JPH0295261A - Glycolysis blocking agent - Google Patents

Abstract

PURPOSE:To immediately block the decrease of blood sugar by glycolysis and to maintain the initial value thereof over a long period of time by incorporating 2-dioxy-D-glucose or its deriv. and a fluoride salt or the metal salt of monohalogenated acetic acid. CONSTITUTION:The 2-dioxy-D-glucose or its deriv. is competitive with D-glucose in the reaction of glucose glucose-6-phosphoric acid by the hexokinase of the glycolysis system. The formation of the glucose-6-phosphoric acid is, therefore, hindered and the quantity of the glucose-6-phosphoric acid entering the erythrocyte membranes decreases. Namely, the sugar transporting mechanism of the erythrocyte membranes is kindered. The immediate effectiveness of blocking the glycolysis for the glycolysis to be inhibited in the initial stage is thus obtd. The sugar value may drop again if the 2-dioxy-D-glucose or its deriv. in the glycolysis blocking agent is consumed as the substrate of the hexokinase. However, the action of the lower enzyme of the glycolysis system is hindered by the presence of the fluoride salt, etc., and, therefore, the glycolysis blocking effect is sustained.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention is capable of rapidly inhibiting a decrease in blood sugar due to glycolysis in the blood, and maintaining its initial value for a long period of time. In particular, the present invention relates to a glycolytic inhibitor capable of measuring glycated hemoglobin A+C.

(Prior art) Measurement of blood glucose (blood sugar) and glycated hemoglobin AI present in red blood cells, especially glycated hemoglobin A1c (In healthy people, 4.
5 to 5.5%) is an important measurement item in diabetes testing.

Generally, when testing blood components, a certain amount of time is required from blood collection to measurement. In particular, in large hospitals and testing centers, where large numbers of samples are handled, it is unavoidable that blood is stored at room temperature for at least several hours after blood collection due to the flow of testing operations, and in some cases, blood is collected and delivered. Samples may be stored for even longer periods of time.

In the case of blood sugar testing, since blood sugar levels tend to drop due to glycolysis during the storage period after blood collection, glycolytic inhibitors are added to suppress this. Conventionally, fluoride salts, monohalogenated acetic acid metal salts, citric acid, D-mannose, and the like are known as glycolytic inhibitors. However, these known glycolytic inhibitors have the following problems.

Fluoride salts and monohalogenated acetic acid metal salts have a glycolysis-inhibiting effect by inhibiting the action of enolase and glyceraldehyde (glyceraldehyde)-s-phosphate dehydrogenase, respectively.
Since these enzymes are lower-level enzymes in the glycolytic system, there is a problem in that there is not enough time for the inhibitory effect to appear, and during that time blood sugar levels drop slightly, resulting in a lack of rapid action. However, once the inhibitory effect occurs, blood sugar levels are maintained continuously thereafter.

Citric acid inhibits phosphofructokinase, an enzyme at the upper level of glycolysis, and therefore exhibits a rapid glycolysis inhibiting effect. However, citric acid is strongly acidic (pH 1-2), so
When mixed with blood, the pH of the blood becomes acidic (pH 4 to 5), red blood cells are denatured, and hemolysis occurs violently. Hemolysis not only gives a sense of anxiety to the laboratory technician handling the specimen, but also poses a problem in that blood sugar measurement using a colorimetric method gives rise to errors. Furthermore, in the measurement of glycated hemoglobin A1c, there is a problem that hemoglobin is denatured at such a low pH, giving rise to errors.

