JPH03224498A - High sensitivity analysis of bile acid and composition therefor - Google Patents

Abstract

PURPOSE:To carry out an accurate analysis of bile acid in a biological sample by using a thionicotinamide adenine dinucleotide(phosphate) as one of two coenzymes for steroid dehydrogenase in enzyme cycling reactions. CONSTITUTION:A reagent containing (A) steroid dehydrogenase capable of catalyzing a reversible reaction for production of oxobile acid using bile acid as the substrate and a thionicotinamide adenine dinucleotide phosphate (a thioNADP) or a thionicotinamide adenine dinucleotide (a thioNAD) and an NADP or an NAD as the coenzymes, (B) a thioNADP or a thioNAD and (C) then, an NADP or an NAD is acted on a sample for carrying out cycling reactions of the formula (A1 is thioNADP or thioNAD; A2 is reduced type product of A1; B1 is NADP or NAD; B2 is oxidized type product of B1), thus measuring the amount of A2 or B1 which changes in the course of the above- mentioned reactions.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to bile acids, particularly a highly sensitive method for quantifying bile acids in biological samples, and a composition for quantifying bile acids.

[Prior art and problems to be solved by the invention] Measuring bile acids in biological samples such as serum is important for liver function tests and for elucidating the mechanisms of jaundice and cholelithiasis. be.

Conventionally, various methods have been reported for measuring bile acids including cholic acid, deoxycholic acid, and chenodeoxycholic acid, but in recent years clinical tests have generally been conducted using 3α-hydroxysteroid dehydrogenase and nicotine as a coenzyme. Amide adenine dinucleotides (hereinafter referred to as N
Reduced NADs (hereinafter referred to as ADs) or nicotinamide adenine dinucleotide phosphates (hereinafter referred to as NADPs) are produced in proportion to the amount of bile acids.
NADHs) or reduced NADPs (hereinafter referred to as N
A method for measuring ADPHs (called ``ADPHs'')
No. 197) is used. However, this method has the disadvantages that bile acids must be separated in advance because it is easily affected by bilirubin, that a large amount of specimen is required, and that sensitivity is low. Therefore, efforts have been made to use highly sensitive coloring agents [Abstracts from the 33rd Annual Meeting of the Japanese Society of Clinical Pathology, p. 123 (1986) and Abstracts from the 34th Annual Meeting, 1
24 (1989)), but this is also not an essential solution.

In addition, as another method, a kit is commercially available that further converts the generated 3-oxosteroid into 3-△4-steroid through the action of 3-oxo-△4-steroid dehydrogenase to produce twice the amount of formazan dye as before. However, this only doubles the sensitivity.

Furthermore, a method for highly sensitive measurement of NADH or NADPH, which is produced in proportion to the amount of bile acids, using an enzyme cycling reaction has been reported (Japanese Patent Publication No. 367-1983).
No. 58). However, this method also uses NADH or NADP.
In order to amplify H by enzymatic cycling reaction, remaining excess NAD or NADP must be decomposed and removed by alkaline heating treatment, which is a complicated operation, and it is difficult to process multiple samples such as in clinical tests. In some cases, there was no escape from being at a disadvantage.

Therefore, for example, malabsorption syndrome is a disease that causes a decrease in blood bile acids, but it is difficult with current enzymatic methods to accurately measure concentrations below normal values.

In addition, most bile acids have a hydroxyl group at the 3α position, but
Depending on the type of bile acid, in addition to the 3α position, there are hydroxyl groups at the 7α and 12α positions, so in addition to 3α-hydroxysteroid dehydrogenase (3α-H2C), 7α-H2
If bile acids in each sample are measured using C, 12α-H2C, it is theoretically possible to measure the level of each bile acid without using equipment such as gas chromatography. In fact, it has been reported that simultaneous measurement of 12α-hydroxy bile acids in addition to measurement of total bile acids using 3α-H2C may be useful for understanding the degree and transition of liver parenchymal damage ( History of medicine, vol. 14
3. N.

10, 775-776 (1987)), and from this point of view, it is desired to increase the sensitivity of bile acid quantification.

