Blood, Vol. 92 No. 10 (November 15), 1998:
pp. 3936-3942
Oxidative Modification of Low-Density Lipoprotein and
Atherogenetic Risk in 
-Thalassemia
By
M.A. Livrea,
L. Tesoriere,
A. Maggio,
D. D'Arpa,
A.M. Pintaudi, and
E. Pedone
From the Istituto di Farmacologia e Farmacognosia e Dipartimento di
Chimica e Tecnologie Farmaceutiche, Università di Palermo,
Palermo; and the Servizio Talassemia, Ospedale Cervello, Palermo.
Italy.
 |
ABSTRACT |
We investigated the oxidative state of low-density lipoprotein (LDL)
in patients with 
-thalassemia to determine whether there was an
association with atherogenesis. Conjugated diene lipid hydroperoxides
(CD) and the level of major lipid antioxidants in LDL, as well as
modified LDL protein, were evaluated in 35 -thalassemia intermedia
patients, aged 10 to 60, and compared with age-matched healthy
controls. Vitamin E and -carotene levels in LDL from patients were
45% and 24% of that observed in healthy controls, respectively. In
contrast, the mean amount of LDL-CD was threefold higher and lysil
residues of apo B-100 were decreased by 17%. LDL-CD in thalassemia
patients showed a strong inverse correlation with LDL vitamin E (r = 
0.784; P < .0001), while a negative trend was observed
with LDL--carotene (r = 0.443; P = .149). In the
plasma of thalassemia patients, malondialdehyde (MDA), a byproduct of
lipid peroxidation, was increased by about twofold, while vitamin E
showed a 52% decrease versus healthy controls. LDL-CD were inversely
correlated with plasma vitamin E (r = 0.659; P < .0001)
and correlated positively with plasma MDA (r = 0.621; P < .0001). Plasma ferritin was positively correlated with LDL-CD (r = 0.583; P =.0002). No correlation was found between the age of
the patients and plasma MDA or LDL-CD. The LDL from thalassemia
patients was cytotoxic to cultured human fibroblasts and cytotoxicity
increased with the content of lipid peroxidation products. Clinical
evidence of mild to severe vascular complications in nine of the
patients was then matched with levels of LDL-CD, which were 36% to
118% higher than the mean levels of the patients. Our results could
account for the incidence of atherogenic vascular diseases often
reported in -thalassemia patients. We suggest that the level of
plasma MDA in -thalassemia patients may represent a sensitive index
of the oxidative status of LDL in vivo and of its potential
atherogenicity.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
THE THALASSEMIAS ARE genetic disorders,
which encompass a wide variety of clinical phenotypes, ranging in
severity from clinically silent heterozygous -thalassemia to severe
transfusion-dependent thalassemia major. Among others, clinical
features of thalassemias include vascular complications, such as
pulmonary thromboembolism, cerebral thrombosis, and leg
ulcers.1-3 Consistent with the pathogenesis of such events,
the data indicate that injury of vascular endothelial cells is present
in thalassemia patients.4
In recent years increasing evidence suggests that the oxidative
modification of low-density lipoprotein (LDL) is the key step in the
sequence of events leading to atherogenesis-related vascular alterations.5-7 Modified LDL are internalized in
monocyte-derived macrophages through cell surface scavenger receptors,
an event that leads to foam cell formation. Infiltration and deposition of these cells in the arterial wall are considered the initiating steps
to develop atherosclerotic plaque.
Oxidation of polyunsaturated fatty acids (PUFA) is considered to be an
important initiating factor in the alteration of LDL.8,9 Decomposition products of PUFA gradually spread over to the protein moiety of apo B-100 and neutralize the positively charged lysil 
-amino groups.10 Little is known about the mechanism by
which LDL then becomes oxidized in vivo. However, in -thalassemia, such conditions as alteration of iron homeostasis,11
interactions between ruptured erythrocytes and LDL,12,13
depletion of antioxidant defenses14,15 and decrease of HDL
particles16 might promote oxidative damage to circulating
LDL. A recent report from our laboratory showed that LDL from
-thalassemia intermedia patients is more susceptible to the in vitro
oxidation, as compared with LDL from healthy donors.15 All
of the above evidence lends credence to the idea that circulating LDL
from thalassemia patients may bear marked oxidative modifications.
