JPS648305B2 - - Google Patents

Description

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

BACKGROUND ART Conventionally, one of the tests for liver function has been the determination of trace amounts of bile acids contained in blood. Blood contains cholic acid, deoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, and lithocholic acid as free forms, glycine conjugates, and taurine conjugates as bile acids. Separating and quantifying each bile acid is useful for pathological analysis and differential diagnosis of hepatobiliary diseases.

Conventionally, high-performance liquid chromatography was used to separate bile acids by injecting a biological sample such as serum mixed with an eluent into a separation column, and then separating bile acids from nicotinamide adenine dinucleotide (hereinafter referred to as nicotinamide adenine dinucleotide).
Supply the reaction solution containing NAD ⁺ (abbreviated as NAD +), and
The separated bile acids are passed through an immobilized enzyme column packed with a carrier on which 3α-hydroxysteroid dehydrogenase (hereinafter abbreviated as 3α-HSD) is immobilized, and the separated bile acids are reacted with NAD ⁺ to generate a fluorescent substance. Each of the 15 types of bile acids in biological samples is quantified based on the results obtained using a fluorescence detector.

However, since biological samples are subjected to pretreatment to remove separation-inhibiting substances before being subjected to liquid chromatography, bile acids in the sample may be lost during this process, which may affect the detection results. , a known third component is added as an internal standard substance.

Conventionally, pregnanetriol has been used as such an internal standard substance. However, the retention time of pregnanetriol in a separation column is longer than that of all bile acids, so it takes time to analyze, and the detection peak also has a broad width, which has the disadvantage of increasing errors when used as a reference peak. It was hot.

The present invention was made with the aim of eliminating such drawbacks, and uses bile acids as an internal standard substance that has a retention time shorter than that of all bile acids, but has the property of eluting more slowly than solvents and other contaminants. This invention was achieved by finding a method that has a sharp peak during detection, and is a method for quantifying bile acids that allows bile acids to be quantified in a short time and with less error. It provides:

That is, the gist of the present invention is to separate bile acids in a biological sample by liquid chromatography, introduce the bile acids into a column containing an immobilized enzyme, and bring the bile acids into contact with the immobilized enzyme within the column to separate the bile acids and the bile acids. In a method for quantifying bile acids by causing a reaction with a substance that can react with the bile acid and measuring the reaction product with a detector, it is recommended to use an acidic amino acid conjugate of deoxycholic acid or chenodeoxycholic acid as an internal standard substance. The method consists in the quantitative determination of bile acids.

In designing the internal standard substance in the present invention, an ionic group was introduced to increase hydrophilicity in order to shorten the retention time in the column for reversed-phase separation, and the reactivity to enzymes was reduced by bile acid. In order to achieve a similar result, we considered using bile acid derivatives. We have also discovered an acidic amino acid conjugate of deoxycholic acid or chenodeoxycholic acid as an internal standard substance that meets this requirement. The acidic amino acid conjugate of deoxycholic acid or chenodeoxycholic acid is chemically bonded by an amide bond that is resistant to hydrolysis in dilute alkaline conditions, and a carboxyl group is used as the ionic group. Further, as the acidic amino acid, those having more carboxyl groups than amino groups, such as glutamic acid and aspartic acid, are used.

To synthesize an acidic amino acid conjugate of deoxycholic acid or chenodeoxycholic acid, for example, the hydrochloride of deoxycholic acid or chenodeoxycholic acid and an acidic amino acid ester is mixed with 1-ethyl-3-hydrochloride.
The reaction may be carried out by stirring in the presence of (3-dimethylaminopropyl)carbodiimide, and further hydrolysis may be carried out using a potassium hydroxide solution. When quantifying bile acids using the above-mentioned internal standard substance, for example, an apparatus as shown in FIG. 1 is used.

