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Department of Biology, National Cheng Kung University, Tainan, Taiwan and * School of Nutrition, Chung Shan Medical University, Taichung, Taiwan
2To whom correspondence should be addressed. E-mail: sjchang{at}mail.ncku.edu.tw.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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30 nmol/L, indicating an adequate vitamin B-6 status. Daily dietary vitamin B-6 intakes of boys and girls were 0.80 ± 0.16 and 0.74 ± 0.16 mg/d, respectively. Daily dietary vitamin B-6 intakes of children who had adequate urinary 4-pyridoxic acid (4-PA) (>3.0 µmol/L), erythrocyte alanine aminotransferase activity coefficient (EALT-AC) (<1.25) and aspartate aminotransferase activity coefficient (EAST-AC) (<1.8) were not different from those of children who had adequate plasma PLP, although the percentages of adequacy for urinary 4-PA, EALT-AC and EAST-AC ranged from 20 to 91%. Vitamin B-6 status indicators were strongly correlated with vitamin B-6 intake. Adequate values of PLP, EALT-AC, EAST-AC and urinary 4-PA were used to determine the EAR according to Dietary Reference Intake (DRI) committee methodology. We determined the vitamin B-6 EAR (RDA) for boys and girls aged 7–12 y to be 0.84 (1.01) and 0.75 (0.89) mg/d, respectively.
KEY WORDS: • vitamin B-6 • nutritional status • Estimated Average Requirement • Recommended Dietary Allowance • school children
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). However, due to a lack of vitamin B-6 content in food composition tables, this nationwide survey did not provide vitamin B-6 intake results (2
). The nutritional risk group was suggested to be selected from the nationwide surveys to find deviations from the overall population (3
).
In 1998, the scientific evaluation of Dietary Reference Intakes (DRI) indicated that the Estimated Average Requirement (EAR) and Recommended Dietary Allowance (RDA) for children and adolescents aged 1–18 y were extrapolated from adult values due to limited information (4
). Therefore, the EAR and RDA of vitamin B-6 for children aged 7–12 y were studied first in Taiwan and those for the 13–18 y age group are currently on-going in our laboratory.
The study of dietary intake, nutritional status and functional consequences of a certain range of intake is essential in determining nutrient requirements (5
). This study was undertaken to investigate the recommendations of vitamin B-6 intake for children aged 7–12 y. We evaluated the effect of vitamin B-6 intake on adequate vitamin B-6 status indicators in plasma, erythrocyte and urine of children determined to be healthy by anthropometric measurements. The EAR and RDA were estimated using the DRI committee’s methodology (4
).
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Healthy elementary school children (102 boys and 111 girls) in Tainan, the southern part of Taiwan, voluntarily participated in this study during the fall of 1999. Informed consent was obtained from both the students and their parents. Students who were not in good health, had any diseases interfering with vitamin B-6 metabolism or taking medicines and/or supplements were not included in the study. A total of 168 children (79 boys and 89 girls) finished the study. The rest of the children withdrew and had only anthropometric measurements. The study protocol was approved by the Department of Health, Executive Yuan, Taiwan, and was explained to the parents before they gave their informed consent.
Dietary intake assessment.
A trained dietary interviewer obtained food intake information from the subjects via three 24-h dietary recalls at the beginning and the end of the semester which is 
5 mo. One recall was for a weekend day and the other two for weekdays. A computer program, Nutritional IV (6
), was used to calculate the daily energy and nutrient intakes.
Anthropometric measurements.
Anthropometric measurements including height, weight, midarm circumference (MAC), triceps skinfold thickness (TSF) and the percentage of body fat were made at the beginning and end of the semester. Body weight and height were taken with subjects wearing school uniform but without shoes. Body weight and the percentage of body fat were measured simultaneously using a Body Fat Monitor/Scale (TBF-531, Tanita, Tokyo, Japan). Weight and height measurements were used to calculate the body mass index as weight (kg)/height2 (m2). Midarm muscle circumference was calculated from the MAC. TSF was measured by skinfold calipers (Lafayette Instrument, Lafayette, IN). Anthropometric data were compared with those of NAHSIT, 1993–1996 (7
) for the appropriate age group.
Sample collection.
