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Transcript of OKORO, ONYINYECHI RUTH (PG/MSc/07/42487) Ruth.pdf · SUPERVISORS: DR. (MRS) C. A. EZEOKONKWO DR. V....
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OKORO, ONYINYECHI RUTH
(PG/MSc/07/42487)
EFFECT OF DURATION OF STARVATION ON
OXIDATIVE STRESS IN ALBINO RATS
BIOCHEMISTRY
A THESIS SUBMITTED TO THE DEPARTMENT OF BIOCHEMISTRY, FACULTY OF
BIOLOGICAL SCIENCES, UNIVERSITY OF NIGERIA NSUKKA
Webmaster
Digitally Signed by Webmaster’s Name
DN : CN = Webmaster’s name O= University of Nigeria, Nsukka
OU = Innovation Centre
OCTOBER, 2009
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TITLE
EFFECT OF DURATION OF STARVATION ON
OXIDATIVE STRESS IN ALBINO RATS
A DISSERTATION SUBMITTED IN PARTIAL FULFILMENT OF THE
REQUIREMENTS FOR THE AWARD OF THE DEGREE OF MASTER
OF SCIENCE (M.Sc) IN NUTRITIONAL BIOCHEMISTRY,
UNIVERSITY OF NIGERIA, NSUKKA.
BY
OKORO, ONYINYECHI RUTH
(PG/MSc/07/42487) DEPARTMENT OF BIOCHEMISTRY
UNIVERSITY OF NIGERIA
NSUKKA
SUPERVISORS: DR. (MRS) C. A. EZEOKONKWO
DR. V. N. OGUGUA
OCTOBER, 2009.
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CERTIFICATION
Okoro, Onyinyechi Ruth, a postgraduate student of the Department of Biochemistry with the
Reg. No PG/M.Sc/07/42487, has satisfactorily completed her requirement for research work for
the degree of Master of Science (M.Sc) in Nutritional Biochemistry. The work embodied in this
project (dissertation) is original and has not been submitted in part or full for any other diploma
or degree of this or any other university.
DR. (MRS) C. A. EZEOKONKWO DR. V. N. OGUGUA (Supervisor) (Supervisor)
PROF. I. N. E. ONWURAH EXTERNAL EXAMINER (Head of Department)
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DEDICATION
This project work is wholly dedicated to JEHOVAH God who mercifully granted me health,
strength, ability and wisdom to produce it and also I dedicate it to my beloved family members.
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ACKNOWLEDGEMENT
I firstly thank Jehovah God for guiding me throughout this project work. I most sincerely
express my appreciation and gratitude to my project supervisors: Dr (Mrs) Ezeokonkwo, who
encouraged me immensely and Dr. V.N. Ogugua who guided, supported and had a better
understanding towards me and helped in bringing this study to a successful completion. My
unreserved gratitude goes to Prof O. Obioda, Prof L.U.S Ezeanyika, Dr H. A. Onwubiko and
Dr B. C. Nwanguma who showed reasonable level of interest towards the correction and
editing of this work.
I very much appreciate the goodwill and encouragement of all the lecturers and
technological staff of the Department of Biochemistry. I thank the head of department Prof,
Onwurah , Prof. P. Uzoegwu, Mr. P. Egbuna, Prof. F.C. Chilaka, Prof O. F. C. Nwodo, Prof O.
Njoku, Prof I. C Ononogbu, Dr. S.O. Eze, Dr E. O. Alumunah ,Mr. Enechi, and Mr. O.E.
Ikwuagwu for their valuable support to this work.
There is this saying by philosophers “that he who can does it and he who cannot forms a
committee”. No one is an island unto himself. So, I appreciate the work of Dr. V.O Shoyinka of
Department of Microbiology and parasitological, Faculty of Veterinary Medicine, University of
Nigeria, Nsukka (UNN), for the pains he took for the detailed analysis of my histopathological
slides. The assistance given to me by Mr Felix Eze, Dr Parker Elijah Joshua, Mr Chekwube
and Mr Austin Eze are highly appreciated.
More to that, I appreciate the help of my fellow Postgraduate students and colleagues. A
personality whom I will never forget is Ada Umeji, who was always there for me. Again are
Adaeze Akuwudike, and Mrs Osueke.
I also acknowledge the help of NAFDAC, Ministry of Health, Ajayi Crowther
University, Oyo, Safety Diagnostic Laboratory, Animal Research House and Home Science
Animal House, UNN.
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I wish to extend my gratitude to Mr Amadife, Mr C. M. Ude (ESUT) and Mr & Mrs
Benson Eluwa for their assistance .With thanks, I appreciate in a special way the love and
encouragement of my friends: Mr Chinedu Nnamdi Nwanyanwu, Miss Eunice Eze, Mr
Kingdom Nwanyanwu, Mr Collins Egwele, Miss Ifeoma .I. Okafor. In fact, I lack space to
mention their names and I can’t thank them enough.
I thank immensely all my brothers & sisters of Jehovah’s Witness for their support and
prayers.
I also owe gratitude to my Uncles, Aunts, and Brothers-in-law; Mr Lucas Asaba also
Mrs Nwanyanwu for their encouragement when I was almost giving up this study.
My family has been supportive and encouraging. I am indebted to my dear parents: Mr
and Mrs N.E. Okoro and Prof. Sylvester Okoro- my dear Uncle for the time, love and resources
they put to see that the project succeeded. Special thanks go to Mrs Esther Asaba, Jethro
Okoro, Trust Okoro, Joy Okoro and Samuel Okoro for their understanding and cooperation.
Finally, this work was supported by grant from the Sa-tome Principe under Chevron
Company of Nigeria.
It is not easy to forget. Thank you all.
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ABSTRACT
This study was carried out using forty Wistar rats of both sexes and the test groups were
differently starved according to time duration. Blood samples were collected from the rats
through ocular puncture at intervals and was used for the analyses, of blood glucose level, lipid
peroxidation index (MDA), vitamin C, glutathione, total protein and lipid profile. There was no
significant increase (p>0.05) in the body weights of the test animals compared with the body
weights of the animals in control group at 0 hour of the experiment. The concentrations of
MDA, ascorbic acid, glutathione, total protein, total cholesterol, triacylglycerol, HDL and LDL
of animals of the test groups were not significant (p<0.05) compared with the control at 0 hour
of the experiment. However, the glucose concentration increased significantly (p<0.05) in
group 3 animals administered water after starvation compared with the control animals at the 0
hour duration of the study. It also shows significant decrease (p<0.05) in the glucose
concentration of animals (group 4) fed fruit after starvation compared with the animals (group
3) administered water after starvation at 0 hour of the experiment. The results revealed that no
significant difference (p>0.05) in the relative weight of rats in the test groups was observed
when compared with control after 6 to 48 hours. There was a general significant decrease
(p<0.05) of blood glucose concentrations in all test groups compared with the control. There
was no significant change (p>0.05) in the concentrations of total cholesterol of the test group
animals compared with the control at 6 and 12 hours duration, while groups 3 (animals starved
and received water) and 4 (animals starved and received fruits only) had elevated levels of
total cholesterol (p<0.05). In triacylglycerol, a trend of results not significant (p>0.05) was
observed at starvation intervals of 6 to 48 hours when a comparison was made between the test
groups and control. When the high density lipoprotein and low density levels of the test groups
were compared with the control, there was no significant difference (p>0.05). Total protein and
glutathione levels between the experimental test groups and control showed no significant
difference (p>0.05). The elevation of MDA levels in all the test groups when compared with
the control group, had no significant difference (p>0.05). The vitamin C concentrations of the
treated groups (2 and 3) were found to be non-significantly lower when compared with the
control group after 6 to 24 hours of starvation while for 48 hours, a significant decrease
(p<0.05) was seen when compared with group 1. It was observed that the MDA concentration
increased as vitamin C decreased in animals in groups 2 (animals starved of feed and water)
and 3 (animals starved and received water). In all, these recent results suggest that starvation is
characterized by a decreased availability of antioxidants and thus results to oxidative stress-
mediated tissue damage.
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TABLE OF CONTENT
PAGE
Title Page .. .. .. .. .. .. .. .. .. .. i
Certification .. .. .. .. .. .. .. .. .. .. ii
Dedication .. .. .. .. .. .. .. .. .. .. iii
Acknowledgement .. .. .. .. .. .. .. .. .. iv
Abstract .. .. .. .. .. .. .. .. .. .. vi
Table of Content .. .. .. .. .. .. .. .. .. vii
List of Figures .. .. .. .. ... .. .. .. .. .. xi
List of Tables .. .. .. .. .. .. .. .. .. .. xii
CHAPTER ONE: INTRODUCTION
1.1 Starvation and malnutrition … … … … … … … 1
1.2 Oxidative stress in starvation and malnutrition … … … … … 2
1.3 Oxidative stress … … … … … … … … 3
1.3.1 Free radicals and their damage … … … … … … 4
1.3.2 Reactive oxygen species … … … … … … … 4
1.3.3 Chemical and biological roles of reactive oxygen species … … … 5
3.4 Excess production of reactive species … … … … … … 7
1.4 Oxidative stress biomarkers … … … … … … … 7
1.4.1 Malondialdehyde (MDA) … … … … … … … 7
1.5 Lipid peroxidation … … … … … … … … 8
1.5.1 Types of lipid peroxidation … … … … … … … 8
1.5.1.1 Non-enzymatic lipid peroxidation … … … … … … 8
1.5.1.2 Enzymatic lipid peroxidation … … … … … … 11
1.6 Antioxidants … … … … … … … … … 11
1.6.1Glutathione … … … … … … … … … 14
1.6.2 Vitamin C as a chain breaker … … … … … … … 16
1.7 Protein metabolism … … … … … … … … 17
1.8 Cholesterogenesis … … … … … … … … 19
1.8.1 Total cholesterol and starvation … … … … … … 20
1.8.2 Lipoproteins … … … … … … … … 20
1.8.3 Functions of lipoproteins … … … … … … … 21
1.9 Rationale of study … … … … … … … 22
1.10 Research objectives … … … … … … … 22
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CHAPTER TWO: MATERIALS AND METHODS
2.1 Materials … … … … … … … … … 23
2.1.1 Animals … … … … … … … … … 23
2.1.2 Instruments/Equipment … … … … … … … 23
2.1.3 Chemicals/ Reagents/Samples … … … … … … 23
2.1.4 Experimental design … … … … … … … 24
2.2 Methods … … … … … … … … … 25
2.2.1 Lipid peroxidation assay … … … … … … … 25
2.2.1.1 Principle … … … … … … … … … 25
2.2.1.2 Reagents … … … … … … … … … 26
2.2.1.3 Procedure … … … … … … … … … 26
2.2.2 Total cholesterol determination … … … … … … 27
2.2.2.1 Principle … … … … … … … … … 27
2.2.2.2 Procedure … … … … … … … … … 27
2.2.2.3 Calculation … … … … … … … … … 28
2.2.3 High density lipoproteins cholesterol determination … … … … 28
2.2.3.1 Principle … … … … … … … … … 28
2.2.3.2 Procedure … … … … … … … … … 29
2.2.4 Low density lipoprotein cholesterol determination … … … … 29
2.2.4.1 Principle … … … … … … … … … 29
2.2.4.2 Procedure … … … … … … … … … 30
2.2.4.3 Calculations … … … … … … … … 30
2.2.5 Determination of serum triacylglycerol … … … … … 31
2.2.5.1 Reagents … … … … … … … … … 31
2.2.5.2 Procedure … … … … … … … … … 32
2.2.6 Total protein determination … … … … … … … 33
2.2.7 Determination of glutathione (GSH) … … … … … 34
2.2.7.1 Principle … … … … … … … … … 34
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2.2.7.2 Preparation of reagent for glutathione … … … … … 34
2.2.7.3 Procedure … … … … … … … … … 34
2.2.7.4 Preparation of glutathione standard curve … … … … … 35
2.2.8 Determination of vitamin C concentration … … … … … 35
2.2.8.1 Principle … … … … … … … … … 35
2.2.8.2 Reagents for vitamin C … … … … … … … 35
2.2.8.3 Procedure … … … … … … … … 36
2.2.9 Blood glucose determination … … … … … … 36
2.2.9.1 Principle … … … … … … … … … 37
2.2.9.2 Reagents … … … … … … … … … 37
2.2.9.3 Procedure … … … … … … … … … 37
2.2.10 Body weight … … … … … … … … 38
2.3 Statistical analysis … … … … … … … … 38
CHAPTER THREE: RESULTS
3.1 Effect of starvation on body weight of Wistar albino
Rats at various time intervals … … … … … … … 39
3.2 Effect of starvation on mean blood glucose concentrations of Wistar
Albino rats at various time intervals … … … … … … 41
3.3 Effect of starvation on mean malondialdehyle (MDA)
concentrations of Wistar albino rats at various time intervals … 44
3.4 Effect of starvation on mean ascorbic concentrations of
Wistar albino rats at various time intervals … … … … … 46
3.5 Effect of Starvation on mean Glutathione concentrations of
Wistar albino rats at various time intervals … … … 48
3.6 Effect of Starvation on mean total protein concentrations of
Wistar Albino rats at various time intervals … … … … … 50
3.7 Effect of starvation on mean total cholesterol concentrations
of Wistar albino rats at various time intervals … … … … 52
3.8 Effect of starvation on mean triacylghycerol concentrations of
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Wistar albino rats at various time intervals … … … … … 55
3.9 Effect of Starvation on mean high density lipoprotein
concentrations of Wistar Albino rats at various time intervals … … 57
3.10 Effect of starvation on mean low density lipoprotein
concentrations of Wistar albino rats at various time intervals … … 59
CHAPTER FOUR: DISCUSSION
4.1 Suggestions for further research … … … … … … 64
REFERENCES … … … … … … … … 65
APPENDICES … … … … … … … … … 79
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LIST OF FIGURES
Fig 1.1 Mechanism of non-enzymatic lipid peroxidation … … … 10
Fig 1.2 The structure of glutathione … … … … … … 15
Fig 1.3 Schematic representation of neutralization of a
free radical by vitamin C. … … … … … … … 17
Fig 1.4 Structures of steroid and cholesterol … … … … … 19
Fig 3.1 Effect of starvation on body weight of Wistar albino Rats
at various time intervals … … … … 40
Fig 3.2 Effect of starvation on mean blood glucose concentrations
of Wistar albino rats at various time intervals … … … … 43
Fig 3.3 Effect of starvation on mean malondialdehyle (MDA)
concentrations of Wistar Albino Rats at various time intervals …… … 45
Fig 3.4 Effect of starvation on mean ascorbic concentrations of
Wistar albino rats at various time intervals … … … … 47
Fig 3.5 Effect of starvation on mean glutathione concentrations of
Wistar albino rats at various time intervals … … … … 49
Fig 3.6 Effect of starvation on mean total protein concentrations of
Wistar albino rats at various time intervals … … … … 51
Fig 3.7 Effect of starvation on mean total cholesterol concentrations
of Wistar albino rats at various time intervals … … … … … 54
Fig 3.8 Effect of starvation on mean triacylghycerol concentrations of
Wistar albino rats at various time intervals … … … … 56
Fig 3.9 Effect of starvation on mean high density lipoprotein
concentrations of Wistar albino rats at various time intervals …… … 58
Fig 3.10 Effect of starvation on mean low density lipoprotein
concentrations of Wistar albino rats at various time intervals … … 60
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LIST OF TABLES
Table 1.1 Intracellular antioxidants … … … … … 12
Table 1.2 Extracellular antioxidants … … … … … … 12
Table 1.3 Lipoprotein antioxidants … … … … … 13
Table 2.1 Reaction mixture for MDA assay … … … … … 27
Table 2.2 Reagents of low density lipoprotein determination … … … 30
Table 2.3 Serum triacylglycerol determination … … … … 32
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CHAPTER ONE
INTRODUCTION
In their natural environments, most organisms are faced with limited food supplies; the
ability of organisms to withstand food deprivation is therefore critical to their survival (Gray et
al., 2004; Wang et al., 2005). Survival during fasting depends on a number of finely
coordinated hormonal and biochemical adjustments including initial maintenance of blood
sugar by mobilization of stored glycogen (Savendahl and Underwood., 1999).
