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ORIGINAL ARTICLE
Year : 2016  |  Volume : 12  |  Issue : 2  |  Page : 634-639

A non-invasive study to estimate and compare salivary sialic acid level as tumor marker in patients with pre-cancer and oral cancer


1 Department of Oral and Maxillofacial Pathology, Sri Sankara Dental College, Trivandrum, Kerala, India
2 Department of Oral Pathology, Vinayaka Missions, Sankarachariyar Dental College, Salem, Tamil Nadu, India
3 Department of Pedodontics and Preventive Dentistry, Vinayaka Missions, Sankarachariyar Dental College, Salem, Tamil Nadu, India
4 Department of Oral and Maxillofacial Pathology, College of Dentistry, University of Hail, Kingdom of Saudi Arabia
5 Department of Biochemistry, Vinayaka Missions Krupananda Variyar Medical College, Salem, Tamil Nadu, India

Date of Web Publication25-Jul-2016

Correspondence Address:
Maya Ramesh
Department of Oral Pathology, Vinayaka Missions, Sankarachariyar Dental College, National Highway 47, Sankari Main Road, Ariyanoor, Salem - 636 308, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.148697

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 > Abstract 


Introduction: Sialic acids are important terminal sugars of the carbohydrate chains of cell membrane glycoproteins and glycolipids. The progressive rise in total sialic acid (TSA) levels has been significantly associated with tumor progression and metastasis.
Materials and Methods: Samples of 2 ml of unstimulated whole saliva were collected from 20 healthy patients, and 20 cases of histopathologically confirmed oral pre-cancer patients and 20 cases of histopathologically confirmed oral squamous cell carcinoma (OSCC) patients, using the method of Navazesh. Estimation of total sialic acid was done by the histochemical method of Yao et al.
Results: The study revealed significantly elevated total sialic acid (TSA) levels in the saliva of oral precancer and OSCC patients, yielding a mean value of 59.75 ± 7.29 mg/dl and 204.85 ± 60.38 mg/dl, respectively, against the mean value of 21.65 ± 5.71 mg/dl of the healthy controls. Also, high total sialic acid (TSA) levels in the well-differentiated squamous cell carcinoma cases were appreciable to those of moderately differentiated ones.
Discussion and Conclusion: The result suggests the correlation of elevated salivary TSA levels with the progression of OSCC. In conclusion, the study indicates that the glycoprotein metabolism is significantly altered in the saliva of patients, with both oral pre-cancer and OSCC.

Keywords: Non-invasive, oral squamous cell carcinoma, pre-cancer, saliva, total sialic acid levels


How to cite this article:
Jacob TV, Ramesh M, Murali S, Ramesh K, Sanjay P R, Abraham P. A non-invasive study to estimate and compare salivary sialic acid level as tumor marker in patients with pre-cancer and oral cancer. J Can Res Ther 2016;12:634-9

How to cite this URL:
Jacob TV, Ramesh M, Murali S, Ramesh K, Sanjay P R, Abraham P. A non-invasive study to estimate and compare salivary sialic acid level as tumor marker in patients with pre-cancer and oral cancer. J Can Res Ther [serial online] 2016 [cited 2019 Sep 21];12:634-9. Available from: http://www.cancerjournal.net/text.asp?2016/12/2/634/148697




 > Introduction Top


Oral cancer is a major problem in India and accounts for 50-70% of all cancers diagnosed, as compared to 2-3% in the UK and USA. It is currently the most frequent cause of cancer-related deaths among Indian men, which is usually preceded by oral pre-cancerous states, i.e., most often a persistent leukoplakia or oral submucous fibrosis (SMF). Oral leukoplakias and SMF have been reported to show an increased risk of conversion to malignant transformation varying from 0.13% to 6%, and the risk further increased to 14% or higher in dysplastic lesions.[1] The most common form of tobacco chewing in India is the betel quid which usually consists of the betel leaf, areca nut, lime and tobacco. Saliva, a complex fluid consisting of a wide variety of organic and inorganic constituents, collectively acts to modulate the oral environment.[2] It is a unique fluid, and the growing interest in its use as a diagnostic tool has advanced exponentially in the recent years. Because of its anatomical proximity to both premalignant and malignant oral neoplasms, saliva is ideal for the screening of these lesions.[3] Very few studies have reported the altered levels of salivary glycoproteins such as p53 and CA125, in various types of malignancies including oral squamous cell carcinoma (OSCC).[4] Biological markers can be used to monitor cancer, predict the therapeutic response and prognosis of cancer and in some certain situations, even diagnose cancer. These are referred to as tumor markers, which are naturally occurring or modified molecules that can be measured in serum, plasma or other body fluids, and their concentration becomes changed in the presence of cancer.[5]

