JPH0761280B2 - Simultaneous measurement of glucose and 1,5-anhydroglucitol - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION <Industrial Field of Application> The present invention relates to glucose, which is a diagnostic marker for diabetes, and 1,5-anhydroglucitol (hereinafter, 1,5), which is expected to be put to practical use as a new diagnostic marker. -AG) and a simultaneous measurement method using a biosensor.

<Prior Art> Glucose has long been known as a diagnostic marker for diabetes. In particular, the blood glucose level is a measurement item that is most frequently used for diagnosing diabetes or determining the therapeutic effect, and various measurement methods including the biosensor method have been put to practical use.

On the other hand, 1,5-AG is a compound which is present in human cerebrospinal fluid, plasma and urine and has been reported to decrease in plasma in diabetes. Conventionally, the method for quantifying 1,5-AG has been mainly performed by gas chromatography (diabetes, 25, 1115-1118, 1982, Yoshioka et al.). Recently, a method has been devised in which glucose is removed from plasma, 1,5-AG is oxidized with pyranose oxidase (hereinafter referred to as PROD), and hydrogen peroxide produced at that time is quantified by colorimetry.

<Problems to be Solved by the Invention> Glucose and 1,5-AG in body fluids are the same marker for diabetes, but are measured separately.

That is, glucose is measured by a glucose sensor, dry chemistry or the like, and 1,5-AG is measured by a gas chromatography method, a colorimetric method or the like. 1,5-AG requires pretreatment of the test solution and labeling of 1,5-AG in the measurement by gas chromatography. Therefore, the analysis requires a long time, and it is difficult to measure a large number of test liquids, which is a problem as a clinical quantification method. Also, using PROD 1,5
-The colorimetric method of AG also requires complicated pretreatment, and remains a problem as a clinical measurement method. Also glucose and 1,5-AG
Is a marker having a different clinical significance, is useful for diagnosing diabetes and determining the therapeutic effect, and a more rigorous determination can be made by simultaneously measuring both. However, there is no example in which both are easily measured simultaneously.

<Means for Solving Problems> PROD is conventionally known as an enzyme that oxidizes saccharides mainly containing glucose.

Recently, this enzyme is an anhydrous cyclic sugar alcohol, 1,5-AG
Was also found to be oxidized, and a colorimetric assay for 1,5-AG was developed. In this method, 1,5-AG alone is quantified by completely removing sugars in blood.

The present inventors positively utilized the above findings and investigated various species of methods for simultaneous quantification of glucose and 1,5-AG.

PROD has low substrate specificity as an enzyme and is known to react with various kinds of saccharides. For example, D-xylose and L
-Reacts with sorbose, D-gluconolactone, etc. (Enzyme Handbook, P66, Asakura Shoten).

Therefore, it is difficult to detect only glucose and 1,5-AG in body fluid, and it is very difficult and not easy to isolate each body fluid component.

However, as a result of various studies, when measuring glucose and 1,5-AG in body fluid such as blood, glucose and 1,5-AG were separated using a column and quantified by a biosensor using PROD. In this case, it was found that glucose and 1,5-AG can be quantitatively and accurately detected even when other saccharides coexist, and the present invention has been completed.

That is, the present invention relates to glucose and 1,5-A in a body fluid sample.
The present invention relates to a simultaneous measurement method for glucose and 1,5-AG, which comprises quantifying G using a biosensor using PROD after separating G on a column.

By the way, the blood glucose level, that is, the blood glucose level, changes depending on the state at the time of measurement, so the fasting blood glucose level, which is relatively stable, is used as an index, and 100 mg / dl or 1 mg / ml or less in a normal person. It is known to go up above. Also,
The 1,5-AG value does not change much depending on the condition, and is 20 to 50 in normal people.
It is said to be 10 μg / ml or less in diabetes. In order to measure both of them simultaneously, the dynamic range of the detector needs to be 1000 to 10000 times, which is impossible by the measuring method such as colorimetry. However, the biosensor using PROD according to the present invention has a wide dynamic range and can measure both at the same time. That is, the present invention has been accomplished by skillfully combining a specific separation method, a wide dynamic range and a specificity of a specific biosensor.

