JPS58760A - Separation of catabolic hemoglobin - Google Patents

Abstract

PURPOSE:To separate and determine catabolic hemoglobin in high accuracy, by using a copolymer constituting of a specific rate of three kinds monomers, each having the hydrophobic parameter lower than the specific value, of a monomer having COOH radical in a molecule less than a specific value, a cross-linking polymeric monomer and a monomer not containing COOH as packings. CONSTITUTION:The fine particles of a copolymer copolymerized three kinds of monomers, each having <=2.3 hydrophobic parameter are used for packings of liquid chromatography. That is, copolymer particles constituted by 5-95wt% monomer A having >=one COOH radical and one polymeric double bond, 10- 95wt% cross-linking monomer B having >=2 polymeric double bond and 0- 85wt% monomer C which is other than the group A and having one polymeric double bond are obtained by crushing after said copolymer is obtained by a suspension polymerization method or bulk polymerization method. Catabolic hemoglobin in blood serum of a diabetic etc. is separated and is determined in a short time in high accuracy by using this particle as packings.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for separating and quantifying hemoglobin in blood, particularly catabolic hemoglobin, for purposes such as diagnosis. Adult hemoglobin (hereinafter abbreviated as III) is a protein with a molecular weight of s o o o, and exists in a round shape with four hemoglobins bound to one globin protein. However, under conditions of alkaline pH (PIIILli)
When Hb is subjected to electricity &
a%HbA1 b, etc. have been separated and their existence has been recognized. Furthermore, from the results of more precise electrical turbulence tests, HbAld.

The presence of HbAlc groups has also been demonstrated. By comparing these abnormal hemoglobins with normal hemoglobin (HbAo), we can determine whether the primary structure of the protein is blocking the protein or whether it is related to FiHhAo.
It is a compound containing K111. Separation and quantification of these catabolic hemoglobins is known to be important as an indicator for various diseases.For example, quantification of HbF is essential for the diagnosis of hereditary hyperHbF syndrome, and HbAI
Checking the increase or decrease of C is essential for the treatment of stationary disease. or,
It is hereditary like HbF, which is called Hb Am, and is also inherited by acetylated hemoglobin (Ac, l1
Abnormal hemoglobins called b) have been found to be produced by taking drugs with acetyl groups, such as aspirin, and the separation and quantification of these catabolic hemoglobins is becoming increasingly important from a medical perspective. The degree of this is increasing.

However, to date, there have been no methods available for separating and quantifying the catabolic substances mentioned above in a short time and with high precision.
i It wasn't suggested.

In view of the current state of affairs as described above, the present invention has been made with the object of providing a method capable of separating and quantifying catabolic globin with high precision and in a short time.
Its gist is that it has one polymerizable double bond and one or more carboxyl groups in the molecule, and has a hydrophobic beak of 23
The following polymerizable monomers: 5 to 90 parts by weight, a cross-linked tumorigenic monomer having two or more polymerizable double bonds in the molecule and a hydrophobicity parameter of 23 or less t-@lO to 95 parts by weight, molecule It has one polymerizable double bond in it and is hydrophobic (with a ramester of z3 or less and the above polymerizability 7) - a monomer that can be dissolved 9-c) from O to Hatake 5. A method for separating catabolic hemoglobin, characterized in that it is carried out by wave body chromatography using a hydrophilic ion exchanger as a stationary phase, which is a copolymerized monomer mixture.

