JPH0115024B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for separating and quantifying hemoglobin in blood, particularly catabolic hemoglobin, for purposes such as diagnosis. Adult hemoglobin (Hb)
) is a protein with a molecular weight of approximately 68,000, and exists in the form of four hemes bound to one globin protein. However, when normal human Hb is electrophoresed under alkaline pH (PH8.6) conditions, in addition to Hb, which accounts for 90 to 95%, namely HbAo, there are also HbF and HbAlc, which have slightly slower mobility than HbAo. Later, HbAla, HbAlb, etc. were separated, and their existence was recognized. Furthermore, the presence of HbAld, HbAle, etc. has also been confirmed from the results of more precise electrophoresis tests. These abnormal hemoglobins have a different primary protein structure compared to normal hemoglobin (HbAo), or have sugars attached to HbAo. Separation and quantification of these catabolic hemoglobins is known to be important as a pathological indicator for various diseases.For example, quantification of HbF is essential for the diagnosis of hereditary hyperHbF syndrome.
Also, checking the increase or decrease in HbAlc is essential for diabetes treatment. Also, like HbF, the component called HbA ₂ is hereditary, and abnormal hemoglobin called acetylated hemoglobin (Ac, Hb) is produced by taking drugs with acetyl groups, such as aspirin. It has become clear that the separation and quantification of these catabolic hemoglobins is becoming increasingly important from a medical standpoint. However, up to now, no method has been proposed that can accurately separate and quantify catabolic hemoglobin as described above. In view of the above-mentioned current situation, the present invention was made with the aim of providing a method that can accurately separate and quantify catabolic hemoglobin in a short time. 5 to 90% by weight of a polymerizable monomer (A) having a polymerizable double bond and one or more carboxyl groups and a hydrophobic parameter of 2.3 or less, having two or more polymerizable double bonds in the molecule and hydrophobicity parameter is 2.3
10 to 95% by weight of the following crosslinking polymerizable monomer (B), a monomer (C) having one polymerizable double bond in the molecule and having a hydrophobicity parameter of 2.3 or less and different from the above polymerizable monomer (A). ) A method for separating catabolic hemoglobin, characterized in that it is carried out by liquid chromatography using as a stationary phase a hydrophilic ion exchanger copolymerized with a monomer mixture consisting of 0 to 85% by weight. In the present invention, a hydrophilic ion exchanger formed by copolymerizing a monomer mixture with a specific composition is used as a stationary phase for liquid chromatography. In order to use the copolymerized ion exchanger as a stationary phase, that is, a filler, for example, the copolymer particles obtained by copolymerization may be used as a filler after adjusting the particle size, etc. In the case of bulk polymerization, a pulverized copolymer may be used with uniform particle size, or the surface of an appropriate core material such as a glass bulb may be coated with the above copolymer by an appropriate method. Good too. However, the copolymer constituting the filler of the present invention is
5 to 90% by weight of a polymerizable monomer (A) having one polymerizable double bond and one or more carboxyl group in the molecule and having a hydrophobicity parameter of 2.3 or less;
The cross-linking polymerizable monomer (B) has 10 or more polymerizable double bonds and has a hydrophobicity parameter of 2.3 or less.
95% by weight, 0 to 85% by weight of a monomer (C) that has one polymerizable double bond in the molecule and has a hydrophobic parameter of 2.3 or less and is different from the above polymerizable monomer (A).
It is a copolymerized product of a monomer mixture consisting of The suspension polymerization method is preferable in that it can be used as it is as a filler. Also, during suspension polymerization,
If a small amount of an organic solvent that dissolves the monomer but does not dissolve the polymer is added, the resulting copolymer particles become porous, and a filler with an increased contact area with the mobile phase can be obtained. Here, the hydrophobic parameter is the logarithm of the solubility ratio of a certain compound in an octyl alcohol-water system, and is a value specific to that compound. Although this parameter can be determined by experiment, the contribution of each fragment in the molecule (hydrophobic fragment constant) can be determined by calculation, and the hydrophobic parameter value of the compound is calculated as the sum of these. can. For information on how to calculate hydrophobicity parameter values, see
“The Hydrophobic” by R.F. Rekker
Fragmental Constant” (Elsevier Scientifie
Publishing Co., 1977), Chapter 3, and the parameter values in the present invention are based on this calculation method. In other words, in this calculation method, the hydrophobic fragment constant (f value) is calculated for each fragment constituting the compound, such as methyl group, carboxyl group, etc.
is determined, and for a particular compound, the hydrophobicity parameter of the compound can be determined by summing the above constants for each fragment of the compound. For example, according to Table 2 on page 48 of Chapter 3, the f value of each fragment is as shown in the following table.

