JP2701176B2 - Sugar alcohol quantification methods, columns and kits - Google Patents

Description

The present invention relates to a method for quantifying a sugar alcohol, a column, and a kit. Sugar alcohols such as 1,5-anhydroglucitol (hereinafter, referred to as AG) and myo-inositol (hereinafter, referred to as MI) are substances that have attracted attention as markers of diabetes in recent years.

[Conventional technology]

When quantifying a sugar alcohol in a human body fluid, a sample from which an interfering substance which affects the quantification of saccharides (especially glucose) and proteins usually contained in a large amount is used.

[Problems to be solved by the invention]

The removal of the above interfering substances, particularly the removal of proteins, conventionally requires a centrifugal separation operation, and is complicated. Particularly, when considering clinical application, simplification of the operation is desired. In addition, it is preferable to be able to simultaneously remove saccharides and proteins in order to quantify sugar alcohols in a large number of samples.

[Means for solving the problem]

The present invention provides: (1) A sample containing a sugar alcohol, a protein, and a saccharide is passed through a column packed with a basic anion exchange resin having a protein-removing ability and a saccharide-removing ability. A method for quantifying a sugar alcohol.

(2) A column for removing proteins and saccharides packed with a strongly basic anion exchange resin having a quaternary ammonium group introduced into a hydrophilic gel carrier and an aqueous boric acid solution (3) A basic compound having a protein removing ability and a saccharide removing ability A kit consisting of a column filled with an anion exchange resin and an aqueous solution of boric acid, a reagent for quantifying sugar alcohol and a sugar alcohol for preparing a calibration curve. (4) Protein insolubilization that causes oily precipitate to be formed in a sample containing sugar alcohol, protein, and saccharide The protein is precipitated as an oily precipitate to obtain a supernatant, and then the supernatant is passed through a column packed with a protein insolubilizing agent-adsorbing resin and a basic anion exchange resin, or the protein precipitating agent is used. And a protein denaturant to precipitate the protein as a water insolubilized substance. A method for quantifying sugar alcohol, which comprises passing the solution through a column filled with a resin, and then quantifying the sugar alcohol in the obtained permeate; and (5) quantifying 1,5-anhydroglucitol in blood In this regard, the present invention relates to a method for quantifying 1,5-anhydroglucitol, which comprises sequentially injecting a large number of analytes into a device provided with an interfering substance removal column, an injector, and a biosensor.

The sample used in the present invention is not particularly limited as long as it is expected to be contaminated with proteins and saccharides and contains a sugar alcohol. Examples thereof include body fluids such as serum, plasma, urine, and cerebrospinal fluid, plants, and animals. Extract of the tissue of the above.

Examples of the sugar alcohol include a linear polyhydric alcohol which is a reduced form of a saccharide, an anhydro compound in which these compounds are dehydrated and closed in a molecule, and a cyclic polyhydric alcohol.
Among these sugar alcohols, AG and MI are useful as diagnostic markers for diabetes, and it is known that the serum AG concentration of diabetic patients is significantly reduced and the urine MI concentration is increased. The saccharide means a monosaccharide such as aldose or ketose or an oligosaccharide thereof, and does not include a sugar alcohol.

In the first invention of the present invention, the basic anion exchange resin having a protein-removing ability and a saccharide-removing ability packed in a column does not have a sugar alcohol-removing ability, and has a hydrophilic property without an aromatic ring. A strongly basic anion exchange resin in which a quaternary ammonium group is introduced as an exchange group into a polymer carrier, for example, a hydrophilic gel carrier (polymer gel having a molecular sieve action) is preferable. Examples of the hydrophilic gel carrier include polysaccharides such as dextrin, agarose, cellulose, and chitosan, polyvinyl alcohols, polyethylene glycols, polyacrylamides, and poly (meth).
Acrylic resins and the like, preferably polyvinyl alcohol resins or poly (meth) acrylate resins are mentioned. As the quaternary ammonium group, a trimethylammonium group, a tri-lower (C ₁ -C ₄₎ alkyl ammonium groups such as triethylammonium group, also hydroxy such as hydroxyethyl dimethyl ammonium group lower (C ₁ -
C ₄ ) alkyl di-lower (C ₁ -C ₄ ) ammonium groups.

The ionic form of these strong basic resins is used as a weak acid salt form such as OH form or boric acid form. Preferred resins are Mono Q (Pharmacia), Shodex ™ (Showa Denko), Nucleosil 100-SB (Nagel), Par
tisil 10 SAX (Whatman), Sepralyte SAX, Bon
d Elute SAX (manufactured by Analytical International, Inc.), QAE-Toyopearl (manufactured by Tosoh Corporation), QA-Trisacryl (I
BF), Sepabeads FP-QA (Mitsubishi Kasei), QAE-Sep
hadex, Q-Sepharose (Pharmacia), QAE-Cellu
lose (manufactured by Chisso). More preferable resin is a polyvinyl alcohol-based resin and QAE-Toyopearl having a triethylammonium group.
When using the biosensor described below, poly (meth)
Shodex ™, which is an acrylate resin and has a trimethylammonium group, may also be used.

The amount of these resins used is 1 ml or more, preferably about 2 to 6 ml per 1 ml of the sample. Further, these resins may be used in combination of two or more kinds according to the purpose, and when the saccharide removal information is not sufficient, the resin may be used in combination with a resin having saccharide removal ability described later.

