JPH071277B2 - Unstable glycated hemoglobin dissociator - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to glycated hemoglobin (hemoglobin) in blood.
A _1), in particular to the dissociation agent labile glycated hemoglobin A _1C used in the measurement of 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 H
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. This is because the amount of L-HbA _1C changes depending on the amount of glucose (that is, blood glucose), and the blood glucose changes drastically and significantly 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, David M. Nathan et al., Clinical Chemistry, 28 512-515.
(1982), semicarbazide and aniline were added to L-HbA.
It has been disclosed that a blood sample is treated for 30 minutes at pH 5.0 and 38 ° C. by using it as a _{1 C} removing agent. Semicarbazide is
It captures glucose, acts as a nucleophile, and competes with the amino group of Hb. Aniline acts as a catalyst.
As a result, L-HbA _1C is substantially removed. However, L-HbA
_{Since the 1C} removal reaction 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, when the elution pattern by ion exchange chromatography is observed, a decrease in peak intensity due to fading due to deheme and an increase in peaks due to HbA _1a and HbA _1b are observed.

JP-A-58-210024 discloses a dihydroxypolyl compound (boric acid compound) as a remover of L-HbA _1C . This dihydroxypolyl compound
Glucose is removed from the system to esterify the OH group, and as a result, L-HbA _{1 C} dissociation proceeds. However, L-HbA
_A high concentration of dihydroxypolyl compound is required to remove _1C . For example, when a hemolysate is eluted using an eluent containing the dihydroxypolyl compound in a sample solution containing hemolyzed blood, the eluent containing 0.01 to 0.15 M It is necessary to be contained in a ratio. When such a high concentration of dihydroxypolyl 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 and the measurement becomes difficult or difficult. It is necessary to correct the conditions. Furthermore, since the optimum pH is about 4.5 to 6.5, preferably 5.0 to 6.0, Hb denaturation may occur.

Another known method for removing L-HbA _1C is to dilute a blood sample to reduce the glucose concentration and accelerate the dissociation of L-HbA _1C . 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, none of these methods are suitable for clinical examination because they require a long time.

(Problems to be Solved by the Invention) The present invention solves the above-mentioned conventional drawbacks, and an object thereof is to obtain stable hemoglobin A in a blood sample.
The _1C only, and accurately simple and can be determined without denaturing the hemoglobin is to provide a removing agent or releasing agent of labile hemoglobin A _1C, and measurement of hemoglobin A _1C methods.

(Means and Actions for Solving Problems) The labile glycated hemoglobin dissociator of the present invention is used for measuring hemoglobin A _{1 in} blood, and the dissociator produces phytic acid and / or a salt of phytic acid. As a main component, it has a function of dissociating unstable hemoglobin A _1C into hemoglobin and glucose, thereby achieving the above object.

The method for measuring glycated hemoglobin of the present invention comprises a sample containing at least red blood cells and / or hemoglobin in blood,
A step of contacting with a dissociation medium containing phytic acid and / or a salt of phytic acid to dissociate unstable hemoglobin A _1C contained in the red blood cells and / or hemoglobin into hemoglobin and glucose, and a sample after the dissociation The method includes the step of separating the hemoglobin therein to each hemoglobin component by ion exchange chromatography, and measuring the stable hemoglobin A _1C present in the sample, thereby achieving the above object.

Phytic acid in the present invention is represented by the following structural formula.

Phytic acid has the same effect even if it is a salt thereof. The type of salt is not particularly limited, but examples thereof include salts of alkali metals and alkaline earth metals, and Na salts and K salts are preferable.

