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Determination of phylloquinone and menaquinones in food. Effect of food matrix on circulating vitamin K concentrations

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Fluctuations in international normalized ratio values are often ascribed to dietary changes in vitamin K intake. Here we present a database with vitamin K(1) and K(2) contents of a wide variety of food items. K(1) was mainly present in green vegetables and plant margarins, K(2) in meat, liver, butter, egg yolk, natto, cheese and curd cheese. To investigate the effect of the food matrix on vitamin K bioavailability, 6 healthy male volunteers consumed either a detergent-solubilized K(1) (3.5 micromol) or a meal consisting 400 g of spinach (3.5 micromol K(1)) and 200 g of natto (3.1 micromol K(2)). The absorption of pure K(1) was faster than that of food-bound K vitamins (serum peak values at 4 h vs. 6 h after ingestion). Moreover, circulating K(2) concentrations after the consumption of natto were about 10 times higher than those of K(1) after eating spinach. It is concluded that the contribution of K(2) vitamins (menaquinones) to the human vitamin K status is presently underestimated, and that their potential interference with oral anticoagulant treatment needs to be investigated.
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Original Paper
Haemostasis 2000;30:298–307
Determination of Phylloquinone and
Menaquinones in Food
Effect of Food Matrix on Circulating Vitamin K Concentrations
Leon J. Schurgers Cees Vermeer
Department of Biochemistry and Cardiovascular Research Institute, Maastricht University,
Maastricht, The Netherlands
Received: July 27, 2000
Accepted in revised form: September 27, 2000
Cees Vermeer, PhD
Department of Biochemistry, University of Maastricht
PO Box 616, NL–6200 MD Maastricht (The Netherlands)
Tel. +31 43 388 1682, Fax +31 43 388 4160
E-Mail c.vermeer@bioch.unimaas.nl
ABC
Fax + 41 61 306 12 34
E-Mail karger@karger.ch
www.karger.com
© 2001 S. Karger AG, Basel
0301–0147/00/0306–0298$17.50/0
Accessible online at:
www.karger.com/journals/hae
Key Words
Phylloquinone
W Menaquinone W Vitamin K W
Food composition W Bioavailability W
Anticoagulant, oral
Abstract
Fluctuations in international normalized ratio
values are often ascribed to dietary changes
in vitamin K intake. Here we present a data-
base with vitamin K
1
and K
2
contents of a
wide variety of food items. K
1
was mainly
present in green vegetables and plant mar-
garins, K
2
in meat, liver, butter, egg yolk, nat-
to, cheese and curd cheese. To investigate
the effect of the food matrix on vitamin K
bioavailability, 6 healthy male volunteers
consumed either a detergent-solubilized K
1
(3.5 Ìmol) or a meal consisting 400 g of
spinach (3.5 Ìmol K
1
) and 200 g of natto
(3.1 Ìmol K
2
). The absorption of pure K
1
was
faster than that of food-bound K vitamins (se-
rum peak values at 4 h vs. 6 h after ingestion).
Moreover, circulating K
2
concentrations after
the consumption of natto were about 10
times higher than those of K
1
after eating spi-
nach. It is concluded that the contribution of
K
2
vitamins (menaquinones) to the human
vitamin K status is presently underestimated,
and that their potential interference with oral
anticoagulant treatment needs to be investi-
gated.
Copyright © 2001 S. Karger AG, Basel
Introduction
Vitamin K is an essential dietary micronu-
trient that facilitates the synthesis of specific
blood coagulation factors and of proteins in-
volved in bone metabolism and vascular biol-
ogy [1, 2]. It serves as a cofactor for the mem-
brane-bound microsomal enzyme Á-glutamyl-
carboxylase [3]. Dietary vitamin K is ab-
sorbed and transported in blood in its most
stable form, i.e. as a quinone. Vitamin K
occurs in two biologically active forms namely
phylloquinone (also known as vitamin K
1
)
Assessment of Menaquinones
Haemostasis 2000;30:298–307
299
and the menaquinones (known by their group
name vitamin K
2
) [2–4]. All K vitamins have
2-methyl-1,4-naphthoquinone (also known as
menadione) as a common ring structure, but
differ from each other in the length and satu-
ration degree of the polyisoprenoid side chain
attached to the 3-position. Phylloquinone is
produced by green plants, where it is tightly
associated with the thylakoid membranes of
the chloroplasts. It is a single compound con-
taining 4 isoprenoid residues (one of which is
unsaturated) in its aliphatic side chain. Mena-
quinones contain side chains of varying
length; they are designated as MK-n where n
denotes the number of isoprenoid residues, all
of which are unsaturated. Long chain mena-
quinones (MK-7 through MK-10) are exclu-
sively synthesized by bacteria [5, 6]. Mena-
dione is often added to fortified animal food
and must be converted in the liver into MK-4
before being active as a cofactor for Á-gluta-
mylcarboxylase [7, 8]. In addition, a number
of other tissues (notably pancreas, testis and
vessel wall) are capable of converting phyllo-
quinone into MK-4 [9, 10]. For these reasons
animal products (meat, dairy, eggs) may con-
tain relatively high concentrations of MK-4. It
is well known that the bacterial flora in the
colon produces large amounts of higher mena-
quinones (notably MK-10) [11], but since at
the site of synthesis absorption seems to be
unlikely, the question of whether and to which
extent the intestinal flora contributes to the
human vitamin K status is still unclear.
Warfarin and other 4-hydroxycoumarin
derivatives are antagonists of vitamin K ac-
tion and are effective antithrombotic agents
(the so-called oral anticoagulants). They block
the conversion of KO into K by inhibiting the
enzyme KO reductase, thus hampering the
recycling of vitamin K [12]. Under these con-
ditions there is a 1:1 stoichiometric relation
between KO formation and the number of
Gla residues synthesized. It is known that
25% of the patients on oral anticoagulant
treatment are not within their therapeutic
range because of fluctuating international
normalized ratio values [13]. Besides interfer-
ing drugs, age, poor compliance and concur-
rent diseases [14–18], unstable levels of anti-
coagulation are often ascribed to dietary in-
fluences, mainly fluctuating vitamin K intake
[19–23].
