This cross-sectional study was conducted between November 2018 and October 2019. Subjects, who underwent overnight polysomnography or polygraphy at Split Sleep Medicine Center (SMC), were invited to participate in this study. The 209 subjects included in this study signed an informed consent form for the use of personal data when accessing the survey and were provided with written information about the study. Included in this study (N = 205) were subjects older than 18 years who underwent overnight polysomnography (PSG, N = 51) or polygraphy (PG, N = 154) and refrained from consuming any food or drink 2 h before collection of saliva samples. ). Subjects under 18 years of age and those who did not comply with the recommendation of fasting for 2 h before taking saliva samples (N = 4) (Fig. 1) were excluded from this study. This study was approved by the Ethics Committee of the Faculty of Medicine of the University of Split (Class: 003–08/14-03/0001, No: 2181–198-03-04-14-0027) and agreed with the Declaration of Helsinki. This study was conducted following the Strengthening Observational Study Reporting in Epidemiology (STROBE) guidelines for cross-sectional studies.
All-night PSG (Alice 5LE, Philips Respironics, Eindhoven, The Netherlands) or all-night PG (Alice NightOne, Philips Respironics, Eindhoven, The Netherlands; SOMNOcheck2, Weinmann, Germany) were performed in the CMS. All data was stored on a computer, manually scored, and evaluated according to the published guidelines of the American Academy of Sleep Medicine (AASM) and the European Society for Sleep Research (ESRS) by a certified physician and technician from the sleep.42.
Based on overnight PSG/PG results, OSA severity was based on AHI value, according to AASM diagnostic criteria and ESRS guidelines42. After polysomnography and/or overnight polygraphy, we divided the total number of events (apneas and hypopneas) by the total number of hours the patient was asleep. Apnea was defined as complete cessation of airflow for 10 s or more, whereas hypopnea was defined as a decrease in airflow of more than 50% for 10 s or more, both followed by desaturation of 3% or more. .42. Therefore, according to the severity of OSA, the subjects were classified into groups: without OSA (IAH < 5; N = 17), AOS de leve a moderada (AHI 5-29,9; N = 109) y AOS grave (AHI > 30; N = 79). Following sleep assessment, subjects who consented to participate in this study were referred for saliva collection (N = 205) and periodontal examination (N = 142) (Fig. 1).
All subjects were interviewed by an examiner who was blinded to the PSG/PG reports. Data on medications (antidepressants, diuretics, antihypertensives, sedatives, bronchodilators, analgesics, antihistamines, anticonvulsants, antiparkinsonians, anticholinergics, retinoids, anorectics, muscle relaxants, and decongestants) and concomitant diseases were collected from the patients’ medical records.
The questionnaire collected demographic data and an evaluation of the subjective assessment of dry mouth on awakening with responses on a Likert scale that included never, rarely, sometimes, often, and almost always, as well as 5 more questions related to dry mouth. subjective assessment of dry mouth. mouth during the day with yes/no answers and questions about oral hygiene habits43.44.
Collection, pH measurement and storage
Unstimulated saliva was collected from all subjects between 9 and 12 am. Subjects were instructed not to eat food, drink beverages, smoke, or chew gum 2 hours before testing. During the test, subjects were advised to rinse their mouths with water and relax for 1 min, swallow to clear the mouth of saliva, and then minimize movement for the next 15 min while collecting saliva and gently spitting it into the mouth. pre-weighed test tubes, as previously described by Navazesh and Kumar4. Immediately after collection, salivary pH was determined with the PICCOLO Plus pH meter with an accuracy of ±0.01 and a HI1295 16 cm (6.3″) replaceable electrode with a temperature sensor (Hanna Instruments HI98113/ Spectrum 240-73492, Smithfield, RI, United States) Prior to the pH measurement, the electrode was calibrated using standards at pH 4.0 and pH 7.0, then the saliva samples were weighed with an analytical balance (KERN ALJ 220-4M) , the weight of the pre-weighed empty test tubes was subtracted from the total weight of the saliva and the test tube to calculate only the mass of the collected saliva that was measured in g/min, which was considered equal to mL/min4. All saliva samples were frozen at -18°C and stored until biochemical analysis.
