Markers of vitamin K status

Coagulation assays such as prothrombin time lack the
sensitivity to detect subclinical vitamin K defi ciency.
More sensitive tests are based on the detection in plasma
of undercarboxylated species of vitamin K-dependent
proteins that are the product of protein synthesis
when either vitamin K is in low supply or its action is
blocked by antagonists. These species are sometimes
called PIVKA (proteins induced by vitamin K absence
or antagonism). Assays to measure undercarboxylated
species in plasma have been developed for two vitamin
K-dependent proteins, prothrombin and osteocalcin,
allowing independent assessment of two different
functional roles of vitamin K (Shearer, 1995a).
Sokoll & Sadowski (1996) evaluated biochemical
markers for assessing vitamin K nutritional status in
healthy adult humans and found that undercarboxylated
serum osteocalcin is the most sensitive marker.
Both serum native osteocalcin and undercarboxylated
osteocalcin can be quantitated by radioimmunoassay
using a rabbit polyclonal antibody raised against purifi
ed bovine bone osteocalcin (Sokoll et al., 1995). The
degree of γ-carboxylation of osteocalcin can also be assessed
by determining the in vitro binding capacity of
serum osteocalcin to hydroxyapatite (Jie et al. 1992).

10.5.4 Role of vitamin K in blood
coagulation
Background information can be found in Section
4.4.3.
The liver synthesizes a group of Gla proteins that
have a regulatory function in blood coagulation: factor
II (prothrombin) and factors VII, IX and X have
a coagulant function, while proteins C and S have an
anticoagulant function.
The chick bioassay for vitamin K is based upon the
degree of lowering of elevated blood clotting times
in vitamin K-depleted chicks. Blood clotting measurements
(actually prothrombin times) are rapidly
determined following the addition of a clotting agent
(thromboplastin) and calcium chloride solution to
oxalated or citrated blood. The chick is the animal of
choice because its vitamin K requirement is fi ve-fold
that of the rat, it is readily depleted of vitamin K, and
coprophagy (faecal recycling) is easier to control. The
chick’s higher requirement for vitamin K compared
with the rat is at least partly attributable to the short
length of its colon and rapid transit time.
Matschiner & Doisy (1966) determined the molar
activities of several forms of vitamin K using the
chick bioassay. Compared to phylloquinone, which
was arbitrarily assigned an activity of 100, MK-4 had
the highest activity (156) followed by MK-7 (122) and
MK-5 (116).

