Three-year low-dose menaquinone-7 supplementation helps decrease bone loss in healthy postmenopausal women

Abstract

We have investigated whether low-dose vitamin K2 supplements (menaquinone-7, MK-7) could beneficially affect bone health. Next to an improved vitamin K status, MK-7 supplementation significantly decreased the age-related decline in bone mineral density and bone strength. Low-dose MK-7 supplements may therefore help postmenopausal women prevent bone loss.
Introduction
Despite contradictory data on vitamin K supplementation and bone health, the European Food Safety Authorities (EFSA) accepted the health claim on vitamin K’s role in maintenance of normal bone. In line with EFSA’s opinion, we showed that 3-year high-dose vitamin K1 (phylloquinone) and K2 (short-chain menaquinone-4) supplementation improved bone health after menopause. Because of the longer half-life and greater potency of the long-chain MK-7, we have extended these investigations by measuring the effect of low-dose MK-7 supplementation on bone health.
Methods
Healthy postmenopausal women (n = 244) received for 3 years placebo or MK-7 (180 μg MK-7/day) capsules. Bone mineral density of lumbar spine, total hip, and femoral neck was measured by DXA; bone strength indices of the femoral neck were calculated. Vertebral fracture assessment was performed by DXA and used as measure for vertebral fractures. Circulating uncarboxylated osteocalcin (ucOC) and carboxylated OC (cOC) were measured; the ucOC/cOC ratio served as marker of vitamin K status. Measurements occurred at baseline and after 1, 2, and 3 years of treatment.
Results
MK-7 intake significantly improved vitamin K status and decreased the age-related decline in BMC and BMD at the lumbar spine and femoral neck, but not at the total hip. Bone strength was also favorably affected by MK-7. MK-7 significantly decreased the loss in vertebral height of the lower thoracic region at the mid-site of the vertebrae.
Conclusions
MK-7 supplements may help postmenopausal women to prevent bone loss. Whether these results can be extrapolated to other populations, e.g., children and men, needs further investigation.

Full text

ORIGINAL ARTICLE
Three-year low-dose menaquinone-7 supplementation helps
decrease bone loss in healthy postmenopausal women
M. H. J. Knapen &N. E. Drummen &E. Smit &
C. Vermeer &E. Theuwissen
Received: 29 November 2012 /Accepted: 12 February 2013 / Published online: 23 March 2013
#International Osteoporosis Foundation and National Osteoporosis Foundation 2013
Abstract
Summary We have investigated whether low-dose vitamin
K2 supplements (menaquinone-7, MK-7) could beneficially
affect bone health. Next to an improved vitamin K status,
MK-7 supplementation significantly decreased the age-
related decline in bone mineral density and bone strength.
Low-dose MK-7 supplements may therefore help postmen-
opausal women prevent bone loss.
Introduction Despite contradictory data on vitamin K supple-
mentation and bone health, the European Food Safety Author-
ities (EFSA) accepted the health claim on vitamin K’srolein
maintenance of normal bone. In line with EFSA’s opinion, we
showed that 3-year high-dose vitamin K1 (phylloquinone) and
K2 (short-chain menaquinone-4) supplementation improved
bone health after menopause. Because of the longer half-life
and greater potency of the long-chain MK-7, we have extended
these investigations by measuring the effect of low-dose MK-7
supplementation on bone health.
Methods Healthy postmenopausal women (n=244) received
for 3 years placebo or MK-7 (180 μg MK-7/day) capsules.
Bone mineral density of lumbar spine, total hip, and femoral
neck was measured by DXA; bone strength indices of the
femoral neck were calculated. Vertebral fracture assessment
was performed by DXA and used as measure for vertebral
fractures. Circulating uncarboxylated osteocalcin (ucOC)
and carboxylated OC (cOC) were measured; the
ucOC/cOC ratio served as marker of vitamin K status.
Measurements occurred at baseline and after 1, 2, and
3 years of treatment.
