CA1148455A

CA1148455A - SUBSTRATE FOR THE DETECTION OF THE .delta.-GLUTAMYLTRANSPEPTIDASE

Info

Publication number: CA1148455A
Application number: CA000351131A
Authority: CA
Inventors: Helmut Heber; Helmut Kohl
Original assignee: Individual
Current assignee: Siemens Healthcare Diagnostics GmbH Germany
Priority date: 1979-05-04
Filing date: 1980-05-02
Publication date: 1983-06-21
Also published as: AU5803280A; DE3067415D1; EP0018628A1; AU544691B2; ES8102358A1; ES490961A0; DE2917999A1; ATE7049T1; JPS55153600A; EP0018628B1

Abstract

SUBSTRATE FOR THE DETECTION OF THE ?-GLUTAMYLTRANSPEPTIDASE

Abstract of the disclosure:
A substrate suitable for the detection of the ?-glu-tamyltranspeptidase in aqueous systems is described. It can be used for the diagnosis of liver diseases and for assessing the course of such diseases.

Description

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2 ~ Jk~5 ~ HOE 79/B 005 The present invention relates to a substrate for thedetection of the ~glutamyltranspeptidase in aqueous systems, in particular in biological fluids, especially in blood se-rum. It is used mainly for the diagnosis of liver diseases and for assessing the course of these diseases.
The ~-glutamyltranspeptidase ( ~-GT)-activity may be determined according to the following known reaction scheme:
2~-glutamyl-p-nitranilide + glycylglycin ~;;;=~ p-nitra-niline + glutamylglycylglycin.
10 The release of the yellow-colored p-nitraniline can be ob-served optically. The change in optical density relates to the 2~-GT activity in the test sample.
This way of detecting ~-GT enzyme activity is rather simple. It has, however, the disadvantage that the substrate 15 ~-glutamyl-p-nitranilide has a very low solubility in aqueous solutions at pH values that have to be maintained in the test tube during the determination of the ~-GT. This is known inter alia from the patent applications DE-OS
2,042,829 published March 16, 1972 and DE-OS 2,259,512 published 20 June 27, 1974. Several trials have been made to solve this problem. For exaTrple, the substrate has been dissolved in dilute hydrochloric acid or in organic solvents in a high concentration and the resulting solution has been added in low quantities to the mixtures to be analyzed, thus preventing a significan. displacement of the pH value in the 25 test tube. However, this method is rather complicated and the substrate is not very stable in highly acidic, aqueous media. Therefore solutions of this type can be used for measurement only within a short period of time.
A further known process comprises dissolving the ~glu-30 tamyl-p-nitranilide at a temperature of 50 to 60C and cooling the resulting solution subsequently to room tempe-rature. This method, too, involves the danger of a sponta-neous hydrolysis of the chromophoric group.
Moreover this procedure is time-consuming and even 35 fails sometimes because of recrystallization of the sub-strate. These methods are unsatisfactory for a routine app-lication of the test in clinical laboratories. It was the-refore a task to surmount the above~mentioned disadvantages .
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- 3 - HOE 79/B 005 and to provide a 3 glutamyl-p-nitranilide substrate 'chat is sufficiently soluble in the buffer solution at a temperatu-re of approximately 25C, which is necessary for carrying out the determination.
It has now been found surprisingly according to the invention that dry Y~-glutamyl p-nitranilide, preferably in lyophilized form, dissolves instantaneously in the test buf-fer at 20 to 25C, if the substrate is present in protoniz-ed form, that means, as a salt. Consequently, an acid has to be added to the ~-glutamyl-p-nitranilide prior to drying, in a stoichiometrical ratio or in an excess, relative to the base equivalents.
The subject of the present invention is therefore a dry and well soluble preparation of the ~-glutamyl-p-nitranilide ~5 substrate suitable for the detection of the t'-GT, containing at least one equivalent of an acid per base equivalent of the substrate.
It has moreover been found that the reagent, u?on re-dissolution, is distinguished by a higher color stability, if a quaternary polycation is present in the substrate so-lution. The dry preparation therefore comprises, if appro priate, a high molecular weight detergent which is a fur-ther feature of the subject of the present invention.
In the narrow sense it is an object of the present in-ven~ion to use the ammonium salt of the ~-glutamyl-p-ni-tranilide as a substrate optionally with an excess of an acid for the detection of ~-glutamyltranspeptidase.
Suitable acids for this purpose are nearly all organic or inorganic acids, that are able of forming an ammonium salt with the ~-glutamyl-p-nitranilide. Examples of these acids are : sulfuric acid, phosphoric acids, all hydrohalic acids, oxyhydrohalic acids, acetic acid and trifluoroacetic acid. Combinations of an i.norganic and an organic acid such as di-, tri- and tetra-carboxylic acids or hydroxycarboxylic acids, for example malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, maleic acid, fumaric acid, malic acid, tartaric acid, citric acld, ascorbic acid, glu-curonic acid, have pro~ed advantageous. A combination of `

