JPH0248238B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for analyzing bile acids.

Conventionally, in order to analyze bile acids including free, glycine-conjugated, and taurine-conjugated types, it has been difficult to detect fractionated bile acids by simply separating them using liquid chromatography. Each bile acid component undergoes a chemical change to the keto form by the enzyme 3α-hydroxysteroid dehydrogenase (hereinafter referred to as 3α-HSD) in the presence of the coenzyme nicotinamide adenisine dinucleotide (hereinafter referred to as NAD ⁺ ). , and at the same time NAD ⁺ is an easily detectable fluorescent substance
The amount of NADH produced in this reaction, that is, the amount of existing bile acids, has been quantified using optical methods such as ultraviolet absorption measurement and fluorescence measurement. In the above quantitative method, in order to effectively use 3α-HSD, it is known to be used by immobilizing it on a support such as glass beads.

However, although glass beads have the advantage of being strong, on the other hand, they cannot be used in alkaline solutions, so they usually have a pH
Must be used with 8.5 or lower. Under these conditions, it is similar to alkaline conditions with a pH of 9 or higher.
Since 3α-HSD does not exhibit high enzymatic activity, it is difficult to improve the sensitivity of the analysis. In addition, non-specific adsorption of organic substances, particularly proteins, is likely to occur on the support, which leads to a decrease in enzyme activity due to surface contamination, resulting in a shortened lifespan of the immobilized enzyme.

In addition, silica-denatured gel-based columns are known for separation of bile acid components in liquid chromatographs, but these columns are difficult to separate from other components such as proteins. It adsorbs non-specifically and the separation performance deteriorates rapidly, so it requires pre-treatment to remove these non-specifically adsorbed substances and is difficult to use. Therefore, in order for bile acid components after separation to react with 3α-HSD with high enzymatic activity, it is necessary to adjust the pH so that it is maintained at a high constant value after flowing out of the separation column. There are drawbacks such as insufficient separation of components.

In view of the current situation in the analysis of bile acids as described above, the present invention provides a method that can easily analyze samples containing bile acids without requiring pretreatment, stably over a long period of time, and with excellent accuracy. It was made for the purpose of providing monomer A represented by tetramethylolmethane x acrylate (or methacrylate) (however,
x is 4 or 3) 100 parts by weight and monomer B represented by the following general formula (In the formula, R ₁ and R ₂ are hydrogen or methyl groups, and n is 3 to 18
A column packed with small polymer particles formed by aqueous suspension polymerization of a monomer mixture consisting of 100 parts by weight or less is used as a separation column, and if necessary, 80 parts by weight or less Contains water-soluble organic solvents
Bile acid components are separated by liquid chromatography using a buffer solution with a pH of 4.0 to 10.5 as an eluent, and the separated liquid flowing out from the separation column is
A mixed solution containing a reaction solution containing NAD ⁺ and whose pH was adjusted to 9.0 or higher is passed through a column filled with 3α-HSD immobilized on a cellulose support, and the effect of the 3α-HSD is The method of analyzing bile acids is characterized by sequentially reacting each bile acid component with NAD ⁺ and measuring a fluorescent substance produced by the reaction.

In the present invention, small polymer particles formed by aqueous suspension polymerization of a monomer mixture of monomer A and monomer B are used as a packing material in a separation column. , the ratio of monomers A and B in the monomer mixture is as follows:
If the amount of monomer B is too large, the resulting copolymer will become soft, causing problems in mechanical strength when used as a packing material for high-performance liquid chromatography, and also reducing the ability to separate bile acids. So,
It is used in an amount of 100 parts by weight or less per 100 parts by weight of monomer A, and it is particularly preferable that the ratio of monomers A to B is 10:1 to 10:3. Furthermore, if the number n of (-CH ₂ -CH ₂ -O)- repeating units in monomer B is 19 or more, problems will arise in terms of mechanical strength of the copolymer particles, and if it is too small, separation will occur. Since the performance decreases, n is set to 3 to 18, more preferably 3 to 14.

