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Articles

Assessment of Vitamin B-6 Status in Young Women Consuming a Controlled Diet Containing Four Levels of Vitamin B-6 Provides an Estimated Average Requirement and Recommended Dietary Allowance^{1 ,2}

Department of Food Science and Human Nutrition, Washington State University, Pullman, WA 99164-6376 and ^* Department of Nutrition and Food Management, Oregon State University, Corvallis, OR 97331-5103

³To whom correspondence should be addressed. E-mail: shultz{at}wsu.edu

	ABSTRACT

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
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.

KEY WORDS: • vitamin B-6 • status assessment • Estimated Average Requirement • Recommended Dietary Allowance • women

	INTRODUCTION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
In the 1998 report of the Standing Committee on the Scientific Evaluation of Dietary Reference Intakes (DRI),⁴ the Recommended Dietary Allowance (RDA) of vitamin B-6 for adult women was reduced from 1.6 to 1.3 mg/d (1) . The Committee based their recommendation primarily on data from six studies (2 3 4 5 6 7) . Data from these studies were reevaluated to determine the vitamin B-6 intake required for a plasma pyridoxal phosphate (PLP) concentration of 20 nmol/L. Plasma PLP concentration was chosen as the standard for adequate status because this measure appears to reflect tissue stores (8) . In the absence of evidence linking a particular concentration to favorable or unfavorable health outcomes, a concentration of 20 nmol/L was chosen as the standard for adequate status. The DRI Committee stated that a plasma PLP concentration of 20 nmol/L is "not accompanied by observable health risks" (1) and was suggested by one group of investigators (8 ,9) .

The study reported here was undertaken to investigate the relationship between vitamin B-6 intake and measures of immune function to determine whether these measures could be used to establish vitamin B-6 requirements. The immune function results will be published separately. In this paper, recommendations for vitamin B-6 intake will be assessed on the basis of the effect of vitamin B-6 intake and the dietary vitamin B-6 to protein ratio on several vitamin B-6 status indicators in plasma, erythrocytes and urine of young women. Following DRI Committee methodology, the Estimated Average Requirement (EAR) will be determined and the RDA calculated. In addition, data from this study and several other recent studies (3 ,5 6 7) will be combined and recommendations for vitamin B-6 intake proposed on the basis of the combined data.

	SUBJECTS AND METHODS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Subjects.

Premenopausal women (n = 8) were recruited from the Washington State University community. Potential subjects completed a health history questionnaire, gave a fasting blood sample for clinical chemistry evaluation, underwent xylose absorption testing (10) and were examined by the study physician. Subjects were in good health, nonsmokers and not taking prescription medications or nutritional supplements. Three-day dietary records were obtained from each subject 4 wk before the beginning of the study to evaluate subjects’ usual nutrient intakes. Subjects’ characteristics are listed in Table 1 . The screening and experimental procedures were reviewed and approved by the Institutional Review Board of Washington State University, and informed consent was obtained from each subject. Subjects were housed in the Human Metabolic Unit at Washington State University throughout the 49-d study and were instructed to maintain their usual activity level.

The protocol consisted of a 7-d adjustment period and three successive 14-d experimental periods (Table 2 ). Subjects consumed a nonvegetarian, 3-d rotating menu providing 1 mg/d (5.91 µmol/d) vitamin B-6 and 1.2 g protein/kg body weight. After the 7-d adjustment period, vitamin B-6 was supplemented as a pyridoxine (PN) hydrochloride (Sigma Chemical, St. Louis, MO) solution given at breakfast. Total vitamin B-6 intake for the three 14-d experimental periods (diet plus supplement) was 1.5, 2.1 and 2.7 mg/d (8.86, 12.41 and 15.95 µmol/d), respectively.

The basal diet fed throughout the four periods (Table 3 ) consisted of natural plant and animal foods, and was adapted from diets previously described by Hansen et al. (5) and Huang et al. (6) . Average daily nutrient composition of the 3-d rotating menu is detailed in Table 4 . Meals were prepared in the kitchen of the Metabolic Unit and eaten in the Unit’s dining room, except for occasional take-out lunches. Food portions and recipe ingredients were weighed accurately to within 0.1 g, and recipes and cooking times were standardized. Food items, except perishables, were purchased in a single lot to minimize variability in nutrient composition. Subjects consumed only those foods and beverages prepared for them or permitted. Egg white powder (reconstituted and cooked) was added to menu items to adjust protein intake according to individual body weight. To provide the 1989 RDA (11) for all nutrients except vitamin B-6, subjects were supplemented with 4 mg nicotinic acid and 200 µg folic acid (Sigma Chemical), 8.8 mg elemental iron (Feosol; Smith Kline Consumer Products, Philadelphia, PA), 333 mg calcium, 133 mg magnesium and 5 mg zinc (Cal-Mag Zinc; Thrifty PayLess, Wilsonville, OR). Additional energy sources that contained very little or no vitamin B-6 [i.e., sugar, margarine, jam, soft drinks, hard candy and cookies (providing <0.01 mg/d)] were offered as required to maintain body weight, and intakes of these foods were recorded daily. Tea and instant coffee were provided at an amount selected by each subject at the beginning of the study as their usual daily consumption.

