SALIVARY GLUCOSE CONCENTRATION IN TYPE-II DIABETIC PEOPLE: A CASE–CONTROL STUDY

Objective: The objective of the present study was to find a non-invasive method of sample collection that can be used to diagnose and monitor diabetes mellitus (DM). Methods: In this study, saliva as a diagnostic fluid was collected noninvasively from subjects with modest training and this offers a cost-effective method for screening diabetes. To evaluate the association of blood glucose level with salivary glucose in Type-II diabetic mellitus (Type-II DM) pattients, a case–control study was conducted on 200 test and 200 healthy control people in selected study village in Kanchipuram (District). The glucose level was measured in saliva and blood plasma by glucose oxidase and peroxidase method. Results: A highly significant positive correlation between fasting salivary glucose (69.377±14.329 mg/dl) and plasma glucose (249.935±64.65 mg/dl) in diabetic patients and in control group, plasma glucose level 117.545±10.595 and saliva glucose level 49.271±13.795 mg/dl was observed. Conclusion: From this study, it can be concluded that fasting salivary glucose level can be used as a non-invasive diagnostic, as well as monitoring tool to assess the glycemic status of Type-II DM patients.


INTRODUCTION
Diabetes mellitus (DM) is a heterogeneous (clinically and genetically) metabolic disease characterized by hyperglycemia and dysregulation of carbohydrate, protein, and lipid metabolism [1]. The main feature of DM is chronic hyperglycemia, resulting from either a defect in insulin secretion from pancreas or resistance of body's cells to produce insulin or both. The characteristic symptoms of diabetes are polyuria, polydipsia, polyphagia, and unexpected weight loss [2]. DM in India is gaining the status of a potential epidemic disease with more than 62 million people diagnosed with DM [3,4]. In the year 2000, India topped the world with the highest number of people having DM (31.7 million) followed by China (20.8 million) in the second place and the United States (17.7 million) in the third place [5][6][7][8].
Blood has been the most commonly used diagnostic fluid for the analysis of its various constituents. This study is focused on the fact that saliva being the principle and defensive fluid in the mouth with informative components can serve as diagnostic tool for human diseases. Collection of saliva is cost effective and non-invasive when compared to blood. The present study is proposed to measure the correlation between salivary and blood glucose levels in diabetic and non-diabetic subjects.

Sample collection, processing, and storage
Diabetes camp was conducted to collect the sample from 200 control group and 200 Type-II diabetic people with different age groups from the selected study area of Thiruporur, Kanchipuram (District). The study was approved by the members of the Institutional Ethical Committee. Exclusion criteria for the control group were pregnancy, alcohol dependency, smoking, chronic diseases, and history of diabetes. After explaining and obtaining an informed consent, all subjects were advised to come on overnight fasting (12 h) before collection of unstimulated whole saliva samples. Denture wearers removed their dentures before saliva collection. Before collection, the subjects rinsed their mouth with water. After 5 min, the saliva was collected in the graduated collection test tube [9][10][11][12][13][14]. Flow rate was measured by recording the reading in the tube. The saliva samples were centrifuged at 4000 rpm for 15 min to remove any particulate material and then supernatant was transferred to a sterile vial immediately, stored at −20°C for further investigations.

Principle
Glucose is oxidized to gluconic acid and hydrogen peroxide in the presence of glucose oxidase. Hydrogen peroxide further reacts with phenol and 4-aminoantipyrine by the catalytic action of peroxidase to form a red-colored quinoneimine dye complex. Intensity of the color formed is directly proportional to the amount of glucose present in the sample.

Reagent
Ready to use reagents provided by the kit.

Sample
The sample was plasma and saliva.

Statistical analysis
Statistical analysis was performed using GraphPad software. Descriptive statistics on each study variable including mean and standard deviations were analyzed. The results obtained from the analysis were tabulated expressed using mean±SD with their level of significance at p<0.0001.

RESULTS
The results were tabulated (Tables 1 and 2).
The observed results were given as mean±SD with their level of significant at p-value.
The fasting and postprandial mean plasma glucose concentration in the test group (155.48±45.577 and 249.935±64.65 mg/dl) were higher than the control subjects. The mean of fasting plasma glucose concentration was 92.495±8.053 mg/dl and postprandial plasma glucose concentration was 117.545±10.595 mg/dl in the control group (Table 1). Significantly elevated plasma glucose concentration was observed in diabetic patient than the control people at p<0.0001.

DISCUSSION
Monitoring the blood glucose by the physician is important for monitoring diabetic control. The routine diagnostic sample is usually blood being invasive, painful, causing discomfort, and may also limit frequent testing of blood sugar level in diabetic patients. The increased blood glucose readily diffuses through the semipermeable membrane and can be detected in saliva also. Salivary glucose assessment may provide a cost-effective approach as saliva is convenient diagnostic fluid for screening large population. In the present study, glucose concentration in blood and saliva of selected study population (control and diabetic people) was analyzed and compared.
Fasting blood sugar and postprandial blood sugar and fasting salivary glucose concentration were measured in control and diabetic people. The fasting and postprandial blood mean sugar level in diabetic patients are significantly higher (p<0.0001) than control subjects. A significantly elevated salivary glucose was observed in diabetic patients than the control subjects. The present study result was found to be similar with other scientific reports [15][16][17][18][19][20][21][22]. Kortuem was the first person to study correlation between blood glucose level and salivary glucose level in 1944 [23] followed by Shannon et al. [24]. Later on, Englander et al. [25] and Campbell, in 1965, shed light on it [26]. Carlson and Ryan (1908) reported the presence of sugar in saliva of diabetic patients [27] and other authors have also reported the increase in salivary glucose levels in diabetics over the non-diabetics [15][16][17][18][19][20][21][22].
Darwazeh et al. in their study correlated the salivary glucose concentrations with the plasma glucose concentration and indicated the presence of glucose in saliva of diabetics reflects the high plasma glucose concentrations [11]. However, Reuterving et al. (1987) reported that the salivary glucose concentration was low during better metabolic control period [15]. Similar to our study, Mahdavi et al. [28] have reported the presence of sugar in significant amount in saliva of diabetic patients and many other authors including Panchbhai et al. [29], Priya et al. [30], Amer et al. [19], Agrawal et al. [31], Abikshyeet et al. [32], and Ivanovski et al. [33] have also reported the increase in salivary glucose concentration in DM patients when compared to non-diabetics. However, contradictory to our study, Bakianian Vaziri et al. [34] concluded that there was no statistically significant difference in salivary glucose concentration between diabetic patients and control subjects. Vasconcelos et al. (2010) also reported a significantly higher salivary glucose level in diabetics when compared to nondiabetics irrespective of periodontal disease [35]. In this study, salivary glucose concentration is higher in diabetics when compared to that of healthy controls.

CONCLUSION
A significant positive correlation was established between blood glucose and salivary glucose levels. Hence, it can be concluded that fasting salivary glucose level can be used as a non-invasive diagnostic tool to monitor diabetes. It is possible that tests based on saliva can have a substantial role in diagnosis and also early detection of DM.

ACKNOWLEDGMENT
The authors are thankful to Aarupadai Veedu Institute of Technology, Vinayaka Missions Research Foundation and Meenakshi Ammal Dental College for providing laboratory facility and their support.