JPS6322262B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a reagent for direct bilirubin measurement. There are two types of bilirubin in body fluids: indirect and direct. Indirect bilirubin is unconjugated bilirubin (free bilirubin), and direct bilirubin is conjugated bilirubin, in which the propionic acid in the side chain of free bilirubin is glucuronic acid. It is a glucuronide (monoglucuronide, diglucuronide, etc.) that has an ester bond with. Total bilirubin is a general term for direct bilirubin and indirect bilirubin. The blood bilirubin concentration and its fractions (direct bilirubin and indirect bilirubin) are measured in clinical tests during various pathological conditions of bilirubin in body fluids.
The main components are bilirubin and urobilin bodies in urine and urobilin bodies in feces, and among these, the dynamics of bilirubin in blood is the most important and has important significance in the classification and differentiation of various types of jaundice. In addition, in recent years, analysis of bilirubin in amniotic fluid has become important for determining the severity of fetal hemolytic disease due to Rh incompatibility and for countermeasures. The measurement method using the diazotization reaction of bilirubin is
It was invented by HIJMANS VAN DEN BERGH and others in the 1910s, and MALLOY, EVE in the 1930s.
Since it was established as a quantitative method by LYN, JENDRASSIK, GROF, and others, many improved methods have been proposed, but methods that utilize this measurement principle still dominate clinical analysis. This classical measurement method is a method in which diazonium salts are prepared by reacting sulfanilic acid with nitrous acid. And this improvement method 1
As one, KULHANEK et al.
There is a method using a stabilized diazonium salt proposed by ERTHINGHAUSEN et al. This improved method is an excellent method in that the reagent can be prepared simply by dissolving the stabilized diazonium salt powder prepared in advance in a buffer solution. However, this improved method measures only total bilirubin and does not directly measure bilirubin. Here, the stabilized diazonium salt is a diazo compound and a naphthalene sulfonate, a double salt with zinc chloride, a fluoroborate, etc., which are separated in a solid state. On the other hand, as a fractional measurement method for total bilirubin and direct bilirubin, Abe et al. have proposed a method using one of the stabilized diazonium salts, p-sulfobenzenediazonium and 1,5-naphthalenedisulfonate. However, this direct bilirubin measuring method using 3.4 mmol of the stabilized diazonium salt has the disadvantage that indirect bilirubin develops a strong color, giving an excessively high measured value as the direct bilirubin value. The inventors of the present invention have made extensive studies in order to improve these drawbacks, and have found that by adjusting the concentration of the stabilizing diazonium salt to a specific concentration for the reagent, the direct reaction of indirect bilirubin can be prevented. The present invention has been achieved based on the discovery that this can be suppressed to an extent that does not occur. That is, the present invention provides a direct bilirubin measurement reagent containing p-sulfobenzenediazonium-1,5-naphthalenedisulfonate and hydrochloric acid, in which p-sulfobenzenediazonium-1,5-naphthalenedisulfonate is included in the measurement reagent. This reagent for direct bilirubin measurement is characterized in that it contains 0.05 to 1.0 mmol per bilirubin. In addition to being able to accurately measure direct bilirubin by suppressing the direct bilirubin reaction of indirect bilirubin to a non-problematic level, the present invention can also be performed using conventional methods, such as the MALLOY-EVELYN method using alcohol as a coupling reagent, and the method using caffeine as a coupling reagent. -JENDRASSIK using benzoic acid as a coupling reagent and adding Fehling's solution after diazo color development to compare the color as alkaline azobilirubin-GROF
In the methods and their improved methods, the concentration of the diazonium salt is determined by the amount of sodium nitrite at the time of adjustment, and is usually about 1.8 to 2.9 mmol/millimol/m; in contrast, in the present invention, the stabilized diazonium salt is It becomes possible to measure by concentration. usually,
In clinical analysis, total bilirubin and direct bilirubin in serum are generally determined in the range of 0.1 to 40 mg/dl, and the reagent of the present invention can satisfactorily measure amounts in this range. The reagent for direct bilirubin measurement of the present invention contains a stabilized diazonium salt and hydrochloric acid. Hydrochloric acid is preferably in the range of 0.01 to 0.10 normal. The stabilized p-sulfobenzenediazonium chloride used in the present invention is p-sulfobenzenediazonium-1,5-naphthalenedisulfonic acid salt stabilized with 1,5-naphthalenedisulfonic acid. The present invention is characterized in that the concentration of the stabilized diazonium salt is 0.05 to 1.0 mmol per reagent for direct bilirubin measurement. In this concentration range, the influence of indirect bilirubin on the direct bilirubin reaction can be suppressed. If it is less than 0.05 mmol/, direct bilirubin cannot be measured, and if it exceeds 1.0 mmol/, the influence of indirect bilirubin will be large and accurate direct bilirubin cannot be measured. Reference example: Preparation of indirect bilirubin solution EDTA・Na ₂ 0.1g, anhydrous sodium carbonate 1.06g
was dissolved in 800 ml of distilled water, 0.2 g of crystalline bilirubin was dissolved, and then 55 g of human serum albumin was dissolved, and distilled water was added to make a constant volume of 1000 ml. 3 of this liquid
Each ml was dispensed into vials, freeze-dried in vacuum, sealed tightly, and stored in a cool, dark place. Freeze-dried product at the time of use
ml of distilled water. Example 1 Reactivity of indirect bilirubin in direct bilirubin measurement reagent As a direct bilirubin measurement reaction reagent, various amounts of p shown in Table 1 were added to a 0.025N hydrochloric acid aqueous solution.
It was prepared by dissolving -sulfobenzenediazonium-1,5-naphthalenedisulfonate (hereinafter abbreviated as NDDS). Indirect bilirubin solution prepared in reference example 50μ
3 ml of the above reaction reagent was added, heated at 37°C for 15 minutes, and absorbance at a wavelength of 560 nm was measured. The absorbance difference ΔODd with respect to the reagent blank was determined. On the other hand, as a reaction reagent for measuring total bilirubin, 0.1
In an aqueous solution 1 containing normal hydrochloric acid and 30% dimethyl sulfoxide, various amounts of
Prepared by dissolving NDDS. Add this reaction reagent 3 to 50μ of the indirect bilirubin solution prepared in Reference Example 1.
ml was added, heated at 37°C for 15 minutes, and absorbance at a wavelength of 560 nm was measured. The absorbance difference ΔODt with respect to the reagent blank was determined. Direct reaction rate of indirect bilirubin is △ODd／△ODt
It is expressed by The results are shown in Table 1.

