JPH0568240B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to a method for quantifying conjugated bilirubin using the enzyme bilirubin oxidase (EC1.3.3.5). Bilirubin is one of the important measurement items in clinical chemistry tests. Prior Art Bilirubin is a yellow substance produced by the metabolism of hemoglobin, and is secreted from the liver as a bile pigment. Bilirubin mainly exists in three forms in biological body fluids such as serum. That is, it is classified into three types: conjugated bilirubin, unconjugated bilirubin, and delta bilirubin, and the sum of these is called total bilirubin. Conjugated bilirubin is produced when bilirubin undergoes glucuronidation by the action of UDP glucuronyl transferase in the liver, and is a monoglucuronide type in which one molecule of glucuronic acid is bound, or a diglucuronide type in which two molecules of glucuronic acid are bound together. Exists as a type. Unconjugated bilirubin is not affected by the above-mentioned conjugating enzymes and is also called free bilirubin. Conjugated and unconjugated bilirubin exist in serum relatively loosely bound to albumin. A third form, delta bilirubin, exists irreversibly bound to serum albumin [J.Lab.Clin.Med. 67 ,
294−306, (1966) and Clin.Chem., 28 , 629
−637, (1982)]. Differential determination of each of the above forms of bilirubin is important as an indicator for the diagnosis of liver diseases. Conventional methods for quantifying bilirubin include the diazo method, which decolorizes the azobilirubin produced by the reaction between bilirubin and a diazo reagent (written by Izumi Kanai, Introduction to Clinical Testing Methods, Vol.
28th edition, Kanehara Publishing, p.
(1982); Selected Topics in Clin.Enzymol.
2, 97-107, (1984); JP-A-59-17999] and methods using column chromatography or high-performance liquid chromatography [J.Lab.Clin, cited above.
Med. 67 , 294-306, (1966) and J.
Chromatogr., 226 , 391-402, (1981)] is known. Bilirubin in biological body fluids is separated by the diazo method into indirect bilirubin, which requires the addition of a reaction accelerator such as methanol, and direct bilirubin, which does not require the addition of a reaction accelerator. The names indirect and direct are based on the difference in reactivity of bilirubin to diazo reactions, and do not correspond to unconjugated and conjugated bilirubin, respectively.
Bilirubin measured directly includes conjugated and digital bilirubin. On the other hand, in the differential determination method of bilirubin using bilirubin oxidase, the difference in the reactivity of bilirubin oxidase to direct and indirect bilirubin is basically used.
Then, the total amount of direct and indirect bilirubin is measured using bilirubin oxidase together with an anionic surfactant [Clinical Pathology, cited above, 30 (Supplementary),
123, (1982) and Japanese Patent Publication No. 59-17999]. Furthermore, conditions for differentially quantifying bilirubin with higher precision using bilirubin oxidase have recently been reported. For example, a method for measuring direct bilirubin and total bilirubin by allowing bilirubin oxidase to act on a sample at pH 3.5 to 4.5 and pH 6 to 9 in the presence of an anionic surfactant (JP-A-59-125899 and JP-A No. 59-59- 130198), a method for measuring conjugated bilirubin by performing a reaction at PH 4.4 [Clinical Pathology, 32 (Supplementary), 373, (1984)], a method for measuring conjugated bilirubin by performing a reaction at PH 5 to 6 in the presence of an anionic surfactant. A method for measuring bilirubin (Japanese Unexamined Patent Publication No. 152955, 1983) and a method for measuring free bilirubin by conducting a reaction at pH 8 or higher in the presence of an anionic surfactant (Japanese Unexamined Patent Publication No. 60-15295).
