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research paper

Oxidative stress and inflammation in iron-overloaded patients


with b-thalassaemia or sickle cell disease

Patrick B. Walter,1,2 Ellen B. Fung,3 Summary


David W. Killilea,1 Qing Jiang,1 Mark
Hudes,4 Jacqueline Madden,2 John Blood transfusion therapy is life-saving for patients with b-thalassaemia and
Porter,5 Patricia Evans,4 Elliott sickle cell disease (SCD), but often results in severe iron overload. This pilot
Vichinsky3 and Paul Harmatz2 study examined whether the biomarkers of tissue injury or inflammation
1
Children’s Hospital Oakland Research Institute, differ in these two diseases. Plasma malondialdehyde (MDA) was significantly
2
Department of Gastroenterology, Children’s increased 1Æ8-fold in thalassaemia relative to control patients. In contrast,
Hospital and Research Center, Oakland, MDA in SCD was not significantly different from controls. In multivariate
3
Department of Hematology/Oncology, Children’s analysis, the strongest predictors of elevated MDA were liver iron
Hospital and Research Center Oakland, Oakland, concentration (P < 0Æ001) and specific diagnosis (P ¼ 0Æ019). A significant
CA, USA, 4Pediatric Clinical Research Center,
2-fold elevation of non-transferrin bound iron (NTBI) was observed in
Children’s Hospital and Research Center,
thalassaemia relative to SCD. NTBI was not a significant predictor of high
Oakland, CA, USA and 5Department of
MDA in multivariate analysis. SCD patients showed a significant 2Æ2-fold
Haematology, University College London, London,
UK
elevation of the inflammatory marker interleukin (IL)-6 relative to controls,
and a 3Æ6- and 1Æ7-fold increase in IL-5 and IL-10 relative to thalassaemia.
Although a-tocopherol was significantly decreased by at least 32% in both
thalassaemia and SCD, indicating ongoing oxidant stress and antioxidant
consumption, c-tocopherol, a nitric oxide-selective antioxidant, was
increased 36% in SCD relative to thalassaemia. These results demonstrate
that thalassaemia patients have increased MDA and circulating NTBI relative
to SCD patients and lower levels of some cytokines and c-tocopherol. This
supports the hypothesis that the biology of SCD may show increased
Received 5 April 2006; accepted for publication inflammation and increased levels of protective antioxidants compared with
12 July 2006 thalassaemia.
Correspondence: Paul Harmatz, Children’s
Hospital & Research Center Oakland, 747 52nd Keywords: iron overload, thalassaemia, sickle cell disease, oxidative stress,
Street, Oakland, CA 94609, USA. E-mail: inflammation, cytokines, non-transferrin bound iron, malondialdehyde, lipid
pharmatz@mail.cho.org peroxidation, vitamin E.

Although the prognosis for patients with thalassaemia and 2004). In pilot data from a recent study, Vichinsky et al
sickle cell disease (SCD) has greatly improved in recent (2005a) confirmed this finding and demonstrated that the
decades with the use of modern newborn screening, prophy- strongest predictors of organ injury in thalassaemia were
lactic penicillin (and the availability of newer antibiotics), duration of iron exposure and the specific diagnosis.
blood transfusions and iron chelation therapy, patients con- Biomarkers of oxidative damage are increased in both
tinue to be at high risk for iron overload and iron-induced thalassaemia and SCD (Rachmilewitz et al, 1976; Repka &
toxicities. Iron-related cardiac disease remains the most Hebbel, 1991). In spite of the iron overload in both diseases,
common cause of death in thalassaemia (Zurlo et al, 1989; oxidants originate from sources other than the iron loaded
Borgna-Pignatti et al, 1998). In addition, over 70% of patients tissues. For example, in b-thalassaemia the excess unpaired
with thalassaemia suffer from primary or secondary amenor- a-haemoglobin chains denature and autoxidise, contributing
rhoea, hypogonadism, osteoporosis and other endocrine to increased oxidants, ineffective erythropoiesis, haemolysis
disorders (Bronspiegel-Weintrob et al, 1990). However, trans- and shortened erythrocyte survival (Scott et al, 1993). Mal-
fused patients with SCD have a conspicuous absence of iron ondialdehyde (MDA), a product of lipid peroxidation and
overload-related organ injury (Finch et al, 1982; Wood et al, protein carbonyls, representing oxidation of the circulating

