Aging alters global hepatic DNA hydroxymethylation in mice, as determined by a novel LC/MS‐MS method

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Aging is associated with changes in global DNA methylation (mC), but changes in DNA hydroxymethylation (hmC) are not established due to a relative lack of methods available to measure hmC. Both modifications can affect gene expression and may be important in aging. We have developed a method to measure 5′‐methyl‐2′‐deoxycytidine and 5′‐hydroxymethyl‐2′‐deoxycytidine in DNA using liquid chromatography/tandem mass spectrometry (LC/MS‐MS) and used this method to assess global hmC and mC levels in hepatic tissue from young (9 months; n=10) and old (23 months; n=6) C57BL/6 male mice. DNA was enzymatically hydrolyzed and isotopomers [15N3]‐2′‐deoxycytidine and (methyl‐d3, ring‐6‐d1)‐5‐methyl‐2′‐deoxycytidine were added as internal standards. The mixture of DNA hydrolysates and internal standards were separated through LC, and nucleosides of interest were detected by MS‐MS in positive ion mode using multiple reaction monitoring. Global mC and hmC levels were calculated as a percentage of total deoxycytidine residues in genomic DNA. Liver tissue from old mice had significantly higher global hmC relative to young mice (0.32±0.02% vs 0.24±0.01%; p=0.02), while mC levels did not change. Using this new method we have found that aging is associated with an increase in global DNA hmC, a finding that may be useful in understanding the physiological mechanisms behind aging.Grant Funding Source: NIH grant R01 AG025834