In absolute amounts K
1
forms well over
80% of the total amount of vitamin K in the
human diet, and most of our present knowl-
edge on vitamin K concerns K
1
. It is known,
however, that the absorption from green vege-
tables is poor and that only 10–15% of the
vitamin is bioavailable, whereas for K
2
vita-
mins this may be higher [24, 25]. Here we
present a database on both dietary forms of
vitamin K, phylloquinone and the menaqui-
nones in a wide range of foods available on
the Dutch market. Since the specimens select-
ed formed a representative sample from the
common Dutch foods the data presented here
can be used in nutritional studies in The
Netherlands. Furthermore, we compared the
efficacy of absorption of phylloquinone and
menaquinones as deduced from their serum
profiles following oral ingestion.
Materials and Methods
Materials
Phylloquinone was obtained form Sigma (St.
Louis, Mo., USA). The menaquinones (MK-4 through
MK-10) and 2,3-dihydrophylloquinone were kind gifts
from Hoffmann-La Roche (Basel, Switzerland). All
common foods were obtained at local supermarkets.
Konakion
®
(detergent-solubilized vitamin K
1
pharma-
ceutical product) was obtained from Hoffmann-La
Roche. For the nutrition experiment we used creamed
cooked spinach from Iglo Ola (Utrecht, The Nether-
lands), and natto, which was bought as a ready-to-use
product at a local oriental store. Silica Sep-Pak car-
tridges were purchased from Millipore (Milford,
Mass., USA). All other chemicals used were of the
highest analytical grade.
Age, years
300
Haemostasis 2000;30:298–307
Schurgers/Vermeer
Extraction of Food
The procedure for extraction and purification of
vitamin K from beverages and dairy produce (except
butter and cheese) was performed as described earlier
[25] using 2,3-dihydrophylloquinone as an internal
standard. Vegetables were bought as precooked deep-
frozen products. Cooked vegetables and raw fruits
were homogenized in a blender (Ultra Turrax; Janke &
Kunkel, Staufen, Germany), and processed as de-
scribed for cooked spinach [25]. Aliquots of 1 g of
cheese, butter or margarine were extracted with 4 ml of
2-propanol, 20 ng internal standard (MK-6 for marga-
rine, 2,3-dihydrophylloquinone for other products)
and 2 ml of distilled water. The mixture was homoge-
nized with a blender, warmed to a temperature of
60
°
C and extracted with 8 ml of hexane. Raw meat
and fish were cut into pieces, 1 g of which was supple-
mented with 2 ml of distilled water, 5 ng of internal
standard (2,3-dihydrophylloquinone) and 4 ml of etha-
nol. Homogenization took place with a blender at
room temperature, and 8 ml of hexane were used for
extraction. Bread was dried and ground to powder in
a mortar, 1-gram aliquots were supplemented with
5 ng internal standard (2,3-dihydrophylloquinone) and
4 ml of ethanol. After homogenization in a blender ex-
traction took place with 8 ml of hexane. In all cases, the
hexane phase was evaporated and redissolved in 2 ml
of hexane. After prepurification over silica Sep-Pak
cartridges the samples were ready to measure on
reversed-phase HPLC. All samples were measured in
duplicate.
Vitamin K Detection
Vitamin K was analyzed by HPLC using a C-18
reversed phase column and fluorometric detection af-
ter postcolumn electrochemical reduction as described
previously [25]. Phylloquinone and the menaquinones
were recorded in the same run. Because of the long
retention times for the long-chain menaquinones the
flow was increased from 0.5 to 1.0 ml/min at 11 min
after injection. The interday variation was 6–8%.
Human Volunteer Study
A panel of 6 male volunteers took part in this proto-
col. Their mean age was 33.5 years, and their body
mass index was 24.3 kg/m
2
(table 1). All participants
were apparently healthy, and their serum lipid profiles
were in the normal range. Neither medications nor
vitamin supplements (other than the experimental
supplements) were taken. The experimental protocol
started at 8 a.m. after an overnight fast. At that time
the participants received a breakfast containing either
a diet low in vitamin K, a similar diet with additional
Table 1.
Characteristics of the subjects
Mean SEM
33.5 2.57
Body mass index, kg/m
2
24.3 0.82
Triacylglycerol, mmol/l 0.87 0.14
Cholesterol, mmol/l 3.96 0.28
Vitamin K
Phylloquinone, nmol/l 1.48 0.19
Menaquinones, nmol/l n.d.
Mean values B SEM of 6 healthy male volunteers.
n.d. = Not detectable.
detergent-solubilized phylloquinone, or a diet contain-
ing 400 g of spinach and 200 g of natto. All diets con-
tained 30 g of fat. During the rest of the day partici-
pants were only allowed to have a lunch low in vitamin
K (toast, marmalade, bananas, apples), and to drink
orange juice and water ad libitum. After 6 p.m. and
during the rest of the experiment only consumption of
vitamin K-rich foods (spinach, broccoli, brussels
sprouts, kale, natto and cheese) was prohibited. Blood
samples were drawn by venipunctures at 0, 1, 2, 3, 4, 5,
6, 7, 8, 10, 11, 24, 48 and 72 h after start. Serum was
prepared and 1-ml aliquots were kept frozen at 80
°
C
until vitamin K determination. The study design was
approved by the local Medical Ethics Committee, and
informed consent was obtained from all subjects ac-
cording to the institutional guidelines.
Data Analysis
Serum vitamin K concentrations during 72 h after
oral ingestion were recorded at indicated intervals. At
each time point mean values B SE for the 6 partici-
pants were calculated and plotted as a function of time.
Blank values (no vitamin K ingested) were subtracted
throughout the study.
