COMPOSE POUR LA DETERMINATION ENZYMATIQUE DU GLUCOSE

COMPOSITION FOR THE ENZYMIC DETERMINATION OF GLUCOSE

Abrégé anglais

COMPOSITION FOR THE ENZYMIC DETERMINATION
OF GLUCOSE . -

ABSTRACT OF THE DISCLOSURE . -

For the determination of glucose in biological
samples such as plasma, urine, deproteinated blood and
the like, a reactant system is disclosed which can be
stored in dry powder condition and diluted immediately
before use without risk of instability. The system
comprises the peroxidase and glucose oxidase enzymes,
a compound having a ?CH group in the alpha position
of an enolic group with double Bonds conjugated to the
enolic double bond and a compound containing at least one
reactive group bound to a structure deriving from
1-phenyl-5-pyrazoline-one. Such a system is easy to
use, reliable and cheap.

Revendications

C L A I M

A composition adapted to the enzymic determination
of glucose, comprising a single reactant system in the
dry state composed by the glucose-oxidase enzyme, the
peroxidase enzyme, a buffer, a compound having a ?CH
group in the alpha position of an enolic group with double
bonds conjugated to the enolic double bond and a compo-
nent with at least a reactive group bound to a structure
deriving from 1-phenyl-5-pyrazoline-one.

Description

~ 10796~5
. . :.
CASE 1016

This invention relates ~o a composition~ more exactly
a set of reactants~ which permits to perform a method~ for :
the enzymic determination of the glucose~ said method being .
.: an integral part of this invention.
~ It is known from the Italial Patent N ';986 838 that : .
it is possible to determine glucose enzymically by employ- .
ing a particular set of reactants which exploits the trans-
formation that glucose undergoes in the presence of glucose- : :
oxidase enzyme and the subsequent reaction steps which ~ake :.
place at the expense of the as-produced substances~ among
which the most important is the oxidation by the as-formed
hydrogen peroxide of an appropriate substrate by the agency
of another enz~me, that is, peroxidase.
Apart from the enzymic compositions and the possible ~ :
use of chromogenic substances, the reactant set the subject
of the above cited patent is essentially comprised of a ~ .
component which contains in its molecule a compound having .;
a ~ CH group in the alpha position of an enolic group with
conjugated double bonds, and a second component having at
least a reactive group bound to a structure which derives :
from l-phenyl-5-pyrazolinone.
According to the patent aforementioned, the reactant :`
system is thus comprised of phenol or an analogous compound
and 4-aminoantipyrin or an analogous compound. Said react-
ants system can be prepared as a ready-for-use solution, or,
as an alternative, phenol and 4-amino-antipyrin can be :
present in discrete solutions to be admixed and diluted prior
to use. As a further alternative, the substances can be
stored in a dry condition for being dissolved and thus for
obtaining the ready-for-use solutions.
. However, the use of phenol as such does not permit
the formulation of a single reactant with the substances
adrixed in the dry condition, to be dissolved, or diluted

2. ~
.

`I 107g6~5


into a single ready-for-use solu~ion~ inasmuch as the
presence of phenol is not such as to warrant stability.
It has now been found, and this ifi the subject-matter
of the present invention, that is the use of phenol as such
is dispensed with and there is used, in its stead in the
formulation of the reactant a compound having a group ~ CH .
in the alpha position of an enolic group with double bonds
conjugated to the enolic double bond, it becomes possible
to formulate a single reactant which comprises all the com-
ponents of interest~ in the dry state~ that is~ in the form
of a powder to be dissolved prior to use without suffering
from the unstability defects enumerated above. .-
More particularly, the present invention relates to
a composition adapted to the enzymic determination of glucose~
which comprises, as a single reactant system in the dry state,
the glucose-oxidase enzyme, the peroxidase enzyme, a buffer,
a compound having a ~CH group in the alpha position of an
enolic group with double bonds conjugated to the enolic
. bond and a component having at least a reactive group to a
structure derived from l-phenyl-5-pyrazoline-one.
As regards the chemi.cal nature and the exact formula-
tion of the two latter substances indicated above, reference : -
is invited to the specification of the above patent with
the obvious indication that, of all the substances reported
there, only those which are solid at room temperature can
be used in the single formulation which is the subject of .
the present application.
It is also desired to emphasize that, in order that
a reactant system may be obtained which is easy to be pre-
pared and is cheap and adequately stable, it is suggested
to select, among all the indicated compounds, those which
simultaneously possess the following properties :
- they must be high-melting and non-deliquescent
- they must be in the form of amorphous or a crystal-
line powder
_ .
. 3

