JPS6083598A - Method for measuring substrate or enzyme - Google Patents

Abstract

PURPOSE:To make it possible to carry out automatic analysis of a substrate or enzyme, by leading reduced form of nicotinamide adenine dinucleotide (NADH) or reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) produced in the dehydrogenase reaction through an electron carrier to the coloration with hydrogen peroxide. CONSTITUTION:In a method for measuring a substrate or enzyme through reduced form of nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphate (NADPH), an electron carrier, e.g. phenazine methosulfate (PMS), 1-methoxyphenazine methosulfate (1-methoxyPMS) or phenazine ethosulfate (PES), is present therein. These electron carriers capable of producing H2O2 when the reduced form thereof is returned to the original oxidized form by oxygen dissolved in the reaction solution. The H2O2 is colored by the well-known enzymic or nonenzymic method to measure the substrate or enzyme. According to this method, the automation of the measurement is easily carried out.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method that enables accurate and rapid measurement of substrates or enzymes without contaminating tubes or cells.

From the perspective of color development methods, methods for measuring metabolized substances in visible areas in clinical laboratories include methods that generate hydrogen peroxide and develop a red to green color using peroxidase or non-enzymatically, and dehydrogenation. Using enzymes, NΔD (cotinamide adenine dinucleotide)/NADH (cotinamide adenine dinucleotide reduced form) or NADP (cotinamide adenine dinucleotide phosphate 11/NADI)H
Method for determining changes in cotinamide adenine dinucleotide phosphate (reduced form) under ultraviolet light and NA
It is roughly divided into methods that lead DH and NADPH to formazan color development.

On the other hand, in recent years, along with advances in automatic analysis, many enzymes that can generate hydrogen peroxide, such as glucose oxidase, uricase, cholesterol oxidase, pyruvate oxidase, etc., have been developed, and biological materials can be easily and quickly processed. It has become possible to measure

For measurement using an automatic analyzer, of the above-mentioned color methods, both the hydrogen peroxide color method and the ultraviolet absorption method using NADP/NADPI-1 as a detection system are applicable.

Furthermore, the formazan coloring method described above is applied to reactions that utilize dehydrogenases that cannot lead to hydrogen peroxide coloring, for example, L l) 11 (lactate dehydrogenase) in the visible region. When measuring, as shown in the reaction formula below, NADH produced by LDH is treated with PMS (phenazine methosulfate), 1-methoxy I) MS (
1-methoxyphenazine methosulfate) etc., which leads to the development of a purple formazan color.

LDH N T B N T B H2 (wherein, NTB is nitrotetrazolium blue, N T
B H2 is the reduced form of nitrotetrazolium blue. ) However, the formazan coloring method had the disadvantage that it was rarely used for automatic analysis because the formazan coloring substance deposited strongly on tubes and cells, causing problems with contamination, and manual methods had to be used. .

The present invention aims to eliminate the drawbacks of the above-mentioned formazan coloring method.
This makes it possible to lead H (or NADPI-1) to hydrogen peroxide color development via an electron carrier. The main points of the method of the present invention can be expressed as follows.

The following margin dehydrogenase NAD (P) NAD (P) H (In the above formula, NAD (P) is NAD or NADP, and NAD (P) H is NADH or NADPI. ) This is characterized in that the generated I4□q is colored enzymatically or non-enzymatically using a chromogen.In short, the reduced electron carrier is converted to its original oxidized form by dissolved oxygen in the reaction solution. The present invention was completed by paying attention to the fact that H and O are generated when returning to .

The present invention eliminates the drawbacks of the conventional formazan method, eliminates the need for tubes and cells, and improves efficiency. The purpose of the present invention is to provide a method for measuring substrates or enzymes that can be easily applied to I/O analysis.

The gist of the present invention is that N A D H or N A D I) H
Alternatively, in a method for measuring substrates or enzymes via these derivatives, hydrogen peroxide is generated via an electron carrier and the generated hydrogen peroxide is colored enzymatically or non-enzymatically to quantify it. It consists of a method for measuring substrates or enzymes with six characteristics.

What is a derivative of N A D H or N A D P H?
3 Acetylpyridine adenine dinucleotide 1-reduced form, such as BRUCE M, ANDIER3ON.

et al J, Biol, Chem, 2:14, 1
219 (1959), 131 UCI': M
, Δ NDER3ON, eL a l J, +3i o
1. Chem, 234.1226 (1959), etc., refers to the reduced form.

Examples of the electron carrier used in the present invention include phenazine methosulfate (PMS), 1-methoquinophenazine methosulfate (1-methoxyPMS), phenazine ethosulfate (PES), and diabolase. Also included are enzymes such as.

