This proposal is aimed at elucidation of the biochemical pathways for the formation and biotransformation of 12-hydroxy- and 12-keto- eicosanoids, particularly 12-mono-hydroxy-5,8, 10, 14 eicosatetraenoic acid enantiomers (12-HETEs) and 12-keto- 5, 8, 10, 14 eicosatetraenoic acid (12-KETE), as well as the dihydro 12-R-HETE but not 12-S-HETE have been found in psoriasis and in view of our work showing that +12-HETE causes epidermal hyperproliferation, we have proposed that 12-R-HETE is a key proinflammatory mediator in psoriasis. This skin disease is characterized by infiltration of polymorphonuclear (PMN) leukocytes and epidermal hyperplasia. Both 12-HETE and exhibit chemotactic activity for PMN; in fact, the former appears comparable in potency to LTB4 in this regard. The 12-S- and 12-R-HETE enantiomers differ in biological profile; such that 12- S-HETE exerts stereospecific effects on cell adhesion and has been implicated in tumor cell metastasis. 12-R-HETrE possesses angiogenic and vasodilatory properties and its biosynthesis is stimulated during inflammation of the eye, suggesting that 12-R-HETrE is a novel mediator of inflammation. The objectives of this proposal are fourfold: First priority will be the chiral synthesis of potential precursors of 12-HETE (isomers of 11, 12-EET and 11, 12-diols) and synthesis of 12-KETE and reduced KETE derivatives which may give rise to 12-R and 12-S-HETEs via the 12-KETE reductase pathway recently identified by us in liver (see Progress Report). Secondly, the synthetic products (including tritiated and deuterated forms) will be used to identify and measure the levels of C12/C11 oxidation products formed from endogenous AA in liver (hepatocytes and subcellular fractions). The information on the chemical identity of the actual intermediates will enable us to reconstruct the principal pathways for biosynthesis of 12-HETEs, 12-KETE and 12-HETrEs. For this analytical work we will rely heavily on radio-HPLC and HPLC-RIA, enzymatic derivatization prior to HPLC and UV. Thirdly, we will extend these studies to another tissue type - normal skin, in order to elucidate the pathways normally operative in skin. The fourth objective is to examine epidermal preparations from psoriatic individuals in terms of possible disturbances in the metabolism of C12/C11 oxidative products. Structure-activity relationships (SAR) will be explored with in vitro models (PMN chemotaxis/chemokinesis and binding to LTB4 receptors on PMN membranes) to clarify the importance of the 12-R-hydroxyl for chemotaxis and receptor- binding. The contribution of C12/C11 oxidative products to the epidermal hyperproliferation of psoriasis will be studied with cultured keratinocytes. The receptor-binding studies could lead to identification of a new chemotactic receptor on PMNs, and the studies of biological activity (leukocyte functions, cell proliferation) will help to clarify the exact contribution of specific 12-hydroxy- and 12-keto-eicosanoids to the pathological changes of psoriasis.
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