Duration: 10/01/2018 to 09/30/2023

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

Diet, including specific nutrients and bioactive food components, play a vital role in improving health and reducing the risk for developmental and chronic diseases. This collaborative research project brings together scientists with expertise in biomolecular, community, and international nutrition experience to test hypotheses that ultimately will enhance the health of the global population through reduced risk and incidence of chronic diseases driven by malnutrition. We plan to advance the understanding of the requirements for nutrients and bioactive food components in maintaining and achieving 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 aspects of bioavailability and bioactivity of dietary constituents for optimal health. Our collaborative goal is to define interactions among nutrients and phytochemicals in relation to human health outcomes, and requires transdisciplinary approaches to advance knowledge that will lead to effective preventative and therapeutic dietary strategies, especially in vulnerable groups. Both the factors determining the absorption/bioavailability and targeted mechanism of actions will be addressed through preclinical and translational approaches in this multi-state effort. A major outcome of this approach is to establish the foundation for evidence-based dietary recommendations for communities and individuals.

Nutrition plays an important role in the promotion of optimal health and the prevention of chronic disease. Optimal nutrition is a fundamental for good health and the prevention, treatment and management of disease. The relationship between food, nutrition and health, however, is complex, dynamic, and multi-faceted and highly affected by biological as well as environmental, socioeconomic, cultural and behavioral factors (Saarloos et al., 2009). Global population growth, climate change and pressure on natural resources, poor access to healthy foods, unhealthy lifestyles, and growing consumer demand, all present are contributing to malnutrition. Malnutrition refers to deficiencies, excesses, or imbalances in a person’s intake of energy and/or nutrients. Malnutrition, in all its forms, includes undernutrition (wasting, stunting, and underweight), inadequate vitamins or minerals, overweight, obesity, and resulting diet-related non-communicable diseases (Lee et al., 2016). Susceptibility to environmental toxins such as air and water pollutants, subgroup differences (age, gender, race and disease-state) may influence bioavailability and absorption of micronutrients.

Paradoxically, while approximately 800 million people in the developing world suffer from chronic undernourishment, both developed and emerging economies face the problem of rising levels of obesity and diet related disease (heart disease, type 2 diabetes, cancer, high blood pressure, inflammatory bowel disease, poor bone health, osteoporosis and osteoarthritis, Mainka et al., 2005). This is largely due to changes in patterns of consumption and the type of food consumed, as well as more sedentary lifestyles. Epidemiological evidence suggests that regular consumption of fruits, vegetables and whole grains is associated with reduced risk of developing chronic diseases. This association has been partly ascribed to the presence of a variety of bioactive food components naturally occurring in plant-based foods (Dillard and German, 2000). Phytochemicals, as plant biologically active compounds have ability to provide health benefits. However, it is still difficult to make nutritional recommendations for these bioactive compounds because of their limited bioavailability, complex structure, specificity and their targeted effects on a limited number of markers (Manach et al., 2009). New approaches are needed to take into account both the bioavailability, diversity of phytochemicals found in the diet and the complexity of their biological effects. One of novel approaches is using exosomes as therapeutic drug carriers, it is reported that bioactive molecules are selectively sorted in exosomes that are then released from donor cells in a regulated manner. Exosomes are specifically internalized by recipient cells via ligand-receptor interactions. Thus, exosomes are promising natural vehicles that stably and specifically transport bioactive molecules to recipient cells (Suzuki et al., 2016). Our scientists have demonstrated that exosomes and their ribonucleic acid (RNA) cargos can be absorbed from foods such as bovine milk and chicken eggs. We seek to identify the exosome cargos and the mechanisms that elicit the above phenotypes. Chronic diseases are often preventable, and frequently manageable through early detection, improved diet and treatment therapy. Currently, our investigative research team is collaborating on several dietary constituents including, but not limited to, folate, exosomes and their RNA cargos, zinc, vitamin E, vitamin D isothiocyanates, polyphenols, soy isoflavones, lycopene and citrus monoterpenes. All of which have important roles in human health and protection against chronic disease.

The global prevalence of obesity and overweight has markedly increased over the past several decades in both developed and developing nations. Excess body weight, a burgeoning problem worldwide, is a major risk factor for cardiovascular disease (CVD) and diabetes. Diabetes affects >180 million people around the world, and the number of patients is anticipated to increase to 300 million by 2025 (International Diabetes Federation Atlas, 2009). Efforts to reduce the clinical consequences of obesity through dietary and behavioral interventions are not only warranted, but provide significant promise. Novel approaches including green tea polyphenols, milk exosomes and their RNA cargos, grape anthocyanins, 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.

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 (Scherer and Hill, 2016). The evidence suggests that proper diet can play an important role in building and maintaining bone mass throughout life, primarily by providing bone-building nutrients and by influencing absorption and retention of these nutrients. We will continue to expand research on calcium and isoflavones and will expand into other nutrients and phytochemicals including vitamin D and non-digestible carbohydrates (i.e., fructooligosaccharides). 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.

Cancer is also 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. In 2017, there will be an estimated 1,688,780 new cancer cases diagnosed and 600,920 cancer deaths in the US (Siegel et al., 2017). Numerous nutrients and phytochemicals have been shown to increase or decrease cancer risk. We aim to identify and target the various phytochemicals that prevent cancer. Chronic inflammation, aging, specific morbidities, dietary patterns and specific bioactive food components have been associated with improved or decreased brain function, as evidenced by changes in cognition, memory and learning that lead to various neurological diseases. There are more than 600 diseases of the nervous system, such as brain tumors, epilepsy, Parkinson's disease and neural tube defects (NTD) as well as less familiar ones such as frontotemporal dementia (Essa et al., 2016). 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. 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. Our efforts to elucidate the optimal intake and/or exposure levels of select dietary constituents (vitamins and phytochemicals) will play a critical role in advancing knowledge resulting in the maintenance of optimal cognitive function in the elderly.

Importance of Work: There is significant need to further understand the independent and interactive role of nutrients and bioactive food constituents in promoting optimal health. Our goal is to provide a clear understanding of the factors that determine their bioavailability and mechanisms of action. This will establish the foundation for evidence-based dietary recommendations to stakeholders that ultimately affect the health of our nation and the world. The opportunity for our experts to share knowledge, techniques and resources is central to achieving our collective goal in a timely, resource-efficient, and strategic manner. For example, while researchers in bone health focus on anti-inflammatory phytochemicals of relevance to bone accretion, others have assimilated this knowledge to apply towards other inflammatory disorders relating to liver and cardiovascular health, and metabolic disorders. Our transdisciplinary approach thereby rapidly advances our understanding of health-promoting dietary approaches having the potential for broader impact on human health, which will be made possible through this multi-state collaboration.

Technical Feasibility of Studying Bioavailability and Bioactivity of Food Components: This W3002 Multi-state group is an extremely diverse group with complementary expertise that is ideally poised to identify the mechanisms and bioavailability of nutrients and other bioactive food components. All of the proposed projects involve established methodologies in the respective investigators’ laboratories and through this collaborative effort, are extremely feasible (see related and previous work). W3002 researchers have established a national and international reputation in studying the mechanisms and bioavailability of nutrients and dietary bioactive components important in chronic disease prevention. We have extensive experience in pharmacokinetic modeling of nutrients, bioavailability studies in preclinical models and humans, epigenetic and genetic polymorphism studies, as well as significant expertise in the study of chronic diseases in relation to malnutrition and metabolic disorders in translational models. Many of the proposed methodologies were pioneered by W3002 members and will be leveraged in this multi-state effort.

