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?
Problem: Energy restriction diets can have deleterious effects on immune function, bone density, and cognitive function. These functions are also impaired by deficiencies in specific minerals and vitamins, including iron, zinc, copper, magnesium, calcium and B-vitamins. This project will focus on defining the effects of energy restriction on mineral status and metabolism and on exploring dietary patterns that optimize mineral and vitamin utilization and minimize or reverse the adverse functional effects.

Objectives: The primary goals of this project are to evaluate the effects of energy restriction on mineral and vitamin homeostasis and several functional endpoints, including cognitive function, immunity, bone metabolism and B-vitamin status, and to identify specific dietary patterns or interventions that ameliorate the adverse effects of energy restricted diets.

Objective 1: To determine the effects of energy restriction diets on mineral (iron and zinc) status and the attentional modality of cognitive function in overweight premenopausal women. We hypothesize that the adverse effects of energy restriction on cognitive function are due in part to alterations in iron utilization that result in a decrease in both iron and zinc status. Further, we hypothesize that restoration of the attentional domain of neuro-psychological function in premenopausal women requires simultaneous supplementation with iron, zinc, and vitamin B-6. The primary hypothesis is based upon findings from two independent studies: 14 obese premenopausal women and 8 normal-weight men. Iron status declined in the dieting women and was strongly associated with attention task performance, i.e. hemogloblin (r= 0.76, p<0.01) and transferrin saturation (r=0.82, p< 0.01). Consistent with this, strong correlations between attention task performance and serum transferrin receptors (r= - 0.92) and zinc (r= 0.86, p , 0.01) have been reported by others, in men whose iron status was lowered by phlebotomy.

Objective 2: To determine effects of energy restriction diets and diets high in soy isoflavones on mineral (copper, zinc, magnesium, and calcium) status and homeostasis. We hypothesize that the adverse effects of energy restriction diets on immune function and bone density are due in part to alterations in mineral metabolism and that these effects can be alleviated by changes in dietary constituents. If changes in mineral status and/or kinetics are demonstrated during a human weight loss study in postmenopausal women, we will conduct a follow-up study to determine if the addition of soy isoflavones to the weight loss diet ameliorates the adverse effects. The hypothesis is based on the observation that adverse effects similar to those of energy restriction diets can results from inadequate and/or excess intakes of minerals we propose to study.

Objective 3: To determine the efficacy of two doses of soy isoflavones in attenuating bone loss in early postmenopausal women and soy safety profile during a three-year period. We hypothesize that soy isoflavones will decrease bone loss in postmenopausal women by maintaining bone mass, with this effect being modulated by endogenous hormones, growth factors, and isoflavone metabolism. Additionally, we hypothesize that the bone-sparing effects will be greater in women receiving the higher dose, but that both groups will benefit from treatment. The hypothesis is based on findings in 69 perimenopausal women: those consuming isoflavone-rich soy protein for 24 weeks had no bone loss (-0.2%; NS), whereas women who consumed whey protein control) experienced significant bone loss (-1.3%), and those who consumed isoflavone-poor soy had intermediate bone loss (-0.7%) from the lumbar spine. Analyses indicated that the isoflavone treatment had a positive (p=0.016) effect on percentage change in lumbar spine bone mineral density (BMD), whereas the other treatments had no effect.

Objective 4: To determine if there are adverse health effects of the vitamin B-12 deficiency that is widespread in population groups such as Mexican adults and US elderly. We hypothesize that B-12 supplementation of Mexican women with deficiency or marginal plasma vitamin B-12 concentrations will result in improvements in hematology, markers of vitamin B-12 status, immune function, bone turnover markers, and DNA hpyomethylation and damage. The hypothesis is based on our previous findings showing that 15% of women in a rural Mexican population have plasma B-12 concentrations indicating severe deficiency, while in an additional 25% they suggest marginal deficiency. About 40% of children and adults in Latin America are vitamin B-12 depleted, due to low consumption of animal products. Marginal deficiency occurs also in 22% of Hispanic adults aged >60 years in the Sacramento region, due to malabsorption. The goal is to determine if there are any adverse health effects of the vitamin B-12 depletion, and if malabsorption of B-12 from food in the elderly can be reversed by treating Helicobacter pylori infection.

