Simultaneous analysis of saturated and unsaturated oxylipins in 'ex vivo' cultured peripheral blood mononuclear cells and neutrophils.

Oxylipins are a family of saturated and unsaturated fatty acids peroxidation products with bioactive properties. We have developed an improved method for the measurement of ex vivo oxylipin production by peripheral blood mononuclear cells (PBMCs) and neutrophils. We aimed to develop an analytical method to determine the production rates of polyunsaturated fatty acids (PUFAs), PUFA-oxylipin, and saturated-oxylipins by stimulated PBMCs and neutrophils based on solid phase extraction and HPLC-MS/MS technology. A UHPLC system coupled to a Q-Exactive Hybrid Quadrupole-Orbitrap mass spectrometer was used to identify and quantify oxylipin production. For each oxylipin and PUFA their differential response was calculated with respect to a deuterated internal standard factor (ISF). To calculate oxylipin and PUFAs in the culture samples, the individual ISF was used for each oxylipin and PUFA with respect to the deuterated internal standard. PBMCs and neutrophils showed a different pattern of oxylipin production and fatty acid secretion. Lipopolysaccharide (LPS) did not stimulate oxylipin production or fatty acids secretion in PBMCs, whereas phorbol myristate acetate (PMA) stimulation increased the production rate of 5-HETE, 15-HETE, 15-HEPE, 17-DoHE, PGE2, AA, and DHA. LPS stimulation decreased 16-hydroxyl-palmitatte (16-OHPAL) production and DHA secretion in neutrophils, while PMA stimulation increased the production rate of AA and its derivate oxylipins, 5-HETE, 15-HETE, and PGE2. In conclusion, we have developed a new method to determine oxylipins derived from both saturated and unsaturated fatty acids in culture cell media. This method has enough sensitivity, and accuracy, to determine oxylipin production and fatty acid secretion by PBMCs and neutrophils.