Does vitamin K2 play a role in the prevention and treatment of osteoporosis for postmenopausal women: a meta-analysis of randomized controlled trials

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1 DOI /s ORIGINAL ARTICLE Does vitamin K2 play a role in the prevention and treatment of osteoporosis for postmenopausal women: a meta-analysis of randomized controlled trials Z.-B. Huang & S.-L. Wan & Y.-J. Lu & L. Ning & C. Liu & S.-W. Fan Received: 19 August 2014 /Accepted: 4 December 2014 # International Osteoporosis Foundation and National Osteoporosis Foundation 2014 Abstract Summary To identify the role of vitamin K2 for the prevention and treatment of osteoporosis in postmenopausal women, we conducted this meta-analysis of 19 randomized controlled trials. Our results showed that vitamin K2 might play a role in maintaining the bone mineral density and in reducing the incidence of fractures for postmenopausal women with osteoporosis. Introduction Vitamin K2 has been revealed to be effective in the prevention and treatment of osteoporosis in Japan, which was not confirmed in western countries. Thus, we conduct this meta-analysis to verify the hypothesis that vitamin K2 plays a role in the prevention and treatment of osteoporosis for postmenopausal women. Methods We searched the Cochrane Library, Pub Med, EMBASE, and ISI web of knowledge (until December 1, 2013) and reference lists of eligible articles. A meta-analysis of all-including randomized controlled trials was then performed. Results Nineteen randomized controlled trials encompassing 6759 participants have met the inclusion criteria. Subgroup analysis of postmenopausal women with osteoporosis revealed a significant improvement of vertebral BMD for both medium-term and long-term results favoring vitamin K2 Z.<B. Huang Department of Orthopaedics, Hangzhou Xiasha Hospital, 368 Xiasha Road, Hangzhou, Zhejiang, China Z.<B. Huang: S.<L. Wan (*) : L. Ning : C. Liu : S.<W. Fan Department of Orthopaedics, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, 3 East Qingchun Road, Hangzhou, Zhejiang , China wanshuanglin@aliyun.com Y.<J. Lu Department of Orthopaedics, Shangyu People s Hospital, Shaoxing, Zhejiang, China group (p< and p=0.0005). However, no significant difference in BMD changes was revealed for the nonosteoporosis subgroup analysis. As for the incidence of fractures, pooled analysis of the seven related studies demonstrated no significant difference in the incidence of fractures favoring vitamin K2 (RR=0.63, p=0.08). However, sensitivity analysis by rejecting the study inducing heterogeneity demonstrated a significant difference in the incidence of fractures favoring vitamin K2 (RR=0.50, p=0.0005). Significant differences were found in undercarboxylated osteocalcin reduction and osteocalcin increment. The result of adverse reaction analysis showed that vitamin K2 group seemed to have a higher adverse reaction rate (RR=1.22, p=0.06). Conclusions This meta-analysis seemed to support the hypothesis that vitamin K2 plays kind of a role in the maintenance and improvement of vertebral BMD and the prevention of fractures in postmenopausal women with osteoporosis. The reduction of undercarboxylated osteocalcin and increment of osteocalcin may have some relation to the process of bone mineralization. However, the effect of vitamin K2 for postmenopausal women without osteoporosis had not been identified. Further high-quality RCTs with large sample size are needed to confirm the role of vitamin K2 in osteoporosis for postmenopausal women. Keywords Bone mineral density. Fracture. Meta-analysis. Osteoporosis. Vitamin K2 Introduction Osteoporosis and osteopenia are public health problems worldwide, especially for postmenopausal women. About 54 million Americans have osteoporosis and low bone mass, which results in 2 million broken bones and $19 billion in related costs every year; by 2025, experts predict that

2 osteoporosis will be responsible for approximately 3 million fractures and $25.3 billion in costs each year [1], and these conditions are possibly worse in other countries. Prevention and treatment of osteoporosis, aiming to reduce the related fractures and increase the quality of life, is challenging and requires critical efforts worldwide. Several pharmaceutical treatments have been proven to be effective for the prevention and treatment of osteoporosis, such as bisphosphonate (alendronate and risedronate), parathyroid hormone, raloxifene, nasal calcitonin, and strontium ranelate [2 7]. Nevertheless, the optimal drug therapy for postmenopausal women with or without osteoporosis has not yet been established. Vitamin K is a