Presented by Kiran KrishnanChief Science Officer, Microbiome Labs, LLC.

• Discovered in 1929

• K stands for “koagulation”

• Fat-soluble vitamin (A, D, E and K)

• Activates vitamin K-dependent proteins (VKD)

• Current RDI is 90-120 µg/day for blood clotting

Vitamin K

Natural Sources of Vitamin K

Vitamin K2

(Menaquinone)O

O

H

n

Vitamin K1

(Phylloqinone)O

O

H

3

Vitamin K2 as

Menaquinone-7

Dietary Sources of Vitamin K2 - Natto

Tissues in need for Vitamin K

clotting factorsII, VII, IX, X, C, S, Z

Vitamin KBone (Osteocalcin)

Arteries (Matrix Gla Proteins)

K1

K2

K2

Function of Vitamin K2

Osteocalcin (OC) and Matrix Gla Protein (MGP) need vitamin K to become

biologically active ……. Carboxylation

- Glu

- Gla

CA

RB

OX

YLA

SE

Glu - glutamic acidGla - gamma-carboxyglutamic acid

KH2- vitamin K hydroxyquinoneKO- vitamin K epoxideK- vitamin

protein

protein

KH2

KO

K

ucOC & ucMGP

cOC & cMGP

● Under-carboxylated Osteocalcin and MGP cannot function adequately in absorbing Calcium in the bones andinhibiting Calcium build up in the arteries respectively until carboxylated.

● Vitamin K2 is effective in the carboxylation of these VKD proteins.

Vitamin K2 and bone health

Bone

Vitamin K2 and Osteocalcin

● Osteocalcin is an vitamin K-dependent bone protein synthesized by

Osteoblasts (bone building cells)

● Osteocalcin – provides the bone matrix upon which calcium

crystallizes

● Osteocalcin - provides the “glue” that holds calcium in the bone –

giving structure and order to bone tissue; without it bone would be

fragile and easily broken

● Vitamin K2 “activates” osteocalcin through a process called

“carboxylation”

● Without carboxylated osteocalcin, calcium cannot be properly

utilized for bone structure

Bone

Eastern Japanese

Woman

5.26 ± 6.13

ng/mL

Western Japanese

Woman

1.22± 1.85

ng/mL

British

Woman

0.37 ± 0.20 ng/mL

Kaneki et al. Journal of Nutrition 2001

Vitamin K2 Intake and Fracture Rates

MK-7

Fracture

Rate

Epidemiology

Conclusion:

High Natto consumption means high Vitamin K2 blood levels and decreased hip fracture rates.

Vitamin K2 levels are inversely related to hip fracture rates.

● 2001 Kaneki, et al – high consumption of MK-7 levels from natto resulted in better levels of activated osteocalcin and a reduced risk fracture

● 2006 Ikeda, et al - natto consumption helps prevent the development of osteoporosis

● 2008 Yaegashi et al - showed that better vitamin K status attributed to Natto resulted in the reduction of hip fracture risk.

BoneNatural Vitamin K2 as MK-7 from

Natto is Effective

Osteocalcin carboxylation, BMD

and fracture rate

• Szulc et al: ucOC inversely correlates with BMD

• Knapen et al: ucOC inversely correlated with BMD

• Vegnaud et al: serum ucOC predicts hip fracture

• Luukinen et al: serum ucOC predicts hip fracture

• Szulc et al: high ucOC // 6-fold increased

fracture risk

Bone

Vitamin K2 and vascular health

CVD

Vitamin K2 and vascular health

CVD

“First it was blood

cholesterol that could give

you an early warning that a

heart attack might be around

the corner. Then came C -

reactive protein. And now

that doctors can get a better

look at what's inside your

heart and arteries, they are

taking a new interest in

something they have always

known was present in

problem vessels: calcium”.

