JP7547952B2 - Hemoglobin preparation and method for measuring hemoglobins using the hemoglobin preparation - Google Patents

Description

The present invention relates to a hemoglobin preparation containing a surfactant for use in measuring hemoglobins by liquid chromatography, and to the quantitative measurement of hemoglobins using the hemoglobin preparation.

Hemoglobin, the main protein in blood, transports oxygen molecules in the body via heme, which binds to it as a cofactor. Hemoglobin molecules are red in color because they absorb blue light due to the presence of protoporphyrin IX and divalent iron ions that compose the heme. Liquid chromatography is one method of analyzing hemoglobins that takes advantage of this property.
Analysis of hemoglobins using liquid chromatography provides highly accurate measurements and is widely used as a diagnostic method for diabetes and hemoglobinopathy (thalassemia), a type of congenital hemolytic anemia.

Normally, adult hemoglobin is a heterotetramer (α2β2) consisting of two types of globin chains, α and β, called hemoglobin A0, which accounts for more than 90% of hemoglobins. Other examples of hemoglobins include hemoglobin A1, which is hemoglobin A0 bound to glucose or various derived sugars, fetal hemoglobin F consisting of two types of globin chains, α and γ, (α2γ2), and hemoglobin A2 consisting of two types of globin chains, α and δ, (α2δ2). Components belonging to hemoglobin A1 include hemoglobin A1a, A1b, and A1c.

In the diagnosis of diabetes, the percentage of total hemoglobin that is hemoglobin s-A1c, a stable glycated component of hemoglobin A0 (HbA1c%), is used as one of the diagnostic indicators. In the diagnosis of thalassemia, the percentage of total hemoglobin that is hemoglobin F and hemoglobin A2 (F% and A2%) are used as diagnostic indicators.

In the analysis of hemoglobins using liquid chromatography, samples such as freeze-dried samples and blood containing hemoglobin are generally hemolyzed and diluted using a hemoglobin preparation containing a surfactant. The hemoglobin preparation used in this case is required to be able to hemolyze the blood sufficiently in a short time and dissolve the sample components containing hemoglobin. In addition, hemoglobin analyzers using liquid chromatography generally omit pretreatment steps such as manual preparation of the measurement sample and are equipped with a function to mix and dilute the sample and hemoglobin preparation within the device. Furthermore, in order to perform continuous measurements of the above samples with high accuracy, it is more preferable that the hemoglobin preparation has the effect of cleaning the sample loop and sampler within the device after each measurement.

A surfactant is added to the hemoglobin preparation to promote hemolysis of blood and rapid dissolution of sample components.

In Patent Document 1, 0.05 M phosphate buffer (pH 7.0) containing 0.1 wt % polyoxyethylene octylphenyl ether dissolved therein is used as a hemoglobin preparation solution.

In Patent Document 2, a hemoglobin preparation containing 0.1 wt% polyoxyethylene sorbitan monolaurate is used as the hemoglobin preparation.

Patent Document 3 evaluates various alkyl ether groups that do not contain polyoxyethylene octylphenyl ether, and claims that polyoxyethylene alkyl ethers with a specific structure are capable of hemolyzing blood samples at the same level as polyoxyethylene octylphenyl ether, and can be used to measure hemoglobin levels with the same accuracy.

The polyoxyethylene octylphenyl ether described in Patent Documents 1 and 3 is widely used not only for the preparation of hemoglobins but also for blood preparation solutions. However, when discharged into rivers, oceans, etc., it is decomposed into alkylphenols, etc., which are classified as environmental hormones, and it has been pointed out that it has a negative impact on the biological environment. In particular, in recent years, the European Chemical Agency (ECHA) in Europe has added a series of substances with octylphenol ethoxylate as a partial structure to the target candidate list of the Registration, Evaluation, Authorization and Restrictions of Chemicals (REACH regulation), so it is necessary to avoid the use of surfactants containing octylphenol ethoxylate structures as much as possible.

The hemoglobin preparation containing polyoxyethylene sorbitan monolaurate described in Patent Document 2 has excellent stability of hemoglobins after preparation, but has a weak hemolytic effect and requires more time for complete hemolysis and dissolution than polyoxyethylene octylphenyl ether. For this reason, when a hemoglobin preparation containing polyoxyethylene sorbitan monolaurate is used in the analysis of hemoglobins using liquid chromatography, if a large amount of blood sample is measured continuously in a short period of time, components that are insufficiently hemolyzed and dissolved may accumulate in the column, causing contamination.

