Nutrition 22 (2006) 845– 852

www.elsevier.com/locate/nut
Review article

Pleiotropic actions of vitamin K: protector of bone health and beyond?

Masao Kaneki, M.D., Ph.D.,a,b,* Takayuki Hosoi, M.D., Ph.D.,c
Yasuyoshi Ouchi, M.D., Ph.D.,d and Hajime Orimo, M.D., Ph.D.e
a
Department of Anesthesia and Critical Care, Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts, USA
b
Shriners Hospital for Children, Boston, Massachusetts, USA
c
Department of Advanced Medicine, National Center for Geriatrics and Gerontology, Aichi, Japan
d
Department of Geriatric Medicine, Graduate School of Medicine, University of Tokyo, Tokyo, Japan
e
University of Health Science, Yamanashi, Japan

Manuscript received December 6, 2005; accepted May 4, 2006.

Abstract Vitamin K is a nutrient that was originally identified as an essential factor for blood coagulation.
Recently, vitamin K has emerged as a potential protector against osteoporosis, atherosclerosis, and
hepatocarcinoma. Accumulated evidence indicates that subclinical non-hemostatic vitamin K defi-
ciency in extrahepatic tissues, particularly in bone and possibly in vasculature, exists widely in the
otherwise healthy adult population. Vitamins K1 and K2 have been shown to exert protective effects
against osteoporosis, although it is important that the beneficial effects will be further confirmed by
large-scale, randomized, clinical trials. Increasing evidence implicates a role for vitamin K in
calcification of arteries and atherogenesis. Moreover, the therapeutic potential of vitamin K2 as an
antihepatoma drug has recently been highlighted. Most of the new biological functions of vitamin
K in bone, vasculature, and hepatoma cells are considered attributable to promotion of
␥-carboxylation of glutamic acid residues in vitamin K– dependent proteins, which is shared by
vitamins K1 and K2. In contrast, vitamin K2–specific, ␥-carboxylation– unrelated functions have
also been demonstrated. Thus, biological differences between vitamins K1 and K2 and potential
involvement of ␥-carboxylation–independent actions in the new roles of vitamin K remain open
issues. Molecular bases of coagulation-unrelated pleiotropic actions of vitamin K and its implica-
tions in human health deserve further investigations. © 2006 Elsevier Inc. All rights reserved.

Keywords: Menaquinone; Phylloquinone; Subclinical vitamin K deficiency; Undercarboxylated osteocalcin; Osteoporosis;

Hepatocellular carcinoma; Atherosclerosis

Introduction min K– deficiency bleeding, and therefore prophylactic vi-

Vitamin K was originally identified as a fat-soluble nu-
trient required for coagulation and then discovered to be an
essential cofactor for post-translational modification of glu-
tamic acid (Glu) residues to ␥-carboxyglutamic acid (Gla)
residues of vitamin K– dependent hepatic blood-coagulating
proteins including prothrombin and factors II, VII, IX, and
X [1]. Hence, vitamin K deficiency results in a bleeding
tendency due to malfunction of vitamin K– dependent clot-
ting factors. In particular, neonates are susceptible to vita-
This work was supported by grant R01DK058127 from the National
Institutes of Health (M.K.).
* Corresponding author. Tel.: ⫹617-726-8122; fax: 617-726-8134.
E-mail address: mkaneki@partners.org (M. Kaneki).
tamin K supplementation has been successfully employed in
neonates [2,3]. Recommended dietary intake of vitamin K
has been determined based on ␥-carboxylation status of
coagulation factors.
Recently, however, coagulation-unrelated functions of
vitamin K have attracted scientific attention [4 – 6]. These
pleiotropic actions of vitamin K include potential protective
effects against osteoporosis, hepatocarcinoma, and athero-
sclerosis. In contrast to newborn babies, in the absence of
aggravating factors such as chronic gastrointestinal disor-
ders or parental feeding in critically ill patients, vitamin K
deficiency in terms of blood coagulation, referred to as
“classic (clinical)” vitamin K deficiency, is rare. Nonethe-
less, a growing body of evidence indicates that “subclinical”
vitamin K deficiency in extrahepatic tissues, particularly in

0899-9007/06/$ – see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.nut.2006.05.003
846 M. Kaneki et al. / Nutrition 22 (2006) 845– 852

Table 1
Vitamin K Deficiency
Classic (clinical) Subclinical

␥-Carboxylation of coagulation factors Impaired Normal

Bleeding tendency (⫹) (⫺)
Marker Blood clotting factors (e.g., prothrombin time) Undercarboxylated osteocalcin
Infants Relatively common Unknown
Adults Rare Relatively common
Vitamin K target tissue Liver Bone (possibly vasculature, hepatoma cells)
Actions (roles) of K1 versus K2 Quite similar Different (?)
Mechanisms of vitamin K action ␥-Carboxylation dependent ␥-Carboxylation dependent and independent (?)
Category of vitamin K function Classic Pleiotropic actions

bone, is not uncommon in the adult population. Classic
(clinical) vitamin K deficiency causes hemorrhage. In con-
trast, subclinical vitamin K deficiency is related to pleiotro-
pic actions in bone, possibly in the vasculature, and locally
in hepatoma cells. It has been proposed to contribute to
osteoporosis, aortic calcification, atherosclerosis, and hepa-
toma development (Table 1).
