JP2643205B2 - Highly sensitive colorimetric method - Google Patents

Description

The present invention relates to a highly sensitive colorimetric method for glycerol-3-phosphate and dihydroxyacetone phosphate.

More specifically, in an enzymatic cycling reaction used for sensitive quantification of glycerol-3-phosphate and dihydroxyacetone phosphate, hydrogen peroxide produced by the enzymatic cycling reaction is converted to a chromogen by peroxidase. The colorimetric reaction for oxidizing to form a color body is performed to make colorimetric determination practical.

"Conventional technology" An analysis method using an enzyme is still one of the important measurement methods because of its easy operation.

In recent years, there has been a demand for the development of a highly sensitive quantification method because of the desire to measure trace substances and the economic merit of reducing the amount of enzyme used for the measurement. Cycling reactions), NADP cycling reactions, CoA cycling reactions, and many other enzymatic cycling reactions have been used.

When the measurement method is used for a clinical test, it is necessary to obtain high sensitivity as described above, while also taking into account restrictions such as simplicity and economy such as the fact that a device that is already widely used can be used. This is considered to be the reason why the absorbance measurement method using the enzymatic cycling reaction is still a powerful method.

Glycerol-3-phosphate and dihydroxyacetone phosphate are produced by various enzymes and their substrates, and are used as precursors thereof by combining reaction systems for converting glycerol-3-phosphate and dihydroxyacetone phosphate. It is a clinically important measurement target that can quantify the activity of the enzyme involved in a certain mass or reaction.

The present inventors have previously shown an enzymatic cycling reaction of glycerol-3-phosphate or dihydroxyacetone phosphate (shown in Formula I. Hereinafter, the enzymatic cycling reaction (I)
Abbreviated. ) Was invented (JP-A-59-140).
No. 900).

(Where G3P is glycerol-3-phosphate, DHAP is dihydroxyacetone phosphate, GPO is glycerol-3-phosphate oxidase, G3PDH is glycerol-3-phosphate dehydrogenase, and H ₂ O ₂ is hydrogen peroxide. In JP-A-59-140900, as a measurement by the absorbance measurement method, for example, the reduced form consumed by the reaction of the enzymatic cycling reaction (I) using a large excess of reduced form NAD
It discloses to measure glycerol-3-phosphate or dihydroxyacetone phosphate based on the NAD amount. The amount of reduced NAD remaining after the reaction of the enzymatic cycling reaction (I) is measured by detecting a change in absorbance at a specific absorption wavelength possessed by reduced NAD, or by using reduced NAD and tetrazolium salt as substrates. A formazan dye is formed by a transferase such as diaphorase, and a change in absorbance at a specific absorption wavelength is detected. Because these methods of measuring the amount of components consumed by the enzymatic cycling reaction (I) are depletion methods, the incremental methods of directly measuring the amount of components generated by the enzymatic cycling reaction (I) When measuring the amount of low-concentration components, it tends to be inaccurate when measuring the amount of components, and the method of forming a formazan dye leaves the troublesome face that the hardly soluble formazan dye adheres to the equipment used and takes time to clean. I was

"Problems to be Solved by the Invention" To solve the above-mentioned problems, the present inventors have proposed a method for measuring absorbance using hydrogen peroxide generated in the enzymatic cycling reaction (I) as a substrate, using peroxidase It was considered that various chromogens were oxidized into color bodies in the presence of, and colorimetrically determined.

While reduced NAD is an essential substrate for the enzymatic cycling reaction (I), it competes with the chromogen of the peroxidase chromogenic system as a hydrogen donor, and hydrogen peroxide generated by the enzymatic cycling reaction (I) Is not fully utilized in the color forming system that makes the chromogen into a color body, and this high-sensitivity colorimetric method is not practical because it results in no quantitativeness, poor sensitivity, or a very narrow range of concentrations that can be measured. There was no one that withstands the above (JP-A-62-14799). That is, there was no choice but to adopt a high sensitivity or an absorbance measurement method using a peroxidase color developing system.

"Means for Solving the Problems" The present inventors have made intensive studies to solve the above-mentioned problems, and as a result, it has been found that reduced NAD essential for the enzymatic cycling reaction (I) for obtaining high sensitivity is reduced. The present inventors have found a measurement method having high sensitivity and quantification without obstructing the peroxidase coloring system, and have completed the present invention.

Conventionally, in the enzymatic cycling reaction (I), reagents involved in the cycling reaction, ie, oxygen and reduced NAD, have been added in an excessive amount so as not to lower the cycling rate during the reaction (JP-A-59-140900). Gazette). When an excessive amount of reduced NAD is added, a considerable amount of reduced NAD remains even after the reaction of the enzymatic cycling reaction (I) and the peroxide formed by the enzymatic cycling reaction (I) Since hydrogen reacts with residual reduced NAD, it is not possible to directly measure hydrogen peroxide generated using a peroxidase coloring system. The present inventors have found that a reduced NAD that immediately supplements the consumed amount of reduced NAD
The amount of reduced NAD required for the enzymatic cycling reaction (I) is reduced to a concentration that does not substantially interfere with the peroxidase coloring system. The present inventors have found that hydrogen peroxide produced using a peroxidase coloring system can be accurately and directly measured by reducing the amount, and the present invention has been completed.

