JPH076989B2 - Method for measuring glycated hemoglobin - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to glycated hemoglobin (hemoglobin) in blood.
A ₁ ), especially concerning the method for measuring hemoglobin A _1C .

(Prior Art) Glycated hemoglobin (hemoglobin A ₁ ) in blood, particularly hemoglobin A _1C, is known as one of the indicators of diabetes.
That measurement is taking place. Hemoglobin A _1C is a complex in which one glucose molecule is non-enzymatically bound to the amino group of valine existing at the N-terminal of the β chain of hemoglobin (hereinafter, hemoglobin is represented by Hb) as shown in the following formula. . When glucose binds to the above amino group of Hb, a Schiff base, unstable hemoglobin A _1C (I), is first formed as shown in the following formula: In the above formula (1), β-A-NH ₂ represents Hb, and NH ₂ therein represents the amino group of valine at the N-terminal of β chain of Hb.

The formation reaction of this unstable HbA _1C (I) is a reversible reaction, and the equilibrium of HbA _1C (I) is unstable depending on the glucose concentration.
_1C (I) is inclined toward the production or dissociation direction. (I)
Are irreversibly stable HbA through the Amadori rearrangement.
Change to _1C (II).

HbA _1C can be isolated from HbA _1C by high performance liquid chromatography, for example.
It can be quantified by separating b into each Hb component and measuring its OD, but separate and measure the stable HbA _1C (S-HbA _1C ) and unstable HbA _1C (L-HbA _1C ). I can't. Therefore, HbA _1C cannot be obtained at a stable value. Because L-HbA _1C is
This is because the amount thereof changes and the blood glucose level changes abruptly and drastically due to diet and exercise.

In order to eliminate the above drawbacks, attempts have been made to remove L-HbA _1C from blood samples and measure only S-HbA _1C . For example, Dsvid M. Nathan et al., Clinical Chemistry, 28 512-515.
(1982) using semicarbazide and aniline, p
A method of treating a blood sample for 30 minutes at H5.0, 38 ° C is disclosed. Semicarbazide captures glucose and acts as a nucleophile, competing with the amino group of Hb. Aniline acts as a catalyst. As a result, L-HbA _1C is substantially removed. However, since the removal reaction of L-HbA _1C is carried out on the acidic side (pH 5.0) and at a relatively high temperature (38 ° C), Hb denaturation (eg deheme) occurs. For example, observing the elution pattern by ion exchange chromatography shows a decrease in peak intensity due to discoloration due to deheme and
An increase in peaks due to HbA _1a and HbA _1b is observed.

JP-A-58-210024 discloses the use of a dihydroxyboryl compound (boric acid compound). Since this dihydroxyboryl compound esterifies the OH group of glucose, glucose is removed from the system, and as a result, dissociation of L-HbA _{1 C} proceeds. However, high concentrations of dihydroxyboryl compounds are required to remove L-HbA _1C . For example, in a sample solution containing hemolyzed blood,
When the hemolysate is eluted using an eluent containing 0.1 to 1.0 M of the dihydroxyboryl compound, 0.
It is necessary to be contained at a ratio of 01 to 0.15M. When such a high concentration of dihydroxyboryl compound is contained, the ionic strength when subjected to ion exchange chromatography is different from the usual case, and as a result, the separation conditions change, making the measurement difficult or difficult. Will need to be corrected. Furthermore, the optimum pH is about 4.5 to 6.5, preferably 5.0.
Since it is ~ 6.0, Hb may be denatured.

As a method of removing L-HbA _{1C and} others, a method of diluting a blood sample to lower the glucose concentration and promoting dissociation of L-HbA _1C is known. To carry out this, for example, a method of incubating red blood cells with a large excess of physiological saline or dialysis of hemolysate is adopted. However, each of these methods requires a long time and is not suitable as a method for clinical examination.

(Problems to be Solved by the Invention) The present invention solves the above-mentioned conventional drawbacks, and an object thereof is to provide unstable hemoglobin in a blood sample.
Removing the A _1C, only stable hemoglobin A _1C, may be measured without denaturing accurately simply and hemoglobin is to provide a method for measuring hemoglobin A _1C.

