Abstract
Blood contains a poorly characterized pool of labile iron called non-transferrin-bound iron (NTBI). In patients with iron-overload diseases such as hemochromatosis, NTBI accumulates in the liver, heart, and other organs. This material is probably nonproteinaceous and low molecular mass (LMM). However, the number, concentration, mass, and chemical composition of NTBI species remain unknown despite decades of effort. Here, solutions of plasma from humans, pigs, horses, and mice were passed through a 10 kDa cutoff membrane, affording flow-through solutions (FTSs) containing ∼1 μM iron. The FTSs were subjected to size-exclusion liquid chromatography at pH 8.5, 6.5, and 4.5. Iron was detected by an online inductively-coupled-plasma mass spectrometer. LC-ICP-MS chromatograms of the FTSs exhibited 2-6 iron-containing species with apparent masses between 400 and 2500 Da. Their approximate concentrations in plasma were 10-8-10-7 M. Not every FTS sample contained every LMM iron species, indicating individual variations. The most reproducible iron species had apparent masses of 400 and 500 Da. Chromatograms of the FTSs from established hemochromatosis patients exhibited no significant differences relative to controls. The peak positions and intensities depended on column pH. Some FTS iron adsorbed onto the column, especially at higher pH. Column-adsorbing-iron coordinated apo-transferrin whereas the more tightly coordinated iron species did not. Ferric citrate standards exhibited LMM iron peaks that were similar to but not the same as those obtained in FTSs. The results indicate that the LMM iron species in healthy blood plasma is not primarily ferric citrate; however, this may be one of many contributing complexes.