Advantages of a Multi-State Effort for the Study of Bioavailability and Bioactivity of Dietary Bioactive Components: Through this multi-state effort we are able to examine mechanisms at the molecular cellular level and directly translate these benefits to susceptible populations. This type of effort and scope would not be possible without the collaborative expertise of each multi-state station. Indeed, defining the bioavailability and function of dietary bioactive components is complex and requires a transdisciplinary scientific approach. Upon the successful completion of this multistate effort, we expect to synergistically address knowledge gaps of importance to human health by combining efforts and expertise, and sharing resources unique to each individual station. The issues to be addressed and benefits to be achieved will exceed those needed by a single state, and are expected to have national and global impact. Since several of the issues are being addressed through complementary, multi-disciplinary approaches among researchers in several states, information exchange and collaborations will facilitate goal achievement, limit scientific duplication among members’ respective units, and potentially produce innovative methodologies for understanding the metabolism and roles for these dietary bioactive constituents in promoting human health. The breadth and depth of expertise (from basic science to human population studies to outreach/extension) among faculty at major land-grant universities facilitates translational studies. Knowledge gained from W3002 collaborative activities will be disseminated to the scientific community and relevant stakeholders thereby having maximal benefit to public health. Importantly, efforts and focus specific to W3002 are not duplicated in any other multi-state project.

Impact of Research/Endpoints: We expect the multidisciplinary, translational research supported by the W3002 investigators to have significant impact on health, which will be identified as measurable endpoints. In a general sense, we expect select aspects of this research to result in the identification of evidence-based strategies using foods and/or their dietary bioactive compounds to contribute to optimal health and reduce chronic disease risk. Specifically, we expect this work will help to establish validated biomarkers for nutrient status, and by extension, redefine dietary requirements for healthy humans including susceptible populations. For example, W3002 researchers have been conducting research in folate and/or fat-soluble vitamin metabolism and requirements aimed at re-defining dietary nutrient requirements (DRIs). Further, collaborations in the area of cancer are expected to result in the development and testing of new chemoprevention agents and dietary recommendations having the potential to reduce oncology patient care burden. Without the research proposed by this multistate group, our understanding of the metabolism and mechanistic role of these health-promoting dietary agents would be hampered, thereby precluding timely dietary recommendations to achieve optimal human health. Other endpoints for the W3002 group include collaborative research grants, publications, and efforts directed at public outreach and support of local extension leaders in developing important educational materials that translate our research findings.

This multi-state team of researchers are a transdisciplinary team spanning fields of agriculture, food science and technology, molecular/cellular nutrition, clinical nutrition and population health. Together, the team is able to address key issues relevant to food and its bioactive compounds and human health, both in the United States and across the globe.

This research is innovative and novel in that it utilizes a translational approach to understand the role of dietary bioactive compounds, 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.

National Information Management and Support System (NIMSS) Search: A NIMSS search with keyword “bioavailability” 12 active 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, W4122” Beneficial and adverse effects of natural chemicals on human health and food safety.” However, there are several important distinctions between the projects. First, this project focuses on optimal health whereas W4122 addresses issue related to cancer, foodborne toxins, and antimicrobials. Our project focuses much more heavily on micronutrients compared to the W4112. Also, food safety/toxicology is the central theme of their research and is not a in our focus of our project. Moreover, issues regarding global health, bioavailability and potential interactions among food, nutrients and processing are not addressed by the W4112 group.

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 and endpoints/clinical outcomes.

Literature Cited

Dillard, C. J., & German, J. B. (2000). Phytochemicals: nutraceuticals and human health. J. Sci. Food Agric., 80(12), 1744-1756.

Essa, M. M., Akbar, M., & Guillemin, G. (Eds.). (2016). The Benefits of Natural Products for Neurodegenerative Diseases. Advances in Neurobiology.

International Diabetes Federation Atlas. Global Burden of Diabetes. http://www.diabetesatlas.org/content/global-burden (Accessed on November 16, 2009).

Lee, J.L.C., Leong, L.P. & Lim, S.L. (2016). Nutrition intervention approaches to reduce malnutrition in oncology patients: a systematic review. Support Care Cancer. 24: 469.

Mainka, S., McNeely, J. A., & Jackson, W. J. (2005). Depend on nature: ecosystem services supporting human livelihoods (1st ed., Vol. 1). Switzerland: IUCN - the World Conservation Union. p37.

Manach, C., Hubert, J., Llorach, R. and Scalbert, A. (2009). The complex links between dietary phytochemicals and human health deciphered by metabolomics. Mol. Nutr. Food Res., 53: 1303–1315.

Saarloos, D., Kim, J.-E., & Timmermans, H. (2009). The Built Environment and Health: Introducing Individual Space-Time Behavior. International Journal of Environmental Research and Public Health, 6(6), 1724–1743.

Related, Current and Previous Work

W3002 researchers have worked collaboratively and have made significant contributions to understanding the role of nutrients and bioactive food components in optimal health. These efforts have focused on optimizing health in vulnerable populations both domestically and globally. To continue this work, our research team will focus its efforts on examining bioavailability of dietary bioactive components, identifying human and environmental factors that modulate their absorption and metabolism, innovative approaches to improve bioavailability and delivery. In addition, an important objective will be to examine activity of dietary bioactive components and understanding their mechanism of action. This group has advanced these areas utilizing multi-disciplinary and translational approaches that span in vitro, in vivo animal models and human populations using both laboratory-based and bioinformatics/computational methodologies.

1. Absorption and Metabolism Modeling

W3002 scientists have developed state of the art techniques for assessing all components of bioavailability and metabolism for several nutrients and food components. These techniques address the interactions between members of the gut microbiome, nutrient bioavailability and metabolism.

Novel methodologies include use of rare and stable isotopes for calcium (IN), and 15nitrogen-labelled nitrate and nitrite (OR) and vitamins such as folate (UCD) and vitamin E (OH). The use of these isotopes will allow W3002 scientists to precisely monitor each nutrient, its uptake, distribution into plasma and elimination in urine & feces across different life stage and physiological conditions. Novel methodologies also include protocols for the purification of exosomes from milk and eggs, the transfection of exosomes with synthetic, fluorophore-labeled RNAs and a transgenic mouse that allows to track the trafficking of exosomes and tissue-specific cargo analysis (NE). W3002 scientists can examine alterations in nutrient metabolism during different physiological conditions, including malnutrition, inflammation, spatial learning and memory, and physical activity. Researchers in UC-B together with UC-D investigated the influence of folate and vitamin B12 status in our experimental animals.

Kinetic modeling and compartmental analysis will be used to assess vitamin, mineral and bioactive compound turnover rates, pool sizes, and metabolism (IN, UCD, OR). 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.

OH is also active in developing liquid chromatography–mass spectrometry (LC-MS) methodologies for the assessment of catechin bioavailability and metabolism, especially those metabolites generated by the gut microbiota that have putative anti-inflammatory activities. The use of these procedures will enable an understanding of the bioactivities of green tea catechins mediated along the gut-liver axis that are responsible for mitigating the risk of nonalcoholic steatohepatitis. This will facilitate the potential translation of preclinical studies demonstrating anti-inflammatory activities of catechins in rodent models into clinical efficacy for the management of nonalcoholic steatohepatotitis (NASH) risk, especially in obesity cohorts that are especially vulnerable to developing this disorder that currently has no Food and Drug Administration (FDA)-approved therapies. Similar approaches have been employed in OR, where LC-MS methodologies for isothiocyanate assessment have been developed to understand differences in the bioavailability of isothiocyanates from supplements or cruciferous vegetable sources.

2. Development and validation of methods for assessing status and bioavailability of nutrients and bioactive food components

Methods are under development to assess nutrient/ bioactive food component bioavailability and/or to validate them in different conditions, such as malnutrition and inflammation, as well as life stage. For example, potassium bioavailability from salts is high and assumed to be high from foods but the effects of the food matrix on K bioavailability is not known. At IN, using accelerator mass spectrometry (AMS) and compartmental modeling for 41Ca, the effect of physiological and dietary interventions on bone calcium retention observed in postmenopausal women are being validated in other life stages. Genetic polymorphisms are being screened as predictors of folate status (UC-D and UC-B). At the end of the project we will have kinetic models of the interactions of single nucleotide polymorphisms (SNPs) on L-5-methyltetrahydrofolate (5-MTHF) versus fatty acid (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 Replication factor C subunit 1 (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.

The primary diagnostic platform employed to measure biomarkers of several nutrient deficiencies is the enzyme-linked immunosorbent assay (ELISA). Although their high sensitivity and staple use in high resource settings, they are laborious, require expensive laboratory equipment and trained personnel, and promote biological waste. At IL, work has been focused on leveraging photonic crystal technology to establish a sensing platform for the detection of iron biomarkers including ferritin and soluble transferrin receptor. Iron oxide nanoparticles were functionalized with antibodies for these biomarkers, which allowed for their selection and rapid separation using a magnet. Once separated, their detection took place on a photonic crystal platform, which was conditioned to detect the complex without any more signals beyond binding. NE has developed new technologies to assess the bioavailability and distribution of milk and egg exosomes and their RNA cargos in mice. These technologies include the use of fluorophore-labeled exosomes and RNAs and species-specific polymerase chain reaction (PCR) protocols.

3. Innovative use of model systems for nutrient and bioactive food component bioavailability

NE has developed an exosomes and cargo tracking (ECT) mouse, which allows to track the trafficking of tissue-specific exosomes (labeled with an endogenously expressed fluorescent protein) among tissues; the mouse also allows to harvest tissue-specific exosomes and assess the effects of physiological and pathological conditions on the cargo of the exosomes. NE is also identifying motifs in dietary microRNAs that predict their bioavailability and tissue distribution. MA has initiated efforts of applying non-vertebrate animal models, such as Caenorhabditis elegans and Drosophila melanogaster, in dietary bioactive compound research. OR has utilized zebrafish models to understand the transgenerational impact of zinc status on developmental processes, susceptibility to environmental stresses and chronic disease. OR is also utilizing machine learning methods to help predict dietary intake patterns from models and pre-existing metabolomic data. KS has used in vitro digestion/Caco-2, rats and chickens models to study iron and vitamin A bioavailability. UC-B has continued to study how genetic polymorphisms and nutrient intake influence biomarkers of vitamin status and one carbon metabolism and the role of genetic polymorphisms in the susceptibility to folate-responsive birth defects.

4. Factors that influence absorption, distribution, metabolism, and excretion (ADME)

A. Subgroup differences (age, gender, race, disease-state)

OH is understanding the influence of obesity-related disorders (e.g. metabolic syndrome, nonalcoholic steatohepatitis) on the bioavailability and pharmacokinetics of vitamin E and green tea catechins. Completion of these studies is expected to provide critical knowledge to guide dietary recommendations to effectively manage these disorders that are characterized by inflammatory stress.

B. Microbiome

NE is taking innovative approaches to assessing the interactions between dietary exosomes (and their RNA cargos) and the gut microbiome, including selection cultures, quorum sensing, delivery of microbial RNAs through bovine milk exosomes, mutational analysis of gut microbiota and interactions of microbial RNAs with Toll-like receptors. OH is also examining the reciprocal benefits of catechins and microbiota composition the generation and absorption of microbial-derived catechin metabolism that are potentially responsible, at least in part, for the anti-inflammatory activity of green tea. In OR, the impact of age on the microbiome and subsequent impact on age-related changes in nutrient utilization is an emerging area of research. PA is assessing complex interaction of dietary bioactive compounds, gut bacteria and the host in low-grade chronic inflammation models to better harness the anti-inflammatory potential of plant foods to promote gut health. PA has also established the perinatal rabbit model of chronic intestinal and liver inflammation to study the anti-inflammatory activity and gut bacterial dysbiosis ameliorating properties bioactive compounds and probiotics. PA also established the pig model to study the gut microbiome during development of low-grade inflammation and intestinal stem cell dysregulation.

C. Environmental interactions

MA has investigated the potential interactive role of environmental contaminants and dietary components on development of obesity and type 2 diabetes. OR is investigating the interaction of micronutrient deficiencies, like zinc with susceptibility to arsenic toxicity. This work is being investigated using in vitro models, in vivo rodent and zebrafish models, and in human populations with high risk for zinc deficiency and arsenic well-water contamination.

D. Nutrient/gene interactions

NE has assessed the microRNA cargos in bovine milk exosomes and chicken eggs. The effects of microRNA cargos on gene expression and the metabolome has been assessed in human peripheral blood mononuclear cells and murine liver, brain, skeletal muscle and placenta in milk and egg (exosome) feeding studies. OR has a strong interest on the impact of zinc deficiency on deoxyribonucleic acid (DNA) integrity and genomic stability, as well as its impact on inflammatory gene expression. Nutrient-epigenome interactions are also being investigated with particular emphasis on bioactive compounds derived from cruciferous vegetables (indoles and isothiocyanates) on DNA methylation, non-coding RNA and histone modifications.

E. Processing

The food and nutrition industry are constantly seeking strategies that result in products containing key nutrients and dietary bioactive compounds, expanding healthy alternatives for specific consumers. Incorporation of nutrients and dietary bioactive compounds into formulations often leads to food deterioration and rapid spoilage, changes in rheology, less favorable sensory characteristics, modified preparation and consumption habits, and interactions with other food components, thereby reducing the intended benefit. Encapsulation has emerged as a promising technology for delivery of many bioactives, nutrients and flavors in in foods. Micro- or nano-encapsulation benefits include the stabilization and protection of labile components, an enhanced functionality of key ingredients, and modification of the release of target compounds. Encapsulation strategies vary due to both the system of dispersal and the bioactive component itself. Despite the different encapsulating strategies using synthetic or natural wall materials for protection and delivery of bioactive food components, their influence on bioavailability is not fully understood, or at best, it is often extrapolated from in vitro or cell studies. IL is using legume-based proteins functionalized after ultrasound and pH shifting treatments to obtain nanoaggregates capable of protecting and dispersing fat soluble compounds. Research has focused on understanding the physico-chemical characteristics of particles with and without oils or other compounds, before and after drying, alone and mixed in foods, and after different phases of in vitro digestion. KS has been looking at the impact of extrusion of food aid product on micronutrient bioavailability. PA has strong interest in novel processing methods to increase the levels of bioactive compounds in food products. For example, we are using color-fleshed potatoes as a model system to study the vacuum frying versus conventional frying on the bioactive compounds in fried chips.

Bioavailability of nutrients and bioactive food components

1. Vitamins

A. Folate and Vitamin B12

Folate and vitamin B12 function 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. Erythrocyte folate is the best indicator of folate status because it reflects the intracellular pool. Current knowledge of folate bioavailability is based largely on research conducted by W3002 scientists. UC-B continues to evaluate genetic risk factors for neural tube defects and to identify putative modifier genes which influence folate status, homocysteine levels, and methylation potential using a number of mouse strains and a cohort of students at Trinity College, Dublin.

Methylmalonic acid (MMA) is a by-product of propionic acid metabolism through the vitamin B12 (cobalamin)-dependent enzyme methylmalonyl CoA mutase. Elevated MMA concentrations are a hallmark of several inborn errors of metabolism and indicators of cobalamin deficiency in older persons. In a genome-wide analysis of 2,210 healthy young Irish adults (median age 22 years) UC-B identified a strong association of plasma MMA with SNPs in 3-hydroxyisobutyryl-CoA hydrolase (HIBCH) and acyl-CoA synthetase family member 3 (ACSF3). These loci accounted for 12% of the variance in MMA concentration. The most strongly associated SNP (HIBCH rs291466) causes a missense change of the initiator methionine codon to threonine. Surprisingly, the resulting variant is associated with increased expression of HIBCH mRNA and encoded protein. These homozygotes had, on average, 46% higher MMA concentrations than methionine-encoding homozygotes in young adults with generally low MMA concentrations. The association between MMA levels and HIBCH rs291466 was also highly significant in a replication cohort of 1,481 older individuals (median age 79 years) with elevated plasma MMA concentrations (0.34 [0.24–0.51] mmol/L. Additionally, in a longitudinal study of 185 pregnant women and their newborns, the association of this SNP remained significant across the gestational trimesters and in newborns.

B. Vitamin E

Vitamin E continues to be a nutrient of public health concern. Indeed, greater than 90% of Americans fail to meet the estimated average requirements for dietary vitamin E (Maras et.al., 2004), and populations characterized by high a magnitude of inflammation are especially susceptible to inadequate vitamin E status. Therefore, continued studies at OH are aimed at assessing vitamin E absorption, distribution, and metabolism in healthy individuals and cohorts of increased chronic disease risk. OH 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 in adults with metabolic syndrome. 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

C. Vitamin A

Vitamin A is one of the most common micronutrients deficiencies particularly in low income countries with little access to animal source foods. KS is examining the bioavailability of vitamin A from food aid products made from different commodities such as corn, soybean, sorghum, and cowpea.

2. Minerals

A. Calcium and Vitamin D

Standard of care for people with low bone mass or of fracture is to ensure adequate calcium and vitamin D. Research by W3002 investigators continued to study these nutrients, but increasingly many dietary factors are associated with bone health and interest related to cardiovascular health will be more prominent in the next 5 years. A large RCT of vitamin D supplementation in adolescent white and black pubertal boys and girls from a Northern and Southern site showed that vitamin D status increased with dose of vitamin D supplementation, but was not associated with any functional benefit including calcium absorption or muscle strength (IN). Use of prebiotics to enhance calcium absorption and bone calcium retention was shown to help correct the common dietary calcium deficiency in adolescents and postmenopausal women.

B. Potassium

Fruits and vegetables add minerals including potassium to the diet which has been associated with improved blood pressure and bone health. The first work on bioavailability of potassium from food sources was begun in IN.

C. Iron

Iron is one of the most common micronutrients deficiencies worldwide. KS is examining the bioavailability of iron from food aid products made from different commodities such as corn, soybean, sorghum, and cowpea.

D. Zinc

Zinc deficiency is a highly prevalent deficiency across the world, yet there is still no reliable human biomarker for zinc deficiency. Research in OR and OK have established that damage to DNA is one of the early hallmarks of zinc deficiency in humans, that precedes decreases in zinc plasma zinc. Zinc deficiency has an important impact health, affecting processes important in growth and development, bone health, immune function, cancer prevention and many others. Work in OR and OK in zinc nutrition and metabolism examines factors that affect bioavailability and health parameters in both model systems and in populations at risk for zinc deficiency. Work at MT has led to a soil strip of seeds that each detect a different soil deficiency for smallholder farmers and zinc, iron, selenium, sulfur soil deficiencies are all detectable by such an inexpensive assay. Field trials are slated for the coming year.

3. Bioactive Food Components

A. Polyphenols - Green tea catechins, flavonoids, anthocyanins, stilbenoids, cranberry phenolics

In progress studies at OH 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. Continued studies in this area in OH 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 extracts on lipid absorption. Collaborative intellectual interactions among investigators working with tea research should promote greater understanding of the potential use of tea and tea polyphenols in obesity prevention. Work in MA station indicated that stilbenoids, such as resveratrol and piceatannol, often found in grapes, and cranberry phenolics reduced fat accumulation and extend lifespan in C. elegans. Other polyphenol rich sources soy, plum, and blueberries were studied for their protective role in ameliorating bone loss (IN) and/or obesity (MA).

PA found that purple-fleshed potato extracts, which are high in anthocyanins, decrease in vitro human colon cancer stem cell proliferation, elevate apoptosis and suppresses cytoplasmic and nuclear levels of β-catenin, the critical downstream effector of Wnt pathway. We have also shown that purple-fleshed potatoes, even after baking, significantly suppress colon tumor number, colonocyte proliferation (ki-67; immunofluorescence) and concomitantly suppressed nuclear beta-catenin expressing oncogenic stem cells (lgr5 positive) in rodent-model of colon carcinogenesis. Recently PA found that only purple-fleshed potatoes, even after processing, prevent and reverse high-fat diet induced colonic-systemic inflammation and oxidative stress markers in pig model.

B. Carotenoids

Serum carotenoids are associated with dietary fruit and vegetable intake. These carotenoids are the plant pigments that are responsible for the vibrant orange, yellow and green colors of cantaloupe, carrots, sweet potato, sweet red peppers, broccoli and green leafy vegetables. PA station found that α- and β-carotenoid concentrations were inversely correlated with obesity measures and triglyceride levels and positively correlated with high density lipoprotein (HDL) (good cholesterol) levels in Mexican-American youth (n = ~570). In addition, the investigators found that the inverse correlations between β-carotene and obesity-related traits and the positive correlation between β-carotene and HDL-C levels may themselves be influenced by genetic factors.

C. Nitrates/isothiocyanates/indole-3-carbinol

Isothiocyanates and indoles derived from cruciferous vegetables such as sulforaphane and indole-3-carbinol have been studied extensively for their potential health benefits including cancer prevention, anti-inflammatory activity and immune dysfunction (Ho et al., 2011). However, the bioavailability of these compounds and their metabolites in humans, especially to target tissues, is relatively unknown. Researchers in OR developed sensitive mass spectrometry methods to better understand the distribution of sulforaphane and indole-3-carbinol and their respective metabolites using pre-clinical models and human subjects. Researchers have also studied effects of whole foods versus supplement sources and have recently found that supplements that lack myrosinase, a key enzyme the helps release sulforaphane, markedly decreases bioavailability of the compounds. Several cancer clinical trials are also underway to test the effects of sulforaphane supplementation. Factors such as polymorphisms in metabolizing enzymes such as glutathione-S-transferases and the gut microbiota may also alter bioavailability and determine individual responses to these phytochemicals (Clarke et al., 2011). As fruit and vegetable consumption are associated with enhanced indices of bone health across the lifespan, W3002 scientists have evaluated the efficacy of dietary nitrate to contribute to bone growth or slow bone loss after surgical ovariectomy in a female rat model system (OR). While ovariectomy in these rats altered the plasma metabolome and fecal microbiome, it was demonstrated that dietary nitrate has no effect on indices of bone health or the community structure of the microbiome in young or ovariectomized rats (Conley et al. 2017). Research at MA found that 3,3’-diindolylmethane, a major metabolite of indole-3-carbinol, inhibits adipogenesis and reduces overall fat accumulation in C. elegans. Research in this area is important for determining optimal dietary recommendations for disease prevention.

D. Bioactive fatty acids (n-3 or conjugated linoleic acid)

MA has investigated the tools to improve bioavailability and/or stability of bioactive fatty acids, such as fish oil and conjugated linoleic acid. This includes application of antioxidant and/or encapsulation to protect polyunsaturated fatty acids and/or improving bioavailability on obesity and aging. Work in MT has led to selective breeding of Camelina sativa varieties that are low in glucosinolates and high in omega 3 fatty acids and high in gamma-tocopherol and these have been released to a food company in Illinois that is testing their desirability in food processing and as chicken feed for high omega-3 egg production.

E. Exosome and RNA cargos

NE has discovered that exosomes and their RNA cargos do not exclusively originate in endogenous synthesis but can also be absorbed from foods such as bovine milk and chicken eggs.

