*Comparison of absorption and efficacy of 3 different menaquinone-7 delivery systems; casein powder, Arabic gum powder, or linseed oil.*

The two most important forms of vitamin K are phylloquinone (vitamin K1) and
the group of K2 vitamins (menaquinones, MK-n). In previous studies, the
different forms of vitamin K have been compared with respect to their
intestinal absorption, biologic half-life time, and their effect on a number of
circulating biochemical markers. These studies have suggested that
menaquinone-7 (MK-7) is the most effective form of vitamin K (1). MK-7 is a
natural product that is found in cheese, curd, and the Japanese food natto.
Supplementation studies have shown the importance of vitamin K1 as well as
menaquinone-4 (MK-4) for optimal bone health and vascular health. Effects have
been reported at pharmacological doses ranging between 1 and 45 mg/d (2, 3).
However, no clinical data on the health effects of MK-7 are presently
available. Based on its longer half-life time and extra-hepatic tissue
distribution, similar effects are to be expected with MK-7 in nutritional doses
as compared to K1 and MK-4 in pharmacological doses (1). Therefore, MK-7 is
recommended as the obvious choice for enrichment of dietary supplements and
functional foods to improve well-being and health.
For dietary supplements, the choice of delivery vehicle is important in order
to achieve maximal absorption. So far, casein (*80% of proteins in cow*s
milk)-enriched powder has been used routinely for MK-7 delivery. However, cow*s
milk allergy is the most common cause of food allergy affecting a minimum of
2-3% of infants. To overcome this problem, we will compare this
protein-delivery system to an alternative delivery system (Arabic gum; 98%
polysaccharides).
Arabic gum is widely used in both the pharmaceutical and food industries as an
emulsifier and stabilizer of various products for human consumption (4). Arabic
gum also has pharmacologic effects related to the gastrointestinal absorption
of nutrients. It enhanced zinc absorption when orally administered in isotonic
solutions to animals (5). In a rat model of chronic osmotic diarrhea, Arabic
gum showed pro-absorptive properties by increased sodium and water absorption
(6, 7). Our hypothesis is that the absorption of MK-7 from Arabic gum will not
be significantly different from its absorption from casein.
The difference in absorption of MK-7 between powder and oil is currently
unknown. We will therefore also investigate the difference between powder and
oil as MK-7 delivery system. Our hypothesis is that the absorption of MK-7 from
powder will not be significantly different from its absorption form oil.
To test these hypotheses, we will determine serum MK-7 concentrations to
compare the absorption profiles between the different delivery vehicles. In
addition, several circulating biochemical markers (cMGP, ucMGP, cOC, ucOC) will
be measured to determine the biological functionality of these delivery
systems. Previous research (unpublished data) indicated that 8-wk treatment of
MK-7 (90 µg/d) significantly influenced the carboxylation of these biochemical
markers. In addition, serum ETP levels will be measured to investigate possible
effects on coagulation.

The main purpose of the study is to compare the absorption of MK-7 from casein
capsules (conventional MK-7 delivery system), Arabic gum capsules (alternative
MK-7 delivery system), and linseed oil capsules in healthy subjects. The
primary endpoint will be the serum level of MK-7.

The study will be a randomized, double-blind, parallel study. Sixty-nine
healthy men and women between 20 and 40 y will be recruited in the southern
region of Limburg. Before the actual start of the study and after an initial
informative meeting, a screening (= blood sampling) will take place to select
subjects with a low vitamin K status [marker = ucMGP; min cutoff value of 60
pmol/L (8)]. Eligible participants will be randomly divided over 3 treatment
groups to receive either casein capsules, Arabic gum capsules, or linseed oil
capsules. All capsules will be enriched with MK-7 (90 µg/d).
• Group 1: 20 subjects will receive daily 1 casein capsule (MK-7 intake = 90 µg/
d).
• Group 2: 20 subjects will receive daily 1 Arabic gum capsule (MK-7 intake =
90 µg/d).
• Group 3: 20 subjects will receive daily 1 linseed oil capsule (MK-7 intake =
90 µg/d).
A first blood sample will be taken after an overnight fast (t = 0 h) on the
first test day. Next, the participants will consume the capsules (1
capsule/subject) together with a standard breakfast [including fat,
carbohydrates, and protein; 3 slices of brown bread with chocolate paste or
margarine and jam, and 2 glasses of milk (milk or fruit&milk beverage)]. The
subsequent samples will be taken at the first test day (t = 0, 2, 4, 6, and 8
h) and several follow-up days (t = 1, 4, and day 7) to determine the absorption
profile. Starting from day 8, subjects will take daily 1 capsule together with
breakfast during the remaining 7 weeks of the study. Blood sampling will be
taken at days 14, 28, 42, and 56 to determine the biological functionality of
MK-7.

Participants will be randomly divided to receive; casein capsules (1
capsule/d), Arabic gum capsules (1 capsule/d), or linseed oil capsules (1
capsule/d). All capsules will be enriched with 90 µg MK-7. One week before the
start of the study and during the 8-wk treatment, participants will be asked to
refrain from consuming foods rich in vitamin K, including both K1-rich foods
(e.g. spinach, kale, broccoli, Brussels sprouts) and MK-rich foods (e.g. curd
cheese, cheese, natto).

One week before the start of the study and during the 8-wk treatment,
participants will be asked to refrain from consuming foods rich in vitamin K,
including both K1-rich foods (e.g. spinach, kale, broccoli, Brussels sprouts)
and MK-rich foods (e.g. curd cheese, cheese, natto).
The major burden for the subjects will consist of 13 venipunctures during the
8-wk study (fasting state). The venipunctures will be made by experienced
coworkers. Nevertheless, blood sampling may cause bruising or hematoma. The
daily dose of MK-7 (90 µg) will not cause adverse side-effects.