Elevated O2 tensions are employed in the treatment of pulmonary insufficiency, and inhaled nitric oxide (NO) is used for treatment of pulmonary vascular disease. Although useful, both of these drugs are potentially reactive chemicals that may cause significant adverse effects, most probably through oxidation or nitration of critical tissue molecules. The purpose of the present study was to test the hypothesis that oxidized proteins, assessed by western analysis after derivatization with 2,4-dinitrophenylhydrazine (DNPH), or nitrotyrosine-containing proteins could function as biomarkers of the respective oxidations. Identification of the specific proteins and modifications would provide important insight into the mechanisms responsible and the biomarkers could be used to guide efforts to minimize the adverse effects of these therapies. Three groups of infants born at greater than 36 wk and mechanically ventilated were studied. Infants were ventilated with: 1) < 30% O2; 2) > 60% O2; and 3) NO, which is preceded by 100% O2. Tracheal aspirate fluids were obtained by suctioning, at times indicated clinically, and aliquots were frozen at -70oC and analyzed by treatment with DNPH, separation by SDS-PAGE, and probed with anti-DNP antibodies. Anti-nitrotyrosine antibodies were employed in samples not treated with DNPH. The major fractions of both modes of protein oxidation were observed uniformly in proteins migrating at molecular weights >87 kDa. In infants ventilated with hyperoxia, the anti-nitrotyrosine-reactive proteins were higher than in infants ventilated with room air in initial samples, but levels in the former group fell during the first week of life, whereas in the latter group the levels did not change markedly. Surprisingly, infants exposed to inhaled NO studied to date showed no distinctly greater levels or differences in distribution of nitrated proteins. The highest levels were observed in the initial sample from each child, with decreases through the first week of life. In infants that required longer ventilation, increased levels of DNPH-reactive proteins were observed. These studies indicate that NO therapy in neonates does not necessarily increase protein oxidation or nitration markedly, and that prolonged mechanical ventilation may contribute significantly to protein oxidation in these infants.