**2. Intervention studies**

#### **2.1 Factors affecting the urinary excretion of water-soluble vitamins**

Urinary excretion of water-soluble vitamins varied among subjects more than blood levels did (Shibata et al., 2009). One possible explanation is that one or more of several factors such as nutrient requirements, energy expenditure, tissue turnover, intestinal absorption, kidney reabsorption, and physical characteristics differ between individuals. In fact, urinary excretion of vitamin B1 is varied with the urine volume (Ihara et al., 2008), and furosemideinduced diuresis increases vitamin B1 excretion rate (Rieck et al., 1999). Physical characteristics also affect the amount of urinary compounds. For example, individuals excreting higher urinary nitrogen had greater weight and body mass index (BMI) than those excreting average or lower nitrogen (Bingham et al., 1995), and creatinine clearance is positively correlated with BMI (Gerchman, 2009). In this context, the physical characteristics and urine volume may affect urinary excretion of B-group vitamins. We measured urinary excretion of B-group vitamins in free-living, healthy human subjects, and determined the correlations between each of the urinary B-group vitamins and factors such as physical characteristics and urine volume (Fukuwatari, 2009).

Twenty four-hr urine samples were collected from 186 free-living Japanese females aged 19– 21 years, and 104 free-living Japanese elderly aged 70–84 years, and correlations were determined between urinary output of each B-group vitamin and body height, body weight, body mass index, body surface area, urine volume, and urinary creatinine. Only urinary excretion of vitamin B12 showed strong correlation with urine volume in both young female and elderly subjects (*r* = 0.683, p < 0.001 and *r* = 0.523, p < 0.001, respectively). All factors such as urine volume, urinary creatinine and physical characteristics such as body height, body weight, BMI and body surface area showed weak or no correlations with other 7 urinary B-group vitamins including thiamin, riboflavin, pyridoxal metabolite 4-pyridoxic acid, sum of nicotinamide metabolites, pantothenic acid, folate and biotin. To determine how urinary vitamin B12 is affected by its intake and urine volume, healthy Japanese adults (10 men; mean age, 25.9 ± 1.0 years; 10 women; mean age, 23.5 ± 6.4 years) orally administrated 1.5 mg cyanocobalamin, which is 500-fold higher daily intake. The Twenty Japanese adults consumed similar foods for 3 days and took a 1.5-mg cyanocobalamin tablet after breakfast on day 2. The 24-hour urine sample was collected for 3 successive days, and Pearson correlation coefficients between urinary vitamin B12 and urine volume on each day were determined.

Pharmacologic dose of cyanocobalamin increased Urinary vitamin B12 only 1.3-fold, and its concentration was not affected (Fig. 1A). Urinary vitamin B12 was always strongly correlated

In the present review, recent findings from our intervention and cross-sectional studies are described to contribute to the establishment and effective use of urinary water-soluble vitamins as potential nutritional biomarkers. Furthermore, we propose the reference values for urinary water-soluble vitamins to show adequate nutritional status based on the findings. Our findings suggest that urinary water-soluble vitamins can be used as nutritional biomarkers to assess their mean intakes in groups. More accurate estimation of individuals' water-soluble vitamin intakes based on urinary excretion requires additional, precise biological information such as the bioavailability, absorption rate, and turnover rate.

Urinary excretion of water-soluble vitamins varied among subjects more than blood levels did (Shibata et al., 2009). One possible explanation is that one or more of several factors such as nutrient requirements, energy expenditure, tissue turnover, intestinal absorption, kidney reabsorption, and physical characteristics differ between individuals. In fact, urinary excretion of vitamin B1 is varied with the urine volume (Ihara et al., 2008), and furosemideinduced diuresis increases vitamin B1 excretion rate (Rieck et al., 1999). Physical characteristics also affect the amount of urinary compounds. For example, individuals excreting higher urinary nitrogen had greater weight and body mass index (BMI) than those excreting average or lower nitrogen (Bingham et al., 1995), and creatinine clearance is positively correlated with BMI (Gerchman, 2009). In this context, the physical characteristics and urine volume may affect urinary excretion of B-group vitamins. We measured urinary excretion of B-group vitamins in free-living, healthy human subjects, and determined the correlations between each of the urinary B-group vitamins and factors such as physical

Twenty four-hr urine samples were collected from 186 free-living Japanese females aged 19– 21 years, and 104 free-living Japanese elderly aged 70–84 years, and correlations were determined between urinary output of each B-group vitamin and body height, body weight, body mass index, body surface area, urine volume, and urinary creatinine. Only urinary excretion of vitamin B12 showed strong correlation with urine volume in both young female and elderly subjects (*r* = 0.683, p < 0.001 and *r* = 0.523, p < 0.001, respectively). All factors such as urine volume, urinary creatinine and physical characteristics such as body height, body weight, BMI and body surface area showed weak or no correlations with other 7 urinary B-group vitamins including thiamin, riboflavin, pyridoxal metabolite 4-pyridoxic acid, sum of nicotinamide metabolites, pantothenic acid, folate and biotin. To determine how urinary vitamin B12 is affected by its intake and urine volume, healthy Japanese adults (10 men; mean age, 25.9 ± 1.0 years; 10 women; mean age, 23.5 ± 6.4 years) orally administrated 1.5 mg cyanocobalamin, which is 500-fold higher daily intake. The Twenty Japanese adults consumed similar foods for 3 days and took a 1.5-mg cyanocobalamin tablet after breakfast on day 2. The 24-hour urine sample was collected for 3 successive days, and Pearson correlation coefficients between urinary vitamin B12 and urine volume on each day

Pharmacologic dose of cyanocobalamin increased Urinary vitamin B12 only 1.3-fold, and its concentration was not affected (Fig. 1A). Urinary vitamin B12 was always strongly correlated

**2.1 Factors affecting the urinary excretion of water-soluble vitamins** 

characteristics and urine volume (Fukuwatari, 2009).

**2. Intervention studies** 

were determined.

with urine volume even on the day before, the day of, and the day after intake (Fig. 1B-D). These results clearly showed that urinary excretion of vitamin B12 was dependent uponurine volume, but not on intake of vitamin B12.Vitamin B12 is different from other B-group vitamins with respect to main excretion route, which is through the bile, and <10% of the total loss of vitamin B12 from the body is through urine (Shinton, 1972). These results suggest that the change in the level of urinary vitamin B12 is too small to evaluate intake of vitamin B12, and thus urinary vitamin B12 was unavailable to be used as biomarker for estimation of its intake. To excrete vitamin B12 into urine, vitamin B12 binds to carrier protein transcobalamin (TC) in serum (Allen, 1975), the TC–vitamin B12 complex is filtered in the glomeruli, and the proximal convoluted tubule reabsorbs this complex via a receptormediated system (Birn, 2006). Megalin is an essential receptor for reabsorption of the TC– vitamin B12 complex in the proximal tubule (Birn et al., 2002), binds to the TC–vitamin B12 complex with an estimated affinity (*K*d) of ~183 nmol/L (Moestrup et al., 1996). This high affinity may explain why urinary loss of vitamin B12 is very low. However, little is known about how water regulation mediated by regulatory factors such as aquaporin, vasopressin and angiotensin is linked to reabsorption of vitamin B12.

Fig. 1. Effect of administration of a pharmacologic dose of cyanocobalamin on urinary concentration of vitamin B12 (A) and the correlations between urinary vitamin B12 and urine volume on the day before cyanocobalamin intake (B), the day of intake (C) and the day after intake (D) (Fukuwatari et al., 2009).

Urinary Water-Soluble Vitamins as Nutritional Biomarker to Estimate Their Intakes 91

Fig. 2. Regression and 95% CI of oral dose and urinary excretion of vitamin B1 (A), vitamin B2 (B), vitamin B6 (C), niacin (D), pantothenic acid (E), folate (F), biotin (G) and vitamin C

Japanese elderly females aged 70–84 years were participated (Tsuji et al., 2010a, 2010b, 2011). The subjects performed 4-day dietary assessment by recording all food consumed during the consecutive 4-day period with a weighed food record, and collected 24-hr urine samples on the fourth day. The results showed that the correlation between the urinary excretion and the dietary intake on the same day as urine collection was highest compared with the correlations on other days in each generation (Table 1-3). Moreover, the correlations between the urinary excretion and the mean dietary intakes during the recent 2–4 days

(H) (Fukuwatari et al., 2008).

#### **2.2 Determination of urinary water-soluble vitamins as biomarkers for evaluating its intakes under strictly controlled conditions**

As mentioned above, it is well known that pharmacological dose of water-soluble vitamin intake dramatically increase urinary vitamin levels, but a few study had studied about the relationship between several oral dose correspond to dietary intake and urinary excretion of vitamin C (Levine et al., 1996, 2001). We also determined whether urinary levels of watersoluble vitamins and their metabolites can be used as possible markers for estimating their intakes in the intervention study (Fukuwatari & Shibata, 2008). Six female Japanese college students participated to the intervention study, and their age, body weight, height and BMI (mean ± SD) were 21.0 ± 0.0 years old, 161.7 ± 1.7 cm, 51.2 ± 2.8 kg and 19.6 ± 1.2, respectively. They were given a standard Japanese diet in the first week, same diet with synthesized water-soluble vitamin mixture as the diet as approximately one-fold vitamin mixture based on DRIs for Japanese in the second week, with three-fold vitamin mixture in the third week, and six-fold mixture in the fourth week. The 24-hr urine was collected on each week, and the relationships were determined between oral dose and urinary vitamin levels. All urinary vitamin and their metabolites levels except vitamin B12 increased linearly in a dose-dependent manner, and highly correlated with vitamin intake (*r* = 0.959 for vitamin B1, *r* = 0.927 for vitamin B2, *r* = 0.965 for vitamin B6, *r* = 0.957 for niacin, *r* = 0.934 for pantothenic acid, *r* = 0.907 for folic acid, *r* = 0.962 for biotin, and *r* = 0.952 for vitamin C; Fig. 2). These findings show that water-soluble vitamin and their metabolite levels in 24-hr urine reflect the vitamin intakes under strictly controlled conditions.

Humans can synthesize the vitamin nicotinamide from tryptophan in the liver, and the resultant nicotinamide is distributed to non-hepatic tissues. The purpose of the synthetic pathway in the liver is not the supply of NAD+ but the supply of nicotinamide for nonhepatic tissues. The conversion pathway of nicotinamide from tryptophan is affected by various nutrients (Shibata et al., 1995, 1997a, 1998; Kimura et al., 2005), hormones (Shibata, 1995; Shibata & Toda, 1997), exercise (Fukuwatari et al., 2001) and drugs (Shibata et al., 1996, 1997b, 2001; Fukuwatari et al., 2004), based on data concerning the urinary excretion of metabolic intermediates in the tryptophan–nicotinamide pathway. However, the intervention study showed that administration of nicotinamide did not affect de novo nicotinamide synthesis from tryptophan (Fukuwatari & Shibata, 2007).

#### **3. Cross-sectional studies: Determination of urinary water-soluble vitamins as biomarkers for evaluating its intakes in free-living subjects**

The intervention study showed that urinary water-soluble vitamin levels are correlated highly with their intake in a strictly controlled environment (Fukuwatari & 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. Thus, we conducted the Values are individual points of six subjects in each dose. 4-PIC signifies 4-pyridoxic acid, a catabolite of pyridoxal, and the Nam metabolites signify the total amount of nicotinamaide metabolites, *N*1-methylnicotinamide (MNA), *N*1-methyl-2-pyridone-5-carboxamide (2-Py) and *N*1-methyl-4-pyridone-3-carboxamide (4-Py).

Cross-sectional studies, and free-living healthy subjects who were 216 university dietetics students aged 18-27 years, 114 Japanese elementary school children aged 10-12 years and 37

**2.2 Determination of urinary water-soluble vitamins as biomarkers for evaluating its** 

As mentioned above, it is well known that pharmacological dose of water-soluble vitamin intake dramatically increase urinary vitamin levels, but a few study had studied about the relationship between several oral dose correspond to dietary intake and urinary excretion of vitamin C (Levine et al., 1996, 2001). We also determined whether urinary levels of watersoluble vitamins and their metabolites can be used as possible markers for estimating their intakes in the intervention study (Fukuwatari & Shibata, 2008). Six female Japanese college students participated to the intervention study, and their age, body weight, height and BMI (mean ± SD) were 21.0 ± 0.0 years old, 161.7 ± 1.7 cm, 51.2 ± 2.8 kg and 19.6 ± 1.2, respectively. They were given a standard Japanese diet in the first week, same diet with synthesized water-soluble vitamin mixture as the diet as approximately one-fold vitamin mixture based on DRIs for Japanese in the second week, with three-fold vitamin mixture in the third week, and six-fold mixture in the fourth week. The 24-hr urine was collected on each week, and the relationships were determined between oral dose and urinary vitamin levels. All urinary vitamin and their metabolites levels except vitamin B12 increased linearly in a dose-dependent manner, and highly correlated with vitamin intake (*r* = 0.959 for vitamin B1, *r* = 0.927 for vitamin B2, *r* = 0.965 for vitamin B6, *r* = 0.957 for niacin, *r* = 0.934 for pantothenic acid, *r* = 0.907 for folic acid, *r* = 0.962 for biotin, and *r* = 0.952 for vitamin C; Fig. 2). These findings show that water-soluble vitamin and their metabolite levels in 24-hr urine

Humans can synthesize the vitamin nicotinamide from tryptophan in the liver, and the resultant nicotinamide is distributed to non-hepatic tissues. The purpose of the synthetic pathway in the liver is not the supply of NAD+ but the supply of nicotinamide for nonhepatic tissues. The conversion pathway of nicotinamide from tryptophan is affected by various nutrients (Shibata et al., 1995, 1997a, 1998; Kimura et al., 2005), hormones (Shibata, 1995; Shibata & Toda, 1997), exercise (Fukuwatari et al., 2001) and drugs (Shibata et al., 1996, 1997b, 2001; Fukuwatari et al., 2004), based on data concerning the urinary excretion of metabolic intermediates in the tryptophan–nicotinamide pathway. However, the intervention study showed that administration of nicotinamide did not affect de novo

**3. Cross-sectional studies: Determination of urinary water-soluble vitamins** 

The intervention study showed that urinary water-soluble vitamin levels are correlated highly with their intake in a strictly controlled environment (Fukuwatari & 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. Thus, we conducted the Values are individual points of six subjects in each dose. 4-PIC signifies 4-pyridoxic acid, a catabolite of pyridoxal, and the Nam metabolites signify the total amount of nicotinamaide metabolites, *N*1-methylnicotinamide (MNA), *N*1-methyl-2-pyridone-5-carboxamide (2-Py)

Cross-sectional studies, and free-living healthy subjects who were 216 university dietetics students aged 18-27 years, 114 Japanese elementary school children aged 10-12 years and 37

**intakes under strictly controlled conditions** 

reflect the vitamin intakes under strictly controlled conditions.

nicotinamide synthesis from tryptophan (Fukuwatari & Shibata, 2007).

**as biomarkers for evaluating its intakes in free-living subjects** 

and *N*1-methyl-4-pyridone-3-carboxamide (4-Py).

Fig. 2. Regression and 95% CI of oral dose and urinary excretion of vitamin B1 (A), vitamin B2 (B), vitamin B6 (C), niacin (D), pantothenic acid (E), folate (F), biotin (G) and vitamin C (H) (Fukuwatari et al., 2008).

Japanese elderly females aged 70–84 years were participated (Tsuji et al., 2010a, 2010b, 2011). The subjects performed 4-day dietary assessment by recording all food consumed during the consecutive 4-day period with a weighed food record, and collected 24-hr urine samples on the fourth day. The results showed that the correlation between the urinary excretion and the dietary intake on the same day as urine collection was highest compared with the correlations on other days in each generation (Table 1-3). Moreover, the correlations between the urinary excretion and the mean dietary intakes during the recent 2–4 days

Urinary Water-Soluble Vitamins as Nutritional Biomarker to Estimate Their Intakes 93

of methylenetetrahydrofolate reductase (MTHFR) gene affects folate metabolism (Bagley & Selhub, 1998). When estimated intake of water-soluble vitamins was calculated using mean recovery rate and urinary excretion values, estimated water-soluble vitamin intakes except vitamin B12 were correlated with 3-day mean intakes, and showed 91–107% of their 3-day mean intakes, except vitamin B12 (61-79%) (Table 2). These findings showed that urinary water-soluble vitamins reflected their dietary intake over the past few days, and could be

> Vitamin intake at Day 3

mean ± SD mean ± SD *rb* mean ± SD *rb* mean ± SD *rb* mean ± SD *rb*

0.41§ 2.90 ± 0.85 (μmol/d)

0.36§ 3.75 ± 1.13 (μmol/d)

0.42§ 5.96 ± 1.65 (μmol/d)

0.18 4.85 ± 5.93 (nmol/d)

0.28§ 101.7 ± 38.2 (μmol/d)

0.28‡ 218 ± 56 (μmol/d)

0.23\* 30.1 ± 7.4 (μmol/d)

0.27‡ 615 ± 423 (nmol/d)

0.35§ 448 ± 313 (μmol/d)

aUrinary 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

<sup>b</sup>*r* means a correlation between urinary excretion and dietary intake of vitamin, for which values are

Table 2. Measured values for 24-hr urinary excretion collected on Day 4 and daily vitamin intake for each water-soluble vitamin, and correlation between 24-hr urinary excretion and

Vitamin intake at Day 2

0.25‡ 2.60 ± 0.74 (μmol/d)

0.36§ 3.59 ± 1.00 (μmol/d)

0.32§ 5.97 ± 1.69 (μmol/d)

0.14 4.76 ± 4.29 (nmol/d)

0.11 105.3 ± 31.3 (μmol/d)

0.23‡ 218 ± 52 (μmol/d)

0.20\* 27.0 ± 6.3 (μmol/d)

0.12 491 ± 123 (nmol/d)

0.23\* 403 ± 289 (μmol/d) Vitamin intake at Day 1

0.07

0.23\*

0.17

0.11

0.23\*

0.25‡

0.25‡

0.24\*

0.18

0.22\* 2.75 ± 0.92 (μmol/d)

0.33§ 3.60 ± 1.17 (μmol/d)

0.36§ 6.00 ± 2.41 (μmol/d)


0.21\* 101.4 ± 32.5 (μmol/d)

0.16 218 ± 56 (μmol/d)

0.31§ 28.7 ± 7.8 (μmol/d)

0.18 532 ± 164 (nmol/d)

0.26‡ 445 ± 328 (μmol/d)

used as biomarkers to assess their intakes in groups.

Vitamin intake at Day 4

3.13 ± 1.01 (μmol/d)

3.47 ± 0.94 (μmol/d)

5.93 ± 1.86 (μmol/d)

3.15 ± 1.97 (nmol/d)

(μmol/d)

214 ± 56 (μmol/d)

27.6 ± 6.9 (μmol/d)

575 ± 170 (nmol/d)

477 ± 225 (μmol/d)

*P*<0.001

reduced and oxidized ascorbic acid and 2,3-diketogluconic acid for vitamin C.

daily vitamin intake in Japanese school children (n=114) (Tsuji et al., 2010b).

24-h urinary excretion of vitamina

(μmol/d)

(μmol/d)

(μmol/d)

(nmol/d)

Niacin --- 97.0 ± 32.3

65.6 ± 27.6 (μmol/d)

11.6 ± 5.5 (μmol/d)

(nmol/d)

(μmol/d)

*P*<0.01, §

Vitamins

Vitamin B1 0.766 ± 0.383

Vitamin B2 0.290 ± 0.209

Vitamin B6 2.36 ± 0.92

Vitamin B12 0.026 ± 0.015

Folate 16.8 ± 6.6

Vitamin C 161 ± 221

denoted as \**P*<0.05, ‡

Niacin equivalent

Pantothenic acid


aUrinary 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.

<sup>b</sup>*r* means a correlation between urinary excretion and dietary intake of vitamin, for which values are denoted as \**P*<0.05, ‡ *P*<0.01, § *P*<0.001

Table 1. Measured values for 24-hr urinary excretion collected on Day 4 and daily vitamin intake for each water-soluble vitamin, and correlation between 24-hr urinary excretion and daily vitamin intake in young Japanese (n=148) (Tsuji et al., 2010a).

showed higher correlations, except for vitamin B12, than those for daily intakes (Table 4-6). However, these correlations ranged from 0.27 to 0.59, and these modest correlations were not enough to use urinary vitamins as biomarkers to estimate their intakes in individuals. Several factors are known to affect water-soluble vitamin metabolism. For example, alcohol, 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

0.29§ 2.46 ± 1.06 (μmol/d)

0.32§ 3.47 ± 1.35 (μmol/d)

0.26‡ 5.62 ± 2.38 (μmol/d )

0.05 3.59 ± 3.86 (nmol/d)

0.32§ 96.5 ± 45.7 (μmol/d)

0.29§ 191 ± 70 (μmol/d)

0.33§ 23.9 ± 8.5 (μmol/d)

0.15 591 ± 321 (nmol/d)

0.29§ 476 ± 354 (μmol/d)

aUrinary 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

<sup>b</sup>*r* means a correlation between urinary excretion and dietary intake of vitamin, for which values are

Table 1. Measured values for 24-hr urinary excretion collected on Day 4 and daily vitamin intake for each water-soluble vitamin, and correlation between 24-hr urinary excretion and

showed higher correlations, except for vitamin B12, than those for daily intakes (Table 4-6). However, these correlations ranged from 0.27 to 0.59, and these modest correlations were not enough to use urinary vitamins as biomarkers to estimate their intakes in individuals. Several factors are known to affect water-soluble vitamin metabolism. For example, alcohol, 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

Vitamin intake at Day 3

mean ± SD mean ± SD *rb* mean ± SD *rb* mean ± SD *rb* mean ± SD *rb*

Vitamin intake at Day 2

0.35§ 2.46 ± 1.00 (μmol/d)

0.28§ 3.43 ± 1.35 (μmol/d)

0.37§ 5.83 ± 2.14 (μmol/d )

0.01 3.49 ± 5.16 (nmol/d)

0.26‡ 98.8 ± 39.5 (μmol/d)

0.24‡ 196 ± 63 (μmol/d)

0.44§ 24.3 ± 9.6 (μmol/d)

0.24‡ 610 ± 423 (nmol/d)

0.34§ 546 ± 435 (μmol/d) Vitamin intake at Day 1

0.12

0.11

0.21‡

0.10

0.22‡

0.21\*

0.10

0.07

0.22‡

0.27§ 2.09 ± 0.84 (μmol/d)

0.31§ 3.17 ± 1.46 (μmol/d)

0.21‡ 5.25 ± 2.37 (μmol/d )


0.17\* 93.4 ± 49.0 (μmol/d)

0.20\* 184 ± 74 (μmol/d)

0.28§ 22.7 ± 11.2 (μmol/d)

0.19\* 569 ± 515 (nmol/d)

0.16 388 ± 276 (μmol/d)

Vitamin intake at Day 4

2.27 ± 0.92 (μmol/d)

3.32 ± 1.09 (μmol/d)

5.30 ± 2.15 (μmol/d )

2.88 ± 3.42 (nmol/d)

(μmol/d)

184 ± 65 (μmol/d)

23.6 ± 8.2 (μmol/d)

569 ± 338 (nmol/d)

425 ± 362 (μmol/d)

*P*<0.001

reduced and oxidized ascorbic acid and 2,3-diketogluconic acid for vitamin C.

daily vitamin intake in young Japanese (n=148) (Tsuji et al., 2010a).

Vitamins

Vitamin B1 0.425 ± 0.286

Vitamin B2 0.382 ± 0.321

Vitamin B6 3.68 ± 1.31

Vitamin B12 0.028 ± 0.018

Folate 23.1 ± 8.8

Vitamin C 139 ± 131

denoted as \**P*<0.05, ‡

Niacin equivalent

Pantothenic acid

24-h urinary excretion of vitamina

(μmol/d)

(μmol/d)

(μmol/d )

(nmol/d)

Niacin --- 90.8 ± 39.4

84.5 ± 28.1 (μmol/d)

16.5 ± 5.2 (μmol/d)

(nmol/d)

(μmol/d)

*P*<0.01, §

of methylenetetrahydrofolate reductase (MTHFR) gene affects folate metabolism (Bagley & Selhub, 1998). When estimated intake of water-soluble vitamins was calculated using mean recovery rate and urinary excretion values, estimated water-soluble vitamin intakes except vitamin B12 were correlated with 3-day mean intakes, and showed 91–107% of their 3-day mean intakes, except vitamin B12 (61-79%) (Table 2). These findings showed that urinary water-soluble vitamins reflected their dietary intake over the past few days, and could be used as biomarkers to assess their intakes in groups.


aUrinary 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.

<sup>b</sup>*r* means a correlation between urinary excretion and dietary intake of vitamin, for which values are denoted as \**P*<0.05, ‡ *P*<0.01, § *P*<0.001

Table 2. Measured values for 24-hr urinary excretion collected on Day 4 and daily vitamin intake for each water-soluble vitamin, and correlation between 24-hr urinary excretion and daily vitamin intake in Japanese school children (n=114) (Tsuji et al., 2010b).

Vitamins

Vitamin B1

Vitamin B2

Vitamin B6

Niacin equivalent

Pantothenic acid

f

\**P*<0.05, ‡

Vitamin B12 3.24 ± 2.62

Niacin 93.6 ± 33.7

Folate 583 ± 243

Vitamin C 446 ± 285

*P*<0.01, §

2 days mean vitamin intake (Days 3–4)

2.37 ± 0.79 (μmol/d)

3.04 ± 0.87 (μmol/d)

5.46 ± 1.85 (μmol/d)

(nmol/d)

(μmol/d)

189 ± 54 (μmol/d)

23.7 ± 7.0 (μmol/d)

(nmol/d)

(μmol/d)

*P*<0.001.

Urinary Water-Soluble Vitamins as Nutritional Biomarker to Estimate Their Intakes 95

4 days mean vitamin intake (Days 1–4)

mean ± SD *ra* mean ± SD *ra* mean ± SD *ra* mean ± SD mean ± SD *re*

0.42§ 2.32 ± 0.63 (μmol/d)

0.43§ 3.00 ± 0.81 (μmol/d)

0.40§ 5.50 ± 1.54 (μmol/d )

0.02 3.23 ± 2.84 (nmol/d)

0.33§ 94.9 ± 28.7 (μmol/d)

0.32§ 190 ± 47 (μmol/d)

0.46§ 23.6 ± 7.0 (μmol/d)

0.27‡ 588 ± 273 (nmol/d)

0.42§ 455 ± 244 (μmol/d)

Table 4. Correlations between 24-hr urinary excretion and mean vitamin intakes, recovery

rates, and mean estimated intakes in young Japanese (n=148) (Tsuji et al., 2010a).

Recovery ratec (%)

0.39§ 17.8 ± 11.4 2.38 ± 1.61

0.39§ 12.4 ± 10.0 3.08 ± 2.59

0.39§ 69.6 ± 28.6 5.29 ± 1.88

0.07 1.4 ± 1.5 2.04 ± 1.33

0.32§ 45.8 ± 16.0 184 ± 61

0.41§ 71.6 ± 23.3 23.0 ± 7.3

0.24‡ 4.3 ± 1.9 540 ± 206

0.41§ 31.3 ± 29.6 446 ± 420

(μmol/d)

(μmol/d)

(μmol/d)

(nmol/d)

(μmol/d)

(μmol/d)

(nmol/d)

(μmol/d)

0.33§ --- --- --- ---

Mean estimated vitamin intaked

> % ratiof

0.40§ 100 %

0.38§ 101 %

0.40§ 95%

0.06 61%

0.33§ 96%

0.47§ 96%

0.24‡ 91%

0.44§ 93%

3 days mean vitamin intake (Days 2–4)

0.40§ 2.40 ± 0.73 (μmol/d)

0.39§ 3.05 ± 0.83 (μmol/d)

0.40§ 5.58 ± 1.62 (μmol/d )

0.06 3.32 ± 2.60 (nmol/d)

0.35§ 95.4 ± 28.7 (μmol/d)

0.33§ 192 ± 47 (μmol/d)

0.47§ 23.9 ± 6.7 (μmol/d)

0.24‡ 593 ± 243 (nmol/d)

0.44§ 478 ± 267 (μmol/d)

aMean dietary intake was calculated using daily dietary intake for each individual. <sup>b</sup>*r* means a correlation between 24-h urinary excretion and mean dietary intake.

% ratio means a ratio between 3-day mean intake and mean estimated intake.

cRecovery rate was derived from 24-h urinary excretion/3-Days mean intake. dMean estimated intake was calculated using 24-hr urinary excretion and recovery rate. <sup>e</sup>*r* means a correlation between 3-day mean dietary intake and mean estimated intake.


aUrinary 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.

*br* means a correlation between urinary excretion and dietary intake of vitamin, for which values are denoted as \**P*<0.05, ‡*P*<0.01, §*P*<0.001

Table 3. Measured values for 24-hr urinary excretion collected on Day 4 and daily vitamin intake for each water-soluble vitamin, and correlation between 24-hr urinary excretion and daily vitamin intake in elderly Japanese (n=35) (Tsuji et al., 2011).

Vitamin intake at Day 3

mean ± SD mean ± SD *rb* mean ± SD *rb* mean ± SD *rb* mean ± SD *rb*

0.47‡ 2.50 ± 0.73 (μmol/d)

0.49‡ 3.60 ± 1.08 (μmol/d)

0.37\* 7.04 ± 2.35 (μmol/d)

0.15 5.89 ± 5.31 (nmol/d)

0.35\* 127 ± 57 (μmol/d)

0.37\* 232 ± 73 (μmol/d)

0.59§ 25.5 ± 8.9 (μmol/d)

0.55§ 845 ± 360 (nmol/d)

0.46‡ 620 ± 407 (μmol/d)

aUrinary 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

*br* means a correlation between urinary excretion and dietary intake of vitamin, for which values are

Table 3. Measured values for 24-hr urinary excretion collected on Day 4 and daily vitamin intake for each water-soluble vitamin, and correlation between 24-hr urinary excretion and

Vitamin intake at Day 2

0.54§ 2.62 ± 0.85 (μmol/d)

0.46‡ 3.69 ± 1.12 (μmol/d)

0.13 7.57 ± 2.71 (μmol/d)


0.38\* 129 ± 65 (μmol/d)

0.45‡ 239 ± 94 (μmol/d)

0.49‡ 25.6 ± 6.4 (μmol/d)

0.24 854 ± 301 (nmol/d)

0.43‡ 722 ± 423 (μmol/d) Vitamin intake at Day 1

0.42\*

0.34\*

0.16


0.32

0.26

0.30

0.28

0.53§

0.28 2.37 ± 0.74 (μmol/d)

0.52§ 3.54 ± 1.14 (μmol/d)

0.34\* 7.45 ± 2.41 (μmol/d)

0.12 6.75 ± 8.43 (nmol/d)

0.39\* 121 ± 47 (μmol/d)

0.39\* 223 ± 71 (μmol/d)

0.46‡ 24.5 ± 7.1 (μmol/d)

0.48‡ 818 ± 366 (nmol/d)

0.39\* 642 ± 356 (μmol/d)

Vitamins

Niacin equivalent

Pantothenic acid

Vitamin B1 0.459 ± 0.494

Vitamin B2 0.852 ± 0.828

Vitamin B6 4.45 ± 2.26

Vitamin B12 0.034 ± 0.035

Folate 36.6 ± 16.9

Vitamin C 214 ± 271

denoted as \**P*<0.05, ‡*P*<0.01, §*P*<0.001

24-h urinary excretion of vitamina

(μmol/d)

(μmol/d)

(μmol/d)

(nmol/d)

Niacin --- 113 ± 49

89.7 ± 30.8 (μmol/d)

15.1 ± 6.2 (μmol/d)

(nmol/d)

(μmol/d)

Vitamin intake at Day 4

2.51 ± 0.91 (μmol/d)

3.47 ± 1.22 (μmol/d)

7.06 ± 2.78 (μmol/d)

5.81 ± 4.91 (nmol/d)

(μmol/d)

213 ± 72 (μmol/d)

26.1 ± 8.9 (μmol/d)

792 ± 305 (nmol/d)

627 ± 310 (μmol/d)

reduced and oxidized ascorbic acid and 2,3-diketogluconic acid for vitamin C.

daily vitamin intake in elderly Japanese (n=35) (Tsuji et al., 2011).


aMean dietary intake was calculated using daily dietary intake for each individual.

<sup>b</sup>*r* means a correlation between 24-h urinary excretion and mean dietary intake.

cRecovery rate was derived from 24-h urinary excretion/3-Days mean intake. dMean estimated intake was calculated using 24-hr urinary excretion and recovery rate.

f % ratio means a ratio between 3-day mean intake and mean estimated intake.

\**P*<0.05, ‡ *P*<0.01, § *P*<0.001.

Table 4. Correlations between 24-hr urinary excretion and mean vitamin intakes, recovery rates, and mean estimated intakes in young Japanese (n=148) (Tsuji et al., 2010a).

<sup>e</sup>*r* means a correlation between 3-day mean dietary intake and mean estimated intake.

Vitamins

Vitamin B12

Niacin equivalent

Pantothenic acid

\**P*<0.05, ‡

Vitamin B1 2.51 ± 0.66

Vitamin B2 3.53 ± 1.03

Vitamin B6 7.05 ± 2.17

Niacin 120 ± 42

Folate 819 ± 279

Vitamin C 624 ± 337

*P*<0.01, §

(μmol/d)

(μmol/d)

(μmol/d)

5.85 ± 3.55 (nmol/d)

(μmol/d)

222 ± 58 (μmol/d)

25.8 ± 8.1 (μmol/d)

(nmol/d)

(μmol/d)

*P*<0.001.

2 days mean vitamin intake (Days 3–4)

Urinary Water-Soluble Vitamins as Nutritional Biomarker to Estimate Their Intakes 97

mean ± SD *ra* mean ± SD *ra* mean ± SD *ra* mean ± SD mean ± SD *re*

0.58§ 2.50 ± 0.59 (μmol/d)

0.57§ 3.57 ± 0.95 (μmol/d)

0.35\* 7.58 ± 1.95 (μmol/d)

0.01 5.85 ± 3.16 (nmol/d)

0.54§ 122 ± 36 (μmol/d)

0.54§ 227 ± 55 (μmol/d)

0.57§ 25.4 ± 6.5 (μmol/d)

0.47‡ 828 ± 266 (nmol/d)

0.50‡ 653 ± 334 (μmol/d)

aMean dietary intake was calculated using daily dietary intake for each individual. b*<sup>r</sup>* means a correlation between 24-h urinary excretion and mean dietary intake.

Table 6. Correlations between 24-hr urinary excretion and mean vitamin intakes, recovery

Relatively low correlations were found between urinary folate and dietary intake in the cross-sectional studies, whereas a high correlation was found in the intervention study (Fukuwatari & Shibata, 2008). The relatively low correlation of folate in free-living subjects may be explained by several reasons. Urinary folate excretion responds slowly to change in dietary folate intake, and is reduced significantly in people who consume a low-folate diet (Kim & Lim, 2008). Some Japanese subjects consumed Japanese green tea and liver well, and these foods contain 16 μg/100 g and 1000 μg/100 g folate, respectively, in the Japanese Food Composition Table (The Ministry of Education, Culture, Sports, Science and Technology, 2007). 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, since the Food

cRecovery rate was derived from 24-h urinary excretion/3-Days mean intake. dMean estimated intake was calculated using 24-hr urinary excretion and recovery rate. e*<sup>r</sup>* means a correlation between 3-day mean dietary intake and mean estimated intake. f

rates, and mean estimated intakes in elderly Japanese (n=35) (Tsuji et al., 2011).

% ratio means a ratio between 3-day mean intake and mean estimated intake.

4 days mean vitamin intake (Days 1–4)

Recovery ratec (%)

0.59§ 16.9 ± 17.7 2.71 ± 2.92

0.55§ 23.1 ± 22.9 3.69 ± 3.58

0.33 64.2 ± 31.7 6.93 ± 3.5


0.49‡ 40.1 ± 12.3 224 ± 77

0.56§ 59.6 ± 24.2 25.3 ± 10.4

0.43‡ 4.5 ± 2.0 805 ± 372

0.53§ 32.0 ± 39.3 682 ± 847

(μmol/d)

(μmol/d)

(μmol/d)2

(nmol/d)

(μmol/d)

(μmol/d)

(nmol/d)

(μmol/d)

0.52§ --- --- --- ---

Mean estimated vitamin intaked

> % ratiof

0.58§ 107 %

0.52§ 103 %

0.35\* 96 %

0.12 65 %

0.54§ 98 %

0.46‡ 98 %

0.48‡ 97 %

0.51‡ 101 %

3 days mean vitamin intake (Days 2–4)

0.62§ 2.55 ± 0.62 (μmol/d)

0.53§ 3.59 ± 0.99 (μmol/d)

0.30 7.22 ± 2.01 (μmol/d)


0.46‡ 123 ± 37 (μmol/d)

0.50‡ 228 ± 56 (μmol/d)

0.58§ 25.8 ± 7.1 (μmol/d)

0.42\* 831 ± 257 (nmol/d)

0.50‡ 657 ± 339 (μmol/d)


aMean dietary intake was calculated using daily dietary intake for each individual b*<sup>r</sup>* means a correlation between 24-h urinary excretion and mean dietary intake.

cRecovery rate was derived from 24-h urinary excretion/3-Days mean intake.

dMean estimated intake was calculated using 24-hr urinary excretion and recovery rate.

<sup>e</sup>*<sup>r</sup>* means a correlation between 3-day mean dietary intake and mean estimated intake f

% ratio means a ratio between 3-day mean intake and mean estimated intake.

\**P*<0.05, ‡ *P*<0.01, § *P*<0.001.

Table 5. Correlations between 24-hr urinary excretion and mean vitamin intakes, recovery rates, and mean estimated intakes in Japanese school children (n=114) (Tsuji et al., 2010b).


aMean dietary intake was calculated using daily dietary intake for each individual. b*<sup>r</sup>* means a correlation between 24-h urinary excretion and mean dietary intake.

cRecovery rate was derived from 24-h urinary excretion/3-Days mean intake. dMean estimated intake was calculated using 24-hr urinary excretion and recovery rate. e*<sup>r</sup>* means a correlation between 3-day mean dietary intake and mean estimated intake. f

% ratio means a ratio between 3-day mean intake and mean estimated intake.

