Improved quantification for non-transferrin-bound iron measurement using high-performance liquid chromatography by reducing iron contamination

Non-transferrin-bound iron (NTBI) refers to all forms of iron in the plasma that bind to ligands other than transferrin, and is considered to be a marker of iron toxicity. A variety of analytical approaches for measuring NTBI have been reported; however, a clinically relevant level of sensitivity has yet to be achieved. In addition, insufficient values of NTBI in some patients and healthy subjects have led to the assumption that there may be contamination of reagents with background iron. The present study re-evaluated the analytical procedures of the assay with regard to the potential points of iron contamination in each step. NTA and tris carbonatocobaltate (III) solutions were prepared with removal of iron contamination, and then quantification of NTBI was performed. As a result, the sensitivity of the high-performance liquid chromatography (HPLC)-based NTBI method was improved by the successful reduction of background iron contamination. Application of our modified method proved that NTBI was detected even in healthy volunteers, although the concentrations were extremely low; the average NTBI levels were 0.206±0.091 µM (males, n=20) and 0.212±0.095 µM (females, n=16). Thus, our modification of the NTBI assay may be clinically meaningful, and may contribute to the understanding of the clinical significance of relatively low, but elevated concentrations of NTBI in diseases other than typical iron overload.