Separation method and separation device of protein binding toxin and plasma transport protein and application of separation method and separation device

Abstract

The invention provides a method and device for separating protein binding toxin from plasma transport protein and application of the method and device, and relates to the technical field of separation. The method for separating the protein-binding toxin from the plasma transport protein comprises the following steps: dialyzing a solution containing the protein-binding toxin and the plasma transport protein, so that the protein-binding toxin is separated out of a dialysis membrane; the solution is subjected to ultrasonic treatment or ultrasonic cavitation treatment in the dialysis process. The research of the inventor finds that ultrasonic or ultrasonic cavitation treatment is carried out in the dialysis process of the protein-binding toxin, so that the separation of the protein-binding toxin and plasma transport protein can be remarkably promoted, and the dialysis efficiency of the protein-binding toxin is improved. The separation method is simple, convenient and low in cost, and the dialysis efficiency of the protein binding toxin can be effectively improved.

Description

Technical Field

[0001] The present invention relates to the technical field of separation, and in particular to a method and device for separating protein-bound toxins from plasma transport proteins and their applications. Background Art

[0002] In patients with advanced chronic kidney disease (uremic stage), due to significant impairment of renal function, small molecule metabolites in the body are retained due to excretion disorders. Among them, protein-bound uremic toxins (PBUTs) are mostly products of the decomposition of food proteins by intestinal bacteria, and more than 96% of them exist in the form of binding to albumin (molecular mass 68,000 D) in the blood circulation. Common protein-bound toxins include indoxyl sulphate (IS), p-cresyl sulphate (PCS), hippuric acid (HA), etc. PBUTs have gradually been considered to be closely related to cardiovascular and cerebrovascular complications, infections, chronic kidney disease-mineral and bone metabolism disorders, malnutrition, etc., and thus have received increasing attention in clinical practice.

[0003] Traditional hemodialysis cannot achieve good clearance effects on PBUTs. The clearance of PBUTs in healthy kidneys largely depends on tubular secretion, while conventional hemodialysis (HD) mainly simulates the filtration function of the kidneys and can only remove the free toxin part. Therefore, reducing the adsorption of PBUTs to blood albumin and increasing the free fraction of PBUTs are the main methods for improving the clearance of protein-bound toxins in blood purification at present.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] The first object of the present invention is to provide a method for separating protein-bound toxins from plasma transport proteins to solve the above technical problems.

[0006] The second object of the present invention is to provide a device for separating protein-bound toxins from plasma transport proteins.

[0007] The third object of the present invention is to provide the application of the above device for separating protein-bound toxins from plasma transport proteins in the preparation of products for treating chronic kidney disease.

[0008] In order to achieve the above objects, the following technical solutions are specifically adopted: In the first aspect, the present invention provides a method for separating protein-bound toxins from plasma transport proteins, including the following steps: Dialyze a solution containing a protein-bound toxin and a plasma transport protein so that the protein-bound toxin is separated outside the dialysis membrane; During the dialysis process, the solution is subjected to ultrasonic treatment or ultrasonic cavitation treatment.

[0009] As a further technical solution, the protein-bound toxin includes indoxyl sulfate, p-cresol sulfate, and hippuric acid.

[0010] As a further technical solution, the plasma transport protein includes albumin.

[0011] As a further technical solution, the dialysis membrane is used to retain the plasma transport protein and permeate the protein-bound toxin.

[0012] As a further technical solution, the ultrasonic cavitation includes the following steps: Add microbubbles to the solution and then perform ultrasonic treatment.

[0013] As a further technical solution, the addition amount of the microbubbles is 10 5 -10 10 / mL.

[0014] As a further technical solution, the solution includes blood.

[0015] In a second aspect, the present invention provides a separation device for a protein-bound toxin and a plasma transport protein, including a dialysis device and an ultrasonic device; The dialysis device is used to perform dialysis treatment on a solution containing a protein-bound toxin and a plasma transport protein so that the protein-bound toxin is separated outside the dialysis membrane; The ultrasonic device is used to perform ultrasonic treatment on the solution in the dialysis device.

[0016] As a further technical solution, it further includes a microbubble device; The microbubble device is used to add microbubbles to the solution in the dialysis device.