Vitamin K exists in two forms in nature: vitamin K1
(phylloquinone) and vitamin K2 (menaquinones). Vitamin
K1 is produced by plants and algae and is widely distributed
in green and leafy vegetables; vitamin K2 is of microbial
origin and is contained in meats, eggs, curd, cheese, and
fermented soybeans. Menaquinones comprise a family of
molecules distinguished from phylloquinone by unsaturated
side chains of isoprenoid units varying in length from 1 to
14 repeats (Fig. 1). With regard to hemostasis, actions of
vitamins K1 and K2 have been considered quite similar or
essentially the same. In contrast, several lines of evidence
Vitamin K1
(Phylloquinone)
2 &+
+ &+ + &+
&+
2 cussion on the guideline of subclinical vitamin K defi-
Vitamin K2
(Menaquinone-n)
2 &+
+ [11], suggesting that, although OC seems to be a regulator
Q paired ␥-carboxylation may not be necessarily associated
&+
2
Fig. 1. Chemical structure of vitamins K1 and K2.
suggest that vitamins K1 and K2 may also have distinct
roles in the pleiotropic actions. Vitamin K2 may also have
␥-carboxylation–independent functions. In this review, we
provide an overview of recent studies on the emerging new
roles for vitamin K in bone, vasculature, and hepatoma cells
and attempt to clarify questions to be answered for future
research.
Subclinical vitamin K deficiency in bone
Among vitamin K– dependent proteins in bone, osteocal-
cin (OC, also termed bone Gla protein), matrix Gla protein
(MGP), and protein S, ␥-carboxylation of OC has been
extensively studied. In healthy adults, a very small portion
of blood clotting factors is undercarboxylated. In contrast, a
substantial portion of circulating OC is undercarboxylated
[7,8]. Thus, circulating undercarboxylated OC (ucOC) is a
more sensitive measurement of vitamin K status than are the
conventional blood coagulation tests [9].
The cutoff value of ucOC for subclinical vitamin K
deficiency has not been established, although Shiraki et al.
[10] proposed an ucOC level of 4.0 ng/mL. They found that
&+ postmenopausal women with a serum ucOC level ⱖ4.0
&+ ng/mL displayed lower serum vitamin K concentrations,
higher bone resorption markers, deoxypyridinoline, and in-
creased vertebral fracture incidence. To facilitate the dis-
ciency, however, ucOC values measured by distinct assay
systems will need to be standardized, because there are
substantial variations in ucOC values among immunoas-
says.
A pathogenic role for ucOC in osteoporosis is poorly
understood. OC knockout mice exhibited increased bone
formation and resistance to ovariectomy-induced bone loss
of bone formation, decreased function of OC due to im-
with osteopenia. In contrast, MGP knockout mice displayed
short stature, osteopenia and fractures, and accelerated cal-
cification of arteries and cartilage [12]. These findings sug-
 M. Kaneki et al. / Nutrition 22 (2006) 845– 852
gest that ␥-carboxylation of MGP may have an important
role in bone formation and mineralization.
Epidemiologic evidence of a link between vitamin K
and osteoporosis
Accumulating epidemiologic evidence suggests that sub-
clinical vitamin K deficiency contributes to age-related bone
loss and osteoporotic fractures. First, an increased circulat-
ing level of ucOC is associated with an increased fracture
incidence [13–15] and low bone mineral density (BMD)
[16]. The Epidémiologie de l’osteoporose (EPIDOS) pro-
spective cohort study followed 7598 healthy elderly women
for 22 mo and demonstrated that circulating levels of ucOC,
but not of total OC, predicted hip fracture risk [15]. Second,
cross-sectional studies have shown that low circulating lev-
els of vitamin K (phylloquinone and menaquinones) were
associated with low BMD and bone fractures [17–22].
Third, prospective cohort studies have reported that vitamin
K1 (phylloquinone) intake is also correlated with fracture
risk and BMD [23–25].
In contrast to phylloquinone, a relation between mena-
quinone intake and bone mass or fracture has not yet been
investigated. In Western populations, phylloquinone consti-
tutes a major part of vitamin K intake. However, previous
studies have suggested that menaquinones may be more
efficiently absorbed than phylloquinone [26] and therefore
argued that phylloquinone and menaquinones may contrib-
ute to nutriture to a comparable extent even in Western
populations, in which phylloquinone intake is much greater
than that of menaquinones [27]. Moreover, particularly in
Japan, intake of natto, a Japanese fermented soybean food
containing a large amount of menaquinone-7 (⬃10 ␮g/g),
has a great effect on circulating vitamin K status [28].
Collectively, therefore, relative contributions of phylloqui-
none versus those of menaquinones to pleiotropic actions of
vitamin K remain an open issue.
Osteoporosis is characterized by decreased bone mass
and deranged microarchitecture, which contribute to bone
fragility. In many previous studies, a significant correlation
between bone fracture and various indices of vitamin K has
been found [19 –21,23–25]. However, with regard to its
relation to BMD, controversial results have been reported.
In some studies, phylloquinone intake was correlated with
fracture risk, but not with BMD [24], whereas phylloqui-
none intake was significantly associated with BMD in other
studies [25]. These apparent discrepancies may be ex-
plained by differences in methodology in measurements and
target population, as discussed by the investigators [25].
Moreover, although Delmas et al. [16] showed a significant
inverse correlation between circulating ucOC and BMD,
they also demonstrated that circulating ucOC predicted frac-
ture risk independent of BMD [15]. It has been suggested,
therefore, that subclinical vitamin K deficiency may con-
tribute to increased fracture incidence through BMD-
847
dependent and -independent mechanisms, the latter of
which is assigned to decreased bone quality [29]. Further
studies are required to clarify this issue.
Treatment of involutional osteoporosis with vitamin K
A 48-wk, double-blind study found that menaquinone-4
(45 mg/d, n ⫽ 272) significantly improved metacarpal
BMD compared with vitamin D3 (0.75 ␮g/d, n ⫽ 274), as
judged by the microdensitometric method, in patients with
involutional (primary) osteoporosis, i.e., postmenopausal
and senile osteoporosis [30]. A 24-wk, randomized, open-
label study [31] compared the effects of the combination of
menaquinone-4 (45 mg/d) plus calcium supplementation
(150 mg/d, n ⫽ 120) with those of calcium alone (n ⫽ 121)
in patients with involutional osteoporosis and found that
menaquinone-4 prevented age-related decrease in lumber
BMD and decreased fracture incidence, with a simultaneous
decrease in ucOC and increase in total OC [31]. A double-
blind, placebo-controlled study that enrolled 80 subjects
found that menaquinone-4 (90 mg/d) increased metacarpal
BMD in patients with involutional osteoporosis. These find-
ings of beneficial effects of menaquinone-4 in involutional
osteoporosis are consistent with results of other randomized
clinical studies performed in Asia, particularly in Japan, by
several independent groups [32–37]. Thus, these relatively
small, randomized, intervention studies have consistently
demonstrated the effectiveness of menaquinone-4 in invo-
lutional osteoporosis. However, it is important that the ef-
ficacy of menaquinone-4 be further confirmed by larger-
scale, randomized, clinical trials. A 2003 report from the
World Health Organization classified the efficacy of
menaquinone-4 on BMD and vertebral fracture into the
evidence B level, i.e., positive evidence from smaller, non-
definitive, randomized, controlled trials [38], whereas the
efficacy of estrogens and some bisphosphonates (alendro-
nate and risedronate) was categorized into the evidence A
level, i.e., positive evidence from at least one adequately
powered, randomized, controlled trial.
