Surface fluorometry with 40 microM
hydroethidine (HE) as a probe was used to detect
oxidant generation in isolated, ventilated rat lungs during lung
ischemia.
Ethidium fluorescence due to HE oxidation was continuously monitored with 470 nm excitation and 610 nm emission. Fluorescence increased with
ischemia in O2-ventilated lungs [0.98 +/- 0.08 arbitrary fluorescence units (AFU)/min vs. 0.58 +/- 0.07 with control perfusion]. HE oxidation during
ischemia was prevented by N2 ventilation but was unaltered by preperfusion with
superoxide dismutase.
Ethidium fluorescence in homogenate prepared from lungs subjected to 1 h of nonhypoxic
ischemia was increased (16.8 +/- 1.5 vs. 9.8 +/- 0.4 AFU/mg
protein in control) but was unchanged in lungs that had been N2 ventilated. Microfluorographs of HE perfused and fixed lung sections demonstrated marked generalized increases in
ethidium fluorescence with
ischemia compared with control perfusion.
Ischemia resulted in significant increases in tissue
thiobarbituric acid reactive substance (176 +/- 13 vs. 44 +/- 3 pmol/mg
protein for control) and in lung conjugated dienes (0.90 +/- 0.07 vs. 0.48 +/- 0.06 U/mg
protein for control), indicating peroxidation of lung
lipids. These results indicate that lung
ischemia leads to intracellular
oxidant generation that can be continuously monitored by surface fluorometry.