2011 Annual Report
1a.Objectives (from AD-416)
Objective 1: Determine the absorption of dietary calcium, magnesium, iron and zinc in children and the influence of other nutrients and dietary factors on the absorption.
Sub-objective 1.A: Evaluate the effects of supplemental vitamin D in enhancing calcium absorption in healthy children 4 to 8 yrs of age.
Sub-objective 1.B: Assess the absorption of magnesium and zinc in healthy children 4 to 8 yrs of age.
Sub-objective 1.C: Determine the effects of a diet lacking in meat (lacto-ovo vegetarian diet) on iron status in small children using a highly precise stable isotope method to measure iron status as determined by absorption of iron (reference dose).
Objective 2: Determine the effect of dietary components on the upregulation of intestinal iron transporter genes in human models.
Sub-objective 2.A: Test the quantity of a food component that must be provided and the duration of exposure to the Caco-2 cells that is needed to obtain a change in expression of the divalent metal transporter (DMT1) and the brush border ferrireductase (Dcytb) genes involved in iron absorption.
Sub-objective 2.B: Apply micro-array technology, once the appropriate conditions have been determined, to identify other intestinal genes that are affected by dietary components.
Sub-objective 2.C. Evaluate foods that contain the food component(s) in question to assess if the food component is able to alter gene expression in a food matrix. This would indicate that the digestion procedure is able to release the food component(s).
Objective 3: Evaluate multiple roles of Vitamin C and ocido-reductases in molecular regulation.
Objective 3: Characterize dynamic indices of bone formation by quantitative histomorphometry and micro computed tomography in 7 mouse models developed in our laboratory.
Objective 4: Quantitate specific gene expression by qRT-PCR in calvarial osteoblasts derived from appropriate models to clarify the specific roles of each knockout gene.
Objective 5: Determine the effects of castration, iron loading, ASC feeding and plant derived antioxidants on bone parameters in vivo.
1b.Approach (from AD-416)
The goal of our research is to provide data to enhance the development of nutritional guidelines, especially as related to mineral nutrition, in children. Using both human experimentation and cell culture models, we are studying methods of delivering the key minerals of calcium, zinc, and iron in the diet so as to optimize health outcomes. This is done by evaluating enhancers of mineral absorption, such as ascorbic acid, prebiotic fibers, and vitamin D and by considering nutrient:nutrient interactions that may limit mineral absorption such as an excess in the zinc:copper intake ratio. 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. These studies will utilize stable isotope techniques so as to provide accurate, practically applicable information that may be obtained from the study populations in a safe manner. In vitro studies will seek to identify genetic basis for mineral absorption and to develop appropriate models for evaluation of mineral absorption. Taken together, this project will provide novel information directly useful to government, industry, and the consumer related to dietary requirements. These data will have global application and provide a strong basis for evidence-based nutritional recommendations to be developed. Additional studies will explore the roles of aldose reductase and aldehyde reductase in modulating oxidative stress in cells, as well as their separate role in providing the starting substrates for the ascorbate synthesis pathway in mice. As a result we will have a better understanding of the role and importance of vitamin C in our diet.
For Obj. 1, we completed recruitment for the controlled trial of vitamin D supplementation in 4- to 8-year-old children and the absorption of calcium, zinc, and magnesium in these subjects. All samples have been analyzed on the mass spectrometer and data entered for interpretation. We have completed the IRB submission for the last phase of the study and begun the recruitment process. We anticipate enrollment of subjects in November 2011 for this part of the study. We cannot finalize results until all analysis of the controlled trial are complete and the study unblended which will occur in early 2012. In Obj. 2 we conducted several experiments in which we treated the mammalian cell line, Caco-2 cells, with different concentrations of iron for different durations of exposure at 6, 24, 48, 72 and 96 hrs. We then measured DMT1 expression (DMT1 is a protein located in the brush border of the cell that is involved in iron uptake) under different iron concentrations/exposures using a scientific machine called a qPCR. Unfortunately the qPCR experienced several issues this year and a replacement machine is being obtained for faster, more reliable samples. We will measure the expression of two proteins located on the brush border of the cell, also involved in iron uptake called Dcytb1 and Dcytb2 under the same conditions when able. In Obj. 3-5, we began our efforts to define the structures and mode of interactions of Osterix (which is a novel transcription factor that is essential for bone formation) with N066. N066 is an ascorbate dependent enzyme that acts as a repressor of Osterix. N066 is also a histone demethylase, a reaction which requires an ascorbate-dependent hydroxylation. Ascorbate deficiency leads to the accumulation of inactive Osterix in immature cells that are responsible for bone formation. Thus we hypothesize that N066-Osterix interaction is the key to cell differentiation for bone formation. We have now succeeded in expressing N066 and Osterix. Also during this period we have moved our laboratory to the CNRC and are now in full operation.
The ADODR monitors project activities by visits, review of purchases of equipment, review of ARS-funded foreign travel, and review of ARS funds provided through the SCA.