We evaluated the extent of protein and lipid oxidation products and
lipid antioxidants in LDL from patients with -thalassemia intermedia, in which transfusion-dependent secondary iron overload is
not a prominent cause of toxicity. In addition, because oxidized LDL
are cytotoxic,17-19 cytotoxicity of LDL from thalassemia
patients on human cultured fibroblasts was investigated as one basic
parameter of its atherogenic potential. A relationship between the
oxidative status of LDL and atherosclerotic vascular lesions observed
in a number of patients is also reported.
| |
MATERIALS AND METHODS |
Subjects.
Twenty female and 15 male thalassemia intermedia patients, age 10 to 60 years (mean, 32 ± 14), who had been previously characterized for
-globin gene mutation were recruited for this study. Consent was
obtained and individuals were under observation for 1 year. All
patients were regularly interviewed and examined by a staff of
physicians at intervals of 15 days to 1 month. Hemoglobin levels were
6.5 to 11.3 g/dL (mean, 8.33 ± 1.28) and patients received occasional transfusions (<3 to 6 per year). Ferritin was measured every 4 months, and cardiac, endocrinologic, and hepatologic
evaluations were performed regularly. No patient was diabetic or
hepatitis C virus positive or showed abnormal levels of serum alanine
or aspartate aminotransferases. Some patients had experienced one or
more of the following vascular complications: pulmonary hypertension (10 patients), cerebral ischemia (three patients), retinal vasculopathy (three patients), and ulcerative peripheral vasculopathy (one patient).
Two of the patients were smokers. Patients were not on lipid-altering
medications.
Clinical chemistry analyses.
After an overnight fast, blood from thalassemia patients was collected
in EDTA (1 mg/mL-1). Blood samples from 35 apparently
healthy individuals, aged 22 to 59, who were nonsmokers and who were
not taking any medication, were used for the control group. Plasma was
separated by centrifugation and divided in suitable aliquots to prepare
LDL and perform the analytical determinations described below.
Total bilirubin, total cholesterol, and high-density lipoprotein (HDL)
cholesterol were evaluated by using commercial analytical kits from
Sigma (St Louis, MO). Concentration of plasma LDL cholesterol was
calculated by the Friedwald formula.20 Ferritin was
determined by an enzyme-immuno assay (Abbott Labs, North Chicago, IL).
Preparation of LDL.
LDL (1.019 to 1.063 g/mL) was isolated from EDTA plasma by stepwise
ultracentrifugation at 4°C in a Beckman L8-70M
ultracentrifuge fitted with a 50 Ti rotor using potassium
bromide for density adjustments (Beckman, Palo Alto, CA), according to
Kleinveld.21 The LDL fraction was shown to be free of other
lipoproteins by electrophoresis on an agarose gel. EDTA and salts were
removed from LDL by gel filtration on Sephadex G-25 Medium (Pharmacia Biotech, Milano, Italy).22 Proteins were determined by the
Bio Rad colorimetric method.23 In typical preparations, 0.6 mg apo B-100 was obtained from 1 mL plasma. To prevent autoxidation
reactions, LDL were used immediately or after an overnight storage at
70°C. Preliminary assays after overnight storage at
70°C showed that this treatment did not modify LDL
composition as compared with freshly prepared LDL.
Apo B-100 analysis.
Apo B-100 lysine residues were evaluated after delipidation and acidic
hydrolysis of the protein in 12 N HCl, for 18 hours at 100°C.
Briefly, LDL samples (1.0 mg protein) in 1.0 mL 0.15 mol/L NaCl, mixed
with 0.7 mL of a mixture of CHCl3:MeOH (2:1, vol:vol) in a
Pyrex tube, were vortexed and then centrifuged at 3,000g for 10 minutes. The bottom CHCl3 layer was removed with a Pasteur
pipette and discarded, and the extraction was repeated three times. The
MeOH:water phase (and the residual CHCl3 were) was
evaporated by placing the tubes in a boiling water bath, then the apo B
precipitated on the tube walls was removed by the aid of 1.0 mL of 12 N
HCl. Screw caps were tightened and hydrolysis was performed for 18 hours at 100°C in a boiling water bath. Residues in the hydrolysate
were analyzed by a Beckman 6003 amino acid analyzer equipped with a
Shimadzu Chromatopac C-R3A integrator (Shimadzu, Kyoto, Japan).
