## 2 Materials and Methods
### Study Participants and Grouping  
The sample size was calculated (OpenEpi, v3) at a 5% margin of error, 95% confidence interval, and 80% expected levels of amylase compared with known levels in saliva from a previous study in diabetic patients. The total sample size was calculated to be 48 for both case and controls. The total sample was raised to 60 in order to compensate for dropouts [27]. The participants who fulfilled the inclusion criteria were recruited using consecutive sampling techniques from the dental outpatient department of Bahria University Dental College, Karachi, Pakistan. The participants were age-matched to rule out the influence of age on the study variables. The study population was divided into three groups, with 20 subjects in each group (\(n=20\)): healthy controls and diabetic and hypertensive patients. The patients were assessed by the PI and SA and were recruited for the study after explaining the procedures and requirements. Informed consent was obtained, and the participants were then allotted to each group.  
Healthy controls: Healthy patients above 40 years of age with no history of chronic illness (such as diabetes mellitus, hypertension, cardiac problems, and prolonged use of anti-inflammatory medications) or use of antibiotics within the last six months. Those who had had recent periodontal treatments, orthodontic treatments, implants, or other dental diseases were also excluded. The age and gender were kept similar between the other two groups to maintain the uniformity of the data. After taking a complete history of the subjects, a HbA1C test was conducted for T2 DM. Furthermore, chairside evaluation of blood pressure was conducted for 3 days at different timings to rule out hypertension.  
Diabetic patients: According to the prevalence data of Pakistani Population, T2 DM is most often seen in middle-aged and older patients greater than 40 years old [28]. Therefore, the inclusion criteria were patients above 40 years of age with a history of T2 DM for more than 3 years with HBA1C values \(\geq\)7% and \(\leq\)10%. This was done to maintain the homogeneity of the sample. The patients may or may not have been taking hypoglycemic medicines for controlling T2 DM. Subjects with a recent history of use of antibiotics, anti-inflammatory medicines, or periodontal or orthodontic treatment were excluded from the study. The patients who were suffering from any complication related to T2 DM or any other chronic diseases such as hypertension or metabolic or nutritional disorders were excluded. Chairside evaluation of blood pressure using a sphygmomanometer was done on three different days to rule out hypertension.  
Hypertensive patients: Hypertension is also prevalent in patients above 40 years of age in Pakistan [29]. Therefore, the inclusion criteria for the subjects were patients above 40 years of age with a history of hypertension for more than 3 years. Subjects with systolic pressure \(\geq\) 140 mmHg and diastolic pressure \(\geq\) 90 were included. Subjects who had a recent history of hospitalization due to any complications related to hypertension and cardiac issues, use of antibiotics, anti-inflammatory medications, blood thinners, or periodontal or orthodontic treatment were excluded. They were age-matched with the control and the diabetic group to maintain the homogeneity of the data. HbA1C was tested to rule out T2 DM. Chairside measurement of the blood pressure was also performed on three different days and times using a sphygmomanometer.  
The patients who were suffering from any other chronic illness, such as cardiac or renal disorders, were excluded from the study. After the consent of the patients was received, the oral examination was performed, and oral hygiene status and periodontal status were checked using the probe and recorded in the data collection proforma.