Nakajima Miki, Tanaka Eriko, Kwon Jun-Tack, Yokoi Tsuyoshi
Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Japan.
Drug Metab Dispos. 2002 Dec;30(12):1484-90. doi: 10.1124/dmd.30.12.1484.
The nicotine and cotinine N-glucuronidations in human liver microsomes were characterized. The Eadie-Hofstee plots of nicotine N-glucuronidation in human liver microsomes were clearly biphasic, indicating the involvement of multiple enzymes. The apparent K(m) and V(max) values were 33.1 +/- 28.1 micro M and 60.0 +/- 21.0 pmol/min/mg and 284.7 +/- 122.0 micro M and 124.0 +/- 44.0 pmol/min/mg for the high- and low-affinity components, respectively, in human liver microsomes (n = 4). However, the Eadie-Hofstee plots of cotinine N-glucuronidation in human liver microsomes were monophasic (apparent K(m) = 1.9 +/- 0.3 mM, V(max) = 655.6 +/- 312.3 pmol/min/mg). The nicotine and cotinine N-glucuronidations in the recombinant human UDP-glucuronosyltransferases (UGTs) (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, and UGT2B15) expressed in baculovirus-infected insect cells or human B-lymphoblastoid cells that are commercially available were determined. However, no recombinant UGT isoforms showed detectable nicotine and cotinine N-glucuronides (the concentrations of nicotine and cotinine were 0.5 and 2 mM, respectively). Nicotine and cotinine N-glucuronidations in pooled human liver microsomes were competitively inhibited by bilirubin as a substrate for UGT1A1 (K(i) = 3.9 and 3.3 micro M), imipramine as a substrate for UGT1A4 (K(i) = 6.1 and 2.7 micro M), and propofol as a substrate for UGT1A9 (K(i) = 6.0 and 12.0 micro M). The nicotine N-glucuronidation (50 micro M nicotine) in 14 human liver microsomes was significantly (r = 0.950, P < 0.0001) correlated with the cotinine N-glucuronidation (0.2 mM cotinine), indicating that the same isoform(s) is involved in both glucuronidations. Furthermore, weak correlations between imipramine N-glucuronidation and nicotine N-glucuronidation (r = 0.425) or cotinine N-glucuronidation (r = 0.517) were observed. In conclusion, the involvement of UGT1A1 and UGT1A9 as well as UGT1A4 in nicotine and cotinine N-glucuronidations in human liver microsomes was suggested, although the contributions of each UGT isoform could not be determined conclusively.
对人肝微粒体中尼古丁和可替宁的N-葡萄糖醛酸化进行了表征。人肝微粒体中尼古丁N-葡萄糖醛酸化的伊迪-霍夫斯泰因（Eadie-Hofstee）图明显呈双相，表明有多种酶参与。人肝微粒体中高亲和力和低亲和力组分的表观K(m)值和V(max)值分别为33.1±28.1 μM和60.0±21.0 pmol/min/mg以及284.7±122.0 μM和124.0±44.0 pmol/min/mg（n = 4）。然而，人肝微粒体中可替宁N-葡萄糖醛酸化的伊迪-霍夫斯泰因图呈单相（表观K(m) = 1.9±0.3 mM，V(max) = 655.6±312.3 pmol/min/mg）。测定了在杆状病毒感染的昆虫细胞或市售人B淋巴母细胞中表达的重组人尿苷二磷酸葡萄糖醛酸基转移酶（UGTs）（UGT1A1、UGT1A3、UGT1A4、UGT1A6、UGT1A7、UGT1A8、UGT1A9、UGT1A10、UGT2B7和UGT2B15）中的尼古丁和可替宁N-葡萄糖醛酸化。然而，没有重组UGT同工型显示出可检测到的尼古丁和可替宁N-葡萄糖醛酸化物（尼古丁和可替宁的浓度分别为0.5和2 mM）。汇集的人肝微粒体中的尼古丁和可替宁N-葡萄糖醛酸化受到胆红素（作为UGT1A1的底物，K(i) = 3.9和3.3 μM）、丙咪嗪（作为UGT1A4的底物，K(i) = 6.1和2.7 μM）和丙泊酚（作为UGT1A9的底物，K(i) = 6.0和12.0 μM）的竞争性抑制。14个人肝微粒体中尼古丁的N-葡萄糖醛酸化（50 μM尼古丁）与可替宁的N-葡萄糖醛酸化（0.2 mM可替宁）显著相关（r = 0.950，P < 0.0001），表明相同的同工型参与了两种葡萄糖醛酸化。此外，观察到丙咪嗪N-葡萄糖醛酸化与尼古丁N-葡萄糖醛酸化（r = 0.425）或可替宁N-葡萄糖醛酸化（r = 0.517）之间存在弱相关性。总之，提示UGT1A1、UGT1A9以及UGT1A4参与了人肝微粒体中尼古丁和可替宁的N-葡萄糖醛酸化，尽管每种UGT同工型的贡献尚不能最终确定。
