JPH04341198A - Process for high-sensitivity determination of alcohols or aldehydes and composition therefor - Google Patents

Abstract

PURPOSE:To enable quick and accurate determination of the subject compounds useful for clinical examination, etc., with a small amount of specimen by treating a specimen with alcohol dehydrogenase, a thio NAD(P) and an NAD(P), thereby performing an enzymic cycling reaction. CONSTITUTION:A specimen containing one or more kinds of the objective compounds such as alcohols and aldehydes is treated with a reagent containing (A) a coenzyme A1 such as thionicotinamide adeninedinucleotide (thio NAD) and thionicotinamide adenine dinucleotide phosphate (thio NADP), (B) reduction product of nicotinamide adeninedinucleotide (NAD), nicotinamide adenine dinucleotide phosphate (NADP), etc., and (C) an enzyme such as alcohol dehydrogenase. A cycling reaction of formula (A2 is reduction product of the component A1; B2 is oxidized product of the component B1) takes place by the above process. The variation of the amount of the component A2 or B1 caused by the reaction is measured to enable the highly sensitive quantitative determination of the objective compounds.

Description

[Detailed description of the invention]

0001

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a highly sensitive method for quantifying alcohols or aldehydes, or substances containing these as reaction products, which are important in the fields of clinical testing, food testing, etc., and a composition for quantitative determination.

0002

[Prior Art] Generally, it is difficult to measure alcohols and aldehydes, which are metabolites of alcohols by alcohol dehydrogenases, in clinical tests and food chemistry.
It is extremely important, and in recent years, enzymatic measurement methods with higher specificity have become popular in place of chemical methods for analyzing these components.

Conventionally, methods using alcohol dehydrogenase (EC 1.1.1.1), which catalyzes the following reactions, have been used for enzymatic measurement of alcohols and aldehydes.

0004

[Chemical 2]

The measurement method using this enzyme is also used in clinical testing and forensic medicine other than food chemistry, and kits for measuring blood ethanol are commercially available from Sigma and other companies. In addition, a kit for analyzing ethanol in foods is commercially available from Boehringer Mannheim, and a kit for quantifying acetaldehyde using this reverse reaction is also commercially available from Boehringer Mannheim as a food analysis enzymatic method reagent. .

[0006] Alcohol dehydrogenase (EC1.1.1.1) used in these measurement methods has a loose substrate specificity and is characterized by acting on many primary and secondary alcohols and aromatic alcohols. however,
The above reaction in this enzyme has a large equilibrium biased toward the alcohol side, so in order to advance the reaction in the direction of aldehyde production, the pH of the reaction solution must be adjusted to the alkaline side of 9 to 10, and the product aldehyde must be It is necessary to add a scavenger such as semicarbazide to the reaction solution in order to remove it from the reaction system, which has the drawback of complicating operations.

Therefore, in the above reaction, acetylpyridine adenine dinucleotide (acetyl NAD), which is an analog of NAD, was used instead of NAD as a coenzyme.
Methods of Enzymatic A
analysis, 3rd ed. , vol. 3,
1052, Academic Press, Inc.
In addition, reduced NAD (P) produced in the above reaction has been measured as a formazan dye using diaphorase and nitro blue tetrazolium.

[0008] Generally, when performing an analysis using an enzyme, the target substance to be measured is converted into spectroscopically detectable hydrogen peroxide, reduced NAD (P), etc., as in the method described above. There are many cases. However, reduced NAD(P)
As mentioned above, when measuring by absorbance at 340 nm, the reaction equilibrium shifts in the opposite direction to the production of reduced NAD (P) due to the accumulation of reduced NAD (P), so strict pH control is required. The problem is that it should not be. Furthermore, measurement methods that utilize color reactions using diaphorase and nitroblue tetrazolium also detect reduced NAD (P
) Although it is possible to prevent product inhibition due to accumulation, formazan dye is poorly soluble and tends to adhere to test tubes and tubes, making it difficult to apply to automatic analyzers.

[0009]Currently, the most popular method for measuring this detectable substance is to use a spectroscopic analyzer, but this also has a limit in sensitivity and is not suitable when the content of the substance to be measured is small. There was a drawback. Therefore, alcohol dehydrogenase is currently only used for the determination of ethanol or acetaldehyde, despite its loose substrate specificity.

[0010] On the other hand, when the content of the object to be measured is small,
Fluorescence analysis, luminescence analysis, etc., which are more sensitive than spectroscopic analysis, are used when the amount of the sample containing the object to be measured is small. However, these methods are not very suitable for general-purpose tests such as clinical tests from the viewpoint of widespread use of the equipment.

