JPS59107264A - Measuring method of total bilirubin in blood - Google Patents

Abstract

PURPOSE:To enable exact measurement in a measuring method of total bilirubin using a stabilized diazonium salt by using diphiline as a reaction accelerator and using an org. acid for a buffer system. CONSTITUTION:A stabilized diazomium salt which is heretofore used is usable and the amt. of the diphiline to be used as a reaction accelerator is preferably 2-10g/dl, more preferably 4g/dl. The larger amt. results in much blank reaction and the smaller amt. in retarded acceleration of reaction. A preferred org. acid has strong buffer power at around 3pH and the acid having about 2-5pKa is preferable. Citric acid, succinic acid, maleic acid, tartaric acid, acetic acid and their salt and other org. acid are used. The pH of the buffer system is preferably 2-4, and around 3pH is best preferred. Excessively high pH results in poor stability and excessively low pH results in poor reactivity.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an accurate and efficient method for measuring total bilirubin in blood.

Bilirubin is produced by the destruction of old red blood cells within the reticulum, and some bilirubin is produced directly from heme proteins without going through the bone marrow. Throat rirubin in the living body is
It is classified into indirect bilirubin and direct bilirubin.

Indirect bilirubin is bilirubin itself, and is extremely poorly soluble in water, but is made water-soluble by binding to albumin in the blood. On the other hand, direct bilirubin, also called conjugated bilirubin, is bilirubin mainly bound to glucuronic acid, and has a structure that is easily soluble in water. Direct bilirubin is measured by direct reaction with a diazo reagent. Total bilirubin is measured by adding an organic solvent such as guiphylline, sodium benzoate-caffeine, surfactant, ethylene glycol, or dimethyl sulfoxide as an accelerator. Total bilirubin includes indirect bilirubin and direct bilirubin.

In this bilirubin measurement method, the classical Eve l
As an alternative to the yn-Malloy method, Jendrassik method, and alkaline azobilirubin method, the stabilized diazonium salt method, which has good reagent stability and is easy to operate, has recently come into use. The stabilized diazonium salts used in this method include 2.
4-dichlorophenylnaphthalene disulfonate, 2,
5-dichloride, lorphenyl tetrafluoroborate, and the like are known, and these are used as coloring agents for reaction with bilirubin.

Looking at the various stabilized diazonium bulk method reagents currently available on the market, they are superior in operability and measurement accuracy to classical methods when dealing with normal samples, but they produce extremely high values when used for artificial dialysis samples. There were many problems, and the drawback was that it could not be measured. This point was considered to be the only major problem when using stabilized diazonium salts. The cause of high values in artificial dialysis specimens is thought to be related to indican and its similar substances, which accumulate in the blood due to abnormal kidney function. It is true that the reactivity of artificial dialysis specimens is similar to that of indica and can. Bilirubin reaction takes several minutes to 5
The reaction is completed within minutes, whereas artificial dialysis samples show a slow reaction similar to indican. This is considered to suggest that the artificial dialysis specimen contains an indican-like substance.

The present inventor has arrived at the present invention as a result of intensive research aimed at eliminating the drawbacks of the stabilized diazonium salt method as described above.

The purpose of the present invention is to provide a measurement method that can accurately measure total bilirubin in an artificial dialysis sample using the stabilized diazonium salt method. The method for measuring total bilirubin is characterized by using guiphylline as a reaction accelerator and using an organic acid as a buffer system.

As the stabilizing diazonium salt, those conventionally used can be used; for example, those disclosed in Japanese Patent Application No. 57-64190
2,4-dichlorophenyldiazonium- shown in No.
1,5-naphthalene disulfonic acid and its salts (e.g., sodium salt, potassium salt, etc.), p-sulfanylbenzenediazonium-1,5-naphthalene sulfonic acid and its salts, varanitrobenzenediazonium tetrafluoroboric acid and its salts, Diazotized sulfanilic acid, tetrafluoroboric acid, salts thereof, and the like are used.

The amount of guiphylline used is preferably about 2 to 10 g/Lil, and optimally about 4 g/lu. If the amount is large, the blank reaction will increase, and if the amount is small, the promotion of the reaction will be weak.

