Method for Measuring Free Fatty Acid Concentration and Reagent Chip for Measuring Free Fatty Acid Concentration

Abstract

In a measuring room 7, coenzyme A, adenosine triphosphate, magnesium ions, acyl coenzyme A synthesizer, pyrophosphatase, maltose, maltose phosphorylase, beta-phosphoglucomutase, oxidized nicotinamide adenine dinucleotide, glucose-6- dehydrogenase phosphate, tetrazolium salt, and diaphorase are prepared at an appropriate concentration. Since the reaction using the abovementioned reagents leads to a color reaction even in an environment in which oxygen supply is restricted, the concentration of free fatty acids can be measured even in a very small closed space.

Description

Technical Field

[0001] This invention relates to a method for measuring free fatty acids, and particularly to a simple measurement method.

Background Art

[0002] In recent years, regarding dairy cows, particularly those after parturition, the excessive mobilization of free fatty acids to the liver has been regarded as a problem. Therefore, in the dairy industry, a method that can easily measure free fatty acids in the blood is desired.

[0003] Therefore, the inventors considered whether they could measure the free fatty acids of cows using the reagent chip described in Patent No. 5931709.

Summary of the Invention

Problems to be Solved by the Invention

[0004] However, there were the following problems in measuring the free fatty acids of cows using the said reagent chip.

[0005] Generally, as a method for measuring free fatty acids, a method for measuring non-esterified fatty acids (NEFA) using the enzymatic reactions of acyl coenzyme A synthetase (ACS) and acyl coenzyme A oxidase (ACOD) is known.

[0006] An explanation of this measurement method will be given. Non-esterified fatty acids (NEFA) are converted into acyl coenzyme A, pyrophosphate, and adenosine monophosphate (AMP) by the action of acyl coenzyme A synthetase (enzyme) activated by magnesium ions in the presence of coenzyme A and adenosine triphosphate (ATP). This acyl coenzyme A reacts with oxygen by acyl coenzyme A oxidase (enzyme), thereby producing trans-2,3-dehydroacyl coenzyme A and hydrogen peroxide (H 2 O 2) is generated. Hydrogen peroxide oxidizes a leuco dye (developer) by the action of peroxidase (enzyme). As a result, the leuco dye is converted into methylene blue. The color density of such methylene blue depends on the concentration of free fatty acids in the sample. Therefore, the concentration of free fatty acids in the sample can be calculated by optically measuring the color of methylene blue (at a wavelength of 600 to 670 nm).

[0007] In the above reaction, the free fatty acid concentration is related to the amount of hydrogen peroxide (H 2 O 2 ). In order for such hydrogen peroxide to be generated, acyl coenzyme A needs to react with a necessary amount of oxygen. Therefore, when using a reagent chip that reacts with a reagent in a minute space having a sealed structure as described above, there is a problem that an accurate free fatty acid concentration cannot be calculated due to the limited supply of oxygen required for the reaction.

[0008] The above problem is similarly a problem not only for dairy cows but also for other animals.

[0009] It is also conceivable to solve the above problem by diluting the specimen by an appropriate multiple (several times to several tens of times). However, for that purpose, it is necessary to dilute it to an accurate magnification, which becomes complicated accordingly.

[0010] An object of the present invention is to solve the above problems and provide a reagent chip that can easily measure the concentration of free fatty acids in a minute space where oxygen supply is restricted.

Means for Solving the Problems

[0011] 1) The method for measuring the free fatty acid concentration according to the present invention is a method for measuring the concentration of free fatty acids in blood by the following steps in a minute closed space where oxygen supply is restricted. 1) A first step of generating pyrophosphoric acid from the free fatty acids contained in the blood, 2) A second step of reacting the pyrophosphoric acid with a plurality of enzymes to generate reduced nicotinamide adenine dinucleotide or reduced thionicotinamide adenine dinucleotide, 3) A third step of calculating the concentration of free fatty acids based on the reduced nicotinamide adenine dinucleotide or reduced thionicotinamide adenine dinucleotide.

[0012] Since the above reaction causes a color reaction by a reduction system reaction, the concentration of free fatty acids can be measured even in a minute closed space where oxygen supply is restricted.

[0013] 2) In the method for measuring the free fatty acid concentration according to the present invention, the first step is a pyrophosphoric acid generation step of reacting the free fatty acids contained in the blood with a first enzyme to generate pyrophosphoric acid, the second step is a phosphoric acid generation step of reacting the pyrophosphoric acid with a second enzyme to generate two molecules of phosphoric acid, a glucose-1-phosphate generation step of reacting the phosphoric acid with a third enzyme to generate glucose-1-phosphate, a glucose-6-phosphate generation step of reacting the glucose-1-phosphate with a fourth enzyme to generate glucose-6-phosphate, and a reduced nicotinamide adenine dinucleotide generation step of reacting the glucose-6-phosphate with a fifth enzyme to generate reduced nicotinamide adenine dinucleotide. The third step is a concentration calculation step of calculating the concentration of free fatty acids by measuring the color development of the generated formazan for the reduced nicotinamide adenine dinucleotide.

[0014] Since the above reaction causes a color reaction by a reduction system reaction, the concentration of free fatty acids can be measured even in a minute closed space where oxygen supply is restricted.

[0015] 3) In the method for measuring the free fatty acid concentration according to the present invention, the first step is a pyrophosphate generation step of reacting the free fatty acids contained in the blood with a first enzyme to generate pyrophosphate, the second step is a hypoxanthine generation step of reacting the pyrophosphate with a sixth enzyme to generate hypoxanthine, and a reduced nicotinamide adenine dinucleotide generation step of reacting the hypoxanthine with a seventh enzyme to generate two molecules of reduced nicotinamide adenine dinucleotide. The third step is a concentration calculation step of calculating the concentration of the free fatty acids by measuring the color development of the generated formazan for the reduced nicotinamide adenine dinucleotide.

[0016] Since the above reaction causes a color development reaction by a reaction of a reducing system, the concentration of the free fatty acids can be measured even in a minute closed space where oxygen supply is restricted.

[0017] 4) In the method for measuring the free fatty acid concentration according to the present invention, the first step is a pyrophosphate generation step of reacting the free fatty acids contained in the blood with a first enzyme to generate pyrophosphate, the second step is a phosphate generation step of reacting the pyrophosphate with a second enzyme to generate two molecules of phosphate, a glucose-1-phosphate generation step of reacting the phosphate with a third enzyme to generate glucose-1-phosphate, a glucose-6-phosphate generation step of reacting the glucose-1-phosphate with a fourth enzyme to generate glucose-6-phosphate, and a reduced thionicotinamide adenine dinucleotide generation step of reacting the glucose-6-phosphate with an eighth enzyme to generate reduced thionicotinamide adenine dinucleotide. The third step is a concentration calculation step of calculating the concentration of the free fatty acids from the absorbance of the reduced thionicotinamide adenine dinucleotide.

[0018] Since the reaction using the above reagent is a reaction of a reducing system, the concentration of the free fatty acids can be measured without being affected by the restriction of the oxygen supply even in a minute closed space.

