Clin Chem Lab Med 2016; aop

Review

Maria Fusaro*, Maurizio Gallieni, Maria Antonietta Rizzo, Andrea Stucchi, Pierre Delanaye,
Etienne Cavalier, Rosa M.A. Moysés, Vanda Jorgetti, Giorgio Iervasi, Sandro Giannini,
Fabrizio Fabris, Andrea Aghi, Stefania Sella, Francesco Galli, Valentina Viola
and Mario Plebani

Vitamin K plasma levels determination in human

health
DOI 10.1515/cclm-2016-0783 measurement of vitamin K status in humans is still a criti-
Received June 19, 2016; accepted September 6, 2016 cal issue. Along with indirect assays, such as the under-
carboxylated fractions of vitamin K-dependent proteins
Abstract: Vitamin K (phylloquinone or vitamin K1 and
[prothrombin, osteocalcin (OC), and matrix gla protein],
menaquinones or vitamin K2) plays an important role as a
the direct analysis of blood levels of phylloquinone and
cofactor in the synthesis of hepatic blood coagulation pro-
menaquinones forms might be considered a more inform-
teins, but recently has also aroused an increasing interest
ative and direct method for assessing vitamin K status.
for its action in extra-hepatic tissues, in particular in the
Different methods for direct quantification of vitamin K
regulation of bone and vascular metabolism. The accurate
serum levels are available. High-performance liquid chro-
matography (HPLC) methods coupled with post-column
reduction procedures and fluorimetric or electrochemical
*Corresponding author: Maria Fusaro, National Research Council detection are commonly used for food and blood analysis
(CNR) – Institute of Clinical Physiology (IFC), Pisa Via G. Moruzzi 1, of phylloquinone, but they show some limitations when
56124, Pisa, PI, Italy; and Department of Medicine, University of
applied to the analysis of serum menaquinones because
Padova Italy; Via Giustiniani 2, 35128, Padova, PD, Italy,
E-mail: [email protected]
of interferences from triglycerides. Recent advancements
Maurizio Gallieni: Nephrology and Dialysis Unit, Ospedale San include liquid chromatography tandem mass spectrome-
Carlo Borromeo, Department of Clinical and Biomedical Sciences try (LCMS/MS) detection, which assures higher specificity.
“Luigi Sacco”, University of Milan, Milan, Italy The optimization and standardization of these methods
Maria Antonietta Rizzo: Nephrology and Dialysis Unit, Ospedale di requires specialized laboratories. The variability of results
Circolo di Busto Arsizio, ASST Valle Olona, Italy
observed in the available studies suggests the need for
Andrea Stucchi: Nephrology and Dialysis Unit, IRCCS Multimedica,
Sesto San Giovanni (Milano), Milan, Italy further investigations to obtain more accurate analytical
Pierre Delanaye: Department of Nephrology, Dialysis, and results.
Transplantation, University of Liège, Centre Hospitalier Universitaire
du Sart Tilman (ULg CHU), Liège, Belgium Keywords: human health; metabolism; plasma levels;
Etienne Cavalier: Department of Clinical Chemistry, University vitamin K.
of Liège, Centre Hospitalier Universitaire du Sart Tilman, Liège,
Belgium
Rosa M. A. Moysés: Universidade Nove de Julho, UNINOVE,
São Paulo, Brazil; and Universidade de Sao Paulo, São Paulo, Brazil Introduction
Vanda Jorgetti: Universidade de Sao Paulo, São Paulo, Brazil
Giorgio Iervasi: Institute of Clinical Physiology, National Council of Vitamin K has important biological actions, some of which
Research, Pisa, Italy
are still being discovered. Vitamin K plays a key role in
Sandro Giannini, Fabrizio Fabris, Andrea Aghi and Stefania Sella:
Department of Medicine, Clinica Medica 1, University of Padova, the synthesis of several blood coagulation factors, but it is
Padova, Italy also strongly connected to bone metabolism and vascular
Francesco Galli: Department of Pharmaceutical Sciences, University calcifications [1].
of Perugia, Italy Vitamin K exists in two main natural forms: K1 (or
Valentina Viola: Azienda Ospedaliera Universitaria Policlinico Tor
phylloquinone, PK) and K2 (including several differ-
Vergata, Rome, Italy
Mario Plebani: Laboratory Medicine Unit, Department of Medicine,
ent vitamers called menaquinones, MKs) (Figure  1). In
University of Padova, Padova, Italy. http://orcid.org/0000-0002- addition to the naturally occurring phylloquinone and
0270-1711 menaquinones, there is as a synthetic form of vitamin K:

