JPH0675517B2 - Method for quantifying reduced nicotinamide coenzyme - Google Patents

Description

TECHNICAL FIELD The present invention relates to a novel method for quantifying reduced nicotinamide coenzyme. More specifically, it is a method for quantifying reduced nicotinamide coenzyme capable of highly accurately quantifying reduced nicotinamide coenzyme present in biological fluids such as urine and serum.

[Prior Art and Problems] The quantification of reduced nicotinamide coenzyme is generally applied to the quantification of substances in biological fluids. For example, as a method for quantifying bile acid contained in biological fluid, the bile acid and oxidized nicotinamide coenzyme are reacted with dehydrogenase to quantify reduced nicotinamide coenzyme produced, and Methods for estimating the amount of bile acids are known. In addition, the quantitative determination of reduced nicotinamide coenzyme can be applied to the measurement of polyamine contained in biological fluid. That is, polyamine is oxidized by an oxidase to be converted into an aminoalkyl aldehyde, and the aldehyde is reacted with an oxidized nicotinamide coenzyme and a dehydrogenase, and the reduced nicotinamide coenzyme produced is quantified to determine the amount of polyamine. This is a method of estimating.

Conventionally, the quantification of such reduced nicotinamide coenzyme is carried out by allowing a tetrazolium-based color developing agent composed of a tetrazolium salt and an electron transfer system such as diaphorase to act on a sample solution and measuring the degree of color formation of the generated formazan. There is.

However, this quantification method has a problem that the stability of coloration is poor due to the deposition and aggregation of the generated formazan on the measuring instrument (cell, test tube, etc.). In addition, there is also a problem of contamination of measuring instruments due to the deposition of formazan.
Further, in the case of the reaction in an aqueous solution, since the formazan is hardly soluble, the apparent molecular absorption coefficient calculated from the absorbance of the formazan produced by the reaction is significantly smaller than the theoretical molecular absorption coefficient of the formazan, and is also measured. It also has a problem that it easily changes depending on the conditions.

Heretofore, various measures have been taken to solve these problems in the determination of reduced nicotinamide coenzyme using a tetrazolium color former.

For example, as a method of increasing the molecular extinction coefficient, the pH of the sample solution on which the tetrazolium-based color-developing agent acts is adjusted to the alkaline side (PH8
~ 10), and as a formazan dispersant, a nonionic surfactant of about 0.01 to 0.1 or bovine serum albumin.
A general method is to add about 0.1% to make the formazan formation reaction as easy as possible.

Further, as a method for preventing the deposition of the generated formazan, there is a method of adding gelatin or the like into the reaction system.

Although the above problems can be solved to some extent by the above method, there is still room for improvement. Moreover, in the case where the sample solution is a biological fluid such as urine or serum, in addition to the above-mentioned problems, the following problems are further caused. That is, when the sample solution is a biological fluid, the tetrazolium salt is reduced by reducing substances in the sample, such as ascorbic acid, bilirubin, uric acid, and reduced glutathione, and formazan other than reduced nicotinamide coenzyme is produced. Therefore, there is a problem that the blank value is high and it fluctuates, which gives a large error to the measurement.

Such a problem can hardly be solved by the above-mentioned conventional method.

Therefore, it has been desired to develop a method for accurately quantifying reduced nicotinamide coenzyme, which has high coloration stability, the generated formazan shows a high molecular extinction coefficient, and the increase in blank value due to a reducing substance is small. .

[Means for solving problems]

In order to achieve the above-mentioned problems in the method for colorimetrically determining the produced formazan, the present inventors act on a sample solution containing a reduced nicotinamide coenzyme with a tetrazolium-based color former, and as a result, earnest studies have been conducted. The present invention has been completed by finding that all such objects can be achieved by causing a tetrazolium color-developing agent to act on the sample solution in the presence of a specific PH and a specific amount of a nonionic surfactant.

