JPS62272995A - Measurement of bile acid and apparatus used therefor - Google Patents

Abstract

PURPOSE:To measure bile acid in a humor with a good accuracy in a short time at a low cost by simple operation, by preparing a test paper capable of measuring the amount of the bile acid in the humor and measuring the coloring using the test paper. CONSTITUTION:Bile acid is measured in the following steps (1) and (2). That is (1) a step of applying a sample solution to a test paper carrying a reagent for measuring bile acid and reacting the bile acid in the sample solution with the reagent for measuring the bile acid to color the test paper to a color unique to the resultant reaction product and (2) a step of irradiating the above- mentioned colored test paper with light having the maximum wavelength part of the reaction product and measuring the intensity of the reflected light from the test paper. The reagent for measuring the bile acid is constituted of nicotinamide adenine dinucleotide, 3alpha-hydroxysteroid dehydrogenase, diaphorase and a tetrazolium salt.

Description

Detailed Description of the Invention 3. Amount of ν (Field of Industrial Application) The present invention relates to a method for measuring bile acids in body fluids using a test strip, and an apparatus used therefor.

(Prior art) Bile acids contained in bile are also present in body fluids such as blood and urine.
Contains gold. The amount of bile acids in body fluids changes depending on hepatobiliary diseases, and it is known that the amount of bile acids in blood is a particularly sensitive marker for such diseases. For example, it is known that the amount of bile acids in the blood or urine increases in infants with biliary tract obstruction. Biliary obstruction occurs at a high incidence of approximately 1 in 10,000 infants, and patients require prompt surgery. Mortality rates are also high when the disease is recognized late. In order to detect such diseases early, it is desirable to accurately measure bile acids in body fluids, particularly in blood and urine, during mass medical examinations.

Methods for measuring the amount of bile acids in a sample solution containing bile acids are described in 1, for example, Japanese Patent Publication No. 13197-1983 and Japanese Patent Application Laid-open No. 1983-13197;
It is disclosed in No. 144096 and Japanese Patent Application Laid-open No. 151499/1983. According to this, first, a sample containing bile acid is heat-treated in an acidic environment (Japanese Patent Publication No. 13197/1988 fg), or oxamic acid, pyruvic acid, etc. Showa 5
6-151499) Lactate dehydrogenase (LDll
) to inactivate enzymes that interfere with measurement. Next.

This is combined with 3α-hydroxysteroid dehydrogenase (
A reaction solution containing 3α-H2O), nicotinamide adenine dinucleotide (NAD"), diaphorase, and a tetrazolium salt is reacted under alkaline conditions at pH 8 to 9. The hydroxyl group of bile acid reacts with NAD in the presence of 3α-H2O. It reacts with ゝ to form a carbonyl group, producing a keto-type bile acid as shown below.

NADH reacts with a tetrazolium salt, which is an electron-accepting chromogen, in the presence of diaphorase to produce formazan as follows. NADH is oxidized again to NAD''.Resazurin may be used instead of the tetrazolium salt, in which case resorufin is produced.

The number of moles of formazan (resorufin) produced is NA[)
It corresponds to the number of moles of H (that is, the number of moles of bile acid).

Therefore, it is possible to quantify bile acids by measuring the absorbance of this formazan (resorufin fluorescence intensity).

Although bile acids in a sample can be measured with high sensitivity by such a method, it requires complicated operations because it is a measurement method that utilizes a reaction in a solution system.

Therefore, it is unsuitable for mass screening or convenient detection of bile acids. Another disadvantage is that an expensive absorbance measuring device is required for measurement.

In order to solve these drawbacks, the inventor dissolved the reagent used in the above method in an appropriate buffer solution, impregnated it onto a carrier made of a polymeric material such as filter paper, and lyophilized it to form a test paper. We prepared this product and attempted semi-quantification of bile acids. but,
Since the above-mentioned tetrazolium salt is unstable, even if it is freeze-dried, the test paper itself develops a reddish-purple to purple color in about 16 hours. moreover.

Bile acids cannot be detected using test strips obtained by this method because the degree of coloration increases over time.

(Problem that the invention attempts to solve) The present invention solves the above-mentioned conventional drawbacks.

The aim is to prepare a test strip that can measure the amount of bile acids in body fluids, which is an important marker for hepatobiliary diseases, and to use this to measure bile acids in body fluids in a short time with a simple operation. Another object of the present invention is to provide a method for measuring at low cost and with high accuracy. Another object of the present invention is to provide an apparatus that can be used in the above method.

