ABSTRACT
Aspartame is a non-nutritive sweetener particularly used in ‘diet’ and ‘low calorie’ products and also in a variety of foods, drugs and hygiene products. The research work aimed at assessing the effect of L-arginine and vitamin C on some antioxidant status and histology of liver and brain tissues of the rats administered aspartame. Wistar albino rats (50-70) g were divided into six (6) group with five (5) rats in each groups. Animals were administered aspartame (1000 mg/kg) and treated with either vitamin C (100 mg/kg) or L-arginine (20 mg/kg and 40mg/kg) respectively. After twenty one (21) days, blood and organ samples were collected for biochemical and histological studies. The results showed that the administration of aspartame caused a significant (p<0.05) increase in liver function indices in the serum, liver and brain homogenates when compared to the normal control group while the co-administration of aspartame with L-arginine and vitamin C showed a significant reduction when compare to the positive control. Total protein concentration showed a significant (P<0.05) increase in the aspartame administered group and co-administration of aspartame and 40 mg/kg of L-arginine when compared with the normal control. While the co-administration of L-arginine 20 mg/kg and 40 mg/kg showed significantly (p<0.05) reduction when compared with the aspartame administered group in the serum, liver and brain. Also a significant (P<0.05) increase in FRAP of serum, brain and liver homogenate of aspartame administered rats was observed when compared with the normal control. The co-administration of aspartame and L-arginine (40 mg/kg) significantly (P<0.05) reduced the FRAP level in the both organs when compared with the normal and positive control. TBARS level significantly (p<0.05) increase in the serum, liver and brain homogenate of aspartame administered rats when compared with the normal control. However, the co-administration of aspartame with vitamin C or different doses of L-arginine (20 mg/kg, 40 mg/kg) respectively caused a significant (P<0.05) reduction. Catalase significantly (p>0.05) increased in the serum, brain and liver homogenate of aspartatme administered rats when compared with the normal control, while the co-administration of aspartame with vitamin C or co-administration of L-arginine (20 mg/kg) and L-arginine administered groups respectively caused a significant (P<0.05) reduction when compared with the positive control and co-administration of L-arginine (40 mg/kg) groups. More so a significant (P<0.05) increase was observed in superoxide dismutase activity of liver and brain homogenate of aspartame administered rats when compared with the normal control, while the co-administration of aspartame with vitamin C (100 mg/kg) and L-arginine (20 mg/kg, 40 mg/kg) respectively caused a significant (P<0.05) reduction when compared with the positive controls. GSH showed a significant (P>0.05) increase in the liver and brain homogenate of aspartame administered rats when compared with the normal control. The co-administration of aspartame with L-arginine (40 mg/kg), vitamin C (100 mg/kg) and L-arginine administration alone showed a significant reduction in the GSH concentration when compared with the positive control group. The histological observation of liver and brain sections administered rats substantiated the biochemical findings. It can be concluded from these observations that consumption of aspartame leads to hepatocellular injury and alterations in brain and serum of antioxidant status and the two antioxidant work to ameliorate adverse effects arising from aspartame administration.
TABLE OF CONTENTS
Title Page i
Declaration ii
Certification iii
Dedication iv
Acknowledgements v
Table of Contents vi
List of Tables x
List of Figures xi
List of Plates xii
Abstract xiii
CHAPTER 1:
INTRODUCTION 1
1.1 Background
of the Study 1
1.2 Aim
of the Study 4
1.3 Objectives
of the Study 5
1.4 Justification
of the Study 5
CHAPTER 2: LITERATURE
REVIEW 7
2.1 Overview
of Artificial Sweetener 7
2.2 Saccharin 8
2.2.1 Properties
of saccharin 9
2.3 Cyclamate 10
2.4 Neotame 11
2.5 Sucralose 12
2.6 Acesulfame-potassium 13
2.7 Aspartame 14
2.7.1 History
of aspartame 15
2.7.2 Metabolism
of aspartame 16
2.7.3 Components
of aspartame 20
2.7.3.1 Aspartic acid 20
2.7.3.2 Phenylalanine 22
2.7.3.3 Methanol 23
2.8 Role
of Oxidative Stress in Inflammation 24
2.9 Antioxidants 25
2.9.1 Superoxide
dismutases (SODS) 26
2.9.1.1 Mechanism of action 27
2.9.2 Catalase 27
2.9.2.1 Mechanism of action 28
