Effect of dietary magnesium supplementation on bone loss in rats fed a high phosphorus diet

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1 Magnesium Research 2005; 18 (2): 91-6 ORIGINAL ARTICLE Experimental paper Effect of dietary magnesium supplementation on bone loss in rats fed a high phosphorus diet S.-I. Katsumata 1, H. Matsuzaki 2, M. Uehara 1, K. Suzuki 1 1 Department of Nutritional Science, Faculty of Applied Bioscience, Tokyo University of Agriculture, Sakuragaoka, Setagaya-ku, Tokyo , Japan; 2 Department of Nutrition, Junior College of Tokyo University of Agriculture, Sakuragaoka, Setagaya-ku, Tokyo , Japan Correspondence: K. Suzuki, Department of Nutritional Science, Faculty of Applied Bioscience, Tokyo University of Agriculture, Sakuragaoka, Setagaya-ku, Tokyo , Japan. <kazu@nodai.ac.jp> Abstract. The purpose of this study was to investigate the effect of dietary magnesium (Mg) supplementation on bone loss in rats fed a high phosphorus (P) diet. Weanling Wistar strain rats were randomly divided into four dietary groups of 6 rats each and fed their respective diets; a diet containing 0.3% P and 0.05% Mg (C), a diet containing 1.5% P and 0.05% Mg (HP), a diet containing 0.3% P and 0.15% Mg (HMg), or a diet containing 1.5% P and 0.15% Mg (HPMg), for 21 days. Compared to the C and HMg groups, serum parathyroid hormone (PTH) concentration was significantly higher in the HP and HPMg groups. Serum osteocalcin concentration and urinary excretion of C-terminal telopeptides of type I collagen (CTx), markers of bone turnover, were significantly higher in the HP and HPMg groups than in the C and HMg groups. Dietary Mg supplementation had no significant effects on serum PTH and osteocalcin concentrations, while urinary excretion of CTx was significantly lower in the HPMg group than in the HP group. These results suggested that dietary Mg supplementation suppressed bone resorption due to high P diet. Key words: dietary Mg supplementation, high P diet, Mg availability, bone resorption, rat It is well known that an excess of phosphorus (P) intake impairs bone mineralization. A high P diet accelerates bone resorption via an elevated parathyroid hormone (PTH) secretion. Previous reports have shown that elevated PTH resulted from changes in serum concentrations of calcium (Ca) and P due to high P diet [1, 2]. In both animal [3-5] and human subjects [6], consumption of high P resulted in bone loss as expected given the results of an increase in the bone resorption marker. Magnesium (Mg) is also one of the constituents of bone, and plays important roles in bone metabolism. Some studies have shown that serum Mg concentration is decreased in postmenopausal osteoporosis [7, 8]. In addition, Mg deficiency caused bone loss [9, 10], and is one of the risk factors for osteoporosis. Recently, some researchers examined the effect of dietary Mg supplementation on bone metabolism. Our previous study showed that dietary Mg supplementation increased the dynamic strength of bone by promoting bone formation and preventing bone resorption in ovariectomized rats [11]. Dimai et al. reported that daily oral Mg supplementation suppressed bone turnover in healthy young adult males [12]. In postmenopausal women, Mg supplementation significantly increased bone density or arrested bone loss [13]. These observations suggest that Mg supplementation is beneficial for the prevention of bone loss. Accordingly, we predicted that dietary Mg supplementation would be beneficial for bone loss induced by high P diet. The purpose of this study was to 91

