JPH03175997A - Determination of total bilirubin and reagent used therefor - Google Patents

Abstract

PURPOSE:To accurately determine total bilirubin of aqueous specimen in a short time by promoting enzymatic reaction with bilirubin oxidase by using a specific phenol and aniline. CONSTITUTION:Enzymatic reaction with bilirubin oxidase is promoted by using one or more compounds selected from a group consisting of phenols shown by formula I (R<1> to R<5> are hydrogen atom, halogen atom, specific alkyl group, specific alkoxyl group, hydroxyl group or carboxyl group and R<3> is other than carboxyl group and hydroxyl group) and anilines shown by formula II (R<6> and R<7> are hydrogen atom, specific alkyl group, hydroxyalkyl group, sulfoalkyl group, hydroxysuloalkyl group or sulfonamidoalkyl group; R<8> and R<9> are hydrogen atom, halogen atom, specific alkyl group or specific alkoxyl group). Consequently, total amounts of bilirubin are measured in a short time.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field 1] The present invention relates to a method for enzymatically quantifying bilirubin and a quantitative reagent used therefor, and particularly relates to a method for quantifying total bilirubin in serum and the like.

[Prior art] Bilirubin is the most abundant pigment in bile, and is mainly produced from hemoglobin produced by the breakdown of old red blood cells.In serum, it is mainly produced as conjugated bilirubin (direct lupine) bound to glucuronic acid or albumin. It exists as unconjugated bilirubin (indirect bilirubin) bound to The sum of conjugated and unconjugated bilirubin is called total bilirubin.

Quantification of bilirubin is clinically extremely important because it is possible to differentially diagnose liver dysfunction and determine the degree of liver dysfunction from the amount of conjugated bilirubin and unconjugated bilirubin present in serum.

Conventionally, the method for quantifying bilirubin is to colorimetrically quantify azobilirubin, which is produced by the reaction of bilirubin and a diazo reagent (Izumi Kanai: Clinical Test Method Required Metals Publishing Page X
Chemical measurement methods such as ll-24 (1978) are mainly used. However, these methods have problems such as a lack of accuracy because the diazo reagent reacts with biological components other than bilirubin present in the specimen.

On the other hand, as a method for quantifying bilirubin using enzymes, bilirubin is oxidized using the fungal mushroom-derived throat oxidase produced by Tie-Wing Tsura, and the decrease in absorbance at this time is measured and quantified (Japanese Patent Application Laid-Open No. 1986-1999). 27656)
Myrothecium (Myrotheci) by Attachment et al.
A method for similarly measuring and quantifying the decrease in the absorbance of bilirubin using bilirubin oxidase derived from a microorganism (Japanese Patent Application Laid-Open No. 159487/1983) has been reported.

[Problems to be Solved by the Invention] The method for quantifying bilirubin using such an enzyme is superior to the method using the diazo reagent in terms of accuracy and analytical precision, but it requires the use of a large amount of enzyme. Since the reagents are required, they are expensive, and the long reaction time (approximately 15 to 30 minutes) is required, making it difficult to automate the measurement.

On the other hand, methods for improving the quantitative method using the enzymatic reaction described above are also being studied. For example, the enzymatic reactivity of bilirubin is improved by using reaction accelerators such as sodium cholate and sulfa drugs together with bilirubin oxidase (Japanese Patent Publication No. 62 -33880, etc.). However, the effect of promoting the reaction by these is insufficient, and although it is possible to shorten the reaction time to some extent, it has not reached the point where it can be adapted to automation (reaction time of about 10 minutes is required).

The present invention has been made in view of the above-mentioned state of the prior art, and provides a method and reagent that can accurately quantify total bilirubin based on an enzyme reaction in a short time.

[Means for Solving the Problems] In order to overcome the above-mentioned problems, the present inventors have conducted extensive research and found that the enzyme They discovered that the reaction could be significantly accelerated and completed the present invention.

