Lipid Mediator Quantification in Isolated Human and Guinea Pig Airways: An Expanded Approach for Respiratory Research
Lipid Mediator Quantification in Isolated Human and Guinea Pig Airways: An Expanded Approach for Respiratory Research
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DOI：
10.1021/acs.analchem.8b01651
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发表时间：
2018-09-04
影响因子：
7.4
通讯作者：
Wheelock, Craig E.
中科院分区：
文献类型：
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作者：
Kolmert, Johan;Fauland, Alexander;Wheelock, Craig E.
在哮喘的临床重要性是公认的，但是，其他脂质介质（通常称为氧化脂质）在气道紧张度和炎症的调节的生化作用仍然不清楚。因此，我们开发了一个工作流程，在两个体外模型（完整的人支气管和豚鼠气管）中研究氧脂肽的生理学和药理学。分离气道，并在用抗IgE或卵清蛋白刺激后的器官浴中测量平滑肌收缩。使用能够共同测量130种化合物的三种开发的LC-MS/MS方法定量氧脂素随时间释放到器官浴中。氧代利平提取回收率平均为71%，方法准确度为90- 98%，变异系数为4.3- 9.4%，基质效应平均为11%。在基线时，在两种组织制备物中均观察到低水平的主要是木兰素和相关代谢物。肥大细胞诱导的气道收缩引起白三烯的释放和前列腺素水平的进一步升高。在器官浴中刺激后60 min，共检测到57种来自人支气管的氧脂素和42种来自豚鼠气管的氧脂素。手性分析表明，与12（S）-HETE、14（5）-和17（S）-羟基二十二碳六烯酸（HDoHE; ee = 100%）相反，人支气管制剂中的5-羟基二十碳四烯酸（5-HETE）不是由5-LOX酶活性产生的（对映体过量[ee] = 10%），这突出表明手性色谱法对于正确的生物学解释是必要的。出乎意料的是，前列腺素D2及其代谢产物在激发后24小时仍然升高，表明先前未描述的肥大细胞的持续活化。报道的翻译方法提供了一个新的平台，全面的研究，以阐明各种气道反应的起源和功能的个别氧化脂质。
The clinical importance of prostaglandins and leukotrienes in asthma is well recognized; however, the biochemical role of other lipid mediators (often termed oxylipins) in the regulation of airway tone and inflammation remains unclear. We therefore developed a workflow to investigate oxylipin physiology and pharmacology in two in vitro models, the intact human bronchus and the guinea pig trachea. Airways were isolated and smooth muscle contraction was measured in an organ bath following stimulation with either anti-IgE or ovalbumin. The associated release of oxylipins over time into the organ bath was quantified using three developed LC-MS/MS methods capable of collectively measuring 130 compounds. Oxylipin extraction recoveries were 71% on average, method accuracy was 90-98%, coefficient of variation was 4.3-9.4%, and matrix effects were on average 11%. At baseline, low levels of primarily prostaglandins and associated metabolites were observed in both tissue preparations. The mast cell induced airway constriction caused release of leukotrienes and further elevations in prostaglandin levels. In total, 57 oxylipins from the human bronchus, and 42 from guinea pig trachea, were detected at 60 min post-stimulation in the organ bath. Chiral analysis demonstrated that 5-hydroxyeicosatetraenoic acid (5-HETE) in the human bronchus preparation was not produced by 5-LOX enzymatic activity (enantiomeric excess [ee] = 10%), as opposed to 12(S)-HETE, 14(5)-, and 17(S)-hydroxy docosahexaenoic acid (HDoHE; ee = 100%), highlighting that chiral chromatography is necessary for correct biological interpretation. Unexpectedly, prostaglandin D2 and its metabolites remained elevated 24 h after the challenges, suggesting a sustained activation of mast cells not previously described. The reported translational methodology provides a new platform for comprehensive studies to elucidate the origin and functions of individual oxylipins in various airway responses.