JPH0912629A - Resin for lipoprotein separation - Google Patents

Abstract

(57) [Summary] [Purpose] Lipoproteins, especially high-density lipoproteins (HD
L), low-density lipoprotein (LDL), ultra-low-density lipoprotein (VLDL) and denatured lipoprotein are provided in a short time and with high precision. [Structure] A lipoprotein separating resin comprising an anion exchanger in which an anion exchange group is introduced into a water-insoluble hydrophilic matrix, wherein the matrix has a weight fraction of units derived from a vinyl monomer of 0 to 80%, the weight fraction of the units derived from the crosslinkable vinyl monomer is 20 to 100%, and at least one of the monomers is a crosslinked copolymer having a hydrophilic group, The anion exchange capacity is 0.2 to 1.2 milliequivalents per gram of dry resin weight.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a resin for separating lipoproteins. More specifically, the present invention relates to a resin useful for separating high density lipoprotein (HDL), low density lipoprotein (LDL), very low density lipoprotein (VLDL) and denatured lipoprotein with high accuracy.

[0002]

BACKGROUND OF THE INVENTION Cholesterol, phospholipids and neutral fats (triglycerides) are contained in plasma and serum,
Of these lipids, the quantification of cholesterol is arteriosclerosis,
It is used as a screening test for liver disease, diabetes, or temporary lipid metabolism disorder, and the quantification of triglyceride has become an indispensable test for distinguishing abnormal lipid metabolism along with cholesterol. The ultracentrifugation method is widely used for the fractionation of lipoproteins, but this method not only takes a long time for treatment, but also in animal species such as rats and mice in which the properties of lipoproteins are different from humans. , Its accurate fractionation is difficult. Further, in the method of separating by high performance liquid chromatography, for example, Journal of Chr
As described in omatography, 570 , 382-389 (1991), a gel filtration method in which fractionation is performed by molecular size has been generally used until now. However, even in that case, there has been a problem that mixing occurs between the lipoprotein fractions and complete separation cannot be achieved.

[0003]

SUMMARY OF THE INVENTION The present invention aims to provide a resin for separating lipoproteins which does not have the above-mentioned problems, and particularly lipoproteins and high-density lipoproteins (H
(DL), low-density lipoprotein (LDL), ultra-low-density lipoprotein (VLDL), and denatured lipoprotein.

[0004]

MEANS FOR SOLVING THE PROBLEMS The present inventors have obtained a porous particle obtained by impregnating seed particles composed of organic polymer particles with a vinyl monomer having a hydrophilic group by a seed polymerization method and polymerizing the same. Porous uniform-sized polymer particles having a structure and having a hydrophilic surface on the surface of the particles including the surface of the pores, and particles having an ion exchange group can separate lipoproteins with high accuracy. I found things and achieved the present invention. That is, the gist of the present invention resides in a resin for separating lipoproteins, which comprises an anion exchanger in which an anion exchange group is introduced into a water-insoluble and hydrophilic matrix. In such a water-insoluble and hydrophilic matrix, the weight fraction of units derived from a monovinyl monomer is 0 to 80%, the weight fraction of units derived from a crosslinkable polyvinyl monomer is 20 to 100%, and A porous cross-linked polymer composed of at least one kind of monomer having a hydrophilic group, and having an average pore radius of 10
~ 2000 angstrom, pore volume 0.2-1.8
It has characteristics of ml / g and pore surface area of 1 to 1000 m ² / g. The anion exchange capacity of the anion exchanger is 0.2 to 1.2 milliequivalents per 1 g of dry resin weight.

Hereinafter, the present invention will be described in detail. The lipoprotein separating resin of the present invention uses seed particles having a narrow particle distribution (particle size monodisperse), which is further impregnated with a vinyl monomer having a hydrophilic group to perform polymerization, a so-called seed polymerization method. It can be easily obtained by utilizing and manufacturing. Further, the presence of the porosifying agent during the seed polymerization makes it possible to produce a separating resin having further excellent performance. In the seed polymerization of the present invention, the organic polymer particles used as seed particles are emulsion polymerization, soap-free emulsion polymerization, dispersion polymerization,
It can be produced by a generally well-known ball-forming polymerization such as suspension polymerization. Among them, polymer particles obtained by emulsion polymerization, soap-free emulsion polymerization, and dispersion polymerization have a narrow particle size distribution and are preferable as seed particles as compared with those produced by suspension polymerization.

