To estimate nutrient intake and to determine nutritional status are important to maintain one's health. Although dietary assessment 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 [1]. 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 [2], same as urinary potassium and potassium intake [3], and urinary sugars for sugar intake [4].

Water-soluble vitamins are absorbed from the digestive tract after ingestion, stored in the liver, delivered to peripheral, and then excreted to urine [5]. Urinary water-soluble vitamins or their metabolites decrease markedly as vitamin status declines, and they are affected by recent dietary intake [5]. 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 [5,6]. 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 [7]. Although pharmacological dose of water-soluble vitamin intake such as vitamin B2 [8], nicotinamide [9] and biotin [10] 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 [11,12].

Recent studies have conducted to establish urinary water-soluble vitamins as nutritional biomarkers to assess their intakes. In the present review, recent findings from 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, the reference values for urinary water-soluble vitamins are proposed to show adequate nutritional status based on the findings.

Intervention Studies

Factors affecting the urinary excretions of water-soluble vitamins

It is well known that urinary excretion of these vitamins varied among subjects more than blood levels did. 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 [13], and furosemide-induced diuresis increases vitamin B₁excretion rate [14]. 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 [15], and creatinine clearance is positively correlated with BMI [16]. In this context, the physical characteristics and urine volume may affect urinary excretion of B-group vitamins. There is a report that urinary excretion of B-group vitamins was measured in free-living, healthy human subjects, and the correlations were determined between each of the urinary B-group vitamins and factors such as physical characteristics and urine volume [17].

In the report, 24-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. 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. Orally administration of 1.5 mg cyanocobalamin, which is 500-fold higher daily intake, increased vitamin B₁₂ content in the urine by only 1.3-fold, and urinary vitamin B₁₂ was always strongly correlated with urine volume (Figure 1). 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 [18]. 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 [19], the TC-vitamin B₁₂ complex is filtered in the glomeruli, and the proximal convoluted tubule reabsorbs this complex via a receptor-mediated system [20]. Megalin is an essential receptor for reabsorption of the TC-vitamin B₁₂ complex in the proximal tubule [21], binds to the TC- vitamin B₁₂ complex with an estimated affinity (Kd) of ~183 nmol/L [22]. 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.

Figure 1: Correlations between urinary vitamin B₁₂ and urine volume [17]. The 24-h urine was collected from 20 Japanese adults, and Pearson’s correlation coefficients were determined between urinary vitamin B₁₂ and urine volume.

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 [11,12]. There is also a report to determine whether urinary levels of water-soluble vitamins and their metabolites can be used as possible markers for estimating their intakes in the intervention study [23]. In the report, six female Japanese college students 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; Figure 2). These findings show that water-soluble vitamin and their metabolite levels in 24-hr urine reflect the vitamin intakes under strictly controlled conditions.

Figure 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) [23]. Values are individual points of six subjects in each dose. 4-PIC signifies 4-pyridoxic acid, a catabolite of vitamin B₆ vitamers, the Nam metabolites does the total amount of nicotinamide metabolites, MNA, 2-Py and 4-Py, the PaA does pantothenic acid, and the AsA does ascorbic acid.

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 non-hepatic tissues. The conversion pathway of nicotinamide from tryptophan is affected by various nutrients [24-27], hormones [28,29], exercise [30] and drugs [31-34], 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 [35].

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 [23]. 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 context, 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 Japanese elderly females aged 70-84 years were participated to the cross-sectional studies [36-38]. 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. Moreover, the correlations between the urinary excretion and the mean dietary intakes during the recent 2-4 days showed higher correlations, except for vitamin B₁₂, than those for daily intakes (Table 1). 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 [39-41]; bioavailability of pantothenic acid in food is half that of free pantothenic acid [42]; and the single nucleotide polymorphism of methylenetetrahydrofolate reductase (MTHFR) gene affects folate metabolism [43]. 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 1). 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 1: Correlations mean water-soluble vitamin intakes in recent 3-days with 24-hr urinary excretion, recovery rates and mean estimated intakes in free-living Japanese young adults (n=148) in cross sectional studies [36].

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 [23]. 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 lowfolate diet [44]. 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 [45]. 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 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 [46].

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 [47]. The study revealed the mean values and ranges for each water-soluble vitamin except vitamin B₁₂ in the subjects with vitamin mixture based on DRIs for Japanese. Based on these results, the reference values for urinary water-soluble vitamins are proposed to show adequate nutritional status in Table 2. When urinary excretion of some vitamins is lower than the lower reference value, subject may not intake its vitamin enough to 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.

Table 2: Proposed reference values for urinary water-soluble vitamins in adults.

Recent studies have induced great advances for urinary watersoluble 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.

The preparation of this review 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).