Direct eicosanoid profiling of the hypoxic lung by comprehensive analysis via capillary liquid chromatography with dual online photodiode-array and tandem mass-spectrometric detection
Direct eicosanoid profiling of the hypoxic lung by comprehensive analysis via capillary liquid chromatography with dual online photodiode-array and tandem mass-spectrometric detection
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DOI：
10.1007/s00216-007-1718-9
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发表时间：
2008-01-01
影响因子：
4.3
通讯作者：
Grimminger, Friedrich
中科院分区：
文献类型：
--
作者：
Kiss, Ladislau;Roeder, Yasmin;Grimminger, Friedrich
二十碳二烯类化合物是花生四烯酸衍生的中间体，具有部分矛盾的、不完全阐明的作用。因此，环氧二十碳三烯酸(EETs)在心血管系统中被认为是血管扩张的内皮衍生超极化因子，但在肺中被报道为血管收缩因子，这是有争议的。关于二十烷类化合物生理学的不一致发现可能是因为以前的方法缺乏敏感性和鉴定可靠性，和/或只关注特殊的二十烷类化合物，而忽略了整个介体背景，从而限制了海螺形成和生物活性图谱之间的关联准确性。因此，我们开发了一种方法，能够在一次50分钟的色谱运行中同时评估44个二十烷基类化合物，包括花生四烯酸级联的所有代表，即细胞色素P450、脂氧合酶、环氧合酶产物和游离异前列腺素作为体内氧化应激的标志物。该方法结合了(I)特定来源的样品提取，(Ii)坚固的等度和高灵敏度的毛细管液相色谱分离，以及(Iii)可靠的双在线光电二极管阵列和电喷雾电离串联质谱学鉴定和定量。由于内径小，并且由于流速在低微升范围内，物质几乎完全转移到质谱仪，因此使用具有典型高峰效率的毛细管柱，而不是使用大内径柱，具有低的色谱信号和仅部分分析物转移，从而实现了高灵敏度和在飞秒范围内的定量限制。这一快速、全面和灵敏的技术通过直接比较所有二十烷类化合物的生成情况，为在不同氧化应激条件下所有相关血管活性耳石的情况下，对特定介质(例如EETs)的生理学提供了新的、准确的见解。事实上，在缺氧性通风的兔肺中应用全面的“EicoProfile”，一目了然地揭示了在整个介质生成环境中，游离EET的生物合成增强，从而表明它们对缺氧性肺血管收缩的假说贡献。
Eicosanoids are arachidonic acid-derived mediators, with partly contradictory, incompletely elucidated actions. Thus, epoxyeicosatrienoic acids (EETs) are controversially discussed as putative vasodilatative endothelium-derived hyperpolarizing factors in the cardiovascular compartment but reported as vasoconstrictors in the lung. Inconsistent findings concerning eicosanoid physiology may be because previous methods were lacking sensitivity, identification reliability, and/or have focused on special eicosanoid groups only, ignoring the overall mediator context, and thus limiting the correlation accuracy between autacoid formation and bioactivity profile. Therefore, we developed an approach which enables the simultaneous assessment of 44 eicosanoids, including all representatives of the arachidonic acid cascade, i.e., cytochrome P450, lipoxygenase, cyclooxygenase products, and free isoprostanes as in vivo markers of oxidative stress, in one 50-minute chromatographic run. The approach combines (i) source-specific sample extraction, (ii) rugged isocratic and high-sensitivity capillary liquid-chromatographic separation, and (iii) reliable dual online photodiode-array and electrospray ionization tandem mass-spectrometric identification and quantitation. High sensitivity with limits of quantification in the femtogram range was achieved by use of capillary columns with typical high peak efficiency, due to small inner diameters, and virtually complete substance transfer to the mass spectrometer, due to flow rates in the low microliter range, instead of large inner diameter columns with low chromatographic signal and only partial analyte transfer employed by previous methods. This expeditious, global and sensitive technique provides the prerequisite for new, accurate insights regarding the physiology of specific mediators, for example EETs, in the context of all relevant vasoactive autacoids under varying conditions of oxidative stress by direct comparison of all eicosanoid generation profiles. Indeed, application of comprehensive "eicoprofiling" to hypoxically ventilated rabbit lungs revealed at a glance the enhanced biosynthesis of free EETs in the overall mediator generation context, thus suggesting their hypothetical contribution to hypoxic pulmonary vasoconstriction.