JPH05304997A - Determination of 1,5-anhydroglucitol - Google Patents

Abstract

PURPOSE:To provide an enzymatic measurement which can be applied to an automated analyzer because 1,5-anhydroglucitol can be determined promptly. CONSTITUTION:An enzymatic measurement for 1,5-anhydroglucitol which is characterized by that the pyranose oxidase originating from a Basidiomycetous fungi No. 52 deposited to Microorganism Institute as FERM No. 1016 is used as an enzyme for allowing to act on anhydroglucitol in the sample. The application of this method has become possible to establish an automatic system and enabled prompt determination and improved accuracy.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to 1,5-anhydroglucitol (hereinafter, 1,5-anhydroglucitol) which is a diagnostic marker for diabetes.
AG) which is simple and rapid and can be applied to an automatic analyzer.

[0002]

2. Description of the Related Art 1,5-AG is a compound which is present in human cerebrospinal fluid and blood and has been reported to have a markedly decreased concentration in certain diseases, particularly in diabetes (Yasuo Akanuma, Kazuyuki Tobe: Japanese Internal Medicine). Journal of the Society, 80 1198 to 1204 19
91), which is regarded as important as a diagnostic marker for diabetes.

As a conventional method for measuring 1,5-AG, a method by gas chromatography (diabetes 25
Vol., 1115-1118, 1982, Yoshioka. Below, G
It is called the C method. ) And pyranose oxidase (hereinafter,
It is called PROD. ) Or a method using L-sorbose oxidase (Japanese Patent Laid-Open No. 185397/1988; hereinafter, such a measuring method is referred to as an enzyme method).

Specimens to be subjected to 1,5-AG measurement are mainly serum or plasma of diabetic patients. The glucose concentration in the blood of the diabetic patient is higher than that of a healthy person, and the value is about 60 to 100 mg / dl in the healthy person, whereas it is 100 to 1,000 in the diabetic patient.
It is widely distributed in the range of mg / dl. On the other hand, the blood 1,5-AG concentration was 1.64 to 2.68 mg in healthy subjects.
/ Dl, whereas in diabetic patients it is 0.18
It shows a remarkably low value of ~ 0.21 mg / dl (Japanese Clinical 47 Volume 1989 Special Issue Wide Range Blood / Urine Chemistry Immunological Test Vol. 439-442 Kawai). Therefore, the concentration of 1,5-AG in blood of diabetic patients is about 1/470 or less of glucose. In addition, since glucose and 1,5-AG are close in structure, selective measurement in the coexistence is not possible according to the current state of the art, and glucose is selectively removed or modified appropriately. Sample pretreatment is essential.

In its pretreatment, the GC method requires labeling of 1,5-AG in addition to glucose removal,
This is a complicated procedure and requires a long time for analysis. Therefore, it is difficult to measure a large number of samples by the GC method, and there is a problem in applying it as a clinical quantitative method.

In the measurement by the enzymatic method, glucose is removed as a pretreatment by using an ion exchange column, or glucose is modified by phosphorylation. The disadvantage of these operations is that the removal by the ion exchange column is extremely complicated. On the other hand, in the glucose modification by phosphorylation, the optimal conditions for the phosphorylation reaction and the optimal conditions for the 1,5-AG quantitative reaction are different, including the difference in the optimum pH. Must be carried out under different reaction conditions. In addition, adenosine-5'-triphosphate used for phosphorylation (hereinafter referred to as AT
(Referred to as P) has an inhibitory effect on PROD,
There is a concentration limit to the addition of ATP to the measurement system in order to accelerate the phosphorylation reaction, and it was difficult to terminate the phosphorylation reaction promptly. In any case, rapid quantification is impossible, and in particular, it has not been applied to an automatic analyzer widely used for various clinical tests.