These resins are not stable in the OH form, and when stored for a long period of time, their ability to remove proteins and saccharides decreases.
Normally, the commercially available Cl form is stable, but it is necessary to wash it until neutral after removing Cll with an alkali, which is not preferable because it requires time and effort. The present inventors have found that the use of the boric acid form improves the stability and requires only washing and washing when used. That is, by immersing the OH type or boric acid type resin in a boric acid aqueous solution of such an amount that a portion where the resin is not immersed, preferably a boric acid aqueous solution having a concentration of 0.05-1 M, is obtained for at least one year. Can be prevented from decreasing. Therefore, the quantification method of the present invention can be easily carried out by producing a column packed with both. In addition, since these resins have an ion exchange action, if there is an ionic substance in the sample, ion exchange will occur and the effluent will be alkaline, so a strong cation exchange resin will be used in combination for pH adjustment, for example, for the column. The lowermost layer may be filled, for example, AG50W-x8 (manufactured by Bio-Rad) or A
An ion exchange resin into which sulfonic acid is introduced, such as mberlite CG-120 Type I (manufactured by Rohm & Haas) is used.

For example, the first invention may be implemented as follows. That is, 50 to 100 μl of a sample is passed through the column as it is, and the column is washed 2 to 4 times with 0.5 to 1.0 ml of water, and the sugar alcohol quantitatively recovered in the obtained permeate is subjected to gas chromatography or the like. It may be determined by a conventional method such as an enzymatic method. Depending on the quantification method, it may not be necessary to completely remove proteins and sugars, especially proteins,
In this case, they may be removed to the extent that they do not interfere with the quantitative determination.

Explaining the enzymatic method, an enzyme that specifically reacts with the sugar alcohol to be measured is used. For example, when measuring AG in serum, 1,5-AG oxidation such as pyranose oxidase or L-sorbose oxidase is used. An enzyme is used, and hydrogen peroxide generated by the oxidation reaction of AG may be detected by various methods. When measuring MI in urine, myo-inositol dehydrogenase is used, and each of the coenzyme β-nicotinamide adenine dinucleotide (NAD) or β-nicotinamide denine nucleotide phosphate (NADP) generated by the reaction is used. Reductant NA
What is necessary is just to detect DH or NADPH.

To explain the quantification method in more detail, for example, in the case of AG,
You can do as follows. That is, an electron acceptor and pyranose oxidase or L-sorbose oxidase are added to a sample in an amount of 0.5 to 10 units, preferably 1 to 5 units per 1 ml of the sample, and then at 4 to 50 ° C., preferably 25 to 40 ° C. for 0.5 to 3 hours. It is preferable to incubate for 0.5 to 1 hour, measure the amount of hydrogen peroxide produced, and determine the amount of AG from a separately prepared calibration curve. The details are as follows.

As a method for detecting hydrogen peroxide used in the present invention, any method can be used as long as it can be detected with high sensitivity, and many methods can be used. Of these, the most commonly used method is to oxidize various HRP substrates with hydrogen peroxide using horseradish peroxidase (HRP) as a catalytic enzyme. The light substance and the chemiluminescence may be measured by absorbance measurement, fluorescence measurement and luminescence measurement, respectively. Substrates for HRP that form a dye include 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS), o-phenylenediamine (OPD), and 5-aminosalicylic acid (5-A
S), 3,3 ', 5,5'-tetramethylbenzidine (TMB),
There are 4-aminoantipyrine and various Trinder reagents. Examples of the Trinder reagent include phenol, phenol derivatives such as 3-hydroxy-2,4,6-triiodobenzoic acid (HTIB), N-ethyl-N- (2-hydroxy-
3-sulfopropyl) -m-toluidine (TOOS) and 3,5
-Dimethoxy-N-ethyl-N- (2-hydroxy-3
Aniline derivatives such as -sulfopropyl) aniline (DAOS).

Substrates for HRP that form a fluorescent substance include p-hydroxyphenylacetic acid and 3- (p-hydroxyphenyl).
And propionic acid (HPPA). Also emits chemiluminescence
Luminol, isoluminol and the like are known as HRP substrates. Specifically, for example, as follows: 0.3 ml of sodium phosphate buffer (1/15 M, pH 5.6), 4 mM
2,2'-azinobis (3-ethylbenzthiazoline-
Coloring solution containing 6-sulfonic acid) (ABTS) and 12 units / ml horseradish peroxidase 0.5 ml, 25 units / ml
0.1 ml of a pyranose oxidase or L-sorbose oxidase solution and 0.1 ml of an AG solution are placed in a container at 25 ° C.
After reacting for an hour, the absorbance at 420 nm is measured.
A calibration curve is prepared using an AG solution having a known concentration, and the AG concentration is calculated from the absorbance of the sample.

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

For example, in the case of MI, the following may be performed. That is, after passing through the column, the coenzyme and myo-inositol dehydrogenase were added in an amount of 0.1 to 10 units, preferably 0.5 to 10 units per ml of the sample.
５5 units, 0.5 to 40.550 ° C., preferably 252540 ° C.
Incubate for ~ 3 hours, preferably 0.5-2 hours,
Next, the produced coenzyme reduced form is measured, and the MI amount may be determined from a separately prepared calibration curve.

The reduced form of the coenzyme used in the present invention, for example, NADH or
As a method of detecting NADPH, any method can be used as long as it can be detected with high sensitivity, and many methods can be used. Of these, the most commonly used method is to measure the absorbance or fluorescence intensity of these reduced forms themselves. Further, the reduced form of the generated coenzyme is air-oxidized in the presence of an electron carrier such as femedin methosulfate, 1-methoxy-5-methylphenadium methyl sulfate, and methylene blue, and the generated hydrogen peroxide is treated as described above. Can also be detected. Specifically, for example, as follows.

