Note Urinary Excretion of Water-Soluble Vitamins Increases in Streptozotocin-Induced Diabetic Rats without Decreases in Liver or Blood Vitamin Content

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1 J Nutr Sci Vitaminol, 58, 54 58, 2012 Note Urinary Excretion of Water-Soluble Vitamins Increases in Streptozotocin-Induced Diabetic Rats without Decreases in Liver or Blood Vitamin Content Eri IMAI, Mitsue SANO, Tsutomu FUKUWATARI and Katsumi SHIBATA* Department of Food Science and Nutrition, School of Human Cultures, The University of Shiga Prefecture, 2500 Hassaka, Hikone, Shiga , Japan (Received May 30, 2011) Summary It is thought that the contents of water-soluble vitamins in the body are generally low in diabetic patients because large amounts of vitamins are excreted into urine. However, this hypothesis has not been confirmed. To investigate this hypothesis, diabetes was induced in male Wistar rats (6 wk old) by streptozotocin treatment, and they were then given diets containing low, medium or sufficient vitamins for 70 d. The contents of 6 kinds of B-group vitamins, namely vitamin B 1, vitamin B 2, vitamin B 6, vitamin B 12, folate and biotin, were determined in the urine, blood and liver. No basic differences among the dietary vitamin contents were observed. The urinary excretion of vitamins was higher in diabetic rats than in control rats. The blood concentrations of vitamin B 12 and folate were lowered by diabetes, while, those of vitamin B 1, vitamin B 2, vitamin B 6, and biotin were not. All liver concentrations of vitamins were increased in diabetic rats above those in control rats. These results showed that streptozotocin-induced diabetes increased urinary excretion of watersoluble vitamins, though their blood and liver concentrations were essentially maintained in the rats. Key Words streptozotocin, diabetes, vitamins, urine It is well known that patients with diabetes mellitus exhibit polyuria, and it is considered that polyuria causes increased urinary excretion of water-soluble vitamins and lowers vitamin content in the body. As a result, diabetes may cause multiple vitamin deficiencies. However, this hypothesis has not yet been confirmed. There are several reports that have studied the relationships between diabetes and water-soluble vitamins in the diabetic patients and animal models. Low levels of whole blood vitamin B 1 (1 3), plasma vitamin B 1 (4) and serum pyridoxal (5, 6) were reported in diabetes, although Hobara et al. (7) have reported that the level of vitamin B 12 contents was increased. Several investigators (8, 9) reported normal whole blood value for vitamin B 1 in diabetes. They showed that the liver vitamin contents in diabetic rats were not always lower than those in control rats. As mentioned above, previous findings are inconsistent. In the present study, to confirm the effects of diabetes on water-soluble vitamin status, we comprehensively determined the contents of urine, blood and liver water-soluble vitamins in streptozotocin-induced diabetic rats. Male Wistar rats aged at 5 wk were purchased from CLEA Japan, Inc. (Tokyo, Japan), immediately placed in individual metabolic cages and fed a 20% casein diet containing 1.0% vitamin mixture for 7 d. They were * To whom correspondence should be addressed. kshibata@shc.usp.ac.jp maintained at around 22 C and about 60% humidity and a 12-h light/dark cycle. After adaptation, the rats were divided into two groups, a control and a diabetic group. The diabetic group was intraperitoneally injected with STZ (70 mg/kg body weight) in 0.5% NaCl (ph 4.4), and the control group was administered a vehicle. The rats in both groups were then subdivided into the three groups, and fed a 20% casein diet containing either 0.3, 0.5 or 1.0% AIN-93-VX vitamin mixture without nicotinic acid, corresponding to low, medium or sufficient vitamins, respectively. A preliminary study showed that the food intake in the diabetic rats gradually increased during the first 10 d, and reached twice that of the control rats. Therefore, to maintain equivalent vitamin intake, the vitamin content of the diets provided to the diabetic rats was adjusted daily depending on the previous day s food intake of the respective control rats and diabetic rats for 70 d. The six groups of rats are hereafter referred to as follows: the,,,, Vitamin Group and. Urine samples were collected on day 70 (09:00 am 09:00 am) and were stored at 20 C until needed. The rats were killed by decapitation after collecting urine samples. Blood was collected, and each liver was dissected. The care and treatment of the experimental animals 54