That is, the present invention relates to an enzymatic cycling reaction (I) for measuring glycerol-3-phosphate or dihydroxyacetone phosphate in a test solution. (Wherein G3P is glycerol-3-phosphate, DHAP is dihydroxyacetone phosphate, GPO is glycerol-3-phosphate oxidase, G3PDH is glycerol-3-phosphate dehydrogenase, H ₂ O ₂ is hydrogen peroxide, POD Is a peroxidase, A is a reducing substance, and B is an oxidation product.) The components in the test solution are reacted with the components forming the enzymatic cycling reaction (I) to form NAD generated by the enzymatic cycling reaction in a highly sensitive colorimetric method having a peroxidase chromogenic system (II) in which hydrogen peroxide amplified and produced by the enzymatic cycling reaction is converted into a colorant by a peroxidase and a chromogen. Highly sensitive colorimetric method characterized by having a regeneration system (III) for regenerating to reduced NAD, and having a reduced NAD concentration of 80% or more to develop a color intensity. It is.

The high-sensitivity colorimetric method of the present invention (hereinafter referred to as the present cycling reaction) comprises the enzymatic cycling reaction (I), a peroxidase coloring system (II), and a reduced NAD regeneration system (I).
II), and each is described below.

The enzymatic cycling reaction (I) is described in JP-A-59-1.
As disclosed in Japanese Patent No. 40900, glycerol-3-phosphate (hereinafter abbreviated as G3P) or dihydroxyacetone phosphate (hereinafter abbreviated as DHAP) is also measured, and glycerol-3-phosphate oxidase (hereinafter abbreviated as GPO), Glycerol-3-phosphate dehydrogenase (hereinafter abbreviated as G3PDH), oxygen and reduced NAD may be combined. In this enzymatic cycling reaction (I), each cycling produces one molecule of hydrogen peroxide and NAD. In addition, the dissolved oxygen dissolved in the reaction solution is usually sufficient.

Next, a regeneration system of reduced NAD for reducing the amount of reduced NAD to a fixed amount will be described.

In order to reduce the amount of the reduced NAD in the enzymatic cycling reaction (I) to a fixed amount, a second dehydrogenase that converts the NAD into a reduced NAD and its substrate (reducing substance) are used. The substrate is not particularly limited as long as it can serve as a substrate for the second dehydrogenase, and an excess may be added. Although the dehydrogenase enzyme usually catalyzes both the forward reaction and the reverse reaction, in the present invention, in order to efficiently construct a regeneration system for NAD → reduced NAD, it is used as a second dehydrogenase.
The objective is to select an enzyme in which the reverse reaction of reduced NAD → NAD is less likely to occur than the normal reaction of NAD → reduced NAD.

(A represents a reducing substance, and B represents an oxidation product.) More specifically, in the equilibrium reaction in the second dehydrogenase, the equilibrium conditions under which reduced NAD is present at least 100 times as large as NAD are tentatively preferable. Then, the substrate concentration and pH may be set so as to satisfy Equation 1.

In reality, extreme pH and substrate concentration cannot be made infinite, and from the viewpoint of cost, it is necessary to reduce the amount of substrate used as much as possible, so select an enzyme with a large equilibrium constant (Keq). Preferably, Keq should be about 10 ^-9 to 10 ^-8 or more at ordinary pH and substrate concentration.

If the above conditions are met, a written copy (enzyme analysis, p20,
Kodansha; Shoichi Shimizu, Takeshi Kobayashi, Jun Okuda, Sugimoto according to _{Etsuro), V m = (20~50)} K m ( minimum enzyme amount of the second dehydrogenase to be added to the V _m (U / ml), and the K _m NAD second dehydrogenase
Means the Michaelis constant (mM) for )
Is shown. As can be seen from this formula, regardless of the type of the second dehydrogenase to be used and the reaction in which G3PDH acts (ie, the enzymatic cycling reaction (I)), the optimum amount of addition for each of the second dehydrogenases used is It is determined independently of the enzymatic cycling reaction (I). The amount to be added fit to the equation by measuring the K _m value of the enzyme can be used as the second dehydrogenase if those may be added physically. The enzymes commonly used as the second dehydrogenase, their _Km values and their substrates are shown below, but it goes without saying that they are not limited to these.

Clinical Enzyme Handbook, Kodansha (1982) Next, the peroxidase coloring system is described.

The term "peroxidase coloring system" refers to a reaction in which hydrogen peroxide produced by the enzymatic cycling reaction (I) is oxidized and condensed or oxidized with chromogens in the presence of peroxidase to form a colored body. What is a chromogen? Formula: (Wherein, R ₁ and R ₄ are each a hydrogen atom, a lower alkyl group or a lower alkoxyl group, R ₂ and R ₃ are each a hydrogen atom,
A lower alkyl group, a hydroxy lower alkyl group, a lower alkyl group containing acetylamide, a lower alkyl group containing a sulfonic acid group, or a hydroxy lower alkyl group containing a sulfonic acid group). Combining 4-aminoantipyrine with the formula: (Wherein X is a hydrogen atom, a halogen atom, a sulfonic acid group,
A nitro group, a lower alkyl group, a lower alkoxyl group, a lower alkylcarbonyl group, a carboxyl group or a lower alkoxycarbonyl group, and Y and Y 'are each a hydrogen atom, a halogen atom, a sulfonic acid group, a nitro group, a lower alkyl group, a lower alkoxyl group , A lower alkylcarbonyl group,
Carbamoyl group or lower alkoxycarbonyl group, Z
And Z ′ are each a hydrogen atom, a sulfonic acid group, a nitro group,
Represents a lower alkyl group, a lower alkoxyl group or a carboxyl group), and a combination of one selected from the group of phenols represented by 4-aminoantipyrine,
Or a leuco compound such as 10-[(3-methoxycarbamoylmethyl) phenylmethylcarbamoyl] 3,7-dimethylaminophenothiazine or bis [3-bis (4-chlorophenyl) methyl-4-dimethylaminophenyl] amine. is there.

