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Mansy et al
2849
Tropical Journal of Pharmaceutical Research December 2017; 16 (12): 2849-2856
ISSN: 1596-5996 (print); 1596-9827 (electronic)
© Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria.
Available online at http://www.tjpr.org
http://dx.doi.org/10.4314/tjpr.v16i12.8
Original Research Article
Effects of chronic consumption of energy drinks on liver
and kidney of experimental rats
Wael Mansy1,2*, Deema M Alogaiel3, Mona Hanafi4, Enas Zakaria5
1Clinical Pharmacy Department, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia, 2Pharmacology
Department, Faculty of Medicine, Cairo University, Cairo, Egypt, 3Health Sciences Department, College of Health and
Rehabilitation, Princess Nourah Bint Abdulrahman University, 4Department of Community Health Sciences. College of Applied
Medical Sciences, 5Pharmaceutics Department, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
*For correspondence: Email: whsayed@hotmail.com; Tel: +966567253275
Sent for review: 26 June 2017 Revised accepted: 19 November 2017
Abstract
Purpose: To investigate the effects of chronic intake of a brand of energy drink (ED) on the liver and
kidney of rats.
Methods: Sixty male adult Sprague Dawley albino rats were randomly assigned to four groups (15 rats
per group). Three groups received ED at different doses (0.4, 1.1 and 2.2 ml/100 g body weight/day) for
12 weeks. The fourth group (control) received distilled water. All treatments were administered by oral
gavage. Blood samples were withdrawn at the start of the experiment, and at the 6th and 12th weeks for
assay of hepatic and renal biochemical parameters. Histopathological studies were done at the end of
the exposure period.
Results: Exposure to ED doses of 1.1 and 2.2 ml/100g body weight/day for 12 weeks induced highly
significant increases in serum aspartate transaminase (AST), alanine transaminase (ALT), alkaline
phosphatase (ALP), blood urea nitrogen (BUN), creatinine and uric acid, when compared with the
control group (p < 0.001). On the other hand, the activities of the antioxidant enzymes, viz, superoxide
dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) significantly decreased (p < 0.001)
by exposure to these two ED doses, relative to controls. Pronounced histopathological changes were
observed in hepatic and renal tissues of the ED-treated rats.
Conclusion: Oral exposure of rats to ED for 12 weeks produced noticeable hepatic and renal damage,
probably due to increased free radical production and oxidative stress.
Keywords: Energy drink, Reactive oxygen species, Liver function, Kidney function, Histopathological
changes
This is an Open Access article that uses a funding model which does not charge readers or their institutions
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(http://www.budapestopenaccessinitiative.org/read), which permit unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited.
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INTRODUCTION
First appearance of energy drinks (EDs) was in
Europe and Asia in the 1960 as a result of
customer requirements for dietary supplements
that give energy [1]. Many Saudi studies found
that more than half of the consumers were
young (13 - 35 years old), more than half
consumed it for over a year, and over 40 %
used to drink more than 3 cans per week [2].
Centers for Disease Control and Prevention
reported that high school students consume
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© 2017 The authors. This work is licensed under the Creative Commons Attribution 4.0 International License
Mansy et al
2850
EDs almost at the same rate as they consume
soda [1]. Indeed, the rate of ED consumption
might be higher than estimated levels in this
self-reporting survey, since such surveys
usually have high probability of under-
estimation.
It has been revealed that EDs contain mainly
taurine, glucuronolactone, caffeine, ginseng
and guarana [3]. These substances, most of
which act as stimulants, are not included in the
list of materials under regulation by the Food
and Drug Administration (FDA) of the United
States of America. The levels of these
stimulants vary amongst different brands of
EDs, and in most cases, are higher than values
allowable [4]. A study has shown that the
caffeine levels in EDs are between 50 and 505
mg/ can, which are much higher than the
caffeine content of one can of Coke (34 mg) [5].
