The sixth DNA base 5-hydroxymethylcytosine (5hmC) is the major oxidation product of the epigenetic modification 5-methylcytosine (5mC), mediating DNA demethylation in mammals. Reduced 5hmC levels are found to be linked with various tumors and neurological diseases; therefore, 5hmC is an emerging biomarker for disease diagnosis, treatment, and prognosis. Due to its advantages of being sterile, easily accessible in large volumes, and noninvasive to patients, urine is a favored diagnostic biofluid for 5hmC analysis. Here we developed an accurate, sensitive, and specific assay for quantification of 5mC, 5hmC, and other DNA demethylation intermediates in human urine. The urinary samples were desalted and enriched using off-line solid-phase extraction, followed by stable isotope dilution HPLC-MS/MS analysis for 5hmC and 5mC. By the use of ammonium bicarbonate (NH4HCO3) as an additive to the mobile phase, we improved the online-coupled MS/MS detection of 5mC, 5hmC, and 5-formylcytosine (5fC) by 1.8-14.3 times. The recovery of the method is approximately 100% for 5hmC, and 70-90% for 5mC. The relative standard deviation (RSD) of the interday precision is about 2.9-10.6%, and that of the intraday precision is about 1.4-7.7%. By the analysis of 13 volunteers using the developed method, we for the first time demonstrate the presence of 5hmC in human urine. Unexpectedly, we observed that the level of 5hmC (22.6 ± 13.7 nmol/L) is comparable to that of its precursor 5mC (52.4 ± 50.2 nmol/L) in human urine. Since the abundance of 5hmC (as a rare DNA base) is 1 or 2 orders of magnitude lower than 5mC in genomic DNA, our finding probably implicates a much higher turnover of 5hmC than 5mC in mammalian genomic DNA and underscores the importance of DNA demethylation in daily life.
Global 5hmC and 5fC contents were decreased significantly in the very early stage of HCC. The decrease of 5hmC and 5fC was mainly due to the decrease of 5mC, and was also associated with HBV infection, decreased TET enzyme activity and uncoordinated expression of DNA methylation-related enzymes. This article is protected by copyright. All rights reserved.
Dear Editor, DNA N 6-methyladenine (6mA), one of the most prevalent epigenetic base modifications in prokaryotes, 1 is recently found in multicellular eukaryotes. 2-8 This nucleobase may have epigenetic roles in regulation of retrotransposons, chromatin organization, and so on. 2-8 However, both our group 9 and Greer's group 10 noticed that eukaryotic DNA is easily contaminated with a minute of bacterial DNA, which carries overwhelmingly abundant 6mA (~2% 6mA/dA). 1 This brings great challenges for accurate detection of DNA 6mA in eukaryotes in terms of both sample pretreatments and analytical technologies. 9,10 For example, inconsistent with the report of Wu et al., 8 Schiffers et al. failed to detect 6mA above background levels in mouse embryonic stem (mES) cells using sensitive ultra-high-performance liquid chromatography-quadruple mass spectrometry (UHPLC-MS/MS) analysis. 11 To date, it is of intensive interest to seek conclusive evidence to support the prevalence of this post-replicative adenine modification in mammals.
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