Iron tibc reagent set

All iron-related measurements were obtained at the time of euthanasia. Non-heme iron of spleen, liver, and stool pellets were determined using a wet ashing procedure.^{58 (link)} In brief, the wet weight of samples obtained at necropsy was quantified and portions of liver, spleen, or stool pellets, were placed in 2 ml micro-tubes. Following desiccation at 65 °C for 24 h, 1 ml of acid digestion mixture (3 M hydrochloric acid, 10% trichloracetic acid) was added and samples were heated at 65 °C for an additional 24 h. The acidified sample (50 μl) was then incubated for 30 min with 200 μl of chromogen (1.6 M bathophenanthroline, 2 M sodium acetate, 11.5 M thioglycolic acid). Absorbance at 535 nm of samples and iron standards was measured in duplicate and mean values used for calculating total iron. Iron transferrin saturation in plasma was calculated using Iron/TIBC Reagent Set (Pointe Scientific, Canton, MI). Plasma ferritin, plasma hepcidin, and liver ALT levels in liver were measured by ELISA following the manufacturer’s instructions (Kamiya Biomedical, Seattle, WA; Intrinsic LifeSciences, La Jolla, CA; and Biomatik, Wilmington, DE, respectively). AST was measured in liver using the AST activity assay kit (Sigma Aldrich, St. Louis, MO).