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US2982700A - Diagnostic composition - Google Patents

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US2982700A
US2982700A US2982700DA US2982700A US 2982700 A US2982700 A US 2982700A US 2982700D A US2982700D A US 2982700DA US 2982700 A US2982700 A US 2982700A
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glucose
titanium
oxalate
urine
diagnostic composition
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/904—Oxidoreductases (1.) acting on CHOH groups as donors, e.g. glucose oxidase, lactate dehydrogenase (1.1)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/805—Test papers

Definitions

  • This invention relates'to a new and improved diagnostic composition and is particularly concerned with a glucose indicator which is useful for the qualitative detection and quantitative determination of glucose in biological fluids, such as mine, and wherein the reagent composition is incorporated upon a bibulous carrier U
  • a glucose indicator which is useful for the qualitative detection and quantitative determination of glucose in biological fluids, such as mine
  • the reagent composition is incorporated upon a bibulous carrier U
  • the detection of glucose in urine as well as the determination of its concentration therein is of great importance for. diabetic patients who must control their diets so as to regulate their sugar intake andwho must frequently be guided in this regard by a regular check on urine glucose. But beyond its usefulness in regular urine testing on known diabetics by both patients and. physicians, this glucose indicator may also beused efliciently in routine urinalyses in hospitals and physicians ofiices,
  • a urine-sugar test to beof greatest value, must be conveniently rapid, simple enough for any patient to learn with ease, accurate enough to serve the clinician, and sensitive enough to reflect variations in the patieuts condition.v Moreover, the reagent composition must be adequately stable. r
  • a diagnostic composition according to the present invention comprises then as essential constituents glucose 'in the art pertaining to glucose diagnostics.
  • glucose indicator of titanium potassium oxalate which is commercially available, represents a substantial and significant advance I Glucose indicators presently on the market depend for the detection of hydrogen peroxide on complex oxidation-reduction reactions which occur only in the presence of enzymes (e.g. peroxidases) and involve the formation of intermediate compounds.
  • the glucose indicator of the present invention is based, as stated above, on a plain and straightforward chromogenic addition reaction of hydrogen peroxide with titanium potassium oxalate.
  • Titanium sulfate (titanyl sulfateTiOSO has in the past been used in analytical chemistry for detecting hydrogen peroxide (Eisenberg, Ind. Eng. Chem, Anal. Ed,
  • titanium sulfate in accordance with the method just described or any other method in 1 conjunction with a formulation which includes glucose oxidase for two reasons: first, a titanium sulfate solution is, due to the ready hydrolysis, and consequent decomposition, of titanium sulfate, extremely acidic and therefore destructive of glucose oxidase; secondly, with titani- -um sulfate thus not being highly soluble in water, it
  • titanium potassium oxalate is very soluble inwater and gives upon dissolution therein the requisite neutral solution.
  • titanium potassium oxalate it is also possible, and therefore contemplated, to employ other equivalent, i.e. highly soluble and non-acidic, titanium oxalates .such as titanium sodium oxalate, titanium amvmonium oxalate, etc.
  • an especially preferred pH is 4.8.
  • the glucose oxidase and titanium potassium oxalate (titanyl potassi- 'um oxaiate-riogcoocooxn
  • the glucose oxidase (glucose aerodehydrogenase), as is well known, catalyzes the aerobic oxidation of glucose to produce gluconic acid (gluconolactone) and hydrogen peroxide as reaction products.
  • the titanium potassium oxalate in turn, reacts with the hydrogen peroxide formed in the above reaction to yield a titanium addition compound of a characteristic yellow color. which is oxidase would be destroyed, while at a pH substantially higher than 5.2, the development of a distinctive bright yellow color would be thwarted since the yellow co'lor obtained on reacting titanium potassium oxalate with hydrogen peroxide is media.
  • test device itself may comprise the reagent composition in the form of a tablet, powder, or other embodiment
  • bi-bulous base materials or carriers such as strips or sticks of filter paper
  • test portion of these strips is of abufi color.
  • Other porous or absorbing materials such as small sticks. of wood, etc., may likewise be used.
  • EXAMPLE II About 20 ml. of water, 400 mg. of gelatin in 10 'rnLof water, 4,000 mg. of titanium potassium oxalate in 20 ml. of water, 24 ml. of buffer solution (prepared as described in Example I) and 1,200 mg. of glucose oxidase (16,000 units per gram) in 10 ml. of water were mixed with stirring to give a uniform impregnating solution.
  • Test strips. were then prepared in accordance with the a procedure recited in Example 1.
  • Example III To an impregnating solution obtained as describedin Example I there was added as an inertdye 1 mg. of FD&C Blue No. l in 0.2 ml, of water. For the preparation of the test strips the procedure given in Example I was followed.
  • an impregnated strip made as described'in any one of the above examples is dipped'into the liquid specimen to be tested.
  • a test strip When contacted with urine contaming glucose, a test strip will give a positive reaction in about one-half to one and one-half minutes evidenced by various shades of yellow color as follows:
  • this invention pertains to a diagnostic test for the detection of glucose in body fluids, and especially in urine, consisting of a bibulous strip or stick that has been impregnated with a composition comprising glucose oxidase and titanium potassium oxalate. The latter reactions with the hydrogen peroxide. formed when glucose is oxidized in the presence of glucose oxidase to yield a yellowtitaniumaddition compound.
  • a diagnostic composition for detecting glucose which comprises glucose oxidase and a titanium oxalate selected from the group consisting of titanium potassium oxalate, titanium ammonium oxalate and titanium sodium oxalate, said titanium oxalate being capable of reacting with hydrogen peroxide formed when glucose is aerobically oxidized in the presence of glucose oxidase to produce a yellow addition compound 2.
  • a diagnostic composition for detecting. glucose which comprisesv glucose, oxidase, a titanium oxalate selected from the groupconsisting of titanium potassium oxalate, titanium ammonium, oxalate and titanium sodium oxalate and a buffer for maintaining the pH of said composition in the presenceof urine at between 3.5 and 5.2.
  • a diagnostic composition for detecting glucose according to claim 3whereinsaid titan um oxalate is titanium potassium oxalate.
  • a diagnostic composition for detecting glucose according to claim 3 wherein the pH maintained'at 4.8.
  • a diagnostic composition for detecting glucose which comprises glucose oxidase, titanium potassium oxalate capable of reacting with hydrogenfiperoxide formed when glucose is aerobically oxidized in the presence of glucose oxidase to produce a yellow addition compound, and a bullet for maintaining thepH of said composition in the presence of urine at 4.8.
  • a diagnostic "composition for. detecting glucose which comprises:

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Description

, 2,982,700 DIAGNOSTIC coMrosmoN i Ernest C. Adams, In, Elkhart, Ind., assignor to Miles Laboratories, Inc., Elkhart, Ind., a corporation of Indiana No Drawing Filed Apr. 22, 1959, Ser. 1 510,807,993
8 Claims. or. 195-1035 I r This invention relates'to a new and improved diagnostic composition and is particularly concerned with a glucose indicator which is useful for the qualitative detection and quantitative determination of glucose in biological fluids, such as mine, and wherein the reagent composition is incorporated upon a bibulous carrier U The detection of glucose in urine as well as the determination of its concentration therein is of great importance for. diabetic patients who must control their diets so as to regulate their sugar intake andwho must frequently be guided in this regard by a regular check on urine glucose. But beyond its usefulness in regular urine testing on known diabetics by both patients and. physicians, this glucose indicator may also beused efliciently in routine urinalyses in hospitals and physicians ofiices,
in diabetes detection screening programs-in the differentiation of glucosuria from other meliturias, and the like.
Because early diagnosis and continued control are so important in diabetes, a urine-sugar test, to beof greatest value, must be conveniently rapid, simple enough for any patient to learn with ease, accurate enough to serve the clinician, and sensitive enough to reflect variations in the patieuts condition.v Moreover, the reagent composition must be adequately stable. r
Procedures for the detection of sugar in urine are well known in clinical chemistry. One such procedure utilizes Benedicts copper reduction test, another employs a selfheating alkaline copper reduction test in tablet form (US. Patent No. 2,387,244), still another involves a test which depends solely on the action of enzymes (U.S.
apphcation No. 514,395, filed June 9, 1955, by Alfred, H. Free and assigned to the assignee of the present invention). None of these procedures, however, has entirely satisfied the above-mentioned requirements.
I have now found a novel and highly useful glucosedetectrng means which represents an important improvement and a fresh approach to the problem of determining glucose in various materials including'body fluids, such'as urine, by a technique that utilizes a diagnostic composition which is better than even the latest and most commonly used glucose tests.
Specifically, I have now found that a unique combination of a glucose oxidase system for determining glucose with a novel titanium potassium oxalate system for detecting hydrogen peroxide offers a superior means for testing biological fluids for their glucose contents.
.A diagnostic composition according to the present invention comprises then as essential constituents glucose 'in the art pertaining to glucose diagnostics.
J 2,982,700 Patented May: 2,
2 immediately indicative of the glucose content of the fluid being tested. The latter reaction may be represented by the following equation:
000K coon 'n=o+n,o,-- Ti (Yellow) 000 000 on door: 00K
The incorporation and use in this new glucose indicator of titanium potassium oxalate, which is commercially available, represents a substantial and significant advance I Glucose indicators presently on the market depend for the detection of hydrogen peroxide on complex oxidation-reduction reactions which occur only in the presence of enzymes (e.g. peroxidases) and involve the formation of intermediate compounds. The glucose indicator of the present invention, on the contrary, is based, as stated above, on a plain and straightforward chromogenic addition reaction of hydrogen peroxide with titanium potassium oxalate.
Titanium sulfate (titanyl sulfateTiOSO has in the past been used in analytical chemistry for detecting hydrogen peroxide (Eisenberg, Ind. Eng. Chem, Anal. Ed,
