JP3204557B2 - Method for continuous measurement of 1,5-anhydroglucitol - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for continuously measuring 1,5-anhydroglucitol. In a biological fluid by flow-ins Jefferies comb tio emission analysis method is more 1,5
The present invention relates to a method for measuring anhydroglucitol.

[0002]

BACKGROUND OF THE INVENTION 1,5-Anhydroglucitol (hereinafter also referred to as AG) is one of the most concentrated polyols in human serum, and is sharply and specifically reduced in serum of diabetic patients. It is known to be a marker for diabetes.

Conventionally, a method using gas chromatography [Scand. J. Clin. Invest., 42 (1982) 445] has been known for the measurement of AG, but the pretreatment of the sample is complicated and a large number of methods are used. Unsuitable for measuring samples. As a simpler measuring method, an enzymatic method using a specific pyranose oxidase has been developed (JP-A-62-79780). Specifically, a specific enzyme having the ability to oxidize AG is allowed to act on AG in the presence of an electron acceptor, and the resulting 1,5-anhydrofructose or hydrogen peroxide is measured by a known method. Things. Further, saccharides as contaminants in the sample are removed in advance, and pyranose oxidase or L-sorbose oxidase is allowed to act as an enzyme,
Means for measuring the generated hydrogen peroxide with a color-developing system has also been developed (JP-A-63-185397).

[0004]

However, when pyranose oxidase is used as an oxidase, the pyranose oxidase has substrate specificity for saccharides other than AG, and contaminants such as glucose are removed from the sample. There is a need to. Therefore, Japanese Patent Application Laid-Open
In the technique disclosed in -185397, after removing the protein by centrifuging the serum sample or the like,
The contaminants were removed by passing through a column filled with an anion exchange resin and a cation exchange resin (further, a boric acid binding resin). Furthermore, in order to improve the detection sensitivity, the solution passed through the column was concentrated and dried together with the washing solution, then redissolved in distilled water, etc., and pyranose oxidase was allowed to act as a sample solution. A complicated operation such as guiding and measuring was required. Therefore, it takes a long time for the measurement, and it is not satisfactory when multiple samples are processed quickly. The present inventors have conducted intensive studies on a method of continuously treating a series of samples from the step of separating AG and contaminants to the step of detecting AG. Unexpectedly, the difference in elution time between ion exchange resins was found. Can be used to continuously and effectively separate AG from contaminants. When the sample thus separated is continuously passed through an immobilized oxidase column, the reaction between AG and oxidase can be achieved. Progresses to quantitatively generate hydrogen peroxide, and furthermore, when the thus obtained hydrogen peroxide is measured by a light-emitting system, it is possible to continuously measure AG with high sensitivity, precision and accuracy. I found it. The present invention is based on these findings.

[0005]

Accordingly, the present invention provides: (1) 1,5-anhydroglucitol and contaminants;
Passing a biological fluid sample through an eluting ion exchange resin column
1,5-anhydrogluci free from contaminants
Eluted fractions containing Toll and other eluted fractions
Separated by elution, followed by (2) contains no Kyozatsu substance 1,5 Anhidorogu
Elution fractions other than the above-mentioned elution fractions containing lucitol were cut out.
The replacement valve, sent to disposal route, (3) On the other hand, free of Kyozatsu substance 1,5 Anhidoroguru
The above-mentioned elution fraction containing siltol is transferred to the above-mentioned switching valve.
More specifically, an enzyme that reacts with 1,5-anhydroglucitol to generate hydrogen peroxide is sent to an immobilized column, and the eluate from the enzyme-immobilized column reacts with hydrogen peroxide to emit light. The present invention relates to a method for continuously measuring 1,5-anhydroglucitol in a biological fluid, which comprises adding a compound to be tested and measuring the amount of luminescence obtained.

