Trace Quantification of the Oxidative Damage Products,meta- andortho-Tyrosine, in Biological Samples by Gas Chromatography–Electron Capture Negative Ionization Mass Spectrometry

Abstract

Oxygen radicals damage biomolecules and may contribute to cellular aging and degenerative disease. We describe a sensitive method for the quantification of two endogenous biomarkers of oxidative damage:meta-tyrosine (m-Tyr) andortho-tyrosine (o-Tyr). The assay can be applied to direct analysis of free amino acids or protein-bound amino acids following hydrolysis. The assay involves derivatization with pentafluorobenzyl bromide and extraction inton-decane, followed by gas chromatography–mass spectrometry. Stable isotope labeledm- ando-Tyr (2H4) and phenylalanine [i.e., Phe (2H5)] were added as internal standards to improve analytical accuracy. Quantification of as little as 50 pg ofm- ando-Tyr in 100 μg protein is possible and the data are expressed as a molar ratio ofm- ando-Tyr to native Phe. The assay was used to determine the levels ofm- ando-Tyr in freshly isolated human plasma protein (4.05 ± 0.67m-Tyr per 104Phe, 0.35 ± 0.07o-Tyr per 104Phe). Exposure of human plasma to reactive oxygen species significantly increased the levels ofm-Tyr (56.4 ± 1.1m-Tyr per 104Phe,P< 0.0001) ando-Tyr (48.9 ± 1.3o-Tyr per 104Phe,P< 0.0001). The mild hydrolysis and derivatization conditions caused no artifactual formation of eitherm- oro-Tyr.