The present invention is a method of acting a tetrazolium color-developing agent on a sample solution containing reduced nicotinamide coenzyme, and quantifying the reduced nicotinamide coenzyme according to the degree of color development, wherein the pH of the sample solution is 4 to 7 And a tetrazolium-based color former is allowed to act in the presence of 0.3 to 10% by weight of a nonionic surfactant, which is a method for quantifying reduced nicotinamide coenzyme.

In the present invention, the reduced nicotinamide coenzyme to be measured includes reduced nicotinamide adenine dinucleotide (hereinafter abbreviated as NADH) or reduced nicotinamide adenine dinucleotide phosphonate (hereinafter abbreviated as NADPH). . The concentration of reduced nicotinamide coenzyme in a sample solution to which the method of the present invention is favorably applied is slightly different depending on reaction conditions and the like and cannot be unconditionally limited.
A range of 0.001 mM to 50 mM is preferred.

The sample solution containing the reduced nicotinamide coenzyme to which the method of the present invention is applied is not particularly limited, but it exerts a remarkable effect particularly on the sample solution containing the reduced nicotinamide coenzyme in the biological fluid. As a specific example of such a sample solution, it is possible to use 3α in biological fluids such as serum, urine and bile which contain bile acids.
-Hydroxysteroid dehydrogenase (hereinafter 3
α-HSD) and oxidized nicotinamide coenzyme obtained by reacting polyamine oxidase with polyamine oxidase in a sample fluid containing reduced nicotinamide coenzyme, biological fluid such as urine, serum Examples thereof include a sample solution containing a reduced nicotinamide coenzyme obtained by converting the aminoalkyl aldehyde and reacting the oxidized nicotinamide coenzyme with the dehydrogenase. Known conditions are adopted without particular limitation in the method for producing the sample solution containing the reduced nicotinamide coenzyme.

In the present invention, the tetrazolium color former comprises a tetrazolium salt and an electron transfer system. The tetrazolium salt may be any tetrazolium salt such as monotetrazolium salt or ditetrazolium salt. For example, 3- (P-
Iodophenyl) -2- (P-nitrophenyl) -5-
Phenyl-2H tetrazolium chloride (hereinafter INT
Abbreviated) Nitrotetrazolium Blue (Nitro-TB),
Examples include tetrazolium salts such as neotetrazolium blue (Neo-TB), tetrazolium blue (TB), and tetranitrotetrazolium blue (TNTB). Among them, INT is preferable as the monotetrazolium salt and Notorotetrazolium blue is preferable as the ditetrazolium salt, although it varies depending on the pH of the reaction. The concentration of the tetrazolium salt varies depending on the reaction conditions, etc., but is usually 0.
1 mM to 50 mM is used. Further, the electron transfer system is a system that causes a tetrazolium salt to give and receive hydrogen of a reduced nicotinamide coenzyme to give a resonance structure, which is converted into a formazan dye. Examples of such an electron transfer system include diaphorase and 1-methoxyphenazine methosulfate (1-methoxy PMS), and the method using diaphorase is preferable. The amount of diaphorase at this time varies depending on the reaction conditions and the like, but is usually about 0.1u to 50u.

In the method of the present invention, a sample solution containing the above-mentioned reduced nicotinamide coenzyme has a PH of 4 to 7, preferably PH5.
.About.6.5 and 0.3 to 10% by weight of the nonionic surfactant, and this phrase is characterized by the action of the tetrazolium color former in the presence of 0.5 to 2% by weight. That is, when the pH of the sample solution when the tetrazolium color-developing agent acts is lower than 4, the reduced nicotinamide coenzyme becomes unstable, and at the same time, sufficient coloration is not exhibited. In addition, when PH is higher than 7, coloration occurs sufficiently, but at the same time, the reduction reaction of the tetrazolium salt by the reducing substance in the sample rapidly increases,
Accurate quantification becomes difficult. On the other hand, when the concentration of the nonionic surfactant is lower than 0.3% by weight, a sufficient molecular extinction coefficient of formazan cannot be obtained, and further formazan deposition or aggregation occurs. On the contrary, the concentration of nonionic surfactant is
When it is higher than 10% by weight, a sufficient molecular extinction coefficient of formazan can be obtained, but the reduction reaction of the tetrazolium salt of the reducing substance in the sample also proceeds at the same time, and accurate quantification is difficult.

