Leukotriene B4 is the most potent derivative of the arachidonic acid cascade in stimulating the recruitment and activation of leukocytes. Previously we studied the metabolic fate of LTB4 in vivo, to provide a rational basis for the quantitation of endogenous LTB4 production. We now propose to develop methods for the monitoring of LTB4 biosynthesis in vivo to explore the role of this mediator in inflammatory and hypersensitivity reactions, and to determine the effect of pharmacological interventions. Two approaches will be taken. The first involves quantitation of a characteristic endogenous metabolite in peripheral plasma or urine. The metabolism studies indicate the importance of Omega-oxidation in the inactivation of LTB4; we propose to develop and apply specific mass spectrometric methods for analysis of the proximal metabolite of this pathway, Omega-OH-LTB4. This method will be used to study endogenous LTB4 production in two defined models of the inflammatory response, viz reaction of the awake sheep to endotoxin and the response of asthmatic subjects to allergen challenge. The second approach will be to determine the capacity of leukocytes to synthesize LTB4 when stimulated in whole blood ex vivo. This is analogous to the measurement of thromboxane B2 levels in serum. The endogenous biosynthesis of eicosanoids is not activated under normal conditions. However, by withdrawing blood and stimulating ex vivo, the effects of systemically administered pharmacological agents can be quantified. We will investigate the dose/response relationships of three stimuli for LTB4 production and determine the effects of changes in cell counts, disease processes (septicemia, chronic myelogenous leukemia) and pharmacological agents on its synthesis. Pharmacological evidence points to LTB4 as an important mediator of inflammation. The studies we propose will address the role of LTB4 in inflammatory reactions and provide a biochemical index of the effects of drugs on the 5-lipoxygenase pathway of arachidonic acid metabolism.
