JPH0244520B2 - GURUKOOSUSOKUTEIYOSOSEIBUTSU - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a composition for measuring glucose containing an enzyme.

Quantification of glucose in body fluids such as urine and blood is one of the extremely important clinical test items used for diagnosing various diseases, such as diagnosing and treating diabetes, observing its progress, and discovering hypoglycemia.

Conventionally, various chemical methods that utilize the reducing power of glucose have been used to quantify glucose in biological samples, but recently enzymes have been used because the reaction reagents are carcinogenic. The law came into frequent use.

As the enzymatic method, glucose oxidase method and hexokinase method are used.
However, enzymes are generally extremely unstable, and when stored under measurement conditions, they remain effectively active for about 3 days when using a commercially available kit for the glucose oxidase method, and for only 1 day when using a commercially available kit for the hexosinase method. cannot hold.

Therefore, the reagent solution applied to the multi-channel automatic analyzer must be replaced frequently, which is complicated and time-consuming, and results in large losses of reagents. Therefore, measures to overcome these shortcomings are strongly desired.

In order to meet such demands, the present inventors searched for reagents that are highly stable and can be stored for a long period of time, and found that they contain sulfhydryl compounds and/or chelating agents as well as ATP, NAD, Mg ions, HK, and NAD as coenzymes. The resulting composition containing glucose-6-phosphate dehydrogenase (G6PDH), a bactericide, and a PH buffer is extremely stable, and the composition is mixed with a sample and left at room temperature for a certain period of time.
We discovered that by measuring absorbance at 340 nm, it is possible to accurately quantify glucose in a sample, and that the lifespan of an aqueous solution of the composition at room temperature is significantly extended compared to that of conventional commercially available kits. Completed the invention.

That is, the present invention is characterized in that it is an aqueous solution containing a sulfhydryl compound and/or a chelating agent, as well as ATP, NAD, Mg ions, HK, G6PDH, a bactericide, and a PH buffer, wherein the sulfhydryl compound has a concentration of 1 to 50 mM. , the chelating agent is 0.1
A composition for measuring glucose at a concentration of ~20mM. ”
It is.

However, the above ATP means adenosine triphosphate, NAD means oxidized beta-nicotinamide adenine dinucleotide, HK means hexokinase, and G6PDH means glucose-6-phosphate dehydrogenase which can use NAD as a coenzyme.

An object of the present invention is to provide a fixed-dose composition of glucose that has a long life even at room temperature after being dissolved in water, and to streamline glucose quantification.

Preferred concentrations of each component of the composition of the invention are:
It is as follows. PH buffer 0.025~0.25M concentration,
ATP 0.4-5mM concentration, Mg ion 1-25mM concentration, HK 0.3-5u/ml, G6PDH 0.3-5u/ml, fungicide 0.1-30mM concentration, sulfhydryl compound 1-
Preferably, the concentration is 50mM, the chelating agent is 0.1-20mM, and 0-0.1% by weight of a nonionic surfactant is added at the time of use.

In addition, freeze-drying stabilizers such as proteins such as albumin, sugars, sugar alcohols, and polyols such as glycerol can also be added to the composition of the present invention. The PH buffer added to the composition of the present invention is preferably one that has a buffering capacity for part or all of the PH range of 6.5 to 8.5 during use.

From a different perspective, the idea of the present invention can also be understood as a method for analyzing glycose in a specimen using the aqueous solution of the composition of the present invention.

The principle of glucose determination according to the present invention can be expressed as follows.

Glucose + ATP Glucose-6-phosphate + ADP ↑ Hexokinase Glucose-6-phosphate 6-phosphogluconate +NAD ↑ +NADH Glucose-6-phosphate, dehydrogenase In the above reaction formula, NAD represents oxidized beta-nicotinamide adenine dinucleotide. or
NADH stands for reduced beta-nicotinamide adenine dinucleotide.

Through the above reaction, glucose is consumed, and the amount of glucose decreased is equal to the amount of NADH produced.
Therefore, the increase in absorbance of NADH at 340 nm can be measured using a spectrophotometer, and the glucose concentration in the sample can be determined.

