Correlation between salivary glutathione, total antioxidant, and periodontal status among smokers and nonsmokers: A cross-sectional study

Kiran Iyer; Laliytha K Bijai; Swapna Munaga

doi:10.4103/jioh.jioh_215_22

ORIGINAL RESEARCH

Year : 2023 | Volume : 15 | Issue : 2 | Page : 200-205

Correlation between salivary glutathione, total antioxidant, and periodontal status among smokers and nonsmokers: A cross-sectional study

Kiran Iyer¹, Laliytha K Bijai², Swapna Munaga³
¹ Preventive Dental Science Department, College of Dentistry, King Saud bin Abdulaziz University for Health Sciences, King Abdullah International Medical Research Centre, Riyadh, Saudi Arabia
² Maxillofacial Surgery and Diagnostic Sciences, College of Dentistry, King Saud bin Abdulaziz University for Health Sciences, King Abdullah International Medical Research Centre, Riyadh, Saudi Arabia
³ Faculty of Dentistry, Department of Restorative and Prosthetic Dental Sciences, College of Dentistry, King Saud bin Abdulaziz University for Health Sciences, King Abdullah International Medical Research Centre, Riyadh, Saudi Arabia

Date of Submission	18-Oct-2023
Date of Decision	25-Feb-2023
Date of Acceptance	04-Mar-2023
Date of Web Publication	28-Apr-2023

Correspondence Address:
Dr. Kiran Iyer
Preventive Dental Science Department, College of Dentistry, King Saud bin Abdulaziz University for Health Sciences, King Abdullah International Medical Research Centre, Prince Mutib Ibn Abdullah Ibn Abdulaziz Road, Ar Rimayah, P.O. Box 22490, Riyadh 11426
Saudi Arabia

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/jioh.jioh_215_22

Abstract

Aim: To estimate glutathione (GSH) and total antioxidant (AO) levels in the saliva of smokers and nonsmokers and to establish a correlation with periodontal status. Materials and Methods: A total of 60 males (30 smokers and 30 nonsmokers) were part of the study. Salivary samples were collected by unstimulated method and were stored at 20°C. Salivary GSH concentration was assessed using the enzymatic recycling method and AO levels by phosphomolybdate method spectrophotometrically. Periodontal status was assessed based on the CPITN index. Kruskal–Wallis H test, unpaired “t”-test, and Spearman’s correlation coefficient were used to analyze the statistical significance. Results: The salivary GSH levels in smokers were lower than in nonsmokers. The mean salivary GSH levels of smokers were 10.22 µM, whereas among nonsmokers was 12.99 µM. The mean total AO level of smokers and nonsmokers was 181.18 and 162.58 µgm/mL, respectively. The difference was statistically significant (P = 0.02). Kruskal–Wallis H test showed to be statistically significant between the periodontal status of smokers and nonsmokers (P < 0.05). Conclusion: AO levels were significantly lower in smokers than in nonsmokers. The periodontal status showed a higher prevalence of calculus and shallow pockets among the smokers. The correlation of all three parameters showed statistical significance between salivary GSH and AO and periodontal status.

Keywords: Antioxidants, Glutathione, Nonsmokers, Periodontal Status, Smokers

How to cite this article:
Iyer K, Bijai LK, Munaga S. Correlation between salivary glutathione, total antioxidant, and periodontal status among smokers and nonsmokers: A cross-sectional study. J Int Oral Health 2023;15:200-5

How to cite this URL:
Iyer K, Bijai LK, Munaga S. Correlation between salivary glutathione, total antioxidant, and periodontal status among smokers and nonsmokers: A cross-sectional study. J Int Oral Health [serial online] 2023 [cited 2023 Jun 1];15:200-5. Available from: https://www.jioh.org/text.asp?2023/15/2/200/375365

Introduction

“Oxidative stress” forms the interlink between free radicals and disease.^{[1],[2],[3],[4]} The human body has an equally impressive defense mechanism in the form of antioxidants (AOs).^[5] Glutathione (GSH) is one such essential tripeptide thiol AO produced in the human liver. It has a critical role in protecting cells from oxidative stress.^[6],[7],[8]

Cigarette smoking has long been associated with several chronic pathological changes including periodontium. Studies have shown there is a decrease in the GSH and AO levels in the body fluids and its components of smokers compared with nonsmokers.^{[9],[10],[11],[12]}

Periodontal health and disease-related research has gained momentum (biomarkers, metagenomics, dental loupes, and baicalin) in the last decade since a high prevalence of the periodontal disease is observed in populations; its early identification and appropriate intervention become more necessary than ever.^{[13],[14],[15]}

