1 Senior Lecturer, Department of Public Health Dentistry, Rama Dental College Hospital and Research Centre, Kanpur, Uttar Pradesh, India.
Find articles by Mitali Raja2 Senior Lecturer, Department of Pedodontics and Preventive Dentistry, Jaipur Dental College, Jaipur, India.
Find articles by Aarti Garg3 Assistant Professor, Department of Periodontology and Public Health Dentistry, Dr ZA Dental College, AMU, Aligarh, India.
Find articles by Pramod Yadav4 Senior Lecturer, Department of Public Health Dentistry, Kalinga Institute of Dental Sciences, KIIT University, Bhubaneshwar, India.
Find articles by Kunal Jha5 Senior Lecturer, Department of Public Health Dentistry, College of Dental Science and Hospital, Rau, Indore, Madhya Pradesh, India.
Find articles by Sahil Handa1 Senior Lecturer, Department of Public Health Dentistry, Rama Dental College Hospital and Research Centre, Kanpur, Uttar Pradesh, India.
2 Senior Lecturer, Department of Pedodontics and Preventive Dentistry, Jaipur Dental College, Jaipur, India.
3 Assistant Professor, Department of Periodontology and Public Health Dentistry, Dr ZA Dental College, AMU, Aligarh, India.
4 Senior Lecturer, Department of Public Health Dentistry, Kalinga Institute of Dental Sciences, KIIT University, Bhubaneshwar, India.
5 Senior Lecturer, Department of Public Health Dentistry, College of Dental Science and Hospital, Rau, Indore, Madhya Pradesh, India.
Corresponding author.NAME, ADDRESS, E-MAIL ID OF THE CORRESPONDING AUTHOR: Dr. Mitali Raja, M.I.G 24 Awas Vikas Scheme No.1 Kailash Vihar Kalyanpur Kanpur-208017, India. E-mail: moc.liamg@ajarilatimrd
Received 2015 Oct 16; Revisions requested 2015 Dec 2; Accepted 2016 Jan 17. Copyright © 2016 Journal of Clinical and Diagnostic ResearchThe greatest disease-producing product known to man is tobacco. It is a cause of many oral diseases and adverse oral conditions. In India, tobacco is available in smokeless and smoking form. Tobacco contains nicotine which metabolises to form a toxic alkaloid i.e. cotinine. It stimulates autonomic ganglia and central nervous system. Cotinine is the best indicator of tobacco smoke exposure. Various methods are used to measure cotinine level in blood, saliva and urine such as high performance liquid chromatography, colorimetric assay, gas chromatography, NicAlert saliva test, etc.
Thus such wide range of methods for cotinine detection in tobacco users requires a detailed discussion regarding their utility. This review will help readers to compare various methods for cotinine detection and enable them to make scientifically informative decision.
Keywords: Nicotine, Tobacco products, Toxic alkaloidTobacco is a harmful, addictive chemical responsible for many oral diseases and adverse oral conditions [1]. It can be consumed in two forms, smoking and non smoking [2]. Tobacco consumption in any form is responsible for diseases like oral cancer, adult periodontal diseases, and congenital defects such as cleft lip and palate in children whose mother consumed tobacco during pregnancy [1].
Virtually all tobacco products contain nicotine in substantial concentration [3]. Cotinine, a major metabolite of nicotine, can be easily detected in various body fluids like blood, urine and saliva [4]. It is most commonly used marker to distinguish between tobacco users and non users because of its greater sensitivity and specificity than other biochemical tests [5,6].
There is a high correlation between blood and saliva cotinine concentrations. A widely used biomarker is urine cotinine level since cotinine concentrations are four to six times higher in urine than that in blood or saliva [3,6]. This makes quantitative methods (like gas chromatography/mass spectrometry or high performance liquid chromatography, colorimetric assays and immunoassays) which measure urine cotinine more valid and reliable. The disadvantages of higher cost and time consumption have been addressed by recently developed semi quantitative methods [5,7].
Cotinine: Every individual is exposed to nicotine either directly or indirectly [8].
Nicotine’s major metabolite is cotinine which is oxidized in the liver by CYP2A6 (Cytochrome P450, Family 2, Subfamily A, Polypeptide 6) and is distributed in various body fluids including the blood, saliva and urine [9]. Cotinine is the main biomarker which is used to distinguish tobacco users from non users and reflects the extent of exposure [10]. Longer half-life of cotinine makes it a useful short-term marker of nicotine exposure. During tobacco smoking its level fluctuates relatively to lesser extent than other tobacco products [11].
