JPH0248059B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field of the Invention] The present invention relates to a pyruvate measuring device, and more particularly to a pyruvate measuring device in which the enzymatic activity of immobilized pyruvate oxidase is stabilized.

[Technical background of the invention and its problems] In recent years, clinical analysis has rapidly developed, and it has become possible to accurately understand pathological conditions by measuring the activities of various enzymes present in the blood. For this reason, clinical analysis has come to make a significant contribution to the advancement of medicine.

Conventionally, enzyme activity is measured by carrying out an enzymatic reaction in an enzymatic reaction system consisting of an enzyme, a substrate, a coenzyme, and if necessary a coloring reagent, and measuring the change in absorbance before and after the reaction for colorimetric determination. was. For example, when measuring the enzymatic activity of glutamate pyruvate transaminase (GPT), which is a measure of liver function, GPT is applied to a substrate to generate pyruvate, which is then converted to lactate dehydrogenase (LDH). When you reduce it,
Nicotinamide-adenine dinucleotide (NADH) coexisting in the reaction system is oxidized.
Since it generates NAD, its potential at a wavelength of 340 nm
indirectly by measuring the absorbance of NADH
The enzymatic activity of GPT can be measured. However, in this absorbance measurement method,
NADH, LDH, various coloring reagents, etc. are expensive, and since these reagents are discarded after measurement, the cost of measurement is extremely high. Also,
If suspended substances such as platelets, blood cells, and dust are mixed in the reaction system, it becomes difficult to measure the absorbance, so a pretreatment to remove the non-measurable substances is required prior to measurement.

In order to overcome these drawbacks of the absorbance measurement method, a method was proposed for quantifying pyruvic acid, an enzymatic reaction product of GPT, by an electrode method (Japanese Patent Application Laid-Open No. 122947/1983). An immobilized enzyme membrane consisting of pyruvate oxidase immobilized on a porous polymer membrane is attached to the surface of the enzyme electrode, and the amount of pyruvate is calculated from the amount of dissolved oxygen decreased due to the decomposition of pyruvate by pyruvate oxidase. It is used to measure However, the activity of the prepared immobilized enzyme membrane was unstable, so it had the disadvantage that it could not be used for a long time (50 times at most). In addition, since pyruvate oxidase is generally an unstable enzyme, its lifespan (half-life of activity) was at most 1 to 2 weeks even when various immobilization methods and carriers were used.

In addition, as a method to measure the enzyme activity of GPT,
When glutamic acid, which is an enzymatic reaction product of GPT, is decomposed by dehydrogenase, the coenzyme reduced by the enzymatic reaction is oxidized with an electrode to which a constant voltage is applied. There is a method to measure the
Since this method is susceptible to interference by other reducing substances (vitamins, etc.) present in the blood, it also has the disadvantage that it cannot withstand long-term use.

[Object of the Invention] An object of the present invention is to provide a pyruvic acid measuring device that does not have the above-mentioned drawbacks, has a long life, and is stable.

[Summary of the invention] Conventionally, pyruvate was measured using a buffer containing 0.01mM FAD, but the present inventors
It was discovered that the lifetime of the enzyme activity of pyruvate oxidase can be stabilized by bringing a high concentration flavin adenine dinucleotide solution of 0.1 mM or more into contact with the immobilized pyruvate oxidase disposed within the electrode, and this led to the completion of the present invention. Ivy.

That is, the present invention is a pyruvate measuring device using immobilized pyruvate oxidase, which is characterized by having a mechanism for bringing a 0.1 to 50 mM flavin adenine dinucleotide solution into contact with the immobilized pyruvate oxidase.

The present invention will be explained in detail below.

The pyruvic acid measuring device according to the present invention is a conventionally used pyruvic acid measuring device newly equipped with the above-described mechanism, and all other structures other than the mechanism may be the same as the conventional device.

The immobilized pyruvate oxidase used in the present invention refers to pyruvate oxidase (POP) immobilized on a carrier capable of holding the enzyme. The immobilization carrier may be one that can immobilize pyruvate oxidase.
It can be anything. Specific examples include collagen, polyacrylamide, carrageenan, agarose gel, porous glass beads, albumin, and the like. Usually, pyruvate oxidase is immobilized on a carrier in a range of 0.01 to 30% by weight. Note that the method for immobilizing pyruvate oxidase on a carrier is not particularly limited. However, when incorporating immobilized pyruvate oxidase into the flow system of a pyruvate measurement device, it is preferable to immobilize it on a membrane, granular, or gel-like carrier by an entrapment method or a covalent bonding method, and further attach it to the electrode surface. In this case, it is preferable to apply a comprehensive method in which an enzyme membrane is created using a fibrous protein such as collagen as a carrier.

