Analysis of 5-Hydroxymethyluracil Levels Using Flow Cytometry

5-hydroxymethyluracil was originally identified as an oxidatively modified DNA base derivative. Recent evidence suggests that its formation may result from the oxidation of thymine in a reaction that is catalyzed by TET proteins. Alternatively, it could be generated through the deamination of 5-hydroxymethylcytosine by activation-induced cytidine deaminase. The standard method for evaluating 5-hydroxymethyluracil content is the highly sensitive and highly specific isotope-dilution automated online two-dimensional ultraperformance liquid chromatography with tandem mass spectrometry (2D-UPLC-MS/MS). Despite many advantages, this method has one great limitation. It is not able to measure compounds at a single-cell level. Our goal was to develop and optimize a method based on flow cytometry that allows the evaluation of 5-hydroxymethyluracil levels at a single cell level in peripheral leukocytes.