health risk of osteoporosis is increasing in korean women and there were many trials to reduce and prevent osteoporotic fracture risk ( 1 , 2 ) .
vitamin k plays a role in bone metabolism and may decrease the risk of fracture ( 3 , 4 ) .
vitamin k also acts synergistically with vitamin d on bone mineral density ( bmd ) and positively influences the balance of calcium , a key mineral in bone metabolism . as a cofactor for carboxylase activity
, vitamin k facilitates the gamma - carboxylation of osteocalcin to increase the formation of bone ( 5 ) .
if the process of gamma - carboxylation is hindered by lack of vitamin k , the concentration of undercarboxylated osteocalcin ( ucoc ) , which has low affinity to hydroxyapatite in bone , increases .
an inverse relation between ucoc and bmd , and high serum ucoc levels in women in their 20 sec and 50 sec was recently reported ( 6 ) .
since high ucoc level may make women in their 50 sec vulnerable to bone fracture , vitamin k supplementation may be worthwhile for women of this age group to improve bmd .
vitamin k supplementation provided protection against fractures , age related bmd decline , and osteoporosis in some studies ( 7 - 9 ) , while other studies do not support a bmd benefit of vitamin k supplementation ( 10 - 14 ) .
although studies examining the effects of vitamin k supplementation on bmd or osteoporosis have been conducted in many asian countries ( 15 - 18 ) , the role of vitamin k in bone health in korean postmenopausal women remains unclear .
the present intervention study was designed to test the effects of vitamin k supplementation with vitamin d and calcium for 6 months on bmd and ucoc among the advanced postmenopausal korean women over sixty years old .
we enrolled the advanced postmenopausal women over 60-yr - old , who did not want to take anti - resorptive agent such as bisphosphonate .
therefore , postmenopausal women over sixty years old living in seoul , korea , who visited the out patients clinic at cha hospital in seoul from january - may 2010 were recruited ( n = 385 ) ( fig .
103 women were excluded for taking vitamin k , anti - lipid medications , hormone replacement therapy , or medications influencing bone metabolism aspects such as bisphosphonate , calcitonin , steroids , phenytoin , carbamazepine , rifampicin , heparin , and warfarin . as well , 199 women with diabetes , hypertension , body mass index ( bmi ) > 30 kg / m , or metabolic bone diseases were excluded .
the remaining 78 women were randomly assigned to either the treatment group ( vitamin k , n = 40 ) or a control group ( n = 38 ) . for 6 months , the treatment group received 15 mg of vitamin k2 ( menatetrenone ) three times a day after every meal , combination form containing 400 iu of active vitamin d3 once a day , and 315 mg of calcium carbonate twice daily . during the same period ,
the control group received 400 iu of vitamin d and 315 mg of calcium twice daily .
during the course of the study , 33 women dropped out due to relocation or loss to follow - up .
fasting blood ( 10 hr of fasting in the morning ) was collected at baseline and at months 3 and 6 .
total cholesterol , triglyceride and high density lipoprotein ( hdl ) cholesterol were measured by an enzymatic colorimetric assay using a model 7600 apparatus ( hitachi , tokyo , japan ) .
the bmd in the lumbar spine ( l1-l4 ) and femur neck , and ward measurements were performed using a qdr 4500 apparatus ( hologic , waltham , ma , usa ) at baseline and 6 months .
the serum ucoc was assayed by an enzyme - linked immunosorbent assay using two monoclonal antibodies .
anti - osteocalcin ( oc ) antibody and a solid - phase anti - glu 21 , 24-oc antibody with recombinant human ucoc ( takara shuzo , shiga , japan ) were used .
the coefficients of variation ( cvs ) in the intra- and inter - assay were 7.3% and 9.7% , respectively .
the study participants reported their medications to the principal investigators at screening , and at months 3 and 6 , and were interviewed about side effects at every visit . for assess study compliance ,
the remaining pill count was checked at all visits , and blood samples were collected at baseline and months 3 and 6 .
an independent t test was used to compare the baseline demographics and any changes that occurred after 6 months of intervention between the vitamin k group and the control group . to compare any changes in each group after 6 months , the paired t test was used . an intention to treat ( itt ) analysis
in addition , perprotocol ( pp ) analysis of the 45 subjects was also conducted .
these analyses were done using spss version 18.0 ( spss , chicago , il , usa ) .
all participants provided written informed consent and this study was reviewed and approved by the institutional review board of cha hospital ( eki - gla-06 - 32 ) .
we enrolled the advanced postmenopausal women over 60-yr - old , who did not want to take anti - resorptive agent such as bisphosphonate .
therefore , postmenopausal women over sixty years old living in seoul , korea , who visited the out patients clinic at cha hospital in seoul from january - may 2010 were recruited ( n = 385 ) ( fig .