Next, a mechanism for bringing the flavin adenine dinucleotide solution into contact with immobilized pyruvate oxidase will be explained, but in the present invention, any mechanism that can supply the solution may be used. An example of this mechanism is shown in FIG. The figure is a schematic diagram of the improved electrode section, in which 1 is a flow cell, 2 is an ultrafiltration membrane, 3 is a buffer layer, 4 is an O-ring, 5 is an oxygen or hydrogen peroxide permeable membrane, and 6 teeth
POP immobilization membrane, 7 is platinum cathode, 8 is lead anode, 9
is a pipe, and 10 is a small inner diameter pipe. The mechanism shown in the figure is a mechanism for bringing the flavin adenine dinucleotide (FAD) solution into contact with pyruvate oxidase during measurement. , an electrode section having the same mechanism as above except that the small inner diameter piping 10 is not provided may be used. As the electrode, an oxygen electrode (using an oxygen permeable membrane 5) is usually used to measure the amount of reduction in dissolved oxygen during the enzymatic decomposition process of pyruvic acid, or an oxygen electrode is used to measure the amount of hydrogen peroxide generated in this process. A hydrogen peroxide electrode (using a hydrogen peroxide permeable membrane 5) is used.

In the present invention, as a method for stabilizing the enzymatic activity of pyruvate oxidase using the above mechanism, after measuring pyruvate (when not measuring),
Examples include a method in which a FAD solution is brought into contact with immobilized pyruvate oxidase. In this case, the usual method is to fill the pipe with FAD solution after the pyruvic acid measurement is completed and to clean the inside of the pipe, and to store it overnight in this state at room temperature. In addition, as another method, each time one measurement is completed,
An example of this method is to clean the inside of the pipe using a cleaning solution containing FAD, and then fill the pipe with the cleaning solution every time a day's measurement is completed and store the pipe in this state overnight at room temperature. Still another method is to supply the FAD solution into the pipe at a rate of 10 ^-9 to ^{10 -6} mol/min while pyruvic acid is being measured. In this case, there is an advantage that the measurement system can be cleaned and the POP activity can be prevented from decreasing at the same time. In the above measurement method, 0.1 to 50m
It is necessary to use a FAD solution of M or a washing solution containing FAD. The longer the contact time between these solutions and the immobilized pyruvate oxidase, the more preferable results will be obtained, and it is desirable to carry out the contact treatment on consecutive days rather than completing the contact treatment only once. Note that the above methods may be performed alone or in combination. By the way, the conventional low concentration of about 0.01mM
If only an FAD-containing buffer solution is used, the effects of the present invention cannot be achieved because FAD cannot be sufficiently diffused through the permeable membrane 5 and is discharged outside the measurement system.

Next, a specific example of the pyruvic acid measuring device will be shown and a method of using the device will be explained. An example of the pyruvate measuring device of the present invention, in which the enzymatic activity of pyruvate oxidase is stabilized by bringing the FAD solution into contact with immobilized pyruvate oxidase during non-measurement periods, is shown in a block diagram in FIG. In the figure, 11
is a storage tank for buffer or substrate solution, 12 is a concentrated
FAD solution storage tank, 13 is automatic solvent changer, 1
4 is a sample injection port, 15 is an electrode section, 16 is a constant flow pump, 17 is an automatic three-way valve, 18a and 18b
1 is a pipe, 19 is a drain tank, 20 is a recorder, 21 is a controller, 22 is a stirrer, and 23 is an agitator.

In this measuring device, measurements are normally performed as follows. First, a measurement command is issued from the controller 21 (in actual operation, simply press the measurement start switch on the display panel of the controller 21), and the automatic solvent changer 13
Alternatively, after the valve F and A are connected and the automatic three-way valve 17 is connected to the pipe 18a, the constant flow pump 16 is operated to fill the measurement system with the buffer solution. When it is completely filled, the RUN display lights up on the display panel of the controller 21. The buffer solution is
It always flows at a constant flow rate during measurement. In this state, when an appropriate amount of sample liquid is injected from the sample injection port 14 (the arrow in the figure indicates the injection direction), pyruvic acid in the sample is detected by the electrode section 15, and the signal is sent to the recorder 20. recorded. usually,
It takes about 5 minutes from sample injection to recording on the recorder (at a flow rate of 0.6 ml/min). Incidentally, the waste liquid that has been detected or quantified is stored in the waste liquid tank 19 via the pipe 18a.

