THE COMPARISON OF MALONDIALDEHIDE LEVEL BEFORE

CHEMOTHERAPY BETWEEN GRADE III AND GRADE IV

Rahmi Utami 1,Dasril David 2,Nadirah Rashid Rida 3

1
Departement of child health, Faculty of Medicine, University of Hasanuddin, Makassar
(Email: rahmioetami@gmail.com)
2 Departement of child health, Faculty of Medicine, University of Hasanuddin, Makassar
(Email: drdasril@gmail.com)
3 Departement of child health, Faculty of Medicine, University of Hasanuddin, Makassar
(Email: Nadirah.bika@gmail.com)

Correspondence
address:

Rahmi Utami Pediatric Health Sciences,

Faculty of Medicine, Hasanuddin
University Makassar, 90245 Mobile:
082187777336 Email:
rahmioetami@gmail.com
Abstract

Retinoblastoma is a neoplasm derived from neuroretina (cone stem cells), and is an intraocular malignant
tumor found in children, especially at the age under five years old. This study aims to compare
malondialdehyde levels before and after chemotherapy between retinoblastoma grade III and grade IV.
The research was a cross sectional study based on primary data conducted from children ranging from 1
to 18 months old who had a medical treatment and was listed in the Hematology division of the Public
Hospital of Dr. Wahidin Sudirohusodo from October 2018 to February 2019. The sample was selected
using consecutive sampling technique divided into two groups, ie children groups with retinoblastoma
grade III and grade IV. The patients' vein blood as sample was centrifuged to take its serum. The data on
the level of malondialdehyde were analyzed using bivariate by means of chi square, Mann-Whitney, and
Kolmogorov smimov. The results of the study indicate that there are 43 children suffering from
retinoblastoma grade III and grade IV. Four of them were executed so 39 of them were analyzed
consisting of 13 children suffering from grade III retinoblastoma and the rest 26 suffering from grade IV
retinoblastoma. The result of the Mann-Whitney test shows that there is a significant difference between
malondialdehyde levels of retinoblastoma grade III and grade IV based on nutrition status, sepsis, and
dehydration with a consecutive p value of 0.364, 0.731, and 0.910 (p> 0.05) . The result of Mann-Whitney
also indicates that there is a significant difference between MDA retinoblastoma grade III and
retinoblastoma grade IV with a value of p = 0.799 (p> 0.05).

Keywords: retinoblastoma, free radicals, malondialdehyde

INTRODUCTION

Retinoblastoma is a neoplasm derived from neuroretina (conecells stem).

