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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。
The intracellular contents of adenosine triphosphate (ATP) and its metabolites and the effects of 2-tert-butyl-1,4-benzoquinone (TBBQ) on the intracellular contents of ATP and its metabolites were studied. A high performance liquid chromatography (HPLC) was developed for the rapid separation and detection of intracellular ATP and its metabolites (adenosine diphosphate (ADP) and adenosine monophosphate (AMP)). Shimadzu HPLC system and Aeger Venusil MP C18 column were used, and the isocratic elution method was used. The mobile phase A was 50 mmol / L sodium phosphate dibasic and 15 mmol / L trimethylamine (TEA). The pH was adjusted to 7.88 with acetic acid (HAc); the mobile phase B was methanol. The working curves of the three metabolites were obtained by HPLC. The correlation coefficient was high (R ~ 2≥0.9996). The contents of three metabolites in MRC-5 cells were in the linear range of 0.1 ~ 100μmol / L). The method detection limit is low. Pre-cooled 80% (volume fraction) aqueous methanol was used to extract intracellular metabolites. The method established in this study was successfully applied to detect the levels of ATP, ADP and AMP in MRC-5 cells. The results showed that TBBQ could affect the contents of ATP, ADP and AMP in cells, but the concentrations of TBBQ and ATP, ADP And the relationship between AMP concentration in MRC-5 cells is more complicated.