JPS6233880B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for enzymatically quantifying bilirubin in a test fluid, particularly in a biological fluid such as blood or urine, and a reagent composition for the assay. Specific examples include a method for directly quantifying bilirubin using bilirubin oxidase produced by Myrocesium genus bacteria, and a method selected from the group consisting of bilirubin oxidase produced by such strains, surfactants, aromatic carboxylic acids, sulfur drugs, and proteases. One or more reaction promoters (hereinafter simply referred to as reaction promoters)
The present invention relates to methods for quantifying total bilirubin and reagent compositions for such quantitative determination. Bilirubin is the most abundant pigment in bile, and is present in serum at approximately 1 mg/dl or less in normal people. In serum, it mainly exists as direct bilirubin bound to glucuronic acid (conjugated bilirubin) or indirect bilirubin bound to albumin (unconjugated, free bilirubin). (PH7.4), it is insoluble in water, so it hardly exists. Direct bilirubin is predominant in urine. The significance of separately quantifying direct bilirubin and indirect bilirubin is important in understanding what causes an increase in bilirubin in biological fluids such as serum and urine. In other words, an increase in direct bilirubin is expected to result from hepatocellular damage, intrahepatic cholestasis, extrahepatic cholestasis, etc., and an increase in indirect bilirubin is thought to be due to increased bilirubin production, abnormal liver processing function, etc. ing. Conventionally, the method for differentially quantifying bilirubin has been to colorimetrically quantify the red color of azobilirubin produced by the repulsion of bilirubin and diazo reagent (Kanai Izumi: Clinical Testing Methods, 28th Edition, Kanehara Publishing, Page XII-24, Chemical methods such as (1973) are mainly used. This method may lack accuracy because the diazo reagent reacts with serum components other than bilirubin. Also, examples of enzymes that react with bilirubin include:
European Journal of Biochemistry (Eur.J.Biochem), Vol. 10, pages 468-473 (1969) describes hemoglobin, cytochrome C, and oxidase in tissues such as the brain, liver, and heart of Guinea Pizugu. It has been reported that peroxidase from horseradish and xanthine oxidase from milk have the ability to oxidize bilirubin.
These enzymes, except xanthine oxidase, are activated by the presence of hydrogen peroxide. There is no mention of whether hydrogen peroxide is included in the reaction product.
Furthermore, JP-A-54-151193 describes an enzyme composition having activity against bilirubin produced by Agaricus bisporus. The enzyme composition reacts with bilirubin, and the reaction product has a maximum absorption at 510 nm and an excitation wavelength of 450 nm at 525 nm.
m fluorescent substance and hydrogen peroxide. However, there is no description of whether it acts on direct or indirect bilirubin. In view of these current circumstances, the present inventors continued intensive research and discovered a new enzyme, bilirubin oxidase (this enzyme was recognized by the International Union of Biochemistry Committee).
Registered as EC1.3.3.5. ), and also discovered a method for differentially quantifying bilirubin in living organisms, especially in serum, by enzymatic measurement. That is, the method of the present invention
A direct method for quantifying bilirubin, characterized in that bilirubin oxidase produced by bacteria of the genus Myrocesium is allowed to act on a test solution; This invention relates to a method for quantifying total bilirubin. The amount of indirect bilirubin is determined by subtracting the amount of direct bilirubin from the total amount of bilirubin. The quantitative method and composition for quantitative determination of the present invention will be explained in detail below. As mentioned above, the presence of bilirubin in biological fluids is due to direct bilirubin bound to glucuronic acid etc. and indirect bilirubin bound to albumin. Broadly classified. Direct bilirubin can be quantified by measuring the decrease in bilirubin caused by the action of bilirubin oxidase produced by bacteria of the genus Myrocesium or the increase in biliverdin, a reaction product. This enzyme does not act on indirect bilirubin. An example of this enzyme is Myrocesium Berkaria MT-1 shown in Japanese Patent Application No. 57-25613.
