JPH024301B2 - - Google Patents

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention is a low-density lipoprotein that selectively adsorbs and removes low-density lipoproteins, which are thought to be closely related to various diseases caused by increased plasma lipids. This invention relates to a method for regenerating a specific gravity lipoprotein adsorbent.

As is well known, an increase in blood lipids, particularly low-density lipoproteins, is thought to be closely related to the cause or progression of arteriosclerosis. As arteriosclerosis progresses, the possibility of developing serious circulatory system symptoms such as myocardial infarction and cerebral infarction becomes extremely high, and the mortality rate is also high.

Therefore, by selectively adsorbing and removing low-density lipoproteins from body fluid components such as blood and plasma,
It was expected that it would prevent the progression of the above-mentioned diseases, alleviate their symptoms, and even hasten their healing.

(Prior art) Existing technologies that can be used for the above purpose include adsorption using an adsorbent in which heparin is immobilized on agarose gel (Lupien, P-J, et.al.: A new
approach the management of familial
hypercholesterolemia.Removal of plasma−
cholesterol based on the principle of affinity
chromatography.Lancet, 2:1261-1264,
1976.) and chromatography using glass powder or beads (Carlson, L.
A.：Chromatographic separation of serum
lipoproteins on glass powder columns.
Description of the method and some
applications.Clin.Chim.Acta, 5:528-538;
1960.).

In addition, recently, an improved invention suitable for the adsorption and removal of low-density lipoproteins by extracorporeal circulation (Japanese Patent Application Laid-open No. 59-1998) has been developed.
-102436) and the invention of adsorbents with even higher adsorption capacity for low-density lipoproteins (Japanese Patent Applications 1983-80777 and 1983-80778), and technological progress has been made.

Additionally, attempts have been made to treat body fluids with these adsorbents, adsorb and remove low-density lipoproteins, and then desorb the low-density lipoproteins from the adsorbents to regenerate and reuse the adsorbents. .

For example, in the case of an adsorbent with immobilized heparin, lipoproteins are washed with 5% sodium chloride (Ddwin A. Burgstaler et.al.:
Laboratory Study, Removal of plasma
lipoproteins from circulating blood with a
Heparin−Agarose column.Mayo Clin Proc
55:180-184, 1980), and when using glass, potassium bicarbonate-carbonate
How to elute lipoproteins with buffer (PH8.8, 9.6, 9.8 and _CO3 ) (Carlson, LA:
Chromatographic separation of serum
lipoproteins on glass powder column.
Description of the method and some
applications.Clin.Chim.Acta, 5:528-538.
1960.).

(Problems to be Solved by the Invention) However, although these adsorbent regeneration methods are sufficient as a regeneration means for conventional adsorbents whose adsorption performance is not very high, the recent low-density lipoprotein adsorption performance Even when the above-described adsorbent regeneration method is applied to a polyanionic adsorbent with improved properties, the adsorbent is not regenerated sufficiently, and improvements have been desired.

(Means for Solving the Problems) As a result of intensive research to solve the above-mentioned problems and provide a simple, safe, and efficient adsorbent regeneration method, the present inventors have found that metal ions are used as the eluent. The present inventors have discovered that adsorbents can be easily and safely regenerated with surprisingly high efficiency by using a liquid containing a substance that reacts to form metal chelates, leading to the completion of the present invention.

That is, the present invention provides a low-density lipoprotein adsorbent that has adsorbed low-density lipoproteins in body fluids and has a polyanion moiety on its surface, and is washed with a solution containing at least one type of chelating agent. This is a method for regenerating specific gravity lipoprotein adsorbent.

The target adsorbent of the present invention is low-density lipoprotein.
2.2×10 ⁶ to 3.5×10 ⁶ , hydrated density 1.003 to 1.034
(g/ml), levitation coefficient (1.063) from 0 to 20×10 ^-13
cm・sec ^-1・dyh ^-1・g ^-1 , diameter from 20.0 to 30.0nm
lipoprotein (SCANU, AM: plasma
lipoproteins：an introduction.
Biochemistry of Atherosclerosis”ed.by
SCANU A≧M., 1979, P.3-8).

