Determination of cytidine modifications in human urine by liquid chromatography - Mass spectrometry analysis

Both DNA cytosine methylation (5-methyl-2'-deoxycytidine, m⁵dC) and RNA cytosine methylation (5-methylcytidine, m⁵rC) are important epigenetic marks that play regulatory roles in diverse biological processes. m⁵dC and m⁵rC can be further oxidized by the ten-eleven translocation (TET) proteins to form 5-hydroxymethyl-2'-deoxycytidine (hm⁵dC) and 5-hydroxymethylcytidine (hm⁵rC), respectively. 2'-O-methyl-5-hydroxymethylcytidine (hm⁵rCm) was recently also identified as a second oxidative metabolite of m⁵rC in RNA. Previous studies showed that the dysregulation of cytidine modifications in both DNA and RNA are closely related to a variety of human diseases. These cytidine modifications are generally excreted from cell into urine. If these cytidine modifications exhibit specific features related to certain diseases, determination of the cytidine modifications in urine could be utilized as non-invasive diagnostic of diseases. Here, we established a solid-phase extraction in combination with liquid chromatography-mass spectrometry (LC-MS/MS) analysis for simultaneous detection of these cytidine modifications in human urine samples. The developed method enabled the distinct detection of these cytidine modifications. We reported, for the first time, the presence of hm⁵rCm in human urine. Furthermore, we found that compared to the healthy controls, the contents of hm⁵dC, hm⁵rC, and hm⁵rCm showed significant increases in urine samples of cancer patients, including lymphoma patients, gastric cancer patients, and esophageal cancer patients. This study indicates that the urinary hydroxylmethylation modifications of hm⁵dC, hm⁵rC, and hm⁵rCm may serve as potential indicator of cancers.