**5. Intervention studies**

independently associated with aortic calcification, and a concentration greater than 921 pM

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

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

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

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

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-

was a predictor of all-cause mortality in a crude analysis.

of 179 patients with chronic heart failure [35].

all of the other studies examined.

risk of CVD.

groups [33].

176 Vitamin K2 - Vital for Health and Wellbeing

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 toward maturity.

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].

Patients with chronic kidney disease and those undergoing dialysis have been shown repeatedly to suffer with high levels of uncarboxylated vitamin K–dependent proteins and have high levels of arterial calcification as well. (The most deficient group is the same patients taking vitamin K antagonist drugs concurrently.) In order to prepare for a RCT with a disease endpoint of stabilizing or reversing arterial calcification in this patient group, a supplement trial was conducted in the Netherlands with 50 hemodialysis patients [44]. An age-matched healthy control group was selected also for comparisons. The hemodialysis patients were randomized into groups taking 45, 135, or 360 µg/day of vitamin K2 as MK-7 for 6 weeks. Measurements were taken for the levels of uncarboxylated osteocalcin (ucOC), dp-ucMGP, and PIVKA-II. PIVKA-II is a prothrombin liver protein that is only seen in the circulation under situations of severe vitamin K deficiency, such as found in hemodialysis patients. At baseline hemodialysis, patients, compared to healthy controls, had 4.5-fold higher dp-ucMGP levels and 8.4-fold higher uncarboxylated osteocalcin levels. PIVKA-II levels were detectable in 49 of the 50 hemodialysis patients. There was a dose-dependent response to the MK-7 treatment, with the 45 µg dose being little different than a placebo, the 135 µg dose giving 37%, 11%, and 34% changes in dp-ucMGP, ucOC, and PIVKA-II, respectively, which was almost significant, and the 360 µg dose yielding statistically significant changes of 61%, 34%, and 42% decreases in dp-ucMGP, ucOC, and PIVKA-II. This short trial showed both the severity of the vitamin K deficiency in this hemodialysis patient group as well as the effectiveness of a relatively high dose (360 µg/day) of MK-7 in bringing down functional markers of vitamin K deficiency.

age group are close to the range at which excess heart disease mortality occurred in other studies, >977 pM in [40] and >921 pM in [32]. Calcification of arteries takes time and finally

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

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

179

In one of the first RCT studies to report a disease endpoint, Knapen and coworkers examined arterial stiffness in 244 women after supplementation for 3 years with 180 µg/day of MK-7 [47]. Previous work by this research group had linked increased levels of dp-ucMGP with arterial stiffness [48]. Compared to the placebo group, dp-ucMGP levels dropped by about 50%. The K2 treatment resulted in improvements in the whole group, but the best results for improving stiffness were seen in those women who started with the worse condition, with a stiffness index β above the median of 10.8. For these women, there were improvements in distention, compliance, distensibility, Young's Modulus, and the local carotid pulse wave velocity. Not all became normal, as acute phase markers interleukin-6, C-reactive protein, and tumor necrosis factor-α remained abnormal as well as the markers of endothelial dysfunction vascular cell

A Polish RCT study examined disease endpoints for vascular calcification and progression of atherosclerosis in 42 women who had chronic kidney disease but were not undergoing dialysis [49]. The women were split into three groups followed for 270 days: one taking 90 µg/ day of MK-7 (K), one group taking 90 µg/day of MK-7 plus 10 µg/day of vitamin D as cholecalciferol (K+D), and one taking only 10 µg/day of vitamin D (D). The results were that the intervention slowed the progression of atherosclerosis but did not significantly slow the increase in coronary artery calcium score. The reason for this lack of success is in the dose of MK-7 used. As noted in the studies by Westenfeld et al. [44] and Caluwé et al. [45], a daily dose of around 360 µg is needed to significantly reduce dp-ucMGP. Changes in dp-ucMGP were reported to be significant in this Polish trial, but actual change was from 1077.1 ± 507.7 to 961.5 ± 506.7, or a change of only 115.6 pM. While this change may have been statistically significant, it was clinically irrelevant. This study also reveals why it is important to develop research stepwise to ensure that interventions will be successful. If the treatment is not expected to yield

a large change in biomarker studies, how would it yield a successful clinical result?

available, which is discussed in the section on plant-based diets below.

**6. Prostate health and vitamin K2**

A rationale and study protocol has been published for the VitaK-CAC trial [50]. For this 2-year trial, the intervention will be 360 µg/day of MK-7. Patients with coronary artery disease will be monitored for coronary artery calcification as the primary endpoint. Secondary endpoints will be arterial structure and function. This study appears to be well designed to yield good results, as long as the arterial calcification seen in coronary arteries is susceptible to the MGP mechanism of action. Nutrients have a way of working in complimentary fashion, if not synergistically. The best chance for successful intervention is to use all of the known tools

While the focus of this chapter has been on arterial calcification and cardiovascular health, it has come to the attention of this author that there is evidence that supports a hypothesis that

takes a toll on the elderly if not protected against through the years.

adhesion molecule and E-selectin.

