Measurements of free radicals in isolated, ischemic lungs and lung mitochondria

Previous studies in isolated, ventilated lungs have demonstrated by indirect measurements that oxidant generation occurs during pulmonary ischemia before reperfusion. To identify and quantify the types of free radical species generated during ischemia, we used electron paramagnetic resonance (EPR) spectroscopy in the presence and absence of the spin trap, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). EPR spectra obtained from the vascular effluent of isolated ferret lungs, contained a doublet signal (g = 2.005) indicative of ascorbyl radical. This signal doubled in magnitude after 180 min of ischemia, providing evidence of oxidant formation during ischemia. When DMPO, which reacts with radicals including superoxide anions and hydroxyl radicals, was added to the perfusate, the spectra contained ascorbyl radical signals but no DMPO-adducts. To clarify the relationship between ascorbyl radical and DMPO-adduct formation, additional studies were conducted in the presence and absence of ascorbate with isolated lung mitochondria as the source of free radicals. The results showed that in the presence of ascorbate, oxygen free radicals were not detected by EPR spin trapping with DMPO because of the formation of prominent ascorbyl radical signals. These data suggest that DMPO may be useful for the detection of reactive oxygen species in isolated lungs, provided the ascorbate can be sufficiently depleted. Alternatively, as shown by our results, EPR studies that directly monitor ascorbyl radical formation may be used as a marker of oxidative stress in the lung.