[Means to solve the problem]

Therefore, the present inventors first used steroid dehydrogenase derived from a specific microorganism in a reversible reaction to generate oxobile acids using bile acids as substrates, and used NADs (or NADPs) as a coenzyme to convert bile acids into oxobile acids. If a reversible cycling reaction between bile acids and oxybile acids is performed using a reaction system that produces bile acids and a small amount of NADPHs (or NADHs) different from the coenzyme, NADHs ( We discovered that the production amount of bile acids (or NADPHs) increases linearly with the passage of time, and that the rate of increase is proportional to the amount of bile acids, and we filed an application earlier (Patent Application No.
-98443).

However, this method also produces NADPH and NAD
Since the maximum absorption wavelength of H is the same in both cases at 340 nm, there was a problem in that an error occurred when only the reduced form produced in proportion to the amount of bile acid was measured.

On the other hand, thionicotinamide adenine dinucleotide (hereinafter referred to as thioNAD) and thionicotinamide adenine dinucleotide phosphate (hereinafter referred to as thioNADP), which are analogs of NAD and NADP, have a reduced maximum absorption wavelength around 400 nm, and NAD and NADP
It is known that the maximum absorption wavelength of the reduced form of

Under these circumstances, the present inventors conducted further research and found that when performing the above enzyme cycling reaction, thio-NADPs or thio-NADs were used as one of the two types of coenzymes to determine which one. We have discovered that hydroxysteroids or oxosteroids can be quantified with high sensitivity by quantifying only the amount of change in one of the coenzymes, and we have further discovered that this method allows accurate quantification of bile acids.
The invention has been completed.

That is, the present invention provides a subject with: (i) one selected from the group consisting of thio-NADPs and thio-NADs, and one selected from the group consisting of NADPs and NADs as coenzymes, and at least bile acid as a substrate; A reagent containing steroid dehydrogenase, ■A1, and ■B1, which undergoes a reversible reaction to produce oxobile acids, is reacted with the following reaction formula (I) (where A1 is thioNADP, thioNAD, NADP). or NADs, A2 represents the reduced product of A1, B1 represents reduced NADPs or reduced NADs when A1 is thioNADPs or thioNADs, and A represents NA
In the case of DPs or NADs, it indicates reduced thioNADPs or reduced thioNADs, and B1 indicates the oxidized product of B), and A2 changed by the reaction. or a highly sensitive method for quantifying bile acids characterized by measuring the amount of B1, and the above-mentioned ■ and ■
The present invention provides a composition for quantifying bile acids characterized by containing (1) and (2).

In the present invention, any steroid dehydrogenase can be used as long as it meets the above requirements, but a specific example of a steroid dehydrogenase that uses NADs and thioNADs as coenzymes is Pseudomonas testosteronii.
ii, J, B, C,, 227, 37-52 (195
7)), Bacillus sphaericus
3α-hydroxysteroid dehydrogenase (3α-H2C) (EC1,1,1,50) derived from P. sphaericus; Japanese Patent Application Laid-open No. 157894/1983)
; Bacteroides fragilis
es fragilis, Biochim.

Biophys, Acta, 384.12-21 (
1975)) and Pseudomonas sp, B-0831 (
7α-H2C derived from Pseudomonas sp, B-0831, Toyo Jozo Catalog No. T-28)
(EC1, 1, 1, 159); Bacillus 5phaericus, Toyo Jozo Catalog No.

12α-H2C (EC1,1,1,
176), etc. In addition, examples of coenzymes containing NADs, NADPs, thioNADs, and thioNADPs include rat liver, prostate (J,
dBiochem, 8.4l-46 (1977)
), Pseudomonas sp, B-0831 (Pse
udomonas sp, B-0831, Toyo Jozo Catalog No. T-27).

In addition, A and B2 are thioNADPs, thioNADs,
Examples of NADPs or NADs include nicotinamide adenine dinucleotide phosphate (NADP), acetylpyridine adenine dinucleotide phosphate (acetylNADP), and nicotinamide hypoxanthine dinucleotide phosphate (deamino). NADP); and nicotinamide adenine dinucleotide (NAD), acetylpyridine adenine dinucleotide (acetyl NADP);
D) and nicotinamide hypoxanthine dinucleotide (deamino NAD).