With respect to degree of effectiveness, small clinical
studies have shown that menaquinone-4 (45 mg/d) de-
creases the relative risk of vertebral fracture to an extent
comparable to that of bisphosphonate (etidronate), hormone
replacement, and calcitonin in postmenopausal osteoporosis
[33,37], although larger clinical trials will be required for
conclusive clarification of the efficacy of menaquinone-4
compared with other antiosteoporotic drugs. Although
menaquinone-4 significantly preserved BMD compared
with the no-treatment group, the effects of other treatments
on BMD seemed to be more pronounced than that of
menaquinone-4 [37]. With regard to the effects on bone
turnover, menaquinone-4 has been shown to decrease bone
resorption by osteoclasts and to promote bone formation by
osteoblasts [31,39,40]. In comparison with other antiosteo-
porotic agents, such as estrogen and bisphosphonates, pro-
848 M. Kaneki et al. / Nutrition 22 (2006) 845– 852
motion of bone formation by menaquinone-4 is more prom-
inent than the anti– bone resorption action [33,41]. Based on
in vitro studies, direct actions on cell survival/apoptosis,
differentiation, and functions of osteoclasts, osteoblasts and
their precursors have been proposed to mediate the salutary
effects of menaquinone-4 [42,43].
In contrast to a relatively large dose of menaquinone-4
(45 mg/d), a much lower dose of phylloquinone (1 mg/d)
has been demonstrated to exert protective effects on BMD
in postmenopausal women [44], although the effects of
phylloquinone on fracture incidence has not been investi-
gated. Vermeer et al. [44] compared Caucasian postmeno-
pausal women who received a placebo (n ⫽ 600), a sup-
plement containing minerals (500 mg of calcium, 10 mg of
zinc, 150 mg of magnesium) plus vitamin D (8 ␮g, n ⫽ 46),
or minerals plus vitamin D with additional phylloquinone (1
mg/d, n ⫽ 56) and followed up BMD for 3 y. The double-
blind intervention study found that supplementation with
minerals, vitamin D, and phylloquinone significantly atten-
uated femoral neck bone loss compared with placebo and
with minerals plus vitamin D [44]. In contrast, minerals plus
vitamin D alone did not exhibit beneficial effects on bone
mass.
Vitamin K has a wide safety range [5,40], although no
sufficient data are available to define an upper tolerable
level for vitamin K. No adverse side effects of vitamin K2
have been reported thus far, whereas menaquinone-4 (45
mg/d) has been used in patients with osteoporosis in Japan,
Korea, Thailand, and Taiwan on a large scale since 1995.
Animal and clinical studies have indicated that vitamin K
administration does not result in a hypercoagulable state
[31,45– 47], although use of vitamin K is contraindicated in
patients on anticoagulant (e.g., warfarin) therapy. Overall,
the safety of vitamin K, up to 45 mg/d of menaquinone-4,
has been well established in the adult population, except in
pregnant women.
Menaquinone-4 versus phylloquinone
The beneficial effects of phylloquinone and menaquinone-4
are consistent. Nevertheless, the difference in doses of vitamin
K used in the studies, namely 45 mg/d of menaquinone-4
versus 1 mg/d of phylloquinone, raises new questions: (1)
What dose of vitamin K supplement is needed to revert sub-
clinical vitamin K deficiency in bone? (2) Can the protective
effects of menaquinone-4 (45 mg/d) be accounted for by re-
versal of subclinical vitamin K deficiency alone? (3) Do as-
yet-undetermined ␥-carboxylation–independent mechanisms
also contribute to the protective effects of vitamin K, especially
menaquinone-4? Considering that dietary vitamin K intake is
an order of ⬃100 – 400 ␮g/d, 45 mg/d of menaquinone-4
appears to be far beyond the level required for reversal of
subclinical vitamin K deficiency. Further, administration of
phylloquinone (1 mg/d) significantly decreased ucOC [44].
Therefore, 1 mg/d of vitamin K supplement may be sufficient
to correct subclinical vitamin K deficiency in bone. Nonethe-
less, evidence arguing against this presumption also exists.
In a randomized, open-label, clinical trial [48], patients with
osteoporosis were administered with different doses of
menaquinone-4 (15, 45, 90, and 135 mg/d) or vitamin D3
(0.75 ␮g/d). As expected, all doses of menaquinone-4 treat-
ment significantly increased urinary excretion of Gla resi-
due, a surrogate marker of total vitamin K– dependent
␥-carboxylation as compared with vitamin D3. However,
urinary Gla excretion was greater in patients who received
higher doses of menaquinone-4 (45, 90, and 135 mg/d) than
in those who received 15 mg/d of menaquinone-4 (urinary
Gla [nanomoles per milligram of creatinine] : vitamin D
[n ⫽ 38], 54 ⫾ 4 [mean ⫾ SEM]; 15 mg of menaquinone-4,
[n ⫽ 41], 62 ⫾ 3; 45 mg of menaquinone-4, [n ⫽ 40], 71
⫾ 4; 90 mg of menaquinone-4 [n ⫽ 41], 71 ⫾ 4; 135 mg of
menaquinone-4, [n ⫽ 34], 74 ⫾ 5; P ⬍ 0.01, vitamin D
versus 15, 45, 90, or 135 mg of menaquinone-4; P ⬍ 0.05,
15 mg versus 45 or 90 mg of menaquinone-4; P ⬍ 0.01, 15
versus 135 mg of menaquinone-4). Undercarboxylated OC
was not examined in the study. These observations suggest
that oral administration of 15 mg/d of menaquinone-4 may
be insufficient to achieve maximal generation of vitamin
K– dependent Gla residues in patients with involutional
osteoporosis.
