Evaluation of Oxidative Stress and Methylated Arginine Levels in Individuals With Periodontitis

Primary outcomes

1. Power analysis

   Time frame: Baseline

   Sample size analysis was performed a priori using G*Power software before the initiation of the study. The analysis applied a large effect size (Cohen's d = 0.8) for independent two-group comparisons, with a significance level (α) of 0.05 and a statistical power of 80%, resulting in a calculated total sample size of 40 participants.

2. Plaque Index (PI)

   Time frame: Baseline

   Separate intergroup comparisons will be conducted for each clinical outcome measure to allow precise and independent reporting. Thea plaque index (PI), assessed using the Silness and Löe plaque index, which evaluates the thickness of plaque along the gingival margin on a scale from 0 (no plaque) to 3 (abundant plaque). All cl

3. Probing depth (PD)

   Time frame: Baseline

   The probing depth (PD), measured in millimeters at six sites per tooth (mesiobuccal, midbuccal, distobuccal, mesiolingual, midlingual, and distolingual) using a standardized manual periodontal probe (e.g., UNC-15 probe) with markings at 1 mm increments to ensure accuracy.

4. Bleeding on probing (BOP)

   Time frame: Baseline

   The bleeding on probing (BOP), recorded as the presence or absence of bleeding within 15 seconds of probing at each site and reported as the percentage of positive sites per participant.

5. Clinical attachment loss (CAL)

   Time frame: Baseline

   Separate intergroup comparisons will be conducted for each clinical outcome measure to allow precise and independent reporting. The clinical attachment loss (CAL), also measured in millimeters at the same six sites per tooth, calculated as the distance from the cementoenamel junction (CEJ) to the base of the periodontal pocket.

6. Determination of salivary and serum oxidative stress index

   Time frame: Baseline

   Total antioxidant status (TAS) and total oxidant status (TOS) were measured at Baseline in saliva and serum samples using Erel's validated method. TAS was based on the reduction of the ABTS⁺ (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) radical by antioxidants, causing a decrease in color intensity read at 600 nm with a Varioskan Multimode Reader. Calibration used ascorbic acid; results were expressed in mmol ascorbic acid equivalents/L. Higher TAS indicates stronger antioxidant capacity. TOS involved oxidation of Fe²⁺ to Fe³⁺, forming a colored complex with xylenol orange, read at 530 nm; results were in μmol H₂O₂ equivalents/L. Higher TOS reflects elevated oxidant levels. The oxidative stress index (OSI) was calculated as (TOS/TAS) × 10, a unitless index where higher values indicate greater oxidative stress. All assays were performed in duplicate for reliability.

7. Determination of Salivary and Serum Methylated Arginine Metabolites

   Time frame: Baseline

   Salivary and serum levels of arginine, asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA) were measured at the baseline time point. Quantification was performed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with an electrospray ionization (ESI) source (Thermo Scientific Accessmax). Samples underwent protein precipitation and derivatization before injection. Chromatographic separation was achieved on a reversed-phase column under optimized gradient conditions, and mass detection was performed in multiple reaction monitoring (MRM) mode for high specificity. Calibration curves with known standards ensured quantitative accuracy, and results were expressed in micromoles per liter (µmol/L). All measurements were performed in duplicate for reliability.