JPH0853444A - New water-soluble tetrazolium salt compound - Google Patents

Abstract

PURPOSE:To provide a new water-soluble compound useful as a reagent for quantitative analysis of a dehydrogenase and a substrate. CONSTITUTION:This compound is expressed by formula I (R<1> is H or methoxy; R<2> is H, carboxyl or sulfonic acid). The compound is synthesized by oxidizing formazan of formula II with an oxidizing agent such as butyl nitrite in an alcohol solvent. Quantitative analysis of the concentration of a dehydrogenase is carried out by using this compound of formula I, reacting the dehydrogenase therewith and measuring the absorbance of the produced formazan. This compound improves water solubility of formazan, prevents precipitation onto a measuring equipment, has the maximum absorption band in a long wavelength region of 510 to 550nm and, therefore, is not interfered by a biological component such as bilirubin or hemoglobin. Accordingly, this compound is useful in clinical examination since it enables measurement by an autoanalyzer.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a water-soluble tetrazolium salt compound, and more particularly to a method for quantitatively analyzing a dehydrogenase and a substrate by the absorbance of formazan produced by reacting with a dehydrogenase.

Various dehydrogenases such as lactate dehydrogenase (hereinafter abbreviated as LDH), alcohol dehydrogenase, and glutamate dehydrogenase have conventionally been quantitatively analyzed using tetrazolium salt compounds.

That is, the tetrazolium salt compound receives hydrogen released by the action of these various dehydrogenases via the intermediate electron carrier to become formazan. The dehydrogenase can be quantified by measuring the absorbance of the formazan.

In particular, among these dehydrogenases, LDH is distributed in all somatic cells, especially in myocardium, liver, skeletal muscle, and kidney, and myocardial infarction, malignant tumor, liver disease, progressive muscle atrophy,
It is known that serum LDH activity is markedly increased in cases of diseases such as intravascular hemolysis and megaloblastic anemia. Therefore, by measuring LDH activity in blood, clinically,
Very meaningful findings for diagnosis can be obtained.

In recent years, there has been a demand for a method using a dehydrogenase which is less likely to be interfered with by biological components even in the measurement of uric acid and bile acids in blood.

Conventional hydrogen acceptors for this purpose are
3 '-[3,3'-Dimethoxy- (1,1'-biphenyl) -4,4'-diyl] -bis [2- (4-nitrophenyl) -5-phenyl-2H
Tetrazolium chloride] (hereinafter abbreviated as nitro TB)
Are generally used.

[0007]

However, the formazan formed by receiving hydrogen by the nitro TB is not soluble in water and is practically inconvenient. Particularly in the automatic analysis, there is a problem that the generated formazan adheres to the measuring system such as the tube and the cell. In order to eliminate these drawbacks, it has become necessary to use tetrazolium salts which produce water-soluble formazan.

In order to solve such problems, some water-soluble tetrazolium compounds have been proposed (JP-A-56-61366 and JP-A-56-61367). However, in each case, the maximum absorption wavelength of formazan is 400 to 500.
Since it is in nm and shorter than that of nitro TB formazan (550 nm), it was difficult to put into practical use due to interference of biological components such as bilirubin and hemoglobin.

[0009]

Means for Solving the Problems As a result of further research on a compound capable of forming a highly soluble formazan, the present inventors have found that a specific water-soluble tetrazolium compound different from the above is an excellent hydrogen acceptor. It was also found that the formazan produced from this is water-soluble, has a long-wavelength maximum absorption, and can be put to practical use without causing adhesion to an automatic analyzer or precipitation.

That is, such a compound has the general formula (1):

[Chemical 3] (In the formula, R ¹ is a hydrogen atom or a methoxy group, and R ² is a hydrogen atom, a carboxyl group or a sulfonic acid), and is a novel water-soluble tetrazolium salt compound.

The formazan derived from the above general formula (1) is
It has a maximum absorption at 510 to 550 nm, is less likely to be disturbed by biological components such as bilirubin and hemoglobin, and enables stable measurement, which is significant in clinical examination.

The compound of the general formula (1) of the present invention can be produced by a conventional method. For example, the following general formula (2)

[0013]

[Chemical 4] The hydrazinobenzothiazole represented by the general formula (3)

[0014]

[Chemical 5] By reacting an aldehyde represented by

[0015]

[Chemical 6] To obtain a hydrazone represented by the formula (1), and then react the corresponding diazonium salt under basic conditions in an organic solvent / water to give a compound of the general formula (5)

[0016] Obtain formazan represented by.

Here, sodium hydroxide, potassium hydroxide or the like is used as the basifying agent.

Then, the obtained formazan of the general formula (5) is oxidized in an alcohol solvent using an oxidizing agent such as butyl nitrite to obtain the tetrazolium salt compound of the general formula (1).

[0019]

EXAMPLES In the following examples, R ^{1 of the} general formula (1)
Is a methoxy group and R ² is a carboxyl group (compound a) and R ¹ is hydrogen and R ² is a carboxyl group (compound b), and reduced nicotinamide adenine dinucleotide (hereinafter abbreviated as NADH) is synthesized. However, the present invention is not limited to these examples of utilization.

Example 1 (Synthesis of Compound a) 25 g of p-formylbenzoic acid was heated under reflux in 200 ml of thionyl chloride for 2 hours. The reaction solution was concentrated and then crystallized with petroleum ether to obtain acid chloride in a yield of 90%. 10 g of taurine was dissolved in 200 ml of water, and 200 ml of chloroform was added thereto. This was cooled to 0 ° C., and a chloroform solution of acid chloride and a 2N aqueous sodium hydroxide solution were added dropwise with vigorous stirring so that the pH of the reaction solution was maintained at 7 to 8. After completion of the reaction, the aqueous phase was separated, adjusted to pH 2 by adding 2N hydrochloric acid, concentrated to dryness, and then crystallized by adding isopropanol to obtain an aldehyde in 100% yield.

5.9 g of 2-hydrazinobenzothiazole and 10 g of the aldehyde obtained above were mixed with methanol and heated under reflux for 4 hours. The precipitate formed is filtered off and the hydrazone is removed.
Obtained in a yield of 66%.

5 g of the obtained hydrazone was dissolved in 50 ml of water and 50 ml of N, N-dimethylformamide and cooled to 0 ° C. Separately, 4-amino-3-methoxybenzoic acid was diazotized by a conventional method and added to this hydrazone solution. An aqueous solution in which 2.1 g of NaOH was dissolved in 20 ml of water was added dropwise while maintaining the reaction mixture solution at -5 to 0 ° C, and after completion of the addition, the mixture was stirred overnight at room temperature.

Hydrochloric acid was added to the reaction mixture, and the resulting precipitate was collected by filtration. The obtained crude product was dissolved in an aqueous NaOH solution, hydrochloric acid was added to reprecipitate the product, and the product was purified to obtain formazan at a yield of 53%.

3.9 g of the obtained formazan was added to 150 m of methanol
The mixture was suspended in l, 13 ml of concentrated hydrochloric acid and 6.7 g of butyl nitrite were added, and the mixture was stirred overnight at room temperature. The deposited precipitate was collected by filtration, and the obtained crude product was recrystallized from methanol-water to obtain a tetrazolium salt (compound a) in a yield of 30%.

Example 2 (Synthesis of Compound b) In Example 1, 4-aminobenzoic acid was used in place of 4-amino-3-methoxybenzoic acid to obtain a compound b in which R ¹ was hydrogen.

The results of elemental analysis of the obtained compound are shown in Table 1.

[0027]

[Table 1]

Example 3 5 ml of 50 mM phosphate buffer (pH 8.0) containing 0.1 mM of compound a and 5 μM of 1-methoxy-5-methylphenazinium methosulfate (hereinafter abbreviated as 1-methoxyPMS)
In addition, 5 mM NADH was added to each of 0, 20, 40, 60, 80 and
After adding 100 μl and reacting at room temperature for 5 minutes, the absorbance was measured. The obtained absorption spectrum is shown in FIG. In FIG. 1, a to f are 0 to 100 of the above NADH addition amount, respectively.
Corresponds to μl.

The relationship between the NADH concentration and the absorbance at 550 nm is shown in FIG. A linear calibration curve passing through the origin was obtained between the NADH concentration and the absorbance.

[0030]

EFFECTS OF THE INVENTION By using the water-soluble tetrazolium compound of the present invention, the water solubility of the obtained formazan is improved, there is no deposition on measuring equipment, and the maximum absorption wavelength is a long wavelength, so that the interference of biological components is prevented. It is possible to measure with an automatic analyzer without receiving the measurement.

[Brief description of drawings]

FIG. 1 is an absorption spectrum of formazan produced by the reaction of a tetrazolium salt compound of the present invention with NADH.

FIG. 2 shows the relationship between the concentration of NADH and the absorbance of the produced formazan.

Claims (2)

[Claims] 1. A compound represented by the general formula (1): (In the formula, R ¹ is a hydrogen atom or a methoxy group, and R ² is a hydrogen atom, a carboxyl group or a sulfonic acid.) A novel water-soluble tetrazolium salt compound. 2. A tetrazolium salt compound represented by the general formula (1): Wherein R ¹ is a hydrogen atom or a methoxy group, and R ² is a hydrogen atom, a carboxyl group or a sulfonic acid, and a water-soluble tetrazolium salt compound is used. Quantification method.