JPH02259467A - Unstable type saccharified hemoglobin dissociation agent - Google Patents

Abstract

PURPOSE:To accurately and simply perform measurement without modifying hemoglobin by using a releasing agent based on phytinic acid and/or a salt thereof and dissociating unstable type hemoglobin A1C into hemoglobin and glucose. CONSTITUTION:This dissociation agent is used in the measurement of hemoglobin A1C in blood and based on phytinic acid and/or a salt thereof and has function for dissociating unstable type hemoglobin A1C into hemoglobin and glucose. In the measurement of this saccharified hemoglobin, a sample containing at least an erythrocyte and/or hemoglobin in blood is brought into contact with the dissociation medium containing phytinic acid and/or the salt thereof to dissociate the unstable type hemoglobin A1C contained in the specimen into hemoglobin and glucose. Thereafter, hemoglobin in the specimen is separated into respective hemoglobin components by ion exchange chromatography to measure unstable type hemoglobin present in the specimen.

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 Ate used in the measurement of hemoglobin 8lc.

(Prior Art) Glycated hemoglobin (hemoglobin A) in the blood, particularly hemoglobin Aic, is known as one of the indicators of diabetes, and its measurement is being carried out. Hemoglobin Ate, as shown in the following formula, is a complex 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 (hereinafter hemoglobin is referred to as llb). 1] When glucose binds to the above amino group of b, first.

Unstable hemoglobin^1c(I), a Schiff base, is formed as shown in the following formula:)1c=D HCDH HC=N-βA C0H CH2-NH,"-βA C=0 HCOHHCOHHCOH CII20HC112011CH20H Glucose
(I) (n) The above formula (
In 1), β-A-N)+2 represents Hb.

NH, therein represents the amino group of valine at the N-terminus of the β chain of 1b.

This reaction of producing unstable Hb^1c (I) is a reversible reaction, and the equilibrium changes depending on the glucose concentration.
, c(■) tends toward the production direction or dissociation direction.

(I) further undergoes Amatoli rearrangement and irreversibly changes to stable HbA, c(n).

)1bA+c can be quantified by, for example, separating Hb into each )1b component by high-performance liquid chromatography and measuring the OD of the stable HbA, c.
(S-HbA, c) and unstable HbA+c (L-H
bAIC) cannot be separated and measured. Therefore, HbAlc cannot be obtained at a stable value.
This is because the amount of L-HbA,c changes depending on the amount of glucose (that is, blood sugar), and the blood sugar level changes rapidly and significantly with one meal or exercise.

In order to eliminate the above drawbacks, L-HbA,
Attempts have been made to remove C and measure only S-11bAlc. For example, David M., Natha
n et al., C11nicalChen+1stry, 28
512-515 (1982) using semicarbazide and aniline as removal agents for L-HbA, c;
p)I 5. Processing blood specimens for 30 minutes at 0.38°C is disclosed. Semicarbazide scavenges glucose and acts as a nucleophile, competing with the amino groups of Hb. Aniline acts as a catalyst. As a result, L-HbA,c is substantially removed. However, the removal reaction of L-HbA,c is on the acidic side (p85.0).
and at a relatively high temperature (38°C).

Denaturation of Hb (eg dehemization) occurs. For example, when observing the elution pattern by ion-exchange chromatography, we find that the peak intensity decreases due to fading due to heme removal, and the H
An increase in the peaks due to bA , , and HbA+b is observed.

JP-A-58-210024 discloses a dihydroxyboryl compound (boric acid compound) as a remover for L-HbA lc. Since this dihydroxyboryl compound esterifies the DH 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-HbAIC. For example, in a sample solution containing hemolyzed blood, approximately 0.
1 to 1, OL When a hemolysate is eluted using an eluent containing the dihydroxyboryl compound, it is necessary that the dihydroxyboryl compound be contained in the eluent at a ratio of 0.01 to 0.15M. If such a high concentration of dihydroxyboryl compound is contained, the ionic strength when subjected to ion exchange chromatography will be different from normal, resulting in changes in the separation conditions, making measurements difficult or changing the measurement conditions. correction is required. Furthermore, the optimum pH is approximately 4.5.
~6.5°, preferably 5.0 ~ 6.0, so H
There is a risk that degeneration of b.

Another known method for removing L-HbAlc 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 hemolysates. However, all of these methods require a long time and are therefore not suitable as methods for clinical testing.

