**4. Biomarker research studies**

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

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

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

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 "anticoa-

velocity [19].

174 Vitamin K2 - Vital for Health and Wellbeing

only for a couple of years.

± 47.1 months of use.

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 are also met.

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 independently associated with aortic calcification, and a concentration greater than 921 pM was a predictor of all-cause mortality in a crude analysis.

stroke surveys, 799 patients were examined who had already experienced a myocardial infarction, coronary revascularization, or first ischemic stroke. After a median follow-up of 5.6 years, 159 patients died. In the fully adjusted model, the patients in the highest quartile of dpucMGP (≥977 pM) had higher risk of all-cause and cardiovascular mortality, HRR 1.89 (95% CI 1.32–2.72) and 1.88 (95% CI 1.18–2.61), respectively [40]. For those subjects in the upper quartile of dp-ucMGP who also had heart failure, indicated by an elevated circulating brain natriuretic peptide level >100 ng/L, mortality risk was further increased, HRR 4.86 (95% CI 3.15–7.49) [41]. In a random sample from the general population from the Czech post-MONICA study, Mayer et al. [42] found that aortic stiffness, as measured by pulse wave velocity, was related to vitamin K status. Compared to the lowest quartile, the upper quartile of dp-ucMGP

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

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

177

One of the first intervention types was to confirm the utility of various species of MGP as biomarkers for vitamin K status. If you improve someone's status by oral supplementation, the biomarker should reflect this improvement in a dose-dependent manner. So, in 2012, Dalmeijer and coworkers [43] reported a randomized, double-blind placebo-controlled trial (RCT) of 60 people taking 0, 180, or 360 µg/day of vitamin K2 as menaquinone 7 (MK-7) for 12 weeks. Assays were performed for three different species of MGP: desphospho-uncarboxylated MGP (dp-ucMGP), desphospho-carboxylated MGP (dp-cMGP), and total uncarboxylated MGP (t-ucMGP). Vitamin K status was also measured using the ratio of uncarboxylated to carboxylated osteocalcin. (Note that the research field on the role of vitamin K2 in bones matured earlier than the field of cardiovascular effects of K2, so osteocalcin was well established as a vitamin K2 marker by this time.) After 12 weeks of the supplements, the osteocalcin ratio decreased significantly, with a 60% drop at 180 µg dose and a 74% drop at the 360 µg dose. The amount of dp-ucMGP decreased significantly and dose-dependently as well, by 31% and 46% at 180 and 360 µg, respectively. There were no changes in the placebo group, as expected. Changes in other species of MGP (dp-cMGP and t-ucMGP) were not different between placebo and the supplement groups. This study was one of the first intervention trials to validate the usefulness of dp-ucMGP as a biomarker for vitamin K status. Observational studies had been carried out, but the intervention studies took the research one more step

In the same year, another RCT was reported of 42 Dutch men and women randomized to receive 0, 10, 20, 45, 90, 180, or 360 µg/day of vitamin K2 as MK-7. The ratio of uncarboxylated to carboxylated osteocalcin was determined along with the concentration of dp-ucMGP. The upper three doses (90, 180, and 360 µg/day) increased the carboxylation of osteocalcin and decreased the amount of dp-ucMGP. In these healthy adults aged 18–45, no adverse effects were seen on the generation of thrombin, indicating that coagulation factors were not perturbed by the additional supply of vitamin K2. This is reasonable, for the coagulation factors are generally all carboxylated. Only the extrahepatic vitamin K–dependent proteins seem to suffer

when there is a shortage of vitamin K, as explained by the triage theory [29].

(≥671 pM) has an increased odds ratio of 1.73 (95% CI 1.17–2.5).

**5. Intervention studies**

toward maturity.

What about when people on dialysis are also taking oral anticoagulants? Among 160 hemodialysis patients in Belgium, the 23 who were treated with anticoagulants had much higher circulating concentrations of dp-ucMGP, 5604 pM (interquartile range: 3758, 7836 pM) and 1939 pM (interquartile range: 1419, 2841pM) for the anticoagulant treated and non-treated groups [33].

