Dry analysis element for bile acid analysis and method for measuring bile acids

Abstract

The present invention addresses the problem of providing: a simple and inexpensive dry analysis element for bile acid analysis that can be used as a quantitative reagent for bile acid levels, not limited to sulfuric acid conjugates; and a method for measuring bile acids using the dry analysis element for bile acid analysis. Provided according to the present invention is a dry analysis element for bile acid analysis in which at least one water-soluble polymer layer and at least one development layer are provided in the stated order on a support, at least one of the water-soluble polymer layer and the development layer containing 3α-hydroxysteroid dehydrogenase, thionicotinamide coenzyme, reduced nicotinamide coenzyme, and a buffer.

Description

Dry analytical element for bile acid analysis and bile acid measurement method

[0001] The present invention relates to a dry analytical element for analyzing bile acids using 3α-hydroxysteroid dehydrogenase, thionicotinamide coenzyme and reduced nicotinamide coenzyme, and a method for measuring bile acids.

[0002] Bile acids are synthesized in the liver by the oxidation of cholesterol. The synthesized bile acids are conjugated with taurine, glycine, sulfuric acid, and glucuronic acid to form various structures. The bile acids synthesized in this way are stored and concentrated in the gallbladder. Bile acids act as surfactants, and therefore play roles in promoting fat absorption, killing bacteria, and regulating cholesterol metabolism.

[0003] Measuring bile acids as a screening test for liver and biliary tract diseases is highly clinically significant, and bile acid levels are generally measured in body fluids, such as blood (whole blood, plasma, serum), cerebrospinal fluid, lymph, saliva, and urine. However, because bile acids are released into the blood due to liver and biliary tract diseases, measuring bile acid concentrations in the blood is useful for liver function testing.

[0004] Bile acids are measured in solution (solution method) using enzymatic cycling, and Patent Document 1 discloses a method in which 3α-hydroxysteroid dehydrogenase (hereinafter also referred to as "3α-HSD") is reacted with reduced nicotinamide coenzymes (hereinafter referred to as "NADHs") and thionicotinamide coenzyme (thio-NAD) (or thio-NADP) as coenzymes, and the amount of thio-NADHs produced is measured. Meanwhile, Patent Document 2 describes a dry chemistry reagent for sulfate-conjugated bile acids.

[0005] JP-A-3-224498 JP-A-8-131193

[0006] The measurement of bile acids by the solution method described in Patent Document 1 requires calibration using a dedicated calibrator before measurement in order to quantify the amount of bile acids based on dye concentration, and requires piping facilities for water supply and drainage, etc., which in turn requires a large and expensive measurement device. Therefore, there is a need for the development of a simple and inexpensive measurement reagent for bile acids.

[0007] However, the method described in Patent Document 2 is limited to "sulfate-conjugated bile acids," which are synthesized in the liver and excreted primarily in urine. While measuring sulfate-conjugated bile acids is useful for testing for diseases such as liver cirrhosis, it has been reported that the proportion of sulfate-conjugated bile acids present in blood is only a few percent of the total bile acids in blood. Therefore, testing for bile acids other than sulfate-conjugated bile acids is important for liver function and intestinal absorption tests in acute hepatitis, chronic liver disease, and the like. Furthermore, while the method described in Patent Document 2 is promising for the qualitative measurement of "sulfate-conjugated bile acids," it lacks linearity in the detected signal concentration relative to the amount of "sulfate-conjugated bile acids," making it difficult to use it as a quantitative reagent.

[0008] An object of the present invention is to provide a dry analytical element for analyzing bile acids that is simple and inexpensive and can be used as a quantitative reagent for determining the amount of bile acids, not limited to sulfate-conjugated bile acids, and a method for measuring bile acids using the dry analytical element for analyzing bile acids.