Results
Vitamin K Content of Various Nutrients
For the determination of dietary phyllo-
quinone and menaquinones we subdivided
common foods into six categories: meat, fish,
vegetables and fruits, dairy, oils and marga-
Assessment of Menaquinones
Haemostasis 2000;30:298–307
301
Table 2.
Mean of K vitamins (Ìg/100 g or Ìg/100 ml) in various foods
Type of food n K
1
MK-4
Meat
Beef 7 0.6 (0.6–0.7) 1.1 (0.7–1.3)
Chicken breast 7 8.9 (6.4–11.3)
Chicken leg 7 8.5 (5.8–10.5)
Pork steak 7 0.3 (0.2–0.4) 2.1 (1.7–2.4)
Pork liver 7 0.2 (0.1–0.3) 0.3 (0.3–0.4)
Minced meat 7 2.4 (2.2–2.5) 6.7 (6.5–6.7)
Salami 7 2.3 (2.1–2.5) 9.0 (8.2–10.1)
Luncheon meat 7 3.9 (3.8–4.2) 7.7 (7.4–9.1)
Hare leg 7 4.8 (4.5–5.3) 0.1 (0.0–0.2)
Deer back 7 2.0 (1.9–2.2) 0.7 (0.6–0.7)
Goose leg 5 4.1 (3.5–4.8) 31.0 (28.2–33.1)
Goose liver paste 5 10.9 (9.3–12.1) 369 (317–419 )
Duck breast 7 1.9 (1.7–2.2) 3.6 (3.3–3.9)
MK-5 MK-6 MK-7 MK-8 MK-9
––––
––––
––––
0.5 (0.4–0.7) 1.1 (0.9–1.2)
––––
––––
––––
––––
––––
––––
––––
––––
Fish
Prawn 7 0.1 (0.0–0.1)
Mackerel 7 2.2 (1.8–2.6) 0.4 (0.3–0.5)
Herring 7 0.1 (0.0–0.2)
Plaice 7 0.2 (0.1–0.3)
Eel 7 0.3 (0.2–0.5) 1.7 (1.4–2.1)
Salmon 7 0.1 (0.1–0.2) 0.5 (0.4–0.6)
Fruits and vegetables
Kale 4 817 (752–881)
Spinach 6 387 (299–429)
Broccoli 5 156 (139–189)
Green peas 4 36.0 (31.2–39.4)
Sauerkraut 7 25.1 (23.8–27.5) 0.4 (0.3–0.5)
Natto 5 34.7 (31.2–36.7)
Banana 4 0.3 (0.2–0.4)
Apple 4 3.0 (2.7–3.4)
Orange 4 0.1 (0.1–0.2)
+
––––
––––
––––
0.3 (0.2–0.3) 0.1 (0.0–0.1) 1.6 (1.3–1.8)
0.1 (0.0–0.2) 0.4 (0.2–0.6)
––––
––––
––––
––––
––––
0.8 (0.6–1.0) 1.5 (1.4–1.6) 0.2 (0.1–0.3) 0.8 (0.6–0.9) 1.1 (0.9–1.3)
7.5 (7.1–7.8) 13.8 (12.7–14.8) 998 (882–1,034) 84.1 (78.3–89.8)
––––
––––
302
Haemostasis 2000;30:298–307
Schurgers/Vermeer
Table 2
(continued)
Type of food n K
1
MK-4
Dairy produce
Whole milk 6 0.5 (0.4–0.6) 0.8 (0.7–0.9)
Skimmed milk 6
Buttermilk 6 0.2 (0.2–0.3)
Whole yoghurt 6 0.4 (0.3–0.5) 0.6 (0.5–0.7)
Skimmed yoghurt 6
Whipping cream 6 5.1 (4.9–5.5) 5.4 (5.2–5.6)
Chocolate 6 6.6 (6.4–6.7) 1.5 (1.4–1.6)
Hard cheeses 15 10.4 (9.4–12.1) 4.7 (4.2–6.6)
Soft cheeses 15 2.6 (2.4–2.9) 3.7 (3.3–3.9)
Curd cheese 12 0.3 (0.2–0.4) 0.4 (0.3–0.6)
Egg yolk 8 2.1 (1.9–2.3) 31.4 (29.1–33.5)
Egg albumen 8 0.9 (0.8–1.0)
MK-5 MK-6 MK-7 MK-8 MK-9
0.1 (0.0–0.1) ––––
––––
0.1 (0.1–0.2) 0.1 (0.0–0.2) 0.1 (0.1–0.3) 0.6 (0.5–0.6) 1.4 (1.2–1.6)
0.1 (0.0–0.2) 0.2 (0.2–0.3)
0.1 (0.0–0.2)
––––
––––
1.5 (1.3–1.7) 0.8 (0.6–1.0) 1.3 (1.1–1.5) 16.9 (14.9–18.2) 51.1 (45.3–54.9)
0.3 (0.2–0.4) 0.5 (0.6–0.7) 1.0 (0.9–1.1) 11.4 (10.7–12.2) 39.6 (35.1–42.7)
0.1 (0.0–0.2) 0.2 (0.1–0.3) 0.3 (0.2–0.5) 5.1 (4.8–5.4) 18.7 (18.1–19.2)
0.7 (0.6–0.8)
––––
Oils and margarines
Margarine 6 93.2 (85.6–98.3)
Butter 6 14.9 (13.2–15.9) 15.0 (13.5–15.9)
Corn oil 6 2.9 (2.7–3.1)
Sunflower oil 6 5.7 (5.5–5.9)
Olive oil 6 53.7 (49.9–57.2)
Bread
Rue bread 6 0.7 (0.5–0.9)
Wheaten bread 6 1.1 (1.0–1.2)
Sourdough bread 6 1.0 (0.9–1.1)
Buckwheat bread 6 3.0 (2.8–3.4)
Beverages
Tea 4 0.3 (0.2–0.4)
Coffee 4
Orange juice 4
All samples were assessed in duplicate. Values are mean values. Highest and lowest values are given in parentheses. Foods were bought from shops in
and around Maastricht. MK-10 was not detectable in any of the foods. N = Number of different samples tested; = not detectable.