(Means for Solving the Problems) The method for measuring bile acids of the present invention includes: (1) applying a sample solution to a test strip for bile acid measurement on which a carrier supports a reagent for measuring bile acids; (2) reacting the bile acids in the sample with the reagent for bile acid measurement, and causing the test paper to develop a color unique to the resulting reaction product; and (2) applying light having the maximum wavelength of the reaction product. The method includes a step of irradiating a colored test paper and measuring the intensity of light reflected from the test paper, thereby achieving the above object.

The bile acid measurement device of the present invention comprises (1) a test strip for bile acid measurement in which a carrier supports a reagent for bile acid measurement, and (2) maximum absorption of a reaction product produced by the reaction between the reagent and bile acid. a light-emitting element that emits light having a specific wavelength region to the test paper; (3)
(4) a light-receiving element that is sensitive to the wavelength region and receives the reflected light from the test paper of the light from the light-emitting element; The intensity of the reflected light received by the light receiving element is calculated based on the relationship between the rate of change over time of the intensity of the reflected light irradiated and received by the light receiving element and/or the rate of change over time of the intensity of reflected light obtained in advance and the bile acid concentration. The present invention has a display means for displaying the concentration of bile acid calculated by applying the rate of change over time of , thereby achieving the above object.

The test strip for bile acid measurement used in the present invention has a reagent for bile acid measurement supported on a carrier made of a polymeric material. As a carrier made of polymeric material.

In addition to paper and nonwoven fabrics made of natural or synthetic fibers, membrane filters are used. When the sample is urine or serum, paper or nonwoven fabric made of natural or synthetic fibers, such as commercially available filter paper, are preferably used. When the sample is whole blood, paper, nonwoven fabric, membrane filter, etc. made of two synthetic fibers are preferably used. As the membrane filter, a cellulose acetate membrane with a pore diameter of 0.1 to 0.4 μm is preferable. As the synthetic paper, for example, Cellpore (hydrophilic type) manufactured by Building Block Chemical Industry Co., Ltd. is suitable.

The reagent for bile acid measurement supported on the carrier is nicotinamide adenine dinucleotide (NAD") +'3α-
Hydroxysteroid dehydrogenase (3α-H2O
), contains diaphorase and tetrazolium salts. To manufacture test strips, first, NAD.

Prepare an aqueous solution of 3α-H2O and diaphorase dissolved in an alkaline buffer. After impregnating this into the carrier, freeze-drying is performed. The origin of the enzyme used here is not particularly limited, but enzymes with excellent organic solvent resistance, stability over time, etc. are preferred.

As such an enzyme, 3α-11sD is Pseudomonas testosterone.
tes tos teron i), and the diaphorase derived from Bacillus stearothermophilus.
philus) is preferably used. N.A.D.
Nicotinamide adenine dinucleotide phosphate (NADP) may be used instead of "NADP". N
ADP" also acts as a coenzyme like NAD+, and when reduced, it produces reduced nicotinamide adenine dinucleotide phosphate (NADPll). 3α-H2O
and diaphorase at a rate of 0.1 to 100 OOIU per 100 cal of carrier, respectively.

"NAD" (hereinafter, "NAD" may be "NADP".

NADH may be N A D P 11) is 0
．． It is supported at a ratio of 1 to 100 mg. If the amount is too low, color development by the bile acid will not occur sufficiently, and if it is in excess, the enzymatic reaction will be inhibited by its decomposition products.

Additives may be contained in the aqueous solution.

Examples of additives include activators and stabilizers for enzymes and coenzymes. Examples of enzyme activators include 1, such as Triton X-
Surfactants such as 100 (trade name) are preferably used. And as an enzyme stabilizer.

For example, proteins such as bovine serum albumin (BSA) are preferably used. When the above-mentioned surfactants and proteins are added, NAD" and enzymes (3α-H2O and diaphorase) are included in these compounds.

Since such surfactants and proteins do not dissolve in the non-aqueous solvent used in the step of supporting the tetrazolium salt described below, the tetrazolium salt, which is the chromogen in the non-aqueous solution, and the enzymes and coenzymes mentioned above come into direct contact. can be avoided. As a result, coloring of the base is more effectively suppressed.

