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 Slide 2 guidelines In
Science, try to make things as simple as possible, but not simpler
Albert Einstein (18791955) One enlarges science in two ways: by
adding new facts and by simplifying what already exists(Le Cahier
Rouge) Claude Bernard (1813-1878) Slide 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 Slide 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 Slide 5 Cell Damage (proteins, DNA,
membranes) OH. O2O2 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+ Slide 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
Slide 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 Slide 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(IIIII): 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 Slide 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 Slide 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 M LPI and LCI are the direct targets of
chelators LPI ~1 M LCI ~1 M TBI ~50 M Slide 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 00 10 30 20 con H2O2H2O2 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 bcac DFP pretreated quenched Fe fluorescent
Fe turn-off/on probe turn-on probe Slide 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 (M) 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- Slide 13 How can shifts in CALG fluorescence
intensity F 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 F 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 F [LCI] -chelator +chelator -CALG 0- F intensity CALG
beads b0= 0.35 0.01; b1= 87 7; r 2 =0.997 bd4-ova--CALG FL (a.u.) K
/ Fm = 0.005 0.0004 1/ Fm = 0.011 0.0003 r 2 =0.999 quenched Fe
chelator fluorescent FF bead Slide 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 transferrins 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 Slide 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 Slide 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 M Fe 3+ Hershko el 1978
; Hider, Porter et al 01) extraction & filtration Slide 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 ~110 % of
the (~ 40 M) TIBC Slide 18 LPI High throughput fluorescence assays
Fe 3+ 40 M 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 M Turn-on Probe Turn-off Probe
Fe chelator blocks ROS oxidizes probe LPI: labile plasma iron,
Espsito 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 Slide 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
Slide 20 LPI as early indicator of chelation efficacy Slide 21
Istanbul Deferasirox on -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 Slide 22 Slide 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% Slide 24 All patients with iron toxicity-related
cardiomyopathies have NTBI/LPI; however, the reverse is not the
case NTBI M (> 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 M (< 10 h washout) 1 Transferrin saturation (%) Do
NTBI/LPI levels correlate with established clinical parameters?
Slide 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 M) 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 Slide 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?
