U.S. patent number 3,773,626 [Application Number 05/170,968] was granted by the patent office on 1973-11-20 for reagent composition and process for determining gamma-glutamyltranspeptidase.
This patent grant is currently assigned to Boehringer Mannheim GmbH. Invention is credited to Hans Ulrich Bergmeyer, Erich Bernt, Wolfgang Gruber, Werner Lippert.
United States Patent |
3,773,626 |
Bernt , et al. |
November 20, 1973 |
REAGENT COMPOSITION AND PROCESS FOR DETERMINING
GAMMA-GLUTAMYLTRANSPEPTIDASE
Abstract
A composition comprising .gamma.-glutamyl-p-nitroaniline,
glucyl-glycine, a buffer, a surface-active agent and
polyvinyl-pyrrolidone is an exceptionally stable reagent for
determining .gamma.-glutamyl-transpeptidase.
Inventors: |
Bernt; Erich (Munich,
DT), Bergmeyer; Hans Ulrich (Tutzing/Upper Bavaria,
DT), Gruber; Wolfgang (Garatshausen, DT),
Lippert; Werner (Seeshaupt, DT) |
Assignee: |
Boehringer Mannheim GmbH
(Mannheim, DT)
|
Family
ID: |
5781014 |
Appl.
No.: |
05/170,968 |
Filed: |
August 11, 1971 |
Foreign Application Priority Data
|
|
|
|
|
Aug 28, 1970 [DT] |
|
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P 20 42 829.9 |
|
Current U.S.
Class: |
435/24 |
Current CPC
Class: |
C12Q
1/48 (20130101) |
Current International
Class: |
C12Q
1/48 (20060101); G01n 031/14 () |
Field of
Search: |
;195/13.5R,99 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Shapiro; Lionel M.
Assistant Examiner: Hensley; Max D.
Claims
What is claimed is:
1. Reagent composition for the determination of
.gamma.-glutamyl-transpeptidase which composition comprises
.gamma.-glutamyl-p-nitroaniline, glycyl-glycine, a tris buffer, a
hydroxypolyethoxyalkane surface-active agent and
polyvinylpyrrolidone.
2. Composition as claimed in claim 1 additionally containing an
azide.
3. Composition as claimed in claim 2 wherein said azide is an
alkali metal azide.
4. Composition as claimed in claim 1 wherein the surface-active
agent is an hydroxypolyethoxydodecane.
5. Composition as claimed in claim 1 wherein the buffer is a tris
-(hydroxyalkylamine).
6. Composition as claimed in claim 1 comprising:
A. 0.05 to 0.2M tris buffer, pH 7.8 to 8.2,
20 to 100 grams polyvinyl-pyrrolidone,
0.75 to 5 grams of a surface-active agent,
0.5 to 4 grams of an azide, and
B. a lyophilized mixture of,
1 to 1.25 grams .gamma.-glutamyl-p-nitroaniline and
5 to 6.2 grams glycyl-glycine
per liter of buffer solution.
7. Composition as claimed in claim 1 comprising:
A. 0.1 to 0.2M tris buffer, pH 8.0,
30 grams polyvinyl-pyrrolidone,
1 gram hydroxypolyethoxyalkane,
1 gram sodium azide, and
B. 1.13 grams .gamma.-glutamyl-p-nitroaniline and
5.6 grams glycyl-glycine,
per liter of buffer solution.
8. Composition as claimed in claim 6 wherein the azide is sodium
azide.
9. Method for determining .gamma.-glutamyl-transpeptidase which
comprises contacting a test sample suspected of containing
.gamma.-glutamyl-transpeptidase with a diagnostic reagent
composition comprising .gamma.-glutamyl-p-nitroaniline,
glycyl-glycine, a tris buffer, a hydroxypolyethoxyalkane
surface-agent and polyvinyl-pyrrolidone, and following the
liberation of p-nitroaniline as a measure of any activity of
.gamma.-glutamyl-transpeptidase present in the test sample.