In the present invention, a hydrophilic ion exchanger formed by copolymerizing a monoe mixture with a specific composition is used as a stationary phase for liquid chromatography. In order to use the above-mentioned copolymerized ion exchanger as a stationary phase, that is, a filler, for example, the copolymer particles obtained by copolymerization may be made to have a uniform particle size, and then used as a filler. In the case of bulk polymerization, pulverized copolymers with the same particle size may be used, or the surface of an appropriate core material such as a plastic ball may be coated with the above copolymer by an appropriate method. You may also use

However, the copolymer constituting the filler of the present invention has one polymerizable double bond and one or more carboxyl groups in the molecule, and has a hydrophobicity parameter of 23 or less. It has two or more polymerizable 211 bonds in ~90 weight molecules and is hydrophobic! The crosslinking polymerizable monomer (2) of 3 or less has a weight of 10 to 9 seconds, has one polymerizable double bond in the molecule, and has a hydrophobic parameter of z3 or less, which is different from the above polymerizable monomer (4). A monomer mixture consisting of 0 to 85 parts by weight of monomers is copolymerized, and conventional polymerization methods such as suspension polymerization, emulsion polymerization, solution polymerization, and bulk polymerization are used for this copolymerization. Although this is possible, suspension polymerization is preferred because the polymer particles can be used as they are as a filler. Additionally, if a small amount of an organic solvent is added during suspension polymerization, which dissolves the polymer but does not dissolve the polymer, the resulting copolymer particles will become porous, increasing the contact area with the mobile phase. A filler containing a mixture of

S- Here, it is the logarithm of the solubility ratio in the octyl alcohol-water system of a compound that differs from the hydrophobic q-rameter, and is a value specific to that compound. The parameters can also be found through experiments, but the contribution of each 72 segment in the molecule (1
1m aqueous 7 fragmentation constant) can be obtained by calculation, and as a summation of these, the hydrophobicity/
You can calculate the haze meter value.

For the calculation method of hydrophobicity/4F meter value, please refer to RmF4
Written by ekk@r rThe Hydrophobl
c FragmentalConstant J(E
lsevier 5cieatifle Publicis
hingCo, 11177), and the puff meter values in the present invention are based on this calculation method.

Next, the polymerizable heptamer (
2) @CIIL Mizunohira Meter to Toke Akuri ---
13. The following values in parentheses indicate meter values), methacrylic acid (-19), crotonic acid (-19), maleic acid
Examples include I(-翫7).

6- Also, as the crosslinking polymerizable monomer mixture used in the present invention, Tetramethylolmethane triacrylate (-α7)
3), Tetramethylolmethanetritsutaacrylate ((
LISI), tetramethylolmethane diacrylate (
-170), Tetramethylolmethane dimethacrylate (α82), Tetramethylolmethanetetraacrylate (α24), Tetramethylolmethanetetramethacrylate (α20), ) Limethylolethane triacrylate (α88), ) Limethylolethane trimethacrylate (240) , dibe>p erythritol hexaacrylate (-(LISI)), and other polyethylene glycol diacrylate (or methacrylate) such as tetraethylene glycol dimethacrylate (-(L30)) and polyethylene glycol diacrylate (or methacrylate) 411 Alkylene glycol diacrylate (or methacrylate) can also be used.

Moreover, the monomer capacitors are different from each other.
In 9i, it is possible to use up to a quantitative range of 85 kg as needed, but as one of the seven parts, tf
NN-dimethylmethacrylamide (-〇〇8), 2-
Hydroxyethyl methacrylate (-α41), glycidyl methacrylate (-α48), etc. are preferably used, and NN-dimethylacrylamide, 2-hydroxyethyl acrylate, glycidyl acrylate, etc. can also be used.

In the present invention, as mentioned above, polymerizable monomer particles 5 to 9
A monomer mixture consisting of 0.1 volume of a cross-linking polymerizable monomer @ 10 to 95 volume of a monomer is then copolymerized by adding one of the monomers up to 85 volume of a cross-linking polymerizable monomer @ 10 to 95 volume of a monomer mixture. The resulting copolymer itself becomes an ion exchanger exhibiting an ion exchange action because the polymerizable monomer (2) having a carboxyl group is present in the molecule. And in the monomer mixture, it has a carboxyl group! If the amount of functional monomer (2) is too small, the ion exchange ability will be reduced, so a monomer weight of 5 weight or more is required. If the amount of the monomer (2) is too small, the density of crosslinks in the copolymer will decrease, causing problems in mechanical strength to withstand high pressure, especially when used as a packing material for fast wave chromatography. Since the monomer @H
More than IO weight is required. In addition, monomers having a polymerizable double bond other than the above-mentioned polymerizable monomers, polymerizable monomer (2), and crosslinking polymerizable monomer (2) may be included in the mixed monomer to the extent that the proportion used does not become less than the required amount. I can do it.