[0019] 5) In the method for measuring the free fatty acid concentration according to the present invention, the first step is a pyrophosphate generation step of reacting the free fatty acid contained in the blood with a first enzyme to generate pyrophosphate, and the second step is a hypoxanthine generation step of reacting the pyrophosphate with a sixth enzyme to generate hypoxanthine, and a reduced thionicotinamide adenine dinucleotide generation step of reacting the hypoxanthine with a ninth enzyme to generate two molecules of reduced thionicotinamide adenine dinucleotide. The third step is a concentration calculation step of calculating the concentration of the free fatty acid from the absorbance of the reduced thionicotinamide adenine dinucleotide.

[0020] Since the reaction using the above reagent is a reaction in a reducing system, the concentration of the free fatty acid can be measured without being affected by the limitation of oxygen supply even in a minute closed space.

[0021] 6) In the method for measuring the free fatty acid concentration according to the present invention, the blood is undiluted. Therefore, the concentration of the free fatty acid can be measured without being affected by the limitation of oxygen supply even in a minute closed space with undiluted blood.

[0022] 7) In the method for measuring the free fatty acid concentration according to the present invention, in the pyrophosphate generation step, in the presence of coenzyme A and adenosine triphosphate, acyl coenzyme A synthetase activated by magnesium ions generates acyl coenzyme A, pyrophosphate, and adenosine monophosphate. In the phosphate generation step, the pyrophosphate is decomposed into two molecules of phosphate by pyrophosphatase. In the glucose-1-phosphate generation step, the phosphate is reacted with maltose by maltose phosphorylase to produce glucose and glucose-1-phosphate. In the glucose-6-phosphate generation step, the glucose-1-phosphate is converted into glucose-6-phosphate by beta phosphoglucomutase. In the reduced nicotinamide adenine dinucleotide generation step, the glucose-6-phosphate and oxidized nicotinamide adenine dinucleotide are reduced by glucose-6-phosphate dehydrogenase to produce gluconolactone-6-phosphate and reduced nicotinamide adenine dinucleotide. In the concentration calculation step, the concentration of the free fatty acid is calculated by measuring the color development of formazan generated by reducing a tetrazolium salt with the reduced nicotinamide adenine dinucleotide and diaphorase.

[0023] Since the above reaction causes a color reaction by a reaction in a reducing system, the concentration of free fatty acids can be measured even in a minute closed space where oxygen supply is restricted.

[0024] 8) In the method for measuring the free fatty acid concentration according to the present invention, in the pyrophosphate generation step, the free fatty acid is reacted with coenzyme A and adenosine triphosphate in the presence of magnesium ions by acyl coenzyme A synthetase activated by magnesium ions to generate acyl coenzyme A, pyrophosphate and adenosine monophosphate. In the hypoxanthine generation step, the pyrophosphate is reacted with inosine by purine nucleotide phosphorylase to generate hypoxanthine and D-ribose-1-phosphate. In the reduced nicotinamide adenine dinucleotide generation step, the hypoxanthine and two molecules of oxidized nicotinamide adenine dinucleotide are reduced by xanthine dehydrogenase to produce uric acid and two molecules of reduced nicotinamide adenine dinucleotide. In the concentration calculation step, the concentration of the free fatty acid is calculated by measuring the color development of formazan generated by reducing a tetrazolium salt with the reduced nicotinamide adenine dinucleotide and diaphorase.

[0025] Since the above reaction causes a color reaction by a reaction in a reducing system, the concentration of free fatty acid can be measured even in a minute closed space where oxygen supply is restricted.

[0026] 9) In the method for measuring the free fatty acid concentration according to the present invention, in the pyrophosphate generation step, in the presence of coenzyme A and adenosine triphosphate, acyl coenzyme synthetase activated by magnesium ions generates acyl coenzyme A, pyrophosphate and adenosine monophosphate. In the phosphate generation step, the pyrophosphate is decomposed into two molecules of phosphate by pyrophosphatase. In the glucose-1-phosphate generation step, the phosphate is reacted with maltose by maltose phosphorylase to produce glucose and glucose-1-phosphate. In the glucose-6-phosphate generation step, the glucose-1-phosphate is converted into glucose-6-phosphate by beta-phosphoglucomutase. In the reduced thionicotinamide adenine dinucleotide Generation step then, the Glucose-6-phosphateAnd nicotinamide adenine dinucleotide in its oxidized form is reduced by glucose-6-phosphate dehydrogenase to produce gluconolactone-6-phosphate and nicotinamide adenine dinucleotide in its reduced form.

[0027] Since the reaction using the above reagents is a reaction of a reducing system, the concentration of free fatty acids can be measured without being affected by the limited oxygen supply even in a minute closed space.

[0028] 10) In the free fatty acid concentration measuring method according to the present invention, in the pyrophosphate generation step, the free fatty acid is reacted with coenzyme A and adenosine 3-phosphate in the presence of magnesium ions by acyl coenzyme synthetase activated by magnesium ions to generate acyl coenzyme A, pyrophosphate and adenosine 1-phosphate. In the hypoxanthine generation step, the pyrophosphate is reacted with inosine by purine nucleotide phosphorylase to generate hypoxanthine and D-ribose-1-phosphate. And hypoxanthine and two molecules of nicotinamide adenine dinucleotide in its oxidized form are reduced by xanthine dehydrogenase to produce uric acid and two molecules of nicotinamide adenine dinucleotide in its reduced form.

[0029] Since the reaction using the above reagents is a reaction of a reducing system, the concentration of free fatty acids can be measured without being affected by the limited oxygen supply even in a minute closed space.

[0030] 11) The reagent chip according to the present invention has a reaction chamber in which a plurality of reagents are installed, a dropping port for dropping blood to be examined, and a transport path for transporting the blood to the reaction chamber. By detecting a color reaction in the reaction chamber, it is a reagent chip for measuring the concentration of free fatty acids in the blood. The reaction chamber is a minute closed space filled with the blood when the blood is supplied. The plurality of reagents are coenzyme A, adenosine triphosphate, magnesium ions, acyl coenzyme A synthetase, pyrophosphatase, maltose, maltose phosphorylase, beta phosphoglucomutase, oxidized nicotinamide adenine dinucleotide, glucose-6-phosphate dehydrogenase, tetrazolium salt, and diaphorase.

[0031] Since the reaction using the above reagents causes a color reaction by a reduction system reaction, the concentration of free fatty acids can be measured without being affected by the limited oxygen supply even in a minute closed space.

[0032] 12) The reagent chip according to the present invention has a reaction chamber in which a plurality of reagents are installed, a dropping port for dropping blood to be examined, and a transport path for transporting the blood to the reaction chamber. By detecting a color reaction in the reaction chamber, it is a reagent chip for measuring the concentration of free fatty acids in the blood. The reaction chamber is a minute closed space filled with the blood when the blood is supplied. The plurality of reagents are coenzyme A, adenosine triphosphate, magnesium ions, acyl coenzyme A synthetase, inosine, purine nucleotide phosphorylase, xanthine dehydrogenase, oxidized nicotinamide adenine dinucleotide, tetrazolium salt, and diaphorase.

