Distinctive Oxidized Cardiolipin Species Generated by Singlet Oxygen and Radical Initiator Characterized by LC-MS/MS Analysis

摘要

Cardiolipin (CL) is a phospholipid predominantly found in the mitochondrial inner membrane and especially associated structurally with proteins of the electron transport chain (ETC). Since the mitochondrial ETC is the major reactive oxygen species (ROS) generating site, the proximity to the ETC and the bisallylic methylenes of the polyunsaturated fatty acid chains of CL make it a likely target of ROS in mitochondrial inner membrane. Identifying oxidized cellular CL products uniquely derived from certain ROS could serve as biomarkers for the presence of the ROS and help to understand the mechanism of oxidative stress. We exposed CL, under aerobic conditions, to singlet oxygen, a radical initiator, 2,2'-azobis(2-methylpropionamidine) dihydrochloride, or room air and the oxidized CL species were characterized by LC-MS/MS. Unique products, chromatographically separable and identified as CL dihydroperoxides, were produced by singlet oxygen-mediated oxidation but not present or barely detectable from radical oxidation or air oxidation. Monohydroperoxides and bis monohydroperoxides of CL were generated in all three oxidation conditions. No oxidative cleavage of the acyl chains was observed from any of the oxidant, in contrast to linoleic acid oxidation promoted by exogenous ferrous ion. Our LC-MS/MS method can separate and characterize oxidized CL species by generating distinctive fragment ions from the cleavage of oxidized acyl chains, particularly the dihydroperoxides that is uniquely generated by singlet oxygen.