Premium
1 H NMR and hyperpolarized 13 C NMR assays of pyruvate–lactate: a comparative study
Author(s) -
Hill Deborah K.,
Jamin Yann,
Orton Matthew R.,
Tardif Nicolas,
Parkes Harold G.,
Robinson Simon P.,
Leach Martin O.,
Chung YuenLi,
Eykyn Thomas R.
Publication year - 2013
Publication title -
nmr in biomedicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.278
H-Index - 114
eISSN - 1099-1492
pISSN - 0952-3480
DOI - 10.1002/nbm.2957
Subject(s) - chemistry , lactate dehydrogenase , kinetics , lactate dehydrogenase a , enzyme kinetics , in vitro , analytical chemistry (journal) , enzyme , biochemistry , chromatography , active site , physics , quantum mechanics
Pyruvate–lactate exchange is mediated by the enzyme lactate dehydrogenase (LDH) and is central to the altered energy metabolism in cancer cells. The measurement of exchange kinetics using hyperpolarized 13 C NMR has provided a biomarker of response to novel therapeutics. However, the observable signal is restricted to the exchanging hyperpolarized 13 C pools and the endogenous pools of 12 C‐labelled metabolites are invisible in these measurements. In this study, we investigated an alternative in vitro 1 H NMR assay, using [3‐ 13 C]pyruvate, and compared the measured kinetics with a hyperpolarized 13 C NMR assay, using [1‐ 13 C]pyruvate, under the same conditions in human colorectal carcinoma SW1222 cells. The apparent forward reaction rate constants ( k PL ) derived from the two assays showed no significant difference, and both assays had similar reproducibility ( k PL = 0.506 ± 0.054 and k PL = 0.441 ± 0.090 nmol/s/10 6 cells; mean ± standard deviation; n = 3); 1 H, 13 C assays, respectively). The apparent backward reaction rate constant ( k LP ) could only be measured with good reproducibility using the 1 H NMR assay ( k LP = 0.376 ± 0.091 nmol/s/10 6 cells; mean ± standard deviation; n = 3). The 1 H NMR assay has adequate sensitivity to measure real‐time pyruvate–lactate exchange kinetics in vitro , offering a complementary and accessible assay of apparent LDH activity. Copyright © 2013 John Wiley & Sons, Ltd.