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2      Fusaro et al.: Vitamin K plasma levels determination in human health

MK-7 through MK-10, which are synthesized by bacteria in

humans.
The predominant dietary form of vitamin K in the
USA, Europe, and most Western countries is phylloqui-
none, while the major form in Japan is menaquinones,
especially menaquinone 7 (MK-7), which is a component
of natto [6]. Natto is most popular in the eastern regions
of Japan, including Kanto, Tohoku, and Hokkaido, but it
is consumed in all areas.
Different phylloquinone and menaquinones plasma
concentrations have been reported, with a possible influ-
ence of dietary intake, and a possible analytic interfer-
ence from triglycerides [7]. Mean plasma concentrations
ranging from 0.22 to 8.88 nmol/L have been reported,
although in most studies phylloquinone had a concentra-
tion below 2 nmol/L [8].
Daily intake of vitamin K in a Western diet range is
estimated from 60 μg to 200 μg, of which phylloqui-
none is the larger component (about 90%, versus 10% of
menaquinones) [9].
Recommendations for daily intake of vitamin K are
inconsistent. The Institute of Medicine (US) Panel on
Micronutrients proposed an adequate intake (AI) for men
and women of 120 and 90 μg/day, respectively, based on
representative dietary intake data from healthy individu-
als, because of the lack of data to estimate an average
requirement [10]. Considering that no adverse effect has
been reported for individuals consuming higher amounts
Figure 1: Structures of phylloquinone (vitamin K1) and menaqui-
nones (MKs or vitamin K2). of vitamin K, a tolerable upper intake level was not estab-
Menadione (vitamin K3) represents the basic structure common to lished. In 2012, the Italian LARN (Reference Assumption
K1 and K2. Menadione is available as a synthetic form of vitamin K. Levels for Nutrients and Energy), proposed by the Human
Nutrition Italian Society (SINU), suggested an intake of
vitamin K stratified for age (140 or 170 μg/day for 18–59
menadione, or vitamin K3 (2-methyl-1, 4-naphthoquinone and > 60  years old, respectively). However, in the 2014
nucleus), representing the basic structure common to release of the same recommendations this group stated
phylloquinone and menaquinones. that the available evidence does not allow to define the
Phylloquinone is mainly found in green vegeta- adequate intake for vitamin K [11]. In the UK, a government
bles. The source of menaquinones is more uncertain, backed panel of experts pointed out that although vitamin
and although the contribution of the intestinal bacte- K is known to be essential, recommendations on adequate
rial flora to vitamin K status is still incompletely under- nutritional intakes have not been precisely established,
stood, the literature indicates that menaquinones are because of the unquantified contribution made by the
derived mainly from intestinal bacteria [2, 3] and fer- intestinal bacteria [12]. They reported previous data of
mented food (e.g. cheeses and the Japanese soybean daily intake suggested by the UK Department of Health’s
product known as ‘natto’) [3]. Liver is also a rich source Committee on Medical Aspects of Food Policy (COMA): 1
of menaquinones [4]. μg/kg body weight [13], which is probably adequate for
Menaquinones are classified according to the length blood clotting, but suboptimal for bone health. Vitamin
of their unsaturated side chains. Up to 12 different types K is required to introduce carboxyl groups into glutamic
have been described, from MK-4 to MK-15 [5]. The most acid residues in four of the blood coagulation factors
common MKs in humans are the short-chain MK-4, which (II, VII, IX, X) to yield γ-glutamic carboxyl (Gla) residues
is the only MK produced by systemic conversion of phyl- (Figure 2), while vitamin K hydroquinone is transformed
loquinone to menaquinones, and the long-chain vitamers, into vitamin K 2,3-epoxide [14]. Importantly, warfarin

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 Fusaro et al.: Vitamin K plasma levels determination in human health      3

ucVKD-Proteins cVKD-Proteins COOH

COOH Gla CH
CH2 Glu Vitamin K
dependent COOH
carboxylase

Vitamin K
Hydroquinone Vitamin K
(KH2) 2,3 Epoxide (KO)
Quinone
reductase
in
far
ar W
Disulfide W ar
Vitamin K fa
r in
NADP+ rin Quinone Dithiol
arfa
W Dithiol
Disulfide Vitamin K-epoxide
reductase

