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ORIGINAL RESEARCH Table of Contents   
Year : 2008  |  Volume : 19  |  Issue : 4  |  Page : 288-291
Evaluation of salivary sialic acid, total protein, and total sugar in oral cancer: A preliminary report


1 Department of Oral and Maxillofacial Pathology, SDM College of Dental Sciences and Hospital, Dharwad, Karnataka, India
2 Department of Biochemistry, SDM College of Medical Sciences and Hospital, Dharwad, Karnataka, India

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Date of Submission09-Jan-2008
Date of Decision24-Apr-2008
Date of Acceptance14-May-2008
 

   Abstract 

Aim: Detection of cancer at the early stage is of utmost importance to decrease the morbidity and mortality of the disease. Apart from the conventional biopsy, noninvasive methods like analysis of saliva may provide a cost-effective approach for screening a large population. Thus, this study aimed to estimate salivary levels of sialic acid, total protein, and total sugar in the oral cancer patients and in healthy control group to evaluate their role in diagnosis and prognosis of oral cancer.
Study Design: Unstimulated whole saliva samples were collected from 30 healthy controls (Group I) and 30 squamous cell carcinoma patients (group II). Estimations of salivary levels of sialic acid, total protein, and total sugar were performed. This was correlated histopathologically with the grades of carcinoma.
Statistical Analysis and Results: The Student's ' t ' test and multivariate regression analysis were performed. The results showed that salivary levels of total protein, total sugar, protein-bound sialic acid, and free sialic acid were significantly higher in oral cancer patients compared to those of normal healthy controls ( P values in all the results were less than 0.001). The salivary free sialic acid levels were found to be significantly higher in well-differentiated squamous cell carcinoma than in moderately differentiated carcinoma ( P < 0.001). However, protein-bound sialic acid, total proteins, and total sugars did not show any statistical significance between well and moderately differentiated carcinomas.
Conclusion: Biochemical analysis of saliva can be used in early detection of cancer and is best correlated with histopathological degree of squamous cell carcinoma.

Keywords: Histopathological differentiation, oral cancer, saliva, sialic acid, total protein, total sugar

How to cite this article:
Sanjay P R, Hallikeri K, Shivashankara A R. Evaluation of salivary sialic acid, total protein, and total sugar in oral cancer: A preliminary report. Indian J Dent Res 2008;19:288-91

How to cite this URL:
Sanjay P R, Hallikeri K, Shivashankara A R. Evaluation of salivary sialic acid, total protein, and total sugar in oral cancer: A preliminary report. Indian J Dent Res [serial online] 2008 [cited 2019 Oct 19];19:288-91. Available from: http://www.ijdr.in/text.asp?2008/19/4/288/44529
Aberrant glycosylations are the universal features of cancer. Glycoproteins and glycolipids are important constituents of cell membrane; hence, they play an important role in malignancy. These glycoconjugates are released into the circulation through increased turn over, secretion, and / or shedding from malignant cells. Usefulness of assay of serum glycoconjugates in early detection of cancer and in monitoring the progress of treatment, have been evaluated in previous studies. Earlier workers have reported elevated serum levels of total sialic acid (TSA), lipid-bound sialic acid (LSA), and TSA to total protein ratio, in various malignancies. [1],[2],[3],[4]

Saliva is a complex fluid composed of a wide variety of organic and inorganic constituents that collectively act to modulate the oral environment. [5] Salivary glycoproteins play an important role in the properties and functions of saliva. Interest in saliva as a diagnostic fluid has grown exponentially in recent years. Whole saliva can be collected noninvasively, and by individuals with limited training. No special equipment is needed for the collection of saliva. Salivary levels of various biochemical parameters have been measured in infectious diseases, autoimmune diseases, cancers, and psychiatric disorders. Salivary parameters such as CA-125, CA-15-3, kallikrein, epidermal growth factor, and p53 were estimated as tumor markers in the malignancies of breast, ovary, lung, and colon. The use of saliva in the oral cancer diagnosis is still in its infancy. Molecules mentioned above have been identified as potential tumor markers in oral squamous cell carcinoma (OSCC). [6],[7],[8],[9],[10]

There is paucity of studies using saliva as a diagnostic fluid for oral cancer. Biochemical analysis of saliva is not been evaluated in detail in routine clinical laboratories. So, this prompted us to estimate salivary levels of sialic acid, total protein, and total sugar in OSCC patients in order to develop cost-effective and simple diagnostic methods.


