Calcium, and Vitamin D + K

Quite a few of my patients have been asking about the risk of taking calcium supplements. The more I read the more I think it’s probably safest to get most of your calcium from food and then if you are taking calcium supplements to take no more than 250mg at a time and at the same time insure that you are taking in the vitamins and minerals along with the calcium that will help direct its use for the bones and NOT for calcifying artery walls. Some of these things include Vitamin D, Vitamins K1 and K2, and possibly things such as Magnesium, Boron, Vitamin A. There’s still a lot of controversy. But I think the safest thing to do is err on the side of caution until we know more.

Vitamin D and Vitamin K Team Up to Lower CVD
Risk: Part II

by Lara Pizzorno, MDiv, MA, LMT

Abstract

Strong correlations have been noted between cardiovascular diseases and low bone density / osteoporosis—
connections so strong that the presence of one is considered a likely predictor of the other. This relationship
has led to the hypothesis that these conditions share core pathophysiological mechanisms. Recent advances
in our understanding of the complimentary roles played by vitamin D3 and vitamin K2 in vascular and bone
health provide support for this hypothesis, along with insight into key metabolic dysfunctions underlying
cardiovascular disease and osteoporosis.

Part I of this review, Vitamin D Deficiency – a Non-Traditional Risk Factor for Cardiovascular Disease –
summarized current research linking vitamin D deficiency to cardiovascular disease, the physiological
mechanisms underlying vitamin D’s cardiovascular effects, and leading vitamin D researchers’
recommendations for significantly higher supplemental doses of the pro-hormone. Read Part I: Vitamin D
Deficiency – a Non-Traditional Risk Factor for Cardiovascular Disease

Part II, The Vitamin K Connection to Cardiovascular Health, reviews the ways in which vitamin K regulates
calcium utlization, preventing vascular and soft tissue calcification while complimenting the bone-building
actions of vitamin D, and also discusses vitamin K safety and dosage issues, and the necessity of providing
vitamin K and vitamin A along with vitamin D to preclude adverse effects associated with hypervitaminosis
D.

Part II: The Vitamin K Connection to Cardiovascular Health

Introduction

First recognized by German researchers as a nutrient required for normal blood “koagulation,” vitamin K is
actually a family of structurally similar, fat-soluble compounds, some of which (the K2 forms) play
essential roles in cardiovascular health, primarily through regulating the body’s use of calcium – both
promoting its integration into bone and preventing of its deposition within blood vessels — and also by
exerting anti-inflammatory and insulin-sensitizing actions. 1

In nature, vitamin K appears primarily in two forms: K1 (phylloquinone [phyllo – relating to a leaf] and K2
(the menaquinones [mena – in reference to their methylated napthoquinone ring structure]). While all forms
of vitamin K share 2-methyl-1,4-naphthoqinone as their common ring structure, individual forms differ in
the length and degree of saturation of a variable aliphatic side chain attached to the 3-position.

K1, a single compound that contains a monounsaturated side chain of four isoprenoid residues, is found
primarily in plants and algae in association with chlorophyll. Dietary sources of K1 include green leafy
vegetables, such as broccoli, kale and Swiss chard, and unhydrogenated plant oils, including canola and
soybean oil.

K2, the menaquinones (MKs) are classified based on the length of their unsaturated side chains into 15
different types denominated as MK-n, with “n” denoting the number of isoprenyl residues in the side chain.
The most common MKs in humans are the short-chain menaquinone, MK-4, which is now thought to be
primarily produced via the systemic conversion of K1 to K2 in the body} 2 3 4 and the long-chain
menaquinones, MK-7 through MK-10, which are exclusively synthesized by bacteria and gut microflora in
all mammals, including humans. K2 (primarily its long-chain forms, MK-7, MK-8 and MK-9) is found in
fermented foods, notably cheese and natto (fermented soybean); the latter is the richest dietary source of
vitamin K presently known, almost all of which occurs in the form of MK-7.45

Vitamin K1, MK-4 and MK-7 are available as supplements: MK-4 as a synthetic version called
menatetrenone, and MK-7, as the natural compound extracted from natto. MK-7 has a much longer half-life
than either K1 or MK-4, which share similar molecular structures (both contain 4 isoprenoid residues, 3 of
which are saturated in K1 but contain a double bond in MK-4) and therefore similar physiokinetics. In
contrast, the longer-chain menaquinones, including MK-7, are much more hydrophobic and are handled
differently by the body. In vivo, they have longer half-lives and are incorporated into low-density
lipoproteins in the circulation, resulting in much more stable serum levels and accumulation to 7- to 8-fold
higher levels during prolonged intake. 5

K3 (menadione), a third, much simpler form of the vitamin, is considered a synthetic analogue, although
intestinal bacteria can produce minute amounts from K1.6 K3 has been utilized in research on vitamin K’s
anti-cancer effects because it potentiates the cytotoxic activity of chemotherapeutic agents and vitamin C
(when acting as an antioxidant, vitamin C is oxidized to dehydroascorbate, a potent free radical that is
spontaneously reduced by glutathione as well as in reactions using glutathione or NADPH 7 ; however,
because of its toxicity, the FDA has banned its use in nutritional supplements.8

Although, following intestinal absorption, both K1 and K2 are taken up in the triglyceride fraction from
which they are rapidly cleared by the liver, only the K2 forms are also taken up and systemically
redistributed by low-density lipoproteins. 910 Compared to K1, whose primary activity is the carboxylation of
blood coagulation factors (II [prothrombin], VII, IX, and X, the anticoagulant proteins C, S and Z), which
are synthesized in the liver, K2 has a much wider range of action, playing a significant role in bone
formation and protection against bone loss, arterial calcification, and oxidation of LDL cholesterol. 11 12 In
addition, K2 is a 15-fold more powerful antioxidant than K1 and is the predominant form of vitamin K in all
tissues, except the liver. 13 Finally, K2 is better absorbed than K1 and remains biologically active far longer;
K1 is cleared by the liver within 8 hours, while measurable levels of the MK-7 form of K2 have been
detected up to 72 hours after ingestion.14

Underlying Mechanism of Action: Gamma-carboxylation

Vitamin K is the cofactor for the enzyme, γ-glutamyl carboxylase, which converts specific glutamic acid
residues in a number of substrate proteins to γ-carboxyglutamic acid (Gla) residues, which then serve to
form calcium-binding groups in these proteins and are essential for their biologic activity.

Carboxylation thus activates this family of Gla-proteins, which are involved in numerous essential activities

throughout the body, including blood coagulation, bone metabolism, vascular repair, prevention of vascular
calcification, regulation of cell proliferation, and signal transduction. 15 16

K1 is preferentially utilized in the carboxylation of clotting factors in the liver. K2 is preferentially used in
the rest of the body to carboxylate the other vitamin K-dependent Gla-proteins, including osteocalcin (which
is essential for bone health and primarily synthesized in bone), and matrix-Gla protein (MGP) (which
prevents calcification of soft tissue, [e.g., the vasculature, myocardium, breasts and kidneys], and is
primarily synthesized in cartilage and the vessel wall. Vitamin K2 is also found in high concentrations in
the brain, where it contributes to the production of myelin and other important compounds.17 18

Vitamin K Recycling

The body very efficiently utilizes vitamin K by recycling this nutrient in a cyclic interconversion called the
vitamin K cycle.19 20 In this cycle, the vitamin K quinone form is reduced by the FAD-containing enzyme
DT-diaphorase (a.k.a. NAD(P)H:quinone oxidoreductase ) into the vitamin K hydroquinone (KH2), which
then serves as the cofactor for vitamin K carboxylation of Gla-proteins and, in so doing, is oxidized to
vitamin K epoxide. Vitamin K epoxide is then recycled back to the quinone form by the enzyme vitamin K
epoxide reductase (VKOR), completing the cycle. On a molecular level, vitamin K expoxide is reduced in
two steps: first to the quinone form by VKOR, then to vitamin K hydroquinone (KH2) by DT-diaphorase.

