked adrenoleukodystrophy: Lorenzo's oil effect.

X-linked adrenoleukodystrophy (X-ALD) is an inherited disorder of peroxisomal metabolism, biochemically characterized by deficient β-oxidation of saturated very long chain fatty acids (VLCFA). The consequent accumulation of these fatty acids in different tissues and in biological fluids is associated with a progressive central and peripheral demyelination, as well as with adrenocortical insufficiency and hypogonadism. Seven variants of this disease have been described, being cerebral childhood the most frequent. The recommended therapy consists of the use of the glyceroltrioleate/glyceroltrierucate mixture known as Lorenzo’s Oil (LO), combined with a VLCFA-poor diet, but only in asymptomatic patients will this treatment prevent the progression of the symptomatology. In the present study we evaluated the biochemical course of patients with cerebral childhood (CCER) and asymptomatic clinical forms of X-ALD treated with LO associated with a VLCFA-restricted diet. We observed that hexacosanoic acid plasma concentrations and hexacosanoic/docosanoic ratio were significantly reduced in CCER patients during treatment when compared with diagnosis. Hexacosanoic acid plasma level was significantly reduced when compared with that at diagnosis and achieved the normal levels only in asymptomatic patients under LO treatment. In asymptomatic patients the magnitude of hexacosanoic acid decrease was higher than that of the CCER patients. These results show the good biochemical response of LO treatment in asymptomatic X-ALD patients. It is possible to suppose that this could be correlated with the prevention of the appearance of neurological signals in this group of patients treated with LO.

A chemically-induced acute model of maple syrup urine disease in rats for neurochemical studies.

We report a chemically-induced model of maple syrup urine disease (MSUD) in 10- and 30-day-old rats produced by subcutaneous administration of a branched-chain amino acids (BCAA) pool along with the analyses of plasma and brain amino acid levels by HPLC at 0-120 min after administration. We observed an increase of plasma leucine (Leu), isoleucine (Ile) and valine (Val) concentrations in both 10- and 30-day-old rats. These increases were accompanied by a concomitant reduction of plasma concentrations of methionine (Met), phenylalanine (Phe), tyrosine (Tyr), histidine (His), alanine (Ala), lysine (Lys), and ornithine (Orn) in 10-day-old rats and of Met, Phe, Tyr, tryptophan (Trp), and Orn in 30-day-old rats. These results are similar to those observed in MSUD patients during crises, when plasma levels of large neutral amino acids (LNAA) are also reduced when BCAA concentrations are increased. In the brain, increased concentrations of Leu, Ile and Val were achieved in 10-day-old rats at all times after injection. In contrast, no differences in cerebral concentrations of BCAA were observed in 30-day-old rats. In conclusion, the present MSUD model, using 10- rather than 30-day-old rats, has a similar amino acid profile to that of MSUD untreated patients and is suitable to investigate the mechanisms of brain damage characteristic of this disorder.

Amino acids levels and lipid peroxidation in maple syrup urine disease patients.

OBJECTIVE In the present study we correlated the amino acids, branched-chain alpha-keto acids and alpha-hydroxy acids levels with the thiobarbituric acid-reactive species (TBARS) measurement, a lipid peroxidation parameter, in plasma from treated MSUD patients in order to examine whether these accumulated metabolites could be associated to the oxidative stress present in MSUD. DESIGN AND METHODS TBARS, amino acids, branched-chain alpha-keto acids and alpha-hydroxy acids concentrations were measured in plasma samples from treated MSUD patients. RESULTS We verified that plasma TBARS was increased, whereas tryptophan and methionine concentrations were significantly reduced. Furthermore TBARS measurement was inversely correlated to methionine and tryptophan levels. CONCLUSIONS Considering that methionine and tryptophan have antioxidant activities, the data suggest that the imbalance of these amino acids may be involved with lipid peroxidation in MSUD.