In general, starvation induces a wide range of responses that alter gene expression,
biochemical activities, physiological and behavioral responses (Wang et al., 2005). Starvation
results in a reduction of body and liver weight (Barthel and Grit, 1998). During starvation
essential metabolic processes are maintained at the expense of accumulated endogenous energy
reserves, which sometimes results in a loss of weight (Steffens, 1989).
It is reported that starvation produces a marked accumulation of ROS and results in cell
death (Lynch et al., 2003; Kang et al., 2003). Therefore, starvation studies may be useful
predictors to determine energetic and metabolic requirement (Guderley et al., 2003). Also, it
has been reported that most of the detrimental effects of food deprivation could be mainly
attributed to the participation of ROS generated under such situation (Robinson et al., 1997;
Domenicali et al., 2001).
1.1 Starvation and Malnutrition
According to the World Health Organization, Starvation and malnutrition constitute the
gravest single threat to the world’s public health. As of 2008, malnutrition continued to be a
worldwide problem particularly in lesser-developed countries (Anonymous,2008). The terms
malnutrition and starvation are used interchangeably when in reality, there are specific
definitions for each. Malnutrition is a general term for a medical condition caused by an
improper or insufficient diet. It most often refers to under nutrition resulting from inadequate
consumption, poor absorption, or excessive loss of nutrients. An extended period of
malnutrition can result in starvation, disease and infection. (Anonymous, 2008).
Starvation refers to the physiologic state that results when food intake is chronically
inadequate. Starvation leads to severe reduction in protein, vitamin and energy intake, and is
the most extreme form of malnutrition (Kalm and Semba, 2005). It is the result of a deprivation
of all food, not just of protein and energy. Its clinical diseases are frequently associated with
deficiencies of micronutrients as well as macronutrients (Bachrach et al., 1991).
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Malnutrition and starvation can be caused by diseases, injuries, the range the animal
lives on or the environmental conditions it lives in. Starvation and malnutrition occur in several
wild life species and routinely eliminate the young, old, weak and sick animals. Malnutrition
involves deficiency of not only the macronutrients –fats, proteins and carbohydrates, but also
results in subphysiological concentration of most micronutrients. Many antioxidant defense
systems depend on micronutrients or are micronutrients themselves (Evans and Halliwell,
2001). Therefore, one would expect a gross derangement of the antioxidant defense
mechanisms in malnutrition. The role of oxidative stress is clear and well known in the
pathogenesis of acquired malnutrition (Tatli et al., 2000).
1.2 Oxidative Stress in Starvation and Malnutrition
Hypoxia, immobilization and starvation are among the stressful physical stimuli applied
to experimental animals. Stress is an adaptive response that prepares the organisms towards
threat. It induces strain upon both emotional and physical endurance which has been considered
a basic factor in aetiology of a number of diseases e.g. cardiovascular diseases, cancer, diabetes
mellitus, etc (Halliwell and Gutteridge, 1984). Most investigations concerning the
influence of prolonged starvation on the metabolic responses in mammals report that the
activity of glucose-degrading enzymes and those of lipogenesis was depressed, whereas the
fatty acids derived from triacylglycerols hydrolysis were preferentially used as fuels through
the corresponding oxidative pathways (Shimeno et al., 1997; Dou et al., 2002; Guderley et al.,
2003).
Present research on starvation in vertebrates is connected with studying leptin that
serves as a mediator of the adaptation to fasting. In humans, serum leptin concentrations as well
as plasma levels of metabolic parameters (glucose, cholesterol, lipids) change rapidly after
short term starvation (Boden et al., 1996).
It is known that deprivation of energy supply induces a delay in the development of
some vital functions in mammals: puberty starts later, the reproductive age prolongs, and
ageing starts later and deterioration of immunity and health is delayed (Banks and Lebel,
2002).
Oxidative metabolism of cells is a continuous source of reactive oxygen species (ROS),
resulting from univalent reduction of O2, which can damage most cellular components leading
to cell death. Under severe conditions, the rate of generation of ROS exceeds that of their
removal and oxidative stress occurs (Sies, 1986; Di Giulio et al., 1995; Halliwell and
Gutteridge, 2000; Livingstone, 2001). In this sense, starvation has been reported to have pro-
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oxidant effects in mammals.It is during starvation that increased ROS generation is not
adequately neutralized by antioxidant systems (Robinson et al., 1997; Domenicali et al., 2001).
The pathogenesis of oedema and anaemia commonly found in children with protein
energy malnutrition has been suggested to be caused by an imbalance between the production
of free radicals (Ashour et al., 1999).
1.3 Oxidative Stress
The body has a hierarchy of defense strategies to deal with oxidative stress within
different cellular compartments (Gutteridge, 1995). The disturbance of the balance between the
production of ROS and antioxidant defenses against ROS produces oxidative stress. Therefore,
oxidative stress is the imbalance or the disturbance between antioxidants and pro-oxidants
status in favour of pro-oxidants leading to potential damage (Sies, 1991; Betteridge, 2000).
This imbalance can be an effect of endogenous antioxidants or their low dietary intake and /or
increased formation of free radicals and other reactive species (Sordergren, 2000). Oxidative
stress is considered a possible molecular mechanism involved in toxicity (Moreira. et al.,
2001).
1.3.1 Free Radicals and Their Damage
Stressful conditions lead to formation of excessive free radicals which are a major
internal threat to cellular homeostasis of aerobic organisms (Yu, 1994). Free radicals are
formed in human body both in physiological and pathological conditions in cytosol,
mitochondria, lysosomes, peroxisomes and plasma membranes (Hemnani, and Parihar, 1998).
These free radicals are extremely reactive and highly unstable chemical species, which
react with proteins, lipids, carbohydrates and nucleic acids in the body causing damage
(Sevanian, and Hochstein, 1985). They have been implicated in many diseases such as cancer,
diabetes, hypertension, etc (Tisan et al., 1995). Organic free radicals also are formed via
reduction and also by oxidation reactions of several compounds (Younes, 1999).
In living cells, the biological effects of free radicals are controlled by nonenzymic
antioxidants such as glutathione, tocopherols, ascorbic acid and carotenoids (Akkus et al.,
1996). A free radical overload damages many cellular components: cellular proteins, DNA and
membrane phospholipids (Patockova et al., 2003). These free radicals are fundamental to any
biochemical process and represent an essential part of aerobic life and our metabolism (Tiwari,
2001).
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Oxidative damage to DNA, proteins and lipids can ultimately lead to results like
disorganization, dysfunction and destruction of membranes, enzymes and proteins, (Slater,
1984; Halliwell, 1994; Halliwell, 1997). Specifically, per oxidation of membrane lipids may
cause impairment of membrane bound receptors and enzymes, increased permeability to ions
and possibly eventual membrane rupture (Gutteridge and Halliwell, 1990; Gutteridge, 1995). If
the oxidative stress is particularly severe, it can produce cell death (Dypbukt et al., 1994;
Halliwell, 1997).
1.3.2 Reactive Oxygen Species
Oxygen exists in air as a molecule (O2) known as dioxygen or molecular oxygen
(Gutteridge; 1995). Oxygen is required by all living organisms for their survival (Charttejea
and Shinde, 2005). This same oxygen however, is potentially toxic at high concentrations,
giving rise to reactive oxygen species, ROS (Halliwell, 1991; WUD and Cederbaum, 2003;
Charttejea and Shinde, 2005).
The term “Reactive Oxygen Species (ROS)” collectively describes free radicals such as
O2. , OH
. and other non-reactive oxygen derivatives such as hydrogen peroxide (H2O2), Singlet
oxygen, O3, hypochlorous acid (HOCl) (Halliwell, Guterridge, 2006). These ROS are generated
by metabolic processes and their concentrations can be increased by environmental stimuli.
Reactive Oxygen Species are constantly produced during normal aerobic metabolism
and are safely removal by a variety of biological antioxidants (Chance et al., 1979). To prevent
ROS from damaging cellular components, organisms have evolved multiple detoxification
mechanism e.g super oxide dismutase and glutathione peroxidase:
2O2.-
+ 2H+ Superoxide Dismutase (SOD) H2O2 + O2
ROOH + 2GSH Glutathione Peroxidase ROH + H2O + GS-SG
1.3.3 Chemical and Biological Roles of Reactive Oxygen Species
From an environmental perspective, photochemical reactions involving reactive oxygen
species are attractive for cleaning up pollution given that many ‘self repair’ processes in the
atmosphere and natural waters are driven by light (Rajeshwar, 1996).
Hypochlorous acid is produced by the neutrophil-derived enzyme myeloperoxidase at
sites of inflammation and when activated neutrophils infiltrate reoxygenated tissue (Hazen et
al., 1996). Hypochlorous acid is a potent chlorinating and oxidizing agent (Weiss et al., 1983).
xviii
It attacks many other biological molecules like primary amines and sulfhydryl (SH) groups in
proteins and chlorinates purine bases in DNA (White man et al., 1997).
The hydroxyl radical (OH.) is a highly reactive oxygen-centred radical with an
estimated half life in cells of only 10-9
s. Hydroxyl radical attacks all proteins, DNA,
polyunsaturated fatty acids in membranes and almost any biological molecule it touches. In the
case of ∙OH generation by fenton-type chemistry (Koppenol, 1993), the extent of
∙OH
formation is largely determined by the availability and location of the metal ion catalyst.
The Fenton and Herber Weiss reactions propose a mechanism for the formation of ∙OH
in biological system (Koppenol, 2001).
Fe2+
+ H2O2 Fe3+
+ OH + OH (Fenton reaction)
2O-2 + 2H
+ 2H2O2 + O2
This can be spontaneous or catalyzed by superoxide dismutase
∙O2
- + Fe
3+ Fe
2+ + O2
Fe2+
+ H2O2 Fe3
+ + OH
- + OH
.