Food composites of the basal diet were made on each of the three different menu days several times during the study. Total vitamin B-6 was determined in the composites by microbiological assay (12) and PN glucoside (PNG) by the method of Kabir et al. (13) .

Daily 24-h urine collections were obtained, using toluene as a preservative, throughout the study. Aliquots of urine were stored at -20°C until analysis. Urinary creatinine was assayed to determine completeness of collection (14) . Urinary 4-pyridoxic acid (4-PA) was analyzed by an HPLC procedure (15) omitting the acidification step. Mean (±SD) recovery of added 4-PA was 98 ± 14%. Total vitamin B-6 in urine was determined by a microbiological assay (16) . Urine was tested weekly for glucose, bilirubin, ketones, blood, protein, pH (Bili-Labstix; Bayer Corporation Diagnostics Division, Elkhart, IN) and pregnancy (QuPID; Stanbio Laboratory, San Antonio, TX).

Weekly blood samples were collected into EDTA and heparin anticoagulated Vacutainer (Becton Dickinson, Rutherford, NJ) tubes after an overnight fast, and immediately placed on ice. After whole blood was removed for hematology determinations (T660 Coulter Counter; Coulter Electronics, Hialeah, FL), plasma and erythrocytes were separated by centrifugation (1430 x g at 4°C). Plasma was removed and aliquots stored at -40°C. Erythrocytes were washed three times with saline, an aliquot of packed cells was removed for assay of alanine and aspartate aminotransferase activities and the remainder was frozen at -40°C. Lymphocytes were separated from one tube of heparinized blood for immune function tests and determination of lymphocyte PLP concentration [preliminary results reported elsewhere (17) ].

Serum alkaline phosphatase activity was determined by Pathologists’ Regional Laboratory (Lewiston, ID) as described by Bowers and McComb (18) on samples taken at screening. Vitamin B-6 metabolites [i.e., PLP, pyridoxamine phosphate (PMP), PL, PN and 4-PA] in plasma and erythrocytes were determined by HPLC with fluorometric detection (19) . Recoveries of added vitamers from plasma were 88% and 94% for PLP and PL, respectively. Recoveries of added vitamers from erythrocytes were 64 ± 9, 114 ± 14, 97 ± 24 and 93 ± 11% for PLP, PMP, PL and PN, respectively. Erythrocyte PLP values were corrected for recovery. Erythrocyte alanine and aspartate aminotransferase activities (EALT and EAST) were measured with and without added PLP (20) ; EALT was assayed the same day blood was drawn, and EAST was assayed the next day on cells frozen at -40°C. The EALT and EAST activity coefficients were calculated as the ratio of stimulated (PLP added) to unstimulated (no PLP added) activities.

Urinary, plasma and erythrocyte vitamin B-6 metabolite and aminotransferase activity measurements were carried out under yellow light to prevent photodecomposition. All analyses were performed in duplicate.

Statistical analyses.

Data were analyzed using SAS and JMP statistical analysis computer programs (SAS Institute, Cary, NC). Group means and standard deviations were calculated at each time point for all measurements. The last time point in each experimental period was used in repeated-measures ANOVA. If 24-h urine collections were judged complete on the basis of creatinine excretion, urinary 4-PA and total vitamin B-6 excretion were averaged over the last 3 d of each period before performing statistical analyses, to minimize the effect of day-to-day variation. When repeated-measures ANOVA indicated significant differences among means, multiple comparison tests were performed using least significant difference. Pearson’s product-moment correlation coefficients were computed to determine relationships among vitamin B-6 status measures and vitamin B-6 intake. Student’s paired t test was used to compare mean body weight at the beginning and end of the study. Statistical comparisons were considered significant at P 0.05.

Values for vitamin B-6 status indicators at the end of the adjustment and three experimental periods were regressed on vitamin B-6 intake and the dietary vitamin B-6 to protein ratio (6) . Intakes were adjusted for bioavailability by converting supplemental vitamin B-6 to dietary vitamin B-6 equivalents [dietary vitamin B-6 equivalents = food vitamin B-6 + (1.27 x supplemental vitamin B-6)] (1) . Adequate (21) and baseline values of status indicators were used to calculate estimates and 95% confidence intervals of vitamin B-6 EAR (1) by inverse prediction (22) , using the linear regression model equations that were statistically significant. Weighted means were determined on the basis of the inverse of the confidence interval range. Recommended Dietary Allowances were calculated using the formula: RDA = 1.2 x EAR (1) , which assumes an EAR CV of 10%.

	RESULTS

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 
On d 29, the plasma PLP concentration and urinary 4-PA excretion of one subject were found to be 28- and 8-fold higher, respectively, than the mean values of the other seven subjects. Therefore, we concluded that this subject did not adhere to the study protocol, and her data were eliminated from the statistical analyses. No differences were found at any time point in means of urinary excretion of creatinine or hematologic measures (data not reported). Two subjects were given an additional iron supplement (27 mg/d elemental iron, Fergon; Bayer Corporation, Morristown, NJ) when their hemoglobin concentration fell below 120 g/L, one after wk 1 and the other after wk 5 of consuming the controlled diet. Mean body weight at the end of the study was not significantly different from baseline.

Diet.

