**2. Observational studies of dietary vitamin K intake and health outcomes**

**1. Introduction**

170 Vitamin K2 - Vital for Health and Wellbeing

to be useful only for coagulation of the blood.

until the modern research era.

fully interchangeable.

research together.

The story of vitamin K goes back to the 1930s, when Henrik Dam at the University of Copenhagen isolated an antihemorrhagic vitamin that was fat soluble but different from previously isolated vitamins A, D, and E [1]. He found high concentrations of the vitamin in hog liver fat and hemp seed, but found it to be virtually absent in cod liver oil while testing a variety of animal organs, hen eggs, cereals, seeds, vegetables, and various fats and oils including butter fat. The initial quantification was all based on the time required to coagulate blood from a chicken. The term "vitamin K" was used as short for the German term "koagulation." Because of the type of assay used, all vitamin K factors, whether K1 or K2, were thought

At the same time as Henrik Dam was working out the details of the antihemorrhagic vitamin, there was another investigator working in the USA with a vitamin factor not found in cod liver oil, but which worked synergistically with them to promote proper mineralization, bone growth, and to prevent dental caries [2]. Dr. Weston Price found that the amount of the factor in mammalian milk varied with the "nutrition of the animal," with highest levels from milk of cows that were consuming rapidly growing green grass. Because Dr. Price's assay was based on the release of iodine from hydroiodic acid, a test for peroxides, no connection was made between his discovery and any other vitamin activity. What Price found was the activity of vitamin K2, formed in mammary glands from phylloquinone, vitamin K1, found in abundance in rapidly growing green grass [3]. No connection was made between the anticoagulation activity of K1 and the mineral-directing activity of vitamin K2

Because of the way vitamin K was discovered for its coagulation function, it was assumed that this was its only function for many years. The Recommended Daily Allowance (RDA) for vitamin K is based on its coagulation function. Though both phylloquinone (phyllo—from plants) and menaquinones support coagulation, as we explore in this chapter, there are clear differences between the functioning of phylloquinone and the menaquinones, and they are not

This chapter is a review of vitamin K2 research in the area of cardiovascular health, especially dealing with arterial calcification. It is organized in a loose chronological order, following the themes of the research as the field matured. First, there were observations about dietary intake of K1 and K2 and health outcomes, forming hypotheses to be tested further. Associations were discovered at this stage. Harmful observations with warfarin-type drugs, which are vitamin K antagonists, were seen early on in the research cycle as well. Hypotheses were then explored with animal studies, looking for mechanisms and biomarkers—to help get endpoints that were quicker to develop than mortality and disease. Then came the rise of biomarker studies, using markers for vitamin K status (dp-ucMGP), as well as markers for risk factors, such as arterial calcification, pointing to disease outcomes. As the science matured even further, intervention studies related to K2 and biomarker changes emerged. The final stage is now emerging with intervention studies looking not just at biomarkers but disease outcomes, thus tying all of the Observational studies help form hypotheses by finding associations between factors that may or may not be related to the health outcome of interest. One of the early population-based studies in the 1990s examined the vitamin K status of 113 postmenopausal women [4]. Dietary intake of vitamin K was assessed along with examinations for the presence or absence of aortic calcified lesions. Blood samples were assayed for osteocalcin, a vitamin K-dependent protein that is responsible for proper deposition of minerals in bone tissue. It was thought that some forms of osteocalcin might be a marker for vitamin K status. The first type had low affinity for hydroxyapatite, while the second had high affinity. Women with calcified lesions (*n* = 34) had a lower intake of total vitamin K as well as a higher amount of low-affinity osteocalcin. Together these results indicated that these women had impaired vitamin K status that might be related to their atherosclerotic lesions.

A groundbreaking study took place in the Netherlands which changed the way vitamin K2 was thought of afterward. First, a very careful survey of foods eaten in the Netherlands was carried out by interview-based dietary intakes of over 5400 people, guided by a validated food frequency questionnaire (FFQ). For many animal-based foods, the quantitation of vitamin K2 had not been done, so this was carried out and reported as well. The mean intake of K1 and K2, respectively, varied from 124 and 9.3 µg/day in the lowest quartile to 375 and 45 µg/day in the upper quartile [5]. A report of health outcomes from the Rotterdam Study in 2004 from 4800 of these subjects revealed that even though vitamin K2 was a minor part of the total vitamin K intake, only K2 and not K1 intake was associated with a lower risk of disease [6]. For people in the upper third of intakes of K2 compared to the lowest third, there were decreases in relative risks of coronary heart disease (CHD) mortality (57%), all-cause mortality (26%), and severe aortic calcification (52%). Even though intakes of K2 were only about 10% as much as the amounts of K1, their effect on cardiovascular disease was greater. Cheese was the primary source of menaquinones in this cohort in the Netherlands, not exactly highly regarded as a heart healthy food. This fact made confounding by other "healthy" nutrients less likely and made the results more robust.

Similar results have been seen in a second study, from the Prospect-EPIC cohort, also from the Netherlands [7]. About 16,000 women aged 49–70 were followed for 8 years. Vitamin K1 and K2 intakes were estimated from a FFQ . Vitamin K2 intake varied from <20 to >36 µg/day across quartiles. For every increase of 10 µg of K2, there was a 9% reduction in hazard ratio of risk of CHD, with the effects coming mainly from menaquinone subtypes MK-7, MK-8, and MK-9. There was no association between intake of K1 and CHD, as seen in the Rotterdam Study.

Two large cohort studies have been analyzed for associations between vitamin K intake and CHD. When the Nurses' Health Study (NHS) cohort was analyzed for an association between dietary vitamin K intake and cardiovascular outcomes, it was found that K1 intake was associated with a 21% decrease in multivariate relative risk of total CHD [8]. The association was attenuated by adjustments for other dietary factors and lifestyle patterns, so that it was not apparent to the authors whether the results were due to vitamin K1 or that K1 was just a marker for a lifestyle pattern associated with a high intake of K1. Median intakes of K1 for the lowest and highest quintiles were 87 and 300 µg/day for the NHS cohort. In another large cohort, the Health Professionals' Follow-up Study, there was a decrease in the relative risk of total CHD across increasing quintiles of vitamin K1 intake. However, when the results were adjusted for lifestyle and other dietary factors, the trend was no longer significant [9]. Neither of these large cohorts reported dietary intakes of K2, perhaps because the database for menaquinone concentrations in the USA was not complete at that time, nor is it fully available at the time of writing this chapter (only partial data are available on MK-4 but none on higher menaquinones), 17 years after such data were obtained for the Rotterdam Study [5]. The negative results from these cohorts for vitamin K1 and CHD only reinforce just how striking the results were from the Dutch studies for vitamin K2. The findings from the Netherlands were not expected or anticipated by many.

0.64 for cancer mortality and all-cause mortality. It is possible that enough vitamin K1 and other plant-based protective nutrients such as folate, vitamin C, fiber, potassium, and magnesium were supplied by the diet that even those participants who had low intakes of menaquinones were still not at an elevated risk compared to other subjects in the PREDIMED cohort. Participants who increased their intake of either vitamins K1 or K2 or both during the course of the study experienced decreased risk of cardiovascular mortality (K1 only), cancer mortality, and all-cause mortality. The main conclusion from this observational study perhaps is that eating plants is good for you, and the Mediterranean Diet is generally beneficial, as seen in

Vitamin K2: Implications for Cardiovascular Health in the Context of Plant-Based Diets, with Applications for Prostate...

http://dx.doi.org/10.5772/63413

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Another line of evidence that led to the discovery of the role of menaquinones was the effect of blood thinning drugs such as warfarin and coumarin. While this class of drugs has been very helpful in preventing strokes in the short term, it has also caused damage long term, as people are often prescribed blood thinners for many years. In 1998, it was reported by Price et al. [15] that warfarin caused calcification of the elastic lamellae in rat arteries and heart valves within a period of 2 weeks, with increasing intensity each week. Vitamin K1 was given concurrently to maintain normal blood coagulation. At the time, menaquinone was not even mentioned in the article, not even in the discussion. The discovery of importance was that warfarin had negative side effects for arterial calcification that were not counteracted by

This discovery nearly coincided in time with work on an MGP-deficient mouse model. Matrix γ-carboxylation protein, or matrix Gla protein (MGP), was originally discovered in bone tissue but is actually expressed in many tissues of the body, including vascular smooth muscle cells and chondrocytes in cartilage. MGP requires the activation by vitamin K in order to bind calcium ions and prevent crystallization of calcium. These MGP-deficient mice developed normally to term but died within 2 months as a result of extensive arterial calcification, which led to blood vessel rupture [16]. Also seen was the inappropriate calcification of cartilage, including the growth plate of bones. This MGP-deficient mouse model clearly showed that MGP has a central, active role in preventing calcifications of arterial walls and also of cartilage. This research coupled together with the warfarin-caused calcification pointed to a central role

Further work on the interrelationship between vitamin K1 and K2 was spurred on by feeding studies in rats. When rats were made vitamin K deficient, then fed only K2 as MK-4, they accumulated MK-4 especially in the pancreas, aorta, fatty tissues, and brain. Liver and serum levels of MK-4 were low. When vitamin K–deficient rats were fed only K1, they accumulated K1 in the liver, heart, and fatty tissues, and they also accumulated MK-4 in the same way as the rats that were fed MK-4, indicating that there was conversion from phylloquinone to MK-4 [17]. So, with a warfarin-rat model that was able to induce arterial calcification and knowing that there were differences between K1 and K2 distributions in the rats and that K2 prevented heart disease in the Rotterdam Study, Spronk and coworkers set out to see how to prevent

another report from this cohort [14].

vitamin K1.

**3. Vitamin K antagonist studies**

for MGP in controlling arterial calcification.

The question of whether vitamin K intake is related to arterial calcifications has been probed in two observational studies. In a cross-sectional study of 1689 women, dietary intakes of vitamin K1 and K2 were estimated with an FFQ, and standard screening mammograms were assessed for the presence of breast arterial calcifications [10]. Unadjusted results showed an inverse association between intake of vitamin K2 and breast arterial calcifications, but adjustments for aging, smoking, diabetes, and dietary factors made the association no longer significant. Adjustment for diabetes may have been unwise, as vitamin K2 intake has also been shown to reduce the risk of diabetes among 38,000 Dutch men and women in the Prospect-EPIC cohort mentioned previously [11]. So, this adjustment may have attenuated the results enough to make the association no longer statistically significant.

In another cross-sectional study, 564 postmenopausal women were examined for an association between coronary calcifications and intake of vitamins K1 and K2 [12]. Women were chosen from the Prospect-EPIC cohort study. In this cohort, cheese contributed 54%, milk products contributed 22%, and meat contributed 15% of the K2 intake. The mean intake of K2 ranged from 18.0 ± 4.5 to 48.5 ± 9.0 in the lowest and highest quartiles, respectively. Examinations found that 62% of the women had coronary calcifications. In the model adjusted for age and cardiovascular risk factors, increased menaquinone intakes were associated with a decreased calcification prevalence ratio of 0.80 (95% CI: 0.65–0.98), comparing highest to lowest quartile.

A more recent observational study reported findings contrary to those found in the Prospect-EPIC cohort and the Rotterdam Study. The PREDIMED cohort is a Spanish study to examine the effect of adoption of the Mediterranean Diet on cardiovascular, cancer, and all-cause mortality. The intakes of vitamins K1 and K2 were estimated by FFQ, and endpoints of cardiovascular, cancer, or all-cause mortality were tracked for a median follow-up of 4.8 years. Energy-adjusted intakes of vitamins K1 and K2, respectively, ranged from 170 and 18.4 µg/day in the lowest quartiles for each vitamin to 626 and 57.5 µg/day in the upper quartiles [13]. People in the upper quartile consumed about twice as many vegetables, especially leafy greens, as those in the lowest quartile. The upper quartile of vitamin K intake in this cohort adopting the Mediterranean Diet was substantially higher than seen in the other observational studies of other European or American cohorts. No protective effects for higher intakes of menaquinones were seen in this cohort for cardiovascular mortality, cancer mortality, or all-cause mortality. However, high intakes of phylloquinone lead to a reduced hazard ratio of 0.54 and 0.64 for cancer mortality and all-cause mortality. It is possible that enough vitamin K1 and other plant-based protective nutrients such as folate, vitamin C, fiber, potassium, and magnesium were supplied by the diet that even those participants who had low intakes of menaquinones were still not at an elevated risk compared to other subjects in the PREDIMED cohort. Participants who increased their intake of either vitamins K1 or K2 or both during the course of the study experienced decreased risk of cardiovascular mortality (K1 only), cancer mortality, and all-cause mortality. The main conclusion from this observational study perhaps is that eating plants is good for you, and the Mediterranean Diet is generally beneficial, as seen in another report from this cohort [14].