40 mM sodium carbonate buffer containing 140 mM ammonium sulfate (pH 9.
0) 1.0 ml, 7 mM NAD solution 0.2 ml, 10 units / ml myo-inositol dehydrogenase solution 0.2 ml, distilled water 0.4 ml and MI
0.2 ml of the solution was placed in a container and reacted at 25 ° C for 2 hours.
The fluorescence intensity at an excitation wavelength of 365 nm and a detection wavelength of 450 nm is measured. A calibration curve is prepared with an MI solution having a known concentration, and the MI concentration is calculated from the fluorescence intensity of the sample.

A kit according to the third invention is a column filled with a basic anion exchange resin having the above-described protein-removing ability and saccharide-removing ability and an aqueous boric acid solution, a sugar alcohol quantification reagent comprising a sugar alcohol quantification enzyme and a detection reagent, and One set of sugar alcohols for preparing a calibration curve. The column is a disposable mini-column in which a strongly basic anion exchange resin based on a hydrophilic gelling carrier capable of removing proteins and sugars is filled in an upper layer and a cation exchange resin is filled in a lower layer. The amount of the resin to be packed in this column per use is as follows, for example.

As the enzyme for sugar alcohol quantification, for example, in the case of AG, there is no particular limitation as long as it is an enzyme which can be directly used for quantification of AG, and examples thereof include the aforementioned pyranose oxidase and L-sorbose oxidase. Also, for example,
In the case of MI, there is no particular limitation as long as it is an enzyme that can be directly used for the determination of MI, and examples thereof include the above-mentioned myo-inositol dehydrogenase.

As a reagent for quantifying a sugar alcohol, when the amount of generated hydrogen peroxide is used as an index, peroxidase or a peroxidase-like active substance and a coloring substrate or a coloring agent and a coupler, peroxidase and a fluorescent substrate, peroxidase and a luminescent substrate, Examples include a combination of a ferricyan ion and a luminescent substrate. Specific examples of these reagents are apparent from the description of the above-mentioned "method for detecting hydrogen peroxide".

When the amount of a coenzyme reductant such as NADH or NADPH is used as an index, the coenzyme itself is used, and its own absorbance or fluorescence intensity is measured. On the other hand, if the reduced form of the coenzyme is converted into hydrogen peroxide via an electron carrier, the above-mentioned reagent for detecting hydrogen peroxide can be used.

The amount of the enzyme for sugar alcohol quantification in the kit varies depending on the number of measurable samples such as for 100 samples and 300 samples, but for each sample, for example, when quantifying AG, about 0.5 to 10 units, quantify MI. In this case, about 0.1 to 5 units may be added. When HRP and ABTS are used as reagents for detecting hydrogen peroxide, the amount of
0.10.1 unit, ABTS is 1-20 μM in the kit.

As a method for detecting a reduced form of a coenzyme, when the fluorescence intensity of the coenzyme is measured, the coenzyme is used in an amount of 0.2 to 10 μm per sample.
Put it in the kit so that it becomes M.

In addition, as a sample for preparing a calibration curve, for example, AG
Alternatively, in the case of MI, it is preferable to incorporate 100 to 1000 μM of each into the kit.

In addition, the enzyme for 1,5-AG quantification and the reagent for detection may be mixed to form a single reagent, and if there are components that interfere with each other, the components may be appropriately combined. May be divided. These may be prepared as a solution or a powdery reagent, or may be contained in a suitable support such as paper or film to prepare a test paper or an analysis film.

Next, an automatic quantification method using a biosensor according to the fifth invention will be described. In this method, a sample is injected into a place where water or borate buffer is passed through an interfering substance removal column, and the interfering substance in the sample is adsorbed and removed.
This is a method of directly detecting a passing solution containing a biosensor. Therefore, the device used in the present invention is basically
It consists of an interfering substance removal column through which liquid is passed by using an injector and a pump for injecting a sample, and a biosensor for detecting AG.

As the biosensor used in the present invention, for example,
There is a flow cell type detector in which a membrane on which 1,5-AG oxidase is immobilized is mounted on the surface of a hydrogen peroxide electrode. The AG passing through the flow cell is oxidized by the AG oxidase on the surface of the hydrogen peroxide electrode to simultaneously generate hydrogen peroxide, which is detected by the hydrogen peroxide electrode, and is a sensor for quantifying AG. Also, AG can be quantified at the same time by using a reactor-type sensor using a small column filled with the immobilized enzyme instead of a membrane-type immobilized enzyme.
Furthermore, in the case of this reactor type, since AG is converted into hydrogen peroxide and comes out, it is necessary to use a biosensor using an electrochemical detector capable of detecting hydrogen peroxide instead of the hydrogen peroxide electrode. Can also.

The method for immobilizing 1,5-AG oxidase may be any of the commonly used enzyme immobilization methods such as an adsorption method, an overhead wire method, and a covalent bonding method, but it is a substrate. It is preferable to adopt a method of obtaining a film having good permeability for AG and hydrogen peroxide. In the case of a reactor type, it is preferable to employ a liquid having good liquid permeability.