Furthermore, a thickener may be contained as an additive.
The thickener suppresses the so-called window pane phenomenon. What is the window frame phenomenon?1 For example, when a carrier is impregnated with the above aqueous solution and dried, the solute in the aqueous solution moves to the periphery of the carrier and becomes concentrated, or when the sample solution is dropped onto the obtained test strip, the test NAD”, 3α-H2O contained in paper
If this window frame phenomenon occurs, in which reagents such as reagents migrate to the periphery of the test paper and become concentrated, bile acids cannot be measured accurately. Examples of the thickener include methylcellulose and polyethylene glycol (PEG). When a thickener is included, the viscosity of the liquid phase impregnated into the carrier (test paper) increases, thereby suppressing the movement of solutes, and as a result, the window frame phenomenon is suppressed. The above-mentioned additives such as enzyme activators, enzyme stabilizers, thickeners, etc. are each supported at a ratio of 200 μm or less, preferably 1 to 100 μm per 100 μm of the carrier.

In the freeze-drying process of a carrier impregnated with an aqueous solution containing enzymes, etc., it is important to sufficiently remove water. If water remains in the carrier, the stability of the tetrazolium salt supported in the next step will be extremely reduced.

Next, the freeze-dried carrier is impregnated with a solution of a tetrazolium salt dissolved in a non-aqueous solvent. Tetrazolium salts include 3,3°-(3,3′-dimethoxy-4,4′-biphenylene)-bis[2-(p-nitrophenyl), which is called nitrotetrazolium blue (NTB) or nitroblue tetrazolium (NBT). )-5
-Phenyl-2H-tetrazolium chloride is preferably used.

Any non-aqueous solvent may be used as long as it can dissolve the tetrazolium salt, and alcohols such as methanol and ethanol; ethyl acetate and the like are used.

Tetrazolium salt is 0.1 to 500 per 100 of carrier.
It is supported at a rate of 1 mg. If the amount is too low, color development by the bile acid will not occur sufficiently, and if it is in excess, it will become difficult to dissolve in the solvent and the base color will become dark, making it difficult to distinguish the change in color tone. The carrier impregnated with the tetrazolium salt solution is immediately dried, preferably by lyophilization. The test paper thus obtained is cut into strips of appropriate size and used by adhering them to the end of a stick made of plastic film.

Bile acids in samples such as urine and serum are measured using the above test paper. When using blood as a sample, add oxamic acid, pyruvic acid, etc. in advance. Such pretreatment can inhibit the 100 reaction that inhibits the enzymatic reaction for detecting bile acids. When the test paper is impregnated with the pretreated sample as described above.

Bile acids react with NAD" in the presence of 3α-H2O to form keto-type bile acids, and NAD" becomes NADH. N.A.D.
ll reacts with a tetrazolium salt in the presence of diaphorase to yield formazan. This is similar to the reaction mechanism described in the prior art section. After impregnating the sample, formazan is usually generated and color develops within 1 to 300 seconds. The amount of formazan produced corresponds to the amount of bile acid, so the amount of formazan contained in the sample can be determined by visually observing the degree of color development, or by using a standard colorimetric table prepared by dropping a known concentration of bile acid onto a test paper. You can know the approximate amount of bile acids consumed. Using the standard colorimetric table, it is 25-50 μmol/1
It is possible to measure the above bile acids.

For example, to measure bile acids with higher precision.

The apparatus shown in FIG. 1 is used. This measuring device 1 includes a transparent plate 3 on which a bile acid measurement test strip 2 impregnated with a reagent is placed, a light emitting element 4 disposed below the transparent plate 3,
A light receiving element 6 placed near the light emitting element 4 via a stray light prevention plate 5, and a test paper 2 detected by the light receiving element 6.
and a display means 8 for numerically displaying the intensity of the reflected light. In order to accurately display the intensity of this reflected light on the display means 8, the display means 8 is electrically connected to the light receiving element 6 via an amplification/measuring circuit/AD converter 7. The light emitting element 4 and the amplification/measuring circuit/A-D converter 7 are connected by a measuring switch 9.

As the light emitting element 4, for example, a light emitting diode (for example, EBG5504S manufactured by Stanley Co., Ltd.) having a maximum emission spectrum near 500 to 600 nm is used. The light-receiving element 6 may be, for example, the light-detecting element 1 having sensitivity to light having a wavelength of around 500 to 600 nm.

