*2.1.2 8-Iso-prostaglandin F2α 8-isoprostanes (8-IP)*

The immunoassay reagent kit from Cayman Chemical Company® (Michigan, USA) was used according to the manufacturer's instructions. The limit of detection was 0.8 pg/mL. The 8-IP assay was based on the principle of competitive binding between samples 8-IP, 8-IP acetylcholinesterase (AChE) conjugate, and 8-IP tracer. Then, 50 μL of samples or standard was added to each well, and 50 μL of 8-IP AChE tracer was added to all wells except the total activity and blank wells; and 50 μL of 8-IP enzyme immunoassay antiserum was added to all wells except the total activity and blank wells. At once, 50 μL of 8-IP antiserum was added to all wells except total activity, non-specific binding, and blank wells. The plate was covered and incubated at 4 °C for 18 h and then washed 5 times with buffer. Absorbance was read at 420 nm. The intra-assay CV was 12.5% [10].

#### *2.1.3 Nitric oxide (NO)*

*Antioxidants*

generally have several traditional cardiovascular risk factors like diabetes mellitus (DM), dyslipidemia, and arterial hypertension. These conditions are associated with oxidative stress (OS), and these can trigger and accelerate the progression of renal injury [2]. OS is defined as the imbalance between oxidants and the antioxidant defenses of the body. Reactive oxygen species (ROS) are generated through enzymes such as nicotinamide phosphate adenine dinucleotide (NADPH) oxidase, which reduces oxygen to superoxide anion (O2<sup>−</sup>). This anion is converted to hydrogen peroxide (H2O2) by the enzyme superoxide dismutase (SOD). The O2<sup>−</sup> anion reacts with nitric oxide (NO) by producing peroxynitrite (nitrosative stress). The H2O2 reacts with intracellular iron to form the hydroxyl radical. In addition, H2O2 is catalyzed to hypochlorous acid in the presence of the chloride ion, by myeloperoxidase activity. Uremic toxins also participate by increasing ROS generation. The excessive produc-

DM is a frequent cause of the need for renal replacement therapy (RRT) [4]. In Jalisco (Mexico), nearly half of the patients in dialysis are on peritoneal dialysis (PD), and almost half of these patients have DM [5]. In 2009, it was reported that diabetic nephropathy (DN) causes ~44% of all cases of ESRD in the United States [6]. DM contributes, in large part, to the high costs of health care and the increase in mortality from the increased incidence of DN that leads to ESRD [7]. The purpose of the study was to compare the oxidants and antioxidants state in patients

A single-center, analytical cross-sectional study was performed with ESRD patients on PD. Patients were eligible if they were 16 years old or older and were incident or prevalent in the population of the PD program and never have had a peritoneal equilibrium test (PET) performed before or if the last PET result was older than 1 year. We exclude patients with a current or a previous peritonitis episode in the last 6 months, patients with PD catheter dysfunction, and patients with a current infectious, inflammatory, and malignant process or impaired glycemic control (serum glucose > 200 mg/dL). We collected information about PD treatment and doses, residual diuresis, ultrafiltration, and the value of D/P creatinine (creatinine ratio in the dialysis fluid and plasma), reported at 4 h at the end of the PET. The type of peritoneal transport is determined by the result obtained, modal-

For measurement of OS markers, 10 mL of blood samples were drawn when PET blood samples were taken, 5 mL with 0.1% of ethylenediaminetetraacetic acid (EDTA) tube and other 5 mL in dry tube. The blood was immediately centrifuged at 10,000 rpm for 10 min at room temperature; supernatants were stored in aliquots at −80°C until their final processing. We included 10 mL of extra blood from 10 blood donors (healthy control) that was used to establish the normal value

The levels of LPO in plasma were measured through the FR22 assay kit (Oxford Biomedical Research Inc., Oxford, MI, USA®) according to the

tion of ROS is able to oxidize lipids, proteins, and nucleic acids [3].

ity and the dialysis glucose solution concentration [8].

with PD according to DM status.