N-acetyl neuraminic acid (commonly referred to as sialic acid), is a negatively charged nine-carbon monosaccharide commonly attached by a –glycosidic linkage. Sialic acids are important terminal sugars of the carbohydrate chains of cell membrane glycoproteins and glycolipids.[6] Previous studies have reported elevated serum and salivary levels of sialic acid, sialyltransferases and certain glycoproteins in various carcinomas, including oral pre-cancer and OSCC.[7],[8],[9]

Specific changes in salivary protein composition alter the ecological balance in favor of many pathogenic microbes, initiating the onset of disease process, thus serving as a tumor marker for early non-invasive diagnosis of oral cancer.

Aim

The aim of the study was to estimate and correlate the TSA levels in salivary samples of patients with oral pre-cancer and OSCC with those of healthy subjects and to evaluate their role in diagnosis.

Objectives

To assess the levels of TSA in saliva of healthy subjects and the study group comprising of oral pre-cancer and oral cancer patients.

To compare and correlate the levels of TSA in saliva of the control group with that of oral pre-cancer patients and oral cancer patients.

To evaluate whether there is any significant changes in the levels of TSA in saliva in the different grades of OSCC.


 > Materials and Methods Top


The subjects included in this study were randomly selected from the Department of Oral Medicine and Radiology, and the Department of Oral Pathology of our institution. Ethical Clearance was obtained from Institutional Ethical Committee. Informed consent was explained to each patient and written consent was obtained. Each individual was subjected to routine medical history and personal history. Habits like the use of tobacco in different forms were specifically sought.

The sample for the present study comprised a total of 20 healthy subjects who served as control, 20 patients diagnosed with oral pre cancer and 20 patients diagnosed with OSCC.

They were categorized as follows: [Table 1], [Figure 1] and [Figure 2].
Table 1: Age and sexwise distribution of healthy controls, oral pre-cancer and OSCC patients

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Figure 1: Sex wise distribution of patient groups

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Figure 2: Age wise distribution of patient groups

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Group I (Healthy Subjects): Comprised healthy patients with good oral hygiene, with no systemic disorders detected – 20 cases. Age range varied from 32 to 65 years

Group II (Oral Precancer): Comprised clinically and histopathologically known oral pre-cancer patients, with no systemic disorders detected – 20 cases. (Leukoplakia-10, Oral Sub Mucous Fibrosis-10). Age range varied from 35 to 64 years

Group III (OSCC): Comprised clinically and histopathologically detected oral squamous cell carcinoma patients, with no systemic disorders detected - 20 cases. (Well differentiated OSCC-12, Moderately differentiated OSCC-8).

Age range varied from 47 to 68 years.

Inclusion criteria

The study group comprised both pre-cancer and oral squamous cell carcinoma patients, free from systemic diseases and regular medications.

Exclusion criteria

Patients on regular medications, which might affect the salivary flow and those who were unable to comply with the study and appointment schedules were excluded.

Armamentarium

Instruments for collection of saliva included pre-sterilized containers, distilled water, glass funnel and ice box/refrigerator. Equipments for biochemical estimation included UV- spectrophotometer, centrifuge, automated micropipettes, water bath, test tubes and test tube holders. Chemicals used for total salivary sialic acid estimation included N-acetyl neuraminic acid, Ninhydrin, 0.9% sodium chloride and glacial acetic acid. All the above chemicals were of high quality (analytical grade) and procured from Hi-Media company.

Saliva collection

Unstimulated whole saliva samples were collected between 10 am and 12 noon by the technique suggested by Navazesh [10] (1993), 2 hours after breakfast. This was to minimize the variability in salivary flow and compositions due to diurnal variation. The subject was asked to rinse the mouth with distilled water thoroughly, to remove any food debris and then after 10 minutes, he/she was directed to spit into a sterile plastic container. The subjects were instructed not to spit forcibly so as to avoid blood contamination. Saliva collected in the containers was placed in an ice carrier box/refrigerator and transferred to the laboratory for the biochemical analysis.


