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Eduard A Struys

Publications and source records attributed to Eduard A Struys.

17 recordsLinked to original sources
PubMed2006

D-2-Hydroxyglutaric aciduria: unravelling the biochemical pathway and the genetic defect.

D-2-Hydroxyglutaric aciduria (D-2-HGA) is a neurometabolic inherited disorder first described in 1980. In the following years, it became clear that the clinical phenotype of the disease varies widely from severe neonatal to asymptomatic. However, the sparse biochemical knowledge made D-2-HGA a poorly understood disease. Much progress has been made in the last five years in various studies, revealing two human enzymes that play a role in the metabolism of D-2-hydroxyglutarate (D-2-HG): hydroxyacid-oxoacid transhydrogenase (HOT) and D-2-HG dehydrogenase. HOT is expected to be responsible for the formation of D-2-HG, while D-2-HG dehydrogenase converts D-2-HG into 2-ketoglutarate. We demonstrated pathogenic mutations in the D2HGD gene in patients with D-2-HGA, helping to unravel the primary defect causing D-2-HGA. However, in approximately 50% of the patients with D-2-HGA examined, no pathogenic mutations have yet been found.

Alcohol Oxidoreductases↗
PubMed2006

Metabolism of gamma-hydroxybutyrate to d-2-hydroxyglutarate in mammals: further evidence for d-2-hydroxyglutarate transhydrogenase.

gamma-Hydroxybutyratic acid (GHB), and its prodrugs 4-butyrolactone and 1,4-butanediol, represent expanding drugs of abuse, although GHB is also used therapeutically to treat narcolepsy and alcoholism. Thus, the pathway by which GHB is metabolized is of importance. The goal of the current study was to examine GHB metabolism in mice with targeted ablation of the GABA degradative enzyme succinic semialdehyde dehydrogenase (SSADH(-/-) mice), in whom GHB persistently accumulates, and in baboons intragastrically administered with GHB immediately and persistently. Three hypotheses concerning GHB metabolism were tested: (1) degradation via mitochondrial fatty acid beta-oxidation; (2) conversion to 4,5-dihydroxyhexanoic acid (a putative condensation product of the GHB derivative succinic semialdehyde); and (3) conversion to d-2-hydroxyglutaric acid (d-2-HG) catalyzed by d-2-hydroxyglutarate transhydrogenase (a reaction previously documented only in rat). Both d-2-HG and 4,5-dihydroxyhexanoic acid were significantly increased in neural and nonneural tissue extracts derived from SSADH(-/-) mice. In vitro studies demonstrated the ability of 4,5-dihydroxyhexanoic acid to displace the GHB receptor ligand NCS-382 (IC(50) = 38 micromol/L), although not affecting GABA(B) receptor binding. Blood and urine derived from baboons administered with GHB also accumulated d-2-HG, but not 4,5-dihydroxyhexanoic acid. Our results indicate that d-2-HG is a prominent GHB metabolite and provide further evidence for the existence of d-2-hydroxyglutarate transhydrogenase in different mammalian species.

Alcohol Oxidoreductases↗
PubMed2005

Phenotypic heterogeneity in the presentation of D-2-hydroxyglutaric aciduria in monozygotic twins.

D-2-hydroxyglutaric aciduria (D-2-HGA) is a very rare autosomal recessive metabolic disorder that has recently been associated with mutations in the D-2-hydroxyglutarate dehydrogenase gene. The biochemical phenotype of D-2-HGA is defined by the accumulation of abnormal amounts of D-2-hydroxyglutarate in cerebrospinal fluid, blood, and urine while the clinical phenotype can vary from a severe epileptic encephalopathy to normal. The basis for this phenotypic variation is not well-defined. We report a set of 412-year-old monozygotic (MZ) female twins with D-2-hydroxyglutaric aciduria who are shown to be compound heterozygotes for c.326-327dupTC, p.Glu110ArgfsX19, and c.1123G-->T, p.Asp375Tyr mutations in the D-2-hydroxyglutarate dehydrogenase gene, but who have remarkably different clinical phenotypes. One twin presented with multiple congenital anomalies, severe developmental delay, and abnormal neuroradiological findings, while the other had normal neurocognitive and neuroradiological phenotypes, without concomitant congenital abnormalities. Monozygosity of these twins implies that the differences in the clinical phenotype arise from postzygotic genetic changes, epigenetic differences, or environmental factors that influence the phenotypic response to biochemical perturbation rather than allelic or locus heterogeneity. Though the mechanistic role of these factors in D-2-HGA is far from apparent, the discordance in the phenotypes of these siblings establishes that these factors are at least as important as the nature of the mutant alleles in influencing the progression of the disorder.

