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The metabolic effects of excess iron and its assessment 3rd Pan-European Conference on Haemoglobinopathies & Rare Anaemias Limassol, 24 – 26 October 2012.

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Presentation on theme: "The metabolic effects of excess iron and its assessment 3rd Pan-European Conference on Haemoglobinopathies & Rare Anaemias Limassol, 24 – 26 October 2012."— Presentation transcript:

1 The metabolic effects of excess iron and its assessment 3rd Pan-European Conference on Haemoglobinopathies & Rare Anaemias Limassol, 24 – 26 October 2012 Ioav Cabantchik Institute of Life Sciences The Hebrew University of Jerusalem

2 guidelines “In Science, try to make things as simple as possible, but not simpler” Albert Einstein (1879–1955) “One enlarges science in two ways: by adding new facts and by simplifying what already exists”(Le Cahier Rouge) Claude Bernard (1813-1878)

3 Iron is life essential respiration energy production substrate conversion Hb synthesis O 2 transport DNA synthesis neurotransmitter synthesis Transcription factors Oxidation/reduction Organisms control iron uptake and storage to meet metabolic needs without incurring into regional or systemic inbalance Iron overload/accumulation systemic (primary/secondary) or regional, associated with maldistribution of the metal Iron deficiency nutritional, acquired, inherited labile Fe(II)  Fe(III) O 2 + OH − + OH ● oxidative cell damage systemic (anemia) and/or regional (tissues/ cells)  O 2 ● + H 2 O 2  Respiration Fe Iron is deleterious

4 BUT ALSO TO COPE WITH COLATERAL DAMAGE EXERTED BY IRON AND O 2 IRON HOMEOSTASIS lessons from cellular, animals and human studies ORGANISMS CONTROL IRON LEVELS BY BALANCING iron storage iron uptake post / translational \ co via expression of ferritin systemic via repression of ferroportin via expression of TfR1 and of DMT1 via expression of TfR1 and of DMT1 cells via expression of ferritin IRP Labile cell ironLCI hamp hepcidin duodenum Spleen

5 Cell Damage (proteins, DNA, membranes) OH. O2÷O2÷ Coping with the inevitable Controlling formation of Reactive O Species (ROS) Controlling labile cell iron LCI GR GR glutathione reductase GSH GSSG NADPH NADP + GPX glutathione peroxidase Antioxidants (bilirubin, uric acid, ascorbic acid, vitamin E, GSH) Radical scavengers Chelators are tools designed to safely extract labile iron. Neutralizing/ s toring the excess 1% of the 3 kg of O 2 consumed daily (  33 g/d) turns into Reactive O Intermediates (ROI) labile cell iron LCI H2O2H2O2 respiration SOD superoxide dismutase Catalase H 2 O+O 2 e-e- Limiting the uptake and thereby lead to depletion of cellular iron pools H2OH2O O2O2 H+H+

6 PATHOLOGICAL IRON ACCUMULATION IN VARIOUS DISORDERS Elevated plasma iron levels (>70 % transferrin saturation) & and body (liver) stores (> 500 ng/ml plasma ferritin) Iron accumulates naturally in various organs and excessively in various disorders Systemic iron accumulation Pearl stained biopsy of liver T 2 * MRI (4 msec) of highly IO heart Pearl stained biopsy of heart hypointensity- dentate nuclei hypertrophic cardiomyopathy & interstitial myocardial fibrosis Regional iron accumulation Iatrogenic Liver and spleen iron accumulation in CKD patients supplemented with polymeric iron-saccharate tiger-eye - globus palidus NBIAFRDA Protein, DNA and lipid oxidation products

7 HOW DO WE KNOW WHEN/IF IRON ACCUMULATION LEADS TO DAMAGE Iron accumulates naturally in various organs and excessively in various disorders Regional iron accumulation Systemic iron accumulation Fe(II) Fe(III) LIP (labile iron pool) IRON TOXICITY O 2 ●− + H 2 O 2 ROS derive from metal catalyzed ROI oxido-reductions O 2 + OH − + OH ● ROIs derive from respiration labile forms, can Fe accumulating in labile forms, can prompt (ROI) reactive O intermediates to form excessive toxic (ROS) reactive O species oxidative damage non- enzymatic enzymatic antioxidantschelators coping with ROI & ROS Oxidative damage ensues when ROS formation overrides cell antioxidant measures Inert iron pools Protein, DNA and lipid oxidation products

8 what is labile iron (LI)’s role in the biomedical scence What is labile iron (LI) LI is a form of ionic Fe (+2 or +3) that is chemically active : exchangeable between (bio)ligands and/or (bio)metals redox-active Fe(II↔III): in biosystems, undergoes conversion by bio-redox agents and catalyzes bioreactions engages in the formation of reactive O species (ROS) by reacting with O 2 or with reactive O intermediates (ROI) (O 2 , H 2 O 2 ) that are products of metabolism/respiration chelatable

