JPH0743387B2 - Direct determination of bilirubin - Google Patents

Description

TECHNICAL FIELD The present invention relates to a method for directly quantifying bilirubin in a biological fluid sample, particularly blood or urine.

[Conventional technology]

Bilirubin is the most abundant pigment in bile,
Serum is present in the serum in the amount of about 1 mg / dl or less in the case of a normal person, but it may reach up to 20 mg / dl in a pathological condition such as jaundice disease. Therefore, the quantification is useful for diagnosis of jaundice disease and the like.

Bilirubin exists in serum as direct bilirubin bound to glucuronic acid, sulfuric acid, etc. and indirect bilirubin bound to albumin. In clinical tests, total bilirubin, direct bilirubin, and indirect bilirubin must be quantified in order to diagnose the abnormal bilirubin metabolism in more detail by the mechanism of jaundice.

As a method for quantifying bilirubin, a method for colorimetrically quantifying the dye of azobilirubin produced by the reaction of bilirubin with a diazo reagent [Practical clinical chemistry; edited by Kitamura Motoko, 10 bilirubin (197
4 years)) and other chemical methods, enzymatic methods, etc. are known.
Recently, copper ions and metal complexes have been found to oxidize bilirubin, and a method for quantifying total bilirubin using this reaction has been reported (JP-A-59-160764).
issue).

[Problems to be solved by the invention]

However, in the most commonly practiced conventional quantification method using a diazo reagent, the diazo reagent itself is unstable and the usage period after preparation is short. There was a problem that the quantitative operation was extremely complicated. Further, in the method of oxidizing bilirubin with copper ions, there is a problem that it is impossible to distinguish between direct bilirubin and indirect bilirubin.
Further, in the enzymatic method, there are problems such as stability and high cost due to the large amount of use since the measurement is performed at a pH far from the optimum pH of the enzyme.

[Means for solving problems]

In view of such circumstances, the present inventors have conducted earnest research to develop a simple and accurate method for quantifying bilirubin, particularly direct bilirubin, and as a result, when copper ion and thiourea or a derivative thereof were added to a test solution, direct bilirubin was obtained. The present invention has been completed by finding out that oxidase is selectively oxidized and can be easily quantified.

That is, the present invention provides a direct method for quantifying bilirubin, which comprises reacting a test solution with copper ions and thiourea or a derivative thereof, and optically measuring the change of bilirubin in the test solution. It is a thing.

In the method of the present invention, copper ions refer to Cu ²⁺ , and for example, copper salts such as copper sulfate, cupric chloride, cupric acetate, cupric phosphate, etc. are preferably supplied as the copper ion-generating compound.

The thiourea or its derivative is represented by the following general formula (I) (Wherein R ₁ , R ₂ , R ₃ and R ₄ each represent a hydrogen atom or an organic group), especially thiourea (R ₁ = R ₂ = R ₃ = R ₄ =
H) or a thiourea derivative in which R _{1 to} R ₄ have an organic group that is not an amino group or an imino group is preferred. Thiourea, N-methylthiourea, 1,3-dimethylthiourea or allylthiourea are particularly preferable.

In order to carry out the method of the present invention, the absorption of a test solution in the vicinity of 450 nm is measured in advance, and the copper salt and thiourea or a derivative thereof are added to the test solution to directly add bilirubin in the test solution. After completion of the reaction with copper ions and thiourea or its derivative, the absorption of the reaction solution near 450 nm is measured, and the bilirubin concentration is directly determined from the difference in absorption before and after the reaction.

The present invention is based on the fact that copper ions form a complex with thiourea or a derivative thereof, and that this complex has a function of directly oxidizing bilirubin selectively. Therefore, in order to allow the copper salt to exist as copper ions in the reaction solution,
The pH of the reaction solution is preferably on the acidic side, particularly 6 or less. In order to adjust the pH, it is preferable to add thiourea or its derivative added to a buffer solution. Further, a surfactant, an acid or the like can be further added to this buffer solution.

The order of adding the copper salt and thiourea or its derivative is
There is no particular limitation. When thiourea or a derivative thereof and a copper salt are added in this order, for example, thiourea or a derivative thereof may be prepared in advance as a buffer solution reagent 1 and a copper salt aqueous solution as a reagent 2. In this case, the quantitative determination is performed by comparing the absorbance of the test liquid after adding the reagent 1 with that after adding the reagent 2.

