QUANTITATION OF NITROTYROSINE LEVELS IN LUNG SECTIONS OF PATIENTS ANDANIMALS WITH ACUTE LUNG INJURY

Activated alveolar macrophages and epithelial type II cells release bo th nitric oxide and superoxide which react at near diffusion-limited r ate (6.7 x 10(9) M(-1)s(-1)) to form peroxynitrite, a potent oxidant c apable of damaging the alveolar epithelium and pulmonary surfactant. P eroxynitrite, but not nitric oxide or superoxide, readily nitrates phe nolic rings including tyrosine. We quantified the presence of nitrotyr osine in the lungs of patients with the adult respiratory distress syn drome (ARDS) and in the lungs of rats exposed to hyperoxia (100% O-2 f or 60 h) using quantitative immunofluorescence. Fresh frozen or paraff in-embedded lung sections mere incubated with a polyclonal antibody to nitrotyrosine, followed by goat anti-rabbit IgG coupled to rhodamine. Sections from patients with ARDS (n = 5), or from rats exposed to hyp eroxia (n = 4), exhibited a twofold increase of specific binding over controls. This binding was blocked by the addition of an excess amount of nitrotyrosine and was absent when the nitrotyrosine antibody was r eplaced with nonimmune IgG. In additional experiments we demonstrated nitrotyrosine formation in rat lung sections incubated in vitro with p eroxynitrite, but not nitric oxide or reactive oxygen species. These d ata suggest that toxic levels of peroxynitrite may be formed in the lu ngs of patients with acute lung injury.