JPS6259782B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates generally to the field of clinical testing, and more particularly to tests useful for the qualitative and quantitative determination of uric acid in body fluids such as urine or blood. More specifically, it relates to a test in which uric acid is converted into oxidizing substances such as peroxide. It has long been known that red quinoneimine dyes are obtained by the oxidative coupling reaction of phenol with 4-aminophenazone, also known as 4-aminoantipyrine, and was published in the Journal of Organic Chemistry (J.Org. .
Chem.) Vol. 8, p. 417 (1943) by Emerson. This reaction was described by Trinder as follows:

[Table] It has been popular in clinical chemistry since it was applied to the enzymatic measurement of glucose based on [Ann. Clin. Biochem. 6:24 (1969)]. Emerson Trinder
Color-forming phenols (including substituted phenols) + 4-aminophenazone +
Peroxidases have been used for the quantitative determination of cholesterol and uric acid as well as glucose in serum, plasma or other biological fluids.
The use of this system for glucose measurements is disclosed in US Pat. No. 3,886,045, now reissued as Re29498 to Meiattini. The general scheme of this reaction is as follows. (1)

[Table] (2) Many phenols can be used in the Emerson-Trinder reaction. Specific examples of phenols most commonly used in clinical chemistry include phenols; p-hydroxybenzoic acid esters;
Examples include 2,4-dichlorophenol; 3,5-dichloro-2-hydroxybenzenesulfonic acid. A variety of substituted and unsubstituted naphthols can be used as well. Sample components that can be measured include glucose, cholesterol, uric acid, or other metabolites that can be oxidized with concomitant generation of hydrogen peroxide by specific oxidizing enzymes. The oxidases include glucose oxidase for glucose measurement; cholesterol oxidase for cholesterol measurement (cholesterol ester hydrolase is also added to hydrolyze esterified cholesterol); and uricase for uric acid measurement. be. The amount of dye produced is proportional to the concentration of hydrogen peroxide and thus to the concentration of the components in the sample. The concentration of a component in a sample can thus be obtained by simply measuring the absorbance of the reaction solution and comparing the resulting value with the absorbance of a known standard solution of the component. The produced pigment is in the visible region, generally 500 ~
500nm region (depending on the phenol used)
and can be measured using only a colorimeter or a photometer in the visible range. This Emerson-Trinder coloring system uses an oxidative coupling reaction between a reducing compound and bilirubin.
In other words, it is normally present in serum at a concentration of less than 1 mg/dl, but in some diseases it can reach very high levels (20 mg/dl or less).
ml/dl or more). Bilirubin levels above normal affect enzymatic tests for glucose, cholesterol, and uric acid by attenuating the color of the reaction. The disturbance increases as bilirubin levels increase. The negative interference of reducing substances (e.g. ascorbic acid) means that the reducing substance is primarily a competitor of the chromophore in the peroxidase catalyzed reaction involving hydrogen peroxide or a decolorizing agent (bleaching agent) for the color formed.
It is very clear that it acts as However, interference with reducing substances is not a real problem, and at least 3 mg of ascorbic acid/
It is not a problem in serum that rarely exceeds dl. In contrast, interference with bilirubin is a serious problem when measuring metabolites in serum by the Emerson-Trinder color system, and in routine laboratory practice where hyperbilirubinemic samples are often found. is the main negative aspect of this system. The reaction mechanism of bilirubin is extremely complex and has not yet been fully elucidated. The best research to date on this issue suggests that interference with bilirubin may be due to one or more of the following factors: simple spectral effects acting as another peroxidase substrate or degradation of peroxidase reaction intermediates. Attributing Witte's Clinical Chemistry (Clin.Chem.) 24:
1778 (1978). Therefore, a first object of the present invention is to provide an improved test for the detection of uric acid in liquid samples. A second object of the invention is to provide an improved test for the detection of uric acid that is highly resistant to the interfering effects of bilirubin. Other objects and a fuller understanding of the invention may be obtained by reference to the following description and claims of preferred embodiments thereof. Bilirubin is produced primarily by two different mechanisms: (1) by overlapping the spectra of the pigments formed during the reaction, causing a positive interference; and (2) by a chemical mechanism causing a negative interference, as described above. It has therefore been found as part of the present invention that types with substituted or unsubstituted phenols and 4-aminophenazones greatly interfere with the color test. While the positive interference resulting from the first mechanism can be reduced by reading the absorbance at wavelengths of 520 nm or higher, the same is not true for the second mechanism. This second mechanism plays a very important role and gives inaccurate results in the above-mentioned tests when bilirubin is present in abnormal concentrations in the sample. In contrast to the compositions of the prior art, the compositions of the invention are highly sensitive to uric acid present in body fluids, while being highly resistant to bilirubin interference. According to the invention, in a liquid sample containing uricase and peroxidase, phenol and 4-aminophenazone, the liquid sample is rendered resistant to bilirubin interference by the inclusion therein of reagent means consisting of ferrocyanide ions. This surprising result is achieved with a composition for detecting uric acid. In the following description, certain terminology is used for clarity; however, these terms are intended to refer only to particular embodiments of the invention, selected for exemplary illustration. , is not intended to define or limit the scope of the invention. The compositions of the present invention can take many physical forms and include phenols, including substituted and unsubstituted phenols known for use with 4-aminophenazone indicator compositions, in combination with reagent means consisting of ferrocyanide ions. contains. Additionally, materials such as stabilizers and other conventional additives can be added to these, if desired. Preferred reagent means comprising ferrocyanide ions include alkali metal salts _{of ferrocyanide, such as sodium ferrocyanide or potassium ferrocyanide, as well as other salts or systems containing or capable of releasing Fe(CN)-46} ^ions . Examples include ferrocyanide ion sources. The composition of the invention comprises means for providing an oxidizing substance in response to uric acid present in a liquid sample together with a reagent means according to the invention. Such a uric acid response means preferably has enzymatic properties, and preferably comprises uricase and a peroxidizing active substance. Concentrations and types of reagents useful in uric acid response measures are known in the art. The test means can be used as a solution for measuring uric acid. The solvent used when preparing the solution is
