Methylmalonic acidemia is an inherited metabolic disorder biochemically characterized by tissue accumulation of methylmalonic acid (MMA) and clinically by progressive neurological deterioration and kidney failure, whose pathophysiology is so far poorly established. Previous studies have shown that MMA inhibits complex II of the respiratory chain in rat cerebral cortex, although no inhibition of complexes I–V was found in bovine heart. Therefore, in the present study we investigated the in vitro effect of 2.5mM MMA on the activity of complexes I–III, II, II–III and IV in striatum, hippocampus, heart, liver and kidney homogenates from young rats. We observed that MMA caused a significant inhibition of complex II activity in striatum and hippocampus (15–20%) at low concentrations of succinate in the medium, but not in the peripheral tissues. We also verified that the inhibitory property of MMA only occurred after exposing brain homogenates for at least 10min with the acid, suggesting that this inhibition was mediated by indirect mechanisms. Simultaneous preincubation with the nitric oxide synthase inhibitor Nω-nitro-l-arginine methyl ester (l-NAME) and catalase (CAT) plus superoxide dismutase (SOD) did not prevent MMA-induced inhibition of complex II, suggesting that common reactive oxygen (superoxide, hydrogen peroxide and hydroxyl radical) and nitric (nitric oxide) species were not involved in this effect. In addition, complex II–III (20–35%) was also inhibited by MMA in all tissues tested, and complex I–III only in the kidney (53%) and liver (38%). In contrast, complex IV activity was not changed by MMA in all tissues studied. These results indicate that MMA differentially affects the activity of the respiratory chain pending on the tissues studied, being striatum and hippocampus more vulnerable to its effect. In case our in vitro data are confirmed in vivo in tissues from methylmalonic acidemic patients, it is feasible that that the present findings may be related to the pathophysiology of the tissue damage characteristic of these patients.

We have previously demonstrated that octanoic (OA) and decanoic acids (DA) inhibit Na+, K+ ATPase activity in synaptic plasma membranes from rat brain. The objective of the present study was to investigate the in vitro effects of the other metabolites that accumulate in tissues of medium-chain acyl-CoA dehydrogenase (MCAD)-deficient patients, namely cis-4-decenoic acid (cDA), octanoylcarnitine (OC), hexanoylcarnitine (HC), hexanoylglycine (HG), phenylpropionylglycine (PPG) and suberoylglycine (SG), on Na+, K+ ATPase activity in synaptic plasma membrane from cerebral cortex of 30-day-old rats. cDA, the pathognomonic compound found in this disorder, provoked the strongest inhibition on this enzyme activity at concentrations as low as 0.25 mM, whereas OC inhibited this activity at 1.0 mM and higher concentrations in a dose-dependent manner. In contrast, HC, HG, PPG and SG did not affect Na+, K+ ATPase activity. Furthermore, pre-treatment of cortical homogenates with the antioxidant enzymes catalase plus superoxide dismutase totally prevented cDA-induced Na+, K+ ATPase inhibition. We also provided evidence that cDA, as well as OA and DA, caused lipid peroxidation, which may explain, at least in part, the inhibitory properties of these compounds towards Na+, K+ ATPase. Considering that Na+, K+ ATPase is a critical enzyme for normal brain development and functioning, it is presumed that these findings, especially those regarding to the marked inhibitory effect of cDA, may be involved in the pathophysiology of the neurological dysfunction of MCAD-deficient patients.

