JP2590124B2 - Water-soluble tetrazolium compound and method for measuring reducing substance using the compound - Google Patents

Description

The present invention relates to a novel water-soluble tetrazolium compound and a method for measuring a reducing substance using the compound, and more particularly, to 2,3,5-triphenyl. The present invention relates to a water-soluble tetrazolium compound having sulfamoylethanesulfonic acid in the phenyl group of -2H-tetrazolium, and a method for measuring a reducing substance using the same.

Tetrazolium compounds can be used to measure the activity of dehydrogenase, thereby measuring the substrate and the substrate that is the target of the oxidase that produces superoxide ions, that is, the quantification of additives in food, or the clinical analysis of biological fluid components. It has been put to practical use as a reagent.

(B) Conventional technology Since the water-soluble tetrazolium compound is a stable water-soluble compound, the formazan generated when the compound is reduced is
By quantifying this formazan, it is used for measuring a reducing substance in an aqueous solution.

Conventional tetrazolium compounds include, for example, 2- (4
-Phenyl iodide) -3- (4-nitrophenyl) -5
-Phenyl-2H-tetrazolium salt (INT), 3- (4,5
-Dimethylthiazolyl-2) -2,5-diphenyl-2H-
Tetrazolium salt (MTT), 2,2 ', 5,5'-tetrakis (4-nitrophenyl) -3,3'-(3,3'-dimethoxy-4,4'-diphenylene) -2H, 2'H -Ditetrazolium salt (NTB), 2,2'-p-diphenylene-3,3 ', 5,5'
-Tetophenyl-2H, 2'H-ditetrazolium salt (Neo-
TB) is typically used. However, these tetrazolium salts and their reduced forms, formazan, are hardly soluble in water, and the use of a large amount of surfactants or organic solvents is unavoidable at present. In particular, in the field of clinical examination, formazan produced causes pigmentation at the photometric site of the measuring instrument, which significantly affects the test results. In addition, with the rapid progress in automation of inspections in recent years, erroneous measurement due to contamination of an automatic analyzer has been caused.

For the reasons described above, even in the case of a test item in which it is preferable to use a tetrazolium salt, it has been unavoidably forced to use another measurement principle.

To deal with such a situation, Japanese Patent Publication No. 56-38154,
Nos. 56-61366, JP-A-56-61367, Japanese Pharmaceutical Association 102nd Annual Meeting Proceedings, 341 page 4K2-4, etc., as disclosed in the phenyl group substituted on the tetrazole ring, sulfone directly Attempts have been made to introduce side chains containing acid or carboxylic acid groups or quaternary ammonium salts.

However, these tetrazolium compounds do not form formazan within the optimum pH range where the enzyme function essential for actual measurement is exhibited due to these acid groups directly introduced into the phenyl group for the purpose of solubilization. It is difficult to apply in the field of clinical examination. Also, the quaternary ammonium salt-introduced compound is insufficient in water solubility.

Recently, as disclosed in JP-A-59-106476, a method of containing two water-soluble groups (sulfonic acid groups and / or carboxylic acid groups) that do not directly bond to a phenyl group has also been attempted.

However, formazan, which is a reduced form of the tetrazolium compound proposed here, has a remarkable fluctuation in the molecular extinction coefficient due to a change in pH.

To stop the enzymatic reaction, it is common in clinical tests to shift the pH of the reaction solution to an acid or alkali.However, when using the tetrazolium compound proposed here, there is a disadvantage that the measurement sensitivity is significantly reduced. Have.

The present inventors have studied to solve such disadvantages of the prior art, and have one sulfonic acid group via a sulfamoyl group in the phenyl group of 2,3,5-triphenyl-2H-tetrazolium. A water-soluble tetrazolium compound and a method for measuring a reducing substance using the same have been invented, and a patent application has already been filed (Japanese Patent Application Laid-Open No. 61-1).
48169).

 A typical example is represented by the following formula (A).

The tetrazolium compound (A) has sufficient water solubility in alkalinity, and its reducing substance, formazan compound, shows good water solubility in the whole pH range. Has been evaluated. However, this tetrazolium compound (A) is slightly soluble in water and under acidic conditions, and thus may not be sufficiently compatible with acidic conditions in clinical tests.