[0033] Since the reaction using the above reagents causes a color reaction by a reduction system reaction, the concentration of free fatty acids can be measured without being affected by the limited oxygen supply even in a minute closed space.

[0034] 13) The reagent chip according to the present invention has a reaction chamber in which a plurality of reagents are installed, a dropping port for dropping blood to be inspected, and a transport path for transporting the blood to the reaction chamber. By detecting a color reaction in the reaction chamber, it is a reagent chip for measuring the concentration of free fatty acids in the blood. The reaction chamber is a minute closed space that is filled with the blood when the blood is supplied. The plurality of reagents are coenzyme A, adenosine triphosphate, magnesium ions, acyl coenzyme A synthetase, pyrophosphatase, maltose, maltose phosphorylase, beta phosphoglucomutase, glucose-6-phosphate dehydrogenase, and oxidized thionicotinamide adenine dinucleotide.

[0035] Since the reaction using the above reagents is a reaction in a reducing system, the concentration of free fatty acids can be measured without being affected by the limited oxygen supply even in a minute closed space.

[0036] 14) The reagent chip according to the present invention has a reaction chamber in which a plurality of reagents are installed, a dropping port for dropping blood to be inspected, and a transport path for transporting the blood to the reaction chamber. By detecting a color reaction in the reaction chamber, it is a reagent chip for measuring the concentration of free fatty acids in the blood. The reaction chamber is a minute closed space that is filled with the blood when the blood is supplied. The plurality of reagents are coenzyme A, adenosine triphosphate, magnesium ions, acyl coenzyme A synthetase, inosine, purine nucleotide phosphorylase, xanthine dehydrogenase, and oxidized thionicotinamide adenine dinucleotide.

[0037] Since the reaction using the above reagents is a reaction in a reducing system, the concentration of free fatty acids can be measured without being affected by the limited oxygen supply even in a minute closed space.

[0038] 15) In the reagent chip according to the present invention, the blood is undiluted. Therefore, the concentration of free fatty acids can be measured without being affected by the limited oxygen supply even in a minute closed space with undiluted blood.

[0039] 16) In the reagent chip according to the present invention, the reaction chamber has a first reaction chamber and a second reaction chamber. In the first reaction chamber, as the plurality of reagents, coenzyme A, adenosine triphosphate, magnesium ions, acyl coenzyme A synthetase, pyrophosphatase, maltose, maltose phosphorylase, beta phosphoglucomutase, oxidized nicotinamide adenine dinucleotide, glucose-6-phosphate dehydrogenase, tetrazolium salt, and diaphorase are installed. In the second reaction chamber, as the plurality of reagents, maltose, maltose phosphorylase, beta phosphoglucomutase, oxidized nicotinamide adenine dinucleotide, glucose-6-phosphate dehydrogenase, tetrazolium salt, and diaphorase are installed. In the second reaction chamber, color development corresponding to the phosphate concentration contained in the blood can be detected. Therefore, by obtaining the difference from the color development in the first reaction chamber, the concentration of free fatty acids can be accurately measured.

[0040] 17) In the reagent chip according to the present invention, the reaction chamber has a first reaction chamber and a second reaction chamber. In the first reaction chamber, as the plurality of reagents, coenzyme A, adenosine triphosphate, magnesium ions, acyl coenzyme A synthetase, inosine, purine nucleotide phosphorylase, xanthine dehydrogenase, oxidized nicotinamide adenine dinucleotide, tetrazolium salt, and diaphorase are installed. In the second reaction chamber, as the plurality of reagents, inosine, purine nucleotide phosphorylase, xanthine dehydrogenase, oxidized nicotinamide adenine dinucleotide, tetrazolium salt, and diaphorase are installed. In the second reaction chamber, color development corresponding to the phosphate concentration contained in the blood can be detected. Therefore, by obtaining the difference from the color development in the first reaction chamber, the concentration of free fatty acids can be accurately measured.

[0041] 18) In the reagent chip according to the present invention, the reaction chamber has a first reaction chamber and a second reaction chamber. In the first reaction chamber, as the plurality of reagents, coenzyme A, adenosine triphosphate, magnesium ions, acyl coenzyme A synthetase, pyrophosphatase, maltose, maltose phosphorylase, beta phosphoglucomutase, glucose-6-phosphate dehydrogenase, and oxidized thionicotinamide adenine dinucleotide are installed. In the second reaction chamber, as the plurality of reagents, maltose, maltose phosphorylase, beta phosphoglucomutase, glucose-6-phosphate dehydrogenase, and oxidized thionicotinamide adenine dinucleotide are installed.

[0042] In the second reaction chamber, reduced thionicotinamide adenine dinucleotide corresponding to the phosphate concentration contained in the blood can be detected. Therefore, by obtaining the difference from the reduced thionicotinamide adenine dinucleotide in the first reaction chamber, the concentration of free fatty acids can be accurately measured.

[0043] 19) In the reagent chip according to the present invention, the reaction chamber has a first reaction chamber and a second reaction chamber. In the first reaction chamber, as the plurality of reagents, coenzyme A, adenosine triphosphate, magnesium ions, acyl coenzyme synthetase, inosine, purine nucleotide phosphorylase, xanthine dehydrogenase, and oxidized thionicotinamide adenine dinucleotide are installed. In the second reaction chamber, as the plurality of reagents, inosine, purine nucleotide phosphorylase, xanthine dehydrogenase, and oxidized thionicotinamide adenine dinucleotide are installed.

[0044] In the second reaction chamber, reduced thionicotinamide adenine dinucleotide corresponding to the phosphate concentration contained in the blood can be detected. Therefore, by obtaining the difference from the reduced thionicotinamide adenine dinucleotide in the first reaction chamber, the concentration of free fatty acids can be accurately measured.

Brief Description of the Drawings

[0045]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Explanation of Signs

[0046] 1 ····· reagent chip 2 ····· second plate 3 ····· third plate 4 ····· fourth plate 7 ····· Measurement chamber (reaction chamber) 8 ····· Flow path (transport path) 11 ····· Specimen supply port (dropping port)

Best Mode for Carrying Out the Invention

[0047] 1. First Embodiment 1.1 Regarding the reagent chip 1 Hereinafter, the reagent chip 1 used in the measurement method according to the present invention will be described with reference to the drawings.

[0048] The reagent chip 1 has a reaction chamber in which a plurality of reagents are installed, a dropping port for dropping blood to be inspected, and a transport path for transporting the blood to the reaction chamber. By detecting the color development reaction in the reaction chamber, it is a reagent chip for measuring the free fatty acid concentration in the blood. The reaction chamber is a minute closed space that is filled with the blood when the blood is supplied. This will be described in detail below.

