JPS592280B2 - 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 enzyme activity involved in purine metabolism in blood, erythrocyte lysate, and culture medium.

Purine metabolism is known as a major pathway of nucleic acid metabolism along with pyrimidine metabolism, and the metabolic pathway from adenoine to uric acid is well known.

Furthermore, the urea group involved in purine metabolism includes 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) that catalyzes the decomposition of hypoxanthine to xanthine and then to acid.

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 tests. be. However, on the other hand, blood A
There are reports that DA activity is increased in cancer patients and liver diseases.

Methods for measuring the activity of these enzymes include l) a method for measuring the decrease in substrate; I) a calorimetric or spectrophotometric method for colorimetrically quantifying ammonia produced as a result of an enzymatic reaction; XOD
A method is known in which the uric acid produced is determined colorimetrically at 293 nm. However, these methods, l
) method, the absorbance of the purine base used as a substrate is easily affected by serum components, and (11) method, the absorbance of the purine base used as a substrate is easily affected by blood ammonia, so the blank value fluctuates.Especially in the calorimetric method, centrifugation 111 requires manual operation, which makes the operation complicated and has poor sensitivity.
) method has many drawbacks, such as the absorbance (293 nm) of uric acid being easily influenced by serum components and the blank value fluctuating due to existing uric acid. I can't find it either. On the other hand, with regard to PNP activity in blood, there are no particular reports on the method for measuring it.

Therefore, the present inventors conducted various studies to establish a simple and accurate method for measuring the activity of these two enzymes.
Discovery of a new method for measuring enzyme activity in which hydrogen peroxide produced by the OD reaction acts on a reduced chromogen in the presence of peroxidase (hereinafter abbreviated as POD) to produce a colored oxidation product, which is then colorimetrically quantified. It was very successful.

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 quantitative determination 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 test agent.

The gist of the present invention is shown by a reaction formula. XOD Substrate → xanthine → uric acid + H2O2 H2O H2O, 10-reduced color formula raw material coification product + H2O.

Next, the features of the present invention and the points where the effects of the present invention could not be obtained with the prior art will be described.

In the conventional technology, when XOD derived from bovine milk is made to act on a substrate and the generated H2O2 is introduced into the dye system, once the dye is generated, it is immediately decomposed and it is not possible to determine the change in absorbance in the visible region. It was said that

In other words, in principle, XOD derived from bovine milk
The type of electron transfer in the reaction is a mixed type of two-electron reduction and one-electron reduction. This electron reduction reaction has the disadvantage that in addition to H2O2, Oi (superoxide anion) is produced, which forms a compound (complex) with POD and inactivates peroxidase. In contrast, the present invention uses a 0.1-0.3M citric acid solution or a 0.1-0.3M citric acid solution.
The method is characterized in that a 2N hydrochloric acid solution is used as the enzymatic reaction termination solution, thereby allowing Oi to be converted to H2O2 (disproportionation reaction) under acidic conditions. In this way, by removing the drawbacks of the conventional technology, X
This is a simple reagent for measuring the activity of PNP and ADA that allows the use of OD. The reagent of the present invention includes 1 substrate solution, 2 enzyme solutions,
It consists of a combination of 3 reaction stop solutions, 4 color developing solutions (a) and 5 color developing solutions (b).

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, A.D.A.
Adenosine is used for PNP, and inosine is used for PNP. Further, the purity of adenosine and inosine can be measured by chemical purity, preferably biochemical purity. As a buffer solution for dissolving these substrates, AD
A and PNP, 0.05-0.2M, . pH7
8, preferably around 7.5, and for example, phosphate buffer, Britton-Lopinson buffer, etc. are suitable. In both cases, the addition of sodium azide is advantageous as a catalase inhibitor or preservative. The minimum amount of PNP and XOD used as the conjugate enzyme is 1.75 mu for the former and 10 mu for the latter to keep the absorbance constant.
Since it was u, it is appropriate to use more than that, preferably 5 times or more.

For the purification method of bovine milk-derived XOD, see 1.
．． 2. Only by ammonium sulfate fractionation. 3. By ammonium sulfate fractionation followed by hydroxyanomatite chromatography. Ammonium sulfate fraction,
After hydroxyapatite graphing, Sephadex G2OO was used [BiOchem. BlOphys
．． 526, 328 (1978) 4,}-1art et al.'s method [BlOchem. J. ll6,85l (1970)
] etc. However, XOD obtained by any method can be measured regardless of purity.

Regarding the purity of PNP, ammonium sulfate suspension of 20 U/SPrOteln PH6. It can be sufficiently measured with O.

Further, as a result of an investigation using 50 μl of serum as a sample, a straight line passing through the origin up to 90 minutes was observed for both ADA and PNP, and therefore 20 to 30 minutes was judged to be appropriate.

