Dissertation “COMPARISON OF SERUM MALONDIALDEHYDE LEVELS IN PATIENTS WITH ORAL SUBMUCOUS

Transcription

“COMPARISON OF SERUM MALONDIALDEHYDE
LEVELS IN PATIENTS WITH ORAL SUBMUCOUS
FIBROSIS AND CONTROL GROUP –
A HOSPITAL BASED STUDY”
By
DR.ALAKA HEBBAR
REG. NO. IG0208001
Dissertation
Submitted to
K.L.E University, Belgaum, Karnataka
In partial fulfillment
Of the requirements for the degree of
MASTERS OF DENTAL SURGERY
In
ORAL MEDICINE AND RADIOLOGY
(BRANCH – IX)
Under the guidance of
DR. ARVIND SHETTI M.D.S
Professor
DEPARTMENT OF ORAL MEDICINE AND RADIOLOGY
KLE VISHWANATH KATTI INSTITUTE OF DENTAL SCIENCES
BELGAUM, KARNATAKA
MAY 2011
I
K.L.E UNIVERSITY, BELGAUM
KARNATAKA
DECLARATION BY THE CANDIDATE
I hereby declare that this dissertation/thesis entitled “COMPARISON OF SERUM
MALONDIALDEHYDE LEVELS IN PATIENTS WITH ORAL SUBMUCOUS
FIBROSIS AND CONTROL GROUP – A HOSPITAL BASED STUDY” is a
bonafide and genuine research work carried out by me under the guidance of
DR.ARVIND SHETTI
M.D.S
Professor and Head, Department of Oral Medicine and
Radiology, K.L.E. V.K Institute of Dental Sciences.
DATE:
PLACE: Belgaum
DR.ALAKA HEBBAR
II
KARNATAKA
CERTIFICATE BY GUIDE
This is to certify that the dissertation entitled “COMPARISON OF SERUM
bonafide research work done by DR.ALAKA HEBBAR, in partial fulfillment of the
requirement for the degree of MASTERS OF DENTAL SURGERY in Oral
Medicine and Radiology.
DATE:
PLACE: Belgaum
DR.ARVIND SHETTI M.D.S
Professor,
Department of Oral Medicine & Radiology,
KLES VK Institute of Dental Sciences,
Belgaum
III
KARNATAKA
CERTIFICATE BY CO-GUIDE
bonafide research work done by DR.ALAKA HEBBAR, in partial fulfillment of the
requirement for the degree of MASTERS OF DENTAL SURGERY in Oral
Medicine and Radiology.
DATE:
PLACE: Belgaum
DR.P.B.DESAI MD
Professor and Head
Department of Biochemistry,
JN Medical College,
Belgaum.
IV
KARNATAKA
ENDORSEMENT BY THE HOD, PRINCIPAL/HEAD OF THE
INSTITUTION
bonafide research work done by DR.ALAKA HEBBAR, post graduate student under
the guidance of DR.ARVIND SHETTI
M.D.S
Professor, Dept. of Oral Medicine and
Radiology, K.L.E V.K Institute of Dental Sciences, Belgaum.
HEAD OF DEPARTMENT
PRINCIPAL
DR.VAISHALI KELUSKAR M.D.S
Professor and Head
Department of Oral Medicine
and Radiology,
K.L.E. V.K Institute of
Dental Sciences,
KLE University
Belgaum -590010.
DR.ALKA D.KALE M.D.S
Principal
K.L.E. V.K Institute of Dental Sciences,
K.L.E University
Belgaum-590010.
Date:
Place: Belgaum
Date:
Place: Belgaum
V
KARNATAKA
COPYRIGHT
DECLARATION BY THE CANDIDATE
I hereby declare that K.L.E University, Belgaum, Karnataka shall have the rights to
preserve, use and disseminate this dissertation/thesis titled “COMPARISON OF
SERUM MALONDIALDEHYDE LEVELS IN PATIENTS WITH ORAL
SUBMUCOUS FIBROSIS AND CONTROL GROUP – A HOSPITAL BASED
STUDY” in print or electronic format for academic/research purpose.
DATE:
PLACE: Belgaum
DR.ALAKA HEBBAR
© KLE University, Belgaum, Karnataka
[Established under section 3 of the UGC Act, 1956 vide GOI. Notification No.F.9-19/2000-U.3(A)]
VI
ACKNOWLEDGEMENT
“A teacher is a mother of knowledge”
“Gurur Brahma, Gurur Vishnu, Gurur Devo Maheshwaraha, Gurur Sakshaath
Parabrahma tasmai Shree Gurave Namaha”
“Teaching is a profession that teaches all the other professions”
I dedicate these words to express my profound respect, deep sense of gratitude
and indebtness to my beloved teacher Dr. Vaishali Keluskar
M.D.S
Professor and
Head, Department of Oral Medicine, Diagnosis and Radiology, K.L.E. V.K Institute
of Dental Sciences, Belgaum, for her immense support, patient hearing and constant
motivation which boosted up my confidence during tough times.
“A Good teacher Explains… A Superior teacher Demonstrates…
A Great teacher Inspires”
A teacher is like a compass that activates the magnets of curiosity, knowledge
and wisdom amongst the people. Words are indeed inadequate to express my heartfelt
gratitude and indebtedness to my guide Dr. Arvind Shetti
M.D.S
Professor and,
Department of Oral Medicine, Diagnosis and Radiology, K.L.E.’s Vishwanath Katti
Institute of Dental Sciences, Belgaum. He with his ever inspiring presence, critical
observation and deep understanding helped me to shape my ideas in a coherent
fashion. Without his enthusiastic guidance, constant encouragement, constructive
criticism and valuable suggestions, it would not have been possible for me to
complete this study.
VII
“Good teaching is more a giving of right questions than a giving of right answers”
I owe an immense debt of gratitude to Dr. Anjana Bagewadi, M.D.S Professor,
Department of Oral Medicine, Diagnosis and Radiology, K.L.E.’s Vishwanath Katti
Institute of Dental Sciences, Belgaum, for her comforting presence and ever helpful
hand during the study.
“Teachers open the door, but you must enter by yourself”
Words do not suffer to express my sincere and humble gratitude to my
respected teachers Dr. Renuka Ammanagi M.D.S and Dr. Zameera Naik M.D.S Reader,
Department of Oral Medicine and Radiology, K.L.E.’s Vishwanath Katti Institute of
Dental Sciences, Belgaum, for their valuable suggestions during the study.
I am highly obliged to our beloved Principal Dr. Alka. D. Kale M.D.S Professor,
Department of Oral Pathology and Microbiology, K.L.E.’s Vishwanath Katti Institute
of Dental Sciences, Belgaum for allowing me to utilize the clinical material, facilities
in this institution and also providing me with all the necessary support whenever
needed.
I take the opportunity to express my profound gratefulness to Dr. P . B. Desai
M.D.
Professor and Head, Department of Biochemistry, Jawaharlal Nehru Medical
College, Belgaum, for his constant encouragement and help in conducting the
biochemical analysis during the period of my study.
I extend my heartfelt thanks to Dr.Vasanti Jirge, M.D.S Dr. Shivyogi
M.D.S
and
Dr. Ambika M.D.S Senior lecturer and for their valuable suggestions during my study.
I express my sincere thanks to Professor Dr.H.N. Nagaraj and Professor Mr.
Mallapur for providing help in statistical analysis for this study.
VIII
“Side by side or miles apart, dear friends are always close to the heart”
I wish to acknowledge and thank my colleagues, Dr. Archana, Dr. Kumud,
Dr. Vishlesh, Dr. Poonam, Dr. Ashish, Dr. Ankur, Dr. Sapna, Dr.Anshul, Dr.
Rakhi, Dr.Nidhi, Dr. Ishita, Dr. Hina, Dr. Sugandha and Dr. John for their cooperation and help throughout the course of my study.
“The virtue of parents is in itself a great legacy”
I am blessed to have loving families, who have been a constant support and
encouragement to me through thick and thin. I am infinitely obliged to express my
feeling of pride for my most cherished treasure – My Father Dr. H.R.Hebbar, mother
Dr. Pushpagandhini and sister Malavika Hebbar, for their love, affection, support
and advice.
I am thankful to my aunties Sumangala Bha, Anuradha Navada and my
uncles H.N. Krishnaraj and Ramachandra Bhat who always stood by me and
supported me in this endeavour.
I am thankful to my friends Dr. Shweta Advani and Dr. Raveena Thapar and
Dr. Tejaswini Gauripur and all those people who have offered me genuine words of
advice and encouragement during the period of my study.
This list will be incomplete if I do not acknowledge the debt that I owe to all
the subjects who consented to be part of the study.
Lastly and above all, I thank God Almighty for making all these wonderful
people happen to me and giving me strength and courage to complete this project.
Dr. ALAKA HEBBAR.
IX
Dedicated to my
beloved Parents and
Grandmother
X
LIST OF ABBREVIATIONS
ANOVA
One way Analysis of Variance
OSMF
Oral Submucous Fibrosis
ROS
Reactive Oxygen Species
MDA
Malondialdehyde
H2O2
Hydrogen Peroxide
RAR
Retinoic Acid Receptor
PAS
Periodic Acid Schiff
ANA
Antinuclear Antibody
SMA
Anti Smooth muscle Antibody
CTLA
Cytotoxic T-lymphocytes Associated Antigen
Ig
Immunoglobulin
SGOT
Serum Glutamic Oxaloacetic Transaminase
SGPT
Serum Glutamic Pyruvic Transaminase
RBC
Red Blood Cells
HLA
Human Leukocyte Antigen
WHO
World Health Organization
O2
Oxygen
PR
Pyrogallol red
H2
Hydrogen
HOCl
Hypochlorous acid
SOD
Superoxide Dismutase
NADPH
Nicotinamide Adenosine Dinucleotide Phosphate
MPO
Myeloperoxidase
Fe
Iron
XI
DNA
Deoxyribonuclic Acid
UV
Ultraviolet
•
OH
Hydroxyl Radical
NO
Nitric Oxide
PUFA
Poly Unsaturated Fatty Acid
ATP
Adenosine Triphosphate
LPO
Lipid Peroxidation
GECA
Anti Gastric Perietal Cell Antibodies
M
Macroglobulin
HOO•
Hydroperoxyl Radical
ROO•
Lipid Peroxyl Radical
R•
Free Radical
MPO
MyeloPeroxidase
O2•-
Superoxide Radical
RSH
Thiol Compounds
CCl4
Carbon Tetrachloride
•
CCl3
Trichloromethyl radical
CH2
Methylene
ROOH
Hydroperoxide
R-OOH
Lipid hydroperoxide
ALE
Advanced lipoxidation end products
AGE
Advanced glycation end products
TBA
Thiobarbituric Acid
TBARS
Thiobarbituric acid reactive substances
GR
Glutathione Reductase
XII
GPX
Glutathione Peroxidase
G6PDH
Glucose-6 Phosphate Dehydrogenase
GST
Glutathione-S-transferase
M1 G
Melondialdehyde guanine
COX2
Cyclooxygenase 2
M1-dG
Melondialdehyde deoxyguanosine
NO3
Nitrate
NO2
Nitrite
HCl
Hydrochloric acid
XIII
ABSTRACT
Background and Objectives:
Oral submucous fibrosis is a chronic, insidious and debilitating condition with a high
malignant potential and a high prevalence rate in India and South East Asian
countries. Studies have shown that the process of carcinogenesis occurs in OSMF by
the generation of reactive oxygen species (ROS), which act by initiating lipid
peroxidation (LPO). Malondialdehyde (MDA) is one of the important and easily
detectable biomarker of LPO and a indicator of oxidative stress.
The objective of this study was to assess MDA levels in the serum of OSMF patients
and to compare it with the Control group.
Method:
36 patients with OSMF and 24 age matched controls were included in the study and
the serum MDA levels were estimated using Thiobarbituric acid (TBA) reagent.
Results:
There was a statistically significant difference in serum MDA levels among the
patients with OSMF and the control group. Serum MDA levels varied in different
stages of OSMF also. The levels were increased in stage III OSMF followed by stage
II and stage I.
Interpretation & Conclusion:
It is evident from the present study that serum MDA levels were significantly
increased in patients with OSMF indicating the increased amount of oxidative stress.
Keywords: OSMF, Malondialdehyde, Oxidative stress, lipid peroxidation
XIV
TABLE OF CONTENTS
SI. NO.
PARTICULARS
PAGE
01
INTRODUCTION
1
02
AIM AND OBJECTIVES
3
03
REVIEW OF LITERATURE
4
04
METHODOLOGY
49
05
RESULTS
58
06
DISCUSSION
66
07
CONCLUSION
74
08
SUMMARY
75
09
BIBLIOGRAPHY
76
10
ANNEXURES
ANNEXURE -
I: PROFORMA
90
ANNEXURE - II: CONSENT FORM
92
ANNEXURE - III: MASTER CHART- CASES
93
ANNEXURE - IV: MASTER CHART – CONTROLS
95
XV
LIST OF TABLES
SI.
NO.
TABLES
PAGE NO.
01
Laboratory procedure for MDA estimation by TBA method
53
02
Mean value for age among various groups
58
03
ANOVA Test for ages among different study groups
59
04
Mean value for MDA among various groups
59
05
ANOVA Test for MDA among various groups
60
06
Mean MDA value comparison
61
07
Multiple comparisons of serum MDA values in various groups
61
using Tukey Kramer Test
XVI
LIST OF GRAPHS
SI.
NO.
GRAPHS
PAGE NO.
01
Distribution of number of subjects in various study groups
63
02
Distribution of subjects in various study groups according to age
63
03
Mean age of patients among different study groups
64
04
Mean serum malondialdehyde levels among various study groups
64
05
Comparison of mean MDA levels among various study groups
65
XVII
LIST OF FIGURES
SI.
FIGURES
NO.
PAGE NO.
01
Molecular pathogenesis of OSMF (1)
14
02
15
03
16
04
16
05
The cellular sources of free radicals
24
06
Generation of oxygen free radicals in respiratory burst
27
07
Lipid peroxidation process
34
08
Structure of malondialdehyde
37
09
Complex multifactorial nature of oxidative damage to cells
39
10
Equipments used in the clinical examination of the patients
54
11
Reagents used for the estimation of serum malondialdehyde
54
12
Boiling water bath
55
13
Centrifuge machine
55
14
Spectrophotometer
56
15
Blanching on labial mucosa
56
16
Blanching on buccal mucosa
57
17
Shrunken uvula
57
18
Reduced mouth opening
57
19
Reduced tongue protrusion
57
XVIII
“COMPARISON OF SERUM MALONDIALDEHYDE
LEVELS IN PATIENTS WITH ORAL SUBMUCOUS
FIBROSIS AND CONTROL GROUP –
A HOSPITAL BASED STUDY”
By
REG. NO. IG0208001
Dissertation
Submitted to
K.L.E University, Belgaum, Karnataka
In partial fulfillment
Of the requirements for the degree of