(C) Problems to be solved by the present invention In order to solve such conventional drawbacks, the present inventors require that the tetrazolium compound be easily soluble in water, and that it undergoes a small pH dependency when subjected to reduction. In particular, when used in a clinical test, it is specifically acted on by reduced nicotinamide amide adenine dinucleotide (hereinafter abbreviated as NADH) or reduced nicotinamide amide dinucleotide phosphate (hereinafter abbreviated as NADPH). The present inventors have focused on that the formed formazan compound must be easily soluble in water, and the like, and as a result, have completed the present invention.

That is, a first object of the present invention is to provide a novel water-soluble tetrazolium compound which forms a water-soluble colored formazan by a reducing substance in the presence of an electron transfer agent.

A second object of the present invention is to provide a method for measuring a reducing substance in an aqueous solution using a novel tetrazolium compound.

Another object of the present invention is to provide a measurement method which is particularly advantageous for NADH and NADPH in a biological body fluid, and which is a method for measuring a reducing substance which is less affected by a change in pH of an aqueous solution and does not contaminate a measuring instrument.

(D) Means for solving the problem The novel water-soluble tetrazolium compound of the present invention is represented by the general formula (1).

Wherein R ₁ and R ₂ are hydrogen, lower alkyl group, lower alkoxy group, halogen, cyano group or nitro group, R ₃ is hydrogen, lower alkyl group, lower alkoxy group or halogen, R ₄ is hydrogen or lower alkyl Group, R ₅ is a lower alkylene group, Y is hydrogen, a lower alkyl group, a lower alkoxy group,
Indicates a halogen or nitro group. ].

The sulfonic acid group forms an inner salt with the tetrazole ring.

The tetrazolium compound of the present invention can be easily produced by known raw materials and methods.

For example, a formula (2) obtained by reducing a condensate of m-tonilobenzenesulfonyl chloride and 2-aminoethanesulfonic acid: Is converted into a hydrazine compound by a conventional method and then condensed with, for example, benzaldehyde to obtain a compound of the formula (3): A phenylhydrazone compound represented by is obtained.

To this, for example, a diazo compound of p-nitroaniline is coupled by a conventional method to obtain a compound of the formula (4): Is obtained.

If the obtained formazan compound is oxidized by a conventional method,
Target tetrazolium compound (5): Can be obtained.

The method for preparing the tetrazolium compound of the present invention is described in detail in Examples below.

The formazan compound in the present invention, such as alcohol,
Since it is dissolved in an organic solvent having a high hydrophilicity, the oxidation is extremely smooth, and the tetrazolium salt compound obtained by the oxidation has practically sufficient water solubility in water at all pH ranges.

A second gist of the present invention resides in a method for measuring a reducing substance in an aqueous solution, comprising using the water-soluble tetrazolium compound represented by the general formula (1).

The tetrazolium compound (1) is a reducing substance, for example,
Acts as a hydrogen receptor for NADH or NADPH. By measuring the degree of color development that is proportional to the amount of formazan that is quantitatively generated during the reduction, and the absorbance thereof, the amount of the reducing substance NADH or NADPH can be measured. Such a measuring method is extremely useful for measuring the activities of oxidase and dehydrogenase and for quantifying the substrate, that is, for quantifying biological components and additives in foods.

An example of these principles for measuring the activity of lactate dehydrogenase (LDH) is as follows: As a result of these reactions, the activity of LDH can be measured by measuring the concentration of formazan that is quantitatively generated and measuring the absorbance.

Next, the measurement of bile acid in a biological component is as follows: In this way, bile acids can be quantified.

The measurement of a biological component using a dehydrogenase with respect to the measurement of cholesterol is as follows: Similarly, cholesterol can be quantified.

Furthermore, creatine phosphate kinase (CPK) in biological components
The following is shown: (NADP: oxidized form of NADPH HK: hexokise ADP: adenosine-2-phosphate ATP: adenosine-3-phosphate G-6-PDH: glucose-6-phosphate dehydrogenase) It is possible.

