JP2618456B2 - Dinucleotide quantification method - Google Patents

Description

Description: TECHNICAL FIELD The present invention relates to a method for quantifying dinucleotides and a composition for quantification. More specifically, a method of reacting dinucleotide with phosphoenolpyruvate in the presence of pyruvate kinase, and quantifying dinucleotide by quantifying hydrogen peroxide produced by reacting pyruvate with pyruvate oxidase. And compositions therefor.

As an application of the present invention, it is possible to quantify a substance or enzyme activity involved in a reaction for quantitatively producing a dinucleotide, particularly an enzyme reaction.

Such reactants include bilirubin, lipopolysaccharide, ceramide and the like, and these components can be quantified by the method of the present invention.

Prior art As a method for quantifying dinucleotides, phosphoenol pyruvate (PEP) and pyruvate kinase (PK) are allowed to act on ADP, and lactate dehydrogenase (LDH) is acted on the resulting pyruvate in the presence of NADH. P by measuring change
A method for quantifying K activity is known.
It is easy to understand that can be determined [The Enzymes 3r
d.ed. 8,353-382 (1973)]. The reaction formula of this reaction is as follows.

In the above literature, PK is a nucleotide other than ADP, such as U
It has also been suggested that DP, GDP, etc. have a slight effect.

Enzymes that produce nucleotides from bilirubin, lipopolysaccharide, ceramide, and the like are known, but a method for quantifying these components via dinucleotides is not known.

A method utilizing a color reaction of bilirubin with a diazonium salt is known, but the effects of coexisting substances and drugs to be administered cannot be ignored, and the intensity of the color is weak, so that it is also affected by turbidity. Also, a method using bilirubin oxidase has been developed, but since it is a method for measuring the degree of color reduction of bilirubin itself, sample blinding is always necessary, and the measurement wavelength is a wavelength that is easily affected by turbidity.

On the other hand, ceramides and lipopolysaccharides are known to use high-performance liquid chromatography, but have problems in terms of sample processing capability and simplicity.

Recently, several specific glycoproteins in the blood of cancer patients that are not found in the normal state have been discovered, and it has been reported that glycosyltransferases are involved in the production process in these organisms. However, the measurement method has not been developed. Therefore, a method for measuring the activity of a substrate or an enzyme involved in the sugar transferase reaction is strongly desired.

Problems to be Solved by the Invention To quantify bilirubin and the like via nucleotides, the final measurement component is very small, and there is no example in which the above-described known method has been applied. A blank test is required, and the sensitivity is poor and cannot be substantially determined.

Means for Solving the Problems A study was conducted on a method of coloring with high sensitivity. As a result, pyruvic acid generated by the reaction formula (1) was decomposed with pyruvate oxidase, and the objective was sufficiently achieved by quantifying the generated hydrogen peroxide. I found what I could do.

According to the present invention, hydrogen peroxide produced by reacting a dinucleotide with phosphoenolpyruvate (PEP) in the presence of pyruvate kinase (PK) and allowing pyruvate to react with pyruvate oxidase (PYOD) By quantifying, the dinucleotide can be quantified. (Hereinafter referred to as Invention 1) The principle of the present invention is represented by the following reaction formula.

In the present invention, if pyruvate is contained in the sample, the total amount of pyruvate in the sample plus pyruvate in the target component-derived pyruvate will be quantified, so a blank test of the sample is separately performed. It is necessary to quantify pyruvic acid in the sample, and the operation is duplicated.

Further, when pyruvic acid is present in a large amount in a sample, when hydrogen peroxide is quantified by a colorimetric method, it is necessary to measure at a position where the absorbance value is high, so that an error easily occurs.

If pyruvate is present in the sample, pyruvate oxidase (PYOD) is added to the sample to generate it.
After elimination of H ₂ O ₂ , dinucleotide can be accurately quantified without applying a sample blank test by applying invention 1 (hereinafter referred to as invention 2).