Individual vitamin B-6 intake of subjects before the study, calculated from 3-d diet records, ranged from 0.9 to 2.1 mg/d (0.013–0.024 mg vitamin B-6/g protein; Table 2 ). Food composites from d 1, 2 and 3 of the basal diet, analyzed by microbiological assay, contained 0.97 ± 0.04, 1.02 ± 0.04 and 0.97 ± 0.03 mg vitamin B-6, respectively. PN glucoside was 19% of the total vitamin B-6 intake in the basal diet for d 1, 21% for d 2 and 9% for d 3, with a mean (±SD) of 16 ± 6%. When vitamin B-6 supplements given during the experimental periods are taken into consideration, PNG was 11, 8 and 6% of the total vitamin B-6 intake during Periods 1, 2 and 3, respectively. The amount of PNG in the typical American diet has been estimated to be 10–15% of the total vitamin B-6 content (23 ,24) .

Urinary vitamin B-6 status measures.

Mean urinary 4-PA and total vitamin B-6 excretion at baseline (d 1) and at the end of each experimental period are presented in Table 5 . Urinary 4-PA excretion at baseline was 3.0 µmol, considered to be indicative of adequate vitamin B-6 status (21) , for all subjects except one (2.5 µmol/d). At the end of the adjustment period (1.0 mg/d vitamin B-6 intake), mean urinary 4-PA excretion had decreased 38% compared with baseline, and only two subjects were excreting 3.0 µmol/d. Mean urinary 4-PA excretion increased significantly with each successive increase in vitamin B-6 intake, and was 3.0 µmol/d for all seven subjects at the end of all three experimental periods. By the end of Period 1 (1.5 mg/d vitamin B-6 intake), mean urinary 4-PA excretion was not significantly different from baseline, but two subjects were excreting 29 and 35% less than baseline. Mean urinary 4-PA excretion represented 59, 48, 55 and 58% of total vitamin B-6 intake during adjustment and the three experimental periods, respectively.

Plasma vitamin B-6 status measures.

Plasma PLP concentrations (Table 5) at baseline for all subjects except one (27.6 nmol/L) were 30 nmol/L, indicating adequate vitamin B-6 status (21) . Mean plasma PLP concentration decreased 36% and was significantly different from baseline at the end of the adjustment period; three of seven subjects had plasma PLP concentrations <30 nmol/L. After 2 wk of consuming 1.5 mg vitamin B-6/d, plasma PLP concentrations remained 29–60% lower than baseline in four subjects, but <30 nmol/L in only one subject. At the end of Period 2 (2.1 mg/d vitamin B-6 intake), plasma PLP concentrations for four subjects were 21–35% less than baseline, but all subjects had concentrations 30 nmol/L. By the end of Period 3 (2.7 mg/d vitamin B-6 intake), mean plasma PLP concentration had increased 89% and was significantly different from the adjustment period. Although mean plasma PLP concentration at the end of Period 3 was greater than the mean baseline concentration, three subjects had not achieved baseline concentrations of plasma PLP. No subjects had plasma PLP concentrations <20 nmol/L during any of the experimental periods.

There were no significant differences in mean plasma PL, PN or 4-PA concentrations among the periods. Mean plasma total vitamin B-6 (i.e., PLP + PL + PN) concentrations were significantly different at the end of the adjustment and three experimental periods. At the end of the adjustment period, mean plasma total vitamin B-6 was significantly lower (32%) than baseline, reflecting the observed changes in plasma PLP concentration. Six of seven subjects’ plasma total vitamin B-6 reached baseline by the end of Period 3.

Erythrocyte vitamin B-6 status measures.

Erythrocyte vitamin B-6 metabolite concentrations and aminotransferase activities are given in Table 6 . Erythrocyte PLP concentration increased significantly (25%) from the adjustment period and surpassed baseline by the end of Period 2; two of seven subjects remained below their baseline concentration. At the end of Period 3, mean erythrocyte PLP was significantly greater (26%) than at the end of Period 2, and all subjects reached or exceeded baseline. Erythrocyte PMP concentration was significantly increased from baseline (29%) and adjustment period (18%) at the end of Period 3. Erythrocyte PL concentration was significantly increased (39%) from the adjustment period at the end of Period 1, but did not differ significantly from baseline during any of the periods. Erythrocyte PN concentration was not significantly different at any time point during the study. Erythrocyte total vitamin B-6 (i.e., PLP + PMP + PL + PN) concentrations were never significantly different from baseline, but were significantly higher than the adjustment concentration at the end of Periods 1 (16%) and 3 (19%). Erythrocyte alanine and aspartate activity coefficients did not vary significantly throughout the study. Basal activity for EALT was significantly increased at the end of Period 3 compared with all the other periods.

Correlations among vitamin B-6 status indicators.

Correlations among vitamin B-6 intake, the dietary vitamin B-6 to protein ratio and vitamin B-6 status indicators are listed in Table 7 . Vitamin B-6 intake and the dietary vitamin B-6 to protein ratio were significantly correlated with urinary 4-PA and total vitamin B-6, plasma PLP, total vitamin B-6 and 4-PA, and erythrocyte PLP. In addition, vitamin B-6 intake was significantly correlated with erythrocyte PMP and EAST basal activity.

Vitamin B-6 requirement.

