Biomedical and Biotechnology Research Journal (BBRJ)

: 2021  |  Volume : 5  |  Issue : 3  |  Page : 342--346

Comparative evaluation of Salivary-Soluble Receptor for Advanced Glycation End Product (sRAGE) levels among betel chewers and chronic periodontitis patients

Rajkumar Chandini, V Vasanthi, K Raghavendhar Karthik, Krishnan Rajkumar 
 Department of Oral Pathology and Microbiology, SRMIST, SRM Dental College, Chennai, Tamil Nadu, India

Correspondence Address:
V Vasanthi
Department of Oral Pathology and Microbiology, SRMIST, SRM Dental College, Ramapuram, Chennai - 600 089, Tamil Nadu


Background: Advanced glycation end products (AGE) are heterogeneous groups of molecules formed from the nonenzymatic reaction of reducing sugars with free amino groups of proteins, lipids, and nucleic acids. Activation of receptor for advanced glycation end products (RAGE) by ligands in a variety of cell types and tissues may play a role in oral-systemic associations. The aim of the study is to estimate the salivary-soluble RAGE (sRAGE) levels in betel chewers and compare with salivary sRAGE levels of chronic periodontitis and normal healthy controls. Methods: Detailed case history and clinical examination was done for the study participants based on the inclusion and exclusion criteria. Study participants were grouped as Group I (chewers), Group II (chronic periodontitis), and Group III (normal controls). Unstimulated saliva samples were collected and salivary sRAGE levels were calculated using the sandwich ELISA technique. Results: Individuals with pan chewing habits have demonstrated increase in salivary sRAGE compared to chronic periodontitis and normal controls. Conclusions: Understanding AGE formation and biochemistry, cellular receptors for AGE, and AGE-induced effects on extracellular and intracellular functions will serve to expedite the process of finding effective therapies that block the excessive accumulation of these species and their interaction with the signal transduction receptor RAGE.

How to cite this article:
Chandini R, Vasanthi V, Karthik K R, Rajkumar K. Comparative evaluation of Salivary-Soluble Receptor for Advanced Glycation End Product (sRAGE) levels among betel chewers and chronic periodontitis patients.Biomed Biotechnol Res J 2021;5:342-346

How to cite this URL:
Chandini R, Vasanthi V, Karthik K R, Rajkumar K. Comparative evaluation of Salivary-Soluble Receptor for Advanced Glycation End Product (sRAGE) levels among betel chewers and chronic periodontitis patients. Biomed Biotechnol Res J [serial online] 2021 [cited 2022 Jan 27 ];5:342-346
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Aging, diabetes mellitus, and neurodegenerative disorders are characterized by the formation of sugar-derived substances called advanced glycation end products (AGEs). AGEs are a heterogeneous group of molecules formed from the nonenzymatic reaction of reducing sugars with free amino groups of proteins, lipids, and nucleic acids. AGEs form at a constant but slow rate in the normal body, starting in early embryonic development, and accumulate with time.[1] AGEs may be stimulated by a range of distinct triggering mechanisms and accumulate in a wide variety of environments, thereby accounting for their roles in multiple settings and disease states.[2] AGE formation is markedly accelerated in conditions such as diabetes because of the increased availability of glucose and in certain other diseased conditions characterized by chronic inflammation. Several compounds, for example, carboxy methyl lysine, pentosidine, or methylglyoxal derivatives, serve as examples of well-characterized and widely studied AGEs.

In addition to those endogenously formed, AGEs can also be introduced in the body from exogenous sources such as in diet (preheated packaged food), where synthetic AGEs (pre-AGE's) are incorporated to boost the flavor of natural foods. There is apparently a direct correlation between circulating AGE levels and those consumed. Tobacco smoke also is a well-known exogenous source of AGEs.[3],[4],[5] The combustion of various pre-AGEs in tobacco during smoking gives rise to reactive and toxic AGEs. Diabetic smokers, as a result, are reported to exhibit greater AGE deposition in their arteries and ocular lenses.[1]

The RAGE (receptor for AGEs) is a multi-ligand member of the immunoglobulin superfamily of transmembrane cell surface molecules, is the best-characterized receptor for AGEs, and activation of the RAGE by AGEs plays a major role in the pathogenesis of diabetic vascular complications. RAGE is minimally expressed in normal tissue and vasculature. However, RAGE is upregulated when AGE ligands accumulate, an example of positive-feedback activation. Upregulation of RAGE occurs on cells such as endothelial cells, smooth muscle cells, and mononuclear phagocytes in diabetic vasculature. A number of RAGE isoforms have been found in both human and murine models recently. Soluble RAGE (sRAGE) exists in the circulation of humans in serum. sRAGE corresponds to the extracellular domain of RAGE lacking cytosolic and transmembrane domains.[5],[6]

