The Journal of Nutrition Nutrient Requirements and Optimal Nutrition Age Group and Sex Do Not Influence Responses of Vitamin K Biomarkers to Changes in Dietary Vitamin K 1 3 Jennifer T. Truong, 4 Xueyan Fu, 4 Edward Saltzman, 4 Ala Al Rajabi, 4 Gerard E. Dallal, 4 Caren M. Gundberg, 5 and Sarah L. Booth 4 * 4 USDA Human Nutrition Research Center on Aging at Tufts University, Boston, MA; and 5 Yale University School of Medicine, Department of Orthopaedics, New Haven, CT Abstract Inadequate vitamin K intake has been associated with abnormal soft tissue calcification. Older adults may have insufficient intakes of vitamin K and respond less to vitamin K supplementation compared with younger adults. However, little is known about the determinants that influence the response to vitamin K supplementation. Our primary objective was to assess dietary and nondietary determinants of vitamin K status in healthy younger and older adults. In a nonrandomized, nonmasked study, 21 younger (18 40 y) and 21 older (55 80 y) men and women consumed a baseline diet (200 mg phylloquinone/d) for 5 d, a phylloquinone-restricted diet (10 mg phylloquinone/d) for 28 d, and a phylloquinonesupplemented diet (500 mg phylloquinone/d) for 28 d. Changes in vitamin K status markers in response to vitamin K depletion and repletion were studied and the influences of BMI, body fat, and circulating TG were assessed by including them as covariates in the model. Despite baseline differences in measures of vitamin K status, plasma phylloquinone tended to increase (P = 0.07) and the percentage of uncarboxylated osteocalcin and uncarboxylated prothrombin both improved with phylloquinone supplementation (P, 0.007), regardless of age group or sex. Only the excretion of urinary menadione, a vitamin K metabolite, was greater among younger adults in response to depletion than in older adults (P = 0.012), regardless of sex. Adiposity measures and circulating TG did not predict response of any measures. In conclusion, poor vitamin K status can be similarly improved with vitamin K supplementation, regardless of age group or sex. J. Nutr. 142: 936 941, 2012. Introduction Vitamin K is an emerging factor in regulation of soft tissue calcification and other chronic diseases (1). However, there is a lack of data from which to estimate an average dietary vitamin K requirement and very little is known about the fundamentals of vitamin K metabolism. Although the observational data suggest that age, body fat, and sex are determinants of vitamin K (2,3), there is a lack of consensus among metabolic studies regarding the effects of vitamin K supplementation on markers of vitamin K status and bone turnover when the elderly are compared with young adults. Prior studies (4 7) are inconclusive, because they lack a wide range of dietary vitamin K intakes, sufficient duration and adequate representation of young and older adults within the same study. There is also controversy regarding the 1 Supported by the USDA Agricultural Research Service under Cooperative Agreement No. 58-1950-7-707 and the NIH (5R01DK69341-4). Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the authors and do not necessarily reflect the view of the USDA. 2 Author disclosures: J. T. Truong, X. Fu, E. Saltzman, A. Al Rajabi, G. Dallal, C. Gundberg, and S. Booth, no conflicts of interest. 3 This trial was registered at clinicaltrials.gov as NCT0036232. * To whom correspondence should be addressed. E-mail: sarah.booth@tufts. edu. interdependence of plasma vitamin K with plasma lipids and the implications when assessing vitamin K status. Such a limited understanding about vitamin K metabolism currently impedes the ability to establish optimal dietary recommendations for vitamin K and to interpret the results from clinical trials conducted primarily in women of a fairly narrow age range (55 80 y). We conducted a metabolic study to assess the role of dietary and nondietary components, including 2 age groups, sex, body fat, and plasma lipids, that influence the response of measures of vitamin K status to vitamin K depletion and repletion in younger (18 40 y) and older men and women (55 80 y). These data are critical for the interpretation of the epidemiologic and clinical data used to determine associations between vitamin K nutritional status and chronic disease prevention, including bone and cardiovascular health. As a secondary objective, we compared the effects of vitamin K depletion and repletion in younger and older men and women on markers of bone turnover. Participants and Methods Study participants. Healthy, ambulatory men and women in a younger age group (18 40 y) and an older age group (55 80 y) were recruited ã 2012 American Society for Nutrition. 936 Manuscript received November 7, 2011. Initial review completed December 5, 2011. Revision accepted February 13, 2012. First published online March 21, 2012; doi:10.3945/jn.111.154807.