 > Procedure Top


  • Acidic Ninhydrin reagent: 250mg Ninhydrin was dissolved in 6ml glacial acetic acid and 4ml concentrated sulfuricacid, by thorough vortexing for 30 minutes
  • N-acetyl neuraminic acid (NANA) standard: 10mg NANA was dissolved in 100ml distilled water
  • 0.9% NaCl
  • 0.1ml of saliva was added to 0.9ml of normal saline to make it 1ml and centrifuged at 3000 rpm for 30 min
  • To this 1ml of glacial acetic acid and 1ml of acid Ninhydrin reagent (freshly prepared) was added
  • The test tubes were kept in boiling water bath for 10 minutes, cooled under tap water and absorbance was read at 470nm. NANA standards ranging in concentration from 20 to 100mg/ml were run simultaneously.


Estimation of total sialic acid

Salivary total sialic acid levels were estimated by the method of Yao et al.[11] using the UV-spectrophotometer (Elico). Estimation was based on the reaction of sialic acid with Ninhydrin reagent in acidic medium. The absorbance of blue-colored complex was measured at 470 nm.

A standard curve was prepared by plotting absorbance readings of standards (S1-S5) against their concentrations. Concentrations of total sialic acid were calculated from the standard curve [Table 2].
Table 2: Total sialic acid (tsa) levels in healthy controls, oscc and oral precancer groups (in mg/100ml)

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 > Results Top


[Table 3] and [Figure 3] shows the values of TSA levels in saliva of the three different groups of control, pre-cancer and oral cancer in mg/100 ml.
Table 3: Kruskal-wallis test

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Figure 3: Total sialic acid (tsa) levels in healthy controls, oral precancer and oscc groups

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The test for mean score analysis (Kruskal-Wallis Test) procedure is used to compare mean scores of more than two groups. The significant difference between the mean scores is tested with respect to the sialic acid levels. The table displays the descriptive statistics of the sample size, mean, standard deviation and standard error along with the Chi-square statistics. [Table 3], [Figure 3] The column P value shows the probability value from the chi square distribution. Since the P value is less than 0.01, the result is highly significant.

The non-parametric Mann-Whitney U Test procedure is used to compare mean scores of sialic acid levels of two squamous cell carcinoma groups. The Group III TSA levels also showed a relative increase in the values of well-differentiated squamous cell carcinoma (WDSCC) against moderately differentiated squamous cell carcinoma (MDSCC) [Table 4] and [Figure 4]. The table displays the descriptive statistics of the sample size, mean, standard deviation and standard error. Since the P value is less than 0.01, it is highly significant. Hence, there is significant difference in the mean sialic acid among the two groups of squamous cell carcinoma patients.
Figure 4: Total sialic acid (tsa) levels in well differentiated and moderately differentiated squamous cell carcinoma

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Table 4: Mann–whitney u test-tsa levels in well differenciated scc with moderately differentiated scc

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From the results of the above the mean and SD of the TSA levels of Group I (Healthy Controls), Group II (Oral Precancer) and Group III (OSCC) were 21.65 ± 5.71, 59.75 ± 7.29 and 204.85 ± 60.38, respectively. The TSA levels of Group III had the most significant high values in comparison to Groups I and II. Also Groups II and III showed increased TSA levels than the healthy controls.


 > Discussion Top


The term 'Sialic Acid', derived from the Greek word “Sialos” meaning “Saliva”was first introduced by Swedish biochemist, Gunnar Blix in 1952. The predominant sialic acid is N-acetyl neuraminic acid (Neu 5AC) or NANA.[12] The name “neuraminic acid” was introduced by German scientist E. Klerk [13] in 1941. He said that the negative charge of this ubiquitous chemical is responsible for the slippery feel of saliva and mucins coating the body's organs.

Sialic acid as a tumor marker

Various studies have reported the significance of sialic acid as a tumor marker.

Our study showed lowest values of total salivary sialic acid levels (TSA) for healthy people, moderate value for pre cancer and very high values for oral squamous cell carcinoma.

Lipton et al.[14] (1979) studied the total protein and sialic acid levels in the serum of patients with either localized or advanced metastatic malignancy and reported elevated mean serum protein and NANA levels. The study conducted by Plucinsky et al.[15] in 1986 in the serum of cancer patients with different primary sites and differing degrees of metastatic involvement, showed significant increase in the total serum sialic acid (TSA) and total protein (TP) values when compared to the normal controls. This was similar to our study though he did not study pre-cancerous lesions. He suggested that TSA/TP was the best of the tested markers and even though it is quite sensitive, but lacks specificity.