Next, after the measurement is completed, controller 2
When you press the measurement end switch on the display panel 1,
A command is issued from the controller 21, and any one of valves C, E, G, or I of the automatic solvent changer 13 is connected to A, and the buffer solution and sample solution filled in the system are all discharged. After a certain period of time (the time required for the buffer to be completely drained), the valve of the automatic solvent changer 13 is switched to D or H, and at the same time the automatic three-way valve 17 is also switched,
The valve 17 is connected to a pipe 18b. By the above operations, the measurement system is filled with a concentrated FAD solution, and the enzyme membrane of the electrode section 15 is prevented from being deactivated.

An example of the pyruvate measuring device of the present invention, in which the enzymatic activity of immobilized pyruvate oxidase is stabilized by containing FAD in the washing solution after measurement, is shown in a block diagram in FIG. In the figure, 12a
is a storage tank for a cleaning solution containing concentrated FAD, but the other configuration is almost the same as the device shown in FIG.

In this measuring device, measurements are normally performed as follows. After the measurement is completed and the buffer solution and sample solution filled in the system are discharged, the valve of the automatic solvent changer 13 is switched to D or H, and then the FAD-containing cleaning solution in the liquid storage tank 12a is transferred to the measuring device. Inactivation of the enzyme membrane can be prevented by flowing the enzyme into the membrane.

An example of the pyruvate measuring device of the present invention, in which the enzymatic activity of pyruvate oxidase is stabilized by bringing the FAD-containing washing solution into contact with immobilized pyruvate oxidase during measurement, is shown in a block diagram in FIG. In the figure, 12 is a storage tank for concentrated FAD solution, 24 is a micro-liquid pump, and 10 is a small inner diameter pipe, but the other configurations are almost the same as the apparatus shown in FIG.

In this measuring device, measurements are normally performed as follows. After the constant flow pump 16 is activated and the buffer solution and concentrated FAD solution begin to flow into the measurement system at a constant flow rate, when an appropriate amount of sample solution is injected from the sample injection port 14, the electrode section 15 Pyruvate is detected.

[Effects of the Invention] According to the present invention, the enzymatic activity of immobilized pyruvate oxidase can be stabilized, so it is possible to provide a long-life and stable pyruvate measuring device. Furthermore, when measuring pyruvic acid, the reproducibility of measured values is also significantly improved.

[Examples of the Invention] Example 1 Using the apparatus and reagents shown below, immobilized pyruvate oxidase was brought into contact with a FAD solution during non-measurement.

(A) Equipment (see Figure 2) (1) Automatic solvent changer: Model SV-5008A, manufactured by Gascro Industries Co., Ltd. (2) Automatic three-way valve: Model MPV-3A, manufactured by Gascro Industries Co., Ltd. (3) Controller: manufactured by Tokyo Shibaura Electric Co., Ltd. (equipped with a microcomputer) (B) Reagent Pyruvate oxidase manufactured by Toyo Jozo Co., Ltd. was used. Thiamine pyrophosphate and FAD
The one manufactured by Tokyo Kasei Co., Ltd. was used. Other reagents were commercially available products (special grade) and were used as they were without purification. In addition, collagen was prepared from cowhide, stored frozen, and thawed before use.
Note that ion-exchanged water was used throughout the entire operation.

(C) Preparation of buffer solution, POP-immobilized collagen membrane, electrode part, and sample solution As the buffer solution, 0.05M phosphate buffer (PH
7.5) containing 0.01mM FAD, 0.045mM manganese chloride, and 0.1mM thiamine pyrophosphate was used.

A POP-immobilized collagen membrane was prepared as follows. 0.6% collagen suspension (PH 4.0)
After stirring 10g well, POP (21units/mg)
After mixing 100 mg and stirring lightly, defoaming was performed for 1 minute using a vacuum pump. Next, the obtained suspension was spread on a Teflon (registered trademark) plate (4 cm x 5 cm) and air-dried at 28°C for 3 hours. Next, the membrane peeled from the Teflon plate was cut into 1 cm x 1 cm pieces, and this was cross-linked in a gas phase at 28°C for 10 minutes using a 0.1% glutaraldehyde aqueous solution (PH 8.0) to form a POP-immobilized collagen membrane. Prepared.