A silicon photodiode (for example, 51226-58Q manufactured by Hamamatsu Photonics) is used. The means 8 for numerically displaying the intensity of reflected light has a built-in microprocessor, and its memory (ROM or RAM) stores in advance a relational expression between the rate of change over time of reflected light intensity and bile acid concentration. It is possible to display the bile acid concentration in the sample obtained by inputting the rate of change in light intensity over time.

Using the above measurement value W1, bile acids are measured, for example, as follows. A test paper 2 for bile acid measurement is attached to the tip of a strip 21 made of white plastic, and a sample (urine, serum, standard solution for preparing a calibration curve, etc.) is dropped onto the test paper 2. At the same time as the sample is dropped, a timer (not shown) is turned on, and at the same time the test paper 2 is placed on the transparent plate 3 with the side facing the transparent body 3. Close the light shielding cover 22. and,
The measurement switch 9 is turned on over time to cause the light emitting element 4 to emit light. The light reflected by the carrier 2 is received by the light receiving element 6, passes through an amplification circuit, a measuring circuit, and an A-D converter 7, and then is sent to a display means 8.
The reflected light intensity and/or bile acid concentration are displayed numerically. Using such a measuring device, 10 μmol/
It is also possible to measure bile acids below 1.

(Function) According to the present invention, bile acids can be easily measured using a bile acid measuring test strip that utilizes an enzymatic reaction. Since the test strip can support tetrazolium salt, which is a chromogen that produces formazan, in a non-aqueous state on a carrier, the tetrazolium salt will not change during storage of the test strip and the base will not develop color. Since the degree of color development changes depending on the amount of bile acids in the sample, the amount of bile acids can be determined by measuring this using the measuring device of the present invention. Since a measuring device is used, there are no individual differences compared to visual observation, and bile acids can be measured accurately and accurately down to the low concentration range. By such a method, the amount of bile acids can be measured with simple operations, in a short time, and at low cost. Therefore, it is suitably used for mass medical examinations and emergency bedside examinations. It is possible to detect hepatobiliary diseases early by measuring bile acids in body fluids.

(Example) The invention will be explained below with reference to examples.

(A) Preparation of test strip for bile acid measurement = 3α-11sD 1
63 IU.

Diaphorase 680010, β-NAD" 10
．． 2mg, PEG 16mg, BSA 100mg and Triton X (trade name) 10μβ at 10mmo
l/l pyrophosphate buffer (pH 8,0) 10 m
Dissolved in l. This aqueous solution was filtered using 300 c+a filter paper (W
It was impregnated with hatman No. 3) and freeze-dried. Next, 0.034 w/w% of nitro blue tetrazolium
An ethanol solution was prepared, and the freeze-dried filter paper was impregnated with it, and then quickly dried. The test paper thus obtained was cut into small pieces of 5 mm x IQu+ and adhered to the end of a stick made of polystyrene film measuring 6 mm x 5 Q with double-sided tape to obtain test paper pieces.

(B) Creating a colorimetric table: Bile acid (sodium glycocholate) was added to a solution of 1% human serum albumin in physiological saline at a concentration of 0.25.5.
Standard sample solutions were obtained by adding 0.100 and 200 u mol/1. Apply 20μ of each of these standard sample solutions of known concentration to the test paper strips obtained in section (A).
It was impregnated with β. When the chromaticity was visually observed after 1 to 2 minutes, the results shown in Table 3 were obtained. It was found that bile acids of 50 μmol/1 or more could be accurately measured using this test paper.

(Margins below) (C) Measurement of bile acids using a measuring device: on the stick-shaped test strip for measuring bile acids obtained in section (A).

20 μl of a bile acid solution of known concentration was added dropwise. At the same time as the bile acid solution was dropped, a timer (not shown) of the bile acid measuring device shown in FIG. I placed it. The light shielding cover 22 was closed, and the measurement switch 9 was turned on over time to cause the light emitting element 4 to emit light. The light reflected by the carrier 2 is received by the light receiving element 6, and the light is amplified and
The reflected light intensity was numerically displayed on the display means 8 through the measuring circuit/A-D converter 7.

FIG. 2 shows the relationship between elapsed time and reflected light intensity.

As is clear from FIG. 2, after the addition of the bile acid solution.

The reflected light intensity becomes lower as time passes.

