Arachidonic acid is oxidized by several cytochrome P450 monooxygenases, yielding epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids (HETEs). Epoxide hydrolases can subsequently oxidize each of the EETs to the corresponding dihydroxyeicosatrienoic acids (DHETs). EETs and DHETs exhibit a wide array of biological actions and are found in most mammalian tissues, blood and urine. However, in contrast to cyclooxygenase and lipoxygenase metabolites, for which facile immunoassays are available, analysis of EETs and DHETs in biological specimens presently requires a complex, tedious and costly analytical methodology. We propose to develop enzyme-linked ImmunoSorbent (ELISA) assays specific for EETs and DHETs. In Phase I, we will develop solid phase competitive ELISAs for all 4 racemic EETs and their DHETs, and determine the sensitivity and specificity of each ELISA, including validation of results by HPLC/GC/MS. In subsequent Phase II studies, we will: (a) Develop ELISAs specific for each EET and DHET stereoisomer, (b) Develop monoclonal antibodies if polyclonal antibodies are not satisfactory (c) Optimize methods for processing biological samples needed for DHET and EET immunoassays, and (d) Investigate the extent of conjugation of EETs and DHETs in biological fluids and develop ELISA kits specific for conjugates if needed.
