Alterations in Substrate Utilization in the Reperfused Myocardium: A Direct Analysis by ¹³C NMR

An alternative ¹³C NMR method which allows direct determination of substrate oxidation in tissue for up to three competing ¹³C-enriched substrates is presented. Oxidation of competing substrates can be measured by ¹³C NMR spectroscopy under non-steady-state conditions if the relative areas of the glutamate C3 and C4 resonances can be determined. The accuracy of this measurement is limited during brief exposure to ¹³C-enriched substrates because of the low enrichment in the C3 carbon. The glutamate C4 resonance from a tissue sample which has oxidized a combination of [1, 2-¹³C]acetate (or a uniformly enriched fatty acid mixture) and [3-¹³C]lactate appears as a nine-line resonance consisting of four multiplet components : a singlet (C4S), two doublets with differing one-bond coupling constants (C4D34 and C4D45), and a quartet (C4Q). It is shown that the sum of the C4S + C4D34 resonance areas versus the C4D45 + C4Q resonance areas directly reports the relative utilization of [3-¹³C]lactate versus [l, 2-¹³C]acetate, respectively, regardless of citric acid cycle intermediate pool sizes or carbon flux through anaplerotic reactions. We also show that homonuclear ¹³C decoupling of the glutamate C2 resonance collapses the C3 resonance multiplet into an apparent triplet (actually, a singlet plus a doublet); the relative area of the singlet component reflects the amount of unlabeled acetyl-CoA entering the cycle. The method has been used to determine the contribution of lactate/acetate/glucose to acetyl-CoA in normoxic and reperfused rat hearts. ¹³C spectra of freeze-clamped heart extracts show quite directly that lactate oxidation is depressed after a 10-min period of global ischemia while acetate becomes the predominant source of energy (60% of the total substrate oxidized through the citric acid cycle). Substrate utilization returns to basal levels (34% acetate, 28% lactate, and 38% unlabeled sources) during 25 min of reperfusion. Ischemic hearts also switch to acetate as the principle source of energy in the presence of a pyruvate/acetate/glucose mixture but not when presented an acetoacetate/acetate/glucose mixture. These results likely reflect control at the level of the pyruvate dehydrogenase complex, which is known to become phosphorylated in heart mitochondria during ischemia.