Saliva acts as an essential biomarker for the progression of many oral diseases^[7]; literature evidence suggests that very few studies have attempted to assess the role of salivary GSH as a biomarker in periodontal disease progression. Further, these studies that assessed salivary GSH and total AO capacity among smokers and nonsmokers did so independent of its role in periodontal status or periodontal disease progression, as well as there is no concurrence on the method used for the estimation of GSH in any of the studies.^{[12],[16],[17]} The present study was undertaken with a rationale to address the above-mentioned literature gaps on salivary GSH and its role as a potential biomarker for periodontal disease, based on the novel enzymatic recycling method put forward for the assessment of salivary GSH.

The present study aimed to estimate the salivary GSH using the kinetic enzymatic recycling essay method and total AO levels with ammonium molybdate method and to assess its correlation with the periodontal status of smokers and nonsmokers, respectively.

Material and Methods

Setting and design

This cross-sectional study was conducted on subjects 20–60 years old at the public health dentistry department, Yenepoya Research Centre, and Yenepoya Dental College during the period of May–June 2021.

Ethical approval for the was obtained on March 24, 2021, from the Institutional Review Board of Yenepoya University (YDC24-2021) Mangalore, India. Written informed consent was obtained from all the subjects prior to enrollment in the study. All procedures in the study at all times adhere to the ethical guidelines as per the Declaration of Helsinki.

Sampling criteria

Nonprobability convenient sampling method was used to recruit the patients in the study. The sample size was derived using the “t” test difference between two independent means formulas with a 95% confidence interval, 80% power, and 0.5 effect size. The sample allocation was nonrandomized since they are naturally allocated into groups of smokers and nonsmokers.

The study included subjects with a history of smoking for 5 years and above and was considered in the group of smokers. Investigators ensured that those categorized as “nonsmokers” were subjects with absolutely no history of smoking; to avoid enrolling “ever” smokers. The subjects were allocated based on their history of smoking into smokers and nonsmokers. The subjects were age- and gender-matched to avoid bias arising due to confounding. Bias arising due to confounders such as alcohol consumption was not controlled in the study, as both smokers and nonsmokers gave gender-matched alcohol consumption, and hence, investigators considered it to be confounders distributed between the groups. Subjects with systemic illness, “ever” smokers, who underwent oral prophylaxis in the last 3 months and those not willing to provide informed consent were excluded from the study.

A total of 60 male subjects (30 smokers and 30 nonsmokers) were part of the study. Data collection involved a two-step process, at the department clinic: first, salivary samples from subjects were collected by the unstimulated method in a vial, which was immediately sent (in a thermal ice box) for centrifugation at the research center and stored at (−20°C) until the time of processing; second, periodontal status (CPITN Index) was evaluated by the principal investigator (PI) among subjects after the salivary sample collection for each subject using the CPITN-E probe. The PI was priorly calibrated based on interexaminer reliability along with the subject expert at the department, for periodontal status assessment, with a kappa score of 0.8 (substantial).

Reagent setup

Buffer used is as follows: 0.1 M potassium phosphate buffer with 5-mM ethylenediamine tetraacetic acid disodium salt, pH 7.5 (Potassium Phosphate- ethylenediamine tetraacetic acid (KPE)).

The materials used to estimate GSH were potassium dihydrogen orthophosphate (KH₂PO₄), dipotassium hydrogen orthophosphate (K₂HPO₄), ethylenediamine tetraacetic acid sodium salt, 5,5’-dithio-bis (2-nitrobenzoic acid), β-nicotinamide adenine dinucleotide phosphate, GSH reductase, and GSH reduced form. All chemical reagents were obtained from Sigma-Aldrich, St. Louis, Missouri, United States for the study.

GSH standards^[11]: preparation of GSH solution (stock) was done by dissolving 1-mg GSH mL⁻¹ in KPE, aliquot, and this solution was stored at (−20°C) until the time of processing. Stock was diluted at 1:100 with KPE to make a working solution of 10 µg mL⁻¹ and later 800 µL of the working solution with 200 µL of KPE to make the top standard concentration.

