US 3791988 A
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United States Patent 3,791,988 DIAGNOSTIC TEST FOR GLUCOSE Dieter Josef, Pratteln, and Alfred Lampart, Magden,
Switzerland, and Pirmin Schwartz, Weil am Rhine, Germany, assignors to Hoifmann-La Roche Inc., Nutley,
NbDrawing. Filed Mar. 23, 1972, Ser. No. 237,528 Int. Cl. com 33/16 US. 01. 252-408 13 Claims ABSTRACT OF THE DISCLOSURE An improved diagnostic composition for the quantitative determination of glucose in biological fluids comprising glucose oxidase, a phenazin and/or phenoxazin-derivative and a tetrazolium salt as well as the incorporation thereof upon a bibulous carrier are described.
DETAILED DESCRIPTION OF THE INVENTION The present invention relates to new and improved diagnostic compositions which are useful in both the qualitative and quantitative determination of glucose in fluids, particularly body fluids such as blood, serum, plasma, urine and the like.
The quantitative determination of glucose in body fluids is of great importance to diabetic individuals who must have frequent checks on the level of glucose in their body fluids as a means of regulating the sugar intake in their diets. The qualitative determination of glucose in body fluids is of importance in routine testing of patients in doctors offices, clinics, institutions, and hospitals as well as in the mass screening of individuals for the presence of the disease.
The ideal diagnostic composition for the detection of glucose in fluids must be simple so as not to require a high degree of technical skill on the part of the technician administering the test, sufliciently rapid to be utilized in large scale qualitative screening determinations and be sensitive and accurate enough to be useful to the clinician. Additionally, such a composition must be sufliciently stable to meet all situations such as prolonged storage and the advance preparation of solutions for large scale screening operations. The present invention pertains to such a composition.
In recent years the methods of determining glucose concentration in fluids have been primarily based on the enzymatic oxidation of glucose with glucose oxidase and oxygen with subsequent detection and measurement of the resulting peroxide or gluconic acid oxidation products by means of a color reaction. Such methods suffer from the disadvantage of having fluctuations in results due to variations in the oxygen content of different samples. Further, with such methods it can never be guaranteed that oxidation process proceeds quantitatively.
One attempt to overcome the above described disadvantage is the addition of an endogeneous oxygen donor, e.g., hydrogen peroxide or the like to the oxidation solution, thereby limiting the measurement of glucose to the relatively insensitive gluconic acid determination. The disadvantage of this approach is the comparative insensitivity of the gluconic acid determination.
The method and composition of the present invention aflords a means whereby the enzymatic oxidation may be utilized to quantitatively determine glucose content Without the disadvantages of prior art processes. Additionally, the method of the invention has the advantage over the prior art of being equally accurate under aerobic and anaerobic conditions and by not being hampered by false positive results due to the presence of ascorbic acid.
In the method of the present invention, the phenazinand/or the phenoxazin derivative enters into the enzy- 3,791,988 Patented Feb. 12., 1974 matic oxidation reaction as a hydrogen acceptor. The reduced form of the phenazinand/or the phenoxazin-derivative(phen, thus formed reacts rapidly in a nonenzymatic reaction with the tetrazolium salt to regenerate the original phenazin and/or phenoxazin-derivative (phen and form a formazan compound which is stable and possesses an intense color. The amount of glucose present is then measured by colorimetrically reading the color of the formazan compound. The reaction may be represented as follows:
glucose phen gluconic acid p red phen d tetrazolium phen formazan Phenazin-derivatives which can be present in the agent provided by the present invention include phenazinium salts in particular methoand etho-compounds. Phenazinium methosulfate is particularly preferred as it is commercially available.
Phenoxazin-derivatives include phenoxazinium salts in particular 7-dimethylamino 1,2 benzophenoxazinium chloride (meldola blue) or 7-dimethyl-amino-2'-hydroxy- 1,2-benzophenoxazinium chloride.