Like glucose, D-mannose is a substrate for hexokinase, and thus competes with glucose in the glycolytic action of hexokinase on glucose. This prevents glycolysis and is fast-acting, but once all of the %D-mannose is consumed, blood sugar decreases again due to glycolysis, so it has a long-term glycolysis inhibiting effect. The problem was that it could not be maintained. Meanwhile, in order to solve the M point, it has been proposed to use D-mannose in combination with a heptafluoride salt or a monohalodanated acetate metal salt to obtain sustainability (Japanese Patent Application Laid-Open No. 106563/1983). However, even in this case, the system using D-mannose still has the following problems. Among the blood sugar measurement methods, the method using glucose theory hydrogen enzyme, which has been widely used recently,
If an enzyme with low substrate specificity is used, not only glucose but also the isomer D-mannose is degraded. Therefore, it causes a positive error. For example, if the substrate specificity of glucose dehydrogenase is low, the glucose measurement value will be 500 to 10001 ng/d/,
This results in a positive error of 5 to 10 times the normal value. Furthermore, when D-mannose is used as a specimen for measuring hemoglobin, Asc, -1-crobin binds to D-mannose and it undergoes hekinohylation.

Bin A1c is produced, which is glycated hemoglobin AIC
There was also the problem that it was added to the measured value, giving a positive error.

(Problem to be solved by the invention) Original F! A solves the above-mentioned conventional problems, and its purpose is to quickly prevent the decrease in blood sugar due to glycolysis in the blood, maintain its initial value for a long time, and further The object of the present invention is to provide a glycolytic inhibitor that allows measurement of glycated hemoglobin A1, particularly glycated hemoglobin AIc.

(Means for Solving the Problems) The glycolytic inhibitor of the present invention is characterized by containing 2-deoxy-〇-glucose or its derivative, and a fluoride salt or a monohalogenated acetic acid metal salt, This achieves the above objective.

The 2-deoxy-D-gluco-
Daoxy-D-glucose, etc.

As the fluoride salt, an alkali metal salt of hydrogen fluoride or an ammonium salt is preferable, and for example, sodium fluoride, potassium fluoride, tin fluoride, ammonium fluoride, etc. are used.

As the monohalogenated acetic acid metal salt, the halogen is preferably chlorine, bromine or iodine, and the metal is preferably an alkali metal, such as monochlorosodium acetate, motuprom sodium acetate, motuprom potassium acetate, monoiodosodium acetate, monoiodopotassium acetate, etc. be used.

The dosage of these drugs is 2.5 to 2.5 to 2-deoxy-D-glucose or its derivatives per ml of whole blood.
151119, preferably 5-10 mg. 2.
When the amount is less than 5.5 cm, the immediate glycolysis inhibiting effect is incomplete, and when it is more than 1 g of 15 W, the osmotic pressure of the blood may be abnormal and hemolysis may occur. Fluoride salts or monohalogenated metal acetate salts are used in amounts of 1 to 2 mDa of each component alone or the sum of both components per 1 d of blood. 11ng
When it is less than 2, it is not possible to maintain the blood sugar drop for a long time.
■When the amount is higher than that, side effects such as hemolysis occur and the condition becomes insular.

In addition, the glycolytic inhibitor of the present invention may further include heparin salt, ethylenediaminetetraacetic acid (hereinafter referred to as EDT).
It is optional to add blood anticoagulants such as salts of A), blood coagulation promoters such as silica, thrombin.

The glycolytic inhibitor may be added directly to the sample blood in the form of powder, granules, tablets, or aqueous solution, or the glycolytic inhibitor powder, granules, tablets, or aqueous solution may be placed in a glass or synthetic resin blood collection tube. may be added to the blood sample in advance, and then the sample blood may be added thereto.

Alternatively, coat a pellet or beads made of glass or synthetic resin with powder or granules of a glycolytic inhibitor dispersed in a suitable solvent, add the pellet or beads to a blood collection tube, and add the sample blood. may be added. Alternatively, the sample blood may be added to the blood collection tube wall coated with a dispersion in the above-mentioned solvent.

The blood collection tube used in these cases may be either a normal pressure or a vacuum blood collection tube, and it is optional to contain blood IjIt or a serum separating agent therein.

(Function) 2-deoxy-D-glucose or its derivatives contained in the glycolytic inhibitor inhibits D-glucose in the hexokinase reaction of glycolysis → glucose-6-phosphate.
competes with glucose. Therefore, the production of glucose-6-phosphate is inhibited, and glucose-6-phosphate enters the red blood cell membrane.
The amount of 6-phosphoric acid is drastically reduced. In other words, the sugar transport mechanism in the red blood cell membrane is inhibited. In this way, since glycolysis is inhibited at an early stage, it has a rapid effect on inhibiting glycolysis.