(Myrothecium verrucaria MT-1), FERM-
One example is bilirubin oxidase NS-1 produced by BP No. 653 (FERM-P No. 5918).
The present bilirubin oxidase NS-1 has the following properties. (1) Action: Bilirubin is acidified in the presence of molecular oxygen, producing a green substance, a pale green substance, and then a pale purple substance, but does not produce hydrogen peroxide. (2) Substrate specificity: Reacts with direct bilirubin.
It does not react with indirect bilirubin. It reacts slightly with biliverdin, hemin, hematoporphyrin, and chlorophyllin. Does not react with hemoglobin, chlorophyll, or vitamin _B12 . (3) Temperature and PH stability: 90% or more remained after treatment at PH8.0 (0.1M phosphate buffer) at 50°C for 15 minutes;
It is inactivated in 15 minutes at ℃. More than 95% remains between PH5 and PH10.5 after treatment at 37°C for 60 minutes. (4) Optimum temperature: around 40℃. (5) Optimum pH: 6-7 (6) Molecular weight: Approximately 52,000 (gel filtration method) (7) Isoelectric point: PI=4.1 (8) Effect of metal ions: Significantly inhibited by F ²⁺ _e . (9) Effects of inhibitors, etc.: Inhibited by potassium cyanide, sodium azide, and thiourea. (10) Visible absorption: There is no absorption maximum near 375nm and 460nm. A method for quantifying direct bilirubin will be explained using bilirubin oxidase NS-1 as an example. That is, it contains 0.1 to 1.0 units of this enzyme.
3ml of 0.1M Tris-HCl buffer (PH8.4) and 100ml of test solution
μ was reacted at 37℃ to reduce bilirubin.
The increase in biliverdin resulting from the reaction is determined from the decrease in absorption at 440 nm or from the increase in absorption at 330 nm or 380 nm. Figure 1 shows the changes in the absorption pattern before and after the reaction when a standard bilirubin solution (reagent bilirubin dissolved in an alkaline aqueous solution and adjusted to pH 8.4) was used as the test solution.
The maximum absorption wavelength of bilirubin is around 440 nm, and the maximum absorption wavelength of biliverdin produced as a result of the reaction is around 330 nm and 380 nm. As can be seen from the figure, measurement based on changes in absorption at 330 nm or 380 nm is not preferred for accurate measurements because the molecular extinction coefficient is less than half of the absorption at 440 nm that bilirubin has, and the sensitivity is low. Next, the method for quantifying total bilirubin will be explained in detail. Bilirubin oxidase NS-1 produced by the Myrocesium genus bacteria does not act on indirect bilirubin bound to albumin. To quantify total bilirubin, it is necessary to measure all types of bilirubin, including direct and indirect types. As a result of our research on a method for quantifying total bilirubin using bilirubin oxidase NS-1, we found that by coexisting a surfactant, aromatic carboxylic acid, sulfur drug, or protease in the reaction system, indirect bilirubin can be It was discovered that this function makes it possible to quantify total bilirubin. It is not clear what kind of action these additives have, but in any case, they have the effect of promoting the reaction between bilirubin oxidase NS-1 and indirect bilirubin.
Specific examples of these reaction accelerators include sucrose fatty acid esters, bile salts, and surfactants.
