European Journal of Clinical Nutrition (2010) 64, 800–807
& 2010 Macmillan Publishers Limited All rights reserved 0954-3007/10
www.nature.com/ejcn
ORIGINAL ARTICLE
Twenty-four-hour urinary water-soluble vitamin
levels correlate with their intakes in free-living
Japanese university students
T Tsuji1,2, T Fukuwatari2, S Sasaki3 and K Shibata2
1
Department of Health and Nutrition, School of Health and Human Life, Nagoya Bunri University, Aichi, Japan; 2Department of Food
Science and Nutrition, Graduate School of Human Cultures, The University of Shiga Prefecture, Shiga, Japan and 3Department of Social
and Preventive Epidemiology, School of Public Health, The University of Tokyo, Tokyo, Japan
Background/Objectives: We examined the association between 24-h urinary excretion of water-soluble vitamin levels and their
intakes in free-living Japanese university students. The design used was cross-sectional study.
Subjects/Methods: A total of 216 healthy, free-living male and female Japanese university students aged 18–27 years
voluntarily participated in this study, of which 156 students were eligible for this assessment. All foods consumed for
4 consecutive days were recorded accurately by a weighed food record method. A 24-h urine sample was collected on the
fourth day, and the urinary levels of water-soluble vitamins were measured.
Results: Each urinary water-soluble vitamin level, except for vitamin B12, was correlated positively with its mean intake in the
recent 2–4 days (vitamin B1: r ¼ 0.42, Po0.001; vitamin B2: r ¼ 0.43, Po0.001; vitamin B6: r ¼ 0.40, Po0.001; vitamin B12:
r ¼ 0.06, P ¼ 0.493; niacin: r ¼ 0.35, Po0.001; niacin equivalents: r ¼ 0.33, Po0.001; pantothenic acid: r ¼ 0.47, Po0.001;
folate: r ¼ 0.27, P ¼ 0.001; vitamin C: r ¼ 0.44, Po0.001). Mean estimated water-soluble vitamin intakes calculated from urinary
levels and recovery rates showed 91–101% of their 3-day mean intakes, except for vitamin B12 (61%).
Conclusions: These results showed that urinary water-soluble vitamin levels, except for vitamin B12, reflect their recent intakes in
free-living Japanese university students, and could be used as a potential biomarker to estimate mean vitamin intake.
European Journal of Clinical Nutrition (2010) 64, 800–807; doi:10.1038/ejcn.2010.72; published online 26 May 2010
Keywords: urinary water-soluble vitamins; biomarker; vitamin intake; free living
Introduction
To assess the nutritional status of healthy free-living human
beings, a weighed food record method has been used widely
to record the dietary intake and calculate nutrient intake
(Willett, 1998). Although this method can provide relatively
precise information regarding the dietary intake compared
Correspondence: Dr K Shibata, Department of Food Science and Nutrition,
Graduate School of Human Cultures, The University of Shiga Prefecture, 2500
Hassaka, Hikone, Shiga 522-8533, Japan.
E-mail: [email protected]
Contributors: TT designed the study, performed the experiments, completed
the statistical analysis, and prepared the manuscript. TF helped to design the
study, performed the experiments, and assisted with data analysis. SS
reviewed the study and assisted with data analysis. KS contributed to the
study design and supervised the study. All authors critically reviewed the
manuscript.
Received 7 April 2009; revised 27 December 2009; accepted 11 January 2010;
published online 26 May 2010
with other dietary assessments (Bingham et al., 1997),
substantial effort is required for respondents to complete
the dietary records and to weigh all food consumed. This
often leads to errors in the records, which reveal the
limitation of a weighed food record method in terms of
accuracy (Livingstone and Black, 2003). Alternatively, other
methods using quantitative biological information, such as
urinary excretion or concentrations of nutrients or their
metabolites in plasma or erythrocytes, as biomarkers to
assess dietary intake or nutritional status have been well
studied in recent years.