10. Method as claimed in claim 9 which comprises forming a reagent
composition by contacting, per liter of solution,
A. 0.05 to 0.2M tris buffer, pH 7.8 to 8.2,
20 to 100 grams polyvinyl-pyrrolidone,
0.75 to 5 grams of a surface-active agent,
0.5 to 4 grams of an azide
in the form of an aqueous solution with
B. a lyophilized mixture of buffer
1to 1.25 grams .gamma.-glutamyl-p-nitroaniline and
5 to 6.2 grams glycyl-glycine;
per liter of solution, and then contacting a test sample with the
resulting mixture and observing the liberation of yellow-colored
.gamma.-glutamyl-p-nitroaniline.
11. Method as claimed in claim 10 which comprises forming a reagent
composition by contacting, per liter of solution,
A. 0.1 to 0.2M tris buffer, ph 8.0
30 grams polyvinyl-pyrrolidone,
1 gram hydroxypolyethoxyalkane,
1 gram sodium azide, and
in the form of an aqueous solution with
B. 1.13 grams .gamma.-glutamyl-p-nitroaniline and
5.6 grams glycyl-glycine;
per liter of solution, and then contacting a test sample with the
resulting mixture and observing the liberation of yellow-colored
.gamma.-glutamyl-p-nitroaniline.
Description
The present invention is concerned with a reagent composition and a
process for the determination of .gamma.-glutamyl-transpeptidase
with the use of .gamma.-glutamyl-p-nitroaniline and glycyl-glycine
as the substrate, in the presence of a suitable buffer.
To an increasing extent, the determination of
.gamma.-glutamyl-transpeptidase (.gamma.-GT) is carried out in the
clinical chemical laboratory for the diagnosis of liver diseases.
This determination takes place, via a known reaction, according to
the equation:
.gamma.-glutamyl-p-nitroaniline + glycyl-glycine .revreaction.
p-nitroaniline + glutamyl-(glycyl-glycine)
The velocity of liberation of the yellow-colored p-nitroaniline can
be followed optically and is a measure of the activity of
.gamma.-GT present.
For a routine use of this process, the instability and poor
solubility of the substrate .gamma.-glutamyl-p-nitroaniline is a
severe disadvantage. This compound can only be brought into
solution with difficulty and solutions thereof are only stable for
a very short time. Therefore, it was previously necessary to
prepare a small amount of solution sufficient only for relatively
few determinations, which had to be carried out within a short
period of time. It was not possible to store the reagent
solution.
The present invention substantially overcomes this drawback of the
prior art compositions by providing a reagent which does not
exhibit the above described instability.
The present invention is based upon the surprising discovery that
the use of polyvinyl-pyrrolidone, together with a surface-active
agent, substantially and decisively improves the solubility
properties and the stability of
.gamma.-glutamyl-p-nitroaniline.
Thus, according to the present invention, there is provided a
reagent composition for the determination of
.gamma.-glutamyl-transpeptidase which comprises
.gamma.-glutamyl-p-nitroaniline, glycyl-glycine and a buffer,
together with polyvinyl-pyrrolidone and a surface-active agent.
The reagent according to the present invention preferably also
contains an azide, especially an alkali metal azide, for example
sodium azide.
As surface-active agent, the reagent according to the present
invention can contain any such agent which does not exert an
inhibiting or inactivating influence on the
.gamma.-glutamyl-transpeptidase. The hydroxypolyethoxyalkanes,
especially hydroxypolyethoxydodecane, have proved to be especially
useful.
The agent according to the present invention preferably contains
the .gamma.-glutamyl-p-nitroaniline in lyophilized form, optionally
together with the other components of the reagent in solid form.
More preferably, however, the reagent according to the present
invention consists of two mixtures, one of which contains
.gamma.-glutamyl-p-nitroaniline and glycyl-glycine in lyophilized
form and the other consists of an aqueous solution, which contains
the buffer, polyvinyl-pyrrolidone, the surface-active agent and
optionally an azide. Both of the mixtures are stable for a long
time and, by simple mixing, with gentle warming, permit the rapid
preparation of a relatively stable solution, which remains usable
for a comparatively long time.
In a preferred specific embodiment, the aqueous solution
comprises:
0.05 to 0.2 M tris (tris-(hydroxyalkylamine)) buffer,
pH 7.8 to 8.2,
20 to 100 grams/liter polyvinyl-pyrrolidone,
0.75 to 5 grams/liter surface-active agent
and optionally
0.5 to 4 grams/liter of an azide.
