The classical signs of severe deficiency are seldom observed with respect to B-group vitamins. Instead, an increase in non-specific symptoms, general morbidity and a loss of appetite related to impaired vitamin status are commonly seen. The aim of the present study was to determine a ‘pre-sign’ that can be observed before the appearance of an obvious vitamin deficiency such as beriberi, pellagra or ariboflavinosis. In general, vitamin deficiencies appear if vitamin intake is inadequate. However, these deficiencies can be caused by malabsorption and the reduced synthetic ability of coenzymes. Therefore, the classical assessment, which is undertaken by comparing the intakes of B-group vitamins with Dietary Reference Intakes( 1 – 3 ), is of limited effectiveness. Biochemical methods may be more suitable to use for assessment of B-group vitamin nutritional status. They include measuring, in erythrocytes, the activity of transketolase for vitamin B1 status( Reference Boni, Kiechens and Hendrikx 4 ), glutathione reductase for vitamin B2 status( Reference Sauberlich 5 ), and aspartate aminotransferase and alanine aminotransferase for vitamin B6 status( Reference Kretsch, Sauberlich and Skala 6 ). Measurement of the activities of these enzymes represents a functional test of the adequacies of vitamins B1, B2 and B6. Relative enhancements of the activities of these enzymes by in vitro saturation with thiamine diphosphate( Reference Sauberlich, Herman and Stevens 7 ), flavin adenine dinucleotide( Reference Tillotson and Baker 8 ) and pyridoxal phosphate( Reference Kretsch, Sauberlich and Skala 6 ) have been shown to be a sensitive and specific measurement for the detection and evaluation of these vitamins. Suitable biological methods are not available for the detection of other B-group vitamins such as niacin, pantothenic acid, folate, biotin and vitamin B12.
Inadequate intake of vitamins typically causes a reduction in the concentration of vitamins in the cells and body fluid. So far, the most commonly used procedure to assess the nutritional status of B-group vitamins has been measurement of the urinary levels of these vitamins( Reference O'Neal, Johnson and Schaefer 9 – Reference Fry, Fox and Tao 13 ). Our research team( Reference Shibata, Fukuwatari and Ohta 14 – Reference Shibata, Fukuwatari and Yoshida 21 ) and other investigators( Reference Eissenstat, Wyse and Hansen 22 – Reference Kim and Lim 27 ) have shown that the urinary excretion of B-group vitamins provides an indication of the recent dietary intakes of these vitamins. However, the questions remain as to whether the urinary excretion of B-group vitamins in animals and human subjects decreases immediately before they become B-group vitamin insufficient or when fed a diet low in vitamins.
In the present study, we investigated the changes in urinary excretion of water-soluble vitamins and those of blood and liver concentrations using rats fed a vitamin-free diet. Our findings suggest that a urine sample is the biological sample most suitable to use as a surrogate indicator of B-group vitamin insufficiency.
Methods and materials
Ethical approval of study protocol
The care and treatment of experimental animals conformed to the guidelines for the ethical treatment of laboratory animals set by The University of Shiga Prefecture (Shiga, Japan).
Chemicals
Vitamin-free milk casein, sucrose and l-methionine were purchased from Wako Pure Chemical Industries. Maize oil was purchased from Ajinomoto. Gelatinised maize starch, a mineral mixture (American Institute of Nutrition (AIN)-93G mineral mixture)( Reference Reeves 28 ), and a vitamin mixture (nicotinic acid-free AIN-93 vitamin mixture containing 25 % choline bitartrate)( Reference Reeves 28 ) were obtained from Oriental Yeast Co., Ltd.
Thiamine hydrochloride (C12H17ClN4OS-HCl; molecular weight (MW) = 337·27 Da), riboflavin (C17H20N4O6; MW = 376·3 Da), pyridoxine hydrochloride (C8H11NO3-HCl; MW = 205·63 Da), cyanocobalamin (C63H88CoN14O14P; MW = 1355·40 Da), nicotinamide (C6H6N2O; MW = 122·13 Da), calcium pantothenate (C18H32N2O10-Ca; MW = 476·54 Da), folic acid (C19H19N7O6; MW = 441·40 Da) and d(+)-biotin (C10H16N2O3S; MW = 244·31 Da) were purchased from Wako Pure Chemical Industries. 4-Pyridoxic acid (C8H9NO4; MW = 183·16 Da) manufactured by ICN Pharmaceuticals was obtained through Wako Pure Chemical Industries.
