JPH1010129A - Measurement of stable saccharified hemoglobin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To shorten the measuring time as a result by suppressing the denaturation of a blood sample and removing unstable saccharified hemoglobin quickly and to provide the accurate measuring method at the same time. SOLUTION: The diluted blood sample, wherein a blood sample, a hemolysis reagent (e.g. polyethylene glycol octylphenyl ether) and unstable saccharogenic hemoglobin removing reagent (e.g. tetrapolyphospheric acid) are contained and the blood undergoes hemolysis and dilution at the same time, is processed by ultrasonic waves. Thus, the unstable saccharogenic hemoglobin is removed and then injected into a separating column, and the measurement is performed by the high-speed liquid chromatography.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for measuring stable glycated hemoglobin by high performance liquid chromatography.

[0002]

2. Description of the Related Art Glycated hemoglobin is formed by sugar in blood, which enters red blood cells in proportion to its concentration and then binds to hemoglobin. It is said to reflect the average sugar concentration. Since the blood sugar level and the urine sugar level are less susceptible to physiological factors, the concentration of glycated hemoglobin in the blood is widely used as an optimal index for diagnosis of diabetes or follow-up of diabetic patients. The main component of the glycated hemoglobin is a hemoglobin β chain with glucose bound to the N-terminus, and its production proceeds in a two-step reaction by a non-enzymatic reaction. That is, glycated hemoglobin generated in the first-stage reaction is reversibly returned to a free form and is called unstable glycated hemoglobin. On the other hand, the second reaction is an irreversible reaction, and stable glycated hemoglobin is produced.
The measured value of the stable glycated hemoglobin is used as an index of a longer-term average blood glucose level.

[0003] In order to selectively measure only the stable glycated hemoglobin, a method of measuring after removing the unstable glycated hemoglobin is adopted. For example, Tokuho 5
JP-A-59380 discloses that a blood sample is diluted by adding a hemolytic agent containing a reagent for removing unstable glycated hemoglobin, and the diluted sample is heated to 45 to 70 ° C.
A method is described in which unstable glycated hemoglobin is removed, subsequently injected into a separation column, and the stable glycated hemoglobin is measured by high performance liquid chromatography. However, in this method, the unstable glycated hemoglobin is removed in a shorter time as the heating temperature becomes higher, but there is a problem that the sample is easily denatured and measurement cannot be performed accurately.

[0004]

DISCLOSURE OF THE INVENTION The present invention has been made to solve the above problems, and an object of the present invention is to suppress the denaturation of a blood sample as compared with the conventional method, and to quickly remove unstable glycated hemoglobin, and as a result, Another object of the present invention is to provide a method for accurately measuring stable glycated hemoglobin, which can shorten the measurement time and also accurately.

[0005]

The method for measuring stable glycated hemoglobin of the present invention comprises a blood sample, a hemolytic reagent and a reagent for removing unstable glycated hemoglobin, wherein the blood is hemolyzed and diluted. The sample is subjected to ultrasonic treatment to remove unstable glycated hemoglobin, then injected into a separation column and measured by high performance liquid chromatography (hereinafter, high performance liquid chromatography is sometimes referred to as HPLC). And

As the reagent for removing unstable glycated hemoglobin used in the present invention, any reagent can be used as long as it can remove unstable glycated hemoglobin. For example, a reagent described in JP-A-63-298064 can be used. Phosphoric acid condensates and salts thereof; and other known boric acids and potassium phthalates.

The above-mentioned phosphoric acid condensate is (HPO ₃ )
metaphosphoric acid represented by _n (n is an integer of 2 or more); polyphosphoric acid containing phosphorus of 2 atoms or more and having a P—O—P bond; and the like, for example, trimetaphosphoric acid, tetrametaphosphoric acid, pyrophosphoric acid, tetraphosphoric acid And polyphosphoric acid.
In addition to these cyclic or linear phosphoric acid condensates, PO
Those having a side chain connected by a -P bond or those having a complex condensation such as ultrapolyphosphoric acid are also included. Further, compounds which form the above-mentioned phosphoric acid condensate when dissolved in water, such as phosphorus oxide, can also be used. When dissolved in water, the phosphorus oxide changes to ultrapolyphosphoric acid, tetrametaphosphoric acid, tetrapolyphosphoric acid, and the like by hydrolysis, and exhibits an unstable glycated hemoglobin removing effect.

