Assessment of Salivary Flow Rate and Salivary PH in Subjects with Smoked, Smokeless, and Combined form of Tobacco Habits (Epidemiological-Based Study)

Abstract

Background:

Saliva is a complicated secretion of the oral cavity and is the first fluid to be exposed to the toxic components of tobacco products. Saliva undergoes structural and functional changes due to tobacco use throughout time. As a result, measurements of salivary flow rate (SFR) and pH were made in subjects with habit of smokeless, smoked, and combined forms of tobacco habits.

Methodology:

The study was conducted in a dental institute. Subjects with the habit of smoking, smokeless, and combined forms of habits were included in this study. Schirmer tear strips and pH strips were used to assess SFR and pH.

Results:

On comparison of SFR between control and habit groups, a statistically significant reduction of SFR was observed in habit groups. On comparison of salivary pH, a statistically significant decrease was observed in smokeless and smoked form of tobacco usage group when compared with the control group. Duration, frequency, and exposure also alter SFR and pH. Significant gradual decline in resting salivary flow rate levels with increase in duration of tobacco usage and increase in number of packs consumed/day was observed.

Conclusion:

The role of salivary flow and pH is to maintain oral and dental health. Based on the results of this study, we concluded that long-term habits, whether smokeless, smoking, or combined tobacco habit, significantly reduce SFR and pH. These alterations can render oral mucosa more vulnerable to various oral diseases.

INTRODUCTION

Saliva is a crucial oral fluid that is sometimes overlooked. It is vital to keeping and sustaining dental health, yet it is often overlooked until the quantity or quality is compromised. Much recent research has been conducted on the subject of salivary dysfunction as it relates to disease or as a side effect of particular drugs. Saliva is also to be an effective noninvasive systemic sampling method for medical diagnosis and research. As a result, doctors must have an adequate grasp of the normal range of salivary flow and function.[1]

METHODOLOGY

The study was conducted in a workplace setting. A total of 212 patients were enrolled from the Department of Oral Medicine and Radiology with an age range of 18–70 years, with the habit of smoking, smokeless tobacco, or combined habits of smoking and smokeless tobacco were included in this study. The study was approved by Institutional Ethical Committee (OMR/01/112021/IEC). The study was performed in accordance with Declaration of Helsinki 1975, as revised in 2013. Informed consent from the patient was obtained well before the study began. Subjects with systemic disease, who were on medication, who consume alcohol, and with a history of radiotherapy, trauma to the head and neck, dentures wearer, and pregnant or postmenopausal women were excluded from the study. Further, the study group was categorized into four groups such as:

• Group I (smoking form): Subjects with a habit of smoking.

• Group II (smokeless form): Subjects with the habit of using smokeless tobacco.

• Group III (combined): Subjects with combined habits of smoking and smokeless tobacco.

• Group IV (control): Healthy subjects with no habits as controls.

The investigation was conducted between 9 and 12 a.m. to eliminate diurnal variation. To diagnose lesions, a complete oral examination was performed. Subjects were asked to swallow saliva in their mouth before the test and to rest their tongue on the hard palate so that the test strip did not touch the tongue during the test. For 3 minutes, the rounded end of Schirmer tear strips was put in the floor of the mouth region, and salivary flow rate (SFR) measurements were taken by noting the wet calibrations on the Schirmer strips [Supplementary Figures 1 ^{(309.1KB, tif)} and 2 ^{(385.6KB, tif)} ].

Inference[2]

• Normal SFR: 25–30 mm

• Dry mouth: 10–15 mm

• Mild dryness: 6–10 mm

• Moderate dryness: 2–5 mm

• Severe dryness: 0–1 mm

Furthermore, the pH strips were placed in the mouth’s floor, and any change in color was matched with the color coding on the pH strip and recorded accordingly.

Inference [Supplementary Figures 3 ^{(330.5KB, tif)} and 4 ^{(472.7KB, tif)} ][3,4]

• Normal salivary PH ranges from 6.5 to 7.5.

• If the PH value is below 6.5, it indicates an acidic pH.

• If the value is above 7.5, it indicates an alkaline pH.

Statistical analysis

Sample size estimation was done by using GPower software (version 3.0). Sample size was estimated for F test and ANOVA: Omnibus fixed one way, for 4 groups with equal sample size (tobacco smokers, nonsmoking tobacco users, combined smoking and chewing tobacco users, and health individuals without any tobacco habit) was chosen.

A minimum total sample size of 212 (53 per group) was found to be sufficient for an alpha of 0.05, power of 95%, and 0.23 as effect size (assessed for difference in salivary flow rate among tobacco smokers, nonsmoking tobacco users, combined smoking and chewing tobacco users, and health individuals without any tobacco habit).

