aliva and serum sampling Saliva were collected to analyze the selected markers as unstimulated samples during the early hours of the day. The saliva was centrifuged and then transferred into Eppendorf tubes. Venous puncture was performed after saliva collection and 10 mL of blood samples were collected by qualified staff (MY,EY) from each participant. Saliva and serum were then stored at -80 °C until analysis.

IL-6 levels in collected samples were determined by ELISA kits and analyzed according to manufacturers' instructions, with colorimetric assessment performed using a microplate reader at 450 nm with the assay detection range between 7.8 and 500 pg/mL. Concentrations were determined based on the respective assay standard curve. All samples were analyzed in duplicate, and the average was used in subsequent calculations.

Determination of methylated arginine metabolites:

The ADMA, SDMA, homoArg, arginine and L-NMMA levels in saliva and serum were assessed by a liquid chromatography-mass spectrometry (LC MS/MS)* method, which was a modification of the method of Di Gangi et al.

Saliva were collected to analyze the selected markers as unstimulated samples during the early hours of the day. The saliva was centrifuged and then transferred into Eppendorf tubes. Venous puncture was performed after saliva collection and 10 mL of blood samples were collected by qualified staff (MFD) from each participant. Saliva and serum were then stored at -80 °C until analysis.

Measurement of the depth of a sulcus or periodontal pocket determined by measuring distance from a gingival margin to the base of the sulcus or pocket with a calibrated periodontal probe

Clinical attachment level (or loss, CAL) is a more accurate indicator of the periodontal support around a tooth than probing depth alone. CAL is measured from a fixed point on the tooth that does not change, the CEJ.

To calculate CAL, two measurements are needed: distance from the gingival margin to the CEJ and probing depth.

In recession: probing depth (+) gingival margin to the CEJ (add). In tissue overgrowth: probing depth (-) gingival margin to the CEJ (subtract)

referring to bleeding that is induced by gentle manipulation of the tissue at the depth of the gingival sulcus, or interface between the gingiva and a tooth

Phenomenex (Torrance, CA, USA) 75 x 4.6 mm x 4 µm polar-RP column, Dionex HPLC and Access MAX LC-MSMS (Thermo Scientific, USA) device were used; in the gradient phase mobile phase A (40 mM ammoniumformate, 3% formic acid) and mobile phase B (acetonitrile) were utilized. A flow chart with increasing in steps of 2-8 minutes for 10% B phase, 30% B phase for 8-11 minutes, and finally 10% B phase for 11-20 minutes was applied at a flow rate of 0.3 ml/min. MRM and CE values were as follows: Arginine 231.3-70.1 CE: 15 V; Homoarginine 245.2-84.2 CE: 24 V; L-NMMA 245.3-70.2 CE:15V; ADMA 259.3-214.0; CE:25V; SDMA 259.3-228.0 CE:15V; ADMA-D7 266.610-221.000 CE:15V; capillary temperature 210 °C; Vaporizer temperature 350 °C; Sheath gas pressure: 30 Arb.; Aux Gas pressure:10 Arb.; Ion Sweep Gas Pressure: 0; Spray Voltage: 3000 V; Polarity: (+).