As the non-enzymatic coloring method in the method of the present invention, known methods can be adopted, such as the Ti-PAR method (Chiyo Matsubara et al., Analytical Chemistry, Vol. 29, 1980, 759-7
page 64) and Ti-X0 method, Ti-CAS method,
T i -E D T A method and C.

-EDTA method (Chiyo Matsubara et al., Pharmaceutical Journal, Vol.

97. 1977, pp. 41-45).

Examples of reactions to which the method of the present invention is applicable include the following.

■ Determination of glucose (al mutarotase α-glucose β-glucose glucose dehydrogenase (bl hemokinase glucose-6-phosphate dehydrogenase ■ Measurement of cholesterol (a) Measurement of total cholesterol cholesterol esterase ester type -n free cholesterol Cholesterol Cholesterol Dehydrogenase NAD (P) NAD (P) H (bl Measurement of Free Cholesterol Cholesterol Dehydrogenase NAD (P) NAD (P)H Below margin ■ Measurement of Bile Acids 3α-Hydroxysteroid Dehydrogenase NAD
(P) NAD (P)Ll ■Measurement of lactic acid Lactate dehydrogenase NAD (P) NAD (P)II ■Lactic dehydrogenase (LDH) /I11 [Constant LDf I NAD (P) NAD (1))lI ■G ○ Go・
G l) "Determination of F - Aspartic acid 10α ~ Ketoglucour OT -> Oxaloacetic acid + L - Glutamic acid L - Alanine + α - K1 - K1 - Kultal CI) T -> Pyruvate 10 - Glutamic acid glutamate Tehydrogenase NAD (P ) NAD (P)H Below margin ■ Taleatine kinase (CK)?) Ill constant K Creatine phosphate -111 → Creadine + A T+) +
ADP hexokinase ATP + glucose ΔD l-' + glucose-6-phosphate glucose-6-phosphate dehydrogenase (in the above formula, ATP is adenosine l-ribosphae-1,
ADP is adenosine sibosphate. ) N A l) II or NΔ1]P produced as in the above reaction
It leads I-1 to hydrogen peroxide color development via an electron carrier. That is, hydrogen peroxide is reacted with 4-aminoantipyrine and a hydrogen donor in the presence of peroxidase to produce a colored substance. As this hydrogen donor, phenol, 2.

4-Sochlorophenol, p-chlorophenol, N.
N-dimethyl metatoluidine, N,N-dimethyl metatoluidine, TOOS (N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine sodium), etc. are used. It is also possible to use combinations of MBTH (3-methyl-2-henzothiazolinone hydrazone hydrochloride), dimethylaniline, diethylaniline, etc., which do not cause hydrogen peroxide to develop color. Furthermore, it is also possible to use a coloring agent (phenylenediamine and its derivatives, MCDP, BCMA, etc.) that can color hydrogen peroxide in the presence of peroxidase without a third factor (4-aminoantipyrine, etc.).

With the above configuration, the method of the present invention eliminates the drawbacks of the conventional formazan coloring method, eliminates the possibility of tubes and cells being contaminated by coloring substances, and facilitates automated analysis of substrates or enzymes. It is effective.

The method of the present invention will be further explained below with reference to Examples.

Example I NADH quantitative reagent composition R 1-liquid phosphate buffer --- 50 mM, pH 7,
01-MethoxyP M S -1---0. 0
7 r mouth MR/2 liquid phosphate buffer --- 5 00 m M.
pH 6, 04-aminoantipyrine---1
．． 081rIMT O O S------------
--1. 8 mM perleoxidase------
- - A sample containing about 5 mM NADH at 12 U/p was diluted in 5 steps from 115 to 515 with a solution of sodium bicarbonate, and linearity was examined.

50 μl of the sample was added to R・l p (12m II), and after leaving it at room temperature for 10 minutes, 1 mβ of R・2 solution was added.
The absorbance was measured at 555 nm after being left for 5 minutes.

The measurement results are shown in FIG. 1, and showed good linearity until the absorbance was less than 1.5.

Example 2 Glucose quantitative reagent composition R・1 liquid phosphorus Mi2fjjllll ----------50m
M, pH 7, 0A T P ------------------------
−−−−−−−−−−−−−−−−−−−−−−−−−
---2m MR-N A D ------
----------2m M hexokinase---
----0,440/m (1 glucose-6-phosphate-dehydrogenase----1-4,5
U/m 1 Magnesium Chloride---------
------------------2m Ml-MethoxyP M S -------0, 015
m MR 2-liquid phosphate buffer---500m M, pH 6
, 04-aminoantivirin-1-1, 08111MT
OOS--1--------------・-----
----21,8mN1 peroxidase---
----■2 U/m 7! A 300 mg/d1 glucose aqueous solution was diluted in 5 steps from 115 to 515 with water,
The linearity was inspected.