Venous blood was collected from fasting subjects in vacutainer tubes containing EDTA between 0800 and 0900 h at the beginning and end of the semester. The samples were kept in crushed ice and protected from light. Blood samples were centrifuged at 3000 xg, 4°C for 10 min. Plasma was removed and aliquots frozen at -40°C for plasma PLP analyses. Erythrocytes were washed three times with saline, and an aliquot of packed cells was removed for assay of erythrocyte alanine aminotransferase activity coefficient (EALT-AC) and aspartate aminotransferase activity coefficient (EAST-AC) according to the method of Woodring and Storvick (8
). A 24-h urine collection was obtained, using toluene as a preservative, on the same day as the blood sample collection. Aliquots of urine were stored at -40°C for urinary 4-pyridoxic acid (4-PA) analyses.
Laboratory analysis.
Plasma PLP concentrations were determined by HPLC with fluorometric detection (9
). The recovery (mean ± SD) of added PLP from plasma was 107.5 ± 4.2%. Within- and between-day reproducibilities were 4.6 and 4.2%, respectively. Erythrocyte alanine and aspartate aminotransferase activities (EALT and EAST) were measured with and without added PLP (8
). The EALT and EAST were assayed the same day blood was drawn. The EALT and EAST activity coefficients (EALT-AC and EAST-AC) were calculated as the ratio of stimulated (PLP added) to unstimulated (no PLP added) activities. Urinary 4-PA was analyzed by HPLC with fluorometric detection (10
). The recovery (mean ± SD) of added 4-PA from urine was 101.2 ± 2.1%. Within- and between-day reproducibilities were 4.8 and 4.6%, respectively.
Statistical analyses.
Data were analyzed using SAS statistical analysis computer program (version 6.12; SAS Institute, Cary, NC). Results were expressed as means ± SD unless otherwise stated. The general linear model was performed to determine the differences between group means at the beginning and end of the semester, and between boys and girls for daily dietary intakes and vitamin B-6 status measures. One-way ANOVA was used to test the differences among the means of vitamin B-6 intakes of children who had adequate vitamin B-6 status indicators. Pearson correlation coefficients were computed to determine relationships among vitamin B-6 status measures and vitamin B-6 intakes. The level of statistical significance was considered at P < 0.05. The percentages of subjects with adequate (11
) plasma PLP, EALT-AC, EAST-AC and urinary 4-PA were calculated. Adequate (11
) status indicators were used to calculate estimates and 95% confidence intervals of B-6 EAR (4
). RDA were calculated using the formula RDA = 1.2 x EAR, which assumes an EAR CV of 10% (4
).
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Anthropometric measurements were made at the beginning and the end of the semester and averaged to determine the semester data for each child (Table 1
). Most data were similar to those of NAHSIT, 1993–1996 (7
), indicating that the children were within normal ranges and were healthy.
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Dietary information obtained at the beginning and end of the semesters was averaged to determine the semester daily intakes. The majority of children reported that these were typical of their usual intakes. Energy, protein and vitamin B-6 intakes did not differ between boys and girls or from the beginning to the end of the semester (Table 2
).
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Plasma PLP concentration, EALT-AC, EAST-AC and urinary 4-PA excretion measured at the beginning and end of the semester were averaged to obtain the individual biochemical indicators of vitamin B-6 (Table 3
). Plasma PLP concentrations for all children were 30 nmol/L (Table 4
), indicating adequate vitamin B-6 status (11
), and meeting the more conservative cut-off value of 20 nmol/L for EAR (4
). The percentages of adequate vitamin B-6 status evaluated by EALT-AC < 1.25, EAST-AC < 1.8 and urinary 4-PA excretion > 3.0 µmol/d (11
) were quite different (Table 4)
. However, the vitamin B-6 intakes of the children who had adequate values of the different indicators were not different from one another in both boys and girls (Table 4)
.
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Vitamin B-6 intake and the dietary vitamin B-6:protein ratio were positively correlated with plasma PLP and urinary 4-PA, and negatively correlated with EALT-AC and EAST-AC (Table 5
). Plasma PLP was positively correlated with urinary 4-PA and negatively correlated with both EALT-AC and EAST-AC (Table 5)
. Urinary 4-PA excretion was also negatively correlated with both EALT-AC and EAST-AC (Table 5)
. EAST-AC was positively correlated with EALT-AC (Table 5)
.