The effect of Lorenzo's oil on oxidative stress in X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD) is a peroxisomal disorder biochemically characterized by the accumulation of very long chain fatty acids (VLCFA), particularly hexacosanoic acid (C(26:0)) and tetracosanoic acid (C(24:0)), in tissues and biological fluids. Although patients affected by this disorder predominantly present central and peripheral demyelination as well as adrenal insufficiency, the mechanisms underlying the brain damage in X-ALD are poorly known. The current treatment of X-ALD with glyceroltrioleate (C(18:1))/glyceroltrierucate (C(22:1)) (Lorenzos oil, LO) combined with a VLCFA-poor diet normalizes VLCFA concentrations, but the neurological symptoms persist or even progress in symptomatic patients. Considering that free radical generation is involved in various neurodegenerative disorders and that in a previous study we showed evidence that oxidative stress is probably involved in the pathophysiology of X-ALD symptomatic patients, in the present study we evaluated various oxidative stress parameters, namely thiobarbituric acid reactive species (TBA-RS) and total antioxidant reactivity (TAR) in plasma, as well as the activities of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) in erythrocytes from symptomatic and asymptomatic X-ALD patients and verified whether LO treatment and a VLCFA restricted diet could change these parameters. We observed a significant increase of plasma TBA-RS in symptomatic and asymptomatic X-ALD patients, reflecting induction of lipid peroxidation even before the disease was manifested. In addition, LO treatment did not alter this profile. Furthermore, plasma TAR measurement of X-ALD patients was not different from that of controls. Similarly, the antioxidant enzyme activities CAT, SOD and GPx were not altered in erythrocyte from X-ALD patients as compared to controls. We also examined the in vitro effects of hexacosanoic acid (C(26:0)) and tetracosanoic acid (C(24:0)) alone or combined with oleic (C(18:1))/erucic (C(22:1)) acids on various oxidative stress parameters in cerebral cortex of young rats, namely chemiluminescence, TBA-RS, TAR, CAT, SOD and GPx in order to investigate whether those fatty acids were able to induce oxidative stress. We found that there was a significant increase of TBARS and of chemiluminescence in rat cerebral cortex exposed to C(26:0)/C(24:0), and that the addition of C(18:1)and C(22:1) to the assays did not prevent this effect. Furthermore, TAR measurement was not altered by C(26:0) and C(24:0) acids in rat cerebral cortex. Taken together, our results indicate that lipid peroxidation occurs in X-ALD and that LO treatment does not attenuate or prevent free radical generation in these patients. Therefore, it may be presumed that antioxidants should be considered as an adjuvant therapy for X-ALD patients.

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.

Acute administration of 5-oxoproline induces oxidative damage to lipids and proteins and impairs antioxidant defenses in cerebral cortex and cerebellum of young rats

Abstract5-Oxoproline accumulates in glutathione synthetase deficiency, an autossomic recessive inherited disorder clinically characterized by hemolytic anemia, metabolic acidosis, and severe neurological symptoms whose mechanisms are poorly known. In the present study we investigated the effects of acute subcutaneous administration of 5-oxoproline to verify whether oxidative stress is elicited by this metabolite in vivo in cerebral cortex and cerebellum of 14-day-old rats. Our results showed that the acute administration of 5-oxoproline is able to promote both lipid and protein oxidation, to impair brain antioxidant defenses, to alter SH/SS ratio and to enhance hydrogen peroxide content, thus promoting oxidative stress in vivo, a mechanism that may be involved in the neuropathology of gluthatione synthetase deficiency.

