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Associations between Vitamin K Biochemical Measures and Bone Mineral Density in Men and Women

Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University (S.L.B., J.W.P., B.D.-H.), Boston, Massachusetts 02111; the Framingham Heart Study (P.W.F.W.), Framingham, Massachusetts 01702; Beth Israel-Deaconess Hospital, Hebrew Rehabilitation Center for Aged Research and Training Institute (K.E.B., D.P.K.) and Harvard Medical School Division on Aging (D.P.K.), Harvard Medical School, Boston, Massachusetts 02131; Departments of Medicine and Preventive Medicine and Epidemiology (P.W.F.W.) and the Department of Biostatistics (D.M.C., L.A.C.), Boston University, Boston, Massachusetts 02118; and Department of Orthopedics and Rehabilitation (C.M.G.), Yale University, New Haven, Connecticut 06510

Address all correspondence and requests for reprints to: Sarah L. Booth, Ph.D., Vitamin K Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts 02111. E-mail: sarah.booth{at}tufts.edu.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Few data exist on the association between vitamin K status and bone mineral density (BMD) in men and women of varying ages. We examined cross-sectional associations between biochemical measures of vitamin K status and BMD at the hip and spine in 741 men and 863 women (mean age, 59 yr; range, 32–86 yr) who participated in the Framingham Heart Study (1996–2000). Vitamin K status was assessed by plasma phylloquinone and percentage undercarboxylated osteocalcin (%ucOC). Among the men, low plasma phylloquinone concentrations adjusted for triglycerides and elevated serum %ucOC levels were associated with low BMD at the femoral neck (P = 0.03 and 0.009, respectively). Among postmenopausal women not using estrogen replacements, low plasma phylloquinone concentrations were associated with low spine BMD (P = 0.007), with a nonsignificant trend of an elevated serum %ucOC with low spine BMD (P = 0.08). In contrast, there were no significant associations between biochemical measures of vitamin K and BMD in either premenopausal women or postmenopausal women using estrogen replacements. Clinical trials are required to isolate any putative effects of vitamin K on rates of bone loss. The target population in these trials, particularly in regard to estrogen use, may be critical, as suggested by the findings of this study.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
THE PRESENCE OF vitamin K and vitamin K-dependent proteins in the skeleton supports the hypothesis that vitamin K has a role in bone biology (1, 2). Vitamin K, a fat-soluble vitamin, is a cofactor specific to the formation of -carboxyglutamyl (Gla) residues from specific glutamate residues in certain proteins (3). Osteocalcin (OC), matrix Gla protein, and protein S are three vitamin K-dependent proteins that have been identified in bone (4). The Gla residues in these vitamin K-dependent proteins, also termed Glacontaining proteins, are thought to confer mineral-binding properties. The amount of OC that is not carboxylated [under-carboxylated (uc)OC] is considered a sensitive measure of vitamin K status in bone (5), with an elevated percentage of ucOC associated with low dietary intakes of vitamin K (6).

Low dietary phylloquinone (vitamin K₁) intakes have been associated with increased hip fracture risk, most notably among postmenopausal women (7, 8). However, the associations between dietary phylloquinone intake and bone mineral density (BMD) are equivocal. Among elderly men and women participating in the Framingham Heart Study, low dietary phylloquinone intakes were not associated with low BMD at either the hip or spine (either cross-sectionally or prospectively), even though low intakes were associated with increased hip fracture risk (8). In contrast, low dietary phylloquinone intakes were associated with low BMD at the hip and spine in pre- and postmenopausal women, but not men, among mostly younger participants in the Framingham Offspring Study (9).

Biochemical indicators of vitamin K status have been measured in several observational studies examining risk factors for age-related bone loss, with an emphasis on postmenopausal women. In prospective studies, elevated ucOC has been associated with an increased risk of hip fracture in elderly institutionalized (10, 11, 12) and free-living women (13, 14) and men (14). Elevated serum ucOC has also been associated with low BMD in postmenopausal women (10, 15); similarly, low plasma phylloquinone concentrations have been associated with low BMD at the spine (16). To the best of our knowledge, there are no studies examining associations between biochemical markers of vitamin K status and BMD in men.