[0007]

DISCLOSURE OF INVENTION Problems to be Solved by the Invention By solving the above-mentioned drawbacks of the conventional 1,5-AG measuring methods,
It is an object of the present invention to provide a method that allows simple and rapid measurement of -AG without the need for a separation operation such as filtration, centrifugation, or adsorption, and a method that can be performed using an automatic analyzer.

[0008]

Means for Solving the Problems The inventors of the present invention have made extensive studies to solve the above problems, and as a result, have found that PROD to act on 1,5-AG in the measurement by an enzymatic method is more suitable than a conventionally used enzyme. By selecting such a compound, we succeeded in solving the conventional difficulties such as avoiding the inhibition reaction of PROD by ATP and synchronizing the phosphorylation reaction condition with the condition of 1,5-AG quantitative reaction,
The present invention has been completed.

That is, the present invention uses Basidiomycetus Fungi as PROD.
ous fungi ) No. The first gist is a method for quantifying 1,5-AG using PROD derived from 52, and saccharides other than 1,5-AG in a sample are selected by phosphorylation so that 1,5-AG remains. Of the 1,5-AG contained in the sample obtained by removing the basidiomycetus fungi No. The second gist is a method of quantifying 1,5-AG using PROD derived from 52, and the selective removal of saccharides other than 1,5-AG described above and the subsequent quantification of 1,5-AG are carried out at pH6. The third gist is the method for quantifying 1,5-AG carried out in the range of 1 to 9, and the selective removal of saccharides other than 1,5-AG is carried out using a large excess of ATP, and then 1,
The fourth gist is a method for quantifying 1,5-AG for quantifying 5-AG.

The present invention will be described in detail below. The sample in the present invention is not particularly limited as long as it is desired to measure the 1,5-AG concentration, and examples thereof include serum or plasma as already mentioned. PROD used in the present invention is Basidiomycetus fungi ( Basidiomyc)
et . fungi ) No. 52 derived PROD
(Hereinafter, particularly referred to as Bf-PROD). Basidiomycetaus Fungi No. 52 is Japanese Patent Laid-Open No. 2-429
June 27, 1988 as described in Japanese Patent Publication No. 80
A basidiomycete strain deposited at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology as Micromachine Research Institute No. 10106
gric. Biol. chem. , 54 (3) 799-
801 (1990) and Agric. Biol. che
m. , 54 (6) 1393-1399 (1990), which is a well-known strain. This strain grows aerobically at pH 3-8 and has an optimum growth temperature of 30.
It is a strain in which asexual spores such as conidia and sexual spores such as basidispores are not observed. Bf-PROD obtained from these cells had a molecular weight of about 300,000 as measured by gel filtration method and an isoelectric point of 6.2 as measured by focus electrophoresis method, and was stained red-purple by PAS staining method. 1,5-AG, which is a glycoprotein that has been confirmed by the present inventors.
Enzymatic properties of the optimum pH of 7.0-8.0,
The enzyme has an active pH range of 6 to 9, and the enzyme has an active temperature range of 25 ° C to 40 ° C. In addition, the inhibitory effect of ATP on the 1,5-AG oxidation reaction of the enzyme It is difficult to receive.

When quantifying 1,5-AG in a sample, first, saccharides other than 1,5-AG present in the sample are selectively removed. The saccharides other than 1,5-AG in the sample mainly refer to glucose, but other saccharides phosphorylated by hexokinase are also targeted. For selective removal of saccharides in a sample, it is preferable to select a method capable of eliminating saccharides such as glucose in a reaction solution without depending on any solid phase for application to an automatic analyzer. As such a method, conventionally known methods include acid decomposition of sugars with 6N hydrochloric acid, reduction treatment with sodium borohydride, conversion of glucose to gluconic acid using glucose oxidase, and hexokinase. There is a method of phosphorylating saccharides by using them. Among them, substances and reaction products involved in the selective removal reaction of saccharides can be used without affecting the 1,5-AG quantitative reaction. Most preferred is a method of phosphorylating saccharides using hexokinase, which allows the reaction to be terminated in a short time. It should be noted that what is collectively referred to as an automatic analyzer in the present invention is, for example, Hitachi 7050 type, Hitachi 705 type, Hitachi 736 type, etc., but not limited to the exemplified models. Any of these may be used.

