JPH02104298A - Quantification of 1,5-anhydroglucitol - Google Patents

Abstract

PURPOSE:To accomplish the title quantification conveniently, accurately and quickly by making, in advance, a reagent containing e.g., glucokinase act on a sample followed by making a determination using e.g., pyranose oxidase. CONSTITUTION:A reagent containing 4-400U/ml of glucokinase or hexokinase, 0.5-12U/ml of pyruvic acid kinase, 1-10mM of phosphoenolpyruvic acid and 0.3-1.4mM of adenosine triphosphoric acid is added to a sample such as 1,5- anhydroglucitol(1,5-AG)-contg. human serum to make a reaction to convert the glucose in the sample into glucose-6-phosphoric acid and allow the 1,5-AG to be left. Thence, this 1,5-AG is incorporated with 10-500U/ml of pyranose oxidase or L-sorbose oxidase and, if needed, 3-70mM of Mg ion followed by reaction at 5-40 deg.C for 1-60min to form H2O2. The H2O2 is then quantified with peroxidase to quantify the corresponding 1,5-anhydroglucitol.

Description

Detailed Description of the Invention [Field of Application] The present invention relates to 1,5-anhydroglucitol (hereinafter referred to as 1.5-anhydroglucitol).
- AC) is accurate, rapid, and applicable to automatic analyzers.

[Prior art]

1.5-AC is a compound that exists in human cerebrospinal fluid and serum, and it has been reported that the amount in serum decreases in certain diseases, particularly diabetes.

Conventionally, 1.5-AG has been measured mainly by gas chromatography. Recently, 1 in the sample
．． A method for measuring 5-AG using a 1.5-AC specific enzyme (Japanese Unexamined Patent Publication No. 79780/1983) was developed.

In addition, 1.5-AC can be used with pyranose oxidase or L-
A method of measuring using sorbose oxidase (Japanese Unexamined Patent Publication No. 185397/1983) has been reported. However, in the latter method, the substrate specificity of pyranose oxidase or L-sorbose oxidase is not strict, so the amount of pyranose oxidase or L-sorbose oxidase is usually 100 mg/a, I! Urinary patient Tehalo001ng/clJ! I coexist like glucose, etc. that reaches Nemo! It reacts frequently and cannot be measured. Therefore, for the purpose of removing Ti such as glucose, there is a method in which a sample is passed through a column packed with ion exchange resin, and then L5-AG is measured using pyranose oxidase or L-sorbose oxidase, or A method of treating glucose with glucose oxidase, decomposing the resulting hydrogen peroxide with catalase, and measuring the remaining 1,5-AC with pyranose oxidase or L-sorbose oxidase, and further after pretreating the sample. There is a method in which, after converting glucose to glucose-6-phosphate using hexokinase, remaining 1,5-AC is measured using pyranose oxidase or L-sorbose oxidase.

[Problem that the invention seeks to solve]

However, the method using a 1.5-AG specific enzyme is likely to cause errors during measurement because its substrate specificity is not very strict, and after removing sugars using a column, The method of using pyranose oxidase or sorbose oxidase requires complicated operations, and is difficult to apply to automation with automatic analysis equipment that has been implemented in recent years for various clinical test items. In addition, the method using glucose oxidase for the removal of glucose involves the use of pyranose oxidase or 1. - Since the reaction of sorbose oxidase is inhibited and the Km value of glucose oxidase is large, glucose in the sample cannot be completely processed. Using hexokinase to remove glucose, remaining 1,5
When trying to measure -AC using pyranose oxidase, there were problems such as the inability to completely remove glucose with hexokinase and the long reaction time of pyranose oxidase, making it impossible to perform accurate quantification quickly.

(Means for solving the problems and their effects) The present inventors investigated a simple and practical method for measuring 1.5-AC, that is, a method that can be applied to automatic analysis equipment, and completed the present invention. did.

The present invention provides a method for efficiently removing glucose coexisting in a sample and accurately and quickly measuring 1.5-AC in the sample.
After reacting the sample with adenosine triphosphate (hereinafter referred to as ATP), phosphoenolpyruvate, pyruvate kinase, hexokinase, or glucokinase to convert glucose in the sample into glucose-6-phosphate, the remaining 1, 5-AC with pyranose oxidase or 1. -Relates to a method of reacting sorbose oxidase and measuring generated hydrogen peroxide using peroxidase and various substrates.

Next, the present invention will be explained in detail. Conversion of glucose to glucose-6-phosphate involves magnesium ions, ATP
The reaction is carried out using hexokinase or glucokinase in the presence of ATP, but according to the research conducted by the present inventors, it was found that the degree of ATP greatly influences the reaction. In other words, when ATP is small, it is difficult to convert glucose sufficiently, and when ATP is large, it is necessary to use pyranose oxidase, L-sorbose oxidase, or peroxidase.

It was found that the reaction of the hydrogen peroxide detection reagent was suppressed.

