JPH01257499A - Quantification of nad(p)h using nad(p)h oxidase - Google Patents

Abstract

PURPOSE:To accurate, inexpensively and simply perform the quantitative analysis of reduced nicotinamide adenine dinucleotide (phosphate), by employing an enzyme which specifically catalyzes the oxidation of the reduced nicotinamide adenine dinucleotide (phosphate). CONSTITUTION:When reduced nicotinamide adenine dinucleotide (NADH) and/or reduced nicotinamide adenine dinucleotide phosphate (NADPH) existing in a specimen or produced by an enzymatic reaction is quantitatively analyzed, an enzyme specifically catalyzing the oxidation of the NADH and/or NADPH is added to the specimen or reaction system and adjusted to a pH of 7-11 to produce hydrogen peroxide. A color-forming substance and a peroxidase are added to the reaction solution and the formed coloring substance is measured. The specifically oxidation-catalyzing enzyme is preferably originated from the genus Bacillus bacteria.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to an enzymatic method for quantifying reduced nicotinamide adenine dinucleotide (NADH) and/or reduced nicotinamide adenine dinucleotide phosphate (NADPH).

(Prior Art) In various biological researches, clinical tests, etc., many substances derived from living organisms and substances existing in the natural world are measured.

Some of the substances to be measured are difficult to measure directly using one device, and when measuring such substances to be measured, for example, using an enzyme reaction involving the substance to be measured,
Methods for measuring substances produced by this enzymatic reaction and coenzymes involved in this enzymatic reaction can be effectively used. especially.

Nicotinamide adenine dinucleotide (the liquefied form is NAD), which is a coenzyme involved in many enzyme reactions.

The reduced form is represented by NADH) or nicotinamide adenine dinucleotide phosphate (the oxidized form is represented by NADP).
The reduced form is represented by NADPH) is widely used. For example, the alcohol can be measured by measuring NADH produced by oxidation of alcohol by an alcohol dehydrogenase reaction. NA
DH and/or NADPH (hereinafter referred to as NAD(P)
[denoted as H] can be quantified with relatively high sensitivity, so it is also used for the quantification of trace substances.

The method for quantifying NAD (P) H is as follows (1)
The methods of ~(5) are known: (1) A specific wavelength in the ultraviolet region of NAD(P)H (for example, 340, which is the maximum absorption wavelength of NAD(P)II)
(2) A method of directly measuring the absorption at 1 nm); (2) Making NAD (P) H fluoresce by reacting with diaphorase (or a chemical electron acceptor) in the presence of a substance that can be changed by a reaction to produce fluorescence; A method for producing a substance and measuring fluorescence generated from the fluorescent substance; (3) NAD
(P) II is reacted with diaphorase in the presence of a tetradurium salt to produce diformazan, and the diformazan is determined colorimetrically; (4) NAD (P) H is reacted with NAD (P) H oxidase.

A method for measuring the consumption of dissolved oxygen, which is a hydrogen acceptor; and (5) NAD(P)H in the presence of oxygen.
(P) A method in which hydrogen peroxide is produced by the action of H oxidase, and luminol is oxidized by the hydrogen peroxide in the presence of potassium ferricyanide (alkaline region), thereby measuring the chemiluminescence produced.

However, the methods (1) to (5) above have the following problems: In the method (1), NAD(P)II
Since it is measured in the ultraviolet region, it is susceptible to interference substances contained in the biological sample, especially when analyzing biological samples such as blood or biological tissue; method (2) is a fluorescence measurement method. method, it requires expensive equipment and is complicated to operate; in method (3), the diformazane dye produced is poorly soluble in water, so it is easily adsorbed to the colorimeter cell; In the method (4), dissolved oxygen is measured using, for example, a polarographic oxygen electrode, but this device is expensive and cannot measure many samples at the same time; P) In the method using H oxidase, the oxidase used has high activity in the neutral to acidic region, so it is possible to measure the target substrate in combination with an enzyme reaction whose optimal reaction condition is 2, for example, slightly alkaline. (e.g., to measure bile acids in combination with a dehydrogenase reaction); furthermore, because it measures chemiluminescence, it is not suitable for general-purpose automatic analyzers that perform colorimetric measurements. It can hardly be used in routine testing.

