Hexose monophosphate shunt measurement in cultured cells with [1-13C]glucose: correction for endogenous carbon sources using [6-13C] glucose

Hexose monophosphate shunt (HMPS) activity can be measured with 1H nuclear magnetic resonance spectroscopy or gas chromatography--mass spectrometry by monitoring the differential production of 3-13C]lactate and 3-12C]lactate from the degradation of 1-13C]-glucose. Errors in measurement of HMPS activity can arise from unlabeled lactate precursors, by recycling of HMPS products, and by incomplete fractional enrichment of labeled glucose. A method utilizing cultured cells incubated with 1-13C]glucose in parallel with incubations using 6-13C]glucose to correct for all these problems is presented. In cultured rat C6 glioma and 9L gliosarcoma cells, failure to apply this correction results in an approximately twofold overestimation of HMPS activity.