Annals of Hematology

https://doi.org/10.1007/s00277-020-03987-7

ORIGINAL ARTICLE

Oxidative stress assessment in sickle cell anemia patients treated

with hydroxyurea
Cristiane O. Renó 1 & Amanda Rodrigues Barbosa 2 & Sara Santos de Carvalho 2 & Melina B. Pinheiro 3 &
Danyelle Romana Rios 2 & Vanessa F. Cortes 1 & Leandro A. Barbosa 1 & Hérica L. Santos 1

Received: 26 August 2019 / Accepted: 1 March 2020

# Springer-Verlag GmbH Germany, part of Springer Nature 2020

Abstract
Hydroxyurea (HU) is used as a therapy in sickle cell anemia (SCA). Many studies have established that HU improves patient
quality of life by reducing symptoms. However, the effect of HU on erythrocytes is not well-described. We evaluated several
parameters related to oxidative stress and total lipid content of erythrocytes in patients with SCA. The patient cohort consisted of
7 SCA patients treated with HU, 17 untreated SCA patients, and 15 healthy subjects. Erythrocytes from patients with SCA
displayed increased oxidative stress relative to the control group, including higher thiobarbituric acid reactive substances
(TBARS), Fe3+ content, and osmotic fragility, and decreased total cholesterol. We observed that treatment of SCA patients with
HU increased Fe3+ content and activity of glutathione peroxidase, and decreased glutathione reductase activity, glutathione
levels, total cholesterol, and phospholipid content comaperaded to patients untreated with HU. Thus, HU alters biochemical
characteristics of erythrocytes; future studies will determine whether they are beneficial or not.

Keywords Sickle cell anemia . Hydroxyurea . Erythrocyte membrane . Oxidative stress

Introduction chains of hemoglobin tetramer. Thus, SCA erythrocytes have

Sickle cell anemia (SCA) originated in Africa, but is now
widely distributed on all continents, with a high prevalence
in the black population and their descendants [1]. SCAwas the
first monogenetic disorder characterized and is caused by a
single point mutation in the adult β-globin gene, resulting in
hemoglobin S (HbS) [2]. The single base A to T transversion
alters the GAG codon encoding the amino acid glutamate,
which is negatively charged, to a GTG codon, which encodes
the neutrally charged valine at position 6 in the β-globin
* Hérica L. Santos
hlima@ufsj.edu.br
1
Laboratório de Bioquímica Celular, Universidade Federal de São
João del-Rei, Campus Centro-Oeste Dona Lindu, Av Sebastião
Gonçalves Coelho 400, Divinópolis 35501–296, Brazil
2
Laboratório de Hematologia Clínica, Universidade Federal de São
João del-Rei, Campus Centro-Oeste Dona Lindu, Av Sebastião
Gonçalves Coelho 400, Divinópolis 35501–296, Brazil
3
Laboratório de Análises Clínicas, Universidade Federal de São João
del-Rei, Campus Centro-Oeste Dona Lindu, Av Sebastião Gonçalves
Coelho 400, Divinópolis 35501–296, Brazil
a neutrally charged hydrophobic site that catalyzes a HbS
polymerization process during low oxygen tension, resulting
in a proclivity towards fiber formation and observable sickling
of red blood cells [3–5]. The biochemical and physical prop-
erties of the sickle erythrocyte result in pathophysiology
relevant to SCA. The clinical presentation of the disease
is due to processes related to vaso-occlusion, which lead to
painful crises and other complications. These events occur
because sickled erythrocytes lead to increased cell adhe-
sion molecules in the vascular endothelium for both plate-
lets and erythrocytes. Sickle cells are also fragile and read-
ily undergo hemolysis, releasing hemoglobin and arginase
into the plasma. These two factors contribute to high oxi-
dative stress and low availability of nitric oxide (NO), re-
spectively [6, 7]. In SCA, erythrocytes are constantly ex-
posed to oxidative stress. In response, they release prod-
ucts from HbS degradation, such as Fe2+ and Fe3+, which,
in turn, cause high levels of O2−, H2O2, and OH−, that
attack the erythrocyte membrane causing lipid peroxida-
tion and generating malondialdehyde (MDA) [8, 9].
Hydroxyurea (HU) has many characteristics of an ideal
drug for treating SCA. The major therapeutic benefit of HU
is to increase the level of fetal hemoglobin (HbF), which

