ORIGINAL Aggarwal ARTICLE et al
Salivary Thiocyanate: A Biochemical Indicator of Cigarette Smoking in Adolescents Anshul Aggarwala/Vaishali Keluskarb/Rati Goyalc/Parveen Dahiyad Purpose: Saliva is considered to be critical for the maintenance of healthy oral mucosa, and oral fluids provide an easily available, non-invasive medium for the diagnosis of a wide range of diseases and clinical situations. The purpose of this study was to estimate the thiocyanate (SCN) level in saliva of cigarette smokers and compare it with that of nonsmokers. Materials and Methods: The present study comprised 100 subjects, of which 50 had a habit of tobacco smoking. The other 50 neither smoked nor chewed tobacco and comprised the healthy control group. The age and sex (all males) of both groups of subjects were matched. All the patients were in the age group of 25 to 40 years. The group of smokers was divided into subgroups according to duration and frequency of smoking. Duration group 1: smoking for a period of 4–7 years; duration group 2: smoking for a period of 8–15 years; duration group 3: chronic smokers, smoking for a period of more than 15 years. Frequency group 1: patients smoked half pack of cigarettes, i.e. 4–6 per day; frequency group 2: patients smoked one pack of cigarettes, i.e. 7–11 per day; frequency group 3: patients smoked more than one pack, i.e. >11, per day. Saliva was collected by the spitting method. Unstimulated whole saliva was refrigerated at 4°C and processed within 24 h. The estimation of thiocyanate in saliva was done according to Densen et al (1967). Results: The present study clearly indicates a significant increase in salivary thiocyanate level in tobacco smokers as compared to nonsmokers (P < 0.0001). Comparing salivary SCN in different duration groups, the salivary SCN level was significantly lower in group 1 vs groups 2 and 3, with P < 0.0001. In terms of smoking frequency, the salivary SCN level was significantly lower in group 1 vs group 3, P < 0.0001. It is also evident that there was an increase in salivary thiocyanate levels with increased duration and frequency, thus showing a positive correlation between them. Conclusion: The findings of this study suggest that salivary thiocyanate can be used as a safe and acceptable prevalence measurement for cigarette smoking behaviour. Key words: saliva, salivary thiocyanate, tobacco smoking Oral Health Prev Dent 2013;11:221-227 doi: 10.3290/j.ohpd.a30169
S
aliva has many properties which play an important role in maintaining the normal function of the mouth. Saliva plays a major role in the protec-
a
Senior Lecturer, Department of Oral Medicine and Radiology, Himachal Institute of Dental Sciences, Paonta Sahib, Himachal Pardesh, India.
b
Professor, Department of Oral Medicine and Radiology, Vishwanath Katti Institute of Dental Sciences, Belgaum, Karnatatka, India.
c
Postgraduate Student, Department of Anatomy, Shree Guru Ram Rai Institute of Medical and Health Sciences, Dehradun, Uttaranchal, India.
d
Reader, Department of Periodontics, Himachal Institute of Dental Sciences, Paonta Sahib, Himachal Pardesh, India.
Correspondence: Dr. A. Aggarwal, Dept. of Oral Medicine and Radiology, Himachal Institute of Dental Sciences, Rampur Ghat Road, Paonta Sahib-173025, Distt. Sirmour (Himachal Pardesh), India. Tel: +91980-574-5041. Email:
[email protected] or draggarwal.
[email protected]
Vol 11, No 3, 2013
Submitted for publication: 29.01.12; accepted for publication: 19.07.12
tion of oral tissues, food preparation, digestion, lubrication and speech (Mandel, 1990). The use of tobacco is harmful to general health; it is a common cause of addiction, illness, disability and death. The use of tobacco also causes an increased risk for oral pre-cancer (mostly leukoplakia), oral cancer, periodontal diseases and other deleterious oral conditions (acute necrotising ulcerative gingivitis, oral candidiasis) and thus it adversely affects the outcome of oral care (Sajith et al, 2007). Accurate measurement of cigarette smoking is critical for understanding patterns of adult smoking behaviour and essential for evaluating health education programmes aimed at reducing or preventing the habit. Chemical measures such as salivary SCN show promising results in obtaining accurate, quantitative information on smoking habits (Gimenez and Adame, 2003). Hence, the
221
Aggarwal et al
purpose of this study was to determine the salivary SCN level in the cigarette smokers and nonsmokers and compare the two.
was asked to accumulate saliva in the mouth for about 2 minutes, after which he was asked to spit the accumulated saliva into a sterile plastic container. The unstimulated saliva thus collected was refrigerated at -20°C and processed within 24 h.