154162) has been reported. Problems to be Solved by the Invention The above-mentioned conventional diazo method or method using bilirubin oxidase makes it impossible to completely separate and quantify conjugated, unconjugated, and delta bilirubin; I had to. The chromatography method is a complicated method that cannot be used in the field of daily clinical chemistry testing. The direct bilirubin measured by the diazo method described above includes conjugated bilirubin and delta bilirubin, and it is not clear which type of bilirubin is measured by the conventional method of differentially quantifying bilirubin using bilirubin oxidase. The object of the present invention is to provide conjugated forms present in biological body fluids,
The present invention provides a method for specifically measuring conjugated bilirubin among unconjugated and delta bilirubin. The delta bilirubin value can be calculated from the difference between the conjugated bilirubin value obtained by the present invention and the direct bilirubin value obtained by the conventional diazo method or the method using bilirubin oxidase. Means and Effects for Solving the Problems According to the present invention, conjugated bilirubin can be obtained by applying bilirubin oxidase to a sample containing bilirubin in a buffer solution in the pH range of 9 to 11 and measuring the resulting change in absorbance. is specifically quantified. The present inventors investigated the differences in the reactivity of bilirubin oxidase to conjugated, unconjugated, and delta bilirubin, and found that the reactivity of bilirubin oxidase to unconjugated and delta bilirubin is minimum in the pH range of 9 to 11. We found that the difference in reactivity to conjugated bilirubin is greatest in the PH range. The method of the present invention utilizes the reaction characteristics of bilirubin oxidase to bilirubin. Bilirubin oxidase used in the present invention is known as an enzyme that catalyzes a reaction that oxidizes yellow bilirubin to produce green biliverdin. Bilirubin is a substance that has an absorption maximum at a wavelength of 410 to 460 nm, although it differs depending on the bond state described above, and bilirubin can be quantified by measuring the decrease in absorption due to the action of bilirubin oxidase. Conjugated bilirubin is
It has an absorption maximum at 450nm. Although bilirubin oxidase can be obtained from Deuteromycetes, Basidiomycetes, and plants, the enzyme most preferably used in the present invention is derived from the Myrothecium genus. This enzyme is commercially available and readily available. The bilirubin-containing sample measured in the present invention is not particularly limited as long as it is an aqueous sample such as serum, plasma, or urine. The composition of the buffer used in the present invention is not particularly limited as long as it has a pH in the range of 9 to 11, but when bilirubin oxidase is allowed to act on bilirubin in the buffer, the reactivity toward conjugated bilirubin is high; It is preferable to choose a buffer that has as large a difference in reactivity and reactivity towards unconjugated and delta bilirubin as possible. Preferred buffers include glycine-sodium hydroxide buffer, Tris-sodium hydroxide buffer, borax-sodium hydroxide buffer, ethanolamine-sodium hydroxide buffer, universal buffer [citric acid, monopotassium phosphate, Analyst, 64 , 490 (1939)], carbonate-sodium hydroxide buffer, diethanolamine-hydrochloric acid buffer and disodium phosphate-sodium hydroxide buffer. can be mentioned. Particularly preferred are glycine-sodium hydroxide buffers and Tris-sodium hydroxide buffers in which the ratio of the reactivity of bilirubin oxidase to conjugated bilirubin to that to unconjugated bilirubin and delta bilirubin is large. A preferred example of the operation for quantifying conjugated bilirubin in the present invention will be described below. The sample containing bilirubin, the buffer and bilirubin oxidase are mixed at 25-45°C, particularly preferably at about
Incubate at 37°C and remove 450n of reaction solution.
Measure the decrease in absorbance at m. The blank test is performed in the same manner as above except that water or the buffer used to dissolve bilirubin oxidase is used instead of bilirubin oxidase. The calibration curve is created by using standard solutions of conjugated bilirubin at various concentrations as samples and performing the same procedure as above. As a standard solution for conjugated bilirubin, it is convenient to use a solution of ditaurobilirubin dissolved in pooled serum with a direct bilirubin value of 0.1 mg/dl or less measured by the diazo method. When the reaction reaches equilibrium, the value of conjugated bilirubin in the sample is determined by comparing the difference in absorbance between the sample and blank reaction solution with a calibration curve. The amount of bilirubin oxidase used in the present invention is preferably 0.3 to 10 units per 0.5 ml of sample. The present invention will be explained in detail below with reference to Examples.
However, the reagents used in Examples, the preparation of bilirubin samples, and the definition of bilirubin oxidase activity are as follows. For human serum albumin, a reagent manufactured by Sigma was used. As bilirubin oxidase, a product manufactured by Amano Pharmaceutical Co., Ltd. was used. Conjugated bilirubin is 1mM ditaurobilirubin disodium salt (manufactured by US Biochemical)
and 1mM human serum albumin were mixed at a ratio (volume ratio) of 1:4. A standard solution of conjugated bilirubin was prepared by dissolving ditaurobilirubin disodium salt in pooled serum whose direct bilirubin value measured by the diazo method was 0.1 mg/dl or less. Unconjugated bilirubin was prepared by mixing crystalline bilirubin (manufactured by Sigma) with human serum albumin in the same manner as in the preparation of conjugated bilirubin. Delta bilirubin was determined by the method of Lauff et al. [Clin.
Chem., 28 , 629-637, (1982)] from human serum. That is, 100 ml of serum from a patient with high direct bilirubin was prepared, 400 ml of methanol adjusted to pH 2.0 with phosphoric acid was added thereto, and the resulting precipitate of the globulin fraction was removed by centrifugation at -10°C. The obtained supernatant was concentrated to 1/5 volume using polyethylene glycol, and after dialysis against pure water,
Caffeine (37 g/), sodium benzoate (56 g/), disodium ethylenediaminetetraacetic acid (1 g/) and anhydrous sodium acetate (56 g/),
Add an albumin-dissociating agent consisting of 1.5 g/g/) to reversibly dissociate bilirubin bound to human serum albumin, and then pass this solution through a hollow fiber membrane (Asahi Kasei Co., Ltd. C5P type, 0.8 mmφ x 1.2 m) to release the bilirubin. removed bilirubin and other low molecular weight substances. Water was added to the obtained non-permeable fraction, and the above membrane filtration was further continued to sufficiently remove low molecular weight substances. The resulting aqueous solution was subjected to high performance liquid chromatography for separation, and a delta bilirubin fraction was collected, dialyzed, and freeze-dried to obtain a delta bilirubin sample. The operation of high performance liquid chromatography is
Nucleosil 300−7C18 column (10φ×250mm; M・
Using a chromatography device (Model LC-4A, manufactured by Shimadzu Corporation) equipped with a chromatography system (manufactured by Nagel), methanol (PH 2.0): dimethyl sulfoxide: water: 1 mol/tetra n-butylammonium hydrogen sulfate (40 :19:40:1) to (80:
This was carried out using a reversed phase partition method using a gradient of 19:0:1). The unit of bilirubin oxidase is 0.02mg/ml
of crystalline bilirubin and 0.2M Tris-HCl buffer (PH8.4) containing 1mM disodium ethylenediaminetetraacetic acid at 37°C and measuring the decrease in absorbance at 440nm for 1 minute. The amount of enzyme that oxidizes 1 micromole of bilirubin was defined as 1 unit. Example 1 1.6 ml of 0.1 M glycine-sodium hydroxide buffer or 0.1 M universal buffer with various pH values, 0.05 ml each of the above-mentioned conjugated bilirubin, unconjugated bilirubin, or delta bilirubin solution as a sample, and 5 mM Tris-HCl buffer. 15 units/ml bilirubin oxidase solution dissolved in liquid (PH8.0)
0.1 ml of each was mixed and incubated at 37° C. for 5 minutes, and the decrease in absorbance at 450 nm was measured. Blank test is 5m instead of enzyme solution.
The same procedure as above was performed using M Tris-HCl buffer (PH8.0). Figure 1 shows the relationship between the difference in absorbance between the obtained sample and the blank reaction solution, that is, the reactivity (relative value) of bilirubin oxidase, the pH of the buffer used, and its type. As can be seen from Figure 1, bilirubin oxidase has maximum activity against conjugated bilirubin near PH4-5 and around PH10, while its activity against unconjugated bilirubin has a maximum around PH5-6; Significantly lower activity than type bilirubin. Activity against delta bilirubin is PH
The lower the activity, the higher the activity, but it is still lower than the activity against unconjugated bilirubin. In the end, it was found that the reactivity of bilirubin oxidase to both unconjugated bilirubin and delta bilirubin reached a minimum around pH 10, and that conjugated bilirubin could be specifically measured if bilirubin was measured around this pH. Regarding the type of buffer solution, a glycine-sodium hydroxide buffer is preferable because its reactivity with conjugated bilirubin is greater than that with unconjugated bilirubin and delta bilirubin. Example 2 In Example 1, the buffer solutions shown in Table 1 were used as buffer solutions.