The hexokinases used for phosphorylating sugars, including the conversion of glucose to glucose-6-phosphate, are ECs according to the classification of the International Union of Biochemistry and EC
It is preferable to use those classified as 2.7.1.1. In this conversion reaction, ATP and magnesium ions are used together with the enzyme. As a source of magnesium ions, an organic acid salt such as a fatty acid salt of magnesium, a halogenated salt, a sulfate, a nitrate, an inorganic acid salt such as a phosphate can be used, and among them, acetate is preferable,
Hydrochloride and the like.

Suitable amounts of hexokinase, ATP and magnesium ion used in the above reaction are 5 to 100 U / ml for hexokinase and 5 to 500 for ATP.
mM and magnesium ion are 5 to 50 mM. The optimum pH of the reaction is 7.5, but a pH range of 6-9 is acceptable. In the present invention, as PROD, Basidiomycetaus Fungi No. 52-derived PROD 1,5-
It is used for the quantification of AG, and PROD is less susceptible to the inhibitory action of ATP. Therefore, during the saccharide removal reaction, a large excess of ATP, for example, 100 mM to 500
The removal reaction was performed using the amount of mM. Then AT as it is
It can be subjected to an enzymatic reaction with PROD without removing P. Further, Basidiomycetus fungi No. used in the present invention. Since 52-derived PROD enzymatically reacts with 1,5-AG in the pH range of 6 to 9, it is possible to quantify 1,5-AG in the same pH range as the above-mentioned saccharide removal reaction. Therefore, according to the present invention, the saccharide removal reaction and the 1,5-AG enzymatic reaction can be carried out under the same pH conditions, for example, in the range of pH 7.0 to 8.0, and both reactions can be carried out for an extremely short time. Can be carried out continuously. Therefore, the method for quantifying 1,5-AG of the present invention can be carried out extremely efficiently by an automatic analyzer.

The 1,5-AG remaining in the sample from which sugars have been selectively removed is measured by using PROD.
Hydrogen peroxide is generated by causing PROD to act on 1,5-AG. The hydrogen peroxide is converted into peroxidase, that is, EC 1.
Using the enzyme classified as 11.1.7, 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid), o-phenylenediamine, 5-aminosalicylic acid, 3,3 ′, 5 5'-tetramethylbenzidine and 4-aminoantipyrine and N-ethyl-N- (2-
Hydroxysulfopropyl) -m-toluidine and other known substrates for peroxidase.
The dye produced from the substrate is measured for absorbance. The peroxidase used to measure hydrogen peroxide is preferably horseradish peroxidase. Substrates that produce the dye used for the absorbance measurement are 4-aminoantipyrine and N-ethyl-N- (2-hydroxysulfopropyl)-
A combination of m-toluidine is preferred. The wavelength of the absorbance measurement range when using 4-aminoantipyrine and N-ethyl-N- (2-hydroxysulfopropyl) -m-toluidine is 500 nm to 800 nm, and in this range, two or more wavelengths are used. It can also be measured.

A suitable amount of PROD, horseradish peroxidase, 4-aminoantipyrine and N-ethyl-N- (2-hydroxysulfopropyl) -m-toluidine used in the above reaction is 5 for PROD.
~ 500 U / ml, horseradish peroxidase 2-20 U / ml, 4-aminoantipyrine 0.
1-10 mM, N-ethyl-N- (2-hydroxysulfopropyl) -m-toluidine is 0.1-10 mM. Further, the pH range effective for the reaction is around 7.5, that is, a range of 6 to 9 as with hexokinase.

