JPS5911196A - 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 an 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 lower acyl, lower alkoxy, etc.; R2 is H, halogen, lower alkyl, lower acyl, lower alkoxy, etc.) to give 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, as a standard method for determining the amount of substrate or enzyme activity in body fluids in clinical tests, methods have been widely used in which oxidizing enzymes are applied to substrates or substances produced by enzymatic reactions, and the hydrogen peroxide produced is measured. It is being

As a method for measuring hydrogen peroxide, (1) 4-aminoantipyrine,
A method involves oxidative condensation of a coupler such as 3-methyl-2-benzothiazolinone hydrazine and (2) a chromogen such as a phenol derivative, aniline derivative, or naphthol derivative to form a coloring material, and then measuring the optical absorbance of the chromogen. It is used. This method is characterized by easy operation.

However, recently, this measurement method has been used to measure cholesterol esters, triglycerides, amylase, GOT, and GOT.
In clinical diagnosis such as PT, it has become a problem to measure free cholesterol, free glycerol, glucose, pyruvic acid, etc. in body fluids as measurements.

The present inventors prevent the inclusion of free cholesterol, free glycerol, glucose, pyruvate, etc. in body fluids,
After conducting various studies with the aim of accurately quantifying the target substrates such as cholesterol esters and triglycerides, or enzyme activities such as amylase, glutamic acid pyruvate transaminase (GPT), and glutamic acid p-acetate transaminase (GOT), we found that
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. 1
1) Peroxidase and (lli) the following general formula (I)
A substrate or enzyme characterized in that a test solution containing a phenol derivative represented by the following formula is used as a first test solution, a test solution containing a (lv) 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 (D: OH R9 (in the formula, Mr.) represents ■ a lower acyl group having 1 to 5 carbon atoms, ■ a lower alkyl ether group having 1 to 5 carbon atoms, or ■ a hydroxyl group or a sulfonic acid group. Have the above ■
Indicates a group of ~■. R2 is hydrogen, halogen, or ■lower alkyl group having 1 to 5 carbon atoms, ■having 1 carbon atom
~5 lower acyl group, ■ lower alkyl ether group having 1 to 5 carbon atoms, ■ lower alkoxycarbonyl group having 1 to 5 carbon atoms, or ■ hydroxyl group or sulfonic acid group of the above ■ to ■ Indicates the group. n=0 to 4. ,) In the present invention, by adding the second test solution after adding the first test solution, it is possible to prevent the measurement of free cholesprol, free glycerol, glucose, pyruvic acid, etc. in body fluids, and to easily and accurately measure the intended purpose. It has now become possible to measure the substrate or enzyme activity. free cholesterol in body fluids,
Free glycerol, glucose, pyruvic acid, etc. react with phenol derivatives, aniline derivatives, or naphthol derivatives (self-condensation reaction) and convert them into chemical substances that do not show absorption in the visible region.This reaction is used to measure the target substrate or enzyme activity. It is thought that it is not involved in the system. Conversely, if the first test solution is added after the second test solution, the objective will not be achieved.

Furthermore, even if a test solution containing 4-aminoantipyrine together with an oxidase and peroxidase is used as the first test solution, a test solution containing a phenol derivative is used as the 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 produced using glycerokinase and glycerophosphate oxidase is reacted with 4-7 minoantibilin in the presence of peroxidase.
After the self-condensation reaction, we added lipase and a Funiol 5-storey conductor to generate a chromophore, and attempted to colorimetrically measure only true triglycerides. However, in this method, a substance that has absorption in the visible region (a substance that casts a shadow of 9 on the measurement wavelength) is produced in the first reaction between 4-aminoantipyrine and free glycerol (self-integration reaction), and the second reaction A problem arose in the colorimetric measurement of the chromophoric material produced by the method.

In addition, as a method for measuring true triglycerides, there is a method of measuring the total triglyceride value, which includes the free glycerol value, and the free glycerol value, respectively, and finding the difference between them, but this method has problems in terms of simplicity. Ta. The present invention is superior to the above methods in terms of simplicity.

The method of the present invention can also be applied to substrates! j This is a method for quantifying the substrate or yeast activity in a sample by allowing an oxidizing enzyme to act on a substance produced by an enzymatic reaction and measuring the produced hydrogen peroxide.

Substrates to be quantified include phsterol ester, triglyceride, creatinine, creatine/
F Doga Sai・ru.

Enzymes to be quantified include aminotransaminases in body fluids, such as glutamic acid truncated transaminase (GOT), glutamic acid pyruvate transaminase (GPT), and amylase.

Substances produced by enzymatic reactions include cholesterol produced by the action of cholesterol esterase on cholesterol ester, glycerol produced by the action of lipase on triglyceride, and glycerin 17-ru3 produced by the action of glycerol with glycerol kinase.
- Glucose produced by the action of amylase and glucoamylase using pyruvate, tenbun or γ-cyclodextrin, which is produced by the action of glutamic acid pyruvate transaminase (GP'r) on phosphoric acid, α-ketoglutarate and alanine, and so on.

