JPH0362400B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for quantifying reduced coenzymes. Measuring enzyme activity and the amount or concentration of substances in biological samples is very important for diagnosing diseases, understanding therapeutic effects, and understanding disease mechanisms. Among body fluid components such as serum or urine, lactate dehydrogenase (LDH) creatine phosphokinase (CPK)
When measuring the activity of dehydrogenases such as α-hydroxybutyrate dehydrogenase (α-HBD), or when measuring the activity of other enzymes through the mediation of these dehydrogenases, or measuring the activity of cholesterol, triglyceride, glucose, etc. When quantifying cholesterol, triglyceride, glucose, etc. by intervening a dehydrogenase that specifically acts on each substance, it is necessary to use a coenzyme to exert the action of the dehydrogenase.
NAD or NADP is used, and the determination is made by quantifying NADH or NADPH produced by the action of dehydrogenase. The methods for quantifying NADH or NADPH include measuring the absorbance at 340 nm, or reacting a tetrazolium salt with NADH or NADPH to generate a colored formazan, followed by colorimetric determination in the visible region. However, when measuring absorption at 340 nm, it is affected by substances in the sample that have absorption near 340 nm, so sample blind testing is required, and the measuring device also needs special specifications for ultraviolet measurement. . In addition, in the visible color method using a tetrazolium salt, the colored formazan produced by the reaction between the reduced coenzyme and the tetrazolium salt has low solubility in water and has strong staining properties, so the dye may precipitate and stain cells or tubes. This often resulted in staining and contamination, which caused measurement problems. Furthermore, methods that utilize the reducibility of reduced coenzymes and measure them as colored substances, such as the visible color method using tetrazolium salts, are susceptible to errors due to the influence of reducing impurities in the sample, such as ascorbic acid and uric acid. It often occurs,
A solution to these problems was desperately needed. As a result of intensive research in order to solve these problems, the present inventors found that by reacting a reduced coenzyme in the presence of glutathione reductase in a medium containing oxidized glutathione at pH 3 to 10, oxidized glutathione was converted into reduced glutathione. By quantitatively changing the color of coenzyme, and then quantitatively developing color using a thiol group (-SH group) coloring agent, it was possible to colorimetrically quantify the reduced coenzyme without being affected by ascorbic acid or uric acid. Heading: The present invention has been completed. That is, in quantifying reduced coenzyme, the present invention allows oxidized glutathione and reduced coenzyme to interact at pH 3 to 10 in the presence of glutathione reductase, and quantitatively reduces the produced reduced glutathione using thiol group coloring. This is a method for quantifying reduced coenzyme, which is characterized by colorimetric measurement using a coloring agent. The reaction of the present invention is shown in the following reaction formula. (1) Oxidized glutathione + reduced coenzyme Glutathione reductase――――――――――――→ pH3~10 Reduced glutathione + oxidized coenzyme (2) Reduced glutathione Thiol group coloring agent―― ――――――――→ Color development→measurement The enzymatic reaction of formula (1) and the color development reaction of formula (2) are each well-known reactions, but the combination of these reactions is not disclosed in this application. There is no previous example of this, and all known examples of the enzymatic reaction in the first step are related to the measurement of the activity of glutathione reductase, so the amount of reduced coenzyme used should be compared with the amount used in the present invention. It is a large amount. In other words, regarding NADPH, which is one of the representative examples of the reduced coenzyme used in the present invention, according to Clinical Pathology, Special Special Issue No. 33, pages 102 and 103 (Japanese Society of Clinical Pathology), The amount used is 0.1m
MOl, but the amount of NADPH used in the present invention is
According to Example 1, cholesterol standard solution (200
