JPH06308120A - Controlling substance for measurement of saccharized hemoglobin and its manufacture - Google Patents

Abstract

PURPOSE:To measure with high reliability by using a controlling substance for measuring saccharized hemoglobin obtained by bringing hemoglobin in touch with carbon monoxide. CONSTITUTION:A suitable amount of distilled water is added calmly to erythrocyte which is sufficiently cleansed by physiological saline water, thereby breaking the membrane of erythrocyte. A part of erythrocyte including a solid content such as the membrane or the like is removed by the centrifugal separation at about 2000-10000G. Alternatively, a suitable amount of hemolytic agent may be added to separate the erythrocyte. Then, carbon monoxide gas is calmly blown into a solution containing hemoglobin while it is stirred under the nitrogen atmosphere. The shifting rate of carbon monoxide of the generated carbon monoxide hemoglobin can be confirmed from the shift of the absorbance spectrum. As the value is larger, it is more suitable for use in measuring saccharized hemoglobin. Thereafter, the hemolysate is distributed every predetermined amount into vials which are in turn dipped in liquid nitrogen to freeze the hemolysate. The hemolysate is dried in high vacuum. Accordingly, even when measuring persons, devices or the time are different, reliable measuring values can be obtained.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a glycated hemoglobin used as an index for a diabetes test, and more particularly to a glycated hemoglobin control substance used for measuring hemoglobin Alc and / or hemoglobin Al.

[0002]

2. Description of the Related Art Glycated hemoglobin in blood, particularly hemoglobin Alc (hereinafter referred to as HbAl), is one of the indicators of diabetes test.
abbreviated as c) and hemoglobin Al (hereinafter abbreviated as HbAl) are known and widely measured in the field of clinical examination. Since these glycated hemoglobins, especially HbAlc, correlate well with the blood glucose level of patients about two months ago, their clinical significance is increasing as one of the important indicators of diabetic condition.

Various methods are known as methods for measuring these glycated hemoglobins, and in particular, methods applying various chromatographic techniques such as mini-column and high performance liquid chromatography (HPLC) have been widely spread in the clinical laboratory field. ing. On the other hand, when measuring the same sample, quality control is performed so that reliable measurement values can be obtained even if the measurer, device, measurement time, etc. differ, ensuring consistent measurement results. There is a need to.

In general, institutional control is carried out using a standard substance showing that the level of the obtained measured value is constant, that is, a glycated hemoglobin measuring controlled substance which should serve as a standard of the measuring system. By measuring this substance in the same manner as the measurement sample, it is possible to correct the error caused by the difference between the measuring devices, the difference in various conditions of the measuring method, the reproducibility of the day difference, the reproducibility of the day, and the like. In addition, when the characteristic value of the glycated hemoglobin measurement controlled substance, for example, the HbAlc value, is confirmed and guaranteed by a measurement method that serves as a reference, the glycated hemoglobin measurement controlled substance can be specified as a standard substance, and the HbAlc measurement It is also possible to use this for the purpose of standardization, calibration and the like.

However, when the HbAlc value or HbAl value obtained is not reliable because the controlled substance for measuring glycated hemoglobin, which is to be the standard, is unstable over time, quality control is performed using such a controlled substance. Even if you do it, it will be meaningless. Further, in this case, if the reliability of the measured HbAl value is poor, it may cause a big problem in the social role of using it as an index of diabetes.

From the above, it has been widely desired to use the controlled substance for measuring glycated hemoglobin, which has excellent stability over time and should serve as a standard for the measuring system, for quality control. In clinical laboratories, in order to obtain a controlled substance for measuring glycated hemoglobin, it is customary to freeze or lyophilize hemolyzed blood and prepare it in-house in the past. There were few.

