F₃‐Isoprostanes as a Measure of in vivo Oxidative Damage in Caenorhabditis elegans

Abstract

Oxidative stress has been implicated in 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 excellent biomarkers of in vivo oxidative damage. Eicosapentaenoic acid (EPA, C20:5, ω-3) is the most abundant PUFA in C. 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 has been optimized and validated to provide the best sensitivity and selectivity for quantification of F3-IsoPs from C. elegans lysates.

Scavengers of reactive γ-ketoaldehydes extend Caenorhabditis elegans lifespan and healthspan through protein-level interactions with SIR-2.1 and ETS-7

“…To quantify oxidant injury, we measured F 3 -isoprostanes (F 3 -IsoPs). F 3 -IsoPs are the products of free radical-mediated peroxidation of eicosapentaenoic acid (EPA), and are known to be a highly sensitive and accurate marker of oxidative damage in Caenorhabditis elegans [ 63 – 65 ]. F 3 -IsoPs were collected from WT N2 and from VC199 (SIR-2.1 deficient strain) nematodes grown on OP50-NGM agar plates containing increasing concentrations of SA from Day 1 of adulthood until collection at Day 15.…”

Application of Caenorhabditis elegans in Lipid Metabolism Research

“…In general, the fatty acid methyl ester (FAME) derivatization of whole worms has been applied to analyze the individual fatty acid abundances of all lipid species [ 227 ]. However, to analyze the abundance of fatty acids within specific lipids and calculate the neutral lipid stores, the separation and enrichment of one type of lipid can be achieved by a solid phase extraction (SPE) pretreatment and preparative chromatography such as TLC [ 53 , 228 ], high performance liquid chromatography (HPLC) [ 229 ], and ultra-performance liquid chromatography (UPLC) [ 230 ]. The chromatographically separated lipids are subsequently subjected to FAME derivatization.…”