Plasma 8-Ohdg Levels Correlate With Endothelial Dysfunction and Atherosclerosis in Peritoneal Dialysis Patients

Background: Accelerated atherosclerosis is a major cause of increased cardiovascular morbidity and mortality in patients on chronic peritoneal dialysis (PD). This study aims to investigate the relationship among oxidative stress, endothelial dysfunction and atherosclerosis in PD patients. Methods: Eighty non-diabetic PD patients and twelve healthy controls were enrolled in the trial. Plasma 8-OHdG and ET-1 levels were quantified using enzyme-linked immunosorbent assay and radioimmunoassay respectively. The eNOS activities were measured by nitrate reductase assay. Carotid intimal-medial thickness (CIMT) was assessed by quantitative carotid artery ultrasonography. Results: The mean age of the PD patients was 41.6±14.03 years and that of controls 40.92 ±5.32 years respectively (P >0.05). Plasma levels of 8-OHdG and ET-1 increased in patients on maintenance peritoneal dialysis. The mean plasma 8-OHdG concentrations were 39.33±9.34 ng/ml in PD patients and 7.25±0.97 ng/ml in the control group while the mean plasma ET-1 were 139.35±31.12pg/ml in PD patients and 16.00±1.41 pg/ml in the control group respectively, (P <0.01). The eNOS activities were significantly lower in PD patients compared to controls (12.57±1.76 u/ml vs 14.25±1.86 u/ml, P <0.01). CIMT was higher in PD patients as compared to controls (1.21±0.40mm vs 0.92±0.29mm, P <0.01). Linear regression analysis showed that the levels of plasma 8-OHdG had a significant positive correlation with plasma ET-1 and CIMT, but a negative correlation with plasma eNOS or KT/V. Conclusion: The endothelial dysfunction in PD patients was significant and it was positive correlated with the oxidative stress indicted by the plasma levels of 8-OHdG. The levels of plasma 8-OHdG could serve as an independently risk factor for endothelial dysfunction and atherosclerosis in PD patients. J Biom Tech Res Vol. 1. Issue. 1. 6000104 Exclusion criteria: patients with any systemic illness such as diabetes, malignancy, lupus, vasculitis were excluded. Patients with intravenous iron treatment or with any infection disease in the past month were also excluded. Laboratory Examinations Measurement of plasma 8-hydroxydeoxyguanosine (8-OHdG) levels 8-OHdG levels were measured by using a competitive in vitro enzyme-linked immunosorbent assay (ELISA) kit (Bioxytech, OXIS Health Products, Inc., Portland, OR, USA) according to the manufacture’s protocol. In brief, 50μl plasma sample and 50μl of reconstituted primary antibody were added to each well of 8-OHdGcoated microtitre plates and incubated at 37°C for 1 h. Plates were washed three times with phosphate-buffered saline (PBS) followed by adding horseradish peroxidase-conjugated secondary antibody. After incubation at 37°C for another hour, unbound secondary antibody was removed, and the plate was washed again three times. The amount of antibody bound to the plate was determined by the development of color intensity after the addition of substrate containing 3,3,5, ′-tetramethyl-benzidine using a computer-controlled spectrophotometric plate reader at the wavelength of 450 nm. The concentration of 8-OHdG was interpolated from a standard curve. The detection range of the ELISA assay was 0.5–200ng/ml. Measurement of plasma Endothelin-1 levels Plasma ET-1 concentrations were determined by use of a radioimmunoassay kit (Beijing North Institute of Biotechnology, Beijing, China) according to its manufacture’s protocol. In short, 100μl of plasma sample were added into duplicate tubes followed by adding primary antibody. After incubation for 16-24 hours at 4°C, 100μl of 125I-peptide tracer solution were added followed by incubation for 16-24 hours at 4°C. Then the tubes were incubated for 90 minutes at room temperature after adding Goat Anti-Rabbit IgG serum and normal Rabbit serum. Tubes were centrifuged at 1700xg for 20 minutes at 4°C. All the supernatants were carefully removed immediately following centrifugation. Counts Per Minute (Cpm) of all the pellets were counted by using a γ -counter. Plasma ET-1 concentrations were calculated from the stand curve. The detection range of the RIA assay was 10–1280pg/ml. Measurement of plasma endothelial nitric oxide synthase activity Plasma eNOS activities were measured using colorimetric nitrate reductase assay by Nanjing Jiancheng Biochemical Co (Nanjing, China). Measurement of carotid artery intimal-medial thickness Quantitative ultrasound measurements for both sides of carotid artery intimal thickness were performed using ATL HDI 5000 SonoCT/XRES Color Doppler System (Bothell, WA) by a single expert sonographer. The sonographer was blinded to the subject’s identity. Astandard protocol [4], scanning the near and far walls of the right and left common, internal and external carotid arteries, and bifurcations in three different projections (anterior, lateral, and posterior), was performed. Eight segments of the right and left carotid arteries in each projection were examined, and the 48 CIMT measurements were averaged to calculate the average CIMT for each subject. The carotid artery thickness is defined as CIMT≥1.0mm at carotid sinus below 10cm and/or CIMT≥1.2mm at the carotid bifurcation according to the standard of AHA (American Heart Association). The subjects were assigned to two groups after CIMT measurement: those who did not have atherosclerosis (CIMT<1.0mm) and those who had atherosclerosis (CIMT≥1.0mm and/or plaque formation). Statistical analysis Age, sex, medications, blood pressure, hemoglobin (Hb), albumin, cholesterol, Calcium (Ca), phosphate (Phos), blood urea nitrogen (BUN) and serum creatinine (Scr) were recorded. Clinical characteristics were reported as means ± standard deviations for continuous variables and frequencies for categorical variables. Statistical analysis was performed on SPSS (IBM, Armonk, NY) version 13.0. Differences between the groups were examined by one-way ANOVA or unpaired Student′s t-test when necessary and chi-square tests for continuous and categorical variables, respectively. A multiple linear regression analysis was used to determine the independent predictor of CIMT. Results with p<0.05 were considered statistically significant in all analyses.