Glucose uptake suppressor against red blood cells, inhibitor for reducing glucose concentration in blood collection tube, and blood collection tube provided with same

Abstract

The purpose of the present invention is to provide: a glucose uptake suppressor for red blood cells; and a blood collection tube for suppressing a decrease in glucose concentration. According to the present invention, it is possible to provide a glucose uptake suppressor for red blood cells, which contains inosine as an active ingredient. The repressor according to the present invention can be used to suppress a decrease in the glucose concentration of whole blood collected into a blood collection tube. According to the present invention, it is also possible to provide a blood collection tube having inosine in the internal space. The blood collection tube can be used for measuring the concentration of glucose in blood. The blood collection tube can further comprise a glycolytic system suppressor.

Description

[0001] REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the priority benefit of Japanese Patent Application No. 2022-174451 (filing date: October 31, 2022) as a prior application, and the entire disclosure thereof is incorporated herein by reference as part of this specification. TECHNICAL FIELD

[0003] The present invention relates to an inhibitor of glucose uptake by red blood cells. The present invention also relates to an inhibitor of glucose concentration reduction in a blood collection tube and a blood collection tube having the inhibitor of glucose concentration reduction. BACKGROUND ART

[0004] Blood glucose level is an essential item for the diagnosis criteria of diabetes and gestational diabetes (glucose 126 mg / dL or more), and its measurement requires extremely high precision and accuracy. The Japanese Society of Clinical Chemistry stipulates that the allowable variation range of blood glucose level is ±4 mg / dL or less. If the blood after blood collection is stored at room temperature in a blood collection tube in the state of whole blood, it may cause a decrease in glucose concentration. One of the reasons is that glucose is taken up by red blood cells in the blood, and glucose is decomposed by the enzymes of the glycolysis system in red blood cells, and over time, the glucose concentration in the blood decreases.

[0005] After blood collection, immediately centrifuging the blood to separate red blood cells and supernatant, and measuring the blood glucose level of the supernatant can avoid the decrease in glucose concentration as described above. However, when blood collection is performed by a self-employed doctor or at a clinic site, the time from blood collection to arrival at a clinical examination facility often takes up to about 12 hours. Since there are almost no centrifuges in the clinics of self-employed doctors or at clinic sites, when blood collection is performed at these facilities, the whole blood after blood collection is stored in a blood collection tube in the state of whole blood for up to about 12 hours from blood collection to centrifugation, and the decrease in glucose concentration cannot be avoided. Therefore, when blood collection is performed by a self-employed doctor or at a clinic site, there is a possibility of misdiagnosis of diabetes.

[0006] To date, a blood collection tube having sodium fluoride as a glycolytic inhibitor in the tube has been put into practical use, and further, a blood collection tube having adenosine triphosphate (ATP) as a glycolytic inhibitor in the tube has been proposed (Patent Document 1).

[0007] On the other hand, since sodium fluoride cannot sufficiently inhibit the decrease in glucose concentration over time until 4 hours in the initial stage, as a countermeasure, it is recommended in the guidelines of the American Association for Clinical Chemistry to immerse it in an ice slurry immediately after blood collection, and when plasma separation cannot be achieved within 30 minutes, a citrate buffer solution is used (Non-Patent Document 1). However, HbA1c measurement cannot be performed in a blood collection tube containing a citrate buffer solution, and it is hardly used in Japan from the viewpoints of efficiency and economy.

[0008] Prior Art Documents

[0009] Patent Documents

[0010] Patent Document 1: Japanese Unexamined Patent Application Publication No. 2019-2821

[0011] Non-Patent Documents

[0012] Non-Patent Document 1: David B. Sacks et al., Guidelines and Recommendations for Laboratory Analysis in the Diagnosis and Management of Diabetes Mellitus, Clinical Chemistry 57(6); e1 - e7 (2011) Summary of the Invention

[0013] An object of the present invention is to provide an inhibitor of glucose uptake into red blood cells. Another object of the present invention is to provide an inhibitor of the decrease in glucose concentration in a blood collection tube. Another object of the present invention is to provide a blood collection tube that inhibits the decrease in glucose concentration of the collected blood.

[0014] The present inventors have found that inosine inhibits glucose uptake into red blood cells, the decrease in glucose concentration of whole blood can be avoided by incorporating inosine into a blood collection tube, and HbA1c, insulin, C-peptide, etc. can be measured using a blood collection tube incorporated with inosine. The present invention has been completed based on these findings.

[0015] According to the present invention, the following inventions can be provided.

[0016] [1] An inhibitor of glucose uptake into red blood cells, which contains inosine as an active ingredient.

[0017] [2] An inhibitor of the decrease in glucose concentration in a blood collection tube, which contains inosine as an active ingredient.

