Duration: 10/01/2008 to 09/30/2013

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

Statement of Issue(s) and Justification

Diet, including specific nutrients and bioactive food components, plays a vital role in improving health and reducing risk for developmental and chronic disease. This collaborative research project brings together scientists with broad nutrition science expertise and experience to test hypotheses that ultimately will enhance the well-being of the population through reduced risk and incidence of several chronic diseases including cancer, obesity and osteoporosis. We plan to advance the understanding of the requirements for nutrients and bioactive food components in maintaining optimal health and to translate our findings to public health practice. There are numerous chronic diseases and developmental disorders for which risk may be modified in relation to exposure to bioactive dietary constituents. However, for the purpose of this collaborative research project the investigators are targeting cancer, bone health, obesity, birth defects and cognitive function and neurological disease. These diseases, and the associated interactions with nutrients and bioactive food components, are complex and require diverse approaches to advance knowledge that will lead to effective preventive and therapeutic strategies.

The numbers are staggering when one reviews disease incidence for these chronic diseases in the United States. Cancer is the second leading cause of death in the U.S. with 1 in 2 Americans receiving a cancer diagnosis in their lifetime and over 550,000 cases diagnosed annually. Specific to our efforts, breast cancer accounts for over 180,000 cancer cases annually and prostate cancer accounts for 9% of all cancer diagnosed in males. Numerous nutrients and bioactive food components have been shown to increase or decrease cancer risk. Currently, our investigative research team is collaborating on several dietary constituents including, but not limited to, folate, zinc, vitamin E, isothiocyanates, polyphenols, soy isoflavones, lycopene and citrus monoterpenes. All of which have important roles in human health and protection against chronic disease.

Osteoporosis, another leading chronic illness in older Americans, has a prevalence rate in people over age 50 years of over 12,000,000 cases (2002) is expected to increase to an estimated 14 million cases by 2020. Consequences of osteoporosis are severe with a mortality rate of 24% in the 12 months following newly diagnosed hip fracture. We will continue to expand research on calcium, and isoflavones and will expand into other nutrients and bioactive food components (BAFC) including iron, vitamin D and non-digestible carbohydrates (i.e., fructooligosaccahrides). Application of new and novel techniques and approaches to assess the impact of dietary interventions on bone outcomes, through the collaborative and complementary expertise within the research group will afford an unprecedented opportunity for research in this area.

Perhaps no chronic illness is of greater concern to the American public than obesity. Over 66% of Americans are overweight or obese (Ogden et.al., 2006) and nearly 40 million have some form of non-alcoholic liver disease (Angulo, 2002), a clinical condition which is strongly and significantly associated with the rising incidence of obesity. Efforts to reduce the clinical consequences of obesity through dietary interventions are not only warranted, but provide significant promise. Novel approaches including green tea polyphenols and/or vitamin E supplementation may help to control the undesirable biological changes including inflammation and oxidative stress responses  that further impair the health status of obese individuals.

Neural tube defects (NTD), including spina bifida and anencephalus, are diagnosed in nearly 30 per 100,000 live births in the U.S. It is well established that increased folate during early pregnancy will reduce birth defects by over 70%; however, the mechanism for protection, which is believed to be genetic, is not fully understood. Because of this dramatic effect of folate the U.S. food supply was fortified for folic acid in 1998. Concerns have been raised about potential adverse effects of excess folic acid intake in U.S. population as well as other concerns about the level of fortification being insufficient to maximize the reduction in birth defects. Current work on the genetic basis for the folate effect on NTDs will be a primary effort of our investigators and is expected to result in the identification of at-risk individuals.

One concern expressed in relation to high folic acid intake is the masking of anemia in the elderly with the consequent development of neurological disease in B12-deficient individuals. Impaired status of various vitamins including B vitamins and antioxidants as well as antioxidant bioactive food components have been implicated in impaired cognitive function. Our efforts to elucidate the optimal intake and/or exposure levels of select dietary constituents (vitamins and BAFC) will play a critical role in advancing knowledge resulting in the maintenance of optimal cognitive function in the elderly.

Thus, there is significant need to further understand the role of bioactive dietary constituents in reducing the risk of chronic disease. The opportunity for our experts to share knowledge, techniques and resources is central to advancing our understanding in a timely, resource-efficient and strategic manner. For example, while researchers in bone health focus on vitamin D as a nutrient of relevance to bone formation, others in cancer research are able to appreciate the role of vitamin D and to rapidly advance our understanding of its functional potential in cancer prevention through collaborations only possible through this project. Other examples of this rich cross-fertilzation is the dual preventive potential of folate in cognitive disease and cancer risk reduction, or iron as a modifier of disease risk in both cancer and bone-related disease.

Technical Feasibility of Studying Bioavailability and Bioactivity of Food Components: This W1002 multistate group is an extremely diverse group with unique expertise that is ideally poised to identify the mechanisms and bioavailability of nutrients and other bioactive food components on chronic disease prevention. All of the proposed involves ongoing methodologies in the respective investigators laboratories and through this collaborative effort, are extremely feasible (see related and previous work). W1002 researchers have established an international reputation in studying the mechanisms and bioavailability of nutrients and other bioactive food components important in chronic disease prevention. We have extensive experience in kinetic modeling of nutrients, bioavailability studies in rodents and humans, epigenetic and genetic polymorphism studies, as well as significant expertise in the study of osteoporosis, cancer and inflammatory disorders in both model systems and in the human population. Many of the proposed methodologies were pioneered by W1002 members, and have been recognized.

Advantages of a Multi-state effort for the study of bioavailability and bioactivity of food components: Through this multi-state effort we are able to examine mechanisms at the molecular cellular level and directly translate these effects to the population at large. This type of effort and scope would not be possible without the collaborative expertise of each multistate station. Defining the bioavailability and function of bioactive dietary chemicals in chronic disease is a complex and requires multifactorial approach. No single station has the capacity, resources, or equipment to undertake the proposed work in isolation. Through this multistate effort, we are able to combine efforts and share resources that are unique to each individual station. The issues to be addressed and benefits to be achieved are common to the nation as a whole, not a single state. Since several of the issues are being addressed in different ways among researchers in several states, information exchange and collaborations will facilitate goal achievement, limit duplication of effort among member's respective units, and potentially produce new and innovative methodologies for understanding the metabolism and roles for these bioactive food compounds.in human health. The diversity of expertise (from basic science to human population studies to outreach/extension) in faculty at major land-grant universities facilitate translational studies that data arising from W1002 collaborative activities will be disseminated to the greatest extent possible among stakeholders and will thus provide maximum benefits to the U.S. public. W1002 efforts and focus are not duplicated in any other regional project.

