JPH06343492A - Method for determining 1,5-anhydroglucitol - Google Patents

Abstract

PURPOSE:To provide a determination method applicable to accurate, rapid and automatic analysis of 1,5-anhydroglucitol (1,5-AG) used as a diagnostic morker for diabetes mellitus. CONSTITUTION:In determining 1,5-AG in a sample such as a serum using pyranose oxidase or L-sorbose oxidase, pH at which glucose mixed in the sample is convertgd to a substance which does not react with pyranose oxidase or L-sorbose oxidase is changed to <7 or an acidic substance is previously added to the sample and then, the glucose is converted to a substance which does not react with pyranose oxidase or L-sorbose oxidase.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention is applied to an accurate, rapid and automatic analyzer for 1,5-anhydroglucitol (hereinafter abbreviated as 1,5-AG) used as a diagnostic marker for diabetes. It concerns a possible measurement method.

[0002]

2. Description of the Related Art 1,5-AG is a cyclic polyol having a structure similar to glucose. This 1,5-AG is present in the blood of healthy people at the next highest concentration (after glucose has a constant value for each individual) in glucose, but in diabetic patients, 1,5-AG is present.
Since the concentration of AG specifically decreases and the rate of decrease is remarkable, it has come to be used for the diagnosis of diabetes.

Conventionally, to measure 1,5-AG, 1,
An enzyme that oxidizes 5-AG (hereinafter referred to as 1,5-AG oxidase) was found, and a measurement method using this enzyme (Japanese Patent Publication No.
-24200) was developed. Also, 1,5-A
Although it is a G-oxidizing enzyme, pyranose oxidase or L-sorbose oxidase whose substrate specificity is not strict is used to selectively select sugars other than 1,5-AG having reactivity with these enzymes (hereinafter referred to as contaminating sugars). A measurement method in combination with one of several methods of removal (Japanese Patent Laid-Open No. Sho 6-96
3-185397) was developed.

In Japanese Patent Laid-Open No. 63-185397, as a method for removing contaminant saccharides (mainly glucose in human blood), (1) adsorption with an ion exchange resin (minicolumn), (2) decomposition with hydrochloric acid , (3) sodium borohydride reduction, (4) glucose oxidase oxidation, and (5) hexokinase phosphorylation. Of these methods, the method (1) is the most rigorous and simple, and currently a 1,5-AG assay kit using this method is developed and put on the market as a diagnostic agent. However, considering a busy clinical scene, a kit of a technique requiring column manipulation is not always simple enough, and there is a strong expectation for the development of an automated method.

[0005] Therefore, attention is paid to the enzymatic phosphorylation method of contaminating sugars (mainly glucose) of (5), which does not require column operation and can be applied to a general-purpose automatic analyzer commercially available for biochemical examination. JP-A-1-320998, JP-A-2-104298, and JP-A-3-27.
An improved method is proposed in Japanese Patent Publication No. 299.

[0006]

By the way, glucose in the blood is controlled to an amount of 800 to 1,200 mg / L on an empty stomach and about 1,400 mg / L after a meal.
However, when it becomes diabetic, it rises to more than 3,000.
It is not uncommon to increase from 1 to 4,000 mg / L. 1
It is possible to increase to 50,000 mg / L. On the other hand, 1,
The amount of 5-AG is distributed in the range of 0 to 50 mg / L, and an average of 23 to 25 mg / L in healthy subjects (about 1/4 of glucose)
0), much lower in diabetic patients 4-7 mg on average
/ L, which may be 1 mg / L or less (one-thousandth of glucose). Therefore, it cannot be said that 1,5-AG can be measured with sufficient accuracy unless it is a method capable of completely removing 10,000 mg / L of glucose. That is, in the blood of a diabetic patient with a glucose concentration of 10,000 mg / L, even if 99.99% of glucose can be removed, 1 mg / L of glucose remains and the measured value of 1,5-AG increases.
It cannot be measured accurately.

Therefore, using pyranose oxidase or L-sorbose oxidase, which has a low specificity for 1,5-AG and rather strongly reacts with glucose,
In order to develop a 1,5-AG automatic measuring method that can be measured by a general-purpose biochemical analyzer, a method for completely removing glucose is required.

Further, in JP-A-1-320998, JP-A-2-104298, JP-A-3-27299, JP-A-5-76397, etc., glucose 6-position phosphatase is used for treating glucose. (Glucokinase or hexokinase) is used, and in order to complete this reaction, a scheme has been made to completely shift the equilibrium of the phosphorylation reaction of glucose to glucose-6-phosphate.

