大鼠心肌内源性代谢物测定方法的建立及在心肌缺血大鼠模型代谢组学研究中的应用

目的: 建立测定大鼠心肌中内源性代谢物的方法,并将其应用于心肌缺血模型的代谢组学研究。 方法: 利用实验设计(DOE)方案,以甲醇、乙醇、异丙醇、丙酮、三氯甲烷不同配比的混合溶剂为提取溶剂,研究心肌组织中小分子化合物的最佳提取方案,根据心肌组织中所能测定的化合物特点和种类优化了GC/TOF-MS测定方法,数据经过偏最小二乘法(PLS)进行分析。利用该方法测定心肌缺血模型和正常对照大鼠心肌的内源性小分子代谢物。 结果: 大鼠心肌组织最佳的提取方案是将心肌组织研磨成细粉(50 mg)后用甲醇-水(900 μL∶50 μL)进行提取。心肌比血浆含有更多种类的糖类、核苷类等细胞生命活动所必需的小分子化合物。利用该方法可从偏最小二乘-判别式法(PLS-DA)得分图中很好地区分正常对照组和模型组。 结论: 本研究建立了全面测定大鼠心肌中内源性代谢物的方法,该方法可成功用于大鼠心肌缺血模型代谢组学研究。

Objective: To develop an extraction method and comprehensive profiling technique for rat myocardial endogenous metabolites and apply to metabonomics research of myocardial ischemia model. Methods: According to the design of experiments(DOE),solvents mixed at different proportions of methanol,ethanol,isopropanol,acetone and chloroform were used to screen the optimal extraction method.Based on the characteristics of metabolites determined in cardiac muscle,the GC/TOF-MS method was optimized and the data was processed by partial least squares projection(PLS).Then the method was used for the determination of endogenous metabolites in cardiac muscle of myocardial ischemia model and normal controlled rats. Results: The optimal extraction method was developed,in which cardiac muscle was pounded into powder(50 mg)and then extracted by methanol-water(900 μL∶50 μL).A comparison of cardiac muscle to the plasma data from the same rat showed that cardiac muscle contained more saccharides and nucleosides than plasma.Myocardial ischemia model animals could be separated clearly from the normal controlled rats in PLS-DA scores plots. Conclusions: The comprehensive method for the determination of rat cardiac muscle metabolites is developed in this study,which can be successfully used for the metabonomics study in myocardial ischemia rat model.