Impact of Research/Endpoints: We expect the multidisciplinary , translational research supported by the W1002 investigators to have significant impact on health, which will be identifiable as measurable endpoints. In a general sense, we expect select aspects of this research to result in the identification of new nutrition based prevention strategies for cancer, obesity, osteoporosis and other significant chronic diseases. More specifically, we expect this work help establish more effective biomarkers for nutrient status and new dietary requirements in the population. For example, W1002 researchers have been conducting research in folate and/or vitamin D metabolism and requirements which are expected to re-define requirements (DRIs) for these nutrients in the context of chronic disease risk reduction in the future. Further, collaborations in the area of cancer are expected to result in the development and testing of new chemoprevention agents and dietary recommendations that will impact oncology patient care in the future. If this work is not completed, this would markedly hamper our understanding of the metabolism and mechanistic role of each nutrient in maintaining optimal health. Without the research proposed by this multistate, optimal requirements for the nation cannot be adequatly assessed and could impact the prevalence of many chronic diseases, such as cancer, obesity and osteoporosis. Other endpoints for the W1002 group include more tangible items such as collaborative research grants, publications as well as outreach endpoints such as support of local extension staff and faculty in developing important patient/public-oriented educational materials integrating our research findings.

Because of the interactions and collaborations of W-1002 scientists, agriculture and health related entities have been provided with considerable information for use in the prevention of nutrition-related chronic diseases, consistent with regional and federal priorities. This group has published over 290 articles in the last 5 years. Data related to previous priorities addressed in W-1002 research has served, in part, as the basis for:
* the fortification of cereal grains with folate to decrease the risk of neural tube defects;
* the inclusion of health statements about calcium and osteoporosis, and hyperhomocystinemia and heart disease on food labels; and
* the increased DRIs for calcium for young adults.

This research is innovative and novel in that it utilizes a translational approach to understand the role of nutrients, especially those consumed as a whole food on optimal health. The collaboration among basic, clinical, epidemiological and extension faculty from diverse academic institutions across the U.S. affords the opportunity to advance scientific understanding of the role of, and requirements for, nutrients and bioactive food components in maintaining optimal health. Further our robust scientific interactions, which are possible through this collaborative agreement, afford an opportunity to translate research findings to public health practice in a time and cost-efficient manner.

CRIS Search: A CRIS search with key words bioavailability & nutrients and phytochemical or phytonutrient revealed 30 active projects. The committee is familiar with all projects. There are no similar regional projects.
A recent review of active multi-state projects suggests only one project has the potential for overlap with our efforts, W2122,beneficial and adverse effects of natural, bioactive dietary chemicals on human health and food safety. However, there are several important distinctions between the projects. First, this project focuses on cancer, obesity, osteoporosis, neural tube defects and cognitive function while W2122 addresses issue related to cancer, foodborne toxins, immunity and antimicrobials. Food safety is the central theme of their research and is not included in our focus in any definitive way. In fact, the one area of potential overlap (cancer) is approached very differently in the two applications. Our proposed project contuation uses a collaborative translational model to identify and implement effective dietary strategies involving select BAFC to reduce risk while the W2112 investigative team employs basic science techniques alone to more clearly characterize the role of select bioactive food compounds (BAFC) in promoting cancer risk or modulating risk associated with exposure to food-based toxins such as aflatoxin or heterocyclic amines. Another potential overlap is in the area of neural tube defects (NTD); however, here we are addressing the role of B vitamins in prevention and the W2112 group is identifying potential neurotoxins in crop feeds that contribute to risk. Further, a search of related research within the USDA CRIS database suggests there are individual investigators completing related research, including work on BAFC in cruciferae (UCBerkeley), chlorophyll (UHawaii), bitter melon (UHawaii), soy (UIllinois), bioidentification methods for BAFC (UCDavis), and several projects related to quantification of BAFC in select crops/foods (Beltsville). The vast majority are basic science related with few indicating the translational approach to BAFC research proposed here.

Without question, the information derived from the proposed research is innovative and unique, and the proposed dissemination of information to both the scientific community and lay public will provide part of the framework on which future nutrient recommendations can be based. Table 1 outlines a list of proposed nutrients and phytochemicals for initial analysis, their biomarkers & endpoints/clinical outcomes.

Related, Current and Previous Work

RELATED, CURRENT, AND PREVIOUS WORK

W1002 researchers have worked collaboratively and have made significant contributions to the understanding the role of nutrients and bioactive food components in optimal health. To continue in these efforts our research team will focus efforts in reducing the risk of chronic disease and developmental/degenerative disorders through the examination of nutrient/phytonutrient bioavailability, their mechanisms of action and the extent to which genetic and epigenetic alterations effect individual nutrient/phytochemical needs and chronic disease risk.
Bioavailability of nutrients and bioactive food components.

A. Absorption and Metabolism

Calcium. Dairy products supply 72 % of the calcium in the adult US diet, grain products about 11 %, and vegetables and fruits about 6 % (USDA, DHHS-89-1255). Although beans and corn tortillas processed with lime provide the bulk of dietary calcium for some ethnic groups, it is difficult for most to ingest sufficient calcium from foods in a cereal-based diet without liberal consumption of dairy products. Many have turned to dietary supplements to meet their calcium needs. In response, food manufacturers have developed calcium-fortified products (e.g., orange juice; rice; breads; low or non caloric beverages) that have had a range of market success.

Although calcium absorption efficiency varies inversely with load, its fractional absorption from various dairy products is similar at ~30 % (Nickel et.al., 1996). Calcium from most supplements is absorbed as well as that from milk, as solubility of its salts at neutral pH has little impact on calcium absorption (Heaney et.al, 1990). Absorption of a very soluble salt, calcium-citrate-malate (CCM), is better than for other salts (Andon et.al., 1996). Although most plant consitituents have been associated with inhibiting calcium absorption, some nondigestible oligosaccharides have been associated with enhancing Ca absorption through formation of short chain fatty acid formation in the lower gut.

