Sun X, Karlsson A, Bartsch H, Nair J
Division of Toxicology and Cancer Risk Factors, German Cancer Research Center (DKFZ), Heidelberg, Germany.
Biomarkers. 2006 Jul-Aug;11(4):329-40. doi: 10.1080/13547500600709606.
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,N6-ethenodeoxyadenosine (epsilondA) and 3,N4-ethenodeoxycytidine (epsilondC) 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 32P-postlabelling/thin-layer chromatography (TLC) method for the analysis of epsilondC 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 epsilondC detectable in 500 microl of human urine. The analysis of human urine samples from 15 healthy volunteers revealed a mean epsilondC level of 2.49+/-1.76 (SD) fmol micromol-1 creatinine (range 0.66-6.42). By this non-invasive method, epsilondC in human urine could be explored as a biomarker for oxidative stress-related human diseases.
乙烯基-DNA加合物可由外源性致癌物如氯乙烯和聚氨酯产生，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。本作者及其他研究人员已证实，1,N6-乙烯基脱氧腺苷（εdA）和3,N4-乙烯基脱氧胞苷（εdC）存在于人类尿液中，可作为监测全身氧化应激的生物标志物进行研究。本研究报告了一种新的超灵敏32P后标记/薄层色谱（TLC）方法，用于分析人类尿液中作为脱氧核苷的εdC。尿液样本在固相硅胶C-18柱上进行纯化和富集，然后进行半制备反相高效液相色谱分析。纯化后的样本在5'-溴-2'-脱氧尿苷（BrdU）作为内标的情况下，用来自黑腹果蝇的多底物脱氧核糖核苷激酶（Dm-dNK）进行标记。该方法的绝对灵敏度为在500微升人类尿液中可检测到0.1飞摩尔的εdC。对15名健康志愿者的人类尿液样本分析显示，εdC的平均水平为2.49±1.76（标准差）飞摩尔/微摩尔肌酐（范围为0.66 - 6.42）。通过这种非侵入性方法，人类尿液中的εdC可作为与氧化应激相关的人类疾病的生物标志物进行研究。
