US3042496A - Diagnostic composition - Google Patents

Description

United States Patent Ofifice 3,642,496 Patented July 3, 1962 This invention relates to a new and improved diagnostic composition and is particularly concerned with a diagnostic test which is useful for the qualitative detection and quantitative determination of glucose in biological fluids, such as urine, and wherein the reagent composition is incorporated upon a bibulous carrier.

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 and who 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 be used efiiciently in routine urinalyses in hospitals and physicians oflices, in diabetes detection screening programs, in the difierentiation of glucosuria from other meliturias, and the like.

Because early diagnosis and continued control are so important in diabetes, a urine-sugar test, to be of 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 patients condition. Moreover, the reagent composition must be adequately stable.

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 coppe 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. application 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.

We have now found a novel and highly useful glucosedetecting 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 which utilizes a diagnostic composition that surpasses even the latest and most commonly used glucose tests in stability and quantitation.

Specifically, we have now found that metalloporphyrins, which are not operable per so, are, however, when activated by certain nitrogenous substances, surprisingly and unexpectedly capable of acting as catalysts to induce the oxidation of indicators by hydrogen peroxide formed when glucose is aerobically oxidized in the presence of glucose oxidase. The principles underlying these reactions are well known and need not be expounded.

A diagnostic composition according to the present invention comprises as essential constituents glucose oxidase, a metalloporphyrin, such as hemin, complex-forming compounds containing amino-donor groups, such as Z-aminobenzothiazole, pyridine, bipyridyl, bipyridylpyridine, nicotinic acid or the like, and color-forming substances or indicators, such as 2,7-diaminofluorene, o-tolidine, leucoindophenols, etc.

The metalloporphyrin used in the preferred embodiments of this invention is hemin, which is commercially available and whose full chemical name is 1,3,5,8-tetramethyl-2,4-diviny1porphine-6,7-dipropionic acid ferrichloride, (C H N O FeCl). The following structural formula characterizes its complex chemical nature:

H3C CH=CH n 1 CH3 l /N Fe 4% nooccn cn 2 Haw H nooccn ca CH3 Hernin and the other 'metalloporphyrins that can be 0 employed in accordance with the inventive concept react wherein the 4-membered ring represents an octahedral porphyrin ring system, M represents Fe, Co, Ni, Mn, Mg,

stands for a nitrogen atom of a complex-forming aminodonor group.

In place of a metalloporphyrin it is also contemplated to employ other equivalent metallic complexes or chelates, such as phthalocyanines, the structure of which is closely analogous to that of the porphine nucleus present in hemin. Further catalytic materials useful in the practice of this invention are ferrocene compounds, the ferrous salt of dimethylglyoxime and the like.

The diagnostic composition of our invention is prepared, due to incompatibility of ingredients, by way of two separate solutions, one comprising the glucose oxidase and the indicator substance dissolved in 1:1 alcohol-water and the other containing hemin and the nitrogenous substance in an alcoholic medium.

It is understood that such additives as suitable protective agents and wetting agents may be incorporated therein. Furthermore, this glucose test may contain a suitable inert dye to impart to the composition a uniform background color as well as an appropriate buffer system to maintain a desired pH range (pH 3 to 7 with a pH of 5.4 being preferred).

The following examples illustrate in greater detail a number of embodiments of our invention as well as their method of manufacture. These examples demonstrate the flexibility of our invention and the various modifications possible within the purview of the inventive concept and should therefore not be construed as limiting the ambit of the appended claims.

EXAMPLE I 0 of water until a clear solution resulted. Then 20 ml. of

a pH 5.4 buffer solution (prepared by dissolving 352.8 g. of sodium citrate monohydrate and 25.6 g. of anhydrous citric acid in water and diluting to 1000 ml.) was added with stirring. A solution of 2 g. of gelatin and 150 mg. of ED. & C. Red No. 3 (disodium salt of 9-o-carboxyphenyl 6 hydroxy 2,4,5,7 tetraiodo-3-isoxanthone) in 100 ml. of hot water was prepared and 20 ml. of this solution was added to the algin-bufier solution with stirring. Then a solution of 200 mg. of 2,7diamino-fiuorene dihydrochloride in 20 m1. of hot 50% ethanol was added. A solution of 400 mg. of glucose oxidase (16,000 units per gram which had been heated at 100 for hours to destroy catalase) in 5 ml. of Water was added. The resulting solution was kept at 4045 by warming as necessary.

