The Recommended Dietary Allowance (RDA) of vitamin B-6 for young women was recently reduced from 1.6 to 1.3 mg/d based on an adequate plasma pyridoxal phosphate (PLP) concentration of 20 nmol/L. To assess vitamin B-6 requirements and suggest recommendations for intake, seven healthy young women consumed a controlled diet providing 1.2 g protein/kg body weight for a 7-d adjustment period (1.0 mg vitamin B-6/d) and three successive 14-d experimental periods (1.5, 2.1 and 2.7 mg/d, respectively). Direct and indirect vitamin B-6 status indicators were measured in plasma, erythrocytes and urine. Indicators most strongly correlated with vitamin B-6 intake [i.e., plasma and erythrocyte PLP, urinary 4-pyridoxic acid (4-PA) and total vitamin B-6] were regressed on vitamin B-6 intake and the dietary vitamin B-6 to protein ratio. Inverse prediction using adequate and baseline values estimated vitamin B-6 requirement. Adequate values were determined for plasma PLP and urinary 4-PA from baseline values of 60 previous subjects, using the statistical method suggested by Sauberlich. The current study suggests a vitamin B-6 Estimated Average Requirement (EAR) for young women of 1.1 mg/d or 0.016 mg/g protein, and a RDA of 1.5 mg/d or 0.020 mg/g protein. When results from this study are combined with data from four other recent studies, the combined data predict an EAR of 1.2 mg/d or 0.015 mg/g protein, and a RDA of 1.7 mg/d or 0.018 mg/g protein. This study suggests that the current vitamin B-6 RDA may not be adequate.

Changes in vitamin B-6 status indicators were evaluated in vitamin B-6-replete subjects. Ten young women consumed diets providing 85 g protein/d and 1.03, 1.33, 1.73, and 2.39 mg vitamin B-6/d for 12 or 15 d during four successive diet periods; in a second study, six women were fed diets providing 85 g protein/d and 0.84, 1.14, and 2.34 mg vitamin B-6/d for 10 or 12 d during three successive diet periods. Vitamin B-6 status indicators showing significant differences among intakes included urinary excretion of 4-pyridoxic acid and total vitamin B-6, pyridoxal 5'-phosphate and total vitamin B-6 in plasma, and xanthurenic acid excretion after a 2-g L-tryptophan load. Significant correlations were found between vitamin B-6 intake and 4-pyridoxic acid, total vitamin B-6, plasma pyridoxal 5'-phosphate, plasma total vitamin B-6, erythrocyte alanine aminotransferase percentage stimulation and postload excretion of xanthurenic acid and volatile amines (kynurenine plus acetylkynurenine). Depending on the indicator, between 20% and 70% of the subjects had inadequate values for 4-pyridoxic acid, total vitamin B-6, plasma pyridoxal 5'-phosphate, and erythrocyte alanine aminotransferase percentage stimulation at a vitamin B-6 intake of 1.33 mg/d (0.016 mg vitamin B-6/g protein). A ratio of dietary vitamin B-6 to protein > 0.016 mg/g is required for adequate vitamin B-6 status in women.

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.

Previous research has shown that the pyridoxine glucoside (PNG) form of vitamin B-6 has a reduced bioavailability compared with pyridoxine, but its effect on vitamin B-6 status has not been assessed. Following an 8-d adjustment period, nine women consumed diets containing a high or low amount of PNG for 18 d each, in a crossover design. The high and low PNG diets provided 1.52 mg/d (8.98 micromol/d) and 1.44 mg/d (8.57 micromol/d) of vitamin B-6, of which 27% and 9% was PNG, respectively. The dietary vitamin B-6 to protein ratio of both diets was 0.017 mg/g. Urinary excretions of 4-pyridoxic acid and total vitamin B-6 were significantly lower (P < 0.05) during the high PNG diet period than when the low PNG diet was consumed. Urinary PNG excretion was equal to about 9% of the total PNG intake during both periods. Plasma total vitamin B-6 (P < 0.01) and red blood cell pyridoxal 5'-phosphate (PLP) (P < 0.05) were significantly lower when the high PNG diet was consumed than during the low PNG diet period. Fecal total vitamin B-6 excretion was significantly higher (P < 0.001) when the high PNG diet was consumed. Women consuming a diet containing a higher percentage of the total vitamin B-6 intake as PNG exhibited a decrease in vitamin B-6 status indicators, consistent with the reduced bioavailability of PNG demonstrated in other studies, equal to a loss of 15-18% of the total vitamin B-6 intake. During the determination of Recommended Dietary Allowances, the reduced bioavailability of PNG and its presence in higher amounts in some diets should be considered.

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.