(Means and Actions for Solving Problems) The method for measuring glycated hemoglobin according to the present invention uses a sample containing at least red blood cells and / or hemoglobin in blood,
Contacting with a dissociation medium containing a salt of a phosphoric acid condensate and / or a phosphoric acid condensate to dissociate the unstable hemoglobin A _1C contained in the red blood cells and / or hemoglobin into hemoglobin and glucose, and The method includes the step of separating hemoglobin in the sample after dissociation into each hemoglobin component by ion exchange chromatography, and measuring the stable hemoglobin A _1C present in the sample, whereby the above object is achieved.

The phosphoric acid condensate used in the present invention functions as a dissociator for L-HbA _1C . Examples of such phosphoric acid condensates include (HPO
₃ ) Metaphosphoric acid represented by _n (n is an integer of 2 or more), polyphosphoric acid containing phosphorus of 2 atoms or more and having a POP bond, and analogs thereof. Examples of metaphosphoric acid include trimetaphosphoric acid (III) and tetrametaphosphoric acid (IV), and examples of polyphosphoric acid include pyrophosphoric acid (V) and tetrapolyphosphoric acid (VI).

Analogues related thereto include compounds in which the above compounds have side chains and compounds in which they are condensed in a more complex manner. For example, the following (VII), (VIII) and (IX)
Compounds of

In addition to the above compounds, compounds that form these phosphoric acid condensates when dissolved in water are also included. For example, phosphorus pentoxide may also be used in the present invention. When phosphorus pentoxide is dissolved in water, it changes into ultrapolyphosphoric acid (IX), tetrametaphosphoric acid (IV), tetrapolyphosphoric acid (VI), etc. by hydrolysis. However, the hydrolysis end product, mono-orthophosphoric acid (H
₃ PO ₄ ) has no such effect.

The degree of condensation of the phosphoric acid condensate is most preferably 2 to 6 in view of the effect of removing L-HbA _1C . However, even compounds with a higher degree of polymerization can be used because they generate P _{2 to} P ₆ phosphoric acid condensates by hydrolysis in aqueous solution.

These phosphoric acid condensates have the same effect even if they are salts thereof. The type of salt is not particularly limited, and examples thereof include salts of alkali metals and alkaline earth metals, Na salt, K
Salt and the like are preferred.

The amount of the phosphorus acid condensate and / or phosphorus acid condensate salts when measuring the HbA _1C (L-HbA _1C dissociating agent), the該解eluant the type and at the time of measurement conditions (such as the hemolysis of blood It is usually used at a rate of 1 to 6000 mg per 1 ml of blood (whole blood), although it depends on the pretreatment conditions, pH, etc. For example, if pyrophosphoric acid is contained in a sample solution obtained by hemolyzing 3 μl of a blood sample with 450 μl of a hemolytic agent solution, about 0.1 to 4.0 W / V%
(W / V% represents the weight ratio per unit volume), preferably in the ratio of 0.5 to 2.0 W / V%, and in the case of tetrapolyphosphoric acid, about 0.001 to 1.0 W / V%, preferably Is 0.0
It is contained at a rate of 1 to 0.2 W / V%. When it is contained in the chromatographic eluent, for example, tetrapolyphosphoric acid is contained at a rate of 0.001 to 0.1 W / V%. If it is too small, the dissociation / removal effect of L-HbA _1C cannot be obtained, and if it is too large, separation during chromatographic separation becomes difficult.

When Hb in blood is measured, the Hb is usually present in red blood cells, and therefore blood is usually hemolyzed in advance in many cases. A surfactant is preferably used as the hemolytic agent. Examples of hemolytic agents include higher aliphatic alcohols, alkylaryl polyether alcohols, polyoxyethylene ethers of sulfonate compounds, polyoxyethylene ethers of sulfate compounds, and polyoxyethylene adducts of sorbit aliphatic esters. The amount of hemolytic agent used varies depending on the type, but usually 1 m of blood
It is 10 to 2000 mg per l. For example, 0.01 to 2 hemolytic agents
Hemolysis can be performed by adding a treatment solution for hemolysis contained at a volume ratio of 2 to 400 ml per 1 ml of blood and incubating. Separation of Hb by chromatography with excess hemolytic agent becomes difficult.