Results MK-7 intake significantly improved vitamin K
status and decreased the age-related decline in BMC
and BMD at the lumbar spine and femoral neck, but
not at the total hip. Bone strength was also favorably
affected by MK-7. MK-7 significantly decreased the
loss in vertebral height of the lower thoracic region at
the mid-site of the vertebrae.
Conclusions MK-7 supplements may help postmenopausal
women to prevent bone loss. Whether these results can be
extrapolated to other populations, e.g., children and men,
needs further investigation.
Keywords Bone mineral density .Bone strength .
Osteocalcin .Postmenopausal .Ve r t e b ra l f r a c t u r e .Vit am in K
Introduction
The prevalence of osteoporosis increases markedly with
age, and despite advances in diagnosis and treatment, a
minority of patients at high fracture risk is identified for
treatment. Long-term vitamin K inadequacy has been
indicated as an independent but modifiable risk factor
for the development of age-related diseases, including
osteoporosis and cardiovascular disease [1]. Vitamin K
is required for the posttranslational carboxylation of glu-
tamate into γ-carboxyglutamate (Gla) residues in so-
called Gla-proteins [2]. Seventeen Gla-proteins have been
identified to date, and the vitamin K-dependent carbox-
ylation is essential for their function. Dietary vitamin K
intake is too low, however, to support full carboxylation
of at least some of these Gla-proteins, including
osteocalcin (OC) and matrix Gla-protein [3]. OC is gen-
erally regarded as a local inhibitor of mineralization in
bone [4], and the uncarboxylated form (ucOC) is an
M. H. J. Knapen :N. E. Drummen :E. Smit :C. Vermeer :
E. Theuwissen (*)
VitaK, Maastricht University, Oxfordlaan 70,
6229 EV Maastricht, The Netherlands
e-mail: e.theuwissen@vitak.com
Osteoporos Int (2013) 24:2499–2507
DOI 10.1007/s00198-013-2325-6

accepted marker for poor (bone) vitamin K status. Low
vitamin K intake and high circulating ucOC have been
associated with low bone mass and increased fracture
risk [5–9]. Improvement of vitamin K status as measured
by increased carboxylation of OC is readily achievable
by dietary supplementation with vitamin K [10–14]. Con-
sistently, the European Food Safety Authorities (EFSA)
accepted the health claim on vitamin K’s role in mainte-
nance of normal bone [15]. Unfortunately, the molecular
mechanism of vitamin K and/or OC on bone health has
yet to be elucidated.
Despite the observational link between vitamin K status
and bone health, intervention studies have been contradic-
tory regarding supplemental effects on BMD and fracture
risk. Most studies have been carried out in Japan with
pharmacological doses of synthetic vitamin K2 (short-chain
menaquinone-4, MK-4). As summarized in the meta-
analysis by Cockayne, these studies showed an overall
benefit on reducing fracture risk [16]. A more recent meta-
analysis on K1 and K2 supplementation showed modest
treatment effects for vitamin K on BMD [17]. Next to the
high-dose MK-4 trials, four studies with nutritional amounts
of K1 and the long-chain menaquinone-7 (MK-7) were in-
cluded; these studies showed conflicting results [11,18–20].
Even with the contradictory findings on BMD, all studies
reported a reduction in circulating ucOC in response to
vitamin K supplementation. The utility of ucOC as a
marker of bone health has however been questioned as
improved OC carboxylation did not give concomitant
effects on BMD [21]. Still, it could be that improving
vitamin K status may have greater effects on bone qual-
ity than on bone mass. On the other hand, carboxylation-
independent actions have also been reported for vitamin
K’sactiononbonehealth[22].
The efficacy of MK-7 to improve bone health has re-
ceived far less attention than K1 and MK-4. In view of its
longer half-life in the circulation and its higher efficacy [23,
24], it is important to study the effects of long-term low-
dose MK-7 supplementation on bone health. Small amounts
of MK-7 can be found in fermented (curd) cheeses, but large
amounts occur in natto, i.e., Japanese fermented soybeans.
