Global DNA methylation measured by liquid chromatography-tandem mass spectrometry: analytical technique, reference values and determinants in healthy subjects. Clin Chem Lab Med
Alterations in global DNA methylation are implicated in various pathobiological processes. We describe a liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for determination of cytosine and 5-methylcytosine in DNA.
DNA was hydrolyzed using formic acid. Cytosine and 5-methylcytosine were separated by gradient-elution reversed-phase chromatography with a mobile phase containing nonafluoropentanoic acid (NFPA) as ion-pairing reagent and quantified using stable isotope dilution LC-ESI-MS/MS. The method was applied to DNA isolated from leukocytes of healthy volunteers.
Linear calibration curves were obtained in the range 0.111-4.422 ng/microL [mean correlation co-efficient 0.9983 (SD=0.0011), n=9] for cytosine and 0.0048-0.1936 ng/microL [mean correlation coefficient 0.9991 (SD=0.0010), n=9] for 5-methylcytosine. The intra- and inter-assay CVs for the 5-methylcytosine/total cytosine ratio (mCyt/tCyt) was 1.7% (n=9) and 3.5% (n=8) for calf thymus DNA (mean mCyt/tCyt ratio 6.5%), and 4.5% (n=6) and 6.5% (n=14), respectively for pBR322 DNA (mean mCyt/tCyt ratio 0.48%). The limit of detection (signal-to-noise ratio 3) was 2 pg on-column for cytosine and 5-methylcytosine. In healthy subjects (n=109), the mCyt/tCyt ratio varied from 2.6% to 4.8% (median 4.1%). DNA methylation was negatively correlated to age, but only in subjects with the methylenetetrahydrofolate reductase (MTHFR) 677 TT genotype (p=0.046). No association with B-vitamin status was observed.
This LC-ESI-MS/MS method is easy to perform and offers reproducibility, selectivity and sensitivity for studying DNA methylation. The method allows a sample throughput of approximately 200 samples/week. The MTHFR C677T genotype influences age-related changes in DNA methylation.
Available from: PubMed Central
- "Hence, one can calculate as a first estimation that damage at C will be 15.6-fold (100/6.4) higher than damage at 5mC based on the known percentage of 5mC to C in CT-DNA (6.4%; (38,39)). In comparison, the total damage at C was 5.9 lesions/106 bases and that at 5mC was 1.0 lesions/106 bases in CT-DNA (Table 1), giving a difference of 5.7-fold (5.9/1.0). "
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ABSTRACT: The methylation and oxidative demethylation of cytosine in CpG dinucleotides plays a critical role in the regulation of genes during cell differentiation, embryogenesis and carcinogenesis. Despite its low abundance, 5-methylcytosine (5mC) is a hotspot for mutations in mammalian cells. Here, we measured five oxidation products of 5mC together with the analogous products of cytosine and thymine in DNA exposed to ionizing radiation in oxygenated aqueous solution. The products can be divided into those that arise from hydroxyl radical (•OH) addition at the 5,6-double bond of 5mC (glycol, hydantoin and imidazolidine products) and those that arise from H-atom abstraction from the methyl group of 5mC including 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC). Based on the analysis of these products, we show that the total damage at 5mC is about 2-fold greater than that at C in identical sequences. The formation of hydantoin products of 5mC is favored, compared to analogous reactions of thymine and cytosine, which favor the formation of glycol products. The distribution of oxidation products is sequence dependent in specific ODN duplexes. In the case of 5mC, the formation of 5hmC and 5fC represents about half of the total of •OH-induced oxidation products of 5mC. Several products of thymine, cytosine, 5mC, as well as 8-oxo-7,8-dihydroguanine (8oxoG), were also estimated in irradiated cells.
Nucleic Acids Research 05/2014; 42(7450-7460). DOI:10.1093/nar/gku334 · 9.11 Impact Factor
Available from: Henk J Blom
- "The relationships between B vitamin concentrations and global DNA methylation in leukocytes are unclear. Results from several feeding trials in healthy subjects have been inconclusive    . Additionally, to our knowledge, data describing the relationships between circulating levels of B vitamins and global DNA methylation in leukocytes of subjects at relatively high risk for CRC, i.e. those with previous adenomas   and/or those with a family history of CRC in a first-degree relative , do not yet exist. "
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Low concentrations of folate, other B vitamins, and methionine are associated with colorectal cancer risk, possibly by changing DNA methylation patterns. Here, we examine whether plasma concentrations of B vitamins and methionine are associated with methylation of long interspersed nuclear element-1 (LINE-1) among those at high risk of colorectal cancer, i.e. patients with at least one histologically confirmed colorectal adenoma (CRA) in their life.
Methods and results:
We used LINE-1 bisulfite pyrosequencing to measure global DNA methylation levels in leukocytes of 281 CRA patients. Multivariable linear regression was used to assess associations between plasma B vitamin concentrations and LINE-1 methylation levels. Plasma folate was inversely associated with LINE-1 methylation in CRA patients, while plasma methionine was positively associated with LINE-1 methylation.
This study does not provide evidence that in CRA patients, plasma folate concentrations are positively related to LINE-1 methylation in leukocytes but does suggest a direct association between plasma methionine and LINE-1 methylation in leukocytes.
Molecular Nutrition & Food Research 04/2013; 57(4). DOI:10.1002/mnfr.201200069 · 4.60 Impact Factor
Available from: Cristina Florindo
- "DNA purity was confirmed by the ratio of absorbance at 260 nm and 280 nm, which was always greater or equal to 1.8. DNA was hydrolyzed and cytosine (C) and 5-methylcytosine (mC) were analysed by LC-MS/MS, as previously described , . In short, 1 µg of genomic DNA was hydrolyzed using 1 molL−1 formic acid. "
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ABSTRACT: Methyltransferases use S-adenosylmethionine (AdoMet) as methyl group donor, forming S-adenosylhomocysteine (AdoHcy) and methylated substrates, including DNA and proteins. AdoHcy inhibits most methyltransferases. Accumulation of intracellular AdoHcy secondary to Hcy elevation elicits global DNA hypomethylation. We aimed at determining the extent at which protein arginine methylation status is affected by accumulation of intracellular AdoHcy. AdoHcy accumulation in human umbilical vein endothelial cells was induced by inhibition of AdoHcy hydrolase by adenosine-2,3-dialdehyde (AdOx). As a measure of protein arginine methylation status, the levels of monomethylarginine (MMA) and asymmetric and symmetric dimethylated arginine residues (ADMA and SDMA, respectively) in cell protein hydrolysates were measured by HPLC. A 10% decrease was observed at a 2.5-fold increase of intracellular AdoHcy. Western blotting revealed that the translational levels of the main enzymes catalyzing protein arginine methylation, protein arginine methyl transferases (PRMTs) 1 and 5, were not affected by AdoHcy accumulation. Global DNA methylation status was evaluated by measuring 5-methylcytosine and total cytosine concentrations in DNA hydrolysates by LC-MS/MS. DNA methylation decreased by 10% only when intracellular AdoHcy concentration accumulated to 6-fold of its basal value. In conclusion, our results indicate that protein arginine methylation is more sensitive to AdoHcy accumulation than DNA methylation, pinpointing a possible new player in methylation-related pathology.
PLoS ONE 02/2013; 8(2). DOI:10.1371/journal.pone.0055483 · 3.23 Impact Factor
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