Vol.4, No.04, April 2023
E-ISSN: 2723-6692
P-ISSN: 2723-6595
http://jiss.publikasiindonesia.id/
Journal of Indonesian Social Science, Vol. 4, No. 04, April 2023 328
The Effect of High Dosage of Iron Supplementation (Fe)
Against Histopathology of Gastric Mucosa in Pregnant Strain
Wistar White Rats (Rattus norvegicus).
Fatmawati
1
, Dyah Aulia Perwitasari
2
1,2
Program Study Pendidikan Profesi Bidan, Fakultas Kedokteran, Universitas Brawijaya
Email: fatmawati@ub.ac.id
Corresponding Author: fatmaw[email protected]
ARTICLE INFO
ABSTRACT
Submitted
:02-04-2023
Received
:05-04-2023
Approved
:15-04-2023
Pregnant women are encouraged to consume iron tablets during
their pregnancy or at least 90 tablets during pregnancy. Side effects
of iron tablets include nausea, vomiting, diarrhea, constipation. If the
tablets is consumed in excess it will cause toxicity because excess
iron will cause oxidative stress which can affect the state of the
stomach. Purpose: to determine the effect of high-dose iron (Fe)
supplementation on the histopathology of gastric mucosa in
pregnant strain Wistar white rats (Rattus norvegicus). This study
used 24 pregnant rats divided into 4 groups, that is, negative
controls; treatment 1 with a dose of 0.54 mg; treatment 2 with a dose
of 1.08 mg; and treatment 3 with a dose of 2.16 mg. Iron (Fe)
supplementation was started from the 1st day of pregnancy until the
18th day, then on the 19th day the rats were dissected and the
gastric organ was taken as a preparation with Hematoxylin Eosin
(HE) staining and then observed with a light microscope. This study
showed the histopathological results of gastric mucosa damaged in
the treatment group which was assessed using the Barthel Manja
score. The One Way ANOVA test results showed a significant
difference from gastric mucosa (p = 0.000) in the control and
treatment groups of this study. High doses of iron (Fe)
supplementation affect the histopathology of gastric mucosa.
Keywords: Iron
Supplementation,
histopathology of gastric
mucosa, pregnant white rats.
Attribution-ShareAlike 4.0 International (CC BY-SA 4.0)
1. Introduction
Anemia in pregnancy has an effect on the mother, both in pregnancy, childbirth, and
in the postpartum period and thereafter. Problems that can arise due to anemia are
miscarriage (abortion), premature birth, prolonged labor due to fatigue of the uterine
muscles in contracting (uterine inertia), postpartum bleeding due to the absence of uterine
muscle contractions (uterine atony), shock, infection both during childbirth and postpartum,
and severe anemia can cause cardiac decompensation (Wiknjosastro et al., 2010).
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One of the government's efforts to prevent anemia is by administering Ferrous coated
tablets. Use in pregnant women is recommended every day for 3 trimesters or at least 90
tablets for 3 trimesters. Apart from blood booster tablets, to meet the needs of iron during
pregnancy can be obtained from animal protein such as liver, fish and meat. Consuming
blood-boosting tablets can cause side effects on the gastrointestinal tract in some people,
such as discomfort in the pit of the stomach, nausea, vomiting and diarrhea, in some women
experiencing constipation (Alimul, 2008).
Excess iron can cause the formation of free radicals (Perdana & Jacobus, 2015). The
formation of free radicals can cause an increase in Reactive Oxygen Species (ROS) which
causes oxidative stress. Excessive or increased ROS will cause lipid peroxidation which can
affect the structure and function of the membrane. This affects the gastric mucosa, and
initiates the formation of lesions in the gastric mucosa (Powers & Jackson, 2008).
