i

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

ii

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.

iii

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)

iv

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.

v

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.

vi

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.

vii

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.

viii

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

ix

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

x

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

xi

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

xii

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

xiii

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

xiv

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).

xv

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-

xvi

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).

xvii

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

xx

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

xxiii

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

xxiv

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).

xxv

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

REFERENCES

Adibi, S. A. (1976). Metabolism of branched-chain amino acids in altered nutrition.

Metabolism. 25: 1287.

Akkus, I., Saglam, N.I., Caglayan, O., Wral, II., Kalak, S. and Saglam, M. (1996). Investigation

of the erythrocyte membrane lipid peroxidation and antioxidant defense systems of

patients with coronary artery disease (CAD) documented by angiography. Clinical

Chemistry Acta. 244: 173-180.

Albers, J. J., Warmick, G. R. and Cheng, M. C. (1978). Determination of High density

lipoprotein (HDL)–Cholesterol. Lipids, 13: 926–932.

Allain, C. C., Poon, L. S., Chan, C. S. G., Richmond, W. and Fu, P. C. (1976).Total cholesterol

determination. Clinical Chemistry, 20: 470–475.

Anderson, T.A., Beauchamp, J.J. and Walton, B.T.(1991). Organic chemicals in the

environment. Fate of volatile and semivolatile organic chemicals in soil: Abiotic versus

biotic looes. Journal of Environmental Quality, 20: 420-424.

Anonymous (2008). Malnutrition the Starvelings. http://www.who.int/child. adolescent. health/

topics/ prevention. Care/child. Retrieved, 12th, March, 2008.

Anonymous (2007). Vitamin C. University of Maryland medical center.www//Wikipedia.

Retrieved 31th, March, 2008.

lxxiv

Anonymous, (2009). Plasma lipoproteins. Composition, structure and biochemistry. http//www.

Lipid library.co.uk. Retrieved 31th, March, 2008.

Anyia, Y. and Naito, A. (1993). Oxidative stress-induced activation of microsomal glutathions

transferase in isolated rat liver. Biochemical Pharmacology. 45: 37-42.

Ashour, M. N., Salem, S. I. and.El-Gadban, H. M. (1999). Antioxidant status in children with

protein energy malnutrition (PEM) living in Cairo. European Journal of Clinical

Nutrition. 53: 669-673.

Assmann, G., Jabs, H. U., Kohnert, U., Nolte, W., and Schriewer, H. (1984). Determination of

Low density lipoprotein (LDL)–Cholesterol. Clinical Chimica. Acta. 140: 77–83.

Bachrach, L.K., Katzman, D.K. and Litt, I.F. (1991). Starvation. Journal of Clinical

Endocrinology and Metabolism. 72: 602-606.

Banks, W. A. and Lebel, C. P. (2002). Strategies for the delivery of Leptin to the CNS. Journal

of drug Targeting. 10:297-308.

Barthel and Grit (1998). Hepatotoxicity of Valproate in isolated Rat Hepatocytes: Influence of

prooxidative Agents and starving. Freien Universitat Berlin. Objekt-metadaten @

Dissertation line, Berlin. Pp. 2.

Bast, A. (1993). Oxidative stress and calcium Hemeostasis, in DNA and free Radicals,

edited by B. Halliwell and O.I. Aruoma, Ellis Horwood, London. Pp. 95-108.

Betteridge, D. J. (2000). What is oxidative stress? Metabolism. 4:3-8.

Bijlani, R. L., Sud, S. Gandhi, B. M. and Tandon, B. N. (1985). Relationship of examination

stress to serum lipid profile. Indian Journal of Physiology and Pharmacology. 30:22-

30.

Blokhina, O., Virolainen, E. and Fagerstedt, K.V. (2002). Antioxidants, oxidative damage and

oxygen deprivation stress: A Review. Annals of Botany. 91:17-194.

Boden, G, Chen, X. and Mozzol, M (1996). Effect of fasting on serum leptin in normal human

subjects. Journal of Clinical Endocrinology and Metabolism. 81:265-275.

Boswell, T, LI, Q and Takeuchi, S. (2002). Neurons expressing neuropeptide Y mRNA in

infundibular hypothalamus of Japanese quait are activated by fasting and co-express

agouti-related protein mRNA. Molecular Brain Research. 100:31-42.

