Research Project: Novel Functions and Biomarkers for Vitamins and Minerals

Location: Obesity and Metabolism Research

2018 Annual Report

Objectives
The goal of the research is to identify novel functions and biomarkers of vitamins and minerals. OBJECTIVE 1: Determine by using metabolomic approaches in animal and cell models, as appropriate, novel functions of zinc related to energy metabolism, insulin resistance in skeletal muscle, and adipose tissue and immune function. Sub-Objective 1A: Determine the mechanism of insulin resistance induced by marginal zinc deficiency in Znt7 KO mice. Sub-Objective 1B: Investigate the mechanisms underlying insulin resistance in the skeletal muscle of Znt7 KO mice. Sub-Objective 1C: Investigate the impact of mild zinc deficiency induced by the Znt7-null mutation on CD40-mediated signaling pathway activation and gene expression. OBJECTIVE 2: Discover novel functions of vitamin B12 related to energy, carbohydrate and 1-C metabolism by measuring metabolomic responses to vitamin B12 supplementation of B12 deficient humans. OBJECTIVE 3. Measure and validate novel functional biomarkers of Zn and vitamin B12 status in response to supplementation of deficient human subjects. OBJECTIVE 4. Evaluate in human intervention trials the impact of dairy consumption on measures of bone, endocrine and immune function. OBJECTIVE 5. Develop Reference Values for mineral and vitamin concentrations in human milk, which will improve estimates of recommended nutrient intakes for breastfeeding infants and their mothers.

Approach
OBJECTIVE 1: Hypothesis: Altered lipid metabolism induced by zinc insufficiency in muscle and adipose tissues contributes to glucose intolerance and insulin resistance. Advanced metabolomic, molecular and cellular technologies will be employed to determine blood and tissue signatures reflective of Zn status and pathways affected by Zn that lead to insulin resistance in muscle and mechanisms underlying Zn effects on adiposity and immune function. Tissue culture and animal models will be used, e.g., marginally Zn deficient mouse model Znt7 knockout. OBJECTIVE 2. Hypothesis: B12 supplementation of those with compromised status will alter pathways of TCA cycle, mitochondrial function, fatty acids, 1-C, amino acid and CHO metabolism. To investigate these pathways, samples from a randomized B12 supplementation trial in deficient Chilean elders will be analyzed using two metabolomic platforms. Relationships among B12 markers, metabolites and physiological functions will be evaluated before and after B12 supplementation. OBJECTIVE 3. Hypothesis: Znt7-null associated metabolite profiles of fatty acid metabolism will be used as biomarkers of zinc status. Serum collected from a Zn depletion/repletion/supplementation study will be measured for target metabolite abundance changes related to oxidative stress from zinc deficiency. Hypothesis: A combined B12 biomarker will be a better predictor of functional B12 status than single or paired biomarkers. Samples used from deficient women and their infants in Bangladesh in a randomized trial of B12 supplementation during pregnancy and lactation plus samples from Chilean elders supplemented for 18 months with B12. Responses in markers of immune function, bone turnover, and breast milk B12 will be measured, and compared to new marker of B12 status with plasma B12, homocysteine, methylmalonic acid and holotranscobalamin in Bangladeshi women. Chilean elders outcomes are neurological function, markers of inflammation, and metabolomics. The combined B12 biomarker will be evaluated in response to supplementation and associated with functional outcomes. OBJECTIVE 4. Evaluate in human intervention trials the impact of dairy consumption on measures of bone, endocrine and immune function. Hypothesis: Inclusion of 4 servings of dairy foods per day will improve bone profile, reduce fractures, improve muscle density and endocrine and inflammation profiles in elderly adults. Serum samples from a dairy interventionl in 600 ambulatory elders will be used to examine the interrelationships among bone, endocrine, immune systems and the responsiveness to dairy intake when diets with insufficient vs. adequate calcium are consumed. Objective 5. Hypothesis: Reference Values for vitamins and minerals in human milk can be established by measuring the range of concentrations in milk from well-nourished women who are not consuming additional micronutrients through supplements or fortified foods.