Of the various biochemical indicators, there is a lack of consensus regarding the best single measure of an individual’s vitamin K status (17). There are also nondietary factors, such as plasma triglycerides and smoking status, that influence these biochemical markers independent of dietary phylloquinone intakes and that need to be accounted for (6). A stronger line of evidence for a protective role of vitamin K in age-related bone loss would be the demonstration of consistent associations among multiple measures of vitamin K status and BMD or hip fracture risk within the same population.

The objective of this study was to examine associations between biochemical measures of vitamin K status and BMD in men and women of varying ages. Vitamin K status was defined by two biochemical measures: 1) plasma phylloquinone, an indicator of recent dietary phylloquinone intake, and 2) serum percent undercarboxylated OC, a sensitive indicator of vitamin K availability in bone. BMD was measured at the hip (femoral neck and trochanter) and the spine (L2–L4).

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Study sample

The Framingham Offspring Study is a longitudinal, community-based study of cardiovascular disease among the children and the spouses of the participants in the original Framingham Heart Study cohort (18). In 1971, 5124 participants were enrolled in the Offspring Study and have returned every 3–4 yr for an extensive physical examination, comprehensive questionnaires, anthropometric measurements, blood chemistries, and assessment of cardiovascular and other risk factors by trained clinical personnel. Between 1996 and 2000, there were 3532 participants in the sixth examination cycle of the Framingham Offspring Study. Of these, 3035 participants had BMD measures between 1996 and 2001, and of those, 1691 (766 men and 925 women) had corresponding fasting blood samples that had not been subjected to multiple freeze-thaw cycles and were available for analyses of vitamin K biochemical measures used for this study. Participants were excluded if they did not have valid measurements of both vitamin K measures (n = 26) or were taking anticoagulants, including the vitamin K antagonist warfarin (n = 28), or osteoporosis-treatment medications (n = 33), thereby reducing the final sample to 1604 (741 men and 863 women) (mean age, 59 yr; range, 32–86 yr). The Institutional Review Boards for Human Research at Boston University, Hebrew Rehabilitation Center for Aged, and Tufts-New England Medical Center approved the protocol.

Methods

At the time of the sixth examination, information regarding medication use, medical history, smoking status, and dietary intake was collected. Height and weight were measured while the subjects stood. Body mass index (BMI) was calculated as the weight in kilograms divided by the square of the height in meters. Current smokers were defined as subjects who reported smoking cigarettes on a regular basis during the previous year. Physical activity was assessed by using a validated questionnaire of self-reported activity in the past 7 d (19). Menopause status was defined as having no menstrual periods for at least 1 yr or currently using postmenopausal estrogens (oral, patch, or cream). Estrogen use was classified as either current use or no use at the time of the examination. Usual dietary intakes of energy, total protein, calcium, vitamin D, vitamin K, alcohol, and caffeine for the previous year were assessed using a semiquantitative food frequency questionnaire, as described elsewhere (6, 20). Information about vitamin and mineral supplements use was also reported in the food frequency questionnaire.

Fasting blood samples (>10 h) were collected as part of the sixth examination cycle. Plasma and serum samples were stored at –70 C for no more than 2 yr and were analyzed upon the first thaw. Plasma phylloquinone concentrations were determined by reversed-phase HPLC with use of postcolumn reduction and fluorometric detection (21). The lower limit of detection for phylloquinone with this assay was 0.05 nmol/liter, so samples with phylloquinone concentrations below the limit of detection were entered as 0.05 nmol/liter for the purposes of statistical analysis. Low and high control specimens had average values of 0.56 and 3.15 nmol/liter, with total coefficients of variation (CVs) of 15.2 and 10.9%, respectively. Plasma 25-hydroxyvitamin D [25(OH)D] was determined by RIA (Diasorin, Stillwater, MN). To minimize seasonal influences of vitamin D on BMD in this cross-sectional analysis, 25(OH)D was measured in the same samples as plasma phylloquinone for use as one of the terms in the multiple linear regression. The limit of detection for 25(OH)D using this assay was 3.8 nmol/liter; however, no 25(OH)D samples had concentrations below the limit of detection. Total CVs for control values of 36 and 137 nmol/liter were 8.5 and 13.2%, respectively. Triglyceride concentrations were measured enzymatically, as described elsewhere (22). Serum total OC and ucOC were analyzed using procedures described elsewhere (5). A RIA for OC uses human OC for standard and tracer and a polyclonal antibody directed to intact human OC (23). The antibody recognizes intact OC and the large N-terminal-mid molecule fragment. The ucOC is a marker of extrahepatic vitamin K status and is determined in this assay as plasma OC that does not bind in vitro to hydroxyapatite. Binding in vitro to hydroxyapatite varies with the amount of total OC in the sample, so ucOC was expressed as the percentage of total OC (%ucOC) to minimize this discrepancy (5). The limit of detection for total OC for this assay was 0.6 ng/ml; however, for any sample with a concentration that was below the limit of detection, it was not feasible to calculate the %ucOC and therefore was excluded from the statistical analysis (n = 3). Total CVs for the three control sera with average total OC results of 3.4, 7.1, and 11.9 µg/liter were 22.3, 12.8, and 7.8%, respectively.

