Reactive nitrogen species mediate oxidative stress and astrogliosis provoked by in vivo administration of phytanic acid in cerebellum of adolescent rats: A potential contributing pathomechanism of cerebellar injury in peroxisomal disorders.

Phytanic acid (Phyt) accumulates in various peroxisomal diseases including Refsum disease (RD) and Zellweger syndrome (ZS). Since the pathogenesis of the neurological symptoms and especially the cerebellar abnormalities in these disorders are poorly known, we investigated the effects of in vivo intracerebral administration of Phyt on a large spectrum of redox homeostasis parameters in the cerebellum of young rats. Malondialdehyde (MDA) levels, sulfhydryl oxidation, carbonyl content, nitrite and nitrate concentrations, 2',7'-dichlorofluorescein (DCFH) oxidation, total (tGS) and reduced glutathione (GSH) levels and the activities of important antioxidant enzymes were determined at different periods after Phyt administration. Immunohistochemical analysis was also carried out in the cerebellum. Phyt significantly increased MDA and nitric oxide (NO) production and decreased GSH levels, without altering tGS, DCFH oxidation, sulfhydryl oxidation, carbonyl content and the activities of glutathione peroxidase (GPx), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PD). Furthermore, immunohistochemical analysis revealed that Phyt caused astrogliosis and protein nitrosative damage in the cerebellum. It was also observed that the NO synthase inhibitor Nω-Nitro-L-arginine methyl ester (L-NAME) prevented the increase of MDA and NO production as well as the decrease of GSH and the immunohistochemical alterations caused by Phyt, strongly suggesting that reactive nitrogen species (RNS) were involved in these effects. The present data provide in vivo solid evidence that Phyt disrupts redox homeostasis and causes astrogliosis in rat cerebellum probably mediated by RNS production. It is therefore presumed that disequilibrium of redox status may contribute at least in part to the cerebellum alterations characteristic of patients affected by RD and other disorders with Phyt accumulation.

In vitro effect of genistein on DNA damage in leukocytes from mucopolysaccharidosis IVA patients.

Mucopolysaccharidosis IVA is a lysosomal storage disorder leading to an increase in glycosaminoglycans storage. Genistein is an isoflavone capable to inhibit glycosaminoglycans production. The objective of this study was to analyze the in vitro effect of different concentrations of genistein on DNA injury in mucopolysaccharidosis IVA patients. The lower concentration tested (10 µM) showed a significant increase on DNA injury in vitro, although higher concentrations (30 µM and 50 µM) showed higher DNA damage.

Oxidative stress in phenylketonuric patients.

Phenylketonuria is the most frequent disturbance of amino acid metabolism. Untreated patients present mental retardation whose pathophysiology is not completely established. In this work we discuss the oxidative stress in phenylketonuric patients. Several studies have shown reduction in antioxidant defenses, possibly due to dietary restriction of nutrients with antioxidant properties and increase in oxidative damage to biomolecules, probably secondary to increased formation of reactive species. Therefore, antioxidants could be considered an adjuvant therapy in phenylketonuria.

Evidence that L-carnitine and selenium supplementation reduces oxidative stress in phenylketonuric patients.

It is well established that the involvement of reactive species in the pathophysiology of several neurological diseases, including phenylketonuria (PKU), a metabolic genetic disorder biochemically characterized by elevated levels of phenylalanine (Phe). In previous studies, we verified that PKU patients (treated with a protein-restricted diet supplemented with a special formula not containing L-carnitine and selenium) presented high lipid and protein oxidative damage as well as a reduction of antioxidants when compared to the healthy individuals. Our goal in the present study was to evaluate the effect of Phe-restricted diet supplemented with L-carnitine and selenium, two well-known antioxidant compounds, on oxidative damage in PKU patients. We investigated various oxidative stress parameters in blood of 18 treated PKU patients before and after 6 months of supplementation with a special formula containing L-carnitine and selenium. It was verified that treatment with L-carnitine and selenium was capable of reverting the lipid peroxidation, measured by thiobarbituric acid-reactive species, and the protein oxidative damage, measured by sulfhydryl oxidation, to the levels of controls. Additionally, the reduced activity of glutathione peroxidase was normalized by the antioxidant supplementation. It was also verified a significant inverse correlation between lipid peroxidation and L-carnitine blood levels as well as a significant positive correlation between glutathione peroxidase activity and blood selenium concentration. In conclusion, our results suggest that supplementation of L-carnitine and selenium is important for PKU patients since it could help to correct the oxidative stress process which possibly contributes, at least in part, to the neurological symptoms found in phenylketonuric patients.

