A quantitative LC-MS/MS method for analysis of mitochondrial -specific oxysterol metabolism.

Oxysterols are critical regulators of inflammation and cholesterol metabolism in cells. They are oxidation products of cholesterol and may be differentially metabolised in subcellular compartments and in biological fluids. New analytical methods are needed to improve our understanding of oxysterol trafficking and the molecular interplay between the cellular compartments required to maintain cholesterol/oxysterol homeostasis. Here we describe a method for isolation of oxysterols using solid phase extraction and quantification by liquid chromatography-mass spectrometry, applied to tissue, cells and mitochondria. We analysed five monohydroxysterols; 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 27-hydroxycholesterol, 7α-hydroxycholesterol, 7 ketocholesterol and three dihydroxysterols 7α-24(S)dihydroxycholesterol, 7α-25dihydroxycholesterol, 7α-27dihydroxycholesterol by LC-MS/MS following reverse phase chromatography. Our new method, using Triton and DMSO extraction, shows improved extraction efficiency and recovery of oxysterols from cellular matrix. We validated our method by reproducibly measuring oxysterols in mouse brain tissue and showed that mice fed a high fat diet had significantly lower levels of 24S/25diOHC, 27diOHC and 7ketoOHC. We measured oxysterols in mitochondria from peripheral blood mononuclear cells and highlight the importance of rapid cell isolation to minimise effects of handling and storage conditions on oxysterol composition in clinical samples. In addition, in vitro cell culture systems, of THP-1 monocytes and neuronal-like SH-SH5Y cells, showed mitochondrial-specific oxysterol metabolism and profiles were lineage specific. In summary, we describe a robust and reproducible method validated for improved recovery, quantitative linearity and detection, reproducibility and selectivity for cellular oxysterol analysis. This method enables subcellular oxysterol metabolism to be monitored and is versatile in its application to various biological and clinical samples.