Net reaction
∙O2
.- + H2O2
∙OH
- OH
– + O2
Reactive Oxygen Species play important roles in cell signaling, a process termed redox
signaling (Schafer and Buettner, 2001). Thus, to maintain proper cellular homeostasis, a
balance must be struck between reactive oxygen production and consumption. ROS such as
superoxide anion, hydroxyl radicals and H2O2 are unwanted and toxic by products formed
during aerobic metabolism. ROS can cause cell death via apoptosis and/ or necrosis in many
cell types, which can be blocked or delayed by various antioxidants and antioxidative proteins/
enzymes (Carmody and Cotter, 2001; Kim et al., 2001; Jang and Surh, 2003). Ozone is a pale
blue gas, which is not produced in vivo. It serves as an important protective shield against solar
radiation in the atmosphere. Close to the earth’s surface, O3 is an unwanted oxidant and is often
regarded as the most toxic air pollutant (Mustafa, 1990). The tissue most susceptible to damage
upon exposure to O3 is the lung. The biological effect of O3 is often attributed to its ability to
cause oxidation or peroxidation of biomolecules either directly or via free-radical mechanisms
(Pryor, 1994).
xix
1.3.4 Excess Production of Reactive Species
Reactive Oxygen Species (ROS) are associated with tissue damage and are the
contributing factors for inflammation, aging, cancer, arteriosclerosis, hypertension and diabetes
(Uzoegwu, 2001; Nwanjo and Oze, 2007; Ogugua and Alumanah, 2007).
Oxidative stress is as a result of the mismatched equilibrium between the production of
ROS and ability of the cells to defend against ROS. Overproduction of ROS results in oxidative
stress which is an important mediator of damage to cell structures (Carnelio et al., 2007).
The respiratory Chain has been reported to be a main intracellular source of ROS
(Ramasarma, 1982; Lenaz, 1998), since under physiological condition, 1-4% of oxygen
reacting in the respiratory chain is incompletely reduced to superoxide radical (Tiidus and
Houston, 1994; Lee et al., 1997). Therefore, under a situation that enhances oxidative
metabolism, it may be expected that an increase in both ROS generation and ROS-scavenging
mechanisms occur.
1.4 Oxidative Stress Biomarkers
Some parameters (Malondialdehyde, antioxidant enzymes like superoxide dismutase,
catalase and Glutathione peroxidase) have been described as valuable biomarkers of pro-
oxidant situations in mammals (Robinson et al., 1997; Gomi and Matsuo, 1998; Domenicali et
al., 2001).
Among different markers of oxidative stress, malondialdehyde (MDA) and the natural
antioxidants, metaloenzymes Cu, zn-superoxide dismutase (Cu, Zn-SOD) and selenium
dependent glutathione peroxidase (GSHPx), are currently considered to be the most important
(Guichardant et al., 1994).
1.4.1 Malondialdehyde (MDA)
Malondialdehyde is a three carbon compound formed from peroxidized polyunsaturated
fatty acids, mainly arachidonic acid. MDA is also an acytotoxic aldehyde. It is considered as a
valuable indicator of oxidative damage of cellular components.
MDA is one of the end products of membrane lipid peroxidation and the extent of lipid
peroxidation is measured by estimating MDA levels more frequently (Draper et al., 1986).
MDA levels are increased in various diseases with excess of oxygen free radicals , (Ohkawa et
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al., 1979, Guichardant et al., 1994). Increased MDA levels is also reported in stress models of
rabbits like starvation stress and ischaemic limb injury in rabbits (Hem lata et al., 2002).
1.5 Lipid Peroxidation
Exposure of lipids in cell membrane to free radicals stimulate the process of lipid
peroxidation (Halliwell and Gutteridge, 1984). The products of lipid peroxidation are
themselves reactive species and lead to extensive membrane, organellar and cellular damage
(Cotran et al., 1999). The free radical activity and the extent of tissue damage are related
qualitatively to the amount of lipid peroxide level in the blood (Yagi, 1987).
Lipid peroxidation is important in vivo for the stability of processed foods. It
contributes to the development of cardiovascular diseases such as pre-eclampsia and
atherosclerosis, and MDA being its end product can cause damage to proteins and DNA.
Peroxidation causes impairment of biological membrane functioning, for example, it decreases
fluidity, inactivates membrane bound enzymes and receptors and may change non-specific
calcium permeability (Orrenius et al.; 1989, Bast, 1993).
Lipid peroxidation is a source of free radicals. It is probably the most extensively
investigated free radical-induced process (Gutteridge and Halliwell, 1990). The detection and
measurement of lipid peroxidation is the evidence mostly frequently cited to support the
involvement of free-radical reactions in toxicology and disease.
1.5.1 Types of Lipid Peroxidation
Lipid peroxidation can be non-enzymatic and enzymatic (Sodergren, 2000).
1.5.1.1 Non Enzymatic Lipid Peroxidation
Polyunsaturated fatty acids (PUFAS) are particularly susceptible to peroxidation and
once the process is initiated, it proceeds as any other free radical mediated chain reaction
involving initiation, propagation and termination. Initiation of lipid peroxidation is caused by
the attack of any species that has sufficient reactivity to abstract a hydrogen atom from a
methylene group of a PUFA (More and Reberts, 1998; Halliwell and Gutteridge, 1999; De
Zwart et al., 1999). The carbon-centred radical produced is stabilized by a molecular
rearrangement to form a conjugated diene, followed by reaction with oxygen to give a peroxyl
radical. Peroxyl radicals are capable of abstracting a hydrogen atom from another adjacent fatty
acid side chain to form a lipid hydroperoxide, but can also combine with each other or attack
membrane proteins. When the peroxyl radical abstracts a hydrogen atom from fatty acid, the
xxi
new carbon-centred radical can react with oxygen to form another peroxyl radical, and so the
propagation of the chain reaction of lipid peroxidation continues (Gutteridge, 1995).
Loss of H to a free radical
Molecular rearrangement
-H
+O2 Uptake of oxygen
PUFA
RCarbon
centred
radical
R
Conjugated
diene
ROO
Peroxyl
radical
xxii
(Gutteridge, 1995)
1.5.1.2 Enzymatic Lipid Peroxidation
By virtue of the processes that are enzymatically catalyzed, peroxidation of PUFAs can
also occur. Enzymatic lipid peroxidation should refer only to the generation of lipid
hydroperoxides at the active site of an enzyme (Gutteridge, 1995). Free radicals are probably
important intermediates in the enzymatically-catalysed reaction, but are localized to the active
sites of the enzyme.. The hydroxides and endoperoxides produced from enzymatic lipid
peroxidation becomes stereo specific and have important biological functions when they are
converted to stable active compounds. During the formation of endoperoxides by
cyclooxygenase, a powerful oxidant is generated that is amenable to scavenging by some
antioxidants (Kuehl and Egan, 1980).
1.6 Antioxidants
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An antioxidant may be defined in a number of ways. For example, as a substance which
when present at low concentrations compared with those of an oxidizable, substance, such as
fats, proteins, carbohydrates or DNA, significantly delays or prevents the oxidation of the
substrate (Halliwell, 1990). Acidic compounds (including phenols) usable in foods which can
readily donate an electron or a hydrogen atom to a peroxyl or alkoxyl radical to terminate a
lipid peroxidation chain reaction or to generate a phenolic compound, or which can effectively
chelate a pro-oxidant transition metal (Sies et al., 1992).
A compound might exert antioxidant reactions in vivo or in food by inhibiting
generation of ROS, or by directly scavenging free radicals. Additionally, in vivo, an antioxidant
might act by raising the levels of endogenous antioxidant defense (e.g. by up regulating
expression of the genes encoding SOD, catalase or glutathione peroxidase).
There are a number of antioxidants present in the body and derived from the diet
(sodergren, 2000). Based on their location in the body they can be divided into intracellular and
extracellular antioxidants and membrane and lipoprotein antioxidants (Rice and Burdon, 1993;
Chaudiere and Ferrari-Iliou, 1999;Gutteridge, 1995).
Super oxide dismutase (SOD), catalase and glutathione peroxidase constitute
intracellular antioxidant enzymes that converts free radicals (Superoxide anion radicals, and
H2O2) to less reactive forms in the body (Janzen, 1990;Rice and Burdan, 1993, Halliwell et al.,
1995;). Below is a table showing antioxidant activities.
Table 1.1: Intracellular antioxidants
Superoxide dismutases (Cu, Zn, Mn) Remove O2 catalytically.
Catalase; contains 4 NADPH molecules
(Fe)
Removes H2O2 when present in high
concentrations.
Glutathione peroxidase (Se) Removes H2O2 when present at low steady-
state concentrations; can remove organic
hydroperoxides.
Prevention of O2, H2O2, OH; formation by
cytochrome oxidase (Cu)
No release of active oxygen’s during
reduction of O2 to H2O
(Gutteridge, 1995).
Table 1.2: Extracellular antioxidants
Transferrin Binds ferric ions (2 per mole of protein)
Lactoferrin Binds ferric ions at lower pH (2 per mole of protein)
Haptoglobins Bind hemoglobin
Hemopexin Binds heme
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Albumin Binds copper, heme and scavenges HOCl
Ceruloplasmin Ferroxidase activity-stoichiometric O2 scavenging, binds copper ions
(nonspecific), utilizes H2O2 for reoxidation of coppers
EC-SOD
Removes O2 catalytically
EC-GSHPx Removes H2O2 and hydroperoxides catalytically. Little GSH available
in plasma.
Bilirubin Scavenges peroxyl radicals (<0.09µ mol/L)
Mucus Scavenges OH radicals
Urate Radical scavenger and metal binder (0.08 µ mol/L)
Glucose OH radical scavenger (4-6 mmo/L)
Ascorbic acid OH radical scavenger (65 µ mol/L)
(Gutteridge, 1995).
Table 1.3: Lipoprotein antioxidants
Lipoprotein Antioxidants
Vitamin E
Β-Carotene
Retinyl Stearate
Lycopene
Erythrocytes Diffusion of H2O2 into the cell and passage
of O2 through the anion channel
(erythrocytes contain catalase and SOD)
(Gutteridge, 1995).
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Extracellular antioxidants include proteins (transferrin, lactoferin, albumin,
ceruloplasmin) and urate sequesters transition metals by chelation. Other scavengers of free
radicals include albumin and bilirubin (Gutteridge, 1995; Rice and Burdon, 1993).
Antioxidants from dietary sources include fat soluble vitamins E and carotenoides as
well as water soluble vitamin C (Burton and Ingold, 1989; Gutteridge, 1995; Halliwell et al.,
1995; Halliwell, 1996). Others include flavonoids and other plant phenolics, taurine and alpha
lipolic acid (Gate et al., 1999).
1.6.1 Glutathione
Glutathione is a ubiquitous antioxidant (GSH). It is a small protein molecule found in
almost every cell. Glutathione cannot enter most cells directly and therefore must be made
inside the cell from its three constituent amino acids: glycine, glutamate and cysteine.
Furthermore, the cysteine molecule has a sulphur containing portion which gives the whole
glutathione molecule its biochemical activity, that is its ability to carry out the vitally important
functions of glutathione (Lomaestro and Malone, 1995) In normal livers prolonged fasting is
known to affect the antioxidant capacity of the cell (Martensson, 1986) because of the lack of
cysteine and the precursor amino acids for the glutathione (GSH) synthesis (Shimizu and
Morita, 1992). Total GSH was reduced after 18 hours of starvation by 39% in mouse liver (Di
Simplicio et al., 1997). The term glutathione is typically used as a collective term to refer to the
tripeptide L-gamma-glutamy L-cycteinylglycine in both its reduced and dimeric forms.
xxvi
Monomeric glutathione is also known as reduced glutathione and its dimmer is also known as
oxidized glutathione, glutathione disulfide and diglutathione.
Glutathione is widely found in all forms of life and plays an essential role in the health
of organisms, particularly aerobic organisms. In animals, including humans, and in plants,
glutathione is the predominant non-protein thiol and function as a redox buffer, keeping with its
own SH groups those of proteins in a reduced condition (Sies, 1999).
N
O
HO
NH2
SH
HOH
O O
N
H
O
Fig. 1.2: The structure of glutathione
Glutathione is present in tissues in concentrations as high as one millimolar. It plays
roles in catalysis, metabolism, signal transduction, gene expression and apoptosis (Anderson et
al., 1991). It is a cofactor for glutathione S-transferase, an enzyme involved in the
detoxification of xenobiotics, including carcinogenic genotoxicants and for glutathione
peroxidases,which are crucial selenium-containing antioxidant enzymes. It is also involved in
the generation of ascorbate from its oxidized form, dehydroascorbate. (Samiec et al., 1998).
