Quantification of 5-(hydroxymethyl)uracil in DNA by gas chromatography/mass spectrometry: Problems and solutions

Oxidation of the thymine methyl group results in the formation of 5- (hydroxymethyl)uracil (HmU). HmU is a recognized endogenous DNA damage product, and HmU levels in DNA are increased by oxidant stress. Previous studies have reported substantially conflicting values for HmU levels in DNA. In studies utilizing postlabeling methods, HmU levels have been reported to be as high as or higher than the levels of some of the more commonly described DNA oxidation damage products such as 8-oxoguanine. In some studies utilizing GC/MS methods, however, HmU has been undetectable. In acid solution, the hydroxymethyl group of HmU can undergo condensation reactions with carboxylic acids, alcohols, and amines. While HmU can be accurately measured by GC/MS, the first step in the preparation of samples for GC/MS analysis is acid hydrolysis of the DNA. Such hydrolysis would be expected to result in substantial derivatization of HmU. We have utilized chemically synthesized oligonucleotides containing a known amount of HmU as well as an isotopically enriched standard to investigate the chemical modification of HmU during the acid hydrolysis of DNA. We conclude that HmU levels reported by GC/MS following acid hydrolysis may be up to an order of magnitude lower than the actual levels. Further, we propose modifications to the standard hydrolysis protocols which maximize recovery of HmU prior to silylation and analysis by GC/MS.