Low-dose menaquinone-7 supplementation improved extra-hepatic vitamin K status, but had no effect on thrombin generation in healthy subjects

Abstract

Vitamin K is required for the carboxylation of Gla-proteins in the liver (coagulation factors) and extra-hepatic tissues, such as bone (osteocalcin, OC), and arterial wall (matrix Gla-protein, MGP). Although the coagulation factors are essentially fully carboxylated under normal conditions, 10-40 % of OC and MGP remains undercarboxylated. We were therefore interested to study the dose-response effects of extra intake of menaquinones on the carboxylation of the extra-hepatic Gla-proteins. A total of forty-two healthy Dutch men and women aged between 18 and 45 years were randomised into seven groups to receive: placebo capsules or menaquinone-7 (MK-7) capsules at a daily dose of 10, 20, 45, 90, 180 or 360 μg. Circulating uncarboxylated OC (ucOC), carboxylated OC (cOC) and desphospho-uncarboxylated MGP were measured by ELISA. The ucOC:cOC ratio was calculated from circulating ucOC and cOC values. Endogenous thrombin potential and peak height were determined by calibrated automated thrombography. To increase the statistical power, we collapsed the treatment groups into three dosage groups: placebo, low-dose supplementation (doses below RDA, Commission Directive 2008/100/EC), and high-dose supplementation (doses around RDA, Commission Directive 2008/100/EC). MK-7 supplementation at doses in the order of the RDA (Commission Directive 2008/100/EC) increased the carboxylation of circulating OC and MGP. No adverse effects on thrombin generation were observed. Extra MK-7 intake at nutritional doses around the RDA (Commission Directive 2008/100/EC) improved the carboxylation of the extra-hepatic vitamin K-dependent proteins. Whether this improvement contributes to public health, i.e. increasing the protection against age-related diseases needs further investigation in specifically designed intervention trials.

Full text

Low-dose menaquinone-7 supplementation improved extra-hepatic vitamin
K status, but had no effect on thrombin generation in healthy subjects
Elke Theuwissen
1
*, Ellen C. Cranenburg
1
, Marjo H. Knapen
1
, Elke J. Magdeleyns
1
, Kirsten J. Teunissen
1
,
Leon J. Schurgers
1,2
, Egbert Smit
1
and Cees Vermeer
1
1
VitaK & Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Oxfordlaan 70, 6229 EV Maastricht,
The Netherlands
2
Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Universiteissingel 50, 6229 ER Maastricht,
The Netherlands
(Received 28 September 2011 – Revised 25 November 2011 – Accepted 28 November 2011)
Abstract
Vitamin K is required for the carboxylation of Gla-proteins in the liver (coagulation factors) and extra-hepatic tissues, such as bone (osteo-
calcin, OC), and arterial wall (matrix Gla-protein, MGP). Although the coagulation factors are essentially fully carboxylated under normal
conditions, 10–40 % of OC and MGP remains undercarboxylated. We were therefore interested to study the dose–response effects of extra
intake of menaquinones on the carboxylation of the extra-hepatic Gla-proteins. A total of forty-two healthy Dutch men and women aged
between 18 and 45 years were randomised into seven groups to receive: placebo capsules or menaquinone-7 (MK-7) capsules at a daily
dose of 10, 20, 45, 90, 180 or 360 mg. Circulating uncarboxylated OC (ucOC), carboxylated OC (cOC) and desphospho-uncarboxylated
MGP were measured by ELISA. The ucOC:cOC ratio was calculated from circulating ucOC and cOC values. Endogenous thrombin potential
and peak height were determined by calibrated automated thrombography. To increase the statistical power, we collapsed the treatment
groups into three dosage groups: placebo, low-dose supplementation (doses below RDA, Commission Directive 2008/100/EC), and high-
dose supplementation (doses around RDA, Commission Directive 2008/100/EC). MK-7 supplementation at doses in the order of the RDA
(Commission Directive 2008/100/EC) increased the carboxylation of circulating OC and MGP. No adverse effects on thrombin generation
were observed. Extra MK-7 intake at nutritional doses around the RDA (Commission Directive 2008/100/EC) improved the carboxylation of
the extra-hepatic vitamin K-dependent proteins. Whether this improvement contributes to public health, i.e. increasing the protection
against age-related diseases needs further investigation in specifically designed intervention trials.
