Baati Tarek, Horcajada Patricia, Gref Ruxandra, Couvreur Patrick, Serre Christian
UMR CNRS 8180, Institut Lavoisier, Université de Versailles Saint Quentin en Yvelines, France.
J Pharmacol Toxicol Methods. 2012 Jul;66(1):29-34. doi: 10.1016/j.vascn.2012.05.006. Epub 2012 May 26.
CYP3A4 is one of the most important of all drug-metabolizing enzymes. Although several direct or indirect quantification methods have been proposed for the determination of the CYP3A4 activity, the sample preparation is mostly tedious and usually requires time consuming separate extraction steps and solid-phase extraction.
Here, we developed a simple and selective HPLC method, coupled to photodiode array detection for direct determination of CYP3A4-mediated testosterone hydroxylase activity in hepatic microsomes of rats. After microsome incubation, a single-step liquid-phase extraction of specific substrates, testosterone and its metabolite 6-hydroxytestosterone, together with the salicylamid acid used as an internal standard, was applied. The analytical method was fully validated by the determination of different parameters (intra- and inter-day variability of 6-β-OH-testosterone concentration, accuracy and limit of detection). Finally, this method was applied to quantify the CYP3A4 activity of rats intravenously administered with either the mesoporous iron(III) trimesate MIL-100 nanocarrier (MIL stands for Materials from Institut Lavoisier) or with its corresponding organic linker.
All analytes were simultaneously separated from a single run shorter than 10 min, reaching relative standard deviations of intra- and inter-day precision <18.5% and an accuracy of estimated 6-β-OH-testosterone concentrations ranging from 95 to 111%. The mean±standard deviation absolute recoveries of 6-β-OH-testosterone at 0.01, 1.00 and 20.00 μg/mL were 97±4%, 101±3% and 99±2%, respectively while it reached 98±3% for the internal standard.
The developed HPLC-PDA method enables the accurate and sensitive determination of the CYP3A4 activity through the quantification of the 6-β-OH-testosterone produced by the CYP3A4-mediated testosterone hydroxylase in rat hepatic microsomal suspensions. Additionally, the rapid procedure offers an economical advantage with respect to resources and operator time. Finally, the determination of CYP3A4 activity in hepatic microsomes of rats administered with nanoparticles of the porous iron(III) trimesate nanocarrier shows no significant differences between control, trimesic and nanoparticle groups, evidencing that the linker is not metabolized by CYP3A4.
细胞色素P450 3A4（CYP3A4）是所有药物代谢酶中最重要的酶之一。尽管已经提出了几种直接或间接的定量方法来测定CYP3A4的活性，但样品制备大多繁琐，通常需要耗时的单独提取步骤和固相萃取。
在此，我们开发了一种简单且具有选择性的高效液相色谱（HPLC）方法，并结合光电二极管阵列检测，用于直接测定大鼠肝微粒体中CYP3A4介导的睾酮羟化酶活性。微粒体孵育后，采用一步液相萃取特定底物睾酮及其代谢物6-羟基睾酮，同时将水杨酰胺酸用作内标。通过测定不同参数（6-β-羟基睾酮浓度的日内和日间变异性、准确度和检测限）对分析方法进行了全面验证。最后，该方法应用于定量静脉注射均苯三甲酸介孔铁（III）纳米载体（MIL代表来自拉瓦锡研究所的材料）或其相应有机连接体的大鼠的CYP3A4活性。
所有分析物在一次运行时间短于10分钟的情况下同时分离，日内和日间精密度的相对标准偏差<18.5%，估计的6-β-羟基睾酮浓度的准确度在95%至111%之间。6-β-羟基睾酮在0.01、1.00和20.00μg/mL时的平均±标准偏差绝对回收率分别为97±4%、101±3%和99±2%，而内标的回收率达到98±3%。
所开发的HPLC-光电二极管阵列方法能够通过定量大鼠肝微粒体悬浮液中CYP3A4介导的睾酮羟化酶产生的6-β-羟基睾酮来准确、灵敏地测定CYP3A4活性。此外，该快速程序在资源和操作人员时间方面具有经济优势。最后，对用均苯三甲酸多孔铁（III）纳米载体纳米颗粒给药的大鼠肝微粒体中CYP3A4活性的测定表明，对照组、均苯三甲酸组和纳米颗粒组之间没有显著差异，证明连接体不会被CYP3A4代谢。