Time Magazine 2005

Vitamin K2 and vascular health

CVD

Synergia Life Sciences P Ltd.Ashit Vora

Predictors of outcome in severe, asymptomatic aortic stenosis

Rosenhek, et al. New England Journal of

Medicine 2000

calciumMGP

MGP (Matrix Gla Protein) when

activated is only known inhibitor

of arterial calcification

Inactive MGP “leads” to calcification

Von Kossa staining. In black calcificationIn red localization of inactive MGP

Schurgers et al, Arterial Thrombosis and Vascular Biology 2005

CVD

K-vitamins in healthy and

atherosclerotic human aorta’s

(means from 3 donors)

0

50

100

150

200

250

Healthy aorta Atherosclerotic aorta

Vit

am

in K

(n

mo

l/g

tis

su

e)

Vitamin K1

Vitamin K2

CVD

Vitamin K2 and vascular health

Rotterdam Study

CVD

Objective To study the association of dietary intake of K1 and K2 with aortic calcification, CVD, and total death

Design cross-sectional analysis in healthy adults

Setting 4807 men and women aged 55 and older;

10 years follow-up

Measurements → calcification

→ end point (death)

Geleijnse et al. Journal of Nutrition 2004

When consuming daily 45 µg

dietary K2 you have:

50% reduction of arterial

calcification

50% reduction of

cardiovascular death

25 % reduction of all cause

mortality

as compared to low intake of dietary K2!

There was no correlation for vitamin K1 in this study!

Geleijnse et al. Journal of Nutrition 2004

Conclusion – Rotterdam

Study

0

20

40

60

80

100

120

<21 21-32 >32

Dietary vitamin K2 intake (µg/day)

Ris

k f

or

even

t (H

azard

rati

o)

Aorta calcification

Cardiovascular mortality

All cause mortality

CVD

G.C.M. Gast : Nutrition, Metabolism & Cardiovascular Diseases (2008), 1-7

Study Design Analysis of food intake using food frequency questionnaire

Subjects 16057 women, aged 49-70 yrs free from CVD

Treatment Vitamin K2 intake was 29.1 ± 12.8 mg/d

Outcome With every 10 µg/day increment of Vitamin K2 intake, an

inverse association between Vitamin K2 and risk of CHD

was observed.

A high intake of only Menaquinones, especially MK-7,

MK-8 and MK-9, could protect against CHD.

Synergia Life Sciences P Ltd.Ashit Vora

Osteoporosis & Cardiovascular

Disease

were once thought to be

unrelated conditions.

Studies indicate the common factor may be

ineffective metabolism and utilization of

Calcium.

Men and women with cardiovascular diseases and peripheral arterial disease tend to have lower areal and

volumetric bone mineral density (BMD) as well as faster bone loss, although findings vary according to skeletal site.

On one hand, severe forms of cardiovascular diseases (heart failure, myocardial infarction, hypertension, severe

AAC) are associated with higher risk of osteoporotic fracture, especially hip fracture.

Szulc P. Association between cardiovascular diseases and osteoporosis—reappraisal. BoneKEy reports. 2012;1:144. doi:10.1038/bonekey.2012.144.

Patients affected with osteoporosis, for example, have a higher risk of cardiovascular diseases than subjects with

normal bone mass.

Sprini D, Rini GB, Di Stefano L, Cianferotti L, Napoli N. Correlation between osteoporosis and cardiovascular disease. Clinical Cases in Mineral and Bone Metabolism. 2014;11(2):117-119.

HYPERVITAMINOSIS D – VITAMIN D TOXICITY

British Medical Journal (BMJ 2005): “Prevention of fractures with Calcium and

Vitamin D is not enough….combining Vitamin K, Vitamin D and Calcium seems

ideal…..”

Vitamin D

Osteoblasts

Uncarboxylated Osteocalcin

Stimulation

Catches calcium and puts on bone

Unable to put calcium on bone

Uncarboxylated MGPVitamin

K2-7

Unable to Prevent Arterial Calcification

Carboxylated Osteocalcin & MGP

Prevents arterial calcification

Stimulation

Diabetes and Insulin Resistance

Diabetes affects 194 million of the world’s population as estimated

by 2003 WHO survey with 10% of death accounted. The number of

deaths is expected to increase by more than 50% in the next 10

years. (Siegel & Narayan, 2008)

Vitamin K2 and Diabetes

“….we have demonstrated for the first time that vitamin K2 supplementation for 4 weeks increased insulin sensitivity in healthy young men, which seems to be related to increased cOC rather than modulation of inflammation.”

“Although our study could not provide the underlying mechanism, we speculate that cOC or vitamin K could modulate adipokines or inflammatory pathways other than the IL-6 pathways. Alternatively, cOC can directly regulate glucose disposal at skeletal muscle or adipose tissues.”

Conclusions—

With each 10 µg increment of menaquinone intake, a linear, inverse

association with risk of type 2 diabetes was observed.

Improvement in blood lipid profile.

This study shows that the intake of Menaquinones reduces the risk of

type 2 diabetes and helps in relieving Insulin Resistance.

Diabetes Care 33:1699–1705, 2010

• Objective To investigate whether dietary phylloquinone and

menaquinones intake are related to risk of type 2 diabetes.

• Design Prospective Cohort study in 38,094 Dutch men & Women,

aged 20-70 years

• Setting 918 incident cases of diabetes. 10.3 years follow-up

Conclusions—

Homeostasis model assessment of insulin resistance (HOMA-IR) & plasma

insulin concentrations were statistically significantly lower at the 36-month

visit.

Vitamin K supplementation for 36 months at doses attainable in the diet

may reduce progression of insulin resistance.

Diabetes Care 31:2092–2096, 2008

• Objective To investigate the hypothesis that vitamin K

supplementation for 36 months will improve insulin

resistance in older men and women.

• Design An ancillary study of randomized, double-blind, controlled

trial.

• Setting 355 men & women aged 60-80 years with supplementation

of 500 µg/day Vitamin K for 36 months.

Prognostic value of coronary artery calcium screening in

subjects with and without diabetes

Raggi, et al. J Amer Coll Cardio 2004

High

risk of

death

low risk

of death

•Vitamin K2 shown to reduce diabetes risk by 20% in a Dutch population

study over 10 years – 38,000 patients

•Vitamin K2 shown to decrease cancer risk. Study on 23,000 German

adults. Showed higher K2 intake associated with a lower likelihood of

developing and dying of cancer.

•Vitamin K2 shown to reduce prostate cancer risk by 35% in a epic study

in 11,319 men taking part in the Heidelberg cohort.

ADDITIONAL CLINICALLY PROVEN INDICATIONS

NEW DISCOVERIES IN VITAMIN K2 FUNCTION

A SEEMINGLY OMNIPRESENT VITAMIN…..

NEW MECHANISM OF K2 ACTION

➢Redox Function of Vitamin K2 – A significant role in the mitochondria

▪ Function in Nerve Health▪ Function in Muscle Contraction ▪ Function in Cardiac Function and Output

YET ANOTHER MECHANISM– 1950’s

Redox cycle activity of vitamin K was proposed by Martius, et al.

– 1960’s

At a later date Johnson et al, refuted this claim.

NADH

NAD+

ADP

ATP

Vitamin K-Mitochondrial Respiration

NADH

NAD+

ADP

ATP

BasalRespiration

ATPProduction

Proton Leak

MaximalRespiration

Non-mitochondrial Respiration

Oligomycin

X

FCCP

H+

Antimycin

X

Rotenone

X

Mitochondrial Respiration: Test sequence in sea horse XF-96 platform

NADH

NAD+

ADP

ATP

Mitochondrial Bioenergetics

Vitamin K BioenergeticsCellular Experiments

10 20 30 40 50

K2-7 Natural (10 µM)Control

0

OC

R (

PM

OL/

MIN

)

MINS

50

100

150

200

250

FCCPOLIGOMYCIN AA/ROT

NADH

NAD+

ADP

ATP

Mitochondrial Respiration: Test sequence in sea horse XF-96 platform

Neuroblastoma cell line

NADH

NAD+

ADP

ATP

“Neuroscientist Patrik Verstreken, associated with VIB and KU Leuven, succeeded in undoing the effect of one of the genetic defects that leads to Parkinson's using vitamin K2. His discovery gives hope to Parkinson's patients.”

www.sciencedaily.com/releases/2012/05/120511101240.htm

Science. 2012 Jun 8;336(6086):1306-10. doi: 10.1126/science.1218632. Epub 2012 May 10.

Vitamin K2 is a mitochondrial electron carrier that rescues pink1 deficiency.

Vos M1, Esposito G, Edirisinghe JN, Vilain S, Haddad DM, Slabbaert JR, Van Meensel S, Schaap O, De Strooper B, Meganathan R, Morais VA, Verstreken P. Author information Abstract

Human UBIAD1 localizes to mitochondria and converts vitamin K(1) to vitamin K(2). Vitamin K(2) is best

known as a cofactor in blood coagulation, but in bacteria it is a membrane-bound electron carrier.

Whether vitamin K(2) exerts a similar carrier function in eukaryotic cells is unknown. We identified

Drosophila UBIAD1/Heix as a modifier of pink1, a gene mutated in Parkinson's disease that affects

mitochondrial function. We found that vitamin K(2) was necessary and sufficient to transfer electrons in

Drosophila mitochondria. Heix mutants showed severe mitochondrial defects that were rescued by

vitamin K(2), and, similar to ubiquinone, vitamin K(2) transferred electrons in Drosophila mitochondria,

resulting in more efficient adenosine triphosphate (ATP) production. Thus, mitochondrial dysfunction was

rescued by vitamin K(2) that serves as a mitochondrial electron carrier, helping to maintain normal ATP

production.

Mitochondrial data

gave us a clue that

perhaps vitamin K2

could regenerate

dysfunctional

mitochondria in

tissue – a great

example would be

neurodegenerative

diseases.

N=30Dosing: 100mcg/dayDuration: 8 weeksResults: Well tolerated and significant alleviation of neuropathy

Vit K2-7 reduces severity of neuropathy

Results

Dose K2-7, 100mcg twice daily

1 2 3 40

1

2

3

4

5

Weeks

0

Seve

rity

of

neu

rop

ath

y

5 6 7 8

6

7

8

“Vitamin K2 and Vitamin K2 block 12-lypoxigenase (12-LOX) activation and inhibit

accumulation of reactive oxygen species (ROS). Vitamin K does not directly inhibit 12-

LOX enzyme activity when evaluated with purified 12-LOX in vitro, suggesting that

vitamin K prevents oxidative cell death indirectly by blocking activation of 12-LOX and

ROS generation.”

“The MK-4 or MK-7 can suppress IL6 and MK-4 can suppress prostaglandin E2 by inhibition of cox2; therefore, these compounds can reduce inflammation and the consequences of autoimmune diseases”

RESULTS: ➢ MS subjects had 1/4th the blood vitamin K2 levels compared to age-matched healthy

cohort. ➢ Female patients had significantly lower VK2 levels than males and a decrease with age

by approximately 10% per decade was found.

NADH

NAD+

ADP

ATP

Experimental Pharmacology

Muscle Relaxant activity

In vitro studies in smooth muscle and heart muscle

A) Guinea Pig ileum

Effect of MyoMax on smooth muscle contraction.

Acetylcholine: Inhibition by 21.62%, 40%, 81.81% at 1,2,4, concentration.

Barium Chloride: Vitamin K2 shows 30% inhibition

Histamine: Vitamin K2 shows 33% inhibition of histamine induced contraction

Muscle Relaxant activity

Frog Heart

Adrenalin: Vitamin K2 failed to inhibit adrenaline-induced contractions.

Inference: Vitamin K2 did not show inhibition of the adrenalic (beta

receptor agonist) response, which indicated that Vitamin K2 does not

exhibit beta blocker activity. Thus suggesting that the peripheral

perfusion effect is not due to a reduction in the arteriolar tone viz.

decreasing peripheral resistance.

Study Results: Therapeutic Activity and Safety of Vitamin K 2-7 in Muscle Cramps

Dose K2-7, 100mcg/day

Month

Group A

Dose K2-7, 0mcg

No

. of

cram

ps/

day

Dose K2-7, 100mcg/day

Month

Group B

Dose K2-7, 0mcg

No

. of

cram

ps/

we

ek

NADH

NAD+

ADP

ATP

CARDIAC OUTPUTStroke volume (SV) X Heart Rate (HR)

During endurance racing or high intensity exercise, increased fractional use of VO2max and HRmax causes a decrease in speed and performance. The increased fractional use of HRmax and subsequent decrease I speed is counter intuitive and is likely associated with metabolic limitations of the mitochondria.

➢ Studies on Marathon runners have shown that over the course of the race, the athletes increase fractional use of HRmax from around 80% of HRmax at the start to around 90% at the finish. This HR increase was associated with a continuous speed decrease.

➢ The upward drift in HR is one component of so-called “cardiovascular drift,” which is also characterized by a decrease in Stroke Volume (SV), overall Cardiac Output and in arterial and pulmonary pressures.

➢ Crandall and Gonzal´ez-Alonso, et al. have reported that a drop in SV (thus Cardiac Output) may be a major limiting factor in exhaustive exercise

A common problem in individuals with a high-degree of aerobic training is that they tend to experience decreases in maximal heart rate, which is a side effect of training induced expansion of blood volume and possibly increased oxidative stress. Unfortunately, reduced maximal heart rate negatively influences the ability to maximize cardiac output and performance

CONCLUSION: Vitamin K2-7 supplementation was

associated with a cardiovascular profile

characterized by increased cardiac output,

increased heart rate, and decreased blood lactate

compared to placebo following 8-weeks of exercise

training with the supplementation

10

15

20

25

Ma

x C

ard

iac

Ou

tpu

t (L

/min

)

Myomax-Base

Myomax-Post

Placebo-Base

Placebo-Post

12% increase of Cardiac Output

Equivalent to 60 liter more blood in 1 hour of work

“Without [vitamin K2-7] supplement, training induced

changes in maximal cardiac output could take 6

months of continuous training to achieve.”

“Supplementation with [vitamin K2-7] during training

appeared to reduce this training window by ~60%.”

ARGUABLY THE MOST IMPORTANT ANTIAGING NUTRIENT

The Mitochondrial Free Radical Theory of Aging (MFRTA) proposes that mitochondrial free radicals, produced as by-products during normal metabolism, cause oxidative damage. According to MFRTA, the accumulation of this oxidative damage is the main driving force in the aging process.

Especially Cardiac Mitochondrial Oxidant Production

“In a revealing study, a team of researchers showed that muscle tissue of a 90-year-old man contained 95% damaged mitochondria compared to almost no damage in that of a 5-year-old.”

By Kirk Stokel

Linnane AW, Kovalenko S, Gingold EB. The universality of bioenergetic disease: age-associated cellular bioenergetic

degradation and amelioration therapy. Ann N Y Acad Sci. 1998 Nov 20;854:202-13.

“Studies indicated a decrease of cardiac output with aging

at rest and with exercise.”

Melvin D. Cheitlin, MD. Am J Geriatr Cardiol. 2003;12(1)

ARGUABLY THE MOST IMPORTANT ANTIAGING NUTRIENT

“A substantially reduced output was a consistent finding

in older subjects.”

MARTIN BRANDFONBRENER, M.D., MILTON LAN DOW-NE, M.D. AND NATHAN W. SHOCK, PH.D.

CIRCULATION . October 1, 1955

CONCLUSIONBONE

HEALTH/OSTEOCALCIN: OSTEOPOROSIS,

DIABETES

ARTERIAL CALCIFICATION:

CARDIOVASCULAR DISEASE

BONE STRENGTH AND FORMATION:

ORAL HEALTH

INHIBITION OF SOFT TISSUE CALCIFICATION:

ELASTIN AND COLLAGEN

NEURONAL MITOCHONDRIA

RESCUE:NEURODEGENERATIVE DISORDERS SUCH AS PARKINSON’S. MS,

NEUROPATHY

MUSCLE MITOCHONDRIA

FUNCTION:CVD, CRAMPS,

CARDIAC OUTPUT (AGING AND

PERFORMANCE)

IL6- AND ROS INHIBITION:

AUTOIMMUNE CONDITIONS, AGE-RELATED DECLINE,

CANCERS, DIABETES