GB1593735A

GB1593735A - Composition suitable for use in the enzymatic determination of glucose

Info

Publication number: GB1593735A
Application number: GB4605777A
Authority: GB
Original assignee: SCLAVO INST SIEROTERAPEUT
Current assignee: SCLAVO INST SIEROTERAPEUT
Priority date: 1976-11-08
Filing date: 1977-11-04
Publication date: 1981-07-22
Also published as: JPS5358293A; FR2370282A1; GR63608B; NL7712310A; CA1079615A; BE860607A; BR7707498A; AU3010377A; AR217266A1; IT1123670B; DE2749991A1; ES464038A1; AU503975B2

Description

(54) COMPOSITION SUITABLE FOR USE IN THE ENZYMIC DETERMINATION OF GLUCOSE (71) We, ISTITUTO SIEROTERAPICO E VACCINOGENO TOSCANO "SCLAVO" S.p.A., an Italian company, of Via Fiorentina 1, Siena, Italy, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: This invention relates to a composition which allows there to be carried out a method for the enzymic determination of glucose.

Our British Patent Specification No. 1,564,467 describes and claims a dry composition which, when dissolved in water, forms a reagent solution suitable for use in the determination of peroxides, the composition comprising (a) a 4-hydroxybenzoate, and (b) a compound containing at least one reactive group bound to a structure derived from 1-phenyl-pyrazolin-5-one.

The present invention provides a composition suitable for use in the enzmic determination of glucose, the composition being in the dry state and comprising (a) a glucose-oxidase enzyme, (b) peroxidase enzyme, (c) a compound having a > CH group in the alpha position of an enolic group with double bonds conjugated with respect to the enolic double bond, and (d) a compound with at least one reactive group bound to a structure derived from 1-phenyl-pyrazolin-5-one, the compounds (c) and (d) being solid at room temperature.

The composition of the invention is dissolved prior to use in the enzymatic determination of glucose, which determination exploits the transformation that glucose undergoes in the presence of the glucose-oxidase enzyme and the subsequent oxidation of the resulting hydrogen peroxide due to the presence of the peroxidase enzyme, the resulting intensity of colour, which is proportional to the glucose content, being determined.

Preferably the composition of the present invention includes a buffer.

Examples of compound (c) are 5-hydroxy-1, 3-dimethylbenzene; 3-hydroxy-1,4 dimethylbenzene; thymol; o-hydroxydiphenyl; o-cyclohexylphenol; o-hydroxyacetophenone; 2-(4' hydroxybenzo)-phenol; 2-chloro-5-hydroxytoluene; p-chlorophenol; p-bromophenol; 2,6dibromophenol; 2,4,6-tribromophenol; guaiacol; the monomethyl ether of resorcinol; guaiacol sulphonate; o-phenol sulphonic acid; p-phenol sulphonic acid; salicylic acid; sodium salicylate; m-hydroxybenzoic acid; p-hydroxybenzoic acid; sodium 4hydroxybenzoate; potassium 3-hydroxybenzoate; methyl-p-hydroxybenzoate; phenyl salicylate; salicylsalicylic acid; salicylamide; vanillic acid; 3,5-dibromo-4-hydroxybenzoic acid; catechol; resorcinol, orcinol; epinephrine, floroglucinol; pyrogallol; m-aminophenol; 1-naphtholsulphonic acid; chromotropic acid; 1-naphthol-7-amino-3-sulphonic acid; 2naphthol; Schaffer's acid; 3-hydroxy-2-naphthoic acid; an N-substituted 3-hydroxy-2naphthamide; 6-hydroxyquinoline; 8-hydroxyquinoline; 8-hydroxyquinoline citrate; 7methyl-8-hydroxyquinoline; barbituric acid or a derivative thereof (non-substituted at the 5-position); a pyrazolone; diketohydrindene; and 2,4-diamino-6-hydroxypyrimidine.