Next, the constituent elements of the adsorbent targeted by the present invention will be explained in detail.

The polyanion part of the low-density lipoprotein adsorbent in the present invention has a molecular weight of 600 or more,
Functional groups that show negative charges in one molecule, such as carboxyl groups (COOH, COO ^- ) and sulfonic acid groups (SO ₃ H, SO ₃ ^- ), have many functional groups that show negative charges in plasma. To tell. Examples include vinyl-based synthetic polyanions such as polyacrylic acid, polyvinyl sulfonic acid, polyvinyl phosphoric acid, and polymethacrylic acid; styrene-based polyanions such as polystyrene sulfonic acid, poly-α-methylstyrene sulfonic acid, and polystyrene phosphoric acid; and vinyl anions. Copolymers of, for example, polyanions such as styrene-maleic acid copolymers, synthetic peptide polyanions such as polyglutamic acid and polyaspartic acid, synthetic nucleic acid polyanions such as poly U and poly A, and polyphosphoric acid and polyphosphate esters. , alginic acid, heparin, and other natural polyanions.

Among them, synthetic polyanions have excellent chemical stability, are stable against high-pressure steam sterilization, gamma ray sterilization, ethylene oxide sterilization, etc., and can be relatively easily adjusted. It is superior to natural products in this respect and can be recommended. In addition, polyanions obtained by synthesis are preferable because polyanions that do not easily cause complement activation as seen in natural polysaccharides can be easily obtained. Furthermore, those that can be graft-polymerized directly onto the carrier, such as vinyl anions, give more favorable results in that a polyanion having a large molecular weight can be fixed to the carrier in a high retention amount.

In addition, since the low-density lipoprotein, which is the target substance for adsorption, is a huge lipoprotein with a diameter of about 200 Å, it is preferable that the structure of the polyanion part is a chain structure, and it is better to extend it long from the surface of the adsorbent. preferable. Moreover, the negative charge density in the polyanion moiety is preferably at least one per 300 molecular weight. More preferably, the number is one or more per molecular weight of 200, and desirably one per unit of molecular weight of 70 to 150. The molecular weight referred to here includes the molecular weight of a functional group that exhibits a negative charge. The molecular weight of the polyanion moiety needs to be at least 600, since it will not adsorb low-density lipoproteins as much as it becomes small. Preferably 2500 or more, from 10,000
A range of 5 million is preferred.

It is thought that the polyanion moiety's many negatively charged functional groups recognize multiple points on the low-density lipoprotein, thereby binding the low-density lipoprotein with strong Coulomb force.

Among the functional groups exhibiting a negative charge, carboxyl groups (COOH, COO ^- ) give particularly favorable results.
Since it is a weaker acid than sulfonic acid groups (SO ₃ H, SO ₃ ^- ), it has low adsorption to useful proteins such as albumin. In addition, blood coagulation system proteins are less adsorbed and less likely to be activated. Furthermore, complement system proteins are also difficult to adsorb.

Among the above-mentioned polyanion moieties, polycarboxylic acids such as polyacrylic acid and polymethacrylic acid are particularly stable and can be recommended.

The density of negative charge is 1μeq to 1meq per ml of adsorbent.
This range is an appropriate range that provides good adsorption performance for low-density lipoproteins, good adsorption selectivity, and little influence on the coagulation fibrinolytic system and the complement system. When the negative charge density is lower than 1 μeq/ml, the adsorption ability of low-density lipoproteins falls short of practical performance, and when it exceeds 1 meq, non-selective adsorption increases, adversely affecting the coagulation fibrinolytic system and the complement system. A more preferable range is from 5μeq/ml
700μeq/ml, more preferably from 10μeq/ml
It is 500 μeq/ml.

The negative charge density can be measured according to a conventional method for measuring the ion exchange capacity of a cation exchange resin.

Examples of the method for producing the low-density lipoprotein adsorbent according to the present invention include a method of activating a carrier and covalently bonding a polyanion, and a method of graft polymerizing an anion monomer to the carrier to form a polyanion graft chain. It will be done.

