J Nutr Sci Vitaminol, 51, 211-215, 2005 Comparative Effects of Vitamin K and Vitamin D Supplementation on Calcium Balance in Young Rats Fed Normal or Low Calcium Diets Jun IWAMOTO1, James K. YEH2, Tsuyoshi TAKEDA1 and Yoshihiro SATO3 1 Department of Sports Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan 2 Metabolism Laboratory, Department of Medicine, Winthrop-University Hospital, NY 11501, USA 3 Department of Neurology, Hitate Hospital, Fukuoka 826-0041, Japan (Received April 28, 2004) Summary We examined the effect of vitamin K and vitamin D supplementation on cal cium balance in young rats fed a normal or low calcium diet. Eighty female Sprague Dawley rats, 6wk of age, were randomized by the stratified weight method into eight groups with 10 rats in each group: 0.5% (normal) or 0.1% (low) calcium diet, 0.5% or 0.1% calcium diet+vitamin K (vitamin K2, menatetrenone, 30mg/100g, food intake), 0.5% or 0.1% cal cium diet+vitamin D (25ƒÊ/100g, food intake), and 0.5% or 0.1% calcium diet+vitamin K+vitamin D. The duration of the study was 10wk. Vitamin K supplementation promoted the reduction in urinary calcium excretion and retarded the abnormal elevation of serum PTH level in rats fed a low calcium diet, and stimulated intestinal calcium absorption in rats fed a normal calcium diet. Vitamin D supplementation stimulated intestinal calcium absorp tion with prevention of the abnormal elevation of serum PTH levels and prevented hypocal cemia in rats fed a low calcium diet, and stimulated intestinal calcium absorption in rats fed a normal calcium diet. The stimulation of intestinal calcium absorption was associated with increased serum 1,25-dihydroxyvitmain D levels. An additive effect of vitamin K and vita min D on intestinal calcium absorption was found only in rats fed a normal calcium diet. This study shows the differential effects of vitamin K and vitamin D supplementation on cal cium balance in young rats fed a normal or low calcium diet. Key Words vitamin K, vitamin D, intestinal calcium absorption, urinary calcium excre tion Calcium intake during growth influences peak bone mass and is a subsequently important factor in prevent ing postmenopausal and age-related osteoporosis (1). Adolescent girls and young adult women are the most likely to have calcium-deficient diets (2), and do not ingest adequate calcium. Inadequate calcium intake during growth may hinder the attainment of peak bone mass. It is known that vitamin D supplementation in creases intestinal calcium absorption via the endocrine system, especially 1,25-dihydroxyvitamin D. Recently, several reports have demonstrated the effects of vitamin K on calcium balance in rats; vitamin K2 improves poor calcium balance in 20-wk-old ovariectomized rats (3), and also improves abnormal declines in serum calcium and magnesium levels and abnormal increases in serum parathyroid hormone (PTH) level and renal cal cium concentration in 7-wk-old calcium/magnesium deficient rats (4). Thus, vitamin K and vitamin D may have the potential to improve calcium imbalance induced by calcium deficiency. However, less attention has been paid to the importance of vitamin D and vita min K during growth in retaining calcium in the intes E-mail: jiwamoto@sc.itc.keio,ac,jp tine and kidney and attaining bone health. Although an additive effect of vitamin K and vitamin D supplementation on increasing peak bone mass in young rats has been demonstrated (5), its mechanism in terms of their additive effect on intestinal and renal calcium retention has not been clarified. Furthermore, it is not determined yet whether vitamin K and vitamin D act on bone mass as well as calcium balance addi tively under calcium deficiency. The purposes of this study were to examine the effects of vitamin K and vita min D supplementation on calcium balance in young rats fed a normal or low calcium diet by nutritional cal cium balance study, and to determine whether com bined supplementation of vitamin K and vitamin D would have additive effects on calcium balance. MATERIALS AND METHODS Treatment of animals. Eighty female Sprague-Dawley rats, 4wk of age, were purchased from Hilltop Lab. Ani mals, Inc. (Scottdate, PA, USA). They were fed a pelleted standard chow diet containing 0.5% calcium, 11.25ƒÊg/ 100g vitamin D, and 3mg/100g vitamin K (Rodent Laboratory Chow; Ralston Purina Co., St. Louis, MO, USA). The form of vitamin K in this chow standard diet was menadione. Animals were housed under local 211