In a study of 147 patients with symptomatic severe calcific valvular aortic stenosis, the levels of dp-ucMGP were associated with cardiac function and long-term mortality in multivariate analysis [34]. Increasing severity of disease was related to dp-ucMGP concentrations in a study of 179 patients with chronic heart failure [35].

The dp-ucMGP assay was checked for correlation with vitamin K status and coronary artery calcification in a study of older adults without cardiovascular disease [36]. While the assay did correlate well with plasma phylloquinone, uncarboxylated prothrombin, and serum uncarboxylated osteocalcin, there was no association between dp-ucMGP levels and coronary artery calcification. Shea and coworkers [36] presented data that are not consistent with the other reports on this assay. Perhaps the assay works better for much higher levels of dp-ucMGP, such as found in disease states. This study looked at older adults without clinical cardiovascular disease, whose levels of dp-ucMGP were much lower than subjects with cardiovascular disease (CVD). As suggested by the authors, the coronary artery calcification analyzed in this report may have been more in the intimal layer, rather than in the medial layer, where MGP has a greater role [28]. A more recent study involving 200 health women found a borderline statistically significant relationship between dp-ucMGP and coronary artery calcification, as well as a strong relationship between dp-ucMGP and vitamin K status (ratio of carboxylated osteocalcin) [37]. The results in [36] appear to the exception, as there is a consistent relationship between dp-ucMGP, vitamin K status, and health outcomes involving arterial calcification in all of the other studies examined.

Other studies have generally found that the biomarker dp-ucMGP does correlate with vitamin K status and disease outcomes related to arterial calcification. In the EPIC-NL cohort, 518 participants were identified as diabetic at baseline [38]. After 11.2 years of follow-up, incidence of CVD was significantly associated with baseline concentrations of dp-ucMGP, but not other species of MGP. The hazard ratio per standard deviation (HRSD) of dp-ucMGP for all CVD was 1.21 (95% CI 1.06–1.38), for peripheral artery disease HRSD = 1.32 (95% CI 1.07–1.65), and for heart failure HRSD = 1.85 (95% CI 1.42–2.17). The prospective Longitudinal Aging Study, Amsterdam (LASA) examined 577 people aged >55 years who were free of CVD at the baseline [39]. There were 40 incident cases of CVD during the 5.6 years of follow-up. For the highest tertile compared to the lowest tertile of dp-ucMGP, there was a hazard ratio of 2.69 (95% CI 1.09–6.62) for being diagnosed with CVD. The carboxylated form of MGP was not related to risk of CVD.

Two Czech Republic prospective studies have examined the usefulness of the dp-ucMGP as a biomarker to predict cardiovascular mortality. From the EUROASPIRE III and EUROASPIRE- stroke surveys, 799 patients were examined who had already experienced a myocardial infarction, coronary revascularization, or first ischemic stroke. After a median follow-up of 5.6 years, 159 patients died. In the fully adjusted model, the patients in the highest quartile of dpucMGP (≥977 pM) had higher risk of all-cause and cardiovascular mortality, HRR 1.89 (95% CI 1.32–2.72) and 1.88 (95% CI 1.18–2.61), respectively [40]. For those subjects in the upper quartile of dp-ucMGP who also had heart failure, indicated by an elevated circulating brain natriuretic peptide level >100 ng/L, mortality risk was further increased, HRR 4.86 (95% CI 3.15–7.49) [41]. In a random sample from the general population from the Czech post-MONICA study, Mayer et al. [42] found that aortic stiffness, as measured by pulse wave velocity, was related to vitamin K status. Compared to the lowest quartile, the upper quartile of dp-ucMGP (≥671 pM) has an increased odds ratio of 1.73 (95% CI 1.17–2.5).
