Characterization of the adduct formed from the reaction between homocysteine thiolactone and low-density lipoprotein: Antioxidant implications

Homocysteine thiolactone is a cyclic thioester that is implicated in the de velopment of atherosclerosis. This molecule will readily acylate primary am ines, forming a homocystamide adduct, which contains a primary amine and a thiol. Here, we have characterized and evaluated the antioxidant potential of the homocystamide-low-density lipoprotein (LDL) adduct, a product of the reaction between homocysteine thiolactone and LDL, Treatment of LDL with h omocysteine thiolactone resulted in a time-dependent increase in LDL-bound thiols that reached approximately 250 nmol thiol /mg LDL protein. The thiol groups of the homocystamide-LDL adduct were labeled with the thiol-reactiv e nitroxide, methanethiosulfonate spin label. Using paramagnetic relaxing a gents and the electron spin resonance spin labeling technique, we determine d that the homocystamide adducts were predominately exposed to the aqueous phase. The homocystamide-LDL adduct was resistant to myoglobin- and Cu2+-me diated oxidation (with respect to native LDL), as measured by the formation of conjugated dienes and thiobarbituric acid reactive substances, and the depletion of vitamin E. This antioxidant effect was due to increased thiol content, as the effect was abolished with N-ethylmaleamide pre-treatment. W e conclude that the reaction between homocysteine thiolactone and LDL gener ates an LDL molecule that is more resistant to oxidative modification than native LDL. The potential relationship between the homocystamide-LDL adduct and the development of atherosclerosis is discussed. (C) 1999 Elsevier Sci ence Inc.