Incidence of 3-hydroxy-3-methylglutaryl-coenzyme A lyase (HL) deficiency in Brazil, South America.

3-Hydroxy-3-methylglutaryl-CoA lyase (HL) deficiency (3-hydroxy-3-methylglutaric aciduria, 3-HMG) is a rare autosomal recessive inborn error of metabolism involving the final step of leucine degradation. HL is the key enzyme for the production of glucose-sparing ketone bodies for brain. Positive biochemical findings are metabolic acidosis, hyperammonaemia, and hypoketotic hypoglycaemia in the neonatal period or infancy. In the present study we report 15 Brazilian patients with HL deficiency and present their clinical and biochemical findings. Urine from all patients contained large amounts of 3-hydroxy-3-methylglutaric, 3-methylglutaconic, 3-hydroxyisovaleric and 3-methylglutaric acids, and 3-methylcrotonylglycine was also observed in 13 patients. The main features at clinical presentation were hypoglycaemia (12 patients), seizures (10 patients), metabolic acidosis (9 patients), vomiting (6 patients), and hepatomegaly (5 patients). All but two patients were of Portuguese ancestry. HL deficiency comprised 7.3% of total organic acidurias detected in our laboratory during a 13-year time span, indicating a high incidence of this disorder in Brazil. Limited molecular characterization (4/15 patients only) revealed two mutations common for individuals of Portuguese/Spanish (Iberian Peninsula) ancestry (E37X and V168fs(-2)). Our findings increase the number of HL-deficient patients and reinforce the characteristic phenotypic picture of the disease. Effective dietary interventions based on mild protein restriction and avoidance of fasting and possibly alternative C5 ketone body generating therapy for this disorder may provide further impetus and rationale for expanded newborn screening of HL deficiency.

Investigation of oxidative stress parameters in treated phenylketonuric patients.

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

Oxidative stress in patients with phenylketonuria.

Phenylketonuria (PKU) is an autossomal recessive disease caused by phenylalanine-4-hydroxylase deficiency, which is a liver-specific enzyme that catalyzes the hydroxylation of l-phenylalanine (Phe) to l-tyrosine (Tyr). The deficiency of this enzyme leads to the accumulation of Phe in the tissues and plasma of patients. The clinical characterization of this disease is mental retardation and other neurological features. The mechanisms of brain damage are poorly understood. Oxidative stress is observed in some inborn errors of intermediary metabolism owing to the accumulation of toxic metabolites leading to excessive free radical production and may be a result of restricted diets on the antioxidant status. In the present study we evaluated various oxidative stress parameters, namely thiobarbituric acid-reactive species (TBA-RS) and total antioxidant reactivity (TAR) in the plasma of PKU patients. The activities of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were also measured in erythrocytes from these patients. It was observed that phenylketonuric patients present a significant increase of plasma TBA-RS measurement, indicating a stimulation of lipoperoxidation, as well as a decrease of plasma TAR, reflecting a deficient capacity to rapidly handle an increase of reactive species. The results also showed a decrease of erythrocyte GSH-Px activity. Therefore, it is presumed that oxidative stress is involved in the pathophysiology of the tissue damage found in PKU.

Evidence that oxidative stress is increased in patients with X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD) is a hereditary disorder of peroxisomal metabolism biochemically characterized by the accumulation of very long chain fatty acids (VLCFA), particularly hexacosanoic acid (C26:0) and tetracosanoic acid (C24:0) in different tissues and in biological fluids. The disease is clinically characterized by central and peripheral demyelination and adrenal insufficiency, which is closely related to the increased concentrations of these fatty acids. However, the mechanisms underlying the brain damage in X-ALD are poorly known. Considering that free radical generation is involved in various neurodegenerative disorders, like Parkinson disease, multiple sclerosis and Alzheimer's disease, in the present study we evaluated various oxidative stress parameters, namely chemiluminescence, thiobarbituric acid reactive species (TBA-RS), total radical-trapping antioxidant potential (TRAP), and total antioxidant reactivity (TAR) in plasma of X-ALD patients, as well as the activities of the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) in erythrocytes and fibroblasts from these patients. It was verified a significant increase of plasma chemiluminescence and TBA-RS, reflecting induction of lipid peroxidation, as well as a decrease of plasma TAR, indicating a deficient capacity to rapidly handle an increase of reactive species. We also observed a significant increase of erythrocytes GPx activity and of catalase and SOD activities in fibroblasts from the patients studied. It is therefore proposed that oxidative stress may be involved in pathophysiology of X-ALD.