[0017] In a third aspect, the present invention provides the application of the above separation device for a protein-bound toxin and a plasma transport protein in the preparation of a product for treating chronic kidney disease.

[0018] Compared with the prior art, the present invention has the following beneficial effects: Through research by the inventor, it is found that during the dialysis process of the protein-bound toxin, ultrasonic treatment or ultrasonic cavitation treatment can significantly promote the separation of the protein-bound toxin and the plasma transport protein and improve the dialysis efficiency of the protein-bound toxin. Based on this, the separation method of the protein-bound toxin and the plasma transport protein of the present invention is proposed. This separation method is simple and convenient, with low cost, and can effectively improve the dialysis efficiency of the protein-bound toxin. Description of the Drawings

[0019] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0020] Figure 1 For ultrasonic cavitation to accelerate the removal of p - cresol sulfate; Figure 2 For ultrasonic cavitation to accelerate the removal of indoxyl sulfate. Specific embodiments

[0021] The following will describe the embodiments of the present invention in detail in combination with the embodiments and examples. However, those skilled in the art will understand that the following embodiments and examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention. Those not specifying specific conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. Those reagents or instruments not indicating the manufacturer can all be obtained as conventional products through commercial purchase.

[0022] Plasma transport proteins are a class of proteins in the blood circulation responsible for binding, carrying, and transporting specific substances.

[0023] In a first aspect, the present invention provides a method for separating a protein - bound toxin from a plasma transport protein, comprising the following steps: Dialyze a solution containing a protein - bound toxin and a plasma transport protein so that the protein - bound toxin is separated outside the dialysis membrane; During the dialysis process, ultrasonic treatment or ultrasonic cavitation treatment is performed on the solution.

[0024] Through research by the inventor, it is found that during the dialysis process of the protein - bound toxin, ultrasonic treatment or ultrasonic cavitation treatment can break the non - covalent binding between the plasma transport protein and the protein - bound toxin, promote the separation of the protein - bound toxin from the plasma transport protein, and improve the dialysis efficiency of the protein - bound toxin. Based on this, the method for separating a protein - bound toxin from a plasma transport protein of the present invention is proposed. This separation method is simple, convenient, and low - cost, and can effectively improve the dialysis efficiency of the protein - bound toxin.

[0025] In some alternative embodiments, the protein - bound toxin includes, but is not limited to, indoxyl sulfate, p - cresol sulfate, and hippuric acid, and may also be other protein - bound toxins well - known to those skilled in the art.

[0026] In some alternative embodiments, the plasma transport protein includes, but is not limited to, albumin, and may also be other plasma transport proteins well known to those skilled in the art.

[0027] In some alternative embodiments, the dialysate is pure water, PBS solution, or a commercial dialysate used in clinical hemodialysis, etc.

[0028] In some alternative embodiments, the dialysis membrane is used to retain plasma transport proteins and permeate protein-bound toxins.

[0029] In some alternative embodiments, the molecular weight cut-off of the dialysis membrane is 500 - 10,000 Da.

[0030] In some alternative embodiments, the ultrasonic cavitation includes the following steps: Microbubbles (in the present invention, microbubbles refer to bubbles with a particle size of 2 - 8 μm) are added to the solution, and then ultrasonic treatment is performed.

[0031] In some alternative embodiments, the addition amount of the microbubbles is 10 5 -10 10 / mL (ultrasonic cavitation can be achieved as long as microbubbles exist in the solution, and the addition amount of microbubbles can be selected according to the actual situation).

[0032] In some alternative embodiments, the solution includes blood.

[0033] In a second aspect, based on the above separation method, the present invention provides a separation device for protein-bound toxins and plasma transport proteins, including a dialysis device and an ultrasonic device; The dialysis device is used to perform dialysis treatment on a solution containing protein-bound toxins and plasma transport proteins, so that the protein-bound toxins are separated outside the dialysis membrane; The ultrasonic device is used to perform ultrasonic treatment on the solution in the dialysis device.

[0034] This separation device has a simple structure and can achieve efficient separation of protein-bound toxins and plasma transport proteins.