Lower fracture rates were reported in Japanese regions with
high natto intake [25]. Additionally, observational studies
suggest that natto may prevent osteoporosis in Japanese
women [26]. Only two 1-year intervention studies are
known on supplemental MK-7 intake and bone health in
healthy elderly, but both lacked a beneficial effect. Similar
to other forms of vitamin K, MK-7 supplementation signif-
icantly improved OC carboxylation in these studies. The
short treatment period may explain the lack of effect of
supplemental MK-7 intake on BMD. We hypothesized that
long-term supplementation with MK-7 at a nutritionally
relevant dosage will beneficially affect bone health.
Methods
Study participants
Healthy postmenopausal women aged between 55 and
65 years were recruited from the southern region of the
province of Limburg (The Netherlands). Exclusion criteria
were <2 years postmenopausal, BMI >30 kg/m
2
,osteoporotic
at baseline (T-score≤−2.5 SD), coagulation disorders, chronic
diseases, metabolic bone diseases, gastrointestinal diseases,
medication that interferes with vitamin K and/or blood coag-
ulation, use of corticosteroids, bisphosphonates, or hormone
replacement therapy, use of supplements containing vitamin
K, participation in a clinical study 3 months prior to this study,
and soy allergy. Based on these exclusion criteria and a prior
health check (interviews and questionnaires), 244 women
were included in the study and randomly assigned to either
nontreatment (n=124) or treatment (n=120). Figure 1repre-
sents the flow diagram of the study.
This study was conducted according to the guidelines
laid down in the Declaration of Helsinki, and all procedures
involving human subjects were approved by the Medical
Ethics Committee of the Maastricht University (Maastricht,
The Netherlands). Written informed consent was obtained
from all subjects before entering the study. Trial registration
code: clinicaltrials.gov NCT00642551.
Study design
The study had a double-blind, randomized, placebo-
controlled, parallel design. Participants were randomized
into two groups to receive either placebo capsules or cap-
sules containing 180 μgMK-7(MenaQ7,NattoPharma
ASA, Høvik, Norway) per capsule. One capsule was taken
daily either with breakfast or dinner during a period of
36 months. Participants came to the research site every year
for measurements of body weight and height, blood sam-
pling, and DXA measurements. During the visits to our
research site, all noticeable changes in health, dietary
pattern, physical activity, and/or medication use were
recorded.
Study products
The capsules were manufactured by EuroPharma Alliance
(Wroclaw, Poland) for Nattopharma (Høvik, Norway). The
study products, containing 180 μg MK-7 in the form of
MenaQ7 (Nattopharma, Høvik, Norway) and matching pla-
cebo capsules, were delivered to VitaK (Maastricht, The
Netherlands) as complete ready-to-use end products (closed
bottles). The proprietary product MenaQ7 utilized in the
study was provided by NattoPharma. To verify the stability
of MK-7, three capsules of each batch were analyzed at the
2500 Osteoporos Int (2013) 24:2499–2507

start, half-way, and at the end of the intervention period.
Before, during, and after the study, the MK-7 content of the
capsules was determined to be stable, and the mean content
was 180±3 μg/capsule.
Blood sampling
Fasting venous blood was collected once a year for the
preparation of serum and plasma (Vacutainers, Greiner
Bio-One BV, Alphen a/d Rijn, The Netherlands). All blood
samples were drawn between 8:00 a.m. and 11:00 a.m. by
experienced research nurses. For plasma preparation, blood
(10 ml) was collected in citrate tubes, centrifuged for 15 min
at 3,000×g, aliquoted, and stored at −80 °C until analysis.
For serum preparation, blood (10 ml) was allowed to clot for
30 min at room temperature, centrifuged, and stored as
described above.
Circulating markers
Serum uncarboxylated and carboxylated OC (ucOC and
cOC) concentrations were determined by separate com-
mercial dual-antibody ELISA tests (Takara Shuzo Co.
Ltd., Otsu, Shiga, Japan). An in-house control serum
pool was run on all ELISA plates. The ucOC/cOC ratio
was calculated from circulating ucOC and cOC values,
andusedasmarkerfor(bone)vitaminKstatus.To
minimize scattering, samples from four different time
points of each subject were analyzed on the same
ELISA plate. Baseline plasma 25(OH)D and serum
iPTH were determined by using the automated chemilu-
minescent immunoassays on an iSYS system (IDS Ltd.,
Boldon, UK).
