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A longitudinal study of the effect of vitamin K2 on bone mineral density in postmenopausal women a comparative study with vitamin D3 and estrogen–progestin therapy
Maturitas 31 (1999) 161 – 164
A longitudinal study of the effect of vitamin K2 on bone mineral density in postmenopausal women a comparative study with vitamin D3 and estrogen–progestin therapy Ichiro Iwamoto a,*, Shoichiro Kosha a, Shin-ichi Noguchi a, Masato Murakami a, Toshinori Fujino b, Tsutomu Douchi a, Yukihiro Nagata a a
Department of Obstetrics and Gynecology, Faculty of Medicine, Kagoshima Uni6ersity, 8 -35 -1 Sakuragaoka, Kagoshima 890 -8520, Japan b School of Allied Medical Sciences, Kagoshima Uni6ersity, 8 -35 -1 Sakuragaoka Kagoshima 890 -8520, Japan Received 18 May 1998; received in revised form 25 November 1998; accepted 25 November 1998
Abstract Objecti6es: To investigate the effect of vitamin K2 treatment for a year on spinal bone mineral density (BMD) in postmenopausal women, comparing with vitamin D3 hormone replacement therapy and to determine the factors which affect the efficacy of vitamin K2 therapy. Subjects and methods: Seventy-two postmenopausal women were randomized into four groups and treated with respective agents. Before the therapy, 6 and 12 months after the treatment, their lumbar spine BMD were measured by dual energy X-ray absorptiometry. The rates of change in BMD (DBMD) were calculated. Correlations of BMD with age, year since menopause and the initial BMD were determined. Results: Vitamin K2 suppressed the decrease in spinal BMD as compared with no treatment group. BMD in women treated with vitamin K2 was inversely correlated with their age (r = −0.54; PB 0.05). Conclusions: Vitamin K2 therapy may be a useful method for preventing postmenopausal spinal bone mineral loss. In addition, the therapy should be started early in postmenopausal period. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Vitamin K2; Bone mineral density; Treatment
1. Introduction
* Corresponding author. Tel.: +81-99-2755423; fax: + 8199-2650507.
Several agents including vitamin D3, estrogen, calcitonin, and bisphosphonate, etc have been used for preventing postmenopausal osteoporosis. But, in clinical practice, there are some people
0378-5122/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 5 1 2 2 ( 9 8 ) 0 0 1 1 4 - 5
162
I. Iwamoto et al. / Maturitas 31 (1999) 161–164
who respond poorly or little to those medicines. Recently, much attention have been paid to vitamin K2 because of its action on bone; vitamin K2 both increases bone formation and decreases bone resorption. Since Bouckaert and Said first reported the effect of vitamin K on fracture healing in 1960 [1], many studies have suggested that vitamin K is closely related with bone metabolism [2,3]. In our country, vitamin K2 (menatetrenone) has been accepted for clinical application in the treatment of osteoporotic osteopenia from 1995. Orimo et al. [4] reported that vitamin K2 increases the bone mineral density (BMD) of cortical bones in elderly osteoporotic patients. However, to prevent bone fracture, cancellous bones should be examined than the cortical ones. In addition, to prevent osteoporosis effectively, the therapy ought to be started earlier than elderly age. There are very few reports about vitamin K2 therapy so far. Our study is the first report which investigated the BMD of cancellous bones in women just after menopause. In the present study, we performed a longitudinal study of the effect of vitamin K2 on vertebral bone in postmenopausal women for 12 months, comparing with vitamin D3 or estrogenprogestins treatment (HRT). Moreover, we investigated the relationship of the rates of change in BMD (BMD) with age, years since menopause (YSM) and initial BMD of the women who were treated by each regimen.
