Figure 7.

Oxidized linoleic acid production and oxidized cardiolipin consumption in wild-type, iPLA₂γ^−/−, and cardiac myocyte-specific iPLA₂γ transgenic mitochondrial homogenates in the presence of exogenous oxTLCL. A and B, mitochondria were isolated from wild-type and iPLA₂γ^−/− mouse liver and homogenized by sonication. Mitochondria homogenates (1 mg protein/ml) were incubated with 20 μm oxTLCL or ethanol vehicle alone at 37 °C for 15 min. The reactions were terminated by the addition of methanol (25% total volume) containing internal standards (13-HODE-d₄, 12(13)-DiHOME-d₄). The released oxidized fatty acids were purified by reversed phase solid phase extraction, derivatized with AMPP, and finally analyzed by LC-MS/MS. C and D, the same experiments were performed with heart mitochondria isolated from wild-type and cardiac myocyte specific iPLA₂γ transgenic mice. E, heart mitochondria were isolated from wild-type and cardiac myocyte-specific iPLA₂γ transgenic mice. Mitochondria homogenates (1 mg protein/ml) were incubated with 20 μm oxTLCL or ethanol vehicle alone at 37 °C for 15 min. The reactions were terminated by adding chloroform/methanol 1:1 (v/v) in the presence of TMCL internal standard. The chloroform phase was separated, dried, and redissolved in chloroform. The oxidized cardiolipin was purified by aminopropyl solid phase extraction column and analyzed by LC-MS/MS in the negative ion mode. The results here show the consumption of oxidized cardiolipin per mg of protein in 30-min incubations. Values are the average of three independent preparations ± S.E. *, p < 0.05; **, p < 0.01; ***, p < 0.001.