SU1301841A1 - Method for determining activity of succinatedehydrogenase in boiological objects - Google Patents

Abstract

This invention relates to medicine. The purpose of the invention is to increase the accuracy of the method. Blood, phosphate buffer mixture, sodium succinate solution and tetrazol solution are introduced into the test tube with distilled water. Samples are incubated and centrifuged. The precipitate is purified from blood protein structures, dissolved in acetone and centrifuged. The centrifugate is photometric and the enzyme activity is calculated. 1 tab. sh as

Description

113

The invention relates, to medicine, to the measurement of the enzymatic activity of succinate dehydrogenase (LDH) and can be used in studies of the influence of various environmental factors on the human body and animals.

The aim of the invention is to improve the accuracy by further purifying the reaction product by centrifuging, followed by dissolving the resulting precipitate in acetone.

The method is carried out as follows.

0.2 ml of blood and 1 ml of a 0.15 M solution of phosphate buffer mixture, a 0.2 M solution of sodium succinate and a 0.1% solution of tetrazole are added to a tube with 1 ml of distilled water. Samples incubated at 37-38 ° C for 60-90 minutes. The reaction product is separated from the incubation medium by centrifuging the samples at 3000 minutes for 15 minutes, the precipitate is washed in 5-10 ml of distilled water and centrifuged for 5 minutes at 3000 and purified from hemoglobin and other blood protein structures, then the precipitate is dissolved in 5 ml of acetone and centrifuged at 3000 for 3-5 minutes,

A centrifugate, which is a purified solution of formazan in acetone, is photometric, for example, on a spectrophotometer (AfTi 475 nm).

The enzyme activity is calculated by the formula

.-

Dj

one

., 8Л „-----; - 5 fj MOL / ML-min,

wed

(one)

50

formazan content in stan- 45

Dart test, rg / ml; the optical density of the standard and test samples at Yn = 475 nm; blood samples, ml incubation time

37-38 ° C, min; formazan molecular weight

(U g / mol;

constant for the proposed method coefficient taking into account the loss of formazan during centrifugation.

55

12

When measuring the optical density of standard samples on an SF-18 spectrophotometer, the dependence of D on the formazan content in a standard sample is linear in the range of values from 0 to 100.

When determining the activity of LDH in tissues, instead of a mixture of t ml of distilled water with a blood sample, 1 ml of 5-10% tissue homogenate is used. In this case, the operation of cleaning the reaction product from hemoglobin and protein structures can be omitted, since the absorption spectrum of the tissue homogenate solution does not contain the Soret spectrum. The enzyme activity is calculated by formula (1), in which the volume of blood volume V is replaced by the weight (mass) of tissue in 1 ml of the homogenate solution in rpaMi iax. The amount of activity in this case is expressed in / J mol / / g min.

The table shows a comparison of the results of determining the activity of SDH according to the proposed method and the Kuna-Ebud method (known). The analysis was performed in rat liver tissue samples.

thirty

 Due to the high optical density, before photometry, the sample was diluted with acetone in a ratio of 1: 4 (1 ml of sample — f 4 M.P. of acetone),

Form-A of the invention

The method of determining the activity of succinate dehydrogenase in biological objects, including their incubation with the substrate in the presence of tetrazole in a buffer medium, followed by centrifugation and photometric

313018414

by measuring the reaction product, about the reaction by centrifuging, in that for about 10-15 minutes at 3000 rpm to increase the accuracy of the method, then dissolving the resulting water to further purify the precipitate in acetone.

Claims (1)

Claim A method for determining the activity of succinate dehydrogenase in biological objects, including their incubation with a substrate in the presence of tetrazole in a buffer medium followed by centrifugation and photometric measurement of the reaction product, characterized in that, in order to increase the accuracy of the method, the reaction product is further purified by centrifugation for 10 -15 min at 3000 rpm, followed by dissolution of the obtained precipitate in acetone.