Sources and Targets of Systemic Iron Overload. Iron Absorbed by the Organism Or Recycled

Red cells BloodMed exclusives: Can labile plasma iron (LPI) and labile cell iron (LCI) levels serve as early indicators of chelation efficacy in iron overload? Ioav Cabantchik1, Eitan Fibach2 and William Breuer1, 1Department of Biological Chemistry, Institute of Life Sciences, Hebrew University of Jerusalem, Israel 91904 and 2Department of Hematology, Hadassah - Hebrew University Medical Center, Ein Kerem, Jerusalem, Israel.

LCI and LPI under normal and iron overload conditions

The appearance of excess labile iron in the plasma and tissues is a characteristic feature of iron overload (IO) associated with transfusional or primary hemosiderosis [1].

The term 'labile' refers to the redox activity and chemical mobility of the metal, often associated with a propensity to catalyze the generation of reactive oxygen species (ROS) and undergo exchange between ligands. Two forms of clinically relevant labile iron will be discussed here, extracellular 'labile plasma iron' LPI) and intracellular 'labile cell iron' (LCI) (Fig. 1).

LPI is considered to be a component of "non-transferrin bound iron' (NTBI) (2,3), a term designating a mixture of iron complexes found in plasma when the binding capacity of transferrin is overwhelmed by excess iron in the circulation. Although NTBI complexes might not be toxic per se, their reduction to iron(II) could facilitate permeation of iron into cells by default pathways, causing a marked rise in the pools of labile cell iron (LCI) [4].

Unlike LPI, which is detected almost exclusively in pathological conditions, LCI is a normal component of virtually all cells distributed among the various organellar labile iron pools (LIPs) [5].

LCI's primary function is to provide a ready and continuous source of iron for immediate metabolic utilization by enzymes, such as those required for DNA synthesis and oxygen sensing, ribonucleotide reductase and proline hydroxylase, respectively. The bound iron cofactor in both enzymes is labile (i.e. redox-active and chelatable) and it is therefore likely to be in dynamic equilibrium with LCI [6], similarly to the iron-sensing regulatory proteins (IRPs) that control LCI levels by ensuring that iron uptake via transferrin receptors is synchronous with iron sequestration by ferritin [7].

In IO conditions, LCI exceeds steady state levels apparently due to its ability to gain entry into cells via transmembrane pathways that are not under IRP regulation [8,9]. High LCI levels lead to increased ROS generation (through the Haber-Weiss and Fenton reactions), which may eventually override the cell antioxidant capacity, resulting in tissue oxidative damage and vital organ dysfunction.

LCI and LPI as targets of chelators in IO

According to the above scenario, excessive LCI and LPI levels have a central role in the generation of the pathology of IO, making them prime targets of chelator-based therapies. Indeed, the application of high affinity iron chelators (desferrioxamine, deferiprone, desferasirox) has proven clinically successful in treating secondary IO [1]. All three agents efficiently access and neutralize the redox activity of LPI (5), but their effects on LCI in critical cells of the liver, heart and endocrine glands are more difficult to estimate, requiring biopsies or MRI.

Importance of early assessment of chelator efficacy

The main parameter that has been clinically used for assessing iron overload in general and for following chelation efficiency in particular, is the level of ferritin protein in the serum, a surrogate marker of body iron stores (when not confounded by inflammation). There is now increased recognition that serum ferritin levels often fail to predict impending cardiac iron overload and ensuing cardiomyopathies [10].

The advent of non-invasive proton relaxation assays of liver and cardiac iron (by NMR R2* or T2*) has provided a significant advance in monitoring chelator effectiveness, although, similarly to serum ferritin, substantial changes in these parameters are usually seen only weeks or even months after the initiation of chelator treatment.

Furthermore, in most clinics treating IO patients, particularly those in underdeveloped countries, the requisite instrumentation is not always available, leaving diagnosis of IO entirely dependent on serum ferritin assays and iron staining of liver biopsies. In these situations, alternative, early clinical indicators for monitoring IO and adequacy of chelation regimens are required.