Base Sequence↗
PubMed2005

Intracellular S-adenosylhomocysteine increased levels are associated with DNA hypomethylation in HUVEC.

Hyperhomocysteinemia is a risk factor for atherosclerosis and vascular disease; however, the mechanism underlying this association remains poorly understood. Increased levels of intracellular S-adenosylhomocysteine (AdoHcy), secondary to homocysteine-mediated reversal of the AdoHcy hydrolase reaction, have been associated with reduced DNA methylation patterns and pointed as responsible for the hyperhomocysteinemia-related endothelial dysfunction. Methylation is an epigenetic feature of genomic DNA, which leads to alterations in gene expression. So far, the effect of intracellular AdoHcy accumulation on DNA methylation patterns has not yet been fully substantiated by experimental evidence. The present study was designed to evaluate, in cultured endothelial cells, the effect of AdoHcy accumulation on genomic global DNA methylation status. Experimental intracellular accumulation of AdoHcy was induced by adenosine-2,3-dialdehyde (ADA), an inhibitor of AdoHcy hydrolase. Increased concentrations of inhibitor were tested, and unsupplemented medium incubations were used as controls. Cytosolic and nuclear fractions were obtained from trypsinized cells after 72 h of incubation. Total homocysteine concentration was quantified (culture medium and cytosolic fractions) by high-performance liquid chromatography (HPLC). S-Adenosylmethionine and AdoHcy concentrations were measured (cytosolic fractions) by stable-isotope dilution LC-tandem mass spectrometry method. Genomic DNA was obtained from the nuclear fraction, and global DNA methylation status was evaluated by the cytosine extension assay. The results showed that supplementation of the culture medium with ADA had no cytotoxic effect and increased the intracellular AdoHcy concentration in a dose-dependent manner. A significant negative correlation was observed between intracellular AdoHcy and genomic DNA methylation status. These findings strongly point to the importance of AdoHcy as a pivotal biomarker of genomic DNA methylation status.

Adenosine↗
PubMed2005

Stable-isotope dilution liquid chromatography-electrospray injection tandem mass spectrometry method for fast, selective measurement of S-adenosylmethionine and S-adenosylhomocysteine in plasma.

BACKGROUND: It has been postulated that changes in S-adenosylhomocysteine (AdoHcy), a potent inhibitor of transmethylation, provide a mechanism by which increased homocysteine causes its detrimental effects. We aimed to develop a rapid and sensitive method to measure AdoHcy and its precursor S-adenosylmethionine (AdoMet). METHODS: We used stable-isotope dilution liquid chromatography-electrospray injection tandem mass spectrometry (LC-ESI-MS/MS) to measure AdoMet and AdoHcy in plasma. Acetic acid was added to prevent AdoMet degradation. Solid-phase extraction (SPE) columns containing phenylboronic acid were used to bind AdoMet, AdoHcy, and their internal standards and for sample cleanup. An HPLC C(18) column directly coupled to the LC-MS/MS was used for separation and detection. RESULTS: In plasma samples, the interassay CVs for AdoMet and AdoHcy were 3.9% and 8.3%, and the intraassay CVs were 4.2% and 6.7%, respectively. Mean recoveries were 94.5% for AdoMet and 96.8% for AdoHcy. The quantification limits were 2.0 and 1.0 nmol/L for AdoMet and AdoHcy, respectively. Immediate acidification of the plasma samples with acetic acid prevented the observed AdoMet degradation. In a group of controls (mean plasma total Hcy, 11.2 mumol/L), plasma AdoMet and AdoHcy were 94.5 and 12.3 nmol/L, respectively. CONCLUSIONS: Stable-isotope dilution LC-ESI-MS/MS allows sensitive and rapid measurement of AdoMet and AdoHcy. The SPE columns enable simple cleanup, and no metabolite derivatization is needed. The instability of AdoMet is a serious problem and can be prevented easily by immediate acidification of samples.