Glutathione is present in the diet in amounts usually less than 100 milligrams daily. The liver is
the principal site of glutathione synthesis. In healthy tissues, more than 90% of the total
glutathione pool is in the reduced form and less than 10% exists in the disulfide form. The
enzyme, glutathione disulfide reductase is the principal enzyme that maintains glutathione in its
reduced form. The latter enzyme uses as its cofactor NADPH (reduced nicotinamide adenine
dinucleotide phosphate) (Griffith, 1999). The consequences of a functional glutathione
deficiency, which results in tissue oxidative stress, can be seen in some pathological conditions.
For example, those with glucose 6-phosphate dehydrogenase deficiency produce lower
amounts of NADPH and hence, lower amounts of reduced glutathione. Oxidative stress caused
by glutathione deficiency results in fragile erythrocyte membranes(Hayes and Mclellan,1999).
xxvii
Chronic functional glutathione deficiency is also associated with immune disorders, an
increased incidence of malignancies. Depletion of glutathione in the hepatocytes, leads to liver
failure and death, if not promptly treated (Hayes and Mclellan, 1999).
1.6.2 Vitamin C as a Chain Breaker
Vitamin C is one of the most ubiquitous vitamins ever discovered. It is perhaps the most
popular vitamin among the common nutrients. Vitamin C also known as ascorbic acid is a
water soluble vitamin found in plants and animals alike (Iqbal et al., 2004). Ascorbic acid
(C6H8O6) is a white to light-yellow crystalline sugar (similar to that of the sugar L-glucose) that
naturally occur in chemical forms of L-xyloscorbic acid and D-xyloarscorbate (Iqbal et al.,
2004; Anonymous, 2008). The L-enantiomer (form) of this acid is commonly known as
vitamin C (Charttejea and Shinde, 2005; Anonymous, 2008). Most animals and probably all
plants can synthesize vitamin C but it is required in the diet of humans and a few other
vertebrates (Anonymous, 2008).
Dietary sources of vitamin C are fruits and vegetables such as citrus fruits (e.g. orange,
lemon, lime) and other fruits like pawpaw, pineapple, banana, strawberry, leafy vegetables,
like cabbage, cauliflower, green peppers, red peppers, broccoli and turnip (Iqbal et al., 2004;
Chattejea and Shinde, 2005).
The biological functions of vitamin C are numerous, but the main biochemical role
played by ascorbic acid is related to its characteristic reducing ability (Padh, 1990; Chaney,
1992). Its importance is reflected in its involvement in several enzymatic hydroxylation
reactions and enzymatic reactions.
In addition to this, vitamin C plays a vital role as an antioxidant. It has been known to
interact with free radicals, an important biological function that leads to the destruction of the
radicals derived from oxygen (Rose and Bede, 1993). The critical role of vitamin C in
ameliorating the adverse effects of reactive oxygen and nitrogen radicals has been well
established (Kelly, 1998). In addition, numerous epidemiological studies strongly support the
protective role of vitamin C in decreasing the incidence of chronic diseases like atherosclerosis
where oxidative stress caused by excessive oxygen or nitrogen radicals may play a causal role
(Mayne,2003).
Furthermore, vitamin C has been shown to facilitate iron absorption by its ability to
reduce inorganic ferric iron to the ferrous form (Padh, 1990; Iqbal et al., 2004; Chattejea and
Shinde, 2005). Ascorbic acid plays a role in the synthesis of the aminoacids carnithine and the
xxviii
catecholamines that regulate the nervous system (Gaby and Singh, 1991; Chatterjea and
Shinde, 2005) as well as in tryptophan, tyrosine, folic acid and cholesterol metabolism (Rath,
1993; Chatterjea and Shinde, 2005). Ascorbic acid is a chain breaking antioxidant. When
vitamin C acts as a chain breaker, it deactivates highly reactive ascorbyl free radicals by
donating one electron leading to the formation of a less reactive ascorbic free radical,
quenching the reactive species. The ascorbyl free radical can be regenerated to ascorbic acid or
oxidized to dehydroascorbic acid.
R + ASC R + AFR
ASCDHAA
Fig 1.3: Schematic representation of neutralization of a free radical by vitamin C
(R = free radical species AFR = Ascorbyl free radical, ASC = Ascrobic acid and DHAA =
dehydroascorbic acid). (source: Sodergren,2000).
1.7 Protein Metabolism
During digestion, proteins are hydrolysed into amino acids, which are then absorbed by
the capillaries of villi and enter the liver via the hepatic portal vein. Proteins, especially from
skeletal muscle, supplies most of the carbon needed for net glucose synthesis. Proteins are
hydrolysed within muscle cells and most amino acids are partially metabolized. Before amino
acids can be catabolised, they must be converted to substances that can enter the TCA cycle.
These conversions involve deamination, decarboxylation, and hydrogenation.
Protein anabolism involves the formation of peptide bonds between amino acids to
produce new proteins. Protein synthesis is carried out on the ribosomes of almost every cell in
the body, directed by the cells ‘DNA and RNA’ (Nelson and Cox, 2005).
Proteins are essential for maintaining lean body mass. Protein breakdown continues
during periods of stress or trauma and it may be minimized by provision of dietary protein
(Collier et al., 1996).
In semistarvation or starvation, it has been reported that plasma proteins and amino
acids are normally unchanged or slightly increased (Kekwick and Pawan, 1957). For instance,
xxix
its average of increase was from 6.8 to 7.3g/ 100 ml and returned to a normal of 6.6g/100ml
after 4 days of rehabilitation in humans.
Infectious illness and starvation are associated with a protein catabolic state
(Wannemacher, 1975). In a fed rat, protein catabolism is not constant throughout the day but
appears to be high during the dark hours when the rat has access to food and low during the
light hours when the rat is inactive and fasting. Thus, starvation is associated with an
increase in protein catabolic rate and a gradual loss in periodicity (Wannemacher, et al., 1997).
The mechanism by which the body regulates protein stores is through alterations in
protein synthesis and breakdown. In animal models, with as little as 12hours without food, the
rate of muscle protein synthesis falls although this can be reversed within 1 hour of refeeding
(Nurlan and Garlick, 1989). Although protein breakdown may increase initially (Young and
Marchini, 1990) there is eventually a decrease in protein breakdown in protein deficient rats
(Hoerr et al., 1993).
With decreased protein intake, protein synthesis and breakdown eventually fall so that
the body can reattain balance (Hoerr et al., 1993; Yang , 1986).
During protein deprivation in rats, both rates of protein turnover (synthesis and
degradation) decrease as the length of time in starvation decreases (Mortimore and Poso, 1987).
For example, in one study rats were given an essentially protein free-diet. After 1 day, protein
synthesis had dropped by 25-40%. After 3 days, protein breakdown and oxidation had
decreased by 30-45%.
During starvation in rats, the first proteins to be lost are from the liver, with 25-40% of
liver protein being lost after 48 hours (Kettelhut et al., 1988, Mortimore and Poso, 1987).
Furthermore, not only does the body appear to first replete those proteins which are first lost
(liver and other organ proteins) but by the time those proteins are repleted, the body has
readapted to the current level of protein intake. In all likelihood, the net result of protein
cycling will be no change in total body protein stores.
1.8 Cholesterogenesis
Sterols are structural lipids present in the membranes of most eukaryotic cells. Their
characteristic structure is the steroid nucleus consisting of four fused rings, three with six
carbons and one with five.
xxx
HO
H3C
CH3
C
H
C
H
H
CH3
H3C
C
H
H
C
CH3
C
H
H
H
Cholesterol
3
2
1
45
6
7
8
910
11
1213
1415
16
17
Steroid
Fig 1.4: Structures of steroid and cholesterol
Cholesterol, the major sterol in animal tissues, is amphipathic, with a polar head group
(the hydroxyl group at C-3) and a non- polar hydrocarbon body (the steroid nucleus and the
hydrocarbon side chain at C-17) about as long as a 16-carbon fatty acid in its extended form.
Similar sterols are found in other eukaryotes: stigmasterol in plants and ergosterol in fungi, for
example. The sterols of all eukaryotes are synthesized from simple five-carbon isoprene
subunits, as are the fat-soluble vitamins, quinones and dolichols (Nelson and Cox, 2005)
Cholesterol is probably the bestknown steroid because of its association with
atherosclerosis. However biochemically, it is also significance because it is the precursor of a
large number of equally important steroids which include the bile acids, adrenocortical
hormones, sex hormones, D Vitamins, cardiac glycosides, sitosterols of the plant kingdom, and
some alkaloids (Mayes, 2000).
Body cholesterol is from both exogenous and endogenous sources. Approximately half
cholesterol of the body comes from its biosynthesis (about 500mgldl) which takes place mainly
in the liver (about 50% of total synthesis) also in the gut, which accounts for about 15% and the
skin for large proportion of the remainder (Mayes, 2000).
Exogenous source of cholesterol is mainly from the diet especially food of animal
origin. Thus, food like egg yolk, cream full fat milk, cheese, liver, kidneys and prawns are
source of cholesterol. However, also food high in saturated fat may lead to an increase in
synthesis of cholesterol by the liver of varying degree with susceptibility of the individual and
genetic factor playing important roles.
xxxi
Cholesterol is transported in lipoproteins, mainly low density lipoproteins (LDL)
known as ‘bad’ cholesterol since excessive amount of it increases the risk of heart disease.
High density lipoproteins (HDL) are referred to as ‘good’ cholesterol since high amount of
HDL are associated with reduced risk of heart disease.
1.8.1 Total Cholesterol and Starvation
Complete fasting is accompanied by substantial lipolysis (Samra et al., 1996, Vaisman
et al., 1990). The report of (Savendahl and Underwood, 1999) shows that in normal weight
subjects, increased serum cholesterol associated with the amount of weight loss was observed
between 2days to 1wk fasting. Food deprivation can cause a shift from lipogenesis to lipolysis
increased fatty acid turnover and reduction in protein anabolism (Buyse et al., 2002).
1.8.2 Lipoproteins
Lipoproteins are complex aggregates of lipids and proteins that render the lipids
compatible with the aqueous environment of body fluids and enable their transport throughout
the body of all vertebrates and insects(Anonymous,2009). They are synthesized mainly in the
liver and intestine. Lipoprotein aggregates are described in terms of the different protein
components or apoproteins (apolipoproteins), as these determine the overall structures and
metabolism (Jonas,2002). They are not able to cross the blood-brain barrier (Jonas, 2002).
In addition to free fatty acids (FFA), four major groups of lipoproteins based on the
relative densities of the aggregates on ultracentrifugation have been identified , which are
important physiologically and in clinical diagnosis.
These are:
Chylomicrons (CM), derived from intestinal absorption of triacylglycerol; Very low
density lipoproteins (VLDL, or pre β-lipoproteins), derived from the liver for the export of
triacylglycerol; Low-density lipoprotein (LDL, or β-lipoproteins) representing a final stage in
the catabolism of VLDL, and High-density lipoproteins (HDL; or α-Lipoproteins) are involved
in VLDL and chylomicron metabolism and also in cholesterol transport (Vance, 2002).
Triacylglycerol is the predominant lipid in chylomicrons and VLDL, whereas
cholesterol and phospholipids are the predominant lipids in LDL and HDL, respectively. In
addition to the use of techniques depending on their density, lipoproteins may be separated
according to their electrophoretic properties into α-, β- and pre –β-lipoproteins and may be
identified more accurately by means of immunoelectrophoresis (Murray et al., 2003).
xxxii
1.8.3 Functions of Lipoproteins
Triacylglycerols: These are blood fats. Triacylglycerols are the most energy-dense
molecules available to the body as a source of fuel. They tend to contain a high proportion
of saturated and monoenoic fatty acids (Skipski,1972). Some suggest that high
triacylglycerol (triglyceride) levels might increase the risk of heart disease.
Very Low Denisty Lipoproteins (VLDL): These are the combination of cholesterol,
proteins and fats (including triacylglycerides). These particles got their name from the
relatively low weight or density of their protein. Most of the plasma VLDL is of hepatic
origin (Fainaru et al.; 2002). They are the vehicles of transport of triacylglycerol from the
liver or intestines to the extra-hepatic tissues. VLDL serve to buffer the plasma free fatty
acids released following lipolysis in adipose tissue in excess of the requirements of muscle
and liver (Anonymous, 2009).
Low Density Lipoproteins (LDL): These are small particles containing mostly cholesterol
and proteins. Many are removed from the blood stream by cells throughout the body, used
for essential body functions. Some people’s systems remove LDL more slowly than others,
which cause high blood LDL. Low-density lipoproteins build up in their blood with a
tendency to deposit the cholesterol and other fatty substances in the walls of the arteries
(Anonymous,2009). Higher concentrations of LDL cholesterol have been associated with
increasing severity of cardiovascular disease (Havonoja, 2000).
