Peroxidative stress and antioxidant enzymes in children with BThalassemia Major
Ahmed M Ezzat (1), MD, Ghada S Abdelmotaleb (1), MD, Ashraf M Shaheen (1) ,MD,
Yasser M Ismail (2), MD, Aliaa M Diab(1)( MSc)
Pediatrics (1) and Clinical Pathology Departments (2), Faculty of medicine, Benha University, Egypt,
Correspondence author: Aliaa M. Diab. email.
Abstract
Background: Beta-thalassaemias are a group of hereditary human diseases caused by more
than 200 mutations of the human β-globin gene. Regular blood transfusions and secondary
iron overload make thalassemic erythrocytes prone to peroxidative injury.
Aim: to evaluate the extent of lipid peroxidation and study the state of antioxidant enzyme in
thalassemic children, to assess oxidative status in β- thalassemia major patients.
Patients and Methods: this is a case control study conducted on forty patients previously
diagnosed as β-thalassemia major and forty healthy age and sex matched children as control
group .The following investigation were done for all children; complete blood picture,
reticulocytic count, serum ferritin level and hepatitis B and C markers. Measurement of
lipoperoxides ; malondialdehyde (MDA), analysis of antioxidant enzymes ; superoxide
dismutase (SOD), glutathione peroxidase (GPx) and Vitamin E levels.
Results: There were significant difference in oxidative status values between cases and
controls. Increased MDA level was ranging (2.3 – 4.6 nmol/ml) in thalessemic patients
comparing to control (p value <0,001). Significantly lowered activity of Super Oxide
Dismutase was 50-78 U/mL in thalessemic as compared to control(P value< 0.001). Also,
GPX level range (2.85-5.85 U/L) was significantly lower in thalessemic patients (p value
<0.001). Vitamin E was significantly lower among patients with β-Thalessemia(0.1 to 0.5
mg/dl) compared to controls (P value <0.001).
Conclusion: increased level of lipid peroxidatation product MDA was detected in βthalessemia major patients accompanied with significantly lower activity level of antioxidant
enzymes and Vitamin E.
Key words: antioxidant enzymes, b-thalassemia children, oxidative stress.
Introduction
Β-Thalassemia is a well known hereditary anemia caused by a decrease in the production of
β-globin chains, affects multiple organs and is associated with considerable morbidity and
mortality.(1)
The term ‘‘antioxidant’’ can be labeled for any substance whose availability, even in
minute concentration inhibits or delays the oxidation of a substrate. There are several species
or molecules, endogenous (internally synthesized) or exogenous (consumed), that plays a role
in antioxidant defense and may be considered as biomarkers of oxidative stress. (1). Oxidative
stress is caused by prolonged imbalance and antioxidant depletion as well as hyperproduction of ROS (2)
Different types of biological antioxidants include, for instance: Glutathione
(oxidized/reduced), SOD (superoxide dismutase) activity is an indirect method for registration
of the content of primary ROS (O2 radicals), Vitamin C & E, cystine, etc. (3)
Oxygen radicals are capable of reversibly or irreversibly damaging compounds of all
biochemical classes including nucleic acid, protein, free amino acid, lipids, lipoprotein,
carbohydrate, and connective tissue macro-molecules. These species may have impact on cell
activities as membrane function, metabolism, and gene expression (4).Under normal
physiological conditions; antioxidant defenses ensure that basal fluxes of ROS do not
negatively affect the body (5).
In addition Thalassemic erythrocytes may be more sensitive to peroxidative damage owing
to their low MCHC values ,which allows the free radicals to find their way to the cell
membrane since the amount of hemoglobin, which they would otherwise attack is not
adequate to act as sump for these reactive species (6).
Aim of our study to evaluate the level of lipid peroxidation product malondialdehyde (MDA)
as an indicator of oxidant stimuli and to assess specific antioxidants including Superoxide
dismutase (SOD), Glutathione Peroxidase(GPX) and vitamin E as biomarkers of antioxidants
capacity in patients with β-Thalessemia major.