In the present invention, either A or B is thioN
It is necessary to be ADs or thioNADPs.

Examples of thioNADPs or thioNADs include thionicotinamide adenine dinucleotide phosphate (thioNADP), thionicotinamide hypoxanthine dinucleotide phosphate, thionicotinamide adenine dinucleotide (thioNAD), and thionicotinamide hypoxanthine. Examples include dinucleotides.

In addition, the steroid dehydrogenase used for quantitative determination is NA
When only D class is used as a coenzyme, thio-NADs and the above-mentioned NADs are used, and when the steroid dehydrogenase used uses both NADs and NADPs as coenzymes,
It may be used by appropriately selecting from thioNADs, thioNADPs, and the above-mentioned NADs and NADPs.

It is necessary that the amounts of A1 and B are in excess compared to the amount of bile acids in the subject, and also in excess compared to the Km values of steroid dehydrogenase for A1 and B1, respectively, Especially the amount of bile acids between 20 and 10,000
Double molar ratio is preferred.

In the composition for quantifying bile acids of the present invention, A1 and B1
The concentration of is 0.02-100mM, especially 0.05-30mM
M is preferred, and the concentration of steroid dehydrogenase is 0.
．． The strength is preferably 05 to 100 U/all, particularly 1 to 50 U/ml, but the amount can be appropriately determined depending on the type of subject, etc., and larger amounts can also be used.

In addition, in the quantitative method of the present invention, steroid dehydrogenase is
When both ADs and NADPs are used as coenzymes, thioNADs and NADs or NA are added to the two coenzymes.
Combination with DPs or thioNADPs and NAD
When choosing a combination with a class or NADP class,
In addition, in the test subject, ① does not act on component bile acids and A1, B,
→ By allowing the second dehydrogenase that forms the reaction B1 and the substrate of the second dehydrogenase to act, B
A bile acid cycling reaction can also be formed by providing a reaction system for the regeneration of bile acids. In this case, during quantitative determination, the amount of A2 produced by the reaction is measured.

B2 B□C In the formula, A1 is thioNADPs, thioNADs, NAD
PQ or NADs, A2 represents the reduced product of A, B represents the reduced NADPs or reduced NADs when A is thioNADPs or thioNADs, and A represents NADP. or NEs indicates reduced thio-NADPs or reduced thio-NADs, B indicates the oxidized product of B, and B1→B1 represents an enzymatic reaction that produces B1 using B2 as a coenzyme. In other words, the second dehydrogenase is added as an auxiliary for the regeneration of B1, which makes it possible to reduce the amount of B1 used.
This is particularly effective when B is expensive. Further, the reaction may be performed using B or a mixture of B and B2 instead of Bl. In this case, the amount of B1 and/or B used is not particularly limited;
/10 mol or less is preferable.

In the composition for quantifying bile acids using this component (2), A,
The concentration of is 0.02-100mM, special 1:0.05-30
mM; 6<preferably, the concentration of B and/or B1 is 0.
05-5000μM1 Especially preferably 5-500μM,
The concentration of steroid dehydrogenase is O,OS~100
U/7d, especially 1 to 50 U/7d is preferred, and the second dehydrogenase has a Km value (in mW) of 2 for B.
It may be adjusted so that it is 0 times the amount (U/customer unit) or more, for example, 1 to 100 U/Tnf1 is preferable, and the second
The dehydrogenase substrate of
20mM is preferred. These amounts can be appropriately determined depending on the type of subject, etc., and larger amounts can also be used.

