Cigarette smoking and infection

EFFECTS OF SMOKING ON

SERUM TOTAL ANTIOXIDANT CAPACITY

Dissertation submitted to

THE TAMIL NADU DR. M.G.R. MEDICAL UNIVERSITY In partial fulfilment of the Regulations for the award of the degree of

(M.D. PHYSIOLOGY) BRANCH-V

THANJAVUR MEDICAL COLLEGE AND HOSPITAL THE TAMILNADU Dr. M.G.R. MEDICAL UNIVERISTY

CHENNAI, INDIA MAY – 2020

Registration Number: 201715201

CERTIFICATE

This dissertation entitled “EFFECTS OF SMOKING ON SERUM TOTAL ANTIOXIDANT CAPACITY” is submitted to The Tamil Nadu Dr. M.G.R. Medical University, Chennai in partial fulfilment of the regulations for the award of M.D., Degree in Physiology in the Examinations to be held during May 2020.

This Dissertation is a record of fresh work done by the candidate Dr.AMRITHA.R.S, during the course of the study (2017-2020). This work was carried out by the candidate herself under my supervision.

Prof.Dr.KUMUDHA LINGARAJ, Prof. Dr.R.VINODHA, M.D.,

M.D (ANAES) D.A., Professor & HOD

The Dean, Department of Physiology,

Thanjavur Medical College, Thanjavur Medical College,

Thanjavur – 613004. Thanjavur – 613004.

Place: Thanjavur Date:

DECLARATION

I solemnly declare that the Dissertation titled “EFFECTS OF SMOKING ON SERUM TOTAL ANTIOXIDANT CAPACITY” is done by me at Thanjavur Medical College, Thanjavur

The Dissertation is submitted to the Tamil Nadu Dr. M.G.R. Medical University, Chennai, in partial fulfilment of requirements for the award of M.D. Degree (Branch V) in Physiology.

Place : Thanjavur Dr.Amritha.R.S

Date : Post Graduate in Physiology,

Thanjavur Medical College, Thanjavur

ANTI PLAGIARISM REPORT

CERTIFICATE – II

This is to certify that this dissertation work titled “EFFECTS OF SMOKING ON SERUM TOTAL ANTIOXIDANT CAPACITY” of the candidate Dr.AMRITHA.R.S with registration Number 201715201 for the award of M.D., in the branch of PHYSIOLOGY. I personally verified the urkund.com website for the purpose of plagiarism Check. I found that the uploaded thesis file contains from introduction to conclusion pages and result shows 3 percentage of plagiarism in the dissertation

Place: Thanjavur Prof. Dr.R.Vinodha, M.D.,

Date: Professor & HOD

Department of Physiology,

Thanjavur Medical College,

Thanjavur-613004

ACKNOWLEDGEMENT

I express my sincere thanks to my guide PROF. DR. R. VINODHA, M.D., Professor and Head of the Department of Physiology, Thanjavur Medical College, Thanjavur for the constant guidance, suggestions and for being a great source of inspiration for my entire duration of the study.

I would like to thank Dr.KUMUDHA LINGARAJ, Dean, Thanjavur Medical College, Thanjavur, for permitting me to do this work at Thanjavur Medical College, Thanjavur.

I would like to thank all of my subjects who participated and for their kind co- operation for this study.

I owe my sincere gratitude to my ever loving parents, husband, in-laws, my dear son and my colleagues for the continued encouragement and support.

Finally, I thank almighty God for giving me the strength and patience especially during all the challenging moments in completing this thesis. I am truly grateful for your exceptional love and grace during this journey.

S.NO CONTENTS PAGE NO

1 ABSTRACT

2 INTRODUCTION ^1-3

3 AIMS & OBJECTIVES ⁴

4 REVIEW OF LITERATURE 5-48

5 METHODS AND MATERIALS ^49-54

6 RESULTS ^55-71

7 DISCUSSION ^72-78

8 CONCLUSION ^78-79

9 BIBLIOGRAPHY 10

ANNEXURES

i ABBREVIATION ii PROFORMA

iii INFORMED CONSENT FORM

iv MASTER CHART

ABSTRACT TOPIC:

EFFECTS OF SMOKING ON SERUM TOTAL ANTIOXIDANT CAPACITY AIM:

The Aim of the study was to evaluate the effects of smoking on Serum Total Antioxidant Capacity in smokers and to compare the levels of TAC between adult male smokers and non-smokers.

METHODS:

This study was a case control study that included 80 subjects of which 40 were non-smokers and 40 were smokers in the age group of 30-50 years. The non- smokers were in control group and smokers in cases group. All the participants were non-athletes, non-alcoholics and had not participated in regular exercise / diet programs for the period preceding 6 months. Smoking history of at least 10 cigarettes a day for 5 years was considered as inclusion criteria in smokers group. Patient with Type II Diabetes mellitus, Respiratory diseases, Cardio-vascular diseases, Cancer, Kidney dysfunction and other chronic diseases were excluded in this study. Blood samples were collected from 8-9 AM after fasting 12 hours overnight. Serum TAC was measured by FRAP spectrophotometric assay.

RESULTS:

The results were considered statistically significant if the p value was < 0.05. In our study, serum TAC was evaluated in smokers and non-smokers. The study findings showed that serum TAC levels were lower in smokers when compared to non-smokers. A negative correlation was found between Serum TAC and duration of smoking.

CONCLUSION:

Our study revealed decreased TAC levels in smokers which may be helpful in predicting oxidative stress related disease earlier and can reduce the progression of the disease. Hence we conclude that quitting smoking, exercise, good nutrition and use of antioxidants supplementation can improve the TAC levels in smokers.

Key Words: Smoking, Cigarette, Oxidative stress, TAC

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INTRODUCTION

Cigarette smoking is one of the most important cause of early and preventable death and a significant public health concern throughout the globe. It has so many serious effects on human health such as chronic obstructive pulmonary disease, atherosclerosis and cancer ⁽¹⁾

Cigarette smoke is highly complex aerosol and composed of 7,357 chemical compounds of different classes ^{(1, 2)}

Two main phases have been identified in cigarette smoke: tar phase and gas phase.

Both phases are rich in oxygen, carbon, nitrogen and free radicals. Analysis has shown that a puff of cigarette contains about 1014 free radicals in the tar phase and 1015 free radicals in the gas phase ⁽³⁾

When we smoke cigarettes, many chemicals in the tar and gas phase enter in our body through our lungs and reach the tissues ⁽¹⁾

Imbalance between antioxidants and oxidants formed in the body is a condition called oxidative stress. Examples of oxidants are Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) ⁽⁴⁾

Smoking produces a large amount of reactive oxygen species which has an influence on normal cellular function and causes changes in the inflammatory markers.

