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A Protocol for Quantifying Lipid Peroxidation in Cellular Systems by F2-Isoprostane Analysis

Cellular systems are essential model systems to study reactive oxygen species and oxidative damage but there are widely accepted technical difficulties with available methods for quantifying endogenous oxidative damage in these systems. Here we present a stable isotope dilution UPLC-MS/MS protocol f...

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Autores principales: Labuschagne, Christiaan F., van den Broek, Niels J. F., Postma, Pjotr, Berger, Ruud, Brenkman, Arjan B.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2013
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3828286/
https://www.ncbi.nlm.nih.gov/pubmed/24244726
http://dx.doi.org/10.1371/journal.pone.0080935
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author Labuschagne, Christiaan F.
van den Broek, Niels J. F.
Postma, Pjotr
Berger, Ruud
Brenkman, Arjan B.
author_facet Labuschagne, Christiaan F.
van den Broek, Niels J. F.
Postma, Pjotr
Berger, Ruud
Brenkman, Arjan B.
author_sort Labuschagne, Christiaan F.
collection PubMed
description Cellular systems are essential model systems to study reactive oxygen species and oxidative damage but there are widely accepted technical difficulties with available methods for quantifying endogenous oxidative damage in these systems. Here we present a stable isotope dilution UPLC-MS/MS protocol for measuring F2-isoprostanes as accurate markers for endogenous oxidative damage in cellular systems. F2-isoprostanes are chemically stable prostaglandin-like lipid peroxidation products of arachidonic acid, the predominant polyunsaturated fatty acid in mammalian cells. This approach is rapid and highly sensitive, allowing for the absolute quantification of endogenous lipid peroxidation in as little as ten thousand cells as well as damage originating from multiple ROS sources. Furthermore, differences in the endogenous cellular redox state induced by transcriptional regulation of ROS scavenging enzymes were detected by following this protocol. Finally we showed that the F2-isoprostane 5-iPF(2α)-VI is a metabolically stable end product, which is excreted from cells. Overall, this protocol enables accurate, specific and sensitive quantification of endogenous lipid peroxidation in cellular systems.
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spelling pubmed-38282862013-11-16 A Protocol for Quantifying Lipid Peroxidation in Cellular Systems by F2-Isoprostane Analysis Labuschagne, Christiaan F. van den Broek, Niels J. F. Postma, Pjotr Berger, Ruud Brenkman, Arjan B. PLoS One Research Article Cellular systems are essential model systems to study reactive oxygen species and oxidative damage but there are widely accepted technical difficulties with available methods for quantifying endogenous oxidative damage in these systems. Here we present a stable isotope dilution UPLC-MS/MS protocol for measuring F2-isoprostanes as accurate markers for endogenous oxidative damage in cellular systems. F2-isoprostanes are chemically stable prostaglandin-like lipid peroxidation products of arachidonic acid, the predominant polyunsaturated fatty acid in mammalian cells. This approach is rapid and highly sensitive, allowing for the absolute quantification of endogenous lipid peroxidation in as little as ten thousand cells as well as damage originating from multiple ROS sources. Furthermore, differences in the endogenous cellular redox state induced by transcriptional regulation of ROS scavenging enzymes were detected by following this protocol. Finally we showed that the F2-isoprostane 5-iPF(2α)-VI is a metabolically stable end product, which is excreted from cells. Overall, this protocol enables accurate, specific and sensitive quantification of endogenous lipid peroxidation in cellular systems. Public Library of Science 2013-11-14 /pmc/articles/PMC3828286/ /pubmed/24244726 http://dx.doi.org/10.1371/journal.pone.0080935 Text en © 2013 Labuschagne et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Labuschagne, Christiaan F.
van den Broek, Niels J. F.
Postma, Pjotr
Berger, Ruud
Brenkman, Arjan B.
A Protocol for Quantifying Lipid Peroxidation in Cellular Systems by F2-Isoprostane Analysis
title A Protocol for Quantifying Lipid Peroxidation in Cellular Systems by F2-Isoprostane Analysis
title_full A Protocol for Quantifying Lipid Peroxidation in Cellular Systems by F2-Isoprostane Analysis
title_fullStr A Protocol for Quantifying Lipid Peroxidation in Cellular Systems by F2-Isoprostane Analysis
title_full_unstemmed A Protocol for Quantifying Lipid Peroxidation in Cellular Systems by F2-Isoprostane Analysis
title_short A Protocol for Quantifying Lipid Peroxidation in Cellular Systems by F2-Isoprostane Analysis
title_sort protocol for quantifying lipid peroxidation in cellular systems by f2-isoprostane analysis
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3828286/
https://www.ncbi.nlm.nih.gov/pubmed/24244726
http://dx.doi.org/10.1371/journal.pone.0080935
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