Calcium excretion and retention are influenced by dietary sodium, age or life stage, and race. Urinary calcium losses account for 50% of the variability in calcium retention (Heaney, RP, 1994) IN have developed a nonlinear regression model using data in adolescent girls to study the relationship between calcium intake and calcium retention (Jackman et al,, 1997). Recently, IN has studied this relationship in adolescent boys and racial differences (Braun et al, a&b). The effect of other dietary constituents and exercise on this relationship and predictors of variance in calcium retention needs to be addressed. During growth, calcium retention is high. Average calcium retention in white female adolescents of ~300 mg/day in contrast to ~0 mg/day in women just a few years older at peak bone mass has been measured (Wastney et.al, 1996). After menopause, with estrogen insufficiency, calcium balance is typically negative (Heaney et.al, 1978). It is not clear whether racial differences in calcium metabolism in response to dietary salt occur at these other life stages because it has only been studied in children.

Men have greater bone mass than women, their vertebrae have larger diameters, and their trabecular bone architecture remains better preserved. To achieve that greater bone mass, peak calcium accretion rates are higher in boys than girls (peak bone mineral content velocity was 282 mg/d in boys vs 212 mg/d in girls determined by cross-sectional analysis of Canadian children), although it occurred 1.5 y later in boys (aged 14.5 y for boys vs. 13 y for girls) (Martin et.al, 1997). The difference in calcium accretion between girls and boys increased through puberty.

Folate: Folate functions in transporting single carbon fragments from one metabolic intermediate to another in the synthesis of nucleic acids and in the metabolism of amino acids. Folate deficiency leads to impaired cell division that presents as neural tube defects and anemia and to dysfunctional amino acid metabolism that presents as hyperhomocystinemia that is now widely accepted as a major risk factor for heart disease. So folate nutrition status is constantly monitored, but measuring erythrocyte folate has been a vexing problem for a very long time until the CA-D station developed an accurate, precise, reliable, and high throughput method, 5000 samples in 21 days (Owens JE et al JAFC 2007;55:3292-7). Erythrocyte folate is the best indicator of folate status because it reflects the intracellular pool. The NHANES has 5000 samples analyzed each year to monitor changes in folate status over time, now this can be completed in three weeks. The CA-D station also developed and validated a folate specific folate screener that assess folate intake, it can be completed in 10 minutes (Owens JE et al JAFC 2007; 55:3737-40.

Current knowledge of folate bioavailability is based largely on research conducted by W-1002 scientists. Current estimates of the efficiency of dietary folate absorption are variable depending on the experimental protocols used to measure absorption (Rodriguez et.al, 1978).
Now a better understanding of factors that affect the bioavailability and dynamics of folate metabolism among individuals is needed for future revisions of the DRI/AI for folate. Current work in CA-Berkeley and CA-Davis have developed novel technologies and modeling systems to address these issues with folate and other B vitamins.

Folate and its role in methylation have also been a topic of active research for cancer research. Growing epidemiological evidence suggests that requirements for folate may be u-shaped in terms of protection against cancer, suggesting that intake at either extreme could lead to cancer promotion. Existing datasets will provide further insight into this complex pattern of optimal nutrient exposure.

Vitamin E Bioavailability:
Greater than 90% of Americans fail to meet the estimated average requirements for dietary vitamin E (Maras et.al., 2004). Therefore, continued studies at CT and UC-D are aimed at assessing vitamin E absorption, distribution, and metabolism. CT has utilized LC/MS techniques for assessing stable isotopic vitamin E administration in humans to evaluate vitamin E absorption, antioxidant recycling, and oxidative stress-mediated vitamin E pharmacokinetics. UC-D has constructed a kinetic model of human vitamin E distribution, fluxes, pool size distribution and P450-mediated metabolism as it might occur in vivo. These stations will initiate collaborations in this area to extend their observations and ascertain future dietary recommendations.

Green tea catechin bioavailability:
In progress studies at CT suggest that green tea extract, presumably through the bioactivities of polyphenolic catechins, may regulate intestinal lipid absorption, lipid metabolism and may serve as a dietary strategy to attenuate the development of nonalcoholic fatty liver disease (NAFLD). Continued studies in this area are aimed at evaluating the bioavailability and metabolism of green tea catechins in experimental animal models of obesity. Continued studies in this area in CT are aimed at evaluating the bioavailability, metabolism, and mechanisms of action of green tea catechins in experimental animal models of obesity and defining the mechanisms underlying the inhibitory effect of green tea on lipid absorption. Pilot work at AZ indicate that overweight, post-menopausal women provided with decaffeinated green tea had a significant, short-term (30 minute) increase in resting energy expenditure that while of importance is too short in duration to suggest any long term role in weight control. Collaborative intellectual interactions among investigators working with tea research should promote greater understanding of the potential use of tea, tea polyphenols in obesity prevention.

Bioactive peptides/lipids:
Carnosine (b-alanyl-L-histidine) is a dipeptide found in the muscle foods that has been postulated to be a bioactive food component. Research in MA has determined the concentration of carnosine in human plasma after ingestion of beef. Subjects fasted for 12 hr and then had blood withdrawn prior to a meal containing 200 g of ground beef. Additional blood samples were collected over the following 24 hr and carnosine concentrations were determined by HPLC. No plasma carnosine was detected in subjects before the consumption of the beef. Carnosine was detected in plasma 15 min after beef consumption. Plasma carnosine concentrations continued to increase with a maximum (32.7 mg carnosine/L plasma) being recorded 2.5 hr after consumption. Carnosine concentrations then decreased until no carnosine could be detected at 5.5 hr post consumption (Park et al, 2005). These results indicate that dietary carnosine is absorbed into human plasma after the consumption of beef. Since carnosine has several potential health benefits, evidence of its bioavailability suggests that it could be a bioactive food component.