MATERIALS AND METHODS Subjects
Spectrophotometry protocol
A total of 100 patients who attended the Department of Oral Medicine and Radiology, Vishwanath Katti Institute of Dental Sciences, Belgaum, India, were included in the study. Group 1 was the study group of cigarette smokers (n = 50) who met the inclusion criteria of male gender, age 25 to 40 years with a history of cigarette smoking at least 4 to 5 times a day for a period of 4 to 5 years or more. Informed consent was obtained from the subjects and ethical clearance was granted by the institutional review board. Exclusion criteria were: a history of tobacco chewing, a history of both smoking (cigarette, bidi) and tobacco chewing and/or a high intake of foods rich in thiocyanate, such as cooked broccoli, Brussels sprouts, raw cabbage and cauliflower. The group of smokers was divided into subgroups according to duration and frequency of smoking. Duration group 1: smoking for a period of 4–7 years; duration group 2: smoking for a period of 8–15 years; duration group 3: chronic smokers, smoking for a period of more than 15 years. Frequency group 1: patients smoked half pack of cigarettes, i.e. 4–6 per day; frequency group 2: patients smoked one pack of cigarettes, i.e. 7–11 per day; frequency group 3: patients smoked more than one pack, i.e. >11, per day. Group 2 was the control group of nonsmokers (n = 50). The inclusion criteria were: age- and sexmatched subjects without a history of consumption of tobacco in any form who also gave informed consent to participate in the study. The exclusion criterion was a history of present or past consumption of tobacco in any form.
Principles
Collection of saliva samples The estimation of salivary SCN was carried out in the Department of Biochemistry, KLE Prabhakar Kore Hospital and Medical Research Centre, Belgaum, India. Subjects were asked to not to eat, drink or smoke an hour prior to collection of saliva. Each subject
222
Tobacco smoke contains HCN gas. Once this gas is inhaled, it is dissociated into its components according to the following equilibrium equation: HCN(aqueous) H+(aqueous) + CN-(aqueous) The CN- ion is then converted into the SCN ion when it reaches the liver. The SCN ion reacts with the Fe3+ ion in the reagent to yield the FeSCN2+ complex that can be easily detected by the spectrophotometric method. In this study, the absorbance of several solutions containing varying concentrations of FeSCN2+ complexes were measured at 405 nm to construct a standard curve.The unknown concentration of FeSCN2+ ion in the saliva samples was determined by using the linear relationship between absorption and concentration as established by a standard curve (Fig 1). To standardise the technique, a MODULAB 4010 spectrophotometer (Metavision; Mumbai, India) was used. Salivary SCN content was measured according to the Densen method (Densen et al, 1967). Reagents
Stock solution was made by dissolving 2 g of KSCN in 1 litre of distilled water. This solution was titrated against 20 ml of AgNO3 solution (2.924 g/litre) plus 5.0 ml of concentrated HNO3 using 1.0 ml of a saturated solution of ferric ammonium sulphate as indicator. The dilution of the SCN solution needed to make 20 ml of its equivalent to 20 ml of the silver nitrate solution was calculated. After making this dilution, it was checked by another titration to be sure that the potassium SCN solution was exactly equivalent to the silver nitrate solution. The final stock solution contained 100 mg of SCN ions per 100 ml. The working standard solution was made by diluting 10 ml of the stock solution with 10 ml of distilled water. This solution contained 10 mg of SCN ions per 100 ml. To make ferric nitrate solution, 50 g of crystalline ferric nitrate was dissolved in 500 ml of distilled water and 25 ml of concentrated HNO3 was added to make 1 litre of solution with distilled water.
