Source: AGRICULTURAL RESEARCH SERVICE submitted to
BONE HEALTH IN THE ELDERLY
Sponsoring Institution
Agricultural Research Service/USDA
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0408148
Grant No.
(N/A)
Project No.
1950-51000-057-00D
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
May 1, 2004
Project End Date
Mar 15, 2009
Grant Year
(N/A)
Project Director
DAWSON-HUGHES B
Recipient Organization
AGRICULTURAL RESEARCH SERVICE
(N/A)
BOSTON,MA 02111
Performing Department
(N/A)
Non Technical Summary
(N/A)
Animal Health Component
100%
Research Effort Categories
Basic
0%
Applied
100%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
70260101010100%
Goals / Objectives
LAB:Bone Health Refine the approach to determining the vitamin D requirement for optimal musculoskeletal and dental health in men and women. Define the impact of dietary protein and the acid/base balance of the diet on musculoskeletal health in men and women. Define the impact of calcium and vitamin D intake on insulin resistance and risk of developing Type 2 diabetes in ment and women. LAB:Vitamin K Identify and measure the amounts of individual dietary forms of vitamin K in key foods and estimate their relative contribution to total dietary vitamin K intake. Define endogenous and exogenous factors affecting bioavailability of phylloquinone, develop stable isotope measurement techniques and determine if age, lipid profile and current vitamin K nutritional status influence the absorption and transport of phylloquinone in younger and older women. Determine the impact of vitamin K supplementation, in an amount that is expected to be nutritionally optimal and safe, on bone, joint and vascular health in elderly men and women.
Project Methods
LAB:Bone Health The Bone Metabolism Laboratory will perform a randomized, controlled trial (RCT) to evaluate the impact of calcium intake on the vitamin D requirement. In the same study, the impact of endogenous hormone levels, gender, and other clinical characteristics on vitamin D metabolism will be examined by linear regression and other analyses. A series of three protein and amino acid intervention studies will be conducted in humans to define the impact of protein and the acid-base balance of the diet on short-term indicators of bone strength. These studies should provide the data needed to devise a dietary supplement to promote bone health. The effect of this supplement on rates of bone loss will be evaluated in a large RCT in older men and women. LAB:Vitamin K This project will identify the distribution of vitamin K in the food supply, and evaluate dietary and non-dietary determinants of the bioavailability of vitamin K from these foods. Specifically, stable isotopes will be used in metabolic studies to compare the absorption and transport of vitamin K from a green vegetable in a vitamin K-deplete state vs. a vitamin K-replete state. The impact of vitamin K supplementation on changes in bone density and vascular calcification will be examined in clinical trials with elderly men and women. Vitamin K food composition data generated from food analysis will be incorporated into the national nutrient databases for the purpose of menu design, dietary assessment and national intake surveys. Information will be collected on the physiological significance of the measures currently used to assess vitamin K nutritional status and the impact of vitamin K supplementation on bone and vascular health.

Progress 05/01/04 to 03/15/09

Outputs
Progress Report Objectives (from AD-416) LAB:Bone Health Refine the approach to determining the vitamin D requirement for optimal musculoskeletal and dental health in men and women. Define the impact of dietary protein and the acid/base balance of the diet on musculoskeletal health in men and women. Define the impact of calcium and vitamin D intake on insulin resistance and risk of developing Type 2 diabetes in ment and women. LAB:Vitamin K Identify and measure the amounts of individual dietary forms of vitamin K in key foods and estimate their relative contribution to total dietary vitamin K intake. Define endogenous and exogenous factors affecting bioavailability of phylloquinone, develop stable isotope measurement techniques and determine if age, lipid profile and current vitamin K nutritional status influence the absorption and transport of phylloquinone in younger and older women. Determine the impact of vitamin K supplementation, in an amount that is expected to be nutritionally optimal and safe, on bone, joint and vascular health in elderly men and women. Approach (from AD-416) LAB:Bone Health The Bone Metabolism Laboratory will perform a randomized, controlled trial (RCT) to evaluate the impact of calcium intake on the vitamin D requirement. In the same study, the impact of endogenous hormone levels, gender, and other clinical characteristics on vitamin D metabolism will be examined by linear regression and other analyses. A series of three protein and amino acid intervention studies will be conducted in humans to define the impact of protein and the acid-base balance of the diet on short-term indicators of bone strength. These studies should provide the data needed to devise a dietary supplement to promote bone health. The effect of this supplement on rates of bone loss will be evaluated in a large RCT in older men and women. LAB:Vitamin K This project will identify the distribution of vitamin K in the food supply, and evaluate dietary and non-dietary determinants of the bioavailability of vitamin K from these foods. Specifically, stable isotopes will be used in metabolic studies to compare the absorption and transport of vitamin K from a green vegetable in a vitamin K-deplete state vs. a vitamin K-replete state. The impact of vitamin K supplementation on changes in bone density and vascular calcification will be examined in clinical trials with elderly men and women. Vitamin K food composition data generated from food analysis will be incorporated into the national nutrient databases for the purpose of menu design, dietary assessment and national intake surveys. Information will be collected on the physiological significance of the measures currently used to assess vitamin K nutritional status and the impact of vitamin K supplementation on bone and vascular health. Significant Activities that Support Special Target Populations This project includes the work of two subordinate projects at the HNRCA funded through a Specific Cooperative Agreement with Tufts University. For further information and progress reports, see 1950-51000-057-03S, Bone Metabolism Laboratory and 1950-51000-057-04S, Vitamin K Laboratory. Significant Activities that Support Special Target Populations LAB: Bone Metabolism Laboratory - NONE LAB: Vitamin K Laboratory - NONE

Impacts
(N/A)

Publications


    Progress 10/01/06 to 09/30/07

    Outputs
    Progress Report Objectives (from AD-416) LAB:Bone Health Refine the approach to determining the vitamin D requirement for optimal musculoskeletal and dental health in men and women. Define the impact of dietary protein and the acid/base balance of the diet on musculoskeletal health in men and women. Define the impact of calcium and vitamin D intake on insulin resistance and risk of developing Type 2 diabetes in ment and women. LAB:Vitamin K Identify and measure the amounts of individual dietary forms of vitamin K in key foods and estimate their relative contribution to total dietary vitamin K intake. Define endogenous and exogenous factors affecting bioavailability of phylloquinone, develop stable isotope measurement techniques and determine if age, lipid profile and current vitamin K nutritional status influence the absorption and transport of phylloquinone in younger and older women. Determine the impact of vitamin K supplementation, in an amount that is expected to be nutritionally optimal and safe, on bone, joint and vascular health in elderly men and women. Approach (from AD-416) LAB:Bone Health The Bone Metabolism Laboratory will perform a randomized, controlled trial (RCT) to evaluate the impact of calcium intake on the vitamin D requirement. In the same study, the impact of endogenous hormone levels, gender, and other clinical characteristics on vitamin D metabolism will be examined by linear regression and other analyses. A series of three protein and amino acid intervention studies will be conducted in humans to define the impact of protein and the acid-base balance of the diet on short-term indicators of bone strength. These studies should provide the data needed to devise a dietary supplement to promote bone health. The effect of this supplement on rates of bone loss will be evaluated in a large RCT in older men and women. LAB:Vitamin K This project will identify the distribution of vitamin K in the food supply, and evaluate dietary and non-dietary determinants of the bioavailability of vitamin K from these foods. Specifically, stable isotopes will be used in metabolic studies to compare the absorption and transport of vitamin K from a green vegetable in a vitamin K-deplete state vs. a vitamin K-replete state. The impact of vitamin K supplementation on changes in bone density and vascular calcification will be examined in clinical trials with elderly men and women. Vitamin K food composition data generated from food analysis will be incorporated into the national nutrient databases for the purpose of menu design, dietary assessment and national intake surveys. Information will be collected on the physiological significance of the measures currently used to assess vitamin K nutritional status and the impact of vitamin K supplementation on bone and vascular health. Accomplishments LAB: BONE HEALTH 1) Calcium and vitamin D intake may lower risk of developing type 2 diabetes. We recently observed that higher calcium and vitamin D intakes were associated with a reduced incidence of type 2 diabetes in older women. In this study, we compare the effects of combined vitamin D and calcium supplementation versus placebo on blood sugar levels and insulin sensitivity in 314 healthy adults 65 years of age or older. These subjects participated in our randomized, placebo-controlled, calcium and vitamin D intervention trial in which they received either 700 IU of vitamin D3 and 500 mg of calcium (as citrate malate) or placebos daily for 3 years. We found that among participants with glucose intolerance at entry into the trial (defined as pre-diabetics), those who took combined vitamin D-calcium supplements had a significantly smaller increase in fasting blood sugar levels and a smaller increase in insulin resistance over 3 years than did those who took the placebos. These findings indicate that adequate vitamin D and calcium nutrition may attenuate the progression to diabetes that occurs in adults with mildly elevated blood sugars, or glucose intolerance. This research falls under NP107 Human Nutrition, Component 6 Prevention of Obesity and Disease: Relationship between Diet, Genetics, and Lifestyle. 2) Testosterone levels affect risk of falling in both men and women. Falls are a common source of serious injury, including fractures, in older subjects. Little is known about how sex hormone levels affect the risk of falling. We examined the association between testosterone and estrogen levels and risk of falling in 199 men and 246 women, age 65 or older, who participated in our 3-yr randomized, placebo-controlled, calcium and vitamin D intervention trial. Compared to the lowest quartile, men and women in the highest sex-specific quartile of total testosterone had a 78% and 66% decreased risk of falling. These benefits appeared to be amplified by vitamin D plus calcium treatment (84% decreased risk in men and 85% in women). Estrogen levels did not predict falling in men or women. These findings suggest that adequate calcium and vitamin D nutrition may allow sex hormones to have more favorable effects on muscle strength and risk of falling. This research falls under NP107 Human Nutrition, Component 4:Nutrient Requirements. 3) Mechanisms by which dietary protein may affect calcium and bone health. Dietary protein has varied and complex effects on calcium metabolism and specific types of amino acids may affect calcium metabolism selectively. In contrast to branched-chain amino acids, aromatic amino acids bind to the calcium sensing receptor and thus have an increased potential to affect calcium homeostasis. In this study we compare the effects of increased intake of aromatic versus branched-chain amino acids on calcium excretion, calcium absorption, serum IGF-1 (a bone growth factor), and markers of bone turnover in 30 healthy adults. Increasing intake of aromatic amino acids increased calcium excretion, calcium absorption, and serum IGF-1 when compared with similar increases in intake of branched- chain amino acids. Neither group of amino acids had an impact on indices of bone turnover. These findings suggest a role for the calcium sensing receptor as a mediator of several of the beneficial effects of dietary protein on calcium metabolism. This research falls under NP107 Human Nutrition, Component 4 Nutrient Requirements. LAB: VITAMIN K 1) Vitamin K and Bone Health Vitamin K intake may lower risk of bone loss and progression of coronary calcification, but little is known about the optimal nutritional dose required. We compared the effects of vitamin K supplementation on bone loss and coronary calcification in 452 healthy men and women, 60 to 80 years of age. All the men and women participating in the study received 400 IU of vitamin D and 600 mg of calcium, and half of them also received a daily vitamin K supplement that contained 500 micrograms of phylloquinone, which is the primary dietary form of vitamin K. At the end of three years, there were no differences in bone mineral density at either the hip or the spine in those men and women receiving vitamin K supplementation compared to those who were not receiving vitamin K supplementation. Similarly, there were no differences in the coronary calcification between the two groups. The men and women participating in this study had healthy diets, which included adequate amounts of supplemental calcium and vitamin D, nutrients known to improve bone health in the elderly. These findings suggest that the addition of 500 micrograms of vitamin K to a calcium and vitamin D supplement does not further reduce bone loss for men and women already eating a healthy diet. This research falls under NP107 Human Nutrition, Component 4 Nutrient Requirements. 2) Determinants of Vitamin K Little is known about the determinants and heritability of vitamin K status in humans. We assessed the genetic and non-genetic determinants of biochemical measures of vitamin K status in a community-based sample of older men and women. The primary non-genetic determinants of vitamin K status, in descending order of impact, were triglyceride concentrations, dietary vitamin K intake, age, smoking and among women, menopause status. A substantial portion of the variation in vitamin K status among individuals was not explained by known biologically plausible genetic or non-genetic factors. These findings suggest that dietary vitamin K intake is one of many factors influencing vitamin K status in older men and women. This research falls under NP107 Human Nutrition, Component 6 Prevention of Obesity and Disease: Relationship between Diet, Genetics, and Lifestyle. 3) Vitamin K and Joint Health We have reported that older men and women with low intakes of vitamin K have greater risk for osteoarthritis. Little is known about the effect of vitamin K supplementation on joint health. We compared the effects of vitamin K supplementation with no vitamin K supplementation on hand osteoarthritis in 378 healthy men and women, 60 to 80 years of age, who had participated in a 3-year study on the effect of vitamin K on bone health. Overall, vitamin K supplementation did not reduce the risk of having osteoarthritis in the hand. However, among those men and women with low plasma vitamin K concentrations before starting the study, daily vitamin K supplementation of 500 micrograms appeared to reduce the risk of developing osteoarthritis in the hand. These findings suggest that adequate vitamin K intake may have a role in the maintenance of joint health in older men and women. This research falls under NP107 Human Nutrition, Component 4 Nutrient Requirements. Technology Transfer Number of Non-Peer Reviewed Presentations and Proceedings: 13 Number of Newspaper Articles,Presentations for NonScience Audiences: 19