2.9.3 Glutathione
reductase 28
2.9.4 Vitamin E (α-tocopherol)
and its antioxidant activity 29
2.9.5 Beta-carotene 30
2.9.6 Vitamin
C 30
2.9.7 L-arginine 32
2.9.7.1 Nitric oxide 33
2.10 Effects
of Aspartame on Human Organs 35
CHAPTER 3:
MATERIALS AND METHODS
3.1 Materials 38
3.1.1 List
of chemicals/reagents used 38
3.1.2 Lists of equipment used 39
3.2 Methods 40
3.2.1 Acclimatization
of animals 40
3 .2.2 Preparation of test agents 40
3.2.3 Experimental
design 40
3.2.4 Liver
and brain tissue homogenization 41
3.2.5 Determination
of thiobarbituric acid (TBAR) in liver and brain
homogenate 41
3.2.6 Determination
of catalase activity 42
3.2.8 Estimation
of superoxide dismutase (SOD) 42
3.2.8 Determination of reduced glutathione
concentration 43
3.2.9 Liver enzyme biomarker 44
3.2.9.1 Assay of aspartate aminotrnsferase (AST)
activity 44
3.2.9.2 Alkaline phosphatase (ALP) 45
3.2.9.3 Assay on alanine aminotransferase (ALT) 45
3.2.10 Total protein estimation 46
3.2.11 Ferric reducing antioxidant power (FRAP) 46
3.2.12 Histopathological
examination 47
3.2.12.1
Tissue preparation 47
3.2.12.2 Slide examination 47
3.2.13 Data analysis 47
CHAPTER 4: RESULTS
AND DISCUSSION 48
4.1 Results 48
4.1.1 Effects
of L-arginine on liver function indices of aspartame-intoxicated
rats 48
4.1.2 Effects
of L-arginine on total protein concentration of serum, liver and
brain
homogenates of aspartame-intoxicated rats 49
4.1.3 Effects
of L-arginine on FRAP status of serum, liver and brain
homogenates
of aspartame-intoxicated rats 50
4.1.4 Effects
of L-arginine on TBARS concentration of serum, liver and brain
of
aspartame intoxicated rats 51
4.1.5 Effects
of L-arginine on catalase activity of serum, liver and brain
homogenates
of aspartame-intoxicated rats 53
4.1.6 Effects
of L-arginine on superoxide dismutase activity of serum, liver
and
brain homogenates of aspartame-intoxicated rats 55
4.1.7 Effects
of l-arginine on reduced glutathione concentration of liver and
brain
homogenates of aspartame-intoxicated rats 56
4.1.8 Histopathology
examination 58
4.1.8.1 Photomicrograph
of rat liver section (H & E, X 100) 58
4.1.8.2 Photomicrograph of rat cerebellum (H & E, X
400) 60
4.2 Discussion 61
CHAPTER 5:
CONCLUSION AND RECOMMENDATIONS 65
5.1 Conclusion 65
5.2 Recommendations 65
References 66
Appendices 78
LIST
OF TABLES
PAGE
3.1 List of chemicals/reagents
used 39
3.2 List of equipment 40
LIST
OF FIGURES
PAGE
2.1 Structure of saccharin 8
2.2 Structure of cyclamate 10
2.3 Structure of sucralose 12
2.4 Structure of acesulfame-potassium 13
2.5 Structure of aspartame 14
2.6 Metabolism of aspartame 16
2.7 Structure of aspartic acid 20
2.8 Structure of phenylalanine 22
2.9 Superoxide dismutase 26
2.10 Chemical structure of
vitamin E 30
2.11 Chemical
structure of vitamin C 32
2.12 L-Arginine Metabolisms 32
4.1 Effects
of L-arginine on liver function indices of aspartame-
intoxicated rats 48
4.2 Effects
of L-arginine on total protein concentration of serum, liver
And
brain homogenates of aspartame-intoxicated rats 49
4.3 Effects
of L-arginine on frap status of serum, liver and
brain
homogenates of aspartame-intoxicated rats 50
4.4 Effects
of L-arginine on TBARS concentration of serum, liver
and
brain of aspartame intoxicated rats 51
4.5 Effects
of L-arginine on catalase activity of serum, liver
and
brain homogenates of aspartame-intoxicated rats 53
4.6 Effects
of L-arginine on superoxide dismutase activity of serum,
liver
and brain homogenates of aspartame-intoxicated rats 55
4.7 Effects
of L-arginine on reduced glutathione concentration of
liver
and brain homogenates of aspartame-intoxicated rats 56
4.8 Effects
of aspartame on cerebellum 60
LIST
OF PLATES
PAGE
4. 1 Photomicrograph
of rat liver section of group 1 rats that received
standard
feed and drinking water only 58
4.2 Photomicrograph
of liver section of aspartame administered rats
(group
2) 58
4.3 Photomicrograph
showing liver section of aspartame treated with
Vitamin
C (group 3) 59
4.4 Photomicrograph
of liver section of aspartame with co-administration
of
L-arginine (20 mg/kg) rats 59
CHAPTER
1
INTRODUCTION
1.1 BACKGROUND OF THE STUDY
Special
attention is paid to sweeteners among food additives, as their utilization
enables both a spontaneous reduction in sugar consumption and a significant
decrease in caloric intake without altering the desirable palatability of foods
and soft drinks (Butchko et al.,
2002). The adverse and toxic effects of some sweeteners upon consumption are
also significant as they react relatively with some food substance.