randomly assigned to one of four groups: (1) the control group was untreated; (2) the hormonal replacement therapy (HRT) group was treated with conjugated equine estrogen (CEE), 0.625 mg/ day, together with medroxyprogesterone acetate, 2.5 mg/day for a year; (3) the vitamin D3 group was treated with 1(OH)D3, 1.0 g/day for a year; (4) the vitamin K2 group was treated with menaquinone-4, 45 mg/day for a year. BMD in the lumbar spine was determined by dual energy X-ray absorptiometry (DEXA: QDR2000, Hologic, USA) at three points, that is, before the treatment, 6 and 12 months after the initiation of the treatment. Scans were made along three lumbar vertebrae (L2–L4). BMD was expressed in g/cm2. Rates of change in BMD (DBMD) were calculated. The correlations of BMD with age, years since menopause (YSM) and initial BMD were investigated. Oral or written informed consent was obtained from each subject before study entry. This study was also conducted with institutional guideline. Data were expressed as the mean standard error of the mean (S.E.M.) when indicated. The data were analyzed statistically by the one-way analysis of variance (ANOVA). Spearmans and Pearsons correlation coefficient was calculated as appropriate. PB 0.05 was considered statistically significant.
3. Results 2. Subjects and methods A total of 72 healthy, non-hysterectomized postmenopausal women who had visted at the outpatient clinic of the Department of Obstetrics and Gynecology, Kagoshima University in Kagoshima, between 1994 and 1996 were selected. Postmenopausal state was defined as the absence of menstruation for at least 12 months. All subjects had no medications and complications, which affected bone metabolism and had no unbalanced diet for vitamin K rich food, e.g. green vegetables and fermented foods. All candidates were non-smokers, non-drinkers and led active lives, but did not practice sports. Subjects were
Table 1 shows the baseline characteristic of the subjects. There was no significant difference in age, BMI, YSM, and the initial BMD among the groups. Fig. 1 presents the rates of change in BMD (BMD) in each group during the treatment study. The mean9 S.E.M of BMD (%) at 12 months were (− 2.879 0.51) in control, (0.239 0.47) in vitamin K2 group, (−0.4790.86) in vitamin D3 group and (4.0 9 1.12) in HRT group. There was significant difference in BMD between HRT group and control group (PB0.01). However, the differences between other groups were not statistically significant. The correlations of BMD with age, YSM, and initial BMD in each treatment group were shown in Table 2. In vita-
I. Iwamoto et al. / Maturitas 31 (1999) 161–164
163
Table 1 Baseline characteristic of the groupsa Group
Control (n= 19)
Vitamin K2 (n =17)
Vitamin D2 (n =16)
HRT (n =23)
Age (years) BMIb YSMc BMD (g/cm2)d
53.69 0.84 22.5 9 0.58 4.689 0.84 0.829 0.02
55.9 9 1.55 22.09 0.70 6.18 9 1.15 0.78 9 0.02
52.6 91.76 23.2 90.76 6.0 91.3 0.81 90.02
53.3 90.76 21.4 9 0.68 4.39 9 0.69 0.82 9 0.02
Data were expressed as mean 9 S.E. BMI, body mass index. c YSM, years since menopause. d BMD, bone mineral density. a
b
min K2 group, there was inverse correlation of BMD with age (r = − 0.54; P B0.05), but no significant correlation was observed between initial BMD and YSM. In vitamin D3 group, no significant correlation of BMD with age, YSM, and initial BMD was observed. In HRT group, there was significant correlation of BMD with initial BMD (r= − 0.43; P B 0.05), but not with age and YSM.