Many preceding studies have investigated urinary excretion as a biomarker for assessing dietary intake. For example,
24-h urinary nitrogen is established as a marker for protein
intake (Bingham, 2003), urinary potassium for potassium
intake (Tasevska et al., 2006), urinary sugars for sugar
intake (Luceri et al., 1996; Bingham et al., 2007; Tasevska
et al., 2005, 2008), and urinary thiamine for thiamine intake
Correlation of urinary vitamins and their intakes
T Tsuji et al
801
(Tasevska et al., 2007). These studies can be classified into
two categories in terms of whether or not an intervention
was used. Many studies have been performed under a strictly
controlled environment with interventions, but few in a
free-living environment. In the latter case, for example,
Chang et al. (2007) have reported that urinary 4-pyridoxic
acid (4-PIC), a metabolite of vitamin B6, reflects current
intake in free-living high-school students.
Vitamin deficiencies cause various disorders; therefore,
a method to evaluate vitamin intake easily and accurately
can be used for early screening at a primary preventive
stage. Methods using a biomarker for vitamin intake offer
an effective approach to evaluate vitamin status. Recently,
we have reported that urinary water-soluble vitamin levels
are correlated highly with their intake in a strictly controlled
environment with interventions (Shibata et al., 2005;
Fukuwatari and Shibata, 2008). Performance of a study
under a free-living environment without any interventions
is the next step to confirm the applicability of methods using
a biomarker.
In this study, we examined the association between
urinary water-soluble vitamins and their intakes in freeliving individuals. To measure dietary intake precisely, we
used a weighed food record method, which was shown to
be of the highest quality in Japan at this time, and based
on extensive research (Sasaki et al., 2003; Murakami et al.,
2006). This is believed to be the first study to show that
seven urinary water-soluble vitamin levels are correlated
with their intakes in free-living Japanese university students
aged 18–27 years.
Materials and methods
Participants
A total of 216 healthy free-living male and female university
dietetics students aged 18–27 years voluntarily participated
in this study. The purpose and protocol of this study was
explained to all participants before joining the study, and
written informed consent was obtained from each participant, and from the parents of participants aged o20 years.
We excluded participants diagnosed with cold or influenza,
and those who had taken multi-vitamin supplements
at least once during the earlier month. In addition, we
excluded participants whose 24-h urine collection or
dietary records were considered as incomplete, with a
collection time outside the 22–26-h range, urine volume
o250 ml, creatinine excretion in relation to body weight
outside the 10.8–25.2 mg/kg range (Stamler et al., 2003;
Murakami et al., 2007), or extremely low or high energy
intake (o2092 or 416 736 kJ/day) (Ministry of Health,
Labour, and Welfare of Japan, 2005). After these screenings, 156 participants (26 male and 130 female) were
found to be eligible. This study was reviewed and approved
by The Ethical Committee of The University of Shiga
Prefecture.
Dietary records
This was a 4-day dietary assessment in which the participants
were living freely in college life and consuming their normal
diet. The first day (Monday) of the experimental period was
defined as Day 1, the second day as Day 2, the third day as
Day 3, and the fourth day as Day 4. All food consumed
during the 4-day period was recorded using a weighed food
record method (Imai et al., 2000). A digital cooking scale
(1 g unit; Tanita Inc. Japan), a set of dietary record forms,
a dietary record manual, and a disposable camera were
distributed to the participants in advance. On entry of the
dietary record, the status of food at oral intake was identified
as ‘raw,’ ‘boiled,’ ‘cooked,’ ‘the presence of skin,’ ‘a part
of cooking ingredients,’ or ‘with or without seasoning,’ and
coded according to the Fifth Revised and Enlarged Edition of
the Standard Tables of Food Composition in Japan (Ministry
of Education, Culture, Sports, Science and Technology,
2007). The participants took photographs with a disposable
camera of the dishes before and after eating. Several experienced dietitians used the photographs to complete the data,
and asked the participants to resolve any discrepancies or
to obtain further information when needed. The food that
remained after eating was measured by a digital scale and
was deducted from the dietary record. Food, nutrient, and
energy intakes were calculated using SAS statistical software,
version 6.12 (SAS Institute, Cary, NC, USA), based on the
Standard Tables of Food Composition in Japan. For vitamin
intake, eight water-soluble vitamins, vitamin B1, vitamin
B2, vitamin B6, vitamin B12, niacin, pantothenic acid, folate,
and vitamin C, were assessed; biotin was excluded because
it was not designated in the current Standard Table of
Food Composition in Japan. Niacin is synthesized from
tryptophan; therefore, the amount of niacin equivalent was
handled separately from niacin. One milligram of nicotinamide is synthesized from 60 mg tryptophan (Fukuwatari
et al., 2004a); therefore, niacin equivalent was calculated as
sum of niacin and 1/60 tryptophan intakes.