Chromatography, Liquid↗
PubMed2005

Quantification of sugar phosphate intermediates of the pentose phosphate pathway by LC-MS/MS: application to two new inherited defects of metabolism.

We describe a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to quantify pentose phosphate pathway intermediates (triose-3-phosphates, tetrose-4-phosphate, pentose-5-phosphate, pentulose-5-phosphates, hexose-6-phosphates and sedoheptulose-7-phosphate (sed-7P)) in bloodspots, fibroblasts and lymphoblasts. Liquid chromatography was performed using an ion pair loaded C(18) HPLC column and detection of the sugar phosphates was carried out by tandem mass spectrometry using an electron ion spray source operating in the negative mode and multiple reaction monitoring. Reference values for the pentose phosphate pathway intermediates in blood spots, fibroblasts and lymphoblasts were established. The method was applied to cells from patients affected with a deficiency of transaldolase. The transaldolase-deficient cells showed an increased concentration of sedoheptulose-7-phosphate. (Bloodspots: 5.19 and 5.43 micromol/L [0.49-3.33 micromol/L]; fibroblasts 7.43 and 26.46 micromol/mg protein [0.31-1.14 micromol/mg protein]; lymphoblasts 16.03 micromol/mg protein [0.61-2.09 micromol/mg protein].) The method was also applied to study enzymes of the pentose phosphate pathway by incubating fibroblasts or lymphoblasts homogenates with ribose-5-phosphate or 6-phosphogluconate and the subsequent analysis of the formed sugar phosphates.

Cells, Cultured↗
PubMed2005

Mutations in phenotypically mild D-2-hydroxyglutaric aciduria.

D-2-hydroxyglutaric aciduria is a neurometabolic disorder with mild and severe phenotypes. Recently, we reported pathogenic mutations in the D-2-hydroxyglutarate dehydrogenase gene as the cause of the severe phenotype of D-2-hydroxyglutaric aciduria in two patients. Here, we report two novel pathogenic mutations in this gene in one patient with a mild presentation and two asymptomatic siblings with D-2-hydroxyglutaric aciduria from two unrelated consanguineous Palestinian families: a splice error (IVS4-2A-->G) and a missense mutation (c.1315A-->G;p.Asn439Asp). Overexpression of this mutant protein showed marked reduction of the enzyme activity.

Alcohol Oxidoreductases↗
PubMed2005

Kinetics of homocysteine metabolism after moderate alcohol consumption.

BACKGROUND: Moderate alcohol consumption is associated with a decreased risk of cardiovascular disease. Because plasma homocysteine (tHcy) is considered an independent risk factor for cardiovascular disease and associated with alcohol consumption, the authors investigated the effect of moderate alcohol consumption on kinetics of plasma tHcy concentration, vitamin B status, and other parameters involved in tHcy metabolism. METHODS: Ten healthy men and nine healthy postmenopausal women (aged 45-65 years) participated in a randomized, diet-controlled, crossover trial. They consumed beer or alcohol-free beer (men: 4 units/day; women: 3 units/day) during 3 weeks, separated by a 1-week washout. On days 5, 10, 15, and 20 of each period, fasting blood samples were taken. RESULTS: Plasma tHcy (microM) and S-adenosyl methionine/S-adenosyl homocysteine ratio were not affected by consumption of beer or alcohol-free beer (p = 0.33 and p = 0.14, respectively). Plasma pyridoxal-5-phosphate (microg/liter) increased during consumption of beer (+11.0%), whereas it decreased during consumption of alcohol-free beer (-34.0%; p = 0.042). Changes over time of plasma vitamin B6 (microg/liter) were similar to changes in plasma pyridoxal-5-phosphate (p = 0.10). Serum vitamin B12 was higher (p < 0.001) after 3 weeks consumption of alcohol-free beer (382.8 +/- 23.7 pg/liter) as compared with beer consumption (327.5 +/- 22.2 pg/liter). Changes in serum methionine, cysteine, cystathionine, and plasma folate were not different between beer-drinking and alcohol-free beer-drinking periods. CONCLUSIONS: This study shows that moderate alcohol consumption does not affect plasma tHcy concentrations or S-adenosyl methionine/S-adenosyl homocysteine ratio. However, it does increase plasma vitamin B6 and decrease serum vitamin B12.

