高效液相色谱法测定细胞内三磷酸腺苷及其代谢物的含量（英文）

Quantification of intracellular adenosine 5′-triphosphate and its metabolites by high performance liquid chromatography analysis

研究了三磷酸腺苷(ATP)及其代谢物在细胞内的含量以及2-叔丁基-1,4-苯醌(TBBQ)对ATP及其代谢产物在细胞内含量的影响。建立了一种高效液相色谱法(HPLC)用于快速分离、检测细胞内ATP及其代谢产物(二磷酸腺苷(ADP)和一磷酸腺苷(AMP))的含量。使用岛津高效液相系统及艾杰尔Venusil MP C18柱,采用等度洗脱的方式。流动相A相为50 mmol/L磷酸氢二钠和15 mmol/L三甲胺(TEA),用醋酸(HAc)调节pH至7.88;流动相B相为甲醇。采用建立的高效液相色谱法得到了3种代谢物的工作曲线,相关系数高(R^2≥0.999 6),MRC-5细胞中3种代谢物的含量均在线性范围(0.1~100μmol/L)内。该方法检出限低。采用预冷的80%(体积分数)甲醇水溶液提取细胞内的代谢物。该研究建立的方法成功地应用于检测MRC-5细胞中的ATP、ADP和AMP的含量,结果表明,TBBQ会对ATP、ADP、AMP在细胞内的含量产生影响,但TBBQ浓度和ATP、ADP以及AMP在MRC-5细胞内浓度的关系比较复杂。

This study was aimed to provide insight regarding the intracellular metabolites of adenosine 5′-triphosphate（ATP）and whether 2-tert-butyl-1,4-benzoquinone（TBBQ）affects cell metabolites. A rapid high performance liquid chromatography（HPLC）protocol was developed for the separation and quantitation of ATP and its metabolites（adenosine diphosphate（ADP）and adenosine monophosphate（AMP））in cells. Chromatographic separation was performed using a Shimadzu HPLC system equipped with an Agela Venusil MP C18column;isocratic elution was adopted. The mobile phase comprised solvent A（50 mmol / L disodium hydrogen phosphate and 15 mmol / L trimethylamine（TEA）;pH adjusted to 7. 88 using acetic acid（HAc））and solvent B（methanol）. The correlation coefficients of the three analytes were very high（R^2≥0. 999 6）,and the contents of the three metabolites in the MRC-5 cells were within the linear ranges（0. 1- 100 μmol / L）. The limits of detection for the detected three compounds were low. Samples were extracted from cells（after exposure and non-exposure to quinones）using 80%（v / v）methanol aqueous solution. The method developed in this study was successfully applied to detect ATP,ADP and AMP in MRC-5 cells,and the results demonstrated that ATP,ADP,AMP levels in cells were affected by TBBQ,but the relations between the concentration of TBBQ and the level of ATP,ADP and AMP were complex.