212 IWAMOTO J et al. vivarium conditions (temperature 23.8 Ž and 12h on/ off light cycle) with free access to water and chow diet. After 2wk of adaptation to their new environment, the rats, 6wk of age, were randomized by the stratified weight method into eight groups of 10 animals in each group: 05% (normal) or 01% (low) calcium diet (NC or LC, respectively) group, 0.5% or 01% calcium diet+vitamin K (vitamin K2, menatetrenone, 30mg/ 100g, food intake) (NCK or LCK, respectively) group, 0,5% or 0.1% calcium diet+vitamin D (25ƒÊg/100g, food intake) (NCD or LCD, respectively) group, and 05% or 0.1% calcium diet+vitamin K+vitamin D (NCKD or LCKD, respectively) group. These special syn thetic diets were formulated by and purchased from Harlan Teklad (Madison, WI, USA), The supplemented vitamin K (menatetrenone) was provided by the Eisai Pharmaceutical Co., Ltd. (Tokyo, Japan), The chow diet of the NC and LC groups contained 11.2 5µg/ 100 g vitamin D and 3mg/100g vitamin K; the chow diet of the NCK and LCK groups contained 11.25ƒÊg/100g vitamin D; and the chow diet of the NCD and LCD groups contained 3mg/100 g vitamin K. The body weight of the rats was monitored weekly, and the exper imental period was 10wk, This study was carried out at Winthrop-University Hospital. Animals were maintained according to the National Institutes of Health (NIH) Guide for Care and Use of Laboratory Animals, and the animal protocols were approved by the Laboratory Ani mal Care Committee of Winthrop-University Hospital. Serum calcium, phosphorus, creatinine, and calciotropic hormones. All rats were processed for death at 10wk after the start of the experiment. They were anesthe tized with 80mg/kg ketamine injected intraperito neally together with 12mg/kg xylazine, and killed by exsanguination. Upon death, serum was collected from the rats in all groups. Serum calcium, phosphorus, and creatinine levels were measured by an automated instrument (Dada Behring Model RXL, Bakersfield, CA, USA): Serum bioactive intact PTH level was measured using a commercial enzyme linked immunosorbent assay (ELISA) kit that was specific to rat PTH (ALPCO Diagnostics, Windham, NH, USA). Serum 25-hydroxy vitamin D [25(OH) D] and 1,25-dihydroxyvitamin D [1,25 (OH)2 D] levels were measured by a radioreceptor binding assay with calf thymus receptor using the two different kits manufactured by DiaSorin Inc. (Stillwater, MN, USA). Intestinal calcium absorption efficiency, calcium balance, and calcium retention. During the eighth and ninth week, food intake for 3 consecutive days was measured and urine and feces for 3 consecutive days were col lected from 8 rats in each group to carry out nutritional balance studies. Urinary calcium, phosphorus, and cre atinine levels were measured by an automated instru ment (Dada Behring Model RXL), and fecal calcium level was measured by a colorimetric method using a diagnostic kit (Sigma, St. Louis, MO, USA). Daily cal cium intake was estimated from daily food intake. Intes tinal calcium absorption efficiency, calcium balance, and calcium retention were calculated by the following equations: intestinal calcium absorption efficiency= (calcium intake-fecal calcium)/calcium intake; cal cium balance=calcium intake-(urinary calcium+fecal calcium); and calcium retention=calcium balance!cal cium intake. Statistical analysis. All data are presented as mean and standard deviation (SD), Comparisons of data among the groups were performed by analysis of vari ance (ANOVA) with Fisher's protected least significant difference (PLSD) test, The effects of vitamin K and vita min D supplementation were analyzed by two-way ANOVA. All statistical analyses were performed using the Stat View J-5.0 program (SAS Institute, Cary, NC, USA) on a Macintosh computer. A significance level of p<0.05 was used for all comparisons. RESULTS Serum calcium, phosphorus, and calciotropic hormones Table 1 shows the levels of serum