The pH of the sample solution described above may be adjusted by any method, but a method of adjusting with a buffer solution is preferable. As the buffer solution, any buffer solution having a buffering effect on PH 4 to 7 may be used. For example, an amino acid-based buffer solution or a female buffer solution in a good-type buffer solution, a bis-tris buffer solution, a pipes buffer solution, or the like is preferable. Also, the PH range is PH4
The pH may be determined within the range of ˜7, but PH5 to 6.5 is particularly preferable in view of the stability of the electron transfer system diaphorase and the like. Further, the concentration of the buffer solution is not particularly limited as long as the pH of the reaction solution can be adjusted stably, but usually 0.1 M to 1.0 M is preferable.

The nonionic surfactant in the present invention is not particularly limited as long as it is a nonionic surfactant that does not inhibit the reaction. However, stabilization of the generated formazan, magnitude of the molecular extinction coefficient of the generated formazan, From the viewpoint of the effect of preventing precipitation and the like, polyoxyethylene alkylallyl ether, polyoxyethylene styrenated phenol and the like are preferably used as the nonionic surfactant. For example, Emulgen 935 (trade name), Penerol N-100N /
C (trade name), Penerol SP-24 (trade name), Emuljet 25 (trade name), Triton X-100 (trade name) and the like. As described above, the effect is recognized when the concentration of the nonionic surfactant is 0.3 to 10% by weight during the reaction. However, preferably 0.5 to 6.0% by weight of nonionic surfactant is present.

In the present invention, when quantifying the reduced nicotinamide coenzyme in the sample solution, the reaction for producing the reduced nicotinamide coenzyme and the reaction for quantifying the produced reduced nicotinamide coenzyme according to the present invention are carried out simultaneously. Well,
Further, these reactions may be carried out as a two-step or multi-step reaction under conditions suitable for each reaction. As a specific example of simultaneously performing a reaction for producing reduced nicotinamide coenzyme and a reaction for quantifying reduced nicotinamide coenzyme, a sample solution containing polyamine is reacted with an oxidase that oxidizes polyamine and converted into aluminum alkyl aldehyde. Then, an oxidized nicotinamide coenzyme, a dehydrogenase, a tetrazolium color-developing agent and a predetermined amount of the nonionic surfactant according to the present invention are added to the sample solution, and the pH is adjusted to 4 to 7. As an example of a dehydrogenase suitable as an enzyme for effectively performing a dehydrogenation reaction in PH of such a reaction,
The ω-aminoalkyl aldehyde dehydrogenase filed on Apr. 4, 1987 by the applicant of the present application may be mentioned. The enzyme has the following physicochemical properties.

Action: acts on 4-aminobutanal in the presence of oxidized nicotinamide dinucleotide or oxidized nicotinamide dinucleotide phosphite, 4-aminobutyric acid and reduced nicotinamide dinucleotide or reduced nicotinamide dinucleotide Generates Phosphate.

Substrate specificity: Acts on oxidized nicotinamide dinucleotide and oxidized nicotinamide dinucleotide phosphite as oxidized nicotinamide coenzyme, and ω
Acts as an aminoalkyl aldehyde on 4-aminobutanal and 5-aminopentanal.

Optimal PH: PH 7.7 to 8.3 PH stability: When stored at 5 ℃ for 24 hours, it has 90% or more existing activity at PH 4.5 to 8.5.

