JPH0432987B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH).
This relates to a method for quantifying. In more detail, test samples, especially body fluid components,
NADH or NADPH, or NADH or
When quantifying components that generate NADPH directly or indirectly through coupling reactions, NADH or NADPH can be easily and quantitatively determined by the action of electron carriers or electron carriers and metal ions. The present invention relates to a method of oxidizing to NAD or NADPH and hydrogen peroxide. BACKGROUND ART Measuring the enzyme activity and the amount or concentration of substances (substrates) in biological samples, where the test sample is a body fluid component, is very important for diagnosing diseases, understanding therapeutic effects, and understanding the mechanisms of diseases. Body fluid components such as serum or urine are used as test samples, and dehydrogenases in these body fluid components, such as lactate dehydrogenase (LDH) and α-hydroxybutyrate dehydrogenase (α
When measuring the activity of dehydrogenases such as -HBD), or when measuring the activity of other enzymes in a test sample using these dehydrogenases,
Similarly, the substances (substrates) in the test sample are quantified using a dehydrogenase that specifically acts on substances (substrates) such as cholesterol, triglyceride, glucose, formalin, aldehyde, and bile acids in the test sample. In such cases, oxidized coenzymes are generally used to exert the action of dehydrogenase, and these oxidized coenzymes are converted and produced by the action of dehydrogenase. A commonly used method is to quantify the enzyme activity and substrate amount in a test sample by quantifying the reduced coenzyme. As the reduced coenzyme, reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) is usually used. NADPH
When quantifying these, it is common to measure their absorbance at 340 nm, or to react a tetrazolium salt with these NADH or NADPH to form a colored formazan, which is then colorimetrically quantified in the visible region. However, when measuring the absorbance at 340 nm, it is affected by substances that have absorption near 340 nm that coexist in the sample, such as bilirubin and hemoglobin, so it is necessary to measure the sample blind value. The equipment also requires special specifications for measuring ultraviolet absorption. Furthermore, in the visual coloring method using a tetrazolium salt, there is a problem with the colored formazan produced by the reaction between the reduced coenzyme and the tetrazolium salt. In other words, the produced colored formazan has low solubility in water and has strong dyeing and staining properties, so the dye may precipitate and stain cells and tubes, causing serious defects in measurement. It was becoming. Therefore, the inventors of the present invention came up with the following idea to eliminate such drawbacks and solve the problems. That is, in general, oxidizable coloring reagents have a wide variety of redox potentials, and the solubility in water and color tone can be selected relatively freely, and there are also many that do not have dyeing properties. There are features that can be selected depending on the intended use. Moreover, oxidizable coloring reagents are known to readily develop color quantitatively with hydrogen peroxide. If it is possible to quantitatively convert the reduced coenzyme into hydrogen peroxide, this hydrogen peroxide will easily quantitatively color the oxidizable coloring reagent, so if this is quantitatively determined,
The drawbacks and problems of the above conventional colorimetric method using tetrazolium salt can be easily overcome and solved. Based on this idea, the present inventors
As a result of intensive research into a method for quantitatively converting NADH or NADPH into hydrogen peroxide, NADH or NADPH
It was discovered that when an electron carrier is applied to hydrogen peroxide, hydrogen peroxide is produced quantitatively, leading to the completion of the present invention. That is, the present invention is characterized in that NADH or NADPH is treated with an electron carrier to quantitatively determine the amount of hydrogen peroxide produced.
This invention is a method for quantifying NADPH. NADH and phenazine methosulfate (PMS), an electron carrier, react at around pH 8 according to the following formula: NADH + PMS → NAD ⁺ + + PMSH ₂ PMSH ₂ +2O ₂ →2O ₂・−+2H ⁺ +PMS It is known that O ₂ - is produced, and it is used as a superoxide ion production system. However, superoxide ion O ₂ is generated by PMSH ₂ generated by the reaction of NADH and PMS.