Intraocular malignant tumors found in children, especially under the age of five years.
Tumors originate from embryonal retinal tissue. It can occur unilateral (70%) and bilateral
(30%) (Dharmawidiarini et al., 2010). Retinoblastoma is associated with mutations in the
RB1 tumor suppressor gene in the long arm of chromosome 13 (13q). RB1 tumor
suppression gene works at the G1 / S checkpoint, resulting in failure of cell repair
mechanisms (Kodrat & Soehartati, 2013).
Malignant cell conversion is normal in three stages - initiation, promotion and
progression. Oxidation can involve all three stages of the carcinogenesis process.
Oxidants can produce genetic lesions, which are poisonous to the cells themselves.
Rearrangement of the promoter region, deletion and inactivation or loss of tumor
suppressor genes can lead to the initiation of a carcinogenic process. In retinoblastoma,
tumor suppressor genes are inactive with changes in structural vital pairs, generally CG
(Cytosine-Guanine) in retinoblastoma.When cellular genes are converted to oncogenes,
base pairs GC and AT present two targets that are vulnerable to attack by oxidants.
Copper ions which are bound to deoxyribonucleic acid (DNA) can increase oxidative DNA
damage. Oxidative stress also can produce 5-hydroxy cytosine. Thymine glycol is toxic to
DNA if it is not released by DNA-glycosylate before replication. This basic division can
cause an abnormal location, which in turn can increasemutagenesis) (Mukhopadhyay et
al., 2012). Increased ROS production in cells cancer is chemically capable of damaging
DNA, protein, and fat (Birben et al.,2012).
Previous research by Mukhopadhyay et al (2012), regarding the expression of
stress oxidative in retinoblastoma metastasis showed results that decreased levels of
enzymes antioxidant along with increased ROS activity in patients with retinoblastoma
metastatic reflecting increased oxidative stress compared with patients primary intraocular
retinoblastoma. The process of fat oxidation by free radicals is called fat peroxidation
which is characterized by an increase in malondialdehyde (MDA), a product peroxidation
polyunsaturated fatty acid (PUFA). Where MDA is the final product in the process of fat
peroxidation caused by free radical reactions to unsaturated fats in cell membranes and is
generally used as a biomarker biological of peroxidation fat to assess oxidative stress
(Jovanovic et al., 2010).
Research by Deepa et al (2009), on oxidative stress in retinoblastoma: correlation
with tumor invasion and overview of clinical pathology results show that the level of MDA
was significantly higher in tumors with invasion of choroid, optic nerve, and orbit,
compared with tumors without invasion. No significant correlation was noted between MDA
immunoreactivity and tumor differentiation. Lipid peroxidation rates increase significantly in
tumors with choroid invasion, optic nerve, and orbit than in tumors without invasion.
Retinoblastoma produces high amounts of ROS as a result of high metabolic
activity, mitochondrial dysfunction, peroxisomal activity, increased signaling cell receptor,
oncogen activity, oxidase activity, cyclooksigenase, lipooksigenase and thymidine
phosphorylase, or through cross reaction with immune cell infiltration (Izyumov et et.
al.,2010).
In Indonesia, according to research by Miranda (2015), reports of patients visiting
retinoblastoma Medan's Haji Adam Malik Hospital who came to health services were
more in extraocular cases (stages III and IV). This result is also supported research by
Nelly that more cases of stage III and IV are found compared to cases of stage I-II. This
shows that most patients who come are already in advanced stages of intraocular and
proptosis (Dharmawidiarini et al, 2010). This is different from in developed countries that
are diagnosed when the disease is still intraocular. This is thought to be related to the low
level of education and socioeconomic conditions and the progress of health facilities. Yet
according to research by Lastariana et al (2018), the mortality rate of retinoblastoma
reaches 68%. The patient's prognosis is influenced by the speed of diagnosis and the
rate of treatment.
High levels of ROS will damage cell components (fat, protein, DNA,
carbohydrates) and disrupt the normal cell cycle, which can inhibit the activities normal of
the cell cycle phase. Of all these molecules, the most vulnerable to attack free radical is
fat, where if ROS reacts with the fatty acid component of the cell membrane, there will be
a chain reaction known as fat peroxidation, where the end result is MDA. The presence of
 MDA shows the presence of oxidative stress on fat cell components and also oxidative
stress on proteins, DNA and carbohydrates. When components of protein, fat or
carbohydrate react to free radical attack, fat cells do not necessarily have cell damage.
Based on the background above, this study aims to compare the levels of
malondialdehyde (MDA) before chemotherapy between patients with grade retinoblastoma
III with grade IV.

MATERIALS AND METHODS

Research Design and Variables
This study is an observational study with approach cross sectional. The research
variables consisted of: independent variables (patients with retinoblastoma grade III and
grade IV), dependent variables (levels of malondialdehyde), intermediate variables
(biological processes that occur due to retinoblastoma until the onset of malondialdehyde),
random variables (status nutritional and sex), control variables (age), and moderating
variables (sepsis and dehydration)
Location and Time of the Research The
Study was conducted at Dr. RSUP Wahidin Sudirohusodo Makassar from October
2018 to July 2019.
Population and Samples
The study population was retinoblastoma sufferers who had not undergone
chemotherapy aged 1 month to 18 years who underwent treatment and were registered in
the division Hematology-oncology at Dr. RSUP Dr. Wahidin Sudirohusodo. The research
sample is all affordable populations that meet the inclusion and exclusion criteria.
Data Collection Method
At the time of hospital admission, patients aged 1 month-18 years and were
diagnosed with grade III and grade IV retinoblastoma. Patients who met the inclusion
criteria blood samples were taken for examination of Malondialdehyde levels. In addition,
age, sex, nutritional status, clinical symptoms and routine laboratory examinations
Data Analysis
All data obtained were recorded in the research data form, then grouped according
to the purpose and type of data. Then the appropriate statistical method was chosen,
namely: univariate analysis, bivariate analysis, and correlation test.