Molecular weight: 102,000 ± 5,000 Molecular weight of subunits: 50.000 ± 5,000 Number of subunits: 2 The above 4-aminobutanal dehydrogenase can be obtained by culturing a bacterium belonging to the genus Micrococcus that is capable of producing this enzyme. The medium used is not particularly limited as long as it contains a carbon source such as glucose, a nitrogen source such as polypeptone, and inorganic salts.
In addition to these components, it is advantageous to contain a polyamine such as putrescine, spermidine, diaminopropane, or calzine in order to increase the productivity of the enzyme. As a culturing condition, a method of culturing at a culturing temperature of 15 to 40 ° C, preferably 20 to 35 ° C is suitable. PH conditions during the culture are in the range of 4.0 to 9.0, and preferably PH 5.0 to 8.0. The culturing time is not particularly limited, but in consideration of the economic efficiency such as the productivity of the enzyme, it is appropriate that the culturing time is within the range from the time when the latter stage of growth is reached to 10 hours after entering the resting state.

As a method for separating bacterial cells from the culture obtained by culturing, conventionally used methods such as centrifugation and filtration can be used, but the method of centrifugation is preferable.

Since 4-aminopentanal dehydrogenase is usually contained in the cells, it is necessary to extract the enzyme from the cells by some method. As the extraction method, microbial cell disruption by ultrasonic waves, which has been performed conventionally, or glass
Examples of the method include cell disruption with a Dynomill disruptor rotated with beads, or disruption of cell membranes with an enzyme such as lysozyme or an organic solvent such as toluene. The enzyme can be collected by selecting an appropriate method from these and extracting the enzyme from the bacterial cells.

When it is necessary to further purify 4-aminobutanal dehydrogenase from the crude enzyme solution extracted by these methods, ammonium sulphate precipitation method, ion exchange column chromatography, which is a commonly practiced purification method for enzymes, is performed. Purification can be carried out by appropriately combining or repeating methods such as the Frauey method, gel filtration method, hydroxyapatite column chromatographic chromatography method, and preparative electrophoresis method.

The method for measuring the enzyme activity of 4-aminobutanal dehydrogenase and the method for displaying the enzyme activity value are as follows.

2.5 ml of 0.1 M tris (hydroxymethyl) aminomethane-hydrochloric acid buffer solution (PH8.0) containing 10 mM putrescine dihydrochloride was placed in a tube with an optical path width of 1 cm and further 20
After adding and mixing μl (5 units) of putrescine oxidase derived from the genus Micrococcus and incubating at 30 ° C. for 10 minutes, 0.5 ml of 20 mM NAD aqueous solution was added to this reaction solution, and then 50 μl An analyte containing 4-aminobutanal dehydrogenase is added and mixed.
Immediately measure the rate of increase in absorbance at 340 nm at 30 ° C.
The enzyme activity value of 4-aminobutanal dehydrogenase per 1.0 ml of the test substance is calculated from the increase amount (A) of the absorbance per minute using the following conversion formula (1). The enzyme activity value is displayed as 1 unit (μmole / mm) of the enzyme value that produces 1 μmole of NADH per minute.

The specific activity value of 4-aminobutanal dehydrogenase according to the present invention is 120 to 140 (unit / mg-protein). In addition, a single protein band is observed on polyacrylamide gel electrophoresis in the presence or absence of sodium dodecyl sulfate.

In the present invention, the addition timing of the nonionic surfactant to be used may be added from the pretreatment step of preparing a sample solution containing reduced nicotinamide coenzyme, or may be added to the adjusted sample solution. Good. That is, the timing of addition of the nonionic surfactant is not limited as long as the nonionic surfactant is present in the sample solution in an amount of 0.3 to 10% by weight when the tetrazolium coloring agent is allowed to act. Further, in the present invention, the reaction of causing the tetrazolium-based color developing agent to act in the presence of the sample liquid containing reduced nicotinamide coenzyme in the presence of PH of 4 to 7 and 0.3 to 10% by weight of the nonionic surfactant, is terminated, Even after the formazan is produced, the PH can be lowered to 4 or less and the produced formazan can be quantified to stably measure.