High Density Lipoproteins (HDL): These act as scavengers in the blood stream, attracting
cholesterol and carrying it back to the liver, where it is either reprocessed in new VLDL or
broken down into substances called bile acids and removed from the body. HDL helps to
reduce the amount of cholesterol that is present in the blood. The higher the HDL level, the
less the risk of developing heart disease-atherosclerosis (Brown, 2007). HDL is synthesized
and secreted from both liver and intestine. A major function of HDL is to act as a repository
for apo C and apo E that are required in the metabolism of chylomicrons and VLDL
(Murray et al., 2003)
1.9 Rationale of Study
Voluntary fasting is practiced by many humans in an attempt to lose body weight. Food
deprivation induces a delay in the development of some vital functions in mammals, producing
accumulation of ROS mediated Oxidative stress (Domenicali et al., 2001).
xxxiii
With the above observations in mind, the present study is aimed at evaluating the effect
of short starving periods (6, 12, 24, 48 hours) on the oxidative stress parameters of rats.
1.10 Research Objectives
This study was primarily designed to determine:
1) The effect of starvation on lipid peroxidation using malondialdehye (MDA) as an index.
2) The biochemical effect of starvation on serum lipid profile of rats.
3) The effect of starvation on total protein level in rats.
4) The antioxidant status of rats during starvation using antioxidants such as vitamin C and
glutathione.
5) The effect of starvation on blood glucose and body weight.
CHAPTER TWO
MATERIALS AND METHODS
2.1 MATERIALS
2.1.1 Animals
The experimental animals used for this study were Wistar albino rats of both sexes. The
average age of the rats was 12 weeks old. The rats were obtained from the animal house of the
Faculty of Biological Sciences, University of Nigeria, Nsukka (UNN). The animals were
acclimatized for two weeks under standard laboratory conditions. They were housed in wire-
meshed cages at ambient temperature with 12 hour day–night cycle and fed with commercial
rat chow (pelletised growers feed) and water ad libitum.
2.1.2 Instruments/Equipment
Water Bath (DK 8A) Gallenkamp, England
Chemical Balance (MB-1610) Gallenkamp, England
Centrifuge (C1415; 3,500 rpm) PIC, England
Refrigerator Kelvinator, Germany
Digital Photocolorimeter EI (312 Model), Japan
Microscope Slides Unescope, U.S.A
Micropipette Perfect, U.S.A
One Touch Ultraglucometer Lifescan, U.S.A
xxxiv
2.1.3 Chemicals/Reagents/Samples
All chemicals used in this study were of analytical grade and products of May and
Baker, England; BDH, England and Merck, Darmstadt, Germany. Reagents used for all the
assays were commercial kits and products of Randox, USA; QCA, Spain; Teco (TC), USA;
Biosystem Reagents and Instruments, Spain. Blood samples were collected at intervals through
ocular puncture. The blood was allowed to clot and serum separated, which was then used for
assaying of some parameters.
2.1.4 Experimental Design
Forty male Wistar rats of both sexes were housed in separate cages, acclimatized for
fourteen days and then divided into Control group of four rats and three groups of three rats
each.
The first, being control (n = 4) was maintained on normal rat chow and water ad
libitum. The experimental animals formed the second, third and fourth groups. Each group
contained 3 rats and had 4 types of time period associated with it. So, each group contained 12
rats (n = 12).
Group 1 animals were the Control, fed with the normal rat diet and water ad libitum.
Group 2 animals were starved of feed and water.
Group 3 animals were starved but received water.
Group 4 animals were starved but received fruits (carrots).
The Proximate Composition of the normal rat diet given to the control was:
Crude protein - 14.50g %
Crude fat - 4.80g %
Crude fibre - 7.20g %
Crude ash - 8.00g %
Phosphorus - 0.62g %
Lysine - 0.60g %
Methionine - 0.29g %
Methionine + Cystine - 0.52g %
Calcium - 0.80g %
Vitamin E - 15mg/100g
xxxv
Vitamin C - 50mg/100g
Manganese - 30mg/100g
Zinc - 30mg/100g
Sodium - 0.15g %
The normal rat diet was purchased from (Bendel Feed and Flour Mills Limited , Enugu
State). Groups 2, 3 and 4 were differently starved according to time duration and then blood
samples were collected through ocular puncture i.e. by orbital bleeding technique and the blood
samples were used for analyzing all parameters and blood glucose level.
The body weight and glucose levels of the rats were taken before and after the
starvation. Several parameters were assayed using the serum of the rats from the various groups
gotten by allowing whole blood to clot and spinning it for its separation. Enough blood was
collected at intervals through ocular puncture for all the below mentioned parameters. The rats
were kept under anaesthesia and sacrificed.
The parameters assayed were:
Lipid peroxidation products using MDA as index.
Total serum cholesterol.
Low density lipoprotein (LDL).
High density lipoprotein (HDL).
Triacyglycerol (TAG).
Total protein
Glutathione (GSH).
Vitamin C.
Blood glucose.
2.2 METHODS
2.2.1 Lipid Peroxidation Assay (Wallin et al., 1993)
2.2.1.1 Principle
Measurement of the extent of lipid peroxidation was determined using the thiobarbituric
acid reactive substance (TBARS) assay described by Wallin et al. (1993). Thiobarbituric acid
xxxvi
reacting substances e.g. malondialdehyde (MDA) reacts with thiobarbituric acid to give a red or
pink colour, which absorbs maximally at 532nm.
Biological specimens contain a mixture of thiobarbituric acid reacting substances
(TBARS) including lipid hydroperoxide and aldehydes which increase as a result of oxidative
stress.
2.2.1.2 Reagent preparation
Thiobarbituric acid was prepared by dissolving 1.0g in 83ml of distilled water on
warming. After complete dissolution the volume was made up to 100ml with distilled water.
A 25% Trichloroactic acid (TCA): A 12.5g of trichloroacetic acid was dissolved is
distilled water and made up to 50ml in a volumetric flask with distilled water.
Normal saline solution: 0.9g of NaCl was dissolved in 10ml of distilled water and make
up to 100ml with distilled water.
2.2.1.3 Procedure
To 0.1ml plasma in the test tube was added 0.45ml of normal saline and mixed
thoroughly before adding 0.5ml of 25% trichloroacetic acid (TCA) and 0.5ml of 1%
thiobarbituric acid.
To the blank was added volume of trichloroactie acid, thiobarbituric acid and saline but
0.10ml of distilled water instead of serum.
The mixture was heated in the water bath at 95oC for 40 minutes. If the content was
turbid, this was by centrifuging or adding chloroform. Otherwise, the mixture was allowed to
cool before reading the absorbance of the clear supernatant against reagent blank at 532nm and
600nm , which are the peak of absoroptions. Thiobarbituric acid reacting substances were
quantified as lipid peroxidation product by referring to a standard curve of malondialdehyde
(MDA) concentration (ie) equivalent generated by acid hydrolysis 1,1,3,3–tetraethoxypropane
(TEP) prepared by serial dilution of a stock solution.
Table 2.1: Reaction Mixture for MDA Assay.
xxxvii
Blank Test
Plasma --- 0.10 ml
Distilled water 0.10 ml ---
Normal saline 0.45 ml 0.45 ml
25% TCA 0.50 ml 0.50 ml
1% TBA 0.50 ml 0.50ml
2.2.2 Total Cholesterol Determination [Using QCA Commercial Kit; Allain et al.
(1976)]
2.2.2.1 Principle
The total cholesterol determination using QCA Commercial Enzyme kit is based on the
assay principle that total cholesterol is determined after enzymatic hydrolysis and oxidation.
The indicator, coloured quinonic derivative is formed from hydrogen peroxide and 4-
aminoantipyrine in the presence of p-hydroxybenzoic acid and peroxidase.
acidsFatty lCholestero OH esters-lCholesteroesterase Chol.
2
22
oxidase Chol.
22 OH neCholesteno O OH 2/1 lCholestero
04H derivated quinonic Coloured acid zoicHydroxyben-p yrineAminoantip-4 OH 2
Peroxidase
22
2.2.2.2 Procedure
Blank (BL), Sample (SA) and Standard (ST) were the three sets of labelled test tubes. A
quantity of 0.01 ml of the serum sample was pipetted into the sample (SA) test tube. Also, 0.01
ml of the standard was introduced into the standard (ST) test tube with a corresponding
addition of 1 ml of working reagent into all the test tubes. The solutions in the different sets of
the test tubes were well mixed and allowed to stand for 5 minutes at 37oC (or 10 minutes at
room temperature). The absorbance was read at the wavelength of 546 nm.
2.2.2.3 Calculations
The total cholesterol concentration in the sample was calculated using the following
general formula:
Cholesterol
esterase
xxxviii
lCholestero Total of mg/dl 200 x O.D.ST
O.D.SA
Where SA is Sample
ST is Standard
OD is Optical density
SI Units
(mg/100 ml) × 0.0259 = mmol/L
2.2.3 High Density Lipoproteins (HDL) –Cholesterol Determination [Using QCA
Commercial Kit; Albers et al. (1978)]
2.2.3.1 Principle
Low density lipoprotein (LDL) and Very low density lipoprotein (VLDL) are
lipoproteins precipitated from serum by the action of a polysaccharide, in the presence of
divalent cations. Then, the high density lipoprotein–cholesterol (HDL–Cholesterol) present in
the supernatant, is determined.
acidFatty lCholestero OH esters-lCholestero esterase chol.
2
22
oxidase chol.
22OH neCholesteno OH O
2
1 lCholestero
04H neQuinoneimi DCFS yrineAminoantip-4 OH2 2
eperoxidase
22
2.2.3.2 Procedure
The procedure took two steps:
(A) Precipitation Step
The serum sample (0.3 ml) was pipetted into labelled centrifuge tubes. Also, one drop
of the precipitant solution or reagent (10g/L of Dextran sulphate, 1M of Magnesium acetate and
stabilizers) was added to the same sets of centrifuge tubes.
(B) Colorimetric Step
xxxix
The contents in the various tubes were thoroughly mixed and allowed to stand for 15
minutes at room temperature (20–25oC); then centrifuged at 2,000 × g for 15 minutes (or
10,000 × g for 2 minutes). The concentration of cholesterol in the supernatant was determined.
2.2.3.3 Calculations
The HDL cholesterol concentration in the sample was calculated using the following
general formula:
lCholestero - HDL mg/dl 52.5 x A
A
standard
sample
Or
lCholestero - HDL mmol/dl 1.36 x A
A
standard
sample
2.2.4 Low Density Lipoprotein-Cholesterol Determination [Using QCA Commercial
Kit; Assmann et al. (1984)]
2.2.4.1 Principle
Low density lipoprotein–Cholesterol (LDL–Cholesterol) can be determined as the
difference between total cholesterol and cholesterol content of the supernatant after
precipitation of the LDL fraction by polyvinyl sulphate (PVS) in the presence of
polyethyleneglycol monomethyl ether.
LDL-Cholesterol = Total Cholesterol – Cholesterol in the Supernatant
Table 2.2: Reagents of low density lipoprotein determination
Content Initial Concentration of Solutions
1. Precipitation Reagent:-
Polyvinyl sulphate 0.7 g/L
EDTA Na2 5.0 mM
Polyethyleneglycol monomethyl ether 170 g/L
Stabilizers
2.2.4.2 Procedure
(1) Precipitation Reaction
xl
The precipitation solution (3 drops or 0.1 ml) was carefully measured into test tubes
labelled accordingly. The serum sample (0.2 ml) was added to the labelled test tubes. The
contents were thoroughly mixed and left to stand for 15 minutes approximately at room
temperature (20–25oC). Then, the mixture was centrifuged at 2,000 × g for 15 minutes and the
cholesterol concentration in the supernatant was determined.
(2) Cholesterol Assay
The concentration of the serum total cholesterol was determined according to the
QCA(Quimica Clinica Aplicada S.A) CHOD–PAP method.
2.2.4.3 Calculations
The LDL–Cholesterol concentration in the sample was calculated using the following
general formula:
LDL–Cholesterol (mg/dl) = Total Cholesterol (mg/dl) – 1.5 × Supernatant Cholesterol (mg/dl).
Clinical Interpretation for Human:
Low Risk < 150 mg/dl
Risk > 190 mg/dl.
2.2.5 Determination of Serum Triacylglycerols (Colorimetric Method of Tietz, 1990).
2.2.5.1 Principle
This method is based on the fact that triacylglycerols undergo enzymatic hydrolysis to
yield H2O2. This hydrogen peroxide produces a quinoneimine which when reacted with 4-
aminophenazone which absorbs light at 500 nm.
TAG + H2O Glycerol + Fatty acids
Glycerol + ATP Glycerol-3-phosphate + ADP
Glycerol-3-phosphate + O2 Dihydroxyacetone + Phosphate + H2O2
2H2O2 + 4-aminophenazone + 4-Chlorophenol Quinoneimine + HCl + H2O
Lipases
Glycerol Kinase
Glycerol-3-(P)
Oxidase
Peroxidase
xli
2.2.5.2 Reagents
The contents of the commercially available Randox Diagnostic Kit include:
a) 40 mmol/l pipes buffer; pH 7.6
b) 5.5 mmol/l 4-Chlorophenol
c) 17.5 mmol/l magnesium ions
d) 0.5 mmol/l 4-Aminophenazone
e) 1.0 mmol/l Adenosine triphosphate
f) ≥ 150 µ/ml Lipases
g) 0.4 µ/ml Glycerol kinase
h) ≥ 1.5 µ/ml Glycerol-3-phosphate oxidase
i) ≥ 0.5 µ/ml Peroxidase
j) 2.229 mmol/l Triacylglycerol standard.
