JPS5913197B2 - Bile acid measurement method - Google Patents

Description

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

More specifically, the present invention involves acidifying a sample containing bile acids, and then treating the sample with 3α-hydroxysteroids.
Dehydrogenase (hereinafter abbreviated as 3α-HSD), diaphorase, nicotinamide adenine dinucleotide (hereinafter abbreviated as NAD), tetrazolium salt, and a base or buffer are added to make the sample alkaline, and the resulting pigment compound is analyzed by spectrophotometry. The present invention relates to a method for measuring bile acids, characterized by measuring bile acids. Conventionally, methods using gas chromatography and enzymes are known as methods for measuring bile acids.

The method using the enzyme will be described in detail below.

0 For example, bile acids in serum are
The eluate was evaporated to dryness and extracted with methanol.

A known method is to add 3α-HSD' to this methanol extract, react the produced cholestenone with a fluorescent substance (for example, hydrazine 15-late), and then quantify it by fluorescence analysis. [Abstracts of the 22nd Annual Meeting of the Japanese Society of Clinical Pathology, page 91 (1975)]. however,
When using this method to quantify bile acids in serum, there are drawbacks such as the need for 1 ml or more of serum and the need for complicated operations such as evaporation to dryness and extraction (90 steps). Furthermore, a method for measuring bile acids by fluorescence analysis is known, which improves the above-mentioned drawbacks. [Clinical Chemistry, Volume 4, Nos. 3 and 4, 31
2-318 (1976)] According to this improved method, 0.2 g of serum is first subjected to high temperature treatment (for example, 67° C. for 20 minutes). After cooling, a solution obtained by dissolving 3α-HSD diaphorase, NAD, and resazurin in O, IM-Tris-HCl buffer (pH 9, O) is added to this solution. After treating the mixture at room temperature (for example, 20°C for 40 minutes),
0 This is a method in which the treatment solution is measured at a fluorescence wavelength of 580 nm. However, even with this modified method, the recovery rate of bile acids is only about 90%, and since a fluorescent substance is used to measure bile acids, impurities in the sample and the reagents used may cause measurement errors. It is susceptible to interference from above, and the measurement values vary widely. It also requires complex equipment called a fluorescence analyzer. The present inventors have conducted various studies with the aim of conducting bile acid analysis using simple equipment that increases the recovery rate of bile acids, improves analytical accuracy, and requires a fluorescent substance for measurement. As a result, the biological components containing bile acids were acidified, then 3α-HS diaphorase, NADl tetrazolium salt, and a base or buffer were added to make them alkaline, and the resulting pigment compounds were analyzed by visible absorption spectrometry. The present invention was completed by discovering a method for measuring bile acids with a high recovery rate and high analysis accuracy.

Hereinafter, the present invention will be explained in detail.

First, the principle of the present invention will be described.

V) 3-ketosteroid and reduced NAD (hereinafter abbreviated as NADH2) are produced by the reaction of bile acid and NAD in the presence of 3α-HSD.

Next, the generated NADH2 and the tetrazolium salt react in the presence of diaphorase to generate NAD and a dye compound. Bile acids in the corresponding biological components can be quantified by measuring the absorbance in the visible region (wavelength 400-600 nm) of this pigment compound, which is produced in 1 mole or 1/2 mole per mole of bile acid. .

Furthermore, during the reaction between bile acids and NAD, enzymes in the blood (e.g. lactic dehydrogenase, male [■■...idrogenase, etc.) interfere with this reaction, so these enzymes in the blood should not be used. It is necessary to deactivate it by some kind of treatment.

In the present invention, the enzymes are inactivated with acid, or the inactivated blood enzymes are further decomposed with enzymes (eg, acidic protease, peptidase, etc.). According to the method of the present invention, the biological component is made acidic (PHO.l~6.0) and heat treated (temperature 20~45°C, time 1~30 minutes).

Through this treatment, the blood enzymes in the biological component 5 are deactivated.
Furthermore, during this treatment, it is preferable to further decompose the deactivated blood enzymes in the biological components by adding an acidic and effective enzyme (for example, acidic protease, peptidase, etc.).
A coloring reagent solution consisting of, for example, diaphorase NAD, a tetrazolium salt, a surfactant, and a buffer is added to the heat-treated solution, and then heat-treated (temperature 20-50°C, time 1
~30 minutes).

In this case, the heat-treated liquid may be added to the solid coloring reagent to dissolve it, and then heat-treated. Next, after adding 3α-HSD to this mixed solution, the pH was made alkaline (PH
7.5 to 12.5). Of course, an alkaline solution containing 3α-HSD may also be used. Thereafter, the absorbance of this liquid is measured at a wavelength of 400 to 600 nm. The amount of bile acid in the biological component can be calculated using the molecular extinction coefficient of the coloring agent or a calibration curve. Further, in the present invention, 3α-HSD may be mixed with the coloring reagent solution.