In the present invention, as a method for estimating the amount of reduced nicotinamide coenzyme from the color development of a tetrazolium color-developing agent, a known colorimetric assay method is adopted without particular limitation. The most general method is a method using a calibration curve.

One of the features of the present invention is that an acidic agent of PH4 to 7 is used, and the reduced nicotinamide coenzyme is colorimetrically determined by a tetrazolium color former. That is, at PH7 or less, the reduction reaction of the tetrazolium color-forming agent due to the reducing substance in the biological sample is suppressed even in the presence of a high concentration of nonionic surfactant, but at PH7 or above, the tetrazolium due to the reducing substance in the biological sample is suppressed. The reduction reaction of the color formation of the system begins to proceed rapidly. This phenomenon is probably due to the reaction system normally used.
Contrary to the pH (alkali side), the redox potential of both the tetrazolium-based color former and the reducing substance in the biological sample changes due to color development on the acidic side, resulting in the tetrazolium-based reducing substance in the biological sample. It is presumed that the reduction reaction of the color former did not occur. However, this alone cannot provide stable and accurate quantification of reduced nicotinamide coenzyme with a tetrazolium-based color former.

In the present invention, the nonionic surfactant is allowed to exist in a high concentration (0.3 to 10% by weight) under the above-mentioned conditions to enable accurate quantification. That is, by using the nonionic surfactant in the above range, the reduction reaction of the tetrazolium color-forming agent by the reducing substance in the biological sample does not proceed, and the reduced nicotinamide coenzyme mediated by an electron carrier such as diaphorase. Therefore, only the reduction reaction of the tetrazolium-based color-developing agent due to the reaction proceeds, and the generated formazan has a high molecular extinction coefficient. It is speculated that this phenomenon plays a role as a dispersant for formazan produced by the nonionic surfactant, and does not affect the redox potentials of the tetrazolium salt and the reducing substance in the biological sample so much. .

Therefore, it becomes possible to colorimetrically determine reduced nicotinamide coenzyme in a biological sample using a tetrazolium-based color former only by combining the two features of the present invention. That is, the present invention, under a low PH condition and the presence of a high concentration of a nonionic surfactant, the blank value is low because the reduction reaction of the tetrazolium salt by the reducing substance in the biological sample can be suppressed, In addition, since the molecular extinction coefficient of the generated formazan is high, it is possible to quantify with high sensitivity.

Further, according to the method of the present invention, since there is no reduction reaction of the tetrazolium salt by the reducing substance in the biological sample, the tetrazolium salt can be used over a wide concentration range.

In the present invention, the addition of the high concentration nonionic surfactant is
It is also soluble in the precipitation of denatured proteins and the like that precipitate and precipitates from biological fluids, and is a very excellent method for quantifying reduced nicotinamide coenzyme present in a sample solution containing such a substance.

According to the present invention, the quantification of reduced nicotinamide coenzyme with a tetrazolium-based color developing agent is stable, accurate, and highly sensitive, and can be performed without contaminating the measuring device or instrument. It is possible.

According to the present invention, it is considered that in the future, a method for quantifying a reduced nicotinamide coenzyme using a tetrazolium-based color developing agent will be widely spread in daily clinical tests. Therefore, the contribution of the present invention to the clinical examination field is expected to be very large.

〔Example〕

Hereinafter, the present invention will be described more specifically by showing examples, but the present invention is not limited to these examples.

Example 1 Table 2 shows the relationship between the measured absorbance values obtained by the following method using the reagents having the following compositions and the reduced nicotinamide coenzyme concentration corresponding to the measured values.

a) Reagent Ascorbic acid oxidase 45u (manufactured by Toyo Brewing Co., Ltd.) Emulgen 935 0.4 g (manufactured by Kao Ishi Ken Co., Ltd.) 0.4M-Tris-hydrochloric acid buffer solution (PH7.8) to make the total volume 10 ml. thing.

