Trace quantification of the oxidative damage products, meta- and ortho-tryrosine, in biological samples by gas chromatography - Electron capture negative ionization mass spectrometry

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) and ortho-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 into n-decane, followed by gas chromatography-mass spectrometry. Stable isotope labeled m- and o-Tyr (H-2(4)) and phenylalanine [i.e., Phe (H-2(5))] were added as internal standards to improve analytical accuracy. Quantification of as little as 50 pg of m- and o-Tyr in 100 mu g protein is possible and the data are expressed as a molar ratio of m- and o-Tyr to native Phe. The assay was used to determine the levels of m- and o-Tyr in freshly isolated human plasma protein (4.05 +/- 0.67 m-Tyr per 10(4) Phe, 0.35 +/- 0.07 o-Tyr per 10(4) Phe). Exposure of human plasma to reactive oxygen species significantly increased the levels of m-Tyr (56.4 +/- 1.1 m-Tyr per 10(4) Phe, P < 0.0001) and o-Tyr (48.9 +/- 1.3 o-Tyr per 10(4) Phe, P < 0.0001). The mild hydrolysis and derivatization conditions caused no artifactual formation of either m- or o-Tyr. (C) 1997 Academic Press