RESULTS
An observational study with approach was used cross sectional to compare levels
of malondialdehyde (MDA) before chemotherapy between patients with retinoblastoma
grade III and grade IV. The study was conducted at the Dr. Wahidin Sudirohusodo
Makassar from October 2018 to July 2019.
During the study period, 43 patients were diagnosed with retinoblastoma, who met
the inclusion criteria of 39 retinoblastoma patients, consisting of 13 patients with grade III
retinoblastoma and 26 patients with grade IV retinoblastoma.
In the study sample there were 26 (66.7%) women with an average value of
malondialdehyde (MDA) above the normal value (1.3 nmol / l) of 11.26 nmol / ml and a
range of 4 to 29 nmol / l and there were 13 (33.3%) men with an average value of
malondialdehyde (MDA) also above the normal value (1.3 nmol / l) which was 17.38 nmol /
ml and a range of 4 to 68 nmol / l. Mann-Whitney U test results showed no significant
difference between MDA levels of patients with sex with a value of p = 0.094 (p > 0.05)
(attachment, Table1).
In the study sample there were 11 (28.2%) patients with malnutrition with a value
Average of malondialdehyde (MDA) above the normal value (1.3 nmol / l) which was 9.72
nmol / ml and a range of 5 to 21 nmol / 1, there were 6 (15.4%) patients with malnutrition
with a value average of malondialdehyde (MDA) also above the normal value (1.3 nmol /
l) which was 12.67 nmol / ml and a range of 6 to 27 nmol / l and there were 22 (56.4%)
patients with malnutrition with an average value of malondialdehyde (MDA) also above
the normal value (1.3nmol / l) which was 15.27 nmol / ml and a range of 4 to 68 nmol / l.
Mann-Whitney U test results showed no significant difference between MDA levels of
patients with nutritional status with a value of p = 0.364 (p > 0.05) (appendix, Table 2).
 In the study sample there were 16 (41%) sepsis patients with an average value of
malondialdehyde (MDA) above the normal value (1.3 nmol / l) which was 11.87 nmol / ml
and a range of 3 to 34 nmol / l and there were 23 (59%) without sepsis with an average
value of malondialdehyde (MDA) also above the normal value (1.3 nmol / l) which is
14.30 nmol / ml and a range of 5 to 68 nmol / l. Mann-Whitney U test results showed no
significant difference between the MDA levels of patients with sepsis and without sepsis
with a value of p = 0.731 (p > 0.05) (appendix, Table 3).
In the study sample there were 18 (46.2%) dehydrated patients with an average
value of malondialdehyde (MDA) above the normal value (1.3 nmol / l) which was 13.66
nmol / ml and a range of 6 to 68 nmol / l and there were 21 (53.8%) without dehydration
with an average value of malondialdehyde (MDA) also above the normal value (1.3 nmol /
l) which was 13.00 nmol / ml and a range of 4 to 34 nmol / l. Mann-Whitney U test results
showed no significant difference between the MDA levels of patients with dehydration and
withoutdehydration with a value of p = 0.910 (p > 0.05) (appendix, Table 4).
In the study sample there were 13 (33.3%) patients with grade 3 retinoblastoma
with a value average of malondialdehyde (MDA) above the normal value (1.3 nmol / l)
which was 11.61 nmol / ml and a range of 6 to 21 nmol / l and there were 26 (66.7%)
grade 4 retinoblastomas with an average value of malondialdehyde (MDA) also above the
normal value (1.3 nmol / l) ie 14.15 nmol / ml and a range of 4 to 68 nmol / l. Mann-
Whitney U test results showed no significant difference between grade 3 retinoblastoma
MDA levels and retinoblastoma grade 4 with a value of p = 0.799 (p > 0.05) (appendix,
Table 5).

