Quantitation of erythrocyte pentose pathway flux with [2-13]Glucose and 1H NMR analysis of the lactate methyl signal

A simple and sensitive NMR method for quantifying excess C-13-enrichment in positions 2 and 3 of lactate by H-1 NMR spectroscopy of the lactate methyl signal is described. The measurement requires neither signal calibrations nor the addition of a standard and accounts for natural abundance C-13-contributions. As a demonstration, the measurement was applied to similar to3 mumol of lactate generated by erythrocyte preparations incubated with [2-C-13]glucose to determine the fraction of glucose metabolized by the pentose phosphate pathway (PP). PIP fluxes were estimated from the ratio of excess C-13-enrichment in lactate carbon 3 relative to carbon 2 in accordance with established metabolic models. Under baseline conditions, PP flux accounted for 7 +/- 2% of glucose consumption while in the presence of methylene blue, a classical activator of PIP activity, its contribution increased to 27 +/- 10% of total glucose consumption (P < 0.01). (C) 2004 Wiley-Liss, Inc.