Urinary Measurement of Epigenetic DNA Modifications: A Non‐Invasive Assessment of the Whole‐Body Epigenetic Status in Healthy Subjects and Colorectal Cancer Patients

Abstract

Active mechanism of DNA demethylation can be responsible for the activation of previously silenced genes. Products of 5‐methylcytosine oxidation are released into the bloodstream and eventually excreted with urine. Therefore, whole‐body epigenetic status can be assessed non‐invasively on the basis of the urinary excretion of a broad spectrum of epigenetic modifications: 5‐hydroxymethylcytosine (5‐hmCyt), 5‐formylcytosine (5‐fCyt), 5‐carboxycytosine (5‐caCyt), and 5‐hydroxymethyluracil (5‐hmUra). We have developed a specific and sensitive, isotope‐dilution, automated, online, two‐dimensional ultra‐performance liquid chromatography system with tandem mass spectrometry (2D UPLC–MS/MS) to measure 5‐hmCyt, 5‐fCyt, 5‐caCyt, and their deoxynucleosides in the same urine sample. Human urine contains all of the modifications except from 5‐formyl‐2′‐deoxycytidine (5‐fdC) and 5‐carboxy‐2′‐deoxycytidine (5‐cadC). A highly significant difference in the urinary excretion of 5‐(hydroxymethyl)‐2’‐deoxycytidine (5‐hmdC) was found between healthy subjects and colorectal cancer patients (3.5 vs. 7.8 nmol mmol−1 creatinine, respectively), as well as strong correlations between the majority of analyzed compounds.

Advances in detection and quantification of methylcytosine and its derivatives

“…With this approach, hydrolyzed DNA samples (hydrolysates) are spiked with a mixture of isotope internal standards to enrich the samples . Using chromatographic separation methods, this approach has been used to quantify all the intermediate products of DNA demethylation pathway in the form of deoxynucleosides in various samples .…”

“…Using chromatographic separation methods, this approach has been used to quantify all the intermediate products of DNA demethylation pathway in the form of deoxynucleosides in various samples . This method is effective in distinguishing 5‐methyl‐2′‐deoxycytidine, 5‐(hydroxymethyl)‐2′‐deoxycytidine, 5‐formyl‐2′‐deoxycytidine and 5‐carboxy‐2′‐deoxycytidine as well as 5‐(hydroxymethyl)‐2′‐deoxyuridine using automated isotope‐dilution online two‐dimensional ultra high performance liquid chromatography with tandem mass spectrometry . This approach also helps to reliably identify and quantitate base modifications in DNA and RNA .…”

“…With the enrichment technique together with LC‐MS, all known cytosine modifications have been separated, distinguished and independently quantitated . Importantly, this approach allows DNA modifications to be assessed non‐invasively .…”

“…A family of bifunctional DNA glycosylases remove 5mC and begin its replacement by unmethylated cytosine via a base excision repair (BER) process in plants . In animals, however, no 5mC DNA glycosylases have been specifically identified.…”

Bioinformatics analysis of the interactions among lncRNA, miRNA and mRNA expression, genetic mutations and epigenetic modifications in hepatocellular carcinoma

“…Genetic mutations are one of the principal factors involved in tumor occurrence and may lead to the activation of proto-oncogenes or the loss of tumor suppressor genes. Furthermore, accumulating evidence demonstrated that genetic modifications are associated with tumorigenesis (8)(9)(10)(11). DNA methylation is a mechanism underlying epigenetic modifications, and is able to affect the DNA conformation, the chromatin structure and the stability of DNA, altering the interactions between DNA and proteins regulating gene expression (12).…”