Department of Biomedical and Pharmaceutical Sciences, School of Pharmacy, Chapman University, Irvine, USA.
Department of Biomedical and Pharmaceutical Sciences, School of Pharmacy, Chapman University, Irvine, USA.
J Chromatogr B Analyt Technol Biomed Life Sci. 2018 Oct 1;1096:66-72. doi: 10.1016/j.jchromb.2018.08.011. Epub 2018 Aug 18.
Formation of dextrorphan (DXT) from dextromethorphan (DXM) has been widely used to assess cytochrome P450 2D (CYP2D) activity. Additionally, the kinetics of CYP2D activity have been well characterized in the liver microsomes. However, studies in brain microsomes are limited due to the lower microsomal content and abundance of CYP2D in the brain relative to the liver. In the present study, we developed a micro-scale enzymatic incubation method, coupled with a sensitive UPLC-MS/MS assay for the quantitation of the rate of DXT formation from DXM in brain microsomes. Rat brain microsomes were incubated with different concentrations of DXM for various times. The reaction was stopped, and the proteins were precipitated by the addition of acetonitrile, containing internal standard (d-DXT). After centrifugation, supernatant (2 μL) was injected onto a UPLC, C18 column with gradient elution. Analytes were quantitated using triple-quadrupole MS/MS with electrospray ionization in positive ion mode. The assay, which was validated for accuracy and precision in the linear range of 0.25 nM to 100 nM DXT, has a lower limit of quantitation of 0.125 fmol on the column. Using our optimized incubation and quantitation methods, we were able to reduce the incubation volume (25 μL), microsomal protein amount (5 μg), and incubation time (20 min), compared with reported methods. The method was successfully applied to estimation of the Michaelis-Menten (MM) kinetic parameters of dextromethorphan-O-demethylase activity in the rat brain microsomes (mean ± SD, n = 4), which showed a maximum velocity of 2.24 ± 0.42 pmol/min/mg and a MM constant of 282 ± 62 μM. It is concluded that by requiring far less biological material and time, our method represents a significant improvement over the existing techniques for investigation of CYP2D activity in rat brain microsomes.
右美沙芬（DXM）转化为右啡烷（DXT）已被广泛用于评估细胞色素 P450 2D（CYP2D）的活性。此外，CYP2D 活性的动力学在肝微粒体中已得到很好的描述。然而，由于大脑相对于肝脏而言，其微粒体含量和 CYP2D 的丰度较低，因此在脑微粒体中的研究有限。在本研究中，我们开发了一种微尺度酶孵育方法，结合超高效液相色谱-串联质谱法（UPLC-MS/MS）灵敏检测，用于定量测定脑微粒体中 DXM 转化为 DXT 的速率。用不同浓度的 DXM 孵育大鼠脑微粒体不同时间。停止反应后，通过加入含有内标（d-DXT）的乙腈沉淀蛋白。离心后，取上清液（2μL）注入超高效液相色谱，C18 柱，梯度洗脱。采用电喷雾正离子模式三重四极杆 MS/MS 定量分析。该方法在 0.25 nM 至 100 nM DXT 的线性范围内进行了准确性和精密度验证，其柱上的定量下限为 0.125 fmol。使用我们优化的孵育和定量方法，与报道的方法相比，我们能够减少孵育体积（25 μL）、微粒体蛋白量（5 μg）和孵育时间（20 min）。该方法成功应用于估计大鼠脑微粒体中美金刚烷-O-脱甲基酶活性的米氏动力学参数（平均值±标准差，n=4），其最大速度为 2.24±0.42 pmol/min/mg，米氏常数为 282±62 μM。结果表明，与现有的技术相比，我们的方法需要更少的生物材料和时间，是研究大鼠脑微粒体中 CYP2D 活性的一个显著改进。
