A 13C mass isotopomer study of anaplerotic pyruvate carboxylation in perfused rat hearts.

Anaplerotic pyruvate carboxylation was examined in hearts perfused with physiological concentrations of glucose, [U-13C3]lactate, and [U-13C3]pyruvate. Also, a fatty acid, [1-13C]octanoate, or ketone bodies were added at concentrations providing acetyl-CoA at a rate resulting in either low or substantial pyruvate decarboxylation. Relative contributions of pyruvate and fatty acids to citrate synthesis were determined from the 13C labeling pattern of effluent citrate by gas chromatography-mass spectrometry (see companion article, Comte, B., Vincent, G., Bouchard, B., and Des Rosiers, C. (1997) J. Biol. Chem. 272, 26117-26124). Precision on flux measurements of anaplerotic pyruvate carboxylation depended on the mix of substrates supplied to the heart. Anaplerotic fluxes were precisely determined under conditions where acetyl-CoA was predominantly supplied by beta-oxidation, as it occurred with 0.2 or 1 mM octanoate. Then, anaplerotic pyruvate carboxylation provided 3-8% of the OAA moiety of citrate and was modulated by concentrations of lactate and pyruvate in the physiological range. Also, the contribution of pyruvate to citrate formation through carboxylation was equal to or greater than through decarboxylation. Furthermore, 13C labeling data on tissue citric acid cycle intermediates and pyruvate suggest that (i) anaplerosis occurs also at succinate and (ii) cataplerotic malate decarboxylation is low. Rather, the presence of citrate in the effluent perfusate of hearts perfused with physiological concentrations of glucose, lactate, and pyruvate and concentrations of octanoate leading to maximal oxidative rates suggests a cataplerotic citrate efflux from mitochondria to cytosol. Taken altogether, our data raise the possibility of a link between pyruvate carboxylation and mitochondrial citrate efflux. In view of the proposed feedback regulation of glycolysis by cytosolic citrate, such a link would support a role of anaplerosis and cataplerosis in metabolic signal transmission between mitochondria and cytosol in the normoxic heart.

Effects of N-acetylcysteine in the rat heart reperfused after low-flow ischemia: evidence for a direct scavenging of hydroxyl radicals and a nitric oxide-dependent increase in coronary flow.

The capacity of N-acetylcysteine to directly scavenge hydroxyl radical produced by rat hearts reperfused after 90 min of low-flow ischemia was assessed by the hydroxylation of 4-hydroxybenzoate into 3,4-dihydroxybenzoate using a gas chromatography-mass spectrometric assay. Reperfused hearts showed a massive release of 3,4-dihydroxybenzoate, lactate dehydrogenase, and total glutathione, contained less reduced and oxidized glutathione, but maintained spontaneous beating and coronary flow rates close to preischemic values. Compared to untreated hearts: reperfused hearts treated with N-acetylcysteine from the start of ischemia (i) released four times less 3,4-dihydroxybenzoate, but similar amounts of lactate dehydrogenase or glutathione, (ii) showed a nitric oxide-dependent increase in coronary flow rate, and (iii) contained less oxidized glutathione, but similar amounts of reduced glutathione. Reperfused hearts receiving N-acetylcysteine since the last 5 min of ischemia had also a four-times lower 3,4-dihydroxybenzoate release, but their coronary flow rate response was similar to that of untreated hearts. These results indicate that N-acetylcysteine can directly scavenge hydroxyl radicals produced by reperfused ischemic hearts, although this effect is not associated with any protective effects as indicated by the lactate dehydrogenase and glutathione release and cannot explain the nitric oxide-dependent reperfusion hyperemia.