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Year : 2020  |  Volume : 31  |  Issue : 4  |  Page : 531-536
Estimation and comparison of serum cotinine level among individuals with smoking and tobacco chewing habit

Department of Oral Pathology, Meenakshi Academy of Higher Education and Research, Faculty of Dentistry, Meenakshi Ammal Dental College, Maduravoyal, Chennai, India

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Date of Submission11-Jul-2018
Date of Decision16-Aug-2018
Date of Acceptance28-May-2019
Date of Web Publication16-Oct-2020


Aims and Objectives: The present study was aimed to estimate and compare tobacco exposure in smokers and chewers. The levels of cotinine, one of the constituents of tobacco were considered as indicative of tobacco exposure. Serum Cotinine levels in individuals with the habit of smoking and tobacco chewing were estimated and compared. Base line values of cotinine levels in normal subjects were established. Materials and Methods: The study groups comprised about 30 individuals with the habit of smoking (Group A); these 30 individuals with the habit of tobacco chewing (Group B), and 20 individuals who were never exposed to any form of tobacco as control group (Group C). Serum cotinine levels were estimated using a commercially available enzyme-linked immunosorbent assay in both test and control groups and the difference in the levels were compared. Results: In individuals with the habit of smoking, cotinine levels ranged between 11 ng/ml and 215 ng/ml with a mean concentration of 87.56. In tobacco chewers, the levels ranged between 11 ng/ml and 128 ng/ml with a mean concentration of 73.66. In control group, the levels ranged between 0.34 ng/ml to 2.5 ng/ml with a mean concentration of 0.93. Cotinine levels between smokers and tobacco chewers were compared and there was no statistically significant difference. Conclusion: Difference in serum cotinine levels between smokers and tobacco chewers is not significant. The fact that cotinine level is influenced by age of the individual, frequency and duration of the habit seems to be irrelevant from the results obtained from this study. Prospective studies considering all the factors and variables, with a preferable larger sample size can probably eradicate the chaos on the reliability of cotinine as a predictive biomarker for the amount of tobacco exposure.

Keywords: Nicotine, serum cotinine, smoking, tobacco chewing

How to cite this article:
Prabhakar M, Bottu K, Sivapathasundharam B. Estimation and comparison of serum cotinine level among individuals with smoking and tobacco chewing habit. Indian J Dent Res 2020;31:531-6

How to cite this URL:
Prabhakar M, Bottu K, Sivapathasundharam B. Estimation and comparison of serum cotinine level among individuals with smoking and tobacco chewing habit. Indian J Dent Res [serial online] 2020 [cited 2022 Aug 19];31:531-6. Available from:

   Introduction Top

Tobacco, both in smoking and chewing form attributes to the development of oral premalignant lesions and conditions, eventually leading to oral squamous cell carcinoma. Tobacco is smoked mainly in the form of cigarette, bidi, cigar, hookah and chutta, and also consumed as various chewable products such as pan masala, gutkha, khaini, zarda, and snuff.[1] About 70 known chemicals contribute to the carcinogenic potential of tobacco and is considered as the prime causative agent of cancer in humans. Chemicals in tobacco smoke include nicotine, polycyclic aromatic hydrocarbons (PAHs), N-Nitrosamines, and other organic and inorganic compounds. In smokeless tobacco, chemicals such as tobacco specific nitrosamines (TSNAs), benzo[a] pyrene and other polycyclic aromatic hydrocarbons (PAHs) are found to be carcinogenic.[2] In addition, Food and drug administration U.S (FDA) has established a list of 93 harmful and potentially harmful constituents in tobacco products and tobacco smoke.[3] Smokers are exposed to a toxic mixture of over 7,000 chemicals, among which, nicotine, which is a colourless, volatile liquid alkaloid is the most abundant and principle ingredient. Oral snuff and pipe tobacco contain nicotine concentrations similar to cigarette tobacco, whereas it is only about half of the nicotine concentration in cigar and chewable form of tobacco.[4],[5] All tobacco products, both combustible and non-combustible are 'packages' that are highly engineered to optimise the delivery of nicotine. Nicotine is a major tobacco component responsible for tobacco addiction. Nicotine addiction depends on the amount of nicotine delivered and the way in which it is been delivered. Nicotine augments the addiction and craving for tobacco products, by releasing variety of neurotransmitters and stimulating central nAChRs (nicotine acetyl choline receptors).[6] Nicotine can be rapidly absorbed in the lungs through cigarette smoking because of the large surface area of the alveoli and the dissolution of nicotine in pulmonary fluid.[7] Nicotine from oral products with alkaline pH can be readily but gradually absorbed through the oral mucosa and is poorly absorbed from the stomach due to the acidic environment. The bio-availability of nicotine is greater when absorbed through the lung or through the oral mucosa because nicotine reaches systemic circulation before passing through the liver ( first-pass metabolism).[7] Metabolism of nicotine is primarily carried out by the liver Cytochrome P450 2A6 (CYP2A6), UDP-glucuronosyltransferase (UGT), and flavin-containing monooxygenase (FMO).[5] Six primary metabolites of nicotine have been identified and the most important metabolite in most mammalian species is the lactam derivative, cotinine.[5] Cotinine, a major metabolite of nicotine, is a reliable biomarker for estimating both active and passive exposure to tobacco because of its stability in body fluids, long half-life (10-40 hrs), low plasma protein binding (2.6%), and dose independent disposition kinetics.[1] Due to its longer half-life (30 hrs), which is longer than nicotine (2-3 hrs), the level of cotinine would not have declined over the time course of tobacco use and thereby serve as a reliable biomarker for daily consumption, both in cigarette smokers and in those exposed to any form of tobacco.[5] It can be estimated at relatively lesser concentrations in the physiological fluid samples of humans like serum, saliva, plasma and urine.[5]