[0011] Another method for measuring trace amounts of a substance is the so-called enzyme cycling method, in which the coenzyme is amplified using two types of enzymes, if the substance can be converted into an equal amount of coenzyme. It is being done. For example, N.A.D.
Although cycling methods, CoA cycling methods, ATP cycling methods, etc. have been used, the current situation is that none of these methods is used in routine analyzes such as clinical tests because the operations are too complicated.

Therefore, it is desired to develop a highly sensitive method for measuring alcohols or aldehydes that can also be applied to spectroscopic analysis instruments.

[0013] The advantage of the high-sensitivity measurement method is that it is possible to reduce the amount of sample required for measurement, not only when the content of the target substance is small, but also when it is possible to reduce the amount of sample required for measurement. When used for a specimen, the influence of coexisting substances on the measurement system can be reduced. In addition, it is possible to increase the number of items that can be tested with a limited amount of sample, and furthermore, in cases where the sample is human blood, the amount of blood collected can be reduced, which reduces the psychological impact on the person receiving blood. It can also reduce the burden. In this way, increasing the detection sensitivity is an inevitable requirement in view of the use of precious specimens such as blood in clinical tests and the necessity of measuring trace components.

[0014]

[Problems to be Solved by the Invention] As mentioned above, various methods for measuring alcohols or aldehydes using alcohol dehydrogenase have been reported, but none of them are yet satisfactory, and it is difficult to measure accurately and with high sensitivity. Law was desired.

[0015]

[Means for Solving the Problems] Under these circumstances, the present inventors have made extensive studies to solve the above problems, and have found that alcohol is In a reaction system using dehydrogenases, thionicotinamide adenine dinucleotides (hereinafter referred to as thioNADs) and thionicotinamide adenine dinucleotide phosphates (hereinafter referred to as thioNADPs) are used as coenzymes.
one selected from the group consisting of nicotinamide adenine dinucleotides (hereinafter referred to as N
We have discovered a cycling reaction that uses two specific types of coenzymes, one selected from the group consisting of ADs) and nicotinamide adenine dinucleotide phosphates (hereinafter referred to as NADPs).

Furthermore, in this reaction, the absorption wavelength of the reduced forms of thio-NADs and thio-NADPs is around 400 nm, and the absorption wavelength of the reduced forms of NADs and NADPs is around 340 nm. By quantifying only the amount of change in the coenzyme by measuring the absorbance at one of the wavelengths, it is possible to perform an enzyme cycling reaction that avoids crowding of the absorption wavelengths of other substances when measuring absorbance. We have confirmed that it is possible to quantify aldehydes and aldehydes, and have completed the present invention.

That is, the present invention provides a test substance containing one or more test components selected from the group consisting of alcohols and aldehydes; (1) thioNADPs and thioNADs; and one or more alcohol dehydrogenases that perform a reversible reaction that uses one coenzyme and one selected from the group consisting of NADPs and NADs to produce aldehydes corresponding to alcohols using at least alcohol as a substrate. By reacting with a reagent containing the enzyme, (2) A1 (3) B1, the following reaction formula is obtained.

0018
]

[C3]

(In the formula, A1 is thioNADP, thioNA
Class D indicates NADPs or NADs, A2 indicates the reduced product of A1, B1 indicates reduced NADPs or reduced NADs when A1 is thioNADPs or thioNADs, and A1 indicates NADP. or NADs indicates reduced thioNADPs or reduced thioNADs, and B2 indicates the oxidized product of B1), and A2 changed by the reaction
or a highly sensitive quantitative method for one kind of test component selected from the group consisting of alcohols and aldehydes, and the above (1), (2) and (3). The present invention provides a composition for highly sensitive quantitative determination of one or more test components selected from the group consisting of alcohols and aldehydes.

[0020] In the present invention, alcohols include primary and secondary alcohols.
alcohols and aromatic alcohols, and aldehydes refer to aldehydes corresponding to the alcohols.

The alcohol dehydrogenases used in the present invention catalyze a reversible reaction that produces aldehydes using at least alcohol as a substrate, and are selected from the group consisting of thioNADPs and thioNADs. Any coenzyme containing one selected from the group consisting of NADPs and NADs can be used.

