Identification and quantification of metabolites of arachidonic acid from cultures of endothelial cells by HPLC-MS²

Abstract

Epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids (HETEs) are oxidative products of arachidonic acid, some of which participate in the regulation of vascular tone. Little is known about the production of EETs and HETEs in cultures of endothelial cells. This paper reports an assay for the simultaneous quantification of isomers of EETs and HETEs from endothelial cell culture supernatants by employing solid-phase extraction and liquid chromatography-mass spectrometry. The method enabled measurement of 5,6-EET, 8,9-EET, 11,12-EET, 14,15-EET, 5-HETE, 8-HETE, 11-HETE, 12-HETE and 15-HETE. The metabolites were chromatographically separated by reversed-phase HPLC and identified by negative ESI tandem mass spectrometry and this method was used to investigate the metabolism of arachidonic acid with an endothelial cell line. For quantification, the sum of signal intensities of characteristic fragment ions was used. The detection limits for 5,6-EET and of other EET and HETE isomers were 2.0, 0.64 and 8 ng ml⁻¹ culture medium, respectively. The precision of the method was determined with spiked culture medium (three concentrations, n = 5) and the average RSD ranged from 6.0 to 24.2%. The dynamic range was 0.6–23.5 ng ml⁻¹ culture medium for EETs and 8.0–200 ng ml⁻¹ for HETEs. Arachidonic acid was mainly metabolised to HETEs with product levels ranging from 59.3 to 460 ng 10⁻⁶ cells. The median of 8,9-EET and 14,15-EET was 14.5 and 17.7 ng 10⁻⁶ cells, respectively, whereas 5,6-EET and 11,12-EET were below 2 ng 10⁻⁶ cells in a 5-min incubation assay at a 30 µM arachidonic acid substrate concentration.

Keywords :

• Arachidonic acid
• hydroxyeicosatetraenonic acids (HETEs)
• epoxyeicosatrienoic acids (EETs)
• endothelial cell culture
• liquid chromatography/tandem mass spectrometry