Key words: Matrix Gla-protein: Osteocalcin: Menaquinone-7: Endogenous thrombin potential
Overall, seventeen vitamin K-dependent proteins, also known
as Gla-proteins, have been identified to date. Of these, one
group of Gla-proteins is involved in haemostasis and includes
clotting factors II, VII, IX, X and proteins C, S, Z
(1)
. These pro-
teins are almost exclusively synthesised by the liver; with the
exception of protein S. Mutant mice revealed that endothelial
cells synthesise about 45 % of circulating protein S
(2)
. The
extra-hepatic group is formed among others by Gla-proteins
involved in extracellular matrix mineralisation, e.g. osteocalcin
(OC; bone), matrix Gla-protein (MGP; cartilage and arterial
vessel wall) and periostin (bone).
Whereas incompletely carboxylated coagulation factors are
seldom seen in healthy subjects, substantial fractions of OC
and MGP circulate in their uncarboxylated forms, suggesting
a suboptimal vitamin K status in extra-hepatic tissues. Elevated
concentrations of uncarboxylated OC (ucOC) have been
associated with osteoporotic fractures
(3)
and high circulating
concentrations of uncarboxylated MGP (ucMGP) have been
shown to predict CVD mortality
(4)
. Inadequate menaquinone
intake has also been associated with increased risk of
cancer
(5)
. The triage theory postulates that long-term vitamin
insufficiency is a risk factor for the development of age-related
diseases
(6)
. In most cases, risk reduction of these diseases
requires vitamin intakes well above the present RDA
(RDA ¼75 mg; Commission Directive 2008/100/EC; Commis-
sion of the European Communities, 2008).
We previously demonstrated that menaquinone-7 (MK-7)
supplementation at a low dose, but above the RDA (Commis-
sion Directive 2008/100/EC), mediates OC carboxylation with
substantially higher efficacy than phylloquinone
(7)
. Here, we
*Corresponding author: Dr E. Theuwissen, fax þ31 433885889, email e.theuwissen@vitak.com
Abbreviations: CAT, calibrated automated thrombography; cOC, carboxylated osteocalcin; dp-ucMGP, desphospho-uncarboxylated matrix Gla-protein; ETP,
endogenous thrombin potential; MGP, matrix Gla-protein; MK-7, menaquinone-7; NP, normal plasma; OC, osteocalcin; PH, peak height; TG, thrombin
generation; ucOC, uncarboxylated osteocalcin.
British Journal of Nutrition, page 1 of 6 doi:10.1017/S0007114511007185
qThe Authors 2012
British Journal of Nutrition

hypothesised that increased carboxylation of OC and MGP can
be obtained with MK-7 administration at nutritional doses. OC
helps in structuring bone by restricting calcification within the
bone fibrils and MGP is the most important local inhibitor of
vascular calcification; a diet leading to increased carboxylation
of both proteins may contribute to public health. Since the key
role of vitamin K in regulating haemostasis is generally
known, an obvious question is whether increased vitamin K
intake may lead to increased thrombosis tendency. Whereas
this is certainly true for subjects receiving vitamin K antagon-
ists, there is no evidence for vitamin K-induced hypercoagul-
ability in healthy subjects. The recent development of
calibrated automated thrombography (CAT) enabled us to
measure the effects on thrombin generation (TG). The objec-
tives of the present study were therefore to study the
dose–response effects of extra MK-7 supplementation on
the carboxylation of OC and MGP and the effects on TG as
an indicator of safety.
Experimental methods
Subjects
Healthy men and women aged between 18 and 45 years were
recruited from the Maastricht University community (Maas-
tricht, The Netherlands) through mailings and small advertise-
ments. Exclusion criteria were BMI .30 kg/m
2
, coagulation
disorders, metabolic or gastrointestinal diseases, chronic dis-
eases (diabetes mellitus, CVD, cancer), the use of supplements
containing vitamin K, routine medication that interferes with
vitamin K and/or coagulation metabolism, participation in a
clinical study 3 months before this study, and soya allergy.
Based on these exclusion criteria and a previous health
check (based on interviews and questionnaires), forty-two
volunteers were selected for this intervention study. There
were no participant dropouts during the period 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 Maastricht University Medical Centre
(Maastricht, The Netherlands). Written informed consent was
obtained from all subjects before entering the study. Trial
registration code: clinicaltrials.gov NCT00483431.
Study design
This study was designed as an exploratory pilot study to
estimate the effective dose of MK-7 (to improve carboxylation
of both OC and MGP) for a more elaborate clinical trial to
investigate the effect of MK-7 supplementation on clinical
endpoints, including bone mineral density, bone mineral
content and vascular characteristics.
The study had a double-blind, randomised, controlled
design. Participants were randomised into seven groups to
receive one of the following supplements (capsules): placebo
or MK-7 at a daily dose of 10, 20, 45, 90, 180 or 360 mg. The
supplements were taken once daily with either breakfast or
dinner during a period of 12 weeks. During the intervention
period, participants returned to the VitaK BV laboratories at
days 0, 1, 3, 7, 14, 28, 42, 56, 70 and 84 for blood sampling.
During the study period, subjects were asked to consume
no more than normal amounts of green vegetables (,200 g/d
of spinach, broccoli, Brussels sprouts and green cabbages)
and (curd) cheese (,50 g/d), and no natto during the study.
The participants were also asked to refrain from consuming
these food items and alcohol for at least 24 h before blood
sampling. Compliance was measured by capsule counts at
the end of the intervention period; the mean compliance for
the MK-7 supplement was 92 %. Any noticeable changes in
health, dietary pattern, physical activity and/or medication
use were recorded.
Study products
The capsules were produced in four different MK-7 (MenaQ7)
dosages (0, 10, 45 and 360 mg/capsule) by NattoPharma ASA
and delivered directly to VitaK BV. The capsules contained
linseed oil, natto oil, potato starch, sunflower oil, carnauba
wax (coating) and ferric oxide (colouring agent). Capsules
with 0, 10, 45 and 360 mg of MK-7 contained 0, 7, 30 and
240 mg of natto oil and 240, 233, 210 and 0 mg of linseed
oil, respectively. All other ingredients were equal for the
different MK-7 capsules.
Blood sampling
Fasting venous blood was collected at days 0, 1, 3, 7, 14, 28,
42, 56, 70 and 84 for the preparation of serum and citrate
plasma (Greiner Bio-One BV). At days 0 and 84, 10 ml of
venous blood was collected for the preparation of platelet-
poor plasma. For serum preparation, blood (10 ml) was
allowed to clot for 30 min at room temperature, centrifuged
at 3000 gfor 15 min, aliquoted and stored at 2808C. Plasma
was prepared immediately after blood sampling (10 ml) by
centrifugation at 3000 gfor 15 min. Also, platelet-poor
plasma was prepared after sampling (10 ml) by a two-step
centrifugation: (1) 3000 gfor 15 min and (2) 10 000 gfor
10 min. Plasma was aliquoted and partly stored at 2808C.
Serum and plasma samples were stored at 2808C until
analysis. All analyses were performed in duplicate (except
for vitamin K analyses), and mean values are given throughout
the present paper.
