ANALYSIS OF PLASMA-CHOLESTEROL OXIDATION-PRODUCTS USING GAS-CHROMATOGRAPHY AND HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY MASS-SPECTROMETRY

The application of gas chromatography and high-pressure liquid chromat ography/mass spectrometry techniques for analysis of plasma cholestero l oxidation products is described. Cholesterol oxides that are widely identified in biological samples were subjected to gas (GC) and high-p ressure liquid chromatographic (HPLC) separations, and their detection and characterization by mass spectrometry (MS) were compared. Analysi s of cholesterol oxides from plasma samples revealed distinct advantag es for each method according to the specific cholesterol oxide in ques tion. Whereas HPLC/MS analysis of cholesterol oxides provided less res olution and lower sensitivity as compared to GC/MS, a distinct advanta ge 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 analy tical conditions used for CC analysis, which are minimized or avoided using the HPLC/MS conditions described. Analysis of human and rabbit p lasma samples identified cholest-5-ene-3 beta, 7 beta-diol (7 beta-hyd roxycholesterol); 5,6 alpha-epoxy-5 alpha-cholestan-3 beta-ol (cholest erol-5 alpha,6 alpha-epoxide); 5 alpha-cholestane-3 beta,5,6 beta-trio l (cholestanetriol); 3 beta-hydroxycholest-5-ene-7-one (7-ketocholeste rol); and 5,6 beta-epoxy-5 beta-cholestan-3 beta-ol (cholesterol-5 bet a,6 beta-epoxide) as commonly occurring components (trivial names indi cated in parentheses). The latter two compounds were dramatically incr eased 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 dieta ry 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 le vels of the above cholesterol oxides, as well as other less prominent oxides, there was no evidence of cholesterol-7-hydroperoxides associat ed with plasma free cholesterol. Although several of the plasma choles terol oxides may derive from cholesterol-7-hydroperoxides, it appears that the latter are either unstable and decompose in plasma, are metab olized to other cholesterol oxidation products, or break down during t heir isolation.