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New ultrasensitive 32P-postlabelling method for the analysis of 3,N4-etheno-2′-deoxycytidine in human urine

Abstract 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 (εdA) and 3,N4-ethenodeoxycytidine (εdC) 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 εdC 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 εdC detectable in 500 µl of human urine. The analysis of human urine samples from 15 healthy volunteers revealed a mean εdC level of 2.49±1.76 (SD) fmol µmol−1 creatinine (range 0.66–6.42). By this non-invasive method, εdC in human urine could be explored as a biomarker for oxidative stress-related human diseases.