Nishi Rie, Yamauchi Takahiro, Ueda Takanori
Division of Hematology and Oncology, Faculty of Medical Sciences, University of Fukui, 23-3, Shimoaizuki, Matsuoka, Fukui 910-1193, Japan.
Cancer Sci. 2006 Nov;97(11):1274-8. doi: 10.1111/j.1349-7006.2006.00323.x.
A deoxycytidine analog, gemcitabine (dFdC), is effective for treating solid tumors and hematological malignancies. After being transported into cancer cells, dFdC is phosphorylated to dFdC triphosphate (dFdCTP), which is subsequently incorporated into the DNA strand, thereby inhibiting DNA synthesis. Intracellular dFdCTP is the critical determinant for dFdC cytotoxicity, so therapeutic drug monitoring or in vitro testing of the capability of cancer cells to accumulate dFdCTP may be informative for optimizing dFdC administration. We have developed a new isocratic-elution high-performance liquid chromatography method for quantifying dFdCTP in cancer cells. Samples (500 microL) were eluted isocratically using 0.06 M Na(2)HPO(4) (pH 6.9) containing 20% acetonitrile, at a constant flow rate of 0.7 mL/min and at ambient temperature. Separation was carried out using an anion-exchange column (TSK gel DEAE-2SW; 250 mm x 4.6 mm inside diameter, particle size 5 microL) and monitored at 254 nm. The standard curve was linear with low within-day and interday variability. The lower detection limit (20 pmol) was as sensitive as that of the previous gradient-elution method. dFdCTP was well separated from other nucleoside triphosphates. The method could measure dFdCTP in cultured or primary leukemic cells treated in vitro with dFdC. The method was also applicable to simultaneous determination of dFdCTP and cytarabine triphosphate, the results of which demonstrated ara-CTP production augmented by dFdC pretreatment. Thus, our isocratic high-performance liquid chromatography assay method will be of great use because of its sensitivity and simplicity as well as its applicability to biological materials.
一种脱氧胞苷类似物吉西他滨（dFdC）对实体瘤和血液系统恶性肿瘤有效。进入癌细胞后，dFdC被磷酸化为三磷酸脱氧胞苷（dFdCTP），随后被掺入DNA链，从而抑制DNA合成。细胞内的dFdCTP是dFdC细胞毒性的关键决定因素，因此治疗药物监测或体外检测癌细胞积累dFdCTP的能力可能有助于优化dFdC给药。我们开发了一种新的等度洗脱高效液相色谱法来定量癌细胞中的dFdCTP。样品（500微升）使用含有20%乙腈的0.06 M磷酸氢二钠（pH 6.9）进行等度洗脱，流速恒定为0.7毫升/分钟，温度为室温。使用阴离子交换柱（TSK凝胶DEAE - 2SW；内径250毫米×4.6毫米，粒径5微米）进行分离，并在254纳米处进行监测。标准曲线呈线性，日内和日间变异性低。最低检测限（20皮摩尔）与之前的梯度洗脱法一样灵敏。dFdCTP与其他三磷酸核苷能很好地分离。该方法可以测量体外经dFdC处理的培养白血病细胞或原代白血病细胞中的dFdCTP。该方法也适用于同时测定dFdCTP和阿糖胞苷三磷酸，结果表明dFdC预处理可增加阿糖胞苷三磷酸的产生。因此，我们的等度高效液相色谱测定方法因其灵敏度、简便性以及对生物材料的适用性而具有很大的用途。