[0049] FIG. 1 is a longitudinal sectional view of a blood inspection instrument, and FIG. 2 is an exploded perspective view thereof. The inspection instrument 100 shown in each of these figures is configured by adhering a substrate 1 and a laminated plate 20. A circular specimen supply port 11 is formed on the substrate 1, and a weir 12 is provided around the upper part thereof. The weir 12 is provided with an annular protrusion 12a that further protrudes from the upper surface. Also, an arcuate protrusion 13 is provided inside the weir 12. This weir 12 is configured such that a plug body 5 made of an elastic material such as butyl rubber is in close contact to seal the internal space. Specifically, when the plug body 5 abuts against the weir 12, the annular protrusion 12a of the weir 12 bites into the edge 5a of the plug body 5, so that the internal space is reliably sealed. When the plug body 5 is pressed in that state, a pressure is applied to the specimen (blood) supplied to the specimen supply port 11. The plug body 5 is pressed by a pressing mechanism (not shown).

[0050] On the lower surface side of the substrate 1, a circular recessed portion 14 is provided at a position facing the specimen supply port 11, and a blood cell separation membrane 6 made of an asymmetric pore diameter membrane for separating and removing blood cells is inserted therein. This blood cell separation membrane 6 is fixed to the substrate 1 with an adhesive (not shown).

[0051] As the material of the substrate 1, a resin material that is easy to process is used. For example, AS resin (styrene-acrylonitrile resin) is preferably used.

[0052] On the upper surface of the substrate 1, protrusions 16 and 17 are provided. These protrusions 16 and 17 are used for positioning the inspection instrument 100 during optical measurement or electrical measurement.

[0053] The laminated plate 20 includes three first plates 2, a second plate 3, and a third plate 4 that are laminated and integrated in the vertical direction via an adhesive (not shown). Inside the laminated plate 20 on the side of the specimen supply port 11, a measurement chamber 7 for measuring the components of the specimen is formed, and a flow path 8 (transport path) for transferring the specimen is formed between this measurement chamber 7 and the specimen supply port 11.

[0054] The flow path 8 will be described with reference to FIG. 2.

[0055] A through hole 81 extending in the vertical direction is formed at a position concentric with the specimen supply port 11 in the first plate 2. A circular through hole 82 is provided in the second plate 3 disposed below the first plate 2 so as to face the through hole 81, and a linear through groove 83 is drilled laterally therefrom. The through hole 81, the through hole 82, and the through grooves 83a to 83c form the flow path 8.

[0056] The measurement chamber 7 will be described. In the second plate 3, eight circular through holes 71 for forming the measurement chamber 7 are formed. A through hole 82 facing the through hole 81 provided in the first plate 2 is provided. A single through groove 83a extending from this through hole 82 branches into eight through grooves 83c at the branch portion 83b, and through holes 71 are formed at the tip ends of these through grooves 83c.

[0057] In this way, the eight measurement chambers 7 are formed by the through holes 71 that constitute the side surfaces and the first plate 2 and the third plate 4 that constitute the upper and lower planes. The same applies to the flow path 8.

[0058] The first plate 2 and the third plate 4 are water-impermeable and air-impermeable plates. As the material thereof, a resin material that is easy to process is used. For example, AS resin (styrene-acrylonitrile resin) is preferably used.

[0059] On the other hand, as the second plate 3, a porous material such as a PTFE (tetrafluoroethylene resin) membrane that is water-impermeable and air-permeable is preferably used. In this way, when the stopper 5 is pressed to transfer the specimen from the flow path 8 to the measurement chamber 7, the air accumulated in the spaces of the flow path 8 and the measurement chamber 7 escapes to the outside through the second plate 3, and the specimen is sequentially transferred so as to fill the space, and a predetermined amount of the specimen is quickly transferred to the measurement chamber 7.

[0060] A reagent filling portion 9 is provided in the measurement chamber 7. A plurality of reagents described later are filled in the reagent filling portion 9.

[0061] When measuring the specimen in the measurement chamber 7, optical measurement using transmitted light or reflected light is employed. A color reaction is caused between the reagent and the specimen in the measurement chamber 7, light is irradiated from below the third plate 4, and the transmitted light is received above the first plate 2 to measure the absorbance in the measurement chamber 7.

[0062] Considering the resolution in the low concentration range and the optical saturation in the high concentration range, the thickness of the second plate 3 is preferably 0.5 mm or less so as to fall within the assumed absorbance range (Abs. 0.001 to 2.000). More preferably, it is 0.1 mm to 0.2 mm. The width of the flow path 8 is 0.25 mm. In this embodiment, the shape of the measurement chamber 7 is an oval shape of 1.8 mm × 2.0 mm, and the light beam for measurement has a diameter of 0.7 mm. Therefore, after the blood is dropped, the measurement chamber 7 becomes a minute closed space with limited oxygen.

[0063] Also, in this embodiment, the flow path 8 has a thickness of 0.1 mm to 0.2 mm and a width of 0.25 mm.

[0064] 1.2 Regarding the reagent In the measurement chamber 7, "coenzyme A", "adenosine triphosphate", "magnesium ion", "acyl coenzyme A synthetase", "pyrophosphatase", "maltose", "maltose phosphorylase", "beta phosphoglucomutase", "oxidized nicotinamide adenine dinucleotide", "glucose-6-phosphate dehydrogenase", "tetrazolium salt", and "diaphorase" are charged at appropriate concentrations as reagents.

[0065] In this embodiment, the reagent was placed as follows.

[0066] On one side of the measurement chamber 7, 50 - 200 mM of maltose from FUJIFILM Wako Pure Chemical Corporation, 5 - 20 mM of adenosine triphosphate (ATP) from Oriental Yeast Co., Ltd., 100 - 500 U / mL of beta phosphoglucomutase (β-Phosphoglucomutase) from Kikkoman Corporation, 50 - 300 U / mL of maltose phosphorylase from Kikkoman Corporation, 10 - 50 U / mL of acyl coenzyme A synthetase (ACS) from Asahi Kasei Corporation, 50 - 200 U / mL of pyrophosphatase from Roche Diagnostics Corporation, 50 - 100 U / mL of glucose-6-phosphate dehydrogenase (G6PDH) from Nipro Corporation, 100 - 500 U / mL of diaphorase (Di-1) from Nipro Corporation, and MgCl 2 ·6H 2 O of 5 - 30 mM are used as reagents, and 100 - 400 mM of HEPES buffer pH7.5 from Dojindo Laboratories and 0.005 - 0.05% of Triton X-100 from FUJIFILM Wako Pure Chemical Corporation are mixed, then dropped and dried (0.5 μL), and this is naturally dried at 20 - 30°C in an environment of 20 - 50% RH.

[0067] On the other hand, on the other side of the measurement chamber 7, 5 to 30 mM of oxidized nicotinamide adenine dinucleotide (NAD) from Oriental Yeast Co., Ltd., 20 to 100 mM of tetrazolium salt (WST-8) from Dojindo Laboratories Co., Ltd., and 2 to 15 mM of coenzyme A from Oriental Yeast Co., Ltd. were used as reagents. 0.5 to 3.0% of D-Sorbitol from Fujifilm Wako Pure Chemical Corporation, 10 to 50 mM of Malate buffer pH4.0 from Fujifilm Wako Pure Chemical Corporation, and 0.005 to 0.05% of triton X-100 from Fujifilm Wako Pure Chemical Corporation were mixed together, and then drop-dried (0.5 μL), and this was naturally dried at 20 to 30 °C under an environment of 20 to 50% RH.

