title: Prostaglandin extraction and analysis in Caenorhabditis elegans.
abstract: Caenorhabditis elegans is emerging as a powerful animal model to study the biology of lipids(1-9). Prostaglandins are an important class of eicosanoids, which are lipid signals derived from polyunsaturated fatty acids (PUFAs)(10-14). These signalling molecules are difficult to study because of their low abundance and reactive nature. The characteristic feature of prostaglandins is a cyclopentane ring structure located within the fatty acid backbone. In mammals, prostaglandins can be formed through cyclooxygenase enzyme-dependent and -independent pathways(10,15). C. elegans synthesizes a wide array of prostaglandins independent of cyclooxygenases(6,16,17). A large class of F-series prostaglandins has been identified, but the study of eicosanoids is at an early stage with ample room for new discoveries. Here we describe a procedure for extracting and analyzing prostaglandins and other eicosanoids. Charged lipids are extracted from mass worm cultures using a liquid-liquid extraction technique and analyzed by liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The inclusion of deuterated analogs of prostaglandins, such as PGF2 α-d4 as an internal standard is recommended for quantitative analysis. Multiple reaction monitoring or MRM can be used to quantify and compare specific prostaglandin types between wild-type and mutant animals. Collision-induced decomposition or MS/MS can be used to obtain information on important structural features. Liquid chromatography mass spectrometry (LC-MS) survey scans of a selected mass range, such as m/z 315-360 can be used to evaluate global changes in prostaglandin levels. We provide examples of all three analyses. These methods will provide researchers with a toolset for discovering novel eicosanoids and delineating their metabolic pathways.
authors.affiliation: Department of Pharmacology and Toxicology, University of Alabama at Birmingham, AL, USA.
authors.forename: Jeevan K
authors.initials: JK
authors.lastname: Prasain
authors.forename: Hieu D
authors.initials: HD
authors.lastname: Hoang
authors.forename: Johnathan W
authors.initials: JW
authors.lastname: Edmonds
authors.forename: Michael A
authors.initials: MA
authors.lastname: Miller
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000737:chemistry*; D002853:Chromatography, Liquid / Q000379:methods; D015777:Eicosanoids / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D011453:Prostaglandins / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D015777:Eicosanoids; D011453:Prostaglandins