Assessment of serum hepcidin levels and iron status

Serum hepcidin concentrations were estimated in all participants using a commercial ELISA kit (DRG Instruments, GmbH, Marburg, Germany), as previously mentioned in the literature [31]. The markers of iron status in this study were iron concentration, Hgb, serum ferritin (SF), and soluble transferrin receptor (sTfR).

Serum concentrations of iron were determined by inductively coupled plasma mass spectrometry (ICP-MS). Non-hemolyzed lipids and jaundice-free serum samples were used for the ICP-MS analysis. In this test, one millilitre of 5% HNO3 (Trace Metal Grade, Thermo Fisher, USA) solution was added to 0.5 ml serum sample to obtain a mixed solution, which was centrifuged at 12,000 rpm for 5 min to obtain the supernatant. After that, 3.5 ml 1% HNO3 solution was added to 0.5 ml supernatant, and then the mixture was centrifuged for 2 min at 2500 rpm and incubated at room temperature for 1 min to obtain the samples to be analyzed by ICP-MS (ICPMS-2030, Shimadzu, Japan). Finally, a certified commercial element standard solution (1000 mg/L) was applied to identify the accuracy and precision of the iron analysis [44–46]. Serum ferritin was measured by the Roche Tina-quant Ferritin immunoturbidimetric assay on the Hitachi 912 clinical analyzer (Roche Diagnostics)” [47]. SF “was estimated with Vitros Immunodiagnostic Products Ferritin Reagent Pack (Quest Diagnostics), sTfR was assessed with nephelometry (Quest Diagnostics). Hgb was assessed with Beckman Coulter LH 780 hematology analyzer (Quest Diagnostics)” [47]. “All three biomarkers (Hgb, SF, and sTfR) are commonly measured in studies assessing iron status during iron intake interventions” [48–50]. “Total body iron stores were also measured to evaluate tissue iron deficiencies status in many iron interventions” [2, 7, 48, 51, 52]. “It was estimated by quantifying the amount of SF and sTfR and is calculated from a validated previously reported method” [53] as follows;

{Total Body Iron Stores (mg/kg) = − [log 10 (sTfR × 1000/ SF-2.8229]/0.1207} [53]

Specifically, based on the status of iron concentrations, our participants were classified into four groups: iron deficiency group (n = 50; < 75 μg/dl), iron deficiency with anaemia group (n = 20; < 75 μg/dl and Hgb < 12.0), Normal group (n = 250; 75–175 μg/dl), and iron overload group (n = 30; high iron levels ˃ 175 μg/dl) as reported previously [54, 55].