Information regarding whether the _production of . , which has not been disclosed to date and is a completely unknown phenomenon. The present inventors believe that if the reaction between NADH and PMS described above quantitatively produces hydrogen peroxide, we can use this reaction to generate NADH and NADPH. Based on the idea that NADH or
As a result of intensive research into a method for quantitatively converting NADPH into hydrogen peroxide, we found that electron carriers such as PMS
It was found that hydrogen peroxide is quantitatively produced when NADH or NADPH is acted upon. That is,
The reaction of the present invention is shown by the following reaction formula. NADH + PMS (NADH) → NAD ⁺ + PMSH ₂ (NADP +) PMSH ₂ + O ₂ → PMS + H ₂ O _2If an electron carrier coexists in the reaction system in which hydrogen peroxide is produced from NADH or NADPH as described above, quantitative excess Hydrogen oxide is produced. At the same time as hydrogen peroxide is produced, if an oxidizable coloring reagent is present, this hydrogen peroxide can be oxidized and colored by a known oxidative coloring reaction.
It exhibits absorbance proportional to the concentration of NADH and NADPH. Therefore, in the present invention, it can be said that the electron carrier plays an important role in quantitatively generating hydrogen peroxide. The electron carriers used in the method of the present invention are phenazine methosulfate (PMS), 1-methoxyphenazine methosulfate (1-methoxy
PMS), 9-dimethylaminobenzo-α-phenazoxonium chloride (Meldra Blue), or any electron carrier having an action equivalent to these can be used. The concentration of this electron carrier is not particularly limited, but is usually 0.01% to 0.0001%.
A concentration of is preferably used. Furthermore, in the present invention, it has been found that the hydrogen peroxide production reaction is promoted by coexisting an electron carrier with divalent manganese ions or divalent cobalt ions. Next, embodiments of the present invention will be described. The present invention provides NADH or NADPH with an electron carrier,
or electron carrier and divalent manganese ion or 2
The method can be easily carried out according to a method known per se, except that hydrogen peroxide is quantitatively produced by the action of cobalt ions. The present invention enables a dehydrogenase to act on a substrate in the presence of oxidized nicotinamide adenine dinucleotide (NAD) or oxidized nicotinamide adenine dinucleotide phosphate (NADP) to quantitatively produce NADH or NADPH, Since it can also be carried out by acting with an electron carrier or an electron carrier and a divalent manganese ion or a divalent cobalt ion, such NAD,
NADP, substrate and dehydrogenase, or enzymes involved in enzymatic reactions that can be linked to these, NAD, NADP
and substrates can also be easily quantified. The electron carrier used in the present invention is PMS,
Refers to 1-methoxy PMS, Meldra Blue, or an electron carrier having an action equivalent to these. Compounds that give divalent manganese ions or divalent cobalt ions include inorganic acid salts of these metals, such as chlorides, sulfates, and nitrates, and organic acid salts, such as acetates, citrates, and tartrates. , ethylenediaminetetraacetate and the like are used, but it is needless to say that the compound is not limited to these compounds. Furthermore, the concentration of divalent manganese ions or divalent cobalt ions used is not particularly limited, but a concentration of 0.5mM/L to 10mM/L is usually preferably used. In the present invention, in order to quantitatively determine the amount of hydrogen peroxide produced, a method for quantifying hydrogen peroxide and a quantitative reagent that are known per se are sufficient. As such methods and reagents for quantifying hydrogen peroxide, which are known per se, there are methods for quantifying hydrogen peroxide and reagents for quantifying hydrogen peroxide, which measure the coloration of an oxidizable color reagent caused by hydrogen peroxide. The present invention allows dehydrogenase to act on a substrate in the presence of NAD or NADP to quantitatively quantify NADH or NADP.
It can also be used in enzymatic reactions known per se to produce NADPH. In such enzymatic reactions, the substrates are cholesterol, bile acids, glycerin, glycerin-3-phosphate, and glucose-6-phosphate.
