JPH06331629A - Unstable saccharified hemoglobin dissociating agent and measuring method for saccharified hemoglobin using same - Google Patents

Abstract

PURPOSE:To measure only S-HbA1c in a blood sample with ease and high accuracy without denaturing hemoglobin by composing an unstable saccharified hemoglobin dissociating agent chiefly of a compound in which hydrogen atoms of ammonia are replaced by a methylene phosphoric acid group. CONSTITUTION:A fundamental structure comprises ammonia or a primary or secondary amine of 1 to 6 carbon atoms, the amine being preferably a straight chain alkylene diamine of 1 to 6 carbon atoms, etc. At least one hydrogen atom on nitrogen atoms included in a compound having the fundamental structure is replaced by a methylene phosphoric acid group or its salt. The substitution products of a kind comprising a methylene phosphoric acid group introduced into the fundamental structure can be synthesized using various manufacturing methods and are each used as a dissociating agent. Of the substitution products, compounds represented by specific formulae and their salts, e.g. nitrilotrimethyl phosphoric acid and ethylene diaminedimethyl phosphoric acid, etc., are preferably used, and the greater the proportion of a methylene phosphoric acid group, the stronger the dissociating effect.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a dissociation agent for unstable glycated hemoglobin used in the measurement of glycated hemoglobin in blood, particularly hemoglobin A _lc , and a method for measuring glycated hemoglobin using the same.

[0002]

2. Description of the _Related Art Glycated hemoglobin contained in blood, particularly hemoglobin A _lc (hereinafter abbreviated as HbA _lc ) is known as one of indicators of diabetes, and its measurement is widely performed in the field of clinical examination. There is.

Since HbA _lc correlates well with the blood glucose level of a patient about 2 months ago, its clinical significance is increasing as one of the important indicators of the pathological condition of diabetes.

This HbA _lc is expressed by the following equation:
This is a complex in which glucose is non-enzymatically bound to the amino group of valine existing at the N-terminal of the β chain of hemoglobin. When glucose binds to the amino group of hemoglobin, first,
As shown in the following reaction formula, unstable H which is a Schiff base
bA _lc (I) (hereinafter abbreviated as L-HbA _lc ) is formed.

[0005]

[Chemical 1] In the above formula, β-A-NH ₂ represents hemoglobin A _o, and NH ₂ in it represents the amino group of valine, which is the N-terminal of the β chain of hemoglobin.

This L-HbA _lc (I) production reaction is a reversible reaction, and since the reaction rate is fast, the equilibrium is dependent on the glucose concentration in the L-HbA _lc (I) production direction (→
Direction) or dissociation direction (← direction). That is, the amount thereof largely depends on the glucose concentration in blood (that is, blood glucose level). L-HbA _lc (I) is further irreversibly converted to stable hemoglobin A _lc (II) (hereinafter abbreviated as S-HbA _lc ) after further undergoing Amadori transition. This S-HbA _lc (II) is stable without being affected by the glucose concentration like L-HbA _lc (I).

HbA _lc can be quantified by, for example, separating hemoglobin into each hemoglobin component by high performance liquid chromatography and measuring the absorbance thereof.
At present, it is difficult to measure S-HbA _lc and L-HbA _lc completely separated on a chromatogram. Therefore, it is not possible to obtain a stable HbA _lc measurement value. This is because the amount of L-HbA _lc changes by glucose (ie glucose) weight, blood glucose level is to change rapidly and significantly by diet and exercise.

Thus, HbA_lcIs usually L-Hb
A_lcAnd S-HbA_lcBoth components are included, but as described above
L-HbA from blood samples_lcExcluding
Leave, S-HbA_lcAttempts have only been made to measure
It For example, David M. Nathan et al., Clin. Chem., 28,512 (1
982), semicarbazide and aniline were added to L-HbA.
_lcUsed as a scavenger for the
It has been reported to process blood samples for 0 minutes. Only
Then, like this, L-HbA_lcThe removal reaction of is under relatively high temperature
(38 ° C) and for a long time, the hemoglobin
Denaturation (eg deheming) can occur. For example,
Observe elution pattern by on-exchange chromatography
Then, the peak intensity decreases due to fading due to deheming.
Or HbA _laAnd HbA_lb(HbA_{la + b}) At the elution position
An increase in the resulting peak is seen. Also, the reagent is unstable
Therefore, it is necessary to prepare each time before use, and the operation is complicated.
It's miscellaneous.