    Impacts
    (N/A)

    Publications

    • Burge, R., Dawson-Hughes, B., Solomon, D.H., Wong, J.B., King, A., Tosteson, A. 2006. Incidence and economic burden of osteoporotic fractures in the United States, 2005-2025. Journal of Bone and Mineral Research. 22(3):465-475.
    • Pittas, A.G., Harris, S.S., Stark, P., Dawson-Hughes, B. 2007. The effects of combined vitamin D and calcium supplementation on glycemia, insulin resistance and systemic inflammation in non-diabetic adults age 65 and older. Diabetes Care. 30(4):980-6.
    • Dawson-Hughes, B., Harris, S.S., Rasmussen, H.M., Dallal, G.E. 2007. Comparative effects of oral aromatic and branched-chain amino acids on urine calcium and excretion. Osteoporosis International. 18: 955-961.
    • Dawson-Hughes, B. 2007. Protein intake and calcium absorption ¿ Potential role of the calcium sensor receptor. In: Elsevier, B.V. International Congress Series. 1297. 217-227.
    • Seeman, E., Compston, J., Adachi, J., Brandi, M.L., Cooper, C., Dawson- Hughes, B., Jonsson, B., Pols, H., Cramer, J.A. 2007. Non-compliance: the Achilles heel of anti-fracture efficacy. Osteoporosis International. 18:711-19.
    • Kakarala, R.R., Chandana, S., Harris, S.S., Kocharla, L., Dvorin, E. 2007. Prevalence of Vitamin D Deficiency in Uninsured Women. Journal of General Internal Medicine. 22:1180-83.
    • Harris, S.S., Dawson-Hughes, B. 2007. Reduced Sun Exposure Does Not Explain the Inverse Association of 25-Hydroxyvitamin D with Percent Body Fat in Older Adults. Journal of Clinical Endocrinology and Metabolism. doi:10.1210/jc.2007-0722.
    • O'Donnell, C., Price, P.A., Shea, K.M., Gagnon, D.R., Wilson, P.W., Larson, M.G., Kiel, D.P., Hoffman, U., Ferencik, M., Clouse, M.E., Williamson, M. K., Cupples, A.L., Dawson-Hughes, B., Booth, S.L. 2006. Matrix Gla Protein is Associated with Risk Factors for Atherosclerosis but not with Coronary Artery Calcification. Arteriosclerosis Thrombosis and Vascular Biology. 26(12):2769-2774.
    • Trovar, A., Ameho, C.K., Blumberg, J., Peterson, J.W., Smith, D., Booth, S. L. 2006. Extrahepatic Tissue Concentrations of Vitamin K are Lower in Rats Fed a High Vitamin E Diet. Nutrition and Metabolism. 3:29.
    • Booth, S.L. 2007. Vitamin K Status in the Elderly. Current Opinion in Clinical Nutrition and Metabolic Care. 10(1):20-23.
    • Harrington, D.J., Booth, S.L., Card, D.J., Shearer, M.J. 2007. Excretion of the urinary 5C- and 7C-aglycone metabolitesof vitamin K in response to changes in dietary phylloquinone and dihydrophyliquinone intake. Journal of Nutrition. 137:1763-68.
    • Dawson-Hughes, B. 2006. Calcium and Vitamin D. In: Favus MJ. Book Chapter. Sixth Edition. Washington, D.C.: American Society for Bone and Mineral Research. p.257-259.
    • Custodio Das Dores, S.M., Booth, S.L., Martini, L.A., De Carvalho Gouvea, V.H., Padovani, C.R., De Abreu Maffei, F.H., Campana, A.O., Rupp De Paiva, S.A. 2007. Relationship between diet and anticoagulant response to warfarin ¿ A factor analysis. European Journal of Nutrition. 46(3):147-154.
    • Pilkey, R.M., Morton, A., Boffa, M.B., Noordhof, C., Day, A.G., Su, Y., Miller, L.M., Koschinsky, M.L., Booth, S.L. 2007. Subclinical vitamin K deficiency in hemodialysis (HD) patients. Journal of the American Society of Nephrology. 49(3):432-9.
    • Felson, D.T., Niu, J., Clancy, M., Aliabadi, P., Sack, B., Guermazi, A., Hunter, D.J., Amin, S., Rogers, G., Booth, S.L. 2006. Low Levels of Vitamin D and Worsening of Knee OA: Results from Two Longitudinal Studies. Arthritis and Rheumatism. 56(1): 129-136.
    • Shea, K.M., Booth, S.L. 2007. Role of Vitamin K in Calcification. Book Chapter. 1297: 165-178.
    • Lukacs, J.L., Booth, S.L., Kleerekoper, M., Ansbacher, R., Rock, C.L., Reame, N.E. 2006. Differential associations for menopause and age in measures of vitamin k, osteocalcin, and bone density: a cross-sectional exploratory study in healthy volunteers. The Journal of the North American Menopause Society. 13(5):799-808.