Brown, W. V. (2007). High-density lipoprotein and Transport of cholesterol and triglyceride in

blood. Journal of Clinical Lipidology. 1:7-19.

lxxv

Burton, G. W. and Ingold, K. U. (1989). Vitamin E as in vitro and in vivo antioxidant. Annals

of New York Academy Science. 570:7-22.

Buyse, J, Janssens, Ka, Vander Geyten, S, Vanas, P, Decuypere, E, Darras, VM (2002). Pre and

post-prandial changes in plasma hormone and metabolic levels and hepatic deiodinase

activities in meal fed broiler chickens. British Journal of Nutrition. 88:641-653.

Cahill, G. F. Jr (1970). Starvation in man. New England Journal of Medicine. 282: 668-675.

Carmody, R. J., Cotter TG (2001). Signaling apoptosis: A radical approach. Redox Report. 6:

77-90.

Carnelio, S., Khan, S. A. and Rodrigues, G.S (2007). Free Radicals and antioxidant therapy in

clinical practice. To Be or not to Be? JCPSP. 17(3):173-174.

Cederbaum, A.I. (1989). Role of Lipid Peroxidation and Oxidative stress in alcohol toxicity.

Free Radicals. Biology and Medicine. 7: 537-539.

Chance B, Sies H and Boveris A (1979). Hydroperoxide metabolism in mammalian organs.

Physiology Review. 59: 527-605.

Chaney S.G. (1992). Principles of nutrition II: Macronutrients;in textbook of Biochemistry

with chemical correlations. Thomas Delin John Wilby and Sonsinc. Pp. 115-147.

Charttejea, M.N. and Shinde, R. (2005). Textbook of medical biochemistry. 6th

ed. Jaypee

Brothers. Pp. 102-108.

Chaudiere J, and Ferrari-Iliou R. (1999). Intracellular antioxidants: from chemical to

biochemical mechanisms. Food chemical Toxicology. 37: 949-962.

Chield, R., Brown, S. and Day, S. (1999). Changes in indices of antioxidant status, lipid

peroxidation and inflammation in human skeletal muscle after eccentric muscle action.

Clinical Science Calch. 96: 106-115.

Collier S, Forchielli M.l, and Lo, C.W (1996). Parentereal nutrition requirements. In: Baker

JR RD, Baker S, Davis Am – pediatric parenteral nutrition. New York: Chapman and

Hall. Pp. 64.

Cotran, R.S., Kumar, V. and Collins, T. (1999). Robbin’s pathological basis of disease. 6th

edn.

Thomson press (1) Ltd, Noida. Pp. 1-31.

De la vega JM, Diazj, Serrano E, and Carbonell LE (2002). Oxidative stress in critically ill

patients with systemic inflammatory response syndrome. Critical Care Medicine. 30:

1782-1786.

De Zwart, L.L, Meerman, J.H, Commaduer, J.N and Vermuelen, N.P (1999). Biomarkers of

free radical damage applications in experimental animals and in humans. Free Radical

Medicine. 26: 202-226.

lxxvi

Di Giulo, R.T., Benson, W.H., Sanders, B.M and Van Veld, P.A(1995). Biochemical

mechanismsof contaminant Metabolism, adaptation and toxicity In: Rand, G. (Ed.),

Fundamentals of Aquatic Toixcology Effects Environmental fate, and Risk Assessment.

Taylor and francis, London. Pp. 523-561.

Di Simplicio, P., Rossi, R.,Falcinelli, S., Ceserani, R. and Formento, M.L (1997). Antioxidants

status in various tissue of the mouse after fasting and swimming. European Journal of

Applied Physiology. 76:302-307.

Disbrey, B.D. and Rack, J.H. (1970). Histological Laboratory Methods. E and S livingstone,

Edinburgh and London, Pp. 1-40.

Domenicali, M., Caraceni, P., Vendemiale, G., Grattagliano, L, Nardo, B., Dall’ Agata, M.,

Santomi, B., Trevisani, F., Cavallari, A., Altomare, E., and Bernardi, M. (2001). Food

deprivation exacerbates mitochondrial oxidative stress in rat liver exposed to ischemia-

reperfusion injury. Journal of Nutrition. 131:105-110.

Dou, S., Masuda, R., Tanaka, M and Tsukamoto, K. (2002). Feeding resumption,

morphological changes and mortality during starvation in Japanese flounder larvae.

Journal of Fish Biology. 60: 1363-1380.

Draper, H.H., McGirr, L.G. and Haldey, M (1986). The metabolism of Malondialdehyde.

Lipids. 21: 305-307.