Factors that modulate absorption and metabolism

1. Identification of susceptible populations- biomonitoring, biomarkers

Identification of populations suffering from micronutrient deficiencies is of critical importance and is one of the most significant limitations that has hindered nutrition, health improvement and human development in low- and middle-income countries. Millions of families in villages, towns, and cities around the world lack ready access to health care and the sophisticated laboratories that produce reliable diagnoses in wealthy countries. In poor countries, health care facilities are mostly centralized, but can be far away, serving widely dispersed populations. Specialized equipment, personnel, and safe waste-disposal systems are simply not there. Currently, a point-of-care, easy-to-use, low-cost diagnostic tools to quickly and accurately determine micronutrient status of at risk populations does not exist. Most tests use ELISA type determinations that are expensive and impractical considering the high cost of equipment (reader, glassware), use of unavailable resources in the field (electricity) and highly trained personnel (nurses). Moreover, the World Health Organization has supported the use of ASSURED (affordable, sensitive, specific, user-friendly, rapid and robust, equipment-free, and deliverable to end-users) criteria to guide scientist in the development of technologies that better address the needs of populations living in low- and middle-income countries. At IL, scientist and engineers are developing sensing prototypes to detect and quantify biomarkers of micronutrient status. Work has also been focused on the development and validation of assays for the detection of nutrients in foods.

A. Environmental factors

Investigation at MA has revealed that high fat diet may potentiate absorption of lipid-soluble environmental contaminants, thus aggravating high fat diet induced responses in animals.

B. Optimizing delivery systems

Nanotechnology continues to be an innovative and rich research area with tremendous potential for designing foods of superior quality, that are safe for human consumption, and that enhance nutrition and health. While nanoparticles are ubiquitous in nature and our food supply, the directed use of nanotechnology in foods is yet to reach its potential and limitations exist. Micro- and nano-encapsulation have received significant attention as feasible strategies to increase product functionality, facilitating delivery of key nutrients and bioactives. Nanoemulsion systems could prove useful to provide stability and facilitate delivery of critical nutrients via innovative foods to at-risk and frail populations. Among suitable nano-materials, legume proteins offer significant advantages within formulations but new technologies must undergo rigorous physical and chemical characterization with further stability, efficacy, and sensory evaluation before their widespread use in food applications.

C. Nutrient-Gene interactions

Research in NE has shown that dietary depletion of exosomes and their RNA cargos elicits phenotypes such as impaired spatial learning and memory, loss of fecundity, changes in the gut microbiome, aberrant purine metabolism, changes in immune function, and changes in the hepatic and muscle transcriptome. These phenotypes are consistent with effects of dietary exosomes in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways in human peripheral blood mononuclear cells and murine liver, brain, skeletal muscle and placenta and milk/egg (exosome) feeding studies.

The ability to study complex food/metabolic interactions that determine nutrient/phytochemical availability is an additional strength of W3002. Previous work in OH and IN have established the role of salivary enzymes and gut microbes on phytochemical metabolism and bioavailability. Studies in OR and CO have also established differences in bioavailability from whole food sources compared to individual supplements (Hsu et al., 2010 and Clarke et al., 2011), and have determined that food processing also alters phytochemical bioavailability, respectively.

Summary

We have also included specific expertise each station member brings to this collaborative effort (Table 1). A summary of proposed collaborations and interactions are listed (Table 2). This W3002 team is a unique group of researchers with an interest in advancing the science in defining the role of dietary bioactive compounds for optimal human health. This group includes experts in a wide variety of nutrients and dietary bioactive components – 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. The value of collaborative activity of the W3002 Multi-state group has allowed its members, or teams they are apart of, to obtain more than $54 million in extramural funding during the W3002 period.

Objectives

1. Determine the bioavailability (absorption, distribution, metabolism, elimination) of nutrients and bioactive food components.
   
2. Determine the efficacy and mechanisms of action of nutrients and dietary bioactive compounds towards improved health.
   

Methods

Objective 1: Determine the bioavailability (absorption, distribution, metabolism, elimination) of nutrients and bioactive food components.

1. Absorption and Metabolism Modeling

W3002 scientists have developed state of the art techniques for assessing all components of bioavailability and metabolism for several nutrients and food components. These techniques address the interactions between the gut microbiome, and nutrient bioavailability and metabolism. Novel methodologies include use of rare and stable isotopes for calcium, and 15N-labelled nitrate and nitrite and vitamins such as folate (UC-D) and vitamin E (OH). The use of these isotopes will allow W3002 scientists to precisely monitor each nutrient, its uptake, distribution into plasma and elimination in urine & feces across different life stage and physiological conditions, including physical activity. W3002 scientists can examine alterations in nutrient metabolism during different physiological conditions, including malnutrition, inflammation, and physical activity. Researchers in UC-B together with UC-D investigated the influence of folate and vitamin B12 status in our experimental animals including some novel mouse models that recapitulate some of the symptoms and metabolic disturbances observed in human B12 deficiency. Some of the proposed studies in zebrafish will allow for the quantification of effect of gut microbial metabolism on the physiological effects of dietary polyphenols. NE will study the absorption and distribution of bovine milk exosomes and their RNA and protein cargos in mice and humans with a particular focus on the blood/brain barrier and placental transfer.

This group has developed sophisticated approaches to understanding transport rates, pool sizes, and metabolism parameters using isotopic tracers, kinetic modeling and compartmental analysis as well as statistical models. This approach will continue to be used to understand metabolism of vitamin, mineral and bioactive compound (IN, OR). For example, potassium bioavailability is being studied from foods for the first time and used kinetic modeling to compare dose and source of potassium from potatoes and a salt on parameters of metabolism (IN). One novel approach using isotopic tracers is the use of the rare isotope, 41Ca, measured by Accelerator Mass Spectrometry to determine net bone calcium retention during interventions and age-related bone loss (IN). Modeling will be used to predict outcomes and design experiments to test predicted outcomes.

OH will also examine inflammation and host-microbial interactions on green tea catechin bioavailability. Using newly established LC-MS procedures, studies in adults with NASH will examine pharmacokinetic parameters of green tea catechins compared with healthy adults, and the role of gut microbiota composition in regulating the generation of microbial-derived catechin metabolites that are potentially responsible for the anti-inflammatory activities of green tea. Studies will also consider time-dependent changes in catechin pharmacokinetics following chronic ingestion along a trajectory of clinical translation of an anti-inflammatory dietary strategy to address the growing concern of metabolic disorders on human health outcomes. Similar studies by OH are also planned to (re)define human dietary requirements of vitamin E (alpha-tocopherol) and novel recommendations for specialized cohorts including those with metabolic syndrome and NASH. These studies will leverage deuterium-labeled alpha-tocopherol in conjunction with LC-MS analytical approaches to assess dose-response relationships of alpha-tocopherol on vitamin E status, specifically by evaluating alpha-carboxyethyl-hydroxychromanol (alpha-CEHC). This novel biomarker of alpha-tocopherol status is generated by the liver in cytochrome P450-dependent in response to adequate hepatic vitamin E status.

2. Development and validation of methods for assessing status and bioavailability of nutrients and bioactive food components

Methods are under development to assess nutrient/ bioactive food component bioavailability and/or to validate them in different conditions, such as malnutrition and inflammation, as well as life stage. The effect of dietary and other interventions on bone calcium retention using 41Ca developed for postmenopausal women are being validated in other life stages (IN). Genetic polymorphisms are being screened as predictors of folate and B12 status, and as modifiers or confounders of bio-markers of nutritional status (UC-D and UC-B).  Importantly, many of the goals proposed in Objective 2 are translational studies performed in human subjects, which is a major strength of this project.