Besides being a cofactor in the vitamin K-dependent carboxylation, KH2 also possesses antioxidant activity
and is highly sensitive to free radicals, which may oxidize (and thus inactivate) KH2 before it can take part
in the carboxylation reaction. KH2’s reactivity to free radicals may increase need for K2 in arteries
burdened by atherosclerotic plaque, where high levels of oxidized LDL can contribute to a local vitamin K
deficiency, further exacerbating the atherosclerotic process.21

As noted, VKOR is a crucial enzyme in vitamin K metabolism, enabling its re-utilization after it has been
oxidized in the carboxylase reaction through which it activates Gla-proteins. Because of VKOR recycling,
the human requirement for vitamin K is extremely low—just 45 mcg/day is suggested to be all that is
needed of its most potent form, MK-7. VKOR is also the target for warfarin and related coumarin
derivatives, which block the recycling of vitamin K by inhibiting this enzyme, thereby decreasing vitamin K
available for the activation of Gla-proteins. The gene for VKOR has recently been identified, and it appears
that most of the variability observed in patients’ response to warfarin is attributable to variability in the
VKOR gene. 21 22 23

Click here for a PDF version of the chart

K2 Regulates Calcium Deposition: Mineralizing Bone, Preventing
Vascular Calcification Mineralizing Bone

Mechanisms

Only after its carboxylation by vitamin K is osteocalcin, the major non-collagenous protein responsible for
inducing bone mineralization in human osteoblasts, able to attract calcium ions and incorporate them into
hydroxyapatite crystals forming the bone matrix. When vitamin K2 levels are insufficient, osteocalcin
remains uncarboxylated with the result that bone mineralization is impaired.24

Not only is vitamin K2 a key inducer of bone mineralization in human osteoblasts, but this form of vitamin
K also inhibits osteoclast differentiation and is necessary to bring to fruition the bone-building effects of
vitamin D3′s upregulation of osteoblast’s expression of osteocalcin.24 25 26 27 28

Research Evidence

Numerous epidemiologic and intervention studies have shown that vitamin K insufficiency, with associated
high levels of undercarboxylated osteocalcin, causes reductions in bone mineral density (BMD) and
increases fracture risk. Conversely, supplementation with vitamin K2 has been shown to increase osteocalcin
activation(carboxylation), promote bone mineralization and lessen risk of fracture.1 29 30 31 32

A further consideration is that a number of clinical trials have demonstrated that the combination of K2 and
vitamin D3 is more effective in preventing bone loss than either nutrient alone.33 34 In a study of 173
osteoporotic/osteopenic women, those given both K2 and D3 experienced an average 4.92% increase in
bone mineral density (BMD), while average BMD increase was just 0.13 in those receiving K2 alone.35 In
another study evaluating the effects of vitamin D or K singly or in combination, 92 postmenopausal women
were assigned to one of four groups: K2 (45 mg/day), D3 (0.75 mcg/day [1 mcg D3 = 40 IU, so this was a
3,000 IU dose), both K2 and D3 at the aforementioned dosages, or calcium lactate (2 g/day). In the women
receiving only calcium, lumbar BMD decreased. Those given either D3 or K2 experienced a slight increase
in BMD. In those taking both, K2 and D3, lumbar BMD increased an average of 1.35%. 35

K2 has also been shown to work with D3 to lessen the risk of osteoporosis in Parkinson's disease, which is
thought to be related in part to immobilization as well as a deficiency of vitamin D caused, not by a lack of
vitamin D, but rather to suppression of D3 by the high blood levels of calcium seen in Parkinson's. When
K2 (MK-4) (45 mg/day for 12 months), was given to 54 female Parkinson's patients with osteoporosis, only
one hip fracture occurred, compared to 10 fractures in a control group of 54 women with Parkinson's who
were not treated with K2. Average bone loss in the untreated group was 4.3% compared to 1.3% in those
given K2.8

Preventing Arterial Calcification

Mechanisms

Matrix Gla-protein (MGP) is the strongest inhibitor of tissue calcification presently known. Its importance
for vascular health was first demonstrated in animals bred to be MGP-deficient, all of which died of
massive arterial calcification within 6–8 weeks after birth. 71

MGP is produced by small muscle cells in the vasculature where—once carboxylated by vitamin K2—it
protects against calcification through several mechanisms, including inhibiting bone morphological protein-
2 (BMP-2), upregulating the gene for DT-diaphorase, and downregulating the gene for osteoprotegerin:

Bone Morphological Protein-2 (BMP-2)

MGP inhibits calcification by binding to and inhibiting the activity of BMP-2, a potent bone morphogen
whose expression triggers the induction of an osteogenic gene expression profile in vascular smooth muscle
cells (VSMC), which causes them to transform into osteoblast-like cells, a transformation known to precede
arterial calcification. BMP-2 is expressed by cells in atherosclerotic lesions, and its expression can be
induced by oxidative stress, inflammation or hyperglycemia. 36 37 67 Overexpression of non γ-carboxylated

MGP, as is seen in calcified lesions in the aorta, results in unopposed BMP-2 activity, which promotes
osteoblastic differentiation of VSMC and the laying down of a calcified matrix.38

DT-diaphorase

DT-diaphorase (a.k.a. NAD(P)H:quinone oxidoreductase) is a FAD-containing enzyme (i.e., incorporates
riboflavin as its cofactor) that plays a key role in vitamin K recycling by reducing vitamin K to vitamin K
hydroquinone, which then serves as the cofactor for vitamin K carboxylation of Gla-proteins. Specifically,
K2’s effects on the gene expression of DT-diaphorase increase the enzyme’s activity in the vasculature 4.8-
fold, greatly increasing levels of activated MGP.19 38

Osteoprotegerin

Osteoclast-like cells have been identified in calcified human aortic plaques.38 Their activation is inhibited
by osteoprotegerin but upregulated by activated MGP. Specifically, osteoprotegerin promotes vascular
calcification by acting as a RANKL (RANK/receptor activator of NF-kappa B ligand) decoy receptor, thus
preventing RANKL from binding to the transmembrane receptor RANK on osteoclast precursors, where it
induces the differentiation and activation of osteoclasts. 39 40 By lessening the production of osteoprotegerin
in the vessel wall, activated MGP increases RANKL concentrations, thus increasing osteoclastic activity and
the removal of calcified areas from the vasculature.

Safeguarding Elasticity

While oxidized cholesterol's contribution to atherosclerosis has been treated as the primary issue in
cardiovascular disease, arteriosclerosis, the calcification of the arterial intima, is just as lethal. The elasticity
characteristic of a healthy artery is what enables it to accommodate increases in blood flow. Enough calcium
deposition and that pliability is lost: blood pressure rises, damaging the vasculature and contributing to
atherosclerosis. The two pathologies—arteriosclerosis and atherosclerosis—are synergistic. By preventing
arterial calcification, vitamin K2 also provides protection against atherosclerosis.

In addition, K2 directly promotes blood vessel elasticity by safeguarding elastin, the core protein in the
muscle fibers primarily responsible for the elasticity of the arterial wall. Calcium deposition not only
damages existing elastin, but inhibits new elastin production. 41

Clinical Evidence

The question of whether high vitamin K-intake is protective against arterial calcification was first addressed
in the Rotterdam Study, a massive European clinical trial following 4,807 subjects aged ≥55 over a 7-10
year period. Dietary intake of vitamin K2 (but not K1) was inversely correlated with cardiovascular
calcification and cardiovascular death. Elderly people in the highest tertile of vitamin K2 intake had 52%
reduction in severe aortic calcification, a 57% reduced risk of cardiovascular disease, and a 26% decreased
risk for all-cause mortality. K1 intake correlated with none of these beneficial outcomes. 41

Sudden death from heart attack is even more highly correlated with calcification of the aorta than
cholesterol. In Framingham study research, aortic calcification was associated with double the risk of death
from cardiovascular disease in men and women younger than 65, even after other risk factors (e.g.,
cholesterol) were taken into account. In men younger than 35, aortic calcification increased risk of sudden
coronary death 7-fold.42 43

Coronary Artery Calcification – A Key Biomarker of Functional
Age

Research involving more than 100,000 men and women in California revealed that aortic calcification
increased risk of coronary heart disease 127% in men and 122% in women. Among women, risk of stroke
also increased concurrently by 146%. 44

A high coronary artery calcium score on electron beam tomography has been found to be a better predictor
of mortality than age. A calcium score of less than 10 confers a reduction in functional age by 10 years in
subjects older than 70, while a calcium score of >400 adds as much as 30 years of functional aging to
younger patients.45 46 47

Vitamin K-dependent Gla-proteins have been shown to inhibit calcification in the heart and arteries; in the
kidneys, where K2 prevents the calcification that typically accompanies dialysis and diabetes; and in the
breast. Women whose diets provide the most vitamin K2 have significantly less breast calcification
compared to those whose diets provide the least. 48 In women, calcification of breast tissue (which several
studies have correlated with vitamin K2 insufficiency49 50) is associated with a 132% increased risk of
cardiovascular disease, a 141% increased risk of stroke, and a 152% increased risk of heart failure.51

Uncarboxylated MGP has also been identified as a key player in the increased calcification seen in the
development of varicosis, as well as in other vascular diseases. Researchers compared healthy veins from 36
male patients (aged 30 to 83) and varicose veins from 50 male patients (aged 40 to 81). In the men with
varicose veins, levels of uncarboxylated MGP, were high, indicating the local vascular vitamin K status in
varicose veins is insufficient to mediate full carboxylation of all newly formed MGP. Vitamin K
supplementation inhibited the mineralization process in varicose small muscle cell cultures, suggesting that
in vitro, carboxylation of MGP could be induced and that its inhibitory effect on varicosis could be
restored.52

In a clinical intervention study in which 78 women between 55 and 65 years of age received either vitamin
K2 (1 mg/day) or placebo for three years, vascular characteristics were assessed (elasticity and
distensibility). In subjects in the placebo group, vascular elasticity had decreased by 10–13%, which is
consistent with what has been considered a “normal” decrease during a three year time period for women in
this age group; in the vitamin K group, however, vascular characteristics remained unchanged, suggesting
that the process of vascular aging can be retarded by increased vitamin K intake. 53

Intra-cranial atherosclerosis, a newly identified risk factor for ischemic stroke 54, has been shown to be an
age-independent risk factor for cerebral atrophy.55 Given the protective effects of carboxylated MGP against
calcification the heart, vasculature, kidneys, and breasts, and the fact that K2 is concentrated in the brain
where it has been shown to completely block free radical accumulation and cell death in cell cultures of
developing fetal cortical neurons} 56 it does not seem unreasonable to hypothesize that K2 may also play a
protective role against calcification in the brain.

