Effects of vitamin K2 (menatetrenone) and alendronate on bone mineral density and bone strength in rats fed a low-magnesium diet

Bone 35 (2004) 1136 – 1143 www.elsevier.com/locate/bone

Effects of vitamin K2 (menatetrenone) and alendronate on bone mineral density and bone strength in rats fed a low-magnesium diet M. Kobayashi *, K. Hara, Y. Akiyama Pharmacological Evaluation Section, Department of Applied Drug Research, Eisai Co., Ltd., Bunkyo, Tokyo 112-8088, Japan Received 8 December 2003; revised 1 April 2004; accepted 13 May 2004 Available online 8 September 2004

Abstract In this study, we examined changes in bone parameters and bone strength in rats fed low-Mg diets (experiment 1) and the effects of vitamin K2 (MK-4, experiment 3) and alendronate (ALN, experiment 2) in this model. In experiment 1, 5-week-old male Wistar rats were fed three low-Mg diets (Mg 9, 6, 3 mg/100 g diet) for 4 weeks. Although the cortical bone mineral content (CtBMC) and cortical thickness (CtTh) of the femoral diaphysis in all low-Mg-diet groups were the same as or greater than those in the intact group (Mg: 90 mg/100 g diet), the maximum load and elastic modulus were significantly reduced in the 3-mg-Mg group. In experiment 2, 4-week-old Wistar rats were fed a 6-mg-Mg diet for 8 weeks, and the effect of ALN (2, 20, and 200 Ag/kg twice a week) was evaluated. The administration of ALN at 200 Ag/kg increased the cortical bone mineral content (CtBMC), CtTh, and maximum load, but had no effect on the elastic modulus, as compared with the low-Mg-control group. In experiment 3, the effect of MK-4 was evaluated under the same conditions as in experiment 2. The administration of MK-4 had no effect on CtBMC, CtTh, or bone components of the femoral diaphysis. However, MK-4 inhibited the decreases in maximum load and elastic modulus due to the low-Mg diet. Since there is no other experimental model in which there is a decrease in bone mechanical properties without a decrease in bone mineral content, the low-Mg diet model is considered to be an excellent model for examining bone quality. Our results from this model suggest that MK-4 and ALN affect bone mechanical properties by different mechanisms. D 2004 Elsevier Inc. All rights reserved. Keywords: Alendronate; Vitamin K2; Magnesium; Bone quality; Rat

Introduction Recent progress in research has helped prevent incidental fractures in patients with osteoporosis by reducing bone turnover and increasing bone density. Though success has been achieved in the prevention of osteoporotic fractures using anti-resorption agents such as bisphosphonates [1,2] and selective estrogen receptor modulators [3,4], the complete resolution of osteoporosis has still to be realized. The recent concept of bone fragility and quality developed at the NIH consensus meeting suggests that bone strength consists of many factors including bone mineralization, architecture, turnover, and concentration of organic proteins [5]. However, there is still no experimen* Corresponding author. Pharmacological Evaluation Section, Department of Applied Drug Research, Eisai Co., Ltd., Koishikawa 4-6-10, Bunkyo, Tokyo 112-8088, Japan. Fax: +81-3-3811-9207. E-mail address: [email protected] (M. Kobayashi). 8756-3282/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bone.2004.05.012

tal model in which a state of low bone quality is created, though there are many models having decreased bone density and increased bone turnover, such as the oophorectomized [6] rat model or steroid-induced osteoporosis model [7]. It has been reported that a low-Mg diet or restriction of Mg absorption by colectomy reduces the blood Mg concentration and decreases bone strength [8,9], and that a decrease in bone strength due to ovariectomy is reversed by the administration of Mg [10], suggesting that Mg plays some role in bone strength. However, there have been few studies on the relationship of the blood Mg levels and bone Mg content and the mechanical properties of bones. In a previous study, we fed rats a low Ca (0.01%) diet with reduced Mg (0.003%, 0.015%, and 0.09% Mg) to rats and found that, concerning parameters of bone strength, maximum load decreased for all Mg concentrations, the elastic modulus varied by the dietary Mg concentration [11]. These results suggest that the