Correlation exists between urinary cotinine and daily tobacco use. A value of urine cotinine ranges from 20-550 ng/ml. A change in the cut-off value is directly proportional to sensitivity of detection [6].
Pharmacokinetics and Pharmacodynamics Properties: In 24 hour urine sample analysis it is found that 5% of nicotine dose is excreted as it is while 10% in the form of cotinine and 35% as hydroxy cotinine. It is stable in body fluids, low plasma protein binding, has a long half-life 15-40 hour, it is directly proportional to the quantity of nicotine absorbed and dose independent disposition kinetics [8]. Thus cotinine is useful marker as it helps in estimation of exposure to active as well as passive smoke [8].
There are certain limitations in measuring cotinine level in tobacco users: it requires expensive laboratory instrumentation requires relatively large sample volume; time to store, ship and analyse; and well-trained personnel, the high cost of analysing the samples, the cost of collecting, handling and arranging for shipment of specimens [5].
Cotinine in Saliva: The small molecules, minimal protein binding in blood and water solubility increases concentration of cotinine in saliva by 15% to 40%. Thus cotinine measurement in saliva becomes a non invasive, easy and well tolerated collection procedure when multiple samples are required over a limited period [3].
Various studies were conducted by authors to detect cotinine in saliva to distinguish tobacco users from non users. [ Table/Fig-1 ] shows studies conducted on salivary cotinine.
Studies conducted by authors on salivary cotinine.
| Authors | Year | Findings and conclusion |
|---|---|---|
| Montalto NJ, Wells WO | 2007 | Saliva NicAlertR assay found to be highly specific, sensitive and valid method for verifying smoking status [12]. |
| Nuca C, Amariei C, Badea V, Zaharia A, Bucur L, Arendt C | 2012 | By using NicAlert™ Saliva tests it was found that 44.06% were active smokers, 16.43% were non-smokers and 39.50% were passive smokers [11]. |
| Kulza M, Wozniak A, Przybylowska SM, Czarnywojtek A, Flisykowska KA, Florek E | 2012 | The concentration of salivary cotinine was detected using high performance liquid chromatography with diode array detection. Mean concentrations of cotinine was found to be 240.9 ng/ml of saliva determination of saliva cotinine is useful in the assessment of tobacco [4]. |
| Asha V, Dhanya M | 2015 | Immunochromatographic assay using NicAlert saliva test was used to detect salivary cotinine in tobacco chewer. Levels found were as follows: 0 for 2 subjects (2.67%), 1 for 16 subjects (21.33%), 2 for 16 subjects (21.33%), 3 for 18 subjects (24.00%), 4 for 21 subjects (28.00%) and 5 for 2 subjects (2.67%). For tobacco chewers this method is convenient and useful for studying the nicotine dependence [13]. |
Cotinine in blood: Cotinine and 3-HC (3- hydroxycotine), nicotine can all be measured in blood but cotinine has longer half-life compared to others therefore it is considered as a preferred [3]. [ Table/Fig-2 ] shows studies conducted on blood cotinine.
Studies conducted by authors on blood cotinine.
| Hsieh SJ, Ware LB, Eisner MD, Lisa Yu, Jacob P, Havel C, Goniewicz ML, Matthay MA, Benowitz NL, Calfee CS | 2011 | More active smokers were identified by using the combination of serum cotinine and urine 4-(methylnitrosamino)-1-(3pyridyl)-1-butanol than did smoking history, secondhand smoke exposure prevalence was high in a critically ill population [14]. |
| Xu X, Su Y, Fan ZH | 2014. | Micellar electro kinetic chromatography with enrichment techniques was used to measure cotinine in serum samples of mice. It was found that there was an association between the concentration in serum cotinine and tobacco smoke-induced emphysema in mice and in future this method can be used to detect cotinine [15]. |
Advantages: Long half life, no requirement for adjusting hydration difference among individuals, uniform matrix measurement though it has a lower sensitivity compared to urine cotinine [3].
Cotinine in urine: The most widely used biomarker in tobacco users is urine cotinine.
Advantages: High sensitivity compared to blood cotinine, collecton is non- invasive, relatively acute exposure [3]. [ Table/Fig-3 ] shows studies conducted on urine cotinine.
