Thiol-oxidizing agent diamide and acidic pH enhance lipid peroxidation of rat heart mitochondria and cardiolipin-cytochrome c complex.

Abstract

In this study, we investigated lipid peroxidation in rat heart mitochondria hydrolyzed by phospholipase A2 (PLA2) and lipid peroxidation in a mitochondrial-mimetic lipid peroxidation system, where phospholipids such as cardiolipin (CL) and cytochrome c (Cyt c) were first mixed together and then PLA2 and calcium chloride were added to the mixture (CL-Cyt c-PLA2 system). Production of hydroperoxy and hydroxy compounds of linoleic acid (LA) in the mixture was measured by high performance liquid chromatography. The ratio of the total amount of hydroperoxy and hydroxy compounds of LA to that of LA was calculated as an index for lipid peroxidation (1000 x mol/mol). The index for lipid peroxidation in the rat heart mitochondria hydrolyzed by PLA2 at the physiological pH of 7.4 was 22.8 +/- 2.2 (mean +/- SD, n = 4) and that at the acidic pH of 6.7 was 41.8 +/- 2.0. In the presence of the thiol (SH)-oxidizing agent diamide, the index was 47.0 +/- 2.6 (pH 7.4). In the CL-Cyt c-PLA2 system, lipid peroxidation seemed to be due to three mechanisms: (1) oxidation of the LA (nonreleased form) constituent of CL by Cyt c (oxidation of CL by Cyt c); (2) oxidation of free LA, released from CL, involving the oxidation of CL by Cyt c (free LA oxidation by the CL-Cyt c complex); and (3) oxidation of free LA, released from CL, by Cyt c and calcium ions (LA-Cyt c-Ca system). The lipid peroxidation of the CL-Cyt c-PLA2 system was also enhanced by the addition of diamide and by an acidic pH of 6.7. The fact that the SH-oxidizing agent enhanced the lipid peroxidation in the CL-Cyt c-PLA2 system suggested that SH groups in the hemoprotein played an inhibitory role in lipid peroxidation in the system.