METHOD FOR DETERMINING SUPEROXIDE ANION RADICALS GENERATION IN BIOLOGICAL MATERIAL BY USING CELLS Russian patent published in 1996 - IPC

FIELD: medicine. SUBSTANCE: method involves taking samples of microbioptates from human individual or animal in performing endoscopic examination of cavitary organs (stomach, intestine, bronchi e.t.c.) or taking pieces of noncavitary organs (liver, kidney e.t.c.) by means of biopsy needle, washing the samples with a pH 7.35 buffer mixture, drying with filtering paper, performing high precision (up to 10^-5 g) bioptates weighing, comminuting them with eye surgery scissors, thermostating with 0.2% water alcohol solution of triton X-100. Then the so produced comminuted bioptates are washed from triton traces and thermostated with superoxide transmutase for experimental subset and with albumin for control subset. The experimental and control comminuted bioptates are thermostated with reduced nicotinamide adenine dinucleotide phosphate stimulating superoxide anion radical production by cells and nitro blue tetrazole detecting superoxide anion radicals as formazanes at 36.7-37.0 C during 40-60 min. The reaction is stopped by adding 5M hydrochloric acid. Next to it, centrifuging is carried out and formazanes precipitate is extracted with a mixture of dimethyl sulfoxide and chloroform taken in 2:1 proportion at 55.0-59.0 C with following spectrophotometric determination of superoxide anion radical. The obtained spectrophotometric data of optical density are recalculated for 1 mg of bioptate. The difference between the experimental and control sample values shows the superoxide anion radical concentration based on optical density of formazanes being the extracted reaction products due to superoxide anion radical and nitro blue tetrazole interaction expressed in optical density units. The prototype is not usable for the purposes of determining superoxide anion radical generation as a sum using parenchymal and interstitial cells of visceral tissues. EFFECT: enhanced effectiveness of method.