title: A sensitive LC-MS/MS method for the quantification of regioisomers of epoxyeicosatrienoic and dihydroxyeicosatrienoic acids in human plasma during endothelial stimulation.
abstract: Epoxyeicosatrienoic acids (EETs) are vasodilating lipid mediators metabolized into dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase. We aimed to develop a LC-MS/MS method to quantify EETs and DHETs in human plasma and monitored their levels during vascular endothelial stimulation. Plasma samples, collected from 14 healthy and five hypertensive subjects at baseline and during radial artery endothelium-dependent flow-mediated dilatation, were spiked with internal standards. Lipids were then extracted by a modified Bligh and Dyer method and saponified to release bound EETs and DHETs. Samples were purified by a second liquid-liquid extraction and analyzed by LC-MS/MS. The assay allowed identification of (±)8(9)-epoxy-5Z,11Z,14Z-eicosatrienoic acid (8,9-EET); (±)11(12)-epoxy-5Z,8Z,14Z-eicosatrienoic acid (11,12-EET); (±)14(15)-epoxy-5Z,8Z,11Z-eicosatrienoic acid (14,15-EET); (±)8,9-dihydroxy-5Z,11Z,14Z-eicosatrienoic acid (8,9-DHET); (±)11,12-dihydroxy-5Z,8Z,14Z-eicosatrienoic acid (11,12-DHET); and (±)14,15-dihydroxy-5Z,8Z,11Z-eicosatrienoic acid (14,15-DHET). (±)5(6)-epoxy-5Z,11Z,14Z-eicosatrienoic acid (5,6-EET) was virtually undetectable due to its chemical instability. The limits of quantification were 0.25 ng/mL for DHETs and 0.5 ng/mL for EETs. Intra- and inter-assay variations ranged from 1.6 to 13.2%. Heating induced a similar increase in 8,9-EET, 11,12-EET, and 14,15-EET levels and in corresponding DHET levels in healthy but not in hypertensive subjects. We validated a sensitive LC-MS/MS method for measuring simultaneously plasma EET and DHET regioisomers in human plasma and showed its interest for assessing endothelial function.
authors.affiliation: Department of Pharmacology, Rouen University Hospital, 1 rue de Germont, 76031, Rouen, France.|Institut National de la Santé et de la Recherche Médicale (INSERM) U1096, 76183, Rouen, France.|Institute for Research and Innovation in Biomedicine, University of Rouen, 22 Boulevard Gambetta, 76183, Rouen, France.
authors.forename: Thomas
authors.initials: T
authors.lastname: Duflot
authors.affiliation: Department of Pharmacology, Rouen University Hospital, 1 rue de Germont, 76031, Rouen, France.
authors.forename: Tony
authors.initials: T
authors.lastname: Pereira
authors.affiliation: Institut National de la Santé et de la Recherche Médicale (INSERM) U1096, 76183, Rouen, France.|Institute for Research and Innovation in Biomedicine, University of Rouen, 22 Boulevard Gambetta, 76183, Rouen, France.
authors.forename: Clothilde
authors.initials: C
authors.lastname: Roche
authors.affiliation: Department of Pharmacology, Rouen University Hospital, 1 rue de Germont, 76031, Rouen, France.
authors.forename: Michèle
authors.initials: M
authors.lastname: Iacob
authors.affiliation: Normandie Université, Laboratoire SMS - EA3233, Université de Rouen, 76281, Mont Saint Aignan, France.
authors.forename: Pascal
authors.initials: P
authors.lastname: Cardinael
authors.affiliation: Normandie Université, Laboratoire SMS - EA3233, Université de Rouen, 76281, Mont Saint Aignan, France.
authors.forename: Najla El-Gharbi
authors.initials: NE
authors.lastname: Hamza
authors.affiliation: Department of Pharmacology, Rouen University Hospital, 1 rue de Germont, 76031, Rouen, France.|Institut National de la Santé et de la Recherche Médicale (INSERM) U1096, 76183, Rouen, France.|Institute for Research and Innovation in Biomedicine, University of Rouen, 22 Boulevard Gambetta, 76183, Rouen, France.
authors.forename: Christian
authors.initials: C
authors.lastname: Thuillez
authors.affiliation: Department of Pharmacology, Rouen University Hospital, 1 rue de Germont, 76031, Rouen, France.
authors.forename: Patricia
authors.initials: P
authors.lastname: Compagnon
authors.affiliation: Department of Pharmacology, Rouen University Hospital, 1 rue de Germont, 76031, Rouen, France.|Institut National de la Santé et de la Recherche Médicale (INSERM) U1096, 76183, Rouen, France.|Institute for Research and Innovation in Biomedicine, University of Rouen, 22 Boulevard Gambetta, 76183, Rouen, France.
authors.forename: Robinson
authors.initials: R
authors.lastname: Joannidès
authors.affiliation: Department of Pharmacology, Rouen University Hospital, 1 rue de Germont, 76031, Rouen, France.|Institut National de la Santé et de la Recherche Médicale (INSERM) U1096, 76183, Rouen, France.|Institute for Research and Innovation in Biomedicine, University of Rouen, 22 Boulevard Gambetta, 76183, Rouen, France.
authors.forename: Fabien
authors.initials: F
authors.lastname: Lamoureux
authors.affiliation: Department of Pharmacology, Rouen University Hospital, 1 rue de Germont, 76031, Rouen, France. jeremy.bellien@chu-rouen.fr.|Institut National de la Santé et de la Recherche Médicale (INSERM) U1096, 76183, Rouen, France. jeremy.bellien@chu-rouen.fr.|Institute for Research and Innovation in Biomedicine, University of Rouen, 22 Boulevard Gambetta, 76183, Rouen, France. jeremy.bellien@chu-rouen.fr.
authors.forename: Jérémy
authors.initials: J
authors.lastname: Bellien
mesh_terms: D001095:Arachidonic Acids / Q000097:blood*; D016022:Case-Control Studies; D002853:Chromatography, Liquid / Q000379:methods*; D004730:Endothelium, Vascular / Q000378:metabolism; D006801:Humans; D006973:Hypertension / Q000378:metabolism; D057230:Limit of Detection; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D001095:Arachidonic Acids
keywords: Dihydroxyeicosatrienoic acids; Endothelial cells; Epoxyeicosatrienoic acids; LC-MS/MS; Soluble epoxide hydrolase; Vascular biology