Hereinafter, the present invention will be described in detail. The present invention is particularly implemented by a flow injection analysis method (hereinafter, also referred to as FIA method). In the FIA method, a carrier liquid composed of an appropriate solution or a reaction solution is continuously supplied at a constant flow rate into a pipe, and a constant amount of a sample is intermittently or intermittently injected into the continuous carrier flow to continuously perform the injection. An intermittent sample zone is formed in the carrier stream, and the carrier stream containing this sample zone is passed through, for example, a coiled tube to cause an extraction process and a detectable reaction, and finally sent to a detector for quick and simple operation. In order to accurately detect the test substance (or the substance derived from the test substance).

In the method of the present invention, a general FIA method can be used. First, a biological fluid sample is intermittently injected into a continuous carrier flow, and a biological fluid sample is injected into the continuous carrier flow. Form a zone. In the method of the present invention, as a liquid sample, a biological fluid sample, that is, a biological fluid itself or a sample derived from a biological fluid, such as blood, serum, plasma, urine, spinal fluid, or cell extract, or a treatment solution thereof (eg, centrifugation treatment) Solution) is optionally diluted with purified water or a buffer. Further, purified water or a buffer solution is used as the carrier liquid. For injecting a biological fluid sample, any conventional means in the FIA method, such as a manual method using a syringe or a method of intermittently and continuously injecting a plurality of sample liquids with an autosampler, may be used. it can.

Next, the continuous flow of the carrier liquid containing the sample liquid zone is passed through an ion exchange resin column, and the 1,5-anhydroglucitol elution fraction (or AG elution fraction; that is, a fraction containing only AG substantially free of contaminants as a substrate for the immobilized enzyme used in the next step) and isolated from other contaminants. The contaminants in the method of the present invention are substances other than AG, which generate hydrogen peroxide by the action of the immobilized enzyme used in the next step,
It is a substance that generates hydrogen peroxide in an amount that substantially affects the measurement of G. Specifically, sugars having a chemical structure similar to AG, for example, glucose, xylose,
Sorbose or galactose.

[0009] Separation of AG from the above contaminants is carried out by utilizing the difference in elution time between ion exchange resin columns. As the ion exchange resin column, a column packed with an anion exchange resin, particularly, a strongly basic ion exchange resin is preferably used. The retention time of AG and various contaminants in the column to be used can be determined by a simple preliminary test, and from the retention time, the elution time at which an AG elution fraction is obtained is determined.

[0010] The carrier liquid containing the AG elution fraction zone is then sent to a column on which an enzyme that reacts with AG to generate hydrogen peroxide is immobilized. This enzyme is in particular an oxidase, such as pyranose oxidase or sorbose oxidase, which are not restricted by the microorganism from which they originate. AG in the AG elution fraction zone
Is present, hydrogen peroxide and 1,5-anhydrofructose are produced according to the amount of AG by the action of the immobilized enzyme. In order to prepare this immobilized enzyme column, any carrier generally used as a carrier for immobilizing an enzyme, that is, a synthetic material or a natural polymer compound, or an inorganic material such as glass or silica gel can be used. A known method such as a glutaraldehyde method can be used as the method. The immobilized enzyme-carrying carrier thus obtained is
It is used by filling in a column made of glass or stainless steel. In order to increase the reaction efficiency, when performing this enzyme reaction step, it is preferable to heat the enzyme within a range where the enzyme is not deactivated.

A switching valve is provided between the ion exchange resin column and the immobilized enzyme column, and only the carrier liquid containing the AG elution fraction zone is sent to the immobilized enzyme column, and other contaminant fraction zones and the like are provided. We discard the carrier liquid from another route, including. When purified water or the like containing no buffer solution is used as the carrier liquid up to the ion exchange resin column, when the AG elution fraction zone is sent to the immobilized enzyme column, the AG elution fraction zone and the carrier liquid And another carrier solution containing a suitable buffer (eg, phosphate buffer, acetate buffer or citrate buffer) that maintains the optimal pH for the enzymatic reaction can be used. .

Finally, a compound that emits light by reacting with hydrogen peroxide is added to the eluate from the immobilized enzyme column, and the amount of emitted light is measured. Conventionally known various means by a chemiluminescence system can be used for the detection of hydrogen peroxide,
For example, a luminol luminescence reaction, a lucigenin luminescence reaction, a peroxyoxalate / fluorescent inclusion luminescence reaction, or the like can be used. The obtained luminescence amount is detected by a known detector (for example, a luminometer). Thus, the AG in the biological fluid sample can be detected or measured.

