Effects of chronic administration of indomie noodles on the activity of

JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL SCIENCES
Sanni et al. Effects of chronic administration of indomie noodles on the activity of alanine
aminotransferase of rat kidney. Journal of pharmaceutical and biomedical sciences (J Pharm Biomed
Sci.) 2013 May (Supplement 1); 30(30): S65-S71.
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Original article
Effects of chronic administration of indomie noodles on the activity of alanine
aminotransferase of rat kidney
1SANNI,
Momoh, 1EJEMBI, Daniel, 2EMMANUEL, T. Friday,
1ABBAH, Okpachi C & *OGALA, Emmanuel
Affiliation:1Department
of biochemistry, Kogi State University,
Anyigba, Nigeria.
2Department of medical biochemistry, Kogi State
University, Anyigba, Nigeria.
Author’s contributions-All authors contributed
equally to this paper.
*Correspondence to:EJEMBI Daniel.
Department of Biochemistry, Kogi State University,
Anyigba, Nigeria.
Abstract:
The effects of chronic administration of Indomie
instant noodles on the activities of alanine
aminotransferase (ALT) of rat kidney was
investigated. Fourty-eight (48) rats (both males
and females) of average weight 210g were
divided into seven groups. Each group consists of
three (3) rats each, except for the control, which
consists six (6) rats (i.e both the males and the
females).The rats in group I, for both sex, were
administered cooked indomie spiced with the
seasoning, group II, were given raw indomie
spiced with the seasoning, group III, were given
both the cooked and raw indomie mixed
together and spiced with the seasoning, group
IV, were given cooked indomie without the
seasoning, group V, were given raw indomie
without the seasoning, group VI, were given both
the cooked and raw indomie, mixed together,
without the seasoning and, group VII, which
served as the control, were given the normal
fowl formulation feed. The administration was
carried out for a period of four (4) weeks, the
result showed that, there was a decrease in
kidney activity of the enzyme.
Key words: Indomie instant noodles; Alkaline
phosphatase.
Article citation:Sanni et al. Effects of chronic administration of indomie noodles on the activity of alanine aminotransferase of rat
kidney. Journal of pharmaceutical and biomedical sciences (J Pharm Biomed Sci.) 2013 May(Supplement 1);30(30):
S65-S71.Available at http://www.jpbms.info
INTRODUCTION
I
ndomie, is the world largest instant noodle
produced by Indofood-the world largest
manufacturer of dried instant noodle in Indonesia
(Indofood, 2010)13. It is sold throughout Indonesia,
Malaysia, Australia, Nigeria, the United States, and
among other numerous countries. Indomie noodle is
very nutritious, easy to make and can be eaten both
as snacks, as well, a major meal. It is very versatile
and this make it attracts the patronage of majority of
Courtesy: www.dufil.com
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people both at work, in school, and at home. Indofood
is one the largest pre-packaged food companies
founded in 1972 in Indonesia as Panganjaya
Intikusuma before changing to its current name in
1990 by Sudono by Salim under the Salims group
(Witular, 2004)20.
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Sanni et al.
.
Ingredients:
Noodles: wheat flour, vegetable oil, iodized salt,
sodium polyphosphate, sodium carbonate, potassium
carbonate, guargum, tartrazine (CI 19140),
antioxidant (TBHQ)
Seasoning powder: iodized salt, monosodium
glutamate (621), hydrolyzed vegetable protein, soy
powder, pepper, garlic powder, chicken flavor, Chili
powder.
Indomie noodles contain Tartrazine, a synthetic
lemon yellow azo dye primarily used as food
colouring agent (Food standards agency website,
2011)11. It has a batch category code of E102 C.I.
19140, FD&C Yellow 5, acid yellow 23, food yellow 4
and
trisodium-1-(4-sulfonatophenyl)-4-(4sulfonatophenylazo)-5-(pyrazolone-3-carboxylate).It
is water soluble and has a maximum absorbance in
aqueous solution at 427±2nm14. Tartrazine is a
commonly used colorant all over the world, mainly
for yellow, but can also be used with brilliant blue
(FD&C Blue; E133) or Green (E142) to produce
various green shades.
Tartrazine was believed to cause most of the allergic
and intolerance reaction experienced amongst all the
azo dyes, particularly in asthmatic patients and those
with aspirin intolerance (UK food Guide, 2007).