through direct mailings, newspaper advertisements, and notices in the community. Women in the older age group were postmenopausal for at least 3 y. Exclusion criteria included kidney stones within the past 2 y; smoking within the last 6 mo; therapy with bisphosphonates, hormone replacement therapy, oral contraceptive use, or estrogen or progesterone use within the past 3 mo; oral antibiotic use within the past 3 mo; seizure medications, diuretics, coumadin use within the past 12 mo; use of proton pump inhibitors; diabetes; inflammatory bowel disease or other malabsorption problems; laboratory evidence of kidney or liver disease; atrial fibrillation or flutter; unstable coronary artery disease; pregnancy or planning on becoming pregnant; history of gastritis within the past 6 mo; prior gastric surgery; history of coagulopathy; and International Normalized Ratio.1.2. Study design. Forty-two (21 younger and 21 older) healthy study participants participated in this 62-d metabolic study in the Metabolic Research Unit (MRU) 6 of the Jean Mayer USDA Human Nutrition Research Center on Aging Study participants were provided with effervescent supplements (Hermes Arzeneimittel) to be taken each morning dissolved in a 140- to 170-g glass of water. For 30 d prior to the study start, participants were provided with the daily supplements that contained 600 mg of elemental calcium in the form of calcium carbonate and 10 mg (400 IU) of vitamin D in the form of cholecalciferol. These amounts corresponded to the requirements for calcium and vitamin D at the time the study was initiated (8). Participants continued these supplements while consuming the baseline and restriction diets. On d 34 through to d 61, the daily supplements contained an additional 500 mg phylloquinone. Participants stopped all other supplements before enrolling in this study. The sequence of the phylloquinone restriction supplementation was identical for all participants, with all members of the study team aware of the intervention. All meals were prepared in advance in the MRU and were provided to the participants on an individual basis. During the first 3 study days (d 1 3), participants were in residency within the MRU. During this residency period, energy intakes were adjusted to maintain a constant body weight during the study. During the free-living days, participants had their body weight and blood pressure measured and they consumed meals in the MRU a minimum of 3 d/wk. All participants signed a written informed consent and this study was approved by the Institutional Review Board at Tufts University. Biochemical measurements. All blood samples were drawn between 0700 and 1000 after a 12-h fast. Dedicated aliquots were stored at 280C and protected from light until the time of analysis. Plasma, diet, and supplement concentrations of phylloquinone were determined by reverse-phased HPLC (9). Protein induced in vitamin K absence or antagonism Factor II (PIVKA-II) concentrations were determined by ELISA using commercial kits (Diagnostica Stago). Serum total Osteocalcin (OC) and uncarboxylated OC (ucoc) were measured by RIA using the method of Gundberg (10). Serum cross-linked N- telopeptide of type 1 collagen (NTx) was measured by ELISA (Walpole Laboratories). Plasma 25-hydroxyvitamin D [25(OH)D] was measured by RIA (DiaSorin). An in-house control pool was run on all assays. Plasma was assayed for TG and total, HDL, and LDL cholesterol with a Hitachi 911 automated analyzer (Roche Diagnostics using enzymatic or immunoturbidometric reagents. Urinary g-carboxyglutamic acid (Gla) was determined by derivitizing Gla and using HPLC (11). The total menadione in urine was measured by HPLC, as described elsewhere (12). Other measurements. Total and trunk body fat was measured at baseline using DXA (GE Lunar Prodigy). Scanner software version 6.1 was used for acquisition and version 12.2 was used for analysis. Multiple thickness phantoms were scanned weekly to monitor for instrument stability. BMI (kg/m 2 ) was calculated from baseline measures of height 6 Abbreviations used: Gla, g-carboxyglutamic acid; MRU, Metabolic Research Unit; NTx, cross-linked N-telopeptide of type 1 collagen; OC, osteocalcin; PIVKA-II, protein induced in vitamin K absence or antagonism Factor II; ucoc, uncarboxylated osteocalcin; 25(OH)D, 25-hydroxyvitamin D. and weight. Medical history and medication use were assessed by medical history and examination at baseline. Statistical analysis. Sample size