[0022] This enzyme is widely present in animal tissues, plants, bacteria, etc. Specifically, alcohol dehydrogenases specific for (thio)NADs (EC 1.1) use primary and secondary alcohols as substrates.
．． 1.1) and (thio)NADP-specific alcohol dehydrogenase (EC 1.1.1.2). Alcohol dehydrogenase (EC 1.1.1
．． 1) is present in yeast and mammalian livers, etc., and its properties often differ between species and tissues, making it an enzyme with remarkable diversity. For example, alcohol dehydrogenase derived from yeast acts on ethanol and allyl alcohol to the same extent, propanol, butanol, pentanol, 2-
The relative activity decreases in the order of propanol (Enzyme Handbook, p1, Asakura Shoten, 1983). In addition, it is generally said that yeast-derived enzymes have higher substrate specificity than mammalian-derived enzymes, but enzymes derived from yeast, human liver, and horse liver all have higher substrate specificity than primary alcohols.
-Unsaturated alcohols are more reactive (Arch.
Biochem. Biophys. , 159, 5
0-60, 1973). Alcohol dehydrogenase (EC 1.1.1.2) is also produced by Leuconostoc mesenteroides.
teroides), Saccharomycopsis lipol
(Enzyme Handbook, p1, Asakura Shoten, 1983).

Furthermore, among alcohol dehydrogenases, those that use aromatics as substrates are sometimes called allylic alcohol dehydrogenases, and a specific example of this is allylic alcohol dehydrogenase (EC), which is specific for (thio)NADs. 1.1.1.90) or (thio)
Allyl alcohol dehydrogenase (EC 1.1.1.91) specific for NADPs is mentioned. Allyl alcohol dehydrogenase (EC 1.1.1.90)
is Pseudomonas putida T-2 (Pseudomonas putida T-2)
as putida T-2), Mycobacterium
Mycobacterium tuberculosis
eculosis), etc. The enzyme derived from Pseudomonas putida T-2 contains methanol,
ethanol, propanol, isopropyl alcohol,
Butanol and isobutyl alcohol have no effect on alcohol dehydrogenase (EC 1.1.1).
．． 1) (Biochim. Biop
hys. Acta, 139, 173-176,
1967). Allyl alcohol dehydrogenase (EC 1.1.1.91) is also produced in Neurosupora crassa (wil
d type SY7A), etc. (Enzyme Handbook, p. 34, Asakura Shoten, 198
3).

Among these alcohol dehydrogenases, those derived from EC 1.1.1.1 yeast and horse liver are commercially available from, for example, Oriental Yeast Kogyo Co., Ltd., Boehringa Mannheim, Sigma, etc. There is. When examining the activities of these enzymes, for example, the relative activity of the horse liver-derived enzyme commercially available from Sigma Co., Ltd. with respect to the coenzyme was found to be 40 mM glycine-NaOH buffer (pH 9.
In 5), when the substrate is ethanol and NAD is used as 100%, thio-NAD is about 363%. In addition, the relative activity of the yeast-derived enzyme commercially available from Oriental Yeast Industry Co., Ltd. with respect to the coenzyme is 40mM.
In the case of glycine-NaOH buffer (pH 9.5), when the substrate is ethanol and NAD is used as 100%, thio-NAD is about 1.5%.

[0025] In the present invention, enzymes from other sources other than those mentioned above may be used as long as they are reactive with alcohols as substrates and have specificity for the coenzymes described below. The specificity for the coenzyme can be confirmed using a coenzyme and a substrate as appropriate.

In the present invention, coenzymes of A1 and B2 include thioNADPs, thioNADs, NADPs,
Examples of thioNADPs or thioNADs include thionicotinamide adenine dinucleotide phosphate (thioNADP), thionicotinamide hypoxanthine dinucleotide phosphate, and thionicotinamide adenine dinucleotide (thioNAD). , thionicotinamide hypoxanthine dinucleotide. Further, examples of NADPs or NADs include nicotinamide adenine dinucleotide phosphate (NADP), acetylpyridine adenine dinucleotide phosphate (acetylNADP),
Acetylpyridine Hypoxanthine Dinucleotide Phosphate, Nicotinamide Hypoxanthine Dinucleotide Phosphate (Deamino NADP), Nicotinamide Adenine Dinucleotide (NAD), Acetyl Pyridine Adenine Dinucleotide (Acetyl NAD), Acetyl Pyridine Hypoxanthine Dinucleotide, Nicotinamide Hypoxanthine Examples include dinucleotides (deamino NAD). The reduced forms of these coenzymes are thioNA and
Displayed as DPHs, thioNADHs, NADPHs, and NADHs.

In the present invention, in A1 and B1, for example, when A1 is thioNAD(P), B1 is N
It must be an AD(P)H class, and A1 is NAD
(P), B1 needs to be thioNAD(P)H.