Aged↗
PubMed2004

Mutations in the D-2-hydroxyglutarate dehydrogenase gene cause D-2-hydroxyglutaric aciduria.

d-2-hydroxyglutaric aciduria is a neurometabolic disorder with both a mild and a severe phenotype and with unknown etiology. Recently, a novel enzyme, d-2-hydroxyglutarate dehydrogenase, which converts d-2-hydroxyglutarate into 2-ketoglutarate, and its gene were identified. In the genes of two unrelated patients affected with d-2-hydroxyglutaric aciduria, we identified disease-causing mutations. One patient was homozygous for a missense mutation (c.1331T-->C; p.Val444Ala). The other patient was compound heterozygous for a missense mutation (c.440T-->G; p.Ile147Ser) and a splice-site mutation (IVS1-23A-->G) that resulted in a null allele. Overexpression studies in HEK-293 cells of proteins containing the missense mutations showed a marked reduction of d-2-hydroxyglutarate dehydrogenase activity, proving that mutations in the d-2-hydroxyglutarate dehydrogenase gene cause d-2-hydroxyglutaric aciduria.

Alcohol Oxidoreductases↗
PubMed2004

Measurement of urinary D- and L-2-hydroxyglutarate enantiomers by stable-isotope-dilution liquid chromatography-tandem mass spectrometry after derivatization with diacetyl-L-tartaric anhydride.

BACKGROUND: The differential diagnosis of D-2-hydroxyglutaric aciduria (D-2-HGA), L-2-hydroxyglutaric aciduria (L-2-HGA), and the combined D/L-2-hydroxyglutaric aciduria (D/L-2-HGA) can be accomplished only by the measurement of the corresponding 2-hydroxyglutarate (2-HG). Available methods for the determination of D- and L-2-HG in urine are either time-consuming and expensive or have not been extensively validated. We aimed to develop a method for their rapid and sensitive measurement. METHODS: We used liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the determination of D- and L-2-HG with stable-isotope-labeled internal standards. Urine samples of 20 microL were mixed with 250 microL of methanol containing the internal standards and subsequently dried under nitrogen. The analytes were derivatized by use of diacetyl-L-tartaric anhydride (DATAN) to obtain diastereomers, which were separated on an achiral C18 HPLC column and detected by MS/MS in multiple-reaction-monitoring mode. RESULTS: The use of DATAN as chiral derivatization reagent provided very well separated peaks of the formed diastereomers of D- and L-2-HG, with a total runtime of 5 min. The inter- and intraassay CVs for D- and L-2-HG ranged from 3.4% to 6.2%. Mean recoveries of D- and L-2-HG, evaluated on two concentrations, were 94%. Detection limit of the presented method was 20 pmol for a sample volume of 20 microL. Method comparison of the LC-MS/MS method with a gas chromatography-mass spectrometry method, in which D- and L-2-HG were derivatized with R-(-)-butanol, showed good agreement between the two methods. CONCLUSIONS: Urinary D- and L-2-HG can be analyzed by MS/MS after derivatization with DATAN. The presented method may be suitable for the differential diagnosis of 2-HGA.

Anhydrides↗
PubMed2004

Ribose-5-phosphate isomerase deficiency: new inborn error in the pentose phosphate pathway associated with a slowly progressive leukoencephalopathy.