The polymer particles as the seed particles are preferably a polymer composed of an aromatic monovinyl monomer and / or an aliphatic monovinyl monomer. These may be either homopolymers or copolymers of two or more monomers, and may be 1% by weight.
It may be a copolymer with the following crosslinkable polyvinyl monomer. Particles made of polystyrene or polymethacrylic acid ester are typically preferred. The size of the particles is in the range of 0.1 to 1000 μm and can be arbitrarily selected according to the purpose.

In the present invention, the seed particles composed of the organic polymer particles produced as described above are added to the monovinyl monomer 0-8.
It is impregnated with a crosslinkable polyvinyl monomer containing 0% by weight.
At least one of the monovinyl monomer and the crosslinkable polyvinyl monomer has a hydrophilic group, for example, a hydroxyl group. The crosslinkable polyvinyl monomer is preferably an aromatic polyvinyl monomer or an aliphatic polyvinyl monomer,
The aromatic polyvinyl monomer is preferably divinylbenzene or bisbiphenylalkane, and the aliphatic polyvinyl monomer is preferably poly (meth) acrylate of a polyhydric alcohol or alkylene poly (meth) acrylamide. Specifically, for example, ethylene glycol di (meth) acrylate, polyethylene glycol di (meth) acrylate,
Glycerin di (meth) acrylate, allyl (meth) acrylate, trimethylolpropane tri (meth) acrylate, tetrahydroxybutane di (meth) acrylate, pentaerythritol di (meth) acrylate,
Methylenebisacrylamide, piperazine diacrylamide, diallyl tartaric acid diamide and the like can be mentioned, and these can be used alone or as a mixture, and glycerin di (meth) acrylate is particularly preferable.

Monovinyl monomers are used as vinyl monomers other than the crosslinkable polyvinyl monomers. Examples of the monovinyl monomer include polyhydric alcohol esters of (meth) acrylic acid having one or more hydroxyl groups such as glycerin mono (meth) acrylate and 2-hydroxyethyl (meth) acrylate, and N such as N-isopropylacrylamide.
-Substituted (meth) acrylamide, acrylamide, methacrylamide, glycidyl (meth) acrylate, a mixture thereof, or the like can be given. In particular, a hydrophilic vinyl monomer that is more easily distributed in an aqueous medium than an organic solvent as a porosifying agent used during seed polymerization is preferable,
Glycerin mono (meth) acrylate, glycidyl (meth) acrylate or mixtures thereof are preferred.

The types and amounts of these monovinyl monomers and polyvinyl monomers are appropriately determined in consideration of the size of seed particles used and the size of the target particles. The amount of the crosslinkable polyvinyl monomer in the vinyl monomer with which the seed particles are impregnated is 20 to 100% by weight, preferably 30 to 1
00% by weight. Further, in the present invention, in order to improve the characteristics of the porous crosslinked polymer particles, it is desirable to carry out seed polymerization in the presence of a porosifying agent. For that purpose, as a porosifying agent, that is, as a porosifying solvent for impregnating seed particles,
Aliphatic and aromatic hydrocarbons that are organic solvents that act as a phase separation agent during seed polymerization and promote porosity of particles,
Examples thereof include esters, ketones, ethers and alcohols. Examples of such a solvent include toluene,
Xylene, cyclohexane, octane, butyl acetate, dibutyl phthalate, methyl ethyl ketone, dibutyl ether, 1-hexanol, 2-octanol, decanol, lauryl alcohol, cyclohexanol and the like can be used, and these can be used alone or in combination. . In some cases, desired properties can be imparted to the crosslinked copolymer particles by selecting the type of the porosifying solvent, for example, aromatic hydrocarbons or alcohols.

The radical polymerization initiator at the time of seed polymerization is a peroxide type initiator such as benzoyl peroxide or butyl peroxyhexanoate, or an azo type initiator such as azobisisobutyronitrile or azobisisovaleronitrile. Is preferred.
These polymerization initiators are dissolved in a vinyl monomer or a porosifying solvent, and are impregnated into the seed particles at the same time as or before or after the impregnation of the vinyl monomer. When the seed particles are impregnated with the vinyl monomer, the porosifying solvent, the radical polymerization initiator, etc., they may be diluted with a solvent having a high affinity for the seed particles and then impregnated. Examples of such a solvent include water-miscible solvents such as alcohol and acetone, and halogenated hydrocarbons such as dichloroethane and methylene chloride. It is preferable that these solvents that promote impregnation are distilled off under reduced pressure before the temperature is raised to the polymerization temperature in order to start seed polymerization.

