Characterisation of novel oxysterols by mass spectrometry

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Oxysterols are oxygenated derivatives of cholesterol. They are intermediates in cholesterol excretion pathways and may also be regarded as transport forms of cholesterol. The introduction of additional hydroxyl groups to the cholesterol skeleton facilities the flux of oxysterols between tissue and blood. Oxysterols have been implicated in mediating a number of cholesterol-induced metabolic effects. It has been suggested that by interacting with the orphan nuclear receptors, LXR alpha and LXR beta, oxysterols may be involved in the control of cholesterol balance by affecting cholesterol synthesis, absorption and metabolism. Endogenous oxysterols are difficult to analyse by modern techniques, not only are they present at very low levels, but they are also present amongst a very large excess of cholesterol (approximately 0.1ng oxysterol per micro g cholesterol). In the proposed study we will develop new mass spectrometry (MS) based methods to allow the identification, characterisation and quantification of known and novel endogenous oxysterols. Initially it will be necessary to develop an extraction and purification scheme to obtain an oxysterol rich fraction from rat tissue (or human plasma). The oxysterol fraction will then be analysed by MS. Using modern MS methods oxysterols are very difficult to analyse in that they are poorly ionised by both atmospheric pressure ionisation eg. ESI, and by MALDI. However, we have recently developed a derivatisation protocol suitable for 3-oxo-4-ene steroids which allows their ESI- or MALDI-MS analysis on the sub pg level. Most oxysterols do not posses a 3-oxo-4-ene group, but can easily be converted to 3-oxo-4-ene analogues by the action of cholesterol oxidase. They can then be subsequently derivatised for ESI-(or MALDI-) MS analysis. It will be necessary to optimise both of the oxidation and derivatisation steps for the compatibility of oxysterol analysis with ESI-MS. For oxysterols unsuitable for oxidation with cholesterol oxidase alternative derivatisation methods based on derivatisation of the alcohol group will be employed. The preferred technique for the analysis of low levels of biomolecule mixtures extracted from biological matrixes is nano-scale-LC-ESI-MS MS (this is preferable to GC-MS on account of the higher sample capacity of the LC column, and the compatibility of LC-ESI to the analysis of previously unidentified conjugated oxysterols). It will be necessary to develop a nano-LC-ESI-MS MS method which is suitable for derivatised oxosterols and will differentiate between oxosterol stereoisomers. Once an LC-MS MS method for oxysterol analysis is optimised, quantitative studies will be performed using stable-isotope dilution methodology. Synthesis of stable-isotope labelled and reference oxysterols will be performed where necessary. Once identified, the interaction between oxysterols and the LXR will be investigated using mass spectrometry methods. We have recently developed mass spectrometry based assays to investigate ligand ¿ nuclear receptor interactions, and also to pull-out ligands from biological extracts. We will employ these methods in the study.