Note: Descriptions are shown in the official language in which they were submitted.
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~ 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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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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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
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lyophilized. To perform the test the lyophilized product is
redissolved with the buffer solution b).
b) buffer solution
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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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