The usual method for detecting hydro- ,genperoxide with titanium sulfate is to suspend it in water and then use the supernatant liquid. However, it is not feasible to utilize titanium sulfate in accordance with the method just described or any other method in 1 conjunction with a formulation which includes glucose oxidase for two reasons: first, a titanium sulfate solution is, due to the ready hydrolysis, and consequent decomposition, of titanium sulfate, extremely acidic and therefore destructive of glucose oxidase; secondly, with titani- -um sulfate thus not being highly soluble in water, it
" would not be possible to achieve in the impregnating mixture the high concentration thereof which is required as those who are versed'in the art of preparing bibulous test strips will appreciate.
In contrast, titanium potassium oxalate is very soluble inwater and gives upon dissolution therein the requisite neutral solution.
In place of the titanium potassium oxalate it is also possible, and therefore contemplated, to employ other equivalent, i.e. highly soluble and non-acidic, titanium oxalates .such as titanium sodium oxalate, titanium amvmonium oxalate, etc.
between 3.5 to 5.2, an especially preferred pH is 4.8.
It must be borne in mind that if the diagnostic is too I V acidic, that is, the pH is less than about 3.5, the glucose oxidase and titanium potassium oxalate (titanyl potassi- 'um oxaiate-riogcoocooxn The glucose oxidase (glucose aerodehydrogenase), as is well known, catalyzes the aerobic oxidation of glucose to produce gluconic acid (gluconolactone) and hydrogen peroxide as reaction products. The titanium potassium oxalate, in turn, reacts with the hydrogen peroxide formed in the above reaction to yield a titanium addition compound of a characteristic yellow color. which is oxidase would be destroyed, while at a pH substantially higher than 5.2, the development of a distinctive bright yellow color would be thwarted since the yellow co'lor obtained on reacting titanium potassium oxalate with hydrogen peroxide is media.
Although the test device itself may comprise the reagent composition in the form of a tablet, powder, or other embodiment, I prefer, to insure ease and simplicity oftestprocedure, to afiix the reagent composition on bi-bulous base materials or carriers, such as strips or sticks of filter paper, by dissolving the component in a suitable solvent, impregnating the bibulous'strips with the resulting solution and drying the impregnated tent brighter with more acidic reaction strips. As -willibei-obvious from the examples, it'very" easy to preparethe, instantglucose. ndicator 1P its manifest simplicity.
The invention will now be illustrated in greater detail,
but not limited, by. h o lo i ex mulfit EXAMPLE I' Preparation of impregnating solution About 400 mg. of sodium alginate were dissolved in 20 of water, and to this solution there was added a gelatin solution consisting of 400 mg. of gelatin in ml. of water. Then, 4,000 mg. of titanium potassium oxalate were dissolved in 20 ml. of water, and this solution was mixed with the sodium alginate-gelatin solution. Next, 24 ml. of a pH 4.8 buffer solution (prepared by dissolving 32.6 g. or sodium citrate and 7.4 g. of citric acid, each per 100 ml; of-water) were added with stirring. And lastly, a solution of 1,200 mg. of commercially available glucose oxidase 16 ,0001'111115 per gram) in 10 of water were likewise added, and the mixture-was stirred well.
Preparation of reagent strip- Bibulous strips, such as filter paper out intonarrow strips with a water-impervious barrier of ethyl cellulose,
one-half inch from the tip, were dipped into the impregnating solution so that through the process of submersion and capillary attraction the entire. one-half inch of the strip up to the barrier was completely. impregnated. These strips were then dried, in a hot air oven. at 100 C. for from about a few minutesto several hours.
The
test portion of these strips is of abufi color. Other porous or absorbing materials, such as small sticks. of wood, etc., may likewise be used.
EXAMPLE II About 20 ml. of water, 400 mg. of gelatin in 10 'rnLof water, 4,000 mg. of titanium potassium oxalate in 20 ml. of water, 24 ml. of buffer solution (prepared as described in Example I) and 1,200 mg. of glucose oxidase (16,000 units per gram) in 10 ml. of water were mixed with stirring to give a uniform impregnating solution.
