|Year : 2022 | Volume
| Issue : 4 | Page : 458-463
Salivary and serum acetaldehyde levels in betel quid chewers, pan masala chewers with or without tobacco and tobacco smokers: A comparative study using head space gas chromatography
Dorairaju Pavithra1, Thayalan Dineshkumar2, Krishnan Rajkumar3
1 Department of Oral Pathology and Microbiology, Thai Moogambigai Dental College and Hospital, Chennai, Tamil Nadu, India
2 Department of Oral Pathology and Microbiology, SRM Dental College, Ramapuram Campus, SRM Institute of Science and Technology, Chennai, Tamil Nadu, India
3 Department of Oral Pathology and Microbiology, SRM, Dental College, Ramapuram Campus, SRM Institute of Science and Technology, Chennai, Tamil Nadu, India
|Date of Submission||27-Mar-2021|
|Date of Acceptance||17-Nov-2021|
|Date of Web Publication||22-Dec-2022|
No. 21, Kanni Street, Jaffer Khan Pet, Chennai - 600 083, Tamil Nadu
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Acetaldehyde, a natural by-product of combustion and photo-oxidation, has been detected at low levels in cigarette smoke. Various literature studies have shown increased salivary acetaldehyde production among smokers and heavy drinkers.
Aim: The present investigation was performed to estimate the salivary and serum acetaldehyde levels in pan masala chewers (with or without tobacco), betel quid chewers and tobacco smokers.
Methodology: Blood and Saliva samples were collected from 40 participants. The study participants were categorized into 4 groups of 10 individuals each. Subjects with no habits, betel quid chewing habit, pan masala chewing habit with or without tobacco and tobacco smoking habit. All the samples were centrifuged and subjected to gas chromatography (GC) analysis as per standard protocols provided by the manufacturer.
Results and Discussion: No statistically significant difference in either serum or salivary acetaldehyde levels between Pan Masala chewers (with or without tobacco) and smokers was noted. However, higher mean serum and salivary acetaldehyde levels than the normal subjects and betel quid chewers was observed. It may be speculated that natural acetaldehyde, may not be released in estimated quantity of acetaldehyde by head space GC. Nevertheless, it is possible that addition of acetaldehyde as flavoring agents or preservatives for improving acceptability of the product can be a possible cause for these observations.
Conclusion: Owing to various socio, economic and political reasons, implementation of the ban remains to be a formidable challenge. With these observations, we believe that absolute ban of all pan masala product is the ideal solution for minimizing the oral cancer burden in India.
Keywords: Acetaldehyde, carcinogen, gas chromatography, oral squamous cell carcinoma, preservatives, volatile compound
|How to cite this article:|
Pavithra D, Dineshkumar T, Rajkumar K. Salivary and serum acetaldehyde levels in betel quid chewers, pan masala chewers with or without tobacco and tobacco smokers: A comparative study using head space gas chromatography. J Oral Maxillofac Pathol 2022;26:458-63
|How to cite this URL:|
Pavithra D, Dineshkumar T, Rajkumar K. Salivary and serum acetaldehyde levels in betel quid chewers, pan masala chewers with or without tobacco and tobacco smokers: A comparative study using head space gas chromatography. J Oral Maxillofac Pathol [serial online] 2022 [cited 2023 Feb 3];26:458-63. Available from: https://www.jomfp.in/text.asp?2022/26/4/458/364830
| Introduction|| |
The head and neck tumors most frequently arise from the aerodigestive tract, oral cavity, oropharynx, hypopharynx and larynx. Among these, oral squamous cell carcinoma (OSCC) accounts for more than 90% of head and neck tumors involving orofacial region. The OSCC is currently ranked as the 12th most common cancer across the globe and grouped together with pharyngeal cancer; it is the sixth most common cancer worldwide. OSCC accounts for 19% of all the total malignancies in Indian men and it is the 3rd most common cancer in Indian females., Areca nuts, reverse smoking and smokeless tobacco (snuff and chewable form) are important risk factors. Tobacco and alcohol are independent risk factors, but together they have a synergistic effect. Natural acetaldehyde levels in human blood are <0.5UM,, but tobacco smoke contains high volatile acetaldehyde, which is 1,000 times more toxic and it easily dissolves in saliva during smoking. It is toxic, mutagenic and carcinogenic according to numerous cell culture and animal models. Alcohol as such is not carcinogenic, but its first metabolite acetaldehyde can increase the risk of upper aero digestive tract cancer.,
The International Agency for Research on Cancer (IARC) in 1999 classified acetaldehyde as (group 2B) “possibly carcinogenic to humans.” Exposure to acetaldehyde may occur from the usage of cosmetic products, environmental pollutants and burning fossil fuel., The highest concentrations of acetaldehyde were determined in vinegar (1.06 g/kg), milk products, diverse fruits and vegetables. Several studies have been done for the estimation of the salivary acetaldehyde levels only in tobacco smokers and alcoholics, but no study has been done to estimate the levels of acetaldehyde in tobacco chewers. Hence, the present investigation was performed to find the salivary acetaldehyde levels in pan masala chewers (with or without tobacco), betel quid chewers and to compare the levels in persons without these habits, smokers and alcoholics using Head space gas chromatography (GC).