JP-A-59-183370 discloses an invention relating to a method for producing a hemoglobin standard substance. This is a method for producing an excellent standard substance, but it includes a step of adding hemoglobin to methoxide (oxidizing heme iron [II] to iron [III]) by adding potassium ferricyanide, which is an oxidant. There is. In this case, depending on the conditions, there is a risk that hemoglobin will undergo denaturation associated with methemoglobin formation. The hemoglobin component that has undergone such denaturation may appear as a denaturation peak on various chromatogram analyzes and may adversely affect the measured value.

[0008]

SUMMARY OF THE INVENTION It is an object of the invention to make the accuracy control carried out in order to obtain consistent measurement values and reliable measurement values more reliable. In particular, it supports the social role of using glycated hemoglobin as an index for diabetes tests.

For this purpose, a controlled substance for measuring glycated hemoglobin, particularly HbAl, which is necessary for performing the above-mentioned quality control and has excellent stability over time, which is a standard of the measuring system.
It is necessary to provide a controlled substance for measuring glycated hemoglobin used for the measurement of c value and / or HbAl. If this problem can be solved, the same sample can be measured by
It is possible to obtain highly reliable measurement values even if the device, measurement time, etc. are different.

Further, when the characteristic value of the controlled substance for measuring glycated hemoglobin, for example, the HbAlc value is confirmed and guaranteed by a measuring method which is a standard, by defining the controlled substance for measuring glycated hemoglobin as a standard substance, It can also be used for the purpose of standardizing HbAlc measurements.

[0011]

The present invention has been devised in order to obtain a controlled substance for measuring glycated hemoglobin, which is excellent in stability over time and should serve as a standard for a measuring system. The features of the present invention are as follows. The purpose is to bring hemoglobin into contact with carbon monoxide to form carbon monoxide hemoglobin, and use this as a control substance for measurement of glycated hemoglobin.

[0012] In an air atmosphere, hemoglobin usually combines with oxygen and exists as oxyhemoglobin. However, when hemoglobin comes into contact with carbon monoxide, it quickly binds to carbon monoxide and becomes carbon monoxide hemoglobin. At this time, since the affinity of carbon monoxide for hemoglobin is much stronger than that of oxygen, carbon monoxide bound to hemoglobin is not easily desorbed. For this reason, it is considered that carbon monoxide hemoglobin is less likely to undergo methification and subsequent denaturation of hemoglobin than oxyhemoglobin.

The present inventors have focused their attention on such characteristics of hemoglobin and repeated various investigations. As a result, this carbon monoxide hemoglobin has excellent stability over time in measuring glycated hemoglobin, particularly HbAlc and / or HbAl. It has been found that it can be used as a controlled substance for measuring glycated hemoglobin.

The present invention will be described in more detail below.

The method for producing a controlled substance for measuring glycated hemoglobin according to the present invention comprises three steps: a step of hemolyzing and purifying hemoglobin from blood or red blood cells, a step of carbonizing hemoglobin to carbon monoxide, and a step of freezing or lyophilizing. Become. Of these, steps and are not particularly limited, and various known methods can be used as long as the purpose of these steps can be achieved.

The order of the step and the step is not particularly limited, and hemoglobin may be hemolyzed and purified and then carbon monoxide may be obtained, or hemoglobin may be hemolyzed and purified after the carbon monoxide treatment. The process (1) is performed after the two processes (1) and (2) are completed.

As an example of the step (1), erythrocytes thoroughly washed with physiological saline are gently added with an appropriate amount of distilled water to rupture (hemolyze) the erythrocyte membrane, and then 2000-1.
Examples include a method of removing solid matter such as red blood cell membrane by centrifuging at 0000 G. As a method of hemolyzing red blood cells, there is a method of adding an appropriate amount of hemolytic agent to red blood cells in addition to the above-mentioned distilled water addition method. Although various surfactants can be used as the hemolytic agent, alkylaryl polyether alcohols, sorbit fatty acid ester polyoxyethylene adducts and the like are preferably used. In addition, lipids such as membrane components can be more efficiently removed by centrifugation after washing with an organic solvent immiscible with water. As the organic solvent, benzene, toluene, xylene, hexane, petroleum ether and the like are preferable. In this case, since it is not preferable that the organic solvent remains, it is more preferable to dialyz the obtained hemoglobin solution with an appropriate buffer such as a phosphate buffer.