That is, the present invention provides a method for quantifying total bilirubin in a bilirubin-containing aqueous sample by allowing bilirubin oxidase to act on the sample and optically measuring the resulting change in bilirubin. R @R4 (In the formula, Rl and R8 each independently represent a hydrogen atom, a halogen atom, an alkyl group having 1 to 4 carbon atoms, and 1 to 4 carbon atoms.
represents an alkoxyl group, hydroxyl group or carboxyl group.

However, those in which R3 is a carboxyl group or a hydroxyl group are excluded. ) (In the formula, R6 and R7 each independently represent a hydrogen atom, or an alkyl group having 1 to 4 carbon atoms, a hydroxyalkyl group, a sulfoalkyl group, a hydroxysulfoalkyl group, or a sulfonamide alkyl group. R8 and R
9 each independently represents a hydrogen atom, a halogen atom, an alkyl group having 1 to 4 carbon atoms, or an alkoxyl group having 1 to 4 carbon atoms. ), and a reagent for colorimetric determination of total bilirubin in a bilirubin-containing aqueous sample, which comprises at least a combination of one or more compounds selected from the group consisting of phenols and anilines shown above and bilirubin oxidase. .

According to the present invention, the amount of total bilirubin (hereinafter referred to as bilirubin unless otherwise specified) can be measured stably and accurately in a short time compared to the conventional enzyme-based total bilirubin quantitative method. , it becomes possible to easily automate the measurement.

Phenols represented by general formula (I) and general formula (II)
) are compounds that promote the enzymatic reaction by bilirubin oxidase (hereinafter referred to as enzymatic reaction promoters), but unlike known direct-acting agents such as sodium cholate, these are compounds that promote the enzymatic reaction by bilirubin oxidase. It also has an effective enzyme reaction promoting effect on bilirubin. Although the mechanism of action is unknown, the phenols or anilines have properties as electron donors and are thought to act on the electron transport chain of bilirubin oxidase, thereby promoting the oxidation reaction of bilirubin. It will be done.

The enzyme reaction promoter that can be used in the present invention has the general formula (
Among the phenols and anilines represented by I) and (II), particularly good effects can be obtained with mono- or di-substituted phenol derivatives, phenol, mono- or di-substituted aniline derivatives, and aniline. Examples of these compounds include those shown in the table below.

Phenols In the table, If not specified, it is hydrogen.

Anilines In the table, if there is no description, it is hydrogen.

Next, the method for quantifying bilirubin of the present invention will be explained.

In the present invention, the specimen for quantifying bilirubin is an aqueous specimen containing bilirubin in -M, such as serum, plasma, bile, etc., which may be diluted or pretreated as necessary. Good too.

Bilirubin oxidases that can be used include those derived from microorganisms of the genus Myrocesium, those derived from Agaricus bisporus, and any enzyme that is generally used as bilirubin oxidase regardless of its origin; The origin is easy to obtain and easy to handle.

The method for quantifying bilirubin of the present invention basically involves treating a bilirubin-containing aqueous sample (hereinafter simply referred to as the sample) with bilirubin oxidase, promoting the enzymatic reaction caused by the oxidase with an enzymatic reaction promoter, and elapsed for a predetermined period of time. Total bilirubin is then quantified by measuring changes in absorbance. This is based on the fact that bilirubin undergoes oxidation and changes to biliverdin through an enzymatic reaction, which changes its light absorption properties.

-i-wise, the optimum pH of bilirubin oxidase is 6 to 7,
Although the optimum temperature is around 40°C, by using the accelerator of the present invention, a remarkable reaction accelerating effect can be obtained at pH 5 to 11. On the other hand, since the light absorption characteristics of bilirubin in the sample change depending on the pH, it is necessary to keep the pH of the reaction solution constant during measurement. For this reason, a buffer solution is generally used when bilirubin is quantified by enzymatic reaction.

Buffers that can be used in the present invention have a pH of 5 to 5.
Any buffer having a concentration of about 11 may be used, and for example, Tris-HCl buffer, phosphate buffer, etc. can be used.