The electrode part is a polarographic hydrogen peroxide electrode consisting of a platinum electrode covered with a hydrogen peroxide permeable membrane, and the sensitive surface is covered with a POP-immobilized collagen membrane (thickness:
coated with about 60 μm, enzyme activity: about 100 IU/cm ² ),
Furthermore, the membrane was covered with a dense layer of cellulose acetate ultrafiltration membrane (thickness: about 48 μm). This electrode part was attached to a plastic flow cell, and this was incorporated into a measurement system.

The sample solution used was the above buffer solution to which 0.5 mM sodium pyruvate was added.

(D) Operation After turning on the measurement start switch on the display panel of the controller and filling the system with buffer, the syringe pump was turned on and 200 μl of the sample solution was injected. The reaction temperature at the time of measurement was 37°C, and the flow rate was 0.6 ml/min. Next, hydrogen peroxide produced by the reaction was detected with the electrode section, and the value was recorded with a recorder. Measurements were performed approximately 30 times a day. After the measurement was completed, the measurement end switch on the display panel of the controller was turned on, the buffer solution in the system was discharged, the system was filled with 1mM FAD solution, and the system was stored in this state overnight at room temperature. The next day, after turning on the measurement start switch on the controller and collecting the FAD solution in the system into the storage tank, the buffer solution was allowed to flow into the system again and the same operation as above was performed.

The above operation was repeated for 5 days. The obtained electrode response values are shown in A of FIG. As is clear from the figure, almost no decrease in response value was observed. Furthermore, the deviation of daily response values was within 5%. Although not shown in the figure, when the above measurements were carried out for one month, the electrode response value after one month was approximately 90% of the initial response value.

Example 2 Measurement was carried out in the same manner as in Example 1, except that a 0.1 mM FAD solution was used. The electrode response value after one week was approximately 90% of the initial response value.

Example 3 Glutamate-oxaloacetate transaminase (GOT) converts α-ketoglutarate to oxaloacetate, and oxaloacetate decarboxylase (OAC) degrades this oxaloacetate to pyruvate. Therefore, an OAC-immobilized column was prepared by immobilizing OAC on aminoalkyl porous glass beads and packing the beads into a glass column (diameter 3 mm, length 50 mm). Insert this column between the sample injection port and the electrode section of the device shown in Figure 2.
The enzymatic activity of GOT was measured repeatedly.

In this example, 0.05M was used instead of the buffer solution.
0.01mM FAD in phosphate buffer (PH7.5),
0.045mM manganese chloride, 0.1mM thiamine pyrophosphate, 2.1mM α-ketoglutarate and 30mM
A substrate solution containing L-alanine was used and stored in a liquid storage tank.

In addition, as a sample solution, in the above substrate solution,
10, 50, 100, and 500 units/L GOT, 0.9%
A solution containing NaCl and 1% albumin was used. When GOT activity was measured for one month in the same manner as in Example 1, the electrode response value after one month was about 80% of the initial response value. Furthermore, the deviation of daily response values was within 5%.

Comparative Example 1 Instead of the FAD solution used in Example 1
Measurement was carried out in the same manner as in Example 1, except that a buffer containing 0.01 mM FAD was filled into the system during non-measurement periods. The measurement results are shown as B in FIG.
As is clear from the figure, the electrode response value gradually decreased from the 3rd day after the start of the measurement, and after 5 days only showed a value of about 30% of the initial response value.

Comparative Example 2 The same results as in Comparative Example 1 were obtained when measurements were carried out in the same manner as in Example 1, except that the 0.05 mM FAD solution was filled into the system during non-measurement periods.

Example 4 Using the apparatus and reagents shown below, a washing solution containing FAD was brought into contact with immobilized pyruvate oxidase after measurement.

(A) Equipment (see Figure 3) (1) Automatic solvent changer: Model SV-5008A, manufactured by Gascro Kogyo Co., Ltd. (2) Constant flow pump: Roller pump, manufactured by Flue Scientific Co., Ltd. (3) Controller, Sample injector: Homemade (B) Reagent The same one as in Example 1 was used.

