JP5554000B2 - Method for measuring samples for measuring glycated hemoglobin - Google Patents

Description

  The present invention relates to a method for measuring a sample for measuring glycated hemoglobin by an enzymatic method, and a pretreatment reagent for the sample for measuring glycated hemoglobin.

  Glycated hemoglobin is an unstable aldoimine formed by non-enzymatic combination of the reducing end of glucose in blood cells and the free amino group of the β chain of hemoglobin, and then transferred to a stable ketoamine by Amadori transfer. It is a kind of Amadori compound produced by doing so. The blood concentration of hemoglobin A1c (hereinafter referred to as HbA1c), which is one type of glycated hemoglobin, is considered to reflect the average blood glucose level in the past 1 to 2 months, and is therefore widely measured as a diagnostic marker for diabetes.

As a method for measuring glycated hemoglobin, a high performance liquid chromatography method, a latex agglutination immunoturbidimetric method and the like are widely used. However, although the high performance liquid chromatography method (HPLC method) is an excellent method in terms of measurement accuracy, reproducibility, etc., there is a problem that the throughput is low. In addition, the latex agglutination immunoturbidimetric method is superior to the HPLC method in terms of throughput, but it is inferior to the HPLC method in terms of measurement accuracy and reproducibility, and it imposes a load on the equipment such as contaminating the reaction tank of the analyzer used for the measurement. There is.
Therefore, in recent years, an enzyme method has been newly developed as a method for solving the problems of the conventional measurement methods. Enzymatic method is to generate proteolytic enzyme from glycated hemoglobin to produce glycated amino acid or glycated peptide, and further generate hydrogen peroxide using oxidoreductase that generates glycated amino acid or glycated peptide as substrate. Furthermore, this is a colorimetric determination method in which the hydrogen peroxide is reacted with a chromogenic substrate in the presence of peroxidase to lead to color development (Patent Document 1).

JP 05-192193 A

However, when the enzyme method is applied to the sample used for the measurement of the glycated hemoglobin, there is a problem that the measured value, that is, the glycated hemoglobin concentration is lower in some samples than in other measuring methods. I understood.
Therefore, an object of the present invention is to provide a method for accurately measuring a sample for measuring glycated hemoglobin by an enzymatic method.

The present inventor conducted various studies on the cause of the low measured value of glycated hemoglobin concentration in some samples, and surprisingly found that the cause is in the sample itself. In other words, hemoglobins are unstable substances, and the oxidation of heme iron is divalent to trivalent, producing methemoglobin from bright red to dark brown. The sample may contain a thiol compound as a hemoglobin stabilizer. This thiol compound gave a negative error in the determination of the glycated hemoglobin concentration by the enzymatic method.
Therefore, when further examination was made, if a sample for glycated hemoglobin measurement was mixed with the sample and the thiol blocking agent in advance and then subjected to measurement by an enzymatic method, the sample contained a thiol compound. However, the inventors have found that the influence of thiol compounds can be avoided and the glycated hemoglobin concentration can be measured with high accuracy, and the present invention has been completed.

That is, the present invention is a method for measuring a glycated hemoglobin measurement sample to which a hemoglobin stabilizer is added by an enzymatic method, wherein the sample and a thiol blocking agent are mixed and then subjected to measurement by an enzymatic method. A method for measuring a sample for measuring glycated hemoglobin is provided.
The present invention also provides a pretreatment reagent for a sample for measuring glycated hemoglobin, which contains a thiol blocking agent.

  According to the present invention, a sample for measuring glycated hemoglobin can be accurately measured by an enzyme method having many advantages compared to other measurement methods.

The calibration curve by L-cysteine dilution series is shown.