Role of vitamin K in bone
metabolism
Gla proteins occurring in bone
Three Gla proteins are found in bone tissue: osteocalcin
(also known as bone Gla protein), matrix Gla
protein and protein S. Osteocalcin is a relatively
small molecule (5.5 kDa) containing three Gla residues.
It is synthesized exclusively by osteoblasts and
odontoblasts and comprises about 15% to 20% of
non-collagen protein in bone. Approximately 20%
of the newly synthesized osteocalcin is not bound
to the hydroxyapatite matrix in bone, but is set free
in the bloodstream (Vermeer et al., 1995). Matrix
Gla protein is a larger molecule of 9.6 kDa containing
fi ve Gla residues. This protein is synthesized by
chondrocytes and is present in every cartilaginous
structure; it is expressed in developing bone prior
to ossifi cation. Little or nothing is known about the
precise functions of the Gla proteins. Osteocalcin has
been proposed as a specifi c regulator of the size of the
hydroxyapatite crystals in bone; it is also involved in
osteoclast recruitment (Robey & Boskey, 1996). Matrix
Gla protein inhibits inappropriate calcifi cation of
the epiphyseal (growth) plate (Olson, 1999). Protein S
has been identifi ed as a ligand of tyrosine kinase-type
receptors that modulate cell proliferation (Kohlmeier
et al., 1996). Children with inherited protein S defi -
ciency not only suffer from recurrent thrombosis, but
also have severely reduced bone mass (osteopenia)
(Pan et al., 1990).
Vitamin K status and osteoporosis
Individuals carrying the E4 allele of apoE experience
a higher incidence of bone fractures during their
lifetimes than do individuals without the E4 allele
(Kohlmeier et al., 1998). The increased risk of hip
and wrist fracture in women with the apoE4 allele
is not explained by bone density, impaired cognitive
function or falling (Cauley et al., 1999). This predisposition
toward bone fracture is consistent with
E3/E4 and E4/E4 phenotypes having lower plasma
phylloquinone levels than normal.
Vitamin K suffi ciency of the bone is related to the
degree of γ-carboxylation of osteocalcin and this in
turn is related to the plasma phylloquinone concentration.
As vitamin K intake decreases, circulating
osteocalcin seems to be the fi rst Gla protein to occur
in an undercarboxylated form (Vermeer et al., 1995).
Circulating osteocalcin is about 92% γ-carboxylated
in healthy young adults on a normal diet. Daily supplementation
with 250 μg of phylloquinone increases
osteocalcin carboxylation to 96%, while 1000-μg supplements
are required to achieve 100% carboxylation
(Binkley et al., 2002). These observations reveal that a
diet suffi cient to maintain normal clotting would not
be able to maximize γ-carboxylation of osteocalcin
and probably other vitamin K-dependent proteins.
It remains unknown whether maximal osteocalcin
carboxylation is necessary for optimal bone health.
Most studies have shown that the circulating levels
of total osteocalcin increase with ageing in normal
women, especially after the menopause. This increase
is likely to refl ect an increase in bone turnover, which
is associated with low bone mass in all skeletal regions
(Ravn et al., 1996). The γ-carboxylation of circulating
osteocalcin is signifi cantly impaired in women
over 80 years of age (Plantalech et al., 1991). Also in
elderly women, high concentrations of circulating
undercarboxylated osteocalcin is associated with low
hip bone mineral density (BMD) (Szulc et al., 1994)
and increased risk of hip fracture (Szulc et al., 1993,
1996; Vergnaud et al., 1997). Plasma levels of phylloquinone
and of the menaquinones MK-7 and MK-8
are depressed in elderly women within a few hours
of hip fracture, suggesting that vitamin K is sequestered
from the circulation for use at the fracture site
(Hodges et al., 1993). Vitamin K1 supplementation
(1000 μg per day) corrected undercarboxylation of
osteocalcin in postmenopausal women (Knapen et
al., 1989; Douglas et al., 1995) and decreased two
markers of bone resorption, urinary calcium and
hydroxyproline excretion (Knapen et al., 1989, 1993).
Booth et al. (1999) reported that 15 days of dietary
vitamin K depletion led to increased bone turnover as
measured by serum osteocalcin and urinary NTx (Ntelopeptides
of type I collagen) concentration. These
markers were subsequently normalized by 10 days of
phylloquinone repletion (200 μg per day). As elevated
bone turnover is associated with rapid bone loss,
vitamin K insuffi ciency would be expected to contribute
to the development of osteoporosis. However,
associations do not necessarily imply causation and
no direct evidence for the participation of decreased
plasma vitamin K in osteopenia in the elderly has been
reported.
In a prospective study involving 72 327 women
(Feskanich et al., 1999), dietary vitamin K intakes less
266 Vitamins: their role in the human body
than 109 μg per day were associated with an increased
risk of hip fracture. Booth et al. (2003) assessed dietary
vitamin K intake with a food-frequency questionnaire
in 1112 men and 1479 women (mean ± SD
age: 59 ± 9 years) and measured BMD of the hip and
spine. Women in the lowest quartile of vitamin K intake
(mean: 70.2 μg per day) had signifi cantly lower
BMD at the hip and spine than did those in the highest
quartile of intake (mean: 309 μg per day). No signifi -
cant association was found between dietary vitamin K
intake and BMD in men.
Tamatani et al. (1998) evaluated the possible participation
of circulating levels of testosterone, vitamin
D metabolites and vitamin K in osteopenia in elderly
men. No signifi cant correlation between plasma testosterone
and BMD was observed, despite the agerelated
decrease in plasma testosterone. However,
elderly men with decreased BMD showed signifi cant
decreases in the circulating levels of 25-hydroxyvitamin
D, phylloquinone and MK-7 compared with
elderly men with normal BMD.