Many researchers have found that there is a direct relationship between oxidative stress and pathological diseases ⁽⁵⁾

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Reactive oxygen species are chemically reactive molecules which contains oxygen. It is a highly reactive atom and potentially damaging molecules commonly called as ―free radicals‖. There are so many types of reactive oxygen species such as hydroxyl radical, superoxide anion radical, hydrogen peroxide etc., These reacts with membrane lipids, nucleic acids, proteins and enzymes and small molecules which causes damage to the cells ^(6,7)

Our body‘s first line of defense against these damaging effects of free radicals is the ―Antioxidants‖ ⁽²⁾

Antioxidants include various agents such as enzymes (glutathione peroxidase, superoxide dismutase, and catalase), large molecules (albumin and ferritin), and small molecules (uric acid, glutathione, bilirubin, vitamin C, and vitamin E). They play an important role in the cellular protection cascade against oxidative damage ⁽⁸⁾

1. It prevents the formation of free radicals

2. Radical-scavenging antioxidants eliminate the free radicals and 3. It act as DNA repairing enzymes ⁽⁹⁾

Thus decrease in protective system of anti-oxidants due to cigarette smoking may be a cause of many pathological conditions such as cardiovascular and respiratory disorders. ⁽²⁾

The accurate assessment of oxidative stress in biological systems is a problem for all investigators working on the role of free radical damage in disease ⁽⁹⁾

Total antioxidant capacity is a biomarker of antioxidant protection against free radicals and represents oxygen radical absorbance capacity which ishighly important. ^{(3, 6)}

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Therefore measurement of TAC reflects the antioxidative status of plasma and can be more useful than the measurement of individual antioxidant levels in cells and plasma.

There are several methods to measure the total antioxidant capacity (TAC) in different biological specimens ⁽⁸⁾

The low levels of TAC could be indicative of oxidative stress or increased susceptibility to oxidative damage ⁽⁹⁾

Therefore this study aimed to measure the serum total antioxidant capacity (TAC) in smokers and non-smokers and to compare the above parameter between the two groups. The measurement of serum total antioxidant capacity in smokers helps us to evaluate the risk of many pathological conditions and associated oxidative stress induced by cigarette smoking.

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AIM AND OBJECTIVES AIMS:

To evaluate the effects of smoking on serum Total Antioxidant Capacity in Smokers

OBJECTIVES:

1. To find out whether any correlation exists between TAC and Age 2. To study the correlation between TAC and duration of smoking

3. To study the correlation between TAC and number of cigarettes smoked per day

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REVIEW OF LITERATURE

Cigarette smoking is the most important cause of morbidity and premature mortality in the world ⁽¹⁰⁾

Smoking causes a wide range of diseases, including many types of cancer, coronary heart disease, peripheral vascular disease, chronic obstructive pulmonary disease, stroke and peptic ulcer disease. In addition, smoking during pregnancy adversely affects fetal and neonatal growth and development ⁽¹¹⁾

Various social factors drive the smokers to smoke their first cigarette at early age and many of them are unaware of the harmful effects it brings in to their individual systems ⁽¹²⁾

Out of the total tobacco production, India ranks third and almost 50% of its domestic products are consumed by in itself. Many respiratory diseases, cardiac diseases and asthma related diseases were caused by smoking and have the potential to affect every part of the body ⁽¹³⁾

Epidemiology of Cigarette smoking:

According to WHO estimation there are about 1.1 billion smokers worldwide and this represents about one-third of the global population aged over 15 years. In developing countries about 700 million males and 100 million females are smokers and in industrialized countries there are about 200 million male smokers and 100 million female smokers ⁽¹⁴⁾

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Six million deaths occur in the world every year due to tobacco usage. Out of these five million deaths occur due to direct tobacco usage, while second hand smoke causes more than six hundred thousand deaths in non-smokers. In India more than 1 million people die annually due to tobacco usage ^{(12, 13)}

It has been estimated that the smoking related mortality will rise to 10 million annually by 2030, with 70% of these deaths occurring in developing countries ⁽¹¹⁾

HISTORY

The earliest European explorers of the America observed the practices of smoking, snuffing and chewing of the leaves from Nicotiana tabacum . These practices were geographically widespread in the Americas for ritual, social and medicinal purposes. By this diverse geographical and functional usage, we came to know that Indians had been using the tobacco for a long time. Over 60 million years ago, nicotine molecule was produced by the ancestral of tobacco plant to the insect herbivores. In 1492 Christopher Columbus introduced tobacco to Europe, the following century, tobacco cultivation was spread around the word. In 1565 Sir John Hawkins introduced tobacco to England. In 1573 Jean Nicot (hence nicotine) wrote: Nicotine a herb of marvelos virtue against all wounds and ulcers. In 1870 the hand-rolled cigarettes were slowly replaced by machine – made cigarettes ⁽¹⁵⁾

Mass marketing and mechanization towards the end of 19^th century popularized the cigarette habit ⁽¹⁶⁾

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TOXICOLOGY OF CIGARETTE SMOKING

Cigarette smoking is an aerosol of droplets contain water, nicotine and other alkaloids and tar ⁽⁶⁾

It contains about 7,357 chemical compounds of different classes. Out of this close to 250 chemicals were proven harmful including hydrogen cyanide, carbon monoxide and ammonia. Among 250 harmful chemicals, 69 chemicals were proven carcinogenic ⁽²⁾ The two major groups of carcinogens are

1. Initiators ( those cause genetic damage)

2. Promoters ( those promote the growth of the tumour)

Mostly the tobacco smoke were genotoxicity but it also contains promoters which spur the mutant cells to proliferate ⁽¹⁷⁾

There are two phases in smoking. They are Gas phase and Tar phase. The toxic compounds in both phases are listed in table 1 ⁽¹⁵⁾

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Table-1

Gas phase Tar Phase

Carbon monoxide Particulate matter Carbon dioxide Nicotine

Formaldehyde Phenol

Acrolein Catechol

Acetone Aniline

Pyridine 2-toluidine

3- vinylpyridine 2-naphthylamine Hydrogen cyanide Benz[a]anthracene Nitrogen oxides Benzo[a]pyrene

Ammonia Quinoline

N-nitrosodimethylamine N-nitrosonornicotine N-nitrosopyrrlidine N-nitrosodiethanolamine

Nickel

Polonium-210

ACTIVE SMOKING AND ILL HEALTH ⁽¹⁵⁾

Heavy smokers of two packs a day showed a higher mortality ratio of 2.0. Cigar and pipe smokers showed less excess mortality than cigarette smoking, which is related

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to the less inhalation of cigar/pipe smoke. The major tobacco exposure fore active smokers comes directly from their own.