mg/dl) is 0.034mMol, and its concentration is 1/3. Since the present invention is an invention for quantifying reduced coenzyme, as a result of research on the reactivity of reduced coenzyme and oxidized glutathione, which are usually used at low concentrations in such cases, it was found that a reaction with sufficient quantitative properties was possible. This process was completed after discovering that the enzymatic reaction of formula (1) and the coloring reaction of formula (2) were combined and the quantitative performance was not affected in any way. . As a thiol group coloring agent, the specificity of the reaction toward SH groups is high, it reacts stoichiometrically with SH groups, and the reaction rate is sufficiently high. Reagents with properties such as detectability are used. For example, 5,5'-dithiobis(2-nitrobenzoic acid), N-(1-anilinonaphthyl-4)maleimide, β-hydroxyethyl-2,4-dinitrophenyl disulfide, 2,2'- Examples include, but are not limited to, dithiopyridine. The thiol-based color former also includes fluorescent color formers such as benzimidazolylmaleimide. As the medium with pH 3 to 10 used in the method of the present invention, all commonly used buffers such as 0.01 to 1M Tris buffer, phosphate buffer, citrate buffer, and acetate buffer can be used. can. The concentration of oxidized glutathione is not particularly limited, but is preferably 0.1 mM to 300 mM. The amount of glutathione reductase per sample is preferably 1 to 100 international units (IU), more preferably 5 to 20 IU. To illustrate an example of measuring enzyme activity in serum using the method of the present invention, in measuring LDH activity,
Thirium lactate 0.1M as substrate, NAD 0.2-0.5%
Take 0.5 to 2 ml of 0.1 M Tris buffer (substrate buffer) with pH 9.0 and prewarm it in a 37℃ thermostatic bath for 3 minutes, then add 20 to 50μ of serum, mix, and leave it in a 37℃ thermostatic bath for 10 minutes. do. Next, add 1 to 3 ml of 0.1M acetate buffer (PH4.5) containing 5mM of oxidized glutathione and 10,000IU of glutathione reductase.
After reacting for 5 minutes at °C, a 0.5% ethanol solution of 5,5'-dithiobis(2-nitrobenzoic acid) was added.
Add 0.5 to 3 ml, mix, and measure absorbance at 410 nm. The LDH activity in the sample may be determined from a calibration curve prepared in the same manner using LDH standard serum. To give an example of determining the concentration of a substance in serum, for the measurement of triglycerides, lipoprotein lipase in 100 ml of Tris buffer of pH 7.5 is used.
15000u, glycerokinase 2500u, glycerol-3-phosphate dehydrogenase 2000u, NAD500mg,
Add 2 ml of test solution containing 170 mg of ATP to 20 µ of serum, mix, and then heat in a thermostat at 37°C for 10 minutes. Next, 1 ml of acetate buffer containing the oxidized glutathione used in the LDH activity measurement and glutathione reductase was added and reacted at 37°C for 5 minutes.
Add 0.5 ml of a 0.5% ethanol solution of dithiobis(2-nitrobenzoic acid) to develop color and measure the absorbance at 410 nm. This method is completely unaffected by reducing substances such as ascorbic acid and uric acid in serum. The method of the present invention is not limited to the above-mentioned examples of use, but is applicable to reduced coenzymes such as NADH or NADPH.
This method can be applied to all methods for measuring enzyme activity and substance amount. Examples will be described below. Example 1 Reagent for determining total cholesterol in serum (1) Cholesterol ester hydrolase Tris buffer (CEH solution) Contains 100 u of cholesterol ester hydrolase in 100 ml of 0.01M Tris buffer at pH 7.0. (2) Cholesterol dehydrogenase/NADP
Tris buffer (CDH/NADP solution) NADP200 in 100ml of 0.1M Tris buffer at PH9.0
mg, Contains 100u of cholesterol dehydrogenase. (3) Oxidized glutathione/glutathione reductase/Tris buffer (GSSG/GR solution) Oxidized glutathione 0.2g, glutathione reductase 1300u in 100ml of 0.1M Tris buffer with pH 8.4
Contains. (4) DTNB solution 5,5'-dithiobis(2-nitrobenzoic acid)
Dissolve 0.5g in 100ml of ethanol. Measurement method: Take 20 μl of serum, add 1 ml of CEH solution, and leave it in a constant temperature bath at 37°C for 5 minutes. Add 1 ml of CDH/NADP solution and leave it at 37°C for 5 minutes. Next, add 1 ml of GSSG/GR solution and leave it at 37℃ for 5 minutes, then add DTNB solution 2.
ml and measure the absorbance at 410 nm using the reagent blind as a control. Separately cholesterol 200mg/
Perform the same operation using a standard solution containing dl and measure the absorbance. Calculate the total cholesterol concentration in serum from the following formula. Es/Estd×200=Total cholesterol mg/dl Es: Absorbance when using a serum sample. Estd: Cholesterol standard solution (cholesterol
200mg/dl). Example 2 Reagent (1) NADH standard solution Dissolve NADH in distilled water or ion-exchanged water and prepare NADH0.5mg/ml, 1mg/ml, 1.5mg/ml,
Prepare a solution with a concentration of 2 mg/ml. (2) Oxidized glutathione/glutathione reductase/Tris buffer (GSSG/GR solution) Same as Example 1. (3) DTNB solution Same as Example 1. Measurement method: Take 50 μl of each NADH standard solution, add 2 ml of GSSG/GR solution, leave in a thermostat at 37°C for 5 minutes, then add 2 ml of DTNB solution and mix. Use reagent blind as a control.
Measure the absorbance at 410 nm. absorbance and
Create a calibration curve based on NADH concentration. The calibration curve at this time is shown in FIG. As is clear from the calibration curve, the calibration curve shows a straight line and has good quantitative properties. The results are also shown in Table 1.