The same procedure was performed except that 0.1M various buffer solutions (PH10.0) were used. The obtained measured values (relative values) are shown in Table 1. In the same table, Bc, Bu, and Bδ represent conjugated, unconjugated, and delta bilirubin, respectively, and (Bu/Bc) and (Bδ/Bc) are the measured values of unconjugated and delta bilirubin relative to the measured values of conjugated bilirubin. Each represents the ratio (%) of [Table] As can be seen from Table 1, glycine at pH 10.0 -
It can be seen that when sodium hydroxide buffer or Tris-sodium hydroxide buffer is used, the specific crossover of conjugated bilirubin and delta bilirubin with respect to conjugated bilirubin is 2% or less. Even when other buffers were used, the crossover of unconjugated bilirubin to conjugated bilirubin was about 7% or less, and the crossover of delta bilirubin was about 4% or less. Example 3 0.1M glycine-sodium hydroxide buffer (PH
10.0) 1.6 ml, 0.05 ml each of five human serum samples and a solution of 15 units/ml bilirubin oxidase dissolved in 5 mM Tris-HCl buffer (PH 8.0)
After mixing 0.1 ml and incubating for 5 minutes at 37°C, the decrease in absorbance at 450 nm was measured.
The concentration of conjugated bilirubin in the sample was determined by comparing the obtained measured values with a calibration curve prepared in advance in the same manner as above using a standard solution of conjugated bilirubin as a sample. On the other hand, for reference, using the same serum samples as above, quantification of total bilirubin and direct bilirubin by a known diazo method, quantification of total bilirubin by an enzymatic method (bilirubin oxidase method), and high performance liquid chromatography (HELC).
Conjugated, unconjugated, and delta bilirubin were determined by the method. Quantification of total bilirubin by bilirubin oxidase is 0.1%
The test was carried out by allowing the enzyme to act on the sample in 0.1M Tris-HCl buffer (PH8.6) containing sodium dodecyl sulfate, and measuring the decrease in absorbance at 45 nm. Quantification by HPLC is performed using a 4.6φ
The same method was used except that a ×250 mm column was used. Table 2 shows each measurement value obtained by the above method. The conjugated bilirubin value obtained by the method of the present invention is determined by HPLC.
It can be seen that this agrees well with that of the law. In addition, the direct bilirubin value determined by the diazo method is determined by HPLC.
It can be seen that the sum of the conjugated form and delta bilirubin is approximated by [Table] Effects of the Invention According to the present invention, it has become possible to separately quantify only conjugated bilirubin among the bilirubin of biological fluid insects. The method of the present invention can be easily carried out using a visible photometer, and does not require expensive equipment or complicated operations unlike conventional high performance liquid chromatography methods.

[Brief explanation of the drawing]

Figure 1 shows the reactivity of bilirubin oxidase to conjugated, unconjugated, and delta bilirubin.
It is a drawing showing the relationship between pH and the type of buffer solution used.

Claims (1)

[Claims] 1. A method characterized in that bilirubin oxidase is allowed to act on a sample containing bilirubin in a buffer solution with a pH range of 9 to 11 in the co-presence of an anionic surfactant, and the resulting change in absorbance is measured. A method for quantifying conjugated bilirubin. 2 The buffer solution is glycine-sodium hydroxide buffer, Tris-sodium hydroxide buffer, borax-sodium hydroxide buffer, ethanolamine-sodium hydroxide buffer, universal buffer, and carbonate-sodium hydroxide buffer. The method for quantifying conjugated bilirubin according to claim 1, wherein the buffer is selected from the group consisting of: