JPH0450000B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel enzymatic highly sensitive measurement method that utilizes a cycling reaction in a method for quantifying either an aliphatic alcohol or an aliphatic aldehyde component in a test liquid.

Traditionally, enzymatic cycling involves NAD
(nicotine adenine dinucleotide) cycling, NADP (nicotine adenine dinucleotide phosphate) cycling, and CoA cycling are known. For example, using ethanol as a substrate,
Alcohol dehydrogenase is activated using NAD to generate reduced NAD, and this reduced NAD is converted into NAD by the activation of malate dehydrogenase using oxalacetic acid as a substrate.
It causes the cycling reaction of monoreduced NAD [edited by the Japanese Biochemical Society, "Biochemistry Experiment Course", Vol. 5, Enzyme Research Methods (1), pp. 121-135, Tokyo Kagaku Doujin Co., Ltd., August 1975. Published, edited by Akitane Mori, “Neurotransmitter Measurement Manual” No. 165~
172 pages, Ishiyaku Publishing Co., Ltd., November 1979].
It is also known that cycling uses reduced NAD oxidase in place of the malate dehydrogenase mentioned above to consume oxygen and reduced NAD to produce water molecules and NAD [edited by RIKEN,
“Current status and future of life sciences” pp. 30-32,
Sozo Life Science Research Group Co., Ltd., March 1981
Monthly issue]. Furthermore, hydroxysteroid dehydrogenase is activated using hydroxysteroid as a substrate to convert NAD into reduced NAD, and this reduced NAD undergoes cycling to form formazan and NAD through the action of a tetrazolium salt and a transposase such as diaphorase. Unexamined Japanese Patent Publication 1987-
No. 144096], glutathione dehydroascorbic acid oxidoreductase acts on glutathione and dehydroascorbic acid to convert dehydroascorbic acid into ascorbic acid, and this ascorbic acid consumes oxygen using ascorbate oxidase to form water molecules and dehydroascorbic acid. A cycling reaction of dehydroascorbic acid and ascorbic acid to produce ascorbic acid is also known [JP-A-56-151498]. In addition, NAD cycling using reduced NAD oxidase that consumes oxygen and generates hydrogen peroxide is also known, as described in JP-A-56-78599. In this way, various cycling reaction systems have been reported, and NAD cycling reactions using reduced NAD oxidase that generates hydrogen peroxide have also been reported.
NAD oxidase has not yet been purified well, and purification of the enzyme itself is difficult and expensive.

Conventional alcohol oxidase is an enzyme that oxidizes aliphatic alcohol to aliphatic carboxylic acid, and it was not known that it oxidizes aliphatic alcohol to an aldehyde.

However, when the alcohol oxidase enzyme reaction system and the alcohol dehydrogenase enzyme reaction system are combined, the alcohol oxidase enzyme reaction system produces H ₂ O ₂ , which has an oxidizing effect, and the reduced form, which has a reducing effect, is used for the alcohol dehydrogenase enzyme reaction system. Despite the coexistence of NAD(P), this oxidative effect is completely unexpected.
We have discovered a new cycling reaction that performs a good cycling reaction without causing a reaction between H ₂ O ₂ and the reducing NAD (P). Moreover, in this cycling reaction, using a test solution containing either aliphatic alcohol or aliphatic aldehyde involved in the reaction, alcohol oxidase, which is a necessary component other than the one component of the test solution, Alcohol dehydrogenase, reduced form
By reacting with NAD(P) and O ₂ , the cycling reaction progresses well with a quantitative reaction rate of more than 10 cycles per minute, and the amount of detectable change caused by the reaction can be quantified. By doing this, we were able to easily and accurately measure the components in the test liquid with high sensitivity.

The present invention has been completed based on the above findings, and provides a method for quantifying any one component of an aliphatic alcohol or an aliphatic aldehyde in a test liquid. In order to form the cycling reaction shown in , the components in the test solution are reacted with alcohol oxidase, alcohol dehydrogenase, reduced NAD (P), and O ₂ that form the cyling reaction [], and the cycling reaction is caused by the cyling reaction []. This is a highly sensitive enzymatic measurement method characterized by quantifying the amount of detectable change that occurs.

First, the test liquid of the present invention includes a system that contains one component of an aliphatic alcohol or an aliphatic aldehyde, or that liberates or produces one of the components. In particular, systems for liberating and producing one component in a test solution are used for the purpose of measuring enzyme activity and quantifying substrates using various enzyme reaction systems. Examples of these enzyme reaction systems include the following various reaction systems, but the present invention is not limited to any of them.

First, a system for liberating and producing aliphatic alcohol and aliphatic aldehyde will be exemplified.

Examples of aliphatic alcohols include ethanol,
Examples include lower aliphatic alcohols such as propanol and butanol, and examples of aliphatic aldehydes include lower aliphatic aldehydes such as acetaldehyde, propionaldehyde, and butyraldehyde, and specific examples include the following.

(1) Ethanol and butanol in alcohol fermentation, the amount of ethanol in the breath and blood after drinking alcoholic beverages, and the amount of ethanol in food and drinks.

(2) Enzyme reaction system using aliphatic alcohols and aliphatic aldehydes as substrates.

(3) Quantification of the enzyme activity of the enzyme reaction system that produces RCH ₂ OH and RCHO, and the original substrate.

In these enzyme reaction systems, a test solution containing either a component to be quantified or a component to be measured for activity is used as a test solution, and necessary reagents for carrying out the enzyme reaction system are added thereto and reacted. This reaction solution is mixed with any one of aliphatic alcohol, aliphatic aldehyde, NAD (P), and reduced NAD (P).
It may be used as a test solution containing the components.In this case, the enzyme reaction system may be performed separately and used as the test solution, or the enzyme reaction system may be used as a cycling reaction system in the same medium. The reaction may be carried out in one step. Furthermore, when reacting the enzyme reaction system, for example, 0.001
Using ~5 ml, mix this with a weakly acidic to weakly alkaline buffer containing the necessary reagents at 37°C for 1 hour.
It is sufficient to react for more than a minute. Further, the amount of the reagent used is not particularly limited, as long as it is used in excess of the amount of the target component to be measured in the test liquid.

In the above cycling reaction [ ], 1 molar ratio of aliphatic alcohol is used as a substrate, and 1
A molar ratio of O ₂ is consumed, normally dissolved oxygen is consumed, and a 1 molar ratio of aliphatic aldehyde and 1 molar ratio of H ₂ O ₂ are produced by the action of alcohol oxidase (AOX), resulting in an AOX-based reaction. In addition, 1 molar ratio of aliphatic aldehyde produced is converted into 1 molar ratio of NAD by the action of alcohol dehydrogenase (ADH) in the presence of 1 molar ratio of reduced NAD (P).
(P) and an ADH-based reaction that produces an aliphatic alcohol in a 1 molar ratio and an R aliphatic alcohol in a 1 molar ratio,
Furthermore, this aliphatic alcohol undergoes a cyclic reaction that produces an AOX reaction. Therefore, this cycle reaction [ ] can be carried out with the following six components: aliphatic alcohol, aliphatic aldehyde AOX, ADH, reduced NAD (P), and O ₂ . Alcohol and aliphatic aldehyde undergo a cycling reaction [ ], and if one component is used as the sample solution, the other component will be generated. It can be used as one component in a liquid.

Furthermore, in this cycling reaction []
NAD is produced by reacting H ₂ O ₂ with reduced NAD (P).
A more sensitive measurement method for producing (P) may also be used. That is, in the cycling reaction [],
NAD (P) peroxidase is an enzyme that catalyzes a reaction that consumes one molecule of H ₂ O ₂ and one molecule of reduced NAD (P) to produce two molecules of water (H ₂ O) and one molecule of NAD. (EC.1.11.1.1) [J.Biol.Chem.,
225, 557 (1957)], and is expressed by the following reaction [].

In this cycling reaction [], H ₂ O ₂ generated in the cycling reaction [] is further consumed together with reduced NAD (P) and becomes NAD.
To produce (P), the cycling reaction []
Two molecules of reduced NAD (P) are consumed in one cycle, resulting in twice the amount of change in reduced NAD (P) compared to the cycling reaction [ ], which allows for more sensitive measurement. Become.

Furthermore, this NAD(P) is converted into reduced NAD by a second dehydrogenase that uses NAD(P) as a substrate and a substrate compound for the dehydrogenase.
A cycling reaction of NAD(P) is carried out using a combination of components of NAD(P) to be converted to (P), a second dehydrogenase, and a substrate compound for dehydrogenase, and this cycling reaction and the cycling reaction of the above six components [ ] may be used in combination, and is expressed by the reaction () below.

Therefore, first, one component in the test solution based on the cycling reaction [] and the component [indicated by 〓] that forms the cycling reaction [] will be described.

(a) When one component in the test solution is an aliphatic alcohol or an aliphatic aldehyde: If aliphatic alcohol or aliphatic aldehyde is one of the components in the test solution, such as an enzyme reaction system that contains or liberates or produces aliphatic alcohol or aliphatic aldehyde, cycling reaction [Ia] should be carried out. The forming components are AOX, ADH,
O ₂ and reduced NAD (P) are used. In this cycling reaction [a], for example,
AOX uses aliphatic alcohol in the test solution as a substrate, consumes one molecule of aliphatic alcohol and O ₂ , and produces one molecule of H ₂ O ₂ with the same number of carbon atoms as the aliphatic alcohol in the test solution. A number of aliphatic aldehydes are produced, and this aliphatic aldehyde is further used as a substrate.
ADH is produced by consuming one molecule of aliphatic aldehyde with the same number of carbon atoms and one reduced NAD (P) to generate one molecule of aliphatic alcohol with the same number of carbon atoms and one molecule of NAD (P). aliphatic alcohol
Forms aliphatic aldehyde cycle reaction. However, O ₂ can also be obtained by using dissolved oxygen in the reaction system, thus reducing AOX, ADH, and the reduced form.
An excess amount of NAD(P) may be used as a reagent for measurement. Furthermore, when the component in the test solution is an aliphatic aldehyde, the cycling reaction is similarly formed by using each of the necessary components shown in [a] above. The content of aliphatic alcohol or aliphatic aldehyde in this test liquid is not particularly limited, and if the concentration is high, it may be used after dilution. Also used
The amount of AOX or ADH to be used is not particularly limited, and may be determined as appropriate based on the amount of aliphatic alcohol or aliphatic aldehyde in the test solution, and is relative to the amount of aliphatic alcohol or aliphatic aldehyde. It may be used depending on the situation, and it is usually sufficient to use 0.01 unit or more per test, preferably
The amount of AOX is about 0.1 to 100 units, and the amount of ADH is usually 0.1 or more units, preferably about 5 to 300 units, and larger amounts of each enzyme may be used. Furthermore, the amount of reduced NAD (P) used should be at least the product of the amount of aliphatic alcohol or aliphatic aldehyde in the test solution and the number of cycles determined by the reaction time; A large excess amount is used compared to the amount of, e.g.
It is preferable to use 50 times or more, preferably 100 to 10,000 times, and there is no limitation on using this amount or more. Furthermore, the amount of detectable change caused by the post-reaction of the cycling reaction [a], that is, the aliphatic alcohol-aliphatic aldehyde cycling reaction of reaction formula [a], is as follows:
Examples include the amount of O ₂ consumed by the reaction, the amount of reduced NAD (P) consumed, and the amount of H ₂ O ₂ generated. In addition, in this aliphatic alcohol-aliphatic aldehyde cycling reaction, H ₂ O ₂ is a generated component and reduced NAD is a consumed component.
(P) may be reacted and measured with higher sensitivity. That is, in this cycling reaction, one molecule
2 by consuming H ₂ O ₂ and one molecule of reduced NAD (P).
It is based on the reaction represented by the above reaction [], which uses a combination of NAD (P) peroxidase, which is an enzyme that catalyzes the reaction that produces a molecule of water and one molecule of NAD, and the aliphatic alcohol-aliphatic aldehyde NAD(P) is produced by reacting with reduced NAD(P) in proportion to the amount of H ₂ O ₂ produced in the cycling reaction [a]. In this reaction, NAD(P) peroxidase may be used generally at least 0.1 unit per test, preferably about 1 to 20 units. The amount of detectable change caused by the reaction is preferably measured by the amount of reduced NAD (P) consumed by the reaction.

Furthermore, aliphatic alcohol or aliphatic aldehyde may be quantified using the following reaction system.

In such a case, the components forming the cycling reaction [a] are AOX, ADH, O ₂ , reduced form
NAD(P), the second dehydrogenase, this second
The following dehydrogenase substrate compounds are used.
In this cycling reaction [a], the second dehydrogenase is converted into a reduced form by the aliphatic alcohol-aliphatic aldehyde cycling reaction.
It acts on NAD(P) generated from NAD(P), consumes one molecule of NAD(P) and a substrate compound for dehydrogenase, and generates one molecule of substrate compound and reduced NAD(P). , this reduced NAD(P)
is a cyclic reaction of aliphatic alcohol-aliphatic aldehyde accompanied by the production of NAD(P). This generated reduced NAD (P) is combined with ADH in an equimolar ratio of aliphatic aldehyde per molecule.
Under the action of AOX, one molecule of NAD (P) and aliphatic alcohol are produced, and by the action of AOX, one
It is formed by consuming molecules of RCH ₂ OH and O ₂ to produce one molecule of H ₂ O ₂ and aliphatic aldehyde. However, O ₂ is sufficient by using dissolved oxygen, and therefore,
AOX, ADH, reduced NAD (P), second dehydrogenase, and this second dehydrogenase substrate compound may be used in excess amounts as reagents for measurement. Further, the amount of detectable changes caused by the post-reaction of this cycling reaction [a] includes the amount of O ₂ consumed or the amount of H ₂ O ₂ produced, and the amount of oxide of the substrate compound. . Based on this cycling reaction [a], there is a good advantage that the amount of expensive reduced NAD (P) used can be saved.

In addition, the second dehydrogenase used in this cycling reaction [a] uses the dehydrogenase substrate compound and NAD(P) as substrates, and produces an oxide of the substrate compound and reduced NAD.
Any enzyme that catalyzes the reaction that produces (P) is sufficient. Examples include dehydrogenase that catalyzes the following reaction and a substrate compound for this dehydrogenase. Used in the cycling reaction of NAD(P) formed.

