JPH05236995A - Method for measuring 1,5-anhydroglucitol - Google Patents

Abstract

PURPOSE:To obtain a measuring method of 1,5-anhydroglucitol capable of determining 1,5-anhydroglucitol in good sensitivity and capable of determining 1,5- anhydroglucitol in good sensitivity and high reliability even if pyranose oxidase contains an unknown enzyme capable of decomposing hydrogen peroxide or a superoxide anion as impurity. CONSTITUTION:The objective method for measuring 1,5-anhydroglucitol is carried out by measuring an amount of hydrogen peroxide produced by an enzymic reaction comprising treating 1,5-anhydroglucitol with pyranose oxidase or L- sorbose oxidase. The method is characterized by carrying out the enzymic reaction in the presence of a superoxide dismutase.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for measuring 1,5-anhydroglucitol.

[0002]

2. Description of the Related Art 1,5-Anhydroglucitol is a compound which is present in human cerebrospinal fluid and serum and has been reported to have a decreased amount in serum in certain diseases, particularly in diabetes.

The measurement of 1,5-anhydroglucitol in blood has hitherto been carried out mainly by gas chromatography ("Diabetes", Vol. 25, 1115-11).
Item 18, 1982). However, this method requires treatment of the test liquid and labeling of 1,5-anhydroglucitol, and also requires a long time for measurement, which makes it difficult to measure a large number of samples at the same time. In addition, there are drawbacks such as the need for advanced technology for maintenance and management of analytical equipment, which makes it inconvenient for practical clinical application.

On the other hand, pyranose oxidase and L-sorbose oxidase are known as enzymes for detecting 1,5-anhydroglucitol, and 1,5-, which is a kind of sugar alcohol, is utilized by utilizing these enzymes. A method has been developed which oxidizes anhydroglucitol and quantifies 1,5-anhydroglucitol in a sample from the amount of hydrogen peroxide produced (JP-A-63-185397).
JP-A-2-104298, etc.). However, the efficiency of hydrogen peroxide produced by the oxidation of pyranose oxidase and L-sorbose oxidase used in this method is as low as about 70% or less, and there is a problem in measurement sensitivity. In addition, commercially available pyranose oxidase may contain an unknown enzyme that decomposes hydrogen peroxide or superoxide anion as an impurity, and if such an impurity is contained, the sensitivity or reliability of the measurement may be deteriorated. There is a problem of decrease.

[0005]

Therefore, an object of the present invention is a method for measuring 1,5-anhydroglucitol using pyranose oxidase or L-sorbose oxidase, which is highly sensitive to 1,5-anhydroglucitol. Anhydroglucitol can be quantified, and even if pyranose oxidase contains an unknown enzyme that decomposes hydrogen peroxide or superoxide anion as an impurity, it has good sensitivity and high reliability. 1,
An object of the present invention is to provide a method for measuring 1,5-anhydroglucitol capable of quantifying 5-anhydroglucitol.

[0006]

As a result of earnest research, the inventors of the present invention carry out an oxidation reaction of 1,5-anhydroglucitol by pyranose oxidase or L-sorbose oxidase in the presence of superoxide dismutase. And pyranose oxidase or L
-The superoxide anion produced when 1,5-anhydroglucitol is oxidized by sorbose oxidase can be efficiently converted to hydrogen peroxide by the action of superoxide dismutase, and as a result, 1,5- Anhydroglucitol can be quantified with high sensitivity, and even if pyranose oxidase contains an unknown enzyme that decomposes hydrogen peroxide or superoxide anion, the sensitivity is good. The present invention has been completed by finding that 1,5-anhydroglucitol can be quantified with high reliability.

That is, according to the present invention, 1,5-anhydroglucitol is reacted with pyranose oxidase or L-sorbose oxidase, and the amount of hydrogen peroxide produced by this enzymatic reaction is measured to give 1,5- In the method for measuring anhydroglucitol, there is provided a method for measuring 1,5-anhydroglucitol, characterized in that the enzymatic reaction is carried out in the presence of superoxide dismutase.

The superoxide dismutase used in the method of the present invention may be of any origin as long as it is a superoxide dismutase. It can be produced by mammalian erythrocyte-derived enzyme or microbial-derived enzyme, and further by genetic engineering. The prepared products can also be used.

The concentration of superoxide dismutase in the reaction solution in the method of the present invention is not particularly limited,
About 20,000 U / L to 80,000 U / L is preferable, and more preferably 30,000 U / L to 6000.
It is about 0 U / L.

The reaction temperature is preferably in the range of 20 to 60 ° C., and the buffer solution used as the reaction medium is a phosphate buffer solution, a Tris buffer solution or the like, and a pH of 5 to 8 at which the enzyme easily acts. It is preferable to have. Also,
The reaction time is preferably about 1 minute to 60 minutes, particularly preferably about 1 minute to 10 minutes.

