Redox Biology 36 (2020) 101595 Contents lists available at ScienceDirect Redox Biology journal homepage: www.elsevier.com/locate/redox A quantitative LC-MS/MS method for analysis of mitochondrial -specific T oxysterol metabolism Khushboo Boraha, Olivia J. Rickmanb, Nikol Voutsinab, Isaac Amponga, Dan Gaoc, ∗ Emma L. Bapleb, Irundika HK. Diasd, Andrew H. Crosbyb, Helen R. Griffithsa, a Department of Nutrition, Faculty of Health and Medical Sciences, University of Surrey, Guildford, GU2 7XH, UK b University of Exeter Medical School, RILD Building, RD&E Hospital Wonford, Barrack Road, Exeter, EX2 5DW, UK c Department of Human Anatomy,Histology and Embryology, School of Basic Medical Sciences, Xi'an Jiaotong University Health Science Center, Xi'an, 710061, China d Aston Medical School, Aston University, Birmingham, B4 7ET, UK ARTICLE INFO ABSTRACT Keywords: Oxysterols are critical regulators of inflammation and cholesterol metabolism in cells. They are oxidation pro- Oxysterol ducts of cholesterol and may be differentially metabolised in subcellular compartments and in biological fluids. Cholesterol New analytical methods are needed to improve our understanding of oxysterol trafficking and the molecular Subcellular interplay between the cellular compartments required to maintain cholesterol/oxysterol homeostasis. Here we Metabolism describe a method for isolation of oxysterols using solid phase extraction and quantification by liquid chro- Monocytes matography-mass spectrometry, applied to tissue, cells and mitochondria. Neuroblastoma Mitochondria We analysed five monohydroxysterols; 24(S)-hydroxycholesterol, 25-hydroxycholesterol, 27-hydro- Blood xycholesterol, 7α-hydroxycholesterol, 7 ketocholesterol and three dihydroxysterols 7α-24(S)dihydrox- Liquid chromatography-mass spectrometry ycholesterol, 7α-25dihydroxycholesterol, 7α-27dihydroxycholesterol by LC-MS/MS following reverse phase Whole cell chromatography. Our new method, using Triton and DMSO extraction, shows improved extraction efficiency and Dihydroxycholesterol recovery of oxysterols from cellular matrix. We validated our method by reproducibly measuring oxysterols in Peripheral blood mononuclear cell mouse brain tissue and showed that mice fed a high fat diet had significantly lower levels of 24S/25diOHC, Brain oxysterol 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 oxy- sterol composition in clinical samples. In addition, in vitro cell culture systems, of THP-1 monocytes and neu- ronal-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 lin- earity and detection, reproducibility and selectivity for cellular oxysterol analysis. This method enables sub- cellular oxysterol metabolism to be monitored and is versatile in its application to various biological and clinical samples. 1. Introduction dihydroxycholesterol (27diOHC) [4]. Hydroxylation of the hydrocarbon rings occurs by oxygen free radical attack forming hydroxylated sterols 7α- Oxysterols are biologically important molecules that regulate cell sig- hydroxycholesterol (7αOHC) and 7β-hydroxycholesterol, oxysterols with a nalling, contribute to regulating cholesterol homeostasis and are essential ketone group 7-ketocholesterol (7ketoOHC), epoxy cholesterols [5β, 6β- for bile acid and steroid hormone biosynthesis [1]. Oxysterols are formed epoxy cholesterol (5β, 6β-epox), 5α, 6α-epoxy cholesterol (5α, 6α-epox) by oxidation and addition of hydroxyl groups onto cholesterol hydro- and cholestan-3β, 5α, 6β-triol [5,6]. carbon rings and side chains [2,3]. Enzymatic catalysed additions of hy- The cellular localisation of enzymes responsible for oxysterol gen- droxyl to the side chain by cytochrome P450 (CYP) enzymes generate eration is critical to local oxysterol homeostasis [2]. In addition, the mono- and di-hydroxysterols; 24(S)-hydroxycholesterol (24OHC), 25-hy- redox states of subcellular compartments are different; the endoplasmic droxycholesterol (25OHC), 27-hydroxycholesterol (27OHC), 20-hydro- reticulum (ER) has a strong reducing environment to enable protein xycholesterol, 22-hydroxycholesterol, 7α-24(S)-dihydroxycholesterol folding, whereas the mitochondria stores glutathione but is exposed to (24SdiOHC), 7α-25-dihydroxycholesterol (25diOHC) and 7α-27- greater potential for oxidation from reactive oxygen species (ROS) ∗ Corresponding author. E-mail address: [email protected] (H.R. Griffiths). https://doi.org/10.1016/j.redox.2020.101595 Received 11 April 2020; Received in revised form 15 May 2020; Accepted 22 May 2020 Available online 01 June 2020 2213-2317/ © 