The degree of condensation of the above phosphoric acid condensate is most preferably 2 to 6 in view of the effect of removing unstable glycated hemoglobin. However, even a compound having a higher degree of condensation has a degree of condensation of 2 to 6 due to hydrolysis in an aqueous solution. It can be used because it produces a condensate.

The above phosphoric acid condensate forms salts with many metals such as potassium or sodium. Salts of these phosphoric acid condensates can also be used because they have an effect of removing unstable glycated hemoglobin.

The unstable glycated hemoglobin removal reagent is usually dissolved in water and used in the form of an aqueous solution.
The aqueous solution is a buffer that can adjust the pH of the diluted blood sample to 4.6 to 7.0, preferably 5.3 to 6.5, from the viewpoint of removing unstable glycated hemoglobin. Is preferred.

Although the optimum concentration of the unstable glycated hemoglobin removing reagent in the diluted blood sample varies depending on the type of the removing reagent, it is usually 0.001 to 5% by weight from the viewpoint of the effect of removing the unstable glycated hemoglobin. preferable.

The hemolytic reagent used in the present invention is a reagent for destroying the erythrocyte membrane in a blood sample to elute hemoglobin, and for example, a surfactant is preferable. Examples of the surfactant include higher aliphatic alcohols, alkylaryl polyether alcohols, polyoxyethylene ethers of sulfonate compounds, polyoxyethylene ethers of sulfate compounds, polyoxyethylene derivatives of fatty acid esters of anhydrous sorbite, and the like.

The above hemolytic reagent is usually dissolved in water and used in the form of an aqueous solution. The aqueous solution adjusts the pH of the diluted blood sample to 4.6 to 7.0, preferably 5.3 to 7.0.
Preferably, the buffer is adjusted to 6.5.

The concentration of the hemolytic reagent in the diluted blood sample is
When the amount is low, sufficient hemolytic action is lost, and when the amount is high, the separation of glycated hemoglobin by HPLC is adversely affected. Therefore, 0.1 to 0.5% by weight is preferable.

Hereinafter, the method for measuring stable glycated hemoglobin of the present invention will be described step by step. First, a diluted blood sample containing a blood sample, a hemolytic reagent, and a reagent for removing unstable glycated hemoglobin is prepared by diluting and diluting the blood. This preparation method is, for example, a method of mixing a blood sample with an aqueous solution of a hemolytic reagent and then mixing an aqueous solution of an unstable glycated hemoglobin removing reagent; or a method of mixing an aqueous solution of a hemolytic reagent and an aqueous solution of an unstable glycated hemoglobin removing reagent. Are mixed. In the diluted blood sample, the blood is hemolyzed and diluted.
The suitable concentration range of the unstable glycated hemoglobin removal reagent or the hemolytic reagent in the diluted blood sample is as described above, and the pH of the diluted blood sample is preferably 4.6 to 7.
0, more preferably 5.3 to 6.5.

Next, the diluted glycated hemoglobin is removed by sonication of the diluted blood sample. In addition,
The diluted blood sample does not necessarily have to be completely lysed before the start of the sonication, and may be lysed during the sonication.

[0017] The ultrasonic treatment means that ultrasonic vibration is applied to the sample. The method of ultrasonic treatment is not particularly limited as long as the method can apply ultrasonic vibration to the sample. As an ultrasonic oscillator,
It is preferable to generate an ultrasonic wave having a frequency of 20 to 90 kHz. The ultrasonic treatment can be performed, for example, by storing the sample in an ultrasonic treatment tank; or by placing an ultrasonic oscillator in the sample and generating ultrasonic waves.

In addition, a commercially available glycated hemoglobin automatic measuring device such as a commercially available device (if a blood sample is injected, hemolytic and unstable glycated hemoglobin removal treatment is performed in the device, and then the resulting sample is injected into a separation column and subjected to HPLC. When the stable glycated hemoglobin concentration in a sample is measured using an automatic measuring device that measures the concentration of stable glycated hemoglobin in the sample, for example, hemolytic dilution of the sample and hemolytic dilution in which unstable glycated hemoglobin is removed simultaneously are performed. An ultrasonic treatment tank may be used instead of the tank, and the hemolytic dilution of the sample, the unstable glycated hemoglobin removal treatment, and the ultrasonic treatment may be performed in the ultrasonic treatment tank.

