In vivo behavior of NTBI revealed by automated quantification system.

Non-Tf-bound iron (NTBI), which appears in serum in iron overload, is thought to contribute to organ damage; the monitoring of serum NTBI levels may therefore be clinically useful in iron-overloaded patients. However, NTBI quantification methods remain complex, limiting their use in clinical practice. To overcome the technical difficulties often encountered, we recently developed a novel automated NTBI quantification system capable of measuring large numbers of samples. In the present study, we investigated the in vivo behavior of NTBI in human and animal serum using this newly established automated system. Average NTBI in healthy volunteers was 0.44 ± 0.076 µM (median 0.45 µM, range 0.28-0.66 µM), with no significant difference between sexes. Additionally, serum NTBI rapidly increased after iron loading, followed by a sudden disappearance. NTBI levels also decreased in inflammation. The results indicate that NTBI is a unique marker of iron metabolism, unlike other markers of iron metabolism, such as serum ferritin. Our new automated NTBI quantification method may help to reveal the clinical significance of NTBI and contribute to our understanding of iron overload.

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

BACKGROUND: Iron 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. METHODS: We 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. RESULTS: The assay shows excellent linearity, reproducibility, and compatibility with HPLC, one of the most reliable conventional methods for NTBI quantification. CONCLUSIONS: Our 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.

Global analysis of the expression patterns of transcriptional regulatory factors in formation of embryoid bodies using sensitive oligonucleotide microarray systems.

We manufactured a highly sensitive oligonucleotide microarray system comprised entirely of transcription regulatory factors (a TF oligo microarray) in order to comprehensively analyze the expression profiles of transcription factors in mice. We compared the expression profiles of transcription regulatory factors in mouse embryonic stem (ES) cells and ES-differentiated cells by using this TF oligo microarray, a cDNA microarray, a GeneChip system, and quantitative RT-PCR. The TF oligo microarray was able to comprehensively analyze the expression profile of transcription regulatory factors. In addition, we used the manufactured TF oligo microarray to analyze the expression patterns of transcriptional regulatory factors during the formation of embryoid bodies. The TF array was able to reveal the chronologic expression profile of transcription regulatory factors involved in embryogenesis or the maintenance of pluripotency in ES cells.