Biochemical analyses.
Malondialdehyde (MDA) was evaluated in 50 µL plasma samples by a
colorimetric reaction with thiobarbituric acid (TBA,
Sigma),24 followed by neutralization of samples with
equivalent volumes of a mixture consisting of 4.5 mL 1.0 mol/L NaOH and
45.5 mL methanol. Isocratic high performance liquid chromatography
(HPLC) separation of the MDA adduct was performed using a Supelco
Supelcosil (Bellefonte, PA) LC-18 column (0.46 x 25 cm), eluted with
40% methanol in 50 mmol/L potassium phosphate buffer, pH 6.8, at 1.5 mL min-1. The MDA-TBA adduct was revealed at 532 nm and
quantified by reference to a calibration curve of tetraethoxypropane
(Sigma), submitted to the TBA colorimetric procedure. Butylated
hydroxytoluene (0.03%) was added to the TBA reagent to prevent
artifactual lipid peroxidation during the assay procedure. The
conjugate diene lipid hydroperoxides in the lipid fraction of LDL
(LDL-CD) were extracted from LDL samples (200 µg protein in 1.0 mL
0.15 mol/L NaCl) by 2.0 mL CHCl3:MeOH (2:1, vol:vol). The
organic extract was dried under a nitrogen stream, resuspended in
cyclohexane, and quantitated spectrophotometrically at 234 nm, using a
molar absorption coefficient of 27,000.25 The results are
expressed as nmol/mg LDL protein.
All-trans retinol and 
-tocopherol were extracted from 200 µL of plasma samples, diluted to 1.0 mL with 0.15 mol/L NaCl, by mixing with 2 volumes of absolute ethanol, followed by two successive extractions with 6 and 2 volumes of petroleum ether. The organic extracts were gathered, dried under nitrogen, resuspended in several microliters of suitable solvent, and injected on top of an LC-18 HPLC
column (see above). Analysis was performed by eluting with methanol at
1.0 mL min-1. Detection of all-trans retinol and
-tocopherol were at wavelengths of 320 nm and 290 nm, respectively.
Under the conditions described, all-trans retinol eluted after
5.2 minutes and -tocopherol after 12.8 minutes. Automatic wavelength
change after 9 minutes allowed the detection of both compounds in the
same sample. Alpha-tocopherol was extracted from LDL samples (50 µg
protein in 1.0 mL phosphate-buffered saline [PBS]) and analyzed by
HPLC as described above.
Beta-carotene was extracted from 500 µg LDL protein in a final volume
of 1.0 mL PBS by mixing with 1 volume of methanol and 3 volumes of
hexane:diethyl ether (1:1, vol:vol). The extracts were then dried under
nitrogen, resuspended with several microliters of a mixture of
acetonitrile:methanol:tetrahydrofurane (58.5:35:6.5, vol:vol:vol) and
analyzed with the same solvent26 with an LC-18 Supelco
column as above, at a flow rate of 2.5 mL min-1. Under
these conditions, -carotene eluted at 13.8 minutes. Revelation was
at 450 nm.
Quantitation of all compounds evaluated by HPLC was performed by
reference to standard curves constructed with 5 to 100 ng of each
compound and by relating the amount of the compound under analysis to
the peak area. All procedures were performed under dim red light to
avoid artifactual photooxidation of lipids and to preserve light
sensitive vitamins.
Test of the cytotoxicity of thalassemic LDL.
Human fibroblasts were obtained from small dermal specimens from the
dorsal forearm of healthy donors. The epidermal layers were carefully
removed and portions of the underlying dermis were cut into explants (1 mm3) and placed in flasks in complete medium (CM, GIBCO,
Grand Island, NY) containing 10% heat-inactivated fetal
calf serum, 2 mmol/L glutamine, 100 U/mL penicillin, and 100 U/mL
streptomycin. The flasks were incubated at 37°C with 5%
CO2 and the medium changed twice weekly. When the
fibroblasts were near confluence, the explanted tissue was removed, the
cells trypsinized, and plated into 25 cm2 culture flasks in
CM. Cytotoxicity experiments were performed with cells in passage 3 through 8, with a density of 1.7 to 2.4 × 105 cells
per 35 mm culture dish. For the experiments, fibroblasts were
trypsinized and plated into 35 mm culture dishes in CM with 10%
heat-inactivated fetal calf serum 24 to 36 hours before the start of
experimentation. After this period, all cultures were rinsed and 1 mL
of either CM or CM containing 200 mg LDL protein was added. After 24 to
48 hours incubation, the cell viability was assessed on an aliquot of
cell culture by Trypan blue exclusion test.