Reports of significant, adverse health problems
due to ingestion of EDs have increased in
recent years. Indeed, in 2013, ED-associated
emergency interventions by the US Substance
Abuse and Mental Health Services
Administration doubled from 10,068 in 2017 to
over 20,000 in 2011 [6]. A major constraint in
understanding the link between EDs and the
adverse effects of their consumption is that very
little is known about the toxicity of the various
compounds present in them. However, based
on reported cases of ED-associated health
problems, and the well-established
physiological effects of the active ingredients of
EDs, it is very likely that the observed adverse
effects of EDs are linked to their compositions
[3]. Due to the aforementioned reasons we
purposed this study to explore the toxic effects
of prolonged intake of ED on hepatic and renal
tissues of rats.
EXPERIMENTAL
Energy drink
The brand of ED used in the present study was
“Red bull”, product of Rauch Trading AG,
Switzerland (manufactured for Red Bull GmbH,
Austria). It was purchased from a local store in
Riyadh, Saudi Arabia.
Animal feeding
Sixty (60) adult, male Sprague Dawley rats
(mean weight = 115.5g) were kept in the Animal
House of College of Pharmacy, King Saud
University. The rats were acclimatized under a
12h/12h light/dark photoperiod and under normal,
healthy laboratory conditions at a mean
temperature of 25 ± 2 °C. The experiments were
carried out in line with the recommendations of
International Laboratory Animal Use and Care
[7]. The study protocols and ethics were
approved by the Animal Research and Ethical
Committee of King Saud University (approval No.
CAMS24/3334, June 2013).
Experimental design
Four (4) groups of rats were used (15
rats/group). There was no bias in the allocation
of rats to any group. Rats in the control group
(G1) were maintained on the basal diet and
distilled water throughout the experiment. Three
levels of ED exposure were used: mild,
moderate and high ED. Rats in the mild ED
intake group (G2) was given low dose of ED
(0.4 ml / 100 g body weight / day) to simulate
low human consumption pattern (280 ml/ED
can), while the moderate ED group (G3)
received 1.1 ml /100g body weight / day, to
reflect moderate human consumption level of
770ml (about 3 cans of ED). The high ED intake
group (G4) was given 2.2 ml/100 g body
weight/day to mimic estimated high human
consumption level of 1540 ml (about six cans).
All treatments were given by gavage, and
lasted for 12 weeks.
Sample collection
Prior to the commencement of ED exposure,
blood samples were taken from the retrobulbar
venous plexus for the determination of various
basal biochemical parameters. Blood samples
were also drawn through the same route at the
6th week (mid-way) and at the end (12th week),
for similar assays. The samples were allowed to
coagulate and the sera were stored at -20 °C
prior to assay of AST, ALT, BUN and ALP.
Moreover, plasma samples from blood collected
in anticoagulant bottles were frozen at – 20 °C
and used for assay of CAT activity. The pellet
(erythrocytes) was washed thrice in 3 mL of
physiological saline, and centrifuged for 10 min
at 3000 x g. The erythrocytes were thereafter
hemolyzed with 1.5 volume of distilled water,
and the hemolysate was clarified by
centrifugation for 15 min at 10,000 x g and 4 oC.
The resultant supernatant was used for the
assay of the activities of the antioxidant
enzymes GPx and SOD.
Histological examination
Blood samples were taken at the expiration of
week 12, and all rats were sacrificed by
decapitation. Liver and kidney samples were
immediately excised and processed for light
Mansy et al
2851
microscopy and histological investigation using
standard methods. Specimens were fixed in 10
% neutral formalin and stained with hematoxylin
and eosin.
Biochemical analysis
The activities of ALT, ALP and AST, and the
levels of creatinine, BUN and uric acid were
measured colorimetrically using Randox UV
kinetic method kits (Randox, USA) in line with
the manufacturer’s protocol [8]. Plasma
catalase (CAT) and erythrocyte SOD and GPx
activities were also assayed colorimetrically
using Randox assay kits (Randox, USA)
according to the procedures specified by the kit
manufacturer [9,10].
Statistical analysis
Data are presented as mean ± SE. One-way
analysis of variance (ANOVA) was used for
assessing differences among groups. This was
followed by Bonferroni post-hoc paired
comparison using Windows SPSS version 20.0
(SPSS Inc., Chicago IL, USA). P < 0.05 was
taken as indicating statistically significant
differences.