M. Kobayashi et al. / Bone 35 (2004) 1136–1143

maximum load is dependent on Ca while the elastic modulus is dependent on Mg. In the present study, we examined changes in bone mass, bone strength, and bone components in rats fed a low-Mg diet to clarify the effect of Mg deficiency on bone strength. We also looked at the role of vitamin K. Vitamin K is essential for the g-carboxylation of OC [12], and its connection with bone metabolism has been attracting attention. Clinically, it has been reported that the blood vitamin K level is reduced in osteoporotic patients with fractures [13 – 15] and that the serum undercarboxylated OC level is a risk factor for bone fracture in elderly women and osteoporotic patients [16]. It has also been reported that the administration of vitamin K2 (menatetrenone, MK-4) improved BMD and reduced the incidence of fractures in patients with osteoporosis [17,18], and that MK-4 prevented ovariectomy-induced [19] and steroidinduced decreases [20,21] in BMC and bone strength in animals. In vitro studies have indicated that MK-4 promotes calcification by osteoblasts [22]. Alendronate (ALN), an amino bisphosphonate, has also been reported to be useful for the treatment of postmenopausal osteoporosis [1] and steroid-induced osteoporosis in clinical trials [23]. In addition, the drug was found to markedly increase BMD in low-bone-mass animal models produced by ovariectomy [24] and immobilization [25]. In vitro studies, ALN has been shown to suppress osteoclastic function [26], and markedly inhibit bone resorption [27]. We, therefore, evaluated the effects of two drugs, MK-4 and ALN, in a low-Mg diet model. These days have different mechanisms of action, one of them promotions bone formation and the other inhibiting bone resorption, using lowMg diet models.

Materials and methods Experimental animals Male Wistar rats (CLEA Japan, Inc., Tokyo, Japan) were used in experiments 1, 2, and 3. The animal room was subjected to a 12-h light –dark cycle and maintained at 23 F 3jC and 55 F 10% humidity. During the experiments, the animals were housed individually in stainless steel cages and given deionized water ad libitum. Experimental design Experiment 1 Twenty-four male Wistar rats aged 5 weeks were divided into an intact group (90 mg Mg), a 9-mg-Mg group, a 6-mg-Mg group, and a 3-mg-Mg group (n = 6). Diets with Mg concentrations of 0.09%, 0.009%, 0.006%, and 0.003% were prepared using a purified diet (CLEA Japan, Inc.) having a Ca content of 0.5% and a Pi content of 0.66% and were given to the intact, 9-mg-Mg, 6-mg-

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Mg, and 3-mg-Mg groups, respectively. Intake was restricted to 80% of the ordinary food intake of Wistar rats of the same age for all four groups. After 4 weeks, the animals were sacrificed under pentobarbital (NembutalR, Dinabot, Osaka, Japan) anesthesia, and blood was sampled with heparinization from the abdominal aorta. The blood samples were centrifuged at 4jC and 3000 rpm for 10 min, and the Mg level and ALP activity were measured in the plasma obtained. After blood sampling, the left femur was removed, the muscles detached, femur length was measured, and the femur placed in saline for 1 week. Then bone mass, bone strength, and bone components, and Ca, Mg, and hydroxyproline (Hypro) levels in the femoral diaphysis were measured. Experiments 2 and 3 Male Wistar rats aged 4 weeks were used in both experiments. In experiment 2, rats were divided into five groups (n = 5): intact, low-Mg-control, and three low-MgALN (three different doses) groups. A purified diet with a Ca content of 0.5% and an Mg content of 0.09% was given to the intact group, and a diet in which the Mg content was reduced to 0.006% was given to the low-Mgcontrol and low-Mg-ALN groups. ALN (TeirocR inj., Teijin Inc., Osaka, Japan) was administered subcutaneously at 2, 20, and 200 Ag/kg twice a week. In experiment 3, rats were divided into three groups (n = 10): intact, low-Mgcontrol, and low-Mg-MK-4. A diet with a reduced Mg content (0.006%) supplemented with menatetrenone (MK4, Eisai, Co., Ltd., Tokyo, Japan) at 60 mg/100 g of diet was given to the low-Mg-MK-4 group. The dose of MK-4 per kilogram of body weight was calculated from the intake and the MK-4 concentration in the diet (60 mg/ 100 g). The mean dose of MK-4 during the 8 weeks was 36.l F 1.2 mg/kg bw. The food supply was restricted to 80% of the normal level as in experiment 1, and the body weight and diet intake were measured once every 2 weeks during the experiment. After 8 weeks, blood was sampled by the same method as used in experiment 1, both femurs were removed, and then length was measured. The left femur was placed in saline for 1 week, and then its bone mass and bone strength were measured. In experiment 3, 7 weeks after beginning the experimental diet, the animals were forced to drink 1 ml of distilled water per 100 g of body weight and placed in individual metabolic cages, where their urine was sampled for 16 h while fasting. Urinary deoxypyridinoline (Dpd) and creatinine (Cr) levels were measured. Blood was also sampled, plasma was separated, and Ca, Mg, ALP, parathyroid hormone (PTH), OC, and 1,25(OH)2D3 levels were measured. Bone components (Ca, Mg, Hypro, Dpd, and OC) were measured for the right femur. All the experiments were approved by the Experimental Animals Ethics Committee at our institution and conducted in accordance with guidelines concerning the management and handling of experimental animals.