Japanese Patent Application Publication No. 10-010107 JP 2018-004606 A Patent No. 6651066

When measuring hemoglobin using liquid chromatography, hemolysates containing polyoxyethylene octylphenyl ether as a surfactant have generally been used up to now. However, there are concerns that the above surfactants may have a negative impact on the biological environment, and from the perspective of environmental conservation, there has been a need for an alternative surfactant to polyoxyethylene octylphenyl ether.

The object of the present invention is to provide a surfactant that replaces polyoxyethylene octylphenyl ether, which has been widely used in the measurement of hemoglobins using liquid chromatography, and a hemoglobin preparation containing the surfactant. More specifically, the object of the present invention is to provide a hemoglobin preparation containing a surfactant in which at least one hydrophilic group contains a sugar alcohol or a sugar molecule, and the hydrophilic group and the hydrophobic group are bonded via an ester bond, which realizes hemolysis performance equal to or greater than that of polyoxyethylene octylphenyl ether and highly accurate measurement of hemoglobins, and at the same time, to provide a method for measuring hemoglobins using the hemoglobin preparation.

As a result of intensive research conducted by the inventors to solve the above problems, they discovered that by using a solution containing a surfactant in which the hydrophilic group contains a sugar alcohol or sugar molecule and the hydrophilic group and the hydrophobic group are bonded via an ester bond as a hemoglobin preparation solution, it is possible to obtain a hemoglobin preparation with excellent stability after dissolution, to measure hemoglobin continuously with high accuracy, and to reduce contamination, and thus completed the present invention.

The present invention will now be described in detail.
The present invention relates to a hemoglobin preparation used for the quantitative analysis of hemoglobins by liquid chromatography, characterized in that the preparation contains a surfactant in which at least one or more hydrophilic groups contain a sugar alcohol or a sugar molecule and the hydrophilic group and the hydrophobic group are bonded via an ester bond, and to a method for measuring hemoglobins using the hemoglobin preparation.

The sugar alcohol or sugar molecule of the hydrophilic group of the surfactant includes, but is not limited to, monosaccharides such as glucose, galactose, fructose, ribose, etc., disaccharides such as sucrose, lactose, maltose, trehalose, cellobiose, etc., trisaccharides such as raffinose, melezitose, maltotriose, etc., tetrasaccharides such as acarbose, stachyose, etc., and sugar alcohols such as glycerin, erythritol, sorbitol, xylitol, isomalt, lactitol, maltitol, mannitol, etc.

The surfactant is preferably nonionic, but may have cationic or anionic groups, or both, attached thereto. Examples of sugar molecules having cationic groups include glucosamine, and examples of sugar molecules having anionic groups include glucuronic acid.

The hydrophobic group of the surfactant is characterized by being a fatty acid having 7 to 22 carbon atoms, but there is no particular restriction on the degree of unsaturation in the fatty acid. Examples of fatty acids of the surfactant include enanthic acid, caprylic acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, palmitoleic acid, oleic acid, and linoleic acid.

Samples containing hemoglobins include freeze-dried reagents, controls, calibrators, and blood.

The hemoglobin preparation of the present invention may contain a preservative, such as, but not limited to, sodium azide, lithium azide, streptomycin, kanamycin, puromycin, tetracycline, etc., as a preservative. In addition, the hemoglobin preparation may contain a hemoglobin stabilizer, such as, but not limited to, sodium salt and potassium salt of EDTA, as a preservative.

The surfactant content of the hemoglobin preparation of the present invention can be set to a concentration of 0.01 to 1.00 wt % depending on the measurement conditions. The preferred concentration is 0.01 to 0.10 wt %.

In addition, pH buffers such as carboxylates, phosphates, ammonium salts, and alkylamines can be added. The pH of the hemoglobin preparation of the present invention is preferably in the range of 5.0 to 9.0.

The present invention relates to a method for measuring hemoglobins, which comprises the steps of: mixing the hemoglobin preparation solution with a sample containing hemoglobins to hemolyze and dissolve the sample to obtain a measurement sample; and measuring the obtained measurement sample by liquid chromatography.