Progress Report
The aim of Sub-objectives 1A and 1B was to determine the mechanisms by which insulin resistance in muscle and/or fat was induced by marginal zinc (Zn) deficiency using a mouse model, zinc transporter 7 (Znt7) mice. ARS researchers in Davis, California, completed these studies and discovered several potential target molecules (fatty acid binding protein 3, Fabp3, and serine/threonine-specific protein kinases, Akts) for zinc actions in regulation of glucose and lipid metabolism in muscle and fat tissue. Manuscripts describing the finding were submitted and accepted for publication. Objective 4 was to evaluate in human intervention trials the impact of dairy consumption on measures of bone, endocrine and immune function. The hypothesis was that inclusion of four servings of dairy foods per day will improve bone profiles, reduce fractures, and improve muscle density and endocrine and inflammation profiles in elderly adults. Serum samples from a dairy intervention in 600 ambulatory elders will be used to examine the interrelationships among bone, endocrine and immune systems and the responsiveness to dairy intake when diets with insufficient vs. adequate calcium are consumed. Due to recruitment difficulties in Australia, the project has been significantly behind schedule from the beginning. However, the intervention was completed in April 2018 and ARS is in receipt of data from Australia, including dietary intake and a variety of clinical parameters. ARS researchers have finished all clinical chemistry measures as well as inflammatory markers. A master data file has been created and researchers are in the process of conducting preliminary statistical analyses. The project should be completed by December 2018. Objective 5 was to establish Reference Values for micronutrient concentrations in milk from well-nourished women who are not taking multiple micronutrient supplements or consuming highly fortified foods. These Reference Values will serve as a benchmark against which to evaluate the nutritional quality of human milk in populations around the world. Funding has been obtained from the Bill & Melinda Gates Foundation (2032-51000-004-30T). In FY16 and FY17, researchers completed a major review of the nutrient composition of human milk, including meta-analyses on data for three nutrients, a review and revision of laboratory analysis methods, and a comparison of milk nutrient values currently used to set recommended intakes for mothers and infants by major agencies. Seven papers from this work were published in May 2018. ARS researchers have also started work in four countries, collecting samples that will be analyzed to develop the Reference Values. The field sites are Dhaka, Bangladesh; Fajara, The Gambia; Copenhagen, Denmark; and Rio de Janeiro, Brazil. Sub-agreements and awards have been made with collaborators, a Manual of Procedures was developed, and questionnaires and an electronic data capture base were established. Enrollment is currently 25 to 100 mothers and infants per site (the goal is 250 pairs per site) and collection of milk and other samples is ongoing. Milk analysis methods have been developed and researchers are working on mass spectrometry methods for micronutrients in plasma.

Accomplishments
1. Signature molecules discovered for insulin resistance induced by cellular zinc deficiency. ARS scientists in Davis, California, demonstrated that the fatty acid uptake system in humans was altered by cellular zinc deficiency, leading to excessive accumulation of fatty acids and lipids in skeletal muscle, which subsequently produced lipid mediators that induced strong oxidative stress and inflammation. A key molecule, Fabp3 (fatty acid binding protein 3) was discovered among the others that connected the link between cellular zinc status and insulin resistance in muscle. This novel finding provided new insight into how zinc regulates muscle fatty acid metabolism. It also highlights novel targets in zinc nutrition, which have potential preventive implications of insulin resistance and Type 2 Diabetes. The study also provided evidence that zinc plays a unique and important role in the early stages of metabolic diseases, and a nutritional intervention, such as zinc supplementation, can be given to reduce or prevent progress of the disease.