2 S.-I. KATSUMATA, ET AL. investigate the effect of dietary Mg supplementation on bone loss in rats fed a high P diet. Materials and methods Experimental design Twenty-four 4-week-old male Wistar strain rats (Clea Japan, Tokyo, Japan) were individually housed in stainless-steel metabolic cages in a room maintained at 22 C with a 12-hour light-dark cycle. The Tokyo University of Agriculture Animal Use Committee approved the study and the animals were maintained in accordance with the guidelines for the care and use of laboratory animals of the university. All rats were fed a control diet containing 0.3% P and 0.05% Mg for a 7 day acclimatization period. After the acclimatization period, all rats were randomly divided into four dietary groups of 6 rats each and fed four diets based on the AIN-93G diet [14] (table 1): the control diet (C), a diet containing 1.5% P and 0.05% Mg (HP), a diet containing 0.3% P and 0.15% Mg (HMg), or a diet containing 1.5% P and 0.15% Mg (HPMg). All experimental diets contained 0.5% Ca. The P, Mg and Ca contents, as determined from an analysis of the experimental diets, are shown in table 1. All rats were allowed to eat ad libitum and given free access to distilled water for 21 days. Their feces were collected for the last 3 days of the experimental period. In the last day of the experimental period, their urine was collected for analysis. At the end of the experimental period, all rats were sacrificed, and then blood and femur samples were collected for analysis. Serum minerals and PTH analysis The blood samples were centrifuged at 3000g for 15 minutes, and the supernatants were used as serum samples. Serum samples were stored at -80 C until analysis. Serum P was measured with P-test Wako (Wako Pure Chemical Industries, Osaka, Japan). Serum Mg and Ca were analyzed by atomic absorption spectrophotometry (Hitachi A-2000) [15]. Serum PTH was measured by enzyme-linked immunosorbent assay (ELISA) kit (Immutopics Inc, San Clemente, Calif., USA). Measurements of apparent absorption of minerals For measurements of P, Mg and Ca, feces were dried, ashed and then demineralized with 1 mol/l HCl solution. P was analyzed colorimetrically according to the method of Gomori [16]. Mg and Ca were analyzed by atomic absorption spectrophotometry. Apparent absorption of minerals was calculated as the difference between intakes and fecal excretion. Table 1. Composition of the experimental diets. C HP HMg HPMg Ingrédient g/kg/ diet Casein Corn starch Sucrose Soybean oil Cellulose powder Mineral mixture a Vitamin mixture b L-Cystine Choline bitartrate Ter-butylhydroquinone KH 2 PO MgO Chemical analysis g/kg/ diet P level Mg level Ca level a AIN-93G mineral mixture. b AIN-93 vitamine mixture. 92

3 DIETARY MG SUPPLEMENTATION AND BONE LOSS Measurements of markers of bone turnover Serum osteocalcin was measured with Osteocalcin rat ELISA system (Amersham Pharmacia Biotech, UK). Urinary C-terminal telopeptide of type I collagen (CTx) was measured with RatLaps ELISA (Nordic Bioscience Diagnostics A/S, Denmark). Urinary creatinine was measured with Creatinine-test Wako (Wako Pure Chemical Industries). Measurements of bone mineral content (BMC) and bone mineral density (BMD) Femurs were removed, cleansed of all soft tissues and were frozen at -80 C until analysis. BMC and BMD of the femur were measured by the dual-energy X-ray absorptiometry (DEXA) using DCS-600EX (Aloka, Tokyo, Japan). The mineralization profiles of the specimens were stored as the monitoring images, and the BMC and BMD values for the femur were obtained. Measurements of mineral concentrations in the femur Femurs were extracted for 48 hours with ether, dried for 24 hours and weighed. The dried fat-free femurs were ashed and then demineralized with 1 mol/l HCl solution. P was analyzed colorimetrically. Mg and Ca were analyzed by atomic absorption spectrophotometry. Statistical analysis The data are presented as the means ± SE for each group of six rats. After two-way analysis of variance (ANOVA), Fisher s PLSD was used to determine significant differences in multiple comparisons among groups. A p value of less than 0.05 was considered significant. Results Body weight and food intake Final body weight, weight gain and food intake were significantly lower in the HP and HPMg groups than in the C and HMg groups (table 2). Dietary Mg supplementation had no significant effects on final body weight, weight gain and food intake. Serum minerals and PTH concentrations Serum