Soberman R J, Sutyak J P, Okita R T, Wendelborn D F, Roberts L J, Austen K F
Department of Medicine, Harvard Medical School, Boston, Massachusetts.
J Biol Chem. 1988 Jun 15;263(17):7996-8002.
Microsomes of human polymorphonuclear leukocytes (PMN) in the presence of 100 microM NADPH converted 0.6 microM leukotriene B4 (LTB4) to 20-OH-LTB4 (retention time = 18.0 min) and to two additional compounds designated I (retention time = 16.8 min) and II (retention time = 9.6 min) as analyzed by reverse-phase high performance liquid chromatography (HPLC). Compounds I and II were also formed from the reaction of 1.0 microM 20-OH-LTB4, PMN microsomes, and 100 microM NADPH; the identity of compound II was confirmed as 20-COOH-LTB4 by gas chromatography-mass spectrometry. Equine alcohol dehydrogenase in the presence of 100 microM NAD+ in 0.2 M glycine buffer (pH 10.0) converted 20-OH-LTB4 to 20-aldehyde (CHO) LTB4, which coeluted with compound I on reverse-phase HPLC. In the presence of 100 microM NADH in 50 mM potassium phosphate buffer (pH 6.5), equine alcohol dehydrogenase reduced both 20-CHO-LTB4 and compound I to 20-OH-LTB4, indicating the identity of compound I as 20-CHO-LTB4. Gas chromatography-mass spectrometry of trideuterated O-methyl-oxime trimethylsilyl ether methyl ester derivative of 3H-labeled compound I definitively established compound I as 20-CHO-LTB4. Addition of immune IgG to cytochrome P-450 reductase or 1.0 mM SKF-525A completely inhibited the formation of 20-CHO-LTB4 from 20-OH-LTB4, indicating that the reaction was catalyzed by a cytochrome P-450. LTB5 (3.0 microM), a known substrate for cytochrome P-450LTB and a competitive inhibitor of LTB4 omega-oxidation, completely inhibited the omega-oxidation of 1.5 microM 20-OH-LTB4 to 20-CHO-LTB4, indicating that the cytochrome P-450 was P-450LTB. Conversion of 1.0 microM 20-CHO-LTB4 to 20-COOH-LTB4 by PMN microsomes was also dependent on NADPH and inhibited by antibody to cytochrome P-450 reductase, 1.0 mM SKF-525A, or 5.0 microM LTB5, indicating that this reaction was also catalyzed by cytochrome P-450LTB. These results identify the novel metabolite 20-CHO-LTB4 and indicate that cytochrome P-450LTB catalyzes three sequential omega-oxidations of LTB4 leading to the formation of 20-COOH-LTB4 via 20-OH-LTB4 and 20-CHO-LTB4 intermediates.
在100微摩尔NADPH存在的情况下，人多形核白细胞（PMN）的微粒体将0.6微摩尔白三烯B4（LTB4）转化为20-羟基-LTB4（保留时间 = 18.0分钟）以及另外两种分别命名为I（保留时间 = 16.8分钟）和II（保留时间 = 9.6分钟）的化合物，通过反相高效液相色谱法（HPLC）进行分析。化合物I和II也由1.0微摩尔20-羟基-LTB4、PMN微粒体与100微摩尔NADPH反应生成；通过气相色谱-质谱联用技术确认化合物II为20-羧基-LTB4。在0.2M甘氨酸缓冲液（pH 10.0）中，100微摩尔NAD⁺存在的情况下，马肝醇脱氢酶将20-羟基-LTB4转化为20-醛基（CHO）LTB4，其在反相HPLC上与化合物I共洗脱。在50mM磷酸钾缓冲液（pH 6.5）中，100微摩尔NADH存在的情况下，马肝醇脱氢酶将20-醛基-LTB4和化合物I都还原为20-羟基-LTB4，表明化合物I为20-醛基-LTB4。对3H标记的化合物I的氘代O-甲基-肟三甲基硅醚甲酯衍生物进行气相色谱-质谱联用分析，最终确定化合物I为20-醛基-LTB4。向细胞色素P-450还原酶中加入免疫IgG或1.0mM SKF-525A可完全抑制由20-羟基-LTB4生成20-醛基-LTB4的反应，表明该反应由细胞色素P-450催化。白三烯B5（LTB5，3.0微摩尔）是细胞色素P-450LTB的已知底物以及LTB4 ω-氧化的竞争性抑制剂，它完全抑制了1.5微摩尔20-羟基-LTB4向20-醛基-LTB4的ω-氧化，表明该细胞色素P-450是P-450LTB。PMN微粒体将1.0微摩尔20-醛基-LTB4转化为20-羧基-LTB4的过程同样依赖于NADPH，并受到细胞色素P-450还原酶抗体、1.0mM SKF-525A或5.0微摩尔LTB5的抑制，表明该反应也由细胞色素P-450LTB催化。这些结果鉴定出了新的代谢产物20-醛基-LTB4，并表明细胞色素P-450LTB催化LTB4的三步连续ω-氧化反应，通过20-羟基-LTB4和20-醛基-LTB4中间体生成20-羧基-LTB4。