HEALTH HAZARDS OF TOBACCO USE (RISKS INCREASED BY SMOKING)⁽¹⁸⁾

Smoking causes many types of cancer such as

Lung, Pancreas, Nasal cavity, sinuses, nasopharynx, Stomach, Esophagus, Larynx, Kidney, Uterine cervix, Urinary tract, Liver, Myeloid Leukemia, Oral Cavity, Oropharynx and hypopharynx

Pulmonary disease Emphysema

Lung Cancer

Increased morbidity from viral respiratory infection Asthma

Increased susceptibility to desquamative interstitial pneumonitis Chronic bronchitis

Increased susceptibility to pneumonia and Pulmonary tuberculosis

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Cardiovascular Disease Unstable angina

Stroke

Acute myocardial infarction Aortic aneurysm

Peripheral arterial occlusive disease (including thromboangiitis obliterans) Sudden death

Oral Disease (Smokeless Tobacco) Tooth staining

Oral cancer Gingivitis

Gingival recession Leukoplakia

Reproductive Disturbances Abruptio placentae

Lower birth weight

Increased perinatal mortality Premature rupture of membranes Spontaneous abortion

Premature birth

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Reduced fertility

Gastrointestinal Disease Peptic ulcer

Esophageal reflux Other

Osteoporosis

Aggravation of hypothyroidism Altered drug metabolism or effects Cataract

Tobacco amblyopia (loss of vision) Premature skin wrinkling

Non-insulin-dependent diabetes mellitus Earlier menopause

Age-related macular degeneration

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CIGARETTE SMOKING AND INFECTION ⁽¹⁸⁾

Cigarette smoking is the major risk factor for the respiratory and other systemic infections. Both active and passive smoke exposure increases the risk of infection. Fig 1

FIGURE-1

Influenza 10%

Legionnaire’s disease

15%

Common cold 7%

Periodontal disease

12%

Tuberculosis 20%

Pneumococcal pneumonia

11%

HIV 15%

Meningococcal disease

10%

Cigarette smoking and infection

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PASSIVE SMOKING AND ILL HEALTH ⁽¹⁵⁾

Passive smoking (involuntary) smoking related to the exposure to tobacco combustion products from the smokers to others, often referred to as environmental tobacco smoke (ETS) exposure.

Health Hazards of Environmental Tobacco Smoke in Non-smokers ⁽¹⁸⁾ Children

Wheezing

Middle ear effusion

Sudden infant death syndrome Asthma

Hospitalization for respiratory tract infection in first year of life Adults

Reduced pulmonary function Cough

Irritation of eyes, nasal congestion, headache Lung cancer

Myocardial infarction

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Table 2: Interaction between Cigarette Smoking and Drugs⁽¹⁸⁾

Drugs Interaction (Effects

Compared with

Nonsmokers)

Significance

Antipyrine Caffeine

Chlorpromazine Clozapine

Desmethyldiazepam Estradiol

Estrone Flecainide Fluvoxamine Haloperidol Imipramine Lidocaine Olanzapine Oxazepam Pentazocine Phenacetin Phenylbutazone Propranolol Tacrine

Accelerated metabolism May require higher doses in smokers; reduced doses after quitting

15 Theophylline

Cimetidine and other H2- blockers

Lower rate of ulcer healing, higher ulcer recurrence rates

Consider using mucosal protective agents

Propranolol Less antihypertensive effect, less antianginal efficacy; more effective in reducing mortality after myocardial infarction

Consider the use of cardioselective beta-blockers

Chlorpromazine (and possibly other neuroleptics)

Less sedation; possibly reduced efficacy

Smokers may need higher doses

Oral contraceptives Enhanced thrombosis, increased risk of stroke and myocardial infarction

Do not prescribe to smokers, especially if >35 years old

Diazepam, chlordiazepoxide (and possibly other sedative- hypnotics)

Less sedation Smokers may need higher

doses

Propoxyphene Reduced analgesia Smokers may need higher

doses Nifedipine (and probably

other calcium blockers)

Less antianginal effect May require higher doses and/or multiple drug antianginal theraphy

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NICOTINE ADDICTION

Nicotine is the principle constituent of tobacco responsible for its addictive character. The other smoke constituents contribute to the strength of the addiction ⁽¹⁹⁾ Although nicotine is the main cause of dependence on tobacco, it is not carcinogenic, does not cause respiratory disease and has only minor haemodynamic effects. However, it can delay wound healing, increase insulin resistance and is associated with harmful effects on the fetal brain and lungs ⁽²⁰⁾

Drug addiction is defined as compulsive use of a psychoactive substances, the consequences of which are detrimental to the individual society. When we smoke, the nicotine is absorbed rapidly into the pulmonary circulation and then it moves quickly to the brain then it acts on nicotinic cholinergic receptor to produce its gratifying effects within 10 to 15 seconds after a puff ⁽¹⁸⁾

Nicotine may stimulate inhibitory systems and inhibit stimulatory system by virtue of the secondary release of other transmitters ⁽⁶⁾

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Neurochemical effects of nicotine ^{(18, 20)}

When it is taken for long-term, physical dependence develops which is associated with an increased number of nicotinic cholinergic receptors in the brain.

Therefore when tobacco is not available, even for only a few hours, withdrawal symptoms occur. They are

1. Anxiety

2. Difficulty in concentrating

NICOTINE

DOPAMINE Pleasure, appetite

suppression

NOREPINEOHRINE Arousal, appetite suppression

ACETYCHOLINE Arousal, Cognitive enhancement

GLUTAMATE Learning, Memory

enhancement

SEROTONIN Mood modulation, appetite suppression

BETA-ENDORPHIN Reduction of anxiety and tension

GABA Reduction of anxiety and

tension

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3. Restlessness 4. Irritability 5. Hunger

6. Craving for tobacco 7. Disturbed sleep and 8. Depression ⁽¹⁸⁾

SMOKING CESSATION

Among cigarette smokers, 70% would like to quit and 46% try to quit each year.

1% of smokers quit spontaneously each year. ⁽¹⁸⁾

Long-term smoking cessation substantially reduces health risks and leads to a decrease in the early mortality. ⁽²¹⁾

GUIDELINESS ⁽²²⁾

The strategy recommended by the guidelines involves:

1. Primary health care teams should ensure that their records concerning which of their patients smoke are kept up to date. General physicians opportunistically advising smokers to stop during routine consultations, giving advice on and/or prescribing effective medications to help them and referring them to specialist cessation services;

2. Specialist smokers‘ services providing behavioral support (in groups or individually) for smokers who want help with stopping and using effective medications wherever possible;

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3. Specialist cessation counselors providing behavioral support for hospital patients and pregnant smokers who want help with stopping;

4. All health professionals involved in smoking cessation encouraging and assisting smokers in use of nicotine replacement therapies (NRT) or bupropion where appropriate.

PHARMACOTHERAPY OF SMOKING CESSATION ^{(4, 23)}

Two medications have been approved by FDA for smoking cessation: Nicotine and Bupropion.

