Serum iron levels were quantified using the MAK025 iron assay kit (Sigma-Aldrich), following the manufacturer's protocol.
To quantify liver iron levels, the livers were first digested at 65°C in an acid digest solution [3 M HCl (Sigma-Aldrich), 10% trichloroacetic acid (Sigma-Aldrich)] overnight, with 1 ml of acid digest for every 100 mg of tissue. After digestion, the samples were vortexed and centrifuged at 830g for 5 min, and then 20 µl of the supernatant was added to 1 ml of chromogen reagent [2.25 M sodium acetate (Sigma-Aldrich) pretreated with Chelex 100 (Bio-Rad), 0.01% bathophenanthroline (Sigma-Aldrich), 0.1% thioglycolic acid (Sigma-Aldrich)]. The absorbances were read at 535 nm using a spectrometer (Beckman DU-640, Beckman Coulter) and the iron levels were calculated by comparing the absorbances with a serial dilution of iron standard (Sigma-Aldrich) mixed with the chromogen reagent.
To quantify liver iron levels, the livers were first digested at 65°C in an acid digest solution [3 M HCl (Sigma-Aldrich), 10% trichloroacetic acid (Sigma-Aldrich)] overnight, with 1 ml of acid digest for every 100 mg of tissue. After digestion, the samples were vortexed and centrifuged at 830