4. Discussion There have been many reports in regard to the effect of HRT on BMD; it is generally recognized
that HRT has a suppressive effect on bone loss and an enhancing effect of increasing BMD at lumbar spine in postmenopausal women [5]. In the present study, HRT also prevented bone mineral loss. Thus, it is certain that HRT is a good way for preventing osteoporosis. However, HRT has some adverse effects, for example, increasing the risk of thromboembolism. There are some alternative agents for preventing the bone loss; vitamin K2 is one of the candidates. Vitamin K2 is unique, its actions include enhancing bone formation and suppressing bone resorption. In fact, this vitamin activates osteoblasts to enhance calcification in vitro [6], acts as a coenzyme of g-carboxylase of Gla protein [7] and results in increase of osteocalcin in sera [4]. On the other hand, it has been reported that vitamin K2 inhibited prostaglandin E2 which is a potent bone-resorping factor in human osteoblastlike cells [8,9], and that it inhibited osteclast-like cell formation in mouse bone marrow cultures [10]. Moreover, it has been reported that vitamin K2 suppressed bone loss induced by prednisolone administration [11] or ovariectomy in rats [12].
Table 2 Correlations of DBMD with age, YSM, and initial BMD in each group
Fig. 1. The changes in annual bone mineral density ( BMD) in untreated (control; n =19), vitamin D3 (n= 16) and estrogen– progesteron (HRT; n= 23) groups.
Group
Age
YSM
BMD at baseline
Vitamin K2 Vitamin D3 HRT
−0.54a 0.33 0.32
−0.21 0.16 0.37
0.07 −0.41 −0.43a
a
PB0.05.
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I. Iwamoto et al. / Maturitas 31 (1999) 161–164
From the phase III study, it was elucidated that vitamin K2 administration enhanced BMD of cortical bone in elderly osteoporotic patients [4]. It is thought that vitamin K2 accelerated bone metabolism due to increase in serum concentration of osteocalcin which is regarded as bone formation marker and increase in urinary hydroxyproline which is regarded as bone absorption marker. In that vitamin K2 enhanced bone metabolism, the effect of vitamin K2 on bone may be predominant in elderly women over younger women. Moreover, it was reported that the effects of this vitamin on BMD was in proportion to circulating level of vitamin D3 [4]. In the light of the decrease of serum vitamin K2 and vitamin D3 concentrations with age, the combination of vitamin K2 and vitamin D3 may be effective for the treatment of osteoporosis in elderly women. Therefore, vitamin K2 treatment might be more applicable for elderly women than for postmenopausal women. But, for preventing bone fraction, it would be wise to start the vitamin K2 therapy earlier than elderly age. In the present study, we found that vitamin K2 suppressed the decrease in spinal bone mineral density in postmenopausal women as compared with no treatment group, although the effect did not reach the statistic significance. We also found that BMD was inversely related with age, suggesting that earlier the therapy starts, the more effective it is for preventing bone loss. Recently, Nagasawa et al. [13] reported that vitamin K2 reduced total cholesterol concentration in osteoporotic patient on continuous ambulatory peritoneal dialysis. This finding suggests that vitamin K2 may have a beneficial effect on lipid metabolism. Though the mechanism by which vitamin K2 acts on metabolism is unclear, if it is beneficial for lipid metabolism, the administration of vitamin K2 may be considered to be useful for postmenopausal women to prevent osteoprosis and to improve hyperlipidemia, especially for women who have a history of thromboembolism which is considered to be contraindication for HRT, or women who respond poorly to HRT. .
Based on these results, we conclude that vitamin K2 may be a promising agent for preventing postmenopausal spinal bone mineral loss. The therapy should be started earlier just after menopause.