Salivary GSH concentration was assessed using the enzymatic recycling method. The salivary sample was centrifuged and stored at −20°C until the time of processing. Care was taken to always maintain the temperature at 4°C during the time of processing. Initially, the salivary sample was diluted by adding 100 µL of saliva to 700 µL of KPE later; 200 µL of this mixture was transferred to 1400 µL of KPE for further dilution, to this mixture, 120 µL of glutathione reductase and 120 µL of 5,5’-dithio-bis (2-nitrobenzoic acid) were added, and this mixture was incubated for a period of 3 min. At the end of the incubation period, 120 µL of nicotinamide adenine dinucleotide phosphate was added to the mixture, and immediately, the mixture was transferred to the cuvette and placed in a spectrophotometer to record the absorbance at 412 nm.

Estimation of salivary total antioxidant capacity

The reagents used for estimation were vitamin C solution, 0.6-M sulfuric acid, 4-mM ammonium molybdate, 0.001 N hydrochloric acid, and distilled water. The salivary total AO capacity was measured using the “ammonium molybdate method.”^[12] An aliquot of 0.1 mL of a sample solution containing a reducing species was combined in an Eppendorf tube with 1 mL of reagent solution (0.6 M sulfuric acid, 28-mM sodium phosphate, and 4-mM ammonium molybdate). The tubes were capped and incubated in a thermal block at 95°C for 90 min. After the samples were cooled down sufficiently to room temperature, the absorbance of an aqueous solution of each was measured at 695 nm against blank. Blank solution is composed of 1 mL reagent and appropriate amount of solvent as used for the sample.

Periodontal index (CPITN) recording

It was measured using a CPITN- E probe along with a mouth mirror under the artificial chair light by the PI. The whole procedure was conducted with all infection control measures in place at the time of assessment.

Statistical analysis

The data were entered in a Microsoft Excel spreadsheet and analyzed using the IBM Corp. (2011) SPSS Statistics for Windows, Version 20.0. Armonk, New York. The mean difference in salivary GSH and AO between smokers and nonsmokers was analyzed by unpaired t test. The difference in periodontal status among the groups was analyzed using the Kruskal–Wallis “H” test. Spearman’s correlation matrix was used to assess the correlation of salivary GSH and AO levels with periodontal status among smokers and nonsmokers.

Results

The mean age of participants enrolled in the study was 36 ± 8.4 years. In the present study, the mean salivary GSH levels in smokers (10.22 µM) were lower than that of the nonsmokers (12.99 µM). The mean difference analyzed using the unpaired t test was not statistically significant (P > 0.05) [Figure 1] and [Table 1]. The mean total AO level of smokers and nonsmokers was 162.58 and 181.18 µgm/mL, respectively; the difference in AO levels was statistically significant between the two groups (P = 0.02) [Figure 2].

Figure 1: Salivary GSH levels in smokers and nonsmokers

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Table 1: Comparison of mean glutathione (GSH) and total antioxidant levels between smokers and nonsmokers using unpaired t test

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Figure 2: Total antioxidant levels in the saliva of smokers and nonsmokers

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The periodontal status of the smokers showed a prevalence of a higher percentage of indexed teeth with score 2 (presence of bleeding on probing) and score 3 (presence of periodontal pockets measuring 4–5 mm), and a lower percentage of score 0 (normal periodontium) [Table 2]. Kruskal–Wallis H test showed a statistically significant difference in the periodontal status of smokers and nonsmokers (P < 0.05).

Table 2: Percentage prevalence of periodontal status among smokers and nonsmokers

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Spearman’s correlation matrix ranked AO and GSH values with CPI values for the index tooth assessed. Smokers’ AO had a positive correlation with the periodontal status of most indexed teeth (16/17, 11/21, 26/27, and 31/41), whereas GSH values were negatively correlated with the periodontal status of smokers. Interestingly a significant (P < 0.021) positive correlation was observed overall for the AO values and periodontal status of nonsmokers, when individual index teeth was considered periodontal status of index teeth (46/47) had a positive correlation (P < 0.048) with AO of these subjects [Table 3].

Table 3: Spearman’s correlation matrix between salivary GSH, AO, and periodontal status (CPI)

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Discussion

There is a lacuna in the literature about GSH levels in saliva and its role as an indicator of oxidative stress for tissues within the oral cavity. This study would like to add strength to the existing literature on the topic while assessing additional variables such as total AOs and periodontal status.^[16] In our study, salivary GSH values were not found to be significantly (P > 0.05) different between smokers and nonsmokers, whereas a significant difference in total AO capacity (P = 0.02) and periodontal status (P < 0.05) was observed between the two groups.