Review Publications
Brito, A., Grapov, D., Fahrmann, J., Harvey, D., Green, R., Miller, J.W., Fedosov, S.N., Shahab-Ferdows, S., Hampel, D., Pedersen, T.L., Fiehn, O., Newman, J.W., Uauy, R., Allen, L.H. 2017. The human serum metabolome of vitamin B-12 deficiency and repletion, and associations with neurological function. Journal of Nutrition. 147(10):1839-1849. https://doi.org/10.3945/jn.117.248278.
Huang, L., Tepaamorndech, S., Kirschke, C.P., Newman, J.W., Keyes, W.R., Pedersen, T.L. 2018. Aberrant fatty acid metabolism in the skeletal muscle contributes to insulin resistance in zinc transporter 7 (Znt7) knockout mice. Journal of Biological Chemistry. 293(20):7549-7563. doi:10.1074/jbc.M117.817692.
Hampel, D., Shahab-Ferdows, S., Gertz, E.R., Flax, V.L., Adair, L.S., Bentley, M.E., Jamieson, D.J., Tegha, G., Chasela, C.S., Kamwendo, D., Van Der Horst, C.M., Allen, L.H. 2017. The effects of a lipid-based nutrient supplement and antiretroviral therapy in a randomized controlled trial on iron, copper, and zinc in milk from HIV+ Malawian mothers and associations with maternal and infant biomarkers. Maternal and Child Nutrition. 14(2):e12503. https://doi.org/10.1111/mcn.12503.
Allen, L.H., Dror, D.K. 2018. Introduction to current knowledge on micronutrients in human milk: adequacy, analysis and need for research. Advances in Nutrition. 9:275S-277S. https://doi.org/10.1093/advances/nmy018.
Figueiredo, A., Cocate, P., Adegboye, A., Franco-Sena, A., Farias, D., Trinidade De Castro, M., Brito, A., Allen, L.H., Mokhtar, R., Holick, M., Kac, G. 2018. Changes in plasma concentrations of 25-hydroxyvitamin D and 1,25 dihydroxyvitamin D during pregnancy: a Brazilian cohort. European Journal of Nutrition. 57:1059-1072. https://doi.org/10.1007/s00394-017-1389-z.
Baldiviez, L.M., Keim, N.L., Laugero, K.D., Hwang, D.H., Huang, L., Woodhouse, L.R., Burnett, D.J., Zerofsky, M., Bonnel, E., Allen, L.H., Newman, J.W., Stephensen, C.B. 2017. Design and implementation of a cross-sectional nutritional phenotyping study in healthy US adults. Biomed Central (BMC) Nutrition. 3(79):1-13. https://doi.org/10.1186/s40795-017-0197-4.
Prado, E., Ashorn, U., Phuka, .J., Maleta, .K., Sadalaki, .J., Oaks, B.M., Haskell, M., Allen, L.H., Vosti, S.A., Ashorn, .P., Dewey, K.G. 2018. Associations of maternal nutrition during pregnancy and postpartum with maternal cognition and mother-child interaction. Maternal and Child Nutrition. 14:e12546. https://doi.org/10.1111/mcn.12546.
Dror, D.K., Allen, L.H. 2018. Overview of nutrients in breast milk. Advances in Nutrition. 9:278S-294S. https://doi.org/10.1093/advances/nmy022.
Dror, D., Allen, L.H. 2018. Retinol to fat ratio and retinol in human milk: a systematic review and meta-analysis. Advances in Nutrition. 9:332S-346S. https://doi.org/10.1093/advances/nmy021.
Dror, D., Allen, L.H. 2018. Vitamin B12 in breast milk: a systematic review. Advances in Nutrition. 9:358S-366S. https://doi.org/10.1093/advances/nmx019.
Dror, D., Allen, L.H. 2018. Iodine in breast milk: a systematic review. Advances in Nutrition. 9:347S-357S. https://doi.org/10.1093/advances/nmy020.
Hampel, D., Dror, D., Allen, L.H. 2018. Micronutrients in human milk: analytical methods. Advances in Nutrition. 9:313S-331S. https://doi.org/10.1093/advances/nmy017.
Garrod, M.G., Buchholz, B.A., Miller, J.W., Haack, K.W., Green, R., Allen, L.H. 2018. Vitamin B12 added as a fortificant to flour retains high bioavailability when baked in bread. Nuclear Instruments and Methods. https://doi.org/10.1016/j.nimb.2018.05.042.
Cai, Y., Kirschke, C.P., Huang, L. 2018. SLC30A family expression in the pancreatic islets of humans and mice: cellular localization in the ß-cells. Journal of Molecular Histology. 49(2):133-145. https://doi.org/10.1007/s10735-017-9753-0.
Allen, L.H. 2018. Using dietary reference values to define fortification levels for national programs. Book Chapter. 43-50. https://doi.org/10.1016/B978-0-12-802861-2.00004-3.
Allen, L.H. 2018. Efficacy and safety of vitamin B12 fortification. Book Chapter. https://doi.org/10.1016/B978-0-12-802861-2.00026-2.
Lepsch, J., Eschriqui, I., Rodriguez-Farias, D., Vaz, J.S., Cunha Figueiredo, A.C., Amorim Adegboye, A.R., Brito, A., Mokhtar, R., Allen, L.H., Holick, M.F., Kac, G. 2017. Association between early pregnancy vitamin D status and changes in serum lipid profiles throughout pregnancy. Metabolism. 70:85-97. https://doi.org/10.1016/j.metabol.2017.02.004.
Sutter, C., Ontai, L.L., Shilts, M.K., Lanoue, L., Allen, L.H., Townsend, M.S. 2018. Associations between school readiness and obesity- and inflammation-related biomarkers in low-income preschoolers within the healthy kids study. Mind, Brain, and Education. 12:28-38. https://doi/pdf/10.1111/mbe.12165.
Brito, A., Habevch, E., Silva-Zolezzi, I., Galaffu, N., Allen, L.H. 2018. Methods to assess vitamin B-12 bioavailability and technologies to enhance its absorption. Nutrition Reviews. 76(10):778-792. https://doi.org/10.1093/nutrit/nuy026.
Allen, L.H., Miller, J.W., De Groot, L., Rosenberg, I.H., Smith, D.A., Refsum, H., Raiten, D.J. 2018. Biomarkers of Nutrition for Development (BOND) - vitamin B12 review. Journal of Nutrition. 148(4):1995S-2027S. https://doi.org/10.1093/jn/nxy201.
Williams, A.M., Stewart, C.P., Shahab-Ferdows, S., Hampel, D., Kiprotich, M., Achando, B., Lin, A., Null, C., Allen, L.H., Chantry, C.J. 2018. Infant serum and maternal milk vitamin B-12 are positively correlated in Kenyan infant-mother dyads at 1–6 months postpartum, irrespective of infant feeding practice. Journal of Nutrition. 148:86-93. https://doi.org/10.1093/jn/nxx009.
Allen, L.H., Pawlak, R., Vos, P., Shahab-Ferdows, S., Hampel, D., Perrin, M.T. 2018. Vitamin B12 content in breast milk of vegan, vegetarian and non-vegetarian lactating women in the United States. American Journal of Clinical Nutrition. https://doi.org/10.1093/ajcn/nqy104.