Bioenergetics in glutaryl-coenzyme A dehydrogenase deficiency: a role for glutaryl-coenzyme A

Inherited deficiency of glutaryl-CoA dehydrogenase results in an accumulation of glutaryl-CoA, glutaric, and 3-hydroxyglutaric acids. If untreated, most patients suffer an acute encephalopathic crisis and, subsequently, acute striatal damage being precipitated by febrile infectious diseases during a vulnerable period of brain development (age 3 and 36 months). It has been suggested before that some of these organic acids may induce excitotoxic cell damage, however, the relevance of bioenergetic impairment is not yet understood. The major aim of our study was to investigate respiratory chain, tricarboxylic acid cycle, and fatty acid oxidation in this disease using purified single enzymes and tissue homogenates from Gcdh-deficient and wild-type mice. In purified enzymes, glutaryl-CoA but not glutaric or 3-hydroxyglutaric induced an uncompetitive inhibition of alpha-ketoglutarate dehydrogenase complex activity. Notably, reduced activity of alpha-ketoglutarate dehydrogenase activity has recently been demonstrated in other neurodegenerative diseases, such as Alzheimer, Parkinson, and Huntington diseases. In contrast to alpha-ketoglutarate dehydrogenase complex, no direct inhibition of glutaryl-CoA, glutaric acid, and 3-hydroxyglutaric acid was found in other enzymes tested. In Gcdh-deficient mice, respiratory chain and tricarboxylic acid activities remained widely unaffected, virtually excluding regulatory changes in these enzymes. However, hepatic activity of very long-chain acyl-CoA dehydrogenase was decreased and concentrations of long-chain acylcarnitines increased in the bile of these mice, which suggested disturbed oxidation of long-chain fatty acids. In conclusion, our results demonstrate that bioenergetic impairment may play an important role in the pathomechanisms underlying neurodegenerative changes in glutaryl-CoA dehydrogenase deficiency.     

Recent progress in understanding glutaric acidemias

Glutaric acidemia, which is due to inherited deficiency of glutaryl-CoA dehydrogenase, is characterized clinically by progressive dystonia and dyskinesia in childhood, and pathologically by degeneration of the caudate and putamen. Results using newer imaging techniques (computer tomography and magnetic resonance image scanning) suggest that neurological involvement in this condition begins before birth, and that gliosis of the basal ganglia is a relatively late event. Glutaric acidemia type II is usually due to inherited deficiency of electron transfer flavoprotein (ETF) or ETF:ubiquinone oxidoreductase, but some patients with typical disease may have another, to date undefined, abnormality. There may also be a clinical phenotype of glutaric acidemia type II which, like glutaryl-CoA dehydrogenase deficiency, is characterized by a movement disorder and by degeneration of the basal ganglia.     

Conservation of central nervous system glutaryl-coenzyme A dehydrogenase in fruit-eating bats with glutaric aciduria and deficient hepatic glutaryl-coenzyme A dehydrogenase

The adult fruit-eating bat, Rousettus aegypticus, excretes massive amounts of glutaric acid in the urine (20-70 mumol/mg creatinine) comparable to those of humans affected with the inherited metabolic disorder, glutaric aciduria type I. Glutaric acid was quantified by sequential liquid partition chromatography and gas chromatography. Oral loading with the amino acid precursors of glutaric acid, L-lysine and L-tryptophan, resulted in significant increases in glutaric acid excretion above the base-line values. Glutaryl-CoA dehydrogenase activity was assayed in adult bat tissues and compared with the same tissues in the rat using methods of 14CO2 evolution from 1,5-[14C]glutaryl-CoA. A severe deficiency of glutaryl-CoA dehydrogenase activity was found in the bat liver and kidney, whereas brain and spinal cord levels were similar to those in the rat. Reverse phase high performance liquid chromatography analysis of the metabolites in the assay mixture showed negligible hydrolysis of [14C]glutaryl-CoA to free [14C]glutaric acid and complete conversion of the product [14C]crotonyl-CoA to 3-hydroxy[14C]butyryl-CoA. The adult bat, with its huge glutaric acid excretion and deficient liver glutaryl-CoA dehydrogenase, metabolically mimics patients affected with glutaric aciduria type I. The bat does not, however, display the neurologic manifestations seen in patients. This may be explained by conservation of glutaryl-CoA dehydrogenase activity in the central nervous system of the bat.     