In Japanese Patent Application Laid-Open No. 58-21024, a dihydroxyboryl compound (boric acid compound) is used as L-HbA.
It is disclosed as a _lc remover. Since this dihydroxyboryl compound esterifies the OH group of glucose, glucose is removed from the system, resulting in L-H
Dissociation of bA _lc proceeds. However, in order to remove the L-HbA _lc requires relatively high concentrations of dihydroxy boryl compounds. For example, when an eluent containing the dihydroxyboryl compound is used to elute a hemolysate of about 0.1 to 1.0 M in a sample solution containing hemolyzed blood, the compound is 0% in the eluent. It is necessary to be contained in a ratio of 0.01 to 0.15M. However, when such a high concentration of dihydroxyboryl compound is contained, when the eluent is subjected to ion exchange chromatography, the ionic strength of the eluent used is different from that in the usual case, and the separation conditions change. The measurement becomes difficult or the measurement conditions need to be corrected.

JP-A-63-298063 discloses the use of polyphosphoric acid, which is a phosphoric acid condensate, as a rapid L- _HbAlc dissociation method without denaturation of hemoglobin. Further, JP-A-2-259467 discloses the use of phytic acid. However, polyphosphoric acid and phytic acid are known to be hydrolyzed when stored as an aqueous solution for a long period of time, and there is a problem that they are finally converted into substances having no L- _HbAlc dissociation effect. For example, polyphosphoric acid is hydrolyzed to give L
By hydrolysis of mono-orthophosphoric acid having no —HbA _lc dissociation effect, phytic acid also changes to mono-orthophosphoric acid and inositol having no L-HbA _lc dissociation effect. This hydrolysis during storage has a temperature acceleration property. For example, 0.
1 w / v tetrapolyphosphoric acid aqueous solution (pH 6.0) 4
When stored at ℃, there is almost no change for more than 1 year, but when stored at 37 ℃, 50% or more of polyphosphoric acid is hydrolyzed in several months, and when stored at 60 ℃, Most of the polyphosphoric acid hydrolyzes in ~ 3 weeks,
Totally not exhibit the L-HbA _lc dissociation effect. For this reason, there is a problem in that, when the solution is stored as an aqueous solution for a long period of time, the storage conditions need to be taken into consideration.

As another method for removing L-HbAlc, a blood sample is diluted to reduce the glucose concentration, and L- _HbAlc is removed.
There is a method of promoting dissociation of bA _lc . For example, Klenk.
DC et al, Clin. Chem., In the 28,2088 (1982), 37 ℃ erythrocytes with a large excess of physiological saline, a method of removing the L-HbA _lc by incubation has been reported. Other known methods include incubating erythrocytes with an isotonic phosphate buffer for several hours and dialysis of hemolysates, but these methods all require a long time for treatment. Therefore, it is not suitable as a method for clinical examination.

[0012]

SUMMARY OF THE INVENTION The present invention has been made to solve the above-mentioned conventional drawbacks, and an object thereof is to obtain S-HbA _lc in a blood sample which is a sample to be tested.
L-Hb, which can be measured accurately and simply, without denaturing hemoglobin, and has excellent storage stability.
An object is to provide a dissociation agent for A _lc and a method for measuring HbA _lc using the dissociation agent.

[0013]

The L- _HbAlc dissociation agent of the present invention is used for measuring glycated hemoglobin in blood,
該解releasing agent is ammonia or a substituted with at least one hydrogen atom on the nitrogen atom of the primary or secondary amine having 1 to 6 carbon atoms, methylene phosphonic acid groups (-CH ₂ PO ₃ H ₂₎ or a salt thereof L-
It is characterized by being an L-HbA _lc dissociator capable of dissociating HbA _lc into hemoglobin A _o and glucose.

Further, the method for measuring glycated hemoglobin of the present invention comprises contacting the erythrocyte dispersion liquid and / or the hemolysis product containing hemoglobin, which is a sample to be tested, with a dissociation buffer solution containing the above dissociation agent. The step of dissociating L-HbA _lc present in the red blood cell dispersion and / or its hemolyzate containing hemoglobin into hemoglobin A _o and glucose, and the hemoglobin and glycated hemoglobin present in the sample after the dissociation are chromatographed. The step of separating each hemoglobin component by a graphic method and measuring S-HbA _lc present in the sample is included.

The basic structure of the dissociator is required to be ammonia or a primary or secondary amine having 1 to 6 carbon atoms. As the amine, linear alkylenediamine having 1 to 6 carbon atoms, diaminocyclohexane, diethylenetriamine, triethylenetetraamine, glycol etherdiamine and the like are preferable. Although the alkylmonoamine is also included in the amine, the diamine, triamine, and tetraamine described above are more preferable because the methylenephosphonic acid group or the salt thereof has a relatively large number of substitution sites.

