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 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.
University Medical Center Utrecht, Department of Metabolic Diseases and Netherlands Metabolomics Center, Utrecht, The Netherlands.
Christiaan F
CF
Labuschagne
Niels J F
NJ
van den Broek
Pjotr
P
Postma
Ruud
R
Berger
Arjan B
AB
Brenkman
D016718:Arachidonic Acid / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D028441:F2-Isoprostanes / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D056945:Hep G2 Cells; D006801:Humans; D015227:Lipid Peroxidation / Q000502:physiology; D053719:Tandem Mass Spectrometry
D028441:F2-Isoprostanes; D016718:Arachidonic Acid