JPH0434364A - Unstable saccharifying hemoglobin dissociating agent - Google Patents

Abstract

PURPOSE:To accurately and simply obtain a dissociating agent of L-HbA1c capable of measuring only S-HbA1c in a blood specimen without denaturing hemoglobin by using a specific acid and/or a salt thereof as a main component. CONSTITUTION:Pyridoxal phosphate and/or a slat thereof or nucleotide and/or a salt thereof is used as a main component to obtain a dissociating agent capable of removing L-HbA1c among HbA1c in blood to measure only S-HbA1c within a short time without denaturing hemoglobin.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to glycated hemoglobin (hemoglobin A) in blood.
1), particularly relates to a dissociating agent for unstable glycated hemoglobin used in the measurement of hemoglobin A1c.

(Prior Art) Glycated hemoglobin (hemoglobin A1) in the blood, particularly hemoglobin A1c (hereinafter referred to as HbA+c), is known as one of the indicators of diabetes, and its measurement is being carried out. The HbA r c value is the amount of H that occupies in total hemoglobin.
It is expressed as a ratio (percentage) of bA and c. HbA+
Since c is relatively stable, it is not affected by significant short-term changes in blood sugar levels and reflects well the blood sugar levels from about one or two months ago. Therefore, it is clinically important in long-term glycemic control in diabetic patients.

HbA and C are complexes in which one molecule of glucose is bound non-enzymatically to the amino group of valine present at the N-terminus of the β chain of hemoglobin, as shown in the following formula (1). When glucose binds to the amino group of hemoglobin, an unstable HbA+c (I), which is a Schiff base, is first formed as shown in the following formula (1): Glucose
(1) (II) In the above formula (1), Hb-NHz represents hemoglobin, and NRt therein represents the amino group of valine at the N-terminus of the β chain of the hemoglobin.

The production reaction of unstable HbA, C(1) is a reversible reaction, and depending on the glucose concentration, the parallel is unstable HbA+
c (I) tilts toward the production direction or dissociation direction.

The unstable HbA+c (I) further undergoes the Amatoli transition and irreversibly changes to the stable HbA+c (If).

) 1bA r c can be quantified by separating hemoglobin into each hemoglobin component by high-performance liquid chromatography (HPLC) and measuring its OD.
, c) and unstable HbA, C (hereinafter referred to as L-Hb
The reason is that it is not possible to separate and measure HbAlc (indicated by A and C), and therefore it is not possible to obtain HbAlc at a stable value.

This is because the amount of L-HbAlc changes depending on the amount of glucose (that is, blood sugar level), and the blood sugar level changes rapidly and significantly with one meal or exercise.

In order to overcome the above drawbacks, attempts have been made to remove L4bA+c from blood samples and measure only 5-HbAlc. For example, David M., Nathan
et al., C11nical Chemistry, 2
8.512-515. (1982) discloses the use of semicarbazide and aniline as removal agents for L-HbAlc, and treatment of blood samples at pH 5.0, 38°C for 30 minutes. Samicarbazide scavenges glucose and acts as a nucleophile, competing with the amino groups of hemoglobin. Aniline acts as a catalyst. As a result, L-HbA,C is substantially removed. However, the removal reaction of L-HbAlc is on the acidic side (p
H5,0) and at a relatively high temperature (38° C.), denaturation of hemoglobin (eg dehemization) occurs.

For example, when observing the elution pattern by ion exchange chromatography.

Decrease in peak intensity due to discoloration due to heme removal and HbA
, , and an increase in the peaks due to HbAlb are observed.

JP-A-58-210024 discloses a dihydroxyboryl compound (boric acid compound) as a remover for L-HbA, e. Since this dihydroxyboryl compound esterifies the OH group of glucose, glucose is removed from the system, and as a result, dissociation of L-HbA,c progresses. However, high concentrations of dihydroxyboryl compounds are required to remove L-HbAl. For example, when adding this dihydroxyboryl compound to a sample solution containing hemolyzed blood, approximately 0.1 to 1
．． It is necessary to add it so that it becomes 0M, and when eluating a hemolysate using an eluent containing the dihydroxyboryl compound, it should be contained in the eluent at a ratio of 0.01 to 0.15M. is necessary. When a dihydroxyboryl compound is contained in such a high concentration, the ionic strength when subjected to ion exchange chromatography differs from that in normal cases, and as a result, the separation conditions change.

