A method for determining oxidative modification of blood plasma fibrinogen content of carbonyl groups in the fibrin clot

摘要

A method for determining oxidative modification of blood plasma fibrinogen content of carbonyl groups in the fibrin clot, comprising adding to 1 ml of citrated blood plasma test sample coagulation activator, and incubation at 37 ° C, the allocation of a fibrin clot, drying it on a filter paper, counting the concentration of fibrinogen in mg / ml, processing test sample 20% solution of trichloroacetic acid (TCA) and a solution of 2,4-dinitrophenylhydrazine in 2 M HCl, incubated at room temperature for 1 hour, centrifugation, trehkra peppermint washing the precipitate with a solution of ethanol and ethyl acetate in the ratio 1: 1, drying the precipitate, dissolving it in 2.5 ml of 8 M urea followed by evaluation of the oxidative modification of proteins formed by level dinitrophenylhydrazone using spectrophotometric optical density recording, characterized in that activating plasma coagulation using thrombin, fibrin clot prior to drying on filter paper, washed three times with 0.9% aqueous NaCl, then converted into a soluble state by hydrolyzed and 1 ml of a 1 M solution of NaOH at 100 ° C, the reaction is stopped after 120 ± 1 with 1 ml 2M HCl, vials cooled in the hydrolyzate protein concentration determined by biuret reaction, converted to fibrinogen concentration in mg / ml of fibrinogen calibration , treatment with 20% solution of TCA is performed 1 hour after the start of incubation, 0.5 ml of the hydrolyzate of the fibrin clot with 2 ml of 0.01 M solution of 2,4-dinitrophenylhydrazine in 2 M HCl, wherein the chilled TCA solution is added to said reaction mixture in a volume ratio of 8: 5 and kept at -20 ° C not less 10 minutes before centrifugation, three times washing