There are various other methods for the application of tetrazolium, which are demonstrated by the examples below.

In the actual measurement, in a suitable medium such as a trisphosphate buffer, the test solution contains an enzyme such as an oxidase and a dehydrogenase specific to the substrate to be quantified and the general formula (1)
The reaction is allowed to proceed by incubating, and the absorbance of formazan, which produces color, is measured, and the reducing substance and the substrate in the test solution are quantified.

In particular, the combined use of a surfactant as an additive aid has an effect of shifting the maximum absorption of formazan coloration to a longer wavelength side, and further increases color sensitivity, which is a preferred embodiment in the measurement method of the present invention. . As the surfactant, a polyoxyethylene derivative of an aliphatic or aromatic alcohol is used, and its polymerization degree is generally about 5 to 30. Generally, commercially available nonionic surfactants can be used without inconvenience.

The pH of the test solution during the measurement is usually preferably in the neutral range of 7.0 to 8.0.However, in the measurement method of the present invention, the change in color sensitivity is small even in the range of pH 3.0 to 1.5, and the change is not changed in the acidic range. small.

(E) Action and effect The tetrazolium compound represented by the general formula (1) and the reduced formazane thereof of the present invention can suppress an excessive increase in water solubility by having a substituted sulfamoyl group having an ethanesulfonic acid group, Easy to manufacture and purify.

In addition, the absorption waveform near the neutrality of the formazan obtained by reduction has a longer wavelength (about 50
0 nm), the color component in the body fluid is clearly distinguished from the vicinity of 450 nm, and a measurement error hardly occurs.

In addition, since the pH dependence when the tetrazolium compound of the present invention undergoes reduction is extremely small, the measurement results have good reproducibility,
Moreover, high sensitivity can be obtained.

When measuring the substrate or enzyme activity using these tetrazolium compounds in the field of clinical testing, various reducing substances coexisting with biological components also reduce the tetrazolium compounds at the same time, resulting in a positive error in the measurement results. Become. In order to eliminate this effect, the sample (biological component) is pretreated with a weak oxidizing agent such as potassium iodate to remove reducing substances, and then the substrate or enzyme activity is measured. Compound is NADH or NA
It has an action specific to DPH and can obtain accurate measurement information without being affected by various reducing substances or pretreatment agents coexisting with biological components.

(F) Examples Hereinafter, the present invention will be described in detail with reference to Examples and Comparative Examples, but the present invention is not limited to these Examples.

Example 1 N (p-hydrazinophenylsulfonyl) -2-aminoethanesulfonic acid (29.5 g) was dissolved in water (700 ml), benzaldehyde (10.8 g) was added, the mixture was stirred at 40 to 45 ° C for 2 hours, and then salted out to give a hydrazone compound. I got 75% yield. After dissolving 20 g of the obtained hydrazone compound in 200 ml of water containing 8 g of caustic soda, 125 g of soda ash was added to adjust the temperature to 15 to 20 ° C.

8.6 g of 2-chloro-4-nitroaniline in 70 g of 90% acetic acid
And diazotized by adding dropwise 16 g of nitrosylsulfuric acid at 10 to 15 ° C. The obtained diazo compound solution was added dropwise to the previously prepared hydrazone compound slurry at 15 to 20 ° C., and the mixture was stirred for 4 hours. The resulting crystals of the formazan compound were filtered and then purified with methanol to obtain 20.6 g of a formazan compound (Na salt). 70% yield.

Next, in a hot water bath, 5 g of the above formazan compound was dissolved in a mixed solution of 50 g of acetonitrile and 10 g of water, and 90% at 70 to 75 ° C.
5 g of acetic acid and 5 g of ammonium persulfate were added, and the mixture was stirred at the same temperature for about 2 hours until the oxidation was completed. Activated carbon was added and the mixture was filtered, and acetonitrile was distilled off from the filtrate under reduced pressure. A small amount of water was added to the residue for crystallization, and the residue was filtered, washed with water and dried to obtain 3.6 g of a yellowish white powder.

This product is a tetrazolium compound represented by the formula (6) having an infrared absorption curve as shown in FIG. 1 and the following elemental analysis values.

