Quantitative profiling of inflammatory and pro-resolving lipid mediators in human adolescents and mouse plasma using UHPLC-MS/MS

Abstract

Objectives: Lipid mediators are bioactive lipids which help regulate inflammation. We aimed to develop an ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to quantify 58 pro-inflammatory and pro-resolving lipid mediators in plasma, determine preliminary reference ranges for adolescents, and investigate how total parenteral nutrition (TPN) containing omega-3 polyunsaturated fatty acid (n-3 PUFA) or n-6 PUFA based lipid emulsions influence lipid mediator concentrations in plasma.

Methods: Lipid mediators were extracted from plasma using SPE and measured using UHPLC-MS/MS. EDTA plasma was collected from healthy adolescents between 13 and 17 years of age to determine preliminary reference ranges and from mice given intravenous TPN for seven days containing either an n-3 PUFA or n-6 PUFA based lipid emulsion.

Results: We successfully quantified 43 lipid mediators in human plasma with good precision and recovery including several leukotrienes, prostaglandins, resolvins, protectins, maresins, and lipoxins. We found that the addition of methanol to human plasma after blood separation reduces post blood draw increases in 12-hydroxyeicosatetraenoic acid (12-HETE), 12-hydroxyeicosapentaenoic acid (12-HEPE), 12S-hydroxyeicosatrienoic acid (12S-HETrE), 14-hydroxydocosahexaenoic acid (14-HDHA) and thromboxane B2 (TXB2). Compared to the n-6 PUFA based TPN, the n-3 PUFA based TPN increased specialized pro-resolving mediators such as maresin 1 (MaR1), MaR2, protectin D1 (PD1), PDX, and resolvin D5 (RvD5), and decreased inflammatory lipid mediators such as leukotriene B4 (LTB4) and prostaglandin D2 (PGD2).

Conclusions: Our method provides an accurate and sensitive quantification of 58 lipid mediators from plasma samples, which we used to establish a preliminary reference range for lipid mediators in plasma samples of adolescents; and to show that n-3 PUFA, compared to n-6 PUFA rich TPN, leads to a less inflammatory lipid mediator profile in mice.

Keywords: AA; AEA; ALOX12; COX; CYP; D-series resolvin; DGLA; DHA; DHEA; DPA; DiHETE; EPA; EPEA; EpDPA; EpEDE; EpETE; EpOME; HDHA; HEPE; HETE; HETrE; HODE; LA; LC-MS/MS; LOX; LTB4; LXA4; LXB4; MaR; PD1; PDX; PGD2; PGE2; PGF1α; PUFA; RvD; SPE; SPM; TPN; TXB2; UHPLC-MS/MS; arachidonate 12-lipoxygenase; arachidonic acid; arachidonoyl ethanolamide; cyclooxygenase; cytochrome P450 monooxygenase; dihomo-γ linoleic acid; dihydroxyeicosatetraenoic acid; docosahexaenoic acid; docosahexaenoyl ethanolamide; docosapentaenoic acid; eicosanoids; eicosapentaenoic acid; eicosapentaenoyl ethanolamide; epoxydocosapentaenoic acid; epoxyeicosadienoic acid; epoxyeicosatetraenoic acid; epoxyoctadecenoic acid; hydroxydocosahexaenoic acid; hydroxyeicosapentaenoic acid; hydroxyeicosatetraenoic acid; hydroxyeicosatrienoic acid; hydroxyoctadecadienoic acid; leukotriene B4; linoleic acid; lipid mediators; lipoxin A4; lipoxin B4; lipoxygenase; maresin; oxylipins; polyunsaturated fatty acid; prostaglandin D2; prostaglandin E2; prostaglandin F1α; protectin D1; protectin DX; reference value; solid phase extraction; specialized pro-resolving mediator; thromboxane B2; total parenteral nutrition; ultra-high-performance liquid chromatography-tandem mass spectrometry; α-linolenic acid; αLA.