calcium, phospho rus, creatinine, and calciotropic hormones. Calcium deficiency induced hypocalcemia and increased serum PTH level, resulting in a decrease in serum 25(OH) D level and an increase in serum 1,25(OH)2 D level as compared with no calcium deficiency. Vitamin K sup plementation did not prevent hypocalcemia, but retard ed the abnormal elevation of serum PTH level with no significant influence on serum 1,25(OH)2 D level in rats fed a low calcium diet, and increased serum 1,25(OH)2 D level in rats fed a normal calcium diet. On the other hand, vitamin D supplementation prevented the abnor mal elevation of serum PTH level and hypocalcemia, and increased serum 25(OH) D and 1,25(OH)2 D levels in rats fed a low calcium diet, and increased serum 25 (OH) D and 1,25(OH)2 D levels without alterations in serum calcium and PTH levels in rats fed a normal cal cium diet. Intestinal calcium absorption efficiency, calcium balance, and calcium retention Tables 2 and 3 show the results of the calcium bal ance study. Urine creatinine data allowed us to confirm the consistency of urine collection. Calcium deficiency reduced daily calcium intake, stimulated intestinal cal cium absorption, reduced urinary calcium excretion, and increased intestinal calcium absorption efficiency and calcium retention, but decreased calcium balance. Vitamin K supplementation promoted the reduction in urinary calcium excretion and increased calcium reten tion and calcium balance in rats fed a low calcium diet, and increased intestinal calcium absorption efficiency, calcium retention, and calcium balance in rats fed a normal calcium diet. On the other hand, vitamin D sup plementation stimulated food (calcium) intake and increased intestinal calcium absorption efficiency, cal cium balance and calcium retention in rats fed a low calcium diet, and also stimulated increased intestinal calcium absorption efficiency, calcium retention, and calcium balance in rats fed a normal calcium diet. Vita min D supplementation increased urinary calcium excretion in rats fed a low or normal calcium diet. An additive effect of vitamin K and vitamin D on intestinal
Vitamin K, Vitamin D, and Calcium Balance 213 Table 1. Serum calcium, phosphorus, creatinine, and calciotropic hormones. Data are expressed as mean±sd. ANOVA with Fisher's PLSD test was used to compare the data between the groups. The * Significantly different from NC. Table 2. Urinary calcium, phosphorus, creatinine, fecal calcium, and calcium intake. Data are expressed as mean }SD. ANOVA with Fisher's PLSD test was used to compare the data between the groups. The * Significantly different from NC. calcium absorption was found only in rats fed a normal calcium diet, but no additive renal effect was found in rats fed either a normal or low calcium diet. DISCUSSION The focus of this study was to examine if simulta neous supplementation of vitamin K and vitamin D would enhance calcium retention in growing female rats. Vitamin K supplementation promoted the reduc tion in urinary calcium excretion in rats fed a low cal cium diet and stimulated intestinal calcium absorption in rats fed a normal calcium diet. On the other hand,
214 IWAMOTO J et al. Table 3. Intestinal calcium absorption efficiency, calcium balance, and calcium retention. Data are expressed as mean }SD. ANOVA With Fisher's PLSD test was used to compare the data between the groups. The * significantly different from NC. vitamin D supplementation stimulated intestinal cal cium absorption via increased serum 1,25(OH)2 D level in both rats fed a normal diet and in those fed a low cal cium diet. An additive effect of vitamin K and vitamin D, which resulted in increased intestinal calcium absorption, was found only when calcium intake was at an adequate level. There was no effect, however, when the diet was deficient in calcium. First, calcium deficiency induced hypocalcemia, increased serum PTH and 1,25(OH)2 D levels with decreased serum 25(OH) D level, stimulated intestinal calcium absorption, and reduced urinary calcium excretion. The decrease in serum 25(OH) D level might have been due to an increased serum level of PTH, which converts 25(OH) D into 1,25(OH)2 D, and an increased serum 