In carrying out the invention 2, PYOD and an H ₂ O ₂ scavenger are added to the sample to deactivate the H ₂ O ₂ scavenging ability after the reaction, and then H ₂ formed by adding PK, PEP and phosphoric acid according to the invention 1. O ₂ to H ₂ O ₂
What is necessary is just to determine with a quantitative system.

The dinucleotides that can be measured in the present invention include UDP, GDP
And the like.

In practicing the present invention, a buffer generally having a pH of 5-9, such as phosphate, citrate, borate, tris-hydrochloride, succinate, bis (2-hydroxyethyl) iminotris (hydroxymethyl) Methane (bis-tris), N
-2-Hydroxyethylpiperazyl-N'-2-catanesulfonic acid (HETS), acetate, Good, etc. in 0.01 to 2 mol /, near the optimum action temperature of the enzyme, usually 5 to 50 ° C, preferably
Performed at 25-40 ° C.

PK and PYOD used in the present invention are used at 1 to 200 U / ml, and PEP is used at 0.1 to 100 mg / ml. PK as required
0.1-50mM of magnesium ion as PYOD
The coenzyme co-carboxylase (thiamine pyrophosphate, TPP) is used at 0.01 to 100 mM and FAD is used at 0.01 to 10 mM.

As a method for measuring the production amount or rate of hydrogen peroxide,
Chemiluminescence analysis, electrochemical analysis, fluorescence analysis, and spectroscopic analysis are known (for example, Journal of the Society of Synthetic Organic Chemistry, Vol. 39, No. 7, p659-666 (1981)). reference). In the present invention, any of them can be applied, but for the purpose of the present invention, it is preferable to employ a system for detecting hydrogen peroxide quickly and with high sensitivity.

Some typical methods for measuring hydrogen peroxide are listed below.

First, as a chemiluminescence analysis method, there is a method in which luminol is reacted with hydrogen peroxide in the presence of red blood salt to measure the amount of luminescence accompanying oxidation. It is also known to use isoluminol, pyrogallol, bis (2,4,6-trichlorophenyl) oxalate in place of luminol and peroxidase, hematin, hemin, cobalt chloride in place of red blood salt.

As an electrochemical analysis method, a method using a Clark-type hydrogen peroxide electrode is generally used. A method of reacting hydrogen peroxide with iodine ion in the presence of a catalyst such as peroxidase or molybdate and measuring the reaction with an iodine ion electrode can also be used.

Fluorescence analysis involves the reaction of homovanillic acid with hydrogen peroxide in the presence of peroxidase,
2'-dihydroxy-3,3'-dimethoxybiphenyl-5,
A method for measuring the fluorescence intensity of 5'-diacetate is exemplified. Instead of homovanillic acid, p-hydroxyphenylacetic acid, diacetylfluorescin derivatives (diacetylfluorescin, diacetyldichlorofluorescin, etc.) and the like can also be used.

As a spectroscopic analysis method, a peroxidase method, a catalase method, and the like are generally used. The peroxidase method is
This is a method in which a chromogen is reacted with hydrogen peroxide in the presence of peroxidase (POD), and the resulting dye is colorimetrically determined. Examples of the chromogen include o-dianisidine, 4-methoxy-1-naphthol, 2,2'-azino-bis (3-ethylbenzothiazoline) -6-sulfonic acid (ABTS), 10N
A single reagent such as -methylcarbamoyl-3,7-dimethylamino-10H-phenothiazine (MCDP), 4-aminoantipyrine (4AA) and a phenolic compound (for example, phenol, p-chlorophenol, 2,4,6-tri- Bromophenol, etc.), aniline-based compounds (eg, N, N-dimethylaniline (DMA), N, N-diethylaniline, etc.),
Combination reagent with N-ethyl-N- (3-methylphenyl) -N'-succinylethylenediamine (EMSE) or toluidine-based compound (for example, N, N-diethyl-m-toluidine and the like), 3-methyl-2- A combination reagent of benzothiazolinohydrazone and N, N-dimethylaniline and the like can be used. Examples of the catalase method include a method in which hydrogen peroxide is reacted with an alcohol (methanol or the like) in the presence of catalase, and the resulting aldehyde is led to a coloring system to measure the absorbance of the dye.

