JPH04365500A - Simultaneous analysis of bile acid - Google Patents

Abstract

PURPOSE:To simultaneously analyze bile acids simply in a short time without requiring complicated pretreatment by high performance liquid chromatography through gradient elution with two or more liquids by using a 3alpha-hydroxysteroid dehydrogenase-immobilized enzyme column. CONSTITUTION:In bile acid analyzing method by high performance liquid chromatography using a 3alpha-hydroxysteroid dehydrogenase-immobilized enzyme column, gradient elution with two or more liquids is utilized, a composition of mobile phase is changed from an initial composition obtained by blending at least a lower aliphatic nitrile (e.g. acetonitrile) with a salt-containing aqueous solution having pH 7-12 and a lower alcohol (e.g. methanol) wherein the contents are 1.5-6M lower aliphatic nitrile, 0.5-250mM salt and <=5M lower alcohol to a final composition obtained by blending at least the lower aliphatic nitrile with the salt-containing aqueous solution having pH 4-12 and the lower alcohol, having 0.9-8M lower aliphatic nitrile, 0.5-120mM salt and 2-12M lower alcohol to simultaneously analyze bile acids.

Description

[Detailed description of the invention]

0001

FIELD OF THE INVENTION The present invention relates to a method for analyzing bile acids. In detail, high performance liquid chromatography (hereinafter referred to as HPLC) using a 3α-hydroxysteroid dehydrogenase (hereinafter referred to as 3α-HSD) immobilized enzyme column is used.
It is abbreviated as ) is a method for simultaneous analysis of multicomponent bile acids in living organisms such as experimental animals.

0002

[Prior Art] Human bile and blood contain bile acids such as cholic acid, chenodeoxycholic acid, deoxycholic acid,
Ursodeoxycholic acid and lithocholic acid each exist in a total of 15 types, including a free type, a glycine-conjugated type, and a taurine-conjugated type (hereinafter, these 15 types of bile acids are referred to as "human bile acids"). Separating and quantifying each of these bile acid components is useful in the field of clinical testing, particularly in the diagnosis of hepatobiliary diseases, and various analytical methods have been studied and put into practical use.

[0003] On the other hand, in the development of new drugs, the effectiveness and safety of the drug is mainly confirmed using non-human animals before starting clinical trials on humans. Analysis of each bile acid component in the living body of experimental animals plays an extremely important role in a variety of preclinical tests in the development of therapeutic drugs for hepatobiliary diseases.

[0004] Some of these experimental animals contain bile acid components in their living bodies that are not contained in human bile acids. For example, in the bile of rats and pigs, in addition to the 15 types of human bile acids, α-muricholic acid, β-muricholic acid,
Including hyodeoxycholic acid, hyocholic acid, and their conjugated bile acids, there are a total of 27 types of bile acids (hereinafter, these 27 types of bile acids are referred to as "rat bile acids").

[0005] Analysis of bile acids in the living bodies of these experimental animals requires higher separation power than the analysis method for human bile acids since it involves multiple components, and the human bile acid analysis method cannot be directly applied.

For example, in the analysis of human bile acids, HP
LC methods are becoming mainstream, but when used for rat bile acids, the separation ability is insufficient and satisfactory results have not been obtained. Japanese Patent Publication No. 56-38200 discloses that a sample containing free and conjugated bile acids is separated into each bile acid component by HPLC, and then each bile acid component is detected using a 3α-HSD immobilized enzyme column. A method for the analysis of bile acids is presented. However, this publication only discloses the analysis results for 13 out of 15 human bile acids, and in fact, as will be explained later, the method described in the publication cannot separate and quantify 27 rat bile acids (comparison). Example 8).

[0007] In addition, rat bile acids were pretreated with alkali to hydrolyze all the conjugated forms into free forms, and then free form bile acids (9 components) were analyzed by HPLC using an immobilized enzyme column.
A method has been reported for quantifying the total amount of
. However, this analytical method cannot separate and quantify rat bile acids into free form, glycine-conjugated form, and taurine-conjugated form, and has low practicality as an analytical method in preclinical studies.