2 Vitamin Contents in Liver, Blood and Urine in STZ Rats 55 conformed to The University of Shiga Prefecture guidelines for the ethical treatment of laboratory animals. Urine, whole blood and liver vitamin B 1 levels were determined by the HPLC method (10). Riboflavin, FMN and FAD in whole blood and liver were measured by the HPLC method (11). Urine pyridoxine metabolite, 4-pyridoxic acid was determined by the HPLC method (12). Liver vitamin B 6 vitamers were determined by the microbioassay method using Saccharomyces carlsbergenisis strain 4228 ATCC 9080 (13). Plasma pyridoxal and PLP were determined by the HPLC method (14). Urine, plasma and liver vitamin B 12 levels were measured microbiologically using Lactobacillus delbrueckii subsp. lactis ATCC 7830 (15). Urine, plasma and liver folate levels were measured microbiologically using Lactobacillus casei ATCC 2733 (16). Urine, plasma and liver biotin levels were measured microbiologically using Lactobacillus plantarum ATCC 8014 (17). Plasma and urinary glucose were measured using a FUJI DRY-CHEM 3500V (Fujifilm Medical Co., Ltd., Tokyo, Japan). All data are expressed as means SE. Significant differences among the three groups of control rats or among the three groups of diabetic rats were tested using one-way ANOVA followed by Tukey s multiple comparison tests and those between respective control and diabetic groups were tested using Student s two-tailed t-test. In all analyses, values of p 0.05 were considered to be statistically significant. All analyses were done using Graph Pad Prism software, version 5 (Graph Software, Inc., San Diego, CA, USA). The final body weight was lower in diabetic rats than in the control rats in the respective dietary groups, although food intake was consistently higher in diabetic rats (Table 1). The differences in dietary vitamin contents had little effect on these parameters among the diabetic and control groups. The plasma glucose contents and urine volume was consistently higher in the diabetic rats than in the control rats. Actual vitamin intake was almost the same for the diabetic and control rats in the respective dietary groups, while urinary vitamin excretion was significantly higher in the diabetic rats than in the control rats (Table 2). Whole blood vitamin B 1 and vitamin B 2, and plasma vitamin B 6 and biotin concentrations were not significantly different between the respective dietary groups. The plasma vitamin B 12 concentration was lower in the and groups, and the plasma folate concentration was lower in the rats. The whole blood vitamin B 1, and plasma vitamin B 6 and folate concentrations dose-dependently increased in both the control and diabetic rats. The plasma biotin concentration dose-dependently increased only in the control rats. The concentrations of liver vitamins B 1, vitamin B 2, vitamin B 6 and vitamin B 12, and folate were higher in each group of diabetic rats than in the respective control rats, while that of biotin in diabetic rats was not different from the control rats, as shown in Table 3. The liver vitamin B 1 and vitamin B 6 concentrations dose-dependently increased in the control rats, and vitamin B 1, vitamin B 12 and folate concentrations did in Table 1. Comparisons of characteristics between diabetic and control rats. Initial body weight (g) Final body weight (g) * * * Total food intake (g/70 d) 1, , * 1, , * 1, ,815 81* Urine volume (ml/d) * * * Plasma glucose (mmol/l) * * * Urinary glucose (mmol/d) * * * Data are means SE for 5 rats per group. * p 0.05 versus the respective control group.

3 56 IMAI E et al. Table 2. Comparisons of urinary vitamin excretion and blood vitamin concentrations between the diabetic and control rats. Urinary excretion of vitamins B1 Vitamin (nmol/d) a a *, b *,2 B2 Vitamin (nmol/d) a *, b *, c *,2 B6 Vitamin (nmol/d) a *, b *, c *,2 Vitamin B12 (pmol/d) *, *,2 Folate (nmol/d) a *, a *, b *,2 Biotin(nmol/d) a *, a *, b *,2 Blood vitamin concentrations B1 Vitamin (pmol/ml) a b b *,3 Vitamin B2 (pmol/ml) B6 Vitamin (pmol/ml) a a 1, , b 1, Vitamin B12 (pmol/ml) * * Folate (pmol/ml) a a , b *,2 Biotin (pmol/ml) a b b Data are mean SE for 5 rats. * p 0.05 versus the respective control group. Superscript letters denote significant differences among the groups of control rats; superscript numbers denote significant differences among the group of diabetic rats. Urine thiamin was determined as vitamin B1, riboflavin as vitamin B2, and pyridoxal metabolite 4-pyridoxic acid as vitamin B6. The sum of blood thiamin, TMP and TDP was determined as vitamin B1, lumifravin converted from blood riboflavin, FMN and FAD as vitamin B2, and the sum of plasma pyridoxal and PLP as vitamin B6. Vitamin B12, folate and biotin in plasma were measured. Table 3. Comparisons of liver vitamin concentrations and contents between diabetic and control rats. Liver weight (g) * * * Vitamin concentrations in the liver B1 Vitamin (nmol/g liver) a a *, b *,2 Vitamin B2 (nmol/g liver) * * * Vitamin B6 (nmol/g liver) a * a * b * Vitamin B12 (pmol/g liver) *, *,1, *,2 Folate (nmol/g liver) *, *, *,2 Biotin (nmol/g liver) Liver vitamin contents per 100 g body weights B1 Vitamin (nmol/100 g bw) a *, a *,1, b *,2 Vitamin B2 (nmol/100 g bw) * * * Vitamin B6 (nmol/100 g bw) a * a * b * Vitamin B12 (nmol/100 g bw) * * * Folate (nmol/100 g bw) * * * Biotin (nmol/100 g bw) * * * Data are mean SE for 5 rats. * p 0.05 versus the respective control group. Superscript letters denote significant differences among the groups of control rats; superscript numbers denote significant differences among the group of diabetic rats.

