Salivary nitric oxide, Simplified Oral Hygiene Index, and salivary flow rate in smokers and non-smokers: a cross-sectional study (2024)

Keywords: Salivary Nitric Oxide, Oral Hygiene Index, Salivary Flow Rate, Smoker, Griess Method, Whole Saliva

Inclusion criteria

All participants: 18–24 years old (late adolescence/young adulthood), in good health, absence of systemic disease or history of systemic disease, normal growth and development, not having consumed food for the two hours before sampling. We assessed young adults assuming that they had not suffered any age related disease that could interfere with levels of NO. Smokers: not having smoked in the hour prior to sampling, been smoking for more than six months and consume at least one tobacco cigarette per day (containing no cloves). Non-smokers: never smoked or not smoked for more than 5 years.

Exclusion criteria

All participants: age <18 years or >24 years, having systemic or congenital diseases related to the oral cavity, currently undergoing medical therapy, currently taking local or systemic medication that might affect saliva composition.

All participants were interviewed in order to obtain all data, which were recorded using a data form (available asExtended data)⁶ prior to data entry into a computer spreadsheet. Each interview was conducted by two authors of this study team (ACP and EWB) at the Faculty of Health Science Universitas Indonesia. Depok Indonesia. Participants were instructed not to ingest food for a minimum of two hours before their saliva samples were collected. The smokers were classified into “light,” “moderate,” and “heavy” smokers based on the number of cigarettes consumed in a day (light:1–10 cigarettes, moderate; 11–20 cigarettes and heavy smoker, smoking more than 20 cigarettes), Individuals who had not smoked in the past 5 years were classified as non-smokers. According the study at the Behavioral Endocrinology Laboratory at Penn State University. Newport, one cigarette contains on average 13.4 mg nicotine. Hence we categorized in a day light smoker have consumed less than 134 mg nicotine, moderate smoker between 134–268 mg nicotine and heavy smoker more than 268 mg nicotine.

Oral hygiene index is a method for classifying the oral hygiene status of population that was determined using the Simplified Oral Hygiene Index (OHI-S), comprising two components: plaque (debris) and calculus. The OHI-S was calculated on six tooth surfaces consisting of buccal surfaces in teeth 11, 16, 26, and 31; and lingual surfaces in teeth 36 and 46⁷. The OHI-S categories and their corresponding scores were as follows, good (0.0-1.2) moderate (1.3-3.0) and bad (3.1-6.0)⁷.

In this study, the saliva samples collected were unstimulated whole saliva (UWS). Over a period of three minutes, all saliva was collected in a 14-mL Falcon tube and the resulting volume was divided to 3 to get the salivary flow rate per minute. Following this, a further sample was collected by having individual participants spit 1 ml saliva into a sterile 14 ml tube. It was then transferred and stored in 1.5 ml microcentrifuge tube containing 500 µL phosphate buffer saline (PBS) (Sigma-Aldrich Dorset UK. Cat.P4417) and a protease inhibitor 0.1M Phenyl methane sulfonyl fluoride (PMSF) (Sigma-Aldrich, Cat. 329-98-6). The saliva samples were centrifuged at 5000×g for 5 min at 4°C. This was done, to separate the supernatant and the pellet in the saliva. The supernatant, containing organic components, was then stored at -20°C and the pellets were discarded. The NO concentration of the supernatant was determined by nitrite levels, using the Griess reaction (Sigma-Aldrich, Dorset, UK, Cat. G4410). Briefly, a duplicate of 50 µL of the supernatant and 50 µL of Griess reagents were mixed and incubated in the dark for 10 minutes at room temperature, then the mixture was read at 450 nm by a spectrophotometer (AccuReader. M965/M965+) Nangang, Taipei, Taiwan.

Statistical analysis

Data were analyzed using SPSS version 22.0. The Mann-Whitney U-test was used to analyze differences between smokers and non-smokers, ANOVA was used to analyze the significant difference between four groups (non-smokers, light smokers, moderate smokers, and heavy smokers). Spearman’s correlation was used to analyze the relationship between salivary flow rate or salivary nitric oxide concentration and OHI-S (for smokers only).