To solve this problem, we included phosphoenolpyruvate and pyruvate kinase in the measurement system. That is, the reaction of hexokinase or glucokinase with glucose produces A
TP is converted to ADP, but by the presence of phosphoenolpyruvate and hexokinase, ADP
is supplied as ATP again, so ATP in the measurement system
The concentration is controlled within a certain range. Therefore, it is possible to control the ATP concentration in the measurement system within the optimal range, the above problems are not observed, and the measurement is quick and simple.Furthermore, because the operation is simple, automated measurement is also possible. It became easy.

Glucokinase, hexokinase, pyruvate kinase, pyranose oxidase, L-sorbose oxidase and peroxidase used in the present invention are classified as EC2, 7, 1, 2 and EC2, 7, respectively, according to the classification of the International Union of Biochemistry.
,1,1,EC2,7,1,40,EC1,1,3,i
o , EC1,1,3,11 and EC1,11,1,7
It is preferable that it be purified to the extent that it can be used in clinical tests. Further, reagents such as phosphoenolpyruvate are used after being purified to such an extent that they do not interfere with the reaction. The amounts of each enzyme and reagent to be used depend on the reaction temperature, reaction time, reaction p)I, the origin of the enzyme, and the purity of the reagent, but may generally be in the amounts shown below. Glucokinase or hexokinase is 4-1
00 U/mfl, pyruvate kinase 0.5-12
11/stable, phosphoenolbilpic acid is 1-10mM,
ATP is 0.3-1.4mM, magnesium ion is 3
~70mM, pyranose oxidase or L-sorbose oxidase is used at ~500U/d.

The reaction temperature is 5 to 40°C, preferably 25 to 40"C, and the reaction time is 1 minute to 60 minutes, preferably 1 minute to 10 minutes. Furthermore, in order to efficiently convert glucose, mutarotase is allowed to coexist. In that case, the amount used is approximately 0.
．． 25-4. OU/ml. Any method that can be used to quantify hydrogen peroxide produced by the reaction with high sensitivity may be used, but usually peroxidase is used to oxidize various substrates with hydrogen peroxide, and the produced dye is measured spectrophotometrically. The method of measuring with a meter is simple and suitable for automatic measuring devices. Also, instead of measuring the hydrogen peroxide produced, the consumed oxygen may be detected by an appropriate method.

Next, the present invention will be explained with test examples and examples, but the present invention is not limited thereto.

Test Example 1 Reactivity of hexokinase to 1.5-AC The reactivity of hexokinase to 1.5-AG at various concentrations was investigated under the following conditions. The average plasma 1.5-AG concentration of healthy individuals is about 2/d1 (Japan Clinical Research, Vol. 44, Summer Special Issue (1986)). 1.5-AC concentration: 50mM containing 13mMgC1g in 0.15 and 20 /d1 solution
Tris-hydrochloric acid buffer (pH 8,0) 2.6rn1 with 500u/rrdl hexokinase solution at 0.0.
1ml, 16mM ATP solution at 0, 1 mfl, 4
hM phosphoenolpyruvate solution.

By adding 0.1 d of a solution containing 6 mM NADH solution and 50 u/rtdl of pyruvate kinase and 50 u/rtdl of raftite dehydrogenase, reacting at 37 °C, and measuring the change in absorbance at 340 nm. The reactivity of hexokinase with 1.5-AG was confirmed.

At any concentration, no difference in reaction was observed compared to that containing no 1.5-AC. That is, hexokinase is 1
．． It was found that it did not react with 5-AC.

Test Example 2 Reactivity of glucokinase to 1.5-AC; The same study as above was conducted on the test side, except that a glucokinase solution with a concentration of 1000 u/ml was used instead of the hexokinase solution. As a result, it was found that glucokinase does not react with 1.5-AG, similar to the case of hexokinase.

fi Effect of ATP concentration when treating glucose with hexokinase Sample: 500 mg/a glucose solution or 1.15 u/ml mutarotase in 0.05 ml water;
4.6u/rtrl peroxidase, 0.05
4-aminoantipyrine at 8 mg/d1, 0.37 m
g/related N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine + 13mM MgCl
50mM Tris-HCl buffer (pH 8,
0) for 2.8 mi, 500 u/- concentration of hexokinase solution at 0.1 concentration and ATP solution at various concentrations at 0.1 d.
After adding ゛ and reacting at 37°C for 10 minutes,
u/roll concentration of pyranose oxidase 0.1d
was added and reacted at 37°C for 10 minutes, and the absorbance at 550 tv was measured.

Table 1 shows the difference in absorbance between samples containing and not containing glucose solution. As the ATP concentration increases,
The difference in absorbance at 550 nm decreases.

It was found that a TP concentration of 330 mM or more is required.

Test Example 4 Effect of ATP concentration when treating glucose with glucokinase The procedure was the same as in the wiping box except that a 1000 u/ml glucokinase solution was used instead of the hexokinase solution.

The results are shown in Table 2. 330++ similar to νJ creation example"-
It has been found that an ATP concentration of M or more is required.

