2010 Annual Report
1a.Objectives (from AD-416)
The overall goal of our research is to develop nutritionally enhanced plant foods that provide increased nutrient bioavailability and absorption in children. Ultimately, this plant food research in combination with mineral nutrition research in children will allow researchers to provide guidance regarding food intake and fortification, specifically related to iron, zinc and calcium. Specific objectives of this research include:.
1)use genetic, molecular, and physiological approaches to define the role of specific genes and gene products in the acquisition and whole-organism partitioning of minerals (iron, zinc, calcium, and magnesium) and other factors that inhibit or promote absorption of these minerals in plant foods;.
2)Conduct animal and human feeding studies to determine mineral bioavailability of the nutritionally enhanced crops;.
3)develop new, cost-effective methods for the intrinsic labeling of plant foods for use in nutrient bioavailability studies;.
4)determine the absorption of dietary calcium, magnesium, iron, and zinc in children and the influence of other nutrients and dietary factors on the absorption;.
5)investigate nutrient interactions influencing Tolerable Upper Levels for mineral intakes in children; and.
6)determine the effect of dietary components on the upregulation of intestinal iron transporter genes in human models. These efforts will expand our capabilities for assessing the absorption and metabolism of various plant-derived minerals and phytochemicals and will provide novel information directly useful to government, industry and the consumer related to dietary requirements. The generation of new bioavailability data for various plant-derived nutrients will be established and such data will have global application and provide a strong basis for evidence-based nutritional recommendations to be developed.
1b.Approach (from AD-416)
These research studies will utilize diverse plant species, human cell culture systems, or human subjects. CNRC scientists will focus on characterizing plant genes and gene products that are involved with mineral transport in the plant, with a focus on iron, zinc, calcium, and magnesium. We will use specifically manipulated transgenic lines, various plant mutants, or unique plant genotypes to assess the impact of altered genes on mineral transport and storage throughout various plant tissues. In order to facilitate studies of bioavailability of plant-based nutrients, we will develop new, cost-effective methods for the intrinsic, stable-isotopic labeling of plant foods, by testing different hydroponic strategies and altered timings of isotope application to the plants. Food-based factors associated with the dietary delivery of the essential minerals calcium, iron, and zinc will be investigated using human in vitro cell culture and human subject-based experiments. We will conduct a controlled trial of vitamin D supplementation to assess the effects of vitamin D status on calcium absorption in small children. We will evaluate different types of whole diets (lacto-ovo vegetarian) on iron status and the effects of differing intakes of zinc on zinc and copper absorption. We will determine if benefits previously seen for prebiotic fibers in enhancing calcium absorption also occur for iron absorption. Low abundance stable isotopes of each element will be used to track absorption in each of these human studies. In vitro cell culture models will seek to identify the genetic basis for iron and zinc absorption in intestinal cells, by monitoring mineral absorption in combination with the differential expression of various metal transporter genes.
Project 1. We conducted clinical studies of vitamin D supplementation in children 4 to 8 years of age. We are gearing up for a related study of children 4 to 8 years of age who are vegetarians. Iron absorption will be studied using stable isotopes in these children. We will test how certain food components (such as beta-carotene, insulin, and the so-called meat factor) affect the expression level of DMT1 and Dcytb. DMT1 is a divalent metal transporter that transports ferrous iron into cells. Dcytb is an enzyme that reduces ferric iron into ferrous iron. We characterized gene expression of DMT1 and Dcytb under different iron concentrations, using a human intestinal cell line (Caco-2). Project 2. We determined that calcium oxalate mutant 1 is new and calcium oxalate mutant 2 is similar to one we previously identified. A library of all the genes expressed in Medicago truncatula leaves has been prepared for use in isolating the genes required for calcium oxalate formation. We created numerous carrots expressing higher levels of nutrient transporters. We generated more than 50 primary carrot transformants and greater than 200 Arabidopsis lines and are monitoring growth and fitness. We further characterized nutrient transporters in other biological systems. We measured transport properties and ability of each nutrient transporter to accumulate nutrients within the cell. Genetic diversity studies using unique lines of soybean, or Medicago truncatula, identified regions of the plant's genome associated with higher tissue levels of minerals. We completed growth, mineral analyses, and statistical tests of data using a Medicago population and obtained genomic locations relevant to higher leaf levels of several minerals. Work with soybean shows similar results for seeds. Backcrossing procedures were completed for several Arabidopsis and Medicago lines previously identified as maintaining good copies of genes for elevated levels of seed minerals. Research involving stable isotope labeling of plants was carried out with Arabidopsis and soybean. Studies with soybean were carried out to find a line with high seed concentrations of vitamin K; none found. Other oil seed crops might need to be pursued for future studies. Project 3. Samples of dry bean and snap bean are not yet available for analysis of mineral concentrations and iron bioavailability potential, so we assessed various software and hardware configurations that will allow us to document and inventory the samples. Based on past experiences and the unique situations that we plan, we are evaluating off-the-shelf software packages and bar code readers to purchase and use with this research project. We established new program parameters on our ICP-OES that will allow us to include selenium in our mineral analyses. New standard solutions have been purchased that include Se, along with our other minerals to be assayed; these will be used for instrument calibration. For the Caco-2 in vitro cell culture studies to assess iron bioavailability, we have discussed how to handle small scale processing of samples (e.g., cooking methods) prior to the analyses, and how best to pilot test various procedures.
