SIMULTANEOUS ANALYSIS OF 4-SERIES AND 5-SERIES LIPOXYGENASE AND CYTOCHROME-P450 PRODUCTS FROM DIFFERENT BIOLOGICAL SOURCES BY REVERSED-PHASE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC TECHNIQUE

Quantification of lipoxygenase and cytochrome P450 products of both ar achidonic acid (AA) and eicosapentaenoic acid (EPA) is of broad intere st due to the multiple biological activities of these compounds. We de veloped a method combining (i) solid-phase extraction, (ii) isocratic reversed-phase high-performance liquid chromatographic separation, and (iii) online photodiode array detection with spectrum analysis for id entification and measurement of all main 4- and 5-series eicosanoids ( leukotrienes, hydroxyeicosatetraenoic acids/hydroxyeicosapentaenoic ac ids, epoxyeicosatrienoic acids) within one run. With these procedures, standard mixtures of AA- and EPA-derived lipid mediators were recover ed from different biological Liquids, like lung perfusate, human bronc hoalveolar lavage fluid, and cell supernatant with linear characterist ics for each compound, Recoveries of the different lipid mediators exc eeded 80% showing excellent reproducibility, Application of the method to isolated, perfused, and ventilated human lungs challenged with the calcium ionophore A23187 and to human neutrophils stimulated in the p resence of arachidonic acid and eicosapentaenoic acid with N-formyl-me thionyl-leucyl-phenylalanine demonstrated the generation of a large ar ray of lipoxygenase and cytochrome P450 products. Thus, convenient qua ntification of 4- and 5-series eicosanoids in fluids of biological int erest is achieved by a technique comprising solid-phase extraction, is ocratic reversed-phase high- performance liquid chromatography, and ph otodiode array based online spectrum analysis of eluting compounds. (C ) 1998 Academic Press.