RESULT

In our study, a total of 2800 subjects were screened in a span of 2 years in a hospital setting; 212 subjects were included in the study, according to inclusion and exclusion criteria, and among them, 53 subjects were included in each group.

Gender-wise distribution of study participants among study groups was found to be statistically significant. The proportion of males among all four group was found to be significantly high with (P value < 0.001).

Distribution of study participants among three study groups according to duration of habit was found to be statistically significant. Among the chewers group, the proportion of study participants with a longer duration of habit was significantly more in 5–15 years, whereas in smokers and combined groups, the proportion of participants was more in 3–5 years duration with P value = 0.029 [Table 1].

Table 1.

Intergroup comparison of duration of habit

			Duration					Total
			<1 Year	1-3 year	3-5 year	5-15 year	15-30 year
Group	Gr 1 (smokeless)	n	1	8	7	26	11	53
		%	1.9%	15.1%	13.2%	49.1%	20.8%	100.0%
	Gr 2 (smoked)	n	1	15	17	12	8	53
		%	1.9%	28.3%	32.1%	22.6%	15.1%	100.0%
	Gr 3 (combined)	n	0	8	20	15	10	53
		%	0.0%	15.1%	37.7%	28.3%	18.9%	100.0%
Total		n	2	31	44	53	29	159
		%	1.3%	19.5%	27.7%	33.3%	18.2%	100.0%
P			0.029, S

Maximum frequency of up to 5 times/day was significantly more among Gr 1, 2, and 3 subjects, while minimum frequency was >10 times/day with P value 0.014 [Table 2].

Table 2.

Intergroup comparison of frequency of habit

			Frequency			Total
			Upto 5 times	6-10 times	>10 times
Group	Gr 1 (smokeless)	n	27	20	6	53
		%	50.9%	37.7%	11.3%	100.0%
	Gr 2 (smoked)	n	37	12	4	53
		%	69.8%	22.6%	7.5%	100.0%
	Gr 3 (combined)	n	37	6	10	53
		%	69.8%	11.3%	18.9%	100.0%
Total		n	101	38	20	159
		%	63.5%	23.9%	12.6%	100.0%
P			0.014, S

Exposure of habit 3–5 min was found to be significantly more among Gr 2 and Gr 3 subjects, while >5 min was found to be significantly more among Gr 1 subjects with P value 0.017 [Table 3].

Table 3.

Intergroup comparison of exposure of habit

			Exposure of habit				Total
			<1 min	1-3 min	3-5 min	>5 min
Group	Gr 1 (smokeless)	n	1	6	17	29	53
		%	1.9%	11.3%	32.1%	54.7%	100.0%
	Gr 2 (smoked)	n	0	9	30	14	53
		%	0.0%	17.0%	56.6%	26.4%	100.0%
	Gr 3 (combined)	n	0	14	22	17	53
		%	0.0%	26.4%	41.5%	32.1%	100.0%
Total		n	1	29	69	60	159
		%	0.6%	18.2%	43.4%	37.7%	100.0%
P			0.017, S

Description of post hoc pairwise comparison SFR: SFR was found to be statistically significant among four study groups. It was found to be significantly higher among Gr 1 and 4 as compared to Gr 3, which was significantly higher than that among Gr 2 with P value 0.023.

Similarly, at 2 min, SFR was found to be significantly higher among Gr 4 compared to Gr 1, which was further significantly higher than that among Gr 2 and Gr 3 with P value < 0.001.

Similarly, at 3 min, SFR was found to be significantly higher among Gr 4 as compared to Gr 3, which was further significantly higher than that among Gr 1 and Gr 2 with P value < 0.001 [Table 4].

Table 4.

Intergroup comparison of mean salivary flow rate at 1 min, 2 min, and 3 min

Salivary flow	Group	n	Mean	Std. Deviation	95% Confidence Interval for Mean		P	Post hoc pairwise comparison
					Lower Bound	Upper Bound
Salivary flow 1 min	Gr 1 (smokeless)	53	9.2925	5.55171	7.7622	10.8227	0.023, S	Gr 2 < Gr 3 < Gr 1, Gr 4
	Gr 2 (smoked)	53	6.9340	4.98848	5.5590	8.3090
	Gr 3 (combined)	53	7.8868	4.88093	6.5414	9.2321
	Gr 4 (control)	53	9.3774	3.30633	8.4660	10.2887
Salivary flow 2 min	Gr 1 (smokeless)	53	15.1321	8.73111	12.7255	17.5387	<0.001, S	Gr 2, Gr 3 < Gr 1 < Gr 4
	Gr 2 (smoked)	53	14.2642	6.42157	12.4941	16.0342
	Gr 3 (combined)	53	14.3962	7.62861	12.2935	16.4989
	Gr 4 (control)	53	23.1887	3.05750	22.3459	24.0314
Salivary flow 3 min	Gr 1 (smokeless)	53	19.4151	10.42636	16.5412	22.2890	<0.001, S	Gr 1, Gr 2 < Gr 3 < Gr 4
	Gr 2 (smoked)	53	19.3585	6.40641	17.5927	21.1243
	Gr 3	53	21.3962	8.65212	19.0114	23.7810
	Gr 4	53	31.1509	3.09712	30.2973	32.0046