NADPH

Figure 2: The vitamin K cycle and the interference of warfarin with vitamin K recycling.
Gla-proteins are carboxylated by a specific vitamin K dependent carboxylase, through several rounds of carboxylation. Here a single round of
carboxylation is depicted. (Reprinted with permission from Fusaro et al. [15].) ucVKD-proteins, ­undercarboxylated vitamin K dependent proteins.

interferes with regeneration of vitamin K 2,3-epoxide to femoral and tibial articular cartilages in PXR knockout
vitamin K hydroquinone, thus impairing γ-carboxylation mice, resulting in aging-dependent wearing of knee joints
and the activity of vitamin K dependent proteins, includ- articular cartilage. These findings may indicate that SXR/
ing the extra-hepatic proteins OC (bone Gla-protein; BGP) PXR protects against aging-dependent wearing of articu-
and matrix Gla-protein (MGP), respectively involved in lar cartilage and that ligands for SXR/PXR have a poten-
bone mineralization and inhibition of vascular calcifi- tial role in preventing osteo-articular diseases caused by
cations. If vitamin K is deficient, vitamin K dependent aging [19].
proteins cannot increase their carboxylation status (and
they become significantly undercarboxylated), losing
their capacity to bind calcium, so that bone metabolism
may be impaired and the process of vascular calcification
Metabolism of vitamin K: general
enhanced [15]. principles
Both phylloquinone and MKs may activate the steroid
and xenobiotic receptor (SXR). SXR is a nuclear receptor Most of our knowledge on the metabolism of vitamin
involved in the transcriptional regulation of enzymes such K  –  in particular about its intestinal absorption, trans-
as cytochrome P450 (in particular the CYP3A4 isoform) port, cellular uptake and catabolism – is related to phyl-
[16]. SXR and its murine ortholog, pregnane X receptor loquinone, while data about menaquinones are more
(PXR), are nuclear receptors that are expressed at high limited [2]. Vitamin K forms derived from plants and bac-
levels in the liver and the intestine. They work as xenobi- teria have a poorer bioavailability than those contained
otic sensors that induce expression of genes involved in in oil-based and processed foods. Low plasma concen-
detoxification and drug excretion, but they were recently trations of phylloquinone reflect low tissue reserves.
shown to be also expressed in osteoblasts and involved Vitamin K tissue concentrations have been determined.
in bone metabolism [17]. Hydroxylation of vitamin K is In adults, phylloquinone concentrations are about 10
catalyzed by cytochrome P450 enzymes, which often are pmol/g-wet tissue in liver, hearth and pancreas, while
induced by their substrates themselves via the activation they are lower in brain, kidney and lung (> 2 pmol/g).
of the nuclear receptor PXR [18]. Contrariwise, high MK-4 concentrations were found in
Azuma et  al. described the osteopenic phenotype of human brain and kidney (6 pmol/g) and even higher in
systemic PXR knockout mice; they observed a remark- pancreas (22  pmol/g) than in other tissues, such as in
able reduction of width and an important gap between plasma and liver [20].

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4      Fusaro et al.: Vitamin K plasma levels determination in human health