N 1-Methylnicotinamide chloride (C7H9N2O-HCl; MW = 159·61 Da) was purchased from Tokyo Kasei Kogyo. N 1-Methyl-2-pyridone-5-carboxamide (2-Py, C7H8N2O2; MW = 152·15 Da) and N 1-methyl-4-pyridone-3-carboxamide (4-Py, C7H8N2O2; MW = 152·15 Da) were synthesised employing the methods of Pullman & Colowick( Reference Pullman and Colowick 29 ) and Shibata et al. ( Reference Shibata, Kawada and Iwai 30 ), respectively. All other chemicals used were of the highest purity available from commercial sources.
Animals and treatment
The room was maintained at about 22°C and at a humidity of about 60 %. A 12 h light-12 h dark cycle (06.00–18.00 hours/18.00–06.00) was employed.
To acclimatise them to a new environment, male Wistar rats (6 weeks old; CLEA Japan) were kept in individual rat metabolism cages (CT-10; CLEA Japan) and fed a control diet ad libitum (Table 1) for 7 d. The rats were then divided into two groups. One group was fed a control diet and the other fed a vitamin-free diet (Table 1) for 5 d. The start of the experiment was designated as the time when the diet was changed. Each day, 24 h urine samples were collected (09.00-09.00 hours) in amber bottles containing 1 ml of 1 m-HCl and stored at −25°C until use. Rats were killed at about 09.00 hours; blood was collected and livers (not perfused) were taken to measure the contents of B-group vitamins in the urine, liver and blood. The removed livers were preserved at −25°C until use.
Table 1. Composition of the diets
*The compositions of the mineral mixture and the vitamin mixture are described by Reeves( Reference Reeves 28 ).
Measurement of levels of B-group vitamins in urine, liver and blood
Preparation and measurement of the extracts of B-group vitamins from urine and blood were as previously described( Reference Shibata, Fukuwatari and Ohta 14 , Reference Fukuwatari, Wada and Shibata 31 , Reference Gregory and Kirk 32 ).
Vitamin B1 . Frozen liver samples, about 0·5 g, were thawed, minced, added to ten volumes of cold 5 % TCA and homogenised in a blender (Waring). The acidified homogenate was centrifuged at 10 000 g for 10 min at 4°C. The supernatant was retained and used for the measurement of the levels of vitamin B1 ( Reference Fukuwatari, Suzuura and Sasaki 33 ).
Vitamin B2 . Frozen liver samples, about 0·5 g, were thawed, minced, added to ten volumes of 50 mm-KH2PO4–K2HPO4 buffer (pH 7·0) and homogenised in a blender. To a 0·1 ml aliquot of the homogenate, 0·44 ml of water and 0·26 ml of 0·5 m-H2SO4 were added and kept at 80°C for 15 min. After cooling, 0·2 ml of 10 % TCA were added and the mixture was centrifuged at 10 000 g for 3 min at 4°C. From the supernatant obtained, 0·2 ml were withdrawn and added to 0·2 ml of 1 m-NaOH. The alkalised mixture was irradiated at a height of 20 cm from the liquid with two fluorescent lamps (20 W) for 30 min at room temperature. Then, 0·02 ml of glacial acetic acid were added to the mixture. The neutralised mixture was passed through a 0·45 µm microfilter. The filtrate was injected directly into an HPLC system for measuring lumiflavin levels( Reference Ohkawa, Ohishi and Yagi 34 ).
Vitamin B6 . Frozen liver samples, about 0·5 g, were thawed, minced, added to 90 ml of 55 m-HCl and homogenised in a blender. The homogenate was autoclaved at 121°C for 3 h to convert vitamin B6 coenzyme to the free form of vitamin B6. After cooling, the mixture was adjusted to pH 5·0 with 1 m-NaOH and then brought up to 100 ml with water. The solution was filtered with qualitative filter number 2 (Advantec). The filtrate was used for measuring levels of vitamin B6 as previously described( 35 ).
