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Status of Thiocyanate Levels in the Serum and Saliva of Non-Smokers, Ex-Smokers and Smokers

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Status of Thiocyanate Levels in the Serum and Saliva of Non-Smokers, Ex-Smokers and Smokers Status of thiocyanate levels in the serum and saliva of non-smokers, ex-smokers and smokers Ananya Madiyal1, Vidya Ajila1, Subhas G Babu1, Shruthi Hegde1, Suchetha Kumari2, Medhini Madi3, Sonika Achalli1, Priyadharshini Alva4, Harshini Ullal2 1. A. B. Shetty Memorial Institute of Dental Sciences, Nitte deemed to be University, Oral. Medicine and Radiology. 2. K. S. Hegde Medical Academy, Nitte deemed to be University, Biochemistry. 3. Manipal College of Dental sciences, Manipal, Oral Medicine and Radiology. 4. Oral Medicine and Radiology Specialist. Emails: 1. Ananya Madiyal- Email id: [email protected], 2. Vidya Ajila-Email id: [email protected], 3. Subhas G Ba- bu-Email id: [email protected], 4. Shruthi Hegde-Email id: [email protected], 5. Sucheta Kumari-Email id: [email protected], 6. Medhini Madi-Email id: [email protected], 7. Sonika Achalli-Email id: sonikachalli@gmail. com, 8. Priyadharshini Alva-Email id: [email protected], 9. Harshini Ullal-Email id: [email protected] Abstract Background: Use of tobacco is often implicated in the development of oral diseases. Questionable accuracy of the traditional questionnaires to assess cigarette exposure necessitates the use of biomarkers like thiocyanate which provide a definitive quan- titative measure. Objective: To assess the rise in the level of thiocyanate for measurement of smoking behaviour in adults. Materials and methods: Serum and salivary thiocyanate levels were estimated in 20 non-smokers, 20 ex-smokers and 40 smok- ers. Smokers were divided into two groups based on the presence or absence of oral mucosal lesions. Results: The mean serum and salivary thiocyanate levels were increased significantly in smokers when compared to non-smok- ers and ex-smokers. The levels were not significantly different between ex-smokers and non-smokers and between smokers with tobacco related oral mucosal lesions and those without. Statistically significant correlation was seen between the serum and salivary levels of thiocyanate. Conclusion: This study highlights the high level of thiocyanate in the serum and saliva of smokers when compared to non-smok- ers and ex-smokers. Significant increase in thiocyanate level was also seen in saliva. Hence it can be stated that saliva can be used as a reliable, non-invasive tool to assess smoking behaviour in the population and its changes over time. Keywords: Thiocyanate, Tobacco, Smoking, Cancer, Pre-cancer. DOI: https://dx.doi.org/10.4314/ahs.v18i3.31 Cite as: Madiyal A, Ajila V, Babu SG, Hegde S, Kumari S, Madi M, Achalli S, Alva P, Ullal H. Status of thiocyanate levels in the serum and saliva of non-smokers, ex-smokers and smokers. Afri Health Sci. 2018;18(3): 727-736. https://dx.doi.org/10.4314/ahs.v18i3.31 Introduction Corresponding author: Tobacco use is one of the greatest challenges that is Ananya Madiyal, faced by the global healthcare today. Approximately 33% Department of Oral Medicine and Radiology of the adult population consumes tobacco in one form A. B Shetty Memorial Institute of Dental Sciences or the other.1 Risk factors such as use of tobacco prod- Nitte deemed to be University ucts, alcohol consumption, betel nut chewing and human Deralakatte, Mangalore-575018 papilloma virus have been linked to the development of Contact number: +919538840189 lesions like smoker’s palate, leukoplakia, erythroplakia, Email: [email protected] oral submucous fibrosis and erosive lichen planus. These African © 2018 Madiyal et al. Licensee African Health Sciences. This is an Open Access article distributed under the terms of the Creative commons Attribution Health Sciences License (https://creativecommons.org/licenses/BY/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 727 African Health Sciences Vol 18 Issue 3, September, 2018 potentially malignant disorders along with oral carcino- high when the plasma cyanide levels are high.9 Saliva also mas cause significant morbidity and mortality in patients. shows high levels of thiocyanate and the physiologic role Oral cancer is one of the leading causes of death in India of thiocyanate in saliva maybe due to hypothiocyanate with incidence rates standing at 53842 cases in males and (HOSCN) which produces an antibacterial effect by the 23161 cases in females.2,3 As there exists a long time lapse action of salivary peroxides.8 between the use of tobacco and the appearance of dis- With the current study we aimed to measure the level ease, the effect of today’s globalization of tobacco mar- of thiocyanate in the serum and saliva of non-smokers, ket will be felt for decades to come.1 ex-smokers and smokers to determine its efficacy as a Tobacco diminishes quality of life, beginning with things biochemical marker of smoking behaviour. as modest as halitosis to as intricate as developmental abnormalities in children whose mothers were smok- Materials and methods ers during pregnancy. Tobacco is known to decrease the The present