Measurement of d-2-hydroxyglutarate dehydrogenase activity in cell homogenates derived from d-2-hydroxyglutaric aciduria patients

d-2-Hydroxyglutaric aciduria (d-2-HGA) is a neurometabolic disorder characterized by elevated levels of d-2-hydroxyglutarate (d-2-HG) in physiological fluids. Recent findings revealed that mutations in the D2HGDH gene, encoding d-2-hydroxyglutarate dehydrogenase, cause d-2-HGA. So far, a functionalenzyme assay to determine d-2-hydroxyglutarate dehydrogenase activity, converting d-2-HG into 2-ketoglutarate (2-KG), has been unavailable. We have now developed a unique enzyme assay for the determination of d-2-hydroxyglutarate dehydrogenase activity in cells derived from d-2-HGA patients and controls. The enzyme assay was performed using enantiomerically pure stable-isotope-labelled d-2-hydroxy[3,3,4,4-²H₄]glutarate. This substrate is convertedby d-2-hydroxyglutarate dehydrogenase into 2-[3,3,4,4-²H₄]ketoglutarate, which is subsequently converted into l-[3,3,4,4-²H₄]glutamate by l-glutamate dehydrogenase, present in saturating amounts in cell homogenates. Enzyme activities were quantified using LC-MS/MS. The mean activities in control fibroblast and lymphoblast homogenates were 298 ± 207 and 1670 ± 940 pmol/h per mg protein, respectively. In fibroblast and lymphoblast cell lines derived from patients with pathogenic mutations in the D2HGDH gene, considerably decreased enzyme activities (e.g. <41 pmol/h per mg protein) were found compared with controls. This enzyme assay will have additional utility in further differentiating patients with d-2-HGA and l-2-HGA and in assessing the residual activities linked to pathogenic mutations in the D2HGDH gene.