103 women were excluded for taking vitamin k , anti - lipid medications , hormone replacement therapy , or medications influencing bone metabolism aspects such as bisphosphonate , calcitonin , steroids , phenytoin , carbamazepine , rifampicin , heparin , and warfarin . as well , 199 women with diabetes , hypertension , body mass index ( bmi ) > 30 kg / m , or metabolic bone diseases were excluded .
the remaining 78 women were randomly assigned to either the treatment group ( vitamin k , n = 40 ) or a control group ( n = 38 ) . for 6 months , the treatment group received 15 mg of vitamin k2 ( menatetrenone ) three times a day after every meal , combination form containing 400 iu of active vitamin d3 once a day , and 315 mg of calcium carbonate twice daily . during the same period ,
the control group received 400 iu of vitamin d and 315 mg of calcium twice daily .
during the course of the study , 33 women dropped out due to relocation or loss to follow - up .
fasting blood ( 10 hr of fasting in the morning ) was collected at baseline and at months 3 and 6 .
total cholesterol , triglyceride and high density lipoprotein ( hdl ) cholesterol were measured by an enzymatic colorimetric assay using a model 7600 apparatus ( hitachi , tokyo , japan ) .
the bmd in the lumbar spine ( l1-l4 ) and femur neck , and ward measurements were performed using a qdr 4500 apparatus ( hologic , waltham , ma , usa ) at baseline and 6 months .
the serum ucoc was assayed by an enzyme - linked immunosorbent assay using two monoclonal antibodies .
anti - osteocalcin ( oc ) antibody and a solid - phase anti - glu 21 , 24-oc antibody with recombinant human ucoc ( takara shuzo , shiga , japan ) were used .
the coefficients of variation ( cvs ) in the intra- and inter - assay were 7.3% and 9.7% , respectively .
the study participants reported their medications to the principal investigators at screening , and at months 3 and 6 , and were interviewed about side effects at every visit . for assess study compliance ,
the remaining pill count was checked at all visits , and blood samples were collected at baseline and months 3 and 6 .
an independent t test was used to compare the baseline demographics and any changes that occurred after 6 months of intervention between the vitamin k group and the control group . to compare any changes in each group after 6 months , the paired t test was used . an intention to treat ( itt ) analysis
in addition , perprotocol ( pp ) analysis of the 45 subjects was also conducted .
these analyses were done using spss version 18.0 ( spss , chicago , il , usa ) .
all participants provided written informed consent and this study was reviewed and approved by the institutional review board of cha hospital ( eki - gla-06 - 32 ) .
a total of 385 postmenopausal women over sixty were screened and 78 women were enrolled in the study ( fig .
1 ) . the main reasons for ineligibility were the used of restricted medications and occurrence of diseases that could affect bone metabolism . in the itt analysis comparing the characteristics of the vitamin k group with the control group ( table 1 ) , the age at menopause in the vitamin k group ( 49.6  2.7 yr ) was significantly different from the age at menopause in the control group ( 50.8  1.7 yr , p = 0.03 ) .
other characteristics did not differ in the treatment group in both itt and pp analyses . in the pp analysis ( table 2 ) ,
characteristics of women in the vitamin k group were not different from those in the control group .
the mean (  sd ) age , age at menopause , and duration of menopause were 67.3  6.3 , 50.6  1.9 , and 16.6  6.8 yr , respectively , in the vitamin k group and 67.8  6.4 , 51.0  1.9 , and 16.8  7.0 yr , respectively , in the control group .
bmds in lumbar and femur were similar in both the vitamin k and the control groups .
in addition , the ucoc concentration in the vitamin k group ( 2.1  1.7 ng / dl ) was not significantly different from that in the control group ( 1.5  1.0 ng / dl , p = 0.214 ) .
the mean (  sd ) osteocalcin level was 9.6  6.4 ng / dl in the vitamin k group and 11.7  6.4 ng / dl in the control group .
there were no significant differences in blood urea nitrogen , creatinine , calcium and serum bone - specific alkaline phosphatase ( bsalp ) between the vitamin k and the control groups .
the bmd in l1-l4 and femur neck , and in the ward in the vitamin k group were not different from those in the control group .
the serum ucoc level in the vitamin k group ( 2.1  1.7 ng / dl ) was higher than that in the control group ( 1.5  1.0 ng / dl ) , but showed no significant difference ( p = 0.21 ) .