Glutaric acidemia type I is an inherited metabolic disorder biochemically characterized by tissue accumulation of predominantly glutaric acid (GA). Affected patients present frontotemporal hypotrophy, as well as caudate and putamen injury following acute encephalopathic crises. Considering that the underlying mechanisms of basal ganglia damage in this disorder are poorly known, in the present study we tested the effects of glutaric acid (0.2–5mM) on critical enzyme activities of energy metabolism, namely the respiratory chain complexes I–IV, succinate dehydrogenase and creatine kinase in midbrain of developing rats. Glutaric acid significantly inhibited creatine kinase activity (up to 26%) even at the lowest dose used in the assays (0.2mM). We also observed that CK inhibition was prevented by pre-incubation of the homogenates with reduced glutathione, suggesting that the inhibitory effect of GA was possibly mediated by oxidation of essential thiol groups of the enzyme. In addition, the activities of the respiratory chain complex I–III and of succinate dehydrogenase were also significantly inhibited by 20 and 30%, respectively, at the highest glutaric acid concentration tested (5mM). In contrast, complexes II–III and IV activities of the electron transport chain were not affected by the acid. The effect of glutaric acid on the rate of oxygen consumption in intact mitochondria from the rat cerebrum was also investigated. Glutaric acid (1mM) significantly lowered the respiratory control ratio (state III/state IV) up to 40% in the presence of the respiratory substrates glutamate/malate or succinate. Moreover, state IV respiration linked to NAD and FAD substrates was significantly increased in GA-treated mitochondria while state III was significantly diminished. The results indicate that the major metabolite accumulating in glutaric acidemia type I moderately compromises brain energy metabolism in vitro.

Considering that pyruvate kinase activity, a crucial enzyme for glucose metabolism and energy liberation in brain, may be regulated by some amino acids, it is possible that diminution of this enzyme activity may contribute to the brain damage caused by amino acids accumulated in metabolic diseases, such as phenylalanine, tryptophan and cystine. Therefore, the present study was undertaken to investigate the effect of these amino acids on pyruvate kinase activity in the brain cortex of rats. We also investigated the effect of serine and alanine on pyruvate kinase activity in the same tissue. The results suggested that phenylalanine, tryptophan, cystine, alanine, and serine act at the same site on the enzyme, phenylalanine, tryptophan, and cystine causing inhibition, and alanine and serine preventing this effect. Cystine also inhibited the enzyme activity through a different mechanism, possibly acting on the enzyme thiol groups. Considering that this enzyme is a target for amino acids accumulated in some metabolic diseases of amino acid metabolism, it is possible that its inhibition may contribute to the brain damage found in these diseases.

Cystinosis is a disorder associated with lysosomal cystine accumulation caused by defective cystine efflux. Cystine accumulation provokes a variable degree of symptoms depending on the involved tissues. Adult patients may present brain cortical atrophy. However, the mechanisms by which cystine is toxic to the tissues are not fully understood. Considering that brain damage may be developed by energy deficiency, creatine kinase is a thiolic enzyme crucial for energy homeostasis, and disulfides like cystine may alter thiolic enzymes by thiol/disulfide exchange, the main objective of the present study was to investigate the effect of cystine on creatine kinase activity in total homogenate, cytosolic and mitochondrial fractions of the brain cortex from 21-day-old Wistar rats. We performed kinetic studies and investigated the effects of GSH, a biologically occurring thiol group protector, and cysteamine, the drug used for cystinosis treatment, to better understand the effect of cystine on creatine kinase activity. Results showed that cystine inhibited the enzyme activity non-competitively in a dose- and time-dependent way. GSH partially prevented and reversed CK inhibition caused by cystine and cysteamine fully prevented and reversed this inhibition, suggesting that cystine inhibits creatine kinase activity by interaction with the sulfhydryl groups of the enzyme. Considering that creatine kinase is a crucial enzyme for brain cortex energy homeostasis, these results provide a possible mechanism for cystine toxicity and also a new possible beneficial effect for the use of cysteamine in cystinotic patients.