When 2-deoxy-D-glucose or its derivatives in glycolytic inhibitors are consumed as substrates for hexokinase,
There is a risk that blood sugar may drop again. However, due to the presence of fluoride salts or monohalogenated acetic acid metal salts, enolase and glyceraldehyde-3-
Since it inhibits the action of phosphate dehydrogenase, a lower enzyme in the glycolytic system, the glycolytic inhibiting effect can be sustained.

Since it does not become a substrate for glucose dehydrogenase, which has low isomerism, it does not give a positive error to glucose measurements.

Furthermore, since 2-deoxy-〇-glucose or its derivatives are deoxyhexoses, they do not react with hemoglobin to produce hexohemoglobin Arc, so they do not give positive errors to the measured values of glycated hemoglobin and Ate. .

(*Example) Examples of the present invention will be described below.

Example 1 A plurality of commercially available 2 ml glass blood collection tubes were prepared containing 10.OWIg of p-9"t lefsamine, 3.0 ~ of sodium fluoride, and 4.0 kg of EDTA2 potassium salt as a blood coagulant. To these, 2 ml of fresh surface from a healthy person was collected and mixed by inversion.

This mixture was centrifuged at 3000 rpm for 5 minutes, and a plasma fraction was collected to measure blood sugar.

In addition, glycated hemoglobin A+ and Alc were measured using the above-mentioned mixed whole blood.

Furthermore, the above-mentioned mixture was left at room temperature for 36 and 24 hours after mixing, and blood sugar was measured in the same manner. In addition, the above-mentioned mixture was added at 24 and 4 hours after mixing.
Glycohemoclobin A+ and Alc were measured using whole blood that had been left at room temperature for 8 hours.

Blood sugar was measured by the glucose dehydrogenase method using the glucose quantitative reagent "Determiner-GL-RJ" manufactured by Kyowa Medex Co., Ltd. The principle is as follows.

β-D-glucose
D-glucono-δ-lactone NADPl produced in the above enzymatic reaction was measured at 340 nm by the reaction rate 11 (L/-to assay) method.

Measurement of glycated hemoglobin and protein C is carried out at ■Kyoto Dai-
Measurement was carried out using Hi-Auto A, c HA-8120 manufactured by Kagaku Co., Ltd. by selecting appropriate conditions. This HA-8120 is a dedicated device for measuring glycated hemoglobin using high-speed liquid body chromatography, and each hemoglobin component is separated and eluted from a single cation exchanger in 4 minutes. A dedicated eluent (phosphate buffer) is used as the elution buffer.

The hemoglobin elution time using this device is shown in the general number and in FIG. In FIG. 1, s PI and h are peaks caused by glycated hemoglobin A, a and A, b components, and s PI and P.

is a peak caused by the glycated hemoglobin C component. P6 is a peak due to other hemoglobins. Here, glycated hemoglobin A, A, and c are calculated using the following formula.

In addition, as a sample to be subjected to this liquid chromatography measurement, 3 μt of the above-mentioned whole blood was mixed with α005M phosphate buffer 100W11 and Tri onx.

-100 (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.), mixed with 450 μt of hemolytic reagent (pH 6,3) consisting of 0.1 ml, and heated to 110 μl at room temperature.
It was used after it was allowed to stand for a minute to cause hemolysis.

Example 2 D-glucosamine 15.01119. Sodium fluoride L51n9, sodium monoiodoacetate LsIn? Blood sugar was measured in the same manner as in Example 1, except that a blood collection tube containing 4.0 μl of EDTA2 and potassium salt was used.

Example 3 D-glucosamine/20.0 drops, monoiodo acid esu)'
J Cutam, Om9, and EDTA2 Calicum Salt 4.
Blood sugar was measured at the same time as in Example 1, except that a blood collection tube containing No. 0 was used.

