JPH0413352B2 - - Google Patents

Description

[Detailed description of the invention]

(a) Industrial Application Field The present invention relates to a novel water-soluble ditetrazolium compound and a method for measuring reducing substances using the compound, and more specifically, 3,3'-(3,3'-dihalo-4, 4′-diphenylene)-bis(2,5-
The present invention relates to a water-soluble ditetrazolium compound characterized by having one sulfonic acid group via a sulfamoyl group to the phenyl group of (diphenyl-2H-tetrazolium), and a method for measuring a reducing substance using the same. Tetrazolium compounds can be used to measure the activity of dehydrogenases, their substrates, and the substrates that act on oxidases that produce superoxide ions, in other words, to quantify additives in foods, or clinically analyze biological fluid components. It has been put into practical use as a reagent. (B) Prior art Since the water-soluble tetrazolium compound is a stable water-soluble compound, the formazan produced when it is reduced is used to measure reducing substances in an aqueous solution by quantifying this formazan. Ru. 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'-tetraphenyl-2H,2'H-
Ditetrazolium salt (Neo-TB) is typically used. However, both of these tetrazolium salts and their reduced form, formazan, are sparingly soluble in water, and at present it is necessary to use a large amount of surfactant or organic solvent in combination. Particularly in the field of clinical testing, the generated formazan causes pigmentation in the photometric area of the measuring device, significantly affecting test results. Furthermore, with the rapid advancement of automation in testing in recent years, erroneous measurements may occur due to contamination of automatic analyzers. For the above reasons, even in the case of test items for which it is preferable to use a tetrazolium salt, it has been unavoidable to use other measurement principles. In response to this situation, Special Publication No. 56-38154,
Patent publications of JP-A-56-61366 and JP-A-56-61367, collection of lecture abstracts of the 102nd annual meeting of the Pharmaceutical Society of Japan, page 341, 4K2-
4 and others, attempts have been made to directly introduce a sulfonic acid group, a carboxylic acid group, or a side chain containing a quaternary ammonium salt into the phenyl group substituted on the tetrazole ring. However, due to these acid groups directly introduced into the phenyl group for the purpose of solubilization, within the optimal pH range where essential enzyme functions are exerted in actual measurements,
Since these tetrazolium compounds do not produce formazan, they are difficult to apply in the field of clinical testing.
Further, compounds into which quaternary ammonium salts are introduced are insufficient in terms of water solubility. Recently, as shown in JP-A-59-106476, attempts have been made to include two water-soluble groups (sulfonic acid groups and/or carboxylic acid groups) that are not directly bonded to the phenyl group. However, the molecular extinction coefficient of formazan, which is a reduced form of the tetrazolium compound proposed here, fluctuates significantly with changes in pH. In order to stop enzyme reactions, it is common in clinical tests to shift the pH of the reaction solution to acid or alkali, but when using the tetrazolium compound proposed here, it has the drawback of significantly reducing measurement sensitivity. have. (c) Problems to be Solved by the Invention The present inventors have found that in order to solve these conventional drawbacks, the tetrazolium compound should be easily water-soluble and should have low pH dependence when undergoing reduction. In particular, when used in clinical tests, it is specifically acted on by reduced nicotinamide adenine dinucleotide (hereinafter abbreviated as NADH) or reduced nicotinamide dinucleotide phosphate (hereinafter abbreviated as NADPH). As a result of their studies, they have completed the present invention, focusing on the fact that the produced formazan compound must be easily water-soluble. That is, the first object of the present invention is to provide a novel water-soluble tetrazolium compound that produces a readily water-soluble color-forming formazan in the presence of an electron transfer agent using a reducing substance. A second object of the present invention is to provide a method for measuring reducing substances and superoxide ions in an aqueous solution using a novel tetrazolium compound. Another purpose is to provide a method for measuring reducing substances that is specifically advantageous for NADH and NADPH in biological body fluids, is less affected by PH fluctuations in aqueous solutions, and does not contaminate measuring equipment. (d) Means for solving the problems The novel water-soluble ditetrazolium compound of the present invention is represented by general formula (1): [In the formula, R ₁ and R ₃ are hydrogen, R ₂ and R ₄ are the formula
(2) or (3) (In the formula, X and Y are SO ₂ NH, R ₇ is hydrogen, R ₆ and R ₈ are sulfonic acid groups.), P and Q are hydrogen, and Z is halogen. ] The sulfonic acid group forms an inner salt with the tetrazole ring. The tetrazolium compound of the present invention includes known raw materials,
It can be easily manufactured by this method. For example, the formula (4) obtained by reducing the condensate of m-tonylobenzenesulfonyl chloride and m-aminobenzenesulfonic acid If the amino compound of is converted into a hydrazine compound by a conventional method and then condensed with, for example, benzaldehyde, the formula (5) is obtained. A phenylhydrazone compound represented by is obtained. By coupling a tetrazo compound of 3,3'-dichlorobenzidine to this by a conventional method, formula (6) was obtained. A formazan compound represented by is obtained. By oxidizing the obtained formazan compound by a conventional method, the desired ditetrazolium compound (7) is obtained. can be obtained. The method for producing the ditetrazolium compound of the present invention is detailed in the Examples below. Since the diformazane compound used in the present invention is easily soluble in highly hydrophilic organic solvents such as alcohol, oxidation is extremely smooth, and the ditetrazolium salt compound obtained by oxidation is somewhat difficult to produce under acidic conditions due to intramolecular salt formation in water. It can be isolated with high purity as crystals of the solution, and relatively high purity quality can be obtained without complicated purification such as column purification. These tetrazolium compounds have sufficient water solubility for practical use by making them slightly alkaline in water. The second gist of the present invention resides in a method for measuring a reducing substance in an aqueous solution, which is characterized by using a water-soluble ditetrazolium compound represented by the general formula (1). The ditetrazolium compound (1) acts as a hydrogen acceptor for reducing substances, such as NADH or NADPH, or is reduced by superoxide ions. Measuring the amount of reducing substances NADH, NADPH, or superoxide ions by spectrophotometrically measuring the degree of color development that is proportional to the amount of formazan quantitatively produced during reduction. I can do it. Such a measurement method is extremely useful for measuring the activity of dehydrogenase and thereby quantifying substrates, that is, biological components, additives in foods, and the like. The following is an example of these principles when measuring the activity of lactate dehydrogenase (LDH): (NAD: Oxidized form of NADH) The activity of LDH can be measured by quantifying the concentration of formazan quantitatively produced as a result of these reactions by measuring absorbance. In addition, the measurement of biological components using dehydrogenase for cholesterol measurement is as follows: Similarly, cholesterol can be quantified. Furthermore, the following is shown regarding creatine phosphate kinase (CPK) in biological components: (NADP: oxidized form of NADPH HK: hexokase ADP: adenosine-2-phosphate ATP: adenosine-3-phosphate G-6-PDH: glucose-6-phosphate dehydrogenase) Therefore, CPK activity can be measured in the same way. is possible. There are various other methods for applying tetrazolium, which will be clarified by the examples below. In actual measurements, enzymes such as oxidases and dehydrogenases specific to the substrate to be quantified and enzymes expressed by general formula (1) are added to the test solution in an appropriate medium such as Tris phosphate buffer. A ditetrazolium compound is added thereto, the reaction is allowed to proceed by incubation, and the absorbance of the formazan produced is measured to quantify the reducing substance or superoxide ion, as well as the substrate, in the test solution. When quantifying superoxide ions, in order to further improve the practical measurement effect in terms of measurement time, sensitivity, quantitative performance, etc.,
Conventionally used phenols, thiophenols, amines, etc. can also be added. Furthermore, a chelating agent may be added to prevent autoxidation, which is an undesirable side reaction during measurement. These additive aids may be used alone or in combination as appropriate. In particular, the combined use of a surfactant as an additive auxiliary agent has the effect of shifting the maximum absorption of formazan coloring to the longer wavelength side, further increasing the coloring sensitivity, and is therefore a preferred embodiment in the measurement method of the present invention. .
