FATTY ACID-DERIVED MEDIATORS OF INFLAMMATION

提高对5-脂氧合酶(5-LO)作用的认识 作为细胞损伤的介体或产物的产品和/或 炎症需要准确和精确的技术 量化。对高灵敏度分析的要求 和选择性，以及数据解释的困难 与体外生产有关，表明它是重要的 建立血凝素含量测定的参考分析方法 那些最可靠地反映在体内的5-LO代谢产物 制作。因此，我们建议发展分析 基于质谱学的程序，并评估这些 方法：通过对沙门氏菌提取的生理液体进行分析。 动物模型和正常人志愿者。我们相信是这样的 现在提出对患者群体进行昂贵的研究还为时过早 这一阶段，但我们预计后续应用我们的 调查人类疾病状态的程序 单独出资。白三烯B4的分析方法 (LTB4)和相关化合物将包含固相 提取(包括使用偶联抗LTB4血清)，高效液相色谱法 五氟苯甲酯的提纯与检测 三甲基硅烷醚衍生物的气相色谱分析 电子捕获负离子质谱仪 选择性反应监测。多肽-白三烯将是 使用C18-硅胶盒从生物体液中提取， 反相高效液相色谱分离纯化，连续进样测定。 流动快速原子轰击/串联质谱仪，选择反应 监控。另一种方法也将被采用 氢化还原裂解得5-甲基-4-羟基-4-羟基-4-羟基-4-酮 羟基二十烷酸，用气相色谱-电子联用法测定 捕获阴性的MS的分析程序将是 通过分析来自众所周知的生物流体进行评估 动物模型，即内毒素休克大鼠。 在正常人类志愿者身上的研究将涉及到确定 5-HETE、LTB4和稳定的代谢物(20-羟基LTB4)在 血浆和其他液体。将采用抽样条件 从而最大限度地减少体外生产。体外实验的影响 对20-羟基LTB浓度的刺激也会 评估过了。作为监测5-LO活性的另一种手段 体外生产的干扰，我们将测定LTE4在 尿液。最后，如果个体间存在明显的差异 在正常志愿者的尿液LTE4水平中，直接评估 在注入氚后，将计算出LT的生产率 白三烯的类似物。

Improved understanding of the role of 5-lipoxygenase (5-LO) products as mediators or products of cell injury and/or inflammation requires techniques for their accurate and precise quantification. The requirement for analyses of high sensitivity and selectivity, together with difficulties of data interpretation associated with ex vivo production, indicate that it is important to establish reference analytical methods for the the assay of those 5-LO metabolites that most reliably reflect in vivo production. Accordingly, we propose to develop analytical procedures based on mass spectrometry and to evaluate these methods with the assay of physiological fluids derived from an animal model and from normal human volunteers. we believe it is premature to propose expensive studies of patient populations at this stage but we anticipate subsequent application of our procedures to the investigation of human disease states by separate funding. The method for the analysis of leukotriene B4 (LTB4) and related compounds will incorporate solid phase extraction (including the use of coupled anti-LTB4 serum), HPLC purification and detection of the pentafluorobenzyl ester, trimethylsilyl ether derivatives by gas chromatography (GC)- electron capture negative ion mass spectrometry (MS) with selective reaction monitoring. The peptido-leukotrienes will be extracted from biological fluids using C18-silica cartridges, purified by reverse-phase HPLC and determined by continuous- flow fast atom bombardment/tandem MS, with selected reaction monitoring. The alternative approach will also be followed of hydrogenation and reductive cleavage to yield 5- hydroxyeicosanoic acid, which will be determined by GC-electron capture negative in MS. The analytical procedures will be assessed by analysis of biological fluids from a well understood animal model, namely the rat subjected to endotoxin shock. Studies in normal human volunteers will involve the determination of 5-HETE, LTB4 and a stable metabolite (20-hydroxy LTB4) in blood plasma and other fluids. Sampling conditions will be adopted which minimize ex vivo production. The influence of ex vivo stimulation on the concentration of 20-hydroxy LTB, will also be assessed. As a further means of monitoring 5-LO activity without the interference of ex vivo production, we will determine LTE4 in urine. Finally, if marked inter-individual differences are apparent in urinary LTE4 levels of normal volunteers, direct assessments of LT production rates will be made following infusion of tritiated analogues of the leukotrienes.