Aromatic carboxylic acids such as dodecyl sulfate, p-toluenesulfonic acid, cetylpyridinium chloride, nonylphenol ethoxylates, and polyoxyethylene-polyoxypropylene condensates; salicylic acid and sulfosalicylic acid as sulfur agents; Examples of the protease include anilamide and sodium acetosulfamine, and pronase P (manufactured by Kaken Chemical Co., Ltd.). Table 1 shows the effects of these reaction accelerators. That is, a reaction solution having the following composition was reacted at 37°C, and the decrease in absorption at 440 nm was measured for 3 minutes after the start of the reaction, and the values corrected per minute are shown in the same table. The indirect bilirubin used as a substrate was Bilirubin Control manufactured by Dade. 0.1M Tris-HCl buffer (PH8.4) 3ml Bilirubin control (20mg/dl) 20μ Bilirubin oxidase NS-1 (15μ/ml)
20μ Reaction promoter (10% aqueous solution) 50μ (Pronase P is 0.1% aqueous solution)

[Table] As can be seen from Table 1, particularly preferred reaction accelerators are sodium cholate, sodium dodecyl sulfate, and salicylic acid. Suitable concentrations of these reaction promoters in the reaction solution are as shown in Table 2, using sodium dodecyl sulfate and sodium cholate as examples. The method was carried out in accordance with the method shown in Table 1 above.

Table 2 shows that in the case of sodium dodecyl sulfate, a concentration of 0.05 to 0.1% in the reaction solution is suitable, and in the case of sodium cholate, a concentration of 0.5 to 0.7% is suitable. As a method for quantifying total bilirubin in the method of the present invention, in addition to a method using bilirubin oxidase produced by a bacterium of the genus Myrocesium in combination with a reaction promoter, a method using bilirubin oxidase produced by a bacterium of the genus Coprinus can be provided.
The method for quantifying total bilirubin using bilirubin oxidase produced by Coprinus spp. does not require a reaction accelerator, but it is desirable to add it to speed up the reaction. The reagent composition for quantifying bilirubin used in the method of the present invention will be explained. The amount of enzyme contained in the reagent composition is 0.01 as bilirubin oxidase activity.
Mu/ml to 20u/ml can be used. Most preferably, bilirubin oxidase is present in the reaction solution at a concentration of 0.01~
Although it is 10u/ml, it may be adjusted as appropriate depending on the measurement time. For example, the reaction accelerator is 0.02% to 0.2%, preferably 0.05 to 0.1% in the reaction solution in the case of sodium dodecyl sulfate, and 0.1 to 1.0%, preferably 0.5 to 0.7% in the case of sodium cholate. Other buffers, enzyme stabilizers, etc. may be added as necessary. Hereinafter, a method for measuring enzyme activity, test examples, and examples will be explained. Bilirubin oxidase activity measurement method 0.2M containing 1mM of ethylenediaminetetraacetic acid
Dissolve 5 mg of reagent bilirubin (manufactured by Wako Pure Chemical Industries, Ltd.) in 250 ml of Tris-HCl buffer (PH8.4), react this 2 ml with 0.2 ml of enzyme solution at 37°C, and measure the decrease in absorbance at 440 nm. One unit is the amount of enzyme that oxidizes 1 micromole of bilirubin per minute. Test Example 1 Preparation of bilirubin oxidase NS-1 Myrothecium bercariae
Verrucaria) MT-1, FERM-BP No.653
(FERM-P No.5918) strain was cultured in glucose/potato medium, and bilirubin oxidase
A culture solution containing NS-1 was obtained. The culture solution was sequentially subjected to ammonium sulfate salting out, dialysis, activated carbon treatment, QAE-Sephadex A-50 column chromatography, ultrafiltration, and Sephadex G-100 column chromatography to obtain purified bilirubin oxidase NS-
I got 1. This specimen was unique in polyacrylamide gel disk electrophoresis. Test Example 2 Preparation of bilirubin oxidase produced by a bacterium of the genus Coprinus Coprinus cinereus
cinereus) IFO8371 strain was cultured in potato glucose medium to obtain a culture solution containing bilirubin oxidase activity. The culture solution was sequentially subjected to ammonium sulfate salting out, dialysis,
DEAE-cellulose column chromatography,
Purified bilirubin oxidase was obtained by performing DEAE-Sepharose column chromatography, ultrafiltration, and Sephadex G-100 column chromatography. The properties of this enzyme are similar to the aforementioned bilirubin oxidase NS-1 produced by Myrocesium spp., but the optimum temperature was around 30°C and the isoelectric point was 3.8. Test Example 3 Preparation of Lactucase Produced by Polyporus Bacteria Polyporus versicolor (Polyporus versicolor)
versicolor) IFO9791 strain with glucose, L-asparagine, DL-phenylalanine, adenine,
The cells were cultured in a medium containing thiamine and inorganic salts to obtain a culture solution containing lactcase activity. The culture solution was sequentially subjected to ammonium sulfate salting out, dialysis, ultrafiltration, DEAE-Sephadex A-50 column chromatography, ultrafiltration, and Sephadex G-100 column chromatography to obtain a purified sample. . This specimen was disk electrophoretically single. Test Example 4 Preparation of tyrosinase derived from pine room Using Tyrosinase (Grade) manufactured by Sigma, this was further added to DEAE-Sephadex A-50.