The addition amount of the copper salt is preferably 0.05 to 100 mM, particularly preferably 0.25 to 5 mM as the final concentration of copper ions in the reaction solution. The amount of thiourea or its derivative added is 1.5 mM to 3 as a final concentration.
M, especially 7.5 to 150 mM is preferred.

Further, the method of the present invention, the optical change of the sample over time, and an automatic analyzer capable of quantitatively measuring, for example Hitachi 705, Hitachi 7050, Hitachi 736, Toshiba TBA-80S, Toshiba TBA. It can also be applied to −480 mag.

[Operation and effect of the invention]

The method of the present invention is an assay method using a reagent that is extremely simple, has good reagent stability, and is inexpensive as compared with the conventional assay method for bilirubin. Further, it is possible to directly measure only bilirubin by directly subjecting a sample containing both direct bilirubin and indirect bilirubin, for example, serum to the method of the present invention, which is useful in clinical examination.

〔Example〕

 Next, the present invention will be described in detail with reference to examples.

Example 1 Reagents 1 and 2 shown below were prepared.

Reagent 1: 0.1M citric acid 3Na-lactic acid pH 3.6 1% acetic acid 0.3% Surfactant 50mM thiourea Reagent 2: 7.5mM copper sulfate Reagent 1 Add 0.1ml of the sample to 2.5ml of the sample, then add the final sample blank at 450nm. Correct the amount and measure. Subsequently, 0.5 ml of Reagent 2 is added, the mixture is heated to 37 ° C., and the absorption at 450 nm is measured again. The bilirubin concentration is measured by the degree of absorption decrease before and after the addition of the reagent 2. The above operation was performed by an automatic analyzer (Toshiba TBA-480). FIG. 1 shows the time-dependent change in absorption at 450 nm when ditaurobilirubin (direct bilirubin) was used as a sample. The results obtained when indirect bilirubin was used as the sample are shown in FIG.

As a result, according to the method of the present invention, direct bilirubin can be selectively measured with almost no change in indirect bilirubin (only change due to change in liquid volume).

Example 2 Ditaurobilirubin and indirect bilirubin were used as specimens in a ratio of 1: 1.
The bilirubin measurement was performed in the same manner as in Example 1 except that the stored serum contained at a ratio of 1 was used, and the sample was diluted with a 0.9% NaCl aqueous solution to show the relationship between the dilution rate and the change in absorption (444 nm / 548 nm). investigated. As a result, as shown in FIG. 3, a good linearity of almost the origin regression is shown, which shows that the method of the present invention is excellent as a clinical examination means.

Example 3 Using 50 serum samples as samples, the method of the present invention (described in Example 1), alkaline azo blue method (Bilirubin Kitt K, Nippon Shoji Co., Ltd.) enzymatic method (Nescort D-BL-V _E ,
The concentration of bilirubin was directly quantified by Nippon Shoji Co., Ltd., and the correlation between these measurement methods was examined. As a result, as shown in FIGS. 4 and 5, the method of the present invention showed good correlation with both the conventional alkali azo blue method and the enzyme method.

[Brief description of drawings]

FIG. 1 is a drawing showing the time course of absorption (444 nm / 548 nm) in the method of the present invention when ditaurobilirubin and indirect bilirubin were used as specimens, respectively. FIG. 3 is a drawing showing the relationship between the change in absorption at 444 nm / 548 nm and the dilution rate of the sample in Example 2. FIG. 4 is a drawing showing the correlation between the method of the present invention and the alkaline azo blue method, and FIG. 5 is a drawing showing the correlation between the method of the present invention and the enzymatic method.

Claims (4)

[Claims] 1. A direct method for quantifying bilirubin, which comprises allowing a test solution to act on copper ions and thiourea or a derivative thereof and optically measuring the change in bilirubin in the test solution. 2. The method for quantifying direct bilirubin according to claim 1, wherein the copper ion-forming compound is copper sulfate, cupric chloride, cupric acetate or cupric phosphate. 3. A thiourea or its derivative is represented by the following general formula (I): The method for quantifying direct bilirubin according to claim 1, which is a compound represented by the formula: wherein R ₁ , R ₂ , R ₃ and R ₄ each represent a hydrogen atom or an organic group. 4. The method for quantifying direct bilirubin according to claim 1, wherein the reaction of copper ion and thiourea or its derivative with bilirubin is carried out under the condition of pH 6 or less.