Recently, increasing evidence has suggested that mena-
quinones, but not phylloquinone, have ␥-carboxylation–
independent functions. ␥-Carboxylation–independent ac-
tions have been proposed to contribute to the protective
effects of menaquinone-4 on bone health, although direct in
vivo evidence is lacking. Menaquinones function as ligands
of “orphan” nuclear receptors, steroid and xenobiotic recep-
tor, and pregnane X receptor, whose ligands and/or func-
tions remain to be determined [49]. Phylloquinone exhibits
one order of magnitude lower affinity to these steroid re-
ceptors relative to menaquinones, although the effectiveness
of phylloquinone and menaquinones on ␥-carboxylation in
vitro is quite similar [50].
Another possibility is the antioxidant property of
vitamin K [51]. Menaquinones have been proposed to
protect neuronal cells from apoptosis by decreasing oxi-
dative stress [52]. Phylloquinone and menaquinone-4
inhibited oxidative stress–induced cell death in oligoden-
drocyte precursors with EC50 (50% effective concentra-
tion) values of 30 and 2 nM, respectively, suggesting that
menaquinone-4 is 15-fold more potent than phylloqui-
none as an antioxidant [52]. An earlier study reported that
most phylloquinone is distributed in microsomes in mam-
malian cells, where ␥-carboxylation is catalyzed, but that
menaquinones are preferentially localized to mitochon-
dria [53]. In bacteria, menaquinones play a critical role in
electron transfer in mitochondria and redox signaling, as
does ubiquinone [54]. Moreover, a recent study has dem-
onstrated that menaquinone-4 binds to 17␤-hydroxys-
teroid dehydrogenase-4 and modulates estrogen metabo-
lism [55]. These findings suggest that menaquinones, but
 M. Kaneki et al. / Nutrition 22 (2006) 845– 852
not phylloquinone, may have an additional role, other
than ␥-carboxylation, and that ␥-carboxylation–indepen-
dent mechanisms might contribute to the salutary effects
of a relatively high dose of menaquinone-4. However,
these possibilities have not been explored in vivo in
mammals.
To compare and interpret the difference in doses of
vitamin K in the clinical trials with menaquinone-4 (45
mg/d) and phylloquinone (1 mg/d), which were per-
formed in Japan and in Europe, respectively, it is impor-
tant to note that ethnic differences also exist in vitamin K
status. Postmenopausal women living in Tokyo exhibited
an almost 20-fold greater serum concentration of mena-
quinone-7 (5.26 ⫾ 6.13 ng/mL, mean ⫾ SD) compared
with British women (0.371 ⫾ 0.204 ng/mL) [28]. This
large geographic difference in menaquinone-7 concentra-
tion is largely accounted for by natto intake [28]. Previ-
ous studies have argued that natto intake may have real
effects on bone metabolism: (1) natto intake increases not
only serum menaquinone-7 concentration but also ␥-car-
boxylated OC and decreases ucOC in women [28,56 –58];
(2) regional (prefectural) natto consumption has dis-
played a significant, inverse correlation with prefectural
relative incidence of hip fracture in postmenopausal
women in Japan [28]; (3) the habit of eating natto was
associated with higher BMD in premenopausal women
[58]; (4) natto intake upregulates bone-specific alkaline
phosphatase in premenopausal women [57]; and 5) natto
has prevented bone loss in ovariectomized rats [59].
In addition, regardless of the habit of eating natto,
serum phylloquinone concentration in Japanese women
was also two-fold greater than that in British women
[28]. In line with this observation, more recent studies
have shown that vitamin K intake of elderly people in
China is almost double that of British individuals, and
that ucOC is lower in China than in the United Kingdom
[60]. Moreover, ethnic differences in apolipoprotein-E
genotype, which affects vitamin K transport, have been
proposed to influence OC ␥-carboxylation [60]. How-
ever, the biological effect of these ethnic differences on
vitamin K status to bone metabolism remains to be in-
vestigated.
Vitamin K and atherosclerosis
In addition to bone, the role of vitamin K in athero-
sclerosis has been an issue of investigation for a couple
of decades. Atherosclerotic plaque contains a vitamin
K– dependent protein, MGP. Gene disruption of MGP
results in extensive aortic and coronary calcification and
osteopenia [12]. Warfarin, an antagonist of vitamin
K– dependent ␥-carboxylation, induces calcification in
arteries and heart valves of rats [61,62]. Oral anticoagu-
lant treatment with coumarin was associated with in-
creased aortic valve calcification in humans [63]. MGP
849
binds to bone morphological protein-2 (BMP-2) and in-
hibits BMP-2–induced osteoblast differentiation and cal-
cification in cultured cells. Undercarboxylated MGP does
not retain the capability of binding to BMP-2. Therefore,
it has been proposed that impaired ␥-carboxylation of
MGP increases vascular calcification by allowing BMP-2
to induce mineralization [64]. Recently, undercarboxylated
MGP was detected in the intima of human atherosclerotic
arteries and in media of Mönckeberg atherosclerotic lesions
with calcification, but not in non-atherosclerotic arteries
[65], thus supporting a role for MGP in atherosclerotic
development and vascular calcification.
