Abstract 652: Production of Hyperpolarized 13C-Bicarbonate from 13C-Pyruvate Measures Flux through Pyruvate Dehydrogenase but not the Tricarboxylic Acid Cycle
Recent in vivo studies of metabolism in ischemic, reperfused porcine heart using dynamic nuclear polarization of [1-13C]pyruvate suggested that reduced appearance of 13C-bicarbonate is due to impaired tricarboxylic acid (TCA) cycle function. We tested the hypothesis that the rate of bicarbonate appearance is sensitive to the presence of fatty acids even when TCA cycle flux is constant. Three groups of isolated rat hearts were studied in the Langendorff mode and supplied with either
pyruvate plus the fatty acid octanoate, or
pyruvate, octanoate and propionate, a known stimulator of pyruvate dehydrogenase (PDH).
Each substrate was present at 2 mM. Hearts were supplied with oxygenated Krebs-Henseleit buffered medium and stabilized for 30 min. After preparation of hyperpolarized [1-13C]pyruvate, the sample was diluted and added to the perfusing medium such that the substrate concentrations did not change. The appearance of pyruvate, lactate, alanine and bicarbonate were monitored with 1 second time resolution by 13C NMR. In separate experiments, myocardial O2 consumption was measured, and 13C NMR isotopomer analysis was used to measure TCA cycle flux, pyruvate oxidation through PDH, pyruvate carboxylation, and octanoate oxidation. 13C bicarbonate and 13C lactate were easily observed in hearts supplied solely with pyruvate. However, in the presence of octanoate or octanoate + propionate, the spectra were dramatically different. The appearance of [1-13C]lactate was enhanced but 13C-bicarbonate was not detected. TCA cycle flux was not different between hearts supplied with pyruvate and octanoate compared to pyruvate alone (9.2 ± 0.3 vs. 7.9 ± 1.2 micromoles/gram dw/min). With pyruvate as the sole source of energy, flux through PDH is high, 7.4 ± 0.1 micromoles/gram dw/min. In the presence of pyruvate plus octanoate, PDH flux was reduced sharply to 0.7 ± 0.1 micromoles/gram dw/min (p<0.0001). In these normoxic hearts, appearance of 13C bicarbonate from [1-13C]pyruvate was due exclusively to flux through PDH and exchange of pyruvate into the lactate pool was greater in the presence of fatty acids. Even when TCA cycle flux is constant, appearance of 13C-bicarbonate can vary over a wide range depending on oxidation of alternative energy sources.