ª 2006 The Authors


doi:10.1111/j.1365-2141.2006.06277.x Journal Compilation ª 2006 Blackwell Publishing Ltd, British Journal of Haematology, 135, 254–263
Oxidative Stress in b-Thalassaemia and Sickle Cell Disease

proteins, are elevated in thalassaemia (Graziano et al, 1976; unknown whether the two diseases develop similar inflamma-
Snyder et al, 1981; Ramenghi et al, 1989; Shalev et al, 1995; tory responses.
Cighetti et al, 2002). On the other hand, SCD haemoglobin The present pilot study examined whether biomarkers of
can bind to the RBC membrane and act as a Fenton reagent, oxidant injury (such as MDA), evidence for inflammation [C-
increasing the generation of oxidants such as superoxide and reactive protein (CRP) and cytokines] and antioxidant levels
hydroxyl radical (Repka & Hebbel, 1991). Further sources of (a- and c-tocopherol) differ in SCD and thalassaemia.
oxidants unique to SCD vasculature include xanthine oxidase
released from the liver, nitric oxide and secondary oxides of
Methods
nitrogen (Aslan et al, 2001, 2003; Aslan & Freeman, 2004,
Aslan et al, 2000) and endothelial NADPH oxidase (Wood
Study design
et al, 2005). These increased oxidants and intravascular
haemoglobin from haemolysis (Reiter et al, 2002) may Patients with thalassaemia or SCD and a history of receiving
contribute to the consumption of nitric oxide in SCD (Aslan chronic transfusion therapy were recruited from the hemato-
et al, 2001). In turn, low levels of nitric oxide may result in logy clinic at the Children’s Hospital and Research Center
haemodynamic instability (Aslan et al, 2001) and reduction of Oakland (CHRCO) at the time of a clinically indicated liver
antioxidant capacity (Gladwin et al, 2003). Additionally, some biopsy for evaluation of iron overload. Healthy controls were
studies have shown increased levels of plasma MDA in SCD recruited from CHRCO staff and their families. The study
(Jain et al, 1990; Sess et al, 1992; Sertac et al, 1997). However, received Institutional Review Board approval. Consent for
it is unknown whether MDA levels differ in these two iron adults and assent for children were obtained in accordance
overload diseases that share similar chelation treatments. with the CHRCO Institutional Review Board guidelines.
To protect against oxidative damage, the body has endog- Twenty-eight chronically transfused haemoglobinopathy pa-
enous defence mechanisms that are supported by dietary tients were studied (eight or more transfusions per year). Case
antioxidants. The lipid-soluble vitamin E (tocopherols) anti- subjects included 17 b-thalassaemia patients [7 male, age:
oxidants, including a- and c-tocopherol, are an important 23Æ7 ± 8Æ6 years (mean ± standard deviation (SD), Table I)],
front line defence (Sess et al, 1992; Christen et al, 1997; Jiang 11 SCD patients [seven male, age: 12Æ7 ± 3Æ7 years
et al, 2002; Jiang & Ames, 2003; Kassab-Chekir et al, 2003; (mean ± SD)] and nine healthy controls [six male, age:
Ruhl & Everhart, 2003). These antioxidants are better known 28Æ2 ± 11Æ4 years (mean ± SD); serum ferritin <300 lg/l;
as oxygen radical scavengers. However, c-tocopherol, the eight Caucasian, one Asian]. Clinical information including
primary form of vitamin E in the USA diet, has recently been demographics and history of transfusion, chelation, and
shown to have a unique role in preventing oxidant damage therapy were obtained by interview and chart review.
(Christen et al, 1997; Jiang & Ames, 2003). In addition to
functioning as an oxygen radical scavenger, c-tocopherol
Laboratory analyses
scavenges reactive nitric oxide species and inhibits prostaglan-
din E2-mediated inflammation (Jiang et al, 2000, 2001, 2002; A blood sample (45 ml) was collected from subjects in the
Jiang & Ames, 2003). c-Tocopherol, in contrast to a- morning at least 2 h postprandial; subjects were advised to
tocopherol, is increased in response to inflammatory stimuli abstain from taking any medications (including chelation)/
(Himmelfarb et al, 2003). It is unknown whether c-tocopherol vitamin or mineral supplements for the previous 24 h. The
levels differ in these two diseases or contribute to differences in study design specified that blood from transfused subjects be
oxidative damage. collected as long as possible after transfusion (i.e. in the period
Sickle cell disease is well recognised as a chronic inflamma- just proceeding the next transfusion). The mean days between
tory disease (Belcher et al, 2000, 2003; Klings & Farber, 2001; transfusion and phlebotomy were 20Æ7 ± 10Æ4 d after the last
Jison et al, 2004). Patients with SCD in crisis show elevated transfusion. Blood was transported at room temperature to the
tumour necrosis factor-a (TNF-a) and interleukin-6 (IL-6) analytical laboratory within 20 min of venepuncture for cell
compared with steady state SCD. In addition, IL-1, IL-6 and isolation and oxidation analysis. Serum, plasma and cells were
interferon-c (IFN-c) are elevated in steady state SCD com- separated by centrifugation.
pared with controls. It is unknown from these studies how iron
overload will affect the levels of chronic inflammation in SCD.
Malondialdehyde (MDA)
Elevations of the pro-inflammatory cytokines TNF-a and IL-2
have also been demonstrated in iron overloaded patients with Malondialdehyde was assayed in duplicate 250 ll plasma
thalassaemia (Del Vecchio et al, 2002). These cytokines samples, using gas chromatography-mass spectrometry (GC–
returned to normal after treatment with the iron chelator MS, model 5888; Agilent Technologies, Palo Alto, CA, USA),
deferiprone (L1) (Del Vecchio et al, 2002). Although it is true as previously described (Yeo et al, 1994, 1999). Two hundred
that oxidant stress can stimulate pro-inflammatory responses micro molar diethylenetriaminepentaacetate and 2Æ5 mmol/l
in both diseases, different patterns of cytokine and immuno- butylated hydroxytoluene (BHT) were added to prevent
modulator expression have been described and therefore it is sample oxidation during analysis. Samples were spiked with

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Journal Compilation ª 2006 Blackwell Publishing Ltd, British Journal of Haematology, 135, 254–263 255
P. B. Walter et al

Table I. Demographics.