To give a specific example, examples of anilines include N-
Ethyl-N-sulfopropyl-m-anisidine (ADP
S), N-sulfopropyl-3,5-dimethoxyaniline (HDAPS), N-ethyl-N- (2-hydroxy-3-
Sulfopropyl) -m-anisidine (ADOS), N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,
5-dimethoxyaniline (DAOS), N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (HDAOS), N-ethyl-N- (2-hydroxy-3-
Sulfopropyl) -m-toluidine (TOOS), N, N-dimethylaniline, N, N-dimethyl-m-toluidine, N, N
-Diethylaniline, N, N-diethyl-m-toluidine, N-ethyl-N- (2-hydroxyethyl) -m-
Toluidine, N-ethyl-N- (3-methylphenyl)
-N'-acetylethylenediamine, N, N-dimethyl-
m-anisidine, N-ethyl-N-sulfopropyl-m
-Toluidine, N-ethyl-N-sulfopropyl-3,5
-Dimethoxyaniline (DAPS), 3-methyl-N-ethyl-N-hydroxyethylaniline, N-ethyl-N-
(2-hydroxy-3-sulfopropyl) aniline and the like.

Examples of phenols include phenol, parachlorophenol, 2,4-dichlorophenol, 2,6-dichlorophenol, 3,5-dichloro-2-hydroxybenzenesulfonic acid, 2,4-dibromophenol, and 2,4-dichlorophenol. 4,6−
Tribromophenol, 3-hydroxy-2,4,6-triiodobenzoic acid and the like.

The degree to which reduced NAD interferes is reduced N as hydrogen donor
It is determined by the ratio of the affinity between AD and chromogen for peroxidase and the quantitative relationship between reduced NAD and chromogen. However, these relationships not only change depending on the type of chromogen and peroxidase, but the reaction itself is difficult to predict by theory, so the following experiment was performed. That is, an experimental system in which a reduced NAD regeneration system and a peroxidase coloring system using various chromogens were added to the enzymatic cycling (I), and low to high concentrations of reduced NAD at each concentration step were added. , A fixed amount of G3P (or DHAP) is added to the experimental system, and color development based on hydrogen peroxide generated by the enzymatic cycling (I) is performed. the absorbance and the detection wavelength near lambda _max was plotted for each chromogenic. The absorbance at each concentration of reduced NAD was shown as a percentage with the maximum value of the absorbance taken as 100%. For example, FIG. 2 shows the results when horseradish peroxidase (EC1.11.1.7, manufactured by Sigma) was used as the peroxidase, 4-aminoantipyrine was used as the chromogen coupler, and phenol was used as the hydrogen donor. The concentration of reduced NAD that gives the maximum value of absorbance (hereinafter referred to as the optimum concentration) is 0.0
The concentration was 20 mM (detection wavelength: 500 nm), which was much lower than the excess amount of about 0.3 mM conventionally used.

When 4-aminoantipyrine is used as a coupler and N-ethyl-N-sulfopropyl-m-anisidine (ADPS) is used as a hydrogen donor (detection wavelength: 540 nm;
Figure), when 4-aminoantipyrine is used as a coupler and N-sulfopropyl-3,5-dimethoxyaniline (HDAPS) is used as a hydrogen donor (detection wavelength: 580 nm, 4th
Figure), 4-aminoantipyrine as a coupler, N-ethyl-N- (2-hydroxy-3- as a hydrogen donor
When sulfopropyl) -m-anisidine (ADOS) was used (detection wavelength; 542 nm, FIG. 5), 4-
When aminoantipyrine and N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (HDAOS) are used as a hydrogen donor (detection wavelength: 583 nm, 7th
The optimum concentration in the figure) was 0.020 mM in the same case as when phenol was used.

When 4-aminoantipyrine is used as a coupler and N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (DAOS) is used as a hydrogen donor, the optimum concentration is 0.050 mM (detection wavelength). ; 600nm, 6th
Figure), 4-aminoantipyrine as a coupler, N-ethyl-N- (2-hydroxy-3- as a hydrogen donor
The optimum concentration when using (sulfopropyl) -m-toluidine (TOOS) was 0.016 mM (detection wavelength; 555 nm, FIG. 8).

When 10-[(3-methoxycarbamoylmethyl) phenylmethylcarbamoyl] 3,7-dimethylaminophenothiazine (MMX) is used as the chromogen, the optimum concentration is 0.0
The concentration was somewhat low at 10 mM (detection wavelength; 660 nm, FIG. 9).

The optimal concentrations for each chromogen were fairly close to each other, and were much lower than the concentration of reduced NAD conventionally used in the enzymatic cycling (I).

When performing the colorimetric measurement of the present invention, it is desirable to carry out the measurement at the optimal concentration of reduced NAD determined by the above experiment. If the concentration of the reduced NAD to be added is too low, sensitivity cannot be obtained, and if it is too high, the concentration range that can be determined is narrow, but if the absorbance is about 80% of the optimal concentration, the effect of the present invention is sufficiently recognized. (Hereinafter, the reduced NAD concentration capable of expressing 80% or more of the absorbance with respect to the optimal
% Or more. ). Therefore, the cycling reaction may be performed at a color developing intensity expression concentration of 80% or more. The peroxidase is horseradish peroxidase and shows a color developing intensity expression concentration of 80% or more of reduced NAD in various typical chromogens, but the peroxidase and chromogen of the present invention are not limited thereto.

