Saliva and serum collection of patients and samples molecules analysis Saliva 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 from each participant. Saliva and serum were then stored at -80 °C until analysis.

TNF-α levels in collected saliva and serum samples were measured by commercial ELISA kits (ELISA Cloud Immunoassay, Cloud Clone Corp; cat #SEA079Hu) and analyzed according to manufacturers' instructions. The standard curve of the assay was used to determine the analyte concentrations in each sample. All samples were analyzed in duplicate, and the mean values were used in subsequent calculations.

TMAO levels in saliva and serum were determined by a liquid chromatography-mass spectrometry (LC MS/MS) method (LC-MS/MS, ESI Source, Thermo Scientific Accessmax), which was a modification of the method by Li et al and similar as our previous study.(Yufei Li, Kang, Lee, Chung, & Cho, 2021) To identify and quantify TMAO, samples were analyzed by an LC-MS/MS 1 system equipped with a Thermo Dionex Ultimate 3000 UHPLC system and an TSQ Quantum Access Max quadrupole mass spectrometer (USA).

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 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)

Bleeding on probing 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

TMAO levels were determined in saliva and serum samples using liquid chromatography-mass spectrometry (LC-MS/MS). TNF-α levels were measured by ELISA method.