Tobacco Habit Duration And Its Association With Oral Mucosal Lesions And Salivary Alpha-Klotho Levels: A Cross-Sectional Study

Tobacco Habit Duration And Its Association With Oral Mucosal Lesions And Salivary Alpha-Klotho Levels: A Cross-Sectional Study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Tobacco Habit Duration And Its Association With Oral Mucosal Lesions And Salivary Alpha-Klotho Levels: A Cross-Sectional Study Shanthi M, Ravishankar P.l, Jayaprakash T, Leela K.V, Ravisankar B, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8614921/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Tobacco use is a significant risk factor for oral mucosal lesions (OMLs) and oxidative stress-induced damage to oral tissues. Alpha-Klotho, a cytoprotective and antiaging protein, has surfaced as a prospective noninvasive salivary biomarker; nevertheless, its significance in populations exposed to tobacco has yet to be inadequately investigated. This research evaluated salivary alpha-Klotho levels in connection with demographic factors, tobacco use patterns, length of habits, and oral mucosal lesions among people in the Kanchipuram area. Methods Cross-sectional analytical research involving 200 adult tobacco users (smokers n = 75, smokeless n = 70, mixed n = 55) recruited from dentistry clinics and community screening initiatives was performed. Demographic data, tobacco use patterns, and OML status were documented via a standardized questionnaire and WHO oral examination standards. Unstimulated saliva samples were analysed for alpha-Klotho expression via ELISA. Statistical studies included t-tests, ANOVA, Pearson correlation, and multiple regression (SPSS v26.0; p < 0.05). Results The average salivary alpha-Klotho concentration was 472.1 ± 102.8 pg/mL. Alpha-Klotho exhibited a notable age-associated reduction (r = − 0.256, p = 0.009) and diminished gradually with increasing length of habit (10 years: 412.6 ± 83.7 pg/mL; p = 0.002). Significant variations in habit types were observed (p = 0.018), with smokers exhibiting the highest levels (512.4 ± 107.6), followed by smokeless users (469.1 ± 93.8) and mixed users (422.8 ± 84.5 pg/mL). Compared with OML-free individuals,participants with OMLs had significantly lower alpha-Klotho concentrations (428.3 ± 88.9 pg/mL) (506.7 ± 101.5 pg/mL; p = 0.006). Multiple regression accounted for 39% of the variation (R² = 0.39, p < 0.001), with length of usage (β = −0.304), OML presence (β = −0.264), habit type (β = −0.207), and age (β = −0.182) identified as significant negative predictors. Conclusion Salivary alpha-Klotho concentrations decrease with increasing age, prolonged tobacco exposure, mixed use patterns, and the presence of OMLs. These results underscore alpha-Klotho as a potential noninvasive biomarker for oxidative stress and the early identification of oral disease in high-risk tobacco-using groups. Alpha klotho Oral mucosal lesion Oxidative stress Salivary biomarkers Smoking Tobacco consumption Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION The condition of the oral mucosa is closely linked to overall human health and serves several key functions, including protection, sensation, and secretion (1). Oral cavity health also plays a vital role in determining an individual’s quality of life (2). Oral mucosal lesions (OMLs) are increasingly reported across global populations and are characterized by abnormal changes in color, surface texture, swelling, or loss of integrity of the oral mucosal lining (3). With the increasing incidence of oral cancer and OMLs, the prevalence of premalignant disorders is also increasing, largely due to the widespread use of various forms of tobacco available on the market (3–5). Oral cancer is among the ten most common malignancies worldwide, with a notable frequency in Central and Southeast Asian countries (6). India accounts for one-third of all oral cancer cases worlwide and is projected to have the second greatest number of cases of oral cancer. Research indicates that 50% of oral potentially malignant lesions (OPMLs) are mostly seen in rural areas as well as in some communities of people with a propensity for tobacco use (7, 8). Tobacco causes the deaths of approximately 7 million individuals annually, including approximately 1.6 million nonsmokers exposed to secondhand smoke (9). Tobacco has been widely utilized in the following varieties: smokeless and smoking. In India, smokeless tobacco (SLT) is used in varieties that include gutkha, khaini, mishri, and tobacco, along with betel nuts and lime, and additional forms include bidi, hookah, and cigarette smoking (10). The toxic compounds of tobacco infiltrate cells, causing malignant alterations and leading to neurological and bodily impairments that diminish their value (11). Tobacco use is associated with a significant incidence of dental caries and elevated decayed, missing, and filled teeth (DMFT) scores (12). Tobacco misuse may result in changes to the local environment of the oral cavity and saliva, along with its components (13). The Klotho protein is named after Klotho (Clotho), a character in Greek mythology and one of the Moirai, believed to connect the strands of life and control the course of humanity (14). The Klotho gene class comprises α-Klotho, β-Klotho, and γ-Klotho. The predominant variant of the Klotho protein ,known as α-Klotho, was initially identified in 1997 by Kuro-o et al. Klotho is a 130 kDa single-pass transmembrane glycoprotein mostly found in the distal segment of the nephron (15, 16). α-Klotho is an antiaging protein found in several organs, such as the kidney, lung, choroid plexus, and skeletal muscle (17, 18). A reduction in Klotho levels facilitates the onset and progression of several disorders (19–21). Quitting smoking markedly reduces blood α-Klotho concentrations and may be an overprotective reaction to the stress of smoking. Research has indicated that, in healthy males, serum α-Klotho levels are markedly elevated in smokers compared with nonsmokers (22) and that α-Klotho levels are decreased in non infected smokers during preterm gestation (23). The relationships among smoking, smoking cessation, and serum α-klotho concentrations are still debated. Japanese scientists reported elevated blood α-klotho concentrations among male smokers without underlying illness in comparison with nonsmokers; however, the findings in females were disparate rather than statistically significant (24). Turkish and American researchers reported lower serum klotho concentrations among smokers than among nonsmokers, healthy men and women with preterm pregnancies,(23, 25). Moreover, scientists discovered that individuals who smoked had reduced serum α-klotho concentrations after the habit was stopped compared with their concentrations while actively smoking (22). The evidence linking smoking and smoking cessation to alterations in serum α-Klotho concentrations remains inconclusive. Therefore, this study aimed to evaluate the associations among salivary alpha-Klotho levels, tobacco use patterns, duration of exposure, demographic variables, and oral mucosal lesions among adult tobacco users in Kanchipuram district. METHODOLOGY Study Design and Setting The current population-based cross-sectional study was conducted in the Kanchipuram district, India, at the Department of Oral Medicine and Radiology. Following the principles of the Declaration of Helsinki, the Institutional Review Board granted ethical approval(IEC review number: 0060/IEC/2024). This study was performed to evaluate salivary alpha-Klotho quantities across people with varying tobacco consumption practices and to examine their correlations with socioeconomic variables, time frame, quantity of tobacco products used, various kinds of tobacco products, and the occurrence of OMLs. This methodology facilitated the concurrent evaluation of biomarker concentrations and pertinent exposure factors within a specified group. The research was conducted among residents of the Kanchipuram district, Tamil Nadu, and involved participants sourced from dental outpatient clinics and community-oriented tobacco consumption monitoring initiatives. These recruiting platforms guaranteed sufficient coverage of the varied consumption of tobacco. A total of 200 adult current tobacco consumers, such as smokers, smokeless tobacco users, and mixed consumers, were recruited. Everyone involved in the study completed a preliminary assessment before being included to ensure data integrity and dependability. Inclusion and Exclusion Criteria Participants who were 18 years or older, who consumed tobacco for a minimum of six months, and who provided written consent were allowed to participate. The exclusion criteria included systemic illnesses associated with salivary biomarkers, including diabetes mellitus, chronic renal damage, and hormonal imbalances. Participants on prolonged drugs that alter salivary composition, people who present with salivary gland abnormalities, associated infections of the mouth, or those who were pregnant or breastfeeding were not included to reduce any unpredictable effects on salivary alpha-Klotho concentrations. Sample size and sampling technique The required sample size of 200 participants was determined on the basis of the variability reported in earlier salivary alpha-Klotho studies, assuming a medium effect size, 80% statistical power, and a 5% alpha level. This sample size provided sufficient power to identify differences in salivary alpha-Klotho concentrations according to tobacco use patterns and oral mucosal lesion status and to support multivariable statistical analyses. Eligible participants were enrolled via a successive sampling method until the target sample size was reached. Data collection procedure The data acquisition had