Lipoxins formation by rat basophilic leukemia (RBL-1) cells

Rat basophilic leukemia (RBL-1) cells (1.5 x 10(9) were incubated with (15S), 15-hydroperoxy-5,8,11-cis-13-trans-eicosatetraenoic acid (15-HPETE) (100 microM) and calcium ionophore A23187 (5 microM) for 30 min at 37 degrees C. The reaction products were extracted and separated on reverse-phase high performance liquid chromatography (RP-HPLC). The fraction of interest which showed the characteristic UV spectrum of lipoxins (LXs) (lambda max at 301 nm, shoulders at 289 nm and 317 nm) was further purified using a second RP-HPLC. This step produced four distinct peaks, three of which co-eluted with synthetic lipoxin A4 (LXA4), lipoxin B4 (LXB4), and authentic 6S-LXA4 standards, respectively. These fractions were analyzed by capillary gas chromatography and mass spectrometry (GC/MS) for structural identity. Based on these data (chromatographic profile, UV spectrum, mass spectra) and the reported criteria (Serhan/Samuelsson, 1984-88), these three fractions were identified with LXA4[(5S,6R,15S)-5,6,15-trihydroxy-7,9,13-trans-11-cis-eicosatetra enoic acid)], LXB4[(5S,14R,15S)-5,14,15-trihydroxy-6,10,12-trans-8-cis-eic osa-tetraenoic acid)] and 6S-LXA4[(5S,6S,15S)-5,6,15-trihydroxy-7,9,13-trans-11-cis-eicosate traenoic acid)]. It is concluded that RBL-1 cells have the capacity to generate LXs by metabolizing arachidonic acid (AA) derivatives (i.e., 15-HPETE) and that LXs produced by RBL-1 cells are indistinguishable from those derived from other cells.