A novel method for production of lipid hydroperoxide- or oxysterol-rich low-density lipoprotein

Abstract 

LDL oxidation may be important in atherosclerosis. Extensive oxidation of LDL by copper induces increased uptake by macrophages, but results in decomposition of hydroperoxides, making it more difficult to investigate the effects of hydroperoxides in oxidised LDL on cell function. We describe here a simple method of oxidising LDL by dialysis against copper ions at 4°C, which inhibits the decomposition of hydroperoxides, and allows the production of LDL rich in hydroperoxides (626±98nmol/mg LDL protein) but low in oxysterols (3±1nmol 7-ketocholesterol/mg LDL protein), whilst allowing sufficient modification (2.6±0.5 relative electrophoretic mobility) for rapid uptake by macrophages (5.49±0.75μg ¹²⁵I-labelled hydroperoxide-rich LDL vs. 0.46±0.04μg protein/mg cell protein in 18h for native LDL). By dialysing under the same conditions, but at 37°C, the hydroperoxides are decomposed extensively and the LDL becomes rich in oxysterols. This novel method of oxidising LDL with high yield to either a hydroperoxide- or oxysterol-rich form by simply altering the temperature of dialysis may provide a useful tool for determining the effects of these different oxidation products on cell function.

Abbreviations: 7-keto, 7-ketocholesterol/7-oxocholesterol, apoB-100, apolipoprotein B-100, CA, cholesteryl arachidonate, CL, cholesteryl linoleate, Chol, nonesterified cholesterol, CL-OH, cholesteryl linoleate hydroxide, CL-OOH, cholesteryl linoleate hydroperoxide, HPLC, high performance liquid chromatography, LDL, low-density lipoprotein, OOH, hydroperoxide, REM, relative electrophoretic mobility

Keywords: Atherosclerosis, Cholesterol, Copper, Hydroperoxide, Oxysterol, Macrophage, Oxidised low-density lipoprotein