nutrient that was originally identified as an essential factor for blood coagulation; however, vitamin Ks have been proven to have multiple functions since then, including vitamin K-dependent proteins in hemostasis (such as prothrombin, factor VII, factor IX, factor X, protein C, protein S, and protein Z), vitamin K-dependent transmembrane proteins (such as proline-rich Gla proteins), vitamin K-dependent proteins isolated from bone (such as osteocalcin and matrix Gla protein), and other characterized proteins (such as Gas6, which has been reported to be related to a wide range of cellular alterations) [8]. Among which, vitamin K2 showed an effect of increasing bone mass and reducing fractures both in vivo and in vitro [9 14]. Vitamin K2 plays a role in the g- carboxylating of osteocalcin (OC), which is involved in bone mineralization [15]. Urayama et al. revealed that vitamin K2 inhibits apoptotic cell death and maintains the number of osteoblasts [16], Ichikawa et al. suggested that vitamin K2 modulates its target gene expression in osteoblastic cells through the PKA-dependent mechanism [17]. These findings have added new explanations to the therapeutic efficacy of vitamin K2 in osteoporosis. Despite the encouraging effects of vitamin K2 for osteoporosis, those studies showing the positive effects of vitamin K2 were exclusively done in Japan [11, 18 21]; therefore, this may lower our confidence in the recommendation for the use of vitamin K2. Furthermore, some European and American studies demonstrated that vitamin K2 did not influence bone loss in early postmenopausal women [22, 23]. Since now, there were some systematic reviews evaluating the effect of vitamin K [24 26], providing lots of useful information to identify the role of vitamin K in osteoporosis; however, no systematic review has focused on the effect of vitamin K2 for osteoporosis in healthy postmenopausal women with or without osteoporosis. As a result, it is necessary to conduct this systematic review and meta-analysis aiming to identify the role of vitamin K2 in the prevention and treatment for postmenopausal women with or without osteoporosis. We make a bold hypothesis that vitamin K2 plays a role in the prevention and treatment of osteoporosis in postmenopausal women, and we ask the following questions, which are the principal aspects to evaluate the effect of vitamin K2 for osteoporosis: (1) Can additional oral vitamin K2 supplementation improve bone mineral density (BMD)? (2) Does vitamin K2 reduce the incidence of fractures? (3) Does vitamin K2 influence the bone metabolism process? (4) Is there any adverse effect of additional oral vitamin K2 supplementation? Materials and methods Search strategy PRISMA-compliant searches of the Cochrane Library, Pub Med, EMBASE, and ISI web of knowledge were performed for all peer-reviewed studies published until December 1, 2013 that evaluated the effect of vitamin K2 for the prevention and treatment of osteoporosis in postmenopausal women. The following search terms were adopted for the database search: vitamin K, menatetrenone, menaquinone, osteoporosis, osteoporotic, and postmenopausal. Broad MeSH terms and Boolean operators were used in order to maximize both the search sensitivity and specificity. The reference lists of all the fulltext papers were examined to identify any initially omitted studies. We made no restrictions in the language of publications. Inclusion criteria Studies were considered eligible for inclusion if they met the following criteria: (1) study design: randomized controlled trials; (2) population: postmenopausal healthy women with or without osteoporosis; (3) intervention: additional oral vitamin K2 supplements (group K2); (4) comparator: oral drug supplements without vitamin K2 or placebo group (group C); and (5) outcomes: reported at least one of the following results: bone mineral density (BMD), the incidence of fractures, undercarboxylated osteocalcin (uc-oc), osteocalcin (OC), and complications (or adverse reactions). Exclusion criteria Studies were excluded if (1) studies focused on participants who had Alzheimer s disease or Parkinson s disease or other neurological diseases, for those diseases may reduce daily activities; (2) studies included participants with renal disease, who needed glucocorticoid therapy. In this case, it may cause hormone-induced loss of BMD; (3) studies contained participants who took the drugs that may influence bone metabolism or contraindicate with vitamin K2, such as warfarin or prednisolone; (4) studies included participants with complicated conditions, such as organ-transplanted patients, hemodialysis patients, and so on.