calculations were based on previous phylloquinone depletion and repletion studies in men and women (13,14). Changes in plasma phylloquinone from the end of phylloquinone depletion to the end of phylloquinone repletion were used. The minimum effects (difference in the 30-d change between younger and older adults) that could be detected with 80% power at the 0.05 level of significance by a study of 36 participants (18/age group) were determined by taking the within-group variability of the published data (13,14). We had an 80% chance of detecting an effect of age group on vitamin K supplementation at the 0.05 level of significance if the mean change in plasma phylloquinone differed between the age groups by at least 0.5 nmol/l. To protect against a potential attrition of participants due to scheduling constraints or noncompliance, we included an additional 3 participants/group, for a total of 21/group. The data were examined numerically and graphically for outliers and any other conditions that might affect the validity of the analyses. Repeated-measures 2-way ANOVA with age group and sex as between-participant factors were used to determine whether the markers of vitamin K status and bone turnover markers changed in response to depletion diet and supplementation and if the response differed between the age groups. Circulating TG and measures of adiposity were added as covariates to determine if they were predictive of measures of vitamin K status. Bonferroni adjustments were made when multiple tests were performed. SAS for Windows (version 9.2, SAS Institute) was used for the statistical analyses. Results were judged significant if the 2-sided observed significance level (P value) was,0.05. All values are mean 6 SEM. Results The phylloquinone supplements contained a phylloquinone concentration of 547 6 6.4 mg/tablet. For the phylloquinone depletion diet, 3 different rotating menus were developed. Their phylloquinone concentrations were 0.5 6 0.15, 0.6 6 0.00, and 0.5 6 0.07 mg/100 g, respectively, to achieve a daily target of ~10 mg of phylloquinone intake. Baseline characteristics of the participants were similar among the 4 groups, with the following exceptions: plasma PIVKA-II was higher in the older age groups and in men, urinary Gla was higher in men, percentage body fat was higher in women, and plasma cholesterol and 25(OH)D were higher in the older age groups (Table 1). Vitamin K status measures. Plasma phylloquinone concentrations were 1.19 6 0.17 nmol/l on entry into the study, 2.85 6 0.31 nmol/l in response to 5 d of the baseline diet, declined to 0.33 6 0.10 nmol/l in response to 28 d of the depletion diet (P, 0.001), and increased to 2.29 6 0.22 nmol/l following supplementation (P, 0.001) (Fig. 1A). Supplementation tended to restore phylloquinone concentrations to those achieved with the baseline diet (P = 0.075). The response of phylloquinone concentrations to dietary changes did not differ between the age groups or between men and women, even when adjusted for plasma TG, percentage body fat, and BMI. Plasma PIVKA-II concentrations were within the normal range (,2.4 mg/l) at baseline (15). They increased in response to phylloquinone depletion (P, 0.001) (Fig. 1B) and decreased in response to supplementation (P, 0.001). PIVKA-II concentrations at d 62 were lower than those achieved with the baseline diet (P = 0.007). When adjusted for plasma TG, plasma PIVKA- II decreased more in the older adults compared to younger adults in response to phylloquinone supplementation (P = 0.004). However, the overall changes in plasma PIVKA-II in response to Determinants of vitamin K 937
TABLE 1 Characteristics of the participants at baseline 1 Younger (20 40 y) Older (55 80 y) Two-factor ANOVA P values Characteristics Men Women Men Women Sex Age Sex 3 age n 9 12 12 9 Age, y 30.4 6 3.1 32.9 6 3.0 66.3 6 2.9 68.6 6 3.6 0.31,0.0001 0.96 Measures of vitamin K status Plasma phylloquinone, nmol/l 1.6 6 0.2 0.7 6 0.2 1.3 6 0.2 1.2 6 0.3 0.16 0.83 0.23 Serum ucoc, % 32.7 6 3.6 a 45.8 6 3.5 b 39.7 6 2.6 b 30.9 6 3.3 a 0.68 0.45 0.04 Plasma PIVKA-II, mg/l 1.9 6 0.1 1.7 6 0.1 2.5 6 0.1 1.9 6 0.1 0.02 0.04 0.18 Urinary Gla, mmol/24 h 36.8 6 2.1 b 38.3 6 2.1 b 47.2 6 1.8 c 31.9 6 2.4 a 0.03 0.62 0.009 Urinary menadione, nmol/24h 11.4 6 1.3 14.7 6 1.4 11.6 6 1.2 10.9 6 1.7 0.45 0.57 0.53 Other biochemical measures Plasma 25(OH)D, nmol/l 42.9 6 2.5 42.4 6 2.5 51.7 6 0.2.0 52.2 6 2.8 0.96 0.02 0.89 Plasma TG, mmol/l 1.02 6 0.08 0.89 6 0.08 1.28 6 0.08 1.31 6 0.13 0.05 0.32 0.36 Plasma cholesterol, mmol/l 4.20 6 0.15 4.39 6 0.15 4.57 6 0.13 4.84 6 0.18 0.12 0.03 0.27 Anthropometrics BMI, kg/m 2 27.1 6 1.3 23.3 6 1.1 30.2 6 1.2 21.8 6 1.4 0.72 0.92 0.83 