Oral Health & Preventive Dentistry
Aggarwal et al
Calibration
1.0 ml of working SCN standard, 8.0 ml of distilled water and 1.0 ml of ferric nitrate solution were poured into a test tube. This was done in triplicate and the reading was taken after 5 min against a distilled water blank at zero, with a wavelength of 405 nm in spectrophotometer. An average of the three readings was recorded as the standard reading ‘S’. The value of the standard reading was recorded as 0.26. The absorbance readings were recorded as 0.10, 0.13, 0.26, 0.39 and 0.52 at 0 mg%, 5 mg%, 10 mg%, 15 mg% and 20 mg% of SCN concentration in varying strengths of the standard, respectively. The calibration of the spectrophotometer revealed that the absorbance readings were directly proportional to the SCN in varying strengths of the standard. The calibration curve was a straight line (Fig 1). Therefore, the use of a standard reading ‘S’ obtained from solutions containing 10 mg of SCN ions per 100 ml is valid within the range.
0.6 Absorbance Reading
The blank solution was 25 ml of concentrated HNO3 diluted to 1 litre of solution with distilled water.
0.5 0.4 0.3 0.2 0.1 0.0 0
5
10 15 mg SCN/100ml
20
25
Fig 1 Standard curve with varying concentrations of thiocyanate standard.
Calculating SCN content
The reading of the blank solution was subtracted from the reading of the unknown solution to obtain the true reading of the unknown. Because 10 mg per 100 ml solution was used, the readings were calculated as: Reading of unknown: (U) × 10 = mg of SCN- per 100 ml of saliva
Measuring SCN in saliva samples
For this procedure, a wavelength of 405 nm, a light path of 1 cm and a temperature of 37°C were used. All glass vessels used in the procedure were thoroughly washed with distilled water before use. • The saliva samples were centrifuged at 3000 rpm for 5 min and clear saliva was separated from the impurities. The given saliva sample was divided into halves. The unknown solution (the saliva sample whose SCN concentration has yet to be determined) was made by placing one half of the clear saliva sample – i.e. 0.5 ml of clear saliva – into a separate test tube. 4 ml of distilled water was added to it and mixed thoroughly. Then 0.5 ml of ferric nitrate solution was added to it slowly with shaking. • The other half of the clear saliva sample – i.e. 0.5 ml of clear saliva – was placed into a separate test tube. 4 ml of distilled water was added to it and mixed thoroughly. Then 0.5 ml of blank solution was added to it slowly with shaking. The solutions were protected from bright light by covering the test tubes with black paper. After 5 min, the reading at 405 nm was taken on the spectrophotometer.
Vol 11, No 3, 2013
Reading of standard (S): U/S × 10 = mg of SCN- per 100 ml of saliva where the reading of the standard = 0.26, i.e. U/0.26 × 10 = mg of SCN-per 100 ml of saliva.
Statistical analysis Data were analysed using SPSS software version 13.1 (SPSS; Chicago, IL, USA). The statistical difference between cigarette smokers and nonsmokers was analysed using the unpaired Student t-test (means and standard deviations). The comparisons of salivary SCN level among the duration and frequency subgroups were performed using one-way analysis of variance (ANOVA). Further pair-wise comparisons were made using Tukey’s post-hoc test.
RESULTS Table 1 shows the comparison of salivary SCN level and age between smokers and controls (nonsmokers). The salivary SCN level was significantly higher in smokers than in controls (P < 0.0001). Smokers
223
Aggarwal et al
Table 1 Comparison of salivary SCN and age means between smokers and controls (nonsmokers) using the Student t-test Smokers
Controls
Parameter
Mean
Standard deviation
Mean
Standard deviation
P-value
Significance
Salivary thiocyanate
12.8
3.19
3.6
1.26
8.75061E-35
HS
Age
33.74
4.61
32
4.94
0.065
NS
HS: highly significant; NS: not significant.
