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PubMed1993

A fibroblast glutaryl-CoA dehydrogenase assay using detritiation of 3H-labelled glutaryl-CoA: application in the genotyping of the glutaryl-CoA dehydrogenase locus.

A method described earlier for measuring glutaryl-CoA dehydrogenase activity in fibroblasts has been further developed. This assay uses the detritiation of [2,3,4-3H]glutaryl-CoA both with and without added artificial electron acceptors as a measure of glutaryl-CoA dehydrogenase activity. Fibroblasts from patients with glutaryl-CoA dehydrogenase deficiency, as determined by the 14CO2 release assay, showed very low detritiation of [2,3,4-3H]glutaryl-CoA without added artificial electron acceptor. Obligate heterozgotes for glutaryl-CoA dehydrogenase deficiency showed detritiation activity intermediate between homozygotes and normal individuals. Addition of an electron acceptor to the assay mixture had no influence on the activity in homozygotes for glutaryl-CoA dehydrogenase deficiency but more than doubled the detritiation activity in obligate heterozygotes and in normal individuals. No difference in detritiation activity could be detected between glutaryl-CoA dehydrogenase deficient patients with a high urinary excretion of glutaric acid and patients with almost no excretion of glutaric acid. In all glutaryl-CoA dehydrogenase deficient cell lines tested the loss of decarboxylation activity was also accompanied by a loss of dehydrogenation activity. The detritiation assay was equivalent to, or even better than, the 14CO2 release assay in distinguishing between homozygotes and heterozygotes for glutaryl-CoA dehydrogenase deficiency.

Cells, Cultured↗
PubMed1986

The binding of 1,N6-ethenoNAD to bovine liver glutamate dehydrogenase: studies using the time-correlated single photon counting fluorescence technique.

The time-correlated single photon counting (TCPC) fluorescence technique has been used as a novel approach to investigate ligand-protein interaction, for the case of the binding of the fluorescent coenzyme analogue 1,N6-ethenoNAD (epsilon NAD) to bovine liver glutamate dehydrogenase in the presence of glutarate, a substrate analogue which stabilizes the complex. System calibration was performed using solutions of epsilon ADP and carefully purified epsilon NAD mixed at variable molar ratios (pH 7.0, 0.05 M sodium phosphate buffer, 20 degrees C). The fluorescence lifetimes obtained after deconvolution were 2.4 ns (for epsilon NAD) and 23 ns (for epsilon ADP), in good agreement with literature values obtained under similar conditions. epsilon NAD binds to glutamate dehydrogenase in the presence of 50 mM glutarate, with a fluorescence quantum yield enhancement factor, Q, of about 17-fold, as previously reported (Favilla, R. and Mazzini, A. (1984) Biochim. Biophys. Acta 48-57). For this system, fluorescence lifetime values were obtained after deconvolution as 2.4 ns for free epsilon NAD and 21 ns for bound epsilon NAD. These values did not vary appreciably with enzyme concentration nor with degree of saturation, thus reflecting the existence of only one spectroscopically relevant type of complex. Addition of either GTP or ADP did not affect the lifetime of epsilon NAD bound to the enzyme, but only its affinity, thus allowing calculations of binding strengths. In the case of a simple binding (i.e., in the absence of GTP) the dissociation constant of the complex could be derived from a simple relationship, in which only the ratio between the pre-exponential factors and the parameter gamma, which represents the molar fraction of epsilon NAD molecules free in solution in the open conformation, are to be taken into account. The results are in good agreement with those reported by some of us (reference above) using a steady-state fluorescence technique, which by itself is, however, unable to resolve the number of relevant species present in the system.

Adenosine Diphosphate↗
PubMed1994

Movement disorders in childhood organic acidurias. Clinical, neuroimaging, and biochemical correlations.

Over the last 5 years the Pediatric Neurology service at King Faisal Specialist Hospital and Research Centre (KFSH&RC) has seen 131 infants and children with movement disorders. Forty-nine (37%) had identifiable biochemical defects, 25 of which were organic acidemias. Nineteen of 29 patients with dystonia had organic acidemias, primarily glutaric aciduria type 1 (7 patients), bilateral striatal necrosis (4 patients), and 3-methyl glutaconic aciduria (3 patients). All patients with parkinsonian rigidity (n = 11) had organic acidemias; again, the greatest number accounted for by glutaric aciduria type 1 (7 patients), who had both parkinsonian rigidity combined with dystonia. Myoclonus occurred in only 1 of 25 and chorea in 7 of 25 patients with organic acidemias. At the least all patients had bilateral lesions of putamen and head of caudate, seen best in MRI brain scans as increased T2 signal intensities with normal volume, and later with volume loss.