DISCUSSION
This study shows that there is no significant difference between levels MDA in
retinoblastoma patients with gender, nutritional status and dehydration status. Test results
Mann-Whitney showed no significant difference between MDA levels of retinoblastoma
grade 3 and retinoblastoma grade 4.
Cancer cells produce more free radicals /Reactive Oxygen Species (ROS)
compared to normal cells, partly due to stimulation of oncogenes, increased activity
 Metabolic and mitochondrial damage. ROS is like a double-edged sword. On the one
hand, at low levels, ROS can facilitate apoptosis, play a role in signaling molecular in
several cellular processes and as part of the phagocytic process. Whereas at high levels,
ROS is associated with a significant decrease in defense from antioxidants which can
cause damage to proteins, fats, DNA and impaired function cellular (Udensi & Paul,
2014).
Compared with normal cells, cancer cells have higher ROS levels, which seem to
be an important part in the process of initiation and development of malignancy. In
addition, the accumulation of ROS is thought to play a role in the change of stem to Cells
retinoblastoma cells. High levels of ROS will damage cell components (fat, protein,DNA)
and disrupt the normal cell cycle, which can inhibit the normal activities of the cell cycle
phase. Of all these molecules, the most vulnerable to radical attack free is fat, where if
ROS reacts with the fatty acid component of the cell membrane, there will be a chain
reaction known as fat peroxidation where the end result is malondialdehyde (MDA).
In this study the most sexes in both the grade retinoblastoma groups 3 and grade
4were women with a 2: 1 ratio and the analysis showed results no significant. In the study
of Pandey (2014), it was stated that there was no predisposition to race and sex in the
incidence of retinoblastoma.
In this study, MDA levels in retinoblastoma patients with both men and women did
not show significant differences in our analysis. Based on age, most retinoblastoma
patients in this study were in the range of ages 1-10 years. As for the nutritional status of
retinoblastoma patients, the obtained highest nutritional status was with the results of the
analysis not meaningful.
In this study showed retinoblastoma patients with malnutrition or
without malnutrition with MDA level analysis which was not significant. In the study
of Khare et al (2014), there was a significant increase in serum MDA in malnourished
children compared to controls (P <0.001).
In this study showed retinoblastoma patients with sepsis or without sepsis
experienced an increase in MDA levels. In a study conducted by Lorente et al (2013),
MDA levels in sepsis patients were 2 times higher than patients without sepsis. In a study
 conducted by Lorente et al (2013), and Daga et al (2016), MDA levels inpatients
sepsis were higher in controls with a P value <0.001. High serum MDA levels support the
hypothesis that increases oxidative stress, specifically lipid peroxidation, contributes to the
pathophysiology of sepsis. The combination of increased superoxide and increased
production of nitric oxide in sepsis leads to increased production of peroxynitrite and
reactive nitrogen species (RNS). The results of these reactions cause accumulation of
damage to lipids, proteins, and nucleic acids which can interfere with cellular function.
Specifically, injury oxidative to lipids in plasma and mitochondrial membranes can alter
permeability and damage membrane-bound receptors and enzymes. MDA and other
reactive aldehydes can interfere with the structure and function of proteins. Because
aldehydes are released into the blood when cells are damaged by lipid peroxidation,
serum MDA is an indirect marker of oxidative stress. Our difference with the results of this
study is that in our patient there are other factors that influence an increase in MDA levels,
namely retinoblastoma (Weiss & Deutschman, 2014; Lorente et al., 2013; Daga et al.,
2016).
In this study showed retinoblastoma patients with dehydration or without
dehydration experienced an increase in MDA levels. Dehydrated retinoblastoma patients
have higher MDA levels than retinoblastoma patients with dehydration. In a study
conducted by Ali et al (2013), MDA levels were higher in a dehydrated state with a p value
<0.05. Malondialdehyde (MDA) is an endogenous product of lipid peroxidation and
naturally occurring prostaglandin biosynthesis. MDA production is used as a biological
marker of endogenous DNA damage. Dehydration causes the production of high reactive
oxygen species which triggers high lipid peroxidation which results in increased MDA
formation.
The results showed an increase in MDA levels well above normal in retinoblastoma
group of grade 3 or grade 4 and there are no significant differences in both groups This
shows that there has been formation of stress oxidative(ROS) both grade 3 and grade 4
retinoblastoma. Deepa research et al (2009) in America showed that there was a
significantly increased level of fat peroxidation (MDA) in retinoblastoma patients who were
invasive compared to those who were not invasive with a value of p <0.05. They have
previously reported increased levels of iNOS and nitrotyrosine in retinoblastoma. NO
produced by iNOS can initiate and / or promote tumorigenesis, iNOS is regulated
transcription and induced by oxidative stress, and also by inflammatory cytokines,
endotoxins, and hypoxia. Nitric oxide can react with free radicals oxygen, such as
superoxide anions, to form peroxynitrite which, in turn, can cause DNA and phospholipid
membrane oxidation and protein tyrosine nitration or free (nitrotyrosine). Together, it can
now be said that oxidative stress and nitrosative stress play an important role in
retinoblastoma.
Mukhopadhyay et al (2012), showed an increase in ROS activity in patients with
metastatic retinoblastoma reflecting increased oxidative stress compared to primary
intraocular retinoblastoma patients. In cancer patients there is an increase in lipid
peroxidation, which is characterized by increased levels of MDA as the product final of lipid
peroxidation.
The results of this study can contribute data on MDA levels in grade 3 and 4
retinoblastoma patients that indicate high levels of oxidative stress so that early prevention
can be done by administering antioxidants during chemotherapy. The researcher realizes
that after conducting this research, some of the hypotheses proposed were not proven. In
fact, there are lower MDA levels inpatients retinoblastoma with sepsis than those without
sepsis.
It was realized by researchers that there were limitations in this study, which was
only to assess the level of oxidative stress in retinoblastoma patients, but no examination
of levels was done antioxidant as a comparison. So that further research is needed to
assess levels of antioxidants in retinoblastoma patients. And sampling is only done once.
While the strength of this research is the method cross sectional which is carried out so
that the implementation is quite easy and results in a greater prevalence rate and more
variables that can be observed.