The values of salivary GSH obtained in the present study are higher compared to studies by Saggu et al.,^[17] and Arbabi-Kalati et al.,^[18], glutathione peroxidase values were reported in these studies, while the former used Rotruck method for estimation and the latter used Amini method, inconsistency in method used could possibly explain for this difference in observation, whereas the mean GSH levels reported by these two studies among nonsmokers compared to smokers is in line with findings of our study. Saggu et al.^[17] observed mean glutathione peroxidase values to be 2.7 and 1.5 µM among nonsmokers and smokers, respectively. Similarly, glutathione peroxidase values of 1.5 and 1.0 U/mg among nonsmokers and smokers were reported by Arbabi-Kalati et al.^[18] in their study. The present study used the kinetic enzymatic recycling essay method given by Rahman et al.,^[11] which is new with high specificity and sensitivity. According to Rahman et al.,^[11] variation in GSH levels may be due to differences in principal approaches of the methods and variations in processing. Different studies have reported different levels of GSH in both control and pathological samples.^[17],[18]

The findings of our study on total AO capacity in smokers and nonsmokers are in line with the findings of studies by Mortazavi et al.,^[19] and Ghazi et al.,^[20] who reported that total antioxidant capacity (TAC) of saliva in smokers was significantly lower than that in nonsmokers. The AO levels also depend on various factors such as age, diet, and oral hygiene habits of the subjects.^[21] In the present study, it was also observed that exposure to smoking has a statistically significant correlation with decreased salivary TAC (P < 0.05). Such decreases may have a consistent role in the mechanisms, by which the toxic effects of smoking initiate oral inflammatory diseases, promote precancerous transformations, and destroy oral cavity homeostasis.^[17],[18] As per current literature decrease in AOs, an increase in endothelin-1 and salivary C-reactive protein is evident in periodontitis and smokers.^[15]

The findings of our study related to the periodontal status, GSH, and AO among smokers and nonsmokers are in line with the study by Chang et al.,^[22] who reported a significant decrease in bleeding on probing (bop) and salivary oxidative stress biomarkers among nonsmokers compared with current smokers, after nonsurgical periodontal treatment.

Significantly lower mean salivary AO (superoxide dismutase) values among smokers with chronic periodontitis were observed compared with nonsmokers with chronic periodontitis in a study by Naresh et al.,^[23] and Ramesh et al.,^[24] respectively; this finding corroborates with a significant difference in AO values and periodontal status as observed among smokers and nonsmokers in the present study. Decreased TAC owing to oxidative stress has been reported independent of smoking status in chronic periodontitis patients.^[25],[26] Hence, it is increasingly evident that smoking further contributes to increased oxidative stress, which has a bearing effect on periodontal disease progression.^[27] To ascertain the role of the periodontal microbiome in oxidative stress, it needs to be studied in conjugation and independent of factors such as smoking through metagenomics.^[13]

The present study has limitations, and results need to be interpreted with caution; lower levels of oxidative stress were found in smokers when compared with studies of similar nature.^[19],[28] This finding may be due to healthy physiological conditions, better nutrition among the subjects enrolled, mean age (36 ± 8.4 years) of the subjects, and subjects recruited from outpatient; their awareness of oral care could have been higher, which was not ascertained in the study.

Conclusion

Salivary GSH was found to be lower among smokers; similarly, TAC and periodontal status were significantly poorer among smokers. The correlation of all three parameters reflected a significant association of periodontal status with TAC. Salivary GSH could serve as a potential biomarker if limitations of this study are overcome such as involving a specific age group, and controlling for diet and samples from the general population; this research can be in conjugation with or independent of concepts such as metagenomics to further assess the interplay of microflora based on oxidative stress.

Acknowledgement

We like to thank Dr. Rekha, P.D., Director, Yenepoya Research Centre, Yenepoya University for extending help in utilizing the lab facilities.

Financial support and sponsorship

Self funded.

Conflicts of interest

There are no conflicts of interest.

Authors contributions

K.I. was involved in study conception, data collection, data acquisition and manuscript writing. L.K.B. was involved in study conception, data analysis, data interpretation and manuscript writing. S.M. was involved in data interpretation and manuscript writing. All the authors approved the final version of the manuscript for publication to be mentioned. The manuscript has been read and approved by all the authors that the requirements for authorship as stated earlier in this document have been met, and that each author believes that the manuscript represents honest work if that information is not provided in another form.

Ethical policy and institutional review board statement:

This study was approved by the Ethics Committee, YDC24-2021. All the procedures have been performed as per the ethical guidelines laid down by Declaration of Helsinki (2013).

Patient declaration of consent

The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed

Data availability statement

The data set used in this study is available on request from the corresponding author (Kiran Iyer e-mail: [email protected]).

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