Potential of the Plasma Nutriproteome to Predict Micronutrient Distributions in Undernourished Settings.

Objectives: Micronutrient deficiencies are common but remain ‘hidden' due to difficulty of assessment. We explored the plasma proteome to identify nutrient-correlated biomarkers that may predict multiple micronutrient status and deficiencies, possibly on a single platform in the future. Methods: We measured, in 500 6-8 year old Nepalese children, plasma concentrations of >20 vitamin/mineral indicators and acute phase proteins (APP) by conventional assays, and relative abundance of proteins by quantitative mass spectrometry, bioinformatics and linear mixed effects models (Herbrich S et al, 2012; Cole R et al, 2013). We identified ~980 proteins in >10% of subjects, and evaluated their strength of correlation with micronutrient and APP distributions. Comparisons were corrected for multiple comparisons, with a 10% threshold for false discoveries. Results: 142 proteins were correlated with plasma retinol, 6 with 25(OH) vitamin D, 119 with α- tocopherol, 12 with γ-tocopherol, 6 with PIVKA-II (reflecting vitamin K status), 89 for vitamin B6, 35 for ferritin and 7 for transferrin receptor (reflecting iron status), 232 for copper, 3 for selenium and none for folate, thyroglobulin (reflecting iodine status) or vitamin B12 (q>0.1 for all comparisons). Initial models with up to 6 covariates suggest an ability to explain 60-80% of the variation (R2) in retinol, α-tocopherol, vitamin B6 and copper, ~50% of the variation in ferritin and, the carotenoid, β-cryptoxanthin and 80-85% of variation in CRP and AGP. Other nutrient-protein models will be presented. Conclusions: Plasma nutrient-correlated proteomes exist that, with absolute quantification of candidate proteins, could provide a basis for multiple micronutrient status assessment of populations in the future.

Harnessing New Computing Technologies for Detailed Assessment of Child Diet in Rural Zambia.

Objectives: To integrate tablet-based data collection tools with dietary data processing software for assessment of dietary intakes among children in rural Zambia. Methods: Dietary intakes of 4- to 8-year-old children in rural Zambia (n=1226) were collected by 24 hour recall interview using a customized survey tool on Android tablets. The 24 hour recall tool was pre-programmed with interviewer prompts, contained 1205 locally consumed foods, and collected detailed food descriptions including portion size, cooking method, added ingredients and source. Interview data were uploaded from the tablets to secure servers daily. Compiled data will be formatted for import into CS Dietary software, a program developed by Harvest Plus for entry and analysis of 24 hour recall data. CS Dietary analyzes foods and nutrients based on food composition, recipe, measurement conversion and food group tables which are selected by the researcher to match the research context. Tables corresponding to the food list used in the tabletbased recall tool will be imported into CS Dietary to create reports on diets of the children surveyed. Results: The total number of foods recorded was 8418. Of these, 31% (n=2620) were stiff, white maize porridge, 21% (n=1739) were leafy vegetable dishes and 11% (n=939) were small, whole fish dishes. Other foods consumed included large fish, eggs, beans, fritters, rice, and other maize preparations. Conclusions: This use of a tablet-based survey tool and CS Dietary software enabled paperless data collection and analysis based on context-specific food and recipe tables and shows the way forward for the deployment of customizable, interview-to-intakes tools for dietary assessment.

Micronutrient Status during Pregnancy and Effects of Antenatal Supplementation with Multiple Micronutrients versus Iron-folic Acid in Rural Bangladesh.

Objectives: Women often enter pregnancy with micronutrient deficiencies, exacerbated by demands of pregnancy. Yet, gestational micronutrient status is uncommonly assessed, even as momentum builds toward preventing multiple micronutrient (MM) deficiencies. We assessed micronutrient status of women early in pregnancy in a rural setting in northern Bangladesh. Methods: In a substudy of women participating in a randomized trial of MM versus iron-folic acid (IFA) supplementation we identified a population-based subsample of n=1526 women from whom plasma samples were obtained in the 1st trimester (TM), prior to supplementation, and in the 3rd TM, to evaluate micronutrient status and response to supplementation. Results: In available 1st TM data (n=491-1448 per nutrient assay), 6.8% of women were vitamin A deficient (retinol < 0.70 μmol/L), 41.7% had low β-carotene (<0.09 μmol/L), and 57.7% were vitamin E deficient (α-tocopherol < 12 μmol/L). Folate deficiency (plasma folate < 6.8 nmol/L) was 2.6%, while prevalence of vitamin B12 deficiency (cobalamin < 150 pmol/L) was 29.8%. Anemia affected 20.6% of women, but iron deficiency by TfR (4.7% > 8.3 μg/L) and ferritin (1.8% < 12 μg/L) was uncommon. Plasma zinc was low (<10 μmol/L) in 30.6% of women. Inflammation, by α-1 acid glycoprotein (AGP > 1 g/L), was present in 8.1% of women. Baseline vitamin D and iodine status, and MM versus IFA effects on micronutrient status by the 3rd TM, are being determined. Conclusions: Women in rural Bangladesh experience a variety of micronutrient deficiencies in early pregnancy, supporting the need to address "hidden hunger" with multiple micronutrient supplementation during pregnancy.