two main steps. A previously verified standardized survey was utilized to collect details about the population, including age and sex, as well as data on tobacco consumption, including the type of behavior, length (in years), and consumption rate per day. Second, all of the participants had an oral cavity assessment conducted according to standardized lighting in accordance with the WHO oral wellness evaluation guidelines. A detailed oral cavity examination was performed for all participants under standardized lighting conditions in accordance with WHO oral health assessment guidelines. Examiner calibration was completed before the study, with an intraexaminer reliability score exceeding 0.80 (kappa), ensuring reproducibility in the detection and classification of oral mucosal lesions. Lesions were documented and categorized clinically based on the basis of their morphological characteristics. The existence or nonexistence of oral mucosal lesions was recorded, and lesions were classified clinically on the basis of their attributes. Collection and Processing of Saliva Samples Unstimulated whole saliva samples were obtained between 9:00 a.m. and 12:00 p.m to minimize diurnal changes in biomarker concentrations. The subjects were directed to abstain from consuming food or beverages, smoking, and doing dental cleaning for a minimum of one hour before sampling. Saliva was collected via the passive drooling technique and then placed into sterile polypropylene tubes. The samples were promptly kept on ice and then centrifuged at 3000 rpm for 10 minutes. The transparent supernatant was distributed into sterile vials and preserved at − 20°C until the test was performed. Biochemical assessment of alpha-Klotho Salivary alpha-Klotho levels were measured via a readily accessible human-specific enzyme-linked immunosorbent assay (ELISA) kit. The standard curves were established according to the company's methodology, and all the samples were evaluated in triplicate to guarantee analytical precision. Final levels were reported in pictograms per millilitre (pg/mL). Statistical analysis The data were aggregated and evaluated with the Statistical Package for the Social Sciences (SPSS) version 26.0. Descriptive statistics, such as the means, standard deviations,frequencies, and percentages, were used to describe socioeconomic information and salivary alpha-Klotho concentrations. The Shapiro–Wilk test was used to evaluate the normality of the alpha-Klotho range. Independent t tests were utilized for inferential tests for comparing average alpha-Klotho concentrations between two groups (e.g., gender, presence of oral mucosal lesions), whereas one-way ANOVA, accompanied by Tukey’s post hoc test, enabled comparisons between multiple groups, including various kinds of consumers of tobacco or different durations of tobacco use. Pearson's correlation coefficient was used to analyse relationships among continuous variables, such as age, length of tobacco consumption, and usage frequency. A multiple linear regression model was developed, integrating demographic and behavioral characteristics, to ascertain independent determinants of salivary alpha-Klotho concentrations. Statistical significance was established at p < 0.05 for all analyses. Ethical considerations Ethical approval for the research was acquired from the Institutional Ethics Committee. Before starting the acquisition of data and samples, informed written permission was obtained from every individual who was involved in the study. Participants who had oral mucosal lesions were found to be suitable for diagnostic assessment and therapeutic intervention. Every detail about the participants was kept undisclosed, and rigorous data anonymity was retained throughout the research. RESULTS Salivary alpha-Klotho levels were quantified via ELISA in 200 participants exhibiting diverse tobacco consumption behaviours. The participants were evaluated to determine the impact of demographic variables and tobacco consumption behaviours on salivary alpha-Klotho concentrations. The present study investigated correlations with age, sex, tobacco use type, duration, and frequency of daily consumption and the occurrence of oral mucosal lesions. Comparative and correlational studies were conducted to determine key findings and determinants. The results elucidate the long-term effects of tobacco smoke and associated oral pathologies on salivary alpha-Klotho, a biomarker associated with oxidative and tissue stress. Table 1 Comprehensive alpha-Klotho concentration (N = 200) Parameter Mean ± SD (pg/mL) Minimum Maximum 95% CI for Mean Alpha-Klotho concentration 472.1 ± 102.8 265.4 698.9 457.7–486.5 Table 1 displays the comprehensive salivary alpha-Klotho concentrations for the study cohort. The average level was 472.1 ± 102.8 pg/mL, with a range of 265.4 to 698.9 pg/mL and a 95% confidence interval of 457.7–486.5 pg/mL. The broad spectrum indicates considerable interindividual variation, possibly affected by demographic and tobacco consumption factors. Table 2 Demographic Age Distribution and Alpha-Klotho Concentrations (N = 200) Age Group (Years) Frequency (n) Percentage (%) Mean ± SD of Alpha-Klotho (pg/mL) < 25 years 38 19.0% 511.4 ± 96.2 25–34 years 62 31.0% 489.8 ± 100.7 35–44 years 54 27.0% 463.6 ± 104.3 45–54 years 32 16.0% 442.7 ± 97.9 ≥ 55 years 14 7.0% 418.3 ± 88.5 Total 200 100% — Table 2 Classification of the subjects according to age and their associated mean salivary alpha-Klotho values. The highest percentage was in the 25–34 years age group (31%), followed by 35–44 years (27%), under 25 years (19%), 45–54 years (16%), and 55 years and over (7%). An evident age-related reduction in alpha-Klotho was observed, with levels decreasing from 511.4 ± 96.2 pg/mL in the < 25 years age group to 418.3 ± 88.5 pg/mL in those aged ≥ 55 years. This adverse trend underscores the importance of alpha-Klotho as a biomarker sensitive to ageing and oxidative stress.[insert Fig. 1 ] Table 3 Comparison of Salivary Alpha-Klotho Concentrations by Gender (N = 200) Gender n Mean ± SD (pg/mL) t-value p-value Male 120 463.9 ± 98.4 1.81 0.073 Female 80 508.2 ± 99.7 Table 3 contrasts salivary alpha-Klotho concentrations between men and females. Among the 200 subjects examined, 60% (n = 120) were male, whereas 40% (n = 80) were female. Compared with men,females had elevated mean levels of alpha-Klotho (508.2 ± 99.7 pg/mL) (463.9 ± 98.4 pg/mL). Despite females exhibiting elevated concentrations, this disparity lacked statistical significance (t = 1.81, p = 0.073), indicating that although sex may exert a slight influence on salivary alpha-Klotho expression, the observed higher levels in females were insufficient to substantiate a definitive effect.[insert Fig. 2 ] Table 4 Relationship between age and the alpha-Klotho concentration (n = 200) Variable Pair Correlation Coefficient (r) p value Interpretation Age vs. Alpha-Klotho −0.256 0.009*CC Weak negative correlation Table 4 shows the correlation between salivary alpha-Klotho levels and age among the 200 individuals included in the present study. A modest negative connection was identified (r = − 0.256), which was statistically significant (p = 0.009). These findings suggests that alpha-Klotho levels decrease modestly with increasing age. The decline in concentrations with increasing age may indicate age-related alterations in cellular metabolism and a progressive deterioration in antioxidative ability. Despite the link being weak, the data support the prevalent notion that alpha-Klotho serves as an antiaging biomarker, which decreases with physiological aging and cumulative exposure to environmental and lifestyle variables, such as tobacco consumption. Table 5 Comparison of Salivary Alpha-Klotho Concentrations among Tobacco Habit Groups (N = 200) Habit Type n Alpha-Klotho (pg/mL, Mean ± SD) p-value Smoking 75 512.4 ± 107.6 0.018* Smokeless 70 469.1 ± 93.8 Mixed 55 422.8 ± 84.5 *Significant difference among habit groups (One-way ANOVA). Table 5 shows the salivary alpha-Klotho concentrations in relation to various tobacco-use behaviours. Among the 200 individuals, 75 were smokers, 70 used smokeless tobacco, and 55 indicated mixed usage. Smokers presented the greatest average alpha-Klotho concentration (512.4 ± 107.6 pg/mL), followed by consumers of smokeless tobacco (469.1 ± 93.8 pg/mL), whereas mixed consumers presented a relatively low average concentration (422.8 ± 84.5 pg/mL). One-way ANOVA revealed a statistically significant difference between the groups (p = 0.018), demonstrating that salivary alpha-Klotho concentrations vary by means of tobacco consumption, with multiple uses linked to notably diminished concentrations, potentially caused by compounded oxidative and inflammatory stress from the combination of tobacco varieties.[insert Fig. 3 ] Table 6 Comparison of Alpha-Klotho Levels between Participants with and without Oral Lesions (N = 200) Lesion Status n Mean ± SD (pg/mL) Mean Difference tvalue df pvalue 95% CI for Mean Difference Lesion Present 98 428.3 ± 88.9 78.4 2.78 198 0.006* 22.5–134.3 Lesion Absent 102 506.7 ± 101.5 *Significant difference by Independent t test. Table 6 provides a comparative analysis of salivary alpha-Klotho levels in subjects with oral mucosal lesions versus those without lesions.Compared with those without lesions ,individuals with lesions (n = 98) presented decreased average alpha -Klotho concentrations (428.3 ± 88.9 pg/mL) (506.7 ± 101.5 pg/mL). The mean difference of 78.4 pg/mL was statistically significant (t = 2.78, p = 0.006), with a 95% confidence interval ranging from 22.5 to 134.3 pg/mL. These findings demonstrate a significant inverse relationship between oral mucosal lesions and alpha-Klotho levels, indicating that lower alpha-Klotho levels might be linked to oxidative stress as well as tissue damage related to tobacco-associated oral lesions.