results in a decrease of the HbS level and the inhibition of
polymerization of the abnormal sickle hemoglobin.
However, the mechanisms by which HU increases HbF are
unclear [10–12]. Regardless of the mechanism of action, from
a patient perspective, HU treatment contributes to an en-
hanced quality of life by reducing painful symptoms [13].
HU has minor positive effects on RBC hydration and
deformability that lessen the rate of hemolysis. In addition,
treatment lowers the number of circulating white blood cells,
which likely leads to decreased endothelial inflammation and
vaso-occlusive crises [12]. HU contains an NO group in its
chemical structure that may be released through an unknown
metabolic process. NO has beneficial effects on vascular en-
dothelium leading to a vasodilatation [14]. Moreover, HU also
modulates the activity of the antioxidant system, decreasing
inflammatory markers and stabilizing the integrity of the
erythrocyte membrane [15]. Thus, there is a great interest in
further understanding the physiological effects following drug
administration. This study evaluates the antioxidant system
and lipid modulation of SCA patient erythrocytes with and
without HU treatment.
Material and methods
Ethics statement
This study was approved by the Hemominas Foundation
Research Ethics Committee (protocol number 685.333) and
by the Ethics in Research involving Human Subjects of the
Universidade Federal de São João del-Rei, Campus Centro-
Oeste Dona Lindu - CEPES/CCO (protocol number 610.493).
All of the procedures followed in this study were in accor-
dance with the ethical standards of the responsible committees
on human experimentation (institutional and national) and
with the Helsinki Declaration of 1975, as revised in 2008.
All patients were informed about the study and consented to
participate by signing an informed consent form.
Study population
The study was conducted in Divinópolis, Minas Gerais, south-
east Brazil. The subjects were included in three groups: pa-
tients with SCA (SS) treated with hydroxurea (HU-treated)
(group 1), patients not treated with hydroxyurea (untreated)
(group 2), and a control group (group 3). Groups 1 and 2 were
diagnosed with SCA and followed up on the Hemonúcleo
Regional de Divinópolis/Fundação Hemominas (FH). All
subjects met the inclusion criteria: age over 12 years and no
history of pain, hospitalization, or blood transfusion in the
3 months just prior to the study. A total of 10 patients using
HU were identified during this period, seven of which were
enrolled in the study (among the remaining three, one declined
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to participate, and two did not have an appointment during our
study period).
The control group (group 3) was composed of healthy vol-
unteers, students, and employees of the Universidade Federal
de São João del-Rei. None of them had sickle cell trait or
hematological abnormalities in hematocrit, hemoglobin level,
or red blood cell morphology. The volunteers in the control
group were matched for age and sex for patients with SCA
patients.
Criteria for classification of patients
Patients were classified according to the severity of the clinical
form of their disease: mild, moderate, or severe, as shown in
Table 1. These classification criteria were based on the litera-
ture [16, 17] and on information obtained through the analysis
of the patient medical records. In order to be included in a
particular category, patients needed to have two or more se-
lection criteria within the category. Patients with only one
characteristic were considered to belong to the less severe
category just below. The classification criteria are listed in
Table 1.
Biological samples
Blood (6 mL) was obtained from veins in EDTA-containing
Vacutainer tubes (Becton Dickinson) from patients with SCA,
treated or untreated with HU. Samples were centrifuged at
3000 rpm for 15 min to obtain the plasma.
Lipid peroxidation
Levels of thiobarbituric acid reactive substances (TBARS) in
plasma were determined to determine lipid peroxidation. We
utilized 100 μL of plasma, to which was added 1 mL of a
solution containing 15% trichloroacetic acid, 0.38% thiobar-
bituric acid, and 0.25 N hydrochloric acid. The mixture was
incubated at 100 °C for 30 min. After 10 min of centrifugation
at 3000 rpm, the optical density (OD) was measured at 535 nm
(OD535) using a spectrophotometer (Genesys S10 UV/VIS;
T h e r m o S c i e n t i f i c , Wa l t h a m , M A , U S A ) . T h e
malondialdehyde (MDA) content was determined using a
standard curve that was constructed in the concentration range