Symptoms of tartrazine sensitivity can occur from
either ingestion or cutaneous exposure to substances
containing tartrazine. Symptoms could either be mild
to severe3. A variety of immunological responses
have been attributed to tartrazine ingestion,
including anxiety, migraine3, clinical depression,
blurred vision, itching, general weakness, feeling of
suffocation, purple skin patches and sleep
disturbance17. People who are exposed to the dye
experience symptoms of tartrazine sensitivity even at
extremely small dose, some for periods up to 72
hours after exposure6. In children, asthma attack and
hives have been claimed, as well as supposed link to
thyroid tumors, chromosomal damage and hyper
reactivity7.
Possible health effects of tartrazine:
The British food standards agency on September 6th,
2007, revised, advice on certain artificial food
additives, including tartrazine (BBC news, 2007)5.
Professor Jim Stevenson of Southampton University,
the author of the report said, “This is an important
investigating area of research”. The result suggests
that consumption of certain mixture of artificial food
color and sodium benzoate preservatives are
associated with increase in hyperactive behavior in
children.
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The food standards agency (FSA) in April 10, 2008,
called for voluntary removal of the colors (but not
sodium benzoate). In addition, it recommended that
the colors should be phased out in foods and drinks
in the European Union (EU) over a specific period
(FSA Board, 2008)11.Tartrazine has a noticeable
effect on the behavior of young mice;
1. Tartrazine inflamed the stomach lining (increased
the number of lymphocytes and eosinophils of
rats) when given in the diet for a prolonged
time15.
2. Tartrazine was found to adversely affect and alter
biochemical markers in vital organs e.g. liver,
kidney, small intestines etc of rat not only at
higher doses but also at low doses4.
Organ studied (The kidney):
The Kidney, a perfect example of homeostatic organ,
maintains the constancy of fluid in human internal
environment, much like a water purification plant
that keeps a city’s water drinkable and disposes of its
waste10.
In rat, the right kidney is slightly placed anterior to
the left while in man; the right kidney is slightly
lower than the left18. Every day, the kidney filters
nearly 200 litres of fluid from the bloodstream,
allowing toxins, metabolic wastes, and excess ions to
leave the body in the urine and returning needed
substances to the blood10.
External anatomy:
The bean-shaped kidney lies in a retroperitoneal
position (i.e. between the dorsal body wall and the
parietal peritoneum). In the superior lumber region,
they extend approximately from the level of the
twelfth thoracic vertebra to the third lumber
vertebra. Thus, the kidney receives some protection
from the lower part of the cage. The right kidney is
crowned by the liver and lies slightly lower than the
left kidney.
Internal anatomy:
A frontal section of the kidney reveals distinct
regions, the cortex, medulla and the pelvis. The most
superficial region, which is the renal cortex10, is light
in color and has a granular appearance. Deep down
the cortex is the darker, reddish-brown renal
medulla, which exhibits cone-shaped tissue masses,
called ‘Medularry or Renal pyramid’. The broad base
of each pyramid faces towards the cortex, and its
apex or papilla (nipple) points internally.
The pyramids appear stripped because they are
formed collecting tubules. The renal columns and
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inward extensions of the cortical tissues separate the
pyramids. Each medullary pyramids and its
surrounding capsule of the cortical tissue constitutes
a lobe of a kidney lateral to the hilus within the renal
sinus called the ‘Renal pelvis’. This flat, funnelshaped tube is continuous, with the ureter leaving
the hilus10. Branching extension of the pelvis from
two or three major calyces (singular) each of which is
subdivided into 10, forming several mino-calyces,
cub-shaped areas that enclose papillae of the
pyramids.
The calyces collect urine, which drains continuously
from the papillae, and empty it into the renal pelvis
urine, then flows through the renal pelvis into the
ureter, which transports it to the bladder to be
stored. The walls of the calyces rhythmically and
propels urine along its course by peristalsis10.
Functions:
The Kidney regulates simultaneously the volume and
chemical makeup of the blood, maintaining the
proper balance between water and salts and also
between acids and bases. The following are specific
functions carried out by the kidneys. They are;
Metabolic activities:
The Kidney plays an important role in
gluconeogenesis, a role undertaken only during
prolonged fasting. At such times, the kidney can
supply approximately one-fifth as much glucose to
the liver10.