Each of these hydrogen peroxide detection systems is a known method, and in carrying out the method, known operations and conditions may be referred to.

The most preferred method for quantifying hydrogen peroxide in practicing the present invention is a method for colorimetrically measuring coloring by a dye produced by reacting H ₂ O ₂ with a chromogen in the presence of peroxidase.

In practicing this method, peroxidase and a chromogen are also added to the reagent. The amount or rate of formation of the dye can be determined, for example, by measuring the change in absorption at the maximum absorption value of the dye.

In the present invention, when H ₂ O ₂ is quantified using a peroxidase and a chromogen, hemoglobin, bilirubin and the like are present in a biological sample, and therefore, the maximum absorption wavelength distant from the maximum absorption wavelength, particularly λmax is 600 to 600 μm. A 750 nm color source is preferred. Examples of such a chromogen include compounds described in JP-A-57-29297 and JP-A-59-74713, or compounds of the general formula [Wherein R ₁ and R ₂ are hydrogen, alkyl having 1 to 6 carbons, for example,
Represents methyl, ethyl, propyl, butyl, etc., and R ₃ and R ₄ represent sulfoalkyl (alkyl has the same meaning as R ₁ )]
The compound represented by is preferred.

Catalase is preferable as the H ₂ O ₂ scavenger for carrying out the invention 2, and sodium azide is used for deactivation thereof.

Further, to decompose H ₂ O ₂ reacts with H ₂ O _2, but any compound that does not inhibit the reaction can be used as H ₂ O ₂ scavenger. Examples of such compounds include H ₂ O _{2 in} the presence of peroxidase.
Compounds that do not form a dye by reacting with a compound are known. Phenol derivatives, aniline derivatives, and naphthol derivatives are exemplified as such compounds. Particularly, in the presence of peroxidase, H ₂ O ₂ reacts with a coupling agent composed of two compounds to form a dye. Is used, H ₂ O ₂ is decomposed, but compounds that do not produce a dye
By adding the other compound of the coupling agent as a chromogen for the H ₂ O ₂ quantitative reaction, H ₂ O ₂ derived from the target component can be quantitatively determined, which is convenient (Japanese Patent Publication No. 62-21517,
Id. 61-23998).

The method of the present invention can be applied not only to the dinucleotide in a sample but also to the quantification of a substrate or an enzyme activity involved in a reaction that generates a dinucleotide by an enzyme reaction or the like. The reaction is exemplified below.

UDP-GB: UDP-glucose bilirubin glucosyltransferase UDP-XB: UDP xylose bilirubin xylidyltransferase UDP-GLB: UDP-glucuronate bilirubin glucosyltransferase UDP-GS: UDP-glucose ceramide glucosyltransferase UDP-NA: UDP- N-acetyllactosamine synthetase UDP-GP: UDP glucose lipopolysaccharide glucosyltransferase UDP-N-AP: UDP-N-acetylgalactosamine protein acetylgalactosaminyltransferase UDP-GK: UDP galactose collagen galactosyltransferase UDP-N-AG : UDP-N-acetylprotein acetylgalactosaminyltransferase N-AL: N-acetyllactosamine synthetase FGAT: Fucosyl-galactose acetylgalacto Saminyltransferase BG-α-GT: Blood group substance-α-galactosyltransferase UDP-GNA: UDP galactose N-acetylfingosine galactosyltransferase UDP-GUK: UDP glucose collagen glucosyltransferase B-N-AG: B-N-acetylglucosaminyl saccharide fucosyltransferase GDP-FL: GDP fucose lactose fucosyltransferase Prevents turbidity due to fats in samples, etc., and inhibition by reducing substances such as ascorbic acid and cysteine that inhibit the coloration of hydrogen peroxide Solubilization of fats with surfactants and enzymes [lipoprotein lipase (LPL)], degradation of ascorbic acid with ascorbate oxidase (AOD) and potassium iodate, and N-ethylmaleimide (NEM) Means such erasure Tin is used effectively.
Surfactants include fatty acid salts, alkyl sulfate salts, alkyl benzene sulfonate salts, dialkyl sulfosuccinate esters, alkyl phosphate esters,
Anionic compounds such as polyoxyethylene alkyl sulfate, polyoxyethylene alkyl ether, polyoxyethylene alkyl phenol, polyoxyethylene fatty acid ester, sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, glycerin fatty acid ester, oxyethylene oxy Nonionic ones such as propylene block polymer, and cationic ones such as alkylammine salts, quaternary ammonium salts, and polyoxyethylene alkylamines are used. Some biological fluid components are conjugated with glucuronic acid, sulfuric acid, or the like, and these can be released by glucuronidase or sulfatase as needed.

