Measurement of urinary excretion of 5-hydroxymethyluracil in human by GC/NICI/MS:

5-Hydroxymethyluracil (5-HMU) is derived from radiation in addition to endogenous oxidative DNA damage and it is one of the most abundant DNA adducts. Human 5-HMU-DNA-glycosylase has been shown to repair this lesion. Whether urinary levels of 5-HMU is a valid biomarker for oxidative DNA damage in vivo has been investigated. However, controversial results on its relation to cigarette smoking were reported. To facilitate analysis of urinary 5-HMU in epidemiological studies, a highly sensitive and specific assay based on stable isotope dilution gas chromatography/negative ion chemical ionization/mass spectrometry was developed. The limit of detection for N1,N3-bis(pentafluorobenzyl)-HMU is 10 fg (20 amol) (S/N = 4) injected on column and the limit of quantification in urine was 0.7 nM of 5-HMU. Using as little as 10 μL of human urine samples, levels of urinary 5-HMU in 21 healthy volunteers were accurately quantified. No correlation was observed between urinary 5-HMU levels and cigarette smoking. However, there was a statistically significant association between urinary levels of 5-HMU and thiobarbituric acid-reactive substances (r = 0.71, p = 0.0003). In addition, urinary 5-HMU levels also correlated with urinary levels of 1,N6-ethenoadenine (r = 0.54, p = 0.01). These findings suggest that this assay should be valuable in assessing the role of urinary 5-HMU as a biomarker of oxidative DNA damage and repair.