Secretion:
The Kidneys produce the enzyme ‘Renin’, which
helps to regulate blood pressure and kidney function,
also the hormone ‘Erythropoietin’, which stimulates
the production of red blood cells (RBC) in bone
marrow.
Excretion:
They filter blood plasma; separate wastes from the
useful chemicals, and also eliminates the wastes
while returning the rest to the bloodstream.
Acid-Base homeostasis:
Two organ systems, the kidneys and lungs, maintain
acid-base homeostasis, which is the maintenance of
pH around a relatively stable value. The lungs
contribute to acid-base homeostasis by regulating
carbon dioxide (CO2) concentration. The kidneys
have two very important roles in maintaining the
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acid-base balance: to reabsorb bicarbonate from
urine, and to excrete hydrogen ions into urine.
Osmolarity regulation:
Any significant rise in plasma osmolarity is detected
by the hypothalamus, which communicates directly
with the posterior pituitary gland. An increase in
osmolarity causes the gland to secrete antidiuretic
hormone (ADH), resulting in water reabsorption by
the kidney and an increase in urine concentration.
The two factors work together to return the plasma
osmolarity to its normal levels.
Enzymes studied:
Alanine amino-transferase:
Alanine transaminase (ALT) test measure the
amount of the enzyme in the blood. It is found mainly
in the liver, but also in small amount in the kidneys,
heart, muscles, and pancreas. Alanine transferase
(ALT) was formally called serum glutamic pyruvic
transferase (SGPT) or Alanine aminotransferase
(ALAT). It plays a role in protein metabolism;
therefore, an abnormal high blood level of ALT
signifies damage in some organs of the body, mostly
the liver (Liver inflammation), (McPherson et al.,
2011)15.
Distribution of alanine aminotrnasferase:
Alanine aminotransferase (ALAT) is mainly found in
the liver, but in small amount in the kidneys, heart,
and muscles. It is less abundant compared to
aspartate transaminase (AST) in all tissues and
mostly occur in the cytosol and mitochondria of the
cells.
Clinical importance of alanine aminotransferase:
It is commonly measured clinically as part of a
diagnostic evaluation of hepatocellular injury, to
determine liver’s state. It is usually measured in
international units/litre (U/L)12,20 .
While sources vary on specific normal range values
for patients, 10-40 U/L is the standard normal range
for experimental studies20. Alanine transaminase
shows a mark diurenal variation.For female, it ranges
between 5-38 U/L, while for males; it ranges
between 10-50 U/L.
Functions: It catalyses the transfer an amino group
from alanine to α-oxoglutarate, the products of this
reversible transamination reaction is pyruvate and
glutamate.
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ALAT
Glutamate+ Pyruvate ⇌ α-Oxoglutarate + alanine
Glutamate
Pyruvate
α-Oxoglutarate
Alanine
It also helps in maintaining a balance in the supply of amino acid building blocks for protein synthesis.
MATERIALS AND METHODS
Enzyme substrate:
The enzyme has already been prepared and ready for
use.
Alanine aminotransferase enzyme (RANDOX KIT)
consists of Phosphate buffer, L-alanine and 2, 4dinitrophenylhydrazine as substrate.
Animal sacrifice:
Test animals:
Fourty eight (48) albino rats of average weight, 210g
were obtained from Faculty of Agriculture farm, Kogi
State University, Anyigba, Nigeria.
Preparation of tissue homogenate:
Methods:
Animal groupings:
Forty eight (48) rats (Rattus novergicus) were
randomly grouped into seven (7) groups. Groups 1-6
being the test groups consists of 6 rats each, each
group was further sub-grouped by sex into 3 males
and 3 females respectively. Group 7 is the control
group and was fed with normal rat feed; the control
group had twelve rats of which six were males and
six females. The experimental design is as follows;
 Group 1 was fed with indomie instant noodles
cooked and spiced with the seasoning.
 Group 2 was fed with raw indomie instant
noodle spiced with the seasoning.
 Group 3 was fed with a mixture of both
cooked and raw indomie instant noodle
spiced with seasoning.
 Group 4 was fed with indomie instant noodles
cooked without seasoning.