[Table] Example 3 Influence of ascorbic acid and uric acid on NADH determination Reagents (1) NADH solution Dissolve 200 mg of NADH in distilled water to make 100 ml. (2) GSSG/GR solution Same as Example 1. (3) DTNB solution Same as Example 1. (4) Ascorbic acid solution Dissolve ascorbic acid in distilled water to prepare ascorbic acid solutions with concentrations of 100 mg/dl and 200 mg/dl. (5) Uric acid solution Take 100 mg of uric acid and 60 mg/dl of tirium carbonate, add distilled water, and dissolve by heating to make a total volume of 100 ml.
Take these 10 ml and 20 ml and add water to make 100 ml, respectively, to prepare solutions with concentrations of uric acid of 10 mg/dl and 20 mg/dl. Measurement method Same as Example 2. When examining the effects of ascorbic acid and uric acid, the same volume of each solution is added at the same time as the NADH solution, and the NADH concentration is calculated from the absorbance after correcting the liquid volume. The results are shown in Table 2.

【table】

[Table] As is clear from the measurement results, the method of the present invention is not affected by ascorbic acid or uric acid at all. Example 4 NADH quantitative reagent (1) NADH standard solution Dissolve NADH in distilled water or ion-exchanged water and prepare NADH1mg/, 2mg/, 5mg/, 7
Prepare solutions with concentrations of mg/, 10 mg/. (2) GSSG/GR solution Same as Example 1. (3) BIPM Reagent Dissolve 5 mmol of N-[p-(2-benzimidazolyl)phenyl]maleimide in 1 ethanol. Measurement method: Take 50μ of NADH standard solution and GSSR/GR solution 3
ml of BIPM sample solution and leave it in a thermostat at 37℃ for 5 minutes.
Add 0.5 ml and measure fluorescence at 365 nm after 5-10 minutes. The excitation wavelength is 315nm. The calibration curve at this time is shown in FIG. The calibration curve is linear and has good quantitative properties. Example 5 NADPH quantitative reagent (1) NADPH standard solution NADPH0.5mg/ml, 1mg/ml, 1.5mg/ml,
Prepare a solution with a concentration of 2 mg/ml. (2) GSSG/GR solution Same as Example 1. (3) DTNB solution Same as Example 1. Measurement method: Take 50 μl of each NADPH standard solution and proceed in the same manner as in Example 2. The calibration curve at this time is shown in FIG. The calibration curve is linear and has good quantitative properties. The results are shown in Table 3.

