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. Introduction Non-transferrin-bound iron (NTBI) refers to all forms of iron in the plasma that bind to ligands other than transferrin (Tf), and was first measured by Hershko et al in 1978 (1). NTBI is increased during iron overload, and is considered to be a marker of iron toxicity (2). In addition, NTBI is decreased to the detection limit by long-term therapies with iron chelators such as deferoxamine and deferasirox in thalassemic patients, and by venesection in hemochromatotic patients (3,4). Therefore, the determination of NTBI is crucial for evaluating and monitoring the risk of iron toxicity (5). A variety of analytical approaches for measuring NTBI have been reported (6-14). In certain methods, NTBI is initially mobilized and chelated by shuttle molecules such as nitrilotriacetic acid (NTA), followed by separation of the chelated iron from serum proteins using ultra-filters. The ultra-filtrate was then analyzed by atomic absorption spectrometry (AAS) (6,7), the colorimetric reaction with bathofenanthroline (8) or highperformance liquid chromatography (HPLC) (9,10). In other methods, NTBI was mobilized and detected in the same reaction mixture without separation by ultra-filtration. Bleomycin (11), iron-sensitive fluorescence probes such as calcein (12), fluorescein-labeled desferrioxamine (13) and fluorescencelabeled apo-transferrin (apo-Tf) (14) were reported to be used for the latter methods. Among these different systems for measuring NTBI, the HPLC method is one of the most convenient methods that was developed initially and was widely used. Consequently, HPLC has been considered to be a ‘gold standard method’ for NTBI measurement. Moreover, HPLC is more sensitive than most methods, with the exception of AAS. However, even using the HPLC method, it has not been possible to fully quantify NTBI at concentrations lower than 0.47 μM, which was reported as the limit of detection (LOD) (9). In addition, various studies on NTBI values of healthy individuals described NTBI concentrations as being negative values. For example, the ranges of NTBI determined by the HPLC method in healthy volunteers were reported to be -0.94±0.08 (15) or -0.30 μmol/l (-1.5 to 0.6 μmol/l) (16). Furthermore, the ability to monitor Improved quantification for non-transferrin-bound iron measurement using high-performance liquid chromatography by reducing iron contamination KATSUNORI SASAKI1, KATSUYA IKUTA2, HIROKI TANAKA1, TAKAAKI OHTAKE2, YOSHIHIRO TORIMOTO3, MIKIHIRO FUJIYA2 and YUTAKA KOHGO2 1Department of Gastrointestinal Immunology and Regenerative Medicine; 2Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University; 3Oncology Center, Asahikawa Medical University Hospital, Asahikawa, Hokkaido 078-8510, Japan Received March 4, 2011; Accepted June 23, 2011 DOI: 10.3892/mmr.2011.518 Correspondence to: Dr Katsuya Ikuta, Division of Gastroenterology and Hematology/Oncology, Department of Medicine, Asahikawa Medical University, 2-1-1-1 Midorigaoka-Higashi, Asahikawa, Hokkaido 078-8510, Japan E-mail: ikuta@asahikawa-med.ac.jp