# Influence of Myeloperoxidase Levels on Periodontal Disease: An Applied Clinical Study
## 2 Materials and Methods  
### Study Design  
For the present study, 311 healthy controls and patients with periodontitis or CHD were chosen at the School of Odontostomatology of the University of Catania, Italy, from October 2018 to December 2019. Patients were chosen in a specific range of age (35-65 years old) and on gender in order to have similar proportion of patients in each category characterized by the selection variable. 50% of the patients and controls were males with 45-54 year of age. The study was performed following the 2016 revision of the Helsinki declaration on medical research. Ethical approval was obtained by the local International Review Board (IRB) (#18-18). The study was registered at clinicaltrials.gov (NCT04152023). An informed written consent was obtained from each enrolled patient. The trial was performed in accordance to the guidelines for the strengthening of reporting of observational studies (STROBE) [27]. The inclusion criteria for the subjects enrolled in the periodontitis group were: (1) at least 16 teeth, (2) at least of 40% of periodontal sites with clinical attachment level (CAL) \(\geq\)2 mm and probing depth (PD) \(\geq\)4 mm [28]; (3) at least one periodontal site with \(\geq\)2 mm of crestal alveolar bone loss confirmed on digital periapical x-rays; (4) at least \(\geq\)40% sites with bleeding on probing (BOP) [29]. Healthy controls had any systemic disorder, at least \(\leq\)10% sites with BOP, and no periodontal sites with PD or CAL \(\geq\)4 mm, or x-ray signs of bone loss. For the CVD group, the inclusion criteria were: at least \(\geq\)18 years; a diagnosis of CVD with \(\geq\)50% of stenosis of at least one coronary artery verified by coronary angiography, or past or current percutaneous coronary intervention [30]. Each type of previous disease, taking drugs, or previous CVD exams (e.g., electrocardiography, etc.) were recorded. In all patients, the diagnosis of CVD was performed by the same operator from medical record information. For the periodontitis plus CVD subjects the inclusion criteria were the same of the single disease (periodontitis and CVD). The exclusion criteria of all subjects, were (1) consumption of contraceptive drugs; (2) consumption of antibiotics, anti-inflammatory or immunosuppressive drugs during the three months previous the trial; (3) presence of gestation or suction; (4) intake of alcohol; (5) anesthetic allergy; (6) intake of nifedipine, hydantoin or cyclosporin a drugs; (7) any type of periodontal treatment in the three months before baseline. Then, 187 subjects were left out from the study because they did not meet the inclusion criteria (n = 129), failed to join in the study (n = 37), or were lost at the first assessment (n = 21). For these reasons, for the present study, 31 healthy subjects, 31 periodontitis patients, 31 CHD patients, and 31 patients with both diseases (periodontitis plus CHD) were enrolled in the end (Figure 1). In each patient, every demographic characteristic (such as educational level) and demographic indices such as age, gender, body mass index (BMI), diabetes and other systemic events were recorded together with the type of drug taken. Diabetes was recorded on the patient's medical story or on fasting blood glucose \(\geq\)125 mg/dL. The BMI was recorded by calculating the patient's weight divided by the square of his height in kg/m\({}^{2}\). All enrolled subjects were also classified on their smoking history, such as normal smokers, ex-smokers (subjects who have not smoked for \(\geq\)5 years), and non-smokers. The periodontal evaluation comprised clinical attachment loss (CAL), probing depth (PD), bleeding on probing (BOP), and plaque score (PI) [31]. CAL was verified, such as PD plus gingival recession using the cemetonemal junction as a reference. All periodontal indexes were registered, in all patients, by two independent calibrated examiners (a principal examiner and a control examiner), exonerated in the subsequent study steps, using a periodontal probe (UNC-15, Hu-Friedy, Chicago, IL, USA).