JPS61173799A - Method of determining activity of substrate or enzyme - Google Patents

Abstract

PURPOSE:In determining activity of substrate or enzyme in a test specimen by measuring formed hydrogen peroxide with peroxidase, to improve measuring accuracy and to make automatic analysis applicable, by using catalase having <=10 times activity value as that of peroxidase. CONSTITUTION:In determining activity of substrate or enzyme in a test specimen by treating a substance formed by a reaction of substrate or enzyme, and measuring formed hydrogen peroxide with a peroxidase system, the first reagent containing oxidase and catalase and the second reagent containing a detecting agent, wherein peroxidase is added to both or one of them, are prepared, the first reagent is added to the test specimen, then the second reagent is added to it, and change in absorbance produced is measured. Preferably peroxidase is added to the first reagent, and the amount of catalase is preferably <=10 times as much as (the activity value) of peroxidase.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a method for quantifying substrate or enzyme activity in body fluids.

Measuring substrate or enzyme activity in body fluids using enzymatic methods has great clinical significance as it provides useful information in clinical diagnosis.

(Conventional technology) In recent years, quantification of substrates surrounding enzymes or enzyme activity has become increasingly important.

In particular, a method of measuring hydrogen peroxide produced using an oxidase is widely used.

However, recently, when quantifying substrate or enzyme activity in body fluids using these measurement methods, it has become a problem that endogenous substances in body fluids involved in the reaction system give a positive error in the measurement of the target substrate or enzyme activity. It is becoming. For example, free glycerol when measuring triglycerides, creatine when measuring creatinine,
Free cholesterol when measuring ester cholesterol, glucose when measuring amylase activity, GOT, biluvic acid when measuring GPT activity, V
Pyruvic acid when measuring alkaline acid.

Examples include uric acid when measuring guanase activity.

There is a way to subtract it. Although this method can prevent errors, it requires a separate reagent for the sample, and additional measurement operations are required, making it cumbersome and impractical as it has low applicability to automatic analyzers.

The second method is to act on endogenous substances directly or in the coexistence of other enzymes with an oxidizing enzyme, and the resulting hydrogen peroxide is self-condensed in the presence of peroxidase and absorbed into the visible region. There is a method in which the target substrate or enzyme activity is measured after hydrogen peroxide is consumed by using a compound that exhibits little to no oxidation. For example, the special public service in 1977-
There is a method of using 4-aminoantipyrine or phenol as a scavenger (a compound that self-condenses hydrogen peroxide and shows almost no absorption in the visible region) as shown in Japanese Patent No. 29159, but in reality, it is In the presence of hydrogen oxide,
The condensate of self-condensed 4-aminoantipyrine is a substance that has a slight absorption in the visible region (a substance that affects the measurement wavelength) and gives a positive error to the colorimetric measurement of the target substrate or enzyme activity.

Particularly when measuring trace amounts of substances in body fluids, there is a drawback that even a small amount of absorption in the visible region can give a large error in the measured value.

In addition, although this method can reduce the error in colorimetric determination in the visible region, it is not effective in quantifying the ultraviolet region because the absorption of the self-condensing compound itself exists in the ultraviolet region. It is not a method.

(Problems to be Solved by the Invention) An object of the present invention is to provide a method for measuring substrate and enzyme activity in body fluids that is simple, highly accurate, and has good applicability to automatic analyzers.

(Ninth way to solve the problem) Conventionally, in a system that uses peroxidase to quantify hydrogen peroxide, using catalase leads to the decomposition of hydrogen peroxide and gives a negative error to the measurement.
Normally, it would be unthinkable for them to be used together. For this reason, it has been more common to use catalase inhibitors such as sodium azide when the influence of catalase is considered to be a peroxidase system. However, the present inventors conducted extensive studies and found that, unexpectedly, catalase in a be-oxidase system was
It has been found that the above objective can be achieved by using the amount within twice as much. In other words, a first reagent containing an oxidase and a catalase is used, a second reagent is used containing a detection agent, and the first reagent is first applied to the sample, and then the second reagent is applied to the sample. By measuring the change in absorbance caused by the action of a reagent, it is possible to accurately and easily measure the substrate or enzyme activity in a sample, and the present invention has been achieved which is highly applicable to automatic analyzers. Ta.

That is, the present invention provides a method for quantifying substrate or enzyme activity in a starved 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 using a poxidase system. , (+) oxidase and (
-) a reagent containing catalase is used as the first reagent, (1) a reagent containing the detection agent is used as the second reagent, and (iiV) peroxidase is contained in the first reagent and/or the second reagent, and the sample contains peroxidase. Add the first reagent and after death, add the second reagent,
This is a method for quantifying substrate or enzyme activity, which is characterized by measuring the generated absorbance.

Substrates and enzymes to be quantified in the present invention include:

Any sample can be applied as long as it simultaneously contains the substrate and endogenous substances involved in the reaction system for measuring the enzyme. For example, triglyceride, creatinine, creatine, ester cholesterol, sialic acid, amylase, GOT.