In other words, to quantify total bilirubin, it is necessary to quantify both direct and indirect bilirubin, but indirect bilirubin hardly undergoes enzymatic reaction in a low pH range (for example, pH 4 or less), so pH
is preferably set to about 5 to 11, preferably about 7 to 10.

In the present invention, the reagent used to quantify the amount of bilirubin in a specimen is basically composed of three components: (i) a buffer, (ii) an enzyme reaction promoter, and (ii) bilirubin oxidase.

There are no particular restrictions on the procedure and form in which the above three reagents are added to the sample, but if the enzyme reaction accelerator and bilirubin oxidase come into contact for a long time, the accelerator will be oxidized and colored by the enzymatic reaction. Therefore, the two reagents need to be separated before being added to the sample.

Examples of embodiments in which the above reagents are used in the present invention include the following.

(1) Add a buffer solution containing an enzyme reaction promoter to the sample, pre-warm it, and then add bilirubin oxidase solution (2
reagent system).

(2) Add 5 buffer solution to the sample and prewarm it, then add the enzyme reaction promoter solution and bilirubin oxidase solution at the same time (
3 reagent system).

Particularly, when bilirubin is to be quantified using an automatic analyzer, the two-reagent system (1) is preferable and can speed up the measurement. Here, the p required for the enzymatic reaction is
Since the H region is 5 to 11 for total bilirubin, the buffering effect of the buffer must be maintained in the reaction solution (ie, all necessary reagents added to the sample). For this reason, if an enzyme reaction promoter or bilirubin oxidase is added separately from the buffer solution and they are present in large amounts as a solution, it will not be possible to maintain a predetermined pH range. It is recommended to dissolve it in an appropriate buffer and add it. In any case, the enzyme reaction promoter needs to be dissolved in the reaction system, and is appropriately selected and used depending on the p)I band of the reaction solution. In addition, bilirubin oxidase may be in an aqueous solution, and if a buffer solution is used, p! It is preferable to set it in the range of about 16 to 9.

When quantifying total bilirubin, the dose of phenols may be any amount that has the effect of promoting the enzyme reaction, but the concentration in the reaction solution (final concentration) is usually 0.1 to 50 mM.
, preferably 1 to 15 mM. Anilines are similarly 0.001-50mM, preferably 0.05-10mM
Good.

The amount of bilirubin oxidase used basically varies depending on the amount of bilirubin present in the sample, but the final concentration is usually 0.05 to 1. , OU/m1 is preferably used.

The final concentration of each of the above is the concentration in the reaction solution,
The concentration in the reagent solution and the amount of the reagent solution are not particularly limited. Since bilirubin oxidase is dissolved in a considerable amount in an aqueous solution (for example, 100 U/ml or more), there is no restriction from solubility, and in this sense, the proportion of each reagent solution is adjusted to the desired pH.
Once the range is reached, it can be set according to the operational convenience of measurement. When using an automatic analyzer, the two-reagent system described in (1) above is used, and the ratio of the enzyme reaction promoter-containing buffer to the bilirubin oxidase solution is usually 5:1 to 1=1.
The degree is practical.

In the present invention, a reaction accelerator such as a known directing agent may be added to the reaction solution. For example, sodium cholate, sodium dodecyl sulfate, etc. can be present in the buffer solution. When using these reaction accelerators, the amount may be generally about 0.1 to 1%.

In the case of the two-reagent system described in (1) above, the specific operation of the above measurement method is to add a predetermined amount of an enzyme reaction promoter-containing buffer to a bilirubin-containing aqueous sample, and after prewarming (for example, 37°C,
3 minutes), add a predetermined amount of bilirubin oxidase solution, and maintain the pH of the reaction solution in the range of 5 to 11 to oxidize bilirubin present in the sample by enzymatic reaction. At this time, by colorimetrically quantifying the decrease in bilirubin in the reaction solution, the amount of bilirubin in the sample can be measured as a result. According to the method of the present invention, after charging bilirubin oxidase, 1
Accurate quantification is possible in about 2 minutes.

