|Year : 2021 | Volume
| Issue : 1 | Page : 39-46
Non-invasive estimation of sialic acid in saliva among patients with oral potentially malignant disorders and oral cancer
Krithigaa Sivaraman1, C Sreeja1, K Murugavel2, Serena Florence Francis1, R Sathish Muthukumar1, Merlin Jayaraj1
1 Department of Oral and Maxillofacial Pathology, Chettinad Dental College and Research Institute, Chennai, Tamil Nadu, India
2 Department of Biochemistry, Chettinad Health City and Research Institute, Chennai, Tamil Nadu, India
|Date of Submission||27-Aug-2020|
|Date of Decision||17-Feb-2021|
|Date of Acceptance||23-Mar-2021|
|Date of Web Publication||19-May-2021|
Dr. Krithigaa Sivaraman
Department of Oral Pathology, Chettinad Dental College and Research Institute, Chettinad Health City, Rajiv Gandhi Salai, Kelambakkam, Kanchipuram District, Chennai - 603 103, Tamil Nadu
Source of Support: None, Conflict of Interest: None
Background: Oral potentially malignant disorders (OPMDs) account for about 2.5% of all the oral lesions, among which 37.5% show malignant transformation. Clinical appearances of any lesion which are deceived are finally confirmed and diagnosed using routine gold standard histological H and E stains. However, with newer technologies, cancer biomarkers in saliva are found to be highly sensitive. So, the early detection of cellular alterations at a non-lesional stage is of utmost importance to prevent its progression.
Aim: To analyze the salivary sialic acid levels in the spectrum of patients having habits with no clinically visible lesion, leukoplakia, oral submucous fibrosis (OSMF) and oral squamous cell carcinoma (OSCC) along with the healthy groups and compare the levels with clinical and histological parameters.
Materials and Methods: A total of 60 subjects were enrolled in this study. Sialic acid levels, total protein (TP) and total carbohydrate (TC) levels were estimated in saliva using appropriate biochemical analysis.
Results: Results were analyzed using mean, standard deviation and Student's t test. Salivary sialic acid levels were significantly increased in leukoplakia, OSMF and OSCC. There is a significant difference in sialic acid levels in smokers and chewers yielding a mean value of 18.97 mg/dL and 23.4 mg/dL, respectively, against the mean value of 15.29 mg/dL of healthy controls. The sialic acid levels showed a statistical difference between clinical staging and histological grading of leukoplakia, OSMF and OSCC. Salivary sialic acid, protein and carbohydrate levels also showed significance.
Conclusion: The present study highlights the altered expression of sialic acid among various groups suggesting that aberrant glycosylation in cell surface molecules during malignant transformation can give a clue on the tumor burden.
Keywords: Oral cancer, oral pre-cancerous lesion, salivary sialic acid
|How to cite this article:|
Sivaraman K, Sreeja C, Murugavel K, Francis SF, Muthukumar R S, Jayaraj M. Non-invasive estimation of sialic acid in saliva among patients with oral potentially malignant disorders and oral cancer. J NTR Univ Health Sci 2021;10:39-46
|How to cite this URL:|
Sivaraman K, Sreeja C, Murugavel K, Francis SF, Muthukumar R S, Jayaraj M. Non-invasive estimation of sialic acid in saliva among patients with oral potentially malignant disorders and oral cancer. J NTR Univ Health Sci [serial online] 2021 [cited 2021 Dec 6];10:39-46. Available from: https://www.jdrntruhs.org/text.asp?2021/10/1/39/316313
| Introduction|| |
Oral mucosa serves as a barrier against foreign substances by harboring immunological and chemical receptors of definite structural and functional signal molecules. During malignant transformation, a healthy cell adapts, alters and strives to endure before transforming into a neoplastic cell by modulating the cell surface molecules. The perception of developing oral squamous cell carcinoma (OSCC) from the pre-existing oral potentially malignant disorders (OPMD) has been well established of which leukoplakia and oral submucous fibrosis (OSMF) being the most common.