[0035] In some alternative embodiments, the protein-bound toxins include, but are not limited to, indoxyl sulfate, p-cresol sulfate, and hippuric acid, and may also be other protein-bound toxins well known to those skilled in the art.

[0036] In some alternative embodiments, the plasma transport protein includes, but is not limited to, albumin, and may also be other plasma transport proteins well known to those skilled in the art.

[0037] In some alternative embodiments, a microbubble device is further included; The microbubble device is used to add microbubbles to the solution in the dialysis device.

[0038] In some alternative embodiments, the dialysis device includes a dialysis bag.

[0039] In some alternative embodiments, the ultrasonic device includes an ultrasonic irradiator.

[0040] In a third aspect, the present invention provides the use of the above-mentioned separation device for protein-bound toxins and plasma transport proteins in the preparation of products for the treatment of chronic kidney disease.

[0041] The separation device provided by the present invention can effectively separate protein-bound toxins and plasma transport proteins, and thus can be used for dialysis treatment of patients with chronic kidney disease.

[0042] The present invention will be further described below through specific examples and comparative examples. However, it should be understood that these examples are only for more detailed description and should not be construed as limiting the present invention in any way.

[0043] In the following examples and comparative examples, the 0 time point is the immediate sampling after the dialysis fluid is replaced, the 1 time point is the immediate sampling 10 minutes after the first sampling or 10 minutes after the first ultrasound, the 2 time point is the sampling after 1 hour of dialysis, the 3 time point is the immediate sampling 10 minutes after continuous dialysis or 10 minutes after the second ultrasound, and the 4 time point is the sampling after 2 hours of dialysis.

[0044] Comparative Example 1 Taking the relative ratio of plasma albumin to PBUTs in patients with chronic renal function as a reference, 0.66 g of BSA and 4.52 mg of PCS were dissolved in water to prepare the solution to be separated 1.

[0045] The solution to be separated 1 was dialyzed using a 1000 Da filter membrane. Samples of the liquid outside the dialysis membrane were taken at 0, 1, 2, 3, and 4 time points respectively. The sampling solution was detected by HPLC, and the concentration of PCS in the dialysis fluid was measured. The results are as Figure 1 shown. After calculation, after 2 hours of dialysis, the protein binding rate of PCS in this system was 86.7%.

[0046] Example 1 Taking the relative ratio of plasma albumin to PBUTs in patients with chronic renal function as a reference, 0.66 g of BSA and 4.52 mg of PCS were dissolved in water to prepare the solution to be separated 1.

[0047] The solution to be separated 1 was dialyzed using a 1000 Da filter membrane (PBS solution), and ultrasonic irradiation was performed for 10 min at the 0 h and 1 h time points. Samples of the liquid outside the dialysis membrane were taken at the 0, 1, 2, 3, and 4 time points respectively. The sampled solution (the solution outside the dialysis membrane) was detected by HPLC, and the PCS concentration in the dialysis solution was calculated. The results are as Figure 1 shown.

[0048] It was found that, compared with Comparative Example 1, the dialysis efficiency of PCS in Example 1 was increased by 1.68 times.

[0049] Example 2 Taking the relative ratio of plasma albumin to PBUTs in chronic renal failure patients as a reference, 0.66 g BSA and 4.52 mg PCS were dissolved in water to prepare the solution to be separated 1.

[0050] The solution to be separated 1 was dialyzed using a 1000 Da filter membrane (PBS solution), and microbubbles were added to the filter membrane (final concentration 10 7 / mL), and ultrasonic irradiation (ultrasonic conditions were the same as those in Example 1) was performed for 10 min at the 0 h and 1 h time points. Samples of the liquid outside the dialysis membrane were taken at the 0, 1, 2, 3, and 4 time points respectively. The sampled solution (the solution outside the dialysis membrane) was detected by HPLC, and the PCS concentration in the dialysis solution was calculated. The results are as Figure 1 shown.

[0051] It was found that, compared with Comparative Example 1, the dialysis efficiency of PCS in Example 1 was increased by 3.42 times.

[0052] Comparative Example 2 Taking the relative ratio of plasma albumin to PBUTs in chronic renal failure patients as a reference, 0.66 g BSA and 2.51 mg IS were dissolved in water to prepare the solution to be separated 2.