Drury, R.A., Wallington, A. and Cameron, S.R. (1967). In: Carlleton’s Histological

Techniques. Oxford University Press. New York. Pp. 1-420.

Dypbukt J.M. Ankarcrona M, Burktt M, Sjtholm A, Strom-Orrenius, S and Nicotera, P (1994).

Different proxidant leelks stimulate growth, trigger apoptosis or produce necrosis of

insulin-secreting RINM5f cells: the role of intracellular polyamines. Journal Biological

Chemistry. 269:30553-30560.

Evans P, and Halliwell, B (2001). Macronutrient: oxidants/antioxidant status. Journal of

Nutrition. 85(2): 567-574.

Fainaru M, Funke H, Boyles J.k , and Ludwig E.H(1988). Metabolism of Canine beta- very

low density lipoproteins in normal and cholesterol fed dog. Arteriosclerosis. 8: 130-

139.

Farinati F, Carin R, Degan P, Rugge M, Mario FD, Bonvicini P and Naccarato R (1998).

Oxidative DNA damage accumulation in gastric carcinogenesis. Gut. 42:351-156.

Fields AL, Falk N, Cheema-Dhadli S, and Halperin ML (1987). Accelerated loss of lean body

mass in fasting rats due to activation of pyruvate dehydrogenase by dichloroacetate.

Metabolism and Clinical Experience. 36: 621-624.

Frei, B., England, L and Ames, B.N. (1989). Ascorbate is an outstanding antioxidant in human

blood plasma. Product of National Academy of Science. 86: 6377-6381.

lxxvii

Frieri, B., Stocker, R and Ames, B.N. (1988). Antioxidant defenses and lipid peroxidation in

human blood plasma. Product of National Academy of Science. 85: 9748-9752.

Gaby, S.K. and Singh,V.N (1991). “Vitamin C” – Vitamin intake and Health: A Scientific

Review, Gaby, S.K. Bendich, A; Singh, V. and Machlin, L.(eds). Marcel Dedder,N.Y.

Clinical Nutrition Review. 5: 110-129.

Gardner, H.W (1989). Oxygen radical chemistry of Polyunsaturated fatty acids. Free Radicals.

Biology and Medicine. 7: 65-86.

Gate L, Paul J.Ba GN, Tem KD, and Tapiero H. (1997). Oxidative stress induced in

pathologies: The role of antioxidants. Biomedical Pharmacotherapy. 53: 169-180.

Gaudin, A.J. (1991). Cardiovascular system anatomy. In: Encyclopedia of human biology. Vol

1. Renato Dulbeccoo Ed. Academic press Incorporated, NewYork. Pp. 50.

Gomi, F., and Matsuo, M. (1998). Effects of starving and food restriction on the antioxidant

enzymes activity of rat livers. Journal of Gerontology, Applied Biological Sciences and

Medical Science. 53: 1361-1367.

Goode, H.F, Cowley, H.C, Walker B.E., Howdle, P.D, and Webster N.R (1995). Decreased

antioxidant status and increased lipid peroxidation in patients with septic shock and

secondary organ dysfunction. Critical Care Medicine. 23: 646-661.

Goodhart, R.S. and Shils, M.E. (1973). Modern Nutrition in health and disease dictotherapy.

Lea and febiger. Pp. 245-253.

Gray, J.V., Petsko, G.A., Johnston. G.C. Ringe, D., Singer, R.A. and Werner-Washburne, M.

(2004). Microbial and Molecular Biology Revision. 68: 187-206.

Griffith, O.W. (1999). Biologic and Pharmacologic regulation of mammalian glutathione

synthesis. Free Radical in Biology and Medicine. 27: 922-935.

Guderley, H., Lapoinite, D., Bedard, M., and Dutil, J.D. (2003). Metabolic priorities during

starvation: enzyme sparing in liver and white muscle of Atlantic cod, Gadus Morhua L.

Comp. Biochemical Physiology. 135: 347-356.

Guichardant M, Vallete – TalBil, Cavadinic, Crozier, G and Berger M (1994).

Malondialdehyde measurment in urine. Journal of Chromatography and Biomedicine

Applied. 655: 112-116.

Gutteridge J M.C. (1995). Lipid peroxidation and Antioxidants as Biomarkers of tissue

damage. Clinical Chemistry. 4112: 1819-1828.

Gutteridge J.M and Halliwell, B (1990). The measurement and mechanism of Lipid

peroxidation in biological systems. Trends in Biochemical Science. 15: 129-135.

