Disclosure of Invention
The first objective of the present invention is to provide a method for separating protein-bound toxins from plasma transport proteins, which solves the above-mentioned problems.
A second object of the present invention is to provide a separation device for protein-bound toxins from plasma transport proteins.
A third object of the present invention is to provide the use of the above-mentioned separation device for protein-bound toxins and plasma transport proteins for the preparation of a product for the treatment of chronic kidney disease.
In order to achieve the above object, the following technical scheme is adopted:
in a first aspect, the present invention provides a method of separating a protein-binding toxin from a plasma transporter, comprising the steps of:
dialyzing the solution containing the protein-bound toxin and the plasma transport protein so that the protein-bound toxin is separated out of the dialysis membrane;
And carrying out ultrasonic treatment or ultrasonic cavitation treatment on the solution in the dialysis process.
As a further technical scheme, the protein-binding toxins include indoxyl sulfate, p-cresol sulfate, and hippuric acid.
As a further aspect, the plasma transporter comprises albumin.
As a further technical solution, the dialysis membrane is used for intercepting plasma transport proteins, and is used for binding toxins through proteins.
As a further technical scheme, the ultrasonic cavitation comprises the following steps:
microbubbles are added to the solution and then sonicated.
As a further technical scheme, the addition amount of the microbubbles is 105-1010/mL.
As a further aspect, the solution comprises blood.
In a second aspect, the invention provides a separation device for protein-bound toxins from plasma transport proteins, comprising a dialysis device and an ultrasound device;
the dialysis device is used for carrying out dialysis treatment on a solution containing protein-bound toxins and plasma transport proteins, so that the protein-bound toxins are separated out of the dialysis membrane;
the ultrasonic device is used for carrying out ultrasonic treatment on the solution in the dialysis device.
As a further technical scheme, the device also comprises a micro-bubble device;
the microbubble device is used for adding the microbubbles to the solution in the dialysis device.
In a third aspect, the invention provides the use of a device for separating a protein-bound toxin from a plasma transport protein as described above in the manufacture of a product for the treatment of chronic kidney disease.
Compared with the prior art, the invention has the following beneficial effects:
The inventor researches find that the ultrasonic treatment or ultrasonic cavitation treatment is carried out in the dialysis process of the protein-bound toxin, so that the separation of the protein-bound toxin and plasma transport protein can be obviously promoted, and the dialysis efficiency of the protein-bound toxin can be improved. The separation method of the protein-bound toxin and the plasma transport protein is simple and convenient, has low cost and can effectively improve the dialysis efficiency of the protein-bound toxin.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to embodiments and examples, but it will be understood by those skilled in the art that the following embodiments and examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The specific conditions are not specified, and the process is carried out according to conventional conditions or conditions suggested by manufacturers. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Plasma transport proteins are a class of proteins responsible for binding, carrying and transporting specific substances in the blood circulation.
In a first aspect, the present invention provides a method of separating a protein-binding toxin from a plasma transporter, comprising the steps of:
dialyzing the solution containing the protein-bound toxin and the plasma transport protein so that the protein-bound toxin is separated out of the dialysis membrane;
And carrying out ultrasonic treatment or ultrasonic cavitation treatment on the solution in the dialysis process.
The inventor researches find that the non-covalent combination of the plasma transport protein and the protein-binding toxin can be opened by carrying out ultrasonic treatment or ultrasonic cavitation treatment in the dialysis process of the protein-binding toxin, the separation of the protein-binding toxin and the plasma transport protein is promoted, and the dialysis efficiency of the protein-binding toxin is improved. The separation method of the protein-bound toxin and the plasma transport protein is simple and convenient, has low cost and can effectively improve the dialysis efficiency of the protein-bound toxin.
In some alternative embodiments, the protein-binding toxins include, but are not limited to indoxyl sulfate, p-cresol sulfate, and hippuric acid, as well as other protein-binding toxins that are well known to those of skill in the art.
In some alternative embodiments, the plasma transporter includes, but is not limited to, albumin, but may also be other plasma transporters known to those of skill in the art.
