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.
Highlights
Reactive oxygen species (ROS) are formed during normal cellular metabolism and it is becoming increasingly apparent that they have an important, yet complicated role in biology and pathology [1,2]
F2-isoprostanes are formed from Arachidonic acid (AA) esterified to phospholipids and to confirm this, we subjected arachidonyl PAF-C16, a glycerol-3-phosphocholine that contains AA to in vitro copper-induced lipid peroxidation and monitored F2isoprostane formation using mass spectrometry
Copperinduced lipid peroxidation of arachidonyl PAF-C16 leads to the formation of lipid peroxyl radicals which initiate a chain of lipid peroxidation reactions resulting in F2-isoprostane formation [2,16,17]
Summary
Reactive oxygen species (ROS) are formed during normal cellular metabolism and it is becoming increasingly apparent that they have an important, yet complicated role in biology and pathology [1,2]. The mitochondria are the main source of ROS production during the process of oxidative phosphorylation [3]. Other sources of ROS include fatty acid oxidation in the peroxisomes and enzyme complexes like NADPH oxidase [4]. As a result of the high reactivity of these molecules, they readily react with DNA, proteins and lipids to cause oxidative damage, thereby altering their function towards pathology. It has recently become clear that ROS and even ROS damage can act as secondary messengers in signal transduction in important metabolic pathways [5,6,7]
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