[0008] Human bile acids were also treated with a hydrophobic gel having an ion exchange group to fractionate them into free, glycine-conjugated, and taurine-conjugated bile acid groups (hereinafter referred to as ``conjugated fraction'').
That's what it means. ), a method of quantifying each fraction by HPLC using an immobilized enzyme column is known (Japanese Patent Publication No. 30588/1988, etc.). Rat bile acids cannot be quantified using this method as is, but by combining this method with the alkali treatment method described above, rat bile acids can be conjugated and fractionated, and the conjugated fraction can be further analyzed as described above. A conceivable method is to deconjugate each fraction by an alkali treatment method, and then quantify each fraction by HPLC using an immobilized enzyme column. However, this method requires 4 to 5 days for pretreatment such as conjugation fractionation and deconjugation, and requires measurement for each fraction, which is complicated and not suitable for general use.

Furthermore, methods such as gas chromatography and gas chromatography mass spectrometry that have been conventionally used to analyze human bile acids can only analyze free bile acids, and require complicated pretreatments such as trimethylsilylation. There are many problems such as necessity, and it cannot be said to be an effective method.

0010

[Problems to be Solved by the Invention] As described above, it is extremely important to obtain information on bile acids in vivo in experimental animals in preclinical studies. Although there is a desire to establish an analytical method that can quantify each substance, an analytical method with excellent quantitative performance and high versatility has not yet been found.

The object of the present invention is to provide a method for easily and quickly separating and quantifying multicomponent bile acids such as rat bile acids into each bile acid component with high sensitivity, without requiring complicated pretreatments such as conjugate fractionation. Our goal is to provide the following.

0012

[Means for Solving the Problems] As a result of intensive research, the present inventors have developed an HPL method using a 3α-HSD immobilized enzyme column.
We have completed a simultaneous analysis method for multicomponent bile acids such as rat bile acids using C.

That is, in the present invention, a sample solution containing free, glycine-conjugated, and taurine-conjugated bile acids is introduced into a separation column together with an eluent, separated into each bile acid component by HPLC, and then fixed. 3α-HSD was passed through an immobilized enzyme column to react with nicotinamide adenine dinucleotide (hereinafter abbreviated as NAD+), and the generated reduced NAD (hereinafter abbreviated as NADH) was measured by ultraviolet absorption measurement. and/or a method of analyzing bile acids by detecting each bile acid component by performing fluorescence measurement, in which (a) at least a lower aliphatic nitrile, a salt containing a From an initial composition A which is a mixture of an aqueous solution and a lower aliphatic nitrile and contains 1.5 to 6M of a lower aliphatic nitrile, 0.5 to 250mM of a salt, and 5M or less of a lower alcohol, (b) at least a lower aliphatic nitrile and a salt. Contains pH 4-12
The composition of the mobile phase was continuously and/or Alternatively, it is a simultaneous analysis method for bile acids, which is characterized by separating and quantifying each bile acid component through stepwise changes.

According to the analytical method of the present invention, cholic acid, chenodeoxycholic acid, deoxycholic acid, ursodeoxycholic acid, lithocholic acid, and α contained in rat bile acids, etc.
- A total of 27 types of bile acids, including muricholic acid, β-muricholic acid, hyodeoxycholic acid, and hyocholic acid, respectively, in free form, glycine-conjugated form, and taurine-conjugated form, can be easily and quickly processed without pretreatment such as conjugated fractionation. can be separated and quantified.

The analytical method of the present invention will be explained in more detail below. The present invention is achieved in HPLC by changing the composition of the mobile phase from a specific initial composition A to a final composition B continuously and/or stepwise by gradient elution (gradient) of two or more liquids.

The mobile phase in the initial composition A is a mixture of at least a lower aliphatic nitrile, a salt-containing aqueous solution having a pH of 7 to 12, and a lower alcohol. As the lower aliphatic nitrile, acetonitrile, propionitrile, etc. are used. As the salt, inorganic or organic salts such as ammonium, sodium, potassium salts of phosphoric acid, carbonic acid, acetic acid, etc. are used. Further, the pH of the salt aqueous solution needs to be 7 to 12. If the pH is less than 7, the separation of each component will be insufficient, and if the pH exceeds 12, the column will deteriorate rapidly and is not practical. The pH of the aqueous solution is adjusted either directly as an aqueous solution of these salts or by adding a suitable alkali to an aqueous solution of the salt. As the lower alcohol, methanol, ethanol, n-propanol, etc. are used.