of 1 to 100 nM [18].
Determination of Fe3+ in plasma samples
Fe3+ quantification was performed as described by Adams
[19] with minor modifications. A total of 150 μL of plasma
was mixed with 1.5 mL of 1 M KSCN, and the volume was
adjusted to 3.0 mL with 0.9% NaCl. The samples were ho-
mogenized, and the optical density at 480 nm (OD480) was
read using a spectrophotometer. A standard curve was
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Table 1 Criteria for classification
of patients according to clinical Criteria
form of the disease
Hb average
HbF%
Painful crises•
Hospitalization•
Complications
CVA Cerebrovascular accident, CRF Chronic renal failure, ACS Acute chest syndrome
•Recorded in the last 1 year
constructed using a 1 M FeCl3 solution as a standard. The OD
was stable for 15 min while using the spectrophotometer.
Osmotic fragility tests
Osmotic fragility tests were performed on erythrocytes from
all subjects. A NaCl working solution was serially diluted to
make a series of 0.1 to 0.9% salt solutions in 0.1% increments.
A total of 25 μL of whole blood was added to each tube and
mixed. The tubes were incubated at 37 °C for 30 min, and then
centrifuged at 1500 rpm for 10 min. The OD of each super-
natant was read at 540 nm (OD540) using distilled water as a
blank in a spectrophotometer. Percent hemolysis was calculat-
ed at each concentration of NaCl setting 0.9%, NaCl as the
100% hemolysis standard. A graph of percent hemolysis ver-
sus salt concentration was plotted.
Determination of catalase activity
Catalase activity was determined in erythrocyte hemolysates
based on the method of Aebi [20] with some modifications.
Hemolysate supernatants (20 μL) were added to a cuvette
containing 2 mL of 50 mM phosphate buffer (pH 7.0). The
reactions were initiated by the addition of 3.4 μL of freshly
prepared 30% H2O2. The decomposition of H2O2 was follow-
ed spectrophotometrically at 240 nm (OD240). The activity
was estimated from the straight of the slope and expressed as
micromoles of H2O2 decomposed per minute.
Superoxide dismutase activity
This method was adapted in our laboratory using epinephrine
in alkaline medium, since this is converted into adrenochrome
producing O−2 which is the SOD substrate. Thus, SOD activ-
ity was defined by assessing its ability to inhibit the oxidation
of epinephrine. A 100% oxidation was defined in a tube con-
taining plasma in a medium consisting of glycine (50 mM, pH
10) and 25 μL of epinephrine (60 mM, pH 2). Other tubes
contained the same medium but with different plasma vol-
umes added (10, 20, and 30 μL). The oxidation reaction was
Severity of clinical signs
Mild Moderate Severe
Greater than 7 g/dL Greater than 7 g/dL Less than 7 g/dL
Greater than 10% 5% ≤ HbF ≥ 10% Less than 5%
Up to 1 Up to 2 Up to 3
_ Up to 3 Greater than 3
_ _ CVA,CRF and ACS
measured by reading the OD at 480 nm (OD480) in a spec-
trophotometer. The enzymatic activity was expressed in units
of enzyme required to inhibit 50% of the adrenochrome for-
mation speed [21].
Determination of the content of reduced glutathione
The GSH concentration was evaluated by adding 25 μL of
plasma to a reaction medium containing 0.1 M phosphate
buffer (pH 8.0) and 10 mM DNTB/EDTA. After incubating
for 15 min at room temperature, the solution was read in a
spectrophotometer at a wavelength of 412 nm (OD412). The
GSH concentration was measured by comparing with a stan-
dard curve to determine nanomole GSH/μL plasma [22].
Determination of glutathione peroxidase enzymatic
activity
Glutathione peroxidase (GPx) enzymatic activity is deter-
mined by following the oxidation of NADPH at a 340 nm
wavelength using hydrogen peroxide. Flohe et al. [23] and
Nakamura and Hosada [24] methods were adapted using a
reaction medium containing 50 mM phosphate buffer, 4 mM
EDTA, 1 mM GSH, 1.25 mM NaN3, 0.16 mM NADPH, and
50 μL of plasma. The total volume was adjusted to 1 mL with
water. The reaction was initiated by the addition 50 μL of
4 mM H 2 O 2 . Enzymatic activity was measured at OD
340 nm (OD340) within 2 min following substrate addition.
The units of enzyme activity were expressed as ρmol
NADPH.min−1.μL of plasma.
Determination of glutathione reductase activity
The method described by Racker et al. [25] was utilized. The
reaction medium contained phosphate buffer, pH 7.6 50 mM,
EDTA 0.4 mM, 0.1 mM NADPH, 50 μL plasma, and 1 mM
glutathione disulfide (GSSG). The volume was brought to
1 mL with distilled water, and the mixture was read in a spec-
trophotometer at a wavelength of 340 nm (OD34) after 2-min