P concentration was significantly higher in the HP group than in the C group, and was significantly lower in the HPMg group than in the HP group (table 3). Serum Mg concentration was significantly lower in the HP group than in the C group, and was significantly higher in the HPMg group than in the HP group. There was no significant difference in serum P and Mg concentrations between the C and HMg groups. Serum Ca concentration was significantly lower in the three experimental groups compared to the C group. There was no significant difference in serum Ca concentration between the HP and HPMg groups. Serum PTH concentration was significantly higher in the HP and HPMg groups than in the C and HMg groups. Dietary Mg supplementation had no significant effect on serum PTH concentration. Apparent absorption of minerals Apparent P absorption was significantly higher in the HP group than in the C group, and was significantly lower in the HPMg group than in the HP group (table 4). There was no significant difference in apparent P absorption between the C and HMg groups. Apparent Mg absorption was significantly Table 2. Body weight and intakes of food and minerals. C HP HMg HPMg Two-way ANOVA 1 Initial body weight (g) ± ± ± ± 0.8 Final body weight (g) ± 2.9 a ± 5.1 a ± 3.2 a 215 ± 6.1 b P Food intake (g/day) 16.8 ± 0.2 a 13.5 ± 0.3 b 17.2 ± 0.2 a 13.5 ± 0.3 b P P intake (mg/day) 52.6 ± 0.7 a ± 4.7 b 53.3 ± 0.6 a ± 5.0 b P Mg intake (mg/day) 8.2 ± 0.1 a 6.4 ± 0.1 b 24.8 ± 0.3 c 20.5 ± 0.5 d P, M, P M Ca intake (mg/day) 81.9 ± 1.0 a 65.1 ± 1.5 b 82.2 ± 1.0 a 64.4 ± 1.6 b P a,b,c,d Values with different superscript letters in the same row are significantly different (p < 0.05). 93

4 S.-I. KATSUMATA, ET AL. Table 3. Serum mineral and PTH concentrations. C HP HMg HPMg Two-way ANOVA 1 P (mg/dl) 9.47 ± 0.25 a ± 0.56 b 9.37 ± 0.20 a ± 0.37 c P, M, P M Mg (mg/dl) 2.47 ± 0.05 a 2.05 ± 0.06 b 2.38 ± 0.04 b,c 2.32 ± 0.05 c P, P M Ca (mg/dl) ± 0.08 a 9.50 ± 0.13 b ± 0.08 c 9.42 ± 0.08 b P, M, P M PTH (mg/dl) ± a ± b ± ± b P a,b,c Values with different superscript letters in the same row are significantly different (p < 0.05). Table 4. Apparent absorption of minerals. C HP HMg HPMg Two-way ANOVA 1 P (mg/day) 42.0 ± 1.2 a ± 4.2 b 37.7 ± 1.1 a ± 6.0 c P, M, P M Mg (mg/day) 7.1 ± 0.1 a 5.1 ± 0.1 b 15.5 ± 0.4 c 10.4 ± 0.5 d P, M, P M Ca (mg/day) 59.5 ± 2.0 a 48.6 ± 1.6 b 58.9 ± 1.1 a 45.6 ± 1.6 b P a,b,c,d Values with different superscript letters in the same row are significantly different (p < 0.05). lower in the HP and HPMg groups than in the C and HMg groups, respectively. Compared to the C and HP groups, apparent Mg absorption was significantly increased in the HMg and HPMg groups, respectively. Apparent Ca absorption was significantly lower in the HP and HPMg groups than in the C and HMg groups. Dietary Mg supplementation had no significant effect on apparent Ca absorption. Markers of bone turnover Serum osteocalcin concentration was significantly higher in the HP and HPMg groups than in the C and HMg groups (table 5). Dietary Mg supplementation had no significant effect on serum osteocalcin concentration. Urinary excretion of CTx was significantly higher in the HP and HPMg groups than in the Table 5. Markers of bone turnover and BMC, BMD and mineral concentrations of the femur. C HP HMg HPMg Two-way ANOVA 1 Serum Osteocalcin (ng/ml) ± 4.49 a ± 9.72 b ± 5.32 b ± 7.21 b P Urine CTx (lg/mmol creatinine) ± 1.96 a ± 8.30 b ± 2.13 a ± 4.02c P, M, P M Femur BMC (mg) ± 2.7 a ± 4.1 b ± 3.0 a ± 2.9 b P Femur BMD (mg/cm 2 ) 97.3 ± 1.1 a 90.6 ± 0.9 b 97.5 ± 0.9 a 90.3 ± 1.0 b P Femur P (mg/g) ± ± ± ± 0.8 Femur Mg (mg/g) 4.4 ± 0.1 a 4.0 ± 0.1 b 4.5 ± 0.1 a,c 4.6 ± 0.1 c M, P M Femur Ca (mg/g) ± ± ± ± 1.0 a,b,c Values with different superscript letters in the same row are significantly different (p < 0.05). 94