As the second dehydrogenase and its substrate, for example, when B2 is NADs or thioNADs, alcohol dehydrogenase (EC1,1,1,1) and ethanol, glycerol dehydrogenase (EC1,1°1) are used.
．． 6) (E, derived from Co11) and glycerol, glycerol-3-phosphate dehydrogenase (EC1,1,
1,8) (derived from rabbit muscle) and L-glycerol-3-
Phosphate, malate dehydrogenase (EC1,1,1,
37) (derived from porcine cardiac muscle, bovine cardiac muscle) and L-malic acid, glyceraldehyde phosphate dehydrogenase (EC1゜1
．． 1.12) (Rabbit skeletal muscle, liver, yeast, E, Co
11) and D-glyceraldehyde phosphoric acid and phosphoric acid,
When B is NADPs or thioNADPs, glucose-6-phosphate zohydrogenase (EC1,1,1
, 49) (derived from yeast) and glucose-6-phosphate,
Incitrate dehydrogenase (EC1,1,1,42
) (yeast, derived from pig myocardium) and incitric acid, glyoxylate dehydrogenase (EC1, 2, 1, 17)
(derived from Pseudomonas OXalati CuS), CoA and glyoxylic acid, phosphogluconate dehydrogenase (EC, 1, 1, 1, 44) (derived from rat liver, brewer's yeast, E, Co1f), 6-phospho-D-gluconic acid, glyceraldehyde phosphate Dehydrogenase (
EC1,2,1,13) (derived from plant chloroplasts) and D-
Glyceraldehyde-3-phosphate and phosphoric acid, benzaldehyde dehydrogenase (EC1,2,1,7) (
Pseudomonas fluorescens) and benzaldehyde.

Furthermore, in the case where steroid dehydrogenase uses both NADs and NADPs as coenzymes, the assay method of the present invention uses a combination of thioNADs and NADs or NADPs as the two coenzymes, or a combination of thioNADPs and NADs as the two coenzymes.
When a combination with ADs or NADPs is selected, the test subject will be given the following:
By allowing the third dehydrogenase that forms the reaction A3→A and the substrate of the third dehydrogenase to act, the regeneration of A1 occurs between A and A as shown in reaction formula (II) below. By providing a reaction system for bile acids, bile acid cycling reactions can be formed. In this case, the consumption amount of B1 is measured for quantitative determination.

(In the formula, A1 is thioNADPs, thioNADs, NAD
Ps or NADs, A2 represents the reduced product of A, B represents the reduced NADPs or NADs when A is thioNADPs or thioNADs; NADPs or NADs indicate reduced thio-NADPs or reduced thio-NADs, B indicates the oxidized product of B1, and A2 → A1 represents an enzymatic reaction that uses A2 as a coenzyme to produce A1. In other words, the third dehydrogenase is added as an auxiliary for the regeneration of A1,
This makes it possible to reduce the amount of A□ used, which is particularly effective when A is expensive. Also, A1
Instead of A, or a mixture of A and A may be used to carry out the reaction. In this case, the amount of A1 and/or A2 used is not particularly limited;
The amount is preferably 1/10 mole or less.

In the composition for quantifying bile acids using this component (2), B1
Concentration 0.02 to 10100 III Special 4: 0.05
~30mM is preferred, and the concentration of A and/or A1 is 0
, 05-5000 μM1, particularly preferably 5-500 μM, and the concentration of steroid dehydrogenase is 0.05-10
0U/mQ, especially 1 to 50U/nil is preferable, and the third
The dehydrogenase has a Km value (in mM) for A2.
The amount may be adjusted to 20 times the amount (U/ml unit) or more, for example, preferably 1 to 100 U/Tnf1,
The third dehydrogenase substrate may also be present in excess, e.g.
．． 05-20mM is preferred. These amounts can be appropriately determined depending on the type of subject, etc., and larger amounts can also be used.

