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

The traditional approach of nutritional assessment is to survey the amount of nutrients consumed by dietary assessment. Although this method can provide approximate intake, this approach often makes misreporting, and can’t determine nutritional status. Especially, to determine micronutrient intake by dietary assessment is difficult because of high variations in habitual micronutrient intake. A nutritional biomarker can be an indicator of nutritional status with respect to intake or metabolism of dietary constituents. The nutritional biomarkers can be designated into one or more of three categories, 1) a means of validation of dietary instruments, 2) surrogate indicators of dietary intakes, or 3) integrated measures of nutritional status for a nutrient (Potischman & Freudenheim, 2003). Recent validation studies have developed the urinary compounds as nutritional biomarkers to estimate nutrient intakes. For example, 24-hr urinary nitrogen has been established as a biomarker for protein intake (Bingham, 2003), same as urinary potassium and potassium intake (Tasevska et al., 2006), and urinary sugars for sugar intake (Tasevska et al., 2005).


Introduction
The traditional approach of nutritional assessment is to survey the amount of nutrients consumed by dietary assessment. Although this method can provide approximate intake, this approach often makes misreporting, and can't determine nutritional status. Especially, to determine micronutrient intake by dietary assessment is difficult because of high variations in habitual micronutrient intake. A nutritional biomarker can be an indicator of nutritional status with respect to intake or metabolism of dietary constituents. The nutritional biomarkers can be designated into one or more of three categories, 1) a means of validation of dietary instruments, 2) surrogate indicators of dietary intakes, or 3) integrated measures of nutritional status for a nutrient (Potischman & Freudenheim, 2003). Recent validation studies have developed the urinary compounds as nutritional biomarkers to estimate nutrient intakes. For example, 24-hr urinary nitrogen has been established as a biomarker for protein intake (Bingham, 2003), same as urinary potassium and potassium intake (Tasevska et al., 2006), and urinary sugars for sugar intake (Tasevska et al., 2005).
Water-soluble vitamins are absorbed from the digestive tract after ingestion, stored in the liver, delivered to peripheral, and then excreted to urine (Food and Nutrition Board, Institute of Medicie, 1998). Urinary water-soluble vitamins or their metabolites decrease markedly as vitamin status declines, and they are affected by recent dietary intake (Food and Nutrition Board, Institute of Medicie, 1998). Urinary excretion of water-soluble vitamins such as thiamin, riboflavin and niacin has been used for setting Dietary Reference Intakes (DRIs) in USA and Japan (Food and Nutrition Board, Institute of Medicie, 1998;The Ministry of Health, Labour, and Welfare, 2009). Although pharmacological dose of watersoluble vitamin intake such as vitamin B 2 (Zempleni et al., 1996), nicotinamide (Shibata & Matsuo, 1990) and biotin (Zempleni & Mock, 1999) dramatically increase urinary vitamin levels, 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(Levine et al., , 2001. Thus, little attention had been paid to assess the quantitative relationships between intakes and urinary excretion of water-soluble vitamins. However, only a single study had investigated urinary vitamin as a possible marker for intake until 2007. Individuals' 30-day means of thiamin intake are highly correlated with their mean 24-hr urine thiamin levels under strictly controlled condition, showing 24-hr urinary thiamin as a useful marker for thiamin intake under strictly controlled conditions (Tasevska et al., 2007).

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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 B 12 was dependent uponurine volume, but not on intake of vitamin B 12 .Vitamin B 12 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 B 12 from the body is through urine (Shinton, 1972). These results suggest that the change in the level of urinary vitamin B 12 is too small to evaluate intake of vitamin B 12 , and thus urinary vitamin B 12 was unavailable to be used as biomarker for estimation of its intake. To excrete vitamin B 12 into urine, vitamin B 12 binds to carrier protein transcobalamin (TC) in serum (Allen, 1975), the TC-vitamin B 12 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 TCvitamin B 12 complex in the proximal tubule (Birn et al., 2002), binds to the TC-vitamin B 12 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 B 12 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 B 12 . Fig. 1. Effect of administration of a pharmacologic dose of cyanocobalamin on urinary concentration of vitamin B 12 (A) and the correlations between urinary vitamin B 12 and urine volume on the day before cyanocobalamin intake (B), the day of intake (C) and the day after intake (D)

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(Levine et al., , 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 B 12 increased linearly in a dose-dependent manner, and highly correlated with vitamin intake (r = 0.959 for vitamin B 1 , r = 0.927 for vitamin B 2 , r = 0.965 for vitamin B 6 , 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., , 1997a(Shibata et al., , 1998Kimura et al., 2005), hormones (Shibata, 1995;Shibata & Toda, 1997), exercise  and drugs (Shibata et al., 1996(Shibata et al., , 1997bFukuwatari 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).

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).
Japanese elderly females aged 70-84 years were participated (Tsuji et al., 2010a(Tsuji et al., , 2010b(Tsuji et al., , 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- 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 B 12 , 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 B 1 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 (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 B 12 were correlated with 3-day mean intakes, and showed 91-107% of their 3-day mean intakes, except vitamin B 12 (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.  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). a Mean dietary intake was calculated using daily dietary intake for each individual. b r means a correlation between 24-h urinary excretion and mean dietary intake. c Recovery rate was derived from 24-h urinary excretion/3-Days mean intake. d Mean estimated intake was calculated using 24-hr urinary excretion and recovery rate. e r 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 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).
www.intechopen.com Mean estimated vitamin intake d mean ± SD r a mean ± SD r a mean ± SD r a mean ± SD mean ± SD r e % ratio f a Mean dietary intake was calculated using daily dietary intake for each individual b r means a correlation between 24-h urinary excretion and mean dietary intake. c Recovery rate was derived from 24-h urinary excretion/3-Days mean intake. d Mean estimated intake was calculated using 24-hr urinary excretion and recovery rate. e r 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).
www.intechopen.com b r means a correlation between 24-h urinary excretion and mean dietary intake. c Recovery rate was derived from 24-h urinary excretion/3-Days mean intake. d Mean estimated intake was calculated using 24-hr urinary excretion and recovery rate. e r 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 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 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, pacetamidobenzoylglutamate, which are excreted into the urine (Wolfe et al., 2003).

Reference values for urinary water-soluble vitamins
Urinary water-soluble vitamins can be used as potential biomarker not only for estimation of its intake but also evaluation for its nutritional status. The intervention study comprehensively investigated urinary water-soluble vitamin values in subjects consuming semi-purified diet with vitamin mixture for 7 days . The study revealed the mean values and ranges for each water-soluble vitamin except vitamin B 12 in the subjects with vitamin mixture based on DRIs for Japanese. Based on these results, we propose the reference values for urinary water-soluble vitamins to show adequate nutritional status in Table 7. When urinary excretion of some vitamins is lower than the lower reference value, subject may not intake its vitamin enough for DRIs. When urinary vitamin is higher than the upper value, subject may intake its vitamin supplement. These reference values may be useful for first screening to check one's vitamin nutritional status and vitamin supplement intake.

Conclusion
Recent studies have induced great advances for urinary water-soluble vitamins as biomarkers for its intakes. Measuring urinary water-soluble vitamin levels can be the good approach for assessing dietary vitamin intake in groups, and for simply evaluation of its nutritional status in individuals. However, there is limitation for its use; urinary vitamins have not been suitable biomarker to estimate its intake in individuals yet. 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. 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.

Acknowledgement
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, Labour and Welfare of Japan (Principal Investigator, Katsumi Shibata).