Carbon flux through citric acid cycle pathways in perfused heart by 13C NMR spectroscopy

AbstractMathematical models of the TCA cycle derived previously for 14C tracer studies have been extended to 13C NMR to measure the 13C fractional enrichment of [2-13C]acetyl-CoA entering the cycle and the relative activities of the oxidative versus anaplerotic pathways. The analysis is based upon the steady-state enrichment of 13C into the glutamate carbons. Hearts perfused with [2-13C]acetate show low but significant activity of the anaplerotic pathways. Activation of two different anaplerotic pathways is demonstrated by addition of unlabeled propionate or pyruvate to hearts perfused with [2-13C]acetate. In each case, the amount of [2-13C]acetate being oxidized and the relative carbon flux through anaplerotic versus oxidative pathways are evaluated

NMR indirect detection of glutamate to measure citric acid cycle flux in the isolated perfused mouse heart

Abstract13C-edited proton nuclear magnetic resonance (NMR) spectroscopy was used to follow enrichment of glutamate C3 and C4 with a temporal resolution of ∼20 s in mouse hearts perfused with 13C-enriched substrates. A fit of the NMR data to a kinetic model of the tricarboxylic acid (TCA) cycle and related exchange reactions yielded TCA cycle (Vtca) and exchange (Vx) fluxes between α-ketoglutarate and glutamate. These fluxes were substrate-dependent and decreased in the order acetate (Vtca=14.1 μmol g−1 min−1; Vx=26.5 μmol g−1 min−1)>octanoate (Vtca=6.0 μmol g−1 min−1; Vx=16.1 μmol g−1 min−1)>lactate (Vtca=4.2 μmol g−1 min−1; Vx=6.3 μmol g−1 min−1)