S.Sathya priya et al.

/ Journal of Pharmacy Research 2011,4(7),2228-2229

Research Article ISSN: 0974-6943

Available online through www.jpronline.info

Potential of Salivary Protein as a Biomarker in prognosis of Diabetes mellitus.

1.

S. Sathya priya 1, Dr. GO.Bharani2, M.Nagalingam1, M.Jayanthi 1, Ms. U. Kanagavalli* PG and Research Department of Biochemistry, Adhiparasakthi college of arts and science, Kalavai, Vellore district 632506. Tamilnadu.,India. 2. Consultant Physician & Diabetologist, Asst. Prof., Govt. Medical College Hospital,Vellore, 632506. Tamilnadu.India.

Received on: 12-04-2011; Revised on: 18-05-2011; Accepted on:21-06-2011 ABSTRACT

Assessment of blood sugar control is vital in managing diabetes. To assess the glycemic level of the body, blood sugar estimation and glycosylated hemoglobin (HbA1c) are done routinely all over the world. Diabetes is known to influence salivary composition and function. A total of 60 patients, comprising 60 diabetic patients and 25 healthy controls were selected. Salivary investigations were performed using unstimulated whole Saliva. A significant correlation was found between salivary and blood concentrations in the diabetes. Mean salivary glucose levels were found to be significantly elevated in uncontrolled diabetics when compared to healthy non-diabetics. There were significant Increase in mean salivary amylase, Protein & Potassium in diabetic patients when compared to healthy non-diabetics. Furthermore, in this study the protein profiles of whole saliva of diabetic and healthy non-diabetic were compared using SDS-PAGE considerable variations between individuals in the protein profiles were observed. The saliva from diabetic patients appeared to have more of proline-rich protein bands. This findings suggests that saliva can be used reliably for reflecting and monitoring the blood glucose concentration in the patients of diabetes mellitus. Key words: Whole Saliva, Uncontrolled Diabetics, Polyacrylamide gel electrophoresis (PAGE), Proline-rich proteins. Introduction Saliva is the watery and frothy substance produced in the mouths of humans and most other animals. [1] Human saliva contains informative components that can be used as diagnostic markers for human disease. [2] Biomarkers detected in saliva can be valuable in a wide range of clinical pathology, forensic medicine and sport medicine [3]. Knowledge of the effects of diabetes on salivary composition and function remains equivocal. Basement membrane permeability of the parotid gland is reported to be higher in diabetes mellitus, and this result in raised percolation of components such as glucose, amylase and protein from blood, thus raising their levels in saliva [4-7]. The proline rich proteins (PRPs) constitute about 70% of human salivary proteins and unusual in their Amino acid compositions, with Proline comprising 25%-45% of the Amino acid residues [8]. If the glycation of salivary proteins is linked with glycated proteins in blood and blood glucose, it can be used to detect diabetes at an early stage. Hence the aims of this study were as follows; First to estimate the constituents of saliva (glucose, amylase, total protein, sodium and potassium) in order to aid in reaching firm conclusions about their alterations in diabetics as compared to healthy non-diabetics; second to compare and correlate these parameters in uncontrolled and controlled diabetics, finally, to assess the significant variations of protein profiles in diabetic and healthy non-diabetics by SDS-PAGE[9] using coomassie-blue R 250 staining were studied and compared. Subjects and Method: The study population was composed of 25 healthy patients (non diabetic and without oral pathology), aged 20-60 years and 60 diabetic individuals from Mothers care, Vellore. Based on their blood sugar level, they were divided into 5 groups. Group 1: Consist of 25 individuals who are clinically healthy their clinical Characteristics matched the patient groups Group 2: Consist of 15 patients whose blood sugar level was below 100 mg/dl Group 3: Consist of 15 patients whose blood sugar level was below 100-150 mg/dl Group 4: Consist of 15 patients whose blood sugar level was below 150-250 mg/dl Group 5: Consist of 15 patients whose blood sugar level was above 250mg/dl

Collection of sample: Saliva: Unstimulated saliva (5 ml) from the diabetic and control groups was collected [10]. Salivary sample collection was performed in the morning, at 8 oclock in an ice-chilled sterile container bearing the appropriate preservatives. Once the saliva was collected, it was centrifuged at 3000 rpm for 20 min to remove any particulate material. The clean supernatants were processed immediately for estimation of glucose, amylase, total protein, sodium & potassium [1113]. Serum: 10 ml of venous blood was drawn and the serum was separated by centrifugation, supernatant were aspirated. Measurement methods: Glucose Estimation: Serum and salivary glucose estimation was performed using the glucose oxidase end-point method [14, 15] at the wavelength of 505 nm. Serum Amylase Estimation: Salivary -amylase estimation was performed using the direct substrate kinetic enzymatic method [15, 16]. Total Protein Estimation: Serum salivary protein estimation was performed using Bradford method [17] using bovine serum albumin as a standard at the wavelength of 595 nm. SDS PAGE: SDS PAGE was carried out according to the method of Laemli et al [18]. Sodium & Potassium estimation by Flame Photometry: Salivary sodium and potassium was estimated by flame photometry [10]. Data and Statistical Analysis: All data were analyzed with SPSS program ver.10.0[19] comparison between groups were made by analysis of variance(ANOVA) test. The statistically significant level was set to p<0.05. Pearsons correction coefficients were used to examine the relation between the variables. RESULTS: Considering the prevalence of diabetes mellitus and its oral manifestations, it has become of paramount importance to study the levels of some crucial parameters in diabetic saliva.The demographic characteristics of the subjects are shown in Table 1.