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Table 1 Effects of restricted dietary magnesium (Mg) intake on body weight, plasma Mg level, alkaline phosphatase activity (ALP), and femoral concentrations of Mg, calcium (Ca), and hydroxyproline in rats

Intact L-Mg-9 L-Mg-6 L-Mg-3

Body weight (g)

Plasma Mg (mg/dl)

ALP (KA-U/dl)

Mg (Ag/mg)

Ca (Ag/mg)

Hydroxyproline (Ag/mg)

285 273 226 207

1.680 0.706 0.599 0.427

18.30 14.04 8.82 8.91

5.08 3.14 2.32 1.96

362.0 334.8 336.2 329.5

24.8 24.9 24.2 25.5

F F F F

3 6 4** 7**

F F F F

Femur

0.060 0.021** 0.062** 0.036**

F F F F

1.20 0.47** 0.74** 0.54**

F F F F

0.14 0.09** 0.06** 0.07**

F F F F

9.5 3.6* 3.5* 5.5**

F F F F

0.2 0.3 0.5 0.4

Each value represents the mean F SEM (n = 6). Intact: 0.09% Mg, L-Mg-9: 0.009% Mg, L-Mg-6: 0.006% Mg, L-Mg-3: 0.003% Mg. * P < 0.05 vs. intact (Dunnett’s multiple comparison test). ** P < 0.01 vs. intact (Dunnett’s multiple comparison test).

Methods of measurement Plasma components The Ca and Mg levels were measured by the atomic absorption method (atomic absorption spectrophotometer AA-6200, Shimadzu, Kyoto, Japan), ALP activity by the phenylphosphate substrate method (Alkaline Phospha KTest Wako, Wako Pure Chemicals, Osaka Japan), PTH by ELISA (RAT PTH ELISA KIT, Amersham Biosciences Corp., Piscataway NJ, USA), Cr by the Jaffe´ method (Creatinine-Test Wako, Wako Pure Chemicals), OC by ELISA (BIOTRAK, Amersham Biosciences Corp., ), and 1,25(OH)2D3 by radioreceptor assay [28]. Urinary components Dpd was measured by EIA (OsteolinksDpd, Sumitomo Pharmaceutical, Tokyo, Japan), and Cr was measured by the same method as for blood. Bone mass parameters The bone mass was measured using peripheral quantitative computed tomography (pQCT, XCT-AScope, Stratec, Birkenfeld, Germany) at the diaphysial region of the femur. The tomography conditions were: diameter 15, voxel size 0.1, CT speed 10, block number 1. As for the procedure, after a scout scan was obtained and the growth plate identified, transverse images of the slices were obtained at a point 16.0 mm distal to the growth plate, and then the BMC, BMD, and total area of the region were measured. The cortical region was extracted from the total region using an algorithm (separation mode 3 and contour mode 2), and both cortical bone area (CtAr) and cortical thickness (CtTh) were measured. Quality assurance measurements were made daily using a hydroxyapatite standard embedded in acrylic plastic to check all system components before sample scans were performed. Mechanical properties of bone Three-point bending strength of the femur. The bone strength of the femur was measured using a tester (AG500E, Shimadzu). The femur was placed on a sample