D-2-Hydroxyglutaric aciduria in a patient with a severe clinical phenotype and unusual MRI findings.

We report an infant with intermittent urinary excretion of D-2-hydroxyglutaric (D-2-OHG) acid who died at the age of 10 months from cardiogenic shock due to cardiomyopathy. High urinary concentrations of D-2-OHG and succinic acid, as well as increased levels of lactic acid were detected on three different occasions, whereas a normal urinary profile of organic acids was found on one occasion. The clinical findings of our patient consisted of generalized hypotonia, irritability, developmental delay, generalized tonic seizures, lethargy, cardiomyopathy, and respiratory distress. Cerebral MRI revealed bilateral lesions in the substantia nigra, the periaqueductal area, the medial part of the thalamus, the hypothalamus, the caudate nucleus, putamen and globus pallidus. This pattern is suggestive of a mitochondriopathy. However, respiratory chain enzyme activities were normal in fibroblasts. Exogenous supplementation of D-2-OHG acid strongly inhibited cytochrome-c oxidase activity in fibroblasts from the patient and from normal controls in vitro. The results suggest that our patient has an unusual form of D-2-hydroxyglutaric aciduria (D-2-OHGA), different from the patients published so far, and that the increase of lactic acid and some citric acid cycle intermediates encountered in some patients with D-2-OHGA may be due to a functional defect of the respiratory chain caused by D-2-OHG acid.

Reduction of large neutral amino acid levels in plasma and brain of hyperleucinemic rats.

Neurological dysfunction is common in patients with maple syrup urine disease (MSUD). However, the mechanisms underlying the neuropathology of this disorder are poorly known. In the present study we investigated the effect of acute hyperleucinemia on plasma and brain concentrations of amino acids. Fifteen-day-old rats were injected subcutaneously with 6 micromol L-leucine per gram body weight. Controls received saline in the same volumes. The animals were sacrificed 30--120 min after injection, blood was collected and their brain rapidly removed and homogenized. The amino acid concentrations were determined by HPLC using orthophtaldialdehyde for derivatization and fluorescence for detection. The results showed significant reductions of the large neutral amino acids (LNAA) L-phenylalanine, L-tyrosine, L-isoleucine, L-valine and L-methionine, as well as L-alanine, L-serine and L-histidine in plasma and of L-phenylalanine, L-isoleucine, L-valine and L-methionine in brain, as compared to controls. In vitro experiments using brain slices to study the influence of leucine on amino acid transport and protein synthesis were also carried out. L-Leucine strongly inhibited [14C]-L-phenylalanine transport into brain, as well as the incorporation of the [14C]-amino acid mixture, [14C]-L-phenylalanine and [14C]-L-lysine into the brain proteins. Although additional studies are necessary to evaluate the importance of these effects for MSUD, considering previous findings of reduced levels of LNAA in plasma and CSF of MSUD patients during crises, it may be speculated that a decrease of essential amino acids in brain may lead to reduction of protein and neurotransmiter synthesis in this disorder.

[Organic aciduria: diagnosis in high-risk Brazilian patients] / Acidúrias orgânicas: diagnóstico em pacientes de alto risco no Brasil.

OBJECTIVE: To determine the prevalence of organic acidurias in high-risk Brazilian patients. METHODS: Laboratory techniques for the detection and quantification of organic acids by gas chromatography/mass spectrometry were implemented in Porto Alegre, Brazil. We investigated 1,480 patients suspected of organic aciduria between January 1994 and June 2000. RESULTS: Seventy three (4.9%) cases of organic acidemias (acidurias) were diagnosed among the tested individuals. In most of these patients, prompt therapy resulted in rapid symptom improvement; these results are completely different from our previous cases diagnosed in other laboratories in Europe and the United States, where several patients died before any measures could be taken. CONCLUSIONS: These results demonstrate the importance of diagnosing organic acidurias in loco even in developing countries, in spite of the extra costs involved.

Reduction of large neutral amino acid concentrations in plasma and CSF of patients with maple syrup urine disease during crises.