The function of Arg-94 in the oxidation and decarboxylation of glutaryl-CoA by human glutaryl-CoA dehydrogenase

Glutaryl-CoA dehydrogenase catalyzes the oxidation and decarboxylation of glutaryl-CoA to crotonyl-CoA and CO(2). Inherited defects in the protein cause glutaric acidemia type I, a fatal neurologic disease. Glutaryl-CoA dehydrogenase is the only member of the acyl-CoA dehydrogenase family with a cationic residue, Arg-94, situated in the binding site of the acyl moiety of the substrate. Crystallographic investigations suggest that Arg-94 is within hydrogen bonding distance of the gamma-carboxylate of glutaryl-CoA. Substitution of Arg-94 by glycine, a disease-causing mutation, and by glutamine, which is sterically more closely related to arginine, reduced k(cat) of the mutant dehydrogenases to 2-3% of k(cat) of the wild type enzyme. K(m) of these mutant dehydrogenases for glutaryl-CoA increases 10- to 16-fold. The steady-state kinetic constants of alternative substrates, hexanoyl-CoA and glutaramyl-CoA, which are not decarboxylated, are modestly affected by the mutations. The latter changes are probably due to steric and polar effects. The dissociation constants of the non-oxidizable substrate analogs, 3-thiaglutaryl-CoA and acetoacetyl-CoA, are not altered by the mutations. However, abstraction of a alpha-proton from 3-thiaglutaryl-CoA, to yield a charge transfer complex with the oxidized flavin, is severely limited. In contrast, abstraction of the alpha-proton of acetoacetyl-CoA by Arg-94 --> Gln mutant dehydrogenase is unaffected, and the resulting enolate forms a charge transfer complex with the oxidized flavin. These experiments indicate that Arg-94 does not make a major contribution to glutaryl-CoA binding. However, the electric field of Arg-94 may stabilize the dianions resulting from abstraction of the alpha-proton of glutaryl-CoA and 3-thiaglutaryl-CoA, both of which contain gamma-carboxylates. It is also possible that Arg-94 may orient glutaryl-CoA and 3-thiaglutaryl-CoA for abstraction of an alpha-proton.     

Partial purification and characterization of glutaryl-coenzyme A dehydrogenase, electron transfer flavoprotein, and electron transfer flavoprotein-Q oxidoreductase from Paracoccus denitrificans.

Glutaryl-coenzyme A (CoA) dehydrogenase and the electron transfer flavoprotein (ETF) of Paracoccus denitrificans were purified to homogeneity from cells grown with glutaric acid as the carbon source. Glutaryl-CoA dehydrogenase had a molecular weight of 180,000 and was made up of four identical subunits with molecular weights of about 43,000 each of which contained one flavin adenine dinucleotide molecule. The enzyme catalyzed an oxidative decarboxylation of glutaryl-CoA to crotonyl-CoA, was maximally stable at pH 5.0, and lost activity readily at pH values above 7.0. The enzyme had a pH optimum in the range of 8.0 to 8.5, a catalytic center activity of about 960 min-1, and apparent Michaelis constants for glutaryl-CoA and pig liver ETF of about 1.2 and 2.5 microM, respectively. P. denitrificans ETF had a visible spectrum identical to that of pig liver ETF and was made up of two subunits, only one of which contained a flavin adenine dinucleotide molecule. The isoelectric point of P. denitrificans ETF was 4.45 compared with 6.8 for pig liver ETF. P. denitrificans ETF accepted electrons not only from P. denitrificans glutaryl-CoA dehydrogenase, but also from the pig liver butyryl-CoA and octanoyl-CoA dehydrogenases. The apparent Vmax was of similar magnitude with either pig liver or P. denitrificans ETF as an electron acceptor for these dehydrogenases. P. denitrificans glutaryl-CoA dehydrogenase and ETF were used to assay for the reduction of ubiquinone 1 by ETF-Q oxidoreductase in cholate extracts of P. denitrificans membranes. The ETF-Q oxidoreductase from P. denitrificans could accept electrons from either the bacterial or the pig liver ETF. In either case, the apparent Km for ETF was infinitely high. P. denitrificans ETF-Q oxidoreductase was purified from contaminating paramagnets, and the resultant preparation had electron paramagnetic resonance signals at 2.081, 1.938, and 1.879 G, similar to those of the mitochondrial enzyme.     