––––
––––
––––
––––
––––
––––
––––
––––
1.1 (1.0–1.2)
––––
––––
––––
Assessment of Menaquinones
Haemostasis 2000;30:298–307
303
Fig. 1.
Serum vitamin K following the oral intake of either Konakion or a meal containing
spinach and natto. The ingested Konakion contained 3.5 ÌM K
1
, the mixed meal contained
3.5 ÌM of K
1
and 3.1 ÌM of MK-7. Points represent mean values from 6 volunteers, error bars
represent SEM. [ = K
1
after Konakion; $ = K
1
after mixed meal; P = MK-7 after mixed
meal.
rines, bread, and beverages. At least three to
six different samples or brands were obtained
in various local supermarkets, and mean val-
ues for each product are given in table 2
together with their ranges for each product.
High amounts of K
1
were found in green leafy
vegetables, broccoli, sauerkraut and marga-
rines based on plant oils. Meat, fish, dairy
produce and eggs contained both K
1
and MK-
4 with relatively high MK-4 concentrations in
goose meat and liver, butter and egg yolk.
Long-chain menaquinones were mainly found
in curd cheese, hard (Dutch) and soft (French)
cheeses, probably derived from the bacterial
starter fermentation. Very rich in menaqui-
nones was the Japanese food natto, which
consists of fermented soy beans. No substan-
tial differences were found between free-range
products (eggs, chicken, meat) and those from
factory farms. The fact that fermented bever-
ages like beer and wines did not contain
detectable amounts of menaquinones is prob-
ably due to the fact that moulds do not synthe-
size menaquinones [26].
Bioavailability of K Vitamins from Food
To examine the blank values (serum vita-
min K at low vitamin K intake) 6 male volun-
teers received a vitamin K-poor breakfast
with blood sampling (up to 72 h) as indicated.
These blank values (data not shown) were
subtracted from those obtained after con-
trolled vitamin K intake. Based on the analy-
ses summarized in table 2 we have prepared
meals consisting of 400 g cooked spinach
(equivalent to 3.5 Ìmol of K
1
), 200 g natto
(3.1 Ìmol of MK-7), supplemented with corn
oil to a total fat content of 30 g. Postprandial
304
Haemostasis 2000;30:298–307
Schurgers/Vermeer
Fig. 2.
Serum vitamin K
1
and MK-7 following the separate intake of either spinach (3.5 ÌM
K
1
) or natto (3.1 ÌM MK-7). Points represent mean values from 6 volunteers, error bars
represent SEM. $ = K
1
; P = MK-7.
serum vitamin K concentrations are given in
figure 1. One week later the volunteers re-
ceived a vitamin K-poor breakfast supple-
mented with 3.5 Ìmol of Konakion. Peak val-
ues for serum vitamin K (both K
1
and MK-7)
were found at 6 h following the meal, and at
4 h after intake of the pure compound. The
very poor absorption from green vegetables
becomes clear by comparing the difference
between the curves for K
1
pure compound
and the similar amount of K
1
from spinach.
Remarkably, MK-7 from natto was absorbed
extremely well with peak values even higher
than those for detergent-solubilized K
1
. After
having reached their peak levels a rapid disap-
pearance of both K
1
and MK-7 was observed,
but MK-7 showed complex pharmacokinet-
ics, with slow disappearance during the sec-
ond part of the curve, while it remained
detectable for at least 72 h. The half-life times
for both K
1
and MK-7 between 6 and 8 h post-
prandially were about 1.5 h, whereas during
the later phases of MK-7 disappearance the
half-life time was about 50 h. To exclude
mutual interference of absorption (e.g. by
competition for the same binding protein),
the above experiment was repeated in a de-
sign in which spinach and natto were given in
two separate meals with a 1-week interval.
The serum curves are shown in figure 2 and
are comparable to those obtained after the
combined meal.
The above absorption curves were re-
peated for other foods: broccoli as source for
K
1
and curd cheese and egg yolk as sources for
higher menaquinones (MK-8 and MK-9) and
MK-4, respectively [Schurgers, unpubl. data].
In all cases it was found that K
1
absorption
from vegetables was very poor (5–10% with-
out concomitant fat intake and 10–15% if tak-
Assessment of Menaquinones
Haemostasis 2000;30:298–307
305
en together with 30 g fat), whereas menaqui-
none absorption from dairy produce and nat-
to was much better, probably almost com-
plete.
Discussion
In this paper we describe the phylloqui-
none and menaquinone content of various
foods available on the Dutch market. All K
vitamins were quantified in the same run
after a slight modification of our previously
reported procedure [25]. It was confirmed
that phylloquinone is mainly present in green
vegetables, margarins and some plant oils
such as olive oil. Since these data are similar
to those reported by others [27–29] we have
focussed on the menaquinones in food. MK-4
was present in nearly all animal products
(meat, dairy produce, eggs), but the fact that
there were no substantial differences between
game (hare, deer), free-range animals and
those from factory farms suggests that conver-
sion of menadione from fortified animal food
(used at factory farms) does not contribute
substantially to the total tissue MK-4 stores.
