GCF Annexin-A1, Carbonic Anhydrase-1, and Elongation Factor-1 Gamma Levels in Periodontitis
Condition: Periodontitis; Periodontal Inflammation
MeSH terms: Periodontitis; Inflammation
Study type: Observational

Summary: 
      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.
    

Detailed description: 
      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.
    

Intervention name(s): Peirodontal clinical measurements and gingival crevicular fluid sampling
Intervention type(s): Other
Intervention description(s): 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.
Observational model: Case-Control
Time perspective: Cross-Sectional
Sampling method: Probability Sample
Study population: 
        A total of 80 systemically healthy and non-smoker individuals(39 males, 41 females) aged
        20-61 years were consecutively included.
      
Biospecimen description: 
      Gingival crevicular fluid