Karu, Kersti; (2009) The analysis of oxysterols by capillary liquid chromatography tandem mass spectrometry. Doctoral thesis (Ph.D.), University College London (United Kingdom).

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Abstract

Oxysterols are oxygenated metabolites of cholesterol. They are present in very low concentrations in mammalian systems, always accompanied by high excess of cholesterol. Therefore, their analysis can be challenging. The traditional method for the analysis of sterols is by gas-chromatography (GC)-mass spectrometry (MS), however the absence of molecular ions in GC-MS spectra, can make the identification of novel oxysterols difficult. A capillary liquid chromatography multi-stage fragmentation mass spectrometry (cap-LC-MSn) methodology has been developed for the identification, with high sensitivity (0.8 pg on-column) and specificity of oxysterols in mammalian central nervous system (CNS), and cortical neurons. Oxysterols were extracted with ethanol, separated from cholesterol by straight-phase chromatography, 3?- hydroxysterols were oxidised with cholesterol oxidase to their corresponding 3-ketones, which were then derivatised with Girard P (GP) hydrazine. The oxidised/GP-derivatised oxysterols were separated by reversed-phase LC, and ES-MS, -MS2 -MS3 were recorded to allow their characterisation. Forty authentic reference sterols were oxidised, GP-derivatised, and analysed by cap-LC-MSn. The identification of sterols in tissues/cells was based on comparison of their retention time, MS2, MS3 spectra with authentic standards. Twenty four sterols were identified in rat brain. 24S-Hydroxycholesterol was confirmed as a major oxysterol (17-24 μg/g). 24S- Hydroxycholesterol is biosynthesised exclusively in brain by the action of CYP46A1, other oxysterols identified in brain tissue included 24,25-, 24,27-, 25,27-, 6,24-, 7α,25-, and 7α,27- dihydroxycholesterols. Nineteen sterols were identified in three regions of embryonic (E11) mouse CNS. Hydroxycholesterols were expressed at low levels (estimated 4-165 ng/g) in the developing CNS, including 24S-hydroxycholesterol. 24S,25-Epoxycholesterol was present at relatively high levels, suggesting that the mevalonate pathway is significantly active at E11. Seventeen sterols were identified in rat cortical neurons, including 24S,25-epoxycholesterol. The methodology was further evaluated for the prenatal diagnosis of Smith-Lemli-Opitz syndrome (SLOS) by measuring sterols in amniotic fluids and blood spots from SLOS affected patients. In conclusion, the profiling of oxysterol/sterol levels may offer important information for a better understanding of neurodegenerative disorders and to diagnose disease.

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