   Materials and Methods Top


All procedures performed in this study involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Ethical clearance for the study was obtained from Institutional review board, Meenakshi Ammal Dental College, on 2nd May 2014, with the clearance number MADC/IRB/2014/029.

Study design

The study included a total of 80 samples between the age group of 20-40 years with about 30 individuals having the habit of smoking (Group A), these 30 individuals with the habit of tobacco chewing (Group B), and 20 individuals who have not smoked or used any form of tobacco in their lifetime as control group (Group C). Individuals in Group A and Group B were selected as per the inclusion criteria of having the habit of smoking at least 1-2 cigarettes per day and chewing 1-2 packets of tobacco per day for a minimum of 1 year. Furthermore, individuals who have smoked at least 100 cigarettes in their lifetime were also included in Group A. Subject with any underlying systemic diseases and disorders were excluded from the study. A questionnaire specifying the type of the habit and its frequency, and duration of consumption was given to the individuals. (Annexure 1), and the informed consent for the same was obtained from all individual participants included in the study.

Estimation of serum cotinine in the samples was carried out by Solid Phase Competitive ELISA Technique. 10 μl of the samples were collected in the wells with the use of micropipette and 100 μl of the enzyme conjugate was added to each well, which is coated with anti-cotinine antibody. The wells are agitated thoroughly for 10-30 seconds and incubated for 60 minutes at room temperature (18-26°C) preferably in the dark. Cotinine in the samples competes with the cotinine enzyme conjugate HRP (Horseradish Peroxidase) for binding sites. The wells were then washed six times with 300 μl distilled water to remove the unbound cotinine and enzyme conjugate, and were allowed to dry on absorbent paper to ensure the removal of residual moisture. 100 μl of substrate reagent (TMB – Tetra methyl benzidine) was added to each well and incubated for 30 minutes at room temperature. Bound cotinine enzyme conjugate is measured by the reaction of HRP enzyme to the substrate TMB. Substrate solution reacts with the peroxide of enzyme conjugate and yields a blue colour by-product having maximum absorbance at 605 nm. The colour intensity is directly proportional to the amount of HRP activity. 100 μl of stop solution (0.16 M sulphuric acid) was then added to each well and the wells were agitated gently to mix the solution. Addition of sulphuric acid inactivates the enzyme activity and the reaction is brought to stop. A yellow colour is formed after stopping the reaction with an acidic solution with the maximum absorbance of 450 nm. Absorbance is the measure of the capacity of a substance to absorb light of specific wavelength and is dependent on the optical density of the sample in the well. The intensity of the colour is inversely proportional to the concentration of cotinine.

The microtiter wells along with the samples were then placed on the ELISA reader and the absorbance at 450 nm was read within 15 minutes after adding the stop solution. ELISA or Microplate reader works by emitting the light of specific wavelength and measuring the amount of light absorbed and reflected by the object. Cotinine concentration in the sample was then calculated by obtaining specific absorbance value for each sample in the microtiter well.

