Maybaum J, Klein F K, Sadee W
J Chromatogr. 1980 Jan 25;188(1):149-58. doi: 10.1016/s0021-9673(00)88425-3.
High-performance liquid chromatographic (HPLC) assay for determining tissue pools of uridine, deoxyuridine, cytidine, deoxycytidine, and thymidine mono-, di-, and triphospates is presented. The method utilizes anion-exchange and, after conversion of nucleotides to nucleosides by acid phosphatase, reversed-phase chromatography on a preparative column with UV detection at 254 and 280 nm. The yield of this procedure is 80 +/- 2% with a sensitivity limit of 100 pmole nucleotide per sample. A sensitivity of 10 pmole can be achieved for each compound by rechromatographing appropriate nucleoside fractions on analytical columns. The recovery, including this step, is 66 +/- 7%. The assay is reproducible and highly selective, with a lower sensitivity limit of approximately 0.1 muM using 150--250 mg (wet weight) tissue samples. Nucleotide pools have been determined in Balb/c mouse liver and in mouse lymphoma (S-49) cell culture, the latter with and without addition of 5-fluorouracil (5-FUra) to the medium. Data obtained with this assay are similar to those using alternative methodologies. Observed depletion of dTXP pools and expansion of dUMP and dCXP pools after 5-FUra treatment are in agreement with published observations. Pools of dUDP and dUTP were not detectable (less than 10 pmole/10(8) cells) in any tissue sample. These data illustrate the utility of the present method in studying actions of pyrimidine antimetabolites.
本文介绍了一种用于测定尿苷、脱氧尿苷、胞苷、脱氧胞苷以及胸苷单磷酸、二磷酸和三磷酸组织池的高效液相色谱（HPLC）分析方法。该方法利用阴离子交换，在酸性磷酸酶将核苷酸转化为核苷后，通过制备柱上的反相色谱法，并在254和280nm处进行紫外检测。该方法的产率为80±2%，每个样品的灵敏度极限为100皮摩尔核苷酸。通过在分析柱上对适当的核苷馏分进行再色谱分析，每种化合物可实现10皮摩尔的灵敏度。包括此步骤在内的回收率为66±7%。该分析方法具有可重复性和高选择性，使用150 - 250mg（湿重）组织样品时，灵敏度下限约为0.1μM。已测定了Balb/c小鼠肝脏和小鼠淋巴瘤（S - 49）细胞培养物中的核苷酸池，后者在培养基中添加或不添加5 - 氟尿嘧啶（5 - FUra）。用该分析方法获得的数据与使用其他方法的数据相似。5 - FUra处理后观察到的dTXP池的消耗以及dUMP和dCXP池的扩大与已发表的观察结果一致。在任何组织样品中均未检测到dUDP和dUTP池（小于10皮摩尔/10⁸细胞）。这些数据说明了本方法在研究嘧啶抗代谢物作用方面的实用性。