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

Its purpose is to measure only stable hemoglobin^1c in a blood sample with high accuracy (simply and without denaturing hemoglobin).A remover or dissociator for unstable hemoglobin A1c.

and to provide a method for measuring hemoglobin A1c.

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

It is used to measure hemoglobin A1 in blood, and the dissociative abrasion mainly contains phytic acid and/or a salt of phytic acid, and has the function of dissociating unstable hemoglobin Alc into hemoglobin and glucose.

This achieves the above objective.

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

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

(Left below) Phytic acid can have the same effect even in its salt form. The type of salt is not particularly limited, but examples include salts of alkali metals and alkaline earth metals, with Na salts, K salts, etc. being preferred.

The amount of L-HbA dissociating agent used in the present invention varies depending on the type of compound used and the conditions at the time of measurement (pretreatment conditions such as blood hemolysis, pH, etc.).
whole blood) at a rate of 1 to 1500 mg per mf. For example, phytic acid is about 0.001 to 1.0 μl in a sample solution obtained by hemolyzing 3 μl of a blood sample with a 450 μm hemolytic agent solution.
0W/V% (1'l/V%l;! Unit volume! [Atari (7
1) weight), preferably 0.01 to 0.211/
It is added in a proportion of V%. L-HbA, if too low.

The effect of dissociation and removal cannot be obtained, and if it is in excess, separation during chromatographic separation becomes difficult.

When measuring Hb in blood, [because Hb exists in red blood cells9] the blood is usually hemolyzed in advance. A surfactant is preferably used as the hemolytic agent. 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.

Usually 10-2000 mg per rnl of blood.

for example.

A hemolysis treatment solution containing a hemolytic agent at a ratio of 1% by volume is 2 to 400 rnf per 1 of blood! Hemolysis can be achieved by adding and incubating at a ratio of . If the hemolytic agent is in excess, separation of Hb by chromatography becomes difficult.

To measure glycated TLB according to the present invention, first.

If necessary, pre-treat the blood sample with a hemolytic agent to lyse it,
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 above dissociation agent of the present invention. This dissociating agent.

The sample may be contacted with red blood cells and/or Hb before being separated by ion exchange and eluted in a chromatography column described below. Therefore.

The dissociating agent may be included in a diluent for diluting a blood sample to an appropriate concentration, a hemolytic agent solution containing a hemolytic agent, an eluent used in chromatography, etc. Such solutions and eluents may also be included. Also. It is considered to be the above-mentioned dissociation medium. In addition, the dissociating agent may be added as a solid to, for example, the sample red blood cell dispersion, its hemolysate, or Hb solution. This dissociating agent dissolves rapidly, and Hb in the sample is dissociated upon contact with the dissociating medium.

Among the methods for measuring glycation 11b using the dissociating agent of the present invention,
About a method of dissociating a sample with the dissociating agent and then subjecting it to chromatography for separation and measurement.

This 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-HbA,c, but if the pH is too low, denaturation of llb occurs. Furthermore, separation of Hb by chromatography becomes difficult under extremely high or low pH conditions.

Therefore,9 usually the pH of the mixed solution containing the sample and dissociating agent is between 4.6 and 7. O1 is preferably 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.

Usually at room temperature for 10 minutes or more, preferably 10 to 3 minutes
It is 0 minutes. It is also possible to shorten the contact time by increasing the temperature; for example, about 3 to 7 minutes at 37°C.

Inking can also be performed at 50°C for about 1-3 minutes. The sample treated in this manner is subjected to ion exchange chromatography (eg, 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 using solution m1& containing the dissociating agent, the pH of the eluent is such that Hb is not denatured and HbAlc is easily separated by chromatography. Any range that can be measured is sufficient. Usually pH5
, 0 to 6.5. L-HbAI contained while the sample passes through the chromatography column
C is dissociated and each Hb acid component is then separated, eluted, and measured. The dissociating agent.

It is also possible to include it in both the sample diluent or hemolysis pretreatment solution and the chromatography eluent, and in this case L-HbA,c is particularly effectively dissociated.

Such dissociation and removal action of L-HbA,e by the dissociating agent of the present invention is considered to be due to the properties of the 2,3-DPG pocket on Hb. Regarding the 2.3-DPG pocket, Benesch et al. (Bioch
em, Biophys, Res.

Commun, , 26.162.1967)
; Chanutin et al. (Arch.

