Assay of unesterified 7-oxocholesterol in human serum by isotope dilution-mass spectrometry.
A sensitive and accurate assay was developed for 7-oxocholesterol, one of the major autooxidation products of cholesterol. The assay is based on mass spectrometry with use of 2H7-labeled 7-oxocholesterol as internal standard. A fixed amount of internal standard (500 ng) is added to a fixed amount of sample (serum, 1 ml). After extraction with chloroform and purification by thin-layer chromatography, the isolated nonesterified 7-oxocholesterol is reduced by sodium borohydride to give a mixture of 7 alpha- and 7 beta-hydroxycholesterol, mainly 7 beta-hydroxycholesterol. After derivatization with trimethylsilyl reagent, the ratio between unlabeled and deuterium-labeled 7 beta-hydroxycholesterol is determined by selected monitoring of the ions at m/z 456 (corresponding to the M-90 fragment in the mass spectrum of the derivative of unlabeled 7 beta-hydroxycholesterol) and m/z 463 (corresponding to the same fragment in the mass spectrum of the derivative of 2H7-labeled 7 beta-hydroxycholesterol). The amount of 7-oxocholesterol is calculated with use of a standard curve obtained by analyses of standard mixtures of unlabeled and 2H7-labeled 7-oxocholesterol carried through the whole procedure. The detection limit of the assay was found to be about 15 ng/ml. The coefficient of variation was 7-8% in the concentration range 60-340 ng/ml. Serum collected in the presence of antioxidants and analyzed immediately contained less than 70 ng/ml of 7-oxocholesterol, and in some cases the concentration was below the detection limit of the assay. It is concluded that the concentration of 7-oxocholesterol in serum is low under normal conditions in vivo, probably due to presence of effective antioxidative and/or metabolizing systems.
D000328:Adult; D002784:Cholesterol / Q000031:analogs & derivatives*; D003903:Deuterium; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D007201:Indicator Dilution Techniques; D007653:Ketocholesterols / Q000097:blood*; D008297:Male; D010084:Oxidation-Reduction
D007653:Ketocholesterols; D002784:Cholesterol; D003903:Deuterium; C003001:7-ketocholesterol