*2.1. Study Design*

The study population consisted of 309 patients with periodontitis, CAD, and healthy controls selected among those who attended the Department of Periodontology, School of Dentistry, from June 2016 to October 2018. Groups were selected from a prespecified age range (40–60) and sex so that a similar proportion to the cases fall into the categories defined by the selection variable (sex and age in this study). Fifty percent of the cases and controls were males aged 45–58 years.

The study was performed by the Declaration of Helsinki, revised in 2016 by medical research. Ethical approval was obtained from the local IRB of the University of Messina (012-2016). The study was registered at clinicaltrials.gov (NCT03873789). Written informed consent was obtained from each patient about the study characteristics and possible risks of the study. This study followed the STROBE guidelines for the strengthening of reporting of observational studies (Table S1) [27].

Inclusion criteria for the periodontitis group were: (1) Presence of at least 16 teeth; (2) a minimum of 40% of sites with clinical attachment level (CAL) ≥2 mm and probing depth (PD) ≥4 mm [28]; (3) presence of at least one site for each quadrant with ≥2 mm of crestal alveolar bone loss verified on digital periapical radiographs; and (4) presence of ≥40% sites with bleeding on probing (BOP) [29]. Healthy individuals presented no systemic disease, ≤10% sites with BOP, no sites with PD ≥4 mm or CAL ≥4 mm, no sites with BOP [29] or radiographic signs of bone loss.

Inclusion criteria for the CAD group were: At least ≥18 years old with a diagnosis of CVD, ≥50% of stenosis of at least one coronary artery verified by coronary angiography or a coronary artery bypass surgery, or past or current percutaneous coronary intervention [30]. Moreover, information on previous medical conditions, cardiovascular risk factors, medications, electrocardiography, echocardiography, and coronary angiogram results were collected. The inclusion criteria for the periodontitis + CAD group were based on the same criteria of the single periodontitis and CAD groups but combined.

The exclusion criteria for all patients were: (1) Use of contraceptives; (2) use of antibiotics, immunosuppressive or anti-inflammatory drugs throughout the last three months prior to the study; (3) status of pregnancy or lactation; (4) previous history of excessive drinking; (5) allergy to local anaesthetic; (6) use of drugs that may potentially determine gingival hyperplasia such as Hydantoin, Nifedipine, Cyclosporin A, or similar drugs; (7) periodontal therapy throughout the last three months prior to the study.

After a first screening, 166 patients were excluded from the final sample because they did not meet the inclusion criteria (*n* = 141), declined to participate (*n* = 14), or did not attend the first appointment (*n* = 11). Finally, for this study, 36 patients with periodontitis, 35 patients with CAD, 36 patients with periodontitis plus CAD, and 36 healthy subjects were finally enrolled (Figure 1).

**Figure 1.** Flowchart of the study.

The demographic (level of education), clinical and medical characteristics (sex, age, body mass index, hypertension, diabetes, dyslipidemia, previous CVD events), and medications were assessed in all enrolled subjects. The presence of diabetes mellitus was based on the history of the patient or a fasting blood glucose ≥126 mg/dL. Body Mass Index (BMI) was estimated on the weight of the patient divided by the square of the patient's height, i.e., kilogram per square meter (kg/m2).

The periodontal evaluation comprised probing depth (PD), clinical attachment loss (CAL), bleeding on probing (BOP), and plaque score (PI) [31], and the presence of bleeding was recorded up to 30 s after probing. CAL was recorded as PD plus recession, with the cementoenamel junction as a reference for CAL measurements. All clinical periodontal parameters were recorded, in all patients, at six sites per tooth on all teeth present, excluding third molars, by two independent calibrated examiners (a

principal examiner and a second control examiner) not involved in the subsequent data analysis with a manual periodontal probe (UNC-15, Hu-Friedy, Chicago, IL, USA). The inter- and intra-examiner reliability of the outcomes PD and CAL were assessed using the intraclass correlation coe fficient (ICC). The inter-examiner reliability resulted in an agreemen<sup>t</sup> for PD (ICC = 0.817) and CAL (ICC = 0.826), denoting a reasonable degree of reliability for both parameters. The intra-examiner reliability of PD and CAL was performed only on 20 selected patients (five patients per group chosen randomly) for both examiners. The intra-examiner reliability for the first examiner resulted in an agreemen<sup>t</sup> for PD (ICC = 0.834) and CAL (ICC = 0.809), and for the second examiner, it resulted in an agreemen<sup>t</sup> for PD (ICC = 0.851) and CAL (ICC = 0.819), denoting a reasonable degree of reliability for both parameters.