Body fat, % 32.9 6 1.4 34.3 6 1.4 34.5 6 1.1 35.3 6 1.8 0.005 0.71 0.62 Body fat, kg 22.0 6 2.6 23.6 6 2.3 23.6 6 2.1 20.7 6 1.6 0.94 0.56 0.62 Trunk fat, kg 12.9 6 1.6 11.8 6 1.3 14.6 6 1.3 10.6 6 1.6 0.09 0.86 0.32 1 Values are means 6 SEM. Means in a row with superscripts without a common letter differ, P, 0.05. Gla, g-carboxyglutamic acid; PIVKA-II, protein induced in vitamin K absence or antagonism Factor II; ucoc, uncarboxylated osteocalcin; 25(OH)D, 25-hydroxyvitamin D. depletion were similar among all participants (P = 0.29). There was no direct sex effect nor did adjustment for covariates affect the PIVKA-II response. The serum percentage of ucoc increased in response to phylloquinone depletion (P, 0.001) and decreased in response to phylloquinone supplementation (P, 0.001) (Fig. 1C). After phylloquinone supplementation the percentage of ucoc was lower than it was following the baseline diet (P, 0.001) (Fig. 2C). There were no significant age or sex effects on the percentage of ucoc, even after adjustment for covariates. Urinary menadione excretion responded to manipulation of phylloquinone intake (P = 0.049) (Fig. 2A). During depletion, younger adults had lower menadione excretion compared with older adults (P = 0.012). However, there was no age group effect on the response to phylloquinone supplementation (P = 0.11). There were no sex effects on menadione excretion, even after adjustment for covariates. In all participants, urinary Gla tended to decrease in response to phylloquinone depletion (P = 0.06) and increased in response to repletion (P = 0.005) (Fig. 2B). There were no significant age or sex effects on urinary Gla excretion, even after adjustment for covariates. Bone markers. The serum concentrations of NTx and total OC did not change in response to manipulation of phylloquinone intake nor did any of the nondietary factors affect the response of either bone marker, even after adjustment for covariates (Fig. 3). Discussion Adults over the age of 70 y have self-reported vitamin K intakes below the current Adequate Intake for vitamin K (16), and older adults are considered less responsive to vitamin K supplementation compared with younger adults (7). In this study of younger and older adults, the overall magnitude of change in response to manipulation of phylloquinone intake did not differ between age groups. These data suggest that vitamin K status can be similarly improved, regardless of the age group. However, there were large intra- and inter-individual variations in measures of vitamin K status in response to dietary manipulation of vitamin K that remain unexplained. Of the nondietary determinants selected based on previous observational data (2), such as age group, sex, and plasma TG, none explained the large variation in response to diet. Similarly, the data do not support previous reports that older adults have greater dietary-induced subclinical deficiency than younger adults (7) or that vitamin K-depleted older women are not responsive to phylloquinone supplementation (14). One caveat to this study was the 55 80 y age range for the older age group. These findings are not necessarily generalizable to the elderly, especially those older than the age group studied, and who have lower self-reported vitamin K intakes compared with other age groups (16). In contrast to previous studies, the strength of this study is the inclusion of younger and older men and women using the same study design of short-term phylloquinone depletion and supplementation. Similarly, the dose of 500 mg/d of phylloquinone used in this study successfully restored vitamin K status to predepletion levels unlike lower doses of 45 200 mg/ d used in previous studies (7,13). Therefore, it is plausible that higher doses of phylloquinone obviate any potential age effects in response to phylloquinone supplementation. In a previous study conducted by this research team, older women did not respond to up to 450 mg/d of phylloquinone following an extended period of phylloquinone depletion. However, in this previous study of older women, efforts to replete with phylloquinone were in the context a low-calcium diet (14). Although there is no direct evidence supporting a role of calcium in vitamin K metabolism, we speculate that calcium and possibly vitamin D status may influence the response of vitamin K status to repletion. All participants in the current study consumed calcium and vitamin D supplements to ensure that their calcium and vitamin D status was adequate based on the requirements at the time the study was conducted (8). Similarly, the majority of clinical trials examining a potential role of vitamin K in bone and cardiovascular health have 938 Truong et al.