Comparative example I D-glucosamine 4.0~, sodium fluoride A 15
ml, sodium monoiodoacetate 15 ~ and EDTA
Blood sugar was measured at the same time as in Example 1, except that a blood collection tube containing 4 Tam of Z-caricum salt was used.

Comparative Example 2 D-glucosamine 4.01n9. Sodium fluoride ALG
&, monoiodoacetate sodium oda and EDTA
Blood sugar was measured in the same manner as in Example 1, except that a blood collection tube containing TOQ-2 potassium salt was used.

Comparative Example 3 Sodium fluoride & 0 ■ and EDTA 2 potassium salt 4
．． Blood sugar, glycated hemoglobin A, and glycated hemoglobin A and C were measured in the same manner as in Example 1, except that a blood collection tube containing 0.0 cm was used.

Comparative Example 4 D-mannose 1&orn9, sodium fluoride A3.0
1"fi' edict and EDTA2 potassium salt 4.Q rn
Blood sugar, glycated hemoglobin A1, and glycated hemoglobin Atc were measured in the same manner as in Example 1, except that a blood collection tube containing No. 9 was used.

Comparative Example 5 Citric acid 15, Oq and EDTA2 potassium salt 4.0
Blood sugar, glycohemoglobin At, and glycohemoglobin C were measured in the same manner as in Example 1, except that a blood collection tube containing 1n9 was used.

Comparative Example 6 Blood sugar was measured in the same manner as in Example 1, except that a blood collection tube containing 15.0 to 15.0 D-glucosamine and 4 Tam of EDTAz potassium salt was used.

Blood glucose measurement values and glycohemoglobin A1 and glycohemoglobin A in each example and comparative example above
, c are shown in Tables 1 and 2.

(The following is a margin.) From Table 1, it can be seen that the initial blood sugar level was maintained in Examples 1 to 3 even if the samples were left at room temperature for 24 hours. Comparative example 1 is D
- Since there is less glucosamine, the blood sugar level decreases with time. Comparative Example 2 had no problem in terms of blood sugar level, but hemolysis occurred due to the large amount of D-glucosamine. Comparative Example 3 does not contain D-glucosamine, so
No fast-acting glycolytic inhibitory effect is observed. Comparative Example 4 had an abnormally high value because glucose dehydrogenase, which has low substrate specificity, also dehydrogenated D-mannose, which is an isomer. Comparative Example 5 had no problem with blood sugar level, but had severe bleeding due to the influence of citric acid. Since Comparative Example 6 uses only D-glucosamine, it can be seen that the initial blood sugar level cannot be maintained for a long time.

From Table 2, it can be seen that in Example 1 and Comparative Example 3, the initial values of glycated hemoglobin, A, and c were maintained even after being left at room temperature for 48 hours. Comparative Example 4 shows that hexohemoglobin A, A, and C were produced in D-mannose, and therefore the measured value was high.
Nru. In Comparative Example 5, PI (PI) decreased due to the influence of citric acid, resulting in denaturation of hemoglobin and a high value.

(Effects of the Invention) The present invention is a glycolytic inhibitor containing 2-deoxy-D-glucose or a derivative thereof and a heptafluoride salt or a monologenated metal acetate, so it has a rapid effect on lowering blood sugar due to glycolysis. The initial value can be maintained for a long time. Therefore, even for collected and delivered samples that may be stored at room temperature for long periods of time, accurate blood sugar levels are low and the substrate for glucose dehydrogenase is 1+1, so blood glucose measurement methods using glucose dehydrogenase cannot be used. can be used.

In addition, hexohemoglobin A1 reacts with hemoglobin.
Since it does not produce AsC, glycohemoglobin, A, and c can be measured accurately without giving any error to the measured values.

[Brief explanation of drawings]

FIG. 1 shows an elution pattern obtained by measuring glycated hemoglobin in blood by liquid chromatography. that's all

Claims (1)

[Claims] (1) A glycolytic inhibitor comprising 2-deoxy-D-glucose or a derivative thereof and a fluoride salt or a monohalogenated acetic acid metal salt.