The study was conducted in the Department of Oral Pathology and Microbiology, Yenepoya Dental College, Deralakatte, Mangalore in the year 2012.

Sixty healthy individuals with various tobacco related deleterious oral habits were included in the study, chosen from the local residents by random sampling method. Age and sex matched, 30 healthy volunteers without tobacco habits were selected for the control Group. The individuals with systemic diseases, abnormal endocrinal & immunological status and those exposed to chemicals as a result of occupation were excluded from the study.

Ethical approval to carry out this study was obtained from the Yenepoya University Ethics committee. The purpose of the study, and the procedures to be carried out, were explained to the study subjects and only those who agreed to give a written consent were included in the study.

The selection of the subjects for the study was done, based on the history of habits. After obtaining detailed history of their habits, a total of 60 individuals who were using tobacco in different forms from more than 5 years were selected as study Group. Selected individuals were categorized into the following Groups –

Group I (Betel quid chewers): Those with traditional betel quid chewing habit (tobacco along with betel leaf and slaked lime). (n =15).

Group II (Gutkha chewers): Those using commercial tobacco products (gutkha sachets) (n =15).

Group III (Smokers): Those with habit of smoking tobacco (beedi/cigarette). (n =15).

Group IV (Mixed): Those with combination of tobacco habits. (n =15).

Control Group: Individuals without any tobacco related habits. (n=30).

After recording detailed habit history, oral examination of the study subjects was carried out using diagnostic instruments (using mouth mirror & probe). If tobacco related mucosal lesions are present, it was noted. After explaining the details of the study, blood samples were collected for analysis.

A 5ml of peripheral blood sample were collected from each patient with disposable syringes under aseptic conditions through venipuncture. Serum was separated from the blood by centrifugation at 3000 rpm. The samples thus collected were stored below zero degrees until analysed for albumin and IMA.

To estimate serum albumin levels Bromocresol Green (BCG) kit was used [3]. IMA was estimated in serum using Albumin Cobalt Binding Assay [4]. It is a manual colorimetric assay to assess the ability of binding exogenous cobalt Co (II) to human albumin in serum. A 50 μL water solution of 0.1% cobalt chloride (CoCl₂.6H₂O) was added to 200 μL of serum, gently mixed and kept in dark for 10 minutes (for adequate cobalt albumin binding), then 50 μL of dithiothreitol (DTT) solution (1.5 mg/mL H₂O) was added as a colorizing agent. The reaction was quenched two minutes later by adding 1.0 mL of 0.9% NaCl. The blank was prepared similarly with the exclusion of DTT. Specimen absorbencies were analysed at 470nm using a spectrophotometer. IMA was calculated from the difference between samples measured with and without DTT. The results were reported as absorbance units (ABSU) [4].

The mean serum IMA levels in control, Group I, Group II, Group III and Group IV were 0.52547 ABSU, 0.68767 ABSU,0.47433 ABSU,0.36540 ABSU and 0.54593 ABSU respectively [Table/Fig-1]. Comparison of the result between the Groups using Tukey HSD Multiple Comparisons [Table/Fig-2] shows that the difference in IMA levels was found to be statistically significant (p≤0.05) between Group I and Group II) and highly significant (p≤ 0.001) between Group I and Group III. The mean serum Albumin levels in control, Group I, Group II, Group III and Group IV were 4.733 gm/dL, 4.7467 gm/dL, 4.7467 gm/dL, 4.9067 gm/dL and 4.5733 gm/dL respectively [Table/Fig-3]. The mean serum IMA/Albumin ratio in Group I, Group II, Group III, Group IV and Group V were 0.1123, 0.1460, 0.1007, 0.0744 and 0.1202 respectively [Table/Fig-4].

Co-relation between tobacco usage and occurrences of oral potentially malignant diseases and oral cancer is well-recognised. Heavy tobacco users have a significantly increased incidence of these lesions [5]. Tobacco products which were usually consumed by a small section of the population are today part of the modern urban and rural lifestyle and are consumed in various forms such as smoking (beedis/cigarettes/pipes), chewing (betel quid/gutkha), snuff, etc. Therefore in the present study subjects with these habits were included.

It is a well-established fact that use of tobacco in various forms is an important aetiologic factor in the development of oral cancer [6]. Possible mechanisms by which tobacco induces carcinogenesis are TSN’s and the related genotoxicity and oxidative stress induced by various chemicals leached out during smoking and chewing [7]. Two popular chewing habits among Indian population are: (i) betel quid chewing, a mixture of areca nut (Areca catechu), catechu (Acacia catechu), tobacco and slaked lime {calcium oxide (CaO) and calcium hydroxide (Ca(OH)₂)} wrapped in a betel leaf (Piper beetle); and (ii) Gutkha which is available in sachets is a mixture of areca nut, catechu, slaked lime and tobacco with added sweeteners and flavouring agents. Studies have shown that the ROS, implicated in multistage carcinogenesis, are generated in ample quantities in the oral cavity during tobacco chewing [8]. Nair et al., first demonstrated that in the alkaline pH(>9.5), aqueous extracts of areca nut and catechu will produce superoxide anion and hydrogen peroxide [7]. The presence of Ca(OH)₂ in slaked lime leads to alkaline conditions in the oral cavity, favouring ROS generation.

Studies have shown that one puff of tobacco smoke contains approximately 10¹⁵ radicals (alkyl & peroxyl types and nitric oxide), thus exposing the oral environment to a state of oxidative stress [9]. Hence, it is well-understood that, the levels of oxidative stress created in the oral cavity by different tobacco related habits vary to a greater extent and may be detectable as a localized or systemic alteration in oxidative biomarker.

IMA is a protein that is proved as a marker of oxidative stress by many studies [10,11]. Serum albumin with a decreased cobalt binding capacity as a result of ischemic events is referred to as IMA. The generation of ROS, modifies the N-terminal region of albumin to yield increased levels of IMA [4]. It is conceivable that greater the magnitude of ROS greater is the levels of IMA. Even though several LPO products and antioxidant enzyme activities have been studied to determine the degree of oxidative stress induced by tobacco [12,13], so far no attempt has been made to estimate IMA as a marker for tobacco induced oxidative stress.

Therefore, the present study was conducted to assess the extent of the oxidative stress induced by different tobacco habits by estimating serum levels of this novel oxidative stress biomarker, IMA. An attempt is also made to compare and establish a relationship between the levels of Albumin, IMA, and their ratio in serum in order to explore the possibility of one or more of the above mentioned parameters being an early marker for tobacco related oxidative stress.

The results show that serum IMA levels were increased in betel quid chewers and mixed Group compared to the controls, while, lesser in other study Groups. It has been reported that elevated IMA levels will return to normal within a few hours, with the removal of free radicals or an accelerated clearance [14]. As the raise in IMA levels is transient and it has a short half life, the IMA level which would increase within few minutes after use of tobacco and related substances may come down after clearing the free radicals. Therefore, it is reasonable to consider, the variation in the time gap between the last use of tobacco and sample collection as a reason for inconsistency in the result obtained, which was a limitation in this study. To overcome this problem standardization of the time gap between the use of tobacco and collection of samples is required to find out the exact relationship between these parameters and oxidative stress induced by tobacco use.

Slight increase in serum IMA noted in betel quid chewers could be due to the high concentration of slaked lime used in betel quid which acts to keep the active ingredients in its freebase or alkaline form, enabling it to enter the bloodstream via sublingual absorption and therefore imparting a systemic effect [15].

From the results noted in this study we suggest that IMA can be used as an early marker for tobacco related oxidative stress. Although utmost care was taken in all steps of the study process including collection, transportation, storage and analysis, there is possibility of some errors. To the best of our knowledge there are no studies reported in the literature where a comparison of serum IMA levels and their relationship to the levels of albumin between the different tobacco user Groups is done. This is a preliminary report in which we attempted to investigate whether IMA can be used as a tobacco induced oxidative stress biomarker. Therefore there is necessity for further research in this direction using more sensitive techniques and more standardized conditions to confirm the results obtained in this study and substantiate that IMA can be early marker for tobacco related oxidative stress.

To confirm the prognostic significance of this marker, further studies are required including a larger sample size in each of study Groups, subjects with long term tobacco use, and patients with tobacco related potentially malignant oral diseases and oral cancer.

Serum IMA levels in different study subjects did not show much alteration from controls. However, slight alteration was noted in betel quid chewers, which may be related to high concentration of slaked lime causing increased alkalinity, favouring better sublingual absorption of chemical constituents. Since IMA levels were increased after exposure to tobacco which is known to cause oxidative stress, it can be considered as a reliable marker for tobacco induced oxidative stress. However, further research works are required to support our observations.