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

We introduce a method for monitoring non-transferrin-bound iron (NTBI), a labile and potentially toxic form of serum iron associated with imbalanced iron metabolism. The assay employs fluorescein-labeled apotransferrin (Fl-aTf), which undergoes fluorescence quenching upon binding iron. It has the advantages of simplicity, high sensitivity, and detection of those forms of NTBI that persist in sera with low transferrin saturations. Since NTBI is not readily available for detection, it is mobilized by 10 mM oxalate. Endogenous serum apotransferrin, capable of binding oxalate-mobilized NTBI, is blocked by 0.1 mM gallium(III). This metal, like iron, binds to Fl-aTf, but it neither quenches its fluorescence nor interferes with quenching by iron. Serum and reagent containing oxalate, Ga(Cl)(3), and Fl-aTf are mixed in multiwell plates and fluorescence is determined after 1 h in a microplate reader. To compensate for artifactual fluorescence changes caused by serum color, parallel samples are prepared with excess unlabeled apotransferrin, which scavenges all iron in the sample. Sera from eight hemochromatosis patients were tested for NTBI by the present assay and by an established alternative method, with qualitatively similar results. A potential application of the test is for screening large numbers of samples from patients at risk of developing NTBI.