A biosensor using a hydrogen peroxide electrode electrochemically detects the oxidation-reduction reaction, so if it is a reducing or oxidizing substance, a signal appears and it is detected in the same way as hydrogen peroxide. If such a substance coexists in the sample, only AG cannot be detected. In particular, AG alone cannot be measured because it is disturbed by ascorbic acid, uric acid and the like present in blood. Therefore, in general, a protective film having no permeability to these substances is attached to prevent such substances. However, in the method according to the present invention, an interference substance removal column packed with the above-mentioned strong basic anion exchange resin can be used, and these interference substances can be adsorbed and removed. It became possible to measure with. In addition, the measurement with the biosensor has almost no effect, but since there is a large amount of protein in the sample, the protein may cover the functional group that adsorbs saccharides, which may significantly reduce the processing capacity. Care must be taken with the filler material. Preferably, a hard resin in which a quaternary ammonium group is introduced into a relatively hydrophilic material such as an acrylic material having no strong aromatic ring and a polyvinyl alcohol material as a hydrophilic material is preferable. In addition, the OH-type or boric-acid type resins of these resins also remove blood components that interfere with the biosensor measurement, as described above. It is a removal column. In addition, since these resins have an ion exchange action, if there is an ionic substance in the sample, ion exchange will occur and the effluent will be alkaline, so filling with a strong cation exchange resin for pH adjustment is also used. Alternatively, an ion exchange resin into which sulfonic acid is introduced is used.

The flow rate of the column is related to the contact time of the biosensor part, and the sensitivity and measurement time vary depending on the contact time. A flow rate of the order of ml / min is preferred. At this time, the liquid may be caused to flow down using a pressure difference, but it is difficult to control the flow rate, so it is better to use a pump. It is desirable that the pump used has little pulsation, and the cylinder type and the plunger type are excellent. The injector may be either a cylinder type or a fixed loop type. However, in order to improve the reproducibility of a sample having a low AG concentration, it is better to use an injector with little fluctuation. It goes without saying that a fully automatic system uses an auto injector. In addition, the injection amount of the sample depends on the measurement sensitivity, but the smaller the amount, the better the influence on the durability of the interfering substance removal column.
About 5 to 50 μl is preferable. As the sample (specimen) used in the present invention, serum or plasma which has not been deproteinized can be used as it is, but as described below, a sample obtained by adding a protein insolubilizing agent to serum or plasma to insolubilize the protein in water is used as a sample. May be used.

For passing through the column, water or borate buffer is used, but if the concentration of borate buffer is high, saccharides may not be adsorbed, and a low concentration of borate buffer is desirable, and 0.2 M or less, preferably 0.1 M or less, more preferably
A concentration of 0.001-0.05M is preferred. In this case, the pH of the borate buffer needs to be changed depending on the type of the packing material of the column for removing an interfering substance.When a strong cation exchange resin is used in combination, cations are adsorbed. Rather, boric acid alone is preferred.
When the strong anion exchange resin is used alone, any pH may be used because the cation passes therethrough, and the optimum pH of the biosensor enzyme, for example, pH 5 to 9, can be adopted. Water or low-concentration boric acid has a small buffering action, so that ionic substances in the sample are ion-exchanged by an interference substance removal column and the pH fluctuates. Therefore, the optimum pH of the enzyme reaction cannot be maintained, and the durability of the enzyme film of the biosensor may be deteriorated, which may cause noise or shock. Therefore, mixing a buffer having a strong buffering action after the column for removing the interfering substance to maintain the optimum pH gives a good result to the durability of the biosensor.
The buffer used here may be any buffer as long as it can maintain the optimal pH of the AG oxidase, for example, pH 5 to 9.
For example, a phosphate buffer may be mentioned.

Preferred methods of the method according to the present invention include distilled water or low-concentration borate buffer in a hydrogen peroxide electrode type biosensor equipped with an auto injector, an interfering substance removal column, and a 1,5-AG oxidase immobilized membrane. The solution is allowed to flow, a mixing joint is provided between the interfering substance removal column and the biosensor, the phosphate buffer is mixed, and the mixture is connected to the biosensor via a mixing coil.
The biosensor is connected to a recorder or a data processor, and the signal from the hydrogen peroxide electrode is peaked and quantified as an area or height. A standard AG and serum or plasma as a sample are injected from the auto injector, and only the AG in the liquid passing through the column for removing the interfering substance is quantified by the biosensor as the amount of hydrogen peroxide. A calibration curve is created from the standard AG, and AG in the sample is quantified based on the calibration curve.

 Next, a fourth invention will be described.

The protein insolubilizing agent that forms an oily precipitate, which is used in the fourth invention, is that the protein is precipitated as an oily precipitate by adding the protein insolubilizing agent, and a deproteinized supernatant is obtained without centrifugation. To make it possible. For example, there is acrinol. The used amount is 2 to 50 mg, preferably about 5 to 20 mg per 1 ml of the sample, and it is preferable to add the solution as an aqueous solution to the sample.

The protein-insolubilizing agent-adsorbing resin is not particularly limited as long as it can adsorb the protein-insolubilizing agent excessively added for protein removal. When Acrynol is used, Diaion HP-20SS (Mitsubishi Kasei) Is preferred. These protein-insolubilizing agent-adsorbing resins may be added to the upper layer of the basic anion-exchange resin, and both the removal of the protein-insolubilizing agent and the removal of saccharides can be performed simultaneously by one chromatographic operation.

