Introduction 1 1 2 2 1 3 4 1 1 5 1 1 6 1 7 8 1 1 9 2 2 3 4 1 2 1 10 1 11 13 14 15 9 16 1 17 1 18 1 2 19 11 14 16 20 21 22 26 27 28 28 26 29 2 Subjects and methods Subjects The study was designed for apparently healthy, non-osteoporotic women. Participants were recruited by local newspapers. Inclusion criteria were female gender, age between 55 and 75 years at intake, Caucasian race, apparently healthy, at least 2 years postmenopausal and willingness to sign informed consent. Exclusion criteria were: a history of metabolic bone disease(s) or recent bone fractures (less than one year), low bone density (T-score < −2.5), ovariectomy, hysterectomy, oral anticoagulant treatment, hormone replacement therapy, treatment with bisphosphonates, calcitonin, prednisone, heparin, or vitamin K-containing vitamin concentrates and food supplements. Also subjects who had received an investigational new drug within the last 12 months were excluded from the trial. In total 325 women met the criteria and were randomized into our study. The participants were pre-stratified according to age: 105 in the age of 55–65 years, and 220 in the age of 65–75 years. The study protocol was approved by the University Hospital medical ethics committee. Masking Participants were identified by a single randomization number according to the randomization schedule generated by the University Hospital Pharmacy using a computer-generated random permutation procedure in the software package SPSS. Participants were randomly assigned to treatment with either 45 mg/day of MK-4 (menatetrenone, EISAI Co, Tokyo, Japan) or placebo, and compliance with treatment was decided from pill counts during home visits. The daily dose of MK-4 was given in three capsules of 15 mg each, which had to be taken at three time points spread over the day, preferably after the meal. The participant randomization codes were allocated sequentially in the order in which the participants were enrolled. After completion of all analyses the randomization code was disclosed to the investigator. Measurements 29 2 \documentclass[12pt]{minimal}
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\begin{document}$${CSI = {\left( {DA - BMD*FNW} \right)}} \mathord{\left/ {\vphantom {{CSI = {\left( {DA - BMD*FNW} \right)}} {weight}}} \right. \kern-\nulldelimiterspace} {weight}$$\end{document} \documentclass[12pt]{minimal}
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\begin{document}$${BSI = {\left( {DXA - BMD*FNW^{2} } \right)}} \mathord{\left/ {\vphantom {{BSI = {\left( {DXA - BMD*FNW^{2} } \right)}} {{\left( {HAL*weight} \right)}}}} \right. \kern-\nulldelimiterspace} {{\left( {HAL*weight} \right)}}$$\end{document} \documentclass[12pt]{minimal}
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\begin{document}$${{ISI = {\left( {DXA - BMD*FNW*HAL} \right)}} \mathord{\left/ {\vphantom {{ISI = {\left( {DXA - BMD*FNW*HAL} \right)}} {{\left( {height*weight} \right)}}}} \right. \kern-\nulldelimiterspace} {{\left( {height*weight} \right)}}}$$\end{document} Sample collection Fasting blood was taken and 2-h fasting urine was collected at baseline and after 3, 6, 12 and 36 months of treatment. After blood was taken, it was allowed to clot at room temperature for half an hour. Serum was prepared after centrifugation at 3,000 rpm, during 15 minutes. It was subdivided in small aliquots and kept at −80°C until use. Urine was collected as the second morning void after an overnight fast. Biochemical measurements t-OC uc-OC c-OC BAP sNTX 25-OH D DPD Ca creat Statistics The required numbers of participants in both age groups were assessed using separate power analyses (power of 90% and a significance level of 0.05). In the younger age group it was assumed that in the placebo group the mean bone loss is approximately 1.5% (4.5% in 3 