Statistical analysis.
All results are expressed as means ± standard deviation (SD).
Comparison between controls and thalassemia patients was performed by
the unpaired Student's t-test. Pearson's correlations were used to determine the relationships between covariates.
| |
RESULTS |
Hematologic data and values for major plasma lipid antioxidants of our
thalassemia intermedia patients and healthy controls are summarized in
Table 1. The mean concentration of serum
ferritin was eight times and that of bilirubin four times higher than
the control values, thus indicating the rather large hemolysis and increased iron absorption in the thalassemic patients (Table 1). Total
cholesterol, as well as HDL and LDL cholesterol, appeared markedly
lower than relevant controls, which is peculiar of the disease,27,28 whereas triglycerides were not significantly varied (Table 1). A marked decrease of lipid antioxidants such as
vitamin A and vitamin E was observed. However, because of the strong
fall in the cholesterol level, when normalized to plasma lipids
(cholesterol + triglycerides), the lipid-corrected vitamin E and
vitamin A did not appear significantly different with respect to
control (Table 1).
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Table 1.
Hematologic Data and Major Lipid Antioxidants in Plasma
From -Thalassemia Intermedia Patients and Controls
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Oxidation parameters in plasma and LDL and the major lipid
LDL-antioxidants of thalassemia patients are shown in
Table 2. Plasma lipid peroxides measured as
MDA were about twofold that of healthy controls. CD lipid
hydroperoxides in LDL from -thalassemia patients ranged from 4.63 to
49.34 nmol/mg LDL protein (mean amount, 22.60 ± 12.84) and were
significantly higher than the CD found in control LDL (6.25 ± 3.04 nmol/mg LDL protein, range, 4.03 to 10.5, Table 2).
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Table 2.
Plasma MDA and Oxidation Indices and Lipid Antioxidants
in LDL From -Thalassemia Patients and Control Subjects
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LDL oxidation involves the loss of lysine -amino groups from apo
B-100. Quantitative analysis of lysine residues in LDL samples showed a
mean loss of 17% (Table 2) in LDL in the thalassemia patients. Table 2
also shows the concentration of the major LDL-associated antioxidants.
The mean amount of vitamin E and -carotene in LDL from patients was
48% and 24% of controls, respectively.
The amount of CD hydroperoxides in LDL from thalassemia patients showed
a strong inverse correlation with both plasma vitamin E (r = 0.659; P < .0001) and vitamin E in LDL (r = 0.784; P < .0001) (Fig 1).
A negative trend was observed with -carotene in LDL (r = 0.443; P = .149, not shown). A positive correlation was
found between LDL-CD and plasma MDA (r = 0.621; P < .0001, Fig 2). Plasma ferritin positively
correlated with CD hydroperoxides in LDL (r = 0.583; P = .0002, Fig 3). No correlation existed between either LDL-CD or plasma MDA and the age of patients.
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| Fig 1.
Correlation between conjugated diene lipid hydroperoxides
in LDL (LDL-CD) and the amounts of vitamin E in plasma (A) and in LDL
(B) from -thalassemia intermedia patients. Each blood sample was
simultaneously processed for isolating and analyzing LDL and for the
analysis of plasma vitamin E (n = 35; A: r = .659; P < .0001; B: r = .784; P < .0001).
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| Fig 2.
Correlation between conjugated diene lipid hydroperoxides
in LDL (LDL-CD) and plasma malondialdehyde (MDA) from -thalassemia
intermedia patients. Each blood sample was simultaneously processed for
isolating and analyzing LDL and for the analysis of plasma MDA (n = 35; r = .621; P < .0001).
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| Fig 3.
Correlation between ferritin and LDL-CD in
-thalassemia intermedia patients. Each blood sample was
simultaneously processed for isolating and analyzing LDL and for the
analysis of ferritin (n = 35; r = .583; P = .0002).
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Content of lipid peroxidation products in LDL has been linked to the
cytotoxic potency of LDL.29,30 The cytotoxicity of LDL from
thalassemia patients was therefore assayed by incubating LDL with
cultured fibroblasts for 24 to 48 hours. When compared with the
fibroblasts incubated with the culture medium alone, the viability of
fibroblasts did not appear affected by incubation with 200 µg protein
of control LDL (Fig 4). No significant
toxicity was demonstrated by control LDL even at a protein amount of
300 µg (not shown). On the contrary, incubation with LDL from
thalassemia patients caused a decrease in cell viability, which
increased with the extent of the oxidative modification of LDL. Minimum oxidized LDL (min-ox LDL, average LDL-CD 10.41 ± 1.58 nmol/mg LDL
protein; range, 4.63 to 13.16) caused a decrease of cell viability of
about 14% after a 24-hour incubation and of 35% after 48 hours. Exposure of cultured cells to medium-oxidized LDL (med-ox LDL, average
LDL-CD 20 ± 3.41 nmol/mg LDL protein; range, 14.58 to 27.45)
determined a loss of viable cells of 37% and 60% after 24 and 48 hours, respectively. After incubation with maximally modified LDL
(max-ox LDL average LDL-CD 32.91 ± 4.7 nmol/mg LDL protein; range,
30.7 to 49.37), the survival of fibroblasts was about 10% after 24 hours and did not differ significantly after 48 hours (Fig 4).
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| Fig 4.
Cytotoxicity of LDL to cultured human fibroblasts.
Treatment of fibroblasts was as in Materials and Methods. Bars refer to
the percent of viable fibroblasts remaining after incubation with 200 µg protein of either control LDL, or LDL from -thalassemia
intermedia patients, for 24 hours (full bars) or 48 hours (shaded
bars). Each value is the mean ± SD of values obtained with
n LDL samples from different healthy controls or
patients, each examined in duplicate. Control LDL, n = 12; minimum
oxidized LDL (min-ox LDL), n = 11; medium oxidized LDL (med-ox LDL),
n = 14; maximum oxidized LDL (max-ox LDL), n = 10. With
respect to fibroblasts incubated for the relevant time * with control
LDL, P < .001; ** with min-ox LDL, P < .001; ***
with med-ox LDL, P < .001; Student's t-test.
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Atherosclerotic vascular lesions are frequent in -thalassemia
intermedia.1-4 Nine of our patients showed evidence of
atherogenesis-related vascular complications. A description of the
patients including plasma MDA and LDL-CD values are reported in
Table 3. It is noteworthy that the lipid
peroxidation products in LDL are 36% to 118% higher than the mean
level of the thalassemia patients as a group.
| |
DISCUSSION |
Oxidative stress is a consequence of the disease process in
-thalassemia.31-34 This is evident as an increase of
plasma lipid peroxidation products such as MDA and a marked decrease of
plasma lipid antioxidants such as vitamin E and vitamin A, as compared with healthy controls.14 The presence of MDA in plasma
suggests that circulating lipoprotein particles are enriched in
oxidatively modified components.35 In accordance with this
hypothesis, our quantitative evaluation of LDL oxidation shows high
amounts of CD lipid hydroperoxides in LDL from patients. At the same
time, oxidation of the apo B is indicated by the loss of specific lysil residues.
If LDL is exposed to prooxidative conditions, it becomes depleted of
its antioxidants, with -tocopherol being the first to be lost and
-carotene the last.36 We found a strong depletion of
these antioxidants in LDL from thalassemia patients, which was
inversely correlated with the level of conjugated diene lipid hydroperoxides in LDL. Hence, due to the ongoing oxidative stress in
-thalassemia, plasma antioxidant defenses are overwhelmed and LDL is
no longer adequately protected and undergoes oxidation. On the basis of
the conjugated dienes and lysil residues measured in LDL from
thalassemia intermedia patients and from healthy controls, the mean
amount of oxidized LDL in the patients may be calculated in the range
17% to 27%.
The characteristics of oxidized LDL are under extensive investigation,
as oxidation of lipoproteins and damage to vascular wall constituents
have been identified as early events in the pathogenesis of
atherosclerosis.29,30 A number of studies have focused on
the capacity of oxidized LDL to injure cultured vascular cells,
fibroblasts and macrophages, and rat endothelial cells in
vivo,17-19,37 pointing to its role in the atherogenetic
disease. In accordance, we found that LDL from thalassemia patients is cytotoxic to cultured human fibroblasts, with the level of cytotoxicity well correlated to the content of CD lipid hydroperoxides.
Risk factors for high levels of oxidized LDL are not well established
and may be important in identifying individuals who may benefit from
antioxidant supplementation. The suggestion that plasma MDA may be
taken into account as a biomarker of oxidative stress in exposed
populations has been recently put forward.38 Because plasma
MDA correlates positively with LDL-CD in thalassemia patients, an
interesting suggestion from our analysis is that this plasma lipid
peroxidation marker can be useful for predicting the potential
cytotoxicity and possibly the atherogenicity of thalassemic LDL. This
may be recommended in thalassemia patients, in that traditional lipid
and lipoprotein risk factors could be biased because of the altered
lipid pattern. According to our prediction, nine of our -thalassemia
intermedia patients, with clinical evidence of severe
atherogenesis-related vascular complications exhibit very high levels
of LDL-CD and have plasma MDA levels twofold to threefold higher than
control.
Intervention to impair oxidative modifications of LDL may be proven of
benefit in the attenuation of atherosclerotic processes. Vitamin E
administration to selected thalassemia intermedia patients has recently
started at our center.
Such features of thalassemia as hemolysis, iron loading, and the
increased iron absorption due to ineffective erythropoiesis could have
a role in the observed LDL oxidation. Although the correlation between
plasma ferritin and LDL-CD suggests an involvement of high iron levels,
it is difficult to decide whether this is the only factor or the most
prominent factor that promotes oxidized LDL production in thalassemia
patients.
Iron accumulation is involved in cardiac injury,39 but its
role in the oxidation of LDL and development of atherogenesis-related pathologies is doubtful.40 Serum iron and iron stores,
expressed as elevated ferritin levels, have been implicated in coronary artery disease.41-44 The interaction between iron, oxygen
free radicals and LDL, leading to oxidized LDL particles, progression of atherosclerosis, and finally to acute myocardial infarction has been
hypothesized to account for this evidence. However, recent epidemiologic studies showed that moderately elevated serum ferritin concentrations (200 to 500 µg/mL) are a strong risk factor for acute
myocardial infarction,45 a finding that was not associated with atherogenic LDL. In addition, premature atherosclerosis is not a
prominent feature in hemochromatosis,46 a common genetic disease causing severe iron accumulation in plasma and liver, although
congestive heart failure is characteristic of these patients. This
suggests that the elevation of iron alone may not bring about the free
radical reactions causing oxidative stress to LDL and would indicate
that additional factors are required. Unpaired hemoglobin chains and
red blood cell hemolysis products may have more
importance.12 This is supported by very recent in vitro studies47 in which oxidative interactions of hemoglobin
-chains with LDL apo B serve as triggers of oxidative modification
of LDL. It is also supported by consideration of the increased in vivo
oxidation of LDL in uremic patients undergoing
hemodialysis,48,49 a practice in which chronic hemolysis
has been demonstrated in vitro and in vivo.50,51 As further
evidence, although the oxidative status of LDL has not been
investigated in subjects affected by sickle cell anemia, another
hemolytic disorder in which reactive iron is produced, clinical
parameters establish these patients to be at risk for
atherogenesis.52,53 It may be worthwhile to investigate to
what extent the transfusion-dependent secondary iron overload would
affect the oxidative status of circulating LDL in thalassemia major
patients. Oxidized LDL could further contribute to the pathogenesis of
the heart disease related to the myocardial iron storage.
| |
ACKNOWLEDGMENT |
The cooperation of the staff of the "Servizio Talassemia,"
Ospedale V. Cervello di Palermo is gratefully acknowledged.
| |
FOOTNOTES |
Submitted November 11, 1997;
accepted July 13, 1998.
Supported by Assessorato Sanità Regione Sicilia and Grant No. CNR
95.04669.ST75.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to M.A. Livrea, PhD, Istituto di
Farmacologia e Farmacognosia, Via C. Forlanini, 1, 90134 Palermo,
Italy; e-mail mal96{at}mbox.unipa.it.
| |
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Abstract
Introduction