2.2.5.3 Procedure
Table 2.3: Serum triacylglycerol determination
Reagent Blank (µl) Standard (µl) Sample (µl)
Sample – – 10
Standard – 10 –
Reagent 1000 1000 1000
The contents of each tube were mixed and incubated for 10 minutes at 20 – 25oC or 5
minutes at 37oC and the absorbance of the samples (ASample) were measured against the reagent
blank within 60 minutes at 500 nm and 1cm light path.
2.2.5.4 Calculation
The concentration of triacylglycerols is calculated using the relationship:
Triacylglycerols Concentration = )/(229.2tan
lmmolA
A
dardS
Sample
xlii
Triacylglycerol Concentration = )/(200tan
dlmgA
A
dardS
Sample
2.2.6 Total Protein Determination (Biuret Commercial Kit Method)
REAGENTS
Contents Concentration of Solutions
1. Biuret Reagent
Sodium hydroxide
Na – K – tartrate
Potassium iodide
Cupric sulphate
100 mmol/l
16 mmol/l
15 mmol/l
6 mmol/l
2. Blank Reagent
Sodium hydroxide
Na – K – tartrate
100 mmol/l
16 mmol/l
3. Standard
Protein
60 g/l (6.0 g/dl)
This method is based on the principle that cupric ions, in an alkaline medium, interact
with peptide bonds of proteins resulting in the formation of a coloured complex.
Distilled water (0.02 ml) was pipetted into reagent blank (B) test tubes only. Standard
solution (0.02 ml) was added to another set of test tubes labelled ST (standard) only. After
which 0.02ml of the sera from the different rats were added to different test tubes labelled SA
xliii
(sample) only. Biuret reagent (1.0ml) was added to all the three sets of test tubes. The contents
of the tubes were mixed thoroughly and incubated for 30 minutes at 25OC.
Absorbance of the Sample (Asample) and of the Standard (Astandard) against the reagent
blank was read at a wavelength of 530nm.
The Total Protein concentration was calculated as follows:
Total Protein Conc. = A sample x Standard Conc.
A standard
Where:
A sample = Absorbance of the Sample
A standard = Absorbance of the Standard
2.2.7 Determination of Glutathione (GSH)
2.2.7.1 Principle
Serum glutathione levels were determined by the method of Snell and Snell (1962).
This method is based on the colorimetric determination of glutathione. According to the
method, glutathione in an alkaline solution reacts with phospho-18-tungstic acid to produce a
purple blue colour.
2.2.7.2 Preparation of Reagent for Glutathione
PHOSPHO-18-TUNGSTIC ACID REAGENT
Sodium tungstate (10g) was dissolved in a little quantity of distilled water and made up
to 70ml and 7.5ml of phosphoric acid was added. After refluxing for 24 hours, 5 drops of
hydrogen peroxide was added and then boiled for 10 minutes. Then, it was diluted to 100ml
with distilled water and later stored in a brown bottle.
20% SODIUM SULPHITE SOLUTION
Sodium sulphite (20g) was dissolved in 100ml of distilled water.
20% SODIUM CARBONATE
Sodium carbonate (20g) was dissolved in 100ml of distilled water.
2.2.7.3 Procedure
A quantity (0.1ml) of each serum sample was measured into test tubes and was made up
to 1ml with distilled water. To each of these, 0.02ml of 20% sodium sulphite solution was
added and properly shaken. After 2 minutes, 0.02ml of 20% of lithium sulphate and 0.2ml of
20% sodium carbonate were added and properly shaken . 20g of lithium sulphate was dissolved
xliv
in 100ml of water and 2g of sodium carbonate was dissolved in 100ml 0f distilled water. This
was followed by addition of 0.2ml phospho- 18 tungstic acid reagent. Again, the tubes were
shaken and allowed to stand for 4 minutes to develop maximum colour. These were then made
up to 2.5ml with 2% sodium sulphite to prevent reoxidation and absorbance read against the
blank at 610nm in less than 10 minutes to avoid bleaching. The concentration was then derived
from the standard curve of glutathione.
2.2.7.4 Preparation of Glutathione Standard Curve
Different dilutions of the glutathione were made 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70,
0.80 and 0.90 all in mg/ml.
To each of the test tubes 0.2 ml of phosphor-18-tungstate acid reagent was added and
shaken for 4 minutes. It was then made up to 2.5 ml with 20% sodium sulphate solution. Read
the absorbance at 610 nm.
2.2.8 Determination of Vitamin C Level (Goodhart and Shils, 1973)
2.2.8.1 Principle
This method involves the oxidation and conversion of ascorbic acid to diketogluconic
acid in strong acid solution. A diphenylhydrazine is formed by the reaction with 2,4-
dinitrophenylhydrazine. Cupric ions act as the oxidizing agent, followed by hydrazone
formation. The hydrazone dissolves in strong sulphuric acid solution to produce a light red
colouration, whose intensity gives a measure of the concentration of ascorbic acid. The addition
of thiourea as a reducing agent adds specificity by avoiding interference from non-ascorbate
chromogens.
2.2.8.2 Reagents for Vitamin C
i) Trichloroacetic acid (TCA) (10% w/v): Trichloroacetic acid (10g) was dissolved in
20ml distilled water and the volume made up to 100ml.
ii) 2,4-dinitrophenylhydrazine Reagent: Concentrated sulphuric acid (25ml) was added
to 75ml of chilled water to give 9.0N H2SO4. Crystalline 2,4-dinitrophenylhydrazine
(2g) was dissolved in 100ml of 9.0N H2SO4 and the resultant solution was filtered
and stored in a brown bottle in the refrigerator.
iii) Thiourea Solution: Thiourea (10g) was dissolved in 100ml of 50% ethanol and the
solution stored in the refrigerator.
iv) Cupric Sulphate (1.5% w/v): Cupric sulphate (1.5g) was dissolved in 10ml of
distilled water and made up to 100ml.
xlv
v) Combined Colour Reagent: This was prepared fresh each day by mixing:
2,4-dinitrophenylhydrazine reagent (5ml)
Cupric sulphate solution (0.1ml) and Thiourea solution (0.1ml)
vi) Sulphuric acid (85%): Concentrated H2SO4 (180ml) was added to 20ml of distilled
water. The solution was thoroughly mixed, cooled and stored in a glass-stoppered
bottle in the refrigerator.
vii) Ascorbic Acid Standard: Ascorbic acid (1g) was dissolved and diluted to 100ml.
This was further diluted with distilled water just before use to give a working
standard of 2mg/100ml.
2.2.8.3 Procedure
To serum (1ml) pipetted into a test tube was added 10% trichloroacetic acid (1ml) and
chloroform (0.5ml). The test tube was stoppered, shaken vigorously for 15 seconds and
centrifuged at 10,000 rpm for 5 minutes. The clear supernatant (1ml) was pipetted into another
sample test tube. A volume of 0.5ml of trichloroacetic (10%) was added to 0.5ml distilled
water and 0.5ml of freshly prepared ascorbic acid standard to give the blank and the working
standard respectively.
Freshly prepared colour reagent (0.4ml) was added to the blank, working standard and
test sample. The resulting solution was thoroughly mixed, stoppered and placed in a water bath
for 1 hour at 56oC. The test tubes were cooled in an ice-bath for 5 minutes.
Ice-cold 85% sulphuric acid (2ml) was slowly added to each test tube with mixing and
allowed to stand at room temperature for 30 minutes. Absorbance of test sample and standard
was measured against blank at 500 nm.
Ascorbic acid concentration is calculated thus:
2)(
)(
SA
TAmg Ascorbic acid/100ml
Where A (T) = Absorbance of test sample
A (S) = Absorbance of standard
2.2.9 Blood Glucose Assay (Marks and Dawson, 1965)
ONE TOUCH (™
) blood glucose monitoring system/meter and test strips (Lifescan Inc,
Johnson-Johnson Company, Milpitas California, USA) was used for the assay.
xlvi
2.2.9.1 Principle
Glucose Gluconic acid + H2O2
H2O2 H2O + O
O + Acceptor Coloured Complex + H2O
The method is based on the reaction of glucose and oxygen in the presence of glucose
oxidase to yield gluconic acid and hydrogen peroxide. Hydrogen peroxide subsequently
oxidizes the dyes in a reaction mediated by peroxidase producing a blue coloured form of the
dyes. The intensity of the blue colour is proportional to the glucose concentration in the sample
and is measured and read by the ONE TOUCH meter.
The One-Touch glucometer was essentially a reflectance meter. The amount of light
reflected in reagent area of the dextrostix measured in a readout meter scale was a measure of
the concentration of glucose in the blood. Snips were made on the tail of the animal to release
blood on the sensitive spot on the glucometer.
2.2.9.2 Reagents
ONE TOUCH Glucometer (Lifescan Inc. Johnson – Johnson Company, USA) and test
strips were used. The composition of the test strips is:
Glucose oxidase (14/U)
Peroxidase (11/U)
3-methyl-2-benzothiazolinonehydrazone hydrochloride (0.06mg)
3-dimethylaminobenzoic acid (0.12mg).
2.2.9.3 Procedure
i) Insert the code key into the glucometer code key opening.
ii) Insert a test strip to make sure that the code on the glucometer matches the code on
the test strip.
iii) Insert a fresh new strip with the orange pad facing up until it goes no further into the
glucometer opening for test strips.
Glucose oxidase
Peroxidase
O-toluidine
xlvii
iv) Wait until the image of a flashing blood appears on the glucometer screen; that
signifies that the glucometer is ready. Then put a drop of blood collected with a
capillary tube on the centre of the square of the orange pad.
v) An hour glass symbol appears on the glucometer screen followed after 5 seconds by
the test result.
vi) Copy the test result as the blood glucose level in g/dl.
2.2.10 Body Weight
According to duration of study both control and the experimental animals were weighed
before the stress was forced on them and after the stress. Therefore, the difference in body
weight was recorded and compared.
2.3 STATISTICAL ANALYSIS
The results were expressed as mean SD and tests of statistical significance were
carried out using student t-test and both one-way and two-way analysis of variance (ANOVA).
The means were separated using Duncan Multiple Test. The statistical package used was
Statistical Package for Social Sciences (SPSS); version 17.
CHAPTER THREE
RESULTS
3.1 EFFECT OF STARVATION ON BODY WEIGHT OF WISTAR ALBINO RATS
AT VARIOUS INTERVALS
The results of the mean body weights of rats in all the test groups were not significantly
different (p>0.05) compared with the control at 0 hour of the experiment as shown by Fig. 3.1.
Results in Fig. 3.1 show that there was no significant difference (p>0.05) between the control
and the treated groups after 6 hours. However, there was a significant increase (p<0.05) in the
body weights of treated rats after 12 hours when compared with the control group. After 24
hours treatment, group 2 animals had significant increase (p<0.05) in their mean body weight
when compared with control. For 48 hours treatment, no significant difference (p>0.05) in
body weight was observed. There was significant increase (p<0.05) in the rats’ body weights of
group 4 treated animals compared with the control group after a period of 6 hours. There was
also a significant increase (p<0.05) in the body weights of treated group 4 rats after 12 hours
xlviii
when compared with the control group. After 24 hours treatment, group 2 had a significant
increase (p<0.05) when compared with control. For 48 hours treatment, no significant
difference (p>0.05) in body weight was observed.
xlix
Fig. 3.1: Effect of starvation on the body weights of rats at various
time intervals
0
50
100
150
200
250
Group 1 Group 2 Group 3 Group 4
Experimental Group
Me
an
Bo
dy
Weig
ht
(g)
0 Hour
6 Hours
12 Hours
24 Hours
48 Hours
3.2 EFFECT OF STARVATION ON MEAN BLOOD GLUCOSE
CONCENTRATIONS OF WISTAR ALBINO RATS AT VARIOUS INTERVALS
Group 1: Control (Normal feed and water
Group 2: Starved of feed and water
Group 3: Starved but given water
Group 4: Starved but fed with fruit
l
The glucose concentration increased significantly (p<0.05) in group 3 test animals
administered water after starvation compared with the control animals at the 0 hour duration of
the study (Fig. 3.2). Fig. 3.2 shows significant decrease (p<0.05) in the glucose concentration
of animals (group 4) fed fruit after starvation compared with the animals (group 3)
administered water after starvation at 0 hour of the experiment. However, the glucose
concentrations of the animals in group 2 (starved of feed and water) and group 4 (starved +
fruit) were not significant (p>0.05) compared with the control.
The blood glucose concentrations of rats in groups 2 and 3 increased significantly
(p<0.05) when compared with the control (group 1) within the duration of 6 hours. But there
was no significant difference (p>0.05) in the glucose concentrations between the control
animals and group 4 animals after 6 hours. Significant increase (p<0.05) in the blood glucose
concentrations of animals in group 4 after 6 hours of starvation was also observed when
compared with the control. Non-significant differences (p>0.05) as shown in Fig. 3.2 were
observed between the control and test groups 2 (starved of feed and water) and 3 (starved but
received water) after a duration of 12 hours. However, Fig. 3.2 shows that there was no
significant difference (p>0.05) between the blood glucose concentrations of the control and the
test groups after 24 and 48 hours.