To whom is it relevant: The project relates directly to the Human Nutrition Program (107). The project falls within the Human Nutrition Program goals 3.1.1, Nutrient Requirements, and 3.1.3, Health Promoting Properties of Plant and Animal Foods. The dietary intervention studies planned will help in defining effects of energy restriction on mineral homeostasis, cognition, and immunity; in determining the effects of soy isoflavones on mineral availability; in evaluating the efficacy of soy isoflavones in ameliorating effects of energy restriction on bone health; and in evaluating the effects of B-12 deficiency, caused by low intake of animal source foods, on human health and function. This research will aid in establishing optimal dietary regimes for good health.

Potential Impact: Attaining the above objectives will provide the information needed to.
1)better assess the adequacy of weight-reducing diets of the US population for iron, zinc, copper, magnesium, and calcium;.
2)determine the benefits of soy isoflavones in reducing bone loss in postmenopausal women; and.
3)understand the role of B-12 supplementation to improve hematological health outcomes in US and Latin American populations. With this research, it will be possible to make realistic dietary recommendations based on scientific data. Results will also help to define dietary patterns that are beneficial or inadequate for optimal cognitive function, immunity, and bone health.

This project is aligned with National Program 107, Human Nutrition.

2.List by year the currently approved milestones (indicators of research progress)
FY 2005: Conduct run-in and intervention trials. Analyze cognitive tests and MEG scan data. Conduct run-in and intervention trials. Analyze cognitive tests and MEG scan, and blood and urine samples. Expand and refine kinetic model of copper metabolism. Prepare manuscript. Prepare facilities for human study. Cohort enrollment and initial testing. Initiation of intervention. Screen Mexican adults for B-12 deficiency (funded by UC Mexus). Establish SCA with Universidad Autonoma de Queretaro. Start vitamin B-12 supplementation trial on 200 subjects.

FY 2006: Conduct statistics & prepare manuscript. Write protocol and complete human studies approvals. Develop standardized operating procedures for all methods. Conduct intervention in the soy isoflanones study. Complete B-12 supplementation trial. Analyze samples at WHNRC. Start manuscript preparation. Using exiting data from Dr. Van Loan, identify osteopenic adults with B-12 deficiency, start vitamin B-12 supplementation study, measure effects on bone turnover. Start study of vitamin B-12 absorption in elderly using ultra low dose radioactivity.

FY 2007: Recruit and screen subjects. Conduct intervention study. Analyze cognitive tests. Begin lab analyses. Complete sample and data analysis. Model mineral metabolism. Conduct intervention in the soy isoflavones study. Complete publication of B-12 intervention. Complete supplementation study on osteopenic women, analyze samples, start publications. Continue studies of B-12 absorption in the elderly and younger adults including H. pylori treatment.

FY 2008: Complete lab analyses. Complete data processing and statistics. Prepare manuscripts. Prepare manuscripts. Recruit subjects. Conduct intervention study. Begin sample analysis Complete sample and data analysis. Model mineral metabolism. Complete publications on osteopenic women. Complete studies of treatment of B-12 malabsorption in the elderly and adults. Complete analyses and publications from malabsorption studies. Sample analysis, data analysis, manuscript preparation of soy study.

4a.List the single most significant research accomplishment during FY 2006.
Association of Inflammatory Markers and Calcium Intake to Bone Density in Early Postmenopausal Women National Program 107 Human Nutrition, Component 3.1.3 Health Promoting Properties of Plant and Animal Foods. Why and Who: To better understand the role of dietary intake and inflammation on bone health, we examined the association among calcium intake, inflammatory markers, and bone density in early postmenopausal women. What was done: These data were part of a multi-year clinical trial in early postmenopausal women conducted in Davis, Ca and in collaboration with Dr. Lee Alekel at Iowa State University. Specific findings in the reporting period (FY 2006): We found that inflammatory markers, i.e. c-reactive protein (c-reactive protein), Interlukin-1 (IL-1) Interlukin-6 (IL-6), Tumor Necrosis Factor-a (TNF-a) etc., were not related to bone density but, current dietary intake of calcium was associated with bone density in early postmenopausal women. Impact: Although high calcium intake during childhood is important for bone growth, these data suggest that bone density in early postmenopausal women is related to current intake of calcium. Furthermore, inflammatory markers may not be the mechanism for reduced bone mass in healthy early postmenopausal women.