Due to the influence of interfering substances, the development of a NAD(P)+1 quantitative method that does not require expensive equipment, is easy to operate, can be automated, and can measure multiple samples simultaneously. desired.

(Problem to be solved by the invention) The present invention solves the above-mentioned conventional problems.

Does the target exist in the sample?

Another object of the present invention is to provide a method for accurately, simply, and inexpensively quantifying NADH and/or NADPI+ produced by an enzymatic reaction. Another object of the present invention is to provide a method for quantifying N A D II and/or N A D P II that can be applied to automatic analyzers.

(Means for Solving the Problems) As a result of repeated research on a method for effectively quantifying NAD(P)H, the inventors found that a method that specifically acts on NAD(P)H and releases hydrogen peroxide. NAD that has the property of generating
The present inventors have discovered that a method using (P)+1 oxidase is advantageous and have completed the present invention.

The present invention relates to N A D It and/or N A D present in a sample or generated by an enzymatic reaction.
A method for quantifying DP II, comprising: (A) adding an enzyme that specifically catalyzes the oxidation of NADH and/or NADPH to the sample and a reaction system containing oxygen;
The reaction was carried out in the pH range of 7 to 11, and NAD and/or
Alternatively, the method includes a step of generating NADP and hydrogen peroxide, and (B) a step of quantifying the generated hydrogen peroxide.

The above objective is thereby achieved.

The enzyme that specifically catalyzes the oxidation of NAD(P)H used in the method of the present invention is an NAD that generates hydrogen peroxide (H2O.) through the reaction catalyzed by the enzyme.
(P)H oxidase is used. NA like this
As D(P)11 oxidase, NAD(P)H oxidase isolated from a microorganism belonging to the genus Bacillus is preferably used. Such microorganisms include Bacillus licheniformis, Bacillus subtilis, and Bacillus subtilis.
Bacillus pumilus, Bacillus anoirinoritias, Hecilus sphericus, Bacillus amyloliquefaciens, Bacillus cereus, and Bacillus thuringiensis, all of which are easily available from public institutions such as the Microtech Institute. . Among the NAD(P)H oxidases obtained from these microorganisms, Bacillus
Taking the enzyme derived from P. thuringiensis as an example, its properties are shown below.

(1) Action and substrate specificity: It specifically catalyzes the oxidation of NADH and/or NADPH, producing NAD and/or NADP and hydrogen peroxide. F.A.D.
By adding , the activity against NADH increased to 35
Double.

And the activity towards NADPH is increased 5 times.

(2) Optimum pH: Shows high activity at pH 6.5 to 8.0.

(3) p11 stability: When kept at 30°C for 24 hours, more than 90% activity remains at pH 7-9.

(4) Km value: 3.2XIO-'M (relative to NADH) (5) Molecular weight and structure Consists of two subunits with a molecular weight of approximately 50,000. Contains one molecule of FAD per subunit.

The principle of the method of the present invention is shown by the following equation (1).

The above NAD (
P) By acting with II oxidase, NA
D(P)II is oxidized to NAD(P) and 0. is H2
It is reduced to 0□. By measuring this 11□0□ using an appropriate method.

NAD (P) H present in the system is measured. The NAD(P)+1 oxidase used in the present invention includes:

As mentioned above, those derived from the genus Bacillus are preferred. The optimal pH of such NAD (P) H oxidase is:

For example, the oxidase derived from Bacillus thuringiensis is 6.5 to 8.0.

Generally, it can act in a wide pn range of about 6 to 11. Until now, NAD that can be used in such enzymatic reactions has
As (P) H oxidase, only NAD (P) I (oxidase), which is active in acidic to neutral ranges, was used, but by using the above oxidase, enzymatic reactions in neutral to alkaline regions are also possible. In the method of the present invention, measurements are particularly advantageously carried out in the pH range of 7 to 11.As mentioned above, the activity of the enzyme toward NAD(P)H is enhanced in the presence of FAD.Therefore, the reaction It is advantageous to co-exist FAD in the system.

The method for measuring the hydrogen peroxide produced above is not particularly limited, but one reaction system includes a chromogen and peroxidase (PO
Preferred is the method of the following formula (II) in which D) is added to oxidize the chromogen to produce a coloring substance, which is then measured.