[0068] The reason for dispersing the reagents into two parts is to separate reagents that are not preferably mixed together.

[0069] Also, as storage conditions, it is preferable to store it under an environment of 20 to 30 °C and 10% RH or less after drying.

[0070] 1.3 About the reaction by reagents The reaction by the above reagents is shown in Fig. 3A. Free fatty acids (NEFA) in blood, in the presence of coenzyme A and adenosine triphosphate (ATP), are converted into acyl coenzyme A, pyrophosphate, and adenosine monophosphate (AMP) by the action of acyl coenzyme A synthetase (enzyme) activated by magnesium ions. The generated pyrophosphate is decomposed into two molecules of phosphate by the action of pyrophosphatase (enzyme). Phosphate reacts with maltose by the action of maltose phosphorylase (enzyme) to produce glucose and glucose-1-phosphate. Glucose-1-phosphate is converted into glucose-6-phosphate by beta phosphoglucomutase (enzyme). Glucose-6-phosphate and oxidized nicotinamide adenine dinucleotide (NAD) are reduced by glucose-6-phosphate dehydrogenase (enzyme) to produce gluconolactone-6-phosphate and reduced nicotinamide adenine dinucleotide (NADH).

[0071] The generated reduced nicotinamide adenine dinucleotide reduces the tetrazolium salt (chromogenic agent) by the action of diaphorase (enzyme) and forms formazan, resulting in color development.

[0072] Since the color development of this formazan depends on the free fatty acid concentration in the sample (blood), the free fatty acid concentration in the sample can be calculated by irradiating light with a wavelength absorbed by formazan and measuring the amount of transmitted light.

[0073] 1.4 Regarding measurement Regarding the method for measuring the specimen in the measurement chamber 7, optical measurement using transmitted light, which has been conventionally known, can be adopted. For example, for the color reaction in the measurement chamber 7, light B with a wavelength of 450 nm can be irradiated from below the fourth plate 4 toward the second plate 2, and the amount of color development can be measured based on the amount of light that has passed through the measurement chamber 7.

[0074] In this embodiment, by pressurizing the plug body 5, the air present in the flow path 8 and the measurement chamber 7 is discharged to the outside, and the specimen is smoothly guided into the measurement chamber 7. However, as long as the air present in the flow path 8 and the measurement chamber 7 can be discharged to the outside, any method may be used. For example, the inspection instrument may be placed in a sealed space and the pressure of the sealed space may be set to a negative pressure state.

[0075] Also, the shapes of the measurement chamber and the flow path are not limited to those of this embodiment and may be any shape.

[0076] 1.5 Regarding the measurement results The measurement results in this embodiment will be described with reference to FIGS. 4 and 5.

[0077] As shown in FIG. 4A, in the conventional method using an oxidation reaction, in the 5-minute measurement method, it is possible to measure up to NEFA: 0.79 mEq / L. However, looking at the shape of the reaction time course, it can be seen that even for a specimen with NEFA: 0.57 mEq / L, the end-point measurement at 5 minutes is in a delicate state.

[0078] This is due to the following reasons. In the conventional reaction, the only source of oxygen required is the dissolved oxygen in the sample. Generally, the dissolved oxygen concentration in the liquid of undiluted blood is about 0.5 mmol / L. In contrast, the blood concentration of free fatty acids is in the normal range: 0.1 - 0.8 mEq / L (required measurement range: 0.05 - 2.5 mEq / L). Therefore, in the said micro-closed space, the reaction only proceeds to about half of the normal range.

[0079] In contrast, as shown in FIG. 4B, in the reaction of the first embodiment, sufficient linearity is ensured up to NEFA: 2.02 mEq / L with the 5-minute measurement method, and it can be seen that endpoint measurement is also possible from the shape of the reaction time course.

[0080] Thus, by changing to a reaction system that does not require oxygen, it is possible to ensure a sufficient reaction rate and measurement range even in a micro-closed space.

[0081] Thus, since the present measurement method does not require oxygen for the reaction, it can accurately measure up to a high concentration of NEFA even in a micro-closed space under oxygen supply limitation for undiluted donors.

[0082] 1.6 Others In the above reaction, NEFA is converted to phosphoric acid, and the NEFA concentration is detected by a color reaction based on the concentration of the phosphoric acid. However, the blood already contains phosphoric acid in advance. Therefore, it is necessary to remove such an amount of phosphoric acid. For this purpose, different reagents may be charged into the first measurement chamber and the second measurement chamber as follows, and the difference between the two may be taken.

[0083] In the first measurement chamber, reagents such as "coenzyme A", "adenosine triphosphate", "magnesium ions", "acyl coenzyme A synthetase", "pyrophosphatase", "maltose", "maltose phosphorylase", "beta phosphoglucomutase", "oxidized nicotinamide adenine dinucleotide", "glucose-6-phosphate dehydrogenase", "tetrazolium salt", and "diaphorase" are charged at appropriate concentrations.

[0084] And in the second measurement chamber, reagents such as "maltose", "maltose phosphorylase", "beta phosphoglucomutase", "oxidized nicotinamide adenine dinucleotide", "glucose-6-phosphate dehydrogenase", "tetrazolium salt", and "diaphorase" are charged at appropriate concentrations.

[0085] In this way, in the first measurement chamber, the color development based on the phosphate generated from free fatty acids in the blood and the phosphate originally contained in the blood is detected, and in the second measurement chamber, the color development based only on the phosphate in the blood is detected. Furthermore, by obtaining the difference between the two, the concentration of free fatty acids can be easily measured.

[0086] In this case, the method of dividing the upper and lower reagents may be the same as that in the above embodiment.

[0087] 2. Second Embodiment 2.1 Regarding Reagents As a reaction of the reduction system, in measurement chamber 7, reagents such as "coenzyme A", "adenosine triphosphate", "magnesium ions", "acyl coenzyme A synthetase", "inosine", "purine nucleotide phosphorylase", "xanthine dehydrogenase", "oxidized nicotinamide adenine dinucleotide", "tetrazolium salt", and "diaphorase" may be charged at appropriate concentrations.

[0088] In this case, the reagents may be placed as follows.

[0089] On one side of the measurement chamber 7, 20 - 80 mM of inosine from FUJIFILM Wako Pure Chemical Corporation, 5 - 20 mM of adenosine triphosphate (ATP) from Oriental Yeast Co., Ltd., 10 - 50 U / mL of acyl-CoA synthetase (ACS) from Asahi Kasei Corporation, 20 - 150 U / mL of purine nucleotide phosphorylase (PNPL II) from Asahi Kasei Corporation, 20 - 150 U / mL of xanthine dehydrogenase (XDH II), 100 - 400 U / mL of diaphorase (Di-3) from Nipro Corporation, and 5 - 30 mM of MgCl 2 ·6H 2 O from FUJIFILM Wako Pure Chemical Corporation were used as reagents, 100 - 400 mM of pH shock agent (TAPSO pH8.0) from Dojindo Laboratories, 0.5 - 3.0% of D-Sorbitol from FUJIFILM Wako Pure Chemical Corporation, 0.005 - 0.05% of Triton X-100 from FUJIFILM Wako Pure Chemical Corporation were mixed, and then drop-dried (0.5 μL), and this was naturally dried at 20 - 30 °C in an environment of 20 - 50% RH.