Phosphoric acid, an aldehyde such as formaldehyde or acetaldehyde, and the dehydrogenases that act on the substrate in the presence of NAD and NADP are cholesterol dehydrogenase, bile dehydrogenase (3α-hydroxysteroid hydrogenase), glycerol dehydrogenase, and glycerol dehydrogenase, respectively. It can also be used for enzymatic reactions known per se, such as -3-phosphate dehydrogenase, glucose-6-phosphate dehydrogenase, formaldehyde dehydrogenase or aldehyde dehydrogenase; In such enzymatic reactions, the dehydrogenase is lactate dehydrogenase or α-hydroxybutyrate dehydrogenase, and the substrate on which the dehydrogenase acts in the presence of NAD or NADP is lactic acid or α-hydroxybutyrate, respectively. It can also be used for enzymatic reactions known per se. In the present invention, the test sample is a body fluid component, and hydrogen peroxide is quantitatively produced by a reaction in a solution, or hydrogen peroxide produced in this manner is quantitatively determined. can also be easily implemented. The present invention provides hydrogen peroxide that is quantitatively produced by a reaction in one solution, or hydrogen peroxide that is quantitatively produced in this way, by a reaction in the same solution, typically by a reaction in the same solution. Quantification can be performed by a color reaction of an oxidizable color reagent present in hydrogen peroxide. In the present invention, the first liquid is a liquid containing an electron carrier, an electron carrier, a divalent manganese ion, a divalent cobalt ion, or these and an oxidizable coloring reagent, but does not contain peroxidase. A liquid containing an oxidizable color reagent but not necessarily an electron carrier or an electron carrier and divalent manganese ions or divalent cobalt ions is used as the second liquid, and the first liquid is first added to the test sample. It can also be easily carried out by a two-liquid method, in which the mixture is incubated, a second liquid is added thereto, the incubation is carried out, and the resulting coloration is measured. To carry out the invention in a two-component method, for example:
Add an electron carrier to a sample containing NADH or NADPH, react for several minutes at room temperature or 37°C, then add a reagent containing peroxidase and an oxidizable coloring reagent, and react at room temperature or 37°C to develop color. Absorbance is measured using a reagent blind control as a control. At this time, if divalent manganese ions or divalent cobalt ions are present in the electron carrier, the hydrogen peroxide production reaction will be accelerated and the time required until the next addition of peroxidase and oxidizable coloring reagent will be shortened. can do. As the oxidizable coloring reagent used in the method and reagent of the present invention, it goes without saying that those commonly used in the H ₂ O ₂ -POD (peroxidase) system and known per se can be used. For example, when 4-aminoantipyrine/phenol is used as an oxidizable color reagent, absorbance at 505 nm may be measured, or 4-aminoantipyrine/3-methyl-N-ethyl-N-(β- hydroxyethyl)
When using aniline as an oxidizable color reagent, absorbance at 550 nm can be measured; when leucomalachite green is used as an oxidizable color reagent, absorbance at 625 nm can be measured. , 3-methyl-2-benzothiazoline hydrazone chromotropic acid system is used as an oxidizable coloring reagent, it is sufficient to measure the absorbance at 570 nm, and 2,2'-azino-bis-(3- When ethylbenzothiazoline-6-sulfonic acid) is used as an oxidizable color reagent, absorbance at 660 nm may be measured. There is usually no problem with the liquid nature of the reaction as long as it is within the range of PH4 to 10, but a pH range of 6 to 9 is often preferred. In this case, divalent manganese ions and divalent cobalt ions have a pH of 7.5.
If the temperature is higher than that, precipitation of hydroxides or basic compounds may occur, but in such cases,
Solubilization can be achieved by adding EDTA, tartrate, or citrate. In order to maintain the desired pH during the solution reaction, it is sufficient to use a buffer solution known per se.