The selection of a particular tetrazolium salt is not critical to the present invention. Examples of suitable compounds include thiazolyl blue [2,5-diphenyl-3-(4,5-dimethyl-2-thiazolyl) mono-tetrazolium bromide],
nitrotetrazolium blue chloride [3,3-dianisyl-4,4'-bis[2- (4-nitrophenyl)-5-phenyl]-tetrazolium chloride],
iodonitrotetrazolium chloride [2-(p-iodophenyl)-3-(pnitrophenyl -5-phenyl-tetrazolium chloride] neotetrazolium chloride [2,2,5,S'-tetraphenyl-3-(4,4'-biphenylene) -ditetrazolium chloride] tetranitrotetrazolium blue [2,2,5,5-tetranitrophenyl-3- (3 ,3 -dimethoxy-4,4'-biphenylene -ditetrazolium chloride],
triphenyltetrazolium chloride, tetrazolium violet [2,5-
diphenyl-3-(a-naphthyD-tetrazolium chloride] and tetrazolium blue [3,3'-dianisyl-4,4'-bis-(3,5-diphenyl) tetrazolium chloride].
Preferred among these are thiazolyl blue, nitrotetrazolium blue chloride and iodonitrotetrazolium chloride.
The diagnostic compositions of the present invention can be provided in a solid form,, e.'g., tablets or powder or, preferably, as a solution in a suitable solvent. Such solutions may be utilized per se or impregnated onto a bibulous carrier such as filter paper, cardboard and the like.
Wherein solutions of the diagnostic compositions of the invention are contemplated, aqueous solutions are preferred. It is expedient, however, to add an organic solvent, e.g., a lower alkanol such as ethanol or a solubilizing agent, e.g., polyethylene glycol, polyoxysorbitanic acid ester or the like to hold in solution the water-insoluble formazans which are formed in the determination reaction. A preferred polyethylene glycol is polyethylene glycol 1500 and a preferred polyoxysorbitanic acid ester is that marketed by Atlas Chemical Industries, Inc., Wilmington, Del. under the trademark Tween 81. This substance chemically is polyoxyethylene(5)sorbitan monooleate. In place of a solvent, a suspending agent such as gelatin or an alginate may be utilized to form a suspension of the formazans as they are produced.
The compositions of the present invention are prepared by simply mixing together the phenazin and/or phenoxazin-deriyative, the tetrazolium salt and glucose oxidase. While the relative concentrations of the components is not particularly critical, it is preferred to have from about 0.1 to about 2.5 millimoles of phenazin and/or phenoxazin-derivative and from about 1.0 to about 10.0 millimoles of tetrazolium salt present for each 0.5 to about 5.0 mg. of glucose oxidase present.
It is preferred in the practice of the present invention to have the pH of the fluid to be tested for glucose content at between about pH 4 and 8, preferably between pH 5.5 and pH 7.5 when the diagnostic composition of the invention is admixed therewith. It is therefore preferred to add a suitable buffer to the diagnostic compositions of the invention. Preferred among the buffering agents common to the art is a phosphate buffer system. Wherein the diagnostic compositions of the invention are in the form of a solution, said solution is preferably from about 0.1 to about 0.4 molar with respect to the buffer. Where the compositions of the invention are in a dry form, a suflicient amount of buffer is added so that a solution formed therefrom will have the desired concentration of buffer. Alternately, the buffer may be added as a solution to the solution of diagnostic composition before conducting the diagnostic determination.
The selection of the particular tetrazolium compound in the practice of the present invention may be determined by the type of test to be conducted. For example, a tetrazolium compound producing a formazan which absorbs light in the spectral range sensitive to the eye can be utilized for qualitative or semi-quantitative glucose determinations such as by the use of bibulous strips impregnated with reagent. Where quantitative determinations are to be carried out utilizing a photometer, the selection of a tetrazolium compound should be made so that the light absorbance of the formazan produced therefrom in the reaction is maximal at the wavelength capacity of the particglar apparatus being utilized. The following table illustrates the wavelength absorption maximum of the formazan produced from preferred tetrazolium compounds in accordance with the invention.
TABLE Wavelength of the absorption maximum of the formazan in Color of the form- Tetrezolium component azan Thlazolyl blue 570 nm Violet. Tetranitrotetrazolium blue 530 nm Blue-red. Nltrotetrazolium blue chloride Blue. Iodonitrotetrazolium chloride. Neotetrazolium chlorlde 560 nm Triphenyltetrazolium chloride 588 mu Tetrazolium violet 510 m Tetrazollum blue 570 nm B e.
polyethylene glycol The following examples serve to further illustrate the invention.
EXAMPLE 1 The following solution was prepared for the quantitative determination of glucose:
of solution thereof Amount of solution Component used Buffer-potassium hos hate pH 7.0 Phenazinium met 05 ate Thlnzolyl blue Glucose oxldase 4 EXAMPLE 2 The procedure of Example 1 was repeated utilizing a buffer solution containing in each ml. 1.0 g. of gelatin. As the formazan compound formed in the reaction was held in suspension treatment with ethanol was not necessary. The photometer reading was taken directly after the two minute incubation period.
Example 3 The procedure of Example 2 was repeated replacing the gelatin with an equal amount of polyoxysorbitanic acid ester (Tween 81, Atlas Powder Co.).
Example 4 The following solution was prepared and impregnated on strips of bibulous material, such as filter paper. The paper was soaked in the solution and dried with the exclusion of light. The thus-formed srips were utilized to Example 5 The following solution was prepared for the quantitative determination of glucose.
Component: Amount, ml. Glucose oxidase (20 mg./ml.) 0.05 Meldola blue (2 mg./ml.) 0.05 Iodonitrotetrazolium chloride (2.5 mg./ml.) 0.40 Buffer (0.2 M potassium phosphate pH 7.0 1%
polyethylenglycol 1500) 1.50
To this solution is added 1 ml. of a sample solution.
-The content of glucose is determined by measuring in a photometer the extinction dilference in comparison with a blank test in the wavelength range of about 492 nm.
What is claimed is:
1. A diagnostic composition for the detection of glucose in fluids consisting essentially of glucose oxidase, a tetrazolium salt and a substance selected from the group consisting of a phenazinium salt, a phenoxazinium salt and mixtures thereof and one or more non-reactive adjuvant materials selected from the group consisting of buffers, solubilizing agents and suspending agents.
2. The composition of claim 1 wherein said buffer is suitable for maintaining the pH of said fluids between pH 4 and 8.
3. The composition of claim 1 wherein said phenazinium salt is selected from the group consisting of phenazinium methoand phenazinium etho-salts.
4. A test indicator for detecting glucose in fluids consisting essentially of a bibulous carrier impregnated with the composition of claim 1.
5. A test indicator for detecting glucose in fluids consisting essentially of a bibulous carrier impregnated with the composition of claim 1 wherein said phenazinium salt is phenazinium methosulfate and said tetrazolium salt is thiazolyl blue.
6. A test indicator for detecting glucose in fluids consisting essentially of a bibulous carrier impregnated with the composition of claim 1 wherein said phenazinium salt is 7-dimethylamino-1,2- benzophenoxazinium chloride and said tetrazolium salt is iodonitrotetrazolium chloride.
7. A diagnostic composition for the detection of glucose in fluids consisting essentially of a solution of the composition of claim 1 in a solvent consisting essentially of water and a lower alkanol.
8. A diagnostic composition for the detection of glucose in fluids consisting essentially of an aqueous solution of the composition of claim 1 wherein said solubilizing agent is selected from the group consisting of polyethylene gly- 001 1500 and po1yoxyethylene(5)sorbitan monooleate.
9. A diagnostic composition for the detection of glucose in fluids consisting essentially of an aqueous solution of the composition of claim 1 wherein said suspending agent is selected from the group consisting of gelatin and an alginate.
10. The composition of claim 2 wherein said phenoxazinium salt is selected from the group consisting of 7-dimethylamino-1,2-benzophenoxazinium chloride and 7- dimethylamino l,Z-benzophenoxazinium chloride.
11. A test indicator for detecting glucose in fluids consisting essentially of a bibulous carrier impregnated with the composition of claim 2.
12. A diagnostic composition in accordance with claim 13. The composition of claim 3 wherein said phenazinium metho-salt is phenazinium methosulfate.
References Cited UNITED STATES PATENTS 3,123,443 3/1964 Smeby 252-408 X 3,233,974 2/1966 Bradley 252-408 X MAYER WEINBLA'IT, Primary Examiner US. Cl. X.R.
23-230 B, 253 TP; 424-7