Low-temperature electron paramagnetic resonance (EPR) spectroscopy and spin traps were used to measure paramagnetic species generation in rat hearts and isolated mitochondria. The hearts were freeze-clamped at 77 K during control perfusion by the Langendorff procedure, after 20–30 min of normothermic ischemia or 10–30 s of reperfusion with oxygenated perfusate. All EPR spectra measured at 4.5–50 K exhibited signals of both mitochondrial free radical centers and FeS proteins. The analysis of spectral parameters measured at 243 K showed that free radicals in heart tissue were semiquinones of coenzyme Q10 and flavins. The appearance of a typical “doublet” signal at g = 1.99 in low-temperature spectra indicated that a part of ubisemiquinones formed a complex with a high potential FeS protein of succinate dehydrogenase. Ischemia decreased the free radical species in myocardium 50%; the initiation of reflow of perfusate resulted in quick increase of the EPR signal. Mitochondria isolated from hearts during control perfusion and after 20–30 min of ischemia were able to produce superoxide radicals in both the NADH-coenzyme Q10 reductase and the bc1 segments of the respiratory chain. The rate of oxyradical generation was significantly higher in mitochondria isolated from ischemic heart.
electron paramagnetic resonance; oxygen paradox; oxyradicals; rat heart; semiquinones
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