Considering that MK-4 derives from the endogenous The liver is also the site of vitamin K catabolic pathway,
conversion of phylloquinone, the specific tissue distribu- common to phylloquinone and MKs. The poly-isoprenoid
tion of MK-4 is suggestive of local synthesis from phyl- side chains are shortened, undergoing ω-oxidation fol-
loquinone. MK-4 is synthetized in testes, pancreas and lowed by β-oxidation leading to two major aglycone metab-
blood vessels. In particular, the conversion of phylloqui- olites with side chain lengths of five and seven carbon
none into MK-4 occurs either directly or by interconver- atoms (5C and 7C metabolites, respectively). Finally, after
sion to menadione (K3), followed by prenylation to MK-4 conjugation with glucuronic acid, the metabolites are
[21]. Therefore, the biological activity of K3 is entirely excreted in the bile and urine, mainly as glucuronides [2].
dependent on its prenylation to MK-4.
However, the molecular mechanisms of conversion
remain unclear. Nakagawa et al. identified a human MK-4
biosynthetic enzyme known as UbiA prenyltransferase
Review of the assessment of
containing 1 (UBIAD1), by screening the human genome vitamin K status and the possible
database. UBIAD1 is localized in the endoplasmic reticu-
lum and ubiquitously expressed in several mouse tissues.
role of vitamin K plasma levels
Short interfering RNA against the UBIAD1 gene inhibited measurement
the conversion of deuterium-labeled phylloquinone mol-
ecules into deuterium-labeled-MK-4 (MK-4-d7) in human One barrier to gaining a better understanding of vitamin
cells. An additional proof that the UBIAD1 gene encodes K function has been the difficulty in developing methods
an MK-4 biosynthetic enzyme derives from its expres- for the measurement of vitamin K. Vitamin K was the last
sion in cells infected with UBIAD1 baculovirus, which of the four fat-soluble vitamins to be measured at endog-
can convert deuterium-labeled vitamin K derivatives into enous levels. The degree of this analytical challenge is a
MK-4-d7 [22]. consequence of vitamin K being the most lipophilic and
Phylloquinone is probably converted into MK-4 within least abundant of the fat soluble vitamins. These proper-
the tissues themselves, rather than via hepatic meta- ties do not easily lend themselves to the development of
bolism. After phylloquinone administration, the MK-4 assays suitable for current generation automated chemis-
concentration increased much more slowly in each of the try platforms, unlike vitamin D.
tissues than that of phylloquinone, and the MK-4 concen- Vitamin K status can be assessed by indirect func-
tration in plasma and liver reached much lower levels tional tests such as the prothrombin time or by measure-
than those observed in other tissues [23]. ment of undercarboxylated proteins (Table 1), such as OC
Vitamin K is transported in plasma by lipoproteins and matrix Gla protein (MGP), which are more sensitive
[24]. After digestion in the intestinal tract, dietary vitamin in detecting subclinical vitamin K deficiency than pro-
K and triglycerides (TG) are emulsified by bile salts to form thrombin time [28]. The amount of undercarboxylated
mixed micelles in the enterocytes and processed into chy- OC could represent a sensitive marker of vitamin K status
lomicrons (CR), containing apolipoprotein A (apoA) and in humans [29]. However, although a high percentage of
apoB, and then secreted into the lymph ducts and blood undercarboxylated OC indicates poor vitamin K status,
circulation. CR are modified peripherally, in adipose or this value better reflects recent vitamin K intake and not
muscular tissues, by the action of lipoprotein lipase (LPL) the long-term vitamin K status.
and re-enter in the circulation, but they continue trans- Osteocalcin levels are also influenced by vitamin D,
porting vitamin K in their lipophilic core [25]. which is required for the production of undercarboxy-
The uptake of vitamin K into the liver seems to follow lated OC, whereas vitamin K is required for the conversion
the same pathway of lipoprotein [26]. In fact, CR enter into of undercarboxylated to mature OC. Data from healthy
the liver by endocytosis and they are processed to finally volunteers indicate a weak but significant correlation
obtain smaller LDL molecules; vitamin K is presumed to between undercarboxylated OC and vitamin D, suggest-
remain still located in the lipophilic core of lipoproteins. ing that the serum level of undercarboxylated OC may
Concerning the uptake of vitamin K into bone tissue be an insufficiently reliable marker for vitamin K status
[27], it is known that osteoblasts obtain most of their phyl- [30]. OC is also influenced by PTH, which is significantly
loquinone by CR pathway and most of their MK-7 by LDL elevated in many CKD patients. Therefore, CKD patients
pathway. Osteoblasts express lipoprotein receptors, which with hyperparathyroidism will present high serum uOC,
interact with CR and LDL and start the process of endocy- but this does not necessarily mean that they are vitamin
tosis of the particles and the vitamin K. K deficient.

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 Fusaro et al.: Vitamin K plasma levels determination in human health      5

Table 1: Measurement of vitamin K status in humans: summary of direct and indirect methods.

Methods   Main characteristics

Indirect methods
 Prothrombin time   Cannot be used as a reliable indicator of vitamin K status
 Undercarboxylated osteocalcin (ucOC) or matrix Gla protein   Vitamin K deficiency is associated with reduced carboxylation of vitamin
(ucMGP) K dependent proteins and higher levels of ucOC and ucMGP. Vitamin D
regulates osteocalcin gene expression
 PIVKA-II   Elevated in vitamin K deficiency
 Urinary vitamin K metabolites (7C-aglycone and 5C-aglycone)   Vitamin K metabolites mainly tested in pediatric population
Direct methods
 High-performance liquid chromatography (HPLC) with   Lower sensitivity and selectivity
ultraviolet (UV) detection
 HPLC with fluorescence detection   Provides greater sensitivity and selectivity than UV detection. Most
common method used in laboratories
 HPLC with electrochemical detection (ECD)   Post-column reduction is used to convert the quinone structure of vitamin K
in the corresponding hydroquinones, measured in oxidation mode
 Liquid chromatography tandem mass spectrometry   Provides higher sensitivity and selectivity in comparison with other
(LC-APCI-MS/MS) techniques