FIRST LINE OF DRUGS Nicotine patch

Sustained- release bupropion hydrochloride Nicotine gum

Nicotine inhaler Nicotine nasal spray

NICOTINE PATCH ⁽²³⁾ ADVANTAGES

 Easy for patients to use

 Can be obtained without prescription

 Patient receives more consistent nicotine levels throughout the day to alleviate continuous cravings

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 ―Step down‖ therapy allows patients to receive decreasing levels of nicotine every few weeks

DISADVANTAGES

 Many patients experience pruritis as allergic reaction to adhesive

 used and may have to use another brand

 Must avoid use in patients with dermatological conditions (i.e., eczema, dermatitis, psoriasis)

NICOTINE GUM/LOZENGE ⁽²³⁾ ADVANTAGES

 Adjustable use; should follow a specific schedule for can use for ―breakthrough (lozenge is a little easier to use) cravings‖

 Good for adjunct therapy with a longer-acting medication, such as patch or oral medication lozenge

 Can be obtained without prescription

 ―Step down‖ therapy allows the patients to receive decreasing levels of nicotine every few weeks

 Gum use may delay weight gain DISADVANTAGES

 Proper chewing technique must be use but used to decrease adverse effects

 Cannot be used by denture wearers

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 Cannot use acidic beverages (orange juice, colas) for 15 minutes before or while using gum/ lozenge

 Adverse effects are mainly gastrointestinal: nausea, hiccups and heartburn

NICOTINE INHALER ⁽²³⁾ ADVANTAGES

 Adjustable use

 Great choice for patients who cannot seem to get past the hand-to-mouth ritual of smoking

 Provides ―step down‖ therapy DISADVANTAGES

 Obtained by prescription only

 Patient could become dependent

 Initial throat or mouth irritation

 Cannot be used by patients with severe reactive airway

 Patients should avoid eating or drinking within 15 minutes of using inhaler

NICOTINE NASAL SPRAY ADVANTAGES

 Adjustable use

 Provides ―step down‖ therapy

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DISADVANTAGES

 Patients with severe reactive airway diseases should avoid using spray

 Obtained by prescription only

 Potential for mouth/throat irritation is significant

 Patients could become dependent

BUPROPION SR ⁽²³⁾ ADVANTAGES

 The only non-nicotine medication

 Easy to use: twice daily by mouth

 Good choice for combination nicotine-replacement therapy

 May delay weight gain

 Can also be used for depression

SECOND LINE OF DRUGS ⁽⁴⁾

 Clonidine

 Notriptyline

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REACTIVE OXYGEN SPECIES (24, 25, 26)

Reactive oxygen species are produced normally in cells during mitochondrial respiration and energy generation and they are degraded by cellular defense system. Thus the cells are able to maintain a steady state in which free radicals may be present transiently at low concentration but do not cause damage. ROS can be divided into two groups: free radicals and non radicals. Molecules containing one or more unpaired electrons and thus giving reactivity to the molecule are called free radicals. The non radical forms are created when two free radicals share their unpaired electrons.

ENDOGENOUS SOURCES OF ROS ⁽²⁶⁾

 Superoxide anion

 Hydrogen peroxide

 Hydroxyl radical

 Hypochlorous acid

 Peroxyl radicals

 Hydroperoxyl radical

EXOGENOUS SOURCE OF ROS

 Cigarette smoke

 Ozone Exposure

 Hyperoxia

 Ionizing Radiation

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 Heavy Metal Ions

Cigarette smoke contains many oxidants, free radicals and organic compounds.

Inhalation of cigarette smoke also activates some endogenous mechanism such as accumulation of neutrophils and macrophages, which further increase the oxidant injury.⁽²⁶⁾

ROS IN NORMAL PHYSIOLOGY ⁽²⁷⁾

ROS, at low concentration, is essential for normal physiological function like gene expression, cellular growth and defense against infection.

1. They act as the stimulating agents for biochemical processes within the cell.

2. They can also cause indirect induction of transcription factors by activating signal transduction pathways.

3. They appear to serve as secondary messengers in many developmental stages and also participate in the biosynthesis of molecules such as thyroxin, prostaglandin that accelerate the developmental process.

4. ROS are used by the immune system. Macrophages and Neutrophils generate ROS in order to kill the bacteria that they engulf by phagocytosis.

5. Furthermore, tumor necrosis factor mediates the cytotoxicity of tumor and virus infected cells through ROS generation and induction of apoptosis.

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OXIDATIVE STRESS ^{(24, 28)}

When there is imbalance between the free radicals and the scavenging system then the condition is called oxidative stress.

Oxidative stress are involved in a wide variety of pathologic process such as cell injury, cancer, aging and some degenerative disease like Alzheimer disease.

GENERATION OF FREE RADICALS ⁽²²⁾

Free Radicals may be generated in several ways. Few are given below

1. The reduction-oxidation reactions that occur during metabolic processes 2. Absorption of radiant energy

3. Rapid bursts of ROS

4. Enzymatic metabolism of exogenous chemicals or drugs

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5. Transition metals catalyze free radical formation- fenton reaction 6. Nitric oxide can act as a free radical

PATHOLOGIC EFFECTS OF FREE RADICALS ⁽²⁴⁾

 Lipid peroxidation in membranes

 Oxidative modifications of proteins

 Lesions in DNA

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1. Lipid peroxidation in membranes ⁽²⁴⁾

Free radicals cause peroxidation of lipids within plasma and organellar membranes. Oxidative damage is initiated when the bonds in unsaturated fatty acids of membrane lipids are attacked by O_2-derived free radicals, particularly by OH. The lipid free radical interactions yield peroxides, which themselves are unstable and reactive, and autocatalytic chain reaction which is called propagation. This results in extensive damage to the cell.

2. Oxidative modification of proteins ⁽²⁴⁾

Free radicals promote oxidation of amino acid side chains, formation of protein- protein cross-linkages and oxidation of protein backbone. This modification may damage the active sites of enzymes, disrupt the conformation of structural proteins, and enhance proteasomal degradation of unfolded or misfolded protein, raising havoc throughout the cell.

3. Lesions in DNA ⁽²⁴⁾

Free radicals are capable of causing single- and double- strand breaks on DNA, cross-linking of DNA strands and formation of adducts. Oxidative DNA damage has been implicated in cell aging and in malignant transformation.

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The production of ROS is a frequent prelude to necrosis. Free radicals can trigger apoptosis as well. ROS has a role in signaling by a variety of cellular receptors and biochemical intermediates. ⁽²⁴⁾

REMOVAL OF FREE RADICALS ⁽²⁴⁾

They are inherently unstable and generally decay spontaneously. Antioxidants either block the initiation of free radicals formation or inactivate free radicals.