osteocalcin level was nonsignificantly lower in the vitamin k group ( 9.6  6.4 ng / dl ) than in the control group ( 11.7  6.4 ng / dl , p = 0.29 ) .
no significant differences in bmd and biomarker values between the vitamin k and the control groups indicated that the randomization worked well .
after 6 months of treatment , the l3 bmd in the vitamin k group compared to the control group increased statistically significantly ( 0.01  0.03 g / cm vs -0.008  0.04 g / cm , p = 0.049 ) .
compared to bmd at baseline , bmd in femur at month 6 was significantly increased in both the vitamin k and the control groups . however , after 6 months of treatment , bmd in the vitamin k group was not statistically different from bmd in the control group .
in addition , compared to the baseline , the vitamin k group significantly decreased ucoc concentration ( -1.6  1.6 ng / dl , p < 0.01 ) , whereas the ucoc level in the control group did not change ( -0.4  1.1 ng / dl ) . the change in ucoc concentration in the vitamin k group , but not in the control group , confirmed the compliance of the participants in the vitamin k group with the study protocol .
osteocalcin was also non - significantly increased in the vitamin k group ( 1.6  5.8 ng / dl ) , but not in the control group ( -1.1  6.0 ng / dl ) .
triglyceride level decreased in the vitamin k group ( -10.0  59.1 ng / dl ) ( table 3 ) .
three people complained of nausea sensation twice after taking vitamin k. no other participants including people in the control group reported adverse events during the study period . in summary , after 6 months treatment of vitamin k to the postmenopausal women over sixty , the l3 bmd increased significantly compared to the control group .
in addition , the changes in some biochemical markers differed by treatment . compared to the control group , the vitamin k group significantly decreased serum ucoc concentration ( p = 0.008 ) .
osteocalcin level was higher in the vitamin k group than in the control group , but the difference was not significant ( p = 0.14 ) .
in this study , supplementation with vitamins k and d , and with calcium for 6 months of treatment significantly improved the l3 bmd compared to supplementation with vitamin d and calcium in postmenopausal korean women over sixty years old .
in addition , femur bmd was increased in the vitamin k group ( vitamin k + vitamin d + calcium ) , but a similar increase was observed in the control group ( vitamin d + calcium supplementation ) .
the ucoc concentration significantly decreased in the vitamin k group compared to the control group ( p < 0.01 ) , and the osteocalcin level increased nonsignificantly in the vitamin k group ( p = 0.14 ) .
the triglyceride level decreased in the vitamin k group , but was not statistically different from the control group .
when vitamin k concentration in the body is low , insufficient amount of osteocalcin completes -carboxylation ; consequently , ucoc concentration increases and the affinity to calcium to bone matrix decreases .
gla - residues of osteocalcin combine with calcium and require vitamin k to activate the reaction site .
therefore , measuring ucoc concentration is a more accurate method of monitoring vitamin k than prothrombin in the liver ( 19 ) . in a previous cross - sectional study in postmenopausal women ,
elevated serum ucoc and low spinal bmd were observed in the low plasma phylloquinone ( vitamin k1 ) group ( 20 ) . in another study ,
healthy elderly scottish women who took vitamin k1 with vitamin d and calcium showed a sustained increase in bmd at the site of the ultradistal radius in comparison with women taking calcium and vitamin d ( 21 ) . a combined vitamin k2 therapy with vitamin d3 for postmenopausal japanese women produced low serum ucoc and improved bmd values ( 22 ) .
in an examination of the effects of vitamin k2 ( 45 mg / day ) supplementation with calcium ( 1,500 mg / day ) on lumbar bmd in postmenopausal women in indonesia , a meaningful increase in bmd and a decrease in ucoc level were reported ( 18 ) . on the other hand ,
a study reported no vitamin k effect on bmd in north american women , prompting the suggestion that women receiving calcium and vitamin d supplements do not need additional vitamin k supplementation to prevent osteoporosis ( 23 ) .
another recent study of vitamin k1 supplementation for 2 - 4 yr in canadian women also reported no benefits on age - related decline in bmd ( 12 ) .
our study showed that compared to the control group that took vitamin d and calcium , additional vitamin k supplementation to vitamin d and calcium improved l3 bmd in korean postmenopausal women over sixty .
our previous cross - sectional study involving korean women reported an inverse association between ucoc and bmd ( 6 ) .
this dichotomy may partially be due to an ethnic difference and subjects ' age in the bone metabolism or response to vitamin k or ucoc .
