Ransel kit

In another set of experiments, blood samples (1 mL) were collected in CLT (n = 6) and L‐NAME‐treated rats (n = 6), and superoxide dismutase (SOD) and glutathione peroxidase (GPx) enzyme activities in the serum were determined using RANSOD and RANSEL kits (Randox Laboratories, Crumlin, UK), respectively. Catalase (CAT) activity was assayed as previously described (Aebi, 1984). Lipid peroxidation was estimated based on serum levels of malondialdehyde (MDA) (Karatas et al., 2002). The protein concentration was measured by the Bradford method.

In the study, tissue SOD and GSH-Px enzyme activities were measured respectively with RANSOD and RANSEL kits (Randox Laboratories Ltd., Crumlin, UK) by colorimetric assay on the Architect c16000 chemistry analyzer (Abbott Diagnostics, USA). GSH-Px and SOD activity values were calculated as mU/mg protein.

The SOD activity was determined using the RanSOD kit from Randox, County Antrim, UK. The measurement of fumarase activity involved the conversion of fumarate to malate, with detection performed at 240 nm [32 (link)]. The enzymatic kinetics of glutathione peroxidase (GPx) were evaluated using the Ransel Kit from Randox, County Antrim, UK. The GST antioxidant assay was used to measure the formation of S-(2,4-dinitrophenyl)-glutathione through the enzymatic activity of GST via the conjugation of 1-chloro-2,4-dinitrobenzene (CDNB) and glutathione (GSH). The measurement of GST activity was carried out by determining the absorbance at a wavelength of 340 nm [33 (link)]. All enzyme activities are quantified as U/mg of protein.
The consumption of H₂O₂ was evaluated with a protocol adapted for microplates [34 (link)]. First, 30% H₂O₂ was diluted in 10 mL of sodium phosphate buffer (50 mmol/L, pH 7) and added into the sample to trigger the reaction, measuring the rate of H₂O₂ consumption via absorbance at 240 nm. H₂O₂ consumption was reported because there are multiple H₂O₂ detoxification mechanisms (mainly CAT and peroxiredoxins) and the test is not specific for any of them [35 (link)]. The activity is expressed as μmol of H₂O₂ consumption/min/mg of protein.
All assays were independently performed in triplicate.

SOD is involved in the first step of the antioxidant enzymatic cascade catalysing the dismutation of superoxide radical into oxygen and hydrogen peroxide. The enzymatic activity of SOD in plasma (diluted 1:6) and RBC lysate (diluted 1:500) was measured with the SOD activity kit (Enzo® Life Sciences, USA) following manufacturer instructions. This test quantifies in vitro the kinetics of inhibition in superoxide formation resulting from SOD antioxidant activity. SOD activity is expressed as U (units of enzymatic activity).mL⁻¹. Intra-individual coefficient of variation based on duplicates was 12.00 ± 1.96%.
Glutathione is used as a reductant by the GPx enzyme to scavenge deleterious hydrogen peroxide. The enzymatic activity of GPx in plasma (diluted 1:10) and RBC lysate (diluted 1:60) was measured with the RANSEL kit (Randox Laboratories, Crumlin, UK) following manufacturer instructions. GPx activity is expressed as U.L⁻¹. Intra-individual coefficient of variation based on duplicates was 9.09 ± 1.40%.

Erythrocyte and plasma selenium concentrations were measured using hydride generation flame atomic absorption spectrometry as previously described [19 (link),23 (link)]. The references for plasma selenium concentration were values between 1.07 to 1.27 μmol/L (84 to 100 μg/L), and those for erythrocyte selenium concentration were values between 0.76 to 1.52 μmol/L (60 to 120 μg/L) [24 (link),25 (link)].
The glutathione peroxidase activity of red blood cell hemolysate was assessed using the method described by Paglia and Valentine using the Ransel kit (Randox Laboratories Ltd, Crumlin, County Antrim, UK) [26 (link)].