[0018] [3] The inhibitor of the decrease in glucose concentration as described in [2] above, which further contains a glycolysis system inhibitor.

[0019] [4] The glucose concentration reducing inhibitor as described in [3] above, wherein the glycolysis system inhibitor is one or more selected from the group consisting of fluorides, and adenosine phosphates or salts thereof.

[0020] [5] The glucose concentration reducing inhibitor as described in any one of [2] to [4] above, which further contains an anticoagulant.

[0021] [6] The glucose concentration reducing inhibitor as described in [5] above, wherein the anticoagulant is one or more selected from the group consisting of: ethylenediaminetetraacetic acid (EDTA), ethylenediaminetetraacetate and their hydrates; citric acid and its salts; and heparin and its salts.

[0022] [7] The glucose concentration reducing inhibitor as described in any one of [2] to [6] above, wherein 0.10 mg or more of inosine is used in every 1 mL of the collected blood.

[0023] [8] A blood collection tube for measuring the glucose concentration in blood, which is a blood collection tube having the glucose concentration reducing inhibitor as described in any one of [2] to [7] above in its internal space.

[0024] [9] The blood collection tube as described in [8] above, wherein in every 1 mL of the collected blood, the glucose concentration reducing inhibitor contains 0.10 mg or more of inosine.

[0025]

[10] The blood collection tube as described in [8] or [9] above, which is also used for measuring one or more selected from the group consisting of HbA1c, insulin and C-peptide.

[0026]

[11] A method for inhibiting glucose uptake by red blood cells in whole blood or a method for inhibiting the reduction of glucose concentration in whole blood, which includes mixing whole blood with inosine in vitro.

[0027]

[12] The method as described in

[11] above, which further includes mixing a glycolysis system inhibitor and / or an anticoagulant with whole blood.

[0028]

[13] The method as described in

[11] or

[12] above, wherein 0.10 mg or more of inosine is mixed in every 1 mL of whole blood.

[0029]

[14] The method as described in

[11] or

[12] above, which is implemented in a blood collection tube.

[0030] According to the present invention, a blood collection tube can be provided which can avoid a decrease in glucose concentration even when whole blood is stored in the blood collection tube for a long time. When measuring the glucose concentration using a conventional blood collection tube containing sodium fluoride and ethylenediaminetetraacetic acid (EDTA) in the tube, the glucose concentration in the blood tends to decrease even after about 4 hours. Therefore, for example, even if a person has diabetes, it may sometimes be judged negative (false negative). According to the present invention, accurate blood glucose value measurement can be performed regardless of the blood collection facility, which is advantageous in helping to detect diabetes at an early stage and avoid missed diagnoses of diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Figure 1 FIG. is a graph showing the relationship between the change in glucose concentration after 12 hours and the hematocrit value of whole blood stored in a blood collection tube. (A) shows the case of using blood collection tube C, and (B) shows the case of using blood collection tube K.

[0032] Figure 2 Figure 2 FIG. is a graph showing the relationship between the change in glucose concentration after 12 hours and the hematocrit value of whole blood stored in a blood collection tube. (A) shows the case of using blood collection tube I, and (B) shows the case of using blood collection tube J.

[0033] Figure 3 Figure 3 FIG. is a graph showing the change in insulin concentration after 24 hours of whole blood stored in a blood collection tube. (A) shows the case of using a conventional blood collection tube (blood collection tube C), and (B) shows the case of using the blood collection tube of the present invention (blood collection tube K) containing inosine.

[0034] Figure 4 Figure 4 FIG. is a graph showing the change in c-peptide concentration after 24 hours of whole blood stored in a blood collection tube. (A) shows the case of using a conventional blood collection tube (blood collection tube C), and (B) shows the case of using the blood collection tube of the present invention (blood collection tube K) containing inosine. DETAILED DESCRIPTION OF THE INVENTION

[0035] <<Glucose uptake inhibitor for red blood cells>>

[0036] According to the present invention, an inhibitor for glucose uptake by red blood cells can be provided, which contains inosine as an active ingredient. Glucose transporter protein (GLUT1) exists on the cell membrane of red blood cells, which is responsible for the uptake of glucose into red blood cells. It is considered that the inhibitor of the present invention acts on GLUT1 and inhibits glucose uptake based on GLUT1. That is, according to the present invention, an inhibitor of glucose transport based on GLUT1 can be provided.

[0037] ​​​​​​​​According to another aspect of the present invention, a method for inhibiting glucose uptake by red blood cells in whole blood can be provided, which includes mixing whole blood with inosine in vitro. In addition to inosine, a glycolysis system inhibitor and / or an anticoagulant can also be mixed in the whole blood. Mixing whole blood with inosine means including a method of collecting whole blood into a blood collection tube containing inosine inside and turning it over for mixing. That is, the method for inhibiting glucose uptake of the present invention can be implemented in a blood collection tube. The method for inhibiting glucose uptake of the present invention can be implemented according to the description of the inhibitor of the present invention.