Water, a physiological solution, an organic solvent such as methanol, or a mixture thereof. To detect uric acid, this composition must be used in urine,
cerebrospinal fluid, tissue culture supernatant and preferably serum;
It is preferable to use it in addition to samples such as plasma or whole blood. When the composition is used in the form of a solution, the reagent means consisting of ferrocyanide ions is added in an amount of approximately 1.0 μmol/ml.
It is preferable to use the concentration of a saturated solution.
This preferred range is about 5 μmol/ to about 50 μmol/
It is mol/. The concentration of uricase is preferably about 10 I.U./ to about 200 I.U./. The concentration of peroxidase is approximately 10 I.U./~200 I.U.
/ is preferred. Enzyme activity is expressed in international units (IU), 1I.
U. is 1 μmol of substrate under specified pH and temperature conditions.
is the amount of enzyme activity required to catalyze the conversion of . Horseradish peroxidase and uricase used in the examples were obtained from Research Products Division, Miles Laboratories, Inc., Elkhart, Indiana.
Laboratories, Inc., Elkhart, Indiana). Also provided is a test device comprising a composition of the invention and a method of manufacturing such a test device, which comprises incorporating the composition into a carrier such as a matrix. If such an inclusion operation is carried out by impregnation with a solution of the composition according to the invention, the carrier impregnated in this way is then dried. In addition to impregnation, the test devices of the invention can be made by other suitable inclusion techniques, such as printing or spraying the composition onto a substrate or matrix. Alternatively, the compositions of the invention may be used in a carrier in the form of a compressed tablet containing conventional carrier materials. The term carrier refers to a matrix that is insoluble and retains its structure intact when in contact with physiological or other fluids. Suitable matrices that can be used include paper,
Various organic polymers such as cellulose, wood, synthetic fleece, glass fibers, woven and non-woven fabrics, gelatin, polypropylene and other organic materials known to those skilled in the art as film-forming materials may be mentioned. Conveniently, the carrier or test device can be combined with an inert support or handle member, which may be made of polystyrene, for example. If the test composition is used for detecting uric acid in blood, the surface of the impregnated carrier matrix is advantageously coated with a semi-permeable transparent coating film of ethylcellulose or other suitable material. be. This is achieved, for example, by applying a layer of ethylcellulose dissolved in benzene to the surface of the impregnated carrier matrix and then removing the solvent by evaporative drying. Uric acid indicators in the form of treated carrier matrices or test devices are often stored for a considerable period of time before use. Therefore, it is desirable that the selected reagents not be easily autoxidized in air. Preferably, the test device should not be exposed to light;
In some cases, it may be desirable to keep one or more test devices enclosed in a moisture-proof container except for the purpose of removing them immediately before use. If necessary, the carrier matrix can be treated with a background dye of a particular color, such as yellow, so that the color produced by the test reaction mixes with the background color and corresponds to the concentration of the sample components. It produces a variety of color tones. Preferably, the test device is prepared by a single dip method. The concentration of the reagent used for immersion is 10 ^-3
Ranges from mM to saturated solutions. 4-aminophenazone is generally most useful at a concentration of about 0.2mM. The concentration of peroxidase in the soaking solution is approximately
It is 0.1 mg/dl to about 20 mg/dl. Solutions used in preparing the impregnating solution include water, physiological solutions, organic solvents, or mixtures thereof. Advantageously, the test device is momentarily immersed in the test sample or the test sample is otherwise introduced onto the carrier matrix, so that when uric acid is present, a detectable color appears on the matrix. changes occur. The test device can be used in a similar manner when testing plasma, serum or body fluid samples. However, when testing whole blood,
Preferably, one drop of blood is brought into contact with the surface of the test device. The examples shown are merely illustrative and are not intended to limit the invention. Those skilled in the art will be able to modify, substitute, and change components and parameters as desired. Example 1 Uricase and peroxidase/3,5-dichloro-2-hydroxybenzenesulfonic acid/4
- Determination of uric acid in serum using aminophenazone A test solution was prepared according to the prior art and the present invention,
The resistance to the interfering effect of bilirubin during uric acid measurement was compared. A test solution was prepared according to the prior art with the following composition. Phosphate buffer 150 mmol, PH7.0 Uricase 60 I.U./ Peroxidase 140 I.U./ 4-aminophenazone 0.24 mmol/ 3,5-dichloro-2-hydroxybenzenesulfonic acid 2.0 mmol/ Composition of the present invention included The test solution is 20μ
mol/potassium ferrocyanide [K ₄ Fe
(CN) ₆ ] was added as described above. 2.0 ml of the prior art test solution was pipetted into each of the first group of test tubes, and 2.0 ml of the test solution of the composition of the invention was pipetted into each of the second group of test tubes. A series of similar samples were placed in each group of test tubes. Serum samples were obtained and collected and tested for uric acid and bilirubin content. Add 6.0 mg of uric acid to the collected serum.
was added to a concentration of dl and then the solution was divided into aliquots. Bilirubin concentration is 0.7, 1.4,
2.2, 3.6, 5.0, 6.5, 8.8, 12.1, 16.7 and 23.4
The amount of bilirubin was added to give mg/dl to obtain uric acid test solution samples having respective concentrations. Other uric acid solutions without bilirubin were used as standard solutions. Each group of tubes was injected with a series of similar 0.05 ml samples containing various concentrations of bilirubin, and the reactions in these tubes were allowed to run for 15 minutes at room temperature. Absorbance was read at 520 nm against a corresponding sample blank obtained by omitting uricase from this reagent formulation. Results are shown in Table 1 reporting the percent recovery of uric acid when tested using the prior art and test compositions of the present invention at various concentrations of bilirubin.

Table: Significant chemical interference (approximately 6%) is observed in the uric acid test in the absence of ferrocyanide even at bilirubin levels as low as 2 mg/dl, and this is even more pronounced at levels of 5 mg/dl. (20
% or more). When using ferrocyanide, the chemical interference of bilirubin is greatly reduced (statistically insignificant at 2 mg/dl bilirubin, 30% at 5 mg/dl, and about 10% at higher bilirubin of 15-20 mg/dl). ). Example 2 Uricase and peroxidase/3,5-dichloro-2-hydroxybenzenesulfonic acid/4
- Test device for measuring uric acid in urine using aminophenazone Test devices including the prior art and the composition according to the invention were prepared and their resistance to the interfering effect of bilirubin when testing for uric acid present in urine was compared. did. A prior art impregnation solution was prepared having the following composition. Phosphate buffer 150 mmol, PH7.0 Uricase 150 I.U. Peroxidase 860 I.U. 4-aminophenazone 0.24 mmol 3,5-dichloro-2-hydroxybenzelsulfonic acid 2 mmol Water was added to make 1000 ml. The impregnating solution containing the composition of the invention is 20μ
mol/potassium ferrocyanide [K ₄ Fe
(CN) ₆ ] was added in the same manner as above. A sheet of Whatman No. 17 paper (Whatman, Inc. Clif, NJ) was impregnated with the impregnating solution to saturation and dried at 60°C. These sheets containing the dried residue of the impregnating solution were cut into 2.5 x 2.5 mm to make test devices. The test device was then lined with double-sided adhesive tape and secured to a plastic handle. Urine samples were obtained, collected and tested for uric acid and bilirubin content. The collected warm urine was then diluted with 10 times the volume of distilled water. Uric acid, concentration 6
It was added to the diluted urine collected until it reached mg/dl. A test solution was then prepared by adding bilirubin to the collected diluted urine until its concentration was 10 mg/dl.
This test solution was divided into portions. A test device according to the invention was briefly dipped into a uric acid test solution, and a test device according to the prior art was briefly dipped into another solution. After about 10 minutes, the test device was visually inspected for color change. The test device prepared according to the present invention showed a clear color change indicating the presence of uric acid, whereas the test device containing the prior art composition showed no color change and was therefore indicative of the presence of uric acid. gave an incorrect negative value. Although the invention has been described with some particularity, it will be understood that this description is merely exemplary and that many changes may be made in detail without departing from the scope of the invention.

Claims (1)

[Scope of Claims] 1. A composition for measuring uric acid in a liquid sample of the type consisting of uricase and peroxidase, phenol or naphthol and 4-aminophenazone, further comprising reagent means consisting of ferrocyanide ions. Composition. 2. The composition according to claim 1, wherein the phenol is an unsubstituted phenol. 3. The composition according to claim 1, wherein the phenol is 3,5-dichloro-2-hydroxybenzenesulfonic acid. 4. The composition of claim 1, wherein said reagent means is a ferrocyanide ionic salt. 5. The composition according to claim 4, wherein the ferrocyanide ion salt is sodium ferrocyanide. 6. The composition according to claim 4, wherein the ferrocyanide ion salt is potassium ferrocyanide.