Parameter Thalassaemia SCD Control P value

Age (years) 23Æ6 ± 8Æ6 (17) (a) 12Æ7 ± 3Æ7 (11) (b) 26Æ5 ± 12Æ3 (9) (a) 0Æ002*
Height (cm) 154Æ2 ± 9Æ1 (16) (a) 150Æ4 ± 23Æ8 (10) (a) 164Æ8 ± 21Æ8 (9) (a) 0Æ207*
Weight (kg) 52Æ3 ± 8Æ9 (17) (a) 45Æ4 ± 20Æ2 (11) (a) 62Æ2 ± 26Æ1 (9) (a) 0Æ125*
BMI (kg/m2) 22Æ3 ± 4Æ7 (16) (a) 19Æ1 ± 3Æ8 (10) (a) 21Æ7 ± 4Æ6 (9) (a) 0Æ201*
Sex M/F 7/10 (a) 7/4 (a) 4/5 (a) 0Æ489
Splenectomy, n 12/17 (67%) (a) 3/11 (27%) (b) – 0Æ025
Chelation, n– 16/17 (94%) (a) 7/11 (64%) (a) – 0Æ062
Transfusion, years 16Æ1 ± 8Æ6 (17) (a) 4Æ6 ± 2Æ9 (11) (b) – <0Æ001§
HCV, n 5/17 (29%) (a) 0 (0%) (a) – 0Æ125

Values are means ± SD. Number of patients analysed are shown in parentheses (n) to the right of the SD. Within a row groups sharing the same
lowercase letter are not significantly different from each other (5% procedure wise error rate).
BMI, body mass index; HCV, hepatitis C virus; SCD, sickle cell disease.
*The P value was obtained from an anova.
The P value was obtained from a chi-square test.
The P value was obtained from a Fisher’s exact test.
§The P value was obtained from a two-sample t-test.
–Chelation was by deferoxamine.

1 lmol/l 2H2-MDA as an internal standard. To hydrolyse had negative NTBI values. A negative NTBI value, a common
protein-bound MDA, 10 ll of 6Æ6 n H2SO4 was added for finding by several groups independent of the method used to
10 min at room temperature. MDA was derivatised to measure NTBI (Singh et al, 1990; Porter et al, 1996; Bradley
pentafluorophenylhydrazine at room temperature for 1 h. et al, 1997; Gosriwatana et al, 1999; Wickramasinghe et al,
Derivatised MDA was extracted with isooctane, and 70 ll was 1999; Jacobs et al, 2005), arises when the transferrin saturation
injected into the GC–MS for detection. in samples is less than 50% (Jacobs et al, 2005). Negative NTBI
values can be caused when transferrin is less than 50%
saturated and iron NTA loads rapidly onto transferrin. Hence,
Vitamin E
normal control samples, which contain around 30% saturated
Plasma a-tocopherol and c-tocopherol were analysed as transferrin, bind iron bound to NTA and thus appear negative
previously described (Jiang et al, 2002; Jiang & Ames, 2003). with respect to the experimental zero iron controls used with
Briefly, tocopherols were extracted using a mixture of standards to generate calibration curves where no transferrin is
methanol/hexane (2:5, v/v) in the presence of 0Æ8 mmol/l present.
BHT added to prevent oxidative losses during work-up.
Tocopherols were separated on a 150 · 4Æ6 mm, 5 lm Supe-
C-reactive protein
lcosilTM LC-18-DB column (Supelco, Bellefonte, PA, USA) and
monitored by coulometric detection (Model Coulochem II; Plasma high sensitivity C-reactive protein (hsCRP) was deter-
ESA Inc., Chelmsford, MA, USA) using a Model 5011 mined in plasma samples by a nephelometric method utilising
analytical cell (Jiang et al, 2002; Jiang & Ames, 2003). latex particles coated with (CRP) monoclonal antibodies by
Quest Diagnostics Inc., San Juan Capistrano, CA, USA.
Non-transferrin bound iron (NTBI)
Cytokines
Non-transferrin bound iron was determined by nitrilotriacetic
acid (NTA) capture of NTBI and quantified by high Plasma levels of 10 cytokines (IL-1b, IL-2, IL-4, IL-5, IL-6, IL-
performance liquid chromatography (HPLC) (Singh et al, 8, IL-10, granulocyte–macrophage colony-stimulating factor
1990). This assay uses a large excess of a low affinity ligand (GM-CSF), IFN-c and TNF-a) were determined in samples by
NTA, which removes and complexes low molecular mass iron a multiplex antibody bead assay by BioSource International,
and iron non-specifically bound to serum proteins. Iron bound Camarillo, CA, USA.
to transferrin, ferritin, desferrioxamine, and its metabolites is
not accessed by the NTA ligand. The Fe–NTA complex present
Liver iron
in the ultrafiltrate represents the NTBI and is then quantified
using an HPLC procedure including on-column derivatisation Tissue iron concentration was determined by inductively
with a high affinity iron chelator (3-hydroxy-1-propyl-2- coupled plasma mass spectrometry (ICP-MS, Mayo, Rochester,
methyl-pyridin-4-one). Using this method, normal individuals NY, USA) as described previously (Vichinsky et al, 2005a).