The present article describes the first patient with a deficiency of ribose-5-phosphate isomerase (RPI) (Enzyme Commission number 5.3.1.6) who presented with leukoencephalopathy and peripheral neuropathy. Proton magnetic resonance spectroscopy of the brain revealed highly elevated levels of the polyols ribitol and D-arabitol, which were subsequently also found in high concentrations in body fluids. Deficient activity of RPI, one of the pentose-phosphate-pathway (PPP) enzymes, was demonstrated in fibroblasts. RPI gene-sequence analysis revealed a frameshift and a missense mutation. Recently, we described a patient with liver cirrhosis and abnormal polyol levels in body fluids, related to a deficiency of transaldolase, another enzyme in the PPP. RPI is the second known inborn error in the reversible phase of the PPP, confirming that defects in pentose and polyol metabolism constitute a new area of inborn metabolic disorders.

Aldose-Ketose Isomerases↗
PubMed2004

Investigations by mass isotopomer analysis of the formation of D-2-hydroxyglutarate by cultured lymphoblasts from two patients with D-2-hydroxyglutaric aciduria.

D-2-Hydroxyglutaric aciduria is an inborn error of metabolism first described in 1980. To date, more than 40 patients have been diagnosed with this disease. To identify the metabolic precursor of D-2-hydroxyglutarate (D-2-HG), cultured human lymphoblasts from two patients with D-2-HG aciduria were grown in culture medium supplemented with [U-(13)C(6)]glucose or [(2)H(5)]glutamate. Mass isotopomer distribution measurements of D-2-HG, 2-ketoglutarate (2-KG) and citrate were performed by gas chromatography-mass spectrometry. The mass isotopomer distributions in D-2-HG, 2-KG and citrate, following [U-(13)C(6)]glucose and [(2)H(5)]glutamate incubations, revealed that 2-KG interconverts rapidly to D-2-HG and that D-2-HG is formed within the mitochondria.

Cell Line↗
PubMed2003

Disease-related metabolites in culture medium of fibroblasts from patients with D-2-hydroxyglutaric aciduria, L-2-hydroxyglutaric aciduria, and combined D/L-2-hydroxyglutaric aciduria.

BACKGROUND: D-2-Hydroxyglutaric aciduria (D-2-HGA), L-2-hydroxyglutaric aciduria (L-2-HGA), and the combined D/L-2-hydroxyglutaric aciduria (D/L-2-HGA) are poorly understood organic acidurias. To investigate the usefulness of cultured human skin fibroblasts for both diagnostic and research purposes, we measured disease-related metabolites in the cell culture medium. METHODS: We measured D-2-hydroxyglutarate (D-2-HG), L-2-hydroxyglutarate (L-2-HG), succinate, 2-ketoglutarate, and citrate in fibroblast cell medium by stable-isotope-dilution gas chromatography-mass spectrometry and glutamine, glutamic acid, and lysine with an amino acid analyzer. We used six cell lines from patients with D-2-HGA, two from patients with L-2-HGA, three from patients with D/L-2-HGA, and seven control cell lines. Culture medium was analyzed after a 96-h incubation period. RESULTS: Culture media from cell lines from D-2-HGA patients contained D-2-HG at concentrations 5- to 30-fold higher than media from controls, whereas the concentration of L-2-HG in media was not increased. Media from L-2-HGA cell lines showed a fivefold increase in L-2-HG compared with controls. Media containing fibroblasts from D/L-2-HGA patients contained moderately increased amounts of both D-2-HG and L-2-HG. For all cell lines, succinate concentrations in the blank medium were higher than after 96 h of incubation with the exception of two of three D/L-2-HGA cell lines. Media of D-2-HGA cell lines had 2-ketoglutarate concentrations that were 40% of that for controls. Glutamic acid concentrations in media of these cell lines were 60% lower than in controls. CONCLUSIONS: Cell culture media from fibroblasts from patients with D-2-HGA, L-2-HGA, or D/L-2-HGA contain increased amounts the corresponding 2-HGs, demonstrating the suitability of fibroblasts for both diagnosis of and research concerning 2-HGAs.

Biomarkers↗
PubMed2003

Increased homocysteine and S-adenosylhomocysteine concentrations and DNA hypomethylation in vascular disease.