This may make measurement difficult or require correction of measurement conditions.

Furthermore, the optimum pH is about 4.5-6.5. Preferably 5.
Since it is between 0 and 6.0, there is a possibility that hemoglobin may be denatured.

Another known method for removing L-HbA,c is to dilute the blood sample to lower the glucose concentration and promote the dissociation of L-HbA,C. This can be accomplished, for example, by incubating red blood cells in a large excess of physiological saline or by dialyzing the hemolysate. However, all of these methods require a long time and are not suitable as methods for PA floor inspection.

JP-A-63-29 reports a method using condensed phosphoric acid as a rapid method for removing L-HbA and c without denaturation of hemoglobin. However, there is no simple measurement method for quantifying condensed phosphate in phosphate buffer, and HPLC
It is necessary to quantify using ion exchange chromatography using ion exchange chromatography, etc., and the operation is complicated.

(Problem a that the invention seeks to solve) The present invention solves the above-mentioned conventional drawbacks.

The purpose is to detect 5-ab^, C in blood samples.
can be easily and accurately measured without denaturing hemoglobin. Another object of the present invention is to provide a dissociating agent for L-HbAIc, which facilitates the preparation and quality control of a dissociating medium containing the dissociating agent.

(Means and effects for solving the problems) The unstable glycated hemoglobin dissociating agent of the present invention is.

It is used for measuring glycated hemoglobin in blood, and the dissociating agent mainly contains pyridoxal phosphate and/or its salt, or nucleotide and/or its salt,
The labile Hb^,C can be dissociated into hemoglobin and glucose, thereby achieving the above objective.

(Left below) Pyridoxal phosphoric acid contained in the dissociating agent of the present invention is represented by the following structural formula.

Eight The nucleotide contained in the dissociation agent of the present invention is.

It is a compound in which the sugar moiety of the nucleoside forms an ester with phosphoric acid, and the position of esterification is not particularly limited. Examples of the above-mentioned nucleotides include adenylic acid, guanylic acid, uridylic acid, cytidylic acid, and the like. Nucleotides include those with different degrees of phosphoric acid condensation. For example, adenylic acid includes adenosine diphosphoric acid,
Adenosine trisonic acid, guanylic acid, guanosine diphosphoric acid, guanosine trisonic acid, cytidylic acid,
Examples include cytidine diphosphoric acid and cytidine diphosphoric acid. L-H
The dissociation ability of bAIc becomes stronger as the degree of condensation of phosphoric acid contained in the nucleotide increases.

Pyridoxal phosphate and nucleotides absorb in ultraviolet light because they contain heterocycles. Therefore, by using a spectrophotometer, the amount of dissociating agent (pyridoxal phosphate, nucleotide or salt thereof) in the dissociating medium can be easily determined.

These pyridoxal phosphates and nucleotides are
The same effect can be obtained even with the salt.

The type of salt is not particularly limited, but examples include salts of alkali metals and alkaline earth metals, including Na salts.

K salt etc. are suitable.

The amount of the L-HbA+cM dissociating agent of the present invention used varies depending on the type of the dissociating agent used and the conditions at the time of measurement (pretreatment conditions such as blood hemolysis, 2 pH, etc.). Usually, 1 to 1500 nucleotides of pyridoxal phosphate or their salts are used per 1 i of blood (whole blood). about 0. ．． 001
~1.0 W/V%, preferably 0.01-0.2 W/
It is added at a ratio of V%.

If it is too small, the effect of dissociating and removing L-flbA,c cannot be obtained, and if it is too large, separation during chromatographic separation becomes difficult.

When measuring hemoglobin in blood, it is normal because hemoglobin is present in red blood cells.

Surfactants are preferably used as the hemolytic agent, which is often used to hemolyze blood in advance.

Examples of the hemolytic agent include higher aliphatic alcohols, alkylaryl polyether alcohols, polyoxyethylene ethers of sulfonate compounds, polyoxyethylene ethers of sulfate compounds, and polyoxyethylene adducts of sorbitol fatty acid esters. The amount of hemolytic agent used varies depending on the type of hemolytic agent, but is usually 10 to 20,011 g per 1 d of blood. For example, the hemolytic agent is 0.
Add a hemolytic agent solution containing 0.01 to 2% by weight to 1 d of blood.
Hemolysis can be achieved by adding and incubating at a rate of 2 to 400 d/day. Excessive hemolysing agent makes it difficult to separate hemoglobin by chromatography.

