Comparison of Overt and Cryptic Labile Plasma Iron In Thalassemia Patients In Israel and Gaza-Palestine
Authors: Breuer William , Ghoti Hussam , Jeadi Hesham , Goldfarb Ada , Rachmilewitz Eliezer A. , Cabantchik Zvi Ioav 
Published: October 2019

Abstract
Abstract 5166

Background.
Systemic iron overload (SIO) is characterized by persistently high levels of plasma iron that often surpass transferrin's (Tf) binding capacity and generate chemical forms identified as non-Tf bound iron (NTBI). These forms have been perceived as: a. clinically important indicators of SIO per se and of impending organ damage, because cells chronically exposed to iron overloaded plasma attain iron levels and ensuing ROS formation that override their antioxidant capacities and b. as pharmacological targets for chelation and thereby of prevention of tissue iron overload. However, NTBI determination in the clinical setting has been confounded by the chemical heterogeneity of iron forms found in fluids like plasma/sera of SIO patients, the presence of residual amounts of undefined chelates or chelators and the need to dislodge NTBI from native ligands with agents that facilitate its detection.
We have assayed the overt forms of NTBI that represent the native pool of labile (= redox-active, chelatable and membrane permeant) iron in plasma/serum. We defined it as ‘labile plasma iron' or LPI and analyzed it by the Aferrix FeROS™ test (1) and used it to asses chelation regimens in their ability to maintain patients' plasma at relatively low (basal) LPI levels (<0.4 μ M, ref. 2). Detection of NTBI forms with both low redox activity and poor chelator accessibility (defined as cryptic LPI) can also be done with the FeROS™ test by supplementing samples with an agent (nitrilotriacetate= NTA < 0.5 mM) that in plasma “extracts” iron from native NTBI. Thus whereas LPI measures overtly labile NTBI in native plasma (i.e. LPI), LPIplus detects both overt + cryptic forms, as in classical NTBI assays that involve either mobilization + filtration (3) or in the DCI (directly chelatable iron) assay that measures deferrioxamine chelatable NTBI (4).

Aim.
To compare SIO parameters in polytransfused thalassemia major patients, chelated and non-chelated, as revealed by measurements of overt and cryptic LPI.

Methods.
The studies involved: 1. The Hadassah Medical Center (HMC) in Jerusalem, where 15–20 (randomly selected, age 14–35) patients were under regular transfusion/chelation treatment and 2. The European Medical Center in Gaza (EMC), where regularly transfused patients (age 10–22) were only sporadically chelated. NTBI assays were performed on sera prepared from blood, (where applicable taken after >10 hrs drug washout, as described for LPI (1,2) and DCI (4); for LPIplus, the LPI test was conducted in the presence of 0.5 mM NTA.

Results.
As shown previously (2,4), LPI was detected only in patients with >70% Tf-saturation.
In HMC, the mean LPI of n=18 patients rose from 0.51±0.41 μ M to 1.00 ±0.46 μ M in the presence of NTA, matching the DCI level of 0.91±0.7 μ M. The LPI rise was detected in 12/15 (= 80%) of samples with LPI>0.4 μ M (≂p 66% of the entire cohort). Thus, despite chelation, a substantial number of patients had relatively low but significant levels of both overt and cryptic NTBI. Among the 3 patients with no significant LPI or DCI (0.2-0.4 μ M), 2/3 became LPI positive (0.6-0.8 μ M) when tested with NTA.
Unexpectedly, in EMC-Gaza, among 20 transfused unchelated patients with serum ferritins > 5000 ng/ml and Tf saturation >100%, 8/20 of them (≂p 40%) had undetectable levels of overt LPI but substantial cryptic NTBI. In the remaining 12/20, the mean overt LPI of 0.69±0.65 μ M rose significantly (p<0.01) to 2.05 ±1.56 μ M when the cryptic component (NTA-extractable) was added.

Discussion.
Overt and cryptic NTBI components were detected by two modalities of the LPI assay in both regularly chelated and unchelated thalassemia patients, although to different extents and proportions. Compared to chelated patients, those unchelated had significantly higher mean values of both overt and cryptic NTBI components, despite the higher proportion of patients with only cryptic NTBI. On an individual basis, the persistent appearance of either/both LPI component(s) of NTBI could provide a measure of SIO and/or the success of individual chelation regimens. However, remaining to be established is the pathophysiological role of each component of NTBI to SIO, disease progression and treatment success. Supported by ISF and the Canadian Friends of HUJI.
1. Esposito et al. Blood 102:2670-7 (2003); 2. Zanninelli et al. Br. J. Hematol. 147: 744–51(2009); 3. Hider R. Eur J Clin Invest 32:S50–4 (2002); 4. Pootrakul et al. Blood 104: 1504–10 (2004).

Disclosures:
Cabantchik: Aferrix Ltd: Consultancy, Membership on an entity's Board of Directors or advisory committees.