4b.List other significant research accomplishment(s), if any.
Physiological Adaptations to Molybdem: National Program 107 Human Nutrition, Component 3.1.1 Nutrient Requirements. Why and Who: To examine the importance of molybden on human health this research examined the physiological response to diets with differing level of molybdem. What was done: A human intervention study was conducted in which differing levels of molybden were fed. Specific findings in the reporting period (FY 2006); We found, based on kinetic modeling of experimental data in humans, that physiological adaptations to changing molybdenum intakes help prevent molybdenum deficiency and toxicity. Impact: We also established that the usual molybdenum intake is well above the dietary requirement.

4c.List significant activities that support special target populations.
The collaborative project on B-12 deficiency with Universidad de Queretaro, Mexico has been conducted with Mexican women. Also the project on B-12 absorption in elderly and young with H. pylori treatment targets the Hispanic community.

4d.Progress report.
Subordiate Project 5306-51520-006-04S. This report serves to document the progress of research conducted under reimbursable agreement between ARS and the Universidad of Queretaro, Mexico. To determine the effects of vitamin B-12 deficiency on human function, 80 women with marginal B-12 deficiency were randomly assigned to B-12 supplementation for 4 months or a placebo control group. The supplementation trial has been completed, blood samples for B-12, folate, and BAP have been analyzed at the WHNRC. Funds have been obtained from the Program in International Nutrition at UC Davis to measure metabolomic and gene expression changes, and evaluate the relationship with common gene polymorphisms in folate and B-12 dependent enzymes. These samples are being analyzed. Data analysis has started for evaluate the effects of supplementation on B-12 and folate status as well as bone turnover.

5.Describe the major accomplishments to date and their predicted or actual impact.
The project was initiated in 2004. In our study of the long-term high copper intake, we discovered that though copper status did not change much with high intake, some indices of immune function and antioxidant status changed, suggesting this intake which was lower than the current upper limit of safe intake, may need to be revised downward. We developed and expanded model of molybdenum metabolism and discovered that the intake of molybdenum required for "steady state" is 43ug/d. This added scientific support for the new dietary recommendation for molybdenum. In the first large scale multi-center intervention to improve calcium intake through targeted behavioral intervention during adolescence, Dr. Van Loan and colleagues found that Asian girls had more lean mass and less total and gynoid fat mass than Hispanic or white girls, however, Asian girls had a greater proportion of fat mass in the android region. These ethnic differences in fat distribution may partially explain ethnic differences in risk for chronic diseases. Furthermore, cut-offs for BMI classifications for health may be different among ethnic groups. Dr, Allen's research has revealed the widespread high prevalence of vitamin B-12 deficiency in developing countries, and the fact that it is due to a lack of adequate intake of animal source foods. Previously, it was assumed that complete avoidance of animal products was necessary to cause this vitamin deficiency. Because of these findings the Pan American Health Organization now supports vitamin B-12 as well as folate fortification of food, the World Health Organization has established a data bank on prevalence of B-12 and folate deficiency and a WHO Consultation on these deficiencies was held in 2006. Our research will obtain additional support for the need to fortify cereals with B-12 in the US.

Calcium intake during early postmenopausal years was more related to current bone density is a group of early postmenopausal women than was childhood calcium take. These results reinforce the importance of the dietary reference intake for high levels of calcium intake during the menopausal period. Measurement of blood molybdenum levels indicate usual intake and can be used to determine if diets are adequate or too high in molybdenum.

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?
None.

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).
Coppers Healthy Limits Probed. ARS News Service press release reported by Marcia Wood on research of J.R. Turnlund, June 21, 2005.

Better Choices in Youth Can Lower Osteoporosis Risk. ARS, Agriculture Research Magazine. January, 2005.

Health at Every Size: New Hope for Obese Americans? ARS. Agriculture Research Magazine. March 2006.

Turnlund JR, Domek JM, Keyes WR. 2004. Zinc, iron, calcium, and magnesium metabolism with high copper intake (abstract). Journal of Trace Elements in Experimental Medicine 17: 198.

Turnlund JR, Keyes WR, Domek JM. 2005. High copper intake and iron status (abstract). Federation of American Societies for Experimental Biology 19: A983.

Turnlund JR, Keyes WR, Domek JM. 2005. Copper turnover and retention in humans with increasing copper intake (abstract). Trace Elements in Man and Animals-12, p.8.

Turnlund JT 2005. Mineral bioavailability and metabolism determined using stable isotope tracers (abstract). Journal of Animal Science. 83: 243.