Such chromogens include combinations of 4-aminoantipyrine and phenolic compounds, naphthol compounds, or aniline compounds; combinations of 3-benzothiazolinone hydrazone (MBTH) and aniline compounds; leucomethylene blue. ; etc. are used. A combination of 4-aminoantipyrine and dimethylaniline is particularly suitable. The type of peroxidase mentioned above is not particularly limited,
Horseradish peroxidase and the like are preferably used.

In the above method, the pH of the 9 reaction system is preferably within the optimum pHjJ range of 5 to 8.5 for peroxidase.

By quantifying NAD (P) H using the method of the present invention, it is possible to indirectly measure the amount of substrate in the enzymatic reaction that produces NAD (P) H. For example, NAD (P) H is added to a substrate that undergoes a dehydrogenase reaction.

When dehydrogenase is applied to this, NAD (P) H is produced. By measuring this NAD (P) H, the above-mentioned amount of substrate is calculated. Substrates that can be measured with such a system include lactic acid, glucose-6-phosphate, alcohol, cholesterol, glucose, glutamic acid, isocitric acid, bile acids, and the like.

The dehydrogenase used is lactate dehydrogenase.

Glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, cholesterol dehydrogenase, glycerol dehydrogenase, glucose dehydrogenase, glutamate dehydrogenase, isocitrate dehydrogenase.

Examples include 3α-hydroxysteroid dehydrogenase.

Enzyme activity can also be measured by quantifying NAD (P) H; for example, taleatin phosphokinase and amylase can be easily measured.

To quantify NAD(P)H by the method of the present invention, for example, follow the order of formulas (I) and (U) above.

NAD (P) I (and NAD (P) F in a system containing oxygen
Add l oxidase and react) 1.0. By adding a dichromogen and peroxidase into the system after producing NAD(P)H oxidase,
This is done by simultaneously adding a chromogen and peroxidase to the system and allowing them to react. The produced colored substance is measured by a colorimeter.

The method of the present invention is generally used to indirectly measure the enzyme activity that catalyzes the enzymatic reaction that produces NAD(P)H, or the substrate in the reaction, by quantifying NAD(P)H as described above. used. Therefore.

Reagents (a), (b) and (C) such as primary
Enzyme activity or substrate amount can be easily measured using these preparations: (a) Dehydrogenase, NAD(P) and NAD(P)H oxidase.

(b) NAD(P) and NAD(P)11 oxidase.

(C) Reagents 2 for measuring 11□0□ produced by the formula (,I), such as peroxidase, and chromogens (for example, 4-aminoantipyrine and aniline compounds).

For example, by combining (a) and (C).

Substrates such as bile acids, glucose, and cholesterol can be measured. By combining (b) and (C), enzyme activities such as 3α-hydroxysteroid dehydrogenase, glucose dehydrogenase, and cholesterol dehydrogenase can be measured. The forms of the above reagents (a) to (C) are not specified, and are supplied, for example, as liquid reagents or as lyophilized products. Even if the liquid reagent contains each component at a concentration that can be used as is.

Alternatively, each component may be contained at a high concentration so that it can be used diluted. When the concentration is such that it can be used as is, the concentration of NAD(P)II oxidase, for example, is preferably 7 ml (o, oi to 100 units Cu). Here, 1 unit of NAD(P)H oxidase produces NAD per minute at pH 7, 0, and 30°C.
This is the amount of enzyme required to oxidize 1 μmol of H.

It is preferable that PAD is added to the above reagent at the same time.

Quantification by the method of the present invention may be performed on individual samples, or it is also possible to perform measurements by sequentially processing a large number of samples by automatic analysis using the above-mentioned appropriate reagents as appropriate.

(Function) According to the present invention, NAD(P)H is quantitatively converted to NAD(P)H using NAD(P)H oxidase.
) and )120□ and measuring this H20□, NAD(P)H can be quantified with high accuracy. H
As a method for quantifying 2O2, as described above, it is advantageous to use peroxidase and a chromogen to generate a pigment according to 20□, and then measure the pigment. This is because the pigments produced in this way have absorption in the visible region.

Measurements are taken in this region without interference from substances in the biological sample. Furthermore, the dyes have the advantage of being water-soluble. In other words, like the formazan dye mentioned in the prior art section.

