A fluorescence-based one-step assay for serum non-transferrin-bound iron

Area of Science:

• Biochemistry
• Analytical Chemistry
• Clinical Chemistry

Background:

• Non-transferrin-bound iron (NTBI) is a labile and potentially toxic serum iron species linked to imbalanced iron metabolism.
• Existing methods for NTBI detection can be limited in sensitivity and scope, particularly in conditions with low transferrin saturation.

Purpose of the Study:

• To introduce a novel, sensitive, and simple assay for monitoring non-transferrin-bound iron (NTBI) in serum.
• To enable the detection of NTBI forms that persist even at low transferrin saturations, improving the assessment of iron metabolism.

Main Methods:

• The assay utilizes fluorescein-labeled apotransferrin (Fl-aTf) that exhibits fluorescence quenching upon binding iron.
• NTBI is mobilized using oxalate, and endogenous apotransferrin is blocked with gallium(III) to prevent interference.
• Fluorescence is measured using a microplate reader after incubation, with adjustments for serum color artifacts.

Main Results:

• The developed assay demonstrates high sensitivity and simplicity for NTBI detection.
• It successfully detects NTBI forms in sera with low transferrin saturations.
• Qualitative agreement was observed between the novel assay and an established method when tested on hemochromatosis patient sera.

Conclusions:

• This assay provides a reliable method for quantifying NTBI, a marker of potentially harmful iron dysregulation.
• Its sensitivity and ability to detect NTBI in low transferrin saturation conditions make it suitable for screening individuals at risk of iron overload disorders.
• The assay offers a valuable tool for clinical diagnostics and research in iron metabolism and related diseases.