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

✍ Scribed by Tiago B. Rodrigues; Sebastián Cerdán

• Publisher: John Wiley and Sons
• Year: 2005
• Tongue: English
• Weight: 309 KB
• Volume: 54
• Category: Article
• ISSN: 0740-3194
• DOI: 10.1002/mrm.20620

✦ Synopsis

Abstract

A fast and sensitive procedure to determine the turnover of the H2 hydrogen of lactate and quantify its ^2^H‐enrichment by ^1^H NMR is illustrated using C6 cells metabolizing (3‐^13^C) lactate in 50% ^2^H~2~O (vol/vol). ^2^H 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~βH‐αH~ = 7.0 Hz), and 2) an upfield isotopic shift derived from the vicinal ^2^H2 substitution (Δ~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(^2^H). Thus, analysis of the observed shifted and unshifted H3 lactate resonances from the incubation medium allows the discrimination of the perprotonated (3‐^13^C) lactate added as substrate, and the (3‐^13^C, 2‐^2^H) lactate recycled to the incubation medium after passage through the cytosol. Magn Reson Med, 2005. © 2005 Wiley‐Liss, Inc.