Analysis of Intra- and Extracellular Levels of Purine Bases, Nucleosides, and Nucleotides in HepG2 Cells by High-performance Liquid Chromatography
Analysis of Intra- and Extracellular Levels of Purine Bases, Nucleosides, and Nucleotides in HepG2 Cells by High-performance Liquid Chromatography
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DOI：
10.2116/analsci.31.895
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发表时间：
2015-09-01
影响因子：
1.6
通讯作者：
Kaneko, Kiyoko
中科院分区：
文献类型：
--
作者：
Fukuuchi, Tomoko;Yamaoka, Noriko;Kaneko, Kiyoko
为了评估细胞摄取和嘌呤运输，我们开发了一种高效液相色谱法来定量细胞内和细胞外嘌呤。我们的目的是建立一种有效的方法，同时定量底物和代谢产物的高灵敏度。不同厂家的C18色谱柱用于同时定量22种不同的嘌呤碱基、核苷和核苷酸。我们使用了YMC-Triart C18色谱柱。优化分析条件，包括细胞提取液和细胞培养基，以达到良好的定量。对该方法的线性度、准确度、检出限和回收率进行了评价，结果良好。该方法成功地应用于22种不同的细胞内和细胞外嘌呤的定性分析，表明该方法可用于研究嘌呤代谢的整体模式。该方法也可用于评价培养细胞在各种刺激条件下嘌呤的代谢动力学。
To evaluate cellular uptake and purine transport, we developed a high-performance liquid chromatography method for intra- and extracellular purine quantification. Our aim was to develop an effective method for simultaneously quantifying the substrate and metabolites with high sensitivity. C18 columns from different manufacturers were tested for simultaneous quantification of 22 different purine bases, nucleosides, and nucleotides. We used a YMC-Triart C18 column. The analysis conditions, including extraction solutions for the cells and cell culture medium, were optimized to achieve good quantification. Linearity, accuracy, determination limits, and recovery were assessed and showed good performance. The developed HPLC method was successfully applied to the qualitative analysis of 22 different intra- and extracellular purines, demonstrating that it is useful for studying the overall pattern of purine metabolism. This method could also be useful for evaluating metabolic dynamics of purines under a variety of stimulatory conditions of culture cells.