Studies conducted on urinary cotinine.
| Balhara YPS, Jain R | 2013 | Value of urinary cut off was kept greater than or equal to 2.47 ng/ml to detect the highest sensitivity and specificity of 100% for smoking using ELISA kits of Calbiotech. Inc., USA. Receiver operating characteristic (ROC) curve. Authors concluded that urinary cotinine cut off value was used to distinguish tobacco users and nonusers [6]. |
| Kotandeniya D, Carmella SG, Ming X, Murphy SE, Hecht SS | 2015 | Liquid chromatography-electrospray ionization-tandem mass spectrometry method was used for detection of total cotinine (cotinine + glucuronide) and total NNAL (NNAL + glucuronide). Thus authors concluded that this method quantifies naturally occurring (13C) cotinine [16]. |
Very few studies have been conducted on COT one step cotinine test device a lateral flow chromatographic immunoassay which is used to detect human urine cotinine at a cut off value of 200ng/ml. There are two lines in the device one is test line and other is control line. Test line consist of mouse monoclonal anti cotinine antibody coupled particles and cotinine protein conjugates while goat antibody is present on control line. Urine sample must be stored at least 2-8 degree Celsius for 48 hours before assay [17], If two lines appear in control and test region the result is said to be negative which means that patient’s urine cotinine concentration is below cut- off value of 200ng/ml, if only one coloured line appears in control and no line in test line region the result is said to be positive and if no line appears means the result is invalid that may be due to incorrect procedure [17]. [ Table/Fig-4 ] shows different diagnostic method for assessing tobacco/nicotine.
Different diagnostic method for assessing tobacco/nicotine dependence.
| Cotinine | Fagestrom Test for Nicotine Dependence (FTND) | Tobacco Dependence Screener (THS) | Cigarette Dependence Scale. (CDS) | Nicotine Dependence Syndrome Scale (NDSS) |
|---|---|---|---|---|
| The major metabolite of nicotine is cotinine, has low plasma protein binding, long half-life and is a good marker for assessment of nicotine dependence in saliva, blood, urine in tobacco users [10,11]. | Fagerström Test for Nicotine Dependence (FTND) is a six-item questionnaire that has scoring from 0-10 which is classified according to patient nicotine addiction as very low (0–2), low (3–4), medium (5), high (6–7), or very high (8–10) [18,19]. | Tobacco dependence screener was developed on 10 symptoms its score was correlated with the years of smoking, the number of cigarettes smoked per day, breath carbon monoxide levels [20]. | CDS is a valid and reliable method of cigarette. Dependence criteria are: withdrawal symptom, compulsion to smoke, loss of control, allocation of time to smoking, neglect of other activities to smoke, and persistence of use despite harm, except tolerance [21]. | NDSS is 19 item questionnaire which includes five factors drive (craving and withdrawal to smoke), priority, tolerance continuity and stereotypy [22]. |
A variety of techniques has been used to measure cotinine including gas chromatography, high performance liquid chromatography and colorimetric assays Immunoassays, NicAlert™ Saliva tests etc. Cotinine can be widely used in future compared to other diagnostic tools because of its higher sensitivity, specificity, long half life as well as it is the best indicator for distinguishing the tobacco users from non users.
[1] Petersen PK. Tobacco and oral health – the role of the World Health Organization. Oral Health Prev Dent. 2003; 1 :309–15. [PubMed] [Google Scholar]
[2] Digambar B, Rajan U, Sidharath M. Urinary levels of nicotine & cotinine in tobacco users. Indian J Med Res. 2003; 118 :129–33. [PubMed] [Google Scholar]
[3] Tang EA, Delaimy WKA, Ashley DL, Benowitz N, Bernert JT, Kim S, et al. Assessing second hand smoke using biological markers. Tobacco control. 2013; 22 (3):164–71. [PMC free article] [PubMed] [Google Scholar]
[4] Kulza M, Wozniak A, Przybylowska SM, Czarnywojtek A, Kurhanska-Flisykowska A, Florek E. Saliva cotinine determination using high-performance liquid chromatography with diode - array detection. Przegl Lek. 2012; 69 (10):837–40. [PubMed] [Google Scholar]
[5] Parker DR, Thomas M, Richard W, Jeff W, Upegui DI, Heimdal J. The accuracy of self-reported smoking status assessed by cotinine test strips. Nicotine & Tobacco Research. 2002; 4 :305–09. [PubMed] [Google Scholar]
[6] Balhara YPS, Jain R. A receiver operated curve-based evaluation of change in sensitivity and specificity of cotinine urinalysis for detecting active tobacco use. Journal of Cancer Research and Therapeutics. 2013; 9 (1):84–89. [PubMed] [Google Scholar]
[7] Acosta M, Buchhalter A, Breland A, Hamilton D, Eissenberg T. Urine cotinine as an index of smoking status in smokers during 96-hr abstinence: comparison between gas chromatography/mass spectrometry and immunoassay test strips. Nicotine Tob Res. 2004; 6 (4):615–20. [PubMed] [Google Scholar]
[8] Peach H, Ellard GA, Jenner PJ, Morris RW. A simple near patient test for nicotine and its metabolites in urine to assess smoking habit. Clinica Chimica Acta. 1996; 256 :135–46. [PubMed] [Google Scholar]
[9] Jung S, Lee IS, Kim SB, Moon CS, Jung JY, Kang YA, et al. Urine cotinine for assessing tobacco smoke exposure in korean: analysis of the korea national health and nutrition examination survey (KNHANES) Tuberculosis and Respiratory Diseases. 2012; 73 (4):210–18. [PMC free article] [PubMed] [Google Scholar]
[10] Benowitz NL, Bernert JT, Caraballo RS, Holiday DB, Wang J. Optimal serum cotinine levels for distinguishing cigarette smokers and nonsmokers within different racial/ethnic groups in the united states between 1999 and 2004. Am J Epidemiol. 2009; 169 :236–48. [PubMed] [Google Scholar]
[11] Nuca C, Amariei C, Badea V, Zaharia A, Bucur L, Arendt C. Salivary cotinine - biomarker of tobacco consumption in the assessment of passive smoking prevalence. FARMACIA. 2012; 60 (5):662–74. [Google Scholar]
[12] Montalto NJ, Wells WO. Validation of Self-reported smoking status using saliva cotinine: a rapid semiquantitative dipstick method. Cancer Epidemiol Biomarkers Prev. 2007; 16 (9):1858–62. [PubMed] [Google Scholar]
[13] Asha V, Dhanya M. Immunochromatographic assessment of salivary cotinine and its correlation with nicotine dependence in tobacco chewers. Journal of Cancer Prevention. 2015; 20 (2):159–63. [PMC free article] [PubMed] [Google Scholar]
[14] Hsieh SJ, Ware LB, Eisner MD, Lisa Yu, Jacob P, Havel C, et al. Biomarkers will be important for future studies investigating the relationship between active smoking and secondhand smoke exposure and critical illness. Crit Care Med. 2011; 39 (1):40–45. [PMC free article] [PubMed] [Google Scholar]
[15] Xu X, Su Y, Fan ZH. Cotinine Concentration in serum correlates with tobacco smoke-induced emphysema in mice. Scientific reports. 2014; 4 :1–5. [PMC free article] [PubMed] [Google Scholar]
[16] Kotandeniya D, Carmella SG, Ming X, Murphy SE, Hecht SS. Combined analysis of the tobacco metabolites cotinine and 4(methylnitrosamino)-1-(3-pyridyl)-1butanol in human urine. Anal Chem. 2015; 87 (3):1514–17. [PMC free article] [PubMed] [Google Scholar]
[17] COT one step cotinine test device pacakage insert [Internet] [cited 2015 Oct 10]. Available from: http://www.mossmanassociates.com/SmokeCheck%20Cassette%20Instructions.pdf
[18] Heatherton TF, Kozlowski LT, Frecker RC, Fagerstrom KO. The fagerstrom test for nicotine dependence: a revision of the fagerstrom tolerance questionnaire. Br J Addict. 1991; 86 :1119–27. [PubMed] [Google Scholar]
[19] Schnoll RA, Rothman RL, Wielt DB. A randomized pilot study of cognitive-behavioral therapy versus basic health education for smoking cessation. Among Cancer Patients. Ann Behav Med. 2005; 30 (1):1–11. [PubMed] [Google Scholar]
[20] Kawakami N, Takatsuka N, Inaba S, Shimizu H. Development of a screening questionnaire for tobacco/nicotine dependence according to ICD-10, DSM-III-R, AND DSM-IV. Addictive Behaviors. 1999; 24 (2):155–66. [PubMed] [Google Scholar]
[21] Etter JF, Houezec JL, Perneger TV. A Self-administered questionnaire to measure dependence on cigarettes: The Cigarette Dependence Scale. Neuropsychopharmacology. 2003; 28 :359–70. [PubMed] [Google Scholar]
[22] Shiffman S, Waters AJ, Hickcox M. The nicotine dependence syndrome scale: a multidimensional measure of nicotine dependence. Nicotine and Tobacco Research. 2004; 6 (2):327–48. [PubMed] [Google Scholar]
Articles from Journal of Clinical and Diagnostic Research : JCDR are provided here courtesy of JCDR Research & Publications Private Limited