20] High-performance liquid chromatography separation and determination of lipoxins

Abstract

The chapter presents a study on high-performance liquid chromatography (HPLC) separation and determination of lipoxins. This chapter describes the isolation, reversed-phase high-performance liquid chromatography (RP-HPLC) separation, and determination of lipoxins of the four series. The lipoxins are a series of biologically active, acyclic eicosanoids, which contain a conjugated tetraene structure as a characteristic feature. The following two main compounds are positional isomers: one is designated lipoxin A4 (LXA4) and the other lipoxin B4 (LXB4). Multiple pathways exist for lipoxin biosynthesis that are substrate-, cell type-, and species-specific. The chapter outlines the route that involves the transformation of 15- hydroxyeicosatetraenoic acid (HETE) and formation of a 5(6)-epoxytetraene by human leukocytes. Other routes can involve the transcellular metabolism of various substrates including leukotriene A4, arachidonic acid, 5,15-DHETE, and 5-HETE. LXA4 and LXB4 each display biological activities, which can be distinguished from those of other eiscosanoids. Several isomers of LXA4 and LXB4 have been identified, including a novel 7-cis- 11-trans-LXA4. Because LXA4, LXB4, and their isomers differ in biological actions and potencies, their identification within biologically derived materials is of interest. As is the case when identifying other eicosanoids, lipoxins should be identified from biological sources by fulfilling several criteria that may include the following: (1) coelution of the product with authentic materials in more than one HPLC system (retention time); (2) coelution of the derivatized material with that of authentic standard (determination of C value); (3) UV spectral analyses of the isolated material; (4) the presence of diagnostic ions in the mass spectra of several derivatives (that is, n-butyl boronate- trimethylsilyl derivatives); and (5) bioassay of the isolated products and comparison with authentic materials.