title: Liquid chromatography-mass spectrometry utilizing multi-stage fragmentation for the identification of oxysterols.
abstract: In humans, the brain accounts for about 20% of the body's free cholesterol, most of which is synthesized de novo in brain. To maintain cholesterol balance throughout life, cholesterol becomes metabolized to 24S-hydroxycholesterol, principally in neurons. In mouse, rat, and probably human, metabolism to 24S-hydroxycholesterol accounts for about 50% of cholesterol turnover; however, the route by which the remainder is turned over has yet to be elucidated. Here, we describe a novel liquid chromatography (LC) multi-stage fragmentation mass spectrometry (MS(n)) methodology for the identification, with high sensitivity (low pg), of cholesterol metabolites in rat brain. The methodology includes derivatization to enhance ionization, exact mass analysis at high resolution to identify potential metabolites, and LC-MS(n) (n=3) to allow their characterization. 24S-hydroxycholesterol was confirmed as a major oxysterol in rat brain, and other oxysterols identified for the first time in brain included 24,25-, 24,27-, 25,27-, 6,24,- 7alpha,25-, and 7alpha,27-dihydroxycholesterols. In addition, 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al and its aldol, two molecules linked to amyloidogenesis of proteins, were characterized in rat brain.
authors.affiliation: The School of Pharmacy, University of London, 29-39 Brunswick Square, London, WC1N 1AX, UK.
authors.forename: Kersti
authors.initials: K
authors.lastname: Karu
authors.forename: Martin
authors.initials: M
authors.lastname: Hornshaw
authors.forename: Gary
authors.initials: G
authors.lastname: Woffendin
authors.forename: Karl
authors.initials: K
authors.lastname: Bodin
authors.forename: Mats
authors.initials: M
authors.lastname: Hamberg
authors.forename: Gunvor
authors.initials: G
authors.lastname: Alvelius
authors.forename: Jan
authors.initials: J
authors.lastname: Sjövall
authors.forename: John
authors.initials: J
authors.lastname: Turton
authors.forename: Yuqin
authors.initials: Y
authors.lastname: Wang
authors.forename: William J
authors.initials: WJ
authors.lastname: Griffiths
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism; D001923:Brain Chemistry; D002784:Cholesterol / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D006888:Hydroxycholesterols / Q000032:analysis*; D013058:Mass Spectrometry / Q000379:methods*; D051381:Rats
chemical_list: D006888:Hydroxycholesterols; C044563:24-hydroxycholesterol; D002784:Cholesterol