Dry analytical element for total ketone body analysis and method for measuring total ketone bodies

Abstract

The present invention addresses the problem of providing: a simple and inexpensive dry analytical element for total ketone body analysis that can be used as a reagent for quantitating a total amount of ketone bodies; and a method for measuring total ketone bodies using the dry analytical element for total ketone body analysis. According to the present invention, provided is a dry analytical element for total ketone body analysis, comprising at least one water-soluble polymer layer and at least one development layer formed in this order on a support, wherein at least one of the water-soluble polymer layer and the development layer contains a 3-hydroxybutyrate dehydrogenase, a thionicotinamide coenzyme, a reduced nicotinamide coenzyme, and a buffer.

Description

Dry analytical element for total ketone body analysis and method for measuring total ketone bodies

[0001] The present invention relates to a dry analytical element for analyzing total ketone bodies using 3-hydroxybutyrate dehydrogenase, thionicotinamide coenzyme and reduced nicotinamide coenzyme, and a method for measuring total ketone bodies.

[0002] Generally, energy is obtained in the body through glucose metabolism, but in cases of diabetes and starvation, which are disorders of glucose metabolism, another substance called ketone bodies is used as an alternative energy source. Ketone bodies are synthesized from acetyl-CoA in the liver. There are three types of ketone bodies: acetone, acetoacetic acid (AcAc), and 3-hydroxybutyric acid (3-HB), which are collectively referred to as "ketone bodies." Of these, acetone is volatile and easily excreted in the breath, so the total amount of acetoacetic acid and 3-hydroxybutyric acid present in the blood is measured as "total ketone bodies."

[0003] When total ketone bodies are elevated, energy metabolism is biased toward fatty acids, and the amount of total ketone bodies is useful as a metabolic indicator. When total ketone bodies in the blood increase due to a lack of sugar caused by abnormal glucose metabolism, the pH of the blood becomes acidic because ketone bodies are acidic, resulting in ketoacidosis. Ketoacidosis can lead to dehydration, central nervous system disorders, coma, and even death. In this sense, it is necessary to immediately and accurately measure total ketone bodies as a way to monitor energy restriction.

[0004] As a method for measuring total ketone bodies in a solution (solution method), a method using enzymatic cycling is known. Patent Documents 1 and 2 disclose a method for measuring the amount of total ketone bodies by reacting reduced nicotinamide coenzymes (hereinafter referred to as "NADHs") and thionicotinamide coenzymes (thio-NAD) (or thio-NADP) as coenzymes and measuring the amount of thio-NADHs produced.

[0005] JP-A-4-158799 JP-A-6-253895

[0006] Reagents for measuring total ketone bodies have been established as reagents for measurement using large-scale automated analyzers. However, it is known that blood ketone body levels decrease with time, such as transportation to a testing center, and therefore it is necessary to measure blood ketone body levels by performing plasma separation promptly after blood collection. Therefore, there is a need for reagents and measurement systems that can perform measurements immediately after blood collection.

[0007] An object of the present invention is to provide a dry analytical element for analyzing total ketone bodies that can be used as a reagent for rapidly quantifying the amount of total ketone bodies in blood after blood collection, and a method for measuring total ketone bodies using the dry analytical element for analyzing total ketone bodies.

[0008] As a result of extensive research to solve the above-mentioned problems, the present inventors have found that a dry analytical element for analyzing total ketone bodies can be provided, 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 3-hydroxybutyrate dehydrogenase, thionicotinamide coenzyme (thio-NAD), reduced nicotinamide coenzyme (NADH), and a buffer, which is simple, does not require water supply and drainage equipment, and can be used quickly as a reagent for quantifying total ketone bodies after blood collection and plasma separation. The present invention was completed based on the above findings. The present invention provides the following:

[0009] <1> A dry analytical element for analyzing total ketone bodies, 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 3-hydroxybutyrate dehydrogenase, thionicotinamide coenzyme, reduced nicotinamide coenzyme, and a buffer. <2> The dry analytical element for analyzing total ketone bodies according to <1>, wherein the water-soluble polymer layer is a gelatin layer. <3> The dry analytical element for analyzing total ketone bodies 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 total ketone bodies according to any one of <1> to <3>, wherein the buffer is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 2-hydroxy-3-morpholinepropanesulfonic acid, or 2-[N,N-bis(2-hydroxyethyl)amino]-1-ethanesulfonic acid. <5> The dry analytical element for analyzing total ketone bodies 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 total ketone bodies according to any one of <1> to <4>, wherein the content of the thionicotinamide coenzyme is 0.6 to 2.6 g / m ２ <7> The dry analytical element for analyzing total ketone bodies according to any one of <1> to <5>, wherein the content of reduced nicotinamide coenzyme is 0.4 to 1.80 g / m ２ <8> The dry analytical element for analyzing total ketone bodies according to any one of <1> to <6>, wherein the content of 3-hydroxybutyrate dehydrogenase is 1,000 to 8,000 KU / m ２ <9> A method for measuring total ketone bodies, comprising spotting a sample on the dry analytical element for analyzing total ketone bodies according to any one of <1> to <8> and measuring color development.

[0010] According to the dry analytical element for analyzing total ketone bodies and the method for measuring total ketone bodies of the present invention, it is possible to provide a dry chemistry reagent that does not require water supply and drainage facilities and can be used as a quantitative reagent for the amount of total ketone bodies.

[0011] Fig. 1 shows the relationship of the ΔOD / min value to the total ketone body concentration in Example 1. Fig. 2 shows the relationship of the ΔOD / min value to the total ketone body concentration in Example 2. Fig. 3 shows the relationship of the ΔOD / min value to the total ketone body concentration in Example 3. Fig. 4 shows the relationship of the ΔOD / min value to the total ketone body concentration in Example 4. Fig. 5 shows the relationship of the ΔOD / min value to the total ketone body concentration in Example 5.

[0012] 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.

[0013] The present invention relates to a dry analytical element for analyzing total ketone bodies, which comprises 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 3-hydroxybutyrate dehydrogenase, thionicotinamide coenzyme, reduced nicotinamide coenzyme, and a buffer.

[0014]

[0015] The method of the present invention utilizes the above-described reaction system in which 3-hydroxybutyrate dehydrogenase (3-HBD) acts as a catalyst. When 3-hydroxybutyrate (3-HB) is present in a sample, it is specifically oxidized by 3-hydroxybutyrate dehydrogenase (3-HBD) in the presence of nicotinamide coenzyme (thio-NAD) to produce acetoacetate (AcAc) and reduced thionicotinamide coenzyme (thio-NADH). Meanwhile, acetoacetate (AcAc) is specifically reduced by 3-hydroxybutyrate dehydrogenase (3-HBD) in the presence of reduced nicotinamide coenzyme (NADH) to produce 3-hydroxybutyrate (3-HB) and nicotinamide coenzyme (NADH). In this way, by cycling 3-hydroxybutyrate (3-HB) and acetoacetic acid (AcAc), which are substrates of 3-hydroxybutyrate dehydrogenase (3-HBD), and measuring the rate of production of the reduced thionicotinamide coenzyme (thio-NADH) produced in the enzyme cycling reaction or the amount produced over a fixed period of time, it is possible to measure the total amount of ketone bodies, which is the sum of 3-hydroxybutyrate (3-HB) and acetoacetic acid (AcAc), in a sample.