Many different food components could have health benefits. However, for foods to be useful delivery systems for these nutrients, the nutrient must be stable during the normal shelf life of the food and the nutrient must be bioavailable. Evidence that carnosine is not destroyed during the cooking of meat shows that it is present in processed meat products. Absorption of carnosine into blood after consumption indicates that it could have potential benefits against mental disorders and heart disease.

B. Nutrient/Gene interactions affecting nutrient requirements.

Inefficiencies in folate metabolism and inadequate intake lead to poor folate nutriture with increased risk of degenerative and developmental diseases. The determination of folates needs in humans has been problematic and in particular, there is a need to understand the effects of common single nucleotide polymorphisms (SNP) on B-vitamin metabolism (Clifford et.al, 2004, Lin et.al, 2004). Previously at CA-Davis, they constructed a kinetic model of in vivo human folate metabolism in 13 adults administered a 0.5 nmol oral dose of 14C-folic acid. The model estimated that apparent absorption of folate was 80%, enterohepatic recycling was 5350 nmol/d, and total body folate stores were 225 mol. All the estimates were larger than previous ones. So, in a preliminary experiment (study 1), we tested whether single nucleotide polymorphisms (SNPs) in folate-relevant enzymes affect folate metabolism by genotyping the 13 subjects SNPs in the following enzymes: MTHFR C677T and A1298C, MTR A2756G, MTRR A66G, CBS T833C, GCP II C1561T, TC II C776G, and RFC1 A80G and testing their effects on folate metabolism. Results of study 1 suggested that SNPs in MTHFR C677T, MTRR A66G, TC II C776G, and RFC1 A80G had significant effects on folate metabolism as reflected by RBC folate, plasma homocysteine and serum B12. To confirm our results, 370 additional subjects were genotyped for SNPs in the same enzymes out of which 234 Caucasian subjects were used to evaluate main and two-way interaction effects of age, gender, BMI, and genotype on plasma normalized homocysteine (nHcy) and on serum vitamin B12. Four main and two interaction effects affected nHcy, whereas two main and one interaction effects affected serum B12. Kinetic and statistical modeling of metabolic and genetic data can refine folate needs for optimal health and potential interactions with genotype. Research by W1002 scientists have created cutting edge methodologies to understand the impact of polymorphism on folate metabolism to help understand individual folate needs.

Nutrients and other food components- Mechanisms of bioactivity

A. Bioactive food components and maintenance of optimal health

Cancer prevention (cruciferous foods, biotin, tea, soy):
The role of diet and bioactive food components to modify cancer risk is an area of active research yet improvements in dietary measurement, increased evaluation of dietary interventions to reduce risk for cancer and more frequent use of surrogate biomarkers of disease risk in the context of dietary intervention research are needed. Foods rich in bioactive food components, including carotenoid-rich yellow-orange vegetables and fruit, cruciferous vegetables and citrus have been hypothesized to reduce cancer risk through a variety of bioactivities including reduction in oxidative stress.

In order to improve measures of cruciferous vegetable exposure we developed and validated a cruciferous vegetable food frequency food questionnaire using urinary biomarkers of cruciferous vegetable exposure, dithiocarbamate, (Thomson et.al, 2007) and have recently described cruciferous vegetable intake in a large population of breast cancer survivors (Thomson et.al 2007). Along these lines we have assessed the effect of a plant-based diet on plasma carotenoid concentrations in breast cancer survivors (Pierce et.al,, 2006) and also explored measurement error associated with self-report instruments (Natarajan et.al., 2006).

Green tea has been studied for its chemopreventive properties for well over a decade including its role in modulating oxidative stress, inflammation, apoptosis, etc. Researchers in this group are involved in a number of studies testing the bioactivity of green tea. AZ has recently completed a short-term green tea intervention trial among overweight breast cancer survivors to assess the effect of intake of 4 cups green tea daily for 6 months, as compared to placebo tea, to test if it will reduce body weight, body fat, insulin resistance and inflammation. OR have also found a decrease in inflammatory signal pathways with green tea consumption in a hormone-induced model for prostate cancer.

Epigenetics is the study of heritable features not associated with changes in the nucleotide sequence of DNA. These features are potentially reversible and may affect genomic stability and expression of genes, including tumor suppressor genes and oncogenes. Hence, epigenetic mechanisms have crucial roles in cancer risk. NE has demonstrated that biotinylation of K8 and K12 in histone H4 plays a role in the cellular response to double-strand breaks in DNA and chromosome condensation during the M phase of the human cell cycle (Zempleni, J, 2005, Kothapalli et.al, 2005, Narang et.al, 2004). Importantly, Zempleni at NE has shown that biotin deficiency and HCS deficiency are associated with decreased DNA repair activity and increased chromosomal damage and is uniquely positioned to investigate roles of histone biotinylation in cancer risk. Research in OR suggests that targeting histone modifications by dietary bioactives is a novel mechanism for prostate cancer prevention (Myzak et al, 2005, Myzak et.al 2006, Dashwood et.al, 2006). In particular, OR has found that sulforaphane, an isothiocyanate derived from cruciferous vegetables is also an HDAC inhibitor slows prostate cancer growth in cell, animals and humans. The identification of dietary HDAC inhibitors, and their use either alone or in combination, may increase efficacy of anti-cancer therapies/prevention strategies.

