INVOLVEMENT OF PREEXISTING LIPID HYDROPEROXIDES IN CU2-STIMULATED OXIDATION OF LOW-DENSITY-LIPOPROTEIN()

Oxidative modification of human low-density lipoprotein (LDL) is thoug ht to play an important role in the development of atherosclerosis. LD L oxidizability is believed to be strongly influenced by factors such as (a) content of preexisting lipid hydroperoxides (LOOHs) and (b) con tent of endogenous antioxidants such as alpha-tocopherol and beta-caro tene. The purpose of this study was to examine the prooxidant role of preexisting LDL-LOOHs, using a recently developed method for ultrasens itive and selective LOOH analysis: high-performance liquid chromatogra phy with mercury drop electrochemical detection (HPLC-EC). Exceedingly low detection limits for LDL-LOOHs have been achieved by HPLC-EC, e.g ., similar to 100 fmol for cholesteryl ester hydroperoxide (CEOOH). Th is sensitivity has allowed us to monitor LDL-LOOHs at levels that are undetectable by most other methods. Fresh LDL prepared with the utmost care to prevent autoxidation was found to contain small, yet signific ant amounts of CEOOH, 6-12 pmol/mg protein. Our data suggest that thes e peroxides could not have arisen during LDL isolation or sample work- up for HPLC-EC. Incubation with GSH and phospholipid hydroperoxide glu tathione peroxidase resulted in nearly complete reduction of the CEOOH . This LDL was found to be much more resistant to Cu2+-induced peroxid ation than starting material, exhibiting a lag period that was at leas t six times greater. We have also determined that LDL becomes progress ively more susceptible to Cu2+-induced Lipid peroxidation (as evidence d by a shortened lag) when it is preloaded with increasing amounts of photochemically generated LOOHs. Taken together, these results provide strong support for the idea that preexisting LOOHs in LDL are importa nt determinants of its overall oxidizability. (C) 1994 Academic Press, Inc.