A power analysis was performed to calculate the minimum sample size required. The sample size was established considering a number of groups equal to 4, an e ffect size of 0.30 for vitamin C (that represented the primary outcome variable), an expected standard deviation of 1.5 [25], a 2-sided significance level of 0.05, and a power of 80%. It was determined that approximately 32 patients per group would be needed. Thus, it was estimated that 128 subjects were needed to ensure a power level of 80%. One hundred and forty-three patients were enrolled so that the study achieved a power of 83%. Power and sample size calculations were performed using statistical software (G\*Power version 3.1.9.4, Universitat Dusseldorf, Germany).

#### *2.2. vitamin C Assessment in Saliva and Serum*

Fasting samples were collected in all subjects between 8:00 and 10:00 am. Participants were asked to refrain from eating, drinking, chewing gum, brushing teeth, as well from using any mouthwashes, in the last 12 h before the sampling.

The venous puncture was performed, and blood samples were collected, cooled on ice immediately, and centrifuged at 4 ◦C (800× *g* per 10 min). Serum samples were stabilized immediately using metaphosphoric acid in order to avoid oxidization of vitamin C. To collect saliva, subjects were asked to chew on a cotton roll for 2 min, and saliva samples were collected using Salivette collection devices (Sarsted, Verona, Italy) and immediately centrifuged at 4 ◦C (1000× *g* per 2 min). Serum and saliva samples were stored at −20 ◦C until analysis.

Levels of vitamin C and antioxidants (retinol, α-carotene, β-carotene, γ-tocopherol, β-cryptoxanthin, lutein, zeaxanthin, and lycopene) were assessed by use of the commercially available kit for high-performance liquid chromatography (HPLC) measurements (Eureka, Ancona, Italy). In fasting conditions, levels of C-reactive protein (hs-CRP) were assessed by a commercially available nephelometric assay. An hs-CRP level higher than 3 mg/<sup>L</sup> was associated with an increased risk of CAD. Plasma lipids and glucose were determined by routine methods.

## *2.3. Statistical Analysis*

The numerical data are expressed as median, 25% and 75% percentiles, and categorical variables as number and percentage. The Kruskal–Wallis test was applied in order to compare the four groups with regard to all numerical variables, and the Mann–Whitney test in order to perform two-by-two comparisons between groups. Since most of the examined variables (e.g., triglycerides, fasting glucose, and all periodontal variables) did not present normal distribution, as verified by a Kolmogorov–Smirnov test, the analysis was performed by non-parametric tests. For these multiple comparisons, Bonferroni's correction was applied, for which the significant alpha level 0.050 was divided by the number of possible comparisons (*n* = 6), so the "adjusted" significance level for this analysis was equal to 0.050/6 = 0.008. A *p*-trend was performed with the Jonckheere–Terpstra Test for serum and salivary vitamin C levels to assess whether the vitamin C levels were significantly increased in healthy, periodontitis, CAD, and periodontitis + CAD patients. The Spearman correlation test was applied to determine the existence of any significant interdependence between hs-CRP, serum, and salivary vitamin C.

In all enrolled subjects, univariate and multivariable linear regression models were performed in order to assess the dependence of salivary and serum vitamin C levels on potentially explicative variables such as age, gender, education, socioeconomic status (SES), BMI, CRP, triglycerides, total cholesterol, and antioxidants. In the final multivariate model, only age, gender, education, and SES were included as confounders, and tests were carried out to analyze if periodontitis, CAD, and hs-CRP influenced serum vitamin C levels. The same analysis was performed for salivary vitamin C as an outcome. Statistical analyses were performed using statistical software (SPSS 22.0 for Windows package (SPS Srl, Bologna, Italy)). A *p*-value < 0.05 was considered statistically significant.