Prenatal diagnosis of propionic acidemia by measuring methylcitric acid in dried amniotic fluid on filter paper using GC/MS

Propionic acidemia is a frequent inborn error of metabolism. Methylcitric acid, a key indicator of propionic acidemia, increases in the amniotic fluid of affected fetuses. For prenatal diagnosis, the methylcitric acid in amniotic fluid can be measured by stable-isotope dilution GC/MS. Here, we quantified this indicator in samples of amniotic fluid that had been dried on filter paper and transported at ambient temperatures, and compared the results with data obtained from the original amniotic fluid. We then used the filter-paper method to screen at-risk fetuses and obtained a clear-cut diagnosis in each case.     

Rapid and sensitive method for prenatal diagnosis of propionic acidemia using stable isotope dilution gas chromatography-mass spectrometry and urease pretreatment

Propionic acidemia is one of the most frequent inborn errors of metabolism caused by a deficiency of propionyl-CoA carboxylase. Methylcitric acid, a key indicator of this disorder, is increased in amniotic fluid when a fetus is affected. Therefore, the direct chemical analysis of cell-free amniotic fluid for methylcitric acid, using stable isotope dilution gas chromatography-mass spectrometry, was carried out for the prenatal diagnosis of propionic acidemia. We developed a simple, highly sensitive, and accurate method for quantitation of this polar methylcitric acid in amniotic fluids by applying a simplified urease pretreatment which we devised earlier for urine. As the recovery of methylcitric acid from amniotic fluid was as high as 91% with a coefficient of variation lower than 3% in this procedure, only 0.02 ml of sample was required for the analysis of the affected fetus. This new procedure takes 1 h for sample pretreatment, including derivatization, and 15 min for GC-MS measurement and provides final results within 1.5 h.     

Cofactors and metabolites as potential stabilizers of mitochondrial acyl-CoA dehydrogenases

Protein misfolding is a hallmark of a number of metabolic diseases, in which fatty acid oxidation defects are included. The latter result from genetic deficiencies in transport proteins and enzymes of the mitochondrial β-oxidation, and milder disease conditions frequently result from conformational destabilization and decreased enzymatic function of the affected proteins. Small molecules which have the ability to raise the functional levels of the affected protein above a certain disease threshold are thus valuable tools for effective drug design. In this work we have investigated the effect of mitochondrial cofactors and metabolites as potential stabilizers in two β-oxidation acyl-CoA dehydrogenases: short chain acyl-CoA dehydrogenase and the medium chain acyl-CoA dehydrogenase as well as glutaryl-CoA dehydrogenase, which is involved in lysine and tryptophan metabolism. We found that near physiological concentrations (low micromolar) of FAD resulted in a spectacular enhancement of the thermal stabilities of these enzymes and prevented enzymatic activity loss during a 1h incubation at 40°C. A clear effect of the respective substrate, which was additive to that of the FAD effect, was also observed for short- and medium-chain acyl-CoA dehydrogenase but not for glutaryl-CoA dehydrogenase. In conclusion, riboflavin may be beneficial during feverish crises in patients with short- and medium-chain acyl-CoA dehydrogenase as well as in glutaryl-CoA dehydrogenase deficiencies, and treatment with substrate analogs to butyryl- and octanoyl-CoAs could theoretically enhance enzyme activity for some enzyme proteins with inherited folding difficulties.     

Glutaric acid stimulates glutamate binding and astrocytic uptake and inhibits vesicular glutamate uptake in forebrain from young rats