Vitamin K
Circulating phylloquinone and MK-7 concentrations were
measured at the end of the intervention period. Compliance
was also checked by means of circulating MK-7. Plasma
phylloquinone and MK-7 concentrations were assayed using
HPLC
(8)
.
Extra-hepatic Gla-proteins
Serum ucOC and carboxylated OC (cOC) concentrations were
determined by separate commercial dual-antibody ELISA tests
(Takara Shuzo Company Limited). An in-house control pool
E. Theuwissen et al.2
British Journal of Nutrition

was run on all ELISA plates. The ucOC:cOC ratio was calcu-
lated from circulating ucOC and cOC values, and used as a
marker for vitamin K status in bone. Time-point samples
of one subject were analysed on the same ELISA plate.
The intra- and inter-assay variations for ucOC were 4·8 and
7·7 %, respectively. For cOC, these variations were 2·3 and
10·6 %, respectively.
Plasma desphospho-uncarboxylated MGP (dp-ucMGP) was
measured by an in-house dual-antibody ELISA test
(9)
. Briefly,
the capture antibody specifically recognising dp-MGP is
bound to the microtitre plate. After incubation with the
sample, a biotinylated conformation-specific antibody against
ucMGP is used as a second detecting antibody. Staining was
performed with streptavidine using 3,30,5,50-tetramethylbenzi-
dine as a substrate. An in-house control pool was run on all
ELISA plates. Time-point samples of one subject were
analysed on the same ELISA plate. The intra- and inter-assay
variations were 5·6 and 9·9 %, respectively.
Thrombin generation
TG in tissue factor-triggered platelet-poor plasma was
measured by means of CAT (Thrombinoscope BV), which
employs a low-affinity fluorogenic substrate to continuously
monitor thrombin activity in clotting plasma
(10)
. Fluorescence
was read with a Fluoroskan Ascent reader (Thermo Labsys-
tems) using a 390/460 filter set. TG curves were calculated
with the Thrombinoscope software (Thrombinoscope BV).
Following this, two parameters were derived from the TG
curves: endogenous thrombin potential (ETP, area under the
TG curve) and thrombin peak height (PH). Each TG measure-
ment included pooled normal plasma (NP), and both ETP
and PH are expressed as the ratio of a patient value to the
NP value; value
test
/value
NP
. Pooled NP was prepared at
the Departments of Hematology and Clinical Chemistry of
the Academic Hospital Maastricht (AZM) by pooling plasma
from eighty-five healthy volunteers who did not use medi-
cation. The intra- and inter-assay variations were ,10 %.
Statistical analyses
At baseline, sex differences were tested by the independent-
samples ttest (P,0·05 was considered statistically significant).
Data are presented as means with standard deviations.
Because of small group sizes, the non-parametric Mann –
Whitney test was used to study differences in circulating
vitamin K concentrations after 3-month supplementation.
End values were compared to the end value of the placebo
group after adjustment for multiple comparisons (P,0·008
was considered statistically significant). Data are presented
as medians with ranges. A paired samples ttest (baseline
values as reference values) was used to study the effects of
MK-7 supplementation on ETP and PH values (comparison
between baseline and end values). Correlation analysis was
performed with the Pearson test (P,0·05 was considered
statistically significant).
To increase the statistical power, we collapsed the
seven treatment groups into three dosage groups: placebo,
low-dose MK-7 (doses below the RDA, Commission Directive
2008/100/EC) and high-dose MK-7 (doses around the RDA,
Commission Directive 2008/100/EC). Associations between
extra MK-7 intake and the end value of the measure of interest
(ucOC, cOC, ucOC:cOC, dp-ucMGP) were investigated with
ANCOVA including sex as a fixed factor and the following
variables as covariates: baseline value, age and BMI. Only
the main effects were tested, since the interaction terms with
the measure of interest were not significant. Multiple compari-
sons were made using Bonferroni adjustment (P,0·05 was
considered statistically significant). Baseline and end values
are presented as medians with ranges for the originally
designed treatment groups (seven groups). Mean percentage
changes as compared to baseline are illustrated for the com-
piled groups: placebo, low-dose MK-7 and high-dose MK-7.
