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Mass Spectrometric Quantification of Markers for Protein Oxidation by Tyrosyl Radical, Copper, and Hydroxyl Radical in Low Density Lipoprotein Isolated from Human Atherosclerotic Plaques* (Received for publication, October 10, 1996, and in revised form, October 28, 1996) Christiaan Leeuwenburgh‡, Jane E. Rasmussen‡, Fong Fu Hsu‡, Dianne M. Mueller‡, Subramaniam Pennathur‡, and Jay W. Heinecke‡§ From the Department of Medicine and § Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110 Lipoprotein oxidation has been implicated in the pathogenesis of atherosclerosis. However, the physio- logically relevant pathways mediating oxidative dam- age have not yet been identified. Three potential mech- anisms 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 lipopro- tein (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 ox- idized with tyrosyl radical. Both o -tyrosine and o , o * -di- tyrosine 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 oxi- dized 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 in- crease 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. An elevated plasma level of low density lipoprotein (LDL) 1 is an important risk factor for the development of atherosclerotic vascular disease (1). Many lines of evidence suggest that LDL must be oxidatively modified before it can initiate atheroscle- rosis (2–7). LDL is oxidized in vitro by several different mech- anisms, although the physiologically relevant pathways have not yet been identified (2–10). The most widely studied model involves free metal ions. LDL oxidation by cultured arterial
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This note was uploaded on 05/19/2011 for the course BCH 3218 taught by Professor Johnsteward during the Fall '08 term at University of Florida.

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