The above glucose measurement method is called the hexokinase method (abbreviated as HK method), and is highly specific for glucose as a glucose quantitative method.

Accurate and does not require a calibration curve.

Less interference.

Can be done quickly.

This is an excellent measurement method with the following advantages. However, the major drawback was that the composition of the liquid used for measurement was unstable and it could not be stored for a long time at room temperature.

The present invention has successfully solved this problem, and by the presence of a sulfhydryl compound and/or a chelating agent, a bactericide, and a PH buffer, the storage stability of the above-mentioned analytical solution is significantly improved. Extended lifespan at room temperature.

Conventionally, various sulfhydryl compounds or chelating agents have been used to purify enzymes from living organisms. This is used to remove enzymes by grinding living cells under cooling, and to prevent enzymes from being destroyed during enzyme purification and storage. Therefore, even though it is known that sulfhydryl compounds or chelating agents are used during enzyme production, there is no evidence that they have the effect of extending the life of the measurement solution when quantifying glucose by the hexokinase method. This was not expected.

In addition, since sulfhydryl compounds or chelating agents may be used when producing enzymes, hexokinase or
It is not inconceivable that G6PDH may be accompanied by these compounds. However, in practice, the amount of these compounds used when producing the enzyme is commensurate with the amount of the enzyme, and when preparing a composition for measuring glucose using them, sulfhydryl compounds and /or the chelating agent is diluted by other ingredients to be outside the concentration range required in the composition of the present invention, and the life-extending effect of the composition of the present invention is not found. This was confirmed by a random investigation of a large number of commercially available HK method glucose analysis kits.

The first advantage of the present invention is that the composition maintains a long service life at room temperature. The second effect of the present invention is that the absorbance of the blank is stable, which improves measurement accuracy, which is also important. When analyzing glucose by the hexokinase method, if a sulfhydryl compound and/or a chelating agent are not present, the absorbance of the reagent blank changes significantly over time, causing variations in measured values.

In the present invention, the presence of either a sulfhydryl compound or a chelating agent is sufficient, but
The effect is particularly remarkable when both are present. Also, preferred concentrations of the sulfhydryl compound and the chelating agent when using the composition of the present invention are 1 to 50 mM and 0.1 to 20 mM, respectively, as described above.

Room temperature as described herein means 0 to 30°C unless otherwise specified.

The principle of measuring glucose in a specimen using the composition of the present invention has already been described. To actually measure glucose, mix the aqueous solution of the composition of the present invention and the sample, leave it at room temperature for a certain period of time, and then
Measure the absorbance of the generated NADH at 340 nm.

Therefore, the various compounds, bactericidal agents, PH buffers, etc. used in the composition of the present invention, as well as nonionic surfactants, freeze-drying stabilizers, etc., may significantly interfere with absorbance measurement at 340 nm. must not. Of course, even if these components have absorption at 340 nm, there is no problem as long as it does not actually affect the measurement of the glucose analysis value by subtracting the blank test value.

Examples of preferred disinfectants added to the compositions of the present invention include alkali metal azides, methylene glutaronitrile bromides, and the like. Examples of sulfhydryl compounds used in the composition of the present invention include reduced glutathione, cysteine, N-
acetylcysteine, thiolactoylglycine,
Thiomalic acid and thioglycerol. Further, examples of chelating agents used in the composition of the present invention include EDTA (ethylenediaminetetraacetic acid), EGTA
(ethylene glycol ether diamine tetraacetic acid)
It is. All of these exemplified compounds are 340nm
does not significantly interfere with glycose measurements.

The hexokinase used in the present invention is
Bergmyer, ed., “Method of Enzymatic
Analysis”2nd.Engl.Ed.Volume 1 p473, p459
Academic Press (1974) and G6PDH, which can use NAD as a coenzyme, are described in “Methods in
Enzymology” Volume 41 p196 Academic Press
(1975). These measurement methods are commonplace and routinely used by those skilled in the art today.

The hexokinase and G6PDH data herein are supported by the above measurement methods. However, the measurement temperature was 30°C.

