My involvement in iron research started in the laboratories of Clem Finch in 1970 after receiving an NIH International Post Doctoral fellowship. Clem was puzzled at that time by the apparent difference in toxicity of iron accumulated in the RE system following multiple transfusions, as against parenchymal siderosis typical of heredirary hemochromatosis. Comparable amounts of iron in RE cells were relatively non-toxic whereas parenchymal siderosis was associated with serious damage to the liver, heart and endocrine organs.
I started working in the laboratory under the guidance of Jim Cook and was trying to establish an experimental model of storage iron regulation in rats. We identified a number of radioiron labelled compounds that, when injected intravenously, homed to iron storage pools. Heat denatured erythrocytes were taken up mainly by splenic RE cells. By contrast, radioiron labeled ferritin accumulated almost exclusively in the liver. This gave us an opportunity to explore systematically the difference in behavior of the two cellular systems under various physiologic and pathologic stimuli such as increased or suppressed erythropoiesis, inflammation, hypoxia, hypertransfusion and response to iron chelators.
In 1972 I returned to Israel and in addition to my clinical responsibilities, continued my animal studies expanding subsequently to studies in patients with transfusional iron overload. This could not have been done without the support of my research associates Gabriela Link, Appie Konijn and more recently Ioav Cabantchik. I was fortunate enough to be able to acquire long term support through NIH RO1 grants acting as PI, an almost impossible mission for non US residents. In Jerusalem I focused my studies on iron toxicity, and the mechanism of action of iron chelators. In 1972, research on iron chelating therapy was extremely limited. Deferoxamine (DF) was available but there was no convincing evidence of its clinical usefulness. The only additional iron chelator available for use in humans was DTPA, a compound intended for treating lead poisoning. DTPA is hydrophilic, poorly soluble in lipids and unable to penetrate cells.
Studies in our animal model have shown that DF was able to promote the excretion of both parenchymal and RE iron. Chelated RE iron was excreted by the kidneys. By contrast, chelated parenchymal iron was excreted in the bile. Studies in thalassemic patients confirmed the concept of this dual pathway of in vivo iron excretion following chelation therapy with DF. However, studies with DTPA yielded a very different picture. Although DTPA infusion was able to promote iron excretion in the urine with an efficiency almost equal to DF, it failed to interact with either RE or hepatocellular iron stores. Iron bound in vitro to DTPA and injected IV was recovered almost completely in the urine. These findings implied that the urinary excretion of iron following DTPA was not directly derived from tissue iron stores, but most likely from a chelatable extracellular iron pool.
Our problem was that it was universally accepted that all extracellular iron is tightly bound to transferrin, and that transferrin-bound iron is not available for interaction with chelators
in vivo or in vitro. Hence, in order to test our hypothesis that iron chelators were able to promote iron excteretion from a chelatable extracellular iron pool, we decided to examine whether in addition to iron bound to transferrin a fraction of plasma iron may not be transferrin-bound namely non-transferrin bound plasma iron or NTBI.
First, we examind in detail the state of plasma iron binding in 35 patients with β thalassemia major and intermedia with severe transfusional siderosis . In studies performed in collaboration with George Bates in patients receiving regular blood transfusions since infancy, we found that plasma transferrin was completely saturated and that about 2.7-7.I μmol/1 of the total plasma iron could be removed by dialysis or ultrafiltration in the presence of a weak chelating agent. By contrast, less than 1.0 μmol/1 of transferrin bound iron was removed when plasma from normal subjects was subjected to the same procedure. The non-transferrin iron of thalassemic plasma could no longer be demonstrated after preincubation with normal plasma containing unsaturated transferrin. These findings implied that non-transferrin iron found in the plasma of patients with severe transfusional siderosis is a chelatable compound which is readily available for transferrin binding. In view of the known toxicity of unbound iron, we hypothesized that its identification in thalassemic sera might be of relevance to the pathogenesis of tissue damage and the protective effect of iron chelating therapy in this disease.
Although our original description of the existence of a chelatable, low molecular weight plasma iron fraction in patients with severe iron overload has been confirmed by many subsequent studies using a variety of methods , the concept of NTBI met with considerable initial skepticism. This attitude has changed gradually, in large part due to the important contributions of the groups led by John Porter and by Ioav Cabantchik.
