Analysis of intracellular nucleoside triphosphate levels in normal and tumor cell lines by high-performance liquid chromatography
Analysis of intracellular nucleoside triphosphate levels in normal and tumor cell lines by high-performance liquid chromatography
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高效液相色谱法分析正常细胞系和肿瘤细胞系的细胞内三磷酸核苷水平
DOI：
10.1016/s1570-0232(02)00780-8
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发表时间：
2003-01-25
影响因子：
3
通讯作者：
Chen, XG
中科院分区：
文献类型：
--
作者：
Huang, D;Zhang, YH;Chen, XG
提出了一种直接同时测定三氯乙酸细胞提取物中核糖核苷三磷酸及其相应脱氧核糖核苷三磷酸的反相离子对高效液相色谱方法。使用该系统，获得了 16 种正常或肿瘤细胞系中 9 种酸溶性化合物的高分辨率，包括 ADP、CTP、dCTP、GTP、UTP、dGTP、dTTP、ATP 和 dATP。该方法基于用 6% 三氯乙酸溶液从细胞中提取核苷酸，然后在 HPLC 分析之前添加 5 M K2CO3 进行中和。使用配有 NovaPak(18) Sentry 保护柱并在 254 nm 处进行 UV 检测的 Symmetry C-18 3.5 mum (150 x 4.6 mm) 柱 (Waters) 进行色谱分离。 HPLC柱保持在27℃。流动相以1.0ml/min的流速输送，采用以下逐步梯度洗脱程序：A-B(60:40)在0分钟-->4(40:60)在30分钟-->4(40:60)在60分钟。溶剂A含有10mM氢氧化四丁基铵、10mM KH2PO4和0.25%MeOH，并用1M HCl调节至pH 6.9。溶剂 B 由 5.6 mM 氢氧化四丁基铵、50 mM KH2PO4 和 30% MeOH 组成，并用 1 M NaOH 中和至 pH 7.0。细胞提取物中各成分的校准曲线（r > 0.99）是用其水性标准品建立的。九种化合物的平均日内精密度为 0.9%，平均日间精密度为 5.0%。 9种试剂的检测限（pmol）分别为1.39（ADP）、4.32（CTP）、15.5（dCTP）、2.38（GTP）、4.42（UTP）、9.45（dGTP）、14.6（dTTP）、2.44（ATP）和11.8（dATP）。该方法对标准品的回收率为 82.4% 至 120.5%。展示了 16 种正常细胞系和肿瘤细胞系中核苷酸库的检测结果。总之，这种简化的分析方法能够同时定量细胞或组织提取物中的 NTP 和 dNTP，并且可能成为检测抗癌/抗病毒药物和疾病引起的细胞内 NTP/dNTP 池微小变化的有价值的工具。 (C) 2002 Elsevier Science B.V. 保留所有权利。
A reversed-phase ion-pair high-performance liquid chromatographic method for the direct and simultaneous determination of ribonucleoside triphosphates and their corresponding deoxyribonucleoside triphosphates in trichloroacetic acid cell extracts is presented. Using this system, high resolution of nine acid-soluble compounds, including ADP, CTP, dCTP, GTP, UTP, dGTP, dTTP, ATP and dATP in 16 normal or tumor cell lines, is obtained. The method is based on an extraction of nucleotides from cells with a solution of 6% trichloroacetic acid followed by neutralization with the addition of 5 M K2CO3 just prior to HPLC analysis. Chromatographic separations were performed using a Symmetry C-18 3.5 mum (150 x 4.6 mm) column (Waters) equipped with a NovaPak(18) Sentry guard column with UV detection at 254 nm. The HPLC columns were kept at 27 C. The mobile phase was delivered at a flow-rate of 1.0 ml/min, with the following stepwise gradient elution program: A-B (60:40) at 0 min-->4(40:60) at 30 min-->4(40:60) at 60 min. Solvent A contained 10 mM tetrabutylammonium hydroxide, 10 mM KH2PO4 and 0.25% MeOH, and was adjusted to pH 6.9 with 1 M HCl. Solvent B consisted of 5.6 mM tetrabutylammonium hydroxide, 50 mM KH2PO4 and 30% MeOH, and was neutralized to pH 7.0 with 1 M NaOH. The calibration curves (r > 0.99) of the components in cell extracts were established with their aqueous standards. The average within-day precision for the nine compounds was 0.9%, and the average day-to-day precision was 5.0%. The detection limits (pmol) of the nine reagents were 1.39 (ADP), 4.32 (CTP), 15.5 (dCTP), 2.38 (GTP), 4.42 (UTP), 9.45 (dGTP), 14.6 (dTTP), 2.44 (ATP) and 11.8 (dATP). The recovery of this method for the standards ranged from 82.4 to 120.5%. The results for the detection of nucleotide pools in 16 normal and tumor cell lines were presented. In conclusion, this simplified analytical method enables the simultaneous quantitation of NTP and dNTP in cell or tissue extracts and may represent a valuable tool for the detection of minute alterations of intracellular NTP/dNTP pools induced by anticancer/antiviral drugs and diseases. (C) 2002 Elsevier Science B.V. All rights reserved.