JPS6028279B2 - Reagent for measuring enzyme activity involved in purine metabolism - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a reagent for measuring the activity of an enzyme involved in purine metabolism in a dish, in a lysed red blood cell, or in a culture medium.

Purine metabolism is known as a major pathway of nucleic acid metabolism along with pea-J midine metabolism, and the metabolic pathway from adenosine to uric acid is well known.

In addition, enzyme groups involved in purine metabolism include adenosine deaminase [hereinafter referred to as ADA], which catalyzes the decomposition of adenosine to inosine, and purine nucleoside phosphorylase [hereinafter referred to as PNP], which catalyzes the decomposition of inosine to hypoxanthine. ] and xanthine oxidase (hereinafter abbreviated as XOD), which catalyzes the decomposition of hypoxanthine to xanthine and then to uric acid, is known.

However, today, among the various metabolites and enzyme groups mentioned above related to purine metabolism, only the metabolic end product uric acid (high levels are shown in gout, kidney disease, etc.) is used in daily clinical diagnosis. Only.

However, on the other hand, there are reports that ADA activity in the blood is increased in fly diseases and liver diseases.

As methods for measuring the activity of these enzymes, (i) a method to measure the decrease in the substrate, (ii) a calorimetric or spectrophotometric method to colorimetrically quantify the ammonia produced as a result of the enzymatic reaction, and (iii) a method using XOD as a conjugated enzyme. , a method of colorimetrically quantifying uric acid produced in 29 square meters is known. However, in these methods, the absorbance of the purine base used as a substrate is easily affected by serum components in the i method, and it is easily affected by blood ammonia in the 'ii' method, so the blank value fluctuates.
In particular, the calorimetric method requires centrifugation, which makes the operation complicated and has poor sensitivity.In the iii method, the absorbance of uric acid (29 m) is easily affected by serum components, and the blank is blanked by existing uric acid. It has a number of drawbacks, such as variable values, and there is no known method for directly reducing and measuring tetrazolium salts instead of measuring hydrogen peroxide.

On the other hand, with regard to PNP activity in blood, there are no particular reports on the method for measuring it.

Therefore, as a result of various studies in order to establish a simple and accurate method for measuring the activity of these two enzymes, the present inventors used the XOD derived from cow's milk as a conjugate enzyme in the final stage of the reaction, and coexisted by the XOD reaction. We discovered and succeeded in developing a new enzyme activity measurement method for colorimetrically quantifying the reduced form of tetrazolium dye (formazan).

According to this measurement method, ADA and PNP activities were measured using serum from patients with various diseases. ADA activity showed high values in myeloid leukemia and hepatitis, and PNP activity showed high values in lymphocytic leukemia and hepatitis. Therefore, the quantification of these two enzymes has clinical significance, and since the measurement method is simple and accurate, the reagent of the present invention is useful as a daily clinical diagnostic agent.

The gist of the present invention is illustrated by a reaction formula. Next, the features of the present invention, the technical aims of the present invention, and the points that could not be obtained with conventional techniques will be described.

In the conventional technology, XOD acts on xanthine to generate uric acid, while the production of reduced NAD (NADH) from nicotinamide adenine dinucleotide (NAD) is used to reduce the tetrazolium salt, and in the presence of diaphorase, I did a colorimetric assay.

However, the present application aims to obtain a reagent that is simpler and cheaper than the prior art, and has made this possible.

In other words, we use XOD derived from vushimilk, which is relatively easy to obtain, and let it act on the substrate, while directly reducing tetrazolium salt (INT*1 or NBT*2) and producing formazan in the visible region (50 Mm or 56 mm). ) was invented for colorimetric determination.

*1mT: 3-(P-iodophenyl)-121(P-nitrophenyl)-5-phenyl-2 days Tetrazolium chloride *2 NBT: 2,2-di-Pnitrophenyl-5,5'-diphenyl-3,3'-(3,3-dimethoxy4,4'-diphenylene)ditetrazolium chloride The reagent of the present invention consists of a combination of (1) substrate solution, (2) coloring solution, (2) enzyme solution, and (2) reaction stop solution.