[0038] <<Inhibitor for reducing glucose concentration in blood collection tube>>

[0039] The inhibitor of the present invention can inhibit glucose uptake into red blood cells, and thus can prevent the decomposition of glucose caused by the enzymes of the glycolysis system present in red blood cells. Therefore, the inhibitor of the present invention can be used to inhibit the decrease in glucose concentration of whole blood stored in a blood collection tube. That is, according to the present invention, an inhibitor for reducing glucose concentration in a blood collection tube can be provided, which uses inosine as an active ingredient.

[0040] The lower limit value of the usage amount of inosine can be set to 0.10 mg, 0.125 mg, 0.25 mg, 0.375 mg, 0.50 mg, 0.75 mg, 1.0 mg, 1.25 mg or 1.5 mg relative to 1 mL of the collected blood, and the upper limit value of the usage amount of inosine can be set to 5.0 mg, 4.0 mg, 3.0 mg relative to 1 mL of the collected blood. The range of the usage amount of inosine relative to 1 mL of the collected blood can be set by combining the above-mentioned lower limit value and upper limit value, for example, it can be set to 0.10 - 5.0 mg, 0.125 - 5.0 mg, 0.10 - 4.0 mg or 0.125 - 4.0 mg.

[0041] The inhibitor for reducing glucose concentration of the present invention can use inosine alone or in combination with a glycolysis system inhibitor. As the glycolysis system inhibitor, fluorides, adenosine phosphates or their salts can be mentioned. By using inosine in combination with a glycolysis system inhibitor (especially fluorides), the effect of inhibiting the decrease in glucose concentration can be exerted more strongly.

[0042] There is no particular limitation on the fluoride, for example, sodium fluoride and potassium fluoride can be mentioned, and sodium fluoride is preferred. They can be used alone or in combination of two or more. Sodium fluoride exerts an effect of preventing the decomposition of glucose in the blood collection tube by inhibiting enolase in the glycolysis system.

[0043] When using a fluoride salt as a glucose concentration reducing inhibitor in a blood collection tube, relative to 1 mL of the collected blood, it is preferable to use the fluoride salt in the range of 0.2 to 3.0 mg, more preferably in the range of 1.0 to 2.5 mg, and even more preferably in the range of 1.5 to 2.0 mg.

[0044] As the adenosine phosphate and its salts of the present invention, ATP (adenosine triphosphate), ADP (adenosine diphosphate), AMP (adenosine monophosphate) and their salts can be cited, and ATP and its salts are preferred. They can be used alone or in combination of two or more.

[0045] As salts of adenosine phosphate, for example, alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as calcium salt and magnesium salt, etc. can be cited, alkali metal salts are preferred, and sodium salt is more preferred. As a salt of ATP, disodium salt is preferred.

[0046] When using adenosine phosphate or its salt as a glucose concentration reducing inhibitor in a blood collection tube, relative to 1 mL of the collected blood, it is preferable to use adenosine phosphate or its salt in the range of 0.1 to 1000 mg, more preferably in the range of 0.5 to 200 mg, and even more preferably in the range of 1 to 100 mg.

[0047] The glucose concentration reducing inhibitor of the present invention can be used in combination with an anticoagulant. As the anticoagulant, the following can be cited: ethylenediaminetetraacetic acid (EDTA) or its salt or their hydrates; citric acid or its salt (for example, an alkali metal salt of citric acid such as sodium citrate); heparin or its salt (for example, an alkali metal salt of heparin such as sodium heparin).

[0048] As the salt of EDTA, there is no particular limitation, and alkali metal salts of EDTA (for example, disodium EDTA and dipotassium EDTA) can be cited. EDTA and its salts and their hydrates can be used alone or in combination of two or more.

[0049] When using EDTA or its salt or their hydrates as an anticoagulant for blood, relative to 1 mL of the collected blood, it is preferable to use anhydrous EDTA in the range of 0.5 to 2.5 mg, more preferably in the range of 0.75 to 2.25 mg, and even more preferably in the range of 1 to 2 mg or 1.2 to 2 mg.

[0050] One of the purposes of the glucose concentration reducing inhibitor of the present invention is for application to a blood collection tube. Therefore, in addition to inosine, it may also contain a glycolysis system inhibitor and / or an anticoagulant, or it may be a combination of inosine and a glycolysis system inhibitor and / or an anticoagulant.

