JPS5911197A - Determination of substrate or enzymatic activity - Google Patents

Abstract

PURPOSE:To measure a true substrate or enzymatic activity simply, by reducing the admixture of materials occurring in a specimen causing a measuring error. CONSTITUTION:In the determination of substrate or enzymatic activity in a specimen by treating a substance formed by a substrate or enzymatic reaction with oxidase, measuring formed hydrogen peroxide, the specimen is reacted with the first reagent containing the oxidase, peroxidase, and a phenol derivative shown by the formula (R1 is halogen; R2 is lower alkyl, lower acyl, lower alkoxy, lower alkoxycarbonyl, etc.; n is 1-4) to form hydrogen peroxide, which is reacted with the phenol derivative in the presence of peroxidase, reacted with the second reagent containing a coupler such as 4-aminoantipyrine, colored, and it is determined by colorimetry.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for quantifying substrate or enzyme activity in body fluids.

In recent years, methods for measuring the amount of radical (ii) in body fluids or hydrogen peroxide produced have been widely used in clinical tests.

As a method for measuring these hydrogen peroxides, the enzymatic action of peroxidase is used to detect s (1) couplers such as 4-aminoantipyrine\3-methyl-2-benzothiacyly/'hydrazine and (2) phenol derivatives, aniline derivatives, or naphthol derivatives. A method is used in which a color former is formed by oxidative condensation with a chromogen such as, and the optical absorbance of the color former is measured. This method is characterized by easy operation.

However, recently, cholesterol ester, triglyceride, amylase-GOT, G
In clinical diagnosis such as PT, free cholesterol in body fluids is
There is a problem in calculating errors such as free glycerol, glucose, and pyruvic acid.

The present inventors prevent the inclusion of free cholesterol, free glycerol monoglucose, pyruvate, etc. in body fluids,
We conducted various intensive studies with the aim of accurately quantifying target substrates such as cholesterol esters and triglycerides, or enzyme activities such as amylase, glutamic acid pyruvate transaminase (GPT), and glutamic acid oxyaloacetate transaminase (GOT). We have arrived at the present invention. That is, the present invention provides a method for quantifying substrate or enzyme activity in a sample by causing an oxidase to act on a substrate or a substance produced by an enzymatic reaction and measuring the produced hydrogen peroxide. 11) Peroxidase and (110 the following general formula (
A substrate or enzyme characterized in that a test solution containing a phenol derivative represented by I) is used as a first test solution, a test solution containing a 0ψ coupler is used as a second test solution, and the second test solution is added after the first test solution is added to the sample. This is a method for quantifying activity.

General formula (I): H R.

(In the formula, R8 represents a halogen. R2 is a lower alkyl group having 1 to 5 carbon atoms, ■ a lower acyl group having 1 to 5 carbon atoms, and ■ a lower acyl group having 1 to 5 carbon atoms. A lower alkyl ether group, (i) a lower alkyl dicarbonyl group having 1 to 5 carbon atoms, or (i) a group of the above (i) to (i) having a hydroxyl group or a sulfonic acid group. n = 1 to 4.) This invention By adding the second test solution after adding the above-mentioned first test solution, it is possible to prevent the measurement of free cholesterol, free glycerol, glucose, pyruvic acid, etc. in body fluids, and easily and accurately measure the target substrate or enzyme. It became possible to measure activity. Free cholesterol, free glycerol, glucose 1-pyruvate, etc. in body fluids react with phenol derivatives-aniline derivatives or naphthol derivatives (self-integration reaction) and are converted into chemical substances that do not absorb in the visible region, and are converted to the target substrate or It is thought that it is not involved in the reaction system for measuring enzyme activity. On the contrary, the second
If you add the test solution and then add the first test solution, the objective will not be achieved. In addition, a test solution containing 4-aminoantipyrine together with oxidase and peroxidase was used as the first test solution, and 7 e/-
Even if a test solution containing a fluorine derivative is used as a second test solution and the first test solution is added and then the second test solution is added, the objective will not be achieved. For example, in triglyceride measurement, hydrogen peroxide generated using glycerokinase and glycerophosphate oxidase is reacted with -4-aminoantipyrine in the presence of peroxidase (self-condensation reaction), and then lipase and a heptanol derivative are added to form a color former. An attempt was made to colorimetrically measure only the true triglyceride by producing \\true triglyceride. However, in this method, a substance that has absorption in the visible region (a substance that affects the measurement wavelength) is produced in the first reaction between 4-aminoantipyrine and free glycerol (self-condensation reaction), and the second reaction A problem arose in the colorimetric measurement of the chromophoric substance produced by the method, which resulted in a correct error.

In addition, one method for measuring true triglycerides is to measure the total triglyceride value and the free glycerol value, which include the free glycerol value, and find the difference, but this method has a problem in terms of simplicity. The present invention is superior to the above methods in terms of simplicity.

The method of the present invention is a method for quantifying substrate or enzyme activity in a sample by allowing an oxidizing enzyme to act on a substrate or a substance produced by an enzymatic reaction and measuring the produced hydrogen peroxide.