(1) Combination reaction in which the dehydrogenase is D-arabinitol dehydrogenase (A Dase: EC.1.1.1.11) and the substrate compound for dehydrogenase is D-arabitol: D-arabitol + NAD ---→ ADase D-xylose + Reduced NAD (2) Combination reaction in which the dehydrogenase is lactate dehydrogenase (EC.1.1.1.27) and the substrate compound for dehydrogenase is L-lactic acid: L-lactic acid + NAD ―――――――――――――― -→ Lactate dehydrogenase pyruvate + reduced NAD (3) Dehydrogenase is maleate dehydrogenase (decarboxylating)
(EC.1.1.1.28), combination reaction in which the substrate compound for dehydrogenase is L-malic acid: L-malic acid + NAD ――――――――――――――――→ Maleate dehydrogenase malate Dehydrogenase (Decarboxylating) Pyruvate + CO ₂ + Reduced NAD (4) Combination reaction in which the dehydrogenase is glycose dehydrogenase (EC.1.1.1.47) and the substrate compound for dehydrogenase is glucose: β-D-glucose + NAD --- ――――――――――――――→ ――――――――――――――――→ Glucose dehydrogenase D-glucono δ-lactone + reduced NAD Next, such aliphatic alcohol The target component is quantified in a test solution containing one of the following components: or aliphatic aldehyde, or in a test solution of an enzyme reaction system that liberates or produces one of the components. can be carried out based on the reaction between the target component and the above-mentioned cycling reaction, and since each cycling reaction generates 10 or more cycles of reaction for a given amount of the component in the test solution, at least the target component is In comparison, it is sufficient to use a reagent in an amount corresponding to a molar ratio greater than or equal to the number of cycles, and furthermore, an extremely small amount of the test liquid or a diluted test liquid may be used. The medium for this reaction may be one in the pH range where the activity of each enzyme used is stable, and is usually weakly acidic or weakly alkaline, such as phosphate buffer with a pH of 6.5 to 8.5, Tris-HCl buffer, imidazole-based HCl buffer, dimethylglutaric acid-NaOH buffer, pipece
A standard buffer such as NaOH buffer is used. Further, the reaction may be carried out usually at around 37°C for 1 minute or more. This cycling reaction [ ] varies depending on the amount of enzyme used and the Km value, but usually the reaction is performed at 10 cycles or more per minute, and preferably the enzyme amount is such that the reaction is performed at 20 cycles or more per minute. , other reagents may be used.

After reacting in this way, the amount of a detectable change caused by the reaction is then quantified. In a single cycle reaction of producing or consuming O 2 , O ₂ consumed, reduced form consumed,
Each component of NAD (P) and _H2O2 produced _| generated is mentioned. First, in quantifying the amount of O ₂ consumed,
Usually, it can be quantified as the amount of electrochemical change using an oxygen electrode. In addition, when quantifying the consumption amount of reduced NAD (P), the amount of reduced NAD (P) used in advance is
Reduced NAD remaining after reaction from NAD(P)
This is done by finding the difference in the amounts of (P).
To quantify the amount of reduced NAD (P) remaining after this reaction or the amount of reduced NAD (P) used in advance, various known methods for quantifying reduced NAD (P) are used. As a method for quantifying this reduced NAD (P), for example, there is no coexisting NAD (P), but the reduced form
The absorbance may be measured based on the wavelength in the specific absorption range of NAD(P). NAD (P) has a specific maximum absorption wavelength around 260 nm, and reduced NAD (P)
has specific maximum absorption wavelengths around 260 nm and 340 nm, so the absorption wavelength range that is the specific absorption wavelength for quantifying reduced NAD(P) is
Around 320nm to 360nm, preferably 340nm
It's nearby. The reduced form that remains due to this wavelength
It is quantified using NAD (P) as an absorbance value. Another method for quantifying reduced NAD (P) is
A method using a hydrogen atom transport system chromogen having the ability to accept the hydrogen atom of NAD(P) may be used. As this hydrogen atom transport system chromogen, for example, 3-
(p-iodophenyl)-2-(p-nitrophenyl)-5-phenyl-2H-tetrazolium chloride, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, 3,3'-(4,4'-biphenylylene)-bis(2,5-diphenyl-2H-tetrazolium chloride), 3,3'-(3,3'-dimethoxy-4,4'-biphenylylene)- Screw [2-
(p-nitrophenyl)-5-phenyl-2H-tetrazolium chloride] (also known as nitrotetrazolium: NTB), 3,3'-(3,3'-dimethoxy-4,4'-biphenylylene)-bis[2, 5
-bis(p-nitrophenyl)-2H-tetrazolium chloride], 3,3'-(3,3'-dimethoxy-4,4'-biphenylylene)-bis(2,5-
diphenyl-2H-tetrazolium chloride)
Tetrazolium salts such as, 2,6-dichlorophenol indophenol, etc. are used, and preferably those made by using a combination of a water-soluble tetrazolium salt and diaphorase or phenazine methosulfate to improve electron transfer are used. This hydrogen transport chromogen forms a formazan dye that develops color by receiving hydrogen atoms from reduced NAD (P), and converts this formazan dye into its absorption wavelength range, for example in the maximum absorption wavelength range of 500 nm to 550 nm. What is necessary is just to measure the absorbance based on this. As another measurement method, for example, reduced NAD (P) may be reacted with diaphorase in the presence of a fluorescent reagent such as resazurin, and the amount of the component that fluoresces due to the reaction may be quantified. This type of means for quantifying reduced NAD (P) is available. In particular, when quantifying reduced NAD (P), it is preferable to use catalase to decompose and eliminate H ₂ O ₂ since the cycling reaction [ ] involves the production of H ₂ O ₂ .
In order to further improve the sensitivity, NAD(P) peroxidase (EC.1.11.1.1, EC.1.11.1.2.) is further added to the cycling reaction system.
Convert the generated H ₂ O ₂ into reduced NAD (P),
Measurement may be performed with twice the sensitivity. Furthermore, H ₂ O ₂ , which is a generated component, can be quantified as the amount of electrochemical change using a hydrogen peroxide electrode.