MASTERS OF DENTAL SURGERY
In
ORAL MEDICINE AND RADIOLOGY
(BRANCH – IX)
DEPARTMENT OF ORAL MEDICINE AND RADIOLOGY
KLE VISHWANATH KATTI INSTITUTE OF DENTAL SCIENCES
BELGAUM, KARNATAKA
MAY 2011
I
KARNATAKA
ENDORSEMENT BY THE HOD, PRINCIPAL/HEAD OF THE
INSTITUTION
bonafide research work done by
Candidate Reg. No.
IG0208001 of Oral
Medicine and Radiology, K.L.E V.K Institute of Dental Sciences, Belgaum.
HEAD OF DEPARTMENT
DR.VAISHALI KELUSKAR M.D.S
Professor and Head
Department of Oral Medicine
and Radiology,
K.L.E. V.K Institute of
Dental Sciences,
KLE University
Belgaum -590010.
PRINCIPAL
DR.ALKA. D.KALE M.D.S
Principal
K.L.E. V.K Institute of Dental Sciences,
K.L.E University
Belgaum-590010.
Date:
Place: Belgaum
Date:
Place: Belgaum
II
LIST OF ABBREVIATIONS
ANOVA
One Way Analysis of Variance
OSMF
ROS
Reactive Oxygen Species
MDA
Malondialdehyde
H2O2
Hydrogen Peroxide
RAR
Retinoic Acid Receptor
PAS
Periodic Acid Schiff
ANA
Antinuclear Antibody
SMA
Anti Smooth muscle Antibody
CTLA
Cytotoxic T-lymphocytes Associated Antigen
Ig
Immunoglobulin
SGOT
Serum Glutamic Oxaloacetic Transaminase
SGPT
Serum Glutamic Pyruvic Transaminase
RBC
Red Blood Cells
HLA
Human Leukocyte Antigen
WHO
World Health Organization
O2
Oxygen
PR
Pyrogallol red
H2
Hydrogen
HOCl
Hypochlorous acid
SOD
Superoxide Dismutase
NADPH
Nicotinamide Adenosine Dinucleotide Phosphate
MPO
Myeloperoxidase
Fe
Iron
III
DNA
Deoxyribonuclic Acid
UV
Ultraviolet
•
OH
Hydroxyl Radical
NO
Nitric Oxide
PUFA
Poly Unsaturated Fatty Acid
ATP
Adenosine Triphosphate
LPO
Lipid Peroxidation
GECA
Anti Gastric Perietal Cell Antibodies
M
Macroglobulin
HOO•
Hydroperoxyl Radical
ROO•
Lipid Peroxyl Radical
R•
Free Radical
MPO
MyeloPeroxidase
O2•-
Superoxide Radical
RSH
Thiol Compounds
CCl4
Carbon Tetrachloride
•
CCl3
Trichloromethyl radical
CH2
Methylene
ROOH
Hydroperoxide
R-OOH
Lipid hydroperoxide
ALE
Advanced lipoxidation end products
AGE
Advanced glycation end products
TBA
Thiobarbituric Acid
TBARS
Thiobarbituric acid reactive substances
GR
Glutathione Reductase
IV
GPX
Glutathione Peroxidase
G6PDH
Glucose-6 Phosphate Dehydrogenase
GST
Glutathione-S-transferase
M1 G
Melondialdehyde guanine
COX2
Cyclooxygenase 2
M1-dG
Melondialdehyde deoxyguanosine
NO3
Nitrate
NO2
Nitrite
HCl
Hydrochloric acid
V
ABSTRACT
Background and Objectives:
Oral submucous fibrosis is a chronic, insidious and debilitating condition with a high
malignant potential and a high prevalence rate in India and South East Asian
countries. Studies have shown that the process of carcinogenesis occurs in OSMF by
the generation of reactive oxygen species (ROS), which act by initiating lipid
peroxidation (LPO). Malondialdehyde (MDA) is one of the important and easily
detectable biomarker of LPO and a indicator of oxidative stress.
The objective of this study was to assess MDA levels in the serum of OSMF patients
and to compare it with the Control group.
Method:
36 patients with OSMF and 24 age matched controls were included in the study and
the serum MDA levels were estimated using Thiobarbituric acid (TBA) reagent.
Results:
There was a significant difference in serum MDA levels among the patients with
OSMF and the control group. Serum MDA levels varied in different stages of OSMF.
Serum MDA levels were found to be highest in stage III OSMF followed by stage II
and stage I.
Interpretation & Conclusion:
It is evident from the present study that serum MDA levels were significantly
increased in patients with OSMF indicating the increased amount of oxidative stress.
Keywords: OSMF, Malondialdehyde, Oxidative stress, lipid peroxidation
VI
TABLE OF CONTENTS
SI. NO.
PARTICULARS
PAGE
01
INTRODUCTION
1
02
AIM AND OBJECTIVES
3
03
REVIEW OF LITERATURE
4
04
METHODOLOGY
49
05
RESULTS
58
06
DISCUSSION
66
07
CONCLUSION
74
08
SUMMARY
75
09
BIBLIOGRAPHY
76
10
ANNEXURES
ANNEXURE -
I: PROFORMA
90
ANNEXURE - II: CONSENT FORM
92
ANNEXURE - III: MASTER CHART- CASES
93
ANNEXURE - IV: MASTER CHART – CONTROLS
95
VII
LIST OF TABLES
SI.
NO.
TABLES
PAGE NO.
01
Laboratory procedure for MDA estimation by TBA method
53
02
Mean value for age among various groups
58
03
ANOVA Test for ages among different study groups
59
04
Mean value for MDA among various groups
59
05
ANOVA Test for MDA among various groups
60
06
Mean MDA value comparison
61
07
Multiple comparisons of serum MDA values in various groups
62
using Tukey Kramer Test
VIII
LIST OF GRAPHS
SI.
NO.
GRAPHS
PAGE NO.
01
Distribution of number of subjects in various study groups
63
02
Distribution of subjects in various study groups according to age
64
03
Mean age of patients among different study groups
64
04
Mean serum malondialdehyde levels among various study groups
65
05
Comparison of mean MDA levels among various study groups
65
IX
LIST OF FIGURES
SI.
NO.
FIGURES
PAGE NO.
01
14
02
15
03
16
04
16
05
The cellular sources of free radicals
24
06
Generation of oxygen free radicals in respiratory burst
27
07
Lipid peroxidation process
34
08
Structure of malondialdehyde
37
09
Complex multifactorial nature of oxidative damage to cells
39
10
Equipments used in the clinical examination of the patients
54
11
Reagents used for the estimation of serum malondialdehyde
54
12
Boiling water bath
55
13
Centrifuge machine
55
14
Spectrophotometer
56
15
Blanching on labial mucosa
56
16
Blanching on buccal mucosa
57
17
Shrunken uvula
57
X
18
Reduced mouth opening
57
19
Reduced tongue protrusion
57
XI
Introduction
INTRODUCTION
Oral submucous fibrosis is a chronic insidious and progressive disease involving oral
mucosa. Recent studies reveal that there is an increase prevalence of Oral submucous
fibrosis in different states of India.1 This condition has been shown to be
precancerous2 and carries a high risk for malignant conversion even after the cessation
of areca nut use, which is known to play a major role in the development of the
disease.3 There is no report suggesting spontaneous regression nor there is any no
effective or widely accepted treatment, making this condition worse.4
Epidemiological studies have shown that the process of carcinogenesis
occurs by the generation of Reactive Oxygen Species (ROS).5 Free radicals can be
defined as molecules or molecular fragments with an unpaired electron which imparts
certain characteristics to the free radicals such as reactivity.7,8 Reactive free radicals
are able to produce chemical modifications and damage proteins, lipids, carbohydrates
and neucleotides in the tissues. Reactive oxygen species (ROS) may damage the cells
by initiation of lipid peroxidation (LPO)6 that causes profound alteration in the
structural integrity and functions of the cell membranes. Free radical induced LPO has
been implicated in the pathogenesis of several diseases. The concentration of LPO
product malondialdehyde (MDA) is most widely used in this regard.
The etiology and malignant transformation potential of Oral submucous
fibrosis is poorly understood. Literature available suggests it to be multifactorial in
origin, which includes arecanut chewing, heredity, nutritional deficiencies and
immunological factors.
1
Introduction
OSMF being the potentially malignant condition and associated with
carcinogens like tobacco was thought to have some relation with ROS. Hence this
study was designed to estimate serum LPO product malondialdehyde in various stages
of OSMF patients and to compare the same with control group.
2
Aim and Objective
AIM AND OBJECTIVES
AIM:
1. To estimate and compare the levels of serum lipid peroxidation product
malondialdehyde in patients with oral submucous fibrosis and the control
group.
OBJECTIVES:
1. To estimate serum lipid peroxidation product malondialdehyde levels in patients
with oral submucous fibrosis.
2. To estimate serum malondialdehyde levels in age & sex matched controls with
chewing habit without the disease.
3. To estimate serum malondialdehyde levels in age and sex matched healthy
controls without chewing habit.
4. To compare serum malondialdehyde levels in patients with oral submucous
fibrosis & controls.
3
Review of literature
ORAL SUBMUCOUS FIBROSIS
Oral submucous fibrosis is a chronic debilitating disease of the oral cavity. It is a
disease predominantly of Indian population, although there is worldwide distribution.
It is a well recognized potentially malignant condition which predominantly affects
the oral cavity, but may extend to pharynx, oesophagus and even to larynx. It is
characterized by juxta-epithelial inflammatory reaction followed by fibro-elastic
changes in the lamina propria with epithelial atrophy leading to stiffness of oral cavity
and causing trismus and inability to eat. OSMF also results in difficulty in speech,
swallowing, pain in the throat and relative loss of auditory acquity.9
HISTORICAL REVIEW
Oral submucous fibrosis has been well established in medical literature since the time
of Sushruta – a renowned Indian physician who lived in the era of 2500-3000 B C. He
described a condition resembling OSMF and named it as ‘VIDHARI’ having features
of progressive narrowing of mouth, depigmentation of oral mucosa and pain on taking
food.10
Schwartz (1952) was the first person to describe OSMF as a fibrosing condition
among five Indian women from Kenya and east Africa and named it as “atophica
idiopathica tropica mucosae oris”.11
Joshi S G (1953) first described the condition in India and suggested the name
“submucous fibrosis” of palate and pillars’12.
Lal D (1953) suggested the notation “diffuse OSMF” to denote the complete
replacement of the subepithelial layer of dense, acellular, nonelastic collagenous
material with collections of lymphocytes and plasma cells.13
4
Sui P (1954) termed it as “idiopathica scleroderma of mouth”.14
Paymaster J C (1956) was the first to describe it as a premalignant condition and its
association in development of slow growing squamous cell carcinoma in one third of
the cases of OSMF in Mumbai.15
Rao B N (1962) termed it as “idiopathic palatal fibrosis” in his study of 46 cases of
palatal fibrosis in Hyderabad.16
Pindborg J J et al (1965) in their study of OSMF among 100 South Indians with oral
cancer reported the finding of OSMF in 40 out of 100 patients.17
Mohd. Akbar (1976) in his study reported the occurrence of OSMF associated with
leukoplakia of buccal mucosa in 4 cases and of tongue in 1 case.18
George Laskaris et al (1981) reported a case of OSMF in a 67 year old Greek female
who later developed squamous cell carcinoma of the tongue which later progressed to
a fetal termination.19
Pindborg J J et al (1984) found that the rate of malignant transformation to be 4.5%
out of 89 patients with the disease in Ernakulum district, Kerala17.
Murti P R et al (1985) in their seventeen year old follow up study of OSMF noted
that the rate of malignant transformation was 4.5 at the end of 15 years and 7.6 at the
end of 17 years, they have stressed that OSMF had a high degree of malignant
potential.20
DEFINITIONS
According to WHO (1978), OSMF is defined as “A slow growing progressive disease
in which fibrous bands form in the blanched oral mucosa resulting in severe
restriction of movement of mouth”.
5
The most accepted definition is the one stated by Pindborg and Sirsat (1966) “OSMF
is an insidious chronic disease affecting any part of oral cavity and sometimes
pharynx although occasionally preceded by and/ or associated with juxta epithelial
inflammatory reaction followed by fibroelastic changes in the lamina propria with
epithelial atrophy leading to stiffness of oral mucosa and causing trismus and inability
to eat”.9
EPIDEMIOLOGY
Worldwide estimates in 1996 indicated that this disease affected 2.5 million people. In
2002 statistics from India alone were about 5 million people that is 0.5% of the Indian
population. OSMF is very common in South East Asian countries such as Bangladesh,
Bhutan, Pakistan, India, Sri Lanka. The prevalence rate of OSMF in these countries
ranges from 0 to 1.2%. Only in India incidence is 0.2 to 0.5%. Prevalence is higher in
south compared to north India. The incidence in Ernakulum was reported as 8 per 1
lakh men and 19 per 1 lakh women.5
ETIOLOGY AND RISK FACTORS
Although various etiological agents are proposed, the exact etiology of oral
submucous fibrosis has not yet been identified. Various etiological agents and
predisposing factors have been studied and current evidence suggests that arecoline in
areca nut plays a major role in initiating the disease process. However role of different
etiological agents and factors so far studied are:

Chillies

Genetic Predisposition
6

Tobacco and areca nut

Immunological factors

Nutritional factors

Autoimmune mechanism
More recently collagen related genes are implicated in the susceptibility and
pathogenesis of OSMF.
i)
Chillies
OSMF is more prevalent in South India and Sri Lanka where chillies are eaten dried,
powdered or raw at meal.25,23 The chillies which contain capsaicin promote changes
of OSMF.
A study showed chilli extract to be mutagenic and it has been found to enhance the
tumorogenicity of tobacco in experimental animals.23,24
ii)
Arecanut
Chewing of areca nut wrapped in betel leaf and smeared with the paste of crude lime
and spices was incriminated in one study as the causative agent. The pathological role
was attributed to high tannic acid, slaked lime, and the continuous and prolonged
action of nicotine contained in the betel nut, these have neurotropic effect on the oral
mucosa.14,21
Experimental evidence for the etiological role of betel nut in OSMF came from in
vitro studies in which it was shown that ethanolic extracts of the nut stimulated
collagen synthesis in human fibroblasts.15
In a study on histological effect of arecoline on the palate and buccal mucosa of 28
vistar rats of varying periods of time was observed. It was noted that OSMF of palate
7
was produced by the habit of chewing betel nut. Based on the findings it was
suggested that arecoline played an important role in the causation of OSMF of palate
in human being also.26, 22
The pharmacological effect of different betel nut preparations was studied in relation
to OSMF. The variations in nut alkaloids and tannin content probably due to plant
variability and different cooking procedures were noted. Thus variations in the
pharmacologically active constituents of the betel nut may contribute to the regional
differences in the incidence of the disease.27
iii)
Nutritional deficiency
OSMF has been proposed as an Asian version of sideropenic dysphagia wherein the
chronic iron deficiency leads to mucosal susceptibility to irritants such as chilli and
areca nut. Deficiency of vitamins and iron has been implicated as being of etiological
importance in the oral submucous fibrosis.28
iv)
Genetic predisposition
In a study on HLA frequencies in 50 OSMF and 50 controls were studied, three
antigens (A10, B7, and DR3) were significantly raised in the patients suggesting
genetic linkage of the disease.13
Researchers analyzed the expression of retinoic acid receptor (RAR beta) and p53 by
immunohistochemistry in 50 cases of OSMF and 30 histologically normal oral tissues.
The study concluded that altered expression of either of these proteins in majority of
OSMF cases or associated with disease pathogenesis. Follow up is required to
determine if these cases harboring concomitant alterations in (RAR beta) and p53 are
at a high risk of transition to malignancy.28
8
v)
Immunological factors
OSMF have similarity in clinical presentation to collagen disorders like scleroderma
that has an autoimmune pathogenesis. The term ‘idiopathic scleroderma of the mouth’
has been coined for it because of the same.13
Histologically eosinophilic material and marked increase in PAS positive material
with metachromasia in the ground substance, indicative of presence of fibroid like
material as seen in number of cases of connective tissue disorders involving dense
collagen proliferation is seen. The ultrastructural changes were also similar to those
seen in rheumatoid arthritis and scleroderma.26
In view of the association of the human leukocyte antigen (HLA) DR3 with
scleroderma a study was conducted and increase in DR3 antigen among 44 patients
with OSMF was noted. An increase in serum IgG immunoglobulins and auto
antibodies were also noted among these subjects.13
Incidence of auto antibodies in 109 Tiwanese patiens with OSMF was studied and a
high frequencies of antinuclear antibodies (ANA) (23.9%), anti-smooth muscle
antibodies (SMA) (23.9%) and anti gastric parietal cell antibodies (GECA) (14.7%)
was observed compared to that of healthy controls(9.2, 7.3 and 5.5%) respectively
suggesting that OSMF may be an autoimmune disease.30
In a recent study the role of cytotoxic T- lymphocytes – associated antigen 4 (CTLA4) in the maintenance of immune tolerance was highlighted. A higher frequency of G
allele at position +49 in exon 1 of CTLA-4 was noticed in 64 OSMF patients as
compared to controls. Such defect in cellular immunity is also seen in scleroderma as
a result of autoimmune phenomenon. The authors thus suggested CTLA-4
polymorphism might play a role in susceptibility to OSMF.31
9
In a review regarding pathogenesis of OSMF multifactorial etiology was proposed
stating the role of genetic alteration, carcinogen like areca nut, tobacco, nutritional
factors and immunological factors.32
The serum protein, ascorbic acid, iron and tissue collagen were studied in OSMF
patients. The patients were categorized into mild, moderate and severe based on the
severity of the clinical manifestation. Biopsy samples were obtained for tissue
collagen estimation. Authors reported significant rise of protein and tissue collagen,
early albumin: globulin ratio was decreased with significantly raised levels of
globulin while ascorbic acid and iron content was decreased.33
In a study quantifying the number of mast cells in different grades of oral submucous
fibrosis it was found that in grade I at an average 4.5 mast cells per unit microfield
were present, in grade II 4.9 cells per unit microfield and 0.5 cells per unit microfield
in grade III cases as compared to the average number of mast cells per unit microfield
in normal buccal mucosa which is 1.02.34
It was later hypothesized that sensitized mast cells coming in contact with arecoline
act as an allergen resulting in degranulation causing release of histamine and heparin
which produces fibrosis. It was further suggested that OSMF may be hypersensitivity
type I reaction to arecoline and the reaction represents a genetically controlled
predisposition to the production of specific IgE antibodies.34
Hypersensitivity caused by local irritants and the resultant persistent juxtaepithelial
inflammatory response may act as initiating factors. Thus defective inflammatory
reparative response culminates in fibrotic healing.35
Increase in the serum mucoprotien and mucopolysaccharide levels in patients with
OSMF has been observed. It was suggested that these represent the reactants in the
active stage of the disease where breakdown of tissues and collagen degeneration is
10
occurring. Rise in anti-streptolysin titre suggested a possible role of immunological
response in the form of a localized collagen disorder to streptococcal toxicity in the
etiology of oral submucous fibrosis.36
One of the studies conducted in 34 patients of OSMF and 20 age and sex matched
controls showed elevated levels of total globulin in OSMF patients and this was
highly significant when compared to control group. The author also reported a
significant increase in total immunoglobulin levels in OSMF patients as compared to
control group. The IgG fraction showed a significant increase in OSMF than controls
but IgA levels were comparable to control group. IgM did not show any alterations in
OSMF. Thus in view of hyperglobulinemia and hyperimmunoglobulinemia the author
concluded that OSMF may be an autoimmune disorder.37
A study was conducted to know the possible rate of immunological factors in the
OSMF by the evaluation of the 113 OSMF cases and 25 controls, male: female ratio
was 1.5:1. Serum IgA, IgG and IgM levels were elevated significantly as compared to
the controls. Circulating autoantibodies and tissue deposited antibodies were also
found in 33% and 40% of patients and serum globulin levels in 47% of patients, thus
indicating a immunological basis.38
vi)
Immunoglobulins
According to WHO, immunoglobulins are the proteins of animal origin endowed with
antibody activity and for certain other proteins related to them by chemical structure.
The definition includes, besides antibody globulins, the subnormal proteins found in
myeloma, macroglobulinemia, cryoglobulinemia and naturally occurring subunits of
immunoglobulins.
11
They are synthesized by plasma cells and to some extent by lymphocytes and
constitute 20-25% of the total serum proteins. Immunoglobulins are glycoproteins,
each molecule consisting of 2 pairs of polypeptide chains of different sizes and is the
basis of classifying 5 types, i.e. IgG, IgA, IgM, IgD and IgE in order of decreasing
concentration in the serum
vii)
Arecoline and immunity
Short term effects of general parameters on the adrenal and the lymphoid organs were
screened to explore in detail its immunomodulatory influence in murine model
system. Arecoline was administered subcutaneously to male mice at subtoxic dose
levels (5, 10, 20 mg/Kg body weight) for 1, 2 and 3 weeks on a daily basis, while total
protein, albumin, glucose, phosphatase and hemoglobin concentrations were not
altered, increase in SGOT and SGPT levels were observed at the high dose. The white
and red blood cell counts decreased in a dose dependent manner. These observations
demonstrated the effect of arecoline on the lymphoid organs which may be due its
direct action or through the elevation of corticosterone39.
B cell mediated immune response in mice after short term exposure to arecoline
examined to explore its modulatory influence on B cell mediated immune response in
murine model system. The in vivo and in vitro effects of arecoline were evaluated at a
subtoxic concentration. Arecoline exposure for a week involved the dose dependent
effect on primary antibody forming cells to the sheep RBC’s with the maximum
reduction at the dosage of 20 mg/Kg bw, a moderate reduction at 10mg/Kg body
weight and no effect at 5 mg/Kg body weight was observed. HA and HL titres to
sheep RBC’s were suppressed markedly at arecoline dosage of 20mg/Kg body weight
12
and moderately at a dose of 10mg/Kg body weight, given daily for 1, 2 and 3 weeks.
The inhibitory effect of arecoline was not dependent on the duration of treatment.40
viii)
Allergic role
The presence of eosinophilic cells as constant finding in most of the histologic
pictures of OSMF made to remark that the condition is probably an allergic reaction.
It was hypothesized that sensitized mast cells coming in contact with arecoline act as
an allergen resulting in degranulation causing release of histamine and heparin results
in fibrosis. It was further suggested that OSMF may be hypersensitivity type I
reaction to arecoline and the reaction represents a genetically controlled
predisposition to the production of specific IgE antibodies. Thus only those
individuals who are active to sensitive ingredients of betel nut are affected. This also
could explain why majority of betel nut chewers in India don’t suffer from OSMF.
Hypersensitivity caused by local irritants and the resultant persistent juxtaepithelial
inflammatory reparative response culminating in fibrotic healing.
ix)
Defective iron metabolism
The first reported case of anemia in patients of OSMF was described by Moosa and
Madan. It is postulated that the changes brought about by anemia in tissues would
make the individuals more susceptible to OSMF. It is a well recognized fact that the
histological changes observed in oral mucosa of patients with OSMF lack of iron in
the tissue would also result in the improper function of vascular channels and
concomitant decrease in the vascularity. The percolation of ester of arecoline would
be made easier which in turn results in fibrosis. It is also noted that occurrence of iron
deficiency anemia could be due to the clinical nature of OSMF. The initial burning
sensation, vesiculations and ulcerations render the consumption of solid food difficult.
13
This lack of consumption of normal diet would possibly initiate anemia in females.
After frank establishment of the lesion anemia may be further perpetuated by
inadequate intake of food due to fibrosis and trismus. Thus the relationship of iron
deficiency anemia with OSMF seems to be a vicious circle.
PATHOGENESIS
The most important histopathological characteristic of OSMF is the deposition of
collagen in the submucosa. The arecanut component of betel quid especially alkaloid
called arecoline plays a role in pathogenesis of OSMF by causing an abnormal
increase in collagen production. The flavanoid component is believed to have some
direct effect on the collagen metabolism. Alkaloid exposure to buccal mucosal
fibroblast results in accumulation of collagen. A decreased degradation of collagen
due to increased cross-linking of fibers and reduced collagenase activity are found in
OSMF mucosa compared to normal oral mucosa. The etiopathogenesis of OSMF is
simplified by the following flow charts.41
Oral Mucosa
Increased susceptibility due to deficiency of iron and
Vit B12
Betel quid habit
Duration and frequency of the habit
Constant irritation
Chronic inflammation
Activated T-cell and macrophages at site
Increase in cytokines- IL6, TNF, IF-α
Increase in growth factor- TGF-β
Figure No.1: Molecular Pathogenesis of Oral Submucous Fibrosis
14
TGF-β
Procollagen
activation
PNP
BMP1/PCP
Increased Procollagen
ProLOX
Increased collagen
(soluble form)
LOX
Increased copper
in arecanut
Increase LOX
Increased collagen
(insoluble form)
Increase in collagen
production
Pro-LOX: Pro Lysyl oxidase
LOX: Lysyl oxidase
PNP: Pro-collagen N-Protein
PCP: Pro-collagen C-Protein
BMP: Bone Morphogenic Protein 1
15
TGF-β
Activation of TMP
gene
Actvation of PAI
gene
Increase TMPs
Increase PAI
plasminogen
Inhibits activated
collagenases
procollagenase
Decrease in collagenase activity
plasmin
collagenase
Flavinoids in arecanut
Decrease in collagen degradation
Decreased collagen
destruction
Increased collagen
production
Increased collagen insoluble form – Cross linking of
insoluble collagen
Fibrosis
16
CLINICAL FEATURES
The majority of patients of OSMF are between the age group of 20 to 40 years. The
most frequent location is the buccal mucosa and retromolar area. It also involves the
palate, faucial pillars, uvula, tongue and labial mucosa. In some cases even the floor
of the mouth and gingival are involved.
SYMPTOMS
The symptoms of OSMF can be broadly divided under two headings

Prodromal symptoms

Later symptoms
Prodromal symptoms:
The onset of this condition is insidious and is often of 2-5 years duration. The most
common initial symptom is burning sensation of the mouth often experienced when
the patient is eating hot and spicy food. The other frequent early symptom is blisters,
ulcerations or recurrent stomatitis. Excessive salivation, defective gustatory sensation
and dryness of the mouth may also occur in early stages of the disease. Loss of taste
sensation may be due to reduced contact surface of the tongue mucosa during
chewing or due to persistent fibrosis of the taste fibres.42,32
Later symptoms:
After varying periods of time, in some cases a few years after the appearance of initial
symptoms, patient complains of stiffness of certain areas of the oral mucosa leading to
difficulty in mouth opening, inability to blow, difficulty in swallowing. Pain in the ear
is the later symptom of the disease. This is attributed to pharyngeal fibrosis and
occlusion of “eustachian tube”. Later nasal twang of the voice may develop.16
17
SIGNS
The most common and earliest sign is blanching of the mucosa caused by impairment
of local vascularity. The blanched mucosa becomes opaque and white. The whitening
often takes place in spots so that the mucosa acquires a marble like appearance. As the
disease progresses the mucosa becomes stiff and the vertical fibrous bands appear.
Labial mucosa is blanched, rubbery and is characterized by the presence of the fibrous
bands around the rima oris. In severe cases opening of the mouth is altered to an
elliptical shape and lips are difficult to evert.
Soft palate and uvula- they become fibrotic and clear delineation of the soft and hard
palate is seen. Mobility of the soft palate is restricted. Uvula when involved is
shrunken and in extreme cases they become bud shaped.
Tongue- the initial change is depapillation, usually in the lateral margins of the
tongue. The surface of the tongue becomes smooth, its mobility also decreases.
Floor of the mouth becomes elastic and rubbery.
Gingival when affected becomes fibrotic, blanched and inelastic.
Associated features:
Hyperpigmentation or loss of pigmentation is very common in association with
OSMF.43 It is usually found in the areas of redness in the soft palate, the anterior
faucial pillars, buccal mucosa or the mucosal surface of the lip particularly lower
labial mucosa. The vesicles are painful and they soon rupture leaving behind
superficial ulcerations.17 Ulcerations often develop in advanced cases. The epithelium
becomes atrophic which render it fragile and vulnerable to ulcerations. Petechiae or
small raised reddish blue spots which sometimes occur in OSMF they are most
common on the tongue, labial mucosa and buccal mucosa due to focal vasodialation.35
18
CLINICAL STAGING OF OSMF
Various staging/grading for OSMF are given by many authors which are as below:
I .Pindborg (1989)44, divided OSMF based on the physical findings into three stages
as follows:
a) Stage I: Stomatitis includes erythematous mucosa, vesicles, mucosal ulcers,
melanotic mucosal pigmentation and mucosal petechiae
b) Stage II: Fibrosis occurs in healing vesicles and healing ulcers, which is the
hallmark of this stage.