Add 10 μβ sample to 2 ml of R-1 solution and incubate at 37°C.
After reacting for 0 minutes, 1 ml of R.2 IIE was added. Next, the absorbance was measured at 555 nm after being left at room temperature for 5 minutes. The results are shown in Figure 2.Glucose 300
Good linearity was shown up to (2)/d1.

The correlation with the conventional method (glucose oxitase/hydrogen peroxide coloring method) in this example is Y=0.95x+10. r=0
．． 987 (n=30).

Example 3 Reagent composition for measuring free cholesterol 1k/11 Tris-phosphate buffer---100mM, pH 8, 6 NAD--------------------- ---
−−−−−−−−−−−−20m M cholesterol dehydrogenase (3β-hydroxysteroid NAD
(P) Oxidoreductase 3---------・--
−1−−−−−−−−−−−−−−−−−−−−−0
, 8U/mβ1-methoxyPMS------
−−−−−−−−−−−0, 06m M Triton X=
100 −−−−−−−−−−−−−0, 4%
R・Two-liquid phosphate buffer---500mM, pH
6,04-aminoantivirine---1,
08m MT OOS -1------------
−−−−−−−−−−−−−−−−−−−−1, 8m
M peroxidase-------12U
/m 1800 mg/d1 cholesterol solution was diluted with water in 5 steps from 115 to 515 to check linearity.

Add 10 μβ sample to 1 ml of R・1 solution, react at 37°C for 15 minutes, and then add 2 ml of R・2 solution! added. Next, the absorbance was measured at 555 nm after being left at room temperature for 5 minutes. The results are shown in Figure 3.ml L-sterol 600q
I lost good iJr linearity until /d1.

Example 4 Reagent composition for measuring total cholesterol R・17-night Tris-phosphoric acid buffer solution---400mM, I) H8, (INAD
−−〜−”'−'−−−−−−−−−−−−−−−2[
) m M cholesterol esterase 2 L-J
/ m /! Cholesterol dehydrogenase (3β-
Hydroxysteroid NAD (1)) oxidoreductase) -'-'-"-"-'-"-"--0,8U
/ m 7!1-methoxyP M 5--0. 0ram
M Triton X -100=--0.4% Thorium cholate -3701111? / eR・
21 night phosphate buffer -----500mM, p II et al., 04-aminoantipyrine --1, 08m MT
OO5--------------------
--1,8m M peroxidase-=---m---
−=−−−−−・−−−12U / mβ500mg1
Human serum with cholesterol of 115 to 5 dl is mixed with water.
15 in 5 steps, and linearity was examined in the same manner as in Example 3. The results are shown in the 4th M, and showed good linearity with total cholesterol being 500 mg/d1 or less.

Example 5 LDH measurement reagent composition R, 1-liquid TrisIJ buffer----------------------
-------------50m ML-lactic acid---
1------------------
−−−−−−−−−−−−−−−−−−・26
mMN A D −−−−−−−−−−−−−−
-------------0, 8m Ml-methoxy P
M S --------------0, 05m
MpH8,4 1 and 2-liquid phosphate buffer ----------------
m------500m MT OO'S -----
−−−−−−−−−−−−−−0,9m M peroxidase−−−−−−−G U / m j' Sodium oxalate−−−−−−−19m M4‐Amine Antipyrine ---0, 5411I MpH6.0 Add LDH standard serum equivalent to about 3500 with water (15-5
The linearity was examined by diluting the sample in 5 steps to 15%.

Add sample 50p (4 to 1rnJ of R・1 solution, 37° (:
After heating for exactly 10 minutes, R.2 solution 2Inβ was added.

The absorbance was then measured at the 555th point 11 immediately after leaving it at room temperature for 5 minutes. The results are shown in Figure 5, but the good linearity was poor at absorbances below 0.3.

[Brief explanation of drawings]

Figure 1 is a scrap diagram showing linearity with respect to the amount of N A D H, Figure 2 is a diagram showing linearity with respect to the amount of glucose,
FIG. 3 is a diagram showing the linearity with respect to the amount of free cholesterol, FIG. 4 is a diagram showing the linearity with respect to the total cholesterol self-call r11, and FIG. 5 is a diagram showing the linearity with respect to Ll, ) II A. HAD Day, Figure 2, Tsunoshifu Alone, Figure 3, Mysterious Pear-shaped Collection, Tyrol Figure 4! Full 0-full with r ball/coin

Claims (1)

[Claims] (1) In a method for measuring substrates or enzymes via NADH or NADPH or their derivatives, hydrogen peroxide is generated via an electron carrier and the generated hydrogen peroxide is colored enzymatically or non-enzymatically. - A method for measuring a substrate or enzyme, which is characterized in that it is quantified in a continuous manner.