Examples of the monomer A include tetramethylolmethanetetraacrylate, tetramethylolmethanetetramethacrylate, tetramethylolmethanetriacrylate, and tetramethylolmethanetrimethacrylate, particularly tetramethylolmethanetetraacrylate and tetramethylolmethanetriacrylate. , tetramethylolmethane trimethacrylate is 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 tetradeca. Examples include ethylene glycol diacrylate, tetradecaethylene glycol dimethacrylate, and particularly preferred are tetraethylene glycol diacrylate, triethylene glycol diacrylate, and tetraethylene glycol dimethacrylate.

Polymerization of the monomer mixture is carried out by aqueous suspension polymerization by adding a radical-generating catalyst as necessary according to a conventional method.During this polymerization, the monomers used are dissolved, but the polymer In the presence of an organic solvent in which polymer particles do not dissolve, the resulting polymer particles become porous and have an increased surface area, making them preferable as fillers.

Examples of the above organic solvents include aromatic hydrocarbons such as toluene, xylene, diethylbenzene, and dodecylbenzene, saturated hydrocarbons such as hexane, heptane, octane, and decane, and alcohols such as isoamyl alcohol, hexyl alcohol, and octyl alcohol. The amount used is 15 to 200 parts by weight per 100 parts by weight of the monomer mixture.
Preferably, parts by weight are used, more preferably
20 to 150 parts by weight. Further, in the present invention, it is preferable that the particle size of the small polymer particles packed in the separation column be uniform in the range of 3 to 40 microns.

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

Next, the column packed with 3α-HSD immobilized on a cellulose support used in the present invention is used to prepare fine particles or powdered cellulose with a size of several microns to several hundred microns, for example, by coating the surface of the cellulose in an appropriate manner. A reagent such as cyanide bromide is activated, the 3α-HSD is brought into contact with the reagent, the 3α-HSD is chemically bonded to the cellulose surface by adsorption or covalent bonding, and immobilized, and this is applied to a column using a conventional method. It can be prepared by filling.

FIG. 1 is a flowchart showing an example of the analytical method of the present invention. The method of the present invention will be explained based on the diagram. In the figure, 1 is an eluent, and the eluent is pumped into the liquid chromatograph by a pump 2. Separation column 4
is transferred to the separation column 4 in a flow path connected to the separation column 4. As the eluent 1, a buffer solution with a pH of 4.0 to 10.5 is used, but this may contain up to 80% by weight of a water-soluble organic solvent, if necessary. Further, the separation column 4 is filled with the above-mentioned specific polymer small particles.

Before the eluent enters the separation column 4, a sample containing bile acids is injected by the injector 3,
It merges with the eluent and passes through the separation column 4. Here, the bile acids contained in the sample are separated into each component by the separation action of the separation column 4.
More leakage.

A reaction solution 5 containing NAD ⁺ is mixed into this separated solution using a pump 6, and this mixed solution is added to a column 7 filled with 3α-HSD immobilized on a cellulose support.
When bile acids come into contact with 3α-HSD in the presence of NAD ⁺ in column 7, the catalytic action of 3α-HSD causes
Bile acids and NAD ⁺ react to produce a fluorescent substance (NADH). Since the amount of this fluorescent substance produced depends on the amount of bile acids contained in the sample solution, the fluorescence detector 8 measures the amount of fluorescent substance using optical methods such as ultraviolet absorption measurement and fluorescence measurement. It is possible to quantify acids. Further, the measurement results of the fluorescence detector 8 may be recorded by a recorder 9. In addition, in the present invention, when the mixed liquid passes through the column 7, the pH of the mixed liquid is
is adjusted to be 9.0 or higher, and this pH adjustment can be achieved by using buffers of appropriate composition as eluent 1 and reaction solution 5 containing NAD ⁺ , or by adjusting their mixing ratio. It can be done easily.

As mentioned above, the reason for adjusting the pH of the mixed liquid in the present invention is that the cellulose support used in the present invention can be used even at a pH of 9 or higher without fear of being attacked, and that the 3α-HSD catalyst This is based on the reason that enzyme activity reaches its maximum in the alkaline range of pH 9 to 11.According to the present invention, glass bead carriers, etc., which were only used in the pH range of about 8.5 or lower, are used. This makes it possible to analyze with much higher sensitivity than the conventional bile acid analysis method used.