Tissue accumulation of high amounts of d-2-hydroxyglutaric acid (DGA) and l-2-hydroxyglutaric acid (LGA) is the biochemical hallmark of the inherited neurometabolic disorders d-2-hydroxyglutaric aciduria (DHGA) and l-2-hydroxyglutaric aciduria (LHGA), respectively. Patients affected by DHGA predominantly present neurological and cardiomuscular symptoms, while those with LHGA have mainly severe neurological symptoms. Lactic aciduria and/or lactic acidemia may also occur in both disorders, suggesting mitochondrial dysfunction. We have previously reported that cytochrome c oxidase (COX) activity is severely inhibited by DGA in rat cerebral cortex and human skeletal muscle. In the present study, we initially evaluated the role of DGA and LGA on the mitochondrial respiratory chain complex activities, as well as CO2 on production in cardiac and skeletal muscle from 30-day-old Wistar rats. DGA significantly inhibited COX and ATP synthase (F0F1-ATP synthase) activities, in contrast to the other activities of the respiratory chain enzymes which were not affected by DGA in both muscular tissues. In addition, CO2 production was also markedly reduced by DGA in rat skeletal and cardiac muscles. On the other hand, LGA did not interfere with any of the respiratory chain complex activities studied, neither with CO2 generation. We also measured mitochondrial respiratory parameters in rat brain mitochondrial preparations in the presence of DGA and LGA. Both metabolites significantly lowered the respiratory control ratio in the presence of glutamate/malate and succinate. Since the metabolites stimulated oxygen consumption in state IV and compromised ATP formation, it can be presumed that these organic acids might act as endogenous uncouplers of mitochondria respiration. Moreover, COX activity linked to TMPD-ascorbate was significantly reduced by DGA in the brain mitochondrial enriched fractions. Finally, DGA and LGA reduced cell viability of rat cerebral cortex slices, as determined by the MTT assay. In case our in vitro data also occur in vivo, it may be presumed that impairment of energy metabolism may contribute to the understanding of the clinical features mainly of patients affected by DHGA.

Quinolinic acid (QA), the major metabolite of the kynurenine pathway, is found at increased concentrations in brain of patients affected by various common neurodegenerative diseases, including Huntington's disease and Alzheimer's disease. Recently, a role for QA in the pathophysiology of glutaric acidemia type I (GAI) was postulated. Considering that oxidative stress has been recently involved in the pathophysiology of the brain injury in these neurodegenerative disorders; in the present study, we investigated the in vitro effect of QA on various parameters of oxidative stress, namely total radical-trapping antioxidant potential (TRAP), total antioxidant reactivity (TAR), glutathione (GSH) levels, thiobarbituric acid-reactive substances (TBA-RS) measurement and chemiluminescence in cerebral cortex of 30-day-old rats. QA diminished the brain non-enzymatic antioxidant defenses, as determined by the reduced levels of TRAP, TAR and GSH. We also observed that QA significantly increased TBA-RS and chemiluminescence. Therefore, in vitro QA-treatment of rat cortical supernatants induced oxidative stress by reducing the tissue antioxidant defenses and increasing lipid oxidative damage, probably as a result of free radical generation. In addition, we examined the effect of QA on TBA-RS levels in the presence of glutaric acid (GA) and 3-hydroxyglutaric acid (3HGA), which are accumulated in GAI, as well as in the presence of 3-hydroxykynurenine (3HK), a tryptophan metabolite of the kynurenine pathway with antioxidant properties. It was verified that the single addition of QA or GA plus 3HGA to the incubation medium significantly stimulated in vitro lipid peroxidation. Furthermore, 3HK completely prevented the TBA-RS increase caused by the simultaneous addition of QA, GA and 3HGA. Taken together, it may be presumed that QA induces oxidative stress in the brain, which may be associated, at least in part, with the pathophysiology of central nervous system abnormalities of neurodegenerative diseases in which QA accumulates.

Organic acidurias represent a group of inherited disorders resulting from deficient activity of specific enzymes of the catabolism of amino acids, carbohydrates or lipids, leading to tissue accumulation of one or more carboxylic (organic) acids. Patients affected by organic acidurias predominantly present neurological symptoms and structural brain abnormalities, of which the aetiopathogenesis is poorly understood. However, in recent years increasing evidence has emerged suggesting that oxidative stress is possibly involved in the pathology of some organic acidurias and other inborn errors of metabolism. This review addresses some of the recent developments obtained mainly from animal studies indicating oxidative damage as an important determinant of the neuropathophysiology of some organic acidurias. Recent data showing that various organic acids are capable of inducing free radical generation and decreasing brain antioxidant defences is presented. The discussion focuses on the relatively low antioxidant defences of the brain and the vulnerability of this tissue to reactive species. This offers new perspectives for potential therapeutic strategies for these disorders, which may include the early use of appropriate antioxidants as a novel adjuvant therapy, besides the usual treatment based on removing toxic compounds and using special diets and pharmacological agents, such as cofactors and L-carnitine.

