PLASMA PROTEIN MODIFICATIONS AS BIOMARKERS OF OXIDATIVE

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Oxidative stress is considered to be a significant component in the pathogenesis of bronchoplumonary dysplasia (BPD) and adult respiratory distress syndrome (ARDS). Oxidative stress can be defined as the pathogenic outcome created by the oxidation of critical tissue targets by reactive species which are generated at rates exceeding tissue antioxidant capacity. However, only few studies that directly measure reactive species or oxidative stress in patients with either BPD or ARDS are available. A major limitation for measuring reactive species is their short half life in biological systems. Since reactive species modify biological molecules such as proteins, lipids and DNA measurement of the modified targets provide the experimental tools for their detection and quantification. Preliminary data in this application indicate that serum protein modifications measured as 2, 4 dinitrophenylhydrazine reactive carbonyls and nitration of tyrosine residues may be indicate oxidative stress in BPD and ARDS. Protein carbonyls are derived by the direct oxidation of amino acid residues conjugation of aldehydes that formed by the oxidation of unsaturated lipids or sugars. Overall, plasma protein carbonyls indicate the formation of oxidants. Nitration of protein tyrosine residues results in the formation of 3-nitrotyrosine. Previously we found that the reaction of peroxynitrite with C02 provides the necessary nitrating agent that explains the formation of plasma protein 3-nitrotyrosine. Peroxynitrite is formed by the nearly diffusion limited reaction of nitric oxide and superoxide. Therefore this application will systematically examine the changes in these biological markers in BPD and ARDS. We propose that these protein modifications indicate generation of oxidants and nitrating species and correlate with the severity of patient's illness and fatal outcome. The critical aspects of our hypothesis will be tested by: 1) correlating the plasma protein levels of 3-nitrotyrosine in BPD and ARDS patients with the severity of clinical illness and outcome. 2) measuring the changes in plasma protein levels of 3-nitrotyrosine and carbonyls in ARDS and BPD patients after inhalation of therapeutic levels of nitric oxide. This application is strengthened by the established protocols for patient blood collection and processing, the experience with measuring these biological markers and the understanding of the biochemical origin of these biological markers.