[Table] And, for example, acrylic acid (CH ₂ = CH-
Regarding the hydrophobic parameters of COOH), CH ₂ , CH,
Since one COOH exists, using the constants in Table 1, the hydrophobicity parameter can be calculated as follows: 0.528 x 1 + 0.260 x 1 + (-0.987) x 1 ≒ -0.20. Next, the polymerizable monomers (A) suitably used in the present invention include acrylic acid (hydrophobicity parameter = -0.20, below the parameter values are shown in parentheses), methacrylic acid (0.39), crotonic acid (0.23),
Examples include maleic acid (-1.46). Moreover, the crosslinking polymerizable monomer (B) used in the present invention
Examples include tetramethylolmethane triacrylate (-0.73), tetramethylolmethane trimethacrylate (0.59), tetramethylolmethane diacrylate (-1.70), tetramethylolmethane dimethacrylate (0.82), and tetramethylolmethanetetraacrylate (0.24). , tetramethylolmethanetetramethacrylate (0.20),
Trimethylolethane triacrylate (0.88),
Examples include trimethylolethane trimethacrylate (2.20), dipentaerythritol hexaacrylate (-0.09), and others such as polyethylene glycol diacrylate (or methacrylate) and polypropylene glycol diacrylate such as tetraethylene glycol dimethacrylate (-0.30). Alkylene glycol diacrylates (or methacrylates) such as (or methacrylates) can also be used. In addition, the monomer (C) different from the monomer (A) can be used in the present invention up to a quantitative range of 85% by weight if necessary.
as NN-dimethylmethacrylamide (-
0.88), 2-hydroxyethyl methacrylate (-0.41), glycidyl methacrylate (-
0.48) and the like are preferably used, and NN-dimethylacrylamide, 2-hydroxyethyl acrylate, glycidyl acrylate, etc. can also be used. In the present invention, as mentioned above, the polymerizable monomer
A monomer mixture consisting of (A) 5 to 90% by weight and a crosslinking polymerizable monomer (B) 10 to 95% by weight, or a monomer mixture in which the above monomer (C) is added up to 85% by weight as necessary, is copolymerized. However, the resulting copolymer itself becomes an ion exchanger exhibiting an ion exchange action due to the presence of the polymerizable monomer (A) having a carboxyl group in the molecule. In the monomer mixture, if the monofunctional monomer (A) having a carboxyl group is too small, the ion exchange ability will decrease, so the monomer (A) is required to be at least 5% by weight. If the amount of the cross-linked polymerizable monomer (B) having a polymerizable double bond is too small, the density of cross-linked bonds in the copolymer will decrease, making it difficult to withstand high pressure, especially when used as a packing material for high-performance liquid chromatography. Since problems arise in terms of mechanical strength, the monomer (B) is required to be present in an amount of 10% by weight or more. In addition, the monomer (C) having a polymerizable double bond other than the polymerizable monomer (A) may be mixed within a range in which the usage ratio of the polymerizable monomer (A) and the crosslinking polymerizable monomer (B) does not become less than the required amount. It can be included in the monomer. All monomers contained in the monomer mixture produced by copolymerization of the hydrophilic ion exchanger used in the present invention have a hydrophobic parameter of 2.3 or less; This invention is based on the knowledge of the present invention that a packing material made of an ion exchanger copolymerized with Since divinylbenzene, which has been commonly used as a divinylbenzene, has a high hydrophobicity parameter of 3.8, the effects of the present invention cannot be achieved even if an ion exchanger containing divinylbenzene as a copolymerization component is used as a filler. In addition, the hydrophobicity parameters of the polymerizable monomer (A), the cross-linking polymerizable monomer (B), and the monomer (C) added as necessary are 0 or less or on the negative side when analyzing the substance to be analyzed in the present invention. This is preferable in terms of preventing adsorption to the filler. The above-mentioned hydrophilic ion exchanger is packed into a column by a conventional method and used for the separation of the present invention by liquid chromatography, and exhibits analytical behavior based on an ion exchange mechanism. In the separation according to the present invention, the column filled with the above-mentioned hydrophilic ion exchanger is connected to a liquid chromatograph, preferably a high-performance liquid chromatograph. This can be carried out by injecting into the chromatograph and flowing the eluent. The optimal elution conditions should be determined depending on the type of catabolic hemoglobin to be separated and the type of column packing material used, but generally an aqueous solution with a pH of 5.0 to 10.0 and an ionic strength of 0.01 or higher is used as the eluent. It is preferable that the pH is controlled during the separation operation.
Various types of catabolic hemoglobin can be successfully separated and quantified by changing the ionic strength. Further, detection is preferably performed using visible light with a wavelength of 415 nm. In addition, the elution order of catabolic hemoglobin generally does not change much; HbAla, HbAlb, HbF, HbAlc,