 The embodiments of the present invention will be described with reference to examples.

Example 1 (Quantitative determination of UDP) Reagent solution A pH = 6.75 Monopotassium phosphate 100 mM Triton X-100 (surfactant) 10 mg / ml Magnesium chloride 2 mg / ml Peroxidase 10 units / ml β-glucuronidase 5 units / mg phosphoenolpyruvate 0.4 mg / ml TPP 0.5 mg / ml FAD 0.01 mg / ml PK 10 units / mg PYOD 9 units / ml MCDP (chromogen) 0.1 mg / ml UDP-2-sodium 10,20, 30,50mg / dl solution each
3.0 ml of reagent solution A was added to 0.05 ml, the mixture was kept at 37 ° C. for 10 minutes, and the change in the absorption of the reaction solution at 666 nm was measured, and 0.357, 0.716, 1.06
An absorbance of 8,1.780 was measured. It is understood that there is a proportional relationship between the absorbance and the UDP content in the sample.

Example 2 (quantification of bilirubin) Reagent solution B pH = 6.75 Monopotassium phosphate 100 mM Triton X-100 10 mg / ml Magnesium chloride 2 mg / ml UDP glucose 1 mg / ml UDPGB 5 units / ml Reagent solution A and reagent of Example 1 A liquid obtained by mixing the liquid B at a ratio of 3: 1 is referred to as a reagent liquid C.

0.1 ml of a sample containing 2.5, 5, 10, 20 mg / dl of bilirubin
After adding 3.0 ml of Reagent C to the mixture at 37 ° C for 10 minutes,
The absorbance of the reaction solution at 66 nm is 0.137, 0.274, 0.541, 1.08
It was 0. This calibration curve is shown in FIG.

Human serum containing 2.5 mg / dl of bilirubin was measured ten times using reagent solution C, and the results shown in Table 1 were obtained.

As a control, measurement was performed 10 times using a commercially available sun assay T-BIL (azobilirubin method, Sanko Junyaku), and the results in the following table were obtained.

It can be seen that the method of the present invention has a higher absorbance (higher sensitivity) and a better CV% as an index of reproducibility.

Example 3 (Quantification of N-acetylsphingosine) UDP glucose ceramide glycosyltransferase (UDP-GS) was used instead of UDPGB of Example 2, and 2.5, 5, 10, or 20 mg / d as ceramide instead of bilirubin.
The same test as in Example 2 was repeated using 1 l of N-acetylfingosine as a standard solution, and the corresponding absorbance was set to 0.
234, 0.462, 0.937 and 1.874 were obtained.

 FIG. 2 shows this calibration curve.

