F₃-Isoprostanes as a measure of in vivo oxidative damage in Caenorhabditis elegans

Oxidative stress has been implicated in the development of a wide variety of disease processes, including cardiovascular disease, cancer, and neurodegenerative diseases, as well as progressive and normal aging processes. Isoprostanes (IsoPs) are prostaglandin-like compounds that are generated in vivo from lipid peroxidation of arachidonic acid (AA, C20:4, ω-6) and other polyunsaturated fatty acids (PUFA). Since the discovery of IsoPs by Morrow and Roberts in 1990, quantification of IsoPs has been shown to be an excellent source of biomarkers of in vivo oxidative damage. Eicosapentaenoic acid (EPA, C20:5, ω-3) is the most abundant PUFA in Caenorhabditis elegans and gives rise to F₃-IsoPs upon nonenzymatic free-radical-catalyzed lipid peroxidation. The protocol presented is the current methodology that our laboratory uses to quantify F₃-IsoPs in C. elegans using gas chromatography/mass spectrometry (GC/MS). The methods described herein have been optimized and validated to provide the best sensitivity and selectivity for quantification of F₃-IsoPs from C. elegans lysates.