[0051] According to another aspect of the present invention, a method for inhibiting a decrease in glucose concentration in whole blood can be provided, which includes mixing whole blood with inosine in vitro. In addition to inosine, a glycolysis system inhibitor and / or an anticoagulant can also be mixed in the whole blood. Mixing whole blood with inosine means including a method of collecting whole blood into a blood collection tube having inosine inside and turning it over for mixing. That is, the method for inhibiting a decrease in glucose concentration of the present invention can be implemented in a blood collection tube. The method for inhibiting a decrease in glucose concentration of the present invention can be implemented according to the description of the glucose concentration decreasing inhibitor of the present invention.

[0052] <<Blood collection tube>>

[0053] The blood collection tube of the present invention includes: a cylindrical body having an upper end with an opening and a lower end with a bottom in the longitudinal direction; and a tube stopper that closes the opening at the upper end. In addition, the internal space of the cylindrical body contains the inhibitor of the present invention or the glucose concentration decreasing inhibitor of the present invention. That is, the blood collection tube of the present invention contains at least inosine in the internal space of the cylindrical body.

[0054] The upper end with an opening and the lower end with a bottom refer to both end portions in the longitudinal direction of the cylindrical body. When using the blood collection tube, the upper end is located higher than the lower end relative to the ground, blood is collected from the opening, and the lower end is located lower than the upper end relative to the ground, and the collected blood is received in a bottomed manner. In addition, it is preferable that the cross section of the cylindrical body is annular. The cross section being annular means that the cross section only needs to be circular or substantially circular.

[0055] The material of the cylindrical body is not particularly limited, and examples thereof include plastics such as glass and polyethylene terephthalate. In order to visually recognize the internal state, a colorless and transparent material is preferred.

[0056] The tube stopper that closes the opening at the upper end of the cylindrical body is not particularly limited, and examples thereof include a rubber stopper and a membrane stopper. The central portion of the tube stopper can be set thinner than the portion other than the central portion of the tube stopper so that a blood collection needle can be easily and safely punctured into the tube stopper. In addition, it is preferable that in the blood collection tube, the tube stopper closes the opening at the upper end of the cylindrical body and decompresses the internal space of the cylindrical body. By decompressing the internal space of the cylindrical body, it has a tendency to make blood collection into the blood collection tube easier. The degree of decompression can be appropriately set according to the blood collection volume and the sealing degree of the tube stopper for the internal space of the cylindrical body.

[0057] In the present invention, when the position of the internal space of the cylindrical body containing additives such as inosine is on the lower end side relative to the upper end of the cylindrical body, it is easy to come into contact with the collected blood, so it is preferred.

[0058] In order to inhibit glycolysis of red blood cells in the collected blood and / or to prevent hemolysis of the collected blood (which sometimes occurs when the pH is below 4), in addition to the inhibitor of the present invention or the glucose concentration reducing inhibitor of the present invention, the blood collection tube of the present invention may further contain a pH regulator.

[0059] The pH regulator is not particularly limited, and examples thereof include citric acid, succinic acid, and their salts. Depending on the purpose, they may be used alone or in combination of two or more.

[0060] In the present invention, additives such as inosine can be accommodated in the internal space of the blood collection tube in any form such as granules, powders, sheets, tablets, etc., or can be spray-coated on the inner wall surface of the blood collection tube and dried to be fixed on the wall surface. Regarding additives such as inosine, two or more kinds can be made into a mixture and accommodated in the internal space of the blood collection tube, or can be separately accommodated in the internal space of the blood collection tube.

[0061] As shown in the following examples, HbAlc was measured for blood samples collected using the blood collection tube of the present invention. As a result, the blood collection tube of the present invention did not affect the measurement results. In addition, insulin and c-peptide were measured for blood samples collected using the blood collection tube of the present invention. As a result, a certain decrease was confirmed compared to the serum immediately after blood collection. However, since a highly correlated relationship was confirmed, it is considered that it can be fully used clinically as long as a coefficient is used. That is, the blood collection tube of the present invention can be used for the measurement of all four items of glucose, HbAlc, insulin, and c-peptide. Therefore, the present invention is an advantageous invention in terms of helping to improve inspection efficiency and reduce costs.

[0062] Examples

[0063] The present invention will be described more specifically based on the following examples, but the present invention is not limited to these examples.

[0064] Blood collection tube

[0065] Commercially available blood collection tubes (blood collection tubes A, B, C, D) used in the examples are as follows.

[0066] [Table 1]

[0067] Table 1: Overview of blood collection tubes used in the examples (1)

[0068] Type of blood collection tube Common name Content 1 Content 2 Blood collection tube A For biochemistry: High-speed coagulation · Contains separator Thrombin Separator Blood collection tube B For complete blood count (EDTA-2k) EDTA-2k (4.5 mg) Blood collection tube C For blood glucose EDTA-2Na (8.2 mg) NaF (3.0 mg) Blood collection tube D For hematological examination (EDTA--2Na) EDTA-2Na

[0069] Blood collection tube A: InsepackII-D for biochemical and serological examinations, high-speed coagulation, blood collection tube with separator SIM-L1008SQ3, Tokuyama Sekisui Kogyo Co., Ltd.