Table 2 Comparison of salivary SCN levels (mg of SCN- per 100 ml of saliva) by duration of smoking (means and standard deviation, SD) Parameter
Salivary thiocyanate
Subgroup
Duration in years
No. of subjects
Mean
SD
1
4–7
17
10
2.13
2
8–15
22
13.7
2.68
3
>15
11
15.4
2.25
Table 3 ANOVA for subgroups of duration of smoking Parameter
Salivary thiocyanate
Source of variation
Sum of squares
Degrees of freedom
Mean square
F
P-value
Between groups
223.82
2
111.91
19.16
P < 0.05
Within groups
274.58
47
5.84
Total
498.40
49
and controls did not differ statistically significantly in terms of age (P = 0.065). The salivary SCN levels between the smoking duration (Tables 2 and 3) and frequency (Tables 5 and 6) subgroups were statistically compared using one-way ANOVA. The P-values among the three duration subgroups in show non-homogeneity, as is also the case among the three frequency subgroups. Pairwise comparison on the basis of duration (Table 4) and frequency (Table 7) were performed using Tukey’s post-hoc test. Table 4 shows that the salivary SCN level was significantly lower in duration subgroup 1 vs subgroups 2 and 3 (P < 0.0001). The salivary SCN level was not significantly different between duration subgroups 2 and 3. Table 7 shows that the salivary SCN level was significantly lower in frequency subgroup 1 than in subgroup 3 (P < 0.0001). However, the salivary SCN level was not significantly different between frequency subgroups 1 and 3 or between subgroups 2 and 3.
224
DISCUSSION According to Sanchez-Perez et al (2007), saliva plays an important role in oral health monitoring, regulating and maintaining the integrity of oral mucosa. Saliva is necessary for the protection and lubrication of oral mucosal tissues, remineralisation of teeth, digestion, taste sensation, stimulation, wash-out effect, pH balance and phonation (Genco, 1996). Human saliva contains a large number of solid (organic and inorganic) constituents such as proteins, sodium, potassium, SCN, immunoglobulins etc. Salivary diagnosis is an increasingly important field in dentistry, as samples are easily obtainable and it is noninvasive. SCN levels in saliva are the most frequently used biochemical tests for establishing the incidence or prevalence of tobacco consumption among smokers. As SCN is a metabolite of a combustion product of hydrogen cyanide, it is not useful for the detection of smokeless tobacco;
Oral Health & Preventive Dentistry
Aggarwal et al
Table 4 Comparison of the salivary SCN levels in subgroups of duration of smoking (Tukey’s test) Parameter
Comparison between subgroups
Salivary thiocyanate
Mean difference (i-j)
P-value
Significance
Subgroup 1
Subgroup 2
3.7245
< 0.05
HS
Subgroup 1
Subgroup 3
5.354
< 0.05
HS
Subgroup 2
Subgroup 3
1.6295
0.1723
NS
HS: highly significant; NS: not significant.
Table 5 Comparison of means of salivary SCN levels (mg of SCN- per 100 ml of saliva) of frequency of smoking subgroups Parameter
Subgroup
Frequency per day (no. of cigarettes)
No. of subjects
Mean
SD
1
4–6
18
11.22
3.05
2
7–11
20
13.13
3.04
3
>11
12
14.83
2.49
Salivary thiocyanate
Table 6 ANOVA for subgroups of frequency of smoking Parameter
Salivary thiocyanate
Source of variation
Sum of squares
Degrees of freedom
Mean square
F
P-value
Between subgroups
96.14
2
48.07
5.62
0.0065*
Within subgroups
402.25
47
8.56
Total
498.40
49
*Very significant (P < 0.05).
Table 7 Comparison of the salivary thiocyanate levels in subgroups of frequency of smoking (Tukey’s test) Parameter
Salivary thiocyanate
Source of variation
Sum of squares
Degrees of freedom
Mean square
F
P-value
Between subgroups
96.14
2
48.07
5.62
0.0065+
Within subgroups
402.25
47
8.56
Total
498.40
49
*Very significant (P < 0.05).
however, it plays a critical role in understanding patterns of adult smoking behaviour and for evaluation of health education programmes aimed at reducing or preventing the habit (Foss and Lund-Larsen, 1986). It is apparent that many systemic diseases affect salivary gland function and salivary composi-
Vol 11, No 3, 2013
tion. Any alteration in the production or composition of saliva can increase the mucosal permeability, especially with the use of tobacco, and predispose it to oral cancer. Salivary diagnosis is anticipated to be particularly useful in cases where repeated samples of body fluid are needed but where drawing blood is impractical, unethical or both.