Bone Health (Calcium, iron, vitamin D, isoflavones, prebiotics):
Several types of botanical products containing isoflavones are being marketed as an alternative to estrogen replacement therapy to prevent osteoporosis in postmenopausal women. Estrogen suppresses bone resorption. Efficacy of isoflavones on bone health has mixed results which may depend on the dose and interaction of isoflavones and the ability of individuals to produce bioactive metabolites through gut microflora. IN will use 41Ca, a long-lived isotope, to label the skeleton and an Accelerator Mass Spectrometry (AMS) to monitor perturbations by diet. This innovative approach to study bone resorption is a direct measure of bone resorption in contrast to biochemical markers of bone turnover and traditional calcium kinetics, rapid compared to studying changes in bone density, and is not as invasive as bone histomorphometry. The effect of various types and dosages of isoflavone-containing botanical products sequentially in the same postmenopausal women for their effectiveness in suppressing bone resorption by measuring changes in 41Ca excretion in the urine. IN published the first clinical trial using this method, a dose response study of soy isoflavones in postmenopausal women (Cheong et al., 2007). A dose response effect of genistein, kudzu, and equol on bone and bone quality in an ovariectomized rat model and in postmenopausal women and the influence of gut microflora will be determined in IN Genistein is one of the most abundant and estrogenic phytoestrogens in soy (Rickard et al, 2003). The amount of genistein in most of the soy food materials ranges from 0.2-1 mg/g as various forms of glycosidic conjugates (Polkowski et al, 2000). Morabito et al. reported that administration of 54 mg of genistein/day was as effective as HRT in protecting against bone loss in postmenopausal women. Genistein increased femur and spine bone mineral density (BMD), reduced bone resorption as assessed by a decrease in urinary pyridinium cross-links, and increased bone formation as assessed by serum bone specific alkaline phosphatase and osteocalcin in postmenopausal women. This strong result may be due to the high dose of a single bioactive isoflavone comparable to the total isoflavone dose in other studies which have utilized isolated soy protein (Potter et al, 1998, Alekel et al, 2000) or it may be because genistein was unopposed by antagonistic components in soy such as daidzein.. Daidzein is one-fourth as estrogenic as genistein as assessed by uterine weight (Farmakalidis et. al, 1985). Equol [7-hydroxy-3-(4¢- hydroxyphenyl)-chroman] is the end product of intestinal bacterial metabolism of daidzein (Baker et al, 2000). The general structure of equol is similar to estrogen. However, equol lacks the lipophilic moiety that is found in estrogen (Notoya et al, 1992). Equol is excreted in the urine after consumption of soy foods which contain daidzin and daidzein (Axelson et al, 1984). Only 30-50% of adults excretes equol in the urine and has serum equol levels above 83 nmol/L. The ability to produce equol likely depends on the profile of intestinal microflora since it can be manipulated by polysaccharide substrates for the bacteria. Setchell et al. put forward a hypothesis that equol production is the key to clinical effectiveness of isoflavones in various tissues including heart and bone.

IN and UCD have developed the ability to prepare C-14 and C-13 labeled plant tissue culture. Pharmacokinetics and the metabolic fate of bioactive compounds throughout the body can be measured and kinetic and compartmental models developed.

The role of iron in bone accrual has received little attention and its mechanisms of action remain unclear. Work done in KS by Medeiros et al (Mederios et al, 2004, 2002, 1997) hypothesized that iron exerts its influence on bone through collagen synthesis. Iron is a required cofactor for prolyl and lysyl hydroxylase enzymes and this step is essential for lysyl oxidase activity, which then catalyzes cross linking of adjacent collagen fibers. In iron deficiency there may be less iron available to the prolyl and lysyl hydroxylase enzymes, which could result in decreased cross-linking activity and subsequently weaker collagen fibers (Tuderman et al 1977). Alternatively, cell culture studies suggest that iron influences the deposition of minerals in bone rather than changes in Type 1 collagen.

We previously reported that severe iron deficiency in the growing female rat has deleterious effects on bone morphometry and microarchitecture (Medeiros et al,2002) and severe calcium restriction exacerbates this effect (Medeiros et al, 2004). Previous studies of iron deficiency in rats found significantly decreased growth rates compared with iron adequate controls (Mederios et al, 2004, 2002, 1997). Medeiros et al (2004) reported results of a severe iron and calcium deficiency study that included a pair-fed group to determine whether the negative impact seen on bone was caused by the iron deficiency alone or by decreased caloric intake that accompanies iron deficiency anemia. Overall, the pair-fed group behaved similarly to the control group, which indicated that compromised bone integrity was caused by iron deficiency rather than decreased caloric intake. Harris et al (2003) showed in a study with post-menopausal women that there was a threshold effect regarding the influence of iron on bone mineral density (BMD). A maximal increase in BMD was reported with increasing iron intake only when Ca intake was between 800-1200 mg. BMD also was influenced by hormone replacement therapy (Maurer et al, 2005), which suggested a complex relationship between Ca, Fe, and bone.

More recently we determined whether marginal iron restriction that reflects some human consumption patterns could have similar consequences to that described above (Maurer et al, 2005). Rats were fed diets similar to above but containing 12 mg of iron per Kg diet. We also studied marginal calcium intake in addition to marginal iron. Rats fed either restricted iron or calcium diets showed several differences in femur and vertebral bone morphology. Vertebrae BMD was lower in all treatment groups compared to the control group and for BMC, the calcium restricted groups were lower than the calcium adequate groups and the marginal iron groups were lower that the iron adequate groups. BMC were lower in iron and calcium restricted groups. The microarchitecture of the L-4 vertebrae was compromised in both the iron and calcium restricted groups either singly or when both nutrients were low together. For the femurs, the cortical thickness of the femur was reduced by both marginal iron and marginal calcium feeding. Finite element analysis revealed that L-4 vertebrae from the marginal iron group had greater internal stress with an applied force than the control group, thus would be more likely to break.

Obesity (tea, vitamin E):
The role of green tea in reducing obesity and the detrimental health affects thereof has been an additional interest of these investigators. In addition, to assessing efficacy in a sample population of breast cancer survivors, Thomson (AZ) has also tested the short-term effects of decaffeinated green tea on oxidative stress and resting energy expenditure in overweight post-menopausal women. This pilot data indicated a significant rise in REE that was of only 30 minutes duration and was not associated with a increase in catecholamine levels as has been suggested when caffeinated green tea is consumed. Further, oxidant stress was not significantly reduced except among women with the highest waist:hip ratio (unpublished).

CT and OR are actively investigating the role of green tea extract in regulating the inflammatory and oxidative stress triggered processes in the development of non-alcoholic fatty liver disease (NAFLD). Preliminary results suggest that green tea extract attenuates hepatic steatosis and hepatic injury in a mouse model of obesity-triggered NAFLD. Continued studies are underway to unravel the protective mechanisms by which green tea attenuated hepatic steatosis and injury in obese mice. Additional studies are in progress to evaluate specific vitamin E forms (a- vs. g-tocopherol) in attenuating the oxidative stress and inflammatory processes implicated in the pathogenesis of NAFLD.