(Margins below) Table 2 Test Example 5 Effect of ATP concentration when measuring 1.5-AG using pyranose oxidase, peroxidase, and hydrogen peroxide detection reagents 20 mg/d of 1.5-AC?
Peroxidase of 4.6u/kura per 0.05d,
4-aminoantipyrine at 0.058n+g/if;
0.31 mg/ml N-ethyl-N-(2-hydroxy-3-sulfopropyl)-m~toluidine, 13
50mM) Lis-HCl buffer containing mM MgCl□ (pH 8,0) 2.8mA and various concentrations of ATP
0.1 mfl of the solution was added with 0.1 mfl of pyranose oxidase at a concentration of 1 ooOu/ml and 1 degree, and the reaction was carried out at 37°C, and the change in absorbance at 550 nm was measured. As shown in FIG. 1, it was found that when the concentration of ATP 1 degree is high, the increase in absorbance is suppressed. As shown in Precedent 3 and Test Example 4, an ATP concentration of 330mM or more is required to remove glucose from a sample], but such a high concentration inhibits the reaction of pyranose oxidase. However, it turned out that accurate measurements could not be made.

2 Effect of concentration of ATP when measuring 1.5-AC when phosphoenolpyruvate, pyruvate kinase is added to the reagent of ``4 precedent'' - As an ATP solution of 16.5 mFI ATP
.

As a result of using 0.1 d of a test solution containing 49 mM phosphoenolpyruvate and 50 u/d pyruvate kinase solution, the results showed that the sample contained glucose and the sample did not contain glucose, as in the case of the 330 mM ATP solution in Table 2. No difference in absorbance was observed, indicating that ATP completely reacts with glucose at a small concentration of ATP. This is because after ATP is converted to ADP by hexokinase, by coexisting phosphoenolpyruvate and pyruvate kinase, ADP is recycled and supplied as ATP, so that no ATP deficiency occurs.

Measurement of 1.5-AC in serum ■Reagent A when using hexokinase for glucose treatment
1.25u/mj! mutarotase.

4.92u/peroxidase.

0.062 mg/ml 4-aminoantipyrine.

19.9u/-hexokinase.

0.66mM ATP.

1, 96mM phosphoenolpyruvate.

2.4u/ml pyruvate kinase.

Reagent B 200u/++j! Add 2.5rnl of reagent A to 0.05mf of pyranose oxidase human serum,
The reaction was carried out at 37°C for 5 minutes. Then reagent B-t-0.
The absorbance was measured at 550 nm after 5 minutes. At the same time, a blank using purified water instead of human serum was also measured. When the 1,5-AC concentration in serum was measured using a 1,5-AC standard calibration curve prepared in advance, it was found to be 2.3 mg/d1.

(2) When glucokinase was used for glucose treatment The procedure was the same as (2) except that 33 u/d glucoginase was used instead of hexokinase.

The serum L5-AC concentration was found to be 2.1 mg/d1.

Example 1 - Comparison of gas chromatography method and final method Conventional gas chromatography method and reverse side of 30 human serum samples I 1 ■ and ■
1.5-AGi 1 degree was quantified using the method of .

The CL-7000 model manufactured by Shimadzu Corporation was used as an automatic analyzer, and 250 µm of reagent A used in 1 case 2 was used.
! , using 50uf of reagent B and 5μ of serum as a sample.
! used.

(2) When hexokinase is used for glucose treatment The results of the gas chromatography method and the terminal method are shown in Figure 2. The correlation coefficient was 0.89, indicating a high correlation between both quantitative methods.

(2) When glucokinase was used for glucose treatment, the correlation coefficient was 0.91, and similar to when hexokinase was used, a high correlation with gas chromatography was observed.

[Effects of the Invention] The present invention relates to a method for measuring 1.5-AC that is accurate and capable of automated measurement. That is, due to its nature, it has become possible to perform measurements using automatic analyzers that have recently been implemented for various clinical test items, and it has become possible to process a large number of samples at the same time.

[Brief explanation of drawings]

Figure 1 shows the difference in reaction due to the difference in ATP concentration, and the second
The figure shows the correlation between the method of the present invention using hexokinase for glucose treatment and the method using gas chromatography in various serum samples. Figure 3 shows the method of the present invention using glucokinase for glucose treatment and the method using gas chromatography. FIG. 2 is a correlation diagram of various serum types. Patent applicant Amano Pharmaceutical Co., Ltd. Figure 1 Reaction time (minutes) Figure 2 The present invention (additional g,/dll) Figure 3 The present invention (mg/dl)

Claims (1)

[Claims] (1) In a method for measuring 1,5-anhydroglucitol using pyranose oxidase or L-sorbose oxidase, glucokinase or hexokinase and adenosine triphosphate, pyruvate kinase, or phosphoenolpyruvate are added to the sample in advance. 1. A method for quantifying 1,5-anhydroglucitol, which comprises reacting with a reagent containing 1,5-anhydroglucitol.