<scp>Plasma nontransferrin bound iron–</scp>nontransferrin bound iron <scp>revisited:</scp> Implications for systemic iron overload and in iv iron supplementation
Authors: Ioav Z. Cabantchik, Chaim Hershko
Journal: American Journal of Hematology (2021)

Nontransferrin bound iron (NTBI) has been classically used to denote a potentially toxic component of plasma detected in systemic iron overload and implicated in end-organ iron accumulation and biological damage. A chelatable labile component of plasma NTBI has also served as target for novel therapeutics. However, as the term NTBI per se does not exclude nontoxic forms of iron that can be detected in plasma, its liberal use without additional pharmacological attributes might be misleading, especially in the clinical context, as for infusible iron supplements. In keeping with the historical and commonly used NTBI term, we propose here to use "labile/nonlabile" plasma NTBI to indicate the toxic potential of the iron species in question. Iron overload (IO) is a pathological condition caused by the accumulation of iron in excess of physiological needs, ultimately resulting in oxidative damage to vital functions. The early diagnosis of the most prevalent IO pathologies has classically relied on the two systemic iron plasma markers, ferritin, and transferrin saturation (TSAT). The end-organ accumulation of iron and ensuing damage were attributed to the opportunistic ingress of nontransferrin bound iron (NTBI) species from plasma to cells, via undefined membrane pathways. The early definition of NTBI in IO plasma (TSAT ≥ 100%) was of an iron species of relatively low molecular weight iron that is not associated with the physiological iron carrier transferrin, insofar as it was captured by a chelator and filterable via dialysis.1, 2 The putative NTBI species was subsequently referred to as plasma catalytic iron, based on its propensity to generate reactive oxygen species (ROS), a property commonly implicated in peroxidative damage.3 Although NTBI per se implies generically all forms of iron not-bound to transferrin, its usage in biology and medicine (especially in hematology), has been confined to either chelatable or catalytic iron in internal biological fluids (reviewed in Brissot et al., 2012. doi: 0.1016/j.bbagen.2011.07.014 and Cabantchik, 2014. doi: 10.3389/fphar.2014.00045).*4, 5 As NTBI was detected in plasma of patients with IO (TSAT>70%), it was viewed as a marker of systemic IO but also inferred as source of impending tissue IO, namely as a potential source of unregulated tissue iron ingress and ensuing accumulation to toxic levels (see also review by Porter & Garbowski, 2014. doi: 10.1016/j.hoc.2014.04.003).6 Although the suggested origin of tissue IO and peroxidative damage was identified with the fraction of plasma iron not associated with transferrin, the role of plasma NTBI in IO pathology was essentially deduced from the protective effect afforded by iron chelators with limited permeation to cells. The ability of chelators to complex plasma NTBI, was subsequently used in the treatment of systemic IO to assess chelation efficacy by different chelation regimens. The generation of NTBI in human plasma reflects the failure of transferrin to capture all iron outpouring into circulation via the ferroportin export system. Transferrin's failure results primarily from a "local insufficiency" of apotransferrin (measurable as UIBC-unsaturated iron-binding capacity) due primarily to the lack of transferrin as in hereditary aceruloplasminemia, liver failure, or insufficient apotransferrin or when iron is under-utilized by the hematopoietic system (see also review by Coates, 2014. doi: 10.1016/j.freeradbiomed.2014.03.039).7 As NTBI is generally removed from circulation by the liver, its plasma levels reflect the dynamics of NTBI generation vis a vis its removal from plasma. Thus, NTBI builds up whenever production exceeds removal from circulation, as found in various types of IO of transfusional origin but also in nontransfusional overload that results from hyperabsorption of iron as in hereditary hemochromatosis or thalassemia intermedia, various forms of ineffective or suppressed erythropoiesis and especially in atransferrinemia). Plasma NTBI was originally detected in patients with systemic IO from different origins and the peroxidative organ damage caused was generally attributed to the uncontrolled infiltration of NTBI into cells. However, the threshold levels of plasma NTBI potential toxicity still remain undefined in the different clinical settings. First and foremost, it has been difficult to identify the chemical composition of the plasma NTBI toxic species that infiltrate different cells opportunistically, their concentrations, and the periods required for them to accumulate in different cell types to the respective toxic levels (as those depend also on the individual routes of iron infiltration and cell defense mechanisms). Second, various forms of NTBI can be detected in plasma not only in overt IO8 but also in normal individuals,9 clearly indicating that the putative NTBI per se need not be pathological or necessarily noxious, paraphrasing Paracelsus.† Notably, the existence of detectable NTBI in the plasma of normal individuals might explain why some NTBI assays, particularly those that assess labile plasma iron (LPI), detect a basal level of labile NTBI in normal plasma.9, 10 Although the use of NTBI was initially confined to a potentially toxic entity in plasma of IO individuals, the term NTBI per se did not exclude plasma ferritin-iron detected in IO plasma, nor iatrogenic NTBI-chelates resulting from the treatment of IO with iron chelators or NTBI-complexes given by intravenous infusions (primarily ferric oxyhydroxide copolymerized with carbohydrate moieties).‡ All those compounds detected in human plasma fall in the category of plasma NTBI, irrespective of their chemical reactivity or toxic potential.