Reference numerals 1 and 2 are eluent tanks, and the eluent in eluent tank 1 is, for example, an ammonium carbonate-acetonitrile mixture, and the eluent 2 is acetonitrile, and a gradient is applied to the addition of acetonitrile. It is assumed that the content of acetonitrile gradually increases. 4 is a gradient programmer for this purpose.

Reference numeral 5 denotes a sample injector, into which a sample solution in which a certain amount of an acidic amino acid conjugate of deoxycholic acid or chenodeoxycholic acid is added to a pretreated serum sample is injected and mixed with the eluent. In addition, as a sample solution, as a method of correcting the loss of blood bile acids during pretreatment, a serum sample may be pretreated and extracted after adding a certain amount of internal standard substance. . The sample liquid is led from the flow path 3 to the separation column 6, where bile acids are separated. The separation column 6 is filled with particles of, for example, porous silica into which octadecyl groups have been introduced, divinylbenzene-(meth)acrylic acid copolymer, or the like.

Bile acids are separated in the separation column 6 by high performance liquid chromatography. A reaction solution containing NAD ⁺ is supplied from the reaction solution tank 10 to the separated solution flowing out from the separation column 6, and is guided to the immobilized enzyme column 7 filled with 3α-HSD, and then
The action of 3α-HSD causes each bile acid component to react with NAD ⁺ in sequence, and the reaction produces a fluorescent substance.

Since the amount of this fluorescent substance produced depends on the amount of bile acid contained in the sample solution, it is detected by a fluorophotometer 8 and the result is recorded by a recorder 9.

In this way, first, the peaks of a mixture of proteins and the like in the sample solution, the peaks due to the acidic amino acid conjugates of deoxycholic acid or chenodeoxycholic acid as internal standard substances, and the peaks due to each bile acid are recorded.

Based on the results thus obtained, the amount of each bile acid can be determined.

According to the present invention, by using an acidic amino acid conjugate of deoxycholic acid or chenodeoxycholic acid as an internal standard substance, the retention time in the separation column is shorter than that of each bile acid.
The analysis time for each bile acid can be shortened, and the peaks at the time of detection will be sharper.
Quantitative errors can be reduced.

Example 1 A glutamic acid conjugate of deoxycholic acid was synthesized as follows.

L-glutamic acid diethyl ester hydrochloride (323 mg) and deoxycholic acid (399 mg) are stirred and suspended in dichloromethane (100 ml), and triethylamine (0.19 ml) is added dropwise. Subsequently, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (293 mg) was added, and the mixture was stirred and reacted at room temperature. After 2 hours, the resulting solution was mixed with water (50 ml x
3) After washing with 1M hydrochloric acid (50 ml x 3) and saturated sodium bicarbonate solution (50 ml x 3), dehydrate over anhydrous magnesium sulfate and filter. The solvent of the solution was distilled off, and then the solution was dissolved in a 1% potassium hydroxide-methanol-water (3:1 V/V) solution (50 ml) for hydrolysis. After stirring at room temperature for 2 hours, methanol was distilled off under reduced pressure, and while cooling the resulting aqueous solution with ice, hydrochloric acid was added dropwise to adjust the pH to 4. After leaving it in the refrigerator and collecting the precipitate, washing it with ice water,
Lyophilized. The yield at this time was 424 mg (80%). This lyophilized powder was used as an internal standard.

Dissolve 20 mg of the above-mentioned internal standard substance (e.g., glutamate conjugate of deoxycholic acid) in ethanol (100 ml), and add this solution to 1 ml of sample serum.
Add 10 μl of ethanol (2.5 μl) to 0.5 ml of this serum.
ml) and stirred for 1 minute in a water bath at 85°C.
This was centrifuged and the supernatant was collected.

Ethanol (2.5 ml) was added to the residue and the above operation was repeated, the resulting supernatant was evaporated to dryness, 100 μm of methanol was added and dissolved, and this 10 μm was injected through the sample injection port 5.