Vitamin K: Key to the Osteoporosis – Atherosclerosis Connection

Osteoporosis and arterial calcification have been thought to be unrelated conditions, but a number of recent
studies suggest a connection.31 In the U.S., ~75–95% of men and women have some degree of coronary
artery calcification on autopsy; 54% of postmenopausal women have osteopenia, and 30% have

osteoporosis.31 It has been noted that patients with low bone mass, osteopenia or osteoporosis frequently
also exhibit vascular calcification, which has been shown to predict both cardiovascular morbidity/mortality
and osteoporotic fractures.57

Aortic calcifications, specifically, have been positively associated with osteoporotic fractures, and the
progression of aortic calcification has been positively associated with the rate of decline in lumbar spine
BMD. 58 In a study of 195 postmenopausal women, the association between echogenic carotid artery
plaques, low bone mass and vertebral fractures was so strong that researchers suggested it could partly
explain why osteoporotic vertebral fractures are linked to increased mortality.59 Similar associations have
been found in men. In a 10 year prospective study of 781 men ≥ 50, calcifications in the abdominal aorta
increased fracture risk 2 to 3-fold, regardless of subjects’ BMI, comorbidities and medications. 60

An explanation for this correlation between osteoporosis and atherosclerosis is being developed in studies
analyzing the two conditions’ underlying pathophysiological mechanisms, which appear to coincide in one
common factor: vitamin K deficiency.

It is becoming apparent that the development of arterial calcification resembles the process of
osteogenesis.61

Both involve the same cell types, proteins and cytokines that lead to tissue mineralization.
More than 90% of atherosclerotic plaques undergo calcification. Ectopic bone tissue has been
identified in calcified plaques, and bone-specific cells have been found in the arterial wall, with
evidence that endothelial cells have transdifferentiated into osteoblasts.
Calcified arteries have also been shown to contain osteoclast-like cells.
Local and serum lymphocytes, monocytes and macrophages are involved in both osteoporosis and
vascular calcification.
Chemical mediators of bone metabolism including osteocalcin, bone morphogenetic protein (BMP),
osteopontin (OPN), osteonectin, osteoprotegerin (OPG), receptor activator of nuclear factor kappa B
ligand (RANKL), and inflammatory cytokines are also present in atherosclerotic arteries.
The vitamin K-dependent Gla proteins, osteocalcin and MGP, are mainly expressed in bone and
vascular cells, and are mediators and inhibitors of osteoid formation.
Although osteocalcin does not appear to play a significant role in the process of vascular calcification,
MGP (if carboxylated) is a key protective factor. Not only do MGP-knockout mice form extensive
and lethal arterial calcifications, they also present with osteopenia, fractures, short stature, and erratic
mineralization of the growth plates.
As noted earlier in this review, carboxylated MGP protein inhibits mesenchymal differentiation into
osteogenic cell lines by blocking the action of bone morphogenic protein (BMP), a potent factor of
bone maturation that initiates the differentiation of vascular mesenchyme into bone cells, thus
increasing calcification.
MGP isolated from calcified atherosclerotic plaques of mice shows incomplete carboxylation.

The Calcification Paradox – Another Iteration of the Same Theme

Upon entering menopause, women simultaneously lose calcium from bone and increase its deposition in
arteries—a negative double whammy called the "calcification paradox," which greatly increases their risk of
both osteoporosis and cardiovascular disease. 31 The drop in estrogen causes both problems; vitamin K2 can
help rectify them.

Estrogen impacts calcium regulation metabolism through several different pathways. Estrogen is involved in

the conversion of vitamin D to its active bone-building form (1,25-dihydroxycholecalciferol [1,25(OH)2D]
or calcitriol). When estrogen levels drop, osteoclasts become more sensitive to parathyroid hormone, which
signals them to increase their activity. Plus, the decline in estrogen allows production of the cytokine,
interleukin-6, to increase, and IL-6 stimulates the production of even more osteoclasts. 27 62

Among postmenopausal women not using estrogen replacement, low levels of vitamin K or high levels of
uncarboxylated osteocalcin are associated with low spine BMD. 63 Supplementation with vitamin K2,
however, has been shown to prevent bone loss associated with estrogen decline. In a 3-year study, 325
postmenopausal women were given either K2 (in the form of MK-4 or menatetrenone, for which the dosage
is 45 mg/day, specifically 15 mg/tid) or placebo. In those receiving K2, bone mineral content increased, and
hip and bone strength remained unchanged, whereas in the placebo group, both bone mineral content and
bone strength decreased significantly.64

Estrogen also protects premenopausal women from cardiovascular disease by increasing endothelial
production of prostacyclin, PG12, which inhibits platelet aggregation and promotes vasodilation. When
estrogen levels drop in menopause, these protective effects are lost. 65

Fortunately, MGP (if carboxylated) both inhibits vascular calcification and, as noted above, helps maintain
blood vessel elasticity. In a 3-year study of 181 postmenopausal women, one-third were given a supplement
containing vitamin D, one-third got a supplement providing both vitamin K and D, and one-third were
given a placebo. In both the vitamin D and the placebo group, elasticity of the common carotid artery
decreased; in those receiving K along with D, elasticity was maintained. 69

Vitamins K and A: Essential for the Prevention of Vitamin D
Toxicity

Vitamin D upregulates the expression of Gla-proteins, whose activation depends on vitamin K-mediated
carboxylation. Vitamin D thus increases both the demand for vitamin K and the potential for benefit from
K-dependent proteins, including osteocalcin in bone and MGP in blood vessels. 25

Another way of looking at this, however, is that by increasing the need for vitamin K2, increased levels of
vitamin D may actually induce a functional vitamin K2 deficiency, with the result that levels of
uncarboxylated osteocalcin and matrix-Gla protein rise in the circulation and vasculature. In this case, not
only is calcium not delivered to the bones, which become porous, but it is deposited in the arteries, which
become calcified. 31 66 11 67 68 69 70 71

It has recently been proposed that vitamin D toxicity is the result of precisely such induction of vitamin K2
deficiency.25. As vitamin D induces levels of Gla proteins to rise, the pool of available vitamin K available
to carboxylate them becomes depleted, so vitamin K-dependent processes that retain minerals in the bone
matrix, protect the soft tissues from calcification, and support the nervous system can no longer be
performed.

In support of this hypothesis, warfarin, a coumadin derivative that induces a functional vitamin K deficiency
by inhibiting the recycling of the vitamin, has definitively been shown to produce extensive
hypervitaminosis D-like calcification of the soft tissues and to exert toxicity synergistically with vitamin D
when the two are combined. 72 73 74 75

Uncarboxylated MGP is abundant in calcified arterial plaque, where its presence is thought to reflect a

reactive attempt by the local tissue to protect itself from calcification—an attempt rendered futile by
inadequate supplies of vitamin K2. In addition, vitamin K alone has been shown to fully reverse the
calcification induced by warfarin} 76 both confirming that the drug’s inhibition of vitamin K is directly
responsible for its induction of calcification, and also adding to the likelihood that vitamin D toxicity is due
to the same or a similar mechanism.2531

Vitamin A — Balancing the Actions of Vitamin D

The hypothesis further proposes, and a number of recent studies suggest, that vitamin A protects against
possible vitamin D toxicity by downregulating the expression of MGP, thus exerting a vitamin K-sparing
effect, which counteracts the depletion of vitamin K potentially induced by increased levels of vitamin D.77

78 79

A number of animal experiments have shown that high doses of vitamin A protect against the growth
retardation, soft tissue calcification and bone resorption induced in rats by dietary vitamin D3, and that
vitamin A completely protects against renal calcification induced by dietary vitamin D3 in turkeys. Vitamin
A has also been shown to decrease MGP expression in human cells.25 Retinoic acid and 1,25(OH)2D3
compete for the same nuclear partners; both the retinoic acid receptor and the VDR must form heterodimers
with retinoid X receptors (RXRs) to binding to response elements and initiate transcription. For this reason,
1,25(OH)2D3 and retinoic acid naturally balance one another’s effects.80

Also, in relation to the efficacy of vitamin D at potentially lower doses or in individuals carrying VDR
SNPs with impaired binding efficacy, recent research has shown that 9-cis-retinoic acid, a derivative of
vitamin A, increases the affinity of VDR/RXR to its DNA recognition site, induces recruitment of
coactivators by the DNA-bound heterodimer and potentiates vitamin D-dependent transcriptional
responses. 81

Thus, the proposed model suggests vitamin D toxicity is actually due, not to higher supplemental doses of
vitamin D, but results from an imbalance among vitamins D, A and K. Proper consideration of the
synergistic relationship among these vitamins could allow vitamin D to be therapeutically effective at lower
doses or to be administered in higher therapeutic doses without incurring the risks associated with
hypervitaminosis D.