The effect of AGEs in the body includes cross-linking of the extracellular matrix proteins thus altering the cellular structure along with interaction with their receptor (RAGE) on the cell surfaces, and thus, altering cellular function through signaling. Activation of the RAGE by AGEs plays a major role in triggering a signaling cascade that results in oxidative stress and results in the production of pro-inflammatory substances and also favors the further formation of AGE. The binding of various pro-inflammatory molecules to the cell-surface RAGE triggers an intracellular signaling pathway. The target is nuclear factor-κB which promotes enhanced transcription of pro-inflammatory genes and also upregulates the transcription of RAGE.[7],[8]

The habit of chewing areca nut in any form is reported to have been associated with obesity, hyperglycemia, and metabolic syndrome in literature. Numerous studies have been done evaluating the decreased sRAGE levels in several chronic inflammatory disorders including diabetes. Relatively little is known about factors that regulate sRAGE levels in human subjects and whether circulating levels reflect tissue RAGE expression and activity is unclear.

The current study proposed to estimate the salivary sRAGE levels in commercial pan chewing subjects and compare them with chronic periodontitis subjects and normal healthy controls. The objective was also to estimate the dose–response relationship of habits and salivary sRAGE based on duration and frequency of chewing betel quid.


The participants of the study were recruited from the outpatient department of the institution after obtaining informed consent. The study was approved by the Institutional Ethical Committee (SRMDC/IRB/2014/MDS/No. 602). A total of 120 participants were categorized into three groups of 40 numbers each. Patients with the habit of chewing betel (pan masala) were considered in Group I, patients with chronic periodontitis were categorized as Group II, and normal healthy controls were included in Group III. The participants included in the study belonged to the age group of 25–40 years. Patients with a history of systemic diseases, long-term medication, and habit of alcohol intake were excluded from the study. Patients with the habit of chewing betel (pan masala) for >5 years were included in Group I, whereas Group II and III patients were without the chewing habit. The commercial pan preparation contained areca nut, sweetening, and flavoring agents but did not contain tobacco. For the purpose of this study, to exclude confounding values in units of measurement of pan consumption per day, we included only subjects consuming one particular brand of pan masala.

Complete case history was obtained from all patients enrolled in the study. Clinical parameters such as age, sex, height, weight, body mass index, and waist: hip ratio were recorded. Random blood sugar levels were measured in subjects of all the three groups to exclude diabetes, minimize the possibility of the presence of occult inflammation, and reduce the confounding factors influencing sRAGE levels. Furthermore, intraoral periodontal examination for measurement of probing pocket depth and clinical attachment loss was done in subjects of all three groups.

Unstimulated saliva samples were collected from the patients in a sterile centrifuge tube. Following collection, the saliva was immediately centrifuged in a cooling centrifuge at 2500 rpm for 15 min at 4°C to remove squamous cells and cell debris. The resulting supernatant was separated into 1 ml aliquots and stored at −80°C freezer for further analysis using ELISA. No more than one freeze–thaw cycle was allowed for each sample. Sandwich ELISA was done for saliva samples for quantification of sRAGE using Human Advanced Glycosylation End Product-Specific Receptor ELISA Kit, from Elabscience.

All samples and standards were assayed in duplicate. One hundered microliter standard or sample was added to each well and incubated for 90 min at 37°C. The liquid was removed and 100 μL biotinylated detection Ab was added and incubated for 1 h at 37°C. Each well was aspirated and washed three times. One hundred microliter horseradish peroxidase conjugate was added and incubated for 30 min at 37°C. Each well was aspirated and washed five times. Ninety microliter substrate reagent was added and incubated for 15 min at 37°C. Fifty microliter stop solution was added and read at 450 nm immediately.

Statistical analysis was done using GraphPad InStat Software version 3 (La Jolla, CA, USA). Following the derivation of the optical density value and subsequent calculation of the quantity of sRAGE in the saliva samples, the resultant values were tabulated and expressed as mean value. Pearson coefficient of correlation was done for correlation between mean age and mean sRAGE in all three groups. One-way analysis of variance (ANOVA) followed by Tukey post hoc test was done for analyzing the significance of mean sRAGE values among three groups. Correlation between frequency, duration, pouch year index and mean sRAGE levels in subjects of pan chewer group was done using Pearson correlation coefficient test. Correlation between pocket probing depth, clinical attachment loss, and mean sRAGE levels in subjects of chronic periodontitis group was done using Pearson correlation coefficient test.


On comparing the age and mean sRAGE among the study groups, Group I (chewers) had positive correlation (correlation score 0.03), Group II (chronic periodontitis) had negative correlation (correlation score − 0.21), and Group III (healthy controls) had negative correlation (correlation score − 0.09). All the three groups did not have significant difference (Group I: P =0.85, Group II: P =0.19, and Group III: P =0.55).