L-carnitine supplementation decreases dna damage in treated MSUD patients

Maple syrup urine disease (MSUD) is an inherited disorder caused by severe deficient activity of the branched-chain α-keto acid dehydrogenase complex involved in the degradation pathway of branched-chain amino acids (BCAAs) and their α-ketoacid derivatives. MSUD patients generally present ketoacidosis, poor feeding, ataxia, coma, psychomotor delay, mental retardation and brain abnormalites. Treatment consists of dietary restriction of the BCAA (low protein intake) supplemented by a BCAA-free amino acid mixture. Although the mechanisms of brain damage in MSUD are poorly known, previous studies have shown that oxidative stress may be involved in the neuropathology of this disorder. In this regard, it was recently reported that MSUD patients have deficiency of l-carnitine (l-car), a compound with antioxidant properties that is used as adjuvant therapy in various inborn errors of metabolism. In this work, we investigated DNA damage determined by the alkaline comet assay in peripheral whole blood leukocytes of MSUD patients submitted to a BCAA-restricted diet supplemented or not with l-car. We observed a significant increase of DNA damage index (DI) in leukocytes from MSUD patients under BCAA-restricted diet as compared to controls and that l-car supplementation significantly decreased DNA DI levels. It was also found a positive correlation between DI and MDA content, a marker of lipid peroxidation, and an inverse correlation between DI and l-car levels. Taken together, our present results suggest a role for reactive species and the involvement of oxidative stress in DNA damage in this disorder. Since l-car reduced DNA damage, it is presumed that dietary supplementation of this compound may serve as an adjuvant therapeutic strategy for MSUD patients in addition to other therapies.

Protective effect of antioxidants on DNA damage in leukocytes from X-linked adrenoleukodystrophy patients.

Toxic metabolites accumulation and oxidative stress have been associated to the pathophysiology of X‐linked adrenoleukodystrophy (X‐ALD), an inborn error of peroxisome metabolism. Parameters of oxidative damage to proteins and lipids in X‐ALD patients were already described in literature; however, DNA injuries were not studied yet. Considering that, the aims were to investigate DNA damage by comet assay in heterozygotes and symptomatic X‐ALD patients, to look for associations between DNA damage and lipid peroxidation as measured by urinary 15‐F2t‐isoprostane; and to evaluate the in vitro effect of N‐acetyl‐l‐cysteine (NAC), trolox (TRO) and rosuvastatin (RSV) on DNA damage in leukocytes from symptomatic patients. Symptomatic patients presented higher DNA damage levels than those found in heterozygotes and controls; heterozygotes and controls showed similar results. In order to investigate the in vitro antioxidant effect on DNA damage, whole blood cells from symptomatic patients were incubated with NAC (1 and 2.5 mM), TRO (25 and 75 μM) and RSV (0.5, 2 and 5 μM) before DNA damage analysis. NAC, TRO and RSV, at all tested concentrations, were all capable to reduce DNA damage in symptomatic X‐ALD patients until control levels. Finally, DNA damage correlated with urinary isoprostanes and plasmatic levels of TBA‐RS and DCFH‐DA, allowing to hypothesize that DNA damage might be induced by lipid peroxidation in symptomatic patients. The present work yields experimental evidence that NAC, TRO and RSV reduce the in vitro DNA injury in symptomatic X‐ALD patients, what may suggest that the administration of these antioxidants might be considered as an adjuvant therapy for X‐ALD.

Experimental evidence of oxidative stress in patients with l-2-hydroxyglutaric aciduria and that l-carnitine attenuates in vitro DNA damage caused by d-2-hydroxyglutaric and l-2-hydroxyglutaric acids

d-2-hydroxyglutaric (D-2-HGA) and l-2-hydroxyglutaric (L-2-HGA) acidurias are rare neurometabolic disorders biochemically characterized by increased levels of d-2-hydroxyglutaric acid (D-2-HG) and l-2-hydroxyglutaric acid (L-2-HG) respectively, in biological fluids and tissues. These diseases are caused by mutations in the specific enzymes involved in the metabolic pathways of these organic acids. In the present work, we first investigated whether D-2-HG and L-2-HGA could provoke DNA oxidative damage in blood leukocytes and whether l-carnitine (LC) could prevent the in vitro DNA damage induced by these organic acids. It was verified that 50μM of D-2-HG and 30μM of L-2-HG significantly induced DNA damage that was prevented by 30 and 150μM of LC. We also evaluated oxidative stress parameters in urine of L-2-HGA patients and observed a significant increase of oxidized guanine species and di-tyrosine, biomarkers of oxidative DNA and protein damage, respectively. In contrast, no significant changes of urinary isoprostanes and reactive nitrogen species levels were observed in these patients. Taken together, our data indicate the involvement of oxidative damage, especially on DNA, in patients affected by these diseases and the protective effect of LC.