Glutaric acidemia type I (GA I) is an inherited neurometabolic disorder caused by glutaryl-CoA dehydrogenase deficiency, which leads to accumulation in body fluids and in brain of predominantly glutaric acid (GA), and to a lesser extent of 3-hydroxyglutaric and glutaconic acids. Neurological presentation is common in patients with GA I. Although the mechanisms underlying brain damage in this disorder are not yet well established, there is growing evidence that excitotoxicity may play a central role in the neuropathogenesis of this disease. In the present study, preparations of synaptosomes, synaptic plasma membranes and synaptic vesicles, as well as cultured astrocytes from rat forebrain were exposed to various concentrations of GA for the determination of the basal and potassium-induced release of [(3)H]glutamate by synaptosomes, Na(+)-independent glutamate binding to synaptic membranes and vesicular glutamate uptake and Na(+)-dependent glutamate uptake into astrocytes, respectively. GA (1-100 nM) significantly stimulated [(3)H]glutamate binding to brain plasma membranes (40-70%) in the absence of extracellular Na(+) concentrations, reflecting glutamate binding to receptors. Furthermore, this stimulatory effect was totally abolished by the metabotropic glutamate ligands DHPG, DCG-IV and l-AP4, attenuated by the ionotropic non-NMDA glutamate receptor agonist AMPA and had no interference of the NMDA receptor antagonist MK-801. Moreover, [(3)H]glutamate uptake into synaptic vesicles was inhibited by approximately 50% by 10 and 100 nM GA and Na(+)-dependent [(3)H]glutamate uptake by astrocytes was significantly increased (up to 50%) in a dose-dependent manner (maximal stimulation at 100 microM GA). In contrast, synaptosomal glutamate release was not affected by the acid at concentrations as high as 1 mM. These results indicate that the inhibition of glutamate uptake into synaptic vesicles by low concentrations GA may result in elevated concentrations of the excitatory neurotransmitter in the cytosol and the stimulatory effect of this organic acid on glutamate binding may potentially cause excitotoxicity to neural cells. Finally, taken together these results and previous findings showing that GA markedly decreases synaptosomal glutamate uptake, it is possible that the stimulatory effect of GA on astrocyte glutamate uptake might indicate that astrocytes may protect neurons from excitotoxic damage caused by GA by increasing glutamate uptake and therefore reducing the concentration of this excitatory neurotransmitter in the synaptic cleft.     

Induction of Oxidative Stress by Chronic and Acute Glutaric Acid Administration to Rats

1. Glutaric acidemia type I (GA I) is a neurometabolic disorder caused by deficiency of glutaryl-CoA dehydrogenase, which leads to tissue accumulation of predominantly glutaric acid (GA) and also 3-hydroxyglutaric acid to a lesser amount. Affected patients usually present progressive cortical atrophy and acute striatal degeneration attributed to the toxic accumulating metabolites. 2. In the present study, we determined a number of oxidative stress parameters, namely chemiluminescence, thiobarbituric acid-reactive substances (TBA-RS), total antioxidant reactivity (TAR), glutathione (GSH) levels, and the activities of catalase and glutathione peroxidase (GPx), in various tissues from rats chronically exposed to GA or to saline (controls). High GA concentrations, similar to those found in glutaric aciduria type I, were induced in the brain by three daily subcutaneous injections of saline-buffered GA (5 μmol/g body weight) to Wistar rats of 5–22 days of life. The parameters were assessed 12 h after the last GA administration in different brain structures, skeletal muscle, heart, liver, erythrocytes, and plasma. The lipid peroxidation parameters chemiluminescence and/or TBA-RS measurements were found significantly increased in midbrain, liver, and erythrocytes of GA-injected rats. The activity of GPx was significantly reduced in midbrain and markedly increased in liver. TAR measurement was significantly reduced in midbrain and liver. Furthermore, GSH levels were reduced in liver and heart. We also investigated the acute in vivo effect of GA administration on the same oxidative stress parameters in cerebral structures and erythrocytes from 22-day-old rats. We found that TBA-RS values were significantly increased in erythrocytes, TAR levels were markedly decreased in midbrain and cerebellum, and GPx activity mildly reduced in the midbrain. 3. These data showing an imbalance between antioxidant defences and oxidative damage, particularly in midbrain, liver, and erythrocytes from GA-injected rats, indicate that oxidative stress might be involved in GA toxicity and that the midbrain, where the striatum is located, is the brain structure more susceptible to GA chronic and acute exposition.     

l-Carnitine prevents oxidative stress in striatum of glutaryl-CoA dehydrogenase deficient mice submitted to lysine overload