The basic anion exchange resin mainly removes saccharides and is usually used as an OH form or a boric acid form. In the method using the OH form of the anion exchange resin, the sample is slowly passed through the OH form of the strongly basic resin to adsorb and remove saccharides. Preferred strongly basic resins are resins having a quaternary ammonium salt as an exchange group, and include strongly basic trimethylamino groups (type I) and hydroxyethyldimethylamino groups (II
Anion exchange resin having a triethylamino group (QAE-resin). In addition, in this method, the particle size of the resin is reduced (200 to 400 mesh) because it is affected by the passing speed of the processing liquid through the resin.
However, it is preferable to let it pass slowly. Further, in order to neutralize the treatment solution, the treatment solution may be further treated with a cation exchange resin.

Cation exchange resins are the H forms of various anionic resins. Anionic resins include all types of cation exchange resins, from strongly acidic cation exchange resins to weakly acidic cation exchange resins. Particularly preferably,
There is an H-form of a strongly acidic cation exchange resin having a sulfonic acid group.

In the method using the boric acid form of the anion exchange resin, the sugar is adsorbed and removed as a complex with boric acid. However, there is no particular limitation as long as the boric acid form of the anion exchange resin is used. Includes all types of anion exchange resins from ion exchange resins to weakly basic anion exchange resins.
Particularly preferably, a strongly basic trimethylamino group (I
), Borate form of anion exchange resin having strong basic hydroxyethyldimethylamino group (form II), anion exchange resin having triethylamino group (QAE -Resin) borate form,
There is a borate form of a resin having a basic property and two kinds of ion-exchange groups of a dimethylamino group and a hydroxyethyldimethylamino group (manufactured by Bio-Rad, Bio-Rex 5). Further, in order to re-adsorb borate ions coming off by the treatment of the sample, the sample may be further treated with an anion exchange resin. Further, a treatment with a cation exchange resin may be added to neutralize the treatment liquid.

Anion exchange resin is an OH type or weak acid salt type of various cation resins, and as a cationic resin,
It includes all types of anion exchange resins, from strongly basic anion exchange resins to weakly basic anion exchange resins. Organic acids such as carbonic acid, formic acid and acetic acid are preferable as the weak acids constituting the weak acid salt form of these cationic resins. Particularly preferred cationic resins include anion exchange resins having a strongly basic trimethylamino group (type I) or hydroxyethyldimethylamino group (type II).

Examples of the cation exchange resin include those described above. In a combination requiring many kinds of resins to remove an interference substance mainly composed of saccharides, one column may be filled with all the resins stacked so as to form a layer or mixed and filled. When filling in layers, it is preferable to fill the upper layer with the resin from which saccharides are removed and fill the lower layer with the neutralizing resin.

In order to carry out the fourth invention, a protein insolubilizing agent capable of precipitating the protein is added to the sample, and after the protein is precipitated, the supernatant is passed through the above-mentioned column. What is necessary is just to determine the sugar alcohol in it.

In the fourth invention, examples of the protein precipitant include strong acids such as trichloroacetic acid, perchloric acid, sulfuric acid, and hydrochloric acid. The addition amount of the precipitant is 0.5-10%,
The optimal amount of trichloroacetic acid when SDS is added to serum varies from 2 to 6%, depending on the type and amount of the sample and denaturant used. The protein denaturant is added to prevent the protein precipitated by the precipitant from re-dissolving, and includes sodium dodecylbenzenesulfonate (hereinafter referred to as SDS), sodium tungstate,
There are sodium molybdate, benzene alcohol and the like, among which SDS is more preferable. The amount of addition when SDS is used may be any amount sufficient to completely denature the protein in the sample, and is 0.2% to 5% in the case of serum, and more preferably 0.5 to 2%.

The above protein denaturing agent and precipitating agent are each used alone.
One solution is formed and added to the sample sequentially, but depending on the case, a solution obtained by mixing them may be added at once.

As the basic anion exchange resin to be filled in the column, the same one as used in the second invention is used,
Further, a cation exchange resin may be used as a neutralizing agent.

In order to carry out the method of the invention 4, a protein denaturing agent and a precipitant are added to the sample to make the protein a water-insoluble precipitate, and the precipitate is passed through a resin-filled column without centrifugation without centrifugation. After washing with water, the sugar alcohol in the passing solution may be quantified. The column used here may be of any type as long as it can remove the insolubilized protein, but it is better to use paper, a filter such as a sintered sheet of surface-hydrophilized polyethylene resin attached to the upper part of the resin. preferable.

The amount used per sample when carrying out the fourth invention is, for example, as follows: The amounts used per sample when carrying out the fourth invention are, for example, as follows: a protein insolubilizing agent which causes an oily precipitate; Or Next, the present invention will be specifically described with reference to experimental examples and examples.

Experimental Example 1. (Calibration curve of AG using pyranose oxidase) Pyranose oxidase (specific activity to glucose 5 units / mg, Takara Shuzo) 2 mg / ml in 0.25 M phosphate buffer (pH 5.6), HRP 0.24 Units / ml were dissolved to give ABTS 4 mM to prepare an AG detection reagent. 2 ml of distilled water was added to 0.1 ml of the AG standard solution to form an AG solution, and 0.5 ml of the above-mentioned AG detection reagent was further added, followed by a reaction at 25 ° C. for 1 hour. The absorbance at 420 nm of this reaction solution was measured to prepare a calibration curve, which is shown in FIG. 1-1.

Experimental Example 2. (AG using L-sorbose oxidase
The calibration curve of AG was prepared in exactly the same manner as in Example 1, except that pyranose oxidase was replaced with L-sorbose oxidase (specific activity for glucose was 4.3 units / mg), and a calibration curve of AG was prepared. Shown in the figure.

