RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA, BANGALORE
ANNEXURE II.
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1 NAME OF THE CANDIDATE Dr. SANGEETH SIDDABASAPPA AND ADDRESS POST-GRADUATE STUDENT (IN BLOCK LETTERS) DEPARTMENT OF ORAL MEDICINE & RADIOLOGY, BAPUJI DENTAL COLLEGE & HOSPITAL DAVANGERE
2 NAME OF THE INSTITUTION BAPUJI DENTAL COLLEGE & HOSPITAL DAVANGERE
3 COURSE OF STUDY AND MASTER OF DENTAL SURGERY- SUBJECT ORAL MEDICINE AND RADIOLOGY
4 DATE OF ADMISSION TO 30 MAY 2007 COURSE
5 TITLE OF THE TOPIC ESTIMATION OF UNSTIMULATED SALIVARY FLOW RATE , pH, COPPER & IRON IN GUTKHA CHEWERS WITH & WITHOUT ORAL SUBMUCOUS FIBROSIS -A PRELIMINARY STUDY
6. BRIEF RESUME OF THE INTENDED WORK:
6.1: Need for the study:
Oral submucous fibrosis (OSMF) is cause for concern because it is one of the preventable, prevalent, premalignant condition of oral mucosa in India. Based on the clinical & epidemiological studies it has been noted that OSMF is increasingly associated with gutkha chewing habit. Gutkha is readymade mixture of arecanut, tobacco, slaked lime, sweetening, flavoring agents. Saliva is important in maintenance of the oral health. Any changes in its quality & quantity may alter the oral health status. Since OSMF is localized condition, saliva may play an important role in its pathogenesis. During gutkha chewing lot of chemicals & metals like copper, iron are leached out into saliva, which in turn alter the property & composition of saliva. In betel quid chewers variations in the saliva flow rate, pH has been reported.
Copper released from arecanut is absorbed by oral mucosa & activates copper dependent enzyme, lysyl oxidase which in turn catalyses cross linking of collagen fibers. Thus copper is implicated in the pathogenesis of OSMF.
Iron in saliva is involved in catalytic process of reactive oxygen species generation which is involved in multistage carcinogenesis. Production of reactive oxygen species is enhanced by the presence of alkaline pH of saliva.
The purpose of this study is to determine saliva flow rate, pH, copper & iron in unstimulated whole mouth saliva of gutkha chewers.
To the best of our knowledge this is the first study in gutkha chewers where salivary parameters of patients with OSMF & without OSMF are correlated.
6.2 Review of literature:
The formation of reactive oxygen species from betel quid ingredients, namely arecanut, catechu & tobacco was studied using a chemiluminescence technique & it was observed that reactive oxygen species generation was enhanced by alkaline pH as well as by the presence of iron & copper.1
In a study, effects of long term use of tobacco on salivary secretion were assessed. Subjects of study were divided into smokers, tobacco chewers, niswar dippers & non tobacco users as controls. Each group was comprised of 20 healthy adult males. It was observed that salivary flow rate of tobacco users was not much different from that of non tobacco users.2
A study on salivary flow rate & pH was done on 110 arecanut chewers & 50 non chewers. The difference in pH was statistically significant among chewers & non chewers & salivary flow rate was statistically insignificant between chewers & non chewers. Salivary flow rate & pH also varied in different types of arecanut chewers.4 A study was conducted on 15 volunteers who were regular arecanut chewers, before & after their habitual chew. 6 non chewing volunteers acted as controls. In this study total copper concentration in saliva was measured by Atomic Absorption Spectrophotometer. 50% of subjects had salivary copper concentration higher than the range seen in normal controls. These data indicated that soluble copper found in arecanut is released into oral environment of habitual chewers.5
6.3: Aims & Objectives of study:
Aims:
1. To estimate & correlate salivary flow rate, pH, copper, iron in gutkha chewers with & without OSMF.
2. To correlate gutkha chewing habits with salivary flow rate, pH, copper, iron
Objectives:
1. The variations in salivary parameters might provide an evidence in the pathogenesis of OSMF. 2. To demonstrate the hypothesis that saliva can be used as a reliable diagnostic medium in predicting the occurrence of OSMF in gutkha chewers.
7. MATERIALS & METHODS: 7.1 SOURCE OF DATA: 1. Patients attending the out-patient department of oral medicine & radiology, Bapuji Dental College & Hospital, Davangere. 2. Patient selection is done under three groups A, B, C. Group A & Group B patients will be selected based on following criterias. Patients between the age group of 15-30 years. Gutkha chewing habits for more than six months. Not having any known systemic disorder & not on any drugs like anticholinergics, diuretics, antihistamines, antihypertensives & psychoactive substance that might alter the salivary parameters. Patients with clinically proven OSMF (burning sensation, restricted mouth opening, palpable fibrous bands) will be included in group A.
Patients without OSMF will be included in group B.
Group C includes controls,
Inclusion criterias :
Subjects between the age group of 15-30 years.
No history of habits like gutkha/pan/arecanut chewing, smoking, consumption of alcohol.
Apparently healthy patients.
7.2 METHODS OF SELECTION OF DATA:(INCLUDING PROCEDURES IF ANY)
60 individuals satisfying the inclusion criteria will be included in the study with 20 in each group.
Patients consent will be obtained for all cases & controls. Detailed analysis of gutkha chewing habits will be done. Comprehensive clinical history will be obtained in the predetermined form.
Saliva Collection: Saliva collection will be done between 10.00 a.m. to 11.30a.m.
Each subject is requested not to eat, drink or chew 60 mins before collection.
Each patient is made to sit on a dental chair and is asked to spit in a graduated container every 1min for 10 mins. During collection instruction is given not to speak or swallow. After collection salivary flow rate is measured & expressed in ml/min. Saliva is preserved at 4 degree centigrade and transported to the laboratory within 30 minutes and is analysed. The pH determination of saliva is done by pH analyzer Elico-L1-612. The ph is determined by electrometric method which comprises the measurement of electromotive force of cell consisting of an indicator electrode immersed in saliva solution & a reference electrode. Contact between the test solution and reference electrode is usually achieved by means of liquid junction. The emf of this cell is measured with pH meter. This is a high impedance electrometer calibrated in terms of pH.
The estimation of copper, iron in saliva is done by an instrument atomic absorption spectrophotometry, AAS-203-CHEMITO.
Basic principle of atomic absorption spectrophotometry: Combustion flames provide a simple means of converting inorganic substances in solution into free atoms when introduced in the form of aerosol. A large number of these free atoms remain in unexcited state or in the ground state. The vaporized atoms are then exposed to radiation from a light source. The light source, a discharge tube is made of very same element being analyzed. It emits only those frequencies of light that are in the emission spectrum of the element. The vaporized atoms in the flame will only absorb these frequencies when it contains this element. Once the radiation has passed through the sample, a monochromator, consisting of a diffraction grating & a slit isolates the desired radiation frequency & transmits it to a detector.
First stock solutions are prepared & analysis is done by Atomic Absorption spectrophotometry. Later saliva sample analysis is done.
Procedure for preparation of stock solutions: All the chemical reagents must be of analytical reagent grade. The weights indicated are to be dissolved in solvent indicated & finally made upto 1000ml with 2% acid solution, hydrochloric acid or nitric acid.
For copper, dissolve 1gm of copper metal in 1:2 nitric acid. For iron, dissolve 1 gm iron powder in 1:2 hydrochloric acid. These are standard solutions which are used first before testing saliva sample & the absorbance of these solutions are recorded for reference.
For copper, optimize with copper hollow cathode lamp & check with copper 5ppm solution to produce a minimum of 0.6 absorbance. Record absorbance for copper at 1ppm, 2ppm, 3ppm, 4ppm, 5ppm. Then 10 ml of saliva sample is aspirated directly into Atomic absorption spectrophotometer recording the absorbance for 10ml of saliva. These readings are transmitted to the detector then given by computer using the software AAS 201/203.
For iron, optimize with iron lamp & check with 10ppm iron solution to produce a minimum of 0.5 absorbance. Record the absorbance at 2, 4, 6, 8, 10 ppm solution & then record the absorbance for saliva which is aspirated. In this also readings are obtained directly by computer using software AAS 201/203.
Statistical analysis: One way ANOVA will be used for multiple group comparisons & students ‘t’ test for two group comparisons.
7.3 Does the study require any investigations or other intervention to be conducted on patients or humans or animals? If so describe briefly. YES
7.4 Has the ethical clearance been obtained from your institution? YES 8. LIST OF REFERENCES:
1. Nair UJ., Floyd R.A, Nair J, Bussachini V, Friesen M, Bartsch H “Formation of reactive oxygen species & of 8-hydroxydeoxyguanosine in DNA in vitro with betel quid ingredients” Chem. Biol. Interact., 1987; 63:157-169.
2. Khan GJ, Mehmood R, Salah-ud-Din, Ihtesham-ul-haq “Effects of long term use of tobacco on taste receptors and salivary secretion” Journal Ayub Medical College Abottabad, 2003;15(4):37-9.
3. Urmila Nair, Helmut bartsch, J Nair “Alert for an epidemic of oral cancer due to use of the betel quid substitutes gutkha & panmasala: a review of agents causative mechanisms” Mutagenesis, 2004; 19(4):251-262.
4. Rooban T, Mishra G, Elizabeth J, Ranganathan K, Saraswathi TR “Effect of habitual betelnut chewing on resting whole mouth salivary flow rate and pH” Indian Journal of Medical Sciences 2006; 60(3):95-105.
5. Kishore B. Raja, Vinay K. Hazarey, Timothy J. Peters & Saman Warnakulasuriya “Effect of areca nut on salivary copper concentration in chronic chewers” Biometals 2007; 20:43-47.