The amount of the L-HbA _1C dissociator used in the present invention varies depending on the type of compound used and the measurement conditions (conditions of pretreatment such as hemolysis of blood, pH, etc.), but usually 1 ml of blood (whole blood)
The ratio is 1 to 1500 mg per unit. For example, blood sample 3μ
Phytic acid is approximately 0.001 to 1.0 W / V% (W / V% indicates weight per unit volume), preferably 0.01 to 0.2 W / V, in a sample solution obtained by hemolyzing 1 of 1 with 450 μl of hemolytic agent solution. % Is added. If it is too small, the dissociation / removal effect of L-HbA _{1 C} 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 fatty acid esters. The amount of hemolytic agent used varies depending on the type, but usually blood
It is 10 to 2000 mg per ml. For example, 0.01 to
Hemolysis can be carried out by adding a treatment solution for hemolysis containing 2% by volume at a rate of 2 to 400 ml per 1 ml of blood and incubating. Excessive hemolytic agent makes it difficult to separate Hb by chromatography.

In order to measure glycated Hb according to 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 of the present invention. 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. Liquid is also considered as the dissociation medium. In addition, the dissociation agent may be added as a solid to, for example, a red blood cell dispersion liquid of a sample, a hemolysate thereof, or an Hb solution. This dissociator dissolves quickly, and Hb in the sample is dissociated when it comes into contact with the dissociation medium.

Among the methods for measuring glycated Hb using the dissociating agent of the present invention, a method of separating and measuring a sample by dissociating the sample with the dissociating agent and then subjecting it to chromatography will be described below. 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 when the pH is too low, Hb
Degeneration occurs. Furthermore, it becomes difficult to separate Hb by chromatography under extremely high or low pH conditions. Therefore, the pH of the mixture containing the sample and the dissociator is usually 4.6.
It is adjusted to be ~ 7.0, preferably 5.3-6.5. The contact time between the sample and the dissociation agent varies depending on the type and concentration of the dissociation agent, pH conditions, etc.
It is preferably 10 to 30 minutes. It is possible to shorten the contact time by raising the temperature.
It is also possible to incubate at 50 ° C. for about 1 to 3 minutes. The sample treated in this manner is subjected to ion exchange chromatography (for example, high performance liquid chromatography), and each component of Hb is separated and eluted, and then 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. 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.

It is considered that the dissociation / removal action of L-HbA _1C by the dissociation agent of the present invention is due to the property of 2,3-DPG pocket on Hb. For 2,3-DPG pockets, see Benesch et al.
ochem.Biophys.Res.Commun., 26,162,1967); Chanutin
(Arch.Biochem.Biophys., 121: 96, 1967) and the like. This 2,3-DPG pocket is a basic amino acid residue such as histidine and lysine in the β chain of Hb,
And HbA _1C is formed by the N-terminal valine of the Hbβ chain and is known to be cationic.
Phytic acid and its salts, which are the main components of the dissociation agent of 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, it competes with glucose and binds to the N-terminal of the β chain of Hb. As a result, dissociation of L-HbA _1C is promoted.

Thus L-HbA _1C by dissociation action of L-HbA _1C salts of phytic acid and / or phytic acid are removed from the sample, only the S-HbA _1C remain in the sample. This dissociator is H
It does not affect the elution pattern of b 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 _1C was measured using Hi-AutoA _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 Trit as a hemolytic agent in 100 ml of 0.005 M phosphate buffer of pH 6.3
onX-100 (manufactured by Wako Pure Chemical Industries, Ltd.) was added to prepare a hemolytic reagent. 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 is 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 SH in blood
It is considered to be equivalent to bA _1C .

Example 1 Triton X-100 as a hemolytic agent in 100 ml of 0.005 M phosphate buffer
(Wako Pure Chemical Industries) 0.1 ml and phytic acid 40 as a dissociator
% Aqueous solution (manufactured by Aldrich) was dissolved and a pH was adjusted to 6.3 by adding a base to obtain a dissociation medium containing a hemolytic agent.
To 450 μl of this dissociation medium containing hemolytic agent, 3 μl of fresh blood was added, and the mixture was allowed to stand at room temperature for about 10 minutes to allow hemolysis and L-HbA _1C.
Was dissociated. The HbA _1C of this sample was measured and found to be 4.4%. Separately, a hemolytic agent solution containing no phytic acid (dissociation agent) was prepared, and HbA _1C was measured by the same method.