As the surfactant, polyoxyethylene derivatives of aliphatic or aromatic alcohols are used, and the degree of polymerization thereof is generally about 5 to 30. Generally commercially available nonionic surfactants can be used without any inconvenience. The PH of the test liquid during measurement is normally preferably in the neutral range of 7.0 to 8.0, but in the measurement method of the present invention, the PH
There is little change in color sensitivity even in the range of 6.0 to 10.5, and even in the acidic range. (E) Action and Effect The ditetrazolium compound represented by the general formula (1) of the present invention and its reduced formazan,
By having two substituted sulfamoyl groups having one sulfonic acid group, excessive water solubility can be suppressed, and production and purification are easy. In addition, the absorption waveform of the formazan obtained by reduction near neutrality has a longer wavelength (approximately 500 nm) than that of conventional formazan, so it is markedly distinguishable from the colored components in body fluids around 450 nm.
Less likely to cause measurement errors. Further, since the ditetrazolium compound of the present invention has extremely low pH dependence upon reduction, measurement results can be obtained with good reproducibility and high sensitivity. When these ditetrazolium compounds are used to measure substrate or enzyme activity in the field of clinical testing, various reducing substances that coexist in biological components may also reduce the tetrazolium compounds, resulting in positive errors in the measurement results. becomes. In order to eliminate this effect, the specimen (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. Tetrazolium compounds are
It has specific action on NADH and NADPH,
Accurate measurement information can be obtained without being affected by various reducing substances and pretreatment agents that coexist with biological components. (F) Examples The present invention will be explained in detail below using Examples and Comparative Examples, but the present invention is not limited to these Examples. Example 1 34 g of phenylhydrazine-3-sulfo-3'-sulfonic acid phenylamide was dissolved in 1000 ml of water, and 10.8 g of benzaldehyde was added. Next, the mixture was stirred at 40 to 45°C for 2 hours and then salted out to obtain a hydrazone compound in a yield of 70%. 2.3 g of the obtained hydrazone compound was added to 40 ml of water containing 6 g of soda ash and dissolved at 5 to 10°C, and separately 3,3'-dichlorobenzicine was added.
0.63 g was tetrazonated using a conventional method, and a solution adjusted to a pH of about 5 was added, and after stirring in an alkaline environment for 7 hours, the obtained diformazan compound was neutralized, crystallized, and filtered. Purified with methanol, diformazan compound cake 7
I got g. Next, dissolve the above diformazan compound cake in 70 ml of methanol in a hot water bath, and heat it at 70 to 75°C for 70 minutes.
Add 10g of % hydrochloric acid, then add 2g of ammonium persulfate crystals, and simmer for 70~70 minutes until oxidation is complete.
Stir at 75℃ for about 2 hours. After adding activated carbon, the mixture was filtered under heat, and the filtrate was diluted with water to 300 ml for crystallization. The precipitated pale yellowish white crystals were filtered, thoroughly washed with cold water, and then dried. 1.3 g of pale yellowish white powder was obtained. This product is a ditetrazolium compound represented by the following formula and has an infrared absorption curve as shown in FIG. 1 and the following elemental analysis values. Elemental analysis value (%) C H N S Calculated value 52.96 3.02 12.35 11.31 Analysis value 52.88 3.00 12.47 11.41 Example 2 Instead of the hydrazine compound of Example 1,
The infrared absorption curve shown in Figure 2 and the following elemental analysis values were obtained by the same synthesis method except that phenylhydrazine-4-sulfo-3'-sulfonic acid phenylamide obtained according to the reference example below was used. 1.5 g of a ditetrazolium compound represented by the following formula was obtained. Elemental analysis value C H N S Calculated value 52.96 3.02 12.35 11.31 Analysis value 52.86 3.01 12.38 11.34 Reference Example: Process for producing phenylhydrazine-4-sulfo-3'-sulfonic acid phenylamide: 25 g of 1-acetyl-aminobenzene-4-sulfonyl chloride and 18 g of m-aminobenzenesulfonic acid were added to 150 ml of water in the presence of 15 g of soda ash. It is gradually added to the dissolved solution at 20-25°C, stirred for 5-6 hours, acidified with hydrochloric acid, and the precipitate is filtered. The resulting cake was dissolved in 150ml of 5% caustic soda.
