Measurement of D: -2-hydroxyglutarate dehydrogenase activity in cell homogenates derived from D: -2-hydroxyglutaric aciduria patients.

ABSTRACT

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-(2)H(4)]glutarate. This substrate is convertedby D -2-hydroxyglutarate dehydrogenase into 2-[3,3,4,4-(2)H(4)]ketoglutarate, which is subsequently converted into L -[3,3,4,4-(2)H(4)]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.