Functional Validation and Clinical Significance Of a Newly Established Non-Transferrin-Bound Iron (NTBI) Assay System Utilizing Conventional Automated Analyzer
Authors: Ito Satoshi , Ikuta Katsuya , Addo Lynda , Hatayama Mayumi , Toki Yasumichi , Tanaka Hiroki , Inamura Junki , Shindo Motohiro , Torimoto Yoshihiro , Kohgo Yutaka 
Published: October 2019

Abstract

Introduction
Iron is an essential metal in the body. However, iron overload is toxic, as excess ‘free’ reactive iron produces damaging free radicals which can lead to cellular and organ damage. Iron homeostasis is therefore tightly regulated. However, when iron balance collapses (as in prolonged transfusion), transferrin (Tf) becomes fully saturated and non-Tf-bound iron (NTBI) appears in serum. NTBI levels are increased in various iron overload states, and decreased after treatment with iron chelators (example deferasirox) in thalassemia and hemochromatosis, and is therefore important in evaluating and monitoring iron toxicity risks. Although several NTBI measurement methods have been reported, they are extremely complicated and low in sensitivity, thus very few laboratories can quantify NTBI. Consequently, NTBI research has not progressed significantly to date. We previously established a novel assay system utilizing automated analyzers (used widely in clinical laboratories for diagnostic testing), which we presented at ASH 2012 poster session. Using this assay, we sought to measure NTBI in iron overloaded animals, healthy volunteers and patients’ sera.

Methods
Data was analyzed using the HITACHI 7700 auto analyzer. Human serum was obtained from 41 healthy volunteers (16 males, 25 females) and 118 patients (61 males, 57 females) receiving treatment at the Asahikawa Medical University Hospital. Average age of healthy volunteers was 34.4 years and 60.6 years in patients. The primary diagnosis in patients included malignant lymphoma, acute myeloid leukemia, myelodysplastic syndromes, multiple myeloma and others. Patient data, including hemoglobin, biochemical markers including C-reactive protein (CRP), serum iron (sFe), unsaturated iron binding capacity (UIBC) and serum ferritin were obtained from the patients’ records or determined for the healthy volunteers. Mice were administered intraperitoneal injections of physiological saline solution or iron-dextran (Fe 1 mg/day or Fe 10 mg/day) for 5 days, after which serum was collected. Rats received intravenous injections of physiological saline or iron sucrose. Serum was collected after 1, 3 and 6 hours iron injection. Informed consent was obtained from all study subjects, and study protocol and experimental procedures were approved by the Ethical and Animal Experiments Committee of Asahikawa Medical University and Hospital. Statistical analysis was done using Mann-Whitney U-test and Student paired t-test.

Results and Conclusion
Median NTBI in healthy volunteers was 0.45 μM; no statistical difference was found between the sexes. Median NTBI in the patient group was 0.38 mM, a slight decrease to that of the healthy volunteers (statistical significance p=0.0144). In transferin saturation (TSAT) and NTBI measurement in the patients, NTBI increased markedly as TSAT reached over 80%. A slightly positive correlation was found between sFe and NTBI, but no significant correlation was observed between serum ferritin and NTBI. CRP>0.3 mg/dL is a positive indicator of inflammation, so median NTBI was compared with CRP-positive and -negative groups; NTBI decreased significantly in the CRP-positive group (p<0.05). On the other hand, median serum ferritin significantly increased in the CRP-positive group (p<0.05). This data shows NTBI is an unmistakably unique marker of iron metabolism unlike serum ferritin. This characteristic of NTBI may be helpful in overcoming problems with serum ferritin use as a marker of iron metabolism (serum ferritin is affected by inflammation), and provide additional information that directly reflects changes in iron metabolism, even in inflammatory states.
Compared to the control group, a statistically significant increase in NTBI was observed in the Fe 10 mg/day mice group. After intravenous iron administration in the rats, NTBI was 0.16±0.04 μM at pre-treatment, and rapidly increased to 2.78±0.62 μM after 1 hour iron injection; this increase decreased over time, indicating that NTBI can be used not only as a marker to evaluate iron overload but also to precisely monitor dynamic changes in iron in serum. Our novel system revealed new findings and it indicates that this system must be useful for studying the physiological and clinical importance of NTBI.

Disclosures:
No relevant conflicts of interest to declare.