DIRECT MEASUREMENT OF LIPID-PEROXIDATION IN SUBMITOCHONDRIAL PARTICLES

The susceptibility of the polyunsaturated fatty acid parinaric acid (c is-PnA) to peroxidative damage with concomitant loss of its fluorescen t character can be used to detect lipid peroxidation in a direct and s ensitive way. The procedure, originally developed to measure peroxidat ion in lipid vesicles and erythrocyte membranes, has been adapted for the study of submitochondrial particles. Optimal conditions for the co ncentrations of cis-PnA (0.8 mol %), mitochondrial membrane (100 mu M membrane phospholipid), and the radical generating system (50 mu M NAD H and 10 mu M:1 mM Fe(III)-ADP) were established. In the absence of pe roxidation inducing compounds, a stable fluorescent signal can be dete cted. Upon addition of NAD(P)H and ADP-Fe(III), lipid peroxidation sta rts, and the observed fluorescence decrease is a measure of peroxidati on. Both NADH and NADPH were able to induce lipid peroxidation in subm itochondrial particles in the presence of an iron chelate. The use of NADH resulted in higher rates of peroxidation compared with NADPH at t he same concentration. Whereas the rate of NADH-induced lipid peroxida tion was maximal at very low NADH concentrations (2.5 mu M) and decrea sed when the concentration became higher, the NADPH-induced Lipid pero xidation reaches saturation at 100 mu M. NADH-induced lipid peroxidati on in submitochondrial particles from different rat tissues (heart, sk eletal muscle, and liver) resulted in a clear difference in peroxidati on rates. The highest rates were observed in heart submitochondrial pa rticles, while the lowest rates were obtained in submitochondrial part icles derived from liver. Skeletal muscle submitochondrial particles s howed intermediate rates of lipid peroxidation. The differences in per oxidation rates showed a clear correlation with the activity of NADH:c ytochrome c oxidoreductase, and a relation between the electron flow t hrough the respiratory chain and the peroxidation rate is demonstrated . Al the results obtained with the PnA assay were compared with peroxi dation rates measured via oxygen consumption. Both methods gave identi cal results, demonstrating that the PnA assay is a reliable method to measure lipid peroxidation in submitochondrial particles.