Twenty-four-hour urine sampling
A single 24-h urine sample was collected on the fourth day to
measure urinary water-soluble vitamins and their metabolites. The urine samples were kept cold in a refrigerator after
collection to avoid degradation of water-soluble vitamins.
In the morning, participants were asked to discard the first
specimen and to record the time on the sheet. The next
morning, participants were asked to collect the last specimen
at the same time as when the specimen was discarded
the previous morning and to record the time on the sheet.
After the urine sample was collected, the volume of the
sample was measured. Aliquots of the urine were stabilized
to avoid destruction of water-soluble vitamins and their
metabolites. For analysis of urinary thiamin, riboflavin,
4-PIC, N1-methylnicotinamide, N1-methyl-2-pyridone-5carboxamide, and N1-methyl-4-pyridone-3-carboxamide,
1 ml of 1 mol/l HCl was added to 9 ml urine. For analysis
European Journal of Clinical Nutrition
Correlation of urinary vitamins and their intakes
T Tsuji et al
802
of urinary pantothenic acid and biotin, urine samples were
not treated. For analysis of urinary folic acid, 1 ml of 1 mol/l
ascorbic acid was added to 9 ml urine. For analysis of
urinary ascorbic acid as the sum of reduced ascorbic acid,
oxidized ascorbic acid, and 2,3-diketogluconic acid, 4 ml of
10% free metaphosphoric acid was added to 4 ml urine. All
treated urine samples were then stored at –20 1C until analysis.
Urinalysis
Urinary thiamin was determined by high performance
liquid chromatography (HPLC)-post-labeled fluorescence
(Fukuwatari et al., 2004b). Urinary riboflavin was determined
by HPLC (Ohkawa et al., 1983). Urinary vitamin B6
metabolite, 4-PIC was determined by HPLC (Gregory and
Kirk, 1979). To measure urinary vitamin B12, urine samples
were added to 0.2 mmol/l acetate buffer (pH 4.8), vitamin B12
was converted to cyanocobalamin by boiling with 0.0006%
potassium cyanide at acidic pH, and cyanocobalamin was
determined by a microbioassay using Lactobacillus leichmanii
ATCC 7830 (Watanabe et al., 1999). Urinary N1-methyl-2pyridone-5-carboxamide, N1-methyl-4-pyridone-3-carboxamide (Shibata et al., 1988), and N1-methylnicotinamide
(Shibata, 1987) were determined by the HPLC method, and
the sum of these compounds was determined as nicotinamide metabolites. Urinary pantothenic acid was determined
by a microbioassay using Lactobacillus plantarum ATCC 8014
(Skeggs and Wright, 1944). Urinary folate was determined
by a microbioassay using Lactobacillus casei ATCC 2733 (Aiso
and Tamura, 1998). Urinary reduced and oxidized ascorbic
acid and 2,3-diketogluconic acid were determined by HPLC
(Kishida et al., 1992).
Statistical analysis
To exclude extraordinarily abnormal urinary vitamin levels
that might be caused by taking unexpected fortified foods,
we applied a quasi-trimmed mean, that is all mean values of
urinary excretion of each vitamin were calculated after
trimming the highest 5% of responses. In consequence,
a total of 148 participants were identified to be valid for
data analysis for each water-soluble vitamin. Similar to an
earlier free-living study (Chang et al., 2007), male and
female subjects were not separated for analysis. SPSS for
Windows version 16 (SPSS Inc., Chicago, IL, USA) was used
for statistical analysis. Values are presented as means±s.d.
Daily measurements of urinary and dietary water-soluble
vitamins were not normally distributed; therefore, the data
were converted logarithmically. Pearson correlation coefficients were calculated to determine the association between
urinary and dietary measurements, and between dietary and
estimated water-soluble vitamin intakes. For calculating
the mean dietary intake, at first, the individual mean value
for target days was calculated, and the mean value of the
subjects was calculated based on the resulting individual
mean values. An ANOVA random effects model was used to
European Journal of Clinical Nutrition
quantify inter- and intra-individual coefficient of variance
(%CV), which was used to estimate the variability in vitamin
intake.
Results
The basic characteristics of the 156 eligible participants
are presented in Table 1. Each value was similar to those
reported for adolescents aged 18–29 years in the Dietary
Reference Intakes for Japanese in 2005 (Ministry of Health,
Labour, and Welfare of Japan, 2005). Therefore, the participants were considered as typical university students in
Japan, who were characterized by relatively low body mass
index (mean ¼ 20.8 kg/m2) and low fat intake (mean ¼ 28.9%
of energy). During the experimental period, all participants
were living freely, and none of the participants were drinking
or smoking. Inter- and intra-individual variations in dietary
intake of water-soluble vitamins for the consecutive 4-day
period are shown in Table 2. For intra-individual variations,
values were 30–40%, except for vitamin B12 and vitamin C.
For inter-individual variations, vitamin B1 and vitamin B6
also showed high variation, exceeding 50%, in addition to
vitamin B12 and vitamin C.
The measured values for 24-h urinary excretion
collected on Day 4, daily vitamin intake for each watersoluble vitamin, and the correlations between 24-h urinary
excretion and daily vitamin intake are shown in Table 3.
For vitamin B2, niacin, and niacin equivalent, the most
significant positive correlation (‘r’ in row 4 in Table 3) was
found between dietary intake on Day 4 and urinary excretion. For vitamin B1, vitamin B6, pantothenic acid, folate,
and vitamin C (‘r’ in row 6 in Table 3), the most significant
Table 1 Basic characteristics of eligible 156 Japanese university students
aged 18–27 years
Variables
Values
Anthropometric variable
Age (years)
Body height (cm)
Body weight (kg)
Body mass index (kg/m2)
20.2±2.3
160.4±6.7
53.7±7.5
20.8±2.2
Dietary intake a
Total energy (kJ/day)
Protein (% of energy)
Fat (% of energy)
Carbohydrate (% of energy)
7443±1589
13.8±3.1
28.9±8.3
56.4±13.1
% Energy intake b
Breakfast
Lunch
Supper
A snack
21.1
31.1
33.6
14.2
a
Dietary intake assessed from the consecutive 4-day dietary records. Values
were expressed as mean±s.d.
b
Average starting time of each meal—breakfast: 0705 hours; lunch: 1220
hours; supper: 1945 hours.
Correlation of urinary vitamins and their intakes
T Tsuji et al
Abbreviations: MNA, N1-methylnicotinamide; 2-Py, N1-methyl-2-pyridone-5-carboxamide; 4-Py, N1-methyl-4-pyridone-3-carboxamide; 4-PIC, 4-pyridoxic acid.
a
Urinary excretion for each vitamin corresponds to thiamin for vitamin B1, riboflavin for vitamin B2, 4-PIC for vitamin B6, the sum of nicotinamide, MNA, 2-Py, and 4-Py for niacin equivalent, the sum of
reduced and oxidized ascorbic acid and 2,3-diketogluconic acid for vitamin C.
b
r means a correlation between urinary excretion and dietary intake of vitamin, for which values are denoted as *Po0.05, **Po0.01, ***Po0.001.