Examples of biological components containing bile acids targeted by the present invention include serum, urine, and bile.

Of course, any target sample may be used as long as it contains bile acids. The amount of biological components used for quantitative determination is 0.01 for blood and urine and 0.01 for bile.
A range of 0.001 to 0.1 m1 is preferable.

Examples of acids used to acidify biological components include mineral acids such as hydrochloric acid and sulfuric acid, and organic acids such as citric acid and malic acid.

Examples of the tetrazolium salt (coloring agent) used in the coloring reagent solution include 3-(p-iodophenyl)-2-(p
-nitrophenyl)-5-phenyl-2H1 tetrazolium chloride (hereinafter abbreviated as INT), 3,35-
(3,35-dimethoxy-4,4-biphenylene)-bis[2-(p-nitrophenyl)-5-phenyl-2H
e-tetrazolium chloride] (hereinafter abbreviated as NTB)
etc.

Examples of the surfactant used in the coloring reagent solution include Triton X-100 (manufactured by Rohm and Haus).

Examples of the buffer used in the coloring reagent solution include boric acid buffer, Tris buffer, and the like.

Further, the concentrations of each component constituting this coloring reagent solution are as follows. The concentration of diaphorase is 0.11U to 10
A range of 10,000 IU/t is preferred.

The concentration of NAD is preferably in the range of 0.1mM to 50mM.

The concentration of tetrazolium salt is 0.1n1M to 50m
A range of M is preferred.

The concentration of the surfactant is preferably in the range of 0.001 to 5%.

The concentration of 3α-HSD can be appropriately selected depending on the reaction completion time, but for example, 0.00011U to 1001u/t.
A range of is preferred.

Examples of the base that makes the measurement liquid alkaline include caustic soda and caustic potash.

Examples of the present invention will be shown below.

Example 1 One-reagent coloring reagent solution: The following four components were contained in a 0.1M borate buffer (PH9.O) in the following proportions.

Diaphorase (manufactured by Worthington Biochemical Company,
301U/W) 0.0055%@/v) NAD (manufactured by Kyowa Hakko Co., Ltd.) 0.32% (w/v) INT (manufactured by Sigma) 0.026% (w/v) Triton X (product name, Rohm・Manufactured by A.N.D. House) 0.1%
(w/v) 2 operations Collect 0.2 ml of blood from a liver cirrhosis patient and use it as the test solution,
each with 0.1N-HCtO. 2ml and distilled water 0.3
Add m1 and heat at 3rC for 10 minutes.

Next, 2.0 m of the above coloring reagent solution was added to these treated test solutions.
After adding 1, add 0.1M borate buffer (PH
9. 3α-HSD (manufactured by Kyowa Hakko Co., Ltd., 0.0) dissolved in
9841U/M9) [0.1% (hoof 4)] 0.3 ml was added to the sample, and 0.3 ml of distilled water was added to one side to form a blank. Then, after vortexing at 37°C for 15 minutes, the absorbance at 500 nm was measured using a blank as a control.
0.352 was obtained. The concentration of bile acids in serum is determined by the above absorbance and the molecular extinction coefficient (at 500 nm) of the coloring agent 1NT (
It was calculated from 1.5×104) and was 352 μM.

Example 2 In Example 1, instead of 0.2 ml of serum from a liver cirrhosis patient, 10 ml of serum from the same liver cirrhosis patient was added with the standard substance glycocholic acid (C26H43NO6.1.5He02M724
93) 0.493η (concentration 100 μM) was added, and the same procedure as in Example 1 was performed, except that 0.2 ml of each sample and blank were collected, and an absorbance value of 0.447 was obtained.

Therefore, the increase in absorbance due to glycocholic acid is determined by the absorbance when a certain standard is added to the sample (value obtained in this example) and the absorbance when no standard is added to the sample (value obtained in this example). 1) and the Ω difference (i.e. 0.447-0.
352), it was calculated as 0.095. The concentration of glucocholic acid detected is therefore 95 μM. Therefore, the recovery rate of glycocholic acid is as follows. Recovery rate of glycocholic acid Example 3 1-reagent coloring reagent solution: The following five components are contained in the following ratio in a 0.1M boric acid buffer (PH9.O).