   Result Top

Serum cotinine levels in smokers, tobacco chewers and control group were compared and analyzed statistically. Mean cotinine concentration in smokers (Group A), and tobacco chewers (Group B) was calculated by descriptive statistical analysis using Kruskal Wallis test and was found to be 87.56 and 73.66 respectively [Table 1] and [Figure 1]. Cotinine levels in smokers ranged from 11 ng/ml – 215 ng/ml; 11 ng/ml – 128 ng/ml in tobacco chewers and 0.34 ng/ml - 2.5 ng/ml in control group.
Table 1: Cotinine level comparison (Kruskal Wallis Test)

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Figure 1: Comparison of cotinine levels between test and control groups

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Pairwise comparison of cotinine concentration between groups was done using Mann Whitney U test and the difference in the level of cotinine between smoking and tobacco chewing groups was found to be minimal and was not statistically significant (P value = 0.501). Difference in cotinine levels between smoking and control group as well as tobacco chewing and control group were found to be statistically significant (P value = 0.001)[Table 2].
Table 2: Pairwise comparison of cotinine levels (Mann Whitney U Test)

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The comparison of frequency and duration of the habit was done by Mann Whitney U test between smokers and tobacco chewers group and it was observed that the frequency of exposure and duration of the habit was found to be almost similar in both the groups and the difference was statistically insignificant. P value = 0.161 (duration) and 0.086 (frequency) [Table 3].
Table 3: Comparison of frequency and duration of habit in Group A (Smokers) and Group B (Smokeless Tobacco)

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The correlation of cotinine concentration with increase in age, duration and frequency in smokers group using Spearman's Correlation test revealed no significant variation [Table 4]. Similarly, in tobacco chewers, no significant difference in levels was seen between age of the individual and duration of the habit. Whereas, Correlation coefficient (r value) andP value were found to be nearly significant when frequency and cotinine levels were taken into consideration, suggesting increase in cotinine levels with increase in frequency of the habit [Table 5] and [Figure 2].
Table 4: Correlation of cotinine levels with age, frequency and duration of habit in Group A (Smokers)

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Table 5: Correlation of cotinine levels with age, frequency and duration of habit in Group B (Tobacco Chewers)

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Figure 2: Positive correlation between cotinine levels and frequency of habit in Group B (tobacco chewers)

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   Discussion Top

Nicotine is a principle and active element in tobacco and is extensively metabolized to a number of metabolites in liver.[8] It is slowly and well absorbed from smokeless tobacco and the levels remain elevated for a longer duration compared to smoking tobacco.[4] There are about six primary metabolites of nicotine identified, among which the most important in mammalian species is the lactam derivative, cotinine.[1],[4] In humans, about 70–80% of nicotine is converted to cotinine.[9] It has been widely used as a biomarker of tobacco exposure due to its longer half-life and stability in body fluids compared to nicotine.[9] The metabolism of cotinine is much slower than that of nicotine and the clearance averages about 45 ml/min. In the present study, exposure of tobacco was estimated by measuring cotinine levels in individuals with smoking and tobacco chewing habit and serum was considered as an acceptable biological fluid due to the discrepancies that emerged in estimating cotinine levels in saliva and plasma.[9] Although there are various methods for estimating cotinine levels like High Performance Liquid chromatography and Gas chromatography, the Enzyme Linked Immunosorbent Assay (ELISA) stands as a precise method for cotinine estimation as the values are estimated using microtiter wells with enzyme substrate.[5],[6],[7],[8],[9]

The serum cotinine levels were estimated in these 30 individuals with the habit of smoking and tobacco chewing as per the inclusion and exclusion criteria. The selection criteria of the subjects and questionnaire incorporated in this study were based on the guidelines given by Global Tobacco Surveillance System (GTSS) in 2011[10] and 100 cigarette criterion for tobacco surveys in 2009.[11]

Significant elevation in serum cotinine levels were observed in smokers (87.56 ng/ml) and tobacco chewers (73.66 ng/ml) compared to the control group (0.093 ng/ml). Serum cotinine levels were higher in smokers compared to chewers (both groups with almost equal exposure) but the difference was not statistically significant. Factors such as pH, nicotine concentration of the product, method of consumption, rate of nicotine absorption and elimination, surface area exposed, size of the tobacco cuttings, chemical coatings are the various reasons that could be attributed to the mild increase in cotinine levels in smokers compared to chewers. Moreover, since considerable amounts of nicotine are swallowed, its bioavailability from chewing tobacco is lower than that with smoking which leads to its unavailability in the bloodstream beyond the liver where it is metabolized to cotinine.[12]

Although levels of cotinine were found to be higher as a whole in smokers, variation of cotinine levels among different brands of cigarettes need to be considered. It was documented in the literature that the difference in nicotine concentration between unfiltered (15.6 mg/gm) and filtered cigarettes (14.5 mg/gm) was very minimal and insignificant, whereas nicotine concentration from bidis averaged 26.9 mg/gm. It was also elucidated that nicotine concentration of different brands of pan masala was found to be an average of 3.4 mg/gm.[13] Since the present study was localized and institutional based, most of the subjects were from middle socio economic status and they reported the use of only cigarettes and not bidis along with the chewing group revealing the use of a common brand of pan masala. Based on the previous findings from the literature, there are possibilities for minimal variation of cotinine levels among groups using different brands of cigarette and chewing tobacco. Specific studies of cotinine on individual brand of tobacco products in future could be useful in determining the values, which was a drawback in this study.

Individual correlation of cotinine levels with age, frequency and duration of the habit showed no significance in smokers. Cotinine levels were not influenced with age as the metabolism of cotinine is unaffected with age.[14] Increase in frequency and duration of the habit normally influences cotinine levels. No such finding was noted in this study and the reason could be attributed to under reporting by the patients, the quantity of the cigarette smoked and the way the cigarette had been smoked; whether inhaled directly into the lungs or kept for a moment inside the mouth or frenched. Similarly, there was no correlation between cotinine levels with age and duration of the habit in tobacco chewers whereas, significant correlation was noticed between cotinine levels and frequency of the habit alone. This could be again due to under reporting by the patients, variation in type, constituents and concentration of nicotine in chewing tobacco. Though, many reported literature states that cotinine level has been influenced by age, frequency and duration of the habit, results from this study found a disassociation between cotinine levels and these variables. A higher sample size will be helpful in substantiating this cause. Gender based variations in cotinine levels were also reported in recent studies with men having higher concentration of cotinine compared to women and the reason would be attributed towards the factors such as height, weight and nicotine metabolism.[15] The present study comprises only male subjects and future studies with equal number of population involving both genders will provide clarity in cotinine level variation among different genders.

The selection of an optimal cotinine cut off value for distinguishing true smokers from non-smokers shows a lack of standardization among studies. Based on the previous studies in the literature, the average cotinine cut off value in saliva ranges between 10–25 ng/mL, 10–20 ng/mL in serum and 50–200 ng/mL in urine.[16] Recently, a cut-off value of 3 ng/ml is recommended to discriminate smokers from non-smokers and the present study strongly supports the finding of all individuals in test group with cotinine levels above 3 ng/ml.[16]

This level of reduction in cut off value is due to the decreased second hand smoke exposure in the environment compared to the previous times where the cut off values seems to be higher (>10 ng/mL) due to heavy second hand smoke exposure.[17] Dietary nicotine from sources such as black tea, tomatoes and potatoes also attribute to the minimum cotinine level of 3 ng/ml in non-tobacco users.[18] Cotinine cut-off points may also range between 1-6 ng/ml according to race and ethnicity as mentioned by Neal L Benowitz in 2008.[17]

In the present study, the average serum cotinine levels in smokers ranged from 11 ng/ml to 215 ng/ml with the mean concentration of 87.56 ng/ml which is lesser than the studies conducted earlier by O'Connor et al. in 2006 (230 ng/ml)[19] and Neal L Benowitz et al. in 2008 (122 ng/ml).[17] The possible reason for this discrepancy could be restriction in age between 20-40 years, type of tobacco smoked and also limited sample size. Similarly, concentration of cotinine in chewer's group ranged from 11 ng/ml to 128 ng/ml with a mean value of 73.66 ng/ml which is in near accordance with the study conducted by David Siegel in 1992 with 82.1 ng/ml as mean concentration.[20]

Individual studies on estimation of cotinine levels in smokers, and chewers have been done but comparative analysis of these levels between these groups are not reported in literature and the present study is the first of its kind. Nicotine, even though is a main ingredient in both smoking and chewing form of tobacco, chewing tobacco have only about half the nicotine concentration (2.6-4.1 mg) of cigarette tobacco (10-14 mg).[5] On contrary, an average of about 4.5 mg of nicotine is absorbed systemically when chewing 4 gms (1 packet) of chewing tobacco and is about 1–1.5 mg while smoking one cigarette.[5] These findings reflect the fact that the effects caused by tobacco, either in its smoking or chewing form are alike except for the direct effect of smoking on lungs leading to lung carcinogenesis.

This study was an attempt to delineate the reasons for differences in cotinine concentrations between smoking and chewing form of tobacco. Findings from this study, reflects the fact that cotinine is a marker for tobacco exposure but its precision on the severity of exposure and pathological correlation seems to be questionable.