5 DIETARY MG SUPPLEMENTATION AND BONE LOSS C and HMg groups. Although there was no significant difference in urinary excretion of CTx between the C and HMg groups, urinary excretion of CTx was significantly lower in the HPMg group than in the HP group. BMC and BMD of the femur BMC and BMD of the femur were significantly lower in the HP and HPMg groups than in the C and HMg groups (table 5). Dietary Mg supplementation had no significant effect on BMC and BMD of the femur. Mineral concentrations in the femur P and Ca concentrations in the femur were not significantly different among all four groups (table 5). Mg concentration in the femur was significantly lower in the HP group than in the C group, and was significantly higher in the HPMg group than in the HP group. There was no significant difference in Mg concentration between the C and HMg groups. Discussion A high P diet may lead to secondary hyperparathyroidism, which in turn results in high bone turnover and bone loss. In this study, serum PTH concentration was elevated in rats fed the high P diet. Furthermore, the high P diet increased both serum osteocalcin concentration and urinary excretion of CTx, which indicated an increase in bone turnover. Consequently, BMC and BMD of the femur were decreased in rats fed the high P diet. These results suggested that an increase in bone turnover caused the bone loss observed in rats fed a high P diet. This conclusion is supported by a previous report that high bone turnover has been implicated as a significant etiological factor for bone loss [17]. Recently, researchers have begun to examine the effect of dietary Mg supplementation on bone metabolism. They suggested that Mg supplementation might be beneficial for the prevention of osteoporosis [11-13]. In this study, dietary Mg supplementation had no influence on BMC and BMD of the femur and serum osteocalcin concentration. However, dietary Mg supplementation decreased urinary excretion of CTx in rats fed the high P diet. These results suggested that dietary Mg supplementation suppressed the bone resorption that was increased by a high P diet. The results of this study suggest that increased dietary Mg supplementation (above the levels used in this study) may inhibit the bone loss induced by a high P diet. With regard to Mg availability, Mg concentrations of serum and femur were decreased in rats fed the high P diet. Furthermore, a high P diet decreased apparent Mg absorption and caused a negative Mg balance. Consequently, Mg availability was decreased in rats fed a high P diet. On the other hand, dietary Mg supplementation increased apparent Mg absorption and Mg concentrations of serum and femur in rats fed the high P diet. These results suggested that dietary Mg supplementation improved Mg availability in rats fed the high P diet, and might suppress bone resorption. Dietary Mg supplementation decreased apparent P absorption and serum P concentration, though the high P diet increased them. Dietary Mg supplementation may decrease P absorption by forming insoluble salts between P and Mg in the intestinal lumen [18]. Our results suggest that dietary Mg supplementation decreased serum P concentration via an inhibition of P absorption. In spite of a decrease in serum P concentration, dietary Mg supplementation did not change serum PTH concentration in rats fed the high P diet. Therefore, dietary Mg supplementation inhibited the influence of excess P on bone resorption, although it showed no effects on serum PTH concentration at the Mg levels used in this study. As stated above, the cause of bone loss by a high P diet is believed to result from secondary hyperparathyroidism that is related to changes in Ca and P metabolism [1, 2]. In this study, a high P diet elevated serum PTH concentration, which increased bone turnover. Furthermore, Mg availability was decreased in rats fed the high P diet. In other words, a high P diet mimicked the effects of Mg deficiency on Mg availability and bone metabolism. Many studies have shown that Mg deficiency induces bone loss, due to increased bone resorption [9, 10]. In this study, dietary Mg supplementation suppressed bone resorption in rats fed the high P diet, regardless of circulating PTH concentration. We suggest that suppression of bone resorption with dietary Mg supplementation resulted from increasing Mg availability which had decreased due to a high P diet. In other words, these results suggest that bone resorption induced by a high P diet is related to a decrease in Mg absorption as well as changes of Ca and P metabolism. Conclusion This study investigated the effects of dietary Mg supplementation on bone loss in rats fed a high P diet. A high P diet increased serum PTH and osteo- 95

6 S.-I. KATSUMATA, ET AL. calcin concentrations and urinary excretion of CTx. Dietary Mg supplementation did not affect serum PTH and osteocalcin concentrations. However, dietary Mg supplementation decreased urinary excretion of CTx in rats fed the high P diet. These results suggested that dietary Mg supplementation suppresses the bone resorption enhanced by a high P diet, regardless of serum PTH concentration. Acknowledgements The authors thank Dr. Yoshiko Ishimi for supporting to measure BMC and BMD, and Ms. Melissa Melby for critical reading of this manuscript. References 1. Almaden Y, Canalejo A, Hernandez A, Ballesteros E, Garcia NS, Torres A, Rodriguez M. Direct effect of phosphorus on PTH secretion from whole rat parathyroid glands in vitro. J Bone Miner Res 1996 ; 11 : Masuyama R, Uehara M, Suzuki K. High P diet induces acute secretion of parathyriodhormone without alteration of serum calcium levels in rats. Biosci Biotechnol Biochem 2000 ; 64 : Bauer KD, Griminger P. Long-term effects of activity and of calcium and phosphorus intake on bones and kidneys of female rats. J Nutr 1983 ; 113 : Draper HH, Sie TL, Bergan JG. Osteoporosis in aging rats induced by high phosphorus diets. J Nutr 1972 ; 102 : Katsumata S, Masuyama R, Koshihara M, Matsuzaki H, Uehara M, Suzuki K. High phosphorus diet changes phosphorus metabolism regardless of PTH action in rats. Biosci Biotechnol Biochem 2004 ; 68 : Bell RR, Draper HH, Tzeng DY, Shin HK, Schmidt GR. Physiological responses of human adults to foods containing phosphate additives. J Nutr 1977 ; 107 : Cohen L. Recent data on magnesium and osteoporosis. Magnesium Res 1988 ; 1 : Reginster JY, Strause L, Deroisy R, Lecart MP, Saltman P, Franchimont P. Preliminary report of decreased serum magnesium in postmenopausal osteoporosis. Magnesium 1989 ; 8 : Rude RK, Kirchen ME, Gruber HE, Stasky AA, Meyer MH. Magnesium deficiency induces bone loss in the rat. Miner Electrolyte Metab 1998 ; 24 : Rude RK, Kirchen ME, Gruber HE, Meyer MH, Luck JS, Crawford DL. Magnesium deficiency-induced osteoporosis in the rat : uncoupling of bone formation and bone resorption. Magnesium Res 1999 ; 12 : Toba Y, Kajita Y, Masuyama R, Takada Y, Suzuki K, Aoe S. Dietary magnesium supplementation affects bone metabolism and dynamic strength of bone in ovariectomized rats. J Nutr 2000 ; 130 : Dimai HP, Porta S, Wirnsberger G, Lindschinger M, Pamperl I, Dobnig H, Wilders-Truschnig M, Lau KHW. Daily oral magnesium supplementation suppresses bone turnover in young adult males. J Clin Endocrinol Metab 1998 ; 83 : Stendig-Lindberg G, Tepper R, Leichter I. Trabecular bone density in a two-year controlled trial of peroral magnesium in osteoporosis. Magnesium Res 1993 ; 6 : Reeves PG, Nielsen FH, Fahey GC. AIN-93 purified diets for laboratory rodents : final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J Nutr 1993 ; 123 : Gimblet EG, Marney AF, Bonsnes RW. Determination of calcium and magnesium in serum, urine, diets and stool by atomic absorption spectrophotometry. Clin Chem 1967 ; 13 : Gomori G. A modification of the colorimetric phosphorus determination for use with the photoelectric colorimeter. J Lab Clin Med 1942 ; 27 : Ravn P, Rix M, Andreassen H, Clemmesen B, Bidstrup M, Gunnes M. High bone turnover is associated with low bone mass and spinal fracture in postmenopausal women. Calcif Tissue Int 1997 ; 60 : Hardwick LL, Jones MR, Brautbar N, Lee DB. Magnesium absorption : mechanisms and the influence of vitamin D, calcium and phosphate. J Nutr 1991 ; 121 :