Fernandez-Salguero P, Gonzalez F J, Etienne M C, Milano G, Kimura S
Laboratory of Molecular Carcinogenesis, National Cancer Institute, Bethesda, MD 20892, USA.
Biochem Pharmacol. 1995 Sep 28;50(7):1015-20. doi: 10.1016/0006-2952(95)00231-n.
A TLC procedure was developed to determine dihydropyrimidine dehydrogenase (DPD) activity in human peripheral lymphocytes. The assay, which used radiolabeled uracil as a substrate, was validated using recombinant pig DPD in which it was demonstrated to yield kinetic constants similar to those found by methods that rely on either spectroscopic determination of NADPH oxidation or HPLC. DPD activity was measured in a group of human lymphocyte extracts, including an extract from a subject that actually presented toxicity to 5-fluorouracil treatment. Measurements of DPD protein content using western immunoblots revealed a significant correlation with activity levels in human lymphocytes. Thus, this correlation could be used to determine not only the levels of expression of this enzyme, which is the cause of an inherited genetic deficiency in pyrimidine catabolism, but also to estimate the degree of sensitivity to pyrimidine-based cancer drugs such as 5-fluorouracil.
开发了一种薄层层析（TLC）程序来测定人外周血淋巴细胞中的二氢嘧啶脱氢酶（DPD）活性。该测定法以放射性标记的尿嘧啶为底物，使用重组猪DPD进行了验证，结果表明其产生的动力学常数与依靠NADPH氧化的光谱测定法或高效液相色谱法（HPLC）所得到的动力学常数相似。在一组人淋巴细胞提取物中测量了DPD活性，其中包括一名实际上对5-氟尿嘧啶治疗呈现毒性的受试者的提取物。使用蛋白质免疫印迹法测量DPD蛋白含量，结果显示与人淋巴细胞中的活性水平存在显著相关性。因此，这种相关性不仅可用于确定这种酶的表达水平（这种酶是嘧啶分解代谢中遗传性基因缺陷的原因），还可用于估计对基于嘧啶的抗癌药物（如5-氟尿嘧啶）的敏感程度。
