JPS6240300A - Activity measurement of adenosine deaminase in humor - Google Patents

Abstract

PURPOSE:To solve problems, e.g. sensitivity of error, by decomposing xanthin, hypoxanthin and uric acid using adenosine as a substrate and carrying out the activity measurement in measuring the activity of the titled substance by measuring inosine. CONSTITUTION:Xanthin, hypoxanthin and uric acid present in the humor are decomposed with xanthin oxidase, uricase peroxidase, phenol or amine. Adenosine is used as a substrate to form inosine by action of adenosine deaminase in the humor, and purine nucleoside phosphorylase is reacted to form hypoxanthin, which is reacted with xanthin oxidase, uricase, reducing type coenzyme, peroxidase, phenol or amine and coloring reagent to be reduced to colorimetrically determine the formed dyestuff.

Description

[Detailed description of the invention] Described measurement method.

3. Detailed description of the invention
[Industrial Application Field], 1 The present invention is directed to the use of 7 denosine denominations in 1 body fluid 1, for example, serum.
:゛.

7°7-5(”T, ADA&111t6.) (7
)in.

Regarding measurement methods.
)・、□. □31 ADA participates in nucleic acid metabolism and releases adenosine.
, an enzyme that generates inosine and ammonia by 1-amination, and is useful for various malignant tumors, liver diseases, and inflammatory diseases.
2, because it is involved in cellular immunity, etc.
1. Measuring ADA activity in fluid is said to have clinical significance.

Methods for measuring the activity of ADA include a method of measuring inosine produced in the reaction of ADA (adenosine +) 120 - - -> inosine + NH3 and a method of measuring NH3.

At present, ADA activity is based on N produced by an enzymatic reaction.
The mainstream method is to quantify the amount of H3 by indophenol reaction, but this measurement method requires separate enzymatic reaction and indophenol reaction.
The procedure is complicated and takes a long time, so it is not necessarily a good method for routine testing.Also, serum blinding is necessary because there is a component that causes an indophenol reaction in serum, and the serum blinded value It also has drawbacks such as a large error due to its high value. Another method for measuring N)13 is to use glutamate dehydrogenase (hereinafter abbreviated as GLDH) in the presence of α-ketogel tar acid and reduced nicotinamide 7-denine dinucleotide phosphorus (NADPH). A method is known in which the decrease in NADPH is measured at a wavelength of 340 nm, but this method is also an ultraviolet absorption measurement method and has drawbacks such as being subject to equipment limitations.

- Recently, various methods for measuring inosine ADA activity have been studied, including purine nucleoside phosphorylase (hereinafter abbreviated as PNP) and xanthine oxidase (hereinafter abbreviated as PNP).

It is abbreviated as XOD. ) has been developed as a conjugate enzyme, and a method has been developed for colorimetrically quantifying the generated H2O2 by oxidizing and coloring a reducible coloring reagent in the presence of peroxidase. However, this method does not allow the superoxide anion (Ol) generated when XOD is conjugated.
This method has a serious drawback in that the dye is reduced and the amount of H2O2 is not proportional to the amount of the dye. In addition, in order to completely convert 02 produced by the enzymatic reaction into H2O2, the reaction solution was made tactile with citric acid or hydrochloric acid to stop the enzymatic reaction, and at the same time 01 was converted to H2O2, and then color development was performed. A method for colorimetric determination by adding a reagent has been developed [the method disclosed in Japanese Patent Publication No. 59-2280] requires a large number of measurement steps and cannot be applied to the late method.

Mata et al., ADA Enzyme Test 1. : Direct colorimetric determination of 02 generated when PNP and X0De conjugated using nitrotetrazolium blue (hereinafter abbreviated as NTB) [Clinical Examination, Vol. 28, 705-708, 198
5 years ago] has also been developed, but this method is still unsatisfactory as it has drawbacks such as low sensitivity, requiring a large sample (usually 50 u), and the need for blind testing of reagents. ′ cannot be said to be true.