With either method, 1,5-AG can be accurately quantified when only 1,5-AG and glucose are used as samples. However, when measuring 1,5-AG in human serum or plasma by these methods,
The measured value varies depending on the storage period from the collection of serum or plasma to the measurement, and the measured value rises over time,
Further, the measured value tends to increase as the glucose amount increases, which is a serious problem.

[0010]

The present inventors have completed a method of the present invention by studying a method for solving the above-mentioned problems by a method applicable to a general-purpose automatic analyzer. That is, the present invention is
(1) When 1,5-anhydroglucitol in a sample is measured using pyranose oxidase or L-sorbose oxidase, glucose in the sample is adjusted to pH 7 with a compound that does not react with pyranose oxidase or L-sorbose oxidase in advance. A method for quantifying 1,5-anhydroglucitol, which comprises enzymatically converting under less than

(2) When measuring 1,5-anhydroglucitol in a sample using pyranose oxidase or L-sorbose oxidase, after adding an acidic substance to the sample to reduce the pH of the sample to less than 7. The present invention relates to a method for quantifying 1,5-anhydroglucitol, which comprises enzymatically converting glucose in a sample into a compound that does not react with pyranose oxidase or L-sorbose oxidase.

The 1,5-AG quantification method of the present invention will be described in detail below. In the present invention, when the glucose in the sample is converted into a compound that does not react with pyranose oxidase or L-sorbose oxidase, or before the conversion, the sample is acidified (pH less than 7) and converted. The present inventors have found that the standard sample does not change with time, but when a sample containing a protein such as serum is used,
We found a phenomenon in which glucose-dependent interference occurs, estimated its cause, and found a solution.

A method in which a sample obtained by adding 10000 mg / L glucose to bovine serum albumin as a sample not containing 1,5-AG can be accurately quantified with a standard sample containing 1,5-AG and glucose (10000 mg / L). When measured with
Quantitative values with large variations were obtained between the measured values of 1 to 10 mg / L. Therefore, when the pH of the sample at this time was changed variously, the quantitative value was low on the acidic side and high on the alkaline side. Since the bovine serum albumin used as a sample does not contain 1,5-AG at all, this quantitative value is estimated to be a measurement of glucose.

A method for removing glucose and protein using a column packed with an ion exchange resin (Lana AG
(Anhydroglucitol) kit (Nippon Kayaku Co., Ltd.)
Manufactured)), the measured values are almost 0
It was mg / L. Therefore, it was found that the substance that raises the measured value can be removed by the column treatment.

As described above, the cause of variation in measured values when a sample containing protein such as serum is used is that protein and glucose are reversibly bound to each other and are affected by the enzyme during the enzyme conversion reaction of glucose. However, it is considered that a part of glucose was dissociated during the quantitative reaction of 1,5-AG and the color was quantitatively determined. The reversible bond between this protein and glucose is presumed to be a Schiff base bond between the amino group on the lysine side chain of the protein or the amino group at the amino terminus of the protein and the aldehyde group of glucose. It is known that the Schiff base is alkaline and its equilibrium is larger when it is produced, and that it is deviated in the opposite direction when it is acidic.

Therefore, under conditions of high pH, protein and glucose form a Schiff base bond, are not reacted during the glucose conversion elimination reaction, and are regenerated by the quantitative reaction of 1,5-AG. Therefore, the measured value is expected to rise. On the other hand, it is known that the pH of serum, plasma, etc. increases when left standing. From this fact,
The 5-AG measurement value increases with time and also increases with the amount of glucose.

In the present invention, since the pH during the glucose conversion reaction in a sample such as serum or plasma is low,
Alternatively, since the pH is lowered by adding an acid to the sample, the bond of the Schiff base is dissociated and separated into protein and glucose, so that the glucose conversion reaction proceeds completely and the 1,5-AG
Can be measured accurately.

In the quantification method (1) of the present invention, the pH range during the glucose conversion reaction is less than 7, preferably 5, 5 to 6, 5. In this case, as the substance for controlling the pH, it is preferable to use a substance having a buffer action rather than simply using an acid. For example, a phosphate buffer, an acetate buffer, a citrate buffer, a succinate buffer, a Good Buffers (eg MES, Bis-
Tris, ADA, PIPES, ACES, MOPS
O, MOPS, etc.), but is not particularly limited as long as it has a buffering action in the range of pH less than 7. The concentration is preferably within the range of 5 to 200 mM which is usually used.

In the quantification method (2) of the present invention, when the pH of the sample is lowered, the pH range of the sample is less than 7, preferably 2 to 6. The acidic substance used for lowering the pH is not particularly limited, and examples thereof include inorganic acids such as hydrochloric acid, sulfuric acid and phosphoric acid, and organic acids such as acetic acid, propionic acid, citric acid, malic acid, tartaric acid, succinic acid and maleic acid. Although it can be used, an acidic substance having an ion that causes enzyme inhibition such as glucose converting enzyme, 1,5-AG oxidase or peroxidase cannot be used.

In the present invention, the glucose conversion reaction can be carried out according to a known method and is not particularly limited, but using glucose 6-position phosphatase (glucokinase or hexokinase) and ATP (adenosine triphosphate), The method of converting to glucose-6-phosphate is preferred. In addition, in order to allow this reaction to proceed completely, a device for regenerating ATP from ADP using another enzyme and a substrate, or a product glucose-6-phosphate using another enzyme, 6-phosphoglucose was used. Nolactone or fructose-6-phosphate and further fructose-2,6
Although any method for converting into diphosphoric acid has been devised, any method can be adopted.

These reactions are usually carried out at 5 to 40 ° C., but they are often carried out at 37 ° C. in an automatic measuring machine. The reaction time is about 1 minute to 1 hour in relation to the reaction temperature, and 3 to 10 minutes is adopted in the automatic measuring machine.

After eliminating glucose in the sample by the above reaction, 1,5-AG was added to pyranose oxidase or L.
-Quantify using sorbose oxidase. This one
A method for quantifying 5-AG is known and disclosed in JP-A-3-24200.
JP-A-63-185397, JP-A-1-3209
It can be carried out according to the method described in Japanese Patent Publication No. 98, etc.

For example, after the glucose in the sample is eliminated by the above reaction, an electron acceptor such as oxygen and pyranose oxidase or L-sorbose oxidase are added thereto, and the temperature is 4 to 50 ° C., preferably 25 to 40 ° C. So 30
After incubating for 2 seconds to 3 hours, preferably for 2 minutes to 1 hour, the reductant of the electron acceptor such as hydrogen peroxide produced is measured, and the 1,5-AG amount can be determined from a separately prepared calibration curve. .

Since the 1,5-AG measurement reaction is carried out subsequent to the glucose elimination reaction, the reaction temperature is often kept as it is. The reaction time is about 1 minute to 1 hour, but it is selected from the relationship between the color development intensity and the measuring method, and 3 to 15 minutes is adopted in the automatic measuring machine.

Pyranose oxidase and L-sorbose oxidase are not particularly limited as long as they can be classified into EC 1.1.3.10 and EC 1.1.3.11 by the nomenclature of IUPAC-IUB, respectively. You can use one. These enzymes are usually 0.5-500 U / mL
Preferably 1 to 50 U / mL is used.

A specific example is as follows. For example, when the reduced form of the electron acceptor is hydrogen peroxide, the method for detecting hydrogen peroxide may be any method as long as it can be detected with high sensitivity, and many methods can be used. The most commonly used method among these is to oxidize various HRP substrates with hydrogen peroxide using horseradish peroxidase (HRP) as a catalytic enzyme. The light substance and chemiluminescence may be measured by absorbance measurement, fluorescence measurement and luminescence measurement, respectively.

As a substrate for HRP which produces a dye,
2'-azinobis (3-ethylbenzothiazoline-6-
Sulfonic acid) (ABTS), o-phenylenediamine (OPD), 5-aminosalicylic acid (5-AS), 3,
3 ', 5,5'-tetramethylbenzidine (TMB),
N- (carboxymethylaminocarbonyl) -4,4 '
-Bis (dimethylamino) -diphenylamine sodium salt (DA-64), 4-aminoantipyrine and phenols, N-ethyl-N-sulfopropyl-m-toluidine or N-ethyl-N- (2-hydroxy-3-)
There is a so-called Trinder-based color-developing agent which is a combination of sulfopropyl) -m-toluidine (TOOS) and the like. As a substrate for HRP that produces a fluorescent substance, p-
Examples include hydroxyphenylacetic acid and 3- (p-hydroxyphenyl) propionic acid (HPPA). Luminol, isoluminol, and the like are known as substrates for chemiluminescent HRP.

