The study protocol was approved by Ankara Yıldırım Beyazıt University and complied with the principles of the Declaration of Helsinki. An informed consent form was signed by all the participants. Patients over 18 years of age with T2DM were included in the study. Those with systemic conditions, such as chronic kidney failure, chronic liver failure, pregnancy, hypertension, hypothyroidism, hyperthyroidism, cardiovascular disease, and rheumatological diseases, and a history of malignancy were excluded from the study to eliminate any possible effect on thiol–disulfide homeostasis [17]. In addition, patients who received laser therapy or intravitreal injection; patients who had diabetic macular edema (DME), PDR, myopia, hyperopia or astigmatism ≥2 diopters, history of intraocular surgery, or chronic ocular diseases (glaucoma, uveitis, dry eye syndrome, or conjunctivitis); and contact lens wearers were not included in the study. A total of 69 patients with T2DM meeting the inclusion and exclusion criteria and 21 healthy controls were included in this study. Retinal layer thickness in the right eye was measured in the patient and control groups. The patients were grouped according to disease duration (<10 years and ≥10 years) to analyze early-stage changes [18,19].
Oculer evaluation
All participants were examinated by two retina specialists. Evaluation included biomicroscopic examination to assess the macula and fundus, non-mydriatic photographs, visual acuity and OCT. Severity of DRP was classified according to the scale of DRP severity [20,21].
SD-OCT
SD-OCT was performed with a dilated pupil. Whole process was conducted by the same experienced operator and no manual correction was performed on OCT scans. The SD-OCT system automatically provided Early Treatment Diabetic Retinopathy Study (ETDRS) thickness maps for the following seven layers: retinal pigment epithelium (RPE), outer nuclear layer (ONL), outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), ganglion cell layer (GCL), retinal nerve fiber layer (RNFL) with the caliper measurement tool embedded in the system. [22]. In this study only the scans with sufficient quality (Q > 25) were included in image analysis
Laboratory evaluation
Glucose, albumin, C-reactive protein (CRP), spot urinary albumin/kreatin ratio were measured using commercially available assay kits (Roche Diagnostics, Mannheim, Germany) and an auto analyzer (Cobas 501, Roche Diagnostics, Mannheim, Germany) as well thiol-disulfide tests. HbA1c was quantified by standardized immunoturbidimetry on a Cobas 502 analyzer (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions.
Thiol/disulfide homeostasis
Venous blood samples from the patients and healthy controls were collected into EDTA tubes after 12 hours of fasting. The blood samples were immediately centrifuged for 10 minutes at 1500 rpm, and plasma and serum were separated. Separated serum samples were stored at -80°C and all samples were run at once. During the thiol/disulfide homeostasis tests, first, the reducible disulfide bonds were reduced to form free functional thiol groups. Formaldehyde was used to remove unused and consumed sodium borohydride. Total thiol (–SH+–S–S–) and native thiol (–SH) concentrations in the samples were measured using Ellman's and modified Ellman's reagent. The natural thiol content was subtracted from the total thiol content and half of this difference gave the amount of dynamic disulfide bonds (–S–S). Using these parameters, disulphide/native thiol, disulphide/total thiol, native thiol/ total thiol ratio were calculated.
Statistical evaluation
All statistical analyses were performed with the SPSS 15.0 software package (SPSS, Inc., Chicago, Illinois). Descriptive statistics for the constant variables were expressed as mean ± SD or median (range), and categorical variables were noted as numerics and percent (%). Student T test was used for comparison between independent groups conforming to normal distribution, and Mann Whitney U test for those, who did not comply with normal distribution. Chi-square was used to compare categorical variables. Analysis of variance (ANOVA) was used to compare normally distributed interval data between patients with DM with and without DR and healthy individuals, and the Kruskal Wallis test was used to compare non-normally distributed interval data. Pearson correlation analysis was used to analyze correlations between normally distributed variables, and Spearman correlation analysis was used to analyze correlations between non-normally distributed variables. To compare interval data between the groups with a DM duration of <10 years and those with a DM duration of ≥10 years, Student's t-test was used for normally distributed data and Mann–Whitney U test was used for non-normally distributed data. The significance level was determined as p < 0.05.