1) When 4-aminoantipyrine is used as a coupler and phenol is used as a hydrogen donor, the color intensity developing concentration of 80% or more is 0.008 to 0.07 mM, and the optimum concentration is 0.020 mM (second
Figure).

2) When 4-aminoantipyrine is used as a coupler and N-ethyl-N-sulfopropyl-m-anisidine (ADPS) is used as a hydrogen donor, the color developing intensity is 80% or more, the color development intensity is 0.006 to 0.07 mM, and the optimum concentration is 0.020. mM (FIG. 3).

3) When 4-aminoantipyrine is used as a coupler and N-sulfopropyl-3,5-dimethoxyaniline (HDAPS) is used as a hydrogen donor, the color developing intensity expression concentration is 80% or more, 0.006 to 0.1 mM, and the optimum concentration is 0.020 mM. (FIG. 4).

4) When 4-aminoantipyrine is used as a coupler and N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-anisidine (ADOS) is used as a hydrogen donor, a color intensity developing concentration of 80% or more is 0.007. 0.050.05 mM, optimal concentration is 0.020 mM (FIG. 5).

5) When 4-aminoantipyrine is used as a coupler and N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (DAOS) is used as a hydrogen donor, 80% or more of color intensity is developed. The concentration is 0.01-0.13m
M, the optimum concentration is 0.050 mM (Fig. 6).

6) When 4-aminoantipyrine is used as a coupler and N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline (HDAOS) is used as a hydrogen donor, the color intensity developing concentration is 80% or more and 0.008 to 0.11 mM, optimal concentration 0.020 mM (Fig. 7).

7) When 4-aminoantipyrine is used as a coupler and N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine (TOOS) is used as a hydrogen donor, the color developing intensity expression concentration is at least 80% and 0.005. 0.00.06 mM, optimal concentration 0.016 mM (FIG. 8).

8) When 10-[(3-methoxycarbamoylmethyl) phenylmethylcarbamoyl] 3,7-dimethylaminophenothiazine (MMX) is used as the chromogen, 80% or more, the color developing intensity expression concentration is 0.003 to 0.04 mM, the optimum concentration. Is 0.010mM
(FIG. 9).

In the present invention, it is a principle that the color development intensity expression concentration is determined to be 80% or more by the above-mentioned experiment, but since the experiment sometimes cannot avoid time, the concentration of the reduced NAD is determined in advance.
The present invention can be implemented with various chromogens if the ratio is about 0.003 to 0.013.

Next, when a typical amount used in the present invention is exemplified, glycerol-3-phosphate oxidase is 5 to 50 U /
ml, preferably 8-30 U / ml, glycerol-3-phosphate dehydrogenase 0.3-20 U / ml, preferably 1-10 U / m
l, peroxidase is 1-20U / ml, preferably 2-10U
/ ml, the concentration of the chromogen has no particular upper limit, but is usually 2 to 100 times, preferably 5 to 20 times by mole ratio to hydrogen peroxide which is another substrate of peroxidase. Good. The pH of this cycling reaction is 7.0 to 8.2
It is preferably 7.2 to 8.0, and the preferred amounts of the second dehydrogenase and its substrate of the regeneration system and reduced NAD are as described above.

In the present invention, an extremely large number of compounds or enzymes can be quantified by combining G3P and DHAP themselves and an enzyme reaction system that produces them.
As the enzyme reaction system for producing G3P and DHAP, either a method of terminating the reaction before performing the present cycling reaction or a method of reacting simultaneously with the present cycling reaction may be selected.

Generally, the measurement may be performed by an advantageous endpoint method. The typical reaction examples are described below.

1) Enzyme reaction system that releases and generates G3P (1) Enzyme reaction system of glycerol kinase (EC 2.7.1.30) that releases and generates ADP and G3P using ATP and glycerol as substrates. By quantifying the amount of G3P released, any one of ATP, glycerol or glycerol kinase activity is measured.

(2) Glycerol in the enzyme reaction system of the above (1) is phosphatidylglycerol and phospholipase D (EC3.
A glycerol derived from the enzyme reaction system of 1.4.4), and an enzyme reaction system for measuring phosphatidylglycerol or phospholipase D activity.

(3) ATP in the enzyme reaction system of the above (1) is ATP derived from the enzyme reaction system of creatine phosphate, ADP, and creatine kinase (EC 2.7.3.2), and is used for measuring creatine phosphate or creatine kinase activity. Enzyme reaction system.

Measurement of the activity of creatine kinase in serum is used for diagnosis of, for example, muscular diseases, neurological diseases, myocardial infarction, myxedema, storage diseases of carbohydrates and lipids, and the like.

(4) ATP in the enzyme reaction system of the above (1) is phosphoenol pyruvate, ADP, pyruvate kinase (EC 2.7.
ATP derived from the enzyme reaction system of 1.40), and an enzyme reaction system for measuring phosphoenolpyruvate or pyruvate kinase activity.

Measurement of pyruvate kinase activity in erythrocytes is used to diagnose congenital hemolytic anemia.

(5) ATP in the enzyme reaction system of (1) above is ATP derived from an enzyme reaction system of adenylate kinase (EC 2.7.4.3) using ADP as a substrate, and an enzyme reaction for measuring adenylate kinase activity. system.

The measurement of the activity of adenylate kinase is used for diagnosis of muscle diseases, particularly muscular dystrophy, Kugelberg-Welander syndrome, and polymyositis.

(6) ATP in the enzyme reaction system of the above (1) is ATP derived from the enzyme reaction system of acetyl phosphate, ADP and acetate kinase (EC 2.7.2.1), and is used for measuring acetyl phosphate or acetate kinase activity. Enzyme reaction system.