The concentration of pyranose oxidase or L-sorbose oxidase used in the method of the present invention is 1,000 U / L to 500,000 U as in the conventional case.
/ L is preferable.

As the method for measuring hydrogen peroxide produced by the oxidation of 1,5-anhydroglucitol by pyranose oxidase or L-sorbose oxidase, any method known in the art can be adopted. For example, in the presence of peroxidase, the amount of hydrogen peroxide produced by reacting with a coloring reagent such as triphenylmethane-based leuco dye and quantifying the resulting dye by colorimetric determination such as absorbance measurement, and by extension, in the sample. The amount of 1,5-anhydroglucitol can be measured. The enzyme and the color-developing reagent for quantifying these hydrogen peroxides can also be included in the reaction mixture from the beginning.

[0013]

EXAMPLES Hereinafter, the present invention will be described more specifically by showing Examples and Comparative Examples of the present invention. However, the present invention is not limited to the following examples.

Example 1 and Comparative Example 1 Pyranose oxidase 4000 U / L, peroxidase (POD); 6000 U / L, various concentrations of superoxide dismutase (SOD), coloring reagent ([bis [4- [N-ethyl-N -(3-Sulfopropyl) aminophenyl]] [4- (N, N-diethylamino) phenyl] methane disodium salt) 100 μM in 1 ml of Tris-succinate buffer (pH = 7.0, 50 mM). 10 μl of a test solution containing 25 μg / ml of 5-anhydroglucitol was added, and the mixture was allowed to stand in a thermostat at 25 ° C. for 20 minutes. After the reaction was completed, the absorbance at a wavelength of 595 nm was measured (Example 1). On the other hand, for comparison, the same experiment as in Example 1 was performed except that SOD was not included (Comparative Example 1).

The results are shown in FIG. FIG. 1 is a plot of SOD activity per liter on the horizontal axis and absorbance on the vertical axis. As shown in FIG. 1, when SOD is added, when SOD is not added (that is, SOD activity 0 U /
Compared to L), the absorbance increased depending on the concentration of SOD, and when 60000 U / L was added, the absorbance increased by 40% or more. From this, 1,5 in the presence of SOD
-It has been clarified that the accuracy of measurement of 1,5-anhydroglucitol is increased by carrying out the oxidation reaction of anhydroglucitol.

Example 2, Comparative Example 2 Commercially available pyranose oxidase known to contain an unknown enzyme decomposing hydrogen peroxide or superoxide anion as an impurity; 4000 U / L; POD; 6
000 U / L, SOD; 60,000 U / L, 0 to 50 μg of 1,5-anhydroglucitol in 1 ml of Tris-succinate buffer (pH = 7.0, 50 mM) containing 100 μM of the same coloring reagent as in Example 1. 10μ including / ml
1 was added and the mixture was allowed to stand in a constant temperature bath at 25 ° C. for 20 minutes. After the reaction was completed, the absorbance at a wavelength of 595 nm was measured (Example 2). On the other hand, for comparison, the same experiment as in Example 2 was performed except that SOD was not included (Comparative Example 2).

The results are shown in FIG. As is clear from FIG. 2, the method of the present invention in which SOD was added showed a clear increase in absorbance and high linearity as compared with the conventional method. From this, it was clarified that the quantification of 1,5-anhydroglucitol can be performed with high sensitivity and reliability even when pyranose oxidase contains impurities.

[0018]

INDUSTRIAL APPLICABILITY According to the present invention, 1,5-anhydroglucitol can be quantified with high sensitivity, and even if pyranose oxidase contains an unknown enzyme that decomposes hydrogen peroxide or superoxide anion as an impurity. The present invention provides a method for measuring 1,5-anhydroglucitol capable of quantifying 1,5-anhydroglucitol with good sensitivity and high reliability even in the case of the above.

[Brief description of drawings]

FIG. 1 shows the concentration of SOD on the horizontal axis and the absorbance (595 n on the vertical axis).
It is a figure which compared with SOD addition and non-addition taking m).

FIG. 2 is a diagram showing the results of examining the effect of adding SOD, with the horizontal axis representing the amount of 1,5-anhydroglucitol and the vertical axis representing the absorbance (595 nm).

Claims (1)

[Claims] 1. 1,5-Anhydroglucitol is prepared by allowing pyranose oxidase or L-sorbose oxidase to act on 1,5-anhydroglucitol and measuring the amount of hydrogen peroxide produced by this enzymatic reaction. In the method for measuring Toll, the method for measuring 1,5-anhydroglucitol is characterized in that the enzymatic reaction is carried out in the presence of superoxide dismutase.