In some alternative embodiments, the dialysate is pure water, PBS solution, or commercial dialysate used in clinical hemodialysis, or the like.
In some alternative embodiments, the dialysis membrane is used to entrap plasma transport proteins, permeate proteins, and bind toxins.
In some alternative embodiments, the dialysis membrane has a molecular weight cut-off of 500-10000Da.
In some alternative embodiments, the ultrasonic cavitation comprises the steps of:
Microbubbles (in the present invention, microbubbles refer to bubbles having a particle size of 2 to 8 uM) were added to the solution, followed by ultrasonic treatment.
In some alternative embodiments, the amount of microbubbles added is 105-1010/mL (ultrasonic cavitation can be achieved by the presence of microbubbles in the solution, and the amount of microbubbles added can be selected according to the actual situation).
In some alternative embodiments, the solution comprises blood.
In a second aspect, based on the above separation method, the present invention provides a separation device for protein-bound toxins from plasma transport proteins, comprising a dialysis device and an ultrasound device;
the dialysis device is used for carrying out dialysis treatment on a solution containing protein-bound toxins and plasma transport proteins, so that the protein-bound toxins are separated out of the dialysis membrane;
the ultrasonic device is used for carrying out ultrasonic treatment on the solution in the dialysis device.
The separation device has a simple structure, and can realize the efficient separation of protein-bound toxin and plasma transport protein.
In some alternative embodiments, the protein-binding toxins include, but are not limited to indoxyl sulfate, p-cresol sulfate, and hippuric acid, as well as other protein-binding toxins that are well known to those of skill in the art.
In some alternative embodiments, the plasma transporter includes, but is not limited to, albumin, but may also be other plasma transporters known to those of skill in the art.
In some alternative embodiments, a microbubble device is also included;
the microbubble device is used for adding the microbubbles to the solution in the dialysis device.
In some alternative embodiments, the dialysis device comprises a dialysis bag.
In some alternative embodiments, the ultrasound device comprises an ultrasound illuminator.
In a third aspect, the invention provides the use of a device for separating a protein-bound toxin from a plasma transport protein as described above in the manufacture of a product for the treatment of chronic kidney disease.
The separation device provided by the invention can effectively separate the protein-bound toxin from the plasma transport protein, so that the separation device can be used for dialysis treatment of chronic kidney disease patients.
The invention is further illustrated by the following specific examples and comparative examples, but it should be understood that these examples are for the purpose of illustration only and are not to be construed as limiting the invention in any way.
In the following examples and comparative examples, the 0 time point is the immediate sampling after the dialysate replacement, the 1 time point is the immediate sampling after 10min of the first sampling or 10min of the first ultrasound, the 2 time point is the sampling after 1 hour of dialysis, the 3 time point is the immediate sampling after 10min of continuing dialysis or 10min of the second ultrasound, and the 4 time point is the sampling after 2 hours of dialysis.
Comparative example 1
Solution 1 to be separated was prepared by dissolving 0.66g BSA and 4.52 mg PCS in water, taking the relative ratio of plasma albumin to PBUTs of a patient with chronic renal function as a reference.
And dialyzing the solution 1 to be separated by using a 1000Da filter membrane, sampling liquid outside the dialysis membrane at time points of 0,1, 2,3 and 4 respectively, detecting the sampled liquid by using HPLC, and measuring and calculating the PCS concentration in the dialysis liquid, wherein the result is shown in figure 1. The protein binding rate of PCS in this system was calculated to be 86.7% after 2 hours of dialysis.
Example 1
Solution 1 to be separated was prepared by dissolving 0.66g BSA and 4.52 mg PCS in water, taking the relative ratio of plasma albumin to PBUTs of a patient with chronic renal function as a reference.
The solution 1 to be separated is dialyzed (PBS solution) by a 1000Da filter membrane, ultrasonic irradiation is carried out for 10min at the time points of 0h and 1h, liquid outside the dialysis membrane is sampled at the time points of 0,1, 2, 3 and 4 respectively, the sampled liquid (the solution outside the dialysis membrane) is detected by HPLC, and the PCS concentration in the dialysis liquid is calculated. The results are shown in FIG. 1.
