Novel 3-hydroxylated leukotriene b4 metabolites from ethanol-treated rat hepatocytes

Coincubations of radiolabeled leukotriene B4 (LTB4) and ethanol with isolated rat hepatocytes led to formation of one dihydroxylated and two novel β-oxidized metabolites of LTB4. The major radioactive peaks from reverse-phase-high performance liquid chromatography (RP-HPLC) eluted with material absorbing UV light maximally at 270 nm, with shoulders at 260 and 280 nm, indicating retention of the conjugated triene structure of the parent molecule in each metabolite structure. Following purification, catalytic reduction, and derivatization, mass spectrometric analysis revealed that all three metabolites were hydroxylated at the C-3 carbon atom based on characteristic ions at m/z 201 and 175 in the electron ionization mass spectra of the metabolites. Negative-ion electron capture mass spectrometry of the metabolites as pentafluorobenzyl (PFB) ester, trimethylsilyl ether derivatives aided structural characterizations while revealing interesting fragmentations. A ketene-containing ion appeared to result from the loss of both PFB groups (one as PFB alcohol), while a lactone alkoxide ion appeared to result following loss of PFB and bis (trimethylsilyl) ether. From these data three novel LTB4 metabolites were suggested to be 3,20-dihydroxy-LTB4 (3,20-diOH-LTB4), 3-hydroxy-18-carboxy-LTB4 (3-OH-18-COOH-LTB4), and 3-hydroxy-16-carboxy-LTB3 (3-OH-16-COOH-LTB3). The significance of the almost exclusive formation of these 3-hydroxylated LTB4 metabolites in the presence of ethanol is currently unknown, but may result from interrupted β-oxidation from the C-1 carboxyl moiety.