Spectrocopical uv vis analysis

MDA production was measured following the method previously described by Esterbauer and Cheeseman [22 ] to assess lipid peroxidation. The peritoneal fluid samples were diluted in Tris HCl buffer (TRIZMA HCl, Sigma Aldrich, São Paulo, Brazil) in distilled water (20 mM, pH 7.4), following homogenization and centrifugation at 10,000 rpm for 10 min at 4°C. To each sample, chromogenic reagent (10.3 mM 1-methyl-2-phenylindole in 3:1 acetonitrile) and HCl (37%) were added. Subsequently, they were incubated for 40 min at 45°C and centrifuged at 10,000 rpm for 5 min at 4°C. Absorbance was recorded at 586 nm using Spectrocopical UV/VIS analysis (Biotek, São Paulo, Brazil) and interpolated in a standard curve with 1,1,3,3-tetraethoxypropane. The results are shown in nmol/μl.

MPO activity was measured according Krawisz et al. [20 (link)]. An aliquot (100 μL) of each sample was diluted in hexadecyltrimethylammonium bromide buffer (HTAB, Sigma Aldrich, São Paulo, Brazil) and homogenized. The duplicate samples were sonicated for 5 min and then centrifuged at 10,000 rpm for 15 min at 4°C and freeze-thaw three times. To the samples, 200 μl of the staining reagent (o-dianisidine dihydrochloride) was added and the absorbance values at 450 nm were recorded by Spectrocopical UV/VIS analysis (Biotek, São Paulo, Brazil). The absorbance data were interpolated from a standard curve of human neutrophil myeloperoxidase and horseradish peroxidase. One unit of MPO (U) was defined as the activity that degrades 1 nmol/min of hydrogen peroxide at 25°C. The results were expressed as U/μL.

Determination of total glutathione content was performed according to the method described by Anderson [15 (link)]. The joint wash intended for analysis was preconditioned in an Eppendorf tube and then frozen at − 80 °C with a volume of trichloroacetic acid (TCA 5% w/v in distilled water) in the ratio of 1:20 v/v to avoid degradation of GSH by gamma-glutamyltranspeptidase. Next, a 20 μl of sample was used for 400 μl of TCA. For determining this content, the samples were thawed and taken to an automatic homogenizer for about 2 min in the cold. Then the samples were centrifuged at 2000 G for 5 min at 4 °C. Next, 100 μl of the sample was withdrawn into an Eppendorf tube, then 100 μl of aqueous solution of sodium phosphate and EDTA (143 mM PBS-EDTA-PBS and 6.3 mM EDTA, pH 7.5) were added, along with 700 μl of the solution of NADPH (0.289 mM β-NADPH in PBS-EDTA) and 100 μl of the DTNB solution (6 mM DTNB in ​​PBS-EDTA). The solutions were taken to a water bath for 5 min at 30 °C, then transferred to ELISA plate wells, and 25 μl of the enzyme solution (glutathione reductase in PBS-EDTA 266 IU/ml) was added to each well. The absorbance was determined at a wavelength of 412 nm by Spectrocopical UV/VIS analysis (Biotek®, São Paulo, Brazil) at 0- and 3-min time. Total glutathione content was calculated by standard curve interpolation and the results were expressed as nmol/ml.