Further, as an example of the process of 1,
Examples include a method of dispensing a few ml each into a vial bottle, immersing in liquid nitrogen and freezing, a method of freezing at -40 ° C. under high vacuum for several hours to several days, and freeze-drying. At this time, a frost damage protector is added if necessary. A polyhydroxy compound is preferably used as the frost damage protector, and examples thereof include inositol, sorbitol, saccharose, glycerol, alkylene glycol, monosaccharides and polysaccharides. Although the amount of the frost damage protector used varies depending on the type and the like, it is usually added at a ratio of 2 to 20 w / v% to the hemoglobin solution. In the case of freeze-drying, it is more preferable to replace the space in the vial bottle with an inert gas such as high-purity nitrogen or argon and then tightly stopper the bottle.

The method of (1) is not particularly limited as long as it results in the production of generalized carbonized hemoglobin, but the most general and simple method is to use carbon monoxide gas in blood, red blood cell dispersion and This is a method of directly blowing into a hemoglobin-containing solution such as a hemoglobin solution. For example, while the hemoglobin solution is gently stirred with a magnetic stirrer under a nitrogen atmosphere, 10 ml to 1 carbon monoxide gas is added to the hemoglobin solution using a Pasteur pipette or the like.
There is a method of blowing at 000 ml / min.

The carbon monoxide hemoglobin produced has a shift in its absorbance spectrum, and its presence and the carbon monoxide ratio can be confirmed by measuring this. For example, in a normal air atmosphere, hemoglobin normally exists as oxyhemoglobin by combining with oxygen. If it is completely carbon monoxide,
The absorption maxima of 5 nm, 541 nm, and 576 nm shift to 419 nm, 540 nm, and 569 nm, respectively.

As a method for more accurately measuring the content of carbon monoxide hemoglobin, the method reported by Matsubara (protein, nucleic acid, enzyme, 32,6,671-674,1) is reported.
987). Using these methods, the quality of the obtained controlled substance for measuring glycated hemoglobin can be checked.

For example, while stirring 10 ml of blood gently with a magnetic stirrer under a nitrogen atmosphere, 100 ml of carbon monoxide gas was added to this with a Pasteur pipette or the like.
When it is blown at a rate of ml to 1000 ml / min, the reaction rate of carbon monoxide is very fast, although it depends on other experimental conditions such as the concentration of hemoglobin, and about 30 to 80% of hemoglobin can be obtained in 20 to 30 seconds. In 1 to 2 minutes, 90% or more of hemoglobin is converted into carbon monoxide. When carbon monoxide gas is blown for several minutes or longer, the production of carbon monoxide hemoglobin is completed, and the content reaches a plateau at about 98 to 99.5%. After that, even if the carbon monoxide gas is further blown in, the content of the carbon monoxide hemoglobin hardly changes. This is because even fresh blood of a healthy person contains 0.5 to 2.0% of methemoglobin from the beginning, and this methemoglobin remains without binding to carbon monoxide. is there.

The higher the content of carbon monoxide hemoglobin, that is, the higher the rate of carbon monoxide, the more preferable the carbon monoxide hemoglobin as the control substance for measuring glycated hemoglobin. For example, if the carbon monoxide conversion rate is 50% or more, the carbon monoxide hemoglobin can be used as a controlled substance. The higher the carbon monoxide rate, the higher the stability over time. It is preferable to carry out carbon monoxide conversion to reach 90% or more, and most preferably to reach a plateau.

In the method for producing a hemoglobin standard substance disclosed in JP-A-59-183370, first, an oxidizing agent such as potassium ferricyanide is added to methemoglobin (heme iron [II] to iron [III]. Oxidation). At this time, depending on the conditions, there is a risk of causing serious denaturation of hemoglobin other than methemoglobin formation. That is, the remaining excess oxidizing agent induces an unnecessary oxidation reaction in hemoglobin, which causes the appearance of a denaturation peak on various chromatograms. For example, abnormally increasing the elution fractions of HbAla and HbAlb,
The target HbAlc separation is insufficient, which adversely affects the measured values, making it difficult to obtain reliable data.