4 Vitamin Contents in Liver, Blood and Urine in STZ Rats 57 the diabetic rats. The vitamin content in the whole liver reflects vitamin storage. As the final body weights were significantly lower in the diabetic rats than in the control rats, whole liver content of vitamins were represented per 100 g of body weight. In a comparison of diabetic and control groups, the liver vitamin contents per 100 g of body weight were higher in the diabetic rats compared with the control rats. Liver vitamin contents per 100 g of body weight were almost the same for the respective dietary groups. Since diabetes causes polyuria, it is thought that water-soluble vitamins are carried out via a large amount of urine. To investigate this hypothesis, the effects of STZ-induced diabetes on urinary vitamin excretion, and the blood and liver vitamin contents were comprehensively determined in rats. To determine water-soluble vitamin status and dynamics precisely, the rats consumed diets containing low, medium or sufficient vitamin levels. As results, the urinary excretion of each water-soluble vitamin was increased by diabetes, even though most blood vitamin concentrations and liver vitamin contents were not affected. From the present data, the nutritional status of water-soluble vitamins can be classified into three patterns. 1) Although the urinary vitamin excretion was high in the diabetic rats, the liver vitamin content was not decreased and the blood vitamin level was normal; vitamin B 1, vitamin B 2, vitamin B 6 and folate are classified under this pattern. 2) The urinary excretion of vitamins and the liver vitamin content were high in the diabetic rats, while the blood vitamin level was low; vitamin B 12 is classified under this pattern. 3) No change was found in blood or liver vitamin levels between the two groups, while the urinary excretion of vitamins was significantly increased in the diabetic rats; biotin is classified under this pattern. In the present study, STZ-treatment rats increased urine volume and urinary excretion of water-soluble vitamins. Although diuretic treatment increases urinary vitamin B 1 (18, 19), the effects of the drug on urinary excretion of other water-soluble vitamins remains to be elucidated. From the present findings, the increase of urine volume by STZ treatment is considered to be one of the reasons for the increase of urinary water-soluble vitamins. Generally, excess water-soluble vitamins are excreted into the urine, when sufficient water-soluble vitamins are stored in the body tissues. Another possibility is that excess water-soluble vitamins were excreted into urine in the diabetic rats. The present findings showed that blood and liver vitamin contents were maintained at normal levels, and urinary excretion of vitamins dose-dependently increased in the diabetic rats. Although the liver weights were 70% with STZ treatment, the liver vitamin concentrations (per g of liver) were approximately 1.5 to 2.0 fold higher, suggesting that the increased urinary excretion of vitamins indicated saturation of those stores. Thus, excessive loss of vitamins into the urine was not simply the result of the increased urine flux due to STZ treatment. Increased urinary excretion of these vitamins did not cause a decreased in tissue concentrations. We first considered that requirement of B-group vitamins in the STZ rats is increased due to several factors, for example, abnormally increased urine volume, and accelerated metabolism of fatty acids and amino acids. But our findings obtained here were that urinary excretion of water-soluble vitamins increased in STZ-induced diabetic rats without decreases in liver or blood vitamin contents. Acknowledgments This investigation was part of the project Studies on the Dietary Reference Intakes for Japanese (principal investigator, Katsumi Shibata), 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. REFERENCES 1) Giarnieri D, Lumia V Action of insulin on blood disphosphothiamine (DPT) in young and aged diabetic patients. Clin Chim Acta 6: ) Haugen HN The blood concentration of thiamine in diabetes. Scand J Clin Lab Invest 16: ) Siliprandi N, Navazio F The action of insulin on blood cocarboxylase in diabetes mellitus. Acta Med Scand 142: ) Thornalley PJ, Babaei-Jadidi R, Al Ali H, Rabbani N, Antonysunil A, Larkin J, Ahmed A, Rayman G, Bodmer CW High prevalence of low plasma thiamine concentration in diabetes linked to a marker of vascular disease. Diabetologia 50: ) Davis RE, Calder JS, Curnow DH Serum pyridoxal and folate concentrations in diabetics. 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