After treatment at 90-95°C for 2 hours, it is neutralized to obtain an aqueous 1-aminobenzene-4-sulfo-3'-sulfonic acid phenylamide solution with a yield of about 70%. 1-Aminobenzene- obtained by the following conventional method
4-Sulfo-3'-sulfonic acid phenylamide is used as a hydrazine compound. Next, color development tests using NADH and water-solubility examples using the compounds of the present invention obtained in the above examples will be shown. Examples 3 to 4 The ditetrazolium compounds obtained in Examples 1 and 2 were mixed with Triton X-405, 0.4%, NAD 2mM/
, to 3 ml of 0.1 M Tris phosphate buffer (PH 7.5) containing 2 U/ml of diaphorase to give a concentration of 0.2 mM/ml, mix, add 50 μ of an aqueous solution containing 2.8 mM/ml of NADH, and incubate at 35-40°C. After heating for 5 minutes, absorbance was measured. The water solubility and color development are shown in the table below.

[Table] The ditetrazolium compound of the present invention has sufficient water solubility for practical use, and also exhibits good color development in a low reduction potential system such as the NADH-diaphorase system. Example 5 Ditetrazolium compound obtained in Example 2
0.2mM/, Triton X-405, 0.4%, NAD
2mM/0.1M containing diaphorase 2U/ml
Take 3 ml of Tris phosphate buffer (PH7.5). on the other hand,
Take 3 ml of a buffer of similar composition without the addition of Triton X-405. NADH2.8mM/aqueous solution in both solutions
After adding 50μ and heating at 37°C for 5 minutes, each absorption spectrum was measured. The results are shown in Figure 3. As shown in FIG. 3, when a surfactant is used in combination, the maximum wavelength of the ditetrazolium compound of the present invention obtained in Example 2 shifts from 520 nm to 525 nm.
Its absorbance increases approximately twice. Next, the PH dependence of color development by the compound of the present invention will be shown using the measurement of NADH as an example. Example 6 In 0.1M Tris phosphate buffer containing 0.4% Triton
8.0, 8.4, 9.0, 9.4, 9.8, and 10.5 were prepared, each containing 0.2 mM/ml of the ditetrazolium compound of the present invention obtained in Example 2 and 2 U/ml diaphorase.
was added, and 50μ of a 2.5mM NADH/aqueous solution was added to 2.5ml of the solution, and after heating at 37°C for 5 minutes, the maximum absorbance of formazan produced at each pH was plotted. The results are shown in Figure 4. That is, there is no change in color sensitivity at least in the pH range of 7.2 to 10.5, and there is very little change in the acidic range. This means that the measurement of NADH and the optimum pH of various dehydrogenases can be used over a significantly wide range, and stable measurement results with good reproducibility and high sensitivity can be obtained. Example 7 and Comparative Example 1 0.1M Tris phosphate buffer containing 0.4% Triton X-405 (adjusted to PH5.8, 7.5, 8.4, 9.0, 9.8)
0.2mM/each of the ditetrazolium compound (Example 7) or NTB (Comparative Example 1) obtained in Example 2,
Add diaphorase to a concentration of 2 U/ml to prepare a reaction solution. 50μ of a 50mg/d ascorbic acid aqueous solution was added to 2.5ml of this reaction solution, heated at 37°C for 5 minutes, and the absorbance was measured at the maximum absorbance of each formazan produced.The results were plotted and We obtained the results shown in the figure. As can be seen from Figure 5, the conventionally used
Compared to NBT, the ditetrazolium compound of the present invention produces significantly less formazan at each pH. This shows that it is difficult to reduce ascorbic acid and shows that it acts specifically on NADH compared to NTB. Example 8 Ditetrazolium compound of the present invention obtained in Example 1