Malaria Exacerbates Inflammation-induced Bias in Ferritin and Soluble Transferrin Receptor Values.

Objectives: In the context of malaria and inflammation, the utility of ferritin and soluble transferring receptor (sTfR), as indicators of iron status may be compromised. In this study, we evaluated the effects of correcting for malaria and inflammation on the prevalence of iron deficiency (ID) as estimated by a) ferritin and b) sTfR. Methods: The analyses used baseline data from 1085 children, 4-8 y, who participated in a carotenoid biofortified maize flour trial in rural Zambia. For each biomarker, we compared the prevalence of ID with the prevalence corrected for a) CRP and AGP only; and b) CRP, AGP and concurrent malaria. Inflammation was defined as CRP>5mg/L and/or AGP>1g/L. Malaria was defined by microscopy. Children were first stratified into groups defined by inflammation and malaria status. Correction factors were then generated by dividing the group geometric means by that of the reference group (those free of both malaria and inflammation). Correction factors were applied to each individual concentration to generated corrected concentrations. Results: For ferritin, the unadjusted prevalence of ID (WHO age-specific cut-offs) increased from 7.3% to 9.5% (p<0.01) and 10.3 %( p<0.01), respectively, after correcting for CRP/AGP only, and CRP, AGP and concurrent malaria combined. For sTfR, the unadjusted ID prevalence (cutoff >8.3 mg/l) decreased from 28% to 21% (p<0.01) after correcting CRP/AGP only, and 19% (p<0.01) after correcting for CRP, AGP and concurrent malaria. Conclusions: Our findings highlight the need to account for both malaria and inflammation when interpreting ferritin and sTfr concentrations in malaria endemic regions.

Effect of Antenatal Micronutrient Supplementation on the Plasma Proteome in School Aged Children in Nepal.

Objectives: Antenatal micronutrient interventions may influence maternal and offspring health in chronically undernourished settings; however, molecular mechanisms remain largely unexplored. We examined effects of multiple combinations of antenatal micronutrients as supplements on the plasma proteome of offspring at 6-8 years of age. Methods: We applied quantitative mass spectrometry to measure plasma protein abundance in 500 children whose mothers had been randomized to receive daily supplements of folic acid (FA), iron-folic acid (IFA), iron-folic acid-zinc (IFAZn), multiple micronutrient (MM), or placebo (control) from 1st trimester to 3 months postpartum (all tablets contained vitamin A). We identified differentially abundant proteins and sets of proteins sharing a common biological function by enrichment analysis using the Gene Ontology (GO) database. Results: With a relaxed discovery threshold (false discovery rate <0.25), maternal FA supplementation increased the abundance of insulin-like growth factor-1 (IGF1) by 33.7 (95% CI: 14.7-55.8)%; maternal IFA supplementation increased tissue inhibitor of metalloproteinase 1 by 12.5 (5.9-19.6)%. All supplements containing iron-folic acid increased IGF1, IGF2, and IGFbinding protein 5 by 23.9 (9.1-40.7)%, 28.6 (10.7-49.4)%, and 23.7 (10.5-38.5)%, respectively, and decreased stromal interaction molecule 1 by 63.3 (36.7-78.8)%. With a discovery threshold of 0.05, maternal IFA supplementation negatively enriched proteins localized in microtubules (GO:5874) with an enrichment score (ES) of -0.62 and maternal IFA and MM supplementation positively enriched proteins with growth factor activity (GO:8083) with ES of 0.70 and 0.75, respectively (all p-values <0.0001). Conclusions: Antenatal micronutrient supplementation exerts subtle metabolic effects on proteins involved in regulating growth/development and intracellular structure in school-aged children.

The Effect of Antenatal Multiple Micronutrient Supplementation on Placental Angiogenesis and Fetal Growth Factors in Rural Bangladesh.

Objectives: Pathways by which micronutrients may influence birth size are not well understood. To elucidate these, we assessed markers of placental angiogenesis and fetal growth factors in a substudy of a community-based, double blinded, cluster-randomized trial of maternal multiple micronutrient (MM) supplementation versus iron and folic acid (IFA) in rural Bangladesh. Methods: We collected maternal blood (n=395) at 10 and 32 weeks gestation and cord blood (n=325) at home deliveries, where infant and placental weight were measured. Angiogenic factors assessed in maternal plasma were placental growth factor (PlGF), angiopoietin 2 (Ang-2), vascular endothelial growth factor (VEGF), and VEGF receptor 1 (VEGFR-1). Insulin, insulin-like growth factor-1 (IGF-1), and IGF binding protein-1 (IGFBP-1) were measured in cord plasma. Results: Mothers had a mean (SD) BMI in early pregnancy of 19.5 (2.5) kg/m2 and infants weighed 2.68 (0.41) kg at birth. Ang-2 at 32 weeks gestation was 3.1 (95% CI: 0.20, 6.0) ng/mL higher in mothers taking MM vs. IFA suggesting improved vascular remodeling. Other angiogenic factors and insulin, IGF-1, and IGFBP-1 did not differ by maternal supplementation, but significant interactions were observed with infant sex and maternal height. For male fetuses, change in VEGF from 10 to 32 weeks was lower by 7.5 (95% CI: -13.3, -1.7) pg/mL for MM vs. IFA groups reflecting improved angiogenesis across pregnancy. Among shorter women (<145 cm), MM increased insulin by 1.8 (95% CI: 1.0, 3.1) μIU/ML indicating improved fetal energy metabolism. Conclusions: MM supplementation has a biologic basis for improving fetal growth beyond improved micronutrient status of the fetus.

