Neutrophil Elastase, Elastase Alpha-1 Proteinase, Periodontitis, Gingival Crevicular Fluid and Acute Coronary Syndrome

This research was approved by the human participants ethics board of Selcuk University Faculty of Dentistry (project number=301) and was conducted in accordance with the Helsinki Declaration of 1975, as revised in 2013. Informed consent was obtained from patients prior to oral diagnostic examination.

Inclusion criteria

for ACS group :

• Patients diagnosed with both ACS and localized or generalized periodontitis of stage II or III with grade B or C.

The diagnosis of ACS was performed by a consultant cardiologist based on clinical signs and symptoms of cardiac ischemia, cardiac enzyme levels, changes in electrocardiography, and results of echocardiography and coronary angiography, according to the guidelines of American College of Cardiology/American Heart Association . The test group of ACS patients myocardial infarction (NSTEMI), and ST-segment elevation myocardial infarction (STEMI) were hospitalized in Selcuk University Meram Medical Faculty Department of Cardiology (Konya/Turkey).Unstable angina (UA) was defined as the new onset or a changed pattern of angina over the past 2 months and Canadian Cardiovascular Society (CCS) class IV angina at the time of presentation coinciding with appropriate objective evidence of myocardial ischemia on an electrocardiogram (ECG) or myocardial perfusion imaging. ACS with ST-segment elevation was defined as AMI characterized by: chest pain, radiating or not to the upper extremities, lower jaw, upper back or epigastrium lasting 30 minutes or more, associated or not with sweating, nausea or pallor; presence of ST-segment elevation of 1 mm in two or more contiguous peripheral leads or 2 mm in two or more contiguous precordial leads on electrocardiogram (ECG); and elevation in serum markers of myocardial injury and necrosis (CK, CK-MB) three times their reference value28. Non-ST-segment elevation ACS on ECG was defined as a clinical condition similar to that mentioned above but with chest pain lasting less than 30 minutes, with or without elevation of serum enzyme markers of myocardial injury and necrosis (CK, CK-MB, cardiac troponins I and T). ACS patients were referred to the Department of Periodontology, Faculty of Dentistry, Selcuk University (Konya/Turkey) for periodontal examination in a one- year period.

Inclusion criteria

for control group :

• Patients diagnosed with localized or generalized periodontitis of stage II or III with grade B or C.

Exclusions for both groups included

• metabolic disorders,
• high blood pressure,
• treated with antihypertensive medications,
• periodontal therapy antibiotic treatment until 3 months prior the study. Smoking habits and education status were also recorded. ACS patients take aspirin (1 × 100 mg per day) during 1 month.This study was supported by grants from the Selcuk University Scientific Research Projects (05401018).

Clinical Periodontal Examination All surviving individuals with ACS underwent a thorough periodontal examination approximately 1 month after treatment and hospital release. All participants were evaluated for oral health clinically and radiographically. The periodontal condition was graded based on the current classification method for periodontal diseases and subjects with localized or generalized periodontitis of stage II or III with grade B or C were selected. Periodontal health status of patients was evaluated by plaque index (PI), papilla bleeding index , probing depth (PD) and clinical attachment level (CAL). All probing scores were measured with Williams periodontal probe calibrated in millimeters. Clinical attachment loss was determined relative to the cementoenamel junction. Periodontal index was evaluated for risk of infectiousness (PIRI) scores.

Gingival Crevicular Fluid Samples:

At baseline, GCF samples were collected for each subject from 4 distinct inter-proximal sites (one site for each quadrant). Sampling sites were chosen only at the buccal directions of the inter-proximal regions of molar teeth with PD more than 5 mm. The four GCF samples for each subject in the two research groups were pooled to ensure sufficient sensitivity for each subject. Consequently, site-specific differences were not evaluated. The same sites were sampled at 1 and 3 months following treatment. Sites were isolated with cotton rolls, gently dried with air syringe and the obvious supra-gingival plaque was carefully removed with a curette. GCF was collected with prefabricated paper strips . The strip was inserted into the pocket until mild resistance was felt and kept there for 30 seconds. Contaminated samples with blood or saliva were discharged. The volume of GCF was determined with Periotron 8000 previously calibrated with serum . Four strips were pooled in a single tube containing 500microliter phosphate buffered saline (PBS), pH 7.4 and, repeated, for each patient. The tubes were vortexed for 10 min and stored at -80 C until assayed.

Inclusion criteria

for ACS group :

• Patients diagnosed with both ACS and localized or generalized periodontitis of stage II or III with grade B or C. ACS patients myocardial infarction (NSTEMI), and ST-segment elevation myocardial infarction (STEMI) were hospitalized .

Unstable angina (UA) was defined as the new onset or a changed pattern of angina over the past 2 months and Canadian Cardiovascular Society (CCS) class IV angina at the time of presentation coinciding with appropriate objective evidence of myocardial ischemia on an electrocardiogram (ECG) or myocardial perfusion imaging. ACS with ST-segment elevation was defined as AMI characterized by: chest pain, radiating or not to the upper extremities, lower jaw, upper back or epigastrium lasting 30 minutes or more, associated or not with sweating, nausea or pallor; presence of ST-segment elevation of 1 mm in two or more contiguous peripheral leads or 2 mm in two or more contiguous precordial leads on electrocardiogram (ECG); and elevation in serum markers of myocardial injury and necrosis (CK, CK-MB) three times their reference value28. Non-ST-segment elevation ACS on ECG was defined as a clinical condition similar to that mentioned above but with chest pain lasting less than 30 minutes, with or without elevation of serum enzyme markers of myocardial injury and necrosis (CK, CK-MB, cardiac troponins I and T). ACS patients were referred to the Department of Periodontology, Faculty of Dentistry, Selcuk University (Konya/Turkey) for periodontal examination in a one- year period.

Inclusion criteria

for control group :

• Patients diagnosed with localized or generalized periodontitis of stage II or III with grade B or C.

Twenty-six age and sex-matched patients (43.04±8.35 years) with periodontitis (Group P) otherwise systemically healthy were recruited as control group in the Department of Periodontology, Faculty of Dentistry, Selcuk University (Konya/Turkey).

Exclusions for both groups included

• metabolic disorders,
• high blood pressure,
• treated with antihypertensive medications,
• periodontal therapy antibiotic treatment until 3 months prior the study. Smoking habits and education status were also recorded. ACS patients take aspirin (1 × 100 mg per day) during 1 month.