Enzymatic Reactions in Leukotriene Biosynthesis
The leukotrienes are a family of polyunsaturated fatty acid metabolites of arachidonic acid. Leukotrienes are known to be the active components of the so-called slow-reacting substance of anaphylaxis (SRS-A). They are potent spasmogens which greatly intensify the effects of an acute allergic reaction. As such, they have been implicated in asthmatic symptoms as well as inflammatory responses of the body. Modulation or inhibition of leukotriene formation may be a useful tool in treating diseases of hypersensitivity such as asthma and allergies, but a methodical approach to finding such a method of modulation suggests the need to be certain of structures and biosynthetic pathways involved in leukotriene production. Recently the structures of the various leukotrienes have been elucidated and a biosynthetic route for their production from arachicbnic acid postulated. However, there exist few reliable methods for quantitatively, extracting and detecting leukotrienes from biological samples. In addition, much of the work with leukotriene biosynthesis has involved use of rat basophilic leukemia- 1 cells as a model system, since they can be obtained rather easily and inexpensively in large quantities when needed, factors which have precluded use of human polymorphonuclear leukocytes in many studies. The purpose of this study was to investigate further methods for extraction and chromatography of leukotrienes and use them to investigate leukotriene production by human PMNLs and subsequently attempt to characterize the biosynthetic pathway of leukotriene formation therein. Leukotriene production by human PMNLs after stimulation with calcium ionophore A23187 was investigated as one segment of this project. In addition, the hypothesized biological intermediates of the leukotriene pathway were incubated in the presence of broken cell preparations containing the enzymes responsible for leukotriene production. Using reverse phase high performance liquid chromatography and extraction procedures tested initially, the products of interest of these reactions were separated, identified and quatitated, yielding support for the proposed biosynthetic pathway of leukotrienes, as well as giving some insight into the competing enzymatic and non-enzymatic reactions which take place.