    Progress 10/01/05 to 09/30/06

    Outputs
    Progress Report 1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter? This five-year research plan represents a continuation of interest on the part of the Bone Metabolism Laboratory in improving the understanding of how diet impacts bone health in older men and women. Serum 25-hydroxy vitamin D [25(OH)D] is the best clinical measure of vitamin D status. One objective of the Bone Metabolism Laboratory is to define factors that influence the 25(OH)D concentrations achieved at a given vitamin D intake level in the wintertime. We will assess the effect of body mass index on the utilization of dietary vitamin D in 389 men and women age 65 and older who participated in a placebo-controlled calcium and vitamin D intervention study. We will also test the hypothesis that the increase in serum 25(OH)D in response to oral dosing with vitamin D is blunted at low calcium intakes. This will be studied in a new randomized controlled trial in which 800 IU/day of vitamin D will be given to older subjects with high and low calcium intakes. We will also determine the impact of endogenous gonadal-hormone levels on 25(OH)D responses to supplemental vitamin D. Falls are a common source of serious injury, including fractures, in older subjects. Recent evidence suggests that low vitamin D levels may contribute to muscle weakness and increased risk of falling in this population. We will examine this possibility in the 389 older men and women who participated in our 3-year calcium and vitamin D intervention study. The impact of lean body mass, gonadal hormone levels, and usual activity levels on risk of falling will also be assessed. The vitamin D studies described above will improve the scientific basis for determining the vitamin D intake requirements for older men and women. A second research initiative of the Bone Metabolism Laboratory is to define the impact of dietary protein and the acid-base balance of the diet on bone health. Several small protein intervention studies will be conducted to define the relative roles of protein, added aromatic or branched-chain L-amino acids, calcium, vitamin D, and alkaline foods (fruits and vegetables) or supplements, such as potassium bicarbonate, on short-term indices of bone metabolism including biochemical markers of bone turnover, calcium absorption, urinary calcium excretion, and insulin- like growth factor-1. A supplement (expected to contain protein, calcium, vitamin D, alkali, and perhaps added branched-chain amino acids) will be tested in a large longitudinal intervention trial in older men and women to determine its impact on rates of change in bone mineral density (BMD). This research falls under National Program 107 - Human Nutrition, and is related to component 4: Nutrient Requirements. The ability to establish dietary recommendations for vitamin K is limited by our inadequate understanding of the dietary sources and metabolic fate of phylloquinone from the diet. Studies are being conducted to identify the forms, amounts and biological activity of vitamin K in the food supply. Vitamin K food composition data generated from food analysis will be incorporated into the national nutrient databases for the purpose of menu design, dietary assessment, and national intake surveys. Stable isotopes will be used to elucidate the absorption and transport of vitamin K from vegetables, the primary dietary source of vitamin K in the United States (US). Information is also being collected on the physiological significance of measures used to assess vitamin K nutritional status, with a focus on the impact of vitamin K supplementation on bone and vascular health. The largest of these studies is a 3-year randomized, placebo-controlled vitamin K intervention trial in 452 men and women, age 60-80, who are calcium and vitamin D replete. The main analysis will determine if there are bone density gains among those receiving vitamin K supplementation. Secondary analysis will determine if there is a reduction in the progression of age- related vascular calcification among those receiving vitamin K supplementation. This research falls under National Program 107 - Human Nutrition, program components 1: Composition of Foods, 2: Bioavailability of Nutrients and Food Components, and 4: Nutrient Requirements. 2. List by year the currently approved milestones (indicators of research progress) Bone Metabolism Laboratory Objective 1: Refine the approach to determining the vitamin D requirement for optimal musculoskeletal and dental health in men and women. Objective 2: Define the impact of dietary protein and the acid/base balance of the diet on musculoskeletal health in men and women. Objective 3: Define the impact of calcium and vitamin D intake on insulin resistance and risk of developing Type 2 diabetes in men and women. 2005 1. To determine the impact of body mass index on the serum 25(OH)D response to supplemental vitamin D. Objective 1 2. To determine the impact of calcium intake on the 25(OH)D response to supplemental vitamin D. Objective 1 3. To assess the long-term effect of vitamin D plus calcium on physical function and on the risk of falling in older men and women. Objective 1 2006 1. To determine the association between serum vitamin D levels and risk of gingivitis. Objective 1 2. To define and compare patterns of calcium and vitamin D supplement use in a random sample of women with and without osteoporosis. Objective 1 3. To determine whether the level of calcium intake influences any association between the ratio of nitrogen to potassium in the urine and rates of bone loss in older men and women. Objective 2 4. To determine the association between the ratio of nitrogen to potassium in the urine, an index of the acidogenicity of the diet, and 3- year rates of bone loss in healthy older men and women. Objective 1 5. To determine the impact of stimulating the calcium sensor receptor on gastric acid secretion and calcium excretion. Objective 2 6. To determine the association between intakes of calcium and vitamin D and incidence of developing Type 2 diabetes. Objective 3 7. To determine and compare the effects of selected aromatic and branched-chain amino acids on urinary calcium excretion in healthy older men and women. Objective 2 2007 1. To study the association between endogenous gonadal hormone levels and physical function and risk of falling in elderly men and women. Objective 1 2. Determine mechanisms by which vitamin D influences muscle strength and risk of falling. Objective 1 3. To determine whether alkalinization of the diet will attenuate the urine calcium losses induced by high protein diets. Objective 2 4. To determine the effect of supplementation with calcium and vitamin D on fasting insulin and glucose levels in healthy older men and women. Objective 3 2008 1. Define mechanisms by which specific amino acids affect intestinal calcium absorption. Objective 2 2. To begin a randomized, controlled longitudinal trial to determine whether alkalinization of the diet will lower the bone turnover rate, and thus lower risk of fracture, in healthy men and women who are being supplemented with calcium and vitamin D. Objective 2 2009 1. Continue the randomized, controlled longitudinal trial to determine whether alkalinization of the diet will lower the bone turnover rate, and thus lower risk of fracture, in healthy men and women who are being supplemented with calcium and vitamin D. Objective 2 Vitamin K Laboratory Objective 1: Identify and measure the amounts of individual dietary forms of vitamin K in key foods representative of the national food supply, and estimate their relative contribution to total dietary vitamin K intake. Objective 2: Define endogenous and exogenous factors affecting bioavailability of phylloquinone, the major form of vitamin K in circulation. Stable isotope techniques will be developed to measure phylloquinone in individual lipoprotein fractions in a fasting and fed state. These stable isotope techniques will be used to determine if age, lipid profile and current vitamin K nutritional status influence the absorption and transport of phylloquinone in younger and older men and women. Apolipoprotein E genotype will be examined as a potential influence on vitamin K nutritional status in men and women. Objective 3: Determine the impact of vitamin K supplementation, in an amount that is expected to be nutritionally optimal and safe, on bone, joint and vascular health in elderly men and women. 2005 1. Characterize and quantify multiple forms of vitamin K in food samples representative of the US food supply. Objective 1 2. Create an expanded, yet targeted vitamin K database using the aforementioned food analysis data, to be used for a food frequency questionnaire. Objective 1 3. To assess the dietary and non-dietary determinants of circulating levels of dihydrophylloquinone, a form of dietary vitamin K, in the Framingham Heart Study. Objective 1 4. To develop stable isotope techniques using liquid chromatography mass spectrometry (LC/MS) to measure vitamin K absorption and transport. Objective 2 2006 1. Quantify multiple forms of vitamin K in dietary supplements representative of the US food supply. Objective 1 2. To relate the dietary intakes of dihydrophylloquinone, a form of dietary vitamin K, to physiological outcomes, such as bone health, in the Framingham Heart Study. Objective 2 3. To assess the role of apolipoprotein E genotype in modulating vitamin K status in the Framingham Heart Study. Objective 2 4. To develop a rat model to examine the effects of vitamin K supplementation on the transport of vitamin K to various tissues. Objective 2 2007 1. To determine the impact of vitamin K supplementation, in an amount that is expected to be nutritionally optimal and safe, on bone, joint and vascular health in 452 elderly men and women. Objective 3 2. To estimate the usual dietary intakes of menaquinone-4, a form of vitamin K present in animal products, among men and women participating in the Framingham Heart Study. Objective 1 2008 1. To complete enrollment of a bioavailability study in younger and older men and women to examine the role of age, lipid profile and current vitamin K status on absorption and relative bioavailability of vitamin K from a vegetable. Objective 2 2. To conduct a cross-sectional analysis of phylloquinone, dihydrophylloquinone and menaquinone-4 intakes and plasma levels in the baseline data from 452 elderly men and women participating in a vitamin K supplementation trial to provide information on the dietary and non- dietary determinants of vitamin K that are specific to the elderly. Objective 2 2009 1. To examine the roles of apolipoprotein E, vitamin K epoxide reductase, and vitamin K-dependent gamma carboxylase genotypes in modulating vitamin K status among participants in a vitamin K supplementation trial. Objective 2 2. To determine the impact of vitamin K supplementation on measures of inflammation, as they relate to bone and vascular health. Objective 3 4a List the single most significant research accomplishment during FY 2006. Vitamin D, Calcium and Type 2 Diabetes Mellitus Higher intakes of vitamin D and calcium significantly lowered risk of developing type 2 diabetes mellitus in the Nurses Health Study, a prospective cohort of 83,779 women age 30 to 55 years who were followed for 20 years. A combined daily intake of over 800 IU of vitamin D and 1200 mg of calcium was associated with a 33% lower risk of developing type 2 diabetes as compared with an intake of less than 200 IU and 600 mg of vitamin D and calcium, respectively. The results of this prospective cohort study suggest a potential beneficial role for both vitamin D and calcium intake in reducing the risk of type 2 diabetes. Type 2 diabetes mellitus is associated with considerable morbidity and mortality and its prevalence has been increasing in the United States and worldwide. Potentially modifiable environmental risk factors for type 2 diabetes have been identified, the major one being obesity. However, although weight-loss (achieved by any means) has been shown to be successful in delaying diabetes, it is difficult to achieve and maintain long-term. Therefore, identification of weight-independent and easily modified dietary risk factors is urgently needed to prevent the increase in the incidence of type 2 diabetes. This research falls under National Program 107 Human Nutrition, Component 1: Nutrient Requirements. 4b List other significant research accomplishment(s), if any. Bone Metabolism Laboratory Vitamin D Supplementation Can Lower Fracture Risk A meta-analysis of randomized, controlled vitamin D intervention studies revealed that vitamin D lowers risk of fracture when the intervention is sufficient to raise the serum 25-hydroxyvitamin D level to 75 nmol/L or higher. Studies in which lower circulating levels of 25-hydroxyvitamin D were achieved, either because of administration of a lower dose of vitamin D or because of non-compliance with the supplements, showed no effect of the vitamin on fracture risk. Vitamin D appears to lower fracture risk by increasing bone mass and by lowering risk of falling. Increasing intake of this vitamin is a safe and cost-effective approach to reducing fractures in elderly men and women. This research falls under National Program 107 Human Nutrition, Components 1: Nutrient Requirements. Vitamin K Laboratory Vitamin K in Grains, Cereals, Fast-Food Breakfasts, and Baked Goods Accurate estimates of dietary vitamin K intake is in the diet requires accurate food composition data. Three forms of vitamin K, phylloquinone, dihydrophylloquinone and menaquinone-4, were determined in representative grains, cereals and baked goods in the U.S. food supply. Key foods were collected as part of the National Food and Nutrient Analysis Program. Overall, breads, grains and breakfast cereals provided limited sources of phylloquinone, which is the primary form of vitamin K in the U.S. diet. In contrast, processed foods, such as fast-food sandwiches and baked goods, contain wide ranges of phylloquinone and dihydrophylloquinone. Low concentrations of menaquinone-4 were detected in those breakfast foods that contained cheese and meat. These findings suggest that processed foods that contain phylloquinone-rich plant oils are an unexpected source of phylloquinone and dihydrophylloquinone in the US food supply. These results are aligned to National Program 107 - Human Nutrition, Component 1: Composition of Foods. 5. Describe the major accomplishments to date and their predicted or actual impact. Bone Metabolism Laboratory As we complete the second year of this 5-year program cycle, the main accomplishments of the Bone Metabolism Laboratory are the findings that supplementation with calcium and vitamin D lowers risk of falling in older subjects. Falls are common occurrences in the elderly 30% of subjects age 65 and 40 to 50% of those age 80 fall each year. These falls are a major precipitant of fractures. As expected, increasing vitamin D intake, by lowering risk of falling, can also lower risk of fracture in older men and women. Dissemination of these findings in the scientific and medical community should lead to increases in vitamin D and calcium intakes and a reduction in falls and fractures in older men and women. This finding is related to Milestone 3, 2005: To assess the long-term effect of vitamin D plus calcium on physical function and on the risk of falling in older men and women. The second major finding is that increased intakes of calcium and vitamin D are associated with lower risk of developing type 2 diabetes. This provides a safe and easy approach to lowering risk of developing this devastating disease. This finding is related to Milestone 6 (2006): To determine the association between intakes of calcium and vitamin D and incidence of developing Type 2 diabetes. These findings are related to National Program 107 - Human Nutrition program component 4: Nutrient Requirements. Vitamin K Laboratory As we complete the second year of a 5-year program period, the major accomplishment of the Vitamin K Laboratory has been to demonstrate that high dietary intakes and biochemical measures of vitamin K are associated with a reduced risk for certain chronic diseases associated with abnormal calcification in older men and women. Of concern is the consistent observation that dietary intakes of vitamin K are lower among U.S. adults than previously assumed. This finding is related to our 2007 milestone 1, which is to determine the impact of vitamin K supplementation in an amount that is expected to be nutritionally optimal and safe, on bone, joint and vascular health in 452 elderly men and women. This finding is aligned with National Program 107 - Human Nutrition, program component 4: Nutrient Requirements. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? Bone Metabolism Laboratory The finding that higher intakes of calcium and vitamin D reduce risk of developing type 2 diabetes has been presented at a scientific meeting and published in the peer-reviewed literature. This information will be useful in determining the calcium and vitamin D requirements. No intellectual property rights issues apply. Vitamin K Laboratory The finding that dihydrophylloquinone, a marker of a non-healthy diet is associated with less bone mineral density has been presented at scientific meetings, and a manuscript describing these findings has been submitted for peer-review. The Vitamin K food composition data were electronically transferred to ARS scientists at Beltsville USDA Nutrient Data Laboratory for input into the USDA Nutrient Databank System. These data will be used by scientists conducting nutrition-related research, by the food industry, and by consumers. The vitamin K food composition data have been published in peer-review food journals. No intellectual property rights issues apply. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Bone Metabolism Lab Environmental Nutrition Calcium and vitamin D help tame blood sugar (Dr. Dawson-Hughes findings on calcium, vitamin D , and risk of developing type 2 diabetes) June, 2006. National Public Radio interview Better bone health with more vitamin D, June 22, 2006. ABC World News Tonight Dr. Dawson-Hughes view on the role of calcium in lowering fracture risk, Febuary 15, 2006. Good Morning America Dr. Dawson-Hughes view on the importance of calcium in bone health. Febuary 16, 2006. Bloomberg News Dr. Dawson-Hughes interpretation of the Womens Health Initiative study of calcium, vitamin D and fracture risk. Febuary 21, 2006. Washington Post - Dr. Dawson-Hughes interpretation of the optimal vitamin D intake for bone health. August 9, 2005. Vitamin K Laboratory American Medical News Susan Landers Vitamin K and healthy bones; August, 2005 Eating Well Magazine Peter Jaret Vitamin K; December 2005.

