JPS59106299A - Determination of reducing coenzyme - Google Patents

Abstract

PURPOSE:To determine the reducing enzyme accurately and rapidly, by reacting oxidized glutathione with reducing coenzyme in the presence of glutathione reductase, and determining the produced reduced glutathione by colorimetry. CONSTITUTION:1-100IU of glutathione reductase [e.g. reduced nicotinamide adenine dinucleotide (phosphate)] is made to react with 0.1-300mM of oxidized glutathione and a reducing coenzyme in a medium of 3-10pH (e.g. 0.01-1M tris buffer solution) to effect the quantitative reduction to form a reduced glutathione. The reduced glutathione is added with a thiol-group color-developing agent [e.g. 5,5'-dithiobis(2-nitrobenzoic acid)] to effect the color development, and the absorption of 410nm wavelength is determined.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for quantifying reduced coenzymes.

Measuring the total amount or concentration of enzyme activity or substances in a biological sample is very important in diagnosing a disease, understanding therapeutic effects, or the mechanism of a disease.

Among body fluid components such as face and urine, lactate dehydrogenase (L
When measuring the activity of dehydrogenases such as DH) creatine phosphokinase (CPK) and α-hydroxybutyrate dehydrogenase (α-)IBD), or when measuring the activity of other enzymes through the mediation of all these dehydrogenases, When quantifying cholesterol, triglyceride, glucose, etc. by intervening a dehydrogenase that acts specifically on each substance, a coenzyme is used to exert the action of the dehydrogenase. as NA
D 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 absorbance at 340 nm, or reacting a tetrazolium salt with NADH or NADPHi to generate colored formazan, which is then colorimetrically determined in the visible region.

However, when measuring absorption at 3401m, 3
Because it is affected by substances that have absorption in the vicinity of 40 nm, it is necessary to perform a sample blind test, and the measuring device is also designed for ultraviolet measurement! 11. Separate specifications are required.

In addition, in the visible color method using tetrazolium salt, the colored formadine produced by the reaction between reduced coenzyme and tetrazolium 11 has low solubility in water and has strong staining properties, so the dye may precipitate or stain cells. This often resulted in staining and contamination of the tubes and 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. These problems often occur, and a solution to these problems has been desperately needed.

As a result of intensive research to solve these problems, the present inventors have found that oxidized glutathione is produced by reacting a reduced coenzyme in a medium containing oxidized glutathione with a pH of 3 to 10 in the presence of glutathione-containing enzyme. Ascorbin Tf was quantitatively converted into reduced glutathione, and then quantitatively colored using a thiol group (-8H group) coloring agent.
The present invention has been completed based on the finding that colorimetric determination of redundant coenzymes can be carried out without being affected by uric acid or uric acid.

That is, the present invention provides a method for quantifying a reduced coenzyme, which is characterized in that the reduced coenzyme is quantitatively reduced and produced reduced glutathione is colored with a thiol-based coloring agent for colorimetric determination. be.

The overall reaction formula of the present invention is as follows.

(1) Oxidized glutathione + reduced coenzyme pH 3-10 Reduced glutathione + oxidized coenzyme (The enzymatic reaction of formula (2) and the coloring reaction of formula (2) are each well-known reactions, but There were no examples of combinations of these reactions prior to the filing of this application, and the known examples of the first-stage enzymatic reactions all relate to the measurement of the activity of glutathione terminal enzymes, so the amount of reduced coenzyme used is is large compared to the amount used in the present invention.

That is, according to the Japanese Society of Pathology (Japan Society of Pathology) as a representative example of the reduced coenzyme used in the present invention, its use ht ld O, 1mMo I
However, the amount of NADPHO used in the present invention is as shown in Example 1.
According to the following, cholesterol standard solution (200■//dt)
is 0.034mMol, and its concentration jW is 1/3.

