CS195756B1 - Method of determination of the efficacy of the glycogenphosphorylasis in the blood - Google Patents

Abstract

1451004 Isophosphorylase detection ARZNEIMITTELWERK DRESDEN VEB 29 Jan 1974 [7 Feb 1973] 04061/74 Heading GIB A process for the determination of the activity of total phosphorylase or of individual isophosphorylases in body fluids, particularly in blood, comprises contacting concentrated body fluid (or a body fluid pre-treated with starch) with a glycogen-containing substrate for phosphorylases, the enzyme activity being determined by determining the inorganic phosphates or amylose-like side chains of glycogen formed. The process may comprise mixing a concentrated body fluid with a solution containing glycylglycine, mercaptoethanol and ethylene diamine tetra acetic acid in a 1:1 ratio or body fluid pre-treated on a starch column by adsorption and washing with a solution of sodium fluoride, sodium #-glycerophosphate and mercaptoethanol (pH 6À1, 4‹C), followed by elution of the phosphorylase at 30 C and pH 6À8 with a solution of sodium #-glycerophosphate, mercaptoethanol and ethylene diamine-tetra acetic acid, and mixing the resulting fluid in a 5:3 ratio with a glycogen-containing solution (the substrate), then adding 2 parts of a solution containing glucose-1-phosphate and adenosine monophosphate, the mixture being incubated for 60 minutes, thereafter denaturing with trichloroacetic acid, the phosphate content being subsequently determined. An alternative process comprises pretreating a body fluid on a starch column as above, diluting the eluate with 1À2M saccharose in a 2:1 ratio, applying the diluted eluate to an acrylamide separation gel containing 0À1% glycogen (the substrate) and buffered with a solution containing tris-(hydroxymethyl)-amino methane, ethylenediamine-tetra acetic acid and borric acid, separating by disc electro-phoreses and subsequently, after incubation in an aqueous solution containing glucose-1-phosphate, adenosine monophosphate, sodium #-glycerophosphate, ethylene diaminetetra acetic acid, mercaptoethanol and sodium fluoride, the formation of the amylose-like side chains on the glycogen being made visible by iodine-potassium iodide coloration.

Description

The invention relates to a method for determining the efficacy of isosophosphorylase in blood. These are in particular glycogen phosphorylase.

It is an object of the present invention to provide a method for determining glycogen phosphorylase so that both total phosphorylase and certain isophosphorylase can be determined.

Methods are known for quantitatively determining glycogen phosphorylase, for example, the efficacy of muscle phosphorylase by measuring glucose-1-phosphate or orthophosphate in tissue homogenates.

1. Pfannemilller, B .: In: Handbuch der Physiologisch- und Pethologisch-Chemlschen Analyze (Hrg. Hoppe-Beyler u. Thierfelder), Springer Verlag, Berlin-Getttingen-Heidelberg, Vol. VI / B, pp. 225-253 ( 1966),

2. Will, H. urid, R.G. Krause: Acta biol. copper. german. 28, 919-933 (1972).

These methods consist in detecting various reaction products of an enzyme-catalyzed reaction. These are the release of inorganic phosphate, the extension of the glycosyl chains of the glycogen molecule, or the formation of glucose-1-phosphate. A number of methods are known for detecting these products which differ only insignificantly in their sensitivity. The known methods are more or less

It is suitable for the quantitative determination of enzyme activity, regardless of the qualitative differences between the various phosphorylase isoenzymes. Determination of efficacy in blood serum is associated with the disadvantage that:. only a very small amount of phosphorylase is present with a large excess of other proteins.

Determining the efficacy of phosphorylase, in particular the specific serum phosphorylase in the blood serum of humans, is of great diagnostic importance since, after damage to muscle cells, for example in a heart attack, this enzyme appears in blood serum [Krause, EG, H. Behhm, H. Will and A. Wollenberger: Dtsch. Ges. Res. 27, 903 (1972)). However, the method for determining the efficacy of blood glycophosphorylase is not yet known.

SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide a method for determining the efficacy of glycogen phosphorylase in blood serum.

The present invention provides a method for determining the efficacy of glycogen phosphorylase in blood serum, characterized in that, after pretreatment, the blood serum is mixed with the enzyme substrate and the effector-containing solution and after incubation the enzymatic activity is measured according to the reaction products formed.

Blood serum is pretreated by concentration by membrane filtration in a ratio of 2: 1 to 3: 1 and optionally pretreated with starch. For starch pre-treatment, serum is applied to a starch-filled column at 0-5 ° C. The column is equilibrated in a solution containing 1 to 10 ³ mol / liter sodium fluoride, 0.1 to 10 ⁴ mol / liter sodium β-glycerophosphate and 0.1 to 10 ⁴ mol / liter mercaptoethanol, pH 5, 5 to 7.0. Then the solution is passed through the column and then eluted phosphorylase at a temperature of 10-35 ° C and pH 6.0 to 7.5 second solution comprises 0.1 to 10 ^"4 mole / liter of sodium β-glycerophosphate, 1 to 10 ~ ⁴ mol / l of ethylenediaminetetraacetic acid and 0.1 to 10 mole ^_4 / l mercaptoethanol.

The serum, concentrated by membrane filtration, is mixed with a solution containing 1 to 10 ³ mol / liter glycylglycine, 0.1 to 10 ⁴ mol / liter mercaptoethanol and 1 to 10 ⁴ mol / liter ethylenediaminetetraacetic acid at pH 6 to 7.5.