    Impacts
    (N/A)

    Publications

    • Pro-Risquez, A., Harris, S.S., Song, L., Rudicel, S., Barnewolt, B., Dawson-Hughes, B. 2004. Calcium supplement and osteoporosis medication use in women and men with recent fractures. Osteoporosis International. 15:689- 694.
    • Goussous, R.Y., Song, L., Dallal, G., Dawson-Hughes, B. 2004. The effect of calcium intake on the vitamin D requirement. [abstract] Journal Bone and Mineral Research. 19(Suppl 1):S202.
    • Bischoff-Ferrari, H.A., Orav, J.E., Dawson-Hughes, B. 2004. Effect of vitamin D3 plus calcium on fall risk in older men and women: a 3-year randomized controlled trial. [abstract] Journal of Bone and Mineral Research. 19(Suppl 1):S57.
    • Booth, S.L., Golly, I., Sacheck, J.M., Roubenoff, R., Dallal, G.E., Hamada, K., Blumberg, J.B. 2004. Effect of vitamin E supplementation on vitamin K status in adults with normal coagulation status. American Journal of Clinical Nutrition. 80:143-148.
    • Schulze, K.J., O'Brien, K.O., Germain-Lee, E.L., Booth, S.L., Leonard, A., Rosenstein, B.J. 2004. Bone calcium deposition is associated with availability of dietary calcium and cirulating leptin and osteocalcin concentrations in girls with cystic fibrosis. Journal of Clinical Endocrinology and Metabolism. 89(7)3385-3391.
    • Cury, R.C., Ferencik, M., Hoffmann, U., Ferullo, A., Moselewski, F., Abbara, S., O'Donnell, C.J., Booth, S.L., Brady, T.J., Achenbach, S. 2004. Epidemiology and association of vascular and valvular calcium quantified by multi-detector computed tomography in elderly asymptomatic subjects. American Journal of Cardiology. 94:348-351.
    • Braam, L.A., Mckeown, N.M., Jacques, P.F., Lichtenstein, A.H., Vermeer, C., Wilson, P.W., Booth, S.L. 2004. Dietary phylloquinone intake as a potential marker for a heart-healthy dietary pattern in the Framingham Offspring Cohort. Journal Of The American Dietetic Association. 104(9) :1410-1414.
    • Damon, M., Zhang, N., Haytowitz, D.B., Booth, S. 2005. Phylloquinone (Vitamin K1) content of vegetables. Journal of Food Composition and Analysis. 18:751-758. Available online at http://www.sciencedirect.com doi:10.1016/j.jfca.2004.07.004
    • Erkkila, A.T., Lichtenstein, A.H., Jacques, P.F., Hu, F.B., Wilson, P.W., Booth, S.L. 2005. Determinants of plasma dihydrophylloquinone in men and women. British Journal of Nutrition. 93:701-708.
    • Goussous, R., Song, L., Dallal, G.E., Dawson-Hughes, B. 2005. Lack of effect of calcium intake on the 25-hydroxyvitamin D response to oral vitamin D3. Journal of Clinical Endocrinology and Metabolism. 90(2): 707- 711.
    • Bischoff-Ferrari, H.A., Dietrich, T., Orav, J.E., Hu, F.B., Zhang, Y., Karlson, E., Dawson-Hughes, B. 2004. Higher 25-hydroxyvitamin D levels are associated with better lower extremity function in both active and inactive adults 60+ years of age. American Journal of Clinical Nutrition. 80:752-758.
    • Booth, S.L., Broe, K.L., Peterson, J.W., Cheng, D.M., Dawson-Hughes, B., Gundberg, C.M., Cupples, L.A., Wilson, P.W., Kiel, D.P. 2004. Associations between vitamin k biochemical measures and bone mineral density in men and women. Journal of Clinical Endocrinology and Metabolism. 89(10):4904-4909.
    • Hyten, D.L., Song, Q., Costa, J., Cregan, P.B. 2003. Linkage disequilibrium in four soybean populations [abstract]. BARC Poster Day. Abstract 14.
    • Vazquez, E., Song, L.Y., Dawson-Hughes, B. 2004. Precision of bone mineral density scans at the proximal tibia in osteoarthritic subjects. Journal of Clinical Densitometry. 7(4):364-367.
    • Bischoff-Ferrari, H.A., Willett, W.C., Wong, J.B., Giovannucci, E., Dietrich, T., Dawson-Hughes, B. 2005. Fracture prevention by Vitamin D supplementation: A meta-analysis of randomized controlled trials. Journal of the American Medical Association. 293:2257-2264.
    • Dawson-Hughes, B. 2004. Dietary protein and the skeleton. In: Burckhardt, P., Dawson-Hughes, B., Heaney, R.P. editors. Nutritional Aspects of Osteoporosis. 2nd edition. San Diego, CA.: Elsevier, Inc. p. 399-409.
    • Bischoff, H.A., Dawson-Hughes, B. 2004. Defining optimal 25-hydroxyvitamin D levels in younger and older adults based on hip bone mineral density. In: Burckhardt, P., Dawson-Hughes, B., Heaney, R.P. editors. Nutritional Aspects of Osteoporosis. 2nd edition. San Diego, CA: Elsevier, Inc., p. 235-244.
    • Dawson-Hughes, B., Heaney, R.P., Holick, M., Lips, P., Meunier, P.J., Vieth, R. 2004. Vitamin D round table. In: Burckhardt, P., Dawson-Hughes, B., Heaney, R., editors. Nutritional Aspects of Osteoporosis. 2nd edition. San Diego, CA.: Elsiever, Inc. p. 263-270.
    • Dawson-Hughes, B. 2004. Racial/ethnic considerations in making recommendations for vitamin D for adult and elderly men and women. In: Proceedings of the NIH Vitamin D Conference, October 10, 2003. American Journal of Clinical Nutrition. 80(Suppl):1763S-1766S.
    • Dawson-Hughes, B., Heaney, R.P., Holick, M.F., Lips, P., Meunier, P.J., Vieth, R. 2005. Estimates of optimal vitamin D status. Osteoporosis International [on-line]. Available at http://www.springerlink.com
    • Dawson-Hughes, B. 2005. The role of vitamin d in fracture prevention. BoneKEy-Osteovision. 2(4):6-10. (Epub) Available at http://www.bonekey- ibms.org/cgi/content/full/ibmske;2/4/6 DOI: 10.1138/20050156
    • Wilsey, B.J., Polley, H.W. 2004. Realistically low species evenness does not alter grassland species-richness-productivity relationships. Ecology. 85(10):2693-2700.
    • Jajoo, R., Song, L., Ramussen, H., Dawson-Hughes, B. 2004. Effect of change in dietary acid-base balance on bone turnover and urine calcium excretion. [abstract] Journal of Bone and Mineral Research. 19(Suppl 1) :S448-449.