Statistical analyses were performed using SPSS for
Windows, version 15 (SPSS, Inc.).
Results
Baseline characteristics
As summarised in Table 1, baseline characteristics were similar
for men and women, with the exception of higher body
weight (P,0·001) and higher cOC concentrations (P¼0·005)
in men. Both ETP (P¼0·008) and PH (P¼0·001) values were
lower in men than in women. A total of eighteen women
were using oral contraceptives, and none of the participants
smoked.
Vitamin K
Plasma phylloquinone concentrations were within the normal
range (0·2 –3·2 ng/ml) and did not differ between the interven-
tion groups (Table 2). Plasma MK-7 concentrations increased
with increasing amount of MK-7 supplementation, indicating
compliance to the intervention. As compared to the placebo
group, the increase in circulating concentrations became
significant from an intake of 90 mg/d.
Table 1. Baseline characteristics†
(Mean values and standard deviations)
All (n42) Men (n20) Women (n22)
Mean SD Mean SD Mean SD
Anthropometric data
Age (years) 28 7 28 7 28 7
Weight (kg) 74 12 81 11 68* 7
BMI (kg/m
2
)243243233
Biochemical markers
ucOC (ng/ml) 4·11 2·71 4·44 3·27 3·82 2·13
cOC (ng/ml) 5·59 2·39 6·64 2·65 4·64* 1·68
ucOC:cOC ratio 0·82 0·54 0·67 0·39 0·95 0·63
dp-ucMGP (pmol/l) 429 140 432 113 427 164
ETP (nmol/l £min) 1516 366 1362 331 1655* 346
PH (nmol/l) 333 85 291 83 372* 67
ucOC, uncarboxylated osteocalcin; cOC, carboxylated osteocalcin; dp-ucMGP,
desphospho-uncarboxylated matrix Gla-protein; ETP, endogenous thrombin
potential; PH, peak height.
* Mean values were significantly different (P,0·05).
† Sex differences were tested by the independent-samples ttest.
Extra-hepatic vitamin K requirement 3
British Journal of Nutrition

Osteocalcin and matrix Gla-protein
Circulating OC and MGP values at baseline and after 3-month
supplementation are described in Table 3. As compared to the
placebo group, MK-7 supplementation at doses around the
RDA (Commission Directive 2008/100/EC; doses described
as high-dose MK-7) significantly improved the carboxylation
of OC and MGP (Fig. 1); significant decreases were seen in cir-
culating ucOC and dp-ucMGP levels as well as in the ucOC:
cOC ratio. Circulating cOC increased significantly after the
highest MK-7 intake. Supplementation at doses below the
RDA (Commission Directive 2008/100/EC; doses described
as low-dose MK-7) had no significant effects on the circulating
levels of both Gla-proteins.
Correlations
At baseline, significant correlations were found between sex
and cOC concentrations (r20·42, P¼0·005). In addition,
age correlated significantly with ucOC concentrations
(r20·36, P¼0·020) and the ucOC:cOC ratio (r20·34,
P¼0·026). At baseline, a significant correlation was also
found between sex and ETP values (r0·40, P¼0·008).
Circulating MK-7 concentrations measured at the end of the
intervention period negatively correlated with changes in
ucOC (r20·37, P¼0·017), ucOC:cOC ratio (r20·45,
P¼0·003) or dp-ucMGP concentrations (r20·63, P,0·001).
End plasma phylloquinone concentrations did not correlate
with changes in ucOC, ucOC:cOC ratio or dp-ucMGP
concentrations.
ETP and PH values highly correlated both at baseline (r0·88,
P,0·001) and at the end of the intervention (r0·91, P,0·001).