Early lesions demonstrates the blanching of the oral mucosa

Oral lesions include vertical and circular fibrous bands in association with
the blanched mucosa. Specific findings include reduction of mouth
opening, stiff and small tongue, blanched and leathery floor of the mouth,
fibrotic and depigmented gingiva, rubbery soft palate with decreased
mobility, blanched and atrophic tonsils, shrunken bud like uvula and
sunken cheeks not commensurate with age and nutritional status.
c) Stage III: Sequelae of OSMF are as follows

Leukoplakia is found in more than 25% of individuals with OSMF

Speech and hearing deficits may occur because of involvement of tongue
and the eustachian tube.
II. LAI D R45 (1995) gave the clinical staging of OSMF based on the interincisal
distance

Group A : Mouth opening greater than 35mm

Group B : Mouth opening between 30-35mm

Group C : Mouth opening between 20-30mm
19

Group D : Mouth opening less than 20 mm
III. S M. Haider47 et al (2000) gave clinical staging based on the presence of fibrous
bands on various anatomic sites and gave functional staging based on the mouth
opening
Clinical staging:

Stage 1: Fibrous bands only

Stage 2: Faucial and buccal bands

Stage 3: Faucial, buccal and labial bands
Functional staging:

Stage 1: Mouth opening >20mm

Stage 2: Mouth opening 11-19mm

Stage 3: Mouth opening <10mm
IV. Bailoor DN and Nagesh KS48 (2001) suggested the following stages
-
-
Stage I: Early OSMF

Mild blanching

Normal mouth opening (35-45mm)

Normal tongue protrusion (5-6cm)

Burning sensation on taking only hot or spicy food
Stage II: Moderate OSMF

Moderate to severe blanching

Mouth opening and tongue protrusion reduced by 33%
20
-

Burning sensation even in the absence of stimuli

Palpable bands present

Unilateral or bilateral lymphadenopathy
Stage III: Severe OSMF

Severe burning sensation

Mouth opening and tongue protrusion reduced by 66%

Ulcerative lesions may appear on the cheek

Thick palpable fibrous bands

Lymphadenopathy bilaterally evident
V. Ranganathan K46 et al (2001) used a baseline study on the mouth opening
parameters of normal patients and divided the OSMF patients as:

Grade I: Only symptoms, with no demonstrable restriction of mouth opening

Grade I: Limited mouth opening and above

Grade III: Mouth opening less than 20mm

Grade IV: Advanced OSMF with limited mouth opening. Precancerous and
cancerous changes seen throughout the mucosa.
VI. Rajendran R35 (2003) reported the clinical features of OSMF as follows

Early OSMF: Burning sensation in the mouth, blisters especially on the
palate, ulcerations or recurrent generalized inflammation of oral mucosa,
excessive salivation, defective gustatory sensation and dryness of mouth.
21

Advanced OSMF: Blanched and slightly opaque mucosa, fibrous bands in
the buccal mucosa running in vertical direction. Palate and the faucial
pillars are the areas first involved. There is gradual impairment of tongue
movement and difficulty in mouth opening.
VII. Sani N K42 and Bhama L K (2006) gave clinical staging based on the
involvement of different anatomical areas

Early: Fibrous bands present, no trismus, no involvement of tongue

Moderate: Trismus present but no gross tongue involvement, fibrosed soft
palate

Advanced : Marked trismus, marked fibrosis stiff soft palate and restricted
tongue movements

OSMF advanced with limited mouth opening. Precancerous and cancerous
changes seen throughout the mucosa
In this study 2.34%, 40%, 10% and 26.67% of the biopsies were in stage I, stage II,
stage III and stage IV respectively.51
MALIGNANT POTENTIAL
Oral cancer originates in submucous fibrosis from diverse intra-oral location, without
any noticeable predilection for any particular site.48 Atrophic epithelium first becomes
hyperkeratotic and later, intracellular edema and basal cell hyperplasia develops
eventually, following epithelial atypia with moderate epithelial hyperplasia and then
carcinoma can develop at any time.49 To substantiate the precancerous nature of the
condition, following points are noted, high occurrence of sub mucous fibrosis in oral
22
cancer patient, higher incidence of oral cancer in patient with OSMF, histological
diagnosis of carcinoma, without the clinical suspicion of it, higher prevalence of
leukoplakia among OSMF patients, higher frequency of epithelial dysplasia. The
WHO Collaborating Centre for Oral Precancerous Lesions has concluded that
although OSMF predisposes to cancer, it is not absolutely conclusive. It is highly
probable that such relationship does exist. Following facts support this hypothesis:51
1) The frequency of oral leukoplakia in OSMF patient is 6-8 times higher than
control group.
2) Carcinoma patients exhibiting OSMF have a frequency of fibrosis in general
population.
3) Immunological alterations observed in OSMF are almost similar to that
observed in oral cancer.
ROLE OF OXIDATIVE STRESS IN ORAL SUBMUCOUS FIBROSIS
FREE RADICALS
A free radical is a species capable of independent existence that contains one or more
unpaired electrons. An unpaired electron is being one that is alone in the orbital.52 The
unpaired electron gives certain characteristic properties to the free radical such as
paramagnetism. The chemical reactivity of free radicals is usually high. They may be
positively charged, negatively charged or electrically neutral.53 A free radical is
conventionally represented by a superscript dot (R•).
A compound becomes free radical by gaining an additional electron as in the case of
reduction of molecular oxygen to superoxide anion radical (O2•). Other free radicals
23
are hydroxyl radical (•OH), hydroperoxyl radical (HOO•), lipidperoxyl radical
(ROO•). The sequential univalent reduction steps of oxygen may be represented as.54
O2
(+)e- O2•-
(+)e-,2H
H2O2
(+)e-, H+
•
OH
(+)e-, H+ H2O
-H2O
Generation of free radicals: Free radicals may be formed by;

Cleavage of covalent bond of a normal molecule

Loss of single electron from a normal molecule

Addition of single molecule to the normal molecule
Sources of free radicals:
Oxidants related to human diseases are derived from three sources

Those generated via normal intracellular biological processes but in a
exaggerated, inappropriate fashion or in a milieu where the normal
defences that serve to protect the tissue are inadequate.

Those released by inflammatory cells into their local environment.
Figure No.5: The Cellular Sources of Free Radicals
24

Those secondary to xenobiotics, either because the xenobiotics include
oxidants (eg: inhaled oxidant gases) or because these xenobiotics induce
oxidant generation within cells, eg: drugs that injure tissues through
oxidant mechanisms.55
1 Endogenous sources:
a) Endogenous sources of free radicals include those that are generated and act
intracellularly and those that are formed within the cell and are released into
the surrounding area.56 Oxidation-reduction reactions generate free radicals
constantly within the body. These can be mediated by the action of the
enzymes or non-enzymatically, often through the redox chemistry of transition
metal ions.57
Oxidants and electron transport systems are prime, continuous sources of
intracellular reactive oxygen free radicals. Electron transfer from transition
metals such as iron to oxygen can initiate free radical reactions. 56 An
important source of superoxide anion radical is the “univalent leak” of
superoxide anion radical from the mitochondrial electron transport system.58
b) Intracellular free radicals are generated from the auto oxidants and consequent
inactivation of the small molecules such as reduced flavins and thiols, and
from the activity of certain oxidases, cycloxygenases, lipoxygenases,
dehydrogenases and peroxidases.56 A variety of enzyme systems catalyze the
univalent reduction of the molecular oxygen to superoxide anion radical. Such
univalent reduction of molecular oxygen also occurs in vivo in non-enzymatic
electron transfer oxidation-reduction reactions.
25
(E.g: hydroquinone + O2
superoxide + O2•- + H+) and during auto
oxidation reactions including those that involve catecholamines, flavins and
reduced ferridoxins.59
c) NADPH oxidase in the inflammatory cells (neutrophils, eosinophils,
monocytes and macrophages) produces superoxide anion by the process of
respiratory burst during phagocytosis. The superoxide is converted to
hydrogen peroxide and to hypochlorous acid (HOCl) with the help of
superoxide dismutase (SOD) and myeloperoxidase (MPO). This superoxide
and hypochlorous ions are the final effectors of bactericidal action. The gene
for myeloperoxidase is chromosome 17. The enzyme myeloperoxidase has a
molecular weight 156 Kilo Dalton and contains two iron atoms per molecule.
This is deliberate production of the free radicals by the body. About 10% of
oxygen uptake by macrophage is used for free radical generation. Along with
the activation of macrophages, the consumption of the oxygen by the cell is
increased drastically; this is called the respiratory burst.60 (Figure 6)
d) Hydroxyl radicals are formed in the “Fenton reactions” whenever hydrogen
peroxide comes into contact with ferrous or cupric ions. An iron catalyzed
Haber-weiss type of reaction may also form this radical, the net effect of
which is an interaction between hydrogen peroxide and superoxide anion
radical in the presence of traces of transition metal ions to form hydroxyl
radical. Finally the OH• .radical is also the product of ionizing reaction.59, 61
Fe2+ + H2O2
O2•- + H2O2
Fe3+ + •OH + OH- (Fenton Reaction)
Fe salt catalyst
O2 + •OH + OH-
(Haber Weiss reaction)
26
The chelated Fe3+ can be reduced to Fe2+ by thiols, ascorbate and most of other
reductants. Fe2+ can then auto oxidize producing O2•.
Figure No. 6: Generation of oxygen free radicals in respiratory burst
2 Exogenous sources:
a) Cigarette smoke contains high concentration of various free radicals
other toxic compounds such as carbon tetrachloride, drugs and
inhalation of the pollutants, anaesthetics, organic solvents, hypertoxic
environments and pesticides will increase the production of free
radicals.56
b) Ionizing radiation damages the tissue by producing haemolytic fission
of background water.
H2O
(gamma, UV radiation)
H• + OH•
c) Light of appropriate wavelengths can cause excitation of oxygen in
presence of photosensitizers to produce singlet oxygen.
27
Sites of free radical generation:
Main sites of free radical generation are mitochondria, lysosomes, peroxisomes,
nuclei, endoplasmic reticulum, plasma membrane and the cytosol.56
i) Endoplasmic reticulum, nuclear membrane and electron transport
systems:
Free radicals produced by the endoplasmic reticulum and nuclear membrane can
undergo both intraorganelle and cytosolic reactions. In case of nuclear membrane
generated radicals DNA would be particularly susceptible to free radical damage.
ii) Plasma membrane:
Plasma membrane is the site of action of extracellularly generated free radicals. They
must cross the plasma membrane before reacting with other cell components and may
initiate toxic reactions at the membrane. The unsaturated fatty acids present in the
membrane and transmembrane proteins containing oxidizable amino acids are
susceptible to free radical damage. Increased membrane permeability caused by lipid
peroxidation or oxidation of structurally important proteins can cause breakdown of
transmembrane ion gradients, resulting in loss of secondary functions and inhibition
of integrated cellular metabolic processes.
The interior of the biological membranes is hydrophobic and O2•- produced in the
environment could be extremely damaging. Much of the O2•- generated within the
cells comes from membrane bound systems and it is certainly possible that some of it
is formed in the membrane interior.62
iii) Peroxisomes:
Peroxisomes are the potential sources of cellular hydrogen peroxide because of the
high concentration of oxidases.
28
REACTIVE OXYGEN SPECIES (ROS)
Oxygen derived free radicals and other non radical species are collectively known as
reactive oxygen species (ROS).
Several reactive oxygen species are known, prominent among them include:
i)
Superoxide radical (O2•- ):
This ROS is formed when oxygen takes up one electron and as leaks in the
mitochondrial electron transport but its formation is easily increased with exogenous
components (redox cycling compounds) are present. Its first production site is the
internal mitochondrial membrane (NADH ubiquinone reductase and ubiquinone
cytochrome-c-reductase).63 This species is reduced and forms hydrogen peroxide
(H2O2). The production of superoxide radicals at the membrane level (NADPH
oxidase) is initiated in specialized cells with phagocytic functions (macrophages) and
contributes to their bactericidal action (oxidative burst).64 The flavin cytosolic enzyme
xanthine oxidase found in quite all tissues and in milk fat globules generates
superoxide radicals from hypoxanthine and oxygen and is supposed to be at the origin
of vascular pathologies.
ii)
Hydrogen peroxide (H2O2):
Hydrogen peroxide is mainly produced by enzymatic reactions. These enzymes are
located in the microsomes, peroxisomes and mitochondria. Even in normal conditions
the hydrogen peroxide production is relatively important and leads to a constant
cellular concentration between 10-9 and 10-7 M, in plant and animal cells superoxide
dismutase is able to produce H2O2 by dismutation of O2.-, thus contributing to the
lowering of oxidative reactions. The neutral combination dismutase and catalase
29
contributes to remove H2O2 and thus has a true cellular antioxidant activity. H2O2 is
also able to diffuse easily through membranes.65
iii)
Hydroxyl radical (•OH) :
In the presence of Fe2+, H2O2 produces the very active species •OH by Fenton
reaction.
Fe2+ + H2O2
Fe3+ +
•
OH + OH-
This iron-catalyzed decomposition of oxygen peroxide is considered the most
prevalent reaction in the biological systems and the source of various deleterious lipid
peroxidation products.
iv)
Nitric oxide (NO•):
Nitric oxide is produced in vascular endothelium. This species is not too reactive
(poorly oxidizing function), it reacts readily with O2•- and gives the extremely reactive
peroxynitrite (ONOO-). This ROS is naturally found in activated macrophages66 and
endothelial cells67 and considered as an active agent in several pathologies based on
inflammation, organ reperfusion and also may play an important role in
atherosclerosis.
v)
Singlet oxygen (1O2 ):
This chemical form of oxygen is not a true radical but is reported to be an important
ROS in reactions related to ultraviolet exposition (UVA 320-400 nm). Its toxicity is
reinforced when appropriate photoexcitable compounds (sensitizers) are present with
molecular oxygen.68 Several natural sensitizers are known to catalyze oxidative
reactions such as tetrapyroles (bilirubin), flavins, chlorophyll, hemoprotiens and
reduced pyridine molecules (NADH), some of these sensitizers are also found in
30
foods and cosmetics. Some are used for therapeutic purposes (anti cancer treatment)
and are sensitive to visible light. The presence of metal contributes to increase the
production of single oxygen as well as anion superoxide, and thus accelerates the
oxidation of unsaturated lipid generating hydroperoxides. It has been suggested that
the singlet oxygen may be formed during the degradation of lipid peroxides and thus
may cause the production of other peroxide molecules. This singlet oxygen formation
may account for the chemiluminescence observed during lipid peroxidation.69
vi)
Thiyl radical:
Thiol compounds (RSH) are frequently oxidized in the presence of iron or copper
ions.
RSH + Cu2+
RS• + Cu+ + H +
These thiyl radicals have strong reactivity in combining with oxygen.70, 71
RS• + O2
RSO•2
Furthermore they are able to oxidize NADH into NAD, ascorbic acid and to generate
various free radicals (•OH and O2.•-). These thiyl radicals may also be formed by
homolytic fission of disulfide bonds in protiens.
vii)
Carbon centered radicals:
The formation of this reactive free radical is observed in calls treated with carbon
tetrachloride (CCl4). The action of the cytochrome P450 systems generates the
trichloromethyl radical (•CCl3) which is able to react with oxygen to give several
peroxyl radicals (i.e. •O2CCl3).72
31
DAMAGE PRODUCED BY FREE RADICALS
Free radicals are extremely reactive. Their mean effective nidus of action is only
30Ao. and their half life is only a few milliseconds. When a free radical reacts with a
normal compound, other free radicals are generated. This chain reaction leads to
thousands of events. Peroxidation of polyunsaturated fatty acids (PUFA) severely
damages the cell membrane leading to loss of membrane functions like absoption,
secretion etc. almost all biological macromolecules are damaged by the free radicals,
e.g.




