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\begin{document}

\title{The association Between Serum Peroxiredoxin 2 and Iron Overload in
Pediatric Patients with Beta Thalassemia: Single center study}



\author[1]{Fady El-Gendy}%
\author[2]{Amira Shehata}%
\author[1]{Sally   El-Fiky}%
\author[1]{Mahmoud El-Hawy}%
\affil[1]{Menoufia University Faculty of Medicine}%
\affil[2]{Menoufia University}%


\vspace{-1em}



  
  \date{May 08, 2024}


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\selectlanguage{english}
\begin{abstract}
Background: Oxidative stress is fundamental in initiating
pathophysiological mechanisms leading to premature hemolysis resulting
in iron overload in patients with beta thalassemia. Peroxiredoxin 2
(Prdx2) is one of the crucial cytoprotective and antioxidant systems
that play a key role against oxidation. Our aim was to investigate serum
Prdx2 levels in children with beta-thalassemia and to explore its
possible relations with iron overload. Methods: The patients were
divided into two groups: beta thalassemia major (BTM) group (n=40) and
beta thalassemia intermedia (BTI) group (n=20). To compare serum Prdx2
and iron status parameters levels, a control group (n=20) was include in
the study. Serum Prdx2 levels were determined by enzyme linked
immunosorbent assay technique. Serum levels of iron and ferritin were
measured using automated chemistry analyzer and electrochemiluminescence
immunoassay respectively. Results: Serum Prdx2 concentrations in
thalassemia major patients were significantly lower than those in
thalassemia intermedia patients (P= 0.026); and Prdx2 concentrations in
thalassemia intermedia patients were significantly lower than those in
control group (P\textless{}0.001). In both thalassemia major and
intermedia groups, serum Prdx2 concentration was positively correlated
with serum iron (r = 0.558, P=0.002; r = 0.718, P=0.004, respectively)
and ferritin levels (r = 0.422, P=0.007; r = 0.550, P=0.012,
respectively). Conclusions: Our results demonstrate the positive
association between Prdx2 and iron overload in thalassaemia patients.
These findings may suggest unconventional therapeutic approach to
control consequences of iron overload through modification of Prx2
activity.%
\end{abstract}%



\sloppy


\textbf{Introduction}

Beta-thalassemia (\selectlanguage{greek}β-\selectlanguage{english}thalassemia) is a highly diverse group of inherited
chronic anemia in which beta-globin chain synthesis is defective.
Consequently, free unpaired alpha-globin chains are accumulated and
precipitated, leading to erythrocytes destruction and ineffective
erythropoiesis {[}1{]}.

Beta thalassemia major (BTM) patients demand frequent packed red blood
cells transfusion to maintain adequate hemoglobin levels and to reduce
hepatosplenomegaly due to extramedullary erythropoiesis. Both chronic
hemolysis and transfusional excess iron load cause exaggerated
generation of free radicals and oxidative damage to biological
macromolecules {[}2{]}.

Both transfusion dependent and transfusion independent thalassemia
patients are susceptible to iron overload {[}3{]}. This necessarily
leads to considerable damage to many vital organs, such as the heart and
liver {[}4{]}. There are no physiological mechanisms to eliminate excess
iron and to prevent its deposition into end organs, hence it is very
essential to remove it with a pharmacological tool and limit the serious
clinical consequences of its overload {[}5{]}.

Peroxiredoxins (Prdxs) are thiol-specific antioxidant proteins, which
act as reactive oxygen species scavengers. In mammalian, they include
six distinct isoforms (Prdx1--6) that are distributed over diverse
cellular compartments {[}6{]}. Prdxs 1, 2, 3, 5 and 6 are found in the
cytoplasm, Prdx 1 in the nucleus, Prdxs 3, 5 and 6 in mitochondria, and
Prdx 5 in mitochondria and peroxisomes {[}7-9{]}.