BMD was measured in the hip and spine by using dual-energy x-ray absorptiometry (DPX-L; Lunar, Madison, WI) as part of the 6^th examination cycle. CVs for the dual-energy x-ray absorptiometry measurements of the hip were 1.7% for the femoral neck, 2.5% for the trochanter, and 0.9% for the lumbar (L2–L4) spine (24).

Statistical analysis

The dependent variables were BMD measured at three sites: femoral neck, trochanter, and lumbar (L2--L4) spine. We assessed the linearity of the relationships between measures of BMD and the independent variables plasma phylloquinone and %ucOC before using linear regression methods. Locally weighted regression smoothing (25) was used to fit nonparametric curves to the data. The resulting curves suggested a linear relationship between BMD and phylloquinone and between BMD and %ucOC. Thus we fit linear regression models to the data.

Subgroups of subjects with differential risks for bone loss were identified based on known predictors of bone density: gender, menopausal status, and estrogen use (24). Separate analyses were therefore conducted for each of the following four subgroups: men (n = 741), premenopausal women (n = 170), postmenopausal women on estrogen (n = 269), and postmenopausal women not on estrogen (n = 424).

Pearson’s correlation coefficients were used to assess associations between biochemical measures of vitamin K and triglycerides in each of the subgroups. Multiple linear regression analyses were performed to evaluate the associations between measures of BMD and plasma phylloquinone. For each outcome, the models included terms for age, BMI, height, current smoking (yes/no), physical activity, plasma 25(OH)D, caffeine and alcohol intake, and dietary and supplemental intakes of calcium. Because transport of vitamin K into osteoblasts is dependent on triglycerides in vitro (26), and plasma triglycerides influence plasma phylloquinone concentrations independent of dietary phylloquinone intakes (6), a separate model was run to include an additional adjustment for triglycerides. The same analyses were used to assess the associations between measures of BMD and %ucOC. All reported P values are based on two-sided tests. The outcomes of interest were specified a priori. All analyses were performed with the use of SAS software (version 8.1; SAS Institute, Cary, NC).

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Subject characteristics are summarized in Table 1. Average (mean) plasma phylloquinone concentrations for all four comparison groups were within the normal range (0.3–2.6 nmol/liter) using this assay (27). Plasma phylloquinone concentrations were positively correlated with fasting triglyceride concentrations in men and postmenopausal women (R = 0.45, P < 0.0001; R = 0.38, P < 0.0001; R = 0.35, P < 0.0001 for men and postmenopausal women using, and not using, estrogen replacements, respectively). There was also a nonsignificant trend for a positive correlation between plasma phylloquinone concentrations and triglycerides among premenopausal women (R = 0.14; P = 0.07). Mean plasma phylloquinone concentrations were lower in premenopausal women (1.05 nmol/liter) compared with the other three comparison groups (1.41–1.54 nmol/liter), consistent with patterns observed for mean plasma triglycerides across the four groups (i.e. lowest among premenopausal women) (Table 1).