Acidosis↗
PubMed2004

Low molecular weight dicarboxylic acids in PM10 in a city with intensive solid fuel burning.

In this work, PM(10) samples were collected in a winter and a summer in Christchurch, a New Zealand city having intensive wood and coal burning and a serious air pollution problem in winter. Oxalic, malonic, succinic, maleic, glutaric and adipic acids in the samples were analysed using ion chromatography. It was suggested that solid fuel burning had large influence on the occurrence of these low molecular weight dicarboxylic acids resulting in significantly higher wintertime concentrations of maleic acid, oxalic acid and glutaric or adipic acid. The most pronounced feature observed was that maleic acid was the second most abundant species of the detected DCAs in the winter (with a mean of 74 ngm(-3) and the highest concentration ever reported of 231 ngm(-3)). In contrast, malonic acid experienced a low abundance in both seasons. The observed seasonal patterns and molecular distribution were inconsistent with those in most other urban areas. On an average, the total detected dicarboxylic acids accounted for about 0.5% of PM(10) mass with a maximum of 1.4% in the winter. The relative importance of different sources to individual dicarboxylic acids varied with seasons and is discussed in detail.

Adipates↗
PubMed2005

Quinolinic acid reduces the antioxidant defenses in cerebral cortex of young rats.

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.

Analysis of Variance↗
PubMed2004

3-Hydroxyglutaric acid fails to affect the viability of primary neuronal rat cells.

Glutaric aciduria type I (GA I) is an autosomal recessive inherited metabolic disorder caused by deficiency of glutaryl-CoA dehydrogenase (GCD) resulting in the accumulation of 3-hydroxyglutaric acid (3OHG), glutaric acid and glutaconic acid in body fluids. GA I is characterized by a specific age- and brain region-dependent neuropathology. Previous studies using organotypic slice cultures of rats and primary chick embryo telencephalon cell cultures indicated that death of neurons is a consequence of an excitotoxic mechanism induced by 3OHG. We used primary neuronal cells of neonatal rats as a model system to test cell viability after treatment with 3OHG. Western blot analysis was used to prove the expression of functional N-methyl-D-aspartate (NMDA) receptors revealing no alteration in the expression of NMDA-2a and -2b receptor subtypes in response to 3OHG. When neuronal cells cultured for 10 or 20 days were treated with 1 mM glutamate, the viability of cells was reduced by 40%. This effect could be prevented by coincubation with the NMDA receptor antagonist MK801. In contrast, incubation of cells with 3OHG for up to 24 h in concentrations of 4-8 mM did not cause increased cell death as compared with untreated control cultures. These results indicate that 3OHG is not excitotoxic in this model of neuronal rat cell cultures despite the presence of functional NMDA receptors. Therefore, alternative or additional pathomechanisms than excitotoxicity may be relevant for neurodegeneration in GA I.

Acids↗
PubMed2006

Riboflavin-responsive glutaryl CoA dehydrogenase deficiency.

We report here riboflavin responsiveness in a patient with glutaryl CoA dehydrogenase (GCDH) deficiency, compound heterozygous for the S139L and P248L mutations and with 20% residual GCDH enzyme activity in vitro. Our results suggest the mitochondrial GCDH homotetramer remains intact with one of these mutations associated with the binding site of the single FAD cofactor and that pharmacological doses of the cofactor precursor may be sufficient to induce an increase in activity in the mutant GCDH enzyme, although not sufficient to normalise urinary organic acid excretion. Serine139 is one of nine conserved amino acid residues that line the binding site of the protein and is in close proximity to both substrate and FAD cofactor. It is possible that steric alterations caused by substitution of serine with leucine at this position may be overcome with high cofactor concentrations. P248L is also associated with some residual GCDH activity in other patients and the unique combination of S139L with P248L may also explain the results in our patient. Responsiveness to riboflavin in our patient has been compared with two other patients with glutaric aciduria type 1 and minimal residual GCDH activity, one with homozygosity for the R257Q mutation and one with heterozygosity for the G354S mutation and a novel G156V mutation. A low lysine diet reduced glutaric acid excretion in our riboflavin-responsive GCDH-deficient patient almost to control values. She is now 21 years of age and clinically and neurologically normal.