The deficiency of the enzyme glutaryl-CoA dehydrogenase leads to predominant accumulation of glutaric acid (GA) in the organism and is known as glutaric acidemia type I (GA1). Despite the mechanisms of brain damage involved in GA1 are not fully understood, oxidative stress may be involved in this process. Treatment is based on protein/lysine (Lys) restriction and l-carnitine (L-car) supplementation. L-car was recently shown to have an important antioxidant role. A knockout mice model (Gcdh^?/?) submitted to a dietary overload of Lys was developed to better understand the GA1 pathogenesis. In this study, we evaluated L-car and glutarylcarnitine levels, the lipid and protein damage, reactive oxygen species (ROS) production and antioxidant enzymes activities in striatum of Gcdh^?/? and wild-type (WT) mice. We also determined the effect of the L-car treatment on these parameters. Thirty-day-old Gcdh^?/? and WT mice were fed a normal chow (0.9% Lys) or submitted to a high Lys diet (4.7%) for 72 h. Additionally, these animals were administered with three intraperitoneal injections of saline or L-car in different times. Gcdh^?/? mice were deficient in L-car and presented a higher glutarylcarnitine levels. They also presented lipid and protein damage, an increased ROS production and altered antioxidant enzymes compared to WT mice. Additionally, mice exposed to Lys overload presented higher alterations in these parameters than mice under normal diet, which were significantly decreased or normalized in those receiving L-car. Thus, we demonstrated a new beneficial effect of the L-car treatment attenuating or abolishing the oxidative stress process in Gcdh^?/? mice.     

Glutaric aciduria type 1 metabolites impair the succinate transport from astrocytic to neuronal cells

The inherited neurodegenerative disorder glutaric aciduria type 1 (GA1) results from mutations in the gene for the mitochondrial matrix enzyme glutaryl-CoA dehydrogenase (GCDH), which leads to elevations of the dicarboxylates glutaric acid (GA) and 3-hydroxyglutaric acid (3OHGA) in brain and blood. The characteristic clinical presentation of GA1 is a sudden onset of dystonia during catabolic situations, resulting from acute striatal injury. The underlying mechanisms are poorly understood, but the high levels of GA and 3OHGA that accumulate during catabolic illnesses are believed to play a primary role. Both GA and 3OHGA are known to be substrates for Na(+)-coupled dicarboxylate transporters, which are required for the anaplerotic transfer of the tricarboxylic acid cycle (TCA) intermediate succinate between astrocytes and neurons. We hypothesized that GA and 3OHGA inhibit the transfer of succinate from astrocytes to neurons, leading to reduced TCA cycle activity and cellular injury. Here, we show that both GA and 3OHGA inhibit the uptake of [(14)C]succinate by Na(+)-coupled dicarboxylate transporters in cultured astrocytic and neuronal cells of wild-type and Gcdh(-/-) mice. In addition, we demonstrate that the efflux of [(14)C]succinate from Gcdh(-/-) astrocytic cells mediated by a not yet identified transporter is strongly reduced. This is the first experimental evidence that GA and 3OHGA interfere with two essential anaplerotic transport processes: astrocytic efflux and neuronal uptake of TCA cycle intermediates, which occur between neurons and astrocytes. These results suggest that elevated levels of GA and 3OHGA may lead to neuronal injury and cell death via disruption of TCA cycle activity.     

Glutaric aciduria type I in the Arab and Jewish communities in Israel.

Mutation analysis was performed in eight families (16 patients) with glutaric aciduria type I (GA-I), which were all the families diagnosed in Israel in the years 1987-1994. Six families were of Moslem origin and two were non-Ashkenazi Jews. The entire coding region of the cDNA of the glutaryl-CoA dehydrogenase gene was sequenced in one patient of each family. Seven new mutations were identified in 15 of 16 mutated alleles, including six point mutations: T416I (4 alleles), G390R (1 allele), and S305L, A293T, L283P, and G1O1R (2 alleles each). In addition, a 1-bp deletion at position 1173 was identified in two alleles. These findings do not provide a molecular basis for the clinical variability in GA-I families. The occurrence of multiple novel mutations in a small geographic area may be explained by their recent onset in isolated communities with a high consanguinity rate.     

Binding, hydration, and decarboxylation of the reaction intermediate glutaconyl-coenzyme A by human glutaryl-CoA dehydrogenase.