Examples of oxidizing enzymes that act on the substrates described above or on the substances produced by the enzymatic reactions mentioned above include cholesterol oxidase, glucose oxidase, glycereptate oxidase, pyruvate oxidase, sarcosine oxidase, and glucose oxidase. and so on.

Examples of the phenol derivatives represented by the general formula (I) used in the present invention include (1) p-acetylphenol (p-oxyacetophenone), p-ethylcarbonylphenol (p-oxypropiophenone), 4n-brol, 3- Chloro-4-
Acetylphenol, 3-methyl-4-acetylphenol, 3.4-diacetyltubonol, 3-methoxy-
4-acetylphenol, 3-methoxycarbonyl-4
-acetylphenol, 3-hydroxymethyl-4-acetylphenol, 3-sulpobrobyl-4-acetylphenol, etc.; 1) p-methoxyphenol, p-ethoxyphenol, 4-n-propyloxyphenol, p-isopropyloxyphenol, 3. 4-dime)-(=diphenol, 3-chloro-4-methoxyphenol, 3-methyl-4-methoxyphenol, 3-methoxycarbonyl-
4-methoxyphenol, 3-acedel-4-methoxyphenol, 3-hydroxymethyl-4-ethoxyphenol, 3-sulpobrobyl-4-ethoxyphenol, 3-hydroxyacedel-4-methoxyphenol, etc. ■ p-hydroxyacetylphenol , 4-(hydroxyethoxy)-phenol, 4-(hydroxyconitoxycarbonyl)phenol, 4-(sulfopropylcarbonyl)phenol, 4-(sulfopropyloxy)
Phenol, 3-talolo, 4-hydroxyacetylphenol, 3-methyl-4-(hydroxyethoxy)phenol, 3-acetyl-4-hydroxyacetyl 7et/
-ol, 3-methoxy-4-(hydroxyethoxycarbonyl)phenol, 3-ethoxycarbonyl-4-hydroxyacetylphenol Couplers used in the present invention include 4-aminoantipyrine, 3-methyl-2-benzothiazoline hydrazone, etc. be.

In the test S used in the present invention, a test solution containing (1) an oxidase, (11) a peroxidase, and (Hl) a phenol derivative is used as a first test solution, and a test solution containing (1) a coupler is used as a second test solution. The second reagent solution may contain an aniline derivative.

Examples of aniline derivatives include aniline, N,N-dimethylaniline, N,N-diethylaniline, N,N-diethyl-In-)luidine, N,N-dimethyl-m-anisidine, N-ethyl-N-(3 -methylphenyl)-N
'-acetylethylenediamine, N-ethylene N-(β
-hydroxyethyl)-m-toluidine, N-ethyl-
N-(2-hydroxy-3-sulfopropyl) -m
-) Ruinin, N-ethyl-N-sulfopropyl-m
-) luidine, N-ethyl-N-sulfopropyl-3,
5-”dimethoxyaniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl) = 3.5-dimethoxyaniline, N-ethyl-N-sulfopropyl-m-
Anisitane, N-ethyl-N-(2-hydroxy-3-
Examples include sulfopropyl)-m-anisidine.

In addition to the above 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 one that adjusts the pH of the first reagent solution and the second reagent solution to usually 5 to 10 is preferable.

The content of phenol derivative is lXl0 in the first test solution.
"~IX10"M. The content of the coupler in the second test solution is 1X10' to 1X10'M.

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.

To determine the substrate, for example, cholesterol esterase, a first test solution containing cholesterol oxidase, peroxidase, a phenol derivative, and a buffer is prepared, and a second test solution contains cholesterol esterase, 4-aminoantipyrine, and a buffer. Prepare the test solution. A first test solution was prepared for the sample, and hydrogen peroxide generated from free cholesterol was reacted (self-condensed) with a phenol derivative in the presence of peroxidase.
Peroxide 71 produced only from cholesterol produced from cholesterol esters is reacted with a test solution and phenol-derived 4-aminoantipyrine is reacted with peroxidase, which is produced from cholesterol esters. .

For example, to measure triglyceride as a substrate, the first test solution is a test solution containing glycerol oxidase, peroxidase, a phenol derivative, and a buffer, or a test solution containing glycerol kinase, glycerol kinase, bermexidase, a phenol derivative, and a buffer. A second reagent solution containing ribo r7 tein lipase, 4-aminoantipyrine, and a buffer is prepared. The first test solution is applied to the sample to react (self-synthesize) the peroxide cloud K produced from the reflected glycerol with the phenol derivative in the presence of peroxidase.
Using L solution, hydrogen peroxide produced from triglyceride and only glycerol was measured colorimetrically.
.

# To measure the activity of glutamate pyruvate transaminase (GPT), for example, prepare a test solution containing pyruvate oxidase, vermexidase, a phenol derivative, and a buffer as the first test solution, and prepare a test solution containing α as the second test solution. - Prepare a test solution containing ketoglutaric acid, D, L-aniline, 4-aminoantipyrine, and a buffer, and apply the first test solution to the sample to convert hydrogen peroxide generated from free pyruvic acid into phenol derivative in the presence of peroxidase. After reacting (self-condensation) with GP, the second test solution is applied to
Hydrogen peroxide produced only from pyruvic acid produced by the action of T is measured 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 the quantitative method using a mixture of the first test solution and the second test solution, the method of the present invention reduces the amount of substances present 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.

1. Sample; Glycerin (26mg/de) solution
■Glycerin (52Tng/d/lte) solution ■Glycerin (104■/lte,) solution ■Serum a: water =
9:1 ■ r + tt quenching a: glycerin aqueous solution ■ (1, t + 4
oyy/d+, ) = 9: 1 serum b: water = 9; 1
■ Serum b: Glycerin aqueous solution ■ (1, o, iamp/de) -92 12 Reagent: The first sample in which the phenol derivative is a compound shown in the following No. 11; Test solution
Tris buffer (pl 7.0) glyceroquina H'
2.0 children/Iglycerorin? Oxidase 6.0 single (stand/ml) Peroxidase 10.0 single stand/me Fugonol induction rest 5rn/d1! ¥2 test solution Tris buffer (pH 7, (+)) Lipoprotein lipase 600 square positions/rae 4-aminoantipyrine 1
omtz/deN-Ezaru N-(3-s 90rl
/dllupoblovir)-m-anisidine Table 1 3 Measurement method Add 2 mg of mtEff to each sample 2011t,
After reacting at 37°C for 5 minutes, 1 ml of the second test solution was added and reacting at 37°C for 10 minutes, resulting in a reaction between 4-aminoantipyrine and N-ethyl-N-(3-sulfopropyl)-m-anisidine. The chromophore was measured at a wavelength of 540 nrn.

The results are shown in Table 2.

Table 2 In Table 2, distilled water is sample ■-(2), C
Sample 3) is a control example of Sample 2, and Sample 2 is a control example of Sample 2.

p-Arhythylphenol used in Reagent A is superior in glycerol scavenging ability compared to m-bromophenol, p-chlorophenol, phenol, and 2,4-dichlorophenol used in other reagents.

Triglyceride in the skin tract side 2 sample was measured using the following drug and the following method.

1. Sample: Glycerin (26■/d1.) solution ■Glycerin (52Tn!i/dl) solution
C2) Glycerin (104■/dl) solution ■ Serum a: water = 9; 1 ■ Serum miniglycerin aqueous solution ■ (1, o4 omv/al) = 9: 1 Serum b: water = 9:1 ■ Blood mb: glycerin aqueous solution (x, o4omy/de) = 9: 1 2. Reagents: Reagents A to D and reagents a to e, in which the phenol derivative was a compound shown in Table 3 below, were prepared.

First test solution Tris buffer (pH 7.0) Glycerokinase 2.0 units/ml Glycerophosphate oxidase 6.0 units/rnl.

Peroxidase 10.0 units/'Phenol derivative 5mg/d12 2nd test solution Tris buffer (pH 7,0) Riboprotein lipase 6
00 units/ml 4-amino/antipyrine 10■
/dlN, N-diethyl-m-90mfld(!
Toluidine Table 3 3, Measurement method 2 ml of the first test solution for each 20 μt sample! Add 37
After reacting for 5 minutes at ℃, 1 ml of the second test solution was added and the mixture was allowed to react for 10 minutes at 37℃. It was measured with The results are shown in Table 4.

Table 4: p-Cyprobiophenone, p-oxybutylphenone, and 3,4-diacetylphenol are superior to O-chlorophenol and 3I+-dimerate used in other reagents.

1 person for patent Toyobo Co., Ltd. 55

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, (11) peroxidase, and (II) a test solution containing a phenol derivative represented by the following general formula (D) is the first test solution, and a test solution containing the (1v) coupler is the second test solution.
A method for quantifying substrate or enzyme activity, which comprises adding a first test solution to a sample and then adding a second test solution. General formula (I): OH R. (In the formula, R2 is ■ a lower acyl group having 1 to 5 carbon atoms, ■ a lower alkyl ether group having 1 to 5 carbon atoms, or ■ the above ω to ■ having a hydrogen group or a sulfonic acid group). represents a group.R1 is hydrogen, halogen, or ■lower alkyl group having 1 to 5 carbon atoms, ■1 carbon number.
-5 lower acyl group, ■lower alkyl ether group having 1 to 5 carbon atoms, ■lower alkoxycarbonyl group having 1 to 5 carbon atoms, or ■a hydroxyl group or sulfonic acid group as described above. Indicates the group of n=o~4. )