Examples of such buffers include phosphate buffer,
Examples include Tris buffer and borate buffer. When using a phosphate buffer, a chelating agent such as EDTA may be added in order to prevent divalent manganese ions from being precipitated as manganese phosphate. Although the present invention provides a method that can easily quantify NADH or NADPH, it uses a tetrazolium salt, which has conventionally been used for quantitative determination to the extent that it is no exaggeration to say that it is fixed. Utilizes a reaction that quantitatively produces hydrogen peroxide, which is completely unrelated to and in contrast to the colorimetric method.
This invention is especially groundbreaking in that it provides a method for quantifying NADH or NADPH, and of course,
This is completely unrelated to the drawbacks and problems of colorimetric assay using tetrazolium salts, and NADH or
Quantitatively generate hydrogen peroxide from NADPH,
By quantifying this, NADH or NADPH can be determined.
This invention is of great significance in that it provides a method for quantifying the amount of quantification, and it has made an extremely significant contribution to this field. Furthermore, as shown in the Examples below, the present invention can be used to quantify body fluid components such as serum and urine, such as cholesterol, triglycerides, glucose, formalin, aldehydes, and bile acids, using enzymatic reactions. , the activity of dehydrogenase when NADH or NADPH is produced by a dehydrogenase that acts specifically on each substance (substrate), and how it is linked to the enzymatic reaction. The purpose of the present invention is to provide methods and reagents that can easily quantify the amount and activity of substances (substrates) involved in enzymatic reactions known per se, enzymes, NAD, NADP, etc. There are some things that make a huge contribution to the work. Example 1 Quantification of NADH 1-methoxyphenazine methosulfate
The first test solution was 0.05M phosphate buffer (PH7.5) containing 0.001%, 4-aminoantipyrine 0.01%,
A 0.1M phosphate buffer (PH7.5) containing 0.1% phenol and 600 u/dl of peroxidase is used as the second test solution. NADH 100, 200, 300, 400, 500, 600 respectively
Take 50μ of 0.1M phosphate buffer (PH7.5) containing mg/dl (hereinafter referred to as standard solution), and
ml and leave at room temperature for 3 minutes, immediately add 3 ml of the second reagent solution, heat in a thermostat at 37°C for 5 minutes, and measure the absorbance at a wavelength of 505 nm using a reagent blind test as a control. The calibration curve connecting the absorbance plotted for each NADH concentration (mg/dl) is a straight line passing through the origin, as shown in FIG. 1, and the calibration curve shows good quantitative properties. Example 2 Quantification of NADH 1-methoxyphenazine methosulfate
The first test solution was 0.05M phosphate buffer (PH7.5) containing 0.001%, and leucomalachite green 0.017
%, peroxadase 600u/dl, Triton X-
100 (Rohm & Haas product name) 0.7%,
A 0.05M phosphate buffer (PH7.5) containing 2% volume/volume ethanol is used as the second test solution. NADH standard solution (100, 200,
300, 400 mg/dl), add 1 ml of the first test solution, leave at room temperature for 3 minutes, immediately add the second test solution, leave at room temperature for 5 minutes, and then immediately set the sample at a wavelength of 625 nm using a reagent blind control. Measure the absorbance. The calibration curve connecting the absorbance plotted for each NADH concentration (mg/dl) is as shown in Figure 2.
Up to NADH300mg/dl, it becomes a straight line passing through the origin,
The calibration curve shows good quantitative properties. Example 3 Quantification of NADH 0.05M Tris-HCl buffer (PH7.0) containing Meldora blue 0.001%, manganese chloride (tetrahydrate) 5mM/L was used as the first test solution, 4-aminoantipyrine 0.01%, A 0.05M Tris-HCl buffer (PH7.0) containing 0.1% phenol and 600 u/dl of peroxidase is used as the second test solution. 50μ of 0.05M Tris-HCl buffer (PH7.0) containing 100, 200, 300, 400, 500mg/dl of NADH.
