Analysis of plasma cholesterol oxidation products using gas- and high-performance liquid chromatography/mass...

The application of gas chromatography and high-pressure liquid chromatography/mass spectrometry techniques for analysis of plasma cholesterol oxidation products is described. Cholesterol oxides that are widely identified in biological samples were subjected to gas (GC) and high-pressure liquid chromatographic (HPLC) separations, and their detection and characterization by mass spectrometry (MS) were compared. Analysis of cholesterol oxides from plasma samples revealed distinct advantages for each method according to the specific cholesterol oxide in question. Whereas HPLC/MS analysis of cholesterol oxides provided less resolution and lower sensitivity as compared to GC/MS, a distinct advantage was evident for direct measurements of cholesterol-7-hydroperoxides and 7-ketocholesterol. These two cholesterol oxides are particularly sensitive to storage in solvents, derivatization procedures, and analytical conditions used for GC analysis, which are minimized or avoided using the HPLC/MS conditions described. Analysis of human and rabbit plasma samples identified cholest-5-ene-3 beta, 7 beta-diol (7 beta-hydroxycholesterol); 5,6 alpha-epoxy-5 alpha-cholestan-3 beta-ol (cholesterol-5 alpha, 6 alpha-epoxide); 5 alpha-cholestane-3 beta, 5,6 beta-triol (cholestanetriol); 3 beta-hydroxycholest-5-ene-7-one (7-ketocholesterol); and 5,6 beta-epoxy-5 beta-cholestan-3 beta-ol (cholesterol-5 beta,6 beta-epoxide) as commonly occurring components (trivial names indicated in parentheses). The latter two compounds were dramatically increased in hypercholesterolemic samples and were found in approximately equal amounts in the free cholesterol and cholesteryl ester fractions. Although most of the plasma cholesterol oxides are found in the dietary cholesterol, others are not, particularly cholesterol-5 beta,6 beta-epoxide, suggesting that at least some of these compounds are formed by in vivo oxidation of cholesterol. Despite the readily measurable levels of the above cholesterol oxides, as well as other less prominent oxides, there was no evidence of cholesterol-7-hydroperoxides associated with plasma free cholesterol. Although several of the plasma cholesterol oxides may derive from cholesterol-7-hydroperoxides, it appears that the latter are either unstable and decompose in plasma, are metabolized to other cholesterol oxidation products, or break down during their isolation.