incubation following substrate addition. The enzyme activity
was quantitated in ρmol NADPH.min−1.μL plasma.
Lipid quantitation in red blood cells
The total lipids of the erythrocytes were extracted with water/
isopropanol/chloroform mixture (5:11:14, final proportions)
as described by Rose and Oklander [26] and Vokurková
et al. [27]. In a capped cup tube, 5 mL of water, then 11 mL
of isopropanol were added to 1 mL of red blood cells and
incubated for 60 min on ice with occasional stirring.
Subsequently, 14 mL of chloroform was added, and the mix-
ture was incubated for an additional 60 min on ice. After phase
separation, the organic phase was completely dried in the ni-
trogen stream and resuspended in 1 mL. Total phospholipids
and total cholesterol were quantified as described by Chen
et al. [28] and Higgins [29], respectively.
Statistical analyses
The results were analyzed using the GraphPad Prism 5 pro-
gram. For the analysis of data normality, the Shapiro-Wilk test
was used (for any sample size). For data with normal distri-
bution, an analysis of variance (ANOVA) followed by
Tukey’s multiple comparison test were utilized.
Results
Participant profile and classification criteria
Twenty-four of the 29 SCA patients recruited in the the Minas
Gerais Hemonúcleo Regional de Divinópolis/FH were evalu-
ated in our study. The distribution was 7 HU-treated and 17
untreated patients. For the control group, 15 healthy subjects
were recruited; the average age of patients with SCA was 30
years and the majority were male (55%). Of the 7 HU-treated
SCA patients, one was classified as severe (14.3%), three were
classified as moderate (28.6%), and four were classified as
mild clinical (57.1%) forms. The untreated SCA group had
two patients that were classified as severe clinical form
(11.8%), two were classified as moderate (11.8%), and four-
teen were classified as mild (76.4%).
Oxidative stress evaluation
The evaluation of lipid peroxidation showed a significant in-
crease in HU-treated (87.1%) and untreated (63%) patients
compared with that of the control group. HU-treated patients
showed an increase of 14.2% MDA compared with that of the
untreated patients, although this increase is not significant
(p = 0.42) (Fig. 1a).
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The level of Fe3+ is an indirect indicator of ROS. When
assessing the concentration of Fe3+ in the plasma of HU-
treated and untreated patients, we observed that both groups
showed a significant increase in the content of Fe3+ compared
with that of the control group. HU-treated patients showed an
increase of 345% (p < 0.001), and untreated patients displayed
an increase of 209% (p < 0.001) (Fig. 1b). By comparing HU-
treated and untreated patients, it was observed that HU-treated
patients had a significant increase of 66.55% in Fe3+ content
(p = 0.05) compared with that of the untreated patients
(Fig. 1b).
When assessing the osmotic fragility of red blood cells
from HU-treated and untreated patients (Fig. 1c), we observed
that, in fact, SCA patients with or without HU treatment have
higher osmotic fragility compared with that of the control
group. This increase in osmotic fragility was significant in a
saline solution of 0.5%, where the groups of HU-treated and
untreated patients exhibited 32.65% (p < 0.05) and 25%
(p < 0.05), respectively, more hemolysis than that of the con-
trol group. No significant differences were observed between
the HU-treated and untreated SCA patients.
Antioxidant enzymes
Antioxidant enzyme activities showed significant changes be-
tween the control and the HU-treated and untreated patient
groups. When evaluating the enzymatic activity of SOD, we
observed that HU-treated and untreated patients showed a
significant increase when compared with that of the control
group, 44.60% (p < 0.001) and 42.14% (p < 0.001), respec-
tively. Again we did not observe a significant difference in
SOD activity between both groups of SCA patients
(Fig. 2a). Additionally, no significant changes were found
(p = 0.068) in the catalase enzyme activity among the groups
(Fig. 2b).
The plasma of both untreated and HU-treated patients ex-
hibited a significant increase in the GSH content of 89.42%
(p < 0.001) compared with that of the control group.
Moreover, an increase of 41.15% (p < 0.05) GSH content
was observed in the untreated patients relative to that of HU-