The sample for measuring glycated hemoglobin to which a hemoglobin stabilizer is added in the present invention is a sample mainly used as a calibration sample or a quality control sample in a clinical test of glycated hemoglobin, to which a hemoglobin stabilizer is added. Sample. A calibration sample is a sample that contains a known concentration of glycated hemoglobin, which is the standard for determining the concentration of glycated hemoglobin. An accuracy control sample is used to manage the accuracy of measurement results in clinical tests of glycated hemoglobin. The sample used. Since the sample is used for a long time, a lyophilized product or a frozen product is preferable, and a lyophilized product is more preferable. When the sample is a lyophilized product, it is used after being dissolved in purified water, a buffer solution or the like at the time of use.
As said buffer solution, Preferably it is pH 4.0-10.0, More preferably, it has buffer capacity in the range of pH 5.0-9.0, for example, a citric acid, a succinic acid, tartaric acid, malic acid Amphoteric buffers such as Good's buffer such as organic acids and salts thereof; amino acids such as glycine, taurine and arginine; inorganic acids such as hydrochloric acid, nitric acid, sulfuric acid, phosphoric acid, boric acid and acetic acid; Can be mentioned.
The sample for measuring glycated hemoglobin may contain a preservative and the like. There are no restrictions on the pH, composition, etc. of the sample.

  Examples of the hemoglobin stabilizer added to the glycated hemoglobin measurement sample include thiol compounds such as sulfur-containing amino acids such as cysteine, methionine, and cystine; thiobenzoic acid, thioglycolic acid, 1-thioglycerin, thiodi Examples include glycol, mercaptoethanol, glutathione, dithiothreitol, and analogs thereof. Usually, the concentration of the thiol compound in the sample is 0.03 μmole or more as the amount of the thiol compound per 1 mg of hemoglobin (Hb). In addition, even if the sample does not contain a thiol compound, it does not affect the measured value of glycated hemoglobin.

The thiol blocking agent used in the present invention blocks the SH group of the thiol compound contained in the glycated hemoglobin measurement sample. Examples of such a thiol blocking agent include iodoacetamide, monoiodoacetic acid, N-ethylmaleimide (NEM), N-4-dimethylamino-3,5-dinitrophenylmaleimide (DDPM), and benzimidazolyl maleimide (BIPM). N- (9-acridinylmaleimide) (NAM), 4-vinylpyridine, 2-vinylquinoline, 9-vinylacridine, 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), 2,2 Examples include '-dithiodipyridine (2PDS), 4,4'-dithiodipyridine (4PDS), 6,6'-dithionicotinic acid (6NDS), thiamine disulfide (TDS), and the like. These can be used alone or in combination of two or more.
The thiol blocking agent is preferably a maleimide compound from the viewpoint of solubility, and particularly preferably NEM.

The mixing amount of the thiol blocking agent with respect to the sample for measuring glycated hemoglobin is not particularly limited as long as it can avoid the influence of the thiol compound contained in the sample on the measurement value. It is preferable to mix in a concentration of ˜100 times, preferably 1 to 50 times.
The sample for measuring glycated hemoglobin may contain 0.03 μmole to 0.5 μmole of thiol compound per 1 mg of hemoglobin (hereinafter referred to as Hb). For example, depending on the thiol compound concentration in the sample, thiol blocking It is preferable to mix the agent so as to be 0.03 μmole to 50 μmole, preferably 0.03 μmole to 25 μmole per 1 mg of Hb.

  Measurement of a sample for measuring glycated hemoglobin by an enzymatic method is performed by a known method (for example, JP-A-2-195899, JP-A-2-195900, JP-A-5-192193, JP-A-6-46846, JP-A-7-289253, JP-A-8-154672, and JP-A-8-336386). For example, a glycated amino acid or glycated peptide is excised from glycated hemoglobin using a protease, The glycated hemoglobin may be colorimetrically determined by causing the oxidase that reacts with the glycated amino acid or the glycated peptide to generate hydrogen peroxide and then leading the chromogenic substrate to color in the presence of peroxidase. .

The pretreatment reagent of the present invention treats a sample for measuring glycated hemoglobin before measuring the glycated hemoglobin concentration by an enzyme method, and the reagent contains the thiol blocking agent.
The content of the thiol blocking agent in the pretreatment reagent is preferably 1 to 300 mM, particularly 10 to 200 mM.