The EAR of vitamin B-6 was calculated by inverse prediction, using both adequate and baseline values (Table 8 ). Linear regression analyses of plasma PLP vs. vitamin B-6 intake (adjusted for bioavailability) and the dietary vitamin B-6 to protein ratio (adjusted for bioavailability) are depicted in Figure 1A and B , respectively. Similar analyses were performed for each of the vitamin B-6 status indicators listed in Table 8 . Regression analysis equations for the other status indicators (y) vs. vitamin B-6 intake (x) and the dietary vitamin B-6 to protein ratio (x), respectively, are as follows: for urinary 4-PA, y = 2.967x + -0.2680 and y = 197.5x + 0.05004; for urinary total vitamin B-6, y = 0.1789x + 0.2847 and y = 13.02x + 0.2719; for erythrocyte PLP, y = 8.950x + 20.36 and y = 626.0x + 20.44. The weighted mean of the predictions yielded an EAR and RDA of 1.1 mg/d (0.016 mg/g protein) and 1.3 mg/d (0.018mg/g protein) vitamin B-6, respectively, using adequate values (21) , and, 2.0 mg/d (0.028 mg/g protein) and 2.4 mg/d (0.031 mg/g protein) vitamin B-6, respectively, using baseline values.

View larger version (24K): [in this window] [in a new window]   Figure 1. Linear regression analysis of plasma pyridoxal phosphate (PLP) concentration vs. vitamin B-6 intake (A) and the dietary vitamin B-6 to protein ratio (B) for 7 women consuming a controlled diet with four levels of vitamin B-6. Intakes were adjusted by converting supplemental vitamin B-6 to dietary vitamin B-6 equivalents [dietary vitamin B-6 equivalents = food vitamin B-6 + (1.27 x supplemental vitamin B-6)] (1) . Inverse regression calculations using adequate (21) and baseline values of PLP concentration predict Estimated Average Requirements for vitamin B-6 of 1.1 and 2.5 mg/d, respectively, and dietary vitamin B-6 to protein ratios of 0.017 and 0.035 mg/g, respectively.

	DISCUSSION

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

Sauberlich (26) proposed that interpretation of biochemical results in nutritional status assessment use a statistical approach in classifying the following three states of risk: 1) high, 2) borderline or moderate, and 3) low. Subjects with values below the 2.5th percentile would be classified as high risk, those between the 2.5th and 30th percentile would be considered borderline (marginally or subclinically deficient), and those above the 30th percentile would be considered to have acceptable values or adequate status. Applying Sauberlich’s approach to baseline data obtained in our laboratories (3 4 5 ,27 28 29) from 60 healthy, unsupplemented women ages 19–50 y consuming self-selected diets with a plasma PLP concentration (mean ± SD) of 42.8 ± 19.1 nmol/L (range: 14.2–109 nmol/L) and urinary 4-PA excretion of 5.25 ± 2.59 µmol/d (range: 2.24–20.22 µmol/d), we determined an acceptable value for plasma PLP concentration of 31.1 nmol/L, indicating adequate status, and 4.07 µmol/d for urinary 4-PA excretion. Thus, when calculating an EAR for vitamin B-6 we used the adequate values published by Leklem (21) , e.g., 30 nmol/L for plasma PLP, rather than the 20 nmol/L plasma PLP concentration used by the DRI Committee.

Many of the studies that have assessed vitamin B-6 requirements have determined the intake that returns status indicators to their prestudy baseline values (6 ,7 ,30 ,31) . This approach has been criticized because the subjects have been motivated healthy individuals consuming self-selected diets or diets that contained 1.5–2.0 mg vitamin B-6, and the assessed requirements generally are similar to or higher than the baseline vitamin B-6 intake (1) . The subjects in the study reported here had a baseline intake of 1.4 ± 0.6 mg/d (0.020 ± 0.007 mg/g protein) and the assessed requirement predicted by baseline status indicators was 2.0 mg/d (0.028 mg/g protein), which is higher than the predicted requirement based on adequate values. Therefore, following the guidelines suggested by the DRI committee, we base our recommendations on inverse predictions using adequate values rather than baseline values.

In the 1989 Recommended Dietary Allowances (11) , the RDA for vitamin B-6 was based on protein intake (i.e., 0.016 mg/g protein). Increased protein intake has been shown to decrease several measures of vitamin B-6 status (3 ,32 33 34 35) . Although Pannemans et al. (36) failed to show an effect of dietary protein on vitamin B-6 status indicators in elderly subjects, young subjects consuming a high protein diet excreted less urinary 4-PA compared with subjects consuming a low protein diet. Another study (31) reported that higher vitamin B-6 intakes were necessary to normalize plasma PLP concentrations in elderly subjects consuming a high protein diet vs. subjects consuming a low protein diet. In the study reported here, because protein intake was based on body weight, we could separate subjects into two body weight/protein intake groups. The lower body weight/protein group (n = 4) had a mean (±SD) body weight of 54 ± 2 kg and protein intake of 65 ± 3 g/d, whereas the higher body weight group (n = 3), had values of 66 ± 1 kg and 79 ± 2 g/d, respectively. At baseline, the higher body weight/protein group had higher plasma PLP concentrations (52.5 ± 8.3 nmol/L vs. 42.2 ± 16.6 nmol/L for the lower body weight/protein group). By the end of Period 3, the higher body weight/protein group had a plasma PLP concentration of 43.0 ± 1.9 nmol/L compared with 65.9 ± 6.9 nmol/L for the lower body weight/protein group. Thus, a 14 g higher protein intake resulted in 23 nmol/L lower plasma PLP concentration. There was no similar effect on other measures of vitamin B-6 status.

