| Research Abstract |
Reactive nitrogen oxides derived from NO produce nitration adducts of various biological molecules. We have developed a series of methods for identification and quantification of nitrated adducts of amino acids and nucleic acid bases, such as 3-nitro-L-tyrosine, nitro-L-tryptophan, and 8-nitroguanine. The assay couples an HPLC system with electrochemical (EC) analysis. Each nitrated compound in Pronase-digested samples, being separated by reverse-phase HPLC, was first reduced electrochemically at -900 mV, followed by detection with an analytical cell at +300 mV. Detection limit for 3-nitrotyrosine reached 10^<-15>mol. Increased protein-bound 3-nitrotyrosine was demonstrated in bronchoalveolar lavage from mice infected with influenza virus; whereas protein-bound 3-nitrotyrosine was not identified with mice deficient in inducible NO synthase. Similarly, appreciable amount of 3-nitrotyrosine was detected in sputum from patients with bronchial asthma and chronic bronchitis. The level of nitrotryptophan was, however, below detection limit in both murine and human samples. Also, our immunohistochemical analysis with a specific anti-3-nitroguanine antibody clearly showed formation of 3-nitroguanosine in the bronchial epithelial cells of the virus-infected mouse lungs. This is the first demonstration of a nitrated nucleotide base formed endogenously in biological systems. The present methods may help us to better understand the biological relevance and the mechanism of biological nitration.
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