*Corresponding author.
Ms.U.Kanagavalli Assistant Professor, Dept. of Biochemistry Adhiparasakthi college of Arts and Science G.B.Nagar, Kalavai 632 506,TamilNadu E-mail:kanagaumapathy@rediffmail.com

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S.Sathya priya et al. / Journal of Pharmacy Research 2011,4(7),2228-2229

Table 1: Distribution of the groups studied according to sex and age of the patients
Experimental group Male Female Age 30 30 56.514.3 Control group 15 15 46.618.2

Levels of significance was p<0.05

Salivary total protein: Mean salivary total protein were higher in uncontrolled diabetics when compared to controlled diabetics and healthy non-diabetics. SDS-PAGE also revealed the presence of total protein content to be higher in uncontrolled diabetics and was found to be consistent with the extent of diabetes mellitus. Salivary potassium were not significantly different when diabetics and healthy non-diabetics were compared. But there increased significant variation in salivary sodium content with the extent of disease. DISCUSSION: The present study was undertaken with the aim of suggesting the possibility of using salivary protein as a biomarker for assessing the control of blood sugar. The salivary samples of the non-diabetic control subjects did not show the presence of glucose in higher concentrations, while the samples obtained from the diabetics showed significant concentrations of glucose in the saliva along with their serum glucose concentration and is found to correlate[20,21]. Salivary glucose, amylase and total protein values in diabetic patients and control groups are shown In table 2 and salivary sodium and potassium levels in table 3 .
Table 2: Intergroup comparisons of mean of salivary parameters in the diabetic group and healthy non-diabetic group.
Blood sugar level (mg/dl) <100 <100-150 <150-250 >250 Control Salivary glucose (mg/dl) Mean SD 7.30 5.84 7.64 6.44 8.09 6.45 9.04 7.17 5.91 2.19 Salivary amylase (U/ml) Mean SD 102.32 67.61 106.83 60.77 108.48 6.37 111.12 11.94 96.72 10.70 Salivary total protein (mg/dl) Mean SD 87.51 40.26 88.91 49.71 90.01 44.22 105.28 45.11 98.25 49.59