1,25(OH)2 D level might have resulted in increased intestinal calcium absorption. The changes in these parameters in response to calcium deficiency are considered to be physiologically reasonable. Vitamin K supplementation promoted the reduction of urinary calcium excretion, increased calcium reten tion and calcium balance, and retarded the increase in serum PTH level despite the absence of a significant effect on hypocalcemia in rats fed a low calcium diet, and stimulated intestinal calcium absorption by increasing serum 1,25(OH)2 D level in rats fed a nor mal calcium diet. The response of calciotropic hor mones and renal and intestinal calcium retention to vitamin K supplementation is apparently different between low and normal calcium diet conditions in rats. Several studies have demonstrated the effects of vitamin K supplementation on intestinal and renal cal cium excretion in animals. Kobayashi et al. (3) showed that vitamin K supplementation in severe calcium/ magnesium-deficient rats improved the abnormal decreases in serum calcium and magnesium levels and abnormal increases in serum PTH level and renal cal cium concentration. Tomiuga et al. (6) showed that vitamin K supplementation in vitamin K-deficient and sodium-loading rats prevented the reduction in intesti nal calcium absorption. Thus, vitamin K supplementa tion may have the potential to increase renal and intes tinal retention. We have found that vitamin K supple mentation acts more strongly on renal calcium reten tion system under calcium deficiency, while vitamin K supplementation increases intestinal calcium absorp tion under calcium sufficiency, although the responses to vitamin K supplementation alone were fairly unre markable with the exception of decreased urinary cal cium excretion in rats fed a low calcium diet. Vitamin D supplementation stimulated intestinal cal cium absorption via increased serum 1,25(OH)2 D lev els with prevention of the abnormal elevation of serum PTH levels and prevented hypocalcemia in rats fed a low calcium diet, and also stimulated intestinal calcium absorption via increased serum 1,25(OH)2 D levels without alterations in serum calcium and PTH levels in rats fed a normal calcium diet. Vitamin D supplementa tion increased urinary calcium excretion in rats fed a low or normal calcium diet. In contrast to vitamin K supplementation, vitamin D supplementation similarly increased intestinal calcium absorption and urinary calcium excretion both in rats fed a low or normal cal cium diet. In particular, this increase in urinary cal cium excretion in rats fed a low calcium diet might sim ply result from increased serum calcium levels. These
Vitamin K, Vitamin D, and Calcium Balance 215 results are supported by several previous reports (7-9). The responses to vitamin D supplementation appeared to be more striking but could be confirmed by numer ous existing studies. The potential additive effects of vitamin K and vita min D on intestinal calcium absorption, calcium bah ance, and calcium retention in rats fed a normal cal cium diet, but not in rats fed a low calcium diet, seem to be very interesting. Available evidence suggests that the effect of vitamin K2 on mineralllization of human peri osteal osteoblasts is enhanced in the presence of 1,25 dihydroxyvitain D in vitro (10). The effect of vitamin K2 on BMD in ovariectomized rats is affected by the plasma 25-hydroxyvitamin D level in vivo, and is signif icant only when rats are fed a diet containing vitamin D (11). Furthermore, since vitamin K2 is a cofactor in the y-carboxylation of Gla proteins, it was expected to improve bone matrix, while vitamin D was expected to improve matrix mineralization. Based on this line of evi dence, combined treatment with vitamin D3 and vita min K2 for osteopenia is surmised to be more effective than either treatment alone. In the present study, how ever, an additive effect on intestinal calcium absorption or renal calcium retention was not found in rats fed a low calcium diet, but that on intestinal calcium absorp tion was found in rats fed a normal calcium diet, Matsu naga et al. (12) and Hirano and Ishii (5) demonstrated the additive effect of vitamin K and vitamin D on