Generation of superoxide anion by mitochondria and impairment of their functions during anoxia and reoxygenation in vitro

A small portion of the oxygen consumed by aerobic cells is converted to sup eroxide anion at the level of the mitochondrial respiratory chain. If produ ced in excess, this harmful radical is considered to impair cellular struct ures and functions. Damage at the level of mitochondria have been reported after ischemia and reperfusion of organs. However, the complexity of the in vivo system prevents from understanding and describing precise mechanisms and locations of mitochondrial impairment. An in vitro model of isolated-mi tochondria anoxia-reoxygenation is used to investigate superoxide anion gen eration together with specific damage at the level of mitochondrial oxidati ve phosphorylation. Superoxide anion is detected by electron paramagnetic r esonance spin trapping with POBN-ethanol. Mitochondrial respiratory paramet ers are calculated from oxygen consumption traces recorded with a Clark ele ctrode. Respiring mitochondria produce superoxide anion in unstressed condi tions, however, the production is raised during postanoxic reoxygenation. S everal respiratory parameters are impaired after reoxygenation, as shown by decreases of phosphorylating and uncoupled respiration rates and of ADP/O ratio and by increase of resting respiration. Partial protection of mitocho ndrial function by POBN suggests that functional damage is related and seco ndary to superoxide anion production by the mitochondria in vitro. (C) 1998 Elsevier Science Inc.