As a follow-up to the Westenfeld Study [44] just reviewed, another larger study with hemodialysis patients was carried out with slightly higher doses, but administered three times a week by a nurse after dialysis [45]. This method was used to increase patient compliance. Doses for the 200 patients were 360, 720, and 1080 µg of MK-7 three times as week for 8 weeks. This works out to equivalent daily doses of 154, 309, and 463 µg/day. After 8 weeks, levels of dpucMGP decreased by 17, 33, and 46% in the three dosage groups, respectively. Results here were similar to those in [44], but with less decrease in relative change in dp-ucMGP at the highest dose (46% vs. 61%). However, the absolute differences before and after intervention were greater in [45]. Absolute changes in dp-upMGP in the Westenfeld Study were −404, −730, and −978 pM at 45, 135, and 360 µg/day [44]. In a study by Caluwé and coworkers [45], absolute changes in dp-ucMGP were −566, −962, and −1487 pM for the equivalent daily doses of 154, 309, and 463 µg/day. So, the outcomes were very similar, especially given that the second trial was 8 weeks long rather than just 6 weeks.

How widespread is extrahepatic vitamin K insufficiency? A cross-sectional sample of 896 healthy individuals showed that dp-ucMGP levels increased with age, staying around 200 pM until about age 40 and then increasing up to over 600 pM for those >70 years of age [46]. Levels of dp-ucMGP decreased in children and adults when given supplemental MK-7, again showing that dp-ucMGP was a biomarker that responded to extrahepatic vitamin K status, and that many adults had less than optimal levels of vitamin K. These levels are not optimal, neither are they as severe as hemodialysis patients, who averaged around 3000 pM but ranged to over 7000 pM in some individuals [44, 45]. Hemodialysis patients who took vitamin K antagonists had a mean dp-ucMGP of about 5600 pM [33]. But the values >600 pM in the >70 age group are close to the range at which excess heart disease mortality occurred in other studies, >977 pM in [40] and >921 pM in [32]. Calcification of arteries takes time and finally takes a toll on the elderly if not protected against through the years.

In one of the first RCT studies to report a disease endpoint, Knapen and coworkers examined arterial stiffness in 244 women after supplementation for 3 years with 180 µg/day of MK-7 [47]. Previous work by this research group had linked increased levels of dp-ucMGP with arterial stiffness [48]. Compared to the placebo group, dp-ucMGP levels dropped by about 50%. The K2 treatment resulted in improvements in the whole group, but the best results for improving stiffness were seen in those women who started with the worse condition, with a stiffness index β above the median of 10.8. For these women, there were improvements in distention, compliance, distensibility, Young's Modulus, and the local carotid pulse wave velocity. Not all became normal, as acute phase markers interleukin-6, C-reactive protein, and tumor necrosis factor-α remained abnormal as well as the markers of endothelial dysfunction vascular cell adhesion molecule and E-selectin.

A Polish RCT study examined disease endpoints for vascular calcification and progression of atherosclerosis in 42 women who had chronic kidney disease but were not undergoing dialysis [49]. The women were split into three groups followed for 270 days: one taking 90 µg/ day of MK-7 (K), one group taking 90 µg/day of MK-7 plus 10 µg/day of vitamin D as cholecalciferol (K+D), and one taking only 10 µg/day of vitamin D (D). The results were that the intervention slowed the progression of atherosclerosis but did not significantly slow the increase in coronary artery calcium score. The reason for this lack of success is in the dose of MK-7 used. As noted in the studies by Westenfeld et al. [44] and Caluwé et al. [45], a daily dose of around 360 µg is needed to significantly reduce dp-ucMGP. Changes in dp-ucMGP were reported to be significant in this Polish trial, but actual change was from 1077.1 ± 507.7 to 961.5 ± 506.7, or a change of only 115.6 pM. While this change may have been statistically significant, it was clinically irrelevant. This study also reveals why it is important to develop research stepwise to ensure that interventions will be successful. If the treatment is not expected to yield a large change in biomarker studies, how would it yield a successful clinical result?

A rationale and study protocol has been published for the VitaK-CAC trial [50]. For this 2-year trial, the intervention will be 360 µg/day of MK-7. Patients with coronary artery disease will be monitored for coronary artery calcification as the primary endpoint. Secondary endpoints will be arterial structure and function. This study appears to be well designed to yield good results, as long as the arterial calcification seen in coronary arteries is susceptible to the MGP mechanism of action. Nutrients have a way of working in complimentary fashion, if not synergistically. The best chance for successful intervention is to use all of the known tools available, which is discussed in the section on plant-based diets below.