    Progress 10/01/04 to 09/30/05

    Outputs
    1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? This CRIS consists of the Bone Metabolism Laboratory and the Vitamin K Laboratory where scientists are working to improve the understanding of how diet impacts bone health in older men and women. Serum 25-hydroxy vitamin D [25(OH)D] is the best clinical measure of vitamin D status. The Bone Metabolism Laboratory is working to define factors that influence the 25(OH)D concentrations achieved at a given vitamin D intake level in the wintertime. Scientists are conducting a study of the effect of body mass index on the utilization of dietary vitamin D in 389 men and women age 65 and older who participated in a placebo-controlled calcium and vitamin D intervention study. They will also test the hypothesis that the increase in serum 25(OH)D in response to oral dosing with vitamin D is blunted at low calcium intakes. This will be studied in a new randomized controlled trial in which 800 IU/day of vitamin D will be given to older subjects with high and low calcium intakes. Scientists will also examine the impact of endogenous gonadal-hormone levels on 25(OH)D responses to supplemental vitamin D. Falls are a common source of serious injury, including fractures, in older subjects. Recent evidence suggests that low vitamin D levels may contribute to muscle weakness and increased risk of falling in this population. Scientists will examine this possibility in the 389 older men and women who participated in a 3-year calcium and vitamin D intervention study. The impact of lean body mass, gonadal hormone levels, and usual activity levels on risk of falling will also be examined. These vitamin D studies will improve the scientific basis for determining the vitamin D intake requirements for older men and women. A second research initiative of the Bone Metabolism Laboratory is to define the impact of dietary protein and the acid-base balance of the diet on bone health. Several small protein intervention studies will be conducted to define the relative roles of protein, added aromatic or branched-chain L-amino acids, calcium, vitamin D, and alkaline foods (fruits and vegetables) or supplements, such as potassium bicarbonate, on short-term indices of bone metabolism including biochemical markers of bone turnover, calcium absorption, urinary calcium excretion, and insulin- like growth factor-1. A supplement (expected to contain protein, calcium, vitamin D, alkali, and perhaps added branched-chain amino acids) will be tested in a large longitudinal intervention trial in older men and women to determine its impact on rates of change in bone mineral density (BMD). This research falls under National Program 107 - Human Nutrition, and is related to component 4: Nutrient Requirements. The ability to establish dietary recommendations for vitamin K is limited by an inadequate understanding of the dietary sources and metabolic fate of phylloquinone from the diet. Studies are being conducted to identify the forms, amounts and biological activity of vitamin K in the food supply. Vitamin K food composition data generated from food analysis will be incorporated into the national nutrient databases for the purpose of menu design, dietary assessment, and national intake surveys. Stable isotopes will be used to elucidate the absorption and transport of vitamin K from vegetables, the primary dietary source of vitamin K in the United States (US). Information is also being collected on the physiological significance of measures used to assess vitamin K nutritional status, with a focus on the impact of vitamin K supplementation on bone and vascular health. The largest of these studies is a 3-year randomized, placebo-controlled vitamin K intervention trial in 452 men and women, age 60-80, who are calcium and vitamin D replete. This study is currently underway, with all subjects enrolled. The main analysis will determine if there are bone density gains among those receiving vitamin K supplementation. Secondary analysis will determine if there is a reduction in the progression of age- related vascular calcification among those receiving vitamin K supplementation. This research falls under National Program 107 - Human Nutrition, program components 1: Composition of Foods, 2: Bioavailability of Nutrients and Food Components, and 4: Nutrient Requirements. 2. List the milestones (indicators of progress) from your Project Plan. Bone Metabolism Laboratory Objective 1: Refine the approach to determining the vitamin D requirement for optimal musculoskeletal and dental health in men and women Objective 2: Define the impact of dietary protein and the acid/base balance of the diet on musculoskeletal health in men and women Objective 3: Define the impact of calcium and vitamin D intake on insulin resistance and risk of developing Type 2 diabetes in men and women Milestones: 2005 1. To determine the impact of body mass index on the serum 25(OH)D response to supplemental vitamin D. Objective 1 2. To determine the impact of calcium intake on the 25(OH)D response to supplemental vitamin D. Objective 1 3. To assess the long-term effect of vitamin D plus calcium on physical function and on the risk of falling in older men and women. Objective 1 2006 1. To determine the association between serum vitamin D levels and risk of gingivitis. Objective 1 2. To define and compare patterns of calcium and vitamin D supplement use in a random sample of women with and without osteoporosis. Objective 1 3. To determine whether the level of calcium intake influences any association between the ratio of nitrogen to potassium in the urine and rates of bone loss in older men and women. Objective 2 4. To determine the association between the ratio of nitrogen to potassium in the urine, an index of the acidogenicity of the diet, and 3- year rates of bone loss in healthy older men and women. Objective 1 5. To determine the impact of stimulating the calcium sensor receptor on gastric acid secretion and calcium excretion. Objective 2 6. To determine the association between intakes of calcium and vitamin D and incidence of developing Type 2 diabetes. Objective 3 7. To determine and compare the effects of selected aromatic and branched-chain amino acids on urinary calcium excretion in healthy older men and women. Objective 2 2007 1. To study the association between endogenous gonadal hormone levels and physical function and risk of falling in elderly men and women. Objective 1 2. Determine mechanisms by which vitamin D influences muscle strength and risk of falling. Objective 1 3. To determine whether alkalinization of the diet will attenuate the urine calcium losses induced by high protein diets. Objective 2 4. To determine the effect of supplementation with calcium and vitamin D on fasting insulin and glucose levels in healthy older men and women. Objective 3 2008 1. Define mechanisms by which specific amino acids affect intestinal calcium absorption. Objective 2 2. To begin a randomized, controlled longitudinal trial to determine whether alkalinization of the diet will lower the bone turnover rate, and thus lower risk of fracture, in healthy men and women who are being supplemented with calcium and vitamin D. Objective 2 2009 1. Continue the randomized, controlled longitudinal trial to determine whether alkalinization of the diet will lower the bone turnover rate, and thus lower risk of fracture, in healthy men and women who are being supplemented with calcium and vitamin D. Objective 2 Vitamin K Laboratory Objective 1: Identify and measure the amounts of individual dietary forms of vitamin K in key foods representative of the national food supply, and estimate their relative contribution to total dietary vitamin K intake. Objective 2: Define endogenous and exogenous factors affecting bioavailability of phylloquinone, the major form of vitamin K in circulation. Stable isotope techniques will be developed to measure phylloquinone in individual lipoprotein fractions in a fasting and fed state. These stable isotope techniques will be used to determine if age, lipid profile and current vitamin K nutritional status influence the absorption and transport of phylloquinone in younger and older men and women. Apolipoprotein E genotype will be examined as a potential influence on vitamin K nutritional status in men and women. Objective 3: Determine the impact of vitamin K supplementation, in an amount that is expected to be nutritionally optimal and safe, on bone, joint and vascular health in elderly men and women. Milestones: 2005 1. Characterize and quantify multiple forms of vitamin K in food samples representative of the US food supply. Objective 1 2. Create an expanded, yet targeted vitamin K database using the aforementioned food analysis data, to be used for a food frequency questionnaire. Objective 1 3. To assess the dietary and non-dietary determinants of circulating levels of dihydrophylloquinone, a form of dietary vitamin K, in the Framingham Heart Study. Objective 1 4. To develop stable isotope techniques using liquid chromatography mass spectrometry (LC/MS) to measure vitamin K absorption and transport. Objective 2 2006 1. Quantify multiple forms of vitamin K in dietary supplements representative of the US food supply. Objective 1 2. To relate the dietary intakes of dihydrophylloquinone, a form of dietary vitamin K, to physiological outcomes, such as bone health, in the Framingham Heart Study. Objective 2 3. To assess the role of apolipoprotein E genotype in modulating vitamin K status in the Framingham Heart Study. Objective 2 4. To develop a rat model to examine the effects of vitamin K supplementation on the transport of vitamin K to various tissues. Objective 2 2007 1. To determine the impact of vitamin K supplementation, in an amount that is expected to be nutritionally optimal and safe, on bone, joint and vascular health in 452 elderly men and women. Objective 3 2. To estimate the usual dietary intakes of menaquinone-4, a form of vitamin K present in animal products, among men and women participating in the Framingham Heart Study. Objective 1 2008 1. To complete enrollment of a bioavailability study in younger and older men and women to examine the role of age, lipid profile and current vitamin K status on absorption and relative bioavailability of vitamin K from a vegetable. Objective 2 2. To conduct a cross-sectional analysis of phylloquinone, dihydrophylloquinone and menaquinone-4 intakes and plasma levels in the baseline data from 452 elderly men and women participating in a vitamin K supplementation trial to provide information on the dietary and non- dietary determinants of vitamin K that are specific to the elderly. Objective 2 2009 1. To examine the roles of apolipoprotein E, vitamin K epoxide reductase, and vitamin K-dependent gamma carboxylase genotypes in modulating vitamin K status among participants in a vitamin K supplementation trial. Objective 2 2. To determine the impact of vitamin K supplementation on measures of inflammation, as they relate to bone and vascular health. Objective 3 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Bone Metabolism Laboratory Milestone # 1 To determine the impact of body mass index on the serum 25(OH)D response to supplemental vitamin D. Milestone Fully Met 2. Bone Metabolism Laboratory Milestone # 2 To determine the impact of calcium intake on the 25(OH)D response to supplemental vitamin D. Milestone Fully Met 3. Bone Metabolism Laboratory Milestone # 3 To assess the long-term effect of vitamin D plus calcium on physical function and on the risk of falling in older men and women. Milestone Fully Met 5. Vitamin K Laboratory Milestone # 1 Characterize and quantify multiple forms of vitamin K in food samples representative of the US food supply. Milestone Fully Met 6. Vitamin K Laboratory Milestone # 2 Create an expanded, yet targeted vitamin K database using the aforementioned food analysis data, to be used for a food frequency questionnaire. Milestone Fully Met 7. Vitamin K Laboratory Milestone # 3 To assess the dietary and non-dietary determinants of circulating levels of dihydrophylloquinone, a form of dietary vitamin K, in the Framingham Heart Study. Milestone Fully Met 8. Vitamin K Laboratory Milestone # 4 To develop stable isotope techniques using liquid chromatography mass spectrometry (LC/MS) to measure vitamin K absorption and transport. Milestone Fully Met 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to accomplish, year by year, over the next 3 years under each milestone? Bone Metabolism Laboratory 2006 1. To determine the association between serum vitamin D levels and risk of gingivitis. Objective 1 Anticipated Results: We expect to find that adults with optimal 25(OH)D concentrations are at decreased risk for gingivitis compared with those with lower levels. 2. To define and compare patterns of calcium and vitamin D supplement use in a random sample of women with and without osteoporosis. Objective 1 Anticipated Results: We expect to find that patterns of calcium and vitamin D supplement use differ between women with and without osteoporosis and that the differences are influenced by their medical treatment. 3. To determine whether the level of calcium intake influences any association between the ratio of nitrogen to potassium in the urine and rates of bone loss in older men and women. Objective 2 Anticipated Results: We expect to find that a higher nitrogen to potassium ratio is less strongly associated with bone loss in older men and women with high calcium intakes than in those with low calcium intakes. 4. To determine the association between the ratio of nitrogen to potassium in the urine, an index of the acidogenicity of the diet, and 3- year rates of bone loss in healthy older men and women. Objective 2 Anticipated Results: We expect to find that a high ratio of nitrogen to potassium predicts increased rates of bone loss in older men and women. 5. To determine the impact of stimulating the calcium sensor receptor on gastric acid secretion and calcium excretion. Objective 2 Anticipated Results: We expect to find that a calcimimetic agent will increase gastric acid secretion and urinary calcium excretion compared with placebo. 6. To determine the association between intakes of calcium and vitamin D and incidence of developing Type 2 diabetes. Objective 3 Anticipated Results: We expect to find that poor intakes of both calcium and vitamin D and a low 25(OH)D level is associated with a higher incidence of type 2 diabetes. 