Objectives

1. Determine the bioavailability (absorption, distribution, metabolism, elimination) of nutrients and other food components and their environmental and genetic determinants
   
2. Evaluate the bioactivity of nutrients and other food components in order to elucidate their underlying protective mechanisms.
   

Methods

Objective 1: Determine the bioavailability (absorption, distribution, metabolism, elimination) of nutrients and other food components and their environmental and genetic determinants

1. Absorption and Metabolism

W-1002 scientists have developed state of the art techniques for assessing all components of bioavailability for several nutrients and for bone turnover. Novel methodologies include use of rare and stable isotopes for calcium, and 14C labeled bioactives (IN) and vitamins such as folate (UCD) and vitamin E (CT). The use of these isotopes allow for W1002 scientists to precisely monitor each nutrient, its uptake, distribution into plasma and elimination in urine & feces across different lifestage and physiological conditions. For example, work done in IN has effectively modeled calcium intake & its metabolisms during adolescence. This data will be critical in helping establish DRI for calcium in this age group. W1002 scientist can also examine alterations in nutrient metabolism during different physiological conditions. Research done in CT has found altered metabolism of vitamin E with smokers. Researchers in UCB together with UCD investigated the influence of folate and vitamin B12 status in our experimental animals using cDNA array technology and have identified a number of inflammatory response genes that are responsive to vitamin status. W1002 scientists will continue development of these methods and use them to study bioavailability and factors affecting vitamin, mineral, and bioactive compound distribution and metabolism at IN, UCD, UCB, AR, and OK.

2. Modeling and model development

Kinetic modeling and compartmental analysis will be used to assess vitamin, mineral and bioactive compound turnover rates, pool sizes, and metabolism (IN, UCD). Bone turnover rates will be used to evaluate predictors of bone formation and resorption during growth and age-related bone loss (IN). Modeling will be used to predict outcomes and design experiments to test predicted outcomes. For example, we will determine the bioavailability (absorption, distribution, metabolism, elimination) of 5-MTHF by quantifying the dynamic and kinetic behavior of tracer doses of 14C-Folic acid and 14C-5-methyltetrahydrofolate (14C-5-MTHF) using a test/re-test design in adults (UCB, UCD). Using AMS and compartmental modeling, differences in the dynamic and kinetic behavior of each folate form as it might occur in vivo in humans will be determined. This approach to bioavailability (absorption, distribution, metabolism, elimination) is unique because we will quantify the kinetic behavior of 14C-folates under steady-state conditions for long time periods to ensure that the administered tracers are fully equilibrated with the most slowly turning over pools; these pools determine the kinetic behavior in plasma. This can only be done by utilizing AMS to analyze the amount of 14C in plasma, urine, feces, and RBCs. Key parameters that we will determine include whole body turnover, mass balance, half-lives for absorption, distribution and elimination in plasma, and the cell population kinetics of RBCs followed over their entire life-span of 120 days. Because we have already constructed a kinetic model of FA metabolism, we are positioned to quantitatively compare the metabolism of the major naturally occurring food folate 5-MTHF to FA. Direct comparisons of 5-MTHF to FA will aid in the development of dietary recommendations for optimal health.

3. Genetics

Gene polymorphisms that affect nutrient flux through metabolic pathways may well be important determinants of nutrient requirements for optimal health. Genetic polymorphisms that are associated with traits such as hyperhomocysteinemia, hypercholesteremia, etc., may be well normalized (over-ridden) by appropriate dietary adjustments. Initially, folate and vitamin B12 will be further studied (UCD and UCB). The influence of polymorphisms in glutathione-s-tranferases and cytochrome P450, on bioavailability of phytochemicals found in cruciferous vegetables will also be examined in collaboration between AZ and OR. Modeling has already suggested that genetic polymorphisms involved in folate metabolism affect the flux through several pathways and these model changes will be tested in experimental systems (UCB and UCD). Tissue specific and cell compartmental models are being used to predict effects of polymorphisms in folate genes on metabolic fluxes (UCB). We will the study subjects for the folate relevant SNPs and search for additional folate-relevant SNPs of RFC1 using a SNP discovery platform developed by re-sequencing candidate genes in panels of 24 subjects from a population of human subjects screened for eight SNPs, diet, and blood chemistries.

4. Development of facile methods for assessing nutrient status and bioavailability

Equations for simple methods to predict true absorption of nutrients such as Ca (IN) against full kinetic models are being developed. Genetic polymorphisms are being screened as predictors of folate status (UCD and UCB). At the end of the project we will have kinetic models of the interactions of SNPs on 5-MTHF versus FA metabolism in humans that will clarify inter-individual responses for the development of improved strategies to minimize the risk of NTDs, age-related diseases and poor folate status. We may also have newly identified SNPs of the RFC1 that affect folate homeostasis. In summary we will know whether the main and interactive effects of genetic polymorphisms in folate relevant enzymes are significant determinants of the dynamic and kinetic behavior of folate (natural versus synthetic folate) metabolism as it might occur in vivo in humans. We will also have models for absorption of other minerals, such as calcium, that will be used to help define absorption of these nutrients in humans and help develop DRIs for optimal health. Importantly, all of the goals proposed in Objective 2 are translational studies performed in human subjects, which is a major strength of this project.

Objective 2. Evaluate the bioactivity of nutrients and other food components in order to elucidate their underlying protective mechanisms.

