JP4183484B2 - Fractionated measurement of conjugated bilirubin - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for differential measurement of conjugated bilirubin in which conjugated bilirubin is enzymatically measured in bilirubin.
[0002]
[Prior art]
Bilirubin is the most abundant pigment in bile, made mainly from hemoglobin produced by waste red blood cells. In serum, conjugated bilirubin mainly bound to glucuronic acid and unconjugated bilirubin and δ-bilirubin bound to albumin Exists as. A combination of conjugated bilirubin, unconjugated bilirubin and δ-bilirubin is called total bilirubin.
Since it is possible to know the differential diagnosis of liver dysfunction and the degree of liver dysfunction from the abundance of conjugated bilirubin and unconjugated bilirubin in serum, the differential quantification of these bilirubins occupies an important position in clinical diagnosis. ing. In particular, conjugated bilirubin is considered to be a prognostic indicator of liver dysfunction, cholestasis, and those diseases.
[0003]
Conventionally, as such a bilirubin measurement method, a chemical measurement method such as a colorimetric method using a diazo reagent (for example, see Non-Patent Document 1) has been mainly used.
However, in these chemical measurement methods, diglucuronide form of conjugated bilirubin reacts quickly, but monoglucuronide form reacts slowly, so accurate measurement of conjugated bilirubin is not possible unless the reaction time is specified accurately. There was a problem.
[0004]
On the other hand, Raul et al. Reported an analysis method of bilirubin in serum by high performance liquid chromatography (HPLC) (see, for example, Non-Patent Document 2).
According to this report, serum bilirubin was mainly divided into 4 fractions of α to δ, and it was revealed that bilirubin in the δ position, that is, δ-bilirubin, is bilirubin covalently bound to albumin. .
Thereafter, many groups proposed a method for quantifying bilirubin by HPLC (see, for example, Non-Patent Documents 3, 4, and 5).
However, this HPLC method has many problems such as the inability to measure a large amount of sample at a time and the high running cost.
[0005]
Furthermore, as a method for quantifying conjugated bilirubin using an enzyme, a measurement method using bilirubin oxidase derived from Myrothecium (see, for example, Patent Document 1) or bilirubin oxidase acts in a pH range of 3.5 to 4.5. The method (for example, refer patent document 2) etc. which are made to measure is reported, and all were excellent in the convenience compared with the conventional diazo method.
[0006]
However, according to the follow-up test of the present inventor, among the enzyme quantification methods, there is a problem that the reaction of unconjugated bilirubin and δ-bilirubin is recognized in the former and lacks accuracy, and the latter is a reaction of δ-bilirubin. Is recognized and lacks accuracy, and a long reaction time is required, making it difficult to automate the measurement.
[0007]
For this reason, this inventor mix | blended aniline, a pentacyano iron complex salt, and a cobalt (II) complex as a reaction accelerator with bilirubin oxidase, and set the measurement pH to 2.5-4.0. A conjugated bilirubin colorimetric method and reagent by an enzymatic method that can be measured selectively and rapidly have already been proposed (see, for example, Patent Document 3).
[0008]
[Patent Document 1]
JP 59-17999 A [Patent Document 2]
JP 59-125899 A [Patent Document 3]
Japanese Patent Laid-Open No. 3-175998
[Non-Patent Document 1]
Izumi Kanai, “Proposal for Clinical Laboratory Methods”, 1978, Kanbara Publishing, p.XII-24
[Non-Patent Document 2]
John J. Lauff, et.al.``J. Chromatogr '', 1981, 226, p.391-402
[Non-Patent Document 3]
Ohnishi S, et.al.``Biochem.J '' 1980, 190, p.527-532
[Non-Patent Document 4]
Nakamura H, et.al. "Analytical Chemistry" 1987, 36, p.352-355
[Non-Patent Document 5]
Adachi Y, et.al. "Clin. Chem." 1988, 34, 385-388
[0010]
[Problems to be Solved by the Invention]
However, in the measurement method using the enzyme, as in the case of using a diazo reagent, the bilirubin is not sufficiently fractionated, so that the reaction with respect to the conjugated bilirubin is not complete, or not only the conjugated bilirubin but also the non-conjugated. There was a problem that it also reacted with a part of bilirubin and δ-bilirubin.
Moreover, in order to improve the fractionation property of the conjugated bilirubin, it is necessary to perform the reaction in a low pH range (pH 2.5 to 4.0), which is not preferable for the analyzer.
[0011]
Therefore, the present invention has been made paying attention to such conventional problems, and can accurately measure only conjugated bilirubin without reacting with unconjugated bilirubin and δ-bilirubin in a sample containing bilirubin. The object is to provide a method for differential measurement of conjugated bilirubin.
[0012]
[Means for Solving the Problems]
As a result of intensive studies to solve the above problems, the present inventors have found that at least one selected from the group consisting of hydroquinones, 6-hydroxy-chromans, and 5-hydroxybenzo-2,3-dihydrofurans. By coexisting, the inventors have found that only conjugated bilirubin can be accurately measured even under mild conditions of pH 4.5 to 7.0, and the present invention has been completed.
[0013]
That is, the present invention has the following configuration.
(1) In a method for measuring the amount of conjugated bilirubin in a specimen by allowing bilirubin oxidase to act on the specimen containing bilirubin and optically measuring the change in bilirubin caused thereby, hydroquinones, 6-hydroxy- A method for differential measurement of conjugated bilirubin in a specimen, wherein at least one selected from the group consisting of chromans and 5-hydroxybenzo-2,3-dihydrofurans coexists.
[0014]
(2) Hydroquinones are hydroquinone, methylhydroquinone, methoxyhydroquinone, 2,3-dimethylhydroquinone, chlorohydroquinone, bromohydroquinone, 3 ', 8'-dihydroxybenzonorbornane, 2,5-dihydroxybenzoic acid, 2,5-dihydroxy Fractionation of conjugated bilirubin in a specimen according to (1), which is at least one selected from the group consisting of benzenesulfonic acid, 2,5-dihydroxy-4′-methyldiphenyl, and 1,2,4-trihydroxybenzene Measurement method.
[0015]
(3) The method for differential measurement of conjugated bilirubin in a specimen according to (1), wherein the 6-hydroxy-chromans are 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid and / or tocopherol .
[0016]
(4) At least one concentration selected from the group consisting of hydroquinones, 6-hydroxy-chromans, and 5-hydroxybenzo-2,3-dihydrofurans is in the range of 0.001 to 10 mM (1 ) To (3) The method for differential measurement of conjugated bilirubin in the specimen.
[0017]
(5) The method for differential measurement of conjugated bilirubin in a specimen according to (1) to (4), wherein the reaction is performed in a pH range of 4.5 to 7.0.
[0018]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in more detail.
In the measurement of bilirubin by an enzymatic method, it is known that when only conjugated bilirubin is to be measured, measurement is performed by changing the pH of the specimen (Akira Kosaka, Clin. Biochem., 20, 451-458; 1987). In other words, unconjugated bilirubin and δ-bilirubin have lower enzyme reactivity than conjugated bilirubin and are less susceptible to enzymatic reaction in a low pH range. For example, when reacted near pH 3.5, conjugated bilirubin reacts preferentially. To come. When the pH is further lowered, unconjugated bilirubin and δ-bilirubin are hardly affected. On the other hand, the optimum pH of bilirubin oxidase is 5 to 7, and the enzyme activity is significantly reduced. It takes a long time or the conjugated bilirubin is not oxidized enough. Moreover, there are similar characteristics even at pH 8-10, and satisfactory measurement results cannot be obtained.
[0019]
The present invention uses at least one selected from the group consisting of hydroquinones, 6-hydroxy-chromans, and 5-hydroxybenzo-2,3-dihydrofurans as a fractionating agent for conjugated bilirubin. It solves the problem.
This fractionating agent has an effect that conjugated bilirubin can react in a pH region where the enzyme activity of bilirubin oxidase is the highest, and further selectively inhibits the reaction of unconjugated bilirubin and δ-bilirubin.
[0020]
The hydroquinones used as a fractionating agent for conjugated bilirubin in the present invention can be appropriately selected from known ones. Specific examples thereof include hydroquinone, methylhydroquinone, methoxyhydroquinone, 2,3-dimethylhydroquinone, chlorohydroquinone, bromohydroquinone, 3 ′, 8′-dihydroxybenzonorbornane, 2,5-dihydroxybenzoic acid, 2,5-dihydroxy. Examples include at least one selected from the group consisting of benzenesulfonic acid, 2,5-dihydroxy-4′-methyldiphenyl, and 1,2,4-trihydroxybenzene.
[0021]
Further, 6-hydroxy-chromans may be appropriately selected from known ones, but 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid and / or tocopherol are particularly preferable.