7. To determine and compare the effects of selected aromatic and branched-chain amino acids on urinary calcium excretion in healthy older men and women. Objective 2 Anticipated Results: We expect to find that aromatic L-amino acids have a greater calciuric effect than branched chain L-amino acids in healthy older men and women. 2007 1. To study the association between endogenous gonadal hormone levels and physical function and risk of falling in elderly men and women. Objective 1 Anticipated Results: We expect to find that elderly men and women who have higher levels of endogenous gonadal hormones have better physical function and fall less often that those with lower levels. 2. Determine mechanisms by which vitamin D influences muscle strength and risk of falling. Objective 1 Anticipated Results: We expect to find that vitamin D increases muscle strength and reduces falling by influencing vitamin D signaling in muscle tissue. 3. To determine whether alkalinization of the diet will attenuate the urine calcium losses induced by high protein diets. Objective 2 Anticipated Results: We expect to find that neutralizing the acid load will reduce urinary calcium losses associated with a high protein diet. 4. To determine the effect of supplementation with calcium and vitamin D on fasting insulin and glucose levels in healthy older men and women. Objective 3 Anticipated Results: We expect to find that supplementation with vitamin D and calcium will reduce fasting insulin and glucose levels in healthy older men and women, consistent with improved glucose control and insulin sensitivity. 2008 1. Define mechanisms by which specific amino acids affect intestinal calcium absorption. Objective 2 Anticipated Results: We expect to find differences between branched- chain L-amino acids and aromatic L-amino acids in their effects on biochemical factors that regulate intestinal calcium absorption. 2. To begin a randomized, controlled longitudinal trial to determine whether alkalinization of the diet will lower the bone turnover rate, and thus lower risk of fracture, in healthy men and women who are being supplemented with calcium and vitamin D. Objective 2 Anticipated Results: We expect to find that neutralizing the acid load will lower the bone turnover rate and reduce fracture risk in healthy men and women. Vitamin K Laboratory 2006 1. Quantify multiple forms of vitamin K in dietary supplements representative of the US food supply. Objective 1 Anticipated Results: We plan to measure vitamin K, in the form of phylloquinone, in approximately 200 dietary supplements currently available in the US. These samples will be collected as part of the National Food and Nutrient Analysis Program, and the results will be disseminated through a national database. 2. Relate the dietary intakes of dihydrophylloquinone, a form of dietary vitamin K, to physiological outcomes, such as bone health, in the Framingham Heart Study. Objective 2 Anticipated Results: We will use the standard food frequency questionnaire (FFQ) that has now been validated for dihydrophylloquinone to determine the associations between dietary dihydrophylloquinone intake and bone health in men and women participating in the physical examination cycle 6 for the Framingham Offspring Heart Study. Dihydrophylloquinone is primarily found in snack foods and fast foods, so this study will test the hypothesis that dihydrophylloquinone consumption is associated with poor bone health in men and women. 3. To assess the role of apolipoprotein E genotype in modulating vitamin K status in the Framingham Heart Study. Objective 2 Anticipated Results: Vitamin K is transported by triglyceride-rich lipoproteins, therefore we predict that apoE genotype will be a non- dietary determinant of bone mineral density in the Framingham Offspring cohort through its role in regulation of vitamin K transport in circulation. 4. To develop a rat model to examine the effects of vitamin K supplementation on the transport of vitamin K to various tissues. Objective 2 Anticipated Results: A rat model will be developed that compares the response of various tissues to equivalent amounts of dietary dihydrophylloquinone and phylloquinone. We predict that certain tissues, such as brain, will not be able to use dihydrophylloquinone as efficiently as phylloquinone. If successful, we will have developed a rat model in which we can compare the effects of vitamin K deficiency with vitamin K sufficiency while maintaining normal blood clotting. In the unlikely event that this rat model is not successful, we will focus on other strategies, such as manipulation of the amount of phylloquinone in the diet. 2007 1. To determine the impact of vitamin K supplementation, in an amount that is expected to be nutritionally optimal and safe, on bone, joint and vascular health in 452 elderly men and women. Objective 3 Anticipated Results: We expect that participation of 452 older men and women will be completed for the 3-year clinical trial to examine the effect of vitamin K supplementation on bone mineral density and vascular calcification. We project that those individuals receiving vitamin K supplementation will have less bone loss and less progression of vascular calcification compared to those individuals not receiving vitamin K. Should there be no differences between the two groups, we will conclude that vitamin K in an amount that can be achieved in the diet, does not confer any beneficial effects on bone or vascular health in this age group. 2. To estimate the usual dietary intakes of menaquinone-4, a form of vitamin K present in animal products, among men and women participating in the Framingham Heart Study. Objective 1 Anticipated Results: Food composition data for menaquinone-4 generated from milestone # 1 achieved in FY 2005 will be assigned to food items listed in a standard food frequency questionnaire (FFQ). Usual dietary intake for the previous year during the physical examination cycle 6 for the Framingham Offspring Heart Study will be assessed using this FFQ. Descriptive statistics will be used to determine mean menaquinone-4 intakes by category of sex and age group. 2008 1. Complete enrollment of a bioavailability study in younger and older men and women to examine the role of age, lipid profile and current vitamin K status on absorption and relative bioavailability of vitamin K from a vegetable. Objective 2 Anticipated Results: We expect to obtain external funding to leverage CRIS funds in order to initiate a metabolic study in younger and older men and women that will use a stable isotope tracer (deuterium-labeled phylloquinone in plants) to determine absorption efficiency and transport of phylloquinone. Production of deuterium-labeled phylloquinone in collards will be completed, and subject recruitment will be completed. In the event that subject recruitment and adherence is below expectation, we will extend the anticipated duration of the study for another fiscal year. 2. To conduct a cross-sectional analysis of phylloquinone, dihydrophylloquinone and menaquinone-4 intakes and plasma levels in the baseline data from 452 elderly men and women participating in a vitamin K supplementation trial to provide information on the dietary and non- dietary determinants of vitamin K that are specific to the elderly. Objective 2 Anticipated Results: Because the participants in this study are of a narrow age range, and limited to elderly, the proposed analysis will provide information on the dietary and non-dietary determinants of vitamin K that are specific to the elderly. 4a What was the single most significant accomplishment this past year? Vitamin K and Osteoarthritis - Osteoarthritis is a common degenerative disease that increases in prevalence with age. In animal models, an inadequate amount of vitamin K has been associated with abnormal development of cartilage and excessive calcification in joints. Scientists in the Vitamin K Laboratory tested the hypothesis that adequate vitamin K status, as measured by intake and plasma levels, is associated with less hand and knee osteoarthritis in older men and women participating in the Framingham Heart Study compared to those with poor vitamin K status. Among the 1276 men and women who completed dietary assessment and had hand and knee radiographs, those with higher dietary intakes of vitamin K had less abnormal calcification in their hand joints compared to those with low dietary intakes of vitamin K. Among the 672 men and women who had vitamin K biochemical measures and hand and knee radiographs, individuals with higher plasma vitamin K levels had fewer joints with osteoarthritis and abnormal calcification compared to those with low plasma vitamin K levels. These data suggest that vitamin K may be a potentially modifiable factor in the prevention of this age-related form of arthritis. 4b List other significant accomplishments, if any. Vitamin D, Calcium and Falls Supplementation with vitamin D and calcium in frequently recommended amounts significantly lowered risk of falling in our cohort of 389 healthy older men and women. Relatively sedentary women appeared to have the greatest risk reduction with supplementation. Falls are the largest single cause of injury in the elderly and 90% of fractures result from falls. In a placebo-controlled calcium and vitamin D intervention trial, scientists in the Bone Metabolism Laboratory acquired information on fall rates every 6 months for 3 years and compared the fall rates among the supplemented and placebo groups. Broad- based increases in vitamin D and calcium intake were shown to have the potential to substantially reduce fractures and other injuries in the elderly by lowering the risk for falling. Vitamin K in Meats and Dairy Menaquinone-4 is a form of vitamin K that is formed by conversion of phylloquinone, the primary form in the diet, in certain tissues. Menaquinone-4 may have a physiological role that is independent of the universal role of a cofactor that is attributed to all forms of vitamin K. Until now, there has been no systematic analysis of menaquinones in the US food supply. Scientists in the Viitamin K Laboratory analyzed the menaquinone-4 content of over 2,000 key foods collected as part of the National Food and Nutrient Analysis Program. This form of vitamin K was present in a variety of meats and dairy foods, although no single food item was identified as a rich dietary source of vitamin K. The presence of menaquinone-4 in meat and dairy foods could be important as physiologic functions unique to menaquinone-4 are identified. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. As we complete the first year of this 5-year program cycle, the main accomplishment of the Bone Metabolism Laboratory is finding that supplementation with calcium and vitamin D lowers risk of falling in older subjects. Falls are common occurrences in the elderly 30% of subjects age 65 and 40 to 50% of those age 80 will fall in one year and falls are a major precipitant of fractures. Dissemination of this finding in the scientific and medical community should lead to increases in vitamin D and calcium intakes and a reduction in falls and fractures in older men and women. This finding is related to Milestone #3: To assess the long-term effect of vitamin D plus calcium on physical function and on the risk of falling in older men and women, and to National Program 107 - Human Nutrition program component 4: Nutrient Requirements, and ARS Strategic Plan Performance Measure 4.1.2.: Improve Human Health by Better Understanding the Nutrient Requirements of Individuals and the Nutritional Value of Foods. The major accomplishment of the Vitamin K Laboratory has been to demonstrate that high dietary intakes and biochemical measures of vitamin K are associated with a reduced risk for certain chronic diseases associated with abnormal calcification in older men and women. Of concern is the consistent observation that dietary intakes of vitamin K are lower among U.S. adults than previously assumed. This finding is related to our FY 2007 milestone 1, which is to determine the impact of vitamin K supplementation in an amount that is expected to be nutritionally optimal and safe, on bone, joint and vascular health in 452 elderly men and women, and to National Program 107 - Human Nutrition, program component 4: Nutrient Requirements, and ARS Strategic Plan Performance Measure 4.1.2.: Improve Human Health by Better Understanding the Nutrient Requirements of Individuals and the Nutritional Value of Foods. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? The Bone Metabolism Laboratorys finding that the calcium intake does not influence the increase in serum 25(OH)D that occurs with vitamin D supplementation (Milestone 2) has been presented at a scientific meeting and published in the peer-reviewed literature. This information will be useful in determining the calcium and vitamin D requirements. No intellectual property rights issues apply. The research findings of the Vitamin K Laboratory have been presented to the scientific community at a number of meetings and have also been published in prestigious peer- reviewed scientific journals. Results of this research reach the public directly through the NIH National Resource Center on Osteoporosis and Related Bone Diseases at the National Osteoporosis Foundation, through press releases, print and electronic media, and the web. Vitamin K food composition data were electronically transferred to ARS scientists at Beltsville USDA Nutrient Data Laboratory for input into the USDA Nutrient Databank System. These data will be used by scientists doing nutrition- related research, by the food industry, and by consumers. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below). Journal of American Medical Association Better osteoporosis management a priority (describing Dr. Dawson-Hughes views on vitamin D and Bone health) - May 25, 2005. Nutrition Action magazine Bonnie Liebman Breaking up: strong bones need more than calcium - April 2005. Balance Tufts Daily Newspaper Erica Nakamota Nutrigenomics studies how DNA individualizes our diet - April 27, 2005. US News World Report National Issues Briefing, Bone Health and Osteoporosis (Calcium and vitamin D in the diet) - October 20, 2004.