References [1] Bouckaert JT, Said AT. Fracture healing by vitamin K. Nature 1960;185:849. [2] Hall JAG, Pauli RM, Wilson KM. Maternal and fetal sequelae of anticoagulation during pregnancy. Am J Med 1980;68:122 – 40. [3] Hart JP, Shearer MJ, Klenerman L, et al. Electrochemical detection of depressed circulating levels of vitamin K1 in osteoporosis. J Clin Endocrinol Metab 1985;60:1268 – 9. [4] Orimo H, Fujita T, Onomura T, Inoue T, Kushida K, Shiraki M. Clinical evaluation of Ea-0167 (menatetrenone) in the treatment of osteoporosis: Phase III. Double-blind multicenter comparative with alfacalcidol (in Japanese). Rinshohyoka (Clin Eval) 1992;20:45 – 100. [5] Christiansen C, Christiansen MS. Bone mass in postmenopausal women after withdrawal of estrogen/gestagen replacement therapy. Lancet 1981;1:459. [6] Koshihara Y, Hoshi K, Shiraki M. Enhancement of mineralization of human osteoblasts by vitamin K2 (menaquinone 4) (in Japanese). Igaku no Ayumi (J Clin Exp Med) 1992;161:439 – 40. [7] Hauschka PV, Reid ML. Vitamin K dependence of a calcium-binding protein containing g-carboxyglutamic acid in chicken bone. J Biol Chem 1978;253:9063 – 8. [8] Koshihara Y, Hoshi K, Shiraki M. Vitamin K2 (menatetrenone) inhibitsprostaglandin synthesis in cultured human osteoblast-like periosteal cells by inhibiting prostaglandin H synthase activity. Biochem Pharmacol 1993;46:1355 – 62. [9] Hara K, Akiyama Y, Tajima T, Shiraki M. Menatetrenone inhibits bone resorption partly through inhibition og PGE2 synthesis in vitro. J Bone Miner Res 1993;8:535 – 42. [10] Akiyama Y, Hara K, Tajima T, Murota S, Morita I. Effect of vitamin K2 (menatetrenone) on osteoclast-like cell formation in mouse bone marrow cultures. Eur J Pharmacol 1994;263:181 – 5. [11] Hara K, Akiyama Y, Ohkawa I, Tajima T. Effects of menatetrenone on prenisolone-induced bone loss in rats. Bone 1993;14:813 – 8. [12] Akiyama Y, Hara K, Ohkawa I, Tajima T. Effects of menatetrenone on bone loss induced by ovariectomy in rats. Jpn J Pharmacol 1993;62:145 – 53. [13] Nagasawa Y, Fujii M, Kajimoto Y, Imai E, Hori M. Vitamin K2 and serum cholesterol in patients on continuous ambulatory peritoneal dialysis. Lancet 1998;351:724.
A longitudinal study of the effect of vitamin K2 on bone mineral density in postmenopausal women a comparative study with vitamin D3 and estrogen–progestin therapy Ichiro Iwamoto a,*, Shoichiro Kosha a, Shin-ichi Noguchi a, Masato Murakami a, Toshinori Fujino b, Tsutomu Douchi a, Yukihiro Nagata a a
Department of Obstetrics and Gynecology, Faculty of Medicine, Kagoshima Uni6ersity, 8 -35 -1 Sakuragaoka, Kagoshima 890 -8520, Japan b School of Allied Medical Sciences, Kagoshima Uni6ersity, 8 -35 -1 Sakuragaoka Kagoshima 890 -8520, Japan Received 18 May 1998; received in revised form 25 November 1998; accepted 25 November 1998
Abstract Objecti6es: To investigate the effect of vitamin K2 treatment for a year on spinal bone mineral density (BMD) in postmenopausal women, comparing with vitamin D3 hormone replacement therapy and to determine the factors which affect the efficacy of vitamin K2 therapy. Subjects and methods: Seventy-two postmenopausal women were randomized into four groups and treated with respective agents. Before the therapy, 6 and 12 months after the treatment, their lumbar spine BMD were measured by dual energy X-ray absorptiometry. The rates of change in BMD (DBMD) were calculated. Correlations of BMD with age, year since menopause and the initial BMD were determined. Results: Vitamin K2 suppressed the decrease in spinal BMD as compared with no treatment group. BMD in women treated with vitamin K2 was inversely correlated with their age (r = −0.54; PB 0.05). Conclusions: Vitamin K2 therapy may be a useful method for preventing postmenopausal spinal bone mineral loss. In addition, the therapy should be started early in postmenopausal period. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Vitamin K2; Bone mineral density; Treatment
1. Introduction
* Corresponding author. Tel.: +81-99-2755423; fax: + 8199-2650507.