Note that the stability of indirect bilirubin varies depending on the pH and is less susceptible to enzymatic reactions at low pH.
In particular m-toluidine derivatives (above table, At, A5.A
By using IO, A18) and adjusting the pH to 3.0 to 4.0, it can become a very effective reaction accelerator even for the fractional measurement of direct bilirubin.

In order to apply the bilirubin quantitative reagent of the present invention to an automatic analyzer or the like, it is practically very convenient to handle it as a kit. That is, a buffer solution in which at least one or more compounds selected from the group consisting of phenols and anilines represented by the general formulas (I) and (II) is dissolved is used as the first reagent and a bilirubin oxidase solution. The operation can be simplified by using a kit for quantifying bilirubin in which the second reagent is . In addition, since bilirubin oxidase has relatively low stability, it is not preferable to store it in a solution state, and it is preferable to prepare it immediately before measurement. The solution in this case may be a buffer solution or an aqueous solution having a pH of about 6 to 9, as described above. Further, each component is adjusted so that its final concentration is within the range described above, and the pH of the buffer solution is adjusted within a predetermined range. For example, sample 10-100μ
t, the amount of phenols as the first reagent is 0.2 to 1
00 mM or/and anilines from 0.002 to 100
2 ml of pH 5-11 buffer containing mM (may also contain sodium cholate etc.), 0.3-6 U/ml of bilirubin oxidase as a second reagent.
It is preferable to use about 0.3 ml to 2 ml of the aqueous solution containing the liquid. The amount of bilirubin oxidase may be adjusted depending on the amount of bilirubin present in the sample, but when a sample such as serum is used, it is usually within the above range.

In addition, colorimetric determination can be carried out by a conventional method, for example,
Using a commercially available spectrophotometer (for example, Hitachi U-3200 self-recording spectrophotometer), the absorbance is measured at a wavelength of 400 to 480 nm using a reagent blind test as a control. At this time, the absorbance was measured in the same manner using serum containing bilirubin at a known concentration (hereinafter referred to as standard) as a control, and as a sample blind test, a buffer solution containing no bilirubin oxidase was added to the sample serum and standard. Measure the absorbance in the same way, and based on these, calculate the bilirubin concentration (mg
/di), bilirubin can be quantified.

: Absorbance of sample serum (sample blinded) : Absorbance of sample serum (enzyme-reacted thing) Ass: Standard absorbance (sample blinded) AC? : Standard absorbance (enzymatically reacted) In the formula, A.

AtX: Bilirubin concentration in standard (mg/old) [Example] Next, the present invention will be explained in more detail with reference to Examples.

Example 1 Bilirubin solution (crystalline bilirubin manufactured by Merck & Co., Ltd. 15 mg
/dl) 2.4-dichlorophenol 5m to 50μm
Br 1tton-Robinson broad range buffer containing M (first reagent: pH 5, 6, 7, 8, 9, 10
Or 11) Add 2ml and prewarm for 3 minutes, then add 3ml
0.5 ml of a U/ml bilirubin oxidase aqueous solution (second reagent) was added, and a reaction curve was measured at 37° C. at a wavelength of 450 nm. In addition, at this time, Br1tton-Robinso which does not contain 2,4-dichlorophenol
Measurements were made in the same manner when a wide range buffer solution of n was used, and the reaction times were compared. The obtained reaction curve is shown in Figure 1 (
Shown in a) to (g).

2.4-dichlorophenol showed a reaction accelerating effect at p)15-11, especially at pH7-10.

