**2. Patients and methods**

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, modality and the dialysis glucose solution concentration [8].

#### **2.1 Oxidative stress markers**

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 of the reagents.

#### *2.1.1 Lipoperoxides (LPO)*

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

**337**

*Increase of Oxidants and Antioxidant Consumption in Patients with Type 2 Diabetes…*

manufacturer's instructions. The limit of detection for this test was 0.1 nmol/ mL. The chromogenic reagent reacts with malondialdehyde (MDA) and

4-hydroxy-alkenals to form a stable chromophore. First, 140 μL of plasma with 455 μL of N-methyl-2-phenylindole in acetonitrile (Reagent 1) was diluted with ferric iron in methanol. Samples were agitated; after which 105 μL 37% HCl was added followed by incubation at 45°C for 60 min and centrifugation at 12,791 rpm for 10 min. Next, 150 μL of the supernatant was added, and absorbance was measured at 586 nm. The pattern curve with known concentrations of 1,1,3,3-tetramethoxypropane in Tris–HCl was used. The intra-assay CV was

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

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

We followed the kit manufacturer's instructions (SOD No. 706002, Cayman

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

<sup>−</sup> generated by the xanthine

*DOI: http://dx.doi.org/10.5772/intechopen.82880*

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

420 nm. The intra-assay CV was 12.5% [10].

*2.1.3 Nitric oxide (NO)*

procedure [11].

**2.2 Antioxidants**

*2.2.1 Superoxide dismutase (SOD)*

Chemical Company®, USA) for the detection of O2

nm. The levels are reported in IU/mL [12].

8.5% [9].

*Increase of Oxidants and Antioxidant Consumption in Patients with Type 2 Diabetes… DOI: http://dx.doi.org/10.5772/intechopen.82880*

manufacturer's instructions. The limit of detection for this test was 0.1 nmol/ mL. The chromogenic reagent reacts with malondialdehyde (MDA) and 4-hydroxy-alkenals to form a stable chromophore. First, 140 μL of plasma with 455 μL of N-methyl-2-phenylindole in acetonitrile (Reagent 1) was diluted with ferric iron in methanol. Samples were agitated; after which 105 μL 37% HCl was added followed by incubation at 45°C for 60 min and centrifugation at 12,791 rpm for 10 min. Next, 150 μL of the supernatant was added, and absorbance was measured at 586 nm. The pattern curve with known concentrations of 1,1,3,3-tetramethoxypropane in Tris–HCl was used. The intra-assay CV was 8.5% [9].