In order to measure glycated Hb by the method of the present invention, first, a blood sample is pretreated with a hemolytic agent, if necessary, to hemolyze, and a sample containing at least red blood cells and / or Hb is prepared.
This sample is brought into contact with a dissociation medium containing the dissociation agent (phosphoric acid condensate and / or salt of phosphoric acid condensate). This dissociating agent may come into contact with red blood cells and / or Hb before the sample is separated and eluted by ion exchange in the chromatography column described below. Therefore, the dissociating agent may be contained in a diluent for diluting a blood sample to an appropriate concentration, a hemolytic agent solution containing a hemolytic agent, an eluent used for chromatography, and the like. Liquids are also considered as dissociation media in the present invention. Others, such as sample red blood cell dispersion, its hemolysate, H
Even when the dissociating agent is added to the solution b as a solid, the dissociating agent is rapidly dissolved, and Hb in the sample is dissociated when it comes into contact with the dissociation medium.

Among the methods of the present invention, after dissociating a sample with a dissociating agent, the method of separating and measuring by chromatography is
It will be described next. The dissociation rate of L-HbA _1C increases as the pH at the time of contact between the sample and the dissociation agent (dissociation medium) becomes more acidic, but Hb denaturation occurs when the pH is too low. Furthermore, it is difficult to separate Hb by chromatography under extremely high or low pH conditions. Therefore, normally, the pH of the mixed solution containing the sample and the dissociating agent is adjusted to 4.6 to 7.0, preferably 5.3 to 6.5. The contact time between the sample and the dissociation agent varies depending on the type and concentration of the dissociation agent, pH conditions, etc., but is usually 10 minutes or longer, preferably 10 to 30 minutes at room temperature. It is possible to shorten the contact time by raising the temperature.
It is also possible to incubate at 37 ° C for about 3 to 7 minutes and at 50 ° C for about 1 to 3 minutes. Samples treated in this way
It is subjected to ion exchange chromatography (for example, high performance liquid chromatography), and each component of Hb is separated, eluted, and measured.

When the sample solution is directly subjected to ion exchange chromatography and eluted with an eluent containing the above dissociator, the pH of the eluent does not denature Hb, and HbA _{1 C} can be easily separated by chromatography. The range may be such that it can be measured and measured. Usually, a pH range of 5.0 to 6.5 is selected. Since the phosphoric acid condensate and its salt have a buffering action, they also function as a buffer for giving a buffering action to the eluent. Furthermore, phosphoric acid condensates and salts thereof also have a function of sequestering heavy metal ions, which adversely affects the separation by ion exchange chromatography. L-HbA _{1 C} contained in the sample is dissociated while passing through the chromatography column, and then separated into Hb components, eluted, and measured. The dissociation agent can be contained in both the diluting solution of the sample and the pretreatment solution for hemolysis and the eluent for chromatography, and in this case, the dissociation of L-HbA _{1 C} is particularly effectively performed.

The action of dissociation and removal of L-HbA _1C by such a phosphoric acid condensate and / or a phosphoric acid condensate salt is considered to be due to the property of 2,3-DPG pockets on Hb. For 2,3-DPG pockets, see Benesch et al. (Biochem.Biophys.Res.Commu.
n., 26,162,1967); Chanutin et al., (Arch.Biochem.Biophy
s., 121: 96, 1967). This 2,3-DPG pocket is formed by histidine and basic amino acid residues such as lysine of Hb β chain, and N-terminal valine of HbA _1C Hb β chain, and is known to be cationic. ing. The phosphoric acid condensate and the salt of the phosphoric acid condensate used in the present invention have an anionic property and have an appropriate molecular shape, and thus have a strong affinity for the 2,3-DPG pocket. Therefore, they compete with glucose and bind to the β chain end of Hb. As a result, dissociation of L-HbA _1C is promoted.