Furthermore, if plasma PLP concentrations of the body weight/protein groups are regressed separately on adjusted vitamin B-6 intake, the inverse prediction of EAR for the higher group is 1.3 mg/d compared with 1.0 mg/d for the lower group. When plasma PLP concentrations are regressed on the dietary vitamin B-6 to protein ratio, however, the predicted EAR is similar for both groups (0.016 and 0.017 mg/g for the lower and higher groups, respectively). These data suggest a significant effect of protein intake on plasma PLP concentration and the importance of considering dietary protein when establishing EAR and RDA for vitamin B-6. However, because the effects of increased body weight and increased protein intake cannot be separated in the current study, the possibility that increased body weight increases the vitamin B-6 requirement is an alternative explanation of this effect.

When we compared the fit of regression lines relating vitamin B-6 status measures to either adjusted vitamin B-6 intake or the dietary vitamin B-6 to protein ratio, for all measures except urinary 4-PA excretion, the r value was higher using the dietary vitamin B-6 to protein ratio. We then combined data from the current study and four other recent studies involving young subjects (3 ,5 6 7) to investigate whether plasma PLP concentration (Fig. 2A and B) and urinary 4-PA excretion (Fig. 3A and B) were more highly correlated with vitamin B-6 intake or the dietary vitamin B-6 to protein ratio, and to calculate an EAR and RDA based on the combined data using the values for adequate status suggested by Leklem (21) . Intakes were adjusted by converting supplemental vitamin B-6 to dietary vitamin B-6 equivalents. Means of plasma PLP concentration and urinary 4-PA excretion were weighted, using the number of subjects (n) as weights (total sum weights = 177 observations). Regression analysis of urinary 4-PA on vitamin B-6 intake and the dietary vitamin B-6 to protein ratio produced a better fit when urinary 4-PA data were transformed by taking the square root. For the combined data, regression on adjusted vitamin B-6 intake resulted in a better fit (higher r values) than regression on the dietary vitamin B-6 to protein ratio. The EAR of vitamin B-6 was determined to be 1.2 mg/d (0.015 mg/g protein) and 1.3 mg/d (0.015 mg/g protein) by inverse prediction using adequate values of plasma PLP and urinary 4-PA (21) , respectively. The RDA, assuming a CV in vitamin B-6 requirement of 10%, was calculated to be 1.4–1.6 mg/d (0.018 mg/g protein). The predicted EAR and RDA for vitamin B-6 from the current study (1.1 mg/d or 0.016 mg/g protein and 1.3 mg/d or 0.018 mg/g protein) agree well with the predicted EAR using the combined data.

View larger version (19K): [in this window] [in a new window]   Figure 2. Linear regression analysis of plasma pyridoxal phosphate (PLP) concentration vs. vitamin B-6 intake (A) and the dietary vitamin B-6 to protein ratio (B) from the current study and four other recent studies (3 ,5 6 7) of women consuming controlled diets with known amounts of vitamin B-6 and protein. Intakes were adjusted by converting supplemental vitamin B-6 to dietary vitamin B-6 equivalents [dietary vitamin B-6 equivalents = food vitamin B-6 + (1.27 x supplemental vitamin B-6)] (1) . Inverse regression calculations using adequate value (21) of PLP concentration predict an Estimated Average Requirement for vitamin B-6 of 1.2 mg/d and a dietary vitamin B-6 to protein ratio of 0.015 mg/g.

View larger version (19K): [in this window] [in a new window]   Figure 3. Linear regression analysis of the square root (sqrt) of urinary 4-pyridoxic acid (4-PA) excretion vs. vitamin B-6 intake (A) and the dietary vitamin B-6 to protein ratio (B) from the current study and four other recent studies (3 ,5 6 7) of women consuming controlled diets with known amounts of vitamin B-6 and protein. Intakes were adjusted by converting supplemental vitamin B-6 to dietary vitamin B-6 equivalents [dietary vitamin B-6 equivalents = food vitamin B-6 + (1.27 x supplemental vitamin B-6)] (1) . Inverse regression calculations using the sqrt of urinary 4-PA adequate value (21) predict an Estimated Average Requirement for vitamin B-6 of 1.3 mg/d and a dietary vitamin B-6 to protein ratio of 0.015 mg/g.

Another question to be considered is whether plasma PLP concentration is the most appropriate measurement to use as a standard for setting requirements. During abnormal conditions [e.g., acute phase of myocardial infarction (37) ] or treatment with drugs [e.g., theophylline (38) ], and normal life-cycle stages such as pregnancy (39) , plasma PLP concentration may not be indicative of vitamin B-6 status (40) . For the purpose of setting requirements for healthy nonpregnant people, however, using plasma PLP is appropriate because this status measure correlates significantly with vitamin B-6 intake and the dietary vitamin B-6 to protein ratio (Table 7) .