At IL, scientists are leveraging the functionality of photonic crystal biosensors for the determination of biomarkers of iron deficiency (i.e. ferritin and soluble transferrin receptor). Studies will be focused on enhancing the number of assays (e.g., retinol binding protein, C-reactive protein) conducted on a single drop of sample, which can be easily measured with the use of a reading apparatus based on smartphone technology, as well as their validation by measuring total allowable error (systematic and random) using established clinical cutoffs. Using the same development paradigm, focus will be also given to the development of low-cost applications (paper based assays) for the determination of micronutrients (e.g., Fe, Zn, vitamin A) in fortified foods. These sensing applications will be useful of monitoring efforts of populations at risk of micronutrients deficiencies as well as of the quality of foods.

At OH and MA use a simulated digestion system to evaluate phytochemical bioacessibility. Using this model that mimics the oral through small intestinal phases of digestion to predict micellarization, intestinal uptake, and bioavailability. Collaborative efforts between these stations will leverage this model system to assess bioaccessibility of lipophilic nutrients, polyphenols, and bioactive lipid mediators. KS sent a graduate student to OH to learn these methods during the last period so that they could be applied to using the model to determine vitamin A and iron bioavailability. Rats have been criticized as a model for iron bioavailability, but work in KS has found rats to be more advantageous than believed by some. Thus, the plan is to continue to use this model and discuss their relevance compared to other commonly recommended model systems.

Zebrafish models are a premier model utilized by toxicologists to study development, but has been under-utilized to examine the impact of dietary compounds on developmental and transgenerational processes. Zebrafish will be utilized to examine the influence of bioactives such as zinc, dietary nitrate and polyphenols across the lifespan in OR. For polyphenols, these studies will address the hypothesis that members of the gut microbial community catalyze the production of specific, water-soluble metabolites called urolithins from dietary ellagitannins (OR).

The mechanism behind the irreversible demyelination disease (sub-acute combined disease) of vitamin B12 deficiency is unknown because of the lack of a convenient animal model. Although monkeys sometimes display the same symptoms as humans, vitamin B12-deficient rodents do not develop demyelination disease. Researchers at UC-B have developed new gene knockout mouse models that develop megaloblastic anemia and that are impaired in myelin synthesis when fed 5-methyl-folate in their diet. These novel mouse models will be used to investigate the mechanism behind the severe demyelination disease of B12 deficiency. They should also prove useful in investigating some the adverse effects reported in the literature for high intakes of folic acid (increased cancer, asthma, etc).

Owing to similarity in digestive physiology and brain morphometry and comparable brain growth dynamic, the domestic pig had been recognized to be a valuable model for pediatric nutrition and neurodevelopment (Odle et al., 2014; Conrad and Johnson, 2015). More research will be continued at UC-D to validate the pig model with pre-weaning and post-weaning pigs and to evaluate the efficacy of dietary bioactive components and elucidate their mechanisms of action. AZ will use a rodent model to address whether the increase in overall feeding in response to environmental noise exposure is due to a compensatory reduction in absorption or metabolism.

MA will use two small animal models to determine the role of dietary bioactive compounds, particularly in aging, obesity, and/or diabetes; Caenorhabditis elegans and Drosophila melanogaster. C. elegans is a small free-living nematode that lives in temperate soil environment, which has been widely used in research involving obesity, aging, and neuro-degenerated disease. Drosophila is a fruit fly used in various biological research, including obesity, diabetes, and aging. Use of these two small animal models will help determine the bioactivities of dietary bioactive compounds on obesity, aging, and/or diabetes in a timely manner (especially for aging study) and saves use of vertebrate animals.

NE will develop a PCR-based technology that allows to distinguish bovine and human microRNAs that differ by as little as one nucleotide. NE will also develop the following transgenic mice to study the bioavailability of bovine milk exosomes and their RNA and protein cargos: 1) microRNA sensor mice (to assess the tissue delivery of microRNAs), 2) TSG101 conditional knockout mice to study the re-packaging of cargos from milk exosomes into endogenous exosomes, and 3) exosome and cargo tracking mice, which will allow to study the trafficking and cargos of exosomes in murine milk and tissues. The mouse will be used to assess roles of exosomes in obesity and colon cancer, obesity and heart disease, and substance abuse disorders.

3. Factors that influence ADME

Subgroup differences (age, gender, race, disease-state)

Gender and race differences in response to dietary patterns that alter potassium, magnesium, calcium, and sodium on mineral metabolism will be evaluated in adolescents (IN). Gender differences in metabolism of blueberry phenolics is being investigated in IN. Elderly individuals have high susceptibility to micronutrient deficiencies. Researchers in OR will examine age-related differences in zinc absorption and cellular transport in aged mice and zebrafish, and their impact on inflammation and immune function.

Microbiome

The influence of age-related alterations in the microbiome on response to bioactive supplementation and susceptibility to stress will be examined in OR using mouse models (OR). We propose using the zebrafish model to determine if dietary elagitannins, rich in pomegranate and walnuts, improve the efficiency of muscle contraction (as evidenced by lower oxygen cost of physical activity) via the production of specific water-soluble metabolites by the gut microbiome. These studies will involve interrogating the effects of a dietary pomegranate extract on the brain and plasma metabolome, as well as the fecal microbiome, and their association with performance and behavioral phenotypes. The influence of dietary blueberries, dietary patterns, and pre-biotic fibers on the gut microbiome in rodent models and humans is being evaluated in IN.

NE will study the effects of exosomes in bovine milk on the microbiome at four levels: 1) changes in microbial communities, 2) selection of microbes in murine and human fecal samples in exosome-defined minimal media, 3) selection of mutations in microorganisms in exosome-defined minimal media, and 4) the roles of microbial RNA cargos in bovine milk exosomes. MA will also conduct the study determining the role of microbiome in metabolisms of bioactive compounds and their bioactivities using a rodent model.

Environmental interactions

Susceptibility to environmental toxins such as air and water pollutants may influence bioavailability and absorption of micronutrients such as zinc. In OR we will utilize cell culture and animal models (mouse and zebrafish) to examine the impact of polycyclic aromatic hydrocarbons and arsenic exposure on zinc metabolism and transport. It is unknown whether noise pollution, which reduces sleep, increases risk for chronic disease and reduces brain function by modifying nutrient bioavailability equally across gender and lifespan. Work in AZ will use an animal model (rat) to test whether sleep loss due to noise exposure reduces memory and increases weight gain by reducing nutrient bioavailability important for brain function. Along with other factors, persistent environmental contaminants have been positively associated with development of chronic disease, particularly obesity and type 2 diabetes. In addition, it was recently reported the interaction of dietary fat and low doses of pesticides on development of obesity and insulin resistance. MA will further test the role of bioactive dietary compounds in prevention of pesticide-induced dysregulation of lipid and glucose metabolisms.

Nutrient/gene interactions

Gene polymorphisms that affect nutrient flux through metabolic pathways may well be important determinants of nutrient requirements for optimal health. Initially, folate and vitamin B12 will be further studied (UC-D and UC-B). UC-B scientists are studying the influence of common genetic polymorphisms as confounders of biomarkers of vitamin status. Effects of nutrients (i.e. amino acids) and dietary supplementation of other bioactive food components on the expression of genes related to gut and brain health will be evaluated at UC-D. The influence of polymorphisms in glutathione-s-transferases and cytochrome P450, and non-coding RNA expression on bioavailability of phytochemicals found in cruciferous vegetables will also be examined in OR. NE will focus on exosome/RNA-dependent gene pathways that play roles in spatial learning and memory and purine metabolism and pathways that link the gut microbiome with human metabolism. Work in MT has progressed along the line of selection of high free lysine cultivars of spring wheat and potatoes, and these are now in field testing.

Processing

A number of processing techniques and approaches can be used to potentially improve bioavailability. For instance, extrusion is a technique that may improve the bioavailability of minerals by decreasing antinutritional factors. In KS, this technique will be leveraged to produce products with increased bioavailability. Sorghum and cowpea, which are typically viewed as inferior commodities, when processed might be equal or more effective for use in certain applications. Activated charcoal filtration has been demonstrated by a MT scientist to remove patulin (a carcinogen produced by Aspergillus flavus) from apple cider and this process is being adopted on large scale by the cider industry.

