2004 Annual 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? 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. Review 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.