JPS5810385B2 - Tetrazolium salt compound and spectrophotometric determination of dehydrogenase using the compound - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel tetrazolium salt compound that yields water-soluble formazan, and a method for spectrophotometric determination of dehydrogenase using the compound, and more particularly, general formula X represents a halogen atom. .

), and relates to a method for spectrophotometric determination of dehydrogenase using a monotetrazolium salt and its compound.

In the compound having the general formula (I) according to the present invention, the formazan produced by accepting hydrogen is water-soluble.

This is extremely useful in clinical testing and can be used for the same purposes as conventional tetrazolium salts.

To explain in detail, it is released by the action of various dehydrogenases, such as lactate dehydrogenase (hereinafter abbreviated as LDH), succinate membrane hydrogenase, and β-oxybutyrate dehydrogenase, which were conventionally quantified using tetrazolium salts. The compound of the present invention can accept hydrogen via an intermediate electron carrier.

Among these dehydrogenases, LDH is distributed in all body cells, and is particularly abundant in the cardiac muscle, liver, skeletal muscle, and kidney. It is known that serum LDH activity increases markedly in cases of diseases such as blastic anemia.

Therefore, by measuring LDH activity in blood, clinically extremely meaningful findings for diagnosis can be obtained.

Conventional 3,3'-(3,3'-dimethoxy-4,4'-biphenylene)-bisC2-(4-nitrophenyl)-5
-phenyl-2H tetrazolium chloride] and the like are commonly used as hydrogen acceptors for this purpose.

However, the formazan produced when this compound accepts hydrogen is completely insoluble in water, and in order to prevent formazan from precipitating and adhering to tubes, etc., especially in automatic analysis, it is highly desirable to have something that dissolves uniformly. 3-(4-iodophenyl)-2- where formazan is relatively easily soluble in water.
(4-nitrophenyl)-5-phenyl-2H tetrazolium chloride is used.

However, even this formazan dissolves in only a small amount, so there are many difficulties in measuring it spectrophotometrically.

The present inventors have conducted various studies on compounds that produce formazan with good solubility and stable measurement values, and have found that the compound having the general formula (I) is an excellent hydrogen acceptor. I discovered that.

In other words, the formazan produced from this compound has the extremely advantageous feature of being water-soluble, and the method using this compound has made it possible to obtain stable measured values without adhesion to automatic analyzers or precipitation. Ta.

The compound having the general formula (I) according to the present invention can be produced by a conventional method.

For example, pyridine-4-aldehyde and phenylhydrazine are reacted in an organic solvent.

Alcohols such as methanol, ethanol, and inbutanol are preferably used as the organic solvent.

The obtained reaction product was dissolved in an organic solvent and treated under basic conditions for 2
-Amino-4,5-dimethylthiazolediazonium chloride solution is added dropwise and stirred for several hours to obtain formazan.

In addition to alcohols such as methanol and ethanol, pyridine and dioxane are used as organic solvents, and sodium acetate, sodium hydroxide, potassium hydroxide and the like are used as basifying agents.

The obtained formazan is dissolved in an organic solvent and oxidized to obtain the desired product.

Organic solvents such as ethyl acetate, ethanol, and chloroform are used.

As the oxidizing agent, N-bromosuccinimide, N-chlorosuccinimide, N-iodosuccinimide, etc. are used.

Next, a method for spectrophotometric determination of dehydrogenase using a compound having general formula (I) will be explained.

An enzyme and a corresponding substrate are brought into contact in a buffer such as a phosphate buffer or a Tris-HCl buffer, and the liberated hydrogen is once accepted by nicotinamide adenine dinucleotide (hereinafter abbreviated as NAD) to form phenazine methosulfate. (Hereafter P
The tetrazolium salt accepts hydrogen via an intermediate electron carrier such as MS).

This produces water-soluble formazan, which is not found in conventional formazan, so the enzyme activity value can be accurately determined by directly measuring the absorbance of the reaction solution.

EXAMPLES Next, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples unless it exceeds the gist thereof.

In addition, 2- and 3-position isomer compounds of pyridine are also synthesized in the same manner as in Example 1 described below, and provide similar effects.

Synthesis of the compound represented by) When 76 g of phenylhydrazine is dissolved in 200 ml of methanol and 75 g of pyridine-4-aldehyde is added while stirring at room temperature, the reaction generates heat and pyridine-4-aldehyde phenylhydrazone 1351 is obtained. .

2-Amino-4,5-dimethylthiazole hydrochloride 171
was suspended in 180 ml of 4N hydrochloric acid, then cooled to around 0°C, and 7v of anhydrous sodium nitrite was added to 50 ml of water.
A solution dissolved in and cooled to around 0°C is added dropwise to diazotize, and the temperature is maintained at the same temperature.

Pyridine-4-aldehydophenylhydrazone 201 was dissolved in 600 ml of methanol and 120 ml of pyridine, and 120 g of anhydrous sodium acetate was added thereto.The mixture was cooled to 0 to 5 DEG C., and a diazonium salt hydrochloric acid solution was added dropwise while stirring.

After the dropwise addition was completed, stirring was continued for 3 hours, and a large amount of the precipitated formazan was collected.

The obtained formazan was suspended in 100 ml of water and after stirring,
Drain again and dry.

171 formazan is obtained. Elemental analysis value (%) C1
□H16N6S Infrared characteristic absorption (cfrL-') Thin layer chromatography Rf value (developing solvent; chloroform:methanol-2:1) 0.81 Melting point 160-1
61°C Visible absorption spectrum (Figure 2 - Compound A) Concentration = 1.
123X10-4 molar aqueous solution (containing 10% 3N hydrochloric acid), cell thickness = 1 cIrL, λmax - 538 nm, molar extinction coefficient -1,218X04 The obtained formazan 10SF was dissolved in 1 t of ethyl acetate with stirring, and N- Bromosuccinimide 81 was added little by little, and after stirring for 2 hours, the precipitated yellow precipitate was collected.

Wash with ethyl acetate and dry. Purification is carried out using ethanol-ether.

7.41 of 3-(4.5-dimethyl-2-thiazolyl)-2-phenyl-5-(4-pyridyl)-2H tetrazolium bromide are obtained.

Elemental analysis value (%) C1□H15N6SBr thin layer chromatography Rf value (developing solvent; methanol:benzene = 2:1) 0.304 Melting point 178-182°C (decomposition) Ultraviolet absorption spectra (Figure 2 - Compound B of the present invention) ) Concentration - 1,685 x 10' molar aqueous solution, cell thickness - 1c
IrL, λmax = 223 nm, molar extinction coefficient 1.685X10' Example 2 (Usage of the compound obtained in Example 1) Glycine-lactate sodium salt aqueous solution (glycine-0, 72%, di-lactic acid-2,
04%, pH=9.6), add 10.5 to 84 mU/ml of standard serum pre-warmed to the same temperature for 30 minutes and stir.

After another minute, add water to dissolve the 0.07% solution of the tetrazolium salt compound obtained in Example 1 (70~),
00mA! Add 1 ml of the closed solution) and stir to make a 1 minute diameter NAD-PMS solution (NAD=2%, PMS=0.2
%, bovine serum albumin fraction 5 = 0.15%, pH = 7.
Add 1 ml of the phosphate buffer solution in step 4), stir, and keep at the same temperature for exactly 8 minutes.

Add 5 ml of 3N hydrochloric acid solution and stir to stop the enzyme reaction, and measure the absorbance at 538 nm.

As shown in Figure 1, good linearity was obtained as a result, and the enzyme activity of unknown serum could be quantified.

In the case of FIG. 1, purified water was used instead of serum as a control.

[Brief explanation of drawings]

FIG. 1 shows the relationship between the amount of enzyme and the absorbance due to formazan produced by the enzyme reaction in Example 2. Figure 2 shows compounds A and B (
The absorption spectrum of the compound of the present invention) is shown.

Claims (1)

[Claims] 1. General formula X represents a halogen atom. ) monotetrazolium salt. 2 The general formula represents a halogen atom. ) Spectrophotometric determination of dehydrogenase using monotetrazolium salt.