Reagent 2 Nitrotetrazolium blue 5.0 mg (manufactured by Dojindo Kenkyusho Co., Ltd.) Diaphorase 80u (manufactured by Oriental Yeast Co., Ltd.) 0.6 M female buffer (PH6) (total amount 20 ml at Dojindo Kenkyuken Co., Ltd.) And what.

Reagent 3 IM hydrochloric acid solution b) Assay method A urine sample containing 200 μmole / l of reduced nicotinamide coenzyme was prepared. On the other hand, the same urine was prepared by adding purified water instead of reduced nicotinamide coenzyme. A dilution series was created for each sample using purified water. Then, the operation was performed as shown in Table 1. 0.5 ml each of this diluted series solution was collected, 0.5 ml of reagent 1 was added and mixed, and the mixture was reacted at 37 ° C. for 20 minutes. Then add 0.5 ml of Reagent 2 and mix to adjust pH to 6.0.
After adjusting the temperature to 37 ° C for 5 minutes and letting the tetrazolium color developing agent act on it, add 0.5 ml of 1mole / l hydrochloric acid aqueous solution and mix.
The absorbance at m was measured.

The calibration curve prepared from Table 2 is shown in FIG. From FIG. 1, the relationship between the concentration of reduced nicotinamide coenzyme and the obtained absorbance shows a linear relationship up to a concentration of 200 μmole / l, which shows that colorimetric quantification of reduced nicotinamide coenzyme is possible.

Example 2 Quantification of reduced nicotinamide coenzyme in urine Reduced nicotinamide coenzyme was added to 30 μmole / l, 60 μmole / l, 120 μmole / l
A urine sample containing was prepared. On the other hand, the same urine was prepared by adding purified water instead of reduced nicotinamide coenzyme.

The above sample was treated in the same manner as in Example 1 with the absorbance Es and
Got Eb. Further, the absorbance was measured by the same method as the method for measuring Es and Eb, using 30 μM reduced nicotinamide coenzyme aqueous solution as a standard solution and purified water as a blind sample. Such standard solution to the absorbance obtained with a Est and Est 'respectively EH ₂ resulting absorbance for blind specimens were O and EH' ₂ O respectively, for.

The amount of reduced nicotinamide coenzyme in the sample was calculated from the measured value obtained by the above method using the following formula.

The above measurement operation was performed 5 times for each of the 3 types of urine samples, and the results are shown in Table 3. The results show that the method of the present invention exhibits good reproducibility.

It can be seen from Table 3 that the method of the present invention can accurately measure the amount of reduced nicotinamide coenzyme in urine.

Example 3 A urine sample containing 120 μmole / l of reduced nicotinamide coenzyme was prepared. On the other hand, the same urine was prepared by adding purified water instead of reduced nicotinamide coenzyme. Absorbances Es and Eb of the above sample were obtained in the same manner as in Example 1. However, the concentration of the nonionic surfactant Emulgen 935 in the reagent 1 shown in Example 1 was 4th during the color reaction.
Each was prepared so as to have the concentration shown in the table. The concentration of reduced nicotinamide coenzyme in urine was calculated from the obtained absorbance according to the following formula.

The above measurement was performed 5 times under the same conditions, and the results are shown in Table 4. The molecular extinction coefficient of formazan under each condition was calculated from the obtained absorbance. The results are also shown in Table 4.

From the above, it was revealed that the present invention can be quantified at a high concentration because it is a very stable and accurate quantitative quantification method for reduced nicotinamide coenzyme and a sufficient molecular extinction coefficient is obtained.

Comparative Example 1 As a comparative example, Table 5 shows the results when the nonionic surfactant Emulgen 935 concentration was 0.3 wt% or less and when it was 10 wt% or more in Example 3.