When the alcohol dehydrogenase used for quantitative determination uses (thio)NADs as the only coenzyme, the above-mentioned thioNADs and NADs may be used. In addition, when the alcohol dehydrogenase used uses only (thio)NADPs as a coenzyme, the above thioNADPs and NA
Thio-NADPs and NADPs may be used in place of D, and the alcohol dehydrogenases used are (
When using both thio)NADs and (thio)NADPs as coenzymes, thioNADs and thioNADPs and NA
It may be appropriately selected from Class D and NADP, and their oxidized and reduced forms may be used as appropriate.

When performing measurements using the highly sensitive quantitative method of the present invention, the relative activity between each coenzyme of the alcohol dehydrogenases used should be taken into account so that the enzyme cycling reaction proceeds most efficiently. It is preferable to select two types of coenzymes and set the ratio of their use, and further set the reaction pH within the optimum pH range for the forward reaction/reverse reaction.

Furthermore, according to the quantitative method of the present invention, it is possible to quantify not only alcohols but also aldehydes in a sample. Alcohols or aldehydes are not particularly limited as long as they are substrates or products of the enzyme used; examples include ethanol, allyl alcohol (
2-propen-1-ol), propanol, butanol, benzyl alcohol, p-hydroxybenzyl alcohol, anisaldehyde, acetaldehyde, allylaldehyde, propionaldehyde, butyraldehyde, benzaldehyde, p-hydroxybenzaldehyde, anisaldehyde, and the like.

[0031] Furthermore, if the highly sensitive quantitative method of the present invention is used,
It is also possible to measure substrates and their enzyme activities in enzyme systems that liberate and produce alcohols or aldehydes. Furthermore, by using the highly sensitive quantitative determination method of the present invention, it is possible to obtain substrates and their enzymes in enzyme systems consisting of a single or multiple steps that can be linked to enzyme systems that liberate or produce alcohols or aldehydes as described above. Activity can also be measured. These enzyme systems are not particularly limited, and include, for example, the various reaction systems shown below.

(1) Enzyme reaction system between benzylamine and monoamine oxidase (EC 1.4.3.6). In this system, benzylamine can be quantified or monoamine oxidase activity can be measured by quantifying benzaldehyde released or produced. Benzylamine +O2 + H2O
→ Benzaldehyde + NH3 + H2O2

00
33] (2) Tyramine and tyramine oxidase (EC
1.4.3.9) Enzyme reaction system. In this system, tyramine can be quantified or tyramine oxidase activity can be measured by quantifying p-hydroxybenzylaldehyde that is released or produced. Tyramine +O2 + H2O
→ p-hydroxybenzylaldehyde + NH3 + H2O2

(3
) When the tyramine in (2) is derived from an enzyme reaction system of tyrosine and tyrosine decarboxylase (EC 4.1.1.25). In this case, by quantifying finally produced p-hydroxybenzylaldehyde, it is possible to quantify tyrosine or measure the activity of tyrosine decarboxylase. Tyrosine → Tyramine + CO2

003
5] In the quantitative composition of the present invention, A1 and B1
The concentration of is 0.02-100mM, especially 0.05-20mM
M is preferable, and the amount of alcohol dehydrogenase is 1
~1000 u/ml, particularly 2 ~ 400 u/ml is preferable, but the amount can be appropriately determined depending on the type of subject, etc., and a larger amount can also be used.

[0036] In the present invention, the amounts of A1 and B1 are in excess compared to the total amount of alcohols and aldehydes in the test sample, and the Km values for alcohol dehydrogenases for A1 and B1, respectively. It is necessary that the amount is in excess, especially for the two test components.
0 to 10,000 times the molar amount is preferable.

[0037] In addition, in the quantitative method of the present invention, when alcohol dehydrogenases use both (thio)NAD and (thio)NADP as coenzymes either alone or in combination of two or more, thioNAD is added to the two coenzymes. When selecting a combination of NADs and NADPs or a combination of thio-NADPs and NADs or NADPs, a second compound that does not act on alcohols and forms a B2→B1 reaction is added to the analyte. B1 and B2 are reacted with the dehydrogenase and the substrate of the second dehydrogenase, as shown in the reaction formula (Chemical formula 4) below.
The cycling reaction can be formed by providing a reaction system for the regeneration of B1 during the reaction.