As noted in Part I of this review, the body’s ability to utilize cholecalciferol in the numerous roles played by
the vitamin D endocrine system is not optimized until blood levels of 25(OH)D are ≥40 ng/ml (98 nmol/L).
Not until this level is the Vmax, of the 25-hydroxylase enzyme achieved (i.e., are all enzyme sites
saturated). Below this level, chronic substrate deficiency prevents full actualization of the myriad benefits of
vitamin D.82 For some individuals, supplementation of vitamin D3 in the range of 5,000 – 10,000 IU/day
may be necessary to reach and maintain these blood levels, which underscores the concomitant need for
adequate supplies of vitamin A as well as vitamin K. The National Institutes of Health has set the RDI for
vitamin A at 3,000 IU for males ≥ 14 years and 2,310 IU for females ≥ 14 years, and the tolerable upper
limits for retinols in both men and women at 10,000 IU.83

Factors Affecting Vitamin K Deficiency

Assuming that normal, healthy levels of beneficial bacteria are present in the intestines, these bacteria
produce about 75% of the vitamin K2 the body absorbs each day. Thus, even a diet quite rich in leafy greens
when consumed by an individual with healthy gut flora supplies less than half the vitamin K2 needed for

this nutrient’s calcium-regulating activities.

Unlike the other fat-soluble nutrients (vitamins A, D and E), vitamin K1 is cleared from the body within 8
hours, and even the MK-7 form of vitamin K2 is not stored in the body for more than 72 hours, thus this
nutrient is best provided daily. Despite the production of vitamin K2 (specifically MK-4) by healthy
intestinal bacteria, humans can develop a deficiency of the vitamin in as few as 7 days on a vitamin K-
deficient diet.84

Absorption of vitamin K, like that of other fat-soluble nutrients (A, D and E), is dependent upon healthy
liver and gallbladder function. Digestive health is also a factor. Deficiency is more likely in people with
digestive problems such as celiac disease, irritable bowel disease, or who have had intestinal bypass
surgery, all of which increase the likelihood of fat malabsorption.

Vitamin K recycling is dependent upon DT-diaphorase (a.k.a. NAD(P)H:quinone oxidoreductase), a FAD-
containing enzyme that reduces vitamin K to vitamin K hydroquinone, which then serves as the cofactor for
vitamin K carboxylation of Gla-proteins. FAD is derived from riboflavin (B2), thus vitamin K recycling is
dependent upon adequate supplies of riboflavin.

Vitamin K needs increase with age. Older individuals (>70) require higher levels of vitamin K1 or K2 to
maintain low levels of uncarboxylated vitamin-K dependent proteins.85

Bile acid sequestrants (e.g., Cholestyramine, Colestipol), which bind to bile acids, forming large compounds
that are poorly reabsorbed from the gut and eliminated in the feces, also bind and remove fat-soluble
vitamins, including vitamin K.

Canola and soybean oils are the primary source of vitamin K in the American diet. Hydrogenation changes
the vitamin K1 (phylloquinone) in these oils into dihydrophylloquinone, a form that does not carboxylate
osteocalcin and other vitamin-K dependent proteins. In 2,544 men and women (average age 58.5) who
participated in the Framingham Offspring Study, those with the highest intake of vitamin K from
hydrogenated oils had the lowest BMD at the neck, hip and spine.86 If your patient eats a fair amount of
processed or fast foods that contain hydrogenated oils, risk of functional vitamin K deficiency is greatly
increased. 87

While levels of vitamin K (K1, specifically) are rarely insufficient to meet clotting needs, levels of vitamin
K necessary for clotting are much lower than those needed (in the form of K2) for bone and arterial
protection. Studies of healthy adults have found high levels of uncarboxylated osteocalcin and matrix Gla-
protein (MGP) in all subjects tested. 71

Laboratory Assessment of Vitamin K Status

A normal prothrombin time is not an indication that sufficient vitamin K is present to maintain carboxylation
of osteocalcin or MGP.24 68 71

To check vitamin K levels, request an osteocalcin test; this measures how much uncarboxylated osteocalcin
is present in the blood. High levels of uncarboxylated osteocalin (ucOC) indicate insufficient vitamin K for
bone health and indirectly indicate that MGP is insufficiently carboxylated.71

Safety and Efficacy

Even in high doses, neither K1 nor K2 has produced adverse effects in individuals not on coumadin
derivatives. For this reason, the Institute of Medicine at the National Academy of Sciences chose not to set
a Tolerable Upper Limit (UL) for vitamin K when it revised its public health recommendations for this
vitamin in 2000.

Drug Interactions

Anticoagulant Medications

In patients on warfarin or other coumadin derivatives, vitamin K1 can interfere with these drugs’ anti-
clotting activity in amounts as small as 1 mg.

As noted above, oral anticoagulant medications, e.g., warfarin and other coumadin derivatives, promote
arterial calcification by preventing vitamin K from activating matrix Gla-protein. 15 88

These medications decrease clotting by blocking vitamin K epoxide reductase (VKOR), thus preventing
vitamin K recycling and greatly increasing risk of vitamin K deficiency, and have also been shown to block
the conversion of K1 to K2.89

A case report recommended physicians prescribing warfarin consider arterial calcification as a potential
consequence after routine examination of a healthy man on long-term warfarin treatment found his coronary
arteries were highly calcified. 90 Other case reports have noted pathologic tracheobronchial calcification with
long-term warfarin therapy in children, an 18-year-old male, and an elderly woman. 91 92 93 Two recent
studies involving more than 100 subjects have shown that patients treated with oral anticoagulants have
double the calcification of patients not on these vitamin K-blocking drugs.88

When improving vitamin K status, however, patients on these medications must be closely monitored. A
dose of just 1-2.5 mg of oral vitamin K1 reduces the range of the international normalized ratio (INR) from
5.0-9.0 to 2.0-5.0 within 24-48 hours; even eating a vitamin K-rich diet can make anticoagulant medications
less effective.94

On the other hand, recent studies have shown that the INR is more sensitive to vitamin K changes in patients
with a low vitamin K status than in those with a normal or high vitamin K status and that dietary vitamin K
intake in unstable patients is considerably lower than in stable patients.95 96 97

Research conducted by Schurgers et al., sugggests that MK-7 supplements supplying <50 mcg/day are not
likely to affect the INR value; however, doses of >50 mcg/day may interfere with oral anticoagulant
treatment in a clinically relevant way. A 50 mcg dose is comparable to the menaquinone content of 75 to
100 grams (2.6 to 3.5 ounces) of cheese, an amount that should lead to a disturbance of the INR value of no
more than 10%. In addition, the long half-life of MK-7 suggests that regular intake in combination with
properly adapted coumarin doses may result in more stable INR values.5

Other Interactions

K3, the synthetic form of vitamin K, promotes ROS production and glutathione depletion. High doses of K3
have been used in cancer research precisely for its ability to promote oxidative stress and cell death. Even in
lower doses, K3 has produced jaundice and hemolytic anemia in human infants. For these reasons, the U.S.
Food and Drug Administration banned the use of K3 in nutritional supplements.

Considerations when Choosing a Vitamin K Supplement

In animal studies, at very high intakes of K1, (200-fold the daily requirement of the liver), vitamin K1 is
converted to K2 (MK-4) in amounts that may be sufficient to help decrease arterial calcification. 98

It is important to differentiate between the two commercially available forms of K2 (the MK-4 and MK-7
menaquinones) since they differ in clinically significant ways.5 99 100 MK-4 is a short-chain menaquinone
available as a synthetic compound (menatetrenone), while MK-7, a long chain menaquinone, is a natural
menaquinone derived from natto fermentation.

The vast majority of studies evaluating the effectiveness of vitamin K for the prevention of both
osteoporosis and arterial calcification have used K2 (MK-4) at a dosage of 45 mg/day (specifically, 15
mg/tid). Not only has the majority of the research been done using MK-4, but MK-4 is the predominant
form of K2 into which the body converts K1. MK-4 appears quickly in the blood but has a half-life of only
1-2 hours, for which reason, high pharmacological doses (typically 45 mg/day given as 15 mg tid) are
necessary. Such large doses necessitate medical supervision in patients on blood-thinning medications (e.g.,
warfarin).

MK-7 is not only highly bioavailable and bioactive—45 mcg/day was sufficient to activate osteocalcin in
the Rotterdam study—but has a much longer serum half life of 3 days, which enables the body to build up a
buffer that can supply vitamin K2 to all tissues 24 hours a day. At 45 mcg/day (a dose 1,000 times less than
that typically used in the research for MK-4), natto-derived MK-7 is less likely to interact negatively with
blood-thinning medications.

Conclusion

As research documenting the widespread and significant beneficial actions of vitamin D continues to appear
in the peer-reviewed medical literature accompanied by reports that the majority of the U.S. population is
deficient in this nutrient, more clinicians are evaluating their patients’ vitamin D levels and prescribing
supplementation, often in amounts as high as 5,000 to 10,000 IU/day, without awareness of the risk of
provoking an imbalance among vitamins D, K and A. Consideration of the synergistic relationship among
these vitamins could allow vitamin D to be administered in doses of greater therapeutic value without
incurring the risks of osteoporosis and vascular calcification associated with hypervitaminosis D.