Vitamin B12 . Frozen liver samples, about 0·5 g, were thawed, minced and then added to 2·5 ml of 0·57 m-acetic acid–sodium acetate buffer (pH 4·5) plus 5 ml of water and 0·1 ml of 0·05 % KCN. The suspension was homogenised in a blender. The homogenate was then put into a boiling water-bath for 5 min. After cooling, 0·15 ml of 10 % metaphosphoric acid was added and brought up to 10 ml with water. The solution was filtered with qualitative filter number 2 (Advantec). The filtrate was used for measuring levels of vitamin B12 as previously described( Reference Watanabe, Abe and Katsura 36 ).
Nicotinamide. Frozen liver samples, about 0·6 g, were thawed, minced and then added to five volumes of 0·1 g/ml isonicotinamide. The suspension was then homogenised in a blender. The homogenate (1 ml) was withdrawn, added to 4 ml of water, and then autoclaved at 121°C for 10 min to convert pyridine nucleotide coenzymes to nicotinamide. After cooling, the mixture was centrifuged at 10 000 g for 10 min at 4°C. The supernatant was retained, the precipitated materials were extracted again with 5 ml of water, and the resulting supernatant was retained. Both retained supernatants were combined and the extract was used for measuring nicotinamide levels as previously described( Reference Shibata, Kawada and Iwai 30 ).
Pantothenic acid. Frozen liver samples, about 0·2 g, were thawed, minced and then added to ten volumes of 50 mm-KH2PO4–K2HPO4 buffer (pH 7·0). The suspension was homogenised with a Teflon/glass homogeniser. The homogenate was incubated overnight at 37°C to convert free pantothenic acid from the bound type of pantothenate compounds. The reaction was stopped by placing the mixture into a boiling water-bath for 5 min. After cooling, the mixture was centrifuged at 10 000 g for 10 min at 4°C. The supernatant was retained, the precipitated materials were extracted again with 2 ml of water and the resulting supernatant retained. Both retained supernatants were combined, and the extract was used for measuring levels of pantothenic acid as previously described( Reference Skeggs and Wright 37 ).
Folate. Frozen liver samples, about 0·5 g, were thawed, minced and then added to ten volumes of 0·1 m-KH2PO4–K2HPO4 buffer (pH 6·1). The suspension was homogenised in a blender. The homogenate was autoclaved at 121°C for 5 min. After cooling, 2·5 ml of proteinase MS (200 U/ml of water; Kaken Pharmaceutical Co., Ltd.) was added. The mixture was incubated at 37°C for 3 h to digest proteins and then release polyglutamated folates from the protein-bound types. The reaction was stopped by placing the mixture into a boiling water-bath for 10 min. After cooling, 0·5 ml of conjugase (extract from porcine kidney acetone powder (type II); Sigma–Aldrich) was added to the mixture and incubated overnight at 37°C to convert polyglutamated folates to monoglutamated folates. The reaction was stopped by placing the mixture in a boiling water-bath for 10 min. After cooling, the mixture was centrifuged at 10 000 g for 10 min at 4°C. The supernatant was retained, the precipitated materials extracted again with 3 ml of water, and the resulting supernatant retained. Both the retained supernatants were combined, and the extract was used for measuring folate levels as previously described( Reference Aiso and Tamura 38 ). The conjugase solution was made up as follows: 60 ml of 50 mm-KH2PO4–K2HPO4 buffer (pH 7·0) was added to 20 g of porcine kidney acetone powder and stirred for 30 min at 4°C. The suspension was centrifuged at 10 000 g for 10 min at 4°C. The supernatant was dialysed against a large amount of 50 mm-KH2PO4–K2HPO4 buffer (pH 7·0) to remove endogenous folate from the kidney acetone powder. The dialysed conjugase solution was used.
Biotin. Frozen liver samples, about 0·5 g, were thawed, minced, added to two volumes of 2·25 m-H2SO4 and then homogenised in a blender. The suspension was autoclaved at 121°C for 1 h to convert bound biotin to the free form of biotin. After cooling, the suspension was centrifuged at 10 000 g for 10 min at 4°C. The supernatant was used for measuring biotin levels( Reference Fukui, Iinuma and Oizumi 39 ).
Statistical analyses
Statistical significance was determined by one-way ANOVA followed by Tukey's multiple comparison tests. P < 0·05 was considered significant. All statistical analyses were undertaken using GraphPad Prism version 5.0 (GraphPad).