  In the pretreatment reagent of the present invention, for example, a buffer, a preservative, a surfactant, a hemoglobin metting agent and the like can be blended. The pH of the pretreatment reagent is not particularly limited, but when a maleimide compound is used as the thiol blocking agent, the pH is 1.0 to 6.0, and more preferably in the range of pH 2.0 to 4.0 due to the stability of the maleimide compound. Is preferred.

The method for treating a sample for measuring glycated hemoglobin using the pretreatment reagent of the present invention is, for example, when the sample is a lyophilized product, after dissolving the sample with purified water or a buffer solution, There is a method in which the sample is mixed and reacted, or a method in which the sample is directly dissolved in a pretreatment reagent and reacted. Moreover, when the sample for measuring glycated hemoglobin is a frozen product, there is a method in which the sample is thawed, mixed with a pretreatment reagent, and reacted. The pretreated sample is subjected to measurement of glycated hemoglobin by an enzymatic method.
Thus, by measuring the sample for measuring glycated hemoglobin by the enzymatic method, an accurate measured value of the glycated hemoglobin concentration without a negative error can be obtained even when compared with other measurement methods.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

Example 1
1. Measurement of concentration of thiol compound contained in sample (1) Preparation of sample and standard solution (cysteine dilution series)
By diluting 1,000 μM L-cysteine aqueous solution with physiological saline twice, 10 concentration dilution series were prepared (1,000, 500, 250, 125, 62.5, 31.25, 15.625, 7.8125, 3.90125, 1.950125)
・ Glyco Hb control (Sysmex, freeze-dried product)
Two types of freeze-dried products were dissolved in 200 μL of purified water (Level 1 (HbA1c%, 5.6%), Level2 (9.8%)) and then diluted 50-fold with physiological saline for measurement. Provided.
-JCCLS CRM-004a (HECTEF, HbA1c measurement actual sample standard substance (frozen product)) (control)
Five frozen products (Level 1 (HbA1c%, 4.54%), Level 2 (5.27%), Level 3 (6.96%), Level 4 (9.24%), Level 5 (11.58) %)) Diluted 50-fold with physiological saline was used as a sample.

(2) Sample measurement Each sample was measured by the following operations using a Hitachi 7170 automatic analyzer.
<First reagent>
50 mM Tris buffer (pH 7.5)
<Second reagent>
1 mM 5,5′-Dithiobis (2-nitrobenzoic acid) (DTNB, manufactured by Wako Pure Chemical Industries, Ltd.)
50 mM Tris buffer (pH 7.5)
<Measurement parameters>
Analysis method: 2-point end measurement wavelength (sub / main): 505/405
Side light point: 16-34
Reaction time: 10 minutes Sample volume: 15 μL
First reagent amount (R1): 180 μL Third reagent amount (R2): 90 μL

180 μL of the first reagent was added to 15 μL of the sample, and the absorbance after heating at 37 ° C. for 5 minutes was measured (absorbance I). Next, 90 μL of the second reagent was added, and the absorbance after heating at 37 ° C. for 5 minutes was measured (absorbance II). Absorbance was measured at a main wavelength of 405 nm (subwavelength 505 nm), and the same operation (reagent blank) using physiological saline instead of the sample was used as a control.
The absorbance change amount (measured value) of each sample was calculated from the absorbance I and absorbance II of each sample using Formula A.
Formula A: Change in absorbance (measured value) = absorbance II− (absorbance I × (15 + 180) / (15 + 180 + 90))

  Using each measured value of the obtained L-cysteine dilution series, a calibration curve was created to calculate the concentration of the thiol compound in the sample. A calibration curve graph is shown in FIG.

2. Measurement of Hb concentration in sample (1) Preparation of sample / glyco Hb control The above-mentioned two lyophilized products dissolved in 200 μL of purified water were subjected to measurement.
・ JCCLS CRM-004a
The above five frozen products were thawed at room temperature and then subjected to measurement.