[insert Fig. 4 ] Table 7 Evaluation of Alpha-Klotho Levels via Duration of Tobacco Use (N = 200) Duration of Use N Alpha-Klotho (pg/mL, Mean ± SD) p-value 10 years 58 412.6 ± 83.7 *Significant trend of decreasing Alpha-Klotho with longer duration (ANOVA). Table 7 shows the correlation between the frequency of tobacco consumption and salivary alpha-Klotho levels. A noticeable decrease in concentrations was observed: participants with less than 5 years of consumption presented the highest average concentrations (532.8 ± 94.2 pg/mL), followed by those with 5 to 10 years of consumption (474.9 ± 89.4 pg/mL), whereas the minimal concentrations were recorded in participants with more than 10 years of consumption (412.6 ± 83.7 pg/mL). The observed variation was statistically significant (F = 6.42, p = 0.002). The results suggest that extended tobacco consumption leads to a consistent reduction in alpha-Klotho levels, probably resulting from cumulative oxidative and inflammatory effects that gradually undermine salivary protective systems.[insert Fig. 5 ] Table 8 Multiple Regression Model for Predictors of Salivary Alpha-Klotho concentration (N = 200) Predictor Variable Mean ± SD Correlation (r) with Alpha-Klotho β (Standardized Coefficient) t-value p-value Age (years) 36.8 ± 10.9 −0.256 −0.182 −2.09 0.038* Gender (Male = 1) M: — F: — −0.142 −0.114 −1.49 0.138 Duration of tobacco use (years) 8.7 ± 5.3 −0.321 −0.304 −3.81 0.001* Lesion presence (Yes = 1) Yes: — No: — −0.278 −0.264 −3.18 0.002* Habit Type (Smokeless/Mixed) Smokeless: 469.1 ± 93.8 Mixed: 422.8 ± 84.5 −0.218 −0.207 −2.63 0.009* R² = 0.39, indicating that 39% of the variation in salivary α-Klotho levels is explained by the model. Adjusted R² = 0.37, F = 15.84, p < 0.001 (model statistically significant). Table 8 outlines the various regression results that reveal important indicators of salivary alpha-Klotho levels in the cohort of 200 consumers of tobacco products. The model accounted for nearly 39% of the variation (R² = 0.39, adjusted R² = 0.37) and demonstrated statistical significance (F = 15.84, p < 0.001). The period of tobacco use (β = −0.304, p = 0.001), the existence of lesions (β = −0.264, p = 0.002), and the level of consumption (β = −0.207, p = 0.009) were identified as key negative predictors, suggesting that prolonged consumption, lesion-positive conditions, and various or more dangerous consumption patterns correlate with decreased alpha-Klotho levels. Age exhibited a minor but significant negative correlation (β = −0.182, p = 0.038), whereas sex was not a significant predictor (p = 0.138). These data indicate that prolonged tobacco exposure and oral pathology significantly reduce salivary alpha-Klotho concentrations. DISCUSSION Saliva forms a thin film over the oral mucosa and plays multiple roles in the protection of the oral cavity, assisting in digestion through amylase, maintaining the pH,and flow rate, and influencing the redistribution of ions between enamel remineralization and demineralization, leading to localized dissolution and destruction of calcified teeth, and supporting tooth surface integrity (26, 27). Through its constituents, such as salivary proteins, electrolytes, and small molecules, it protects against abrasion, attrition, erosion, and dental caries. prevents injury to the oral mucosa through its clearance properties, and protects against physical damage,and antibacterial and antifungal effects (28). Smoking decreases the commensal population of normal oral cells and increases pathogenic microbes and microbial colonization via biofilm formation on oral epithelial cells (29). Few studies have evaluated the role of tobacco and related substances exposure and oral health status, especially in the assessment of salivary components. Whole unstimulated saliva was collected in the present study, as the basal salivary flow rate is reflected by unstimulated whole saliva, and the same is favoured by most population studies (13, 30, 31). Klotho influences oral health through several mechanisms. It reduces oxidative stress and apoptosis in human periodontal ligament stem cells (hPLSCs) (15, 32), as shown by lowered reactive oxygen species and malondialdehyde levels, restored antioxidant enzyme activity, and decreased expression of apoptotic markers such as BAX and Caspase-3 under H₂O₂-induced stress (33). Therefore, klotho deficiency may promote oxidative damage and apoptosis, contributing to oral deterioration. Klotho is also essential for dentin formation and mineralization, regulating alkaline phosphatase (ALP) and key matrix proteins, including dentin matrix protein-1 (DMP-1) and osteopontin (OPN) (34). Animal studies have reported weak ALP staining and irregular DMP-1 and OPN distribution in klotho-deficient mice (35). Additionally, klotho maintains calcium and phosphorus homeostasis, and reduced levels of these minerals have been observed in teeth lacking klotho. In addition to being involved in oral tissues, klotho participates in broader metabolic and endocrine pathways, influencing energy balance, mineral metabolism, and disease risk across multiple organ systems (15, 35). This cross-sectional study investigated the associations among tobacco use behaviors, demographic variables, oral mucosal lesions, and salivary alpha-Klotho concentrations in adults from the Kanchipuram district. The results reveal specific biomarker patterns that reflect cumulative oxidative stress and tobacco-related oral pathology, which is consistent with recent scientific literature. This study revealed a notable negative correlation between age and salivary alpha-Klotho levels, reinforcing its established role as an anti ageing protein, the expression of which decreases with increasing age and elevated oxidative stress. Bhavsar et al. (2023) reported that a graded reduction in alpha-Klotho was observed with increasing duration of tobacco use, indicating an additional exposure-related effect independent of chronological aging (13). Females presented higher mean alpha-Klotho levels; however, this difference was not statistically significant, which aligns with previously documented but inconsistent sex-related variations (25, 36). Multivariate regression analysis identified duration of tobacco use, presence of lesions, type of habit, and age as significant predictors, collectively explaining 39% of the variability in alpha-Klotho concentrations.These findings indicate that behavioral and pathological factors have a greater impact than demographic characteristics, do which aligns, with recent clinical biomarker studies linking lifestyle exposures to reduced alpha-Klotho activity (22, 37). This study has several limitations. This cross-sectional design precludes conclusions regarding causality. Salivary alpha-Klotho concentrations were measured at a single time point, without parallel assessment of serum levels for comparison. Additionally, tobacco use was self-reported, which may introduce recall bias. Future prospective studies with biochemical verification and longitudinal follow-up are needed to validate these findings. CONCLUSION This study revealed a significant association between the salivary alpha-Klotho concentration and factors such as age, duration, and type of tobacco use, as well as the presence of mucosal lesions. The reduction in alpho-Klotho with increasing age and duration consistently observed with prolonged tobacco exposure and mixed tobacco habits indicates a cumulative effect of inflammation and oxidative stress, while sex-related differences were not statistically significant. The regression model identified duration of use, lesion status, and habit type as significant predictors of salivary alpha-Klotho levels, each contributing meaningfully to the variability observed in these levels. These findings indicate that salivary alpha-Klotho serves as a promising noninvasive biomarker for oxidative stress and early alterations associated with chronic tobacco exposure in oral tissues. Salivary alpha-Klotho may serve as a practical, noninvasive biomarker for the early detection and risk stratification of tobacco-related oral mucosal pathology. Abbreviations ALP alkaline phosphatase (ALP) DMP 1 –dentin matrix protein–1 (DMP–1) and OPN osteopontin (OPN) hPLSCs human periodontal ligament stem cells (hPLSCs) ELISA enzyme–linked immunosorbent assay (ELISA) OPMLs oral potentially malignant lesions (OPMLs) OMLs oral mucosal lesions (OMLs) SLT smokeless tobacco (SLT) DMFT decayed, missing, and filled teeth (DMFT) WHO World Health Organisation (WHO) Declarations Ethics approval and consent to participate Ethical approval for this study was obtained from the Institutional Ethics Committee, SRM Institute of Science and Technology (IEC Review No: 0060/IEC/2024). The study was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants prior to enrolment in the study. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Funding This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. Author Contribution MS conceived and designed the study, performed data collection, statistical analysis, and manuscript preparation. PLR supervised the study design, clinical evaluation, and critically revised the manuscript. TJ contributed to laboratory analysis and interpretation of biochemical data. KVL provided microbiological expertise and reviewed the manuscript. VKR contributed to clinical assessment and manuscript review. All authors read and approved the final manuscript. Acknowledgements The authors sincerely thank the participants for their cooperation and the institutional authorities of SRM Kattankulathur Dental College and Hospital for their support in conducting this study. 