Ransod kit

The method of determination relies on the production of a superoxide anion by the reaction of xanthine with xanthine oxidase (XOD), which, by reacting with 2-(4-iodophenyl)-3(4-nitrophenyl)-5-phenyltetrazole (INT) chloride, forms a red formazan dye. The measurement of the absorbance of the colored product determines the effectiveness of the above reaction. $$\text{Xantine}\stackrel{\text{XOD}}{⟶}\text{Uric}\text{acid}+{{\text{O}}_2}^{-}$$
$$\text{I}.\text{N}.\text{T}.\stackrel{{{\text{O}}_2}^{-}}{⟶}\text{Formazan}\text{dye}$$ CuZnSOD presented in the tested sample (biological material) inhibits this reaction by dismutating O₂⁻ to H₂O₂ and O₂. The degree of inhibition of this reaction is directly proportional to the activity of CuZnSOD. $${{\text{O}}_2}^{-}+{{\text{O}}_2}^{-}+2{\text{H}}^{+}\stackrel{\text{CuZnSOD}}{⟶}{\text{O}}_2+{\text{H}}_2{\text{O}}_2$$ CuZnSOD activity was determined spectrophotometrically using a standard RANSOD kit by Randox (United Kingdom), which includes the following reagents:

Substrate: Xanthine 0.05 mmol/L, INT 0.025 mmol/L;

Buffer: N-cyclohexyl-3-aminopropanesulfonic acid 40 mmol/L, pH 10.2, EDTA 0.94 mmol/L;

Xanthine oxidase 80 U/mL; and

Standard – CuZnSOD 5.5 U/mL.

CuZnSOD activity in the tested biological material was determined in a Shimadzu UV 1202 spectrophotometer (Kyoto, Japan), measuring the absorbance at 0 and 3 min at a wavelength of 505 nm.

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.

The superoxide dismutase (SOD) activity was determined in erythrocytes using the Ransod kit (Randox, Crumlin, UK). The reaction was based on the superoxide radical anion production by the action of xanthine oxidase which reacted with 2-(4-iodophenyl)-3-(4-nitrophenol)-phenyltetrazolium chloride, resulting in the formation of a red formazan dye. The higher SOD activity in the sample, the less formazan dye produced. One unit of SOD was defined as the amount of enzyme resulting in 50% inhibition of dye formation [21 (link)].