107961~

- they must be adèquately stable
- they must be sufficiently nonreactive in the dry condi-
tion, so that they do no-t display a tendency to de-
naturate the enzymic proteins which are contained in
S the powder mix, but they must be sufficiently react-
ive in the solution ready for the intended use.
It will beeasy, at any rate, for a skilled technician,
to select among the large number of chemical formulae report-
ed in the above patent, those which are most suitable for
effecting the formulation of the single powdery reactant
system, also with a view to its intended practical use, that
is, to the reading method one desires to adopt.
The following examples are reported by way of illu-
stration only of a few composition among the many composi-
tions which are possible, but the invention must not be
construed as being in any wise restricted thereto.
EXAMPLES OF POWDER FORMULATIONS (A SINGLE REACTANT SYSTEM )
A) Varying the buffer and the compound having a ~CH
in the alpha position of an enolic group with double
bonds conjugated to the enolic double bond.
`A~) Phosphate buf~er at pH 7.5, 150 millimols/liter
GOD 18,000 U/liter
POD 1~000 U/liter -
4-aminoantipyrin 0.4 millimol/liter
Na-4-hydroxybenzoate 10 miilimol/liter
A2) Phosphate buffer at pH 7.3~ 150 millimols/liter
GOD 18~000 U/liter
POD 1,000 U/liter
4-aminoantipyrin 0.4 millimol/liter
K-3-hydroxybenzoate 10 millimol/liter
A3) Tris-maleate buffer at pH 7.7 300 millimols/liter
GOD 18~000 U/liter
POD 1~000 U/liter
4-aminoantipyrin o.5 millimol/liter
Na-salicylate 12 millimol




:. ~ . :, ,.. :. .. .

l 1079615

A4) 2-amino-2-methyl-1~3-propanediol buffer
at pH 7.8 200 millimols/liter
GOD 25 ,ono U/liter
¦ POD 2~000 U/liter
¦ 4-aminoantipyrin0.6 millimol/liter
¦ salicylamide 7 millimol/liter
¦ A5) Tris-KH2P04 buffer at pH 7.6 150 millimol/liter
GOD 18,000 U/liter
l POD 1~000 U/liter
~ 4-aminoantipyrin 0.4 millimol/liter
l vanillic acid 12 millimol/liter
; ¦ A6) Phosphate buffer a~ pH 6 150 millimol/liter
I GOD 20~000 U/liter
¦ POD 2,000 U/liter
l 4-aminoantipyrin o.35 millimol/liter
¦ A chromatropic acid (4~5-dihydroxy-2~7-naphthalene-
¦ disulfonic acid) 4 millimol/liter
A7) Acetate-maleate buffer at pH 6 100 millimol/liter
¦ GOD 25~000 U/liter
l POD 2~500 U/liter
I 4-aminoantipyrino.6 millimol/liter
¦ 8-hydroxyquinoline
citrate 15 millimol/liter
A8) Phosphate buffer at p~ ~.7 100 millimol/liter
GOD 18,000 U/liter
POD 500 U/liter
4-aminoantipyrin 0.35 millimol/liter
barbituric acid 4 millimol/liter
; B) Varying the 4-aminoantipyrin
Bl) Phosphate buffer at -
pH 7.5 150 millimol/liter
GOD 18~000 U/liter
POD 1~000 U/liter
The sodium salt of Sulfamipyrin (2,3-dimethyl-1-
-phenyl-5-pyrazolone-4-aminomethanesulphonic acid)
1 millimol/liter
. .
5. . .

107961S

Na-4-hydroxy~enzoate 10 millimol/liter
EXAMPLES OF PRACTIC~L USE . -
... ...
C - ~eading on completion of tne color evolution
Sample : serum, plasma~ urine '
(deproteinated blood) 0.02 ml (0.2)
Powdered reactant~dissolved in
2 2.5 mls
Admixture and incubation ac the temperature and
during the time required in order that the evolution
of the color be completed and has attained its maximum.
Photometer reading at the optimum wavelength for the
chromogenic system is used (e.g. at 340 or at 500-510
nm (nanometer) with 4-hydroxybenzoate and 4-amino-
antipyrin~ at 340 or at 600 nm with the chromotropic
15- acid and 4-aminoantipyrin, and so onj.
The calculation can be made by using the exstinction
coefficient of the colored chromogen, or by using
one or more reference solutions (standards) having
a known glucose contents, treated in the same way
as the sample and with reference to these standards
the calculation is made thus :
Concentration of the Standard solution by D.O. of sample
D.O. of the standard ~ concentration of sample
The measurements of volumes and the a~nixture of the
reactants can be made either manually, as indicated, or by
using specially provided machine for carrying out the seve-
ral operations mechanically and automatically.
D - With "kinetic" reading
Sample : serum, plasma, urine (deproteinated
blood) 0.01 mls (0.1)
Powdered reactant,dissolved in H20 mls 2
Admixture and photometer imme- -
diate reading, at 340 nm or at the wavelength which
}s optimum for the chromogenic system used (such
as C). The photometer readings can be taken at




.

1079615

regular time intervals, or continually recorded on
paper.
The measured variation _ - is proportional
to the glucose concentration in the sample.
Also the "kinetic" method can be carried out manually,
as indicated, or by usin$ ~pecially provided machine
for performing mechanically and automatically the .
. operations of reactant metering, admixture, photo-
meter reading~ timing~ thermostatic adjustments~
~O recording~ computing and others.
For the calculation, refer to Example C above~
. The s~mbols_employed have the following meanings :
GOD = glucose oxidase
POD = peroxidase
D.O. = optical density
nm = nanometers
t = time