    Impacts
    (N/A)

    Publications

    • Jajoo, R., Song, L., Ramussen, H., Dawson-Hughes, B. 2004. Effect of change in dietary acid-base balance on bone turnover and urine calcium excretion. [abstract] Journal of Bone and Mineral Research. 19(Suppl 1) :S448-449.
    • Goussous, R.Y., Song, L., Dallal, G., Dawson-Hughes, B. 2004. The effect of calcium intake on the vitamin D requirement. [abstract] Journal Bone and Mineral Research. 19(Suppl 1):S202.
    • Bischoff-Ferrari, H.A., Orav, J.E., Dawson-Hughes, B. 2004. Effect of vitamin D3 plus calcium on fall risk in older men and women: a 3-year randomized controlled trial. [abstract] Journal of Bone and Mineral Research. 19(Suppl 1):S57.
    • Booth, S.L., Golly, I., Sacheck, J.M., Roubenoff, R., Dallal, G.E., Hamada, K., Blumberg, J.B. 2004. Effect of vitamin E supplementation on vitamin K status in adults with normal coagulation status. American Journal of Clinical Nutrition. 80:143-148.
    • Schulze, K.J., O'Brien, K.O., Germain-Lee, E.L., Booth, S.L., Leonard, A., Rosenstein, B.J. 2004. Bone calcium deposition is associated with availability of dietary calcium and cirulating leptin and osteocalcin concentrations in girls with cystic fibrosis. Journal of Clinical Endocrinology and Metabolism. 89(7)3385-3391.
    • Cury, R.C., Ferencik, M., Hoffmann, U., Ferullo, A., Moselewski, F., Abbara, S., O'Donnell, C.J., Booth, S.L., Brady, T.J., Achenbach, S. 2004. Epidemiology and association of vascular and valvular calcium quantified by multi-detector computed tomography in elderly asymptomatic subjects. American Journal of Cardiology. 94:348-351.
    • Braam, L.A., Mckeown, N.M., Jacques, P.F., Lichtenstein, A.H., Vermeer, C., Wilson, P.W., Booth, S.L. 2004. Dietary phylloquinone intake as a potential marker for a heart-healthy dietary pattern in the Framingham Offspring Cohort. Journal Of The American Dietetic Association. 104(9) :1410-1414.
    • Damon, M., Zhang, N., Haytowitz, D.B., Booth, S. 2005. Phylloquinone (Vitamin K1) content of vegetables. Journal of Food Composition and Analysis. 18:751-758. Available online at http://www.sciencedirect.com doi:10.1016/j.jfca.2004.07.004
    • Pro-Risquez, A., Harris, S.S., Song, L., Rudicel, S., Barnewolt, B., Dawson-Hughes, B. 2004. Calcium supplement and osteoporosis medication use in women and men with recent fractures. Osteoporosis International. 15:689- 694.
    • Bischoff-Ferrari, H.A., Dietrich, T., Orav, J.E., Hu, F.B., Zhang, Y., Karlson, E., Dawson-Hughes, B. 2004. Higher 25-hydroxyvitamin D levels are associated with better lower extremity function in both active and inactive adults 60+ years of age. American Journal of Clinical Nutrition. 80:752-758.
    • Booth, S.L., Broe, K.L., Peterson, J.W., Cheng, D.M., Dawson-Hughes, B., Gundberg, C.M., Cupples, L.A., Wilson, P.W., Kiel, D.P. 2004. Associations between vitamin k biochemical measures and bone mineral density in men and women. Journal of Clinical Endocrinology and Metabolism. 89(10):4904-4909.
    • Hyten, D.L., Song, Q., Costa, J., Cregan, P.B. 2003. Linkage disequilibrium in four soybean populations [abstract]. BARC Poster Day. Abstract 14.
    • Vazquez, E., Song, L.Y., Dawson-Hughes, B. 2004. Precision of bone mineral density scans at the proximal tibia in osteoarthritic subjects. Journal of Clinical Densitometry. 7(4):364-367.
    • Bischoff-Ferrari, H.A., Willett, W.C., Wong, J.B., Giovannucci, E., Dietrich, T., Dawson-Hughes, B. 2005. Fracture prevention by Vitamin D supplementation: A meta-analysis of randomized controlled trials. Journal of the American Medical Association. 293:2257-2264.
    • Dawson-Hughes, B. 2004. Dietary protein and the skeleton. In: Burckhardt, P., Dawson-Hughes, B., Heaney, R.P. editors. Nutritional Aspects of Osteoporosis. 2nd edition. San Diego, CA.: Elsevier, Inc. p. 399-409.
    • Bischoff, H.A., Dawson-Hughes, B. 2004. Defining optimal 25-hydroxyvitamin D levels in younger and older adults based on hip bone mineral density. In: Burckhardt, P., Dawson-Hughes, B., Heaney, R.P. editors. Nutritional Aspects of Osteoporosis. 2nd edition. San Diego, CA: Elsevier, Inc., p. 235-244.
    • Dawson-Hughes, B., Heaney, R.P., Holick, M., Lips, P., Meunier, P.J., Vieth, R. 2004. Vitamin D round table. In: Burckhardt, P., Dawson-Hughes, B., Heaney, R., editors. Nutritional Aspects of Osteoporosis. 2nd edition. San Diego, CA.: Elsiever, Inc. p. 263-270.
    • Dawson-Hughes, B. 2004. Racial/ethnic considerations in making recommendations for vitamin D for adult and elderly men and women. In: Proceedings of the NIH Vitamin D Conference, October 10, 2003. American Journal of Clinical Nutrition. 80(Suppl):1763S-1766S.
    • Dawson-Hughes, B., Heaney, R.P., Holick, M.F., Lips, P., Meunier, P.J., Vieth, R. 2005. Estimates of optimal vitamin D status. Osteoporosis International [on-line]. Available at http://www.springerlink.com
    • Dawson-Hughes, B. 2005. The role of vitamin d in fracture prevention. BoneKEy-Osteovision. 2(4):6-10. (Epub) Available at http://www.bonekey- ibms.org/cgi/content/full/ibmske;2/4/6 DOI: 10.1138/20050156
    • Wilsey, B.J., Polley, H.W. 2004. Realistically low species evenness does not alter grassland species-richness-productivity relationships. Ecology. 85(10):2693-2700.
    • Goussous, R., Song, L., Dallal, G.E., Dawson-Hughes, B. 2005. Lack of effect of calcium intake on the 25-hydroxyvitamin D response to oral vitamin D3. Journal of Clinical Endocrinology and Metabolism. 90(2): 707- 711.
    • Erkkila, A.T., Lichtenstein, A.H., Jacques, P.F., Hu, F.B., Wilson, P.W., Booth, S.L. 2005. Determinants of plasma dihydrophylloquinone in men and women. British Journal of Nutrition. 93:701-708.


