Capillary gas chromatography quantification of cholesterol in copper-oxidized low-density lipoprotein.

A simple analytical method, using capillary gas chromatography-mass spectrometry (GC-MS) was used to investigate the oxidative modification of cholesterol in copper-oxidized low-density lipoprotein (oxidized LDL). The incubation of LDL and 5 microM CuSO4 in 0.15 M NaCl/0.02 M Tris-HCl buffer at pH 7.4 led to the peroxidation of LDL as shown by the detection of a thiobarbituric acid-reactive substance (TBA-RS). Oxidized LDL was shown to cause a significant fall in the unesterified and esterified cholesterol content, while oxysterols such as 7-hydroxycholesterol (7-OH) and 7-ketocholesterol (7-Keto) were formed from cholesterol, these conversions occurred in a dose- and time-dependent manner. Both oxysterols were identified by GC-MS on a fused-silica capillary column. In addition, copper-oxidized high-density lipoprotein (oxidized HDL) also resulted in the generation of TBA-RS and oxysterols. In addition, changes in the composition and amount of oxysterols during the incubation of macrophages with oxidized LDL were investigated. The incubation of macrophages with oxidized LDL resulted in the accumulation of cholesterol, 7-OH and 7-Keto derivatives in macrophages and this accumulation also occurred in a dose-dependent manner. These results suggest that the measurement of oxysterols may afford an additional index of oxidized LDL, and that foam cell formation and early atherosclerotic lesions may be responsible for the accumulation of oxysterols in macrophages. In addition, this study shows that HDL is also modified when submitted to an oxidative process.