9 LCI Fe(II) 6. Ingress of chelators 5. Ingress of permeant Fe Fe(II) NADH Infiltration of NTBI hereditary and transfusional siderosis ? 1. ferritin levels Fe(II) 2. [Tf] (& TfR) levels 4. Genes of Fe and heme metabolism 3. Redox status of cells response to signals LCI as bona fide indicator of cell and systemic iron status toxicological phamacological

10 Measuring labile iron in plasma and in cells ● LABILE PLASMA IRON (LPI) the redox-active, chelatable and membrane permeant component of non transferrin bound iron (NTBI) ● LABILE CELL IRON (LCI) the metabolically and redox- active and chelatable component of cellular iron TCI ~60 µM LPI and LCI are the direct targets of chelators LPI ~1 µM LCI ~1 µM TBI ~50 µM

11 Measuring labile cell iron LCI in living cells as redox-active and chelatable iron Breuer, Epsztejn, Glickstein & Cabantchik, 95-98 1- 2- 3- 5- 0- 4- │ 40 min │0│0 │ 10 │ 30 │ 20 con H2O2H2O2 DFR chelator  no chelator DFP DHR oxidation (r.u.) chelator non-fluorescent oxidizable precursors become fluorescent by ROS generated from labile cell iron prompted with H 2 O 2 R DHR H2O2H2O2 as directly chelatable iron (DCI) LCI=  ∆F Fluorescence LCI=  ∆F bc→ac DFP pretreated quenched Fe fluorescent Fe turn-off/on probe turn-on probe

12 -Fe (fluorescent) +Fe (non-fluorescent) Fe(II)/ Fe(III) Fluorescence (485-515) 2’ CALG 0 4- DFO F dequenching ≡ Fe binding F recovery ∆F is ~ [Fe} Fe CALG Labile iron is dynamically monitored y Calcein green (CALG) in fluids and cells CALG-AM cytosol targeting AM CALG-histone nuclear targeting LH ↖RPA CALG↘ 10 1 10 2 10 3 10 4 counts Fe (µM) 0.5   0 120- 80- 40- after addition of chelator L1→ FL1-H CALG-beads flow cytometry microscope imaging Breuer, Epsztejn, Glickstein & Cabantchik, 95-98 2-

13 How can shifts in CALG fluorescence intensity ΔF obtained in cells be converted into [LCI]? Breuer and Cabantchik, 2010 FC analysis of CALG-loaded cells before and after addition of permeant chelator. Breuer and Cabantchik, 2006 Shifts in fluorescence ΔF are higher in blood cells of hyoertranfused patients. Prus and Fibach 2008 (thalass. patients) Doulias..& Galaris 2008 (ox. stress and age) 400- 200- l 10 0 l 10 4 l 10 2 l 10 3 l 10 1 ΔF ≡ [LCI] -chelator +chelator -CALG 0- F intensity CALG beads b0= 0.35 ± 0.01; b1= 87± 7; r 2 =0.997 bd4-ova--CALG ΔFL (a.u.) K ½ / ΔFm = 0.005 ± 0.0004 1/ ΔFm = 0.011 ± 0.0003 r 2 =0.999 quenched Fe chelator fluorescent ΔFΔF bead

14 ● LABILE CELL IRON (LCI): target of sytemic iron overload the metabolically and redox-active and directly chelatable component of cellular iron pools (= LIP). ● NON TRANSFERRIN BOUND IRON (NTBI): mediator of iron overload Iron that outpours into circulation causing plasma iron to rise and surpass transferrin’s binding capacity generates iron forms not bound to transferrin Hershko et al 78, 79: “… NTBI might be relevant to the pathogenesis of tissue damage and the protective effect of chelation…” NTBI LCI Thalassemia major (TM) Thalassemia intermedia (TI) Myelodyplastic syndrome (MDS) Sickle cell disease (SCD) Aplastic anemia (AA) Bone-marrow transplantation Chemotherapy Hereditary hemochromatosis (HH) a variety of chronic metabolic disorders such as diabetes

15 PLASMA NTBI for defining the degree of systemic iron overload (diagnostically/ prognostically) ? for predicting tissue iron overload and end organ toxicity? for assessing the efficacy of chelation in maintaining plasma free of iron sources implicated in tissue iron overload? possible clinical value