Example 2 5mM of 3.5-dimethylphenol in 50μm of sample serum
and 0.2m containing 0.5% sodium cholate
M) Add 2 ml of lithium-hydrochloric acid buffer (first reagent: pt17.8 or 9) and preheat for 3 minutes, then add 30%
ml bilirubin oxidase aqueous solution (second reagent) 0.5
ml was added and the reaction curve was measured at 37° C. at a wavelength of 450 nm. At this time, measurements were also made in the same manner using a buffer solution that did not contain 3,5-dimethylphenol, and the reaction times were compared. The reaction curves obtained are shown in Figure 2 (a) to (
Shown in C).

The same effects as in Example 1 were obtained.

Example 3 N-ethyl-N-(2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline 5 was added to 50 μm of sample serum.
0, containing mM and 0.5% sodium cholate.
2M Tris-HCl buffer (1st reagent: p117.8
, or 9) Add 2 ml and prewarm for 3 minutes, then add 3 ml to this.
0/ml bilirubin oxidase aqueous solution (second reagent) 0
．． 5 ml was added and the reaction curve was measured at 37° C. at a wavelength of 450 nm. In addition, at this time, N-ethyl-N-(
Measurements were made in the same manner using a buffer solution that did not contain 2-hydroxy-3-sulfopropyl)-3,5-dimethylaniline, and the reaction times were compared. The obtained reaction curves are shown in FIGS. 3(a) to (c). The same effects as in Example 1 were obtained.

Example 4 Using the following first reagent and second reagent, a standard solution and a 5-step dilution of high bilirubin serum were each used as specimens to measure,
The linearity of the reagent was investigated.

(Type 3 drug system composition) First reagent: 0.1M Tris-HCl buffer (pi48.5
) Contains 5mM 3.5-dimethylphenol, 0.5% sodium cholate.

Second reagent: 3U/ml bilirubin oxidase aqueous solution (measurement and results) Add 400μ of the first reagent to a 14μ sample and allow to react at 37°C for 5 minutes. Measure the absorbance after 5 minutes (A,). Next, 100 μl of the second reagent is added to the corresponding reaction solution, reacted at 37° C. for 5 minutes, and the absorbance is measured (A:). Determine the absorbance difference = A; A; and use the standard solution (known concentration of bilirubin).
The bilirubin concentration was determined by comparing the absorbance difference between the two. In addition, a Hitachi 705 type automatic analyzer (Hitachi, Ltd.) was used for the measurement, and the measurement wavelength was a main wavelength of 450 nm and a sub wavelength of 660 nm.
The experiment was conducted using two wavelengths of m.

As a result, as shown in FIG. 4, linearity up to about 35 mg/dl was clear.

Example 5 The quantitative value of bilirubin according to the present invention was compared with the quantitative value according to the conventional diazo method. That is, sample (serum) 14
Add 400 μm of the following first reagent to μm and react at 37° C. for 5 minutes. Measure the absorbance after 5 minutes (A,). Next, add 100 μm of the following second reagent to the corresponding reaction solution, and
Let it react for a minute and measure the absorbance (All). The absorbance difference = ABAs was determined, and the total bilirubin concentration was determined by comparing the absorbance difference with a standard solution (known concentration of total bilirubin).

A Hitachi 705 type automatic analyzer (Hitachi, Ltd.) was used for the measurement, and the measurement wavelength was 450 nm as the main wavelength and 66 nm as the sub wavelength.
The experiment was carried out using two wavelengths of 0 nm.

Reagent composition First reagent 0.15M Tris-HCl buffer (pH 8
,5) 10mM 3.5-dimethylphenol 0.5% sodium cholate second reagent 20mM Tris-HCl buffer (pH 9,
0) 4 II/ml Bilirubin oxidase results are shown below and in Figure 5, correlation coefficient r = 0.997
An extremely high correlation with 9 was observed.