After adding 1 ml of the first reagent and heating in a 37°C thermostatic bath for 5 minutes, add 2 ml of the second reagent and further heating in a 37°C thermostatic bath for 5 minutes.
Measure the absorbance at 505 nm. The calibration curve connecting the absorbance plotted for each NADH concentration (mg/dl) is as shown in Figure 3.
A straight line passes through the origin, and the calibration curve shows good quantitative properties. Example 4 Determination of serum free cholesterol 1-methoxyphenazine methosulfate
0.001% NAD 100mg/dl, cholesterol dehydrogenase 200u/dl, Triton shall be. 4-aminoantipyrine 0.01
%, phenol 0.1%, peroxidase 600u/
Add 0.05M phosphate buffer (PH7.5) containing dl to the second
Use as test solution. Take 50 μ of serum, add 1 ml of the first test solution, and incubate at 37℃.
After heating in a constant temperature bath for 10 minutes, add 2 ml of the second test solution.
After heating at 37℃ for 15 minutes, the wavelength was adjusted using a reagent blind control.
Measure the absorbance at 505 nm. Separately, the free cholesterol concentration in serum is calculated from the absorbance obtained using a standard cholesterol solution (cholesterol 100 mg/dl) in the same manner as for serum. Reference example 1 Quantification of serum free cholesterol Phenol 0.1%, 4-aminoatipyrine 0.01
%, cholesterol oxadase 10u/dl, peroxidase 300u/dl, and Triton Use as coloring test solution. Take 50 μ of serum, add 3 ml of coloring reagent, and
After heating for 15 minutes in a thermostatic bath at °C, the absorbance at a wavelength of 505 nm is measured using a reagent blind test as a control. Separately, from the absorbance obtained using a cholesterol standard solution (cholesterol 100 mg/dl) in the same manner as for serum,
Calculate free cholesterol concentration in serum. As shown in Table 1, the values of Example 4 and the values of this reference example agree well, and no significant difference is observed between them.

[Table] Example 5 Determination of serum free cholesterol Meldora blue 0.001%, manganese chloride 5m
M/L, NAD 100mg/dl, cholesterol dehydrogenase 200u/dl, Triton X-100 (Rohm & Haas Company brand name) 0.2%,
These are dissolved in 0.05M Tris-HCl buffer (PH7.0) and used as the first test solution. 4-aminoantipyrine
0.01%, phenol 0.1%, peroxidase
0.05M Tris-HCl buffer (PH
7.0) as the second test solution. Take 50μ of serum and measure the absorbance using the same procedure as in Example 4 to determine the cholesterol concentration. As shown in Table 2, the values of Example 5 and the values of this reference example are in good agreement, and no significant difference is observed between them.

【table】

【table】 [Brief explanation of drawings]

Figures 1, 2, and 3 show the calibration curves obtained in Example 1, Example 2, and Example 3, respectively, and the calibration curves obtained for each NADH concentration (mg/dl) on the horizontal axis. A calibration curve for NADH is shown, which connects the points obtained by plotting the measured absorbance along the vertical axis.

Claims (1)

[Claims] 1. Quantitative determination of hydrogen peroxide produced by acting an electron carrier on reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) NADH or
Quantification method of NADPH. 2 In the presence of oxidized nicotinamide adenine dinucleotide (NAD) or oxidized nicotinamide adenine dinucleotide phosphate (NADP), a dehydrogenase is applied to the substrate to produce it quantitatively.
The quantitative method according to claim 1, wherein NADH or NADPH is made to act with an electron carrier. 3 Electron carriers are phenazine methosulfate (PMS), 1-methoxyphenazine methosulfate (1-methoxyPMS), and 9-dimethylaminobenzo-α-phenazoxonium chloride (Meldra Blue). A quantitative method according to claim 1 or 2. 4. The quantitative method according to any one of claims 1 to 3, wherein divalent manganese ions or divalent cobalt ions are allowed to coexist when an electron carrier acts on NADH or NADPH.