 Group 5 was fed with raw indomie instant
noodle without seasoning.
 Group 6 was fed with a mixture of both
cooked and raw indomie instant noodle
without seasoning.
Water was provided ad libitum. The rats were
examined within a thirty (30) days scheme in order
to ascertain levels of chronic toxicity.
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All experimental rats were starved for 24hours prior
to the day of sacrifice after the thirtieth day of
feeding and then sacrificed. Part of the organ of
interest was cut, removed and put in a 0.25M ice cold
sucrose solution.
The tissue was weighed and homogenized using an
automated homogenizing machine in a 0.25M
sucrose solution; this was kept in a clean dry sample
bottle and kept in the freezer before been used for
enzyme activity determination2.
Enzyme activity determination:
Determination of alanine aminotransferase
(Reitman and frankel, 1957)17
The colorimetric method of Reitman and Frankel was
employed of 1957 was employed.
Principle:
α- oxoglutarate + L-alanine → L- glutamate + pyruvate
Procedure: (Reitman and frankel, 1957)17
The reagent composition consist of 100mmol/L, of
phosphate buffer and 200mmol/L, of L-alanine,
2.0mmol/L of α-oxoglutarate, and 2.0mmol/L of 2, 4dinitrophenylhydrazine, which is the substrate. 0.1ml
of sample was pipette into a clean test tube, 0.5ml of
the solution R1 was also added into both the reagent
blank and the sample, 0.1ml of distilled water was
added into the blank, mixed and incubated for
30mins at 37 °C. 0.5ml of solution R2 was added to
the blank and sample mixed and was allowed to
stand for 20mins at 25°C. Then, 5.0ml of sodium
hydroxides was added into the blank and the sample
mixed, the absorbance of the sample was read
against the reagent blank after 5 minutes.
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RESULTS
Alanine aminotransferase activity
The results obtained were carefully and adequately
examined. From the chat presented below, the
results indicates the effects of the test sample
(indomie) on the activity of the marker enzyme,
alanine aminotransferase (ALT), whose cellular and
extracellular levels serves as indicators of
pathological state. From the data obtained in the
study of the effects of chronic administration of
indomie on the activity of alanine aminotransferase
on rats kidney, in group 2 showed the greatest
average decrease in the enzyme’s activity).
Generally, rats fed with indomie containing spice
showed greater decrease in ALT (Alanine
aminotransferase) activity than those fed without the
spice. Also a greater decrease in the enzyme’s activity
among males than females in group 1, group 3, group
4 and group 5 except in group 2, where there was an
average decrease, and in group 6, with higher
decrease in the enzyme activity in females than in
males was observed.
Figure 1. Graph of Alanine aminotransferase activity
From figure 1 above, the blue charts represents the
values for ALT activity in male rats while the green
charts represent the values of ALT activity in female
rats.
When indomie was administered into the rats, the
following result was observed.
For males, taking control rats as the reference group;
for group1, the activity of ALT was observed to
decrease by 9.98%, in group2, it decreased by 22.0%,
in group3, it decreased by 18.97%, in group4, it
decreased by 46.20%, in group5, it decreased by
23.01% and in group6, it decreased by 70.00%.
For females, taking control as the reference group; in
group1, the activity of ALT was observed to decrease
by 36.10%, in group2, it decreased by 22.00%, in
group3, it decreased by 36.02%, in group4, it
decreased by 50.00%, in group 5, it decreased by
30.02%, in group6, it decreased by 34.15%.
Comparing and contrasting the results of group 1 and
4, in reference to the control results, it was
discovered that the enzyme activity in the kidney of
male rats in group 1 was lower than that of the male
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rats in group 4, indicating the possible role of the
seasoning spice causing more toxic effect.
Likewise, for female rats in group 1, the enzyme
activity in the kidney was also lower than that of the
female rats in group 4, indicating also a possible role
of the seasoning spice causing toxic effect.
Comparing and contrasting the results of group 2 and
group 5 with reference to the control result, it was
observed that there were greater activity of the
enzyme in the kidney of rats in group 5 over the rats
in group 2 irrespective of their sex, this also shows
the possible role of the seasoning spice in causing a
more toxic effect and also reducing the activity of the
enzyme in the kidney over a time.