Bone measurements
BMC and BMD of the femoral neck, total hip, and lumbar
spine (L1–L4) were determined at baseline and at years 1, 2,
and 3 by DXA (Hologic Discovery A, Waltham, MA, USA).
APEX software version 12.7 was used for acquisition and
analysis. The geometry of the hip bone comprising femoral
neck width (FNW) and hip-axis length (HAL) were assessed
by the scanner software. Indices of femoral neck bone
strength (compression strength, CSI; bending strength,
BSI; and impact strength, ISI) were calculated from body
weight and height, BMD, FNW, and HAL data [12,27].
Fig. 1 Flow diagram
Osteoporos Int (2013) 24:2499–2507 2501

Vertebral fracture (VF) assessment (VFA) was performed
immediately after BMC and BMD measurements. Making
use of Hologic’s software, six markers were placed on the
cranial and caudal sides of the vertebral bodies in anterior,
posterior, and middle positions. Subsequently, the anterior,
posterior, and middle vertebral heights were determined
from the marker points. The Genant’s semiquantitative
method was used to define VF as mild (height reduction
20–25 %), moderate (height reduction 25–40 %), or severe
(height reduction >40 %) [28]. Furthermore, a distinction
was made in fracture site (mid thoracic, T7–T9; low thorac-
ic, T10–T12; lumbar, L1–L3) and type of VF (wedge,
biconcave, crush). L4 was excluded from data analyses,
because the accurate positioning of the markers on the sides
of L4 was difficult in 32 women due to the disappearance of
the disk space between L4 and L5.
Statistics
Based on preliminary estimates of SD in bone strength in
postmenopausal women, we determined that 120 partici-
pants were required in each group for the study to have a
statistical power of 90 % to detect clinically meaningful
differences of 20 % between treatment groups while
allowing for a projected withdrawal of 10 % per year.
Normality was tested by the Kolmogorov–Smirnov test.
Data that were not normally distributed were log-
transformed before statistical analyses (ucOC/cOC).
Between-group differences were tested by the independent
samples ttest. The paired samples ttest was used to study
within-group effects. Linear regression analysis was
performed to study associations between supplemental
MK-7 intake and the measures of interest (ucOC, cOC,
ucOC/cOC, BMD, BMC, and bone strength indices). The
measure of interest was used as the dependent variable. The
concomitant baseline value and the treatment code were
included as independent variables. Age and BMI (if appli-
cable) were included as covariates. Years since menopause,
smoking, 25(OH)D, and iPTH were non-significant contrib-
utors to the statistical model and were therefore not included
in the analyses. Data are presented as means with SD (SE in
figures). A p<0.05 was considered statistically significant.
Statistics were performed using SPSS for Windows, version
19 (SPSS Inc., Chicago, Illinois, USA).
Results
Baseline characteristics
Baseline characteristics of the total group as well as the
placebo and MK-7 groups are presented in Table 1. Mean
values of the variables at baseline were similar (p> 0.05) in
both treatment groups.
At the end of the study, twelve women in the placebo
group and nine women in the MK-7 group had withdrawn
from the study. The overall drop-out rate was 8.6 %. Only a
few women reported complaints during the study. The com-
plaints in the placebo group were: hair loss and/or brittle
nails (n=2), hot flashes (n=1), knee pain (n=1), numb
sensation in arms and legs, washed-out (n= 1), and weight
gain (n=2) and in the MK-7 group: bone pain (n=1), hot
flashes (n=1), rash around eyes and ears (n=1), smelly
capsules (n=1), and weight gain (n=1). Five women with-
drew due to these complaints; four women in the placebo
group and one in the MK-7 group. Compliance was mea-
sured by capsule counts at the end of every half-year period;
the mean compliance for both treatment groups was 97 %.
There were no crush deformations at baseline in this
cohort. Within the total group, the prevalence of biconcave
deformations was the highest in the vertebrae T11 and T12,
whereas wedge deformations occurred mainly in T7 and T8.