BaxiBR [16]et al. (1991) found that serum TSA and LSA levels were significantly increased in oral pre-cancer and OSCC patients when compared with controls. The patients with metastasis had higher levels of the biomarkers than in oral pre-cancer. We also obtained a similar result in saliva although we did not check the correlation of salivary sialic levels with metastatic lesions.

Again in 1991, Xing RD [17]et al. found significant elevation of serum sialic acid levels in OSCC patients when compared to those of healthy controls and patients with benign disease. No significant difference was noted between the normal control group and the patients with benign tumor. This can be taken as a study proving that even an early change towards malignancy can be identified using sialic acid as the tumor marker. And this study is replicated in a non-invasive way in our study using salivary sialic acid as the tumor marker.

Rajpura et al.[7] (2005) estimated total and lipid bound serum sialic acid levels in oral pre-cancer, OSCC and healthy subjects, respectively, and found that serum levels of total and lipid bound sialic acid were significantly elevated in untreated OSCC patients as compared to healthy individuals and patients with oral pre-cancer. The progressive rise in total and lipid bound sialic acid was significantly associated with the stage of the malignant disease. This study was also very similar to our present study, but we used the non-invasive technique of sialic acid estimation using saliva. We also correlated the histopathologic grading of oral squamous cell carcinoma with sialic acid levels and found that well differentiated squamous cell carcinoma had higher values of salivary TSA compared to moderately differentiated SCC.

Sanjay et al.[18] reported that salivary levels of total protein, total sugar and sialic acid were significantly higher in OSCC patients compared to those of normal healthy controls. The salivary sialic acid levels were found to be significantly higher in well-differentiated squamous cell carcinomas than in moderately differentiated squamous cell carcinomas. Our present study also showed a similar result.

In 2013, the study done by Dhakar et al.[19] showed a significant increase in serum sialic acid, salivary sialic acid and serum protein from control to OSCC and suggests that these markers may be reliable in diagnosis and predicting prognosis.

In 2013, Vajaria et al.[20] reported that salivary TSA/TP ratio and α-l-fucosidase activity were elevated with higher magnitude than serum levels. These results suggest that a larger study may prove the use of these saliva biomarkers as a non-invasive method for detecting early changes occurring during oral carcinogenesis.

The TSA levels of the oral pre-cancerous states, mainly leukoplakia and oral submucous fibrosis were included in the present study, were emphatically high against those of the healthy subjects. These significant elevations in the important glycoprotein constituents in patients with oral pre-cancer could be indicators of early biochemical changes denoting malignant transformation of the cell. But some of the leukoplakia cases showed higher salivary TSA levels and others lower salivary TSA levels compared to oral submucous fibrosis. When well-differentiated squamous cell carcinoma was compared with moderately differentiated squamous cell carcinoma, well-differentiated SCC showed higher values. More studies with more number of specimens are required to give an explanation to this finding.


 > Conclusion Top


Aberrant glycosylation of glycol-conjugates is among the important molecular changes that accompany malignant transformation.[7] The present study is aimed at estimating and correlating the total sialic acid levels in salivary samples of patients with oral pre-cancer and oral squamous cell carcinoma with those of healthy patients, so as to potentially utilize these parameters for early diagnosis. In conclusion, the study indicates that glycoprotein metabolism is significantly altered in the saliva of patients, with both OSCC and oral pre-cancer. Saliva study offers a bright scope in the future for the detailed research of the applications of salivary parameters in early detection of pre-cancer and oral cancer patients.


 > Acknowledgement Top


Prof. Dr. Saramma Mathew Professor Emeritus. VMSDC, Salem. Dr. Sachu Philips, Reader Department of Biochemistry, Vivekananda Dental College.