[Table] Example 6 α-hydroxybutyrate dehydrogenase (α-
(HBD) activity measurement reagent (1) Substrate coloring reagent solution Contains 0.15M α-hydroxybutyric acid, 500mg NAD, 1g oxidized glutathione, 0.1g DTNB, and 7000u glutathione reductase in 11M Tris buffer at pH 8.5. Measurement method: Take 2 ml of substrate coloring test solution and leave it in a 37°C thermostatic chamber for 3 minutes, then add 50 μl of serum, mix, and place in a 37°C thermostatic cell.
Measure the change in absorbance at 410 nm from 2 minutes to 1 minute. Separately, α-HBD in serum was determined using a standard curve prepared in the same manner using α-HBD standard serum.
Determine HBD activity. Example 7 Total cholesterol determination reagent in serum Reagent (1) Cholesterol ester hydrolase Tris buffer (CEH solution) Same as Example 1. (2) Cholesterol dehydrogenase/NAD Tris buffer (CDH/NAD solution) Same as Example 1. (3) Oxidized glutathione/glutathione reductase/Tris buffer (GSSG/GR solution) Same as Example 1. (4) HEDD solution Dissolve 0.5 g of β-hydroxyethyl-2,4-dinitrophenyl disulfide in 100 ml of ethanol. Measurement method The following example 1 was followed, using a HEDD solution instead of the DTNB solution in Example 1 and setting the measurement wavelength to 408 nm. Example 8 Reagent for determination of total cholesterol in serum (1) Cholesterol ester hydrolase Tris buffer (CEH solution) Same as Example 1. (2) Cholesterol dehydrogenase/NAD Tris buffer (CDH/NAD solution) Same as Example 1. (3) Oxidized glutathione/glutathione reductase/Tris buffer (GSSG/GR solution) Same as Example 1. (4) 2PDS solution 0.5g of 2,2'-dithiopyridine in ethanol
Dissolve in 100ml. Measurement method The following is the same as in Example 1, using a 2PDS solution instead of the DTNB solution in Example 1 and setting the measurement wavelength to 281 nm. Example 9 Total cholesterol determination reagent in serum Reagent (1) Cholesterol ester hydrolase Tris buffer (CEH solution) Same as Example 1. (2) Cholesterol dehydrogenase/NAD Tris buffer (CDH/NAD solution) Same as Example 1. (3) Oxidized glutathione/glutathione reductase/Tris buffer (GSSG/GR solution) Same as Example 1. (4) BIPM solution Dissolve 0.01 g of benzimidazolylmaleimide in 100 ml of ethanol. Measurement method: BIPM solution was used instead of the DTNB solution in Example 1, and the excitation wavelength was measured using a fluorescence spectrophotometer.
Measured at 315 nm and fluorescence wavelength of 365 nm, and the following is the same as in Example 1. Example 10 Glycocholic acid quantitative reagent (1) Glycocholic acid solution Glycocholic acid was dissolved in physiological saline to give glycocholic acid 50uM/1100uM/1150uM/
Prepare a solution with a concentration of 1200uM/1. (2) 3α-hydroxysteroid dehydrogenase・
NAD Tris buffer (3α-HSD/NAD solution) NAD145 in 100ml of 0.05M Tris buffer at PH7.2
mg, contains 12.3 u of 3α-hydroxysteroid dehydrogenase (3α-HSD). (3) Oxidized glutathione/glutathione reductase/Tris buffer (GSSG/GR solution) Oxidized glutathione 184 mg, glutathione reductase in 100 ml of 0.05M Tris buffer at PH7.2
Contains 2050u. (4) DTNB solution 5,5'-dithiobis(2-nitrobenzoic acid)
Dissolve 200mg in 100ml of ethanol. Measurement method: Take 200ul of glycocholic acid solution, add 1.9ml of GSSG-GR solution and 1.9ml of 3α-HSD/NAD solution,
After leaving in a thermostat at 37°C for 10 minutes, add 0.1 ml of DTNB solution, mix, and measure the absorbance at 405 nm using a reagent blind test as a control. Create a calibration curve based on absorbance and glycocholic acid concentration. The calibration curve at this time is shown in FIG. As is clear from FIG. 4, the calibration curve shows a straight line and has good quantitative properties. The results are also shown in Table 4.

