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Abstract No.: MoP-174
Session: Metabolomics, Metabonomics
Presentation date: Mon, Aug 28, 2006
Presentation time: 14:30 – 16:00

Capillary-LC-MSn for the Analysis of Oxysterols Brain

Kersti Karu1, Gunvor Alvelius2, Karl Bodin2, Jan Sjovall2, Yuqin Wang1, William J. Griffiths1

1 The School of Pharmacy, University of London, 29/39 Brunswick Square, London WC1N 1AX, United Kingdom
2 Karolinska Institutet, Stockholm SE 171 77, Sweden

Correspondence address: Kersti Karu, The School of Pharmacy, University of London, Pharmaceutical and Biological Chemistry, 29/39 Brunswick Square, London, WC1N 1AX United Kingdom.

Keywords: Lipid; Low Flow Electrospray; Metabolic Profiling; MS/MS, Liquid Chromatography.

Novel aspect: Identification of new oxysterols in rat brain.

 

Oxysterols are oxygenated derivatives of cholesterol. The formation of oxysterols represents the first step of cholesterol metabolism. The introduction of additional hydroxyl groups to the cholesterol skeleton facilitates the flux of oxysterols across the blood brain barrier and they are further converted to bile acids in the liver. In addition they also behave as ligands for the liver X receptor and have been suggested to have roles in cholesterol homeostasis.

Sterols were extracted from the rat brain and separated by normal-phase chromatography into two fractions (U1, U2). The first fraction (U1) containing sterol esters was hydrolysed then subjected to further stage of normal-phase chromatography generating two fractions (U1i and U2ii). The second fraction from the initial chromatography step (U2), and the second chromatographic fraction following ester hydrolysis (U1ii) were rich in oxysterols. Fractions (U2) and (U1ii) were each divided into two portions. To the first portion oxysterols with a 3beta-hydroxy-5-ene or 5alpha-hydrogen-3beta-hydroxy structure were converted by cholesterol oxidase to 3-oxo-4-ene sterols, which were subsequently derivatised with Girard P reagent. The second portion was derivatised with Girard P directly with out the use of cholesterol oxidase in order to convert endogenous oxosterols to their GP hydrazones. Both fractions were subjected to C18 solid phase extraction and subsequently analysed using capillary liquid chromatography-ion trap mass spectrometry (LC-MSn).

Initially, a panel of synthetic oxysterols were oxidized and derivatised with Girard P hydrazine, then analysed by electrospray ionization tandem mass spectrometry (ESI-MSn) with low-energy collision-induced dissociation to establish structurally informative fragment ion patterns.

Capillary LC-MSn analysis of the oxysterol fractions from rat brain identified 24(S)-hydroxycholesterol as confirmed as the major oxysterol by comparison of its retention time and MS2 or MS3 spectra with synthetic standards. The current study also identified three dihydroxycholesterols, ketocholesterols and two hydroxycholesterols.

In conclusion the analytical method in use is specific, sensitive and robust for the analysis of oxysterols from biological tissues.