Cystinosis is a metabolic disturb associated with excessive lysosomal cystine accumulation secondary to defective cystine efflux. Patients affected by this disease develop a variable degree of symptoms depending of the involved tissues. Accumulation of cystine in the brain may lead to severe neurological symptoms. However, the mechanisms by which cystine is neurotoxic are not fully understood. Considering that pyruvate kinase (PK) is a thiolic enzyme crucial for the glycolytic pathway, and disulfides like cystine may alter thiolic enzymes by thiol/disulfide exchange, the main objective of the present study was to investigate the effect of cystine on PK activity in the brain cortex of developing Wistar rats. We also performed kinetic studies and investigated the effects of GSH, a biologically occurring thiol groups protector, and cysteamine (CysN), the drug used for cystinosis treatment, on the enzyme activity. We observed that cystine inhibited the enzyme activity by two different mechanisms, one through the competition with ADP and phosphoenolpyruvate (PEP), and the other non-competitively, probably through oxidation of the thiol groups of PK. We also observed that GSH and cysteamine fully prevented and reversed the inhibition caused by cystine. Considering that cysteamine is used in patients with cystinosis because it causes parenkimal organ cystine depletion, the present data provides a possible new beneficial effect for the use of this drug.

Maple syrup urine disease (MSUD) is a metabolic disorder caused by the deficiency of the activity of the mitochondrial enzyme complex branched-chain L-2-keto acid dehydrogenase. The metabolic block results in tissue and body fluid accumulation of the branched-chain amino acids leucine (Leu), isoleucine and valine, as well as of their respective alpha-keto acids. Neurological sequelae are usually present in MSUD, but the pathophysiologic mechanisms of neurotoxicity are still poorly known. It was previously demonstrated that Leu elicits oxidative stress in rat brain. In the present study we investigated the possible mechanisms involved in Leu-induced oxidative damage. We observed a significant attenuation of Leu-elicited increase of thiobarbituric acid-reactive substances (TBA-RS) measurement when cortical homogenates were incubated in the presence of the free radical scavengers ascorbic acid plus trolox, dithiothreitol, glutathione, and superoxide dismutase, suggesting a probable involvement of superoxide and hydroxyl radicals in this effect. In contrast, the use of Nomega-nitro-L-arginine methyl ester or catalase (CAT) did not affect TBA-RS values. We also demonstrated an inhibitory effect of Leu on the activities of the antioxidant enzymes CAT and gluthathione peroxidase, as well as a significant reduction in the membrane-protein thiol content from mitochondrial enriched preparations. Furthermore, dichlorofluorescein levels were increased although not significantly by Leu. Taken together, our present data indicate that an unbalance between free radical formation and inhibition of critical enzyme activities may explain the mechanisms involved in the Leu-induced oxidative damage.

d-2-Hydroxyglutaric acid (DGA) is the biochemical hallmark of patients affected by the neurometabolic disorder known as d-2-hydroxyglutaric aciduria (DHGA). Although this disease is predominantly characterized by severe neurological findings, the underlying mechanisms of brain injury are virtually unknown. In the present study, we investigated the effect of DGA on total, cytosolic, and mitochondrial creatine kinase (CK) activities from cerebral cortex of 30-day-old Wistar rats. Total CK activity (tCK) was measured in whole cell homogenates, whereas cytosolic and mitochondrial activities were measured in the cytosolic and mitochondrial preparations from cerebral cortex. We verified that CK activities were significantly inhibited by DGA (11–34% inhibition) at concentrations as low as 0.25 mM, being the mitochondrial fraction the most affected activity. Kinetic studies revealed that the inhibitory effect of DGA was non-competitive in relation to phosphocreatine. We also observed that this inhibition was fully prevented by pre-incubation of the homogenates with reduced glutathione, suggesting that the inhibitory effect of DGA on tCK activity is possibly mediated by oxidation of essential thiol groups of the enzyme. Considering the importance of CK activity for brain metabolism homeostasis, our results suggest that inhibition of this enzyme by increased levels of DGA may be related to the neurodegeneration of patients affected by DHGA.