16 Measuring plasma NTBI total 3. Detection with sensor (  HPLC) Fe 3+ NTA Fe 3+ 1. extraction via “non-mild” chelation (80 mM NTA) 2. filtration Laborious; might mobilize Fe from Tf and Fe-chelates Accurate, sensitive, reproducible. transferrin plasma 40  M Fe 3+ NTBI, mostly protein adsorbed 2 µM Fe 3+ Hershko el 1978 ; Hider, Porter et al 01) extraction & filtration

17 is detected in plasma when transferrin saturation exceeds 70% NTBI is heterogeneous: its composition differs according to sources, levels attained and also following chelation. (Fe-chelate complexes can be measured as NTBI by some assays!). is composed of complexes of : iron-citrate and phosphates iron and/or of iron-ligands bound to proteins NTBI these properties define the labile components of NTBI in plasma the iron in the complexes can be : exchanged with other metals or ligands chelated by agents with high binding affinity for the metal reduced by natural reductants and translocated across membranes via resident transporters/channels represents ~1–10 % of the (~ 40 µM) TIBC

18 LPI High throughput fluorescence assays Fe 3+ 40 µM TBI bead Fe 3+ a fluorescent- chelator binds LPI ascorbate prompts NTBI to redox-cycle  ROS and oxidize a non-fluorescent probe NTBI Fe 3+ 2 µM Turn-on Probe Turn-off Probe Fe chelator blocks ROS oxidizes probe LPI: labile plasma iron, Espósito et al 2003 feROS, DCI: Directly chelatable iron, Breuer et al 98, 02 feRISK, plate reader Measuring plasma NTBI labile components LPI and DCI DHR R plasma factors that affect LPI/DCI (albumin, citrate, uric acid) are eliminated with 0.1-0.5 mM NTA) flow cytometer DCI

19 Pootrakul P. et al. 2004 Blood 104 p. 1504 LPI in non chelated beta-thal/HbE patients correlations LPI appears when transferrin saturation exceeds 70-80% LPI correlates with   RBC membrane Fe  cell accumulated Fe serum ferritin  iron stores

20 LPI as early indicator of chelation efficacy

21 Istanbul Deferasirox on β-thalassemia patients period of treatment required to attain basal LPI levels Daar S., et al. 2009 E.J. Haem. 82 p. 454 week 5204162840 Post administration (2h) Preadministration (trough value) * * p< 0.01 (n=13) * * * * Measure of chelation activity retained in plasma 24 h after drug intake Measure of chelation activity attained 2h after absorption of the drug trough levels attained normal range within 16 weeks Cabantchik May 2012


23 Istanbul Combination ( continued sequential DFP and DFO): maintained low LPI over 24 h Deferiprone (3x 25 mg/k/d po) : diurnal fluctuations in LPI and high stdev Deferasirox (switched from DFO or DFP after 24 h drug washout) show low LPI over 24h Deferrioxamine (O/N sc ): significant rise in diurnal LPI and high stdev Zanninelli G. et al. 2009 Br. J. Haem. 147 p. 744 LPI (group mean of n=20 ± stdev) Which chelation regimen confers daily protection from LPI appearance in thalassemia major patients under different chelation regimens? 95% 88% 45% 50%

24 All patients with iron toxicity-related cardiomyopathies have NTBI/LPI; however, the reverse is not the case NTBI µM (> 24 h washout) 2 1. Zanninelli G, et al. 2009 Brit J. Hematol. 147, 744. 2. Piga A, et al. 2009, Am J Hematol. 84:29-33. LPI µM (< 10 h washout) 1 Transferrin saturation (%) Do NTBI/LPI levels correlate with established clinical parameters?

25 Istanbul Summary A single LPI measurement 2 h after drug intake provided a measure for the ability of the drug to attain levels sufficient for instantaneous elimination of LPI A single LPI measurement taken at trough chelator levels in plasma (~24 h after administration of DFR, 10-12 h of DFP and 12- 14 h of DFO) provided an indication for the ability of a chelation regimen to maintain LPI at basal levels (< 0.2 ± 0.1 µM) at a given day in the course of treatment of thalassemia major, thalassemia intermedia, SCD or MDS patients. Repeated (monthly) LPI measurements in the course of treatment indicated that LPI reached basal levels while serum ferritin continue to decline and was correlated with long term reduction in liver iron concentration

26 Development of early alert markers of emerging iron overload that: respond to changes in iron status with minimal delay are based on readily available technology. The routine treatment is administration of iron chelators, but clinicians must make some important decisions … … when should chelator treatment be initiated? … which chelation regimen to use? … how should chelation efficacy/efficiency be evaluted/modified? These decisions are too often based on indicators of iron overload that have delayed-response, like serum ferritin levels. Iron overload in organs can be imaged by MRI methods, but these are expensive and not routinely available. When and how should iron overload be treated?

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