Figure-2 ROLE OF REACTIVE OXYGEN SPECIES IN CELL INJURY

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ANTIOXIDANTS

Biologically antioxidants are defined as synthetic or natural substances added to products to prevent or delay their deterioration by the action of oxygen in air. For example, enzymes or other organic substances such as vitamin E or β-carotene ⁽²⁴⁾

In the circulatory system, the concentration of human serum albumin in plasma is 35-50g/L. It is important in maintaining endogenous antioxidant function in organisms⁽³⁰⁾

Antioxidants are chemical compounds which bind to free oxygen radicals and prevents these radicals from damaging healthy cells. The chemical compounds, which decrease the rate of lipid oxidation reaction ⁽²⁹⁾

Antioxidants are effective because they can donate their own electrons to ROS and thereby neutralizing the adverse effects of the latter. In general, an antioxidant in the body may work at three different levels:

(a) prevention – keeping formation of reactive species to a minimum e.g.

desferrioxamine

(b) interception – scavenging reactive species either by using catalytic non- catalytic molecules e.g. ascorbic acid, alpha-tocopherol and

(c) repair-repairing damaged target molecules e.g. glutathione ^{(27, 31)} Antioxidants rich foods ^{(32, 33)}

1. B-Caraten Yellow orange fruits such as cantaloupe and vegetable such as carrots and green leafy vegetables

30

2. Vitamin C Fruits such as citrus, vegetables including red and green peppers, fully ripe tomatoes, potatoes, green leafs such as spinach and collard green

3. Vitamin E Vegetable oils such as soybeans, corn, coconut oil, safflowers, margarine, wheat germ and leafy vegetables

4. Polyphenolic components-Tea, coffee, soy, olive oil, cinnamon and redwine

ANTIOXIDANTS RICH FOODS

31

ROLE OF ANTIOXIDANTS ⁽²⁵⁾

It is capable of inhibiting the oxidation of another molecule. It breaks the free radical chain of reactions by sacrificing their own electrons to feed free radicals, without becoming free radicals themselves.

ANTIOXIDANTS PREVENTS AGAINST FREE RADICAL DAMAGE ⁽²⁵⁾

Our body naturally circulates a variety of nutrients for their antioxidant properties and manufactures antioxidant enzymes to control these destructive chain reactions. Examples are vitamin C, vitamin E, carotenes and lipoic acid.

When cells use oxygen to generate energy, free radicals are produced by the mitochondria. These by-products are generally ROS as well as reactive nitrogen species

32

(RNS) that result from the cellular redox process. The free radicals have a special affinity for lipids, proteins, carbohydrates, and nucleic acids. ⁽²⁵⁾

The interior of our cells and the fluid between them are composed mainly of water, but cell membranes are made up of lipids .They are present in aqueous body fluids such as blood and the fluids within and around the cells (the cytosol, or cytoplasmic matrix).Free radicals can strike the watery cell contents or the fatty cellular membrane, so the cell need defense for both. ⁽²⁵⁾

CLASSIFICATION OF ANTIOXIDANTS ^{(29, 32)} DEPENDING ON TH ENZYMES

1. Non-enzymatic antioxidants 2. Enzymatic antioxidants

ENZYMATIC ANTIOXIDANTS ⁽²⁵⁾

1. Primary antioxidants: Superoxide dismutase, Glutathione peroxidase.

2. Secondary antioxidants: Glutathione reductase, Glucose 6 - phosphate dehydrogenase.

Enzymatic antioxidants work by breaking down and removing free radicals. These enzymatic antioxidants cannot be supplemented orally but must be produced in our body.

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NONENZYMATIC ANTIOXIDANTS ⁽³²⁾

Non-enzymatic antioxidants interrupt free radical chain reactions a. Minerals: Zinc, selenium.

b. Vitamins: Vitamins A, C, E.

c. Carotenoids: Beta carotene, lycopene, lutein, zeaxanthin.

d. Low molecular weight antioxidants: Glutathione, uric acid.

e. Organo sulfur compounds: Allium, allyl sulfide, indoles.

f. Antioxidants cofactors: Coenzyme O 10.

g. Polyphenols: Flavonoids, phenolic acid.

ANTIOXIDANT ENZYME ⁽³²⁾

These enzymes mainly act on the free radicals.

(i) CATALASE

Catalase is the first antioxidant enzyme that comes into action and has two functions, one is the conversion of hydrogen peroxide to water and oxygen.

Catalase — Fe (III) + H2O –––– Compound I (hydrogen peroxide) Compound I + H2O –––– Catalase — Fe (III) + 2 H2O +O2

It consists of four protein subunits, each of which contains haem group and molecules of NADPH. This enzyme is mainly present in the peroxisomes within the cell, along with catalase and some other enzymes such as hydrogen peroxide. These catalases which are present in the lysosomes are very easily ruptured during minor exploitation of cells. The

34

greatest activity is present in liver and erythrocytes but some of the catalases are found in all tissues.

(ii) GLUTATHIONE PEROXIDASE AND GLUTATHIONE REDUCTASE ⁽²⁹⁾

GSH (cysteine containing natural antioxidant) is called as the ―master antioxidant‖ and is found in every single cell of your body, maximizing the activity of all the other antioxidants. GSH is regenerated from GSSG by the enzyme GSH reductase (GSR) Glutathione peroxidase catalyzes the oxidation of glutathione at the cost of a hydroperoxidase, which might be hydrogen peroxide. An increased GSSG-to-GSH ratio is considered indicative of oxidative stress.

ROOH + 2 GSH –––– GSSG + H2O + ROH

Lipid hydroperoxides plays an important role in repairing damage cell resulting from lipid peroxidation. Selenium is required for activation of glutathione peroxidase.

Deficiency of selenium causes decrease in concentration of glutathione peroxidase.

Glutathione peroxides mainly synthesis in the kidney.

(iii) SUPEROXIDE DISMUTASE ⁽²⁹⁾ There are three forms of superoxide dismutase:

1. Copper-zinc superoxide dismutase (Cu Zn SOD).

2. Manganese superoxide dismutase (Mn SOD).

3. Extra cellular superoxide dismutase (EC SOD).

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COPPER ZINC SUPEROXIDE DISMUTASE ⁽²⁹⁾

It is mainly seen within cell, especially cytoplasm and other organelles. Cu Zn SOD has a molecular weight of 32,000 KDa with two proteins subunits, each one containing a catalase, which gets activated by copper and zinc molecules.

MANGANESE SUPEROXIDE DISMUTASE ⁽²⁹⁾

Mn SOD is predominantly present in the mitochondria with a molecular weight of 40,000 KDa. The sequence of amino acids of Mn SOD is entirely different from Cu Zn SOD.

EXTRA CELLULAR SUPEROXIDASE DISMUTASE ⁽²⁹⁾

EC SOD first described by Marklund in the year 1982. EC SOD also contains copper and zinc, but is distinct from Cu Zn SOD. EC SOD is synthesized by only a few cell types such as fibroblasts and endothelial cells. EC SOD also expresses heparin sulfate on the cell surface. EC SOD is one of the major SOD traced in extracellular fluids and is released into the circulation from the surface of vascular endothelium. EC SOD plays a role in the regulation of vascular tone, because an endothelial derived relaxing factor is neutralized in the plasma by superoxide.

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NON-ENZYMATIC ANTIOXIDANTS ⁽³⁴⁾ VITAMIN E

It is a potent chain breaking lipid soluble antioxidants. It reacts with lipid peroxyl radicals eventually terminating the peroxidation chain reaction and thereby reducing oxidative damage.

VITAMIN C

It represents the major water-soluble antioxidants in the human body.

VARIES ANTIOXIDANT WITH PROTECTIVE MECHANISM ⁽³²⁾ Varies antioxidants are broadly divided into three categories

 Endogenous antioxidants

 Dietary antioxidants

 Metal binding protection Endogenous antioxidants:

 Uric acid

 Enzymes: Copper/zinc and magnesium, iron dependent

 catalase, Selenium–dependent glutathione peroxidase

 NADPH and NADH

 Thiols eg: Glutathione, lipoic acid, N-acetyl cysteine Dietary antioxidants:

 Vitamin E, Vitamin C, Beta carotene and other carotenoids Eg: Lycopene

 Polyphenols: Eg. Flavonoids, Flavones

37

Metal binding proteins:

 Copper, Cerebroplasmines, Myoglobin, ferritin, Transferrin Synthetic antioxidants are

 Butylated hydroxytoluene (BHT)

 Butylated hydroxyanisole (BHA) and

 Tertbutyl hydroquinone (TBHQ)

In recent years, there is an increasing interest in natural antioxidants and subsequently looking through the literature; it is recognized that the replacement of synthetic antioxidants by natural ones may have several benefits and much of the research on natural antioxidants has focused on phenolic compounds, in particular, flavonoids as potential sources of natural antioxidants. Natural phenolic compounds are widely distributed in plants and are the main contributors to the antioxidant activities of food. ⁽²⁹⁾

Number of naturally existing antioxidant compounds present in fruits, vegetables, and dietary supplements are ascorbic acid, α-tocopherol, phenolic acids (benzoic acid, trans-cinnamic acid, and hydroxycinnamic acid), coumarins, lignans, stilbenes (in glycosylated form), flavonoids, isoflavonoids, and phenolic polymers (tannins) ⁽²⁹⁾

Under flavonoids ⁽³²⁾

a. Xanthones — Mangostin

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b. Flavonoids — Quercein, kaempferol c. Flavanols — Catechin, EGCG d. Flavanones — Hesperetin

e. Flavones — Chrysin, isoflavanoids — genistein f. Anthocyanidines — Cyanidin, pelagonidin Under phenolic acid ⁽³²⁾

a. Hydroxycinnamic acid: Ferulic, p-coumaric b. Hydroxybenzoic acid: Gallic acid, ellagic c. Gingerol, curcumin ⁽⁾

FUNCTIONS OF ANTIOXIDANTS ⁽³²⁾

 It reduces the free radicals

 It stimulates the growth of normal cells

 It protects cells against premature and abnormal ageing

 It helps to fight age related molecular degeneration

 It supports the body immune system

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SOURCES OF FREE RADICALS HELPS IN TISSUE REPAIR MECHANISM ⁽³²⁾

Free Radical Production

O2, H2O2

Transition metals - Fe2, Cu

OH

Metal binding proteins- Transferrin, Ferritin, Lacotoferrin

Enzyme antioxidants – SOD, Catalase, Glutathione peroxidase, Ceruloplasmin

Chain breaking antioxidants -Directly scavenge free radicals, Consumed during scavenging process

Tissue damage Lipid phase:

Tocopherols, Ubiquinol, Carotenoids, Flavonoids

Repair mechanism Aqueous phase:

Ascorbate, Urate,

Glutathione and other thiols

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TOTAL ANTIOXIDANT CAPACITY

Total antioxidant capacity is the measure of the amount of free radical scavenged by a test solution frequently to assess the antioxidant status of biological samples. ⁽³⁵⁾ ADVATAGESOF TAC ASSAY ⁽³⁵⁾

 Simplicity of the techniques

 Low cost per sample

 Speed of reactions and

 Possibility to be performed using automated, semi-automated, or manual methods

LIMITATIONS ⁽³⁵⁾

It provides limited information about the antioxidant status, because TAC assays do not measure all antioxidant components. For example, they do not evaluate the role of important enzymes such as superoxide dismutase, glutathione peroxidase and catalases.

TAC is reduced by alcoholism, smoking, and exposure to radiation, herbicides, carbon monoxide, carbon tetrachloride, lead, arsenic, mercury, cadmium, aluminum, and other toxic elements. ^{(2, 36)}

CLASSIFICATION OF ANTIOXIDANT METHODS ⁽²⁹⁾ 1. IN VITRO ANTIOXIDANT METHODS

Based on the chemical reaction involved between the antioxidant compounds and the free radicals, antioxidant capacity assays are broadly classified into two types.

41

 Hydrogen atom transfer (HAT) reaction based assays

 Electron transfer (ET) reaction based assays- FRAP method 2. IN VIVO ANTI-OXIDANT METHODS

IN VITRO ANTIOXIDANT METHODS ⁽²⁹⁾ Table 3:

ET based assays HAT based assays Other in vitro antioxidant methods DPPH free radical scavenging

assay

Oxygen radical

absorbance capacity

Ascorbic acid content assay

Superoxide anion radical

scavenging assay Crocin Bleaching Assays

Electron paramagnetic resonance spectroscopy investigations

FRAP assay TRAP Phosphomolybdenum

assay

TEAC Hydroxyl radical

scavenging activity

Xanthine oxidase method

CUPRAC assay Hydroxyl radical averting

capacity Metal chelating activity Folin-Ciocalteu reagent, the

total phenols assay

Scavenging of Hydrogen peroxide radicals

Reducing power assay

Nitric oxide radical inhibition

activity

TBARS assay

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Table 4:

In vivo antioxidant methods ⁽²⁹⁾ Ferric reducing ability of plasma Reduced glutathione estimation Glutathione peroxidase estimation Glutathione –S-transferase

Superoxide dismutase method Catalase assay

γ-Glutamyl transpeptidase activity assay Glutathione-Disufide-Reductase assay Lipid peroxidation assay

Low density lipoprotein assay Cellular antioxidant activity assay FRAP METHOD ^{(31, 37)}

The total antioxidant capacity (TAC) of a sample can be measured with a ferric reducing antioxidant power (FRAP) assay.

PRINCIPLE:

The FRAP assay of Benzie and Strain is based on the principle that at low pH, the ferric tripyridyl triazine (FeIII TPTZ) complex gets reduced to the ferrous form, developing an intense blue color with a maximum absorption at 593 nm. ⁽³⁷⁾

The amount of Fe^II-TPTZ produced is measured spectrophotometrically after a fixed time (4 min). Alternatively, iron reduction can be assessed by the potassium

43

ferricyanide-ferric chloride method. This method essentially provides the stoichiometry of antioxidants, which for instance has been determined as two for ascorbic acid, uric acid and alpha α-tocopherol, about four for bilirubin, and zero for albumin. ⁽³¹⁾

The reaction with ascorbic acid is instantaneous, whereas with uric acid it takes about 2 min to occur; thus after 4 min, both antioxidants have the same FRAP power.

