JP2020033288A - Method for stabilizing NADH and NADPH - Google Patents

Abstract

To provide a method for stabilizing NADH and NADPH in a neutral region.SOLUTION: In an aqueous solution with a pH of 6.2-7.8, a material having a cation site coexists with NADH or NADPH.SELECTED DRAWING: None

Description

The present invention relates to a method for stabilizing NADH and NADPH.
INDUSTRIAL APPLICABILITY The present invention is useful in the field of life science such as clinical examination, clinical pathology and medicine, and in the field of chemistry such as analytical chemistry.

NADH (nicotinamide adenine dinucleotide [reduced form]) and NADPH (nicotinamide adenine dinucleotide phosphate [reduced form]) are electron carriers in vivo.
NADH and NADPH (hereinafter sometimes collectively referred to as "NADHs") act as coenzymes for various dehydrogenases.

For example, the following method is known as a method for measuring the activity of aspartate aminotransferase (AST) [GOT] in serum or plasma.
AST in the sample produces oxaloacetic acid and L-glutamic acid by contacting L-aspartic acid and α-ketoglutaric acid as substrates.
This oxaloacetate is converted to L-malate by the action of malate dehydrogenase (MD), and at the same time, NADH as a coenzyme is changed to NAD ⁺ [oxidized form].
Since NADH has an absorption maximum at 340 nm, the AST activity value can be determined by measuring the rate of decrease in the absorbance.

  However, it is known that NADHs have poor stability in an aqueous solution, and in particular, it has been known that stability is particularly poor in a neutral range to an acidic range as compared with a high pH range.

  Therefore, it was common for NADHs to be present in a high pH range.

  For example, in a method of extending the stable life of a reference reagent solution of NADH or NADPH under conditions that include a strong base such that the pH of the reference reagent solution rises to a basic range of at least about 7.9, the amine base Selected from the group consisting of a strong base, a base whose cation is considered to have a positive valence of 1, a base whose cation is considered to have a positive valence of 2, and a basic solution of a salt formed by the reaction of a strong base with a weak acid. There has been proposed a method characterized by containing one or more kinds of compounds (see Patent Document 1).

  A kit for determining a component of a biological sample, which is directly or indirectly affected by an enzymatic reaction in which NADH is a coenzyme, a) a reaction system affected by the component to be determined And b) as additional reagents, a concentration of about 1-4 mmol of NADH and an alkali metal or ammonium carbonate / bicarbonate buffer at a concentration of about 2-15 mmol. And an aqueous solution having a pH of about 9.5 to 11.0 (see Patent Document 2).

  Furthermore, a stabilized aqueous solution of a hydrogentransferase coenzyme, wherein the working solution has a pH value of at least 8.5 and is in the oxidized or reduced form of NAD, NADP or a suitable derivative; An aqueous solution containing ６０60 mM of a water-soluble heavy metal salt has been proposed (see Patent Document 3).

  By the way, many enzymes used in the measurement reaction have an optimum pH in a neutral range, and the stability of the enzyme is also stable in a neutral range.

  That is, there has been a demand for a method for stabilizing NADHs in a neutral region which is optimal for many enzymes.

JP-A-55-42599 JP-A-4-229192 Japanese Unexamined Patent Publication No. Hei 10-513063

NADHs used for coenzymes such as dehydrogenase in the measurement of substances to be measured in a sample are relatively stable in a high pH range, but their stability is not always good in a neutral range. .
However, most of the enzymes used in the measurement reaction have an optimum pH in a neutral range, and the stability of the enzyme is often stable in a neutral range.

  On the other hand, an object of the present invention is to provide a method for stabilizing NADHs in a neutral region.

  The present inventors have repeatedly studied a method for stabilizing NADHs in a neutral region, and found that the above problem can be solved by coexisting a substance having a cation site together with NADHs, and completed the present invention. I came to.