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). K. Borah, et al. Redox Biology 36 (2020) 101595 leakage by the electron transport chain, increasing the potential for Oxysterol content also differs according to the cell type, tissue and local cholesterol autoxidation [2]. Interaction between endosomes, ER, organism [2]. The different structural composition of discrete cells, mitochondria, cell membrane, peroxisomes and lysosomes is necessary tissue and biological fluids requires that the methods for isolation of for cholesterol and oxysterol trafficking and sterol homeostasis in- lipids and oxysterols are tailored for the biological sample under study. tracellularly; for instance, cholesterol is transported to mitochondria To date, no comprehensive method for oxysterol quantification at cel- from the ER either by direct membrane contact between ER and mi- lular and subcellular levels has been reported. In this study, we describe tochondria, or mobilised in lipid droplets (cholesterol esters) [7,8]. In the isolation of mitochondria from whole cells, followed by isolation of mitochondria, the steroidogenic acute regulatory protein (StAR) lipids and oxysterols from mitochondria. We adapted our LC-MS/MS transports cholesterol from the outer to inner membrane and controls method for characterisation of mono and dihydroxycholesterols from mitochondrial sterol trafficking [7,9]. Less is known about oxysterol mitochondria. trafficking between the subcellular compartments [2]. Here we have compared our newly developed method to two pre- Changes to oxysterol levels are implicated in the pathogenesis of viously published methods for extraction of lipids in mitochondrial several diseases and targeting cholesterol and oxysterol dyshomeostasis isolates from tissues by Bird et al., 2013 [38] and for measuring free may provide a platform for development of novel therapeutics. oxysterols in plasma by Dias et al., 2018 [29]. Our newly developed Dysregulation of cholesterol metabolism has been associated with me- method shows improved recovery, selectivity and highly sensitive de- tabolic diseases such as cancer, heart and liver diseases and motor tection and quantification of oxysterols from discrete cellular com- neurone diseases [2,10,11]. partments. Oxysterol distribution in whole cell and mitochondrial iso- Oxysterols are known to be cytotoxic at high concentration; accu- lates were profiled in THP-1 monocytes, SH-SY5Y neuroblastoma cell mulation of 7-ketoOHC in lysosomes triggered membrane destabiliza- and peripheral blood mononuclear cells (PBMC) isolated from whole tion and induced cell death in human monocytic U937 cells [12]. At a blood of healthy individuals. Our analysis showed differential oxysterol subcellular level, accumulation of 7-ketoOHC and 7β-hydro- distribution across cell types indicating cell and tissue specific oxysterol xycholesterol by cholesterol autoxidation caused redox imbalance and metabolism. Furthermore, measurement of mitochondrial specific peroxisomal dysfunction in nerve cells [13,14]. Mitochondrial dys- oxysterol profile indicated local metabolism of oxysterols that was function and aberrant oxysterol metabolism have been recognized in distinct from the whole cell and endoplasmic reticulum. This method the pathogenesis of diseases such as atherosclerosis, carcinogenesis and enables quantification of oxysterols in mitochondria to elucidate com- multiple neurodegenerative diseases including Alzheimer's disease partment specific metabolism in clinical samples. [15–20]. In vitro studies have shown that the addition of 7β-hydro- xysitosterol and 7β-OHC induced loss of mitochondrial membrane po- 2. Materials and methods tential, membrane permeabilisation, oxidative stress and apoptosis in human colon cancer, Caco-2 cells [21]. Again, high concentrations of 2.1. Ethical statement for blood and mouse tissue samples 24OHC caused necroptosis in human neuroblastoma SH-SY5Y cells whereas sublethal concentrations of 24OHC induced protection against Blood samples were collected from healthy volunteers with ethics cytotoxic stress in these cells [22,23]. Similarly, accumulation of committee approval and with informed consent. 25OHC has been shown to inhibit inflammasome activation in macro- The mouse experimental procedures were conducted under the phages [24]. In neurodegenerative conditions, such as Alzheimer's State Council of the People's Republic of China (Decree No. 2 of the disease, oxysterol levels are linked to neuroinflammation, mitochon- State Science and Technology Commission) October 31, 1988. drial oxidative