[0017] In addition, the initial composition A has a lower aliphatic nitrile content of 1.5 to 6M and a salt content of 0.5 to 250mM.
and the lower alcohol content must be 5M or less. If the content of lower aliphatic nitriles or lower alcohols in the initial composition of the mobile phase exceeds the upper limit of this range,
The elution power of the mobile phase becomes excessive and the bile acids are eluted all at once without being separated. On the other hand, if the content of the lower aliphatic nitrile is below the lower limit of this range, the elution power of the mobile phase decreases and the retention power of bile acids increases, so that elution takes a long time. In addition, if the salt content in the initial composition of the mobile phase is 0.5 mM or less, α, β-
Separation of the muricholic acid group becomes impossible, and if the concentration exceeds 250 mM, salts will precipitate in the mobile phase, leading to equipment and column failure.

The mobile phase in the final composition B is a mixture of an aqueous solution with a pH of 4 to 12 containing at least a lower aliphatic nitrile and a salt, and a lower alcohol; It is necessary that it contains 120mM and lower alcohol 2-12M. The lower aliphatic nitriles, salts and lower alcohols in final composition B may be the same as or different from those used in initial composition A, and those listed for initial composition A are used. As with the initial composition, good separation results cannot be obtained if the final composition of the mobile phase is outside this range.

Continuous and/or stepwise changes in the composition of the mobile phase from the initial composition A to the final composition B can be achieved by adjusting the mixing ratio of two or more eluents to achieve a predetermined composition gradient using a gradient device or the like. This is achieved by The gradient method is, for example, an eluent prepared with composition A of 100
It may be a two-liquid gradient from % to 100% of the eluent prepared in composition B, or it may be a three-liquid or more gradient in which eluents having different compositions are combined.

Other gradient conditions are appropriately selected depending on the type of column packing material, the composition of the mobile phase, the mixing ratio, etc.

[0021] Samples used in the present invention include biological samples such as human and animal bile, blood, urine, and liver. These biological samples are subjected to pretreatments such as desalination, protein removal, extraction, concentration, and washing as necessary, and free and conjugated bile acids are recovered and purified as a methanol solution or the like. Also,
When using the internal standard method, an internal standard substance is added to the sample solution. As an internal standard substance, 3α,7β-dihydroxy-
12-oxo-5β-cholanic acid, 3α-hydroxy-7
, 12-dioxo-5β-cholanic acid, 3α,7β,12
α-trihydroxy-5β-cholanic acid, 3α-hydroxy-7-oxo-5β-cholanic acid, 3α-hydroxy-
12-oxo-5β-cholanic acid, 3α,12α-dihydroxy-7-oxo-5β-cholanic acid and their glycine- and taurine-conjugated bile acids, 5β-pregnane-3α,12α,20α-triol, 5β- Androstane-3α, 11α, 17β-triol, 5β
-Androstane-3α,17β-diol and the like are used.

A typical apparatus for carrying out the present invention is shown in FIG. 1a and 1b are eluent tanks, and 2a and 2b are eluent pumps. The sample liquid injected from the sample injector 4 is introduced into the separation column 5 together with the mobile phase adjusted to a desired mixing ratio by the gradient device 3.

As packing materials for separation columns, silica gel or porous polymers modified with functional groups such as octadecyl, octyl, butyl, methyl, phenyl, phenylethyl, cyanopropyl, and aminopropyl groups can be used. Chemically bonded fillers of reversed phase type are used. The particle size of the filler is preferably 3 to 10 μm, and the pore size is 50 to 10 μm.
300 Å is preferred. In addition, the size of the separation column is
A diameter of 4 to 6 mm and a length of 10 to 30 cm are preferred.

The sample solution is subjected to HPLC in the separation column 5.
A reaction solution that is separated into each bile acid component and contains NAD+ in the separated solution flowing out from the separation column is supplied from the reaction solution tank 6.

[0025] Next, the separated liquid is immobilized 3α-HS.
D is introduced into the immobilized enzyme column 8. The immobilized enzyme column used is one in which 3α-HSD is chemically immobilized on a spherical cellulose, aminoalkylated silica carrier, or the like, and packed in a stainless steel column or the like.