study was an observational study conducted body’s immune response and is a well-known cause for on subjects reporting to the Department of Oral Medi- the development of adult periodontitis, oral cancer and cine and Radiology at a Dental Science Institute in South oral cancer recurrence.1 The use of tobacco has been India. The sample population consisted of semi-urban positively linked to the development of leukoplakia and and rural subjects. oral submucous fibrosis which have a potential for malig- Sample size was determined based on the pilot study per- nant transformation.3 It is seen that there exists a clear formed. A sample of 17.5 was determined as the mini- dose-response relationship between smoking and oral mum in each group to estimate and to calculate for the cancer. Some studies show that, after a decade of quit- statistical analysis. We rounded 17.5 into 20 so as to re- ting, the risk of development of cancer was the same as duce errors. Therefore, the study sample included a to- that of non-smokers while other studies found that the tal of 80 subjects who were divided into the following risk in ex-smokers was marginally increased.4,5 groups: Control Group (C): 20 subjects who were non-smokers Traditionally, measurement of exposure to cigarettes has of tobacco products. been done through questionnaires. The exactness of such Group I (G1): 20 subjects who were ex-smokers of to- a method is limited due to cognizant false reporting as bacco products. well as insufficient recognition of the true degree of ex- Group II (G2): 40 subjects who were smokers of tobac- posure.6 To overcome this hurdle, various biochemical co products. markers of cigarette smoke like carbon monoxide, nic- Sub-Group IIa (G2a): 20 subjects who were smokers of otine, cotinine and thiocyanate have been used as mea- tobacco products without oral mucosal lesions associated sures of smoking behaviour in the recent years.6 The long with any tobacco related disorders. biologic half-life of thiocyanate (10-14 days) makes it an Sub-Group IIb (G2b): 20 subjects who were smokers ideal substrate to determine habitual smoking behaviour.7 of tobacco products with oral mucosal lesions associated The combustion of nitrogen containing compounds with tobacco related disorders. found in cigarettes leads to inhalation of hydrogen cy- Strict inclusion criteria were followed for the control anide (HCN) gas which is ultimately assimilated into the group and the study groups. Healthy non-smoker sub- body as cyanide (CN-). This inhibits the activity of cyto- jects in the age group of 18-60 years, without any histo- chrome oxidase.8 Cyanide is gradually released from its ry of oral and systemic diseases were taken as controls. bond with ferric ion and is metabolized to thiocyanate Group I consisted of subjects who were in the age group after accepting a sulfur atom from a sulfur donor such as of 18-60 years who used to smoke one or more than one thiosulfate. This reaction is catalyzed by thiosulfate trans- pack of hand rolled or manufactured tobacco products ferase.9 The thiocyanate level in plasma is considered to per day but had quit since the last 1 year. Group II con- be a good indicator of exposure to cyano-containing or- sisted of subjects who were in the same age group who ganic substances since thiocyanate levels are inexorably regularly smoked one or more than one pack of hand African Health Sciences Vol 18 Issue 3, September, 2018 728 rolled or manufactured tobacco products per day since • p-phenylenediamine the last 1 year or more. This group was subdivided into • 2 g/liter of 0.5 molar HCl GIIa consisting of subjects who did not present with oral • Pyridine reagent mucosal lesions associated with any tobacco related dis- • Pyridine-concentrated HCl-de-ionized water, 6:1:4 by orders. GIIb consisted of subjects who presented with volume oral mucosal lesions associated with tobacco related dis- • Pyridine-p-phenylenediamine reagent orders including, but not restricted to, leukoplakia, eryth- • Pyridine reagent-p-phenylenediamine reagent, 3:1 by roplakia, stomatitis nicotina, smoker’s melanosis and oral volume squamous cell carcinoma. Subjects with a history of any long term systemic diseas- Procedure es or acute infections, subjects who were on any med- Estimation of thiocyanate in serum or saliva: ications like erythromycin, nitroprusside, chlorambucil, Serum (1 ml) was separated and de-proteinized by adding ifosfamide and melphalan, and subjects with a positive trichloroacetic acid (9 ml of a 10 g/100 ml solution). history of tobacco chewing habits were excluded from Portions (1 ml) of the supernatant fluid were then ana- the study. Each patient was thoroughly examined both lyzed for thiocyanate. intra orally and extra orally under artificial illumination. A portion (1 ml) of the test solution (serum or saliva) was acidified (0.5 ml of 1 mol/litre HCl) and bromine water Method of collection of data was added (2 drops of saturated solution), followed, af- Sample collection: ter mixing, by the addition of arsenious oxide solution (3 Informed consent was obtained from the patients in- drops) to remove excess bromine.
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