Undercaboxylated unphosphorylated MGP after prophylaxis. Infants mainly excreted 5C-aglycone,

(ucdpMGP), the inactive form of MGP, may be a good while increased excretion of the 7C-aglycone was associ-
alternative to evaluated vitamin K status of the subjects; ated with metabolic overload because of the exposure to
Indeed, randomized controlled studies have shown that high-tissue phylloquinone concentrations. Measurement
vitamin K therapy decreases ucdpMGP levels [31–34]. of the 5C- and 7C-aglycones was therefore proposed as a
Moreover, AVK treatment increased the amount of inactive good approach to study vitamin K status in neonates and
ucdpMGP [35, 36]. Stopping that treatment has also been as an aid the development of improved prophylactic regi-
shown to decrease ucdpMGP [37]. All these date suggest mens [40]. Urinary vitamin K metabolites have not been
that ucdpMGO could be a good marker of vitamin K status. studied in CKD patients, but deterioration of renal func-
However, ucdpMGP determination is only available on the tion might interfere with the reliability of this marker of
automated IDS iSYS instrument, which is not available vitamin K status.
everywhere. Moreover, measuring inactive MGP may only
reflect the vitamin K status at the vascular level and not
at other organ’s levels (in particular bone and liver). In
this context, direct measurement of vitamin K could be of
Determination of vitamin K plasma
interest by its own, or in combination with ucdpMGP. levels: technical aspects
Moreover, protein induced in vitamin K absence
(PIVKA-II) could be a sensitive marker to predict mild Providing a standardized measurement of plasma levels of
vitamin K deficiency, as reported among newborns. vitamin K forms, in particular for some MKs, is challeng-
PIVKA-II is an inactive precursor of prothrombin and is ing. Measuring vitamin K in plasma is difficult because
elevated in vitamin K deficiency [38]. of the low circulating vitamin K levels and the non-polar
Urinary vitamin K metabolites can be measured in characteristics of vitamin K as well as the interference of
the urine. Harrington et al. tested this analytic approach lipids [41].
in young adults [39] and studied its clinical significance Different methods for direct quantification of
in the pediatric population [40]. Urinary excretion of vitamin K serum levels have been developed and evalu-
7C-aglycone and 5C-aglycone, vitamin K metabolites ated (Table  1). High-performance liquid chromatography
common to both phylloquinone and the menaquinone (HPLC) is considered the elective technique to measure
series, were assessed following restriction or supple- vitamin K subtypes.
mentation with phylloquinone. Results indicate a good HPLC has been developed starting from the second
relationship of urinary metabolites with dietary phylloqui- half of the last century [42], then extended to the meas-
none intake. In newborns, vitamin K urinary metabolites urement of long-chain MKs in the late 1980s [43]. Ini-
excretion was 25 times lower than adults and improved tially, HPLC with ultraviolet detection (UV) was used to

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6      Fusaro et al.: Vitamin K plasma levels determination in human health