[0009] As a result of extensive research to solve the above-mentioned problems, the present inventors have found that a dry analytical element for bile acid analysis, which comprises at least one water-soluble polymer layer and at least one spreading layer provided in this order on a support, and at least one of the water-soluble polymer layer and spreading layer containing 3α-hydroxysteroid dehydrogenase, thionicotinamide coenzyme (thio-NAD), reduced nicotinamide coenzyme (NADH), and a buffer, can provide a dry chemistry reagent that does not require water supply and drainage facilities and can be used as a reagent for quantifying bile acid levels. The present invention was completed based on the above findings. The present invention provides the following:

[0010] <1> A dry analytical element for analyzing bile acids, comprising at least one water-soluble polymer layer and at least one spreading layer provided in this order on a support, wherein at least one of the water-soluble polymer layer and the spreading layer contains 3α-hydroxysteroid dehydrogenase, thionicotinamide coenzyme, reduced nicotinamide coenzyme, and a buffer. <2> The dry analytical element for analyzing bile acids according to <1>, wherein the water-soluble polymer layer is a gelatin layer. <3> The dry analytical element for analyzing bile acids according to <1> or <2>, wherein the buffer has a buffering capacity in the pH range of 6.0 to 10.0. <4> The dry analytical element for analyzing bile acids according to any one of <1> to <3>, wherein the buffer is trishydroxyaminomethane, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, N-[tris(hydroxymethyl)methyl]glycine, or glycine. <5> The dry analytical element for analyzing bile acids according to any one of <1> to <4>, wherein the thionicotinamide coenzyme is thio-NAD and the reduced nicotinamide coenzyme is NADH. <6> The dry analytical element for analyzing bile acids according to any one of <1> to <4>, wherein the content of the thionicotinamide coenzyme is 0.05 to 0.6 g / m ２ <7> The dry analytical element for analyzing bile acids according to any one of <1> to <5>, wherein the content of the reduced nicotinamide coenzyme is 0.05 to 0.6 g / m ２ <8> The dry analytical element for analyzing bile acids according to any one of <1> to <6>, wherein the content of 3α-hydroxysteroid dehydrogenase is 3.0 to 12.0 KU / m ２ <9> A method for measuring bile acids, comprising spotting a bile acid-containing sample on the dry analytical element for bile acid analysis according to any one of <1> to <8> and measuring color development.

[0011] The dry analytical element for bile acid analysis and the method for measuring bile acid of the present invention make it possible to provide a dry chemistry reagent that does not require water supply and drainage facilities and can be used as a reagent for quantifying the amount of bile acid.

[0012] FIG. 1 shows the relationship of ΔOD / min value to bile acid concentration in Example 1. FIG. 2 shows the relationship of ΔOD / min value to bile acid concentration in Example 2. FIG. 3 shows the relationship of ΔOD / min value to bile acid concentration in Example 3. FIG. 4 shows the relationship of ΔOD / min value to bile acid concentration in Example 4. FIG. 5 shows the relationship of ΔOD / min value to bile acid concentration in Example 5. FIG. 6 shows the relationship of ΔOD / min value to bile acid concentration in Example 6.

[0013] Hereinafter, embodiments of the present invention will be described in detail. In this specification, a numerical range indicated using "to" means a range that includes the numerical values ​​before and after "to" as the minimum and maximum values, respectively.

[0014] The present invention relates to a dry analytical element for analyzing bile acids, which comprises at least one water-soluble polymer layer and at least one spreading layer provided in this order on a support, and at least one of the water-soluble polymer layer and the spreading layer contains 3α-hydroxysteroid dehydrogenase, thionicotinamide coenzyme, reduced nicotinamide coenzyme, and a buffer.

[0015]

[0016] The method of the present invention utilizes a reaction system in which 3α-hydroxysteroid dehydrogenase (3α-HSD) acts as a catalyst. As described above, in the presence of bile acids, thionicotinamide coenzyme (thio-NAD) and reduced nicotinamide coenzyme (NADH) are converted to reduced thionicotinamide coenzyme (thio-NADH) and nicotinamide coenzyme (NAD), respectively. In the above reaction system, the color development of thio-NADH correlates with the amount of bile acid reacted, and therefore the amount of bile acid can be quantified by measuring the color development of thio-NADH. Therefore, adjusting the amount of 3α-hydroxysteroid dehydrogenase (3α-HSD) will cause the reaction rate of the analyte amount to vary depending on the amount of bile acid, which is the analyte, resulting in differences in the rate method or the amount of color development (OD) over a fixed time period. In this way, bile acids can be quantified.

[0017] The water-soluble polymer layer used in the present invention refers to a layer containing a water-soluble polymer on a support. The water-soluble polymer layer draws in a bile acid-containing sample spotted on a spreading layer (described later), thereby spreading the sample substantially uniformly throughout the analytical element. Gelatin is a preferred water-soluble polymer contained in the water-soluble polymer layer. That is, the water-soluble polymer layer is preferably a gelatin layer. The water-soluble polymer has a particular swelling property. There is no particular limitation on the amount of water-soluble polymer used, but it is preferred that the amount be 8.0 g / m ２ 40.0g / m or more ２ Preferably, 15.0 g / m or less ２ 30.0g / m or more ２ The following is more preferred:

[0018] The spreading layer used in the present invention is a layer that has the function (metering function) of spreading an aqueous liquid sample that has been spot-applied to the upper surface of the dry analytical element for bile acid analysis laterally without causing the components contained in the aqueous liquid sample to be unevenly distributed, and supplying the sample to a lower layer containing a water-absorbent water-soluble polymer at an approximately constant volume per unit area.

[0019] Examples of the spreading layer used in the present invention include woven spreading layers (e.g., plain weave fabrics such as broadcloth and poplin) described in JP-A-55-164356 and JP-A-57-66359, knitted spreading layers (e.g., tricot knitted fabric, double tricot knitted fabric, Milanese knitted fabric) described in JP-A-60-222769, spreading layers made of paper containing organic polymer fiber pulp described in JP-A-57-148250, and spreading layers made of paper containing organic polymer fiber pulp described in JP-B-53-21677 and U.S. Pat. No. 3,992,119. 58, etc., a non-fibrous isotropic porous spreading layer such as a porous layer containing continuous microvoids in which polymer microbeads, glass microbeads, or diatomaceous earth are held in a water-soluble polymer binder, or a non-fibrous isotropic porous spreading layer consisting of a porous layer containing continuous microvoids (a three-dimensional lattice-like granular structure layer) in which polymer microbeads described in JP-A-55-90859 are bonded in a point-contact manner with a polymer adhesive that does not swell in water. Knitted spreading layers (e.g., tricot knitted fabric, double tricot knitted fabric, Milanese knitted fabric, etc.) are preferred as spreading layers.

[0020] Furthermore, an intermediate layer such as an adhesive layer may be provided between the support and the layer provided thereon, and between each layer provided on the support.

[0021] The support is preferably a water-impermeable support. Examples of suitable materials for the water-impermeable support include polymers such as polyethylene terephthalate, bisphenol A polycarbonate, polystyrene, and cellulose esters (e.g., cellulose diacetate, cellulose triacetate, cellulose acetate propionate, etc.), with polyethylene terephthalate being particularly preferred. The support may be a smooth, flat, transparent support having a thickness of about 50 μm to about 1 mm, preferably about 80 μm to about 300 μm, that transmits electromagnetic radiation having wavelengths in at least a portion of the range from about 200 nm to about 900 nm. A known primer layer or adhesive layer may be provided on the surface of the support to strengthen adhesion to the intermediate layer.

[0022] In the dry analytical element for analyzing bile acids of the present invention, at least one of the water-soluble polymer layer and the spreading layer contains 3α-hydroxysteroid dehydrogenase (3α-HSD). 3α-hydroxysteroid dehydrogenase is an enzyme that oxidizes 3α-hydroxysteroids and is a conjugated enzyme. The content of 3α-hydroxysteroid dehydrogenase (3α-HSD) is 3.0 to 12.0 KU / m to ensure sensitivity. ２ is preferred, and 4.0 to 8.0 KU / m ２ is more preferred.

[0023] The dry analytical element for bile acid analysis of the present invention contains an (oxidized) thionicotinamide coenzyme. The (oxidized) thionicotinamide coenzyme is a coenzyme that acts by binding with various oxidoreductases in the same manner as nicotinamide coenzymes and is involved in hydrogen transport in living organisms, specifically thio-NAD or thio-NADP. The content of the (oxidized) thionicotinamide coenzyme is 0.05 to 1.0 g / m to ensure sensitivity. ２ is preferred, and 0.05 to 0.6 g / m ２ More preferably, 0.1 to 0.5 g / m ２ The content of (oxidized) thionicotinamide coenzyme is more preferably 0.6 g / m ２ A value of less than this is preferable because it reduces the background.

[0024] The dry analytical element for analyzing bile acids of the present invention contains a reduced nicotinamide coenzyme. The reduced nicotinamide coenzyme is a coenzyme for various dehydrogenases, specifically NADH or NADPH. The amount of the reduced nicotinamide coenzyme used is 0.05 to 1.0 g / m to ensure sensitivity. ２ is preferred, and 0.05 to 0.6 g / m ２ More preferably, 0.1 to 0.5 g / m ２ The content of reduced nicotinamide coenzyme is more preferably 0.6 g / m ２ A value of less than this is preferable because it reduces the background.