As described above, the present invention is a novel quantitative determination method based on the aliphatic alcohol-aliphatic aldehyde cycling reaction, which performs a high-cycle reaction of 10 cycles or more per minute, and is capable of detecting analytes in a sample solution with extremely high sensitivity. It is an excellent device for measuring components.

Next, the present invention will be specifically described with reference to Examples, but the present invention is not limited thereto.

Example 1 Determination of acetaldehyde or ethanol 50m
M phosphate buffer (PH7.5) Alcohol dehydrogenase (manufactured by yeast) 40U/ml Alcohol oxidase (manufactured by Candida boidinii, manufactured by Sigma) 40U/ml 1.8mM Reduced NAD 0.2M Kcl A small test of 0.5ml of the reaction solution having the above composition Place in a tube and heat to 37℃, then add 0, 20, 40, 60, 80,
100 μM ethanol or 10 μM acetaldehyde
was added and reacted at 37°C for exactly 5 minutes.
0.5% SDS (dodecyl/sodium sulfate) solution (PH
7.5) The reaction was stopped by adding 2.5 ml, and the absorbance at 340 nm was measured. The results are shown in Figure 1, and the cycling rate was about 30 revolutions/minute.

○−○ethanol, ●−●acetaldehyde.

Example 2 To a reaction solution having the same composition as Example 1
The same reaction as in Example 1 was carried out using a reaction solution to which 1.2 U/ml of NAD-peroxidase (EC.1.11.1.1, StrePtococcus faecalis, manufactured by Sigma) or 400 U/ml of catalase was added. The results are shown in Figure 2, and when NAD-peroxidase was added, the sensitivity was doubled. Furthermore, when catalase was added, good linearity was obtained in the high value area. Note that acetaldehyde was used as the sample.

Example 3 1.0 ml of a reaction solution having the same composition as in Example 1 was placed in a reaction tank equipped with a galvanic oxygen electrode.
After keeping the temperature at 37℃, 0, 50, 100, 150,
200, 300, 400, 500 μM acetaldehyde solution
20μ was added and the oxygen consumption rate at this time was measured. The results are as shown in FIG. 3, and good linearity was obtained.

Example 4 Using 1.0 ml of a reaction solution having the same composition as in Example, a hydrogen peroxide electrode manufactured by YSI was installed, and 37
After heating to ℃, 0, 5, 10, 15, 20, 30, 40,
Add 10μ of 50μM ethanol and react.
The amount of ethanol at that time was measured in relation to the amount of H ₂ O ₂ produced, and the results are shown in ◯ to ◯ in Figure 4, and a good linear relationship was obtained.

Further, when the same procedure was carried out using propanol instead of the above-mentioned ethanol, a good linear relationship was obtained as shown in Figure 4 ●-●.