However, in the present invention, it is not necessary to use an oxidizing agent at all, and there is no fear that the denaturation of hemoglobin by an excessive oxidizing agent is caused. Further, it is not necessary to perform a treatment such as dialysis for removing the excess oxidizing agent, and the manufacturing process can be simplified.

[0026]

The present invention will be described with reference to the following examples.

Measurement Method In the following examples, the HbAlc value and the HbAl value were measured in the hemolysis mode using Auto Alc HA-8121 manufactured by Kyoto Daiichi Kagaku Co., Ltd.

This Auto Alc HA-8121 is a device exclusively for measuring glycated hemoglobin by HPLC, and each hemoglobin component is separated and eluted in 4 minutes by a separation column packed with a cation exchange resin dedicated for this device. As the elution buffer, an eluent (phosphate buffer) exclusively for this device was used. For hemolysis, a processing solution for hemolysis (containing a hemolytic agent) dedicated to this apparatus was used. The details of other usages were in accordance with the instruction manual of this measuring device. An example of hemoglobin elution pattern using this apparatus is shown in FIG. In FIG. 1, P1 and P2 are HbAl
The peaks due to a and HbAlb, P3 in the hatched area are
HbAlc and P4 are peaks due to other hemoglobin (HbAo). P5 is a component called fetal hemoglobin (HbF), which is not normally contained in glycated hemoglobin.

Here, HbAlc% and HbAl% are calculated by the following equations, and the measured value is automatically printed out as a% value with one decimal place.

[0030]

[Equation 1]

[Equation 2] In this embodiment, blood of the same person (healthy person) is used,
Immediately after blood collection, an appropriate amount of heparin was added and used as fresh blood.

Method for Quantifying Carbon Monoxide Hemoglobin For details of the method for quantifying carbon monoxide hemoglobin, see Matsubara's reference (protein, nucleic acid, enzyme, 32, 6, 671-67).
4, 1987).

0.1 ml of hemolyzed blood just before freeze-drying
% Dissolved in ammonia water, hydrosulfite (Na
₂ S ₂ O ₄ ), 20 mg, added at 538 nm and 578
The absorbance at two wavelengths of nm was measured. The absorbance at this time is E
S 538, ES 578, and absorbance ratio QS = ES 538
/ ES 578 was calculated.

Then, the absorbance ratios of the carbon monoxide hemoglobin contents of 0% and 100% were measured in the following manner to prepare a calibration curve.

Immediately before blowing carbon monoxide, 1m of hemolyzed blood
In order to remove carbon monoxide hemoglobin which may be present in the eluted blood in a trace amount from the beginning, oxygen gas was blown with a Pasteur pipette at 100 ml / min for 5 minutes. Next, 0.25 ml of this blood was dissolved in 50.0 ml of 0.1% ammonia water, and 2
Dispensed in 0.0 ml increments.

First, 20 m of Na ₂ S ₂ O ₄ was added to the test tube A.
In the same manner, the absorbance at two wavelengths of 538 nm and 578 nm was measured.

The absorbance at this time was measured with EA 538 and EA 578.
Then, the absorbance ratio QA = EA 538 / EA 578 was calculated (QA corresponds to the absorbance ratio of carbon monoxide hemoglobin 0%.) On the other hand, after adding 20 mg of Na ₂ S ₂ O ₄ to the test tube B, Then, carbon monoxide gas was blown in at a rate of 100 ml / min for 5 minutes, and the absorbance at two wavelengths of 538 nm and 578 nm was similarly measured. Absorbance at this time is EB 538, EB
Then, the absorbance ratio QB = EB538 / EB578 was calculated (QB corresponds to the absorbance ratio of 100% carbon monoxide hemoglobin.) The content of carbon monoxide hemoglobin in the sample at this time was as follows. It is calculated by the formula.