Comparing and contrasting the results of group 3 and
group 6 with reference to the control result, the
result showed a greater activity of the enzyme in the
kidney of male rats in group 6 over the male rats in
group 3, indicating a possible role of the seasoning
spice.
However, in female rats of group 3, the activity of the
enzyme in the kidney was greater than that of group
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6, indicating the other factors such as varied
metabolic patterns between sexes, which could be
responsible for this difference.
DISCUSSION
Defect to metabolic route could possibly be reasons
for causes of several diseases of which hypo activity,
hyperactivity and inhibition of metabolic enzymes
are enormously involved. Increase or decrease in
amount of these metabolic enzymes serves as
biomarker for clinical diagnosis of some illness9.
Biochemical parameter like enzyme assay indicates
that cellular damages could be by conventional
historical techniques1,2.
Only one (1) ‘marker’ enzyme was assayed for this
present study to determine the effects of indomie
instant noodles on the activity of Alanine
aminotransferase (ALT) in rat kidney.
Alanine aminotransferase is a transaminase enzyme
(EC 2.6.1.2). It is mostly associated with the liver, but
also found in the serum and kidney.
Tissue enzyme:
Alanine aminotransferase
The effect of Indomie instant noodles on the activities
of alanine aminotransferase was observed in the
kidney and the result is as follows;
There was a fall in the activity of the enzyme in the
kidney. This could be due to the inhibition of the
enzyme activity by some agents which are present in
the indomie, one of which may be Tartrazine.
The continuous decrease in the enzyme activity could
make the kidney become permeable, thereby
allowing the enzyme to escape into the extra-cellular
fluid, preventing the kidney from maintaining
constancy of fluid in the internal environment.
CONCLUSION
This research work had shown that chronic
consumption of indomie instant noodles could be
dangerous to health. Much intake of it could lead to
diseases like kidney failure, Cancer, and high blood
pressure. The effect also could vary depending on the
mode of preparation, sex of the animal, and also the
physiological state of the animal.
RECOMMENDATION
I therefore, suggest that further research be carried
out on indomie noodles for a more prolong time, with
the same method used above under more favourable
conditions.
I also suggest that, even though indomie should be
consumed, it should be done occasionally, especially
when the need for a fast food arises. This is because it
contains the necessary nutrients for healthy living in
moderate proportion.
Table1. Alanine aminotransferase activity results
GROUP
MALE AST(nm/min) I/U
1
10.40
2
22.85
3
18.02
4
44.72
5
25.50
6
73.10
CONTROL
53.04
FEMALE AST(nm/min) I/U
34.85
22.85
35.70
48.17
30.66
34.85
40.04
Table 2. Analysis of variance (Anova) For ALT activity In male albino rats
Descriptive
male
95% Confidence Interval for Mean
N
Mean
Std.
Deviation
Std. Error
Lower Bound
Upper Bound
Minimum
Maximum
Group1
1
10.4000
.
.
.
.
10.40
10.40
Group2
1
22.8500
.
.
.
.
22.85
22.85
Group3
1
18.0200
.
.
.
.
18.02
18.02
Group4
1
44.7200
.
.
.
.
44.72
44.72
Group5
1
24.5000
.
.
.
.
24.50
24.50
Group6
1
73.1000
.
.
.
.
73.10
73.10
Control
1
53.0400
.
.
.
.
53.04
53.04
Total
7
35.2329
22.44439
8.48318
14.4753
55.9905
10.40
73.10
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Table 3. Analysis of variance (Anova) For ALT activity In female albino rats
Descriptive
Female
95% Confidence Interval for Mean
N
Mean
Std. Deviation
Std. Error
Minimum
Maximum
Group1
1
34.8500
.
.
.
.
34.85
34.85
Group2
1
22.8500
.
.
.
.
22.85
22.85
Group3
1
35.7000
.
.
.
.
35.70
35.70
Group4
1
48.1700
.
.
.
.
48.17
48.17
Group5
1
30.6600
.
.
.
.
30.66
30.66
Group6
1
34.8500
.
.
.
.
34.85
34.85
Control
1
40.0400
.
.
.
.
40.04
40.04
Total
7
35.3029
7.80173
2.94878
28.0875
42.5182
22.85
48.17
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