Peroxiredoxin 2 (Prdx2) is widely expressed in various tissues and is
essential regulator of cell proliferation, differentiation and apoptosis
by modifying the intracellular level of hydrogen peroxide
(H\textsubscript{2}O\textsubscript{2}) level {[}10{]}.

Erythrocytes are extremely exposed to oxidative stress due to plenty of
heme iron and oxygen, which can generate reactive oxygen species (ROS).
Under conditions of excessive oxidative stress, erythrocyte membrane
damage may occur because of the reduced activity of membrane-bound
enzymes, the suppression of transmembrane transport mechanisms, and the
leakage of cellular constituents into circulation. Thus, erythrocytes
have very efficient defense mechanisms against oxidative stress. Prdx2,
the third most abundant protein in erythrocytes, is known to play a
protective function against ROS--induced damage in these cells {[}11,
12{]}. In this study, we aimed to investigate serum peroxiredoxin 2
levels, as a marker of oxidative stress, in children with
beta-thalassemia and to explore its possible relations with iron
overload parameters.

\textbf{Methods}

Beta thalassemia patients included in this study were recruited from
Hematology Unit, Department of Pediatric Hematology, Menoufia
University. The patients were divided into two groups: \selectlanguage{greek}β \selectlanguage{english}thalassemia
major group (n=40, aged 2--18 years, 16 female and 24 male) and \selectlanguage{greek}β
\selectlanguage{english}thalassemia intermedia group (n=20, aged 3--16 years, 9 female and 11
male). All thalassemia major patients were on regular blood transfusion
and received iron chelation therapy, oral deferasirox, according to
their serum ferritin levels.

The control group (n=20, aged 2--18 years, 11 female and 9 male)
included healthy volunteers. Complete blood count (CBC), peripheral
blood smears and high-performance liquid chromatography (HPLC) were
performed to ensure exclusion of thalassemia traits or any hemolytic
anemia for the control.

This study was approved by the Ethics Committee of Menoufia University.
Informed consent was taken from the guardians of healthy controls and
thalassemia patients.

\textbf{Blood sampling}

Blood samples from patients were collected prior blood transfusion.
Venous blood samples were collected in EDTA (for CBC, peripheral smear)
and plain tubes (for iron profile and Peroxiredoxin 2) from patients and
controls. Blood samples collected were sent immediately to Hematology
Unit, Clinical Pathology Department. Blood samples for CBC and
peripheral smear were analyzed within one hour after collection. Samples
collected for iron profile and Peroxiredoxin 2 were left to clot, then
centrifuged at 3000 rpm for 15 min; separated sera for estimation were
kept frozen at --20 \selectlanguage{ngerman}°C until analysis.

\textbf{Methodology}

Automated complete blood counts (CBC) were analyzed by Sysmex XN-1000
hematology analyzer (Sysmex Corporation, Kobe, Japan). Liver
transaminases were measured using AU680 chemistry analyzer (Beckman
Coulter Inc., Brea, California, USA). Iron and TIBC were done by Cobas
Integra 400 plus analyzer (Roche Diagnostics, Mannheim, Germany) and
serum ferritin levels were measured using Cobas e411 immunoassay
analyzer (Roche Diagnostics). Serum peroxiredoxin 2 levels were
determined by enzyme linked immunosorbent assay (ELISA) kit (Elabscience
Biotechnology Inc., Houston, Texas, United States, Catalog No:
E-EL-H1137) in accordance with manufacturer's protocol.

\textbf{Statistical analysis}

Results were statistically analyzed by SPSS statistical package version
23 (Armnok, NY: IBM Corp.). Student's t-test and Mann Whitney's test
were used for comparison of quantitative variables between two groups.
One-way analysis of variance (ANOVA) and Kruskal Wallis tests were used
for comparison of quantitative variables between more than two groups.
Spearman correlation was used for correlation analysis. Chi-square test
(\selectlanguage{greek}\textbf{χ\textsuperscript{2}}\selectlanguage{english} ) was used to study association between
qualitative variables. Two-sided P value less than 0.05 was considered
statistically significant.