The carrier only needs to be able to immobilize the polyanion, and both hydrophilic and hydrophobic carriers can be used. However, when using a hydrophobic carrier, non-specific adsorption of albumin to the carrier sometimes occurs, so a hydrophilic carrier is preferable. gives the desired result.

The shape of the insoluble carrier can be any known shape such as particulate, fibrous, hollow fiber, or membrane, but in terms of the amount of polyanion retained and ease of handling as an adsorbent, particulate or fibrous carriers are preferred. Preferably.

The carrier may be made of any material as long as it can fix the polyanion. Porous carriers that can be used include organic or inorganic porous materials such as cellulose gel, dextran gel, agarose gel, polyacrylamide gel, porous glass, and vinyl polymer gel. All carrier materials can be used, but since the adsorbed substance, low-density lipoprotein, has a diameter of 200 to 300 Å and a molecular weight of 2.2 × 10 ⁶ to 3.5 × 10 ⁶ , the exclusion limit molecular weight of the carrier is 2.2 million or more, and the pore diameter is 200 Å or more for higher adsorption efficiency.

Among the above-mentioned carriers, carriers made of cross-linked copolymers mainly composed of vinyl alcohol units are particularly hydrophilic, so they have little interaction with solutes such as proteins in plasma, and minimize non-specific adsorption. decrease to. Furthermore, it has extremely excellent properties such as not interacting with the complement system and coagulation system in plasma.
In terms of physical properties, it exhibits an excellent pore size distribution, is heat resistant, allows heat sterilization, and has excellent physical and mechanical strength, which is a characteristic of synthetic polymers. When used as a carrier for adsorbent for whole blood, it has extremely excellent properties such as minimal interaction with blood cell components, minimizing thrombus formation, non-specific adhesion of blood cell components, and residual blood. .

A crosslinked polymer having vinyl alcohol units as a main component can be synthesized by polymerizing a monomer having a hydroxyl group or by introducing a hydroxyl group through a chemical reaction of the polymer. It is also possible to synthesize both in combination. As the polymerization method, a radical polymerization method can be used. The crosslinking agent may be introduced by copolymerization during polymerization, or may be introduced by chemical reaction between polymers (between polymers or between polymer and crosslinking agent), or both may be used in combination.

For example, it can be produced by copolymerizing a vinyl monomer and a vinyl or allyl crosslinking agent. Examples of the vinyl monomer in this case include vinyl esters of carboxylic acids such as vinyl acetate and vinyl propionate, and vinyl ethers such as methyl vinyl ether and ethyl vinyl ether.

Examples of crosslinking agents include allyl compounds such as triallyl isocyanurate and triallyl cyanurate, di(meth)acrylates such as ethylene glycol dimethacrylate and diethylene glycol dimethacrylate, butanediol vinyl ether, diethylene glycol divinyl ether, and tetravinylglyoxal. polyvinyl ethers such as diarylidene pentaerythritol,
Polyallyl ethers such as tetraallyloxyethane and glycidyl acrylates such as glycidyl methacrylate can be used. Also,
If necessary, a copolymer of other comonomers can be used.

In the case of vinyl copolymers, triallylisocyanurate of polyvinyl alcohol obtained by copolymerizing a vinyl ester of carboxylic acid and a vinyl compound (allyl compound) having an isocyanurate ring, and then cross-hydrolyzing the copolymer. A crosslinked product provides a particularly good carrier in terms of strength and chemical stability.

All known methods such as covalent bonding, ionic bonding, physical adsorption, embedding, and precipitation insolubilization on the polymer surface can be used to immobilize the polyanion on the surface of the insoluble carrier. Considering this, it is preferable to use it after being fixed and insolubilized by covalent bonding. For this purpose, we usually use immobilized enzymes, known carrier activation methods used in affinity chromatography, binding methods with ligands, and graft polymerization techniques in which the carrier or activated carrier is used as a backbone polymer and polyanions are used as branches. be able to.

