Oxidative modification of proteins has been implicated in a variety of processes ranging from atherosclerosis to aging. Identifying the underlying oxidation pathways has proven difficult, however, due to the lack of specific markers for distinct oxidation pathways. Previous in vitro studies demonstrated that 3-chlorotyrosine is a specific product of myeloperoxidase-catalyzed oxidative damage and that the chlorinated amino acid may thus serve as an index of phagocyte-mediated tissue injury in vivo. Here we describe a highly sensitive and specific analytical method for the quantification of 3-chlorotyrosine content of tissues. The assay combines gas chromatography with stable isotope dilution mass spectrometry, and it detects attomole levels of 3-chlrotyrosine in a single determination. Furthermore, the method is highly reproducible, with inter- and intra-sample coefficients of variance of <3%. The specificity, sensitivity, and reproducibility of 3-chlorotyrosin e determin ation should make this method useful for exploring the role of myeloperoxidase in catalyzing oxidative reactions in vivo.
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