Glutaconyl-coenzyme A (CoA) is the presumed enzyme-bound intermediate in the oxidative decarboxylation of glutaryl-CoA that is catalyzed by glutaryl-CoA dehydrogenase. We demonstrated glutaconyl-CoA bound to glutaryl-CoA dehydrogenase after anaerobic reduction of the dehydrogenase with glutaryl-CoA. Glutaryl-CoA dehydrogenase also has intrinsic enoyl-CoA hydratase activity, a property of other members of the acyl-CoA dehydrogenase family. The enzyme rapidly hydrates glutaconyl-CoA at pH 7.6 with a k(cat) of 2.7 s(-1). The k(cat) in the overall oxidation-decarboxylation reaction at pH 7.6 is about 9 s(-1). The binding of glutaconyl-CoA was quantitatively assessed from the K(m) in the hydratase reaction, 3 microM, and the K(i), 1.0 microM, as a competitive inhibitor of the dehydrogenase. These values compare with K(m) and K(i) of 4.0 and 12.9 microM, respectively, for crotonyl-CoA. Glu370 is the general base catalyst in the dehydrogenase that abstracts an alpha-proton of the substrate to initiate the catalytic pathway. The mutant dehydrogenase, Glu370Gln, is inactive in the dehydrogenation and the hydratase reactions. However, this mutant dehydrogenase decarboxylates glutaconyl-CoA to crotonyl-CoA without oxidation-reduction reactions of the dehydrogenase flavin. Addition of glutaconyl-CoA to this mutant dehydrogenase results in a rapid, transient increase in long-wavelength absorbance (lambda(max) approximately 725 nm), and crotonyl-CoA is found as the sole product. We propose that this 725 nm-absorbing species is the delocalized crotonyl-CoA anion that follows decarboxylation and that the decay is the result of slow protonation of the anion in the absence of the general acid catalyst, Glu370(H(+)). In the absence of detectable oxidation-reduction, the data indicate that oxidation-reduction of the dehydrogenase flavin is not essential for decarboxylation of glutaconyl-CoA.     

Mechanism-based inactivation of human glutaryl-CoA dehydrogenase by 2-pentynoyl-CoA: rationale for enhanced reactivity

2-Pentynoyl-CoA inactivates glutaryl-CoA dehydrogenase at a rate that considerably exceeds the rates of inactivation of short chain and medium chain acyl-CoA dehydrogenases by this inhibitor and related 2-alkynoyl-CoAs. To determine the rate of inactivation by 2-pentynoyl-CoA, we investigated the inactivation in the presence of a non-oxidizable analog, 3-thiaglutaryl-CoA, which competes for the binding site. The enhanced rate of inactivation does not reflect an alteration in specificity for the acyl group, nor does it reflect the covalent modification of a residue other than the active site glutamate. In addition to determining the inactivation of catalytic activity a spectral intermediate was detected by stopped-flow spectrophotometry, and the rate constants of formation and decay of this charge transfer complex (lambdamax approximately 790 nm) were determined by global analysis. Although the rate-limiting step in the inactivation of the other acyl-CoA dehydrogenases can involve the abstraction of a proton at C-4, this is not the case with glutaryl-CoA dehydrogenase. Glutaryl-CoA dehydrogenase is also differentiated from other acyl-CoA dehydrogenases in that the catalytic base must access both C-2 and C-4 in the normal catalytic pathway. Access to C-4 is not obligatory for the other dehydrogenases. Analysis of the distance from the closest carboxylate oxygen of the glutamate base catalyst to C-4 of a bound acyl-CoA ligand for medium chain, short chain, and isovaleryl-CoA dehydrogenases suggests that the increased rate of inactivation reflects the carboxylate oxygen to ligand C-4 distance in the binary complexes. This distance for wild type glutaryl-CoA dehydrogenase is not known. Comparison of the rate constants of inactivation and formation of a spectral species between wild type glutaryl-CoA dehydrogenase and a E370D mutant are consistent with the idea that this distance in glutaryl-CoA dehydrogenase contributes to the enhanced rate of inactivation and the 1,3-prototropic shift catalyzed by the enzyme.     

Mechanistic Effects of Amino Acids and Glucose in a Novel Glutaric Aciduria Type 1 Cell Model

Acute neurological crises involving striatal degeneration induced by a deficiency of glutaryl-CoA dehydrogenase (GCDH) and the accumulation of glutaric (GA) and 3-hydroxyglutaric acid (3-OHGA) are considered to be the most striking features of glutaric aciduria type I (GA1). In the present study, we investigated the mechanisms of apoptosis and energy metabolism impairment in our novel GA1 neuronal model. We also explored the effects of appropriate amounts of amino acids (2 mM arginine, 2 mM homoarginine, 0.45 g/L tyrosine and 10 mM leucine) and 2 g/L glucose on these cells. Our results revealed that the novel GA1 neuronal model effectively simulates the hypermetabolic state of GA1. We found that leucine, tyrosine, arginine, homoarginine or glucose treatment of the GA1 model cells reduced the gene expression of caspase-3, caspase-8, caspase-9, bax, fos, and jun and restored the intracellular NADH and ATP levels. Tyrosine, arginine or homoarginine treatment in particular showed anti-apoptotic effects; increased α-ketoglutarate dehydrogenase complex (OGDC), fumarase (FH), and citrate synthase (CS) expression; and relieved the observed impairment in energy metabolism. To the best of our knowledge, this study is the first to investigate the protective mechanisms of amino acids and glucose in GA1 at the cellular level from the point of view of apoptosis and energy metabolism. Our data support the results of previous studies, indicating that supplementation of arginine and homoarginine as a dietary control strategy can have a therapeutic effect on GA1. All of these findings facilitate the understanding of cell apoptosis and energy metabolism impairment in GA1 and reveal new therapeutic perspectives for this disease.     

Argininosuccinic aciduria: prenatal studies in a family at risk.

We have monitored two successive pregnancies in a family which we found to be at risk for argininosuccinic aciduria. We measured argininosuccinic acid (ASA) concentrations in amniotic fluid and utilized an indirect assay of ASA lyase activity in cultured amniotic fluid cells. The assay procedure is based on the uptake of 14C from [14C]citrulline and of [3H]leucine into protein. ASA was easily measured in amniotic fluid from the first fetus at risk, whereas none was detectable in control fluids. Amniotic fluid cells cultured from this fetus had only 5.5% of control ASA lyase activity. The pregnancy was terminated, and hepatic ASA lyase activity in the fetus was shown to be about 1.3% of control values. In addition, eight fetal tissues were analyzed for ASA, and all had significant accumulation. ASA was not detected in amniotic fluid from the second fetus at risk, and ASA lyase activity in cultured cells was 80% of control activity. Enzymatic analysis of erythrocyte lysate confirmed the diagnosis of an unaffected child (ASA lyase = 46% of control) and indicated heterozygosity. Thus, we provide further evidence that argininosuccinic aciduria can be diagnosed successfully in utero by indirect assay of ASA lyase activity in cultured amniotic fluid cells. In addition, high amniotic fluid ASA concentrations provide strong adjunctive evidence for such a prenatal determination, and may prove to be sufficient for diagnosis.     

Microbial metabolism of the pyridine ring. Metabolic pathways of pyridine biodegradation by soil bacteria.

1. Two bacteria, a Bacillus sp. and a Nocardia sp. (strain Z1) were isolated from soil by enrichment with 0.1 percent (v/v) pyridine and grew rapidly on this compound as sole C, N and energy source. The monohydroxypyridines, tetrahydropyridine, piperidine and some other analogues were not utilized for growth or oxidized by washed suspensions of either bacterium. 2. Cell-free extracts were unable to metabolize pyridine even after supplementation with a variety of cofactors or protecting agents. Treatment of cells with toluene led to rapid loss of the ability to oxidize pyridine. 3. In the presence of 10mM-semicarbazide at pH 6.0, Nocardia Z1 accumulated a semialdehyde idenditied as its 2,4-dinitrophenylhydrazone by chromatography, mixed melting point, mass spectrometry and isotope trapping from [2,6(-14)C]pyridine as glutarate semialdehyde. 4. Extracts of this bacterium prepared from cells grown with pyridine or exposed to the gratuitous inducer 2-picoline, contained high activities of a specific glutarate semialdehyde dehydrogenase. 5. Cells grown with pyridine or glutarate also contained a glutaric dialdehyde dehydrogenase, an acyl-CoA synthetase and elevated amounts of isocitrate lyase but no glutaryl-CoA dehydrogenase. 6. Bacillus 4 accumulated in the presence of 10mM-semicarbazide several acidic carbonyl compounds from pyridine among which was succinate semialdehyde. Extracts of this bacillus after growth of the cells with pyridine contained an inducible succinate semialdehyde dehydrogenase in amounts at least 50-fold over those found in succinate-grown cells. 7. Two mutants of this bacillus, selected for their inability to grow on pyridine were deficient in succinate semialdehyde dehydrogenase. 8. In the presence of 0.2mM-KCN, washed suspensions of Bacillus 4 accumulated formate and possibly formamide from pyridine. The use of [14C]pyridine showed that formate was derived from C-2 of the pyridine ring. 9. The organism had a specific formamide amidohydrolase cleaving formamide quantitatively to formate and NH3. 10. Formate was further oxidized by the particle fraction. There was no soluble formate dehydrogenase in extracts.     

