RU2121690C1 - Method of determining plasminogen and plasmin inhibitors levels in blood plasma - Google Patents

Abstract

FIELD: analytical methods in medicine. SUBSTANCE: chromogenic substrate with chemical formula For-Ala-Phe-Lys-pNA.HBr is used in analysis. After activation with streptokinase, above-named proteins cleave peptide substrate to form p-nitroaniline. EFFECT: extended diagnostic possibilities. 5 ex

Description

 The invention relates to medicine and can be used in the diagnosis and control of the treatment of cardiovascular, surgical, hematological and other diseases in which the protein content of the fibrinolysis system changes in the blood. The level of these proteins characterizes the severity of the disease, so the convenient way to determine them in the blood plasma of patients is relevant. For determination using biochemical methods, based, in particular, on the reaction of interaction of these proteins with chromogenic peptide substrates, as a result of which the substrate is hydrolyzed and a colored substance forms. A number of synthetic peptide substrates are known that are suitable for determining the active one of the components of the fibrinolysis system — the plasmin enzyme, based on which the content of plasminogen and plasmin inhibitors is determined [1,2]. As the closest analogue, we consider a method for determining this activity using the tripeptide H-D-Val-Leu-Lys-pNa • 2HCl. [3] This substrate (manufactured by Kabi, Sweden) is the most widely used and is used to determine the plasminogen content in blood plasma after its activation in plasmin using streptokinase.

The determination is carried out in the following way: 1 ml of streptokinase is added to 0.2 ml of the studied blood plasma, incubated for 5 minutes at 37 ^° C, 100 μl of a 3 mM substrate solution is added, and either the optical density gain is determined spectrophotometrically in 1 minute at 405 nm, or after a two-minute incubation of the sample at 37 ^o C the reaction is stopped by adding 0.5 ml of 20% acetic acid, after which the optical density of the solution is determined at 405 nm. The plasminogen content is expressed in% and determined by a calibration curve constructed by diluting standard plasma. The specified substrate is also used to determine plasma plasmin inhibitors.

 The synthesis of the peptide H-D-Val-Leu-Lys-pNa • 2HCl is carried out by classical peptide chemistry. It is a laborious multi-stage process, including (based on free amino acids, depending on the scheme) 10-12 stages [4].

 Task. To develop a method for determining the activity of plasminogen and plasmin inhibitors using a chromogenic substrate readily available for synthesis. The problem is solved by using the substance For-Ala-Phe-Lys-pNa • HBr as the chromogenic peptide substrate, where the For-formyl group, Ala-alanine, Phe-phenylalanine, Lys-lysine, pNa-p-nitroaniline, HBr hydrobromic acid "

 The method for preparing For-Ala-Phe-Lys-pNa • HBr is much simpler and involves only 7 steps, two of which are enzymatic reactions that proceed in almost quantitative yield [5]. The same substrate can be used to determine the number of plasma plasmin inhibitors.

The method in General
The method for determining plasminogen repeats the existing method and differs only in the use of a substance corresponding to the chemical formula For-Ala-Phe-Lys-pNa • HBr as a plasmin substrate.

To ml of the studied blood plasma, add 1 ml of streptokinase, incubate for 5 min at 37 ^o C, add 100 μl of 3 mm solution of the substrate For-Ala-Phe-Lys-pNa • HBr, incubate for 2 min at 37 ^o C, stop the reaction by adding 0.5 ml of 20% acetic acid and determine the optical density of the solution at 405 nm. The plasminogen content is expressed as a percentage and is determined by a calibration curve constructed by diluting standard plasma.

The method is illustrated by the following examples.
Example 1, (control for example 2). Determination of plasminogen content in the plasma pool of 10 healthy donors using a reagent kit from Kabi, (Sweden).

1 ml of streptokinase is added to 0.2 ml of the studied blood plasma, incubated for 5 minutes at 37 ^° C, 100 μl of 3 mM D-Val-Leu-Lys-pNa • HCl substrate solution is added, incubated for 2 minutes at 37 ^° C, the reaction is stopped by adding 0.5 ml of 20% acetic acid. The optical density of the solution at 405 nm was 0.09 ΔA / min and was taken as 100%.

 Example 2. Determination of plasminogen content in blood plasma of 10 healthy men using our proposed substrate For-Ala-Phe-Lys-pNa • HBr.

 The determination is carried out as in Example 1, but a For-Ala-Phe-Lys-pNa • HBr substrate solution is used. The optical density of the solution at 405 nm was 0.089 ΔA / min, which corresponds to 98.9% plasminogen.

 Examples 3. Determination of plasminogen content in the patient’s blood, in the history of myocardial infarction, accompanied by thrombolytic therapy using a For-Ala-Phe-Lys-pNa • HBr substrate.

 The determination is carried out as in Example 1, but a For-Ala-Phe-Lys-pNa • HBr substrate solution is used. The optical density of the solution at 405 nm was 0.046 ΔA / min, which corresponds to 51.1% plasminogen.

 Example 4. (Control for example 5). Determination of the number of plasmin inhibitors in standard plasma using a reagent kit company Kabi, (Sweden).

 The determination is carried out as in example 1, only the first incubation is carried out for 40 minutes. The optical density of the solution at 405 nm was 0.12 ΔA / min, which is taken as 100%.

 Example 5. Determination of the number of plasmin inhibitors in standard plasma using our proposed substrate For-Ala-Phe-Lys-pNa • HBr.

 The determination is carried out as in example 4, but For-Ala-Phe-Lys-pNa • HBr is used as a substrate. The optical density of the solution at 405 nm was 0.122 ΔA / min, which corresponds to 101.7% plasminogen.

 Thus, from examples 1 and 2 (for plasminogen) and examples 4 and 5 (for plasmin inhibitors) it follows that the claimed method for determining these enzymes in sensitivity is not inferior to the method described in the closest analogue. And example 3 indicates that the method using the For-Ala-Phe-Lys-pNa • HBr peptide is quite sensitive and allows you to reliably determine the amount of plasminogen at lower concentrations in the blood of patients.

Literature
1. Gabriella Cs. Szabo, Marianna Pozsgay, P. Elodi, Trombosis Research, 20, 1980, 199-206.

 2. Yoshio Okada, Yuko Tsuda, Noaki Teno, Keiko Wanaka, Kuniko Sasaki, Akiko Hijikata, Shosuke Okamoto, Int. J. Peptide Protein Res., 27, 1986, 79-85.

 3. EP 264753.

 4. Sushil K.Sharma, Francis J. Castelino, Trombosis Research, 57, 1990, 127-138.

 5. Voyushina T. L., Terent'eva E. Yu., Stepanov V.M., Biomed. Biochim. Acta, 50 1991, 209-212.

Claims (1)

 A method for determining the content of plasminogen and plasmin inhibitors in blood plasma by adding chromogenic peptide substrates to it, followed by determining the degree of their hydrolysis, characterized in that a substance having the chemical structure of For-Ala-Phe-Lys-pNa.HBr is used as a chromogenic substrate, where For is the formyl group, Ala is alanine, Phe is phenylalanine, Lys is lysine, pNa-p-nitroaniline, HBr is hydrobromic acid.