holder, adjusting the distance between the fulcrums to 13 mm, and was pressed at a crosshead speed of 2 mm/min until it broke. A load-deformation curve was drawn. The maximum load and elastic modulus were obtained using software for the measurement of bone strength (Shikibu, Shimadzu). Bone components. A 6-mm segment was cut from the center of the right femur using a circular saw (Hozan, Tokyo, Japan) and freeze-dried for 24 h. After measuring the dry weight, the femur was hydrolyzed with 6 N HCl (130jC, 3 h). Part of the hydrolyzed femur was sampled, and Ca, Mg, Pi, Hypro, OC, and Dpd levels were measured by the same methods as for blood or urine. Inorganic phosphorus was measured with a commercial kit (p-test Wako, Wako Pure Chemical Industries). Hypro was measured by the Kivirikko et al. [29] method. Statistics Values in the figures and tables are shown as the mean F SEM. In experiment 1, comparisons among the intact, 9mg-Mg, 6-mg-Mg, and 3-mg-Mg groups were performed by Dunnett’s multiple comparison test. In experiments 2 and 3, comparisons between the intact group and low-Mg-control group and between the low-Mg-control group and low-Mg-

Table 2 Effects of restricted dietary magnesium (Mg) intake on bone parameters of femoral diaphysis in rats Cortical bone region Bone mineral Bone mineral Bone area content (mg/mm) density (mg/cm3) (mm2) Intact 6.79 L-Mg-9 6.93 L-Mg-6 7.27 L-Mg-3 6.78

F F F F

0.16 0.12 0.14* 0.12

1250 1274 1229 1227

F F F F

7 5 14 10

5.43 5.45 5.93 5.53

F F F F

Cortical thickness (mm)

0.12 0.598 0.11 0.606 0.16* 0.667 0.14 0.608

F F F F

0.012 0.008 0.026* 0.013

Peripheral quantitative computed tomography (pQCT) measurement was performed at a point 16.0 mm distal to the growth plate. Each value represents the mean F SEM (n = 6). Intact: 0.09% Mg, L-Mg-9: 0.009% Mg, L-Mg-6: 0.006% Mg, L-Mg-3: 0.003% Mg. * P < 0.05 vs. intact (Dunnett’s multiple comparison test).

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Fig. 1. Effects of dietary magnesium (Mg) intake on mechanical properties of femurs in rats. The bone strength of femurs was measured using a three-point bending test. Intact: 0.09% Mg, L-Mg-9: 0.009% Mg, L-Mg-6: 0.006% Mg, L-Mg-3: 0.003% Mg. Each value represents the mean F SEM (n = 6). *P < 0.05, **P < 0.01 vs. intact (Dunnett’s multiple comparison test). The number in each column indicates the percentage relative to the intact group.

MK-4 and -ALN group were performed using the Student’s t test.

but the elastic modulus of the femur decreased. In the 3-mgMg group, there was no significant change in the diaphysial bone mass, but the maximum load and elastic modulus decreased.

Results Experiment 1: changes in body weight, blood components, and bone components in rats on low-Mg diets Table 1 shows changes in body weight and plasma and bone components. Body weight, blood Mg levels, ALP activity, and the bone Mg levels decreased in proportion to the Mg content of the diet. The bone Ca level was low in all (9, 6, and 3 mg) Mg groups. The bone Hypro concentration was almost the same in all the groups. Table 2 shows changes in bone parameters and Fig. 1 shows changes in bone strength in the three-point bending test (maximum load, elastic modulus). In the 9-mg-Mg group, there were no significant differences in bone mass or bone strength as compared with the intact group. In the 6-mg-Mg group, the CtBMC, CtAr, and CtTh of the femoral diaphysis increased,

Experiment 2: effects of ALN on bone mass and bone strength in rats on low-Mg diets Table 3 shows changes in the CtBMC, CtBMD, CtAr, and CtTh of the femoral diaphysis. A low-Mg diet increased the CtBMC to 110% of that in the intact group but caused no significant change in the CtBMD, CtAr, or CtTh. The CtBMC, CtAr, and CtTh were greater in the 2 and 20 Ag/kg ALN groups and significantly greater in the 200 Ag/kg ALN group as compared with the low-Mg-control group. The CtBMD was significantly lower in the 200 Ag/kg ALN group than in the low-Mg-control group. Fig. 2 shows changes in femoral bone strength. The maximum load and elastic modulus decreased in the low-Mg group to 77% and 57%, respectively, of the value in the intact group. In the 200 Ag/kg ALN group, the maximum load was higher, but

Table 3 Effects of alendronate (ALN) on bone parameters in the femoral diaphysis after 8 weeks of treatment Cortical bone region

Intact L-Mg-control L-Mg-ALN 2 Ag/kg L-Mg-ALN 20 Ag/kg L-Mg-ALN 200 Ag/kg

Bone mineral content (mg/mm)

Bone mineral density (mg/cm3)