§ However, over the years, the NTBI term has been used more liberally, far from what was originally intended, including any iron-complex applied experimentally for loading cells with iron. In many cases, those compounds were implied to simulate/mimic the genuine pathological NTBIs, whose chemical structures are yet to be fully resolved, especially those germane to pathological tissue IO. We are of the idea that in the biomedical setting, the term NTBI¶ should be used in a way that will reflect its pathological nature and toxic potential. For that, a clear distinction between potentially toxic and nontoxic NTBI species would seem timely and appropriate, for scientific, diagnostic as for therapeutic reasons. Whether any chemical agent is or is not toxic was addressed by Paracelsus for more than 500 years in reference to poisons. The criterion is empirical and mostly based on the dose, but also on the period exposure to the toxic agent in the native (or closely similar) biological milieu, be it cell or whole organism. Iron toxicity stems primarily from its chemical lability when complexed to particular ligands that modulate its redox potential, particularly its reactivity in an oxygenated environment. We propose to continue using the generic NTBI, mainly for historical reasons, but add to NTBI a qualifier to specify its labile or nonlabile character, as that qualifier refers directly to either its toxic or nontoxic potential, respectively. For example: "labile-NTBI" refers to iron that is transferable/chelatable and redox-active, rendering "plasma labile-NTBI" and "labile plasma iron" as equivalent terms applicable to the clinical setting of systemic IO (e.g. NTBI species with potential toxicity, as found in the plasma of IO patients with hemochromatosis, secondary IO resulting from repeated blood transfusions as in thalassemia syndromes, or in patients following myeloablative chemotherapy and others). Similarly, plasma NTBI derived from infusible iron supplements containing ferric oxyhydroxide-carbohydrate, or from fully coordinated iron-chelates following chelation treatment, should be referred to as nonlabile NTBI or rather demonstrably free of labile NTBI, irrespective of their chemical structure. Hitherto the chemical identity of plasma labile-NTBI species remains to be determined in different IO states that differ in their plasma chemical composition, the degree, and speed of IO and the redox activity of the medium. Although NTBI speciation studies indicated a range of iron citrate species as likely candidates,8, 11 the actual forms of labile-NTBI relevant to pathological loading of tissue compartments across disease states need to be elucidated, particularly for characterizing the membrane routes of iron loading into different tissues (reviewed by Knutson, 2019. doi: 10.1016/j.freeradbiomed.2018.10.413).12 The detection of plasma NTBI has relied on analytical assays that monitor iron captured (chelated) by a metal sensitive probe or by measuring iron's redox catalytic activity that is susceptible to chelation. "Total" NTBI detected in plasma provides a measure of IO, while potentially toxic or labile fractions have been detected by LPI assays. Importantly, the notion that tissue IO could be reduced by controlling labile-NTBI levels in plasma, has led to the development of clinical chelator regimens aimed at minimizing daily tissue exposure to plasma labile-NTBI components.11 NTBI remains an important concept in clinical management of systemic IO, because much of the morbidity and mortality in systemic IO have been attributed to excess tissue iron accumulation from overexposure to NTBI. However, its limited use in clinical research stems from outstanding need for validating assays for quantitating plasma NTBI species in different IO pathologies and defining their respective etio-pathological roles (including threshold toxic levels). Those shortcomings are also reflected in the NTBI values reported by different analytical assays performed on the same plasma of patients with different sources of IO13 and different values of labile iron reported for iv-iron formulations using different assays, including values in plasma following iv administration.14 The differences stem not only from variable sensitivity and/or specificity and/or robustness of the NTBI assays, but also from the choice of reference standards used for measuring chemically heterogeneous species taken from a complex-heterogeneous medium, that is, plasma or serum. Hitherto, the major clinical contribution of labile-NTBI assays has been in the design of treatment regimens that aim to minimize tissue exposure to plasma NTBI components (e.g. LPI) and hence provide extended protection from impending IO. Initial attempts have been done to determine the threshold levels of labile NTBI/LPI or TSAT that could serve as indicators for initiating chelation in naïve hypertranfused children with thalassemia major.15 For diagnostic purposes, labile NTBI/LPI > 0.2 μM levels were consistently found in plasma with TSAT levels >70% and maintaining levels below this throughout the day in IO patients can serve as one of the goals in the treatment of IO or vice versa, to start treatment (particularly chelation) when threshold levels of NTBI/LPI or TSAT are surpassed. While plasma NTBI remains an important pathological marker of IO and potential organ toxicity, the lack of clinical validation of "toxic"-plasma levels NTBI/LPI has thus far limited its use in clinical practice. A simple, robust validated assay is still to be established for carrying out large-scale clinical studies in order to determine the degree of IO (as it is reflected in labile NTBI/LPI and TSAT levels), primarily for initiating and/or optimizing therapeutic interventions. As in the case of serum cholesterol, the journey to achieve that goal might be long and treacherous. However, "The danger for most of us lies not in setting our aim too high and falling short, but in setting our aim too low, and achieving our mark."-Michelangelo Buonarroti. As to the biomedical use of the term NTBI for any nonphysiological compound applied to or detected in a transferrin-containing medium, we urge to specify its labile/nonlabile chemical character to denote its toxic potential.