Read Part I: Vitamin D and Vitamin K Team Up to Lower CVD Risk:
Vitamin D Deficiency – a Non-Traditional Risk Factor for Cardiovascular Disease

©2010 Smart Publications. All Rights Reserved. www.lmreview.comVitamin D and Vitamin K Team Up to Lower CVD
Risk: Part II

by Lara Pizzorno, MDiv, MA, LMT

Abstract

Strong correlations have been noted between cardiovascular diseases and low bone density / osteoporosis—
connections so strong that the presence of one is considered a likely predictor of the other. This relationship
has led to the hypothesis that these conditions share core pathophysiological mechanisms. Recent advances
in our understanding of the complimentary roles played by vitamin D3 and vitamin K2 in vascular and bone
health provide support for this hypothesis, along with insight into key metabolic dysfunctions underlying
cardiovascular disease and osteoporosis.

Part I of this review, Vitamin D Deficiency – a Non-Traditional Risk Factor for Cardiovascular Disease –
summarized current research linking vitamin D deficiency to cardiovascular disease, the physiological
mechanisms underlying vitamin D’s cardiovascular effects, and leading vitamin D researchers’
recommendations for significantly higher supplemental doses of the pro-hormone. Read Part I: Vitamin D
Deficiency – a Non-Traditional Risk Factor for Cardiovascular Disease

Part II, The Vitamin K Connection to Cardiovascular Health, reviews the ways in which vitamin K regulates
calcium utlization, preventing vascular and soft tissue calcification while complimenting the bone-building
actions of vitamin D, and also discusses vitamin K safety and dosage issues, and the necessity of providing
vitamin K and vitamin A along with vitamin D to preclude adverse effects associated with hypervitaminosis
D.

Part II: The Vitamin K Connection to Cardiovascular Health

Introduction

First recognized by German researchers as a nutrient required for normal blood “koagulation,” vitamin K is
actually a family of structurally similar, fat-soluble compounds, some of which (the K2 forms) play
essential roles in cardiovascular health, primarily through regulating the body’s use of calcium – both
promoting its integration into bone and preventing of its deposition within blood vessels — and also by
exerting anti-inflammatory and insulin-sensitizing actions. 1

In nature, vitamin K appears primarily in two forms: K1 (phylloquinone [phyllo – relating to a leaf] and K2
(the menaquinones [mena – in reference to their methylated napthoquinone ring structure]). While all forms
of vitamin K share 2-methyl-1,4-naphthoqinone as their common ring structure, individual forms differ in
the length and degree of saturation of a variable aliphatic side chain attached to the 3-position.

K1, a single compound that contains a monounsaturated side chain of four isoprenoid residues, is found
primarily in plants and algae in association with chlorophyll. Dietary sources of K1 include green leafy
vegetables, such as broccoli, kale and Swiss chard, and unhydrogenated plant oils, including canola and
soybean oil.

K2, the menaquinones (MKs) are classified based on the length of their unsaturated side chains into 15
different types denominated as MK-n, with “n” denoting the number of isoprenyl residues in the side chain.
The most common MKs in humans are the short-chain menaquinone, MK-4, which is now thought to be
primarily produced via the systemic conversion of K1 to K2 in the body} 2 3 4 and the long-chain
menaquinones, MK-7 through MK-10, which are exclusively synthesized by bacteria and gut microflora in
all mammals, including humans. K2 (primarily its long-chain forms, MK-7, MK-8 and MK-9) is found in
fermented foods, notably cheese and natto (fermented soybean); the latter is the richest dietary source of
vitamin K presently known, almost all of which occurs in the form of MK-7.45

Vitamin K1, MK-4 and MK-7 are available as supplements: MK-4 as a synthetic version called
menatetrenone, and MK-7, as the natural compound extracted from natto. MK-7 has a much longer half-life
than either K1 or MK-4, which share similar molecular structures (both contain 4 isoprenoid residues, 3 of
which are saturated in K1 but contain a double bond in MK-4) and therefore similar physiokinetics. In
contrast, the longer-chain menaquinones, including MK-7, are much more hydrophobic and are handled
differently by the body. In vivo, they have longer half-lives and are incorporated into low-density
lipoproteins in the circulation, resulting in much more stable serum levels and accumulation to 7- to 8-fold
higher levels during prolonged intake. 5

K3 (menadione), a third, much simpler form of the vitamin, is considered a synthetic analogue, although
intestinal bacteria can produce minute amounts from K1.6 K3 has been utilized in research on vitamin K’s
anti-cancer effects because it potentiates the cytotoxic activity of chemotherapeutic agents and vitamin C
(when acting as an antioxidant, vitamin C is oxidized to dehydroascorbate, a potent free radical that is
spontaneously reduced by glutathione as well as in reactions using glutathione or NADPH 7 ; however,
because of its toxicity, the FDA has banned its use in nutritional supplements.8

Although, following intestinal absorption, both K1 and K2 are taken up in the triglyceride fraction from
which they are rapidly cleared by the liver, only the K2 forms are also taken up and systemically
redistributed by low-density lipoproteins. 910 Compared to K1, whose primary activity is the carboxylation of
blood coagulation factors (II [prothrombin], VII, IX, and X, the anticoagulant proteins C, S and Z), which
are synthesized in the liver, K2 has a much wider range of action, playing a significant role in bone
formation and protection against bone loss, arterial calcification, and oxidation of LDL cholesterol. 11 12 In
addition, K2 is a 15-fold more powerful antioxidant than K1 and is the predominant form of vitamin K in all
tissues, except the liver. 13 Finally, K2 is better absorbed than K1 and remains biologically active far longer;
K1 is cleared by the liver within 8 hours, while measurable levels of the MK-7 form of K2 have been
detected up to 72 hours after ingestion.14

Underlying Mechanism of Action: Gamma-carboxylation

Vitamin K is the cofactor for the enzyme, γ-glutamyl carboxylase, which converts specific glutamic acid
residues in a number of substrate proteins to γ-carboxyglutamic acid (Gla) residues, which then serve to
form calcium-binding groups in these proteins and are essential for their biologic activity.

Carboxylation thus activates this family of Gla-proteins, which are involved in numerous essential activities

throughout the body, including blood coagulation, bone metabolism, vascular repair, prevention of vascular
calcification, regulation of cell proliferation, and signal transduction. 15 16

K1 is preferentially utilized in the carboxylation of clotting factors in the liver. K2 is preferentially used in
the rest of the body to carboxylate the other vitamin K-dependent Gla-proteins, including osteocalcin (which
is essential for bone health and primarily synthesized in bone), and matrix-Gla protein (MGP) (which
prevents calcification of soft tissue, [e.g., the vasculature, myocardium, breasts and kidneys], and is
primarily synthesized in cartilage and the vessel wall. Vitamin K2 is also found in high concentrations in
the brain, where it contributes to the production of myelin and other important compounds.17 18

Vitamin K Recycling

The body very efficiently utilizes vitamin K by recycling this nutrient in a cyclic interconversion called the
vitamin K cycle.19 20 In this cycle, the vitamin K quinone form is reduced by the FAD-containing enzyme
DT-diaphorase (a.k.a. NAD(P)H:quinone oxidoreductase ) into the vitamin K hydroquinone (KH2), which
then serves as the cofactor for vitamin K carboxylation of Gla-proteins and, in so doing, is oxidized to
vitamin K epoxide. Vitamin K epoxide is then recycled back to the quinone form by the enzyme vitamin K
epoxide reductase (VKOR), completing the cycle. On a molecular level, vitamin K expoxide is reduced in
two steps: first to the quinone form by VKOR, then to vitamin K hydroquinone (KH2) by DT-diaphorase.

Besides being a cofactor in the vitamin K-dependent carboxylation, KH2 also possesses antioxidant activity
and is highly sensitive to free radicals, which may oxidize (and thus inactivate) KH2 before it can take part
in the carboxylation reaction. KH2’s reactivity to free radicals may increase need for K2 in arteries
burdened by atherosclerotic plaque, where high levels of oxidized LDL can contribute to a local vitamin K
deficiency, further exacerbating the atherosclerotic process.21

As noted, VKOR is a crucial enzyme in vitamin K metabolism, enabling its re-utilization after it has been
oxidized in the carboxylase reaction through which it activates Gla-proteins. Because of VKOR recycling,
the human requirement for vitamin K is extremely low—just 45 mcg/day is suggested to be all that is
needed of its most potent form, MK-7. VKOR is also the target for warfarin and related coumarin
derivatives, which block the recycling of vitamin K by inhibiting this enzyme, thereby decreasing vitamin K
available for the activation of Gla-proteins. The gene for VKOR has recently been identified, and it appears
that most of the variability observed in patients’ response to warfarin is attributable to variability in the
VKOR gene. 21 22 23

Click here for a PDF version of the chart

K2 Regulates Calcium Deposition: Mineralizing Bone, Preventing
Vascular Calcification Mineralizing Bone