CONCLUSIONS AND SUGGESTIONS

Researchers concluded that the analysis of the mean value of MDA levels in the
grade 4 retinoblastoma group and the grade 3 retinoblastoma group did not have
 differences significant. MDA levels increase above normal starting from grade 3 or
retinoblastoma grade 4 retinoblastoma which indicates that the formation of oxidative
stress has begun to occur when patients are diagnosed with grade 3 retinoblastoma.
Researchers suggest that be conducted further research to determine levels of
antioxidants in both grade retinoblastoma patients 3 and grade 4. thought to provide
antioxidants in retinoblastoma patients both grade 3 and grade 4.

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APPENDIX
Table 1. Comparative Analysis of Malondialdehyde Levels of Patients with Grade 3 and Grade 4
Retinoblastoma Based on Gender

MDA Level Female Male

(nmol/ml) (n = 26) (n = 13)
Mean 11,26 17,38
Median 9,00 13,00
Standard Deviation 6,44 16,68
Range 4,00-29,00 4,00-68,00

Mann-Whitney U Test p = 0,094 (p > 0,05)

Table 2. Comparison Analysis of Content Malondialdehyde Retinoblastoma Patients Based on

Status Nutritional
MDA Level Poor Nourished Under Nourished Well Nourished
(nmol/ml) (n = 11) (n = 6) (n = 22)
Mean 9,72 12,67 15,27
Median 9,00 10,50 12,00
Standard Deviation 4,60 7,52 13,76
Range 5,00-21,00 6,00-27,00 4,00-68,00
Mann-Whitney U Test p = 0,364 (p > 0,05)
Table 3. Comparative Analysis of the Levels of Malondialdehyde in Patients with Grade 3 and Grade
4 Retinoblastoma Based on Sepsis and Without Sepsis
MDA Level Sepsis Not sepsis
(nmol/ml) (n = 16) (n = 23)
Mean 11,87 14,30
Median 9,00 11,00
Standard Deviation 7,26 13,23
Range 4,00-34,00 5,00-68,00
Mann-Whitney U Test p = 0,731 (p > 0,05)

Table 4. Relationship Analysis of Dehydration Status Between Patients with Grade 3

Retinoblastoma and Grade 4
MDA Level Dehydration No dehydration
(nmol/ml) (n = 18) (n = 21)
Mean 13,66 13,00
Median 10,00 11,00
Standard Deviation 13,92 8,37
Range 6,00-68,00 4,00-34,00
Mann-Whitney U Test p = 0,910 (p > 0,05)

Table 5. Comparative Analysis of Levels of Malondialdehyde Patients with Grade 3 Retinoblastoma

and Grade 4
MDA Level Retinoblastoma
Grade 3 Grade 4
(nmol/ml)
(n = 13) (n = 26)
Mean 11,61 14,15
Median 12,00 10,50
Standard Deviation 4,73 13,23
Range 6,00-21,00 4,00-68,00
Mann-Whitney U Test p = 0,799 (p > 0,05)