| Methodology|| |
Selection of study participants
A total of 40 patients were recruited from the Outpatient Department of SRM Dental College, Chennai, India. The study participants were categorized into 4 groups as A, B, C and D. Group A comprises 10 individuals with no history of habits such as Pan-Masala chewing with or without tobacco, betel chewing or tobacco smoking and social drinking (Control Group). Group B comprises 10 cases with betel quid chewing habit (only handmade, indigenous preparations, with no addition of tobacco) with a frequency of more than 5 times a day (Betel quid chewers without tobacco). Group C comprises 10 cases with only Pan-Masala chewing habit with or without tobacco (any commercial preparations) and Group D comprises 10 cases with known tobacco smoking habit. Individuals with known occupational acetaldehyde exposures, xerostomia associated systemic diseases, xerostomia and alcoholics were excluded from the study. The details of the study were explained to all the participants followed by complete case histories and questionnaires; verbal and written consents were obtained before the commencement of study. The present study was done as a preliminary study to determine the sample for the larger estimatory levels and observe the significant correlation.
Blood and saliva were collected from the participants and were transferred into coated centrifuge tubes for serum and noncoated centrifuge tubes for saliva. The nonstimulated saliva was collected by asking the patient to spit in a sterile container and transferred to noncoated centrifuge tube. Cool centrifugation was done at 4-degree temperature with 5000 RPM for 5 min. The supernatant was collected with the help of micropipettes and transferred to the Eppendorf tubes (2 ml) and the samples were stored in the deep freezer at −80°.
Determination of acetaldehyde in saliva and serum sample
GC is the method used for separation of volatile substances from volatile derivatives. GC consisting of 30M DB wax column with 20 ml head space vial aluminum cap, silicone gasket and crimping tool was used for the study. Standard aqueous solution was prepared by weighing 0.03 g, 0.06 g and 0.09 g of pure acetaldehyde solution in a 100 ml standard flask separately followed by addition of De-ionized water. 1 microliter of each prepared standard was injected into the GC using head space vial. The sample vials were placed in head space compartment and heat at 80°C for 5 min and 1 ml of vapor from the head space was directed automatically to the GC with DB-Wax capillary column maintained at 40°C for 10 min. Serum and salivary acetaldehyde levels (in ppm) in individuals belonging to all four groups were noted from the respective report for further analysis using SPSS software ver 20.0 (IBM Corp., Armonk, N. Y., USA). The area obtained from the chromatogram against concentration (μg/mL) is plotted in a graph by G. C software automatically [Figure 1].
| Results|| |
The collected data was analyzed using descriptive and inferential statistical methods. The descriptive statistics seen as mean and standard deviation have been used to express the levels of acetaldehyde in serum and saliva. One-way ANOVA statistical method and Tursky multiple comparison tests had been applied to compare the acetaldehyde in group wise. The estimation of acetaldehyde levels (in ppm) in both serum and saliva is shown in [Table 1] and [Table 2] along with their mean values. The mean acetaldehyde level in serum for the pan masala chewers was 35.76 with a standard deviation of 49.7. For the smokers, the mean acetaldehyde level was 30.21 with a standard deviation of 30.7 while for the other two groups the mean had been nil. The mean values are also shown in [Graph 1] and [Graph 2] for serum and saliva respectively.