225
Aggarwal et al
All the patients in the present study were in the age group of 25 to 40 years. This is consistent with other studies, as the tobacco habit is more prevalent in younger age groups (Reddy et al, 1980). Only male subjects were included in the current study, as the tobacco habit is more prevalent among males in the Belgaum population. In our study, the salivary SCN level was significantly increased in smokers as compared to nonsmokers. This is due to the fact that the main source of SCN is tobacco smoke, which is absorbed in the lungs and later metabolized to SCN (Benfari et al, 1977; Tenovuo, 1978; Borgers and Burckhard, 1979). Borgers and Burckhard (1979) found that the high concentration of cyanide in tobacco smoke may persist in the oral cavity of smokers for some time, and the saliva thus contaminated may artifactually increase the cyanide level. Courant (1967) observed significantly higher concentrations of SCN in the saliva of smokers.12 This was consistent with our results and was also reported in various other studies (Tenovuo and Makinen, 1976; Luepker et al, 1981; Lamberts et al, 1984). In the present study, salivary SCN level was also compared on the basis of duration and frequency of smoking. This study indicates a significant increase in salivary SCN level with increased duration and frequency, thus showing a positive correlation between them. This may be attributed to the fact that each cigarette increases the level of hydrogen cyanide delivered to the mouth of a smoker (approximately 30 to 200 μg) (Armenio et al, 1953). This hydrogen cyanide is metabolised by the liver to SCN. This clearly indicates that the greater the duration and frequency of smoking, the higher is the level of hydrogen cyanide, which in turn increases the level of SCN in the saliva of smokers. Armenio et al (1953) found that chronic smokers clearly had higher values of SCN in saliva than moderate smokers. Tenovuo and Makinen (1976) observed a significant increase in the salivary SCN level with increased duration and frequency, thus showing a positive correlation between them. According to another study, smokers who have smoked longer have significantly higher SCN levels than smokers who have smoked for a shorter duration (Luepker et al, 1981). According to Foss and Lund-Larsen (1986), SCN has a high specificity for heavy smokers, as compared to light smokers. Bliss and O’Connell (1984) indicated that a SCN assay shows excellent sensitivity (over 90%) in chronic smokers who smoke more than 15 cigarettes per day, whereas in light
226
(one to five cigarettes per day) and moderate smokers (five to ten cigarettes per day), the sensitivity of the SCN level was not found to be significant.This is consistent with results of the present study. The present study showed a statistically significant increase in the salivary SCN level in smokers over controls. It was also evident that very high levels of SCN were obtained in salivary samples from heavy smokers; however, the technique is not very useful in detecting low-level smokers, such as experimenters, or those who smoke only a few cigarettes a month (Luepker et al, 1981). Thus, assessing SCN levels may reveal the smoking behaviour of an individual, making it a potentially effective epidemiological tool (given an increased sample size) for evaluating smoking prevalence in populations. In another study (Pojer et al, 1984), cigarette smoking histories were compared by examining carboxyhemoglobin and serum thiocyanate concentrations obtained from 426 smokers and 191 nonsmokers. The mean levels of both carboxyhemoglobin and serum thiocyanate were significantly higher among cigarette smokers and correlated with the number of cigarettes smoked per day. The specificity of both procedures was 81%, but serum thiocyanate had a higher sensitivity than carboxyhemoglobin (93% vs 83%), making it potentially more suitable for use as an index of cigarette smoking. In yet another study (Noland et al,1988), biochemical determinations of plasma and salivary cotinine and thiocyanate were used to distinguish smokers from nonsmokers and to follow daily smoking patterns in smokers. Results indicate that cotinine is better suited than thiocyanate to determine smoking status in large scale epidemiological studies and to follow alterations in smoking behaviour over time. Salivary cotinine is a reliable alternative to plasma for validation of smoking status and for following changes in daily smoking patterns. Vogt et al (1977) evaluated the use of salivary cotinine, salivary thiocyanate and expired-air carbon monoxide as biochemical validation measures for assessing the smoking status of adults. By use of data from known nonsmokers and admitted smokers, the sensitivity and specificity of the validation measures were as follows: salivary cotinine 99% and 100%, expired-air carbon monoxide 96% and 100%, and salivary thiocyanate 67% and 95%, respectively. The salivary cotinine and expired-air carbon monoxide tests confirmed smoking cessation for 55% and 74%, respectively, of the proclaimed quitters. The length of time since quitting was significantly related to the results observed
Oral Health & Preventive Dentistry
Aggarwal et al
with the latter measures. Consideration of these observations along with various practical factors suggests that expired-air carbon monoxide assays may be the validation measure of choice for most clinical trials (Vogt et al, 1977).