0.07
0.22**
569±515 (nmol/day)
388±276 (mmol/day)
0.19*
0.16
610±423 (nmol/day)
546±435 (mmol/day)
0.24**
0.34***
591±321 (nmol/day)
476±354 (mmol/day)
0.15
0.29***
569±338 (nmol/day)
425±362 (mmol/day)
23.1±8.8 (nmol/day)
139±131 (mmol/day)
0.10
22.7±11.2 (mmol/day)
0.28***
24.3±9.6 (mmol/day)
0.44***
23.9±8.5 (mmol/day)
23.6±8.2 (mmol/day)
16.5±5.2 (mmol/day)
0.33***
0.12
0.11
0.21**
0.10
0.22**
0.21*
2.09±0.84 (mmol/day)
3.17±1.46 (mmol/day)
5.25±2.37 (mmol/day)
3.05±5.69 (nmol/day)
93.4±49.0 (mmol/day)
184±74 (mmol/day)
0.27***
0.31***
0.21**
0.06
0.17*
0.20*
2.46±1.00 (mmol/day)
3.43±1.35 (mmol/day)
5.83±2.14 (mmol/day)
3.49±5.16 (nmol/day)
98.8±39.5 (mmol/day)
196±63 (mmol/day)
0.35***
0.28***
0.37***
0.01
0.26**
0.24**
2.46±1.06 (mmol/day)
3.47±1.35 (mmol/day)
5.62±2.38 (mmol/day)
3.59±3.86 (nmol/day)
96.5±45.7 (mmol/day)
191±70 (mmol/day)
2.27±0.92 (mmol/day)
3.32±1.09 (mmol/day)
5.30±2.15 (mmol/day)
2.88±3.42 (nmol/day)
90.8±39.4 (mmol/day)
184±65 (mmol/day)
0.29***
0.32***
0.26**
0.05
0.32***
0.29***
rb
Mean±s.d.
Mean±s.d.
rb
Mean±s.d.
rb
Vitamin intake
at Day 1
rb
0.425±0.286 (mmol/day)
0.382±0.321 (mmol/day)
3.68±1.31 (mmol/day)
0.028±0.018 (nmol/day)
—
84.5±28.1 (mmol/day)
Vitamin B1
Vitamin B2
Vitamin B6
Vitamin B12
Niacin
Niacin
equivalent
Pantothenic
acid
Folate
Vitamin C
In this study, a significant positive correlation was found
between the urinary excretion of seven water-soluble
vitamins and dietary intake in free-living Japanese university
students aged 18–27 years. The correlation calculated for the
day before urine collection was relatively higher than for
the other days. Moreover, the mean intake during the past
2–4 days showed a higher positive correlation with the
Mean±s.d.
Discussion
Mean±s.d.
positive correlation was found between dietary intake on
Day 3 and urinary excretion.
To investigate the influence of dietary intake during
the last few days on urinary excretion, we determined
the association between mean intake and urinary excretion.
Significant positive correlations were found between the
2–4-day mean intake and urinary excretion for eight
water-soluble vitamins, except for vitamin B12. Urinary
water-soluble vitamins showed the highest association with
3-day mean intakes.
To examine the influence of dietary intake period on 24-h
urinary excretion, we determined the correlation between
24-h urinary excretion and mean dietary intake. The
significant positive correlations (‘r’ in row 5 in Table 4) were
found between urinary excretion (row 2 in Table 3) and
3-day mean intake (row 4 in Table 4) for all water-soluble
vitamins, except for vitamin B12. The recovery rate (row 8 in
Table 4) was determined from urinary excretion (row 2 in
Table 3) and 3-day mean intake (row 4 in Table 4). These
values conformed to those reported in an earlier study
(Fukuwatari and Shibata, 2008), except for vitamin B12.
Estimated intake of water-soluble vitamins (row 9 in Table 4)
was calculated using these recovery (row 8 in Table 4) and
urinary excretion (row 2 in Table 3) values. Estimated watersoluble vitamin intakes except for vitamin B12 were correlated with 3-day mean intakes. Mean estimated intakes
showed 91–101% of their 3-day mean intakes, except for
vitamin B12 (61%).
