title: Validation of an isotope dilution gas chromatography-mass spectrometry method for analysis of 7-oxygenated campesterol and sitosterol in human serum.
abstract: High dose daily intake of plant sterols decreases the uptake of cholesterol in the intestine by competitive mechanisms and thus leads to reduced serum levels of total and LDL-cholesterol. By this, the commercialization of plant sterol enriched 'functional food' products is rapidly increasing. Subjects using these kinds of diet present a duplication of their serum plant sterol levels after long-term intake. In analogy to cholesterol, plant sterols such as campesterol and sitosterol can be oxidized to oxyphytosterols and these may counteract the primary anti-atherosclerotic action of cholesterol lowering. In order to investigate the whole spectrum of the consequences following high plant sterol intake a highly sensitive and specific isotope dilution gas chromatography-mass spectrometry method for the analysis of 7-oxygenated campesterol/sitosterol in trace amounts in human serum is presented in this paper. The validation was based on limits for detection and quantification, recovery, precision and minimization of autoxidation during work-up. Our results show an overall coefficient of variation ≤10% for the precision. The lowest limits for detection and quantification for 7α-hydroxy-campesterol were 7 pg/mL and 23 pg/mL, respectively. Data for overall sum recovery ranged from 92% to 115%. We practically used this method for analysis of oxyphytosterols simultaneously with plant sterol concentrations in serum from healthy volunteers. Sixteen subjects were treated with plant sterol enriched margarine (3 g/day) for 28 days. The results showed a significant increase of the oxyphytosterol 7β-hydroxy-sitosterol from 1.19±0.54 (before intake) to 2.24±1.24 ng/mL (mean±SD; +86.7%; P=0.007) after intake of the margarine. There was a highly significant correlation between the serum levels of campesterol and the sum of 7-oxygenated campesterol (R(2)=0.915; P<0.001) and sitosterol and the sum of 7-oxygenated sitosterol (R(2)=0.915; P<0.001). We can conclude from this study that the analytic method is well suited for detection of OPS, even at trace amounts.
authors.affiliation: Institute of Clinical Chemistry and Clinical Pharmacology, University Clinics of Bonn, Bonn, Germany.
authors.forename: Constanze
authors.initials: C
authors.lastname: Husche
authors.forename: Oliver
authors.initials: O
authors.lastname: Weingärtner
authors.forename: Hanna
authors.initials: H
authors.lastname: Pettersson
authors.forename: Tim
authors.initials: T
authors.lastname: Vanmierlo
authors.forename: Michael
authors.initials: M
authors.lastname: Böhm
authors.forename: Ulrich
authors.initials: U
authors.lastname: Laufs
authors.forename: Dieter
authors.initials: D
authors.lastname: Lütjohann
mesh_terms: D000328:Adult; D001774:Blood Chemical Analysis / Q000379:methods*; D002784:Cholesterol / Q000031:analogs & derivatives* / Q000097:blood / Q000378:metabolism; D005260:Female; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006801:Humans; D057230:Limit of Detection; D016014:Linear Models; D008297:Male; D008383:Margarine; D010100:Oxygen / Q000378:metabolism*; D010840:Phytosterols / Q000097:blood* / Q000378:metabolism*; D012855:Sitosterols / Q000097:blood* / Q000378:metabolism*
chemical_list: D010840:Phytosterols; D012855:Sitosterols; C021273:campesterol; C025473:gamma-sitosterol; D008383:Margarine; D002784:Cholesterol; D010100:Oxygen