|HEAD AND NECK SYMPOSIUM: ORIGINAL ARTICLE
|Year : 2014 | Volume
| Issue : 3 | Page : 214-218
Serum and salivary sialic acid as a biomarker in oral potentially malignant disorders and oral cancer
M Dadhich1, V Prabhu1, VR Pai2, J D'Souza2, S Harish2, M Jose1
1 Department of Oral Pathology and Microbiology, Yenepoya Dental College, Mangalore, India
2 Department of Biochemistry, Yenepoya Medical College, Yenepoya University, Mangalore, India
|Date of Web Publication||10-Dec-2014|
Department of Oral Pathology and Microbiology, Yenepoya Dental College, Mangalore
Source of Support: None, Conflict of Interest: None
Background: Aberrant glycosylation is the universal feature of cancer and components of various glycoconjugates, such as sialic acid is found to rise in various malignancies. The objective of this study was to evaluate the serum and salivary sialic acid in oral potentially malignant disorders (OPMD) and oral cancer (OC) to investigate the possibility of using this as a diagnostic marker. Materials and Methods: The study included 85 subjects, who were grouped as control (30), OPMD patients (25), and oral cancer patients (30). Serum and unstimulated whole saliva was collected from subjects of all groups and sialic acid estimation was done using spectrophotometry. The results were tabulated and analyzed statistically. Results: The mean serum sialic acid levels in normal, OPMD, and oral cancer group were 7.515, 19.620, and 55.235 mg/dL, respectively, whereas the levels of salivary sialic acid were 1.5113, 2.3302, and 9.0304 mg/dL, respectively. A very highly significant rise (P < 0.005) in serum and salivary sialic acid was observed in the study subjects compared with that of the control. Conclusions: The present study showed a significant and gradual increase in serum and salivary sialic acid from control to oral potentially malignant disorders to oral cancer. From this study we can suggest that sialic acid can be used as a reliable biomarker. As this monosaccharide is observed in saliva in detectable quantity, saliva can be used as a diagnostic medium for screening and early detection of oral cancer.
Keywords: Saliva, serum, sialic acid, oral cancer, oral potentially malignant disorders
|How to cite this article:|
Dadhich M, Prabhu V, Pai V R, D'Souza J, Harish S, Jose M. Serum and salivary sialic acid as a biomarker in oral potentially malignant disorders and oral cancer. Indian J Cancer 2014;51:214-8
|How to cite this URL:|
Dadhich M, Prabhu V, Pai V R, D'Souza J, Harish S, Jose M. Serum and salivary sialic acid as a biomarker in oral potentially malignant disorders and oral cancer. Indian J Cancer [serial online] 2014 [cited 2020 Aug 10];51:214-8. Available from: http://www.indianjcancer.com/text.asp?2014/51/3/214/146720
| » Introduction|| |
Oral and pharyngeal cancer, grouped together, is the sixth most common cancer in the world.  Oral cancer is on a rise in younger population and a 5-year mortality rate of almost 50%, which has not changed significantly in the last five decades, despite the advances in the multimodality treatment.  Timely intervention can cure the cancer and prevent from more mutilating surgery and also decreases mortality. 
Oral cancer (OC) is generally preceded by oral potentially malignant disorders (OPMD), such as leukoplakia, oral submucous fibrosis (OSMF), and others. OPMD signifies an increased risk of developing OC with malignant transformation rates that vary from 0.6% to 36%.  Estimation of various biomarkers in serum may act as an adjunct to conventional biopsy and salivary biomarker estimation may provide a noninvasive and cost-effective method for screening the oral cancer patients. 
Cell surface is transformed during carcinogenesis and is vital for uncontrolled growth and malignant behavior of the neoplastic cells. Glycoconjugates such as glycoproteins and glycolipids are important constituents of cell membrane and therefore important in malignancy.  N-acetylneuraminic acid (NANA/sialic acid) is a negatively charged 9 carbon monosaccharide. It is commonly attached to the nonreducing residues of the carbohydrate chains of glycoconjugates by a glycosidic linkage.  Sialic acid is found to be significant in determining the surface properties of cells and hence plays a role in cellular invasiveness, adhesiveness, and immunogenicity. The glycoconjugates are released into the circulation and body fluids, through increased turnover, secretion, and/or shedding from malignant cells. 
Usefulness of serum sialic acid as a biomarker for various malignancies, including oral cancer is well established and was found to be well correlated with tumor progression or reduction in tumor mass and recurrence or metastasis in oral cancer. ,,,,,,,,,,,,,,,,,,, Saliva being a biofluid, which is a filtrate of serum from the vasculature that nourishes the salivary glands, any change that occurs in the serum as a result of disease process will be reflected in saliva.  However, studies in this direction particularly related to glycoconjugate components, such as sialic acid, are very few in the literature. Therefore, this study was designed to estimate serum and salivary sialic acid in OPMD and OC patients to verify the possibility of including sialic acid as a biomarker of these conditions and to investigate the use of saliva as a diagnostic fluid for its detection. Such an examination will be of great benefit because of many advantages of saliva over serum.
| » Materials and Methods|| |
Twenty-five patients with oral potentially malignant disorders (group-OPMD) and 30 oral cancer patients (group-OC) who have sought treatment in Dental Hospital of our institution and nearby cancer centers were chosen for the study. Thirty age- and gender- matched healthy volunteers without tobacco-related oral habits, any oral lesions, and any systemic disorder from the university campus were selected for the normal control group (group-NC). Selection of the subjects for the study was done based on the case history, clinical, and histopathologic examination. Patients suffering from any other tobacco-related diseases or other malignancies were excluded from the study. A written consent was obtained from the selected individuals. Ethical clearance was obtained from the institutional ethical committee.