(2) Measurement of Hb concentration Each sample was measured by the following operation using Nescoat Hemokit-N (manufactured by Alfresa Pharma).
<Reagent for measuring hemoglobin concentration>
-Colored solution Used diluted 50 times with purified water.
Standard solution (hemoglobin 16.0 g / 100 mL)
Used as is.

20 μL of the sample and 5 mL of the colored solution were mixed well and allowed to stand at room temperature for 5 minutes, and then the absorbance at 541 nm (absorbance I) was measured with a Hitachi U-3310 spectrophotometer. Simultaneously, the absorbance of physiological saline as a standard solution and a blank (absorbance II and absorbance III, respectively) was measured. The Hb concentration of each sample was calculated from the absorbance of each sample using Formula B.
Formula B: Hb concentration (g / dL) = (absorbance I−absorbance III) ÷ (absorbance II−absorbance III) × 16.0

  From the Hb concentration in each sample and the concentration of the thiol compound calculated above, the concentration of the thiol compound per 1 mg of Hb was determined. Table 1 shows the concentration of the thiol compound per 1 mg of Hb.

3. Measurement of HbA1c content of sample containing thiol compound by enzyme method (1) Sample preparation / glyco-Hb control After dissolving the above two lyophilized products in 200 μL of purified water, 150 mM N-ethylmaleimide (NEM) (Tokyo Kasei) Kogyo Kogyo Co., Ltd.) was mixed with an acidic solution (pH 2.8, prepared with 1N hydrochloric acid) or an equal amount of purified water, and further diluted 7-fold with the following specimen diluent to be used for measurement.
・ JCCLS CRM-004a
After thawing the above five types of frozen products, an equal amount of 150 mM N-ethylmaleimide (NEM) acidic solution (pH 2.8) or purified water is mixed in an equal amount and further diluted 6-fold with the following sample diluent as a sample. It used for the measurement.

(2) Hemoglobin A1c enzymatic quantification method (enzyme method) Reagent <specimen diluent>
10 mM sodium nitrite <Protease-containing substrate reagent (first reagent)>
50 mM sodium phosphate buffer pH 7.0 containing the following ingredients
1.5% Amphitol 20BS (manufactured by Kao Corporation)
2.0 mg / mL Protin PC10F (Daiwa Kasei Kogyo Co., Ltd.)
0.01% sodium azide (Kishida Chemical Co., Ltd.)
50 μM DA-67 (10- (carboxymethylaminocarbonyl) -3,7-bis (dimethylamino) phenothiazine sodium, manufactured by Wako Pure Chemical Industries, Ltd.)
<Coloring reagent (second reagent)>
50 mM sodium phosphate buffer pH 7.0 containing the following ingredients
10 U / mL fructosyl peptide oxidase (Kikkoman)
1U / mL peroxidase (Toyobo)
<Sample for calibration>
Blank sample: 10 U / mL POD aqueous solution (10,000 U peroxidase (Toyobo Co., Ltd.) was dissolved in 1 L of purified water)
・ Hemoglobin A1c calibration sample L (HbA1c (%) 5.27%, lyophilized product), (Sekisui Medical, HbA1c Control L)
・ Hemoglobin A1c calibration sample M (HbA1c (%) 7.64%, freeze-dried product), (Sekisui Medical, HbA1c Control M)
・ Hemoglobin A1c calibration sample H (HbA1c (%) 10.81%, freeze-dried product), (Sekisui Medical, HbA1c Control H)
All were dissolved in 300 μL of sample diluent and used for measurement.