Substrates to be quantified include cholesterol esters, triglycerides, and creatinine-creatine in body fluids.

Examples of enzymes to be quantified include amine transaminases in body fluids, such as glutamate oxyaloacetate transaminase (GOT) and glutamate acetate.

Examples include lupine transaminase (GPT)-amylase.

Substances produced by enzymatic reactions include cholesterol produced by the action of cholesterol esterase on cholesterol esters, cholesterol produced by the action of lipase on 1-noceride, 1-nocerol, and cholesterol produced by the action of glycerokinase on glycerol. Examples include pyruvic acid produced from glycerol-3-phosphate-α-ketoglutaric acid and alanine with glutamic acid pyruvate, and butotsaccharide produced by the action of amylase or lysulfamylase using starch or r-cyclodextrin as a substrate.

The oxidizing enzyme that acts on the substrate or the substance produced by the enzymatic reaction includes cholesterol oxidase,
These include glycerol oxidase, lysyl-1-neptanophosphate oxidase, pyruvate oxidase, sarcosine oxidase, and glucose oxidase.

Examples of the phenol derivatives represented by the general formula (I) used in the present invention include 3-methyl-4-chlorophenol, 3-methyl-4-bromophenol-3-ethyl-4-chlorophenol 113-butyl-4- Chlorophenol-3-acetyl-4-chlorophenol, 3-
Ethylcarbonyl-4-chlorophenol, 3-methoxycarbonyl-4-chlorophenol, 3-ethoxycarbonylchlorophenol, 3-hydroxymethyl-4
-chlorophenol, 3-hydroxyethyl-4-chlorophenol, 3-hydroxypropyl-4-chlorophenol, 3-sulfopropyl-4-chlorophenol, 3-hydroxyacetyl-4-chlorophenol, 3
-sulfopropylcarbonyl-4-chlorophenol,
Examples include 3-hydroxyacetoxycarbonyl-4-chlorophenol.

Couplers used in the present invention include 4-aminoantipyrine S3-methyl-2-benzothiazoline hydrazone and the like.

Use as a test solution. The second reagent solution may contain an aniline derivative.

Aniline derivatives include aniline, N,N-dimethylaniline, N,N-diethylaniline-N,N-diethyl-m-toluidine, N,N-dimethyl-m-anisidine, N-ethyl-N-(3-methylphenyl )-N-acetylethylenediamine\N-ethyl-N-(β-hydroxyethyl)-m-toluidineNN-ethyl-N-(
2-Hydroxy-3-sulfopropyl)-m-)luidine sN-ethyl-N-sulfopropyl-m-)luvidine 1N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline, N-ethyl-N-(2-hydroxy- 3
-sulfopropyl) - 3.5-dimethoxyaniline, N-ethyl-N-sulfopropyl-m-anisidine 1
Examples include N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-anisidine.

In addition to the above-mentioned components, the first reagent solution and the second reagent solution contain a buffer, and if necessary, a surfactant, a stabilizer, and the like. As the buffer, a conventional one is used, and it is preferable to adjust the size of the first reagent solution and the second reagent solution to 5 to 10.

The content of phenol derivative is 1×10 in the first test solution.
~IXIO-2M. The content of Bt in the coupler is 1X10 to IXIO-''M in the second test solution.

The reagent used in the present invention may contain other enzymes, substrates, various stabilizers, reagents for removing harmful substances, surfactants, and the like.

Next, the substrate or enzyme activity will be specifically explained. The present invention is not limited to these specifically described examples.

For example, to measure cholesterol ester as a substrate, a first test solution containing cholesterol oxidase-peroxidase, a 7-ether derivative, and a buffer is prepared, and a second test solution is prepared containing cholesterol esterase, 4-aminoantipyrine, and a buffer. Prepare a test solution containing the agent. The first test solution is applied to the sample, and hydrogen peroxide generated from free cholesterol is reacted with a phenol derivative (self-condensation) in the presence of peroxidase, and then the second test solution is applied to react only with the cholesterol generated from cholesterol ester. The produced hydrogen peroxide is reacted with a phenol derivative and 4-aminoantipyrine in the presence of peroxidase to obtain a colored material, which is then colorimetrically determined.

For example, when measuring triglyceride as a substrate, prepare a first test solution containing glycerol oxidase/peroxidase, a phenol derivative, and a buffer, or a test solution containing glycerol kinase, glycerophosphate oxidase, peroxidase, a phenol conductor, and a buffer; Riboprotein lipase and 4-
Prepare a test solution containing aminoantipyrine and a buffer. The first reagent is applied to the sample, and hydrogen peroxide generated from free glycerol is reacted (self-condensed) with a phenol derivative in the presence of peroxidase, and then the second reagent is applied to react only with the glycerol generated from triglyceride. The hydrogen peroxide produced is determined colorimetrically.