[Table] Comparative examples are shown below. Comparative Example 1 Reagent for quantifying serum total cholesterol (1) Buffer solution 0.1% p-chlorphenol in 0.15M Tris buffer (PH7.0), Triton X-100 (2) Color reagent solution 0.1% p-chlorphenol, Triton X-
100 0.2%, cholesterol oxidase 15u/
dl, cholesterol ester hydrolase
The concentration should be 20u/dl, peroxidase 300u/dl, 4-aminoantipyrine 0.02%.
Dissolve in 0.15M Tris buffer (PH7.0). (3) Standard solution Cholesterol 200 mg/dl Measurement method Take 10 μl of serum, add 3.0 ml of coloring reagent solution (coloring agent dissolved in buffer solution), mix, and leave in a thermostat at 37°C for 5 minutes. After that, use a blind test as a control. Measure the absorbance at a wavelength of 505 nm. Example 1 From the absorbance obtained by performing the same operation using a standard solution containing 200 mg/dl of cholesterol
Calculate the total cholesterol concentration in serum according to the calculation formula. Table 5 shows a comparison of the measurement results of serum total cholesterol by the methods of Example 1 and Comparative Example 1. Further, a correlation diagram at this time is shown in FIG. As is clear from these results, the values measured by the methods of Example 1 and Comparative Example 1 show a very good correlation with a correlation coefficient of 0.994, indicating that the method of the present invention has no practical problems at all. .

【table】

[Table] Comparative Example 2 Reagent for measuring the activity of α-hydroxybutyrate dehydrogenase in serum (1) Substrate buffer Contains α-hydroxybutyrate in 0.1M Tris buffer (PH8.5). (2) Substrate coloring reagent α-ketobutyric acid 20mM, NAD 0.15%, nitrotetrazolium blue 0.05%, phenazine methosulfate 0.1M to give a concentration of 0.01%.
Dissolve in Tris buffer (PH8.5). (3) 1N hydrochloric acid (4) Standard serum Measurement procedure Take 0.5ml of the substrate coloring reagent solution (coloring agent dissolved in substrate buffer) and leave it in a thermostat at 37°C for 3 minutes, then add 50μ of serum, mix, and further boil at 37°C. Leave it in a constant temperature bath for 20 minutes. Next, add 5.0 ml of 0.1N hydrochloric acid (10-fold dilution of 1N hydrochloric acid) and mix, using a reagent blind test as a control.
Measure the absorbance at 560 nm. α−
α-HBD activity in serum is determined from a standard curve created in the same manner using HBD standard serum. Serum α- by the methods of Example 6 and Comparative Example 2
Table 6 shows a comparison of HBD measurement results. However, u in the table indicates Rosalki unit. Further, a correlation diagram at this time is shown in FIG. As is clear from these results, the values measured by the methods of Example 6 and Comparative Example 2 show a very good correlation with a correlation coefficient of 0.9997, indicating that the method of the present invention has no practical problems.

【table】 [Brief explanation of drawings]

Figure 1 shows the calibration curve in Example 2, Figure 2 shows the calibration curve in Example 4, Figure 3 shows the calibration curve in Example 5, and Figure 4 shows the calibration curve in Example 10. 5 is a correlation diagram for Comparative Example 1, and FIG. 6 is a correlation diagram for Comparative Example 2.

Claims (1)

[Scope of Claims] 1. In quantifying reduced coenzyme, oxidized glutathione and reduced coenzyme are allowed to interact at pH 3 to 10 in the presence of glutathione reductase, and the reduced glutathione produced by quantitative reduction is converted into thiol. A method for quantifying a reduced coenzyme, which is characterized by colorimetric measurement using a basic coloring agent. 2. The quantitative method according to claim 1, wherein the reduced coenzyme is NADH (reduced nicotinamide adenine dinucleotide) or NADPH (reduced nicotinamide adenine dinucleotide phosphate).