RESULTS
Liver function
Rats treated with either moderate or high doses
of ED had significant increases in serum AST,
ALT and ALP levels at weeks 6 and 12 of the
experimental period, when compared with their
baseline levels and corresponding levels in the
control group. These results are shown in
Tables 1, 2 and 3 below.
Kidney function
The effects of the three doses of ED on kidney
function of rats are presented in Tables 4, 5 and
6. Rats exposed to high doses of ED had
significant increases in serum creatinine, BUN
and uric acid levels at week 12 when compared
with the baseline levels of these parameters, and
the corresponding values in the control group.
Activity of antioxidant enzymes
The effect of the three doses of ED on levels of
some antioxidant enzymes in rats are
presented in Tables 7, 8 and 9. Exposure to
high dose of ED led to significant decreases in
SOD, GPx and CAT activities at 12 weeks of
the experimental period, relative to their
baseline levels and the corresponding levels in
the control group.
Histological features
The general architecture of the liver and the
kidneys in G4 were distorted with congestion of
central and portal veins and inflammation of
portal areas as shown in Figure 1 I. Proliferation
of bile ducts and starting fibrosis appears in
Figure 1 II.
Table 1: Serum AST (U/L) levels of rats treated daily by different concentrations of energy drinks for 12 weeks
Group
Duration of consumption
P-value
Initial
Half-way (6 weeks) End (12 weeks)
Group 1:(control)
66.20±4.551
68.60±4.70
a1
67.77±3.81
a1
0.83
Group 2:(0.4 ml/100g/day) 78.21±4.76
ab12
91.21±4.76
b2
0.001
Group 3:(1.1 ml/100g/day) 93.43±6.91
b2
122.69±6.86
c3
0.001
Group 4:(2.2 ml/100g/day) 128.57±6.83
c2
190.62±3.61
d3
0.001
P
-value 1.00
0.001
0.001
Data are expressed as mean ± SE (n = 15); mean values with different alphabet superscripts within a column
differ significantly (p<0.05); mean values not sharing a superscript digit in a row indicate significant difference at p
< 0.05
Table 2: Serum ALT (U/L) levels of rats treated daily by different concentrations of energy drinks for 12 weeks
Group
Duration of consumption
P-value
Initial
Half-way (6 weeks) End (12 weeks)
Group 1:(control)
45.27±2.471
54.27±4.62
a2
58.92±3.99
a2
0.04
Group 2:(0.4 ml/100g/day) 59.09±2.66
a2
66.27±3.45
a2
0.001
Group 3:(1.1 ml/100g/day) 61.94±4.23
a2
69.13±4.39
a2
0.001
Group 4:(2.2 ml/100g/day) 90.79±6.73
b2
107.80±7.90
b2
0.001
P
-value 1.00
0.001
0.001
Data are expressed as mean ± SE (n = 15); mean values with different alphabet superscripts within a column
differ significantly (p<0.05); mean values not sharing a superscript digit in a row indicate significant difference at p
< 0.05
Mansy et al
2852
Table 3: Serum ALP (U/L) levels of rats treated daily by different concentrations of energy drinks for 12 weeks
Group
Duration of consumption
P-value
Initial
Half-way (6 weeks) End (12 weeks)
Group 1:(control)
137.27±18.201
142.40±11.37
a1
133.69±8.71
a1
0.91
Group 2:(0.4 ml/100g/day) 158.64±9.16
ab1
182.64±9.16
b1
0.06
Group 3:(1.1 ml/100g/day) 200.29±11.62
b2
237.00±12.41
c2
0.001
Group 4:(2.2 ml/100g/day) 300.86±15.70
c2
421.69±18.25
d3
0.001
P
-value 1.00
0.001
0.001
Data are expressed as mean ± SE (n = 15); mean values with different alphabet superscripts within a column
differ significantly (p<0.05); mean values not sharing a superscript digit in a row indicate significant difference at p
< 0.05
Table 4: Serum creatinine (mg/dL) levels of rats treated daily by different concentrations of energy drinks for 12
weeks
Group
Duration of consumption
P-value
Initial
Half-way (6 weeks) End (12 weeks)
Group 1:(control)
0.33±0.021
0.43±0.03
a12
0.45±0.03
a2
0.02
Group 2:(0.4 ml/100g/day) 0.45±0.02
a2
0.53±0.02
ab2