[Purpose of the invention]

The present invention was made in view of the above-mentioned situation, and it addresses the problem of measurement sensitivity, which has been conventionally required to be improved, in the method of measuring ADA activity by measuring inosine.
The purpose of the present invention is to provide a novel and highly accurate method for quantifying ADA activity that solves all problems such as sample blindness and errors caused by coexisting substances.

[Structure of the invention]

The present invention uses adenosine as a substrate and produces hyboxanthin by the action of purine nucleoside phosphorylase (PNP) on inosine produced by the action of adenosine deaminase (ADA) in body fluids, xanthine oxidase (XOD), uricase, reduced form Adenosine, characterized in that its activity value is determined by colorimetrically quantifying the pigment produced by the action of a coenzyme, peroxidase, phenols (or naphthols) or amines, and a reducible coloring reagent. This is a deaminase activity measurement method.

The method of the present invention is shown by the following reaction formulas (1) to (4).

(adenosine) (inosine) (hypoxanthine) (inosine) (hypoxanthine) 3H202+NA (P)
or amineformazan or diformazan 10 NAD(P)
(4) In detail, the equation (3) consists of the following two equations.

(Hyboxanthin) In this way, the present invention allows XOD and uricase to act on hypoxanthine, which is produced by the action of PNP on inosine produced from adenosine by ADA. XOD from hyboxanthin is decomposed by IR gas
The total amount of H2O2 produced by uric acid and 1202 produced by uricase from uric acid was measured using a reducible coloring reagent in the presence of reduced coenzymes, peroxidase, phenols (naphthols), or amines. According to the measurement method of the present invention, the sensitivity is improved compared to the conventional method, and the sample amount can be reduced from the conventional 50JI7 to 204. Inspection, Vol. 23, 705-708.1985)
Approximately 2.5 times the sensitivity can be obtained.

The H2O2 produced by the enzymatic reaction is produced by the method for which the present inventors previously applied for a patent (Japanese Patent Application No. 80-071832), that is, reduced coenzymes, POD, phenols (or naphthols) or amines, and H using reducing color reagents
It can be measured by applying the quantitative method of 2O2,
This makes it possible to obtain measured values that are less affected by coexisting substances.

In addition, in carrying out the present invention, errors due to urine discoloration, xanthine, etc. present in the specimen (serum) should be considered in advance by XOD,
Since uricase, peroxidase, and amines or phenols (or naphthols) are present in the sample and can be eliminated by reacting for a certain period of time, sample-blind measurements are unnecessary.

In addition, in the method of the present invention, the method of Japanese Patent Publication No. 58-2280,
Unlike the invention, there is no need to temporarily stop the enzymatic reaction using a reaction terminator in order to completely convert oat H2O2.

That is, in the reaction in formula (3a), it is converted to H2O2 via O1, but in order to complete this reaction, it is usually necessary to make the liquid acidic or to use superoxide dismutase ( Although it is necessary to add SOD (hereinafter abbreviated as SOD), the method of the present invention does not require such treatment at all.

Since the color reaction can be carried out simultaneously with the enzymatic reaction of adenosine-ADA, measurement by the late method is also possible. In addition, when measuring with the Endo method, conventionally used acids such as hydrochloric acid and citric acid may be used as the reaction terminator, but if neutral dodecyl sulfate is used, the material of the automatic analyzer It is more preferable because there is no fear of damaging it.

Examples of reduced coenzymes used in the present invention include reduced nicotinamide adenine dinucleotide (NADH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH), but are not limited to these. isn't it.

Further, peroxidase derived from plants, animals, and microorganisms can all be used, but peroxidase derived from horseradish is usually preferably used.

As the reducible coloring reagent used in the present invention, all compounds, existing or unknown, that can be reduced and colored can be mentioned. Existing and commonly used ones include NTB, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (hereinafter abbreviated as INT).
, 3-(4,5-dimethylthiazolyl-2)-2,
5-diphenyltetrazolium bromide (hereinafter referred to as MT
Abbreviated as T □ru・)・2-(2-<7zothiaso゛)'b)-3-.

(2-carboxyphenyl)-5-(4-[hydroxypoly(oxy-1,2-ethanediyl)]phenyl)-
Examples include tetrazolium salts such as 2H tetrazolium chloride (hereinafter abbreviated as BCHT).

