An assay for uracil in human DNA at baseline: effect of marginal vitamin B6 deficiency.
Improvements are made to our gas-chromatography-mass-spectrometry-based assay for quantifying low levels of DNA-uracil. Folate deficiency leads to increased deoxyuridine monophosphate/thymidylate (dUMP/dTMP) ratios and uracil misincorporation into DNA, which may increase cancer risk. Vitamin B6 (B6) deficiency might also result in increased DNA-uracil because B6 is a cofactor for serine hydroxymethyltransferase, which catalyzes the methylation of tetrahydrofolate (THF) to methylene-THF, the folate form that is required to convert dUMP to dTMP. However, the low baseline levels of DNA-uracil in healthy human lymphocytes are difficult to measure accurately. This version of the assay (Uracil assay V3) has an approximately 10-fold increase in signal strength over the previous method and a 10-fold lower detection limit (0.2 pg uracil). Five micrograms of DNA, the amount in about 1 ml of human blood, is a suitable amount for this assay. Using this improved assay, DNA-uracil was measured in lymphocytes from 12 healthy smoking or nonsmoking young men and women who consumed a B6-restricted diet (0.7 mg B6/day, or approximately half the recommended dietary allowance) for 28 days. DNA-uracil concentration was not significantly related to B6 status or smoking. More severe and/or prolonged B6 deficiency may be necessary to detect significant changes in DNA-uracil in humans. The average concentration of DNA-uracil in these subjects was found to be approximately 3,000 uracils per diploid lymphocyte, which is comparable to steady state levels of one of the oxidative adducts of DNA, 8-oxoguanine.
Mashiyama
Susan T
ST
Department of Cell and Molecular Biology, Division of Biochemistry and Molecular Biology, University of California, Berkeley, CA 94720, U.S.A.
Hansen
Christine M
CM
Roitman
Esther
E
Sarmiento
Stella
S
Leklem
James E
JE
Shultz
Terry D
TD
Ames
Bruce N
BN
D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D017931:DNA Primers; D006801:Humans; D014498:Uracil / Q000032:analysis*; D026681:Vitamin B 6 Deficiency / Q000235:genetics*
D017931:DNA Primers; D014498:Uracil; D004247:DNA