Galvão Alyne Fávero, Petta Tânia, Flamand Nicolas, Bollela Valdes Roberto, Silva Célio Lopes, Jarduli Luciana Ribeiro, Malmegrim Kelen Cristina Ribeiro, Simões Belinda Pinto, de Moraes Luiz Alberto Beraldo, Faccioli Lúcia Helena
Departamento de Análises Clínicas, Toxicológicas e Bromatológicas, Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Av. do Café, s/n, Ribeirão Preto, São Paulo, 14040-903, Brazil.
Département de Médecine, Faculté de Médecine Université Laval, Centre de Recherche de l'Institut Universitaire de Cardiologie et de Pneumologie de Québec, 2725, chemin Sainte-Foy, Québec, G1V 4G5, Canada.
Anal Bioanal Chem. 2016 May;408(13):3613-23. doi: 10.1007/s00216-016-9445-8. Epub 2016 Mar 11.
Eicosanoids play an important role in homeostasis and in the pathogenesis of various human diseases. Pharmacological agents such as Ca(2+) ionophores and Ca(2+)-ATPase inhibitors, as well as natural agonists such as formylmethionine-leucyl-phenylalanine (fMLP), can stimulate eicosanoid biosynthesis. The aims of this work were to develop a method to determine the eicosanoid profile of human plasma samples after whole blood stimulation and to assess differences between healthy and sick individuals. For this purpose, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was partially validated for the quantification of 22 eicosanoids using human plasma from healthy volunteers. In addition, we optimized a method for the stimulation of eicosanoids in human whole blood. LC-MS/MS analyses were performed by negative electrospray ionization and multiple reaction monitoring. An assumption of linearity resulted in a regression coefficient ≥0.98 for all eicosanoids tested. The mean intra-assay and inter-assay accuracy and precision values had relative standard deviations and relative errors of ≤15%, except for the lower limit of quantification, where these values were ≤20%. For whole blood stimulation, four stimuli (fMLP, ionomycin, A23187, and thapsigargin) were tested. Results of the statistical analysis showed that A23187 and thapsigargin were potent stimuli for the production or liberation of eicosanoids. We next compared the eicosanoid profiles of stimulated whole blood samples of healthy volunteers to those of patients with sickle cell anemia (SCA) under treatment with hydroxyurea (HU) or after chronic red blood cell (RBC) transfusion. The results indicate that the method was sufficient to find a difference between lipid mediators released in whole blood of SCA patients and those of healthy subjects, mainly for 5-HETE, 12-HETE, LTB4, LTE4, TXB2, and PGE2. In conclusion, our analytical method can detect significant changes in eicosanoid profiles in stimulated whole blood, which will contribute to establishing the eicosanoid profiles associated with different inflammatory and infectious diseases.
类花生酸在体内稳态以及多种人类疾病的发病机制中发挥着重要作用。诸如钙离子载体和钙 -ATP酶抑制剂等药理试剂，以及甲酰甲硫氨酸 - 亮氨酰 - 苯丙氨酸（fMLP）等天然激动剂，均可刺激类花生酸的生物合成。本研究的目的是开发一种方法，用于测定全血刺激后人血浆样本中的类花生酸谱，并评估健康个体与患病个体之间的差异。为此，使用健康志愿者的人血浆对一种液相色谱 - 串联质谱（LC-MS/MS）方法进行了部分验证，以定量22种类花生酸。此外，我们优化了一种在人全血中刺激类花生酸的方法。LC-MS/MS分析通过负电喷雾电离和多反应监测进行。线性假设导致所有测试类花生酸的回归系数≥0.98。除定量下限外，批内和批间的平均准确度和精密度值的相对标准偏差和相对误差≤15%，定量下限处这些值≤20%。对于全血刺激，测试了四种刺激物（fMLP、离子霉素、A23187和毒胡萝卜素）。统计分析结果表明，A23187和毒胡萝卜素是类花生酸产生或释放的有效刺激物。接下来，我们将健康志愿者刺激后的全血样本的类花生酸谱与接受羟基脲（HU）治疗或慢性红细胞（RBC）输血后的镰状细胞贫血（SCA）患者的类花生酸谱进行了比较。结果表明，该方法足以发现SCA患者全血中释放的脂质介质与健康受试者之间的差异，主要体现在5-羟二十碳四烯酸（5-HETE）、12-羟二十碳四烯酸（12-HETE）、白三烯B4（LTB4）、白三烯E4（LTE4）、血栓素B2（TXB2）和前列腺素E2（PGE2）方面。总之，我们的分析方法能够检测到刺激全血中类花生酸谱的显著变化，这将有助于建立与不同炎症和感染性疾病相关的类花生酸谱。