Thrombin generation
When comparing baseline and end values, no differences
were found for both ETP (P¼0·691) and PH (P¼0·844) values.
Table 2. Circulating vitamin K concentrations after 3-month supplemen-
tation†
(Medians and ranges)
MK-7
dose
(mg/d)
Phylloquinone (ng/ml) MK-7 (ng/ml)
Median Range Median Range
Group 1 0 0·2 0·0–0·5 0·4 0·0 –0·5
Group 2 10 0·8 0·3– 1·3 0·6 0·4 – 2·1
Group 3 20 0·5 0·1– 1·0 0·5 0·0 – 1·6
Group 4 45 0·3 0·1– 1·2 1·4 0·4 – 4·6
Group 5 90 0·2 0·1–0·9 1·5* 0·5 –4·2
Group 6 180 0·2 0·1– 0·4 3·4* 1·1– 6·5
Group 7 360 0·4 0·3– 0·8 5·5* 4·1– 10·6
MK-7, menaquinone-7.
*P,0·008 for multiple comparisons.
† A total of forty-two participants were randomised into seven groups (six sub-
jects/group) to receive one of the following supplements (capsules): placebo or
MK-7 at a daily dose of 10, 20, 45, 90, 180 or 360 mg. Circulating phylloquinone
and MK-7 concentrations were measured at the end of the intervention period.
Between-group differences (differences as compared to placebo) were tested by
the non-parametric Mann– Whitney test.
Table 3. Circulating osteocalcin (OC) and matrix Gla-protein (MGP) at baseline and after 3-month
supplementation
(Medians and ranges)*
ucOC (ng/ml) cOC (ng/ml) dp-ucMGP (pmol/l)
MK-7 dose (mg/d) Median Range Median Range Median Range
0
Baseline 3·9 2·1– 6·9 4·7 3·0–8·5 435 203– 702
End 3·7 1·8–10·1 5·2 2·2–8·3 508 271–841
10
Baseline 2·3 0·7– 3·4 5·5 3·1–10·2 471 166– 565
End 3·1 0·9–5·9 5·0 2·9– 12·3 414 235 – 846
20
Baseline 4·7 2·3– 9·0 6·0 3·2–7·5 476 247– 621
End 5·3 1·4– 9·2 5·5 2·7– 8·9 427 153– 993
45
Baseline 3·0 1·4– 6·3 6·5 2·2–10·9 416 349– 568
End 2·5 0·8– 7·9 6·1 2·9– 8·4 365 287– 591
90
Baseline 3·8 1·1– 10·5 4·8 2·8 –9·6 428 175–563
End 2·7 0·7– 8·1 5·3 2·2– 8·5 284 120– 383
180
Baseline 4·5 2·4– 12·4 5·5 2·4 –11·3 426 292– 697
End 2·1 0·8–9·5 8·4 3·4– 15·0 257 230 – 397
360
Baseline 1·9 0·8– 5·3 4·5 2·0–6·2 389 272– 516
End 0·7 0·3–2·2 6·7 3·5– 13·8 171 144 – 198
MK-7, menaquinone-7; ucOC, uncarboxylated osteocalcin; cOC, carboxylated osteocalcin; dp-ucMGP, desphospho-
uncarboxylated MGP.
* A total of forty-two participants were randomised into seven groups (six subjects/group) to receive one of the
following supplements (capsules): placebo or MK-7 at a daily dose of 10, 20, 45, 90, 180 or 360 mg. Circulating
OC and MGP were measured at baseline and at the end of the intervention period.
E. Theuwissen et al.4
British Journal of Nutrition

Discussion
There is some concern that current recommendations for
vitamin K intake may be insufficient to ensure adequate
function of vitamin K-dependent proteins not involved
in coagulation
(6)
. While prothrombin is essentially 100 %
carboxylated under normal conditions, 10– 40 % of circulating
OC and MGP remains undercarboxylated
(6,9,11)
. In line with
this, circulating concentrations of ucOC and dp-ucMGP were
detectable in all our volunteers. On the other hand, relatively
low doses of MK-7 were sufficient to substantially increase
carboxylation. A key question, of course, is whether increased
extra-hepatic carboxylation contributes to better health.
McCann & Ames recently postulated that at suboptimal
supply, vitamins are primarily utilised for functions required
for short-term survival
(6)
. Long-term vitamin insufficiencies
may increase the development of age-related diseases,
including osteoporosis, CVD and cancer. In case of vitamin
K, bleeding is the most immediate threat; so a transport
system has evolved ensuring preferential targeting to the
liver. This would explain why the first signs of vitamin K
insufficiency are seen as incomplete carboxylation of the
extra-hepatic Gla-proteins.
Thus far, only two dose– response studies have been per-
formed with MK-7; one study involved reinforced natto
intake and the other studied the effects of enriched olive oil.
Tsukamoto et al.
(12)
showed that 1-week consumption of
natto containing 0·8, 1·3 or 1·8mg MK-7/100 g natto increased
circulating MK-7 concentrations dose-dependently in eight
healthy Japanese volunteers
(12)
. However, only the highest
dose significantly decreased serum ucOC concentrations. Cir-
culating cOC increased in the two highest dosing groups. It
is possible that their regular natto consumption overran the
benefits of additional natto intake. Recently, Bruge
`et al.
(13)
showed in twelve healthy men and women that 2-week
administration of olive oil enriched with 0, 45 or 90 mg MK-7
resulted in a dose-dependent increase in plasma concen-
trations. Only the highest dose significantly improved the car-
boxylation of OC. The lack of effect in both studies may reflect
the small sample size and/or the short intervention period.
Despite the dose-dependent MK-7 increase in our study, the
lower nutritional doses (#45 mg) had also no significant
effects on carboxylation. In agreement, a much larger
sample size is probably needed to detect significant improve-
ments after administrating such low dosages.
Further, extra menaquinone intake in the form of MK-7 sup-
plements has been studied to date in two intervention trials;
one in healthy adults
(7)
and the other in healthy children
(14)
.
Efficacy of MK-7 (0·22 mmol) was compared with that of
phylloquinone (0·22 mmol) in eighteen healthy adults
(7)
.
MK-7 accumulated during the first 2 weeks to reach a plateau
value; phylloquinone remained slightly above baseline values.
Within 3 d, both vitamins had increased OC carboxylation, but
only during MK-7 intake the improvement continued during
the entire 6-week period. Thus, higher and more stable
plasma concentrations were reached with MK-7, and MK-7
was more effective in carboxylating OC. Preferential tissue
targeting may explain the differences in extra-hepatic degree
of carboxylation. In children, 8-week supplementation
with 45 mg MK-7 significantly decreased circulating ucOC
and the ucOC:cOC ratio, demonstrating an improved vitamin
K status
(14)
. Serum cOC concentrations increased by 11 % as
compared to the placebo group; the change was however
not statistically significant. The dosage of 45 mg was based
on the results of the adult study
(7)
; a dosage of 0·22 mmol,
100
80
60
40
Change (%) as compared to baseline
cOC ucOC ucOC:cOC dp-ucMGP
*
*
*
*
20
0
–20
–40
–60
–80
–100
Fig. 1. A total of forty-two participants were randomised into seven groups (six subjects/group) to receive: placebo capsules or menaquinone-7 (MK-7) capsules at
a daily dose of 10, 20, 45, 90, 180 or 360 mg. Circulating osteocalcin (OC) and matrix Gla-protein (MGP) were measured at baseline and at the end of the inter-
vention period. To increase the statistical power, we collapsed the seven treatment groups into three dosage groups: placebo, low-dose MK-7 (doses below the
RDA, Commission Directive 2008/100/EC) and high-dose MK-7 (doses around the RDA, Commission Directive 2008/100/EC). Associations between extra MK-7
intake and the end value of the measure of interest (uncarboxylated osteocalcin (ucOC), carboxylated osteocalcin (cOC), ucOC:cOC, desphospho-uncarboxylated
matrix Gla-protein (dp-ucMGP)) were investigated with ANCOVA including sex as a fixed factor and the following variables as covariates: baseline value, age and
BMI. Multiple comparisons were made using Bonferroni adjustment. Mean percentage changes as compared to baseline are illustrated for the compiled groups:
placebo ( ), low-dose MK-7 ( ) and high-dose MK-7 ( ). *Mean changes (% as compared with baselie) were significantly different from those in the placebo
group (P,0·05).
Extra-hepatic vitamin K requirement 5
British Journal of Nutrition

i.e. 150 mg MK-7 was used for adults with an estimated weight
of 80 kg. This adult dosage was recalculated to the body
weight of children (aged 6–10 years; estimated average
weight 25 kg), leading to a dosage of 45 mg MK-7 for children.
Given the large inter-individual variability in MK-7 absorp-
tion and in effect size, we studied the association of plasma
MK-7 concentrations with changes in circulating extra-hepatic
Gla-proteins. Significant associations were found; the higher
the end plasma MK-7 values, the larger the decrease in circu-
lating ucOC and dp-ucMGP. Tsukamoto et al.
(12)
found a positive
relationship between serum MK-7 and cOC concentrations,
both in men and in women. For this experiment, both markers
were measured in 134 young adults who were classified
according to their intake of dietary natto: rare, occasional
and frequent. In agreement, Bruge
`et al.
(13)
found a significant
correlation between differences in plasma MK-7 and changes
in the cOC:ucOC ratio. Additionally, a high intra- and inter-
individual biological variability in the cOC:ucOC ratio was
described. Considering the high variability of this index,
older volunteers were postulated to show more consistent
effects due to a higher requirement of vitamin K.
To investigate the potential effects of vitamin K supplements
on coagulation, we have used baseline and end-point samples
to compare the paired ETP curves generated by CAT. CAT is a
more sensitive assay than the routine coagulation assays for
anticoagulation monitoring and reversal with different hepar-
inoids
(15)
. Using this highly sensitive system, no effect on the
blood coagulation system was observed. On a theoretical
basis such an effect is not to be expected, because in healthy
subjects all clotting factors contain the maximum number of
Gla-residues (e.g. ten for prothrombin, twelve for factor IX).
Incompletely carboxylated coagulation factors are seldom
seen in the healthy population, but only in patients on cou-
marin derivates or with malabsorption of fat-soluble vitamins.
In the wide range of MK-7 intake of the present study, no
effect on the blood coagulation system was observed.
In conclusion, daily intake of MK-7 at doses in the order of
the RDA (Commission Directive 2008/100/EC) results in mea-
surable changes in circulating ucOC and ucMGP. Given the
fact that in the Western society the total vitamin K intake
amounts to 100 – 150 mg/d, and that the MK-7 was given on
top of a regular diet, this is remarkably low and demonstrates
the high potency of this vitamer. MK-7 is safe with respect to
the haemostatic system, i.e. in subjects not on oral anticoagu-
lants, it does not increase the thrombosis risk even at doses
above the RDA (Commission Directive 2008/100/EC).
Acknowledgements
The present study was supported by Nattopharma ASA (Oslo,
Norway). None of the authors had any possible conflicts of
interest. The contributions of the authors to this study were
as follows: C. V. and L. J. S. designed the research; M. H. K.,
E. J. M. and K. J. T. conducted the research (M. H. K. and
K. J. T. were responsible for the daily management of the
study, E. J. M. was responsible for the OC and MGP analyses);
E. T. and E. C. C. analysed the data; E. T., E. S. and C. V. wrote
the paper; C. V. had primary responsibility for the final
content. All authors read and approved the final manuscript.
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