Dietary Diversity is Low and Associated with Nutritional Status of Women in Early Pregnancy in Rural Bangladesh.

Objectives: To evaluate the association between nutritional status and other risk factors with dietary diversity in the 1st trimester of pregnancy among women in rural Bangladesh. Methods: A cross-sectional analysis of data among women enrolled during the 1st trimester of pregnancy into the JiVitA-1 weekly vitamin A or beta-carotene supplementation trial in rural Bangladesh from 2001 to 2007. A dietary diversity score (DDS) based on 9-conventionally defined food groups was calculated based on data collected from a 7-day food frequency questionnaire. Mid-upper arm circumference (MUAC) from the main trial (n=67,690), and BMI and serum markers of micronutrient status from a substudy (n=1,869) of women were used as indicators of maternal nutritional status. Other individual, household and seasonality factors were examined for their association with DDS, and as potential confounders in the association between DDS and maternal nutritional status. Results: Overall mean (sd) DDS was low at 2.1 (1.1) (Max. Score=9). In a multivariate model, selected individual (maternal education, being a wage earner, and selected morbidity symptoms), household (living standard index, smaller household size, food security, and ownership of a fruit grove, home garden or fish pond) factors and seasonality (non-lean season) were positively associated with maternal DDS. Maternal MUAC, BMI and serum levels of lycopene (biomarker for vegetable/fruit intake) were also associated with DDS. Conclusions: In this rural setting of northern Bangladesh, dietary diversity is low and maternal nutritional status is poor. Dietary diversity, measured by a simple score summed over one week, was positively associated with maternal nutritional status in early pregnancy.

Bioelectrical Impedance among Rural Bangladeshi Women during Pregnancy and in the Postpartum Period.

Properties of bioelectrical impedance analysis (BIA) reflect body-composition and may serve as stand-alone indicators of maternal health. Despite these potential roles, BIA properties during pregnancy and lactation in rural South Asian women have not been described previously, although pregnancy and infant health outcomes are often compromised. This paper reports the BIA properties among a large sample of pregnant and postpartum women of rural Bangladesh, aged 12-46 years, participating in a substudy of a communitybased, placebo-controlled trial of vitamin A or beta-carotene supplementation. Anthropometry and single frequency (50 kHz) BIA were assessed in 1,435 women during the first trimester (≤12 weeks gestation), in 1,237 women during the third trimester (32-36 weeks gestation), and in 1,141 women at 12-18 weeks postpartum. Resistance and reactance were recorded, and impedance and phase angle were calculated. Data were examined cross-sectionally to maximize sample-size at each timepoint, and the factors relating to BIA properties were explored. Women were typically young, primiparous and lacking formal education (22.2±6.3 years old, 42.2% primiparous, and 39.7% unschooled among the first trimester participants). Weight (kg), resistance (Ω), and reactance (Ω) were 42.1±5.7, 688±77, and 73±12 in the first trimester; 47.7±5.9, 646±77, and 64±12 in the third trimester; and 42.7±5.6, 699±79, and 72±12 postpartum respectively. Resistance declined with age and increased with body mass index. Resistance was higher than that observed in other, non-Asian pregnant populations, likely reflecting considerably smaller body-volume among Bangladeshi women. Resistance and reactance decreased in advanced stage of pregnancy as the rate of gain in weight increased, returning to the first trimester values by the three months postpartum. Normative distributions of BIA properties are presented for rural Bangladeshi women across a reproductive cycle that may be related to pregnancy outcomes and ultimately be used for assessing body-composition in this population.

Validation of Two Portable Instruments to Measure Iron Concentration in Groundwater in Rural Bangladesh.

Iron is ubiquitous in natural water sources used around the world for drinking and cooking. The health impact of chronic exposure to iron through water, which in groundwater sources can reach well above the World Health Organization’s defined aesthetic limit of 0.3 mg/L, is not currently understood. To quantify the impact of consumption of iron in groundwater on nutritional status, it is important to accurately assess naturally-occurring exposure levels among populations. In this study, the validity of iron quantification in water was evaluated using two portable instruments: the HACH DR/890 portable colorimeter (colorimeter) and HACH Iron test-kit, Model IR-18B (test-kit), by comparing field-based iron estimates for 25 tubewells located in northwestern Bangladesh with gold standard atomic absorption spectrophotometry analysis. Results of the study suggest that the HACH test-kit delivers more accurate point-of-use results across a wide range of iron concentrations under challenging field conditions.