Several agents including vitamin D3, estrogen, calcitonin, and bisphosphonate, etc have been used for preventing postmenopausal osteoporosis. But, in clinical practice, there are some people
0378-5122/99/$ - see front matter © 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 5 1 2 2 ( 9 8 ) 0 0 1 1 4 - 5
162
I. Iwamoto et al. / Maturitas 31 (1999) 161–164
who respond poorly or little to those medicines. Recently, much attention have been paid to vitamin K2 because of its action on bone; vitamin K2 both increases bone formation and decreases bone resorption. Since Bouckaert and Said first reported the effect of vitamin K on fracture healing in 1960 [1], many studies have suggested that vitamin K is closely related with bone metabolism [2,3]. In our country, vitamin K2 (menatetrenone) has been accepted for clinical application in the treatment of osteoporotic osteopenia from 1995. Orimo et al. [4] reported that vitamin K2 increases the bone mineral density (BMD) of cortical bones in elderly osteoporotic patients. However, to prevent bone fracture, cancellous bones should be examined than the cortical ones. In addition, to prevent osteoporosis effectively, the therapy ought to be started earlier than elderly age. There are very few reports about vitamin K2 therapy so far. Our study is the first report which investigated the BMD of cancellous bones in women just after menopause. In the present study, we performed a longitudinal study of the effect of vitamin K2 on vertebral bone in postmenopausal women for 12 months, comparing with vitamin D3 or estrogenprogestins treatment (HRT). Moreover, we investigated the relationship of the rates of change in BMD (BMD) with age, years since menopause (YSM) and initial BMD of the women who were treated by each regimen.
randomly assigned to one of four groups: (1) the control group was untreated; (2) the hormonal replacement therapy (HRT) group was treated with conjugated equine estrogen (CEE), 0.625 mg/ day, together with medroxyprogesterone acetate, 2.5 mg/day for a year; (3) the vitamin D3 group was treated with 1(OH)D3, 1.0 g/day for a year; (4) the vitamin K2 group was treated with menaquinone-4, 45 mg/day for a year. BMD in the lumbar spine was determined by dual energy X-ray absorptiometry (DEXA: QDR2000, Hologic, USA) at three points, that is, before the treatment, 6 and 12 months after the initiation of the treatment. Scans were made along three lumbar vertebrae (L2–L4). BMD was expressed in g/cm2. Rates of change in BMD (DBMD) were calculated. The correlations of BMD with age, years since menopause (YSM) and initial BMD were investigated. Oral or written informed consent was obtained from each subject before study entry. This study was also conducted with institutional guideline. Data were expressed as the mean standard error of the mean (S.E.M.) when indicated. The data were analyzed statistically by the one-way analysis of variance (ANOVA). Spearmans and Pearsons correlation coefficient was calculated as appropriate. PB 0.05 was considered statistically significant.