1mM/, lactate lithium salt 25mM/,
NAD 2mM/, diaphorase 2U/ml, EDTA
-2NalmM/ is added to 0.2M Tris-HCl buffer (PH8.2) containing 0.4% Triton X-405 to prepare a coloring reagent. Take 20μ of human serum, add 2.5ml of coloring reagent,
The absorbance at 517 nm, which is the maximum absorbance of formazan continuously produced at a constant temperature of 37°C, was measured. The results are shown in Figure 6. Further, after increasing the amount of human serum to 20, 30, and 40μ, and measuring the absorbance continuously in the same manner, the change in absorbance per minute was plotted to obtain the results shown in FIG. These results show that the measuring method of the present invention is effective in measuring body fluid components and is a quantitative reaction. Next, the determination of superoxide will be explained using the determination of cholesterol as an example. Example 9 0.2mM of the compound obtained in Example 1, cholesterol oxidase 0.2U/ml, peroxidase 10U/ml, N-ethyl (2-hydroxy-3-
sulfopropyl)-m-toluidine 1mM/,
Reduced glutathione 0.5mM/Triton
-100, dissolve in 0.1M Tris-HCl buffer containing 0.2% and use as a coloring reagent. Dissolve cholesterol in isopropanol,
Prepare the concentration to 300mg/d and use it as a sample. Samples 10, 20, 30, 40, and 50μ were each taken, 5 ml of coloring reagent was added thereto, and after incubation at a constant temperature of 37°C for 5 minutes, the absorbance at 517 nm was measured using a reagent blank as a control. The results are shown in FIG. This shows that superoxide ions generated by the action of cholesterol oxidase can be quantitatively measured.

[Brief explanation of drawings]

Figures 1 and 2 are infrared absorption spectra of the ditetrazolium compounds obtained in Examples 1 and 2, Figure 3 is the absorption spectrum obtained in Example 5, and Figure 4 is the absorption spectrum of the ditetrazolium compounds obtained in Example 6. Figure 5 is a diagram showing the absorbance obtained in Example 7, Figures 6 and 7 are diagrams showing the absorbance obtained in Example 8, and Figure 8 is a diagram showing the absorbance obtained in Example 8. is a diagram showing the absorbance obtained in Example 9.

Claims (1)

[Claims] 1 General formula (1) [In the formula, R ₁ and R ₃ are hydrogen, R ₂ and R ₄ are the formula
(2) or (3) (In the formula, X and Y are SO ₂ NH, R ₅ and R ₇ are hydrogen, R ₆ and R ₈ are sulfonic acid groups.) P and Q are hydrogen, and Z is halogen. . ] A novel water-soluble ditetrazolium compound represented by: 2 General formula (1) [In the formula, R ₁ and R ₃ are hydrogen, R ₂ and R ₄ are the formula
(2) or (3) (In the formula, X and Y are SO ₂ NH, R ₅ and R ₇ are hydrogen, R ₆ and R ₈ are sulfonic acid groups.) P and Q are hydrogen, and Z is halogen. . ] A method for measuring a reducing substance in an aqueous solution, characterized by using a water-soluble tetrazolium compound represented by the following. 3. The measuring method according to claim 2, wherein the reducing substance is reduced nicotinamide adenine dinucleotide or reduced nicotinamide dinucleotide phosphoric acid. 4. The measuring method according to claim 2, wherein the reducing substance is a superoxide ion. 5. The measuring method according to any one of claims 2 to 4, in which a tetrazolium compound is used in combination with a surfactant. 6. The measuring method according to any one of claims 2 to 5, wherein a component in an aqueous solution is quantified by measuring a reducing substance in the aqueous solution. 7. The measuring method according to claim 6, wherein the component in the aqueous solution is a component in a biological body fluid.