Since it is insoluble in water, it adheres to measuring instruments and contaminates them. There is no problem. By appropriately selecting the type of chromogen, it is possible to change the measurement wavelength, which allows measurement with higher sensitivity.

By quantifying NAD(P)H, NAD(P)
Measurement of enzyme activity or substrate amount that produces H can be advantageously carried out. In particular, by interacting with NAD (P) and dehydrogenase, N
It is used to quantify substrates that produce AD(P)H and the activity of the dehydrogenase. For example, cholesterol, steroids such as bile acids, lactic acid, glucose, etc. are quantified. NAD(P)H oxidase used in the present invention is stable in a wide pH range.

Measurements can be made over a wide range of pH.

By the way, the following types of NAD(P)H oxidase are known so far.

■From 5tre tococcus faecalis (Dolin, Arch.

Biochem, Biophy, +46+483
+ (1953)); ■Lactobacillus
From Frog Encyclopedia No. 11 (Fukui et al., Agr, B
iol, Chem, 25, 876, (1961))
；■Leuconostoc mesentero
ides (Kobawa et al., J.

Biochem, 97, 1279. (1985))
; ■ Bacillus derived from Hadagumikensha Soki (Saeki et al., J., Bio-chem, 98, 1433.
1985)) ;■ Derived from Bacillus st.

Among these, the enzymes ■～■ and ■ are NAD(P)
Oxidizes H but does not catalyze the reaction that produces hydrogen peroxide. Since the enzyme (2) catalyzes the reaction of the same formula as in the method of the present invention, it is also possible to use this enzyme. However, this enzyme is not practical because it has poor pH stability and thermal stability, and furthermore, the productivity of the enzyme from microorganisms is low.

Therefore, in the method of the present invention, the above-mentioned NAD (P) H oxidase derived from the genus Bacillus is most suitable. For example, using such NAD(P)I+ oxidase,
When the pH is set in the weak alkaline region of 7 to 11, the pH
Determination of the substrates on which dehydrogenases exhibit high activity in the region is advantageously carried out. For example, measurements of steroid compounds such as bile acids and cholesterol are preferably carried out.

Various substrates and enzymes in biological samples can be measured using the measurement system of the method of the present invention.

(Example) The invention will be illustrated with reference to the following examples and reference examples.

NAD from Bacillus thuringiensis (P
) Preparation of H oxidase) 1% glucose, 1% peptone, 0.2% yeast extract,
0.1% sodium citrate, 0.2% (NH4)t
sO4゜0.6% KH2PO4, 1.4%, HPO,
, and pH containing 0.02% MgO<4HzO
A 7.0 medium was prepared, and 5 d of the medium was dispensed into test tubes. Put a cotton stopper into this test tube.

Sterilization was performed by autoclaving at 120''C for 15 minutes.

A single ear of Bacillus thuringiensis was inoculated into the above test tube medium, and cultured with shaking at 30°C for about 20 hours to obtain a seed culture. 50! Medium 301 having the above composition was placed in a jar fermenter, steam sterilized at 120°C for 20 minutes, and 300 tnfl of the above seed culture was inoculated thereto. At a rotation speed of 30 Orpm and ventilation of 13017 minutes,
A culture of Bacillus thuringiensis was obtained by culturing at 30°C for 6 hours. This culture was centrifuged to recover bacterial cells, which were suspended in 30 mM phosphate buffer (pH 7.2) 3.21, and then disrupted using a Dynomil KDL microbial disruptor.

The obtained cell suspension was continuously centrifuged at 12.00 rpm to obtain 3.21 ml of supernatant. Ammonium sulfate powder was added to this supernatant to perform ammonium sulfate fractionation. It did not precipitate with 35% saturated ammonium sulfate, but precipitated with 60% saturated ammonium sulfate. Collect the two parts.
After dissolving in 0mM phosphate buffer (pH 7,2).