\textbf{Results}

The clinical and laboratory characteristics of the thalassemia major and
intermedia patients are summarized in (table 1). There was no
significant difference between both groups regarding TLC and platelets
count (P\textgreater{}0.05). There was significant difference between
both groups in terms of age at first diagnosis, interval between blood
transfusions, pretransfusion Hb and ALT (P= 0.002; P\textless{}0.001;
P=0.017; P=0.017 respectively).

\textbf{Demographic and Biochemical parameters of thalassemia patient
and control groups}

There was no significant difference between ages or gender of the
patients and controls. A statistically significant difference between
thalassemia patients and control group was observed with respect to iron
profile parameters and Prdx2 (P \textless{}0.001) (table 2).

Serum Prdx2 concentrations in thalassemia major patients (65.44 \selectlanguage{ngerman}± 8.17
ng/ml) were significantly lower than those in thalassemia intermedia
patients (70.58 ± 9.44) (P= 0.026); and Prdx2 concentrations in
thalassemia intermedia patients were significantly lower than those in
control group (77.64 ± 7.13 ng/ml) (P\textless{}0.001) (Table 2) (Figure
1).

\textbf{Correlation between Prdx2 and iron overload parameters:}

Correlation analyses revealed a significantly positive correlation
between serum Prdx2 concentration and iron level in both thalassemia
major and intermedia groups (r = 0.558, P=0.002; r = 0.718, P=0.004,
respectively) (table3). A positive correlation was observed between
Prdx2 concentration and transferrin saturation in thalassemia intermedia
group only (r = 0.564, P=0.001) (figure 2). A positive correlation
between Prdx2 concentration and ferritin concentration was detected in
both thalassemia major and intermedia groups (r = 0.422, P=0.007; r =
0.550, P=0.012, respectively) (figure 3).

\textbf{Discussion}

Beta-thalassemia is a typical model of stress erythropoiesis
characterized by intense oxidative stress {[}13{]}. The control of the
deleterious effects of oxidation during erythropoiesis is extremely
substantial to ensure cell differentiation and maturation {[}14, 15{]}.
Recently, the key role of peroxiredoxin-2 was reported in normal and
pathologic erythropoiesis. Prdx2 is a potent antioxidant agent that can
act as a molecular chaperone targeting free heme to control oxidative
insults {[}16{]}.

Prdx2 was reported as a key cyto\selectlanguage{english}-protector against iron overload induced
either by iron administration or due to chronic hemolysis in a mouse
model of \selectlanguage{greek}β-\selectlanguage{english}thalassemia {[}17{]}. As there have been no satisfactory
reports on the relation between human serum Prdx2 and iron status
parameters levels in \selectlanguage{greek}β-\selectlanguage{english}thalassemia; the present study aimed to
investigate serum Prdx2 levels, as a marker of oxidative stress, in
children with beta-thalassemia and to explore its possible relations
with iron status.

Previous studies reported the upregulated gene expression of Prdx2 in
\selectlanguage{greek}β-\selectlanguage{english}thalassemic erythroid cells compared to the expression in controls
both in mouse {[}18{]} and human cell culture models {[}19{]}.

In contrast with results from mouse and in vitro models, no significant
difference was demonstrated between mRNA levels of Prdx2 in
reticulocytes of BTM patients and control. As regard BTI, there was
reduced mRNA levels of Prdx2 in reticulocytes of patients in comparison
to control, but with no differences in Prdx2 of erythrocytes at the
protein level. In addition, the protein levels of Prdx2 were highly
increased in erythrocytes of BTM when compared to BTI patients {[}20{]}.

In this study, we revealed that serum Prdx2 concentrations in BTM and
BTI patients were significantly lower than those in controls. These low
Prdx2 levels indicated that thalassemia patients might require much more
antioxidant levels to overcome oxidative stress caused by iron overload.

