Yukihiro Takada

Milk basic protein: a novel protective function of milk against osteoporosis

Milk is recommended as an excellent calcium source for bone health. Moreover, milk is considered to contain other components effective for bone health. In our previous studies, using an unfractionated bone cell culture system, we found that milk whey protein, especially its basic fraction (milk basic protein [MBP]), suppressed bone resorption. In this present study, we investigated whether MBP could prevent bone loss in aged ovariectomized rats. Twenty-one 51-week-old female Sprague-Dawley rats were ovariectomized (ovx), and another seven rats received a sham operation (sham). After a 4-week recovery period, the ovx rats were separated into three groups, and they were then fed a control diet, a 0.01% MBP diet (0. 01% casein of the control diet replaced with MBP), or a 0.1% MBP diet for 17 weeks. The sham rats were fed the control diet. Bone mineral density (BMD) of the femur was measured by dual-energy X-ray absorptiometry in vivo. The BMD in the ovx-control group noticeably decreased during the experimental period in comparison with that in the sham group. However, the BMD in the OVX-0.1% MBP group was significantly higher than that in ovx-control group at weeks 12 and 16 (p < 0.05). After the 17-week feeding period, the breaking energy of the excised femur of all groups was determined by use of a three-point bending rheolometer. The breaking energy in the ovx-control group was significantly lower than that in the sham group (p < 0.05). However, the breaking energy in the ovx-0.1% MBP group was significantly higher than that of the ovx-control group (p < 0.05). Urinary deoxypyridinoline (D-Pyr) level of the ovx-control group was higher than that of the sham group, whereas the level of D-Pyr excretion in the ovx-0.01% MBP and ovx-0.1% MBP groups was significantly lower than that of the ovx-control group (p < 0.05). These results suggest that MBP suppresses the osteoclast-mediated bone resorption and prevents bone loss caused by ovariectomy. Moreover, we performed an in vitro study using isolated osteoclasts from rabbit bone to investigate the possible mechanism. MBP dose-dependently suppressed the number of pits formed by these osteoclasts. This result indicates that MBP suppresses bone resorption by its direct effects on osteoclasts. To our knowledge, this study provides the first evidence that MBP directly suppresses osteoclast-mediated bone resorption, resulting in the prevention of the bone loss that occurs in ovx rats.

Milk whey protein enhances the bone breaking force in ovariectomized rats

Abstract We studied the effects of whey protein (WP) and fractionated WP (HWP; heat-stable WP, LWP; low M.W. WP, EWP; ethanol-precipitated WP) on calcium and bone metabolism in ovariectomized (OVX) rats. Six-week-old female Sprague-Dawley rats were ovariectomized and fed a low-calcium diet (0.03% Ca, 0.3% P) for 4 weeks. The rats were divided into five groups, Cont, WP, HWP, LWP and EWP group, and were fed a Cont diet (20% casein, 0.3% Ca) or a diet (19% casein, 0.3% Ca) containing 1% WP, HWP, LWP or EWP for 4 weeks. There were no significant differences in the calcium balance, serum calcium and calcitonin levels among the experimental groups. However, serum ALP activity of the HWP and EWP groups at 14 wks. were lower than that of the Cont group. The bone breaking strength and energy of femur of the HWP, LWP and EWP groups were higher than those of the Cont group. As for the amount of calcium, phosphorus, and magnesium in the femur, there were no significant differences among the experimental groups; however, the amounts of total amino acids in the femur of the HWP, LWP and EWP groups were higher than that of the Cont group. The amounts of proline and hydroxyproline (typical amino acids of collagen) in the femur of the HWP, LWP and EWP groups were also higher than those of the Cont group. These data indicate that milk WP contains active components that influence bone metabolism in OVX rats by increasing in bone protein such as collagen and enhance the bone breaking force (strength and energy). These results suggest that the active components are existed in heatstable, low M.W. and 30–70% ethanol-precipitated fraction, respectively.

Milk Basic Protein (MBP) Increases Radial Bone Mineral Density in Healthy Adult Women

We studied the effects of daily intake of milk basic protein (MBP) on radial bone mineral density (BMD) in healthy adult women. Thirty-three healthy women were randomly assigned to a 6-month trial with either placebo or MBP (40 mg per day). The radial BMD of each volunteer was measured at the beginning of and at six months after the trial. The mean BMD value at the 6th month in the MBP group increased significantly at both 1/6 and 1/10 portion from the distal end of the radius, whereas that in the control group did not. The BMD gain of each volunteer in the MBP group was significantly higher than that in the placebo group. Thus a daily MBP supplementation of 40 mg in healthy adult women can significantly increase radial BMD.