(3) Measurement of each sample Using a Hitachi 7170 automatic analyzer, each sample was measured by the following operation.
<Measurement parameters>
Analysis method: 3-point end measurement wavelength (Hb, secondary / main): 800/505
(HbA1c, Deputy / Main): 800/660
Metering point (Hb): 0-14
(HbA1c): 16-34
Reaction time: 10 minutes Sample volume: 12 μL
First reagent: 180 μL (R1) Third reagent (R2): 60 μL

After dispensing 12 μL of each sample and 180 μL of the first reagent into the cell and heating at 37 ° C. for 5 minutes, the difference in absorbance between the main wavelength of 660 nm and the sub wavelength of 800 nm (Abs1), and the difference in absorbance between the main wavelength of 505 nm and the sub wavelength of 800 nm (Abs3) was measured. Subsequently, 60 μL of the second reagent was added, and after heating at 37 ° C. for 5 minutes, the difference in absorbance between the main wavelength of 660 nm and the sub wavelength of 800 nm was measured (Abs2).
The absorbance change amount (measured value) of each sample was calculated from Abs1 (mOD) and Abs2 (mOD) of each sample using the following formula C.
Formula C: Change in absorbance (ΔAbs) = Abs2−Abs1 × 192 ÷ 252

A calibration curve of HbA1c was created from the four points ΔAbs of the blank sample and the calibration sample.
In addition, a calibration curve of Hb was created from two points Abs3 of the blank sample and the calibration sample (HbA1c control L).

Using the prepared calibration curve, the HbA1c concentration (μmol / L) and Hb concentration (μmol / L) of each sample are calculated, and further, an automatic analyzer for converting into Formula D (Glycohemoglobin standardized HbA1c value of the Japan Diabetes Society) Formula of Inter-Items, Japan Society for Clinical Chemistry Diabetes-related Indicators Special Committee: Diabetes-related Indicators Special Committee on Consensus Statement on International Standardization of Hemoglobin A1c Measurement by ADA, EASD, IFCC, IDF, Clinical Chemistry 36, 310 (2007)) to calculate HbA1c%.
Formula D: HbA1c (%) = HbA1c (μmol / L) ÷ Hb (μmol / L) × 96.3 + 1.62

  Table 1 shows the HbA1c amount (%) of each sample.

4). Measurement of HbA1c content of sample containing thiol compound by HPLC method (1) Preparation of sample / glyco Hb control After dissolving the above two lyophilized products in 200 μL of purified water, 101 was further diluted with a sample diluent (manufactured by Tosoh Corporation). The diluted product was measured with a Tosoh automatic glycohemoglobin analyzer HLC-723G8.
・ JCCLS CRM-004a
Measurement was performed with HLC-723G8 as a sample obtained by diluting the above five kinds of frozen products 201-fold with a specimen diluent.

(2) Measurement of sample It measured using HLC-723G8. Table 1 shows the HbA1c amount (%) of each sample obtained by the HPLC method.

As shown in Table 1, it was found that the glyco Hb control contained a large amount of thiol compound as compared with JCCLS CRM-004a.
In addition, as shown in Table 1, in the method in which glyco Hb control containing a large amount of thiol compound is used for measurement without mixing with NEM acidic solution, the enzyme method measurement value is significantly lower than the HPLC method measurement value. On the other hand, in the method used for the measurement after mixing with the NEM acidic solution, the enzyme method was able to obtain a measurement value almost coincident with the HPLC method. In addition, about JCCLS CRM-004a, the enzyme method measured value substantially corresponded with the HPLC method measured value irrespective of the presence or absence of mixing with the NEM acidic solution.

Claims (4)

The enzyme method, a method of the thiol compound to measure glycated hemoglobin glycated hemoglobin measurement sample which is added, after mixing the sample and N- ethylmaleimide, characterized in that subjected to measurement by an enzymatic method , Measuring method. The measurement method according to claim 1 , wherein the glycated hemoglobin measurement sample to which the thiol compound is added is a calibration sample. The measurement method according to claim 1 , wherein the glycated hemoglobin measurement sample to which the thiol compound is added is a quality control sample. The measurement method according to any one of claims 1 to 3 , wherein the glycated hemoglobin is hemoglobin A1c .

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