There are GPT, guanase, etc.

Examples of endogenous substances that interfere with the measurement of substrates and enzyme activities include free glycerol in the case of triglycephyte, creatine in the case of creatinine, sarcoV in the case of creatine, and zacoV in the case of creatine.

Examples include free cholesterol in the case of ester type cholesterol-A/(D), pyruvic acid in the case of sialic acid, glucose in the case of amylase, pyruvic acid in the case of GOT and GPT, uric acid in the case of guanase, etc.

Examples of (1) oxidizing enzymes in the present invention include 1 such as chrycerol oxidase, glycerophosphate oxidase, sarcosine oxidase, cholesterol oxidase,
Examples include pyruvate oxidase, glucose oxidase, uricase, etc., and any oxidizing enzyme that generates hydrogen peroxide may be used.

(1) Catalase used in the present invention may be of any origin. For example, there are those derived from animal organs such as the liver, red blood cells, and kidneys, and those derived from microorganisms such as those belonging to the genus Micrococcus.

The detection agent (1) in the present invention may be any substance that causes a change in absorption spectroscopically by hydrogen peroxide in the presence of peroxidase. For example 4
-Aminoantipyrine (4AA) and aniline derivatives, 4
-Aminoantipyrine and Fu-! -/-z' derivative,
a-Methyl-2-benzothiazolinone hydrazone (MB
TH) and an aniline derivative or 4-aminoantipyrine alone, an aniline derivative alone, a phenol derivative alone, etc. can be considered.

Aniline derivatives include N, N-jetyl-m-toluidine CDET), diethylaniline (DEA), N-
Examples include ethyl-N-(3-sulfopropy/I/)-m-anisidine (ADPS).

71 / -ol derivatives include p-chlorophenol /
Examples include L/, 2-sulfo-V-4-chlorophenol, and the like.

In the present invention, in addition to the above-mentioned components, the first reagent and the second reagent contain peroxidase, a buffer, and if necessary, an oxidizing enzyme and an enzyme other than peroxidase.

It may contain a substrate, a surfactant, a stabilizer, various interfering substance removers, and the like. It is better to use peroxidase as the first reagent.

There are no particular restrictions on the buffering agent, and it can be used at a pH appropriate for the reaction system.
Any type of material may be used as long as it can maintain H, but it is preferable to adjust the amount to 1 to 5 to 10.

The concentration of catalase is controlled by the concentration of coexisting peroxidase, and is suitably within 10 times the concentration of peroxidase. If it is more than that, it is not appropriate because it leads to a decrease in the sensitivity of the detection system.

Next, a specific method for quantifying substrate or enzyme activity and a quantitative reagent will be explained.

After adding the following first reagent to a triglyceride quantitative sample, add the following two reagents and measure the resulting absorbance.

First Reagent Glycerol Kinase L-α-Glycerol Phosphate Oxidase Catalase TP Second Reagent Riboprotein Lipase The above quantitative method follows the following reaction.

Riboprotein lipase triglyceride Glycerol Deca fatty acid glycerol kinase Glycerol + ATP Glycerol-3-phosphate + ADP Dihydroxyacetone + H2O2 +4H*0 After adding the following first reagent to the creatinine quantitative sample, add the following second reagent,
Measure the resulting absorbance.

First reagent Creatine amidinohydrolase Sarcosine oxidase Peroxidase Catalase Second reagent Creatinine amidohydrolase Add the following first reagent to the sample, then add the following second reagent,
Measure the resulting absorbance.

First reagent Sarcosine oxidase peroxidase catalase Second reagent Creatinamide hydrofuse The above quantitative method follows the following reaction.

Creatinine amide hydrolase Creatinine + H20 Creatine + Urea Creatinium amide hydrolase Creatine + H20 Sarcosine + Urea Formaldehyde + Green + H2O2 + 4H20 Ester After adding the following first reagent to the cholesterol quantitative sample, add the following second reagent,
Measure the resulting absorbance.

First reagent cholesterol oxitase peroxidase catalase Second reagent cholesterol esterase The above quantitative method follows the following reaction.

Free cholesterol + fatty acid cholestenone + H2O2 peroxidase 2Hz02 + 4AA + DEA quinone dye + 4H20 After adding the following first reagent to the mifuse quantitative sample, add the following second reagent,
Measure the resulting absorbance.

First reagent glucose oxidase catalase peroxidase β-gluco Vdase Second reagent Starch (substrate) The above quantitative method follows the following reaction.

α-amylase starch maltose β-glucosidase maltose 2 glucose glucose oxidase glucose + 02 gluconic acid + HIQ3 peroxidase 2HzOz + 4AA + DEA
Quinone dye +4H*0 As the substrate, in addition to starch, modified starch, oligoglucopyranoside, etc. can be used.

Furthermore, α-glucosidase may be used in place of β-gluco Vdase.

After adding the following first reagent to the GPT quantitative sample, add the following second reagent,
Measure the resulting absorbance.

