Isotope dilution mass spectrometric quantification of 3-nitrotyrosine in proteins and tissues is facilitated by reduction to 3-aminotyrosine

Oxidative damage by reactive nitrogen species has been implicated in the pathogenesis of atherosclerosis and other inflammatory diseases. The mechanisms of tissue damage are poorly understood, however, because the toxic intermediates are short-lived. Previous in vitro studies have suggested that 3-nitrotyrosine represents a specific marker of protein oxidation by reactive nitrogen species. The detection of this nitrated aromatic amino acid may thus serve as an indicator of tissue injury by nitrogen species in vivo. Here we describe a highly sensitive and specific analytical method for quantifying free and protein-bound 3-nitrotyrosine. The assay involves acid hydrolysis of proteins, isolation of 3-nitrotyrosine by ion exchange chromatography, and reduction of 3-nitrotyrosine to 3-aminotyrosine with dithionite. The reduced amino acid is then converted to its n-propyl, per-heptafluorobutyryl derivative and quantified by isotope dilution gas chromatography negative- ion chemical ionization mass spectrometry. Attomole levels of 3- nitrotyrosine can be reproducibly measured in this manner. Quantifying 3- nitrotyrosine levels of tissues by stable isotope dilution gas chromatography/mass spectrometry should provide a powerful tool for exploring the impact of reactive nitrogen species on oxidative reactions in vivo.