Gutteridge, J.M.C (1988). Lipid peroxidation, some problems and concepts. Editted by B.

Halliwell. Bethesda, MD, FASEB for Upjohn Co. Pp: 9-19.

lxxviii

Halliwell B (1990). How to characterize a biological antioxidant. Free Radical Research

Communication. 9: 163-167.

Halliwell, B (1994). Free radicals and antioxidants: A personal view. Nutritional Review. 52:

253-265.

Halliwell, B and Gutteridge, J.M. (1999). Free radicals in biology and medicine. 3rd

ed.

Oxford: Oxford University Press. Pp. 617-783.

Halliwell, B. and Gutteridge, J. M. C. (2006). Free radicals in Biology and Medicine. 4th

Edition, Oxford University Press, Oxford.

Halliwell, B. (1991). Reactive oxygen species in living systems: Biochemistry and Role in

Human Disease. American Journal of Medicine. 91: 145-195.

Halliwell, B. (1996). Oxidative stress, nutrition and health experimental strategies for

optimization of nutritional antioxidant intake in humans. Free radical resolution. 25:

57-74.

Halliwell, B. (1997). Antioxidants and human diseases: a general introduction. Nutrition

Review. 55: 544-574.

Halliwell, B. and Gutteridge, J.M.C. (1984). Lipid peroxidation, oxygen radicals, cell damage

and antioxidant therapy. Lancet. 11: 1396-1397.

Halliwell, B. and Gutteridge, J.M. (1990). Role of free radicals and catalytic metal ions in

human disease: an overview. Methods in Enzymology. 186: 1-85.

Halliwell, B., and Gutteridge, J.M.C (2000). Free radicals in Biology and medicine, 3rd

ed.

Oxford University Press Oxford. Pp. 225-233.

Halliwell, B., March, M.A., Chirico, S. and Aruoma, O. (1995). Free radicals and antioxidants

in food and in vivo: what they do and how they work. Critical Review Food Science

Nutrition. 35: 7-20.

Hayes, J.D. and Mclellan, L.I. (1999). Glutathione and glutathione dependent enzymes

represent a co-ordinately regulated defense against oxidative stress. Free Radical

Research. 31: 273-300.

Hazen S.l, Hsuff, Mueller D.M, Crowley J.R,and Heinecke J.W (1996). Human neutrophils

employ chlorine gas as an oxidant during phagocytosis. Journal of Clinical Investment.

98: 1283-1289.

Hem Lata, Ahuja, G.K and Narang, A.P.S (2002). Effect of starvation stress on lipid

peroxidation and lipid profile in rabbits. Indian Journal of Physiology and

Pharmacology. 46: 371-374.

Hemnani, T. and Parihar, M.S. (1998). Reactive oxygen species and oxidative DNA damage.

Indian Journal of Physiology and Pharmacology. 42: 440-452.

lxxix

Hevonoja, T., Pentikainen, M. O., Ityvonen, M. T., Kovanen, P. T. and Ala-Korpela, M.

(2000). Structure of low density lipoprotein (LDL) particles: basis for understanding

molecular changes in modified LDL. Biochim. Biophys. Acta, 1488: 189 – 210.

Heyland Dk, Dhaliwal R, Berger M, and Suchner,U. (2003). Low endogenous stores of

antioxidants are associated with increased free radical generation Free Radical

Research. 31:1048-105.

Hoerr, RA ,Matthews.E , Bier D.M and Yong V.R (1993). Effect of protein restriction and

acute refeedin g on leucine and lysine kinetics in young men. American Journal of

Physiology. 264: 567-575.

Iqbal, S.P.Shafian, Mehhoobali, N. and Abbasik,(2004).Deficiency of vitamin C in

South Asia. British Medical Journal. 328: 807-810

Jang H.H, and Surh, Y.J (2003). Protective effects of resveratrol on B-amyloid induced

oxidative Pc12cell death. Free Radical Biology and Medicine. 34: 1100-1110.

Janzen, I.G. (1990). Spin trapping and associated vocabulary. Free Radical Resolution

Communication. 9: 163-167.

Jimena Abiles, Antonio Perezde la cruz, Jose Castano Manuel Rodriguez-Elvira, Eduardo

Aguayo,Rosario Moreno-Torres, Juan Llopis, Pilar Aranda, Sandro Arguelles, Antonio

Ayala, Alberto Machado de la Quintana and Elena Maria Planells (2006). Oxidative

stress in increased in critically ill patients according to antioxidant vitamins intake,

independent of severity: a cohort study. Critical Care. 10:146.