Biochemical and clinical heterogeneity in long chain 3-hydroxyacyl-CoA dehydrogenase deficiency: identification of a new disorder of mitochondrial fi-oxidation. R. J. A. Wanders, L Ijlst, A. Munnich and J.-M. Saudubray Neonatal symptoms are common in medium chain acyl coenzyme A dehydrogenase deficiency. B. Wilcken, K. Carpenter and J. Hammond Choline deficiency in non-ketotic hyperglycinaemia. J. L K. Van Hove, F. Lazeyras, S. C. Garner, C. H. Charles, S. G. Kahler, R.-M. Boustany and S. H. Zeisel Dextromethorpban, a N-methyl-D-aspartate antagonist, in the treatment ofnon-ketotic hyperglycinaemia. B. Schmitt, B. Steinmann, R. Gitzelmann, L. Thun-Hohenstein and G. Dumermuth Complex I (NADH dehydrogenase) activity and biosynthesis in fibroblasts and lymphoblasts from patients with Leber's hereditary optic neuropathy. W. d. Rhead, E. Freneaux, A. Shires and E. Stone Porphyria due to 5-aminolevulinic acid dehydratase deficiency: a novel clinical and biochemical presentation. H. Mandel, M. Jaffe, N. Schoenfeld, A. Aizin and M. Berant Hereditary tyrosinaemia type I: self-induced correction of the genetic defect? E. A. Kvittingen, H. Rootwelt, P. Bradtzaeg, A. Bergan and R. Berger Galactosaemia, a carbohydrate-deficient glycoprotein syndrome. L d. M. Spaapen, T. Vulsma, P. M. V. M. Theunissen, S. B, van der Meer and J. daeken Alanine effect on phenylketonuric rats. M. Wajner, C. G. Pedron, F. C. Moreira, M. H. Xavier, B. d. dos Santos, A. T. S. Wyse and C. M. D. Wannmacher Possible high frequency of tetrahydrobiopterin deficiency in South Brazil. L B. Yardim, M. G. Burin, R. Giugliani and N. Blau Screening of PKU mutations in Spain and South America. B. Perez, L R. Desviat, M. Die, M. J. Garcia and M. Ugarte Evaluation of dental health in phenylketonuric children. M. Roupa, C. Demars and B. Francois A mutation tightly linked to haplotype 6 in the Turkish PKU alleles. M. Ozgiig, E. I~lmaz, H. Erdem, T. Coskun, S. Ayter and I. Ozalp fl-Endorphin system in chemically-induced phenylketonuria. C. M. D. Wannmaeher, A. R. Bolner, J. Engelmann and M. Wajner Is there still a place for the Guthrie test? A. H. Wilcox, J. Stern and J. M. Whitaker Magnetic resonance imaging, biochemical control and genetic analysis in patients with phenylketonuria. J. H. Walter, L A. Tyfield, J. B. Holton and C. Johnson Neuroradiological (NMR) examination in PKU patients: clinical and biochemical correlation. V. Leuzzi, F. Fabbrizi, I. Antonozzi, G. Gualdi, C. Di Biasi and G. Trasimeni Localized proton NMR spectroscopy in patients with phenylketonuria. K. Johannik, P. Van Hecke, B. Francois, M, Smet, J. Jaeken and A. Baert Central motor conduction time in phenylketonuria after cessation of dietary treatment. M. A. Cleary, J. H. Walter, J. E. Wraith and S. M. Alani Supplementation of phenylketonuria children with a bacterial selenosource. M. Calomme, J. Hu, B. Francois, M. Van Caillie-Bertrand and D. A. Van den Berghe Phenylalanine feto-material gradients by cordocentesis at 19 to 33 weeks gestation. W. E. Schoonheyt, W. B. Hanley, J. T. R. Clarke and J. Johnson Fumarylacetoacetate lyase-enzyme stability and its relevance to tyrosinaemia type I and glutathione synthetase deficiency. R. G. F. Gray, A. J. Lloyd and A. Green Cerebrospinal fluid amines and pterins in tyrosinaemia type II. S. J. R. Heales, A. Bowron, I. Smith, R. A. H. Surtees and P. T. Clayton Molecular basis of phenylketonuria in Central and Southern Italian population. C. Carducei, A. Pontecorvi, L. Ellul, M. Scuderi, F. Fabbrizi, V. Leuzzi and I. Antonozzi Maple syrup urine disease diagnosis and treatment in Bulgaria. B. Radeva and I. Sinigerska Intermittent form of maple syrup urine disease in brother and sister. M. Hrebicek, S. Stastna, U. Wendel, P. Verner, J. Zeman, V. Kozich, J. H yanek, Z. Rytir, D. Nemcova and J. Rytir Management and CT-findings in a case of neonatal (classical) maple syrup urine disease. J. G. M. Huijmans, J. B. C. de Klerk, S, G. F. Robben, W. J. Kleijer, R. Jankie, R. Slotema and R. Spritzer Maple syrup urine disease: prevention of postoperative metabolic decompensation by insulin. B. Biggeman, R. Zass and U. Wendel An asymptomatic variant of maple syrup urine disease without organic aciduria. M. L Cabello,