0.3% by weight ammonium carbonate from eluent tank 1
A mixed solution of 20% by volume and 80% by volume of acetonitrile is supplied by the pump 11, and acetonitrile is supplied from the eluent tank 2 by the pump 12 so as to have a gradient from the initial value of 0% by volume to 1% by volume per minute, and the flow rate is adjusted. The mixture was mixed with the sample at a rate of 0.5 ml/min, and the resulting sample solution was introduced into the separation column 6. As the separation column 6, we used a column with an inner diameter of 3.9 mm and a length of 30 cm, filled with spherical silica gel with a particle size of around 10 μm and having octyl groups chemically bonded to its surface. was separated. Each component coming out of the separation column 6 is transferred from the reaction liquid tank 10 to the pump 13.
The immobilized enzyme column 7 was mixed with the reaction solution supplied by the immobilized enzyme column 7.

The reaction solution contained 0.3mM β-NAD ⁺ , 10mM phosphate buffer (PH8.0), 1mM EDTA, and 0.05% by weight of 2
- Consists of mercaptoethanol, supply amount is 0.5
ml/min.

The immobilized enzyme column 7 used was one in which 100 units of 3α-HSD were immobilized on 2 cc of granular cellulose by the cyanogen bromide method and packed into a column with an inner diameter of 4.6 mm and a length of 5 cm.

The bile acid component in the sample solution is oxidized to β-keto form by the enzyme, and at the same time, β-NAD ⁺ in the reaction solution is reduced to fluorescent NADH, and its concentration is measured by the fluorophotometer 8. was measured. The fluorescence photometer 8 was used for measurement under the conditions of an excitation wavelength of 350 mm and a fluorescence wavelength of 450 mm. The measurement result by the fluorescence photometer 8 is recorded by the recorder 9.
Recorded by.

Figure 2 shows an example of measurement using a serum sample from an acute hepatitis patient.
P _B was well separated from peak P _A due to contaminants such as proteins in the serum sample and peak P _C due to bile acids.

Example 2 Glutamic acid conjugate of chenodeoxycholic acid 20
mg was dissolved in 100 ml of ethanol as an internal standard substance. This solution was added in an amount of 5μ per ml of serum sample from a patient with chronic hepatitis. This was added to a pretreatment column packed with hydrophobic gel, washed with 5 ml of distilled water, and eluted with 10 ml of absolute ethanol. After further evaporating the ethanol to dryness, the residue was dissolved in methanol.
Dissolved in 100μ.

Bile acids were quantified in the same manner as in Example 1 using the sample solution treated in this manner.

Figure 3 shows the results. The peak P _B due to the glutamic acid conjugate of chenodeoxycholic acid, which is an internal standard, was well separated from the peak P _A due to contaminants such as proteins in the serum sample and the peak P _C due to bile acids.

[Brief explanation of drawings]

FIG. 1 is an explanatory diagram showing an example of an apparatus used in the method for quantifying bile acids of the present invention, FIG. 2 is a chromatogram in Example 1, and FIG. 3 is a chromatogram in Example 2. 1, 2... Eluent tank, 4... Gradient programmer, 5... Sample injection port, 6... Separation column, 7... Immobilized enzyme column, 8... Fluorescence photometer, 9... Recorder , 10...reaction liquid tank, 11,1
2,13...Pump.

Claims (1)

[Claims] 1. Bile acids in a biological sample are separated by liquid chromatography, introduced into a column containing an immobilized enzyme, and brought into contact with the immobilized enzyme within the column to separate the bile acids and the bile acids. causing a reaction with a reactive substance,
A method for quantifying bile acids by measuring a reaction product with a detector, the method comprising using an acidic amino acid conjugate of deoxycholic acid or chenodeoxycholic acid as an internal standard substance. 2. The method for quantifying bile acids according to claim 1, wherein the internal standard substance is a glutamic acid conjugate of deoxycholic acid or chenodeoxycholic acid. 3. The method for quantifying bile acids according to claim 1, wherein the internal standard substance is an aspartic acid conjugate of deoxycholic acid or chenodeoxycholic acid.