New ultrasensitive 32P-postlabelling method for the analysis of 3,N4-etheno-2′-deoxycytidine in human urine
New ultrasensitive 32P-postlabelling method for the analysis of 3,N4-etheno-2′-deoxycytidine in human urine
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DOI：
10.1080/13547500600709606
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发表时间：
2006-07-01
期刊：
影响因子：
2.6
通讯作者：
Nair, J.
中科院分区：
文献类型：
--
作者：
Sun, X.;Karlsson, A.;Nair, J.
乙烯-DNA加合物由外源性致癌物质如氯乙烯和氨基甲酸乙酯产生，也可由内源性脂质过氧化产物如反式-4-羟基-2-壬烯醛(HNE)产生。1，N-6-亚乙基脱氧腺苷(Oda)和3，N-4-亚乙基脱氧胞苷(Epsilon DC)存在于人体尿液中，可作为监测全身氧化应激的生物标志物。本文报道了一种新的超灵敏的P-32-后标记/薄层层析(TLC)方法，用于人尿中脱氧核苷epsilon DC的分析。尿样在固相硅胶C-18柱上净化和浓缩，然后进行半制备反相高效液相色谱。以5‘-溴-2’-脱氧尿苷(BrdU)为内标，用来自黑腹果蝇的多底物脱氧核糖核苷激酶(DM-DNK)标记纯化的样品。该方法的绝对灵敏度为0.1fmoL epsilon DC，可在500亩L尿液中检测到。对15名健康志愿者的尿样进行分析，结果显示尿Ep DC平均水平为2.499+/-1.76(SD)fmolMol(-1)肌酐(范围0.66-6.42)。通过这种非侵入性的方法，人类尿液中的epsilon DC可以作为人类氧化应激相关疾病的生物标志物。
Etheno-DNA adducts are generated from exogenous carcinogens such as vinyl chloride and urethane and also from endogenous lipid peroxidation products such as trans-4-hydroxy-2-nonenal (HNE). The present authors and others have established that 1,N-6-ethenodeoxyadenosine (odA) and 3,N-4-ethenodeoxycytidine (epsilon dC) are present in human urine and could be explored as biomarkers for monitoring whole-body oxidative stress. The present study reports on a new ultrasensitive P-32-postlabelling/thin-layer chromatography (TLC) method for the analysis of epsilon dC as deoxynucleoside in human urine. The urine samples were purified and enriched on a solid-phase silica C-18 column followed by a semi-preparative reverse-phase high-performance liquid chromatography. The purified sample was labelled with a multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster (Dm-dNK) in the presence of 5'-bromo-2'-deoxyuridine (BrdU) as internal standard. The absolute sensitivity of the method was 0.1 fmol epsilon dC detectable in 500 mu l of human urine. The analysis of human urine samples from 15 healthy volunteers revealed a mean epsilon dC level of 2.499 +/- 1.76 (SD) fmol mu mol(-1) creatinine (range 0.66-6.42). By this non-invasive method, epsilon dC in human urine could be explored as a biomarker for oxidative stress-related human diseases.