Thus L-HbA _1C by dissociation action of L-HbA _1C of phosphorus acid condensate and / or phosphorous acid condensate salts are removed from the sample, only the S-HbA _1C remain in the sample. This dissociator does not affect the elution pattern of Hb by ion exchange chromatography. Therefore, S-HbA _1C is separated by ion-exchange chromatography in the same manner as in the conventional method, and is accurately measured.

(Example) Hereinafter, the present invention will be described with reference to Examples.

Measurement method In the following examples, HbA ₁ was measured using Hi-Auto A _1C HA-8120 manufactured by Kyoto Daiichi Kagaku Co., Ltd. under appropriate conditions. This HA-8120 is a dedicated device for HbA _1C measurement by HPLC and separates and elutes each Hb component in 4 minutes by cation exchange. As the elution buffer, a dedicated eluent (phosphate buffer solution) is used. The elution pattern of Hb using this apparatus is generally shown in FIG. In Fig. 1, P ₁ and
P ₂ is the peak due to the HbA _{1a + b} component, and P ₃ and P ₄
Are peaks due to L-HbA _1C and S-HbA _1C components.
P ₅ is a peak due to other hemoglobin. Here, HbA _1C (total of S type and L type) is calculated by the following equation.

In this embodiment, blood of the same person (healthy person) is used,
Immediately after blood collection, heparin-added one was used as fresh blood.

Measurement of reference value 0.005M phosphate buffer solution (pH 6.3)
A hemolysis reagent was prepared by adding 0.1 ml of iton X-100 (manufactured by Wako Pure Chemical Industries, Ltd.). After lysed by the addition of fresh blood 3μl this hemolysis reagent 450 [mu] l, was measured HbA _1C (including L-type and S-type) by the above process, the total Hb in HbA _1C 5.0
It was found to exist in the ratio of%. This value was used as a blank value.

Next, about 5 ml of blood cells obtained by centrifuging 10 ml of fresh blood was added to 8 / 32i.
The cells were placed in an nch cellophane tube (manufactured by Wako Pure Chemical Industries, Ltd.) and incubated with physiological saline 1 twice each for a total of 5 hours at 37 ° C. After dissociating L-HbA _1C in this manner, about 1.5 μl of this was collected and hemolyzed using 450 μl of the above hemolytic reagent. HbA _1C of this sample was measured by the same method and found to be 4.3%. This value is S-HbA in blood
_It is considered to be equivalent to _1C .

Example 1 Triton X-100 as a hemolytic agent in 100 ml of 0.005 M phosphate buffer
(Wako Pure Chemical Industries, Ltd.) 0.1 ml and tetrapolyphosphoric acid (Wako Pure Chemical Industries, Ltd.) about 0.1 g as a dissociator are dissolved, and a base is added to pH.
Was adjusted to 6.3 to obtain a dissociation medium containing a hemolytic agent. 3 μl of fresh blood was added to 450 μl of this dissociation medium containing a hemolytic agent, and left at room temperature for about 10 minutes to perform hemolysis and dissociation of L-HbA _1C . The HbA _1C of this sample was measured and found to be 4.4.
%Met. Separately, a hemolytic agent solution containing no tetrapolyphosphate (dissociation agent) was prepared, and HbA _1C was measured by the same method.

Next, the pH of each of the hemolytic agent-containing dissociation medium and the hemolytic agent solution (containing no dissociating agent) was adjusted to 5.8, 5.3, 5.0.
And 4.6, and HbA _1C was measured in the same manner. The HbA _1C content in the total Hb when using a dissociation medium containing a hemolytic agent is shown by a solid line (white plot) in Fig. 2, and HbA when a hemolytic agent solution (without a dissociating agent) is used. _{The 1C} content is shown by a solid line (black plot). Furthermore, the total amount of Hb when the dissociation medium containing the hemolytic agent is used is shown in FIG. 2 by the alternate long and short dash line (white plot), and the total amount of Hb when the hemolytic agent solution (without the dissociating agent) is used. It is indicated by a one-dot chain line (black plot).
The total Hb amount was converted with the total Hb amount in the original blood as 100%.