However, in the case of other antioxidants, the reaction is not complete after 4 min, so the result of this test is expected to arbitrarily depend on the reaction time. The reaction is nonspecific, and any compound with a suitable redox potential will drive Fe^III-TPTZ reduction. ⁽³¹⁾

ADVANTAGES ⁽³⁵⁾

1. Quick and simple to perform 2. Can be easily automated

3. No need of highly specialized equipment or skills

4. Reagents are inexpensive and sample pre-treatment is not required 5. Highly reproducible over a wide concentration range

DISADVANTAGES ⁽³⁵⁾

1. It requires an acidic PH to maintain iron solubility

2. It does not measure the antioxidants containing thiol groups and only measure the reducing capability based upon the ferric ion

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SMOKING AND ANTIOXIDANTS

Cigarette smoke is a complex mixture of chemical compound contains many toxic, carcinogenic and mutagenic chemicals, as well as free radicals and ROS in the gas phase and particulate phase with the potential for biological oxidative damage ^{(38, 39)}

Free radicals have unpaired electrons in the outer orbit. They are extremely unstable and become a stable molecules with only paired electrons in the outer shell ⁽²⁵⁾

ROS are free radicals formed as a natural byproduct of the normal metabolism of oxygen and have important role in signally and homeostasis ^{(2, 40)}

Living organisms have developed complex antioxidant system to counteract ROS and to reduce their damage. In healthy subjects with good nutrition these free radicals are eliminated by antioxidant mechanism, including enzymatic and non-enzymatic system ^(41,

42)

45

Cigarette smoking causes production of free radicals and causes an imbalance between free radicals and antioxidant protective mechanism. This imbalance is called oxidative stress ^{(43, 44)}

Cigarette smoke may enhance oxidative stress by the following reasons.

1. They are rich in pro-oxidants

2. These pro-oxidants consume more antioxidants and

3. Smokers have a tendency for low intake of dietary antioxidants ⁽⁴⁵⁾

Therefore free radicals may be the most critical triggering plasma depletion of antioxidants, increase in lipid peroxidation level and protein modification. The products arising from lipid peroxidation and protein modification can react with cigarette smoke constituents and create additional toxic products. Thus causes smoking related oxidative damage. Free radicals in the cigarette smoke deplete some plasma antioxidants in vitro ^(46,

47)

46

Potential pathways and mechanisms for cigarette smoking-mediated cardiovascular dysfunction ⁽⁴⁸⁾

47

Shrivastava et al ⁽³⁸⁾ did a cross sectional study in 45 smokers and 45 non- smokers of age group 20-40 years. The study findings showed lower TAC levels with lower sensory nerve conduction velocity of ulnar and median nerve in smokers compared to non-smokers. The difference was statistically significant. A negative correlation was observed between smoking index and TAC as well as smoking index and sensory nerve conduction velocity of ulnar and median nerve and motor nerve conduction velocity of ulnar and median nerve. A positive correlation was found between TAC and sensory nerve conduction velocity in both the nerves was statistically significant. Cigarette smoking has been implicated in causing subclinical changes in myelin sheath of peripheral nerves and resulting demyelination causes conduction deficit. From this study we come to know that the decrease in TAC indicates oxidative stress in smokers which may be responsible for nerve conduction deficits among smokers.

Salama et al ⁽⁴⁹⁾ performed a case-control study carried on 60 patients with COPD, and on 15 apparently healthy smokers and 15 apparently healthy non-smokers. They assessed the role of antioxidant status in pathogenesis of COPD and in predicted the severity of airway obstruction. Serum TAC level was significantly reduced in COPD patients and healthy smokers compared with healthy non-smokers. And also observed decreased levels of TAC in COPD when compared with healthy smokers, and significantly reduced in severe and very severe COPD patients compared with mild and moderate COPD. This study suggests that Serum TAC is a valuable biomarker in identifying and predicting the severity of the diseases.

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Dr.Swati Digambar Sawant ⁽⁴⁷⁾ et al did a case control study to evaluate the occupational exposure of tobacco and its exposure on TAC in Bidi workers. They observed low levels of TAC in bidi workers compared to that of normal healthy subjects.

The study confirms that occupational exposure to tobacco can decrease the level of TAC which may cause the subjects less resistant to oxidative injuries and subsequent diseases.

Cigarette smoking is associated with an increased incidence of acute MI.

Cessation of smoking significantly reduces this risk over a one to three year period with exponential decline approaching the risk in ex-smokers within five years of cessation.

Recent data indicate an immediate reduction in thrombotic events with cessation. ⁽⁴⁸⁾ One study found that having higher level of tobacco-related knowledge had a significant impact on positive attitudes towards smoking cessation. ⁽⁵⁰⁾

Majority of people in developed countries are aware of the association between smoking and general health, lung cancer and heart disease. However, awareness of the association between smoking and other conditions such as stroke and the impact of environmental tobacco smoking exposure on the health of non-smokers is less frequently reported. ⁽⁵¹⁾

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MATERIALS AND METHODS STUDY DESIGN

This study was a case control study that included 80 subjects of which 40 were non-smokers and 40 were smokers. Only healthy male subjects were taken in the age group of 30-50 years. The non- smoker were in control group and smoker in cases group.

All the participants were non-athletes, non-alcoholics and had not participated in regular exercise / diet programs for the period preceding 6 months.

Based on the age both the groups were divided into two group .The age between 30-40 years in first group and 41-50 years in second group. The Mean TAC values were compared to know whether any age factor influence the TAC levels. Furthermore the smoker group was stratified depending on the duration of smoking and no. of cigarettes.

The duration of smoking ≤ 6years and > 6 years were considered. First group included the subjects who smoke 10-14 cigarettes per day and the second group who smoke 15-20 cigarettes per day. The TAC levels were correlated with duration of smoking (in years) and number of cigarettes per day in the smokers group to check whether any correlation between TAC and the above said factors. And also to know the severity of cigarette smoking on TAC.

INCLUSION CRITERIA

Smoking history of at least 10 cigarettes a day for 5 years for smoker group

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EXCLUSION CRITERIA Type II Diabetes mellitus Respiratory diseases Cardiovascular diseases Cancer

Kidney dysfunction and Other chronic diseases

STUDY SITE

The study groups were selected in Thanjavur medical college, Thanjavur.

Each participant received written and verbal explanations about the nature of the study before signing an informed consent form.

Ethical clearance from the Institutional Ethical Committee was obtained.

ANTHROPOMETRIC MEASUREMENTS

Each subject‘s body weight and height were measured by using a stadiometer.

Body mass index (BMI) was calculated by Quetlet Body Mass Index ( dividing body mass (kg) by height in m² ).

The two group smokers and non-smokers matched for age and anthropometrical markers, Heart rate and Blood pressure.

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LABORATORY ANALYSIS

The total antioxidant capacity samples were collected after an overnight fasting between 8:00 a.m. and 9:00 a.m.

The blood was kept overnight before centrifugation to get serum. Centrifugation was done at 3000 rpm for 5 minutes at room temperature. Serum samples were stored at 4 degree centigrade until required for use. ⁽⁵²⁾

PREPARATION OF FRAP REAGENTS ^{(37, 52)}

 300 mmol/liter acetate buffer (pH 3.6)

 10 mmol/liter TPTZ (2,4,6-tripyridyl-s-triazine) solution

 20 mmol/liter Ferric chloride solution in the ratio 10:1:1

52

REAGENTS USED IN THIS STUDY

INSTRUMENT USED:

SPECTROPHOTOMETRY

53

PROCEDURE ⁽³⁷⁾

20 μL of sample (serum or plasma) was mixed with 300 μL of FRAP reagent; after 10 minutes of incubation at 37o C, the ferric tripyridyl triazine (FeIII -TPTZ) complex is reduced to the ferrous tripyridyl triazine (FeII-TPTZ) form in the presence of antioxidants, developing an intense blue color with a maximum absorption at 593 nm.

