Structure and permeability of dialysis membranes sterilized by various methods

Appropriate design of dialysis membranes requires correct values of structural parameters such as pore radius, surface porosity, water content and tortuosity. It is impossible, however, to find the pore radius of dialysis membranes using a mercury porosimeter or an electron microscope because the pores are only several tens of angstroms in radius. Consequently, diffusional and convectional procedures such as the Lw method that uses both pure water permeability and water content, the Lw and Pm method which uses both solute and pure water permeability, and the sigma method which uses the reflection coefficient, have been employed extensively to determine the pore radius of dialysis membranes. The structural parameters of symmetric membranes are determined from water content, solute and pure water permeability data based on the Lw and Pm method combined with the tortuous pore model we have proposed. Determination of the structural parameters of microfiltration membranes for plasma separation having huge pores by various methods is required to verify the method of characterizing dialysis membranes based on the tortuous pore model. A dyeing method is also suitable for characterizing dialysis membranes of regenerated cellulose. Pore model studies using permeability and water content data for sterilized and treated membranes facilitate optimal design of dialysis membranes for clinical applications.     

利用聚丙烯酰胺凝胶电泳纯化的微量蛋白制备抗血清

目的　以SDS PAGE纯化的微量蛋白制备抗人层粘连蛋白受体前体蛋白 (LRP)的血清。方法　以SDS PAGE分离纯化原核表达的人LRP与 6×组氨酸的融合蛋白 ,切下并粉碎融合蛋白所在凝胶块。将含 10 μg融合蛋白的凝胶颗粒与弗氏佐剂混匀后 ,免疫BALB/c小鼠。以Westernblot检测所获抗血清的滴度和特异性。结果　以胃癌细胞总蛋白为靶抗原时 ,Westernblot显示 ,所获抗血清仅识别 1条Mr 约 4 2 0 0 0的蛋白带 ,将抗血清做 1∶2 0 0 0稀释后 ,仍能清晰地显示其靶抗原所在位置。结论　以聚丙烯酰胺凝胶颗粒为载体 ,用微量蛋白即可成功地制备抗血清     

Amount of adsorbed albumin loss by dialysis membranes with protein adsorption

Polymethylmethacrylate (PMMA) membrane is the first synthetic polymeric hollow fiber used in dialyzers that is known to adsorb β₂-microglobulin. Polyester polymer alloy (PEPA), a blend of two polymers, i.e., polyarylate and polyethersulfone, is another dialysis membrane material with adsorption characteristics. In this study, the adsorption and permeation characteristics of BG-1.6PQ (PMMA) and FLX-15GW (PEPA) dialyzers were investigated by performing ultrafiltration experiments using chymotrypsinogen (molecular weight 25 000) and albumin (molecular weight 66 000) as test solutes. Although PMMA and PEPA had the same sieving coefficient for chymotrypsinogen at steady state, PMMA showed approximately 20% higher fractional adsorption than PEPA under the same initial concentrations. The fractional adsorption for albumin was approximately 20% in PEPA regardless of the ultrafiltration flow rate. The fractional adsorption for albumin in PMMA, however, increased as the ultrafiltration flow rate increased and reached 50%–60% after 10 h. Since PEPA has two skins, one inside and one outside the hollow fiber, proteins may have been adsorbed mainly by these two layers. However, since PMMA is a uniform membrane and since the higher the ultrafiltration flow rate, the higher the fractional adsorption found in PMMA, adsorption may be the result of the occlusion of the dense structure of the membrane. The amount of albumin loss is often clinically evaluated by measuring the amount of permeated albumin in the dialysate. However, when dialyzers with adsorption characteristics are examined, the loss by adsorption should also be taken into account.     

Hydraulic permeability of hollow-fiber membranes

Two experimental methods have been developed to characterize the pressure-driven flow of a liquid through the walls of single hollow-fiber membranes. In one method, the permeation rate is measured directly by following the air-water interface in a pressurized pipet connected to a hollow fiber which is sealed at one end. With the second method, the permeation rate is determined by tracking the decent of an air bubble inside a pressurized hollow fiber sealed at one end. Results obtained by the two experimental methods are in good agreement. Darcy's law is used to analyze the experimental data because the Darcy permeability constant, k is an intrinsic property of the material. The membrane dimensions and the Darcy permeability constant for reconstituted collagen hollow fibers are shown to be quite sensitive to hydrogen ion concentration and ionic strength. A conceptual link between the Darcy permeability constant and the membrane microstructure is obtained by combining the the aligned-rod structural model for reconstituted collagen proposed by Kramer with a hydrodynamic calculation due to Happel and electrical double-layer theory. On the average, the predictions of this model for volume fraction solids and microstructure dimensions are in reasonable agreement with experimental observations.     

高通量透析膜在血液透析中的应用

背景：高通量膜具有很高的通透性及溶质转运性能,透析中能有更多相对分子质量较大的溶质从血液转运到透析液中,故称之为高通量透析,属于一种高效血液净化治疗方法。 目的：总结近年高通量透析膜在血液透析中的应用现状。 方法：由作者应用计算机检索维普数据库,检索时限2000-02/2008-10。检索关键词：高通量透析膜;血液透析;临床应用。纳入标准：①高通量透析膜在血液透析中的应用。②实验结果叙述与材料有关的临床应用问题。排除标准：重复研究或较陈旧文献。根据纳入排除标准共保留相关文献12篇。 结果与结论：与低通量血液透析相比可清除β2微球蛋白,降低透析患者血脂、血磷及甲状旁腺素水平,减轻炎症反应、氧化应激及血管内皮变化,不影响维持性血液透析患者血浆细胞因子及内毒素水平,对尿素氮、肌酐等小分子的清除也优于常规透析。因此,高通量透析可预防血液透析患者的远期并发症如肾性骨病,动脉硬化,并可改善食欲不振,高血压症状,提高透析的充分性。     

Complement activation by cellulosic dialysis membranes.

AIMS--To assess the effect of cellulosic dialysis membranes on the production of complement degradation products to determine to the role of the classical pathway. METHOD--Complement activation was studied in 33 patients during a single haemodialysis session using cellulosic membranes. Pre- and post-dialysis plasma EDTA valves of C3, C4, C3dg, C4d and C reactive protein (CRP) were measured. Statistical analysis was done using the Wilcoxon signed rank test. RESULTS--Post-dialysis C4 (p = 0.0003), C3dg (p < 0.0001), and C4d (p = 0.003) concentrations were increased compared with pre-dialysis values. There was no significant change in C3 (p = 0.095) and CRP (p = 0.13) values. Post-dialysis C3dg and C4d concentrations correlated significantly (p = 0.007). IgG, an undialysed molecule, was quantified and post-dialysis valves were significantly higher than those before dialysis (p = 0.0002), indicating a degree of haemoconcentration. To remove this effect, the C3:IgG, C4:IgG, C3dg:IgG, C4d:IgG and CRP:IgG ratios were calculated. Compared with pre-dialysis values, post-dialysis C3dg:IgG and C4d:IgG ratios were increased and C3:IgG decreased significantly. No change was observed in C4:IgG and CRP:IgG ratios. CONCLUSION--This study confirms that significant complement activation takes place following dialysis with cellulosic membranes. This is denoted by an increase in C3dg. This was paralleled by a rise in C4d, implying a contributory role for the classical pathway. Concomitant post-dialysis increases in IgG and C4 indicate a degree of haemoconcentration; but removal of this effect shows that C3dg and C4d are increased following dialysis--suggesting classical, in addition to alternative, pathway activation.     

Complement activation and hypersensitivity reactions to dialysis membranes

Certain patients receiving hemodialysis experience recurrent chest pain, dyspnea, and hypotension during exposure to new cuprophane-membrane dialyzers (the "first-use syndrome"). Because activation of complement may be involved in these events, we examined in vivo complement activation with new cuprophane membranes and in vitro activation by zymosan in 6 such patients, and compared them with 10 patients who did not have symptoms during dialysis. All patients with the first-use syndrome had maximal complement activation 10 minutes after initiation of dialysis, with C3a des-arginine (desArg), the stable metabolite of C3 activation, equal to 8533 +/- 157 ng per milliliter (mean +/- S.E.M.). In asymptomatic patients the maximal C3a desArg value occurred at 15 minutes and was only 2907 +/- 372 ng per milliliter (P less than or equal to 0.0001). At a concentration of 3.8 x 10(-5) g of zymosan per milliliter, patients with the first-use syndrome had a C3a desArg level of 29.6 +/- 1.4 micrograms per milliliter, whereas it was only 16.6 +/- 2.3 micrograms per milliliter in asymptomatic patients (P less than or equal to 0.0001). Two other patients, who experienced cardiopulmonary collapse during the first two minutes of dialysis, had a C3a desArg level of 18,900 and 7800 ng per milliliter, respectively. We conclude that the occurrence of adverse symptoms associated with new cuprophane-membrane dialyzers correlates with complement activation.     

Ionic liquid-derived blood-compatible composite membranes for kidney dialysis

A novel heparin- and cellulose-based biocomposite is fabricated by exploiting the enhanced dissolution of polysaccharides in room temperature ionic liquids (RTILs). This represents the first reported example of using a new class of solvents, RTILs, to fabricate blood-compatible biomaterials. Using this approach, it is possible to fabricate the biomaterials in any form, such as films or membranes, fibers (nanometer- or micron-sized), spheres (nanometer- or micron-sized), or any shape using templates. In this work, we have evaluated a membrane film of this composite. Surface morphological studies on this biocomposite film showed the uniformly distributed presence of heparin throughout the cellulose matrix. Activated partial thromboplastin time and thromboelastography demonstrate that this composite is superior to other existing heparinized biomaterials in preventing clot formation in human blood plasma and in human whole blood. Membranes made of these composites allow the passage of urea while retaining albumin, representing a promising blood-compatible biomaterial for renal dialysis, with a possibility of eliminating the systemic administration of heparin to the patients undergoing renal dialysis.     

Comparison of hemostasis with two high-flux hemocompatible dialysis membranes

Eight adults with chronic renal failure were dialyzed using polyacrylonitrile (AN 69) or polysulfone (PS) membranes with a high (HHR) or low (LHR) continuous non-fractionated heparin regimen--a total of either 90 or 50 IU/kg body weight. With the HHR, for a mean anti-Xa (aXa) activity of around 0.40 IU/ml, no plasma activation of coagulation was observed; fibrinopeptide A (FPA) was in agreement with the residual blood volume (RBV) and the state of the bubble trap, especially with the PS membrane. With the LHR, for a mean aXa below 0.21 IU/ml, there was only moderate activation of coagulation. The PS membrane gave different results from the AN 69 membrane, RBV values on the HHR and aXa being lower on both the HHR and LHR, with FPA values being regularly lower on the LHR. The decrease in plasma beta-TG on the LHR was more marked with the PS than with the AN 69 membrane due to loss on dialysis or adsorption, as shown by the arterio-venous difference. The increase in plasma PF4 was related to the effect of heparin. However, there was no platelet activation. On the LHR, platelet count and intraplatelet beta-TG and PF4 levels remained very stable. The two high-flux membranes were very hemocompatible and require only low doses of heparin, but the dialyzer with AN 69 membrane need its geometry improving.     

Effect of ionenes on ion permeability of erythrocyte membranes

Ionophore activity of ionenes, like that of polymyxins, is shown to be potentiated by long-chain antiions. Rapid stoichiometric anion equilibration in isoosmotic buffered sugar medium revealed that the amplitude of Cl⁻/OH⁻ exchange in erythrocytes induced by an ionene-detergent complex gradually drops with the decrease of both the charge density and the number of monomeric moieties in the polymeric chain. This is also paralleled by passive K⁺ leakage from the cells. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 7, pp. 44–46, July, 1994     

Investigation of endotoxin adsorption with polyether polymer alloy dialysis membranes

Endotoxin (ET) in the dialysate is known to be adsorbed by dialysis membranes made of polyether polymer alloy (PEPA) and polymethylmethacrylate (PMMA). In the present study, the effect of polyvinylpyrrolidone (PVP) localization of the PEPA dialysis membrane on the adsorption of ET was investigated. The compounding of PVP in the PEPA membrane was changed, and hydrophobic membrane in both blood side and dialysate side, and hydrophilic membrane in only the blood side were used. Adsorption was evaluated by filling the contaminated dialysate inside and outside the membrane after priming with physiological saline, and determining the ET concentration in the blood side and dialysate side of dialysis membrane during the 240 min period from the start of filling the contaminated dialysate. With the PEPA membranes investigated, ET was significantly adsorbed to the hydrophobic side and was not adsorbed to the blood side of hydrophilic type membrane. These results suggest that in addition to electrostatic action attributable to the compounding of hydrophilic agent PVP to the dialysis membrane, the distribution of PVP that was compounded and the potential of the membrane itself may cause differences in adsorption of ET.     

Evaluation by SDS-PAGE and immunoblotting of residual antigenicity in hydrolysed protein formulas

Background Extensively hydrolysed protein formulas are widely used as an alternative diet for children with cow's milk allergy. Partially hydrolysed protein formulas have been noted in some studies as useful in the prevention of allergy in infants at high risk of atopy. Although normally well tolerated, these ‘hypoallergenic’ products have been reported to cause serious immunological reactions in very sensitive subjects. Objective Starting from these considerations, we studied some commercial hydrolysed formulas in search of biological data supporting the observed clinical reactions. Methods We set up an electrophoretic method sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) which allowed us to study the molecular weight of peptides contained in hydrolysed products. Then, using the immunoblotting technique we evaluated the reactivity of circulating IgE (from serum of children allergic to cow's milk proteins) with the residual intact proteins and with the peptides present in these formulas. Results Both group of milk proteins (caseins and whey proteins) were important allergens for children included in this study. The presence of high-molecular polypeptides was shown in partial hydrolysed formulas as such and in extensive hydrolysed products after protein enrichment by trichloroacetic acid (TCA) precipitation. Intact residual proteins were mainly responsible for the formation of FgE-antigen complexes observed in immunoblotting. More rarely, polypeptides of partial hydrolysed formulas were involved in immunological responses. Conclusions Both partial and extensive hydrolysed formulas could induce clinical reactions in very sensitive subjects. These responses are mainly associated with allergy to the small amounts of residual intact proteins.     

Influence of dialysis membranes on the convective transport of middle molecules

Ultrafiltrates from 10 patients in chronic maintenance hemodialysis (7 males and 3 females) were obtained simultaneously using three different membranes: cuprophan, polyacrilonitrile and polysulfone. Middle molecules (MM) chromatographic profiles and total MM amount were determined by gel chromatography and high performance liquid chromatography. The convective transport of MM was similar and not membrane related. Hemofiltration, a predominantly convective solute transfer procedure using high flux membranes such as polyacrilonitrile and polysulfone, in which large amounts of fluids have to be ultrafiltered, is an effective form of MM removal.     

Preparation and permeability properties of chitosan membranes having hydrophobic groups

Membranes consisting of N-acylated chitosan and carrying hydrophobic groups were prepared and the states of water in these membranes were examined by differential scanning calorimetry (DSC). The contents of freezable and non-freezable water were found to be closely related with the structure and properties of the membranes. On the other hand, their permeability properties for sodium salts are found to be affected considerably by modifying the membrane structure as well as the cationically charged groups. With respect to D ,L -amino acids, the selective permeation of the L -isomer was enhanced in the hydrophobic chitosan membrane, suggesting the possibility to separate optical isomers by membrane transport.     

Two dimensional Blue Native-/SDS-PAGE analysis of SLP family adaptor protein complexes

SH2 domain containing leukocyte protein (SLP) adaptor proteins serve a central role in the antigen-mediated activation of lymphocytes by organizing multiprotein signaling complexes. Here, we use two dimensional native-/SDS-gel electrophoresis to study the number, size and relative abundance of protein complexes containing SLP family proteins. In non-stimulated T cells all SLP-76 proteins are in a approximately 400 kDa complex with the small adaptor protein Grb2-like adaptor protein downstream of Shc (Gads), whereas half of Gads is monomeric. This constitutive SLP-76/Gads complex could be reconstituted in Drosophila S2 cells expressing both components, suggesting that it might not contain additional subunits. In contrast, in B cells SLP-65 exists in a 180 kDa complex as well as in monomeric form. Since the complex was not found in S2 cells expressing only SLP-65, it was not di/trimeric SLP-65. Upon antigen-stimulation only the complexed SLP-65 was phosphorylated. Surprisingly, stimulation-induced alteration of SLP complexes could not be detected, suggesting that active signaling complexes form only transiently, and are of low abundance.     

Detection of charges and their distribution on dialysis membranes with cationic/anionic dyes using confocal laser scanning microscopy.

Methods for the detection of positive or negative charges on the surface of biomaterials/membranes and inside a membrane are important for the characterisation of such materials. We tested different dyes and optimized staining procedures. Under standardized conditions negatively charged membranes were stained with cationic triarylmethane compounds such as crystal violet and positively charged membranes with the anionic anthraquinone dye anthralan blue B. There was no staining of uncharged cellulose membranes. The applicability of these methods was demonstrated on membranes coated to varying degrees with charged compounds such as heparin, these changes in charge being detectible quantitatively by photometry. The distribution of charges inside a membrane was detected by optical sectioning across the stained (FITC labelled poly-L-lysine) membrane using confocal laser scanning microscopy (LSM). LSM offers a completely new application possibility in biomaterial and biocompatibility research.