**2. Patients and methods**

**2.1 Oxidative stress markers**

**336**

of the reagents.

*2.1.1 Lipoperoxides (LPO)*

Prior to the determination of the NO levels, the serum samples were deproteinized by the addition of 6 mg of zinc sulfate to 400 μL of sample and vortexed for 1 minute and the samples were centrifuged at 10,000×*g* for 10 min at 4°C. For the determination of ON, the colorimetric method was used according to the kit (Nitric Oxide Assay Kit, NB98, Oxford Biomedical Research®). About 85 μL of the standard or sample was added to the wells of the plate, 10 μL of nitrate reductase was added to each well, and 10 μL of 2 mM NADH was added to the wells. The plate was stirred for 20 min at room temperature. Then 50 μL of dye 1 was added and stirred briefly and then 50 μL of dye 2, and again the samples were vortexed for 5 min at room temperature. Finally, the plate was read at 540 nm in a spectrophotometer within the first 20 minutes of completion of the procedure [11].

#### **2.2 Antioxidants**

#### *2.2.1 Superoxide dismutase (SOD)*

We followed the kit manufacturer's instructions (SOD No. 706002, Cayman Chemical Company®, USA) for the detection of O2 <sup>−</sup> generated by the xanthine oxidase and hypoxanthine enzymes through the reaction of tetrazolium salts. We diluted the serum samples 1:5 in the sample buffer, 200 μL of the radicals' detector (1:400 dilution), and added 10 μL of the sample. After slow agitation, 20 μL of xanthine oxidase was then added to the wells. Then, the microplate was incubated for 20 minutes at room temperature. The absorbency was read at 440 wavelength of nm. The levels are reported in IU/mL [12].

#### *2.2.2 Total antioxidant capacity (TAC)*

The evaluations of TAC were made following the instructions of the kit manufacturer (Total Antioxidant Power Kit, No. TA02.090130, Oxford Biomedical Research®), to obtain the concentration in mM equivalents of uric acid. The detection limit was 0.075 mM. The samples and standards were diluted 1:40, and 200 μL was placed in each well. The plate was read at 450 nm as a reference value, 50 μL of copper solution was added, and the plate was incubated at room temperature for 3 minutes. Afterward, 50 μL of stop solution was added and the plate was read at 450 nm. The dilution factor was considered in the result. The intra-assay CV was 7.8% [13].

#### **2.3 Statistical analysis**

Normally distributed variables were presented as mean ± standard deviation (SD); skewed variables were exhibited as median with interquartile range (IQR). Categorical variables were expressed as frequency and percentage. All demographic and PD-related characteristics were compared between diabetics and nondiabetic patients using Chi2 , Student's *t* test, or Mann-Whitney U test accordingly to the type of data distribution. When significant differences in serum levels of oxidative stress markers were found between groups and were feasible, we conducted a multivariable analysis, to determine the interaction between DM and other factors associated with increased OS. The statistical analyses were performed using the IBM SPSS v.18 software (Chicago, IL, USA). For all the analysis, a *p* ≤ 0.05 value was considered as statistically significant.

#### **2.4 Ethics considerations**

The scientific research study abides by the regulations of the internationally established guidelines of the Declaration of Helsinki 1964, revised in October 2013 at the World Medical Assembly. All procedures were performed according to regulations stipulated in the General Health Legal Guidelines for Healthcare Research in Mexico, 2nd Title, in Ethical Aspects for Research in Human Beings, Chapter 1, Article 17. The study was evaluated and approved by the Local Ethics and Research Committee at the Regional General Hospital No. 46, Mexican Institute of Social Security, Guadalajara, Jalisco, Mexico, with registration number R-2017-1303-117. All patients gave and signed the informed consent form in the presence of signed witnesses. Patients had the right to withdraw from the study at any time without representing harm to the patient-doctor relationship and without affecting their treatment. At all times, total confidentiality was maintained, and the patients were informed of the results throughout the study.