0.001
Group 3:(1.1 ml/100g/day) 0.47±0.02
ab2
0.58±0.05
b2
0.001
Group 4:(2.2 ml/100g/day) 0.57±0.04
b2
0.73±0.03
c3
0.001
P
-value 1.00
0.001
0.001
Data are expressed as mean ± SE (n = 15); mean values with different alphabet superscripts within a column
differ significantly (p<0.05); mean values not sharing a superscript digit in a row indicate significant difference at p
< 0.05
Table 5: Blood urea nitrogen (mg/dL) levels of rats treated daily by different concentrations of energy drinks for
12 weeks
Group
Duration of consumption
P-value
Initial
Half-way (6 weeks) End (12 weeks)
Group 1:(control)
7.57±0.551
10.23±0.33
a2
8.68±0.44
a12
0.84
Group 2:(0.4 ml/100g/day) 10.60±0.48
a2
11.54±0.48
b2
0.001
Group 3:(1.1 ml/100g/day) 18.70±0.60
b2
19.79±0.64
c2
0.001
Group 4:(2.2 ml/100g/day) 17.41±0.59
b2
23.48±0.63
d3
0.001
P
-value 1.00
0.001
0.001
Data are expressed as mean ± SE (n = 15); mean values with different alphabet superscripts within a column
differ significantly (p<0.05); mean values not sharing a superscript digit in a row indicate significant difference at p
< 0.05
Table 6: Serum uric acid (mg/dl) levels of rats treated daily by different concentrations of energy drinks for 12
weeks
Group
Duration of consumption
P-value
Initial
Half-way (6 weeks) End (12 weeks)
Group 1:(control)
2.37±0.101
2.41±0.21
a1
2.31±0.19
a1
0.84
Group 2:(0.4 ml/100g/day) 2.57±0.10
a2
2.71±0.11
b3
0.001
Group 3:(1.1 ml/100g/day) 3.07±0.12
b2
3.28±0.13
c2
0.001
Group 4:(2.2 ml/100g/day) 3.46±0.15
b2
3.89±0.16
d3
0.001
P
-value 1.00
0.001
0.001
Data are expressed as mean ± SE (n = 15); mean values with different alphabet superscripts within a column
differ significantly (p<0.05); mean values not sharing a superscript digit in a row indicate significant difference at p
< 0.05
Mansy et al
2853
Table 7: Superoxide dismutase (SODs, units/mL) level in erythrocytes of male rats orally and daily
administrated different concentration of energy drinks for 12 weeks
Group
Duration of consumption
P-value
Initial
Half-way (6 weeks) End (12 weeks)
Group 1:(control)
0.20±0.021
0.22±0.02
a1
0.23±0.02
a1
0.75
Group 2:(0.4 ml/100g/day) 0.21±0.02
a1
0.21±0.02
a1
0.99
Group 3:(1.1 ml/100g/day) 0.20±0.01
a1
0.19±0.01
a1
0.77
Group 4:(2.2 ml/100g/day) 0.17±0.01
a1
0.09±0.01
b2
0.001
P
-value 1.00
0.26
0.001
Data are expressed as mean ± SE (n = 15); mean values with different alphabet superscripts within a column
differ significantly (p<0.05); mean values not sharing a superscript digit in a row indicate significant difference at p
< 0.05
Table 8: Glutathione Peroxidase (nmol/min/mL) level in erythrocyte of rats treated daily by different
concentrations of energy drinks for 12 weeks
Group
Duration of consumption
P-value
Initial
Half-way (6 weeks) End (12 weeks)
Group 1:(control) 80.30±1.451
78.91±2.08
a1
77.30±1.45
a1
0.47
Group 2:(0.4 ml/100g/day) 76.07±1.98
a12
73.92±0.85
a2
0.02
Group 3:(1.1 ml/100g/day) 56.31±3.19
b2
53.40±3.19
b2
0.001
Group 4:(2.2 ml/100g/day) 45.86±2.49
c2
42.69±2.41
c2
0.001
P
-value 1.00
0.001 0.001
Data are expressed as mean ± SE (n = 15); mean values with different alphabet superscripts within a column
differ significantly (p<0.05); mean values not sharing a superscript digit in a row indicate significant difference at p
< 0.05
Table 9: Plasma catalase (nmol/min/mL) level of rats treated daily by different concentrations of energy drinks for
12 weeks
Group
Duration of consumption
P-value
Initial
Half-way (6 weeks) End (12 weeks)
Group 1:(control) 14.78±1.181
15.69±1.40
a1
14.82±1.37
a1
0.86
Group 2:(0.4 ml/100g/day) 14.46±1.31
a1
13.20±0.99
a1
0.61
Group 3:(1.1 ml/100g/day) 13.96±1.28
a1
12.97±1.29
a1
0.60
Group 4:(2.2 ml/100g/day) 12.74±0.83
a12
10.17±0.73
a2
0.01
P
-value 1.00
0.45 0.07