contrast, we used dietary supplements in this study, consistent with previous clinical trials (4), and although the absolute changes in measures of vitamin K status were similar to previous studies using food sources, the inter-individual variation was considerably larger. According to self-report, there was 100% adherence to supplementation by the participants in this study. We do not currently have an explanation for this, except that plant sources may have considerably less bioavailability compared to dietary supplements; hence, we had a larger signal to noise effect. Given the large variation in individual measures of vitamin K, it is plausible that we were underpowered to detect subtle effects associated with any of the nondietary determinants. However, these effects would be unlikely to have clinical relevance. Interestingly, changes in urinary menadione in response to dietary phylloquinone manipulation differed between the age groups. There is growing evidence that vitamin K has functions in addition to its classic role as an enzyme cofactor for the g-carboxylase. Menadione is a urinary metabolite that reflects overall vitamin K metabolism (12) independently of its role as a cofactor. Changes in menadione excretion in younger adults were greater in response to phylloquinone depletion and repletion compared with older adults. The implications for these age group differences are not known and may simply reflect age group-related differences in renal function. However, similar age group differences in urinary Gla excretion were not observed. FIGURE 1 Circulating phylloquinone (A), percentage ucoc (B), and PIVKA-II (C) in younger (20 40 y) and older (55 80 y) men and women when they consumed 200 mg/d (baseline), 10 mg/d (depletion), and 500 mg/d (supplementation) phylloquinone. Values are means 6 SEM, n = 9 (younger men, older women) or 12 (younger women, older men). In all participants, plasma phylloquinone decreased in response to depletion (P, 0.001) and percentage ucoc and PIVKA-II increased (P, 0.001). All markers reversed direction in response to repletion (P, 0.001). PIVKA-II, protein induced in vitamin K absence or antagonism Factor II; ucoc, uncarboxylated osteocalcin. provided vitamin K with a supplemental amount of calcium and vitamin D (4,5,17,18). Data from rodent studies suggest a potential interaction of vitamin D with vitamin K tissue concentrations, although the mechanism is not known (19). There may also be other dietary components that improve vitamin K metabolism in the current study diet that were not present in previous studies, further emphasizing the need to consider response to single nutrients in the context of the entire diet. Similarly, the observational data may have identified nondietary influences on vitamin K status that were confounded by other dietary and lifestyle factors. In previous studies using food sources (13,14,20,21), the inter-individual variation in the status of vitamin K measures in response to phylloquinone intake was relatively small. In FIGURE 2 Urinary menadione (A) and Gla (B) excretions in younger (20 40 y) and older (55 80 y) men and women when they consumed 200 mg/d (baseline), 10 mg/d (depletion), and 500 mg/d (supplementation) phylloquinone. Values are mean 6 SEM, n = 9 (younger men, older women) or 12 (younger women, older men). In all participants, urinary menadione decreased in response to depletion (P, 0.05). Urinary menadione and Gla reversed direction in response to repletion (P, 0.05). Labeled mean differences in response to phylloquinone depletion (d5 34) or supplementation (d34 61) without a common letter differ by age group, P, 0.05. Gla, g-carboxyglutamic acid. Determinants of vitamin K 939
analysis and contributed to the manuscript; G.E.D. performed statistical analysis and contributed to study design and manuscript; C.G. performed laboratory analysis and contributed to study design and manuscript; and S.L.B. conceived of the study and drafted the manuscript. All authors read and approved the final manuscript. Hermes Arzeneimittel GMBH, Munich, Germany generously donated the supplements used for this study. FIGURE 3 Serum NTx (A) and total OC (B) in younger (20 40 y) and older (55 80 y) men and women when they consumed 200 mg/d (baseline), 10 mg/d (depletion), and 500 mg/d (supplementation) phylloquinone. Values are means 6 SEM, n = 9 (younger men, older women) or 12 (younger women, older men). There were no significant changes in either NTx or total OC in response to depletion or repletion. NTx, cross-linked N-telopeptide of type 1 collagen; OC, osteocalcin. Bone turnover markers did not change in response to vitamin K depletion-repletion. This