In the whole reaction for measuring 1,5-AG in a sample, the reaction temperature is 5 to 40 ° C., preferably 25 to 4
0 ° C., reaction time 2 to 60 minutes, preferably 2 to 3
0 minutes. When preparing the reaction mixture, the entire reaction was adjusted to pH 7.
Since the reaction proceeds from 0 to 8.0, preferably from 7.5 to 8.0, the pH of the reaction solution which is a buffer solution among the reaction solutions constituting the reagents is 7.0 to 8.0. In order to stabilize in the range of preferably 7.5 to 8.0 and the maximum width of 6 to 9, a phosphate buffer, a Tris hydrochloride buffer, a HEPES buffer or the like is used as a buffer. HEP
In the case of ES buffer, a concentration of 50 to 500 mM is preferable. Further, for controlling the ionic strength, an alkali metal halide salt, preferably sodium chloride, can be used. As PROD, Bf-PROD described in JP-A-2-42980 was used, and the whole reaction was carried out at pH6.
-9, preferably pH 7.0-8.0, the phosphorylation reaction of saccharides in the sample such as glucose and the color reaction for measuring the generation of hydrogen peroxide due to the action of PROD are both performed for 5 minutes. It can be finished within.

When measuring 1,5-AG by this method,
The above-mentioned components may be added to one solution and used, or the components may be divided and used so as to form an appropriate combination. Although they may be in the form of a solution or freeze-dried, they are preferably freeze-dried when intended for long-term storage. In addition, a surfactant can be added within a concentration range that does not inhibit the measurement reaction, and when the measurement system is freeze-dried, an appropriate amount of a stabilizer may be added.

[0018]

EXAMPLES The present invention will now be described in more detail with reference to examples. Of course, the invention is not limited by these examples. Example 1 Optimal pH of PROD for 1,5-AG The optimal pH of Bf-PROD for 1,5-AG was measured, and the results are shown in FIG. The reaction conditions at the time of measurement are as follows. 1000 U / 1 PROD solution 10
280 μl of a first reagent having the following composition and 70 μl of a second reagent were reacted with μl to obtain a main wavelength of 546 nm and a sub wavelength of 700 nm by a Hitachi 7150 type automatic analyzer, and the absorbance change between 30 and 40 photometric points by the function of the analyzer. The rate was tracked. Maximum absorbance change rate (p
(At H7.5) was set as 100% and shown as relative activity. As shown in Figure 1, PROD shows maximum activity at pH 7.0-8.0 against 1,5-AG in Good's buffer.

[0019] The first reagent MES 200 mM NaCl 0.99 mM magnesium acetate 10 mM N-ethyl -N- range of the first reagent pH 5.5 to 6.5 (2-hydroxy-sulfopropyl) - m-toluidine 1 mM par Oxidase 5 KU / l hexokinase 20 KU / l pH 1 the first reagent in the range of 6.5-7.5 PIPES 200 mM sodium chloride 150 mM magnesium acetate 10 mM N-ethyl-N- (2-hydroxysulfopropyl) -m- Toluidine 1 mM peroxidase 5 KU / l hexokinase 20 KU / l pH 1 The first reagent in the range of 7.5 to 8.5 HEPES 200 mM sodium chloride 150 mM magnesium acetate 10 mM N-ethyl-N- (2-hydroxysulfopropyl ) -M-Toluidine 1 mM Okishitaze 5 KU / l hexokinase 20 KU / l

Second reagent 4-aminoantipyrine 4 mM sodium chloride 150 mM 1,5-AG 100 mM without buffer

