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Papers In Press, published online ahead of print April 1, 2007
J. Lipid Res., doi:10.1194/jlr.M600497-JLR200

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Journal of Lipid Research, Vol. 48, 976-987, April 2007
Copyright © 2007 by American Society for Biochemistry and Molecular Biology

Methods

Liquid chromatography-mass spectrometry utilizing multi-stage fragmentation for the identification of oxysterols

Kersti Karu^*, Martin Hornshaw, Gary Woffendin, Karl Bodin, Mats Hamberg, Gunvor Alvelius, Jan Sjövall, John Turton^*, Yuqin Wang^* and William J. Griffiths^1,*

^* The School of Pharmacy, University of London, 29-39 Brunswick Square, London, WC1N 1AX, UK
Thermo Electron Corporation, Stafford House, Boundary Way, Hemel Hempstead, HP2 7GE, UK
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm SE-17177, Sweden

The online version of this article (available at http://www.jlr.org) contains additional two tables and twelve figures.

Published, JLR Papers in Press, January 24, 2007.

¹ To whom correspondence should be addressed. e-mail: william.griffiths{at}pharmacy.ac.uk

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 (MSⁿ) 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-MSⁿ (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,- 7,25-, and 7,27-dihydroxycholesterols. In addition, 3ß-hydroxy-5-oxo-5,6-secocholestan-6-al and its aldol, two molecules linked to amyloidogenesis of proteins, were characterized in rat brain.

Supplementary key words sterol • cholesterol • 24S-hydroxycholesterol • dihydroxycholesterol • secosterol • derivatization • Girard P • LTQ-Orbitrap • liver X receptor • Alzheimer's disease

Abbreviations: API, atmospheric pressure ionization; ES, electrospray; GP, Girard P; LC-MS, liquid chromatography-mass spectrometry; LIT, linear ion trap; MS/MS, tandem mass spectrometry; RA, relative abundance; RIC, reconstructed ion chromatogram

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