Measurement of isoprostanes as markers of oxidative stress

Oxidative stress results from an imbalance between production of reactive oxygen and nitrogen species (ROS and RNS, respectively) and endogenous antioxidant defense mechanisms. Increased generation of ROS/RNS is implicated in the pathogenesis of a variety of human diseases, including neurodegenerative disease, atherosclerosis, cancer, and aging. However, measuring oxidative stress in biological systems is complex and requires accurate quantification of either free radicals or damaged biomolecules. One method to quantify oxidative injury is to measure lipid peroxidation. Lipids are readily attacked by free radicals, resulting in the formation of a number of peroxidation products. F₂-isoprostanes (F₂-IsoPs) are one group of these compounds and they are derived by the free radical peroxidation of arachidonic acid (AA). The F₂-IsoPs, prostaglandine F₂-like compounds, provide an accurate measure of oxidative stress both in vitro and in vivo. This protocol details current methodology used to quantify these molecules using gas chromatography-mass spectrometry (GC/MS).