Milk Calcium Taken with Cheese Increases Bone Mineral Density andbonestrengthingrowingrats

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1 Biosci. Biotechnol. Biochem., 66 (11), , 2002 Milk Calcium Taken with Cheese Increases Bone Mineral Density andbonestrengthingrowingrats Ken KATO, 1, Yukihiro TAKADA, 2 Hiroaki MATSUYAMA, 2 Yoshihiro KAWASAKI, 2 Seiichiro AOE, 2 Hideo YANO, 3 and Yasuhiro TOBA 2 1 Product Planning and Development Division, Snow Brand Frozen Foods Co., Ltd., Kawagoe, Saitama , Japan 2 Technology and Research Institute, Snow Brand Milk Products Co., Ltd., Kawagoe, Saitama , Japan 3 Division of Applied Bioscience, Graduate School of Agriculture, Kyoto University, Kyoto , Japan Received May 2, 2002; Accepted July 5, 2002 We investigated the calcium bioavailability of milk calcium, taken with or without cheese. Twenty-four 6- week-old male rats for a meal-feeding experiment were trained to consume an AIN-76 diet within 2 h (2 times perday)for2weeks.theratswerethendividedinto three experimental groups, each fed 2 types of experimental diets: Control group, Cheese group, and Ca- Cheese group. The rats were each alternately given 2 types of experimental diets at 2-h meal-feeding for 31 days. The breaking force and energy of the femur in the Ca-Cheese group were signiˆcantly higher than in the control group. The bone mineral density (BMD) of the lumbar spine and the femur in the Ca-Cheese group was also signiˆcantly higher than in the other two groups. These results indicate that milk calcium taken with cheese increases bone strength and BMD e ciently, results that may be useful for the prevention of osteoporosis. Key words: cheese; milk calcium; bioavailability; bone; rats With the prolongation of the human life span, the incidence of metabolic diseases such as osteoporosis and bone fracture increase in the elderly. Osteoporosis is a disorder characterized by bone loss and is associated with an increased risk of fractures and back or joint pain. It is more frequently observed in the elderly and in women after menopause. In particular, the bone mass of many women after menopause decreases and the risk of fracture increases rapidly, since the bone mass of women is generally lower than that of men. 1,2) Femoral neck fracture is one of the most serious injuries found in elderly patients in orthopaedics. It is important for the prevention of osteoporosis to take enough dietary nutrients, especially foods with a high content of calcium. Dietary calcium, which is more absorbable and bioavailable, is essential when considering calcium requirements in the elderly, since the absorbability and bioavailability of calcium declines with age. Milk is a safe food that can be taken over a long time, and it has been useful for human health. Milk is also an excellent source of calcium, considering the absorption of calcium and its bioavailability. 3 5) Calcium in milk is more absorbable and bioavailable than calcium in ˆsh or vegetables and as calcium carbonate. 3,6,7) Milk also contains lactose, casein phosphopeptides (CPP) that formed by proteolytic digestion of casein in gut, and vitamin D, which accelerate calcium absorption. 8,9) Recently, to increase daily calcium intake, commercially available calcium-fortiˆed foods have been placed on the market. These foods are fortiˆed with many calcium sources such as calcium carbonate, calcium phosphate, cattle bone powder, and milk calcium. In particular, milk calcium is more absorbable than any other calcium source. Also, colloidal calcium and calcium caseinate in milk are considered to be more absorbable and bioavailable than ionic calcium, and the chemical form of milk calcium may ašect calcium absorbability and bioavailability. 10,11) We prepared food consisting basically of skim milk powder, fortiˆed with milk calcium. The calciumfortiˆed food was a good calcium source, considering an increase of bone mineral contents and bone strength. 