Each of these reagents is prepared separately and mixed sequentially when measuring ADA or PNP activity.

Substrates vary depending on the enzyme of interest.

That is, adenosine is used for ADA, and inosine is used for PNP. Further, the purity of adenosine and inosine can be measured using chemical purity, preferably biochemical purity.

The buffer solution for dissolving these substrates is preferably in the range of 0.05 to 0.2 mol and feed 7 to 8, preferably around pH 7.5, for ADA and PNP due to the properties of each enzyme.
For example, phosphate buffer, Britton-Robinson buffer, etc. are suitable. In both cases, the addition of sodium azide is advantageous as a catalase inhibitor or preservative.

The amounts of PNP and XOO used as conjugated enzymes are:
The minimum amount for which the absorbance is constant is 1.79 hU for the former and 1 for the latter.
Since it was the enemy hU, it would be appropriate to use more than that, preferably 5 times more.

For the purification method of bovine milk-derived XOD, see 1.
After ammonium sulfate fractionation, by hydroxyapatite chromatography, after diammonium sulfate fractionation, after hydroxyapatite chromatography, by ammonium sulfate fractionation, after hydroxyapatite chromatography, by Sephadex G200
[Biochem. Bioph side. 526,
328 (1978) 4 Han et al.'s method [BiMhem.
J. 116,851 (1970)], etc.
XOD obtained according to either method can be measured regardless of its purity. Regarding the purity of PNP, 2 melons/machine 9pro
An ammonium sulfate suspension from field n with a pH of 6.0 is sufficient for measurement.

Furthermore, as a result of examining the enzyme reaction time using 50 samples of serum, a straight line passing through the origin up to 90 minutes was observed for both ADA and PNP, so it was determined that 20 to 30 minutes was appropriate. Furthermore, since the maximum amount of serum at which linearity was observed was ADA125 and PNP120, respectively, the amount of serum used was determined to be 50 for ADA and PNP. Note that it is advantageous to store the enzyme solution as a 1.5-4 mol ammonium sulfate suspension or a 40-60% glycerin solution.

As a stop solution for the enzyme reaction, an acidic solution that stops at a PHI of ~4 when added to the reaction solution, for example, 0.1
-0.3 mol citric acid solution, 0.1-1N hydrochloric acid solution, 0.1-0.5 mol acetic acid solution, etc. are suitable.

Also, in this case, a preservative such as 0.02-0.1% sodium benzoate can be added. After the reaction has stopped, each coloring solution becomes stable after being left for 10 minutes, and remains stable for up to 6 times thereafter.

Examples of tetrazolium dyes used as coloring agents include 3-(p-iodophenyl)-2-(p-nitrophenyl)-5-phenyl-tetrazolium chloride [hereinafter abbreviated as mT] or 2,〆-p-nitrophenyl-5,5'-diphenyl- 3,3-(3,3-dimethoxy-4,4'-diphenylene)ditetrazolium chloride [hereinafter NBT
] are preferred, and examples of buffers for dissolving these include Macluvaine buffer, succinate buffer, phosphate buffer, Britton's buffer. Robinson buffer etc. can be used.
In this case, it is advantageous to add a nonionic surfactant such as Triton X-100 or bovine serum albumin as a solubilizer for the formazan produced by the XOD reaction. Also, the measurement wavelength is 50 plates m when using INT, but NB
When using T, 56 plates n is suitable. Furthermore, when we searched for the influence of serum components when measuring each enzyme activity using the reagent of the present invention, no influence of albumin, glucose, uric acid, bilirubin, ascorbic acid, glutathione, etc. was observed.

Furthermore, the simultaneous reproducibility and day-to-day variation in each enzyme activity measurement were both 3% or less when expressed as coefficients of variation. Next, practical examples of the preparation method of the reagent of the present invention and the measurement of ADA and PNP activities in serum will be specifically illustrated.

Example 1 Preparation of reagent ■ Preparation of substrate solution i Dissolve 100 parts of Inosine for measuring PNP activity in 0.1 M, pH 7.5 phosphate buffer to make a total volume of 50 parts.