determinate PK plasma concentration. Afterwards, HPLC low vitamin K concentrations [51]. During ECD, post-col-
with fluorescence after post column reduction demon- umn reduction is used to convert the quinone structure of
strated greater sensitivity and selectivity than UV method vitamin K in the corresponding hydroquinones, measured
[44, 45], but it required extensive sample pre-purification in oxidation mode. This reduction is operated by zinc or
to reduce the chromatographic interference induced by platinum. Reduction can also be achieved electrochemi-
lipids [41]. cally, through a dual electrode approach [52].
Other recent methods for determination of PK and In the VIKI study [53], taking into account the con-
MKs are based on liquid chromatography tandem mass nection between the lipid plasma content and the chro-
spectrometry (LCMS/MS), which present higher sensi- matography resolution, the authors who performed the
tivity and selectivity, but they require longer analytical laboratory analyses used a simple, sensitive and selective
times. Suhara et al. [46] developed a model of liquid chro- reversed phase HPLC method for determination of vitamin
matography-tandem mass spectrometry (LC-APCI-MS/MS) K subtypes in human plasma. The method followed the
method for the measurement of vitamin K plasma levels principle of redox mode electrochemical detection based
(PK, MK-4, and MK-7), characterized by high sensitivity on Wakabayashi’s technology [52]. It is characterized by a
and selectivity. However, the method is excessively long liquid-liquid extraction, followed by a solid-phase extrac-
for routine analysis, because of the pre-purification pro- tion of human plasma using polymeric reversed phase
cedure is based on dual step extraction, chromatographic cartridges; the MKs were measured by an electrochemi-
separation followed by a wash and re-equilibration period. cal detector after post-column reduction with platinum
Gentili et al. simplified the process and reduced the total on alumina powder and using the MK-8 form as internal
run-time [47]. With this method, MK-4 and MK-7 levels standard. The method was able to reduce the percent-
were undetectedable in the analyzed serum samples of age of bad chromatogram resolution in plasma of dialy-
subjects on a Mediterranean diet [47]. Karl et al. [48] devel- sis patients, which is often characterized by increased
oped a method employing HPLC-mass spectrometry with total cholesterol and triglycerides concentrations. The
atmospheric pressure chemical ionization (LC-APCI-MS) authors also observed that some vitamer concentrations
for simultaneous quantification of 11 vitamin K vitamers, resulted higher than the normal reference value previ-
which was applied to biological samples such as serum, ously reported in literature [53].
faeces and food. They suggested that the method can be Because of the complexity of plasma vitamin K
applied in human and animal studies examining the role determination and of possible analytical errors, external
of vitamin K vitamers derived from the diet and gut bacte- quality assurance services could be useful to verify and
ria synthesis in health and disease. harmonize results from different laboratories. One exter-
Riphagen et  al. [49] also described a method based nal quality assurance service (KEQAS) for circulating
on LC-APCI-MS/MS with atmospheric pressure chemical phylloquinone analysis is already active [54].
ionization to detect plasma PK, MK-4 and MK-7 levels, sim-
plifying the sample pre-purification process and reducing
the total run-time without compromising sensitivity and
selectivity. In a study population of 60 renal transplant
Determination of vitamin K levels:
recipients, the authors confirmed that plasma PK con- clinical issues
centrations were significantly associated with recent PK
dietary intake and plasma MK-4 concentrations, and that A large variability of vitamin K levels has been observed
plasma vitamin K levels were strongly correlated with in humans [8]. In addition to the analytical variability,
plasma triglyceride concentrations [49]. dietary and individual factors influence plasma levels
It is known that vitamin K is mainly transported by of vitamin K subtypes. Most studies assessed circulating
triglyceride-rich lipoproteins [50]. This is a critical point to vitamin K in relation to dietary vitamin K intake, and a
consider for obtaining a reliable determination of vitamin smaller but significant number of studies evaluated the
K serum levels, because the interaction of the lipoprotein association of vitamin K levels with chronic diseases. Cur-
and the HPLC-column may affect the sample purification rently, there is not a consensus on a plasma vitamin K
and measurement. level indicating deficiency or insufficiency. Similarly, it is
Considering that UV and fluorimetric detectors appear not clear which vitamer should be considered as reference
to be largely inadequate, selective and sensitive analysis of for determining the vitamin K status. Most studies have
all the vitamin K subtypes can be also achieved by electro- addressed the coagulation system, but other outcomes,
chemical detection (ECD), using small samples containing especially bone metabolism and vascular calcifications,

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 Fusaro et al.: Vitamin K plasma levels determination in human health      7