Effects of treatment on circulating osteocalcin
MK-7 supplementation significantly decreased circulating
ucOC levels by 51± 21 % as compared to placebo (+ 4±
49 %) (p<0.001; Fig. 2a). Circulating cOC was increased by
21± 19 % as compared to placebo (+ 3±16 %) (p<0.001;
Fig. 2b). Vitamin K status of bone, determined as the
ucOC/cOC ratio, was significantly improved by 58± 18 %
after MK-7 supplementation, whereas placebo supplements
did not alter the ratio (+ 2±47 %) (p< 0.001; data not shown).
The maximal effect on osteocalcin carboxylation was already
reached during the first year and was maintained throughout
the next 2 years of supplementation.
Effects of treatment on BMC and BMD
Both the placebo group and the MK-7 group experienced
bone loss (decrease in BMC and BMD) at the site of the
femoral neck (Fig. 2c, d). During the first year, the rate of
bone loss was similar in both groups. After the first year, the
lines started to diverge with slower bone loss in the MK-7
group. Only after 3 years, MK-7 intake beneficially affected
bone health as compared to the placebo group (p= 0.011 for
BMC, p=0.012 for BMD). After adjusting for age and BMI,
the effect of MK-7 was still significant (p=0.023 for BMC
and p=0.014 for BMD). BMC and BMD of the total hip
steadily decreased during the 3-year study period in both
treatment groups with no significant differences between the
groups (data not shown). In the MK-7 group, the decrease in
BMC and BMD at the lumbar spine was less than 1 % after
the 3-year supplementation period compared to baseline (p=
0.001 and p=0.111, respectively; paired samples ttest). The
2502 Osteoporos Int (2013) 24:2499–2507

placebo group confirmed the expected age-related decline in
BMC and BMD (Fig. 2e, f). Between-group analysis
showed significant differences at the site of the lumbar spine
after the first year of supplementation for BMC (p= 0.019)
and after the second year for BMD (p=0.008). After
adjusting for age and BMI, the beneficial effect of MK-7
on BMC remained significant (p= 0.042), whereas the effect
on BMD became borderline significant (p= 0.070).
Table 1 Baseline characteristics
of the subjects
Data are means±SD or percent-
age of total. No significant dif-
ferences were found between the
treatment groups
BSI bending strength index, CSI
compression strength index, cOC
carboxylated osteocalcin, FNW
femoral neck width, HAL hip axes
length, ISI impact strength index,
ucOC uncarboxylated osteocalcin
Total (n=244) Placebo (n=124) MK-7 (n= 120)
Clinical parameters
Age (years) 60± 3 59± 3 60± 4
Years since menopause 9± 6 8± 5 9±6
Weight (kg) 69± 10 68± 9 69± 10
Height (cm) 165±6 165±6 166± 6
BMI (kg/m
2
) 25±3 25±3 25±3
Current smoker (%) 13 (n=32) 15 (n=19) 11 (n=13)
Cigarettes per day 9± 7 11± 7 7±8
Former smoker (%) 55 (n=135) 58 (n=72) 53 (n=63)
Alcohol consumption (%) 76 (n=185) 78 (n=97) 73 (n=88)
Units of alcohol per week 5.6± 5.5 5.6± 5.0 5.5±6.0
Circulating markers
cOC (ng/ml) 5.5± 1.3 5.6 ±1.3 5.4 ±1.2
ucOC (ng/ml) 3.6± 1.9 3.6 ±1.9 3.6 ±1.9
ucOC/cOC ratio 0.69± 0.41 0.69±0.41 0.69± 0.41
