Direct analysis of 5-methylcytosine and 5-methyl-2′-deoxycytidine in human urine by isotope dilution LC-MS/MS

Recent evidence suggests that active DNA demethylation involves base excision repair (BER) and nucleotide excision repair (NER) pathways. We hypothesized that the resulting excision products could be further excreted and present in urine. A highly specific and sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was first developed for simultaneously measuring urinary 5-methylcytosine (5-meC) and 5-methyl-2′-deoxycytidine (5-medC). With the use of isotope internal standards and online solid-phase extraction (SPE), the detection limits of 5-meC and 5-medC were estimated to be 1.2 and 0.3 pg, respectively. This (Figure presented) method was applied to measure urinary samples of 376 healthy males. Urinary samples were also measured for methylated and oxidized DNA lesions, namely, N7-methylguanine (N7-meG), N3-methyladenine (N3-meA), 8-oxo-7,8-dihydroguanine (8-oxoGua), and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), using reported online SPE LC-MS/MS methods. Results showed that mean urinary levels of 5-meC and 5-medC were 28.4 ± 14.3 and 7.04 ± 7.2 ng/mg creatinine, respectively, supporting the possible presence of DNA demethylation through BER and NER mechanisms. Urinary levels of 5-meC were significantly positively correlated with N7-meG, N3-meA, and 8-oxodG. Good correlations between 5-meC and methylated and oxidized DNA lesions may have implied the underlying linkage between genetic (DNA lesions) and epigenetic (DNA methylation) alterations derived from exogenous exposure and/or from endogenous cellular processes in human and require further investigation.