Elemental analysis (%) Calculated for CHNS: 44.64 3.01 19.84 14.88 Analytical value: 44.61 2.98 19.86 14.92 Example 2 In Example 1, 2-chloro-4-nitroaniline 8.
The same procedure was repeated using 8.6 g of 2-methoxy-5-nitroaniline instead of 6 g to obtain the formazan compound 22.0.
g was obtained. Yield 74.8%.

In a hot water bath, 5 g of the above formazan compound was dissolved in 75 g of dimethylformamide, 2.5 g of glacial acetic acid was added, 10 g of electrolytic manganese dioxide was added at room temperature for about 30 minutes, and the mixture was stirred for about 5 minutes, and then filtered. The agent was added and filtered, and the filter cake was washed with a small amount of dimethylformamide and methanol. The obtained solution of the tetrazolium compound was decolorized and filtered with activated carbon, and concentrated under reduced pressure. Dissolve the residue by adding methanol,
The mixture was again decolorized and filtered with activated carbon, and the filtrate was concentrated, cooled, crystallized, and filtered. Drying yielded 3.7 g of a pale yellow powder.

This product is a tetrazolium compound represented by the formula (7) having an infrared absorption curve and the following elemental analysis value as shown in FIG.

CHNS calculated: 44.64 3.01 19.84 14.88 Analytical: 44.58 2.99 19.87 14.81 Thereafter, the tetrazolium compounds shown in the following Examples 3, 4 and 5 were obtained in the same manner. FIGS. 3, 4 and 5 show the respective infrared absorption spectra.

(Example 3) (Example 4) (Example 5) Examples 6 to 10 and Comparative Examples 1 and 2 The following tests were performed to evaluate the water solubility of the tetrazolium compound of the present invention.

Use a 0.2 M Na ₂ HPO ₄ solution and a 0.1 M citric acid solution to adjust the pH to 3.
0 to 6.0 of the buffer, also was prepared buffer pH6.0~8.0 using 0.1M-KH ₂ PO ₄ solution and 1N-NaOH.

To these buffers, the tetrazolium compounds prepared in Examples 1 to 5 were added and dissolved to a final concentration of 1 mM, and the liquid was observed. Table 1 shows the results. NT at the same time
The water solubility of B and the tetrazolium compound represented by the formula (A) was similarly examined, and the results are shown in Table 1 as a comparative example.

As can be seen from the results in Table 1, NTB has poor water solubility in each pH range, and the tetrazolium compound of formula (A)
It becomes soluble from about 7.5, but is insufficient in the acidic range. On the other hand, the tetrazolium compound of the present invention is excellent in water solubility in all pH ranges.

Further, 10 mg of the tetrazolium compound shown in Table 1 was added to 0.05 N-
Dissolve in 2 ml of NaOH, add an appropriate amount of ascorbic acid, 37 ° C
At room temperature for 30 minutes to form each formazan. 0.1 ml of the formazan was added to 5 ml of a 1% to 10% saline solution prepared in advance. Indicated.

From these results, although NTB is poor in water solubility of formazan,
It shows that the water solubility of the tetrazolium reduced formazan of the present invention is also good.

Example 11 2.5 ml of a 0.1 M phosphate buffer (pH 7.5) containing 0.2 mM of the tetrazolium compound obtained in Example 1, 0.4% of Triton X-405, 2 mM of NAD and 2 U / ml of diaphorase is taken. On the other hand, in each case, 2.5 ml of a buffer having the same composition without adding Triton X-405 is used. 50 μl of 2.5 mM aqueous solution of NADH was added to each solution, and the solution was heated at 37 ° C. for 5 minutes, and the absorbance was measured. Each result is shown in FIG.

As shown in FIG. 6, when a surfactant is used in combination, the maximum wavelength of the compound of Example 1 shifts from 495 nm to 505 nm, the absorbance increases 1.2 times or more, and the color sensitivity increases.

Example 12 Next, as an example in a clinical test using the tetrazolium compound of the present invention, lactate dehydrogenase (LDH) in serum
An example of the activity measurement is given below.