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Am J Med Sci. 2021;361(2):169–175. doi: 10.1016/j.amjms.2020.10.003 Tyurenkov IN, Perfilova VN, Nesterova AA, Glinka Y. Klotho protein and the cardiovascular system. Biochemistry (Mosc). 2021;86(2):132–145. doi: 10.1134/S0006297921020026 Kamizono Y, Shiga Y, Suematsu Y, Imaizumi S, Tsukahara H, Noda K, et al. Impact of cigarette smoking cessation on plasma α-klotho levels. Medicine (Baltimore). 2018;97(35):e11947. doi: 10.1097/MD.0000000000011947 Lam-Rachlin J, Romero R, Korzeniewski SJ, Schwartz AG, Chaemsaithong P, Hernandez-Andrade E, et al. Infection and smoking associated with decreased plasma α-klotho. J Perinat Med. 2013;41(5):581–594. doi: 10.1515/jpm-2012-0222 Nakanishi K, Nishida M, Harada M, Ohama T, Kawada N, Murakami M, et al. Klotho-related molecules upregulated by smoking. Sci Rep. 2015;5:14230. doi: 10.1038/srep14230 Onmaz M, Demirbas N, Onmaz DE, Kutlu R, Unlu A. Effect of cigarette smoking on serum methylarginine and α-klotho levels. Nutr Metab Cardiovasc Dis. 2023;33(3):602–609. doi: 10.1016/j.numecd.2022.12.012 Enax J, Fandrich P, Schulze zur Wiesche E, Epple M. Enamel remineralization from saliva. Dent J (Basel). 2024;12(11):339. doi: 10.3390/dj12110339 Pedersen A, Sørensen C, Proctor G, Carpenter G, Ekström J. Salivary secretion in health and disease. J Oral Rehabil. 2018;45(9):730–746. doi: 10.1111/joor.12664 Kumari S, Samara M, Ampadi Ramachandran R, Gosh S, George H, Wang R, et al. Saliva-based health diagnostics. Biomed Mater Devices. 2024;2(1):121–138. doi: 10.1007/s44174-024-00046-9 Kauss AR, Antunes M, Zanetti F, Hankins M, Hoeng J, Heremans A, et al. Influence of tobacco smoking on halitosis. Toxicol Rep. 2022;9:316–322. doi: 10.1016/j.toxrep.2022.02.006 Awan KH, Patil S. Association of smokeless tobacco with oral cancer. J Coll Physicians Surg Pak. 2016;26(9):775–780. Okuyama K, Yanamoto S. Saliva in oral and systemic health. Cancers (Basel). 2024;16(24):4276. doi: 10.3390/cancers16244276 Chen H, Huang X, Fu C, Wu X, Peng Y, Lin X, et al. Recombinant klotho protects periodontal ligament stem cells. Oxid Med Cell Longev. 2019;2019:9261565. doi: 10.1155/2019/9261565 Chen M, Cai W, Zhao S, Shi L, Chen Y, Li X, et al. Oxidative stress biomarkers in saliva. J Clin Periodontol. 2019;46(6):608–622. doi: 10.1111/jcpe.13124 Hikone K, Hasegawa T, Tsuchiya E, Hongo H, Sasaki M, Yamamoto T, et al. Periodontal tissues in klotho-deficient mice. J Histochem Cytochem. 2017;65(4):207–221. doi: 10.1369/0022155417691596 Chen GQ, Duan Y, Wang JF, Lian Y, Yin XL. Serum α-klotho and oral health in U.S. adults. Front Endocrinol (Lausanne). 2022;13:970575. doi: 10.3389/fendo.2022.970575 Nakanishi K, Nishida M, Yamamoto R, Koseki M, Moriyama T, Yamauchi-Takihara K. Sex differences in smoking-related klotho expression. Clin Chim Acta. 2018;476:44–48. doi: 10.1016/j.cca.2017.11.020 Gao W, Yuan C, Zhang J, Li L, Yu L, Wiegman CH, et al. Reduced klotho expression in COPD airway epithelial cells. Clin Sci (Lond). 2015;129(12):1011–1023. doi: 10.1042/CS20140645 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8614921","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":588071020,"identity":"02a37e54-e558-4564-a94d-546f200c3cfb","order_by":0,"name":"Shanthi 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legend\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8614921/v1/7a938ef6feb699821dfc2fa4.jpeg"},{"id":104397285,"identity":"692f9852-ac2b-4a62-b811-662172e25728","added_by":"auto","created_at":"2026-03-11 11:46:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2122973,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8614921/v1/2fb4663e-9f13-464c-935c-9ba0244fdc72.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Tobacco Habit Duration And Its Association With Oral Mucosal Lesions And Salivary Alpha-Klotho Levels: A Cross-Sectional Study","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe condition of the oral mucosa is closely linked to overall human health and serves several key functions, including protection, sensation, and secretion (1). Oral cavity health also plays a vital role in determining an individual\u0026rsquo;s quality of life (2). Oral mucosal lesions (OMLs) are increasingly reported across global populations and are characterized by abnormal changes in color, surface texture, swelling, or loss of integrity of the oral mucosal lining (3). With the increasing incidence of oral cancer and OMLs, the prevalence of premalignant disorders is also increasing, largely due to the widespread use of various forms of tobacco available on the market (3\u0026ndash;5).\u003c/p\u003e \u003cp\u003eOral cancer is among the ten most common malignancies worldwide, with a notable frequency in Central and Southeast Asian countries (6). India accounts for one-third of all oral cancer cases worlwide and is projected to have the second greatest number of cases of oral cancer. Research indicates that 50% of oral potentially malignant lesions (OPMLs) are mostly seen in rural areas as well as in some communities of people with a propensity for tobacco use (7, 8). Tobacco causes the deaths of approximately 7\u0026nbsp;million individuals annually, including approximately 1.6\u0026nbsp;million nonsmokers exposed to secondhand smoke (9).\u003c/p\u003e \u003cp\u003eTobacco has been widely utilized in the following varieties: smokeless and smoking. In India, smokeless tobacco (SLT) is used in varieties that include gutkha, khaini, mishri, and tobacco, along with betel nuts and lime, and additional forms include bidi, hookah, and cigarette smoking (10). The toxic compounds of tobacco infiltrate cells, causing malignant alterations and leading to neurological and bodily impairments that diminish their value (11). Tobacco use is associated with a significant incidence of dental caries and elevated decayed, missing, and filled teeth (DMFT) scores (12). Tobacco misuse may result in changes to the local environment of the oral cavity and saliva, along with its components (13).\u003c/p\u003e \u003cp\u003eThe Klotho protein is named after Klotho (Clotho), a character in Greek mythology and one of the Moirai, believed to connect the strands of life and control the course of humanity (14). The Klotho gene class comprises α-Klotho, β-Klotho, and γ-Klotho. The predominant variant of the Klotho protein ,known as α-Klotho, was initially identified in 1997 by Kuro-o et al. Klotho is a 130 kDa single-pass transmembrane glycoprotein mostly found in the distal segment of the nephron (15, 16). α-Klotho is an antiaging protein found in several organs, such as the kidney, lung, choroid plexus, and skeletal muscle (17, 18). A reduction in Klotho levels facilitates the onset and progression of several disorders (19\u0026ndash;21).\u003c/p\u003e \u003cp\u003eQuitting smoking markedly reduces blood α-Klotho concentrations and may be an overprotective reaction to the stress of smoking. Research has indicated that, in healthy males, serum α-Klotho levels are markedly elevated in smokers compared with nonsmokers (22) and that α-Klotho levels are decreased in non infected smokers during preterm gestation (23). The relationships among smoking, smoking cessation, and serum α-klotho concentrations are still debated. Japanese scientists reported elevated blood α-klotho concentrations among male smokers without underlying illness in comparison with nonsmokers; however, the findings in females were disparate rather than statistically significant (24).\u003c/p\u003e \u003cp\u003eTurkish and American researchers reported lower serum klotho concentrations among smokers than among nonsmokers, healthy men and women with preterm pregnancies,(23, 25). Moreover, scientists discovered that individuals who smoked had reduced serum α-klotho concentrations after the habit was stopped compared with their concentrations while actively smoking (22). The evidence linking smoking and smoking cessation to alterations in serum α-Klotho concentrations remains inconclusive. Therefore, this study aimed to evaluate the associations among salivary alpha-Klotho levels, tobacco use patterns, duration of exposure, demographic variables, and oral mucosal lesions among adult tobacco users in Kanchipuram district.\u003c/p\u003e"},{"header":"METHODOLOGY","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy Design and Setting\u003c/h2\u003e \u003cp\u003e The current population-based cross-sectional study was conducted in the Kanchipuram district, India, at the Department of Oral Medicine and Radiology. Following the principles of the Declaration of Helsinki, the Institutional Review Board granted ethical approval(IEC review number: 0060/IEC/2024). This study was performed to evaluate salivary alpha-Klotho quantities across people with varying tobacco consumption practices and to examine their correlations with socioeconomic variables, time frame, quantity of tobacco products used, various kinds of tobacco products, and the occurrence of OMLs. This methodology facilitated the concurrent evaluation of biomarker concentrations and pertinent exposure factors within a specified group. The research was conducted among residents of the Kanchipuram district, Tamil Nadu, and involved participants sourced from dental outpatient clinics and community-oriented tobacco consumption monitoring initiatives. These recruiting platforms guaranteed sufficient coverage of the varied consumption of tobacco. A total of 200 adult current tobacco consumers, such as smokers, smokeless tobacco users, and mixed consumers, were recruited. Everyone involved in the study completed a preliminary assessment before being included to ensure data integrity and dependability.