To measure glycated hemoglobin using the dissociation agent of the present invention, first, if necessary, a blood sample is pretreated with a hemolysate to cause hemolysis, thereby preparing a sample containing at least red blood cells and/or hemoglobin.

This sample is brought into contact with a dissociation medium containing the dissociation agent of the present invention. This dissociating agent only needs to come into contact with red blood cells and/or hemoglobin before the sample is separated and eluted by ion exchange in the chromatography column described below. It may be included in a diluent for dilution, a hemolytic agent solution containing a hemolytic agent, an eluent used in chromatography, etc., and such solutions are also included. It is considered to be the above-mentioned dissociation medium. In addition, the dissociating agent is 1, for example, a red blood cell dispersion of the sample,
The hemolysate may be added as a solid to the hemoglobin solution. This dissociation agent is interpreted to dissolve rapidly and the hemoglobin in the sample comes into contact with the dissociation medium.

After dissociating a sample with the dissociating agent in the method for measuring glycated hemoglobin using the dissociating agent of the present invention.

The method of separating and measuring by chromatography will be explained next. The more acidic the pH at the time of contact between the sample and the dissociation agent (dissociation medium), the higher the dissociation rate of L-1(bh), but if the pH is too low, denaturation of hemoglobin will occur. The separation of Hb by chromatography becomes difficult under conditions of high or low pH.1 Therefore, it is common practice to
It is 4.6-7.0. Preferably, it is adjusted to 5.3 to 6.5. The contact time between the sample and the dissociating agent varies depending on the type and concentration of the dissociating agent, pH conditions, etc.2, but is usually 10 minutes or more, preferably 10 to 30 minutes at room temperature. It is also possible to shorten the contact time by increasing the temperature; for example, incubation can be carried out at 37°C for about 3 to 7 minutes or at 50°C for about 1 to 3 minutes. Samples treated in this way are then subjected to ion exchange chromatography.
(for example, high performance liquid chromatography, HPLC), and each component of hemoglobin is separated, eluted, and measured.

When a sample solution is directly subjected to ion-exchange chromatography and eluted using an eluent containing the above-mentioned dissociating agent, the pH of the eluent is such that it does not denature hemoglobin and that HbA, c is easily removed by chromatography. It may be within a range that can be measured separately. usually,
A pH range of 5.0 to 6.5 is selected. L- contained while the sample passes through the chromatography column.
HbAIc is dissociated and then separated into each hemoglobin component and eluted.

be measured.

The dissociating agent can also be included in both the sample diluent, hemolytic agent solution containing a hemolytic agent, and the chromatography eluent. In this case, it is particularly effective for L-HbA,
c is dissociated.

Dissociation and dissociation of L-HbA,c by the dissociation agent of the present invention
The scavenging effect is believed to be due to the nature of the 2.3-DPG pocket on hemoglobin. Regarding the 2.3-DPG pocket, see Benesch et al., Bioche
+a, Biophys.

Res, Com5un, 26,162. (1
967); Chanutin et al., Arch.

Biochem, Biophys, 121,96.
(1967) and others. This 2
．． The 3-DPG pocket contains basic amino acid residues such as histidine and lysine in the β chain of hemoglobin, and HbA,
It is formed by the N-terminal valine of the hemoglobin β chain of c.

Pyridoxal phosphoric acid, which is the main component of the dissociating agent of the present invention, is known to have cationic properties.

Because nucleotides and their salts have anionic properties in aqueous solutions and have appropriate molecular shapes.

It has strong affinity for the 2.3-DPG pocket. Therefore, it competes with glucose and binds to the N-terminus of the β chain of hemoglobin. As a result, L-HbA,c
dissociation is promoted.

In this way, due to the dissociative action of L-HbA, c of pyridoxal phosphate, nucleotides or their salts, L
-HbAIc is removed from the sample, 5-HbA,c
only remains in the sample. This dissociating agent does not affect the elution pattern of hemoglobin by ion exchange chromatography. Therefore, the above-mentioned 5-HbA, c is separated by ion exchange chromatography in the same manner as conventional methods and measured with high accuracy.

(Margin below) (Example) The invention will be explained below with reference to examples.