3. Results 2. Subjects and methods A total of 72 healthy, non-hysterectomized postmenopausal women who had visted at the outpatient clinic of the Department of Obstetrics and Gynecology, Kagoshima University in Kagoshima, between 1994 and 1996 were selected. Postmenopausal state was defined as the absence of menstruation for at least 12 months. All subjects had no medications and complications, which affected bone metabolism and had no unbalanced diet for vitamin K rich food, e.g. green vegetables and fermented foods. All candidates were non-smokers, non-drinkers and led active lives, but did not practice sports. Subjects were
Table 1 shows the baseline characteristic of the subjects. There was no significant difference in age, BMI, YSM, and the initial BMD among the groups. Fig. 1 presents the rates of change in BMD (BMD) in each group during the treatment study. The mean9 S.E.M of BMD (%) at 12 months were (− 2.879 0.51) in control, (0.239 0.47) in vitamin K2 group, (−0.4790.86) in vitamin D3 group and (4.0 9 1.12) in HRT group. There was significant difference in BMD between HRT group and control group (PB0.01). However, the differences between other groups were not statistically significant. The correlations of BMD with age, YSM, and initial BMD in each treatment group were shown in Table 2. In vita-
I. Iwamoto et al. / Maturitas 31 (1999) 161–164
163
Table 1 Baseline characteristic of the groupsa Group
Control (n= 19)
Vitamin K2 (n =17)
Vitamin D2 (n =16)
HRT (n =23)
Age (years) BMIb YSMc BMD (g/cm2)d
53.69 0.84 22.5 9 0.58 4.689 0.84 0.829 0.02
55.9 9 1.55 22.09 0.70 6.18 9 1.15 0.78 9 0.02
52.6 91.76 23.2 90.76 6.0 91.3 0.81 90.02
53.3 90.76 21.4 9 0.68 4.39 9 0.69 0.82 9 0.02
Data were expressed as mean 9 S.E. BMI, body mass index. c YSM, years since menopause. d BMD, bone mineral density. a
b
min K2 group, there was inverse correlation of BMD with age (r = − 0.54; P B0.05), but no significant correlation was observed between initial BMD and YSM. In vitamin D3 group, no significant correlation of BMD with age, YSM, and initial BMD was observed. In HRT group, there was significant correlation of BMD with initial BMD (r= − 0.43; P B 0.05), but not with age and YSM.
4. Discussion There have been many reports in regard to the effect of HRT on BMD; it is generally recognized
that HRT has a suppressive effect on bone loss and an enhancing effect of increasing BMD at lumbar spine in postmenopausal women [5]. In the present study, HRT also prevented bone mineral loss. Thus, it is certain that HRT is a good way for preventing osteoporosis. However, HRT has some adverse effects, for example, increasing the risk of thromboembolism. There are some alternative agents for preventing the bone loss; vitamin K2 is one of the candidates. Vitamin K2 is unique, its actions include enhancing bone formation and suppressing bone resorption. In fact, this vitamin activates osteoblasts to enhance calcification in vitro [6], acts as a coenzyme of g-carboxylase of Gla protein [7] and results in increase of osteocalcin in sera [4]. On the other hand, it has been reported that vitamin K2 inhibited prostaglandin E2 which is a potent bone-resorping factor in human osteoblastlike cells [8,9], and that it inhibited osteclast-like cell formation in mouse bone marrow cultures [10]. Moreover, it has been reported that vitamin K2 suppressed bone loss induced by prednisolone administration [11] or ovariectomy in rats [12].
Table 2 Correlations of DBMD with age, YSM, and initial BMD in each group
Fig. 1. The changes in annual bone mineral density ( BMD) in untreated (control; n =19), vitamin D3 (n= 16) and estrogen– progesteron (HRT; n= 23) groups.
Group
Age
YSM
BMD at baseline
Vitamin K2 Vitamin D3 HRT
−0.54a 0.33 0.32
−0.21 0.16 0.37
0.07 −0.41 −0.43a
a
PB0.05.
164
I. Iwamoto et al. / Maturitas 31 (1999) 161–164
From the phase III study, it was elucidated that vitamin K2 administration enhanced BMD of cortical bone in elderly osteoporotic patients [4]. It is thought that vitamin K2 accelerated bone metabolism due to increase in serum concentration of osteocalcin which is regarded as bone formation marker and increase in urinary hydroxyproline which is regarded as bone absorption marker. In that vitamin K2 enhanced bone metabolism, the effect of vitamin K2 on bone may be predominant in elderly women over younger women. Moreover, it was reported that the effects of this vitamin on BMD was in proportion to circulating level of vitamin D3 [4]. In the light of the decrease of serum vitamin K2 and vitamin D3 concentrations with age, the combination of vitamin K2 and vitamin D3 may be effective for the treatment of osteoporosis in elderly women. Therefore, vitamin K2 treatment might be more applicable for elderly women than for postmenopausal women. But, for preventing bone fraction, it would be wise to start the vitamin K2 therapy earlier than elderly age. In the present study, we found that vitamin K2 suppressed the decrease in spinal bone mineral density in postmenopausal women as compared with no treatment group, although the effect did not reach the statistic significance. We also found that BMD was inversely related with age, suggesting that earlier the therapy starts, the more effective it is for preventing bone loss. Recently, Nagasawa et al. [13] reported that vitamin K2 reduced total cholesterol concentration in osteoporotic patient on continuous ambulatory peritoneal dialysis. This finding suggests that vitamin K2 may have a beneficial effect on lipid metabolism. Though the mechanism by which vitamin K2 acts on metabolism is unclear, if it is beneficial for lipid metabolism, the administration of vitamin K2 may be considered to be useful for postmenopausal women to prevent osteoprosis and to improve hyperlipidemia, especially for women who have a history of thromboembolism which is considered to be contraindication for HRT, or women who respond poorly to HRT. .