In the present invention, the vinyl monomer, the porosifying solvent and the polymerization initiator are thus impregnated, the seed particles are enlarged, and then suspended in an aqueous medium for polymerization. A dispersion stabilizer is preferably contained in the aqueous medium in order to prevent aggregation, deformation, and fusion during seed polymerization and increase the dispersion stability. As the dispersion stabilizer, known anionic and nonionic surfactants, polyvinylpyrrolidone, polyethyleneimine, vinyl alcohol / ethyl acetate copolymers and the like are preferable. Seed polymerization is initiated by raising the temperature to the polymerization temperature. The polymerization temperature is preferably 50 ° C to 80 ° C, though it depends on the kind of the polymerization initiator used. Further, the polymerization time of the seed polymerization is preferably around the half-life of the polymerization initiator or longer, and for example, 3 hours to 48 hours is preferable.

In the present invention, the seed polymerization can be carried out by a general method known per se. 0.1-1.5 produced by emulsion polymerization method or soap-free emulsion polymerization method
In the case where a polymer having a particle size of μm is used as seed particles, a method in which porous particles having a uniform particle size up to about 100 μm can be produced by first swelling with a swelling aid such as dimethyl phthalate and then performing seed polymerization (J.Ugelstad et.al.Makromo
leculare Chemie, 80, 737 (1979)) is preferably used.
In addition, a method (see, for example, Japanese Patent Laid-Open No. 64-26617) in which polymer particles having a particle size of 1 to 10 μm produced by dispersion polymerization are used as seed particles can be suitably used. Furthermore,
In the present invention, after the completion of seed polymerization, a good solvent for the organic polymer particles used as seed particles is used to wash the resulting crosslinked polymer particles to remove a part or all of the polymer derived from the seed particles. Also by doing so, a resin having a desired porosity can be obtained.

The preferred porous physical properties of the porous crosslinked polymer particles obtained in the present invention in a dry state measured by the BET method are 10 to 2000 angstroms, preferably 75 to 1500 angstroms, as an average pore radius. The pore volume is 0.2 to 1.8 ml / g, preferably 0.4 to 1.2 ml / g, and the pore surface area is 1
˜1000 m ² / g, preferably 5 to 500 m ² / g.

An anion exchange group is introduced into the porous polymer particles thus obtained to obtain a lipoprotein separating resin. Examples of the anion exchange group to be introduced include various amino groups including dimethylaminoethyl group and diethylaminoethyl group, various quaternary ammonium groups, and the like.
The method of introducing an anion exchange group into the porous polymer particles,
It is performed according to a known method. For example, when the vinyl monomer forming the matrix has an epoxy group, a method of ring-opening modification of the epoxy group with water, glycerin, ethylene glycol or the like, and introducing an anion exchange group into the generated hydroxyl group, or Examples thereof include a method of directly bonding an anion exchange group to an epoxy group. In addition, the amount of the anion exchange group to be introduced is 1 weight of dry resin.
0.1-1.5 milliequivalent per g, preferably 0.2-
It is 1.2 meq.

[0015]

EXAMPLES Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples unless it exceeds the gist thereof.

Example 1 An aqueous suspension of uniform polystyrene seed particles having a particle diameter of 1 μm (9.
5 × 10 ^-2 g / ml) 1.4 ml, swelling aid phthalic acid dibutyl ester 0.95 ml, benzoyl peroxide 0.085 g as a polymerization initiator, and sodium dodecyl sulfate 0.04 g in water 10 ml. It was mixed with the microemulsion dispersed therein and gently stirred at room temperature.
The swelling in the first step was performed until the added microemulsion droplets could not be seen in the observation with the optical microscope. 5.0 g of glycerin dimethacrylate, 5.0 g of glycerin monomethacrylate, 5.0 g of glycidyl methacrylate as monomers, and 30 ml of cyclohexanol as a porosifying solvent were used as suspension stabilizers of 4.80 g of polyvinyl alcohol (polymerization degree 500, The fine dispersion liquid suspended in 225 ml of water containing a saponification degree of 86.5-89 mol% was added to the suspension after the primary swelling described above, and gently stirred overnight at room temperature (secondary swelling). ).