25(OH)D (ng/ml) 30.3± 11.0 30.8±11.2 29.8± 10.9
iPTH (pg/ml) 43.0±15.6 42.5± 15.1 43.4± 16.2
Bone density parameters
BMC femoral neck (g) 3.77± 0.60 3.74± 0.53 3.79 ±0.67
BMC total hip (g) 32.1±4.9 31.9± 4.3 32.4± 5.4
BMC lumbar spine (g) 56.0±8.7 55.5± 7.8 56.5± 9.6
BMD femoral neck (g/cm
2
) 0.732 ±0.101 0.728± 0.085 0.737± 0.115
BMD total hip (g/cm
2
) 0.872± 0.102 0.867±0.088 0.877 ±0.115
BMD lumbar spine (g/cm
2
) 0.933± 0.119 0.926± 0.105 0.939 ±0.132
Hip geometry
FNW (cm) 3.43± 0.27 3.43±0.28 3.43 ±0.26
HAL (cm) 11.2± 0.7 11.2±0.7 11.3 ±0.7
BSI (g/kg.M) 1.13±0.21 1.13± 0.21 1.13± 0.21
CSI (g/kg.m) 3.70 ±0.58 3.69 ±0.54 3.70 ± 0.63
ISI (g/kg.M) 0.25±0.04 0.25 ±0.04 0.25 ± 0.05
Vertebral Fracture Assessment
Height (mm) at the posterior site of
T7–T9 61.5± 3.8 61.3 ±3.9 61.7 ±3.7
T10–T12 71.9± 4.0 71.6 ±3.8 72.1 ±4.2
L1–L3 83.1± 4.6 82.7 ±4.6 83.5 ±4.5
Height (mm) at the midst site of
T7–T9 55.5± 2.9 55.3 ±2.6 55.6 ±3.2
T10–T12 64.4± 3.7 64.3 ±3.3 64.4 ±4.1
L1–L3 75.3± 4.5 75.1 ±4.2 75.6 ±4.7
Height (mm) at the anterior site of
T7–T9 55.4± 3.5 55.5 ±3.4 55.3 ±3.6
T10–T12 66.0± 4.6 65.9 ±4.3 66.1 ±4.6
L1–L3 80.2± 4.5 80.0 ±4.7 80.5 ±4.2
Osteoporos Int (2013) 24:2499–2507 2503

Effects of treatment on bone strength indices
The FNW as well as the HAL did not change signifi-
cantly during the treatment with either placebo or MK-7
(data not shown). For the CSI (Fig. 2g), significant
differences were seen after the second year of MK-7
supplementation (p=0.026) and remained significant
(p=0.045). For the ISI (Fig. 2h), between-group differ-
ences were significant after 1, 2, and 3 years of treatment
(p=0.021, p=0.016, p=0.019, respectively). After
adjusting for age, the difference in ISI between placebo
and MK-7 remained significant (p<0.05), whereas the
effects on CSI became less pronounced (p= 0.022 and
p=0.075 for years 2 and 3, respectively). No significant
effects were found on the BSI (data not shown).
Effects of treatment on vertebral fractures
In six participants of the placebo group, moderate
wedge/biconcave VF occurred in T11 (n=4), in T12 (n=1),
and in T7+ T10 (n=1) during the trial; these vertebrae had
normal heights at baseline. Only in one participant from the
Fig. 2 Effect of MK-7
supplementation on osteocalcin,
BMC and BMD of the femoral
neck and lumbar spine, and bone
strength indices. CSI
compression strength index, ISI
impact strength index, cOC
carboxylated osteocalcin, ucOC
uncarboxylated osteocalcin,
FeNe femoral neck, LS lumbar
spine. Mean (SE) % change in
osteocalcin, BMC and BMD of
femoral neck and lumbar spine
and bone strength indices relative
to baseline during 3 years of
supplementation with MK-7
(white circles) and placebo (black
circles). aand bcirculating ucOC
and cOC; cand dBMC and
BMD of the femoral neck; eand f
BMC and BMD of the lumbar
spine; gand hCSIandISIofthe
femoral neck. *p<0.05,
**p<0.005, #p<0.0001 as
compared to placebo
2504 Osteoporos Int (2013) 24:2499–2507

MK-7 group, T10 showed a moderate wedge VF after 3 years
of treatment. The numbers are however too small to perform
statistics.