Description of post hoc pairwise comparison of Salivary pH: It was statistically significant among four study groups. It was found to be significantly higher among Gr 4 than in Gr 1, Gr 2, and Gr 3. Among different groups of tobacco users, no significant difference could be found among salivary pH (P value < 0.001) [Table 5].

Table 5.

Intergroup comparison of mean salivary pH

Salivary pH	Group	n	Mean	Std. Deviation	95% Confidence Interval for Mean		P	Post hoc pairwise comparison
					Lower Bound	Upper Bound
pH	Gr 1 (smokeless)	53	6.4528	1.02968	6.1690	6.7366	<0.001, S	Gr 1, Gr 2, Gr 3<Gr 4
	Gr 2 (smoking)	53	6.3113	0.78585	6.0947	6.5279
	Gr 3 (combined)	53	6.2264	1.18308	5.9003	6.5525
	Gr 4 (control)	53	7.4264	0.61273	7.2575	7.5953

Distribution of study population among different study groups according to salivary flow at 1, 2, and 3 minutes was found to be statistically significant. The frequency of subjects with extremely low salivary flow, i.e., <2 mm, was found to be significantly more among Gr 1 and Gr 2 as compared to Gr 3 and Gr 4 with P value 0.001.

The salivary flow in the range of 25–30 mm was found to be significantly more common among Gr 4, i.e., control group with P value < 0.001.

In addition, the salivary flow in the range of 11–24 mm was found to be significantly more common among Gr 2 with P value < 0.001.

Distribution of study population among different study groups according to salivary pH was found to be statistically significant. The frequency of subjects with alkaline salivary pH, i.e., above 7.4, was found to be significantly more among Gr 4 as compared to other study groups. The frequency of subjects with acidic salivary pH, i.e., <6.7, was found to be significantly more among Gr 1, 2, and 3 as compared to Group 4 with P value < 0.001.

DISCUSSION

Saliva is a complicated and crucial bodily fluid that is critical to dental health.[5] The etiology of oral and dental disorders is influenced by altered whole-mouth SFR.[6] There is a wealth of clinical and epidemiological data that tobacco has a negative impact on oral health.[7,8] Saliva is the first biological fluid to be exposed to tobacco (smoked or smokeless form), which contains a variety of harmful components that cause structural and functional alterations in saliva.[9]

The pH of saliva is normally between 6.7 and 7.3. SFR (unstimulated whole salivary flow rate) is approximately 0.3–0.5 ml/min. The buffering capacity of saliva is an important element that normally correlates with flow rate; a drop in flow rate tends to decrease buffering capacity, resulting in a decrease in salivary pH (acidic), making the oral mucosa and dental structures more susceptible to changes.[10] The fluid secreted in the mouth cavity in the absence of any external or pharmacological stimuli is referred to as the Resting or Unstimulated Salivary Flow Rate (RSFR). The RSFR in healthy people is reported to be between 0.3 and 0.5 ml/min.[11]

In our study, the tobacco chewing habit was more prevalent in males (84.9%) and females (15.1%). These findings were similar to the study conducted by Y. Kantak et al., (2017)[12] and S J.S Bhat et al., (2010)[13] who observed 33 males and 11 females among chewers and 79.9% males, respectively. This could be because some male occupations involve a significant amount of physical energy and a high level of focus, such as drivers with irregular working hours. This can be stressful, and when combined with peer pressure, it can lead to the development of harmful oral habits.[14] Chewing was also used to relieve boredom and anxiety, to promote focused attention at work, to facilitate social interaction among friends, and as a medicine for digestive and dental health.[13] The habit of smoking was also more prevalent among males (100%), and in females, it was negligible. This was in accordance with the study conducted by Asha. V et al., (2015)[15] with 73 males and 2 females had habit of smoking. Among men 100% smoked and 84.9% chewed tobacco and in females there were 0% smokers and 15.1% chewers which was in accordance with Sen U (2002). In Kolkata, a multistage cluster sample survey of 100 respondents from each of 60 clusters (total sample of 12,000) of adults (over the age of 18) was undertaken. Using conventional WHO standards, data on demographic and socioeconomic variables, as well as tobacco use, were collected. Men smoked 28% of the time and chewed 36% of the time, while women smoked 0.5% of the time and chewed 19% of the time. Smoking was substantially associated with socioeconomic status.[16]

The mean duration of smokeless habit between 15 and 30 years was (20.8%) followed by 1–3 years (15.1%), 3–5 years (13.2%), and <1 year (1.9%), whereas maximum duration was observed in 49.1% among 5–15 years which were in accordance with the study conducted by Shrihari J.S Bhat et al., (2010)[13] who reported <30 years (55%) and studies conducted by Yashashree Kantak et al., (2017)[12] T. Rooban et al., (2006)[5] reported mean duration of 6.41 years in their study subjects, respectively. The mean duration of smoking was minimum between <1 year (1.9%), 15–30 years (15.1%), 5–15 years (22.6%), and 1–3 years (28.3%), and the maximum duration was observed in 3–5 years (32.1%) which were not similar to the findings conducted by Maryam Rad et al., (2010)[17] who observed mean duration of smoking 12.5 years (min = 2, maximum 30 years).

The minimum mean frequency was seen at 11.3% (>10 times), followed by 37.7% (6–10 times), whereas the maximum mean frequency was 50.9% (up to 5 times). These findings were similar to the study conducted by S. J. S Bhat et al., (mean = 1.9),[13] Y. Kantak et al., (2017)[12] reported a mean frequency of 3.90, and Rooban et al., (2006)[5] as 4.58 in their studies, respectively. The mean frequency in subjects who smoked was observed at a minimum of 7.5% (>10 times), followed by 22.6% (6-10 times), and the maximum observed frequency was 69.8% (up to 5 times). These findings were not in accordance with the study conducted by Maryam Rad et al., (2010)[17] who observed 14.8 cigarettes per day (min = 2 and maximum = 40 cigarettes/day).

The minimum mean exposure of habit (smokeless) observed <1 min (1.9%), followed by 1–3 min (11.3%) and 3–5 min (32.1%), and maximum exposure observed is >5 min (54.7%). These findings were similar to the studies conducted by S. J. S Bhat et al., (2010)[13] (no longer than 20 mins), Y. Kantak et al., (2017)[12] (14 min), and T. Rooban et al., (2006)[5] (13.08 min), respectively. The minimum mean exposure of habit (smoking) was observed at 1–3 min (17%), followed by >5 min (26.4%), and maximum exposure was observed in 3–5 min (56.6%).

Nicotine is an addictive component found in cigarettes that can induce dependence and make cigarettes a daily requirement in adulthood. Nicotine can also trigger stimulation and sedation in the central nervous system depending on the quantity of exposure. Each brand of cigarette has a distinct nicotine content, resulting in different levels of nicotine absorbed by everyone although smoking the same number of cigarettes.[18] Nicotine can impact nerve activity by acting on certain cholinergic receptors in the brain, causing changes in salivary pH and flow rates.[19]

Areca nut has habit-forming and dose-dependent effects. The fact that the effects are more severe in new or occasional chewers and less pronounced in frequent chewers shows that tolerance or habituation develops in areca nut consumption. As a result, the receptors become accustomed to the stimuli (T. Rooban et al., 2006; Chu, 2001).[5]

This study found a significant, steady decrease in resting salivary flow rate (RSFR) values as the duration of cigarette use increased. The lowest RSFR was seen in people who had been consuming gutka for more than ten years. Maryam. Rad et al.[17] discovered considerably lower RSFR values in persistent smokers as well. Chewing tobacco increases stimulated salivary flow rates due to the parasympathetic impact, which is absent during nonchewing periods. Furthermore, studies demonstrate that nicotine and areca nut products influence the autonomic nervous system by increasing plasma levels of adrenaline and norepinephrine, which may result in lower flow rates between chewing sessions.[20,21] There was also a significant decrease in RSFR with increasing pack consumption per day and duration of exposure (P = 0.0001) among all categories of tobacco chewers. Gutka, paan, and numerous users who consumed more than 20 packs per day had low RSFR. This was similar to the observations of T. Rooban et al.[5]

Subjects who smoked/chewed for 15–30 years were observed to be at more than three times the risk of those who smoked/chewed for 5 years. This was a statistically significant finding. With higher frequency, there was an increased chance of lesions. When the duration of the habit was compared, individuals who had it for 5–15 years were shown to be at a higher risk. Significant findings were observed for all domains based on frequency, with participants with a habit frequency of 6–10 times/day being at the highest risk of developing lesions.[14]