[0090] On the other side of the measurement chamber 7, 5 - 30 mM of oxidized nicotinamide adenine dinucleotide (NAD) from Oriental Yeast Co., Ltd., 20 - 100 mM of tetrazolium salt (WST-8) from Dojindo Laboratories, and 2 - 15 mM of coenzyme A from Oriental Yeast Co., Ltd. were used as reagents, 0.5 - 3.0% of D-Sorbitol from FUJIFILM Wako Pure Chemical Corporation, 10 - 50 mM of Malate buffer pH4.0 from FUJIFILM Wako Pure Chemical Corporation, and 0.005 - 0.05% of triton X-100 from FUJIFILM Wako Pure Chemical Corporation were mixed, and then drop-dried (0.5 μL), and this was naturally dried at 20 - 30 °C in an environment of 20 - 50% RH.

[0091] 2.2 Regarding the reaction The reaction with the above reagent is shown in Fig. 3B. Free fatty acid (NEFA) produces acyl coenzyme A, pyrophosphate and adenosine monophosphate (AMP) by the action of acyl coenzyme synthetase (enzyme) activated by magnesium ions in the presence of coenzyme A and adenosine triphosphate (ATP).

[0092] Pyrophosphate reacts with inosine by purine nucleotide phosphorylase (enzyme) to produce hypoxanthine and D-ribose-1-phosphate.

[0093] Hypoxanthine and two molecules of oxidized nicotinamide adenine dinucleotide (NAD) are reduced by xanthine dehydrogenase (enzyme) to produce uric acid and two molecules of reduced nicotinamide adenine dinucleotide (NADH).

[0094] The reactions after this are the same as those in the first embodiment.

[0095] 2.3 Regarding Phosphoric Acid in Blood Similar to the first embodiment, in the second embodiment, phosphoric acid in blood is also measured. In this case as well, a reagent obtained by removing the reagent for obtaining phosphoric acid from free fatty acid may be charged into another measurement chamber, and the difference between the two may be obtained.

[0096] In the second embodiment, the reagents necessary for measuring only phosphoric acid in blood are "inosine", "purine nucleotide phosphorylase", "xanthine dehydrogenase", "oxidized nicotinamide adenine dinucleotide", "tetrazolium salt", and "diaphorase" among the above reagents.

[0097] 2.4 Regarding Measurement Results Also in the second embodiment, similar to the first embodiment, as shown in Figs. 4B and 5B, sufficient linearity up to NEFA: 2.02 mEq / L is ensured by the 5-minute measurement method as compared with the conventional method, and endpoint measurement was also possible with the shape of the reaction time course.

[0098] 3. Third Embodiment In the first embodiment, as shown in Fig. 3A, gluconolactone-6-phosphate is used to generate reduced nicotinamide adenine dinucleotide (NADH), and the free fatty acid concentration is calculated by the color reaction caused thereby.

[0099] However, it is not limited to this. Reduced thio-nicotinamide adenine dinucleotide (thio-NADH) may be generated from gluconolactone-6-phosphate, and the free fatty acid concentration may be calculated by measuring this reduced thio-nicotinamide adenine dinucleotide (thio-NADH).

[0100] In this case, as the reagent, oxidized thio-nicotinamide adenine dinucleotide (thio-NAD) may be used instead of NAD. Also, diaphorase and tetrazolium salt are not necessary.

[0101] In this embodiment, from one side of the measurement chamber 7, a plurality of the same reagents as in the first embodiment except for diaphorase are provided, and on the other side of the measurement chamber 7, instead of oxidized nicotinamide adenine dinucleotide (NAD) of Oriental Yeast Co., Ltd. and tetrazolium salt (WST-8) of Dojindo Laboratories, 10 to 60 mM of oxidized thio-nicotinamide adenine dinucleotide (thio-NAD) of Oriental Yeast Co., Ltd. is adopted, and the other plurality of reagents are the same.

[0102] The reaction in this case is shown in Fig. 6A.

[0103] Free fatty acids (NEFA) in the blood, in the presence of coenzyme A and adenosine triphosphate (ATP), are converted by the action of acyl coenzyme A synthetase (enzyme) activated by magnesium ions into acyl coenzyme A, pyrophosphate, and adenosine monophosphate (AMP). The generated pyrophosphate is decomposed into two molecules of phosphate by the action of pyrophosphatase (enzyme). The phosphate reacts with maltose by the action of maltose phosphorylase (enzyme) to produce glucose and glucose-1-phosphate. Glucose-1-phosphate is converted into glucose-6-phosphate by beta phosphoglucomutase (enzyme).

[0104] Gluconolactone-6-phosphate and oxidized thio-nicotinamide adenine dinucleotide (thio-NAD) are reduced by glucose-6-phosphate dehydrogenase (enzyme) to produce gluconolactone-6-phosphate and reduced thio-nicotinamide adenine dinucleotide (thio-NADH).

[0105] This reduced thio-nicotinamide adenine dinucleotide (thio-NADH) can be measured by the absorbance of light at a wavelength of 405 nm.

[0106] 4. Fourth Embodiment Regarding the second embodiment, two molecules of reduced thio-nicotinamide adenine dinucleotide (thio-NADH) may be generated from the generated hypoxanthine and two molecules of oxidized thio-nicotinamide adenine dinucleotide (thio-NAD) to calculate the free fatty acid concentration.

[0107] In this case, as the reagent, oxidized thio-nicotinamide adenine dinucleotide (thio-NAD) may be used instead of NAD. Also, diaphorase and tetrazolium salt are not required.

[0108] In this embodiment, from one side of the measurement chamber 7, a plurality of the same reagents as in the first embodiment are provided except for diaphorase, and on the other side of the measurement chamber 7, the oxidized nicotinamide adenine dinucleotide (NAD) of Oriental Yeast Co., Ltd. is changed to 5 to 30 mM, and the tetrazolium salt (WST-8) of Dojindo Laboratories is changed to 20 to 100 mM, and 10 to 60 mM of oxidized thionicotinamide adenine dinucleotide (thio-NAD) of Oriental Yeast Co., Ltd. is adopted, and the other plurality of reagents are the same.

[0109] The reaction in this case is shown in Fig. 6B.

[0110] Free fatty acid (NEFA) generates acyl coenzyme A, pyrophosphate and adenosine monophosphate (AMP) by the action of acyl coenzyme synthetase (enzyme) activated by magnesium ions in the presence of coenzyme A and adenosine triphosphate (ATP).

[0111] Pyrophosphate reacts with inosine by purine nucleotide phosphorylase (enzyme) to produce hypoxanthine and D-ribose-1-phosphate.

[0112] Hypoxanthine and two molecules of oxidized thionicotinamide adenine dinucleotide (thio-NAD) are reduced by xanthine dehydrogenase (enzyme) to produce uric acid and two molecules of reduced thionicotinamide adenine dinucleotide (thio-NADH).