That is, the present invention provides a test substance containing one or more test components selected from the group consisting of alcohols and aldehydes; (1) one selected from the group consisting of NADPs and thioNADs; and one or more alcohol dehydrogenases, which use one selected from the group consisting of NADPs and NADs as a coenzyme, and perform a reversible reaction that uses at least alcohol as a substrate to produce aldehydes corresponding to the alcohols. an enzyme, (2) A1 (3) B1 or/and B2 (4) a second dehydrogenase that does not act on alcohols and forms a B2→B1 reaction, and a reagent containing a substrate for the second dehydrogenase; At least the following reaction equation

0039
]

[C4]

(In the formula, A1 is thioNADP, thioNA
Class D indicates NADPs or NADs, A2 indicates the reduced product of A1, B1 indicates reduced NADPs or reduced NADs when A1 is thioNADPs or thioNADs, and A1 indicates NADP. or NADs indicates reduced thioNADPs or reduced thioNADs, B2 indicates the oxidized product of B1, and B2→B1 indicates B2
is selected from the group consisting of alcohols and aldehydes, and is characterized by forming a cycling reaction represented by The present invention provides a highly sensitive method for quantifying one or more test components.

In this case, it is preferable that the second dehydrogenase does not substantially act on A1 in this measurement system, or that conditions are set such that it cannot substantially act on A1. a combination of selecting a second dehydrogenase that is not used as a coenzyme;
Examples include combinations in which conditions are selected in which the second dehydrogenase does not substantially act on A1 depending on the quantitative relationship between A1 and B2. For quantitative determination, the amount of A2 produced by the reaction is measured.

[0042] In the quantitative composition using the above components (1) to (4), the concentration of A1 is 0.02 to 100mM;
Particularly preferred is 0.05-20mM, and the concentration of B2 or/and B1 is 0.05-5000μM, especially 5-50mM.
0 μM is preferable, the concentration of alcohol dehydrogenases is preferably 1 to 1000 u/ml, especially 2 to 400 u/ml, and the concentration of the second dehydrogenase is usually 1 to 1
00 u/ml is preferable, and the amount may be prepared to be at least 20 times the Km value (mM unit) for B2 (in u/ml unit), and the substrate for the second dehydrogenase may be added in an excess amount, for example 0.05 to 20mM is preferred. These amounts can be appropriately determined depending on the type of subject, etc., and larger amounts can also be used.

[0043] The second dehydrogenase is added as an auxiliary for the regeneration of B1, and this makes it possible to reduce the amount of B1 used, which is particularly effective when B1 is expensive. Furthermore, the reaction may be performed using B2 or a mixture of B1 and B2 instead of B1. In this case, the amount of B1 and/or B2 used is not particularly limited, but is generally preferably 1/10 mole or less of A1.

As the second dehydrogenase and its substrate, for example, when B2 is NADs or thioNADs, glycerol dehydrogenase (EC 1.1
．． 1.6) (derived from E. coli) and glycerol, L-
Glycerol-3-phosphate dehydrogenase (EC.1
．． 1.1.8) (derived from rabbit muscle) and L-glycerol-3-phosphate, malate dehydrogenase (EC.1.
1.1.37) (derived from pig myocardium, bovine myocardium) and L-malic acid, glyceraldehyde phosphate dehydrogenase (E
C 1.1.1.12) (rabbit skeletal muscle, liver, yeast, E
．． coli origin) and D-glyceraldehyde phosphate and phosphoric acid, and when B2 is NADP or thioNADP, glucose-6-phosphate dehydrogenase (EC 1
．． 1.1.49) (from yeast) and glucose-6-phosphate, isocitrate dehydrogenase (EC 1.1.1
．． 42) (yeast, derived from porcine heart muscle) and isocitrate, glyoxylate dehydrogenase (EC 1.2.1.17)
) (from Pseudomonas oxalaticus) and CoA and glyoxylate, phosphogluconate dehydrogenase (EC 1.1.1.44) (rat liver,
Brewer's yeast, E. coli) and 6-phospho-D-gluconic acid, glyceraldehyde phosphate dehydrogenase (EC 1.2.1.13) (derived from plant chloroplasts) and D-
Examples include glyceraldehyde-3-phosphoric acid and phosphoric acid.

Furthermore, in the case where alcohol dehydrogenases are used singly or in combination of two or more to coenzyme (thio)NAD and (thio)NADP, the method of the present invention is applicable to two coenzymes. NA
When a combination of Class D and NADs or NADPs, or a combination of thioNADPs and NADs or NADPs is selected, the combination of D class and NADs or NADPs is selected. By reacting the third dehydrogenase and the substrate of the third dehydrogenase, A1 and A
The cycling reaction can be formed by providing a reaction system for regeneration of A1 between 2 and 2.