As the third dehydrogenase and its substrate, for example, when At is NAD or thioNAD, alcohol dehydrogenase (EC1,1,1,1) and acetaldehyde, glycerol dehydrogenase (EC1,
1,1,6) (E, derived from CoLi) and dihydroxyacetone, glycerol-3-phosphate dehydrogenase (EC1,1,1,8) (derived from rabbit muscle) and dihydroxyacetone phosphate, malate dehydrogenase (EC1,1,1,8) (derived from rabbit muscle).
EC1,1,1,37) (derived from pig cardiac muscle, bovine cardiac muscle) and oxaloacetate, glyceraldehyde phosphate dehydrogenase (EC1,1,1,12) (derived from rabbit skeletal muscle, liver, yeast, E, Co11) and 1 .3-diphospho D-glyceric acid, A1 is NADPs or thioNAD
For P class, glucose-6-phosphate dehydrogenase (EC1,1,1,49) (derived from yeast) and gluconolactone-6-phosphate, glyceraldehyde phosphate dehydrogenase (EC1,2,1,13) (derived from plant chloroplasts) and 1,3-diphospho D-glyceric acid.

Regarding steroid dehydrogenases that can be used in preparing the composition for quantifying bile acids of the present invention, for example, NADs (preferably NAD), thio-NAD, etc. as coenzymes,
(preferably thio-NAD), or NADP (preferably thio-NAD)
preferably NADP), thioNADPs (preferably!
Any substance that has reactivity to bile acids such as cholic acid as a substrate can be used, and can be confirmed by using these coenzymes and substrates.

Regarding the reaction solution composition, two types of coenzymes are selected appropriately considering the relative activity between each coenzyme of steroid dehydrogenase to be used, and the pH conditions between the optimal pi (in between) for the termination reaction/reverse reaction are determined. The pH conditions may be set so that the reaction rate ratio of forward reaction/reverse reaction approaches 1 by adjusting as appropriate.For example, 3α-)ISD (Pseudomonas sp.
, B-0831, Toyo Jozo Catalog No.

Regarding T-27), when cholic acid is used as the substrate and thio-NAD is used as the coenzyme, the relative activity is about 40% compared to when NAD is used, and the optimum pH for the forward reaction is 9. The optimum pi for the reverse reaction is around 5.5. These enzymes may be used alone or in combination of two or more as appropriate.

In order to measure the amount of bile acids in a subject using the thus prepared composition for quantifying bile acids of the present invention, the above-mentioned components
Add 0.001 to 0.5 ml of the test substance to a composition containing ■, ■ to ■, or ■ to ■ and ■, react at a temperature of about 37°C, and The amount of A produced or the amount of B consumed for several minutes to several tens of minutes, for example, 1 minute after 3 minutes and 4 minutes or 5 minutes after 3 minutes and 8 minutes, is calculated for each It may be measured by a change in absorbance based on the absorption wavelength. For example, At is thioN
When ADH and Bl are NADH, the generation of A is 400n
The bile acids in the test solution can be determined by measuring the increase in the absorbance of B, or by measuring the consumption of B by the decrease in the absorbance at 340 nm, and comparing this with the value measured using known concentrations of bile acids. The amount can be calculated in real time.

In addition, the quantitative method of the present invention directs the bile acids themselves in the test solution to the enzyme cycling reaction, and is less susceptible to the effects of coexisting substances in the test solution, so blank measurements of the test solution can be omitted. can be easily measured using a late assay. Furthermore, by appropriately changing the enzyme for measuring bile acids, we can measure not only total bile acids by 3α-HSD, but also
7α-HSD, 7α-hydroxy bile acid, 12α-
This makes it possible to separately quantify 12α-hydroxy bile acids using HSD.

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

〔Effect of the invention〕

As mentioned above, the present invention uses reduced coenzymes with different absorption wavelengths, so there is no measurement error, and sensitivity can be increased by combining enzymatic cycling reactions, so it can be used with small amounts of samples. Bile acids in a sample can be quantified quickly and accurately.

〔Example〕

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

Example 1 Reagent> 40mM phosphate buffer - (pH 8,0) 1mM Thio-NAD 0.2mM NADH 011% Triton X-100 5U/mQ3a-HSD (Pseudom
onastestosteronii (manufactured by Sigma) (Procedure) Place the above reagent 1 in a cuvette and add 0.10.20.4
10 μQ of 0.60.80.100 μM cholic acid solutions were added, respectively, and the reaction was started at 37°C. The absorbance of the 40-ounce bean paste was read 2 minutes and 3 minutes after the start of the reaction, and the difference was determined. The results are shown in Figure 1.