Mechanisms

Only after its carboxylation by vitamin K is osteocalcin, the major non-collagenous protein responsible for
inducing bone mineralization in human osteoblasts, able to attract calcium ions and incorporate them into
hydroxyapatite crystals forming the bone matrix. When vitamin K2 levels are insufficient, osteocalcin
remains uncarboxylated with the result that bone mineralization is impaired.24

Not only is vitamin K2 a key inducer of bone mineralization in human osteoblasts, but this form of vitamin
K also inhibits osteoclast differentiation and is necessary to bring to fruition the bone-building effects of
vitamin D3′s upregulation of osteoblast’s expression of osteocalcin.24 25 26 27 28

Research Evidence

Numerous epidemiologic and intervention studies have shown that vitamin K insufficiency, with associated
high levels of undercarboxylated osteocalcin, causes reductions in bone mineral density (BMD) and
increases fracture risk. Conversely, supplementation with vitamin K2 has been shown to increase osteocalcin
activation(carboxylation), promote bone mineralization and lessen risk of fracture.1 29 30 31 32

A further consideration is that a number of clinical trials have demonstrated that the combination of K2 and
vitamin D3 is more effective in preventing bone loss than either nutrient alone.33 34 In a study of 173
osteoporotic/osteopenic women, those given both K2 and D3 experienced an average 4.92% increase in
bone mineral density (BMD), while average BMD increase was just 0.13 in those receiving K2 alone.35 In
another study evaluating the effects of vitamin D or K singly or in combination, 92 postmenopausal women
were assigned to one of four groups: K2 (45 mg/day), D3 (0.75 mcg/day [1 mcg D3 = 40 IU, so this was a
3,000 IU dose), both K2 and D3 at the aforementioned dosages, or calcium lactate (2 g/day). In the women
receiving only calcium, lumbar BMD decreased. Those given either D3 or K2 experienced a slight increase
in BMD. In those taking both, K2 and D3, lumbar BMD increased an average of 1.35%. 35

K2 has also been shown to work with D3 to lessen the risk of osteoporosis in Parkinson's disease, which is
thought to be related in part to immobilization as well as a deficiency of vitamin D caused, not by a lack of
vitamin D, but rather to suppression of D3 by the high blood levels of calcium seen in Parkinson's. When
K2 (MK-4) (45 mg/day for 12 months), was given to 54 female Parkinson's patients with osteoporosis, only
one hip fracture occurred, compared to 10 fractures in a control group of 54 women with Parkinson's who
were not treated with K2. Average bone loss in the untreated group was 4.3% compared to 1.3% in those
given K2.8

Preventing Arterial Calcification

Mechanisms

Matrix Gla-protein (MGP) is the strongest inhibitor of tissue calcification presently known. Its importance
for vascular health was first demonstrated in animals bred to be MGP-deficient, all of which died of
massive arterial calcification within 6–8 weeks after birth. 71

MGP is produced by small muscle cells in the vasculature where—once carboxylated by vitamin K2—it
protects against calcification through several mechanisms, including inhibiting bone morphological protein-
2 (BMP-2), upregulating the gene for DT-diaphorase, and downregulating the gene for osteoprotegerin:

Bone Morphological Protein-2 (BMP-2)

MGP inhibits calcification by binding to and inhibiting the activity of BMP-2, a potent bone morphogen
whose expression triggers the induction of an osteogenic gene expression profile in vascular smooth muscle
cells (VSMC), which causes them to transform into osteoblast-like cells, a transformation known to precede
arterial calcification. BMP-2 is expressed by cells in atherosclerotic lesions, and its expression can be
induced by oxidative stress, inflammation or hyperglycemia. 36 37 67 Overexpression of non γ-carboxylated

MGP, as is seen in calcified lesions in the aorta, results in unopposed BMP-2 activity, which promotes
osteoblastic differentiation of VSMC and the laying down of a calcified matrix.38

DT-diaphorase

DT-diaphorase (a.k.a. NAD(P)H:quinone oxidoreductase) is a FAD-containing enzyme (i.e., incorporates
riboflavin as its cofactor) that plays a key role in vitamin K recycling by reducing vitamin K to vitamin K
hydroquinone, which then serves as the cofactor for vitamin K carboxylation of Gla-proteins. Specifically,
K2’s effects on the gene expression of DT-diaphorase increase the enzyme’s activity in the vasculature 4.8-
fold, greatly increasing levels of activated MGP.19 38

Osteoprotegerin

Osteoclast-like cells have been identified in calcified human aortic plaques.38 Their activation is inhibited
by osteoprotegerin but upregulated by activated MGP. Specifically, osteoprotegerin promotes vascular
calcification by acting as a RANKL (RANK/receptor activator of NF-kappa B ligand) decoy receptor, thus
preventing RANKL from binding to the transmembrane receptor RANK on osteoclast precursors, where it
induces the differentiation and activation of osteoclasts. 39 40 By lessening the production of osteoprotegerin
in the vessel wall, activated MGP increases RANKL concentrations, thus increasing osteoclastic activity and
the removal of calcified areas from the vasculature.

Safeguarding Elasticity

While oxidized cholesterol's contribution to atherosclerosis has been treated as the primary issue in
cardiovascular disease, arteriosclerosis, the calcification of the arterial intima, is just as lethal. The elasticity
characteristic of a healthy artery is what enables it to accommodate increases in blood flow. Enough calcium
deposition and that pliability is lost: blood pressure rises, damaging the vasculature and contributing to
atherosclerosis. The two pathologies—arteriosclerosis and atherosclerosis—are synergistic. By preventing
arterial calcification, vitamin K2 also provides protection against atherosclerosis.

In addition, K2 directly promotes blood vessel elasticity by safeguarding elastin, the core protein in the
muscle fibers primarily responsible for the elasticity of the arterial wall. Calcium deposition not only
damages existing elastin, but inhibits new elastin production. 41

Clinical Evidence

The question of whether high vitamin K-intake is protective against arterial calcification was first addressed
in the Rotterdam Study, a massive European clinical trial following 4,807 subjects aged ≥55 over a 7-10
year period. Dietary intake of vitamin K2 (but not K1) was inversely correlated with cardiovascular
calcification and cardiovascular death. Elderly people in the highest tertile of vitamin K2 intake had 52%
reduction in severe aortic calcification, a 57% reduced risk of cardiovascular disease, and a 26% decreased
risk for all-cause mortality. K1 intake correlated with none of these beneficial outcomes. 41

Sudden death from heart attack is even more highly correlated with calcification of the aorta than
cholesterol. In Framingham study research, aortic calcification was associated with double the risk of death
from cardiovascular disease in men and women younger than 65, even after other risk factors (e.g.,
cholesterol) were taken into account. In men younger than 35, aortic calcification increased risk of sudden
coronary death 7-fold.42 43

Coronary Artery Calcification – A Key Biomarker of Functional
Age

Research involving more than 100,000 men and women in California revealed that aortic calcification
increased risk of coronary heart disease 127% in men and 122% in women. Among women, risk of stroke
also increased concurrently by 146%. 44

A high coronary artery calcium score on electron beam tomography has been found to be a better predictor
of mortality than age. A calcium score of less than 10 confers a reduction in functional age by 10 years in
subjects older than 70, while a calcium score of >400 adds as much as 30 years of functional aging to
younger patients.45 46 47

Vitamin K-dependent Gla-proteins have been shown to inhibit calcification in the heart and arteries; in the
kidneys, where K2 prevents the calcification that typically accompanies dialysis and diabetes; and in the
breast. Women whose diets provide the most vitamin K2 have significantly less breast calcification
compared to those whose diets provide the least. 48 In women, calcification of breast tissue (which several
studies have correlated with vitamin K2 insufficiency49 50) is associated with a 132% increased risk of
cardiovascular disease, a 141% increased risk of stroke, and a 152% increased risk of heart failure.51

Uncarboxylated MGP has also been identified as a key player in the increased calcification seen in the
development of varicosis, as well as in other vascular diseases. Researchers compared healthy veins from 36
male patients (aged 30 to 83) and varicose veins from 50 male patients (aged 40 to 81). In the men with
varicose veins, levels of uncarboxylated MGP, were high, indicating the local vascular vitamin K status in
varicose veins is insufficient to mediate full carboxylation of all newly formed MGP. Vitamin K
supplementation inhibited the mineralization process in varicose small muscle cell cultures, suggesting that
in vitro, carboxylation of MGP could be induced and that its inhibitory effect on varicosis could be
restored.52

In a clinical intervention study in which 78 women between 55 and 65 years of age received either vitamin
K2 (1 mg/day) or placebo for three years, vascular characteristics were assessed (elasticity and
distensibility). In subjects in the placebo group, vascular elasticity had decreased by 10–13%, which is
consistent with what has been considered a “normal” decrease during a three year time period for women in
this age group; in the vitamin K group, however, vascular characteristics remained unchanged, suggesting
that the process of vascular aging can be retarded by increased vitamin K intake. 53

Intra-cranial atherosclerosis, a newly identified risk factor for ischemic stroke 54, has been shown to be an
age-independent risk factor for cerebral atrophy.55 Given the protective effects of carboxylated MGP against
calcification the heart, vasculature, kidneys, and breasts, and the fact that K2 is concentrated in the brain
where it has been shown to completely block free radical accumulation and cell death in cell cultures of
developing fetal cortical neurons} 56 it does not seem unreasonable to hypothesize that K2 may also play a
protective role against calcification in the brain.

