Institut d'Investigació Sanitària de les Illes Balears (IDISBA), E-07120 Palma, Spain; Research Group in Community Nutrition and Oxidative Stress, University of Balearic Islands, E- 07122, Palma, Spain.
Poznan University of Physical Education, Poland.
J Pharm Biomed Anal. 2020 Jul 15;186:113258. doi: 10.1016/j.jpba.2020.113258. Epub 2020 Apr 12.
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.
氧化应激产物是一类具有生物活性的饱和和不饱和脂肪酸的过氧化产物。我们开发了一种改进的方法，用于测量外周血单核细胞（PBMC）和中性粒细胞的体外氧化应激产物的产生。我们旨在开发一种分析方法，基于固相萃取和高效液相色谱-串联质谱技术（HPLC-MS/MS）来确定刺激 PBMC 和中性粒细胞产生多不饱和脂肪酸（PUFA）、PUFA-氧化应激产物和饱和-氧化应激产物的速率。使用超高效液相色谱系统（UHPLC）与 Q-Exactive 杂交四极杆轨道阱质谱仪（Q-Exactive Hybrid Quadrupole-Orbitrap mass spectrometer）联用，以鉴定和定量氧化应激产物的产生。对于每种氧化应激产物和 PUFA，根据氘代内标因子（ISF）计算其差异反应。为了计算培养样本中的氧化应激产物和 PUFA，使用每个氧化应激产物和 PUFA 相对于氘代内标物的个体 ISF。PBMC 和中性粒细胞表现出不同的氧化应激产物和脂肪酸分泌模式。脂多糖（LPS）不能刺激 PBMC 中氧化应激产物的产生或脂肪酸的分泌，而佛波醇 12-肉豆蔻酸 13-乙酸酯（PMA）刺激增加了 5-HETE、15-HETE、15-HEPE、17-DoHE、PGE2、AA 和 DHA 的产生速率。LPS 刺激降低了中性粒细胞中 16-羟基棕榈酸（16-OHPAL）的产生和 DHA 的分泌，而 PMA 刺激增加了 AA 及其衍生的氧化应激产物、5-HETE、15-HETE 和 PGE2 的产生速率。总之，我们开发了一种新的方法来确定培养细胞培养基中源自饱和和不饱和脂肪酸的氧化应激产物。该方法具有足够的灵敏度和准确性，可用于测定 PBMC 和中性粒细胞中氧化应激产物的产生和脂肪酸的分泌。