In addition to these, those having a structure in which a hydrogen atom in the molecule is replaced with a lower alkyl group, a hydroxyl group (-OH) or the like can be used for the same purpose. For example, 1,2-diaminopropane in which a methyl group is introduced into a carbon atom is used as a monosubstituted product of ethylenediamine, and 1,3-diamino-2-propanol in which a hydroxyl group is similarly introduced as a monosubstituted product of propylenediamine. Yes, these can also be the basic structure of the dissociator.

The dissociating agent is characterized in that at least one hydrogen atom on the nitrogen atom contained in the compound having these basic structures is substituted with a methylenephosphonic acid group or a salt thereof. As a method for producing a compound substituted with these methylenephosphonic acid groups, various methods have long been known.

For example, Irani.RR et al., J. Org. Chem. 31.16.
03 (1966), which utilizes the Mannich reaction of amine, formalin and phosphorous acid, and is known as one of the excellent synthetic methods for phosphonic acid derivatives. Also, Motekaitis.RJ
The method reported by J. inorg.nucl.Chem.33 3353 (1971) can also be used as a method for synthesizing the dissociating agent. It uses the chloromethylene phosphinic acid _{(Cl-CH 2 PO 2 H} ), a hydrogen atom of an amino group substituted by methylene phosphinic acid group (-CH ₂ PO ₂ H) First, followed by the addition of mercuric chloride heating, a method of oxidizing the methylene phosphinic acid to methylene phosphonic acid _{(-CH 2 PO 3 H 2)} , higher purity of the methylene phosphonic acid substituted compound obtained, preferably.

By utilizing these synthetic methods, various substitution products in which a methylenephosphonic acid group is introduced into the above basic structure can be synthesized, and these can be used as the labile glycated hemoglobin dissociating agent of the present invention.

Among these various substitution products, particularly preferable as the dissociating agent of the present invention are, for example, compounds represented by the following structural formulas and salts thereof.

Nitrilotrimethylene-phosphonic acid (III: Nitrilotris- (methylenephosphonic acid),
Hereinafter abbreviated as NTPO), ethylenediamine dimethylenephosphonic acid (IV: Ethylenediamine-N, N'-di (methylenephosphonic
) Acid, hereinafter abbreviated as EDDPO), ethylenediaminetetramethylenephosphonic acid (V: Ethylenediamine-N, N, N ', N'-tetra (methylenep
hosphonic) acid, hereinafter abbreviated as EDTPO), hexamethylenediamine-tetramethylenephosphonic acid (VI: Hexamethylenediamine-N, N, N ', N'-tetra (methyle
nephosphonic) acid), diaminocyclohexanetetramethylenephosphonic acid (VII: 1,2-diaminocyclohexane-N, N, N ', N'-tetra (met
hylenephosphonic) acid), diethylenetriamine pentamethylenephosphonic acid (VIII: Diethylenetriamine-N, N, N ', N``-N''-penta (me
thylenephosphonic) acid), triethylenetetramine hexamethylenephosphonic acid (IX: Triethylenetetramine-N, N, N ', N``, N''', N '''-he
xa (methylenephosphonic) acid), glycol ether diamine tetramethylenephosphonic acid (X: O, O'-Bis- (2-aminoethyl) ethyleneglycol-N, N,
N ', N'-tetra (methylenephosphonic) acid), diaminopropanol tetramethylenephosphonic acid (XI: 1,3-Diamino-2-hydroxypropane-N, N, N', N'-tetr
a (methylenephosphonic) acid), diaminopropane tetramethylenephosphonic acid (XII: 1,2-Diaminopropane-N, N, N ', N'-tetra (met
hylenephosphonic) acid).

Further, in the dissociator, when a hydrogen atom other than the hydrogen atom substituted with the methylenephosphonic acid group or a salt thereof is substituted with a functional group other than the methylenephosphonic acid group, it is L-HbA. Such compounds can also constitute the dissociation agent of the present invention as long as they are substituents that do not particularly affect the dissociation action of _lc . Examples of these substituents include an acetic acid group (—CH ₂ COOH),
Examples thereof include a hydroxyl group and a lower alkyl group. For example, ethylenediamine diacetate dimethylenephosphonic acid (XIII: Etylenediamine-N, N'-di (acetic) -N, N'-di (met
hylenephosphonic) acid).