[Equation 3] Example 1 10 ml of fresh blood was 1500 g × 10 mi in a cooling centrifuge.
Centrifuge at n to precipitate blood cells and remove upper plasma.
To this red blood cell layer, 5 ml of physiological saline was added and mixed by inversion thoroughly, and then centrifuged again to remove the supernatant. This operation was repeated 3 times to wash the red blood cell layer. 5 ml of distilled water was added to about 5 ml of the washed red blood cell layer thus obtained to cause hemolysis. To this hemolyzed blood, 2 ml of toluene was added and shaken in a separating funnel for 10 minutes. After allowing this to stand for a while, the upper toluene layer was removed, and the eluted blood was centrifuged with a cooling centrifuge at 5000 g × 20 min to remove solid matters. Then, 1000 ml of the eluted blood under refrigeration
It was dialyzed overnight against 50 nM phosphate buffer (pH 6.8). Carbon monoxide gas was blown into the hemolyzed blood at a speed of 100 ml / min for 10 minutes using a Pasteur pipette.

Next, 10 μl of this hemolyzed blood was added to 4 m of distilled water.
It was diluted with 1 and put in a glass cell having a cell length of 1 cm, and an absorption spectrum at 350 to 600 nm was measured with a Hitachi spectrophotometer (U-3200 type). As a result, 419,54
An absorption maximum was confirmed at 0,569 nm. Further, the content of carbon monoxide hemoglobin in the hemolyzed blood was measured based on the above-mentioned quantification method, and was 99.3%. From this, it can be seen that most hemoglobin is carbon monoxide.

Sucrose was added to the hemolyzed blood so that the final concentration was 2.5% (w / v), and the hemolyzed blood was thoroughly stirred and then dispensed in 1 ml aliquots into freeze-drying vials. This was freeze-dried under high vacuum at -40 ° C and then 4 ° C for one day. After the freeze-drying was completed, the inside of the vial was replaced with high-purity nitrogen, and then the bottle was sealed.

In order to carry out a long-term storage stability test on the glycated hemoglobin-controlled substance thus produced, 10 vials were randomly withdrawn, and 5 vials were re-added with 1 ml of distilled water. After dissolving and immediately diluting with a dedicated hemolysis treatment solution to a concentration suitable for measurement, the glycated hemoglobin value was measured according to the measurement method described above. For the remaining 5 vials, store them in a dark place at 4 ° C for 6 months while keeping them tightly closed.
1 was added thereto to redissolve them, and the glycated hemoglobin value was similarly measured. No significant change was observed between the HbAlc value and HbAl value of the controlled substance for measuring glycated hemoglobin before long-term storage and the measurement value measured after long-term storage. The measured values are shown in Table 1. From this, it is understood that the controlled substance for measuring glycated hemoglobin produced by the present invention is sufficiently stable even if stored at 4 ° C. for at least 6 months before dissolution.

Example 2 A controlled substance for measuring glycated hemoglobin was produced by the same production method as in Example 1.

10 μl of this hemolyzed blood was diluted with 4 ml of distilled water, and the absorption spectrum at 350 to 600 nm was measured in the same manner as in Example 1. As a result, 419, 540, 569
Absorption maximum was confirmed at nm. Further, the content of carbon monoxide hemoglobin in the hemolyzed blood was measured based on the above-mentioned quantification method, and was 99.1%. For controlled storage stability test after re-dissolving this controlled substance, 10 vials were randomly withdrawn, and 1 ml of distilled water was added to re-dissolve the controlled substance.

