JPS59151898A - Determination of bile acid - Google Patents

Abstract

PURPOSE:The high-sensitivity determination of constituents of bile acid can be effected by means of liquid chromatography with a filling agent of specific copolymer particles. CONSTITUTION:In the separation and determination of bile acid containing the free type, glycine and taurine clathrate type bile acids by means of liquid chromatography according to the known method, particles of a copolymer from a monomer of x-methylol y-acrylate or methacrylate (4>=x>=y>=3 and both x and y are integers), e.g., tetramethylolmethane tetra-acrylate and another monomer of the formula (R1, R2 are H, methyl; n is integer of 3-18) such as tetraethylene glycol diacrylate are used as a filler for the chromatography column. The elution solution to be introduced in the column is allowed to contain NAD<+>.

Description

[Detailed description of the invention] The present invention relates to a method for measuring bile acids.

BACKGROUND ART Conventionally, as one of the tests for liver function, bile acids contained in trace amounts in blood have been measured.

Bile acids in the blood include cholic acid, deoxycholic acid,
Chenodeoxycholic acid and lithocholic acid are contained in the free form, glycated/conjugated form, and taurine-conjugated form, respectively, and it is important to separate and quantify each of the bile acid components of the decatopes that exist in these forms. It is useful for pathological analysis and differential diagnosis of diseases.

For this reason, in the past, as in Japanese Patent Publication No. 56-38200, high-performance liquid chromatography was used to use a spherical modified silica gel of around 10μ to which phenyl groups were chemically bonded, and the gel was mixed with the eluent in a separation container. A biological sample such as blood serum was injected to remove each bile acid component, and the reaction solution containing NAD+ was mixed with the effluent from the column.
-Hydroxysteroid dehydrogenase (3α-HS
D) After passing through an immobilized enzyme column packed with an immobilized carrier and reacting each bile acid component separated with NAD+ sequentially, the reaction product was subjected to ultraviolet υ absorption measurement and/or fluorescence measurement. Bile acids are measured by detecting each bile acid component.

By the way, in the above-mentioned conventional method for determining σ11j, a silica-denatured gel is used as a separation column in liquid chromatography, but it tends to non-specifically adsorb miscellaneous compounds such as proteins, and its separation performance deteriorates rapidly. Pretreatment is required to remove these non-specifically adsorbed substances, and since the eluent is unstable to alkali and can only be used as an eluent with a pH of 8.5 or less, the bile acid component after separation has a high enzymatic activity of 3α-H5D. In order to carry out the reaction, it was necessary to adjust the pH value after flowing out from the separation column.

In addition, it has been carried out to supply a reaction solution in which NAD+ is added to the effluent from a separation column using threads that react with each bile acid component and NAD+ in an immobilized enzyme column, but in this case, The effluent containing each bile acid component is diluted by the reaction solution, and the liquid is turbulent during mixing, which makes it impossible to measure each bile acid component with high sensitivity.

The purpose of the present invention is to solve the above-mentioned drawbacks, and its gist is to apply a sample solution containing free, glycine-conjugated, and taurine-conjugated bile acids to a separation column together with an eluent. The immobilized 3α-H5D
Each bile acid component is reacted with NAD+ by passing through an immobilized enzyme column having a In the measuring method, the packing material of the separation column is a monomer represented by τ methylol alkyl 1 acrylate (or methacrylate) (where π and 1 are integers in the relationship of 4≧χ≧γ≧3) and the following. A monomer represented by the general formula, (wherein R1 and R2 are hydrogen or methyl groups, and n is 3 to 1
A method for measuring bile acids, characterized in that 1c NAD+ is present in an eluent introduced into a separation column from particles of a copolymer (an integer of 8).

Next, the method for measuring bile acids of the present invention will be explained in more detail.

The sample liquid in the present invention includes biological sample liquids such as blood (serum) and bile. There are free forms in the sample solution,
Contains 15 types of bile acids, including glycine-conjugated and taurine-conjugated types. In order to separate these bile acid components, a sample solution is introduced into a separation column together with an eluent and subjected to liquid chromatography, particularly high performance liquid chromatography.