Cystatin C in milk basic protein (MBP) and its inhibitory effect on bone resorption in vitro

A cystein protease inhibitor was identified in the basic fraction of bovine milk. We have reported in our previous study that the milk basic protein (MBP) fraction suppressed osteoclast-mediated bone resorption in vitro. Since osteoclasts secreted cystein protease to digest collagen in the bone matrix, we identified the cystein protease inhibitor in MBP. A 12-kDa inhibitor was purified from MBP by papain affinity gel chromatography and subsequent Hi-Load Superdex 75 gel filtration chromatography. The N-terminal sequence of the 18 amino acid residues of the inhibitor corresponded to bovine cystatin C. The 12-kDa cystein protease inhibitor in MBP therefore seemed to be cystatin C. Purified cystatin suppressed bone resorption with the use of isolated osteoclasts in vitro. Cystatin in MBP is suggested as one of the factors inhibiting bone resorption.

Comparison of the effects of milk components and calcium source on calcium bioavailability in growing male rats

Abstract We compared the effects of milk components (lactose and milk protein) and calcium source on calcium bioavailability in growing male rats. Six-week-old male Sprague-Dawley rats were divided into four groups (Milk components (MC)+CaCO 3 , CaCO 3 , MC+WheyCa and WheyCa). Each group of rats was fed either a diet containing milk components plus CaCO 3 or whey Ca, or a milk components-free diet containing CaCO 3 or whey Ca for 46 days. Calcium bioavailability was defined by apparent calcium absorption rate and bone mineral density (BMD). Two-way ANOVA was used to estimate the effects of milk components and calcium source on calcium bioavailability. The apparent calcium absorption rate was estimated at 7 and 12 weeks of age. The type of dietary calcium source did not affect the calcium absorption rate in either period. However, the milk components significantly increased the apparent calcium absorption rate at 7 weeks of age, and tended to increase the apparent calcium absorption rate at 12 weeks of age (p=0.09). Moreover, the milk components increased the apparent magnesium absorption rate in both periods. The type of dietary calcium source did not affect the BMD of the femur or lumbar vertebrae (L3–L5). However, the milk components diet increased the BMD of femur and L3–L5. These data indicate that the administration of milk components is more effective for the enhancement of calcium bioavailability rather than the chemical form of calcium in growing rats.

EFFECTS OF DIETARY MAGNESIUM LEVEL ON CALCIUM ABSORPTION IN GROWING MALE RATS

The effects of dietary magnesium level (0.025, 0.05, and 0.15% magnesium) on calcium absorption were studied in growing rats fed the diets containing 0.5% calcium. The apparent calcium absorption rate and the retention rate of rats fed diets containing 0.15% magnesium were significantly lower than those of rats fed diets containing 0.05% magnesium. On the other hand, apparent magnesium absorption and retention were significantly increased dose-dependently. Femoral bone mineral density (BMD) of rats fed diets containing 0.15% magnesium was significantly lower than that of rats fed diets containing 0.025% and 0.05% magnesium. The amounts of femoral calcium of rats fed diets containing 0.15% magnesium also tended to be lower than those of rats fed diets containing 0.025% and 0.05% magnesium. These results suggested that dietary magnesium and calcium competed with each other in intestinal absorption; therefore 0.05% dietary magnesium level was preferable compared with 0.15% dietary magnesium if the diet contained 0.5% calcium. On the other hand, the amounts of femoral magnesium of rats fed diets containing 0.15% magnesium were significantly higher than those of rats fed diets containing 0.025% magnesium, and the femoral breaking energy of rats fed diets containing 0.15% magnesium was significantly higher than that of rats fed diets containing 0.025% magnesium. These results also suggested that magnesium might have an effect on bone metabolism other than calcium absorption.

Milk calcium taken with cheese increases bone mineral density and bone strength in growing rats.

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 per day) for 2 weeks. The rats were then divided into 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 significantly 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 significantly higher than in the other two groups. These results indicate that milk calcium taken with cheese increases bone strength and BMD efficiently, results that may be useful for the prevention of osteoporosis.