[0070] Blood collection tube B: Nipro Neotube A EDTA-2k (granular) NP-EK0205, Nipro Corporation

[0071] Blood collection tube C: Nipro Neotube A sodium fluoride + EDTA-2Na (granular) OP-FN0205, Nipro Corporation

[0072] Blood collection tube D: Insepack II 5 mL blood collection tube for hematological examination SMD750ENA, Tokuyama Sekisui Kogyo Co., Ltd.

[0073] Blood collection tubes made from commercially available blood collection tubes are as follows.

[0074] [Table 2]

[0075] Table 2: Overview of blood collection tubes used in the examples (2)

[0076] Type of blood collection tube Basic blood collection tube Additive 1 Additive 2 Blood collection tube E Blood collection tube B Inosine 1.0 mg Blood collection tube F Blood collection tube B Inosine 2.0 mg Blood collection tube G Blood collection tube B Inosine 3.0 mg Blood collection tube H Blood collection tube C ATP 5.0 mg Blood collection tube I Blood collection tube C ATP 15.0 mg Blood collection tube J Blood collection tube C ATP 20.0 mg Blood collection tube K Blood collection tube C ATP 5.0 mg Inosine 2 mg Blood collection tube L Blood collection tube C Inosine 0.75 mg Blood collection tube M Blood collection tube C Inosine 1.50 mg Blood collection tube N Blood collection tube C Inosine 2.25 mg Blood collection tube O Blood collection tube C Inosine 3.00 mg Blood collection tube P Blood collection tube C Inosine 3.75 mg Blood collection tube Q Blood collection tube C Inosine 4.50 mg Blood collection tube R Blood collection tube C ATP 5.0 mg Inosine 1.50 mg Blood collection tube S Blood collection tube C ATP 5.0 mg Inosine 3.00 mg Blood collection tube T Polystyrene Spitz ※1 Part of the plasma is discarded Blood collection tube U Blood collection tube C ※1 Part of the plasma is discarded Blood collection tube V1 Blood collection tube C ※1 Part of the plasma is discarded Inosine 0.25 Blood collection tube V2 Blood collection tube C ※1 Part of the plasma is discarded Inosine 0.50 Blood collection tube V3 Blood collection tube C ※1 Part of the plasma is discarded Inosine 0.75 Blood collection tube V4 Blood collection tube C ※1 Part of the plasma is discarded Inosine 1.00 Blood collection tube V5 Blood collection tube C ※1 Part of the plasma is discarded Inosine 1.25

[0077] ※1: Blood after discarding 0.85 mL of plasma from 5 mL of blood collected using blood collection tube D.

[0078] After preparing each additive in a solution state, immerse it in filter paper and dry it in a refrigerator (4°C) for 48 hours. Add it to the blood collection tube in this state to prepare the blood collection tubes in Table 2. Specifically, prepare ATP (product number 309-50513, Fujifilm Wako Pure Chemical Corporation) into a 200 mg / mL solution, attach 25 μL (equivalent to 5 mg of ATP) to filter paper cut into a diameter of 6 mm (qualitative filter paper No. 131, Advantech Corporation) and dry it, and put the required number of sheets into the blood collection tube. Prepare inosine (product number 099-00231, Fujifilm Wako Pure Chemical Corporation) into a 10 mg / mL:10 mg / mL solution, attach 25 μL (equivalent to 0.25 mg of inosine) to filter paper cut into a diameter of 6 mm (qualitative filter paper No. 131, Advantech Corporation) and dry it, and put the required number of sheets into the blood collection tube.

[0079] Experimental method

[0080] In the following examples, the determination of blood glucose (glucose concentration) uses an automatic glucose analyzer (Adams glucose GA-1153, GOD electrode method, Arkray Inc.).

[0081] Regarding serum, within 5 minutes after blood collection, centrifuge at 3000 rpm for 5 minutes, transfer the supernatant (serum) to another test tube and use it as a specimen, and measure the glucose concentration. Regarding blood collection tubes other than serum, centrifuge at 3000 rpm for 5 minutes to separate the blood cells and the supernatant (plasma), and use the separated supernatant (plasma) for the measurement of glucose concentration.

[0082] Example 1: Effect of inosine on reducing the measured value of blood glucose concentration in the blood collection tube

[0083] (1) Method

[0084] Collect blood from 10 volunteer subjects into the following various blood collection tubes. After blood collection, keep it in the whole blood state (i.e., not centrifuged) at room temperature for up to 4 hours, and then keep it at 4°C for up to 24 hours. Use blood collection tubes C, I, J, and K 12 hours after blood collection to measure the glucose concentration (mg / dL), and use this measured value as the glucose concentration of the blood collection tube.