Jonas, A. (2002). Lipoprotein structure. In: Biochemistry of lipids, lipoproteins and membranes

(4th

Edn). Pp 483 – 504.

Kalm, L. M. and Semba, R.D. (2005). They starved so that others be better fed. Journal of

Nutrition. 135(6): 1347-1352.

Kang, S., Song, J., Kang, H., Kim, S., Lee, Y. and Park, D. (2003b). Insulin can block

apoptosis by decreasing oxidative stress via phosphatidylinositol 3-kinase and

extracellular signal-regulated protein kinase-dependent signaling pathways in HepG2

cells. European Journal of Endocrinology. 148: 147-155.

Kekwick, A. and Pawan, G. L. S. (1957). Metabolic studies in human obesity with isocaloric

diets high in fat, protein or carbohydrates. Metabolism and Clinical Experiment. 6: 447.

Kelly, F. J. (1998). Use of antioxidants in the prevention and treatment of disease. Journal of

Clinical Chemistry. 10: 21-23.

Kettelhut, I. C., Wing, S. S. and Goldberg, G. (1989). Endocrine regulation of protein

breakdown in skeletal muscle. Diabetes and Metabolism Revision. 5: 227-245.

Kettelhut, IC; Wing, SS and Goldberg, A.L (1988). Endocrine regulation of protein breakdown

in skeletal muscle. Diabetes and Metabolism Revision. 4: 751-772.

lxxx

Keys, A; Brozek, J, and Henschel, A (1950). The biology of Human starvation Minneapolis.

University of Minnesota press. Pp. 174.

Kim H.J, So Y.J, Jang J.H, Lee J.S, OH Y.J, and Surh Y.J (2001). Differential cell death

induced by salsolinol with and without copper. Possible role of reactive oxygen species.

Molecular Pharmacology. 60: 440-449.

Koppenol, W.H (1993). The centennial of the fenton reaction . Free Radical Biology and

Medicine. 15: 645-651.

Koppenol, W. H. (2001). “The Harber-Weiss Cycle – 70 years later”. Redox Report, 6(4): 229–

234.

Kowalski, D.P., Feeley, R.M., and Jones, D.P (1990). Use of exogenous glutathione for

metabolism of peroxidized methyl linoleate in the small intestine.Journal of Nutrition.

120: 1115-1121.

Kuehl F.A, and Egan R.W (1980). Prostaglandins, arachidonic acid and inflammation. Science.

210: 978-984.

Lamarche B, Despres J.P, Moorjani S, Cantin B, Dagenais G.R, Lupien P.J (1996).

Triglycerides and HDL-cholesterol as risk factors for ischemic heart disease. Results

from the quebec cardiovascular study. Atherosclerosis. 119: 235-245.

Lamosova, M. Macajova, M. and Zeman, M (2004). Effects of short term fasting on selected

physiological functions in Adult male and female Japanese Quait. Acta Vet. Brno.73:

9-16.

Laurindo F.R, Da Luzpl, Unit L, Rocha, T.F, Jaeger R.G, and Lopes E.A.(1991). Evidence for

superoxide radical-dependent coronary vasospasm after angioplasty in intact dogs.

Circulation. 83: 1705-1715.

Lee, C.M., Weindruch, R., and Aiken, J.M (1997). Age-associated alterations of the

mitochondrial genome. Free Radical Biology and Medicine. 22: 1259-1269.

Lemberg, A., Schreier, L., Romay, S., Fernandez, M., Roseelo, D., Gonzales, S., Perazzo, J.,

Filinger, E and Tomaro, M (2007). Involvement of serum apolipoprotein AI and B100

and lecithin cholesterol acyl transferase in alcoholic cirrhotics. Annals of Hepatology.

6(4): 227-232.

Lenaz, G. (1998). Role of mitochondria in oxidative stress and aging. Biochemistry Biophysics

Acta. 694: 69-93.

Livingstone, D.R. (2001). Contaminant reactive oxygen species production and oxidative

damage in aquatic organisms. Mar. pollution Bull. 42: 656-666.

Lomaestro, B. and Malone, M (1995). Glutathione in health and disease: Pharmacotherapeutic

Issues. Annals of Pharmacotherapy. 29: 1263-1273.

lxxxi

Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). Protein measurement

with the Folin phenol reagent. Journal of Biology and Chemistry. 193: 265-275.

