色谱 ›› 2017, Vol. 35 ›› Issue (1): 54-58.DOI: 10.3724/SP.J.1123.2016.08031

• 研究论文 • 上一篇    下一篇

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)

朱会宇, 吴丹妮, 汪海林   

  1. 中国科学院生态环境研究中心, 环境化学与生态毒理学国家重点实验室, 北京 100085
  • 收稿日期:2016-08-26 出版日期:2017-01-08 发布日期:2013-05-06
  • 通讯作者: 汪海林,Tel:+86-10-62849010,E-mail:hlwang@rcees.ac.cn.

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

ZHU Huiyu, WU Danni, WANG Hailin   

  1. State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, China
  • Received:2016-08-26 Online:2017-01-08 Published:2013-05-06
  • Supported by:

    National Natural Science Foundation of China (No. 21327006).

摘要:

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(R2≥0.9996),MRC-5细胞中3种代谢物的含量均在线性范围(0.1~100 μmol/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。

关键词: 代谢产物, 高效液相色谱, 醌类, 三磷酸腺苷, 细胞内

Abstract:

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 C18 column; 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 (R2≥0.9996), and the contents of the three metabolites in the MRC-5 cells were within the linear ranges (0.1-100 μ mol/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.

Key words: adenosine 5'-triphosphate (ATP), high performance liquid chromatography (HPLC), intracellular, metabolites, quinones

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