Serum iron assay kit

DFO, DFX, ferrous chloride (FeCl₂), ferric chloride (FeCl₃), and iron dextran were obtained from Sigma Aldrich (USA). Ca‐AM was purchased from the AAT bioquest (USA). CCK‐8, Cremopho EL, and MDA assay kits were purchased from Solarbio (Beijing, China). Serum iron assay kit, alanine aminotransferase assay kit (ALT), aspartate aminotransferase assay kit (AST), lactate dehydrogenase assay kit (LDH), and Cr assay kit were obtained from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). C11‐BODIPY^581/591 probes were obtained from Invitrogen (USA). FerroOrange probes were purchased from Dojindo Molecular Technologies, Inc. (Japan). Methanol and acetonitrile were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Dulbecco's modified Eagle's medium (DMEM), RPMI 1640, and phosphate buffered saline (PBS) were purchased from Corning (USA). Fetal bovine serum (FBS) was purchased from Gibco Inc. (USA).

Serum iron levels were measured with a serum iron assay kit according to the manufacturer's instructions (Nanjing Jiancheng Bio-Engineering Institute, China). Serum hepcidin levels were assessed using enzyme-linked immunosorbent assay (Elisa) following the manufacturer's protocol (Cloud-Clone Corp., China).

The concentrations of hemoglobin in the serum were determined using an automated hematology analyzer (Sysmex K-1000D, Sysmex Inc., Kobe, Japan). The concentrations of serum iron and total iron binding capacity (TIBC) in the serum were measured using commercial kits (serum iron assay kit, No: A039-1; TIBC assay kit, No: A040, Jiancheng, China). The concentrations of hepcidin in the serum and liver were measured by using a hepcidin ELISA kit (NO: H252, Jiancheng, China). Iron concentrations in the tissues and digesta were measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES) according to the procedures described previously [17 (link)]. Briefly, 0.25 g of tissues or digesta (dried samples, the tissues and digesta were put in the freeze dryer for 24 h), 5 mL of 65% HNO₃ and 2 mL of 30% H₂O₂ were mixed in a polytetrafluoroethylene (PTFE) tube. Then, the samples were digested in a microwave sample digestion system (MARS 6, CEM Corporation, Matthews, NC, USA). Afterwards, the PTFE tubes were kept in a heater to remove acid. Five percent of HNO₃ was used to dissolve the tube residues in the PTFE tubes. Finally, the prepared solution was stored in a refrigerator at 4 °C until analysis.

Blood samples for analysis of iron metabolism were drawn as soon as patients were admitted to the hospital. Blood was centrifuged within 1 hour of being drawn at 2500 g for 10 minutes at room temperature, and serum was collected and stored at −80℃.Serum levels of iron were quantified by the serum iron assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Routine biochemical analyses, including TF and the soluble TFR (sTFR), were determined by conventional assays. Bottom and peak levels of these biochemical molecular analyses were defined as the minimum and maximum value measured during hospitalization. The inter‐assay and intra‐assay coefficient of variation were <7% for all the latter analyses.

The day before they were euthanized, the SD rats were fasted overnight with free access to deionized water. Then, the rats’ body weights were recorded, and they were administered 1 mL FeSO₄ or Fe-Gly at a relatively high dose (800 mg/L as iron). Two hours after gavage, the rats were anesthetized with chloral hydrate, and blood was collected from their eyeballs. The whole blood samples were sent to the Laboratory Animal Center of Zhejiang University for hematological measurements. Sera were separated by centrifugation at 3, 000×g for 10 min at 4 °C, and the iron levels were determined by using a serum iron assay kit (Jiancheng Bioengineering Institute, Nanjing, China).
Then, the rats were sacrificed by cervical dislocation, and liver specimens were obtained and fixed in 4% formaldehyde for immunohistochemical analysis. Approximately 3 cm of the duodenum was removed from each rat, washed with normal saline, and packed with sterile and RNase-free silver paper. After being rapidly frozen in liquid nitrogen, the samples were stored at −80 °C until RNA extraction.

The total iron concentration of serum and tissues (heart, liver, spleen, kidney, testis) was measured using Serum Iron Assay Kit and Tissue Iron Assay Kit from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China) according to the manufacturer’s instructions. The serum was obtained by retro orbital bleeding, and the supernatant was collected after centrifugation at 2000× g for 20 min at 4 °C.