As the organic acid, one with a strong buffering capacity at around pH 3 may be selected, and one with a pKa value of 2 to 5 is preferable. Specifically, citric acid and its salts, succinic acid and its salts,
Maleic acid and its salts, tartaric acid and its salts, acetic acid and its salts, and other organic acids are used, and the concentration used is preferably 5 to 2゜Ommol/#, and 10mmol/#.
I/I1. It is optimal.

In addition, p)( of the buffer system is preferably 2 to 4, and most preferably around pH 3.0. As the pH increases, the stability of the stabilized diazonium salt deteriorates, and as the ])H decreases, the reactivity with bilirubin decreases. Deteriorate.

Examples of the present invention are given below.

Example 1 Preparation of reagent 2. Sodium 4-dichlorophenyldiazonium-1,5-naphthalene disulfonate ----
-・-50 呵/dI Guifilin-Illustrated・-------・
-Machi------4g, #/citric acid---
-------・-Yu------1--Song-210■/
dIp)I was adjusted to 3.0.

Measurement method: 3 m of reagent and 0.1 mj of sample! was added and reacted at 37° C. for 20 minutes, and then measurements were performed at 540 nm to 570 nm.

Measurement item (1) Bilirubin (bilirubin 4M?J = substance'R12■
/dl) and indicane (potassium indoxyl sulfate, manufactured by Hanui Chemical Co., Ltd., lomg/dl) after the reaction. The results are shown in FIG.

(2) The amount of bilirubin was kept constant (5 mg/dI), and the amount of indican was varied and measured at 540 nm. The results are shown in Table 1.

(3) Test the original trial sample and the artificial dialysis sample at 540 nm and 57 nm.
Measured at 0 nm. The results are shown in Table 2. Example 2 Succinic acid 118.7 (U) was used instead of citric acid in Example 1.
A reagent was prepared using the same method, and the same measurement was performed for measurement item (1). The results are shown in Figure 2.

Ta.

Example 3 Maleic acid 98 mg/d instead of citric acid in Example 1
Preparation of comparative example reagent 2. Sodium 4-dichlorophenyldiazonium-1,5-naphthalene disulfonate −−−−−−−−−1−−5
0mg Brij35 (manufactured by Kao Atlas) - 4.5g
.

0, '05NHCl total amount 100rr+j! Closed.

Measurement items (1)' (2) and (3) were measured using the above reagents (for measurement item (3), 540
measurement in nm only). The results are shown in FIG. 4 and Tables 1 and 2.

Below is the margin in Table 1. Unit of each numerical value ■/dl Bilirubin amount 5ITf/dI From Table 1, it can be seen that according to the present invention, the influence of indican is clearly avoided compared to the comparative example (conventional method). .

In Table 2 in the margin below, W: Wavelength (nm), A: Genuine sample, B: Artificial dialysis sample, Unit of each measurement value: ■/dl, Amount used for each sample: Q, 1 mj! It is. The Eherin-Malloy method was performed based on quality, but since this method itself is well known, the details are omitted. From Table 2, it can be seen that for normal samples, there is almost no difference in the measured values with either method. However, it was found that the comparative example showed a significantly higher value in artificial dialysis samples. In the present invention, the measured values are in good agreement with those of the conventional Evelin Malloy method, and it can be seen that the present invention is improved compared to the conventional stabilized diazonium salt method as in the comparative example. In addition, in the present invention, the measurement wavelength is 540
It has been shown that there is less error at 570 rim than at 570 nm, which is due to less color cast in indican at 570 nm.

[Brief explanation of drawings]

FIGS. 1 to 4 are drawings showing absorption spectra of Examples 1 to 3 and Comparative Example, respectively.

Claims (5)

[Claims] (1) In the method for measuring total bilirubin using a stabilized diazonium salt, using guiphylline as a reaction accelerator,
A method for measuring total bilirubin in blood, characterized by using an organic acid in the buffer system. (2) The method according to claim 1, wherein the organic acid is citric acid and its salts, succinic acid and its salts, maleic acid and its salts, and tartaric acid and its salts. (3) The method according to claim 1 or 2, characterized in that 2 to 10 g/dl of guiphylline is used. (4) Any one of claims 1 to 3, characterized in that 5 to 200 mmar/l of organic acid is used.
The method described in section. (5) The method according to any one of claims 1 to 4, wherein the pH of the buffer system is 2 to 4.