A novel method for production of lipid hydroperoxide- or oxysterol-rich low-density lipoprotein.
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 degrees C, which inhibits the decomposition of hydroperoxides, and allows the production of LDL rich in hydroperoxides (626+/-98 nmol/mg LDL protein) but low in oxysterols (3+/-1 nmol 7-ketocholesterol/mg LDL protein), whilst allowing sufficient modification (2.6+/-0.5 relative electrophoretic mobility) for rapid uptake by macrophages (5.49+/-0.75 microg (125)I-labelled hydroperoxide-rich LDL vs. 0.46+/-0.04 microg protein/mg cell protein in 18 h for native LDL). By dialysing under the same conditions, but at 37 degrees 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.
Gerry
Andrew B
AB
Cardiovascular Research Group, Biomolecular Sciences Section, School of Biological Sciences, University of Reading, Whiteknights, PO Box 228, Reading, Berkshire RG6 6AJ, UK.
Satchell
Leanne
L
Leake
David S
DS
D000818:Animals; D002478:Cells, Cultured; D003080:Cold Temperature; D003300:Copper / Q000737:chemistry; D003956:Dialysis / Q000379:methods*; D006358:Hot Temperature; D006801:Humans; D007653:Ketocholesterols / Q000138:chemical synthesis*; D008054:Lipid Peroxides / Q000138:chemical synthesis*; D008077:Lipoproteins, LDL / Q000138:chemical synthesis* / Q000737:chemistry; D008264:Macrophages; D051379:Mice
D007653:Ketocholesterols; D008054:Lipid Peroxides; D008077:Lipoproteins, LDL; C099252:oxidized low density lipoprotein; D003300:Copper; C003001:7-ketocholesterol