Since the present invention is an invention for quantifying reduced coenzymes, research on the reactivity of reduced coenzymes, which are usually used at low concentrations in such cases, and acid-rich glutathione has shown that the reactivity is sufficiently constant. He discovered that the reaction progresses and that even if the enzymatic reaction of formula (1) is combined with the coloring reaction of Mr. It is something.

5- As a thiol group coloring agent, the specificity of the reaction with respect to the SH group is high, the reaction with the SH group is stoichiometric, and the reaction amount 1i is sufficiently large, and the equivalence point and the reaction amount must be controlled in some way. Various reagents with properties such as scales that can be sensitively detected by the method are used.

For example, 5,5'-dithiobis(2-nitrobenzoic acid)
, N-(]-]anilinonaphthyl-4maleimide, β-
Examples include hydroxyethyl-2, s') nitrophenyl disulfide, 2,2'-dithiopyridine, etc.
It's a shame that it's limited to these. The thiol-based color former also includes fluorescent color formers such as benzimidazolylmaleimide.

The medium having a pH of 3 to 10 used in the method of the present invention may be 0. All commonly used buffers such as old to IM Tris buffer, phosphate buffer, citrate buffer, acetate buffer, etc. can be used.

The concentration of oxidized glutathione is not particularly limited, but is preferably 0.1 mM to 300 rnM.

6- Glutathione reductase is preferably 1 to 1 per sample.
00 international units (TU), but a more convenient system is 5-2
It is 0 IU.

To give an example of measuring the total enzyme activity in serum using the method of the present invention, L D f (in the measurement of activity, lithium lactate 0.TM, NADo, 2-0.5% as substrate)
pH 9, 0 (1') 0,] M) containing Lith buffer (substrate buffer) 0.5-2 m/! f-tori 37
After prewarming in a thermostatic bath at 37° C. for 3 minutes, 20-50μ of serum is added and the mixed layer is left in a thermostatic bath at 37° C. for 10 minutes. Next, oxidized glutathione 5mM, glutathione reductase 10
,000 TIJ/lf7 in 0.1M acetate buffer (p
H4,5) 1~3m/! Add 'j5 and react for 5 minutes at 37°C, then add 0.5-3me gold solution of 5,5'-dithiobis(2-nitrobenzoic acid) in 0.5% alcohol, mix and measure the absorbance at 410nm. . L D 1 (L D T (activity) can be determined from a standard curve prepared in the same manner using standard serum.

In addition, when determining the concentration of a substance in serum, to give an example, the concentration of triglyceride is 1, and the Tris buffer K of U, H7,5 is
Ii, 100m1. medium lipoprotein lipase 1500
0u, glycerokinase 2500u, glycerol-
3-phosphate dehydrogenase 2000u.

2ml of test solution containing NAD5004, ATP/170nvi
After adding 20 μl of filtrate and mixing, place in a 37°C constant temperature bath.
Warm for 0 minutes. Next, 1 ml of acetate buffer containing the oxidized glutathione and glutathione reductase used in the LDI (activity measurement) was added and reacted at 37°C for 5 minutes. 0
．． 5% ethanol tK+0°5fn1. In addition, color was developed and the absorbance at 410 nm was determined. 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 usage examples, but can be applied to all methods for determining enzyme activity and total amount of substances by measuring reduced coenzymes, such as NADH or NADPH.

Examples will be described below.

Example 1 Reagent for determination of total cholesterol in serum (1) Cholesterol ester hydrolase Tris buffer (CEH solution) 0.01M Tris buffer at pH 7.0 + 00m7! Contains cholesterol ester hydrolase TOul.

(2) Cholesterol dehydrogenase/NADP),
Lis buffer (CI) H-NADP solution) 100 m7 of 0.1M Tris buffer with p'H 9,0! Medium NADP200r
ng, cholesterol dehydrogenase 1000.

