采用特异性探针药物法测定双峰驼CYP3A酶活性  

Determination of the Activities of Bactrian Camel CYP3A Enzyme by Specific Probe Drug

作　　者：裴乐[1] 张文彬 哈斯苏荣[1,3] 

机构地区：[1]内蒙古农业大学兽医学院/农业部动物疾病临床诊疗技术重点实验室,呼和浩特010018 [2]内蒙古阿拉善骆驼科学研究所,内蒙古巴彦浩特750300 [3]内蒙古骆驼研究院,内蒙古巴丹吉林750300

出　　处：《中国农业科学》2015年第16期3266-3274,共9页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31260623)

摘　　要：【目的】探究双峰驼CYP3A酶体外活性及其对外源性药物代谢的影响。【方法】将双峰驼禁食过夜12 h,颈静脉放血处死后,立即于肝门静脉处采集块状肝脏组织,洗去血污称重匀浆后,采用Ca2+沉淀法制备双峰驼肝微粒体,并通过BCA法测定其蛋白含量。同时优化双峰驼肝微粒孵育体系,取不同浓度的双峰驼肝微粒体蛋白悬液(0.016、0.031、0.063、0.125、0.250、0.500、1.000、2.000 mg·m L-1)、不同浓度的探针药物咪达唑仑(MDZ)(7.813、15.625、31.250、62.500、125、250、500、1 000μg·m L-1)分别加入到孵育体系中,37℃预孵育5 min后,加入NADPH溶液后再孵育不同时间(0、2、5、10、15、20、25、30 min)。待反应完全后加入冰冷的甲醇和地西泮(DZP)溶液,涡旋混匀,800μL全部混合液体经14 500 r/min离心20 min后,取20μL上清液进行高效液相色谱紫外检测(HPLC-UV),确定最适底物浓度、最适酶浓度以及最适孵育时间。分别取浓度为7.813、15.625、31.250、62.500、125、250、500、1 000μg·m L-1 MDZ探针药物溶液20μL与双峰驼肝微粒体共同孵育15 min后,以混有内标DZP的冰冷甲醇终止反应,采用高效液相色谱紫外检测法测定反应产物1'-羟基咪达唑仑(1'-OHMDZ)的动态含量,计算出部分药物动力学参数。色谱条件:Sigma-Aldrich/Supelco Discovery C18色谱柱(150 mm×4.6 mm,5μm);流动相为V(乙腈)﹕V(0.01 mol·L-1 PBS缓冲液)=40﹕60;等浓度洗脱20 min;检测波长为254 nm;流速为0.8 m L·min-1;柱温为30℃;进样量为20μL。【结果】使用Ca2+沉淀法制备的双峰驼肝微粒体保持了酶的良好活性,能满足后续体外药物代谢试验的基本要求,经BCA法测定其蛋白含量为(1.936±0.052)mg·m L-1。双峰驼肝微粒体蛋白悬液、探针药物MDZ、PBS缓冲液、Mg Cl2溶液以及NADPH溶液构成双峰驼肝微粒体孵育体系,随着孵育时间的延长,酶和底物进一步反应,当MDZ浓度为125μg·m L-1(终浓度为7.073 nmol·m L【Objective】The objective of this paper is to study the activities of Bactrian camel hepatic CYP3 A enzyme in vitro and its influences on exogenous drug metabolism. 【Method】 The Bactrian camels were sacrificed by bloodletting in jugular veins after fasting for overnight about 12 hours, and liver tissues were collected immediately from the area of hepatic portal vein, washed away the blood by cold normal saline, and preserved in liquid nitrogen and sent to the lab. The Bactrian camel hepatic microsome was prepared by Ca^2＋ precipitation method following weighing and homogenate, and then BCA method was used in this experiment for detecting the protein contents of hepatic microsomes. As well as Bactrian camel hepatic microsomal incubation system was optimized, different concentrations of Bactrian camel hepatic microsomal protein suspensions（0.016, 0.031, 0.063, 0.125, 0.250, 0.500, 1.00, 2.000 mg·m L^-1）, different concentrations of midazolam（MDZ） as probe drug（7.813, 15.625, 31.250, 62.500, 125, 250, 500, 1 000 μg·m L^-1） were, respectively, added to the incubation system, after 5 min for preincubation at 37℃, added the solution of reduced form of nicotinamide-adenine dinucleotide phosphate（NADPH） and incubated again for different times（0, 2, 5, 10, 15, 20, 25, 30 min）. The cold methanol and diazepam（DZP） solution were added to the system for terminating the reaction, and vortex mixing, 800 μL mixed liquids were centrifugated at 14 500 r/min for 20 min, 20 μL supernatant was detected by the high performance liquid chromatography with UV method（HPLC-UV） to determine the optimum concentration of substrate, the optimum concentration of enzyme, and the optimal incubation time. Different concentrations of 20 μL MDZ solution（7.813, 15.625, 31.250, 62.500, 125, 250, 500, 1 000 μg·m L^-1） as probe drug were incubated with Bactrian camel hepatic microsomes for 15 min, respectively, and then the reaction was terminated by ice-cold methanol mixed with the internal standard of

关 键 词：双峰驼 CYP3A酶 高效液相色谱法 探针药物 

分 类 号：S824[农业科学—畜牧学]