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eInclusion and Exclusion Criteria\u003c/h3\u003e\n\u003cp\u003eParticipants who were 18 years or older, who consumed tobacco for a minimum of six months, and who provided written consent were allowed to participate. The exclusion criteria included systemic illnesses associated with salivary biomarkers, including diabetes mellitus, chronic renal damage, and hormonal imbalances. Participants on prolonged drugs that alter salivary composition, people who present with salivary gland abnormalities, associated infections of the mouth, or those who were pregnant or breastfeeding were not included to reduce any unpredictable effects on salivary alpha-Klotho concentrations.\u003c/p\u003e\n\u003ch3\u003eSample size and sampling technique\u003c/h3\u003e\n\u003cp\u003e The required sample size of 200 participants was determined on the basis of the variability reported in earlier salivary alpha-Klotho studies, assuming a medium effect size, 80% statistical power, and a 5% alpha level. This sample size provided sufficient power to identify differences in salivary alpha-Klotho concentrations according to tobacco use patterns and oral mucosal lesion status and to support multivariable statistical analyses. Eligible participants were enrolled via a successive sampling method until the target sample size was reached.\u003c/p\u003e\n\u003ch3\u003eData collection procedure\u003c/h3\u003e\n\u003cp\u003eThe data acquisition had two main steps. A previously verified standardized survey was utilized to collect details about the population, including age and sex, as well as data on tobacco consumption, including the type of behavior, length (in years), and consumption rate per day. Second, all of the participants had an oral cavity assessment conducted according to standardized lighting in accordance with the WHO oral wellness evaluation guidelines. A detailed oral cavity examination was performed for all participants under standardized lighting conditions in accordance with WHO oral health assessment guidelines. Examiner calibration was completed before the study, with an intraexaminer reliability score exceeding 0.80 (kappa), ensuring reproducibility in the detection and classification of oral mucosal lesions. Lesions were documented and categorized clinically based on the basis of their morphological characteristics. The existence or nonexistence of oral mucosal lesions was recorded, and lesions were classified clinically on the basis of their attributes.\u003c/p\u003e\n\u003ch3\u003eCollection and Processing of Saliva Samples\u003c/h3\u003e\n\u003cp\u003eUnstimulated whole saliva samples were obtained between 9:00 a.m. and 12:00 p.m to minimize diurnal changes in biomarker concentrations. The subjects were directed to abstain from consuming food or beverages, smoking, and doing dental cleaning for a minimum of one hour before sampling. Saliva was collected via the passive drooling technique and then placed into sterile polypropylene tubes. The samples were promptly kept on ice and then centrifuged at 3000 rpm for 10 minutes. The transparent supernatant was distributed into sterile vials and preserved at \u0026minus;\u0026thinsp;20\u0026deg;C until the test was performed.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBiochemical assessment of alpha-Klotho\u003c/h2\u003e \u003cp\u003eSalivary alpha-Klotho levels were measured via a readily accessible human-specific enzyme-linked immunosorbent assay (ELISA) kit. The standard curves were established according to the company's methodology, and all the samples were evaluated in triplicate to guarantee analytical precision. Final levels were reported in pictograms per millilitre (pg/mL).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data were aggregated and evaluated with the Statistical Package for the Social Sciences (SPSS) version 26.0. Descriptive statistics, such as the means, standard deviations,frequencies, and percentages, were used to describe socioeconomic information and salivary alpha-Klotho concentrations. The Shapiro\u0026ndash;Wilk test was used to evaluate the normality of the alpha-Klotho range. Independent t tests were utilized for inferential tests for comparing average alpha-Klotho concentrations between two groups (e.g., gender, presence of oral mucosal lesions), whereas one-way ANOVA, accompanied by Tukey\u0026rsquo;s post hoc test, enabled comparisons between multiple groups, including various kinds of consumers of tobacco or different durations of tobacco use. Pearson's correlation coefficient was used to analyse relationships among continuous variables, such as age, length of tobacco consumption, and usage frequency. A multiple linear regression model was developed, integrating demographic and behavioral characteristics, to ascertain independent determinants of salivary alpha-Klotho concentrations. Statistical significance was established at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 for all analyses.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eEthical considerations\u003c/h3\u003e\n\u003cp\u003e \u003cstrong\u003eEthical approval\u003c/strong\u003e \u003cp\u003e for the research was acquired from the Institutional Ethics Committee. Before starting the acquisition of data and samples, informed written permission was obtained from every individual who was involved in the study. Participants who had oral mucosal lesions were found to be suitable for diagnostic assessment and therapeutic intervention. Every detail about the participants was kept undisclosed, and rigorous data anonymity was retained throughout the research.\u003c/p\u003e \u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e Salivary alpha-Klotho levels were quantified via ELISA in 200 participants exhibiting diverse tobacco consumption behaviours. The participants were evaluated to determine the impact of demographic variables and tobacco consumption behaviours on salivary alpha-Klotho concentrations. The present study investigated correlations with age, sex, tobacco use type, duration, and frequency of daily consumption and the occurrence of oral mucosal lesions. Comparative and correlational studies were conducted to determine key findings and determinants. The results elucidate the long-term effects of tobacco smoke and associated oral pathologies on salivary alpha-Klotho, a biomarker associated with oxidative and tissue stress.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComprehensive alpha-Klotho concentration (N\u0026thinsp;=\u0026thinsp;200)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (pg/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMinimum\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMaximum\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e95% CI for Mean\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlpha-Klotho concentration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e472.1\u0026thinsp;\u0026plusmn;\u0026thinsp;102.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e265.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e698.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e457.7\u0026ndash;486.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e displays the comprehensive salivary alpha-Klotho concentrations for the study cohort. The average level was 472.1\u0026thinsp;\u0026plusmn;\u0026thinsp;102.8 pg/mL, with a range of 265.4 to 698.9 pg/mL and a 95% confidence interval of 457.7\u0026ndash;486.5 pg/mL. The broad spectrum indicates considerable interindividual variation, possibly affected by demographic and tobacco consumption factors.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDemographic Age Distribution and Alpha-Klotho Concentrations (N\u0026thinsp;=\u0026thinsp;200)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge Group (Years)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFrequency (n)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePercentage (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of Alpha-Klotho (pg/mL)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;25 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e19.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e511.4\u0026thinsp;\u0026plusmn;\u0026thinsp;96.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e25\u0026ndash;34 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e31.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e489.8\u0026thinsp;\u0026plusmn;\u0026thinsp;100.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e35\u0026ndash;44 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e27.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e463.6\u0026thinsp;\u0026plusmn;\u0026thinsp;104.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e45\u0026ndash;54 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e16.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e442.7\u0026thinsp;\u0026plusmn;\u0026thinsp;97.9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026ge;\u0026thinsp;55 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.0%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e418.3\u0026thinsp;\u0026plusmn;\u0026thinsp;88.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e200\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026mdash;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e Classification of the subjects according to age and their associated mean salivary alpha-Klotho values. The highest percentage was in the 25\u0026ndash;34 years age group (31%), followed by 35\u0026ndash;44 years (27%), under 25 years (19%), 45\u0026ndash;54 years (16%), and 55 years and over (7%). An evident age-related reduction in alpha-Klotho was observed, with levels decreasing from 511.4\u0026thinsp;\u0026plusmn;\u0026thinsp;96.2 pg/mL in the \u0026lt;\u0026thinsp;25 years age group to 418.3\u0026thinsp;\u0026plusmn;\u0026thinsp;88.5 pg/mL in those aged\u0026thinsp;\u0026ge;\u0026thinsp;55 years. This adverse trend underscores the importance of alpha-Klotho as a biomarker sensitive to ageing and oxidative stress.