[0025] The dry analytical element for analyzing bile acids of the present invention contains a buffering agent, which preferably has a buffering capacity in the pH range of 6.0 to 10.0, more preferably in the pH range of 7.0 to 9.0.

[0026] Examples of the buffer include known buffers such as trishydroxyaminomethane, glycine, phosphate, and Good's buffer. The buffer is preferably trishydroxyaminomethane (also referred to as Tris), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (also referred to as HEPES), N-[tris(hydroxymethyl)methyl]glycine (also referred to as Tricine), N,N-bis(2-hydroxyethyl)glycine (also referred to as Bicine), or glycine. The buffer is more preferably trishydroxyaminomethane, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, N-[tris(hydroxymethyl)methyl]glycine, or glycine.

[0027] The content of the buffer is not particularly limited as long as it is not affected by the pH of the sample, but is preferably 1.0 to 10.0 g / m ２ is preferred, and 3.0 to 8.0 g / m ２ is more preferred.

[0028] In the present invention, the amount of bile acid can be quantified by including 3α-hydroxysteroid dehydrogenase (3α-HSD), thionicotinamide coenzyme, reduced nicotinamide coenzyme, and a buffer in at least one of the water-soluble polymer layer and the spreading layer. That is, the 3α-hydroxysteroid dehydrogenase, (oxidized) thionicotinamide coenzyme, reduced nicotinamide coenzyme, and buffer may be included in the water-soluble polymer layer, the spreading layer, or both the water-soluble polymer layer and the spreading layer. A configuration in which these are included in the spreading layer is more preferable.

[0029] The dry analytical element for analyzing bile acids of the present invention may further comprise a reagent layer, a reflective layer, a light-shielding layer, a filtration layer, a primer layer, and other layers. Examples of such analytical elements include those disclosed in U.S. Patent Nos. 3,992,158 and 4,042,335. However, a preferred configuration of the present invention is an integrated multilayer analytical element prepared by sequentially laminating a water-soluble polymer layer having a water-absorbing function and a spreading layer for spreading the sample laterally on a light-transmitting, water-impermeable support.

[0030] The dry analytical element for bile acid analysis of the present invention can be prepared by methods known to those skilled in the art. For example, a coating solution formulated as an intermediate layer coating solution is applied to a support and dried to form a dry film approximately 40 μm thick, and then a woven fabric for the spreading layer is laminated thereon. A coating solution formulated as a reagent holding layer solution is then applied to the woven fabric side of the spreading layer and dried to produce the dry analytical element for bile acid analysis. The dry analytical element for bile acid analysis is preferably cut into square pieces with sides of approximately 15 mm to 30 mm or similar shapes, and then placed in a slide frame as described in, for example, Japanese Patent Publication No. 57-28331, Japanese Utility Model Application Laid-Open No. 56-142454, Japanese Patent Application Laid-Open No. 57-63452, Japanese Utility Model Application Laid-Open No. 58-32350, or Japanese Patent Publication No. 58-501144 for use as a chemical analysis slide, from the viewpoints of various aspects such as production, packaging, transportation, storage, and measurement operations. Depending on the intended use, the tape may be in the form of a long tape stored in a cassette or magazine, or in small pieces attached to or stored in an apertured card.

[0031] According to the present invention, there is provided a method for measuring bile acids, which comprises spotting a bile acid-containing sample on the dry analytical element for bile acid analysis of the present invention and measuring the color development.

[0032] For example, about 5 μL to about 30 μL, preferably about 8 μL to about 15 μL, of an aqueous liquid sample such as whole blood, plasma, serum, lymph, or urine is spotted on the spreading layer and incubated at a substantially constant temperature in the range of about 20° C. to about 40° C., preferably at a substantially constant temperature around 37° C., for about 1 to about 10 minutes, preferably about 2 to about 7 minutes. A detectable change, such as a color change or color development, within the dry analytical element for bile acid analysis is measured by reflection photometry from the support side, and the bile acid content in the liquid sample can be determined based on the principle of colorimetric measurement. In the present invention, the optical density of the spreading layer is measured by reflection photometry using light at or near the absorption maximum wavelength of the bile acid, and the bile acid content in the liquid sample can be determined based on the principle of colorimetric measurement using a previously prepared calibration curve. By maintaining a constant amount of aqueous liquid sample spotted, incubation time, and temperature, quantitative analysis of bile acids can be performed with high accuracy. Measurement can be performed with extremely simple procedures using chemical analyzers described in Japanese Patent Laid-Open Nos. 60-125543, 60-220862, 61-294367, and 58-161867, and highly accurate quantitative analysis can be carried out.

