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Am J Clin Nutr 2009;89:1799807. Printed in USA. 2009 American Society for Nutrition 1799
1800 SHEA ET AL
MGP was assayed by radioimmunoassay (16, 17). Plasma con- skewness in formal analyses. Students t test for independent
centrations of phylloquinone were measured by HPLC (18). samples (for normally distributed continuous outcomes), Wil-
Plasma OPG and IL-6 were assayed by enzyme immunoas- coxons sign-rank test (for nonnormally distributed continuous
say with commercially available high-sensitivity kits (Im- outcomes), and the chi-square test for homogeneity of proportions
munodiagnostics from Biomedica, Vienna, Austria, and R&D (for categorical outcomes) were used to test for baseline differ-
Systems, Minneapolis, MN) (19). Plasma CRP was assayed by ences between treatment groups. Students t test for paired sam-
using high-sensitivity Immulite CRP kits (Diagnostic Products ples and McNemars test for correlated proportions for categorical
Corporation, Los Angeles, CA) and the COBAS MIRA (Roche outcomes were used to examine differences from baseline to year
Instruments, Belleville, NJ) (19). 3. All participants with measures of CAC at baseline and year 3
were used in primary intention-to-treat analyses, regardless of
Covariates adherence (n 200 in the treatment group and 188 in the control
group). Secondary analyses were limited to those who were 85%
Information was collected at baseline and year 3 regarding
adherent to treatment of whom all covariate data were available
medical history, medication use, smoking status, and physical
(n 149 in the treatment group and 146 in the control group).
activity (20). Measurements of weight and height were taken, and
Because CAC progresses more rapidly in individuals who already
body mass index (BMI; in kg/m2) was calculated.
have CAC present, as a hypothesis-generating subgroup analysis,
we examined the effect of vitamin K supplementation on CAC
Statistical analyses progression and serum MGP in individuals who had at least mild
A logarithmic transformation was applied to plasma concen- CAC at baseline (defined as AS . 10) [81 in the treatment group
trations of phylloquinone, IL-6, and CRP and to AS to reduce (32 women) and 89 in the control group (44 women)]. An AS . 10
1802 SHEA ET AL
is predictive of increased risk of CAC progression and for CVD did not change in the control group (P 0.79) (13). Statin medi-
and mortality (21, 22). Interactions between sex and treatment and cation use increased equally in both groups (P , 0.001), although
between sex and baseline Agatston score with respect to the 3-y there was no between-group difference in statin use at year 3 (P
change in CAC were not significant, so all analyses were sex- 0.92). There was also an equal, but nonsignificant decrease, in
pooled and adjusted for sex. antiinflammatory medication use observed in both groups.
In intention-to-treat and secondary analyses, an independent- Although the median AS at baseline was higher in the control
samples t test was used to compare the 3-y change in AS (which group, the difference in baseline AS between the 2 groups was not
was normally distributed) between the phylloquinone-treated significant (P 0.59, Wilcoxons rank-sum test). Of the adherent
group and the control group. Analysis of covariance (ANCOVA, participants, the median (IQR) baseline AS did not differ between
using proc glm; SAS version 9.1, SAS Institute, Cary, NC) was treatment groups [phylloquinone treatment group: 18 (189);
subsequently used to adjust for baseline AS and for known de- control group: 40 (199); P 0.35], nor did other baseline char-
terminants of CAC progression: sex, age, smoking status (yes or acteristics (all P 0.06). Of the adherent participants with AS .
no), statin use (yes or no), prevalent diabetes (yes or no), tri- 10 at baseline (81 in the treatment group and 89 in the control
glycerides, LDL cholesterol, systolic blood pressure, BMI, and group), those in the control group had a significantly higher me-
physical activity (23). Because a natural log transformation was dian (IQR) CRP concentration [2.0 (4.1)] than did those in the
applied to baseline and year 3 AS for formal analyses, we re- phylloquinone treatment group [1.2 (2.4); P 0.02], but there
peated these analyses using the ratio of the natural log of the were no significant differences between treatment groups in any
AS at year 3 to the natural log of the AS at baseline as the other baseline characteristic, including median (IQR) AS [phyl-
outcome. loquinone treatment group: 145 (395); control group: 146 (325);
A similar approach was used to examine for the effect of P 0.90; all other P 0.10].
phylloquinone supplementation on change in serum MGP. An In an intention-to-treat unadjusted analysis, there was no dif-
TABLE 1
Participant characteristics at baseline and year 31
All participants with CAC measures used Adherent participants used in
in intention-to-treat analysis2 secondary analysis2,3
When the change in circulating OPG, IL-6, or CRP was included did those who did not. In contrast, vitamin K supplementation did
as a covariate in the models that assessed the influence of vitamin K not reduce the development of new CAC, of which there was an
supplementation on change in CAC, the significance of the treat- annual increase of 5% in both groups. These estimates are
TABLE 2
Effect of phylloquinone supplementation on the mean (95% CI) 3-y change in Agatston score (AS) and serum matrix Gla
protein (MGP) in the intention-to-treat analyses
Intention-to-treat analyses
concentrations are significantly higher among those adults with coronary artery calcium. Baseline serum OPG concentrations are
a higher Framingham Coronary Heart Disease Risk Score, but are higher among those individuals with higher calcification scores.