|Table 1: Serum acetaldehyde levels (ppm) in individuals in all four groups|
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|Table 2: Salivary acetaldehyde levels (ppm) in individuals in all four groups|
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Spearman rank correlation coefficient between the acetaldehyde level in saliva and in serum with the duration [Table 3] and frequency of the habit by group-wise showed no significant difference in the duration however frequency of the habit has shown statistical significant at P < 0.05 [Table 4]. It was observed that Pan Masala chewers (with or without tobacco) and smokers had higher mean serum acetaldehyde levels than the normal subjects and betel quid chewers. The salivary levels of acetaldehyde appear to be more the serum acetaldehyde level and there were no significant differences existing between the Pan Masala chewers (with or without tobacco) and smokers. For smokers, the frequency of smoking increased the acetaldehyde levels in saliva (P < 0.05) [Table 5]. All other correlations have been nonsignificant [Table 6]. The results indicate that the frequency of habit has no relation with the level of acetaldehyde in serum and saliva for the other two groups in this study.
|Table 3: Spearman rank correlation coefficient between the acetaldehyde level in saliva and in serum with the duration of the habit by group-wise|
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|Table 4: Spearman rank correlation coefficient between the acetaldehyde level in saliva and in serum with the frequency of the habit by group-wise|
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|Table 5: Distribution of frequency of habit of chewing or smoking by group wise|
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[Graph 3] shows that 40% of the betel quid chewers chew the betel quid more than 10 times/day with the maximum of 20 times in a day by 3 subjects. 20% of the Pan masala chewers chews for more than 10 times/day, with the maximum of 20 times. 20% of the smokers smoke for more than 10 times with the maximum of 12 times/day.
| Discussion|| |
Humans are frequently exposed to naturally occurring acetaldehyde that can exist in the air and that can be ingested as a contaminant of food and alcoholic drinks because it is the main compound formed during ethanol metabolism. It has been associated with cancer development and has been classified as Group 1 carcinogenic agent to humans by the IARC of the World Health Organization. Soffritti et al. in 2002 and Woutersen et al. in 1986 experimentally observed that acetaldehyde is the genotoxic carcinogen. Lachenmeier et al. in 2009 found acetaldehyde is added to the food to increase its flavor. Michael et al. established quantitative detection of acetaldehyde in various kinds of food and products using GC with head space analyzer. GC is one of the most useful and popular tools of biochemistry. It is the technique of analyzing the substances of closely related compounds from mixture example proteins, amino acids, carbohydrates, vitamins, drugs.
Determination of salivary and serum acetaldehyde levels in individuals using commercial chewable pan masala products has not been reported. Assessing the genotoxic potential of the serum or salivary acetaldehyde obtained from the samples is of prime importance in this study. The literature informs that acetaldehyde in saliva is only by ethanol metabolism or as a pyrolysis product of the mainstream tobacco smoke., The results of our study have therefore got no comparison with any gold standard or known permissible limits. IARC has labelled acetaldehyde derived from alcohol consumption as a group I carcinogen (definitely carcinogenic to humans) and in the year 2007, IARC labelled smokeless tobacco as a group I carcinogen due to the presence of TSNA's
Our results showed that 8 out of 10 individuals chewing pan masala demonstrated salivary acetaldehyde. This study was primarily focused on determination and estimation of salivary and serum acetaldehyde levels in commercial pan masala chewers, and we did not aim to associate the quantified levels to their frequency and duration of the habit. Nevertheless, the clinical data included the above-mentioned parameters and consequently included chewers with a wide variance of frequency and duration of chewing habit. For the same reason, we have included individuals of all age groups, though there was a sampling bias toward males, possibly because of the high prevalence of the habits in our study population.
The mean serum acetaldehyde levels in normal (Group A) and betel quid chewers (Group B) was nil. Literature also reports that serum acetaldehyde in normal healthy individuals to be negligible levels. In tobacco smokers (Group D, n = 10) 60% of subjects demonstrated serum acetaldehyde. The mean serum acetaldehyde levels were 30.21 ppm, though with wide variance. In pan masala chewers (Group C, n = 10) again 60% of individuals demonstrated serum acetaldehyde. The mean serum acetaldehyde level being 35.76 ppm was higher than the smoker group. But the variance among the same group was very high. These results confirmed statistically that mean serum acetaldehyde levels in pan chewers and smokers are higher than normal group and betel quid chewers. Also statistically, there is no difference between the mean serum acetaldehyde level of pan masala chewers (Group C) and smokers (Group D), with a significance of P = 0.995.