The actual state of the supplementary composition is an aqueous solution. EXAMPLES The present invention will be specifically illustrated below with reference to Examples and Comparative Examples. In addition, for some of these Examples and Comparative Examples, the results of measuring the glucose of specimens using these compositions will be described.

Example 1 ATP 2mM concentration, NAD 1mM concentration, magnesium acetate 10mM concentration, N-acetylcysteine
Contains 10mM concentration, EDTA 2mM concentration, sodium azide 3mM concentration, Triton X100 0.01% by weight, yeast HK1u/ml and lactic acid bacteria G6PDH 1u/ml.
The composition of Example 1 is a 0.1M concentration tris(hydroxymethyl)aminomethane hydrochloric acid buffer (PH7.5).

The composition of Example 1 above was stored at 26°C. Collect 3 ml of this composition, that is, the liquid reagent, and add the sample to this.
After 10 minutes at room temperature, the absorbance at 340 nm was measured, and the absorbance at 340 nm (blank value) of the liquid reagent was subtracted to measure the glucose in the sample. Note that this measurement method is sometimes called the terminal method.

The results are shown in No. 1 in Figure 1.

As shown by this No. 1 line, the measured values showed the same value over 10 days. At this time, the reagent blank value (other than adding the sample to the liquid composition,
Value measured under the same conditions as when the sample was added. The same applies hereafter) is shown in No. 2 of Fig. 1. This blank value also showed a constant measured value over 10 days and was stable.

Comparative Example 1 A commercially available reagent kit for glucose determination using the hexokinase method (UV glucose manufactured by Fujisawa Medical Supply Co., Ltd.; does not contain effective amounts of either sulfhydryl compounds or chelating agents) was dissolved in the specified water, and azide was added to it. The composition to which sodium chloride was added at a concentration of 3mM was stored at room temperature (26°C). Glucose was measured using this solution in the same manner as in Example 1. The result is No. 3 in Figure 1.
is shown on the line. According to this line, the measured value decreased significantly by the 3rd day. At that time, the reagent blank value rapidly increased by the 10th day, as shown by line No. 4 in Figure 1.

Reference Example 1 Using the composition of Example 1, the amount of glucose was measured for samples with various glucose concentrations in the same manner as in Example 1, and the data was plotted on a graph. As a result, a linear relationship as shown in FIG. 2 was obtained between absorbance and concentration.

In addition, the results of comparing the measurement methods for samples with various glucose concentrations using the composition of the present invention and using the same commercially available hexokinase method glucose determination kit as used in Comparative Example 1 are shown below. It is shown in Figure 3. However, when the above commercially available kit is used, all measurements are completed within several hours after preparing the liquid composition for glucose analysis.

[Brief explanation of drawings]

FIG. 1 is a diagram showing changes over time in glucose measurements and reagent blanks performed using the composition of the present invention or a comparative reagent. Figure 2 shows Example 1
FIG. 3 is a diagram showing the linearity between the glucose concentration in the sample (horizontal axis) and the 340 nm absorbance (vertical axis) using the composition of FIG. Figure 3 shows the absorbance obtained using the composition of the present invention (vertical axis) and the absorbance obtained using a commercially available hexokinase method glucose determination kit (horizontal axis).
FIG. In addition, in FIGS. 1 to 3, the absorbance values other than No. 2 and No. 4 in FIG. 1 are values obtained by subtracting the reagent blank.

Claims (1)

[Claims] 1 Sulfhydryl compound and/or chelating agent and ATP, NAD, Mg ion, HK,
A composition for measuring glucose, which is an aqueous solution containing G6PDH, a bactericide, and a PH buffer, wherein the sulfhydryl compound has a concentration of 1 to 50 mM, and the chelating agent has a concentration of 0.1 to 20 mM. However, the above
ATP adenosine triphosphate, NAD
is oxidized beta-nicotinamide adenine dinucleotide, HK is hexokinase, and G6PDH is
It refers to glucose-6-phosphate dehydrogenase that can use NAD as a coenzyme.