Example 2 Inhibition of ATP on PROD
Changes in pH The extent to which the response of Bf-PROD to 1,5-AG was affected by ATP was pH 5.5 in Good's buffer.
It examined in the range of 5. 1000 U / l PROD solution 1
280 μl of 1st reagent of the following composition and 70 μl of 2nd reagent were reacted with 0 μl, and the main wavelength was 546 nm and the sub-wavelength was 700 nm by Hitachi 7150 type automatic analyzer, and the absorbance change between 30-40 photometric points by the function of the analyzer The rate was tracked. The degree of inhibition by ATP is based on the PROD activity in each case where ATP does not exist under the condition that 100 mM ATP is newly added to the buffer of each pH, and the PROD activity in the case where ATP exists in the same pH is relative. The results obtained as activity are shown in FIG. Figure 2
As shown in (3), PROD was not inhibited by ATP at all in the Good's buffer, but rather the activity was enhanced, and this tendency became more remarkable as the pH was increased. Figure 1
From the results of Fig. 2 and Fig.
It is clear that in the optimum pH range of hexokinase from 7.0 to 8.0, Bf-PROD is not inhibited by ATP at all, but rather is enhanced and shows good reactivity to 1,5-AG. Therefore, glucose can be efficiently phosphorylated at this pH range, preferably at pH 7.5, and a good 1,5-AG detection reaction can be performed subsequently.

First reagent MES 200 mM sodium chloride 150 mM magnesium acetate 10 mM N-ethyl-N- (2-hydroxysulfopropyl) -m-toluidine 1 mM per range of pH 5.5-6.5. Oxidase 5 KU / l hexokinase 20 KU / l ATP 0 mM or 100 mM pH 1st reagent in the range of 6.5-7.5 PIPES 200 mM sodium chloride 150 mM magnesium acetate 10 mM N-ethyl-N- (2-hydroxy Sulfopropyl) -m-toluidine 1 mM peroxidase 5 KU / l hexokinase 20 KU / l ATP 0 mM or 100 mM pH 1st reagent in the range of pH 7.5 to 8.5 HEPES 200 mM sodium chloride 150 mM magnesium acetate 10 mM N-ethyl -N- (2-hydroxysulfopropyl) -m-toluidine 1 mM peroxidase 5 KU / l hexokinase 20 KU / l ATP 0 mM or 100 mM

Second reagent 4-aminoantipyrine 4 mM sodium chloride 150 mM 1,5-AG 100 mM without buffer

Example 3 Measurement reaction time in the present invention
When the course physiological saline blank, 5 mg / dl 1,5-AG aqueous solution, and two types of human serum (samples 1 and 2) were used as specimens and reacted with the first and second reagents having the following compositions The reaction time course was measured, and the obtained results are shown in FIG. The reaction conditions are: sample volume 7 μl, first
The reagent volume was 280 μl, the second reagent volume was 70 μl, and the two-point assay was performed using a Hitachi 7150 type automatic analyzer with a main wavelength of 546 nm and a sub-wavelength of 700 nm. The measurement reaction is almost completed in 5 minutes.

First Reagent HEPES 200 mM Sodium Chloride 150 mM Magnesium Acetate 10 mM N-Ethyl-N- (2-hydroxysulfopropyl) -m-Toluidine 1 mM Peroxidase 5 KU / l Hexokinase 20 KU / l ATP 100 mM pH 7.5

Second reagent HEPES 200 mM sodium chloride 150 mM 4-aminoantipyrine 4 mM PROD 62.5 KU / l pH 7.5

Example 4 1,5-AG calibration curve and sample
The glucose scavenging property of 0.5 to 5 mg / dl of 1,5-AG aqueous solution prepared in increments of 0.5 mg / dl was reacted under the measurement conditions of Example 3 and the respective absorbances were measured. The results are shown in Fig. 4. In addition, 0 to 15 prepared in 150 mg / dl steps
Absorbance was measured in the same reaction with a glucose aqueous solution of 00 mg / dl. The results are shown in Fig. 4. As can be seen from FIG. 4, 5 mg was obtained when 1,5-AG was reacted.
It showed good linearity through the origin up to / dl. When glucose was reacted, the absorbance up to 1500 mg / dl was almost the same as that of the reagent blank, and glucose was 150
Almost completely erased up to 0 mg / dl. Therefore, it can be judged that the present measurement conditions have sufficient glucose scavenging properties and can perform favorable 1,5-AG quantification.