By changing the elution conditions during the elution procedure, a _specific catabolic hemoglobin can be made to appear as a well-resolved chromatographic peak, while other catabolic hemoglobins and normal hemoglobin can be separated as clusters that are not well separated. It is also possible to effectively quantify the target catabolic hemoglobin in a short time by making it appear as a peak. The present invention is a method for separating catabolic hemoglobin as described above, in which a hydrophilic ion exchanger copolymerized with a monomer mixture having a specific composition is used as a stationary phase, and the stationary phase has both separation performance and strength. Because of its excellent properties, it can be used as a packing material for high performance liquid chromatography, and the present invention has the excellent effect of allowing catabolic hemoglobin to be separated and quantified with high accuracy and in a short time. Examples of the present invention will be described below. Example 1 400 ml of 4% by weight aqueous polyvinyl alcohol solution, 40 g of tetraethylene glycol dimethacrylate, 10 g of tetramethylolmethane triacrylate, and 50 g of methacrylic acid were placed in two separable flasks equipped with a cooler, stirrer, thermometer, and dropping funnel. , a mixed solution consisting of 40 g of toluene and 1.5 g of benzoyl peroxide was supplied. next
Raise the temperature to 80℃ while stirring at a stirring speed of 400 rpm.
The reaction was carried out for 10 hours and then cooled. After cooling, the polymerization product was separated from the mother liquor and washed with hot water and acetone to obtain a spherical polymer having a particle size of 5 to 20 microns. Particles of 8 to 12 microns, obtained by removing fine particles and coarse particles, were dispersed in 80 ml of ion-exchanged water and placed in a stainless steel column (diameter 7.9 mm, length
30 cm) was filled with the above ion-exchanged water by force-feeding it at a rate of 1.6 ml/min using a high-pressure constant flow pump. The obtained packed column was connected to a high performance liquid chromatograph (trade name: Shimadzu DuPont High Performance Liquid Chromatograph Model 830), and the following analytical operations were performed. Analysis was performed by injecting 10 μl of a hemoglobin solution prepared by mixing 50 μl of blood from a normal person and a diabetic patient with 1 cc of ion-exchanged water and hemolyzing the sample using a microsyringe. As an eluent
A mixture of 30% 0.1N disodium phosphate aqueous solution and 70% 0.1N monopotassium phosphate aqueous solution was used as solution A (PH6.5), and NaCl was dissolved in solution A to a concentration of 3% as solution B. An aqueous solution was used. Elution was started with 100% solution A, and solution B was increased at a rate of 5%/1 minute. The obtained chromatogram is shown in FIG. Note that a 415 nm visible light detector was used for detection. Next, by ion exchange column chromatography using a liquid chromatography packing material (Biorex-70, manufactured by BioRad (USA)), which is well known in this field, Ala, Alb, Alc, Ao,
Each component was fractionated over about 2 hours or more, and each fractionated component was subjected to high performance liquid chromatography analysis according to the present invention under the above conditions, and peaks 1 to 7 in the chromatogram shown in FIG. 1 were identified. However,
Ala, Alb, Alc, Ao and A ₂ are respectively 2,
It was found that peaks 3, 5, 6 and 7 coincided with each other. Moreover, it is thought that peak 1 is a non-hemoglobin component. Furthermore, it was found that peak 4 was HbF as a result of identification using fetal blood as a sample. By the way, HbAlc determined by the baseline method
The amount was 3.2% in normal human blood and 5.9% in diabetic patient blood. Example 2 Using the same column, equipment, and elution conditions as in Example 1, an aqueous solution prepared by mixing neonatal blood with 20 times the volume of ion-exchanged water to cause hemolysis was analyzed as a sample, and the chromatogram shown in Figure 2 was obtained. Obtained. 4 in the chromatogram is HbF, which is separated very sharply. Example 3 Example 1 except that 50 g of trimethylolethane trimethacrylate and 15 g of crotonic acid were used as the monomers, and 40 g of toluene was used as the organic solvent.
A packing material and a packed column were prepared in the same manner as in Example 1, and the same sample as in Example 1 was used.
The chromatograph shown in FIG. 3 was obtained by operating under the elution conditions of flowing a mixture of 90% liquid for 5 minutes and then switching to liquid B. A is a chromatogram of normal human blood, B is a chromatogram of diabetic patient blood, and 5 is a chromatogram of normal human blood.
HbAlc, 6 is the peak of HbAo. In this way, according to this example, it was possible to separate HbAlc in a short time.

[Brief explanation of drawings]

FIG. 1A is a chromatogram of normal human blood, FIG. 1B is a chromatogram of diabetic patient blood in Example 1, FIG. 2 is a chromatogram obtained in Example 2, and FIG. 3A is a chromatogram of normal human blood. Figure 3B is Example 3 of blood of a diabetic patient.
The chromatogram is shown below. 2...HbAla, 3...HbAlb, 4...HbF,
5...HbAlc, 6...HbAo, 7... _HbA2 peaks.

Claims (1)

[Claims] 1 Contains one polymerizable double bond and one or more carboxyl groups in the molecule, and has a hydrophobic parameter
2.3 or less polymerizable monomer (A) 5 to 90% by weight, cross-linked polymerizable monomer (B) that has two or more polymerizable double bonds in the molecule and has a hydrophobicity parameter of 2.3 or less
10 to 95% by weight, and 0 to 85% by weight of a monomer (C) having one polymerizable double bond in the molecule and having a hydrophobic parameter of 2.3 or less and different from the above polymerizable monomer (A). A method for separating catabolic hemoglobin, characterized in that it is carried out by liquid chromatography using a hydrophilic ion exchanger obtained by copolymerizing a mixture as a stationary phase.