Reference numeral 1 denotes an eluent tank, and examples of the eluent used include ammonium carbonate-acetonitrile mixture, phosphoric acid, ammonium-acetonitrile mixture, and the like. NAD+ is added to this dissolved Pt solution.

A sample solution such as serum or bile is injected and mixed with an eluent containing NAD+.

The sample solution is mixed with an eluent containing NAD+ and guided to the separation column 4.

The filler filled in the separation force 2m4 to separate each bile extract component is a monomer represented by π methylolalkyl 1 acrylate (or methacrylate) (where π and y are 44χ≧1). ≧
3) (A) and a monomer (B) represented by the following general formula, R100R2 (wherein R1 and R2 are hydrogen or a methyl group,
(an integer of 8).

Examples of the monomer (A) include tetramethylolmethane diacrylate, tetramethylolmethanetetramethacrylate, tetramethylolmethanetriacrylate, tetramethylolmethanetrimethacrylate, trimethylolmethanetriacrylate, and trimethylol. Examples include methane trimethacrylate, trimethylolmethane triacrylate, trimethylolpropane trimethacrylate, and particularly tetramethylolmethanetetraacrylate, tetramethylolmethane triacrylate, and tetramethylolmethane trimethacrylate are preferably used.

Examples of the monomer (B) include triethylene glycol diacrylate, triethylene glycol dimethacrylate, tetraethylene glycol diacrylate, tetraethylene glycol dimethacrylate, nonaethylene glycol diacrylate, nonaethylene glycol dimethacrylate, and tetraethylene glycol dimethacrylate. Examples include decaethylene glycol diacrylate, tetradecaethylene glycol dimethacrylate, and especially tetraedelene glycol diacrylate), )! 7 ethylene glycol diacrylate and tetraethylene glycol dimethacrylate are preferably used.

In order to obtain particles of a copolymer of monomer (Al and monomer (B)), for example, a radical generating catalyst is added to a mixture of monomer (Al and monomer (B)) as needed to form an aqueous suspension. Perform turbid polymerization.

The ratio of monomers (A) and (Bl) used is preferably such that the amount of monomer (B) is 100 parts by weight or less per 100 parts by weight of monomer.

This is because when the proportion of monomer (B) exceeds the above proportion, the resulting copolymer becomes soft, which causes problems in mechanical strength when it is packed in separation column 4 and subjected to high performance liquid chromatography. This is because the separation function of bile acids is also likely to deteriorate. Also monomer (A)
The optimum filler is obtained when the ratio of the pair (B) is between 10:1 and 10:3.

In addition, if the monomer (the number of repeating units of +CH2-CH2-0+ in Bl 7L ill, 9 becomes 19 or more, problems will occur in terms of mechanical strength of the copolymer particles, and if n is 1 or 2) Since the separation power decreases, n is set in the range of 3 to 18.

During polymerization, if an organic solvent is present that dissolves the monomers used but does not dissolve the resulting copolymer, the resulting polymer particles will become porous and have an increased surface area, making them preferable as fillers. That's it.

Examples of the above organic solvents include aromatic hydrocarbons such as toluene, xylene, diethylbenzene, and dodecylbenzene;
Examples include saturated hydrocarbons such as hexane, hegetane, octane, and decane; alcohols such as inamyl alcohol, hexyl alcohol, and octyl alcohol; ) mixture 1
It is preferably used in an amount of 15 to 200 parts by weight, more preferably 20 to 150 parts by weight.

Furthermore, it is preferable that the particle size of the small polymer particles packed in the separation column used in the present invention be uniform in the range of 3 to 40 μm.

Then, the small polymer particles are dispersed in water and are force-fed and packed into a column made of stainless steel or the like using a constant flow pump or the like, thereby preparing the separation column used in the present invention.

A sample solution mixed with an eluent containing NAD+ is introduced into the separation column 4, and each bile acid component is sequentially separated by liquid chromatography. KO for each bile acid component
The effluent that is separated and flows out from separation force 2m4 contains NAD+
is contained as is.

The effluent is passed through an immobilized enzyme force 5 with immobilized 3α-H2O. Immobilized 3α-H2O
To obtain this, for example, the surface of the cellulose is activated in advance with a suitable reagent such as bromide cyanide, and 3α-H2O is contacted with fine particles or powdered cellulose having a size of several μm to several hundred μm. The 3α-H8D
It is preferable to chemically bond and immobilize it on the cellulose interface by adsorption, covalent bonding, etc.