Data are expressed as mean ± SE (n = 15); mean values with different alphabet superscripts within a column
differ significantly (p < 0.05); mean values not sharing a superscript digit in a row indicate significant difference
at p < 0.05
Marked dilatation and congestion of veins and
severe inflammation of the interstitial as shown
in Figure 1 III. Remnants of destroyed tubules
were seen within areas of inflammation with
signs of degeneration, necrosis, loss of cellular
details and cell boundaries as shown in Figure 1
IV.
DISCUSSION
This study has demonstrated that oral
administration of ED to rats for 12 weeks
resulted in varying degrees of liver and kidney
damage. This was evident in the ED-induced
significant elevations in serum AST, ALT and
ALP, creatinine, BUN and uric acid levels.
Increases in the blood levels of hepatic enzymes
serve as reliable indicators of liver damage by
toxic agents. Similar increases have been
reported in serum AST, ALT and ALP of rats
exposed to caffeinated EDs [11]. It has been
demonstrated that rats administered ED alone or
in combination with alcohol showed higher
serum total bilirubin, ALT, ALP and AST than
untreated controls [12].
In the present study, serum uric acid and
creatinine concentrations were significantly
increased in ED-treated rats. Increases in blood
levels of uric acid and creatinine are usually
associated with impaired kidney function [13].
These results are in agreement with the findings
of Khayyat and his colleagues who reported that
EDs induced elevations in serum urea, uric acid
and creatinine [14]. These researchers
suggested that caffeine induced the elevations
in urea, uric acid and creatinine through
inhibition of A2A adenosine receptors, resulting
in the development of interstitial inflammation,
increased proteinuria and deleterious changes in
renal function and structure [14].
Mansy et al
2854
Figure 1: Light micrographs of liver (I and II) and
kidney (III and IV) sections of rat in G 4 (highest ED-
exposed group). Specimens were fixed in 10%
neutral formalin and stained with H & E,
magnification 100 μm. I: Showing a markedly
dilated and congested portal vein (PV). The portal
area shows starting fibrosis (F), inflammatory
cellular infiltration (I), and bile duct proliferation
(asterisks). Hepatocytes at the periphery of the
lobules show undergoing degeneration and necrosis
with shrunken or disappearing nuclei (arrows). II:
Showing distorted general architecture of the liver,
congestion of a portal vein (PV) and inflammation (I)
in portal areas as well as between hepatocytes
which also show marked vacuolation mainly at the
peripheries of lobules (arrows). III: Showing
distorted general architecture, marked dilatation and
congestion of veins (V) and severe inflammation of
the interstitial tissue (I). Some renal corpuscles are
reduced in size with narrowing of the urinary space
(arrows). IV: Showing a markedly dilated vein (V), a
renal corpuscle with a markedly shrunken
glomerulus (G) and a markedly widened urinary
space (asterisk). The renal corpuscle is surrounded
by starting Fibrosis (F). The tubular cells show signs
of degeneration and necrosis with loss of cellular
details and cell boundaries (arrows)
However, some other ED-based studies
reported findings that are at variance with these
results. For example, it has been reported that
consumption of EDs was associated with higher
plasma total protein and lowered levels of
creatinine, albumin and uric acid [16]. Yet other
researchers found no significant association
between caffeine intake and the serum levels of
urea and creatinine in rats [17]. These
disparities on the effect of ED may be attributed
to lack of uniformity in the composition of these
energy beverages.