OSCC, the disease of oral microenvironment, commonly occurs in people above 40 years of age, with various etiological factors—the most common suspected contributing factors to be consumption of different forms of tobacco, alcohol, drug abuse, viral pathogens like Human papilloma virus (HPV), environmental and genetic factors. Carcinogens present in tobacco, namely, aromatic hydrocarbon, benzo-pyrene, and the tobacco-specific nitrosamines that Enters into saliva can interfere with DNA replication by generating DNA adducts, and thereby, damaging the replicating cells of the immune response.
With advancements in prevention and therapeutics, cancer research is now widely focusing on a less invasive and cost-effective diagnostic tool to provide comprehensive details of the disease to support the clinicians for effective management. Over the years, many studies and reviews have summarized the updates on evaluation of various salivary biomarkers on OPMDs and oral cancer stating their potential for diagnosis, prognosis and monitoring of the therapeutic.
Sialic acid, a negatively charged monosaccharide molecule, present as a terminal branch of cell surface glycans is responsible for cell adhesion, cellular interactions, invasion and immunogenicity. Modification of surface glycans reflects changes in cell behavior, and, its release into the body fluids by tumor cells can be either due to cause or effect of malignant transformation. Due to paucity of studies using sialic acid levels estimated in patients having habits with no clinical lesion in comparison to healthy and diseased groups along with clinical and histological parameters, this prompted us to conduct a study to analyze the significance of sialic acid release and understand its clinical significance., So, early intervention by using a biomarker along with clinical and histological tests would help in predicting the malignant potential, and thereby, minimize the morbidity and mortality rate.
| Aim And Objective|| |
To determine the role of salivary sialic acid as a reliable biomarker for early detection of malignant transformation from a non-lesional stage and predict the biological behavior of malignant lesion with clinical and histological parameters.
To estimate and compare the salivary sialic acid levels in groups.
To assess the salivary sialic acid concentration in patients having tobacco-smoking and areca-nut-chewing habits without any visible clinical lesion.
- Correlate the sialic acid concentration with total protein (TP) and total carbohydrate (TC) levels
- To correlate the salivary sialic acid levels with clinical stage of the disease
- To correlate the salivary sialic acid levels with cellular changes of disease.
| Materials and Methods|| |
The present study is an observational cross-sectional study conducted on patients visiting the Department of Oral Medicine and Radiology in the month of September 2019. Ethical clearance was obtained from the Institutional Ethical Committee (proposal No. –380/IHEC/11-19).
Study comprised of 60 subjects which included 10 subjects each per category. Subjects were clinically and histologically diagnosed with OSMF, dysplasia and OSCC. Detailed case history with thorough oral examination was recorded. Informed consent was obtained from the subjects prior to sample collection.
Study population –
- Areca nut chewers
Study population was categorized into two groups based on the habits.
- Patients between 20 and 60 years of age
- Patients with tobacco products induced OPMDs and oral cancer
- Healthy individuals with no history of systemic illness
- Chewers with the habit of consuming >10 mg of paan packet for at least a year
- Smokers using tobacco products minimum 5-6 per day.
- Patients with known systemic conditions like diabetes, hypertension, pregnancy, allergies, inflammatory and liver disease
- Patient under any medications
- Patients undergoing or previously treated for oral cancer and potentially malignant disorders.
About 2 mL of unstimulated saliva was collected by Navazesh method 2 h after the consumption of food. Unstimulated saliva was collected using sterile container by asking the subject to spit every minute for 5–8 min. Later, the sample was centrifuged at 3000 rpm for 15 min and supernatant was refrigerated at —20° till the sample was used for biochemical analysis. Biochemical analysis was done using spectrophotometry based on the chemicals being detected as follows:
- For sialic acid – Diphenylalanine method
- For TP – Lowry's method
- For TC – Phenol –sulphuric acid method.