By contacting the bile acids with the 3α-IT SD in the presence of NAD+ in the immobilized enzyme column 5, the 3α-IT SD
-1 (By the catalytic action of S'D, bile acids and NAD+ react to produce a fluorescent substance (NhDn).

And the NADH produced by that reaction has a wavelength of 365 nm.
The absorption spectrum shows a maximum near 465 nm, and the fluorescence spectrum shows a maximum near 465 nm. Therefore, the excitation wavelength is 3657
L? It% fluorescent wavelength 465? Bile acids are detected by setting L7FL and performing ultraviolet absorption measurement and/or fluorescence measurement using the detector 6, and the results are recorded on the recorder 7. The sample liquid leaving the detector 6 is introduced into a liquid tank 8.

According to the method of the present invention, the filler having the function of separating each bile acid is composed of particles of a monomer (copolymer of Al and monomer (B)); Non-specific adsorption of miscellaneous compounds such as proteins, and prevention of a decrease in the E ability of each bile acid component due to non-specific adsorption of proteins, and the release of free, glycine-conjugated and taurine-conjugated compounds. A sample solution containing bile acids can be well separated into each bile acid component.

In addition, the particles of the copolymer of monomer (A) and monomer (Bl) are stable up to a pH value of about 10, so 3α-H5
Eluents with high prl values that increase D activity can be used.

Furthermore, in the present invention, since NAD is pre-existing in the eluent introduced into the separation column, it is possible to ensure that the effluent containing gold of each bile acid component is not diluted by the NAD+ solution. Furthermore, since there is no disturbance of the liquid when mixing the ten NAD liquids, each bile acid component can be measured with high sensitivity.

Example 1 In a 2t separable flask equipped with a condenser, a stirrer, a thermometer, and a dropping funnel, a 5% FFI polyvinyl alcohol aqueous solution 40Q tut, tetramethyl i-lmethane triacrylate 84f, and tetraethylene glycol were added. A mixture of 16y diacrylate and 0.15y benzoyl peroxide was fed. Furthermore, 100 y of toluene was added. Then 40 Or, p, m
The temperature was raised to 80° C. with stirring, the reaction was carried out for 10 hours, and the mixture was cooled. After cooling and separating the polymerization product,
A porous spherical polymer having a particle size of 5 to 20 μ9 was obtained by washing with hot water and aqueous solution. Particles of 8 to 10 μm obtained by removing fine particles and coarse particles were dispersed in soml of ion-exchanged water and placed in a stainless steel column (diameter 7.9 μm).
A separation column was prepared by filling a column (with a length of 30 m) with ion-exchanged water using a constant flow pump at a rate of λ0rtl/mIn.

Next, using cellulose fine particles (particle size approximately 80 microns) as a support, 5 m of ion-exchanged water and 10 ml of 2M sodium carbonate aqueous K were added to the cellulose fine particles, and after stirring, 2 y of cyanide bromide was added to this in advance. Add 1 ml of acetonitrile dissolved in and stir vigorously.
The reaction was allowed to take place for 0 seconds. The thus activated cellulose fine particles were quickly mixed with 0.1M carbonate solution (pH 9, s), ion exchange water and 0.1M sodium chloride containing 0.5M sodium chloride.
After washing with carbonate buffer (PH9,6), 3α-H5
Add 5 ml of 0.1 M carbonate buffer (pF[9,5) containing 0.5 M sodium chloride in which D44g was dissolved and stir for 2 hours at room temperature to react. -0.05% of 2
- containing mercaptoethanol, 1M)! The reaction was carried out at 4° C. for 2 hours in I sous-acid buffer (pH 8,0) for 7 nights.

The thus obtained cellulose support on which 3α-HSD was immobilized was heated to 0.1M containing 05λ (sodium chloride).
Acetic acid grade example solution (PH5,0), ion exchange water and 0.5M
0.1λ (carbonate buffer (pH 9) containing sodium chloride
, 5), the diameter was 45. length 10c
The complete eluent for the 0.3% ammonium phosphate aqueous solution in a stainless steel column is a solution prepared by adding NH4OH to a 3% ammonium phosphate aqueous solution to adjust the pH to 9.5, and 18 volts of acetonitrile added thereto (liquid I), or a 0.3% ammonium phosphate aqueous solution with NH4OH.
Add 27 VO to an aqueous solution with 401'1 and adjust the pH to 9.5.
A solution containing 1% acetonitrile was used (Liquid II).

