Abstract
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.
Highlights
Oxysterols are biologically important molecules that regulate cell signalling, contribute to regulating cholesterol homeostasis and are essential for bile acid and steroid hormone biosynthesis [1]
We isolated lipids and oxysterol pools from unfractionated whole cells and subcellular fractions (Fig. 1B and C) with authentic standards incorporated into whole cell (WC) lysate to mimic the effect of a cellular matrix, which may impair extraction efficiency
The first extraction method M1, which had been established for human plasma [29], involved protein precipitation with methanol followed by oxysterol isolation using solid phase extraction (SPE) with polymeric reversed-phase sorbent
Summary
Oxysterols are biologically important molecules that regulate cell signalling, contribute to regulating cholesterol homeostasis and are essential for bile acid and steroid hormone biosynthesis [1]. Oxysterols are formed by oxidation and addition of hydroxyl groups onto cholesterol hydrocarbon rings and side chains [2,3]. Enzymatic catalysed additions of hydroxyl to the side chain by cytochrome P450 (CYP) enzymes generate mono- and di-hydroxysterols; 24(S)-hydroxycholesterol (24OHC), 25-hydroxycholesterol (25OHC), 27-hydroxycholesterol (27OHC), 20-hydroxycholesterol, 22-hydroxycholesterol, 7α-24(S)-dihydroxycholesterol (24SdiOHC), 7α-25-dihydroxycholesterol (25diOHC) and 7α-27-. Hydroxylation of the hydrocarbon rings occurs by oxygen free radical attack forming hydroxylated sterols 7αhydroxycholesterol (7αOHC) and 7β-hydroxycholesterol, oxysterols with a ketone group 7-ketocholesterol (7ketoOHC), epoxy cholesterols [5β, 6βepoxy cholesterol (5β, 6β-epox), 5α, 6α-epoxy cholesterol (5α, 6α-epox) and cholestan-3β, 5α, 6β-triol [5,6]. The cellular localisation of enzymes responsible for oxysterol generation is critical to local oxysterol homeostasis [2]. The redox states of subcellular compartments are different; the endoplasmic reticulum (ER) has a strong reducing environment to enable protein folding, whereas the mitochondria stores glutathione but is exposed to greater potential for oxidation from reactive oxygen species (ROS)
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