The suspension after the secondary swelling was polymerized at 70 ° C. for 24 hours in the presence of a water-soluble polymerization inhibitor in an argon atmosphere. The suspension after polymerization was poured into 250 ml of water, and after the particles were settled, the supernatant was discarded to remove the suspension stabilizer. Further, the particles were redispersed in methanol, and after the particles were settled, the supernatant liquid was removed to wash the particles. This operation was performed once more with methanol and twice with tetrahydrofuran, then the particles were filtered and dried at room temperature. The introduction of ion-exchange groups leads to the particles 5.7 obtained above.
8 g were dispersed in 50 ml of dimethylformamide, and N, N-diethylethylenediamine 10 was added under an argon atmosphere.
ml was added, and the mixture was gently stirred at 70 ° C. for 6 hours. After the reaction was completed, the suspension was poured into water, and after the particles were settled, the supernatant was removed. Further, the particles were redispersed in methanol, and after the particles were settled, the supernatant liquid was removed to wash the particles. This operation was performed once with methanol and twice with acetone, and then the particles were filtered and dried to obtain a granular resin (separating agent). The result of elemental analysis of this separating agent is C = 54.22
%, H = 7.89%, N = 2.23%. Ion exchange capacity was calculated to be 0.8 based on the results of elemental analysis.
It was a meq / g-polymer.

Example 2 A granular resin (separating agent) was obtained in the same manner as in Example 1 except that toluene was used instead of cyclohexanol as the porosifying solvent. The result of elemental analysis is C = 54.27%,
H = 7.64% and N = 1.85%. When calculated from the results of elemental analysis, the ion exchange capacity is 0.7 meq /
It was a g-polymer.

Test Example 1 Using the granular resins obtained in Examples 1 and 2, lipoproteins were separated using ICR mouse serum as a sample. Test method According to the system shown in FIG. 1, an eluent is caused to flow through a separation column into which a sample has been injected to separate lipoproteins, an enzyme solution is added to the separated lipoproteins, the mixture is heated and reacted, and then detected by a detector.

Column: A granular resin was used by being packed in a stainless steel column having an inner diameter of 4.6 mm and a length of 150 mm by a wet method. Eluent: The following two buffers were used, eluent B was added to eluent A according to FIG. 2, and the mixture was run for 45 minutes. A: 20 mM sodium phosphate buffer + 1 mM ethylenediaminetetraacetic acid (pH 7.0) B: (eluent A) +0.5 M sodium chloride Enzyme reagent: Cholesterol C Test Wako (manufactured by Wako Pure Chemical Industries, Ltd.) Column temperature: 25 ° C. Detection: 505 nm Sample: ICR mouse serum 25 μl Results Chromatograms using the granular resins of Example 1 and Example 2 are shown in FIGS. 3 and 4, respectively.

[0021]

By using the resin of the present invention, lipoproteins such as cholesterol in plasma and serum can be separated with extremely high accuracy.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing a system of a lipoprotein separation test method in Test Example 1.

FIG. 2 is a diagram showing a gradient in an eluent in Test Example 1.

FIG. 3 is a chromatogram showing the results of separating lipoproteins with the resin of Example 1.

FIG. 4 is a chromatogram showing the results of separating lipoproteins with the resin of Example 2.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication C08F 220/32 MMP C08F 220/32 MMP 220/56 MNC 220/56 MNC G01N 30/48 G01N 30 / 48 P 30/88 30/88 J // B01D 15/08 B01D 15/08

Claims (5)

[Claims] 1. A resin for separating lipoproteins, which comprises an anion exchanger in which an anion exchange group is introduced into a water-insoluble hydrophilic matrix. 2. A water-insoluble, hydrophilic matrix having a weight fraction of units derived from a monovinyl monomer of 0-8.
0%, containing 20 to 100% by weight of a unit derived from a crosslinkable polyvinyl monomer, and at least one of the monovinyl monomer unit and the crosslinkable polyvinyl monomer unit has a hydrophilic group. The resin for separating lipoproteins according to claim 1, which comprises an anion exchanger having an anion exchange capacity of 0.1 to 1.5 meq / g of dry resin. 3. The matrix has an average pore radius of 10 to 2
000 angstrom, pore volume 0.2-1.8 ml
/ G, water-insoluble hydrophilic porous polymer particles having a pore surface area of 1 to 1000 m ² / g, and the lipoprotein separating resin according to claim 1 or 2. 4. An organic polymer particle having 0 to 10 monovinyl monomers.
After impregnating a crosslinkable polyvinyl monomer containing 80% by weight, a radical polymerization initiator and a porosifying solvent, the particles are suspended in an aqueous medium, and the suspension is heated to polymerize,
The resin for separating lipoprotein according to any one of claims 1 to 3, wherein an anion exchange group is introduced. 5. The resin for separating lipoprotein according to claim 1, wherein the anion exchange group is a substituted amino group.