DISSERTATION SYNOPSIS

Dr. ANUJA JOHN

POST GRADUATE STUDENT
DEPARTMENT OF ORAL MEDICINE
AND RADIOLOGY
2013-2016

A.J. INSTITUTE OF DENTAL SCIENCES,

KUNTIKANA, MANGALORE.

Rajiv Gandhi University of Health Sciences, Karnataka.

Bangalore.
 ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION

1. Name of the Candidate Dr. ANUJA JOHN

POSTGRADUATE STUDENT,
And Address
DEPARTMENT OF ORAL
( in block letters ) MEDICINE AND RADIOLOGY,

A .J. INSTITUTE OF DENTAL

SCIENCES, KUNTIKANA,
MANGALORE 575004.
2. Name of the institution A .J .INSTITUTE OF DENTAL SCIENCES,
MANGALORE

3. Course of study and subject Master of Dental Surgery (M.D.S)

ORAL MEDICINE AND RADIOLOGY

4. Date of admission to course 5th July 2013

5. Title of the topic :

Estimation of serum and salivary iron and ascorbic acid levels: A clinical and
biochemical study in Oral submucous fibrosis patients

6. Brief resume of the intended work :

6.1 Need for the study:

Oral submucous fibrosis (OSMF) is a fibrotic condition of the oral cavity and is always
associated with chronic epithelial inflammation and progressive deposition of
collagenous proteins (extra cellular matrix) in the subepithelial layer of the buccal
mucosa1. Iron and Ascorbic acid are important agents for collagen synthesis1.
Iron
Iron is a necessary trace element found in nearly all living organisms. Iron deficiency is a
common form of malnutrition worldwide. Iron deficiency may cause oral cancer via the
induction of oxidative stress2.
Iron deficiency anaemia in patients with OSMF could be related to the pre cancerous
nature of this condition3. The decreased serum iron can be an important tool for the
diagnosis of this potentially malignant condition.

Ascorbic acid
Ascorbic acid or vitamin C has the potential to protect both cytosolic and membrane
components of cells from oxidative damage1. Ascorbic acid levels have been investigated
in several cancer related studies and many studies have shown a significant decrease in
serum and salivary ascorbic acid in OSMF. This has led to the theory that ascorbic acid
may have been used for the excessive collagen production and cross-linking that occurs
in OSMF1. Thus ascorbic acid could be an important indicator for the formation of
OSMF.

Hence the present study is undertaken to assess the level of serum and salivary Iron and
ascorbic acid in OSMF patients.

HYPOTHESIS:

1. Null hypothesis (H1): There will not be any significant difference in serum and
salivary iron and ascorbic acid in patients with OSMF.

2. Alternate hypothesis (H2): There will be a significant difference between serum

and salivary iron and ascorbic acid in patients with OSMF.

6.2 Review of literature :

1. Sunalis Khanna, Karjodkar FR.(2006)7 Studied the levels of circulating immune

complexes, trace elements (copper, iron, Selenium) in the serum of patients with
OSMF, oral leukoplakia, and oral squamous cell carcinoma. Serum copper levels
showed gradual increase from precancer to cancer patients. However, serum iron
levels were decreased significantly in the cancer group and selenium levels
showed a marked decrease in the cancer group. Their study showed that these
immunological and biological markers may be associated with the pathogenesis
of oral premalignant and malignant lesions and their progressions.

2. Tadakamadla Jyothi, Kumar Santosh. et al (2011) 11 conducted a study for 50

patients clinically diagnosed with OSMF and 50 healthy controls who were
matched for age and gender to estimate and compare the levels of serum copper
 and iron. In their study OSMF patients were categorized by clinical staging and
serum estimation of copper and iron was done using atomic absorption
spectrometry. Mean copper and iron differed significantly between the patients
and controls with patients exhibiting higher copper and lower iron levels as
compared to controls who exhibited lower copper and higher iron levels in serum.
They thus concluded that copper levels increased and iron levels decreased as the
clinical stage of OSMF increased.

3. Aravindh and Jagathesh et al (2012)10 conducted a study on 60 patients out of

which 20 patients were suffering from OSMF, 20 patients from oral cancer, and
20 patients were normal controls to evaluate the levels of antioxidants beta
carotene, Vitamin C and Vitamin E, the most important antioxidant nutrients in
OSMF and oral cancer. Blood samples were collected and antioxidant nutrients:
beta carotene, Vitamin C and Vitamin E analysis was done by standardized
protocols using spectrophotometry. They found that the mean beta carotene, mean
Vitamin C and mean Vitamin E levels were highest in controls followed by
OSMF patients and lowest in oral cancer patients.