Sialic acid estimation – Diphenylalanine method
- Preparation of acidic ninhydrin reagent – 250 mg ninhydrin dissolved in 6 mL of glacial acetic acid followed by 4 mL of concentrated sulphuric acid. Thorough vortexing done for 30 min
- 0.1 mL of saliva was made up to 1 mL with normal saline followed by centrifugation at 3000 rpm for 30 min to which 1 mL of glacial acetic acid and 1 mL of freshly prepared reagent was added. Following the mixture preparation, test tubes were kept in boiling water bath for 10 min, cooled under tap water and absorbance was read at 470 nm
- 10 mg of N-Acetylneuraminic acid (NANA) dissolved in 100 mL distilled water was used as a standard.
TP estimation – Lowry's method
- Biuret reagent was prepared by mixing 0.5 mL of 1% cupric sulfate with 0.5 mL of 2% sodium potassium tartrate, followed by the addition of 50 mL of 2% sodium carbonate in 0.1 N NaOH.
- To 50 μl of a saliva sample, 1 mL of reagent was added and incubated for 10 min at room temperature. Later, to the mixture, 0.1 mL of Folin and Ciocalteu's reagent was added and incubated at room temperature for 30 min. Absorbance was read at 750 nm.
- 2mg of Bovine serum albumin dissolved in 1 mL distilled water was used as standard.
TC estimation – Phenol-sulphuric acid method
- 01 mL of saliva was made up to 1 mL with normal saline. 3 mL of concentrated sulphuric acid was added. The solution was thoroughly vortexed and kept at room temperature for 30 min. To the solution mixture, 0.1 mL of freshly prepared ethanolic o-cresol was added, mixed and incubated for 30 min. Absorbance reading was taken at 500 nm.
- 5 mg of D-mannose dissolved in 1 mL distilled water was used as the standard.
OD – optical density
The targeted analysis of sialic acid in saliva was presented as mean ± SD and P values. Statistical comparison was done using t test and Pearson's correlation to compare the difference between the mean values of the groups. All statistical analysis were performed using SPSS software 2.0 version and P < 0.05 value was considered statistically significant.
| Results|| |
The study was conducted to detect the role of sialic acid as a biomarker for early detection of malignant transformation from a non-lesional stage and predict the biological behavior of malignant lesions.
A total of 60 patients were recruited and grouped based on the study subjects satisfying all the inclusion criteria. The mean age of the subjects was 36.84 ± 13.05 years indicating that the individuals were above 40 years. Study comprised of 35.71% females and 64.28% males [Table 1].
Comparison of salivary sialic acid levels within groups
The mean salivary sialic acid levels among groups with smoking habit: control group was 15.29 mg/dL, followed by 18.97 mg/dL in smokers with no lesions whereas it was 30 mg/dL and 36.5 mg/dL in leukoplakia and oral cancer groups, respectively and when compared were statistically significant.
The mean salivary sialic acid levels in groups with tobacco chewing habit showed 23.4 mg/dL, 46.3 mg/dL and 36.5 mg/dL in tobacco chewers with no lesion, OSMF and oral cancer , respectively, and were also statistically significant.
The mean salivary total sialic acid (TSA) levels of the subgroups in oral habit groups were compared by Student's t test. The t value between tobacco chewers group and control group was found to be 4.97, whereas it was 4.81 between smokers and control group; and was statistically significant (P < 0.05) [Figure 1].
Comparison of salivary sialic acid levels with clinical parameters
The OSMF group was clinically divided into four stages based on Ranganathan et al (2001). criteria. The mean TSA level in stage II was 43.5 mg/dL whereas it was 46.5 mg/dL, and 47.5 mg/dL in stage III and stage IV, respectively. The t values showed marked increase in the sialic acid levels as the disease progressed [Table 2].
|Table 2: Comparison of Sialic Acid with Clinical and Histological Parameters|
Click here to view
The leukoplakia group was clinically correlated with the color of the lesion, where the values observed were 28.3 mg/dL, 30 mg/dL and 35 mg/dL in leukoplakic, erythroleukoplakic and erythroplakic cases , respectively. The sialic acid levels were higher in erythroplakia compared to the other groups and suggested that TSA levels were directly proportional to the redness of the lesion [Table 2].