Examples of activation methods include cyanogen halide method, epichlorohydrin method, bisepoxide method,
Examples include the halogenated triazine method, the bromoacetyl bromide method, the ethyl chloroformate method, and the 1,1'-carbonyldiimidazole method. The activation method of the present invention is not limited to the above examples as long as it can perform a substitution and/or addition reaction with a nucleophilic reactive group having active hydrogen such as an amino group, a hydroxyl group, a carboxyl group, or a thiol group of the ligand. However, in consideration of chemical stability, thermal stability, etc., a method using an epoxide is preferable, and an epichlorohydrin method is particularly recommended.

In addition, for carriers with silanol groups such as silica-based and glass-based, various silicas such as γ-glycidoxypropyltrimethoxysilane, γ-aminopropyltriethoxysilane, γ-mercaptopropyltrimethoxysilane, and vinyltrichlorosilane are used. Coupling agents are preferably used.

Examples of graft polymerization methods include methods that utilize chain transfer reactions, methods that utilize reactions such as dehydrogenation and dehalogenation by radiation, ultraviolet light, etc., and methods that utilize the formation of peroxides.
A method in which an anionic monomer is graft-polymerized onto a carrier having a reducing group such as a hydroxyl group, a thiol, an aldehyde, or an amine using a cerium salt, an iron salt, or the like as an initiator is simple and recommended. In addition, a graft polymerization system is preferably used because a polyanion having a relatively large molecular weight can be fixed to the inside of the carrier.

Two or more types of polyanions may be bound to the carrier.

Above, as a method for manufacturing a low-density lipoprotein adsorbent, a method of activating a carrier and then binding a polyanion has been described, but the method is not limited to this.

The low-density lipoprotein adsorbent is one in which the above-described low-density lipoprotein adsorbent is filled and held in a container equipped with an inlet and outlet for body fluids.

In the present invention, the adsorbent is generally used while being filled in a container equipped with an inlet and outlet for body fluids.

In FIG. 1, reference numeral 1 shows an example of a low-density lipoprotein adsorber containing a low-density lipoprotein adsorbent, and a packing 4 with a filter 3 stretched inside is inserted into the opening at one end of a cylinder 2. A cap having a body fluid inlet is screwed into the cylinder 2, and a cap 8 having a body fluid outlet 7 is screwed into the opening at the other end of the cylinder 2 through a packing 4' having a filter 3' on the inside. A low-density lipoprotein adsorbent is filled and held in the gap between the filters 3 and 3' to form an adsorbent layer 9.

The adsorbent layer 9 may be filled with a low-density lipoprotein adsorbent alone, or may be mixed or laminated with other adsorbents. Other adsorbents include, for example:
Something like activated carbon, which has a wide range of adsorption capacities, can be used. As a result, a wider range of clinical effects can be expected due to the synergistic effect of the adsorbent. The volume of the adsorbent layer 9 is 50 to 400 ml when used for extracorporeal circulation.
The degree is appropriate. When this device is used for extracorporeal circulation, there are roughly two methods as follows. One method is to separate blood taken from the body into plasma components and blood cell components using a centrifuge or membrane plasma separator.The plasma components are passed through the device, purified, and then separated into blood cells. One method is to return the blood to the body together with its components, and the other method is to directly pass blood taken from the body through the device for purification.

The method for passing body fluids may be either continuous or intermittent, depending on clinical needs or equipment conditions.