Experimental Example 3. (Calibration curve of MI using myo-inositol dehydrogenase) 20 mM sodium carbonate buffer (pH 9.0) containing 70 mM ammonium sulfate
In, myo-inositol dehydrogenase (10 units / mg,
Sigma) and NAD were dissolved at 1 unit / ml and 1 mM, respectively, to prepare an MI detection reagent. 0.1 ml of MI standard solution, M
0.9 ml of I detection reagent was added and reacted at 25 ° C. for 2 hours. The fluorescence intensity of this reaction solution was measured at an excitation wavelength of 365 nm and a detection wavelength of 450 nm, and the results are shown in FIG. 1-3.

Example 1-1. (AG using pyranose oxidase
Measurement) Serum sample was subjected to pretreatment shown in Examples 1-4 to 1-6 described below to remove saccharides, and the remaining AG experimental example 1 was carried out.
The measurement was carried out by the method shown in FIG. The quantification of AG was performed by a calibration curve prepared by pretreating each AG standard solution in exactly the same manner as the serum sample. The results are shown in Table 1.

Example 1-2. (Measurement of AG using L-sorbose oxidase) The same serum sample as used in Example 1-1 was subjected to the pretreatment shown in Examples 1-4 to 1-6 described later. The saccharides were removed by annealing, and the remaining AG was measured by the method described in Experimental Example 2. The quantification of AG was performed by a calibration curve prepared by pretreating each AG standard solution in exactly the same manner as the serum sample. The results are shown in Table 1.

Example 1-3. (Measurement of MI using myo-inositol dehydrogenase) A urine sample was subjected to the pretreatment described in Example 1-7 described below to remove saccharides, and the remaining MI was shown in Experimental Example 3. Measured by the method. The quantification of MI was carried out using a calibration curve prepared by pretreating each MI standard solution in exactly the same manner as for a urine sample. The results agreed well with the values measured by gas chromatography on the same urine sample.

Example 1-4. (Pretreatment of serum sample for sugar alcohol analysis using an interference substance removal column to which a protein adsorption resin is added) Ion exchange resin AG50W-X8H, QAE-Toyopearl 55oc
The sample pretreatment columns were prepared by packing the boric acid forms (0.1 ml, 0.5 ml each) in small layers from bottom to bottom in order of 0.1 ml and 0.5 ml, respectively. 0.1 ml of human serum is directly passed through this column and distilled water
After washing four times with 0.5 ml, 2.1 ml of a passing solution was obtained. An example in which AG was quantified among the sugar alcohols recovered in the passing solution was performed in the same manner as in Example 1. The correlation between the same sample and the value measured by the gas chromatography method is shown in FIG. 1-6.

In the above column, when the QAE-Toyopearl 55oc boric acid form was changed to its OH, and the other conditions were the same as above, the same results as in this example were obtained.

Further, the resin to be filled in the pretreatment column is AG50W-X
The same result as in the present example was obtained by changing the mixed resin of the 8 H form and the QAE-Toyopearl boric acid form to a ratio of 1: 5, and the other conditions were the same as above.

The OH type and boric acid type resins of QAE-Toyopearl used in this example are prepared from a commercially available Cl type by the following method. That is, 250 ml of QAE-Toyopearl 55oc (Cl form) is packed in a column, 0.2 M sodium hydroxide solution 1 is slowly flowed to allow Cl ions to flow out, and the column is washed with distilled water, and the column effluent becomes neutral. Wash to obtain the OH-type resin.
A 0.5 M boric acid solution 3 is slowly flowed from above to the OH type resin to neutralize the effluent wastewater until the pH becomes acidic. Subsequently, the effluent is washed with distilled water until the waste liquid becomes neutral, thereby obtaining a boric acid type resin.

The H form of AG50W-X8 is prepared from the commercially available Na form by the following method. That is, 200 ml of AG50W-X8 (Na form) is packed in a column, IM hydrochloric acid 1 is slowly flowed to allow Na ions to flow out, and then washed with distilled water to make the pH of the effluent wastewater neutral.

Example 1-5. (Pretreatment for the analysis of sugar alcohols using an irreversible protein insolubilizing agent for serum samples and an interfering substance removal column with an over-filter) Ion exchange resin AG50W-X8 H form, AG1-X8 OH form (both are Biorad) (Manufactured by Co., Ltd.) in small layers of 0.1 ml and 0.4 ml, respectively, in a layered manner from the bottom, and a sintered filter of hydrophilic polyethylene was set on the top of the filled resin to prepare a sample pretreatment column.

After 0.1 ml of 5% SDS solution was added to 0.2 ml of human serum and shaken, 0.1 ml of a 12% aqueous solution of trichloroacetic acid was further added to shake vigorously. 0.2 ml of the resulting dispersion containing the precipitate was passed through the column for sample pretreatment described above, and washed four times with 0.5 ml of distilled water to obtain 2.2 ml of a passing solution. An example in which AG is quantified among the sugar alcohols recovered in the passing solution is described below. The measurement of AG was performed by the method shown in Experimental Example 1. That is, 0.5 ml of an AG detection reagent was directly added to this AG solution and reacted. Furthermore, quantification was performed using a calibration curve prepared using an AG standard solution. The correlation between the serum AG values of the normal and diabetic patients thus measured and the serum AG values when the same sample was measured by the gas chromatography method is shown in FIG. 1-4. As is clear from this figure, the quantitative value obtained by the method of the present invention showed a high correlation with the quantitative value obtained by the gas chromatography method, and was not affected by the glucose present in a large excess.

