Wheelan P, Murphy R C, Simon F R
National Jewish Center for Immunology and Respiratory Medicine, Denver, Colorado 80206, USA.
J Mass Spectrom. 1996 Mar;31(3):236-46. doi: 10.1002/(SICI)1096-9888(199603)31:3<236::AID-JMS293>3.0.CO;2-U.
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.
用气相色谱/质谱联用仪（GC/MS）分析白三烯B4（LTB4）与肝星状细胞（Ito细胞）孵育后形成的衍生代谢产物，拓展了我们之前关于衍生化羟基取代不饱和脂肪酸裂解机制的认识。LTB4可被大鼠Ito细胞（一种肝血窦周围星状细胞）通过δ10-和δ14-还原酶途径代谢，生成10,11-二氢-LTB4和10,11,14,15-四氢-LTB4。还观察到中间代谢产物12-氧代-10,11-二氢-LTB4和12-氧代-10,11,14,15-四氢-LTB4的形成。对作为五氟苄基酯/三甲基硅醚化合物衍生化的12-氧代代谢产物进行GC/电子轰击（EI）质谱分析，得到了表明氧代取代基和双键位置的独特裂解模式。10,11-二氢-LTB4和10,11,14,15-四氢-LTB4通过羧基末端β-氧化进一步代谢，生成链缩短的单羟基代谢产物。这种代谢过程中可能的中间体，即导致LTB4原有的C-5羟基取代基丢失的中间体，被鉴定为含2,4,6-共轭三烯的C-18代谢产物。在衍生化的10,11,14,15-四氢LTB4代谢产物中，三甲基硅氧基醚的烯丙基双键缺失，显著降低了这些化合物EI质谱中观察到的α-裂解离子的丰度，这表明在这种α-裂解反应中形成烯丙基稳定自由基的重要性。由于缺乏有利的α-裂解反应，对10-羟基-2,4,6-十八碳三烯酸进行GC/EIMS分析，生成了m/z 91的离子，这可能是通过共轭三烯部分的环化产生的。此外，对含有2,4,6-共轭三烯部分的衍生代谢产物进行GC/MS分析，在电子捕获电离质谱中得到了一个独特的碎片离子，它也可能是通过共轭三烯环化形成m/z 121而产生的。