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The EAR of vitamin B-6 was calculated from the daily intake of half of the healthy children who had adequate plasma PLP concentration, EALT-AC, EAST-AC and urinary 4-PA excretion (Table 6
). The mean intake of four vitamin B-6 status indicators resulted in EAR of 0.84 and 0.75 mg/d for boys and girls, respectively. The RDA of 1.01 and 0.89 mg/d for boys and girls, respectively, were calculated according to DRI committee methodology (4
). The dietary vitamin B-6:protein ratio was also calculated as a reference (Table 7
), although it was not recommended for determining the EAR by the DRI committee (4
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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20 nmol/L), urinary 4-PA excretion (>3.0 µmol/L), EALT-AC (<1.25) and EAST-AC (<1.8). The vitamin B-6 RDA of 1.01 and 0.89 mg/d for boys and girls aged 7–12 y, respectively, were calculated from the EAR according to the DRI committee methodology (4
). These values were comparable with those of the DRI committee, which determined a vitamin B-6 EAR of 0.8 mg/d and a RDA of 1.0 mg/d for children aged 9–13 y; these values were extrapolated from adult values due to the limited information (4
). Most earlier studies evaluated the inadequacy of vitamin B-6 status by erythrocyte alanine aminotransferase (
-EALT) ratios (12
–14
). However, inconsistency between the -EALT ratios and clinical indicators of deficiency, and considerable variation in -EALT ratios (11
) limit their usefulness as status indicators to derive the EAR from mean vitamin B-6 intakes in these studies. Recently, plasma PLP, urinary 4-PA, at least one indirect measure, and the intakes of vitamin B-6 and protein have been recommended for proper assessment of vitamin B-6 status (11
). We determined the vitamin B-6 EAR using a combination of plasma PLP, urinary 4-PA, EALT-AC, EAST-AC, and the intake of vitamin B-6.
Plasma PLP is suggested to be the best single indicator (4
,15
) because it appears to reflect tissue stores. We found that the plasma PLP concentration of every child was >30 nmol/L, indicating adequate status (11
). However, a cut-off value of 20 nmol/L for plasma PLP was used as a criterion to calculate the EAR (4
) for boys and girls aged 7–12 y in this study. The intakes of vitamin B-6 for boys and girls were 0.80 ± 0.16 and 0.74 ± 0.16 mg/d, respectively, which were similar to EAR of 0.84 and 0.75 mg/d determined with four vitamin B-6 status indicators.
Recently, plasma PLP concentrations were found to be 59 ± 33 nmol/L, in 39 Indonesian boys, and 50 ± 24 nmol/L, in 38 Indonesian girls aged 8–9 y with vitamin B-6 intakes of 0.62 and 0.53 mg/d, respectively (16
). However, plasma PLP concentrations 
30 nmol/L and vitamin B-6 intakes <0.5 mg/d (the 1998EAR for those 4–8 y) were observed in 25 and 55% of these children (16
), indicating that vitamin B-6 intakes of 0.62 and 0.53 mg/d for boys and girls, respectively, were inadequate. The median intakes of vitamin B-6 for boys and girls participating in Young Hearts cohort at age 12 y were 1.45 and 1.28 mg/d, respectively. Unfortunately, the vitamin B-6 status of these children was not available (17
). Plasma vitamin B-6 concentrations were 194.2 ± 43.8 and 197.2 ± 40.3 nmol/L in Nigerian boys and girls aged 10–12 y, respectively, with vitamin B-6 intakes of 1.52 ± 0.4 and 1.54 ± 0.24 mg/d. Approximately 30% of these children had EALT-AC > 1.25 with a plasma vitamin B-6 concentration of 150 ± 28.4 nmol/L (<150 nmol/L is considered inadequate), indicating that the high vitamin B-6 consumed by these children is of low biological availability because of diets high in plant proteins (18
). Thus, vitamin B-6 requirements of boys and girls for this age group should be <1.52 and 1.54 mg/d, respectively. The protein intake of children in the present study was only slightly above the Recommended Daily Nutrient Allowances (RDNA; 50 g/d) for the age group (19
). In addition, the vitamin B-6 intake was correlated with plasma PLP (r = 0.828, P < 0.001). In combination with the finding of adequate plasma PLP (>30 nmol/L) for every child in the present study, vitamin B-6 intakes of 0.80 ± 0.16 and 0.74 ± 0.16 mg/d for boys and girls, respectively, appeared to be adequate.