Vitamin intake
at Day 2
41.1
38.5
40.6
141.2
35.1
49.2
39.9
52.4
78.4
Vitamin intake
at Day 3
79.3
41.6
52.4
110.4
23.1
33.9
31.4
41.5
165.7
Vitamin intake
at Day 4
Intra-individual variations
24-h urinary excretion
of vitamin a
Vitamin B1
Vitamin B2
Vitamin B6
Vitamin B12
Niacin
Niacin equivalent
Pantothenic acid
Folate
Vitamin C
Inter-individual variations
Vitamins
%CV (n ¼ 148)
Vitamins
Table 3 Measured values for 24-h urinary excretion collected on Day 4 and daily vitamin intake for each water-soluble vitamin, and correlation between 24-h urinary excretion and daily
vitamin intake (n ¼ 148)
803
Table 2 Inter- and intra-individual variations on the dietary intake of
water-soluble vitamins measured for the consecutive 4-days experiment
period
European Journal of Clinical Nutrition
Correlation of urinary vitamins and their intakes
T Tsuji et al
804
Table 4 Summary of derived values from measured values (daily vitamin intake and 24-h urinary excretion in Table), that are calculated mean dietary
intakes and correlations with 24-h urinary excretion, recovery rates and mean estimated intakes (n ¼ 148)
Vitamins
2 days mean vitamin
intake (days 3–4)a
mean±s.d.
2.37±0.79
(mmol per day)
3.04±0.87
Vitamin B2
(mmol per day)
5.46±1.85
Vitamin B6
(mmol per day)
3.24±2.62
Vitamin B12
(nmol per day)
Niacin
93.6±33.7
(mmol per day)
Niacin equivalent 189±54
(mmol per day)
Pantothenic acid 23.7±7.0
(mmol per day)
Folate
583±243
(nmol per day)
Vitamin C
446±285
(mmol per day)
Vitamin B1
rd
3 days mean vitamin
intake (days 2–4)a
mean±s.d.
0.40*** 2.40±0.73
(mmol per day)
0.39*** 3.05±0.83
(mmol per day)
0.40*** 5.58±1.62
(mmol per day)
0.06
3.32±2.60
(nmol per day)
0.35*** 95.4±28.7
(mmol per day)
0.33*** 192±47
(mmol per day)
0.47*** 23.9±6.7
(mmol per day)
0.24** 593±243
(nmol per day)
0.44*** 478±267
(mmol per day)
4 days mean vitamin
intake (days 1–4)a
Recovery
rateb (%)
rd
Mean±s.d.
rd
Mean±s.d.
0.42***
2.32±0.63
(mmol per day)
3.00±0.81
(mmol per day)
5.50±1.54
(mmol per day)
3.23±2.84
(nmol per day)
94.9±28.7
(mmol per day)
190±47
(mmol per day)
23.6±7.0
(mmol per day)
588±273
(nmol per day)
455±244
(mmol per day)
0.39***
17.8±11.4
0.39***
12.4±10.0
0.39***
69.6±28.6
0.43***
0.40***
0.02
0.33***
0.32***
0.46***
0.27**
0.42***
0.07
1.4±1.5
0.33***
—
0.32***
45.8±16.0
0.41***
71.6±23.3
0.24**
4.3±1.9
0.41***
31.3±29.6
Mean estimated vitamin intakec
re
% ratiof
2.38±1.61
(mmol per day)
3.08±2.59
(mmol per day)
5.29±1.88
(mmol per day)
2.04±1.33
(nmol per day)
—
0.40***
100
0.38***
101
0.40***
95
0.06
61
—
—
184±61
(mmol per day)
23.0±7.3
(mmol per day)
540±206
(nmol per day)
446±420
(mmol per day)
0.33***
96
0.47***
96
0.24**
91
0.44***
93
Mean±s.d.
a
Mean dietary intake was calculated using daily dietary intake (Table 3) for each individual.
Recovery rate was derived from 24-h urinary excretion (Table 3)/3 days mean intake.
c
Mean estimated intake was calculated using 24-h urinary excretion (Table 3) and recovery rates.
d
r means a correlation between 24-h urinary excretion (Table 3) and mean dietary intake, for which values are denoted as *Po0.05, **Po0.01 and ***Po0.001.
e
r means a correlation between 3 days mean dietary intake and mean estimated intake, for which values are denoted as *Po0.05, **Po0.01 and ***Po0.001.
f
% ratio means a ratio between 3 days mean intake and mean estimated intake.
b
urinary excretion for each water-soluble vitamin, except for
vitamin B12. These findings show that urinary levels of
water-soluble vitamins are affected by their dietary intake
over the past few days.
