OXYGEN RADICAL GENERATION AND ENZYMATIC-PROPERTIES OF MITOCHONDRIA INHYPOXIA REOXYGENATION/

The time-dependence of oxygen radical formation and development of enz ymatic dysfunction after hypoxia/reoxygenation was investigated in iso lated rat liver mitochondria. Generation of oxygen radicals was studie d by electron paramagnetic resonance (EPR) spectroscopy using the spin trap DMPO (5,5-dimethyl-1-pyrroline-N-oxide). The spin adduct DMPO-OH was found to be formed from the primarily generated adduct of DMPO wi th the superoxide anion radical (DMPO-OOH). Hypoxic storage followed b y reoxygenation at room temperature resulted in an increased decay rat e of the DMPO-OH spin adduct while its steady state concentration rema ined unchanged. This finding strongly suggests an increased rate of DM PO-OH formation which originally derived from enhanced superoxide anio n radical production due to hypoxia/reoxygenation. The enhanced supero xide radical formation seems to be due to dysfunction of respiratory c hain enzymes, resulting in increased levels of reductive components. I n agreement with that, we found the decrease of respiration control an d ATP synthesis activity at a similar time scale as that for DMPO-OH a dduct formation. The increase of superoxide radical formation and of t he reductive capacity of mitochondria was accompanied by a decrease in membrane order at the polar interface. Oxidative phosphorylation was completely abolished after 30 min of hypoxic storage, whereas ATP synt hesis decreased significantly after 15 min of hypoxia.