2. Materials and Methods
In this study, researchers used experimental laboratories (true experiment) with a
Post Test Only Control Group Design. The experimental group will be selected randomly or
randomly, after which it will be given treatment and made observations. Furthermore, the
results of observations of each group will be compared. This research was carried out at the
Laboratory of Pharmacology and Anatomical Pathology, Faculty of Medicine, Brawijaya
University. White female rats are the experimental animals used in the study. The white rats
used had the criteria of age 8-10 weeks, weight 150-250 grams, pregnant, healthy condition
which was marked by active rat movement. This study used 24 rats obtained from the
Pharmacology Laboratory, Faculty of Medicine, Brawijaya University. The division of groups
in this study was 4 groups, namely, the K-negative group, P-1, P-2 and P-3. Each research
group contained 6 rats. The control group was not given iron supplementation, for the
treatment groups 1, 2 and 3 were given iron supplementation at doses of 0.54 mg/200grBW,
1.08mg/200grBW, and 2.16mg/200grBW given once a day.
The dose of iron supplementation in this study was the independent variable, whereas
the histopathology of the gastric mucosa which was given iron (Fe) supplementation was the
dependent variable. Prior to the study, the rats were acclimatized for 7 days. Then the rats
were mated with a ratio of male and female rats, namely, 1:1 and 1:2 each in one cage. If after
mating a female rat has a vaginal plug, then the rat is declared pregnant on day 0 and put into
the K-negative and treatment groups. The K-negative group will not be given iron
supplementation, group P-1 will be given iron supplementation at a dose of
0.54mg/200grBW per day, P-2 will be given iron supplementation at a dose of
1.08mg/200grBW per day, and treatment 3 will be given iron supplementation at a dose of
2.16 mg/200grBB. All rats were given a standard feed of 40 grams per day and drank ad
libitum.
Iron supplementation was given to rats during the 18th day of pregnancy as much as
1 ml by sonde. The rat stomach was taken and then proceeded to make preparations from
the organs using Hematoxylin and Eosin (HE) staining. After that, they were observed using
an Olympus BX 1 light microscope and OlyVIA Scan Dot Slide software with 400x
magnification in 5 fields of view randomly. Observation of gastric preparations was assessed
using a modification of the Bathel Manja mucosal epithelial integrity score (Barthel et al.,
2003).
Table 1. Mucosal Epithelial Integrity Score
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Score
Mucosal Epithelial Integrity
0
No pathological changes
1
Epithelial desquamation
2
Damage 1-10 epithelial cells
3
Damage > 10 epithelial cells
Data analysis technique
The SPSS 16.0 for Windows program is a program used to process data that has been
obtained. Then tested for normality and homogeneity first. If the data is normal and
homogeneous, then proceed with the One Way ANOVA parametric test and proceed with Post
Hoc Tukey analysis.
3. Results and Discussions
The treatment of iron supplementation was given from the first day of pregnancy to
the 18th day. Stomach collection of rats was carried out on the 19th day of pregnancy and
then HE preparations were made. The results of observations of rat gastric epithelial cells
using the mucosal epithelial integrity score were then averaged and processed using a
program for analysis (McCrea & Bates, 1999).
Figure 1. Histopathology of the Rat Gastric Mucosal Epithelium at 400x Magnification
(A) Control has a score of 0, i.e. no pathological/normal changes, (B) Treatment 1 has
a score of 1 seen epithelial desquamation, (C) Treatment 2 has a score of 2 seen damage to 1-
10 epithelial cells, (D) Treatment 3 has a score 3 shows >10 epithelial cell damage.
The following are the results of the average gastric mucosal epithelial integrity score:
Figure 2. Graph of the results of the average mucosal epithelial integrity score
C
D
A
B
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The graph shows that the higher the average value, the higher the epithelial damage.
In the graph it is known that the lowest damage is the control/without treatment group and
the highest damage is in the treatment group 3. This shows that the increasing dose given,
the damage that occurs also increases.
