2.1 Participants
Prior to the implementation of the study, a full-mouth periodontal examination was performed on all participants by an examiner using a sterile periodontal probe (PCPUNC 15, Hu-Friedy, Chicago, IL, USA). Periodontal pocket depth (PPD), gingival Bleeding Index (GBI) and calculus Index (CI) were recorded to identify the periodontal health status. Structured questionnaire was used to collect the information on sociodemographic background (age and sex), smoking habit, oral health-related behaviors (toothbrushing frequency and use of dental floss), and medical history (presence of hypertension or diabetes) of each participant.
Ethical approval was obtained by the Research Ethics Committee of Shanghai Ninth People’s Hospital. Each participant was informed about the purpose and procedure of the study. Each participant signed a written informed consent prior to the implementation of the study.
2.2 Collection of biological samples
2.2.1 Serum
A total of 3 mL peripheral blood was collected in vacutainer tubes in the morning from all participants. The blood samples were incubated at room temperature for not more than 2 h and centrifuged for 10 min at 2,000 × g at 4℃. Serum was collected in the empty tubes and then stored at -80°C.
2.2.2 GCF
All participants were instructed not to eat or drink within 2 h prior to the collection of GCF. PerioPaper Strips® (Oraflow Inc, New York, USA) were used to collect GCF samples from the disto-buccal and mesio-buccal sites of all the first molars. The targeted area was separated with cotton rolls, PerioPaper Strips® were placed in the gingival sulcus for 30 s to collect GCF, and all periopapers strips from different sites were pooled in the same tube. The samples were then stored in an empty tube at -80°C.
2.3 Estimation of inflammatory markers in GCF and serum
Accordingtothekitinstructions, the concentrations of Interleukin-1β (IL-1β), Interleukin-6 (IL-6), and Interleukin-8 (IL-8) in GCF and serum as well as C-reactive protein (CRP) in GCF were measured using Human High Sensitivity T Cell Magnetic Bead Panel (Luminex Corporation, Austin, TX, USA). Further, the concentration of CRP in serum was measured using an CRP ELISA kit (R&D Systems, Minneapolis, MN, USA).
2.4 Metabolite extraction
For metabolite extraction from GCF, 200 μL extraction solution (methanol: acetonitrile: water = 2:2:1, with isotopically labelled internal standard mixture) was added to periopaper strips. The mixture was vortexed for 30 s and grinded at 35 Hz for 4 min. Thereafter, the samples were sonicated for 15 min in ice-water bath. For the extraction of serum metabolites, 50 μL of sample was mixed with 200 μL of extraction solution (acetonitrile: methanol = 1: 1, containing isotopically labelled internal standard mixture). The mixture was vortexed for 30 s and sonicated for 10 min in ice-water bath. Both mixtures were incubated for 1 h at -40oC and centrifuged at 12,000 rpm for 15 min at 4oC. The supernatant was separated and mixed with quality control (QC) samples for machine testing.
2.5 Metabonomics analysis
Liquid chromatography/mass spectrometry (LC/MS) was used for metabonomics analysis. It was performed using a UHPLC system (Vanquish, Thermo Fisher Scientific) containing a UPLC BEH Amide column (2.1 mm × 100 mm, 1.7 μm) with Q Exactive HFX mass spectrometer (Orbitrap MS, Thermo). HPLC contained aqueous phase (25 mmol/L ammonium acetate and 25 mmol/L ammonia) and acetonitrile. The analysis was processed with elution gradient. Other LC parameters were as follows: mobile phase flow rate: 0.5 mL/min, column temperature: 30oC, sample pan temperature: 4oC, injection volume: 4 μL. The QE HFX mass spectrometer was used to collect primary and secondary mass spectrometry data under the control of the acquisition software (Xcalibur, Thermo).
2.6 Data preprocessing and annotation
The raw data were transformed into the mzXML format, using R software to process peak detection, extraction, alignment, as well as integration. Further, an in-house MS2 database (BiotreeDB) was used for metabolite annotation with the cutoff for annotation set at 0.3.
2.7 Sample size calculation
A pilot study (n=15 in each group) was performed to obtain information about inflammatory markers. The levels of IL-8 were significantly different between two groups, which we chose as the primary outcome. We assumed two-sided hypothesis testing under the 5% type I error and 90 % statistical power to detect a difference of 0.815 ng/ml with estimated group standard deviations of 1.331 ng/ml and 0.603 ng/ml. A total of 36 participants were required in each group, which was calculated using PASS11.0(NCSS, LLC. Kaysville,Utah,USA).
2.8 Statistical data analysis
The Kolmogorov–Smirnov test was performed to test the normality of data distribution. Numerical variables were presented as mean (SD) when parameters were normally distributed or as median (interquartile range) when the distribution was skewed. Student’s t-test or Mann–Whitney U test was used to compare the differences between ESRD and HC groups depending on whether variances were homogenous and normal. Chi-square test or Fisher’s exact test was used to compare the differences of sociodemographic background, lifestyle, and medical history between the two groups. Based on SIMCA software V16.0.2 (Sartorius Stedim Data Analytics AB, Umea, Sweden), principal component analysis (PCA) was used to show the sample distribution within the two groups. Orthogonal partial least-squares discriminant analysis (OPLS-DA) was also used to assess total differences of metabolites between the two groups. Multivariate models were performed to determine various metabolites with variable importance (VIP) values >1.0 and P <0.05 as the key metabolites for a further analysis. Pearson correlation coefficient was used to calculate the correlations between metabolites and inflammatory markers. Multiple factor ANOVA analysis was used to assess the association between system diseases and the levels of IL-8 in the CGF. P <0.05 was considered to be statistically significant.