Lynch, AM, Moore M, Craig S, Lonergan, PE; Martin, DS; and Lynch, M.A (2003). Analysis

of interleukin- 1 beta-induced cell signaling activation in rat hippocampus following

exposure to gamma irradiation. Journal of Biology and Chemistry. 278: 51075-51084.

Marks, V. and Dawson, A. (1965). Rapid stick method for determining blood glucose

concentration. British Medical Journal. 1: 25-29.

Martensson, J. (1986). The effect of fating on leukocyte and plasma glutathione and sulfur

amino acid concentrations. Metabolism. 35: 118-121.

Martensson, J. and Meister, A. (1991). Glutathione deficiency decreases tissue ascorbate spares

glutathione and protects. Product of National Academy of Science, USA, 88: 4656-

4660.

Mayes, P.A. (2000). Lipids of physiological significance. Harpers Biochemistry 25th

ed

Appleton and Lange, USA. Pp.169.

Mayne, S. T. (2003). Antioxidant nutrients and chronic disease: Use of biomarkers of exposure

and oxidative status in epidemiologic search. Journal of Nutrition. 133(3): 9339-9409.

Meister, A. (1992). On the antioxidant effects of ascorbic acid and glutathione. Biochemistry

and Pharmacology. 44: 1905-19115.

More, K. and Roberts, L. J. H. (1998). Measurements of lipid peroxidation. Free radical

Resolution. 28: 659-671.

Mortimore, G. E. and Poso, A. R. (1987). Lar protein catabolism and its control during nutrient

deprivation and supply. Annual Review of Nutrition. 7: 539-564.

Murray, R. K., Granner, D. K., Mayers, P. A. and Rodwel, V. W. (2003). Harper’s

Biochemistry. 26th

ed. New York: Mc Grawhill Publishers. Pp. 109.

Mustafa, M. G. (1990). Biochemical Basis of ozone Toxicity. Free Radical Biology and

Medicine. 9: 245-265.

Nelson, D. L. and Cox, M. M. (2005). Leninger’s Principles of Biochemistry.4th

ed. New York.

Worth Publishers. Pp. 256.-257.

Nishikim, M. (1975). Oxidation of ascorbic acid with superoxide anion generated by the

xanthine-xanthine oxidase system. Biochemistry and Biophysics Acta, 876: 294-299.

Nurlan,M. C. and Garlick, P. J. (1989). Influence of nutrient intake on protein catabolism in

starved and infected rats. Journal of Clinical Nutrition. 30: 1510-1511.

Nwanjo, H. U. and Oze, G. O. (2007). Oxidative Imbalance and Non-Enzymatic Antioxidant

status in pulmonary tuberculosis infected subjects: Carcinogenic potential. Pakistan

Journal of Nutrition. 6(6): 590-592.

lxxxii

Ogugua, V. N. and Alumunah, E. O. (2007). Does oxidative stress involve in diabetes mellitus?

A case study of lipid peroxidation, Antioxidants and lipid levels in Alloxan Induced

Diabetic Rabbits. Bio-Research. 5(2): 265-268.

Ohkawa, H., Ohishi N., and Yagi, K., (1979). Assay for lipid peroxides in animal tissues by

thiobarbituric acid reaction. Annals of Biochemistry. 95: 351-358.

Orrenius, S., D.J. Mc Conkey, G. Bellowmo, and P. Nicotera (1989). Role of Ca2+

in Toxic cell

killing .Trends in Pharmacological Science. 10: 281-285.

Padh, H. (1990). Cellular functions of Ascorbic acid. Biochemistry of cell Biology. 68: 1163-

1173.

Patockova, J. Marhol, P. and Andel,M(2003).Oxidative stress in the Brain Tissue of Laboratory

mice with acute post Insulin Hypoglycemia. Physiology Research. 52:131-135.

Pryor, W. A. (1994). Mechanism of Radical formation from reactions of ozone with target

molecules in the lung. Free Radical Biology and Medicine, 17: 451-465.

Rajeshwar, K. (1996). Photochemical strategies for abating environmental pollution. Chemistry

Industrial. 12: 454-458.

Ramasarma, T. (1982). Generation of H2O2 in biomembranes. Biochemistry Biophysics Acta.,

694: 69-93.

Rath, M. (1993). Eradicating heart disease. Health Now. San franciso, CA. Pp: 153-162.

