Deppd

The concentration of RC-DEPPD was determined as described by Alberti et al. (2000) [24 (link)]. The estimation of RC-DEPPD formed in the reaction of alkoxy and peroxy radicals derived from hydroperoxides (present in the sample) was performed using an indirect estimation of the level of hydroperoxides. The incubation mixture contained 1 ml of acetate buffer (pH 4.8), 10 μL of an aqueous solution of DEPPD (Sigma, Poznan, Poland) (0.37 mol/l) and 20 μL of plasma. After 1.5 h incubation at 37^o C absorbance was read at 505 nm against distilled water (Ultrospec 2000, Pharmacia, Uppsala, Sweden). In the control sample, 20 μL of distilled water replaced the plasma. Calculations were based on a standard curve prepared with six different dilutions of H₂O₂. The results were expressed as mmol/g protein.

Chemical reagents such as acetylthiocholine iodide, thiobarbituric acid, sulphanilamide, reduced glutathione, DEPPD, DMSO, DPPH, trichloroacetic acid, and sodium acetate were sourced from Sigma-Aldrich (now Merck KGaA, Darmstadt, Germany). Hydrogen peroxide, methanol, acetic acid, hydrochloric acid, aluminium chloride, potassium acetate, sodium dodecyl sulphate, iron (II) sulphate, manganese chloride, potassium ferrycyanide, and ferric chloride were sourced from BDH Chemicals Ltd., (Poole, England). Except stated otherwise, all other chemicals and reagents were of analytical grades and the water was glass distilled.

The method was based on the estimation of radical cation formed in the reaction of alkoxy and peroxy radicals derived from the hydroperoxides by use of N,N-diethyl-para-phenylene diamine (DEPPD, Sigma, Poznan, Poland). Incubation mixture contained 1 ml acetate buffer (pH = 4.8), 10 μl aqueous solution of DEPPD (0.37 mol/dm³) and 20 μl supernatant or plasma. After 1.5 h incubation at 37 °C absorbance was read at 505 nm (Ultrospec 2000, Pharmacia, Sweden) against distilled water. Control sample contained distilled water instead of supernatant. Calculations were based on standard curve prepared with different dilutions of H₂O₂. The results were expressed as μmol/g protein.