That is, the present invention includes the following inventions.
(1) A method for stabilizing NADH and NADPH, comprising coexisting a substance having a cation site together with NADH or NADPH in an aqueous solution of pH 6.2 to pH 7.8.
(2) The method for stabilizing NADH and NADPH according to the above (1), wherein the cation site is a Group 1 element or a Group 2 element.

  ADVANTAGE OF THE INVENTION The stabilization method of NADH and NADPH of this invention can maintain NADH and NADPH stably for a long period of time in aqueous solution of pH6.2-pH7.8.

  That is, the method for stabilizing NADH and NADPH of the present invention can keep NADH and NADPH stable for a long period of time in a neutral pH range where the enzyme used in the measurement reaction has an optimum pH and stability.

1. General The method for stabilizing NADH and NADPH of the present invention is characterized by allowing NADH or NADPH to coexist with a substance having a cation site in an aqueous solution of pH 6.2 to pH 7.8.

  The method for stabilizing NADH and NADPH of the present invention keeps NADH and NADPH stable for a long period of time by allowing NADH or NADPH and a substance having a cation site to coexist in an aqueous solution of pH 6.2 to pH 7.8. Is what you can do.

2. NADH in the Present Invention NADH and NADPH in the method for stabilizing NADH and NADPH of the present invention are not limited to NADH and NADPH, and include analogs and derivatives of NADH and NADPH.
For example, thioNADH and thioNADPH are also included in NADH and NADPH in the present invention.

The NADHs may be in a free form, or may be in a salt form.
Examples of the form of the salt include a salt with a Group 1 element, a Group 2 element, or the like.

  In this case, as the Group 1 element, for example, lithium, sodium, potassium, rubidium, or the like can be given.

  In addition, examples of the Group 2 element include beryllium, magnesium, calcium, strontium, and barium.

  As the form of the salt, a salt with a Group 1 element is preferable, a sodium salt or a potassium salt is more preferable, and a sodium salt is particularly preferable.

The concentration of NADHs in the aqueous solution is not particularly limited.
It can be applied from a trace area to a high concentration area.

3. Substance Having a Cation Site in the Present Invention (1) General In the method for stabilizing NADH and NADPH of the present invention, the substance having a cation site may be any substance having a cation site, and is not particularly limited.

(2) Cation Site In the method for stabilizing NADH and NADPH of the present invention, the cation site is a site consisting of a cation, and is not particularly limited.

  Examples of the cation site include a metal ion, an ammonium ion, and a guandium ion.

  Examples of the metal ion include ions of a Group 1 element, a Group 2 element, and other metals.

  Examples of the Group 1 element include lithium, sodium, potassium, rubidium and the like.

  Examples of Group 2 elements include beryllium, magnesium, calcium, strontium, barium, and the like.

  Examples of other metals include aluminum, gallium, indium, and germanium.

  As the cation site, for example, an ammonium ion, a guadinium ion, an ion of a Group 1 element, or an ion of a Group 2 element is preferable.

(3) Substance Having a Cation Site In the present invention, the substance having a cation site is not particularly limited, and may be any substance having a cation site.
Examples of the substance having a cation site include a hydroxide at the cation site and a salt at the cation site.

  Examples of the salt at the cation site include a salt with a halogen ion and a salt with an acid group.

  Examples of the halogen ion include a fluorine ion, a chlorine ion, a bromine ion, and an iodine ion.

  Examples of the acid group include a sulfuric acid group, a hydrochloric acid group, and a nitric acid group.

  Specific examples of the substance having a cation site include, for example, potassium chloride, calcium chloride, sodium chloride, aluminum chloride, potassium nitrate, sodium sulfate, sodium bromide, lithium chloride, beryllium sulfate, magnesium chloride, sodium decyl sulfate, sodium dextran sulfate. , Disodium EDTA, benzalkonium chloride, ammonium chloride, guanidine hydrochloride, arginine, lauryl dimethylamino acetate betaine, or carnitine.

  Preferred examples of the substance having a cation site include benzalkonium chloride, sodium chloride, calcium chloride, aluminum chloride, magnesium chloride, guanidine hydrochloride, arginine, and potassium chloride.

