Inhaled nitric oxide (NO), commonly administered with 100% oxygen, is increasingly being used as a therapy for treatment of pulmonary hypertension. The present study examines exposure to inhaled NO/hyperoxia and oxidation of specific proteins, which could be used as biomarkers for monitoring potentially adverse effects of inhaled NO therapy.

Methods: Male Fischer-344 rats were placed in 50ppm NO at FiO2>0.95 for 48 hr, with controls exposed to FiO2>0.95 or to room air. To assess protein oxidation, bronchoalveolar lavage (BAL), lung subcellular fractions, and lung homogenates were derivatized with 2,4-dinitrophenylhydrazine (DNPH), separated by gel electrophoresis, transferred, and DNPH-reactive proteins detected with anti-DNPH antibodies. Statistics were performed by ANOVA.

Results: Although no rats died prematurely, the rats exposed to hyperoxia or inhaled NO/hyperoxia had marked respiratory distress. The animals exposed to NO/hyperoxia showed markedly greater protein concentrations in their lavage fluids (see table). Overall, there were more DNPH-reactive proteins, “protein carbonyls,” observed in the NO/hyperoxia exposed animals than in the hyperoxia or room air animals. N-terminal amino acid sequencing of a unique DNPH-reactive protein at MW 54kDa, found only in the BAL from NO/hyperoxic rats, matched Vitamin D-binding protein precursor. There were also oxidized proteins, found only in NO/hyperoxia exposed animals, seen in the nuclear and microsomal subcellular fractions of the lung.

Table 1 No caption available.
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Conclusions: In the present experimental model, NO enhanced hyperoxic lung injury. The oxidation of the Vitamin D-binding protein precursor is interesting. This protein is responsible for transport of Vitamin D and depolymerization of actin at sites of tissue necrosis, suggesting a possible role in resolution of lung injury. The identity of the other oxidized proteins is currently being investigated.