GAS-CHROMATOGRAPHIC MASS-SPECTROMETRIC ANALYSIS OF OXO AND CHAIN-SHORTENED LEUKOTRIENE B-4 METABOLITES, LEUKOTRIENE B-4 METABOLISM IN ITO CELLS

Analysis by gas chromatography/mass spectrometry (GC/MS) of derivatize d metabolites formed following incubation of leukotriene B-4 (LTB(4)) incubation with Ito cells extends previous knowledge concerning fragme ntation mechanisms for derivatized hydroxy-substituted unsaturated fat ty acids. LTB(4) was metabolized by rat Ito cells, a hepatic perisinus oidal stellate cell, by the Delta(10)- and Delta(14)-reductase pathway s, resulting in the formation of 10,11-dihydro-LTB(4) and 10,11,14,15- tetrahydro-LTB(4). Formation of the intermediate metabolites, 12-oxo-1 0,11-dihydro-LTB(4) and 12-oxo-10,11,14,15-tetrahydro-LTB(4), was also observed. GC/electron impact (EI) MS analysis of the 12-oxo metabolit es, derivatized as the pentafluorobenzyl ester/trimethylsilyl ether co mpounds, resulted in unique fragmentations indicative of the oxo subst ituent and double bond positions. Further metabolism of 10,11-dihydro- LTB(4) and 10,11,14,15-tetrahydro-LTB(4) by carboxy terminus beta-oxid ation resulted in chain-shortened monohydroxy metabolites. Possible in termediates in this metabolism, which resulted in loss of the original C-5 hydroxy substituent from LTB(4), were identified as 2,4,6-conjuga ted triene-containing C-18 metabolites. The absence of a double bond a llylic to the trimethylsiloxy ether in derivatized 10,11,14,15-tetrahy dro LTB(4) metabolites strikingly reduced the abundance of alpha-cleav age ions observed in the EI mass spectra of these compounds, thus sugg esting the importance of formation of an allylic stabilized radical in such alpha-cleavage reactions. Lacking a favorable alpha-cleavage rea ction, GC/EIMS analysis of 10-hydroxy-2,4,6-octadecatrienoic acid resu lted in the formation of m/z 91, which may arise via cyclization of th e 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 for mation of m/z 121.