LC-MS/MS method for rapid and concomitant quantification of pro-inflammatory and pro-resolving polyunsaturated fatty acid metabolites

Abstract

Lipid autacoids derived from n-3/n-6 polyunsaturated fatty acids (PUFA) are some of the earliest signals triggered by an inflammatory reaction. They are acting also as essential regulators of numerous biological processes in physiological conditions. With regards to their importance, a robust and rapid procedure to quantify a large variety of PUFA metabolites, applicable to diverse biological components needed to be formulated. We have developed a simple methodology using liquid chromatography-tandem mass spectrometry allowing quantification of low-level of PUFA metabolites including bioactive mediators, inactive products and pathway biomarkers. Solid phase extraction was used for samples preparation with an extraction yield of 80% ranging from 65% to 98%. The method was optimized to obtain a rapid (8.5min) and accurate separation of 26 molecules, with a very high sensitivity of detection and analysis (0.6-155pg). When applied to biological samples, the method enabled characterization of eicosanoids and docosanoids production in epithelial cells or foam macrophages stimulated with LPS, in biological fluids and tissues from mouse models of peritonitis or infectious colitis. Our results demonstrate that this new method can be used in cultured cells, in fluids and in colonic tissues to quantify pro-inflammatory and pro-resolving PUFA metabolites mediators.

Keywords: 10(S),17(S)-protectin; 18-HEPE; 18-hydroxyeicosapentaenoic acid; 5,6-DiHETE; 5-oxo-ETE; 5-oxoeicosatetraenoic acid; 6-keto-prostaglandin F1α; 6kPGF(1α); 7(S)-maresin; 7-MaR1; AA; ACN; BHT; COX; CYP; Citrobacter rodentium; DHA; Docosanoid; EET; EPA; Eicosanoid; FA; Foam macrophage; HBSS; HDoHE; HETE; Hank's Balanced Salt Solution; IBU; IS; LC–MS/MS; LOD; LOQ; LOX; LPS; LTB(4); LTB(4)-d4; LTB(5); LxA(4); LxA(4)-d5; LxB(4); MH Quant; MPO; Mass Hunter Quantitative; MeFor; MeOH; PBMC; PDx; PGE(2); PGE(3); PMA; Peritonitis; RvD1; S/N; SFM; SPE; SRM; Selected Reaction Monitoring; TXB(2); ZIL; ZYM; acetonitril; arachidonic acid; butylated hydroxytoluene; cyclooxygenase; cytochrome P450; dihydroxy-eicosatetraenoic acid; docosahexaenoic acid; eicosapentaenoic acid; epoxyeicosatrienoic acid; formic acid; hydroxy-docosahexaenoic acid; hydroxyeicosatetraenoic acid; ibuprofen; internal standard; leukotriene B4; leukotriene B4 deuterated; leukotriene B5; limit of detection; limit of quantification; lipopolysaccharides; lipoxin A4; lipoxin A4 deuterated; lipoxin B4; lipoxygenase; methanol; methyl formate; myeloperoxidase; oxLDL; oxidized LDL; peripheral blood mononuclear cells; phorbol myristate acetate; prostaglandin E2; prostaglandin E3; resolvin D1; serum-free medium; signal to noise ratio; solid-phase extraction; thromboxane B2; zileuton; zymosan A.