Several methods for chemiluminescence detection without HRP are also known. For example, a method of causing luminol to emit light with hydrogen peroxide in the presence of ferricyan ion, a method of emitting lucigenin with hydrogen peroxide in the presence of metal ion, and an allyl oxalic acid such as bis (2,4,6-trichlorophenyl) oxalate. There is known a method of reacting an ester compound with hydrogen peroxide in the presence of a fluorescent substance to excite the fluorescent substance with the decomposition energy of oxalate ester to emit light. Further, as a method for directly detecting hydrogen peroxide, a hydrogen peroxide electrode may be used.

Various samples can be used as the sample used in the present invention, and there is no particular limitation as long as 1,5-AG is to be quantified. For example, body fluids such as cerebrospinal fluid, plasma, serum, urine and plants, Examples include extracts such as animal tissues and deproteinized products thereof.

According to the present invention, glucose and protein can be reversible by performing the glucose conversion reaction in the sample under acidic conditions of less than 7 or by lowering the pH of the sample with an acidic substance. Since glucose is converted by shifting the specific bond in the dissociation direction, glucose is less likely to be dissociated from the protein and push up the measured value during the measurement of 1,5-AG using pyranose oxidase or L-sorbose oxidase. Therefore, an accurate 1,5-AG measurement value can be obtained. Therefore, according to the present invention, even if the sample is stored or measured immediately after collection, the measured value does not change, and 1,5-AG can be quantified with high accuracy.

[0029]

EXAMPLES The present invention will be described in detail below with reference to comparative examples and examples, but the present invention is not limited to these examples.

The following serum (specimen) was used as a sample in both Comparative Examples and Examples. 1,5-AG concentration (mg / L) Glucose concentration (mg / L) Specimen A 28.4 820 Specimen B 14.5 1250 Specimen C 4.6 3120 (The 1,5-AG concentration is Rana AG (anhydroglucan Sitol) kit (manufactured by Nippon Kayaku Co., Ltd.)

After separation of serum, these were dispensed into plastic sample tubes, frozen and stored at -20 ° C, and thawed at the time of use to prepare fresh samples. In addition, the dispensed serum was stored in a refrigerator for one month, and used as stored samples, which were used as Sample D, Sample E, and Sample F, respectively. For the measurement, an automatic analyzer 7150 (manufactured by Hitachi, Ltd.) was used, and the amount of the sample was 10 μl.
Addition and treatment at 37 ° C. for 5 minutes, then 130 μl of reagent 2
The mixture was further treated at 37 ° C. for 5 minutes and measured. The calibration curve has 1,
A standard solution having a 5-AG concentration of 50 mg / L and a physiological saline solution (1,5-AG concentration of 0 mg / L) were used.

Comparative Example 1 The following reagents were prepared and samples A to F were measured. The results are shown in Table 1. The pH of the system when treating the sample with the reagent 1 was about 8. Reagent 1 (pH 8.0) 4-aminoantipyrine 1.5 mM MgCl ₂ 5 mM KCl 50 mM ATP 5 mM glucokinase 2 U / mL ascorbate oxidase 5 U / mL pyruvate kinase 3 U / mL phosphoenolpyruvate 4 mM HEPES buffer 50 mM

Reagent 2 (pH 8.0) TOOS 4.5 mM Pyranose oxidase 100 U / mL HRP 3.75 U / mL HEPES buffer 50 mM

Comparative Example 2 The following reagents were prepared and the samples A to F were measured. The results are shown in Table 1. The pH of the system when treating the sample with the reagent 1 was about 8. Reagent 1 (pH 8.0) 4-aminoantipyrine 1.5 mM MgCl ₂ 5 mM KCl 50 mM ATP 5 mM glucokinase 2 U / mL ascorbate oxidase 5 U / mL glucose-6-phosphate dehydrogenase 12 U / mL lactate dehydrogenase 2 U / mL Pyruvate 2mM NAD 1mM HEPES buffer 50mM

Reagent 2 (pH 8.0) TOOS 4.5 mM Pyranose oxidase 100 U / mL HRP 3.75 U / mL HEPES buffer 50 mM

Comparative Example 3 The following reagents were prepared and samples A to F were measured. The results are shown in Table 1. The pH of the system when treating the sample with the reagent 1 was about 8. Reagent 1 (pH 8.0) 4-aminoantipyrine 1.5 mM MgCl ₂ 10 mM KCl 25 mM KH ₂ PO ₄ 40 mM CaCl ₂ 1.5 mM ATP 15 mM glucokinase 3.5 U / mL ascorbate oxidase 5 U / mL glucose phosphate isomerase 3. 5 U / mL 6-phosphofructokinase 5 U / mL pyruvate kinase 3 U / mL phosphoenolpyruvate 4 mM HEPES buffer 20 mM