Rather, it seems that the major part of MK-4
in animal products originates from conver-
sion of K
1
as was also reported to occur in
rats [10]. Relatively high concentrations of
long-chain menaquinones were found in all
cheeses. As was suggested by Shearer [26],
they probably originate from bacteria present
in the starter cultures used to induce fermen-
tation. On the basis of food frequency ques-
tionnaires and the data in table 2 it has been
calculated that phylloquinone forms almost
90% of the total dietary vitamin K intake in
the Dutch population, whereas menaqui-
nones account for less than 12% [6]. Phyllo-
quinone, however, is tightly bound to the thy-
lakoid membranes of plant chloroplasts, and
the efficacy of its liberation therefrom in the
digestive tract is poor [24, 25]. This was con-
firmed in an experiment in which we com-
pared the serum concentration vitamin K
profiles after ingestion of similar amounts of
K
1
from spinach and from a detergent-solubi-
lized pharmaceutical product. To compare
the efficacy of absorption of phylloquinone
and menaquinone we have chosen a design in
which K
1
was obtained from spinach and
MK-7 from natto. In this way the molar con-
centrations of both K vitamers could be kept
similar. As is shown in figure 1, the postpran-
dial serum concentrations of MK-7 were
much higher than those of K
1
, with a peak
height difference of more than 10-fold. Both
absorption peaks occurred 2 h later than that
for the detergent-solubilized product. From
the curves obtained, it may be concluded that
the contribution of MK-7 from natto to the
total bioavailable pool of vitamin K is much
higher than estimated on the basis of intake.
Menaquinones from other sources (cheeses,
egg yolk) were absorbed with comparable effi-
cacy as was MK-7 [Schurgers, unpubl. data],
suggesting that the contribution of menaqui-
nones to the total human vitamin K status is
much higher than generally assumed, and
may equal that of K
1
.
Another remarkable difference between K
1
and menaquinones was that the former had a
disappearance curve with an apparent half-
life time of 1.5 h, whereas the long chain men-
aquinones (not MK-4) had more complex dis-
appearance curves with a very long half-life
time. Rapid clearance is consistent with the
previously reported uptake and transport of K
vitamins in chylomicrons, from where they
are cleared by the liver during the first 8 post-
prandial hours. The very long half-life times
of the higher menaquinones suggest that these
vitamers (and not K
1
and MK-4) are redis-
tributed by the liver and set free in the circula-
tion in low and high density lipoproteins. It is
well known that LDL may be present in the
306
Haemostasis 2000;30:298–307
Schurgers/Vermeer
circulation for several days. The long resi-
dence times of higher menaquinones in the
circulation implies that they are available for
extrahepatic tissue uptake for much longer
periods than is phylloquinone. Both because
of their high postprandial serum concentra-
tion and their slow clearance, the importance
of higher menaquinones for extrahepatic tis-
sues such as bone and arterial vessel wall may
be underestimated if only dietary intake is
regarded. Since vitamin K-dependent pro-
teins have been reported to be involved in the
regulation of calcium deposition in bone [30]
and in the prevention of arterial calcification
[31], intake of higher menaquinones may be
important for functions of vitamin K not
related with blood coagulation.
The high efficacy of menaquinone absorp-
tion may also have consequences for subjects
on oral anticoagulant treatment. In attempts
to identify potential causes of unstable anti-
coagulation, menaquinone intake has been ig-
nored thus far. Our data demonstrate that this
is not justified. Their efficient absorption
combined with long serum and tissue half-life
times [32] suggests that menaquinones from
curd and cheese may accumulate at repeated
intake and are a potential cause of distur-
bance of anticoagulant therapy. This is even
more so for subjects consuming natto. Al-
though in general natto is not eaten by Cauca-
sians, dietary habits may survive after migra-
tion of subjects from Asiatic countries so that
hematologists in western countries may be
confronted with this unsuspected source of
highly bioavailable vitamin K.
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... The main known biological function of vitamin K1 is played in blood clotting, since it acts as a cofactor for the enzymatic conversion of glutamic acid (Glu) residues to gamma-carboxyglutamic acid (Gla) in vitamin K-dependent proteins (VKDPs), through vitamin K-dependent gamma-glutamyl carboxylase, localized in the endoplasmic reticulum of the cells of all mammalian tissues [7][8][9], and for the conversion of protein-bound glutamate in carboxy-glutamate, needed for II, VII, IX, and X coagulation cascade factors, and for the natural anticoagulants proteins S and C [10,11]. The source of vitamin K1 is mainly represented by leafy or flowering vegetables (spinach, lettuce, broccoli, cabbage, Brussels sprouts, turnip greens), but chickpeas, peas, soya, green tea, eggs, pork, and beef liver also contain vitamin K1 [12]. Vitamin K2 is synthetized essentially by intestinal microbiota and is denoted as menaquinone (MK); according to the length of the isoprene chain attached to the methylated naphthoquinone ring, several different forms could be identified, as numbered from 4 to 13. MK-4 is obtained from the conversion of phylloquinone or menadione and is found mainly in meat and animal by-products such as eggs, cow's milk and yoghurt [13][14][15]. ...
... Multiple factors can affect vitamin K stores in CKD patients, and the main causes of its deficiency include food restriction, uraemia-associated dysbiosis, and drugs [29][30][31]. Moreover, dietary restriction due to the high potassium content in most vitamin K-rich, green vegetables contributes to its deficiency [12]. Alongside dietary intake, vitamin K is recycled through the "vitamin K cycle", which includes vitamin K epoxide reductase, DT-diaphorase and g-glutamylcarboxylase. ...
Article
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Chronic kidney disease (CKD) is commonly associated with vitamin K deficiency. Some of the serious complications of CKD are represented by cardiovascular disease (CVD) and skeletal fragility with an increased risk of morbidity and mortality. A complex pathogenetic link between hormonal and ionic disturbances, bone tissue and metabolism alterations, and vascular calcification (VC) exists and has been defined as chronic kidney disease–mineral and bone disorder (CKD-MBD). Poor vitamin K status seems to have a key role in the progression of CKD, but also in the onset and advance of both bone and cardiovascular complications. Three forms of vitamin K are currently known: vitamin K1 (phylloquinone), vitamin K2 (menaquinone), and vitamin K3 (menadione). Vitamin K plays different roles, including in activating vitamin K-dependent proteins (VKDPs) and in modulating bone metabolism and contributing to the inhibition of VC. This review focuses on the biochemical and functional characteristics of vitamin K vitamers, suggesting this nutrient as a possible marker of kidney, CV, and bone damage in the CKD population and exploring its potential use for promoting health in this clinical setting. Treatment strategies for CKD-associated osteoporosis and CV disease should include vitamin K supplementation. However, further randomized clinical studies are needed to assess the safety and the adequate dosage to prevent these CKD complications.
