The importance of
reactive nitrogen species in
atherosclerosis remains poorly understood, despite the semi-quantitative evidence for the presence of
3-nitrotyrosine provided by immunohistochemical staining studies. At this time, there appear to be no data describing the prevalence of nitration relative to oxidation in
atherosclerotic plaque proteins. The present study used
3-nitrotyrosine and
dityrosine as markers of nitration and oxidation respectively to examine the relative abundance of each process. Substantial methodological improvements were required to overcome problems associated with sensitivity and artefactual production of
3-nitrotyrosine when quantified by GLC-MS. It was shown that careful selection of hydrolysis vessel, sample reduction and the use of the
oxazolinone derivative provided sample stability and exquisite sensitivity. Using these methods, it was observed that the frequency of nitration was 92+/-15 micro mol/mol of
tyrosine (0.01%).
Dityrosine was present at 1.5+/-0.14 mmol/mol of
tyrosine (0.30%) using HPLC/fluorescence; thus nitration accounted for approx. 3% of the
tyrosine modifications measured. Given that other modifications of
tyrosine are known to occur in carotid plaque
proteins, the contribution of nitration to the total pool of modified
tyrosine is very small. However, the possibility of metabolic processes or chemical agents modifying
3-nitrotyrosine to secondary oxidation products remains an alternative explanation for the low levels demonstrated in this study.