In the present study, urinary 4-PA excretion and erythrocyte PLP concentration were also strongly correlated with both vitamin B-6 intake and the dietary vitamin B-6 to protein ratio. The DRI Committee rejected using urinary 4-PA excretion for status assessment because 4-PA excretion responds rapidly to changes in dietary intake (8) , thus reflecting only recent intake (1) . Erythrocyte PLP has been suggested as a more relevant measure because the site of PLP coenzyme function is intracellular (40) . However, few studies assessing vitamin B-6 requirements have measured erythrocyte metabolites (5 ,6) ; consequently, adequate values for erythrocyte PLP concentration have not been established.

Erythrocyte aminotransferase activation by PLP (i.e., EALT and EAST activity coefficient or percentage stimulation) is useful as a long-term measure of vitamin B-6 status (21) , but its use in assessing requirements is limited in the present study because of the short length of the experimental periods. Although EAST basal activity showed a significant correlation with vitamin B-6 intake, it was paradoxically negative. One possible explanation is a lag time between changes in intake and changes in erythrocyte enzyme activity.

Plasma PL has also been suggested as an indicator of vitamin B-6 status (21) . In agreement with a previous study conducted in our laboratory (6) , plasma PL concentration was not significantly correlated with vitamin B-6 intake or the dietary vitamin B-6 to protein ratio. Therefore, it appears that plasma PL has limited usefulness as a measure of vitamin B-6 status.

Ideally, the best status measure to use when determining vitamin B-6 adequacy would be a functional measure related to a specific health outcome. In epidemiologic studies, low vitamin B-6 intake has been associated with increased risk of heart disease (41 42 43) and cancer (44 45 46 47) . Future research may further elucidate the mechanisms for these associations and help define a functional measure upon which recommendations for vitamin B-6 intake can be based. Until such a measure is determined, selecting adequate cut-off values for vitamin B-6 status measures will remain controversial.

In conclusion, predicting the EAR and RDA on the basis of adequate values of commonly measured status indicators calculated by the method suggested by Sauberlich (26) is the best approach available in the absence of status indicators linked to specific health outcomes (e.g., prevention of heart disease, cancer or other chronic diseases). The data presented here combined with previously published data suggest an EAR of 1.1–1.2 mg/d (0.015–0.016 mg/g protein) and an RDA of 1.5–1.7 mg/d (0.018–0.020 mg/g protein) for young women.

	ACKNOWLEDGMENTS

 
We gratefully acknowledge the technical assistance of Hedy Herrick, Jim Ridlington and Karin Hardin. We thank Marc Evans for his statistical advice, and the subjects for their invaluable cooperation.

	FOOTNOTES

 
¹ Presented in part at Experimental Biology 99 (Washington, DC) and 2000 (San Diego, CA) [Kwak, H. K., Hansen, C., Hardin, K., Ridlington, J., Leklem, J. E. & Shultz, T. D. (1999) A positive effect of vitamin B-6 on the immune response of young women. FASEB J. 13: A699 (abs.); Hansen, C., Kwak, H. K., Memon, H. S., Shultz, T. & Leklem, J. (1999) Plasma total homocysteine concentrations in women consuming four levels of vitamin B-6 intake. FASEB J. 13:A889 (abs.); Shultz, T. D., Hansen, C. M., Memon, H. S., Kwak, H. K. & Leklem, J. E. (2000) Urinary and erythrocyte vitamin B-6 (B6) status indicators of women consuming four levels of B6 intake. FASEB J. 14: A242 (abs.)].

² Supported by U.S. Department of Agriculture NRICGP grant #97–35200–4238.

⁴ Abbreviations used: DRI, Dietary Reference Intake; EALT, erythrocyte alanine aminotransferase; EAR, Estimated Average Requirement; EAST, erythrocyte aspartate aminotransferase; 4-PA, 4-pyridoxic acid ; PL, pyridoxal; PLP, pyridoxal phosphate; PMP, pyridoxamine phosphate; PN, pyridoxine; PNG, PN glucoside; RDA, Recommended Dietary Allowance.

Manuscript received November 17, 2000. Initial review completed January 4, 2001. Revision accepted March 8, 2001.

	REFERENCES

TOP ABSTRACT INTRODUCTION SUBJECTS AND METHODS RESULTS DISCUSSION REFERENCES

 

1. Institute of Medicine DRI Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B₆, Folate, Vitamin B₁₂, Pantothenic Acid, Biotin, and Choline 1998 National Academy Press Washington, DC

2. Brown R. R., Rose D. P., Leklem J. E., Linkswiler H., Anand R. Urinary 4-pyridoxic acid, plasma pyridoxal phosphate, and erythrocyte aminotransferase levels in oral contraceptive users receiving controlled intakes of vitamin B₆. Am. J. Clin. Nutr. 1975;28:10-19[Abstract/Free Full Text]

3. Hansen C. M., Leklem J. E., Miller L. T. Vitamin B-6 status of women with a constant intake of vitamin B-6 changes with three levels of dietary protein. J. Nutr. 1996;126:1891-1901

4. Hansen C. M., Leklem J. E., Miller L. T. Vitamin B-6 status indicators decrease in women consuming a diet high in pyridoxine glucoside. J. Nutr. 1996;126:2512-2518