Store at 2-8℃.

ii. Dissolve 100% of Adinosine for measuring ADA activity in 0.1M, pH 7.5 phosphate buffer to make a total volume of 50%.

Store at 2-8℃.

■ Preparation of coloring solution i For PNP and ADA activity measurement 1 1NT16 o and 10% Triton X-100
Dissolve 0.2' of the solution in 0.1M, pH 7.5 phosphate buffer to make a total volume of 100%.

Stored in an amber bottle at 2-8℃.
M, FH Dissolve the total volume in 7.5 phosphate buffer to 100%
Let's say '.

Store in a brown bottle at 2-8℃.

3 NBT26 9 and 10% Triton X-100
Dissolve 0.2' of the solution in 0.1 M, pH 7.5 phosphate buffer to make a total volume of 100'.

Store in a brown bottle at 2-8℃.

4. Dissolve 9 of NBT26 and 12 of bovine serum albumin in 0.1 M, pH 7.5 phosphate buffer to make a total volume of 100.

Store in a brown bottle at 2-8℃.

■ Preparation of enzyme solution i For PNP activity measurement I Add XODIOU to 2.3M ammonium sulfate solution
Suspend in 'oo'.

Dissolve 2×ODIOU in 100 ml of 50% glycerin solution.

ii PNPI.I for measuring ADA activity. 75U and XODIOU are suspended on top of a 2.3M ammonium sulfate solution.

2 PNPI. Dissolve 75 U and XODIOU in 100 ml of 50% glycerin solution.

In both cases, the sample is stored in a brown bottle at a temperature of 0 to 400.

■ Preparation of reaction stop solution (common to all enzymes) 1. Dissolve 4.2 parts of citric acid monohydrate and 50 parts of sodium benzoate in water to make a total volume of 100 parts.

2. Dissolve 50 parts of sodium benzoate in a pH 2.0 hydrochloric acid solution to make a total volume of 100 ships. Example 2 Quantification of PNP activity Using each of the prepared reagents described in Example 1, measurement was performed according to the following procedure.

However, 1:1 was used for the substrate solution, 1:1 was used for the coloring solution, 1:1 was used for the enzyme solution, and 2 was used for the reaction stop solution. 1) Add 100 ml to the mixture of 0.2 and 2.5, and pre-incubate at 370 °C for 5 minutes.

Next, add 50 ml of serum and react at 370 ml for 30 minutes. Afterwards, ① 0.5' is added and the colorimetry is determined at a wavelength of 50 Mm. To convert absorbance to PNP activity, calculate the enzyme activity (IU/
This can be done by reading the words (evening). However, the PNPI unit is the enzyme activity that produces 1 Amole of hypoxanthine per minute in the above reaction system. Quantification of ADA activity Using each of the prepared reagents described in Example 1, ADA activity was measured according to the following procedure.

However, (ii) was used for the substrate solution, (ii) was used for the coloring solution, (i) was used for the enzyme solution, and (ii) was used for the reaction stop solution. ■0.2 skin [
Add ■100 Sanyu to the mixed liquid of ■2.5 m', and add 37
Pre-incubate at 0 for 5 minutes.

Next, add 50% of the serum and allow to react at 370°C for 30 minutes. Afterwards, add 0.5% of the mixture and perform colorimetric determination at a wavelength of 50 m. Conversion from absorbance to ADA activity can be performed by reading the enzyme activity (IU/unit) corresponding to the absorbance of the sample from a previously prepared calibration curve. However, the ADAI unit is the enzyme activity that produces 1 mole of hypoxanthine per minute in the above reaction system. Next, PNP and ADA in serum of various diseases were quantified by the above method using the reagent of the present invention.
The activity is shown in the table.

table

Claims (1)

