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Background: Female athletes and active women require adequate nutrition for optimal health and performance. Nutrition assessments are needed to identify potential nutrients of concern. Folate, vitamin B6, and vitamin B12 function in important pathways used during physical activity and female athletes may be at risk for poor status of these micronutrients. This cross-sectional study described a comprehensive nutrition assessment of the B-vitamins (folate, vitamin B6, and vitamin B12) using both dietary (food and dietary supplements) and biochemical assessments among highly active and sedentary women. Methods: Highly active (n = 29; age 20 ± 2 years; body mass index (BMI) 23.8 ± 3.5 kg/m2) and sedentary (n = 29; age 24 ± 3 years; BMI 22.6 ± 3.0 kg/m2) women were recruited for this study. Participants completed 7-day weighed food records and a fasting blood draw. Results: Although the highly active women reported higher intakes of energy (p < 0.01), folate (p < 0.01), vitamin B6 (p < 0.01), and vitamin B12 (p < 0.01), no significant differences were found between the groups for biomarkers of folate, vitamin B6, and vitamin B12. All of the highly active women had biomarkers within the desired reference ranges, suggesting good status. In general, most participants were able to meet the 1998 Recommended Daily Allowance (RDA) from food alone. For the women that reported using dietary supplements, micronutrient intakes met the 1998 RDA and in some cases, exceeded the Tolerable Upper Intake Level. Conclusion: This nutrition assessment documented good status for folate, vitamin B6, and vitamin B12 in the highly active women. Similar assessment approaches (food, dietary supplements, and biomarkers) should to completed with other nutrients of concern for the female athlete.

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.

The bootstrap procedure is a versatile statistical tool for the estimation of standard errors and confidence intervals. It is useful when standard statistical methods are not available or are poorly behaved, e.g., for nonlinear functions or when assumptions of a statistical model have been violated. Inverse regression estimation is an example of a statistical tool with a wide application in human nutrition. In a recent study, inverse regression was used to estimate the vitamin B-6 requirement of young women. In the present statistical application, both standard statistical methods and the bootstrap technique were used to estimate the mean vitamin B-6 requirement, standard errors and 95% confidence intervals for the mean. The bootstrap procedure produced standard error estimates and confidence intervals that were similar to those calculated by using standard statistical estimators. In a Monte Carlo simulation exploring the behavior of the inverse regression estimators, bootstrap standard errors were found to be nearly unbiased, even when the basic assumptions of the regression model were violated. On the other hand, the standard asymptotic estimator was found to behave well when the assumptions of the regression model were met, but behaved poorly when the assumptions were violated. In human metabolic studies, which are often restricted to small sample sizes, or when statistical methods are not available or are poorly behaved, bootstrap estimates for calculating standard errors and confidence intervals may be preferred. Investigators in human nutrition may find that the bootstrap procedure is superior to standard statistical procedures in cases similar to the examples presented in this paper.

Plasma total homocysteine concentrations were measured in 8 healthy premenopausal women who, after a 9-d adjustment period with a vitamin B-6 intake of 1.60 mg/d, were marginally depleted of vitamin B-6 (0.45 mg/d) for 27 d, and then repleted with three levels of vitamin B-6 (1.26, 1.66 and 2.06 mg/d) for periods of 21, 21 and 14 days, respectively. Significant increases in plasma homocysteine concentrations were seen at the end of repletion periods 1 and 2, with a return to the adjustment concentration by the end of repletion period 3. Plasma homocysteine concentrations were negatively correlated with two measures of vitamin B-6 status, urinary 4-pyridoxic acid and plasma pyridoxal phosphate. Additional research is needed to determine whether plasma homocysteine concentration may be used as a functional, or indirect, measure of vitamin B-6 status.

Pyridoxal 5'-phosphate (PLP) stimulation of erythrocyte alanine and aspartate aminotransferase (EALT, EAST) activities is a frequently used functional measure of vitamin B-6 status. Stability of enzyme activities and activity coefficients (AC, stimulated / unstimulated) was assessed in packed erythrocytes frozen at -20, -80 degrees C and under liquid nitrogen (-196 degrees C). Activities of EALT and EAST, with and without added PLP, were determined in fresh erythrocytes (d 0) and frozen samples on d 1, 7, 14, 28, 58 and 84. In -20 degrees C samples, EALT basal activity decreased 17 and 22% (P < or = 0.05 for both) by d 58 and 84, respectively, and EAST basal activity decreased 40% (P < or = 0.05) by d 58. In -80 and -196 degrees C samples, EALT and EAST basal activities did not change significantly. Activity coefficients did not differ significantly from d 0 at any storage temperature, but EAST-AC increased 9-19% (nonsignificant) in samples stored at -20 and -80 degrees C for 7 to 84 d. Additionally, EAST-AC was significantly higher in -20 than -80 and -196 degrees C samples on d 1 and 58, respectively. Erythrocytes may be frozen for 28 d at -20 degrees C and 84 d at -80 degrees C before analysis for EALT; for EAST, activity should be measured on fresh erythrocytes.

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.

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.

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.