Biochem, Biophys, 121:96.
(1967) and others. This 2.
3-[)PG pocket contains basic amino acid residues such as histidine and lysine in the β chain of Hb, and Hb in HbAIc.
It is formed by the N-terminal valine of B-chain, and is known to have cationic properties. Since phytic acid and its salts, which are the main components of the dissociating agent of the present invention, have anionic properties and have appropriate molecular shapes, the above-mentioned 2.3-DPG
Has a strong affinity for pockets. Therefore, it competes with glucose and binds to the N-terminus of the β chain of Hb. As a result, dissociation of L-HbA,c is promoted.

In this way, L-HbAIc is removed from the sample by the dissociation effect of L-HbA,c of phytic acid and/or a salt of phytic acid, and only 5-HbA,c remains in the sample. This dissociating agent does not affect the elution pattern of Hb by ion exchange chromatography. Therefore, the above-mentioned 5-HbA,c was separated by ion exchange chromatography in a conventional manner.

Measured accurately.

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

Measurement method In the following examples, HbA was measured by selecting appropriate conditions using the old Auto A+cHA-8120 manufactured by Kyoto Dai-Kagaku. This HA-8120 is available on the HP
This is a dedicated device for measuring HbA+ using LC, and each Hb acid 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 Hb using this device is generally shown in FIG. In Figure 1, +Pl and P2 are HbA
The peak is due to the la+b component and +P3 and P4
are peaks caused by L-HbA, c and 5-HbA lc components. P is a peak due to other hemoglobins. Here, HbA,c (total of S type and L type) is calculated by the following formula.

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

Triton
A hemolysis reagent was prepared by adding 1 ml of O. After adding 3 μβ of fresh blood to 450 μl of this hemolytic reagent and hemolyzing it, HbA1c (including L type and S type) was measured by the above method. 2 1 in total Hb (bA + c is 5.0%
It was found that there is a proportion of This value was used as a blank value.

Next, about 5 ml of blood cells were obtained by centrifuging the fresh surface 10rd.
8/321nch cellophantium tube (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.)
2 times each using 11 saline solutions for a total of 5 hours37
Incubated at ℃. In this way, L-HbA
, c was dissociated, about 1.5 μl of this was collected and hemolyzed using 450 μl of the above-mentioned hemolytic reagent.

When 1lbA,c of this sample was measured in the same manner, it was 4.3%. This value is 5-
It is thought to correspond to HbA,c.

Example 1 T as a hemolytic agent in 0.005M phosphate buffer 100-
ritonX-100 (Wako Tsumugi Pharmaceutical) O, lrn! ! and a 40% aqueous phytic acid solution (Aldri
Dissolve about 0-1 g of the product (manufactured by Ch.
．． 3 to obtain a dissociation medium containing a hemolytic agent. 3 μl of fresh blood was added to 450 μl of this hemolytic agent-containing dissociation medium and left at room temperature for about 10 minutes to cause hemolysis and L-HbA.
, c was dissociated. When the HbA1c of this sample was measured, it was 4.4%. Separately, prepare a hemolytic agent solution without adding phytic acid (dissociation agent), and use the same method to
A. Measurements were made.

Next, ρ1] for each of the hemolytic agent-containing dissociation medium and the hemolytic agent solution (not containing a dissociating agent) is set to 5.8.
5.3.5.0 and 4.6, as well as HbA,
c was measured. 1 lbA, c-containing, and total Hb in total Hb when using a dissociation medium containing a hemolytic agent and when using a hemolytic agent solution (without a dissociating agent)
The amounts are shown in Table 1. The total b amount was calculated using the original total b amount in the blood as 100%.

(Left below) From Table 1, as the pH condition decreases) lbA, c
The value becomes lower, and it is clear that L-11bA and c are dissociated and removed. However, when the pH drops below 5.3, discoloration due to heme removal is observed, and the amount of mHb decreases. In the above system, the amount of mHb at pH 4.6 is about 75% of that at pH 6.3.

When measuring using the dissociating agent of the present invention, I]86.
It is clear that in the region of 3 to 5.3, L-HbA,c is dissociated and removed without any change in Hb.

