Characterization of lipoxins by combined gas chromatography and electron-capture negative ion chemical ionization mass spectrometry: Formation of lipoxin A4 by stimulated human whole blood

Abstract

The lipoxins are a recent addition to the family of bioactive products derived from arachidonic acid. Here, we have prepared pentafluorobenzyl ester, trimethylsilyl ether derivatives of lipoxin A₄, lipoxin B₄ and pentadeuterolipoxin A₄ and have characterized these products by electron-capture negative ion chemical ionization gas chromatography/mass spectrometry (NICI GC/MS). Lipoxin A₄ (5S,6R,15S-trihydroxy-7,9,13-trans-11-cis-eicosa-tetra-enoic acid; LXA₄) was quantified following extraction from whole blood by stable isotopic dilution utilizing deuterium-labeled LXA₄ as internal standard and selected ion monitoring of the [M - pentafluorobenzyl] anions. Studies with a second tritiated internal standard (e.g. [11,12-³H]LXA₄) also showed that the recovery of LXA₄ was > 80% following solid-phase extraction from whole blood, and > 90% from isolated cells. In addition, neither isolated neutrophils nor platelets oxidatively metabolized [11,12-³H]LXA₄ when incubated in the presence or absence of stimuli. Whole blood incubated with either the ionophore of divalent cations (A23187), thrombin, or thrombin plus the chemotactic peptide formylmethionyl-leucine-phenylalanine generated both LXA₄ and thromboxane, which were quantified by stable isotope dilution. The ratio of thromboxane to LXA₄ formed by stimulated whole blood ranged from ∼2:1 to 20:1. These results indicate that the lipoxins display suitable characteristics as their respective pentafluorobenzyl ester, trimethylsilyl ether derivatives for quantification by electron-capture NICI GC/MS. Moreover, they provide evidence that LXA₄ can be generated from endogenous sources in whole blood following exposure to physiologically relevant stimuli.