Fig. 3.2 shows significant elevation (p<0.05) of blood glucose concentration in the
animals of group 3 (starved but received water) compared with the animals in the control group
after 6 hours’ starvation. The blood glucose level decreased significantly (p<0.05) in group 3
animals compared with the control after 12 hours of starvation as recorded at the end of the
experiment (Fig. 3.2). Significant reduction (p<0.05) in the concentrations of blood glucose
after the experiment was observed under 24 hour duration in groups 2 (starved of feed and
water) and 3 (starved but received water) when compared with the group 1 Control (Fig. 3.2).
The concentrations of blood glucose after the experiment were significantly (p<0.05) elevated
in the group 3 compared with the group 4 (starved but received fruit only). Under the 48-hour
period of starvation, significant (p<0.05) reduction of glucose concentrations was noticed in all
the test groups (groups 2, 3 and 4) as compared with the Control (Fig. 3.2b).
On the other hand, non-significant difference (p>0.05) in the glucose concentrations was
noticed among the control within the durations of 0, 6, 12, 24 and 48 hours respectively. Group
2 under 6-hour duration was found to be significant (p<0.05) when compared with group 2 of
12, 24 and 48 hours (Fig. 3.2b). Group 2, under 12 hours showed significant increase (p<0.05)
in blood glucose concentrations compared with group 2 under 48-hour duration. The blood
li
glucose concentrations of group 2 within 48 hours showed significant reduction (p<0.05) when
compared with group 2 during 12 and 24 hours respectively.
lii
Fig. 3.2: Effect of starvation on the blood glucose concentration of
rats at various time intervals
0
20
40
60
80
100
120
140
Group 1 Group 2 Group 3 Group 4
Experimental Group
Me
an
Blo
od
Glu
co
se
Co
nc
. (m
g/d
l)
0 Hour
6 Hours
12 Hours
24 Hours
48 Hours
3.3 EFFECT OF STARVATION ON MEAN MALONDIALDYHYDE (MDA)
CONCENTRATIONS OF WISTAR ALBINO RATS AT VARIOUS INTERVALS
Group 1: Control (Normal feed and water
Group 2: Starved of feed and water
Group 3: Starved but given water
Group 4: Starved but fed with fruit
liii
The finding in Fig 3.3 shows neither significant increased nor decrease (p>0.05) in the
concentrations of malondialdehyde (MDA) of animals in the different test groups compared
with the control at 0 hour of the experiment.
It was observed in Fig. 3.3 that there was neither a significant increase nor decrease
(p>0.05) in malondialdyhyde (MDA) level of rats in all the experimental test groups (groups 2,
3 and 4) compared with the control at 6 hours interval of starvation . Also, there was no
significant difference (p>0.05) between control (group 1) and all the test groups observed in
Fig. 3.3 at 12 hours interval of starvation.
Results in Fig. 3.3 show elevated concentrations of MDA in all the test groups when
compared with the control but such increase were not significant (p>0.05) at the 24th
hour
interval of starvation. At the 48th
hour interval of starvation, the same trend as recorded in 24th
hour of starvation was also observed (Fig. 3.3).
There were no significant differences (p>0.05) in MDA concentrations of the
experimental rats within the different time intervals (6 to 48 hours) under each group.
liv
Fig. 3.3: Effect of starvation on the malondialdehyde concentration
of rats at various time intervals
0
5
10
15
20
25
30
Group 1 Group 2 Group 3 Group 4
Experimental Group
Me
an
MD
A C
on
c.
(mm
ol/
ml)
0 Hour
6 Hours
12 Hours
24 Hours
48 Hours
3.4 EFFECT OF STARVATION ON MEAN ASCORBIC ACID CONCENTRATIONS
OF WISTAR ALBINO RATS AT VARIOUS INTERVALS
Group 1: Control (Normal feed and water
Group 2: Starved of feed and water
Group 3: Starved but given water
Group 4: Starved but fed with fruit
lv
There was no significant (p>0.05) time-dependent decrease in ascorbic acid
concentrations in the animals in both groups 2 (starved of feed and water) and 3 (starved but
water). Here, concentrations of ascorbic acid decreased with corresponding increase in the time
of the experiment. At 0 hour, the ascorbic acid concentration neither showed significant
(p>0.05) increase nor decreasing in the test group compared with the ascorbic acid
concentrations in the control group.
The Vitamin C concentrations of the treated groups were not significantly lower
(p>0.05), (Fig. 3.4) when compared with the control group after 6 hours of starvation.
However, after the 12th
hour interval of starvation, vitamin C concentrations of group 4 was
high but not significant (p>0.05) when compared to control. For 24 hours, the ascorbic acid
concentrations of treated groups (groups 2 and 3) were statistically not significant (p>0.05)
while for 48 hours, groups 3 and 4 decreased significantly (p<0.05) when compared with the
control group (group1). On the other hand, there was no significant difference (p>0.05) among
the control within the 6th
, 12th, 24th and 48th
hours of starvation respectively. Group 2 was not
significantly different (p<0.05) under 6 to 48 hours. Group 3 also shows a significant increase
(p<0.05) under the 6th
period in starvation when compared with group 3 of 24th
and 48th
hours.
Significant (p<0.05) elevation was observed when group 4 under 6 hours of starvation was
compared with group 4 of 24 and 48 hours respectively.
lvi
Fig. 3.4: Effect of starvation on the ascorbic acid concentration of
rats at various time intervals
0
0.5
1
1.5
2
2.5
3
3.5
Group 1 Group 2 Group 3 Group 4
Experimental Group
Me
an
As
co
rbic
Ac
id C
on
c.
(mg
/dl)
0 Hour
6 Hours
12 Hours
24 Hours
48 Hours
3.5 EFFECT OF STARVATION ON MEAN GLUTATHIONE CONCENTRATIONS
OF WISTAR ALBINO RATS AT VARIOUS INTERVALS
Group 1: Control (Normal feed and water
Group 2: Starved of feed and water
Group 3: Starved but given water
Group 4: Starved but fed with fruit
lvii
Fig. 3.5 shows no significant (p>0.05) differences in the concentrations of glutathione of
animals in the test groups (groups 2, 3 and 4) compared with the glutathione concentrations of
animals in the control group at 0 hour of the experiment. On the other hand, the concentrations
of glutathione of group 2 animals starved of feed before water administration was found to
have time-dependent response; the glutathione concentrations of the group 2 animals did not
decrease significantly (p>0.05) as the time of the experiment progressed.
Results from (Fig 3.5) show that no significant difference (p>0.05) exist between the
test groups of 3 and 4 when compared with control group after a duration of 6 hours. GSH
concentrations did not decrease significantly (p>0.05) in the 12th
hour period of starvation.
Under 24 hours, an increase in the GSH concentrations were found between the test group
(group 4 -starved and received fruits only) when compared with control; though it was not
significant (p>0.05). Then, the 48th
period showed no significant difference (p>0.05) between
the treated groups and the control. There were no time-dependent differences in groups 2, 3 and
4 glutathione concentrations of the rats after the starvation experiment. No significant
difference (p>0.05) was noticed among the control within the starvation periods of 6, 12, 24
and 48 hours respectively. Group 2 under 48 hours showed significant reduction (p<0.05) in
glutathione concentrations when compared with group 2 of the 24 hours duration (Fig. 3.5).
Again, group 3 was not significantly different (p>0.05) for all the time intervals. Finally, a
significant decrease (p>0.05) occurred in group 4 under 48 hours when compared with group 4
of the 6th, 12
th, 24
th and 48
th hour periods.
lviii
Fig. 3.5: Effect of starvation on the glutathione concentration of rats
at various time intervals
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Group 1 Group 2 Group 3 Group 4
Experimental Group
Me
an
Glu
tath
ion
e C
on
c.
(mm
ol/
ml)
0 Hour
6 Hours
12 Hours
24 Hours
48 Hours
3.6 EFFECT OF STARVATION ON MEAN TOTAL PROTEIN
CONCENTRATIONS OF WISTAR ALBINO RATS AT VARIOUS INTERVALS
Group 1: Control (Normal feed and water
Group 2: Starved of feed and water
Group 3: Starved but given water
Group 4: Starved but fed with fruit
lix
Decrease in the total protein concentrations of animals in all the test groups was
observed (Fig. 3.6) compared with the control at 0 hour; though such decrease in the total
protein concentrations was not significant (p>0.05). There was no significant difference
(p>0.05) in the levels of total protein between the group 1 (control) and all the experimental
test groups (groups 2, 3 and 4) at 0, 6, 12, 24 and 48th
hour intervals of starvation as shown in
Fig. 3.6. Similarly, there was no significant difference (p>0.05) in the levels of total protein
between the test groups at 6, 12, 24 and 24 hours of starvation.
The same pattern of result in the concentrations of total protein of rats at different time
intervals (0, 6, 12, 24 and 48 hours of starvation) was observed in individual experimental
groups.
lx
Fig. 3.6: Effect of starvation on the total protein concentration of
rats at various time intervals
0
1
2
3
4
5
6
Group 1 Group 2 Group 3 Group 4
Experimental Group
Me
an
To
tal
Pro
tein
Co
nc
. (g
/dl)
0 Hour
6 Hours
12 Hours
24 Hours
48 Hours
3.7 EFFECT OF STARVATION ON MEAN SERUM TOTAL CHOLESTEROL
CONCENTRATIONS OF WISTAR ALBINO RATS AT VARIOUS INTERVALS
Group 1: Control (Normal feed and water
Group 2: Starved of feed and water
Group 3: Starved but given water
Group 4: Starved but fed with fruit
lxi
There was no significant (p>0.05) decrease in the concentration of serum total
cholesterol of the control animals at 12, 24 and 48 hours compared with the serum total
cholesterol concentration of the control animals at 0 hour (Fig. 3.7). Generally, there were no
significant differences (p>0.05) in the concentrations of serum total cholesterol of animals in
the test groups compared with the animals in the control group at 0 hours as shown in Fig. 3.7.
Fig. 3.7 shows no significant difference (p>0.05) in the concentrations of serum total
cholesterol between the control and all the test groups at the 6th
-hour interval of starvation.
However, the serum total cholesterol concentrations of all test experimental groups at the 12th
-
hour interval of starvation increased significantly (p<0.05) compared with the control. There
was no significant difference (p>0.05) in the concentrations of serum total cholesterol between
the test groups (groups 2, 3 and 4) within the duration of 12 hours.
When considering starvation at the 24-hour interval, significant (p<0.05) elevated
concentrations of serum total cholesterol was observed (Fig. 3.7) in all the test groups in
comparison with the control. There was also significant difference (p<0.05) between group 2
(starved of feed and water) and group 4 (starved and received fruits only) at the interval of 24
hours of starvation (Fig. 3.7). In the same vein, significant increase (p<0.05) in total cholesterol
concentrations was observed in all the test groups compared with the control at the 48th
hour
interval of starvation (Fig. 3.7).
Considering differences between the different time intervals of starvation in each
experimental group, no significant differences (p>0.05) were observed as recorded in Fig. 3.7
between the different time intervals of starvation in group 1. Significant difference (p<0.05)
exists in the cholesterol concentrations between 6th
hour interval of starvation and other time
intervals (12, 24 and 48 hours of interval of starvation) in the group 2 (starved of feed and
water). Similar trend was observed in the same group 2 when comparing 48 hours and other
hours (6, 12 and 24 hours) of starvation. There was a significant difference (p<0.05) in serum
total cholesterol concentrations between 6 hours of starvation and other intervals (12, 24 and
hours) of starvation under group 3. In group 3, significant difference (p<0.05) in the
concentrations of total cholesterol was observed between 48 hours of starvation and that of 6
and 12 hours of starvation (p<0.05; Fig. 3.7). No significant difference (p>0.05) was observed
in group 4 serum total cholesterol concentrations between all the intervals of starvation with
exception of 6 hours of starvation. However, Fig. 3.7 shows significant difference (p<0.05) in
the concentrations of serum total cholesterol between 6 hours of starvation and other intervals
(12, 24 and 48 hours) of starvation as observed in group 4 (starved but received fruits only).
lxii
lxiii
Fig. 3.7: Effect of starvation on the serum total cholesterol
concentration of rats at various time intervals
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Group 1 Group 2 Group 3 Group 4
Experimental Group
Me
an
To
tal
Ch
ol.