Aspartame
is the most highly utilized artificial sweetener out of over 6000 products
including beverages and pharmaceuticals (Sahelian, 2016). It is an odorless, highly-sweet,
white, crystalline powder (Magnuson et
al., 2007). Aspartame can be hydrolyzed into its constituent amino acid
under condition of elevated temperature or pH (Sahelian, 2016).
James
Schlatter accidentally discovered the sweetener, aspartame, in his effort to produce
a gastric ulcer drug in 1965 (Stegink, 1987). This sweetener is a methyl ester which
is comprised of natural amino acid; L-aspartic and L-phenylalanine. Hydrolysis
of aspartame by digestive esterases and
peptidases in the intestinal lumen yields two components amino acid and alcohol;
40% of aspartate, an excitatory amino acid, 50% of phenylalanine the precursor
for two neurotransmitters of the catecholamine family, and 10% of methanol
(Yagasaki and Hashimoto, 2008). Production of formaldehyde and later formate
from the oxidation of methanol, results in the liver cell damages. The
concentrations of the metabolites of aspartame in the blood are elevated, subsequent
to its consumption (Stegink 1987).
Food
and Drug Administration (FDA), in 1981, approved the use of aspartame in dry
applications; in 1983, this was followed by approval for its use in carbonated
soft drinks and as a general sweetener in 1996 (Butchko et al., 2002). It is now a worldwide knowledge that aspartame
represents 62% of the value of the intense sweetener marketed as regards to its
world consumption (Butchko et al.,
2002). For instance, in the United State, more than 70% of aspartame sales are associated
with soft drinks (American Dietetic Association, 2004). Ever since aspartame
was approved for use as an artificial sweetener, it has been subjected too much
debate, especially, with its relation to health effects including increase in brain
cancer rates (Olney et al., 1996).
High doses of aspartame have also been reported to produce major neurochemical
changes in rats (Coulombe and Sharma, 1986). In animal studies, the toxic
effect of aspartame has been attributed to the pro-oxidative effects produced
by it (Prokic et al., 2014).
Overproduction
of reactive oxygen species (ROS) by
aspartame has resulted to a significant increase in pro-apoptotic marker (Bax),
as well as decrease in anti-apoptotic marker (Bcl-2) in rats’ brains, which
indicates that aspartame produces an
adverse effect at cellular level (Ashok and Sheeladevi, 2014). Most of them were attributed to the production
of metabolites of aspartame, especially, to methanol metabolites, including
formaldehyde and formate. Hence the FDA and European Food Safety Authority
(EFSA) approved a recommended daily intake (RDI) of 40 mg/kg b.w. /day
(Magnuson et al., 2007).
Currently,
several studies have justified aspartame as a carcinogenic agent and a
neurotoxin which elevates the risk of leukemia, neurological tumours and urinary
tract tumours, even at a low concentration (Soffritti et al., 2005). Aspartame-induced
toxicity leads to induction of free radical (Walaa and Howida, 2015). Free
radicals are harmful substances generated in the body along with toxins and
wastes which are formed during the body’s normal metabolic process. Over
production of the free radicals could be responsible for tissue injury.
Oxidative stress is fundamental to many diseases (Soffritti et al., 2007).
