CA1079615A - Composition for the enzymic determination of glucose - Google Patents

Composition for the enzymic determination of glucose

Info

Publication number
CA1079615A
CA1079615A CA289,446A CA289446A CA1079615A CA 1079615 A CA1079615 A CA 1079615A CA 289446 A CA289446 A CA 289446A CA 1079615 A CA1079615 A CA 1079615A
Authority
CA
Canada
Prior art keywords
liter
glucose
millimol
group
enolic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired
Application number
CA289,446A
Other languages
French (fr)
Inventor
Franco Meiattini
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
ISTITUTO SIEROTERAPICO E VACCINOGENO TOSCANO "SCLAVO" SpA
Original Assignee
ISTITUTO SIEROTERAPICO E VACCINOGENO TOSCANO "SCLAVO" SpA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by ISTITUTO SIEROTERAPICO E VACCINOGENO TOSCANO "SCLAVO" SpA filed Critical ISTITUTO SIEROTERAPICO E VACCINOGENO TOSCANO "SCLAVO" SpA
Application granted granted Critical
Publication of CA1079615A publication Critical patent/CA1079615A/en
Expired legal-status Critical Current

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/54Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose

Abstract

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.

Description

~ 10796~5 . . :.

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. . .

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 .

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

Claims

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.
CA289,446A 1976-11-08 1977-10-25 Composition for the enzymic determination of glucose Expired CA1079615A (en)

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
IT2911776A IT1123670B (en) 1976-11-08 1976-11-08 COMPOSITION SUITABLE FOR THE ENZYMATIC DETERMINATION OF GLUGOSE

Publications (1)

Publication Number Publication Date
CA1079615A true CA1079615A (en) 1980-06-17

Family

ID=11226323

Family Applications (1)

Application Number Title Priority Date Filing Date
CA289,446A Expired CA1079615A (en) 1976-11-08 1977-10-25 Composition for the enzymic determination of glucose

Country Status (13)

Country Link
JP (1) JPS5358293A (en)
AR (1) AR217266A1 (en)
AU (1) AU503975B2 (en)
BE (1) BE860607A (en)
BR (1) BR7707498A (en)
CA (1) CA1079615A (en)
DE (1) DE2749991A1 (en)
ES (1) ES464038A1 (en)
FR (1) FR2370282A1 (en)
GB (1) GB1593735A (en)
GR (1) GR63608B (en)
IT (1) IT1123670B (en)
NL (1) NL7712310A (en)

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5520471A (en) * 1978-08-01 1980-02-13 Kyowa Hakko Kogyo Co Ltd Hydrogen peroxide quantifying method
US4460684A (en) * 1982-08-02 1984-07-17 Miles Laboratories, Inc. Ascorbate-resistant broad range glucose test composition, test device and method
US4935346A (en) * 1986-08-13 1990-06-19 Lifescan, Inc. Minimum procedure system for the determination of analytes
DE102004050209A1 (en) * 2004-10-15 2006-04-20 Merck Patent Gmbh Means and methods for detecting furfurals

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IT986838B (en) * 1972-05-12 1975-01-30 Sclavo Inst Sieroterapeut COMPLEX OF REAGENTS FOR THE ENZYMATIC DETER MINATION OF GLUCOSE GLUCOSE SYSTEM OXIDASE PEROXIDASE DASES WITH MANUAL AND AUTOMATED METHODS CO WITH READING AT TERM OR IN KINETICS

Also Published As

Publication number Publication date
IT1123670B (en) 1986-04-30
AR217266A1 (en) 1980-03-14
AU3010377A (en) 1979-05-03
GB1593735A (en) 1981-07-22
DE2749991A1 (en) 1978-05-11
ES464038A1 (en) 1978-07-16
JPS5358293A (en) 1978-05-26
BR7707498A (en) 1978-07-18
FR2370282A1 (en) 1978-06-02
GR63608B (en) 1979-11-26
NL7712310A (en) 1978-05-10
AU503975B2 (en) 1979-09-27
BE860607A (en) 1978-05-08

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