Journal of Biological Chemistry
Volume 272, Issue 6, 7 February 1997, Pages 3520-3526
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Protein Chemistry and Structure
Mass Spectrometric Quantification of Markers for Protein Oxidation by Tyrosyl Radical, Copper, and Hydroxyl Radical in Low Density Lipoprotein Isolated from Human Atherosclerotic Plaques*

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Lipoprotein oxidation has been implicated in the pathogenesis of atherosclerosis. However, the physiologically relevant pathways mediating oxidative damage have not yet been identified. Three potential mechanisms are tyrosyl radical, hydroxyl radical, and redox active metal ions. Tyrosyl radical forms o,o′-dityrosine cross-links in proteins. The highly reactive hydroxyl radical oxidizes phenylalanine residues to o-tyrosine 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 developed a highly sensitive and quantitative method for measuring levels of o,o′-dityrosine, o-tyrosine, and m-tyrosine in proteins, lipoproteins, 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 o-tyrosine and o,o′-dityrosine were major products when LDL was oxidized with hydroxyl radical. Only o-tyrosine was formed in LDL oxidized with copper. Similar profiles of oxidation products were observed in bovine serum albumin oxidized with the three different systems. Applying these findings to LDL isolated from human atherosclerotic lesions, we detected a 100-fold increase in o,o′-dityrosine levels compared to those in circulating LDL. In striking contrast, levels of o-tyrosine and m-tyrosine were not elevated in LDL isolated from atherosclerotic tissue. Analysis of fatty streaks revealed a similar pattern of oxidation products; compared with normal 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 with the hypothesis that oxidative damage in human atherosclerosis is mediated 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.

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*

This work was supported by Awards R01 AG12293 and RR00954 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

    The abbreviations used are:

    LDL

    low density lipoprotein

    BSA

    bovine serum albumin

    DTPA

    diethylenetriamine pentaacetic acid

    GC-MS

    gas chromatography-mass spectrometry

    m/z

    mass-to-charge ratio.