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

A simple and sensitive NMR method for quantifying excess 13 C‐enrichment in positions 2 and 3 of lactate by 1 H 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 13 C‐contributions. As a demonstration, the measurement was applied to ∼3 μmol of lactate generated by erythrocyte preparations incubated with [2‐ 13 C]glucose to determine the fraction of glucose metabolized by the pentose phosphate pathway (PP). PP fluxes were estimated from the ratio of excess 13 C‐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). Magn Reson Med 51:1283–1286, 2004. © 2004 Wiley‐Liss, Inc.