Adult↗
PubMed1998

Functional characterization of the rat multispecific organic anion transporter OAT1 mediating basolateral uptake of anionic drugs in the kidney.

The functional characteristics of rat organic anion transporter OAT1 were investigated using Xenopus laevis oocytes. Uptake of p-aminohippurate (PAH) by the oocytes expressing OAT1 was markedly inhibited by glutarate, alpha-ketoglutarate and probenecid, moderately inhibited by folate and methotrexate, but not inhibited by taurocholate or tetraethylammonium. Methotrexate and folate were transported by OAT1, but probenecid, a typical inhibitor of organic anion transporter, was not transported. Inhibition of PAH uptake by aliphatic dicarboxylates with various alkyl chain lengths was maximal at 5 (glutarate) and 6 (adipate) carbon atoms. OAT1-mediated PAH uptake was markedly inhibited by phorbol 12-myristate 13-acetate (PMA), phorbol 12,13-dibutyrate and mezerein, but not by 4alpha-phorbol 12,13-didecanoate. The inhibitory effect of PMA was attenuated in the presence of staurosporine, suggesting that OAT1 is regulated by protein kinase C. These results suggest that the substrate recognition of OAT1 is comparable to that of renal basolateral organic anion transporter, and the transport activity is regulated by protein kinase C.

Animals↗
PubMed1994

Prophylaxis and reversal of ifosfamide encephalopathy with methylene-blue.

The antineoplastic ifosfamide produces dose-dependent signs of neurotoxicity. After ifosfamide overdose in a patient, we found excessive urinary excretion of glutaric acid and sarcosine, which is compatible with glutaric aciduria type II, a defect in mitochondrial fatty acid oxidation that results from defective electron transfer to flavoproteins. We therefore used the electron-accepting drug methylene-blue as an antidote for ifosfamide encephalopathy. In one patient, ifosfamide neurotoxicity was rapidly reversed by methylene-blue 50 mg intravenously. In another patient with previous episodes of ifosfamide encephalopathy, methylene-blue was administered orally prophylactically. No symptoms of neurotoxicity were noted.

Administration, Oral↗
PubMed2002

Quantification of 3-hydroxyglutaric acid in urine, plasma, cerebrospinal fluid and amniotic fluid by stable-isotope dilution negative chemical ionization gas chromatography-mass spectrometry.

This paper describes a stable isotope dilution method for quantification of 3-hydroxyglutaric acid (3-HGA) in body fluids. The method comprises a solid-phase extraction procedure, followed by gas chromatographic separation and negative chemical ionization mass spectrometric detection. This method is selective and sensitive, and enables measurement of 3-HGA concentrations in urine-, plasma-, and CSF- samples of controls. The control ranges for 3-HGA were: urine 0.88-4.5 mmol/mol creatinine (n=12); plasma 0.018-0.10 micro mol/l (n=10), CSF 0.022-0.067 micro mol/l (n=10). We applied this method to measure 3-HGA in body fluids of three patients with glutaric aciduria type I. We also quantified 3-HGA in amniotic fluid of controls (range 0.056-0.11 micro mol/l; n=12) and in two samples from fetuses affected with glutaric aciduria type I.

Amniotic Fluid↗
PubMed2004

Correlation of genotype and phenotype in glutaryl-CoA dehydrogenase deficiency.

We have investigated the correlation between genotype and phenotype in a large number of patients with glutaric aciduria type I (GA I). The deficiency of glutaryl-CoA dehydrogenase has been confirmed in the Rigshospitalet's laboratory in 215 patients since 1975. Most of the patients were of European ancestry. Complete absence of enzyme activity was found in more than half of the patients, while 34% of patients had a residual activity up to 5% and a few patients had a residual activity of 5-15%. In four exceptional cases, a very high residual activity of up to 30% was found. Enzyme studies are thus a reliable method for confirming the diagnosis of GA I, although it may be difficult to distinguish exceptional 'mild' cases from heterozygous carriers for GA I. Three of the patients with very high residual activity are compound heterozygous for the missense mutations R227P and V400M, both of which are associated with residual enzyme activity of 8-10% in homozygous patients. Patients with a mild mutation on at least one chromosome frequently show unusual biochemical findings such as low or normal urinary excretion of glutaric acid and mild or only slightly increased excretion of 3-hydroxyglutaric acid. In contrast, patients with severe mutations such as R402W or A293T on both alleles have no residual activity and show the typical urinary metabolite pattern. Clinical data were available for a subgroup of 79 patients. No correlation with the biochemical phenotype or the genotype could be established.