That is, the present invention provides a test substance containing one or more test components selected from the group consisting of alcohols and aldehydes; (1) one selected from the group consisting of NADPs and thioNADs; and one or more alcohol dehydrogenases, which use one selected from the group consisting of NADPs and NADs as a coenzyme, and perform a reversible reaction that uses at least alcohol as a substrate to produce aldehydes corresponding to the alcohols. enzyme, (2) A1 or/and A2 (3) B1 (4) acting on a reagent containing a third dehydrogenase that does not act on alcohols and forms the reaction A2→A1 and a substrate for the third dehydrogenase; At least the following reaction equation

0047
]

[C5]

(In the formula, A1 is thioNADP, thioNA
Class D indicates NADPs or NADs, A2 indicates the reduced product of A1, B1 indicates reduced NADPs or reduced NADs when A1 is thioNADPs or thioNADs, and A1 indicates NADP. or NADs indicates reduced thioNADPs or reduced thioNADs, B2 indicates the oxidized product of B1, and A2→A1 indicates A2
is selected from the group consisting of alcohols and aldehydes, and is characterized by forming a cycling reaction represented by The present invention provides a highly sensitive method for quantifying one or more test components.

In this case, it is preferable that the third dehydrogenase cannot substantially act on B1 in this measurement system, or that conditions are set such that it cannot substantially act on B1. Examples include a combination in which an enzyme that does not essentially use B1 as a coenzyme is selected, and a combination in which conditions are selected so that the third dehydrogenase does not substantially act on B1 due to the quantitative relationship between B1 and A2. At the time of quantitative determination, the consumption amount of B1 is measured.

In the quantitative composition using these components (1) to (3) and (5), the concentration of B1 is 0.02 to 1.
The concentration of A2 or/and A1 is preferably 0.05 to 5000 μM, especially 5 to 500 μM, and the concentration of alcohol dehydrogenases is 1 to 1000 u/ml, especially 2 to 400 μM.
The concentration of the third dehydrogenase is preferably 1 to 100 u/ml, and the Km relative to A2 is preferably 1 to 100 u/ml.
The amount of the third dehydrogenase substrate may be adjusted to be 20 times (in u/ml) or more the value (in mM), and the amount of the third dehydrogenase substrate is preferably in excess, for example, 0.05 to 20 mM. These amounts can be appropriately determined depending on the type of subject, etc., and larger amounts can also be used.

The third dehydrogenase is added as an auxiliary for the regeneration of A1, and this makes it possible to reduce the amount of A1 used, which is particularly effective when A1 is expensive. Further, the reaction may be performed using A2 or a mixture of A1 and A2 instead of A1. In this case, the amount of A1 and/or A2 used is not particularly limited, but is generally preferably 1/10 mole or less of B1.

As the third dehydrogenase and its substrate, for example, when A1 is NADs or thioNADs, glycerol dehydrogenase (EC 1.1
．． 1.6) (derived from E. coli) and dihydroxyacetone, L-glycerol-3-phosphate dehydrogenase (
E.C. 1.1.1.8) (derived from rabbit muscle) and dihydroxyacetone phosphate, malate dehydrogenase (EC
1.1.1.37) (derived from pig cardiac muscle, bovine cardiac muscle) and oxyaloacetic acid, glyceraldehyde phosphate dehydrogenase (EC 1.1.1.12) (derived from rabbit skeletal muscle, liver, yeast, E. Coli). When 1,3-diphospho-D-glyceric acid and A1 are NADPs or thioNADPs, glyceraldehyde phosphate dehydrogenase (EC
1.2.1.13) (derived from plant chloroplasts) and 1,3-diphospho-D-glyceric acid.

In order to measure alcohols or aldehydes in a specimen using the quantitative composition of the present invention thus prepared, the above-mentioned components (1) to (3), (1) to (
4), or add 0.001 to 0.5 ml of the test substance to the composition containing (1) to (3) and (5), and add about 37
The reaction is carried out at a temperature of ℃, and for several minutes to several tens of minutes between two points after a certain time after the start of the reaction, for example, for 3 minutes after 2 minutes and after 5 minutes.
or A2 produced in 5 minutes after 3 and 8 minutes.
The amount of B1 or the amount of B1 consumed may be measured by changes in absorbance based on the respective absorption wavelengths. For example, if A2 is thio-NADH and B1 is NADH, the production of A2 is measured by an increase in absorbance around 400 nm, or the consumption of B1 is measured by a decrease in absorbance around 340 nm, and alcohols or aldehydes of known concentrations are measured. By comparing the values with those measured using a similar method, the amount of each substance in the test liquid can be determined in real time.