Vitamin K: Key to the Osteoporosis – Atherosclerosis Connection

Osteoporosis and arterial calcification have been thought to be unrelated conditions, but a number of recent
studies suggest a connection.31 In the U.S., ~75–95% of men and women have some degree of coronary
artery calcification on autopsy; 54% of postmenopausal women have osteopenia, and 30% have

osteoporosis.31 It has been noted that patients with low bone mass, osteopenia or osteoporosis frequently
also exhibit vascular calcification, which has been shown to predict both cardiovascular morbidity/mortality
and osteoporotic fractures.57

Aortic calcifications, specifically, have been positively associated with osteoporotic fractures, and the
progression of aortic calcification has been positively associated with the rate of decline in lumbar spine
BMD. 58 In a study of 195 postmenopausal women, the association between echogenic carotid artery
plaques, low bone mass and vertebral fractures was so strong that researchers suggested it could partly
explain why osteoporotic vertebral fractures are linked to increased mortality.59 Similar associations have
been found in men. In a 10 year prospective study of 781 men ≥ 50, calcifications in the abdominal aorta
increased fracture risk 2 to 3-fold, regardless of subjects’ BMI, comorbidities and medications. 60

An explanation for this correlation between osteoporosis and atherosclerosis is being developed in studies
analyzing the two conditions’ underlying pathophysiological mechanisms, which appear to coincide in one
common factor: vitamin K deficiency.

It is becoming apparent that the development of arterial calcification resembles the process of
osteogenesis.61

Both involve the same cell types, proteins and cytokines that lead to tissue mineralization.
More than 90% of atherosclerotic plaques undergo calcification. Ectopic bone tissue has been
identified in calcified plaques, and bone-specific cells have been found in the arterial wall, with
evidence that endothelial cells have transdifferentiated into osteoblasts.
Calcified arteries have also been shown to contain osteoclast-like cells.
Local and serum lymphocytes, monocytes and macrophages are involved in both osteoporosis and
vascular calcification.
Chemical mediators of bone metabolism including osteocalcin, bone morphogenetic protein (BMP),
osteopontin (OPN), osteonectin, osteoprotegerin (OPG), receptor activator of nuclear factor kappa B
ligand (RANKL), and inflammatory cytokines are also present in atherosclerotic arteries.
The vitamin K-dependent Gla proteins, osteocalcin and MGP, are mainly expressed in bone and
vascular cells, and are mediators and inhibitors of osteoid formation.
Although osteocalcin does not appear to play a significant role in the process of vascular calcification,
MGP (if carboxylated) is a key protective factor. Not only do MGP-knockout mice form extensive
and lethal arterial calcifications, they also present with osteopenia, fractures, short stature, and erratic
mineralization of the growth plates.
As noted earlier in this review, carboxylated MGP protein inhibits mesenchymal differentiation into
osteogenic cell lines by blocking the action of bone morphogenic protein (BMP), a potent factor of
bone maturation that initiates the differentiation of vascular mesenchyme into bone cells, thus
increasing calcification.
MGP isolated from calcified atherosclerotic plaques of mice shows incomplete carboxylation.

The Calcification Paradox – Another Iteration of the Same Theme

Upon entering menopause, women simultaneously lose calcium from bone and increase its deposition in
arteries—a negative double whammy called the "calcification paradox," which greatly increases their risk of
both osteoporosis and cardiovascular disease. 31 The drop in estrogen causes both problems; vitamin K2 can
help rectify them.

Estrogen impacts calcium regulation metabolism through several different pathways. Estrogen is involved in

the conversion of vitamin D to its active bone-building form (1,25-dihydroxycholecalciferol [1,25(OH)2D]
or calcitriol). When estrogen levels drop, osteoclasts become more sensitive to parathyroid hormone, which
signals them to increase their activity. Plus, the decline in estrogen allows production of the cytokine,
interleukin-6, to increase, and IL-6 stimulates the production of even more osteoclasts. 27 62

Among postmenopausal women not using estrogen replacement, low levels of vitamin K or high levels of
uncarboxylated osteocalcin are associated with low spine BMD. 63 Supplementation with vitamin K2,
however, has been shown to prevent bone loss associated with estrogen decline. In a 3-year study, 325
postmenopausal women were given either K2 (in the form of MK-4 or menatetrenone, for which the dosage
is 45 mg/day, specifically 15 mg/tid) or placebo. In those receiving K2, bone mineral content increased, and
hip and bone strength remained unchanged, whereas in the placebo group, both bone mineral content and
bone strength decreased significantly.64

Estrogen also protects premenopausal women from cardiovascular disease by increasing endothelial
production of prostacyclin, PG12, which inhibits platelet aggregation and promotes vasodilation. When
estrogen levels drop in menopause, these protective effects are lost. 65

Fortunately, MGP (if carboxylated) both inhibits vascular calcification and, as noted above, helps maintain
blood vessel elasticity. In a 3-year study of 181 postmenopausal women, one-third were given a supplement
containing vitamin D, one-third got a supplement providing both vitamin K and D, and one-third were
given a placebo. In both the vitamin D and the placebo group, elasticity of the common carotid artery
decreased; in those receiving K along with D, elasticity was maintained. 69

Vitamins K and A: Essential for the Prevention of Vitamin D
Toxicity

Vitamin D upregulates the expression of Gla-proteins, whose activation depends on vitamin K-mediated
carboxylation. Vitamin D thus increases both the demand for vitamin K and the potential for benefit from
K-dependent proteins, including osteocalcin in bone and MGP in blood vessels. 25

Another way of looking at this, however, is that by increasing the need for vitamin K2, increased levels of
vitamin D may actually induce a functional vitamin K2 deficiency, with the result that levels of
uncarboxylated osteocalcin and matrix-Gla protein rise in the circulation and vasculature. In this case, not
only is calcium not delivered to the bones, which become porous, but it is deposited in the arteries, which
become calcified. 31 66 11 67 68 69 70 71

It has recently been proposed that vitamin D toxicity is the result of precisely such induction of vitamin K2
deficiency.25. As vitamin D induces levels of Gla proteins to rise, the pool of available vitamin K available
to carboxylate them becomes depleted, so vitamin K-dependent processes that retain minerals in the bone
matrix, protect the soft tissues from calcification, and support the nervous system can no longer be
performed.

In support of this hypothesis, warfarin, a coumadin derivative that induces a functional vitamin K deficiency
by inhibiting the recycling of the vitamin, has definitively been shown to produce extensive
hypervitaminosis D-like calcification of the soft tissues and to exert toxicity synergistically with vitamin D
when the two are combined. 72 73 74 75

Uncarboxylated MGP is abundant in calcified arterial plaque, where its presence is thought to reflect a

reactive attempt by the local tissue to protect itself from calcification—an attempt rendered futile by
inadequate supplies of vitamin K2. In addition, vitamin K alone has been shown to fully reverse the
calcification induced by warfarin} 76 both confirming that the drug’s inhibition of vitamin K is directly
responsible for its induction of calcification, and also adding to the likelihood that vitamin D toxicity is due
to the same or a similar mechanism.2531

Vitamin A — Balancing the Actions of Vitamin D

The hypothesis further proposes, and a number of recent studies suggest, that vitamin A protects against
possible vitamin D toxicity by downregulating the expression of MGP, thus exerting a vitamin K-sparing
effect, which counteracts the depletion of vitamin K potentially induced by increased levels of vitamin D.77

78 79

A number of animal experiments have shown that high doses of vitamin A protect against the growth
retardation, soft tissue calcification and bone resorption induced in rats by dietary vitamin D3, and that
vitamin A completely protects against renal calcification induced by dietary vitamin D3 in turkeys. Vitamin
A has also been shown to decrease MGP expression in human cells.25 Retinoic acid and 1,25(OH)2D3
compete for the same nuclear partners; both the retinoic acid receptor and the VDR must form heterodimers
with retinoid X receptors (RXRs) to binding to response elements and initiate transcription. For this reason,
1,25(OH)2D3 and retinoic acid naturally balance one another’s effects.80

Also, in relation to the efficacy of vitamin D at potentially lower doses or in individuals carrying VDR
SNPs with impaired binding efficacy, recent research has shown that 9-cis-retinoic acid, a derivative of
vitamin A, increases the affinity of VDR/RXR to its DNA recognition site, induces recruitment of
coactivators by the DNA-bound heterodimer and potentiates vitamin D-dependent transcriptional
responses. 81

Thus, the proposed model suggests vitamin D toxicity is actually due, not to higher supplemental doses of
vitamin D, but results from an imbalance among vitamins D, A and K. Proper consideration of the
synergistic relationship among these vitamins could allow vitamin D to be therapeutically effective at lower
doses or to be administered in higher therapeutic doses without incurring the risks associated with
hypervitaminosis D.

