A randomized pilot study on probiotics and their effect on vitamin K2 status

Rationale: Vitamin K is mainly known for its function in blood coagulation, but
recently other functions in bone metabolism and vascular health have become
apparent. Vitamin K is a fat-soluble vitamin present in green vegetables in the
form of vitamin K1 (phylloquinone) and as vitamin K2 (menaquinones) in animal
products (meat, cheese). Vitamin K2 is the most active form of vitamin K and
the substantial part of vitamin K2 is derived from gut bacteria biosynthesis.
Besides the production in the gut, vitamin K2 is also present in fermented
dairy (cheese, yoghurt) and fermented soy beans (natto). Vitamin K is a
cofactor involved in the carboxylation (activation) of several proteins, such
as matrix Gla-protein (MGP) and reduces the inactive form of MGP,
dephosphorylated uncarboxylated matrix gla protein (dp-ucMGP), and could
thereby inhibit ongoing calcium deposition in the vascular system and
eventually arterial calcification. Recently, it has been discovered in-vitro
that certain probiotics * normally used for other indications* can also produce
vitamin K2.
Observational studies have shown that a high vitamin K2 intake is associated
with reduced coronary calcification and a reduced risk of coronary heart
disease. The available randomized controlled trials have mainly used vitamin K1
supplements and these studies indicated that vitamin K1 supplements improved
the elastic properties of the vessel wall and inhibit progression of coronary
artery calcium. To date, few randomized controlled trials showed that vitamin
K2 supplementation reduced dp-ucMGP with approximately 40% within 3 months and
among postmenopausal women, vitamin K2 supplementation improved arterial
stiffness among women with high arterial stiffness at baseline. This study will
explore whether probiotics are an effective vehicle to increase vitamin K
status and might be an alternative to diet and supplements. This study will
assess whether supplementation with probiotics that produce vitamin K2 can
improve vitamin K status as measured by inactive MGP in middle-aged adults with
high risk of metabolic disturbances.

Objectives: To investigate the effect of a daily sachet of probiotics on
vitamin K status for 12 weeks vs placebo.

Study design: Double-blind, placebo-controlled, randomized controlled pilot
trial. Participants will be randomized into two equal groups, one group
receives probiotics with bacteria that can produce up to 180 µg vitamin K2
daily and the other group receives placebo sachets daily for 12 weeks. The dose
is based on a previous study that showed a 31% decrease of dp-ucMGP after 12
weeks of supplementation with 180 microgram MK-7 daily. The duration of the
present study is 12 weeks, which will be long enough to achieve similar
reductions in dp-ucMGP levels. Dp-ucMGP can be influenced after 2-4 weeks of
supplementation, however, the effects of probiotics with vitamin K producing
properties are currently unknown.

A daily sachet of 4 gram freeze-dried probiotics producing 180 µg vitamin K2 to
be dissolved in lukewarm water with a slight vanilla taste.

The participants are requested to use a daily sachet dissolved in lukewarm
water with vanilla taste.
The probiotics can lead to changes in bowel frequency and movement, however
this will stabilize after 2 weeks. The dose of the vitamin K producing
probiotics is within the physiologic borders and therefore not considered
harmful. The participants will follow a vitamin K low diet for 2 weeks. This
means low intake of fermented dairy (cheese, buttermilk, yoghurt) and green
leafy vegetables (such as spinach, endive, kale, lettuce). After the 2 week
run-in diet no dietary restrictions are necessary. For the study, 4 visits to
the DCS VUmc will take a total time of 2-3 hours. During the visits the
participants are asked to fill out some questionnaires regarding medical
history including a short diet questionnaire to estimate the main vitamin K
sources via diet. In addition, a physical examination will be performed
including anthropometry (height, weight, blood pressure), blood collection.
Each visit a total amount of up to 25 ml blood will be collected by means of
vena puncture. Vena puncture can cause discomfort and can result in bruising
that continues up to a few days after the examinations. Also during the visits,
participants will hand in a 24-hour urine collection and a frozen stool sample.
Participants gain no individual benefit from their participation in the study.
However, the study is expected to increase our understanding of vitamin K
metabolism and contributes to developing vitamin K requirements and may
ultimately lead to a new therapeutic intervention.