Whereas plasma phylloquinone and %ucOC were not correlated in men or premenopausal women, even after adjustment for triglycerides, there was a significant inverse correlation between plasma phylloquinone and %ucOC in postmenopausal women, regardless of estrogen use (R = –0.22, P = 0.0002; R = –0.11, P = 0.02 for users and nonusers of estrogen replacements, respectively). We currently do not have an explanation for these age and gender differences in correlations between plasma phylloquinone and %ucOC.

Among men, after adjustment for multiple covariates, low plasma phylloquinone concentrations and high serum %ucOC were associated with low BMD of the hip (femoral neck and trochanter) (Table 2). The positive association between plasma phylloquinone and hip BMD was stronger, as indicated by the increase of the ß-coefficient from 0.004 to 0.007, when the regression model also included plasma triglycerides (Table 2). In contrast, adjustment for triglycerides did not change the magnitude of the inverse association between %ucOC and hip BMD, as indicated by the ß-coefficient, which was –0.0008 both for unadjusted and adjusted for triglycerides (Table 2). These findings were consistent with the observed positive correlations between plasma triglycerides and plasma phylloquinone, but not %ucOC, among men.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

Among postmenopausal women not using estrogen replacement, low plasma phylloquinone concentrations were associated with low spine BMD, with a nonsignificant trend of an inverse association between %ucOC and spine BMD. In contrast, there were no significant associations between biochemical measures and BMD in premenopausal women or postmenopausal women using estrogen. Estrogen status has not been systematically studied in terms of its influence on any putative role of vitamin K and bone health. In one study of women aged 20–90 yr, significant inverse associations between serum %ucOC and BMD at the hip were limited to those women within the first 10 yr of menopause (15). To the best of our knowledge, no other studies assessing the association between vitamin K status and BMD in premenopausal women are available for comparison. When stratified by estrogen use in the Nurses’ Health Study, high phylloquinone intake was associated with reduced hip fracture risk only among postmenopausal women not taking estrogen (7). Previous studies from the Framingham Heart Study have described a protective effect of estrogen use on bone loss among postmenopausal women (24), consistent with the findings of other observational studies (30, 31). Therefore, it is plausible that the magnitude of the protective effect of estrogen use on BMD masks any potential contribution of a single nutrient, such as vitamin K, to BMD.

In previous studies among postmenopausal women not taking estrogen replacements, low plasma phylloquinone concentrations have been associated with low BMD at the spine (16), and high serum ucOC has been associated with low BMD at the hip (10). In the only published long-term, placebo-controlled clinical trial investigating the effects of phylloquinone supplementation on bone, a significant reduction in femoral neck bone loss was observed among postmenopausal women, aged 50–60 yr, consuming a phylloquinone, calcium, and vitamin D supplement compared with a placebo or a supplement containing calcium and vitamin D only (32). In contrast to the findings in the current study, no beneficial effect of phylloquinone was observed in the spine (32). Although encouraging, more clinical trials need to be completed before conclusions can be made regarding the efficacy of phylloquinone supplementation in reducing age-related bone loss. Furthermore, the target population in these clinical trials may be critical, as suggested by findings of the current study.