\**P*<0.05, ‡ *P*<0.01, § *P*<0.001.

96 Biomarker

4 days mean vitamin intake (Days 1–4)

mean ± SD *ra* mean ± SD *ra* mean ± SD *ra* mean ± SD mean ± SD *re*

0.42§ 2.85 ± 0.58 (μmol/d)

0.43§ 3.60 ± 0.78 (μmol/d)

0.49§ 5.96 ± 1.35 (μmol/d)

0.10 4.35 ± 2.10 (nmol/d)

0.29‡ 101 ± 20.4 (μmol/d)

0.29‡ 217 ± 39 (μmol/d)

0.32§ 28.3 ± 5.7 (μmol/d)

0.24\* 553 ± 147 (nmol/d)

0.39§ 443 ± 170 (μmol/d)

Table 5. Correlations between 24-hr urinary excretion and mean vitamin intakes, recovery rates, and mean estimated intakes in Japanese school children (n=114) (Tsuji et al., 2010b).

Recovery ratec (%)

0.35§27.6 ± 12.2 2.83 ± 1.42

0.42§ 7.9 ± 5.2 3.66 ± 2.63

0.43§39.8 ± 14.0 5.90 ± 2.30

0.10 0.7 ± 0.6 3.72 ± 2.14

0.32§30.7 ± 12.6 215 ± 91

0.32§41.4 ± 19.5 28.1 ± 13.3

0.27‡ 3.1 ± 1.3 536 ± 211

0.39§36.4 ± 50.3 447 ± 613

(μmol/d)

(μmol/d)

(μmol/d)

(nmol/d)

(μmol/d)

(μmol/d)

(nmol/d)

(μmol/d)

0.32§ --- --- --- ---

Mean estimated vitamin intaked

> % ratiof

0.37§ 10 0%

0.26‡ 10 2%

0.41§ 10 0%

0.06 79 %

0.20\* 99 %

0.27‡ 99 %

0.09 97 %

0.39§ 10 0%

3 days mean vitamin intake (Days 2–4)

0.42§ 2.88 ± 0.63 (μmol/d)

0.41§ 3.60 ± 0.79 (μmol/d)

0.45§ 5.95 ± 1.29 (μmol/d)

0.19\* 4.25 ± 2.55 (nmol/d)

0.24\* 101 ± 21.7 (μmol/d)

0.29‡ 217 ± 43 (μmol/d)

0.26‡ 28.2 ± 5.6 (μmol/d)

0.23\* 560 ± 174 (nmol/d)

0.39§ 442 ± 183 (μmol/d)

aMean dietary intake was calculated using daily dietary intake for each individual b*<sup>r</sup>* means a correlation between 24-h urinary excretion and mean dietary intake. cRecovery rate was derived from 24-h urinary excretion/3-Days mean intake.

dMean estimated intake was calculated using 24-hr urinary excretion and recovery rate. <sup>e</sup>*<sup>r</sup>* means a correlation between 3-day mean dietary intake and mean estimated intake f % ratio means a ratio between 3-day mean intake and mean estimated intake.

Vitamins

Vitamin B1 3.02 ± 0.77

Vitamin B2 3.61 ± 0.85

Vitamin B6 5.94 ± 1.41

Vitamin B12 4.00 ± 3.14

Niacin 99 ± 26

Folate 595 ± 236

Vitamin C 462 ± 200

*P*<0.01, §

Niacin equivalent

Pantothenic acid

\**P*<0.05, ‡

(μmol/d)

(μmol/d)

(μmol/d)

(nmol/d)

(μmol/d)

216 ± 48 (μmol/d)

28.8 ± 6.0 (μmol/d)

(nmol/d)

(μmol/d)

*P*<0.001.

2 days mean vitamin intake (Days 3–4)

> Table 6. Correlations between 24-hr urinary excretion and mean vitamin intakes, recovery rates, and mean estimated intakes in elderly Japanese (n=35) (Tsuji et al., 2011).

Relatively low correlations were found between urinary folate and dietary intake in the cross-sectional studies, whereas a high correlation was found in the intervention study (Fukuwatari & Shibata, 2008). The relatively low correlation of folate in free-living subjects may be explained by several reasons. Urinary folate excretion responds slowly to change in dietary folate intake, and is reduced significantly in people who consume a low-folate diet (Kim & Lim, 2008). Some Japanese subjects consumed Japanese green tea and liver well, and these foods contain 16 μg/100 g and 1000 μg/100 g folate, respectively, in the Japanese Food Composition Table (The Ministry of Education, Culture, Sports, Science and Technology, 2007). 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, since the Food

Urinary Water-Soluble Vitamins as Nutritional Biomarker to Estimate Their Intakes 99

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. Next step in this type of study will be to determine whether vitamin contents in spot urine sample is used to assess water-soluble vitamin intakes in groups.

The preparation of this manuscript was supported by a Research Grant for Comprehensive Research on Cardiovascular and Lifestyle Related Diseases from the Ministry of Health,

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**6. Acknowledgement** 

F408-F416.

**7. References** 

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. Nutrient intakes were calculated using this Food Composition Table which did not take account of cooking loss for the above foods, and thus this might cause potential low level of accuracy. There might be also a technical issue. Urinary intact folates were measured by a microbiological assay in the cross-sectional studies. However, folates are catabolized into *p*-aminobenzoylglutamate and the acetylated form, *p*acetamidobenzoylglutamate, which are excreted into the urine (Wolfe et al., 2003).