[0033] Next, the present invention will be described with reference to examples, but the present invention is not limited to these examples.

[0034] Example 1 (1) Preparation of coated film and dried slide A water-absorbing layer was formed by coating an aqueous solution of the following composition-1 onto a smooth, colorless, transparent, 180 μm polyethylene terephthalate (PET) film that had been subbed with gelatin, to a thickness of 40 μm after drying, and then drying.

[0035] Water absorption layer (composition 1) Gelatin 17g / m ２ Surfactant 0.2 g / m ２ Here, the surfactant used was polyoxy(2-hydroxy)propylene nonylphenyl ether (Surfactant 10G, manufactured by Olin).

[0036] Next, about 30 g / m ２ After wetting the fabric with water in an amount of 1000, a polyester spun yarn tricot knit fabric equivalent to 50 denier was attached to the fabric using a wet lamination method while applying light pressure to form a spread layer.

[0037] Next, an aqueous solution A having the following composition was applied onto the spreading layer so that the amounts of each component were as follows, and then dried to prepare a dry analytical element for bile acid analysis according to the present invention.

[0038] Aqueous solution A: 3α-hydroxysteroid dehydrogenase (3α-HSD) (manufactured by Toyobo Co., Ltd.) 6.4 KU / m ２ Thio-NAD (manufactured by Oriental Yeast Co., Ltd.) 0.32 g / m ２ NADH (manufactured by Oriental Yeast Co., Ltd.) 0.32 g / m ２ Trishydroxyaminomethane (Fujifilm Wako Pure Chemical Industries, Ltd.) 8.7 g / m ２ Polyvinylpyrrolidone (manufactured by BASF) 10.9 g / m ２

[0039] The above dry analytical element for bile acid analysis was cut into a size of 12 mm x 13 mm, and a slide was prepared according to the method described in JP-A-57-63452 to prepare a dry analytical slide (1) for bile acid analysis.

[0040] (2) Measurement of bile acid concentration Human pooled serum samples adjusted to bile acid concentrations of 48.9 μmol / L, 117.8 μmol / L, and 185.8 μmol / L, and a 7% HSA (human serum albumin) aqueous solution as a measurement reagent with a bile acid concentration of zero, were prepared, and 10 μL of each was spotted on the dry analysis slide (1) prepared in Example 1 above. While kept at 37° C., the reflection density at 415 nm was measured every 10 seconds over a period of 3 minutes using a Fuji DryChem 7000 Analyzer (manufactured by Fujifilm Corporation).

[0041] The bile acid concentrations and the change in reflection density per minute (ΔOD / min) in the reflection density that increases during the measurement time from 60 sec to 180 sec are summarized in Table 1, and the relationship between the bile acid concentration and the ΔOD / min value is shown in Figure 1, where the bile acid concentration is plotted on the horizontal axis and the ΔOD value is plotted on the vertical axis.

[0042]

[0043] Example 2 (1) Preparation of coated film and dried slide An aqueous solution of the following composition-2 was applied to a smooth, colorless, transparent PET film of 180 μm thick and undercoated with gelatin, so that the thickness after drying would be 40 μm, and then dried to provide a water-absorbing layer.

[0044] Water absorption layer (composition 2) Polyvinyl alcohol 23 g / m ２ Surfactant 0.2 g / m ２ Here, the surfactant used was polyoxy(2-hydroxy)propylene nonylphenyl ether (Surfactant 10G, manufactured by Olin).

[0045] Next, a spreading layer was formed by wet laminating a polyester spun yarn tricot knit fabric equivalent to approximately 50 denier using the same method as described in Example 1. Furthermore, the same amount of aqueous solution A as in Example 1 was applied onto the spreading layer, and the resulting solution was dried to prepare a dry analysis slide (2) for bile acid analysis according to the present invention.