[insert Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e]\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of Salivary Alpha-Klotho Concentrations by Gender (N\u0026thinsp;=\u0026thinsp;200)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGender\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (pg/mL)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003et-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e120\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e463.9\u0026thinsp;\u0026plusmn;\u0026thinsp;98.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e1.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.073\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFemale\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e508.2\u0026thinsp;\u0026plusmn;\u0026thinsp;99.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e contrasts salivary alpha-Klotho concentrations between men and females. Among the 200 subjects examined, 60% (n\u0026thinsp;=\u0026thinsp;120) were male, whereas 40% (n\u0026thinsp;=\u0026thinsp;80) were female. Compared with men,females had elevated mean levels of alpha-Klotho (508.2\u0026thinsp;\u0026plusmn;\u0026thinsp;99.7 pg/mL) (463.9\u0026thinsp;\u0026plusmn;\u0026thinsp;98.4 pg/mL). Despite females exhibiting elevated concentrations, this disparity lacked statistical significance (t\u0026thinsp;=\u0026thinsp;1.81, p\u0026thinsp;=\u0026thinsp;0.073), indicating that although sex may exert a slight influence on salivary alpha-Klotho expression, the observed higher levels in females were insufficient to substantiate a definitive effect.[insert Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e]\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRelationship between age and the alpha-Klotho concentration (n\u0026thinsp;=\u0026thinsp;200)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable Pair\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCorrelation Coefficient (r)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003ep value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInterpretation\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eAge vs. Alpha-Klotho\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e\u0026minus;0.256\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e0.009*CC\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eWeak negative correlation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows the correlation between salivary alpha-Klotho levels and age among the 200 individuals included in the present study. A modest negative connection was identified (r = \u0026minus;\u0026thinsp;0.256), which was statistically significant (p\u0026thinsp;=\u0026thinsp;0.009). These findings suggests that alpha-Klotho levels decrease modestly with increasing age. The decline in concentrations with increasing age may indicate age-related alterations in cellular metabolism and a progressive deterioration in antioxidative ability. Despite the link being weak, the data support the prevalent notion that alpha-Klotho serves as an antiaging biomarker, which decreases with physiological aging and cumulative exposure to environmental and lifestyle variables, such as tobacco consumption.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of Salivary Alpha-Klotho Concentrations among Tobacco Habit Groups (N\u0026thinsp;=\u0026thinsp;200)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHabit Type\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAlpha-Klotho (pg/mL, Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSmoking\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e512.4\u0026thinsp;\u0026plusmn;\u0026thinsp;107.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" morerows=\"2\" nameend=\"c5\" namest=\"c4\" rowspan=\"3\"\u003e \u003cp\u003e0.018*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSmokeless\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e469.1\u0026thinsp;\u0026plusmn;\u0026thinsp;93.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMixed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e422.8\u0026thinsp;\u0026plusmn;\u0026thinsp;84.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c4\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003e*Significant difference among habit groups (One-way ANOVA).\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows the salivary alpha-Klotho concentrations in relation to various tobacco-use behaviours. Among the 200 individuals, 75 were smokers, 70 used smokeless tobacco, and 55 indicated mixed usage. Smokers presented the greatest average alpha-Klotho concentration (512.4\u0026thinsp;\u0026plusmn;\u0026thinsp;107.6 pg/mL), followed by consumers of smokeless tobacco (469.1\u0026thinsp;\u0026plusmn;\u0026thinsp;93.8 pg/mL), whereas mixed consumers presented a relatively low average concentration (422.8\u0026thinsp;\u0026plusmn;\u0026thinsp;84.5 pg/mL). One-way ANOVA revealed a statistically significant difference between the groups (p\u0026thinsp;=\u0026thinsp;0.018), demonstrating that salivary alpha-Klotho concentrations vary by means of tobacco consumption, with multiple uses linked to notably diminished concentrations, potentially caused by compounded oxidative and inflammatory stress from the combination of tobacco varieties.[insert Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e]\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab6\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 6\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of Alpha-Klotho Levels between Participants with and without Oral Lesions (N\u0026thinsp;=\u0026thinsp;200)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLesion Status\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003en\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (pg/mL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMean Difference\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003etvalue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003edf\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003epvalue\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e95% CI for Mean Difference\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLesion Present\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e428.3\u0026thinsp;\u0026plusmn;\u0026thinsp;88.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e78.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e2.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e198\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.006*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e22.5\u0026ndash;134.3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLesion Absent\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e102\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e506.7\u0026thinsp;\u0026plusmn;\u0026thinsp;101.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e*Significant difference by Independent t test.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab6\" class=\"InternalRef\"\u003e6\u003c/span\u003e provides a comparative analysis of salivary alpha-Klotho levels in subjects with oral mucosal lesions versus those without lesions.Compared with those without lesions ,individuals with lesions (n\u0026thinsp;=\u0026thinsp;98) presented decreased average alpha -Klotho concentrations (428.3\u0026thinsp;\u0026plusmn;\u0026thinsp;88.9 pg/mL) (506.7\u0026thinsp;\u0026plusmn;\u0026thinsp;101.5 pg/mL). The mean difference of 78.4 pg/mL was statistically significant (t\u0026thinsp;=\u0026thinsp;2.78, p\u0026thinsp;=\u0026thinsp;0.006), with a 95% confidence interval ranging from 22.5 to 134.3 pg/mL. These findings demonstrate a significant inverse relationship between oral mucosal lesions and alpha-Klotho levels, indicating that lower alpha-Klotho levels might be linked to oxidative stress as well as tissue damage related to tobacco-associated oral lesions.[insert Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e]\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab7\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 7\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eEvaluation of Alpha-Klotho Levels via Duration of Tobacco Use (N\u0026thinsp;=\u0026thinsp;200)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDuration of Use\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAlpha-Klotho (pg/mL, Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;5 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e532.8\u0026thinsp;\u0026plusmn;\u0026thinsp;94.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" morerows=\"2\" nameend=\"c5\" namest=\"c4\" rowspan=\"3\"\u003e \u003cp\u003e0.002*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u0026ndash;10 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e474.9\u0026thinsp;\u0026plusmn;\u0026thinsp;89.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;10 years\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e412.6\u0026thinsp;\u0026plusmn;\u0026thinsp;83.7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003cb\u003e*Significant trend of decreasing Alpha-Klotho with longer duration (ANOVA).\u003c/b\u003e\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab7\" class=\"InternalRef\"\u003e7\u003c/span\u003e shows the correlation between the frequency of tobacco consumption and salivary alpha-Klotho levels. A noticeable decrease in concentrations was observed: participants with less than 5 years of consumption presented the highest average concentrations (532.8\u0026thinsp;\u0026plusmn;\u0026thinsp;94.2 pg/mL), followed by those with 5 to 10 years of consumption (474.9\u0026thinsp;\u0026plusmn;\u0026thinsp;89.4 pg/mL), whereas the minimal concentrations were recorded in participants with more than 10 years of consumption (412.6\u0026thinsp;\u0026plusmn;\u0026thinsp;83.7 pg/mL). The observed variation was statistically significant (F\u0026thinsp;=\u0026thinsp;6.42, p\u0026thinsp;=\u0026thinsp;0.002). The results suggest that extended tobacco consumption leads to a consistent reduction in alpha-Klotho levels, probably resulting from cumulative oxidative and inflammatory effects that gradually undermine salivary protective systems.