The column for separating glucose and 1,5-AG used in the present invention is a sugar used in a solvent system that does not affect the detection of the biosensor or a solvent system that can be changed to a solvent system that does not affect the detection of the biosensor. A separation column can be used. In the sugar separation column, separation is performed based on the principle of molecular size separation, ion exclusion separation, ligand exchange separation, partition separation, etc., but it is possible to further improve the separation by combining them. Various columns can be used as the column used in the present invention, but preferable examples include anion exchange columns based on the principle of ligand exchange, and specifically, HPIC-AS manufactured by Dionex.
6, AS7, ^# 3013-N manufactured by Hitachi, Ltd., and the like.

The mobile phase (solvent system) is preferably an aqueous system, but a system containing an organic solvent such as alcohol or acetonitrile can be used as long as it does not inactivate the enzyme PROD of the biosensor. Further, even if PROD is inactivated under the separation condition, any one can be used as long as it can be changed to the enzyme reaction condition after the column separation. For example, HPIC-AS6 (Dionex column) uses an aqueous alkaline solution as a mobile phase (solvent system) at the time of separation, but glucose and 1,
It becomes possible to detect 5-AG.

Examples of the biosensor used in the present invention include a flow cell type detector in which a PROD-immobilized membrane is mounted on the surface of a hydrogen peroxide detection electrode, which is glucose or 1,5
-AG is a sensor that oxidizes by PROD in the presence of oxygen and quantifies the hydrogen peroxide generated at that time by the hydrogen peroxide detection electrode. PROD-immobilized membranes can be manufactured by the commonly used membrane immobilization methods, and any of adsorption, cross-linking and covalent bonding methods can be used as the membrane immobilization method. It is desirable to adopt a method of obtaining a film having good properties. For example, a method for immobilizing an enzyme on a triacetylcellulose membrane with good membrane permeability is described in "New Electrochemistry" (Baifukan) P248 edited by the Electrochemical Society, and a membrane with good hydrogen peroxide permeability is It can be easily manufactured by a known method. To mount the obtained PROD membrane on the electrode surface, a method of pressing it with a nylon net or a method of wrapping it with a membrane having a high substrate permeability such as a dialysis membrane is used.

Further, as the biosensor used in the present invention, there is also a type in which an enzyme is immobilized on a water-insoluble granular carrier, a small column is filled with the hydrogen peroxide, and hydrogen peroxide generated by a hydrogen peroxide detection electrode arranged after the small column is detected. Yes, glucose and 1,5-AG can be quantified similarly to the membrane type. Furthermore, as a hydrogen peroxide detection electrode, it is optimal to use a hydrogen peroxide electrode or an electrochemical detector for detecting the generated hydrogen peroxide, but oxygen consumed during enzymatic oxidation is replaced by an oxygen electrode. It is also possible to use and quantify. It is also possible to convert hydrogen peroxide produced by enzymatic oxidation into another compound, for example, potassium ferrocyanide to potassium ferricyanide, and measure it with an electrochemical detector.

PROD used in the present invention is the IUPAC-ICB Nominal Law Commission
There is no particular limitation as long as it can be classified as EC1,1,3,10 or EC1,1,3,11.
-Porus obtusus) those produced by ATCC 26733.

Needless to say, the higher the specific activity of this enzyme, the better it is for quantification, but it does not necessarily require the highest purity.

The test liquid to be quantified in the present invention is not limited as long as it is desired to quantify 1,5-AG and glucose, and examples thereof include plasma, serum, urine, cerebrospinal fluid, which are generally called body fluids. In addition, a sample obtained by removing proteins that coexist in body fluid is also used. Deproteinization methods include methods using acids such as perchloric acid and trifluoroacetic acid, methods using salts and alkalis such as barium chloride and barium hydroxide, methods using organic solvents such as alcohol, or column separation. However, either method may be used, but 1,5-A
A method that does not interfere with the column separation of G and glucose and the enzymatic reaction should be adopted. Further, the pH can be adjusted after deproteinization if necessary.