According to our current understanding, in iron overload caused by multiple blood transfusions or excessive intestinal iron absorption, excess iron will lead to increasing saturation of transferrin and ultimately to the presence of iron species in the plasma that are not bound to transferrin, so called non transferrin bound iron (NTBI). These species are likely to be hereogenous, consisting of iron citrate monomers, oligomers and polymers, as well as protein bound forms. Plasma NTBI (or subfractions of it) have long been known to generate lipid peroxidation and an assay has been developmed for this redox active subfraction, termed labile plasma iron (LPI). There is evidence that some NTBI species are taken into myocardium and endocrine tissues through L-type voltage dependent calcium channels that are not present on all tissues and this is likely to explain the pattern of tissue iron deposition in transfusional iron overload.
A variety of assays are available to measure NTBI yielding variable reference ranges but generally correlating with each other. The assay measuring a labile subfraction (the component capable of accelerating oxidation of a fluorophore, termed LPI assay) is convenient for measuring iron in the presence of chelators. Progressive removal of this subfraction has been seen following treatment with deferiprone and with deferasirox. Removal of LPI is maintained for 24h per day with deferasirox, consistent with the notion that continuous chelation minimises exposure to NTBI species. There are interesting differences in NTBI and LPI values between disease states. Plasma NTBI is lower in sickle cell disease than in thalassemia major at matched levels of iron loading.
In an international inter-laboratory study comparing ten worldwide leading assays for NTBI or LPI, highest assay levels were observed for patients with untreated hereditary hemochromatosis and β-thalassemia intermedia, patients with transfusion-dependent
myelodysplastic syndromes and patients with transfusion-dependent and chelated β-thalassemia major. Absolute levels differed considerably between assays and were lower for labile plasma iron than for non-transferrin-bound iron. Assays were reproducible with high between-sample and low within-sample variation. Increased transferrin saturation, but not ferritin, was a good indicator of the presence of forms of circulating non-transferrin-bound iron. It was concluded that the use of non-transferrin-bound iron and labile plasma iron measures as clinical indicators of overt iron overload and/or of treatment efficacy would largely depend on the rigorous validation and standardization of assays.
An important challenge for future studies employing NTBI and LPI assays is to explore the rates and sites of their production and clearance. From ferrokinetic studies of transferrin-bound iron we already know that measuring the magnitude of serum iron only is of limited value for understanding its importance . The same is true for NTBI and LPI. In the presence of ineffective erythropoiesis such as in thalassemia major, the rates of NTBI production would be extremely fast implying that within a given time period considerable amounts of NTBI may be released into the circulation. Conversely, when increased NTBI is the result of suppressed iron uptake by the erythroid tissues such as after intensive chemo-radiotherapy, the mechanism of NTPI accumulation implies normal or decreased plasma iron turnover involving limited production of NTBI and most probably no significant risk of iron toxicity. Presently only a few studies have attempted to approach such issues. In a recent study, Porter et al compared NTBI measurements in patients with Thalassaemia Major, Sickle Cell Disease and Diamond-Blackfan Anemia , with matched transfusion histories. The most striking differences between these conditions were relationships of NTBI to erythropoietic markers. Based on these findings, the authors proposed three mechanisms of NTBI generation: iron overload (in all), ineffective erythropoiesis (predominantly in thalassemia) and low transferrin-iron utilization (in DBA).
With the improvement in the standardization of NTBI and LPI assays, and the availability of novel assay systems utilizing automated analyzers that are widely used in clinical laboratories, it is possible now to acquire sufficient experience to explore basic issues such as the value of such measurements for predicting survival in iron overload, to evaluate the risk of developing cardiac and endocrine complications, and to monitor and assess the quality of iron chelation therapy.
Aydinok Y, Evans P, Manz CY, Porter JB. Timed non-transferrin bound iron determinations probe the origin of chelatable iron pools during deferiprone regimens and predict chelation response. Haematologica. 2012 Jun;97 (6):835-41
Cabantchik ZI, Breuer W, Zanninelli G, Cianciulli P. LPI-labile plasma iron in iron overload. Best Pract Res Clin Haematol. 2005;18:277-287.