12) Calcium from cheddar cheese and processed cheese is as well utilized as from cow milk. 4) The absorbability of calcium from cheese is high, since cheese contains CPP, which are formed by proteolytic digestion of casein in gut and increases calcium absorption. 13) Ripened cheese is an especially good calcium source for lactase-deˆcient individuals. 14) The purpose of this study is to investigate the calcium bioavailability of milk calcium, taken with To whom correspondence should be addressed. Fax: ; k-ken@mxp.mesh.ne.jp Abbreviations: BMD, bone mineral density; CPP, casein phosphopeptides

2 Bioavailability of Milk Calcium Taken with Cheese 2343 Table 1. Composition of Milk Calcium Milk calcium g W kg Moisture 40 Protein 85 Fat 5 Carbohydrate 60 Ash 810 gwkg Chemical analysis Calcium 163 Phosphorus 100 Magnesium 10 Table 2. Calcium Compositions of Cheese and Cheese Fortiˆed with Milk Cheese Cheese fortiˆed with milk calcium Moisture (gw100 g) Fat Protein Ash Carbohydrate Ca (mg W 100 g) P Mg Na K or without cheese, by examining bone strength and bone mineral density (BMD) in growing male rats. Materials and Methods Preparation of milk calcium. Milk calcium was obtained from milk whey as follows. Milk whey was prepared from fresh milk, which was defatted by centrifugation and adjusted to ph 4.2 with HCl. Milk whey was concentrated by evaporation 10 times and cooled to 49C for 12 h, and the lactose was precipitated and separated by centrifugation at 3,000 g.its supernatant was adjusted to ph 6.8 with 1 N NaOH. The solution was centrifuged at 3,000 g and the residual lactose was removed. Its precipitate was washed with deionized water and the whey protein was separated by ultraˆltration (molecular mass cutoš, 100 kda). Its retentate was collected and spray-dried. The concentrations of calcium, phosphorus, and magnesium in the milk calcium were 163, 100, and 10 g Wkg, respectively (Table 1). Composition of cheese fortiˆed with milk calcium. Cheese fortiˆed with milk calcium used in this experiment was processed cheese, which was prepared from two types of natural cheeses (Gouda and cheddar) and milk calcium. The composition of cheese forti- ˆed with milk calcium is shown in Table 2. The calcium concentrations of cheese fortiˆed with milk calcium and non-calcium fortiˆed cheese, which was processed cheese and prepared from the same natural cheese as cheese fortiˆed with milk calcium, were 1,200 mg W 100 g and 630 mg W100 g, respectively (Table 2). Diets. The composition of the pre-experimental diet is shown in Table 3. The diet was prepared according to the AIN-76 diet. 15) The composition of experimental diets is shown in Table 4. During the experimental period, each group was given 2 types of experimental diets (Type I diet and Type II diet). In the control diet, soy protein isolate was used as the sole source of protein, and calcium carbonate and Table 3. Composition of Pre-Experimental Diet* (z) Casein 20.0 Cornstarch 15.0 Cellulose 5.0 Corn oil 5.0 Sucrose 50.2 DL-Methionine 0.3 Vitamin mixture** 1.0 Mineral mixture*** 3.5 * This diet was that of AIN-76 (American Institute of Nutrition, 1977). ** Identical to AIN-76 vitamin mixture. *** Identical to AIN-76 mineral mixture. milk calcium were used as the sources of calcium (Type I diet: calcium carbonate 0.5z; milk calcium 1.1z. Type II diet: calcium carbonate 0.5z). In the Cheese diet, 25z soy protein isolate and 50z calcium carbonate in the control diet were replaced by non-calcium-fortiˆed cheese, because the same amount of milk calcium and cheese as cheese fortiˆed with milk calcium was fed separately (Type I diet: calcium carbonate 0.5z; milk calcium 1.1z.TypeII diet: cheese 35z). In the Ca-Cheese diet, 25z soy protein isolate, 50z calcium carbonate, and 100z milk calcium in the control diet were replaced by cheese fortiˆed with milk calcium, because milk calcium and cheese were fed at the same time (Type I diet: cheese fortiˆed with milk calcium 35z. TypeII diet: calcium carbonate 0.5z). The amounts of protein, sulfur-containing amino acid, calcium, phosphorus, and magnesium were equal in the three experimental diets of 2 types each. Animals. Twenty-four 6-week-old male Sprague- Dawley rats (Charles River Inc., Kanagawa, Japan) were housed in individual stainless steel wire-mesh cages in a temperature- and humidity-controlled room (239C and60±5z relative humidity) with a 12-h lightwdark cycle. All rats were treated in accordance with the NIH ``Guide for the Care and Use of Laboratory Animals''. 16) In the pre-experimental period, rats for the meal-feeding experiment were

3 2344 K. KATO et al. Table 4. Compositions of Experimental Diets (z) Control Cheese Ca-Cheese Type I Type II Type I Type II Type I Type II Soy protein isolate* Cheese Cheese fortiˆed with milk calcium Cornstarch Cellulose Corn oil Sucrose L-Cystine DL-Methionine Milk calcium CaCO NaCl K 3 C 6 H 5 O 7 H 2 O Citric acid monohydrate KH 2 PO K 2 CO Na 2 CO K 2 SO MgSO MgO Vitamin mixture** Basal mineral mixture*** * Fujipro R, Fujipurina Protein Ltd., Tokyo, Japan. ** Identical to AIN-76 vitamin mixture. *** Contained 17.5z MnCO 3, 30.0z Fe-citrate (Fe 17z), 8.0z ZnCO 3,1.5z CuCO 3,0.05z Na 2SeO 3 5H 2O, 0.05z KIO 3, and 2.75z CrK(SO 4) 2 12H 2O. trained to consume an AIN-76 diet within 2 h (2 times per day) and given free access to deionized water for 2 weeks. 17) After a 2-week period, they were separated into three experimental groups of 8 rats each, and each rat had a similar body weight of 248 g. All rats were alternately given the Type I diet (morning on the odd-numbered days and evening on the even-numbered days) and the Type II diet (evening on the oddnumbered days and morning on the even-numbered days) for each of the 31 times of a 2-h meal-feeding (2 times per day). They were also given free access to deionized water for 31 days. Body weight was recorded once a week, and food intake was monitored daily. After the 31-day feeding period, the rats were deprived of food overnight and anesthetized with diethyl ether. Blood was collected from the vena cava inferior and the serum prepared. The femora and the fourth lumbar spine were both excised from the rats, and the muscles and connective tissues were removed. The bone samples were kept in phosphate bušer before measurement. Serum analysis. Serum calcium was measured by an automatic analyzer (Fuji Drychem Systems, Tokyo, Japan). Measurement of BMD of the fourth lumbar spine and the femur. The BMD of the fourth lumbar spine and the femur were measured by dual-energy X-ray absorptiometry, with a Dichroma Scan DCS-600A (Aloka, Tokyo, Japan) adapted for measuring small animals with beam energies of 22 and 53 kev. The scanning speed was 10 mm Ws, and each scanning step was 1 mm. Measurement of bone strength of the femur. The breaking force and energy of the femoral diaphysis (the center of the femur) were measured by a threepoint bending rheolometer (RX-1600, Aitechno, Japan), as in the methods of Ezawa et al. 18) and Takada et al. 19) The measurement conditions were as follows: sample space, 1.0 cm; plunger speed, 20 mm Wmin; and load range, 40.0 kg. Statistical analysis. Statistical analysis was done by one-way ANOVA. When F ratios were signiˆcant, individual means were compared by a Tukey-Kramer test. All calculations were performed with Super ANOVA software (Abacus Concepts, CA). DiŠerences with Pº0.05 were taken to be signiˆcant. Results Body weight gain, food intake and food e ciency The amounts of daily calcium intake were equal in the three experimental groups (52.5 mgwday). The body weight gain, food intake, and food e ciency were not signiˆcantly dišerent among the experimental groups (Table 5).

4 Bioavailability of Milk Calcium Taken with Cheese 2345 Table 5. Body Weight Gain, Food Intake, and Food E ciency Group Body weight gain (gwday) Food intake (gwday) Food e ciency (z) Control 3.7± ± ±1.7 Cheese 3.4± ± ±2.5 Ca-Cheese 3.3± ± ±2.1 Values are means±sd, Table 6. Concentration of Serum Calcium (Ca) in 12-Week-old Male Rats Control Cheese Ca-Cheese Ca (mgw100 ml) 8.7± ± ±0.2 Values are means±sd, Fig. 2. EŠects of Experimental Diets on Segmental Bone Mineral Density (BMD) of the Femur in 12-Week-old Male Rats. a; Signiˆcantly dišerent from the control group (Pº0.05). b; Signiˆcantly dišerent from the control and the Cheese group (Pº0.05). c; Signiˆcantly dišerent from the Cheese group (Pº0.05). Fig. 1. EŠects of Experimental Diets on Bone Mineral Density (BMD) of the Fourth Lumbar Spine in 12-Week-old Male Rats. * Signiˆcantly dišerent from the control group (Pº0.05). Serum analysis The serum concentration of calcium was normal and not signiˆcantly dišerent among the experimental groups (Table 6). BMD of the fourth lumbar spine and the femur The BMD of the lumbar spine in rats fed the Cheese diet and Ca-Cheese diet were signiˆcantly higher than in rats fed the control diet (Fig. 1). In the 9 segmental BMD of the femur, the segment 2 BMD in rats fed the Ca-Cheese diet was signiˆcantly higher than in rats fed the control diet; the segment 3 BMD in rats fed the Ca-Cheese diet was signiˆcantly higher than in rats fed the control and Cheese diets; and the segment 4 BMD in rats fed the Ca-Cheese and control diets were signiˆcantly higher than in rats fed the Cheese diet (Fig. 2). Fig. 3. EŠects of Experimental Diets on Breaking Force and Breaking Energy of the Femur in 12-Week-old Male Rats. * Signiˆcantly dišerent from the control group in breaking force (Pº0.05). Signiˆcantly dišerent from the control group in breaking energy (Pº0.05). Breaking force and energy of the femur The breaking force and energy of the excised femur in rats fed the Ca-Cheese diet were signiˆcantly higher than in rats fed the control diet (Fig. 3). Signiˆcantly positive correlation was found between the BMD and the breaking force of the femur, and the BMD and the breaking energy of the femur, respectively (breaking force, r=0.60 and Pº0.05; breaking energy, r=0.50 and Pº0.05). Discussion Cheese is an excellent calcium source, which has absorbable calcium. 4) The absorbability of calcium

5 2346 K. KATO et al. from cheese is high, since cheese contains CPP, which are formed by proteolytic digestion of casein in gut and increases calcium absorption. 13) In this study we evaluated the calcium bioavailability of milk calcium, which is well known as a good calcium source, 10,20,21) taken with or without cheese. The breaking force and energy of the femur in rats fed cheese fortiˆed with milk calcium are signiˆcantly higher than in rats fed the control diet (Fig. 3). Milk calcium taken with cheese increased bone strength e ciently. Bone strength is increased when the absorption or retention of calcium is increased. In this study, the BMD of the lumbar spine in rats fed cheese fortiˆed with milk calcium was higher than in rats fed the control diet (Fig. 1). And in segmental BMD of the femur in rats fed cheese fortiˆed with milk calcium was higher than in rats fed the Cheese diet or the control diet (Fig. 2). Also, signiˆcantly positive correlation was found between the BMD and the breaking force of the femur, and the BMD and the breaking energy of the femur. These results indicate that the increase of bone strength in rats fed milk calcium taken with cheese is due to the increased bone mineral. From this study, milk calcium taken with cheese increased bone strength and BMD e ciently. Therefore the increased bone strength and BMD in rats fed cheese fortiˆed with milk calcium is due to their taking milk calcium and cheese, which contain absorbable calcium, at the same time. In conclusion, these results indicate that milk calcium taken with cheese is an excellent calcium source, better than milk calcium taken without cheese. Also, milk calcium taken with cheese increases bone strength and BMD e ciently, which may be useful for the prevention of osteoporosis. References 1) Seeman, E., Cooper, M., and Hopper, J. L., EŠect of early menopause on bone mass in normal women and patients with osteoporosis. Am. J. Med., 85, (1988). 2) Kanis, J. A., Geusens, P., and Christiansen, C., Guidelines for clinical trials in osteoporosis. Osteoporosis Int., 1, (1991). 3) Wong, N. P., and LaCroix, D. E., Biological availability of calcium in dairy products. Nutr. Rep. Int., 21, (1980). 4) Kansal, V. K., and Chaudhary, S., Biological availability of calcium, phosphorus and magnesium from dairy products. Milchwissenschaft, 37, (1982). 5) Poneros-Schneier, A. G., and Erdman, J. W., Jr., Bioavailability of calcium from sesame seeds, almond powder,wholewheatbread,spinachandnonfatdry milk in rats. J. Food Sci., 54, (1982). 6) Kanematsu, S., Studies on the utilization of various foods as calcium source and the calcium requirement in adult men. Eiyo to Shokuryo (in Japanese), 6, (1953). 7) Uenishi, K., Ezawa, I., Kajimoto, M., and Tsuchiya, F., Calcium absorption from milk, ˆsh (pond smelt, sardine) and vegetables (komatsuna-green, jew's marrow, saltwort) in Japanese young women. Nippon Eiyo Shokuryo Gakkaishi (in Japanese), 51, (1998). 8) Sato, R., Noguchi, T., and Naito, H., Casein phosphopeptide (CPP) enhances calcium absorption from the ligated segment of rat small intestine. J. Nutr. Sci. Vitaminol., 32, (1986). 9) Takeuchi, A., Okano, T., Tsugawa, N., Katayama, M., Mimura, Y., and Kobayashi, T., Determination of vitamin D and its metabolites in human and cow's milk. J. Micronutr. Anal., 4, (1988). 10) Igarashi, C., and Ezawa, I., EŠects of whey calcium and lactulose on the strength of bone in ovariectomized osteoporosis model rats. Oyo-yakuri (in Japanese), 42, (1991). 11) Kato, K., Takada, Y., Matsuyama, H., Aoe, S., Sakamoto, T., Yahiro, M., and Nakajima, I., Bioavailability of milk calcium prepared from rennet casein in rats. Nippon Eiyo Shokuryo Gakkaishi (in Japanese), 47, (1994). 12) Kato, K., Toba, Y., Takada, Y., and Aoe, S., Mineral bioavailability of fortiˆed food based on skim milk powder in rats. Jpn. J. Nutr. (in Japanese), 55, (1997). 13) Naito, H., The mechanism of enhancement in intestinal calcium absorption with phosphopeptides derived during casein digestion. Nippon Eiyo Shokuryo Gakkaishi (in Japanese), 39, (1986). 14) Tunick, M. H., Calcium in dairy products. J. Dairy Sci., 70, (1987). 15) American Institute of Nutrition, Report of the American Institute of Nutrition ad hoc committee on standards for nutritional studies. J. Nutr., 107, (1977). 16) National Research Council, Guide for the Care and Use of Laboratory Animals. Publication No (revised), National Institutes of Health, Bethesda, MD (1985). 17) Lee, Y. S., Noguchi, T., and Naito, H., Phosphopeptides and soluble calcium in the small intestine of rats given a casein diet. Br.J.Nutr., 43, (1980). 18) Ezawa, I., Okada, R., Nozaki, Y., and Ogata, E., Breaking-properties and ash contents of the femur of growing rats fed a low calcium diet. Nippon Eiyo Shokuryo Gakkaishi (in Japanese), 32, (1979). 19) Takada, Y., Matsuyama, H., Kato, K., Kobayashi, N., Yamamura, J., Yahiro, M., and Aoe, S., Milk whey protein enhances the bone breaking force in ovariectomized rats. Nutr. Res., 17, (1997). 20) Igarashi, C., Ezawa, I., and Ogata, E., EŠects of whey calcium on bone metabolism in ovariectomized osteoporosis model rats. Nippon Eiyo Shokuryo Gakkaishi (in Japanese), 43, (1990). 21) Tsuchita, H., Sekiguchi, I., and Kuwata, T., Comparison of the ešects of whey mineral complexes on bone metabolism in male growing rats. J. Nutr. Sci. Vitaminol., 39, (1993).