Gas chromatographic-mass spectrometric analysis of hexose monophosphate shunt activity in cultured cells

A GC/MS method is described for monitoring the relative amount of glucose degraded to lactate via the hexose monophosphate shunt (HMPS) in neoplastic cells. C6 glioma cells were incubated in medium supplemented with [1-13 C]glucose and medium containing [1-13C]glucose with 0.001 mM phenazine methosulfate (PMS). The ratio of the [13C]lactate to [12C]lactate determined from the measurement of the m/z 219/220 and 117/118 ions of the trimethyl silyl derivative, was used to calculate HMPS activity. PMS increased HMPS activity in C6 glioma cells by 3.2 and 4.8 fold at 2 and 12 hours of incubation respectively. GC/MS results were compared with 1H NMR measurements of the [3-13C]lactate/[3-12C]lactate ratio. The GC/MS method was found to require less sample size and yielded better sensitivity than the NMR method.