[insert Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e]\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab8\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 8\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMultiple Regression Model for Predictors of Salivary Alpha-Klotho concentration (N\u0026thinsp;=\u0026thinsp;200)\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePredictor Variable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCorrelation (r) with Alpha-Klotho\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eβ (Standardized Coefficient)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003et-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAge (years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e36.8\u0026thinsp;\u0026plusmn;\u0026thinsp;10.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026minus;0.256\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026minus;0.182\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026minus;2.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.038*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGender (Male\u0026thinsp;=\u0026thinsp;1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eM: \u0026mdash; F: \u0026mdash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026minus;0.142\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026minus;0.114\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026minus;1.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.138\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDuration of tobacco use (years)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.7\u0026thinsp;\u0026plusmn;\u0026thinsp;5.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026minus;0.321\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026minus;0.304\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026minus;3.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.001*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLesion presence (Yes\u0026thinsp;=\u0026thinsp;1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYes: \u0026mdash; No: \u0026mdash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026minus;0.278\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026minus;0.264\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026minus;3.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.002*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHabit Type (Smokeless/Mixed)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSmokeless: 469.1\u0026thinsp;\u0026plusmn;\u0026thinsp;93.8 Mixed: 422.8\u0026thinsp;\u0026plusmn;\u0026thinsp;84.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026minus;0.218\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026minus;0.207\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026minus;2.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.009*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003cul\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eR\u0026sup2; = 0.39, indicating that 39% of the variation in salivary α-Klotho levels is explained by the model.\u003c/b\u003e \u003c/p\u003e \u003c/li\u003e \u003cli\u003e \u003cp\u003e \u003cb\u003eAdjusted R\u0026sup2; = 0.37, F\u0026thinsp;=\u0026thinsp;15.84, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 (model statistically significant).\u003c/b\u003e \u003c/p\u003e \u003c/li\u003e \u003c/ul\u003e \u003c/p\u003e \u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab8\" class=\"InternalRef\"\u003e8\u003c/span\u003e outlines the various regression results that reveal important indicators of salivary alpha-Klotho levels in the cohort of 200 consumers of tobacco products. The model accounted for nearly 39% of the variation (R\u0026sup2; = 0.39, adjusted R\u0026sup2; = 0.37) and demonstrated statistical significance (F\u0026thinsp;=\u0026thinsp;15.84, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The period of tobacco use (β = \u0026minus;0.304, p\u0026thinsp;=\u0026thinsp;0.001), the existence of lesions (β = \u0026minus;0.264, p\u0026thinsp;=\u0026thinsp;0.002), and the level of consumption (β = \u0026minus;0.207, p\u0026thinsp;=\u0026thinsp;0.009) were identified as key negative predictors, suggesting that prolonged consumption, lesion-positive conditions, and various or more dangerous consumption patterns correlate with decreased alpha-Klotho levels. Age exhibited a minor but significant negative correlation (β = \u0026minus;0.182, p\u0026thinsp;=\u0026thinsp;0.038), whereas sex was not a significant predictor (p\u0026thinsp;=\u0026thinsp;0.138). These data indicate that prolonged tobacco exposure and oral pathology significantly reduce salivary alpha-Klotho concentrations.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003e Saliva forms a thin film over the oral mucosa and plays multiple roles in the protection of the oral cavity, assisting in digestion through amylase, maintaining the pH,and flow rate, and influencing the redistribution of ions between enamel remineralization and demineralization, leading to localized dissolution and destruction of calcified teeth, and supporting tooth surface integrity (26, 27). Through its constituents, such as salivary proteins, electrolytes, and small molecules, it protects against abrasion, attrition, erosion, and dental caries. prevents injury to the oral mucosa through its clearance properties, and protects against physical damage,and antibacterial and antifungal effects (28).\u003c/p\u003e \u003cp\u003eSmoking decreases the commensal population of normal oral cells and increases pathogenic microbes and microbial colonization via biofilm formation on oral epithelial cells (29). Few studies have evaluated the role of tobacco and related substances exposure and oral health status, especially in the assessment of salivary components. Whole unstimulated saliva was collected in the present study, as the basal salivary flow rate is reflected by unstimulated whole saliva, and the same is favoured by most population studies (13, 30, 31).\u003c/p\u003e \u003cp\u003eKlotho influences oral health through several mechanisms. It reduces oxidative stress and apoptosis in human periodontal ligament stem cells (hPLSCs) (15, 32), as shown by lowered reactive oxygen species and malondialdehyde levels, restored antioxidant enzyme activity, and decreased expression of apoptotic markers such as BAX and Caspase-3 under H₂O₂-induced stress (33). Therefore, klotho deficiency may promote oxidative damage and apoptosis, contributing to oral deterioration. Klotho is also essential for dentin formation and mineralization, regulating alkaline phosphatase (ALP) and key matrix proteins, including dentin matrix protein-1 (DMP-1) and osteopontin (OPN) (34).\u003c/p\u003e \u003cp\u003eAnimal studies have reported weak ALP staining and irregular DMP-1 and OPN distribution in klotho-deficient mice (35). Additionally, klotho maintains calcium and phosphorus homeostasis, and reduced levels of these minerals have been observed in teeth lacking klotho. In addition to being involved in oral tissues, klotho participates in broader metabolic and endocrine pathways, influencing energy balance, mineral metabolism, and disease risk across multiple organ systems (15, 35). This cross-sectional study investigated the associations among tobacco use behaviors, demographic variables, oral mucosal lesions, and salivary alpha-Klotho concentrations in adults from the Kanchipuram district.\u003c/p\u003e \u003cp\u003eThe results reveal specific biomarker patterns that reflect cumulative oxidative stress and tobacco-related oral pathology, which is consistent with recent scientific literature. This study revealed a notable negative correlation between age and salivary alpha-Klotho levels, reinforcing its established role as an anti ageing protein, the expression of which decreases with increasing age and elevated oxidative stress. Bhavsar et al. (2023) reported that a graded reduction in alpha-Klotho was observed with increasing duration of tobacco use, indicating an additional exposure-related effect independent of chronological aging (13). Females presented higher mean alpha-Klotho levels; however, this difference was not statistically significant, which aligns with previously documented but inconsistent sex-related variations (25, 36).\u003c/p\u003e \u003cp\u003eMultivariate regression analysis identified duration of tobacco use, presence of lesions, type of habit, and age as significant predictors, collectively explaining 39% of the variability in alpha-Klotho concentrations.These findings indicate that behavioral and pathological factors have a greater impact than demographic characteristics, do which aligns, with recent clinical biomarker studies linking lifestyle exposures to reduced alpha-Klotho activity (22, 37). This study has several limitations. This cross-sectional design precludes conclusions regarding causality. Salivary alpha-Klotho concentrations were measured at a single time point, without parallel assessment of serum levels for comparison. Additionally, tobacco use was self-reported, which may introduce recall bias. Future prospective studies with biochemical verification and longitudinal follow-up are needed to validate these findings.