An earlier intervention study has shown extremely high
positive correlations between urinary levels of water-soluble
vitamins and their dietary intakes (Fukuwatari and Shibata,
2008). There were some differences in the dietary assessment
protocols between the earlier and present studies. In the
earlier study, all participants consumed exactly the same
defined diets with or without synthesized water-soluble
vitamin mixtures for 4 weeks, vitamin intakes were
controlled by the amount of vitamin mixtures, and the
nutrients in the diets were measured chemically. In this
study, the dietary assessment was performed for 4 consecutive days without any interventions, and the nutrient
composition was derived from a food composition table.
Considering these differences in protocols, this study had a
shorter assessment period and no intervention. Assuming
that the dietary assessment protocol in this study best
contributed to reduce the errors in the dietary records, the
similar results from the different protocols indicate that the
urinary levels of water-soluble vitamins are closely associated
with their intakes.
Correlation coefficients between urinary excretion and
intake of water-soluble vitamins ranged from 0.27 to 0.47.
Generally, it is thought that extra intake of water-soluble
vitamins is excreted rapidly into the urine. However, the
European Journal of Clinical Nutrition
metabolic fate is different for each vitamin. For example,
nicotinamide is metabolized to N1-methyl-2-pyridone-5carboxamide and N1-methyl-4-pyridone-3-carboxamide
through N1-methylnicotinamide by the strong catabolic
pathway (Shibata, 1989). These catabolites are excreted
mainly into the urine, and nicotinamide level is low (Shibata
and Matsuo, 1989). Similarly, vitamin B6 is catabolized to
4-PIC through pyridoxal, and 4-PIC is excreted into the urine
(Lui et al., 1985). In this study, we measured only 4-PIC.
Folates are catabolized into p-aminobenzoylglutamate and
the acetylated form, p-acetamidobenzoylglutamate, which
are excreted into the urine (Wolfe et al., 2003). However, we
measured only intact folates in urine, by a microbiological
assay. Biotin is catabolized into bisnorbiotin and biotin
sulfoxide (Mock et al., 1993). The bioassay organism may
grow equally well on biotin and the biotin catabolites such
as bisnorbiotin and biotin sulfoxide in urine (Mock et al.,
1993). In this study, urinary biotin was measured by
bioassay; therefore, the values of biotin corresponded to
the sum of biotin, bisnorbiotin, and biotin sulfoxide. Little
is known about the catabolism of vitamin B1, vitamin B2,
vitamin B12, pantothenic acid, and vitamin C in human
beings, and the major urinary excretory compound may be
the intact vitamin.
The extensive inter-individual variability in water-soluble
vitamin intakes might also affect those modest correlations
ranged from 0.27 to 0.47. Several factors are known to affect
water-soluble vitamin metabolism. For example, alcohol,
Correlation of urinary vitamins and their intakes
T Tsuji et al
805
carbohydrate, and physical activity are expected to affect
vitamin B1 metabolism (Hoyumpa et al., 1977; Manore,
2000; Elmadfa et al., 2001); bioavailability of pantothenic
acid in food is half that of free pantothenic acid (Tarr et al.,
1981); and the single nucleotide polymorphism of methylenetetrahydrofolate reductase gene affects folate metabolism
(Bagley and Selhub, 1998). However, mean estimated watersoluble vitamin intakes calculated using urinary vitamins
and recovery rates were in exact agreement with 3-day mean
and daily intakes. These findings suggest that urinary watersoluble vitamins can be used as biomarkers to assess their
intakes in groups. In fact, the requirements of vitamin B1,
vitamin B2, and niacin have been determined by assessment
of the relationships between urinary excretion and intake of
these vitamins (Ministry of Health, Labour, and Welfare of
Japan and Food and Nutrition Board, Institutes of Medicine,
USA). The requirement of vitamin C was settled on mainly
from the data for the relationship between the plasma
concentration and intake of vitamin C (Ministry of Health,
Labour, and Welfare of Japan and Food and Nutrition Board,
Institutes of Medicine, USA), although the data on urinary
excretion were also considered. Levine et al. (1996) have
reported that urinary vitamin C was almost undetectable in
volunteers who took o50 mg vitamin C. However, the
present data showed that the urinary excretion of ascorbic
acid (sum of reduced ascorbic acid, oxidized ascorbic acid,
and 2,3-diketogluconic acid) was detectable in the subjects
who took o50 mg of ascorbic acid, and there was no point in
the dietary intake when excretion was zero.