   Conclusion Top

In the present study serum cotinine levels in smokers and tobacco chewers have a wide range of distribution. Moreover, the fact that cotinine level is influenced by age of the individual, frequency and duration of the habit seems to be irrelevant from the results obtained from this study. Variations in cotinine levels in the present trial and other previously reported trials in the literature concludes that the type of tobacco product and the method of consumption influence the values. Prospective studies considering all the factors and variables, with a preferable larger sample size can probably eradicate the chaos on the reliability of cotinine as a predictive biomarker for the amount of tobacco exposure, thereby making the professionals to alert the patients regarding the hazards and risks involved in continuing tobacco habits.


I would like to acknowledge Mrs. Sharon NS., M. Sc., Ph.D., Assistant Professor, Department of Biochemistry, Meenakshi Ammal Dental College, Maduravoyal, Chennai.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

   References Top

Digambar B, Rajan U, Sidharath M. Urinary levels of nicotine and cotinine in tobacco users. Indian J Med Res 2003;118:129-33.  Back to cited text no. 1
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Janne H, Pleyton J, Benowitz NL. Metabolism and disposition kinetics of nicotine. Pharmacol Rev 2005;57:79-115.  Back to cited text no. 4
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Benowitz NL. Pharmacology of nicotine: Addiction, smoking-induced disease, and therapeutics. Annu Rev Pharmacol Toxicol 2009;49:57-71.  Back to cited text no. 6
Nicotine addiction: Past and Present. How Tobacco Smoke Causes Disease: The Biology and Behavioural Basis for Smoking-Attributable Disease; Centers for Disease Control and Prevention (US); 2010.  Back to cited text no. 7
Benowitz NL. Nicotine and smokeless tobacco. Cancer J Clin 1988;38:244-7.  Back to cited text no. 8
Van Vunakis H, Tashkin DP, Rigas B, Simmons M, Gjika HB, Clark VA. Relative sensitivity and specificity of salivary and serum cotinine in identifying tobacco-smoking status of self-reported nonsmokers and smokers of tobacco and/or marijuana. Arch Environ Mental Health 1989;44:53-8.  Back to cited text no. 9
Global Adult Tobacco Survey Collaborative Group. Tobacco Questions for Surveys. 2nd ed.. Centers for Disease Control and Prevention; 2011.  Back to cited text no. 10
Bondy SJ, Victor JC, Diemert LM. Origin and use of the 100 cigarette criterion in tobacco surveys. Tob Control 2009;18:317-23.  Back to cited text no. 11
Sujatha SR, Radha P. Estimation of serum nicotine levels among tobacco users. Res J Pharm Biol Chem Sci 2013;4:1239-46.  Back to cited text no. 12
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Nosratzehi T, Arbabi-Kalati F, Alijani E, Tajdari H. Comparison of cotinine salivary levels in hookah smokers, passive smokers, and non-smokers. Addict Health 2015;7:3-4.  Back to cited text no. 14
Chen A, Krebs NM, Zhu J, Sun D, Stennett A, Muscat JE. Sex/gender differences in cotinine levels among daily smokers in the Pennsylvania adult smoking study. J Womens Health (Larchmt) 2017; 26:1222-30.  Back to cited text no. 15
Kim S. Overview of cotinine cutoff values for smoking status classification. Int J Environ Res Public Health 2016;13. pii: E1236.  Back to cited text no. 16
Benowitz NL, Bernert JT, Caraballo RS, Holiday DB, Wang J. Optimal serum cotinine levels for distinguishing cigarette smokers and non-smokers within different racial/ethnic groups in the united states between 1999 and 2004. Am J Epidemiol 2009;169:236-48.  Back to cited text no. 17
Davis RA, Stiles MF, deBethizy JD, Reynolds JH. Dietary nicotine: A source of urinary cotinine. Food Chem Toxicol 1991;29:821-7.  Back to cited text no. 18
O'Connor RJ, Giovino GA, Kozlowski LT, Shiffman S, Hyland A, Bernert JT, et al. Changes in nicotine intake and cigarette use over time in two nationally representative cross-sectional samples of smokers. Am J Epidemiol 2006;164:750-9.  Back to cited text no. 19
David S, Benowitz NL, Emster VL, Grady DG, Hauck WW. Smokeless tobacco, cardiovascular risk factors, and nicotine and cotinine levels in professional baseball players. Am J Public Health 1992;82:417-21.  Back to cited text no. 20

Correspondence Address:
Dr. Manoj Prabhakar
Department of Oral Pathology, Meenakshi Academy of Higher Education and Research, Faculty of Dentistry, Meenakshi Ammal Dental College, Alapakkam Main Road, Maduravoyal - 600 095, Chennai
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijdr.IJDR_558_18

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  [Figure 1], [Figure 2]

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]


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