Oxysterols are oxygenated metabolites of cholesterol formed in various tissues by the action of cytochrome P450 enzymes. The formation of oxysterols constitutes the first step in cholesterol metabolism, and they are ultimately converted to bile acids in the liver. Oxysterols are more membrane soluble than cholesterol, can cross the blood-brain barrier, and can be regarded as transport forms of cholesterol [1]. In addition, oxysterols have biological activity and have been shown in vitro to activate the liver X receptor [2]. Oxysterols have been suggested to have roles in cholesterol homeostasis and are implicated in neurological disorders including Alzheimer’s disease [3].

Oxysterols have traditionally been analysed by GC-MS following derivatisation, however their analysis is complicated by the fact that they are present in low levels against a large background of cholesterol. In this regard, analysis of oxysterols extracted from biological tissue would be improved by the introduction of an LC-ES-MS or LC-MALDI-MS method. In this paper, we describe the extraction, purification and analysis of oxysterols from rat brain by LC-ES-low-energy-MS/MS and MALDI-high-energy-MS/MS.

Experimental

Oxysterols were extracted from rat brain homogenate, and purified by a combination of anion-exchange and straight-phase separation steps. The resulting oxysterol fraction was treated with cholesterol oxidase to convert 3ß-hydroxy-Δ⁵ sterols to their 3-oxo-Δ⁴ analogues. These sterols were then derivatised with Girard P (GP) reagent to give Girard P hydrazones [4]. The resulting oxysterol GP hydrazones were separated from excess reagent on a C18 cartridge.

Mass Spectrometry. With out any further treatment 2 μL aliquots of purified GP hydrazones, corresponding to the oxysterols content of 200 μg of brain, were analysed by nanoscale-LC-MS/MS on a Q-TOF Ultima. MS/MS spectra were recorded at a collision energy of 35 eV, with Ar as the collision gas. Samples were further analysed on a 4700 MALDI-TOF/TOF instrument, in which case the collision gas was air, and the collision energy was 1 keV.

Results

The GP hydrazones were found to give three series of peaks corresponding to [M]⁺ ions of singly derivatised oxysterols, [M]²⁺ ions of doubly derivatised oxysterols, and [M-H]⁺ ions of doubly derivatised oxysterols. (Table 1). The oxysterols partially characterised are listed in Table 1.

The ES-low-energy-MS/MS spectra recorded on the Q-TOF give structural information with respect to the A- and B-rings [4] (see Fig. 2). Most of the spectra of the brain derived oxysterol GP hydrazones showed series of peaks characteristic of a 3-oxo-Δ4 GP structure, suggesting no further modification to the A- or B-rings. Samples were additionally analysed by MALDI-high-energy-MS/MS on the TOF/TOF, and the resulting spectra indicated the location of additional hydroxy and/or ketone groups (see Fig. 1).

Fig. 1. MALDI-MS/MS of m/z 534.		Fig. 2. ES-MS/MS of m/z 534.

To date, using the above methodology we have partially characterised eight oxysterols in rat brain.

[1] Björkhem I, Meaney S, Diczfalusy U, Curr Opin Lipidol 1999; 10: 161-165.
[2] Lehmann JM, Kliewer SA, Moore LB, Smith-Oliver TA, Oliver BB et al, J Biol Chem 1997; 272: 3137-3140.
[3] Heverin M, Bogdanovic N, Lütjohann D, Bayer T, Pikuleva I, Bretillon L, Diczfalusy U, Winblad B, Björkhem I. J. Lipid Res 2003: 45: 186-193.
[4] Griffiths WJ, Liu S, Alvelius G, Sjövall J, Rapid Commun Mass Spectrom 2003; 17: 924-935.