FATTY ACID-DERIVED MEDIATORS OF INFLAMMATION

提高对5-脂氧合酶（5-LO）作用的认识 作为介质的产物或细胞损伤的产物和/或 炎症需要技术来准确和精确地 量化 高灵敏度分析的要求 以及数据解释的困难 与离体生产相关，表明它是重要的 建立用于含量测定的参考分析方法 这些5-LO代谢物最可靠地反映了体内 生产 因此，我们建议开发分析 基于质谱的程序，并评估这些 本发明提供了测定来自于人的生理流体的方法， 动物模型和正常人类志愿者。 我们认为 提出对患者群体进行昂贵的研究还为时过早， 但我们预计， 人类疾病状态调查程序， 单独融资。 白三烯B4的分析方法 （LTB 4）和相关化合物将结合固相 提取（包括使用偶联抗LTB 4血清），HPLC 五氟苄基酯的纯化和检测， 三甲基硅醚衍生物气相色谱法（GC）- 电子捕获负离子质谱（MS）， 选择性反应监测 肽-白三烯将 使用C18-二氧化硅柱从生物流体中提取， 通过反相HPLC纯化，并通过连续色谱法测定。 流动快原子轰击/串联质谱，选择反应 监测. 还将采用以下替代方法： 氢化和还原裂解以产生5- 羟基二十烷酸，其将通过GC-电子 在MS中捕获阴性。分析方法将 通过分析生物流体进行评估， 动物模型，即内毒素休克大鼠。 在正常人类志愿者中的研究将涉及测定 5-HETE、LTB 4和稳定代谢物（20-羟基LTB 4）在 血浆和其他液体。取样条件将采用 其使离体产生最小化。 体外影响 刺激20-羟基LTB的浓度，也将是 评估。 作为监测5-LO活性的另一种手段， 体外生产的干扰，我们将确定LTE 4在 尿 最后，如果个体间差异明显， 在正常志愿者的尿LTE 4水平中， LT生产率将在输注氚化的 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.