(7) The ATP in the enzyme reaction system of (1) is D-1,3-
ATP derived from an enzyme reaction system of bisphosphoglycerate, ADP and phosphoglycerate kinase (EC 2.7.2.3), and an enzyme for measuring D-1,3-bisphosphoglycerate or phosphoglycerate kinase activity. Reaction system.

(8) Lysoglycerophospholipid, lysophospholipase (EC
2.7.2.3) Glycerophosphocholinesterase (EC2.
7.2.3) An enzyme reaction system for measuring G3P derived from the enzyme reaction system and for measuring lysoglycerophospholipid or lysophospholipase activity.

(9) The lysoglycerophospholipid in the enzyme reaction system of the above (8) is lysolecithin, and the lysolecithin is lysolecithin derived from the enzyme reaction system of lecithin, cholesterol, lecithin cholesterol acyltransferase (EC2.3.1.43); An enzyme reaction system for measuring cholesterol and lecithin cholesterol acyltransferase activity.

The measurement of lecithin cholesterol acyltransferase activity is used for liver function tests and diagnosis of abnormal lipid metabolism and diabetes.

(10) The lysoglycerophospholipid in the enzyme reaction system of the above (8) is lysolecithin, and the lysolecithin is lecithin, phospholipase A (EC3.1.1.4 or EC3.1.1.3).
2) A lysolecithin derived from the enzyme reaction system of 2), which is used for measurement of lecithin and phospholipase A activities.

2) Enzyme reaction system that liberates DHAP (1) Ketose 1-phosphate and aldolase (EC4.1.2.
7) An enzyme reaction system for quantifying DHAP derived from the enzyme reaction system and for measuring ketose 1-phosphate or aldolase activity.

(2) for quantifying DHAP derived from an enzymatic reaction system of D-glyceroaldehyde-3-phosphate and triosephosphate isomerase (EC 5.3.1.1), wherein D-glyceroaldehyde-3-phosphate or trioselin is used. Enzyme reaction system for measuring acid isomerase activity.

"Examples" Next, the present invention will be specifically described with reference to experimental examples and examples, but the present invention is not limited thereto.

Example 1 Quantification of G3P (1) Preparation of reaction reagent GPO (derived from Aerococcus viridans, manufactured by Toyo Brewery Co., Ltd.) 117
U, G3PDH (EC1.1.1.8, derived from rabbit skeletal muscle, Boehringer Mannheim) 34U, 12α-hydroxysteroid dehydrogenase (EC1.1.1.176, Basillus sphaericus)
50U, peroxidase (EC1.11.
1.7, from horseradish, Sigma) 45U, 0.05mM reduction type
NAD (manufactured by Oriental Yeast), 0.03% 4-aminoantipyrine (manufactured by Wako Pure Chemical), 0.02% phenol (manufactured by Wako Pure Chemical), 0.5 mM cholic acid (manufactured by Wako Pure Chemical), 5 mM calcium chloride (manufactured by Wako Pure Chemical) Kojun Pharmaceutical) and 0.1% Triton X-100
(Sigma, trade name) is dissolved in 50 mM HEPES buffer solution (Trade name of Dojindo Laboratories, trade name (pH 8.0)) to make 10 ml.

(2) Quantification of G3P Each of the above reaction reagents was dispensed in 1 ml portions, and 20 μl of 0, 15, 30, 45, 60 and 75 μM each concentration of G3P aqueous solution was added to a test tube preliminarily heated at 37 ° C. After further reacting at 37 ° C. for 15 minutes, 2.0 ml of 0.5% SDS (sodium dodecyl sulfate, manufactured by Wako Pure Chemical Industries, Ltd.) was added to stop the reaction. The absorbance of the reaction solution at 500 nm was measured (Shimadzu self-recording spectrophotometer UV-250).

The results showed good linearity as shown in FIG. 1 (in the figure,
White circle).

Comparative Example 1 The same measurement was carried out as in Example 1 except that only 12α-hydroxysteroid dehydrogenase was added, and the other components were the same (FIG. 1, black triangle in the figure).

 As a result, almost no quantitative property was obtained.

Comparative Example 2 The reduced NAD concentration added in Example 1 was 0.05 m.
M was set to 0.5 mM, and the other components were the same, and the same measurement was carried out (black circles in FIG. 1 and the figure).

The results are not linear, and it is understood that there is no quantitativeness when reduced NAD is present in excess.

Comparative Example 3 The reduced NAD concentration added in Example 1 was 0.05 m.
M was set to 0, and other components were the same, and the same measurement was performed (FIG. 1, black square in the figure).

 As a result, almost no sensitivity was obtained.

Example 2 Measurement of Free Glycerol in Serum (1) Preparation of Reaction Reagent GPO 300 U, G3PDH 34 U, 12α-hydroxysteroid dehydrogenase 50 U, peroxidase 50 U, glycerol kinase (derived from the genus Streptomyces, manufactured by Toyo Brewery) 5 U, 0.05 mM reduction Type NAD, 0.03% 4-aminoantipyrine, 0.02% phenol, 0.5 mM cholic acid, 0.5 mM ATP,
2mM magnesium chloride, 0.1% Triton X-100 with 50mM HEPE
Dissolve in S buffer (pH 8.0) to make 10 ml.

(2) Measurement of Free Glycerol in Serum To a test tube preliminarily heated at 37 ° C. by dispensing 1 ml of the above reaction reagent, 20 μl of serum sample was added, and further reacted at 37 ° C. for 15 minutes. The reaction was stopped by adding 2.0 ml of 0.5% SDS as a stop solution. After stopping the reaction, the absorbance of the reaction solution at 500 nm was measured.