■, 0.22 ml of N A l) was added to both solutions. In addition, as samples, a total of 15 types of 5 types of bile acids, cholic acid, ursodeoxycholic acid, chenodeoxycholic acid, deoxycholic acid, and lithocholic acid, and glycine conjugates and taurine conjugates of each of these bile acids were collected at 10η. Each sample was dissolved in 1 ton of methanol.

In the analysis operation, 10 μt of the sample was injected into the injector 3 while the eluent (1 solution) was flowing through the constant flow bonder 2 at a flow rate of 1.0 m/min, and taurine-conjugated deoxycholic acid (TDCA) was injected into the injector 3. When eluted, eluent (Jl solution) 1. The flow rate was switched to s d /win.

Thus, all of the 15s of the series shown in Figure 2 are K.
A clear peak was drawn.

Example 2 Using the same equipment as in Example 1, including a separation column, an immobilized enzyme column, and an eluent containing NAD+, normal human serum was pretreated under the same conditions as in Example 1. Each bile acid component was measured by injecting it without treatment. The results are shown in FIG.

Comparative Example 1 In Example 1, NAD+ was not used in the solution, and a reaction solution containing NAD+ was continuously added to the effluent from the separation column at a rate of 1 ml/min. Each bile acid component was measured in the same manner as in Example 1, except that the serum was injected without pretreatment.

The results are shown in FIG. 4, and the sensitivity was significantly lower than the results shown in FIG.

[Brief explanation of drawings]

Fig. 1 is an explanatory diagram showing an example of the apparatus used in the method for measuring bile acids of the present invention, Fig. 2 is a lipid diagram in Example 1, Fig. 3 is a chromato duram in Example 2, and Fig. 4 is These are the two chroma dogs in Comparative Example I. Explanation of symbols 1...Eluent tank, 2...Constant Dw,
% Bong, 3...Sample injector, 4...
... Separation column, 5... Immobilized enzyme column,
6...detector, 7...recorder,
Pl , , , cholic acid , P2 , , , ,
. Glycine-conjugated cholic acid I'+Z, P3... Taurine-conjugated cholic acid % 1'4... Ursodeoxycholic acid, P5... Glycine-conjugated ursodeoxycholic acid, P6... ...Taurine-conjugated ursodeoxycholic acid, 7...chenodeoxycholic acid, P8...deoxychol KR1P9.
...Glycine-conjugated chenodeoxycholic acid, Plo
...Glycine conjugated deoxycholic acid, pH...
...Taurine-conjugated chenodeoxycol a, Pl2・
...Taurine-conjugated deoxycol a, Pl3...
...Litcall TW? ,, Pl4...Glycine-conjugated lithocholic acid, Pl5......Taurine-conjugated lithocholic acid. Patent applicant Sekisui Chemical Co., Ltd. Representative Mototoshi Fujinuma

Claims (1)

[Scope of Claims] 1. A sample solution containing free, glycine/conjugated, and taurine-conjugated bile acids was introduced into a separation column together with an eluent and separated into each bile acid component by liquid chromatography. After that, the effluent containing each bile acid component from the separation column is passed through an immobilized enzyme column containing immobilized 3α-hydroxystetehydrogenase to separate each bile acid component and nicotinamide adenine dinucleotide (NA).
In the method of measuring bile acids by reacting with I)-) and detecting each bile acid component by ultraviolet absorption measurement and/or fluorescence dI determination of the reaction product, the packing material of the separation column However, the northern methylol alkyl γ acrylate (
or methacrylate) (however, χ and 1
is an integer in the relationship of 4≧dr≧1≧3), and a monomer represented by the following general formula, RIO0R2 (wherein R1 and R2 are hydrogen or methyl groups, and 9 is 3 to 1
A measurement of bile acids characterized by the presence of nicotinamide adenine dinucleotide (NAD+) in the eluent introduced into the separation column. Method.