As a result, it was found that the dialysis efficiency of the PCS of example 1 was improved by a factor of 1.68 as compared with comparative example 1.
Example 2
Solution 1 to be separated was prepared by dissolving 0.66g BSA and 4.52 mg PCS in water, taking the relative ratio of plasma albumin to PBUTs of a patient with chronic renal function as a reference.
The solution 1 to be separated is dialyzed (PBS solution) by using a 1000Da filter membrane, microbubbles (the final concentration is 107/mL) are added into the filter membrane, ultrasonic irradiation (the ultrasonic conditions are the same as in the example 1) is carried out for 10min at the time points of 0h and 1h, the liquid outside the dialysis membrane is sampled at the time points of 0, 1, 2,3 and 4 respectively, the sampled liquid (the solution outside the dialysis membrane) is detected by HPLC, and the PCS concentration in the dialysate is calculated. The results are shown in FIG. 1.
As a result, it was found that the dialysis efficiency of the PCS of example 1 was improved by a factor of 3.42 as compared with comparative example 1.
Comparative example 2
Solution 2 to be separated was prepared by dissolving 0.66g of BSA and 2.51 mg IS in water, taking the relative ratio of plasma albumin to PBUTs of a patient with chronic renal function as a reference.
And dialyzing the solution 2 to be separated by using a 1000Da filter membrane, sampling liquid outside the dialysis membrane at time points of 0,1, 2,3 and 4 respectively, detecting the sampled liquid by using HPLC, and measuring and calculating the PCS concentration in the dialysis liquid, wherein the result is shown in figure 2. The protein binding rate of PCS in this system was calculated to be 84.1% after 2 hours of dialysis.
Example 3
Solution 2 to be separated was prepared by dissolving 0.66g of BSA and 2.51 mg IS in water, taking the relative ratio of plasma albumin to PBUTs of a patient with chronic renal function as a reference.
The solution 2 to be separated is dialyzed (PBS solution) by a 1000Da filter membrane, ultrasonic irradiation is carried out for 10min at the time points of 0h and 1h, liquid outside the dialysis membrane is sampled at the time points of 0,1, 2, 3 and 4 respectively, the sampled liquid (the solution outside the dialysis membrane) is detected by HPLC, and the PCS concentration in the dialysis liquid is calculated. The results are shown in FIG. 1.
As a result, it was found that the dialysis efficiency of the PCS of example 1 was improved by 6.1 times as compared with comparative example 1.
Example 4
Solution 2 to be separated was prepared by dissolving 0.66g of BSA and 2.51 mg IS in water, taking the relative ratio of plasma albumin to PBUTs of a patient with chronic renal function as a reference.
The solution 2 to be separated is dialyzed (PBS solution) by using a 1000Da filter membrane, microbubbles (the final concentration is 107/mL) are added into the filter membrane, ultrasonic irradiation (the ultrasonic conditions are the same as in the example 3) is carried out for 10min at the time points of 0h and 1h, the liquid outside the dialysis membrane is sampled at the time points of 0, 1, 2,3 and 4 respectively, the sampled liquid (the solution outside the dialysis membrane) is detected by HPLC, and the PCS concentration in the dialysate is calculated. The results are shown in FIG. 1.
As a result, it was found that the dialysis efficiency of the PCS of example 1 was improved by 11.6 times as compared with comparative example 1.
Experimental studies were performed using BSA with PCS and IS as examples and comparative examples above, and one skilled in the art would reasonably expect that other albumin and protein-bound toxins would also be suitable for isolation by the methods of the present invention based on the results of the present invention.
It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that the technical solution described in the above embodiments may be modified or some or all of the technical features may be equivalently replaced, and these modifications or substitutions do not make the essence of the corresponding technical solution deviate from the scope of the technical solution of the embodiments of the present invention.