Human cytochromes P450 mediating phenacetin O-deethylation in vitro: Validation of the high affinity component as an index of CYP1A2 activity
Human cytochromes P450 mediating phenacetin O-deethylation in vitro: Validation of the high affinity component as an index of CYP1A2 activity
复制标题
DOI：
10.1021/js980255z
复制
发表时间：
1998-12-01
影响因子：
3.8
通讯作者：
Greenblatt, DJ
中科院分区：
文献类型：
--
作者：
Venkatakrishnan, K;von Moltke, LL;Greenblatt, DJ
非那西丁o -去甲基化被广泛用作细胞色素P450 1A2 （CYP1A2）活性的指标反应，在人肝微粒体中显示双相动力学。CYP1A2已被确定为高亲和力部分的贡献者，但尚未被证实为高亲和力阶段的唯一贡献者。此外，低亲和期的人类CYP亚型尚未被确定。我们已经使用异源表达的人类CYP异构体来鉴定、动力学表征和预测介导非那西丁o -去乙基化的主要人类肝脏CYP异构体的相对贡献。CYP1A2 （K-m 31 μ M）是人肝微粒体中唯一的高亲和力非那西丁o -去乙基化酶，而cvp 2A6 （K-m 4098 μ M）、2C9 （K-m 566 μ M）、2C19 （K-m 656 μ M）、2D6 （K-m 1021 μ M）和2E1 （K-m 1257 μ M）均参与了该反应的低亲和力阶段。考虑到人肝脏中各种cypp的相对丰度，在底物浓度为100 μ M时，CYP1A2占净反应速度的86%，而在底物浓度为865 μ M及以上时，CYP2C9成为主要的非那西丁o -去乙基化酶，占反应净V-max的31%。异源表达CYPs的动力学研究预测与磺胺苯唑和α -萘黄酮对人肝微粒体的化学抑制研究一致，表明在较高的底物浓度下，CYP2C9的作用更大，CYP1A2的作用更小。因此，CYP1A2是唯一高亲和力的人肝脏非那西丁o -去乙基化酶，从而验证了高亲和力成分作为人肝微粒体CYP1A2活性指标的使用。
Phenacetin O-deethylation, widely used as an index reaction for cytochrome P450 1A2 (CYP1A2) activity, displays biphasic kinetics in human liver microsomes. CYP1A2 has been identified as contributing to the high affinity component, but is not verified as the sole contributor to the high affinity phase. In addition, the human CYP isoforms accounting for the low affinity phase have not been identified. We have used heterologously expressed human CYP isoforms to identify, kinetically characterize, and predict the relative contribution of the major human liver CYP isoforms mediating phenacetin O-deethylation. CYP1A2 (K-m 31 mu M) is the only high affinity phenacetin O-deethylase in human liver microsomes, while CVPs 2A6 (K-m 4098 mu M), 2C9 (K-m 566 mu M), 2C19 (K-m 656 mu M), 2D6 (K-m 1021 mu M), and 2E1 (K-m 1257 mu M) all contribute to the low affinity phase of the reaction. Considering the relative abundance of the various CYPs in human liver, CYP1A2 accounts for 86% of net reaction velocity at a substrate concentration of 100 mu M, while CYP2C9 becomes the primary phenacetin O-deethylase at substrate concentrations of 865 mu M and higher and accounts for 31% of the net V-max of the reaction. Predictions from kinetic studies on heterologously expressed CYPs are consistent with chemical inhibition studies on human liver microsomes with sulfaphenazole and alpha-naphthoflavone that suggest a greater role for CYP2C9, and a smaller role for CYP1A2, at higher substrate concentrations. Thus CYP1A2 is the only high affinity human liver phenacetin O-deethylase, thereby validating the use of the high affinity component as an index of CYP1A2 activity in human liver microsomes.