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-(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.

Kinetic characterization of human hydroxyacid-oxoacid transhydrogenase: relevance to D-2-hydroxyglutaric and gamma-hydroxybutyric acidurias.

We investigated the presence of hydroxyacid-oxoacid transhydrogenase (HOT), which catalyses the cofactor-independent conversion of gamma-hydroxybutyrate (GHB) to succinic semialdehyde coupled to reduction of 2-ketoglutarate (2-KG) to D-2-hydroxyglutarate (D-2-HG), in human liver extracts employing [2H6]GHB and 2-KG as substrates. We measured incorporation of 2H in D-[2H]2-HG using GC-MS analyses, providing evidence for HOT activity in humans. Kinetic characterization of HOT was undertaken in forward and reverse directions. We employed [2H6]GHB and [2H4]2-KG as cosubstrates in order to develop a HOT activity assay in cultured human fibroblasts derived from patients with D-2-hydroxyglutaric aciduria. HOT activity was quantified in this system by the measurement of D-[2H5]2-HG production. Fibroblasts derived from patients with D-2-hydroxyglutaric aciduria showed normal HOT activities. Our results provide the first demonstration and preliminary kinetic characterization of HOT activity in human tissues.