[0113] The measurement of reduced thionicotinamide adenine dinucleotide (thio-NADH) is the same as in the third embodiment.

[0114] 5. Other Embodiments In this embodiment, bovine free fatty acid is measured, but it can be similarly applied to other animals, humans, etc.

[0115] The method for measuring the free fatty acid of this case is effective because the detection range is sufficient even for undiluted specimens, but it is not limited to this, and it can also be applied when diluting the specimen.

[0116] In the above, the present invention has been described as a preferred embodiment, but it was used for explanation, not for limitation, and it can be changed within the scope of the appended claims without departing from the scope and spirit of the present invention.

Claims

1. A method for measuring the concentration of free fatty acids in blood by the following steps in a minute closed space where oxygen supply is restricted. A first step of generating pyrophosphate from the free fatty acids contained in the blood, A second step of reacting the pyrophosphate with a plurality of enzymes to generate reduced nicotinamide adenine dinucleotide or reduced thionicotinamide adenine dinucleotide, A third step of calculating the concentration of free fatty acids based on the reduced nicotinamide adenine dinucleotide or the reduced thionicotinamide adenine dinucleotide.

2. The method for measuring the concentration of free fatty acids according to Claim 1, wherein the first step is a pyrophosphate generation step of reacting the free fatty acids contained in the blood with a first enzyme to generate pyrophosphate, the second step is composed of a phosphate generation step of reacting the pyrophosphate with a second enzyme to generate two molecules of phosphate, a glucose-1-phosphate generation step of reacting the phosphate with a third enzyme to generate glucose-1-phosphate, a glucose-6-phosphate generation step of reacting the glucose-1-phosphate with a fourth enzyme to generate glucose-6-phosphate, and a reduced nicotinamide adenine dinucleotide generation step of reacting the glucose-6-phosphate with a fifth enzyme to generate reduced nicotinamide adenine dinucleotide, and the third step is a concentration calculation step of calculating the concentration of free fatty acids by measuring the color development of formazan for the reduced nicotinamide adenine dinucleotide.

3. The method for measuring the concentration of free fatty acids according to Claim 1, wherein the first step is a pyrophosphate generation step of reacting the free fatty acids contained in the blood with a first enzyme to generate pyrophosphate, the second step is composed of a pyrophosphate generation step of reacting the free fatty acids contained in the blood with a first enzyme to generate pyrophosphate, a hypoxanthine generation step of reacting the pyrophosphate with a sixth enzyme to generate hypoxanthine, and a reduced nicotinamide adenine dinucleotide generation step of reacting the hypoxanthine with a seventh enzyme to generate two molecules of reduced nicotinamide adenine dinucleotide. The third step is a concentration calculation step of calculating the concentration of free fatty acids by measuring the color development of formazan for the reduced nicotinamide adenine dinucleotide, A method for measuring the concentration of free fatty acids, characterized by the above. **Claim 4** The method for measuring the concentration of free fatty acids according to Claim 1, wherein the first step is a pyrophosphate generation step of reacting the free fatty acids contained in the blood with a first enzyme to generate pyrophosphate, the second step is composed of a phosphate generation step of reacting the pyrophosphate with a second enzyme to generate two molecules of phosphate, a glucose-1-phosphate generation step of reacting the phosphate with a third enzyme to generate glucose-1-phosphate, a glucose-6-phosphate generation step of reacting the glucose-1-phosphate with a fourth enzyme to generate glucose-6-phosphate, and a reduced thio nicotinamide adenine dinucleotide generation step of reacting the glucose-6-phosphate with an eighth enzyme to generate reduced thio nicotinamide adenine dinucleotide, and the third step is a concentration calculation step of calculating the concentration of free fatty acids from the absorbance of the reduced thio nicotinamide adenine dinucleotide, A method for measuring the concentration of free fatty acids, characterized by the above. **Claim 5** The method for measuring the concentration of free fatty acids according to Claim 1, wherein the first step is a pyrophosphate generation step of reacting the free fatty acids contained in the blood with a first enzyme to generate pyrophosphate, the second step is composed of a hypoxanthine generation step of reacting the pyrophosphate with a sixth enzyme to generate hypoxanthine, and a reduced thio nicotinamide adenine dinucleotide generation step of reacting the hypoxanthine with a ninth enzyme to generate two molecules of reduced thio nicotinamide adenine dinucleotide, and the third step is a concentration calculation step of calculating the concentration of free fatty acids from the absorbance of the reduced thio nicotinamide adenine dinucleotide, A method for measuring the concentration of free fatty acids, characterized by the above. **Claim 6** In the method for measuring the concentration of free fatty acids according to any one of Claims 2 to 5, the blood is undiluted, A method for measuring the concentration of free fatty acids, characterized by the above. **Claim 7** In the method for measuring the concentration of free fatty acids according to Claim 2, In the pyrophosphate generation step, in the presence of coenzyme A and adenosine 3-phosphate, acyl coenzyme synthetase activated by magnesium ions generates acyl coenzyme A, pyrophosphate, and adenosine 1-phosphate. In the phosphate generation step, the pyrophosphate is decomposed into two molecules of phosphate by pyrophosphatase. In the glucose-1-phosphate generation step, the phosphate is reacted with maltose by maltose phosphorylase to produce glucose and glucose-1-phosphate. In the glucose-6-phosphate generation step, the glucose-1-phosphate is converted to glucose-6-phosphate by beta-phosphoglucomutase. In the reduced nicotinamide adenine dinucleotide generation step, the glucose-6-phosphate and oxidized nicotinamide adenine dinucleotide are reduced by glucose-6-phosphate dehydrogenase to produce gluconolactone-6-phosphate and reduced nicotinamide adenine dinucleotide. In the concentration calculation step, the concentration of the free fatty acid is calculated by measuring the color development of formazan produced by reducing a tetrazolium salt with the reduced nicotinamide adenine dinucleotide and diaphorase. A method for measuring the concentration of free fatty acid, characterized by the above.

8. In the method for measuring the concentration of free fatty acid according to Claim 3, In the pyrophosphate generation step, in the presence of coenzyme A and adenosine 3-phosphate, the free fatty acid is reacted with acyl coenzyme synthetase activated by magnesium ions to generate acyl coenzyme A, pyrophosphate, and adenosine 1-phosphate. In the hypoxanthine generation step, the pyrophosphate is reacted with inosine by purine nucleotide phosphorylase to produce hypoxanthine and D-ribose-1-phosphate. In the reduced nicotinamide adenine dinucleotide generation step, the hypoxanthine and two molecules of oxidized nicotinamide adenine dinucleotide are reduced by xanthine dehydrogenase to produce uric acid and two molecules of reduced nicotinamide adenine dinucleotide. In the concentration calculation step, the concentration of the free fatty acid is calculated by measuring the color development of formazan generated by reducing a tetrazolium salt with the reduced nicotinamide adenine dinucleotide and diaphorase. A method for measuring the concentration of free fatty acid, characterized by the above.

