Gas chromatographic/mass spectrometric analysis of oxo and chain-shortened leukotriene B4 metabolites. Leukotriene B4 metabolism in Ito cells.

Abstract

Analysis by gas chromatography/mass spectrometry (GC/MS) of derivatized metabolites formed following incubation of leukotriene B4 (LTB4) incubation with Ito cells extends previous knowledge concerning fragmentation mechanisms for derivatized hydroxy-substituted unsaturated fatty acids. LTB4 was metabolized by rat Ito cells, a hepatic perisinusoidal stellate cell, by the delta 10- and delta 14-reductase pathways, resulting in the formation of 10,11-dihydro-LTB4 and 10,11,14,15-tetrahydro-LTB4. Formation of the intermediate metabolites, 12-oxo-10,11-dihydro-LTB4 and 12-oxo-10,11,14,15-tetrahydro-LTB4, was also observed. GC/electron impact (EI) MS analysis of the 12-oxo metabolites, derivatized as the pentafluorobenzyl ester/ trimethylsilyl ether compounds, resulted in unique fragmentations indicative of the oxo substituent and double bond positions. Further metabolism of 10,11-dihydro-LTB4 and 10,11,14,15-tetrahydro-LTB4 by carboxy terminus beta-oxidation resulted in chain-shortened monohydroxy metabolites. Possible intermediates in this metabolism, which resulted in loss of the original C-5 hydroxy substituent from LTB4, were identified as 2,4,6-conjugated triene-containing C-18 metabolites. The absence of a double bond allylic to the trimethylsiloxy ether in derivatized 10,11,14,15-tetrahydro LTB4 metabolites strikingly reduced the abundance of alpha-cleavage ions observed in the EI mass spectra of these compounds, thus suggesting the importance of formation of an allylic stabilized radical in such alpha-cleavage reactions. Lacking a favorable alpha-cleavage reaction, GC/EIMS analysis of 10-hydroxy-2,4,6-octadecatrienoic acid resulted in the formation of m/z 91, which may arise via cyclization of the conjugated triene moiety. In addition, GC/MS analysis of derivatized metabolites containing the 2,4,6 conjugated triene moiety resulted in a unique fragment ion in the electron capture ionization mass spectra that also may arise via cyclization of the conjugated triene with formation of m/z 121.