Enhancing yeast as a research tool. A strategy to increase the calcium concentration in fruits and vegetables is by altering the regulation of plant calcium transporters so they move increased amounts of calcium into the edible tissues of plants. A simple and quick process researchers typically use to study plant calcium transporters is by expressing these transporters in brewer's yeast where they can study their biological properties because yeast is a single celled organism that lacks the complex physiology of a plant. By using yeast to express the transporter, the researcher can study the single transporter and remove the background of the many other plant transporters that may have similar functions; however, when a particular plant transporter is inactive in yeast this creates an obstacle. In Houston, Texas, Children's Nutrition Research Center researchers have generated a process that now makes the transporter active in yeast so that their functions can be readily analyzed in a simple biological system. These findings are important since it will impact how biologists view the use of yeast as a tool to study plant transporters.
A strategy for identifying new genes for enhancing calcium bioavailability in edible plants. Calcium, when present as the calcium oxalate crystal in foods, is unavailable for nutritional absorption. Such crystals are common in edible plant foods thereby reducing their nutritional quality. Researchers at the Children's Nutrition Research Center at Houston, Texas, have determined that using the latest integrative molecular and genetic tools (e.g., transposon-tagged mutant lines) developed in the legume model, Medicago truncatula, will expedite discovery of the genes that compose the pathways of calcium oxalate crystal formation in plants. Segregation and complementation analysis has shown that the gene containing the transposon-tag is linked to the mutant calcium oxalate phenotype. It is anticipated that the genes identified through the use of these tools will provide the molecular targets for a decreased expression strategies to enhance the nutritional value of economically important crop plants.
Chandel, G., Banerjee, S., Vasconcelos, M., Grusak, M.A. 2010. Characterization of the root transcriptome for iron and zinc homeostasis-related genes in indica rice (Oryza sativa L). Journal of Plant Biochemistry and Biotechnology. 19(2):145-152.
Levin, S.R., Chen, Z., Abrams, S.A. 2010. Effect of tannic acid on iron absorption in straw-colored fruit bats(Eidolon helvum). Journal of Zoo Biology. 29(3):335-343.
Mitchell, S.M., Rogers, S.P., Hicks, P.D., Hawthorne, K.M., Parker, B.R., Abrams, S.A. 2009. High frequencies of elevated alkaline phosphatase activity and rickets exist in extremely low birth weight infants despite current nutritional support. BMC Pediatrics. 9:47.
Zappitelli, M., Juarez, M., Castillo, L., Coss-Bu, J., Goldstein, S.L. 2009. Continuous renal replacement therapy amino acid, trace metal and folate clearance in critically ill children. Intensive Care Medicine. 35(4):698-706.
Manary, M.J., Abrams, S.A., Griffin, I.J., Quimper, M.M., Shulman, R.J., Hamzo, M.G., Chen, Z., Maleta, K., Manary, M.J. 2010. Perturbed zinc homeostasis in rural 3-5-y-old Malawian children is associated with abnormalities in intestinal permeability attributed to tropical enteropathy. Pediatric Research. 67(6):671-675.
Sullivan, S., Schanler, R.J., Kim, J.H., Patel, A.L., Trawoger, R., Kiechl-Kohlendorfer, U., Chan, G.M., Blanco, C.L., Abrams, S., Cotten, C.M. 2010. An exclusively human milk-based diet is associated with a lower rate of necrotizing enterocolitis than a diet of human milk and bovine milk-based products. Journal of Pediatrics. 156(4):562-567
Rogers, S.P., Hicks, P.D., Hamzo, M., Veit, L.E., Abrams, S.A. 2010. Continuous feedings of fortified human milk lead to nutrient losses of fat, calcium, and phosphorous. Nutrients. 2(3):230-240.
Willis, T.C., Carter, B.A., Rogers, S.P., Hawthorne, K.M., Hicks, P.D., Abrams, S.A. 2010. High rates of mortality and morbidity occur in infants with perenteral nutrition - associated cholestasis. Journal of Parenteral and Enteral Nutrition. 34(1):32-37.