1. Identify the relationships among nutrient status, dietary intake of nutrients and other food compounds and their association with chronic disease risk in the human population. Collaborating investigators will utilize existing and yet to be developed data sets from cohort studies and clinical trials to assess the relationship between nutrients/ bioactive food components and disease outcomes and/or surrogate biomarkers of disease risk. Clinical and biological samples from the Womens Healthy Eating and Living study (N =3088 breast cancer survivors) will be used (AR) to evaluate the role of cruciferous vegetables and their constitutive bioactive food components (OR) in reducing breast cancer recurrence risk. This data set also affords the opportunity to explore other food sources of bioactive food components (BAFC) and breast cancer recurrence risk including citrus, omega 3 fatty acids, and vitamins and minerals. In addition, dietary and clinical data from over 165,000 post-menopausal women from the Womens Health Initiative, that include sub-groups randomized to a low fat diet, and/or calcium supplementation, will be used to assess relationship between nutrient status and health outcomes. In collaboration with other investigators with expertise in select nutrient metabolism, including iron (AR), calcium (IN), folate (CA-B), vitamin E and tea polyphenols (CT), this longitudinal dataset which includes cancer, cardiovascular, and bone health outcomes, supports our collaborative efforts to assess the role of diet in chronic disease risk reduction through epidemiological data analysis. To advance our capacity to evaluate the relationship between bioactive food components and disease risk, we will also develop and enhance dietary assessment tools of intake and/or exposure to food components. This includes development of nutrient-specific self-reporting instruments (AR) and assessment of biological markers of intake including: carotenoids (AR), calcium (IN), vitamin E and polyphenolic flavonoids (CT), folate (CA-D, CA-D).

2. Identify interactions among dietary compounds and genetic and epigenetic events that influence growth, developmental disorders, and chronic disease. The effect of genetic variation on bioactivity of bioactive food components will be assessed. Initially, folic acid, vitamin B12, and glutathione S-transferase (GST) polymorphisms will be studied (CAD, CAB, and AR).

In addition, a collaboration among NE, OR and AZ will enable the study of epigenetics and cancer risk. NE will examine the effects of biotinylation of histones on cancer risk. Biotinylation will be assessed using chromatin immunoprecipitation and real-time PCR and chromosome painting techniques will be used to quantify effects of altered histone biotinylation in pericentromeric chromatin on chromosomal stability. In addition a, mouse model for disrupted histone methylation has been developed at UC-B and will be used to study nutritional modification of methylation in development. The impact of cruciferous vegetable intake and other bioactive food components on histone acetylation has not been clearly identified. Importantly, collaboration between OR and AR give the opportunity for TRANSLATIONAL studies, using a unique and extensive longitudinal dietary, clinical and demographic dataset, tumor tissue samples, and DNA samples collected for the on-going Womens Healthy Eating and Living (WHEL) Study of 3088 breast cancer survivors participating in a randomized, controlled, plant-based dietary intervention trial. With this group, we will be the first, to our knowledge, to refine our understanding of epigenetic events by describing the interaction between epigenetic alterations (HDAC) and candidate genetic polymorphisms in relevant genes involved in the bioavailability, bioactivation and biotransport of cruciferous vegetable-derived isothiocyanates including sulforaphane, cruciferous vegetable intake and breast cancer recurrence.

3. Identify bioactive food compounds that regulate antioxidant status, inflammation and cell signaling pathways leading to disease prevention. Specific goals for this aim include, defining the bioactivity of green tea extract on lipid metabolism during the development of obesity-triggered NAFLD and defining the redox sensitive mechanisms regulated by bioactive food components such as green tea, soy isoflavones and fruit & vegetable intake on the development of NAFLD (CT) and prostate cancer (OR). This hypothesis will be tested by feeding green tea extract to ob/ob mice (model for NAFLD) or hormone-induced prostate cancer. CT and OR work in collaboration to examine dietary antioxidant status, endogenous antioxidant defenses, inflammatory cytokines and oxidative/nitrative stress, damage biomarkers, redox sensitive transcription factor analysis and gene expression of factors regulating inflammation and cell signaling. Work done other natural phytochemicals, such as those found in blueberries is being investigated in ME.

We will also ssess the effect of nutrients and/or food components on oxidative stress and inflammatory biomarkers in a human population. Markers of oxidative stress (8-OHdG and 8-epi-PGF2±) and inflammatory (hs-CRP, TNF) will be measured in pre- and post dietary intervention groups (AR, IN, OR, CT).

4. Evaluate bioactivity in bone health

Iron Status In Bone Health We will examine the impact of iron status on calcium kinetics and bone health. Rats will be depleted and repleted in iron (KS) and iron status (AR), bone turnover with 45Ca (IN), as well as bone mineral concentration, microarchitecture, and strength (OK) will be measured. Specific endpoints/biomarkers to be utilized include 1. Mechanical testing of femurs A 3 point breakage test will be conducted by using an Instron Universal Testing System. By using a vernier caliper the midpoints of the femurs will be determined by measuring the length of each bone and dividing that length by two. The length of the femur will be determined by measuring from the highest point on the head to the lowest point of the inner condyle. The Instron is interfaced with the companys Series IX software for Windows to measure bone strength and stress. 2. Mineral analysis of bone and diet The right femurs and tibias will be subjected to wet ashing as previously described by our lab (15). Analysis of both Ca and Fe will be conducted by flame atomic absorption. Certified standards for Ca and Fe will obtained from Fischer Scientific Co. MicroCT & Finite Element Analysis The DEXA small animal high-resolution scan module (Hologic QDR-4500A) will used to determine the BMC and BMD of L-4 vertebrae of the L1-6 articulated vertebrae and of left femurs. Micro-computed tomography (Micro-CT 40, Scanco Medical, Switzerland) will be used to evaluate the three dimensional architecture of the vertebrae.. The vertebrae will be placed in 16 mm tubes and images taken at every 0.0165 ¼m. The trabecular area will be outlined and three-dimensional values determined by using a contouring algorithm. Analyses of bone volume fraction, connectivity, and trabecular number, thickness, and separation will be conducted. The cortical bone at the midshaft of the femur will be analyzed by micro-CT. Total volume, bone volume, porosity and cortical thickness will be determined. Finite element software will allow us to simulate compression properties of the vertebrae Total compression force, size-independent-stiffness, and von Mises stresses will be determined for each L-4 vertebra.. Studies will be performed in collaboration with KS , AR (iron analysis) , IN (Calcium kinetics) and OK (bone architecture).. If iron effects with repletion are observed, experimental models where iron status is altered by genetic mutation (AZ) will be used to further explore the effect of iron on bone health.