From the above, it can be seen that when the concentration of the nonionic surfactant Emulgen 935 is 0.3% by weight or less, the obtained molecular extinction coefficient is small and the sensitivity is low, and the measured values also vary greatly. Further, even if it is 10% by weight or more, the sensitivity is sufficient, but the dispersion of the measured values becomes large, and it was proved that it is not suitable as a method for quantifying reduced nicotinamide coenzyme.

Example 4 When the reduced nicotinamide coenzyme was quantified according to the present invention using the reagents having the following compositions, the influence on the quantified value of the reducing substance was examined and the results are shown in Table 6.

a) Reagent Reagent 1 A solution of 40 u of ascorbic acid oxidase in 10 ml of 0.2 M Tris-hydrochloric acid buffer solution (PH8.0).

Reagent 2 Nitrotetrazolium blue 1.5 mg (manufactured by Dojindo Laboratories Co., Ltd.) Diaphorase 40u (manufactured by Oriental Yeast Co., Ltd.) Emulgen 935 0.4 g or more dissolved in 20 ml of 0.2 M female buffer.

b) Assay method A solution containing a known concentration of reduced nicotinamide coenzyme and each reducing substance was diluted with a solution containing an equal concentration of reduced nicotinamide coenzyme to obtain the same reduced nicotinamide concentration. Dilution series with different concentrations of active substances were prepared. Similarly, a dilution series of purified water instead of reduced nicotinamide coenzyme was prepared. Hitachi 70
50 The amount of reduced nicotinamide coenzyme was quantified using an automatic analyzer and the effect of each reducing substance was observed.

20 μl of sample was sampled and Reagent 1 was used as the first reagent.
100 μl was dispensed, and after reacting at 37 ° C. for 5 minutes, 250 μl of each reagent 2 was dispensed as the second reagent, and the main wavelength was 700 nm, and the concentration of reduced nicotinamide coenzyme was determined. As a blind-like sample at this time, physiological saline was used. The absorbance obtained when a sample containing reduced nicotinamide coenzyme was used as the sample was A _1, and the absorbance, that is, the blank increase when the sample containing no reduced nicotinamide coenzyme was used as the sample was A _2. And The concentration conversion from the obtained absorbance is
Pre-blinded physiological saline, standard solution 50μ
The respective absorbances were measured using physiological saline containing mole / l reduced nicotinamide coenzyme. At this time
Absorbance of 50 μmole / l reduced nicotinamide coenzyme is 0.04
It was 75. The concentration conversion from each absorbance was performed by the following formula.

The results are shown in Table 6.

From Table 6, a slight increase in blank was observed in ascorbic acid, but no effect of other reducing substances was observed. Analysis value of reduced nicotinamide coenzyme (C _1-
It was found that C ₂ ) was not affected by any reducing substance. In addition, the coloration stability of formazan was very excellent, and no contamination or deposition was found on the cells of the automatic analyzer.

Example 5 In the same manner as in Example 1, quantification of reduced nicotinamide coenzyme in urine was performed. The reduced nicotinamide coenzyme concentration was 60 μmole / l, and purified water was added as a control sample.The absorbance at 530 nm Es and Eb
Got The reduced nicotinamide concentration was calculated by the following formula.

The same measurement was repeated 5 times to determine the reproducibility. However, the pH during the color development reaction was adjusted to be the pH shown in Table 7. The results are shown in Table 7.

From the above, in PH7.9, the variation of the measured value was large due to the increase of the blank, and in PH3.5, the sensitivity was low and caused the variation.

[Brief description of drawings]

FIG. 1 shows the relationship between the NADH concentration in Example 1 and the absorbance obtained using the method of the present invention.

Claims (1)

[Claims] 1. A method for quantifying the reduced nicotinamide coenzyme by allowing a tetrazolium-based color-developing agent to act on a sample solution containing reduced nicotinamide coenzyme, wherein the pH of the sample solution is A method for quantifying reduced nicotinamide coenzyme, which comprises allowing a tetrazolium color-developing agent to act in the presence of 4 to 7 and 0.3 to 10% by weight of a nonionic surfactant.