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~, _ 1~ _ HOE 79/B_005 an inorganic acid, for example hydrochloric acid and an or-ganic acid, for example tartaric acid, is used preferably.
A ratio of about 0.9 equivalent of the inorganic acid to about 0.1 e~uivalent of the organic acid, relative to the base equivalent of the nitranilide, is particularly suitable.
Any chemical methods~suitable for the preparation of an ammonium salt from the primary amine may be used for preparing the substrate. An excess of acid is not detrimen-tal as far as the substrate can be lyophilized conveniently and the redissolved substrate does not exceed the bu~fer capacity of the test buffer. According to a very simple method of preparing the substrate, an aqueous solution of the ~-glutamyl-p-nitranilide is used as starting product.
Subsequently the stoichiometrical ratio of the anilide to an acid is determined, the appropriate acid is added to the solution so as to obtain the desired ratio, optionally in an excess of about 20 ~ and the resulting product is dried, preferably by lyophilization.
A further simple way to obtain a preparation according to the invention consists in acidifying the aqueous solution of the nitranilide with the appropriate acid prior to dry-ing.
The essential advantage of the dry product according to the invention is to be seen in the fact that no signifi-cant hydrolysis occurs prior to its application in the testand that the test is not impaired by high blank values.
As mentioned above, the substrate properties are im-proved by adding to the substrate solution a quaternary polycation in a concentration range between 0.1 and 100 mg/l. These findings are surprising, for it is known that conventional cationic or anionic detergents inhibit or reduce the ~-glutamyltranspeptidase activity. However, polycations derived from polyvinylpyrrolidone have no detectable influence on the ~T-activity. Ethoxylated alkylphenols or ethoxylated phenols as well as tetrafluor-ethylene polymers having branched perfluoro groups act in analogous manner. These polymers, used alone or in combina~
tions, have a retarding effect on the recrystalization of .
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- 5 - HOE _~/P, 005 the ~ ~lutamyl-p l~itranllide at room temperature.
For the convenience of the user the salt of the ~-glu-tamyl-p-nitranilide, to which the polymer has been added optionally, may be present in the test kit in solid form, for example as a dry powder or in tableted form.
The stability o~ ~le ~xduct ~lmo~e~verbe:~,proved by add-ing compounds to the lyophili~ation solution such as gelatin or gelatin hydrolysates, sugar alcohols or carbohydrates, buffer components, preservation agents and/or compounds promoting dissolution. In a preferred embodiment mannitol is used in a concentration range between 1 and 50 mg/ml.
The glycylglycin participating in the reaction of the transpeptidase may be likewise present in the dry prepara-tion. To perform the ~GT-determination, the dry substrate mixture has simply to be dissolved in water.
The following example illustrates the invention:
Example The substrate according to the invention is prepared in the following manner:
a) Substrate components:
1~ 1.17 g of ~-glutamyl-p-nitranilide (1.176 g/1000 ml = 4.4 mM), 2) 8.7 g of glycylglycin, 3) 16 g of mannitol,

4) 10 g of potassium-sodium tartrate . 4 H20,

5) 40 g of quaternary polyamine,

6) 2 N HCl

7~ distilled water.
Gomponents 1 to 5 are dissolved in about 600 to 700 ml of distilled water and heated to about 40C. Component 6 is added until a pH value between 3 and 4 is reached.
After complete dissolution of all substrate components the solution is cooled to about 20C and the pH is adjusted ` to 2 to 3 with 2 N HCl. Distilled water (7) is added up to a volume of 1 liter.
Suitably the ~ glutamyltranspepti.dase is determined by using 2 ml of this substrate solution. Therefore the~ solu-tion is dispensed to vial5 in portions of 2 ml, which are . -., , lyophilized. To perform the test the lyophilized product is redissolved with the buffer solution b).
b) buffer solution ...... _

8) 24 g of tris-(hydroxymethyl) aminomethane and

9) 5 g of sodium azide are dissolved in about 800 ml of distilled water, the pH is adjusted to 9.2 - 9.5 with 2 N HCl and distilled water is ad-ded up to a volume of 1.0 liter. A pH value of about 8.2 will result, if 2 ml of buffer solution of the above compo-sition are added to the lyophilized substrate mixture ac-cording to a).

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Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE PROPERTY
OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A dry preparatin of ?-glutamyl-p-nitranilide for use as a substrate for the detection of ?-glutamyl-transpeptidase, con-taining an acid in an at least molar ratio, relative to the base equivalent of the nitranilide.

2. A dry preparation as claimed in claim 1 which also con-tains a detergent.

3. A dry preparation as claimed in claim 2 in which the detergent is a quaternary polycation based on polyvinylpyrrolidine or an ethoxylated alkylphenol, an ethoxylated phenol or a tetra-fluoroethylene polymer having branched perfluoro alkyl groups.

4. A dry preparation as claimed in claim 2 or claim 3 in which the detergent is present in a concentration ranging from 0.1 to 100 mg/ml of test solution.

5. A dry preparation as claimed in claim 1, claim 2 or claim 3 in which the acid is selected from the group of organic or inorganic acids capable of forming an ammonium salt with ?-glutamyl-p-nitranilide.