As is clear from FIG. 1, the amount of change in absorbance with respect to the amount of cholic acid showed good linearity.

Example 2 Reagent> 40mM phosphate buffer y-(pH 7,0) 1mM Thio-NAD 1 n+M NADH 0,1% Triton X-100 20U/mQ 7 a-HSD (Pseudomon
as sp, Toyo Jozo Catalog No. T-28) <Procedure> Place the above reagent 1 in a cuvette and add 0.100.200.
, 300, 400, and 500 μM nochenoteoxycholic acid solutions were added in an amount of 10 μa each, and the reaction was started at 37°C. The absorbance at 400 nm was read at 2 minutes and 3 minutes after the start of the reaction, and the difference was determined. The results are shown in Figure 2.
Shown below. As is clear from FIG. 2, the amount of change in absorbance with respect to the amount of chenodeoxycholic acid showed good linearity.

Example 3 <Reagents> 40mM) Lis-○CII (pH 8,5) 1mM Thio-NAD 2mM NADPH 9U/ill 3a-HSD (Pseudo
monas sp, Toyo Jojo Catalog No. T-27) <Procedure> Place 1 ml of the above reagent in a cuvette and add 0.10.20
．． 40, 60, 80, and 50 μl of a 100 μM cholic acid solution were added, respectively, and the reaction was started at 37°C. The absorbance at 400 nm was read at 3 minutes and 4 minutes after the start of the reaction, and the difference was determined. The results are shown in FIG.

As is clear from FIG. 3, the amount of change in absorbance with respect to the amount of cholic acid showed good linearity.

Example 4 <Reagent> 40mM Tris-HCQ (pH 8,0) 1mM ThioN
AD 0.2mM NADH 012% Trito”y
monas sp, Toyo Jozo Catalog No. T=27) <Procedure> 1 ml of the above reagent was placed in a quartz cuvette, 100 μg of each serum diluted in 5 stages was added, and the reaction was started at 37°C. 400n at the 3rd and 4th minute after the start of the reaction
The absorbance at m was read and the difference was determined. The results are shown in FIG.

Example 5 <Reagents> 40mM Tris-HCl2 (pH 8,0) 1mM Thio-NAD 0.2mM NADH 0.2% Triton X-100 2mM Oxamic acid 3.5U/mQ3a-H5C (Pseudo
monas sp, Toyo Jojo Catalog No., T-27
Origin) <Procedure> The above reagent ITrfi was placed in a cuvette, and 50 μg of the sample to which cholic acid had been added to the pooled serum to give a concentration of 10, 20, and 50 μN was added, and the same operation as in Example 4 was performed. The results are shown in Table 1, and the recovery rate is 97.8 to 104.
．． It was 0%.

Margin table 1 below Example 6 <Reagent> 50mM phosphate buffer - (pH 7,0) 2.5mM
ThioNAD 0.35mM NADH 59U/m127 a-H2C (Pseudomona
s sp, Toyo Jozo Catalog No., T-28) <Procedure> Place 0.5 ml of the above reagent in a test tube, and add 0.0.2.
, 4, 06.0, and 8. 25 μQ of cholic acid solutions of 1.0 μM were added, and reacted at 30° C. for 60 minutes.

Then, the reaction was stopped by adding 0.5 ml of 0.5% sodium dodecyl sulfate (SDS) solution, and 400 nI
Absorbance at I+ was measured. The results are shown in FIG. As is clear from FIG. 5, the amount of change in absorbance with respect to the amount of cholic acid showed good linearity.

Example 7 <Reagent> 40mM glycine-NaOH (pH 10,0) 5mM
NADP 50 μM Thio-NAD 0.4 M Ethanol 30 U/m Alcohol dehydrogenase (manufactured by Oriental Yeast Co., Ltd.) 10 U/ml 3 a-H3C (Pseudo
monas sp, B-0831 Toyo Jojo Catalog N
o, derived from T-27) 0.2% Triton 10μ of each acid solution was added, and the reaction was started at 37°C.

The absorbance at 340 nm was read at 3 minutes and 4 minutes after the start of the reaction, and the difference was determined. The results are shown in FIG.