Life stage and health status on skeletal calcium and bone retention. The methodology utilized in rodent studies will be translated to studies performed in human subjects to examine effects of life stages and other nutrient deficiencies on bone health. Adolescents will be studied (IN) for effects of vitamin D, calcium source, and subgroup populations. Postmenopausal women will be studied for effects of plant isoflavones (IN, AR) and gut microflora (IN) on calcium bioavailability and bone loss by DEXA (bone density ) and microCT (bone architecture). Similarly, breast cancer survivors will be studied for plant-based versus meat-based diets (AR), and their influence on bone density. Ethiopian mothers and infants will be studied for zinc and vitamin D status (OK). Biomarkers for zinc status and vitamin D status will be correlated with alterations in bone density and architectural alterations in bone in these populations.

Expected endpoints/outcomes: The expected endpoints resulting from the multidisciplinary, collaborative interactions among the W1002 investigators include: " Establish models of bioavailability for key nutrients involved in optimal health, including calcium, vitamin D, B vitamins, Vitamin E and phytochemicals such as isoflavones and isothiocyanates. " expanded use of existing epidemiological datasets for which the collaborative researchers will have access in order to test hypotheses related to the role of our targeted bioactive food constituents in chronic disease risk reduction, " develop collaborations to translate in vitro and animal studies on nutrients to human populations " develop collaborations to perform mechanistic studies from observational epidemiological studies " collaborative support in grant development to assure optimal research design particularly in regard to bioactive food constituent measurements, - both through self report and biological markers of exposure " Shared methodology regarding measurement of oxidative stress and/or inflammation as an intermediate indicator of chronic disease risk, including strengths and limitations of available biomeasures, understanding of bioactive deposition requirements to modulate these markers and optimal measurement conditions including frequency as well as in relation to timing of exposure " Resource sharing related to new imaging technologies for use in assessing endpoints of feeding and/or bioactive dietary constituent epidemiological trials (i.e. bone health, body composition, nutrient deposition in targeted tissues) " through shared knowledge, advanced understanding by all investigators of the potential role of genetic background in modifying exposure and/or efficacy of bioactive dietary constituents in relation to normal growth as well as chronic disease risk reduction " joint peer-reviewed publications " Support for the development of targeted outreach materials that integrate research findings resulting from this research

Summary: A summary of proposed collaborations and interactions are listed in table 2. We have also included specific expertise each station member brings to this collaborative effort in table 3- Resources. This W1002 team of investigators consists of a unique group of researchers that can advance science in defining the role of bioactive dietary constituents for optimal human health. This group includes experts in a wide variety of nutrients and bioactive food constituents is an appropriate approach given that the food we eat is a complex and integrated mixture of nutrients and bioactives. Further, the research technological expertise of our group is also diverse and includes expertise in cell culture, tissue culture, animal models, imaging, epidemiology and clinical trials. This diversity provides fertile ground for collaborations in the form of informal conversations as well as formal and collaborative grant proposal development.

Measurement of Progress and Results

Outputs

• Additional collaborative projects
• Research data
• Peer-reviewed publications
• Presentations and posters at scientific meetings
• E-files and web-based applications
• Outreach presentations or publications for non-scientific organizations

Outcomes or Projected Impacts

• Increased knowledge of the bioactivity of nutrients and other food components and their underlying protective mechanisms
• Increased knowledge of environmental and genetic determinants of the bioavailability of nutrients and other food components
• Development and publication of models for determining bioactivity and bioavailability of nutrients and other bioactive food components

Milestones

(2008): 1) Development of collaborative proposals for seed money to support proposals for more extensive collaboration. <p> 2) Initiate the collaborative studies as designed under the timeline for year 1. <p> 3) Researchers will write peer-reviewed articles related to research findings. <p> 4) Discuss the design and implementation of a program of e-materials and web-based distribution.

(2009): 1) Initiate the collaborative studies as designed under the timeline for year <p> 2)Report on and assess collaborative efforts on projects initiated in year 1 that extend to all years. <p> 3) Researchers will write peer-reviewed articles related to research findings. <p> 4) Implement the program for e-materials and web-based distribution.

(2010): 1) Initiate the studies as designed under the timeline for year 3-4. <p> 2) Report on and assess further collaborative efforts for bioavailability of calcium, for bioinformatics of genotyping data, for epigenetic cell culture studies and on-going efforts that extend to all years. <p> 3) Researchers will write peer-reviewed articles related to research findings. <p> 4) Implement the program for e-materials and web-based distribution.

(2011): 1) Report on and assess further collaborative efforts for in vivo models, human dietary supplement intervention trials, dietary measurement/validation, cancer survivors dataset and epidemiological and association studies and on-going efforts that extend to all years. <p> 2) Researchers will write peer-reviewed articles related to research findings. <p> 3) Evaluate the program for e-materials and web-based distribution and make changes as appropriate.

(2012): 1) Evaluate the accomplishments of the collaborative efforts for the project. <p> 2) Prepare a report on the web-based applications. <p> 3) Determine goals and prepare a proposal to continue collaborative efforts. <p> 4) Researchers will write peer-reviewed articles related to research findings.

Projected Participation

View Appendix E: Participation

Outreach Plan

An important strength to this proposal is the experience of select participants in community-based outreach and education and/or are registered dietitians with experience in clinical settings (AR, KA, CT, IN) will facilitate translation of research findings beyond the scientific community to clinicians in the venue of grand rounds and clinical conferences or public health thru community-based programming -including Extension programs at the universities involved - and thru media (when appropriate) thru university-based public relations offices. Outreach will also be performed to research peers in the form of publications of original research and presentations at national meetings. In addition, we will create e-files of relevant publications, review articles and methodologies on our website, maintained by Dr. Ann Bock at NM (http://agesvr1.nmsu.edu/agepages/marbock/W1002/index.html). This website will also have links to fact sheets and other relevant information in bioavailability and disease prevention of various bioactive food components. Most people are not aware that 'just because you consume a nutrient, does not mean that your body can digest, absorb and/or utilize it. These materials will offer an excellent avenue for educating the public.

Organization/Governance

The recommended Standard Governance for multistate research activities include the election of a Chair, a Chair-elect, and a Secretary. All officers are to be elected for at least two-year terms to provide continuity. Administrative guidance will be provided by an assigned Administrative Advisor and a CSREES Representative.

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Attachments

Land Grant Participating States/Institutions

AZ, CA, CT, IL, MA, MT, NE, OH, OK, OR, WA

Non Land Grant Participating States/Institutions

California-San Diego