Rice, E.C. and Burdon, R. (1993). Free radical-lipid interaction and their pathological

consequences. Progress lipid Resolution. 32: 71-110.

Robinson, M. K., Rustum, R. R., Chambers, E. A, Rounds, J. D., Wilmore, D. W. and Jacobs,

D. O. (1997). Starvation enhances hepatic free radical release following endotoxemia.

Journal of Surgical Research, 69: 325- 330.

Rose, R. C and Bede A. M (1993). Biology of free radical scavengers: an evaluation of

ascorbate. The FASER Journal, 7: 11135 – 1142.

Samiec, P. S, Drews – Botsch, C. and Flayg, E. W. (1998). Glutathione in human plasma:

Decline in association with aging, age – related macular degeneration and diabetes.

Free Radical Bioliogy and Medicine, 24: 699 – 704.

Samra, J. S., Clark, M. L. Humphrey, S. M., MacDonald, I. A. and Frayn, K. N. (1996).

Regulation of lipid Metabolism in adipose tissue during early starvation. American

Journal of Physiology. 271: 541 – 546.

Sarton, D. R. S., Migliorini, R. H., Veiga, J., Moura, J. L., Kettelhut, I. C. and Linder, C.

(1995). Metabolic adaptations induced by long term Fasting in quails. Communication

in Biochemistry and Physiology. 111: 487 – 493.

lxxxiii

Savendahl, L. and Underwood, L. E. (1999). Fasting increases serum total cholesterol, LDL

cholesterol and Apolipoprotein B in Healthy, Nonobese Humans. Journal of Nutrition,

129: 2005-2008.

Schafer F.Q, and Buettner G.R (2001). “Redox environment of the cell as viewed through the

redox state of the glutathione disulfide/glutathione couple”. Free Radical in Biology

and Medicine. 30(11): 1191 – 1212.

Sevanian, A. and Hochstein, P. (1985). Mechanism and consequences of lipid Peroxidation in

biological system. Annual Review in Nutrition. 5:365 – 370.

Shimeno, S., Shikata, T., Hosokawa, H., Masumoto, T. and Kheyyali, D. (1997). Metabolic

response to feeding rates in common carp, Cyprinus carpio. Aquaculture. 151: 371-377.

Shimizu, M. and Morita, S. (1992). Effects of feeding and fasting on hepatolobular distribution

of glutathione and Cadmium-induced hepatotoxicity. Toxicology. 75: 97-107.

Sies H. (1991). Oxidative stress: introduction. Oxidative stress. xv-xxii London Academic

Press, San Diego. Pp.53.

Sies, H. (1986). Biochemistry of oxidative stress. Angew. Chemistry International Edition. 25:

1058 – 1071.

Sies, H. (1999). Glutathione and its role in cellular functions. Free Radical in Biology and

Medicine. 27: 916 – 921.

Singhal, S. Agarwal, D.K. and Srivastara, U. (1997). Effect of immobilization stress on lipid

profile. Indian Journal Physiology and Applied Science. 51: 138-143.

Skipski, V.P (19720). In: Blood Lipids and Lipoproteins.Quantitation, Composition and

Metabolism. Pp. 471-483

Slater, T. F. (1984). Free radical mechanism in tissue injury. Biochemistry Journal, 222: 1-15.

Snell, F. D. and Snell, C. T. (1962). Colorimeter of Analysis. Vol. 3; D. Van Nostrand Co. Inc.,

New York. Pp: 119,469.

Sodergreen, E. (2000). Lipid peroxidation in vivo: Evaluation and application of methods for

measurement. Comprehensive summaries of Uppsala dissertations from the Faculty of

Medicine, 949: 91-554-4791-0.

Steffens, W. (1989). Principles of fish Nutrition.. Ellis Horwood, Chichestor. Pp. 384.

Stuck, K. C., Watts, S. A. and Wang, S. Y. (1996). Biochemical responses during starvation

and subsequent recovery in post larval pacific white shrimp, Penaeus vannamei. Marine

Biology, 125: 33-45.

Swaner, J. C. and Connor, W. E. (1975). Hypercholesterolemia of total starvation: its

mechanism via tissue mobilization of cholesterol. American Journal of Physiology,

229: 365-369.

lxxxiv

Tampo, Y. and Yonaha, M. (1990). Vitamin E and glutathione are required for preservation of

microsomal glutathione S-transferase from O.S in microsomes. Pharmacology and

Toxicology, 66: 259-265.