Neurological dysfunction is common in patients with maple syrup urine disease (MSUD). However, the mechanisms underlying the neuropathology of this disorder are poorly understood. We determined the concentrations of all amino acids in plasma of patients with MSUD during crises (with severe CNS symptoms) and after recovery in the hope of detecting possible alterations of these levels during metabolic decompensation. Blood samples obtained from 11 children with MSUD aged 1 month to 7 years and from 10 age-matched controls (5 months to 6 years) with no evidence of metabolic disease were examined for their amino acid content by high-performance liquid chromatography. We observed that leucine, isoleucine and valine concentrations were respectively 30, 9 and 3 times higher than normal values, whereas the concentrations of the large neutral amino acids (LNAA) phenylalanine, tyrosine, tryptophan and methionine were significantly lower during metabolic decompensation as compared to the controls. In addition, concentrations of leucine, but not of valine or isoleucine, were inversely related to the LNAA concentrations in plasma. The concentrations of these amino acids in plasma returned to normal values when patients were clinically well. CSF amino acid concentrations also showed decreased amounts of LNAA and increased concentrations of branched-chain amino acids. It is possible that the decrease in plasma concentrations of LNAA may lead to a deficit of these essential amino acids in the brain as well as of their products such as proteins and neurotransmitters, a fact that might be related to the neurological dysfunction of MSUD.

Effect of chemically induced propionic acidemia on neurobehavioral development of rats.

High levels of propionic acid (PPA) comparable to those of human propionic acidemia were achieved in blood (1-5 mmol/l) and brain (1 micromol/g) of rats by administering saline-buffered propionate (pH 7.4) subcutaneously twice a day from the 6th to the 28th day of life. PPA doses ranged from 1.44 to 1.92 micromol/g body weight as a function of animal age. Control rats were treated with saline in the same volumes. Growth and development of physical landmarks were assessed by monitoring the following parameters daily: body weight, upper incisor eruption, eye opening, and hair coat. Development of some reflexes was also monitored, and a specific subset of motor skills was evaluated at days 14 and 21 of life by the free-fall righting test and the spontaneous alternation test. Chronic PPA administration had no effect on body weight, cerebral cortex weight, or cerebellum weight, but caused slight but significant delays in the day of appearance of hair coat and eye opening, indicating an effect of PPA on the development of physical parameters. Free-fall righting was impaired in PPA-treated animals. On the other hand, PPA administration had no effect on the performance of the animals in the spontaneous alternation tests. Long-term effects of early PPA administration were investigated by assessing animal performance in an aversive task (two-way shuttle avoidance task) and in a nonaversive (open-field task) behavioral task at 60 days of age. PPA-treated rats did not habituate to the open field, and presented a lack of retention of the shuttle-avoidance task. Our results suggest that early postnatal PPA administration to rats alters normal development and induces long-term behavioral deficits in aversive and nonaversive tasks.

Selective screening of 10,000 high-risk Brazilian patients for the detection of inborn errors of metabolism.

UNLABELLED: The number of diagnosed inborn errors of metabolism (IEM) is growing constantly due to the improvement and widespread availability of analytical techniques. In 1982, a laboratory for the detection of IEM was set up in Porto Alegre, Brazil, and became a national reference centre for the diagnosis of these disorders. Ten thousand patients with signs and symptoms suggestive of IEM were investigated in our laboratory from 1982 to 1995 using specific protocols which included tests for the detection of glucosaminoglycans (GAGS), amino acids, sugars, oligosaccharides, sialyloligosaccharides, organic acids, as well as various metabolite. The biochemical investigation was completed in 9,901 patients and an IEM was detected in 647 cases (6.5%). Groups of IEM of higher incidence in our sample were lysosomal storage disorders (59.8%) and aminoacidopathies (21.2%). The disorders most frequently diagnosed were classical phenylketonuria, GM1 gangliosidosis, mucopolysaccharidosis type I, mucopolysaccharidosis type VI and metachromatic leukodystrophy. CONCLUSION: This study shows that the establishment of reference centres for the investigation of rare genetic diseases is a suitable approach to the study of IEM in developing countries such as Brazil.

Pyridoxine-dependent seizures associated with white matter abnormalities.

Pyridoxine-dependent seizures are a disorder of GABA metabolism probably due to a defective binding of pyridoxal phosphate coenzyme (PALP) with glutamate decarboxylase (GAD), the rate-limiting enzyme in GABA synthesis. The resulting GABA deficiency causes severe epilepsy in infancy. We report on a boy with seizures starting soon after birth, and only controlled by pyridoxine at pharmacological dosages. After two months without seizures, a CT scan showed hypodense white matter in frontal and occipital lobes suggestive of a retarded or defective myelination. We are not aware of other descriptions of such morphological abnormalities in a patient with this disorder.