Development of a solid phase extraction method for evaluating the production of classic and nonclassic eicosanoids in human and fish cells using liquid chromatography tandem mass spectrometry

Abstract

Eicosanoids are the major metabolites of fatty acids and they are correlated with many kinds of diseases such as Alzheimer's, cancer and cardiovascular diseases. Usually, most of the eicosanoids derived from omega-6 fatty acids have pro-inflammatory properties while those from omega-3 fatty acids are anti-inflammatory. The type and amount of the production of eicosanoids are affected by many factors including availability of fatty acids, activity of cyclo- and lipo- oxygenase and type of cells. In this thesis, a solid-phase extraction method combined with HPLC-MS/MS is first developed to analyze simultaneously PGE2, PGE3, LTB4, 6-keto- PGF1alpha, Δ17-6-keto-PGF1alpha, RvD1 and RvD2 in two types of cell culture medium, specifically EBM-2 medium and cL-15 medium. This method is systematically optimized and validated. The concentrations of internal standards are determined by a Doehlert design to keep the response factors constant in the analytical range. Method validation for the various eicosanoids in EBM-2 medium shows limits of quantification (LOQ) between 0.1-0.6 ng/ml, recovery between 57.1- 127.2% and precision between 0.2- 20.7%. For cL-15 medium, LOQ is between 0.1-8 ng/ml, recovery 63.9- 125.7% and precision 3.5-30.7% for the analyzed eicosanoids. The validated method is then applied to investigate the effect of fatty acids on the production of eicosanoids in fish liver cells, fish head kidney cells and human umbilical vein endothelial cells (HUVEC). The various types of cells are incubated in medium with different combination of fatty acids according to a 24- factorial design and eicosanoids in the medium are analyzed after 24 hours. The results reveal that RvD1 and RvD2 cannot be detected in any type of cells. Addition of arachidonic acid (AA) increases the productions of all the other five eicosanoids except in fish head kidney cells where production of PGE3 is reduced in a small degree. Individual linoleic acid (LA) reduces the productions of 6- keto-PGF1alpha and Δ17-6-keto-PGF1alpha and increases the production of PGE3 in both types of fish cells. Eicosapentaenoic acid (EPA) remarkably increases the production of LTB4 and docosahexaenoic acid (DHA) decreases the production of Δ17-6-keto-PGF1alpha in three types of cells. The effects of interactions are complex and most of them are different in different types of cells.