; Test strips. were then prepared in accordance with the a procedure recited in Example 1.
EXAMPLE III To an impregnating solution obtained as describedin Example I there was added as an inertdye 1 mg. of FD&C Blue No. l in 0.2 ml, of water. For the preparation of the test strips the procedure given in Example I was followed.
Procedure of testing In use, an impregnated strip made as described'in any one of the above examples, is dipped'into the liquid specimen to be tested. When contacted with urine contaming glucose, a test strip will give a positive reaction in about one-half to one and one-half minutes evidenced by various shades of yellow color as follows:
- intermediate intensities, the urine having the greatest glucose content produc ng the brighter yellow. When dipped in urine containing no glucose, the strips undergo no color change. A simple color, chart based onthis phenomenon may be conveniently prepared for use in testing for urine glucose. V
Test strips impregnated with anyof the solutions doscribed in the above examplesare strikingly stable. For
example, after exposure for 90 hours to atemperature of 50 C. and a relativev humidity of 100% no loss in reactivity was. perceptible. The impregnating solution has alsqbeenfound to. be unusually stable and may beleit standing withoutanydeteriorative efieet. I am. not aware of any other diagnostic formulation with glucose oxidase that evidences such surprising stability.
In summary, this invention pertains to a diagnostic test for the detection of glucose in body fluids, and especially in urine, consisting of a bibulous strip or stick that has been impregnated with a composition comprising glucose oxidase and titanium potassium oxalate. The latter reactions with the hydrogen peroxide. formed when glucose is oxidized in the presence of glucose oxidase to yield a yellowtitaniumaddition compound.
What is claimed is:
l. A diagnostic composition for detecting glucose which comprises glucose oxidase and a titanium oxalate selected from the group consisting of titanium potassium oxalate, titanium ammonium oxalate and titanium sodium oxalate, said titanium oxalate being capable of reacting with hydrogen peroxide formed when glucose is aerobically oxidized in the presence of glucose oxidase to produce a yellow addition compound 2. A diagnostic composition for detecting glucose according to claim 1 wherein saidtitanium oxalate is titanium potassium oxalate. f
3. A diagnostic composition for detecting. glucose which comprisesv glucose, oxidase, a titanium oxalate selected from the groupconsisting of titanium potassium oxalate, titanium ammonium, oxalate and titanium sodium oxalate and a buffer for maintaining the pH of said composition in the presenceof urine at between 3.5 and 5.2. v
4. A diagnostic composition for detecting glucose according to claim 3whereinsaid titan um oxalate is titanium potassium oxalate.
5. A diagnostic composition for detecting glucose according to claim 3 wherein the pH maintained'at 4.8.
6. A diagnostic composition for detecting glucose which comprises glucose oxidase, titanium potassium oxalate capable of reacting with hydrogenfiperoxide formed when glucose is aerobically oxidized in the presence of glucose oxidase to produce a yellow addition compound, and a bullet for maintaining thepH of said composition in the presence of urine at 4.8.
7. A diagnostic "composition for. detecting glucose which comprises:
Parts Glucose oxidase 12.00 Titanium potassium oxalate 40.00 Sodium citrate (dihydrate) 78.24 Citric acid (anhydrous) 17.76 Sodium alginate 4.00 Gelatin 4.00 FD&C Blue No. 1 0.01
References Cited in the file of this patent UNITED STATES PATENTS 2,848,308 Free Aug. 19, 1958 FOREIGN PATENTS" 203,451 Australia Sept. 27, 1956 OTHER REFERE CES Chemical Elements and Their Compounds by Sidgwick, vol. 1, pp. 636 and 643 (1950).
Z. fur Anorganische und Allegemeine Chemie, vol. 211, page 402 (1923).

