In our community-based sample, there was an inverse association between vitamin K status, as measured by plasma phylloquinone and phylloquinone intake, and overall circulating markers of inflammation. In the same cohort, vitamin D status, as measured by plasma 25-hydroxyvitamin D was not consistently associated with systemic inflammatory markers.
Our findings are in general agreement with in vitro
studies that report a decrease in production of inflammatory markers, including interleukin-6, by human macrophage and fibroblast cells with vitamin K treatment (11
). The current study expands our knowledge on this putative role of vitamin K because our panel consisted of 14 biomarkers of inflammation, many of which have not been previously studied with respect to vitamin K. In contrast, our findings in this community sample did not support the in vitro
data that suggest treatment with different forms of vitamin D may reduce production of inflammatory cytokines (35
)., although we acknowledge that in in vitro
studies the active vitamin D metabolite (calcitriol) was more effective in influencing cytokine production, while in vivo
25-hydroxyvitamin D was used as the estimate of vitamin D status, since circulating concentrations of calcitriol are tightly controlled. Most studies that report a beneficial effect of vitamin D on inflammatory cytokines were based on individuals who were diagnosed with chronic diseases (19
). The Framingham Offspring study is a generally healthier, older cohort. Our results are similar to those of a single study by Gannage-Yared, which reported that supplementation with 25-hydroxyvitamin D did not influence concentrations of circulating cytokines in a small sample (n=47) of healthy post-menopausal women (21
The mechanisms by which vitamin K influences biomarkers of inflammation are not known, although there is some suggestion that vitamin K suppresses inflammation by decreasing expression of genes for individual cytokines, such as interleukin-6 and osteoprotegerin (13
). Interleukin-6 and osteoprotegerin were the two markers that were inversely associated with both plasma phylloquinone and phylloquinone intake in our study. Vitamin K, interleukin-6 and osteoprotegerin are all implicated in bone resorption and the regulation of vascular calcification (39
). Lower osteoprotegerin concentrations in skeletal and vascular tissue are associated with an increase in bone resorption and vascular calcification (42
), whereas patients with osteoporosis and cardiovascular disease are reported to have increased concentrations of circulating osteoprotegerin (43
). These observed increases in circulating osteoprotegerin concentrations in patients with skeletal and/or vascular pathology are proposed to be an incomplete compensatory response to factors leading to increased bone resorption and/or atherosclerosis (45
It has been assumed that any role of vitamin K in bone or cardiovascular health is mediated through its action as a co-factor in the carboxylation of vitamin K-dependent proteins, including osteocalcin. Whereas we observed an association between phylloquinone intake and concentrations and overall inflammation, there was no association between percent undercarboxylated osteocalcin and overall inflammation. The percent undercarboxylated osteocalcin is used as a measure of the amount of vitamin K available for carboxylation in extra-hepatic tissues. It is possible that vitamin K modulates inflammation by a mechanism that does not involve its role in γ-carboxylation. The in vitro
studies suggest a direct effect of vitamin K on gene expression that is not related to carboxylation of vitamin K-dependent proteins. Further, it has been shown that vitamin K has a protective effect against oxidative stress that is independent of carboxylation, which may be an alternative anti-inflammatory mechanism associated with vitamin K (46
). The variation in biochemical measures of vitamin K status is determined by both dietary and non-dietary factors (Shea et al, submitted; unpublished data), which may explain why the significance of associations between biochemical measures of vitamin K status and phylloquinone intake with individual markers of inflammation were not always consistent.
Calcitriol has been postulated to regulate immune function through the nuclear vitamin D receptor, which is expressed by most cells of the immune system. In vivo
, the reported immunoregulatory effects of active vitamin D occur at very high concentrations (18
). Since the conversion of 25-hydroxyvitamin D to calcitriol is tightly regulated, and range of circulating calcitriol concentrations is narrow in the absence of chronic disease (47
), the reported effects of vitamin D on inflammation may not have been detected by our measures of vitamin D status in this community sample of healthy individuals, similar to the findings of others (21
). Vitamin D status, as measured by plasma 25-hydroxyvitamin D, was inversely associated with urinary isoprostanes, a measure of oxidative stress. This inverse association is in general agreement with Lin et al. who have reported a protective role of vitamin D in reducing oxidative stress by acting to terminate the lipid peroxidation chain reaction (48
Strengths and Limitations
There are several limitations to this study. Importantly, we tested associations between multiple biomarkers of inflammation and measures of vitamin K and D status, and we can not discount that significant associations may be due to chance because of multiple testing. By using a p
≤0.01 as the level of significance, we hoped to decrease this possibility. Certain health conditions, such as prevalent cardiovascular disease, are associated with increases in inflammatory biomarkers and with unhealthy lifestyle patterns that may influence vitamin K and D status. However, results were not substantively changed in analyses excluding individuals with diagnosed cardiovascular disease. Conversely, phylloquinone is in foods generally consumed as part of a healthy diet (49
), which may partially account for the reduction in inflammatory markers associated with higher intakes of phylloquinone. Furthermore, although in vitro
data suggest that calcitriol, the biologically active form of vitamin D, is involved in immunoregulation, we did not measure this form of vitamin D since circulating levels are tightly regulated. The Offspring cohort participants are primarily older, of northern European descent, and predominantly reside in the Northeastern United States; we can not generalize our findings to other ethnic/racial groups, younger individuals, or individuals residing in sunnier climates. We acknowledge that the measurement of some of the vitamin concentrations at examination six (49 percent), about three years prior to the examination seven inflammatory markers may have led to some misclassification of the association; however, it should be noted that we did not observe significant effect modification by examination. The cross-sectional study design precludes causal inferences in interpreting our results. Although we modeled the relation of vitamins to inflammatory markers (dependent variables), we acknowledge that inflammatory markers may influence vitamins concentrations, or both may be related via other unmeasured intermediate factors. Finally, although the associations were statistically significant, the clinical significance of the very modest changes observed is uncertain. Balanced against these limitations are the novelty of the analyses, the routine ascertainment of vitamins, inflammatory biomarkers and covariates, in community-based individuals.
Clinical and research implications
Our findings provide one potential alternative mechanism for a putative, protective effect of vitamin K in the progression of cardiovascular disease and osteoporosis, since both diseases are characterized by inflammation. Limited in vitro data support the inverse association between vitamin K and interleukin-6, and this may influence the association between vitamin K and other cytokines, such as osteoprotegerin. Further research to better elucidate mechanisms underlying the associations between vitamin K and inflammatory cytokines is warranted.