From FIG. 2, it is clear that the HbA _1C value becomes lower as the pH condition becomes lower, and L-HbA _1C is dissociated and removed. However, it can be seen that when the pH is lower than 5.3, discoloration due to deheme is observed and the total Hb amount decreases. This tendency is particularly remarkable in the system of the present invention containing tetrapolyphosphoric acid, and the total Hb amount at pH 4.6 is about 75% of that at pH 6.3. In the method of the present invention, it is clear that L-HbA _{1 C} is dissociated and removed without changing Hb in the pH range of 6.3 to 5.3.

Example 2 According to Example 1, the amount of tetrapolyphosphoric acid was 1.0
A hemolytic agent-containing dissociation medium (all having a pH of about 6.0) containing g, 0.2 g, 0.05 g, 0.005 g and 0.001 g was prepared. Using this, the HbA _{1 C} content was measured in the same manner as in Example 1. The results are shown in the table below.

From the table, it can be seen that the effect of removing L-HbA _{1 C} can be obtained even when the concentration of tetrapolyphosphoric acid is as low as 0.005%. When the concentration of tetrapolyphosphate is 1.0%, the pattern of the chromatogram changes broadly, so it is desirable to lower the concentration slightly.

Example 3 In accordance with Example 1, a hemolytic agent-containing dissociation medium having a pH adjusted to 6.0 was prepared. Using this, temperature conditions were set to 37 ° C, 50 ° C, and 60 ° C, and after incubating for a predetermined time, HbA _1C was measured. The results are shown in FIG.

From Fig. 3, L-HbA increases more rapidly as the reaction temperature is increased.
It can be seen that _1C is removed. However, denaturation of Hb was observed after incubation at 60 ℃ for 3.5 minutes, and the total Hb content was about 90
Fell to%. Therefore, it is considered not desirable to incubate at high temperature for a long time.

Example 4 About 5 ml of blood cells obtained by centrifuging 10 ml of fresh blood was 8/32 inch.
Put in a cellophane tube (manufactured by Wako Pure Chemical Industries) and use 1000 ml of isotonic solution containing 1000 mg / dl of D-glucose for 5 hours at 37 ° C.
The sample was dialyzed with L-HbA _1C to obtain a sample with a high content. A hemolytic agent solution (containing no dissociation agent) having a pH of 6.0 was prepared according to Example 1, and about 1.5 µl of the above sample was added to 450 µl of the hemolytic agent solution to hemolyze. HbA _{1 C} content measured using hemolysate is
It was 6.0%. The elution pattern of the chromatogram at this time is shown in FIG. Next, a hemolytic agent-containing dissociation medium was prepared by adding tetrapolyphosphoric acid to the hemolytic agent solution so that the concentration of tetrapolyphosphoric acid became 0.1%.
A sample with high A _1C content was added. After incubating this at 50 ° C for 2 minutes, the HbA _1C amount was measured.
It was 4%. The elution pattern of the chromatogram at this time is shown in FIG. 4 (b). It can be seen that the peak P ₃ observed in FIG. 4 (a) has almost disappeared in FIG. 4 (b). Thus, it is clear that even in the sample containing a high proportion of L-HbA _1C as in this example, L-HbA _1C is sufficiently removed by the method of the present invention.

Example 5 The same as Example 4 except that a hemolytic agent-containing dissociation medium containing sodium pyrophosphate (manufactured by Hanai Chemical Co., Ltd.) at a ratio of 1.0% was used in place of tetrapolyphosphoric acid. Hb
The A _1C content was 4.5%.

Example 6 The same as Example 4 except that a hemolytic agent-containing dissociation medium containing sodium hexametaphosphate at a ratio of 1.0% was used in place of tetrapolyphosphoric acid. HbA _1C content is 4.
It was 5%.

Example 7 Tetrapolyphosphoric acid was added to a dedicated eluent A for HbA ₁ measurement (manufactured by Sekisui Chemical Co., Ltd.) consisting of a phosphate buffer at 0.005% to obtain a dissociator-containing eluent. Separately, a hemolytic agent solution (containing no dissociation agent) similar to that in Example 1 was prepared, and using this, fresh blood was hemolyzed in the same manner as in Example 1. When this sample was immediately chromatographed using the above eluent and separated and measured, the elution pattern was almost the same as that when the dissociation medium of Example 1 was used, and the HbA _{1 C} content was 4.4%. there were. It can be seen that L-HbA _{1 C} is sufficiently removed by the method of this example.

