Non‐transferrin bound iron in Thalassemia: Differential detection of redox active forms in children and older patients

Abstract

Non-transferrin bound iron (NTBI) is commonly detected in patients with systemic iron overload whose serum iron-binding capacity has been surpassed. It has been perceived as an indicator of iron overload, impending organ damage and a chelation target in poly-transfused thalassemia patients. However, NTBI is a heterogeneous entity comprising various iron complexes, including a significant redox-active and readily chelatable fraction, which we have designated as ''labile plasma iron'' (LPI). We found that LPI levels can be affected by plasma components such as citrate, uric acid, and albumin. However, the inclusion of a mild metal mobilizing agent in the LPI assay (designated here as ''eLPI''), at concentrations that do not affect transferrin-bound iron, largely overcomes such effects and provides a measure of the full NTBI content. We analyzed three distinct groups of poly-transfused, iron overloaded thalassemia patients: non-chelated children (3-13 yrs, Gaza, Palestine), chelated adolescents-young adults (13-28 yrs, Israel), and chelated adults (27-61 yrs, Israel) for LPI and eLPI. The eLPI levels in all three groups were roughly commensurate (r 2 5 0.61-0.75) with deferrioxamine-detectable NTBI, i.e., DCI. In older chelated patients, eLPI levels approximated those of LPI, but in poly-transfused unchelated children eLPI was notably higher than LPI, a difference attributed to plasma properties affected by labile iron due to lack of chelation, possibly reflecting age-dependent attrition of plasma components. We propose that the two formats of NTBI measurement presented here are complementary and used together could provide more comprehensive information on the forms of NTBI in patients and their response to chelation. Am. J. Hematol. 87:55-61, 2012. V

The role of endocytic pathways in cellular uptake of plasma non-transferrin iron

“…17,18 The assay is based on the binding of NTBI in serum to fluorescein-DFO (Fl-DFO), causing quenching of its fluorescence. Briefly, each serum sample is measured under two separate conditions: A, with Fl-DFO only; and B, as in condition A but in the presence of a large excess of non-fluorescent DFO.…”

“…The fact that inclusion of nitrilotriacetic acid in the DCI assay did not reveal additional DFO chelatable material was taken as an indication that the DCI values represented most of the NTBI fraction. 18 …”

“…All sera were initially tested for iron related parameters serum ferritin and transferrin saturation and for non-transferrin bound iron (NTBI) measured as directly chelatable iron (DCI). 18 Sera with DCI over 1.2 μM were used as source of high NTBI (hNTBI) and those with values below 0.4 μM as low NTBI (lNTBI). Model NTBI-containing sera were prepared by supplementing 20 μM ferric citrate to human sera that had originally a lower than 70% transferrin saturation and thereby attained NTBI levels of 10-15 μM (measured as DCI, see below).…”

Second international round robin for the quantification of serum non-transferrin-bound iron and labile plasma iron in patients with iron-overload disorders

“…[1][2][3][4][5] Plasma NTBI apparently comprises several subspecies, which may be classified by their chemical composition, chemical reactivity or susceptibility to chelation. [6][7][8][9][10][11][12][13][14][15] The chemical composition of NTBI is heterogeneous and it is thought that there are several circulating isoforms, that is Fe(III) bound to albumin and citrate and potentially to acetate, malate and phosphate. [6][7][8]14 Of these, citrate has the highest affinity for Fe(III), and under physiological conditions two isoforms dominate, i.e.…”

“…5,13,24 Due to the complexity and potential clinical importance of NTBI, several assays have been developed for its detection. 6,[10][11][12]15,[25][26][27][28][29] In our previous round robin 1 we found that NTBI values of patients with HFE-related hemochromatosis differed considerably depending on which of various assays was used. 30 We concluded that NTBI assays were insufficiently standardized and the most pertinent assay for clinical applications was uncertain.…”