Five milliliters of peripheral blood sample was collected from each subject with disposable syringes under aseptic conditions through venipuncture. Serum was separated by centrifugation at 3000 rpm for 15 min. The samples thus collected were stored at −80°C until analysis of sialic acid was done. Unstimulated whole saliva (1.5-2 mL) was collected under resting condition during the morning hours, to ensure that variability in salivary flow and compositions is minimized due to diurnal variation.  The subjects were asked to thoroughly rinse their mouth with distilled water to remove any food debris. Then after 10 min, directed to spit into a wide-mouthed sterile plastic container, till the required amount of saliva was collected. The subjects were instructed not to spit forcibly to avoid blood contamination. The plastic container was placed in an ice carrier box and transferred to the laboratory, Saliva sample was centrifuged at 3000 rpm for 15 min and the supernatant was separated and stored at −80°C until sialic acid analysis was done.
Estimation of sialic acid was done following the method described by Yao et al.  In the presence of acidic medium, sialic acid reacts with ninhydrin to form a colored product which can be measured spectrophotometrically at 470 nm. Precipitate was obtained after ethanol treatment of the sample. To the precipitate, 1.0 mL of distilled water and 1.0 mL glacial acetic acid was added followed by 1.0 mL of acid ninhydrin reagent. The reaction mixture was vortexed and then heated for 10 min in a boiling water bath. After cooling the mixture under tap water, absorbance was measured at 470 nm using 60S UV-vis spectrophotometer from Thermo Scientificβ . Standard sialic acid was run simultaneously with the test samples to obtain the standard curve. All the chemicals used were of analytic grade and were procured from Hi- Media and Merck companies.
Biochemical values obtained were recorded and statistically analyzed using SPSS software (version 17.0; SPSS, Inc., Chicago, IL, USA). Kruskal-Wallis test was applied to compare all the 3 groups. Comparison among 2 groups was done with Mann-Whitney test, and Wilcoxon test was applied to compare serum and salivary sialic acid. P value ≤ 0.05 was considered significant. Correlation between serum and salivary sialic acid in different groups was verified using Spearman correlation test.
| » Results|| |
A total of 85 cases were analyzed among the 3 groups. The age- and genderwise distribution of the subjects in all the 3 groups is shown in [Table 1].
The mean values of serum sialic acid in the normal group, OPMD group, and OC group are given in [Table 2]. The difference in serum sialic acid in OPMD and OC group was found to be very highly significant as compared with the NC group (P < 0.0005). When comparison of the result was done between the groups using Mann-Whitney test, the difference in the levels was found to be very highly significant between the normal and OPMD groups, normal and OC groups and between the OPMD and OC groups (P < 0.0005) [Table 2]. Mean serum sialic acid was found to rise from normal to OPMD group to OC group as shown in [Figure 1].
|Figure 1: Serum sialic acid levels in normal, oral potentially malignant disorders, and oral cancer groups|
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Similarly, salivary sialic acid values were recorded and mean values in normal, OPMD group, and OC group are tabulated in [Table 3]. The difference in salivary sialic acid in the OPMD and OC groups was found to be very highly significant as compared with NC group (P < 0.0005). On comparing the levels of sialic acid between the groups, the difference in the levels was found to be highly significant between the normal and OPMD groups (P = 0.005) and very highly significant between normal and OC groups and between OPMD and OC groups (P < 0.0005) [Table 3]. Salivary sialic acid was found to rise in OPMD and OC group when compared with normal group as depicted in [Figure 2].
|Figure 2: Salivary sialic acid levels in normal, oral potentially malignant disorders, and oral cancer groups|
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Comparison of serum and salivary sialic acid was done in various groups and very highly significant difference was obtained in all the study groups (P < 0.0005) [Table 4]. The difference in the mean values of sialic acid in serum and saliva is shown in [Figure 3].
|Figure 3: Comparison of salivary and serum sialic acid in the normal, oral potentially malignant disorders, and oral cancer groups|
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Correlation between serum and salivary sialic acid in different groups was verified using Spearman test. In normal control and OPMD groups, the serum and salivary sialic acid values showed mild correlation, whereas the OC group showed a moderate correlation. However, no significant correlation was noted in any of the above groups [Table 5].
| » Discussion|| |
The present study was carried out to estimate the sialic acid levels in serum and saliva and to correlate the concentrations in both these biofluids in an attempt to test the possibility of using this for early detection of potentially malignant oral disorders and oral cancer.