(C) Preparation of buffer solution, POP-immobilized collagen membrane, electrode part, and sample solution The same buffer solution and POP-immobilized collagen membrane as in Example 1 were used. The same electrode part as in Example 1 was used except that an oxygen permeable membrane was used instead of the hydrogen peroxide permeable membrane. In addition, as a sample solution,
Whole blood supplemented with 0.5 mM sodium pyruvate and whole blood supplemented with an appropriate amount of GPT were used.

(D) Operation The same procedure as in Example 1 was performed except that the enzyme activity was measured by measuring the amount of dissolved oxygen that decreased as a result of the enzyme reaction. Each time a measurement was completed, the inside of the system was washed with a washing solution containing 0.5 mM FAD (0.01% Triton solution), and then the buffer solution was allowed to flow down into the piping, and the next measurement was performed. Also 1
After the day's measurements were completed, the system was filled with a cleaning solution and stored in this state overnight at room temperature.

The above operation was repeated for 5 days. The obtained electrode response values are shown as A in FIG. As is clear from the figure, almost no decrease in response value was observed. Furthermore, the deviation of daily response values was within 5%. When the above measurements were carried out for one month, the electrode response value after one month was approximately 90% of the initial response value.
It was hot.

Example 5 Measurements were carried out in the same manner as in Example 4, except that a 0.1 mM FAD washing solution was used. The electrode response value after 5 days of measurement was about 80% of the initial response value.

Example 6 Alanine Ketoglutarate Aminotransferase (GPT) is an enzyme that converts L-alanine to pyruvate. Therefore, in this example, instead of the buffer, 0.01mM FAD, 0.045mM manganese chloride, 0.1
A substrate solution containing mM thiamine pyrophosphate, 0.02M α-ketoglutaric acid, and 0.3M L-alanine was used and stored in a reservoir to measure GPT activity in whole blood.

In addition, as a sample solution, add 10% to the above substrate solution.
Whole blood containing 50, 100, 200 and 500 units/L of GPT was used.

When GPT activity was measured in the same manner as in Example 4, there was a linear relationship between GPT activity and electrode response value within the above range. Therefore, 100 units/L
When measurements were taken for one month using whole blood containing GPT, the electrode response value after one month was approximately the initial response value.
It was 75%. Also, the deviation of the daily response value is 5%.
It was within

Comparative Example 3 Measurement was carried out in the same manner as in Example 4, except that the FAD concentration in the washing solution was 0.01 mM. The measurement results are shown as B in FIG. As is clear from the figure, the electrode response value gradually decreased from the 3rd day after the start of the measurement, and after 5 days only showed a value of about 25% of the initial response value.

Comparative Example 4 Measurements were carried out in the same manner as in Example 4, except that the FAD concentration in the washing solution was 0.05mM, and the same results as in Comparative Example 3 were obtained, and the electrode response value after 5 days was about the same as the initial value. decreased to 30%.

Example 7 Immobilized pyruvate oxidase was brought into contact with a flavin adenine dinucleotide solution during measurement using the apparatus and reagents shown below.

(A) Equipment (see Figure 4) Micro-liquid pump: Manufactured by Shimadzu Corporation, LC-5A (B) Reagent The same material as in Example 1 was used.

(C) Preparation of buffer solution, POP-immobilized collagen membrane, electrode part, and sample solution The same buffer solution and POP-immobilized collagen membrane as in Example 1 were used. Example 1 except that an oxygen permeable Teflon membrane was used instead of the hydrogen peroxide permeable membrane as the electrode part.
I used something similar. Further, the same sample solution as in Example 1 was used, and 100 microns of each solution was injected from the sample injection port 4 with a microsyringe.

(D) Operation First, constant flow pump (flow rate: 0.6ml/min)
Then, the micro-liquid pump (flow rate: 10 μ/min) was started, and the buffer solution and 1 mM FAD solution were injected into the system. After confirming on the recorder that the response of the electrode part was stable, 100 μ of the above sample solution was injected from the sample injection port. After one day's measurements were completed, each device was stopped and stored overnight in this state at room temperature.

Measurements were taken at least 30 times a day, and the above operations were repeated for 7 days. The obtained electrode response values are shown as A in FIG. As is clear from the figure, almost no decrease in response value was observed. Furthermore, the deviation of daily response values was within 5%. In addition,
When the above measurements were carried out for one month, the electrode response value after one month was about 90% of the initial response value.