In the method of the present invention, the body fluid sample is passed through the column as it is or after dilution if necessary, and after deproteinization if necessary (in this case, the flow rate is usually preferably about 0.1 to 2 cc / min), and then this Contact the biosensor. The temperature at which the column effluent is contacted with the biosensor is preferably about 20 to 40 ° C., and
The pH is preferably 5 to 8 positions, more preferably 5 to 7 positions.

<Example> Example 1 The measuring apparatus is a normal high performance liquid chromatograph apparatus,
A 0.1N-NaOH aqueous solution was passed through the column at a flow rate of 0.5 ml / min.
The column is HPIC-AS6 (4.6 mmφ x 25 manufactured by Dionex)
0 mm) was used. The column effluent was mixed with 0.4N-H ₃ PO ₄ aqueous solution supplied at a flow rate of 0.1 ml / min through a mixing joint, and was completely mixed by a mixing coil (0.5 mmφ × 10 m) to a pH of 5.7. . Then, this mixed liquid flow was brought into contact with a biosensor set in a flow cell (volume: 100 μl), and the amounts of 1,5-AG and glucose were quantified by the amount of hydrogen peroxide generated.

The biosensor was used by mounting a PROD immobilization membrane of the same size on a surface (5 mmφ) of a hydrogen peroxide electrode [made by Able Co., Ltd.] with a nylon net. The PROD-immobilized film was produced by the following method.

That is, PROD (5.2U / mg manufactured by Takara Shuzo Co., Ltd.) 10 mg and bovine serum albumin (manufactured by Sigma) 6 mg were added to 1/15 M phosphate buffer (pH 7.
2) Dissolve in 0.6ml and 0.2m of 1% glutaraldehyde aqueous solution
Add l and mix. Immediately after mixing, the mixture was slowly dropped on two nitrocellulose membranes (25 mmφ pore size 3 μm), spread so that the whole was uniform, and air-dried at 4 ° C. overnight.

In the above system, first, 50 μl of a standard solution of 1,5-AG and glucose was injected from an injector provided in front of the column and detected by a biosensor to prepare a calibration curve of 1.5-AG and glucose. The results are shown in FIG. Separately, 15 μl of 60% perchloric acid aqueous solution was added to 200 μl of plasma of diabetic patients and normal persons, and the mixture was shaken and then centrifuged at 3000 rpm × 15 minutes to remove proteins. 40 to 150 μl of the supernatant
A sample was prepared by adding 10 μl of% sodium hydroxide. This sample is 57μ in the same way as when the calibration curve was created.
1 (50 μl as serum content) was injected into the above-mentioned indicator, detected by a biosensor, and quantified from the area value using a calibration curve. The results are shown in Table-1.

In Example 1, passed through a column of 0.05 N-NaOH aqueous solution at a flow rate of 0.5 ml / min as the aqueous NaOH solution also supplies 0.2 N-aqueous H ₃ PO ₄ as aqueous H ₃ PO ₄ at a flow rate of 0.1 ml / min Then, ^# 3013-N (manufactured by Hitachi Ltd., 40φ × 150 mm) was used for the column, and the measurement was performed in the same manner as in Example 1 except for that. The results are shown in Table 1 together with Example 1.

<Effect of the Invention> According to the present invention, glucose and 1, which are markers of diabetes,
5-AG can be measured simultaneously, and diabetes can be easily diagnosed by the indicators of both.

[Brief description of drawings]

 FIG. 1 shows the calibration curve of Example 1.

Claims (1)

[Claims] 1. Glucose and 1,5-anhydroglucitol in a body fluid sample are separated by a column and then quantified by a biosensor using pyranose oxidase. Simultaneous measurement of citrus.