Dialysis was performed against the same phosphate buffer. This dialysate was prepared using DEAE-Sepharose CL-6B (Pharmacia) equilibrated with 30mM phosphate buffer (pH 7,2).
Adsorb onto column (if volume) and add 0.2M NaCl
It was eluted. The elution fractions containing NAD(P)H oxidase activity were collected and diluted with 100mM phosphate buffer (pH
Gel filtration was performed using Ultrogel ACA34 equilibrated with 7,2). The eluted fraction containing NAD(P)H oxidase activity was concentrated by ultrafiltration and dialyzed against 50 mM phosphate buffer (pH 7.2). This dialysate
5 equilibrated with 50mM phosphate buffer (pif7.2)
'-AMP-Sepharose 4BC (Pharmacia) column was adsorbed, and the non-adsorbed fraction was washed and removed.
50+sM phosphate buffer (p
It eluted with H7,2). The NAD(P)H oxidase active fraction was dissolved in 100mM phosphate buffer (pH 7,2
) and gel filtration using Sephacryl S-200 (Pharmacia) equilibrated with ), and both fractions containing NAD(P)II oxidase activity were collected and concentrated by ultrafiltration. This concentrated solution was used as a purified NAD(P)l oxidase enzyme solution.

This purified NAD(P)l oxidase enzyme solution was SO
3-polyacrylamide gel electrophoresis showed a single band. The titer of the purified NAD(P)l oxidase enzyme solution is 60 u/1 expressed as specific activity.
mg protein. The titration method for NAD (P) + (oxidase is as follows.

NAD(P)H oxidase titer measurement method: 250
2.51d in mM phosphate buffer (pi47.0),
Add 0.3 ml of an mM NADH solution and 0.1 ml of a 1 mM FAD solution, and use this as reaction solution 1. This reaction solution 1 was preheated at 30°C, and NAD(P)It
Add 0.1 d of oxidase solution to start the enzyme reaction at 30°C. The amount of NADHO consumed per reaction time by incubating at the same temperature is 34
It is measured as a change in absorbance at 0n... Under the above reaction conditions.

The amount of enzyme required to oxidize 1 μmol of NADH per minute is defined as 1 unit (u).

NAD (P) 11 oxidase producing bacteria belonging to the genus Bacillus other than Bacillus thuringiensis (e.g.

It was also possible to prepare NAD(P)H oxidase from the aforementioned Bacillus licheniformis, Bacillus subtilis, etc.) by the above preparation method.

In the following examples and comparative examples, the purified NAD(P)l oxidase enzyme solution prepared by the method of the above reference example was diluted with 100 mM phosphate buffer to 0.1 u/m
This was used as a NAD(P)l oxidase enzyme solution in various measurements.

NADH (7) by 尤U硼上(NAD(P))I oxidase
Consumption and! 1) Confirmation of production of 02) NADII is consumed by NAD(P)H oxidase.

In order to confirm that 11□0z was produced at the same time, Reagent A and Reagent B having the following compositions were prepared and used.

(Left below) Test person = 2mM NADH solution 0.
3d1mM FAD
0. lll11250wM phosphate buffer, pt1
7.0 2.5ml! μ”sy: 0.3% 4-aminoantipyrine aqueous solution 0.1m0
62% N,N-dimethylaniline o,2Id(0,01
N-hydrochloric acid solution) Peroxidase solution 0. IJ11!
(50u/30mM phosphate buffer (pH 7,0)) 25
0mM'J acid buffer, pH 7,00, 2IIdl
After prewarming reagent A at 30'C, (i) Bacillus
0.1 d of NAD(P)H oxidase solution derived from C. thuringiensis was added and reacted at 30"C for 12 minutes. The absorbance of this reaction solution (this is referred to as reaction system (i)) at 340r++++ was measured over time. NADHI was directly measured by measuring the NA
A system in which purified water was added instead of the D(P)H oxidase solution was prepared and the same measurements were carried out (this was referred to as reaction system (ii)). The results are shown in FIG. There was no change in the absorbance of the reaction system (ii) in which purified water was added (without the addition of NAD(P)H oxidase), but
) The absorbance of reaction system (i) with H oxidase added is 1
゜Preparation becomes 0.0, NADII becomes NAD(P)
It was shown that it was consumed by H oxidase.

Next, 0.25% of the reaction system (i) and (ii) above and 0.15# of purified water were added. 1! The above reagent B O, 6m
and reacted at room temperature for 10 minutes. To this, add 2 bottles of deionized water to make the total amount 3 all, 54
Absorbance at 0 nm was measured. Instead of the above reagent A, use 0.25 mj of HzO□ solution of known concentration! was used to react with reagent B in the same manner as above, and a calibration curve of H20□ was created. The amount of 11□0□ was calculated using this calibration curve. in this way.

