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.

Improved vitamin B-6 status is positively related to lymphocyte proliferation in young women consuming a controlled diet.

To examine the effect of increased intake levels of vitamin B-6 (B-6) on lymphocyte proliferation and interleukin 2 (IL-2) concentration, young women (n = 7) consumed a constant diet containing 1 mg (5.91 micro mol) B-6/d for a 7-d adjustment period, followed by three 14-d experimental periods during which the daily B-6 intake was 1.5, 2.1 and 2.7 mg (8.86, 12.41 and 15.95 micro mol)/d, respectively. Weekly fasting blood and daily 24-h urine samples were collected. Lymphocyte proliferation and IL-2 production were measured in response to phytohemagglutinin. Vitamin B-6 status improved with increased B-6 intake as measured by plasma pyridoxal 5'-phosphate (PLP) and urinary 4-pyridoxic acid. When subjects consumed 2.1 mg B-6/d for 7 d, lymphocyte proliferation increased by 35% (P < or = 0.05) compared with the mean value after consumption of 1.5 mg B-6/d for 14 d. There was no further enhancement after an additional week of 2.1 and 2.7 mg B-6/d for 2 wk. Lymphocyte proliferation was correlated (P < or = 0.01) with vitamin B-6 intake (r = 0.757), plasma PLP (r = 0.456) and erythrocyte aminotransferase activities (r = -0.361). Plasma IL-2 concentration and in vitro production did not change throughout the study, although five of seven subjects showed increases with intakes of 2.1 and 2.7 mg B-6/d, respectively, compared with the 1.5 mg/d intake. Concentrations of PLP in peripheral blood mononuclear cells were correlated (r = 0.357, P < or = 0.01) with plasma PLP, but not with proliferation. These results show that improving vitamin B-6 status by consuming a B-6 intake higher than the current Recommended Dietary Allowance enhances lymphocyte proliferation.