One embodiment of an apparatus that can be used in carrying out the method of the present invention is shown in FIG. For example, the purified water carrier liquid is sent from the pipe 11 by the pump 21, and the biological fluid sample is intermittently injected from the sample injector 31. Subsequently, the sample zone is passed through an ion exchange resin column (separation column) 41, and the sample zone is divided into an AG elution fraction from a predetermined elution time zone and other fractions, and is sent to a switching valve 51. In the separation valve 51, the AG elution fraction is sent from the pipe 16 to the immobilized enzyme column 42 together with the buffer sent from the pipe 12 and the pump 22. Fractions other than the AG elution fraction and the carrier liquid from the pipe 11
To a waste tank (not shown). Immobilized enzyme column 42
Is maintained at an optimum temperature for the enzyme reaction by a suitable heater (not shown). The eluate from the immobilized enzyme column 42 is
Cyanide sent from the pipe 13 and the pump 23 through the pipe 17, and the pipe 14 and the pump 2
Luminol sent from 4 is mixed at the intersection 61 of each pipe and sent to the luminometer 71, the detection data is sent to the recorder 72, and the waste liquid is sent from the pipe 18 to a waste tank (not shown).

In the above-mentioned FIA method, a Teflon tube or a stainless steel tube having an inner diameter of about 0.2 to 1 mm used in a normal FIA method is used as a pipe for feeding a sample zone or the like. The pump has a flow rate of 0.1 to 10 m.
Any pump that can supply a constant flow rate that can be variably adjusted within a range of about 1 / min may be used, and it is preferable to use a rotary pump, a plunger pump, or the like.

2 and 3 show specific examples of the liquid separating valve 51. FIG. As shown in FIG. 2, while the sample zone-containing carrier liquid is passed through the pipe 11 through the ion exchange resin column 41 and the AG elution fraction is eluted from the ion exchange resin column 41, the flow holes 1 and 2 of the valve 51 are used. , Flow hole 3 and flow hole 4,
And the flow hole 5 and the flow hole 6 are connected. Since the flow hole 5 and the flow hole 6 are connected, the AG elution fraction is carried into the loop 52. The volume of loop 52 should be sufficient to accommodate the entire volume of the AG elution fraction. During this time, the buffer solution passing through the pipe 12 is directly transferred to the immobilized enzyme column 4.
2 to wash the column.

Subsequently, the ion exchange resin column 41
While fractions other than the G elution fraction are eluted, as shown in FIG. 3, the flow holes 1 and 6 of the valve 51, the flow holes 2 and 3,
And the flow hole 4 and the flow hole 5 are connected. Since the flow hole 1 and the flow hole 6 and the flow hole 2 and the flow hole 3 are connected, the AG elution fraction contained in the loop 52 is transferred to the immobilized enzyme column 42 by the buffer from the pipe 12. Carried. on the other hand,
Since the flow hole 5 and the flow hole 4 are communicated, fractions other than the AG elution fraction are carried from the pipe 15 to the waste liquid tank. By repeating these connection switching operations, the biological fluid sample continuously sent from the pipe 11 is successively divided into an AG elution fraction and other fractions, and the fraction is separated into the immobilized enzyme column 42 and the waste liquid tank, respectively. Can be sent separately. These connection switching operations can be automatically performed mechanically if various conditions such as elution time are determined.