As is clear from FIG. 6, the amount of change in absorbance with respect to the amount of cholic acid showed good linearity.

Example 8 <Reagent> 40mM PIPES-NaOH (pH 7,0) 0.2
5mM NADPH 0,025mM ThioNAD 5+oM dihydroxyacetone phosphate 10U/-glycerol-3-phosphate dehydrogenase (manufactured by Boehringer; derived from rabbit muscle) 20tJ/mQ 3 a-) ISD (Pseu
domonas sp, B-0831 Toyo Jozo Catalog No. T-27) 0.2% Triton X-10
0 <Operation> Place the above reagent 1 in a quartz cuvette and add 0.10.20
．． 30. 40. 50 μM cholic acid solution, respectively.
μg was added, and the reaction was started at 37°C. The absorbance at 340 nm was read at 3 and 4 minutes after the reaction volume was 4°, and the difference was determined. The results are shown in FIG.

As is clear from FIG. 7, the amount of change in absorbance with respect to the amount of cholic acid showed good linearity.

[Brief explanation of drawings]

Figure 1 shows 400n versus cholic acid amount in Example 1.
FIG. 3 is a drawing showing the results of a rate assay in m. FIG. 2 is a drawing showing the results of a rate assay at 400 nm for the amount of chenodeoxycholic acid in Example 2. Figure 3 shows 400n versus cholic acid amount in Example 3.
FIG. 3 is a drawing showing the results of a rate assay in m. FIG. 4 is a drawing showing the results of a rate assay at 400 nm for serum amount in Example 4. Figure 5 shows 400n versus cholic acid amount in Example 6.
FIG. 3 is a drawing showing the results of a rate assay in m. Figure 6 shows 340n versus cholic acid amount in Example 7.
It is a drawing showing the results of a rate assay in ml. FIG. 7 is a diagram showing the results of a late assay at 340 ml of cholic acid in Example 8. that's all

Claims (1)