Calcium, phosphate and magnesium concentrations were measured at the Clinical Institute for Laboratory Diagnostics, Clinical Hospital Center Zagreb with an Agilent 7500 cx (Agilent Technologies, Waldbronn, Germany) and inductively coupled plasma mass spectrometry (ICP‒MS). Saliva samples (400 μL) were digested with nitric acid (2 mL of 65% HNO3 and 1 ml of HtwoO) by high-pressure microwave digestion (UltraCLAVE, Milestone, Italy). After cooling, samples were diluted with 1% (v/v) HNO.3 up to a total volume of 15 mL, and calcium, magnesium and phosphates were analyzed by ICP‒MS. All standard solutions were prepared from a 1 g/L PlasmaCAL standard (SCP Science, Canada). Seronorm® TraceElements Serum Control Level I and Level II (Sero AS, Billingstad, Norway) were used to control the precision of the measurements. Salivary free cortisol was measured using a commercially available enzyme-linked immunosorbent assay (ELISA) produced by Demeditec Diagnostics GmbH, Kiel, Germany. Specifically, free cortisol from the tested saliva sample competed for binding with a cortisol-enzyme conjugate to a polyclonal antibody in a precoated microtiter well. The addition of a substrate caused color development, which was measured spectrophotometrically at 450 nm and was inversely proportional to the salivary free cortisol concentration in the saliva sample.
Periodontal status was assessed immediately after saliva collection by two experienced periodontists (MR, PS). After examination of the same 10 participants, the achievement of intra-examiner and extra-examiner reliability greater than 95% allowed the examiners to collect data independently. Of 205 subjects, 63 were excluded from periodontal examination: those who were edentulous or had fewer than two teeth (N = 13) and those who refused to participate in this study due to lack of time/interest (N = 50) ( Figure 1 ). The examination included assessment of the number of teeth, dental plaque, bleeding on probing, and periodontal measurements: gingival recession (GR), PPD, and CAL expressed in millimeters. GR was defined as the distance between the cement-enamel junction and the gingival margin, and the distance between the gingival margin and the bottom of the gingival sulcus was defined as PPD. The sum of GR and PPD was calculated as CAL. These periodontal parameters were measured following previously published studies for periodontal evaluation and diagnosis.Four. Five,46.47. Based on those results, PISA scores were calculated using an online calculator available at www.parsprototo.info as previously described48.
Analysis was performed in SPSS (SPSS 14.0 Student Version for Windows) and MS Excel (Microsoft Corporation, 2018, Microsoft Excel). Data normality was tested using the Kolmogorov-Smirnov test or the Shapiro-Wilk normality test. Data are presented as medians with interquartile ranges for continuous variables. Categorical variables are reported as frequencies and percentages for each investigated category. All reported differences were assessed using the appropriate test for statistical significance. The names of all tests are described in the footnotes according to their use in the tables. When more than two comparisons were made, the nonparametric Kruskal-Wallis test for group differences was calculated, followed by the Mann-Whitney test to assess specific differences between groups. Categorical data reported as frequencies (percentages) were compared using the chi-square test or Fisher’s exact test, depending on the variable.
Multiple linear regression was performed when salivary flow rate was included as a dependent variable, while the independent variables included in the model were AHI (events/h), age, gender, BMI, salivary calcium, salivary phosphate, salivary magnesium, Ca/Mg, Ca/PO4mg/PO4 and salivary cortisol. Statistical significance was set at p < 0.05. The sample size was calculated following an analysis in MedCalc (MedCalc for Windows, version 19.1.2.) based on the correlation coefficient of the mean AHI and the salivary flow rate (r = − 0.273) evaluated in a pilot sample of 23 respondents, of which 16 had a diagnosis of OSA (mild to severe) and 7 did not have OSA. In the reported analysis, the α level was 0.05 and the potency was set at 90%. The final sample size calculation was 136 respondents, which was increased towards the end of the study in order to have as many control subjects as possible.