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.