The chelating agent referred to in the present invention refers to a metal chelate (a complex in which one ligand coordinates to the central metal at two or more places to form a cyclic structure containing metal atoms) that reacts with metal ions. ), examples include malonic acid, oxalic acid, phthalic acid, succinic acid, maleic acid, citraconic acid,
Carboxylic acids (dibasic acids) such as itaconic acid, aliphatic amines such as ethylenediamine, diethylenetriamine, and propylene diamine, aromatic amines such as α,α′-dipyridyl and phenanthroline, alanine, aspartic acid, glycine, and glycylglycyl. Natural amino acids and peptides such as glycine and glutamic acid, β-alanine-N,N-diacetic acid, aminobenzoic acid-N,N-diacetic acid, iminodiacetic acid,
Aniline diacetic acid, 1,3-diaminocyclohexane-N,N'-tetraacetic acid, ethylenediaminetetraacetic acid, pentamethylenediaminetetraacetic acid, etc., non-naturally occurring amino acids, citric acid, gluconic acid, glyceric acid, glycolic acid, malic acid, Oxyacids such as 5-sulfosalicylic acid and tartaric acid; condensed phosphoric acids such as pyrophosphoric acid, trimetaphosphoric acid, and triphosphoric acid; nitrocarboxylic acids such as nitroacetic acid and O-nitrobenzoic acid; acids such as oxalacetic acid and pyruvic acid; salicylaldehyde , 3-chlorsalicylaldehyde, 5-sulfosalicylaldehyde and other salicylaldehydes and their derivatives, 2-oxy-1-naphthaldehyde, 2-oxy-3-
Oxyaldehydes such as naphthaldehyde, β-diketones such as acetylacetone, benzoylacetone, 2-furoylbenzoylmethane, and trifluoroacetylacetone, phenol derivatives such as 8-oxyquinoline, catechol-3,5-disulfonic acid, and eriochrome black. Examples include O,O'-dioxyazo dyes such as T, Eriochrome Blue Black R, aminobenzenethiol, O-aminophenol, and ethyl acetoacetate. Also,
Polycarboxylic acids such as polyacrylic acid and polymethacrylic acid, and polysaccharides such as heparin and alginic acid can also be used.

The presence of a high concentration of chelating agent shifts the adsorption equilibrium of cationic substances such as low-density lipoproteins, calcium, and magnesium, which are adsorbed on the adsorbent through interaction with the polyanion moiety of the adsorbent, causing them to be desorbed from the adsorbent. It is thought that it will be released.

Among chelating agents, sodium citrate, heparin sodium, etc. are excellent in terms of safety and can be recommended if the adsorbent is to be reused for extracorporeal circulation treatment.

Next, a method for regenerating a low-density lipoprotein adsorbent will be explained using an example.

As mentioned above, the low-density lipoprotein adsorbent is usually filled into a container and used as a lipoprotein adsorber as shown in FIG. Body fluid is poured into this low-density lipoprotein adsorber, and low-density lipoproteins in the body fluid are adsorbed. After use, regeneration begins. Usually, first, a physiological solution (e.g. Wash with physiological saline). This operation may be omitted if the regenerating liquid used thereafter does not have an adverse effect on body fluids, such as denaturation or aggregation.

Next, the regenerating solution of the adsorbent is passed through a low-density lipoprotein adsorber. Since the efficiency is very high, almost complete desorption can be achieved by flowing 2 to 5 times the capacity of the adsorber. The flow rate can be arbitrarily selected as long as it is not extremely fast, and the temperature can also be arbitrarily selected.

Finally, in consideration of next use, it is replaced with a physiological solution or a preservation solution such as a disinfectant for preservation. If necessary, after replacing with a physiological solution, sterilization operations such as moist heat sterilization, gamma ray sterilization, etc. may be performed.

The low-density lipoprotein adsorbent is regenerated by the operations described above.

Furthermore, if necessary, the low-density lipoprotein adsorbent can be regenerated even during the adsorption and removal of low-density lipoproteins in blood through extracorporeal circulation. In this case, use two or more low-density lipoprotein adsorbers, divide them into two systems, one for adsorbing low-density lipoproteins, and the other for regeneration, and operate them aseptically. is preferred. However, it is also possible to perform one operation intermittently.

When carrying out the method for regenerating a low-density lipoprotein adsorbent of the present invention, particularly when the low-density lipoprotein adsorbent is being regenerated while low-density lipoproteins in the blood are being adsorbed and removed by extracorporeal circulation. As a device for adsorption and regeneration of low-density lipoproteins, a ``low-density lipoprotein adsorbent having a polyanion moiety on the surface'' is stored in a container with two systems of liquid inlets and two systems of liquid outlets. By using this product, the adsorption and regeneration operation of low-density lipoproteins is very simple, and sterile handling is also easy.

That is, one of the liquid inlets and one of the liquid outlets is used for adsorbing low-density lipoproteins, and the other liquid inlet and the other liquid outlet are used for regenerating the low-density lipoprotein adsorbent. Therefore, adsorption and regeneration of the low-density lipoprotein adsorbent can be easily and sterilely performed, which is very convenient.

