Simultaneous Analysis of 4- and 5-Series Lipoxygenase and Cytochrome P450 Products from Different Biological Sources by Reversed-Phase High-Performance Liquid Chromatographic Technique

• Published in: Analytical biochemistry 1998-07, Vol.261 (1), p.16-28
• Main Authors: Kiss, Ladislau, Bieniek, Ewa, Weissmann, Norbert, Schütte, Hartwig, Sibelius, Ulf, Günther, Andreas, Bier, Jens, Mayer, Konstantin, Henneking, Klaus, Padberg, Winfried, Grimm, Helmut, Seeger, Werner, Grimminger, Friedrich
• Format: Article
• Language: English
• Subjects: Arachidonate 5-Lipoxygenase - metabolism; arachidonic acid; Arachidonic Acid - metabolism; Bronchoalveolar Lavage Fluid - chemistry; Chemistry Techniques, Analytical; Chromatography, High Pressure Liquid - methods; Cytochrome P-450 Enzyme System - metabolism; cytochrome P450; eicosanoids; Eicosanoids - analysis; Eicosanoids - chemistry; Eicosanoids - metabolism; eicosapentaenoic acid; Eicosapentaenoic Acid - metabolism; epoxyeicosatrienoic acids; Granulocytes - metabolism; Humans; hydroxyeicosapentaenoic acids; hydroxyeicosatetraenoic acids; In Vitro Techniques; isocratic reversed-phase high-performance liquid chromatography; leukotrienes; lipoxygenase; Lipoxygenase - metabolism; Lung - metabolism; Perfusion; photodiode array detection; solid-phase extraction; Spectrophotometry, Ultraviolet
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.1998.2674

Quantification of lipoxygenase and cytochrome P450 products of both arachidonic acid (AA) and eicosapentaenoic acid (EPA) is of broad interest due to the multiple biological activities of these compounds. Wedeveloped 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 identification and measurement of all main 4- and 5-series eicosanoids (leukotrienes, hydroxyeicosatetraenoic acids/hydroxyeicosapentaenoic acids, epoxyeicosatrienoic acids) within one run. With these procedures, standard mixtures of AA- and EPA-derived lipid mediators were recovered from different biological liquids, like lung perfusate, human bronchoalveolar lavage fluid, and cell supernatant with linear characteristics for each compound. Recoveries of the different lipid mediators exceeded 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 presence of arachidonic acid and eicosapentaenoic acid withN-formyl-methionyl-leucyl-phenylalanine demonstrated the generation of a large array of lipoxygenase and cytochrome P450 products. Thus, convenient quantification of 4- and 5-series eicosanoids in fluids of biological interest is achieved by a technique comprising solid-phase extraction, isocratic reversed-phase high-performance liquid chromatography, and photodiodearray-based online spectrum analysis of eluting compounds.