LIPOXIDATION PRODUCTS AS BIOMARKERS OF OXIDATIVE DAMAGE TO PROTEINS DURING LIPID-PEROXIDATION REACTIONS

Oxidative stress is implicated in the pathogenesis of numerous disease processes including diabetes mellitus, atherosclerosis, ischaemia rep erfusion injury and rheumatoid arthritis. Chemical modification of ami no acids in protein during lipid peroxidation results in the formation of lipoxidation products which may serve as indicators of oxidative s tress in vivo. The Focus of the studies described here was initially t o identify chemical modifications of protein derived exclusively from lipids in order to assess the role of lipid peroxidative damage in the pathogenesis of disease. Malondialdehye (MDA) and 4-hydroxynonenal (H NE) are well characterized oxidation products of polyunsaturated fatty acids on low-density lipoprotein (LDL) and adducts of these compounds have been detected by immunological means in atherosclerotic plaque. Thus, we first developed gas chromatography-mass spectrometry assays f or the Schiff base adduct of MDA to lysine, the lysine-MDA-lysine diim ine cross-link and the Michael addition product of HNE to lysine. Usin g these assays, we showed that the concentrations of all three compoun ds increased significantly in LDL during metal-catalysed oxidation in vitro. The concentration of the advanced glycation end-product N-epsil on-(carboxymethyl)lysine (CML) also increased during LDL oxidation, wh ile that of its putative carbohydrate precursor the Amadori compound N -epsilon-(1-deoxyfructose-1-yl)lysine did not change, demonstrating th at CML is a marker of both glycoxidation and lipoxidation reactions. T hese results suggest that MDA and HNE adducts to lysine residues shoul d serve as biomarkers of lipid modification resulting from lipid perox idation reactions, while CML may serve as a biomarker of general oxida tive stress resulting from both carbohydrate and lipid oxidation react ions.