... Vitamins K 1 (phylloquinone) and K 2 (menaquinones, MK4, MK7, and MK8) in EDTA plasma were analyzed at VitaK laboratories (Maastricht, The Netherlands) by the following method: circulating levels of phylloquinone and menaquinones were measured with a standard HPLC technique using a C18 reversed-phase column and fluorometric detection after post-column electrochemical reduction [44]. Vitamin K1 (G L Synthesis, Worcester, MA, USA) was used as standard [44]. ...
... Vitamins K 1 (phylloquinone) and K 2 (menaquinones, MK4, MK7, and MK8) in EDTA plasma were analyzed at VitaK laboratories (Maastricht, The Netherlands) by the following method: circulating levels of phylloquinone and menaquinones were measured with a standard HPLC technique using a C18 reversed-phase column and fluorometric detection after post-column electrochemical reduction [44]. Vitamin K1 (G L Synthesis, Worcester, MA, USA) was used as standard [44]. ...
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Bovine meat provides healthy nutrients but has also been negatively linked to greenhouse gases and non-communicable diseases. A double-blind intervention study was carried out to compare beef meat from bulls fed with feed supplemented with selenium, vitamin D, E, K (SeDEK-feed), and n-3, or REGULAR feed. Thirty-four young healthy women (19–29 years old) consumed 300 g of these beef types per day for 6 days in a cross-over design. Diet registrations, blood samples, anthropometric measurements, and clinical data were collected four times. Both beef diets were higher than their habitual diet in protein, fat, saturated fat, and several micronutrients; contained more vegetables and fewer carbohydrates and were followed by a higher feeling of satiety. The SeDEK beef had higher amounts of selenium, vitamin 25-hydroxyvitamin D3 (25(OH)D3), E, and K (MK4), and increased serum selenium and 25(OH)D3 from the participants’ normal values if they were below 85 µg/L of selenium and 30 nmol of 25(OH)D3/L, respectively. Our study showed that optimized beef increased serum selenium in young women having moderate selenium levels and improved blood 25(OH)D3 in a woman having low to normal 25(OH)D3. Meat should be optimized to increase specific consumer groups’ needs for selenium and vitamin D.
... One contributor is dietary restriction, given the high potassium content in most vitamin K-rich, green vegetables. 8 Beyond dietary intake, vitamin K is recycled via the vitamin K cycle, encompassing vitamin K epoxide reductase, DT-diaphorase, and g-glutamylcarboxylase. Because of reduced g-glutamyl-carboxylase activity, over a mechanism similar to the action of coumarins, 9 impaired vitamin K recycling has been found in CKD rats. ...
... In addition, among the vitamin K2 species, MK7 is readily available in synthetic form (albeit not drug grade) and exhibits a long half-life in healthy subjects. 8,31 In addition, using the same dose, MK7 induces more carboxylation of osteocalcin than vitamin K1, indicating not only better absorption but also better bioactivity. 32 In healthy subjects of the present study, following a single oral dose, MK4 serum levels hardly changed, whereas MK7 levels rapidly increased in serum, confirming previously published data. ...
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Chronic kidney disease (CKD) is accompanied with extensive cardiovascular calcification, in part correlating with functional vitamin K deficiency. Here, we sought to determine causes for vitamin K deficiency beyond reduced dietary intake. Initially, vitamin K uptake and distribution into circulating lipoproteins after a single administration of vitamin K1 plus K2 (menaquinone 4 and menaquinone 7, respectively) was determined in patients on dialysis therapy and healthy individuals. The patients incorporated very little menaquinone 7 but more menaquinone 4 into high density lipoprotein (HDL) and low density lipoprotein particles than did healthy individuals. In contrast to healthy persons, HDL particles from the patients could not be spiked with menaquinone 7 in vitro and HDL uptake was diminished in osteoblasts. A reduced carboxylation activity (low vitamin K activity) of uremic HDL particles spiked with menaquinone 7 vs. that of controls was confirmed in a bioassay using human primary vascular smooth muscle cells. Kidney menaquinone 4 tissue levels were reduced in 5/6-nephrectomized versus sham-operated C57BL/6 mice after four weeks of a vitamin K rich diet. From the analyzed enzymes involved in vitamin K metabolism, kidney HMG-CoA reductase protein was reduced in both rats and patients with CKD. In a trial on the efficacy and safety of atorvastatin in 1051 patients with type 2 diabetes receiving dialysis therapy, no pronounced vitamin K deficiency was noted. However, the highest levels of PIVKA-II (biomarker of subclinical vitamin K deficiency) were noted when a statin was combined with a proton pump inhibitor. Thus, profound disturbances in lipoprotein mediated vitamin K transport and metabolism in uremia suggest that menaquinone 7 supplementation to patients on dialysis therapy has reduced efficacy.
... 8 In fact, comparing the adsorption between MK7 and K1, administrated through natto and spinach, respectively, menaquinone exhibited 10-fold higher postprandial serum concentration in comparison with phylloquinone, and a half-life of 72 h with respect to 3 h of phylloquinone, lasting up to 144 h in the circulation. 9 In addition, the role of MK7 in bone health has been highlighted by several studies showing how natto consumption reduces the incidence of hip fractures in women in Japan 10,11 and recently confirmed by a large prospective cohort study. 12 This evidence, along with a large set of clinical studies, highlights the beneficial effects of MK7 supplementation in the prevention of osteoporosis [13][14][15] and in the reduction of vascular stiffness, 16,17 making MK7 essential for the prevention of bone and cardiovascular disease (CVD). ...