[0023]

[Chemical 2]

[Chemical 3]

[Chemical 4] However, among these phosphonic acid-substituted substances, high purity ones are difficult to obtain, and there are limited reliable reagents due to difficulties in synthesis and purification.
Some of TPO, EDDPO, and EDTPO are currently commercially available as chelating agents, and they are easily available, and are most preferable as the dissociating agent.

The dissociation agent is ammonia, or at least one hydrogen atom on the nitrogen atom of the primary or secondary amine is a methylenephosphonic acid group (-CH ₂ PO).
₃ H ₂ ) or a salt thereof, but the dissociation effect becomes stronger as the number of methylenephosphonic acid groups increases. For example, ethylenediamine has two amino groups, and EDDPO in which one hydrogen atom of each amino group is replaced with a methylenephosphonic acid group.
And in EDTPO in which hydrogen atoms of all amino groups are substituted with methylenephosphonic acid groups, the latter exhibits a stronger L- _HbAlc dissociation effect.

Further, the methylenephosphonic acid-substituted substance of the dissociation agent exhibits the same L- _HbAlc dissociation effect even if its salt. The type of salt is not particularly limited, but for example, salts of alkali metals or alkaline earth metals are used, and Na salts, K salts and the like are particularly preferable.

These methylenephosphonic acid-substituted substances and salts thereof may be used alone or in combination of two or more.

In these methylenephosphonic acid-substituted substances, when the number of carbon atoms in the molecule exceeds 6, L-Hb
The _Alc dissociation effect decreases and is not very suitable.

In the present invention, the dissociation of L-HbA _lc can be achieved by contacting the erythrocyte dispersion liquid and / or its hemolysis product containing hemoglobin, which is the sample to be tested, with the dissociation buffer containing the dissociation agent. To be achieved.

The dissociating buffer solution is a buffer solution used for maintaining the pH range of the method for measuring glycated hemoglobin according to the present invention, containing the dissociating agent, and is mixed with a sample to be tested. L contained in the sample due to
In which dissociation of -hba _lc is made. The composition of the buffer solution can be used as long as it has a buffering ability in the range of pH 4 to 7, and is not particularly limited, but a phosphate buffer solution, a citrate buffer solution, a phthalate buffer solution and the like are preferable.

As the erythrocyte dispersion liquid, whole blood, washed erythrocytes, erythrocyte concentrated liquid and the like are applicable. The hemolysis product containing hemoglobin means a treatment liquid obtained by hemolyzing the red blood cells in the red blood cell dispersion liquid by any method to elute the hemoglobin outside the red blood cell membrane. The red blood cell dispersion must be hemolyzed before it is introduced into the chromatography column described below.

The dissociating agent is brought into contact with the red blood cell dispersion liquid and / or hemoglobin before each hemoglobin component contained in the test sample is separated by ion exchange in the chromatography column and eluted outside the column. L- _HbAlc dissociation effect is produced by being made to do.
Therefore, the dissociating agent may be contained in a diluent for diluting the test sample to an appropriate concentration, a hemolysis buffer containing a hemolytic agent, an eluent used for chromatography, and the like. The eluent is also the above dissociation buffer.

For example, normally, whole blood is used for measuring glycated hemoglobin, but since whole blood is a red blood cell dispersion liquid, it is necessary to hemolyze erythrocytes first when measuring glycated hemoglobin. In this case, when the hemolytic buffer containing a hemolytic agent is used by containing the dissociating agent, L-HbA _lc
It is most preferable because the dissociation and hemolysis can be performed at the same time. As the hemolytic agent contained in the hemolytic buffer, a surfactant is particularly preferable, and examples thereof include polyoxyethylene alkylaryl ethers, higher aliphatic alcohols, polyoxyethylene ethers of sulfonate compounds and sulfate compounds, and polyoxyethylene of sorbit fatty acid ester. An addition body etc. are illustrated. The amount of hemolytic agent used varies depending on the type, but usually 1 m of blood
It is 10 to 200 mg per liter. For example, 1 hemolytic agent
When a hemolysis buffer containing w / v% is used, the ratio of hemolyzing agent is 1 to 200 ml per 1 ml of blood, and the hemolytic agent is 0.
When using the hemolysis buffer containing 01 w / v%, the hemolysis can be performed by adding the buffer at a rate of 100 to 500 ml per 1 ml of blood and gently stirring. Excessive hemolytic agent makes it difficult to fractionate hemoglobin by chromatography.

