LEUKOTRIENES AND SLOW REACTING SUBSTANCE OF ANAPHYLAXIS

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DESCRIPTION: (Adapted from the applicant's abstract). Leukotrienes are lipid mediators derived form arachidonic acid by enzymatic oxidation catalyzed by 5-lipoxygenase (5-LO). Interest in these molecules stems from their potent activities in causing neutrophil chemotaxis (LTB4) and bronchial smooth muscle constriction (LTC4). Fundamental questions remain concerning their biosynthesis and metabolism and these are the focus of the proposed work. This project consists of four major goals. The first goal will be to utilize mass spectrometry (MS) for the structural characterization of the major human urinary metabolites of LTB4. A MS quantitative assay will be developed to asses in vivo biosynthesis and normal entry rate of LTB4 into the circulation. The second goal is to asses LTC4 and LTB4 biosynthesis in asthmatic patients that are intolerant to aspirin by quantitative analysis of urinary metabolites for each eicosanoid. LTB4 biosynthesis involves 5-LO and LTA4 hydrolase that are inactivated through a mechanism based suicide reaction that results in covalent binding of an electrophilic eicosanoid intermediate to the enzyme. LTA4 also binds covalently to human serum albumin, a carrier protein for this triene epoxide. The application's third goal is to utilize electrospray tandem MS and MALDI-TOF MS in order to determine the site of covalent attachment and structure of the protein bound lipid. It is expected that such information will provide insights into the mechanism of suicide inactivation and the importance of specific amino acids to the catalysis event The fourth goal will be to investigate the autocrine and paracrine effects of LTB4 on leukotriene biosynthesis and metabolism. The mechanisms by which extracellular LTB4 can modulate neutrophil LTB4 biosynthesis will be investigated. LTB4 has been suggested to increase LTB4 metabolism through the interaction with the nuclear peroxisomal proliferation activating receptor (PPARalpha) and by this mechanism, limit the proinflammatory properties of LTB4. This putative effect of LTB4 will be investigated in hepatocytes and other cells by analysis of both kinetics and extent of leukotriene metabolism. An understanding of the metabolic events occurring in vivo is essential to an understanding of the role these molecules play in inflammation.