JPH07114711B2 - Quantitative method for reduced coenzyme - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for quantifying reduced coenzyme and a composition for quantifying.

Quantification of reduced coenzymes such as reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) is NAD (P).
It is useful for quantifying the substrate or enzyme activity of the enzymatic reaction involving the production of H.

Conventional Techniques and Problems Conventional methods for quantifying NAD (P) H, for example, a method of directly measuring absorption of NAD (P) H in the ultraviolet region, NAD (P) H
A method of guiding H to a fluorescent substance via an electron carrier and measuring the intensity of the fluorescence, NAD (P) H is guided from a tetrazolium salt to formazan via an electron carrier, and the amount of the produced dye is colorimetrically determined. And a method for quantifying hydrogen peroxide produced in the presence or absence of superoxide dismutase (SOD) by causing an electron carrier to act on NAD (P) H recently (Clinical Chemistry, Volume 15: Issue 1 (1986)
20-27, JP-A-59-210899, JP-A-62-19100).

These methods have the following disadvantages. That is, with regard to, the sensitivity is low, and when it is applied to a clinical test, since it is measured in the ultraviolet region, it is easily affected by biological components in the sample. For, a fluorometer is required. For, the sensitivity is low in the determination of trace components, the generated formazan is sparingly soluble in water, deposits on equipment, etc., and does not easily fall off, and many tetrazolium salts and formazan are unstable to light. Sometimes be careful. Furthermore, since the λ _{max of the} generated pigment is around 500 nm, it is easily affected by the vital pigment. In addition, it is essential to divide the reagent into two or more types. That is, when measuring NAD (P) H in a sample, first, an electron carrier is allowed to act on NAD (P) H in the sample in the presence or absence of SOD to convert it into hydrogen peroxide. It is necessary to accumulate in this first reagent solution (first reagent solution). Next, a hydrogen peroxide quantitative reagent must be added as a second reagent solution to the first reagent solution for colorimetric determination. Accurate measurement cannot be performed even if NAD (P) H is added to a mixture of the first reagent solution and the second reagent solution. This fact is a fatal drawback particularly when a biological sample is used as a measurement target.

That is, since hydrogen peroxide consuming substances such as ascorbic acid, uric acid, catalase, bilirubin, and hemoglobin are present in a biological sample, when it is necessary to accumulate hydrogen peroxide for a certain period of time as in the method described above, Hydrogen peroxide consuming substances that coexist with the hydrogen peroxide consume the accumulated hydrogen peroxide, which causes a negative error in the measured value.

In order to remove hydrogen peroxide consuming substances as described above,
For example, pretreatment of a biological sample with ascorbic acid oxidase for ascorbic acid, uricase for uric acid, bilirubin oxidase for bilirubin, etc. is conceivable. These pretreatment enzymes and the like are allowed to coexist in the first reagent solution and pretreatment is performed. On the contrary, when it is carried out, the catalase activity and peroxidase activity mixed in these enzymes consume hydrogen peroxide which is still accumulating,
Negative error may occur in the measured value. In addition, enzymes such as uricase that produce hydrogen peroxide cannot be practically used for pretreatment. In order to set up a strict pretreatment system, the first reagent solution,
In addition to the second reagent liquid, reagent liquids for pretreatment are necessary, which makes the operation complicated and is inconvenient. Further, the peroxidase and the chromogen must coexist in the second reagent solution, and a measurement error due to coloring of the reagent during storage cannot be avoided. There are various problems in putting such a method into practical use. In addition, NAD (P) H and a specific color source (i)
NAD by quantifying the dye produced by reaction in the presence of peroxidase or thiol oxidoreductase and (ii) diaphorase or electron carrier
A method for quantifying (P) H is known (JP-A-60-80).
600) Means for Solving Problems According to the present invention, NAD (P) H is a dye produced by reacting NAD (P) H with peroxidase or diaphorase in the presence of a compound represented by the following general formula or a salt thereof. Can be quantified by quantifying.

General formula (I): In the formula, Y is a hydrogen atom or , Z represents an oxygen atom or a sulfur atom, X represents a hydrogen atom, alkyl, alkenyl, allyl, amino or substituted amino, R ₁ represents hydroxyl, amino or substituted amino, and R ₂ represents a hydrogen atom or hydroxyl. , Alkyl, alkoxy, allyl, alkenyl, amino or substituted amino, R ₃ R ₄ , R ₅ and R ₆ may be the same or different, and are hydrogen atom, alkyl, alkenyl, acyl, allyl, halogen atom, nitro and sulfo. , Carboxyl, hydroxyl, alkoxy, and groups represented by the following general formulas III, IV, VI, and VII.