[0017]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto. The FIA system used in the following examples was assembled as shown in FIG. That is, an LC pump (L-6000; Hitachi, Ltd.) is used for feeding the carrier of the pipe 11 and for sending the buffer solution of the pipe 12, respectively, and a reagent pump (for sending the reaction reagent of the pipes 13 and 14) is used. KHU-W-52; Kyowa Seimitsu),
An injector with a 50 μl loop (SV1-6U7; Sanuki) as the injector 31 for sample injection, a hexagonal valve (TVM-6M2; Sanuki) with a loop (inner diameter 0.5 mm; length 1 m) as the liquid separation valve 51, and further as a luminometer 71 A system prepared using a luminometer (LF-500; Nisson) equipped with a flow cell (270 μl) was used. The sample is flowed at 1.0 ml / m from pipe 11
in mobile phase (first carrier; water) and pipe 1
The buffer from (2) (second carrier) has a flow rate of 0.5 ml /
min, a 0.7 mM luminol solution (prepared for use; dissolved in a 0.3 M carbonate buffer at pH 10.5) was sent from the pipe 13 at a flow rate of 0.5 ml / min, and a 200 mM potassium ferricyanide solution (10 Was used at a flow rate of 0.5 ml / min.

Example 1: Preparation of separation column (1) TSKgel SAX as anion exchange resin
(Particle size: 10 μm; Cl type; Tosoh) was packed in a stainless steel column (inner diameter: 4 mm; length: 4 cm) by a slurry method. Next, the packed column was washed with 3 mM NaCl at a flow rate of 0.5 ml / min for 20 minutes, and then with purified water at a flow rate of 1.0 ml / min for 10 minutes. (2) Using the above column, and using water as a mobile phase, AG
(50mg / ml) and glucose (500mg / ml)
The separation of the mixture (both from Sigma) was examined. AG
And glucose were not both retained in Cl form.
Thus, 50 μl of 1 M NaOH was injected five times, and the column was partially converted to the OH form. When this partial conversion column is used, the retention times of AG and glucose are 35 seconds and 3, respectively, at a flow rate of water of 1.0 ml / min.
It was three minutes. A chromatogram using a differential refractometer is shown in FIG. 4 (AG is a peak of 1,5-anhydroglucitol, and GL is a peak of glucose). (3) In order to determine the switching time of the six-way valve in relation to the time change of the AG elution amount, the change of the AG elution amount was examined. As shown in FIG. Since the value reached, the valve switching timing time (switching from FIG. 2 to FIG. 3) was set 45 seconds after the sample injection. FIG. 4 in relation to washing of the separation column
According to the figure, since 5.5 ml of a washing solution is required to completely elute glucose from the separation column, after switching the six-way valve to the state of FIG. 3, washing with purified water at a flow rate of 5.0 ml / min for 100 seconds. Then, the valve was returned to the state shown in FIG. 2, and the flow rate was set to return to the original state. Furthermore, chloride ions are contained in serum samples. If these affect the column, the elution of glucose will be accelerated. Therefore, after processing a certain amount of the sample, it is necessary to flow 1 M NaOH again. In the actual operation, 3 μl of a 30-fold diluted serum was injected 60 times, and then 50 μl of 1 M NaOH was used.
It was also confirmed that l was injected twice.

Example 2 Preparation of Immobilized Enzyme Reactor (1) Pyranose oxidase (derived from Coriolus versicolor; 1150 U / ml; Kyowa Hakko Kogyo Kogyo) was used as the oxidizing enzyme to be immobilized. First, aminopropyl-CPG
(Pore size 59 nm; amine amount 76 μmol / g; particle size 200/400 mesh; CPG) was packed in a stainless steel column (inner diameter 4 mm; length 5 cm) by a slurry method. A 2% glutaraldehyde solution (p
H7.0 in 0.1 M phosphate buffer).
Degassed water flows at a flow rate of 0.5 m at a flow rate of 3 ml / min for 30 minutes.
The column was washed by flowing at 1 / min for 15 minutes. Then
Pyranose oxidase solution (230 U) purified with hydroxyapatite (HCA-100S; Mitsui Toatsu)
/ Ml) at a flow rate of 0.2 ml / min for 3 hours,
Pyranose oxidase was immobilized. Its immobilization rate was about 65%. (2) 0.1M M
The study was carried out using acIlvine buffer (prepared from a 0.1 M sodium monohydrogen phosphate solution and a 0.05 M citric acid solution). The optimum pH was about 4.5. When the relationship between temperature and enzyme activity was confirmed, the enzyme activity gradually increased from 20 to 60 ° C, but was inactivated at 70 ° C. Since the reaction rates were 77%, 66% and 45% at 60 ° C, 40 ° C and 20 ° C, respectively, 50 ° C was selected as the set temperature of the enzyme-immobilized column. For column stability,
When used for 8 hours a day (approximately 200 injections of a sample) and stored at 4 ° C. in 0.1 M phosphate buffer (pH 7.0),
Even after 6 weeks, 90% of the initial activity remained.