Claims (1)

1. A DIAGNOSTIC COMPOSITION FOR DETECTING GLUCOSE WHICH COMPRISES GLUCOSE OXIDASE AND A TITANIUM OXALATE SELECTED FROM THE GROUP CONSISTING OF TITANIUM POTASSIUM OXALATE, TITANIUM AMMONIUM OXAIATE AND TITANIUM SODIUM OXALATE, SAID TITANIUM OXALATE BEING CAPABLE OF REACTING WITH HYDROGEN PEROXIDE FORMED WHEN GLUCOSE IS AEROBICALLY OXIDIZED IN THE PRESENCE OF GLUCOSE OXIDASE TO PRODUCE A YELLOW ADDITION COMPOUND.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3266868A (en) * 1962-01-24 1966-08-16 Miles Lab Diagnostic composition and test indicator
US3466145A (en) * 1965-08-30 1969-09-09 Charles M Van Duyne Urine screening device
US4098574A (en) * 1977-08-01 1978-07-04 Eastman Kodak Company Glucose detection system free from fluoride-ion interference

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2848308A (en) * 1955-12-05 1958-08-19 Miles Lab Composition of matter

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2848308A (en) * 1955-12-05 1958-08-19 Miles Lab Composition of matter

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3266868A (en) * 1962-01-24 1966-08-16 Miles Lab Diagnostic composition and test indicator
US3466145A (en) * 1965-08-30 1969-09-09 Charles M Van Duyne Urine screening device
US4098574A (en) * 1977-08-01 1978-07-04 Eastman Kodak Company Glucose detection system free from fluoride-ion interference

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