As noted in Part I of this review, the body’s ability to utilize cholecalciferol in the numerous roles played by
the vitamin D endocrine system is not optimized until blood levels of 25(OH)D are ≥40 ng/ml (98 nmol/L).
Not until this level is the Vmax, of the 25-hydroxylase enzyme achieved (i.e., are all enzyme sites
saturated). Below this level, chronic substrate deficiency prevents full actualization of the myriad benefits of
vitamin D.82 For some individuals, supplementation of vitamin D3 in the range of 5,000 – 10,000 IU/day
may be necessary to reach and maintain these blood levels, which underscores the concomitant need for
adequate supplies of vitamin A as well as vitamin K. The National Institutes of Health has set the RDI for
vitamin A at 3,000 IU for males ≥ 14 years and 2,310 IU for females ≥ 14 years, and the tolerable upper
limits for retinols in both men and women at 10,000 IU.83

Factors Affecting Vitamin K Deficiency

Assuming that normal, healthy levels of beneficial bacteria are present in the intestines, these bacteria
produce about 75% of the vitamin K2 the body absorbs each day. Thus, even a diet quite rich in leafy greens
when consumed by an individual with healthy gut flora supplies less than half the vitamin K2 needed for

this nutrient’s calcium-regulating activities.

Unlike the other fat-soluble nutrients (vitamins A, D and E), vitamin K1 is cleared from the body within 8
hours, and even the MK-7 form of vitamin K2 is not stored in the body for more than 72 hours, thus this
nutrient is best provided daily. Despite the production of vitamin K2 (specifically MK-4) by healthy
intestinal bacteria, humans can develop a deficiency of the vitamin in as few as 7 days on a vitamin K-
deficient diet.84

Absorption of vitamin K, like that of other fat-soluble nutrients (A, D and E), is dependent upon healthy
liver and gallbladder function. Digestive health is also a factor. Deficiency is more likely in people with
digestive problems such as celiac disease, irritable bowel disease, or who have had intestinal bypass
surgery, all of which increase the likelihood of fat malabsorption.

Vitamin K recycling is dependent upon DT-diaphorase (a.k.a. NAD(P)H:quinone oxidoreductase), a FAD-
containing enzyme that reduces vitamin K to vitamin K hydroquinone, which then serves as the cofactor for
vitamin K carboxylation of Gla-proteins. FAD is derived from riboflavin (B2), thus vitamin K recycling is
dependent upon adequate supplies of riboflavin.

Vitamin K needs increase with age. Older individuals (>70) require higher levels of vitamin K1 or K2 to
maintain low levels of uncarboxylated vitamin-K dependent proteins.85

Bile acid sequestrants (e.g., Cholestyramine, Colestipol), which bind to bile acids, forming large compounds
that are poorly reabsorbed from the gut and eliminated in the feces, also bind and remove fat-soluble
vitamins, including vitamin K.

Canola and soybean oils are the primary source of vitamin K in the American diet. Hydrogenation changes
the vitamin K1 (phylloquinone) in these oils into dihydrophylloquinone, a form that does not carboxylate
osteocalcin and other vitamin-K dependent proteins. In 2,544 men and women (average age 58.5) who
participated in the Framingham Offspring Study, those with the highest intake of vitamin K from
hydrogenated oils had the lowest BMD at the neck, hip and spine.86 If your patient eats a fair amount of
processed or fast foods that contain hydrogenated oils, risk of functional vitamin K deficiency is greatly
increased. 87

While levels of vitamin K (K1, specifically) are rarely insufficient to meet clotting needs, levels of vitamin
K necessary for clotting are much lower than those needed (in the form of K2) for bone and arterial
protection. Studies of healthy adults have found high levels of uncarboxylated osteocalcin and matrix Gla-
protein (MGP) in all subjects tested. 71

Laboratory Assessment of Vitamin K Status

A normal prothrombin time is not an indication that sufficient vitamin K is present to maintain carboxylation
of osteocalcin or MGP.24 68 71

To check vitamin K levels, request an osteocalcin test; this measures how much uncarboxylated osteocalcin
is present in the blood. High levels of uncarboxylated osteocalin (ucOC) indicate insufficient vitamin K for
bone health and indirectly indicate that MGP is insufficiently carboxylated.71

Safety and Efficacy

Even in high doses, neither K1 nor K2 has produced adverse effects in individuals not on coumadin
derivatives. For this reason, the Institute of Medicine at the National Academy of Sciences chose not to set
a Tolerable Upper Limit (UL) for vitamin K when it revised its public health recommendations for this
vitamin in 2000.

Drug Interactions

Anticoagulant Medications

In patients on warfarin or other coumadin derivatives, vitamin K1 can interfere with these drugs’ anti-
clotting activity in amounts as small as 1 mg.

As noted above, oral anticoagulant medications, e.g., warfarin and other coumadin derivatives, promote
arterial calcification by preventing vitamin K from activating matrix Gla-protein. 15 88

These medications decrease clotting by blocking vitamin K epoxide reductase (VKOR), thus preventing
vitamin K recycling and greatly increasing risk of vitamin K deficiency, and have also been shown to block
the conversion of K1 to K2.89

A case report recommended physicians prescribing warfarin consider arterial calcification as a potential
consequence after routine examination of a healthy man on long-term warfarin treatment found his coronary
arteries were highly calcified. 90 Other case reports have noted pathologic tracheobronchial calcification with
long-term warfarin therapy in children, an 18-year-old male, and an elderly woman. 91 92 93 Two recent
studies involving more than 100 subjects have shown that patients treated with oral anticoagulants have
double the calcification of patients not on these vitamin K-blocking drugs.88

When improving vitamin K status, however, patients on these medications must be closely monitored. A
dose of just 1-2.5 mg of oral vitamin K1 reduces the range of the international normalized ratio (INR) from
5.0-9.0 to 2.0-5.0 within 24-48 hours; even eating a vitamin K-rich diet can make anticoagulant medications
less effective.94

On the other hand, recent studies have shown that the INR is more sensitive to vitamin K changes in patients
with a low vitamin K status than in those with a normal or high vitamin K status and that dietary vitamin K
intake in unstable patients is considerably lower than in stable patients.95 96 97

Research conducted by Schurgers et al., sugggests that MK-7 supplements supplying <50 mcg/day are not
likely to affect the INR value; however, doses of >50 mcg/day may interfere with oral anticoagulant
treatment in a clinically relevant way. A 50 mcg dose is comparable to the menaquinone content of 75 to
100 grams (2.6 to 3.5 ounces) of cheese, an amount that should lead to a disturbance of the INR value of no
more than 10%. In addition, the long half-life of MK-7 suggests that regular intake in combination with
properly adapted coumarin doses may result in more stable INR values.5

Other Interactions

K3, the synthetic form of vitamin K, promotes ROS production and glutathione depletion. High doses of K3
have been used in cancer research precisely for its ability to promote oxidative stress and cell death. Even in
lower doses, K3 has produced jaundice and hemolytic anemia in human infants. For these reasons, the U.S.
Food and Drug Administration banned the use of K3 in nutritional supplements.

Considerations when Choosing a Vitamin K Supplement

In animal studies, at very high intakes of K1, (200-fold the daily requirement of the liver), vitamin K1 is
converted to K2 (MK-4) in amounts that may be sufficient to help decrease arterial calcification. 98

It is important to differentiate between the two commercially available forms of K2 (the MK-4 and MK-7
menaquinones) since they differ in clinically significant ways.5 99 100 MK-4 is a short-chain menaquinone
available as a synthetic compound (menatetrenone), while MK-7, a long chain menaquinone, is a natural
menaquinone derived from natto fermentation.

The vast majority of studies evaluating the effectiveness of vitamin K for the prevention of both
osteoporosis and arterial calcification have used K2 (MK-4) at a dosage of 45 mg/day (specifically, 15
mg/tid). Not only has the majority of the research been done using MK-4, but MK-4 is the predominant
form of K2 into which the body converts K1. MK-4 appears quickly in the blood but has a half-life of only
1-2 hours, for which reason, high pharmacological doses (typically 45 mg/day given as 15 mg tid) are
necessary. Such large doses necessitate medical supervision in patients on blood-thinning medications (e.g.,
warfarin).

MK-7 is not only highly bioavailable and bioactive—45 mcg/day was sufficient to activate osteocalcin in
the Rotterdam study—but has a much longer serum half life of 3 days, which enables the body to build up a
buffer that can supply vitamin K2 to all tissues 24 hours a day. At 45 mcg/day (a dose 1,000 times less than
that typically used in the research for MK-4), natto-derived MK-7 is less likely to interact negatively with
blood-thinning medications.

Conclusion

As research documenting the widespread and significant beneficial actions of vitamin D continues to appear
in the peer-reviewed medical literature accompanied by reports that the majority of the U.S. population is
deficient in this nutrient, more clinicians are evaluating their patients’ vitamin D levels and prescribing
supplementation, often in amounts as high as 5,000 to 10,000 IU/day, without awareness of the risk of
provoking an imbalance among vitamins D, K and A. Consideration of the synergistic relationship among
these vitamins could allow vitamin D to be administered in doses of greater therapeutic value without
incurring the risks of osteoporosis and vascular calcification associated with hypervitaminosis D.

Read Part I: Vitamin D and Vitamin K Team Up to Lower CVD Risk:
Vitamin D Deficiency – a Non-Traditional Risk Factor for Cardiovascular Disease

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