Location: Children's Nutrition Research Center2010 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.
3. Progress Report
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.
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.