5. Hansen C. M., Leklem J. E., Miller L. T. Changes in vitamin B-6 status indicators of women fed a constant protein diet with varying levels of vitamin B-6. Am. J. Clin. Nutr. 1997;66:1379-1387[Abstract/Free Full Text]

6. Huang Y. C., Chen W., Evans M. A., Mitchell M. E., Shultz T. D. Vitamin B-6 requirement and status assessment of young women fed a high-protein diet with various levels of vitamin B-6. Am. J. Clin. Nutr. 1998;67:208-220[Abstract]

7. Kretsch M. J., Sauberlich H. E., Skala J. H., Johnson H. L. Vitamin B-6 requirement and status assessment: young women fed a depletion diet followed by a plant- or animal-protein diet with graded amounts of vitamin B-6. Am. J. Clin. Nutr. 1995;61:1091-1101[Abstract/Free Full Text]

8. Lui A., Lumeng L., Aronoff G. R., Li T.-K. Relationship between body store of vitamin B₆ and plasma pyridoxal-P clearance: metabolic balance studies in humans. J. Clin. Lab. Med. 1985;106:491-497[Medline]

9. Lumeng L., Li T-K. Vitamin B₆ metabolism in chronic alcohol abuse. Pyridoxal phosphate levels in plasma and the effects of acetaldehyde on pyridoxal phosphate synthesis and degradation in human erythrocytes. J. Clin. Invest. 1974;53:693-704

10. Harris A. L. Determination of D-xylose in urine for the D-xylose absorption test. Clin. Chem. 1969;15:65-71[Abstract]

11. National Research Council Recommended Dietary Allowances 10th ed. 1989 National Academy Press Washington, DC.

12. Horwitz G.W. Official Methods of Analysis of the AOAC 13th ed. 1980:768 AOAC Washington, DC.

13. Kabir H., Leklem J. E., Miller L. T. Measurement of glycosylated vitamin B-6 in food. J. Food Sci. 1983;48:1422-1425

14. Pino S., Bennotte J., Gordnya H. An automated method for urine creatinine which does not require a dialyzer module. Clin. Chem. 1965;11:664-666[Abstract]

15. Gregory J. F., III, Kirk J. R. Determination of urinary 4-pyridoxic acid using high performance liquid chromatography. Am. J. Clin. Nutr 1979;32:879-883[Abstract/Free Full Text]

16. Miller L. T., Edwards M. Microbiological assay of vitamin B-6 in blood and urine. Leklem J. E. Reynolds R. D. eds. Methods in Vitamin B-6 Nutrition 1981:45-55 Plenum Press New York, NY.

17. Kwak H. K., Hansen C., Hardin K., Ridlington J., Leklem J. E., Shultz T. D. A positive effect of vitamin B-6 on the immune response of young women. FASEB J 1999;13:A699(abs.)

18. Bowers G. N., Jr, McComb R. B. Measurement of total alkaline phosphatase activity in human serum. Clin. Chem. 1975;21:1988-1995[Medline]

19. Sharma S. K., Dakshinamurti K. Determination of vitamin B6 vitamers and pyridoxic acid in biological samples. J. Chromatogr. 1992;578:45-51[Medline]

20. Woodring M. J., Storvick C. A. Effect of pyridoxine supplementation on glutamic-pyruvic transaminase and in vitro stimulation in erythrocytes of normal women. Am. J. Clin. Nutr. 1970;23:1385-1395[Medline]

21. Leklem J. E. Vitamin B-6: a status report. J. Nutr. 1990;120:1503-1507

22. Ott R.L. An Introduction to Statistical Methods and Data Analysis 4th ed. 1993 Wadsworth Publishing Company Belmont, CA.

23. Andon M. B., Reynolds R. D., Moser-Veillon P. B., Howard M. P. Dietary intake of total and glycosylated vitamin B-6 and vitamin B-6 nutritional status of unsupplemented lactating women and their infants. Am. J. Clin. Nutr. 1989;50:1050-1058[Abstract/Free Full Text]

24. Gregory J. F., Trumbo P. R., Bailey L. B., Toth J. P., Baumgartner T. B., Cerda J. J. Bioavailability of pyridoxine-5-ß-D-glucoside determined in humans by stable isotope methods. J. Nutr. 1991;121:177-186

25. Whyte M. P., Mahuren J. D., Vrabel L. A., Coburn S. P. Markedly increased circulating pyridoxal 5'-phosphate levels in hypophosphatasia: alkaline phosphatase acts in vitamin B-6 metabolism. J. Clin. Invest. 1985;76:752-756

26. Sauberlich H. E. Introduction. Laboratory Tests for the Assessment of Nutritional Status 2nd ed. 1999:3-8 CRC Press Boca Raton, FL.

27. Hansen C. M. Effect of Vitamin B-6 Intake, Protein Intake and Bioavailability on Vitamin B-6 Status of Women. Doctoral thesis 1995 Oregon State University Corvallis, OR

28. Shultz T. D. Comparative Nutrient Intake and Biochemical Interrelationships among Healthy Vegetarian and Nonvegetarian Seventh-Day Adventists, Nonvegetarians, and Hormone Dependent Cancer Subjects 1980 Doctoral thesis Oregon State University, Corvallis, OR.