The present study also revealed that ED
exposure led to increased oxidative stress in the
rats. This was evident in the ED-induced
significant decreases in activities of SOD, CAT
and GPx. These enzymes are important
antioxidants which work in concert with the non-
enzymatic antioxidant system to protect cells
from oxidative damage by free radicals [18].
Indeed, the antioxidant enzymes are the first line
of defense which protects cells from oxidative
stress-induced damage. Superoxide dismutase
(SOD) neutralizes the highly reactive superoxide
anion by converting it to hydrogen peroxide,
which is in turn degraded to water by GPx and
CAT [19]. The significant reductions in blood
levels of these enzymes, especially in the rats
that received medium and high doses of ED,
might be due to ED-induced increases in
superoxide radical, thereby overwhelming the
neutralizing capacities of the antioxidant
enzymes.
Studies have shown that exposure of human
cells to high levels of caffeine induced a pro-
oxidant environment in the cells, leading to
increased protein oxidation, while low levels of
caffeine had no effect on the antioxidant
capacity of cells [20]. It has been demonstrated
that caffeine significantly increased BUN levels,
resulting in the activation of xanthine oxidase
which in turn, stimulated the oxidation of
xanthine to uric acid, and generation of
superoxide anion and H2O2 [21]. The interaction
between H2O2 with O2 produces free radicals.
On the other hand, several studies have
independently demonstrated the antioxidant
properties of many components of ED such as
taurine, ginseng, caffeine and guarana [21].
The pattern of variations seen in liver and kidney
function parameters of rats exposed to the
different doses of ED was in agreement with the
lesions in the photomicrographs of these
tissues. The observed lesions are most likely a
consequence of the deleterious effects of ED. It
can be reasonably suggested that the lesions
were brought about by tissue damage arising
from ED-induced oxidative stress. These results
are consistent with a previous report on
evidence of hepatotoxicity and alterations in liver
ultrastructure in rats treated with different types
of EDs [22]. In another study, the lesions in liver
and kidney tissues were attributed to potential
reaction of taurine with some other active ED
ingredient such as caffeine [23]. In addition,
Khayyat and his colleagues found that rats
treated with EDs had hepatic cytoplasmic
vacuolations due to presence of lipid droplets
which were attributed to deteriorative changes
within hepatocytes [22].
Many investigators are in agreement on the
adverse effects of ED as obtained in the present
Mansy et al
2855
study [11]. However, others reported that Power
Horse and Red Bull significantly influenced liver
enzyme activities but had no significant
influence liver histopathology [16]. Some
researchers reported irregular outlines and
pyknosis in the nuclei of hepatocytes, and
numerous mitotic figures [17].These changes
may be attributed to the toxic effects of caffeine,
and the harmful effects of preservative
substances added to EDs, such as sodium
benzoate [24]. However, it has been reported
that ED-induced hepatocyte damage was
reversible as indicated by blood chemistry
analysis and histopathological studies of the
organs of animals in the recovery group [19].
CONCLUSION
The results of this study demonstrate that
exposure of rats to high doses of Red bull for 12
weeks leads to liver and kidney damage. The
pronounced reduction in the blood levels of key
antioxidant enzymes suggests that the harmful
effects of Red bull are mediated through
increased ROS generation and oxidative stress.
If animal-to-man extrapolation is permitted, these
results call for restraint and caution in the
consumption of Red bull and other EDs. Thus,
the need for adequate public awareness cannot
be over-emphasized.
DECLARATIONS
Acknowledgement
The authors greatly appreciate the efforts of Dr
Mohammad Atteya, Assistant Professor of
Histology, Anatomy Department, College of
Medicine, King Saud University in preparing,
interpreting and writing reports for the
histopathological aspects of this work.
Conflict of Interest
No conflict of interest is associated with this
work.
Contribution of Authors
We declare that this work was done by the
authors named in this article and all liabilities
pertaining to claims relating to the content of this
article will be borne by the authors. Mansy and
Hanafi designed the study and drafted the
manuscript, Alogaiel participated in the whole
experimental work and statistical analysis.
Zakaria, Alogaiel and Mansy critically reviewed
the manuscript. All authors read and approved
the final manuscript.
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