2010 Annual Report
1a.Objectives (from AD-416)
Objective 1: Investigate the impact of docosahexaenoic acid (DHA) intake from food and supplemental sources on blood levels, cognitive performance, neurophysiological function, heart rate and blood pressure as well as a lower incidence of allergies and upper respiratory infection in children. Sub-Obj 1A. Determine the DHA content of the plasma and erythrocyte phospholipid fractions and relate these to current as well as previous DHA intake. Sub-Obj 1B. Determine the effect of short- and long-term DHA supplementation on the DHA content of plasma and erythrocyte phospholipid fractions. Sub-Obj 1C. Determine the effect of DHA supplementation on cognitive and neurophysiological function, heart rate and blood pressure, and prevalence of allergies and upper respiratory infection. Obj. 2: Investigate the pathways and nutritional modulation of methyl group production in under- and normal weight pregnant women. Sub-Obj 2A. Determine whole body protein kinetics, methionine kinetics and transmethylation, an index of methyl production and utilization, serine and glycine fluxes, indices of production rates of methyl group precursors, and conversion of serine to glycine, and glycine to CO2, indices of methyl group supply from these precursors to the transmethylation pathway, in under- and normal-weight pregnant women. Sub-Obj 2B. Determine the effect of dietary supplementation with sulfur amino acid-rich whey protein vs. legume/cereal protein on methionine production and transmethylation rate and on serine and glycine fluxes in underweight pregnant women. Sub-Obj 2C. Determine methionine kinetics and transmethylation rates during the first trimester in groups of underweight pregnant women with either normal or low plasma vitamin B12 concentration, after dietary supplementation with Vitamin B12. Sub-Obj 2D. Determine methionine kinetics and transmethylation rates in underweight pregnant women with either normal or low plasma folate concentration after dietary supplementation with folate.Obj. 3: Investigate differences in bowel flora, antioxidant capacity, and mitochondrial integrity between severely malnourished and well-nourished children. Sub-Obj 3A. Measure the populations of bacterial divisions and species in bowel flora populations in children as well as bowel flora diversity with edematous as well as non-edematous SCU and in well-nourished children. Sub-Obj 3B. Measure antioxidant capacity and mitochondrial integrity, as well as characterize the immune system in children with edematous vs. non-edematous SCU. Obj. 4: Initiate a pilot study of genetic susceptibility to ESCM. Obj. 5: Conduct exploratory analyses of the relationship between risk of ESCM and individual genetic variation. Obj. 6: Evaluate population-specific genetic variation. Obj. 7: Characterize the developmental profile of the GI microbiome and transcriptome in healthy, term infants. Obj. 8: Compare the effect of breast versus bottle-feeding on the development of the GI microbiome and lactose digestion/absorption. Obj. 9: Profile changes in the GI microbiome in response to the introduction of weaning foods such as dietary starch in the form of cereal.
1b.Approach (from AD-416)
Studies will be performed in two groups of healthy, well-nourished children between 3 and 6 years of age (n=160) and between 8 and 12 years of age (n=160). A blood sample will be obtained, and the DHA content of the child's plasma and erythrocyte phospholipid fractions will be measured and related to current and prior DHA intakes, to blood pressure, heart rate, and presence of allergies and upper respiratory infections. Whole body protein kinetics, methionine production and transmethylation, serine and glycine fluxes, and conversion of serine to glycine and glycine to carbon dioxide will be measured in groups of Indian women with low (=18.5) and normal (>18.5 = 25) BMI between 10 and 12 weeks of pregnancy and again at 26-28 weeks. These measurements plus maternal gestational weight gain, neonate gestational age, birth weight, length, and head circumference will be repeated in groups with BMIs =18.5 after dietary supplement with more energy and protein and in those women with low blood vitamin B12 and folate, after 16 weeks of supplementation with vitamin B12 and folate. Additional studies will evaluate 6- to 24-month twins who are at high risk for malnutrition. Stool samples will be collected in a disposable diaper for multiplex pyrosequencing of bacterial 16S rRNA genes present in gut microbial communities and pyrosequencing of total community DNA (the gut microbiome). A second study will be performed in 50 severely undernourished, 6- to 12-month-old children who are receiving therapeutic food to promote rapid catch-up growth. Antioxidant capacity will be assessed by whole blood glutathione, erythrocyte superoxide dismutase, erythrocyte glutathione peroxidase, and serum oxidized proteins. Mitochondrial integrity will be assessed by lactate and the copy numbers of mitochondrial DNA/RNA in peripheral monocytes, measured by real time duplex nucleic acid sequence-based amplification. To assess how immune response varies with nutritional state, a panel of 27 cytokines will be assessed.
We recruited and studied 24 subjects to determine methionine kinetics and transmethylation rates during the first trimester in groups of underweight pregnant women with either normal or low plasma vitamin B12 concentration and during the third trimester in the deficient group, after dietary supplementation with vitamin B12. We have now started recruiting subjects to address other sub-objectives, and consent has been obtained from 5 subjects, and they are currently undergoing their first study. We started a collaboration with the Tropical Metabolism Research Unit, University of the West Indies. We applied for and received support to carry out a pilot project to test 1 million genetic markers in 60 children with kwashiorkor and 60 children who developed marasmus after extreme starvation. We have completed the study in 46 children in each category. The DNA samples from the remaining children are undergoing preliminary quality testing. Preliminary data analysis is underway. It indicates that the resulting single nucleotide genotyping data are very high quality. We will use these preliminary data to plan a larger and more definitive study design. Between 2008 and 2010, a total of 317 pairs of twins and 3 sets of triplets were enrolled at 5 different rural health centers in 4 different districts in southern Malawi, as part of a longitudinal study to explore the relationship between the intestinal microbiome and childhood growth and nutritional status. 204 twin families remain actively enrolled in the study, with 70 families having been discharged or dropped out, and 43 pairs of children have graduated at 3 years of age. Through the course of the study, a total of 11,303 patient visits have been recorded and a total of 6,266 stool specimens have been collected from the children in the study during these visits. In terms of severe acute malnutrition, a total of 78 episodes of kwashiorkor and 74 episodes of marasmus have been identified. Sample analyses are pending and data will be interpreted thereafter. Our research protocol on the impact study of DHA intake from food and supplemental sources on blood levels, cognitive performance, and neurophysiological function of children has been approved by the IRB, but start of the project has been delayed awaiting delivery of the DHA supplement. Actual enrollment will begin when the supplement is available. Meanwhile, a newspaper ad has yielded about 90 responses from individuals interested in participating in the study. In addition, we have identified another 90 previous volunteers in our CNRC database who may qualify for the study. Plan is to complete enrollment and determine biochemical assessments during the next 12 months.
The ADODR monitors activities for the project by routine site visits, and review of major purchases of supplies/equipment, use of SCA funds for foreign travel, and submission of grant applications by investigators funded through the SCA.
Unmasking vitamin B12's role in methyl production in pregnant women. Several studies have reported that pregnant women with low levels of vitamin B12 have a higher risk of delivering smaller babies. Vitamin B12 is important for methyl production for DNA and protein synthesis, which are necessary for cell division and growth of the fetus during pregnancy. To determine if women with low vitamin B12 levels may be making smaller babies due to a lack of methyl production, Children's Nutrition Research Center researchers studied methionine metabolism. We found that pregnant women with low vitamin B12 concentrations were producing methyl at the same rate as women with normal vitamin B12 levels. These results indicate that an inability to make methyl does not appear to be the reason why they give birth to smaller babies and further research is necessary to see what are the potential causes.