All monomers contained in the monomer mixture produced by copolymerizing the hydrophilic ion exchanger used in the present invention have a hydrophobicity/hetometer of 23 or less; This is based on the knowledge of the present invention that a packing material made of an ion exchanger copolymerized with a 7-mer with a q-meter of zs or less exhibits excellent separation ability in the analysis of catabolic hemoglobin. Divinylbenzene, which is commonly used as a crosslinking polymerizable hexamer to produce ion exchangers, has a high hydrophobicity parameter of 18, so even if an ion exchanger containing this as a copolymerization component is used as a filler. It is impossible to achieve the effects of an uninvented invention. In addition, the polymerizable monomer particles, the cross-linked polymerizable hexamer (6), and the additive added as necessary csct This hydrophilic ion exchanger is preferred in terms of preventing adsorption of substances to the packing material.The above hydrophilic ion exchanger is packed into a column by a conventional method and used for liquid chromatography. Demonstrates analytical behavior based on mechanism.

In the separation according to the present invention, the column packed with the above-mentioned hydrophilic ion exchanger is connected to a housing chromatograph, preferably a highly insulating body, and then separated and quantified using a liquid chromatograph. This can be performed by injecting blood as a sample into the chromatograph 1) 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 it is also possible to obtain favorable elution conditions for various catabolic hemoglobins by changing KPM and ionic strength during the separation operation. can be separated and quantified. Also, detection is at wavelength 4.
It is preferable to use visible light of 15 nm.

Also, the elution order of catabolic hemoglobin is generally almost l! Without consideration, BbAIa, HbAIb, Hb F, Hb
The order is A 1 c , IHb A o , and Hb As. However, if the elution conditions are changed during the elution operation, a specific catabolic hemoglobin will appear as a good chromatographic peak, and other embodied umbilical globins and It is also possible to effectively quantify catabolic hemoglobin in a short period of time by causing it to appear as a stationary peak with poor separation of normal hemoglobin.

The present invention provides a method for separating catabolic hemoglobin as described above, and in particular, a hydrophilic ion exchanger formed by copolymerizing a heptamer mixture with 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 chromadog 27. According to the present invention, catabolic hemoglobin can be separated and quantified with high precision and in a short time, which is an excellent effect. .

The present invention will be explained below with reference to examples.

Example 1 In a 2 t separable flask equipped with a cooler, stirrer, thermometer and dropping funnel, 400 ml of 4% by weight aqueous polyvinyl alcohol solution, 4°t of tetraethylene glycol dimethacrylate, 16 t of tetraethylene glycol dimethacrylate, 16 t of methacrylic acid were added. acid 50F,) luene 40?
and benzoyl peroxide LIF were supplied. Next, the temperature was raised to 1 lOtl while stirring at a stirring speed of K 4 G Or p m, and the same reaction was carried out at 10 o'clock, followed by cooling. 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.

Of these, fine particles and coarse particles are removed to obtain 9S~1
Particles of 2 microns were dispersed in 80-degree ion-exchanged water, and a stainless steel column (diameter 7.9 mm, length 30 m) K
The ion-exchanged water was pumped at a rate of 16-7 minutes using a high-pressure constant flow pump to fill the container. The resulting 9-packed column was connected to a high-speed wave chromatograph (trade name: Shimadzu DuPont High-Speed Wave Chromatograph 8308), and the following analytical operations were performed.