Garrod MG, Grant RW, Allen LH, Haan MN, Green R, Miller JW. 2005. The ratio holotanscobalamin to total B12: Association with transcobalamin genotype, methylmalonic acid, and homocysteine (abstract). Experimental Biology.

Deegan KL, Jones KM, Graham JM, and Allen LH. 2005. Methodology for Determining the Vitamin B12 Concentration in Breast Milk. (Abstract) Experimental Biology.

Miller JW, Garrod MG, Rockwood AL, Kushnir MM, Allen LH, Haan MN, and Green R. 2006. (review article) Clinical Chemistry.

Teegarden D, White K, Zemel M, Van Loan M, Matkovic V, Lyle RM, Craig D, Schoeller D. 2005. Calcium and vitamin D modulation of lipid utilization and energy expenditure (abstract). Federation of American Societies for Experimental Biology #269.8

Novonty R, Going S, Teegarden D, Van Loan M, McCabe G, McCabe L. Daida Y. 2005. Asian, Hispanic and White adolescent body size, composition and fat distribution (abstract). Federation of American Societies for Experimental Biology # 270.2.

Going S, Novotny R, McCabe G, McCabe L, Teegarden D, Lohman T, Van Loan M, Diada Y, Boushey C. 2005. Relationship of body mass index to percent fat in Asian, Hispanic and non-Hipanic adolescent girls (abstract). Federation of American Societies for Experimental Biology # 270.8.

Teegarden D, Novoty R, Van Loan MD, Going S, Read M, McCabe G, McCabe L, Boushey C, Saviano D. 2005. Calcium intake and body fat measurements in adolesecnt girls (abstract). Federation of American Societies for Experimental Biology # 448.6.

Pappathakis PC, Van Loan M, Rollins N, Chantry C, Bennish M, Brown K. 2005. HIV-infected South African breastfeeding women lose more weight and body fat postpartum than their HIV-uninfected counterparts (abstract). Federation of American Societies for Experimental Biology # 274.8

Allen L, Anaya M, Begin F, Torun B, Brown KH. 2005. High risk of vitamin B-12 deficiency in predominantly breastfed Guatemalan infants is reduced by their consumption of dairy products (abstract). International Society for Research on Human Milk and Lactation.

Turnlund, J. R. 2005. Mineral bioavailability and metabolism determined using stable isotope tracers. Journal of Animal Science. 83: 243 (abstract) and J.Dairy Sci. 88: 243 (abstract).

NE Silverman, E Gertz, J Stewart, DL Alekel, L Clark, C Stephensen, M Van Loan. 2006. Relationship of cytokines to bone mineral density and content in postmenopausal women (abstract). Federation of American Societies for Experimental Biology #378.7.

JT Smilowitz, JB German, M Van Loan. 2006. Dairy food consumption alters lipid responses in humans (abstract). Federation of American Societies for Experimental Biology #387.1.

SN Bhupathiraju, KB Hanson, E Westbrook, JW Stewart, LN Hanson, MB Reddy, K Koehler, M Van Loan, DL Alekel. 2006. Relationship between homocysteine and trabecular bone in postmenopausal women (abstract). Federation of American Societies for Experimental Biology #670.6.

KM Shedd, KB Hanson, DL Alekel, LN Hanson, MD Van Loan. 2006. Bone mineral density and content: dual energy X-ray absorptiometry versus peripheral quantitative computed tomography (abstract). Federation of American Societies for Experimental Biology #670.8.

JW Stewart, KB Hanson, LN Hanson, OA Matvienko, M Van Loan, DL Alekel. 2006. Strength measures are related to bone mineral content and bone mineral density in postmenopausal women (abstract). Federation of American Societies for Experimental Biology #670.9.

K. M. Shedd, K. B. Hanson, D. L. Alekel, L. N. Hanson, M. D. VanLoan. 2006. Associations between bone mineral density using dual energy x-ray absorptiomewtry and bone strength properties using peripheral quantitative computed tomography (abstract). European Calcified Tissue Symposium # P467.

N. E. Silverman, E. Gertz, K. B. Hanson, J. W. Stewart, D. L. Alekel, L. N. Hanson, C. B. Stephensen, M. D. Van Loan. 2006. Relationship of inflammatory cytokines to bone mineral density and content in healthy postmenopausal women. European Calcified Tissue Symposium #P324.

Review Publications
Novotny Dura, J., Turnlund, J.R. 2006. Molybdenum disposition in humans during molybdenum depletion and repletion. Journal of Nutrition 136:953-957.