Hettiarachchi, M., Liyanage, C., Hilmers, D., Griffin, I., Abrams, S.A. 2010. Changing the zinc:iron ratio in a cereal-based nutritional supplement has no effect on percent absorption of iron and zinc in Sri Lankan children. British Journal of Nutrition. 103(7):1015-1022.
Domellof, M., Hernell, O., Abrams, S.A., Chen, Z., Lonnerdal, B. 2009. Iron supplementation does not affect copper and zinc absorption in breastfed infants. American Journal of Clinical Nutrition. 89(1):185-190.
Zhao, J., Shigaki, T., Mei, H., Guo, Y-Q., Cheng, N-H., Hirschi, K.D. 2009. Interaction between Arabidopsis Ca(2+)/H(+) exchangers CAX1 and CAX3. Journal of Biological Chemistry. 284(7):4605-4615.
Hawthorne, K.M., Morris, J., Hotze, T., Hirschi, K.D., Abrams, S.A. 2009. Biotechnologically-modified carrots: Calcium absorption relative to milk. Journal of Bioequivalence and Bioavailability. 1(1):34-38.
Korenkov, V., King, B., Hirschi, K., Wagner, G.J. 2009. Root-selective expression of "AtCAX4" and "AtCAX2" results in reduced lamina cadmium in field-grown "Nicotiana tabacum L". Plant Biotechnology Journal. 7(3):219-226.
Nakata, P.A., He, C. 2010. Oxalic acid biosynthesis is encoded by an operon in Burkholderia glumae. FEMS Microbiology Letters. 304:177-182.
Waters, B.M., Uauy, C., Dubcovsky, J., Grusak, M.A. 2009. Wheat (Triticum aestivum) NAM proteins regulate the translocation of iron, zinc, and nitrogen compounds from vegetative tissues to grain. Journal of Experimental Botany. 60(15):4263-4274.
Sperotto, R.O., Ricachenevsky, F.K., Duarte, G.L., Boff, T., Lopes, K.L., Sperb, E.R., Grusak, M.A., Fett, J.P. 2009. Identification of up-regulated genes in flag leaves during rice grain filling and characterization of OsNAC5, a new ABA-dependent transcription factor. Planta. 230(5):985-1002.
Griffin, I.J., Lynch, M.F., Hawthorne, K.M., Chen, Z., Hamzo, M., Abrams, S.A. 2008. Magnesium retention in 12 to 48 month-old children. Journal of American College of Nutrition. 27(2):349-355.
Hawthorne, K.M., Abrams, S.A., Heird, W.C. 2009. Docosahexaenoic acid (DHA) supplementation of orange juice increases plasma phospholipid DHA content of children. Journal Of The American Dietetic Association. 109(4):708-712.
Balay, K.S., Hawthorne, K.M., Hicks, P.D., Griffin, I.J., Chen, Z., Westerman, M., Abrams, S.A. 2010. Orange but not apple juice enhances ferrous fumarate absorption in small children. Journal of Pediatric Gastroenterology and Nutrition. 50(5):545-550.
Castillo, L., Carcillo, J. 2009. Secondary hemophagocytic lymphohistiocytosis and severe sepsis/systemic inflammatory response syndrome/multiorgan dysfunction syndrome/macrophage activation syndrome share common intermediate phenotypes on a spectrum of inflammation. Pediatric Critical Care Medicine. 10(3):387-392.
Abrams, S.A. 2010. Calcium absorption in infants and small children: Methods of determination and recent findings. Nutrients. 2(4):474-480.
Mohan, P., Abrams, S.A. 2009. Oral lactoferrin for the treatment of sepsis and necrotizing enterocolitis in neonates. Cochrane Database Systematic Reviews. Issue 1:CD007138.
Manohar, M., Mei, H., Franklin, A.J., Sweet, E.M., Shigaki, T., Riley, B.B., MacDiarmid, C.W., Hirschi, K. 2010. Zebrafish ("Danio rerio") endomembrane antiporter similar to a yeast cation/H(+) transporter is required for neural crest development. Biochemistry. 49(31):6557-6566.
Han, J-S., Park, S., Shigaki T., Hirschi, K.D., Kim, C.K. 2009. Improved watermelon quality using bottle gourd rootstock expressing a Ca(2+)/H(+) antiporter. Molecular Breeding. 24(3):201-211.
Zhao, J., Connorton, J.M., Guo, YQ., Li, X., Shigaki, T., Hirschi, K.D., Pittman, J.K. 2009. Functional studies of split "Arabidopsis" Ca(2+)/H(+) exchangers. Journal of Biological Chemistry. 284(49):34075-34083.
Abrams, S.H., Shulman, R.J. 2009. Carbohydrate and dietary fiber. In: Kleinman, R.E., editor. Pediatric Nutrition Handbook. 6th edition. Elk Grove Village, IL: American Academy of Pediatrics. p. 343-356.