Number of data: N = 40 Correlation coefficient: r = 0.9979 Average value; x (enzyme method) = 3.378 mg/dlY (
Diazo method) = 3.010mg/d1 regression equation; Y=Ω
, 967X −0,256S y x = 0.379
0t (calculated value)・0.283 (no significant difference) [Effect of the invention] As explained above, according to the present invention, the enzymatic reaction by bilirubin oxidase is promoted by using specific phenols or anilines. Therefore, the total amount of bilirubin that can be used for diagnosing liver dysfunction can be measured accurately and quickly, and the measurement can be easily automated. In addition, the present invention is very useful in practice, as the reagents of the present invention are made into a kit, which makes handling easier.

[Brief explanation of drawings]

FIG. 1 is a diagram showing the results of total bilirubin measurement conducted in Example 1 as changes in absorbance; (a), (b), (C)
, (d) (e), (f), and (g) are diagrams when p)l of the buffer solution is 5.6.7.8.9.10.11, respectively, and Figure 2 is for Example 2. It is a diagram showing the results of total bilirubin measurement as changes in absorbance, (a), (b), (
C) is a diagram when the pH of the buffer solution is 7.8.9, respectively.
FIG. 3 is a diagram showing the results of total bilirubin measurement conducted in Example 3, and (a), (b), and (C) are diagrams when the pH of the buffer solution was 7.8.9, respectively. Figure 4 shows the linearity of the measurement when the sample was diluted in Example 4, and Figure 5 shows the correlation between the conventional method (diazo method) and the enzyme method of the present invention performed in Example 5. It is a diagram.

Claims (1)

[Scope of Claims] 1. A method for quantifying total bilirubin in a bilirubin-containing aqueous sample by acting bilirubin oxidase on the sample and optically measuring the resulting change in bilirubin, which comprises: is promoted by one or more compounds selected from the group consisting of phenols represented by general formula (I) and anilines represented by general formula (II). . ▲There are mathematical formulas, chemical formulas, tables, etc.▼...(I) (In the formula, R^1 to R^5 each independently represent a hydrogen atom,
Halogen atom, alkyl group having 1 to 4 carbon atoms, 1 to 4 carbon atoms
4 represents an alkoxyl group, hydroxyl group or carboxyl group. However, those in which R^3 is a carboxyl group or a hydroxyl group are excluded. ) ▲There are mathematical formulas, chemical formulas, tables, etc.▼...(II) (In the formula, R^6 and R^7 each independently represent a hydrogen atom, an alkyl group having 1 to 4 carbon atoms, a hydroxyalkyl group, Represents a sulfoalkyl group, hydroxysulfoalkyl group, or sulfonamidoalkyl group.R^8 and R^9 each independently represent a hydrogen atom, a halogen atom,
It represents an alkyl group having 1 to 4 carbon atoms or an alkoxyl group having 1 to 4 carbon atoms. 2) A buffer solution containing one or more compounds selected from the group consisting of phenols and anilines is added to the bilirubin-containing aqueous sample, and after prewarming, bilirubin oxidase is added. The quantitative method described in 1. 3. The quantitative determination method according to claim 1, wherein the phenol is a mono- or di-substituted product. 4. Claim 1, wherein the aniline is mono- or di-substituted.
Quantitative method described in. 5. A reagent for colorimetric determination of total bilirubin in a bilirubin-containing aqueous sample, which comprises at least a combination of one or more compounds selected from the group consisting of phenols and anilines shown below and bilirubin oxidase. ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, R^1 to R^5 each independently represent a hydrogen atom,
Halogen atom, alkyl group having 1 to 4 carbon atoms, 1 to 4 carbon atoms
4 represents an alkoxyl group, hydroxyl group or carboxyl group. However, those in which R^3 is a carboxyl group or a hydroxyl group are excluded. ) ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, R^6 and R^7 each independently represent a hydrogen atom, a C1-C4 alkyl group, a hydroxyalkyl group, a sulfoalkyl group, a hydroxysulfonate group) Represents an alkyl group or a sulfonamide alkyl group.R^8 and R^9 each independently represent a hydrogen atom, a halogen atom,
It represents an alkyl group having 1 to 4 carbon atoms or an alkoxyl group having 1 to 4 carbon atoms. )