Unstimulated whole salivary flow rate in smokers and non-smokers

The results, after measuring the unstimulated salivary flow rate in 25 smokers and 25 non-smokers, showed that the flow rate in smokers was lower at 0.30 ml/minute, with a minimum value of 0.10 ml/minute and a maximum value of 1.67 ml/minute. In comparison, non-smokers had a flow rate of 0.33 ml/minute, with a minimum value of 0.06 ml/minute and a maximum value of 1 ml/minute (Figure 1). Analysis through the Normality test showed that the distribution of data was abnormal. There was no significant difference between unstimulated salivary flow rates in smokers and non-smokers according to the Mann-Whitney statistical test (p = 0.748).

Unstimulated whole salivary flow rate within the smokers’ group, based on the extent of smoking

There was a trend of decreasing salivary flow rate with increasing levels of smoking from “light,” to “moderate,” to “heavy”. The results of the Kruskal Wallis statistical test showed that the average unstimulated salivary flow rate in heavy smokers was lower than that of light and moderate smokers, with no significant difference between the smokers (p = 0.151). As shown inFigure 2, light smokers had an unstimulated salivary flow rate of 0.50 ml/minute (minimum value of 0.13 ml/minute and a maximum of 1.67 ml/minute). The unstimulated salivary flow rate in moderate smokers was 0.2830 ml/minute (minimum 0.10 ml/minute, and maximum value was 0.67 ml/minute). Heavy smokers had a salivary flow rate of 0.20 ml/minute with a minimum value of 0.10 ml/minute and a maximum value of 0.30 ml/minute.

The concentration of salivary nitric oxide in smokers and non-smokers

Based on the results of the Mann-Whitney statistical test, there was a significant difference between the concentrations of NO in smokers and non-smokers (p = 0.040). Smokers displayed higher salivary NO concentrations (185.4 µM; 114.60-295.10 µM) than non-smokers (139.5 µM; 97.70-538.4 µM) (Figure 3).

The concentration of salivary nitric oxide within the smokers’ group, based on the extent of smoking

Amongst the 25 smokers, 14 (56%) were light smokers, 8 (36%) were moderate smokers, and 3 (12%) were heavy smokers.Figure 4 shows an increasing trend in NO concentrations in the saliva with increased extent of smoking. ANOVA analysis found no significant difference in NO concentration (p = 0.600) between the different smoker groups. The concentration of NO in heavy smokers was higher than that for moderate and light smokers; 220.333 µM for heavy smokers, 196.950 µM for moderate smokers, and 183.642 µM for light smokers.

Relationship between unstimulated whole salivary flow rate and the status of oral hygiene indicated by the OHI-S for the smoker group.

Results of the Spearman’s correlation test to determine the relationship between salivary flow rate of the smokers and the status of oral and dental hygiene indicated by OHI-S scores, found an insignificant negative relationship (r = -0.118, p = 0.573). This is depicted inFigure 5.

The relationship between salivary nitric oxide concentration and the status of oral hygiene assessed through the OHI-S of smokers

The results of the analysis of the relationship between NO concentrations and oral hygiene status assessed through the OHI-S of smokers are illustrated inFigure 6. The Spearman’s correlation test showed that there was a moderate positive but insignificant relationship (r = 0.305, p = 0.138) between the concentration of NO and the OHI-S.

Underlying data

Open Science Framework: Salivary nitric oxide, Simplified Oral Hygiene Index, and salivary flow rate in smokers and non-smokers.https://doi.org/10.17605/OSF.IO/WD2GV⁶.

This project contains the following underlying data:

• F1000 Raw data for NO concentrations (salivary NO concentrations of each participant).

• F1000 S-OHIS (raw data for plaque and calculus for each participant).

Extended data

Open Science Framework: Salivary nitric oxide, Simplified Oral Hygiene Index, and salivary flow rate in smokers and non-smokers.https://doi.org/10.17605/OSF.IO/WD2GV⁶.

This project contains the following extended data:

• Quesioner F1000 research (English translation of the questionnaire used in this study).

Data are available under the terms of theCreative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication).

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