bone mass in adult ovariectomized rats or young rats fed a normal calcium diet. Iwamoto et al. (13) also demon strated the efficacy of combined treatment with vitamin K2 and vitamin D3 in postmenopausal women with osteoporosis under calcium supplementation. Thus, we speculate that calcium supplementation may be needed to produce additive effects of vitamin K and vitamin D supplementation on calcium balance in the treatment of osteopenia in rats. Probably because increased uri nary excretion caused by vitamin D supplementation significantly counteracts an increase in renal calcium retention resulting from vitamin K supplementation in rats fed a low calcium diet, their additive effect was not significantly found under calcium deficiency. In conclusion, this study shows the differential effects of vitamin K and/or vitamin D supplementation on cal cium balance in young rats fed a normal or low calcium diet. Vitamin K supplementation promoted the reduc tion in urinary calcium excretion in rats fed a low cal cium diet, and stimulated intestinal calcium absorption by increasing serum 1,25(OH)2 D level in rats fed a nor mal calcium diet, On the other hand, vitamin D supple mentation stimulated intestinal calcium absorption via increased serum 1,25(OH)2 D level with prevention of the abnormal elevation of serum PTH level and pre vented hypocalcemia in rats fed a low calcium diet, and stimulated intestinal calcium absorption via increased serum 1,25(OH)2 D level without alterations in serum calcium or PTH levels in rats fed a normal calcium diet. Vitamin D supplementation increased urinary calcium excretion in rats fed a low or normal calcium diet. An additive effect of vitamin K and vitamin D on intestinal calcium absorption was found only in rats fed a normal calcium diet. REFERENCES 1) Matkovic V, Fontana D, Tominac C, Goel P, Chesnut CH III. 1990. Factors that influence peak bone mass forma tion: A study of calcium balance and the inheritance of bone mass in adolescent females. Am J Clin Nutr 52: 878-888. 2) Matkovic V 1992. Calcium and peak bone mass. J Inter Med 231: 151-160. 3) Kobayashi M, Hara K, Akiyama Y. 2002, Effects of vita min K2 (menatetrenone) on calcium balance in ovariec tomized rats. Jpn J Pharmacol 88: 55-61. 4) Kobayashi M, Hara K, Akiyama Y. 2002. Effect of menatetrenone (Vitamin K2) on bone mineral density and bone strength in Ca/Mg deficient rats. Nippon Yakurigaku Zasshi 120: 195-204 (in Japanese). 5) Hirano J, Ishii Y. 2002. Effects of vitamin K2, vitamin D, and calcium on the bone metabolism of rats in the growth phase. J Orthop Sci 7: 364-369. 6) Tomiuga T, Kobayashi M, Nakajima Y, Bessho M, Katoh Y, Hara K, Akiyama Y, Nakamura T, Tajima T. 1994. Effects of menatetrenone on the decrease in calcium balance induced by vitamin K-deficient diet and sodium loading in rats. Jpn J Pharrnacol 65: 35-43. 7) Ooms ME, Roos JC, Bezemer PD, van der Vijgh WJF, Bouter LM, Lips P 1995, Prevention of bone loss by vita min D supplementation in elderly women: A random ized double-blind trial. I Clin Endocrinol Metab 80: 1052-1058. 8) Baeksgaard L, Andersen KP, Hyldstrup L. 1998. Cal cium and vitamin D supplementation increases spinal BMD in healthy, postmenopausal women. Osteoporosis lot 8: 255-260. 9) Shiraishi A, Higashi S, Ohkawa H, Kubodera N, Hirakawa T, Ezawa I, Ikeda K, Ogata E. 1999. The advantage of alfacalcidol over vitamin D in the treat ment of osteoporosis. Calcif Tissue lot 65: 311-316. 10) Koshihara Y, Hoshi K, Shiraki M. 1992. Enhancement of mineralization on human osteoblasts by vitamin K2 (menaquinone 4). J Clin Exp Med 161: 439-440 (in Japanese). 11) Hara K, Akiyama Y, Tomiuga T, Kobayashi M, Naka mura T, Tajima T. 1994, Influence of vitamin D3 on inhibitory effect of vitamin K2 on bone loss in ovariecto mized rats. Folio Pharmacol Jpn 104: 101-109 (in Japa nese). 12) Matsunaga S, Ito H, Sakou T. 1999. The effect of vita min K and D supplementation on ovariectomy-induced bone loss. Calcif Tissue lot 65: 285-289. 13) Iwamoto J, Takeda T, Ichimura S. 2000. Effect of com bined administration of vitamin D3 and vitamin K2 on bone mineral density of the lumbar spine in postmeno pausal women with osteoporosis. J Orthop Sci 5; 546 551.