In contrast to our results, Prdx2 levels were analyzed in plasma samples
with no difference was reported between thalassemia major patients and
control group {[}21{]}.

Additionally, we reported that serum Prdx2 concentrations in BTM were
significantly lower than those in BTI patients. Consistent with our
results, some studies have shown elevated state of oxidative stress in
BTM more than BTI, both in plasma and erythrocytes {[}22, 23{]}.

In this study, we analyzed the correlation between serum Prdx2 and iron
overload parameters. We detected significant positive correlation
between serum Prdx2 concentration; and iron and ferritin levels in both
thalassemia major and intermedia groups. A positive correlation was
found between Prdx2 concentration and transferrin saturation in
thalassemia intermedia group only. These findings could be explained by
the increased oxidative stress due to iron overload in thalassemia
patients.

Our study provides the first demonstration of the relation between serum
Prdx2 and iron overload parameters in human model with beta thalassemia.

Further studies will be required to clarify Prx2 function and regulation
in both normal and stress erythropoiesis. Also, estimation of Prx2
levels at different stage of erythroid maturation may be useful to
identify the definite role of Prx2 in beta thalassemia erythropoiesis.

\textbf{References:}

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stress in \selectlanguage{greek}β-\selectlanguage{english}thalassaemia and sickle cell disease. Redox Biol. 2015;
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2- Manafikhi H, Drummen G, Palmery M, Peluso I. Total Antioxidant
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3- Leecharoenkiat K, Lithanatudom P, Sornjai W, Smith DR. Iron
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4- Taher AT, Saliba AN. Iron overload in thalassemia: different organs
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5- Kontoghiorghes GJ. The aim of iron chelation therapy in thalassaemia.
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6- Rhee SG, Kil IS. Multiple Functions and Regulation of Mammalian
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8- Jiang H, Wu L, Mishra M, Chawsheen HA, Wei Q. Expression of
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12- Ishida YI, Aki M, Fujiwara S, Nagahama M, Ogasawara Y. Peroxidatic
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13- Oikonomidou PR, Rivella S. What can we learn from ineffective
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al. Glutathione peroxidase 4 and vitamin E control reticulocyte
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16- Matt\selectlanguage{ngerman}é A, Pantaleo A, Ferru E, Turrini F, Bertoldi M, Lupo F. et al.
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homeostasis and senescence. Free Radic Biol Med. 2014; 76:80-88.

17- Matte A, De Falco L, Federti E, Cozzi A, Iolascon A, Levi S. et al.
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18- Matte A, Low PS, Turrini F, Bertoldi M, Campanella ME, Spano D. et
al. Peroxiredoxin-2 expression is increased in beta-thalassemic mouse
red cells but is displaced from the membrane as a marker of oxidative
stress. Free Radic Biol Med. 2010;49(3):457-466.

19- De Franceschi L, Bertoldi M, De Falco L, Santos Franco S, Ronzoni L,
Turrini F et al. Oxidative stress modulates heme synthesis and induces
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erythropoiesis. Haematologica. 2011; 96(11): 1595--1604.

20- Romanello KS, Teixeira KKL, Silva JPMO, Nagamatsu ST, Bezerra MAC,
Domingos IF et al. Global analysis of erythroid cells redox status
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22- Cighetti G, Duca L, Bortone L, Sala S, Nava I, Fiorelli G, et al.
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23- Boudrahem-Addour N, Izem-Meziane M, Bouguerra K, Nadjem N, Zidani N,
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Figures legends:

Figure 1: Serum peroxiredoxin 2 levels in the three studied groups

Figure 2: Correlation between serum peroxiredoxin 2 and transferrin
saturation in thalassemia patients.

Figure 3: Correlation between serum peroxiredoxin 2 and ferritin in
thalassemia patients.

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