In addition, the maximum serum volume at which linearity was observed was
5 μJ. Since it was PNPl2Oμ2, the amount of serum used was determined to be 50 μj for ADA and PNP. The enzyme solution is a 1.5-4M ammonium sulfate suspension or a 4M ammonium sulfate suspension.
0-6001) It is advantageous to store it as a glycerin solution.

As a stop solution for the enzyme reaction, an acidic solution that stops at pH 3 to 4 when added to the reaction solution, for example, 0.1
-0.3M citric acid solution, 0.05-0.2N hydrochloric 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, the color development of each coloring solution becomes stable after being left for 10 minutes, and remains stable for 40 to 90 minutes thereafter.

The coloring agent may be a reduced active substance that is oxidized by hydrogen peroxide in the presence of POD to form a colored oxidation product, preferably one that exhibits an absorbance of 400 nm or more that is not affected by serum components. is preferred.

In addition, as a buffer solution for dissolving each coloring agent, in the case of pH 3 to 5, McAlvaine buffer, succinate buffer, etc.
In the case of H5-8, phosphate buffer, Britton-Robinson buffer, etc. are suitable.

Next, oxidation products, their PH, and absorbance are illustrated.
In addition, when we searched for the influence of serum components when measuring each enzyme activity using the reagent of the present invention, some inhibition was observed by albumin 20 W/ml, but glycose,
Almost no effects of uric acid, bilirupine, ascorbic acid, etc. were observed.

Furthermore, the daily variation in simultaneous reproducibility in each enzyme activity measurement was 3% or less when expressed as a coefficient of variation. Next, practical examples of the preparation method of the reagent of the present invention and the measurement of M8 and PNP activities in serum will be specifically illustrated.

Example 1 Preparation of reagent 1 Preparation of substrate solution 1) Inosine 117η and sodium azide 23η for PNP activity measurement were mixed at 0.
1M. Dissolve the total volume in pH 7.5 phosphate buffer at 35%
0m1.

Store at 2-8℃.

11) Adenosine 117 for measuring ADA activity and sodium azide 23W9 at 0.1M. Dissolve in pH 7.5 phosphate buffer to make a total volume of 350 mt.

Store at 2-8℃.

2 Preparation of enzyme solution 1) For PNP activity measurement 1) Suspend XOD, 1Ou in 100 mt of 2.3M ammonium sulfate solution.

2) XOD, lOu in 100d of 50% glycerin solution
dissolve in

11) For ADA activity measurement 1) PNP, l. 75u and XOD, lOu 2.
Suspend in 100 d of 3M ammonium sulfate solution.

2) PNP,. l. 75u and XOD, 40u for 5
Dissolve in 100 ml of 0% glycerin solution.

In all cases, the samples were stored in brown pins at 0 to 4°C. 3 Reaction stop solution (common to each enzyme) 1) Dissolve 4.29 ml of citric acid l-hydrate and 50 η of sodium benzoate in water to make a total volume of 100 ml.

2) Add 50 watts of sodium benzoate to pH 2. Dissolve in a hydrochloric acid solution of O to make the total volume 100ml.

4. Coloring solution (a) 5. Preparation of coloring solution (b) (common to each enzyme) 1) Coloring solution (a): 3-methyl-2-benzothiazolinone-hydrazone hydrochloride 9.6m9 and PODl4
Dissolve OOu in 0.2M, pH 3.5 McElvain buffer to make a total volume of 50ml.

Store in brown pins at 2-8℃.

Coloring solution (b): Dissolve 0.3 ml of N,N-dimethylaniline in a 0.2 M, pH 3.5 McLuvain buffer solution to make a total volume of 50 ml.

Store in brown pins at 2-8℃.

2) Coloring solution (a): 0-dianisidine 10.5 to 0.
Dissolve the total volume in 2M, pH 6.5 phosphate buffer at 50%
Let it be m1.

Store in brown pins at 2-8℃.

Coloring solution (b): POD, 2.4OOu is suspended in 50ml of 3.2M ammonium sulfate solution.

Store at 2-8°C.

3) Coloring solution (a): 4-aminoantipyrine 8η and POD, 4OOu are dissolved in a 0.1 M, pH 7.4 phosphate buffer solution to make a total volume of 50 ml.

Store in brown pins at 2-8℃.

Coloring solution (b): 0.2M of phenol 20%, pH 8.
0 phosphate buffer to make a total volume of 50 ml.

Store in brown pins at 2-8℃.

4) Coloring solution (a): 12ml of p-methoxyaniline
．． Dissolve in 2M, pH 3.5 McElvain buffer to make the total volume 50ml.

Store in brown pins at 2-8℃.

Coloring solution (b): POD, 4OOu is suspended in 50ml of 3.2M ammonium sulfate solution.