Normality and Homogeneity Test
The results of the normality test of the Shapiro-Wilk Test (Table 2) showed normal or
significant data, namely 0.180 (p>0.05), which can be concluded that the mucosal epithelial
integrity score data was normally distributed. While the homogeneity test used was the
Levene Test (Table 3) which showed a data result of 0.827 (p > 0.05) which can be concluded
that the mucosal epithelium integrity score data has a homogeneous data variant.
One Way ANOVA test
This test was carried out to determine whether there was a significant difference in
the epithelial cells of the four groups. The difference is called significant/meaningful if p
<0.05. The results were 0.0000 (Table 4) so that it can be said that there were significant
differences in the epithelial cells of each group
Post Hoc Tukey Test
Table 5. Post Hoc Tukey Test
Post Hoc Tukey test results (Table 5) showed no significant differences in epithelial
Integritas Mukosa
Tukey HSD
a
6
.3000
6
.7000
6
1.3000
6
1.8667
.184
1.000
1.000
Kelompok
Kontrol
P1
P2
P3
Sig.
N
1
2
3
Subset for alpha = .05
Means for groups in homogeneous subsets are displayed.
Uses Harmonic Mean Sample Size = 6.000.
a.
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cells between the K-negative and P-1 groups. There were significant differences in epithelial
cells seen between the K-negative and P-2 and P-3 groups.
Based on the results of the study, the highest average mucosal integrity score was
treatment 3 with a dose of 2.16 mg/200 g BW. It can be concluded that the higher the dose
given, the higher the score or the higher the damage to the epithelial cells. ANOVA analysis
showed that there was a significant difference with the results p = 0.000 (p <0.05), the
meaning of the results was that there were significant differences between groups P-1, P-2,
and P-3 who were given iron supplementation at a dose of 0.54 mg /200grBB,
1.08mg/200kgBB and 2.16mg/200grBB with the control group seen from the gastric
mucosal epithelium score (Sunkara et al., 2017).
Damage to gastric epithelial cells occurs due to the formation of free radicals caused
by iron supplementation, then causes an increase in Reactive Oxygen Species (ROS) which
will trigger oxidative stress. Free radicals themselves will trigger lipid peroxidation and
tissue damage. Lipid peroxidation can cause damage to the structure and function of the
membranes, one of which is the gastric mucosa, which initiates the formation of lesions in
the gastric mucosa (Powers & Jackson, 2008).
The gastric mucosa naturally produces prostaglandins (PGE2) which can inhibit
gastric acid secretion. Iron can inhibit the production of prostaglandins. Inhibition of
prostaglandin synthesis in the stomach will increase acid secretion, and cut off the defense of
the gastric mucosa (Hisakawa et al., 1998). These conditions can cause irritation to the
stomach and result in damage to the mucosa, parietal cells, and endothelial cells
Nozaki et al., 2011).
This study has results that are in accordance with other existing studies related to iron
(Fe) supplementation can cause damage to the stomach. As in the case study conducted by
Marginean and Melit (2017).
The histopathology of the gastric mucosa also shows inflammation and necrosis.
Damage to gastric epithelial cells can be influenced by external factors that were not studied,
such as the psychological state of the rats, the influence of rat feed, or Helicobacter pylori
bacteria. It could also be caused by the possibility of errors in the technique of organ
harvesting and preparation of preparations which is a weakness in this study.
4. Conclusion
From the research that has been done, it can be concluded as follows: Giving high
doses of iron (Fe) supplementation has an effect on the histopathology of the gastric mucosa
of rats. Provision of iron (Fe) supplementation at a dose of 0.54 mg/200grBB from research
has had a damaging effect on gastric mucosal epithelial cells.
Based on the research that has been done and the conclusions obtained, suggestions
from researchers are as follows. Further research can be carried out regarding iron (Fe)
supplementation for more in-depth gastric histopathology. Further research can be carried
out regarding Helicobacter pylori bacteria in the stomach given iron (Fe) supplementation.
Further research can be carried out regarding iron (Fe) supplementation on variables.
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