Example 4 (Quantification of lipopolysaccharide and lactose) UDP-N-
Acetylglucosamine, using UDP-N-acetyllactosamine synthetase (UDP-NA) as an enzyme,
Lactose quantification, UDP glucose as donor,
Using UDP glucose lipopolysaccharide glucosyltransferase (UDP-GP) as an enzyme, the quantification of lipopolysaccharide (derived from Escherichia coli serotype 026: B6) was performed in the same manner as in Example 2 for 2.5, 5 or 10 mg / dl samples. A similar test was performed, and the corresponding absorbances were 0.233, 0.465, 0.935 for lactose and 0.070, 0.141, 0.285 for lipopolysaccharide. FIG. 3 shows these calibration curves.

Example 5 (quantification of bilirubin and UDP) In place of MCDP of Example 1, 4AA and N-ethyl-N-
Using (3-methylphenyl) N'-succinylethylenediamine (EMSE) at a concentration of 1 mg / ml each in the same manner as in Example 2 (however, the measurement wavelength is 555 nm), and 10,20 mg / dl
The corresponding absorbance for the bilirubin-containing sample of
At 4,0.187, the absorbance for samples containing 10, 20, 30, or 50 mg / dl UDP was 0.062, 0.124, 0.185, 0.309. 4th
The figure shows this calibration curve.

Example 6 (Quantification of BG-α-GT activity in serum) Reagent solution D pH = 7.0 Monopotassium phosphate 100 mM Triton X-100 (surfactant) 10 mg / ml Magnesium chloride 2 mg / ml Peroxidase 10 Unit / ml Phosphoenolpyruvate 0.4 mg / ml TPP 0.5 mg / ml FAD 0.01 mg / ml PK 10 units / mg PYOD 9 units / ml MCDP 0.1 mg / ml 10,20,30,50 mg of UDP-2-sodium dl solution each
3.0 ml of reagent solution D was added to 0.05 ml, the mixture was kept at 37 ° C. for 10 minutes, and the change in absorption at 666 nm was measured, and 0.357, 0.716, 1.068, and 1.780 were measured.
Was measured.

Reagent E pH = 7.0 Monopotassium phosphate 100 mM Triton X-100 10 mg / ml Magnesium chloride 2 mg / ml UDP galactose 1 mg / ml 2'-fucosyllactose 0.5 mg / ml Manganese chloride 1 mM human serum type B 0.025,0.05,0.075 and 0.1ml respectively
/ E = 3/1 (v / v) mixed reagent solution (3.0 ml) was added, the mixture was kept at 37 ° C. for 30 minutes, and the absorbance at 666 nm was measured.
0.372, 0.557 and 0.744 were obtained. The result is shown in FIG. The same procedure was performed for human serum of type A, but there was no change in absorbance.

Example 7 (Quantification of FGAT activity) The same test as in Example 6 was carried out on a 0.05 ml sample using UDP N-acetylglucosamine instead of UDP galactose of Example 6, and the reaction was started for 10, 20, and 30 minutes. Was 0.107, 0.221, 0.332 for type A human serum and almost the same as blank for type B human serum. The calibration curve is shown in FIG. This is due to the fact that the activity of fucosylgalactose acetylgalactosaminyltransferase, an enzyme unique to type A human serum, has been measured.

Example 8 Instead of the UDP standard solution of Example 1, 10, 20, 30, 50 mg / GDP
A standard curve of dl was added, and the same procedure as in Test 1 of Example 1 was performed to obtain a calibration curve in FIG.

Example 9 1.7 μmol / ml pyruvate and 0.51 μmol / m bilirubin
The activity of UDP in the sample containing l was determined by the following method.