Nutrients and bioactives with low polarity require dispersion in biliary micelles before absorption in the small intestine. Reduction and homogenization of particle size of different bioactive molecule formulations can enhance their incorporation into biliary micelles, and thus, their absorption in the gut. This enhanced absorption could reduce the amount of bioactive used in food formulations, which instead saves resources, reduce negative effects of bioactives on important food attributes, and limit the potential for toxicity. Scientists at IL will continue with the design and evaluation of different encapsulation and emulsification techniques aimed at enhancing the bioaccessibility and bioavailability of nutrients and bioactives. Chemical and physical processes such as pH shifting, high power ultrasound, microfluidization, freeze and spray drying will be used to design products with superior functionalities, especially for the incorporation of non-polar molecules such as vitamins A, D, and E, resveratrol and lutein.

Objective 2. Determine the efficacy and mechanisms of action of nutrients and dietary bioactive compounds towards improved health.

Obesity and metabolic diseases

CVD

Specific dietary patterns are associated with either improved or diminished cardiovascular function. In adults, sodium reduction and the Dietary Approaches to Stop Hypertension (DASH) style diet that is rich in fruits, vegetables, dairy, fish, nuts, legumes and low in added fats and sugars are associated with lower blood pressure and blood lipids. These dietary interventions will be tested in adolescents when antecedents for CVD are being established (IN). Human consumption of dietary nitrate primarily comes from vegetables in clinical studies, with reduced blood pressure and decreased the oxygen cost of exercise in humans. W3002 scientists will use the zebrafish model system to elucidate the physiological determinants of improved efficiency of muscle contraction during exercise caused by dietary nitrate (OR). Additional work in zebrafish is proposed to determine of dietary elagitannins, rich in pomegranate and walnuts, improve efficiency of muscle contraction during exercise via the production of metabolites, such as urolithins, by the gut microbiome. MT has released a new cultivar of Camelina sativa selected for low glucosinolate and high omega-3 (ALA) content, and it is being produced by an Illinois based food processing company for the value-added aspect of its extracted oil. Given omega-3 consumption is associated with decreased CVD mortality.

OH is a leader in conducting postprandial studies examining acute hyperglycemia-mediated impairments in vascular endothelial function (VEF). Clinical studies show that postprandial hyperglycemia (PPH) transiently impairs VEF in an oxidative stress-dependent manner that decreases nitric oxide bioavailability, and that dietary interventions can either effectively limit the hyperglycemia stimulus and/or downstream oxidative stress responses that otherwise impair VEF. Planned studies in normoglycemic adults and those with prediabetes will utilize ultrasound-based flow-mediated dilation of the brachial artery to examine bioactive proteins of dairy foods and eggs and dietary phytochemicals (vitamin E, green tea catechins) in regulating postprandial VEF. These studies aim to establish evidence-based recommendations to prevent the accumulation of transient insults to the vascular endothelial that would be expected to contribute to long-term CVD risk.

Cancer

Cruciferous vegetable intake has been associated with decreased incidence of both prostate and breast cancer. OR researchers are examining the impact of bioactive compounds derived from cruciferous vegetables on genetic and epigenetic mechanisms leading to suppression of cancer cell growth. Clinical and biological samples from the clinical trials using broccoli sprout extracts in OR are being used to evaluate the role of cruciferous vegetables and their constitutive bioactive food components (OR) in reducing breast cancer recurrence and prostate cancer risk. In collaboration between OR and OK, we are also examining the impact of zinc status on DNA integrity in populations susceptible to zinc deficiency.

Obesity

A series of cultivars of naked oats have been developed, MT, originally aimed at the gluten free market, however their high protein (20-22% as compared to rice at 8% protein), low glycemic index, lack of arsenate, and low pesticide input (if any) make them an attractive substitute for rice in diets where diabetes is a concern. Production is being ramped up from 1,000 acres in Montana, for domestic and international markets. NE has developed a transgenic mouse that will allow to track study the trafficking of endogenous exosomes from adipose tissue to the colon and mammary glands, and collect the exosomes for cargo analysis. The mouse will be used to study the effects of obesity on colon and breast cancer, and the role of exosomes in increasing cancer risk in obesity.

MA will study the role of dietary bioactive compounds in the development of obesity along with determining the markers of aging using two small animal models, Caenorhabditis elegans and Drosophila melanogaster. These will include determining the role of compounds on adipogenesis for obesity and lifespan for aging including physical activities (as energy expenditure for obesity and healthy life span for aging). AZ will use rodent models to test whether environmental factors modify obesity risk and worsen cognition by increasing efficiency of energy utilization and reducing sensitivity to neuropeptides that promote memory and normal energy homeostasis.

Diabetes, Metabolic Syndrome, Fatty Liver Disease

OH has been active investigating anti-inflammatory activities of green tea catechins in obese models of NASH. Evidence suggests that green tea catechins attenuate hepatic NFkB-mediated liver injury through a mechanism involving the gut-liver axis that limits gut-derived endotoxin translation to the portal circulation to prevent hepatic TLR4/NFkB activation to protect against NASH. OH will lead collaborative efforts to conduct studies examining green tea catechins in modulating gut microbiota composition and metabolomic responses that otherwise disrupt enterocyte signaling leading to impaired tight junction protein expression. Translational studies in humans will also examine the extent to which controlled feeding of green tea regulates gut health in relation to improved microbiota composition using non-invasive probes that effectively evaluate gut barrier permeability. Metabolomics studies in both preclinical and clinical models are expected to identify catechins and/or microbial-derived catechin metabolites that are functionally responsible for improvements in gut health in relation to attenuating hepatic inflammatory responses leading to hepatic injury. Six new cultivars of potatoes selected for their resistant starch characteristics, traits  affecting their rate of digestion, have been developed for consumers (after the Carisma model in Australia) and one cultivar is being grown in experimental community demonstration plots on tribal reservations in MT, because of their interest in diabetes and obesity mitigation at the food level.

Gut Health

CA-D and MA will focus on determine the impact of  dietary bioactive components, such as, plant extracts, on gut health and physiology with in vitro cell culture models and pig as well as rodent models. Dual benefits will be generated from this work for utilizing the bioactive food components to improve both animal and human gut health. At OR, scientists are investigating whether members of the gut microbiome in zebrafish hydrolyze ellagitannins from pomegranates to urolithins. Urolithins are associated with anti-inflammatory, improved vascular function, lowered blood pressure, and increased efficiency of muscle contraction. NE will study the effects of exosomes in bovine milk on the microbiome at five levels: 1) changes in microbial communities, 2) selection of microbes in murine and human fecal samples in exosome-defined minimal media, 3) selection of mutations in microorganisms in exosome-defined minimal media, 4) quorum sensing, and 5) the roles of microbial RNA cargos in bovine milk exosomes.

MT has developed a cultivar of barley (from an Ethiopian landrace population of barley) that shows an unusually high level of resistance to plant viruses, and it appears to be active in curing bacterial plasmids as evidenced by the mitigation of scours syndrome in lambs and calves. As plasmids are a source of toxin, intestinal binding and antibiotic resistance genes, this “remove the messenger” approach is a novel way to tackle the problem of constantly emerging antibiotic resistance.

Inflammation

Chronic inflammation is a common precursor to many chronic disease states. W3002 researchers are examining the impact of bioactive food components on mitigating inflammatory processes. OR is examining the interaction among the microbiome, inflammation and zinc status. A particular focus is on identifying bioactive food components to mitigate age-related chronic inflammation in mouse models and in humans. More research will be conducted at UC-D and MA to examine the influences of bioactive components on gut and systemic inflammation caused by infectious diseases. Research will focus on the regulation of both gut microbiome and immunity. NE will study the activation of toll-like receptors (TLRs) receptors by RNAs, encapsulated in bovine milk exosomes, in murine TLR reporter cells and TLR reporter mice; the latter will be challenged with influenza A virus to elicit a strong TLR response.