Comparative Example 1 Sodium dihydrogen phosphate (manufactured by Wako Pure Chemical Industries, Ltd.) was used in place of the tetrapolyphosphoric acid of Example 1, and the hemolysis reagent was adjusted to pH 6.0 containing about 1.0% of the sodium dihydrogen phosphate. Was prepared. After adding this to fresh blood and incubating at 50 ° C. for 2 minutes after hemolysis, HbA was added in the same manner as in Example 1.
A measurement of _{1 C} was performed. The HbA _1C value was 5.0%, indicating that L-HbA _1C was not removed.

Comparative Example 2 Hemolytic agent similar to that in Example 1 (same concentration as in Example 1), semicarbazide hydrochloride (manufactured by Wako Pure Chemical Industries, Ltd.) 0.01M, aniline (manufactured by Wako Pure Chemical Industries, Ltd.) 0.004M, and 0.005M phosphorus at pH 5.3 A hemolytic agent-containing L-HbA _{1 C} removing agent solution consisting of an acid buffer was prepared. Using this, fresh blood was hemolyzed in the same manner as in Example 1, and the hemolyzed product was treated at 60 ° C.
And incubated for 2 minutes. When I measured this
The HbA _1C content was 4.6%. However, the fractionation pattern became quite broad, and the total Hb peak area was about 70% of the original.

Comparative Example 3 A hemolytic agent-containing L-HbA _1C scavenger solution consisting of the same hemolytic agent as in Example 1 (same concentration as in Example 1) and 0.005M phosphate buffer of pH 5.3 containing 1.0% boric acid was prepared. Prepared. Using this, fresh blood was hemolyzed in the same manner as in Example 1, and the hemolyzed product was incubated at 50 ° C for 2 minutes. When this was measured, Hb
The A _1C content was 4.4%. However, the boric acid concentration is 0.1%
When pH was adjusted to 6.0 and the same measurement was performed, HbA
_{The 1C} content was 4.8%. Thus, when boric acid has a low concentration, L-HbA _{1 C} cannot be removed sufficiently.

(Effects of the Invention) According to the present invention, as described above, LH of HbA _1C in blood is
bA _1C is removed, and only S-HbA _1C can be measured accurately and easily in a short time without denaturing hemoglobin. Since S-HbA _1C exists in blood relatively stably regardless of transient rise or fall in blood glucose, this measurement value may be a more reliable indicator of diabetes. By automating the measurement system, more effective clinical tests will be possible.

[Brief description of drawings]

Fig. 1 is a chart showing the elution pattern when Hb in blood is separated and measured by ion exchange chromatography, and Fig. 2 is the hemolysis of blood under different pH conditions using the method of the present invention and the conventional method. When measured after the
Fig. 3 is a graph showing the relationship between pH condition and HbA _1C content, and the relationship between the pH condition and total Hb content, and Fig. 3 is the dissociation reaction time and HbA _1C content when HbA _1C in blood was measured by the method of the present invention. Graph showing the relationship with, and Fig. 4 (a) and (b)
FIG. 3 is a chart showing a blood sample having a high HbA _1C content and an elution pattern of ion exchange chromatography when the sample is treated by the method of the present invention.

Claims (2)

[Claims] 1. A sample containing at least erythrocytes and / or hemoglobin in blood is brought into contact with a dissociation medium containing a phosphoric acid condensate and / or a salt of a phosphoric acid condensate to contain the red blood cells and / or hemoglobin. The process of dissociating unstable hemoglobin A _1C into hemoglobin and glucose, and separating the hemoglobin in the sample after the dissociation into each hemoglobin component by ion exchange chromatography, and the stable hemoglobin A _1C present in the sample A method for measuring glycated hemoglobin, which comprises the step of measuring. 2. The dissociation medium is at least one of a diluent used for preparing the sample, a pretreatment solution for hemolyzing the sample, and an eluent used for the chromatography. The measuring method according to the first paragraph.