CONCLUSION The present study revealed a significant increase in salivary SCN level in smokers as compared to nonsmokers. The main source of SCN is tobacco smoke which is absorbed in the lungs and later metabolised to SCN. Thus, it can be estimated only in smokers and not tobacco chewers. The study also indicates a significant increase in salivary SCN level with increased duration and frequency of smoking, showing a positive correlation between the two. This could be attributed to the fact that a high concentration of cyanide included in tobacco smoke remains in the oral cavity of chronic smokers for a longer time, contaminating the saliva, which in turn may artifactually increase the cyanide level. Hence, this study also showed that salivary SCN can act as a reliable biochemical indicator for assessing smoking behaviour.
REFERENCES 1. Armenio G, Laforgia PD, Buonsanto M. Variation in concentration of thiocyanates in relation to tobacco smoke. Chem Abst 1953;47:8867. 2. Benfari RC, Mcintyre K, Baldwin A, Ockene J. The use of thiocyanate determination for indication of cigarette smoking status. Eval Quarterly 1977;1:629. 3. Bliss RE, O’Connell KA. Problems with thiocyanate as an index of smoking status: a critical review with suggestions for improving the usefulness of biochemical measures in smoking cessation research. Health Psych 1984;3: 563-581. 4. Borgers D, Burckhard J. Thiocyanate as an indication of tobacco smoke. Prev Med 1979;8:351.
Vol 11, No 3, 2013
5. Courant P. The effect of smoking on the antilactobacillus system in saliva. Odontol Revy 1967;18:251-261. 6. Densen PM, Davidow B, Bass HE, Jones EW. A chemical test for smoking exposure. Arch Environ Health 1967;14: 865–874. 7. Foss OP, Lund-Larsen PG. Serum thiocyante and smoking interpretation of serum thiocyante levels observed in a large health study. Scand J Clin Lab Invest 1986;46:245. 8. Genco RJ. Current view of risk factors for periodontal diseases. J Periodontol 1996;67:1041–1049. 9. Gimenez TJC, Adame ML. Addictive Behaviors 2003;28: 81–89. 10. Lamberts BL, Pruitt KM, Pederson ED, Golding MP. Comparison of salivary peroxidase system components in caries free and caries active naval recruits. Caries Res 1984;18:488–494. 11. Luepker RV, Pechacek TF, Murray DM, Johnson CA, Hund F, Jacobs DR. Saliva thiocyanate: a chemical indicator of cigarette smoking in adolescents. Am J Public Health 1981;71:1321. 12. Mandel ID. The diagnostic uses of saliva. J Oral Pathol Med 1990;19:119-125. 13. Noland MP, Kryscio RJ, Riggs RS, Linville LH, Perrit LJ, Tucker TC. Saliva cotinine and thiocyanate: chemical indicators of smokeless tobacco and cigarette use in adolescents. J Behav Med 1988;11:423–433. 14. Pojer R, Whitfield JB, Richmond R, Hensely WJ. Carboxyhaemoglobin, cotinine and thiocyante assay compared for distinguishing smokers from non-smokers. Clin Chem 1984;30:1377. 15. Reddy MS, Naik SR, Bagga OP, Chuttani HK. Effect of chronic tobacco betel lime quid. Chewing on human salivary secretions. Am J Clini Nutrition 1980;33:77-80. 16. Sajith V, Zdenek F, Smejkalová J, Jacob V, Somanathan R. Smoking related systemic and oral diseases. Acta Medica 2007;50:161–166. 17. Sanchez-Perez A, Moya-Villaescusa MJ, Caffesse RG. Tobacco as a risk factor for survival of dental implants. J Periodontol 2007;78:351–359. 18. Tenovuo J, Makinen KK. Concentration of thiocyante and ionizable iodine in saliva of smokers and nonsmokers. J Dental Res 1976;55:661-663. 19. Tenovuo J. Inhibition by thiocyante of lactoperoxidase catalysed oxidation and iodination reaction. Arch Oral Biol 1978;23:899. 20. Vogt TM, Selvin S, Widdowson G, Hulley SB. Expired air carbon monoxide and serum thiocyanate as objective measures of cigarette exposure. Am J Public Health 1977;67: 545–549.
227