Cabantchik ZI. Labile iron in cells and body fluids: physiology, pathology, and pharmacology. Front Pharmacol. 2014;5:45. 10.3389/fphar.2014.00045. eCollection 2014.
Daar S, Taher A, Pathare A, et al. Plasma LPI in Thalassemia Patients before and after Treatment with Deferasirox (Exjade®, ICL670). Blood. 2005;106:Abstract 2697.
de Swart L, Hendriks JC, van der Vorm LN,et al Second international round robin for the quantification of serum non-transferrin-bound iron and labile plasma iron in patients with iron-overload disorders. Haematologica. 2016 ;101(1):38-45.
de Swart L, Reiniers C, Bagguley T, et al. Labile plasma iron levels predict survival in patients with lower-risk myelodysplastic syndromes. Haematologica. 2018 Jan;103(1):69-79.
Evans RW, Rafique R, Zarea A, et al. Nature of non-transferrin-bound iron: studies on iron citrate complexes and thalassemic sera. J Biol Inorg Chem. 2008;13:57-74.
Grootveld M, Bell JD, Halliwell B,et al . Non-transferrin-bound iron in plasma or serum from patients with idiopathic hemochromatosis. Characterization by high performance liquid chromatography and nuclear magnetic resonance spectroscopy. J Biol Chem. 1989;264:4417-4422.
Gutteridge J, Rowley D, Griffiths E, Halliwell B. Low molecular weight iron complexes and oxygen radical reactions in idiopathic haemochromatosis. Clin Sci. 1985;68:463-467.
Hershko C, Graham G, Bates G, Rachmilewitz E. Non-specific serum iron in thalassaemia; an abnormal serum fraction of potential toxicity. Br J Haematol. 1978;40:255-263.
Ito S, Ikuta K, Kato D, et al Non-transferrin-bound iron assay system utilizing a conventional automated analyzer. Clin Chim Acta. 2014 1;437:129-35.
Jacobs EM, Hendriks JC, van Tits BL, et al. Results of an international round robin for the quantification of serum non-transferrin-bound iron: Need for defining standardization and a clinically relevant isoform. Anal Biochem. 2005;341:241-250.
Oudit GY, Sun H, Trivieri MG, et al. L-type Ca2+ channels provide a major pathway for iron entry into cardiomyocytes in iron-overload cardiomyopathy. Nat Med. 2003;9:1187- 1194.
Pootrakul P, Breuer W, Sametband M, Sirankapracha P, Hershko C, Cabantchik ZI. Labile plasma iron (LPI) as an indicator of chelatable plasma redox activity in iron-overloaded beta-thalassemia/HbE patients treated with an oral chelator. Blood. 2004;104:1504- 1510.
Piga A, Longo F, Duca L,et al. High nontransferrin bound iron levels and heart disease in thalassemia major. Am J Hematol. 2009 ;84(1):29-33.
Porter JB, Abeysinghe RD, Marshall L, Hider RC, Singh S. Kinetics of removal and reappearance of non-transferrin-bound plasma iron with deferoxamine therapy. Blood. 1996;88:705-713.
Porter JB, Cappellini M, El-Beshlawy A, et al. Effect of Deferasirox (Exjade®) on Labile Plasma Iron Levels in Heavily Iron-Overloaded Patients with Transfusion-Dependent Anemias Enrolled in the Large-Scale, Prospective 1-Year EPIC Trial. Blood,. 2008 112 Abstract 3881.
Porter JB, Garbowski M. The pathophysiology of transfusional iron overload. Hematol Oncol Clin North Am. 2014;28(4):683-701 .
Porter JB, Walter PB, Neumayr LD et al. Mechanisms of plasma non-transferrin bound iron generation: insights from comparing transfused diamond blackfan anaemia with sickle cell and thalassaemia patients Br J Haematol. 2014 Dec; 167(5): 692–696.
Shah F, Westwood MA, Evans PJ, Porter JB. Discordance in MRI assessment of iron distribution and plasma NTBI between transfusionally iron loaded adults with sickle cell and thalassaemia syndromes. . Blood 2002;100:468a.
Walter PB, Fung EB, Killilea DW, et al. Oxidative stress and inflammation in iron-overloaded patients with beta-thalassaemia or sickle cell disease. Br J Haematol. 2006;135:254- 263.