We previously reported that low dietary intakes of phylloquinone were not associated with low BMD among elderly men and women (8) and mostly younger men (9) participating in the Framingham Heart Study. Biochemical measures of vitamin K are affected by both dietary and nondietary factors (6), some of which may have attenuated any putative association between dietary phylloquinone and BMD in the diet studies. Of the nondietary factors influencing phylloquinone concentrations, triglycerides may be critical in understanding the role of phylloquinone in bone health. Plasma phylloquinone concentrations were positively correlated with corresponding plasma triglycerides in the current study, an observation that has been reported in other observational (6) and metabolic studies (33). The positive association between plasma phylloquinone and BMD at the hip was strengthened with the adjustment for plasma triglycerides in men, as indicated by the increase in the ßcoefficient, which is consistent with current understanding of vitamin K transport to osteoblasts (26). Vitamin K is not known to have a carrier protein; instead, triglyceride-rich lipoproteins, primarily chylomicron remnants, are thought to be the main transporters of phylloquinone (34, 35, 36). In contrast, adjustment of plasma phylloquinone concentrations for plasma triglycerides weakened the positive association with spine BMD measured in postmenopausal women not taking estrogen, even though plasma triglycerides were positively associated with plasma phylloquinone in this group. One explanation for this difference may be that the men had a wider range of fasting triglyceride concentrations (0.33–15.3 mmol/liter) compared with the postmenopausal women (0.33–7.9 mmol/liter), which may have confounded the positive association between plasma phylloquinone and BMD at the hip. If triglyceride-rich lipoproteins are the major forms of transport of vitamin K to the osteoblasts, as suggested by an in vivo study (26), and carboxylation of OC is determined by the availability of vitamin K, it would be expected that adjustment for plasma triglycerides would also increase the strength of the inverse association between serum %ucOC and hip or spine BMD. However, adjustment of %ucOC for plasma triglycerides did not change the ß-coefficient of the inverse associations between %ucOC and hip BMD in men or the ß-coefficient of the inverse associations between %ucOC and spine BMD in postmenopausal women not using estrogen replacements. This would suggest that the degree of -carboxylation of OC is not linearly related to the amount of phylloquinone transported to the osteoblasts by the triglyceride-rich lipoproteins, and adjustment for triglycerides is not necessary when examining associations between %ucOC and bone health. More systematic investigation into the role of triglycerides in the transport of phylloquinone into the bone and its use in the -carboxylation of OC is required before conclusions can be made regarding the necessity of adjusting measures of vitamin K status for plasma triglycerides.

Interpretation of the results from the current study is limited by the cross-sectional design, which used biochemical measures obtained from a single blood draw. Although plasma phylloquinone concentrations are dependent on recent dietary intake (17), the additional measurement of serum %ucOC at the same time point provided a more robust assessment of vitamin K status. However, the physiological significance of these biochemical measures has yet to be established (17). Also, we currently do not have an explanation for lack of association between the biochemical measures of vitamin K and spine BMD in the men, when low plasma phylloquinone and high %ucOC were associated with low BMD at the hip. It is plausible that given the sum of the age groups studied, spinal osteoarthritis in these men diminished our ability to accurately assess spine BMD. Unfortunately, radiographs from the same time period were not available to assess this possibility. This is to the best of our knowledge the first observational study to assess the associations between vitamin K status and BMD in men and premenopausal and postmenopausal women. One caveat is that the subgroup analysis among women may have resulted in sample sizes that were too small in statistical power to detect significant differences. This may explain why there were differences between men and women in terms of the strength of the associations between markers of vitamin K status (i.e. positive association between plasma phylloquinone and BMD and a negative association between serum %ucOC and BMD) and the BMD site at which the associations were significant. In the current study, the analysis of the men included a broad age range, which limits the ability to draw conclusions of the protective role of vitamin K in bone loss with advancing age.

In conclusion, our data suggest that there are estrogen status differences in the cross-sectional assessment of associations between biochemical markers of vitamin K status and BMD. Among men, poor vitamin K nutritional status, as indicated by low plasma phylloquinone concentrations and high serum %ucOC, was associated with low BMD at the hip. Among postmenopausal women not using estrogen replacements, poor vitamin K nutritional status was associated with low BMD of the spine. However, there were no associations observed among premenopausal women or among postmenopausal women reporting estrogen replacement use. Clinical trials are required to isolate any putative effects of vitamin K on rates of bone loss.

	Acknowledgments

 
We are grateful to the Framingham Study participants and staff, especially the Framingham densitometer technicians, Mary Hogan, and the Framingham Study laboratory chief, Patrice Sutherland, and her staff. We also thank Marian Hannan for her contribution to the study design and data interpretation and Robert McLean and David Gagnon for their contribution to the data analysis.

	Footnotes

 
This work was supported by the National Institute of Aging (AG14759 to S.L.B.), the National Institute of Arthritis and Musculoskeletal and Skin Diseases (AR41398 to D.P.K.), the National Heart, Lung, and Blood Institute’s Framingham Heart Study (N01-HC-25195), and the U.S. Department of Agriculture under agreement 58-1950-001.

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the views of the U.S. Department of Agriculture.

Abbreviations: BMD, Bone mineral density; BMI, body mass index; Gla, -carboxyglutamyl; OC, osteocalcin; 25(OH)D, 25-hydroxyvitamin D; uc, under--carboxylated.

Received September 24, 2003.

Accepted June 25, 2004.

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