HPLC法测定大鼠脑组织磷酸二酯酶活性  

HPLC method for determining phosphodiesterase activity in rats' brain

作　　者：李才明[1] 张鸿炼[2] 王海燕[3] 张成[1] 柳太云[1] 冯善伟[1] 

机构地区：[1]中山大学第一附属医院神经科,广州510080 [2]广东省惠州市中心人民医院药理室,惠州516001 [3]广东省惠州市人民医院妇产科,惠州516002

出　　处：《中国临床药学杂志》2005年第6期348-350,共3页Chinese Journal of Clinical Pharmacy

基　　金：国家自然科学基金资助项目(30370510;30170337);广东省自然科学基金资助项目(31693);卫生部临床学科重点项目基金资助项目(2001321);CMB基金资助项目(98-667);教育部骨干教师基金资助项目(082004);广东省卫生厅医学基金资助项目(B2002124)

摘　　要：目的建立大鼠脑组织磷酸二酯酶(PDE)活性测定的HPLC法。方法大鼠脑组织匀浆后,可溶部分为酶样品,以cAMP和cGMP为反应底物,采用双管法测定。色谱柱为Nova-PakC18(3.9mm×150mm,4μm);流动相为甲醇-磷酸盐缓冲液(10∶90,V/V);流速为0.8mL·min^(-1);检测波长254nm。结果PDE底物(cAMP、cGMP)和系列产物(5’AMP、5’GMP、腺苷、次黄嘌呤和腺嘌呤)分离良好,无干扰。cAMP和cGMP检测限分别为0.125μmoL·L^(-1)和0.0625μmoL·L^(-1);平均回收率91.2%~102.8%;日内及日间RSD均<10%。大鼠脑组织PDE活性为(364.6601±66.6862)nmol·g^(-1)·min^(-1)(n=6)。结论本法能准确测定大鼠脑组织的PDE活性,方法简便、灵敏。AIM To establish a HPLC method for determining phosphodiesterase activity in rats＇ brain. METHODS The rat＇ s brains were homogenated and the parts of dissolution were serviced as enzyme samples, cyclic adenosine monophosphate（cAMP） and cyclic guanylic acid（cGMP） were serviced as substrates, using double-tubes method to determine phosphodiesterase（PDE） activities. Nova-Pak Cl8 （ 150 mm ± 3.9 mm, 4 μm） column was used. The mobile phase consisted of methyl alcohol and phosphate buffer （ 10 ： 90, V/V） ; the flow rate of mobile phase was 0.8 mL·min^-1 ; the UV detection wavelength was 254 nm. RESULTS cAMP, cGMP, 5＇ AMP, 5＇ GMP, adenosine, hypoxanthine and adenine were separated well and not interfered. The lowest detectable limit of cAMP and cGMP was 0. 125 μmol·L^-1 and 0.062 5μmol·L^-1 respectively. The average recovery was 91.2 % - 102.8 %. The RSD of within-day and between-day was less than 10%. Six SD rat brains＇ samples were determined by this method and the phosphodiesterase activity was （ 364. 6601 ± 66. 6862 ） nmol·g^-1·min^-1. CONCLUSION The proposed method is convenient, sensitive and accurate for determining phosphodiesterase activity in rats＇ brain.

关 键 词：磷酸二酯酶 高效液相色谱法 大鼠 

分 类 号：R743.3[医药卫生—神经病学与精神病学]