2.5. Biochemical and Mineral Measurements

The concentrations of HEPC, LTF, HCY, Ft, and ErFe in the collected serum were determined using enzyme-linked immunosorbent assay (ELISA). Commercial ELISA kits were employed (Fine Test, Wuhan Fine Biological Technology, Hubei, China) and absorption spectrophotometry was used (LEDetect96, Labexim, Lengau, Austria). The accuracy of the concentration measurements was checked in each case by the following procedure: 2 standards were run as samples, and their calculated concentrations were compared to nominal ones; additionally, 3 randomly chosen rat samples were run in triplicate and their concentrations were compared. In each procedure, coefficient of variance did not exceed 5%. Reproducibility was verified using the control serum sample provided by the kit producer. Serum C-reactive protein (CRP) was measured at a commercial laboratory.

Liver sample homogenates were prepared using an automatic homogenizer (MagNALyser, Roche, Basel, Switzerland). The concentrations of DMT1, TfR1, TfR2, and ZIP14 in the liver homogenate samples were estimated using commercial ELISA kits (Shanghai Qayee Biotechnology, Shanghai, China, for DMT1, TfR1, and TfR2; Bioassay Technology Laboratory, Shanghai, China, for ZIP14).