Pharmacologic doses of menaquinone-4 have consis-
tently ameliorated vitamin D–induced aortic calcification
in rats [66]. Supplementation with menaquinone-4, but
not with phylloquinone, prevented warfarin-induced ar-
terial calcification in rats [50]. Utilization of menaqui-
none-4 is more efficient than that of phylloquinone in the
aorta, although phylloquinone and menaquinone-4 were
utilized equally in the liver [50]. However, it is important
to note that phylloquinone can be converted into mena-
quinone-4 in rodents [67], and this conversion is tissue
specific [68]. Our preliminary results showed that oral
phylloquinone administration results in increased serum
concentration of menaquinone-4 and phylloquinone in
postmenopausal women (M. Kaneki, T. Hosoi, Y. Ouchi,
and H. Orimo, unpublished observations). Therefore,
menaquinone-4 converted from phylloquinone might me-
diate the protective effects of prolonged supplementation
of phylloquinone in vasculature in humans. As with os-
teoporosis, it is unknown whether the efficiency of phar-
macologic doses of menaquinone-4, which is far beyond
the level of dietary intake, in the vasculature of animals
can be accounted for by reversal of subclinical vitamin K
deficiency or attributed to as yet undetermined pharma-
cologic effects of menaquinone-4.
The Rotterdam Study, a prospective, population-based
cohort study comprising 7983 individuals, demonstrated
that dietary intake of menaquinones, but not of phyllo-
quinone, was associated with aortic calcification and cor-
onary heart disease after adjustment for age, gender, body
mass index, smoking, diabetes, education, and dietary
factors [69]. Compared with the lower tertile, the upper
tertile of menaquinone intake exhibited a lower incidence
of coronary heart disease mortality (relative risk, 0.43;
95% confidence interval, 0.24 – 0.77) and severe aortic
calcification (odds ratio of 0.48; 95% confidence interval,
0.32– 0.71). A 3-y, double-blind, placebo-controlled, in-
tervention study analyzing 108 postmenopausal women
demonstrated that supplementation of vitamin K1
(1 mg/d) with vitamin D (8 ␮g/d) and minerals (calcium,
zinc, and magnesium) significantly preserved the elastic
properties of the carotid artery, although vitamin D plus
minerals alone did not exhibit beneficial effects [70].
850 M. Kaneki et al. / Nutrition 22 (2006) 845– 852
Vitamin K as a potential inhibitor of hepatocarcinoma
development
Des-␥-carboxyprothrombin (PIVKA-II) has been estab-
lished as a marker in the diagnosis and prognosis of hepa-
tocellular carcinoma (HCC) [71]. Prothrombin is a vitamin
K– dependent plasma coagulation factor that is synthesized
in the liver. Vitamin K– dependent ␥-carboxylation of 10
glutamic acid residues in the precursor of prothrombin is
necessary for the coagulation activity. PIVKA-II is an ab-
normal prothrombin that is not fully carboxylated.
PIVKA-II is expressed not only in cancer tissue but also in
the surrounding non-cancer tissue of HCC [72]. Vitamin K
content was decreased in HCC tissues compared with non-
tumorous parts of the liver [73]. However, molecular bases
underlying increased PIVKA-II in HCC remain elusive.
An 8-y, randomized, but not placebo-controlled, study
analyzing 40 cases found that the risk ratio in postmeno-
pausal women with viral liver cirrhosis treated with
menaquinone-4 (45 mg/d) was 0.20 (95% confidence inter-
val 0.04 – 0.91), compared with those who did not receive it
[74]. Moreover, a recent 3-y, randomized, non–placebo-
controlled study that analyzed 61 patients showed that the
hazard ratio of HCC recurrence in patients treated with
menaquinone-4 (45 mg/d) was 0.27 (95% confidence inter-
val 0.12– 0.60) compared with those who did not receive it.
Menaquinone-4 decreased serum PIVKA-II in patients with
HCC [75]. Menaquinone-4 inhibited growth and invasion of
HCC cells in vitro [76,77] and decreased tumor growth and
body weight loss in a mouse model of human HCC [76]. Of
interest, a recent study has demonstrated that PIVKA-II
binds to Met, a receptor for hepatocyte growth factor, and
activates the Janus kinase-1 signal transducers and activa-
tors of the transcription 3 (STAT3) signaling pathway [78].
The investigators proposed that PIVKA-II may have a
pathogenic role as an autologous growth factor for HCC.
These findings warrant a large-scale, placebo-controlled,
randomized, clinical trial to determine the efficacy of
menaquinone-4 against HCC.
Conclusions
An increasing body of work indicates that subclinical
vitamin K deficiency may be associated with osteoporosis
and possibly with hepatocarcinoma and atherosclerosis. The
efficacy of vitamin K in the prevention and/or treatment of
these diseases deserves large-scale intervention studies.
Molecular mechanisms underlying the emerging new roles
for vitamin K await further investigations.
Acknowledgments
The authors apologize to colleagues whose work has not
been cited in this review due to space limitation.
References
[1] Suttie JW. Vitamin K– dependent carboxylase. Annu Rev Biochem
1985;54:459 –77.
[2] Suzuki S, Iwata G, Sutor AH. Vitamin K deficiency during the
perinatal and infantile period. Semin Thromb Hemost 2001;27:93– 8.
[3] Autret-Leca E, Jonville-Bera AP. Vitamin K in neonates: how to
administer, when and to whom. Paediatr Drugs 2001;3:1– 8.
[4] Vermeer C, Shearer MJ, Zittermann A, Bolton-Smith C, Szulc P,
Hodges S, et al. Beyond deficiency: potential benefits of increased
intakes of vitamin K for bone and vascular health. Eur J Nutr 2004;
43:325–35.
[5] Weber P. Vitamin K and bone health. Nutrition 2001;17:880 – 87.
[6] Berkner KL, Runge KW. The physiology of vitamin K nutriture and
vitamin K– dependent protein function in atherosclerosis. J Thromb
Haemost 2004;2:2118 –32.
[7] Bugel S. Vitamin K and bone health. Proc Nutr Soc 2003;62:839 – 43.
[8] Shearer MJ. Role of vitamin K and Gla proteins in the pathophysi-
ology of osteoporosis and vascular calcification. Curr Opin Clin Nutr
Metab Care 2000;3:433– 8.
