A fast and sensitive 1H NMR method to measure the turnover of the H2 hydrogen of lactate

A fast and sensitive procedure to determine the turnover of the H2 hydrogen of lactate and quantify its H-2-enrichment by H-1 NMR is illustrated using C6 cells metabolizing (3-C-13) lactate in 50% (H2O)-H-2 (Vol/Vol). H-2 substitution of the lactate H2 hydrogen resulted in two easily detectable transformations of the vicinal H3 doublet resonance: 1) the formation of an H3 singlet due to the disappearance of the homonuclear coupling to H2 ((3)j(beta H-alpha H) = 7.0 Hz), and 2) an upfield isotopic shift derived from the vicinal 2 H2 substitution (Delta(3) = -0.007 ppm). Only those lactate molecules that have passed through the cell cytosol experience these effects, since H2 deuteration involves lactate dehydrogenase activity and NAD(H-2). Thus, analysis of the observed shifted and unshifted H3 lactate resonances from the incubation medium allows the discrimination of the perprotonated (3-C-13) lactate added as substrate, and the (3-C-13, 2-H-2) lactate recycled to the incubation medium after passage through the cytosol.