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eThis study revealed a significant association between the salivary alpha-Klotho concentration and factors such as age, duration, and type of tobacco use, as well as the presence of mucosal lesions. The reduction in alpho-Klotho with increasing age and duration consistently observed with prolonged tobacco exposure and mixed tobacco habits indicates a cumulative effect of inflammation and oxidative stress, while sex-related differences were not statistically significant. The regression model identified duration of use, lesion status, and habit type as significant predictors of salivary alpha-Klotho levels, each contributing meaningfully to the variability observed in these levels. These findings indicate that salivary alpha-Klotho serves as a promising noninvasive biomarker for oxidative stress and early alterations associated with chronic tobacco exposure in oral tissues. Salivary alpha-Klotho may serve as a practical, noninvasive biomarker for the early detection and risk stratification of tobacco-related oral mucosal pathology.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eALP\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ealkaline phosphatase (ALP)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDMP\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cb\u003e1\u003c/b\u003e\u0026ndash;dentin matrix protein\u0026ndash;1 (DMP\u0026ndash;1) and\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eOPN\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eosteopontin (OPN)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ehPLSCs\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehuman periodontal ligament stem cells (hPLSCs)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eELISA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eenzyme\u0026ndash;linked immunosorbent assay (ELISA)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eOPMLs\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eoral potentially malignant lesions (OPMLs)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eOMLs\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eoral mucosal lesions (OMLs)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eSLT\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esmokeless tobacco (SLT)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDMFT\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003edecayed, missing, and filled teeth (DMFT)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eWHO\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eWorld Health Organisation (WHO)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":" \u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003e Ethical approval for this study was obtained from the Institutional Ethics Committee, SRM Institute of Science and Technology (IEC Review No: 0060/IEC/2024). The study was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants prior to enrolment in the study.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eMS conceived and designed the study, performed data collection, statistical analysis, and manuscript preparation. PLR supervised the study design, clinical evaluation, and critically revised the manuscript. TJ contributed to laboratory analysis and interpretation of biochemical data. KVL provided microbiological expertise and reviewed the manuscript. VKR contributed to clinical assessment and manuscript review. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003e The authors sincerely thank the participants for their cooperation and the institutional authorities of SRM Kattankulathur Dental College and Hospital for their support in conducting this study.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col start=\"1\" type=\"1\"\u003e\n\u003cli\u003eYadav NR, Jain M, Sharma A, Yadav R, Pahuja M, Jain V. 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Recombinant klotho protects periodontal ligament stem cells. \u003cem\u003eOxid Med Cell Longev.\u003c/em\u003e 2019;2019:9261565.\u003cbr\u003e\u003cstrong\u003edoi:\u003c/strong\u003e 10.1155/2019/9261565\u003c/li\u003e\n\u003cli\u003eChen M, Cai W, Zhao S, Shi L, Chen Y, Li X, et al. Oxidative stress biomarkers in saliva. \u003cem\u003eJ Clin Periodontol.\u003c/em\u003e 2019;46(6):608\u0026ndash;622.\u003cbr\u003e\u003cstrong\u003edoi:\u003c/strong\u003e 10.1111/jcpe.13124\u003c/li\u003e\n\u003cli\u003eHikone K, Hasegawa T, Tsuchiya E, Hongo H, Sasaki M, Yamamoto T, et al. Periodontal tissues in klotho-deficient mice. \u003cem\u003eJ Histochem Cytochem.\u003c/em\u003e 2017;65(4):207\u0026ndash;221.\u003cbr\u003e\u003cstrong\u003edoi:\u003c/strong\u003e 10.1369/0022155417691596\u003c/li\u003e\n\u003cli\u003eChen GQ, Duan Y, Wang JF, Lian Y, Yin XL. Serum \u0026alpha;-klotho and oral health in U.S. adults. \u003cem\u003eFront Endocrinol (Lausanne).\u003c/em\u003e 2022;13:970575.\u003cbr\u003e\u003cstrong\u003edoi:\u003c/strong\u003e 10.3389/fendo.2022.970575\u003c/li\u003e\n\u003cli\u003eNakanishi K, Nishida M, Yamamoto R, Koseki M, Moriyama T, Yamauchi-Takihara K. Sex differences in smoking-related klotho expression. \u003cem\u003eClin Chim Acta.\u003c/em\u003e 2018;476:44\u0026ndash;48.\u003cbr\u003e\u003cstrong\u003edoi:\u003c/strong\u003e 10.1016/j.cca.2017.11.020\u003c/li\u003e\n\u003cli\u003eGao W, Yuan C, Zhang J, Li L, Yu L, Wiegman CH, et al. Reduced klotho expression in COPD airway epithelial cells. \u003cem\u003eClin Sci (Lond).\u003c/em\u003e 2015;129(12):1011\u0026ndash;1023.\u003cbr\u003e\u003cstrong\u003edoi:\u003c/strong\u003e 10.1042/CS20140645\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Alpha klotho, Oral mucosal lesion, Oxidative stress, Salivary biomarkers, Smoking, Tobacco consumption","lastPublishedDoi":"10.21203/rs.3.rs-8614921/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8614921/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eTobacco use is a significant risk factor for oral mucosal lesions (OMLs) and oxidative stress-induced damage to oral tissues. Alpha-Klotho, a cytoprotective and \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eantiaging\u003c/span\u003e protein, has surfaced as a prospective noninvasive salivary biomarker; nevertheless, its significance in populations exposed to tobacco has yet to be inadequately investigated. This research evaluated salivary alpha-Klotho levels in connection with demographic factors, tobacco use patterns, length of habits, and oral mucosal lesions among people in the Kanchipuram area.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eCross-sectional analytical research involving 200 adult tobacco users (smokers n\u0026thinsp;=\u0026thinsp;75, smokeless n\u0026thinsp;=\u0026thinsp;70, mixed n\u0026thinsp;=\u0026thinsp;55) recruited from dentistry clinics and community screening initiatives was performed. Demographic data, tobacco use patterns, and OML status were documented via a standardized questionnaire and WHO oral examination standards. Unstimulated saliva samples were analysed for alpha-Klotho expression via ELISA. Statistical studies included t-tests, ANOVA, Pearson correlation, and multiple regression (SPSS v26.0; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe average salivary alpha-Klotho concentration was 472.1\u0026thinsp;\u0026plusmn;\u0026thinsp;102.8 pg/mL. Alpha-Klotho exhibited a notable age-associated reduction (r\u0026thinsp;=\u0026thinsp;\u0026minus;\u0026thinsp;0.256, p\u0026thinsp;=\u0026thinsp;0.009) and diminished gradually with increasing length of habit (\u0026lt;\u0026thinsp;5 years: 532.8\u0026thinsp;\u0026plusmn;\u0026thinsp;94.2; 5\u0026ndash;10 years: 474.9\u0026thinsp;\u0026plusmn;\u0026thinsp;89.4; \u0026gt;10 years: 412.6\u0026thinsp;\u0026plusmn;\u0026thinsp;83.7 pg/mL; p\u0026thinsp;=\u0026thinsp;0.002). Significant variations in habit types were observed (p\u0026thinsp;=\u0026thinsp;0.018), with smokers exhibiting the highest levels (512.4\u0026thinsp;\u0026plusmn;\u0026thinsp;107.6), followed by smokeless users (469.1\u0026thinsp;\u0026plusmn;\u0026thinsp;93.8) and mixed users (422.8\u0026thinsp;\u0026plusmn;\u0026thinsp;84.5 pg/mL). Compared with OML-free individuals,participants with OMLs had significantly lower alpha-Klotho concentrations (428.3\u0026thinsp;\u0026plusmn;\u0026thinsp;88.9 pg/mL) (506.7\u0026thinsp;\u0026plusmn;\u0026thinsp;101.5 pg/mL; p\u0026thinsp;=\u0026thinsp;0.006). Multiple regression accounted for 39% of the variation (R\u0026sup2; = 0.39, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), with length of usage (β = \u0026minus;0.304), OML presence (β = \u0026minus;0.264), habit type (β = \u0026minus;0.207), and age (β = \u0026minus;0.182) identified as significant negative predictors.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eSalivary alpha-Klotho concentrations decrease with increasing age, prolonged tobacco exposure, mixed use patterns, and the presence of OMLs. These results underscore alpha-Klotho as a potential noninvasive biomarker for oxidative stress and the early identification of oral disease in high-risk tobacco-using groups.\u003c/p\u003e","manuscriptTitle":"Tobacco Habit Duration And Its Association With Oral Mucosal Lesions And Salivary Alpha-Klotho Levels: A Cross-Sectional Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-11 16:48:50","doi":"10.21203/rs.3.rs-8614921/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"09c97e88-6683-4a02-8633-ed054574b10a","owner":[],"postedDate":"February 11th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-09T12:56:12+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-11 16:48:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8614921","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8614921","identity":"rs-8614921","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}