This mixture or starch column eluate is added to a solution containing a 5: 3: 2 solution of glycogen, glucose-1-phosphate and adenosine monophosphate, and the resulting mixture is incubated at 37 ° C for 60 minutes. The mixture is then denatured with trichloroacetic acid and the phosphate content is determined optically.

Glycogen phosphorylase is divided into 3 isophosphorylase by electrophoresis to determine the form typical of the cardiac muscle. After incubation in the presence of the substrate, the iso-phosphorylase is stained according to the reaction product with a mixture of iodine and potassium iodide.

The method according to the invention is suitable as an examination method for the diagnosis of heart attack, which has not been possible with the prior art methods. No phosphorylase activity can be measured in the blood serum of healthy persons. In this regard, any determination of serum phosphorylase in principle causes a suspected heart attack.

The following examples describe the determination of phosphorylase as well as the composition of the reagent used to carry out the process of the invention.

He did

The phosphorylase reagent has the following composition:

solution 1

0.1 m sodium fluoride

Sodium J-glycerophosphate 0.001 m, pH 6.1 0.015 m mercaptoethanol solution 2

0.04 m sodium s-glycerophosphate, pH 6.8 0.001 m ethylenediaminetetraacetic acid 0.015 m mercaptoethanol solution 3

0.04 m glycylglycine at pH 6.8

0,01 m mercaptoethanol 0,008 m ethylenediaminetetraacetic acid solution 4

3.3 o / o glycogen solution 5

0.083 m glucose-1-phosphate

0.005 m adenosine monophosphate

The serum is concentrated by membrane filtration in a ratio of 2: 1 to 3: 1, optionally pretreated with insoluble starch. In this case, 3-4 ml of serum are applied at 4 ° C to a 2X1 centimeter starch column, equilibrium, in solution. Then, the column was washed with 3 X 3 mL of solution 1 and eluted with 2 mL of solution 2 at 30 ° C with phosphorylase.

part of the concentrated serum is mixed with 1 part of the solution 3 and 5 parts of the mixture or eluate are added 3 parts of solution 4 and 2 parts of solution 5. After 60 minutes incubation at 37 ° C the solution is denatured with trichloroacetic acid and by known method [Wie-Melherbe , H.: Handbuch der Physiologisch- und Pathologisch-Hemischen Analyze (Hrg. Hoppe-Seyler, Thierfelder), Springer Verlag, Berlin-Götgen-Heidelberg, Vol. III / 1, pp. 457-538 (1955)], the reaction product phosphate is analyzed.

Example 1 a d 2

Enzyme activity is now determined in the presence of specific isophosphorylase inhibitors, so that, unlike the assay of Example 1, the proportion of cardiac muscle-specific phosphorylase is determined.

The reagent for separating the isophosphorylase and determining the enzymatic activity has the following composition:

solution 1

0.042 m tris (hydroxymethyl) aminoethane 0.001 m ethylenediaminetetraacetic acid 0.049 m boric acid pH 8.2 solution 2

0.03 m glucose-1-phosphate

0.002 m adenosine monophosphate

0.002 m @ 2 - sodium glycerophosphate, pH 6.8

0.002 m ethylenediaminotetraacetic acid

0.001 m of mercaptoethanol

0.1 m sodium fluoride solution 3

0.01 m iodine

0.014 m potassium iodide

An aliquot of the eluate from the phosphorylase-containing screw column is diluted with a 2: 1 solution of 2: 1 sucrose and the iso phosphorylase is separated by disk electrophoresis. A gel consisting of 5% acrylamide and 0.2% N, N‘-methylbisacrylamide containing 0.1% glycogen and solution 1 as a buffer, which is also an electrode buffer, is used for separation. buffer. After incubation of this gel in solution 2, solution 3 is applied to stain the amylose side chains resulting from glycogen polymerization.

Claims (4)

A method for determining the efficacy of glycogen phosphoirylase in blood, characterized in that it is blood. After pretreatment, the serum is mixed with the substrate for the enzyme and the solution containing the effector and after incubation the enzymatic activity is measured according to the reaction products formed. 2. The method of claim 1, wherein:. the blood serum is concentrated by membrane filtration in a ratio of 2: 1 to 3: 1 and optionally pretreated with insoluble starch. 3. Method according to claim 2, characterized by: 2. The method of claim 1, wherein, in the starch pretreatment step, blood serum is applied at 4 [deg.] C. to an equilibrated starch column in the solution it contains. 1 to 10-3 moles / liter of sodium chloride, 0.1 to 10-4 moles / liter of sodium glycerophosphate and 0.1 to 10-4 moles / liter of mercaptoethanol at pH 6.1, then the column is washed with the same solution and finally eluted at 30 ° C and pH 6.8 with a solution containing 0.1 to 10-4 moles / liter of sodium β-glycerophosphate, 1 to 10-4 moles / liter of ethylenediaminetetraacetic acid and 0.1 to 10 moles / liter Mole / liter of mercaptoethanol to obtain phosphorylase. 4. A method according to claims 1 and 2, characterized in that the concentrated serum is mixed with a solution containing 1 to 10 -3 moles / liter of glycylglycine, 0.1 to 10 -4 moles / liter of mercaptoethanol and 1 to 10 moles / liter of glycylglycine. 4 mol / l of ethylenediaminetetraacetic acid at pH 6.8, the obtained mixture or the starch column eluate was added to a solution of glycogen and glucose solution · ^{1-1-phosphate} and ^'1 adenosinmono phosphate in a ratio of 5: 3: 2, the resulting mixture was Incubate for 60 minutes at 37 ° C, denature with trichloroacetic acid and determine the phosphate content optically after reduction of the phosphomolybdate complex.