BIOEQUIVALENCE STUDY 2010
Comparison of the absorption of MK-7 after intake of 3 different existing MK-7 supplements

Vitamin K is a group name for a family of related compounds, generally divided
into the naturally occurring phylloquinone (vitamin K1) and menaquinones (MK-n;
K2 vitamins). The latter can be subdivided into the short-chain menaquinones
(e.g. MK-4) and the long-chain menaquinones (e.g. MK-7, MK-8, and MK-9). All K
vitamins share a common 2-methyl-1,4-naphthoquinone nucleus, substituted at the
3-position with different polyisoprenoids. Vitamin K1 (K1) is distributed in
green leafy vegetables and vegetable oils. K2 vitamins (K2) make up about 10%
of vitamin K consumption and are found in animal products, such as meat, egg
yolk, and dairy products. The higher menaquinones can also be synthesized by
gut bacteria; however the vitamin K contribution from this source to general
health is not fully understood. Natto, a Japanese fermented (using Bacillus
subtilis natto) soybean product, contains substantial amounts of MK-7, the most
bioavailable form of K2.
New research showed that increased K2 intake strongly reduced the risk of
coronary heart disease (CHD). In contrast, consumption of K1 had no effect on
vascular health. The reduction in CHD risk was tied to the higher menaquinones.
Higher menaquinones are more efficiently absorbed than K1, suggesting that K1
and K2 may contribute similarly to the body*s vitamin K supply. Most of K1 is
carried by the triacylglycerol-rich lipoproteins (chylomicrons and VLDL) in the
circulation and rapidly cleared to tissue (mainly liver); a small amount is
also carried by LDL and HDL. The higher menaquinones are observed in the same
classes of lipoprotein particles as K1, but appear to have a different
distribution (predominantly LDL). Since LDL has a long half-life time in the
circulation, these menaquinones may have better bioavailability for
extra-hepatic tissue uptake compared to K1. Consumption of higher menaquinones
may therefore be important for vitamin K functions not related to blood
coagulation, such as regulation of calcium deposition in bone and prevention of
arterial calcification.
As the Western diet is likely deficient in vitamin K, supplementation or
enrichment with these higher menaquinones is an obvious choice. At present,
MK-7 is the only natural vitamin K2 on the market; the potential market size
for MK-7 is considered to be extremely large. We are interested to compare the
absorption of MK-7 after intake of 3 different MK-7 products that are already
on the market.

The primary objective of the study is to compare the absorption of MK-7 after
intake of 3 different MK-7 supplements.

After meeting the inclusion criteria, study participants will be randomized
into six groups and will receive three different types of capsules as a single
dose (75 µg of MK-7/day): MenaQ7 M-1500 capsule, Gnosis P-1000 capsule, and
Gnosis M-1500 capsule. Every two weeks, participants switch to another type of
capsule; the washout period (WO) will therefore be two weeks. Each group will
consist of four volunteers with (approximately) equal numbers of men and women.
After meeting the inclusion criteria, 3x four intervention days (for each MK-7
product four visits) will be planned during a period of 9 weeks. On the first
intervention day, the first blood sample will be taken at 08.00 AM. Immediately
after sampling, participants will receive a standardized breakfast [three
slices of brown bread with chocolate paste or margarine&jam, and two glasses of
juice (orange juice or fruit&milk beverage)] and take the first type of MK-7
capsule. Subsequently, blood will be sampled at 10.00 AM, 12.00 PM, 14.00 PM,
16.00 PM, and at 08.00 AM on Tuesday, Wednesday, and Friday. After the sampling
at 12.00 PM, participants will receive a standardized lunch (similar as the
standardized breakfast).

After meeting the inclusion criteria, 3x four intervention days (for each MK-7
product four visits) will be planned during a period of 9 weeks. On the first
intervention day, the first blood sample will be taken at 08.00 AM. Immediately
after sampling, participants will receive a standardized breakfast [three
slices of brown bread with chocolate paste or margarine&jam, and two glasses of
juice (orange juice or fruit&milk beverage)] and take the first type of MK-7
capsule. Subsequently, blood will be sampled at 10.00 AM, 12.00 PM, 14.00 PM,
16.00 PM, and at 08.00 AM on Tuesday, Wednesday, and Friday. After the sampling
at 12.00 PM, participants will receive a standardized lunch (similar as the
standardized breakfast).
Two weeks before the start of the study and during the study, subjects will be
asked to refrain from consuming foods rich in K1 (spinach, kale, broccoli,
Brussels sprouts) and rich in K2 (curd cheese, cheese, natto). The subjects
will also be instructed to report any signs of illness, medication used, and
any deviations from the study protocol.

No adverse effects are to be expected from supplementing MK-7.
The major burden for the subjects will be the 25 venapunctions.
Participants will be asked to arrive for each study visit after a fast of at
least 8 hours. On the morning of sampling, subjects are not allowed to eat or
to drink (except water). In addition, subjects are not allowed to drink alcohol
24 hours before sampling. All venipunctures will be performed by experienced
researchers. The risks for the subjects are minimal. Another major burden is
the abstaining from foods rich in K1 (spinach, green kale, broccoli, Brussels
sprouts) and rich in K2 (curd cheese, cheese, natto) during 9 weeks.
Other minor restrictions:
• Replication of smoking schedule at all sampling visits
• Standardization of physical activity during the 24 hours prior to each study
visit