Peroxidation of PUFA in plasma membrane
Oxidative inactivation of sulfhydryl containing enzymes
Polysaccharide depolymerisation and DNA breaks
DNA damage may directly cause inhibition of protein and enzyme synthesis
indirectly it also causes cell death or mutation and carcinogenesis.

Lipid peroxidation and consequent degradation product such as MDA seen in
biological fluids. Their effect in the serum is often employed to assess the
oxidant stress.73
Proteins:
Protein molecules undergo substantial modifications through reactive reactions with
free radicals. Proteins containing tryptophan, tyrosine, phenylalanine, histamine,
methionine and cysteine can undergo free radical mediated amino acid modifications.
Free radicals promote sulfhydryl mediated cross linkage of such labile amino acids as
well as cause fragmentation of the polypeptide chains. Oxidative modifications
enhance degradation of critical enzymes by cytosolic neutral proteases.74 Enzymes
32
undergo cross-linking with resulting increase in molecular weight, each enzyme
cross-links with their neighbors in a random destructive reaction. The normal
precision arrangement of protein and enzymes in subcellular membrane and
organelles is badly disrupted and their biological properties are lost or impaired75.
Carbohydrates:
Advances in free radical chemistry indicate that no biological substructure is
impervious to free radical attack. Therefore it is most important that glucose and other
related monosaccharides undergo when conditions are appropriate.74 Hyaluronic acid
undergoes polymer fragmentation following exposure to free radical systems, which
leads to destabilization of connective tissue and loss of synovial fluid viscosity.
Nucleic acids:
DNA is readily attacked by oxidizing radicals if they are found in its vicinity has
been clearly demonstrated by radiation biologists. It must therefore be considered as a
vulnerable and important target. Cell mutation and death from ionizing radiation is
primarily due to free radical reactions with DNA. Cell death and mutations arising
from free radicals generated during normal metabolism have also been ascribed to
reactions with DNA.76
Lipids:
All of the major classes of biomolecule may be attacked by free radicals but lipids are
probably the most susceptible.57 Cell membranes are the rich sources of
polyunsaturated fatty acids. Biomembrane and organelles are the major sites of lipid
peroxidation damage. Major constituents of the biological membranes are lipids and
33
proteins. Lipid peroxidation can damage membrane protein as well as lipids.62 The
membrane fluidity is due to the presence of polyunsaturated fatty acid side chain in
many membrane lipids, which lower the melting point of the interior membrane. Lipid
peroxidation decreases membrane fluidity. The conditions which favor lipid
peroxidation are:
i)
A high degree of unsaturation in the lipid substrate
ii)
A rich supply of oxygen and
iii)
The presence of traditional metal catalysts77
LIPID PEROXIDATION
Lipid peroxidation is defined as “oxidative deterioration of polyunsaturated lipids.
Lipid peroxidation is particularly damaging because it proceeds as a self perpetuating
chain reaction”.78
Figure No. 7: The Lipid Peroxidation Process
34
Figure illustrates lipid peroxidation. The reaction is initiated by an existing free
radical (X•), by light or by metal ions. Malondialdehyde is only formed by fatty acids
with three or more double bonds, and is used as a measure of lipid peroxidation
together with ethane from the terminal two Carbon of α-3 fatty acids and pentane
from terminal five carbon of α - 6 fatty acids.79
Peroxidation of polyunsaturated fatty acids usually involves three operationally
defined processes.62
1. Initiation phase:
During this phase the primary event is the abstraction of the hydrogen atom from bisallylic site of PUFA. Initiation peroxidation sequence in membrane or PUFA74 is due
to the attack of any species that has sufficient reactivity to abstract a hydrogen atom
from methylene (CH2) group. This leaves behind an unpaired electron on the carbon
–
CH-. The carbon radical tends to be stabilized by the molecular rearrangement to
produce a conjugated diene, which then easily reacts with an oxygen molecule to give
peroxy radical, R-CO•. the presence of the redox active metals such as iron or copper
can facilitate the initiation process.62
ROOH + Metal (n)+
X• + RH
ROO• + Metal(n-1)+
+ H+
R• + XH
2. Propagation phase:
During this phase lipid peroxidation relies on the interaction of molecular oxygen
with carbon-centered free radicals to form lipid hydroperoxides.74 The peroxy radical
abstract a hydrogen atom from another lipid molecule and once the process begins it
tends to continue. The peroxy radical combines with the hydrogen atom that it
35
abstracts to give lipid hydroperoxides R-OOH. A probable alternative fate of the
peroxy radicals is to form cyclic peroxides. With the help of metal catalysts the
decomposition of the hydroperoxides results in the formation of alkoxyl or peroxyl
radicals. These radicals are capable of further reactions and thus the propagation of
lipid peroxidation continues.62, 74
R• + O2
ROO•
ROO• + RH
ROOH + R•
3. Termination phase:
The propagation reactions of lipid peroxidation will not proceed very far before they
meet a protein molecule., which can then be attacked and damaged, in addition
aldehyde can attack amino groups on the protein molecule to form both
intramolecular cross links and also cross links between different protein molecules,
e.g: malondialdehyde. Any kind of lipid free radical can react with a lipid peroxyl
radical to give non-initiating and non-propagating species.
ROO• + ROO•
ROO• + R
R• + R •
RO-OR + O2
ROOR
RR
TOXIC EFFECTS OF LIPID PEROXIDATION:
The uncontrolled peroxidation of bio-membranes can lead to profound effects on
membrane structure and function and may be sufficient to cause cell death.53 The
toxic products generated during lipid peroxidation may be involved in damage to
specific protein and transport system critical to cell function.82 Malondialdehyde
produced by lipid peroxidation can cause cross linking and polymerization of
36
membrane components. This can alter the intrinsic membrane properties such as ion
transport, enzyme activity. Because malondialdehyde is diffusible, it will also react
with nitrogenous bases of DNA81 lipid hydroperoxides can directly inhibit enzymes.82
MALONDIALDEHYDE
Malondialdehyde is the organic compound with the formula CH2 (CHO)2. The
structure of this species is more complex than this formula suggests. This reactive
species occurs naturally and is a marker for oxidative stress.
IUPAC name[hide]
Propanedial
Figure No 8: Structure of malondialdehyde
Structure and synthesis :
Malondialdehyde mainly exists in the enol form:
CH2(CHO)2 → HOCH=CH-CHO
In organic solvents, the cis isomer is favored, whereas in water the trans-isomer
predominates.
37
Malondialdehyde is a highly reactive compound that is not typically observed in pure
form. In the laboratory it can be generated in situ by hydrolysis of 1,1,3,3tetramethoxypropane, which is commercially available.83 It is easily deprotonated to
give the sodium salt of the enolate (m.p. 245 °C).
Biochemistry:
Reactive oxygen species degrade polyunsaturated lipids, forming malondialdehyde.84
This compound is a reactive aldehyde and is one of the many reactive electrophile
species that cause toxic stress in cells and form covalent protein adducts which are
referred to as advanced lipoxidation end products (ALE), in analogy to advanced
glycation end-products (AGE).85 The production of this aldehyde is used as a
biomarker to measure the level of oxidative stress in an organism.86, 87
Malondialdehyde reacts with deoxyadenosine and deoxyguanosine in DNA, forming
DNA adducts, primarily M1G, which is mutagenic.88 The guanidine group of arginine
residues condense with MDA to give 2-aminopyrimidines.
Human ALDH1A1 aldehyde dehydrogenase is capable of oxidising malondialdehyde.
MDA is reactive and potentially mutagenic.
OXIDATIVE STRESS
The occurrence of ROS known as pro-oxidants is an attribute of normal aerobic life.
The steady state formation of pro-oxidants is balanced by similar rate of their
consumption by antioxidants that are enzymatic and/or non-enzymatic. “Oxidative
stress” results from imbalance in this pro-oxidant-antioxidant equilibrium in favour of
the pro-oxidants.in the context of an increased oxidant burden and/or decreased
38
antioxidant defense, the oxidants are free to interact with and modify normal
biological components. When these components are critical and extent of
modification sufficient injury becomes manifest at the tissue level. Figure depicts
some of the mechanisms by which oxidative stress causes tissue injury.92
DNA
DNA
DAMAGE
DAMAGE
OXIDATIVE
STRESS
Increased lipid
peroxidation
GSH DEPLETION
DIRECT
DAMAGE TO
PROTEINS
CYTOSKELETAL
DAMAGE
RISE IN INTRACELLULAR
FREE Ca2+
INHIBITION OF ATP
SYNTHESIS
MEMBRANE
BLEBBING
NAD(H)
DEPLETION
POLY(ADP) RIBOSE
SYNTHETASE
ACTIVATION
RISE IN INTRACELLULAR FREE IRON
MEMBRANE PEROXIDATION AND
DESTRUCTION
Increased damage to DNA, proteins and lipids
Metal ion release into surrounding tissue,
injury to adjacent cells
Figure No.9: Complex Multifactorial Nature of Oxidative Damage to Cells
39
MALONDIALDEHYDE IN HEALTH AND DISEASES
M. Nishi et al (1986) examined changes in the lipid peroxide levels in the
submandibular gland, blood, and liver after low-dose x-ray irradiation of rats. The
lipid peroxide level in the blood plasma was determined according to Yagi (1976).
The
lipid
peroxide
concentration
was
calculated
in
terms
of
mol
malondialdehyde/mL of blood. The lipid peroxide levels in the blood plasma of the
irradiated group were generally higher than those of controls. Significant differences
were seen at two hours, seven days, and 14 days after irradiation.93
Balwant Rai (2007) conducted a study on salivary lipid peroxidation product
malondialdehyde in periodontal diseases. Lipid peroxidation product MDA was
analyzed in 25 patients of periodontal disease and 30 healthy subjects served as
controls. Significantly elevated levels of salivary MDA was observed in periodontitis
as compared to controls (p<0.05). These findings indicate a role of free radicals in its
pathogenesis.94
Joseph A. Knight et al (1987) measured lipoperoxides, as malondialdehyde
(MDA), by liquid chromatography in plasma from 230 male and 148 female adult
blood donors, to establish reliable reference values and to compare possible sex-, age, and specimen related differences. Their studies showed that men have higher MDA
concentrations in plasma [0.60 (0.21) µmol/L] than do women [0.54 (0.20) µmol/L]
(P <0.05), older men have higher values [0.67 (0.20) µmol/L] than younger men [0.58
(0.17) µmol/L] (p <0.05), and older women have higher values [0.57 (0.20) µmol/L]
than young women [0.47 (0.17) µmol/L] (P<0.001). These age-related results support
40
earlier studies in experimental animals that lipid peroxidation increases with
increasing age.95
J Khalili et al (2008) carried out a study on salivary MDA level in clinically
healthy and periodontal diseased individuals. MDA levels in saliva were measured in
104 subjects aged between 18-65 years. Three groups with different degrees of
severity of generalized chronic periodontitis were established, 30 patients in early
stage , 30 with moderate disease and 14 with severe disease. And 30 with healthy
periodontium served as controls, a significant increase in the salivary MDA level
existed in the samples obtained from the three groups of the patients compared to
control subjects.96
MA Carbonneau et al (1991) performed assay of free and total
malondialdehyde (MDA) in human serum and plasma from healthy subjects and from
patients with high risk of lipoperoxidation. Free MDA averaged 0.042 and 0.043
µmol/L, respectively, in serum and plasma from healthy subjects. Free MDA
increased significantly in the plasma from cancer patients (0.270 ± 0.047 µmol/L) and
from hemodialyzed patients (0.214 ± 0.035 µmol/L).97
Marie-Jeanne Richard et al (1992) determined the MDA-TBA complex by
HPLC method. The normal range (2.51 ± 0.25 µmol/L) determined in 32 normal
subjects showed that there existed no sex-related difference for TBARS: 2.57 (0.28)
in men versus 2.44 (0.20) µmoI/L in women.98
41
Flemming N et al (1997) carried out a study to generate reliable reference
intervals for plasma malondialdehyde. Analysis of variance was used to reveal
relations between normalized plasma MDA concentration in the three 20-year age