Interestingly the mean salivary acetaldehyde levels in normal group (Group A) was 4.65 ppm with one person (out of n = 10) demonstrating a peak in the salivary sample. A possible explanation could be that certain dietary substances may naturally contain high levels of acetaldehyde, and since we did not control the individual dietary habits in this study, 'stray' values are probably expected infrequently and at much lower ppm levels. In the betel quid chewers (Group B) one out of n = 10 demonstrated acetaldehyde peak and the mean value was 2.81 ppm. This result indicates that the indigenous preparations most likely did not contain any source of acetaldehyde, and the reported value in one subject was possibly from the dietary source as observed in the Group A results.
In Pan Masala chewers (Group C), 80% of subjects demonstrated a mean salivary acetaldehyde level of 88.13 ppm, while 80% smokers (Group D) demonstrated a mean salivary acetaldehyde level of 60.81 ppm. Due to high intra group variance in ppm values, the logarithmic transformation was done and ANOVA was tested. Our study results showed that Pan masala chewers (Group C) and smokers (Group D) had higher salivary acetaldehyde levels compared to Group A and B. These results also revealed that there is no statistical difference in either serum or salivary acetaldehyde levels between Pan Masala chewers (with or without tobacco) and smokers. Previous studies show that acetaldehyde was available in saliva and serum only after pyrolysis of the tobacco product or after metabolic transformation of ethanol when consumed as a habit.
In our study, we have excluded subjects with alcohol habits and in the Group C none of the subjects were smokers. From these results, we can only speculate with regards to presence of such high levels of acetaldehyde in the saliva of Group C subjects. Natural tobacco may contain volatile aldehydes such as formaldehyde, acetaldehyde and crotonaldehyde in very small quantities (in nano-grams). Pan Masala with tobacco (Gutkha) even if it contains natural acetaldehyde, may not release this quantity of acetaldehyde as determined and estimated by Head space GC. This emphasis that analytical chemical studies using Head space GC with mass spectrometric analysis have to be undertaken, especially for determining the nature of additives, flavoring agents, preservatives, pH altering substances and for the analysis of acetaldehyde levels. With the above evidence, we may make a guarded speculation that it is possible for the manufacturer to add flavoring agents or preservatives containing acetaldehyde for the purpose of improving acceptability of the product by the user.
Limitations of the study
Epidemiological studies on the effect of smokeless tobacco in pathogenesis of oral diseases need to be done after excluding confounding factors like smoking and alcohol consumption, due to the high prevalence of more than one habit in our population. Designing a study with a larger sample size is mandatory to validate the results we have obtained in this study and to determine the correlation between various smoking and chewing indices and the salivary acetaldehyde levels. Within the limitation of the study based on the frequency of saliva and serum collection saliva sample will be better for the estimation acetaldehyde. Since acetaldehyde is more in saliva due to sustained release of tobacco products due to prolong frequency of habit.
| Conclusion|| |
Our study results revealed that there is no statistical difference in either serum or salivary acetaldehyde levels between Pan Masala chewers (with or without tobacco) and smokers, however showed higher mean serum and salivary acetaldehyde levels than the normal subjects and betel quid chewers. Nonetheless, assessing the genotoxic potential of the salivary acetaldehyde obtained from the samples is of prime importance and a natural evolution of this study. Baseline values of salivary and serum acetaldehyde in normal healthy subjects (with no habits) should be studied from a larger set of individuals to identify the role of various dietary sources in our population. Segregation of so-called Plain pan masala that is supposedly tobacco free and studying the salivary acetaldehyde levels in the users has to be evaluated further. Though for various social, economic and administrative reasons and manipulation by the tobacco products, implementation of the ban is going to be a formidable challenge. Thus, absolute ban of all pan masala products (plain or with tobacco) is the ideal solution for minimizing the oral cancer burden in India.
I would like to thank Dr. Swaminathan of Astha Giri Herbal Research Foundation for his support in the laboratory procedures.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]