1.0 mM tetrazolium compound obtained in Example 1, Triton X-405: 0.4%, NAD 6.5 mM, diaphorase 10 U / ml,
Take 0.5 ml of 0.1 M Tris buffer (pH 8.2) containing 0.5 M lactic acid. To this, add 20 μl of human serum as a sample, and
After allowing the reaction to warm for 10 minutes, the reaction is stopped by adding 1.0 ml of 0.1N-HC, and the absorbance at a wavelength of 510 nm is measured. At this time, a blank is taken using purified water instead of human serum. This is converted into the activity of LDH from a calibration curve prepared using a standard solution in advance.

According to this method, there is no aggregation of the reaction solution, and there is no problem in the measurement by the automatic analyzer.

The correlation between this method and a commercially available kit using NTB as a tetrazolium compound (Tetraform LDH, manufactured by Kokusai Reagent) using 20 human sera was found to be in good correlation with a correlation coefficient of 0.988.

Example 13 Further, as an example in a clinical test, an example of measurement of bile acid in serum will be described.

1.0 mM tetrazolium compound obtained in Example 1, NAD
2.0 mM, diaphorase 10 U / ml, oxamic acid 10 mM, 3α
-Hydroxysteroid dehydrogenase (3α-HSD) 0.1 U /
Take 0.8 ml of a 0.02 M phosphate buffer (pH 7.0) containing 0.1 ml. After adding 0.2 ml of human serum as a sample and heating at 37 ° C. for 10 minutes to react, Triton X-405: 0.1N-HC containing 0.5%
The reaction is stopped by adding 0.5 ml, and the absorbance at a wavelength of 510 nm is measured. At this time, a reaction solution from which 3α-HSD was removed and which was similarly measured was taken as a blank. This is converted into a bile acid amount from a calibration curve prepared using a standard solution in advance.

According to this method, in addition to the advantage that the reaction solution has no aggregation as in Example 12, the tetrazolium compound according to the present invention has a high molecular extinction coefficient, so that trace components such as bile acids in human serum can be measured with high sensitivity. can do.

The correlation between this method and a commercially available kit using NTB as a tetrazolium compound (Embazail Daiichi Kagaku) by 60 human sera was found to have a good correlation with a correlation coefficient of 0.992.

[Brief description of the drawings]

1 to 5 are diagrams showing an infrared absorption spectrum of the tetrazolium compound obtained in Examples 1 to 5, and FIG. 6 is a diagram showing an absorption spectrum of a visible portion obtained in Example 11; The case where X-405 is included indicates the case where Triton X-405 is not included.

Claims (6)

(57) [Claims] 1. The general formula (1) Wherein R ₁ and R ₂ are hydrogen, lower alkyl group, lower alkoxy group, halogen, cyano group or nitro group, R ₃ is hydrogen, lower alkyl group, lower alkoxy group or halogen, R ₄ is hydrogen or lower alkyl Group, R ₅ is a lower alkylene group, Y is hydrogen, a lower alkyl group, a lower alkoxy group,
Indicates a halogen or nitro group. ] The novel water-soluble tetrazolium compound represented by these. 2. The general formula (1) Wherein R ₁ and R ₂ are hydrogen, lower alkyl group, lower alkoxy group, halogen, cyano group or nitro group, R ₃ is hydrogen, lower alkyl group, lower alkoxy group or halogen, R ₄ is hydrogen or lower alkyl Group, R ₅ is a lower alkylene group, Y is hydrogen, a lower alkyl group, a lower alkoxy group,
Indicates a halogen or nitro group. ] A method for measuring a reducing substance in an aqueous solution, comprising using a water-soluble tetrazolium compound represented by the following formula: 3. The method according to claim 2, wherein the reducing substance is reduced nicotinamide amide adenine dinucleotide or reduced nicotinamide amide dinucleotide phosphate. 4. The method according to claim 2, wherein a tetrazolium compound is used in combination with a surfactant. 5. The method according to claim 2, wherein the amount of the component in the aqueous solution is determined by measuring the reducing substance in the aqueous solution.
5. The measuring method according to any one of Items 4 to 4. 6. The method according to claim 5, wherein the component in the aqueous solution is a component in a biological body fluid.