Examples of phenols used in the present invention include phenol itself, as well as methyl and ethyl.

Alkyl substituted phenols such as propyl, e.g. alkoxy substituted phenols such as methoxy, ethoxy, propoxy, etc.
Various derivatives such as phenols substituted with halogens such as chlorine, bromine, fluorine, and iodine can be used. In addition, as naphthols,
In addition to naphthol itself, naphthol derivatives such as naphthol sulfonic acids and naphthol carboxylic acids can be used.

As the amines, any ordinary organic amines can be used, regardless of whether they are -class, secondary, or tertiary, and aromatic amines are generally more effective when used in smaller amounts than aliphatic amines. Specific examples of these amines include, for example,
Aniline, N-methylaniline, N-ethylaniline,
N,N-dimethylaniline, N,N-diethylaniline, N,N-diethyl-m-)luidine, N-ethyl-N-(β-hydroxyethyl)-m-toluidine, 3.5-dimethoxy-N- Ethyl-N-(2-hydroxy-3-sodiumsulfobrobyl)aniline, N-ethyl-N-(2-hydroxy-3-sulfopropyl)-
Examples include m-toluidine sodium (hereinafter abbreviated as TOO5), but it is needless to say that it is not limited to these.

These amines, phenols, and naphthols may be used alone or in any combination of two or more. For example, l-N, N-dimethylamino-4
- Even if one compound is a phenol, such as naphthol and 4-N,N-diethylaminosalicylic acid, h9. It is also possible to use compounds that are also amines.

The method of the present invention usually has three steps in the end method and two steps in the late method. That is, in the first step, xanthine, hyboxanthin, uric acid, and, if necessary, ascorbic acid and bilirubin, which coexist in the sample, are decomposed.The reagents used for this purpose include at least xanthine oxidase, uricase, and peroxidase. , as well as phenols (or naphthols) or amines, but for samples containing inosine, errors caused by inosine can also be eliminated by adding PNP to the sample.

In the case of a sample containing ascorbic acid, if copper ions are allowed to coexist in the first step reagent solution (hereinafter referred to as the first reagent solution), it is possible to use a sample containing ascorbic acid.
As shown in the invention of No. 205999 or JP-A-59-210899, ascorbic acid can be decomposed and removed;
Furthermore, ascorbate oxidase may be allowed to coexist in the first reagent solution. Furthermore, bilirubin can be decomposed and removed by coexisting bilirubin oxidase in the first reagent solution. In the second step, a reagent solution containing the substrate 7-denosine is used, and the enzymatic reaction of ADA and the coloring reaction are carried out simultaneously. In the rate method, the increase in coloration per unit time of this coloration reaction is measured. In the endo method, the ADA enzyme reaction in the second step is carried out for a certain period of time, 4h1, and then 9 reactions are stopped.
111 Elementary Reaction 2 Stop 1st, measure the absorbance. The reaction between H2O2 and the reducible coloring reagent produces reduced coenzymes, POD, and phenols (or naphthols).
Alternatively, since the process proceeds in the coexistence of amines, there is no problem in the presence of either a reducible coloring reagent or a reduced coenzyme in the first reagent.

m1XIKettL6XOD, +! J#--t/,
P.O.

D. The amount of phenols (or naphthols) or amines is not particularly limited as long as it can decompose hypoxanthine, xanthine, and uric acid in the sample, but it is preferably an amount that does not interfere with the reaction in the second step. Needless to say, the second step reagent (hereinafter referred to as the second reagent) further contains xOD, uricase, POD, and pheno-2,a. 1yo+7)-z,,)tyoa5□,.

There is no problem in having any or all of them exist.

The liquid properties of the first test solution are not particularly limited as long as they are within the range where XOD, uricase, and P0D act, but usually have a pH of 5 to 5.
A range of 9 is preferably used.

