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

Abstract

A simple and sensitive NMR method for quantifying excess 13C-enrichment in positions 2 and 3 of lactate by 1H 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 13C-contributions. As a demonstration, the measurement was applied to ∼3 μmol of lactate generated by erythrocyte preparations incubated with [2-13C]glucose to determine the fraction of glucose metabolized by the pentose phosphate pathway (PP). PP fluxes were estimated from the ratio of excess 13C-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 PP activity, its contribution increased to 27 ± 10% of total glucose consumption (P < 0.01). © 2004 Wiley-Liss, Inc.