Hepcidin, Soluble Transferrin Receptor, and Other Biomarkers of Iron Status Distributions in Healthy 2 Years Old Infants from a National Ambulatory Study in France
Authors: Sacri Anne-Sylvia , Lefebvre Thibaud , De Montalembert Mariane , Bocquet Alain , Gembara Piotr , Pinçant Brigitte , Hercberg Serge , Levy Corinne , Ganon Amandine , Gouya Laurent , Chalumeau Martin 
Published: November 2019

Background:
Adequate evaluation of iron status in young children is of paramount importance given the frequency of iron deficiency (ID) and its potential short- and long-term neurocognitive adverse effects when occurs early. Iron metabolism is complex and the correct evaluation of iron status may be difficult, notably when inflammation is present. Soluble transferrin receptor (sTfR) is not modified by inflammation but lacks specificity in ID, and its combination with serum ferritin (SF) by the TfR-F index (TfR/logSF) has been proposed to improve diagnosis performances [Punnonen Blood 1997]. Hepcidin has been identified in the two last decades has the key regulator of iron homeostasis mainly by controlling iron release from macrophages via ferroportin degradation, as well as enterocytes absorption [Ganz Blood 2011]. Scarce studies have been published on hepcidin in healthy children in industrialized countries [Uijterschout Pediatr Res 2014]. The distribution of sTfR and hepcidin in healthy young children is unknown, including according to gender.
Aims:
Our objective was to describe hepcidin, sTfR and other iron status biomarkers (serum ferritin [SF], hemoglobin (Hb), transferrin saturation, zinc protoporphyrin [ZnPP]) distributions in a population of healthy infants aged 2 years old.
Methods:
In a cross-sectional observational study conducted in primary care pediatricians' offices throughout France from 2016 to 2017, infants aged 2 years old were consecutively included to undergo a blood sampling in the morning fasting. They were excluded if they were affected by a chronical disease involving iron metabolism, had fever in the last 15 days or biological inflammation defined as a CRP≥10 mg/L, and had no measurement for hepcidin. Hepcidin and ZnPP in erythrocytes were measured after a <24 hours frozen transport at -80°C in the dark by liquid chromatography-tandem mass spectrometry (LC-MS/MS, limit of detection = 0.75 ng/mL, intra- and inter-assay precision ≤ 12.3% and 9.9% [Lefebvre Clin Chem Lab Med 2015]) and by a fluorimetric method, respectively. Hb and mean corpuscular Hb concentration were measured immediately after the sample by spectrophotometric methods. The other iron biomarkers were immediately measured after refrigerated or frozen transport using immuno-turbidimetric method for transferrin (limit of detection = 0.1 g/L, intra- and inter-assay precision ≤1.2 and 2.6%) and sTfR (limit of detection 0.5 mg/L, intra- and inter-assay precision ≤2.1 and 3.6%), and electro‐chemiluminescence immunoassay for SF (limit of detection = 0.5 μg/L, intra- and inter-assay precision ≤1.1% and 5.7%). The distribution of biomarkers were described, including after classical mathematical transformation.
Results:
Among the 539 included children, the mean age was 24 months (SD 0.6), 49% were girls. Prevalence of ID (SF <10 µg/L) and anemia (Hb <11 g/dL) was 7% (37/539, 95% CI 4.9-9.3) and 3% (19/536, 95% CI 2.1-5.5), respectively. Hepcidin (ng/mL) median was 3.3 (IQR: 1.0-7.1; min-max: 0.75-104.1) and its mean was 6.7 (SD 10.7). Hepcidin was neither normally nor log-normally distributed (Figure 1.A, 1.B). sTfR median was 4.1 (IQR: 3.6-4.8; min-max: 1.2-13.5) and its mean was 4.2 (SD 1.1). TfR-F index median was 1.2 (IQR: 1.0-1.5; min-max: 0.3-8.4) and its mean was 1.4 (SD 0.6). No statistically significant difference was found between girls and boys for hepcidin, sTfR, and TfR-F index distributions (all p t-tests > 0.3). Distributions of other iron biomarkers were closed to those reported in the literature.
Conclusion:
We described for the first time in a nationwide ambulatory study hepcidin and TfR/logSF distributions in a population of 2 year-old healthy infants with a low ID prevalence. Hepcidin had a right-skewed distribution and its normalisation was not obtained by usual transformations. Low values, partly corresponding to the limit of detection, were over-represented despite the low proportion of ID defined by SF level. High values were also observed despite the exclusion of infants with CRP>10 mg/L. We did not find significant variations according to gender. Our results will help define normal values at this age to better interpret iron status.

Disclosures
Sacri: Secteur Francais des Aliments de l Enfance: Research Funding; French Ministry of Health DGOS PHRC regional 2014 no. AOR14053: Research Funding. De Montalembert:Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Addmedica: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Chalumeau:French Ministry of Health DGOS PHRC regional 2014 no. AOR14053: Research Funding; Secteur Francais des Aliments de l Enfance: Research Funding.