In our study, the serum sialic acid in the normal group was in the range of 1.0-27.5 mg/dL with a mean of 7.515 mg/dL as compared with the study done by Joshi et al. (1989) in which the serum sialic acid in the normal group was in the range of 36-60 mg/dL.  Serum sialic acid in the OPMD group in the present study was in the range of 1.8-53.1 mg/dL with a mean of 19.620 mg/dL as compared with a mean of 47.74 mg/dL obtained in the study done by Baxi et al. (1991), which is more than the mean value in our study.  We obtained a value of serum sialic acid in the range of 18.6-168.9 mg/dL in the OC group with a mean of 55.235 mg/dL, whereas Joshi et al. (1989) reported a range of 35-116.7 mg/dL for the same group.  The mean value of serum sialic acid obtained in our study is almost equivalent to the mean value obtained in the study done by Baxi et al. (1991), which is 54.78 mg/dL.  The mean serum sialic acid in the study done by Silvia et al. (2001) in the oral cancer group was 114 mg/dL, which is higher than the value obtained in our study.  There are only few studies reported in the literature where a comparison of serum sialic acid is done in normal, OPMD, and oral cancer groups. In the study done by Raval et al. (2003), the mean values of serum sialic acid reported in oral cancer (63.20 mg/dL), OPMD (52.28 mg/dL) and the normal group (44.62 mg/dL) are higher than the values in our study  The variation in the values obtained in different studies might be due to the diverse methods used for the estimation. However, as reported in previous studies, we also have observed a consistent increase in the serum sialic acid from normal to OPMD to OC, which was statistically significant (P < 0.0005).
The mean salivary sialic acid values obtained in NC, OPMD, and OC group in our study were 1.5113, 2.3302, and 9.0304 mg/dL, respectively, which also has exhibited a consistent, statistically significant raise (P < 0.0005). Although few, the findings of available studies ,, are consistent with our results where significant increase in the levels of salivary sialic acid was found among oral cancer and OPMD subjects compared with normal healthy individuals.
While comparing the serum and salivary sialic acid levels in different groups it was noted that values in serum were significantly higher than in saliva (P < 0.0005). This results reflect the fact that the informative analytes are generally present in lower amounts in saliva than in serum as it is a filtrate of serum.  In addition, on analyzing the values using Spearman correlation test, we could observe only a mild to moderate correlation between serum and salivary sialic acid in various groups without any statistical significance. This suggests the possibility of other modifying factors determining the salivary sialic acid level of which the most important could be enzymatic degradation of sialic acid by oral microbes. Although less in concentration, the sialic acid present in saliva was in detectable amount in OPMD and oral cancer and showed a significant and a consistent increase compared with the NC group.
Sialic acid is an important constituent for the characteristic changes of transformed cells. The alterations in glycoproteins start at an early stage of tumorigenesis.  Glycosyltransferases such as sialyltransferase involved in linking terminating residues on glycans tend to be overexpressed in tumor tissue. The increase in activity of these glycosyltransferases in turn leads to the overexpression of certain terminal glycans involving sialic acid. Examples of terminal glycan epitopes commonly found on transformed cells include sialyl Lewis x (sLex), sialyl Tn (sTn), Globo H, Lewis y (Ley), and polysialic acid.  Increased sialylation helps malignant cells to disguise their immunogenic sites, to increase the negative charge of the outer cell membrane so that the binding and killing by lymphocytes and macrophages can be impaired, and to hide the receptor sites for IgM antibodies, which kill cells by a complement-mediated reaction. , Increased sialylation in tumor tissue is reflected as increased sialic acid levels in serum and other body fluids, therefore it may be considered as a significant biomarker of malignancy.
There are convincing reasons to use saliva as a diagnostic fluid to screen oral cancer patients. For a clinician and laboratory professionals, saliva is easy to collect, store, and ship, can be obtained at low cost in sufficient quantities for analysis, saliva collection is safer than venepuncture, which could expose health care providers to HIV or hepatitis virus and saliva is also easier to handle for diagnostic procedures since it does not clot, lessening the manipulations required. For patients, the noninvasive collecting techniques dramatically reduce anxiety and discomfort and simplify procurement of repeated samples for longitudinal monitoring over time. Saliva-based diagnostics are therefore less invasive, less expensive, and present less risk to both the patient and the health care provider than current methodologies. 
In the present study, we have observed a consistent elevation in the serum and salivary sialic acid level from normal to OPMD to oral cancer. Although lower, the concentration of sialic acid was in detectable amount in saliva and reflected an increasing pattern as in serum among different groups evaluated. Based on our findings and previous reports, we suggest that sialic acid is a valuable early biomarker for OPMD and OC and saliva can be a feasible biofluid for detecting sialic acid. Therefore, salivary sialic acid estimation can be used for screening purposes, which can help in identifying high-risk individuals and detecting oral cancer in early stages.
Further studies are required to identify the value of this biomarker in predicting disease outcome and response to therapy in oral cancer patients. Further advancement in technologies is also required to develop portable, low cost, automated devices for rapid estimation of salivary sialic acid.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]
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