years). The lowest detectable effect was set at an improvement of 30% (=3.15% bone loss after 3 years). Taking into account a drop-out frequency of 12% per year this group should consist of at least 76 subjects. In the older age group the mean bone loss was assumed to amount 1% per year (3% in 3 years). With a lowest detectable effect of 30% (=2.1% bone loss in 3 years) and a drop-out frequency of 19% per year, the minimal size of this group is 218 subjects. Statistical analysis was performed using the statistical package SPSS (SPSS Corp, Chicago, IL). The calculations are based on the intention-to-treat principle. Drop-outs were included in the analyses, with the last data available in all subsequent measuring points. All follow-up measurements were related to the baseline values and the proportional changes were calculated for each subject. Differences between and within the different groups were assessed with the unpaired and paired Student’s t-test, respectively. All data are given as mean values ±SE. The linear regression model was used to investigate the effect of treatment after adjustment for potential confounders. Change of FNW, HAL and bone strength indices after 3 years of treatment were used as dependent variables and age and BMI as independent variables. Results Baseline measurements and follow-up 1 2 1 2 Table 1 Baseline characteristics of all participants subdivided according to treatment   Placebo n = 164 MK-4 n = 161 P-value Anthropomorphic variables:    Age (years) 66.0 ± 0.5 65.9 ± 0.4 0.9 Years since menopause 17.7 ± 0.7 17.2 ± 0.6 0.6 Weight (kg) 71.8 ± 1.0 70.3 ± 0.9 0.3 Height (cm) 162 ± 0.5 161 ± 0.5 0.2 2 27.3 ± 0.3 27.1 ± 0.4 0.7 Calcium intake (mg/day) 811 ± 26 870 ± 32 0.1 Non-smoking (%) 87 87 1.0 Bone density characteristics:    2 0.688 ± 0.007 0.706 ± 0.008 0.1  Total hip 0.842 ± 0.009 0.861 ± 0.008 0.1  Lumbar L2-L4 0.931 ± 0.012 0.929 ± 0.013 0.9 BMC (g) of: femoral neck 3.54 ± 0.04 3.65 ± 0.04 0.06  Total hip 30.6 ± 0.43 31.4 ± 0.39 0.2  Lumbar L2-L4 43.1 ± 0.72 42.4 ± 0.76 0.6 Hip geometry:    Mean FNW (cm) 3.43 ± 0.02 3.45 ± 0.017 0.4 HAL (cm) 11.6 ± 0.07 11.7 ± 0.06 0.2 Bone strength indices:    CSI (g/kg.m) 3.41 ± 0.055 3.52 ± 0.052 0.1 BSI (g/kg.m) 1.00 ± 0.017 1.04 ± 0.017 0.2 ISI (g/kg.m) 0.24 ± 0.004 0.26 ± 0.004 0.03 Serum markers for bone metabolism:    c-OC (ng/mL) 6.1 ± 0.2 6.2 ± 0.2 1.0 uc-OC (ng/mL) 3.3 ± 0.2 3.3 ± 0.2 0.4 t-OC (ng/mL) 13.2 ± 0.4 13.2 ± 0.5 0.7 BAP (ng/mL) 23.1 ± 0.7 23.6 ± 0.8 0.2 NTX (nM) 10.8 ± 0.2 10.7 ± 0.3 0.5 25-OH D (nM) 67.2 ± 2.3 72.3 ± 2.6 0.09 Urine markers for bone metabolism:    Creatinine (mmol/L) 3.5 ± 0.3 3.6 ± 0.2 1.0 DPD / creat (μmol/mol) 7.4 ± 0.2 7.5 ± 0.2 0.7 Calcium / creat (mol/mol) 0.29 ± 0.02 0.29 ± 0.01 0.7 Only the BMC and the impact strength index of the femoral neck in the MK-4 group were slightly higher than in the placebo one. This difference is accounted for by expressing MK-4-induced changes as a percentage of the baseline values. All data are given ± SE. Table 2 Baseline characteristics of participants subdivided according to age   Age 55–65 n = 105 Age 65–75 n = 220 P-value Anthropomorphic variables:    Age (years) 59.0 ± 0.3 69.3 ± 0.2  Years since menopause 9.7 ± 0.5 21.2 ± 0.4  Weight (kg) 67.4 ± 1.0 72.8 ± 0.8 0.0001 Height (cm) 163 ± 0.6 161 ± 0.4 0.003 2 25.4 ± 0.4 28.1 ± 0.3 0.0001 Calcium intake (mg/day) 816 ± 34 858 ± 27 0.4 Non-smoking (%) 88 87 0.9 Bone density characteristics:    2 0.716 ± 0.010 0.689 ± 0.006 0.02 Total hip 0.871 ± 0.012 0.842 ± 0.007 0.03 Lumbar L2-L4 0.940 ± 0.015 0.925 ± 0.011 0.4 BMC (g) of: femoral neck 3.69 ± 0.06 3.55 ± 0.03 0.02 Total hip 31.4 ± 0.6 30.7 ± 0.3 0.3 Lumbar L2-L4 44.0 ± 0.9 42.1 ± 0.6 0.1 Hip geometry:    Mean FNW (cm): 3.44 ± 0.022 3.44 ± 0.016 0.9 HAL (cm): 11.8 ± 0.08 11.6 ± 0.06 0.03 Bone