As the temperature of the diluted blood sample at the time of sonication increases, unstable glycated hemoglobin is removed in a shorter time. However, when the temperature is increased, the hemoglobin in the sample is denatured or decomposed, and separation by HPLC is difficult. The temperature is preferably 50 ° C. or lower, particularly preferably 40 ° C. or lower, since it becomes insufficient.

The optimum time for the ultrasonic treatment varies depending on the treatment conditions such as the temperature during the treatment. For example, when the temperature during the treatment is 30 to 37 ° C., it is preferably 0.5 to 2 minutes, Two minutes is particularly preferred. In this case, the unstable glycated hemoglobin is usually sufficiently removed within 2 minutes.

Next, the ultrasonically treated sample is injected into a separation column, and the stable glycated hemoglobin is measured by HPLC. As the separation column, a column packed with a known cation exchange resin developed for measuring glycated hemoglobin is used. As the cation exchange resin,
For example, coated polymer particles described in JP-A-2-309253, in which a layer of acrylic acid and / or methacrylic acid (co) polymer is formed on the surface of hydrophobic crosslinked polymer particles, are mentioned. Can be The above HPLC method uses a known HPL except that the above-mentioned ultrasonically treated sample is used.
The method may be performed by the method C, for example, in the same manner as in the method for measuring glycated hemoglobin described in JP-A-2-309253.

The concentration of stable glycated hemoglobin after removal of unstable glycated hemoglobin can be determined by a known method.
For example, 415 n of each component eluted from the separation column
The absorbance at m and 500 nm (reference) is measured, and can be calculated from the chromatogram obtained from the difference between the absorbances.

(Effect) In the method for measuring stable glycated hemoglobin of the present invention, a diluted blood sample containing a blood sample, a hemolytic reagent and an unstable glycated hemoglobin removing reagent, wherein the blood is hemolyzed and diluted, By the ultrasonic treatment, the denaturation of the blood sample can be suppressed and the unstable glycated hemoglobin can be rapidly removed as compared with the conventional method. As a result, the stable glycated hemoglobin can be measured accurately in a short time.

[0024]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described in detail with reference to examples. The measuring device, measuring method, hemolytic agent and blood sample used in the following Examples and Comparative Examples are as follows.

Measuring apparatus and measuring method of glycated hemoglobin Stable glycated hemoglobin was measured using Hi-Auto A1c HA-8121 manufactured by Kyoto Daiichi Kagaku Co. under appropriate conditions. This HA-8121 is HPL
C is a device dedicated to glycated hemoglobin measurement by C, which uses a column filled with a cation exchange resin, and separates and elutes each hemoglobin component by cation exchange in 4 minutes. As the eluate, a dedicated eluate 21A (0.10 M phosphate buffer, p
H6), dedicated eluate 21B (0.15 M phosphate buffer,
pH 7), using a dedicated eluent 21C (0.10 M phosphate buffer, pH 6), and the chromatogram was obtained by measuring the absorbance at 415 nm and 500 nm of each component eluted from the separation column.
The absorbance of (Reference) was measured and obtained from the difference in the absorbance.

The solution containing a hemolytic agent hemolysis reagent and labile glycated hemoglobin removal reagent (hereinafter, referred to as hemolytic agent) as, Triton X-100 (polyethylene glycol octylphenyl ether, manufactured by Wako Pure Chemical Industries, Ltd.) as a hemolysis reagent 0 As a reagent for removing unstable glycated hemoglobin at 0.1% by weight, a 0.05M phosphate buffer (pH 6.0) in which 0.1% by weight of tetrapolyphosphate (manufactured by Nacalai Tesque) was dissolved was used.

Blood sample As a blood sample, blood of the same person (healthy person) is used.
Immediately after blood collection, 1 ml of whole blood was supplemented with 10 mg of sodium fluoride as a blood anticoagulant.

