EVALUATION OF SALIVA AND SERUM HEME OXYGENASE, ARYLESTERASE AND NUCLEAR FACTOR ERYTHROID 2-RELATED FACTOR 2 LEVELS IN PATIENTS WITH STAGE III PERIODONTITIS

TAS concentrations were evaluated utilizing an innovative automated colorimetric assay, originally developed by Erel. The outcomes were reported as millimoles of Trolox equivalent per liter (mmol Trolox Eq/L).

Total antioxidant levels are significantly lower in periodontitis patients compared to healthy individuals, suggesting increased oxidative stress associated with periodontal inflammation.

TOS concentrations were determined through a distinct automated colorimetric technique, also described by Erel. Results were expressed as micromoles of hydrogen peroxide equivalent per liter (µmol H₂O₂ Eq/L). Compared to healthy controls, periodontitis patients show higher total oxidant levels, which may contribute to tissue destruction and disease progression.

TAS values (initially in mmol Trolox Eq/L) were converted to µmol Trolox Eq/L. OSI was then computed using the following formula: OSI = [(TOS, µmol/L) / (TAS, µmol Trolox Eq/L)] × 100.

The oxidative stress index is significantly higher in periodontitis patients compared to healthy individuals, reflecting an imbalance between oxidants and antioxidants.

Arylesterase activity was assessed using commercially available assay kits (Rel Assay Diagnostics, Turkey). For the measurement of arylesterase activity, phenylacetate was used as the substrate. The enzymatic activity was calculated based on the molar extinction coefficient of the phenol formed (1,310 M-¹cm-¹). One unit of arylesterase activity was defined as the amount of enzyme required to hydrolyze 1 µmol of phenol per minute under the assay conditions and was also expressed as U/L. Arylesterase activity is significantly decreased in periodontitis patients compared to healthy controls, suggesting impaired antioxidant enzyme function.

The level of NRF-2 was quantified using an enzyme-linked immunosorbent assay (ELISA) kit (BT lab.). In this assay, wells were pre-coated with human NRF-2-specific antibodies. Samples containing NRF2 were added to the wells, allowing target binding. Subsequently, a biotin-conjugated anti-NRF-2 antibody was applied, followed by streptavidin-horseradish peroxidase (HRP) conjugate. After washing to remove unbound components, a substrate solution was added, producing a colorimetric reaction proportional to the amount of NRF-2 present. The reaction was terminated by adding an acidic stop solution, and absorbance was measured at 450 nm. It is suggested that the reduced expression of NRF-2 in periodontitis patients, relative to healthy controls, contributes to increased oxidative stress and tissue damage.

The concentration of HO-1 was measured using an ELISA kit (BT Lab.) Wells were pre-coated with human HO-1-specific antibodies. Following the addition of samples, human HO-1 proteins bound to the immobilized antibodies. A biotinylated anti-HO-1 antibody and subsequently streptavidin-HRP conjugate were introduced. After removal of unbound components via washing, a substrate solution was added to initiate color development, which was proportional to the HO-1 concentration. The reaction was stopped using an acidic solution, and absorbance was recorded at 450 nm. Compared to healthy subjects, HO-1 is considered to be less expressed in periodontitis patients, potentially compromising antioxidant and anti-inflammatory defenses

Gingival Index (GI) (Löe ve Silness) : The presence of gingival inflammation was determined on 4 surfaces of all teeth using the GI score defined by Löe and Silness. The periodontal probe was moved over the tooth and index values between 0-3 were determined for each tooth. Compared to healthy individuals, a high gingival index score is frequently observed in periodontitis patients, indicating greater gingival inflammation.

The presence of plaque and the Silness and Löe plaque index (PI) were obtained from 4 surfaces of the teeth after drying the teeth and index values between 0-3 were given. Plaque accumulation, as reflected by higher plaque index scores, is more prominent in periodontitis patients than in healthy controls.

Bleeding in probing was evaluated as (+) if there was bleeding in the relevant area within 30 seconds after applying pressure to the gingival sulcus with the periodontal probe in 4 regions of all teeth with the weight of the probe itself, and as (-) if there was no bleeding. Compared to healthy controls, periodontitis patients demonstrate a higher incidence of bleeding on probing, indicating active inflammation and compromised periodontal tissue integrity.

The distance between the cementoenamel junction of the tooth and the base of the periodontal pocket is calibrated millimetrically, with the clinical attachment level on 6 surfaces of all teeth. using a Williams periodontal probe. Clinical attachment level is significantly increased in periodontitis patients compared to healthy individuals, indicating the extent of periodontal tissue destruction.

Probing pocket depth was performed on 6 surfaces of all teeth using a millimetrically calibrated Williams periodontal probe. The distance of the free gingival margin to the pocket bottom was measured in millimeters. The probe was held parallel to the long axis and the force applied to the probe was released when resistance was encountered during the measurement. Probing pocket depth is significantly greater in periodontitis patients compared to healthy individuals, reflecting deeper periodontal pockets due to tissue destruction.