Department of Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Friedrich-Schiller-University Jena, D-07743 Jena, Germany.
Prostaglandins Other Lipid Mediat. 2019 Oct;144:106350. doi: 10.1016/j.prostaglandins.2019.106350. Epub 2019 Jul 16.
Glutathione (GSH) conjugates of oxygenated polyunsaturated fatty acids comprise a group of pro-inflammatory and pro-resolving lipid mediators formed in immunocompetent cells. While the pro-inflammatory conjugates such as the cysteinyl leukotrienes (cys-LTs), eoxins (EXs) and five-oxo-GSH conjugate (FOG) derive from arachidonic acid (AA), the group of conjugates in tissue regeneration (CTRs) such as maresin CTRs (MCTRs), protectin CTRs (PCTRs) and resolvin CTRs (RCTRs) are biosynthesized from docosahexaenoic acid (DHA). Here, we present a gradient UPLC-MS/MS method for the analysis of pro-inflammatory and pro-resolving GSH conjugates using positive electrospray ionization (ESI(+)) and collision-induced fragmentation for unambiguous identification and structural information, and a negative ionization (ESI(-)) mode for quantification of the GSH conjugates. The method was employed to detect GSH conjugates in human platelets and macrophages. MCTRs were detected in platelets upon addition of exogenous docosahexaenoic acid (DHA) and the biosynthesis was independent on leukotriene C (LTC) synthase activity. Pathogenic bacteria stimulated the formation of EXs and PCTRs in M2 macrophages, whereas Ca-ionophore activated the biosynthesis of LTC in M1 and M2 macrophage phenotypes. Together, our methodology covers the qualitative and quantitative analysis of GSH conjugates and gives an analytical basis for the detection and structural elucidation of cysteinyl-containing lipid mediators.
谷胱甘肽（GSH）与含氧多不饱和脂肪酸的轭合物包含一组在免疫活性细胞中形成的促炎和促解决的脂质介质。虽然促炎轭合物，如半胱氨酰白三烯（cys-LTs）、eoxins（EXs）和五氧化谷胱甘肽轭合物（FOG）源自花生四烯酸（AA），但组织再生中的轭合物（CTRs）组，如maresin CTRs（MCTRs）、protectin CTRs（PCTRs）和resolvin CTRs（RCTRs）是由二十二碳六烯酸（DHA）生物合成的。在这里，我们提出了一种梯度 UPLC-MS/MS 方法，用于分析使用正电喷雾电离（ESI(+))和碰撞诱导碎裂进行的促炎和促解决的 GSH 轭合物，以进行明确的鉴定和结构信息，并采用负离子化（ESI(-))模式对 GSH 轭合物进行定量。该方法用于检测人血小板和巨噬细胞中的 GSH 轭合物。在外源性二十二碳六烯酸（DHA）存在下，血小板中检测到 MCTRs，其生物合成不依赖于白三烯 C（LTC）合酶活性。致病菌刺激 M2 巨噬细胞中 EXs 和 PCTRs 的形成，而钙离子载体激活 M1 和 M2 巨噬细胞表型中 LTC 的生物合成。总之，我们的方法涵盖了 GSH 轭合物的定性和定量分析，并为检测和阐明含半胱氨酸的脂质介质提供了分析基础。
