Oxygen radical generation and enzymatic properties of mitochondria in hypoxia/reoxygenation

The time-dependence of oxygen radical formation and development of enzymatic dysfunction after hypoxia/reoxygenation was investigated in isolated rat liver mitochondria. Generation of oxygen radicals was studied by electron paramagnetic resonance (EPR) spectroscopy using the spin trap DMPO (5,5-dimethyl-l-pyrroline-N-oxide). The spin adduct DMPO-OH was found to be formed from the primarily generated adduct of DMPO with the superoxide anion radical (DMPO-OOH). Hypoxic storage followed by reoxygenation at room temperature resulted in an increased decay rate of the DMPO-OH spin adduct while its steady state concentration remained unchanged. This finding strongly suggests an increased rate of DMPO-OH formation which originally derived from enhanced superoxide anion radical production due to hypoxia/reoxygenation. The enhanced superoxide radical formation seems to be due to dysfunction of respiratory chain enzymes, resulting in increased levels of reductive components. In agreement with that, we found the decrease of respiration control and ATP synthesis activity at a similar time scale as that for DMPO-OH adduct formation. The increase of superoxide radical formation and of the 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 synthesis decreased significantly after 15 min of hypoxia.