Might nontransferrin-bound iron in blood plasma and sera be a nonproteinaceous high-molecular-mass Fe(III) aggregate?

The HFE (Homeostatic Fe regulator) gene is commonly mutated in hereditary hemochromatosis. Blood of (HFE)(−/−) mice and of humans with hemochromatosis contains toxic nontransferrin-bound iron (NTBI) which accumulates in organs. However, the chemical composition of NTBI is uncertain. To investigate, HFE(−/−) mice were fed iron-deficient diets supplemented with increasing amounts of iron, with the expectation that NTBI levels would increase. Blood plasma was filtered to obtain retentate and flow-through solution fractions. Liquid chromatography detected by inductively coupled plasma mass spectrometry of flow-through solutions exhibited low-molecular-mass iron peaks that did not increase intensity with increasing dietary iron. Retentates yielded peaks due to transferrin (TFN) and ferritin, but much iron in these samples adsorbed onto the column. Retentates treated with the chelator deferoxamine (DFO) yielded a peak that comigrated with the Fe–DFO complex and originated from iron that adhered to the column in the absence of DFO. Additionally, plasma from younger and older (57)Fe-enriched HFE mice were separately pooled and concentrated by ultrafiltration. After removing contributions from contaminating blood and TFN, Mössbauer spectra were dominated by features due to magnetically interacting Fe(III) aggregates, with greater intensity in the spectrum from the older mice. Similar features were generated by adding (57)Fe(III) to “pseudo plasma”. Aggregation was unaffected by albumin or citrate at physiological concentrations, but DFO or high citrate concentrations converted aggregated Fe(III) into high-spin Fe(III) complexes. Fe(III) aggregates were retained by the cutoff membrane and adhered to the column, similar to the behavior of NTBI. A model is proposed in which Fe(II) entering blood is oxidized, and if apo-TFN is unavailable, the resulting Fe(III) ions coalesce into Fe(III) aggregates, a.k.a. NTBI.