Figure 4.

Peroxidation of lipids in isolated rat heart mitochondria in the presence of Fe²⁺. Mitochondria were incubated with MitoCLox (50 nM), and fluorescent spectra were analyzed after subtraction of light scattering as described in Methods. Kinetics of MitoCLox oxidation calculated as a ratio of fluorescence at 521 nm and 596 nm. (a,b) Changes in fluorescent spectra of MitoCLox with time initiated by glutamate (5 mM) and malate (5 mM) in the absence (a) or in the presence (b) of rotenone. (c) Kinetics of MitoCLox oxidation induced by glutamate, malate, and rotenone (G/M + rot) or by succinate (succ, 5 mM) in the absence or in the presence of antimycine A (Ant). No substrates—Contr. (d) The effects of SkQ1 (30 nM, 100 nM, 300 nM) and MB (1 μM) on MitoCLox oxidation induced by glutamate, malate, and rotenone. (e) Hydrogen peroxide production was measured fluorometrically using Amplex Red and horseradish peroxidase. The reaction was initiated by glutamate and malate in the presence of rotenone or succinate in the presence of antimycin. SkQ1 (300 nM) was added 5 min before glutamate and malate. No substrates—control.