Using a normal human serum sample (serum 1), its stock solution, 4 /
Each diluted solution of 5, 3/5, 2/5, 1/5, and 0 was prepared with distilled water, and the absorbance was measured as a serum sample after the reaction according to the above procedure. The results showed good linearity as shown in FIG. 10, confirming that quantification was possible.

At the time of quantification, a blank test in which 20 μl of a serum sample was not separately added and a standard solution test in which 20 μl of a glycerol aqueous solution having a known concentration (for example, 50 μM) was added were performed in the same manner.

The free glycerol concentration in serum can be calculated using the following formula.

A _a; absorbance S of glycerol standard solution Test; absorbance A _c of blank test was not added serum specimen; absorbance A _b of the serum samples were added test solution concentration of the glycerol solution used in the glycerol standard solution Test ([mu] M) Normal human serum samples 1 to 5 were measured by the above operation.
As shown in Table 1, a trace amount of glycerol concentration could be measured.

Example 3 Quantification of lysolecithin (1) Preparation of reaction reagent GPO 150U, G3PDH 34U, 12α-hydroxysteroid dehydrogenase 50U, peroxidase 45U, lysophospholipase (EC3.1.1.5, derived from Vibrio, Toyo Brewery) 6U, glycero Phosphorylcholine phosphodiesterase (EC3.1.4.2, microbial origin, manufactured by Toyo Brewery Co., Ltd.) 3U,
0.05 mM reduced NAD, 0.03% 4-aminoantipyrine, 0.0
2% DAOS (N-ethyl-N- (2-hydroxy-3-propyl) -3,5-dimethoxyaniline, manufactured by Dojin Kagaku), 0.5 mM cholic acid, 5 mM calcium chloride in 50 mM HEPES
Dissolve in buffer (pH 8.0) to make 10 ml.

(2) Quantification of lysolecithin 1 μl each of 0, 30, 60, 90, 120 and 150 μM sizolecithin aqueous solution was added to test tubes preliminarily heated at 37 ° C. by dispensing 1 ml of the above reaction reagent, respectively. 10 at 37 ° C
After reacting for 2 minutes, 2.0 ml of 0.5% SDS was added as a reaction stop solution to stop the reaction. After stopping the reaction, the absorbance of the reaction solution at 600 nm was measured.

The results showed good linearity as shown in FIG.

Example 4 Measurement of Lecithin Cholesterol Acyltransferase Activity in Serum (1) Preparation of Reaction Reagents GPO 150U, G3PDH 34U, 12α-hydroxysteroid dehydrogenase 40U, peroxidase 45U, lysophospholipase (EC3.1.1.5, derived from Vibrio spp. 5U, glycerophosphorylcholine phosphodiesterase (EC3.1.4.2, derived from microorganisms, manufactured by Toyo Brewery) 3U,
0.05 mM reduced NAD, 0.03% 4-aminoantipyrine, 0.0
2% DAPS (N-ethyl-N-sulfopropyl-3,5-dimethoxyaniline, manufactured by Dojindo), 0.25 mM cholic acid, 5 m
M calcium chloride and 0.1% Triton X-100 are dissolved in 50 mM HEPES buffer (pH 8.0) to make 10 ml.

(2) Measurement of lecithin cholesterol acyltransferase activity in serum 0.2 ml of a serum sample was dispensed into each of two test tubes A and B,
Test tube A contains cholesterol hydrolase (EC 3.1.1.1).
(3. Microbial origin, manufactured by Toyo Brewery Co., Ltd.) 1U was added thereto, followed by heating at 37 ° C. for 15 minutes, and 50 μl of 10% Triton X-100 was added. To test tube B, 50 μl of 10% Triton X-100 is added, and then 1 U of cholesterol hydrolase is added. Lecithin cholesterol acyltransferase is activated by cholesterol hydrolase, 10% Triton X-100
Stops the enzyme reaction.

Separately, a new test tube was prepared, and 1 ml of the reaction reagent preliminarily heated at 37 ° C. and 8 μl of the test solution of the test tube A or B obtained above were dispensed at 37 ° C. After reacting for 1 minute, test tubes A 'and B' were obtained. 0.5% SDS in both test tubes 2.
0 ml was added to stop the reaction. After stopping the reaction,
The absorbance at nm was measured.

For quantification, a blank test in which 8 μl of distilled water was used instead of 8 μl of the test solution in test tube A or B by the same operation, and a lysolecithin aqueous solution of known concentration (for example, 150 nmole /
ml), and a standard solution test to which 8 μl was added was performed.

Lecithin cholesterol acyltransferase activity in serum can be calculated using the following formula.

A _a; vitro 'absorbance A of the test solution _b; tube B' A absorbance A of the test solution of _c; absorbance S of the blank test using distilled water; absorbance A _d of standard solution test using lysolecithin solution The concentration of the aqueous lysolecithin solution used in the lysolecithin standard solution test (nmole / ml) 60/15 is a multiplier for converting the activity value for 15 minutes to the activity per hour.

Normal human serum samples 1 to 3 were measured by the above operation (Table 2).

Example 5 Measurement of Phosphatidylglycerol in Amniotic Fluid (1) Preparation of Reaction Reagent Reagent 1 34 U of catalase (manufactured by Sigma, bovine liver) was added to 100 mM P
Dissolve in IPES buffer (manufactured by Dojindo Laboratories, trade name (pH 7.4)) to make 10 ml.

