Carbonyl iron

Ferric pyrophosphate (soluble crystals), ferric sodium EDTA hydrate, ferric ammonium citrate, ferric citrate hydrate, ferrous gluconate hydrate, carbonyl iron, ferrous lactate hydrate, ferric chloride hexahydrate, ferrous fumarate, ferrous sulfate heptahydrate, and ferric sulfate hydrate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ferrous bisglycinate was purchased as a dietary supplement in capsules targeted for consumers from the online store Supersmart (headquarters in Luxembourg). Stock solutions ([Fe] = 5 mM) of each compound were made fresh before each experiment.
In the initial experiments, ferrous fumarate, ferrous bisglycinate, Ferric pyrophosphate, and carbonyl iron were dissolved in hydrochloric acid (50 mM), while the others were dissolved in ultrapure water. In later experiments, dissolution in hydrochloric acid was omitted.

Mice were fed a commercial diet containing ~200 mg of iron per kg (Teklad Global 18% protein rodent diet; Harlan Teklad, Madison, WI). Dietary iron overload was produced by feeding 8-week-old mice the same commercial diet supplemented with 25 g carbonyl iron per kg (2.5% wt/wt carbonyl iron; Sigma-Aldrich, St. Louis, MO, USA) for 2 weeks (Jiang et al., 2008 (link)). All animals were 10-weeks old at the end of the experiments.

Peripheral blood lymphocytes were isolated using the carbonyl iron granulocyte depletion method, followed by density gradient centrifugation with Lymphoprep^TM, as previously described (8 (link), 23 (link)). In brief, blood was collected aseptically via a jugular venipuncture into sterile 60-mL syringes with 17 IU/mL of lithium heparin (Sigma-Aldrich), and plasma was allowed to separate for 20 min at room temperature (RT). Plasma was separately collected into conical tubes using extension sets and incubated with carbonyl iron (Sigma-Aldrich) in agitation for 30 min at 37°C. Then, carbonyl iron was placed at the bottom of the tubes by using a magnet, and supernatant was collected and centrifuged at 310 × g for 5 min. The cellular pellet was resuspended in PBS and overlayed on Lymphoprep^TM. After centrifugation at 690 × g for 15 min (without brake), the lymphocyte layer was recovered and washed with PBS. The cells were counted in a hemocytometer chamber using 0.4% trypan blue as dye exclusion. This isolation technique has been reported to provide an enriched lymphocyte population (95–99%) (8 (link), 24 (link)).

Previously cryopreserved MSCs were thawed and plated at 50,000 cells per well for 24 h prior to inactivation with mitomycin C (Sigma Aldrich) as previously described (23 (link)). Responder and stimulator lymphocytes were isolated from two unrelated donors using a Ficoll (GE Healthcare) gradient with the addition of carbonyl iron (Sigma Aldrich) (24 (link)). Stimulator lymphocytes were inactivated by incubation with 50 μg/ml mitomycin C for 30 min and then added at a density of 1 x 10⁶ stimulator lymphocytes per well. Responder lymphocytes were stained with a commercially available nuclear stain (CellTrace® Violet, Thermo Fisher) and 2 x 10⁶ responder lymphocytes were added to each well. Cultures were maintained for 5 days, after which lymphocytes were collected and stained with anti-equine CD3+ antibody (UC Davis) at a 1:200 dilution. Flow cytometry was then performed on CD3+ T lymphocytes to assess proliferation with the use of commercially available software (FlowJo™ Software). Stained, unstimulated responder lymphocytes were used as a negative proliferation control, Concanavalin A (Sigma Aldrich) stimulated responder lymphocytes were used as a positive proliferation control, and changes in mean fluorescence intensity were evaluated as a percent change from the negative control as previously described (25 (link)).

Weanling male Sprague-Dawley rats (Charles River Laboratories) were randomized (n = 6/group) to receive either iron-deficient (FeD), iron-adequate (FeA), or iron-overloaded (FeO) diets. Purified diets were prepared according to the AIN-93G formulation, but with no added iron (FeD), 35 mg/kg ferric citrate (FeA), or 2% carbonyl iron (Sigma-Aldrich) (FeO). Iron contents of the diets, as determined by inductively coupled plasma mass spectroscopy (ICP-MS), were 5 ppm (FeD), 36 ppm (FeA), and 20,275 ppm (FeO). Diets were also modified to contain Avicel® microcrystalline cellulose instead of cellulose (to minimize contaminant iron) and 20% sucrose instead of 10% sucrose (while reducing the amount of cornstarch accordingly) [11] (link). The amount of sucrose was increased in order to make the iron-loaded diet more palatable. After 3 weeks of feeding, overnight-fasted rats were sacrificed by exsanguination from the descending aorta. Blood was collected into heparinized syringes and then centrifuged to obtain plasma. Pancreases were quickly harvested, immediately frozen in liquid nitrogen, and maintained at −80°C for subsequent analyses. Animal experiments were approved by the Institutional Animal Care and Use Committee at the University of Florida (Protocol # 201101613).

The standard iron chelating drug, deferoxamine (DFO) was obtained from Novartis International AG (Basel, Switzerland). Carbonyl iron, GA, soybean lecithin, ethanol, vitamin C, ferric chloride, ferrous chloride, linoleic acid, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2, 2-azinobis (3-ethylbenzo-thiozoline-6-sulfonic acid) (ABTS), potassium ferricyanide and 30% hydrogen peroxide were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The assay kits for alanine transaminase (ALT) (C009-2-1), aspartate transaminase (AST) (C010-2-1), γ-glutamyl transferase (γ-GT) (C017-2-1), and superoxide dismutase (SOD) (A001-3-2), glutathione (GSH) (A006-2-1), malondialdehyde (MDA) (A003-1-1), total cholesterol (TC) (A111-1-1), total iron-binding capacity (TIBC) (A040-1-1), triglyceride (TG) (A110-1-1) were procured from Nanjing Jiancheng Biotechnology Co., Ltd. (Nanjing, China). Antibodies against transferrin receptor (TfR1) (ab84036), Ferritin (ab153976), ferritin light chain (FL) (ab109373) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (ab198306) were all purchased from Abcam Inc. (Cambridge, MA, USA).