Reagent 2 (pH 8.0) TOOS 4.5 mM Pyranose oxidase 100 U / mL HRP 3.75 U / mL HEPES buffer 50 mM

[0038]

Table 1 Comparative Example 1 Comparative Example 2 Comparative Example 3 Sample A 28.3 28.5 28.4 Sample B 14.6 14.6 14.4 Sample C 4.8 4.6 4.5 Sample D 29.5 30.1 29.3 Specimen E 16.2 16.4 16.9 Specimen F 9.8 9.2 10.1 (1,5-AG concentration, mg / L)

Example 1 The buffer solution of the reagent 1 of the comparative example 1 was 50 mM of MES buffer, and the pH of the reagent 1 was adjusted to 6.3. Others were measured in the same manner as in Comparative Example 1. The results are shown in Table 2. The pH of the system when treating the sample with Reagent 1 was about 6.3 in all cases.

Example 2 The buffer solution of the reagent 1 of the comparative example 2 was 50 mM of MES buffer, and the pH of the reagent 1 was adjusted to 6.0. Others were measured in the same manner as in Comparative Example 2. The results are shown in Table 2. The pH of the system during treatment of the sample with Reagent 1 was approximately 6
Met.

Example 3 The buffer solution of the reagent 1 of the comparative example 1 was 20 mM of MES buffer, and the pH of the reagent 1 was adjusted to 5.8. Others were measured in the same manner as in Comparative Example 1. The results are shown in Table 2. The pH of each system when the sample was treated with the reagent 1 was about 5.8.

[0042]

Table 2 Table 1 Example 1 Example 2 Example 3 Specimen A 28.4 28.5 28.6 Specimen B 14.6 14.7 14.4 Specimen C 4.7 4.9 4.7 Specimen D 28.4 28.2 28.3 Specimen E 14.5 14.6 14.6 Specimen F 5.0 4.6 4.8 (1,5-AG concentration, mg / L)

Examples 4 to 6 Samples were prepared by adding 50 μl of 10% acetic acid aqueous solution to 0.5 ml of each of the samples A to F used in Comparative Examples 1 to 3 to adjust the pH to 4.2 to 4.5. The measurement was performed using the reagents used in Comparative Examples 1 to 3. The standard solution and physiological saline solution having a 1,5-AG concentration of 50 mg / L used for the calibration curve were also treated in the same manner. The measurement results are shown in Table 3.

[0044]

[Table 3] Table 3 Example 4 Example 5 Example 6 Specimen A 28.5 28.5 28.6 Specimen B 14.5 14.8 14.4 Specimen C 4.7 4.9 4.8 Specimen D 28.4 28.6 28.7 Specimen E 14.5 14.7 14.6 Specimen F 4.9 4.7 4.9 (1,5-AG concentration, mg / L)

Examples 7 to 9 Samples were prepared by adding 50 μl of 10% malic acid aqueous solution to 0.5 ml of each of the samples A to F and adjusting the pH to 3.7 to 4.0 in the same manner as in Examples 4 to 6. And Example 4
The measurement was carried out in the same manner as ~ 6. The measurement results are shown in Table 4.

[0046]

[Table 4] Table 4 Example 7 Example 8 Example 9 Specimen A 28.5 28.6 28.9 Specimen B 14.6 14.8 14.8 Specimen C 4.7 4.9 4.8 Specimen D 28.5 28.4 28.3 Specimen E 14.5 14.8 14.6 Specimen F 4.8 4.9 5.8 (1,5-AG concentration, mg / L)

[0047]

EFFECTS OF THE INVENTION According to the present invention, 1,5-AG in a sample can be accurately quantified by a quick and simple method regardless of the storage condition of the sample, and measurement by an automatic analyzer is possible. It is possible.

Claims (2)

[Claims] 1. A compound which does not previously react glucose in a sample with pyranose oxidase or L-sorbose oxidase when 1,5-anhydroglucitol in the sample is measured using pyranose oxidase or L-sorbose oxidase. 1. A method for quantifying 1,5-anhydroglucitol, which comprises enzymatically converting the enzyme under pH 7 conditions. 2. When measuring 1,5-anhydroglucitol in a sample using pyranose oxidase or L-sorbose oxidase, an acidic substance is added to the sample to make the pH of the sample less than 7, A method for quantifying 1,5-anhydroglucitol, which comprises enzymatically converting glucose in the enzyme into a compound that does not react with pyranose oxidase or L-sorbose oxidase in advance.