[9] Price PA. Role of vitamin-K– dependent proteins in bone metabolism.
Annu Rev Nutr 1988;8:565– 83.
[10] Shiraki Y, Aoki C, Shiraki M. Vitamin K insufficiency judging from
serum level of undercarboxylated osteocalcin and it’s clinical signif-
icance. Osteoporos Jpn 2002;10:179 – 82.
[11] Ducy P, Desbois C, Boyce B, Pinero G, Story B, Dunstan C, et al.
Increased bone formation in osteocalcin-deficient mice. Nature 1996;
382:448 –52.
[12] Luo G, Ducy P, McKee MD, Pinero GJ, Loyer E, Behringer RR,
Karsenty G. Spontaneous calcification of arteries and cartilage in
mice lacking matrix GLA protein. Nature 1997;386:78 – 81.
[13] Szulc P, Chapuy MC, Meunier PJ, Delmas PD. Serum undercarboxy-
lated osteocalcin is a marker of the risk of hip fracture in elderly
women. J Clin Invest 1993;91:1769 –74.
[14] Szulc P, Chapuy MC, Meunier PJ, Delmas PD. Serum undercarboxy-
lated osteocalcin is a marker of the risk of hip fracture: a three year
follow-up study. Bone 1996;18:487– 8.
[15] Vergnaud P, Garnero P, Meunier PJ, Breart G, Kamihagi K, Delmas
PD. Undercarboxylated osteocalcin measured with a specific immu-
noassay predicts hip fracture in elderly women: the EPIDOS Study.
J Clin Endocrinol Metab 1997;82:719 –24.
[16] Szulc P, Arlot M, Chapuy MC, Duboeuf F, Meunier PJ, Delmas PD.
Serum undercarboxylated osteocalcin correlates with hip bone min-
eral density in elderly women. J Bone Miner Res 1994;9:1591–95.
[17] Booth SL, Broe KE, Peterson JW, Cheng DM, Dawson-Hughes B,
Gundberg CM, et al. Associations between vitamin K biochemical
measures and bone mineral density in men and women. J Clin En-
docrinol Metab 2004;89:4904 –9.
[18] Kanai T, Takagi T, Masuhiro K, Nakamura M, Iwata M, Saji F.
Serum vitamin K level and bone mineral density in post-menopausal
women. Int J Gynaecol Obstet 1997;56:25–30.
[19] Hodges SJ, Pilkington MJ, Stamp TC, Catterall A, Shearer MJ,
Bitensky L, Chayen J. Depressed levels of circulating menaquinones
in patients with osteoporotic fractures of the spine and femoral neck.
Bone 1991;12:387–9.
[20] Hodges SJ, Akesson K, Vergnaud P, Obrant K, Delmas PD. Circu-
lating levels of vitamins K1 and K2 decreased in elderly women with
hip fracture. J Bone Miner Res 1993;8:1241–5.
[21] Kaneki M, Mizuno Y, Hosoi T, Inoue S, Hoshino S, Akishita M, et
al. [Serum concentration of vitamin K in elderly women with invo-
lutional osteoporosis]. Nippon Ronen Igakkai Zasshi 1995;32:195–
200.
[22] Tamatani M, Morimoto S, Nakajima M, Fukuo K, Onishi T, Kitano
S, et al. Decreased circulating levels of vitamin K and 25-hydroxyvi-
tamin D in osteopenic elderly men. Metabolism 1998;47:195–9.
 M. Kaneki et al. / Nutrition 22 (2006) 845– 852
[23] Feskanich D, Weber P, Willett WC, Rockett H, Booth SL, Colditz
GA. Vitamin K intake and hip fractures in women: a prospective
study. Am J Clin Nutr 1999;69:74 –9.
[24] Booth SL, Tucker KL, Chen H, Hannan MT, Gagnon DR, Cupples
LA, et al. Dietary vitamin K intakes are associated with hip fracture
but not with bone mineral density in elderly men and women. Am J
Clin Nutr 2000;71:1201– 8.
[25] Booth SL, Broe KE, Gagnon DR, Tucker KL, Hannan MT, McLean
RR, et al. Vitamin K intake and bone mineral density in women and
men. Am J Clin Nutr 2003;77:512– 6.
[26] Groenen-van Dooren MM, Ronden JE, Soute BA, Vermeer C. Bio-
availability of phylloquinone and menaquinones after oral and colo-
rectal administration in vitamin K– deficient rats. Biochem Pharmacol
1995;50:797– 801.
[27] Vermeer C, Braam L. Role of K vitamins in the regulation of tissue
calcification. J Bone Miner Metab 2001;19:201– 6.
[28] Kaneki M, Hodges SJ, Hosoi T, Fujiwara S, Lyons A, Crean SJ, et al.
Japanese fermented soybean food as the major determinant of the
large geographic difference in circulating levels of vitamin K2: pos-
sible implications for hip-fracture risk. Nutrition 2001;17:315–21.
[29] Liu G, Peacock M. Age-related changes in serum undercarboxylated
osteocalcin and its relationships with bone density, bone quality, and
hip fracture. Calcif Tissue Int 1998;62:286 –9.
[30] Orimo H, Fujita T, Onnomura T, Inoue T, Kushida K, Shiraki M.
Clinical evaluation of Ea-0167 (menatetrenone) in the treatment of
osteoporosis. Clin Eval (Japan) 1992;20:45–100.
[31] Shiraki M, Shiraki Y, Aoki C, Miura M. Vitamin K2 (menatetrenone)
effectively prevents fractures and sustains lumbar bone mineral den-
sity in osteoporosis. J Bone Miner Res 2000;15:515–21.
[32] Bunyaratavej N, Penkitti P, Kittimanon N, Boonsangsom P, Bonjong-
sat A, Yunoi S. Efficacy and safety of menatetrenone-4 postmeno-
pausal Thai women. J Med Assoc Thai 2001;84(suppl 2):S553–9.