Urinary 4-PA excretion reflects vitamin B-6 intake (15
), is a short-term indicator of vitamin B-6 status and is considered adequate at > 3.0 µmol/d (11
). In the present study, 77% of boys and 69% of girls had urinary 4-PA excretion > 3.0 µmol/d with vitamin B-6 intakes of 0.82 ± 0.17 and 0.77 ± 0.16 mg/d, respectively. These values were also comparable with those evaluated by the adequacy of plasma PLP reported in the present study.
EALT-AC and EAST-AC are commonly used as measures of long-term vitamin B-6 status. In the present study, percentages of adequate EALT-AC for boys and girls were higher than those of adequate EAST-AC (boys: 91 vs. 27%; girls: 85 vs. 20%). These results confirmed another study that EALT-AC is a better indicator of vitamin B-6 status than EAST-AC (11
). For those who had adequate EALT-AC and EAST-AC, mean daily dietary vitamin B-6 intakes ranged from 0.73 ± 0.16 to 0.80 ± 0.17 mg/d, which were also comparable with those that had adequate plasma PLP. A recent study indicated that EALT-AC of Indonesian boys and girls aged 8–9 y were 1.21 ± 0.16 and 1.20 ± 0.15, with vitamin B-6 intakes of 0.62 ± 0.25 and 0.53 ± 0.22 mg/d. However, 30% of these children had inadequate EALT-AC (1.25) (16
), indicating that 0.62 and 0.53 mg/d of vitamin B-6 were inadequate. Driskell et al. (20
) reported that EALT activities were 11.5 and 14.0% in Caucasian and African-American girls aged 12 y with vitamin B-6 intakes of 1.23 ± 0.08 and 1.30 ± 0.08 mg/d. Coenzyme stimulation of EALT activity of >25% was used as a criterion for determining inadequate vitamin B-6 status in their study (20
). Therefore, vitamin B-6 intakes of 1.23 and 1.30 mg/d may be above the requirement.
Vitamin B-6 intake and status indicators were correlated in the present study. In addition, direct biomarkers of vitamin B-6 intake (plasma PLP and urinary 4-PA excretion) were significantly related to functional indicators (EALT-AC and EAST-AC). Therefore, vitamin B-6 intakes resulted in the adequacies of these direct and functional indicators were used to determine the vitamin B-6 requirement of children aged 7–12 y in this study.
In conclusion, EAR and RDA were calculated on the basis of adequate values of plasma PLP, urinary 4-PA, EALT-AC and EAST-AC using the DRI committee methodology (4
). We combined the four indicators and determined EAR of 0.84 and 0.75, and RDA of 1.01 and 0.89 mg/d for boys and girls ages 7–12 y, respectively.
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FOOTNOTES |
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3 Abbreviations used: DRI, Dietary Reference Intake; EALT-AC, erythrocyte alanine aminotransferase activity coefficient; EAR, Estimated Average Requirement; EAST-TC, erythrocyte aspartate aminotransferase activity coefficient; MAC, midarm circumference; NAHSIT, Nutrition and Health Survey in Taiwan; 4-PA, 4-pyridoxic acid; PLP, pyridoxal phosphate; RDA, Recommended Dietary Allowance; TSF, triceps skinfold thickness.
Manuscript received 26 April 2002. Initial review completed 5 June 2002. Revision accepted 12 July 2002.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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1. Chang, S.-J., Fan, H.-J., Yeh, W.-T. & Pan, W.-H. (1999) Vitamin B-6 status in Taiwanese population from the Nutrition and Health Survey in Taiwan (NAHSIT) 1993–1996. NAHSIT (rev. version) 1999:195-203 Executive Yuan, Department of Health Taiwan, ROC. .
