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

Struys, Eduard A.; Jansen, Erwin E.W.; Verhoeven, Nanda M.; Jakobs, Cornelis

doi:10.1373/clinchem.2004.033399

Cited by 138 publications

(107 citation statements)

References 14 publications

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“…In order to separate alpha-hydroxyacid enantiomers, we adapted a liquid chromatography-mass spectrometry (LC-MS) protocol involving derivatization of metabolites with the chiral compound diacetyl-L-tartaric anhydride (Supplementary Results, Supplementary Fig. 1) 28 . The identity of alpha-hydroxyacid enantiomers was confirmed by comparison to similarly derivatized standards (Fig.…”

Section: Resultsmentioning

confidence: 99%

L-2-Hydroxyglutarate production arises from noncanonical enzyme function at acidic pH

et al. 2017

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The metabolite 2-hydroxyglutarate (2HG) can be produced as either a D(R)- or L(S)- enantiomer, each of which inhibits alpha-ketoglutarate (αKG)-dependent enzymes involved in diverse biologic processes. Oncogenic mutations in isocitrate dehydrogenase produce D-2HG, which causes a pathologic blockade in cell differentiation. On the other hand, oxygen limitation leads to accumulation of L-2HG, which can facilitate physiologic adaptation to hypoxic stress in both normal and malignant cells. Here we demonstrate that purified lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) catalyze stereospecific production of L-2HG via ‘promiscuous’ reduction of the alternative substrate αKG. Acidic pH enhances production of L-2HG by promoting a protonated form of αKG that binds to a key residue in the substrate-binding pocket of LDHA. Acid-enhanced production of L-2HG leads to stabilization of hypoxia-inducible factor 1 alpha (HIF-1α) in normoxia. These findings offer insights into mechanisms whereby microenvironmental factors influence production of metabolites that alter cell fate and function.

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Section: Resultsmentioning

confidence: 99%

L-2-Hydroxyglutarate production arises from noncanonical enzyme function at acidic pH

et al. 2017

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“…Additional laboratory work up including lactic acid, pyruvic acid, mitochondrial respiratory enzyme analysis, CoQ10 levels, neurotransmitter profile (5-methyltetrahydrofolate, 5-hydroxyindoleacetic acid, homovanillic acid, 3-O-methyldopa, neopterin, tetrahydrobiopterin, succinyladenosine and pyridoxal 5 phosphate), karyotype, chromosomal microarray (44K custom Agilent platform), plasma amino acids and acylcarnitine profile was normal. Urine organic acid analysis revealed significant 2-hydroxyglutaric aciduria, which was confirmed to be the D-enantiomer by a LC-MS/MS method that separates D-from the L-enantiomer [1]. Enzyme analysis on skin fibroblast cultures showed severely deficient D-2-hyroxyglutarate dehydrogenase activity.…”

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confidence: 93%

Co-morbidity of Sanfilippo Syndrome type C and d-2-hydroxyglutaric aciduria

et al. 2011

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“…Because of the hydroxyl group on the second carbon, 2HG exists under two enantiomeric configurations (L or D) that can be separated by gas or liquid chromatography after derivatization with another chiral compound and that can therefore be differentially assayed in biological samples using GC-MS or LC-MS methods (2,3). The interest in 2HG increased when it was found to accumulate in urine of patients with suspected inborn errors of metabolism (4,5).…”

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confidence: 99%

Saccharomyces cerevisiae Forms d-2-Hydroxyglutarate and Couples Its Degradation to d-Lactate Formation via a Cytosolic Transhydrogenase

Becker-Kettern

Paczia

Conrotte

et al. 2016

Journal of Biological Chemistry

122

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The D or L form of 2-hydroxyglutarate (2HG) accumulates in certain rare neurometabolic disorders, and high D-2-hydroxyglutarate (D-2HG) levels are also found in several types of cancer. Although 2HG has been detected in Saccharomyces cerevisiae, its metabolism in yeast has remained largely unexplored. Here, we show that S. cerevisiae actively forms the D enantiomer of 2HG. Accordingly, the S. cerevisiae genome encodes two homologs of the human D-2HG dehydrogenase: Dld2, which, as its human homolog, is a mitochondrial protein, and the cytosolic protein Dld3. Intriguingly, we found that a dld3⌬ knock-out strain accumulates millimolar levels of D-2HG, whereas a dld2⌬ knock-out strain displayed only very moderate increases in D-2HG. Recombinant Dld2 and Dld3, both currently annotated as D-lactate dehydrogenases, efficiently oxidized D-2HG to ␣-ketoglutarate. Depletion of D-lactate levels in the dld3⌬, but not in the dld2⌬ mutant, led to the discovery of a new type of enzymatic activity, carried by Dld3, to convert D-2HG to ␣-ketoglutarate, namely an FAD-dependent transhydrogenase activity using pyruvate as a hydrogen acceptor. We also provide evidence that Ser3 and Ser33, which are primarily known for oxidizing 3-phosphoglycerate in the main serine biosynthesis pathway, in addition reduce ␣-ketoglutarate to D-2HG using NADH and represent major intracellular sources of D-2HG in yeast. Based on our observations, we propose that D-2HG is mainly formed and degraded in the cytosol of S. cerevisiae cells in a process that couples D-2HG metabolism to the shuttling of reducing equivalents from cytosolic NADH to the mitochondrial respiratory chain via the D-lactate dehydrogenase Dld1. 2-Hydroxyglutarate (2HG)3 is a 5-carbon dicarboxylic acid that was first detected in human urine in the late 1970s (1).Because of the hydroxyl group on the second carbon, 2HG exists under two enantiomeric configurations (L or D) that can be separated by gas or liquid chromatography after derivatization with another chiral compound and that can therefore be differentially assayed in biological samples using GC-MS or LC-MS methods (2, 3). The interest in 2HG increased when it was found to accumulate in urine of patients with suspected inborn errors of metabolism (4, 5). Most 2-hydroxyglutaric aciduria patients present elevations of either L-2HG or D-2HG in their extracellular fluids, and the clinical phenotype depends on the configuration of the accumulated organic acid. More recently, cases of "combined D,L-hydroxyglutaric aciduria" have been reported (6).2-Hydroxyglutaric acidurias remained enigmatic diseases because neither L-2HG nor D-2HG are intermediates of any known metabolic pathway, and the causal gene deficiencies were only discovered many years after the first patient case reports. It is now established that L-2-hydroxyglutaric aciduria is caused by loss-of-function mutations in the L2HGDH gene, encoding a specific L-2HG dehydrogenase, whereas in many cases D-2-hydroxyglutaric aciduria results from loss-of-function mutations in the D2H...

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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

Cited by 138 publications

References 14 publications

L-2-Hydroxyglutarate production arises from noncanonical enzyme function at acidic pH

L-2-Hydroxyglutarate production arises from noncanonical enzyme function at acidic pH

Co-morbidity of Sanfilippo Syndrome type C and d-2-hydroxyglutaric aciduria

Saccharomyces cerevisiae Forms d-2-Hydroxyglutarate and Couples Its Degradation to d-Lactate Formation via a Cytosolic Transhydrogenase

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