[33] Iwamoto J, Takeda T, Ichimura S. Effect of menatetrenone on bone
mineral density and incidence of vertebral fractures in postmeno-
pausal women with osteoporosis: a comparison with the effect of
etidronate. J Orthop Sci 2001;6:487–92.
[34] Ozuru R, Sugimoto T, Yamaguchi T, Chihara K. Time-dependent
effects of vitamin K2 (menatetrenone) on bone metabolism in post-
menopausal women. Endocr J 2002;49:363–70.
[35] Hidaka T, Hasegawa T, Fujimura M, Sakai M, Saito S. Treatment for
patients with postmenopausal osteoporosis who have been placed on
HRT and show a decrease in bone mineral density: effects of con-
comitant administration of vitamin K(2). J Bone Miner Metab 2002;
20:235–9.
[36] Sato Y, Kaji M, Tsuru T, Satoh K, Kondo I. Vitamin K deficiency and
osteopenia in vitamin D– deficient elderly women with Parkinson’s
disease. Arch Phys Med Rehabil 2002;83:86 –91.
[37] Ishida Y, Kawai S. Comparative efficacy of hormone replacement
therapy, etidronate, calcitonin, alfacalcidol, and vitamin K in post-
menopausal women with osteoporosis: the Yamaguchi Osteoporosis
Prevention Study. Am J Med 2004;117:549 –55.
[38] WHO Scientific Group on the Preview and Management of Osteo-
porosis. Prevention and management of osteoporosis: report of a
WHO scientific group. Geneva: World Health Organization; 2003.
[39] Iwamoto J, Takeda T, Sato Y. Effects of vitamin K2 on osteoporosis.
Curr Pharm Des 2004;10:2557–76.
[40] Adams J, Pepping J. Vitamin K in the treatment and prevention of
osteoporosis and arterial calcification. Am J Health Syst Pharm 2005;
62:1574 – 81.
[41] Shiomi S, Nishiguchi S, Kubo S, Tamori A, Habu D, Takeda T, Ochi
H. Vitamin K2 (menatetrenone) for bone loss in patients with cirrho-
sis of the liver. Am J Gastroenterol 2002;97:978 – 81.
[42] Koshihara Y, Hoshi K, Okawara R, Ishibashi H, Yamamoto S. Vita-
min K stimulates osteoblastogenesis and inhibits osteoclastogenesis
in human bone marrow cell culture. J Endocrinol 2003;176:339 – 48.
851
[43] Takeuchi Y, Suzawa M, Fukumoto S, Fujita T. Vitamin K(2) inhibits
adipogenesis, osteoclastogenesis, and ODF/RANK ligand expression
in murine bone marrow cell cultures. Bone 2000;27:769 –76.
[44] Braam LA, Knapen MH, Geusens P, Brouns F, Hamulyak K, Gerich-
hausen MJ, Vermeer C. Vitamin K1 supplementation retards bone
loss in postmenopausal women between 50 and 60 years of age.
Calcif Tissue Int 2003;73:21– 6.
[45] Ushiroyama T, Ikeda A, Ueki M. Effect of continuous combined
therapy with vitamin K(2) and vitamin D(3) on bone mineral density
and coagulofibrinolysis function in postmenopausal women. Maturi-
tas 2002;41:211–21.
[46] Asakura H, Myou S, Ontachi Y, Mizutani T, Kato M, Saito M, et al.
Vitamin K administration to elderly patients with osteoporosis in-
duces no hemostatic activation, even in those with suspected vitamin
K deficiency. Osteoporos Int 2001;12:996 –1000.
[47] Ronden JE, Groenen-van Dooren MM, Hornstra G, Vermeer C.
Modulation of arterial thrombosis tendency in rats by vitamin K and
its side chains. Atherosclerosis 1997;132:61–7.
[48] Orimo H, Fujita T, Onomura T, Inoue T, Kushida K, Shiraki M.
Clinical effects of menatetrenone soft capsule (Ea-0167) in osteopo-
rosis. J N Rem Clin (Japan) 1992;41:1249 –79.
[49] Tabb MM, Sun A, Zhou C, Grun F, Errandi J, Romero K, et al.
Vitamin K2 regulation of bone homeostasis is mediated by the steroid
and xenobiotic receptor SXR. J Biol Chem 2003;278:43919 –27.
[50] Spronk HM, Soute BA, Schurgers LJ, Thijssen HH, De Mey JG,
Vermeer C. Tissue-specific utilization of menaquinone-4 results in
the prevention of arterial calcification in warfarin-treated rats. J Vasc
Res 2003;40:531–7.
[51] Vervoort LM, Ronden JE, Thijssen HH. The potent antioxidant ac-
tivity of the vitamin K cycle in microsomal lipid peroxidation. Bio-
chem Pharmacol 1997;54:871– 6.
[52] Li J, Lin JC, Wang H, Peterson JW, Furie BC, Furie B, et al. Novel
role of vitamin k in preventing oxidative injury to developing oligo-
dendrocytes and neurons. J Neurosci 2003;23:5816 –26.
[53] Reedstrom CK, Suttie JW. Comparative distribution, metabolism, and
utilization of phylloquinone and menaquinone-9 in rat liver. Proc Soc
Exp Biol Med 1995;209:403–9.
[54] Georgellis D, Kwon O, Lin EC. Quinones as the redox signal for the
arc two-component system of bacteria. Science 2001;292:2314 – 6.
[55] Otsuka M, Kato N, Ichimura T, Abe S, Tanaka Y, Taniguchi H, et al.
Vitamin K2 binds 17beta-hydroxysteroid dehydrogenase 4 and mod-
ulates estrogen metabolism. Life Sci 2005;76:2473– 82.
[56] Tsukamoto Y, Ichise H, Kakuda H, Yamaguchi M. Intake of
fermented soybean (natto) increases circulating vitamin K2
(menaquinone-7) and gamma-carboxylated osteocalcin concentra-
tion in normal individuals. J Bone Miner Metab 2000;18:216 –22.