[0085] In addition, for the whole blood state (i.e., not centrifuged) blood collected using blood collection tube B (for complete blood count) within 4 hours after blood collection, use a multi-item automatic blood cell analyzer XN-1000 (Sysmex Corporation) to measure the hematocrit value.

[0086] Regarding serum, keep the supernatant (serum) of the blood collected from 10 volunteer subjects at room temperature for up to 4 hours, and then keep it at 4°C for up to 24 hours. Measure the glucose concentration (mg / dL) up to 24 hours after blood collection, and use this measured value as the serum glucose concentration at each time.

[0087] (2) Results

[0088] The value obtained by subtracting the serum glucose concentration from the glucose concentration of the blood collection tube is shown for each subject in Figure 1 in (A) (blood collection tube C), Figure 1 in (B) (blood collection tube K), Figure 2 in (A) (blood collection tube I) and Figure 2(B) (blood collection tube J) in it. In the conventional blood collection tube C containing sodium fluoride as a glycolysis inhibitor, even when the hematocrit value is within the reference range (40.7 - 50.1%), due to storage, the glucose concentration decreases beyond the allowable variation range (±4 mg / dL) for blood glucose measurement. Four out of five such cases were confirmed. In blood collection tubes I and J supplemented with ATP, even when the hematocrit value is within the reference range (40.7 - 50.1%), due to storage, the glucose concentration decreases beyond the allowable variation range (±4 mg / dL) for blood glucose measurement. Three out of five such cases were confirmed for ATP at 15 mg and one out of five such cases was confirmed for ATP at 20 mg. On the other hand, in blood collection tube K where the amount of ATP was reduced to 5 mg and 2 mg of inosine was added, cases where the hematocrit value exceeded the reference range (40.7 - 50.1%) and the glucose concentration decreased beyond the allowable variation range (±4 mg / dL) were confirmed. However, no cases where the glucose concentration exceeded the allowable variation range for measurement were confirmed in five cases where the hematocrit value was within the reference range.

[0089] Example 2: Effect of various components on reducing the glucose concentration in the blood collection tube

[0090] (1) Method

[0091] Blood was collected from one volunteer subject into the following various blood collection tubes. After blood collection, for serum, immediately centrifuged serum was used. For other blood, it was stored at room temperature in whole blood state (i.e., not centrifuged) for up to 4 hours and then at 4°C for up to 24 hours. The glucose concentration (mg / dL) in blood collection tubes B, C, H, L, M, N, O, P, Q, R, and S was measured immediately after blood collection, and 4 hours, 12 hours, and 24 hours after blood collection. This measured value was taken as the blood collection tube glucose concentration.

[0092] Regarding serum, the supernatant (serum) of the blood collected from one volunteer subject was stored at room temperature for up to 4 hours and then at 4°C for up to 24 hours. Polystyrene Spitz was used as the blood collection tube. The glucose concentration (mg / dL) was measured from the time of blood collection up to 4 hours later. This measured value was taken as the serum glucose concentration at each time.

[0093] (2) Results

[0094] The glucose concentration immediately after blood collection of serum and the time-course changes in the glucose concentration in serum and various blood collection tubes are shown in Table 3 below.

[0095] [Table 3]

[0096] Table 3: Time-course change of glucose concentration in various blood collection tubes

[0097]

[0098] In the conventional blood collection tube C containing sodium fluoride as a glycolysis inhibitor and the blood collection tube H obtained by adding 5 mg of ATP to the blood collection tube C, the glucose concentrations after 12 hours and after 24 hours (mg / dL) were 91, 90 and 90, 90, respectively. On the other hand, in the blood collection tubes L, M, N, O, P, and Q obtained by adding inosine to the blood collection tube C, the glucose concentrations after 24 hours (mg / dL) were all 94 or higher, and a high inhibitory effect on glucose concentration reduction was confirmed. In addition, in the blood collection tubes R and S obtained by adding inosine and ATP to the blood collection tube C, the glucose concentrations after 24 hours (mg / dL) were both 97, and a higher inhibitory effect on glucose concentration reduction was confirmed. According to the present invention, it is possible to achieve a high inhibitory effect on glucose concentration reduction without using expensive ATP or reducing the amount of ATP used, and by using inexpensive inosine.

[0099] Example 3: Effect of inosine on reducing the glucose concentration in the blood collection tube

[0100] (1) Method

[0101] Blood was collected from one volunteer subject into the following various blood collection tubes (blood collection tubes T, U, and V1 to 5).

[0102] Regarding the serum, the supernatant (serum) of the blood collected from one volunteer subject was stored at room temperature for 4 hours and then at 4°C for 24 hours. The blood collection tube used was a polystyrene Spitz. The glucose concentration (mg / dL) was measured until 4 hours after blood collection, and this measured value was used as the serum glucose concentration at each time.