Alleles↗
PubMed1972

A radiometric technique for measuring L-asparagine in picomole quantities.

A radiometric technique for measuring l-asparagine in small samples of biological origin is presented. After removal of l-glutamate and l-aspartate by enzymic decarboxylation, l-asparagine in the sample is hydrolysed to l-aspartate with l-asparaginase from Escherichia coli. The l-aspartate so generated is then enzymically transaminated with 2-oxo[1-(14)C]glutarate yielding l-[1-(14)C]glutamate. After specific chemical removal of unchanged 2-oxo[1-(14)C]glutarate, the l-[1-(14)C]glutamate generated is determined by scintillation counting of the radioactivity. This technique can measure the concentration of l-asparagine present in approx. 1mul of mouse blood, and is capable of detecting as little as 25pmol of the amide. Illustrative applications of the method are presented and discussed, including measurement of the l-asparagine content of the mouse hypophysis.

Animals↗
PubMed2004

Identification of a dehydrogenase acting on D-2-hydroxyglutarate.

Extracts of frozen rat liver were found to catalyse the formation of 3H2O from DL-2-hydroxy[2-3H]glutarate. Three peaks of enzyme activities were observed on separation by chromatography on DEAE-Sepharose. The first and second peaks corresponded to an enzyme acting on L-2-hydroxyglutarate and the third peak corresponded to an enzyme acting on D-2-hydroxyglutarate, as indicated by competitive inhibition of the detritiation of the racemic radioactive compound by the unlabelled L- and D-isomers respectively. The enzyme acting on the D-form was further characterized. It was independent of NAD or NADP and it converted D-2-hydroxyglutarate into a-ketoglutarate, transferring electrons to artificial electron acceptors. It also oxidized D-lactate, D-malate and meso-tartrate and was stimulated by Zn2+, Co2+ and Mn2+, but not by Mg2+ or Ca2+. Subcellular fractionation indicated that it was present in the mitochondrial fraction. The enzyme was further purified by chromatography on Blue Trisacryl and phenyl-Sepharose, up to a stage where only a few bands were still visible by SDS/PAGE. Among the four candidate polypeptides that were identified by MS, one corresponded to a predicted mitochondrial protein homologous with FAD-dependent D-lactate dehydrogenase. The corresponding human protein was expressed in HEK-293 cells and it was shown to catalyse the detritiation of DL-2-hydroxy[2-3H]glutarate with similar properties as the purified rat enzyme.

Amino Acid Sequence↗
PubMed2006

Resorcinarenes are hexameric capsules in solution.

The host-guest complexes of resorcin[4]arenes with small molecules in organic solutions are examined using modern NMR spectroscopic methods. The complexation of glutaric acid and beta-methyl d-glucopyranoside in chloroform were investigated through 2D COSY, 2D NOESY, 1D NOE, and diffusion-ordered NMR spectroscopy (DOSY) techniques. These methods indicate that the complex is a self-assembled capsule composed of six resorcinarenes that surround six guest molecules of glutaric acid or three molecules of beta-methyl d-glucopyranoside inside. The multiplicity of guest proton signals shows that the capsule provides an asymmetric magnetic environment that persists on the (1)H NMR time scale. The encapsulation of these guests and common solvents suggests that the phenomenon of reversible encapsulation in chemistry may be a century old.

Calixarenes↗
PubMed2000

Cocrystallization of a mutant aspartate aminotransferase with a C5-dicarboxylic substrate analog: structural comparison with the enzyme-C4-dicarboxylic analog complex.