groups and gender. The analysis revealed a significant effect from gender (p = 0.033),
but apparently not from age (P = 0.109). No major interaction occurred between
gender and age (p = 0.103). Men had slightly but significantly higher plasma MDA
concentrations than women. In the reference sample group, 92 were smokers and 122
were nonsmokers. Smokers had a significantly higher plasma MDA concentration
(mean 0.66 µmol/L) than nonsmokers (mean 0.60 µmol/L) (p = 0.05). Correlation
analysis revealed an association between plasma MDA and the number of hours of
daily exposure to cigarette smoke (r = 0.162, p = 0.03), but we found no clear
correlation between plasma MDA and the number of cigarettes smoked (r = - 0.065, p
= 0.55). Plasma MDA was significantly correlated with weekly alcohol consumption
(r = 0.153, p = 0.03).9
F Farinati et al (1998) in a study involving 88 consecutive outpatients,
undergoing endoscopy for upper gastrointestinal symptoms, divided the patients into
histological subgroups: 27 with chronic non-atrophic gastritis, 41 with atrophic
gastritis, six with gastric cancer, and 14 unaffected controls. No significant difference
was observed in thiobarbituric acid reactive substance (TBARS) concentrations in the
above mentioned four groups of patients, nor was there any difference in patients with
no, mild, moderate, or severe intestinal metaplasia (no or mild: 25.7 (4.4), moderate,
30.1 (5.5), severe: 25.4 (4.3) mol/g). However, patients with high grade intestinal
metaplasia (moderate and severe cases considered together) showed a trend towards
42
higher TBARS concentrations (28.1 (12.8) versus 24.3 (11.1) mol/g). Similarly, no
difference in TBARS concentrations was detected in patients with no versus moderate
or severe disease activity (20 .8 (8.6) versus 26.0 (12.9) and 26.8 (12.3) mol/g,
respectively) or in patients with no, moderate, or severe H pylori infection (no:
23.5 (7.8), moderate: 29.4 (14.4), severe: 27.8 (8.8) mol/g; NS). However, H pylori
positive patients had significantly higher TBARS concentrations overall (29.1 (13.4)
versus 21.5 (8.7) mol/g, p=0.04).100
G. Gitanjali et al (1999) estimated the level of vitamin E and serum MDA in
patients with cervical carcinoma receiving radical radiotherapy. Control group
comprised of 50 healthy, nonsmoking women. Study group was divided into two
subgroups of 25 each. Group I was given vitamin E supplementation (100 mg orally
daily) in addition to radical radiotherapy. Group II was given placebo. There was no
statistically significant difference in the mean serum MDA levels of healthy controls
(3.40 ± 0.75 mole/ml) and the 50 patients taken together (3.54 ± 1.89 mol/ml). In
group I after completion of radical radiotherapy and vitamin E supplementation, the
mean serum vitamin E levels increased from 9.17 ± 2.69 to 10.88 ± 2.63 µg/ml (p <
0.02) and the mean serum MDA levels decreased from 3.19 ± 1.02 to 0.56 mol/ml (p
< 0.01). In group II the mean serum vitamin E levels increased from 9.18 ± 3.14 to
11.3 ± 2.81 µg/ml (p < 0.01) while serum MDA levels decreased from a mean value
of 3.88 ± 1.66 mol/ml to a mean of 3.23 ± 1.53 mol/ml (p < 0.04). The difference
in serum MDA level in group I and II was statistically significant with p < 0.05.101
43
Sabitha KE et al (1999) studied the effect of radiation on oral cancer patients
using activities of superoxide dismutase (SOD), catalase, glutathione peroxidase
(GPX), glutathione reductase (GR), glutathione-S-transferase (GST), glucose-6phosphate dehydrogenase (G6PDH) and levels of malondialdehyde (MDA). The
levels of MDA showed a significant increase in untreated and radiation treated oral
cancer patients (p < 0.01 and p < 0.001 respectively) when compared with normal
subjects. Radiation treated patients had higher MDA levels than untreated patients.
The activities of red blood cell hemolysate antioxidant enzymes such as SOD,
catalase, GPX, GR, GST and G6PDH showed a significant decrease, representing the
lack of antioxidant defense. Radiation induces lipid peroxidation by inactivating the
antioxidant enzymes, thereby rendering the system inefficient in management of the
free radical attack. Thus, the degree of radiation affects the extent of the depression of
the antioxidant enzyme activities and increases lipid peroxidation.102
Talia Weinstein et al (2000) studied extracellular and intracellular antioxidant defence mechanisms 3 months after recovery from haemolysis in
haemodialysis patients. In 29 patients and 20 controls serum MDA was measured as a
marker of oxidative stress. In the haemodialysis group, MDA levels were higher than
the controls (2.37±0.07 versus 0.97±0.10 mol/ml; p<0.0001. There was a correlation
between MDA levels and the number of years patients were treated by dialysis
(r=0.43; P<0.02).103
Unal Sahin et al (2001) randomly selected 47 male patients with lung cancer
(37 smokers, 10 nonsmokers) in the study group and 35 healthy subjects (20 smokers,
44
15 nonsmokers) were taken as controls. MDA levels levels were studied by
flourometric determination of thiobarbituric acid reactive substances in serum based
on the reaction between Malondialdehyde and thiobarbituric acid described by
Wasowicz et al. The MDA levels of the study and control groups were 20.5 ± 7.9 and
12.6 ± 7.1 mol/ml with statistically significant difference (p < 0.001). MDA levels
of smoker and nonsmoker lung cancer patients were 22.5 ± 8.0 and 16.1 ± 6.7
mol/ml, (p < 0.001).104
Ricky A. Sharma et al (2001) tested the hypothesis that cycloxygenase-2
(COX-2) activity in human colon cells results in formation of MDA and generation of
malondialdehyde-guanine (M1G) adducts. Levels of M1G correlated significantly (r =
0.98, p < 0.001) with those of intracellular MDA determined colorimetrically in the
four malignant cell types, but neither parameter correlated with expression of COX-2
or prostaglandin biosynthesis. Malondialdehyde treatment of human nonmalignant
colon epithelial cells resulted in a doubling of M 1G levels. These results show for the
first time in human colon cells that COX-2 activity is associated with formation of the
endogenous mutagen, MDA. Moreover, they demonstrate the correlation between
MDA concentration and M1G adduct levels in malignant cells.105
Simon M. Everett et al (2001) measured levels of malondialdehydedeoxyguanosine (M1-dG), a DNA adduct derived from lipid peroxidation, using the
immunoslot-blot technique alongside mucosal MDA and plasma, mucosal, and gastric
juice ascorbic acid and total vitamin C. They studied patients with normal and H.
pylori-infected mucosa and followed H. pylori infected patients for up to 12 months
45
after eradication of the organism. The MDA concentration in H. pylori-positive
patients were higher [123.7 mol/g (93.5–157.2)] than H. pylori negative patients
[82.5 mol/g (59.5–104.7)] (p <0.001). After successful eradication of H. pylori from
42 patients. MDA concentrations fell (from 134.9 to 106.2 mol/g; p = 0.007), and
gastric juice ascorbic acid and total vitamin C both increased significantly 6 months
after successful eradication of H. pylori (from 6.7 to 13.0 µg/ml for ascorbic acid;
from 9.4 to 17.0 µg/ml for total vitamin C; p < 0.001 for both). No changes were seen
in plasma or antral ascorbic acid or total vitamin C. Despite these potentially favorable
changes, there was no change in M1-dG concentration 6 months after successful
eradication of H. pylori. Thirteen evaluable patients who had received eradication
therapy returned at 12 months, all of whom were H. pylori negative. There were no
significant changes between 6 and 12 months for gastric juice, antral and plasma
ascorbic acid, and total vitamin C or for MDA. Twenty-one patients received placebo
eradication therapy. Data were available for 12 of these patients at 6 months and for 8
patients at 12 months, all of whom remained H. pylori positive. MDA levels did not
change significantly between pretreatment and 6 months (from 95.9 to 103.6 mol/g;
p = 0.5) but surprisingly, increased significantly by 12 months (from 95.9 mol/g at
pretreatment to 178.7 mol/g at 12 months; p < 0.001).106
Patait M (2002) estimated serum MDA level in patients with oral cancer
undergoing radiotherapy with and without antioxidant therapy. He found that the
mean serum MDA level in the pre-radiotherapy study group was 0.598 ± 0.1609
µmol/L which was significantly higher than the control group 0.3084 ± 0.1016
µmol/L. The mean serum MDA level in the study group after radiotherapy without
46
taking antioxidant was 0.792 ± 0.1157 µmol/L which was significantly higher as
compared to pre-radiotherapy mean serum MDA level of 0.598 ± 0.1609 µmol/L. The
mean serum MDA level in the study group after radiotherapy taking antioxidant
therapy was 0.763 ± 0.0953 µmol/L which was significantly higher as compared to
pre-radiotherapy mean serum MDA level of 0.596 ± 0.0952 µmol/L. No correlation in
lipid peroxides level was observed between degree of differentiation of malignant
lesion with or without antioxidant therapy. Thus he stated that the mean serum MDA
level increases in oral squamous cell carcinoma patients as compared to the healthy
individuals and MDA level further increases after radiotherapy.107
Ebubekir Bakan et al (2002) investigated the levels of malondialdehyde and
total nitrite (NO2–) plus nitrate (NO3–) marker for nitric oxide (NO•) generation in
gastric carcinoma and to correlate their levels with the cancer stage. The pretreatment
plasma samples were obtained from 38 patients with gastric cancer (seven patients at
stage II, 19 at stage III and 12 at stage IV). MDA was measured by the thiobarbituric
acid method. The levels of plasma MDA, NO• and NO3– were significantly higher in
patients with gastric cancer compared with the healthy control group. Higher levels of
MDA, NO• and NO3– were observed as the stage of the disease increased. MDA level
in control group (n = 24) was 5.4 ± 1, in stage II (n = 7) was 6.7 ± 1.0 (p< 0.01), in
stage III (n = 19) was 7.7 ± 1.5 (p< 0.001), in stage IV (n = 12) was 7.8 ± 1.3 (p<
0.001) and total (n = 38) was 7.6 ± 1.4 (p < 0.001). Thus they found that increased
NO• production and MDA levels were present in plasma of patients with gastric
cancer. These increases can be associated with the oxidant–antioxidant status in these
patients.108
47
Soma Gupta et al (2004) estimated the levels of MDA and antioxidants in
plasma and erythrocytes of 34 cases of oral submucous fibrosis. They found that
plasma MDA level was increased in all grades of oral submucous fibrosis cases (3.3 +
0.4 mole/ml, p < 0.001) as compared to controls (2.4 + 0.5 mole/ml), plasma β
carotene and vitamin E levels were decreased significantly in oral submucous fibrosis
(81.7 + 14.3 µg/100 ml, p < 0.001; 9.3 + 0.9 mg/L, p < 0.01 respectively) with respect
to healthy controls (110 + 20.8 µg/100 ml and 10.1 + 1.2 mg/L). The mean MDA
level was more in OSMF grade II (3.5 + 0.8 mol/ml, p < 0.001) than in grade III (3.3
+ 0.1 mol/ml, p < 0.001) and grade I (3.1 +0.8 mol/ml, p <0.001). The decrease in
β-carotene and vitamin E was found to be more significant in OSMF grade II and III
than in grade I. After 6 weeks of oral administration of beta-carotene and vitamin E,
patients showed increase in plasma levels of these two antioxidants along with
decrease in MDA level associated with clinical improvement.109
48
Methodology
MATERIALS AND METHODS
SOURCE OF DATA:
Patients reporting to the Department of Oral Medicine and Radiology, KLE’S.V.K.
Institute of Dental Sciences, Belgaum, with clinically diagnosed Oral submucous
fibrosis were selected in the study after obtaining an informed consent.
METHOD OF COLLECTION OF DATA:
This study included two main groups:
1) Clinically diagnosed cases of OSMF (36)
2) Age and sex matched healthy control group (12)
3) Age and sex matched chewer control group (12)
Diagnosis of OSMF was done on the basis of history and characteristic clinical
features based on case history proforma designed for the same. OSMF was divided
clinically into three stages as per the criteria described by Bailoor DN (1993).
Age and sex matched controls with tobacco and arecanut chewing habit and without
the habit were selected.
After explaining about the study to the subjects, an informed consent was obtained as
per the consent form attached. Subjects were grouped according to age. All cases and
controls underwent detailed history, thorough oral examination and serum
malondialdehyde estimation.
SELECTION CRITERIA:
Inclusion criteria:
1) 36 clinically diagnosed cases of Oral submucous fibrosis

