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

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.