Plasma non-transferrin-bound-iron (NTBI) is believed to be responsible for catalyzing the formation of reactive radicals in the circulation of iron overloaded subjects, resulting in accumulation of oxidation products. We assessed the redox active component of NTBI in the plasma of healthy and ␤-thalassemic patients. The labile plasma iron (LPI) was determined with the fluorogenic dihydrorhodamine 123 by monitoring the generation of reactive radicals prompted by ascorbate but blocked by iron chelators. The assay was LPI specific since it was generated by physiologic concentrations of ascorbate, involved no sample manipulation, and was blocked by iron chelators that bind iron selectively. LPI, essentially absent from sera of healthy individuals, was present in those of ␤-thalassemia patients at levels (1-16 M) that correlated significantly with those of NTBI measured as mobilizer-dependent chelatable iron or desferrioxamine chelatable iron. Oral treatment of patients with deferiprone (L1) raised plasma NTBI due to iron mobilization but did not lead to LPI appearance, indicating that L1-chelated iron in plasma was not redox active. Moreover, oral L1 treatment eliminated LPI in patients. The approach enabled the assessment of LPI susceptibility to in vivo or in vitro chelation and the potential of LPI to cause tissue damage, as found in iron overload conditions. (Blood. 2003;102: 2670-2677)

Persistent levels of plasma nontransferrin bound iron (NTBI) have been associated with tissue iron overload and toxicity. We characterized NTBI's susceptibility to deferoxamine (directly chelatable iron [DCI]) and redox activity (labile plasma iron [LPI]) during the course of long-term, continuous L1 (deferiprone) treatment of patients with hemoglobin E disease and ␤-thalassemia (n ‫؍‬ 17). In 97% of serum samples (n ‫؍‬ 267), the LPI levels were more than 0.4 M (mean ؎ SEM, 3.1 ؎ 0.2 M) and the percent transferrin (Tf) saturation more than 85 (111 ؎ 6), whereas only in 4% of sera were the LPI levels more than 0.4 M for Tf saturation less than 85%. Daily administration of L1 (50 mg/kg) for 13 to 17 months caused both LPI and DCI to decrease from respective initial 5.1 ؎ 0.5 and 5.4 ؎ 0.6 M to steady mean levels of 2.18 ؎ 0.24 and 2.81 ؎ 0.14 M. The steady lowest levels of LPI and DCI were attained after 6 to 8 months, with a half time (t 1/2 ) of 2 to 3 months. Serum ferritin and red cell membraneassociated iron followed a similar course but attained steady basal levels only after 10 to 12 months of continuous treatment, with a t 1/2 of 5 to 7 months. These studies indicate that LPI and DCI can serve as early indicators of iron overload and as measures for the effectiveness of iron chelation in reducing potentially toxic iron in the plasma. IntroductionIron chelation therapy with deferoxamine (DFO) results in a significant improvement in the life expectancy of patients with transfusional iron overload. This is largely attributed to the prevention of heart disease in well-treated thalassemia major patients and, in a few, to the reversal of existing heart disease by aggressive DFO therapy. 1,2 The orally active L1 (deferiprone) is more convenient for patients because, unlike DFO, it does not require a special transfusion pump or costly disposable infusers. Since its introduction in 1987, 3 the orally active L1 has undergone clinical trials for treating thalassemia major patients. 4,5 Although in some patients it might be as effective as DFO given subcutaneously, in others higher doses of DFO or combined therapy with L1 may be needed to provide adequate iron chelation. 6 L1 seems to be particularly useful for patients with thalassemia intermedia, who accumulate iron at a much slower rate in the absence of transfusions. Early studies on those patients indicated a reduction in serum ferritin 7 and liver iron stores 8 following L1 treatment of up to 12 months. A more recent study of L1 has shown significant falls in serum ferritin and red cell membrane iron followed periodically over an 80-week period. 9 Long-term treatment with L1 also led to a major reduction in end-point levels of liver iron and circulating forms of labile iron, collectively known as nontransferrin bound iron (NTBI), 10 that are potentially harmful to the heart and other organs.The present prospective study was undertaken with the aim of assessing the possibility that the basal levels of plasma NTBI could serve as early indicators of iron overload...