To measure the activity of an enzyme, for example, glutamate pyruvate transaminase (GPT), first prepare a test solution containing pyruvate oxidase, peroxidase, a heptanoyl derivative, and a buffer as a first test solution, and prepare a test solution containing α-ketoglutar as a second test solution. A test solution containing acid, D, L-aniline, 4-aminoantipyrine, and a buffer is prepared, and the first test solution is applied to the sample to react hydrogen peroxide generated from free pyruvic acid with a phenol derivative in the presence of peroxidase. (Self-condensation), then apply the second test solution to reduce %G
Hydrogen peroxide produced only from pyruvic acid produced by the action of PT is determined colorimetrically.

In addition to measuring the substrates and enzyme activities described above, the method of the present invention can also be used to measure hydrogen peroxide produced by other oxidizing enzymes.

Compared to quantitative methods that use a mixture of a first test solution and a second test solution, the method of the present invention reduces the amount of substances that coexist in the sample that cause measurement errors, and makes it easy to measure the true substrate or enzyme activity. It became possible to do so.

Next, the present invention will be explained using examples.

Example 1 Triglyceride in a sample was measured using the following reagent and the following method.

L Sample: Glycerin (26■/dl) solution ■
Glycerin (52■/aZ) solution ■Glycerin (1
04■/al) Solution ■Serum a:water = 9:1
■Serum miniglycerin aqueous solution ■(1,
04040 ■/dl971 Serum b: Water = 9 = 1 ■ Serum b: Glycerin aqueous solution ■ (1, o4otIVal) =
9: 1z K drug: Reagents An and reagents a to e, whose phenol derivatives were compounds shown in Table 1 below, were prepared.

First test solution Tris buffer (+) + (7,0) glycerokinase 2.0 units/-glycerophosphate oxidase 6.0 units/peroxidase resistance
10.0 units/-phenol derivative
5kg/dl 2nd test solution Tris buffer (pH 7,0) Riboprotein lipase 600 units/-4-aminoantipyrine 10~/1llN-x Chill-N-
(3-7, Luhopro 90 Ilv/dl Building Lm-Anisidine Technique Measurement method: Add the first test solution 2- to 20 μl of each sample, react at 37°C for 5 minutes, then add the second test solution 1- and heat to 37°C. The chromophore of 4-amino/antipyrine and N-ethyl-N-(3-sulfopropyl)-m-anisidine was measured at a wavelength of 540 nm.
Shown in the table.

Table 2 In Table 2, distilled water is a control example for samples ■, ■, and ■, sample ■ is a control example for sample ■, and sample ■ is a control example for sample ■.

3-Acetyl-4-chlorophenol used in the reagent is superior in glycerol scavenging ability compared to m-bromophenol\p-chlorophenol and phenol% 2.4-dichlorophenol used in other reagents. .

Example 2 Triglyceride in a sample was measured using the following reagent and the following method.

L sample; glycerin (26 mq/al) solution
■Glycerin (52 kg/dl) solution ■Glycerin (104 ■/dl) solution ■Serum a: water - 9 =
1 ■Serum PL: Glycerin aqueous solution
■(1,o4o~/d7) = 9:1 Serum b: Water = 9:1 ■Serum b: Glycerin aqueous solution ■(1,040η/a = 9:
1 2 Reagent: Ta.

1st test solution Tris buffer (7.0) glycerokinase 2.0 units/-glycerophosphate oxidase 6. o Enthronement/-peroxidase
10.0 units/d phenol derivative
5q,'al 2nd reagent solution Tris buffer (g(7,o)
Riboprotein lipase 600 units/-4-aminoantipyrine l0W9/d, i! N, N-
Diethyl-m-90m? /d1 Toluidine & Measurement method Add 2tIl of the first test solution to 20μl of each sample and heat at 37°C.
After reacting for 5 minutes at , the second test solution was added and reacted at 37°C for 10 minutes, and the color forming product of 4-aminoantipyrine and N,N-diethyl-m-toluidine was measured at a wavelength of 540 nm. . The results are shown in Table 4.

Table 4 4-chloro-m-cresol used in test ZA%B, 4-
Bromo-m-cresol is superior in glycerol scavenging ability compared to O-chlorophenol, 3,4-dimethoxyphenol, and 0-isopropylphenol-dimethoxyphenol used in other reagents.

Patent applicant: Toyobo Co., Ltd.

Claims (1)

[Claims] A method for quantifying a substrate or enzyme activity in a sample by causing an oxidizing enzyme to act on a substrate or a substance produced by an enzymatic reaction and measuring the produced hydrogen peroxide, comprising (1)
) oxidase, (II) peroxidase, and (110) a test solution containing a phenol derivative represented by the following general formula (1) as the first test solution, a test solution containing a coupler as the second test solution, and A method for quantifying substrate or enzyme activity, which comprises adding a second test solution after adding a first test solution. General formula (1): (In the formula, R1 represents a halogen. R2 is a lower alkyl group having 1 to 5 carbon atoms.
■Lower acyl group having 1 to 5 carbon atoms, ■Lower alkyl ether group having 1 to 5 carbon atoms, ■Lower alkoxycarbonyl group having 1 to 5 carbon atoms, or ■Hydroxy or sulfonic acid group. The above groups (1) to (2) have the following formulas. n=1~\. )