[Scope of Claims] 1 (1) A solution containing 3.74 to 15.0 mM inosine as a substrate, (2) 0.198 to 1.58 M 3-(P-
iodophenyl)-2-(P-nitrophenyl)-5-
Phenyl-2H-tetrazolium chloride and a nonionic surfactant (or bovine serum albumin) and 0.05~
Coloring solution consisting of 0.2M pH 6-8 buffer, (3) 1
1.5-4M ammonium sulfate suspension of 0-40mU of bovine milk-derived xanthine oxidase or 40-6
0% glycerin solution, (4) 0.1-0.3M citric acid solution, 0.1-1N hydrochloric acid solution or 0.1-0.5
It consists of a combination of a reaction stop solution, which is an acetic acid solution of M, and allows xanthine oxidase derived from bovine milk to act on the produced hyboxanthin and xanthine, while directly reducing the tetrazolium salt to produce formazan, which is then colorimetrically quantified. in blood, red blood cell lysate,
A reagent for measuring purine nucleoside phosphorylase activity involved in purine metabolism in culture fluid. 2 (1) 3.74-15.0mM adenosine as a substrate solution, (2) 0.198-1.58M 3-(P
-iodophenyl)-2-(P-nitrophenyl)-5
- phenyl-2H-tetrazolium chloride and nonionic surfactant (or bovine serum albumin) and 0.05
A coloring solution consisting of a buffer solution of ~0.2M, pH 6-8, (3
) 10-40 mU of bovine milk-derived xanthine oxidase and 1.75-5 mU of purine nucleoside phosphorylase in a 1.5-4 M ammonium sulfate suspension or 40-60% glycerin solution, (4) 0.1-0
．． It consists of a combination of a reaction stop solution, which is a 3M citric acid solution, a 0.1-1N hydrochloric acid solution, or a 0.1-0.5M acetic acid solution, and causes the bovine milk-derived xanthine oxidase to act on the produced hypoxanthine and xanthine, while A reagent for measuring adenosine deaminase activity involved in purine metabolism in blood, red blood cell lysate, and culture medium, which is characterized by directly reducing a tetrazolium salt to generate formazan, and quantifying the same by colorimetry. 3 (1) 3.74-15.0mM inosine-based solution, (2) 0.198-1.58M 2,2'-
di-P-nitrophenyl-5,5'-diphenyl-3,
3'-(3,3'-dimethoxy-4,4'-diphenylene) ditetrazolium chloride and a nonionic surfactant (
or bovine serum albumin) and 0.05-0.2M,
Coloring solution consisting of a buffer solution with a pH of 6 to 8, (3) 10 to 40
1.5 mU of bovine milk-derived xanthine oxidase
~4M ammonium sulfate suspension or 40-60% glycerin solution, (4) 0.1-0.3M citric acid solution,
It consists of a combination of a reaction stop solution, which is a 0.1-1N hydrochloric acid solution or a 0.1-0.5M acetic acid solution, and allows xanthine oxidase derived from bovine milk to act on the produced hypoxanthine and xanthine, while directly reducing the tetrazolium salt. A reagent for measuring purine nucleoside phosphorylase activity involved in purine metabolism in blood, red blood cell lysate, and culture medium, which produces formazan and performs colorimetric determination. 4 (1) 3.74-15.0mM adenosine-based solution, (2) 0.198-1.58M 2,2'
-di-P-nitrophenyl-5,5'-diphenyl-3
, 3'-(3,3'-dimethoxy-4,4'-diphenylene) ditetrazolium chloride and a nonionic surfactant (or bovine serum albumin) and 0.05-0.2M
, a coloring solution consisting of a buffer solution with a pH of 6 to 8, (3) 10 to 4
0 mU of bovine milk-derived xanthine oxidase and 1.75-5 mU of purine nucleoside phosphorylase in a 1.5-4 M ammonium sulfate suspension or 40
-60% glycerin solution, (4) 0.1-0.3M citric acid solution, 0.1-1N hydrochloric acid solution or 0.1-0.
．． consisting of a combination of reaction stop solution, which is a 5M acetic acid solution;
Bovine milk-derived xanthine oxidase acts on hypoxanthine and xanthine produced, while directly reducing tetrazolium salt to produce formazan, which is then colorimetrically quantified. A reagent for measuring adenosine deaminase activity involved in purine metabolism.