Oxysterols can contribute to proliferation of breast cancer through activation of the Estrogen Receptors, and to metastasis through activation of the Liver X Receptors. Endogenous levels of both esterified and free sidechain-hydroxylated oxysterols were examined in breast cancer tumours from Estrogen Receptor positive and negative breast tumours, using a novel fast liquid chromatography tandem mass spectrometry method. Multiple aliquots of five milligram samples of 22 tumours were analysed for oxysterol content to assess intra- and inter-tumour variation. Derivatization was performed with Girard T reagent (with and without alkaline hydrolysis) and sample clean-up was performed using a robust automatic on-line column switching system (“AFFL”). Oxysterols were separated isocratically on a 2.1 mm inner diameter column packed with ACE SuperPhenylHexyl core shell particles using a mobile phase consisting of 0.1% formic acid in H2O/methanol/acetonitrile (57/10/33, v/v/v) followed by a wash out step (0.1% formic acid in methanol/acetonitrile, 50/50, v/v). The total analysis time, including sample clean-up and column reconditioning, was 8 min (80% time reduction compared to other on-line systems). Analysis revealed large intra-tumour variations of sidechain oxysterols, resulting in no significant differences in endogenous oxysterols levels between Estrogen Receptor positive and Estrogen Receptor negative breast cancers. However, a correlation between esterified and free 27-hydroxycholesterol was observed. The same correlation was not observed for 24S-hydroxycholesterol or 25-hydroxycholesterol. The oxysterol heterogeneity of tumour tissue is a critical factor when assessing the role of these lipids in cancer.
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