It was confirmed that nzo□ was produced in the reaction system (ii).

Implementation fl (Quantification of NADH) Reagent I and Reagent ■ having the following compositions were prepared. However, as the NAD (P) H oxidase contained in reagent 1, an enzyme derived from Bacillus lichensformis was used.

MJLL: (Final concentration) NAD(P)+1 oxidase solution 0.1u
/1ffiFAD
1mM phosphate buffer, pH 7,050mM Wipe J↓: (Final concentration) Peroxidase 10u/mn4
-Aminoantipyrine 0.005% disulfobutyl-m-toluidine (DSBmT) 0.0
2% phosphate buffer, pH 7,050mM NADH at 50% each. They were dissolved in 0.2M) Lis-HCl buffer (p118.0) to a concentration of 100 and 200 μH, respectively, and used as standard solutions. 0.3 ml of this standard solution was taken, 2.4 ml of Reagent I was added, and the mixture was incubated in a constant temperature bath at 37°C for 5 minutes. Add 0.6 ml of reagent ■ to this solution, and heat at 37°C for 5 ml.
After incubation for minutes, absorbance at 555 nm was measured.

In this measurement, a reagent-blind test with NAo++ [degree of Oμ] was used as a control. This NADI+ calibration curve is shown in FIG.

Real new elbow (quantification of free cholesterol in serum) To quantify free cholesterol in serum.

Reagent I and Reagent II having the following compositions were prepared and used. However, the NAD(P)+1 oxidase solution contained in Reagent I used an enzyme derived from Bacillus subtilis.

Qiyuetu: (Final concentration) Cholesterol dehydrogenase 0.5u/NA-free
D' 2mM
NAD(P)H oxidase solution 0.1u/
mRFAD
1mM disulfobutyl-m-toluidine (DSBmT) 0.02
% Triton
- Aminoantipyrine o, oos% phosphate buffer, pH 7,050+++M 25 μ2 of serum was collected, 2.4 tnl of Reagent I was added, and the mixture was incubated at 37°C for 5 minutes.
Incubated for minutes. Add reagent ■ to this solution at 0 and 6
After adding ml and incubating for 5 minutes at 37°C, the absorbance at 555rv+ was measured.

This measurement was performed using a reagent blind test using the above phosphate buffer instead of serum as a control. Separately, prepare a cholesterol standard solution (0-100 mg/a).

Measure the absorbance in the same manner as for the serum above.

A calibration curve was created. This cholesterol calibration curve was
As shown in the figure. The serum cholesterol concentration was calculated from this calibration curve. It was found that the serum free cholesterol value measured by this method showed a good correlation with the value measured using the conventional method shown in the following comparative example, and can be used for daily testing in the same way as the conventional method. A correlation diagram between the methods of the present example and the comparative example is shown in the fourth section.

Comparative Example (Quantification of free cholesterol in serum using cholesterol oxidase) Free cholesterol in serum was quantified using a reaction system not containing NAD(P)II oxidase, and Example 3
A comparison was made with the measured values.

A coloring reagent having the following composition was prepared.

Effect: (Final concentration) Cholesterol oxidase 5u/d1 peroxidase 15u/d14-aminoantipyrine 0.025% disulfobutyl-m-)luidine (DSBmT) 0.03% Triton X-1000, 15% Gud buffer, pH6.

Prepare 50 μl of the same serum used in Example 3,
To this, 3 mfl of the above coloring reagent was added and incubated at 37°C for 10 minutes. 600nm of this solution
The absorbance at
6,0) A reagent blind test using 50 μl was used as a control. Separately, a calibration curve was created using a cholesterol standard solution with the same concentration as in Example 3, and the cholesterol concentration in serum was calculated.

1!1l- (Quantification of total bile acids in serum) In order to quantify total bile acids in serum, Reagent I and Reagent ① having the following compositions were prepared. As NAD(P)11 oxidase contained in Reagent I.

An enzyme derived from Bacillus cereus was used.