General formula (III): General formula (IV): General formula (VI): General formula (VII): In the formula, A ₁ represents an alkylene group, A ₂ has the same meaning as R ₂ , B ₁ , B ₂ , B ₃ , B ₄ , B ₅ and B ₆ are the same or different and each is a hydrogen atom, alkyl or alkenyl. , Acyl, allyl,
Represents a halogen atom, nitro, sulfo, carboxyl, hydroxyl, alkoxy or hydroxyalkyl.

Furthermore, superoxide dismutase (hereinafter referred to as SOD)
Said) stabilizes the produced dye.

According to the method of the present invention, the object can be achieved by adding a reagent necessary for quantification to a sample and reacting it in one step, which is extremely simple.

As the substituent of the substituted amino in R ₁ and R _2, alkyl, alkenyl, allyl, hydroxyalkyl, cycloalkyl, acyl, allylalkyl, acylalkyl,
Carboxyl, alkoxy, sulfo, sulfoalkyl and the like are exemplified.

The alkyl in each of the above definitions includes a group having 1 to 5 carbon atoms, and is exemplified by methyl, ethyl, propyl, butyl, pentyl and the like.

The alkoxy group includes a group having 1 to 5 carbon atoms, and is exemplified by methoxy, ethoxy, propoxy, butoxy and the like.

The alkenyl includes a group having 2 to 5 carbon atoms and is exemplified by vinyl, propylene, butylene and the like.

Examples of allyl include phenyl and naphthyl (these groups may have a substituent), and the like.
Alkyl having 4 carbon atoms, halogen atom, amino,
Alkoxycarbonylamino, alkoxycarbonylaminoalkyl, alkoxy, acyl and the like are included.

The acyl includes a group having 2 to 5 carbon atoms and is exemplified by acetyl, propionyl, butyryl and the like.

Examples of the halogen atom include a fluorine atom, a chlorine atom and a bromine atom.

Alkenylene includes a group having 3 to 4 carbon atoms, and -CH = CH
_{-CH 2 -, - CH = CH} -CH = CH- , and the like.

Examples of the reduced coenzyme that can be quantified in the present invention include NA
Examples thereof include DH, NADPH, reduced pyrroloquinoline quinone, and the like, but are not limited thereto.

In carrying out the present invention, a buffer having a pH of 2 to 11, such as phosphate, Tris-hydrochloric acid, succinate, oxalate, acetate, Good, etc., is generally added in an amount of 0.1 to 1000 in a 0.005 to 2 mol / l solution.
IU / ml peroxidase and 0.1-1000 IU / ml diaphorase or NAD (P) H oxidoreductase, 0.1-
500 IU / ml of superoxide dismutase and compound (I) or a salt thereof is added to the reduced coenzyme in the sample in an equimolar amount or more, usually 10 to 1000 times, to prepare a reagent solution. In addition to the sample contained, the reaction is carried out at 5 to 50 ° C, preferably 25 to 40 ° C.

A method known per se that can be quantified can be applied to the quantification of the produced dye, but the absorption by the reaction solution after the reaction is measured at the maximum absorption wavelength of the produced dye using the reagent blank as a control, and the calibration obtained from the test for the known amount in advance. The reduced coenzyme in the sample can be quantified using the line.

The reduced coenzyme in the reaction solution is preferably diluted with a reagent solution or distilled water so as to be 0.0001 to 1 mg / ml.

If necessary, a surfactant such as Triton X-100 (trade name, polyethylene glycol mono-p-iso-octylphenyl ether) is used in the reaction system.

A small amount of phenol can be added to the reaction system to activate peroxidase or to accelerate the pigment formation reaction.

The method of the present invention can be suitably applied to the quantification of a reaction substance or an enzyme activity involved in a reaction system for producing reduced coenzyme as described below. In particular, when the reduced coenzyme production system is an oxygen reaction, the reduced coenzyme production reaction and the dye production reaction can be carried out in the same system, so that the quantitative determination can be performed in a short time.

When the object to be measured is a substrate, an enzyme that decomposes this to produce a reduced coenzyme, and when the object has an enzymatic activity, the enzyme substrate is used as a reagent together with compound (I) or a salt thereof. In addition to the liquid, add this reagent liquid to the sample to react,
The substrate or enzyme activity can be measured by measuring the absorption of the colored reaction solution.

As described above, according to the method of the present invention, when forming a reagent system for analyzing a biological sample, it is natural that the production of NAD (P) H can be colorimetrically determined by one reagent solution system, but it is more accurate. It is also possible to use pretreatment reagents to obtain different measured values.

That is, a pretreatment system containing peroxidase, diaphorase or NAD (P) H oxidoreductase can be incorporated into the first reagent solution, and a reaction system containing compound (I) or a salt thereof can be incorporated into the second reagent solution. For example, in the case of a system for quantifying glycerol in serum using glycerol dehydrogenase, the first reagent solution contains peroxidase, diaphorase or NAD (P) H oxidoreductase, oxidized NAD,
An oscorbate oxidase or the like is placed, and glycerol dehydrogenase, compound (I) or a salt thereof is placed in the second reagent solution. In this system, even if the peroxidase activity is mixed with the ascorbate oxidase, it does not matter at all. It is also noteworthy that when the compound (I) or its salt is used as the color source, it is not affected by hydrogen peroxide consuming substances such as catalase, uric acid and bilirubin. This suggests that even if a side reaction by an enzyme or the like that accompanies the production of hydrogen peroxide is carried out in the first reagent solution, catalase and the like can coexist in the first reagent solution to eliminate the hydrogen peroxide that does not participate in the reaction in advance. is doing. By performing the complete pretreatment in this way, it is possible to very accurately quantify NAD (P) H produced by the reaction of glycerol dehydrogenase, which is first initiated by the addition of the second reagent solution. In this case, in the glycerol in the serum, NAD from the first reagent solution is converted to NADH by glycerol dehydrogenase simultaneously with the addition of the second reagent solution, and further diaphorase or NAD (P) H oxidoreductase, peroxidase, compound (I) or NAD consisting of that salt
(P) Color is developed by the reagent for quantifying H. If the enzymatic activity of diaphorase, NAD (P) H oxidoreductase, or peroxidase is sufficient, NADH instantly develops its color. Therefore, the rate-determining step of this reaction is the reaction of glycerol dehydrogenase. This fact is extremely preferable for the assay method using an enzyme, and shows that the enzyme activity in the sample can also be accurately measured. Further, since a two-reagent system in which the peroxidase and the compound (I) or a salt thereof are separated can be used, a measurement error due to coloring of the reagent during storage can be avoided, which is convenient.

It should also be noted that many of the dyes produced by the reaction are water-soluble and have a λ _max of around 700 nm. This means that colored components derived from biological samples, such as hemoglobin,
It shows that it is extremely unlikely to be affected by bilirubin.

Representative color source materials used in the present invention are exemplified in Table 1.

M: CH ₃ E: C ₂ H ₅ α ₁ : CH ₂ CH ₂ CH ₃ α ₂ :> CH CH ₂ CH ₂ CH ₃ D: CH ₂ CH ₂ CH ₂ SO ₃ H All of R ₄ , R ₆ , B ₂ , B ₃ , B ₅ and B ₆ in the compound represent hydrogen.

Y is H (for compounds 1-29) (For compound 30) is shown.

R ₃ indicates the 2nd position and R ₅ indicates the 9th position.

Alternatively, B ₁ is the 4-position for the compounds Nos. 16 and 17, and the others are all the 3-positions for groups other than H, and B ₄ is the 8-position for groups other than H.

Maximum absorption wavelength (λ _max ) when using these color sources
Table 2 shows the sensitivity, the effect of the components in serum when used in the following reduced coenzyme quantification method, and the degree of solubility of the produced dye in water.

The sensitivity was measured by the following method.

20 U / ml peroxidase, 20 U / ml diaphorase, 10
50 mM N- (2-acetamido) iminodiacetic acid containing U / ml superoxide dismutase, 5 mg / ml Triton X-100, 0.05 mg / ml phenol, 0.05 mg / ml compound (I) (Good PH 7.0 buffer solution) is added to 50 ml of 300 μmol / L NADH solution. The reaction is carried out at 37 ° C for 5 minutes, and the OD at λ _max of the reaction solution is measured.

Nitrotetrazolium blue is shown in the table as a color source.
In the case of nitrotetrazolium blue, an experiment was conducted using a system in which peroxidase, superoxide dismutase and phenol were removed from the above buffer solution.

The effects of serum components are as follows: when bilirubin 10 μg / 3 ml, cysteine 10 μg / 3 ml, or uric acid 10 μg / 3 ml is present, the effect is 5-10% as ±, 10-20% as +, and 5% or less as- Show.

The degree of solubility of the produced dye in water is an evaluation when compared with nitrotetrazolium blue. AA has significantly better solubility in water than nitrotetrazolium blue. A has good solubility in water. B means that it is soluble in water only to the same extent as nitrotetrazolium blue.

High λ _max , high sensitivity, unaffected by biological components,
The more soluble it is in water, the more suitable it is for the quantification of trace biological components.

The chromogen used in the present invention is obtained by subjecting leuco bases obtained by reducing diphenylamines to a condensation reaction with benzhydrols and sulfuric acid. The reaction is completed in a few hours at 30-100 ° C. The desired product can be isolated by adding the reaction solution to an organic solvent capable of crystallizing the desired color source, for example, n-octanol, dissolving the crystallized product in an organic solvent such as methanol, and performing silica gel chromatography.

The method for producing the compounds shown in Table 1 is described in JP-A-60-218069.

INDUSTRIAL APPLICABILITY The method of the present invention can be applied to the measurement of a reactant or enzyme activity in a reaction system that produces NAD (P) H stoichiometrically by a reaction. As such a reaction system, many reaction systems using NAD and dehydrogenase are known. Examples of such substances include glucose, galactose, lactic acid, alcohol, malic acid, aldehyde, xanthine, cholesterol, bile acid, lactate dehydrogenase (LDH) activity, and neutral fat.

These reaction systems are shown schematically below, and the substrate and enzyme activity in the reaction system can be measured.

The composition for quantifying NAD (P) H used in the present invention is (a) peroxidase (b) diaphorase or NAD (P) H oxidoreductase (c) compound (I) or a salt thereof or (d) super Consists of oxidodismutase, which are (a) 2-100 U / ml, (b) 2-50 U / ml (c) 0.01-
It is convenient to use that the amount ratio of 1.0mg / ml, (d) 0.1-50U / ml. In addition, buffers and surfactants can be combined with the composition if desired.

A composition containing a substrate or enzyme required depending on the factor to be measured or a kit prepared separately and combined is extremely convenient and useful for quantifying various substances or enzyme activities.

Hereinafter, embodiments of the present invention will be shown by examples.

Example 1 (Quantification of NADH) Peroxidase 2 was added to 100 ml of 50 mM Good buffer (pH 7.0).
000 units, diaphorase 2000 units, superoxide dismutase 1000 units, surfactant (Triton X-100) 5
Dissolve 5 mg of phenol, 5 mg of phenol and 5 mg of compound No. 5, and prepare a reagent solution.

Separately, prepare NADH solutions of 100, 200, 300, 500 and 1000 μmol / l, divide 50 μ each into a test tube, add 3 ml of each of the above reagent solutions, and leave at 37 ° C. for 5 minutes, then at λ _max 755 nm Absorbance is measured using a test blind as a control.

The results are shown in Table 3.

Example 2. (Determination of total cholesterol) 5 mg of each of Compound Nos. 1, 2, 3, 5, 6 and 22 instead of Compound No. 5
6 kinds of reagent solutions having the same composition as in Example 1 are prepared except that 500 mg of NAD, 500 units of cholesterol dehydrogenase and 100 units of cholesterol esterase are added.

Transfer 10μ of live serum and 3ml of the above color test solution to a test tube, and
After left at 5 ° C for 5 minutes, the absorbance at λ _max of the produced dye is measured using a test blind as a control, and the total cholesterol concentration in serum is calculated from a calibration curve prepared in advance. As a control, cholesterol esterase and cholesterol oxidase are added to the sample, and the produced hydrogen peroxide is quantified using peroxidase and a chromogen.

Example 3. (Quantification of neutral fat) Instead of Compound No. 5, Compound No. 5, Compound No. 23,
Compound No.26, Compound No.27, Compound No.28 Compound N
Six kinds of reagent solutions having the same composition as the reagent solution of Example 1 (b) are prepared except that 5 mg each of o.29 is used and 500 mg of NAD, 50,000 units of lipoprotein lipase, and 5000 units of glycerol dehydrogenase are added.