12 patients with Grade I Oral submucous fibrosis (GROUP 1)

12 patients with Grade II Oral submucous fibrosis (GROUP 2)
49
Methodology

12 patients with Grade III Oral submucous fibrosis (GROUP 3)
2) 24 age & sex matched healthy individuals, 12 among them were chewers and
12 non chewers, without any oral & systemic diseases (CONTROL).

12 chewer controls (GROUP 4)

12 non-chewer controls (GROUP 5)
Exclusion criteria:
Patients with any systemic diseases/conditions which may alter serum
malondialdehyde levels will be excluded from the study like

Patients with chronic systemic diseases like Tuberculosis, AIDS,
Diabetes etc.

Patients with chronic renal failure and on hemodialysis

Patients with carcinoma

Patients on drug therapies such as antioxidants, anticonvulsants, broad
spectrum antibiotics and systemic steroids.
ARMAMENTARIUM:
For clinical examination of the patient:
-
Dental chair with light source
-
Mouth mirror
-
Probe
-
Tweezer
-
Gauze
-
Gloves
-
Mouth mask
-
Scale
-
Divider
50
Methodology
For the collection of blood sample:
-
Disposable syringe
-
Gauze
-
Antiseptic
-
Test tube
-
Tourniquet
For separating serum and storage:
-
Pipette
-
Pipette tips
-
Centrifuging machine
-
Storage vial with a lid
-
Refrigerator
For estimation of malondialdehyde:
-
Gloves
-
Test tubes
-
Test tube stand
-
Pipette
-
Boiling water bath
-
Centrifuging machine
-
Spectrophotometer
METHOD FOR COLLECTION OF BLOOD SAMPLE:
A detailed medical and dental history was recorded as per the case history proforma,
clinical examination was carried out and clinical diagnosis of Oral submucous fibrosis
51
Methodology
was made. Patients were categorized into 3 stages according to Nagesh and Bailoor
classification. Later the purpose and procedure was explained to the patient and
patient’s consent was obtained. 5 ml of blood was drawn by vena puncture from the
OSMF patients and controls under aseptic precautionary measures.
The subject was made to sit in a comfortable position, the patient’s arm was stretched
and tourniquet was applied above the right cubital fossa. Around 5 ml of blood was
withdrawn from the cubital vein under aseptic condition in a disposable syringe. The
blood was then put into a 10 ml glass test tube. Then the test tube along with blood
was subjected to centrifugation for 4-5 mins at 2500 rpm. Later the test tube was
withdrawn from the centrifugation machine and with the help of a pipette serum was
separated in a vial and stored in the refrigerator until it was transferred to the
biochemical laboratory.
The above procedure was repeated for all the patients.
ESTIMATION OF MALONDIALDEHYDE (MDA) IN SERUM110
Principle:
The reaction depends on the formation of pink colored complex between
malonaldehyde and thiobarbituric acid (TBA) having an absorption of maximum at
532nm.
Preparation of thiobarbituric acid reagent:
-
75ml thiobarbituric acid, 15gm trichloroacetic acid, 2.08ml – 0.2N HCl
All the above mentioned components were mixed and volume made upto
100ml with distilled water.
52
Methodology
Procedure:
TABLE 1:
Blank (ml)
Test (ml)
Serum
-
0.75
Distilled water
0.75
-
Thiobarbituric acid reagent 3
3
-
Keep the test tubes in boiling water bath for 15 minutes.
-
Cool, centrifuge for 10 minutes at 10,000 r.p.m.
-
Read absorbance of supernatants of blank and test immediately at 535nm.
0.75ml of serum was mixed with 3 ml of thiobarbituric acid in a test tube and the test
tube was kept in a boiling water bath for 15 mins, then it was removed from the
boiling water bath and was centrifuged for 10 mins at 10,000 rpm. The supernatant
fluid was separated and is read under spectrophotometer at 535nm immediately.
Calculation:
Malondialdehyde (nano moles/ ml)
= Absorbance of test X total volume
Nanomolar extension of coefficient X sample volume X 100
= Absorbance of test X 3.75
1.56 X 105 X 0.75 X 100
= Absorbance of test X 3205
100
53
Methodology
ARMAMENTARIUM USED
Figure No. 10: EQUIPMENTS USED IN CLINICAL EXAMINATION OF THE
PATIENTS
Figure No. 11: REAGENTS USED FOR ESTIMATION OF SERUM
MALONDIALDEHYDE
54
Methodology
Figure No. 12: BOILING WATER BATH
Figure No. 13: CENTRIFUGE MACHINE
55
Methodology
Figure No. 14: SPECTROPHOTOMETER
Figure No. 15: BLANCHING ON LABIAL MUCOSA
56
Methodology
Figure No. 16: BLANCHING ON
BUCCAL MUCOSA
Figure No. 18: REDUCED MOUTH
OPENING
Figure No. 17: SHRUNKEN
UVULA
Figure No. 18: REDUCED TONGUE
PROTRUSION
57
Results & Observation
RESULTS AND OBSERVATION
A total of 60 patients were included in the study. The study group comprised of all male
patients. The age of the patients varied between 18 years to 58 years. The patients were
divided into five groups with 12 patients in each group. There were 36 OSMF patients
who were divided into three groups Grade I, Grade II and Grade III. There were 24
controls who were divided under two groups i.e. tobacco chewers without OSMF or any
other premalignant lesion/condition and healthy controls without chewing habit.
Table 2: Mean value for age among various groups
Level
Minimum
Median
Maximum
Mean
SD
Chewer controls
21
31.5
48
32.0833
9.3172
Healthycontrols
22
34
45
33.6667
8.49996
OSMF stage I
25
32
40
32.6667
4.6775
OSMF stage II
18
26.5
58
30.5000
10.7153
OSMF stage III
22
32.5
46
32.3333
7.8547
The above table shows that mean value of age among the five groups are homogenous
Conclusion: the age distributions are very similar (p-value for comparing the mean ages =
0.9235). Since they are all males, the groups are gender and age matched.
58
Table 3: ANOVA test for ages among various groups
One-way analysis of variance was used to compare the means of the ages of the five
groups.
Source
DF
Sum of
Mean Square
F Ratio
Prob > F
Inf.
Squares
Group
4
63.3333
15.8333
0.2215
0.9253
NS
Error
55
3931.9167
71.4894
-
-
-
C. Total
59
3995.2500
-
-
-
-
From the above table of one way ANOVA the ages in the five groups are homogenous
Table 4: Mean value of MDA among various groups
Means and SD:
Level
Number
Mean
SD
Std Err Mean
Chewer controls
12
5.7250
0.90174
0.26031
Healthy controls
12
4.4017
1.65257
0.47706
OSMF Stage I
12
10.8458
1.21897
0.35189
OSMF Stage II
12
14.1708
1.11169
0.32092
OSMF Stage III
12
14.9817
1.40018
0.40420
59
The above table shows that highest mean serum MDA level observed in group three
followed by group 2, group 1, chewer controls and healthy controls. For healthy controls
the mean value is 4.4017.
Table 5: ANOVA test for MDA among various groups
One way analysis of variance was used to test the equality of the mean MDA values for
the five groups and there was a significant difference (p<0.0001). Then Tukey-Kamer test
was used at a level of significance of 0.05 to simultaneously test the equality of any two
pairs of groups. The conclusion is that there is no significant difference between Stage III
and II (group A), Stage I is significantly different from the rest (group B), and there is no
significant difference between the healthy and chewer controls (group C). See the table
below with highlighted p-values for the pair wise comparisons. There is also a table
below that gives means, standard deviations, and standard errors for each of the groups.
There is also a 95% confidence interval for the mean in that table.
Source
DF
Sum of Squares
Mean Square
F Ratio
Prob > F
Inf.
Group
4
1110.4972
277.624
168.7404
<.0001*
S
Error
55
90.4901
1.645
-
-
-
C. Total
59
1200.9873
-
-
-
-
60
The p-value in the above table indicates non homogenicity in the means of five groups.
To make pair-wise comparisons Tukey-Kramer Test was conducted and following table
is obtained.
Table 6: Mean comparisons
Comparisons for all pairs using Tukey - Kamer HSD
Level
Mean
OSMF Stage III
A
14.981667
OSMF Stage II
A
14.170750
OSMF Stage I
B
10.845833
Chewer controls
C
5.725000
Healthy controls
C
4.401667
Levels not connected by same letter are significantly different.
61
TABLE 7: Multiple comparison of serum MDA among various groups using
Tukey-Kramer test
Level
- Level
Difference
Std Err Dif
p-Value
Inf.
OSMF Stage III
Healthy controls
10.58000
0.5236530
<.0001*
S
OSMF Stage II
Healthy controls
9.76908
0.5236530
<.0001*
S
OSMF Stage III
Chewer controls
9.25667
0.5236530
<.0001*
S
OSMF Stage II
Chewer controls
8.44575
0.5236530
<.0001*
S
OSMF Stage I
Healthy controls
6.44417
0.5236530
<.0001*
S
OSMF Stage I
Chewer controls
5.12083
0.5236530
<.0001*
S
OSMF Stage III
OSMF Stage I
4.13583
0.5236530
<.0001*
S
OSMF Stage II
OSMF Stage I
3.32492
0.5236530
<.0001*
S
Chewer controls
Healthy controls
1.32333
0.5236530
0.0993
NS
OSMF Stage III
OSMF Stage II
0.81092
0.5236530
0.5361
NS
62
The above table shows that in all the pair wise comparison there is statistically significant
difference in all except last two rows that is between chewer control and healthy controls,
between Stage II and Stage III.
Graph 1: Number of patients among various groups
14
No. of patients
12
12
12
12
12
12
10
8
6
Series 1
4
2
0
Grade I
Grade II
Grade III
Chewer Cnt Healthy Cnt
Study Groups
Graph 2: Distribution of patients in study groups in different age
30
24
25
No. of patients
20
15
18
15
Series 1
10
5
2
1
0
16-25
26-35
36-45
46-55
56-65
Age in years
63
Graph 3: Mean age among patients of different study groups
34
33.667
33.5
33
32.667
32.333
Mean age
32.5
32
32.003
31.5
31
30.5
30.5
Series 1
30
29.5
29
28.5
Grade I
Grade II
Grade III
Healthy
controls
Chewer
cintrols
Study Groups
Graph 4: Mean serum MDA levels among various study groups
16
14.1708
14.9817
14
Mean MDA levels
12
10.4858
10
8
6
5.725
Column1
4.4017
4
2
0
Healthy Cnt Chewer Cnt
Grade I
Grade II
Grade III
Study groups
64
Graph 5: One way analysis of MDA by group
The above graph shows the equality of the mean MDA values in five groups which was
tested using ANOVA test. It also shows the degree of equality among any two pairs of
the groups which is denoted by circles.
IMPRESSION: At the end of the study it was observed that there was significant rise in
serum MDA levels in patients with Oral submucous fibrosis with a maximum increase in
Stage III compared to that of control group.
65
Discussion
DISCUSSION
Oral submucous fibrosis is a chronic irreversible disease, etiology of which is
poorly understood. It is mainly found in Indian population and other South East Asian
countries like Sri Lanka, Pakistan, Bangladesh and Nepal. OSMF affects about 0.2 1.2% of Indian population attending dental clinics. OSMF is also seen in Indians
living in other countries like Kenya, Malaysia, Uganda, South Africa, Fiji and UK.
Cases have also been reported from ethnic groups in Taiwan, Thailand and Vietnam.
The disease is predominant in males in their second and third decades of life. The
disease leads to fibroelastic transformation of the lamina propria and epithelial
atrophy of the oral mucosa. Later the mucous membrane becomes stiff leading to
trismus. Although the etiology is not known it has been postulated that the disease is
caused by an irritation of the oral submucosa by irritants such as tannins in betel nut
and capsaicin in chillies. Immunological, genetic and environmental factors have also
been thought to be contributing factors for the disease.
Clinical research in the area of lipid peroxidation has been hampered by the
lack of a valid biomarker. One of the most frequently used biomarkers providing an
indication of the overall lipid peroxidation level is the concentration of
malondialdehyde, one of the several byproducts of lipid peroxidation processes.16
Malondialdehyde is a naturally occurring endogenous product of lipid peroxidation
and prostaglandin biosynthesis, but is mutagenic and tumorigenic. The MDA value in
blood is a measure for the ability of the body to handle the oxidative stress it is
exposed to. Disturbance of balance between oxidative processes and antioxidative
defenses causes the oxidative stress that can damage proteins, lipids, polysaccharides
and nucleic acids. The most frequently used test is the measurement of MDA by the
66
Discussion
TBA reaction. Here, TBA reacts with MDA to form TBA: MDA adduct, which
absorbs maximally at 535 nm. In this study this coloured complex was measured by
photospectrometer. This method is of particular interest because of its procedural
simplicity and nanomolar sensitivity.
This study included 36 subjects with oral submucous fibrosis and 24 subjects
without OSMF as controls. In the OSMF group the largest number of patients
belonged to the age group of 20-45. This finding correlates with the report of Chiang
CP et al (2002)111, Anuradha CD et al (1993)112 and Gupta S et al (2004).113
The mean age in our study was 32.3 years which was similar to Ranganathan
K et al (2004)114 who reported a mean age of 32.4 years. Most of the patients in our
study were in the second and third decade of life with 15 patients (25%) in the age
group 16-25 years, 24 patients (40%) in the age group of 26-35 years, 18 patients
(30%) belonged to the age group of 36-45 years and 3 patients (5%) were above 46
years. This is in agreement with Chaturvedi VN et al (1991)115 and Haider SM et al
(2000).47 The higher prevalence of OSMF patients in younger age group is explained
by popularity of refined areca nut products, which are readily available and
introduction of betel nut chewing habit at an early age.
OSMF is seen to be more prevalent among males as per the observation of Kiran
K et al (2007)116, Hazare VK et al (2007)117, Chaturvedi VN and Marathe NG
(1988)118, Shah N et al (1994)40 and Ranganathan K et al (1004).114 The sex incidence
ratio varies from study to study due to varying habits in various populations. As the
habit of chewing betel nut is less prevalent among females in and around Belgaum
city, only male patients with OSMF were included in our study to maintain
homogeneity with respect to gender.
67
Discussion
All the patients in our study had atleast one of the arecanut based chewing habits.
Similar observations were made by Shear et al (1967)119, Van Wyk CW et al
(1990)120. Arecanut chewing is identified as the most important etiological factor.
Some researchers noticed OSMF in patients without any oral habits, such as 0.12%
reported by Gupta S (2004)109 and 1.81% reported by Shah N (1998)121 but this was
not consistent with our study.
This suggests that betel nut chewing could contribute to the development of
oral cancer, but requires a longer period of time to produce the changes in the oral
epithelium. Whereas subjects who chewed tobacco with betel leaf and betel nut
developed carcinoma within a shorter time span, indicating that tobacco and betel
leaf/betel nut together can induce malignant changes earlier than betel nut alone. Betel
leaf and tobacco quid not only irritates the oral mucosa mechanically, but the
chemical irritation (nicotine and arecoline predominantly) is probably much greater
because of the higher concentration of the chemicals kept for prolonged periods at one
site. According to M.C. Downer122, only heavy smoking (> 20 cigarettes per day)
produced a significant odds ratio. One patient who had habit of alcohol (wine/whisky)
intake along with tobacco, betel nut and cigarette developed precancerous changes in
oral mucosa followed by oral cancer within a shorter period of time (8 years) and also
showed high serum MDA level (43.60 nmol/ml).
Literature reveals that the process of carcinogenesis occurs by generation of
reactive oxygen species, which act by initiating lipid peroxidation. The present study
has revealed an intriguing aspect of tumor biochemistry. This study showed
significant differences between serum MDA levels in oral submucous fibrosis
[14.1708 (± 0.32092) nmol/ml], chewer controls [5.7250 (± 0.26031) nmol/ml] and
68
Discussion
healthy controls [4.4017 (±0.47706) nmol/ml] (p < 0.01). Serum MDA increased in
oral submucous fibrosis and chewer controls as compared to normal healthy
individuals.
In cancers other than oral cancer, MDA levels showed marked increase.104, 107
According to Unal Sahin et al33 MDA levels are increased significantly in lung cancer
(20.5 ± 7.9 nmol/ml) as compared to healthy controls (12.6 ± 7.1 nmol/ml) (p <
0.001). Ebubekir Bakan et al108 stated that the levels of plasma MDA were
significantly higher in patients with gastric cancer (7.6 ± 1.4 nmol/ml) as compared to
the healthy controls (5.4 ± 1 nmol/ml). In a study on cervical carcinoma by G.
Gitanjali et al102, there was no statistically significant difference in the mean serum
MDA levels of healthy controls (3.40 ± 0.75 nmol/ml) and patients with cervical
carcinoma (3.54 ± 1.89 nmol/ml).
Other than precancer and cancer, literature reported increased MDA levels in
chronic obstructive pulmonary disease, H. pylori-infected gastric mucosa35, during
cardiopulmonary bypass123 and during hemodialysis.105, 97
Though radiotherapy is one of the clinical means by which oral cancer can be
treated, many biochemical complications, such as damage to cellular DNA and
membrane structures can occur. Both Sabitha K. E. et al102 and Patiat M108 showed
significant increase in MDA levels in untreated and radiation treated oral cancer
patients when compared with normal subjects. Radiation treated patients had higher
MDA levels (0.792 ± 0.1157 nmol/ml) than untreated patients (0.598 ± 0.169
nmol/ml) (p < 0.001).
According to Soma et al38 MDA levels in oral sub mucous fibrosis (3.3 + 0.4
nmol/ml) were more as compared to healthy controls (2.4 + 0.5) (p < 0.001). In our
69
Discussion
study serum MDA levels was found to be increased in oral submucous fibrosis
[14.1708 (± 0.32092) nmol/ml] as compared to normal individuals [4.4017
(±0.47706) nmol/ml] and the increase was statistically significant (p < 0.01). This
shows a possibility of the role of reactive oxygen species in etiopathogenesis of oral
submucous fibrosis.
An attempt was made to study the level of oxidative stress in different stages
of OSMF. In our study serum MDA levels in stage I OSMF was [10.8458 (± 0.35189)
nmol/ml], MDA levels in stage II OSMF was found to be [14.1708 (± 0.32092)
nmol/ml] and in stage III OSMF it was [14.9817 (± 0.40420) nmol/ml]. This shows
that the oxidative stress increases as the disease progresses reflecting the amount of
tissue damage, our study results also showed statistically significant difference in
MDA level of grade I and stage III OSMF (p <0.0001)as well as stage I and stage II
OSMF (p <0.0001) but there was no statistical significance between stage II and stage
III disease (p =0.5361), which is in correlation with the study conducted by Soma
Gupta et al (2004) where plasma MDA levels were found to be increased in all stages
of OSMF (mean = 3.3 + 0.4 nmol/ml) compared to healthy controls (mean= 2.4 +0.5
nmol/ml) and the increase was statistically significant (p <0.001). It is established that
the lipid peroxidation increases with severity of the disease reflecting the extent of the
tissue injury. Their results also showed that mean MDA levels were more in OSMF
stage II than in stage I (3.1 + 0.8 nmol/ml). However it did not vary much between
grade II(3.5 + 0.8 nmol/ml) and grade III (3.3 + 0.1 nmol/ml).
Further attempt was made to study the association of lipid peroxidation and
the habit of either betel nut or betel leaf chewing, or tobacco chewing, in normal
subjects. In the present study 12 controls had betel nut or tobacco chewing habit
70
Discussion
without OSMF and 12 were healthy controls without chewing habit. The MDA levels
were increased in chewer controls [5.7250 (± 0.26031) nmol/lm] as compared to the
healthy nonchewer controls [4.4017 (± 0.47706) nmol/ml] though the increase was
not statistically significant (p = 0.0993).
Cigarette smoke, in both the gaseous phase and condensed particles, contains
alkenes, nitrosamines, aromatic and heterocyclic hydrocarbons and amines. In
addition, it is an excellent source of ROS, such as hydroxyl radicals, superoxides and
peroxides that are capable of initiating or promoting oxidative damage. Unal Sahin et
al104 has reported increased MDA levels in smokers (22.5 ± 8.0 nmol/ml) than non
smokers (16.1 ± 6.7 nmol/ml) (p < 0.001) with lung cancer. Abraham Z. Reznick et
al124 elucidated the outcome of interaction of between cigarette smoke and oral
salivary peroxidase in smokers and nonsmokers. After smoking a single cigarette, a
sharp drop of oral peroxidase activity was observed in both groups: 42.5% in smokers
and 58.5% in nonsmokers (p < 0.05). After 30 minutes, the level of activity returned
to 90 - 100% of the pre smoking level, presumably due to the secretion of new saliva
into the oral cavity. The oral peroxidase activity loss was accompanied by increased
carbonylation of the salivary proteins, an indicator of the oxidative damage to
proteins. According to Flemming Nielsen et al99 daily smokers had a slightly higher
average concentration of plasma MDA than nonsmokers, and plasma MDA correlated
with daily exposure to cigarette smoke. Jason D. Morrow125 proved that Plasma levels
of lipid peroxidation product (F2-isoprostanes) were significantly higher in the
smokers as compared to the nonsmokers. One study by Wendy Y. Craig et al126 was in
contrast with the above studies, which stated that there was no strong relation between
lipid peroxide level (determined by TBARS level) and cigarette smoke exposure,
suggesting that certain interactions related to oxidation status are not measurable in the
71
Discussion
serum compartment. They further stated that serum copper was the major determinant
of serum lipid peroxidation status, indicating that it contributes to lipid peroxidation in
vivo.
Flemming Nielsen et al99 demonstrated a positive correlation between plasma
MDA and alcohol consumption. No major interaction occurred between gender and
age. Men had slightly but significantly higher plasma MDA concentrations than
women.
Reaction of the ROS with cellular DNA results in oxidative damage, including
a number of oxidized bases, single-strand breaks and/or alkali-labile lesion. These
types of DNA damage are considered to be crucial in cancer development, thus
providing an additional possible mechanism for an apparent association between
tobacco chewing, smoking and mouth, lung, pharynx, esophagus, bladder and cervical
cancers. Laura J. Niedernhofer et al127 proved that MDA-induced DNA damage is
mutagenic in human cells. Yujing Zhang et al128 showed that MDA–DNA adducts
may serve a biomarker of DNA damage by lipid peroxidation induced endogenously
or exogenously in oral mucosal cells. Chiara Leuratti et al129 showed that
malondialdehyde - deoxyguanosine (M1-dG), a DNA adduct derived from lipid
peroxidation plays a role in human colorectal carcinogenesis, in combination with
other genetic and environmental factors.
Within-subjects and day-to-day variations of serum MDA levels indicated that
further studies have to be carried out with larger sample size. However, on a group
basis, the present data support that serum MDA levels may be a potential biomarker
for oxidative stress. Still much work is required to study the role of reactive oxygen
72
Discussion
species and lipid peroxidation in oral precancer and cancer. Studies should be
undertaken in more number of subjects to compare age and sex related differences.
73
Conclusion
CONCLUSION
The present study showed significant increase in the serum malondialdehyde
levels, which is a reliable marker and product of lipid peroxidation and this was
increased in all grades of Oral submucous fibrosis and the maximum being in Grade
III. The levels of serum malondialdehyde in healthy controls and chewer controls
were significantly low. There was no statistically significant difference between the
serum MDA levels between healthy controls and chewer controls. This increase in the
serum MDA levels is indicative of increased damage to the tissues caused by lipid
peroxidation due to free radical; it reflects the increased oxidative stress in the tissues.
To conclude the present study suggests a possibility of ROS playing a part in
aetiopathogenesis of the disease. Exogenous supplementation of non-enzymatic
antioxidants may decrease the damage to oral mucosa by quenching and preventing
the free radical action which are responsible for the disease process and malignant
conversion.
74
Summary
SUMMARY
The present study was conducted to estimate serum lipid peroxidation product
malondialdehyde levels in 36 patients with OSMF and 24 age and sex matched
chewer and non-chewer controls without OSMF. Alterations in the serum
malondialdehyde levels were assessed using conventional thiobarbituric acid method,
using a photospectrometer and reading are calculated. The statistical analysis was
done using Tukey Kamer test and ANOVA test. The result of our study depicted the
following outcome.

There was a significant rise in the serum malondialdehyde levels in patients
with OSMF and controls who were arecanut chewers.

The maximum rise of serum malondialdehyde levels were seen in Grade III
OSMF, followed by Grade II, Grade I, chewer controls and finally the healthy
controls.

There was no significant difference in serum malondialdehyde levels among
chewer controls and healthy controls and also between Grade II and Grade III
OSMF.
From the present study it is evident that lipid peroxidation product malondialdehyde
level rises in patients with Oral submucous fibrosis. Malondialdehyde is an indicator
of oxidative stress which in turn indicates increased damage caused by ROS. In order
to scavenge these free radicals antioxidants like vitamin E, vitamin A, vitamin C, zinc
selenium etc should be supplemented to the patients suffering from OSMF which will
reduce the malignant transformation rate and helps to regress the condition to some
extent. However further evaluation in this direction with larger sample size is
necessary to give greater insight into the pathogenesis and management of this
condition and to decrease the rate of malignant transformation rate.
75
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Annexure
ANNEXURE – I
DEPARTMENT OF ORAL MEDICINE AND RADIOLOGY,
K.L.E.’S VISHWANATH KATTI INSTITUTE OF DENTAL SCIENCES
NEHRU NAGAR, BELGAUM.
“COMPARISON OF SERUM MALONDIALDEHYDE LEVELS IN PATIENTS
WITH ORAL SUBMUCOUSFIBROSIS AND CONTROL GROUP- A
HOSPITAL BASED STUDY”.
CASE HISTORY PROFORMA
Patient’s Name:
Case no:
Age:
Date:
Sex:
Occupation:
Address:
Chief complaint:
History of present illness:
Past History:
- Medical History:
- Dental History:
Personal history:
Habits:

Guthka

Tobacco

Betel quid

Cigarette smoking

Beedi smoking
Duration
Frequency
90
Annexure
Extra oral examination:

TMJ:

Lymphnodes:
Intra oral examination:

Hard tissue examination:

Soft tissue examination:

Buccal mucosa

Palate

Tongue

Floor of mouth

Lips

uvula

Mouth opening

Tongue protrusion
Provisional Diagnosis:
Investigations:
Treatment plan:
91
Annexure
ANNEXURE – II
CONSENT FORM
DEPARTMENT OF ORAL MEDICINE AND RADIOLOGY,
INSTITUTE OF DENTAL SCIENCES, BELGAUM
“COMPARISON OF SERUM MALONDIALDEHYDE LEVELS IN PATIENTS
WITH ORAL SUBMUCOUSFIBROSIS AND CONTROL GROUP- A
HOSPITAL BASED STUDY”.
I, _________________________________ aged__________ years has been informed
about my involvement in the study.
1. I agree to give my personal details like Name Age, Sex, Address, Past Dental
and Medical History and any other details required for the study to the best of
my knowledge.
2. I will cooperate with the dentist for examination and also for various
investigations.
3. I permit the operator to utilize the information given by me and the results
obtained from this study for presentation and publication.
4. I permit the dentist to take my photographs, collect blood to utilize it for the
study and presentation purpose.
5. I am participating in this study with my own wish and will and the dentist has
explained the nature and the effect of the procedure in my vernacular
language.
Name of the doctor:
Name of the patient:
Name of the witness:
Signature of Patient:
Signature of witness:
Date:
Date:
92
Annexure
ANNEXURE – III
MASTER CHART – CASES
CASES: OSMF STAGE I
Sl. No.
NAME
AGE
SEX
Hb%
MDA levels
1
Vital
Gundu
Yonus
Vinod
Adivappa
Mumtaz
Amrut
Gangadhar
Basalinga
Ravsaheb
Jyothiba
harishchandra
37yrs
40yrs
29yrs
25yrs
30yrs
32yrs
26yrs
35yrs
32yrs
32yrs
37yrs
37yrs
M
M
M
M
M
M
M
M
M
M
M
M
11.47gm%
11.08gm%
10.69%
9.66gm%
12.30gm%
9.68gm%
7.31gm%
12.3gm%
9.40gm%
12.12gm%
9.68gm%
12.30gm%
10.96
10.86
11.43
8.67
10.93
12.64
9.93
9.96
10.16
12.13
12.65
9.83
2
3
4
5
6
7
8
9
10
11
12
CASES: OSMF STAGE II
Sl. No.
NAME
AGE
SEX
Hb%
MDA levels
1
Somanath M
Ramappa
Pramod
Imthiyaz
ajay H
Immansaab
Abhishek
Thosif
Akram
Elig
Liaquat
Shantinath
23yrs
58yrs
25yrs
29yrs
24yrs
26yrs
18yrs
27yrs
26yrs
31yrs
38yrs
41yrs
M
M
M
M
M
M
M
M
M
M
M
M
11.3gm%
10.23gm%
9.32gm%
10.16gm%
10.28gm%
10.61gm%
9.69gm%
11.11gm%
9.68gm%
9.71gm%
9.70gm%
9.61gm%
14.129
2
3
4
5
6
7
8
9
10
11
12
13.98
12.81
14.62
12.13
14.64
13.62
15.67
13.62
16.15
13.96
14.97
93
Annexure
CASES: OSMF STAGE III
Sl. No.
NAME
AGE
SEX
Hb%
MDA levels
1
Shrinivas
Mahesh
Anarnath
Shafaj
Gangappa
Ravi
Rphan
Krishna
Gajanan
Issrani
Arif
Sharrieff
29yrs
36yrs
22yrs
32yrs
35yrs
44yrs
46yrs
28yrs
33yrs
37yrs
24yrs
22yrs
M
M
M
M
M
M
M
M
M
M
M
M
10.93gm%
11.42gm%
9.35gm%
9.17gm%
10.26gm%
10.24gm%
10.10gm%
9.12gm%
7.69gm%
9.36gm%
9.24gm%
8.36gm%
15.28
13.39
14.33
18.21
16.23
14.31
15.21
13.16
14.45
13.93
15.16
16.12
2
3
4
5
6
7
8
9
10
11
12
94
Annexure
ANNEXURE –I V
MASTER CHART – CONTROLS
HEALTHY CONTROLS
Sl. No.
NAME
AGE
SEX
Hb%
MDA levels
1
Yusuf
Praveen
Jyotjiba
mahaveer
Krishna
Basappa
Nagappa
Deepak
Gopal
ganpathi
Ramesh
Jagendra
22YRS
24yrs
32yrs
36yrs
41yrs
45yrs
25yrs
32yrs
36yrs
24yrs
45yrs
42yrs
M
M
M
M
M
M
M
M
M
M
M
M
12.37gm%
12.31gm%
13.06gm%
13.12gm%
12.40gm%
10.12gm%
12.06gm%
11.62gm%
12.13gm%
13.48gm%
12.43gm%
12.33gm%
5.89
3.85
4.49
3.95
3.39
3.86
2.56
4.32
2.68
4.48
4.52
8.83
2
3
4
5
6
7
8
9
10
11
12
CHEWER CONTROLS
Sl. No.
NAME
AGE
SEX
Hb%
MDA levels
1
mallappa
Dattu
Naziyak
shubhalaxman
Vihaba
padmaraj
Santosh
Yash
raja K S
uttam A
mallappa
Manjith
22yrs
31yrs
26yrs
41yrs
48yrs
21yrs
26yrs
35yrs
37yrs
21yrs
45yrs
32yrs
M
M
M
M
M
M
M
M
M
M
M
M
10.36gm%
10.38gm%
11.14gm%
12.36gm%
13.64gm%
12.28gm%
12.23gm%
15.16gm%
12.24gm%
12.11gm%
11.22gm%
13.06gm%
5.12
6.11
6.11
6.25
6.12
5.51
4.5
4.43
6.91
6.93
6.22
4.49
2
3
4
5
6
7
8
9
10
11
12
95

Dissertation “COMPARISON OF SERUM MALONDIALDEHYDE LEVELS IN PATIENTS WITH ORAL SUBMUCOUS

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