[Scope of Claims] 1. The test subject contains (1) thionicotinamide adenine dinucleotide phosphates (hereinafter referred to as thioNADPs) and thionicotinamide adenine dinucleotides (hereinafter referred to as thioNADPs).
nicotinamide adenine dinucleotide phosphates (hereinafter referred to as NA); and nicotinamide adenine dinucleotide phosphates (hereinafter referred to as NA
steroid dehydrogenase, which performs a reversible reaction to produce oxobile acids using at least bile acids as a substrate, using one selected from the group consisting of nicotinamide adenine dinucleotides (hereinafter referred to as NADs) as a coenzyme; 2) A_1, (3) B_1, is reacted with a reagent containing the following reaction formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (In the formula, A_1 is thioNADPs, thioNADs, NA
DPs or NADs, A_2 represents the reduced product of A_1, B_1 represents reduced NADPs or NADs when A_1 is thioNADPs or thioNADs, and A_1 represents NADPs or NADs. 2 indicates reduced thio-NADPs or reduced thio-NADs, and B_2 indicates the oxidized product of B_1), and the amount of A_2 or B_1 changed by the reaction is calculated. A highly sensitive method for quantifying bile acids. 2. The test subject: (1) One selected from the group consisting of thio-NADPs and thio-NADs and one selected from the group consisting of NADPs and NADs as coenzymes, and at least bile acids as substrates and oxo-enzymes. A steroid dehydrogenase that forms a reversible reaction that produces bile acids, (2) A_1, (3) B_1 or/and B_2, (4) a second dehydrogenase that does not act on bile acids and A_1 and forms a reaction of B_2→B_1. and the substrate of the second dehydrogenase, and react with the following reaction formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (wherein A_1 is thioNADPs, thioNADs, NA
DPs or NADs, A_2 represents the reduced product of A_1, B_1 represents reduced NADPs or NADs when A_1 is thioNADPs or thioNADs, and A_1 represents NADPs or NADs. , B_2 indicates the oxidized product of B_1, B_2→B_1 indicates B_2
1. A highly sensitive method for quantifying bile acids, which comprises forming a cycling reaction (representing an enzymatic reaction that produces B_1 using B_1 as a coenzyme) and measuring the amount of A_2 produced by the reaction. 3. The test subject: (1) One selected from the group consisting of thio-NADPs and thio-NADs and one selected from the group consisting of NADPs and NADs as coenzymes, and at least bile acid as a substrate, oxo A steroid dehydrogenase that forms a reversible reaction that produces bile acids, (2) A_1 or/and A_2, (3) B_1, (5) a third dehydrogenase that does not act on bile acids and B_1 and forms a reaction of A_2 → A_1. and the substrate of the third dehydrogenase, and the following reaction formula ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ (wherein A_1 is thioNADPs, thioNAPs, NA
DPs or NADs, A_2 represents the reduced product of A_1, B_1 represents reduced NADPs or NADs when A_1 is thioNADPs or thioNADs, and A_1 represents NADPs or NADs. When , B_2 indicates the oxidized product of B_1, A_2→A_1 indicates A_2
1. A highly sensitive method for quantifying bile acids, which comprises forming a cycling reaction (representing an enzymatic reaction that produces A_1 using B_1 as a coenzyme) and measuring the amount of B_1 consumed by the reaction. 4. The highly sensitive method for quantifying bile acids according to any one of claims 1 to 3, wherein the steroid dehydrogenase is 3α-hydroxysteroid dehydrogenase. 5. The highly sensitive method for quantifying bile acids according to any one of claims 1 to 3, wherein the steroid dehydrogenase is 7α-hydroxysteroid dehydrogenase. 6. The highly sensitive method for quantifying bile acids according to any one of claims 1 to 3, wherein the steroid dehydrogenase is 12α-hydroxysteroid dehydrogenase. 7.NADPs include nicotinamide adenine dinucleotide phosphate (NADP), acetylpyridine adenine dinucleotide phosphate (acetylNADP)
and nicotinamide hypoxanthine dinucleotide phosphate (deamino NADP). 8. Any one of claims 1 to 3, wherein the NAD is selected from the group consisting of nicotinamide adenine dinucleotide (NAD), acetylpyridine adenine dinucleotide (acetyl NAD), and nicotinamide hypoxanthine dinucleotide (deamino NAD). A highly sensitive method for quantifying bile acids as described in the above section. 9. The highly sensitive method for quantifying bile acids according to any one of claims 1 to 3, wherein the thioNAD is selected from the group consisting of thioNAD and thionicotinamide hypoxanthine dinucleotide. 10. The highly sensitive method for quantifying bile acids according to any one of claims 1 to 3, wherein the thioNADP is selected from the group consisting of thioNADP and thionicotinamide hypoxanthine dinucleotide phosphate. 11. The following components (1) to (3) (1) One selected from the group consisting of thioNADPs and thioNADs and one selected from the group consisting of NADPs and NADs are used as coenzymes, and at least 1. A composition for quantifying bile acids, comprising steroid dehydrogenases (2) A_1, (3) B_1, which perform a reversible reaction to produce oxobile acids using bile acids as substrates. 12. The following components (1) to (4) (1) One selected from the group consisting of thioNADPs and thioNADs and one selected from the group consisting of NADPs and NADs are used as coenzymes, and at least Steroid dehydrogenase, which performs a reversible reaction to produce oxobile acids using bile acids as substrates, (2) A_1, (3) B_1 or/and B_2 (4) Does not act on bile acids and A_1, forming the reaction B_2→B_1 A composition for quantifying bile acids, comprising a second dehydrogenase and a substrate for the second dehydrogenase. 13. Complement the following components (1) to (3) and (5) (1) One selected from the group consisting of thioNADPs and thioNADs and one selected from the group consisting of NADPs and NADs. steroid dehydrogenase, which is an enzyme and performs a reversible reaction to produce oxobile acids using at least bile acids as substrates; (2) A_1 or/and A_2; (3) B_1; (5) A_2→ A composition for quantifying bile acids, comprising: a third dehydrogenase that forms the reaction A_1; and a substrate for the third dehydrogenase.