TCA cycle kinetics in the rat heart by analysis of 13C isotopomers using indirect 1H[13C] detection

This study was designed to test the hypothesis that indirect H-1 [C-13] detection of tricarboxylic acid (TCA) cycle intermediates using heteronuclear multiple quantum correlation-total correlation spectroscopy (HMQC-TOCSY) nuclear magnetic resonance (NMR) spectroscopy provides additional C-13 isotopomer information that better describes the kinetic exchanges that occur between intracellular compartments than direct C-13 NMR detection. NMR data were collected on extracts of rat hearts perfused at various times with combinations of [2-C-13]acetate, propionate, the transaminase inhibitor aminooxyacetate, and 13C multiplet areas derived from spectra of tissue glutamate were fit to a standard kinetic model of the TCA cycle. Although the two NMR methods detect different populations of 13C isotopomers, similar values were found for TCA cycle and exchange fluxes by analyzing the two data sets. Perfusion of hearts with unlabeled propionate in addition to [2-C-13]acetate resulted in an increase in the pool size of all four-carbon TCA cycle intermediates. This allowed the addition of isotopomer data from aspartate and malate in addition to the more abundant glutamate. This study illustrates that metabolic inhibitors can provide new insights into metabolic transport processes in intact tissues.