Example 8 Measurement was carried out in the same manner as in Example 7 except that 0.1 mM FAD solution (flow rate: 10 mol/min) was used instead of 1 mM FAD solution. After repeated measurements for 5 days, the electrode response value was approximately 80% of the initial value.
It was hot. Furthermore, the deviation of daily response values was within 10%.

Example 9 In the same manner as in Example 3, OAC was immobilized on aminoalkyl porous glass beads, which were packed into a glass column (diameter 3 mm, length 50 mm), and OAC
An immobilized column was prepared. Insert this column between the sample injection port and the electrode section of the device shown in Figure 4.
The enzymatic properties of GOT were repeatedly measured.

In this example, 0.05M was used instead of the buffer solution.
0.01mM FAD in phosphate buffer (PH7.5),
0.045mM manganese chloride, 0.1mM thiamine pyrophosphate, 2.1mM α-ketoglutarate and 30
A substrate solution containing mM L-alanine was used and stored in a storage tank.

In addition, as a sample solution, 10,
50, 100, and 500 units/L GOT and additional 0.9%
A solution containing NaCl and 1% albumin was used. GOT activity was measured for one month in the same manner as in Example 7, and the electrode response value after one month was approximately 75% of the initial response value. Furthermore, the deviation of daily response values was within 8%.

Comparative Example 5 Measurement was carried out in the same manner as in Example 7, except that the micro-liquid pump was not operated. The measurement results are indicated by B in FIG. As is clear from the figure,
The electrode response value gradually decreased from the third day after the start of the measurement, and after one week, the electrode response value showed only about 25% of the initial response value.

Comparative Example 6 Measurement was carried out in the same manner as in Example 7, except that 0.05mM FAD solution (flow rate: 5 × 10 ^-10 mol/min) was used instead of 1mM FAD solution, and the same results as in Comparative Example 5 were obtained. The results were obtained.

[Brief explanation of drawings]

Figure 1 is a partial cross-sectional view of the electrode part in the device of the present invention, and Figure 2 is a POP that immobilizes the FAD solution when not measuring.
FIG. 3 is a block diagram of the device of the present invention equipped with a mechanism for bringing the FAD-containing washing solution into contact with the immobilized POP when not in measurement, and FIG. Figures 5 to 7 are block diagrams of the device of the present invention equipped with a mechanism for bringing POP into contact with immobilized POP, and Figures 5 to 7 show the results of measurements in the example and comparative example using the devices shown in Figures 2 to 4, respectively. It is a relationship diagram between the number of measurement days and electrode response values. 1... Flow cell, 2... Ultrafiltration membrane, 3...
Buffer layer, 4... O-ring, 5... Oxygen or hydrogen peroxide permeable membrane, 6... POP immobilization membrane, 7...
...Platinum cathode, 8...Lead anode, 9...Piping, 10...
...Small inner diameter piping, 11...Liquid storage tank for buffer solution or substrate solution, 12...Liquid storage tank for concentrated FAD solution, 13...
...Automatic solvent changer, 14...Sample injection port, 15...
... Electrode part, 16 ... Constant flow pump, 17 ... Automatic three-way valve, 18a and 18b ... Piping, 19 ...
... Drainage tank, 20 ... Recorder, 21 ... Controller, 22 ... Stirrer, 23 ... Stirrer, 2
4...Minor liquid feed pump.

Claims (1)

[Scope of Claims] 1. A pyruvate measuring device using immobilized pyruvate oxidase, characterized in that it is equipped with a mechanism for bringing a 0.1 to 50 mM flavin adenine dinucleotide solution into contact with the immobilized pyruvate oxidase. Acid measuring device. 2. The pyruvate measuring device according to claim 1, comprising a mechanism for bringing the flavin adenine dinucleotide solution into contact with the immobilized pyruvate oxidase during non-measurement. 3. The pyruvate measuring device according to claim 1, comprising a mechanism for bringing the washing solution containing flavin adenine dinucleotide into contact with the immobilized pyruvate oxidase after the measurement is completed. 4. The pyruvate measuring device according to claim 1, comprising a mechanism for bringing the flavin adenine dinucleotide solution into contact with the immobilized pyruvate oxidase at a supply rate of 10 ^-9 to ^{10 -6} mol/min during measurement.