Ammonium Accumulation and Cell Death in a Rat 3D Brain Cell Model of Glutaric Aciduria Type I

Glutaric aciduria type I (glutaryl-CoA dehydrogenase deficiency) is an inborn error of metabolism that usually manifests in infancy by an acute encephalopathic crisis and often results in permanent motor handicap. Biochemical hallmarks of this disease are elevated levels of glutarate and 3-hydroxyglutarate in blood and urine. The neuropathology of this disease is still poorly understood, as low lysine diet and carnitine supplementation do not always prevent brain damage, even in early-treated patients. We used a 3D in vitro model of rat organotypic brain cell cultures in aggregates to mimic glutaric aciduria type I by repeated administration of 1 mM glutarate or 3-hydroxyglutarate at two time points representing different developmental stages. Both metabolites were deleterious for the developing brain cells, with 3-hydroxyglutarate being the most toxic metabolite in our model. Astrocytes were the cells most strongly affected by metabolite exposure. In culture medium, we observed an up to 11-fold increase of ammonium in the culture medium with a concomitant decrease of glutamine. We further observed an increase in lactate and a concomitant decrease in glucose. Exposure to 3-hydroxyglutarate led to a significantly increased cell death rate. Thus, we propose a three step model for brain damage in glutaric aciduria type I: (i) 3-OHGA causes the death of astrocytes, (ii) deficiency of the astrocytic enzyme glutamine synthetase leads to intracerebral ammonium accumulation, and (iii) high ammonium triggers secondary death of other brain cells. These unexpected findings need to be further investigated and verified in vivo. They suggest that intracerebral ammonium accumulation might be an important target for the development of more effective treatment strategies to prevent brain damage in patients with glutaric aciduria type I.     

Proton abstraction reaction, steady-state kinetics, and oxidation-reduction potential of human glutaryl-CoA dehydrogenase

Glutaryl-CoA dehydrogenase catalyzes the oxidation of glutaryl-CoA to crotonyl-CoA and CO(2) in the mitochondrial degradation of lysine, hydroxylysine, and tryptophan. We have characterized the human enzyme that was expressed in Escherichia coli. Anaerobic reduction of the enzyme with sodium dithionite or substrate yields no detectable semiquinone; however, like other acyl-CoA dehydrogenases, the human enzyme stabilizes an anionic semiquinone upon reduction of the complex between the enzyme and 2,3-enoyl-CoA product. The flavin potential of the free enzyme determined by the xanthine-xanthine oxidase method is -0.132 V at pH 7.0, slightly more negative than that of related flavoprotein dehydrogenases. A single equivalent of substrate reduces 26% of the dehydrogenase flavin, suggesting that the redox equilibrium on the enzyme between substrate and product and oxidized and reduced flavin is not as favorable as that observed with other acyl-CoA dehydrogenases. This equilibrium is, however, similar to that observed in isovaleryl-CoA dehydrogenase. Comparison of steady-state kinetic constants of glutaryl-CoA dehydrogenase with glutaryl-CoA and the alternative substrates, pentanoyl-CoA and hexanoyl-CoA, suggests that the gamma-carboxyl group of glutaryl-CoA stabilizes the enzyme-substrate complex by at least 5.7 kJ/mol, perhaps by interaction with Arg94 or Ser98. Glu370 is positioned to function as the catalytic base, and previous studies indicate that the conjugate acid of Glu370 also protonates the transient crotonyl-CoA anion following decarboxylation [Gomes, B., Fendrich, G. , and Abeles, R. H. (1981) Biochemistry 20, 3154-3160]. Glu370Asp and Glu370Gln mutants of glutaryl-CoA dehydrogenase exhibit 7% and 0. 04% residual activity, respectively, with human electron-transfer flavoprotein; these mutations do not grossly affect the flavin redox potentials of the mutant enzymes. The reduced catalytic activities of these mutants can be attributed to reduced extent and rate of substrate deprotonation based on experiments with the nonoxidizable substrate analogue, 3-thiaglutaryl-CoA, and kinetic experiments. Determination of these fundamental properties of the human enzyme will serve as the basis for future studies of the decarboxylation reaction which is unique among the acyl-CoA dehydrogenases.