  The lower limit of the concentration of the substance having a cation site in the aqueous solution is not particularly limited, but is preferably 0.01 mM or more, more preferably 0.1 mM or more, and particularly preferably 1 mM or more.

  The upper limit of the concentration of the substance having a cation site in the aqueous solution is not particularly limited, but is preferably 10000 mM or less, more preferably 5000 mM or less, and particularly preferably 1000 mM or less in consideration of cost and the like.

(4) Aqueous Solution In the present invention, the aqueous solution having a pH of 6.2 to 7.8 preferably contains a known buffer having a buffering capacity in this pH range.
Examples of the buffer include phosphoric acid, tris (hydroxyl) aminomethane, or MES, Bis-Tris, ADA, PIPES, ACES, MOPSO, BES, MOPS, TES, HEPES, TAPSO, POPSO, HEPPSO, EPPS, Goodine or a good buffer such as TAPS.
In addition, the aqueous solution may contain an enzyme, an enzyme substrate, an enzyme stabilizer, or an enzyme activator.

(5) Measurement reagent An example in which the present invention is applied to a measurement reagent is shown below.

(A) Aspartate aminotransferase (AST) [GOT] activity measuring reagent

(I) First reagent (enzyme coenzyme reagent) [pH 6.2 to pH 7.8]
Sodium L-aspartate (for example, 1 mM to 3000 mM)
NADH.2Na (for example, 0.01 mM to 1 mM)
Substance having a cation site (for example, 0.01 mM to 10000 mM)
Malate dehydrogenase (for example, 100 U / L to 100,000 U / L)

(Ii) Second reagent (α-KG solution) [for example, pH 3.0 to pH 11.0]
Sodium L-aspartate (for example, 0 mM to 3000 mM)
α-Ketoglutaric acid (for example, 1 mM to 500 mM)

(Iii) Measurement principle AST in a sample generates oxaloacetic acid and L-glutamic acid by contacting L-aspartic acid and α-ketoglutaric acid as substrates.
The oxaloacetate is changed to L-malate by the action of malate dehydrogenase (MD), and at the same time, NADH and hydrogen ions as coenzymes are changed to NAD ⁺ .
Since NADH has an absorption maximum at 340 nm, the AST activity value can be determined by measuring the rate of decrease in the absorbance.

(Iv) Measurement example in which measurement is performed using an automatic analyzer 100 μL of the first reagent is added to 5.7 μL of the sample, and the mixture is incubated at 37 ° C. for 5 minutes. Then, 33 μL of the second reagent is added. Incubated at 5 ° C for 5 minutes. After the addition of the second reagent, the decrease rate of the absorbance of NADH is measured at the main wavelength of 340 nm and the sub wavelength of 405 nm, and the AST activity value in the sample is obtained.

(B) Alanine aminotransferase (ALT) [GPT] activity measuring reagent

(I) First reagent (enzyme coenzyme reagent) [pH 6.2 to pH 7.8]
L-alanine (for example, 1 mM to 3000 mM)
NADH.2Na (for example, 0.01 mM to 1 mM)
Substance having a cation site (for example, 0.01 mM to 10000 mM)
Lactate dehydrogenase (for example, 100 U / L to 100,000 U / L)

(Ii) Second reagent (α-KG solution) [for example, pH 3.0 to pH 11.0]
L-alanine (for example, 0 mM to 3000 mM)
α-Ketoglutaric acid (for example, 1 mM to 500 mM)

(Iii) Measurement principle ALT in a sample generates pyruvate and L-glutamic acid using L-alanine and α-ketoglutarate as substrates.
This pyruvate is changed to L-lactic acid by the action of lactate dehydrogenase, and at the same time, NADH and hydrogen ions are changed to NAD ⁺ .
Since NADH has an absorption maximum at 340 nm, the ALT activity value can be determined by measuring the rate of decrease in the absorbance.

(Iv) Measurement example in which measurement is performed using an automatic analyzer 100 μL of the first reagent is added to 5.7 μL of the sample, and the mixture is incubated at 37 ° C. for 5 minutes. Then, 33 μL of the second reagent is added. Incubated at 5 ° C for 5 minutes. After the addition of the second reagent, the decrease rate of the absorbance of NADH is measured at a main wavelength of 340 nm and a sub-wavelength of 405 nm, and the ALT activity value in the sample is determined.

(C) Urea nitrogen (BUN) measuring reagent (i) First reagent (enzyme reagent A) [pH 6.2 to pH 7.8]
NADPH.4Na (for example, 0.01 mM to 1 mM)
Substance having a cation site (for example, 0.01 mM to 10000 mM)
Glutamate dehydrogenase (for example, 100 U / L to 100,000 U / L)
α-Ketoglutaric acid (for example, 1 mM to 500 mM)

(Ii) Second reagent (enzyme reagent B) [for example, pH 3.0 to pH 11.0]
Urease (for example, 100 U / L to 100,000 U / L)
α-Ketoglutaric acid (for example, 0 mM to 500 mM)

(Iii) Measurement Principle First, ammonia, α-ketoglutarate, NADPH, and hydrogen ions are converted into glutamic acid, NADP ⁺ and water by glutamate dehydrogenase to eliminate endogenous ammonia in the specimen.
After eliminating endogenous ammonia in the sample, urea in the sample is decomposed into ammonia and carbon dioxide by the action of urease.
The ammonia and α-ketoglutarate are converted to glutamate by the action of glutamate dehydrogenase, and NADPH is simultaneously converted to NADP ⁺ .
Since NADPH has an absorption maximum at 340 nm, the urea nitrogen value can be determined by measuring the rate of decrease in the absorbance.

(Iv) Measurement example when measuring using an automatic analyzer A sample (1.7 μL) was added with the above-mentioned first reagent (100 μL), incubated at 37 ° C. for 5 minutes, and then the above-mentioned second reagent (25 μL) was added. Incubated at 5 ° C for 5 minutes. After the addition of the second reagent, the rate of decrease in the absorbance of NADPH is measured at a main wavelength of 340 nm and a sub-wavelength of 405 nm to determine the urea nitrogen value in the sample.

(D) Urea nitrogen (UN) measurement reagent [ICDH coexistence method]
(I) First reagent (enzyme reagent A) [pH 6.2 to pH 7.8]
Glutamate dehydrogenase (for example, 100 U / L to 100,000 U / L)
Isocitrate dehydrogenase (ICDH) (eg, 1 U / L to 100,000 U / L)
Potassium L-isocitrate (for example, 0.1 mM to 1000 mM)
NADPH.4Na (for example, 0.01 mM to 1 mM)
Substance having a cation site (for example, 0.01 mM to 10000 mM)
α-Ketoglutaric acid (for example, 1 mM to 500 mM)

(Ii) Second reagent (enzyme reagent B) [for example, pH 3.0 to pH 11.0]
Urease (for example, 100 U / L to 100,000 U / L)
α-Ketoglutaric acid (for example, 0 mM to 500 mM)

(Iii) Measurement Principle First, ammonia, α-ketoglutarate, NADPH, and hydrogen ions are converted into glutamic acid, NADP ⁺ and water by glutamate dehydrogenase to eliminate endogenous ammonia in the specimen.
NADP ⁺ generated at this time is converted to NADPH, α-ketoglutarate and carbon dioxide by the action of isocitrate dehydrogenase together with L-isocitrate.
After erasing endogenous ammonia in the sample, urea in the sample is decomposed into ammonia and carbon dioxide by the action of urease.
The ammonia and α-ketoglutarate are converted to glutamate by the action of glutamate dehydrogenase, and NADPH is simultaneously converted to NADP ⁺ .
Since NADPH has an absorption maximum at 340 nm, the urea nitrogen value can be determined by measuring the rate of decrease in the absorbance.