The oral cancer group was divided clinically based on the site of lesion occurrence. The levels were 32.5 mg/dL in buccal mucosa, 36.5 mg/dL in the floor of the mouth and 40 mg/dL in the lateral border of the tongue. Increased values in the floor of the mouth and lateral tongue border suggests the increase tumor burden at these sites [Table 2] and [Figure 2].
Comparison of salivary sialic acid levels with cytological and histological parameters
Cytologically the OPMDs were subgrouped as Class II, Class III and Class IV with mean TSA values 26 mg/dL, 30 mg/dL and 33.3 mg/dL , respectively. These values was statistically significant although the mean TSA values showed a marked increase with cytological atypia [Table 2].
Histopathologically, oral cancer was divided by Broder's classification into two groups: well-differentiated squamous cell carcinoma (WDSCC) and moderately differentiated squamous cell carcinoma (MDSCC) due to the non-availability of poorly differentiated cases. The means salivary TSA levels in WDSCC were 38.3 mg/dL and MDSCC were 33.3 mg/dL. When compared with Chi-square test, values were found to be statistically not significant [Table 2].
Comparison of TSA levels were done with TC and TP levels in both the group population, which was statistically significant (P < 0.05), suggesting that salivary sialic acid is dependent and has a positive correlation with the glycoprotein levels [Table 3] and [Figure 3].
| Discussion|| |
The incidence of oral cancer accounts for about 3% of all malignancies worldwide. The tendency to detect oral cancer at a later stage of life is responsible for its high morbidity and mortality rate. The clinical and biological behavior of the tumor is assessed histologically based on the number of cellular and architectural changes.
So, diagnosing these changes at an early stage of life may reduce the risk of malignant transformation. Many diagnostic tools have been marketed to aid health care professionals for early diagnosis of the potential cancerous lesion at a non-lesional stage to formulate a treatment protocol to improve the patient's quality of life. Early detection using salivary biomarkers detects the analytes present in the saliva virtually reflecting the entire spectrum of health and disease state. Expanding the utility of saliva in the field of early intervention will help to improve the patient's survival and risk of disease recurrence.
Upregulation or downregulation of biochemical substances at the cell surface is a common contributing factor for the activation of various downstream signaling pathways like HIPPO pathway, Sonic hedgehog (SHH), pathway and Mitogen activated protein kinase (MAPKinase).
Sialic acid, a derivative of neuraminic acid, and a monosaccharide, occurs in combination with other monosaccharides like mannose, glucosamine, fucose and galactose. These carbohydrate moieties are released into the circulation during increased cell turnover, secretion or shedding, which is associated with cell-to-cell adhesion, cellular interactions, tumor progression and metastasis. Many studies have shown to use sialic acid as a therapeutic target for various systemic diseases like atherosclerosis, epilepsy, influenza, pneumonia and chronic periodontitis.,
Similarly, many studies documented in the literature have shown variation of salivary sialic acid levels with various oral diseases. So, in this study, subjects with smoking and chewing habits with no detectable benign lesions, along with clinical parameters like lesion site, color and cytology were taken as a new parameter to compare.
The levels of sialic acid within different subjects in Group I showed a significant increase which was consistent with the study done by Jacob et al. and Sonika Achali et al. having a mean value of 57.56 mg/dL in leukoplakia and 67.022 mg/dL in OSCC. This level of increase observed suggests that the degree of dysplasia increases during disease progression.
Between the different subjects in Group II, there was a decline in the sialic acid level between OSMF (46.3 mg/dL) and OSCC (36.5 mg/dL) which was contrary to the findings of Jacob et al. study reports that showed elevated levels of sialic acid in OSCC (86.21 mg/dL) compared to patients with OSMF (65.02 mg/dL). This variation in salivary sialic acid levels in OSMF and OSCC patients compared to other studies could be a reason for the relative less chance of malignant transformation in OSMF than leukoplakia, according to the literature reports by Zhang et al.