Diaphorase (manufactured by Worthington Biochemical Co., Ltd.,
301 ~) 0.0055% (w/v) NTB (manufactured by Sigma) 0.026% (w/v) NA
D (? Manufactured by Wafunko Co., Ltd.) 0.32% (w/v) 3α-HS
D (? Manufactured by Wafunko Co., Ltd., 0.9841~) 0.1% (w/
v) Triton
This contains 0.1% (w/v) of pepsin (Worthington Biochemical Co., 25001UA).
01N-HCtO. 8 ml was added and heated at 37°C for 10 minutes.

Next, 2 ml of the above coloring reagent solution was added to this mixed solution, and the mixture was further heated at 37° C. for 15 minutes. 0.2 ml of distilled water was used instead of the serum of a liver cirrhosis patient, and the sample was treated in the same manner as the specimen and used as a plank. The absorbance of this reaction solution at 550 nm with respect to the blank was 0.230.
The amount of bile acids in serum is determined by the absorbance obtained and the color former NT.
Molecular extinction coefficient of B at 550 nm (3.64 x 104)
It was calculated from 1901 tM. Example 4 In Example 3, instead of the serum 0.2WL1 of the liver cirrhosis patient, the standard substance glycocholic acid (C26Hl3NO6・1.5H20, Mw=4
93) 0.493η (concentration 100 μM) was added, and the same procedure as in Example 3 was carried out, except that 0.2 ml of each sample and blank were collected, and an absorbance value of 0.348 was obtained.

Therefore, the increase in absorbance due to glycocholic acid is determined by the absorbance when a certain standard is added to the sample (value obtained in this example) and the absorbance when no standard is added to the sample (value obtained in this example). 3) (i.e. 0.348-0
．． 230), it was calculated as 0.118. Therefore, the detected concentration of glycocholic acid is 971 tM. Therefore, the recovery rate of glycocholic acid is as follows. Recovery rate of glycocholic acid Example 5 Color reagent solution (same as used in Example 1) 2ml 10.1M borate buffer (PH9.O) 0.5
wL1 and 3α-HSD (0.9841U/Rflg
) 0.3 ml of 0.1M borate buffer (PH9.O) containing 0.1% (w/v) was mixed and heated at 37°C for 10 minutes.

Then, the standard substances sodium deoxycholate (233.4 μM) (hereinafter abbreviated as SD), quinodeoxycholate (211.4 μM) (hereinafter abbreviated as CDC) were added to this solution.
Glycocholic acid (209.1 μM) (hereinafter abbreviated as GCA) and sodium taurocholate (196.3 μM)
Add 0.2 ml of each solution (hereinafter abbreviated as STC),
The absorbance at 500 nm (measured value and theoretical value) and recovery rate of each solution heated at 37° C. for 5 minutes are as follows. Example 6 Urine 7a from a healthy adult male was adjusted to pH 5 with acetic acid, and β-
Glucuronidase (manufactured by Boehringa and Mannheim, 3)
1U/η) 0.251U was added and heated at 50°C for 15 hours.

This solution was diluted to 10 ml with water, 0.2 ml of it was collected, and the same procedure as in Example 3 was carried out to measure the absorbance at 550 nm. The value was 0.045, from which the amount of bile acids in the urine was calculated to be 53 μM. Example 7
In reproducibility test of measurement values in Example 3, serum 0.2TfL1. of another liver cirrhosis patient was used instead of serum 0.2 The same procedure as in Example 3 was carried out except that 10 samples were used for each test, and the following 10 measurement values were obtained.

The average value, standard deviation, and coefficient of variation from the above values are as follows.

Average value (Ma): 75.2 μM Standard deviation (SD): 1.032 Coefficient of variation (C): 1.3% Reference example 1 The measured values of bile acids obtained by the method of the present invention and the fluorescence method We investigated the correlation.

An experiment was conducted in the same manner as in Example 3 using 10 samples of various serums.

Fluorescence measurements were based on the literature by Ohkan et al.

[Clinical Chemistry, Volume 4, Combined Issues 3 and 4, 312-31
8 (1976)] From the above table, the correlation coefficient and regression line between the method (y) of the present invention and the fluorescence method (x) are as follows.

Correlation coefficient: 0.99 Regression line: y=1.01x-0.35

Claims (1)

[Claims] 1 After making the sample containing bile acids acidic and heat-treating it,
Bile acid and nicotinamide adenine dinucleotide (hereinafter referred to as NAD) are reacted with 3α-hydroxysteroid dehydrogenase to generate 3-ketosteroids and reduced NAD (hereinafter referred to as NADH), and the generated NADH and tetrazolium salt. 1. A method for measuring bile acids, which comprises reacting with and in the presence of diaphorase to produce a dye compound, and measuring the absorbance in the visible region of a reaction solution based on the dye.