In the immobilized enzyme column 8, 3α-HS
Due to the catalytic action of D, bile acids and NAD+ react specifically, converting 3α-hydroxy bile acids into 3-oxo forms,
At the same time, an equal amount of NADH is produced. The generated NADH is excited by light with a wavelength of 340 nm, resulting in 460
It emits fluorescence with a maximum value at nm. By measuring the fluorescence with the detector 9, bile acids are indirectly detected.
The results are recorded by the recorder 10.

[0027]

[Effect of the invention] The analytical method of the present invention has excellent separation ability,
Multi-component bile acids such as rat bile acids can be easily and quickly separated and quantified into each bile acid component without pretreatment such as conjugate fractionation.

That is, according to the analytical method of the present invention, cholic acid, chenodeoxycholic acid, deoxycholic acid, ursodeoxycholic acid, lithocholic acid, α-muricholic acid, β-muricholic acid, hyodeoxycholic acid and hyocholic acid It is possible to separate and quantify a total of 27 types of bile acids, including free, glycine-conjugated, and taurine-conjugated types.
Human, monkey, rat, guinea pig, pig, cow, bear,
It can be used to analyze bile acids in vivo in various animals such as dogs, cats, sheep, goats, rabbits, chickens, geese, pufferfish, and carp.

[0029]

[Examples] The analytical method of the present invention will be explained below with reference to Examples. Example 1 1. Separation analysis (1) Sample solution A total of 27 types of bile acids, including the following free type, glycine-conjugated type, and taurine-conjugated type, which are rat bile acid components, were collected at one time each.
A sample solution was prepared by dissolving 0 mg each in 11 methanol, and 20 μl of the sample solution was used as the injection volume. Note that the parentheses after each bile acid name represent the abbreviation of each bile acid in this specification. ■ Free cholic acid (CA), chenodeoxycholic acid (CDCA)
, deoxycholic acid (DCA), ursodeoxycholic acid (UDCA), lithocholic acid (LCA), α-muricholic acid (α-MCA), β-muricholic acid (β-MCA)
), hyodeoxycholic acid (HDCA), hyocholic acid (HCA) ■ Glycine-conjugated glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA), glycodeoxycholic acid (GDCA)
), glycoursodeoxycholic acid (GUDCA), glycolithocholic acid (GLCA), glyco-α-muricholic acid (G-α-MCA), glyco-β-muricholic acid (
G-β-MCA), glycohyodeoxycholic acid (GH
DCA), glycohyocholic acid (GHCA) ■ Taurine-conjugated taurocholic acid (TCA), taurochenodeoxycholic acid (TCDCA), taurodeoxycholic acid (TDCA)
), tauroursodeoxycholic acid (TUDCA), taurolithocholic acid (TLCA), tauro-α-muricholic acid (T-α-MCA), tauro-β-muricholic acid (
T-β-MCA), taurohyodeoxycholic acid (TH
DCA), taurohyocholic acid (THCA) (2) HP
LC device TRI ROTAR-VI (manufactured by JASCO Corporation) (3) Detector JASCO FP-210 (manufactured by JASCO Corporation) (4) Separation column Inertsil ODS-2 (5 μm), 4. 6mmφ
×15cm (manufactured by GL Science) (5) Immobilized enzyme column Sekisui-E-3α-HSD immobilized enzyme column, 4mm
φ×2cm (manufactured by Sekisui Chemical Co., Ltd.) (6) Column temperature: 30°C (7) Reaction solution: Phosphate buffer containing 0.3mM NAD+ (pH 7.8) (8) Flow rate: 1ml/ml for both mobile phase and reaction solution minutes (9
) Mobile phase 10mM triammonium phosphate aqueous solution (73vol%
), acetonitrile (19vol%) and methanol (
Two eluents were used: a mixture of 8 vol%) (solution A) and a mixture of 20mM triammonium phosphate aqueous solution (40 vol%), acetonitrile (20 vol%) and methanol (40 vol%) (solution B). there was. (10) Gradient conditions First, 100% liquid A was run for 50 minutes, and then the liquid was changed linearly from 100% liquid A to 100% liquid B over 60 minutes. The chromatogram obtained in Example 1 is shown in FIG. Separation of each bile acid component was extremely good.

2. Repeated Measurement Accuracy 200 ng of each bile acid was injected five times, and the reproducibility of retention time and peak height ratio was examined. The results are shown in Table 1.