Examples of compound (d) are aminophenazone (i.e. 4-dimethylamino-2,3-dimethyl-1phenyl-3-pyrazolin-5-one); sulphamylpyrine (i.e. the sodium salt of 2 ,3-dimethyl- 1-phenyl- 5-pyrazolone-4-aminomethanesulphonic acid); dibupyrene (i.e. the sodium salt of (1phenyl-2,3-dimethyl-5-pyrazolon-4-yl) isobutylamino methanesulphonate); propylphenazone (i.e. 4-isopropyl-2, 3-dimethyl-1-phenyl-3-pyrazoline-3-one); thozalinone (i.e. 2dimethylamino-5-phenyl-2-oxazoline-4-one); and methampyrone (i.e. sodium 1-phenyl-2, 3-dimethyl-5-pyrazolonc -4-methylamínomethane-sulphonate) .

It is also tobe appreciated that in order that a composition may be obtained which is easy to be prepared, is cheap and is adequately stable, it is suggested to select, among all the indicated compounds, those which simultaneously possess the following properties:- (i) high-melting point and non-deliquescence; (ii) the form of an amorphous or crystalline powder; (iii) adequate stability; and (iv) sufficient non-reactivity in the dry condition, so that they do not displacy a tendency to denature the enzymic proteins which are contained in the composition, but sufficient reactivity when in solution ready for their intended use.

The following Examples are reported by way of illustration only of a few compositions among the many compositions which are possible.

EXAMPLES OF POWDER FORMULATIONS (with indication of concentration of constituents when diluted with water to make one litre of solution).

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.

Al) Phosphate buffer at pH 7.5, 150 millimoles/litre GOD (i.e. glucose oxidase) 18,000 U/litre POD (i.e. peroxidase) 1,000 U/litre 4-aminoantipyrine 0.4 millimole/litre Na-4-hydroxybenzoate 10 millimoles/litre A2) Phosphate buffer at pH 7.3 150 millimoles/litre GOD 18,000 U/litre POD 1,000 U/litre 4-aminoantipyrine 0.4 millimole/litre K-3-hydroxybenzoate 10 millimoles/litre A3) Tris-maleate buffer at pH 7.7 300 millimoles/litre GOD 18,000 U/litre POD 1,000 U/litre 4-aminoantipyrine 0.5 millimole/litre Na-salicylate 12 millimoles/litre A4) 2-amino-2-methyl-propan -1,3-diol buffer at pH 7.8 200 millimoles/litre GOD 25,000 U/litre POD 2,000 U/litre 4-aminoantipyrine 0.6 millimole/litre Salicylamide 7 millimoles/litre A5) Tris-KH2PO4 buffer at pH 7.6 150 millimoles/litre GOD 18,000 U/litre POD 1,000 U/litre 4-aminoantipyrine 0.4 millimole/litre vanillic acid 12 millimoles/litre A6) Phosphate buffer at pH 6 150 millimoles/litre GOD 20,000 U/litre POD 2,000 U/litre 4-aminoantipyrine 0.35 millimole/litre A chromatropic acid (4,5-dihydroxy 2,7-naphthalenc-disulphonic acid) 4 millimoles/litre A7) Acetate-maleate buffer at pH 6 100 millimoles/litre GOD 25,000 U/litre POD 2,500 U/litre 4-aminoantipyrine 0.6 millimole/litre 8-hydroxyquinoline citrate 15 millimoles/litre A8) Phosphate buffer at pH 6.7 100 millimoles/litre GOD 18,000 U/litre POD 500 U/litre 4-aminoantipyrine 0.35 millimole/litre barbituric acid 4 millimole/litre B) Varying the 4-aminoantipyrine B1) Phosphate buffer at pH 7.5 150 millimole/litre GOD 18,000 U/litre POD 1,000 U/litre The sodium salt of Sulfamylpyrine (2,3-dimethyl-1-phenyl-5-pyrazolon-4-aminomethanesulphonic acid) 1 millimole/litre Na-4-hydroxybenzoate 10 millimoles/litre EXAMPLES OF PRACTICAL USE C - Reading on completion of the reaction and hence the colour evolution Sample: volume of solution of serum, plasma, urine (deproteinated blood) 0.02 ml (0.2) The value in parentheses is the volume of deproteinated blood.

