**3. Vitamin K antagonist studies**

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

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

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

were not expected or anticipated by many.

172 Vitamin K2 - Vital for Health and Wellbeing

enough to make the association no longer statistically significant.

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 vitamin K1.

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 for MGP in controlling arterial calcification.

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 arterial calcification [18]. When warfarin-treated rats were fed K1, the rats got arterial calcification, as shown before [15], even at the highest tested dose of K1. But when the warfarintreated rats were fed K2 as MK-4 or K1 together with MK-4 simultaneously, the arterial calcification was prevented. The picture was becoming clearer. Further studies have shown that in this rodent model warfarin treatment not only causes arterial calcification but functionally augments aortic peak velocity, aortic valve-peak gradient, and carotid pulse-wave velocity [19].

gulants," the preferred term for many of these authors is vitamin K antagonists, for this is their

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

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

175

Calcification of arteries had originally been thought of as a one-way process, without reversibility, similar to the thinking about coronary plaque. However, just as regression can be seen of atherosclerotic plaques [27], so calcification of arteries, too, is a dynamic process. Using the warfarin-treated rat as a model for arterial calcification, Schurgers et al. [28] first fed rats for 6 weeks on the diet to induce calcification. Then the warfarin treatment was stopped and rats were fed normal levels of K1, or high levels of K1 or K2 (as MK-4). Normal levels of vitamin K1 continued to progressively increase calcification, but both forms of vitamin K at high doses reversed arterial calcification by about 50%. Vitamin K1 does not work as long as warfarin is present, as it inhibits the conversion of K1 into MK-4. But when the warfarin treatment is stopped, this research clearly showed that this calcification process could be reversed by high

One difficulty in this field of research is determining the functional vitamin K status of an individual. A blood test of vitamin K levels is not sufficient. The amount of vitamin K in the blood is very small and generally only reflects the vitamin K1 that was consumed within the last 4 hours or so, as K2 levels are too low to assay in blood, and K1 clears from the blood with triglycerides. As research progressed, it became increasingly apparent that there were more functions for vitamin K than originally discovered. Coagulation was only the most immediately obvious function of vitamin K in the liver. But the observation studies and vitamin K antagonist research indicated more functions beyond coagulation, dealing with regulation of calcification throughout the body. McCann and Ames [29] elaborated on this multifunction vitamin, indicating that triage theory helps us understand the distribution of vitamin K to various organs. Triage theory states that the most critical functional needs are met first in the body (coagulation) when there is a shortage of a micronutrient. Then when there is an abundance of the micronutrient, all of the secondary functions important to long-term health

For these reasons, and possibly others, functional tests for vitamin K status for these secondary functions beyond coagulation were sought. Osteocalcin, a vitamin K–dependent protein found in bone, can be measured in the circulation as well. The ratio of carboxylated to undercarboxylated or uncarboxylated osteocalcin is one biomarker for functional vitamin K status. However, this applies more to the status of vitamin K as it applies to bones. Since MGP is involved in arterial calcification, assays for determining the concentrations of various forms of MGP were developed [30, 31]. Of the various forms of MGP, the dephosphorylated, uncarboxylated form has been most closely related to arterial calcification. Among coumarin users an elevated dp-ucMGP level was found compared to controls (1439 ± 481 pM vs. 299 ± 163 pM, respectively) [22]. In a cohort of 101 chronic kidney disease patients, the level of dpucMGP increased with increased severity of the disease [32]. Plasma dp-ucMGP was also

mode of action.

are also met.

doses of vitamin K, especially K2.

**4. Biomarker research studies**

The work in rats spurred investigators to look at the effect of anticoagulants in people. In one study, aortic heart valves were examined that had been replaced during routine surgery. Some patients received preoperative marcoumar treatment, for between 16 and 35 months, with a mean of 25 months. When compared with patients who did not have any blood thinner treatment, there was about twice as much calcification on the valves from patients who had received the marcoumar [20]. The mean calcified area on the valve went from 16% in the untreated group to 37% in the anticoagulant group. In a cross-sectional study, coronary artery calcium scores and valvular calcium scores were compared between patients on long-term use of anticoagulants and patients without any anticoagulant therapy [21]. The Agaston calcium scores were about double in the anticoagulant treatment group, indicating that the effects of anticoagulants seen in mice and rats are also present in people, even when the treatment was only for a couple of years.

These initial results have been confirmed by further studies. Rennenberg et al. [22] examined 19 patients younger than 55 years of age who had used coumarins for more than 10 years but did not have other cardiovascular risk factors. These patients were compared with 18 matched healthy controls. When they examined femoral arteries, they found the coumarin users had 8.5 times the chance of having arterial calcification compared to the healthy controls. Fourteen of 19 coumarin users, but only 4 of 18 controls had femoral arterial calcifications. Another crosssection examination of low-risk atrial fibrillation patients found that both age and use of oral anticoagulants were related to increased coronary calcium score [23]. And as length of time using the anticoagulants increased, the coronary calcium score also increased, going from 53 ± 115 for no use to 90 ± 167 for 6–60 months, and to 236 ± 278 for >60 months of use. These findings were also confirmed in a series of 133 oral anticoagulant users matched by age, gender, and Framingham cardiovascular risk score [24]. Agaston calcium scores increased from 79.6 ± 159.8 for use of 2.5 ± 1.5 months, to 142.4 ± 306.0 for 18.7 ± 8.8 months, to 252.5 ± 399.3 for 86.4 ± 47.1 months of use.

In women undergoing screening mammography who took warfarin, breast arterial calcifications were also more common with increasing length of warfarin treatment [25]. Prevalence of breast arterial calcifications increased from 25.0% for <1 year of therapy to 74.4% for >5 years of therapy. So, these calcifications can appear in peripheral tissues as well, not just in the aorta. To show this peripheral effect further, and in men, after completing the breast arterial calcification study, Han and O'Neill examined radiographs of ankles and feet, retrospectively, and checked records for warfarin use prior to the x-ray [26]. They found a significant increase, from 19% to 38% prevalence, in peripheral arterial calcifications in people who had been using warfarin for at least 5 years prior to their x-ray. While these drugs could be termed "anticoagulants," the preferred term for many of these authors is vitamin K antagonists, for this is their mode of action.

Calcification of arteries had originally been thought of as a one-way process, without reversibility, similar to the thinking about coronary plaque. However, just as regression can be seen of atherosclerotic plaques [27], so calcification of arteries, too, is a dynamic process. Using the warfarin-treated rat as a model for arterial calcification, Schurgers et al. [28] first fed rats for 6 weeks on the diet to induce calcification. Then the warfarin treatment was stopped and rats were fed normal levels of K1, or high levels of K1 or K2 (as MK-4). Normal levels of vitamin K1 continued to progressively increase calcification, but both forms of vitamin K at high doses reversed arterial calcification by about 50%. Vitamin K1 does not work as long as warfarin is present, as it inhibits the conversion of K1 into MK-4. But when the warfarin treatment is stopped, this research clearly showed that this calcification process could be reversed by high doses of vitamin K, especially K2.
