A Method for Defining the Stages of Low-Density Lipoprotein Oxidation by the Separation of Cholesterol and Cholesteryl Ester-Oxidation Products Using HPLC

Abstract

A new high-performance liquid chromatographic system for the identification of some of the lipid oxidation products of low-density lipoprotein (LDL) oxidized by copper is described. Using a reversed-phase C-18 column and an isocratic solvent system of acetonitrile/isopropanol/water (44/54/2, v/v/v), a number of oxidized lipid moieties were resolved and detected simply by their 234-nm absorbance. The nature of several of these compounds was determined by chromatographic criteria, chemiluminescence, and mass spectrometry. The production of compounds within 4 h oxidation corresponded to the production of lipid hydroperoxides, the quantitatively most important of which is cholesterol linoleate hydroperoxide, and to the rapid decrease in the cholesterol ester content of LDL detected at 210 nm. More prolonged copper oxidation (up to 48 h) of LDL resulted in decreased quantities of lipid hydroperoxide moieties and increased amounts of a number of other, nonhydroperoxide, compounds. 7-Ketocholesterol and cholesterol linoleate hydroxide are two of the major products of prolonged oxidation. The detection of oxidation products correlates with the modification of LDL protein, permits a four-stage definition of metal-mediated LDL oxidation, and enables the calculation of a quantitative index of oxidation (lipoprotein oxidation index). This method will be generally applicable to cell-and copper-mediated oxidation, and will enable standardization of, and direct comparison between, different preparations of oxidized LDL.