[0046] (2) Measurement of bile acid concentration Human pooled serum samples adjusted to bile acid concentrations of 48.9 μmol / L, 117.8 μmol / L, and 185.8 μmol / L, and a 7% HSA aqueous solution as a measurement reagent with a bile acid concentration of zero, were prepared, and 10 μL of each was spotted on the dry analysis slide (2) prepared in Example 2 above. While kept at 37° C., the reflection density at 415 nm was measured every 10 seconds over a period of 3 minutes using a Fuji Dry Chem 7000 Analyzer (manufactured by Fujifilm Corporation).

[0047] The bile acid concentrations and the change in reflection density per minute (ΔOD / min) in the reflection density that increases during the measurement time from 60 sec to 180 sec are summarized in Table 2, and the relationship between the bile acid concentration and the ΔOD / min value is shown in Figure 2, where the bile acid concentration is plotted on the horizontal axis and the ΔOD value is plotted on the vertical axis.

[0048]

[0049] The results of Examples 1 and 2 show that both dry analytical elements for bile acid analysis have good linearity and exhibit sufficient performance as test reagents capable of quantitatively measuring bile acids. The use of gelatin as the water-absorbing layer in the dry analytical element is more preferable because a larger signal-to-noise ratio (S / N) can be obtained by using gelatin.

[0050] Examples 3 to 6: The following buffer levels were prepared and slides were prepared for confirmation. A second experiment (Example 3) was conducted using aqueous solution A of Example 1. Dry analytical slides were prepared in the same manner as in Example 1, except that the trishydroxyaminomethane (Tris) in Example 3 was replaced with HEPES (Example 4), Tricine (Example 5), or Glycine (Example 6). Similarly to Example 1, pooled human serum samples were prepared so that the bile acid concentrations (measured by the solution method) were 0 μmol / L, 118 μmol / L, or 186 μmol / L. 10 μL of each sample was spotted on a dry analytical slide using the above buffer solution. The slides were incubated at 37°C, and the reflection density at 415 nm was measured every 10 seconds over a period of 3 minutes using a Fuji DryChem 7000 Analyzer (manufactured by Fujifilm Corporation). The number of samples was N=2.

[0051] The bile acid concentrations and the change in reflection density per minute (ΔOD / min) in the reflection density that increases during the measurement time from 60 sec to 180 sec are summarized in Table 3. The relationship between the bile acid concentration and the ΔOD / min value is shown in Figures 3 to 6, with the bile acid concentration on the horizontal axis and the ΔOD value on the vertical axis.

[0052]

[0053] The results of Examples 3 to 6 show that all dry analytical elements for bile acid analysis have good linearity and exhibit sufficient performance as test reagents capable of quantitatively measuring bile acids.

Claims

1. A dry analytical element for analyzing bile acids, comprising at least one water-soluble polymer layer and at least one spreading layer provided in this order on a support, at least one of the water-soluble polymer layer and the spreading layer containing 3α-hydroxysteroid dehydrogenase, thionicotinamide coenzyme, reduced nicotinamide coenzyme, and a buffer.

2. The dry analytical element for analyzing bile acids according to claim 1, wherein said water-soluble polymer layer is a gelatin layer.

3. The dry analytical element for analyzing bile acids according to claim 1 or 2, wherein the buffering agent has a buffering capacity in the range of pH 6.0 to 10.

0.

4. The dry analytical element for analyzing bile acids according to claim 1 or 2, wherein the buffer is trishydroxyaminomethane, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, N-[tris(hydroxymethyl)methyl]glycine, or glycine.

5. The dry analytical element for analyzing bile acids according to claim 1 or 2, wherein the thionicotinamide coenzyme is thio-NAD and the reduced nicotinamide coenzyme is NADH.

6. The content of thionicotinamide coenzyme is 0.05 to 0.6 g / m ２ 3. The dry analytical element for analyzing bile acid according to claim 1 or 2, 7. The content of reduced nicotinamide coenzyme is 0.05 to 0.6 g / m ２ 3. The dry analytical element for analyzing bile acid according to claim 1 or 2, 8. The content of 3α-hydroxysteroid dehydrogenase is 3.0-12.0 KU / m ２ 3. The dry analytical element for analyzing bile acid according to claim 1 or 2, 9. A method for measuring bile acid, comprising spotting a bile acid-containing sample on the dry analytical element for analyzing bile acid according to claim 1 or 2, and measuring color development.

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