2. Pan, W.-H., Chang, Y.-H., Chen, J.-Y., Wu, S.-J., Tzeng, M.-S. & Kao, M.-D. (1999) Nutrition and Health Survey in Taiwan (NAHSIT) 1993–1996: dietary nutrient intakes assessed by 24-hour recall. Nutr. Sci. J. 24:11-40.
3. Lowik, M.R.H., Brants, H.A.M., Brussaard, J. H., van den Berg, H., Kistemaker, C. & Hermus, R.J.J. (1997) Rational, design and methods of a study on micronutrient status among adults. Eur. J. Clin. Nutr. 51:S4-S11.
4. Institute of Medicine (1998) DRI Reference Intakes for Thiamin, Riboflavin, Niacin, Vitamin B-6, Folate, Vitamin B-12, Pantothenic Acid, Biotin, and Choline 1998 National Academy Press Washington, DC. .
5. King, J. (1996) The need to consider functional endpoints in defining nutrient requirements. Am. J. Clin. Nutr. 63:983S-984S.
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7. Kao, M-D., Tzeng, M-S., Yeh, W-T., Cheng, Y-S. & Pan, W-H. (1999) Nutrition and Health Survey in Taiwan (NAHSIT) 1993–1996: anthropometric measurements and prevalence of obesity NAHSIT (rev. version) 1999:145-163 Executive Yuan, Department of Health Taiwan, ROC .
8. Woodring, M. J. & Storvick, C. A. (1970) Effect of pyridoxine supplementation on glutamic-pyruvic transaminase and in vivo stimulation in erythrocytes of normal women. Am. J. Clin. Nutr. 23:1385-1395.[Medline]
9. Kimura, M., Kanehira, K. & Yokoi, K. (1996) Highly sensitive and simple liquid chromatographic determination in plasma of B-6 vitamers, especially pyridoxal-5'-phosphate. J. Chromatogr. A. 722:269-301.
10. Gregory, J. F., III & Kirk, J. R. (1979) Determination of urinary 4-pyridoxic acid using high performance liquid chromatography. Am. J. Clin. Nutr. 32:879-883.
11. Leklem, J. E. (1990) Vitamin B-6: a status report. J. Nutr. 120:1503-1507.
12. Kirksey, A., Keaton, K., Abernathy, R. P. & Greger, J. L. (1978) Vitamin B-6 nutritional status of a group of female adolescents. Am. J. Clin. Nutr. 31:946-954.
13. Driskell, J. A., Clark, A. J., Bazzarre, T. L., Chopin, L. F., McCoy, H., Kenney, M. A. & Moak, S. W. (1985) Vitamin B-6 status of southern adolescent girls. J. Am. Diet. Assoc. 85:46-49.[Medline]
14. Driskell, J. A., Clark, A. J. & Moak, S. W. (1987) Longitudinal assessment of vitamin B-6 status in southern adolescent girls. J. Am. Diet. Assoc. 87:307-310.[Medline]
15. Lui, A., Lumeng, L., Aronoff, G. R. & Li, T.-K. (1985) Relationship between body store of vitamin B-6 and plasma pyridoxal-P clearance: metabolic balance studies in humans. J. Lab. Clin. Med. 106:491-497.[Medline]
16. Setiawan, B., Giraud, D. W. & Driskell, J. A. (2000) Vitamin B-6 inadequacy is prevalent in rural and urban Indonesian children. J. Nutr. 130:553-558.
17. Robson, P. J., Gallagher, A. M., Livingstone, M.B.E., Cran, G. W., Strain, J. J., Savage, J. M. & Boreham, C.A.G. (2000) Tracking of nutrient intakes in adolescence: the experiences of the Young Hearts Project, Northern Ireland. Br. J. Nutr. 84:541-548.[Medline]
18. Korede, K. & Ajayi, O. A. (1991) Plasma vitamin B-6 concentrations in Nigerian adolescents. Am. J. Clin. Nutr. 45:111-115.
19. Executive Yuan, Department of Health (1993) Recommended Daily Nutrient Allowances (RDNA) 5th ed. 1993 Taipei Taiwan, ROC. .
20. Driskell, J. A. & Moak, S. W. (1986) Plasma pyridoxal phosphate concentrations and coenzyme stimulation of erythrocyte alanine aminotransferase activities of white and black adolescent girls. Am. J. Clin. Nutr. 43:599-603.
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