Example 5 Addition recovery test A sample prepared by using patient sera having a known content of 1,5-AG and adding 1,5-AG to them was used in Example 3 above.
The reaction was carried out under the measurement conditions of, and 1,5-AG was quantified using the calibration curve obtained in the above example. The value obtained by dividing the actually obtained quantitative value (actual measurement value) by the value (theoretical value) which is the sum of the 1,5-AG content of the patient serum and the later added 1,5-AG The recovery rate. The results are shown in Table 1.

[0029]

[Table 1] The average recovery rate is almost 100%, which shows that this measurement method has high accuracy.

Example 6 Correlation with conventional assay method An enzyme method (hereinafter column enzyme) in which glucose is removed by using an ion exchange column, which has been regarded as a highly reliable assay method for 1,5-AG up to now. Method) and the measured values of the enzymatic method of the present invention were investigated. The column enzyme method was carried out by using Nippon Kayaku Co., Ltd. Lana AG (registered trademark) and human serum 57
The sample was measured as described in the instruction manual.
Regarding the enzyme method of the present invention, the calibration curve prepared in Example 4 was used, and the same human serum as used in the column enzyme method was used as a sample to perform the measurement in the same manner as in Example 3. The result is shown in Figure 5.
As shown in. The correlation coefficient was 0.97, and a high correlation was recognized between both measurement methods.

[0031]

As shown in the above examples, the method for measuring 1,5-AG according to the present invention is suitable for application to an automatic analyzer. That is, the method of the present invention enables automation of 1,5-AG measurement, which has been impossible in the past, and can process a large number of specimens rapidly, accurately, and in a large amount like many other clinical test items. Is now possible.

[Brief description of drawings]

FIG. 1 Basidiomycetous fung in Good's buffer.
i ) No. It is a figure which shows the optimal pH with respect to 1,5-AG of PROD derived from 52.

[Figure 2] ATP's Basidiomycetus Fungi ( Ba
sidiomycetousfungi ) No. It is a figure which shows the change in pH of the inhibition degree with respect to 52-derived PROD.

FIG. 3 is a diagram showing a measurement reaction time course in the present invention.

FIG. 4 is a diagram showing a calibration curve of 1,5-AG. It is a figure which also shows the glucose elimination capability of the reaction system.

FIG. 5 is a diagram showing the correlation between the method of the present invention and the column enzyme method.

Claims (5)

[Claims] 1. A method of quantifying using pyranose oxidase 1,5-anhydroglucitol in a specimen, Basi geo My Seta Usu-Funghi as pyranose oxidase (Basidiomycetous fu
ngi ) No. A method for quantifying 1,5-anhydroglucitol using 52-derived pyranose oxidase. 2. Pyranose oxidase is allowed to act on 1,5-anhydroglucitol in a sample at a pH of 6 to 9, and 1,5-anhydroglucitol is converted from the amount of hydrogen peroxide produced. The method for quantifying 1,5-anhydroglucitol according to claim 1, which is quantified. 3. A saccharide other than 1,5-anhydroglucitol existing in a sample is phosphorylated with hexokinase and adenosine-5′-triphosphate to give 1,5-an in the sample. 3. The 1,5-anhydroquinone according to claim 1, wherein the saccharide is selectively removed so that hydroglucitol remains, and then 1,5-anhydroglucitol in the obtained sample is quantified. Determination of hydroglucitol. 4. The method for quantifying 1,5-anhydroglucitol according to claim 3, wherein the selective removal of sugars other than 1,5-anhydroglucitol is carried out within a pH range of 6 to 9. 5. The method for quantifying 1,5-anhydroglucitol according to claim 3, wherein adenosine-5'-triphosphate is used in a large excess.