Non-transferrin-bound iron assay system utilizing a conventional automated analyzer

• NTBI assay reagents that can be used in automated analyzers were established.
• Our automated system can distinguish NTBI from Tf-bound iron.
• Sufficient dynamic range to analyze serum with high NTBI values was obtained.
• Inter- and intra-assay precision were satisfactory.
• The automated system was shown to be compatible with the conventional HPLC method.

BackgroundIron is an essential metal in the body, but its excessive accumulation causes damage in various organs through free radical production. Iron homeostasis is therefore tightly regulated. However, when iron balance collapses, such as in prolonged transfusion, transferrin (Tf) is fully saturated and non-Tf-bound iron (NTBI) appears in the serum. Monitoring serum NTBI levels is therefore crucial in the assessment of the clinical status of patients with iron overload, since NTBI is associated with cellular and organ damage. Several methods for NTBI determination have been reported, but these are extremely complicated and very few laboratories can quantify NTBI at present.MethodsWe established a novel assay system utilizing automated analyzers that are widely used in clinical laboratories for diagnostic testing. In this assay, NTBI is chelated by nitrilotriacetic acid (NTA), after which the iron is reduced and transferred to nitroso-PSAP, a chromogen.ResultsThe assay shows excellent linearity, reproducibility, and compatibility with HPLC, one of the most reliable conventional methods for NTBI quantification.ConclusionsOur novel method for NTBI measurement is high-throughput and may be a useful and powerful tool in the study of the physiological and clinical importance of NTBI.