In an especially preferred specific embodiment, the aqueous
solution comprises:
0.1 to 0.2 M tris buffer, pH 8.0,
30 grams/liter polyvinyl-pyrrolidone,
1 gram/liter hydroxypolyethoxyalkane and
1 gram/liter sodium azide.
The second component of the reagent according to the present
invention, i.e., the solid mixture of
.gamma.-glutamyl-p-nitroaniline and glycyl-glycine, preferably
comprises:
1.0 to 1.25 grams, preferably 1.13 grams,
.gamma.-glutamyl-p-nitroaniline and
5.0 to 6.2 grams, preferably 5.6 grams, glycyl-glycine, per liter
of liquid reagent component.
The reagent according to the present invention permits the
preparation of solutions ready to use for the determination of
.gamma.-glutamyl-transpeptidase, which solutions remain usable for
a comparatively long period of time, in contradistinction to the
previously known solutions of .gamma.-glutamyl-p-nitroaniline
which, on the one hand, were difficult to prepare and from which,
on the other hand, this compound crystallized out again a short
time after preparation of the solution. Thus, for the first time,
the reagent according to the present invention provides the
possibility of carrying out the routine determination of
.gamma.-glutamyl-transpeptidase, for example, in automatic analysis
devices.
The following Examples are given for the purpose of illustrating
the present invention and should not be construed as unduly
limitative thereof.
PREPARATION OF THE REAGENT
a. Lyophilized substrate
6.85 grams .gamma.-glutamyl-p-nitroaniline and 33.8 grams
glycyl-glycine are dissolved, with warming to 70.degree. to
80.degree.C., in 6 liters of double distilled water. The warm
solution was filled in 3.0 ml. portions into 200 small bottles,
frozen therein and lyophilized in the usual way. The preparations
so obtained were stable at ambient temperature for at least 12
months.
b. Preparation of the buffer solution
193.5 grams tris-(hydroxymethylamine)-methane, 240 grams
polyvinyl-pyrrolidone, 8 grams polyethoxydodecane (commercially
available under the trade name "Thesit") and 8 grams sodium azide
were dissolved in 8 liters of double distilled water. The solution
is adjusted to pH 8.0 with dilute hydrochloric acid. The solution
thus obtained was of unlimited stability at ambient
temperature.
c. Activity determination of .gamma.-glutamyl-transpeptidase
The lyophilized prepared was described under (a) above was
suspended in 3 ml. of the solution prepared according to (b) above
and, by brief warming to about 30.degree. to 40.degree.C., was
completely brought into solution. The solution was then allowed to
cool to ambient temperature. To the reagent thus obtained, which
was ready to use, were added 0.2 ml. of a test solution, for
example serum, and, by repeated measurement of the extinction, the
liberation of the yellow-colored p-nitroaniline was followed, the
velocity of which was a measure of the
.gamma.-glutamyl-transpeptidase activity.
d. Influence of polyvinyl-pyrrolidone (PVP) and
hydroxypolyethoxydodecane (HPD) on the solubility of
.gamma.-glutamyl-p-nitroaniline
11.4 grams .gamma.-glutamyl-p-nitroaniline and 56.4 grams
glycyl-glycine were dissolved, with warming to 70.degree. to
80.degree.C., in 10 liters double distilled water. The warm
solution was filled, in 3.0 ml. portions, into small bottles,
frozen in an lyophilized.
Solubility experiments were then carried out with the lyophilizates
thus obtained. The lyophilizates were dissolved at 50.degree. to
70.degree.C. in 0.2M tris buffer which contained (a) no additive
and (b) PVP and HPD in a definite concentration ratio, the solution
cooled to 25.degree.C. and the solubilizing properties of PVP and
HPD determined either by measurement of the turbidity (percent
light permeability) or by observation of the crystallization time.
The results obtained are set out in the following Table:
% PVP 0 3 % HPD 0 1 initially 85% 84% after 3 hours 60% 85% after 6
hours 50% 84% after 24 hours 20% 84% after 2 days crystallizes 82%
after 6 days crystallizes 78%
It will be understood that the specification and examples are
illustrative but not limitative of the present invention and that
other embodiments within the spirit and scope of the invention will
suggest themselves to those skilled in the art.
* * * * *