Elsevier

Atherosclerosis

Volume 225, Issue 2, December 2012, Pages 397-402
Atherosclerosis

The effect of menaquinone-7 supplementation on circulating species of matrix Gla protein

https://doi.org/10.1016/j.atherosclerosis.2012.09.019Get rights and content

Abstract

Objective

To investigate whether menaquinone-7 (MK-7) supplementation increases carboxylation of MGP.

Design

A randomized, double-blind, placebo-controlled trial was performed. Sixty participants (40–65 y) were randomly allocated to supplementation of 180 μg/d, 360 μg/d of MK-7 or placebo during 12 weeks. At baseline, after 4 and 12 weeks, desphospho-uncarboxylated MGP (dp-ucMGP), desphospho-carboxylated MGP (dp-cMGP) and total uncarboxylated MGP (t-ucMGP) were measured by ELISA techniques. Furthermore, the ratio of uncarboxylated osteocalcin (ucOC) to carboxylated osteocalcin (cOC) was used as proxy of vitamin K status and various cardiovascular risk factors were measured.

Results

Dp-ucMGP decreased significantly and dose-dependently in the 180 μg and 360 μg MK-7 supplementation groups (P time*treatment < 0.001) after 12 weeks, by 31% and 46% respectively, while dp-ucMGP levels remained unchanged after placebo treatment. The osteocalcin ratio also decreased significantly after 12-week supplementation with 180 μg (60%) and 360 μg (74%) MK-7 (P time*treatment < 0.001), while levels remained unchanged after placebo treatment. These results indicate improved vitamin K status and good compliance to the study treatment. Changes over time of dp-cMGP (p = 0.42) and t-ucMGP (p = 0.23) levels did not differ between treatment arms. Other cardiovascular risk factors did not differ between treatments arms.

Conclusions

Menaquinone supplementation dose-dependently decreases dp-ucMGP concentrations, but does not affect other MGP species. Dp-ucMGP may serve as a non-invasive marker of vitamin K status.

Highlights

► Vitamin K2 supplementation, dose-dependently decreases dp-ucMGP concentrations. ► Changes in dp-ucMGP occur within 4 weeks of supplementation in healthy adults. ► Vitamin K2 supplementation does not affect dp-cMGP and t-ucMGP concentrations.

Introduction

Vitamin K is a fat-soluble vitamin that occurs in two biologically active forms; phylloquinone (vitamin K1) and menaquinones (vitamin K2). The major dietary source of vitamin K is phylloquinone, found in leafy green vegetables and vegetable oils [1], [2]. The richest dietary sources of menaquinones are meat, eggs and fermented dairy like cheese and curd [3].

Vitamin K functions as a cofactor for the enzyme gamma-glutamyl carboxylase, catalyzing the gamma-glutamyl carboxylation of certain glutamic acid residues (Gla) in proteins [4]. These vitamin K-dependent proteins include hepatic-coagulation factors like prothrombin [4], but also some extra-hepatic Gla-proteins in bone (osteocalcin (OC)) and the vessel wall (matrix Gla protein (MGP)). MGP is a powerful inhibitor of vascular calcification [5]. Coronary calcification is a strong, independent predictor of coronary events [6]. The importance of MGP for vascular health was demonstrated in MGP-deficient mice, who all died of massive arterial calcification within 6–8 weeks after birth [7].

Observational studies have shown that high dietary menaquinone intake is associated with reduced risk of coronary vascular disease (CVD) and coronary artery calcification [8], [9], [10], [11]. This association may be explained by carboxylation of MGP by menaquinones. Human studies investigating the association between circulating MGP levels and CVD, however, showed inconsistent results [5], [6], [7]. Inability of the MGP assays to discriminate between different MGP species might explain these inconsistencies. MGP exists as various species, which differ in their state of phosphorylation and/or carboxylation: phosphorylated (pMGP), non-phosphorylated (dephospho-MGP, dpMGP), carboxylated (cMGP) or uncarboxylated (ucMGP). Development of assays to measure circulating MGP species enabled the investigation thereof in the circulation [12]. Cross-sectional studies suggested that low dephospho-uncarboxylated MGP (dp-ucMGP) levels were associated with high vitamin K status among hypertensive patients and older people (60–80 years) [13], [14]. Consistently, a randomized controlled trial showed a significant reduction in dp-ucMGP levels after supplementation with 500 μg/d of phylloquinone after three years [14]. A 6-week randomized non—placebo-controlled trial [15] and an exploratory pilot study among healthy adults [16] showed similar results. It is, however, unknown whether changes of dp-ucMGP already occur after a shorter period and what the effect is of specific dosages of menaquinone. Finally, the effect of vitamin K supplementation on other MGP species has not been investigated to date.

The aim of this study is to investigate the short-term (after 4 and 12 weeks) effect of menaquinone-7 (MK-7) supplementation on different MGP species, including dp-ucMGP, at different dosages.

Section snippets

Design & participants

The study was performed according to a randomized, double-blind, placebo-controlled trial. Sixty participants were randomly assigned through a web-based application to one of the three treatment arms (placebo, 180 μg/d MK-7, 360 μg/d MK-7), stratified by gender. The participants were recruited through the Julius Center database of subjects who have indicated their interest in participating in studies, and complied with the following criteria: apparently healthy men and healthy postmenopausal

Results

In total, 884 potential participants were invited to take part in the study, of which 284 replied and 148 were interested to participate. During screening, 87 participants were excluded (84 did not meet entry criteria, 3 refused participation). Finally, 61 participants entered the study, of which one participant in the 360 μg/d treatment arm withdrew during the run-in period (Fig. 1). This participant was excluded from the analysis.

Table 1 shows the baseline characteristics across the 3

Discussion

In this 12-week, double-blind, randomized, placebo-controlled trial, we showed that supplementation with MK-7 dose-dependently decreased dp-ucMGP by 31%–46% after 12 weeks. These results were comparable to those of the OC ratio, although that showed larger effect sizes of 60% and 75%. The changes already occurred in the first 4 weeks of intervention. This relatively short period of MK-7 supplementation did not affect other MGP species, nor did it affect any of the cardiovascular risk markers.

Acknowledgments

We thank the personnel of the Clinical Trial Unit of the Julius Center for their excellent contribution to the conduct of the trial and the data collection. This research was supported by a personal Dr. Dekker postdoctoral grant (2008T062) from the Netherlands Heart Foundation (JW Beulens). The authors wish to thank NattoPharma ASA (Lysaker, Norway) for kindly providing the study products. There were no commercial funding involved. The authors declare to have no conflict of interest. Cees

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