Tatli, M. M., Vural, H., Koc, A., Kosecik, M. M., Vural, H., Koc, A., Kosecikm, and Atas, A.

(2001). Altered antioxidant status and increased lipid peroxidation in marasmic

children. Pediatrics International, 42: 289-292.

Thorens, B., Flier, J. S., Lodish, H. F. and Katin, B. B. (1990). Differential regulation of two

glucose transporters in rat liver of fasting and refeeding and by diabetes and insulin

treatment. Diabetes, 39: 712 – 719.

Tietz, N. W. (1990). Clinical Guide to Laboratory Tests. 2nd

ed. W. B. Saunders Company,

Philadelphia, U. S. A. Pp.554 – 556.

Tiidus, P. M. and Houston, M. E. (1994). Antioxidant and oxidative enzyme adaptations to

vitamin E deprivation and training. Medical Science, Sports and Exercise, 26: 354-359.

Tisan, L., Cai, A. and Bowen, R. (1995). Effect of caloric restriction on age related oxidative

modification of macromolecules and lymphocyte proliferation in rat. Free Radical

Biology and Medicine. 19: 859-865.

Tiwari, A. K. (2001). Imbalance in antioxidant defense and Human diseases: Multiple approach

of natural antioxidants therapy. Current Science, 81(9-10): 1179-1187.

Tsaik, Hsu T, Kong C, Link, L.U.F. (2000). Is the endogenous Peroxil radical scavenging

capacity of plasma protective in systemic inflammatory disorder in humans? Free

Radical Biology and Medicine, 28: 926-933.

Uzoegwu, P. N. (2001). Correlation of lipid peroxidation index with concentration of sickle

Haemoglobin of Malaria parasite infected and uninfected subjects of different

Haemoglobin groups in Uga. Nigerian Journal Biochemistry and Molecular Biology,

16(3): 1255-1305.

Vaisman N., Sklan, D. and Dayan, Y. (1990). Effect of moderate semi-starvation on plasma

lipids. International Journal of Obesity, 14: 989-996.

Vance, J. (2002). Assembly and secretion of lipoproteins. In: Biochemistry of lipids,

lipoproteins and membranes (4th

Edn). Pp 505–526.

Wallin, B., Rosengren. B., Shertzer, G. H. and Camejo, G. (1993). Lipoprotein oxidation and

measurement of thiobarbituric acid reacting substances formation in a single microtitre

plate:its use for evaluation of antioxidants. Analytical Biochemistry, 208:10-15.

Wang, T., Hung, C. C. and Randall, D. J. (2005). Annual Review in Physiology, 68:

040104 - 105739; In press published Online October 19, 2005.

Wannemacher, R. W. (1975). In: Total parenteral nutrition: Premises and promises, edited by

H.Ghadimi. New York: John Wiley and Sons, Inc. Pp. 85.

lxxxv

Wannemacher, W. and Dinterman, E (1977). Total body protein catabolism in starved and

infected rats. Journal of Clinical Nutrition, 30: 1510-1511.

Weiss, S. J., Lampert, M. D., Test, S. T. (1983). Long-lived oxidants generated by human

neutrophils characterization and bioactivity. Science, 222: 625-628.

Whiteman, M, Jenner, A. and Halliwell, B. (1997). Hypochlorous acid-induced-based

modification in isolated calf thymus DNA. Chemistry Research. 10: 1240-1246.

Wu, D. and Cederbaum A. I. (2003). Alcohol, oxidative stress, and free radical damage.

Alcohol Research and Health, 27: 277-84.

Yagi, K. (1987). Lipid peroxides and human diseases. Chemistry, Physics and Lipids, 45: 337-

351.

Yang, R. D. (1986). Response of alanine metabolism in humans to manipulations of dietary

protein and energy intakes. American Journal of Physiology, 250: 39- 46.

Younes, M. (1999). Free radicals and reactive oxygen species in: Toxicology, edited by H.

Marquardt, S.G. Schafer, R.O. Mc Clellan and F. Welsch, Academic press USA. Pp.

111-124.

Young, V. R. and Marchini, J. S. (1990). Mechanisms and Nutritional significance of

metabolism response to altered intakes of protein and amino acids, with reference to

nutritional adaptation in humans. American Journal of Clinical Nutrition, 51: 270-289.

Yu, B. P. (1994). Cellular defences against damage from reactive oxygen species. Physiology

Review, 74: 139-162.

APPENDICES

lxxxvi

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