[0103] For the blood collection tube T, 5 mL of blood was collected in each of the blood collection tubes D (a total of 2 tubes), centrifuged for 5 minutes, and 0.85 mL of the supernatant was discarded from each blood collection tube. The contents were combined into one tube and thoroughly mixed, then injected into the blood collection tube T, and stored at room temperature for 4 hours and then at 4°C for 24 hours. The hematocrit value was measured using a multi-item automatic blood cell analyzer XN-1000 (Sysmex Corporation), and the result was that the hematocrit value was 50.1%.

[0104] For blood collection tubes U and V, 5 mL of blood was collected in each of the blood collection tubes D (10 in total), centrifuged for 5 minutes, and 0.85 mL of the supernatant was discarded from each blood collection tube. The samples were combined into one tube and thoroughly mixed to prepare a high hematocrit specimen. 2 mL of this high hematocrit specimen was dispensed into a clean polystyrene tube (without additives) to obtain blood collection tube T. Blood collection tubes were prepared by dispensing 2 mL of this high hematocrit specimen into the blood collection tube U (blood collection tube C) without inosine added in the blood collection tube C (for blood glucose) or into the blood collection tubes V1 - 5 with various concentrations of inosine added in the blood collection tube C (for blood glucose). They were stored at room temperature for up to 4 hours and then at 4°C for up to 24 hours. Using blood collection tube T, the hematocrit value was measured using a multi - item automatic blood cell analyzer XN - 1000 (Sysmex Corporation), and the result was that the hematocrit value was 50.8%.

[0105] (2) Results

[0106] The time - course changes in the glucose concentration in the serum at each time point and in various blood collection tubes are shown in Table 4 below.

[0107] [Table 4]

[0108] Table 4: Time-course change of glucose concentration in various blood collection tubes

[0109]

[0110] In the conventional blood collection tube U (blood collection tube C) containing sodium fluoride as a glycolysis inhibitor and the blood collection tubes V1 - 5 obtained by incorporating various concentrations of inosine into blood collection tube C, the glucose concentration after 24 hours was higher than that in blood collection tube U, and a high inhibitory effect on glucose concentration reduction was confirmed. It should be noted that the hematocrit value of the stored blood was approximately 50%. Although the hematocrit value was near the upper limit of the reference range, a higher inhibitory effect on glucose concentration reduction was confirmed in the blood collection tubes V containing inosine than in the conventional blood collection tube U.

[0111] Example 4: Effect on glucose uptake by red blood cells

[0112] (1) Method

[0113] The blood collected in blood collection tube B from volunteer subjects was centrifuged at 3000 rpm for 5 minutes within 10 minutes, and the plasma was discarded to obtain fresh red blood cells. These red blood cells were washed with PBS(-) and used for the following tests (washed red blood cells).

[0114] Dissolve the test substance in an amount recorded in Table 5 relative to 1 mL of PBS(-). On the other hand, aliquot 5 μL of washed red blood cells impregnated with PBS(-) and allowed to stand for about 15 minutes into a test tube, add 150 μL of the test substance solution to each, and heat at 37 °C for 15 minutes. Then, add 2 μL of the dissolved probe reagent in the Glucose Uptake Assay Kit-Green (Dojindo Laboratories), and heat at 37 °C for 45 minutes. Note that the probe reagent is fluorescently labeled glucose (fluorescently labeled glucose), and a solution obtained by dissolving the powdery reagent in 20 μL of DMSO was used.

[0115] After completion of impregnation into the glucose uptake analysis reagent, wash the red blood cells 3 times with 150 μL of a 50-fold diluted and cooled WI solution (prepared by diluting the WI solution attached to the aforementioned analysis reagent 50-fold with 4 °C PBS(-)), further add 150 μL of the 50-fold diluted and cooled WI solution, stir, then pipette 20 μL onto a glass slide and spread it on the slide. Take a photograph of the slide using a fluorescence microscope.

[0116] Centrifuge the remaining red blood cells, discard the supernatant, add 125 μL of water for injection to the remaining lower-layer red blood cells to cause hemolysis. Measure the fluorescence intensity of 100 μL of the centrifuged supernatant using a microplate reader (MTP-800Lab, Corona Electric Co., Ltd.). The measurement conditions are excitation light wavelength: 530 nm, fluorescence measurement wavelength: 492 nm.

[0117] (2) Results

[0118] The test substance and the observed fluorescence intensity are shown in Table 5 below.

