Contribution of UDP-glucuronosyltransferase 1A1 and 1A8 to morphine-6-glucuronidation and its kinetic properties
Contribution of UDP-glucuronosyltransferase 1A1 and 1A8 to morphine-6-glucuronidation and its kinetic properties
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DOI：
10.1124/dmd.107.019281
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发表时间：
2008-04-01
影响因子：
3.9
通讯作者：
Nakajin, Shizuo
中科院分区：
文献类型：
--
作者：
Ohno, Shuji;Kawana, Kiyoshi;Nakajin, Shizuo
吗啡代谢转化为吗啡-6-葡萄糖醛酸苷（M6 G）可能在吗啡的临床作用中起重要作用，因为M6 G具有上级镇痛作用。因此，阐明UDP葡萄糖醛酸转移酶（UGT）同工酶对吗啡-6-葡萄糖醛酸化的特异性具有重要意义。我们研究了重组人UGT同工酶在杆状病毒感染的昆虫细胞微粒体中催化吗啡-6-葡萄糖醛酸化的特异性。用高效液相色谱荧光检测法测定了重组人UGT同工酶与吗啡和UDP-葡萄糖醛酸孵育的吗啡葡萄糖醛酸化活性。不仅已知催化吗啡-6-葡萄糖醛酸化的UGT 2B 7，而且UGT 1A 1和1A 8在相对低的吗啡浓度（< 100 μ M）下也有效地催化吗啡-6-葡萄糖醛酸化。在低底物浓度下，两种同工酶的动力学均呈双曲线米氏动力学。然而，当吗啡浓度接近100 μ M时，吗啡-6-葡萄糖醛酸化活性逐渐降低，并且动力学与底物抑制Michaelis-Menten动力学行为非常相似。UGT 1A 1和1A 8的Km值分别为67.9和68.1 μ M，Ksi值分别为218.9和88.0 μ M。这些动力学基本上不同于UGT 2B 7对吗啡-6-葡萄糖醛酸化的动力学，这表明了双相Michaelis-Menten动力学行为。此外，为了估计这些UGT同工酶在体内M6 G形成中的贡献，通过逆转录实时聚合酶链反应测定了人肝脏和肠道中UGT 1A 1和1A 8 mRNA的表达水平。结果强烈表明，UGT 1A 1和UGT 1A 8是参与体内吗啡-6-葡萄糖醛酸化的同工酶，人体中的UGT 2B 7也是如此。
The metabolic conversion of morphine to morphine-6-glucuronide (M6G) seems to play a significant role in mediation of the clinical effect of morphine because of the superior analgesic effect of M6G. Therefore, it would be of great interest to clarify the specificity of morphine-6-glucuronidation by UDP glucuronosyltransferase (UGT) isozymes. We investigated the specificity of morphine-6- glucuronidation catalyzed by recombinant human UGT isozymes in microsomes from baculovirus-infected insect cells. The morphine glucuronidation activity of recombinant human UGT isozymes incubated with morphine and UDP-glucuronic acid was determined by high-performance liquid chromatography with a fluorescence detector. Not only UGT2B7, which is well known to catalyze morphine-6-glucuronidation, but also UGT1A1 and 1A8 effectively catalyzed morphine-6- glucuronidation at relatively low morphine concentrations (< 100 mu M). The kinetics of both isozymes at the low substrate concentrations showed hyperbolic Michaelis-Menten kinetics. However, as the morphine concentration approached 100 mu M, morphine-6- glucuronidation activity gradually decreased, and the kinetics closely resembled substrate inhibition Michaelis-Menten kinetic behavior. The Km values were 67.9 and 68.1 mu M and the Ksi values were 218.9 and 88.0 mu M for UGT1A1 and 1A8, respectively. These kinetics are basically different from that of morphine-6-glucuronidation by UGT2B7, which suggested biphasic Michaelis-Menten kinetic behavior. Furthermore, to estimate the contribution of these UGT isozymes in M6G formation in vivo, the expression levels of UGT1A1 and 1A8 mRNA in human liver and intestine were determined by reverse transcription real-time polymerase chain reaction. The results strongly suggest that UGT1A1 and UGT1A8 are isozymes involved in morphine-6-glucuronidation in vivo, as is UGT2B7 in humans.