Of these, 5 vials were immediately diluted with a dedicated hemolyzing treatment solution in the same manner as in Example 1, and the glycated hemoglobin value thereof was measured according to the measuring method described above. The remaining 5 vials were stored in the dark place at 4 ° C. for 7 days as they were, and then similarly diluted with a dedicated hemolyzing treatment solution just before the measurement, and the glycated hemoglobin value was measured in the same manner.
The HbAlc value and HbAl value measured immediately after re-dissolving in distilled water and the measurement value measured after storage at 4 ° C. for 7 days showed no significant change in the measured value. The measured values are shown in Table 2. From this, the glycated hemoglobin measurement control substance produced by the present invention, even after re-dissolution,
It can be seen that it is sufficiently stable even when stored at 4 ° C for at least 7 days.

Comparative Example 1 A controlled substance for measuring glycated hemoglobin was produced by omitting only the step of blowing carbon monoxide in Example 1.

10 μl of this hemolyzed blood was diluted with 4 ml of distilled water, and the absorption spectrum at 350 to 600 nm was measured in the same manner as in Example 1. As a result, 415, 541, 57
An absorption maximum of 6 nm was confirmed. Further, the content of carbon monoxide hemoglobin in the hemolyzed blood was measured based on the above-mentioned quantification method and was found to be 0.0%. By this,
It can be seen that the main hemolyzed blood does not contain carbon monoxide hemoglobin.

In order to carry out a long-term storage stability test in the same manner as in Example 1, 10 vials were randomly withdrawn, and 5 of them were
The vials were immediately stored, and the remaining 5 vials were stored in a dark place at 4 ° C. for 6 months with the stopper tightly closed, and the glycated hemoglobin value was measured according to Example 1. Hb before long-term storage and after long-term storage
Table 1 shows the Alc value and the HbAl value. In this case, compared with the results of Example 1, the HbAlc value and HbAl
It can be seen that the values have changed significantly.

Comparative Example 2 As in Comparative Example 1, a controlled substance for measuring glycated hemoglobin was produced by omitting only the step of blowing carbon monoxide in Example 1.

10 μl of this hemolyzed blood was diluted with 4 ml of distilled water, and the absorption spectrum at 350 to 600 nm was measured in the same manner as in Example 1. As a result, 415, 541, 57
An absorption maximum of 6 nm was confirmed. Further, the content of carbon monoxide hemoglobin in the hemolyzed blood was measured based on the above-mentioned quantification method and was found to be 0.0%.

In order to conduct a storage stability test after reconstitution in the same manner as in Example 2, 10 vials were randomly withdrawn, reconstituted with 1 ml of distilled water, and immediately after 5 reconstitutes with 5 vials.
The remaining 5 vials were stored at 4 ° C for 7 days, and then the HbAlc value and the HbAl value were measured again immediately after dissolution and after being stored at 4 ° C for 7 days according to Example 2. The measured values at this time are shown in Table 2. In this case, it can be seen that the HbAlc value and the HbAl value are significantly different from the results of Example 2.

[0049]

[Table 1]

[Table 2]

[0050]

INDUSTRIAL APPLICABILITY According to the present invention, a controlled substance for measuring glycated hemoglobin having excellent stability over time, which serves as a reference for a measuring system, particularly a controlled substance for measuring glycated hemoglobin used for measuring HbAlc and / or HbAl is supplied. be able to. As a result, it is possible to ensure constant consistency of measured values, and when measuring the same sample, it is possible to obtain reliable measured values even if the measurer, device, measurement time, etc. are different. Consequently, it can support the social role of using glycated hemoglobin as an index for diabetes testing.

When the characteristic value of the controlled substance for measuring glycated hemoglobin, for example, the HbAlc value is confirmed and guaranteed by a measurement method which is a standard, by defining this substance as a standard substance, the standardization of HbAlc measurement can be performed. It can also be used for

[Brief description of drawings]

FIG. 1 is a graph showing an elution pattern of hemoglobin.

Claims (2)

[Claims] 1. A controlled substance for measuring glycated hemoglobin, which comprises carbon monoxide hemoglobin. 2. A method for producing a controlled substance for measuring glycated hemoglobin, comprising the step of bringing hemoglobin into contact with carbon monoxide.