Based on these results, we conclude that vitamin K2 may be a promising agent for preventing postmenopausal spinal bone mineral loss. The therapy should be started earlier just after menopause.
References [1] Bouckaert JT, Said AT. Fracture healing by vitamin K. Nature 1960;185:849. [2] Hall JAG, Pauli RM, Wilson KM. Maternal and fetal sequelae of anticoagulation during pregnancy. Am J Med 1980;68:122 – 40. [3] Hart JP, Shearer MJ, Klenerman L, et al. Electrochemical detection of depressed circulating levels of vitamin K1 in osteoporosis. J Clin Endocrinol Metab 1985;60:1268 – 9. [4] Orimo H, Fujita T, Onomura T, Inoue T, Kushida K, Shiraki M. Clinical evaluation of Ea-0167 (menatetrenone) in the treatment of osteoporosis: Phase III. Double-blind multicenter comparative with alfacalcidol (in Japanese). Rinshohyoka (Clin Eval) 1992;20:45 – 100. [5] Christiansen C, Christiansen MS. Bone mass in postmenopausal women after withdrawal of estrogen/gestagen replacement therapy. Lancet 1981;1:459. [6] Koshihara Y, Hoshi K, Shiraki M. Enhancement of mineralization of human osteoblasts by vitamin K2 (menaquinone 4) (in Japanese). Igaku no Ayumi (J Clin Exp Med) 1992;161:439 – 40. [7] Hauschka PV, Reid ML. Vitamin K dependence of a calcium-binding protein containing g-carboxyglutamic acid in chicken bone. J Biol Chem 1978;253:9063 – 8. [8] Koshihara Y, Hoshi K, Shiraki M. Vitamin K2 (menatetrenone) inhibitsprostaglandin synthesis in cultured human osteoblast-like periosteal cells by inhibiting prostaglandin H synthase activity. Biochem Pharmacol 1993;46:1355 – 62. [9] Hara K, Akiyama Y, Tajima T, Shiraki M. Menatetrenone inhibits bone resorption partly through inhibition og PGE2 synthesis in vitro. J Bone Miner Res 1993;8:535 – 42. [10] Akiyama Y, Hara K, Tajima T, Murota S, Morita I. Effect of vitamin K2 (menatetrenone) on osteoclast-like cell formation in mouse bone marrow cultures. Eur J Pharmacol 1994;263:181 – 5. [11] Hara K, Akiyama Y, Ohkawa I, Tajima T. Effects of menatetrenone on prenisolone-induced bone loss in rats. Bone 1993;14:813 – 8. [12] Akiyama Y, Hara K, Ohkawa I, Tajima T. Effects of menatetrenone on bone loss induced by ovariectomy in rats. Jpn J Pharmacol 1993;62:145 – 53. [13] Nagasawa Y, Fujii M, Kajimoto Y, Imai E, Hori M. Vitamin K2 and serum cholesterol in patients on continuous ambulatory peritoneal dialysis. Lancet 1998;351:724.