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

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.