In addition, the dissociating agent may be added to the erythrocyte dispersion liquid of the sample to be inspected, its hemolyzed product, or hemoglobin solution as it is as a solid. This dissociator dissolves quickly,
Hemoglobin in the test sample comes into contact with the dissociation buffer.

Among the methods for measuring HbA _lc using the dissociation agent of the present invention, the test sample is treated with a dissociation buffer containing the dissociation agent to dissociate L-HbA _lc , followed by a chromatographic method. A method for separating and measuring each component of hemoglobin will be described. In this case, the dissociation buffer solution is used as a diluting solution for diluting the test sample to an appropriate concentration, a hemolysis buffer solution containing a hemolytic agent, or the like.

If the amount of the dissociating agent contained in the dissociation buffer is too _small , the dissociation effect of L-HbAlc cannot be obtained, and if it is excessive, the separation during the chromatographic separation becomes difficult. The content varies depending on the type and the condition at the time of measurement (pretreatment method such as hemolysis of blood, pH condition, reaction temperature, etc.), but in the case of EDTPO and NTPO, the dissociation is usually performed in the dissociation buffer. About 0.01
~ 2.0 w / v%, preferably 0.1-1.0 w / v%
To be added. ED for EDDPO
Since the L- _HbAlc dissociation effect is smaller than that of TPO, it is preferable to add the dissociation agent 2 to 3 times as described above.

P at the time of contact between the test sample and the dissociation buffer
The more H is on the acidic side, the more L-HbA _lcDissociation rate increases
However, if the pH is too low, hemoglobin denaturation will occur.
It In addition, chromatographies should be performed under extremely high or low pH conditions.
Separation of hemoglobin by chromatography becomes difficult. Servant
Therefore, normally, a mixture of the sample to be tested and the dissociation buffer is
pH should be 4.6-7.0, preferably 5.3-6.5.
Is adjusted to Contact time between test sample and dissociator
Depends on the type and concentration of the dissociator, pH conditions, reaction temperature, etc.
Normally, 10 minutes or more at room temperature, preferably
Is 10 to 30 minutes. This contact time raises the temperature
It is possible to shorten it by
Incubation for about 3-7 minutes at 50 ° C for 1-3 minutes
L-HbA_lcCan be dissociated.

The test sample thus treated is subjected to various chromatographic methods, for example, high performance liquid chromatography (HPLC) utilizing ion exchange,
Each component of hemoglobin is separated and eluted and measured.

Next, using an eluent which the dissociation agent is formed by containing as dissociation buffer, subjected to the test sample to chromatography to how to dissociate L-HbA _lc.

The buffer solution constituting the eluent has a pH of 4
It can be used as long as it has a buffering capacity in the range of up to 7.5 and does not affect chromatographic separation, and is not particularly limited, but phosphate buffer, citrate buffer, phthalate buffer, etc. Is preferred.

In this case, the p of the eluent, which is a dissociation buffer, is used.
H may be in a range that does not denature hemoglobin and allows HbA _lc to be easily separated and measured by chromatography, and a range of pH 5.0 to 7.5 is usually selected. Further, the content of the dissociating agent contained in the present eluent varies depending on the type and measurement conditions (pretreatment such as hemolysis of blood, pH conditions and column temperature), but in the case of EDTPO or NTPO, Generally, the dissociating agent is added to the eluent at a ratio of about 0.001 to 1.0 w / v%, preferably 0.01 to 0.2 w / v%. In the case of EDDPO, it is L-Hb compared to EDTPO.
Since the _Alc dissociation effect is small, the dissociation agent should be 2 to 3 above.
It is preferable to add twice.

At this time, while the test sample passes through the chromatography column, the contained L- _HbAlc is dissociated by contact with the dissociating agent, and then separated into each hemoglobin component to be eluted and measured. To be done.

The dissociation agent can be contained in both the hemolysis buffer and the eluent. In this case, L- _HbAlc is dissociated particularly effectively.

The dissociative action of L-HbA _lc according to the present invention is
It is believed to be due to the nature of the 2.3-DPG pocket on hemoglobin. For 2,3-DPG pockets, see Banesch et al. (Biochem.Biophys.Res.Commun., 26,16).
2,1967); Chanutin et al., (Arch. Biochem. Biophys., 12
1: 96,1967) and so on. This 2,3-DPG pocket is formed by histidine of hemoglobin β chain, basic amino acid residues such as lysine, and hemoglobin β chain N-terminal valine,
It is known to be cationic. It is believed that glucose enters this pocket and binds to the β chain N-terminal valine to produce HbA _lc . Since the dissociation agent of the present invention has anionicity and its molecular shape is sterically appropriate, it has strong compatibility with the 2,3-DPG pocket. Therefore, the dissociation agent of the present invention is
It is considered that they compete with glucose that reversibly binds to hemoglobin, enter the 2,3-DPG pocket, and drive it out, and as a result, dissociation of L-HbA _lc is promoted. However, glucose that is irreversibly bound cannot be displaced.