[0119] [Table 5]

[0120] Table 5: Fluorescence intensity of fluorescently labeled glucose taken up into red blood cells

[0121]

[0122] In Test Area 1 where there is no test substance, fluorescently labeled glucose is taken up by red blood cells, and as a result, strong fluorescence is observed due to the fluorescence taken up into the red blood cells. In Test Area 2, compared with the fluorescently labeled glucose, a large amount of unlabeled glucose is present, so competition occurs for the glucose transporter (GLUT1), and the amount of glucose passing through generally increases. As a result, the passage of the fluorescently labeled glucose is inhibited, and as a result, almost no fluorescence in the red blood cells is observed. In Test Area 5 where inosine is present, the uptake of the fluorescently labeled glucose is blocked by inosine, and as a result, almost no fluorescence in the red blood cells is observed, similar to Test Area 2. For sodium fluoride and ATP in Test Areas 3 and 4, although there is a tendency for the uptake of the fluorescently labeled glucose to be inhibited, the degree of uptake inhibition is much lower than that of inosine. From the above results, it was confirmed that inosine has a strong inhibitory effect on the glucose uptake of red blood cells.

[0123] Example 5: Influence on test items other than glucose concentration

[0124] (1) Influence on HbA1c measurement

[0125] In a conventional blood collection tube (Blood Collection Tube C) containing sodium fluoride and the blood collection tube of the present invention (Blood Collection Tube K) obtained by adding 2 mg of inosine and 5 mg of ATP to this blood collection tube, 2 mL of whole blood collected from volunteer subjects was stored at room temperature for 24 hours, and the hemoglobin A1c after storage was measured. As a result, HbA1c was 5.8% in all blood collection tubes. That is, it was confirmed that the blood collection tube of the present invention does not affect the measurement of HbA1c.

[0126] (2) Influence on insulin and c-peptide measurements

[0127] According to the description in (1) of Example 1, blood was collected in a conventional blood collection tube (Blood Collection Tube C) containing sodium fluoride and the blood collection tube of the present invention (Blood Collection Tube K) obtained by adding 2 mg of inosine and 5 mg of ATP to this blood collection tube. Immediately after blood collection and 24 hours after blood collection, the insulin concentration and c-peptide concentration were measured. For the conventional blood collection tube and the blood collection tube of the present invention, the insulin concentrations in the serum and blood collection tube plasma immediately after blood collection are shown in Figure 3 . For the conventional blood collection tube and the blood collection tube of the present invention, the c-peptide concentrations in the serum and blood collection tube plasma immediately after blood collection are shown in Figure 4 . When the measured values of the serum obtained by centrifugation immediately after blood collection were used as the reference for the insulin concentration and c-peptide concentration, as a result, a decrease in the measured values was confirmed in both the conventional blood collection tube and the blood collection tube of the present invention. However, since it was confirmed that the measured values decreased in a certain proportion, the measured values could be converted to the serum measured values by setting a correction coefficient. That is, it was confirmed that the insulin concentration and c-peptide concentration can be measured using the blood collection tube of the present invention.

Claims

1. A glucose uptake inhibitor for red blood cells, which uses inosine as an active ingredient.

2. An inhibitor for reducing the glucose concentration in a blood collection tube, which uses inosine as an active ingredient.

3. The glucose concentration reducing inhibitor according to claim 2, which further contains a glycolysis system inhibitor.

4. The glucose concentration reducing inhibitor according to claim 3, wherein, the glycolysis system inhibitor is one or more selected from the group consisting of fluorides, and adenosine phosphates or their salts.

5. The glucose concentration reducing inhibitor according to claim 2 or 3, which further contains an anticoagulant.

6. The glucose concentration reducing inhibitor according to claim 5, wherein, the anticoagulant is one or more selected from the group consisting of: ethylenediaminetetraacetic acid (EDTA), ethylenediaminetetraacetate and their hydrates; citric acid and its salts; and heparin and its salts.

7. The glucose concentration reducing inhibitor according to claim 2 or 3, wherein, 0.10 mg or more of inosine is used per 1 mL of the collected blood.

8. A blood collection tube for measuring the glucose concentration in blood, which is a blood collection tube having the glucose concentration reducing inhibitor according to claim 2 or 3 in its internal space.

9. The blood collection tube according to claim 8, wherein, per 1 mL of the collected blood, the glucose concentration reducing inhibitor contains 0.10 mg or more of inosine.

10. The blood collection tube according to claim 8 or 9, which is further used for measuring one or more selected from the group consisting of HbA1c, insulin and C-peptide.

11. A method for inhibiting glucose uptake by red blood cells in whole blood or a method for inhibiting reduction of the glucose concentration in whole blood, which includes mixing whole blood with inosine in vitro.

12. The method according to claim 11, which further includes mixing a glycolysis system inhibitor and / or an anticoagulant with whole blood.

13. The method according to claim 11 or 12, wherein, 0.10 mg or more of inosine is mixed per 1 mL of whole blood.

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