Results
Effects of feeding vitamin-free diet on food intake and gain in body weight
Male Wistar rats (aged 7 weeks) were fed the vitamin-free diet or the control diet for 5 d. No significant difference was observed in the daily gain in body weight between the control group and the vitamin-free diet group (Fig. 1(A)). No significant difference in daily food intake between the two groups was observed (Fig. 1(B)). Rats fed the vitamin-free diet for 5 d showed no abnormalities in body appearance.
Fig. 1. Changes in body weight (A) and food intake (B) in male Wistar rats (aged 7 weeks) fed a vitamin-free diet (•) or a control diet (○) for 5 d. Values are means for five rats, with standard errors represented by vertical bars.
Effects of feeding vitamin-free diet on the urinary excretion of B-group vitamins
Urinary excretion of each water-soluble vitamin, except for vitamin B12 and the sum of nicotinamide and its catabolites, decreased steeply upon feeding the vitamin-free diet (Fig. 2). Excreted levels of vitamin B1, vitamin B2, 4-pyridoxic acid (catabolite of vitamin B6), pantothenic acid, folate and biotin steeply decreased and approached zero within 1–2 d. Urinary excretion of vitamin B12 did not decrease because the main elimination route of vitamin B12 is through the bile duct and not the kidney( Reference Shinton 40 ). Urinary excretion of the sum of nicotinamide and its catabolites did not decrease because nicotinamide is biosynthesised from tryptophan in the vitamin-free diet.
Fig. 2. Changes in the urinary excretion of thiamine (A), riboflavin (B), 4-pyridoxic acid (4-PIC) (C), vitamin B12 (cyanocobalamin) (D), nicotinamide (Nam) and its metabolites (E), pantothenic acid (PaA) (F), folate (G) and biotin (H) in male Wistar rats (aged 7 weeks) fed a vitamin-free diet (•) or a control diet (○) for 5 d. Each day, 24 h urine samples (09.00–09.00 hours) were collected. Values are means for five rats, with standard errors represented by vertical bars. a,b,c Mean values with unlike letters for the group fed the vitamin-free diet were significantly different (P < 0·05; one-way ANOVA followed by Tukey's multiple-comparison tests).
Effects of feeding vitamin-free diet on the concentrations of B-group vitamins in blood and plasma
Nicotinamide was not detected in plasma, even in control rats. Hence, we measured the concentration of nicotinamide in whole blood. Its concentration did not change even when the vitamin-free diet was fed to rats for 5 d. This is because nicotinamide is biosynthesised from tryptophan. With respect to blood, only the concentrations of vitamin B1, pyridoxal phosphate (a coenzyme of vitamin B6), and biotin decreased to 15 % (P < 0·0001), 7 % (P < 0·0001) and 2 % (P < 0·0001) on day 5, respectively, compared with the values at the beginning of the experiment, whereas the reduction in the concentrations of vitamin B2 and folate was not so steep (Fig. 3). The plasma concentrations of vitamin B12 and pantothenic acid were not affected.
Fig. 3. Changes in the levels of vitamin B1 (A), vitamin B2 (B), pyridoxal phosphate (PLP) (C), vitamin B12 (D), nicotinamide (Nam) (E), pantothenic acid (PaA) (F), folate (G) and biotin (H) in the plasma or whole blood of male Wistar rats (aged 7 weeks) fed a vitamin-free diet for 5 d. Rats were killed each day and blood was collected. Values are means for five rats, with standard errors represented by vertical bars. a,b,c,d,e Mean values with unlike letters were significantly different (P < 0·05; one-way ANOVA followed by Tukey's multiple-comparison tests).
Effects of feeding vitamin-free diet on liver concentrations of B-group vitamins
The liver contents of all of the B-group vitamins did not decrease even if rats were fed the vitamin-free diet for 5 d (Fig. 4). The liver samples were not perfused, so a proportion of the levels of vitamins might originate from those found in the blood. However, the concentrations of respective B-group vitamins were much lower in blood samples than in the liver samples (Figs. 3 and 4). Therefore, the blood vitamin levels most probably did not have a significant impact on the vitamin levels observed in the liver samples.
Fig. 4. Changes in the levels of vitamin B1 (A), vitamin B2 (B), vitamin B6 (C), vitamin B12 (D), nicotinamide (Nam) (E), pantothenic acid (PaA) (F), folate (G) and biotin (H) in the livers of male Wistar rats (aged 7 weeks) fed a vitamin-free diet for 5 d. Rats were killed every day and their livers were removed. Values are means for five rats, with standard errors represented by vertical bars.
Discussion
We have reported previously that the urinary excretion of B-group vitamins reflects recent dietary intakes of these vitamins( Reference Shibata, Fukuwatari and Ohta 14 – Reference Shibata, Fukuwatari and Yoshida 21 ), and proposed reference values of the urinary levels of B-group vitamins for evaluating nutritional status. This method has been applied to evaluate human nutritional status( Reference Shibata, Fukuwatari and Yoshida 21 , Reference Fukuwatari, Shibata and Khan 41 ). However, the question remains as to whether the urinary excretion of B-group vitamins decreases immediately before animals and human subjects become B-group vitamin insufficient or when they are fed a diet low in vitamins.
In the present study, we investigated the changes in the urinary excretion of B-group vitamins in rats when they were fed a vitamin-free diet. Feeding the vitamin-free diet to growing rats for 5 d did not affect the food intake, body weight and vitamin concentrations in the liver. These findings indicate that this treatment did not induce B-group vitamin insufficiency.
We measured the plasma B-group vitamins in rats during the feeding of the vitamin-free diet. The concentrations of only three vitamins of the eight B-group vitamins (vitamin B1, pyridoxal phosphate and biotin) were decreased by feeding the vitamin-free diet, but the speed of the decrease was slower and the magnitude was lower than urinary excretion levels. For other B-group vitamins, the concentrations of vitamin B2 and folate were slightly reduced, whereas those of vitamin B12, nicotinamide and pantothenic acid were not decreased. These findings suggested that when rats were fed the vitamin-free diet, the concentrations of vitamins are maintained in the body by restriction of the elimination of B-group vitamins into urine. Therefore, the blood samples were not suitable to use for evaluating the nutritional status of B-group vitamins.
As was anticipated, the urinary excretion of B-group vitamins such as vitamin B1, vitamin B2, vitamin B6, pantothenic acid, folate and biotin was steeply decreased by feeding the vitamin-free diet. The decrease was rapid and dramatic (Fig. 2). These findings support our proposal that the urinary excretion of B-group vitamins is a better indicator of B-group vitamin nutritional status than blood concentrations.
In the present study, because sufficient niacin is biosynthesised from tryptophan, the total excretion of nicotinamide and its catabolites was not decreased by feeding the vitamin-free diet. When the rats were fed a tryptophan-limited and niacin-free diet, the urinary excretion of niacin decreased significantly( Reference Shibata, Mitsue and Fukuwatari 42 ). Therefore, the urinary excretion of nicotinamide and its metabolites can be used as an index of niacin nutritional status.
The urinary excretion of vitamin B12 was not affected by feeding the vitamin-free diet. This can be explained by the very slow turnover rate of vitamin B12, and the fact that its elimination route is via the bile( Reference Shinton 40 ) and not via the kidney. In addition, a large amount of vitamin B12 is stored in the liver and kidneys( Reference Shibata, Shimada and Kondo 43 ).
In conclusion, in animals and human subjects consuming diets low in vitamins, the first potential mechanism to prevent insufficiency of B-group vitamins, except for vitamin B12, is to reduce the urinary elimination of B-group vitamins via the kidney( Reference Shibata and Matsuo 44 ). Therefore, the measurement of the urinary B-group vitamins, except for vitamin B12, is a suitable method for individual nutritional status assessment. The present data complement our previous proposal that the urinary excretion of B-group vitamins reflects the nutritional status of these vitamins( Reference Shibata, Fukuwatari and Ohta 14 – Reference Shibata, Fukuwatari and Yoshida 21 , Reference Fukuwatari, Shibata and Khan 41 ).
Acknowledgements
This investigation was part of the project ‘Studies on the Dietary Reference Intakes for Japanese’ (principal investigator, K. S.), which was supported by a Research Grant for Comprehensive Research on Cardiovascular and Life-Style Related Diseases from the Ministry of Health, Labour and Welfare of Japan. K. S., M. S. and T. F. carried out the study design. C. S. and M. S. conducted the experiments. K. S. drafted the manuscript. T. F. provided critical comments on the manuscript. All the authors read and approved the final version of the manuscript. The authors declare that they have no conflicts of interest.
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