not consistently associated with higher CAC (32). Similarly, there However, there was no influence of phylloquinone treatment on
were only nonsignificant trends between baseline serum MGP and change in circulating OPG, IL6, and CRP (19), and controlling for
CAC in this study, which lends further support to previous con- the 3-y change in cytokines did not alter the significance of the
clusions that serum MGP concentrations are not a robust predictor treatment effect on change in AS. This suggests that any putative
of CAC (16). In contrast, serum MGP concentrations were higher effect of phylloquinone treatment on progression of CAC was
in this study among the phylloquinone treatment group than in the independent of changes in individual cytokines.
control group, which was unexpected because the phylloquinone The amount of CAC is a well-known independent predictor of
treatment group had less progression of CAC. Controlling for the CVD risk (34). In several population-based studies, CAC was
3-y change in serum MGP did not attenuate the significance of the incrementally predictive of cardiovascular events beyond tradi-
treatment effect with respect to change in AS, which suggested tional risk factors or the Framingham Risk Score (1, 35). Thus,
that any effect of phylloquinone treatment on CAC progression slowing down the progression of CAC, which leads to lower
was independent of the change in serum MGP. The assay used to absolute CAC, may constitute a favorable result. However,
measure MGP did not differentiate between the c-carboxylated progression of CAC has not yet been established as a surrogate
and uncarboxylated forms of MGP (17). It is assumed that only the marker of therapy success or as an indicator of cardiovascular
carboxylated form of MGP is functional as an inhibitor of calci- event risk. Intervention studies that have assessed the efficacy of
fication, so interpretation of serum total MGP concentrations is standard cardiovascular therapies, such as lipid-lowering medi-
problematic. Novel assays that purportedly measure the un- cations or other supplementation with other nutrients, such as
carboxylated form of MGP may be used in the future to elucidate vitamins C and E, on minimizing the progression of CAC have
the role of functional forms of MGP in response to vitamin K thus far reported no effect (36, 37).
supplementation and CAC (33). Alternatively, it is plausible that Certain limitations of this study should be acknowledged.
serum MGP concentrations, regardless of c-carboxylation status, Known CVD was an exclusion criterion for our study, and our
do not reflect MGP expression in arterial walls, as observed in follow-up was limited to 3 y; therefore, we were not able to show
mice lacking MGP (34). that slowing down the progression of CAC with vitamin K sup-
We also examined associations between changes in proin- plementation reduced cardiovascular event risk, and longitudinal
flammatory markers and changes in calcification as an alternative studies are warranted to test this hypothesis. Although differences
mechanism by which vitamin K may protect against progression of in baseline CAC between treatment groups were not significant, the
1806 SHEA ET AL
CAC was not equally matched between groups, despite the use of 14. Ferencik M, Chan RC, Achenbach S, et al. Arterial wall imaging:
a double-blind randomized controlled study design. However, the evaluation with 16-section multidetector CT in blood vessel phantoms
and ex vivo coronary arteries. Radiology 2006;240:70816.
difference was controlled for in our statistical analyses. Ethnic 15. Hoffmann U, Siebert U, Bull-Stewart A, et al. Evidence for lower variability
variability in CAC progression is reported (24). Because our of coronary artery calcium mineral mass measurements by multi-detector
sample was .90% white, our findings cannot be generalized to computed tomography in a community-based cohortconsequences for
other ethnic groups. Because vitamin K supplementation reduced progression studies. Eur J Radiol 2006;57:396402.
16. ODonnell CJ, Shea MK, Price PA, et al. Matrix Gla protein is associ-
the progression of CAC among men and women who adhered to ated with risk factors for atherosclerosis but not with coronary artery
our intervention, overall and in those with preexisting CAC, our calcification. Arterioscler Thromb Vasc Biol 2006;26:276974.
results should be considered hypothesis-generating. Given the 17. Price PA, Rice JS, Williamson MK. Conserved phosphorylation of
variability in CAC measures, our results would be strengthened by serines in the Ser-X-Glu/Ser(P) sequences of the vitamin K-dependent
matrix Gla protein from shark, lamb, rat, cow, and human. Protein Sci
larger confirmatory studies and an investigation of the effect of
1994;3:82230.
vitamin K supplementation on CAC among those at greater risk of 18. Davidson KW, Sadowski JA. Determination of vitamin K compounds in
progressive CAC. plasma or serum by high-performance liquid chromatography using
In conclusion, vitamin K supplementation reduced the pro- postcolumn chemical reduction and fluorimetric detection. Methods
gression of existing CAC in asymptomatic older men and women Enzymol 1997;282:40821.
19. Shea MK, Dallal GE, Dawson-Hughes B, et al. Vitamin K, circulating
when taken with recommended amounts of calcium and vitamin cytokines, and bone mineral density in older men and women. Am J Clin
D. The mechanisms by which vitamin K conferred a protective Nutr 2008;88:35663.
role are still uncertain. Larger studies in other populations are 20. Washburn RA, Smith KW, Jette AM, Janney CA. The Physical Activity
needed to confirm these findings, and to assess the risks and Scale for the Elderly (PASE): development and evaluation. J Clin Epi-
demiol 1993;46:15362.
benefits of vitamin K supplementation on clinical CVD. 21. Yoon HC, Emerick AM, Hill JA, Gjertson DW, Goldin JG. Calcium
begets calcium: progression of coronary artery calcification in asymp-