(Effects of the Invention) As described above, according to the method for regenerating a low-density lipoprotein adsorbent of the present invention, it is possible to completely regenerate a polyanionic adsorbent that exhibits extremely high adsorption capacity and high selectivity. Since it has become possible to perform this process easily and safely, it has become possible to reuse the low-density lipoprotein adsorbent, and a large amount of low-density lipoprotein can be adsorbed with a single low-density lipoprotein adsorbent. It became possible.

Since a chelating agent is used as a regenerating agent,
By using a chelating agent that has good regeneration efficiency and is safe even if it enters the living body, we have made regeneration possible during extracorporeal circulation treatment.

The present invention is effective for regenerating low-density lipoprotein adsorbents for the treatment of familial hypercholesterolemia,
Unprecedented in the past, it is completely recyclable and safe.

(Example) Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 Using the low-density lipoprotein adsorption device shown in Figure 1,
We conducted adsorption, regeneration, and re-adsorption experiments on low-density lipoproteins.

The low-density lipoprotein adsorber has an inner diameter of 0.8 cm and a length of
A 2.0 cm, internal volume of 1.0 ml filled with 1.0 ml of low-density lipoprotein adsorbent was used.

The low-density lipoprotein adsorbent was manufactured by the method described below.

Vinyl acetate 1000g, triallylisocyanurate 414g, ethyl acetate 1000g, heptane 1000g,
Polyvinyl acetate (degree of polymerization 500) 70g and 2,
A homogeneous liquid mixture consisting of 36 g of 2'-azobisisobutyronitrile, 1% by weight of polyvinyl alcohol, 0.05% by weight of sodium dihydrogen phosphate dihydrate, and 1.5% by weight of disodium hydrogen phosphate dodecahydrate. The dissolved water 4 was placed in a flask, thoroughly stirred, and then heated and stirred at 65°C for 18 hours and then at 75°C for 5 hours to carry out suspension polymerization to obtain a granular copolymer. During the polymerization, the stirring speed was controlled to keep the particle size small. After filtering, washing with water, and then extracting with acetone, the copolymer was transesterified in a solution consisting of 465 g of caustic soda and 20 g of methanol at 40° C. for 18 hours. The average particle size of the obtained particles was 40 μm.

This gel was packed into a stainless steel column with an inner diameter of 7.5 mm and a length of 120 cm, and aqueous solutions of dextran and polyethylene glycol with various molecular weights, albumin, immunoglobulin G, immunoglobulin M, β-lipoprotein, tobacco, mosaic, When the virus was measured in a phosphate buffered salt solution, each virus was eluted in descending order of molecular weight. The exclusion limit molecular weight of dextran was approximately 5×10 ⁶ , and the exclusion limit molecular weight of protein was approximately 2×10 ⁷ .
In addition, human-γ-
When a solution of globulin and human albumin was run, the recovery rate was almost 100%, and non-specific adsorption of the gel was very low. All sample measurements were performed at a flow rate of 1 ml/min.

Next, it is transesterified, washed thoroughly with water,
150g of dried gel in dimethyl sulfoxide 1800g
ml and 1200 ml of epichlorohydrin suspended in a solution consisting of 150 ml of 50% aqueous sodium hydroxide solution.
and react at 30°C for 5 hours with stirring. After the reaction was completed, it was filtered through a glass filter and washed with dimethyl sulfoxide (3) and water (20) to obtain an epoxy group-bonded gel.

The amount of epoxy group bonded in this gel is per ml.
It was 0.11 mmol. Using the epoxy group-bonded gel, polyacrylic acid (weight average molecular weight 9×10 ⁴ ,
(manufactured by Aldrich, USA) as a ligand to create an adsorbent.

4.5 g of polyacrylic acid was made up to 500 ml with distilled water, and this was used as a ligand solution. Add 100 ml of the epoxy group-binding gel to this ligand solution and heat at 50°C for 16 hours.
The ligand binding reaction was performed while shaking.

