ISOTOPE-DILUTION MASS-SPECTROMETRIC QUANTIFICATION OF 3-NITROTYROSINEIN PROTEINS AND TISSUES IS FACILITATED BY REDUCTION TO 3-AMINOTYROSINE

Oxidative damage by reactive nitrogen species has been implicated in t he pathogenesis of atherosclerosis and other inflammatory diseases. Th e mechanisms of tissue damage are poorly understood, however, because the toxic intermediates are short-lived. Previous in vitro studies hav e suggested that 3-nitrotyrosine represents a specific marker of prote in oxidation by reactive nitrogen species. The detection of this nitra ted aromatic amino acid may thus serve as an indicator of tissue injur y by nitrogen species in vivo. Here we describe a highly sensitive and specific analytical method for quantifying free and protein-bound 3-n itrotyrosine. The assay involves acid hydrolysis of proteins, isolatio n 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 derivat ive and quantified by isotope dilution gas chromatography negative-ion chemical ionization mass spectrometry. Attomole levels of 3-nitrotyro sine can be reproducibly measured in this manner. Quantifying 3-nitrot yrosine levels of tissues by stable isotope dilution gas chromatograph y/mass spectrometry should provide a powerful tool for exploring the i mpact of reactive nitrogen species on oxidative reactions in vivo. (C) 1998 Academic Press.