Example 5 0.5M phosphate buffer (PH7.5) 100ml Alcohol dehydrogenase (manufactured by yeast) 40000U Alcohol oxidase (derived from Pichia; manufactured by Fritz Petroleum) 40000U Reduced NAD 200μmo/es Kcl 0.2mo/e Sucrose 10g FAD 5μmo/es Freeze-dry 100ml of the solution with the above composition.
It was used as a reagent for 1000ml. This reagent is used as a reagent for quantifying aliphatic alcohols and aliphatic aldehydes, or for quantifying components of a reaction system that liberate them, or for measuring enzyme activities involved in them.

Example 6 Determination of ethanol and acetaldehyde 50m
M phosphate buffer (PH6.5) Alcohol dehydrogenase (manufactured by yeast) 40U/ml Alcohol oxidase (manufactured by Candida boidinii, manufactured by Sigma) 40U/ml 1.8mM Reduced NAD 0.2M Kcl 0.5ml of the reaction solution having the above composition was tested in a small test. Place in a tube and heat to 37℃, then add 0, 20, 40, 60, 80,
100 μM ethanol or 10 μM acetaldehyde
was added and reacted at 37°C for exactly 5 minutes.
0.5% SDS (dodecyl/sodium sulfate) (PH7.5)
The reaction was stopped by adding 2.5 ml, and the absorbance at 340 nm was measured. The results are shown in FIG. 5, and good linearity was obtained for both ethanol (○-○) and acetaldehyde (●-●), and the cycling rate was 19 revolutions/min.

[Brief explanation of the drawing]

Figure 1 is the quantitative curve for acetaldehyde and ethanol, Figure 2 is the quantitative curve for acetaldehyde, Figure 3 is the quantitative curve for acetaldehyde by enzyme electrode method, Figure 4 is the calibration curve for ethanol and prepanol, and Figure 5 is the analytical curve for ethanol and prepanol. A quantitative curve of acetaldehyde is shown.

Claims (1)

[Scope of Claims] 1. In a method for quantifying one component of an aliphatic alcohol or an aliphatic aldehyde in a test liquid, the formula (However, the number of carbon atoms in aliphatic alcohol and aliphatic aldehyde is the same.) In order to form the cycling reaction shown by A highly sensitive enzymatic measurement method characterized by reacting reduced NAD (P) and O ₂ and quantifying the amount of detectable change caused by the cycling reaction [ ]. 2 In the cycling reaction [], one molecule of
2 by consuming H ₂ O ₂ and one molecule of reduced NAD (P).
Claim 1, which is obtained by allowing NAD (P) peroxidase that catalyzes a reaction that produces one molecule of water and one molecule of NAD (P) to act, and then quantifying the amount of detectable change caused by the reaction. Measurement method described in section. 3 NAD produced in cycling reaction []
In (P), a second dehydrogenase that uses NAD (P) as a substrate and a substrate compound for the dehydrogenase are used to act on the generated NAD (P) to cycle NAD (P) to reduced NAD (P). 2. The measuring method according to claim 1, which comprises forming a second cycling reaction. 4. The measuring method according to claim 1, wherein the amount of change that can be detected is O ₂ consumption. 5. The measuring method according to claim 1, wherein the amount of change that can be detected is the amount of H ₂ O ₂ produced. 6. The measuring method according to claim 1, wherein the amount of change that can be detected is reduced NAD (P) consumption. 7. The measuring method according to claim 1, wherein the aliphatic alcohol is a lower aliphatic alcohol. 8. The measuring method according to claim 7, wherein the lower aliphatic alcohol is ethanol, propanol or butanol. 9. The measuring method according to claim 1, wherein the aliphatic aldehyde is a lower aliphatic aldehyde. 10. The measuring method according to claim 9, wherein the lower aliphatic aldehyde is acetaldehyde, propionaldehyde or butyraldehyde.