50 samples of blood each from a normal person and a diabetic patient
Analysis was performed by injecting a hemoglobin solution 1oPt, which was prepared by mixing pL with ion-exchanged water of lcc and causing hemolysis, using a microsyringe. As the eluent, 30 liters of an aqueous solution of disodium alN phosphate and 1 liter of an aqueous solution of alN phosphoric acid 1 [7
The mixed solution with 0% is called A solution (PRa5), and the B wave is called 13
- An aqueous solution in which KNaCl was dissolved to a total of 3 liters was used.

Elution was started with an A wave of 100 drops, and the B wave was increased at a rate of 5%/1 minute. The obtained chromatogum is the first
As shown in the figure. The detection was carried out using a 415 am visible party detector.

Next, 1 well-known liquid chromatography packing material in this field (Biorex-70, Bio-Rad Co., Ltd.
Alm and Alb were obtained by ion exchange column chromatography using a commercially available product (manufactured in USA).

A I C * A O Each component was fractionated over a time period of about z or more, and each fractionated component was subjected to high performance liquid chromatography analysis according to the present invention under the above conditions, and the peaks 1 to 1 of the chromatograms in FIG. 7 was identified, A1
a%A1bx ^1c, A (1 and mu, tf respectively,
It was found that the peaks corresponded to No. 2, 3, 5, 6, and 7. It is also believed that the beak lFi is a non-hemoglobin component.

In addition, it was found that the peak was 4tiHbF as a result of the identification of most of Yuma's blood. By the way, the amount of HbAIC determined by the base twin method 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 amount of ion-exchanged water to cause hemolysis was analyzed as a sample, and the chromatogram shown in Figure 2 was obtained. Obtained. There are 4 HbF in the 7 black tons, and they are separated very clearly.

Example 3 A packing material and a packed column were prepared under pressure in the same manner as in Example 1, except that 50 f of trinotyrolethane trinotacrylate and 15 t of crotonic acid were used as the sulfuric acid, and 40 f of toluene was used as the organic solvent. Using the same sample as in Example 10 and operating under the elution conditions of flowing a mixture of 10% A solution and 90% B solution for 5 minutes and then switching to B*, chromatograph 7 shown in FIG. 3 was obtained. . 5 is a chromatogram of normal human blood, and 1 is a chromatogram of diabetic patient blood, with the peaks of 5-digit HbAIc and 6-digit HbA·. Like this*
According to the example, it was possible to separate Hb and Alc in a short time.

[Brief explanation of drawings]

Figure 1 Talomadogram of company A normal human blood, Figure 1 B#'lt talomadogram of diabetic patient blood 0% Example IK, Figure 2 Fi chromatogram obtained in Example 2, Figure 3 AFi normal human blood. FIG. 3 shows a chromatogram in Example 3 of BFi sugar W disease patient blood. 2.=HbA lm, 3=HbA1b,
4.-HbF. 5-HbA1c, 6-HbAo,
Each peak of 7-HbAz. Patent applicant Sekisui Chemical Co., Ltd. Representative Mototoshi Fujinuma 3I11

Claims (1)

[Claims] 1 Polymerizable monomer having one polymerizable type 2 packing and one or more carboxyl groups in the molecule and having a hydrophobicity/hydrophobicity of 13 or less (2) 2 in 5 to 90 weight molecules 2 or more polymerizable type 2 bonds and a hydrophobic polymerization parameter of 2 or more.
Cross-linked polymerizable heptads of 3 or less @ 10-9s weight attack, have one polymerizable type 2 packing in the molecule and have a hydrophobic parameter of 23 or less, and the above-mentioned polymerizable monomers are captured and activated. A method for separating catabolic hemoglobin, characterized in that it is carried out by liquid chromatography using as a stationary phase a woody ion exchanger copolymerized with a monomer mixture consisting of 8I.