In the following examples, glycated hemoglobin was measured using ■Hi-Auto AicHA-8120 manufactured by Kyoto Dai-Kagaku.
The measurements were carried out using appropriate conditions. This day ^-812
0 is a dedicated device for measuring HbA and C by HPLC, and each hemoglobin component is separated and eluted in 4 minutes by cation exchange. A dedicated eluent (phosphate buffer) is used as the elution buffer. The elution pattern of hemoglobin using this device is generally shown in FIG. In FIG. 1, P and P2 are peaks caused by the HbA+-b component, and P and P are L-ElbA and P, respectively.
, and 5-HbA, a peak due to the c component. P is a peak due to other hemoglobins.

HbA, C value is HbA+c occupied in total hemoglobin
It is the ratio (percentage) of (the sum of L-HbA+c and 5-HbA, C) and is calculated by a linear equation.

In this example, blood from the same person (W1 normal person) was used, and heparin was added immediately after blood collection and used as fresh blood.

Triton was added to prepare a hemolytic agent solution. After 3 μl of fresh blood was added to 450 μl of this hemolytic agent solution to cause hemolysis, the HbA and e values were measured using the above method and were found to be 5.0%. This value is the total HbA+c in the blood (L-HbA r c and 5-tlbAt
e), and this was used as a blank value.

Next, about 5I blood cells obtained by centrifuging 10Id of fresh blood.
11 in 8/321nch cellophane tube (manufactured by Wako Pure Chemical Industries, Ltd.), and physiological saline 11. The cells were incubated twice each for a total of 5 hours at 37°C. In this way L
- After dissociating HbA,c, this is about 1.5μ! It was collected and hemolyzed using 450 μl of the above hemolytic agent solution. The HbA and c values of this sample were measured in the same manner and were found to be 4.3%. This value is.

It is thought to correspond to 5-HbA and C in blood.

■ Add T as a hemolytic agent in 100 d of 0.005 M phosphate buffer.
0.11 d of riton

A base was added to adjust the pH to 6.3 to obtain a dissociation medium containing a hemolytic agent. This hemolytic agent-containing dissociation medium is 450μ! 3μ of fresh blood! was added and left at room temperature for about 10 minutes to perform hemolysis and dissociation of L-HbA,c. When the HbA, c value of this sample was measured, it was 4.4%. Separately, prepare a hemolytic agent solution without adding pyridoxal phosphate (a dissociating agent and in a similar manner) lbA.

The value was measured.

Next, the pH of each of the hemolytic agent-containing dissociation medium and hemolytic agent solution (not containing a dissociating agent) was adjusted to 5.8.5.
．． 3.5.0 and 4.6, as well as HbA,
The c value was measured. In each pal. The HbA+c value (%) and total hemoglobin amount (total b amount) when using a dissociation medium containing a hemolytic agent and when using a hemolytic agent solution (not including a dissociating agent) are shown in the table (1) below. Shown below.

The total hemoglobin amount was calculated using the original total hemoglobin amount in the blood as 100%.

(Left below) From Table (1), as the pH condition decreases, HbAt
It is clear that the c value decreases and L-HbA+c is dissociated and removed. However, when the pH falls below 5.3, discoloration due to heme removal is observed, and the total hemoglobin amount decreases. When measuring using the dissociation side of the present invention, L-HbA is dissociated and hemoglobin does not change in the pH range of 6.3 to 5.3.
It is clear that it will be removed.

Add 0.1 d of Triton χ-100 (manufactured by Wako Pure Chemical Industries, Ltd.) as a hemolytic agent to 100 d of 0.005M phosphate buffer.
About 0.1 g of sodium pyridoxal phosphate was dissolved therein as a dissociating agent, and a base was added to adjust the pH to 6.3 to obtain a dissociating medium containing a hemolytic agent. Using this hemolytic agent-containing dissociation medium, hemolysis and L-
HbA lc was dissociated, and the HbA r of this sample was
The c value was measured. As a result, the HbA+c value was 4.
It was 4%.

1 vessel ■1 Add hemolytic agent to 100 d of 0.005M phosphate buffer and add Triton I I
d and sodium adenosine diphosphate (manufactured by Sigw+a) as a dissociating agent.