 
 > References Top

1.
Rajendran.R. Benign and malignant tumors of the oral cavity. In, Rajendran R, Sivapathasundaram B (ed). Shafer's Textbook of Oral Pathology, 6th ed. Elsevier,2009;80.  Back to cited text no. 1
    
2.
Streckfus CF, Bigler LR.”Saliva as a diagnostic fluid”. Oral Dis 2002;8:69-76.  Back to cited text no. 2
    
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Cohen RE, Levine MJ. “Salivary glycoproteins. In: Tenovuo JO, editors, Human saliva: Clinical, chemical and microbiology”. Vol. 1. Florida: Publisher CRC Press; 2000. p. 101-30.  Back to cited text no. 3
    
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Navarro MA, Mesia R, Diez-Gilbert O, Rueda A, Ojeda B, Alonso MC. Epidermal growth factor in plasma and saliva of patients with active breast cancer and breast cancer patients in follow-up compared with healthy women. Breast cancer Res Treat 1997;42:83-6.  Back to cited text no. 4
    
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Chan DW, Schwartz MK. Tumor markers-Introduction and general principles. In: Diamandis EP, Fritsche HA, Lilja H, Chan DW, Schwartz MK, editors. Tumor Markers: Physiology, Pathobiology, Technology, and Clinical Applications. Washington, DC: AACC Press; 2002. p. 9-17.  Back to cited text no. 5
    
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Schauer R. Chemistry, metabolism, and biological functions of sialic acids. Adv Carbohydr Chem Biochem 1982;40:131-234.  Back to cited text no. 6
    
7.
Rajpura KB, Patel PS, Chawda JG, Shah RM. Clinical significance of total and lipid bound sialic acid levels in oral pre-cancerous conditions and oral cancer. J Oral Pathol Med 2005;34:263-7.  Back to cited text no. 7
    
8.
Raval GN, Parekh LJ, Patel DD, Jha FP, Sainger RN, Patel PS. Clinical usefulness of alterations in sialic acid, sialyltransferase in sialic acid, sialyl transferase and sialoproteins in breast cancer. Indian J Clin Biochem 2004;19:60-71.  Back to cited text no. 8
    
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Tewarson SL, Mittal VP, Singh M, Gupta GP. Serum sialic acid--an important cancer marker. Indian J Cancer 1993;30:125-31.  Back to cited text no. 9
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Navazesh M. Methods for collecting saliva. Ann NY Acad Sci 1993;20:72-7.  Back to cited text no. 10
    
11.
Yao K, Ubuka T, Masuoka N, Kinuta M, Ikeda T. Direct determination of bound sialic acids in sialoglycoproteins by acidic ninhydrin reaction. Anal Biochem 1989;179:332-5.  Back to cited text no. 11
    
12.
Available from: http://en.wikipedia.org/wiki/Neuraminic_acid [Last accessed on 2014 Nov 10].  Back to cited text no. 12
    
13.
Klerk E. Neuraminic acid. Physiol Chem 1941;268:50-58.  Back to cited text no. 13
    
14.
Lipton A, Harvey HA, Dellong S, Allegra J, White D, Allegra M, et al. Glycoproteins and human cancer. 1. Circulating levels in cancer serum. Cancer 1979;43:1766-71.  Back to cited text no. 14
    
15.
Plucinsky MC, Riley WM, Prorok JJ, Alhadeff JA. Total and lipid associated serum sialic acid levels in cancer patients with different primary sites and differing degrees of metastatic involvement. Cancer 1986;58:2680-5.  Back to cited text no. 15
    
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Baxi BR, Patel PS, Adhavaryu SG, Dayal PK. Usefulness of serum glycoconjugates in precancerous and cancerous diseases of oral cavity. Cancer 1991;67:135-40.  Back to cited text no. 16
    
17.
Xing RD, Wang ZS, Li CQ, Tang QY, Jiang CB, Zang YZ. Total sialic acid as a tumor marker for oral cancer. Int J Biol Markers 1991;9:239-42.  Back to cited text no. 17
    
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Sanjay PR, Hallikeri K, Shivashankara AR. Evaluation of salivary sialic acid, total protein and total sugar in oral cancer: A preliminary report. Indian J Dent Res 2008;19:288-91.  Back to cited text no. 18
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Dhakar N, Astekar M, Jain M, Saawarn S, Saawarn N. Total sialic acid, total protein and total sugar levels in serum and saliva of oral squamous cell carcinoma patients: A case control study. Dent Res J (Isfahan) 2013;10:343-7.  Back to cited text no. 19
    
20.
Vajaria BN, Patel KR, Begum R, Shah FD, Patel JB, Shukla SN, et al. Evaluation of serum and salivary total sialic acid and α-l-fucosidase in patients with oral precancerous conditions and oral cancer. Oral Surg Oral Med Oral Pathol Oral Radiol 2013;115:764-71.  Back to cited text no. 20
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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