(3) Oxidized glutathione/glutathione/reductase/Tris buffer (GSSG-GR bath solution pH 8.4 o, IM) Lis buffer IQOm/! Medium oxidized glutathione 0.27, glutathione reductase 130
Contains 0u.

(,1) DTNB solution 5.5'-dithiobis(2-nitrobenzoic acid) 0°52
Dissolve in 100% of total ethanol.

Measurement method 9 - Take 20 μl of serum and add 1 ml of CEH solution at 37°C.
After leaving it in a constant temperature bath for 5 minutes, add 1ml of CDI (-NADP solution).
Add and leave at 37°C for 5 minutes. Then, add 1 mI of G55G-GR solution! After leaving at 37°C for 5 minutes, 2 ml of DTNB solution was added and the absorbance at 410 nm was measured using a reagent blind test as a control. Separately cholesterol 200■/
The same operation is carried out using all the semi-solutions containing dl f, and the absorbance is measured.

Calculate the total cholesterol concentration in serum from the following formula.

5 EStdX 200 = total cholesterol■/dlES
: Absorbance when using serum sample.

ES td: Absorbance when using cholesterol standard solution (cholesterol 200 μ/d1).

Example 2 NADH quantitative reagent (1) NADH standard solution Dissolve NADH in distilled water or ion exchange water,
Ho, 5■/-91■/d, 1.5■/-210- 2~/m7! Prepare a solution with a concentration of

(2) Oxidized glutathione/glutathione reductase/Tris buffer (GSSG-GR warm solution Same as Example 1).

(3) DTNB solution Same as Example 1.

Measurement method: Take 50 μt of NADT (standard solution), add G55G-GR solution 2-, leave it in a thermostat at 37° C. for 5 minutes, add DTNB solution 2-, mix, and measure the absorbance at 410 nm as a reagent-blind control. Create a calibration curve based on absorbance and NAD)I 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 1 Example 3 Influence of ascorbic acid and uric acid in NADH determination Reagent (+) NADH solution NAD) 1200"l - 100ynl dissolved in distilled water
shall be.

(2) GSSG-GR hot melt Example (3) D T N B solution Same as Example 1.

(4) Ascorbic acid solution Ascorbic acid Dissolved in fully distilled water and ascorbic acid 10
0 mq/dl! , 200 my/dlt 7:4 degree solution is prepared.

(5) Uric acid solution Add 100 ml of uric acid and 60 ml of lithium carbonate and distilled water and dissolve by heating to make a total volume of 100 ml.

This] Oml, 20ml! '(il-add water to 100- and uric acid 1' Omy/di!,
Prepare a solution 12- with a concentration of 20 m9/de (D).

Measurement method Same as Example 2. When looking at the effects of ascorbic acid and uric acid, add the same volume of each solution at the same time as the NADH solution and calculate the NADH from the absorbance after correcting the liquid volume.
Calculate the concentration.

As is clear from the measurement results shown in Table 2, the method of the present invention is not affected by ascorbic acid or uric acid at all.

Example 4 Quantification of NADH 13- Reagent (NADT) (Standard solution NADH is dissolved in distilled water or ion-exchanged water,
T(ITng/ll 2q/z, 5q/ll 7T
A solution with a concentration of IVttlO/l is prepared.

(2) GSSG-GR hot dissolution example (3) BIPM test solution N-[P-(2-pence A midazolyl) phenylcomaleimide is dissolved in 5 mmol of ethanol 11.

Measurement method: Take 2 μt of NADH standard solution, add 3 μt of G55R-GR solution, leave in a thermostat at 37°C for 5 minutes, then leave 0.5 μt of BIPM test solution.
- and measure the fluorescence at 3651 m after 5 to 1o minutes. The excitation wavelength is U315 nm.

The entire calibration curve at this time is shown in Figure 2. The calibration curve is linear and has good quantitative properties.

Example 5 NADPH quantitative reagent 14- (+) N A D P T (standard solution N A D P H0, 5W/me + 1 mg
/met 1. Prepare a solution with a concentration of 5"f/me, 2"/ml.

