Article: article from journal or magazin.
Development and implementation of a novel assay for L-2-hydroxyglutarate dehydrogenase (L-2-HGDH) in cell lysates: L-2-HGDH deficiency in 15 patients with L-2-hydroxyglutaric aciduria.
Journal of Inherited Metabolic Disease
L-2-hydroxyglutaric aciduria (L-2-HGA) is a rare inherited autosomal recessive neurometabolic disorder caused by mutations in the gene encoding L-2-hydroxyglutarate dehydrogenase. An assay to evaluate L-2-hydroxyglutarate dehydrogenase (L-2-HGDH) activity in fibroblast, lymphoblast and/or lymphocyte lysates has hitherto been unavailable. We developed an L-2-HGDH enzyme assay in cell lysates based on the conversion of stable-isotope-labelled L-2-hydroxyglutarate to 2-ketoglutarate, which is converted into L-glutamate in situ. The formation of stable isotope labelled L-glutamate is therefore a direct measure of L-2-HGDH activity, and this product is detected by liquid chromatography-tandem mass spectrometry. A deficiency of L-2-HGDH activity was detected in cell lysates from 15 out of 15 L-2-HGA patients. Therefore, this specific assay confirmed the diagnosis unambiguously affirming the relationship between molecular and biochemical observations. Residual activity was detected in cells derived from one L-2-HGA patient. The L-2-HGDH assay will be valuable for examining in vitro riboflavin/FAD therapy to rescue L-2-HGDH activity.
Alcohol Oxidoreductases/analysis, Alcohol Oxidoreductases/cerebrospinal fluid, Animals, Brain Diseases, Metabolic, Inborn/cerebrospinal fluid, Brain Diseases, Metabolic, Inborn/diagnosis, Calibration, Cell Extracts/analysis, Cell Extracts/chemistry, Cells, Cultured, Chromatography, High Pressure Liquid, Chromatography, Liquid/methods, Enzyme Assays/methods, Enzyme Assays/standards, Fibroblasts/chemistry, Fibroblasts/enzymology, Humans, Lymphocytes/chemistry, Lymphocytes/enzymology, Models, Biological, Models, Molecular, Rats, Research Design, Tandem Mass Spectrometry/methods
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