[0054] Furthermore, if alcohols and aldehydes corresponding to the alcohols coexist in the specimen,
According to the method of the present invention, these total amounts are quantified. Therefore, if you want to measure the values of each component separately, pre-treat the sample with an enzyme that affects the taste of one of the components so that only alcohols or aldehydes are present in the sample. Just leave it as . That is,
Aldehyde dehydrogenase (EC 1.2.1.3,
If pretreatment is performed according to EC 1.2.1.4, EC 1.2.1.5), aldehydes in the specimen will be converted to acids, so the enzymatic cycling reaction according to the present invention can be performed subsequently. By this method, only alcohols can be quantified. Further, by subtracting the amount of the alcohol from the total amount of the alcohol and the aldehyde corresponding to the alcohol, the amount of only the aldehyde can be calculated.

[0055] Furthermore, the quantitative method of the present invention is one in which alcohols or aldehydes themselves in the test solution are introduced into an enzyme cycling reaction, and are not easily affected by coexisting substances in the test solution. Blank measurement can be omitted, and a simple measurement can be performed using a late assay.

In the present invention, when measuring A2 or B1, other known measuring methods can be used instead of absorbance measurement.

[0057]

Effects of the Invention As described above, the present invention uses reduced coenzymes with different absorption wavelengths, so no measurement errors occur, and sensitivity can be increased by combining enzyme cycling reactions. Alcohols or aldehydes in a sample can be determined quickly and accurately.

[0058]

[Examples] Next, the present invention will be specifically described with reference to Examples, but the present invention is not limited thereto in any way.

Example 1 Determination of ethanol <Reagent> 40 mM glycine-NaOH buffer (pH
9.5) 2 mM Thio-NAD (manufactured by Sigma)
0.4 mM reduced NAD (manufactured by Oriental Yeast Co., Ltd.) 5 u/ml alcohol dehydrogenase (manufactured by Sigma, derived from horse liver) <Procedure> Place 1 ml of the above reagent in a cuvette and add 0, 5,
20 μl of each of 10, 15, 20, and 25 μM ethanol solutions were added, and the reaction was started at 37°C. The absorbance at 400 nm was read at 2 minutes and 5 minutes after the start of the reaction, and the difference was determined. The value of concentration 0 was used as a reagent blank, and this value was subtracted from the value of each ethanol of 5 to 25 μM, and the results are shown in FIG. As is clear from FIG. 1, the amount of change in absorbance with respect to the amount of ethanol showed good linearity.

Example 2 Determination of n-butanol <Reagent> 40 mM glycine-NaOH buffer (pH
9.5) 1 mM Thio-NAD (manufactured by Sigma)
0.2 mM Reduced NAD (manufactured by Oriental Yeast Co., Ltd.) 1.92 u/ml Alcohol dehydrogenase (manufactured by Sigma, derived from horse liver) <Procedure> Place 1 ml of the above reagent in a cuvette and add 0, 1,
2, 3, 4, and 5 μM n-butanol solutions, respectively.
0 μl was added and the reaction was started at 37°C. The absorbance at 400 nm was read at 3 minutes and 8 minutes after the start of the reaction, and the difference was determined. As in Example 1, the difference from the reagent blank was determined, and the results are shown in FIG. As is clear from FIG. 2, the amount of change in absorbance with respect to the amount of n-butanol showed good linearity.

Example 3 Quantification of benzaldehyde <Reagent> 40 mM glycine-NaOH buffer (pH
9.5) 1 mM Thio-NAD (manufactured by Sigma)
0.2 mM Reduced deamino NAD (manufactured by Sigma) 18 u/ml Alcohol dehydrogenase (manufactured by Sigma, derived from horse liver) <Procedure> Place 1 ml of the above reagent in a test tube and add 0, 0.5,
40 μl of each of 1.0, 1.5, 2.0, and 2.5 μM benzaldehyde solutions were added, and the reaction was started at 37°C. Twenty minutes after the start of the reaction, 1 ml of 0.5% sodium dodecyl sulfate solution was added to stop the reaction. Thereafter, the absorbance at 400 nm was read, and the results are shown in FIG. As is clear from FIG. 3, the amount of change in absorbance with respect to the amount of benzaldehyde showed good linearity.