Example 1-6. (Pretreatment for Analysis of Sugar Alcohol by Serum Sample by Interaction Substance Removal Column with Addition of Protein Insolubilizing Agent and Insolubilizing Agent Adsorbing Resin Which Causes Oily Precipitation) Ion Exchange Resin AG50W-X8 H Form, AG1-X8 OH Shape (both made by Bio-Rad), hydrophobic resin Diaion HP-
20SS (manufactured by Mitsubishi Kasei) was packed in small columns of 0.1 ml, 0.3 ml, and 0.1 ml, respectively, in order from the bottom to form a sample pretreatment column.

0.2 ml of 2.5% acrinol was added to 0.2 ml of human serum and shaken. Since the protein in the serum precipitates as an oily precipitate, 0.2 ml of the supernatant is passed through the sample pretreatment column, washed four times with 0.5 ml of distilled water and washed with 2.2 ml of the filtrate.
ml was obtained. An example in which AG was quantified among the sugar alcohols recovered in the passing solution was performed in the same manner as in Example 1-4. The correlation between the same sample and the value measured by the gas chromatography method is shown in FIG. 1-5.

Example 1-7. (Pretreatment for Sugar Alcohol Analysis Using Interfering Substance Removal Column with Added Protein Adsorption Resin of Urine Sample) Ion-exchange resins AG50W-X8 H form and QAE-Toyopearl borate form 0.2 ml and 1.8 ml, respectively. The small columns were sequentially packed in layers from the bottom to prepare sample pretreatment columns. After directly passing 0.5 ml of human urine through this column, the column was washed eight times with 1.0 ml of distilled water to obtain 8.5 ml of a permeate. An example in which MI is quantified among the sugar alcohols recovered in the passing liquid is described below.

A total of 8.5 ml of the above passing solution was concentrated to dryness and redissolved in 0.1 ml of distilled water to obtain a treated solution of a pretreatment column. MI was detected in accordance with Experimental Example 3. That is, 0.9 ml of the MI detection reagent was directly added to the treatment solution to cause a reaction. Further, the quantification of MI was performed using a calibration curve prepared using the MI standard solution. The urine MI values of normal people and diabetic patients measured in this way are
Table 2 shows the results of comparison with the values measured by the gas chromatography method for the same sample.
It was shown to.

Example 1-8. (Example of serum AG measurement kit) (1) Preparation of kit AG detection reagent: PROD 200 mg (5 units / mg, manufactured by Takara Shuzo), HRP0.
24 mg (100 U / mg, manufactured by Wako Pure Chemical Industries) and 220 mg of ABTS (manufactured by Boehringer) were treated with 0.25 M sodium phosphate buffer (pH 5.6) 10
It was dissolved in 0 ml, dispensed into vials 16 ml at a time, and freeze-dried by a conventional method.

Restoring solution: Triton X-405 (manufactured by Wako Pure Chemical) 0.6 g in distilled water 1
The mixture was dissolved in 00 ml, dispensed into vials in 16 ml portions, and stoppered.

Standard solution: 5 mg of AG is dissolved in 100 ml of distilled water, and 3
The mixture was dispensed in ml and stoppered.

Pretreatment column: 0.1 ml of AG50W-X8 H form (manufactured by Bio-Rad) and QAE-Toyopearl boric acid form (manufactured by Tosoh Corporation) in a reservoir (1.5 ml capacity, manufactured by Analytical International) equipped with a frit filter in order from the bottom. ) 0.5m
l was filled. Further, a frit filter was attached from above the filled resin, and fixed so that the resin did not move. Also,
The column was filled with a 0.2 M boric acid solution to maintain the stability of the packed resin and prevent drying, and a cap was provided at the outlet, and the inlet was sealed with a seal.

(2) Operation method The cap and seal of the pretreatment column were removed, the boric acid solution in the reservoir was drained, and further washing was performed twice with 1 ml of distilled water to completely wash away excess boric acid in the resin. . The washed pretreatment column was set on a test tube, and 100 μl of a serum sample was directly conducted.
After washing four times with 0.5 ml, 2.1 ml of a passing solution was obtained. AG detection reagent 1
The vial was reconstituted with one vial of reconstitution solution, and 0.5 ml of this solution was used.
Was added to 2.1 ml of the solution passed through the pretreatment column described above, followed by stirring.
The reaction was performed at 5 ° C. for 1 hour. The absorbance of this reaction solution at 420 nm was measured with a usual spectrophotometer. Further, a standard solution was diluted twice with distilled water to prepare a dilution series, which was also processed and reacted in the same manner as in the case of the serum sample, to thereby prepare an AG calibration curve. The AG concentration in the serum sample was calculated from the absorbance value of the sample using this calibration curve. The same results as in Examples 1-4 were obtained by the kit method.

Example 2-1 The measurement was performed using a 0.05M borate buffer (pH 5.8) from a pump.
At a flow rate of 0.5 ml / min, and an auto injector,
The measurement was performed using an apparatus including an interference substance removal column, a biosensor, and a data processor connected to the biosensor.