treated SCA patients. HU-treated patients, when compared
with the control group, showed an increase of GSH
(34.20%) although was not statistically significant (Fig. 2c).
GPx activity in HU-treated and untreated patients was sig-
nificantly increased in both groups compared with that of the
control group. The increase in the untreated SCA patients was
dramatic, with GPx activity increasing 554% (p < 0.001),
whereas HU-treated patients had an even greater increase of
871% (p < 0.001). Comparing GPx activity between SCA pa-
tient groups, an increase in activity of 48.3% (p < 0.001) was
observed in HU-treated patients, reflecting the possibility that
an antioxidant system was operative to combat ROS and de-
crease oxidative stress (Fig. 2d). Finally, glutathione reductase
Ann Hematol
Fig. 1 Oxidative stress indicator determination in groups: control (n =
15), patients with sickle cell anemia untreated (n = 17), and HU-treated
(n = 7). a Lipid peroxidation in plasma, *** p < 0.001. b Fe3 + content in
plasma, (***p < 0.001 and ▲ p < 0.05). c Erythrocyte osmotic fragility
activity was significantly increased 129% (p < 0.001) in HU-
treated patients and 168% (p < 0.001) in untreated patients.
Therefore, the GR activity of HU-treated patients was signif-
icantly decreased by 17.1% (p < 0.018) relative to untreated
patients (Fig. 2e)
Lipids from red blood cells
The concentration of total phospholipids in the red blood cells
in HU-treated patients was significantly lower when com-
pared with that of the other groups (control and untreated
SCA patients), 63.35% and 68.2%, respectively. The group
of untreated patients displayed a reduction of 8% relative to
the control group, but this was not significant (Fig. 3a).
Regarding total cholesterol in red blood cells, a significant
reduction of 56% (p < 0.001) was observed in the group of
untreated patients, whereas HU-treated patients showed a de-
crease of 140%, when compared with that of the control
group. The HU-treated patients also presented a significant
reduction of total cholesterol by 65% (p < 0.05) when com-
pared with that of the untreated patients (Fig. 3b).
Discussion
The objective of this study was to better understand the effect
of HU on erythrocytes of SCA patient, especially on the anti-
oxidant system and lipid modulation, which would be of great
interest in further understanding the physiological effects fol-
lowing drug administration. It was possible to observe in our
study that HU seems to cause changes in the erythrocyte mem-
brane (decreases total cholesterol and phospholipid content)
and in some enzymes of the antioxidant system (increases
activity of glutathione peroxidase, and decreases glutathione
reductase activity and glutathione levels). An important dif-
ference in our study from other studies on the effect of HU
(*p < 0.05). *Significant differences between the patient groups and the
control group. ▲Significant difference between the group of patients
treated and the group of untreated.
treatment of SCD is that we only used SCA patients. Our
choice to assess only SCA patients was based on the availabil-
ity of a homogenous patient set. Although they provide only a
small number of patients treated with HU, they all have the
same disease (HbSS). Patients were classified according to the
criteria shown in Table 1. Most patients were within the mild
to moderate stages of the disease, which was expected given
the frequent medical care that reduces the occurrence of
complications.
The Brazilian Ministry of Health [30] defined the criteria to
initiate public treatment with HU.We observed that 85.7% of
HU-treated patients fell into the moderate or mild clinical form
of the disease, which suggested the HU treatment was effica-
cious. To test this hypothesis, the following tests were per-
formed to evaluate the parameters of oxidative stress in red
blood cells of HU-treated and untreated SCA patients.
TBARS is a biomarker that indirectly measures the
lipid peroxidation process, which makes it a good indi-
cator of pro-oxidant stimuli. Previous studies reported
that SCA patients have higher levels of lipid peroxida-
tion [31, 32]. Our data corroborate this study, because
the plasma MDA content of patients with or without
HU treatment was significantly higher than that of the
control group (Fig. 1a).
Fe3+ is also an indirect ROS indicator, as some iron com-
plexes induce lipid peroxidation by the Fenton reaction [33].
Evaluation of the Fe3+ content present in the plasma of HU-
treated and untreated patients revealed that both groups showed
increased Fe3+ levels compared with that of the control group.
This increase confirmed existing data that patients with SCA
have a higher release of ROS compared with normal individuals,
suggesting that increased oxidative stress could be an important
mediator of the clinical manifestations of the disease [8, 9].
The high level of lipid peroxidation can directly affect the
erythrocyte membrane by compromising its integrity [34]; for
this reason, the sickled red blood cells survive up to 40 days
Ann Hematol
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ƒFig. 2 Antioxidant enzyme activity of erythrocyte in groups: control (n =
15), patients with sickle cell anemia untreated (n = 17), and HU-treated
(n = 7). a CAT activity. b SOD activity in plasma (***p < 0.001). c GSH
plasma content (***p, 0 < 0.001 and ▲ p < 0.05). d GPx activity plasma
content (***p < 0.001 and ▲ p < 0.001). e GR activity plasma content
(***p < 0.001). *Significant differences between the patient groups and
the control group. ▲Significant difference between the group of patients
treated and the group of untreated.
less than normal erythrocytes. In both groups of patients, an
osmotic fragility higher than that of the control group at a
concentration of 0.5% saline was observed, confirming the
increased fragility of the erythrocytes in SCA (Fig. 1c) [35,
36].
HU can increase the bioavailability of NO, converting it to
peroxynitrite (ONOO), which contributes to the release of
ROS. Other studies have shown that HU also directly in-
creases lipid peroxidation, osmotic fragility, and methemoglo-
bin production in normal erythrocytes in vitro [37, 38].
Comparing the significant increase of iron in HU-treated
patients versus untreated patients (Fig. 1b), the possibility ex-
ists that treatment with HU may be contributing to the in-
creased release of iron. Iyamu et al. [38] reported that in blood
treated in vitro with different concentrations of HU, there was
an increase in the oxidation rate of HbS, which in turn led to
the formation of methemoglobin and release Fe3+, contribut-
ing to increased oxidative stress and damage to cell mem-
branes [39]. In our study, no significant differences were
Fig. 3 Total lipid determination in erythrocyte in groups: control (n = 15),
patients with sickle cell anemia untreated (n = 17), and HU-treated (n =
7). a Total phospholipid content (*p, 0 < 0.05 and ▲ p < 0.05). b Total
found in lipid peroxidation between the two groups, although
a 14% increase in lipid peroxidation was observed in the treat-
ed patients (Fig. 1a).
When we compared GSH, GPx, and GR activity (Fig. 2c, d,
and e), among the HU-treated and untreated SCA patients, a
relative decrease of GSH and GR, and an increase of GPx activ-
ities was observed. Altogether, these data suggest that the increase
of GPx activity of HU-treated patients may modulate a positive
response to the treatment and that HU is able to decrease the total
oxidative stress. Cho and collaborators [40] reported that patients
treated with HU not only had a higher GPx activity but also had
an increased expression of this enzyme. In fact, GPx activity was
inversely proportional to hemolysis of erythrocytes, whereas the
preferred enzyme substrate would be the lipid hydroperoxides.
The increased GPx activity protects the erythrocyte membrane
from lipid peroxidation, since GPx activity is increased by
48.3% in HU-treated patients. No difference in erythrocyte stabil-
ity was observed between HU-treated and untreated patients.
Therefore, we suggest that the erythrocytes of patients are less
susceptible to hemolysis after treatment with HU (Figs. 1c and
2d, respectively).
The lipid alterations found in the study were a reduction in
the total cholesterol content in the treated and untreated patient
groups when compared with that of the control group (Fig. 3b)
and a reduction of total phospholipids and cholesterol in the
group treated with HU when compared with that of the other
groups (Fig. 3a). The reduction in cholesterol in the HU-
cholesterol content (***p < 0.001). *Significant differences between the
patient groups and the control group. ▲Significant difference between
the group of patients treated and the group of untreated.