are clinically relevant and might behave differently with evidence of vitamin K biological actions in bone metab-
respect to plasma PK or MKs. olism and cardiovascular disease, exceeding its better
Data obtained in healthy subjects and osteoporotic known involvement in the blood coagulation system [59],
patients supplemented with MK-4 showed a large vari- as reported in Table 3.
ability of vitamin K levels. In healthy subjects, levels of Several studies suggest that low vitamin K levels are
MK-4, PK and MK-7 (reported as ng/mL and mean ± SD) related to osteoporosis, pathological fractures and vas-
were 0.15 ± 0.17, 1.81 ± 1.10 and 16.27 ± 20.58, respectively, cular calcifications. Supplementing MK-7 at the dose of
while in osteoporotic patients receiving MK-4, these levels at least 200 μg per day might help protecting from vas-
were 46.83 ± 46.41, 0.62 ± 0.25 and 4.18 ± 6.28, respectively cular calcification, osteoporosis and cancer [60]. More-
[42]. The influence of supplementation on MK-4 levels was over, supplementation of 5  mg daily phylloquinone in
also observed in another study [55], in contrast with the 440 postmenopausal women with osteopenia for 2 years
low MK-4 bioavailability reported in humans by Sato [56]. in a randomized, placebo-controlled, double-blind trial
Variability of menaquinones levels has also been caused a > 50% reduction in clinical fractures vs. placebo,
reported in a Japanese study in post menopausal women although no protection against the age-related decline in
(5.26 ± 6.13 ng/mL in the Tokyo area, where natto is largely bone mineral density was observed [61].
consumed vs. 1.22 ± 1.85 in the western Japanese area), A meta-analysis has shown that in seven Japanese
indicating that the geographic difference in MK-7 levels trials reporting fractures, menaquinones administration
may be ascribed to natto intake. In this study, the authors significantly reduced the risk of hip (77% reduction), ver-
observed an inverse correlation between natto consump- tebral (60% reduction) and all non-vertebral fractures
tion and fracture risk, suggesting the possibility that natto (81% reduction) [62].
intake might contribute to reduce fractures by increasing Vitamin K administration also significantly delayed
MK-7 levels [6]. the progression of coronary artery calcifications and the
Other authors measured phylloquinone levels, dem- deterioration of arterial elasticity [63]. A lower risk of
onstrating that vitamin K deficiency affects 24% of the coronary heart disease and severe aortic calcifications
general population and 29% of hemodialysis patients was observed with higher menaquinones intake, but not
[57, 58]. with phylloquinone intake. This finding suggests that the
The impact of vitamin K on human health (Table  2) dietary phylloquinone intake, without menaquinones,
has become more and more relevant, considering the may not be sufficient to suppress arterial calcifications
[64, 65].
Table 2: Main vitamin K actions in humans.
Menaquinones have been shown to play an important
role also in cancer. In a small (40 patients) randomized
– Regulation of blood coagulation activity study the administration of menaquinones 45  mg/day
– Bone protection; prevention of osteoporosis and bone fracture reduced the development of hepatocellular carcinoma in
– Prevention of vascular calcifications patients with liver cirrhosis: the risk ratio for the devel-
– Prevention of cancer opment of hepatocellular carcinoma in patients given
– Prevention of inflammation
menaquinones was 0.13 [66].

Table 3: Consequences of inhibition of vitamin K-dependent proteins.

Coagulation   Prothrombin (factor II)   Bleeding

  VII, IX, X factors  
  Protein C, S, Z  
Vessels   MGP   Vascular calcifications
  Osteocalcin  
  GAS-6  
Bone   Osteocalcin   Inadequate bone mineralization
  MGP   Bone fractures
  Periostin  
Cellular proliferation   GAS-6   Action on cellular proliferation, cellular adhesion, inhibition of apoptosis
Others: Inflammation   Not defined   Increase of inflammation

MGP, matrix Gla protein; BGP, bone Gla protein; Gas, growth arrest specific gene.

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8      Fusaro et al.: Vitamin K plasma levels determination in human health