9. In the method for measuring the concentration of free fatty acid according to claim 4, In the pyrophosphate generation step, in the presence of coenzyme A and adenosine triphosphate, acyl coenzyme synthetase activated by magnesium ions generates acyl coenzyme A, pyrophosphate, and adenosine monophosphate. In the phosphate generation step, the pyrophosphate is decomposed into two molecules of phosphate by pyrophosphatase. In the glucose-1-phosphate generation step, the phosphate is reacted with maltose by maltose phosphorylase to produce glucose and glucose-1-phosphate. In the glucose-6-phosphate generation step, the glucose-1-phosphate is converted into glucose-6-phosphate by beta phosphoglucomutase. In the reduced thionicotinamide adenine dinucleotide generation step, the glucose-6-phosphate and oxidized thionicotinamide adenine dinucleotide are reduced by glucose-6-phosphate dehydrogenase to produce gluconolactone-6-phosphate and reduced thionicotinamide adenine dinucleotide. A method for measuring the concentration of free fatty acid, characterized by the above.

10. In the method for measuring the concentration of free fatty acid according to claim 5, In the pyrophosphate generation step, the free fatty acid is reacted in the presence of coenzyme A and adenosine triphosphate with acyl coenzyme synthetase activated by magnesium ions to generate acyl coenzyme A, pyrophosphate, and adenosine monophosphate. In the hypoxanthine generation step, the pyrophosphate is reacted with inosine by purine nucleotide phosphorylase to produce hypoxanthine and D-ribose-1-phosphate. The hypoxanthine and two molecules of oxidized thionicotinamide adenine dinucleotide are reduced by xanthine dehydrogenase to produce uric acid and two molecules of reduced thionicotinamide adenine dinucleotide. A method for measuring the concentration of free fatty acid, characterized by the above.

11. A reaction chamber in which a plurality of reagents are installed A dropping port for dropping the blood to be inspected, A conveyance path for conveying the blood to the reaction chamber, and has, A reagent chip for measuring the free fatty acid concentration in the blood by detecting a color reaction in the reaction chamber, The reaction chamber is a minute closed space filled with the blood when the blood is supplied, The plurality of reagents are coenzyme A, adenosine triphosphate, magnesium ions, acyl coenzyme A synthetase, pyrophosphatase, maltose, maltose phosphorylase, beta phosphoglucomutase, oxidized nicotinamide adenine dinucleotide, glucose-6-phosphate dehydrogenase, tetrazolium salt, and diaphorase, A reagent chip characterized by that.

12. A reaction chamber in which a plurality of reagents are installed, A dropping port for dropping the blood to be inspected, A conveyance path for conveying the blood to the reaction chamber, and has, A reagent chip for measuring the free fatty acid concentration in the blood by detecting a color reaction in the reaction chamber, The reaction chamber is a minute closed space filled with the blood when the blood is supplied, The plurality of reagents are coenzyme A, adenosine triphosphate, magnesium ions, acyl coenzyme A synthetase, inosine, purine nucleotide phosphorylase, xanthine dehydrogenase, oxidized nicotinamide adenine dinucleotide, tetrazolium salt and diaphorase, A reagent chip characterized by that.

13. A reaction chamber in which a plurality of reagents are installed, A dropping port for dropping the blood to be inspected, A conveyance path for conveying the blood to the reaction chamber, and has, A reagent chip for measuring the free fatty acid concentration in the blood by detecting a color reaction in the reaction chamber, The reaction chamber is a minute closed space filled with the blood when the blood is supplied, The plurality of reagents are coenzyme A, adenosine triphosphate, magnesium ions, acyl coenzyme A synthetase, pyrophosphatase, maltose, maltose phosphorylase, beta phosphoglucomutase, glucose-6-phosphate dehydrogenase, and oxidized thionicotinamide adenine dinucleotide, A reagent chip characterized by that.

14. A reaction chamber in which a plurality of reagents are installed, A dropping port for dropping the blood to be inspected, A conveyance path for conveying the blood to the reaction chamber, and has, A reagent chip for measuring the free fatty acid concentration in the blood by detecting a color reaction in the reaction chamber, The reaction chamber is a minute closed space that is filled with the blood when the blood is supplied, The plurality of reagents are coenzyme A, adenosine triphosphate, magnesium ions, acyl coenzyme A synthetase, inosine, purine nucleotide phosphorylase, xanthine dehydrogenase, and oxidized thionicotinamide adenine dinucleotide, A reagent chip characterized by the above.

15. In the reagent chip according to any one of Claims 11 to 14, The blood is undiluted, A reagent chip characterized by the above.

16. In the reagent chip of Claim 11, The reaction chamber has a first reaction chamber and a second reaction chamber, In the first reaction chamber, as the plurality of reagents, coenzyme A, adenosine triphosphate, magnesium ions, acyl coenzyme A synthetase, pyrophosphatase, maltose, maltose phosphorylase, beta phosphoglucomutase, oxidized nicotinamide adenine dinucleotide, glucose-6-phosphate dehydrogenase, tetrazolium salt, and diaphorase are installed, In the second reaction chamber, as the plurality of reagents, maltose, maltose phosphorylase, beta phosphoglucomutase, oxidized nicotinamide adenine dinucleotide, glucose-6-phosphate dehydrogenase, tetrazolium salt, and diaphorase are installed, A reagent chip characterized by the above.

17. In the reagent chip of Claim 12, The reaction chamber has a first reaction chamber and a second reaction chamber, In the first reaction chamber, as the plurality of reagents, coenzyme A, adenosine triphosphate, magnesium ions, acyl coenzyme synthetase, inosine, purine nucleotide phosphorylase, xanthine dehydrogenase, oxidized nicotinamide adenine dinucleotide, tetrazolium salt, and diaphorase are installed, In the second reaction chamber, inosine, purine nucleotide phosphorylase, xanthine dehydrogenase, oxidized nicotinamide adenine dinucleotide, tetrazolium salt, and diaphorase are installed as the plurality of reagents. A reagent chip characterized by the above.

18. In the reagent chip according to claim 13, the reaction chamber has a first reaction chamber and a second reaction chamber, in the first reaction chamber, coenzyme A, adenosine triphosphate, magnesium ions, acyl coenzyme A synthetase, pyrophosphatase, maltose, maltose phosphorylase, beta-phosphoglucomutase, glucose-6-phosphate dehydrogenase, and oxidized thionicotinamide adenine dinucleotide are installed as the plurality of reagents. in the second reaction chamber, maltose, maltose phosphorylase, beta-phosphoglucomutase, glucose-6-phosphate dehydrogenase, and oxidized thionicotinamide adenine dinucleotide are installed as the plurality of reagents. A reagent chip characterized by the above.

19. In the reagent chip according to claim 14, the reaction chamber has a first reaction chamber and a second reaction chamber, in the first reaction chamber, coenzyme A, adenosine triphosphate, magnesium ions, acyl coenzyme synthetase, inosine, purine nucleotide phosphorylase, xanthine dehydrogenase, and oxidized thionicotinamide adenine dinucleotide are installed as the plurality of reagents. in the second reaction chamber, inosine, purine nucleotide phosphorylase, xanthine dehydrogenase, and oxidized thionicotinamide adenine dinucleotide are installed as the plurality of reagents. A reagent chip characterized by the above.

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