29. Shultz T. D., Leklem J. E. Urinary 4-pyridoxic acid, urinary vitamin B₆, and plasma pyridoxal phosphate as measures of vitamin B₆ status and dietary intake in adults. Leklem J. E. Reynolds R. D. eds. Methods in Vitamin B₆ Nutrition 1981:297-320 Plenum Press New York, NY.

30. Meydani S. N., Ribaya-Mercado J. D., Russell R. M., Sahyoun N., Morrow F. D., Gershoff S. N. Vitamin B-6 deficiency impairs interleukin 2 production and lymphocyte proliferation in elderly adults. Am. J. Clin. Nutr. 1991;53:1275-1280[Abstract/Free Full Text]

31. Ribaya-Mercado J. D., Russell R. M., Sahyoun N., Morrow F. D., Gershoff S. N. Vitamin B-6 requirements of elderly men and women. J. Nutr. 1991;121:1062-1074

32. Baker E. M., Canham J. E., Nunes W. T., Sauberlich H. E., McDowell M. E. Vitamin B₆ requirement for adult men. Am. J. Clin. Nutr. 1964;15:59-66[Abstract]

33. Canham J. E., Baker E. M., Harding R. S., Sauberlich H. E., Plough I. C. Dietary protein—its relationship to vitamin B6 requirements and function. Ann. N.Y. Acad. Sci. 1969;166:16-29[Medline]

34. Miller L. T., Leklem J. E., Shultz T. D. The effect of dietary protein on the metabolism of vitamin B-6 in humans. J. Nutr. 1985;115:1663-1672

35. Miller L. T., Linkswiler H. Effect of protein intake on the development of abnormal tryptophan metabolism by men during vitamin B-6 depletion. J. Nutr. 1967;93:53-59

36. Pannemans D.L.E., van den Berg H., Westerterp K. R. The influence of protein intake on vitamin B-6 metabolism differs in young and elderly humans. J. Nutr. 1994;124:1207-1214

37. Vermaak V.J.H., Barnard H. C., Van Daelen E.M.S.P., Potgieter G. M., Van Jaarsveld H., Myburgh S.J.S. Compartmentalization of pyridoxal-5'-phosphate during the acute phase of myocardial infarction. Klin. Wochenschr. 1988;66:428-433[Medline]

38. Ubbink J. B., Delport R., Becker P. J., Bissport S. Evidence of a theophylline induced vitamin B-6 deficiency due to non-competitive inhibition of pyridoxal kinase. J. Lab. Clin. Med. 1989;113:15-22[Medline]

39. Barnard H. C., De Kock J. J., Vermaak W.J.H., Potgieter G. M. A new perspective in the assessment of vitamin B-6 status during pregnancy. J. Nutr. 1987;117:1303-1306

40. Vermaak V.J.H., Ubbink J. B., Barnard H. C., Potgieter G. M., van Jaarsveld H., Groenwald A. J. Vitamin B-6 nutrition status and cigarette smoking. Am. J. Clin. Nutr. 1990;51:1058-1061[Abstract/Free Full Text]

41. Robinson K., Mayer E. L., Miller D. P., Green R., van Lente F., Gupta A., Kottke-Marchant K., Savon S. R., Selhub J., Nissen S. E., Kutner M., Topol E. J., Jacobsen D. W. Hyperhomocysteinemia and low pyridoxal phosphate: common and independent reversible risk factors for coronary artery disease. Circulation 1995;92:2825-2830[Abstract/Free Full Text]

42. Verhoef P., Stampfer M. J., Buring J. E., Gaziano J. M., Allen R. H., Stabler S. P., Reynolds R. D., Kok F. J., Hennekens C. H., Willett W. C. Homocysteine metabolism and risk of myocardial infarction: relation with vitamins B6, B12, and folate. Am. J. Epidemiol. 1995;143:845-859[Abstract/Free Full Text]

43. Rimm E. B., Willett W. C., Hu F. B., Sampson L., Colditz G. A., Manson J. E., Hennekens C., Stampfer M. J. Folate and vitamin B-6 from diet and supplements in relation to risk of coronary heart disease among women. J. Am. Med. Assoc. 1998;279:359-364[Abstract/Free Full Text]

44. Slattery M. L., Potter J. D., Coates A., Ma K. N., Berry T. D., Duncan D. M., Caan B. J. Plant foods and colon cancer: an assessment of specific foods and their related nutrients (United States). Cancer Causes Control 1997;8:575-590[Medline]

45. Key T. J., Silcocks P. B., Davey G. K., Appleby P. N., Bishop D. T. A case-control study of diet and prostate cancer. Br. J. Cancer 1997;76:678-687[Medline]

46. Kaaks R., Tuyns A. J., Haelterman M., Riboli E. Nutrient intake patterns and gastric cancer risk: a case-control study in Belgium. Int. J. Cancer 1998;78:415-420[Medline]

47. Stolzenberg-Solomon R. Z., Albanes D., Nieto F. J., Hartman T., Tangrea J. A., Rautalahti M., Selhub J., Virtamo J., Taylor P. R. Pancreatic cancer risk and nutrition-related methyl-group availability indicators in male smokers. J. Natl. Cancer Inst. 1999;91:535-541[Abstract/Free Full Text]

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