Column chromatography, hydroxyapatite column chromatography, and DEAE-Sephadex A-50 column chromatography were performed to obtain a purified sample. The purity of this specimen was approximately 90% by disk electrophoresis. Test Example 5 Preparation of Urushi laccase Sumac sap was salted out, dialyzed, and ultrafiltered, and then subjected to Sephadex G-200 column chromatography to obtain a partially purified sample. Test Example 6 Substrate specificity of various enzymes In order to clarify the difference between bilirubin oxidase used in the method of the present invention and other similar enzymes, substrate specificity was investigated. The enzymes were bilirubin oxidase NS-1 (abbreviated as A, hereinafter the same) prepared in Test Examples 1 to 4, bilirubin oxidase (B) produced by Coprinus spp., latcase (C) produced by Polyporus spp. Tyrosinase (E), latcase derived from sumac (D), and ascorbic acid oxidase derived from Japanese cucumber (Glade, manufactured by Toyobo) (F) were used. Various substrates shown in Table 3 at a concentration of 7mM (unconjugated bilirubin was used) and a certain amount of the above enzyme were mixed at 37
The reaction was carried out at ℃ and the enzyme activity was measured. The results are shown in Table 3. In the same table, the enzyme showing the highest activity for each substrate is expressed as a relative activity, with the enzyme showing the highest activity as 100.

【table】

[Table] As shown in Table 3, bilirubin oxidase had the strongest effect on bilirubin, but weak reactivity was also observed with other enzymes. Example 1 The effects of bilirubin oxidase NS-1 on various types of bilirubin were examined. For indirect bilirubin, Bilirubin Control manufactured by Dade was used. The conjugated bilirubin used was prepared by the present inventors in the following manner. i.e. 10ml of dog bile
was extracted with twice the amount of chloroform, and the aqueous layer was concentrated and filtered through Sephadex G-25 gel to obtain a purified sample. When this specimen was subjected to a diazo reaction using the bilirubin B test "Wako" (manufactured by Wako Pure Chemical Industries, Ltd.), no indirect bilirubin was detected.
Bilirubin oxidase NS-10.3 units, 0.1M-
A quantitative reagent composition consisting of 3 ml of Tris-HCl buffer and 100 µ of bilirubin at various concentrations were reacted at 37°C for 30 minutes, and then the decrease in absorption at 440 nm was measured. The results are shown in Figure 2. In the figure, (-○-) represents indirect bilirubin, and (-▲-) represents conjugated bilirubin. As can be seen from the figure, bilirubin oxidase NS-1 had almost no effect on indirect bilirubin. Also, conjugated type,
It reacted with bilirubin, and good linearity was obtained. Example 2 A calibration curve for indirect bilirubin quantification was determined using bilirubin oxidase NS-1 and a reaction promoter. Sodium dodecyl sulfate was used as a reaction promoter. i.e. bilirubin oxidase NS-10.3 units, sodium dodecyl sulfate 3
100μ of bilirubin control at various concentrations was reacted with a quantitative reagent composition consisting of 3ml of 0.1M Tris-HCl buffer, and the decrease in absorption at 440nm was measured. The results are shown in Figure 3. Good linearity was obtained between bilirubin concentration and absorbance. Example 3 The same procedure as in Example 2 was performed except that bilirubin oxidase (0.01 unit) of Coprinus sp. was used in place of bilirubin oxidase NS-1.