20 μl of live serum and 3 ml of reagent solution were placed in a test tube and allowed to stand at 37 ° C. for 5 minutes, and the absorbance at λ _max of each chromogen shown in Table 3 was measured in advance using a reagent blind test as a control. The neutral fat concentration in serum is calculated from the calibration curve.

As a control, lipoprotein lipase was added to the sample to produce glycerate from neutral fat, and hydrogen peroxide produced by the action of glycerol oxidase on this was guided to the dye and quantified by the colorimetric reaction solution. The results are shown in Table 5.

Example 4. [Measurement of lactate dehydrogenase (LDH) activity] Compound No. 5, Compound No. 22, instead of Compound No. 5,
A reagent solution having the same composition as that of the reagent of Example 1 was prepared except that 5 mg each of Compound No. 24 and Compound No. 25 was used, and NAD 500 mg and sodium L-lactate 50 mg were added.

Preliminarily warm the reagent solution at 37 ℃ for 10 minutes, and then add serum 20μ
Is added, and the absorbances 1 minute and 3 minutes after the addition are measured at λ _max of each chromogen shown in Table 3 using the test blind as a control. The difference in absorbance after 3 minutes and after 1 minute is calculated, and the LDH activity in serum is calculated from the calibration curve prepared in advance.
The results are also shown in Table 6 in comparison with the values measured using the UV method.

Example 5. (Quantification of total bile acid) A two-reagent system is used to obtain a more accurate measurement value. That is, 4000 units of peroxidase, 4000 units of diaphorase, 2000 units of superoxide dismutase, 10 mg of phenol, 5 mg of surfactant (Triton X-100) and 500 mg of NAD are dissolved in 100 ml of 50 mM Good buffer solution (pH 7.0), Use the reagent solution.

Next, 200 units of 3α-hydroxysteroid dehydrogenase and 5 mg of a surfactant (Triton X-100) were dissolved in 100 ml of 50 mM Good buffer (pH 7.0), and Compound No. 1 and Compound No. .2, compound No. 3, compound
Reagent solutions containing 10 mg each of No. 5 and compound No. 6 dissolved in 5
Prepare a kind and use it as the second reagent solution.

Put 50μ of live serum and 1.5ml of the first reagent solution into a test tube and incubate at 37 ℃.
After preliminarily heating for 5 minutes at 1.
Add 5 ml and leave at 37 ° C for 5 minutes. Then, the absorbance at λ _max of each chromogen shown in Table 3 is measured using a test blind as a control, and the total bile acid concentration in serum is calculated from a calibration curve prepared in advance. The results are shown in Table 7 in comparison with the values measured using the high performance liquid chromatography method (HPLC method).

It is understood that in each of Examples 2 to 5, each of the measuring methods of the present invention is close to and accurate in the results of the other methods.

Claims (2)

[Claims] 1. NAD in the presence of a compound represented by the following general formula (I) [hereinafter referred to as compound (I)] or a salt thereof.
(P) A method for quantifying reduced coenzyme, which comprises reacting peroxidase or diaphorase with H and measuring the absorbance of the reaction solution which has been colored. General formula (I): In the formula, Y is a hydrogen atom or , Z represents an enzyme atom or an ionic atom, X represents a hydrogen atom, alkyl, alkenyl, allyl, amino or substituted amino, R ₁ represents hydroxyl, amino or substituted amino, and R ₂ represents a hydrogen atom or hydroxyl. , Alkyl, alkoxy, allyl, alkenyl, amino or substituted amino, R ₃ , R ₄ , R ₅ and R ₆ may be the same or different,
A hydrogen atom, alkyl, alkenyl, acyl, allyl, halogen atom, nitro, sulfo, carboxyl, hydroxyl, alkoxy and groups represented by the following general formulas III, IV, VI and VII are shown. General formula (III): General formula (IV): General formula (VI): General formula (VII): In the formula, A ₁ represents an alkylene group, A ₂ has the same meaning as R ₂ , B ₁ , B ₂ , B ₃ , B ₄ , B ₅ and B ₆ are the same or different and each is a hydrogen atom, alkyl or alkenyl. , Acyl, allyl,
Represents a halogen atom, nitro, sulfo, carboxyl, hydroxyl, alkoxy or hydroxyalkyl. 2. A composition for quantifying NAD (P) H, which comprises (a) peroxidase (b) diaphorase (c) compound (I) or a salt thereof.