Bone area (mm2)

Cortical thickness (mm)

7.02 7.69 8.07 9.24 9.30

1297 1321 1321 1312 1232

5.42 F 0.07 5.84 F 0.22 6.11 F 0.12 6.29 F 0.15 7.56 F 0.20**

0.607 0.628 0.661 0.691 0.808

F F F F F

0.08 0.22* 0.17 0.17 0.19**

F2 F 15 F 13 F8 F 10**

Peripheral quantitative computed tomography (pQCT) measurement was performed at a point 16.0 mm distal to the growth plate. Each value represents the mean F SEM (n = 5). Intact: Mg 0.09%, L-Mg: Mg 0.006%. * P < 0.05 vs. intact (Student’s t test). ** P < 0.01 vs. L-Mg-control (Dunnett’s multiple comparison test).

F F F F F

0.005 0.024 0.018 0.018 0.019**

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Fig. 2. Effect of alendronate on mechanical properties in the femoral diaphysis after 8 weeks of treatment. The bone strength of femurs was measured using a three-point bending test. Each value represents the mean F SEM (n = 5). *P < 0.05, **P < 0.01 vs. intact (Student’s t test), yyP < 0.01 vs. control (Dunnett’s multiple comparison test). The number in each column indicates the percentage relative to the intact group. Intact: 0.09% Mg, low-Mg diet: 0.006% Mg.

there was no change in the elastic modulus as compared with the low-Mg-control group. Experiment 3: effects of MK-4 on bone mass and bone strength in rats on low-Mg diets Changes in body weight, bone length, and blood and urine components One animal in the low-Mg-control group died after week 5 of the experiment. One animal in the low-Mg-MK-4 group was excluded because its intake of the diet was reduced for 10 days beginning on day 35 of the experiment. Table 4 shows changes in body weight, and blood and urine components. Body weight in the intact group after 8 weeks was 309 F 2 g, and in the low-Mg-control group, it was 84% of the intact group. There were significant decreases in the plasma Mg, ALP, and OC levels in the low-Mg-control group to 5%, 45%, and 69%, respectively, of those in the intact group. The plasma Ca, PTH, and 1,25(OH)2D3 levels were not significantly different between the intact group and

low-Mg-control group. There was also no significant difference between the urinary Dpd excretion in the intact group and low-Mg-control group. The administration of MK-4 had no effect on any of the above parameters. Changes in bone mass Table 5 shows changes in bone mass in the diaphysial regions of the femur. In the low-Mg control group, the CtBMC and CtBMD were significantly higher than those in the intact group. The CtAr and CtTh were not significantly different between the intact and low-Mg group. The administration of MK-4 had no effect on diaphysial bone mass. Changes in bone strength Fig. 3 shows changes in the three-point bending strength of the femur. The maximum load and elastic modulus of the femur were 112.1 F 2.3 N and 1450 F 75 N/mm2, respectively, in the intact group, but were reduced to 85% and 68% of these values in the low-Mg-control group. In the low-Mg-MK-4 group, the maximum load and elastic mod-

Table 4 Effects of menatetrenone (MK-4) on body weight, plasma calcium (Ca), magnesium (Mg), alkaline phosphatase activity (ALP), osteocalcin, parathyroid hormone (PTH), 1,25(OH)2D3 levels and urinary deoxypyridinoline (Dpd) after 8 weeks of treatment Body weight (g)

Intact L-Mg-control L-Mg-MK-4

309 F 2 259 F 4** 269 F 5

Plasma Ca (mg/dl) 9.99 F 0.08 10.27 F 0.12 9.74 F 0.13

Mg (mg/dl)

ALP (KA-U/dl)

Osteocalcin (ng/ml)

PTH (pg/ml)

1,25(OH)2D3 (pg/ml)

Urine Dpd/Cr (nM/mM)

1.51 F 0.02 0.07 F 0.02** 0.1 F 0.02

24.68 F 1.20 11.00 F 0.59** 13.86 F 1.49

81.90 F 5.32 56.85 F 3.70** 63.44 F 4.56

57.9 F 9.2 69.9 F 6.5 72.2 F 14.6

353 F 21 296 F 24 348 F 21

939.9 F 59.2 1015.5 F 70.8 839.0 F 60.0

Each value represents the mean F SEM (n = 9 – 10). Dpd/Cr: deoxypyridinoline/creatinine. Intact: Mg 0.09%, L-Mg: Mg 0.006%. ** P < 0.01 vs. intact (Student’s t test).

