Analysis of native and oxidized low-density lipoprotein oxysterols using gas chromatography - mass spectrometry with selective ion monitoring

Cholesterol oxidation products have been demonstrated to possess a wide variety of biological properties and have been implicated in playing an important role in the development of atherosclerosis, We have developed an analytical method using capillary gas chromatography-mass spectrometry (CC-MS) for the analysis of cholesterol oxidation products in low-density lipoprotein (LDL), The method uses programmed multiple selected ion monitoring (SIM), providing enhanced sensitivity and accuracy of peak detection over full-scan mass spectra, The major oxidation products of cholesterol in oxidized LDL were identified as 7 beta-hydroxy-cholesterol and 7-keto-cholesterol, Minor products included 4 beta-hydroxy-cholesterol, 6 beta-hydroxy-cholesterol and cholesterol-5 alpha,6 alpha-epoxide, Native LDL contains 7-lathosterol, which is a biosynthetic precursor of cholesterol, as well as low levels of 7 beta-hydroxy-cholesterol and 7-keto-cholesterol, 7-Lathosterol was not detected in oxidized LDL, A time course oxidation of native LDL with 8 mu M CuCl2 demonstrated a rapid increase in 7 beta-hydroxy-cholesterol and 7-keto-cholesterol over the first 4 h, Cholesterol -5 alpha,6 alpha-epoxide, and beta 4-hydroxy- and 6 beta-hydroxy-cholesterol levels increased gradually, while 7-lathosterol decreased over the same period, This method was used to measure the levels of 7-lathosterol and cholesterol oxides in the LDL of 20 healthy subjects in order to establish the mean concentration and a reference range, This method can be used for the characterization and quantitation of oxysterols in native and oxidized LDL and may afford an additional index of oxidative modification of plasma lipoproteins.