Co
nc
. (g
/dl)
0 Hour
6 Hours
12 Hours
24 Hours
48 Hours
3.8 EFFECT OF STARVATION ON MEAN TRIACYGLYCEROL
CONCENTRATIONS OF WISTAR ALBINO RATS AT VARIOUS INTERVALS
Group 1: Control (Normal feed and water
Group 2: Starved of feed and water
Group 3: Starved but given water
Group 4: Starved but fed with fruit
lxiv
The concentrations of triacylglycerol (TAG) of animals in the test groups did not alter
significantly (p>0.05) compared with the TAG concentrations of the animals in the control
group at 0 hour of the experiment (Fig. 3.8). Fig. 3.8 shows no significant difference (p>0.05)
in the concentrations of triacylglycerol (TAG) between the control and the test groups at 6
hours interval of starvation. Similar pattern of result was obtained at starvation intervals of 12,
24 and 48 hours.
On the aspect of time-dependent effects in individual groups, there was no significant
difference (p>0.05) in the concentrations of TAG between 6 hours of starvation and other hours
(12, 24 and 48 hours) of starvation and the control (Fig. 3.8) . On the contrary, Fig. 3.8 shows
that significant difference (p<0.05) exists in group 2 (starved of feed and water) between 48
hours of starvation and other intervals of starvation (6, 12 and 24 hours). In group 2, there was
significant difference (p<0.05) in TAG concentrations between 6 hours of interval and 48 hours
of interval of starvation but no significant difference (p>0.05) between other intervals of
starvation (Fig. 3.8). No significant difference (p>0.05) was observed in the concentrations of
TAG between all the time intervals of starvation as found in group 4 (Fig. 3.8).
lxv
Fig. 3.8: Effect of starvation on the triacylglycerol concentration of
rats at various time intervals
0
0.5
1
1.5
2
2.5
Group 1 Group 2 Group 3 Group 4
Experimental Group
Me
an
TA
G C
on
c.
(mm
ol/
L)
0 Hour
6 Hours
12 Hours
24 Hours
48 Hours
3.9 EFFECT OF STARVATION ON MEAN HIGH DENSITY LIPOPROTEIN
CONCENTRATIONS OF WISTAR ALBINO RATS AT VARIOUS INTERVALS
Group 1: Control (Normal feed and water
Group 2: Starved of feed and water
Group 3: Starved but given water
Group 4: Starved but fed with fruit
lxvi
The concentrations of high density lipoprotein (HDL), as observed in Fig. 3.9, showed
no significant (p>0.05) differences in the test groups (groups 2, 3 and 4) compared with the
HDL concentrations of animals in the control group at 0 hour of the experiment. Results (Fig.
3.9) show that the high density lipoprotein (HDL) concentrations of the test groups (Groups 2,
3 and 4) were not significant (p>0.05) when compared with the control after the duration of the
starvation (6 to 48 hours). There were no time-dependent HDL differences in the rats after the
duration of 6 to 48 hours. Also, no significant differences in the HDL concentrations of the rats
in control group as well as groups 2 to 4 under the 6 to 48 hours of experiment was seen (Fig
3.9).
lxvii
Fig. 3.9: Effect of starvation on the high density lipoprotein
concentration of rats at various time intervals
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
Group 1 Group 2 Group 3 Group 4
Experimental Group
Me
an
HD
L C
on
c.
(mm
ol/
L)
0 Hour
6 Hours
12 Hours
24 Hours
48 Hours
3.10 EFFECT OF STARVATION ON MEAN LOW DENSITY LIPOPROTEIN
CONCENTRATIONS OF WISTAR ALBINO RATS AT VARIOUS INTERVALS
Group 1: Control (Normal feed and water
Group 2: Starved of feed and water
Group 3: Starved but given water
Group 4: Starved but fed with fruit
lxviii
Fig. 3.10 shows no significant (p>0.05) concentrations of low density lipoprotein
(LDL), of the test groups (groups 2, 3 and 4) compared with the concentrations of LDL of the
animals in the control group at 0 hour of the experiment. In Fig 3.10, a non- significant
difference (p>0.05) in the low density lipoprotein (LDL) concentrations between the test
groups and the control after 6 to 48 hours duration was observed. Then, under 6 to 48 hours
time intervals, the control did not show statistical difference (p>0.05; Appendix a, b, c, d). Low
density lipoprotein (LDL) concentrations of groups 2 to 4 under 6 hours of starvation were
found to decrease significantly (p<0.05) as compared with group 2 of 12 to 48 hours and
likewise to other time intervals (12, 24 and 48 hours time intervals respectively).
lxix
Fig. 3.10: Effect of starvation on the low density lipoprotein
concentration of rats at various time intervals
0
0.5
1
1.5
2
2.5
Group 1 Group 2 Group 3 Group 4
Experimental Group
Me
an
LD
L C
on
c.
(mm
ol/
L)
0 Hour
6 Hours
12 Hours
24 Hours
48 Hours
Group 1: Control (Normal feed and water
Group 2: Starved of feed and water
Group 3: Starved but given water
Group 4: Starved but fed with fruit
lxx
CHAPTER FOUR
DISCUSSION
The ability to withstand and recover from periods of nutritional stress (starvation) is an
important adaptation for survival of any organism that must sporadically endure periods of limited food
supply (Stuck et al., 1996). Under certain pathological conditions, ROS production is increased and the
level of antioxidant substances and enzymes are reduced (Lemberg, et al., 2007). This creates an
imbalance in the oxidative status of the system. This imbalance between ROS production and its
removal constitutes the process called Oxidative Stress (Wu and Cederbaum, 2003).
This work investigated the effects of the oxidative stress sequel to short starving periods of
Wistar rats. The work showed significant changes in body weight (p<0.05) between normal and starved
states. There was no significant increase (p>0.05) in the body weights of the test animals
compared with the body weights of the animals in control group at 0 hour of the experiment.
This indicates that signal of starvation was received by all the experimental rats; thus making
them to lose weight. Also, the starved rats that were administered fruit increased in weight than
the control because fruits serve as energy source. Food deprivation lasting for 12 hours led to
reduction of body weight in animals starved of feed and water (group 2) and animals starved of water
(group 3) respectively. Body weight of animals starved of water decreased in 24 hours when compared
with the initial body weight. For animals starved of fruits, it decreased in 24 and 48 hours.
During early starvation, weight loss becomes rapid but it gradually slow down without
noticeable changes as starvation progressed. Energy expenditure over the day decreases in starvation
and starving individuals voluntarily diminish their spontaneous movements (Keys et al., 1950). Survival
during starvation is dependent upon mechanisms that limit oxidative loss of pyruvate in non-neuronal
tissues of the body (Cahill, 1970).
However, the glucose concentration increased significantly (p<0.05) in group 3 animals
administered water after starvation compared with the control animals at the 0 hour duration of
the study. It also shows significant decrease (p<0.05) in the glucose concentration of animals
(group 4) fed fruit compared with the animals (group 3) administered water after starvation at 0
hour of the experiment. This could be attributed to the increase in glucose concentration which
stimulates insulin secretion. This suggests that by 0 hour time interval, hepatic gluconeogenesis
was sufficient to drive or maintain the output of the starvation (Thorens et al., 1990).
In this study, the short-term fasting results indicated a general significant decrease (p<0.05) of
glucose concentrations in blood of rats during starvation durations of 12-48 hours when compared with
the initial glucose level. Normalization of plasma glucose concentration results probably from an
increase in gluconeogenesis and a decrease in glucose utilization after a certain short term fasting
(Lamosova et al., 2004). Glucose is normally the sole energy source for certain key tissues, including
lxxi
the central nervous system. According to Adibi (1976), provision of glucose (gluconeogenesis) during
starvation is essential. The trigger that induces the initial metabolic adaptations during starvation is the
arterial glucose level, which begins to fall in humans within 15 hours of fasting.
Furthermore, the findings of this study reveal significant increase (p<0.05) in serum total
cholesterol, low lipoprotein and triacylglycerols after starvation of different durations in rats. The
increase in total serum cholesterol concentration and LDL is in agreement with previous studies of Hem
Lata et al. (2002) and Bijlani et al. (1985). These results support the hypothesis that cholesterol stored
in the adipose tissue cells is released into plasma and is the chief source of the hypercholesterolemia
observed during starvation. Another cause of hypercholesterolemia during starvation may be attributed
to the continued biosynthesis with concomitant decreased or complete absence of intestinal excretion
(Swaner and Connor, 1975).
The main cellular components in the body susceptible to damage by free radicals are lipids
(unsaturated fatty acids in membranes), proteins, carbohydrates and nucleic acids (Blokhina et al.,
2002). The interest for oxidative stress in relation to the development of disease has gained large
attention during the last decade. Animals use their lipid stores to compensate for deficit of energy and
loss of body weight induced by periods of food shortage (Boswell et al., 2002).
The increase in LDL levels is time-dependent in this study. Between 24 and 48 hours, animals
starved of feed and water, animals starved of water and animals starved of fruit increased; thus
indicating that there may be an increased susceptibility of the animals to atherosclerosis as a result of
starvation. The data of this work also suggest that food deprivation causes an increased lipolysis
simultaneously with lowered lipogenesis during the 48 hours period.
Triacylglycerol in this work increased significantly (p<0.05) depending on duration . The
increase in TGs concentrations may be due to the release of TGs from storage sites for the formation of
glucocorticoids in response to starvation (Singhal et al., 1997).
The results of HDL in this study indicate that HDL levels did not show significant difference
(p>0.05) after starvation. Previous studies of Vaisman et al. (1990) and Savendhal and Underwood,
1999) have reported either no change or decrease in HDL concentrations. This may be due to different
experimental conditions and organism differences in susceptibility to stress. Again, the low level
groups 2-4 of HDL may exert anti-atherogenic and antioxidative effects when present in sufficient
amounts and the reduced HDL concentrations in group 1 is often accompanied by elevations in plasma
TG levels (Lamarche et al., 1996).
Dietary antioxidants are known to play a fundamental role protecting against ROS (Jimena et
al., 2006). Different classes of antioxidants play a major role in the organism’s defense system against
the free radicals generated. The idea that diet plays an important role in oxidative stress has been
enhanced in recent years by studies on both natural and synthetic antioxidants (Cederbaum, 1989).
Vitamins such as A, C and E, and other supplementary materials such as carotenoids, cholesterol and
unsaturated fats presumably play important roles in the balance between pro-oxidant and antioxidant
lxxii
systems in humans (Gutteridge, 1995; Halliwell, 1996). Ascorbic acid is known to represent the first
line of antioxidant defense (Frei et al., 1988, 1989) and this vitamin is likely to be most susceptible to
free radical oxidation. Nishikimi (1975) had reported its oxidation by superoxide anion radical.
Goode et al. (1995) in their work observed increased free radical activity, and low antioxidant
vitamins levels in patients with septic shock. Greater depletion of antioxidants has been related to a
greater severity of trauma (De la vega et al., 2002; Tsaik, et al., 2000). Low endogenous stores of
antioxidants are associated with increased free radical generation and vice versa (Heyland et al., 2003).
Therefore, in this study, the results of animals starved of feed and water and animals starved of
water decreased in vitamin C level with an increase in MDA. This agrees with the findings of Lemberg
et al. (2007) who reported an increase in TBARS concentration and a marked decrease in the two
antioxidant molecules (Vitamin C and Glutathione); thereby indicating an oxidative condition.
Prolonged fasting is known to affect the antioxidant capacity of the cell (Martensson, 1986). In
this study on starvation, the stress resulted in significantly diminished GSH values in controls (starved
rats without feed and water and starved rats with water under 24 and 48 hours duration). This really
agrees with the finding in other work (Di Simplicio et al., 1997) who said that total GSH was reduced
after 18 hours of starvation by 39% in mouse liver.
The action of vitamin C and GSH in protecting cellular macromolecules from oxidant damage
is well known (Meister, 1992; Tampo and Yonaha, 1990). Anyia and Naito (1993) had reported that
severe oxidative stress might result in decrease in glutathione. Therefore, this agrees with the result of
this work.
In conclusion, the findings from this study support the hypothesis that: Starvation is
characterized by increased oxidative damage, oxidative injury and oxidative damage to lipids; thus
decreasing the availability of antioxidants. Additionally, that short term starvation still causes
generation of ROS despite the taking of water. Though oxidative stress markers do not worsen when
fruit is taken, still there is an imbalance occurring between plasma oxidant and antioxidant systems in
starved rats and starved rats fed with fruits.
4.2 Suggestions for Further Research
The results of the present study give new and relevant biological information about the
physiological and biochemical responses during starving conditions; hence suggestions for further
studies could be considered thus:
1. The use of vitamin C or fruits as a nutritional supplement in the amelioration of starvation
induced oxidative stress at prolonged periods.
2. The effect of prolonged starvation and refeeding on antioxidant status and some metabolic
parameters in the liver of rats.
lxxiii
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APPENDICES
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Fig. 1: Standard curve of Glutathione
y = 0.9944x
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1
Concentration (mg/ml)
Ab
so
rba
nc
e
Appendix I: Standard curve of glutathione
lxxxvii
Appendix II: Calibration curve of malondialdehyde (MDA)
Fig. 13: Calibration curve for Malondialdehyde (MDA)
y = 0.0064x
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0 20 40 60 80 100
Concentration (nmol/L)
Ab
so
rban
ce