3 Study selection Three reviewers (ZB.H, SL.W, and YJ.L) independently screened the titles and abstracts for the eligibility criteria. Then, full-text intensive reading was performed when those studies might meet the inclusion criteria, and the literature was reviewed to determine the final inclusion. We resolved disagreements by discussion to reach a consensus. Data extraction Three reviewers (ZB.H, SL.W, and YJ.L) independently extracted the following data: study characteristics, country, sample size, age, types of interventions, and outcome parameters. For multiple competitor studies, we extracted the data of the vitamin K2 supplementation group and the controlled group without vitamin K2 supplementation compared to the vitamin K2 group. The extracted data were rechecked by author ZB.H. Outcome The primary outcomes included BMD changes and the incidence of fractures. The following items were included as secondary outcomes: uc-oc, OC, and adverse reactions. We defined time point including medium-term as occurring around 6 months and long-term as occurring around 12 months or even longer [27]. For the result of incidence of fractures, we counted on the final follow-up results. Assessment of methodological quality The risk of bias in the included studies was independently assessed by three authors (ZB.H, SL.W, and YJ.L), in accordance with the Cochrane Handbook for Systematic Reviews of Interventions, version 5.1. Any disagreements were resolved by discussion. We evaluated the included studies using the Cochrane collaboration s tool for assessing the risk of bias, which contains the following domains: adequate sequence generation; allocation concealment; blinding; incomplete outcome data; free from selective reporting; and free from other bias, including baseline balance between groups, no support by funding, and valid sample size estimation. Data analysis We performed all of the meta-analysis within the Review Manager software (RevMan Version 5.1; The Nordic Cochrane Center, The Cochrane Collaboration, Copenhagen, Denmark). For continuous outcomes, the means and standard deviations (SD) were pooled to a weighted mean difference (WMD) and 95 % confidence interval (CI). For dichotomous outcomes, the risk ratio (RR) and the 95 % CI were assessed. A probability of p<0.05 was regarded as statistically significant. Statistical heterogeneity was assessed using Q statistics. A fixed-effects model was used when the effects were assumed to be homogeneous (p>0.05); otherwise, a random effects model was applied when p<0.05 which implied statistical heterogeneity. The subgroup analyses were conducted according to whether the studies included postmenopausal women with osteoporosis or postmenopausal women without osteoporosis. The sensitivity analysis was performed by rejecting the studies which induced the heterogeneity. Because most of the included studies offered the percentage of changes for continuous outcomes; also because the baseline values are instable among studies, our metaanalyses were performed using the percentage of changes to minimize statistical biases. Dealing with studies offering the graph showing mean change percentage and 95 % confidence interval (CI), we calculated the standard deviation (SD) according to SD=N (upper limit lower limit)/t and we retrieve the t value by typing =tinv(1-0.95,n-1) in a cell in a Microsoft Excel spreadsheet [28]. When dealing with studies missing SD of change from baseline, offering only mean and SD of baseline and final result, we retrieve the needed SD according to the method offered in Cochrane Handbook [29]. As for those we could not get the exact SD by any means, we borrowed the needed SD from some other studies included with a similar sample size [29]. Results Search result A total of 811 titles and abstracts were preliminarily reviewed, of which 44 full texts undergone further assessment for eligibility after the removal of duplication and apparently unsatisfied studies. Twenty-eight studies were rejected according to the following reasons: (1) articles focused on vitamin K1 instead of vitamin K2, (2) study participants with various diseases which would influence bone quality, (3) non-randomized controlled trials or retrospective studies or case reports, and (4) less than 20 participants in each group at randomization. Finally, the remaining 16 studies eventually satisfied the eligibility criteria and three more articles were included from the reference lists [11, 18 23, 30 41]. The study selection process was summarized in Fig. 1. Demographic characteristics and quality assessment Eventually, 19 randomized controlled studies with 6759 participants were eligible for inclusion, of which 3364 participants underwent additional oral vitamin K2 supplement, and the remaining 3395 participants received relevant intervention without vitamin K2. However, the intervention of the control group differed among the studies, two studies of which used