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256 Journal Compilation ª 2006 Blackwell Publishing Ltd, British Journal of Haematology, 135, 254–263
Oxidative Stress in b-Thalassaemia and Sickle Cell Disease

Liver biopsies were collected within an average of 15 d (mean cant. Post hoc tests were done by Tukey’s analysis at a 5%
14Æ5 ± 20Æ5) of the phlebotomy for the study. Three samples procedure-wise error rate. As MDA and ferritin values were
were excluded from these analyses because they were collected right skewed, the analyses on these two variables were
more than 2 months from the blood sample. performed on log-transformed data. Logistic regression was
used to create multivariate models to describe relationships
between oxidative injury (MDA, ALT) and factors that may
Blood and liver function tests
influence their development (i.e. diagnosis, duration of
Alanine transaminase (ALT) and ferritin were measured in the chronic transfusion, liver iron concentration, NTBI or age).
clinical lab at CHRCO. Serum ferritin concentrations were Other variables were also examined as potential confounders,
measured by a one step immunoassay (FERR Flex; Dade such as serum ferritin and hepatitis C virus (HCV) and were
Behring Inc., Newark, DE, USA). Serum ALT concentrations found to be non-significant in the model.
were measured on the Dimension clinical chemistry system
(Dade Behring Inc.). The manufacturer’s reference range for
Results
the normal general population is 30–65 U/l (n ¼ 244 adults).
Ferritin and ALT are presented as the mean values obtained
Demographics
during the 6 month prior to the study phlebotomy. Hepatitis
C RNA was identified by polymerase chain reaction. The demographics of the study populations are shown in
Table I. No significant differences were seen in sex, height,
weight, body mass index, chelation or percentage of HCV
Percentage transferrin saturation
infection, among the study groups. Duration of chronic
This was determined on serum samples using the urea gel transfusion was significantly longer in thalassaemia patients
method of Evans and Williams (1978). Twenty-five micro litres (P < 0Æ001) and they were significantly older than the SCD
of serum were treated with an excess of rivanol to remove most group (P ¼ 0Æ002). Splenectomy (P ¼ 0Æ025) was more com-
of the serum proteins excluding transferrin. After centrifuga- mon in thalassaemia patients, but there was no significant
tion, samples of the supernatants were run on polyacrylamide difference in chelation treatment (deferioxamine, P ¼ 0Æ062)
gels containing 6 mol/l urea. Under these partially denaturat- compared with SCD patients.
ing conditions, the four different species of transferrin, namely The caloric intake was not different among the three groups
apo-, diferric-, C- and N-terminal monoferric, denatured to (approximately 2000 ± 1000 kcal) and no difference was
differing extents and were therefore separated on the gel, which observed in total fat, protein, carbohydrate, calcium, iron,
enabled quantification using densitometry from which the per vitamin A, C, E and D intake between groups (including
cent saturation was calculated. supplements).

Protein carbonyls Comparison of iron burden


Oxidative damage to plasma proteins were assayed as previ- Parameters of iron burden are shown in Table II. Although
ously described by measuring carbonyl groups on plasma both thalassaemia and SCD groups had evidence of severe
proteins by reaction with 2,4-dinitrophenylhydrazine to form a haemosiderosis by quantitative liver iron measurements, these
hydrazine complex, which was measured spectrophotometri- were higher in SCD than thalassaemia patients in this study
cally (Houglum et al, 1990, 1997). population (15Æ0 ± 5Æ6 vs. 9Æ2 ± 7Æ2 mg/g dry tissue weight,
P ¼ 0Æ033, Table II). Serum ferritin and transferrin saturation
showed similar high elevation in both thalassaemia and SCD
Diet
patients relative to controls. Despite higher tissue iron burden,
The Block Food Frequency Questionnaire (Block, 1998) was SCD patients had significantly lower NTBI than thalassaemia
completed by a majority of subjects to obtain a semi- patients (Table II).
quantitative assessment of usual food intake over the previous
12 months. The Block Food Frequency Questionnaire has been
Oxidative injury and inflammation
validated in adult (Sinha et al, 1993) and paediatric popula-
tions (Block et al, 1995) and is used commonly in epidemi- In thalassaemia patients, MDA levels were significantly higher
ological trials of oxidative stress. than controls. This difference was not observed for SCD
patients (MDA values were log-transformed prior to analysis;
Table III). Further analysis of MDA comparing it with liver
Statistics
iron concentration in both disease patient groups showed a
Data are reported as mean ± SD. Comparisons among groups positive correlation between liver iron concentration and
were made using one-way analysis of variation (anova) plasma MDA (Fig 1; thalassaemia, R2 ¼ 0Æ695, P ¼ 0Æ003 and
analyses, where P < 0Æ05 was considered statistically signifi- SCD, R2 ¼ 0Æ644, P ¼ 0Æ032). Plasma a-tocopherol was signi-

