大鼠心肌组织中高能磷酸化物的含量测定  

Determination of High Energy Phosphates in Rat Myocardium

作　　者：冯琳[1] 孟丹[1] 陈相健[1] 卞智萍[1] 杨笛[1] 张寄南[1] 

机构地区：[1]南京医科大学第一附属医院,南京210029

出　　处：《江苏药学与临床研究》2004年第5期11-13,共3页Jiangsu Pharmacertical and Clinical Research

基　　金：国家自然科学基金资助项目 (NO :3 0 2 0 0 3 74)

摘　　要：目的　建立了同步测定大鼠心肌组织中 5种高能磷酸化物含量的反相离子对高效液相色谱法 ,并观察异丙肾上腺素致害大鼠心肌损伤模型中能量代谢的变化。方法　色谱柱 :SupelcoODS C18(2 5 0mm× 4 6mm ,5 μm) ;柱温 :2 3℃～ 2 5℃ ;UV检测波长 2 10nm ;流动相 :2 2 0mmol·L 1磷酸盐缓冲液 (含 3mmol·L 1四丁基氢氧化铵和 7%甲醇 ) ,pH6 5 ;流速 1 2ml·min 1和1 3ml·min 1。结果　 5种能量代谢物质ATP、ADP、AMP、CrP和Cr达到良好分离 ,ATP、ADP、CrP在 10～ 10 0 μmol·L 1,AMP在 10 0～ 5 0 0 μmol·L 1,Cr在 10 0～ 10 0 0 μmol·L 1浓度范围 ,峰面积与浓度呈良好线性关系。日内和日间精密度的RSD均 <10 % ,ATP、ADP、AMP、CrP及Cr的提取回收率 (% )平均为 :97 86、10 4 38、10 0 0 0、97 6 4和 98 8。与生理盐水组相比 ,ISO致害大鼠心室组织中ATP含量及能荷 (EC)显著下降 (P <0 0 5 )、ADP呈下降而AMP呈上升趋势 ,CrP含量显著上升 (P <0 0 1)。结论　本法操作简便、结果准确 。Objective To develop a RP-Ion pair HPLC method for separate and determine simultaneously five high energy phosphates(HEPs) in rat myocardium,and to analyse the changes on energy metabolism of rat myocardium injuried by isoproterenol(ISO).Method The column used was Supelco ODS-C_(18)(250 mm×4.6 mm,5μm),the column temperature was controlled at 23 ℃～25 ℃,UV detection wavelength was set at 210 nm,mobile phase consisted of 220 mmol·L^(-1) phosphate buffer with 3 mmol·L^(-1) tetrabutylammonium hydroxide(TBAOH) and 7% methanol in distilled deionized water,pH was adjusted to 6.5 with 5 mol·L^(-1)potassium hydroxide(KOH),at 1.3 ml·min^(-1) and 1.2 ml·min^(-1) flow rate.Results Five HEPs ATP,ADP,AMP,CrP,Cr were successfully separated. There was good liner relationship within the range of 10-100 μmol·L^(-1) in ATP,ADP,CrP,100-500 μmol·L^(-1) in AMP and 100-1000 μmol·L^(-1) in Cr respectively. Both coefficient of variation for within-day and between-day were lower than 10%.The average extraction recovery(%) of ATP,ADP,AMP,CrP and Cr was 97.86,104.38,100.00,97.64 and 98.8 respectively. Compared with saline group,ATP and energy capacity(EC) levels in rat myocardium injured by ISO weredecreased(P<0.05),CrP level was increased significantly(P<0.01),and there was a fall tendency of ADP while a rise of AMP level.Conclusion The method is simple,accurate and suitable for HEPs determination in rat mycardial extracts,so as to analyse the state of energy metabolism.

关 键 词：高效液相色谱法 高能磷酸化物 大鼠心肌 

分 类 号：R977[医药卫生—药品]