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.