[0016] 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 absorbs a sample containing total ketone bodies spotted on a spreading layer (described later), thereby spreading the sample substantially uniformly throughout the analytical element. A preferred water-soluble polymer contained in the water-soluble polymer layer is gelatin. 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:

[0017] 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 spotted and supplied to the upper surface of the dry analytical element for analyzing total ketone bodies laterally without causing the components contained in the aqueous liquid sample to be distributed unevenly, and supplying the sample to a lower layer containing a water-absorbent water-soluble polymer at an approximately constant volume per unit area.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] In the dry analytical element for analyzing total ketone bodies of the present invention, at least one of the water-soluble polymer layer and the spreading layer contains 3-hydroxybutyrate dehydrogenase (3-HBD). 3-Hydroxybutyrate dehydrogenase is an enzyme that oxidizes 3-hydroxybutyrate and is a conjugated enzyme that catalyzes the production of 3-hydroxybutyrate by reducing acetoacetic acid. The content of 3-hydroxybutyrate dehydrogenase (3-HBD) is 1,000 to 8,000 KU / m to ensure sensitivity. ２ is preferred, and 3000 to 5000 KU / m ２ Here, 1 U is defined as the amount of enzyme that can convert 1 μmol of 3-hydroxybutyrate into acetoacetate per minute under optimal conditions.

[0022] The dry analytical element for analyzing total ketone bodies of the present invention contains (oxidized) thionicotinamide coenzyme. The (oxidized) thionicotinamide coenzyme is a coenzyme that acts in combination with various oxidoreductases in the same manner as nicotinamide coenzyme and is involved in hydrogen transfer in vivo, specifically meaning thio-NAD or thio-NADP. The (oxidized) thionicotinamide coenzyme is preferably thio-NAD. The content of the (oxidized) thionicotinamide coenzyme is 0.6 to 2.6 g / m to ensure sensitivity. ２ is preferred, and 0.8 to 2.0 g / m ２ More preferably, 0.9 to 1.7 g / m ２The content of (oxidized) thionicotinamide coenzyme is more preferably 1.5 g / m ２ A value of less than this is preferable because it reduces the background.

[0023] The dry analytical element for analyzing total ketone bodies of the present invention contains a reduced nicotinamide coenzyme. The reduced nicotinamide coenzyme is a coenzyme for various dehydrogenases, specifically NADH or NADPH. The reduced nicotinamide coenzyme is preferably NADH. The amount of the reduced nicotinamide coenzyme used is 0.4 to 1.8 g / m to ensure sensitivity. ２ is preferred, and 0.5 to 1.6 g / m ２ More preferably, 0.6 to 1.2 g / m ２ The content of reduced nicotinamide coenzyme is more preferably 1.8 g / m ２ A value of less than this is preferable because it reduces the background.

[0024] The dry analytical element for analyzing total ketone bodies of the present invention contains a buffering agent, which preferably has a buffering capacity in the pH range of 5.0 to 9.0, more preferably in the pH range of 6.0 to 8.0.

[0025] Examples of the buffer include known buffers such as trishydroxyaminomethane and Good's buffer. The buffer is preferably 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (also known as HEPES), 2-hydroxy-3-morpholinepropanesulfonic acid (also known as MOPSO), or 2-[N,N-bis(2-hydroxyethyl)amino]-1-ethanesulfonic acid (also known as BES). The buffer is more preferably 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES).

[0026] 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.

[0027] In the present invention, the total amount of ketone bodies can be quantified by including 3-hydroxybutyrate dehydrogenase (3-HBD), thionicotinamide coenzyme, reduced nicotinamide coenzyme, and a buffer in at least one of the water-soluble polymer layer and the spreading layer. That is, 3-hydroxybutyrate dehydrogenase, (oxidized) thionicotinamide coenzyme, reduced nicotinamide coenzyme, and a 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.

[0028] The dry analytical element for analyzing total ketone bodies of the present invention may further include 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. Pat. 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 a sample laterally on a light-transmitting, water-impermeable support.