   Materials and Methods Top


The study population consisted of thirty OSCC patients. OSCC was diagnosed clinically and histopathologically during their visit to the Department of Oral and Maxillofacial Surgery, S. D. M College of Dental Sciences and Hospital, Dharwad.The control population consisted of thirty normal, apparently healthy, age and sex matched subjects.

The methodology was reviewed and permission was granted for the study by the ethical committee of our institution. All the subjects were informed about the procedure and study was carried out after their consent.

Histopathological assessment

Formalin fixed, paraffin embedded sections were prepared from biopsies taken from the OSCC group. On histopathological examinations, these were subdivided based on Broder's histopathological grading into well-differentiated (22 cases), moderately differentiated (7 cases), and poorly differentiated (1 case) OSCC.

Unstimulated whole saliva samples were collected, after the histopathological confirmation of the clinical diagnosis of OSCC, between 10 a.m. and 12 p.m., two hours after the subject's usual breakfast time. This was to ensure that the variability in salivary flow and compositions due to diurnal variation were minimized. The subjects were asked to rinse the mouth with distilled water thoroughly to remove any food debris and then to spit into a sterile small plastic container. The subjects were instructed not to spit forcibly so as to avoid blood contamination, if any, from the inflamed gingival tissue or the ulcerated lesion. Once the saliva (2 ml) was collected, the plastic container was placed in a ice carrier box and transferred to the laboratory for biochemical analysis. [11]

Estimation of biochemical parameters in saliva

Saliva sample was centrifuged at 3000 rpm for 15 min. The supernatant was taken for analysis of sialic acid, total proteins, and total sugars. Sialic acid was estimated by the method of Yao et al. [12] Saliva was treated with ethanol to precipitate proteins. Sialic acid contents of both the precipitate and supernatant were estimated on the basis of reaction of sialic acid with ninhydrin reagent. This was compared with N-acetyl neuraminic acid standards ranging in concentration from 20-100 mg/ml. Sialic acid concentration obtained from the precipitate was 'protein-bound sialic acid', and sialic acid concentration in the supernatant was 'free sialic acid'.

Salivary total proteins were estimated by the method of Lowry et al. [13] Bovine serum albumin standards ranging in concentration from 0.05-0.5 mg/ml were run simultaneously with the test samples.

Total sugar content was estimated based on phenol-sulphuric acid reaction of sugars. [14] Glucose standards ranging in concentration from 0.01-0.08 mg/ml, were also run.

Statistical analysis

The data were analyzed using the SPSS statistical software. The value of biochemical parameters were expressed as mean ± SD, the levels of significance were determined by employing Student's 't' test. The multivariate regression analysis was performed to correlate sialic acid, total proteins, and total sugars in OSCC patients. Alterations in each parameter were also correlated with histopathological grades of oral cancer.


   Results Top


Detail data of salivary sialic acid, total proteins, and total sugars are listed in the [Table 1] and [Table 2]. Comparative values of salivary biochemical parameters obtained are presented in [Table 3] and [Table 4].

Salivary levels of total protein, total sugar, protein-bound sialic acid, and free sialic acid were significantly higher in OSCC patients compared to those of normal healthy controls. When we compared the values of above parameters between moderately differentiated and well-differentiated OSCC cases, the free sialic acid levels were found to be significantly higher in well-differentiated cases. However, protein-bound sialic acid, total proteins, and total sugars did not differ significantly among well-differentiated and moderately differentiated cases. We did not find any correlation among the biochemical parameters with respect to their elevation in cancer patients.


   Discussion Top


In the present study, we have made an attempt to evaluate the role of salivary biochemical parameters as markers of OSCC. We observed elevated levels of free sialic acid, protein-bound sialic acid, total protein, and total sugar in the saliva of patients compared to those of healthy controls. We have also found significantly higher levels of free sialic acid in well-differentiated OSCC patients compared to those of moderately differentiated cases. This suggests correlation of elevated salivary sialic acid levels to the progression of OSCC.

Various studies have reported the significance of sialic acid as a tumor marker. Aberrant glycosylation processes in tumor cells contribute to the biosynthesis of certain oligosaccharides; hence, malignant or transformed cells contain increased sialic acid residues on their surfaces. [15] Previous studies revealed elevated serum levels of TSA and LSA in malignancies of lung, breast, skin, colon, prostate, and bladder. Sialic acid levels also served to monitor treatment of cancer. [1],[2],[3],[4],[16],[17],[18],[19] Value of serum sialic acid as a tumor marker in OSCC was demonstrated by few workers. [17],[20],[21],[22] However, sialic acid levels were also found to be increased in noncancerous clinical conditions. [23],[24]