Solution B.Hemin, 50 mg, and 500 mg. of 2-aminobenzothiazole were added to 50 ml. of 95 ethanol and 50 ml. of 10% aqueous polyvinyl alcohol and the mixture was heated to boiling and shaken for two minutes. The excess hemin was removed by filtration, and the filtrate was allowed to cool to room temperature.

Preparation of Diagnostic Strip Bilbulous strips, such as filter paper cut into narrow strips, small sticks of wood, or other porous or absorbing material with a water-impervious barrier of ethyl cellulose, one-half inch from the tip, were dipped into 'solution A so that through the process of submersion Procedure of Testing In use, an impregnated strip made as described above is dipped into the liquid specimen to be tested. When contacted with urine containing glucose, test strips gave positive reactions in about one minute evidenced by various shades of blue color as follows:

With urine containing 1% or 2% glucose an intense blue color developed in one minute, while with urine containing 0.1% glucose a faintly blue color developed in one minute. With urines containing 0.25% and 0.5% glucose the blue colors which developed in one minute were of intermediate intensities, the urine containing the most glucose being the darkest blue. When dipped in urine containing no glucose, the sticks underwent no color change. A simple color chart based on this phenomenon may be conveniently prepared for use in estimating the glucose content of the urine being tested.

Other specific embodiments of our invention are illustrated by the following examples:

EXAMPLE II (A) Twelve ml. of a buffer solution (which was prepared by dissolving 7.4 g. of anhydrous citric acid and 32.6 g. of trisodium citrate dihydrate in 100 ml. of water) was added rapidly with good stirring to 10 ml. of a warm gelatin solution (which was prepared by dissolving 4.8 g. of gelatin in 100 ml. of boiling water). One hundred mg. of o-tolidine dihydrochloride was mixed with 10 ml. of 95% ethanol (2B), and the mixture was added rapidly to the gelatin-buffer solution with good stirring. Then 10 ml. of a glucose oxid-ase solution was added, which was prepared by dissolving 0.5 g. of glucose oxidase (having an activity of 16,000 units per gram) in 50 ml. of water, and the mixture was stirred until well mixed. Paper strips were dipped into this solution and dried at 100 C. for 10 minutes.

(B) Five mg. of hemin was dissolved in two to ten drops of pyridine, and the mixture was diluted with 95% ethanol. The strips were dipped into this solution and then (While still wet) into urine containing 1% glucose.

EXAMPLE III A mixture of 50 mg. of hemin, 500 mg. of nicotinic acid, 50 ml. of ethanol and 50 ml. of 10% aqueous polyvinyl alcohol was warmed and shaken for 5 minutes. The excess hemin was removed by filtration. Strips described in Example IIA were impregnated with this mixture and dried at for 10 minutes. These strips developed a blue-green color after 5 minutes with urine containing 1% glucose and only a very faint green color with urine containing glucose.

EXAMPLE IV When 50 mg. of Z-aminobenzothiazole was substituted for the nicotinic acid in Example III, the strips were a little more active, and a blue-green color developed with 1% glucose in urine in 3 minutes.

EXAMPLE V (A) Twenty ml. of hot buffer solution (which was prepared by dissolving 352.8 g. of sodium citrate monohydrate and 25.6 g. of anhydrous citric acid in water and diluting to 1000 ml.) was added with stirring to a solution of 200 mg. of algin in 20 ml. of water. Then 20 ml. of a hot gelatin-dye solution (Which was prepared by dissolving 2 g. of gelatin and mg. of ED. & C. Red No. 3 dye in 100 ml. of water) was added with stirring and followed by a solution of 2,7-diaminofiuorene dihydrochloride (200 mg.) in 20 ml. of hot 50% aqueous ethanol. Finally a solution of 400 mg. of glucose oxidase (16,000 units per gram) in 5 ml. of water was added with good stirring, and paper strips were impregnated at once with the warm mixture. The strips were dried at 100 for 10 minutes.

(13) These strips were dipped into the hemin-nicotinic acid complex solution as in Example II-I and dried at 100 for 10 minutes. When dipped in urine containing 1% glucose, a moderate blue color developed in 2 minutes.

With glucose in urine only a faint color change was observed in 2 minutes.

In summary, this invention pertains to a diagnostic composition 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, a metalloporphyrin, such as hemin, a complex-forming compound containing aminodonor groups, such as 2-aminobenzothiazole, nicotinic acid or pyridine to activate the metalloporphyrin and a color-forming substance, such as 2,7-diaminolluorene or o-tolidine, which is oxidizable by hydrogen peroxide in the presence of the activated metalloporphyrin. The composition containing glucose oxidase, hemin, Z-aminobenzothiazole and 2,7-diaminofiuorene constitutes the preferred embodiment of this invention.

What is claimed is:

1. A diagnostic composition for detecting glucose which comprises glucose oxidase, a metalloporphyrin, a complexforming compound containing amino-donor groups selected from the class consisting of 2-aminobenzothiazole, nicotinic acid and pyridine to activate said metalloporphyrin and a color-forming substance taken from the group consisting of 2,7-diaminofluorene and o-tolidine, oxidizable by hydrogen peroxide in the presence of the activated metalloporphyrin.

2. A diagnostic composition for detecting glucose which comprises glucose oxidase, hemin, a complex-forming compound selected from the class consisting of Z-aminobenzothiazole, nicotinic acid and pyridine to activate said hemin and a color-forming substance taken from the group consisting of 2,7-diaminofiuorene and o-tolidine, oxidizable by hydrogen peroxide in the presence of the activated hemin.

3. A diagnostic composition for detecting glucose which comprises glucose oxidase, hemin, Z-aminobenzothiazole to activate said hemin by forming a complex therewith and 2,7-diaminofluorene oxidizable by hydrogen peroxide in the presence of the activated hemin.

4. A glucose-detecting means including a bibulous carrier impregnated with a diagnostic composition comprising glucose oxidase, hemin, Z-aminobenzothiazole to activate said hemin, and 2,7-dia-minofluorene oxidizable by hydrogen peroxide in the presence of the activated hemin.

5. A glucose-detecting means including a bibulous carrier impregnated with a diagnostic composition comprising glucose oxidase, hemin, an amino-donor selected from the group consisting of Z-aminobenzothiazole, nicotinic acid and pyridine to activate said hemin and 2,7-diaminofluorene oxidizable by hydrogen peroxide in the presence of the activated hemin.

rier impregnated with a diagnostic composition comprising glucose oxidase, hemin, pyridine to activate said hemin and 2,7-diaminofiuorene oxidizable by hydrogen peroxide in the presence of the activated hemin.

References Cited in the file of this patent FOREIGN PATENTS Australia Sept. 27, 1956 OTHER REFERENCES Fearon, W. B.: Introduction to Biochemistry, 1947, published by Grunt Stratton, N.Y., pp. 212, 251, 459.

Sumner, J. B., and Somers, G. F.: Chemistry and Methods of Enzymes, 1953, Academic Press, N.Y., pp. 225-226.

Claims (1)

1. A DIAGNOSTIC COMPOSITION FOR DETECTING GLUCOSE WHICH COMPRISES GLUCOSE OXIDASE, A METALLOPORPHYRIN, A COMPLEXFORMING COMPOUND CONTAINING AMINO-DONOR GROUPS SELELCTED FROM THE CLASS CONSISTING OF 2-AMINOBENZOTHIAZOLE, NICOTINIIC ACID AND PYRIDINE TO ACTIVATE SAID METALLOPORPHYRIN AND A COLOR-FORMING SUBSTANCE TAKEN FROM THE GROUP CONSISTING OF 2,7-DIAMINOFLUORENE ANND 0-TOLIDINE, OXIDIZABLE BY HYDROGEN PEROXOXIIDE IN THE PRESENCE OF THE ACTIVATED METALLOPORPHYRIN.