[0053] The solution to be separated 2 was dialyzed using a 1000 Da filter membrane. Samples of the liquid outside the dialysis membrane were taken at the 0, 1, 2, 3, and 4 time points respectively. The sampled solution was detected by HPLC, and the PCS concentration in the dialysis solution was calculated. The results are as Figure 2 shown. After calculation, after 2 hours of dialysis, the protein binding rate of PCS in this system was 84.1%.

[0054] Example 3 Taking the relative ratio of plasma albumin to PBUTs in chronic renal failure patients as a reference, 0.66 g BSA and 2.51 mg IS were dissolved in water to prepare the solution to be separated 2.

[0055] The solution to be separated 2 was dialyzed using a 1000 Da filter membrane (PBS solution), and ultrasonic irradiation was performed for 10 min at the 0 h and 1 h time points. Samples of the liquid outside the dialysis membrane were taken at the 0, 1, 2, 3, and 4 time points respectively. The sampled solution (the solution outside the dialysis membrane) was detected by HPLC, and the PCS concentration in the dialysis solution was calculated. The results are as Figure 1 shown.

[0056] It was found that, compared with Comparative Example 1, the dialysis efficiency of PCS in Example 1 was increased by 6.1 times.

[0057] Example 4 Taking the relative ratio of plasma albumin to PBUTs in chronic renal failure patients as a reference, 0.66 g BSA and 2.51 mg IS were dissolved in water to prepare the solution to be separated 2.

[0058] The solution to be separated 2 was dialyzed using a 1000 Da filter membrane (PBS solution), and microbubbles (final concentration of 10 7 / mL) were added to the filter membrane, and ultrasonic irradiation (ultrasonic conditions were the same as those in Example 3) was performed for 10 min at the 0 h and 1 h time points. Samples of the liquid outside the dialysis membrane were taken at the 0, 1, 2, 3, and 4 time points respectively. The sampled solution (the solution outside the dialysis membrane) was detected by HPLC, and the PCS concentration in the dialysis solution was calculated. The results are as Figure 1 shown.

[0059] It was found that, compared with Comparative Example 1, the dialysis efficiency of PCS in Example 1 was increased by 11.6 times.

[0060] The above examples and comparative examples were experimentally studied using BSA, PCS, and IS as examples. Those skilled in the art can reasonably expect that other albumins and protein-bound toxins are also applicable to the method of the present invention for separation according to the results of the present invention.

[0061] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that: they can still modify the technical solutions described in the foregoing examples, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for separating a protein-binding toxin from a plasma transport protein, characterized in that, Comprising the following steps: Dialyze a solution containing a protein-bound toxin and a plasma transport protein so that the protein-bound toxin is separated outside the dialysis membrane; During the dialysis process, perform ultrasonic treatment or ultrasonic cavitation treatment on the solution.

2. The separation method according to claim 1, characterized in that, The protein-bound toxin includes indoxyl sulfate, p-cresol sulfate, and hippuric acid.

3. The separation method according to claim 1, characterized in that, The plasma transport protein includes albumin.

4. The separation method according to claim 1, wherein The dialysis membrane is used to retain the plasma transport protein and permeate the protein-bound toxin.

5. The separation method according to claim 1, wherein The ultrasonic cavitation includes the following steps: Add microbubbles to the solution and then perform ultrasonic treatment.

6. The separation method according to claim 5, characterized in that, The addition amount of the microbubbles is 10 5 -10 10 / mL.

7. The separation method according to claim 1, wherein The solution includes blood.

8. An apparatus for separating a protein-binding toxin from a plasma transport protein, characterized in that, Comprising a dialysis device and an ultrasonic device; The dialysis device is used to perform dialysis treatment on a solution containing a protein-bound toxin and a plasma transport protein so that the protein-bound toxin is separated outside the dialysis membrane; The ultrasonic device is used to perform ultrasonic treatment on the solution in the dialysis device.

9. The separation device according to claim 8, characterized in that, Further comprising a microbubble device; The microbubble device is used to add microbubbles to the solution in the dialysis device.

10. Use of the separation device for protein-bound toxin and plasma transport protein according to claim 8 or 9 in the preparation of a product for treating chronic kidney disease.

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