[0029] The dry analytical element for analyzing total ketone bodies 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 with a thickness of approximately 40 μm, 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 prepare the dry analytical element for analyzing total ketone bodies. The dry analytical element for analyzing total ketone bodies is preferably cut into square pieces with sides of approximately 15 mm to 30 mm or similar shapes, and placed in a slide frame 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.

[0030] According to the present invention, there is provided a method for measuring total ketone bodies, which comprises spotting a sample on the dry analytical element for analyzing total ketone bodies of the present invention and measuring the color development. The sample may be a sample containing at least one of 3-hydroxybutyric acid and acetoacetic acid.

[0031] 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 analyzing total ketone bodies is measured by reflection photometry from the support side, and the total ketone body 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 color developed by total ketone bodies, and the total ketone body 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 total ketone bodies can be performed with high precision. 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.

[0032] Next, the present invention will be described with reference to examples, but the present invention is not limited to these examples.

[0033] Example 1 (1) Preparation of coated film and dry analysis slide An aqueous solution of the following composition-1 was applied to a smooth, colorless, transparent, 180 μm polyethylene terephthalate (PET) film that had been subbed with gelatin, so that the thickness after drying would be 40 μm, and then dried to provide a water-absorbing layer.

[0034] Water absorption layer (composition 1) Gelatin 17g / m ２ Surfactant 0.2 g / m ２Here, the surfactant used was polyoxy(2-hydroxy)propylene nonylphenyl ether (Surfactant 10G, manufactured by Olin).

[0035] 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.

[0036] Next, 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 analyzing total ketone bodies according to the present invention.

[0037] Aqueous solution A: 3-hydroxybutyrate dehydrogenase (3-HBD) (Asahi Kasei Pharma Corporation) 38004 KU / m ２ Thio-NAD (manufactured by Oriental Yeast Co., Ltd.) 1.28 g / m ２ NADH (manufactured by Oriental Yeast Co., Ltd.) 0.90 g / m ２ HEPES (manufactured by Dojindo Institute) 4.0g / m ２ Polyvinylpyrrolidone (manufactured by BASF) 10.9 g / m ２

[0038] The above dry analytical element for total ketone body 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 total ketone body analysis.

[0039] (2) Measurement of Total Ketone Body Concentration Human pooled serum samples adjusted to have total ketone body concentrations of 300 μmol / L and 105 μmol / L, and a 7% HSA (human serum albumin) aqueous solution as a measurement reagent with a total ketone body 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).

[0040] The total ketone body concentration and the change in reflection density per minute (ΔOD / min) in the reflection density that increases between 60 and 180 seconds of measurement are summarized in Table 1. The relationship between the total ketone body concentration and the ΔOD / min value is shown in Figure 1, where the horizontal axis represents the total ketone body concentration and the vertical axis represents the ΔOD value per minute.

[0041]

[0042] Example 2 (1) Preparation of coated film and dry analysis slide An aqueous solution of Composition-2 below 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.

[0043] 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).

[0044] As described above, a dry analysis slide (2) for total ketone body analysis was prepared in the same manner as in Example 1, except that the water-soluble polymer in the water-absorbing layer was changed from gelatin to polyvinyl alcohol and the water-absorbing layer was provided on a PET film.

[0045] (2) Measurement of total ketone body concentration Measurement of total ketone body concentration was also performed in the same manner as in Example 1. The total ketone body concentration and the change in reflection density per minute (ΔOD / min) in the reflection density increasing during the measurement time from 60 seconds to 180 seconds are summarized in Table 2. The relationship between the total ketone body concentration and the ΔOD / min value is shown in Figure 2, where the horizontal axis represents the total ketone body concentration and the vertical axis represents the ΔOD value per minute.

[0046]

[0047] The results of Examples 1 and 2 show that both dry analytical elements for analyzing total ketone bodies have good linearity and exhibit sufficient performance as test reagents capable of quantitatively measuring total ketone bodies. 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.