In terms of the completeness of the dietary assessment in
this study, there are several limitations of using a weighed
food record method. One of the limitations is the reliance
on self-report. To reduce errors associated with self-report,
several dietitians reviewed the collated records along with
the photographs. The selection of participants from among
dietetics students also contributed to reduce the errors in
reporting, because they have nutritional knowledge and are
well trained. Another limitation exists in the present food
composition table developed for Japan. In a dietary assessment of free-living people, potential errors caused by the
quality of the food composition table are inevitable, such as
defects in food composition. For example, the composition
of Japanese tea may vary depending on whether the extract
of tea was made personally or whether it was a bottled tea
beverage, because the present Japanese food composition
table cannot differentiate such products. Similarly, as the
food composition table only describes the composition of
raw liver, an error exists between the quantity of vitamin
intake obtained from the food composition table and the
actual intake from cooked liver. Such restrictions may lower
the accuracy of the data obtained from a weighed food
record. However, identifying the food status at oral intake
and coding the intake according to the food composition
table should help to increase the accuracy of the records. In
terms of complete 24-h urine collection, we used the
INTERMAP criteria (Stamler et al., 2003) as described in
‘Participants’ section. The p-aminobenzoic acid method
requires intervention by taking p-aminobenzoic acid tablets
orally; therefore, we did not use this method. The participants in our study were dietetics students with nutritional
expertise and were well motivated for the study; therefore,
the proportion of participants with incomplete urine
samples was presumed to be small (Murakami et al., 2008).
A significant correlation was not found between urinary
vitamin B12 and dietary intake in the present or in an earlier
study of Fukuwatari and Shibata (2008). This is consistent
with earlier studies that have shown that urinary vitamin B12
increased by only 1.5–2 times when 1 mg vitamin B12, which
is 300 times higher than usual intake, was administered
orally, and by only 2–3 times when 0.45 mg was injected
intramuscularly (Pitney and Beard, 1954; Mehta and Regr,
1964). Foods that included vitamin B12 were very limited;
therefore, its intake showed very high inter- and intraindividual variation in our study.
Relatively low correlations were found between urinary
folate and dietary intake in this study, whereas a high
correlation was found in an earlier study (Fukuwatari and
Shibata, 2008). The relatively low correlation of folate in this
study is consistent with a study that has indicated that
urinary folate excretion responds slowly to changes in
dietary folate intake, and is reduced significantly in people
who consume a low-folate diet (Kim and Lim, 2008).
Otherwise, as mentioned above, consuming Japanese green
tea and liver may affect the accuracy of folate intake
measurement, because Japanese tea and raw beef liver
contain 16 mg/100 g and 1000 mg/100 g folate, respectively,
in the Japanese food composition table (Ministry of Education, Culture, Sports, Science and Technology, 2007). This
potential low level of accuracy might cause lower correlation
between urinary folate and dietary intake of seven watersoluble vitamins.
In this study of free-living Japanese university students, we
found that 24-h urinary levels of water-soluble vitamins,
except for B12, correlated with their recent intakes, and could
be used as a potential biomarker to assess mean estimated
vitamin intake. This biomarker will be useful to assess and
compare mean vitamin intakes between several groups, and
to validate the study. More accurate estimation of the dietary
intake of water-soluble vitamins based on urinary excretion
requires additional, precise biological information such as
the bioavailability, absorption rate, and turnover rate.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
We thank all the volunteers who participated in this
study. This study represents the results of ‘Studies on the
European Journal of Clinical Nutrition
Correlation of urinary vitamins and their intakes
T Tsuji et al
806
Construction of Evidence to Revise the Dietary Reference
Intake for Japanese people—Elucidation of the Balance of
Micronutrients and Major Elements’ (principal investigator,
Katsumi Shibata), which was supported by a research grant
for Comprehensive Related Diseases from the Ministry of
Health, Labour, and Welfare of Japan.
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