Monitoring urinary excretion of 5-hydroxymethyluracil for assessment of oxidative DNA damage and repair

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Abstract
Urinary excretion of oxidized nucleobases and nucleosides has been used as a biomarker of oxidative DNA damage and repair. Most studies have focused on the measurements of 8-oxo-7,8-dihydro-2′-deoxyguanosine; however, the urinary levels of other DNA modifications may represent useful indicators of oxidative stress. We developed a method for the determination of 5-hydroxymethyluraciI (5-HMUra), consisting of the separation of the modified base in urine by HPLC and quantification by GC/MS in the selective ion monitoring mode. This experimental approach was subsequently validated in human samples, with the effect of storage and the inter- and intra-individual variations in 5-HMUra excretion being evaluated. Results showed that 5-HMUra is stable in samples frozen at-80 °C for at least 4 months. Inter-individual variations in 5-HMUra excretion were observed when the results were expressed either as nmoles excreted per kg per day (1.2–2.4) or corrected by creatinine values (7.2–12.2 nmoles 5-HMUra per mmoles creatinine). Intra-individual variability was low, varying slightly at different time collections for several individuals. Differences in the excretion of 5-HMUra in urine collected at three different 8-h intervals during the day were not significant and, in particular, the levels of 5-HMUra calculated from the overnight or the 24-h samples were highly correlated. These results indicate that monitoring urinary levels of 5-HMUra could be a suitable indicator of oxidative damage in human studies.