Thus, the L- by the dissociation agent of the present invention is
HbA_lcL-HbA by dissociation _lcIs in the sample to be inspected
Selectively removed from the S-HbA_lcOnly remains in the sample
To do.

The dissociation agent of the present invention dissociates L-HbA _lc into hemoglobin A _o and glucose in a low concentration and in a short time, and therefore does not adversely affect the elution pattern of hemoglobin by ion exchange chromatography. Therefore, the S- _HbAlc is separated by ion exchange chromatography in the same manner as in the conventional method, and is accurately measured.

The dissociation agent is chemically more stable than polyphosphoric acid and phytic acid disclosed in the prior art, and the dissociation buffer solution, which is an aqueous solution thereof, is also an aqueous polyphosphoric acid solution or phytic acid. It is superior in storage stability to aqueous solutions. Therefore, there is little need to be careful about preservation such as preservation in a cold place.

[0047]

EXAMPLES The present invention will be described below with reference to examples.

Measurement Method In the following examples, HbA _lc value measurement was carried out in a hemolysis mode using Auto-A _lc HA-8121 manufactured by Kyoto Daiichi Kagaku.

This Auto- _Alc HA-8121 was obtained by HPLC
Is a device exclusively for measuring glycated hemoglobin, and a hemoglobin component is separated and eluted in 4 minutes by a separation column packed with a cation exchange resin dedicated to this device. As the elution buffer, an eluent (phosphate buffer) exclusively for this device was used. For hemolysis, a hemolysis buffer (containing a hemolytic agent) dedicated to this apparatus was used. The details of other usages were in accordance with the instruction manual of this measuring device. An example of hemoglobin elution pattern using this device is shown in FIG. In FIG. 1, P
1 and P2 are HbA _lc and HbA _lb (Hb
The peak due to A _{la + b} ), P3 in the hatched part is H
bA _lc , and P4 are other hemoglobins (Hb
This is a peak due to A _lc ). P5 is a component called fetal hemoglobin (HbF) and is usually not included in glycated hemoglobin. L-HbA _lc is contained in a peak that exists between P3 and P5, it can be seen that not completely separated from P3.

Here, HbA _lc % is calculated by the following equation, and the measured value is automatically printed out as a% value with one decimal point.

[0051]

[Equation 1] In this embodiment, blood of the same person (healthy person) is used,
Immediately after blood collection, an appropriate amount of heparin was added and used as fresh blood.

Measurement of Reference Value 0.1 g of polyoxyethylene (10) octyl phenyl ether (obtained from Wako Pure Chemical Industries, Ltd.) as a hemolytic agent was added to 100 ml of 50 mM phosphate buffer solution of pH 6.0 to hemolyze. Buffer solution was prepared. This hemolysis buffer 450μ
3 μl of fresh blood immediately after blood collection was added to 1 to cause hemolysis, and HbA _lc % (L type and S
(Including the mold) was 5.0%. The value, if not removed at all of L-HbA _lc, i.e. the blank value when containing 100% L-HbA _lc.

Next, about 5 ml of erythrocytes obtained by centrifuging 10 ml of fresh blood was put into an 8/32 inch cellophane tube (obtained from Wako Pure Chemical Industries, Ltd.), and physiological saline 1 was added.
00 ml was used and incubated at 37 ° C. for 5 hours. 1.5 μl of this erythrocyte was collected, 450 μl of the above-mentioned hemolysis buffer was added to cause hemolysis, and HbA _lc % was immediately measured by the same method, and it was 4.3%. This value, HbA _lc when L-HbA _lc was completely removed
%, That is, 100% S- _HbAlc %.