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Journal Compilation ª 2006 Blackwell Publishing Ltd, British Journal of Haematology, 135, 254–263 257
P. B. Walter et al

Table II. Measurements of iron status.

Parameter Thalassaemia SCD Control anova P value

Liver iron (mg/g dw) 9Æ2 ± 7Æ2 (16) (b) 15Æ0 ± 5Æ6 (11) (a) 0Æ033
Ferritin (lg/l) 1868 ± 1042 (17) (a) 2515 ± 1153 (11) (a) 65Æ0 ± 70Æ9 (9)(b) <0Æ001
Transferrin saturation (%) 82Æ5 ± 21Æ7 (6) (a) 64Æ5 ± 23Æ3 (4) (a,b) 40Æ2 ± 13Æ1 (5) (b) 0Æ014
NTBI (lmol/l) 4Æ0 ± 1Æ6 (15) (a) 1Æ9 ± 2Æ1 (11) (b) )1Æ0 ± 0Æ4 (9) (c) <0Æ001

Values are means ± SD. Number of patients analysed are shown in parentheses (n) to the right of the SD. Within a row, groups not sharing the same
letter are significantly different from each other (5% procedure wise error rate).
dw, dry weight; NTBI, non-transferrin bound iron; SCD, sickle cell disease.

Table III. Measurements of oxidative injury and inflammation.

Parameter Thalassaemia SCD Control anova P value

MDA, nmol/l* 35Æ3 ± 20Æ0 (17) (a) 26Æ1 ± 9Æ7 (11) (a,b) 19Æ3 ± 13Æ4 (9) (b) 0Æ006
a-tocopherol, lmol/l 14Æ8 ± 4Æ6 (17) (b) 15Æ7 ± 4Æ1 (11) (b) 22Æ7 ± 3Æ4 (9) (a) <0Æ001
c-tocopherol, lmol/l 3Æ3 ± 2Æ1 (17) (b) 5Æ5 ± 1Æ4 (11) (a) 1Æ8 ± 0Æ7 (9) (c) <0Æ001
Protein carbonyls, nmol/mg 0Æ7 ± 0Æ3 (16) (a) 0Æ6 ± 0Æ1 (11) (a) 0Æ6 ± 0Æ1 (9) (a) 0Æ454
ALT, U/l 57Æ5 ± 31Æ1 (17) (a) 36Æ1 ± 11Æ0 (11) (b) 34Æ9 ± 7Æ7 (9) (b) 0Æ020
hsCRP, mg/l 1Æ0 ± 1Æ2 (16) (a) 2Æ7 ± 3Æ2 (10) (a) 0Æ9 ± 0Æ7 (9) (a) 0Æ061
Cytokines: pg/ml
IL-5 0Æ3 ± 0Æ3 (13) (b) 1Æ0 ± 0Æ6 (10) (a) 0Æ6 ± 0Æ6 (8) (a,b) 0Æ010
IL-6 3Æ1 ± 1Æ7 (15) (a,b) 4Æ5 ± 2Æ2 (11) (a) 2Æ0 ± 1Æ0 (8) (b) 0Æ014
IL-10 2Æ1 ± 1Æ2 (17) (b) 3Æ5 ± 1Æ6 (11) (a) 1Æ8 ± 1Æ0 (9) (b) 0Æ007

Values are means ± SD. Number of patients analysed are shown in parentheses (n) to the right of the SD. Within a row, groups not sharing the same
letter are significantly different from each other (5% procedure wise error rate).
*MDA values were log-transformed prior to analysis.
ALT, alanine transaminase; IL, interleukin; hsCRP, high sensitivity C-reactive protein; MDA, malondialdehyde; SCD, sickle cell disease.

100 for normal healthy controls used in other studies (Himmelfarb


et al, 2003)]. The dietary questionnaire found no difference in
SCD
Thalassemia intake of c- or a-tocopherol. Protein carbonyls, a measure of
oxidative damage to amino acid side chains, were not
MDA (nmol/l)

significantly changed in either group in comparison to


controls.
Multivariate analysis using log MDA as the dependent
variable representing oxidant injury was employed to examine
the pattern of several of the following relationships simulta-
neously (Table IV). This analysis examined as co-variants (i.e.
predictors): diagnosis, liver iron concentration, duration of
10
chronic transfusion and age. Two significant predictors of
0 5 10 15 20 25 30 MDA were found, diagnosis of thalassaemia and quantitative
Liver iron concentration (mg/g dw)
liver iron (P < 0Æ001 and ¼0Æ019 respectively). Age was not a
Fig 1. Regression analysis comparing plasma malondialdehyde (MDA) significant predictor in multivariate analysis (Table IV). ALT,
(nmol/l, log scale) with liver iron concentration [mg/g dry weight another marker of tissue injury was significantly increased
(dw)]. Regressions for thalassaemia (top; R ¼ 0Æ695, P ¼ 0Æ003) and (1Æ7-fold, P ¼ 0Æ020) in the thalassaemia group relative to the
sickle cell disease (bottom; R ¼ 0Æ644, P ¼ 0Æ032), show that, in both SCD group (Table III). Further multivariate analysis, with ALT
diseases, higher liver iron concentrations are associated with more
as the dependent variable, and liver iron concentration and
plasma MDA.
diagnosis as independent variables in the model, found that
diagnosis of thalassaemia was a predictor of higher ALT
ficantly reduced in both thalassaemia and SCD patients relative (P ¼ 0Æ025, data not shown).
to controls. In contrast, c-tocopherol levels were significantly Markers of inflammation including hsCRP and 10 cytokines
higher in SCD compared with thalassaemia patients as well as were compared with each of the groups (Table III). A 2Æ5-fold
controls [Table III, the control group was in the range found higher hsCRP level was observed in SCD compared with

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258 Journal Compilation ª 2006 Blackwell Publishing Ltd, British Journal of Haematology, 135, 254–263
Oxidative Stress in b-Thalassaemia and Sickle Cell Disease

Table IV. Oxidant injury analysis model using two groups (thalassaemia and SCD).

Bivariate (crude) Multivariate (adjusted)

P-value P-value Regression coefficient 95% confidence intervals

MDA (dependent variable, nmol/l) R2 for model ¼ 0Æ55, n ¼ 26


Diagnosis [thalassaemia (reference) versus SCD] 0Æ185 0Æ019 )0Æ481 )0Æ870, )0Æ091
Liver iron concentration (mg/g) 0Æ012 <0Æ001 +0Æ045 0Æ024, 0Æ067
Duration of chronic transfusion (years) 0Æ199 0Æ641 +0Æ005 )0Æ016, 0Æ026
Age (years) 0Æ041 0Æ697 +0Æ004 )0Æ017, 0Æ025

MDA values were log transformed prior to analysis.


MDA, malondialdehyde; SCD, sickle cell disease.

thalassaemia patients, which showed a trend toward statistical (Cunningham et al, 2004; Vichinsky et al, 2005b). In order to
significance (P ¼ 0Æ061; Table III). The inflammatory cyto- investigate the relationship of iron overload and specific
kines IL-5, IL-6 and IL-10 were highest in SCD patients, with disease to injury, we examined biomarkers of oxidative stress,
IL-6 and IL-10 being significantly different from controls, inflammation and tissue injury. However, while each class of
while IL-5 and IL-10 were also significantly elevated in SCD biomarker is traditionally thought to represent one of these
compared with thalassaemia patients. There was no significant processes, it is important to note that they are not distinct
difference between the levels of IL-5, IL-6 and IL-10 in entities and have considerable interaction, i.e. oxidative stress
thalassaemia and control patients (Table III). There were no can initiate tissue injury and/or inflammation.
significant differences among groups for the other cytokines Plasma malondialdehyde was studied as a marker of tissue
examined (GM-CSF, IFN-c, IL-1b, IL-2, IL-4, IL-8, and TNF- injury and oxidative stress. MDA is a well-recognised bio-
a, data not shown). marker of lipid peroxidation, but its measurement has been
limited by the specificity of the methodology. The present
study utilises GC–MS, a technique that avoids problems of
Discussion
oxidation during processing (as may be seen in thiobarbituric
Emerging clinical data suggest that iron overloaded SCD acid reactive substance analysis) and specifically measures
patients are at less risk for organ injury than patients with MDA (Liu et al, 1997). Another factor we have taken into
thalassaemia. Wood et al (2004) recently reported increased account is artifactual oxidation during the measurement of
cardiac iron deposition in chronically transfused haemoglob- MDA because of the high NTBI in our samples. We took great
inopathy patients. Decreased cardiac function only occurred in care to prevent any possible enhanced MDA during the
thalassaemia patients, not in iron overloaded SCD patients preparation of our samples by adding chelators and antioxi-
(Wood et al, 2004). In a recent study involving 30 thalassaemia dants. Regression analysis revealed that higher liver iron
and 43 SCD patients with severe haemosiderosis, we confirmed concentrations are associated with more plasma MDA in both
that greater cardiac and endocrine disease occurred in the disease groups. Further multivariate analysis identified specific
thalassaemia patients (Vichinsky et al, 2005a). Although the disease (thalassaemia) and liver iron concentration as signifi-
specific role of iron overload in mediating injury is the primary cant predictors of high MDA. In animal models, high liver iron
focus of this paper, the unique pathophysiologies of SCD and levels induce elevation of lipid peroxides and oxidants
thalassaemia certainly play a critical role in promoting the (Knutson et al, 2000; Walter et al, 2002) presumably through
observed pathology. Specifically, in SCD the tissue injury iron-initiated Fenton chemistry. Increased lipid peroxidation
related to sickling of RBCs and in thalassaemia the surplus of markers have previously been observed in thalassaemia
a-globin chains and intramedullary ineffective erythropoiesis patients (Livrea et al, 1998; Cighetti et al, 2002; Laksmitawati
are certainly important factors. Also important is the enhanced et al, 2003). However, MDA studies in SCD have been largely
gastrointestinal iron absorption seen thalassaemia patients that focused on in vitro studies of sickle-cell erythrocyte membranes
may initiate different iron trafficking compared with SCD. (Buchanan & Holtkamp, 1981; Hebbel & Miller, 1984; Jain &
Clearly, disease diagnosis is important; in fact, multivariate Shohet, 1984; Jain et al, 1989, 1990; Sertac et al, 1997). This is
analysis showed disease diagnosis of thalassaemia to be a the first study to compare oxidative injury with iron overloa-
significant predictor of MDA and this variable may be closely ded SCD and thalassaemia patients.
related to these distinct underlying fundamental pathophysi- Duration of chronic transfusion and NTBI seem to be likely
ologies. Furthermore, concerning the ethnicity difference candidates as predictors of MDA, because duration of
between SCD and thalassaemia; we cannot discount ethnicity transfusion indicates the time the iron stress has been endured
being a factor. However, while this is true, the incidence and and NTBI estimates the possible availability of free iron.
prevalence of iron-induced organ injury was not influenced by However, neither was a predictor of MDA in multivariate
the ethnicity of the patients in multiple large thalassaemia trials analysis. Concerning duration of chronic transfusion, elevated

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Journal Compilation ª 2006 Blackwell Publishing Ltd, British Journal of Haematology, 135, 254–263 259
P. B. Walter et al

MDA is probably a real-time marker of oxidative injury important inflammatory differences that may exist between
(Kadiiska et al, 2005a,b) and indicative of current liver iron these two diseases. SCD is characterised by microvascular
concentration. Duration of chronic transfusion correlates hypoxia-reperfusion leading to inflammation. Human and
better with cumulative tissue injury (Vichinsky et al, 2005a). animal models have found elevated TNF-a, IL-6, IL-10 and
We suggest that this may explain why duration of transfusion hsCRP as markers of inflammation in SCD (Hedo et al, 1993;
is not a predictor of MDA in multivariate analysis. NTBI is also Singhal et al, 1993; Bank et al, 1998; Bourantas et al, 1998;
not a significant predictor of MDA. This is surprising, because Dale & Alberio, 1998; Sess et al, 1998; Belcher et al, 2000, 2003;
with high levels of both plasma MDA and NTBI, it is the Makis et al, 2000; Barbeau et al, 2001; Ludwiczek et al, 2003;
potential free iron in NTBI that could be initiating Fenton Banerjee & Kuypers, 2004). Our results support these
chemistry in vivo, which induces the lipid peroxidation that observations. Inflammation (Fillet et al, 1989) and cytokines
gives increased MDA. However, this is not the case. We suggest (IL-10) stimulate the uptake and retention of iron into
that the increased levels of plasma MDA in thalassaemia may monocytes and reticuloendothelial (RE) cells (Ludwiczek et al,
result from several mechanisms. First, plasma MDA could be 2003). Recently, hepcidin that is increased in inflammation,
enhanced in thalassaemia patients because it may be dependent has been identified as a critical participant in this cellular
on the amount of circulating erythroid precursors and regulation of iron storage (Papanikolaou et al, 2005). In
peripheral blood erythrocytes that have a high density of transfused thalassaemia or SCD patients the hepicidin level
unpaired a-haemoglobin chains (Scott et al, 1993). The excess may vary depending on the severity of anaemia as suggested in
a-chains in thalassemic red blood cells are unstable and prone a recent study (Kearney et al, 2005). Increased cytokines (such
to denaturation and oxidation (Scott et al, 1993) and, in the as IL-10) may explain the lower NTBI levels in SCD patients as
present work, may have contributed more to enhanced plasma compared with thalassaemia patients (Ludwiczek et al, 2003).
MDA than the SCD haemoglobin, which is also capable of We and others suggest that the NTBI of SCD patients may be
producing oxidants (Repka & Hebbel, 1991). The a-chains in taken up by the RE system, sparing the parenchymal tissues of
thalassemic red blood cells can autoxidise, release haem and injury (Pippard, 1994). In contrast, the high NTBI in
generate superoxide (Scott et al, 1993), which can then thalassaemia may also contribute to the increased cardiac iron
increase lipid peroxidation and thus enhance MDA levels. and organ dysfunction observed in thalassaemia patients.
Furthermore, plasma MDA may be increased in thalassaemia Antioxidant capacity is an important determinant of tissue
because of peroxidation of tissues that leak MDA into the injury, especially in patients with increased oxidant stress. We
plasma. For example, plasma ALT, another marker of tissue have found significant differences in antioxidant capacity in
injury, was also increased in thalassaemia relative to SCD and thalassaemia and SCD patients in terms of plasma tocopherol
is indicative of liver dysfunction and leakage of liver meta- levels. Plasma a-tocopherol was decreased in both thalassaemia
bolites into the plasma. Parallel to ALT, plasma MDA may also and SCD patients in ours and other studies (Rachmilewitz
rise partly as a result of possible liver lipid peroxidation and et al, 1976; Sess et al, 1998; De Luca et al, 1999; Kassab-Chekir
leakage into the plasma. It is an attractive suggestion that MDA et al, 2003). Our observed changes in a-tocopherol were not
may leak from the liver because of its strong correlation to liver because of variation in dietary intake and are probably related
iron concentration in multivariate analysis. to its consumption as a scavenger of oxidants. These low levels
We also examined inflammation, a known regulator of iron probably contribute to increased MDA levels. In contrast to
transport and storage, as another possible explanation for the the decreased a-tocopherol, c-tocopherol levels were increased
observed differences in MDA levels, and to determine the in both groups, but levels were significantly higher in the SCD

Table V. Model of transfusional iron overload


Thalassaemia SCD in thalassaemia and SCD (shown are the relative
differences).
General tissue changes:
More organ dysfunction

Changes in specific biomarkers:


More MDA (adjusted), ALT, NTBI More IL-5 and IL-10
More c-tocopherol

Hypothesis:
Transfusional iron load stored Transfusional iron load is taken up by
in the liver, heart and endocrine organs. activated RE cells keeping iron
away from vital organs.
Tissue iron load leads to dysfunction. c-tocopherol is higher which may protect.

ALT, alanine transaminase; MDA, malondialdehyde; NTBI, non-transferrin bound iron; RE,
reticuloendothelial; SCD, sickle cell disease.

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260 Journal Compilation ª 2006 Blackwell Publishing Ltd, British Journal of Haematology, 135, 254–263
Oxidative Stress in b-Thalassaemia and Sickle Cell Disease

patients. Inflammation has been shown to decrease degrada- Banerjee, T. & Kuypers, F.A. (2004) Reactive oxygen species and
tion of c-tocopherol through inhibition of a cytochrome P450 phosphatidylserine externalization in murine sickle red cells. British
3A–dependent process (Parker et al, 2000; Sontag & Parker, Journal of Haematology, 124, 391–402.
2002) and may explain our observed difference. We suggest Bank, N., Kiroycheva, M., Ahmed, F., Anthony, G.M., Fabry, M.E.,
Nagel, R.L. & Singhal, P.C. (1998) Peroxynitrite formation and
that the higher levels of c-tocopherol in SCD may be
apoptosis in transgenic sickle cell mouse kidneys. Kidney Interna-
important in reducing tissue injury related to inflammation
tional, 54, 1520–1528.
and oxidant stress. Barbeau, P., Woods, K.F., Ramsey, L.T., Litaker, M.S., Pollock, D.M.,
Pollock, J.S., Callahan, L.A., Kutlar, A., Mensah, G.A. & Gutin, B.
Conclusions (2001) Exercise in sickle cell anemia: effect on inflammatory and
vasoactive mediators. Endothelium, 8, 147–155.
Malondialdehyde levels were higher in thalassaemia compared Belcher, J.D., Marker, P.H., Weber, J.P., Hebbel, R.P. & Vercellotti,
with SCD (conclusions summarised in Table V). The strongest G.M. (2000) Activated monocytes in sickle cell disease: potential role
predictors of elevated MDA, by multivariate analysis, were in the activation of vascular endothelium and vaso-occlusion. Blood,
diagnosis and liver iron concentration. In contrast, several 96, 2451–2459.
inflammatory markers appeared higher in SCD. This inflam- Belcher, J.D., Bryant, C.J., Nguyen, J., Bowlin, P.R., Kielbik, M.C.,
Bischof, J.C., Hebbel, R.P. & Vercellotti, G.M. (2003) Transgenic
mation in SCD may generate increased levels of c-tocopherol,
sickle mice have vascular inflammation. Blood, 101, 3953–3959.
leading to decreased tissue peroxidation and injury. Inflam-
Block, G. (1998) Invited commentary: comparison of the Block and the
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Bourantas, K.L., Dalekos, G.N., Makis, A., Chaidos, A., Tsiara, S. &
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by the following: NIH grants R01-DK057778, M01 RR01271, blative chemotherapy. British Journal of Haematology, 99, 337–343.
U54HL070583, R01-AT001821, the National Center for Bronspiegel-Weintrob, N., Olivieri, N.F., Tyler, B., Andrews, D.F.,
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