Reagent 2 GPO 600U, G3PDH 68U, 12α-hydroxysteroid dehydrogenase 80U, peroxidase 90U, glycerol kinase (EC 2.7.1.30, derived from Bacillus, Boehringer Mannheim) 10U, 0.05 mM reduced NAD, 0.
06% 4-aminoantipyrine, 0.04% ADOS (N-ethyl- (2-hydroxy-3-sulfopropyl) -m-anisidine, manufactured by Dojin Kagaku), 1 mM cholic acid, 1 mM ATP, 4 mM
Magnesium chloride, 4 mM sodium nitride, 10 mM calcium chloride, 0.2% Triton X-100 in 50 mM PIPES buffer (pH 7.
Dissolve in 5) to make 10 ml.

Reagent 3 Dissolve 100 U of phospholipase D (derived from Streptomyces sp., Manufactured by Toyo Brewery Co., Ltd.) in 10 mM Tris-HCl buffer (manufactured by Wako Pure Chemical Industries, Ltd., (pH 8.0)) to make 2 ml.

(2) Measurement of phosphatidylglycerol in amniotic fluid 50 μl of amniotic fluid sample and reagent 1 were placed in two test tubes A and B, respectively.
Dispense 0.5 ml and preheat at 37 ° C. Further, 15 U of glycerol oxidase (from Aspergillus) was added and reacted at 37 ° C. for 5 minutes to eliminate endogenous glycerol,
0.5 ml of Reagent 2 is added. Test tube A contains 20 µl of reagent 3
Then, add 20 μl of distilled water to test tube B, react at 37 ° C. for 10 minutes, and terminate the reaction with 2.0 ml of 0.5% SDS. The absorbance at 542 nm of each test solution was measured.

Separately, dispens 0.5 ml each of Reagent 1 and Reagent 2 and 37 ° C
Prepare a pre-warmed test tube at
A standard solution test to which 50 µl of a P aqueous solution (for example, 10 µM) was added and a blank test to which 50 µl of distilled water were added were performed.

The concentration of phosphatidylglycerol in amniotic fluid can be calculated using the following formula.

A _a; absorbance A of test solution of the test tube A _b; absorbance of the blank test using distilled water S;; absorbance A _d of standard solution test using a G3P solution; absorbance A _c of test solution of the test tube B G3P Table 3 shows the results of measuring amniotic fluid specimens 1 to 5 in terms of the concentration (μM) of the aqueous G3P solution used in the standard solution test.

Example 6 Measurement of glycerol kinase activity (1) Preparation of reaction reagent GPO 200U, G3PDH 34U, 12α-hydroxysteroid dehydrogenase 50U, peroxidase 50U, 0.05
mM reduced NAD, 0.03% 4-aminoantipyrine, 0.02%
Phenol, 0.5mM cholic acid, 5mM magnesium chloride, 2m
M ATP, 1 mM glycerol in 100 mM PIPES buffer (pH 7.5)
And make up to 10 ml.

(2) Measurement of glycerol kinase activity A test tube into which 1 ml of a reaction reagent has been dispensed is preliminarily heated at 37 ° C., 50 μl of a sample is added, and the mixture is reacted at 37 ° C. for 10 minutes (t). After adding 2.0 ml of 0.5% SDS to stop the reaction, the absorbance of the reaction solution at 500 nm was measured.

At the time of quantification, a blank test was performed by adding 50 μl of distilled water instead of 50 μl of the sample by the same operation. Furthermore, the cycling rate (k _c ) of the enzymatic cycling reaction was measured using a G3P aqueous solution having a known concentration. Under the experimental conditions, the rate was 8 times / min.

Glycerol kinase activity can be calculated using the following formula. The enzyme activity that converts 1 μmole of glycerol into G3P per minute at 37 ° C. is defined as 1 U.

A _s : Absorbance of test solution to which sample was added A _B : Absorbance of test solution of blank test to which distilled water was added t: Reaction time (min) k _c : Cycling rate of the enzymatic cycling reaction Under the experimental conditions, the rate was 8 times / min.

12; Millimol molecular extinction coefficient (cm ² / μmole) of a color body produced as a chromogen using 4-aminoantipyrine and phenol
(Refer to TOYO ENZYMES 1982 p10) 1/2; constant 2 indicating that 1 mole of hydrogen peroxide produces 1/2 mole of the above-mentioned color body 2; constant 3.05 / 0.05 indicates that the sample solution is used as the final reaction solution. The dilution ratio of the sample stock solution is shown, and 10 ³ is a multiplier for converting ml to l.

A glycerol kinase aqueous solution of unknown activity was diluted with distilled water to a stock solution, 4/5, 3/5, 2/5, 1/5, 0 and measured by the above method. It showed linearity.

[Effects of the Invention] The present cycling reaction can easily measure G3P or DHAP by a colorimetric method without requiring a new device. Conventionally, it has been difficult to quantitatively measure hydrogen peroxide produced by the enzymatic cycling reaction (I) using a peroxidase colorimetric system. However, according to the present invention, it is highly sensitive and has good quantitative properties. A measuring method was obtained. The effect of the present invention has been proved by comparing Example 1 with Comparative Examples 1 to 3. That is, when the regeneration system was not added, as shown in Comparative Example 1, the added reduced NA was added.
When the concentration of D is exhausted, quantification cannot be obtained at a higher concentration, and when reduced NAD is excessively added, quantification is poor as shown in Comparative Example 2. Conversely, when no reduced NAD was added, no sensitivity was obtained as shown in Comparative Example 3, and Example 1 was several hundred times more sensitive than Comparative Example 3.