Application of LCI and LPI measurements for monitoring chelator treatment efficacy

The advantage of LPI measurements is not only its technical simplicity, sensitivity and demand for minute serum samples, but also its applicability to blood serum or plasma samples that have been kept frozen for weeks to months [3,11].

The limitation of LPI measurements is its dependence on a fluorescence multi-well plate reader, an instrument not universally available in hematology clinics except those operating in clinical research centers. A recent clinical trial aimed at assessing the adequacy of 4 clinically established protocols of chelation (based on mono and combined therapy) in eliminating LPI on a sustained daily basis, highlighted the heterogeneity in the individual responses to chelation regimens [11,13].

These and more recent studies [13-15] indicated that diurnal monitoring of individual LPI fluctuations can be a useful indicator of both the adequacy of a given chelation regimen and patient compliance, while providing a basis for customizing and optimizing chelation therapy.

A complementary method that measures the LCI (5) has recently been adapted for flow cytometry of blood cells, rendering it applicable in most hematological laboratories. The procedure entails loading peripheral blood or bone marrow cells with the intracellular iron probe calcein and monitoring their fluorescence by flow cytometry [16].

Application of parameters for identifying specific blood cell subpopulations (side-light scattering and expression of surface antigens) showed that the LCI levels decrease in the order monocytes > PMN > lymphocytes > RBC. The results also showed that in thalassemia major patients LCI levels are significantly increased in erythrocytes and reticulocytes suggesting that the procedure might be useful for monitoring the intracellular effects of chelator treatment.

A major potential advantage of blood cell LCI measurements is that they reflect more faithfully the exposure of cells throughout the body to LPI than obtained with traditional assays.

Can LCI and LPI measurements provide a reliable basis for assessing IO in general and of chelation treatment adequacy in particular?

Although a correlation between the appearance of cardiac complications and the presence of sustained levels of NTBI following a period of extended chelator washout might implicate the latter as the source of cardiac iron overload [17], a causative relationship between these properties awaits to be firmly established.

Nonetheless, the primary value of chelation of LPI or NTBI is considered to be the prevention of increases in LCI [11-13, 18]. As chelator treatments do not always eliminate LPI/NTBI or prevent their daily resurgence, assessment of the ability of chelators to reduce plasma and cellular labile iron pools might be of paramount importance for optimizing chelation protocols. Acknowledgements

Research was funded in part by the Israel Science Foundation (ISF) and the EEC Framework 6 (LSHM- CT-2006-037296 Euroiron1). ZIC holds the Adelina and Massimo Della Pergola Chair in Life Sciences. This review is dedicated to the emeriti Professors C. Hershko and E. Rachmilewitz (Jerusalem, Israel), who were the first to recognize NTBI as an important factor in the pathophysiology of iron overload.

Images

Sources and targets of systemic iron overload. Iron absorbed by the organism or recycled by the reticuloendothelial system is normally sequestered by plasma transferrin (referred as transferrin bound iron-TBI). Following blood transfusions (generally thalassemia major-TM and some myelodysplatic-MDS patients) there is a massive influx of iron into plasma, surpassing TBI and generating non-TBI (referred as NTBI).

An analogous rise in NTBI occurs in hereditary hemochromatosis (HH) and Thalassaemia intermedia (TI) where iron is hyperabsorbed by the gut. Physiological TBI is taken up by tissues by regulated pathways, whereas the pathological NTBI, especially its labile component LPI (labile plasma iron) access cells, including red and white blood cells (RBC and WBC) via opportunistic routes, raising the labile cell iron (LCI) levels and in extreme conditions overriding the cellular antioxidant capacities.

Chelators (Ch) can act by suppressing LPI and/or by permeating into cells and chelating LCI and thereby gradually reducing the tissue iron loads, most of which are associated with ferritin and hemosiderin.

References

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