Detection of Human Urinary 5-Hydroxymethylcytosine by Stable Isotope Dilution HPLC-MS/MS Analysis

Abstract

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.

Low level of 5-Hydroxymethylcytosine predicts poor prognosis in non-small cell lung cancer

“…Discussion 5-hmC, as the hydroxylated form of 5-mC, was first discovered in T-even phage DNA in 1952 (15). A previous study indicated that 5-hmC was associated with diseases such as nerve disorders, leukemia and tumors (16). In recent years, 5-hmC has become more extensively studied.…”

Technology and methodology of DNA adducts analysis

“…的准确定量. 此方法还可以提高DNA 5-甲基胞嘧啶 (5-methylcytosine, 5mC)的氧化产物分析的灵敏度, 包 括5-羟甲基胞嘧啶(5hmC)和5-醛基胞嘧啶(5fC) [73,74] .…”

“…ESI-MS方法适用于5mC氧化产物5hmC和5fC的高灵 敏分析, 在此基础上, 结合SPE技术实现了健康人类尿 液中5mC初级氧化产物 5hmC 的检测 [74] . 与哺乳动物 methyladenine, 6mA), 其表观遗传功能也得到了系统 深入的研究 [90] .…”

Detection of 1,N2-propano-2′-deoxyguanosine in human urine by stable isotope dilution UHPLC–MS/MS analysis

“…Among them, salts, which can suppress ionization efficiency and contaminate ion source are present with high abundance [30]. In order to clean up salts and selectively enrich urinary ProdG adducts, C18 and HLB cartridges were used to obtain the optimized extraction.…”

“…Various methods have been developed for the detection of urinary nucleosides, including electrochemical method [18], immunoassays [19,20], capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) [17,[21][22][23][24], matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) [25,26], liquid chromatography tandem mass spectrometry (LC-MS) [27][28][29][30][31][32]. In comparison to other methods, the LC-MS based methodologies take the advantages of high sensitivity and direct analysis with structural information from MS measurements [33].…”