a study of inflammatory bowel disease patients in japanese women reported a decrease in the concentrations of vitamin k and d , and a resulting decrease in bmd ( 24 ) .
the foregoing results imply that ethnic - related diversity in gastrointestinal absorption may explain the different response to vitamin k supplement and its subsequent effects on bmd ( 25 ) .
several studies reported that obesity has been related to the level of vitamin k in adipose tissue , reflecting it 's fat - soluble nature . in one study
, obese patients displayed a lower ucoc concentration and their ucoc / osteocalcin ratio was negatively related with body mass index ( 26 ) .
another study reported that people with a high serum triglyceride level had high bmd in skeletal muscles ( 27 ) . on the other hand ,
adult obesity has been inversely related to circulating indicators of serum vitamin k ( 28 )
. limitations of the study were the small number of participants , relatively high dropout rate , and lack of a placebo group ( i.e. , no supplementation ) .
the dosages of vitamin k , vitamin d and calcium and the duration of treatment may have been insufficient to induce responses in related biochemical markers and total bmd . moreover
also , bone quality was not measured , which might be only a minor limitation . a previous study ( 6 )
suggested that ucoc concentration is inversely associated with age , with higher ucoc concentrations in younger women .
this suggests that the effect of the vitamin k on osteocalcin may also be age - related .
given the advanced age of our study subjects ( mean age 68 yr ) and relatively short treatment period , the study may have not been able to discern an increase all part of bmd .
however , it is noteworthy that changes in ucoc concentration among older women have been reported , with ucoc concentration in women in their 50 sec being higher than that in women in their 60 sec and 70 sec ( 6 ) .
therefore , vitamin k may be more effective in preventing bone loss or improving bmd in women aged 50 - 59 yr , especially in perimenopausal women or women with few years of menopause . a study testing the influence of vitamin k supplementation to perimenopausal women or women with few years of menopause for more than 6 months is needed .
in addition , inconsistent findings from different countries suggest that a comparison of korean data with data from other countries is warranted .
a study design involving a longer duration of vitamin k supplementation and with postmenopausal women having a short duration of menopause may prove valuable .
finally , physical activity was not measured . in conclusion , in this randomized study , supplementation with vitamin k , d and calcium for 6 months improves l3 bmd and reduces ucoc concentration in korean postmenopausal women over sixty .
vitamin k supplement along with vitamin d and calcium reduced serum concentration of undercarboxylated osteocalcin while increasing bone mineral density in korean postmenopausal women over sixty - years - old sang hyeon
je , nam - seok joo , beom - hee choi , kwang - min kim , bom - taeck kim , sat - byul park , doo - yeoun cho , kyu - nam - kim and duck - joo lee this study is to evaluate effects of vitamin k supplementation on bone mineral density ( vmd ) in postmenopausal korean women .
78 postmenopausal women over sixty years old were assigned to either control ( calcium & vitamin d , n = 40 ) or vitamin k - supplemented group ( calcium , vitamin d & vitamin k , n = 38 ) .
after 6 months of treatment , the vitamin k group showed decrease of undercarboxylated osteocalcin and increase of bmd . the intention to treat ( itt )
 there are inconsistent findings on the effects of vitamin k on bone mineral density ( bmd ) and undercarboxylated osteocalcin ( ucoc ) . 
 the present intervention study evaluated the effect in subjects over 60-yr - old . 
 the vitamin k group ( vitamin k + vitamin d + calcium supplement ; 15 mg of vitamin k2 [ menatetrenone ] three times daily , 400 iu of vitamin d once a day , and 315 mg of calcium twice daily ) and the control group ( vitamin d + calcium supplement ) were randomly assigned . during the six months of treatment , seventy eight women participated ( 38 in the vitamin k group and 40 in the control group ) and 45 women completed the study . 
 the baseline characteristics of study participants did not differ between the vitamin k and the control groups . in a per protocol analysis after 6 months 
 , l3 bone mineral density has increased statistically significantly in the vitamin k group compared to the control group ( 0.01  0.03 g / cm2 vs -0.008  0.04 g / cm2 , p = 0.049 ) . 
 ucoc concentration was also significantly decreased in the vitamin k group ( -1.6  1.6 ng / dl vs -0.4  1.1 ng / dl , p = 0.008 ) . in conclusion , 
 addition of vitamin k to vitamin d and calcium supplements in the postmenopausal korean women increase the l3 bmd and reduce the ucoc concentration . 
INTRODUCTION
MATERIALS AND METHODS
Subjects and study design
Statistical analysis
Ethics statements
RESULTS
DISCUSSION
AUTHOR SUMMARY