The mean SFR was found to be low in group 2 (smokers) and group 3 (smoking and smokeless) compared to group 4 (control), which was significant. This drop in SFR in group 2 and group 3 subjects is most likely related to nicotine’s effect on taste sensitivity. This finding is consistent with the findings of T. Rooban et al.[5] Similarly, A. Kanwar et al., (2013)[22] discovered statistically significant variations in mean SFR amongst smokes, chewers, and controls. M. Singh et al., (2015)[23] discovered that smokers have a lower salivary flow rate than controls. A number of studies have shown that smoking causes a noticeable short-term increase in SFRs because it increases the activity of salivary glands in anyone who begins smoking, whereas long-term use has shown that some individuals develop tolerance to the salivary effect of smoking, so it reduces SFRs. Furthermore, smoking is one of the risk factors for decreased saliva and xerostomia.[24] A constant heat blowing into the mouth cavity may potentially produce changes in blood flow and a decrease in salivary secretion.[25] This is due to the fact that smoking practices that involve a large number of cigarette intakes per day over a long period of time might result in a loss in sensitivity to oral receptors, resulting in a decrease in salivary reflex.[26] For more than 6 months, smoking 10–15 cigarettes per day may reduce salivary flow rate to 0.20 ml/min.[23] As a result of the substantial relationship between smoking duration and salivary flow rate, both stimulated and unstimulated salivary flow rates dropped as smoking duration rose; however, the decreased salivary flow rates were not significant when compared to the number of cigarettes ingested daily.[27]

Moreover, in the present study, it was also observed that the mean salivary pH of saliva, was low in groups 1, 2, and 3 compared to group 4, and this difference was statistically significant. In our study group 3 (combined tobacco habit) followed by groups 2 and 1, participants had the lowest salivary pH. The reason could be due to the usage of smokeless lime, which can react with the bicarbonate buffering mechanism, causing a loss of bicarbonate and so making saliva more acidic. As electrolytes and ions interact with the buffering mechanisms of saliva, they change the pH. These findings agree with those of Khan et al.,[26] T. Rooban et al.,[5] and M. Singh et al.[23] In the current study, the mean pH for group 1 (6.45) was followed by group 2 (6.3 pH), group 3 (6.2 pH), and group 4 (7.4) when compared. A lower pH was observed in groups 1, 2, and 3 when compared to group 4. Salivary pH was lowest in Group 3 compared to Groups 1, 2, and 3, most likely because of the usage of smokeless lime, which can react with the bicarbonate buffering system by releasing bicarbonate, making saliva more acidic. As electrolytes and ions interact with saliva’s buffering systems, they change the pH. These findings were consistent with the findings of Neeraj Grover et al., (2016).[28] G. J Khan et al.[26] also found that smokers had lower salivary pH than nonsmokers, which was similar to the current study’s findings.

Nonchewers had a pH of 7.42 on average. Those that chew raw areca nut (RAN) have a higher mean PH (6.45), indicating that the secretion is more serious, as documented by R.K. Chadda et al, (2003).[29] SFR elevates salivary pH via increasing bicarbonate secretion, and so an increase in saliva bicarbonate raises the pH.[30] Lime presumably reacts with the bicarbonate buffering mechanism in habitual BQ (Betel quid) chewers, causing bicarbonate loss and increasing saliva acidity (6.22). The SFR reduces in those who chew BQT and PAN, making the pH less acidic. These were consistent with Rooban et al. (2006)[5]

CONCLUSION

Salivary flow and pH have a role in maintaining oral and dental health. Based on the findings of this study, we concluded that long-term practices, whether smokeless, smoking, or a combination of the two, dramatically diminish SFR and pH. It can indicate if a person is healthy or sick. These changes can make the oral mucosa more susceptible to oral infections and tooth decay. Given the multiple functions of saliva in the oral cavity, as well as its protective influence on mucosa and teeth, patients should be taught and motivated to abandon the habit. Chemical exposure can be detected via qualitative and quantitative changes in saliva.

Further recommendation

A further recommendation is to incorporate it as a behavior intervention technique in patient counseling, education, and motivation for tobacco cessation and overall monitoring of habit abstinence and cessation.

Conflicts of interest

There are no conflicts of interest.

Supplementary Figure 1

Schirmer tear strip

Supplementary Figure 2

Salivary flow rate determination using Schirmer tear strip

Supplementary Figure 3

pH strip

Supplementary Figure 4

Salivary pH determination using pH strip

Funding Statement

Nil.