A Protocol for Quantifying Lipid Peroxidation in Cellular Systems by F2-Isoprostane Analysis
Open Access
- 14 November 2013
- journal article
- research article
- Published by Public Library of Science (PLoS) in PLOS ONE
- Vol. 8 (11), e80935
- https://doi.org/10.1371/journal.pone.0080935
Abstract
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-iPF2α-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.This publication has 27 references indexed in Scilit:
- Redox-Dependent Control of FOXO/DAF-16 by Transportin-1Molecular Cell, 2013
- Isoprostane Generation and FunctionChemical Reviews, 2011
- Unraveling the Biological Roles of Reactive Oxygen SpeciesCell Metabolism, 2011
- A Mitochondrial Superoxide Signal Triggers Increased Longevity in Caenorhabditis elegansPLoS Biology, 2010
- Two modes of mitochondrial dysfunction lead independently to lifespan extension in Caenorhabditis elegansAging Cell, 2010
- How mitochondria produce reactive oxygen speciesBiochemical Journal, 2008
- Biomarkers of Oxidative Stress Study II: Are oxidation products of lipids, proteins, and DNA markers of CCl4 poisoning?Free Radical Biology & Medicine, 2005
- Factors Regulating Isoprostane Formation In VivoAntioxidants and Redox Signaling, 2005
- Control of Cell Cycle Exit and Entry by Protein Kinase B-Regulated Forkhead Transcription FactorsMolecular and Cellular Biology, 2002
- Effects of 2,2′-azobis (2-amidinopropane) hydrochloride in vivo and protection by vitamin EFree Radical Biology & Medicine, 1990