To investigate the effects of MK-7 supplementation on
the vertebral heights, we divided the spine in three parts:
mid thoracic (T7–T9), lower thoracic (T10–T12), and lum-
bar (L1–L3) spine. The losses of height (mean loss in T7–
T9, T10–T12, and L1–L3) at the posterior and anterior sites
of the spine were comparable for both treatment groups
(p>0.05; data not shown). The height loss of the middle site
of the vertebrae at T10–T12 was significantly lower in the
MK-7 group than in the placebo group after 2 (MK-7, −2.5±
1.8 %; placebo, −3.1± 2.2 %; p=0.044) and 3 years
(MK-7, −3.3±2.0 %; placebo, −4.1±2.2 %; p=0.003).
Discussion
In this paper, we demonstrate that 3-year supplementation of
low-dose vitamin K2 as MK-7 significantly decreased the
age-related loss in bone mass. Participants taking the MK-7
supplements showed a 3-year preservation of lumbar spine-
BMD, while the placebo group confirmed the expected age-
related decline in BMD. Though both intervention groups
experienced bone loss at the site of the femoral neck, MK-7
intake beneficially affected the rate of bone loss—but the
difference became only significant after 3 years of supple-
mentation. Also, bone strength of the femoral neck was
positively affected by MK-7 supplementation. These results
confirm the hypothesis that long-term supplementation with
MK-7 beneficially affects bone health. Our findings support
EFSA’s acceptance of the health claim that “a cause and
effect relationship has been established between vitamin K
and maintenance of normal bone”[15]. EFSA’s opinion was
mainly based on observational data showing a link between
vitamin K (status) and bone health, as supplementation
studies showed contradictory results. In view of the increas-
ing retail availability of MK-7 supplements intended for
bone health, it is also important to substantiate their effec-
tiveness. The only comparable MK-7 trials published thus
far are the Postmenopausal Health Study II (PHSII) [29] and
a Norwegian study in healthy postmenopausal women [19],
which were both 1-year trials showing conflicting results.
The PHSII showed that 1-year consumption of milk and
yogurt enriched with CaD and MK-7 (100 μg/day) together
with nutrition and lifestyle counseling significantly im-
proved lumbar spine-BMD as compared to the control group
(no vitamin fortification and no counseling). It should be
noted that similar results were seen with products enriched
with CaD and products fortified with CaD and K1
(100 μg/day). In the Norwegian study, 1-year supplementa-
tion with MK-7 (360 μg/day) capsules did not influence
BMD of total hip, femoral neck, lumbar spine, and total
body. The short follow-up period of 1 year was mentioned
as a major limitation. Indeed, we did show that effects of
MK-7 supplementation on bone mass only became signifi-
cant after at least 2 years of intervention.
Subgroup analysis in the most recent meta-analysis on
vitamin K and bone health revealed that K2, but not K1
supplementation, had favorable effects on lumbar spine-
BMD [17]. It is important to note that only two placebo-
controlled trials with MK-7 supplementation were included
[19,20], and outcomes were mainly based on MK-4 in-
terventions. Most of the MK-4 trials were performed in
Japanese osteoporotic women and/or used a pharmacologi-
cal dose of MK-4 making direct comparison to trials with
nutritional dosages and/or healthy participants difficult. Ad-
ditionally, two supplementation trials with K1 at nutritional
doses were included in the meta-analysis. In healthy older
Scottish women, 2-year supplementation of CaD and K1
(200 μg/day) significantly increased distal radius-BMD, but
this change did not differ from that in the control group [11].
In line, no beneficial effect was seen on BMD of lumbar
spine and femoral neck after 3-year supplementation with
CaD and K1 (500 μg/day) in healthy American elderly [13].
K1 and MK-7 differ with respect to their chemical structure
and pharmacokinetics [23,24], which may account for the
differences in effects on bone mass when administrated in
nutritional dosages. Actually, we previously showed greater
bioactivity of MK-7 as compared to K1 when administrating
the same molar dose to healthy adults: higher serum levels
were reached with MK-7, and MK-7 had a higher efficacy in
both hepatic and extra-hepatic protein carboxylation [23].
Recent in vitro data also suggested superiority of MK-7 as
compared to MK-4 and K1 in inhibiting NF-ĸB activation
related to osteoclast development [22].