Most studies documented in literature lack to analyze the sialic acid levels in tobacco usage individuals without a benign lesion. In the present study, sialic acid levels were estimated in patients having tobacco smoking and chewing habits with no clinical lesion. Here, we found that there was a significant difference between the health and diseased subjects, demonstrating that the carcinogens present in the tobacco-related products cause cellular alterations in the mucosal layer. This can be taken into consideration proving that even an early change towards malignancy can be identified using sialic acid.
TPs and TCs are the routine tests performed in patients suspected of any pathology and if it proved to have any significant change can aid in the diagnosis. We observed a statistically significant positive correlation between TSA, TP and TC which was in accordance with Sanjay PR et al. Other studies (Nidhi Dhakar et al.) have reported negative correlation between TSA and total protein while with TC showed significant correlation.
The clinical and histological parameters of potentially malignant disorders and OSCC are routinely used to assess the risk of disease progression. On correlating with the clinical grades of OSMF, statistically significant difference was noted. These findings were like the study done by Chittemsetti S et al., Joshi M et al. and Sawhney H et al. suggesting that sialic acid increases the tumor cell surface during mucosal change.
Lesion color emphasized by Mashberg et al. stating that redness is an early sign of cancerous change was analyzed by segregating the subjects as leukoplakia, erythroleukoplakia and erythroplakia. We observed that sialic acid level was high in erythroplakia (35.5 mg/dL) compared to others, suggesting that sialic acid released from cell surface increases with mucosal changes.
Warnakulasuriya and Ariyawardana study suggested that lesions present in the tongue and floor of mouth had a poor prognosis compared to other sites. In the present study, the subjects with OSCC lesions were subclassified based on the clinical site as buccal mucosa, tongue and floor of the mouth. Sialic acid levels were significantly higher in patients with lesion present in the tongue and floor of mouth compared to the buccal mucosa. This change could attribute to the mucosal changes taking place due to overexposure of carcinogens present in saliva pooling at these sites and can also provide additional rationale for the role of sialic acid in the prognosis of OSCC.
The cellular changes in OPMDs were correlated with salivary sialic acid levels using cytological grading of the lesion. A significant amount of sialic acid level was increased in Class IV. On comparing the sialic acid levels of OSCC patients using histopathological grading, a reciprocal variation of sialic acid levels was observed. We observed well-differentiated cases having higher values compared to moderately differentiated carcinoma cases. This was in accordance with the study conducted by Jacob et al. and Sonika Achalli et al. suggesting that this variation could be due to a cell losing its identity of parent cell during differentiation in the neoplastic lesion.
Increase or decrease in the sialic acid within the group subjects, comparing with clinical and histological parameters, suggests that sialic acid could be a part of the puzzle that a disorder reflects during progression. Thus, in the present study, the level of sialic acid was significantly elevated from the non-lesional stage suggesting that evaluating sialic acid using non-invasive saliva is helpful in the diagnosis of the disease in the early stage itself.,
Levels of sialic acid in saliva need to be standardized with serum levels. Large sample studies need to be carried out to correlate the association of sialic acid concentration with clinical and all histological grades of the lesion.
| Conclusion|| |
Though awareness about salivary biomarkers as a preliminary screening tool is explicit among dentists, the ability to implement them as a part of routine practice is very less. Various reasons have been quoted for the same, which includes the construction of specialized biosensors or test strips. This lacuna is the key for our future research focusing on construction of biosensors to be used as chairside screening tool to educate and create awareness among patients to understand the tumor burden. Excluding minor errors, alteration in sialic acid levels in individuals with habit, OPMD and OSCC indicate its importance as a diagnostic marker.
Authors wish to thank Prof. C.J. Gunasekaran and Prof. Padma Govindan for their kindly help in preparation of this research.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]