is in contrast to a previous study in which we reported a change in young adults (13) but consistent with subsequent clinical trials that have reported no effect of phylloquinone supplementation on bone measures (4,5,17). Bone turnover markers have wide margins of error, and it may have been a spurious finding in our previous study (13) that was not replicated in this study or in response to phylloquinone supplementation for extended periods of time (4,5,17). In conclusion, markers of vitamin K status similarly responded to phylloquinone restriction and supplementation in younger and older adult age groups. However, nondietary determinants of inter-individual variation in vitamin K response to supplementation remain largely unexplained. There is growing evidence that vitamin K has functions in addition to its classic role as an enzyme cofactor for the g-carboxylase. These data have focused on measures of vitamin K s role as an enzyme cofactor, and more research is required to determine if tissue stores of different vitamin K forms respond similarly across age groups. Acknowledgments J.T.T. carried out the recruitment and oversight of study participant visits and drafted the manuscript; X.F. performed laboratory analysis and contributed to the manuscript; E.S. was the medical doctor on the protocol and contributed to the study design and manuscript; A.R. performed laboratory Literature Cited 1. Booth SL. Roles for vitamin K beyond coagulation. Annu Rev Nutr. 2009;29:89 110. 2. Shea MK, Benjamin EJ, Dupuis J, Massaro JM, Jacques PF, D Agostino RB Sr, Ordovas JM, O Donnell CJ, Dawson-Hughes B, Vasan RS, et al. Genetic and non-genetic correlates of vitamins K and D. Eur J Clin Nutr. 2009;63:458 64. 3. McKeown NM, Jacques PF, Gundberg CM, Peterson JW, Tucker KL, Kiel DP, Wilson PW, Booth SL. 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Food and Nutrition Board, Institute of Medicine. Dietary reference intakes: calcium, phosphorus, magnesium, vitamin D and fluoride. Washington, DC: National Academies Press; 1997. 9. Davidson KW, Sadowski JA. Determination of vitamin K compounds in plasma or serum by high-performance liquid chromatography using postcolumn chemical reduction and fluorimetric detection. Methods Enzymol. 1997;282:408 21. 10. Gundberg CM. Biology, physiology, and clinical chemistry of osteocalcin. J Clin Ligand Assay. 1998;21:128 38. 11. Haroon Y. Rapid assay for gamma-carboxyglutamic acid in urine and bone by precolumn derivatization and reversed-phase liquid chromatography. Anal Biochem. 1984;140:343 8. 12. Al Rajabi A, Peterson J, Choi SW, Suttie J, Barakat S, Booth SL. Measurement of menadione in urine by HPLC. J Chromatogr B Analyt Technol Biomed Life Sci. 2010;878:2457 60. 13. Booth SL, Lichtenstein AH, O Brien-Morse M, McKeown NM, Wood RJ, Saltzman E, Gundberg CM. Effects of a hydrogenated form of vitamin K on bone formation and resorption. Am J Clin Nutr. 2001;74:783 90. 14. Booth SL, Martini L, Peterson JW, Saltzman E, Dallal GE, Wood RJ. Dietary phylloquinone depletion and repletion in older women. J Nutr. 2003;133:2565 9. 15. Grosley BM, Hirschauer C, Chambrette B, Bezeaud A, Amiral J. Specific measurement of hypocarboxylated prothrombin in plasma or serum and application to the diagnosis of hepatocellular carcinoma. J Lab Clin Med. 1996;127:553 64. 16. Institute of Medicine. Dietary Reference Intakes for Vitamin A. Vitamin K, arsenic boron, chromium, copper, iodine, iron, manganese, molybdenum, nickel, silicon, vanadium, and zinc. Washington, DC: National Academies Press; 2001. 17. Cheung AM, Tile L, Lee Y, Tomlinson G, Hawker G, Scher J, Hu H, Vieth R, Thompson L, Jamal S, et al. Supplementation in postmenopausal women with osteopenia (ECKO Trial): a randomized controlled trial. PLoS Med. 2008;5:e196. 18. 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supplementation and progression of coronary artery calcium in older men and women. Am J Clin Nutr. 2009;89:1799 807. 19. Fu X, Wang XD, Mernitz H, Wallin R, Shea MK, Booth SL. 9-Cis retinoic acid reduces 1alpha,25-dihydroxycholecalciferol-induced renal calcification by altering vitamin K-dependent gamma-carboxylation of matrix gamma-carboxyglutamic acid protein in A/J male mice. J Nutr. 2008;138:2337 41. 20. Booth SL, Lichtenstein AH, Dallal GE. Phylloquinone absorption from phylloquinone-fortified oil is greater than from a vegetable in younger and older men and women. J Nutr. 2002;132:2609 12. 21. Booth SL, O Brien-Morse ME, Dallal GE, Davidson KW, Gundberg CM. Response of vitamin K status to different intakes and sources of phylloquinone-rich foods: comparison of younger and older adults. Am J Clin Nutr. 1999;70:368 77. Determinants of vitamin K 941