4 Fig. 1 Summary of the study selection and exclusion process bisphosphonates as comparator [31, 37], seven studies of which used placebo [20 23, 30, 38, 41], and the remaining ten studies used either calcium or vitamin D3 or combined calcium with vitamin D3 as comparator [11, 18, 19, 32 36, 39, 40]. The participants of nine studies were healthy postmenopausal women without osteoporosis, and the participants of the remaining ten studies were postmenopausal women with osteoporosis. The demographic characteristics of the included studies are summarized in Table 1. Of all the included studies, nine studies offered the details of randomization and of which seven studies offered the blinding of result assessment, who suffered less bias [20, 22, 30, 36, 38, 39, 41]. The remaining ten studies did not mention the details of randomization, let alone the blinding of result assessments. The methodological quality of the included studies is presented in Table 2. Primary outcomes Six studies reported lumbar BMD changes at medium-term follow-up, showing a significant difference in the percentage change of lumbar BMD between the two groups for overall effect, favoring the vitamin K2 group (mean difference 2.01, 95 % CI 0.21 to 3.81, p=0.03) (Fig. 2). The subgroup analysis of participants with osteoporosis revealed a significant difference favoring the vitamin K2 (mean difference 2.70, 95 % CI 1.72 to 3.69, p< ); however, the subgroup analysis of participants without osteoporosis demonstrated no difference between the vitamin K2 group and control group (p=0.67) (Fig. 2). Long-term follow-up lumbar BMD percentage change data was available in ten studies, showing that vitamin K2 group could maintain lumbar BMD better for overall effect (mean difference 1.15, 95 % CI 0.22 to 2.07, p=0.02) and so was the result for the subgroup analysis of participants with osteoporosis (mean difference 2.35, 95 % CI 1.02 to 3.68, p=0.0005). Likewise, the subgroup analysis of participants without osteoporosis found no significant difference between the two groups (p=0.35) (Fig. 3). Three studies reported hip BMD changes at mediumterm follow-up, with only non-osteoporotic participants included, showing that there was no difference between the two groups (mean difference 0.35, 95 % CI 0.12 to 0.82, p=0.14) (Table 3). As for the long-term followup result of hip BMD changes, data was available in six studies with non-osteoporotic participants included only, demonstrating that there was no significant difference between vitamin K2 and the control group in hip BMD changing percentage (mean difference 0.38, 95 % CI 0.17 to 0.93, p=0.17) (Table 3). Five studies reported forearm BMD changes at medium-term follow-up, four of which included osteoporotic participants, demonstrating no significant difference between the two groups for both subgroup analysis and overall effect (mean difference 1.95, 95 % CI 1.21 to

5 Table 1 Characteristics of studies included in the meta-analysis Study ID Study design Country Number OS? Age Follow-up Intervention Comparison intervention Shiraki et al.[11] RCT without details Japan 121/120 Y 67.2± M 45 mg/day menatetrenone + C 150 mg/day Ca Iwamoto et al. [18] RCT without details Japan 29/21 Y M 45 mg/day menatetrenone + C 0.75 μg/day VD3 Iwamoto et al. [19] RCT without details Japan 24/23 Y M 45 mg/day menatetrenone 2 g/day Ca Ishida et al. [20] RCT with details Japan 66/66 Y M Menatetrenone 45 mg/dμ Placebo Orimo H et al. [21] RCT without details Japan 41/39 Y W 90 mg/day menatetrenone Placebo Emaus et al. [22] RCT with details Norway 167/167 N M 360 μg MK-7 Placebo Binkley et al. [23] RCT with details America 129/126 N M 45 mg/day menatetrenone + C Placebo Koitaya et al. [30] RCT with details Japan 24/24 N M 1.5 mg/day MK-4 Placebo Kasukawa et al. [31] RCT without details Japan 50/51 Y >60 12 M 45 mg/day menatetrenone + C 17.5 mg/week risedronate Kanellakis et al. [32] RCT with details Greece 38/39 N M 100 μg MK-7 + C 800 mg Ca 10 μg VD3 Moschonis et al. [33] RCT without details Greece 26/24 N M 100 μg MK-7 + C 800 mg Ca 10 μg VD3 Je et al. [34] RCT without details Korea 40/38 N >60 6 M 45 mg/day menatetrenone + C 400 IU VD3qd 315 mg Ca bid Shiraki et al.