Sialic acid is a constituent of many salivary glycoproteins. Studies have reported elevated salivary levels of sialic acid in pregnancy, Down's syndrome, and diabetes mellitus. [25],[26] Dablesteen et al., [27] have reported elevated salivary sialic acid in oral cancer. Baxi et al., studied the sialic acid levels in various cancers and reported elevated levels of sialic acid compared to healthy subjects. [17] The significant elevations of serum sialic acid in oral cancer patients compared to precancerous conditions suggested the potential utility of this parameter in diagnosis as well as determining clinical stage of the malignant disease. [16]

We also observed increased salivary levels of total protein and sugar in OSCC patients. Their role in malignancy is unknown. Further studies are to be conducted to know their diagnostic importance in malignancies with large sample size. Salivary glycoproteins are mainly responsible for the protective qualities of saliva. Contucci et al., [10] observed elevated statherin levels in the saliva of patients with precancerous and cancerous lesions of the oral cavity. Boyle et al., [7] reported elevated levels of salivary p53 in oral cancer. Other studies have reported elevated salivary levels of kallikrein, CA-125, and epidermal growth factor in ovarian and breast cancers. Dablesteen et al., have reported altered carbohydrate expression and elevated protein levels in OSCC.


   Conclusion Top


In this pilot study, we have made an attempt to evaluate the importance of saliva as a diagnostic fluid. A detailed study with larger sample size is needed to establish the clinical importance of salivary glycoconjugates as markers of OSCC. We plan to undertake such a study in the future.

 
   References Top

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2.Baxi BR, Patel PS, Adhavaryu SG. A report on clinical importance of serum glycoconjugates in oral cancer. Indian J Clin Biochem 1990;5:139-44.  Back to cited text no. 2    
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9.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. 9    
10.Contucci AM, Inzitari R, Agostino S, Fiorita A, Cabras T, Scarano E, et al . Statherin levels in saliva of patients with precancerous and cancerous lesions of the oral cavity: A preliminary report. Oral Oncology 2005;11:95-9.  Back to cited text no. 10    
11.Navazesh M. Methods for collecting saliva. Ann NY Acad Sci 1993;20:72.   Back to cited text no. 11    
12.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. 12    
13.Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ. Protein measurement with Folin-phenol reagent. J Biol Chem 193;265-75.  Back to cited text no. 13    
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15.Yogeeswaran G. Metastatic potential is positively correlated with cell surface sialylation of cultured murine cell lines. Science 1981;212:1514-6.  Back to cited text no. 15    
16.Rao VR, Krishnamurthy L, Kumaraswamy SV, Ramaswamy G. Circulating levels in serum total sialic acid, lipid associated sialic acid and flucose in precancerous lesions and cancer of the oral cavity. Cancer Detect Prev 1998;22:237-40.  Back to cited text no. 16    
17.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. 17    
18.Raval GN, Parekh LJ, Patel MM, Patel PS, Rawal RM, Baler DB, et al . Role of sialic acid and alkaline Dnase in breast cancer. Int J Biol Markers 1997;12:61-7.  Back to cited text no. 18    
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20.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. 20    
21.Rajpura KB, Patel PS, Chawda JG, Shah RM. Clinical significance of total and lipid bound sialic acid levels in oral precancerous conditions and oral cancer. J Oral Pathol Med 2005;34:263-7.  Back to cited text no. 21    
22.Plucinsky MC, Riley MW, Prorok JJ. 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. 22    
23.Lindberg G, Eklund G, Gulberg B, Rastram L. Serum sialic acid concentration and cardiovascular mortality. Br Med J 1991;302:143-6.  Back to cited text no. 23    
24.Cvook M, Constale S, Lumb P, Rymer J. Elevated serum sialic acid in pregnancy. J Clin Pathol 1997;50:494-5.  Back to cited text no. 24    
25.Yarat. Salivary sialic acid, protein, salivary flow rate, pH, buffering capacity and caries studies in subjects with Down's syndrome. J Dentist 1999;27:115-8.  Back to cited text no. 25    
26.Dharapur MS. Glycosylated proteins in body fluids and their clinical applications. Thesis submitted to Karnataka University Dharwad, for the award of PhD 1999. p. 97-112.   Back to cited text no. 26    
27.Dabensteen E, Clausen H, Mandel U. Aberrant glycosylation oral malignant and pre-malignant lesions. J Oral Pathol Med 1991;20:361-8.  Back to cited text no. 27    

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Correspondence Address:
Kaveri Hallikeri
Department of Oral and Maxillofacial Pathology, SDM College of Dental Sciences and Hospital, Dharwad, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9290.44529

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    Tables

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

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