[Brief description of the drawings]

FIG. 1 shows glycerol measured by the method of the present invention.
3 shows a calibration curve of 3-phosphate and a calibration curve of glycerol-3-phosphate in Comparative Examples 1 to 3. 2 to 9 show the relationship between the concentration of reduced NAD in various chromogens and the absorbance (percentage where the maximum value of the absorbance is 100%). FIG. 2 shows that the chromogens are 4-aminoantipyrine and phenol. FIG. 3 shows that the chromogens are 4-aminoantipyrine and ADPS. FIG. 4 shows that the chromogens are 4-aminoantipyrine and HDAPS. FIG. 5 shows that the chromogen is 4
-Aminoantipyrine and ADOS. FIG. 6 shows that the chromogens are 4-aminoantipyrine and DAOS. FIG. 7 shows that the chromogens are 4-aminoantipyrine and HDAOS. FIG. 8 shows that the chromogens are 4-aminoantipyrine and TOOS. FIG. 9 shows that the chromogen is MMX. FIG. 10 shows a calibration curve of glycerol in serum. FIG. 11 shows a calibration curve of lysolecithin. FIG. 12 shows a calibration curve for glycerol kinase.

Claims (12)

(57) [Claims] An enzyme cycling reaction (I) for measuring glycerol-3-phosphate or dihydroxyacetone phosphate in a test solution. (Wherein G3P is glycerol-3-phosphate, DHAP is dihydroxyacetone phosphate, GPO is glycerol-3-phosphate oxidase, G3PDH is glycerol-3-phosphate dehydrogenase, H ₂ O ₂ is hydrogen peroxide, POD Is a peroxidase, A is a reducing substance, and B is an oxidation product.) The components in the test solution are reacted with the components forming the enzymatic cycling reaction (I) to form NAD generated by the enzymatic cycling reaction in a highly sensitive colorimetric method having a peroxidase chromogenic system (II) in which hydrogen peroxide amplified and produced by the enzymatic cycling reaction is converted into a colorant by a peroxidase and a chromogen. Highly sensitive colorimetric method characterized by having a regeneration system (III) for regenerating to reduced NAD, and having a reduced NAD concentration of 80% or more to develop a color intensity. . 2. A highly sensitive colorimetric method according to claim 1, wherein the peroxidase is horseradish peroxidase. 3. The chromogen has the formula: (Wherein, R ₁ and R ₄ are each a hydrogen atom, a lower alkyl group or a lower alkoxyl group, R ₂ and R ₃ are each a hydrogen atom,
A lower alkyl group, a hydroxy lower alkyl group, a lower alkyl group containing acetylamide, a lower alkyl group containing a sulfonic acid group, or a hydroxy lower alkyl group containing a sulfonic acid group). Combining 4-aminoantipyrine with the formula: (Wherein X is a hydrogen atom, a halogen atom, a sulfonic acid group,
A nitro group, a lower alkyl group, a lower alkoxyl group, a lower alkylcarbonyl group, a carboxyl group or a lower alkoxycarbonyl group, and Y and Y 'are each a hydrogen atom, a halogen atom, a sulfonic acid group, a nitro group, a lower alkyl group, a lower alkoxyl group , A lower alkylcarbonyl group,
Carbamoyl group or lower alkoxycarbonyl group, Z
And Z ′ are each a hydrogen atom, a sulfonic acid group, a nitro group,
Represents a lower alkyl group, a lower alkoxyl group or a carboxyl group), and a combination of one selected from the group of phenols represented by 4-aminoantipyrine,
2. The method according to claim 1, wherein the method is 10-[(3-methoxycarbamoylmethyl) phenylmethylcarbamoyl] 3,7-dimethylaminophenothiazine. 4. A color development intensity of at least 80% of 0.003 to 0.13 m
2. The high-sensitivity colorimetric method according to claim 1, wherein M is M. 5. The high sensitivity according to claim 3, wherein the aniline is N-ethyl-N-sulfopropyl-m-anisidine and has a color developing intensity of at least 80% of 0.006 to 0.07 mM. Colorimetric method. 6. An aniline comprising N-sulfopropyl-3,5
4. The high-sensitivity colorimetric method according to claim 3, which is dimethoxyaniline and has a color developing intensity expression concentration of 80% or more of 0.006 to 0.1 mM. 7. The method according to claim 1, wherein the aniline is N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-anisidine and has a color developing intensity of not less than 80% of 0.007 to 0.05 mM. Item 4. The high-sensitivity colorimetric method according to Item 3. 8. The aniline is N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethoxyaniline, and has a color developing intensity of at least 80% of 0.01 to 0.13.
4. The high-sensitivity colorimetric method according to claim 3, which is mM. 9. An aniline comprising N- (2-hydroxy-3)
The high-sensitivity colorimetric method according to claim 3, wherein the sulfopropyl) -3,5-dimethoxyaniline has a color developing intensity of 80% or more and a concentration of 0.008 to 0.11 mM. 10. The aniline represented by N-ethyl-N- (2-
4. The high-sensitivity colorimetric method according to claim 3, wherein (hydroxy-3-sulfopropyl) -m-toluidine has a color developing intensity expression concentration of 80% or more and 0.005 to 0.06 mM. (11) the phenol is phenol;
The high-sensitivity colorimetric method according to claim 3, wherein the color development intensity expression concentration is 0.008 to 0.07 mM or more. 12. The method according to claim 1, wherein the chromogen is 10-[(3-methoxycarbamoylmethyl) phenylmethylcarbamoyl] 3,7-dimethylaminophenothiazine and has a color developing intensity of at least 80% of 0.003 to 0.04 mM. 2. A high-sensitivity colorimetric method according to item 1.