Malnutrition

The immediate cause of malnutrition includes an inadequate nutrient intake or a specific disease condition that limits the consumption, absorption and utilization of nutrients. In low-resource settings, soil-transmitted helminths (STHs) and food and waterborne protozoans (FWPs) are among the most widespread infectious agents afflicting millions of people worldwide, particularly in marginalized, low-income and resource-constrained regions. At IL, scientists will focus on the design, development and evaluation of enhanced food formulations for emergency relief. These new food formulas will provide critical nutrients (e.g., omega-3 polyunsaturated fatty acids (PUFAs), through enhanced blends of nutrients of superior quality and absorption profile, while directly addressing STHs and FWPs infections through the functional dispersion of bioactive compounds. This work will transform the ability of world agencies (USAID, WHO, WFP, FAO) to better address undernutrition during emergency episodes or for food supplementation programs. In KS, work will focus on determining the bioavailability of iron and vitamin A from different food aid products such as fortified-blended foods, fortified rice, and couscous type products. Results from this work should better inform food aid providers and distributors. MT scientists have released a high lysine spring wheat cultivar and it currently is being produced in field increase plots for determination of milling and baking and nutritional qualities. Tests are being done by a major grains processing company with an interest in the nutraceutical niche market. UC-D will keep exploring high quality protein food ingredients, which will help to reduce hunger and malnutrition related to protein deficiency.

Neurological Health

Aging, specific morbidities, dietary patterns and specific bioactive food components have been associated with improved or decreased brain function, as evidenced by changes in cognition, memory and learning. W3002 scientists propose to study dietary compounds, including zinc, iron, nitrate and polyphenols, in C. elegans model (MA), the zebrafish model (OR), rodent model (AZ), and pig model (UC-D) to determine the effect on behavior, learning, memory and various other measures of cognitive function. These studies will investigate the effects of these dietary bioactive compounds on the brain and plasma metabolome, as well as the fecal microbiome, and their association with these behavioral phenotypes and physical performance. NE will study the transfer of bovine milk exosomes and their RNA cargos across the blood-brain barrier and their roles in spatial learning and memory, and prevention of seizures.

Impairments of folate-dependent one carbon metabolism, caused by gene disruptions, genetic variants, and nutritional deficiencies have been implicated in vascular disease. Although the initial case-control studies suggesting that mild hyperhomocysteinemia may be a risk factor for CVD have not been supported by RCTs, other studies support a role for hyperhomocysteinemia in the development of cerebral-vascular disease including stroke. Folate has also been shown to retard memory loss in the elderly. Studies at UC-B will continue their studies on the relationship of UCB genetic studies on spina bifida families and, more recently, on a large, healthy young cohort (Trinity Student Study) have suggested novel biomarkers for assessing vitamin status and how common genetic polymorphisms may influence these biomarkers.

Bone

At the CA-B station, more research will be conducted to determine the effects of nutrients and other bioactive food components on digestibility of Ca and P and on modulating the concentrations of osteoblast-related markers in plasma and the expression of genes related to transcellular Ca absorption in the small intestine and kidneys. 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. Scientists are quantifying mineral and their ratios from National Health and Nutrition Examination Survey (NHANES) and risk of blood pressure and indices of bone health (IN).

Measurement of Progress and Results

Outputs

• Additional collaborative projects
• Research data
• Peer-reviewed publications
• Presentations and posters at scientific meetings
• Data sharing
• Outreach presentations or publications for non-scientific organizations
• Annual meeting
• Students advised and postdocs trained

Outcomes or Projected Impacts

• Establish models of bioavailability for bioactive food components involved in improved health, including lysine, iron, zinc, calcium, potassium, vitamin D, B vitamins, vitamin E, vitamin A, exosomes, RNA and plant compounds such as nitrate, flavonoids and isothiocyanates.
• Collaborating researchers will have access to shared datasets in order to test hypotheses related to the role of our targeted bioactive food constituents
• Collaborations to translate in vitro and animal studies to human populations
• Collaborative support in grant development to assure optimal research design particularly in regard to bioactive food constituent measurements
• Shared methodologies regarding measurement of health outcomes and improved human health
• Support for the development of outreach materials that translate research findings for broad based consumption and improved healthcare
• Increased knowledge of the bioactivity of nutrients and dietary bioactive components and their underlying protective mechanisms
• Increased knowledge of biomarkers and determinants of ADME of dietary bioactive components
• Development and publication of models for determining bioactivity and bioavailability of nutrients and other dietary bioactive components
• Building a highly skilled workforce in human nutrition

Milestones

(2019):♦ Report on research activities and discuss collaborative and synergistic opportunities ♦ Submit a conference symposium proposal ♦ Publish peer-reviewed original research articles, review articles, and book chapters. Increase the number of open access journal articles. ♦ Disseminate research findings in seminars, at conferences, and through presentations to professional organizations and the general public. ♦ Train new nutrition/food scientists

(2020):♦ Report on research activities and discuss collaborative and synergistic opportunities ♦ Publish peer-reviewed original research articles, review articles, and book chapters. Increase the number of open access journal articles. ♦ Disseminate research findings in seminars, at conferences, and through presentations to professional organizations and the general public. ♦ Train new nutrition/food scientists

(2021):♦ Report on research activities and discuss collaborative and synergistic opportunities ♦ Publish peer-reviewed original research articles, review articles, and book chapters. Increase the number of open access journal articles. ♦ Disseminate research findings in seminars, at conferences, and through presentations to professional organizations and the general public. ♦ Train new nutrition/food scientists

(2022):♦ Report on research activities and discuss collaborative and synergistic opportunities ♦ Publish peer-reviewed original research articles, review articles, and book chapters. Increase the number of open access journal articles. ♦ Disseminate research findings in seminars, at conferences, and through presentations to professional organizations and the general public. ♦ Train new nutrition/food scientists

(2023):♦ Evaluate the accomplishments of the collaborative efforts of the project. ♦ Determine new goals and prepare a proposal to continue collaborative efforts. ♦ Publish peer-reviewed original research articles, review articles, and book chapters. ♦ Disseminate research findings in seminars, at conferences, and through presentations to professional organizations and the general public. ♦ Report on the success and placement of trained students and postdoctoral research associates

Projected Participation

View Appendix E: Participation

Outreach Plan

All members of this multistate group will engage in various outreach activities. The type of activity will depend on each member’s expertise and environment. For example, some members will make presentations to professional organizations and the lay public, or will have appearances on local TV and radio stations. Other members will offer research and education opportunities for high school students and teachers. All members will disseminate their research findings through publications in science journals, presentations at science meetings, and through invited seminars. Group members will adhere to the practice of making manuscripts available in the public domain no later than 12 months after publication.

We also plan to invite extension specialist to our next meeting scheduled to be held in Arizona, so that they can learn about the research being conducted and there can be discussions on what information is ready for dissemination to the audiences they serve. We hope to continue to try to engage others in our future meeting including extension, industry, and biomedical researchers. If funding and logistics allow, we may also be able to host a symposium, mini-symposium, session or something similar at the American Society for Nutrition’s annual conference, Institute for Food Technologists conference, and/or the Food & Nutrition Conference Expo.

Organization/Governance

The recommended Standard Governance for multistate research activities includes the election of a Chair, a Secretary, who will become the chair the following year. Typically the chair hosts the annual meeting. All officers are to be elected for two-year terms (1 year Secretary, 1 year Chair) 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, FL, HI, IA, IL, IN, KS, MA, ME, MO, MT, NE, NY, OH, OK, OR, RI

Non Land Grant Participating States/Institutions

California-San Diego