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Introduction
Iron deficiency anemia (IDA) affected 1.24 billion peo- ple and was the most common cause of Years Lived with Disability (YLD) in low- and low-middle income countries in 2016.1 Iron deficiency (ID) and anemia disproportion- ately affect young children in such socio-economic set- tings.2 Beyond anemia, ID during infancy is linked to impaired cognitive and behavioral development, poten- tially yielding irreversible long-term individual and socie- tal impacts.3,4
Neonates are born with a maternal iron endowment that is initially largely contained within hemoglobin and influenced by birthweight, gestational age, and the timing and method of cord clamping.5-7 In healthy term infants, this birth endowment meets the iron needs of blood vol- ume expansion, brain development and tissue accretion for several months, compensating for the very low level of iron in breast milk;5 after this, complementary feeding must supply iron.6-9 Iron-poor diets and high infection bur- dens, when combined with the significant physiological iron demands of growth and development during infancy, render young LMIC children especially vulnerable to ID/IDA.
The hepatic hormone hepcidin regulates systemic iron handling.10 Hepcidin inhibits the cellular iron exporter fer- roportin, preventing iron recycling by erythrophagocytic macrophages and dietary iron uptake through entero- cytes.11 Hepcidin induction by iron prevents iron overload, while its production during inflammation/infection gener- ates an acute anti-infective hypoferremia;12,13 moreover, chronically raised hepcidin causes ID and IDA,14 and chronic ID and IDA can protect against malaria.15-17 In con- trast, hepcidin suppression during iron demand, for exam- ple via erythroferrone activity in response to erythropoiet- ic stress, increases serum iron availability.18 In African pre- school children, hepcidin reflects iron status and effective- ly predicts utilization of orally administered iron.19,20 However, its regulation during the first year of life remains poorly characterized.6
The Gambia is an African country characterized by high burdens of infection, undernutrition and anemia, together with extreme seasonality, all of which have the potential to influence iron control through effects on hepcidin.21 In the clearly demarcated wet season, nutritional availability and quality deteriorate, infections are more prevalent, and growth faltering is more prominent. Here, we used two longitudinal birth cohort studies from rural communities in The Gambia to investigate how relative changes in iron, infection and inflammation, erythropoietic drive, season- ality, and growth rate predict changes in hepcidin and the iron stores marker ferritin during the first year of life.
Methods
Details of cohort characteristics, methods and statistics are pre- sented in the Online Supplementary Appendix and Online Supplementary Tables S1 and S2.
Cohorts
We obtained biochemical, anthropometric and infection data at serial time points [birth (cord blood), 2, 5, 9 (VA cohort only) and 12 months of age] from two rural birth-cohort studies in The Gambia, West Africa:
- VPM study: Vaccination and Paediatric Microbiome (VPM) study. Conducted in the Western Region; March 2013-September 2015; n=114;
- VA study: a vitamin A supplementation randomized con- trolled trial.22 Conducted in the West Kiang Region; September 2001-October 2004; n=193.
Definitions
Anthropometric Z-scores (World Health Organization child growth standards): generated using STATA package zscore06.23 Wet season: July-October. Weaning: first recorded occurrence of any feeding other than exclusive breastfeeding. Positive recent infection: occurrence within the previous four weeks of ear infec- tion, chest infection, meningitis/sepsis, or other symptoms associ- ated with infection (including fever, diarrhea, vomiting), or if antibiotics were administered within the previous two weeks (VPM); or of diarrhea, vomiting, fever, cough or clinic attendance (VA). Iron deficiency (ID): ferritin <12 μg/L or ferritin <30 μg/L if CRP >5 mg/L; anemia: Hb <11 g/dL; inflammation: CRP >5 mg/L; iron deficiency anemia (IDA): anemia in the presence of ID.21
Statistical analysis
RStudio (v1.1), Stata14 (Statacorp-LP), R (www.r-project.org) and Prism7 (GraphPad Software) were used for statistical analysis and graphics.
To address the potential bias that may arise when deriving parameter estimates in the presence of missing data,24,25 as dis- cussed in detail in the Online Supplementary Appendix, we imple- mented multiple imputation by chained equations (MICE) to gen- erate 100 independent datasets with missing data imputed;26 after analyzing imputed datasets, estimates were pooled using Rubin’s rules.27 Continuous variables were log10-transformed prior to multiple imputation. Subsequent analyses were performed on both imputed datasets and original pre-imputation datasets; the latter are shown in the Online Supplementary Appendix.
Analyses comprised:
- univariate correlations with hepcidin and ferritin, computing Pearson correlation coefficients;
- multivariate cross-sectional analyses at five and 12 months of age incorporating parameters returning P<0.2 in univariate analy- sis; Seemingly Unrelated Regression analysis,28 enabled simulta- neous modeling of the correlated outcomes, hepcidin and ferritin;
- longitudinal panel fixed effects models [estimating Driscoll- Kraay standard errors29 using Stata command xtscc (st0128)30] to enable investigation of how within-infant changes over time in time-variant explanatory variables predict changes in hepcidin and ferritin addressing any confounding induced by time-invariant characteristics (e.g. sex, genotype, ethnicity), whether measured or unmeasured; to visualize relative effect sizes, data were stan- dardized, fixed effects models were refitted, and forest plots were generated.
Ethical considerations
The Gambia Government/Medical Research Council (MRC) Joint Ethics Committee approved both studies (VPM: SCC1315; VA: SCC844); VA was also approved by the Ethics Committee of the London School of Hygiene and Tropical Medicine (LSHTM), Banjul, The Gambia. All participants were recruited via approved
haematologica | 2019; 104(8)
Rapid growth and hepcidin suppression in infancy
Laboratory analysis
Ferritin, plasma iron, soluble transferrin receptor (sTfR), α(1)- acid glycoprotein (AGP), C-reactive protein (CRP), transferrin (VPM only), and hemoglobin (VA only) concentrations were meas- ured by automated analyzers. Hepcidin was measured by manual ELISA (Bachem/Peninsula Laboratories, San Carlos, CA, USA).21
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