DIRECT MEASUREMENT OF LIPID-PEROXIDATION IN SUBMITOCHONDRIAL PARTICLES

The susceptibility of the polyunsaturated fatty acid parinaric acid (cis-PnA) to peroxidative damage with concomitant loss of its fluorescent character can be used to detect lipid peroxidation in a direct and sensitive way. The procedure, originally developed to measure peroxidation in lipid vesicles and erythrocyte membranes, has been adapted for the study of submitochondrial particles. Optimal conditions for the concentrations of cis-PnA (0.8 mol %), mitochondrial membrane (100 mu M membrane phospholipid), and the radical generating system (50 mu M NADH and 10 mu M:1 mM Fe(III)-ADP) were established. In the absence of peroxidation inducing compounds, a stable fluorescent signal can be detected. Upon addition of NAD(P)H and ADP-Fe(III), lipid peroxidation starts, and the observed fluorescence decrease is a measure of peroxidation. Both NADH and NADPH were able to induce lipid peroxidation in submitochondrial particles in the presence of an iron chelate. The use of NADH resulted in higher rates of peroxidation compared with NADPH at the same concentration. Whereas the rate of NADH-induced lipid peroxidation was maximal at very low NADH concentrations (2.5 mu M) and decreased when the concentration became higher, the NADPH-induced Lipid peroxidation reaches saturation at 100 mu M. NADH-induced lipid peroxidation in submitochondrial particles from different rat tissues (heart, skeletal muscle, and liver) resulted in a clear difference in peroxidation rates. The highest rates were observed in heart submitochondrial particles, while the lowest rates were obtained in submitochondrial particles derived from liver. Skeletal muscle submitochondrial particles showed intermediate rates of lipid peroxidation. The differences in peroxidation rates showed a clear correlation with the activity of NADH:cytochrome c oxidoreductase, and a relation between the electron flow through the respiratory chain and the peroxidation rate is demonstrated. Al the results obtained with the PnA assay were compared with peroxidation rates measured via oxygen consumption. Both methods gave identical results, demonstrating that the PnA assay is a reliable method to measure lipid peroxidation in submitochondrial particles.