Example 1 0.1 g of polyoxyethylene (10) octylphenyl ether (obtained from Wako Pure Chemical Industries, Ltd.) as a hemolytic agent was added to 100 ml of 50 mM phosphate buffer, and EDTPO (as a dissociator) 0.2 g of (obtained from Dojindo Laboratories) was dissolved. The pH was adjusted to 6.0 by appropriately adding a 1N sodium hydroxide aqueous solution and a 1N phosphoric acid aqueous solution,
A dissociation buffer containing a hemolytic agent was obtained. This dissociation buffer 4
3 μl of fresh blood was added to 50 μl for hemolysis, and the hemolyzed product was incubated at room temperature for about 20 minutes to give L-
Dissociation of _HbAlc was performed. The HbA _lc % of this sample was 4.3% when measured according to the above-mentioned measuring method. The eluted blood product prepared in the same manner was incubated at 50 ° C. for 2 minutes, and then HbA _lc % was measured according to the above-mentioned measuring method and found to be 4.3%.

The total area of the fractionated hemoglobin peaks was 97% of the total area at the time of reference measurement,
There was almost no change.

From the above, it can be seen that L- _HbAlc is almost completely dissociated by EDTPO without denaturing hemoglobin.

Example 2 According to Example 1, 0.1 g of polyoxyethylene (10) octylphenyl ether (obtained from Wako Pure Chemical Industries, Ltd.) as a hemolytic agent was added to 100 ml of 50 mM phosphate buffer solution, and dissociated. 0.2 g of NTPO (obtained from Dojindo Laboratories Co., Ltd.) as an agent was dissolved and the pH was adjusted in the same manner as in Example 1 to obtain a dissociation buffer. 450 μl of this dissociation buffer
Fresh blood 3μl added, was dissociation of hemolysis and L-HbA _lc was left for about 20 minutes at room temperature. The HbA _lc % of this sample was measured and found to be 4.4%.
A hemolyzed product prepared in the same manner was incubated at 50 ° C. for 2 minutes, and HbA _lc % was measured.
%Met.

Also, with respect to the total area of the fractionated hemoglobin peaks, the same results as in Example 1 were obtained.

From the above, EDTPO is also applied to NTPO.
It can be seen that it has the same L- _HbAlc dissociation ability as in.

Example 3 According to Example 1, 0.1 g of polyoxyethylene (10) octylphenyl ether (obtained from Wako Pure Chemical Industries, Ltd.) as a hemolytic agent was added to 100 ml of 50 mM phosphate buffer solution, and dissociated. 0.5 g of EDDPO (obtained from Dojindo Laboratories Co., Ltd.) as an agent was dissolved and the pH was adjusted in the same manner as in Example 1 to obtain a dissociation buffer. This dissociation buffer 450
added fresh blood 3μl in [mu] l, was dissociation of hemolysis and L-HbA _lc was left for about 20 minutes at room temperature. The HbA _lc % of this sample was measured and found to be 4.4%. A hemolyzed product prepared in the same manner was incubated at 50 ° C. for 2 minutes, and HbA _lc % was measured.
It was 4.4%.

Also, with respect to the total area of the fractionated hemoglobin peaks, the same results as in Example 1 were obtained.

From the above, EDTP is also used for EDDPO.
It can be seen that it has the same L- _HbAlc dissociation ability as O.

Example 4 0.1 w / EDTPO was added to a dedicated eluent A for measuring HbA _lc (manufactured by Sekisui Chemical Co., Ltd.) consisting of a phosphate buffer.
v% as above to obtain an eluent containing a dissociation agent as a dissociation buffer. Separately, a hemolysis buffer similar to that in Example 1 (containing no dissociation agent) was prepared, and using this, fresh blood was hemolyzed in the same manner as in Example 1. When this sample was immediately subjected to chromatography using this eluent to measure HbA _lc %, the elution pattern was almost the same as when the dissociation buffer of Example 1 was used.
The _Alc % was 4.4%.

Also, with respect to the total area of the fractionated hemoglobin peaks, the same results as in Example 1 were obtained.

[0065] As described above, sufficiently even using an elution solution containing a dissociating agent of the present invention L-HbA _lc is understood to be removed.

Example 5 The dissociation buffer solution prepared in Example 1 was placed in an incubator at 60 ° C. and heated for 20 days. This dissociation buffer 4
3 μl of fresh blood was added to 50 μl to hemolyze, and the hemolyzed product was incubated at room temperature for about 20 minutes,
When the HbA _lc % of this sample was measured according to the above-mentioned measuring method, it was 4.4%. The hemolysate prepared in the same manner was incubated at 50 ° C. for 2 minutes, and then Hb
When A _lc % was measured according to the above measuring method, it was 4.4.
%Met.

Also, with respect to the total area of the fractionated hemoglobin peaks, the same results as in Example 1 were obtained.

From the above, it can be seen that EDTPO has almost no effect on the L- _HbAlc dissociation ability even when left at 60 ° C. for 20 days.