Patients and methods: this was case-control study that conducted at Pediatric Hematology
clinic, Benha University hospitals from December 2015 to march 2016 after obtaining
informed written consents from patients parents. The study was approved by the ethical
committee of Benha University.
Patients: Forty patients with confirmed with β thalassemia major on the basis of severe
anemia and hemoglobin electrophoresis were included in this study. All patients aged 6 to 18
years with mean (9.5+3.1) were examined regularly once or twice monthly .They were
receiving packed RBC transfusion every 2-4weeks and iron chelating drugs. Further 40
apparently healthy children with matching age & sex were included as a control group.
Inclusion criteria: Patients with β-thalassemia major patients less than 18 years.
Exclusion criteria: Patients had Hepatitis B or C infection or HIV infection.
Methods: All patients were subjected to the following: full medical history by direct patient
interviewing including; age, sex, consanguinity and similar family condition. History of
concomitant medical conditions was taken e.g. viral hepatitis (HBV, HCV) and blood
transfusion history including; age of onset, duration and frequency of transfusion and history
of drug therapy e.g. chelation.
*Complete physical examination was performed for all patients by assessing
anthropometric measurements, vital signs, presence of pallor, jaundice, spleen and liver
status.
The following investigation were done for all children: Complete blood picture with blood
indices by Coulter Counter ,Reticulocytic count ,serum ferritin estimated by ELISA ,
Hepatitis B and HCV antbodies( IgG) by ELISA.
Also, 4ml of venous blood sample was collected under complete aseptic technique and
divided for 4parts for the following: 1-Plasma malondialdehyde level estimation
calorimetrically; 1 ml was collected without using an anticoagulant. Blood was allowed to
clot for 30 min. at 25°. Blood was centrifuged at 2,000 for 15 min. Serum was stored on ice
and frozen at -80°C (Thiobarbituric acid (TBA) reacts with malondialdehyde (MDA ) in
acidic medium at temperature of 95°C for 30 min to form thiobarbituric acid reactive product
the absorbance of the resultant pink product can be measured at 534 nm) .
2-Plasma vitamin E level was determined by high performance liquid chromatography
(HPLC). Another 1 ml without using an anticoagulant (Vitamin E is light and temperature
sensitive; therefore samples had been protected from light) then cooled and centrifuged
immediately. The serum samples were stabled at -20 ° for about one month.
3-Superoxide Dismutase(SOD) assay relies on the ability of the enzyme to inhibit the
phenazine methosulphate-mediated reduction of nitroblue tetrazolium dye. Use 1ml of
heparinized whole blood samples. Centrifuge 0.5 ml of whole blood for 10 minutes at 4000
rpm and then aspirate off the plasma. Then wash erythrocytes four times with 3 ml of 0.9 %
NaCl solution with centrifuging for 10 minutes at 4000 rpm after each wash. The washed
centrifuged erythrocytes made up to 2.0 ml with cold redistilled water, mixed and left to stand
at +4 °C for 15 minutes, then store at -70 .The lysate was diluted with distilled water , so that
the % inhibition falls between 30 % and 60 % .
4-Glutathione Peroxidase(GPX) assay is an indirect measure of the activity of c-GPx.
Oxidized glutathione (GSSG), produced upon reduction of an organic peroxide by c-GPx, is
recycled to its reduced state by the enzyme glutathione reductase (GR). Blood sample about 1
ml was collected using an anticoagulant such as heparin. The red cells collected by
centrifugation (e.g., 4000 rpm x 10 minutes at 4°C) and the plasma drawn off then the cells
washed once for 10 volumes of cold saline. Lyse the red cell pellets by adding 4 volumes of
cold deionized water to the estimated pellet volume. Remove the red cell stroma by
centrifuging (e.g., 4000 rpm x 10 minutes at 4°C), collect the resulting clarified supernate for
use in the assay and the sample was frozen at -70°C before use.
Statistical analysis
Data management and statistical analysis were performed using Statistical Package for Social
Sciences (SPSS) vs. 22.Numerical data were summarized using means and standard
deviations and ranges. Categorical data were summarized as numbers and percentages.