Example 3 Measurement of AG Conditions set in Examples 1 and 2 (1 cycle for 3 minutes)
As a result, a calibration curve was prepared using the dilution series of AG.
It showed linearity in the range of 4-200 μM. Further, 5 μM of AG could be quantified with a relative standard deviation of 1.9% (n = 10). The detection limit (S / N = 3) was 0.2 μM (2
ng / 50 μl). A serum sample (3 μl) was diluted 30 times with purified water, ultrafiltered, injected with 50 μl of filtrate, and analyzed for 10 days. 144 μM (2
(3.6 mg / ml) of AG could be quantified with a daily variation of 2.0% and a daily variation of 2.4%. Further, AG was adjusted so that the final concentration of AG was 19.2 to 25.2 mg / ml.
In the analysis of human pooled sera to which was added, the recovery of AG was as good as 97-104%. The FIA method according to the present invention and the conventional batch method [Clin. Chem., 35, (1)
989) 2039]. Correlation plot is y = 1.01x + 0.0
8. The correlation coefficient was 0.9861, showing good agreement over a wide concentration range.

[0021]

According to the method of the present invention, the conventional method for measuring AG in a biological fluid requires a complicated operation and a long time, and is inconvenient for rapidly processing many samples. It is possible to process a multi-sample sample very simply and in a short time (several minutes per sample), which is extremely effective in the field of clinical diagnosis such as a diabetes screening test. Further, since chemiluminescence is used, the measurement sensitivity can be greatly improved, and a more accurate diagnostic index can be provided.

[Brief description of the drawings]

FIG. 1 is an explanatory view schematically showing one example of an apparatus that can be used when carrying out the method of the present invention.

FIG. 2 is an explanatory view of a connected state of a six-way switching valve that can be used in the method of the present invention.

FIG. 3 is an explanatory view of another connection state of the six-way switching valve of FIG. 2;

FIG. 4 is a graph showing the difference in retention time between 1,5-anhydroglucitol and glucose.

FIG. 5 is a graph showing an optimum time for switching a switching valve after a sample is injected.

[Explanation of symbols]

11, 12, 13, 14, 15, 16, 17, 18, ...
Pipes 21, 22, 23, 24 Pump 31 Sample injector 41 Ion exchange resin column 42 Immobilized enzyme column 51 Hexagon switching valve 71 Luminometer 72 Recorder

Continuation of the front page (56) References JP-A-3-47094 (JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12Q 1/25-1/66

Claims (3)

(57) [Claims] (1) 1,5-anhydroglucitol
By passing a biological fluid sample through an ion-exchange resin column that elutes contaminants and 1,5-A
Elution fractions containing hydroglucitol and other
The eluted fractions were sequentially separated and eluted. (2) Subsequently, 1,5-anhydrog
Elution fractions other than the above-mentioned elution fractions containing lucitol were cut out.
The replacement valve, sent to disposal route, (3) On the other hand, free of Kyozatsu substance 1,5 Anhidoroguru
The above-mentioned elution fraction containing siltol is transferred to the above-mentioned switching valve.
More specifically, an enzyme that reacts with 1,5-anhydroglucitol to generate hydrogen peroxide is sent to an immobilized column, and the eluate from the enzyme-immobilized column reacts with hydrogen peroxide to emit light. A method for continuously measuring 1,5-anhydroglucitol in a biological fluid, comprising adding a compound to be tested and measuring the amount of luminescence obtained. 2. The method according to claim 1, wherein the enzyme that reacts with 1,5-anhydroglucitol to generate hydrogen peroxide is pyranose oxidase. 3. The method according to claim 1, wherein luminol is used as the compound that emits light by reacting with hydrogen peroxide.