The liquid properties of the second reagent solution are adjusted so that the liquid properties are pH 6 to 9 when mixed with the first reagent liquid to cause an ADA enzymatic reaction. The concentration of 7 denosine in the second test solution is 0.02 to 2 mm when mixed with the first test solution to perform the second step reaction.
Adjust to a range of ol/l.

PNP is contained in either or both of the first reagent solution and the second reagent solution, but in any case, the concentration in the reaction solution of the second step is adjusted to be 1 to 500 υ/1.

X OD It 1-1000/in the reaction solution of the second step
l.

Uricaseha 1-3001/l, POD (t 100-
10,000u/1, phenols (or naphyl-ols) or amines 0.1-500 anal/1.
N A D (P)H' is 0, O1 ~ 20IIaol/
A range of l is preferably used, but the range is not limited thereto.

As the buffer used to adjust the liquid properties of the first and second test solutions, commonly used buffers such as phosphate buffer, Tris-HCl buffer, Gud buffer, etc. can be used, but they are not limited to these. It is not something that will be done.

In order to smoothly carry out the enzymatic reaction or color reaction, a surfactant may be added to the first reagent solution and/or the second reagent solution.

When dodecyl sulfate is used as a reaction terminator, the concentration in the final color solution should be 1 mmol/1 or more.When acids such as hydrochloric acid or citric acid are used as a reaction terminator, the final color solution should be Make sure that the color liquid has a pH of 3 or less.

The present invention will be explained in more detail with reference to Examples below.
The present invention is not limited to these.

〔Example〕

Example 1゜[Preparation of test solution] ■ 80 U/1. 'F U Curse 1o
Otl/1. POD was dissolved at a concentration of 3,000 U/I, phenol at a concentration of 0.01%, and NTB at a concentration of 0.04%.

■ In the second test solution 0.05M phosphate buffer (p) 17.15)
Adenosine 0.5 ■al/! , PNP 500/
l, NAD PH 1.25 m5ol/l, and h)
U) NX-100 was dissolved to a concentration of 0.1%.

■ Reaction stop solution A 0.2% sodium dodecyl sulfate aqueous solution was prepared.

[Sample] Adenosine deaminase ↑1pe I (From calf 1ntesti) manufactured by Sigma was added to the pooled serum.
nal Mucosa) respectively 2G, 40.80.8
0.100. t50U/I-A: This was used as a sample.

[Measurement method] Take 20 ILl of sample and add the first test solution! , add 0-,
After heating at 37°C for 5 minutes, add 1.0ml of the second test solution,
1.0ml of zero reaction stop solution heated at 37℃ for exactly 10 minutes
After adding and mixing, the reagent blank was used as a control, and wavelength 5
Absorbance at 80 nm was measured.

Figure 1 shows the relationship between adenosine deaminase (ADA) a degree and absorbance.As is clear from Figure 1, each ADA
A calibration curve connecting the absorbance plotted against the activity value (U/l) shows good quantitative properties.

Example 2゜ [Preparation of test solution] ■ 1st test solution Example 1. Same as .

■ Second test solution Example 1. Same as .

■ Reaction stop solution Example 1. Same as .

[Samples] Serum, serum, xanthine, and hypoxa'' were also added to one sample.-1 [Measurement method]
: Example 1. Same as .

3.1□yo□1.38o. (Table 1') As is clear from Table 1, the method of the present invention was not affected at all by uric acid, xanthine, and hyboxanthin in serum.

Example 3゜ [Preparation of test solution] ■ 1st test solution Example 1. Same as .

■ Second test solution Example 1. Same as .

■ Reaction stop solution Example 1. Same as .

[Samples] 10 serum samples and adenosine deaminase (ADA
) A standard solution containing 401 J/l was used as a sample.

〔Measuring method] Example 1. Same as .

The measurement results are shown in Tables 2 and 3.

Reference example 1゜ ■ Substrate solution A 5 anol/l aqueous solution of adenosine was prepared.

■ NTB in enzyme solution 0.2 M phosphate buffer (pH 7.5)
wo IQhg/l, P N P wo8.3 u/l, X
OD 8.8ti/1. 0.1% Triton X-100
It was dissolved to a concentration of .

■ Reaction stop solution 2N hydrochloric acid was prepared.

[Sample fee] Example 3. Same as .

[Measurement method] Mix 0.5 ml of substrate solution and 2 ml of enzyme solution, add sample (serum) 504 to this, heat at 37°C for 10 minutes, add 0.5 d of reaction stop solution, and add water. As a control, the absorbance at a wavelength of 5G0 ns was measured (Et).

Perform the same procedure using distilled water instead of the substrate solution.

The absorbance of the serum blank was measured (Eta).

Using substrate solution 0.5aZ and enzyme solution 2d, serum 504
A reagent blank was measured in the same manner as for serum, using distilled water instead (E9L).

A standard solution containing 40 U/I of ADA was used as a sample, and the absorbance was measured in the same manner as for serum (Estd
).

The measurement results are shown in Tables 2 and 3.

(i) Table 2 shows a comparison of the sensitivities of the present invention (Example 3) and the conventional method (Reference Example 1).

As is clear from Table 2, the method of the present invention is applicable to Reference Example 1 for measuring 0; It was found that the sensitivity was 2.57 times that of the previous one.

(eyes) Serum! Example 3 using 0 specimen. and reference example 1
The results measured at °C are shown in Table 3.

As a result of testing the results of Table 3, the significance level of Example 3 was 5%. and reference example 1. No difference was observed between them.

Table 3 Y = 0.989 X + 0.22 (y = 0.9998
) [Effects of the Invention] As described above, the present invention provides a new and useful method for quantifying ADA.

Medium Sensitivity is higher than conventional methods, and the amount of sample can therefore be reduced to less than half of the conventional method.

■Measurements are carried out in a reducing system using a tetrazolium salt such as nitrotetrazolium blue as a coloring reagent, making it possible to obtain measured values that are less affected by coexisting substances with reducing properties such as ascorbic acid.

■ Xanthine, hypoxanthine,
The influence of uric acid, etc. can be confirmed by pre-preparation of xOD, uricase, peroxidase, and amines or phenols (
or naphthols) and reacted for a certain period of time, eliminating the need for sample-blind measurements.

It is an invention that has a remarkable effect on the industry, and will make a great contribution to the industry.

[Brief explanation of drawings]

FIG. 1 shows Example 1. represents the calibration curve obtained in
The absorbance obtained for each adenosine deaminase activity value (U/I) on the horizontal axis is plotted along the vertical axis, and the points are connected. Patent Applicant: Wako Pure Chemical Industries, Ltd.'J61 (U/1)
) Procedural Amendment October 28, 1986 i Dear Commissioner of the Patent Office:
1. Indication of the case 1985 Patent Application No. 177811 2. Name of the invention Method for measuring the activity of adenosine deaminase in body fluids 3. Person making the amendment Relationship with the case Patent applicant
I postal code 541
f41'T
'*'fij, J? f*km*r, 1=it! 1llll
lT°11”osttu,. Contact: Patent Division (Tokyo) 03-270-1357
14゜Date of amendment order
11'. 5. Detailed description of the invention in the specification to be amended (3) Delete "decauric acid" in reaction formula (3) described in Section 7 of Book 101. (4) The above reaction formula (3a) described on page 7 of the specification

Claims (4)

[Claims] (1) Using adenosine as a substrate, hypoxanthine is produced by the action of purine nucleoside phosphorylase (PNP) on inosine, which is produced by the action of adenosine deaminase (ADA) in body fluids. Adenosine deaminase, characterized in that its activity value is determined by colorimetrically quantifying the pigment produced by the action of an enzyme, peroxidase, phenols (or naphthols) or amines, and a reducible coloring reagent. Activity measurement method. (2) Xanthine and hypoxanthine present in body fluids,
The measuring method according to claim 1, wherein adenosine deaminase activity in body fluids is measured after decomposing uric acid using xanthine oxidase, uricase, peroxidase, phenols (or naphthols), or amines. (3) the reduced coenzyme is reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH),
A measuring method according to claim 1 or 2. (4) The measuring method according to any one of claims 1 to 3, wherein the reducible coloring reagent is a tetrazolium salt.