The biosensor uses a surface (5 m) of a flow cell (a hydrogen peroxide electrode (manufactured by Able Co., Ltd.) with a volume of about 100 μl).
mφ), a PROD fixed membrane of the same size was used with a nylon net. The PROD-immobilized membrane was prepared by the following method. PROD (Takara Shuzo 5.4 U / mg) 10 mg and bovine serum albumin (Sigma) 6 mg in 1/15 M phosphate buffer (pH
7.2) Dissolve in 0.6 ml, 1% glutaraldehyde aqueous solution.
Add 2 ml and mix. Immediately after mixing, the mixture was slowly dropped onto two nitrocellulose membranes (25 mmφ × pore size 3 μm), spread so that the whole became uniform, and air-dried at 4 ° C. overnight to obtain.

The interference substance removal column is a 5 mmφ × 100 mm column in which a polyvinyl alcohol-based QAE-Toyopearl 550C (manufactured by Tosoh Corporation) is converted into an OH form by a usual method, and then into a boric acid form and a strong cation exchange resin AG50W-X8. (H-type, manufactured by Bio-Rad Co., Ltd.) in a ratio of 5: 1.

In this apparatus, the standard 1, 10, and 40 μg / ml of AG was arranged in an auto injector (20 μl fixed loop), and then 10 serum samples were arranged to start measurement. Using a data processor, a calibration curve was created from the standard area values of AG,
AG quantitative values were obtained. FIG. 2-1 shows the correlation between the same sample and a quantitative value measured by the GC method. As is clear from this figure, the quantitative value according to the present invention showed a high correlation with the GC method.

Example 2-2 The measuring device was composed of two pumps, an auto injector, an interfering substance removal column, a biosensor and a data processor connected to the biosensor. at a flow rate of 0.1 ml / min, and the liquid passing through the column is supplied at a flow rate of 0.1 ml / min.
This is a device mixed with an M-phosphate buffer through a mixing joint, mixed completely with a mixing coil (1.0 mmφ × 3 m), and connected to enter the same biosensor flow cell as in Example 2-1.

The interference substance removal column is a 5 mm φ × 100 mm column with QAE
-Toyopearl 550C (borate type) was used.

With this apparatus, measurement was performed in the same manner as in Example 2-1. Also,
The correlation between the same sample and the quantitative value measured by the GC method is shown in FIG. 2-2. As is clear from this figure, the quantitative value according to the present invention showed a high correlation with the GC method.

Example 2-3 The measurement apparatus was performed by the same system as that of Example 2-2.
Add 0.01M-borate buffer (pH 6.0) to the interference substance removal column.
The mixture was flowed at a flow rate of 1.0 ml / min, and 1.1 M-phosphate buffer (pH 5.8) was supplied from the other side at a flow rate of 0.1 ml / min.
The column for removing the interfering substance used was a column of 5 mmφ × 100 mm packed with acrylic Shodex® TM-L (manufactured by Showa Denko KK) (borate type).

In this device, 20 serum samples were measured in the same manner as in Example 2-1. The correlation between the same sample and a quantitative value measured by the GC method is shown in FIG. 2-3. As is clear from this figure, the quantitative value according to the present invention showed a high correlation with the GC method.

〔The invention's effect〕

As is clear from the above, according to the method of the present invention, interference substances such as proteins and saccharides in a sample can be easily removed, and measurement of sugar alcohols has become extremely simple. Although specific sugar alcohols have specificity, it has been revealed that enzymes that react widely with saccharides can be sufficiently used for quantification by combining the saccharide removal pretreatment method of the present invention. .

[Brief description of the drawings]

FIG. 1-1 shows a calibration curve of AG using pyranose oxidase, FIG. 1-2 shows a calibration curve of AG using L-sorbose oxidase, and FIG. 1-3 shows a calibration curve of myo-inositol dehydrogenase. The calibration curve of the MI
The figure shows the correlation between the value of the serum treated according to the fourth invention measured using pyranose oxidase and the gas chromatography method. FIG. 1-5 shows the serum treated according to the fourth invention treated with pyranose oxidase. Fig. 1-6 shows the correlation between the value measured using pyranose oxidase and the value measured using pyranose oxidase in the serum treated according to the first invention, respectively. is there. FIGS. 2-1 to 2-3 show Examples 2-1 to 2 respectively.
3 shows a correlation with the GC method when the serum AG was measured by the measurement system of No.-3.

 ──────────────────────────────────────────────────続 き Continued on the front page (72) Inventor Shigeru Tajima 675-11 Fujioka, Fujioka-shi, Gunma Prefecture (72) Inventor Tadashi Hashiba 804-12 Asami, Kasakemura, Nitta-gun, Gunma Prefecture (72) Inventor Hiroshi Hayami Takasaki, Gunma Prefecture 239 Iwahana-cho, Ichigo (72) Inventor Tomoko Takezawa 1906- 5 Fujioka, Fujioka-shi, Gunma (72) Inventor Masachika Hirayama 136-51 Nara-cho, Omiya-shi, Saitama 7-203 Nara-cho housing complex Examiner Kazuo Hirata

Claims (3)

(57) [Claims] (1) passing a sample containing a sugar alcohol, a protein, and a saccharide through a column filled with a basic anion exchange resin having a protein removing ability and a saccharide removing ability, and then quantifying the sugar alcohol in the passing solution; A method for quantifying a sugar alcohol, comprising: 2. A column for removing proteins and saccharides, which is packed with a strongly basic anion exchange resin having a quaternary ammonium group introduced into a hydrophilic gel filtration carrier and an aqueous boric acid solution. 3. A kit comprising a column filled with a basic anion exchange resin having a protein-removing ability and a sugar-removing ability and a boric acid aqueous solution, a sugar alcohol quantifying reagent and a sugar alcohol for preparing a calibration curve.