Further, by setting the pH of the mixture to 9 or higher, there is also the advantage that non-specific adsorption of organic substances, particularly proteins, to the support is less likely to occur, which would shorten the life of the immobilized enzyme.

The method for analyzing bile acids of the present invention is as described above, and in particular, the column packed with the specific polymer particles is used as a separation column, and the separated liquid flowing out from the separation column contains NAD ⁺ A mixed solution containing the reaction solution and adjusted to a pH of 9.0 or higher is mixed with 3α-
Since this method requires passing through a column packed with HSD, it is possible to sharply separate the bile acids in the sample with high sensitivity and precision, and it is also possible to perform repeated and continuous analysis. This method is possible, and furthermore, it can produce excellent effects such as being able to directly analyze biological samples such as serum without requiring pretreatment such as protein removal.

Examples of the present invention will be described below.

Example 1 400 ml of a 5% by weight aqueous polyvinyl alcohol solution, 84 g of tetramethylolmethane triacrylate, 16 g of tetraethylene glycol diacrylate, and 0.15 g of benzoyl peroxide were placed in a second separable flask equipped with a condenser, stirrer, thermometer, and dropping funnel. A mixed solution consisting of g was supplied. Furthermore, 100 g of toluene was added. Next, the temperature was raised to 80° C. while stirring at 400 rpm, reaction was carried out for 10 hours, and then cooled. After cooling, the polymerization product was separated and washed with hot water and acetone to obtain a porous spherical polymer with a particle size of 5 to 20 μm. Particles of 8 to 10 μm obtained by removing fine particles and coarse particles were dispersed in 80 ml of ion-exchanged water, and ion-exchanged water was added to a stainless steel column (diameter 7.9 mm, length 30 cm) at a rate of 2.0 ml/min using a constant flow pump. The column was packed by pressure-feeding at high speed and a column for separation was prepared.

Next, using cellulose fine particles (particle size of about 80 microns) as a support, add 5 ml of the cellulose fine particles to
5ml of ion exchange water, 10ml of 2M sodium carbonate solution
After stirring, 1 ml of acetonitrile in which 2 g of cyanide bromide had been previously dissolved was added, and the mixture was reacted for 90 seconds with vigorous stirring. After quickly washing the activated cellulose microparticles with 0.1M carbonate buffer (PH9.5), ion exchange water, and 0.1M carbonate buffer (PH9.5) containing 0.5M sodium chloride, 44mg of 3α-HSD was added. 0.1M carbonate buffer containing dissolved 0.5M sodium chloride (PH9.5)
5 ml was added and stirred at room temperature for 2 hours to react.

Next, in order to block the active sites still present on the cellulose support on which 3α-HSD was immobilized by the above treatment, it was added to 0.1M Tris-HCl buffer (PH8.0) containing 0.05% 2-mercaptoethanol. The mixture was reacted at 4°C for 2 hours. 3α thus obtained
-0.5M cellulose support with immobilized HSD
0.1M acetate buffer containing sodium chloride (PH
After repeated washing with ion-exchanged water and 0.1M carbonate buffer (PH9.5) containing 0.5M sodium chloride, the column was packed into a stainless steel column with a diameter of 4 mm and a length of 10 cm.

Using the column obtained above as column 7 in the flow chart shown in FIG. 1, and using a liquid chromatograph incorporating the separation column prepared above as a liquid chromatograph,
Bile acids were analyzed by connecting these columns and a fluorescence detector as shown in FIG.

As an eluent, add NH ₄ OH to 0.3% ammonium phosphate aqueous solution to make the pH 9.5, and add 18 vol% to the aqueous solution.
of acetonitrile (liquid A) or 0.3
% ammonium phosphate aqueous solution by adding NH ₄ OH to PH
An aqueous solution of 9.5 and 27 vol% acetonitrile was used. In addition, as samples, 10 mg of 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. One part of each was dissolved in methanol.

In addition, in FIG. 1, the reaction solution 5 mixed with the separated solution separated by the separation column 4 contains 199.1 mg of NAD ⁺ 2 and 2 ethylenediamine-4-acetic acid.
A 0.026M pyrophosphate buffer with a pH of 9.5 containing 24.5 mg of sodium salt was used.