(Example 1) First, 100 m of the above blood sample was
glucose at a rate of 1000 mg per 1
This was incubated at 37 ° C. for 3 hours to intentionally generate unstable glycated hemoglobin. A blood sample prepared by diluting 3 μl of the unstable glycated hemoglobin to 150 times with the above-mentioned hemolytic agent was used as a diluted blood sample, and the diluted blood sample was treated in an ultrasonic treatment tank of an ultrasonic washer manufactured by Nippon Seiki Seisakusho Co., Ltd.
0.5 °, 1.0, 1.5 or 2.0 at 28 kHz at 28 ° C.
The sonication was performed by changing the sonication time to minutes. Then, the sonicated diluted blood sample is measured for glycated hemoglobin by the above-described measuring method using the above-described measuring device, and the resulting chromatogram is calculated based on the total sum of all hemoglobin (glycated hemoglobin and non-glycated hemoglobin) peaks. Percentage of glycated hemoglobin A1c peak (%)
(This is referred to as glycated hemoglobin A1c concentration), which is shown in FIG. 1 as G1 (●). From the same chromatogram, the ratio (%) of the glycated hemoglobin A1a + b peak to the total sum of all hemoglobin (glycated hemoglobin and non-glycated hemoglobin) peaks in the obtained chromatogram (this is referred to as glycated hemoglobin A1a + b concentration) was determined. 2 is shown as G1 (●). In addition,
Glycated hemoglobin A1a + b is a component that increases as hemoglobin denatures.

(Example 2) The temperature inside the ultrasonic treatment tank in Example 1 was changed to 37 ° C instead of 30 ° C.
In the same manner as in Example 1, the ratio (%) of the glycated hemoglobin A1c peak is G2 (■) in FIG. 1, and the ratio (%) of the glycated hemoglobin A1a + b peak is G2 in FIG.
(■).

Comparative Example 1 The diluted blood sample in Example 1 was subjected to ultrasonic treatment at 30 ° C. for 0.5, 1.0, 1.5 or 2.0 minutes while changing the ultrasonic treatment time. Instead of "diluted blood samples at 0.5C, 0.5, 1.0,
The heat treatment was performed in a constant temperature bath while changing the heating time to 1.5 or 2.0 minutes. 1), the ratio (%) of the glycated hemoglobin A1c peak was determined in the same manner as in Example 1.
The ratio (%) of the glycated hemoglobin A1a + b peak is shown as H1 (○) in FIG.

(Comparative Example 2) The same procedure as in Comparative Example 1 was carried out except that the temperature in the thermostatic chamber in Comparative Example 1 was changed to 37 ° C instead of 30 ° C, and the ratio (%) of the glycated hemoglobin A1c peak was determined. The ratio (%) of the glycated hemoglobin A1a + b peak is shown as H2 (□) in FIG. 1 and H2 (□) in FIG.

(Comparative Example 3) The same procedure as in Comparative Example 1 was carried out except that the temperature in the thermostatic chamber in Comparative Example 1 was changed to 45 ° C instead of 30 ° C, and the ratio (%) of the glycated hemoglobin A1c peak was determined. The ratio (%) of the glycated hemoglobin A1a + b peak is shown as H3 (△) in FIG. 1 and H3 (△) in FIG.

(Comparative Example 4) The same procedure as in Comparative Example 1 was carried out except that the temperature in the thermostatic chamber in Comparative Example 1 was changed to 50 ° C instead of 30 ° C, and the ratio (%) of the glycated hemoglobin A1c peak was determined. The ratio (%) of the glycated hemoglobin A1a + b peak is shown as H4 (◇) in FIG. 1 and H4 (◇) in FIG.

[0034]

The configuration of the method for measuring stable glycated hemoglobin of the present invention is as described above. According to the present invention, the denaturation of a blood sample can be suppressed and the unstable glycated hemoglobin can be rapidly removed as compared with the conventional method. As a result, stable glycated hemoglobin can be measured accurately in a short time.

[Brief description of the drawings]

FIG. 1 shows the relationship between the ultrasonic treatment time and the glycated hemoglobin A1c concentration (%) in Examples 1 and 2, and the heating time and glycated hemoglobin A1c in Comparative Examples 1 to 4.
It is a graph showing the relationship of concentration (%).

FIG. 2 shows the relationship between the ultrasonic treatment time and the glycated hemoglobin A1a + b concentration (%) in Examples 1 and 2, and the heating time and glycated hemoglobin A in Comparative Examples 1 to 4.
It is a graph showing the relationship of 1a + b density (%).

Claims (1)

[Claims] 1. An unstable glycated hemoglobin is removed by sonicating a diluted blood sample containing a blood sample, a hemolytic reagent and a reagent for removing unstable glycated hemoglobin, wherein the blood is hemolyzed and diluted. And then injecting the resulting mixture into a separation column for measurement by high performance liquid chromatography.