Reagent A PH: 6.75 Monopotassium phosphate 100 mM Triton X-100 10 mg / ml Magnesium chloride 2 mg / ml Peroxidase 10 U / ml β-glucuronidase 5 U / ml PEP 0.4 mg / ml TPP 0.5 mg / ml FAD 0.01 mg / ml PK 10 U / ml PYOD 9 U / ml catalase 2.00 U / ml Reagent B PH: 6.75 monopotassium phosphate 100 mM triton X-100 10 mg / ml magnesium chloride 2 mg / ml UDP glucose 1 mg / ml UDP-GB 5 U / ml sodium azide 1 mg / ml MCDP 1.5 mg / ml (1) Add 2.25 ml of reagent solution A to 0.05 ml of serum, and add 5 ml at 37 ° C.
After holding for 0.7 minute, add 0.75 ml of reagent solution B and hold for 10 minutes.
The absorbance of the reaction solution at 666 nm was determined (E). Eb is the case where distilled water is used instead of serum, and Es is the case where a standard solution is used.

(2) A reagent solution was prepared by mixing reagent solution A and reagent solution B at a ratio of 3: 1. 0.05 ml of serum was added to 3.0 ml of this solution, the mixture was kept at 37 for 10 minutes, and the absorbance was determined at 666 nm (E). . Also,
Eb and Es were determined as in (1).

(3) The above samples were quantified by a conventional method for comparison.

Reagent solution (pH = 7.0) Potassium phosphate 100 mM Triton X-100 10 mg / ml Magnesium chloride 2 mg / ml Lactate dehydrogenase (LDH) 5 U / ml β-glucuronidase 5 U / ml PEP 0.4 mg / ml TPP 0.5 mg / ml FAD 0.01 mg / ml PK 10 U / ml NADH 1 mg / ml UDP-glucose 1 mg / ml UDP-GB 5 U / ml 3.0 ml of reagent solution was added to 0.05 ml of serum and added at 37 ° C for 10 minutes. After warming, the absorbance at 340 nm is determined (Ess). In addition, the same operation was performed using a reagent solution from which UDP-GB was removed from the system described above, an inspection blind was determined (Esb), and the value of E = Esb-Ess was determined. The same operation was performed for a standard solution of 1 μmol / ml of pyrilvin separately (Es). The results are shown in the table.

In test 1, the error was 0. In tests 2 and 3, a size error occurs due to pyruvate in the sample.

[Brief description of the drawings]

FIG. 1 shows the relationship between the content of UDP · 2Na (1) or bilirubin (2) and the absorbance. FIG. 2: Relationship between N-acetylsphingosine content and absorbance. FIG. 3 shows the relationship between the content of lactose (1) or lipopolysaccharide (2) and the absorbance. FIG. 4: Relationship between bilirubin (1) or UDP (2) content and absorbance. FIG. 5 shows the relationship between the amount of type B serum (BG-α-GT activity) and absorbance. FIG. 6 shows the relationship between reaction time (FGAT activity in serum) and absorbance. 1: A type, 2: B type Fig. 7: Shows the relationship between GDP · Na content and absorbance.

Claims (3)

(57) [Claims] 1. A dinucleotide and phosphoenolpyruvate are reacted in the presence of pyruvate kinase to produce trinucleotide and pyruvate, and hydrogen peroxide produced by reacting pyruvate with pyruvate oxidase. And a method for quantifying dinucleotides. 2. A phosphoenol pyruvate, a pyruvate kinase, a pyruvate oxidase and the following a) and b)
Or a composition for quantifying dinucleotides comprising c). a) a compound selected from the group consisting of luminol, isoluminol, pyrogallol and bis (2,4,6-trichlorophenyl) oxalate, and a compound selected from the group consisting of red blood salts, peroxidase, hematin and cobalt chloride b) homovanillin Compounds selected from the group consisting of acid, p-hydroxyphenylacetic acid and diacetylfluorescin derivatives and peroxidase c) Chromogen and peroxidase 3. The method according to claim 1, wherein pyruvate in the sample is treated with pyruvate oxidase to eliminate hydrogen peroxide, and then dinucleotide and phosphoenolpyruvate are reacted in the presence of pyruvate kinase to form trinucleotide. A method for quantifying dinucleotides, which comprises producing pyruvic acid and quantifying hydrogen peroxide produced by reacting pyruvic acid with pyruvate oxidase.