Comparative Example 1 Instead of EDTPO of Example 1, 0.01 M of semicarbazide hydrochloride (obtained from Wako Pure Chemical Industries, Ltd.) and 0.004 M of aniline (obtained from Wako Pure Chemical Industries Ltd.) were added. The pH was adjusted to 5.0 in the same manner as in Example 1,
A dissociation buffer containing a hemolytic agent was obtained. This dissociation buffer 4
After adding 3 μl of fresh blood to 50 μl and incubating at 38 ° C. for 30 minutes, HbA _lc % was measured and found to be 4.5%. However, the fractionation patterns varied considerably, with the total area of the hemoglobin peak being 70% of the reference. A hemolyzate prepared in the same manner was incubated at 50 ° C. for 2 minutes, and then HbA
_{The lc} % was measured and found to be 4.8%.

From the above, it is understood that the reaction time cannot be shortened even if the reaction temperature is raised.

Comparative Example 2 Boric acid (obtained from Wako Pure Chemical Industries, Ltd.) was added at 1 w / v% instead of EDTPO of Example 1, and pH was adjusted to 5 by the same method as in Example 1. It was adjusted to 0.0 to obtain a dissociation buffer containing a hemolytic agent. This dissociation buffer 450μ
3 μl of fresh blood was added to 1 for hemolysis. After incubating the hemolyzed product at 60 ° C. for 5 minutes, HbA _lc % was measured in the same manner as in Example 1.
It was 3%. However, the hemolyzed product prepared in the same manner was incubated at room temperature for 20 minutes, and
The A _lc % was measured and found to be 4.7%.

Further, hemolysis was carried out using a dissociation buffer having a boric acid concentration of 0.1 w / v%, and the hemolyzed product was treated at 60 ° C.
When the same measurement was carried out after 5 minutes of incubation at HbAlc, the _HbAlc content was 4.7%. Thus, it can be seen that the L- _HbAlc dissociation ability decreases when boric acid has a low concentration or when the heating temperature is insufficient.

Comparative Example 3 Instead of EDTPO of Example 1, sodium tetrapolyphosphate was used.
Lium (obtained from Taihei Chemical) and dipotassium phytate
0.1 W each (obtained from Wako Pure Chemical Industries, Ltd.)
/ V% so that the pH is the same as in Example 1.
It was adjusted to 6.0 to obtain a dissociation buffer containing a hemolytic agent.
Using this, fresh blood was hemolyzed in the same manner as in Example 1, and lysed.
Blood products were incubated at room temperature for 20 minutes. Fruit
HbA as in Example 1_lcWhen the% is measured,
It was 4.4% in each case. However, this hemolysis buffer
After heating at 60 ° C for 20 days, the same measurement was performed.
HbA _lcThe contents were 4.9% and 5.0%, respectively.

From the above, it can be seen that the polyphosphoric acid and phytic acid contained in the main hemolysis buffer are hydrolyzed by heating at 60 ° C. for 20 days, and the L- _HbAlc dissociation effect is lost. .

[0075]

As described above, when the L-HbA _lc dissociating agent of the present invention is used, L-HbA of HbA _{lc in} the sample is analyzed.
_lc is rapidly removed, and only S-HbA _lc is measured accurately and easily and in a short time without denaturing hemoglobin. For S-HbA _lc is relatively stable irrespective of the rise or descent of the temporary blood glucose present in the blood, this measurement may be indicative of a more reliable diabetes.

[Brief description of drawings]

FIG. 1 is a chart showing an elution pattern when hemoglobin in blood is separated and measured by ion exchange chromatography.

Claims (3)

[Claims] 1. An unstable glycated hemoglobin dissociator comprising a compound in which at least one hydrogen atom of ammonia is replaced with a methylenephosphonic acid group or a salt thereof. 2. A labile glycated hemoglobin dissociator comprising a compound in which at least one hydrogen atom on the nitrogen atom of a primary or secondary amine having 1 to 6 carbon atoms is replaced with a methylenephosphonic acid group or a salt thereof. 3. The unstable hemoglobin A _lc and the hemoglobin A _o are brought into contact with the hemolysis product containing the red blood cell dispersion liquid and / or hemoglobin and the dissociation buffer solution containing the dissociation agent according to claim 1 or 2. A step of dissociating into glucose, and a step of separating hemoglobin and glycated hemoglobin present in the sample after the dissociation into respective hemoglobin components by a chromatography method, and measuring stable hemoglobin A _lc present in the test sample, A method for measuring glycated hemoglobin, which comprises: