Involvement of preexisting lipid hydroperoxides in Cu(2+)-stimulated oxidation of low-density lipoprotein.
Oxidative modification of human low-density lipoprotein (LDL) is thought to play an important role in the development of atherosclerosis. LDL oxidizability is believed to be strongly influenced by factors such as (a) content of preexisting lipid hydroperoxides (LOOHs) and (b) content of endogenous antioxidants such as alpha-tocopherol and beta-carotene. The purpose of this study was to examine the prooxidant role of preexisting LDL-LOOHs, using a recently developed method for ultrasensitive and selective LOOH analysis: high-performance liquid chromatography with mercury drop electrochemical detection (HPLC-EC). Exceedingly low detection limits for LDL-LOOHs have been achieved by HPLC-EC, e.g., approximately 100 fmol for cholesteryl ester hydroperoxide (CEOOH). This 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 significant amounts of CEOOH, 6-12 pmol/mg protein. Our data suggest that these peroxides could not have arisen during LDL isolation or sample work-up for HPLC-EC. Incubation with GSH and phospholipid hydroperoxide glutathione peroxidase resulted in nearly complete reduction of the CEOOH. This LDL was found to be much more resistant to Cu(2+)-induced peroxidation than starting material, exhibiting a lag period that was at least six times greater. We have also determined that LDL becomes progressively more susceptible to Cu(2+)-induced lipid peroxidation (as evidenced 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 important determinants of its overall oxidizability.
Thomas
J P
JP
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226.
Kalyanaraman
B
B
Girotti
A W
AW
D000328:Adult; D002788:Cholesterol Esters / Q000737:chemistry; D003300:Copper / Q000494:pharmacology*; D005979:Glutathione Peroxidase / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008054:Lipid Peroxides / Q000737:chemistry*; D008077:Lipoproteins, LDL / Q000737:chemistry*; D008297:Male; D010084:Oxidation-Reduction; D010777:Photochemistry
D002788:Cholesterol Esters; D008054:Lipid Peroxides; D008077:Lipoproteins, LDL; D003300:Copper; D005979:Glutathione Peroxidase