Example 2 Trit, onX-100 (Wako Tsumugi Pharmaceutical Co., Ltd.) 0.1- as a hemolytic agent and sodium phytate (Aldrich
Dissolve approximately 0.1 g of
A dissociation medium containing a hemolytic agent was obtained. Add 3 μm of fresh blood to 450 μl of this hemolytic agent-containing dissociation medium,
Leave it at room temperature for about 10 minutes to eliminate hemolysis and L-HbA1c.
The dissociation was performed. The HbA,c of this sample was measured and found to be 4.3%.

Example 3 According to Example 2, the amounts of sodium phytate were 1.0 g, 0.2 g, 0.05 g, and 0.005 g, respectively.
and 0.001 g of a hemolytic agent-containing dissociation medium (both with a pu of about 6.0) were prepared. Using this, the content of HbAlc was measured in the same manner as in Example 1. The results are shown in Table 2.

Table 2 According to Example 2, a hemolytic agent-containing dissociation medium whose pH was adjusted to 6.0 was prepared. Using this, the temperature condition was set to 37
℃, 50°C, and 60°C, and after incubation for a predetermined time, HbA and c were measured.

The results are shown in Table 3.

Table 3 From Table 2, sodium phytate is 0.005g (0
,005W/V%) even at a low concentration of L-HbA.
, c can be obtained. Sodium phytate is 1.0/g (approximately 1.0 W/V%) (7
) case 1t, ri07) Since the Durham pattern changes to a broad one, it is desirable to lower the density a little.

Example 4 From Table 3, as the reaction temperature is increased, L-Hb
It can be seen that A and c are removed. However, when incubated at 60° C. for 3.5 minutes, denaturation of Hb was observed, and the total amount of B decreased to about 90%. Therefore.

Incubation at high temperatures for long periods of time is considered undesirable.

Example 5 0.0% sodium phytate was added to a phosphate buffer solution (made by Suikagaku Kogyo ■) exclusively for HtlA+c measurement.
0.005% to obtain a dissociating agent-containing eluent (pH 6.0). Separately, the same hemolytic agent solution as in Example 1 (
(containing no dissociating agent) was prepared and used to hemolyze fresh blood in the same manner as in Example 1. When this sample was immediately subjected to chromatography 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 HbA+c
The content was 4.4%. It can be seen that L-HbA and C can be sufficiently removed by using the eluent containing the dissociating agent of the present invention.

Comparative example 1 Hemolytic agent similar to Example 1 (same concentration as Example 1).

Hemolytic agent-containing L-11 consisting of a 0.005M phosphate buffer with a pH of 5.3 containing 0.01M of semicarbazide hydrochloride (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.) and 0.004M of aniline (manufactured by Wako Tsumugi Pharmaceutical Co., Ltd.)
bAIe remover solution 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 this was measured, HbA, c
The content was 4.7%. However, the 7-fraction pattern became quite broad, and the total area of the Hb peak was about 70% of the original.
Met.

Comparative Example 2 0.005M of pf 15.3 containing the same hemolytic agent as Example 1 (same concentration as Example 1) and 1.0% boric acid
A hemolytic agent-containing L-HbA, c remover solution consisting of a 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 this was measured, the HbA,c content was 4.4
%Met. However, similar measurements were performed with the boric acid concentration adjusted to 0.1% and the pH adjusted to 6.0.

The HbA,c content was 4.8%. As described above, when the concentration of boric acid is low, L-11bA and C cannot be sufficiently removed.

(Effects of the Invention) According to the present invention, L-HbA, c is removed from HbAlc in the blood, and only 5-HbA, e is removed with high precision and in a short time without denaturing hemoglobin. It will be measured within

5-HbA exists in the blood relatively stably, unaffected by the temporal rise or fall of blood sugar.
This measured value can be used as a more reliable indicator of diabetes. By automating the measurement system, more effective clinical tests will become possible.

[Brief explanation of drawings]

FIG. 1 is a chart showing the elution pattern when Hb in blood is separated and measured 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:
An unstable glycated hemoglobin dissociating agent which contains phytic acid and/or a salt of phytic acid as a main component and is capable of dissociating unstable hemoglobin A_1_c into hemoglobin and glucose. 2. A sample containing at least red blood cells and/or hemoglobin in blood is brought into contact with a dissociation medium containing phytic acid and/or a salt of phytic acid to convert unstable hemoglobin A_1_c contained in the red blood cells and/or hemoglobin into hemoglobin. and glucose, and separating the hemoglobin in the sample after the dissociation into each hemoglobin component by ion exchange chromatography, and measuring the stable hemoglobin A_1_c present in the sample. measurement method.