[35] RCT without details Japan 60/62 Y 68.6±7.6 6 M 45 mg/day menatetrenone mg/day Ca Inoue et al. [36] RCT with details Japan 2193/2185 Y >50 48 M 45 mg/day menatetrenone + C Oral Ca Hirao et al. [37] RCT without details Japan 26/22 N M 45 mg/day vitamin K2 + C 5 mg/day alendroate Knapen et al. [38] RCT with details Netherlands 164/161 N M 45 mg/day menatetrenone Placebo Purwosunu et al. [39] RCT with details Indonesia 30/33 Y W 45 mg/day menatetrenone + C 1500 mg Ca Ushiroyama et al. [40] RCT without details Japan 43/43 Y M 45 mg/day menatetrenone + C 1 μg/day VD3 Knapen et al. [41] RCT with details Netherlands 124/120 N M 180 μg MK-7/day Placebo RCT with details randomized controlled trial with details of how random sequence generation was conducted, OS osteoporosis, N no, Y yes, M month, C comparison intervention, MK menaquinone, Ca calcium, VD3 vitamin D3

6 Table 2 Risk of bias assessment of study included in this meta-analysis Study ID Random sequence generation (selection bias) Allocation concealment (selection bias) Blinding of participants and personnel (performance bias) Blinding of outcome assessment (detection bias) Incomplete outcome data (attrition bias) Selective reporting (reporting bias) Other bias Shiraki et al. No No No No Yes Yes Yes [11] Iwamoto No No No No Yes Yes Yes et al. [18] Iwamoto No No No No Yes Yes Yes et al. [19] Ishida et al. Yes Yes Yes Yes Yes Yes Yes [20] Orimo H No No Unclear Unclear No Yes Yes et al. [21] Emaus et al. Yes Yes Yes Yes Yes Yes Yes [22] Binkley Yes No No No Yes Yes Yes et al. [23] Koitaya Yes Yes Yes Yes Yes Yes Yes et al. [30] Kasukawa Unclear No No No No Yes Yes et al. [31] Kanellakis Yes No No No No Yes Yes et al. [32] Moschonis et al. [33] No No No No Yes Yes Yes Je et al. [34] No No No No No Yes Yes Shiraki et al. No No No No Yes Yes Yes [35] Inoue et al. Yes No Yes Yes Yes Unclear Yes [36] Hirao et al. No No No No Yes Yes Yes [37] Knapen Yes Yes Yes Yes Yes Yes Yes et al. [38] Purwosunu Yes Yes Yes Yes Yes Yes Yes et al. [39] Ushiroyama No No No No No Yes No et al. [40] Knapen et al. [41] Yes Yes Yes Yes Yes Yes Yes Yes indicating low risk of bias, No indicating high risk of bias, Unclear risk indicating insufficient data for judgment 5.11, p=0.23) (Table 3). As for the long-term follow-up result of forearm BMD changes, data was available in four studies, showing that there was a significant difference between vitamin K2 group and the control group in forearm BMD changing percentage, favoring vitamin K2 group (mean difference 0.89, 95 % CI 0.39 to 1.39, p= ) (Table 3). As for the incidence of fractures, related data was available in seven studies. Two studies included participants without osteoporosis, showing no difference in the incidence of fractures between the two groups (p=0.45) (Fig. 4a). The participants with osteoporosis subgroup analysis demonstrated that no significant difference was found between the two groups with obvious heterogeneity (RR=0.64, 95 % CI 0.36 to 1.13, p=0.13,i 2 =74%)(Fig.4a). After rejecting the study inducing heterogeneity [36], the pooled analysis of the remaining four studies demonstrated a significant difference in the incidence of fractures for vitamin K2 group over the control group (RR= 0.47, 95 % CI 0.32 to 0.70, p=0.0002, I 2 =0 %), and the test for overall effect also showed a significant difference in the incidence of fractures favoring vitamin K2 (RR=0.50, 95 % CI 0.33 to 0.74, p=0.0005, I 2 =6 %) (Fig. 4b). Secondary outcomes We utilized uc-oc and OC, two important bone metabolism makers, as secondary outcomes. Six studies reported mediumterm follow-up result of uc-oc, showing a significant

7 Fig. 2 Forest plot of the medium-term follow-up of vertebral BMD changes, showing a significant improvement of vertebral BMD in osteoporosis subgroup analysis and overall effect for vitamin K2 over control group decrease of uc-oc in the vitamin K2 group than in the control group (p=0.006). Meanwhile, both the participants with osteoporosis subgroup and the participants without osteoporosis subgroup revealed a significant difference between the two groups (p=0.04 and p=0.01, respectively) (Table 3). Longterm follow-up results of uc-oc were available in nine studies, of which two studies focused on the participants with osteoporosis, showing no significant difference between vitamin K2 group and control group (p=0.19). However, the remaining seven studies consisting of non-osteoporotic participants revealed a significant difference, showing a 52.8 % more decrease in the vitamin K2 group than in the control group (p< ). And the overall effect of the nine studies showed a significant difference between the two groups (p< ) (Table 3). As for the result of OC, six studies reported medium-term follow-up data, demonstrating a significant increase