[0048] Examples 3 to 5: 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, and dry analytical slides were prepared in the same manner as in Example 1, except that the HEPES in Example 3 was replaced with MOPSO (Example 4) or BES (Example 5). As in Example 1, pooled human serum samples were prepared so that the total ketone body concentrations (measured by the solution method) were 0 μmol / L, 105 μmol / L, or 300 μmol / L. 10 μL of each sample was spotted on a dry analytical slide using the above buffer solution. The samples 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.

[0049] The total ketone body concentration and the change in reflection density per minute (ΔOD / min) in the reflection density that increases between 60 seconds and 180 seconds of measurement time are summarized in Table 3. The relationship between the total ketone body concentration and the ΔOD / min value is shown in Figures 3 to 5, with the total ketone body concentration on the horizontal axis and the ΔOD value on the vertical axis.

[0050]

[0051] The results of Examples 3 to 5 show that all dry analytical elements for analyzing total ketone bodies have good linearity and exhibit sufficient performance as test agents capable of quantitatively measuring total ketone bodies.

[0052] Example 6 After informed consent, blood was drawn from volunteer patients, and immediately plasma separation was performed. 10 μL of plasma (human specimen-1, human specimen-2, human specimen-3) was then performed. The plasma was then quickly separated and applied to the dry analysis slide (1) for total ketone body analysis prepared in Example 1. As in Example 1, the change in reflection density per minute (ΔOD / min) in the reflection density that increased between 60 and 180 seconds of measurement was determined, and the total ketone amount (μmol / L) was calculated from the relationship of the ΔOD / min value to the total ketone body concentration shown in FIG. 1. The results are shown in Table 4.

[0053] Additionally, the total ketone amounts (μmol / L) of the above human specimens 1, 2, and 3 were measured using a large automated analyzer that measures in solution (the instrument was a BM6010 manufactured by JEOL Ltd., and the reagent was TKB-L manufactured by Kainos Corporation), and the results are also shown in Table 4. The reference range for total ketone amounts in healthy individuals is 26 to 122 μmol / L.

[0054]

[0055] As shown in Table 4, the measurement results of the total ketone body amount measured using the dry analytical element for analyzing total ketone bodies of the present invention were almost identical to the measurement results of the total ketone amount measured using a large automatic analyzer that measures in a solution state. In other words, it was confirmed that the use of the dry analytical element for analyzing total ketone bodies of the present invention makes it possible to measure total ketone bodies quickly, accurately, and easily after blood collection.

Claims

1. A dry analytical element for analyzing total ketone bodies, 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 3-hydroxybutyrate dehydrogenase, thionicotinamide coenzyme, reduced nicotinamide coenzyme, and a buffer.

2. The dry analytical element for analyzing total ketone bodies according to claim 1, wherein the water-soluble polymer layer is a gelatin layer.

3. The dry analytical element for analyzing total ketone bodies according to claim 1 or 2, wherein the buffer has a buffering capacity in the range of pH 6.0 to 10.

0.

4. The dry analytical element for analyzing total ketone bodies according to claim 1 or 2, wherein the buffer is 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 2-hydroxy-3-morpholinepropanesulfonic acid, or 2-[N,N-bis(2-hydroxyethyl)amino]-1-ethanesulfonic acid.

5. The dry analytical element for analyzing total ketone bodies 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 0.6 to 2.6 g / m ２ 3. The dry analytical element for analyzing total ketone bodies according to claim 1 or 2, 7. The content of reduced nicotinamide coenzyme is 0.4 to 1.8 g / m ２ 3. The dry analytical element for analyzing total ketone bodies according to claim 1 or 2, 8. The content of 3-hydroxybutyrate dehydrogenase is 1000-8000 KU / m ２ 3. The dry analytical element for analyzing total ketone bodies according to claim 1 or 2, 9. A method for measuring total ketone bodies, comprising spotting a sample on the dry analytical element for analyzing total ketone bodies according to claim 1 or 2, and measuring the color development.

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