treated group compared with that of the untreated group sug-
gests that the number of ROS generated in treated patients is
greater than that in untreated patients, since there is a signifi-
cant increase in the content of Fe3+ compared with that of the
group of untreated patients. This increase may lead to a higher
production of reactive oxygen species mainly from the Fenton
reaction that generates the hydroxyl radical (OH.) [33]. Liu
and Shan [41], when performing experiments with normal
erythrocytes treated with ROS in vitro, including OH pro-
duced by the Fenton reaction, observed that cholesterol is
preferentially affected by these, generating three detectable
by-products when attacked by OH, while membrane polyun-
saturated fatty acids remain intact. The reduction in total phos-
pholipids found in the HU-treated group compared with that
of the untreated patients may be because in the untreated
group, phospholipids have been injured in smaller chains but
not completely removed from the cell membrane, keeping a
portion attached to the phosphate group. Another hypothesis
is that in HU-treated patients, the number of ROS is very high
to the point of damaging the membranes promoting the loss of
phospholipids. Membrane fluidity depends on the phospho-
lipid content and its degree of unsaturation. Changes in the
amount and position generate changes in the erythrocyte, in-
cluding flexibility, modulation of enzyme activity, and cellular
signaling [42, 43]. In SCA, the erythrocyte membranes have
an abnormal composition, especially during sickling process-
es, with phosphatidylcholine in the inner membrane leaflet
and phosphatidylserine and phosphatidylethanolamine in the
outer leaflet. In addition, there is a reduction in polyunsaturat-
ed phospholipids, an increase in monounsaturated phospho-
lipids, and a decreased membrane flexibility, all of which con-
tribute to occlusive vascular crises and decreased erythrocyte
survival [44].
The lipid analyses performed in our study determined the
quantity and not the identification of those. However, HU
clearly provides alterations of the lipid profile of the erythro-
cyte membrane which may be due to the increase of Fe3+ in
HU-treated patients. Compared with untreated SCA patients,
such changes may impair the erythrocyte membrane and allow
more flexibility of these cells, in addition to altering mem-
brane enzyme activities and decreasing the erythrocyte life.
While it is possible to identify a change in the total phospho-
lipid content caused by treatment with HU, further studies are
needed to conclude whether this change leads to malfunctions
or benefits for the viability of the erythrocyte.
Acknowledgments We thank the Hemominas Foundation for providing
samples and archives. We are thankful to Dr. Kenneth Peterson (KUMC)
for the helpful evaluation of the manuscript.
Author contributions Conceived and designed the experiments: MBP,
DRR, VFC, LAB, and HLS. Performed the experiments: COR.
Analyzed the data: COR, MBP, DRR, VFC, HLS, and LAB.
Ann Hematol
Contributed reagents/materials/analysis tools: ARB, SSC, MBP, HLS,
and LAB. Wrote the paper: COR, VFC, HLS, and LAB.
Funding information This work was supported by FAPEMIG (Fundação
de Amparo a Pesquisa do Estado de Minas Gerais), CAPES
(Coordenação de Aperfeiçoamento de Pessoal de Nível Superior), and
CNPq (Conselho Nacional de Desenvolvimento Científico e
Tecnológico).
Compliance with ethical standards
Conflict of Interest The authors declare that they have no conflict of
interest.
Informed consent Informed consent was obtained from all patients for
being included in the study.
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