A mutant Escherichia coil aspartate aminotransferase with 17 amino acid substitutions (ATB17), previously created by directed evolution, shows increased activity for beta-branched amino acids and decreased activity for the native substrates, aspartate and glutamate. A new mutant (ATBSN) was generated by changing two of the 17 mutated residues back to the original ones. ATBSN recovered the activities for aspartate and glutamate to the level of the wild-type enzyme while maintaining the enhanced activity of ATB17 for the other amino acid substrates. The absorption spectrum of the bound coenzyme, pyridoxal 5'-phosphate, also returned to the original state. ATBSN shows significantly increased affinity for substrate analogs including succinate and glutarate, analogs of aspartate and glutamate, respectively. Hence, we could cocrystallize ATBSN with succinate or glutarate, and the structures show how the enzyme can bind two kinds of dicarboxylic substrates with different chain lengths. The present results may also provide an insight into the long-standing controversies regarding the mode of binding of glutamate to the wild-type enzyme.

Aspartate Aminotransferases↗
PubMed1994

X-ray crystallographic study of pyridoxamine 5'-phosphate-type aspartate aminotransferases from Escherichia coli in three forms.

The three-dimensional structures of pyridoxamine 5'-phosphate-type aspartate aminotransferase from Escherichia coli and its complexes with maleate and glutarate have been determined by X-ray crystallography at 2.2, 2.1, and 2.7 A resolution, respectively. The enzyme is a dimeric form comprising two identical subunits, each of which is divided into one large and one small domain. The complex with maleate showed that substrate (or inhibitor) binding induced a large conformational change from the "open" to the "closed" form, resulting in closure of the active site by the small domain movement, as was observed in the pyridoxal 5'-phosphate-type enzyme. In the open form, three hydrophobic residues (hydrophobic plug) at the entrance of the active site are exposed to solvent. Maleate binding make the active site more hydrophobic by charge compensation and release of water molecules, facilitating the movement of the hydrophobic plug into the active site pocket to induce a large conformational change in the enzyme. Maleate is fixed rigidly in the active site pocket by extensive salt bridges and a hydrogen bonding network, guaranteeing the stereo-specificity of the catalysis and giving a Michaelis complex model. Contrary to our expectation, the glutarate complex was in the open form, suggesting that the equilibrium between the open and closed forms lies far toward the open form in solution. The water molecules located in the active site pocket were almost completely conserved between Escherichia coli and chicken mitochondrial aspartate aminotransferase with the same type of cofactor and the same conformation.

Aspartate Aminotransferases↗
PubMed1999

Role of the calcium/calmodulin-dependent protein kinase II in the regulation of the renal basolateral PAH and dicarboxylate transporters.

The aim of the present study was to investigate whether the activities of the renal basolateral organic anion transporter (PAH transporter) and the sodium-dependent dicarboxylate transporter are modulated by the calcium/calmodulin-dependent multifunctional protein kinase II (CaM kinase II). The studies were performed on isolated S2 segments of proximal tubules microdissected from rabbit kidneys without the use of enzymatic agents. 3H-PAH was used as marker substance of the anion transporter, and 14C-glutarate as a marker of the sodium/dicarboxylate cotransporter. Because the tubules were not perfused, and hence were collapsed, the tubular uptake of the marker substances reflects transport across the basolateral cell membrane. To obtain uptake rates most closely related to initial transport rates, 30 s tubular uptake measurements were performed. The results show that a selective inhibitor of CaM kinase II, KN93, inhibited tubular PAH uptake. The smallest effective dose was 10(-7) M. An inactive analogue of KN93, KN92, was without effect, even at the high concentration of 10(-5) M. In contrast to PAH transport, tubular 14C-glutarate uptake was not affected by KN93 (10(-5) M). PAH transport was also inhibited after elevation of intracellular Ca2+ by the Ca(2+)-ionophore A 23187 and by the polycationic antibiotic neomycin, but not by the intracellular Ca2+ modulators thapsigargin and ryanodine. The effect of the Ca(2+)-ionophore could be abolished by KN93, but not by Rp-cAMPs, an inhibitor of protein kinase A, indicating that this event was mediated by CaM kinase II, but not by PKA. The results provide the first evidence that, in addition to the protein kinases A and C (previous studies from this lab), CaM kinase II has a role in the regulation of the renal basolateral PAH transporter, whereas the renal basolateral dicarboxylate transporter does not depend on CaM kinase II activity.

Animals↗