M. Kobayashi et al. / Bone 35 (2004) 1136–1143 Table 5 Effects of menatetrenone (MK-4) on bone parameters in the femoral diaphysis after 8 weeks of treatment Cortical bone region Bone mineral Bone mineral Bone area content density (mm2) (mg/mm) (mg/cm3) Intact 7.81 F 0.10 1342 F 6 L-Mg-control 8.15 F 0.11* 1378 F 4** L-Mg- MK-4 8.19 F 0.08 1392 F 2

Cortical thickness (mm)

5.83 F 0.09 0.660 F 0.001 5.91 F 0.09 0.657 F 0.014 5.88 F 0.05 0.671 F 0.005

Peripheral quantitative computed tomography (pQCT) measurement was performed at a point 16.0 mm distal to the growth plate. Each value represents the mean F SEM (n = 9 – 10). Intact: Mg 0.09%, L-Mg: Mg 0.006%. * P < 0.05 vs. intact (Student’s t test). ** P < 0.01 vs. intact (Student’s t test).

ulus of the femur were significantly greater than in the lowMg-control group. Changes in bone components Table 6 shows changes in the bone components of the femur. In the low-Mg-control group, Ca was significantly greater than in the intact group at 104%, and Mg was significantly less at 29%, but there was no difference in Pi. Bone OC, Dpd, and Hypro levels decreased in the low-Mg-control group to 86%, 80%, and 96%, respectively, of levels in the intact group. The Ca/Hypro ratio was 108%, and the OC/ Hypro and Dpd/Hypro ratios were 89% and 84%, respectively, of the ratios in the intact group. MK-4 had no significant effect on these changes.

Discussion Recent clinical studies have shown that increased bone mineral density does not always decrease the occurrence of

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new fractures [2,3]. Therefore, bone quality has become an important issue in osteoporosis research [30] but up till now an experimental model available for the study of low bone quality has not been available. The present study investigated whether rats fed a low-Mg diet could be used as an experimental model to study the issue of bone quality. In experiment 1, we fed rats diets containing 0.003%, 0.006%, and 0.009% Mg for 4 weeks and examined changes in the mechanical properties of bone for the femoral diaphysis. In the 0.003% Mg group, the maximum load and elastic modulus decreased but there were no changes in CtBMC and CtTh. In experiments 2 and 3, though there was an increase in CtBMC for rats fed with the 0.006% Mg diet for 8 weeks, the maximum load and elastic modulus decreased (Figs. 2, 3). In experimental bone loss models induced by steroid treatment or a low-Ca diet, it was reported that both maximum load and elastic modulus decreased along with decreases in CtBMC and CtAr [31,32]. In this low-Mg model, the correlation coefficient between CtBMC and the maximum load was r = 0.366 and that between CtBMC and the elastic modulus was r = 0.132, these low correlation coefficients suggests that the low-Mg diet reduced bone quality, ALP activity, and OC levels also decreased, suggesting reduced of osteoblast function. Osteoblasts are responsible for the synthesis of matrix proteins, such as collagen, and mineralization. We observed no significant difference in urinary Dpd excretion between the low-Mg-control group and the intact group after the 8 weeks of the diets (Table 4), and there was no change in bone resorption. To clarify the mechanism involved in reduction of bone quality, we measured bone component levels, as shown in Table 6. For organic components, there was no change in the Ca level but Mg decreased. Among organic components, the Hypro, OC, and Dpd levels decrease. We therefore speculated that reduction in osteoblast function decreases not only bone levels of Hypro and

Fig. 3. Effect of menatetrenone (MK-4) on mechanical properties in the femoral diaphysis after 8 weeks of treatment. The bone strength of femurs was measured using a three-point bending test. Each value represents the mean F SEM (n = 9 – 10). **P < 0.01 vs. intact yP < 0.05, yyP < 0.01 vs. control (Student’s t test). The number in each column indicates the percentage relative to the intact group. Intact: 0.09% Mg. Low-Mg diet: 0.006% Mg.