BACKGROUND: The pathogenic mechanism of homocysteine's effect on cardiovascular risk is poorly understood. Recent studies show that DNA hypomethylation induced by increases in S-adenosylhomocysteine (AdoHcy), an intermediate of Hcy metabolism and a potent inhibitor of methyltransferases, may be involved in homocysteine-related pathology. METHODS: We measured fasting plasma total Hcy (tHcy), AdoHcy, and S-adenosylmethionine (AdoMet) and methylation in leukocytes in 17 patients with vascular disease and in 15 healthy, age- and sex-matched controls. RESULTS: Patient with vascular disease had significantly higher plasma tHcy and AdoHcy concentrations and significantly lower plasma AdoMet/AdoHcy ratios and genomic DNA methylation. AdoMet concentrations were not significantly different between the two groups. More than 50% of the patients fell into the highest quartiles of plasma tHcy, AdoHcy, and [(3)H]dCTP incorporation/ micro g of DNA (meaning the lowest quartile of DNA methylation status) and into the lowest quartile of the AdoMet/AdoHcy ratios of the control group. Plasma tHcy was significantly correlated with plasma AdoHcy and AdoMet/AdoHcy ratios (n = 32; P < 0.001). DNA methylation status was significantly correlated with plasma tHcy and AdoHcy (n = 32; P < 0.01) but not with plasma AdoMet/AdoHcy ratios. CONCLUSION: Global DNA methylation may be altered in vascular disease, with a concomitant increase in plasma tHcy and AdoHcy.

Cardiovascular Diseases↗
PubMed2003

Profiling of pentose phosphate pathway intermediates in blood spots by tandem mass spectrometry: application to transaldolase deficiency.

BACKGROUND: Recently, several patients with abnormal polyol profiles in body fluids have been reported, but the origins of these polyols are unknown. We hypothesized that they are derived from sugar phosphate intermediates of the pentose phosphate pathway (PPP), and we developed a semiquantitative method for profiling of pentose phosphate pathway intermediates. METHODS: Sugar phosphates in blood spots were simultaneously analyzed by liquid chromatography-tandem mass spectrometry using an ion-pair-loaded C(18) HPLC column. The tandem mass spectrometer was operated in the multiple-reaction monitoring mode. Enzymatically prepared D-[(13)C(6)]glucose 6-phosphate was used as internal standard. The method was used to study sugar phosphates abnormalities in a patient affected with a deficiency of transaldolase (TALDO1; EC 2.2.1.2). RESULTS: In control blood spots, dihydroxyacetone phosphate, pentulose 5-phosphates, pentose 5-phosphates, hexose 6-phosphates, and sedoheptulose 7-phosphate were detected. Detection limits ranged from approximately 100 to approximately 500 nmol/L. Glyceraldehyde 3-phosphate and erythrose 4-phosphate were undetectable. Intra- and interassay imprecision (CVs) were 10-17% and 12-21%, respectively. In blood from the TALDO1-deficient patient, sedoheptulose 7-phosphate was increased. CONCLUSIONS: The new method allows investigation of patients in whom a defect in the PPP is suspected. Measurements of sugar phosphate intermediates of the PPP may provide new insights into metabolic defects underlying the accumulating polyols.

Adolescent↗
PubMed2002

Quantification of plasma S-adenosylmethionine and S-adenosylhomocysteine as their fluorescent 1,N(6)-etheno derivatives: an adaptation of previously described methodology.

A simplified reversed phase HPLC system for the detection of fluorescent 1,N(6)-etheno derivatives of SAM (S-adenosylmethionine) and S-adenosylhomocysteine (SAH) is described. The most important changes from the previously reported method are a shorter derivatization reaction time, the use of a solid-phase extraction resulting in an increase of the method's sensitivity, and the use of only one chromatographic system to separate SAM and SAH (in which the use of an ion-pairing reagent in the mobile phase is avoided). The linearity of the method was established, and the intra-assay coefficients of variation were 10.4 and 4.7% for SAM and SAH, respectively. Normal plasma values (n=8), evaluated with the present methodology, were, for SAM and SAH, respectively, 57+/-12 and 28+/-3 nM (mean+/-SD).

Chromatography, High Pressure Liquid↗