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

Khushboo Borah; Olivia J. Rickman; Nikol Voutsina; Isaac Ampong; Dan Gao; Emma L. Baple; Irundika Hk. Dias; Andrew H. Crosby; Helen R. Griffiths

doi:10.1016/j.redox.2020.101595

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

Khushboo Borah, Olivia J. Rickman, Nikol Voutsina, Isaac Ampong, Dan Gao, Emma L. Baple, Irundika Hk. Dias, Andrew H. Crosby, Helen R. Griffiths

Aston Medical School

Research output: Contribution to journal › Article › peer-review

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.

Original language	English
Article number	101595
Journal	Redox Biology
Volume	36
Early online date	1 Jun 2020
DOIs	https://doi.org/10.1016/j.redox.2020.101595
Publication status	Published - Sept 2020

Bibliographical note

Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)

Keywords

Blood
Brain oxysterol
Cholesterol
Dihydroxycholesterol
Liquid chromatography-mass spectrometry
Metabolism
Mitochondria
Monocytes
Neuroblastoma
Oxysterol
Peripheral blood mononuclear cell
Subcellular
Whole cell

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10.1016/j.redox.2020.101595Licence: CC BY-NC-ND 3.0

A quantitative LC-MS/MS method for analysis of mitochondrial -specific oxysterol metabolism
Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)
Accepted author manuscript, 3.1 MBLicence: CC BY-NC-ND 3.0

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@article{b6af1b0436e946938adafb42282ca614,

title = "A quantitative LC-MS/MS method for analysis of mitochondrial -specific oxysterol metabolism",

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.",

keywords = "Blood, Brain oxysterol, Cholesterol, Dihydroxycholesterol, Liquid chromatography-mass spectrometry, Metabolism, Mitochondria, Monocytes, Neuroblastoma, Oxysterol, Peripheral blood mononuclear cell, Subcellular, Whole cell",

author = "Khushboo Borah and Rickman, {Olivia J.} and Nikol Voutsina and Isaac Ampong and Dan Gao and Baple, {Emma L.} and Dias, {Irundika Hk.} and Crosby, {Andrew H.} and Griffiths, {Helen R.}",

note = "Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)",

year = "2020",

month = sep,

doi = "10.1016/j.redox.2020.101595",

language = "English",

volume = "36",

journal = "Redox Biology",

issn = "2213-2317",

publisher = "Elsevier",

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TY - JOUR

T1 - A quantitative LC-MS/MS method for analysis of mitochondrial -specific oxysterol metabolism

AU - Borah, Khushboo

AU - Rickman, Olivia J.

AU - Voutsina, Nikol

AU - Ampong, Isaac

AU - Gao, Dan

AU - Baple, Emma L.

AU - Dias, Irundika Hk.

AU - Crosby, Andrew H.

AU - Griffiths, Helen R.

N1 - Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0)

PY - 2020/9

Y1 - 2020/9

N2 - 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.

AB - 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.

KW - Blood

KW - Brain oxysterol

KW - Cholesterol

KW - Dihydroxycholesterol

KW - Liquid chromatography-mass spectrometry

KW - Metabolism

KW - Mitochondria

KW - Monocytes

KW - Neuroblastoma

KW - Oxysterol

KW - Peripheral blood mononuclear cell

KW - Subcellular

KW - Whole cell

UR - https://linkinghub.elsevier.com/retrieve/pii/S2213231720305632

UR - http://www.scopus.com/inward/record.url?scp=85086643431&partnerID=8YFLogxK

U2 - 10.1016/j.redox.2020.101595

DO - 10.1016/j.redox.2020.101595

M3 - Article

C2 - 32574926

SN - 2213-2317

VL - 36

JO - Redox Biology

JF - Redox Biology

M1 - 101595

ER -

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

Abstract

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Keywords

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