In particular, the intensity of reflected light decreases significantly up to 30 seconds. Therefore, after dropping the bile acid solution, the change value of the reflected light intensity for 20 seconds from 10 seconds to 30 seconds was calculated as the reflected light intensity decrease rate (Δ
, 3), and this was used as a guideline for the reflected light intensity. Varying the bile acid concentration, the reflected light intensity reduction rate (
Δ1. ) was measured. FIG. 3 shows the relationship between the rate of decrease in reflected light intensity (Δ13) and bile acid concentration (μmol/ 1 ). What is the regression equation?

ΔI:1=19.4+0.534 X (#mol/j
the law of nature .

(Correlation coefficient R=0.993) Met.

(D) Comparison of measurement method using bile acid test strips and conventional method:
Urine from patients with biliary obstruction (sample 1) and normal human serum (samples 2 and 4) were used as test samples.

Sera from liver cirrhosis patients (Samples 3 and 5) were prepared. The bile acids contained in these were measured and determined using the test paper obtained in section (A) and according to the standards defined in section (B). Next, the regression equation obtained in section (C) was input into the memory of the bile acid measuring device, and the bile acid concentration of each sample was measured according to the method in section (C). Separately, each sample above was embazized (
Measured by solution method using Daiichi Chemicals (manufactured by Kiku),
Bile acid concentration was calculated. The results are shown in Table 2.

Table 2 From Table 2, the measurement results using the bile acid measurement method of the present invention correspond well with the measurement results using the conventional solution method. It can be seen that measurement is possible.

(Effects of the Invention) According to the present invention, the amount of bile acids in a sample solution can be measured in a short time and with high precision using a simple method. There is no variation in measurement due to individual differences. The test paper used can be stored stably for a long period of time, and for example, the underlying color will not develop due to changes in components over time. According to the method of the present invention, it is possible to detect hepatobiliary diseases at an early stage through mass medical examinations, etc., and it is also highly useful for bedside emergency testing.

4. Figure 1 is a schematic diagram showing an embodiment of the bile acid measuring device of the present invention, and Figure 2 is a schematic diagram showing an embodiment of the bile acid measuring device of the present invention.
A graph obtained by measuring the rate of decrease in reflected light intensity from the test paper over time using the device of the present invention, and Figure 3 shows the rate of decrease in reflected light intensity measured by the device of the present invention and bile acid concentration. It is a graph showing the relationship between.

1... Bile acid measuring device, 2... Test paper for bile acid measurement.

3... Transparent plate, 4... Light emitting element, 5... Stray light prevention plate, 6... Light receiving element, 7... Width amplification/measurement circuit/A-
D converter.

8...Display means, 9...Measurement switch, 21...
-Plastic strip, 22...shading cover.

that's all

Claims (1)

[Claims] 1. (1) A sample solution is applied to a test strip for bile acid measurement in which a carrier supports a reagent for bile acid measurement, and the bile acids in the sample solution and the reagent for bile acid measurement are separated. and (2) irradiating the colored test paper with light having the maximum wavelength of the reaction product to produce a color unique to the reaction product. A method for measuring bile acids, comprising the step of measuring the intensity of reflected light. 2. Applying the rate of change in reflected light intensity over time obtained from the measurement value to the relational expression between the rate of change in reflected light intensity over time and bile acid concentration obtained in advance from steps (1) and (2) above, The measuring method according to claim 1, which calculates the bile acid concentration in a sample liquid. 3. The measuring method according to claim 1, wherein the bile acid measuring reagent contains nicotinamide adenine dinucleotide, 3α-hydroxysteroid dehydrogenase, diaphorase, and tetrazolium salt. 4. (1) A test strip for measuring bile acids in which a carrier supports a reagent for measuring bile acids; (2) A test strip for measuring bile acids that carries a reagent for measuring bile acids; a light-emitting element that emits light to the test paper, (3) a light-receiving element that is sensitive to the wavelength range and receives the reflected light from the test paper of the light from the light-emitting element, and (4) a sample liquid applied. The test paper is irradiated with the above light from the light emitting element, and the relationship between the rate of change over time of the intensity of reflected light received by the light receiving element and/or the rate of change over time of the intensity of reflected light obtained in advance and the bile acid concentration. A bile acid measuring device comprising: a display means for displaying a bile acid concentration calculated by applying the rate of change over time of the intensity of reflected light received by the light receiving element to the equation. 5. The measuring device according to claim 4, wherein the bile acid measuring reagent contains nicotinamide adenine dinucleotide, 3α-hydroxysteroid dehydrogenase, diaphorase, and tetrazolium salt.