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Carboxylative enzymes are involved in many pathways and their regulation plays a crucial role in many of these pathways. In particular, γ‐glutamylcarboxylase (GGCX) converts glutamate residues (Glu) into γ‐carboxyglutamate (Gla) of the vitamin K‐dependent proteins (VKDPs) activating them. VKDPs include at least 17 proteins involved in processes such as blood coagulation, blood vessels calcification, and bone mineralization. VKDPs are activated by the reduced form of vitamin K, naturally occurring as vitamin K1 (phylloquinone) and K2 (menaquinones, MKs). Among these, MK7 is the most efficient in terms of bioavailability and biological effect. Similarly to other trans isomers, it is produced by natural fermentation or chemically in both trans and cis. However, the efficacy of the biological effect of the different isomers and the impact on humans are unknown. Our study assessed carboxylative efficacy of trans and cis MK7 and compared it with other vitamin K isomers, evaluating both the expression of residues of carboxylated Gla‐protein by western blot analysis and using a cell‐free system to determine the GGCX activity by HPLC. Trans MK7H2 showed a higher ability to carboxylate the 70 KDa GLA‐protein, previously inhibited in vitro by warfarin treatment. However, cis MK7 also induced a carboxylation activity albeit of a small extent. The data were confirmed chromatographically, in which a slight carboxylative activity of cis MK7H2 was demonstrated, comparable with both K1H2 and oxidized trans MK7 but less than trans MK7H2. For the first time, a difference of biological activity between cis and trans configuration of menaquinone‐7 has been reported.
... Because of the amount of cheese present in the diet, it is thought that cheese contributes to a significant amount of MKs in the diet [17,23]. Thus, the focus of this study was to investigate the bioaccessibility of vitamin K in cheese in order to investigate if difference in bioaccessibility should be taken into account when estimating, e.g., dietary intake based on the content of the vitamin K in the food product and the relation to health effects. ...
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Vitamin K is a fat-soluble group of vitamers consisting of phylloquinone (PK) and menaquinones (MKs). To date, only a daily reference intake for PK is set; however, in the last decade, research studying the correlation between MKs intake and improvement of health in regards to cardiovascular diseases, bone metabolism, and chronic kidney disease has been conducted. MKs are synthesised by bacteria in the fermentation process of foods, e.g., cheeses. The content and bioaccessibility of vitamin K vitamers (PK, MK-4, MK-5, MK-6, MK-7, MK-8, MK-9, and MK-10) were assessed in eight different cheese products differing in ripening time, starter culture, fat content, and water content. The bioaccessibility was assessed using the static in vitro digestion model INFOGEST 2.0. Variation of the vitamin K content (<0.5 μg/100 g–32 μg/100 g) and of the vitamin K bioaccessibility (6.4–80%) was observed. A longer ripening time did not necessarily result in an increase of MKs. These results indicate that the vitamin K content and bioaccessibility differs significantly between different cheese products, and the ripening time, starter culture, fat content, and water content cannot explain this difference.
... Vitamin K is a fat-soluble vitamin occurring in two biologically active forms; phylloquinone (vitamin K 1 ) and menaquinones (vitamin K 2 ) [1]. Vitamin K 1 is mainly derived from green leafy vegetables, while vitamin K 2 mainly occur in fermented animal products like cheese and meat. ...
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The vitamin K1 content of 10 commercially available vegetable oils was determined by reversed-phase high-performance liquid chromatography (HPLC). Prior to HPLC, crude lipid extracts were purified by solid-phase extraction on silica. Rapeseed and soybean oils were found to contain the greatest amounts of vitamin K1 (140-200 mug/100 g) followed by olive oil (55 mug/100 g). Almond, sunflower, safflower, walnut, and sesame oils contained between 6 and 15 mug/100 g, while peanut and corn oils provided less than 3 mug/100 g. Vitamin K1 was stable to processing mode, decreased slightly but significantly with heat, and was rapidly destroyed by both daylight and fluorescent light. Amber glass containers protected the oils from the destructive effects of light. Soybean and rapeseed oils are excellent sources of vitamin K1 and can provide greater than 100 % of the required dietary allowance for vitamin K when present in the diet at greater than 15 % of the caloric content.
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A provisional table on the vitamin K1 (phylloquinone) content of foods was compiled in response to the expressed need for tabulated data by the public and professionals in medicine, nutrition, dietetics, and research. Data for vitamin K1 content in foods were evaluated according to criteria set for the analytical method, sampling, and quality control, with a confidence code assigned to each accepted value for each food item. Given the large analytical variation associated with the chick bioassays, only data obtained from methods using HPLC (high-performance liquid chromatography) were used. An effort was made to include vitamin K values that were representative of foods in the retail market. There are few food composition data for this nutrient. The available data do indicate that leafy, green vegetables, and certain legumes and vegetable oils are good dietary sources of vitamin K1. The distribution of vitamin K1 in plants is not uniform, with higher concentrations of the vitamin found in the outer leaves as compared to concentrations in the pale, inner leaves. The peels of fruits and vegetables appear to have higher concentrations of the vitamin than do the fleshy portions. Recent investigations indicate that season, climate, growing location, and soil fertility are sources of natural variation in the vitamin K1 concentration of foods. The limited data on the vitamin K1 content of foods need to be expanded to include other commonly-consumed foods, including prepared foods. One approach would be an improved database for simple foods, which could then be used in the U.S. Department of Agriculture recipe file of the USDA Survey Nutrient Database to calculate the vitamin K1 content of multicomponent foods. Furthermore, investigation is required to differentiate natural variation from that attributable to analytical methodology and sample preparation, such as homogenization and cooking.
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Two forms of vitamin K [phylloquinone (K1) and menaquinone-4 (MK-4)] were added to vitamin K-deficient rat food in varying amounts. These diets were given as the sole source of nutrition to rats for one week. The minimal dietary requirements (MDR) to attain maximal prothrombin synthesis were determined to be 0.6 and 6–10 μg/g of food for K1 and MK-4, respectively. The difference between both vitamers could be explained by the limited hepatic accumulation of MK-4. Next, vitamin K was offered to rats at concentrations ranging between 0.6 and 3000 μg/g of food, and the tissue distribution of vitamin K was investigated after one week of administration. Accumulation of K1 and MK-4 was found in all tissues investigated, but both the absolute tissue concentration and the ratio between K1 and MK-4 were tissue-dependent. Highest values were found in liver and in heart, but since the heart contains no γ-glutamylcarboxylase, the function of vitamin K in this tissue remains obscure. High tissue concentrations of MK-4 were also found in pancreas and testis after a diet containing K1 exclusively. The data indicate that this conversion is tissue-specific, but neither the reason nor its mechanism are known.