of OC in the vitamin K2 group than in the control group for both subgroup analysis and the overall effect (p=0.001) (Table 3). As for the result of long-term follow-up changes for OC, adequate data were available in nine of the trials. There were significant differences between the two groups for both the participants with osteoporosis subgroup analysis and the participants without osteoporosis subgroup analysis (p< and p=0.02, respectively). And the overall effect of the nine studies showed a significant difference between the two groups (p<0.0001) (Table 3). Adverse reactions were reported in ten studies. Pooled result analysis revealed that vitamin K2 group seemed to have a higher rate of adverse reaction (RR= 1.22, 95 % CI 0.99 to 1.49, p=0.06); however, one Fig. 3 Forest plot of the long-term follow-up of vertebral BMD changes, showing a significant improvement of vertebral BMD in osteoporosis subgroup analysis and overall effect for vitamin K2 over control group

8 Table 3 Meta-analysis result of hip BMD, forearm BMD, uc-oc, and OC of the included studies Outcome Subgroup analysis Study number Mean difference 95 % CI P value Model Middle-term hip BMD Osteoporosis 0 Non-osteoporosis to Fixed Overall effect to Fixed Long-term hip BMD Osteoporosis 0 Non-osteoporosis to Random Overall effect to Random Middle-term forearm BMD Osteoporosis to Random Non-osteoporosis to Random Overall effect to Random Long-term forearm BMD Osteoporosis to Fixed Non-osteoporosis to Fixed Overall effect to Fixed Middle-term uc-oc Osteoporosis to Random Non-osteoporosis to Random Overall effect to Random Long-term uc-oc Osteoporosis to Random Non-osteoporosis to < Random Overall effect to < Random Middle-term OC Osteoporosis to Random Non-osteoporosis to Random Overall effect to Random Long-term OC Osteoporosis to < Random Non-osteoporosis to Random Overall effect to < Random BMD bone mineral density, uc-oc undercarboxylated osteocalcin, OC osteocalcin, 95 % CI 95 % confidence interval study contributed a weight of 76.7 % [36] (Fig. 5). Subgroup analyses showed that adverse reactions were more frequently seen in participants with osteoporosis, but there was no difference in adverse reactions between the two groups in participants without osteoporosis (Fig. 5). Pooled result of the remaining nine studies showed no significant difference between the two groups (RR=1.07, 95 % CI 0.69 to 1.67, p=0.75). There are no reported serious adverse events related to oral vitamin K2 supplementation, and minor gastrointestinal symptoms, such as nausea and abdominal pain, were mostly reported adverse reactions. Publication bias The funnel plot for studies reporting the log [RR] of adverse reaction as a measure of treatment effect was conducted to assess the publication bias. The plot is symmetrical, and all Fig. 4 Forest plot of the risk ratio for the incidence of fracture. a Forest plot of the incidence of fracture with obvious heterogeneity. b Result of sensitivity analysis after rejecting the study by Inoue et al. [36]

9 Fig. 5 Forest plot of the incidence of adverse reactions, showing a significant higher incidence of adverse reaction in osteoporosis subgroup analyses for vitamin K2 group over control group studies fall within the 95 % CI axis for a given standard error. There may be few studies missing from the search strategy, and so there is minimal evidence of publication bias (Fig. 6). Discussion This is a meta-analysis of 19 RCTs aiming to estimate the effect of additional supplementation with vitamin K2 for the prevention and treatment of osteoporosis in postmenopausal women. To our knowledge, this is the first meta-analysis of randomized controlled trials focusing on the effect of vitamin K2 in the postmenopausal women with or without osteoporosis. Our result of this meta-analysis showed that vitamin K2 was effective for maintaining the vertebral BMD and forearm BMD in the postmenopausal women with osteoporosis, though no significant effect was found for the improvement of vertebral BMD, hip BMD, and forearm BMD in the Fig. 6 Funnel plot to assess publication bias

