Location: Children's Nutrition Research Center2009 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
We have obtained IRB approval for our randomized study of vitamin D supplementation & calcium absorption. We have recruited our first subject & anticipate clinically beginning the study this summer. At this time, the projects described in 1D & 2A & 2B will not be completed due to a CNRC researcher departure. We conducted experiments with Caco-2 cells grown in 6 well-plates for 12 days in DMEM (a cell media), which were subsequently exposed for 72 hrs to different iron concentrations in MEM ranging from 0 to 800 M Fe. Following 72 hrs of exposure to the different iron treatments, the cells were harvested & RNA was isolated & converted to cDNA which was used in PCR. We found that DMT1 mRNA expression was observed at all Fe concentrations except at 0 Fe. While DMT1 expression was evident from the 25 to the 800 uM Fe treatment, its expression was minimal at 600-800 uM Fe treatment. This finding is consistent with past studies that have found that DMT1 mRNA expression decreases with exposure to iron in a dose-dependent manner. Iron-fed cells don't need to express much of the transport protein as iron-deficient cells. The cells exposed to 0 Fe didn't express any DMT1 mRNA. Dcytb expression was also assessed in Caco-2 cells. (Project 1) Growth of Medicago RILs (recombinant inbred lines) for leaf mineral determinations & quantitative trait locus (QTL) analysis have been initiated. The greenhouse growth of soybean RILs was completed & the field growth of another RIL population is underway. The back-crossing & selfing of Arabidopsis & Medicago RILs to generate candidate NILs (near isogenic lines) is in progress; F1 crosses have been made with selected informative RILs. nce these seeds are mature, they will be planted to produce F2 seeds for subsequent analysis. Deuterium oxide (D2O) labeling of Arabidopsis plants at different concentrations is in progress, to determine which concentrations allow full reproductive development. Vitamin K analysis in seed oil from a broad set of soybean accessions has been completed; vitamin K levels were much lower than anticipated & are presumed to be too low to make it worthwhile to label the plants with D2O for vitamin K studies. Additionally, we have initiated the transformation of Arabidopsis & have just isolated seeds this summer. After obtaining homozygous lines, we should complete phenotype analysis by mid-spring 2010. The transgenic tobacco lines are growing & we are starting to analyze phenotypes & perform transport studies of T2 lines in the early fall. These studies should be concluded by early spring 2010. For the carrots we started the transformation during the spring & should have seeds sometime in 2010. Progress has been made in our effort to understand the process of calcium oxalate crystal formation in plants. Back-crossing & complementation studies are underway to aid in the ID of new genes involved in calcium oxalate formation. Gene transcripts have been purified as a step toward the production of a library from which full length cDNAs will be isolated. (Project 2)