[Table] As is clear from Table 1, the NDDS concentration is approximately 1.0
If it exceeds mmol/millimol/millimol/m, the direct reaction of indirect bilirubin will be more than 30%, which is not preferable. Example 2 Reactivity of direct bilirubin direct reaction A reaction reagent for direct bilirubin measurement was prepared by dissolving 0.15 mmol of NDDS (and 0.02 mmol as a reference example) in 1 part of a 0.01 N hydrochloric acid aqueous solution. A sample (direct bilirubin value 39.3 mg/dl measured by the MALLOY-EVELYN method) was diluted with distilled water to create the dilution series shown in Table 2. Add 3 ml of the above direct bilirubin measurement reagent to 50μ of this diluted sample, heat it at 37°C for 5 minutes, and then
Absorbance at 560 nm was measured. The results are shown in Table 2.

[Table] As is clear from Table 2, the absorbance changes linearly with the degree of dilution. On the other hand, the reference example with a low NDDS concentration has a low degree of color development and poor linearity.

Claims (1)

[Claims] 1 p-sulfobenzenediazonium-1,5-
A reagent for direct bilibin measurement containing naphthalene disulfonate and hydrochloric acid, characterized in that p-sulfobenzenediazonium-1,5-naphthalene disulfonate is contained in a proportion of 0.05 to 1.0 mmol/based on the measurement reagent. A reagent for direct bilirubin measurement.