As a result, the calibration curve was the same as in Example 2. Example 4 The required amount of bilirubin oxidase NS-1 in a reagent composition for quantifying bilirubin was determined. That is, a composition consisting of 0.075 units, 0.15 units, 0.2 units, or 0.3 units of bilirubin oxidase NS-1 and 100μ of 20% sodium cholate and 3 ml of 0.1M Tris-HCl buffer (PH8.4) was mixed with a bilirubin control aqueous solution (20 mg). /dl) at 37°C, and the decrease in wavelength at 440 nm was measured over time.
The results are shown in Figure 4. It was found that approximately 0.15 units or more is sufficient to complete the reaction in 30 minutes. Example 5 Total bilirubin and direct bilirubin in 17 serum samples were quantified. The following reagent composition for quantitative determination was used. (1) Reagent composition for direct bilirubin determination: 3 ml of Tris-HCl buffer (PH8.4) containing the following 0.3 units of bilirubin oxidase NS-1 (2) Reagent composition for determining total bilirubin: Tris-HCl buffer (PH8.4) containing the following: .4) 3ml Bilirubin Oxidase NS-1 0.3 units Sodium cholate 0.6% (3) For blank The same as in (2) above without the enzyme Add 100μ of each sample to the above kit, and after reacting at 37℃ for 30 minutes,
The decrease in absorption at 440 nm was measured, and the content of bilirubin was determined from the calibration curve. As a control, quantitative determination was carried out by the diazo method using Wako Pure Chemical's Bilirubin-B Test Wako. The relationship between this example and the measurement results by the diazo method is shown in Figures 5 and 6.
As shown in the figure. FIG. 5 shows the quantitative results of total bilirubin, and FIG. 6 shows the quantitative results of direct bilirubin.
The correlation coefficient is for total bilirubin measurements.
0.984, 0.983 for direct bilirubin measurements
, and a strong correlation was observed between both quantitative methods.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing changes in the absorption pattern when the enzyme used in the present invention acts on bilirubin. FIG. 2 is a diagram showing calibration curves for various types of bilirubin according to the method of the present invention, and FIG. 3 is a diagram showing a calibration curve for indirect bilirubin. Figure 4 shows bilirubin oxidase in the reagent composition of the present invention.
FIG. 2 is a diagram showing the amount of NS-1 and reaction rate. Fifth
The figure is a diagram showing the relationship between the quantitative results of serum total bilirubin by the method of the present invention and the diazo method.
The figure also shows the relationship between the quantitative results of direct bilirubin.

Claims (1)

[Scope of Claims] 1. Direct bilirubin is oxidized by the action of bilirubin oxidase produced by a strain belonging to the genus Myrocesium on human serum, and the resulting change in bilirubin is optically measured. 1 selected from the group consisting of bilirubin oxidase produced by strains belonging to the genus Myrocesium, surfactants, aromatic carboxylic acids, sulfur drugs, and proteases.
Total bilirubin is quantified by measuring the change in bilirubin that occurs when direct and indirect bilirubin is oxidized by the action of a species or a combination of two or more reaction promoters. A method for quantifying bilirubin, which is characterized by separately quantifying bilirubin. 2. A reagent comprising bilirubin oxidase produced by a strain belonging to the genus Myrocesium, and one selected from the group consisting of bilirubin oxidase produced by a strain belonging to the genus Myrocesium, a surfactant, an aromatic carboxylic acid, a sulfur agent, and a protease; or A reagent composition for quantifying human serum bilirubin comprising a reagent containing two or more reaction promoters.