[0022]
The concentration of the conjugated bilirubin fractionating agent is preferably in the range of 0.001 to 10 mM, more preferably in the range of 0.01 to 0.5 mM.
When the concentration of the fractionating agent is less than 0.001 mM, the reaction of unconjugated bilirubin and δ-bilirubin cannot be selectively inhibited. Therefore, only the conjugated bilirubin cannot be accurately measured, and conversely, when it exceeds 10 mM, The enzyme activity of bilirubin oxidase is inhibited.
[0023]
Since the optimum pH of bilirubin oxidase is 5.0 to 7.0, preferably 5.0 to 6.5, it is necessary to keep the pH of the reaction solution constant during the measurement. For this reason, when measuring a conjugated bilirubin by an enzyme reaction generally, a buffer solution is used.
The buffer that can be used in the present invention is not particularly limited as long as the pH can be maintained in the above range, and can be appropriately selected from known buffer solutions. An acid buffer or the like is preferable.
In the present invention, the reaction can be performed under a mild condition of pH 4.5 to 7.0, which is preferable for the analyzer.
[0024]
In the present invention, the sample for measuring conjugated bilirubin is generally an aqueous sample containing bilirubin, and examples thereof include serum, plasma, bile and the like. These specimens may be diluted or pretreated as necessary.
In addition, the bilirubin oxidase used in the present invention may be any enzyme that is generally used as bilirubin oxidase, but in view of availability and ease of handling, those derived from the genus Miloseces are preferable. The concentration of this bilirubin oxidase during the reaction is 0.01 to 10 units / mL, preferably 0.1 to 2 units / mL.
[0025]
The present invention relates to a buffer solution (first reagent) containing at least one selected from the group consisting of hydroquinones, 6-hydroxy-chromans, and 5-hydroxybenzo-2,3-dihydrofurans, bilirubin oxidase A reagent for measuring conjugated bilirubin consisting of a solution (second reagent) can be provided.
Hydroquinones and 6-hydroxy-chromans may be appropriately selected from those described above.
[0026]
The measurement of the conjugated bilirubin according to the present invention is carried out by using a buffer solution containing at least one selected from the group consisting of the above hydroquinones, 6-hydroxy-chromans, and 5-hydroxybenzo-2,3-dihydrofurans (first Reagent) is added, and after preheating, a bilirubin oxidase solution (second reagent) is added and reacted, and the resulting decrease in conjugated bilirubin is measured optically.
[0027]
Moreover, the effect as a fractionating agent such as hydroquinones can be further enhanced by adding histidine or arginine. In the present invention, although not particularly required, a reaction accelerator such as benzoic acid, a surfactant, a protein such as albumin, and the like can be appropriately added as necessary.
[0028]
【The invention's effect】
The present invention provides a sample containing bilirubin by coexisting at least one selected from the group consisting of hydroquinones, 6-hydroxy-chromans, and 5-hydroxybenzo-2,3-dihydrofurans. It is possible to provide a differential measurement method for conjugated bilirubin capable of accurately measuring only conjugated bilirubin without reacting with unconjugated bilirubin and δ-bilirubin.
Therefore, according to the present invention, since conjugated bilirubin in a sample can be measured with high accuracy, liver function and hematopoietic functional disease can be diagnosed with high accuracy.
[0029]
Further, the present invention can be said to be preferable for an analyzer because only conjugated bilirubin can be accurately measured even under mild conditions of pH 4.5 to 7.0.
[0030]
【Example】
EXAMPLES Hereinafter, although this invention is demonstrated further in detail based on an Example, this invention is not limited by this.
[0031]
Example 1 Inhibitory effect of unconjugated bilirubin reaction 200 mM 2-morpholinoethanesulfonic acid (MES) buffer (first solution containing 50 μM various hydroquinones, 0.05% Triton X-305, and 100 mM sodium salicylate Reagent: pH 5.50) To 0.24 mL, 0.008 mL of unconjugated bilirubin (interference check BIL-F diluted to 15 mg / dL: manufactured by Kokusai Reagent Co., Ltd.) was added, and after preheating at 37 ° C. for 5 minutes, bilirubin Hitachi 7170 automatic analyzer at 37 ° C. with addition of 0.06 mL of 10 mM N-cyclohexyl-3-aminopropanesulfonic acid (CAPS: pH 9.0) buffer (second reagent) containing oxidase 2.0 U / mL Was used to measure the change in absorbance over time at a wavelength of 450 nm.
Moreover, except not containing hydroquinones, it measured by the method completely same as the above, and compared with the said measured value as control. As a result, as shown in FIG. 1, according to the fractionated measurement method of conjugated bilirubin of the present invention, it can be seen that the unconjugated bilirubin is hardly oxidized until a certain time has elapsed after the addition of the second reagent. The time during which oxidation of unconjugated bilirubin can be prevented can be controlled by the type and concentration of hydroquinone.
In addition, in 1,2,4-trihydroxybenzene, although the raise of an absorbance is seen, it turns out that the reaction of unconjugated bilirubin is suppressed also in this case.
[0032]
Example 2 Influence on Conjugated Bilirubin Reaction Other than using ditaurobilirubin sample (interference check BIL-C diluted to 10 mg / dL: conjugated bilirubin manufactured by Kokusai Reagent Co., Ltd.) instead of unconjugated bilirubin sample in Example 1 Measured the change in absorbance over time in exactly the same manner as in Example 1. As a result, as shown in FIG. 2, it can be seen that the oxidation reaction of the conjugated bilirubin is very rapid, and there is almost no influence of hydroquinones.
From the results of Example 1 and Example 2, it can be seen that hydroquinones specifically inhibit only the oxidation reaction of unconjugated bilirubin, so that only conjugated bilirubin can be measured separately.
[0033]
Example 3 Reactivity of Various Bilirubins in Reagents 0.45 mL of the first reagent using hydroquinone as hydroquinones in Example 1 and 0.015 mL of serum sample were preheated at 37 ° C. for 5 minutes and then used in Example 1. Add 0.112 mL of the second reagent, further preheat at 37 ° C. for 5 minutes, add 0.03 mL of 3 g / dL sodium fluoride aqueous solution and 0.01 mL of 10 g / dL ascorbic acid aqueous solution, .1 mL was subjected to HPLC.
HPLC was added so that sodium fluoride was 0.05 g / dL and ascorbic acid was 0.2 g / dL in eluent-1, and Licrospher 100RP-18 (manufactured by Merck) was used as a column. Other than using the Tosoh 8020 analyzer as the apparatus, the procedure was performed according to the method of Raul et al.
The first reagent and the serum sample were preheated at 37 ° C. for 5 minutes, and then an aqueous sodium fluoride solution and an ascorbic acid aqueous solution were added. Then, the second reagent was added, and various solutions in the solution when the bilirubin oxidase reaction was suppressed were added. Bilirubin was used as a control.
As a result, as shown in FIG. 3, the peak corresponding to diglucuronide bilirubin and monoglucuronide bilirubin disappears due to the bilirubin oxidase reaction with the reagent of the present invention, and the peak corresponding to unconjugated bilirubin and δ-bilirubin is It can be seen that 100% and 96% remain, respectively.
From this, it was shown that only the conjugated bilirubin can be separately measured by the reagent according to the present invention.
[Brief description of the drawings]
FIG. 1 is a graph showing the inhibitory effect of unconjugated bilirubin reaction.
FIG. 2 is a graph showing the effect on conjugated bilirubin response.
FIG. 3 is a graph showing reactivity to various bilirubins before and after a reagent reaction.

Claims (3)

In a method for measuring the amount of conjugated bilirubin in the specimen by allowing bilirubin oxidase to act on the specimen containing bilirubin and optically measuring the change in bilirubin caused thereby, hydroquinone, methylhydroquinone, methoxyhydroquinone, 2, 3-dimethylhydroquinone, chlorohydroquinone, bromohydroquinone, 2,5-dihydroxybenzoic acid, 2,5-dihydroxybenzenesulfonic acid, 2,5-dihydroxy-4'-methyldiphenyl, and 1,2,4-trihydroxybenzene A method for fractionated measurement of conjugated bilirubin in a specimen, wherein at least one selected from the group consisting of: Hydroquinone, methylhydroquinone, methoxyhydroquinone, 2,3-dimethylhydroquinone, chlorohydroquinone, bromohydroquinone, 2,5-dihydroxybenzoic acid, 2,5-dihydroxybenzenesulfonic acid, 2,5-dihydroxy-4'-methyldiphenyl, and separating and measuring process of conjugation bilirubin in a specimen according to claim 1, wherein a range of at least one concentration of 0.001~10mM selected from the group consisting of 1,2,4-trihydroxybenzene. The method for differential measurement of conjugated bilirubin in a specimen according to claim 1 or 2 , wherein the reaction is performed in the range of pH 4.5 to 7.0.

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