| Albumin dialysis, known as Molecular Adsorbents Recycling System (MARS), is an effective and widely studied detoxification treatment for liver failure. Albumin dialysis is derived by adding albumin to the dialysate of extracorporeal hemodialysis. Both water-soluble and protein-bound toxins can be simultaneously removed by this treatment. The effectiveness of MARS in eliminating toxins has been confirmed by many clinical studies. Human serum albumin (HSA) is the key to the removal of protein-bound toxins by MARS, but HSA is isolated from human serum, and is therefore expensive as well as in short supply. To reduce the cost, limited dosage of HSA is used in MARS, and it has to be regenerated in situ. Due to the incomplete regeneration of HSA, the capacity and rates of MARS to remove protein-bound toxins decline over time. Furthermore, albumin regeneration system makes MARS very complicated and expensive, which has limited its clinical application, especially in developing countries. Thus developing an inexpensive water-soluble molecular adsorbent as an alternative to HSA is very attractive. This work represents the development of an efficient and inexpensive water-soluble molecular adsorbent for removing plasma bilirubin.Seven bilirubin adsorbents were synthesized by grafting different ligands (quaternary ammonium, hydrophobic chain, β-CD) onto water-soluble polymers (chitosan, dextran, polyethyleneimine). Among the seven adsorbents, β-CD-PEI exhibited the highest adsorption capacity for bilirubin. In a plasma dialysis system,150mL plasma (with300mg/L bilirubin) was dialyzed with200mL of1%(w/v) adsorbent-spiked dialysate, bilirubin adsorption capacity of2.84mg/g was achieved by β-CD-PEI in1h. By optimizing the synthesis of β-CD-PEI, its bilirubin adsorption capacity further increased to3.55mg/g. Therefore, β-CD-PEI was chosen for subsequent studies.13C NMR results indicated that each β-CD-PEI has366branching points and51β-CD functional groups. The average molecular weight of β-CD-PEI was-157kD.In order to understand the mechanism associated with the binding of bilirubin to β-CD at molecular level, dockings of bilirubin to α-, β-, γ-CD were carried out using AutoDock Vina1.1program. The results indicated that Bilirubin was too big to insert into α-CD, so its binding energy with bilirubin was the weakest among the CD family. Bilirubin-(β-CD)2complex was most favorable in term of binding energy. The structural change of β-CD caused by the grafting reaction did not hinder its binding with bilirubin. The best binding energy between bilirubin and HSA was higher than that of bilirubin-(β-CD)2complex. This result demonstrated that two β-CD molecules under the lowest binding energy could competitively bind bilirubin bound by HSA. The1:2binding model for bilirubin and β-CD was also confirmed by Benesi-Hildebrand experiments.The highest bilirubin clearance (35.8%) was achieved by adopting the optimal flow rate of300mL/min for plasma and50mL/min for dialysate. Cross-membrane ultrafiltration enhanced concentration polarization in plasma during the dialysis, and increased bilirubin transfer resistance. The bilirubin clearance of Lengthen LST140dialyzer was stronger than Nipro Sureflux-130G dialyzer. The albumin adsorbed on the surface and pores of membrane can improve the transfer of bilirubin during dialysis. Bilirubin removal was fast in β-CD-PEI-spiked dialysis, with90%of total bilirubin removed in first4h.β-CD-PEI-spiked dialysis could achieve high bilirubin clearance in plasma with different initial concentrations of bilirubin. Bilirubin clearance increased with increases in β-CD-PEI dosage, and high β-CD-PEI concentration was more favorable for bilirubin removal. About44.8%of bilirubin (140.4mg/L) was removed from200mL plasma by1L dialysate spiked with4%β-CD-PEI in6h. β-CD-PEI exhibited a significantly higher bilirubin clearance than BSA (P<0.05), which is an analogue of HSA, therefore demonstrating its strong bilirubin-binding ability. Both TBA and the three aromatic amino acids could be removed by β-CD-PEI-spiked dialysis without influencing the concentrations of plasma proteins and ions.A quantitative description of bilirubin removal during β-CD-PEI-spiked dialysis was established by means of a mathematical model. Model development and validation are based on in vitro data of bilirubin concentration acquired in five sessions at different times during each session. The accuracy of the model in reproducing real data is high, and error of concentration of plasma bilirubin is less than5%. This model could be used to predict treatment outcomes in experiments with different initial concentrations of bilirubin and different dosages of β-CD-PEI.To summarize, the water-soluble molecular adsorbent β-CD-PEI described in this study offers an efficient alternative to albumin for removing bilirubin from plasma. Since β-CD-PEI is much cheaper than HSA, it can reduce the economic burden for the patients. Almost unlimited ability to remove toxins could be achieved by continuously replacing the saturated adsorbent with new ones. No additional adsorbent regeneration devices are needed. Therefore, the equipment for the set up of β-CD-PEI-spiked dialysis can be greatly simplified compared with that of MARS. The results presented in this study demonstrate that β-CD-PEI, a water-soluble adsorbent, can effectively remove plasma bilirubin, and therefore may have potential application in a dialysis system.
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