(Left below) Wiper expert: (Final concentration) 3α-hydroxysteroid dehydrogenase 0.2u/ml
1 N.A.D.
2mM NADH oxidase solution 0.1u/mNFAD
1mM Toridoa X-
1000, 1% phosphate buffer, pH 7,050mM Wipe 11: (Final concentration) Peroxidase 10u/mR4
-Aminoantipyrine o, oi% disulfobuty) Lt-m, ) Luigi 7 (DSBmT)
0.02% phosphate buffer, pH 7,050mM serum 100μ! 3. Add 2 ml of Reagent I to
Incubate for 5 minutes at 7°C. Inl of reagent (1) was added to this solution, and after incubation at 37°C for 5 minutes, the absorbance at 555 nm was measured.

This measurement was performed using a reagent blind test using the above phosphate buffer instead of serum as a control. Separately, glycol M'J
A semi-liquid (25, 50, 75, 100uM) manufactured by OgJl.

Measure the absorbance in the same manner as for the serum above.

A calibration curve was created. This calibration curve is shown in FIG.

The total bile acid concentration in serum was calculated from this calibration curve.

Tip construction (quantification of glucose in blood) In order to quantify glucose in blood, reagent (1) and reagent (2) having the following compositions were prepared. As for the NAD (P) H oxidase contained in Reagent I.

An enzyme derived from Bacillus pumilus was used.

(Left below) LTLL= (Final concentration) Hexokinase 3u/dll8T
P
1mMG-6-PDH10u/
d1 NADP 1 m
MFAD' 1
mMNAD (P) H oxidase solution 0.
1u/mAMgCh
O, 01M triethanolamine-HCl-Na0f (buffer + pl (7,50, 1M buffer) (final concentration) Peroxidase 10u/Id Leucomethylene blue 2mM triethanolamine-HCl-NaOH buffer, pH 7,50, Prepare 5 μ! of 1M hemolyzed blood, add 2.4 ml of reagent ■, and incubate at 37°.
The cells were incubated at C for 10 minutes.

To this solution, 0.6% of reagent (1) was added, and after incubation at 37°C for 5 minutes, the absorbance at 600 nm was measured. This measurement was performed as a control in a reagent blind test using the above buffer solution instead of blood. Especially.

Prepare a glucose standard solution (0-1.000 mg/d1).

Measure the absorbance in the same manner as in the case of blood above.

A calibration curve was created. This calibration curve is shown in FIG.

Blood glucose concentration was calculated from this calibration curve.

(Effects of the Invention) According to the present invention, NAD(P)H can be quantified with high precision and with a simple operation. Since measurements can be made over a wide p)l range using the secondary method, substrates and enzymes that produce NAD (P) H through enzymatic reactions can be advantageously measured. In particular, the hydrogen peroxide produced in the 9-method is applied to the chromogen in the presence of peroxidase.
A method of measuring as a pigment is preferred. Since this method performs measurement by colorimetry, for example, various substrates and enzymes in biological samples can be effectively measured by the method of the present invention using a general-purpose colorimeter.

4. Simple 9'H of Pa Figure 1 is a graph showing the NADH consumption curve obtained in Example 1; Figure 2 is a graph showing the NADH calibration curve obtained in Example 2; Figure 3 is a graph showing the calibration curve of cholesterol obtained in Example 3; Fig. 4 is a graph showing the correlation between the results obtained by the methods of Example 3 and Comparative Example; A graph representing a calibration curve created for quantifying total bile acids; FIG. 6 is a graph representing a calibration curve created for quantifying blood glucose in Example 5.

that's all

Claims (1)

[Claims] 1. Reduced nicotinamide adenine dinucleotide (present in a sample or produced by an enzymatic reaction)
NADH) and/or reduced nicotinamide adenine dinucleotide phosphate (NADPH), the method comprising: (A) adding NADH to the sample and a reaction system containing oxygen;
and/or an enzyme that specifically catalyzes the oxidation of NADPH, and the reaction is carried out in the pH range of 7 to 11 to oxidize NA
A method for quantifying NADH and/or NADPH, which includes the steps of: generating D and/or NADP and hydrogen peroxide; and (B) measuring the generated hydrogen peroxide. 2. Furthermore, (C) adding a chromogen and peroxidase to the reaction system in which the hydrogen peroxide has been generated, oxidizing the chromogen to produce a coloring substance; and (D) measuring the coloring substance. 2. The method of claim 1, comprising the step of: 3. The method according to claim 1, wherein the enzyme that specifically catalyzes the oxidation of NADH and/or NADPH is derived from a bacterium belonging to the genus Bacillus.