Comparisons between the 2 groups with respect to normally distributed numeric variables
were done using the t-test. For categorical variables, differences were analyzed with 2 (chi
square) tests and Fisher’s exact test when appropriate.
Pearson Correlation between variables was done, “r” (pearson correlation coefficient) ranges
from +1 to -1. A value of 0 indicates that there is no association between the two variables; a
value greater than 0 indicates a positive association; a value less than 0 indicates a negative
association. All p-values are two-sided. P-values < 0.05 were considered significant
Results
Forty patients 23 (57.5%) males and 17 (42.5%) females were included in our study. All were
children aged 6 to 18 years with established diagnosis of β-Thalessemia major in a steady
state disease. Further 40 apparently healthy children with matching age & sex were included
as a control group. Both cases and controls were comparable regarding gender, age, weight
and BMI. However, cases were significantly shorter compared to controls. Regarding
features of the disease, pallor was seen in 14 (35%), hepatomegaly in 9 (22.5%) cases and
splenomegaly in 25 (62.5%) cases.
Complete cell count (CBC); showed mean hemoglobin level (8.4 + 0.5 g/dl) below the
normal physiologic levels in β-thalessemia major patients .
In our study serum ferritin level was significantly higher (4654.9 + 2672.4 ng/ml) in patients
than control range (p<0.001) .Table (1)
Then mean liver enzymes AST, ALT were significantly higher in patients with thalessemia
compared with controls (p<0.001). There was significant positive correlation between serum
ferritin and liver enzymes, asparate transaminase (r=0.978, P value<0.001) and alanine
transaminase (r=0.98, P value <0.001).
In this study we observed an increase level of MDA in β-Thalessemia major (P < 0.001)
compared with controls in both male and female patients. MDA mean level was (3.5+0.7
nmol/ml) in thalassemic patients and (1.7+ 0.2 nmol/ml) in control. There was negative
correlation was detected between MDA and Hb level (r=0.35, P =0.001). MDA correlate
positively with serum ferririn (r=0.959, P <0.001). Fig (1)
There was also positive correlation between MDA and liver enzymes, suggesting the role of
the liver damage in the peroxidative damage. MDA positively correlated with liver enzymes;
AST (r=0.935, P <0.001) and ALT (r=0.946, P <0.001) with highly significant. Table (3)
We had significantly low activity of mean Super Oxide Dismutase in thalessemic(60.5 +8.6
U/mL) as compared to control (111.7+ 20.8 U/mL) (P < 0.001)
In our study showed significant reduction in mean Glutathione Peroxidase (GPX) in βThalessemia major (4.27+0.82 U/L) as compared to control (17.37+1.86 U/L) (P <0.001)
Also, Vitamin E mean level was significantly lower among patients with β-Thalessemia major
(0.3+ 0.1 mg/dl) compared with controls (1.3 + 0.2 mg/dl) (P <0.001).
There were significant correlations between vitamin E and MDA & Serum ferritin which
suggests a consumption of the vitamin as a radical scavenger. Vitamin E level correlate
negatively with serum ferritin level (r= - 0.833, P =0.001).There was also significant
correlation between vitamin E level and MDA. Vitamin E correlate negatively with MDA
(r = - 0.907, P = 0.001).
