Trans-3′-hydroxycotinine O- and N-glucuronidations in human liver microsomes
Trans-3′-hydroxycotinine O- and N-glucuronidations in human liver microsomes
复制标题
DOI：
10.1124/dmd.104.001701
复制
发表时间：
2005-01-01
影响因子：
3.9
通讯作者：
Yokoi, T
中科院分区：
文献类型：
--
作者：
Yamanaka, H;Nakajima, M;Yokoi, T
反式-3 '-羟基可替宁是人体内尼古丁的主要代谢产物，主要以O-葡糖苷酸形式经吸烟者尿液排泄。人肝微粒体与UDP-葡萄糖醛酸孵育不仅产生反式-3 '-羟基可替宁O-葡萄糖醛酸苷，还产生N-葡萄糖醛酸苷。在大多数人肝微粒体中，N-葡糖苷酸的形成超过O-葡糖苷酸的形成，尽管从未在人尿液中检测到N-葡糖苷酸。人肝微粒体中的反式-3 '-羟基可替宁N-葡萄糖醛酸化与尼古丁和可替宁N-葡萄糖醛酸化显著相关，尼古丁和可替宁N-葡萄糖醛酸化主要由UDP-葡萄糖醛酸基转移酶（UGT）1A 4催化，并受到UGT 1A 4底物丙咪嗪和尼古丁的抑制。重组UGT 1A 4表现出显著的反式-3 '-羟基可替宁N-葡萄糖醛酸基转移酶活性。这些结果表明，人肝微粒体中的反式-3 '-羟基可替宁N-葡萄糖醛酸化主要由UGT 1A 4催化。在本研究中，对人肝微粒体中的反式-3 '-羟基可替宁O-葡萄糖醛酸化进行了全面表征，因为反式-3'-羟基可替宁O-葡萄糖醛酸苷是尼古丁的主要代谢产物之一。动力学拟合为Michaelis-Menten方程，K-m为10.0 +/- 0.8 mM，V-max为85.8 +/- 3.8 pmol/min/mg。在杆状病毒感染的昆虫细胞中表达的11种重组人UGT亚型中，UGT 2B 7表现出最高的反式-3 '-羟基可替宁O-葡萄糖醛酸基转移酶活性（1.1 pmol/min/mg），随后为UGT 1A 9（0.3 pmol/min/mg），UGT2B15（0.2 pmol/min/mg），和ugt 2b 4（0.2 pmol/min/mg），底物浓度为1 mM。重组UGT 2B 7的葡萄糖醛酸基转移酶活性随着底物浓度增加而增加，直至16 mM（10.5 pmol/min/mg）。重组UGT 1A 9的动力学符合Michaelis-Menten方程，K-m = 1.6 +/- 0.1 mM，V-max = 0.69 +/- 0.02 pmol/min/mg蛋白质。13个人肝微粒体中的反式-3 '-羟基可替宁O-葡萄糖醛酸基转移酶活性范围为2.4 - 12.6 pmol/min/mg，与由UGT 2B 7、UGT 1A 6和UGT 1A 9催化的丙戊酸葡萄糖醛酸化显著相关（r = 0.716，p < 0.01）。丙咪嗪（UGT 1A 4的底物，IC 50 = 55 μ M）、雄甾烷酮（UGT 2B 15的底物，IC 50 = 169 μ M）和丙泊酚（UGT 1A 9的底物，IC 50 = 296 μ M）可抑制人肝微粒体中的反式-3 '-羟基可替宁O-葡萄糖醛酸基转移酶活性。有趣的是，丙咪嗪（IC 50 = 45 μ M）、雄甾烷二醇（IC 50 = 21 μ M）和异丙酚（IC 50 = 41 μ M）也抑制重组UGT 2B 7的反式-3 '-羟基可替宁O-葡萄糖醛酸转移酶活性。这些结果表明，人肝微粒体中的反式-3 '-羟基可替宁O-葡萄糖醛酸化主要由UGT 2B 7催化，在较小程度上由UGT 1A 9催化。
Trans-3'-hydroxycotinine is a major metabolite of nicotine in humans and is mainly excreted as O-glucuronide in smoker's urine. Incubation of human liver microsomes with UDP-glucuronic acid produces not only trans-3'-hydroxycotinine O-glucuronide but also N-glucuronide. The formation of N-glucuronide exceeds the formation of O-glucuronide in most human liver microsomes, although N-glucuronide has never been detected in human urine. Trans-3'-hydroxycotinine N-glucuronidation in human liver microsomes was significantly correlated with nicotine and cotinine N-glucuronidations, which are catalyzed mainly by UDP-glucuronosyltransferase (UGT)1A4 and was inhibited by imipramine and nicotine, which are substrates of UGT1A4. Recombinant UGT1A4 exhibited substantial trans-3'-hydroxycotinine N-glucuronosyltransferase activity. These results suggest that trans-3'-hydroxycotinine N-glucuronidation in human liver microsomes would be mainly catalyzed by UGT1A4. In the present study, trans-3'-hydroxycotinine O-glucuronidation in human liver microsomes was thoroughly characterized, since trans-3'-hydroxycotinine O-glucuronide is one of the major metabolites of nicotine. The kinetics were fitted to the Michaelis-Menten equation with a K-m of 10.0 +/- 0.8 mM and a V-max of 85.8 +/- 3.8 pmol/min/mg. Among 11 recombinant human UGT isoforms expressed in baculovirus-infected insect cells, UGT2B7 exhibited the highest trans-3'-hydroxycotinine O-glucuronosyltransferase activity (1.1 pmol/min/mg) followed by UGT1A9 (0.3 pmol/min/mg), UGT2B15 (0.2 pmol/min/mg), and UGT2B4 (0.2 pmol/min/mg) at a substrate concentration of 1 mM. Trans-3'-hydroxycotinine O-glucuronosyltransferase activity by recombinant UGT2B7 increased with an increase in the substrate concentration up to 16 mM (10.5 pmol/min/mg). The kinetics by recombinant UGT1A9 were fitted to the Michaelis-Menten equation with K-m = 1.6 +/- 0.1 mM and V-max = 0.69 +/- 0.02 pmol/min/mg of protein. Trans-3'-hydroxycotinine O-glucuronosyltransferase activities in 13 human liver microsomes ranged from 2.4 to 12.6 pmol/min/mg and were significantly correlated with valproic acid glucuronidation (r = 0.716, p < 0.01), which is catalyzed by UGT2B7, UGT1A6, and UGT1A9. Trans-3'-hydroxycotinine O-glucuronosyltransferase activity in human liver microsomes was inhibited by imipramine (a substrate of UGT1A4, IC50 = 55 mu M), androstanediol (a substrate of UGT2B15, IC50 = 169 mu M), and propofol (a substrate of UGT1A9, IC50 = 296 mu M). Interestingly, imipramine (IC50 = 45 mu M), androstanediol (IC50 = 21 mu M), and propofol (IC50 = 41 mu M) also inhibited trans-3'-hydroxycotinine O-glucuronosyltransferase activity by recombinant UGT2B7. These findings suggested that trans-3'-hydroxycotinine O-glucuronidation in human liver microsomes is catalyzed by mainly UGT2B7 and, to a minor extent, by UGT1A9.