Oxidative
stress could be defined as a condition where there is imbalance between the
antioxidant and free radical generation (ROS) in the body. When this occurs,
the generated free radicals which are unstable atoms with unpaired valence
electrons (Walaa and Howida, 2015), attack bio-molecules in the body
transforming them into free radical like hydrogen peroxide (H2O2),
hydroxyl radical (OH-) and nitric oxide (NO). Reactive oxygen
species are continuously produced during oxidative metabolism in cells. Some
vital organs in the body are very susceptible to ROS, because of their
metabolic rate, vital biochemical functions and high content of oxidizable
substrates. To prevent the effects caused by ROS, organisms have developed multiple
systems of antioxidant defense that are essential for cellular metabolism and
functions (Poljsak and Fink, 2014)
According
to Valko et al. (2007), the term antioxidant could be
defined as a natural or artificial substance that delays or prevents oxidation
of an oxidizable substrate at low doses, when compared to that of the substrate.
They are found in many foods including fruits and vegetables. Antioxidants can
transfer electron to oxidizing agents, thus inhibiting free radical production
and cell damage (Valko et al., 2006).
They could be enzymatic and non-enzymatic. Sources of non-enzymatic
antioxidants are beta carotene, carotene, hypotaurine, glutathione, selenium,
taurine, vitamins C and E, and zinc. Enzymatic antioxidants include catalase,
glutaredoxin, glutathione reductase, and Superoxide dismutase (SOD) (Willcox
and Ash, 2004).
L-arginine
is a semi-essential, basic amino acid that participates in protein, creatine
synthesis, anabolic hormone simulation and nitrogen balance improvement.
L-arginine is a substrate for nitric oxide synthesis (El Mesallamy et al., 2008). The vasodilator tone which
plays a crucial role in the regulation of blood pressure is a consequent of the
synthesis of nitric oxide by vascular endothelium. Nitric oxide is a
neurotransmitter that supports several functions, including the formation of
memory, which occurs in the central nervous system. It also plays an essential
role in the regulation of platelet aggregation and cardiac contractility. These
actions are all stimulated by the soluble guanylate cyclase activation and the
consequent increase in the concentration of cyclic guanosine monophosphate (cGMP)
in target cells (El Mesallamy et al.,
2008).
Vitamin
C is an essential constituent to human health. It is water soluble (causes simultaneous
elimination and prevention of storage) but cannot be synthesized by humans.
There are numerous reasons why vitamin C is paramount to our health, but many
involve its aspect as essential factor in the biosynthesis of carnitine, collagen,
and norepinephrine (Traber and Stevens, 2011). Vitamin C is able to protect the
low density lipoproteins (LDLs), known as the good cholesterols, from being
oxidized. It is also able to decrease damaging due to oxidation in the stomach,
and stimulate the absorption of iron, because of its antioxidant potential (Treber and Stevens, 2011).
Some previous studies indicated that
L-arginine is a protective agent against chronic disease (Lass et al., 2002) by mechanism that may be
mediated via NO. L-arginine together with aspartame may be present in human
diet warranting this study.
1.2 AIM OF THE STUDY
This
study aimed at investigating the effects of L-arginine and vitamin C on some
antioxidant status in serum, liver and brain homogenates of
aspartame-intoxicated rat models.
1.3 OBJECTIVES OF THE STUDY
The
specific objectives of the study were to;
1. To
assess the effects of L-arginine and vitamin C on some oxidative stress markers
in the serum, liver and brain homogenates of aspartame-intoxicated rats.
2. To
assess the effects of L-arginine and vitamin C in the liver enzyme biomarkers
(alanine amino transferase, alkaline phosphatase and aspartate amino
transferase) in the serum aspartame-intoxicated rats.
3. To
assess the effects of L-arginine and vitamin C on histopathological changes of
selected organs (liver and brain) of aspartame-intoxicated rats.
1.4 JUSTIFICATION OF THE STUDY
Many
food production companies worldwide employ several means of enhancing their
products. Some use chemicals (such as aspartame) without knowing their health
implication. Aspartame is added to varieties of products such as beverages and
pharmaceuticals.
Special attention is paid to aspartame among
food additives, as their utilization enables both a spontaneous reduction in
sugar consumption and a significant decrease in caloric intake without altering
the desirable palatability of foods and soft drinks (Butchko et al., 2002). The adverse and toxic
effects of some sweeteners upon consumption are also significant as they react
relatively with some food substance.
Nature
has endowed humans with variety of endogenous antioxidant to counter the effect
of oxidative stress. It has been recorded that Vitamin C and L-arginine possess
antioxidant properties and are able to quench the work of free radicals.
Since
aspartame consumption is on the rise among people, this study will help create
awareness regarding the usage of this artificial sweetener. It will help to
reveal how beneficial the co-administration of aspartame with antioxidant;
vitamin C and l-arginine can be.
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