Abstract
Iron overload is a key contributor to the pathogenesis of multiple human disorders including the sideroblastic anemias. The specific iron compounds present in tissues or cells that are the targets of excess iron deposition remain poorly understood. Our objective is to further identify the composition of such iron aggregates. As there is evidence that some forms are magnetically active, we have developed a simple and specific method to purify iron-overloaded red blood cells using magnetic affinity columns. RBC derived from mice transplanted with SOD2-deficient hematopoietic stem cells served as a source of iron-laden cells (Friedman et al., Blood 2004; J. Exp. Med. 2001). Purification was based upon the observation that iron deposits in SOD2-deficient cells are magnetically susceptible and allow for retention of iron-laden cells in a strong magnetic field. Peripheral blood from SOD2-deficient chimeric mice was passed through magnetic separation columns; iron-overloaded cells were eluted and subsequently characterized by flow cytometry, western blot and microscopy. We were able to purify 3% of the total red cells as iron-laden siderocytes. More than 93% of the magnetically purified SOD2-deficient cells were identified as reticulocytes; they had numerous siderotic granules, produced enhanced reactive oxygen species levels, and showed increased protein oxidative damage, mitochondrial enrichment and mitochondrial membrane potential. Our method can be used to purify iron-laden cells as well as iron-associated subcellular fractions prepared from iron-loaded tissues, allowing elucidation of the structure, location and protein composition of such iron deposits. We believe that this data will help develop our understanding of the pathogenesis of sideroblastic anemia and other disorders characterized by iron overload.
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