Sodium adenosine diphosphate (manufactured by Sigma) or sodium adenylate (manufactured by Sigma) was added to approximately 0
゜Dissolve 1g, add base to adjust pH to 6.0,
Various hemolytic agent-containing dissociation media were obtained. The procedure was repeated in the same manner as in Example 1 using each of the hemolytic agent-containing dissociation media.

Hemolysis and dissociation of L-11bA,c were performed. When the HbA lc values of these samples were measured, the HbA+c value was 4.3% with the addition of sodium adenosine diphosphate, the HbAtc value was 4.5% with the addition of sodium adenosine diphosphate, and the HbA+c value with the addition of sodium adenosine diphosphate was 4.5%. The HbA+c value was 4.7%.

From this result, each nucleotide is L-HbA+c
It can be seen that nucleotides with a high degree of phosphoric acid condensation have a high ability to dissociate L41bA+c.

Add sodium pyridoxal phosphate to 0.005% to 1 (bA, e eluent A solution (manufactured by Tanemizu Kagaku Kogyo)) consisting of a diphosphate buffer solution and prepare an eluent containing a dissociating agent. Separately, a hemolytic agent solution (not containing a dissociating agent) similar to that in Example 1 was prepared. Using this hemolytic agent solution, fresh blood was hemolyzed in the same manner as in Example 1. When the sample was immediately chromatographed using the above eluent and separated and measured, the elution pattern was as follows.
Almost the same as when using the dissociation medium of Example 1,
HbA and C values were 4.5%. It can be seen that L-HbA+c can be sufficiently removed even when an eluent containing the dissociating agent of the present invention is used.

Northern Comparison ■ Instead of pyridoxal phosphate in Example 1, semicarbazide hydrochloride (manufactured by Wako Pure Chemical Industries, Ltd.) 0. Prepare an L-FlbA, C remover solution consisting of 0.004M aniline (manufactured by Wako Pure Chemical Industries, Ltd.) containing OIM and a 0.005M phosphate buffer with a pH of 5.3 containing the same hemolytic agent as in Example 1. did.

Using this, fresh blood was hemolyzed in the same manner as in Example 1, and the hemolysate was incubated at 60°C for 2 minutes. When the HbA and c values of this sample were measured, they were 4.
．． It was 7%. Furthermore, the pattern of the obtained chromatodalum was quite broad, and the total area of the hemoglobin beak was approximately 70% of the original area.

Technique 5I Pi (5,3 0%
．． An L-tlbA+c remover solution consisting of 005M phosphate buffer was prepared. Using this, fresh blood was hemolyzed in the same manner as in Example 1, and the hemolysate was incubated at 60°C for 2 minutes. When the HbAlc value of this sample was measured, it was 4.4%. However, the boric acid concentration was reduced to 0.
When similar measurements were performed with the concentration adjusted to 1% and the pH adjusted to 6.0, the HbAlc value was 4.8%.

In this way, when the concentration of boric acid is low, sufficient L
-FtbA+e cannot be removed.

(Effects of the Invention) When the L-HbA+c dissociating agent of the present invention is used, L-Hb^1. is removed.

Only 5-HbA can be measured accurately, easily, and in a short time without denaturing hemoglobin.

Furthermore, since the dissociating agent of the present invention has absorption in the ultraviolet region, it can be easily quantified by measuring absorbance. Therefore, preparation and quality control of the dissociation medium containing the L-HbA+e dissociation agent can be easily performed.

S-11bA, e exists relatively stably in the blood without being affected by the temporal rise or fall of blood sugar;
This measured value can be used as a more reliable index of diabetes. By automating the measurement system, more effective clinical tests will become possible.

Figure 1 shows the elution pattern when hemoglobin in blood was separated by ion exchange chromatography.

that's all

Claims (1)

[Scope of Claims] 1. A dissociating agent for unstable glycated hemoglobin used for measuring glycated hemoglobin in blood, the dissociating agent comprising:
The main component is pyridoxal phosphate and/or a salt of pyridoxal phosphate, and unstable hemoglobin A_1_
An unstable glycated hemoglobin dissociating agent capable of dissociating c into hemoglobin and glucose. 2. A dissociating agent for unstable glycated hemoglobin used for measuring glycated hemoglobin in blood, the dissociating agent comprising:
The main component is nucleotides and/or nucleotide salts, and can dissociate unstable hemoglobin A_1_c into hemoglobin and glucose. Unstable glycated hemoglobin dissociating agent.