Powdered reactant (composition), dissolved in H2O 2.5 ml Admixture and incubation at the temperature and during the time required in order that the evolution of the colour be completed and has attained its maximum. Photometer reading at the optimum wavelength for the chromogenic system is used (e.g. at 340 or about 500-510 nm (nanometer) with 4-hydroxybenzoate and 4-aminoantipyrine, at 340 or at 600 nm with the chromotropic acid and 4-aminoantipyrine, and so on).

The calculation can be made by using the extinction coefficient of the coloured chromogen, or by using one or more reference solutions (standards) having a known glucose content, treated in the same way as the sample and with reference to these standards the calculation is made thus: Concentration of sample solution optical density of sample x concentration of standard solution optical density of standard solution The measurements of volumes and the admixture of the reactants can be made either manually, as indicated, or by using specially provided machine for carrying our the several operations mechanically and automatically.

D - With "kinetic" reading, made during the entire course of the reaction, Sample: volume of solution of serum, plasma, urine (deproteinated blood) 0.01 ml (0.1) Powdered reactant (composition, dissolved in 2 ml Admixture and photometer immediate reading, at 340 nm at the wavelength which is 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 AD.O./At is proportional to the glucose concentration in the sample. Here "t" is time.

Also the "kinetic" method can be carried out manually as indicated, or by using specially provided machine for performing mechanically and automatically the operations of reactant metering, admixture, photometer reading, timing, thermostatic adjustments, recording, computing and others. For the calculation, refer to Example C above.

Claims

WHAT WE CLAIM IS:

1. A composition suitable for use in the enzymic determination of glucose, the composition being in the dry state and comprising (a) glucose-oxidase enzyme, (b) peroxidase enzyme, (c) a compound having a > CH group in the alpha position of an enolic group with double bonds conjugated with respect to the enolic double bond, and (d) a compound with at least one reactive group bound to a structure derived from 1-phenyl-pyrazolin-5-one, the compounds (c) and (d) being solid at room temperature.

2. A composition according to claim 1, and also including a buffer.

3. A composition according to claim 1 or 2, wherein the compound (c) is selected from 5-hydroxy-1, 3-dimethylbenzene; 3-hydroxy-1, 4-dimethylbenzene; thymol; o- hydroxydiphenyl; o-cyclohexylphenol; o-hydroxyacetophenone; 2-(4'-hydroxybenzo)phenol; 2-chloro-5-hydroxytoluene; p-chlorophenol; p-bromophenol; 2,6-dibromophenol; 2,4,6-tribromophenol; guaiacol; the monomethyl ether of resorcinol; guaiacol sulphonate; o-phenol sulphonic acid; p-phenol sulphonic acid; salicylic acid; sodium salicylate; m-hydroxybenzoic acid; p-hydroxybenzoic acid; sodium 4-hydroxybenzoate; potassium 3-hydroxybenzoate; methyl-p-hydroxybenzoate; phenyl salicylate; salicylsalicylic acid; salicylamide; vanillic acid; 3,5-dibromo-4-hydroxybenzoic acid; catechol; resorcinol; orcinol. epinephrine; floroglucinol; pyrogallol; m-aminophenol; 1-naphtholsulphonic acid; chromotropic acid; 1-naphthol-7-amino-3-sulphonic acid; 2-naphthol; Schäffer's acid; 3-hydroxy-2-naphthoic acid; and N-substituted 3-hydroxy-2-naphthamide; 6hydroxyquinoline; 8-hydroxyquinoline; 8-hydroxyquinoline citrate; 7-methyl-8hydroxyquinoline; barbituric acid or a derivative thereof (non-substituted at the 5position); a pyrazolone; diketohydrindene; and 2,4-diamino-6-hydroxypyrimidine.

4. A composition according to claim 1, 2 or 3, wherein the compound (d) is selected from aminophenazone; sulphamylpyrine; dibupyrene; propylphenazone; thozalinone; and methampyrone.

5. A composition according to claim 1 suitable for use in the determination of glucose, substantially as hereinbefore described.

6. A method for the determination of glucose in a sample, which comprises reacting the glucose in the sample with a composition according to any preceding claim, and determining the resulting intensity of colour which is proportional to the original glucose content of the sample.