Example 4 Quantification of benzaldehyde <Reagent> 40 mM glycine-NaOH buffer (pH
9.5) 2 mM Thio-NAD (manufactured by Sigma)
0.2 mM Reduced NAD (manufactured by Oriental Yeast Co., Ltd.) 568 u/ml Alcohol dehydrogenase (manufactured by Oriental Yeast Co., Ltd.: derived from yeast) <Procedure> Place 1 ml of the above reagent in a cuvette, add 0, 5,
50 μl of each of 10, 15, 20, and 25 μM benzaldehyde solutions were added, and the reaction was started at 37°C. The absorbance at 400 nm was read at 2 minutes and 5 minutes after the start of the reaction, and the difference was determined. As in Example 1, the difference from the reagent blank was determined, and the results are shown in FIG. As is clear from FIG. 4, the amount of change in absorbance with respect to the amount of benzaldehyde showed good linearity.

[Brief explanation of the drawing]

FIG. 1: 40% relative to the amount of ethanol in Example 1
It is a drawing showing the results of a rate assay at 0 nm.

FIG. 2 is a drawing showing the results of a rate assay at 400 nm for the amount of n-butanol in Example 2.

FIG. 3 is a drawing showing the results of a rate assay at 400 nm for the amount of benzaldehyde in Example 3.

FIG. 4 is a drawing showing the results of a rate assay at 400 nm for the amount of benzaldehyde in Example 4.

Claims (8)

[Claims] Claim 1: (1) Thionicotinamide adenine dinucleotide phosphates (hereinafter referred to as thioNADPs) and One selected from the group consisting of thionicotinamide adenine dinucleotides (hereinafter referred to as thioNADs),
One selected from the group consisting of nicotinamide adenine dinucleotide phosphates (hereinafter referred to as NADPs) and nicotinamide adenine dinucleotides (hereinafter referred to as NADs) as a coenzyme, and at least an alcohol as a substrate to correspond to the alcohol. One or more enzymes selected from alcohol dehydrogenases that perform a reversible reaction to produce aldehydes, (2) A1 (3) B1 are reacted with a reagent containing the following reaction formula [Chemical formula 1] (wherein, A1 is thioNADPs, thioNADs, NAD
Ps or NADs, A2 represents the reduced product of A1, and B1 represents A1 is thioNADPs or thioNADs.
When using reduced NADPs or reduced NADs,
When A1 is NADPs or NADs, reduced thioN
Indicates ADPs or reduced thioNADs, B2 is B1
One type selected from the group consisting of alcohols and aldehydes, characterized by forming a cycling reaction represented by (indicating an oxidized product of A highly sensitive quantitative method for the above test components. 2. The NADPs are nicotinamide adenine dinucleotide phosphate (NADP), acetylpyridine adenine dinucleotide phosphate (acetylNADP), acetylpyridine hypoxanthine dinucleotide phosphate, and nicotinamide hypoxanthine dinucleotide phosphate (deaminoNADP).
The highly sensitive quantitative method according to claim 1, which is selected from the group consisting of: 3. The NADs are selected from the group consisting of nicotinamide adenine dinucleotide (NAD), acetylpyridine adenine dinucleotide (acetyl NAD), acetyl pyridine hypoxanthine dinucleotide and nicotinamide hypoxanthine dinucleotide (deamino NAD). The highly sensitive quantitative method according to claim 1, which is a method according to claim 1. 4. The thioNADPs are thionicotinamide adenine dinucleotide phosphate (thioNADP).
) and thionicotinamide hypoxanthine dinucleotide phosphate. 5. The highly sensitive quantitative method according to claim 1, wherein the thioNAD is selected from the group consisting of thionicotinamide adenine dinucleotide (thioNAD) and thionicotinamide hypoxanthine dinucleotide. 6. The highly sensitive quantitative method according to claim 1, wherein the alcohol is ethanol and the aldehyde is acetaldehyde. 7. The highly sensitive quantitative method according to claim 1, wherein the aldehyde is allylaldehyde, benzaldehyde or p-hydroxybenzylaldehyde. [Claim 8] The following components (1) to (3) (1) NA
One selected from the group consisting of DPs and thioNADs and one selected from the group consisting of NADPs and NADs are used as coenzymes, and aldehydes corresponding to the alcohols are produced using at least alcohol as a substrate. One or more enzymes selected from alcohol dehydrogenases that perform a reversible reaction, (2) A1 (3) B1 (A1 represents thio-NADPs, thio-NADs, NADPs or NADs, and B1 means that A1 is thio-NADPs or When A1 is a thio-NAD, it represents a reduced NADP or a reduced NAD, and when A1 is an NADP or a NAD, it represents a reduced thio-NADP or a reduced thio-NAD. A composition for highly sensitive quantitative determination of one or more test components selected from the group consisting of aldehydes and aldehydes.