4. Shetty SR, Babu S et al. (2012)1 conducted a study to estimate the levels of iron
and ascorbic acid in serum and saliva of 65 clinically diagnosed and
histopathologically confirmed cases of OSMF along with 21 age and sex matched
controls patients. Serum and salivary iron was analysed by the dipyridyl method
and serum and salivary ascorbic acid were analysed by dinitrophenyl hydrazine
method. The serum and salivary ascorbic acid levels were found to consistently
decrease with the progression of histopathological grading of OSMF. Serum and
salivary iron levels were also decreased. They concluded that ascorbic acid and
iron may have been used for the excessive collagen synthesis occurring during the
progression of OSMF and hence their monitoring may play a crucial role in the
early diagnosis and prognosis of OSMF.

5. Ankolekar M, Karjodkar FR (2013)8 conducted a study to compare the levels

of trace elements in patients with gutkha eating habits with or without OSMF and
in healthy patients. A total of 75 patients were include in their study and they
were divided into three groups; the individuals with the history of gutkha intake
with OSMF, the individuals with the history of gutkha intake without OSMF and
healthy individuals without OSMF and without any habits. Blood and saliva were
collected and subjected for analysis using atomic absorption spectrometry and a
differential pulse anodic stripping voltmeter. They found a significant decrease in
the serum Magnesium and iron levels between the patients with habits and
normal healthy individuals. They also found a significant decrease in serum Zinc
in the patients with habits and without OSMF and concluded that trace elements
could be used as potential prognostic and diagnostic markers in OSMF patients.
 6.3 Objectives of the study:

1. To estimate the levels of iron and ascorbic acid in the serum and saliva in patients
with OSMF.

2. To correlate the change in the levels of iron and ascorbic acid with the clinical
grading of OSMF.

7 Material and methods:

7.1 Source of data :

Data will be collected from out patients reporting to the department of Oral medicine
and Radiology, AJ Institute of Dental Sciences, Mangalore after obtaining informed
consent and clearance from the Institutional Ethical Committee.

INCLUSION CRITERIA:

1. Patients clinically and histopathologically diagnosed with OSMF

2. Healthy patients without any systemic disease as controls.

EXCLUSION CRITERIA

1. Patients who are already undergoing treatment for OSMF.

2. Patients with systemic disorders, with history of drug intake containing iron or
ascorbic acid.

7.2 Methods of collection of data (including sampling procedure,

If any )

88 patients (study and control) will be selected from the Department of Oral medicine
and Radiology, AJ Institute of Dental Sciences, Mangalore after obtaining an informed
consent and clearance from the Institutional Ethical Committee. Patients from all groups
will be examined thoroughly. Each patients detailed case history will be recorded.
OSMF will be grouped based on the clinical criteria according to Nagesh and Bailoor
(1993)4. Diagnosis of lesions of groups II, III, IV will be confirmed histopathologically.

The patients will be divided into the following groups

Group I: 22 healthy individuals

Group II: 22 patients with grade I OSMF

Group III: 22 patients with grade II OSMF

Group IV: 22 patients with grade III OSMF

METHODOLOGY

Collection of sample :
5 ml of unstimulated saliva will be collected by spitting method by asking the patient to
spit into a sterile container.

5 ml of venous blood will be withdrawn from the antecubital vein from each patient
using sterile disposable syringe, transferred to plastic test tubes and will be kept standing
for 30 minutes at room temperature.

Serum and salivary iron and ascorbic acid will be analysed in the Department of
Biochemistry, AJ Institute of Medical Sciences, Mangalore.

Estimation of Iron by Colorimetric method by using semi automated

analyser with ferrozine method5 [by Seidel. j; et al]

L1(buffer reagent)- Composition: Acetate buffer 100 mM; Sodium Hydroxyl

Ammonium Chloride 225 mM; detergents and preservatives.

L2(color reagent)- Composition: Ferrozine 15 mM

Standard solution:2 ml of iron in 100g/dl

1. Label the test tubes as Blank (B), Standard (S), Sample blank (SB) and Test (T).
2. Blank (B) will be prepared by mixing 0.2 ml of distilled water with 1 ml of buffer
reagent (L1) and 0.05 ml of colour reagent (L2).

3. Standard (S) will be prepared by mixing 0.2 ml of iron standard(S) solution with 1.0
ml of the buffer reagent (L1) and 0.05 ml of colour reagent (L2)

4.Sample blank (SB) will be prepared by mixing 1.05ml of buffer reagent(L1) with
0.2ml of sample,

5. Test (T) will be prepared by mixing 0.2 ml of serum/saliva sample with 1.0 ml of the
buffer reagent (L1) and 0.05 ml of colour reagent (L2).

Mix well and incubate at room temperature for 5 minutes. Measure the absorbance of the
Blank (Abs.B), Standard (Abs.S), Sample Blank (Abs.SB) and Test Sample (Abs.T)
against D.W using spectrophotometry at 562nm.

CALCULATIONS

Iron in g/dl = Abs.T (Abs.SB + Abs.B)/ Abs.S Abs.B x 100

Estimation of Ascorbic acid by Dinitrophenyl Hydrazine(DNPH)

method6
TCA-Trichloroacetic acid

DTC reagent-2,4 DNPH, thiourea, CuSO4

1.100L of sample Serum/saliva will be taken in a clean test tube

2. 900L of 5% (TCA) will be added to it and allowed to precipitate proteins for about
ten minutes and centrifuged.

3.100L of the supernatant is separated and transferred into another test tube.

3. To this 200L of DTC reagent is added, the tube is plugged and the mixture is
incubated at 600c for 60 minutes in a water bath.

4. Simultaneously a blank with 1mL of TCA and 200L of DTC reagent is also
incubated under similar conditions.
5. Following 60 minutes of incubation, the reaction mixture is cooled in an ice bath and
1mL of 4.5M sulphuric acid is added to it and after it reaches room temperature the
optical density is read at 540nm against blank using spectrophotometry.

Collected data will be analysed by mean, standard deviation, frequency, percentage and
the results obtained will be statistically analysed using Chi Square test and ANOVA test.

7.3 Does the study require any investigations or interventions to be conducted on

patients or other humans or animals? If so, please describe briefly.

Yes, Venous blood withdrawal and Biopsy

7.4 Has ethical clearance been obtained from your institution in case of 7.3?

Yes
 STUDY DESIGN

OUTPATIENTS FROM THE DEPARTMENT OF ORAL MEDICINE

AND RADIOLOGY, AJ INSTITUTE OF DENTAL SCIENCES

SUBJECTS WILL BE DIVIDED INTO FOUR GROUPS WITH

22 SAMPLES EACH

GROUP I: HEALTHY INDIVIDUALS

GROUP II: SUBJECTS WITH GRADE I OSMF

GROUP III: SUBJECTS WITH GRADE II OSMF

GROUP IV: SUBJECTS WITH GRADE III OSMF

SERUM AND SALIVARY IRON AND ASCORBIC

ACID WILL BE ASSESSED

RESULTS

STATISTICAL

ANALYSIS
List of references :

1. Shetty SR, Babu S, Kumari S, Shetty P, Vijay R, Karikal A. Evaluation of

micronutrient status in serum and saliva of oral sub mucous fibrosis patients: A
clinicopathological study. Indian J Med Paediatr Oncol 2012; 33(4):224-6

2. Richie JP Jr, Kleinman W, Marina P, Abraham P, Wynder EL, Muscat JE.

Blood iron, glutathione, and micronutrient levels and the risk of oral cancer. Nutr
Cancer 2008; 60(4): 474-82

3. Karthik H, Nair P, Gharote HP, Agarwal K, Ramamurthy Bhat G, Kalyanpur

Rajaram D. Role of Hemoglobin and Serum Iron in Oral Submucous Fibrosis: A
Clinical Study. ScientificWorldJournal 2012; 2012: 254013

4. More CB, Gupta S, Joshi J, Varma SN. Classification system for oral submucous
fibrosis. J Indian Acad Oral Med Radiol 2012; 24(1): 24-9

5. Stookey LL. Ferrozine. A new spectrophotometric reagent for iron. Analyt Chem
1970; 42 (7):779-781

6. Roe JH, Kuether CA, The determination of ascorbic acid in whole blood and
urine through the 2,4-dinitrophenylhyrazine derivative of dehydroascorbic acid. J
Biol Chem 1943; 147: 399-407

7. Khanna SS, Karjodkar FR. Circulating Immune Complexes and trace elements
(Copper, Iron and Selenium) as markers in oral precancer and cancer: a
randomised, controlled clinical trial. Head & Face medicine 2006; (2)33:1-10

8. Ankolekar KM, Karjodkar FR. Estimation of the serum and salivary trace
elements in OSMF patients. J Clin and Diagn Res 2013; 7(6): 1215-8

9. May JM. Is ascorbic acid an antioxidant for the plasma membrane?. FASEB J
1999;13: 995-1006

10. Aravindh L, Jagathesh P, Shanmugam S, Sarkar S, Kumar PM,

Ramasubramanian S. Estimation of plasma antioxidants beta carotene, vitamin c
and vitamin e levels in patients with osmf and oral cancer-indian population. Int J
Biol Med Res.2012;3(2):1655-1657
11. Tadakamadla J, Kumar S, Mamatha GP. Evaluation of serum copper and iron
levels among oral submucous fibrosis patients. Med Oral Patol Oral Cir Bucal
2011;16 (7):870-3