The study aimed to investigate gingival crevicular fluid (GCF) levels of possible novel biomarkers Annexin-A1 (ANX A1), Carbonic anhydrase- 1 (CA I), and Elongation Factor-1 Gamma (EF1-Ɣ) in health along with different periodontal diseases.

In total, 80 systemically healthy individuals were included in this study; 20 with periodontitis stage 3 grade B , 20 with periodontitis stage 3 grade C (P-Stage III/C), 19 with gingivitis, and 21 with clinically healthy periodontium. Probing depth, clinical attachment level, plaque index, and papillary bleeding index were recorded. GCF ANX A1, CA I and EF1-Ɣ levels were analyzed by enzyme-linked immunosorbent assay (ELISA). Receiver operating characteristics curve was used for estimating the under the curve.

Diagnosis of periodontal diseases and conditions was arranged according to the radiographic and clinical diagnostic criteria proposed by the 2017 World Workshop on Classifcation of Periodontal and Peri-implant Diseases and Conditions; 20 patients with Stage III Grade B generalized periodontitis , 20 patients with Stage III Grade C generalized periodontitis , 19 gingivitis patients and periodontally healthy volunteers were included in this study.

The clinical periodontal examination of the healthy and periodontitis subjects consisted of plaque index (PI), probing pocket depth (PPD), clinical attachment level (CAL), and bleeding on probing (BOP) recorded. CAL was calculated by adding GR to the PPD values. Averages for full mouth PPD, CAL, and the percentage of sites with BOP were calculated for each subject. A single calibrated examiner (VO) conducted a full mouth periodontal examination of all participants. The measurements were performed using a Williams periodontal probe (Hu-Friedy, Chicago, IL, USA). All measurements were performed full mouth and at 4 sites (mesio-buccal, mid-buccal, distobuccal, and mid-lingual) for each tooth. Before clinical measurements, intra-examiner calibration was performed by measuring PPD and CAL values twice on five patients with one day interval resulting in intraclass correlation coefficients were 0.92 for PD and 0.90 for CAL.

GCF samples were taken from the buccal aspects of two nonadjacent interproximal sites in single-rooted teeth. In periodontitis groups, GCF was sampled from two deepest pockets of single-rooted teeth. Samples were collected from the sites with visible signs of inflammation in gingivitis group and without BOP in the healthy group. The selected areas were carefully cleared of supragingival plaque using sterile curettes, isolated with cotton rolls, and slightly air-dried to avoid contamination.

Standardized filter paper strips (PerioPaper, Proflow, Amityville, NY) were used for GCF sampling. Sterile paper strips were gently inserted into the gingival sulcus or pocket until mild resistance was felt and left there for 30 seconds. Mechanical irritation was avoided and strips visually contaminated with blood were discarded. A precalibrated electronic device measured the absorbed fluid volume (Periotron 8010, Oraflow, Amityville, NY). The readings were converted to an actual volume (microliter, μL) by reference to the standard curve. The paper strips were individually placed into a sterile polypropylene tube and stored at -80◦C for further analysis.

Annexin-A1 levels were studied with commercially available kits using the ELISA method (Bioscience SRB, catalog no: 201-12-3158). The optical density was measured spectrophotometrically at a wavelength of 450 nm (Tecan). The assay ranges for the Annexin-A1 kit were 0.20-20 ng/mL, sensitivity 0.2 ng/mL, and the intra- and interassay coefficients of variance (CV%) were<10%. The results were presented as ng.

Carbonic anhydrase 1 levels were studied with commercially available kits using the ELISA method (Bioscience SRB, catalog no: SRB-T-88927). The optical density was measured spectrophotometrically at a wavelength of 450 nm (Tecan). The assay ranges for the CA I kit were 3.12-200ng/mL, sensitivity <1.875ng/mL, and the intra- and interassay coefficients of variance (CV%) were<10%. The results were presented as ng.

EEF1G levels were studied with commercially available kits using the ELISA method (Bioscience SRB, catalog no: 201-12-3732). The optical density was measured spectrophotometrically at a wavelength of 450 nm (Tecan). The assay ranges for the EF1- Ɣ kit were 31.25-2000pg/mL, sensitivity <12.4pg/mL, and the intra- and interassay coefficients of variance (CV%) were<10%. The results were presented as ng.

Statistical analysis was performed using non-parametrical techniques. Comparisons between the study groups were performed using the Kruskal-Wallis test. When there were significant differences (p < 0.05), post-hoc 2-group comparisons were assessed with Bonferroni-corrected Mann-Whitney U tests, and p-values < 0.05 was considered significant. All data analysis was performed using a statistical package (Abacus Concepts Inc., Berkeley, CA, USA). The diagnostic accuracy, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), receiver operating characteristic (ROC) curve, and area under the ROC curve (AUC) of the test dataset were assessed. P values < 0.05 were considered statistically significant, and 95% confidence intervals (CIs) were calculated.

Inclusion Criteria:

• Non-smokers with no history of smoking(determined by self-reporting)
• Individuals who had at least 20 teeth(exclude wisdom teeth)

Exclusion Criteria:

• Pregnant and lacting women
• Patients who under any medication including immunosuppressants, steroids, non-steroidal anti-inflammatory drugs, antibiotics, antiepileptic drugs, calcium channel blockers, beta,blockers, anticoagulants, hormonal contraceptives and nutritional supplements within 6 monthsi topical antiseptic solutions in the last 3 months.
• Patients with diagnosed medical illness such as diabetes mellitus, rheumatoid arthritis, cardiovascular diseases, immunological diseases.

A total of 80 systemically healthy and non-smoker individuals(39 males, 41 females) aged 20-61 years were consecutively included.