The mixing step may be performed by a hemoglobin measuring device or may be performed in advance by a person performing the measurement. The hemoglobin levels can be measured, for example, by the following procedure.
1. The hemoglobin measuring reagent is mixed with a sample containing hemoglobin.
2. The solution obtained in 1. is measured using cation exchange chromatography.
3. Calculate the percentage of the peak to be measured using the following formula A.
[Calculation formula A] Abundance ratio (%) of target component = (peak area of measured component / sum of peak areas of all components)
In the measurement of hemoglobins by liquid chromatography, it is more convenient to use an automatic analyzer that can fully automatically perform the steps from diluting a sample containing hemoglobins to calculating the peak area. Commercially available analyzers include the automatic glycohemoglobin analyzer HLC-723 G11 (manufactured by Tosoh Corporation).

The hemoglobin preparation of the present invention has excellent hemolysis and dissolution capabilities for samples containing hemoglobins, and can achieve continuous reproducibility equal to or greater than that of polyoxyethylene octylphenyl ether, which has been widely used in the field of quantitative measurement of hemoglobins, and can reduce carryover values. In addition, the use of the above hemoglobin measurement reagent is expected to reduce the amount of polyoxyethylene octylphenyl ether used, thereby reducing the environmental burden.

FIG. 2 shows a chromatogram obtained in Example 1. FIG. 2 shows a chromatogram obtained in Comparative Example 1. FIG. 2 shows chromatograms of Example 7 and Comparative Example 2. FIG. 1 shows a chromatogram obtained in Example 8. FIG. 1 shows chromatograms obtained in Examples 9, 10, 11, and 12. FIG. 1 shows chromatograms obtained in Examples 13, 14, and 15.

The following examples are provided to explain the present invention in detail, but the present invention is not limited to these examples.

Example 1
Blood samples were measured using the following hemoglobin preparations.

1. Preparation of Hemoglobin Preparation Solution In pure water, 0.05 wt % sucrose laurate (manufactured by Mitsubishi Chemical Corporation, product name: L-1695) as a surfactant, 0.05 wt % sodium azide (manufactured by Fuji Film Wako Co., Ltd.) as a preservative, and EDTA.4Na (manufactured by Dojindo Laboratories Ltd.) and EDTA.2K (manufactured by Dojindo Laboratories Ltd.) as hemoglobin stabilizers were dissolved to obtain hemoglobin preparation solution A with a pH of 7.5.

2. Measurement of hemoglobins Using blood samples as samples containing hemoglobins, and using hemoglobin preparation solution A, the blood samples were measured 20 times consecutively to measure HbA1c% using an automatic hemoglobin analyzer HLC-723 G11 in STD mode (measurement time: 30 seconds/test), and then pure water was measured to measure the carryover value. The chromatograms obtained for every 5 samples are shown in Figure 1. In addition, the average value of HbA1c% and the coefficient of variation (CV%) were calculated from the results of the consecutive measurements.
The analytical column used was TSK gel G11 (manufactured by Tosoh Corporation), and the eluent used was G11 eluent first, second, and third solutions (manufactured by Tosoh Corporation).
The carryover value was calculated using the following formula B.
[Calculation formula B]: Carryover value (%) = (sum of all peak areas detected during measurement of pure water/sum of all peak areas detected during measurement of a sample immediately before measurement of pure water) x 100
Example 2
The surfactant used in the hemoglobin preparation in Example 1 was changed from sucrose laurate to sucrose myristate (manufactured by Mitsubishi Chemical Corporation, product name: M-1695), to obtain a hemoglobin preparation B. The other conditions were the same as in Example 1.

Example 3
The surfactant used in the hemoglobin preparation in Example 1 was changed from sucrose laurate to sucrose palmitate (manufactured by Mitsubishi Chemical Corporation, product name: P-1670), to obtain a hemoglobin preparation C. The other conditions were the same as in Example 1.

Example 4
The surfactant used in the hemoglobin preparation in Example 1 was changed from sucrose laurate to sucrose oleate (manufactured by Mitsubishi Chemical Corporation, product name: O-1570), to obtain a hemoglobin preparation D. The other conditions were the same as in Example 1.

Example 5
The surfactant used in the hemoglobin preparation in Example 1 was changed from sucrose laurate to sucrose stearate (manufactured by Mitsubishi Chemical Corporation, product name: S-1670), to obtain a hemoglobin preparation E. The other conditions were the same as in Example 1.

