The metabolic effects of excess iron and its assessment

3rd Pan-European Conference on Haemoglobinopathies & Rare AnaemiasLimassol, 24 – 26 October 2012

Ioav CabantchikInstitute of Life Sciences

The Hebrew University of Jerusalem

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)

Iron is life essential•respiration•energy production•substrate conversion•Hb synthesis•O2 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 deficiencynutritional, acquired,

inherited

labile Fe(II) Fe(III)

O2 + OH− + OH●

oxidative cell damage

•systemic (anemia) and/or• regional (tissues/ cells)

O2● + H2O2

Respiration

Fe

Iron is deleterious

•BUT ALSO TO COPE WITH COLATERAL DAMAGE EXERTED BY IRON AND O2

IRON HOMEOSTASISlessons 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

cells

via expression of ferritin

IRP Labile cell ironLCI

hamp

hepcidin

duodenumSpleen

Cell Damage (proteins, DNA, membranes)

OH.

O2÷

Coping with the inevitable

Controlling formation of

Reactive O Species (ROS)

Controlling labile cell iron

LCI

GR glutathione reductase

GSHGSSG

NADPHNADP+

GPX glutathione peroxidase

Antioxidants(bilirubin, uric acid, ascorbic

acid, vitamin E, GSH)

Radical scavengers

Chelators are tools designed to safely extract labile iron.

Neutralizing/ storing the excess

1% of the 3 kg of O2 consumed daily ( 33 g/d)

turns into Reactive O Intermediates (ROI)

Fe(III)

Fe(II)

labile cell iron LCI

H2O2

respiration

SODsuperoxide dismutase

Catalase

H2O+O2

e-

Limiting the uptake

and thereby lead to depletion of cellular iron pools

H2O

O2H+

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

T2* 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

HOW DO WE KNOW WHEN/IF IRON ACCUMULATION LEADS TO DAMAGEIron 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

O2●− + H2O2

ROS derive from metal catalyzed ROI

oxido-reductionsO2 + OH− + OH●

ROIs derive from respirationFe 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

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 ischemically 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 O2 or with reactive O intermediates (ROI) (O2,

H2O2) that are products of metabolism/respiration• chelatable

LCIFe(II)

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

Measuring labile iron in plasma and in cells

● LABILE PLASMA IRON (LPI) the redox-active, chelatable andmembrane 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

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

│10

│30

│20

conH2O2

DFRchelator

no chelator

DFP

DH

R ox

idat

ion

(r.u

.)

chelator

non-fluorescent oxidizable precursors become fluorescent by ROS generated from labile cell iron prompted with H2O2

RDHRH2O2

as directly chelatable iron (DCI)

LCI=∆F

Fluo

resc

ence

LCI=∆F

bc→ac DFP pretreated

quenched Fe fluorescent

Fe

turn-off/on probe

turn-on probe

-Fe (fluorescent) +Fe (non-fluorescent)

Fe(II)/Fe(III)

Fluo

resc

ence

(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↘

101 102 103 104

coun

ts

Fe (µM)0.5 0

120-

80-

40-

after addition of chelator L1→

FL1-H

0 100 200 300 400 500

0.0

0.1

0.2

0.3

0.4

0.5

[Fe]

/F

after 0 conc subtractionR-Square=0.94

0.0008+/-0.00009

0.07+/-0.02

[Fe] nM

FL1-H

CALG-beads flow cytometry

in solution in cells

microscope imaging

Breuer, Epsztejn, Glickstein & Cabantchik, 95-98

2-

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-

l100

l104

l102

l103

l101

ΔF ≡ [LCI]

-chelator +chelator

-CALG

0-

F intensity

CALGbeads

Merav b-d4-ova-calg

delt

a F

0

20

40

60

80

Fe μM0 5 10

[Fe]

/ΔF

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

Fe [µM] 0 0.5 1 1.5 2 2.5 3

CALG-BR 2= 1int = 4.6e-03 ± 3.5e-04slope = 1.1e-02 ±- 3.2e-04

b0= 0.35 ± 0.01; b1= 87± 7; r2=0.997bd4-ova--CALG

ΔFL

(a.u

.)

K½/ ΔFm = 0.005 ± 0.00041/ ΔFm = 0.011 ± 0.0003 r2=0.999

quenched

Fe

Fe

chelator

fluorescentΔFbead bead

● LABILE CELL IRON (LCI): target of sytemic iron overloadthe metabolically and redox-active and directly chelatable component of cellular iron pools (= LIP).

● NON TRANSFERRIN BOUND IRON (NTBI): mediator of iron overloadIron 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

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

Measuring plasma NTBI total

3. Detection with sensor ( HPLC)

Fe3+

NTA

Fe3+

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 mMFe3+

NTBI, mostly protein adsorbed

2 µM Fe3+

Hershko el 1978 ; Hider, Porter et al 01)extraction & filtration

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

LPIHigh throughput fluorescence assays Fe3+

40 µM

TBI

bead

bead

Fe3+

a fluorescent-chelator binds LPI

ascorbate prompts NTBI to redox-cycle ROS and oxidize a non-fluorescent probe

NTBIFe3+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

Pootrakul P. et al. 2004 Blood 104 p. 1504

LPI in non chelated beta-thal/HbE patientscorrelations

LPI appears when transferrin saturation exceeds 70-80%

LPI correlates with

RBC membrane Fe

cell accumulated Fe

serum ferritin

iron stores

LPI as early indicator of chelation efficacy

Istanbul

Deferasirox on β-thalassemia patientsperiod of treatment required to attain basal LPI levels

Daar S., et al. 2009 E.J. Haem. 82 p. 454

week520 4 16 28 40

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

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

0 2 4 6 8 10 12 240.0

0.2

0.4

0.6

0.8

1.0

1.2

DFPx3

0 2 4 6 8 10 12 240.0

0.2

0.4

0.6

0.8

1.0

1.2--DFO--

0 2 4 6 8 10 12 240.0

0.2

0.4

0.6

0.8

1.0

1.2

LPI (mM

)LP

I (mM

)

treatment hr treatment hr

DFR

0 2 4 6 8 10 12 240.0

0.2

0.4

0.6

0.8

1.0

1.2

--DFO--DFPx3

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%

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(%)

6

0

3

40 60 80 100 120−3

0

3

6

40 60 80 100 120

Do NTBI/LPI levels correlate with established clinical parameters?

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

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?