First reagent pyruvate oxidase peroxidase catalase phosphoric acid DL-alanine (substrate) Second reagent α-ketoglutarate (substrate) The above quantitative method follows the following reaction.

PT DL-alanine + α-ketoglutal ll--decapyrupic acid + L-glutamic acid acetyN phosphate + co, + H,O.

+4H20 After adding the following first reagent to the GOT quantitative sample, add the following second reagent,
Measure the resulting absorbance.

First reagent pyruvate oxidase phosphoric acid peroxidase catalase Aspartic acid (substrate) Oxaloacetate decarboxylase Second reagent d-ketoglutarate (substrate) The above quantitative method follows the following reaction.

Oxaloacetic acid + L-glutamic acid + C02 Pyruvate oxidase Pyruvate + 02 + Acetyl phosphate deca CO2 + H2O2 + 4H20 After adding the following first reagent to the quantitative sample of sialic acid, add the following second reagent,
Measure the resulting absorbance.

First reagent pyruvate oxidase catalase peroxidase second reagent neuramindase N-acetylneuraminic acid oxidase The above quantitative method follows the following reaction.

Neuramindase 7A'1lll N-acetylneuraminic acid N-acetyl-D-mannosamine + pyruvic acid + C (h + H2 (h) After adding the following first reagent to the quantitative sample of guanase, add the following second reagent,
Measure the resulting absorbance.

First reagent xanthine oxidase uricase peroxidase catalase Second reagent Guanine (substrate) The above quantitative method follows the following reaction.

Guanase guanine → xanthine + NH3 xanthine oki Vdase xanthine uric acid + H2O2
Quinone color chart (effect) The quantitative method of the present invention eliminates the inclusion of substances (endogenous substances) that coexist in the sample and cause measurement errors, and easily measures the true substrate or enzyme activity with high accuracy. Furthermore, it has become possible to provide a product with good applicability to automatic analyzers.

(Example) The present invention will be explained in detail below using examples.

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

1. Sample glycerin (104 cape/dl) solution ■Serum: water; 9
:l ■Serum: Glycerin (104
0 kg/dl) solution = 9:1 ■ 2. Reagent A: First reagent Tris buffer (pH 7, (ii) Glycerokinase 2.0 units/
- Glycerophosphate oxidase 6.0 units/w
l Veroqui Vdase 10.0 units/d
Catalase 50.0 units/d Second reagent Tris buffer (pH 7, (ii) Lipoprotein lipase 600 units/ml
4-aminoantipyrine 10MP/d
JN-ethyl-N-(3-sulfopropy/I/)-m-
Anisidine 90”W/djB: The first reagent of A, minus p-catalase.

Everything else is the same as humans.

C: Catalase was removed from the first reagent of A and 4-aminoantipyrine of the tX2 reagent was transferred to the first reagent. Others are the same as A.

I removed the catalase from the first reagent of D2A and added 51,197 dl of ``Kawashiniphenol''.

3.Measurement method Add 2 ml of the first reagent to each 20μ sample.

After reacting at 37°C for 5 minutes, add 1 d of the second reagent and
React for 10 minutes at 7℃"? Measurement was performed at a wavelength of 540 nm.

The results are shown in Table 1.

Table 1 In reagent A, distilled water and sample μ■ [glycerin (1041
1P/dJ) solution].

Sample ■ (serum: water = 9:1) and sample ■ [serum: glycerolin (10411P/ctJ)
Since the absorbance of the solution 9:1) is equal to that of the solution, it is clear that there is no measurement of glycerin.

Example 2' Triglycerides in a sample were measured using the following reagents and the following method.

1. Samples ■, ■, ■2 used in Sample Example 1, Reagent A used in Reagent Example 1. The catalase concentrations shown in Table 2 were used.

Table 2 3.Measurement method Table 3 shows the results measured using the same method as in Example 1.

Table 3 It can be seen from Table 3 that when catalase is contained 10 times more than peroxidase (reagent G), there is no effect of glycerin, but a decrease in sensitivity is observed (decreased absorbance). Within 10 times (Reagent A.

In E and F), there is no reduction in sensitivity.

Claims (3)

[Claims] (1) A method for quantifying the substrate or enzyme activity in a sample by causing an oxidase to act on the substrate or a substance produced by an enzymatic reaction, and measuring the produced hydrogen peroxide using a peroxidase system. and (ii) a reagent containing catalase is the first reagent, (iii) a reagent containing the detection agent is the second reagent, and (iv) peroxidase is contained in the first reagent and/or the second reagent, and the sample A method for quantifying substrate or enzyme activity, which comprises adding a first reagent to a liquid, then adding a second reagent, and measuring the resulting absorbance. (2) The method for quantifying substrate or enzyme activity according to claim 1, wherein the first reagent contains (iv) peroxidase. (3) The first reagent contains (iv) peroxidase, and (ii) catalase is one of (iv) peroxidase.
2. The method for quantifying substrate or enzyme activity according to claim 1, wherein the amount is within 0 times (activity value).