Example 6
The surfactant used in the hemoglobin preparation in Example 1 was changed from sucrose laurate to sorbitol caprylate (manufactured by Riken Vitamin Co., Ltd., product name: Poem C-250), to obtain hemoglobin preparation F. The other conditions were the same as in Example 1.

(Comparative Example 1)
The surfactant used in the hemoglobin preparation in Example 1 was changed from sucrose laurate to polyoxyethylene sorbitan monolaurate (manufactured by Fuji Film Wako Co., Ltd., product name: Tween 20), to obtain hemoglobin preparation X. The other conditions were the same as in Example 1. The chromatograms for each of the five specimens measured in Comparative Example 1 are shown in FIG.

As can be seen from Figures 1 and 2, in Comparative Example 1, an unknown peak appeared behind the A0 peak due to contamination of the column during continuous measurements, and gradually grew as the number of measurements increased. In addition, a decrease in HbA1c% was observed as the unknown peak grew. On the other hand, in Example 1, no unknown peak like that seen in Comparative Example 1 appeared, and no decrease in HbA1c% was observed.

Table 1 shows the carryover values obtained in Examples 1, 2, 3, 4, 5, 6 and Comparative Example 1, and the coefficient of variation (CV% value) of HbA1c% in 20 consecutive measurements. The carryover values were calculated using formula B. From the results in Table 1, the carryover values in Examples 1, 2, 3, 4, 5 and 6 were all less than 2%, indicating that no contamination of the column was observed and that the blood sample was sufficiently hemolyzed and dissolved. On the other hand, the carryover value in Comparative Example 1, which used polyoxyethylene sorbitan monolaurate as a surfactant, exceeded 10%, indicating that contamination of the column had occurred. As for the measurement accuracy, the CV% in Examples 1, 2, 3, 4, 5 and 6 was less than 1%, showing good reproducibility. On the other hand, Comparative Example 1 was affected by contamination of the column, resulting in a value of about 2%.

(Example 7)
A blood sample was used as a sample containing hemoglobins, and the blood sample was measured 20 times in succession using the hemoglobin preparation A prepared in Example 1. Then, pure water was measured three times in succession, and the measured value of A2% and the carryover value were measured. After the measurement, the average value of A2% and the coefficient of variation CV% were calculated from the results of the successive measurements.

Hemoglobins were analyzed using an automatic hemoglobin analyzer HLC-723 G11 β-Thalassemia mode (measurement time: 5 minutes/test), a cation exchange column TSK gel G11 β-Thal. (manufactured by Tosoh Corporation), and an eluent G11 β-Thalassemia Elution Buffer No. 1(S), No. 2(S), No. 3(S) (manufactured by Tosoh Corporation).

(Comparative Example 2)
The same evaluation as in Example 7 was carried out except that the hemoglobin preparation X prepared in Comparative Example 1 was used in place of the hemoglobin preparation A.

Chromatograms of the 20th measurement obtained in Example 7 and Comparative Example 2 are shown in Figure 3, and the obtained carryover values and the coefficient of variation CV% of A2% during 20 consecutive measurements are shown in Table 2. The carryover values in Table 2 were calculated using formula B. As shown in Figure 3, there was no clear difference in the chromatograms between the hemoglobin preparations, but the results in Table 2 show that the measurement in Example 7 had a smaller carryover value and less column contamination than Comparative Example 2.

(Example 8)
A blood sample was used as a sample containing hemoglobins, and the blood sample was measured 20 times in succession using the hemoglobin preparation A prepared in Example 1. Then, pure water was measured three times in succession, and the measured value of glycated hemoglobin % and the carryover value were measured. After the measurement, the average value of glycated hemoglobin % and the coefficient of variation CV % were calculated from the results of the continuous measurements.

The analysis was performed using a Tosoh Corporation automatic hemoglobin analyzer HLC-723 G8 Affinity mode (measurement time: 2.2 minutes/test), a TSK gel AF-GHb (Tosoh Corporation) affinity column, and G8 eluent AF-GHb A(S), B(S) (Tosoh Corporation) eluents.