(2) GSSG-Grt solution Same as Example 1.

(3) D T N B 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 Figure @3. The calibration curve is linear and has good quantitative properties.

The results are shown in Table 3.

Table 3 Example 6 α-hydroxybutyrate dehydrogenase (α-HBD) in serum
activity measurement.

Reagents (substrate coloring reagent PH8, s 0.1M Tris mild buffer, air α-hydroxybutyric acid 015M, NAD500), oxidized glutathione 11, DTNBo, 11, glutathione reductase 70
Contains 00ui.

Measurement method: Substrate coloring test solution 2 - Take the entire sample and leave it in a 37℃ thermostatic chamber for 3 minutes, then add 50μt2 of serum, mix, and put in a 37℃ thermostatic cell 4] On
Measure the change in absorbance from 2 minutes to 1 minute at m.

Separately, the α-HBD activity in the serum is determined from a standard curve prepared in the same manner using α-HBD standard serum.

Example 7 Reagent for quantitative 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-NAT) solution) Same as Example 1.

(3) Oxidized glutathione/glutathione reductase/Tris buffer (GSSG-GR warm solution Same as Example 1).

(4) HE D D solution β-hydroxyethyl-2,4-dinitrophenyl disulfide 0.5f'i dissolved in 100% ethanol.

Measurement method Using HEDD solution instead of DTNB solution in Example 1,
The measurement wavelength f: 408 nm, and will be described in Example 1 below.

Example 8 Total cholesterol determination reagent in serum (Licholesterol ester hydrolase Tris buffer (CEH solution) Same as Example 1.

17- (2) Cholesterol dehydrogenase/NADF'J
Buffer solution (CDH-NAD solution) Same as Example 1.

(3) Oxidized glutathione/glutathione reductase/Tris buffer (GSSG-GR warm solution Same as Example 1).

(4) 2P Ds bath solution, 2'-dithiopyridine 0.59f ethanol 100
- dissolves in

Measurement method Using 2PDS solution instead of DTNB solution in Example 1,
The measurement wavelength was set to 281 nm and will be described in Example 1 below.

Example 9 Total cholesterol determination reagent in serum (Licholesterol ester hydrolase Tris buffer (CEI (solution)) Same as Example 1.

(2) Cholesterol dehydrogenase/NAD Tris buffer (CDH-NAD solution) 18- Same as Example 1.

(3) Oxidized glutathione, glutathione reductase,
Tris buffer (GSSG-GR solution) Same as Example 1.

44) BTPM solution benzimidazolylmaleimide 0. O]r'% - Dissolve in 100 me of ethanol.

Measurement method For B T PM solution instead of the DTNI3 solution in Example 1, measurement was performed using a fluorescence spectrophotometer at an excitation wavelength of 315.
4111 was determined at a wavelength of nm1 fluorescence of 365 nm, and the following is referred to in Example 1.

A comparative example is shown below.

Comparative Example l Reagent for quantifying serum total cholesterol (Lis buffer, 1), 15M) P-chlorophenol 0.1% in Liss buffer (PH 7,0), Triton X-, 100 (2) Coloring agent cholesterol ester hydrolase , cholesterol oxidase, peroxidase, 4-aminoantipyrine (3) Standard solution Cholesterol /l / 200 mq / diMeasurement method 10 μl of serum Color reagent solution (color developer dissolved in total buffer solution)3. Add Om/i and mix in a constant temperature bath at 37℃.
The absorbance at a wavelength of 505 nm is measured using a blind test of leaving the sample for a minute as a control.

Separately, the total cholesterol concentration in serum is calculated from the absorbance obtained by performing all of the same operations using a standard solution containing 200 mfl/de-f of cholesterol according to the calculation formula of Example 1.

Table 4 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 problems in practice. ing.