Despite the heterogeneity in bone mass outcomes, all
studies that measured ucOC (expressed as total circulating
ucOC or percentage ucOC of total circulating osteocalcin)
reported a significant reduction of 20–85 % in this bone
marker in response to vitamin K supplementation [10–13,
19,29–32]. In agreement, we found that 3-year MK-7
supplementation at a daily dose of 180 μg significantly
lowered circulating ucOC by ~50 %. The maximal effect
on osteocalcin carboxylation was already reached during the
first year and was maintained over the next 2 years of
supplementation. Recently, we described a similar reduction
in serum ucOC levels after 3-month supplementation with
the same dose of MK-7 [33]. Although significant positive
effects were seen on both osteocalcin carboxylation and
bone mass, no association was found between the vitamin
K-induced changes in serum ucOC levels and bone param-
eters. Such correlation analyses are however lacking in other
published intervention trials studying vitamin K effects on
bone mass. On the other hand, cross-sectional analyses have
shown an inverse relationship between circulating ucOC
Osteoporos Int (2013) 24:2499–2507 2505

and bone health, measured as BMD or hip fracture [5].
However, the physiological implications for (maximally)
improved carboxylation of circulating ucOC have remained
unknown. Next to the suggested primary mechanism
through OC carboxylation, vitamin K was described to have
carboxylation-independent effects on bone health as well.
Next to superiority of MK-7, the in vitro data showed that
MK-7’s action on osteoblast and osteoclast formation and
activity was accomplished by downregulating NF-ĸB acti-
vation in a carboxylation-independent manner [22].
VF are the most common osteoporotic fractures; they are
mostly asymptomatic, but strongly predict risks for subse-
quent fractures independent of BMD [34]. VFA by DXA
can be used to classify vertebral deformity consistent with
fractures, but has not yet become standard practice. Neverthe-
less, this method was considered a new patient-friendly diag-
nostic tool as it can detect unknown VF in one out of each six
patients [35]. In our study population, the thoracic vertebrae
showed more VF as compared to the lumbar spine. Regarding
the thoracic spine, biconcave deformations were mostly seen
in the lower region (T10–T12) and wedge deformations in the
mid region (T7–T9). Low dietary intake of MK-7 significantly
reduced the loss in vertebral height of the lower thoracic
region (T10–T12) at the mid-site of the vertebrae. This could
however be a chance finding. It should be noted that the
number of participants was not calculated to detect changes
in VF (or fracture risk). More studies are warranted to assess
the relation between K2 intake and VF (or fracture risk).
There are certain limitations to consider. Although sev-
eral bone parameters were investigated in this study, we did
not perform measurements of bone quality, including frac-
ture risk or markers of bone formation/resorption. As men-
tioned before, improving vitamin K status may have more
pronounced effects on bone quality than on bone mass.
Further, we targeted participants in a group generally known
to be at increased risk for accelerated bone loss: healthy
early postmenopausal women. Whether these data can be
extrapolated to other study populations, including children,
men, osteoporotic subjects, or other patient groups needs
further investigation. Nevertheless, the targeted study pop-
ulation may explain why we found positive results [12,30]
in contrast to other long-term studies (2–3 years of treat-
ment) in late postmenopausal women [11,18], and
osteopenic/osteoporotic women [10]. Finally, our study
products were not enriched with CaD, while most studied
K1 supplements did contain CaD. A subject’s CaD environ-
ment may influence vitamin K’s effect on bone health.
Conclusions
Long-term use of MK-7 supplements significantly decreased
the age-related decline in bone mass and strength.
Additionally, the loss in vertebral height of the lower thoracic
region was significantly reduced at the mid-site of the verte-
brae after intake of the MK-7 capsules. We conclude therefore
that postmenopausal women may benefit from taking MK-7
supplements to prevent the age-related bone loss. Since the
efficacy of MK-7 supplements is poorly documented, our
results significantly contribute to substantiating the marketed
anti-osteoporotic properties of vitamin K2.
Acknowledgments The study was funded by Nattopharma ASA
(Høvik, Norway).
Conflicts of interest None.
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