Menaquinones have additional properties in certain opening interesting perspectives for research in human
cell and tissue types, particularly in bone tissue and in the health.
immune system. Much of the available evidence relates Finally, vitamin K status was found to be inversely and
specifically to MK-4, which was found to have a role in significantly related to individual inflammatory markers
bone health since the 1990s. Low circulating levels of and to the inflammatory process in a human population
menaquinones are associated with osteoporotic fractures study based on the Framingham Offspring Study cohort
in the elderly [67], and menaquinones improved bone [77]. This finding is supported by studies on rats demon-
mineral density in Japanese women [68]. In an experi- strating that animals with vitamin K-deficient diets had an
mental setting, MK-4 reduced bone losses caused by enhanced expression of genes involved in acute inflam-
either oestrogen withdrawal or corticosteroid treatment matory response compared to those with normal or phyl-
in experimental model on rats [69, 70]. Moreover, other in loquinone-supplemented diets and that a supplemented
vitro studies showed that MK-4 inhibits the synthesis of diet suppressed the inflammatory response [78].
prostaglandin E2 (PGE2), a bone reabsorption-inducing
agent, in cultured osteoblasts [71], and inhibits the for-
mation of osteoclast-like cells in bone marrow-derived
cultures [72]. Finally, experimental data suggests a possi- Conclusions
ble role of MK-4 on pancreatic exocrine cells metabolism.
Stimulation of pancreatic acinar cells with secretagogues There is no homogeneity of data about vitamin K plasma
cholecystokinin-8 and secretin induces secretion of MK-4, levels in healthy subjects. As dietary analytical, nutri-
along with phospholipase and the membrane trafficking tional and metabolic factors play an important role,
protein caveolin-1 [73], although a well-defined function further investigations are necessary to provide more infor-
of MK-4 in this setting remains unclear. mation on vitamin K status.
The frequent use of warfarin enhances the problem In this review, we analyzed the literature regarding
of vitamin K deficiency and its role on bone and vascu- the validity of direct measurement of vitamin K levels.
lar disease [74]. Warfarin may predispose to bone frac- Vitamin K is a molecule with non-polar characteristics and
tures and vascular calcification by different mechanisms: lipids, in particular triglycerides, interfere with vitamin K
directly, by inhibition of γ-carboxylation of OC and other measurement. Sample preparation for vitamin K analysis
bone matrix proteins; indirectly, because patients treated remains difficult and requires highly specialized laborato-
with warfarin may limit their dietary intake of foods rich ries. In addition, circulating vitamin K levels are markedly
in vitamin K. New oral anticoagulant seems to have less lower than those of other lipophilic vitamins.
influence on bone metabolism, but their long-term effects Indeed, not necessarily direct measurement of vitamin
need more studies [75, 76]. K plasma levels are superior to the assessment of vitamin
In the VIKI study [53], a comprehensive assessment of K status through measurement of undercarboxylated frac-
vitamin K status was carried out in a cohort of hemodialysis tions of vitamin K dependent proteins, which could better
patients and in healthy controls, including most vitamin K represent the functional status of the protein. However,
subtypes (in particular PK, MK-4, MK-5, MK-6, and MK-7), if we recognize that phylloquinone and menaquinones
adjusted for triglycerides levels. Vitamin K deficiency was have different actions, carboxylation status of vitamin K
found in 35.4% of hemodialysis patients for MK-7, 23.5% dependent proteins might not distinguish the respective
for PK and 14.5% for MK-4. With the limitations of its roles of the two forms of vitamin K. Thus, developing better
observational nature, this is the first study to relate phyl- analytical techniques for direct vitamin K determination
loquinone and menaquinones deficiency directly both to is certainly desirable for improving selective therapies for
vertebral fractures and vascular calcification in the dialy- menaquinones associated bone and vascular diseases.
sis population. In particular, phylloquinone deficiency Currently, an effective analytical method for assess-
was the strongest predictor of vertebral fractures, while ing circulating vitamin K appears to be the one described
lower MK-4 and MK-7 levels were associated with vas- by Riphagen et al. [49], a liquid chromatography tandem
cular calcification. The results in ­hemodialysis patients mass spectrometry method for determination of three
may point out a possible role of vitamin K deficiency as vitamers (PK, MK-4, and MK-7) with a simplified sample
a cause of bone and vascular disease also in the general pre-purification process and reduced total run-time.
population. We can hypothesize that a diet rich in vitamin Hopefully, this assay should avoid the interference by cir-
K and/or vitamin K supplements might be of help in pre- culating triglycerides and should allow the identification
venting bone disease and avoiding vascular calcifications, of all vitamin K subtypes.

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 Fusaro et al.: Vitamin K plasma levels determination in human health      9

Further efforts are needed for developing a single, 13. Committee on Medical Aspects of Food and Nutrition Policy
rapid and standardized method for evaluating vitamin K (COMA). Dietary reference values for food energy and nutrients
for the United Kingdom. Report of the panel on dietary reference
plasma levels.
values. HMSO, London; 1991.
14. Oldenburg J, Marinova M, Müller-Reible C, Watzka M. The vita-
Author contributions: All the authors have accepted min K cycle. Vitam Horm 2008;78:35–62.
responsibility for the entire content of this submitted 15. Fusaro M, Crepaldi G, Maggi S, Galli F, D’Angelo A, Calò L, et al.
manuscript and approved submission. Vitamin K, bone fractures, and vascular calcifications in chronic
kidney disease: an important but poorly studied relationship.
Research funding: None declared.
J Endocrinol Invest 2011;34:317–23.
Employment or leadership: None declared.
16. Traber MG. Vitamin E and K interactions: a 50-year-old problem.
Honorarium: None declared. Nutr Rev 2008;66:624–9.
Competing interests: The funding organization(s) played 17. Tabb MM, Sun A, Zhou C, Grün F, Errandi J, Romero K, et al.
no role in the study design; in the collection, analysis, and Vitamin K2 regulation of bone homeostasis is mediated
interpretation of data; in the writing of the report; or in the by the steroid and xenobiotic receptor SXR. J Biol Chem
2003;278:43919–27.
decision to submit the report for publication.
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and gene activating routes for vitamins E and K. Mol Aspects
Med 2003;24:337–44.
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