MASS-SPECTROMETRIC QUANTIFICATION OF MARKERS FOR PROTEIN OXIDATION BYTYROSYL RADICAL, COPPER, AND HYDROXYL RADICAL IN LOW-DENSITY-LIPOPROTEIN ISOLATED FROM HUMAN ATHEROSCLEROTIC PLAQUES

Lipoprotein oxidation has been implicated in the pathogenesis of ather osclerosis. However, the physiologically relevant pathways mediating o xidative damage have not yet been identified, Three potential mechanis ms are tyrosyl radical, hydroxyl radical, and redox active metal ions, Tyrosyl radical forms o,o'-dityrosine cross-links in proteins, The hi ghly reactive hydroxyl radical oxidizes phenylalanine residues to o-ty rosine and m-tyrosine, Metal ions oxidize low density lipoprotein (LDL ) by poorly understood pathways. To explore the involvement of tyrosyl radical, hydroxyl radical, and metal ions in atherosclerosis, we deve loped a highly sensitive and quantitative method for measuring levels of o,o'-dityrosine, o-tyrosine, and m-tyrosine in proteins, lipoprotei ns, and tissue, using stable isotope dilution gas chromatography-mass spectrometry, We showed that o,o'-dityrosine was selectively produced in LDL oxidized with tyrosyl radical, Both a-tyrosine and a,o'-dityros ine were major products when LDL was oxidized with hydroxyl radical, O nly o-tyrosine was formed in EDL oxidized with copper, Similar profile s of oxidation products were observed in bovine serum albumin oxidized with the three different systems, Applying these findings to LDL isol ated from human atherosclerotic lesions, we detected a 100-fold increa se in o,o'-dityrosine levels compared to those in circulating LDL, In striking contrast, levels of o-tyrosine and m-tyrosine were not elevat ed in LDL isolated from atherosclerotic tissue, Analysis of fatty stre aks revealed a similar pattern of oxidation products; compared with no rmal aortic tissue, there was a selective increase in o,o'-dityrosine with no change in o-tyrosine, The detection of a selective increase of o,o'-dityrosine in LDL isolated from vascular lesions is consistent w ith the hypothesis that oxidative damage in human atherosclerosis is m ediated in part by tyrosyl radical, In contrast, these observations do not support a role for free metal ions as catalysts of LDL oxidation in the artery wall.