The chromatogram of the 20th measurement obtained in Example 8 is shown in Figure 4, and the obtained carryover values and the CV% for glycated hemoglobin% during 20 consecutive measurements are shown in Table 3. The carryover values in Table 3 were calculated using formula B. As shown in Figure 4, hemoglobin preparation A can be used for quantitative analysis of hemoglobins by affinity chromatography as well as ion exchange chromatography, and the results in Table 3 show that glycated hemoglobin% can be measured with high measurement accuracy, with a CV% of less than 1%.

Example 9
A hemoglobin preparation G was obtained by changing the concentration of the surfactant used in Example 1 from 0.05 wt % to 0.01 wt %. The hemoglobin preparation G was used instead of the hemoglobin preparation A, and the same evaluation as in Example 1 was performed.

Example 10
A hemoglobin preparation H was obtained by changing the concentration of the surfactant used in Example 1 from 0.05 wt % to 0.10 wt %. The hemoglobin preparation H was used instead of the hemoglobin preparation A, and the same evaluation as in Example 1 was performed.

(Example 11)
Hemoglobin preparation I was obtained by changing the concentration of the surfactant used in Example 1 from 0.05 wt % to 0.50 wt %. Hemoglobin preparation I was used in place of hemoglobin preparation A, and the same evaluation as in Example 1 was performed.

Example 12
A hemoglobin preparation J was obtained by changing the concentration of the surfactant used in Example 1 from 0.05 wt % to 1.00 wt %. The hemoglobin preparation J was used instead of the hemoglobin preparation A, and the same evaluation as in Example 1 was performed.

Chromatograms after 20 measurements obtained in Examples 9, 10, 11, and 12 are shown in Figure 5, and carryover values obtained from formula B are shown in Table 4. As shown in Table 4, the carryover value was less than 1% for all hemoglobin preparations, and there was no contamination of the column.

(Example 13)
The pH of the hemoglobin preparation used in Example 1 was adjusted to 5.5 to obtain hemoglobin preparation K. The same evaluation as in Example 1 was carried out using hemoglobin preparation K in place of hemoglobin preparation A.

(Example 14)
The pH of the hemoglobin preparation used in Example 1 was adjusted to 6.5 to obtain hemoglobin preparation L. The hemoglobin preparation L was used in place of hemoglobin preparation A, and the same evaluation as in Example 1 was carried out.

(Example 15)
The pH of the hemoglobin preparation used in Example 1 was adjusted to 8.5 to obtain hemoglobin preparation M. The same evaluation as in Example 1 was carried out except that hemoglobin preparation M was used in place of hemoglobin preparation A.

The chromatograms obtained in Examples 13, 14, and 15 are shown in Figure 6, and the carryover values obtained from formula B are shown in Table 5. The results in Figure 6 show that there is no difference in the chromatograms when the pH of the hemoglobin preparations is in the range of pH 5.5 to 8.5. As shown in Table 5, the carryover value was less than 1% for all hemoglobin preparations, and there was no contamination of the column.

Claims (7)

A hemoglobin preparation used in the quantitative analysis of hemoglobins by liquid chromatography, the preparation comprising at least one type of surfactant having a hydrophilic group containing a sugar alcohol or a sugar molecule and having a hydrophilic group and a hydrophobic group bonded via an ester bond;
A hemoglobin preparation, characterized in that the content of the surfactant in the measurement reagent is 0.01 to 1.00 wt % . The hemoglobin preparation according to claim 1, characterized in that the hydrophilic group constituting the surfactant is sucrose. The hemoglobin preparation according to claim 1 or 2, wherein the hydrophobic group constituting the surfactant is a fatty acid having 7 to 22 carbon atoms. 4. The hemoglobin preparation according to claim 1 , wherein the surfactant has a pH of 5.5 to 9.0. 5. The hemoglobin preparation according to claim 1, further comprising at least one preservative. 6. The hemoglobin preparation according to claim 5, characterized in that the preservative comprises at least one selected from the group consisting of ethylenediaminetetraacetic acid, glycoletherdiaminetetraacetic acid, ethylenediamine-N,N'-disuccinic acid, the sodium salt of ethylenediaminetetraacetic acid, the potassium salt of ethylenediaminetetraacetic acid, the sodium salt of ethylenediamine-N,N'-disuccinic acid, and sodium azide. A method for measuring hemoglobin, comprising the steps of: obtaining a measurement sample by mixing the hemoglobin preparation solution according to claim 1 with a sample containing hemoglobin; and measuring the measurement sample by liquid chromatography.