Metabolite extraction was performed as described earlier (35 (link)), with some modifications. After confirming 80% confluence of the cells, we replaced the media with fresh media for 2 h before metabolite extraction. Media was aspirated and the cells were washed twice with PBS to remove residual media before lysing the cells. The polar metabolites were then extracted with cryogenically cold 80% methanol/water mixture. LC/MS grade water (Fisher Scientific, Pittsburgh, PA, USA) and LC/MS grade methanol (Fisher Scientific, Pittsburgh, PA, USA) were utilized. The cells from the cold plates were scraped with a cell scraper, pipetted into an Eppendorf tube, and centrifuged at 13,000 rpm for 5 min to collect the supernatant. Samples were dried using a speed vacuum evaporator (Savant Speed Vac® Plus, Thermo Electron Corporation, USA) to evaporate the methanol and lyophilized using freeze dry system (Labconco, Kansas City, USA) to remove water. The dried sample was then prepared for mass spectrometry by dissolving in LC/MS grade water. Liquid chromatography/tandem mass spectrometry (LC-MS/MS) was performed as described below.
As a separate measure of D-2 -hydroxyglutarate (D2-HG) levels, the metabolite was independently measured using colorimetric method (BioVision; K213–100), as per the manufacturer’s instructions.
As a separate measure of D-