1 3 : Healthy nondiabetic ; 4 6 : Type 1 diabetes ; 7 9 : Type II diabetes Fig 1: Comparison of Protein profiles using SDS-PAGE We compared salivary parameters based on duration and type presuming that they would provide some fundamental information in terms of the oral manifestations seen in diabetes. Although the results are not entirely conclusive quantitatively, the study made some novel observations that will unquestionably contribute to providing a platform for further research. The observations derived from this study require more comprehensive evaluation with emphasis on broader representation. ACKNOWLEDGMENTS: We are grateful to Mr.A.Mohamed Sadiq, (Principal, Adhiparashakthi College Arts and Science in Kalavai) for his valuable guidance and technical expertise for his timely assistance. And I also thank Dr. GO. BHARANI, MOTHERS CARE DIABETES CENTRE , Vellore for her constant guidance throughout my work. REFERENCES:
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. Johnson,L.R.,ed:Essential medical physiology,1998-739-42. Michael Glceson Ph.D. :Saliva and its use as diagnostic fluid 2009-654-53. Kagami H , Hiramatsu Y , Hishida S, Okazaki Y, Horie K , Oda Y , etal. Salivary growth factors in health and disease . Adv. Dent Res. 2000; 14:99-102. Edgar WM (1992) Saliva : its secretion , composition and functions. Brit Dent J 172, 305-312. Mealey B (2003) Diabetes mellitus. In : Burkets Oral medicine. Diagnosis and treatment, 10th ed , Greenberg MS, Glick Meds , BC Decker , Hamilton, 563-577. Tenovuo J, Lehtonen OP, Viikari J, Larjava H, Vilja P, Tuohimaa P (1986) Immunoglobulins and innate antimicrobial factors in whole saliva of patients with insulin-dependent diabetes mellitus. J Dent Res 65,62-66. Mangos JA (1979) The uptake of sugars by isolated rat parotid acinar cells. J Dent Res 58,1465-1470. Bennick A : Salivary proline-rich proteins. Mol cell Biochem 45:83-99,1982. Henkin R.I., Lippoldt R.E., Bilstad J., Wolf R.O., Lam C.K.L., Edelhoch H. Fractionation of human parotid saliva protein. J. Biol.chem.1980;253;7556-7565. Aydin .S., Halifeoglu.I., Ozercan.I.H., Erman, F.,Kilic .N., Aydin . S.,Ilhan.N., Ozkan.Y., Akpolat.N., Sert.l., and Caylak,(2005) A Comparison of leptin and ghrelin levels in plasma and saliva of young healthy subjects. Peptides 26,647-652. Marder MZ, Abelson DC, Mandel ID (1975) Salivary alterations in diabetes mellitus. J Periodontol 46,567-569. Moore PA, Guggenheimer J, Etzel KR, Weyant RJ, Orchard T (2001) Type 1 diabetesw mellitus, xerostomia and salivay flow rates. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 92,281-291. Chavez EM, Borell LN, Tylor GW, Ship JA (2001) A Longitudinal analysis of salivary flow in control subjects and oral adults with type 2 diabetes. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 91, 166-173. Pal P, Desai NT, Kannan N, Masur VN, Daniel MJ, Bhatt N (2003) Estimation of salivary glucose , salivary amylase, salivary total protein and periodontal microflora in diabetes mellitus. J Indian Dent Assoc 74,143-149. Lopez ME, Colloca ME, Paez RG, Schallmach JN, Koss MA, Chervonagura A (2003) Salivary characteristics of diabetic children. Braz Dent J 14, 26-31. Henskens YM, Van den Keijbus PA, Veerman EC,Van der Weijden GA, Timmerman MF,Snoek CM, Van der Velden U, Nieuw Amerongen AV (1996) Protein composition of whole and parotid saliva in healthy and periodontitis subjects. Determination of cystatins , albumin, amylase and Ig A. J Periodontal Res 31, 57-65. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proline-rich binding. Anal Biochem. 1976; 72: 248-254. .Laemli, W.K. Clevage of structural proteins during the assembly of Head of Bacteriophage T4. Nature . 1970; 227: 680-685. SPSS Inc. SPSS Base 10.0 Breif Guide Chicago; SPSS Inc.; 2000. Darwazeh A.M., Mac Falane T.W., Mc Cuish A. and Lamey P.J. (1991). Mixed salivary glucose levels and candidal carriage in patients with diabetes mellitus. J O Pathol Med. 20 (6): 280-283. Swanljung O., Meurman J.K.,Torkko H., Sandholm L., Kaprio E., Maenpaa J. (1992). Caries and saliva in 12-18 years old diabetics and controls. Scand J Dent Res. 100(6): 310-313. Varletzidis,E., Ikkos,D., Paleologou, G., Pantazopoulos, P., Miras, K. and Adamopoulos, G.(1978). Salivary amylase activity in diabetes mellitus. Panminerva Med. 20, 255-262. Newrick, P.G, Bowman, C., Green, D., OBrien, I.A., Porter , S.R., Scully , C. and Corrall, R.J. (1991) Parotid salivary secretion in diabetic autonomic neuropathy . J Diabet. Complications 5,3537.

Levels of significance was p<0.05 Table 3: Sodium & potassium values of diabetic patients & control groups are shown in tables.
Blood sugar level (mg/dl) <100 <100-150 <150-250 >250 Control Salivary sodium (mmol/L) 4.80 3.33 5.00 1.23 6.24 1.42 7.00 0.74 2.68 0.74 Salivary potassium (mmol/L) 27.0 4.19 32.0 1.02 35.8 1.63 37.5 1.87 39.9 1.06

Levels of significance was p<0.05

Salivary glucose: Mean salivary glucose levels were higher in the uncontrolled and controlled diabetic groups than in the healthy non-diabetic group and the differences were highly significant (table 2) uncontrolled diabetics had higher mean salivary glucose levels than controlled diabetics. Salivary amylase: The salivary amylase levels were significantly higher in controlled diabetics when compared with healthy non-diabetics. A dramatic important of salivary amylase activity has been found in diabetic patients compared to healthy controls. The present study has shown that the alpha-amylase levels in unstimulated saliva from type 2 diabetic patients are higher than in control groups. There is considerable disagreement in the literature about salivary amylase activity in diabetic patients; different rescales have reported that salivary amylase concentrations from diabetics are higher [22], lower [15] or the same [23].with regard to salivary total protein, the present study results are consistent with most previous studies, higher [15] lower or the same. A significantly positive correlation was observed between total protein levels of uncontrolled diabetic group and controlled diabetic group [14]. SDS PAGE RESULT: Also the protein bands appeared during SDS PAGE was found to correlate with the extent of diabetes mellitus and there was a significant correlation between the healthy non-diabetic patients and diabetic patients.

17. 18 19. 20. 21. 22. 23.

Source of support: Nil, Conflict of interest: None Declared

Journal of Pharmacy Research Vol.4.Issue 7. July 2011

2228-2229