For analysis, pump 2 pumps eluent A.
The reaction solution was pumped by pump 6 at a flow rate of 1.0 ml/min.
10μ of the sample while flowing at a flow rate of ml/min.
was injected from injector 3, and when taurine-conjugated deoxycholic acid (TDCA) was eluted, the flow rate was changed to 1.5 ml/min of eluent B.

In this way, a chromatogram with clear peaks for all 15 types as shown in Figure 2 was obtained.

In addition, 11-25 in FIG. 2 are the peaks of the following bile acids, respectively.

11: Cholic acid, 12: Glycine-conjugated cholic acid, 13: Taurine-conjugated cholic acid, 14: Ursodeoxycholic acid, 15: Glycine-conjugated ursodeoxycholic acid, 16: Taurine-conjugated ursodeoxycholic acid, 17: Chenodeoxycholic acid, 1
8: Deoxycholic acid, 19: Glycine-conjugated chenodeoxycholic acid, 20: Glycine-conjugated deoxycholic acid, 21: Taurine-conjugated chenodeoxycholic acid, 22: Taurine-conjugated deoxycholic acid, 2
3: Tricholic acid, 24: Glycine-conjugated lithocholic acid, 25: Taurine-conjugated lithocholic acid Example 2 Using the same equipment and method as in Example 1, 100μ of serum from a patient with obstructive jaundice was directly analyzed without pretreatment. The results were as shown in Figure 3. This analysis was repeated 20 times, but almost no changes were observed from the results obtained in the first run shown in Figure 3.

Next, add 1.0 ml of serum from the above patient to 9 ml of 0.1N NaOH.
The mixture was passed through a glass column packed with 10 ml of synthetic resin adsorbent (Amberlite Add 1.0ml of methanol to
A sample subjected to protein removal treatment was prepared, and 100μ of the sample was used for analysis in the same manner as in the case without pretreatment.

The results are shown in Figure 4.
A chromatogram was drawn in which the peak height was reduced by about 70% compared to the figure.

In addition, in the peaks shown in FIGS. 3 and 4, 12, 13, 19, and 21 refer to bile acids with the same numbers in FIG. 2, and 26 is a component other than bile acids.

[Brief explanation of the drawing]

Figure 1 is a flow chart showing an example of the analytical method of the present invention, Figure 2 is a chromatogram obtained in Example 1 of the present invention, and Figures 3 and 4 are chromatograms obtained in Example 2 of the present invention. This is a chromatogram. 1... Eluent, 2, 6... Pump, 3... Injector, 4... Separation column, 5... Reaction solution containing NAD ⁺ , 7... Column packed with immobilized 3α-HSD,
8...Detector, 9...Recorder, 11-25...Peaks of each bile acid component, 26...Components other than bile acid.

Claims (1)

[Claims] 1 Monomer A represented by tetramethylolmethane x acrylate (or methacrylate) (however,
x is 4 or 3) 100 parts by weight and monomer B represented by the following general formula (In the formula, R ₁ and R ₂ are hydrogen or methyl groups, and n is 3 to 18
A column packed with small polymer particles formed by aqueous suspension polymerization of a monomer mixture consisting of 100 parts by weight or less is used as a separation column, and if necessary, 80 parts by weight or less Contains water-soluble organic solvents
Bile acid components are separated by liquid chromatography using a buffer solution with a pH of 4.0 to 10.5 as an eluent, and the reaction solution containing nicotinamide adenine dinucleotide (NAD ⁺ ) is added to the separated solution flowing out from the separation column. A mixture containing
It is passed through a column filled with 3α-hydroxysteroid dehydrogenase (3α-HSD), and each bile acid component is separated by the action of 3α-HSD.
1. A method for analyzing bile acids, which comprises sequentially reacting with NAD ⁺ and measuring a fluorescent substance produced by the reaction. 2 Monomer A vs. Monomer B in a Monomer Mixture
2. The analytical method according to item 1, wherein the ratio by weight is 10:1 to 10:3. 3. A separation column filled with small porous polymer particles formed by aqueous suspension polymerization of a monomer mixture in the presence of an organic solvent that dissolves the mixture but does not dissolve the polymer. Analytical method described in section.