    Progress 10/01/03 to 09/30/04

    Outputs
    1. What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? What does it matter? This five-year research plan represents a continuation of interest on the part of the Bone Metabolism Laboratory and Vitamin K Laboratory in improving the understanding of how diet impacts bone health in older men and women. Serum 25-hydroxy vitamin D [25(OH)D] is the best clinical measure of vitamin D status. One objective of the Bone Metabolism Laboratory is to define factors that influence the 25(OH)D concentrations achieved at a given vitamin D intake level in the wintertime. We will examine the effect of body mass index on the utilization of dietary vitamin D. This will be studied in the 389 men and women age 65 and older who participated in a placebo-controlled calcium and vitamin D intervention study. We will also test the hypothesis that the increase in serum 25(OH)D in response to oral dosing with vitamin D is blunted at low calcium intakes. This will be studied in a new randomized controlled trial in which 800IU/day of vitamin D will be given to older subjects with high and low calcium intakes. We will also examine the impact of endogenous gonadal-hormone levels on 25(OH)D responses to supplemental vitamin D. Falls are a common source of serious injury, including fractures, in older subjects. Recent evidence suggests that low vitamin D levels may contribute to muscle weakness and increased risk of falling in this population. We will examine this possibility in the 389 older men and women who participated in our 3-year calcium and vitamin D intervention study. The impact of lean body mass, gonadal hormone levels, and usual activity levels on risk of falling will also be examined. The vitamin D studies described above will improve the scientific basis for determining the vitamin D intake requirements for older men and women. A second research initiative of the Bone Metabolism Laboratory is to define the impact of dietary protein and the acid-base balance of the diet on bone health. Several small protein intervention studies will be conducted to define the relative roles of protein, added aromatic or branched-chain L-amino acids, calcium, vitamin D, and alkaline foods (fruits and vegetables) or supplements, such as potassium bicarbonate, on short-term indices of bone metabolism including biochemical markers of bone turnover, calcium absorption, urinary calcium excretion, and insulin- like growth factor-1. A supplement (expected to contain protein, calcium, vitamin D, alkali, and perhaps added branched-chain amino acids) will then be tested in a large longitudinal intervention trial in older men and women to determine its impact on rates of change in bone mineral density (BMD). This research falls under National Program 107 - Human Nutrition, Component 1: Nutrient Requirements. The ability to establish dietary recommendations for vitamin K is limited by our inadequate understanding of the dietary sources and metabolic fate of phylloquinone from the diet. Studies are being conducted to identify the forms, amounts and biological activity of vitamin K in the food supply. Vitamin K food composition data generated from food analysis will be incorporated into the national nutrient databases for the purpose of menu design, dietary assessment, and national intake surveys. Stable isotopes will be used to study the absorption and transport of vitamin K from vegetables, which are the primary dietary source in the United States (US). Information is also being collected on the physiological significance of measures used to assess vitamin K nutritional status, with a focus on the impact of vitamin K supplementation on bone and vascular health. The largest of these studies is a 3-year randomized, placebo-controlled vitamin K intervention trial in 450 men and women, age 60-80, who are calcium and vitamin D replete. This study is currently underway, with all subjects enrolled. The main analysis will determine if there are bone density gains among those receiving vitamin K supplementation. Secondary analysis will determine if there is a reduction in the progression of age-related vascular calcification among those receiving vitamin K supplementation. This research falls under National Program 107 - Human Nutrition, Components 1: Nutrient Requirements, 4; Composition of Foods, and 7: Bioavailability of Nutrients and Food Components 2. List the milestones (indicators of progress) from your Project Plan. Bone Metabolism Laboratory 1. To determine the impact of body mass index on the 25(OH)D response to supplemental vitamin D. 2. To determine the impact of calcium intake on the 25(OH)D response to supplemental vitamin D. 3. To assess the long-term effect of vitamin D plus calcium on physical function and on the risk of falling in older men and women. 4. To study the association between endogenous gonadal hormone levels and physical function and risk of falling in elderly men and women. 5. To determine the association between the ratio of nitrogen to potassium in the urine, an index of the acidogenicity of the diet, and 3- year rates of bone loss in 389 healthy older men and women. 6. To determine whether the level of calcium intake influences any association between the ratio of nitrogen to potassium in the urine and rates of bone loss in older men and women. 7. To determine the impact of increasing protein intake on urinary calcium excretion and the rate of bone turnover in healthy older men and women. 8. To determine whether alkalinization of the diet with potassium-rich foods (fruits and vegetables) will attenuate the urine calcium losses induced by high protein diets. 9. To determine and compare the effects of selected aromatic and branched- chain amino acids on urinary calcium excretion in healthy older men and women. 10. To conduct a randomized, controlled intervention trial to determine whether alkalinization of the diet will lower the bone turnover rate, and thus lower risk of fracture, in healthy men and women who are being supplemented with calcium and vitamin D. Vitamin K Laboratory 1. Characterize and quantify multiple forms of vitamin K in food samples representative of the U.S. food supply. 2. Create an expanded, yet targeted vitamin K database using the aforementioned food analysis data, to be used for a food frequency questionnaire. 3. To assess the relative individual contribution of individual forms of vitamin K to total dietary intakes in the Framingham Heart Study, and among participants in a vitamin K supplementation trial. 4. To develop stable isotope techniques using LC/MS to measure vitamin K absorption and transport. 5. To conduct a bioavailability study in younger and older women to examine the role of age, lipid profile and current vitamin K status on absorption and relative bioavailability of vitamin K from a vegetable. 6. To examine the role of apolipoprotein E genotype in modulating vitamin K status in the Framingham Heart Study, and among participants in a vitamin K supplementation trial. 7. To determine the impact of vitamin K supplementation, in an amount that is expected to be nutritionally optimal and safe, on bone and vascular health in 452 elderly men and women. 3. Milestones: Bone Metabolism Laboratory A. In 2004, we completed milestone #7 above, To determine the impact of increasing protein intake on urinary calcium excretion and the rate of bone turnover in healthy older men and women. In this study, 32 subjects were randomized to low or high protein intakes and consumed food supplements, either meat or carbohydrate-rich foods, daily for 9 weeks. Half of the subjects received calcium supplements and the other half received placebo. In retrospect, we found that the calcium supplements did not disintegrate appropriately, and so the calcium could not be absorbed. From the study, we did find however that the meat supplements did not significantly increase urine calcium excretion, but they were associated with higher levels of the bone growth factor, IGF-1, and lower levels of the bone resorption marker. Thus it appears that the meat supplements had a net favorable effect on bone. Results of this study were recently published. Other parts of this new 5-year plan are not completed, as our 5-year plan was approved in May 2004. Vitamin K Laboratory A. In 2004, we continued phylloquinone and dihydrophylloquinone analysis of representative foods collected as part of the USDA National Food and Nutrient Analysis Program (milestone #1). As an expansion of the project's original aims, we have confirmed the identification of menaquinone-4, another form of vitamin K, in foods of animal origin through use of high performance liquid chromatography (HPLC) followed by gas chromatography-mass spectrometry (GC/MS). We have also initiated the development of a liquid chromatography-mass spectrometry (LC/MS) method to measure stable isotope-labeled vitamin K in order to extend our human studies on vitamin K absorption and transport (milestone #4). An application has been submitted for outside funding to conduct a metabolic study on the absorption and relative bioavailability of vitamin K in younger and older women using this LC/MS method. As projected for milestone #7, recruitment of 452 older men and women has been completed for a 3-year clinical trial to examine the effect of vitamin K supplementation on bone mineral density and measures of bone turnover and vascular calcification in older men and women who are calcium and vitamin D replete. Other parts of this new 5-year plan are underway as this new 5- year plan was approved in May 2004. B. Milestones for 2005-2007 Bone Metabolism Laboratory 2005 (Year 2) 1. Milestone # 1: To determine the impact of body mass index on the 25(OH)D response to supplemental vitamin D. We will examine the rise in 25(OH)D that occurred after one year of supplementation with 700IU/d of vitamin D in the 389 men and women age 65 and older who participated in our STOP/IT study. This analysis of existing variables will be carried out and the findings submitted for publication in 2005. 2. Milestone # 2: Fifty-six men and women will be recruited and randomized to calcium, 1000 mg/day, or placebo. All subjects will take 800 IU of vitamin D per day over the 3-month study period. The findings in this study will help to determine whether calcium intake influences the vitamin D requirement. This study should be completed and the results submitted for publication in 2005. 3. Milestone # 3: Examine the long-term effect of supplementation with calcium and vitamin D on muscle strength and the incidence of falling in 389 healthy men and women age 65 and older. This analysis of existing variables will be started in 2005, and the findings submitted for publication in 2005 or 2006. 4. Milestone # 8: Forty healthy older men and women will be recruited, screened, and randomized to consuming protein supplements as meat, or to protein supplements and fruits and vegetables in amounts needed to neutralize the acid load that accompanies the meat supplements over a 74- day study period. All subjects will receive calcium and vitamin D. The purpose of this study is to determine whether balancing the pH of the diet with fruits and vegetables will improve two short-term indicators of bone health, urine calcium excretion and the rate of bone resorption. We expect to complete the study in 2005. 2006 (Year 3) 5. Milestone # 4: Examine the extent to which the level of usual physical activity and/or endogenous estrogen and testosterone levels influence muscle strength and risk of falling in 389 men and women age 65 and older who participated for 3 years in a calcium and vitamin D or placebo intervention trial. This analysis of existing variables will be started in 2006 and the findings should be submitted for publication in 2006. 6. Milestone # 5: Determine whether the nitrogen to potassium ratio in the urine, an index of the acid-base balance of the diet is a determinant of rates of bone loss in 389 men and women age 65 and older who participated for 3 years in a calcium and vitamin D or placebo intervention trial. Urine potassium measurements are currently available. Nitrogen content will be measured in archived urine samples, to determine the ratio. This analysis should be completed and a manuscript written by the end of 2006. 7. Milestone # 6: In a companion analysis, determine whether the level of calcium intake affects any observed association between the urinary nitrogen to potassium ratio and rates of bone loss. This should be completed by the end of 2006. 8. Milestone # 9: Thirty men and women will be randomized to treatment first with low, then high intakes of either aromatic (phenylalanine and histidine) or branched-chain (leucine and isoleucine) amino acids. The amino acids will be mixed into selected foods in metabolic diets and consumed over a 24-day study period. This study tests the hypothesis that aromatic amino acids cause greater urine calcium losses than branched- chain amino acids. This hypothesis is based on the finding that aromatic amino acids bind with higher affinity to calcium sensor receptors in the kidney, triggering urine calcium losses. 2007 (Year 4) 1. Milestone #10: Several of the studies cited above are directed at determining a nutrient package that will improve bone health in older men and women. This improvement would be reflected in lower rates of bone turnover and ultimately lower fracture rates. We plan to have identified the intervention to be tested in 2006 and to seek funding needed to test the intervention in a large randomized, in healthy older men and women. We hope to begin the trial in late 2007. Vitamin K Laboratory 2005 (Year 2) 1. Milestone # 1: Phylloquinone and dihydrophylloquinone analysis of foods collected as part of the National Food and Nutrient Analysis Program will be ongoing. Menaquinone-4 analysis of key foods and ingredients will be analyzed in the same food samples. Initiate abstract and manuscript preparation upon completion of each series of analyses for individual food groups. 2. Milestone # 2: Submit data from Milestone #1 to ARS scientists at the HNRC at Beltsville, MD for inclusion in the USDA national food composition databases. 3. Milestone # 4: Complete the development of a liquid chromatography- mass spectrometry (LC/MS) method to measure stable isotope-labeled vitamin K in order to extend our human studies on vitamin K absorption and transport (milestone #4). Using archived samples from a previous metabolic study of five men using deuterium-labeled plants, we will then compare the results from the LC-MS assay with those derived from a GC/MS assay. In addition, LC/MS will be used to analyze those samples in which we encountered technical difficulties using GC/MS to determine if the difficulties can be overcome with the LC/MS assay, which if successful, will establish its widespread utility. 4. Milestone # 6: We recently determined that among men and postmenopausal women not on estrogen replacements who are participating in the Framingham Offspring Cohort, low vitamin K status is associated with low bone mineral density. Based on these findings, the scope of this gene-nutrient interaction analysis has been expanded from the project's original aims to include an assessment of the influence of apoE genotype on the association between vitamin K and bone. Obtain approval from the Framingham DNA Committee to initiate studies on the influence of apolipoprotein E (apoE) genotype on measures of vitamin K status in the Framingham Offspring cohort. 5. Milestone # 7: We anticipate that participation of 452 older men and women will be ongoing for the 3-year clinical trial to examine the effect of vitamin K supplementation on bone mineral density and measures of bone turnover and vascular calcification. Laboratory analysis will also be ongoing for this project. 2006 (Year 3) 1. Milestone # 1: Complete phylloquinone and dihydrophylloquinone analysis of foods collected as part of the National Food and Nutrient Analysis Program. Perform menaquinone-4 analysis of key foods and ingredients in archived food samples that have been analyzed for phylloquinone and dihydrophylloquinone. Initiate abstract and manuscript preparation upon completion of each series of analyses for individual food groups. 2. Milestone # 2: Submit data from Milestone #1 to ARS scientists at the HNRC at Beltsville, MD for inclusion in the USDA national food composition databases. 3. Milestone # 3: Food composition data for dihydrophylloquinone generated from milestone # 1 above, will be assigned to food items listed in a standard food frequency questionnaire (FFQ). Usual dietary intake for the previous year during the physical examination cycle 6 for the Framingham Offspring Heart Study will be assessed using this FFQ. Descriptive statistics will be used to determine mean dihydrophylloquinone intakes by category of sex and age group. Statistical significance of trends across quartiles of dihydrophylloquinone intake will be assessed with linear regression models. To examine potential dietary and non-dietary determinants of plasma dihydrophylloquinone concentrations, we will create categories of body mass index and alcohol intake and quartile categories of dihydrophylloquinone intakes, among other variables. 4. Milestone # 5: We anticipate that we will have obtained external funding to initiate a metabolic study in pre- and postmenopausal women that will use a stable isotope tracer (deuterium-labeled phylloquinone in plants) to determine absorption efficiency and transport of phylloquinone. Production of deuterium-labeled phylloquinone in collards will be completed, and subject recruitment will be initiated. 6. Milestone # 6: Complete an assessment of the influence of apoE genotype on the influence of apolipoprotein E (apoE) genotype on measures of vitamin K status in the Framingham Offspring cohort. Submit manuscript for publication. 7. Milestone # 7: Ongoing participation of 452 older men and women in a 3- year clinical trial to examine the effect of vitamin K supplementation on bone mineral density and measures of bone turnover and vascular calcification. Laboratory analysis will also be ongoing for this project. 2007 (Year 4) 1. Milestone # 1: Complete all food analyses of key foods for multiple forms of vitamin K. Submit manuscripts for publication. 2. Milestone # 2: Complete transfer of all food composition data generated as part of milestone # 1 for entry into USDA national food composition databases. 3. Milestone # 3: Assign food composition data for menaquinone-4 generated from milestone # 1, to food items listed in a standard food frequency questionnaire (FFQ). Assess usual dietary intake for the previous year during the physical examination cycle 6 for the Framingham Offspring Heart Study using this FFQ. Use descriptive statistics to determine mean menaquinone-4 intakes by category of sex and age group. Assess statistical significance of trends across quartiles of menaquinone- 4 intake with linear regression models. 4. Milestone # 5: Use a stable isotope tracer (deuterium-labeled phylloquinone in plants) to determine absorption efficiency and transport of phylloquinone in a metabolic study of pre- and postmenopausal women. Enrollment and laboratory analysis will be ongoing. 5. Milestone # 7: Participation of 452 older men and women will be completed for the 3-year clinical trial to examine the effect of vitamin K supplementation on bone mineral density and measures of bone turnover and vascular calcification. Laboratory analysis and data analysis will be ongoing for this project. 4. What were the most significant accomplishments this past year? This project was recently established following completion of the Office of Scientific Quality Review process. Please see the report for 1950- 51000-049-00D Bone Health in the Elderly. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. This project was recently established following completion of the Office of Scientific Quality Review process. Please see the report for 1950- 51000-049-00D Bone Health in the Elderly. 6. What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end- user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products? This project was recently established following completion of the Office of Scientific Quality Review process. Please see the report for 1950- 51000-049-00D Bone Health in the Elderly. 7. List your most important publications in the popular press and presentations to organizations and articles written about your work. There are no media interviews or presentations to report as this project was recently established following completion of the Office of Scientific Quality Review process. Please see the report for 1950-51000-049-00D Bone Health in the Elderly.

    Impacts
    (N/A)

    Publications

    • DAWSON-HUGHES, B., HARRIS, S.S., RASMUSSEN, H.M., SONG, L., DALLAL, G.E. EFFECT OF DIETARY PROTEIN SUPPLEMENTS ON CALCIUM EXCRETION IN HEALTHY OLDER MEN AND WOMEN. JOURNAL OF CLINICAL ENDOCRINOLOGY AND METABOLISM. 2004;89(3):1169-73.