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

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.