[0031]

[Table 1]

The retention time for each bile acid has a relative deviation of 0.
It showed good reproducibility of 0.01 to 0.29%. Furthermore, the ratio of the peak height of bile acid to the peak height of the internal standard substance also showed good reproducibility with a relative deviation of 0.3 to 1.3%.

3. Each bile acid was measured at 6 concentrations between 10 and 2000 ng as a calibration curve injection amount, and a calibration curve was created. The results are shown in Figure 3. Free, glycine-conjugated, and taurine-conjugated bile acids all showed good linearity passing through the origin with a correlation coefficient of 0.999.

4. Each bile acid was measured in the range of 0.5 to 10 ng as the detection limit injection amount, and the detection limit of each bile acid at S/N=2 was determined. The results are shown in Table 2.

[0035]

[Table 2]

[0036] The detection limit of each bile acid at S/N=2 was 1 to 5 ng as an injection amount, and it was found that the detection sensitivity was high.

Examples 2 to 12 and Comparative Examples 1 to 7 Separation analysis was carried out using salt aqueous solutions having various pH values as solutions A and B. In the experiment, in Example 1,
The procedure was carried out in substantially the same manner except that the salt aqueous solution listed in Table 3 was used instead of the triammonium phosphate aqueous solution. The separation results are shown in Table 3.

[0038]

[Table 3]

For reference, Example 4 and Comparative Example 1
The chromatograms are shown in Figures 4 and 5, respectively.

Comparative Example 8 For comparison, 15 components of human bile acids (CA, CDCA, D
CA, UDCA, LCA, GCA, GCDCA, GDC
A, GUDCA, GLCA, TCA, TCDCA, TD
CA, TUDCA, TLCA) and 27 components of rat bile acids were analyzed by conventional methods (Japanese Patent Publication No. 56-38200). (1) Separation column μBONDAPAK/Phenyl, 3.9mmφ
×30cm (manufactured by Waters) (2) Reaction solution Phosphate buffer containing 0.3mM NAD+ (pH 7.0) (3) Flow rate Mobile phase 1ml/min, reaction solution 0.5m
l/min (4) Mobile phase Two eluents were used: a 0.3% ammonium carbonate aqueous solution (solution A) and acetonitrile (solution B). (5) Gradient conditions: from A solution: B solution (5:1) to A solution: B solution (25:1) in 64 minutes.
:11). The other conditions were the same as in Example 1.

A chromatogram of 15 components of human bile acids obtained in Comparative Example 8 is shown in FIG. 6, and a chromatogram of 27 components of rat bile acids is shown in FIG. Separation of rat bile acid components was incomplete and quantification was not possible.

[0042]

[Brief explanation of drawings]

FIG. 1 is an explanatory diagram showing an example of an apparatus used in the bile acid analysis method of the present invention.

FIG. 2 is a chromatogram of 27 components of rat bile acids in Example 1.

FIG. 3 is a calibration curve in Example 1.

FIG. 4 is a chromatogram of 27 components of rat bile acids in Example 4.

FIG. 5 is a chromatogram of 27 rat bile acid components in Comparative Example 1.

FIG. 6 is a chromatogram of 15 human bile acid components in Comparative Example 8.

FIG. 7 is a chromatogram of 27 rat bile acid components in Comparative Example 8.

[Explanation of symbols]

1a, 1b Eluent tank 2a, 2b Eluent pump 3 Gradient device 4 Sample injector 5 Separation column 6 Reaction liquid tank 7 Pump for reaction liquid

Claims (1)

[Claims] Claim 1: In a bile acid analysis method by high-performance liquid chromatography using a 3α-hydroxysteroid dehydrogenase-immobilized enzyme column, gradient elution of two or more liquids is performed to obtain (a) a pH 7 bile acid containing at least a lower aliphatic nitrile and a salt; -12 aqueous solutions and lower alcohols are mixed, and from the initial composition A containing 1.5-6M of lower aliphatic nitrile, 0.5-250mM of salt, and 5M or less of lower alcohol, (b) at least lower aliphatic nitrile , a mixture of an aqueous solution containing a salt with a pH of 4 to 12 and a lower alcohol, and a lower aliphatic nitrile of 0.9 to 12.
8M, salt 0.5-120mM and lower alcohol 2-1
A simultaneous analysis method for bile acids, characterized in that each bile acid component is separated and quantified by changing the composition of a mobile phase continuously and/or stepwise to a final composition B containing 2M.