Venkatakrishnan K, von Moltke L L, Greenblatt D J
Department of Pharmacology and Experimental Therapeutics, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
J Pharm Sci. 1998 Dec;87(12):1502-7. doi: 10.1021/js980255z.
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 (Km 31 microM) is the only high affinity phenacetin O-deethylase in human liver microsomes, while CYPs 2A6 (Km 4098 microM), 2C9 (Km 566 microM), 2C19 (Km 656 microM), 2D6 (Km 1021 microM), and 2E1 (Km 1257 microM) 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 microM, while CYP2C9 becomes the primary phenacetin O-deethylase at substrate concentrations of 865 microM and higher and accounts for 31% of the net Vmax 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.
非那西丁O - 脱乙基反应被广泛用作细胞色素P450 1A2（CYP1A2）活性的指标反应，在人肝微粒体中呈现双相动力学。CYP1A2已被确定为高亲和力组分的贡献者，但尚未被证实是高亲和力阶段的唯一贡献者。此外，导致低亲和力阶段的人CYP同工酶尚未被鉴定出来。我们利用异源表达的人CYP同工酶来鉴定、动力学表征并预测介导非那西丁O - 脱乙基反应的主要人肝CYP同工酶的相对贡献。CYP1A2（Km为31 microM）是人肝微粒体中唯一的高亲和力非那西丁O - 脱乙基酶，而CYP 2A6（Km为4098 microM）、2C9（Km为566 microM）、2C19（Km为656 microM）、2D6（Km为1021 microM）和2E1（Km为1257 microM）均对反应的低亲和力阶段有贡献。考虑到人肝中各种CYP的相对丰度，在底物浓度为100 microM时，CYP1A2占净反应速度的86%，而在底物浓度为865 microM及更高时，CYP2C9成为主要的非那西丁O - 脱乙基酶，占反应净Vmax的31%。对异源表达的CYPs进行动力学研究的预测结果与用磺胺苯吡唑和α - 萘黄酮对人肝微粒体进行化学抑制研究的结果一致，后者表明在较高底物浓度下，CYP2C9的作用更大，而CYP1A2的作用较小。因此，CYP1A2是唯一的高亲和力人肝非那西丁O - 脱乙基酶，从而验证了将高亲和力组分用作人肝微粒体中CYP1A2活性指标的用途。