Aqueous solutions of known Fe² concentration, in the range of 100–1000 mmol/liter were used for calibration ⁽⁵²⁾

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STATISTICAL ANALYSIS

The results were analyzed using statistical analysis software, SPSS version 16 for windows 10. All the data were expressed as mean ± Standard deviation and were evaluated for normality.

Student ‗t‘ test was used to compare between non-smokers and smokers to test for statistical significance. To evaluate the correlation between the variables we used Pearson‘s Correlation Coefficient. For all the tests, P value <0.005 at 95% was considered as statistically significant.

55

RESULTS

This study included 80 healthy subjects of which 40 non-smokers and 40 smokers in the age group of 30-50 yrs. The mean age of control group is 37.13±6.039 and 38.09 ± 6.753. Both groups are matched in terms of age, anthropometric measurements, heart rate and blood pressure. The mean TAC values were compared between smokers and non- smokers. Then based on age, both groups were divided into two sub groups. The first group is between 30-40 years of age and the second group is between 41-50 years of age.

The mean TAC levels were compared within the group as well.

The Smokers group is again divided into two sub groups based on duration of smoking (≤ 6years and > 6 years) and their mean TAC levels were compared. The TAC levels were then correlated with duration of smoking (in years) and with number of cigarettes per day with respect to the smokers group

.

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Table 1: DESCRIPTIVE STATISTICS OF CONTROLS

PARAMETER

A Group (n=40)

Minimum Maximum Mean S.D.

Age(years) 30 50 37.12 6.039

Height(cm) 156 180 167.83 5.755

Weight(kg) 52 81 64.18 7.175

BMI(kg/m²) 19.40 27.00 22.7853 1.79027

HR(bpm) 72 88 78.30 5.014

Systole(mmHg) 110 130 115.00 6.405

Diastole(mmHg) 70 80 73.75 4.903

The mean and standard deviation for age, anthropometric measurements and blood pressure for control group were stated in table- 1. The mean age and standard deviation for controls was 37.12±6.039.

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Table 2: DESCRIPTIVE STATISTICS OF CASES

The mean and standard deviation for age, anthropometric measurements, Heart rate and Blood pressure of smokers group were stated in table 2. The mean age and standard deviation for controls was 38.08±6.753.

PARAMETER

B Group (n=40)

Minimum Maximum Mean S.D.

Age(years) 30 50 38.08 6.753

Height(cm) 156 185 169.18 6.679

Weight(kg) 50 83 65.68 7.888

BMI(kg/m²) 18.70 26.00 22.7625 2.03546

HR(bpm) 72 88 79.20 5.653

Systole(mmHg) 110 130 116.00 5.905

Diastole(mmHg) 70 90 75.00 5.547

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Table 3: DEMOGRAPHIC PROFILE OF SMOKERS AND NON-SMOKERS GROUP A- CONTROLS (NON-SMOKERS)

GROUP B- CASES (SMOKERS)

PARAMETERS n Mean S.D t p value

AGE(years)

A 40 37.13 6.039

-0.66

.509>0.05 Not

Significant

B 40 38.08 6.753

HEIGHT(cm)

A 40 167.8

3 5.755

-0.97

.336>0.05 Not

Significant

B 40 169.1

8 6.679

WEIGHT(kg)

A 40 64.18 7.175

-0.89

.376>0.05 Not

Significant

B 40 65.68 7.888

BMI(kg/m²)

A 40 22.78

5 1.7903

0.053

.958>0.05 Not

Significant

B 40 22.76

3 2.0355

HR(bpm)

A 40 78.3 5.014

-0.75

.454>0.05 Not

Significant

B 40 79.2 5.653

SYSTOLIC

BP(mmHg)

A 40 115 6.405

-0.73

.470>0.05 Not

Significant

B 40 116 5.905

DIASTOLIC

BP(mmHg)

A 40 73.75 4.903

-1.07

.289>0.05 Not

Significant

B 40 75 5.547

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TABLE 3 shows the mean and standard deviation of age, anthropometric measurements, Heart rate and Blood pressure for both non-smokers and smokers. The Age, Anthropometric measurements, Heart rate and Blood pressure were matched with respect to both groups.

Table 4:

PARAMETERS n Minimum Maximum Mean S.D

NO. OF CIGARETTE 40 10 20 12.38 3.469

DURATION OF

SMOKING 40 5 12 7.1 2.061

The mean value and standard deviation for duration of smoking and number of cigarettes per day in smokers group is given in table- 4.

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TABLE 5: COMPARISON OF TAC BETWEEN NON-SMOKERS AND SMOKERS

GROUP n MINIMUM MAXIMUM MEAN S.D t p NON-

SMOKERS 40 318.59 708.26 523.027 97.9746

7.7 0.000<0.05 Significant SMOKERS 40 211.45 550.86 362.149 89.1929

Table 5 show the mean and standard deviation of TAC for both non-smokers and smokers group. Lower levels of TAC in smokers were noted and the difference in mean TAC between both group (non-smokers and smokers) is statistically significant (p value 0.000)

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FIGURE-1

COMPARISON OF SERUM TOTAL ANTIOXIDANT CAPACITY LEVELS IN SMOKING AND NON-SMOKING GROUP

FIGURE-1 show that the mean serum total antioxidant capacity in the smokers group was lower compared to the non-smoker group.

0 100 200 300 400 500 600

Non-Smokers Smokers

523.0268

362.1488

Non-Smokers Smokers

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FIGURE-2

TOTAL ANTIOXIDANT CAPACITY IN SMOKERS AND NON-SMOKERS

Figure 2 show that the smokers had low levels of TAC compared to the non-smokers

Based on their age, both groups were divided into two sub groups (30-40 years and 41-50 years respectively) and the TAC levels were compared between the groups.

The data of both smokers and non-smokers were given in table-6.

0 100 200 300 400 500 600 700 800

1 4 7 10 13 16 19 22 25 28 31 34 37 40

TAC (Smokers vs Non-Smokers)

Non-Smokers Smokers

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Table 6: CROSS TABULATION

AGE

NON-SMOKERS SMOKERS

n % n %

30 to 40yrs 29 72.5% 27 67.5%

41 to 50yrs 11 27.5% 13 32.5%

Total 40 100.0% 40 100.0%

The total number of non-smokers in this study were 40, among them 29 subjects were between the age group of 30-40years and 11 subjects were between 41-50 years. The smoker group also included 40 subjects of which 27 subjects were between the age group of 30-40 years and 13 were between 41-50 years.