Monosialoganglioside (GM1) is a neuroprotective agent that has been reported to scavenge free radicals generated during reperfusion and to protect receptors and enzymes from oxidative damage. However, only a few studies have attempted to investigate the effects of GM1 on enzymatic antioxidant defenses of the brain. In the present study, we evaluate the effects of the systemic administration of GM1 on the activity of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), and on spontaneous chemiluminescence and total radical-trapping potential (TRAP) in cerebral cortex of rats ex vivo . The effects of GM1 on CAT activity and spontaneous chemiluminescence in vitro were also determined.Animals received two injections of GM1 (50 mg/kg, i.p.) or saline (0.85% NaCl, i.p.) spaced 24 h apart. Thirty minutes after the second injection the animals were sacrificed and enzyme activities and spontaneous chemiluminescence and TRAP were measured in cell-free homogenates. GM1 administration reduced spontaneous chemiluminescence and increased catalase activity ex vivo , but had no effect on TRAP, SOD or GSH-Px activities. GM1, at high concentrations, reduced CAT activity in vitro . We suggest that the antioxidant activity of GM1 ganglioside in the cerebral cortex may be due to an increased catalase activity.

Patients affected by medium-chain acyl CoA dehydrogenase (MCAD) deficiency, a frequent inborn error of metabolism, suffer from acute episodes of encephalopathy. However, the mechanisms underlying the neuropathology of this disease are poorly known. In the present study, we investigated the in vitro effect of the medium-chain fatty acids (MCFA), at concentrations varying from 0.01 to 3 mM, accumulating in MCAD deficiency on some parameters of energy metabolism in cerebral cortex of young rats. 14CO2 production from [U14] glucose, [1-14C] acetate and [1,5-14C] citrate was evaluated by incubating cerebral cortex homogenates from 30-day-old rats in the absence (controls) or presence of octanoic acid, decanoic acid or cis-4-decenoic acid. OA and DA significantly reduced 14CO2 production from acetate by around 30–40%, and from glucose by around 70%. DA significantly reduced 14CO2 production from citrate by around 40%, while OA did not affect this parameter. cDA inhibited 14CO2 production from all tested substrates by around 30–40%. The activities of the respiratory chain complexes and of creatine kinase were also tested in the presence of DA and cDA. Both metabolites significantly inhibited cytochrome c oxidase activity (by 30%) and complex II–III activity (DA, 25%; cDA, 80%). Furthermore, only cDA inhibited complex II activity (by 30%), while complex I–III and citrate synthase were not affected by these MCFA. On the other hand, only cDA reduced the activity of creatine kinase in total homogenates, as well as in mitochondrial and cytosolic fractions from cerebral cortex (by 50%). The data suggest that the major metabolites which accumulate in MCAD deficiency, with particular emphasis to cDA, compromise brain energy metabolism. We presume that these findings may contribute to the understanding of the pathophysiology of the neurological dysfunction of MCAD deficient patients.