strength indices:    CSI (g/kg m): 3.75 ± 0.068 3.31 ± 0.039 0.0001 BSI (g/kg m): 1.09 ± 0.022 0.98 ± 0.013 0.0001 ISI (g/kg m): 0.27 ± 0.005 0.24 ± 0.003 0.0001 Serum markers for bone metabolism:    c-OC (ng/mL) 5.9 ± 0.2 6.3 ± 0.2 0.2 uc-OC (ng/mL) 3.3 ± 0.2 3.2 ± 0.1 0.8 t-OC (ng/mL) 13.9 ± 0.6 12.8 ± 0.4 0.1 BAP (ng/mL) 21.8 ± 0.8 24.1 ± 0.7 0.05 NTX (nM) 11.0 ± 0.3 10.6 ± 0.2 0.2 25-OH D (nM) 76.5 ± 2.7 66.3 ± 2.3 0.005 Urine markers for bone metabolism:    creatinine (mmol/L) 3.5 ± 0.3 3.6 ± 0.2 0.8 DPD / creat (nmol/mmol) 8.2 ± 0.2 7.1 ± 0.2 0.0001 Calcium / creat (mmol/mmol) 0.29 ± 0.02 0.29 ± 0.01 0.9 The differences in all age-related variables were highly significant. All data are given±SE. After 3 years 257 women had completed the study: 133 women in the MK-4 group and 124 women in the placebo group. In total 68 women stopped during the study. This was 21% of the total. The largest group of participants (n = 30) stopped during the first 3 months of the trial. The most important reasons for discontinuation in this time period were various health problems (n = 18), low DXA-BMD (n = 17), lack of motivation (n = 10) and complaints about the capsules (n = 9). Subjects who stopped because of low DXA-BMD did so after consultation during the trial with their general practitioner; there were two complaints about the capsules in the MK-4 group and seven in the placebo group. Two participants died during the trial: one brain tumor and one cerebrovascular accident. Both casualties were in the placebo group. In total, 36 negative or positive side effects were mentioned by the participants. In the MK-4 group 16 women experienced negative effects and four experienced positive effects of the capsules; in the placebo group the numbers were 15 and one, respectively. The most frequent negative effects were gain of weight and gastro-intestinal complaints. However, there was no difference in total numbers of complaints between the MK-4 and the placebo group. Effect of MK-4 supplementation 1 2 2 2 2 Fig.1 a d  Fig. 2 Effects of MK-4 on bone after 3 years of treatment. All data are expressed as a percentage of the respective baseline values. Graph A shows the data of DXA-BMD of the femoral neck (DXA-BMDFN), total hip (DXA-BMDtotalhip) and lumbar spine (DXA-BMDLS); graph B shows the BMC of the femoral neck (BMCFN), total hip (BMCtotalhip) and lumbar spine (BMCLS); graph C shows the femoral neck width (FNW) and hip axis length (HAL) and graph D shows the indices of the compression strength (CSI), bending strength (BSI) and impact strength (ISI). Open bars: placebo; hatched bars: MK-4. Error bars represent SE. Significance of change from baseline: *: p < 0.05, **: p < 0.005; significance of difference MK-4 compared to placebo: #: p < 0.05, ##: p < 0.005  3 3 Table 3 Effect of MK-4 treatment on geometrical variables   Unadjusted adjusted mean difference (SE) p Mean difference(SE) p FNW (%) +1.30 (0.50) 0.01 +1.34 (0.50) 0.009 HAL (%) − 0.25 (0.26) 0.3 +0.23 (0.22) 0.9 CSI (%) +2.26 (0.77) 0.004 +2.03 (0.76) 0.008 BSI (%) +3.80 (1.2) 0.002 +3.83 (1.2) 0.001 ISI (%) +1.98 (0.82) 0.02 +1.72 (0.79) 0.03 2 Fig. 3 Effect of MK-4 on bone strength indices in 2 different age groups (55–65 and 65–75 years of age). All data are expressed as the mean percent change relative to the respective baseline values after 3 years of treatment with either MK-4 or placebo. Graph A shows the compact strength index (CSI), graph B the bending strength index (BSI) and graph C the impact strength index (ISI). Open bars: placebo; hatched bars: MK-4. Error bars represent SE. Significance of change from baseline: *: p < 0.05, **: p < 0.005; significance of difference MK-4 compared to placebo: #: p < 0.05  Discussion 2 2 2 30 32 33 18 19 34 21 2 1 2 2 35 36 37 2 1 1 1 2 1 2 2 39 1 10 2 2 38 2 2