Importance of endotoxins in high-flux dialysis.
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Biomedical subjects
Publications and source records attributed to D Falkenhagen.
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As the quality of water in dialysis fluid varies considerably, and, in view of the fact that endotoxin or active derivates can cause acute and chronic side effects in patients under hemodialysis treatment, the dialysis fluid must be sterile and endotoxin-free. The predialyzer fluid in 20 hemodialysis patients was investigated. The bacterial loading was between 5/ml and 12,000/ml, the endotoxin concentration was high and extremely variable. Therefore we introduced the ultrafiltration of the dialysis fluid by a polyamide hollow fiber membrane before entering the dialyzer. All samples were free of bacteria, and the concentration of endotoxin was lower than the detectable limit. With this procedure we can obtain sterile dialysis fluid, which is endotoxin free. Our preliminary results showed that Interleukin-1 in the patients was significantly (p less than 0.005) lower under ultrafiltration of the dialysis fluid than without ultrafiltration.
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.
Leukocyte (PMN) functional capacity has been investigated through evaluation of phagocytosis of opsonised yeast cells in a radiometric test system. The PMN of dialysis patients (DP) had a slightly lower ability to ingest opsonised yeast cells in comparison with normal persons (NP), suggesting that an intrinsic cellular defect may exist. Under the influence of six membranes (cellulose acetate, regenerated cellulose, modified cellulose, cuprophane, polysulphone, and polymethylmethacrylate) the phagocytosis index decreased significantly between 10 and 17% in NP and between 13 and 23% in DP. There is a clear correlation with the membrane surface area. These results are not explained by the number of dead leukocytes (4-6.5% in DP and also in NP independently of membrane contact). The direct membrane effect could be responsible for the diminished phagocytic activity of leukocytes in NP and DP. Aqueous extracts of membranes alone resulted in no change of the phagocytic ability of PMN. Extracellular or 'uraemic factors' were excluded by the test procedure. The killing rate of yeast cells by PMN in NP and DP was not influenced in any of the membranes tested.
Endotoxins are not only important for inducing pyrogenic reactions during haemodialysis but also for the stimulation of different blood cells. This is followed by the release of interleukin-1 and other powerful biological active substances, resulting in a broad spectrum of biological activities. Permeation of LPS (endotoxins) through dialysis and haemofiltration membranes is of great importance. Using a quantitative turbidimetric method based on the LAL (limulus amoebocyte lysate) test LPS concentrations were measured in a closed in vitro system for 11 different dialysers and haemofilters. To assess the passage of endotoxin through the membrane, permeation was measured from the dialysate to the blood side as well as from the blood compartment to the dialysate side.
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Blood compatibility is determined by interactions at the blood-material interface that depend on the material surface chemical structure. Through selective modifications of the chemically reactive hydroxyl groups of cellulose, the aim was to improve the biocompatibility of cellulose membranes. The number of potentially reactive hydroxyl groups on the cellulose membrane surface were reduced through isocyanate cross-links or through the introduction of hydrophilic, hydrophobic, or ionic functionalities by graft copolymerization. To assess blood compatibility, levels of C3a desArg were determined in plasma after membrane contact. Using the electrophoretic mobility test, the release of cytokines were measured after in-vitro incubation of mononuclear cells with membranes. Adsorption of 131J-human fibrinogen was additionally investigated. With respect to the biocompatibility parameters selected, the modified cellulose membranes show improved in-vitro blood compatibility in comparison to unmodified cellulose membranes.
Considering the plasma colloid osmotic pressure (COP) as a possible parameter for the monitoring of dialysis treatment compatibility, a characteristic time course was found. The COP and the total protein concentration very often do not increase significantly during the first treatment hour in spite of ultrafiltration. An increase in the plasma sodium concentration, which was higher than expected, was found to be the reason for a plasma dilution effect. This can be explained by a transcapillary sodium transfer coefficient which is not infinitely high as assumed in single-pool sodium modelling. From a 2-pool model considering the plasma volume as a separate pool and including capillary filtration time courses for plasma sodium, total protein concentration and COP could be calculated, which was very similar to the measured curves.
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Due to their partial permeability and their good mechanical properties, the symplex capsules are technically suited for an application in extracorporal detoxification. By this newly developed procedure the activity of encapsulated enzymes is considerably increased; thus, application for extracorporal detoxification seems to be advisable.
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Rat liver microsomes were microencapsulated in a pure aqueous medium by means of a new technique. The wall of the microcapsules consists of a semipermeable simplex membrane which is stabilized mainly by electrostatic interactions between a polymeric polyanion (sodium cellulose sulphate) and a polymeric polycation (polydimethyldiallylammonium chloride). The metabolic as well as the mechanic parameters of the microcapsules could be markedly improved by separating the metabolic (liver microsomes) from the membrane component (sodium cellulose sulphate) in such a way that two distinct compartments are formed during the preparation of the microcapsules.
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Normal leukocyte functional capacity was investigated by evaluation of phagocytosis of opsonised yeast cells in a radiometric test system. After incubation with dialysis membranes (different cellulosic membranes, polysulfon membrane (PS), polymethylmetacrylate membrane (PMMN), the phagocytosis index, expressed as percent decrease with respect to initial values without membrane, decreased by 10%-25%. The most pronounced effect was observed with PS, cuprophane, modified cellulose and PMMA. The results are not related to differences in the viability of PMN during the test procedure; dead PMN amounted to about 4-6.5%. A significant increase in beta-NAG and beta-Gluc activities was released in the supernatants of the phagocytosis suspensions. This increase activity can be explained by the phagocytosis of PMN but it was not influenced by membrane contact. There was no influence of membrane contact or phagocytosis activity of PMN on the beta 2 M concentration in the supernatant demonstrating that no in vitro generation during incubation with either membrane exists.
The 1-pool-model of sodium kinetics during hemodialysis is based upon the assumption of an immediate compensation of osmotic shifts. This assumption is not supported by measurements of plasma sodium, total protein concentration and colloid osmotic pressure kinetics. When a high dialysate sodium concentration is applied, an inflow of sodium into the plasma space occurs, which results in an osmotic suction and thus a plasma dilution. These conditions can be represented by a 2-pool-model taking into consideration capillary filtration. The results indicate that following the first treatment period the sodium kinetics are sufficiently explained by a 1-pool-model with the total body water as distribution volume. Both the plasma sodium concentration and the eliminated sodium at the end of a hemodialysis treatment can be described to an acceptable level by the 1-pool-model. The input of the measured in-vivo sodium dialysance value (or alternatively the urea clearance) is necessary.
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