10 postmenopausal women without osteoporosis. Vitamin K2 reduced the overall incidence of fractures with a risk ratio of 0.63, which will be discussed later. Supplementation with vitamin K2 showed a significant effect on the decrease of uc-oc and increase of OC, indicating a positive effect for the bone metabolism. The result of adverse reaction analysis showed that vitamin K2 group owned a higher adverse reaction rate. The effect of the prevention of fractures, which is the most important target of a drug for osteoporosis, might not be fully assessed in this meta-analysis due to the lack of data for the incidence of fractures. In total, only seven studies reported the result of incidence of fractures [11, 19, 20, 22, 31, 36, 41], demonstrating no significant reduction of the incidence of fractures for vitamin K2 (RR=0.63, 95 % CI 0.38 to 1.05, p=0.08, I 2 =66 %), after rejecting the study which induced heterogeneity [36]; the pooled analysis of the remaining six studies demonstrated a significant difference in the incidence of fractures for the vitamin K2 group over the control group (RR=0.50, 95 % CI 0.33 to 0.74, p=0.0005, I 2 =6 %). We excluded the OF study by Inoue T in sensitivity analysis [36], which in case would induce selective bias probably. This wellconducted RCT including more than 4000 participants contributed much weight in the meta-analysis of fracture outcomes, showing no significant difference in the incidence of fractures between the two groups. However, this OF study revealed that the incidence rate of new vertebral fractures was significant lower in combined therapy with vitamin K2 among the patients with at least five vertebral fractures. Besides, a significant smaller decrease of height among the older population was observed in the combined group, and a significant higher of activities of daily living (ADL) was observed in the combined therapy group. The OF study had showed a positive effect of vitamin K2 for older population with more serious osteoporosis; however, the conclusion of the positive effect of the fracture rate reduction should be drawn with caution. Stevenson et al. (2009) carried out a systematic review on vitamin K to prevent fractures in older women, demonstrating that vitamin K2 may be associated with a reduced risk of vertebral fractures, but the difference was not significant [42]. An earlier systematic review suggested that vitamin K2 had a strong effect on incidence fractures among Japanese participants [43]; however, four out of the seven studies consisting of participants with stroke, Parkinson disease, and Alzheimer disease and the conclusion might not be applicable in the healthy postmenopausal women population. As for the assessment of BMD, which was presently being the gold standard for definition of osteoporosis, we took the BMD of the vertebra, hip, and forearm for assessment, since these are most commonly seen fracture location in osteoporosis. Our result of this meta-analysis showed that vitamin K2 was effective for maintaining the vertebral BMD; the effect was significant in the postmenopausal women with osteoporosis at both medium-term and long-term follow-up (p< and p=0.0005, respectively). However, the subgroup analysis without osteoporosis revealed no difference in the vertebral BMD change between vitamin K2 and the control group. As for the result of hip BMD, which was reported in studies composed by postmenopausal women without osteoporosis only [22, 23, 30, 34, 37, 38], vitamin K2 did not show the effect of BMD improvement either. However, the study by Knapen MH showed a significant improvement of femoral neck width and bone strength indices (such as compression strength index, bending strength index, and impact strength index), but no changes in BMD [38], explained by the formula BMD=BMC/area, where FNW is one of the dimensions of the surface area. Therefore, the improvement of bone quality does not mean necessary improvement in BMD. A significant difference of forearm BMD changing percentage was found in subgroup analysis of participants with osteoporosis for long-term follow-up, and no significant difference was revealed in non-osteoporotic subgroup analysis. Moreover, the studies showing a positive effect of vitamin K2 were mostly done in Japan [11, 18, 37, 40], and the studies showing negative result were undertaken elsewhere [22, 23, 33, 38, 39], so we conducted another subgroup analysis according to regional difference to explore the potential regional heterogeneity (data not shown), demonstrating significant heterogeneity did exist between study in Japan and elsewhere. In this circumstance, we should take caution to draw the conclusion of the positive effect of vitamin K2 for the improvement of BMD in postmenopausal women, and further studies should be designed to identify the possible factors in this regional heterogeneity. As for the bone metabolism markers, such as uc-oc and OC, supplementation with vitamin K2 showed a significant effect on the decrease of uc-oc and increase of OC. Osteocalcin is a bone matrix component, which may activate the osteoblast and modulate the bone turnover. uc-oc has been identified as a indicator for vitamin K status and