The mean sRAGE levels of the study groups are as follows: chewers (Group I): 3120.19 pg/ml, chronic periodontitis (Group II): 2826.06 pg/ml, and normal controls (Group III): 2288.11 pg/ml [Figure 1]. Test of significance was done for these values using ANOVA and the results were studied with the P value set at 0.05, showing that the derived values were statistically significant (P = 0.04) among the three groups [Table 1]. Due to the significant results, the values were tested further to find out which of the groups showed statistically significant difference, using Tukey's multiple comparison test, and P value was set at 0.05. The results showed that pan masala chewers had statistically significant higher salivary sRAGE levels compared to normal controls [Table 1].{Figure 1}{Table 1}

The habit index of chewers as a product of frequency and duration of chewing per unit value of the product for the corresponding number of years was calculated. Based on the pouch year values obtained, the pan chewer subjects were further grouped into subgroups of 6–13 pouch years [Table 2], 14–21 pouch years [Table 3], and 24–48 pouch years [Table 4], and correlation to salivary sRAGE was attempted. The result for pouch years showed no significant correlation to salivary sRAGE levels in all the subgroups of pan chewers [Table 5]. Only in the last subgroup (24–48 pouch years), there was a weak negative correlation but with no statistical significance.{Table 2}{Table 3}{Table 4}{Table 5}


Within the scope of the current study, from these results, we can only conjecture with regard to the presence of such high levels of sRAGE in the saliva of pan masala chewing subjects. It can be conjectured that pro-inflammatory state from chewing pan masala might trigger upregulation of RAGE on the cell surface of vascular endothelial cells. Overexpression of RAGE also could be followed by proteolytic cleavage of the full-length RAGE, releasing only the extracellular component of full-length RAGE. As mentioned in the literature, the local pro-inflammatory state may consequently result in overexpression of matrix metalloproteinases that is well known to induce proteolytic cleavage of full-length RAGE.[9]

Betel chewing has been shown to be associated with the development of obesity, increased waist size, and Type 2 diabetes in humans. After adjustment for well-established risk factors, a prevalence-based study showed independent predictive dose–response effects of betel chewing, for the metabolic syndrome.[10]

Studies evaluating the presence of pre-AGEs in cigarette smoke and corresponding strong positive association with serum AGE's and negative correlation to serum sRAGE levels have been previously published. Estimation of RAGE expression in gingival tissues in periodontitis (with and without diabetes) has been done,[11],[12] but there exist lacunae in studies evaluating the salivary levels of sRAGE in these subjects.

Yamagishi et al. investigated the association of sRAGE with serum levels of AGEs in humans. The study reported that serum endogenous sRAGE levels were positively associated with circulating AGEs in the nondiabetic general population.[13]

Although in the current study, the correlation between salivary sRAGE levels in pan masala chewers did not show statistical significance to habit indices, it will be pertinent to study the long-term effect of chewing in a well-stratified larger sample with graded dose–response relationship to understand the sRAGE biology in pan masala chewing.

An important limitation of our study is the measurement of total sRAGE because the detection system used cannot discriminate between specific sRAGE splice variants. Therefore, it should be kept in mind that increased sRAGE levels measured by this assay may be caused by an alteration of a distinct circulating sRAGE isoform, which has to be evaluated using more expensive assay kits (estimating both esRAGE and total sRAGE). Second, our results show the limitations of cross-sectional, observational studies, evaluating the association, not prospective, predictive, or cause–effect relationship of sRAGE. Further studies in pan chewers utilizing other inflammatory markers and clinical outcomes have to be corroborated for a better understanding of sRAGE biology. Third, the presence of confounding factors such as the presence of dietary AGEs cannot be ruled out in all three groups. Furthermore, it is remotely possible for some subjects to be harboring occult inflammation without overt manifestation of the disease that can influence the RAGE expression, including sRAGE. The current study also has a limited scope of estimating only salivary sRAGE in all the three groups. It will be prudent to understand the changes in serum sRAGE in the groups for corroborating the findings.


The influence of pan chewing on human health is still an important problem worldwide. In the present study, it has been demonstrated that pan chewers exhibit a statistically significant increased level of salivary sRAGE. The uniqueness in the current study is the estimation of salivary sRAGE levels in normal healthy controls, pan chewers, and chronic periodontitis subjects which have not been previously reported in literature to the best of our knowledge. Studies evaluating the expression level of RAGE and sRAGE in different tissues and biological matrices are required. Moreover, the relationship between sRAGE, esRAGE, and serum AGE also has to be elucidated. Understanding AGE formation and biochemistry, cellular receptors for AGE, and AGE-induced effects on extracellular and intracellular functions will serve to expedite the process of finding effective therapies that block excessive accumulation of these species and their interaction with the signal transduction receptor RAGE.

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Conflicts of interest

There are no conflicts of interest.


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