(Iv) Measurement example when measuring using an automatic analyzer 100 μL of the first reagent is added to 3.8 μL of the sample, and the mixture is incubated at 37 ° C. for 5 minutes. Then, 25 μL of the second reagent is added. Incubated at 5 ° C for 5 minutes. After the addition of the second reagent, the rate of decrease in the absorbance of NADPH is measured at a main wavelength of 340 nm and a sub-wavelength of 405 nm to determine the urea nitrogen value in the sample.

  Hereinafter, the present invention will be described specifically with reference to examples, but the present invention is not limited to these examples.

[Example 1] (Confirmation of stabilizing effect of NADHs by substance having cation site)
The effect of stabilizing NADHs by the substance having a cation site in the present invention was confirmed.

1. Aqueous solution Each aqueous solution was prepared by dissolving a substance having a cation site and a buffer in 0.3 mM disodium NADH or tetrasodium NADPH (tetrahydrate).

  In addition, the substance which has each cation site | part and its density | concentration, the buffer, its density | concentration, and pH were shown in Table 1.

  As substances having a cation site, potassium chloride [Kanto Chemical], calcium chloride (dihydrate) [Kokusan Kagaku], aluminum chloride hexahydrate [Fuji Film Wako Pure Chemical], potassium nitrate [Fuji Film Wako Pure Chemical] , Sodium sulfate [Kanto Chemical], sodium bromide [Fuji Film Wako Pure Chemical], lithium chloride [Kokuyo Chemical], beryllium sulfate (tetrahydrate) [Hayashi Junyaku Kogyo], magnesium chloride hexahydrate [Domestic Chemical] ], Sodium decyl sulfate [Nikko Chemicals], sodium dextran sulfate 5000 [Fuji Film Wako Pure Chemical], disodium EDTA (dihydrate) [Sekisui Medical], CA-101 [benzalkonium chloride] [Nikko Chemicals], chloride Ammonium [Fuji Film Wako Pure Chemical], Guanidine hydrochloride [Fuji Film Wako Pure Chemical], L-argi Emissions [Nacalai Tesque], AM-301 [lauryl betaine] [Nikko Chemicals], and DL- carnitine hydrochloride [Fuji Wako Pure Chemical] were used, respectively.

2. Measurement before storage The absorbance at 340 nm (absorbance 1) of each aqueous solution prepared in 1 above was measured with a spectrophotometer [J-Spectrum Co .; V-630].

3. Storage Each aqueous solution measured in 2 was stored at the temperature and for the period shown in Table 1, respectively.

4. Measurement The absorbance at 340 nm (absorbance 2) of each aqueous solution stored in the above section 3 was measured with a spectrophotometer [J-Spectrum Co .; V-630].

5. Residual absorbance ratio The value of “residual absorbance ratio” (unit: percent) was determined by multiplying 100 by the value obtained by dividing the value of “absorbance 2” in 4 above by the value of “absorbance 1” in 2 above.

  In addition, it is determined that NADH or NADPH is stabilized as the value of the “residual absorbance ratio” is higher.

6. Table 2 shows the measurement results.

From this table, it can be seen that the residual concentration ratio is increased when a substance having a cation site is coexisted as compared with a case where a substance having a cation site is not coexisted.
From this result, it was confirmed that NADH and NADPH can be stabilized by coexisting NADH or NADPH with a substance having a cation site in an aqueous solution of pH 6.2 to pH 7.8.

As the cationic site, for example, a metal ion, ammonium ion, or guar two indium ions.

Examples of the cation sites, for example, ammonium ions, guanidinium-ion, ions of alkali metal, or ion of the second group elements are preferred.

Claims (2)

  A method for stabilizing NADH and NADPH, comprising coexisting a substance having a cation site together with NADH or NADPH in an aqueous solution of pH 6.2 to pH 7.8.   The method for stabilizing NADH and NADPH according to claim 1, wherein the cation site is a Group 1 element or a Group 2 element.