may be a risk factor for femoral fracture [44]. In case of vitamin K insufficiency in bone, OC does not undergo complete g-carboxylated, referred as uc-oc, and uc-oc is not incorporate into bone matrix. However, the function of OC and uc-oc has not been well elucidated. Several included studies reported a significant decrease of uc-oc, but no improvement in BMD measures [23, 38, 39, 41], offering no definite explanations. We found that those studies showing a decrease of uc-oc but no improvement in BMD measures mostly included participants without osteoporosis. In our opinion, the possible explanation for this may include as follows: (1) statistically speaking, the participants without osteoporosis mean higher baseline BMD, which results in less percentage change with a same absolute change value; (2) theoretically speaking, the participants without osteoporosis mean higher bone mineralization at baseline, so further mineralization will be more

11 difficult than those with lower bone mineralization at baseline. High-quality studies are needed to confirm our explanation. We took the adverse reaction rate as safety assessment, the result showed that the vitamin K2 group seemed to have a higher adverse reaction rate (RR=1.2, 95 % CI 0.99 to 1.45, p=0.06). However, the statistically significant difference was mainly drawn by one study [36], which contributed a weight of 76.2 %. Pooling the remaining nine studies, the result showed no difference in the adverse reaction rate between vitamin K2 and control group (RR=1.07, 95 % CI 0.72 to 1.58, p=0.75). None of the studies reported serious adverse reaction caused by vitamin K2, and minor gastrointestinal symptoms (nausea or mind abdominal pain) or itching were most reported symptoms. So far, oral supplementation with vitamin K2 was considered to be safe. Though this is a meta-analysis of 19 randomized controlled trials, there are some limitations: (1) over half of the studies suffered from various kinds of bias, including selection bias, performance bias, and detection bias, which lowered the quality of evidence; (2) relative small sample size (ten studies contained less than 50 participants in each group) and lack of critical outcome data (only seven studies reported the incidence of fractures); (3) some important SD data were not reported directly, by converting the reported data to SD, statistical biases were induced into this meta-analysis; and (4) there were obvious between-study heterogeneity in the effect of vitamin K2 on BMD, incidence of fractures, and so on. And we conducted sensitivity analysis and subgroup analysis to identify and minimize the heterogeneity. Nevertheless, we can still obtain important information from this metaanalysis including as follows: (1) the encouraging result of vertebral BMD change was found, especially in osteoporotic women, which theoretically had some positive effect in the prevention of osteoporotic vertebral compression fractures; (2) our result showed that vitamin K2 could probably reduce the incidence of fracture; (3) the result of significant decrease of uc-oc and increase of OC could improve the status of vitamin K deficiency and promote bone mineralization. Conclusion This meta-analysis seemed to support the hypothesis that vitamin K2 plays kind of a role in the maintenance and improvement of vertebral BMD and the prevention of fractures in postmenopausal women with osteoporosis. The reduction of uc-oc and increment of OC may have some relation to the process of bone mineralization. However, the effect of vitamin K2 for postmenopausal women without osteoporosis had not been identified. Further high-quality randomized controlled trials with large sample size are needed to confirm the role of vitamin K2 in osteoporosis. Conflicts of interest Zhao-Bo Huang, Shuang-Lin Wan, Yin-Jiang Lu, Lei Ning, Chao Liu, and Shun-Wu Fan declare that this manuscript is supported by the Office of Education in Zhejiang Province. Grant Number: Y Also, this manuscript is supported by Science and Technology Agency in Zhejiang Province. Grant Number: 2013C References 1. National Osteoporosis Foundation. Fast facts. connect/get-the-facts. Accessed 24 Jan (2014) 2. Meunier PJ, Roux C, Ortolani S et al (2009) Effects of long-term strontium ranelate treatment on vertebral fracture risk in postmenopausal women with osteoporosis. Osteoporos Int 20: Meunier PJ, Roux C, Seeman E et al (2004) The effects of strontium ranelate on the risk of vertebral fracture in women with postmenopausal osteoporosis. N Engl J Med 350: Delmas PD (2002) Treatment of postmenopausal osteoporosis. 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