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

Summary 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.