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To elucidate the role of intestinal bacteria in the conversion of phylloquinone into menaquinone-4 (MK-4) we investigated the tissue distribution of vitamin K in germ-free rats. The rats were made vitamin K deficient by feeding a vitamin K-free diet for 13 days. In a subsequent period of 6 days, phylloquinone and menadione were supplied via the drinking water in concentrations of 10 and 50 μmol l−1. Menadione supplementation led to high levels of tissue MK-4, particularly in extrahepatic tissues like pancreas, aorta, fat and brain. Liver and serum were low in MK-4. Phylloquinone supplementation resulted in higher phylloquinone levels in all tissues when compared with vitamin K-deficient values. The main target organs were liver, heart and fat. Remarkably, tissue MK-4 levels were also higher after the phylloquinone supplementation. The MK-4 tissue distribution pattern after phylloquinone intake was comparable with that found after menadione intake. Our results demonstrate that the conversion of phylloquinone into MK-4 in extrahepatic tissues may occur in the absence of an intestinal bacterial population and is tissue specific. A specific function for extrahepatic MK-4 or a reason for this biochemical conversion of phylloquinone into MK-4 remains unclear thus far.
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To identify the possible factors determining the dose of warfarin prescribed in patients receiving anticoagulant treatment. The computerised records of 2305 patients maintained on the drug in seven hospitals were amalgamated and classified into one of seven diagnostic groups. The associations with the dose of warfarin prescribed were investigated by univariate and multiple regression analysis. Differences between hospitals were studied with regard to the coagulometric method and the thromboplastin preparation used. The geometric mean dose of warfarin was 4.57 mg and 5% of patients were prescribed 10 mg or greater. There was a noticeable decrease in dose with increasing age, which averaged about 6 mg for patients aged 30 but 3.5 mg for those aged 80. Men required slightly more warfarin than women. Patients with heart disease or atrial fibrillation required lower doses of warfarin, while higher doses were required by patients with deep vein thrombosis. Significant differences in mean warfarin dose among the seven hospitals were evident. These differences could not be explained entirely by the use of different coagulometric methods or thromboplastins. Clinicians should be aware that older patients need reduced doses of warfarin. The considerable differences in doses of warfarin among hospitals indicates that further efforts to improve uniformity are required.
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This article has no abstract; the first 100 words appear below. BLOOD clotting is a host defense mechanism that, in parallel with the inflammatory and repair responses, helps protect the integrity of the vascular system after tissue injury. This system is normally quiescent but becomes active within seconds after injury. Cells (platelets, leukocytes, and endothelial cells) and the plasma blood-clotting proteins are critical in this reaction. The response to vascular injury culminates in the formation of a platelet plug, the generation of a fibrin clot, the deposition of white cells in the area of tissue injury, and the initiation of inflammation and repair. Molecular Basis of Blood Coagulation All the protein . . . Supported by grants (R37 HL38216, P01 HL42443, and R37 HL18834) from the National Institutes of Health. Source Information From the Center for Hemostasis and Thrombosis Research, Division of Hematology—Oncology, New England Medical Center, Boston, and the Departments of Medicine and Biochemistry, Tufts University School of Medicine, Boston. Address reprint requests to Dr. Bruce Furie at the Division of Hematology-Oncology, New England Medical Center, 750 Washington St., Boston, MA 02111.
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The hepatic turnover of phylloquinone and menaquinone-9 (MK-9) and their relative efficacy in satisfying the dietary requirement for vitamin K were compared in male rats. Rats fed 1.1 mumol phylloquinone/kg diet had higher initial liver and serum vitamin K concentrations than rats fed an equimolar amount of MK-9. The initial rate of hepatic turnover of phylloquinone was two to three times as rapid as that of MK-9. After about 48 h of vitamin K restriction there were no significant differences in hepatic vitamin K concentration of rats fed phylloquinone or MK-9. Phylloquinone was much more effective than MK-9 in maintaining normal vitamin K status at low dietary concentrations (0.2 mumol/kg diet), whereas at high dietary concentrations (5.6 mumol/kg diet) they were equally effective.
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Vitamin K functions as a co-factor for the post-translational carboxylation of specific glutamate residues to gamma-carboxyglutamate (Gla) residues in several blood coagulation factors (II, VII, IX and X) and coagulation inhibitors (proteins C and S) in the liver; as well as a variety of extrahepatic proteins such as the bone protein osteocalcin. This review outlines some recent advances in our understanding of the metabolism of vitamin K and its role in human nutriture. The introduction of new methodologies to measure the low endogenous tissue concentrations of K vitamins and circulating plasma levels of des-gamma-carboxyprothrombin (PIVKA-II) have provided correspondingly more refined indices for the assessment of human vitamin K status. The assays for vitamin K have also been used to study the sources, intestinal absorption, plasma transport, storage and transplacental transfer of K vitamins and the importance of phylloquinone (vitamin K1) versus menaquinones (vitamins K2) to human needs. The ability to biochemically monitor subclinical vitamin K deficiency has reaffirmed the precarious vitamin K status of the newborn and led to an increased appreciation of the risk factors leading to haemorrhagic disease of the newborn and how this may be prevented. Biochemical studies are leading to an increased knowledge of the mode of action of traditional coumarin anticoagulants and how some unrelated compounds (e.g. antibiotics) may also antagonize vitamin K and cause bleeding. There is also an awareness of the possible deleterious effects of vitamin K antagonism or deficiency on non-hepatic Gla-proteins which may play some subtle role in calcium homeostasis.