Determination of cellular vitamin C dynamics by HPLC-DAD
Determination of cellular vitamin C dynamics by HPLC-DAD
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DOI：
10.1039/c8an02240b
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发表时间：
2019-06-07
期刊：
影响因子：
4.2
通讯作者：
Miyahara, Yuji
中科院分区：
文献类型：
--
作者：
Miyazawa, Taiki;Matsumoto, Akira;Miyahara, Yuji
抗坏血酸（ASC）和其氧化形式脱氢抗坏血酸（DHA）在细胞内环境中的氧化还原敏感性相互转化已成为最近代谢组学和药物研究的特殊兴趣。我们开发了一种色谱方案，可以立即从细胞提取物中确定这些维生素的每个身份，而无需任何标记和预处理。由于其简单性，人们可以容易地继续测定数小时，否则难以覆盖细胞内DHA-ASC转化开始发挥作用的时间尺度。该方法已被验证用于胰腺癌细胞的分析，据我们所知，有核细胞类型的首次研究，详细追踪其葡萄糖转运蛋白依赖性DHA摄取的动力学，同时，细胞内ASC转化。所有相关技术中最简单的，但具有在高通量基础上提供每个维生素身份的独特能力，这种方法应该为VC涉及的生理和药学研究提供最实用的选择，包括高剂量VC癌症治疗。
A redox-sensitive inter-conversion between ascorbic acid (ASC) and its oxidized form dehydroascorbic acid (DHA) in the intracellular environment has been of exceptional interest to recent metabolomics and pharmaceutical research. We developed a chromatographic protocol to instantly determine these vitamers with each identity from cellular extracts, without any labeling and pretreatments. Owing to its simplicity, one can readily continue the assay for hours, an otherwise difficult to cover timescale at which the intracellular DHA-ASC conversion comes into play. The method was validated for the analysis of pancreatic cancer cells, to our knowledge the first-ever study on a nucleated cell type, to trace in detail their kinetics of glucose transporter-dependent DHA uptake and, simultaneously, that for the intracellular ASC conversion. The simplest of all the relevant techniques and yet with the unique ability to provide each vitamer identity on a high-throughput basis, this method should offer the most practical option for VC-involved physiological and pharmaceutical studies including high-dose VC cancer therapy.