Table (1): Laboratory data of β-thalassemia major patients and the control group
Cases
HB (g/dl)
Ht (%)
Ferritin(ng/ml)
AST(U/L)
ALT(U/L)
Mean
8.4
25.1
4654.9
104.1
110
±SD
0.5
1.7
2672.4
37.8
36.4
Control
Mean
±SD
11.3
1
34
2.9
86.3
15.1
30.5
5.8
34.9
4.9
P value
<0.001*
<0.001*
<0.001*
<0.001*
<0.001*
AST (asparate transaminase) , ALT (alanine transaminase)
Table (2): Antioxidant enzymes and Malonidialdehyde (MDA) levels in patients with βthalassemia major and control
Cases
MDA (nmol/ml)
SOD (U/mL)
GPX(U/L)
Vit E (mg/dl)
SOD (superoxide dismutase),
Mean
3.5
60.5
4.27
0.3
SD
0.7
8.6
0.82
0.1
GPX( Glutathione peroxidase)
Control
Mean
SD
1.7
0.2
111.7
20.8
17.37
1.86
1.3
0.2
MDA (Malonidialdehyde)
P value
<0.001*
<0.001*
<0.001*
<0.001*
Table (3): Correlation between MDA and both serum ferritin and liver enzymes
Ferritin (ng/ml)
AST(U/L)
ALT(U/L)
r coefficient
0.959
0.935
0.946
P value
<0.001*
<0.001*
<0.001*
MDA (Malonidialdehyde) , AST(Asparate transaminase),
ALT(Alanine transaminase)
Figure (1): Correlation between MDA and serum Ferritin
Table (4) :Correlation between Vitamin E and MDA &serum Ferritin
MDA(nmol/ml)
Ferritin(ng/ml)
r coefficient
P value
-0.907
-0.833
<0.001*
<0.001*
Figure (2): Correlation between Vit E and serum Ferritin; it shows negative correlation
Discussion
β-thalassaemia major (βTM), is a type of chronic, inherited and microcytic anemia that is
characterized by impaired biosynthesis of the β-globin leading to accumulation of unpaired αglobin chain . Free α-globin is highly unstable and readily precipitates and release iron in
reactive form. In addition to this, repeated blood transfusions and increased gastrointestinal
iron absorption lead to iron overload in the body. Humans are unable to eliminate the iron,
and the excess iron is deposited as hemosiderin and ferritin in the liver, spleen, endocrine
organs and myocardium. On the other hand, super oxide is the main reactive oxygen species,
which react with nitric oxide radical and forms peroxynitrate, therapy causing oxidative stress
and cellular damage (7).
It appears that oxidative stress is a basic mechanism in β-thalassemia major
pathological alternations. It has already been established that oxidative stress is increased in
patients with iron overload .This is a result of the mounted concentration of highly reactive
Fe2+ions which catalyze the generation of ROS. The deposited iron is responsible for the
formation of reactive oxygen species (ROS) such as superoxide anion (O2–), hydroxyl radical
(OH), singlet oxygen and hydrogen peroxide (H2O2), which induces oxidative stress in
thalassemia major patients (8)
Our study was designed to evaluate the level of lipid peroxidation product
malondialdehyde (MDA) as an indicator of oxidant stimuli and to assess specific antioxidants
including Superoxide dismutase (SOD), Glutathione Peroxidase(GPX) and vitamin E as
biomarkers of antioxidants capacity in patients with β-Thalessemia major.
Forty patients 23 (57.5%) males and 17 (42.5%) females were included in our study. All
were children aged 6 to 18 years mean (9,5 + 3.1) with established diagnosis of βThalessemia major. Further forty apparently healthy children with 25 (62.5 %) males,
15(37.5%) females matching age & sex was served as a control group.
Both cases and controls were comparable regarding gender, age, weight and BMI
(p˃0.05). However, cases were significantly shorter when compared to controls. Regarding
features of the disease, pallor was seen in 14 (35%), hepatomegaly in 9 cases (22.5%),
splenomegaly in 25 cases (62.5%).
In our study serum ferritin mean level was significantly higher (4654.9 + 2672.4 ng/ml)
than control between that agrees with previous studies Naithani et al.,2006 9) Ghone et
al.,2008 10) and Patne et al.,2012 11) and Mahdi ,2014 12)
Then mean liver enzymes AST, ALT were significantly higher in patients with Thalessemia
compared with controls (p<0.001), with positive correlation between serum ferritin and liver
enzymes ; AST& ALT that agrees with previous studies done by Naithani et al.,2006 9)
In this study we observed an increase level of MDA in β-Thalessemia major (3.5+0.7
nmol/ml) compared with controls (3.5+0.7 nmol/ml). Reactive oxygen species can cause
damage to biological macromolecules and membrane lipids readily react and undergo
peroxidation(13). The peroxidative process yields lipid peroxides, lipids alcohol and
aldehydic products and MDA (14). This is also consistent with Naithani et al.,2006 9 results
where MDA was ranging from 2.3+0.96 mmol/L. Ghone et al.,(2008)10 found MDA
level(2.48+0.65 nmol/ml)with, Patne et al.,(2012) 11 MDA level(3.4+1.1 nmol/ml) and
Mahdi ,(2014) 12 MDA level(17.8+6.5 nmol/ml) .