[57] Katsuyama H, Ideguchi S, Fukunaga M, Fukunaga T, Saijoh K,
Sunami S. Promotion of bone formation by fermented soybean
(Natto) intake in premenopausal women. J Nutr Sci Vitaminol (To-
kyo) 2004;50:114 –20.
[58] Katsuyama H, Ideguchi S, Fukunaga M, Saijoh K, Sunami S. Usual
dietary intake of fermented soybeans (Natto) is associated with bone
mineral density in premenopausal women. J Nutr Sci Vitaminol
(Tokyo) 2002;48:207–15.
[59] Yamaguchi M, Kakuda H, Gao YH, Tsukamoto Y. Prolonged
intake of fermented soybean (natto) diets containing vitamin K2
(menaquinone-7) prevents bone loss in ovariectomized rats.
J Bone Miner Metab 2000;18:71– 6.
[60] Beavan SR, Prentice A, Stirling DM, Dibba B, Yan L, Harrington DJ,
Shearer MJ. Ethnic differences in osteocalcin gamma-carboxylation,
plasma phylloquinone (vitamin K1) and apolipoprotein E genotype.
Eur J Clin Nutr 2005;59:72– 81.
[61] Price PA, Faus SA, Williamson MK: Warfarin-induced artery calci-
fication is accelerated by growth and vitamin D. Arterioscler Thromb
Vasc Biol 2000;20:317–27.
[62] Howe AM, Webster WS. Warfarin exposure and calcification of the
arterial system in the rat. Int J Exp Pathol 2000;81:51– 6.
852 M. Kaneki et al. / Nutrition 22 (2006) 845– 852
[63] Schurgers LJ, Aebert H, Vermeer C, Bultmann B, Janzen J. Oral
anticoagulant treatment: friend or foe in cardiovascular disease?
Blood 2004;104:3231–2.
[64] Wallin R, Cain D, Sane DC. Matrix Gla protein synthesis and
gamma-carboxylation in the aortic vessel wall and proliferating vas-
cular smooth muscle cells—a cell system which resembles the system
in bone cells. Thromb Haemost 1999;82:1764 –7.
[65] Schurgers LJ, Teunissen KJ, Knapen MH, Kwaijtaal M, van Diest R,
Appels A, et al. Novel conformation-specific antibodies against ma-
trix {gamma}-carboxyglutamic acid (Gla) protein. Undercarboxy-
lated matrix gla protein as marker for vascular calcification. Arterio-
scler Thromb Vasc Biol 2005;25:1629 –33.
[66] Seyama Y, Kimoto S, Marukawa Y, Horiuchi M, Hayashi M, Usami
E. Comparative effects of vitamin K2 and estradiol on experimental
arteriosclerosis with diabetes mellitus. Int J Vitam Nutr Res 2000;70:
301– 4.
[67] Ronden JE, Drittij-Reijnders MJ, Vermeer C, Thijssen HH. Intestinal
flora is not an intermediate in the phylloquinone–menaquinone-4
conversion in the rat. Biochim Biophys Acta 1998;1379:69 –75.
[68] Davidson RT, Foley AL, Engelke JA, Suttie JW. Conversion of
dietary phylloquinone to tissue menaquinone-4 in rats is not depen-
dent on gut bacteria. J Nutr 1998;128:220 –3.
[69] Geleijnse JM, Vermeer C, Grobbee DE, Schurgers LJ, Knapen MH,
van der Meer IM, et al. Dietary intake of menaquinone is associated
with a reduced risk of coronary heart disease: the Rotterdam Study. J
Nutr 2004;134:3100 –5.
[70] Braam LA, Hoeks AP, Brouns F, Hamulyak K, Gerichhausen MJ,
Vermeer C. Beneficial effects of vitamins D and K on the elastic
properties of the vessel wall in postmenopausal women: a follow-up
study. Thromb Haemost 2004;91:373– 80.
[71] Tamano M, Sugaya H, Oguma M, Iijima M, Yoneda M, Murohisa T,
et al. Serum and tissue PIVKA-II expression reflect the biological
malignant potential of small hepatocellular carcinoma. Hepatol Res
2002;22:261–9.
[72] Tang W, Kokudo N, Sugawara Y, Guo Q, Imamura H, Sano K, et al.
Des-gamma-carboxyprothrombin expression in cancer and/or non-
cancer liver tissues: association with survival of patients with resect-
able hepatocellular carcinoma. Oncol Rep 2005;13:25–30.
[73] Miyakawa T, Kajiwara Y, Shirahata A, Okamoto K, Itoh H, Ohsato
K. Vitamin K contents in liver tissue of hepatocellular carcinoma
patients. Jpn J Cancer Res 2000;91:68 –74.
[74] Habu D, Shiomi S, Tamori A, Takeda T, Tanaka T, Kubo S, Nishi-
guchi S. Role of vitamin K2 in the development of hepatocellular
carcinoma in women with viral cirrhosis of the liver. JAMA 2004;
292:358 – 61.
[75] Sakon M, Monden M, Gotoh M, Kobayashi K, Kanai T, Umeshita K,
et al. The effects of vitamin K on the generation of des-gamma-
carboxy prothrombin (PIVKA-II) in patients with hepatocellular car-
cinoma. Am J Gastroenterol 1991;86:339 – 45.
[76] Otsuka M, Kato N, Shao RX, Hoshida Y, Ijichi H, Koike Y, et al.
Vitamin K2 inhibits the growth and invasiveness of hepatocellular
carcinoma cells via protein kinase A activation. Hepatology 2004;40:
243–51.
[77] Hitomi M, Yokoyama F, Kita Y, Nonomura T, Masaki T, Yoshiji H,
et al. Antitumor effects of vitamins K1, K2 and K3 on hepatocellular
carcinoma in vitro and in vivo. Int J Oncol 2005;26:713–20.
[78] Suzuki M, Shiraha H, Fujikawa T, Takaoka N, Ueda N, Nakanishi Y,
et al. Des-gamma-carboxy prothrombin is a potential autologous
growth factor for hepatocellular carcinoma. J Biol Chem 2005;280:
6409 –15.