HPLC测定心肌梗死大鼠心肌磷酸肌酸和ATP的含量

目的 利用HPLC测定梗死心肌组织中磷酸肌酸（Phosphocreatine,PCr）与ATP浓度。方法 采用C18色谱柱（250 mm×4.6mm,5μm）,以0.1 mol/L磷酸二氢钾缓冲液（含5 mmol/L四丁基氢氧化铵）-乙腈为流动相,梯度分离,检测波长为205 nm。心肌梗死模型采用冠状动脉左前降支结扎的方法,术后8周成模。结果 PCr、ATP标准品与峰面积呈良好的线性关系（R2〉0.999）。重复测定不同浓度PCr、ATP标准品平均日内相对标准偏差（RSD%）均小于1.56%（n=5）,日间RSD%均小于4.22%（n=5）。两者平均加样回收率在95.26%~103.44%之间。心肌梗死后大鼠PCr含量下降（P〈0.001）,ATP浓度不变,PCr/ATP比值下降（P〈0.001）。结论 反相离子对HPLC法可准确、简便、快速测定大鼠心肌组织内PCr与ATP的含量。心肌梗死后心肌能量贮存指数PCr/ATP比值明显下降。

Concentration Determination of Phosphocreatine and Adenosine Triphosphate in Infracted Rats Myocardium by HPLC

Objective To establish a simple HPLC method for measuring the contents of phosphocreatine （PCr） and adenosine triphosphate （ATP） in myocardium tissue. Methods PCr and ATP were separated with gradient by Cls column （250 minX4.6 ram, 5 Ixm） and detected at 205 nm. The mobile phase consisted of 0.1 mol/L monopotassium phosphate buffer （including 5 mmol/L tetrabutyl ammonium bromide） and Methyl Cyanides. The rats were subjected by left coronary artery ligation for 8 weeks to build myocardial infarction model. Results The calibration curve showed good linearity （R2〉0.999） for PCr and ATP. The precision （relative standard deviation, RSD） in 1 d and in 5 d were less than 1.56% （ n = 5） , 4.22% （ n = 5 ） , respectively. The average recoveries of PCr and ATP were 95.26%- 103.44% （ n = 5 ）. Compared with control rats, both infracted rats exhibited a marked decrease in PCr concentration and PCr/ATP ratios （both P〈0.O01 ） , though the ATP contents were not changed. Conclusion The ion-pair reversed-phase HPLC method is accurate and simple. It can rapidly determine the concentration of PCr and ATP in the rat heart at the same time. After myocardial infarction, the myocardial energy reserve index was markedly decreased.