There was negative correlation between MDA and Hb level (r=0.35,P =0.001). MDA
correlated positively with serum ferririn ( r=0.959 ,P <0.001) .There was also significant
correlation between MDA and liver enzyme(AST &ALT),suggesting the role of the liver
damage in the peroxidative damage this agree with Naithani et al.,(2006) 9
Superoxide Dismutase (SOD) is the essential antioxidant that decreases the formation of
ROS and oxidative stress thus protecting the cells from damage. Erythrocyte SOD protects
the erythrocyte from being damage during oxidative stress (11)
We have seen significantly lowered activity of Super Oxide Dismutase is (60.5 +8.6
U/mL) in thalassemic as compared to control(111.7+ 20.8 U/mL) . This agree with Patna et
al.,2012 11 SOD level(111+2,7 U/mL) ,Mahdi,(2014) 12 SOD level (77.5+22.2U/mL)
While our results disagree with authors Kampa et al.,(2002) 15 , Naithani et al.,2006 9 and
Ghone et al.,(2008) 10 who showed increase in SOD level in b-Thalassemia major compared
to control which explained by that frequent blood transfusion and increase in the proportion
of younger erythrocyte as a compensatory mechanism after increase oxidative stress(16)
Glutathion peroxidase (GPX) belongs to group of antioxidant selenoenzymes that
protects the cell damage by catalyzing the reduction of lipid hydroperoxides. This action
requires the presence of glutathione. Glutathione peroxidase levels in the body are in close
relation with the glutathione, which is the most important antioxidant present in the cytoplasm
of the cells17. Decreased level of GPX is due to inactivation by the increased superoxide anion
production leading to an increase in oxidative stress18.
In our study shows significant reduction in Glutathione Peroxidase (GPX) in βThalassemia major (4.27+ 0.82 U/L) as compared to control (17.37+1.86 U/L) (P <0.001
This result agree with Patna et al., 2012 11 GPX level (4053.95+161.9 U/ml) and Mahdi,
2014 12 GPX level (5.35+1.2 U/L) .
Vitamin E plays a key role in protecting cells against oxidative damage. The
antioxidant role of vitamin E is attributed to its ability in quenching highly reactive lipid
peroxide intermediate by donating hydrogen and this prevents extraction of hydrogen from
PUFA. This assists in restricting self perpetuated lipid peroxidation chain reaction (16).
In our study Vitamin E was significantly lower among patients with β-Thalassemia major
(0.3+ 0.1 mg/dl) compared with controls (1.3 + 0.2 mg/dl) with significant (P <0.001).This
observation is consistent with Ghone et al., 2008 10 as Vit E level 0.81+0.16mg/dl.
There was significant correlations were detected between vitamin E and MDA & serum
ferritin which suggests a consumption of the vitamin as a radical scavenger. Vitamin E level
correlate negatively with serum ferritin level (r= - 0.833, P value =0.001).
Conclusion
Based on the results of this study, increased level of lipid peroxidatation product MDA was
detected in β-Thalessemia major patients accompanied with significantly lower activity level
of antioxidant enzymes and Vitamin E. This is an indication that β-Thalessemia major
patients produced greater quantities of reactive oxygen species which are less likely to be
removed effectively with the endogenous mechanism. Thus combination of effective iron
chelating agents with natural or synthetic antioxidants can be very helpful in clinical practice
and the regulation of the antioxidant status of patients with β-thalassemia major.
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