Next, the pH of the dissociation medium containing the hemolytic agent and the hemolytic agent solution (without the dissociating agent) were adjusted to 5.8, 5.3 and 5.0, respectively.
And 4.6, and HbA _1C was measured in the same manner. Table 1 shows the HbA _1C content and the total Hb content in the total Hb when the dissociation medium containing the hemolytic agent was used and when the hemolytic agent solution (without the dissociating agent) was used. The total Hb amount was converted with the total Hb amount in the original blood as 100%.

From Table 1, 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. In the above system, the total Hb content at pH 4.6 is about 75% of that at pH 6.3. When measured using the dissociator of the present invention, pH 6.3 to 5.
It is clear that L-HbA _{1 C} is dissociated and removed in the region 3 without changing Hb.

Example 2 Triton X-100 as a hemolytic agent in 100 ml of 0.005 M phosphate buffer
0.1 ml (manufactured by Wako Pure Chemical Industries, Ltd.) and about 0.1 g of sodium phytate (manufactured by Aldrich) as a dissociating agent were dissolved and a pH was adjusted to 6.3 by adding a base to obtain a dissociating medium containing a hemolytic agent.
To 450 μl of this dissociation medium containing hemolytic agent, 3 μl of fresh blood was added, and the mixture was allowed to stand at room temperature for about 10 minutes to allow hemolysis and L-HbA _1C.
Was dissociated. The HbA _1C of this sample was measured and found to be 4.3%.

Example 3 The amount of sodium phytate was changed according to Example 2.
A hemolytic agent-containing dissociation medium (each having a pH of about 6.0) containing 1.0 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 Table 2.

From Table 2, sodium phytate 0.005g (0.005W / V%
It can be seen that the effect of removing L-HbA _{1 C} can be obtained even at a low concentration (about). Sodium phytate is 1.0 / g (1.0W /
In the case of about V%), the pattern of the chromatogram changes broadly, so it is desirable to lower the concentration a little.

Example 4 According to Example 2, 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 Table 3.

From Table 3, L-HbA _1C becomes faster as the reaction temperature is increased.
It can be seen that is removed. However, when incubated at 60 ° C for 3.5 minutes, Hb denaturation was observed 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 5 Sodium phytate was added to 0.005 in a dedicated eluent A for HbA _{1 C} measurement (manufactured by Sekisui Chemical Co., Ltd.), which was composed of a phosphate buffer.
% To obtain an eluent containing a dissociator (pH 6.
0). 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 is sufficient to use an eluent containing the dissociator of the present invention.
It can be seen that L-HbA _1C is removed.

Comparative Example 1 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 this was measured, the HbA _1C content was 4.7%. However, the fractionation pattern became quite broad, and the total Hb peak area was about 70
%Met.

Comparative Example 2 A hemolytic agent-containing L-HbA _1C scavenger solution consisting of the same hemolytic agent as in Example 1 (the 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 60 ° 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 an elution pattern when Hb in blood is separated and measured by ion exchange chromatography.

Claims (2)

[Claims] 1. A dissociation agent for unstable glycated hemoglobin used for measurement of glycated hemoglobin in blood, wherein the dissociation agent contains phytic acid and / or a salt of phytic acid as a main component, and unstable hemoglobin A _1C. An unstable glycated hemoglobin dissociator that can dissociate into hemoglobin and glucose. 2. A labile hemoglobin contained in red blood cells and / or hemoglobin by contacting a sample containing at least red blood cells and / or hemoglobin in blood with a dissociation medium containing phytic acid and / or a salt of phytic acid. A step of dissociating A _1C into hemoglobin and glucose, and a step of separating hemoglobin in the sample after the dissociation into each hemoglobin component by ion exchange chromatography, and measuring a stable hemoglobin A _1C present in the sample, A method for measuring glycated hemoglobin, which comprises: