**1. Introduction**

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 to be useful only for coagulation of the blood.

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

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

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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

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

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

to their atherosclerotic lesions.

likely and made the results more robust.

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 until the modern research era.

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 fully interchangeable.

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 research together.

Vitamin K2: Implications for Cardiovascular Health in the Context of Plant-Based Diets, with Applications for Prostate... http://dx.doi.org/10.5772/63413 171