Table 4 □ Comparative Example 2 A reagent for measuring the activity of α-hydroxyacetate dehydrogenase in serum (contains α-hydroxybutyric acid in substrate buffer 0.1M Tris buffer (tP 8,5)).

(2) Coloring agent NAD, nitrotetrazolium blue, 2 & 21- Contains ginmethosulfate.

(3) IN Hydrochloric acid (4) Standard serum measurement procedure Substrate coloring reagent solution (all coloring agents dissolved in substrate buffer)
0.5m/full area 37℃ left in constant temperature bath for 3 minutes, then serum 50%
Add μt2, mix, and leave in a constant temperature bath at 37°C for 20 minutes. Then 0. IN Hydrochloric acid (]N salt N total 2110 times dilution 5
．． 0tnli plus 560 as a reagent-blind control after mixing.
Measure the absorbance at nmVC. Separately, the α-)(BD activity in the serum is determined from a standard curve prepared in the same manner using α-HBD standard serum.

Serum α-HBDi11 obtained by the methods of Example 6 and Comparative Example 2
Table 5 shows a comparison of the 11 results.

Further, a correlation diagram at this time is shown in FIG. As is clear from these results, the suppression by the methods of Example 6 and Comparative Example 2 was
The constant value shows a very good correlation with a correlation coefficient of 0.9997, which shows that the method of the present invention has no practical problems at all.

22- Table 5 4 Simple drawing Q'1. f, C Explanation Figure 1 shows the test line 1 in Example 2, Figure 2 shows the calibration curve in Leprosy Example 4, and Figure 3 shows the calibration curve in Example 5. 4 is a correlation diagram for Comparative Example 1, and FIG. 5 is an A11 correlation diagram for Comparative Example 2.

Patent Applicant Wayo Pure Chemical Industries, Ltd. 23- Figure 1 Figure 2 Ox liver Applicant 3 Figures NADP) + (#/resistant) Procedural Amendment 1 Indication of the Case 1982 Patent Application No. 215036 2 Name of the invention Method for quantifying odontoid coenzyme 3 Relationship with the case of the person making the amendment Patent applicant contact information Patent Ml (IK Kyo) No. 03-270-85714
Date of amendment order 5. Detailed description of the invention in the specification subject to amendment.

6. Contents of amendment (1) 1(2) Coloring agent cholesterol ester hydrolase, cholesterol okindase, belox/dase, 4-aminoantipyrine” described from page 19, line 19 to page 20, line 2 of the specification as follows: Correct as follows.

[(2) Color reagent P-chlorophenol o, i%, Triton X-10
00.2%, Cholesterol Okindase 15u Hawk Cholesterol Ester Hydrolase 20uAll.

01 to give a concentration of peroxidase 300 u//dg, 4-aminoantipyrine 0.02%.
Dissolve in 5M Tris buffer (pH 7,0). ”(2
) The statement "(2) Contains the coloring agent NAD, nitrotetrazolium blue, and enazine methosalunate" from page 21, line 19 to page 22, line 1 of the specification is corrected as follows.

(2) Substrate coloring reagent solution α-ketobutyric acid 20mM, NAD O, 15%.

Nitrotetrazolium blue 0.05%, 7enazine methosulfate 0.01% [0,
IM) Dissolve in Lys buffer (pH 8,5). ” (3) “Comparison of serum α-HBD measurement results is shown in Table 5” described from line 14 to line 15 on page 22 of the specification.
Insert the next VC "However, the glue in the table indicates the Rosalki unit." .

that's all

Claims (2)

[Claims] (1) When quantifying reduced coenzyme, in the presence of glutathione reductase, oxidized glutathione and reduced coenzyme f
A method for quantifying a reduced coenzyme, which is characterized in that the reduced glutathione produced by the reaction at pH 3 to 10 is quantitatively reduced, and the produced reduced glutathione is colored with a thiol-based coloring agent for colorimetric measurement. (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).