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Table 6 Effects of menatetrenone (MK-4) on bone compositions in the femoral diaphysis after 8 weeks of treatment Inorganic component Calcium (Ag/mg) Magnesium (Ag/mg) Intact 263.0 F 1.7 L-Mg-control 273.6 F 0.9** L-Mg- MK-4 271.1 F 2.3

Organic component Pi (Ag/mg)

Osteocalcin (ng/mg)

Calcium/Hypro

Dpd (nM/mg) Hydroxyproline (ng/mg)

5.41 F 0.05 128.6 F 0.7 778.0 F 25.1 45.0 F 2.4 1.58 F 0.08** 128.6 F 1.0 668.1 F 34.3* 36.2 F 1.4** 1.71 F 0.08 128.5 F 1.0 735.4 F 34.7 32.8 F 1.9

20.2 F 0.3 19.4 F 0.2* 19.2 F 0.2

Osteocalcin/Hypro (ng/Ag)

13.04 F 0.15 38.56 F 1.31 14.09 F 0.18** 34.27 F 1.47** 14.17 F 0.22 38.39 F 1.77

Each value represents the mean F SEM (n = 9 – 10). Intact: Mg 0.09%, L-Mg: Mg 0.006%. * P < 0.05 vs. intact (Student’s t test). ** P < 0.01 vs. intact (Student’s t test).

OC but also mineralization, but not the level of Ca, a parameter of mineralization. For our results, the bone Ca/ Hypro ratio was high, and the proportion of matrix was less than that of mineral. We thus assumed that when animals are given a low-Mg diet, Ca deposition is impaired the standard process of mineralization in which collagen is synthesized by osteoblasts and mineralization starts at the matrix vesicles embedded in collagen dose not take place. This process may also be involved in the reduction of bone mechanical property parameters, especially the elastic modulus. Since Mg has been reported to affect hydroxyapatite crystallization [33], we expected abnormal calcification to occur in our low-Mg model, and that this would cause divergence between osteoblast function and the CtBMC resulting in deterioration of bone quality. To study the pathogenesis of this model, we used ALN as an anti-bone resorption agent, and MK-4 as a bone formation agent. Clinically, ALN has been reported to reduce the incidence of new fractures in osteoporotic patients by increasing the BMD of the lumbar spine [1,2,34]. And, as possible as the mechanisms by which ALN inhibits bone resorption, promotion of apoptosis in osteoclasts [35], inhibition of osteoclasts attachment to bone [26], and interference with the formation of ruffled borders [36] have been considered. In this study, the administration of ALN dose-dependently increased the CtBMC of the diaphysis in rats fed a low-Mg diet (Table 3), suggesting that ALN prevented the bone resorption that had previously been takes place. ALN also appeared to reverse the reduction in maximum load probably by increasing CtBMC and CtTh, suggesting that CtBMC and CtTh are important factors for maximum load, the improvement in the elastic modulus may have be due to other factors like collagen. Treatment with MK-4 has been reported to be effective in the prevention of new fractures without increasing BMC in osteoporotic patients [17]. It has also been reported to improve bone strength more than BMD in ovariectomized rats [37], and thus the preventive effect is considered to involve improvement of factors other than BMD. In this study, MK-4 improved both the maximum load and elastic modulus, without influencing CtBMC. MK-4 also increased plasma ALP activity and plasma/bone OC levels, though such increases were not significant, suggesting that MK-4

tended to ameliorate osteoblast function in this model. While MK-4 had no significant effect on the changes in bone Ca, Mg Hypro, and Dpd levels caused by the low-Mg diet, the OC/Hypro ratio in the MK-4 group recovered to a level similar to that in the intact group. MK-4 may have increased collagen synthesis by improving osteoblast function, thereby correcting the imbalance between mineral and collagen and reversing the reduction in the mechanical properties of bones. Though the role of osteocalcin in bone remains unclear, a study in OC-knockout mice indicated that bone formation increases in the OC deficient condition [38]. This suggests that the recovery in the OC/Hypro ratio due to MK-4 also contributed to the mechanism by which bone strength in the diaphysis was increased, and that this was not mediated by an improvement in CtBMD. In the present study, OC bone levels decreased and CtTh increased, suggesting that the increase in mineralization might have been due to the decrease in the OC levels. In conclusion, the results of comparing the effects of two drugs with different mechanisms of action on the mechanical properties of bone that we obtained suggest that maximum load and elastic modulus may be regulated by different factors. They further indicate that CtBMC is important to maximum load, while bone matrix protein is important to elastic modulus and improvement in these factors result in an improvement in overall bone quality.

Acknowledgments The authors wish to thank Haruko Yamauchi, Ikiko Mitome, and Rumi Satoh for their technical assistance in this research.

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