JPS6057839B2 - Glucose quantitative agent and method - Google Patents

Description

[Detailed description of the invention] 1 JP → M tooly 1 [ This invention relates to quantitative agents and quantitative methods.

In general, in a healthy person, glucose exists in very small amounts in the urine and a fixed amount in the blood, but when diabetes occurs, the amount of glucose in the blood increases and there is a considerable amount of glucose in the urine. Contains a large amount of glucose.

Therefore, quantifying the amount of glucose in blood and urine is extremely important for diagnosis and follow-up monitoring of these diseases. Conventionally, various methods utilizing the reducing power of glucose have been used to quantify glucose in blood or urine. However, these methods have both advantages and disadvantages, and are not easy or accurate. The present inventors conducted research in search of a method for quantifying glucose that was completely unaffected by metabolites other than glucose in blood and urine, and found that methanol was added to the quantification agent in the presence of glucose oxidase and catalase. By generating formaldehyde, then reducing NAD to NADH by the action of formaldehyde dehydrogenase in the presence of reduced glutathione, and measuring the increase in the amount of NADH, a specific method for measuring glucose could be invented.

The glucose quantitative agent of the present invention contains the following composition.

Methanol 0.5~Hemorrhoid-reduced glutathione 0.5~1.5n1MNAD0.
5-5.0n1M glucose oxidase 1-2
0U/ml catalase 25-1000
U/ml formaldehyde dehydrogenase
0.5~5.0U/1! LtpH7. O~8.
0 Here, the enzyme unit means an international unit, and one unit is the amount of enzyme that transfers 1 micromole of substrate per minute under standard conditions.

Then, the present invention involves treating a glucose-containing sample with glucose oxidase and catalase in the presence of methanol and oxygen, and treating the resulting formaldehyde with formaldehyde dehydrogenase in the presence of reduced glutathione to convert NAD to NADH. increasing N
, is a method of quantifying glucose by measuring the amount of ADH.

The characteristics of the present invention are that formaldehyde, which is not found in living organisms at all, is produced by reacting glucose with glucose oxidase and catalase in the presence of methanol and oxygen, and NAD is converted to NADH by the action of formaldehyde dehydrogenase. The purpose of this method is to measure the amount of increase in NADH by treating glucose in body fluids as a series of reactions, and it is very characteristic that glucose can be quantified reliably in a short period of time.

The reaction for quantifying glucose in the present invention is schematically shown as follows.

One condition for the quantitative method of the present invention is that the reaction pH is 7.0 to 8.0.
Any buffer solution may be used as long as it has a buffering effect at .about.8.0, but phosphate buffer is particularly preferred.

Next, test examples and examples of the present invention will be shown.

Test example 1 Glucose oxidase...10U/m
l Methanol...10%
Reduced glutathione...1mMN
, /1J) ...1
mM catalase...1σu/
ml formaldehyde dehydrogenase
...For compositions of 2 U/ml or more, P
H5. Using 0.1M phosphate buffer, glucose was added and reacted at 37° C., and the reaction was measured using 340 nm to determine the optimum pH for the reaction.

The results are as shown in Figure 1, and from now on,
Reaction pH7. It was found that PH = 8.0 is preferred, and the most preferred is PH = 7.5.

Test Example 2 Phosphate Kalibatsuha (PH = 7.5)...0.1M...1900ml (contains 10% methanol) Catalase...105U/ml...
...0.020m1NAD...1000mM...
...0.020ml reduced glutathione ...10
0mM...0.020ml Formaldehyde dehydrogenase...400U/ml For a composition of 0.010ml or more, 10% of glucose oxidase
A 3 U/ml solution was prepared, 0 to 0.02 mt each was added, glucose was added, 3rc11 powder was reacted, and measurement was performed using 340 nm.

The results are shown in Figure 2, and the amount of glucose oxidase added was approximately 2.5 U/ml and 340 n
It can be seen that the absorbance of m, reaches the maximum.

Example 10.1M potassium phosphate buffer (PH=7.5)...1.9
0 (ml) (contains 10% methanol) 100n1M reduced glutathione...20100n1MNAD
...20400U/ml formaldehyde dehydrogenase ...101C
Pu/m catalase...20103
u/ml glucose oxidase
... 10 or more were mixed to form a mixed solution.

The glucose quantitative agent of the present invention consists of the above-mentioned liquid mixture.

A 10mM glucose solution was appropriately added to this glucose quantitative agent to adjust the glucose concentration in the reaction solution to 0 to 0.1.
Change the concentration to mM and react with 1 powder at 37°C, 3
An increase in absorbance was observed at 40ruT1.

The results are shown in FIG. 3, and it was found that ΔE increased according to the amount of glucose, and there was a proportional relationship between the two. This Figure 3 can be used as a standard graph. ξExample 2 Serum was separated from blood collected from a patient who appeared to have diabetes.

Add 0.01 ml of this serum to 1.99 ml of the mixture of Example 1.
When added to the solution and subjected to a one-powder reaction at 3rC and measured by the increase in 340nn1, the directivity immediately after addition was 0.139.
The value after one error was 0.575. Therefore 340r1
The ΔE value at n1 was read as 0.436. When this was applied to the standard graph prepared in Example 1, it became clear that the serum contained 253 m9/dl of glucose.

[Brief explanation of the drawing]

FIG. 1 is a graph showing the optimum pH of the quantitative agent.

Claims (1)

[Claims] 1. Glucose quantitative agent containing the following composition: methanol 0.5-5mM reduced glutathione 0.5-1.5
mM NAD 0.5-5.0mM Glucose oxidase 1-20u/ml Catalase 25-1000u/ml Formaldehyde dehydrogenase 0.5-5.0u/ml pH 7.0-8.0 2 In the presence of methanol for glucose-containing samples Formaldehyde produced when glucose is quantitatively measured by the action of glucose oxidase and catalase in the presence of oxygen is reacted with formaldehyde dehydrogenase in the presence of reduced glutathione to convert NAD to NADH.
A method for quantifying glucose, characterized by measuring the amount of NADH that increases.