After this, after thoroughly washing with water and dehydrating with suction, 0.1
The polyacrylic acid was immersed in a specified sodium hydroxide solution for 30 minutes to convert it into a sodium form, and then washed with a sufficient amount of water to obtain a low-density lipoprotein adsorbent.

Adsorption experiments were performed using plasma from patients with familial hypercholesterolemia.

First, the low-density lipoprotein adsorbent was primed with physiological saline, and then familial hypercholesterolemia patient plasma was allowed to flow through the low-density lipoprotein adsorbent at a flow rate of 0.17 ml/min. The first 1 ml of physiological saline flowing out from the adsorber was discarded, and 1 ml of the plasma that came out thereafter was collected into test tubes.
After 12 ml of plasma was flowed, 5 ml of a 29.4 g/dl solution of trisodium citrate dihydrate (pH adjusted to 7 with hydrochloric acid) was used as a regenerating solution at the same flow rate as the plasma. Thereafter, after washing the regenerating solution with 5 ml of physiological saline, 12 ml of plasma from a patient with familial hypercholesterolemia was flowed again at the same flow rate to perform a resorption test.

Figure 2 shows the results of measuring the cholesterol concentration of all the effluents during the adsorption, regeneration and re-adsorption tests. The horizontal axis is the amount of effluent, and the vertical axis is the concentration of cholesterol in each effluent. First, in the first adsorption test, the adsorption amount of cholesterol (mostly low-density lipoprotein cholesterol since it is plasma from a patient with familial hypercholesterolemia) is expressed by the area of the shaded area in Figure 2. The calculation results in 25 mg. next,
The amount of cholesterol flowing out with the regenerating liquid is expressed by the area of the shaded area in Figure 2, and when calculated, it matches the area of the shaded area, and all of the adsorbed cholesterol is desorbed with the regenerating liquid and flows out of the adsorber. It turns out that it has leaked. Next, the amount of cholesterol adsorbed in the second adsorption test is expressed by the area of the shaded area in Figure 2, and since this also coincides with the area of the shaded area, the low-density lipoprotein adsorbent is completely absorbed by the regeneration solution. You can see that it has been played.

Comparative Example 1 Except for using 5% saline as the regenerating solution,
All the procedures were carried out in the same manner as in Example 1, and the amount of cholesterol desorbed from the regenerating solution was 73% of the amount of cholesterol adsorbed in the first time, and the amount of cholesterol adsorbed in the second time was also 68% of the first time. Ta.

That is, the low-density lipoprotein adsorbent could not be completely regenerated with 5% saline.

Example 2 A 4.52 g/dl solution of tetrasodium ethylenediaminetetraacetic acid tetrahydrate (pH adjusted to 7.0 with hydrochloric acid) was used as a regenerating liquid.
The procedure was carried out in the same manner as in Example 1, except that the amount of cholesterol adsorbed in the first time, the amount of cholesterol desorbed by the regenerating solution,
The adsorption amount of cholesterol at the second time was all the same. That is, the low-density lipoprotein adsorbent was completely regenerated.

Example 3 Polyacrylic acid with a molecular weight of 2000 was used as a regeneration liquid.
Everything was carried out in the same manner as in Example 1 except that a 0.72 g/dl solution (pH adjusted to 7.0 with sodium hydroxide) was used. Second
The amount of cholesterol adsorbed for the second time completely matched, indicating that the low-density lipoprotein adsorbent was completely regenerated by the regenerating solution.

[Brief explanation of drawings]

FIG. 1 is a schematic cross-sectional view showing a low-density lipoprotein adsorbent filled with a low-density lipoprotein adsorbent having a polyanion portion on the surface, and FIG. 2 is a graph showing the experimental results of Example 1. 1...Low-density lipoprotein adsorber, 2...Cylinder,
3, 3'...Filter, 4...Packing, 5
... Body fluid inlet, 6, 8 ... Cap, 9 ... Adsorbent layer.

Claims (1)

[Claims] 1. A low-density lipoprotein adsorbent that has adsorbed low-density lipoproteins in body fluids and has a polyanion moiety on its surface, and is washed with a solution containing at least one type of chelating agent. How to play.