Store at 2-8°C.

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 substrate solution is 1), 2 enzyme solution is 1) of I], 3 reaction stop solution is 1), 4 color developing solution (a) and 5 color developing solution (b)
1) was used. 2100μ in 11.2m1! Add 3
Brain-cubate at 7°C for 5 minutes.

Next, 50μ2 of serum is added and reacted at 37°C for 30 minutes.
After that, 1 ml each of 4 and 5 was added to 30.5 ml and incubated at 37°C for 10 minutes.
Colorimetrically determined at 0 nm. Conversion from absorbance to PNP activity can be performed by reading the enzyme activity (IU/l) corresponding to the absorbance of the sample from a previously prepared calibration curve.

However, one unit of PNP is 1 μmol h per minute in the above reaction system.
This is the enzyme activity that produces hyboquinanthine. Quantification of ADA activity Using each of the prepared reagents described in Example 1, ADA activity was measured according to the following procedure.

However, 1 substrate solution 11) and 2 enzyme solutions are 1) and 3 of 11).
The reaction stop solution is 1), color developing solution 4 (a) and color developing solution 5 (b).
) used 1). Add 2100 μl to 11.2 ml and brain-incubate at 37° C. for 5 minutes.

Next, 50μ2 of serum is added and reacted at 37°C for 30 minutes.
After that, 30.5m11 was added, then 1m1 each of 4 and 5 was added.
After incubating for 10 minutes at 37°C,
Colorimetrically determined in nm. Conversion from absorbance to ADA activity can be performed by reading the enzyme activity (U/l) corresponding to the absorbance of the sample from a previously prepared calibration curve.

Claims (1)

[Claims] 1. (1) 0.625-5mM inosine as a substrate solution, (2) 10-40mU suspension of bovine milk xanthine oxidase in 1.5-4M ammonium sulfate or 40-60% glycerin. solution, (3) 0.1-0.
It consists of a combination of a reaction stop solution, which is a 3M citric acid solution or a 0.1-0.2N hydrochloric acid solution, (4) a coloring solution (a), and (5) a coloring solution (b), and is produced by a xanthine oxidase reaction derived from bovine milk. Purine nucleoside phosphorylase activity involved in purine metabolism in blood, erythrocyte lysate, and culture fluid, characterized by converting hydrogen peroxide into a colored oxidation product in the presence of peroxidase, and quantifying this colorimetrically. Reagent for measurement of. 2 (1) a solution with 0.625-5mM adenosine as a substrate; (2) a 1.5-4M ammonium sulfate suspension of 10-40mU bovine milk-derived xanthine oxidase and 1.75-5mU purine nucleoside phosphorylase; or 40-60% glycerin solution, (3)0
．． A reaction stop solution which is a 1-0.3M citric acid solution or a 0.1-0.2N hydrochloric acid solution, (4) a color developing solution (a) and (
5) Blood comprising a combination of the coloring liquid (b), characterized in that hydrogen peroxide produced by a reaction with xanthine oxidase derived from bovine milk is converted into a colored oxidation product in the presence of peroxidase, and this is colorimetrically quantified; Reagent for measuring adenosine deaminase activity involved in purine metabolism in erythrocyte lysate and culture medium. 3 The coloring solution is 0.53-2.12mM 3-methyl-2-
Coloring solution (a) consisting of benzothiazolinone-hydrazone hydrochloride, 2-8u peroxidase and 0.1-0.3M, pH 3-4 buffer and 15.8-63.2mM
N,N-diethylaniline and 0.1-0.3M, p
The reagent according to claim 1 or 2, which is a combination of a coloring solution (b) consisting of a buffer solution of H3-4. 4 Coloring solution (a) consisting of 0.410-1.64mM 0-dianisidine and 0.2-0.4M, pH 6-7 buffer and 1.5-8u of peroxidase
3. The reagent according to claim 1 or 2, which is a combination of a color developing solution (b) which is a 4M ammonium sulfate suspension or a 40-60% glycerin solution. 5 Coloring solution contains 0.492-1.97mM 4-aminoantipyrine, 2-8u peroxidase and 0.1-0
．． Coloring solution (a) consisting of 3M, pH 7-8 buffer and 2
．． 13-8.52mM phenol or 2.13-8.
52mM p-chlorophenol and 0.2-0.3M
The reagent according to claim 1 or 2, which is a combination of a coloring solution (b) consisting of a buffer solution having a pH of 8 to 9. 6 Coloring solution (a) consisting of 1.95-7.80mM p-methoxyaniline and 0.2-0.3M, pH 3-5 buffer and 1.5% of 2-8u peroxidase
4M, ammonium sulfate suspension or a 40-60% glycerin solution in combination with coloring solution (b).