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 (FOG7) 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 C4 (LTC4) synthase activity. Pathogenic bacteria stimulated the formation of EXs and PCTRs in M2 macrophages, whereas Ca2+-ionophore activated the biosynthesis of LTC4 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.

中文翻译：

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氧化多不饱和脂肪酸的谷胱甘肽共轭物的液相色谱-质谱联用分析。

氧化的多不饱和脂肪酸的谷胱甘肽（GSH）缀合物包含在免疫活性细胞中形成的一组促炎性和促拆分性脂质介体。虽然促炎性结合物（例如半胱氨酰白三烯（cys-LTs），曙红（EXs）和五-氧代-GSH结合物（FOG7）源自花生四烯酸（AA），但组织再生中的结合物（CTRs）由于maresin CTR（MCTR），protectin CTR（PCTR）和resolvin CTR（RCTR）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，用于使用正电喷雾电离（ESI（+））和碰撞诱导的碎裂来分析促炎和促分解GSH偶联物，以进行明确的鉴定和结构信息，负电离（ESI（-））模式用于定量GSH偶联物。该方法用于检测人血小板和巨噬细胞中的GSH偶联物。加入外源二十二碳六烯酸（DHA）后在血小板中检测到MCTR，其生物合成与白三烯C4（LTC4）合酶活性无关。致病细菌刺激了M2巨噬细胞中EX和PCTR的形成，而Ca2 +离子载体激活了M1和M2巨噬细胞表型中LTC4的生物合成。总之，我们的方法涵盖了GSH共轭物的定性和定量分析，并为检测和半胱氨酸半胱氨酸脂质介体的结构分析提供了分析依据。加入外源二十二碳六烯酸（DHA）后在血小板中检测到MCTR，其生物合成与白三烯C4（LTC4）合酶活性无关。致病细菌刺激了M2巨噬细胞中EX和PCTR的形成，而Ca2 +离子载体激活了M1和M2巨噬细胞表型中LTC4的生物合成。总之，我们的方法涵盖了GSH共轭物的定性和定量分析，并为检测和半胱氨酸半胱氨酸脂质介体的结构分析提供了分析依据。加入外源二十二碳六烯酸（DHA）后在血小板中检测到MCTR，其生物合成与白三烯C4（LTC4）合酶活性无关。致病细菌刺激了M2巨噬细胞中EX和PCTR的形成，而Ca2 +离子载体激活了M1和M2巨噬细胞表型中LTC4的生物合成。总之，我们的方法涵盖了GSH共轭物的定性和定量分析，并为检测和半胱氨酸半胱氨酸脂质介体的结构分析提供了分析依据。