High-performance liquid chromatography as a technique to measure the competitive adsorption of plasma proteins onto latices

Isotherms of human serum albumin (HSA), human immunoglobulin G (HIgG), and human fibrinogen (HFb) onto a polystyrene (PS)-latex were determined by depletion of protein in the solution, which was either followed by radioactivity measurements or by UV spectroscopy. Different adsorption isotherms for the same protein were obtained when either radioactivity measurements or UV spectroscopy was used as a detection technique. In order to obtain reliable results from competitive protein adsorption experiments, a method based on the use of high-performance liquid chromatography was developed. A strong preferential adsorption of HFb was observed when adsorption studies were carried out with mixtures of HSA, HFb, and HIgG. When adsorption studies were carried out with solutions containing HSA monomer and dimer, a strong preferential adsorption of HSA dimer was also observed

Dynamic aspects of contact angle measurements on adsorbed protein layers

Contact angle measurements using drops of paraffin oil have been performed on polystyrene (PS) substrates, coated with human serum albumin (HSA) or human fibrinogen (HFb), immersed in buffer solution. The contact angle appeared to be time dependent. The final value for HSA-coated substrates was 50° ± 5° and for HFb-coated substrates 130° ± 10° (measured through the oil phase). From measurements of the interfacial tension at oil/buffer interfaces on passing HSA-coated substrates and from measurements using radio-labeled HSA in such experiments, it may be concluded that an adsorbed layer of HSA on PS can turnover from the PS substrate to the oil/buffer interface. The difference in behavior between PS substrates coated with HSA and PS surfaces coated with HFb is attributed to association of the HFb molecules upon adsorption at the PS/buffer interface, whereas the stronger intramolecular forces in HSA do prevent this behavior

Characterization of hemodialysis membranes by inverse size exclusion chromatography

Inverse size exclusion chromatography (i-SEC) was used to characterize three different cellulosic hollow fiber hemodialysis membranes, i.e. low-flux cuprophan and hemophan and high-flux RC-HP400A. With the i-SEC technique the pore size distribution and porosity of a membrane can be determined and adsorption phenomena can be studied. The membranes showed clear differences in pore size and porosity, the high-flux RC-HP400A membrane has a larger pore size as well as a higher porosity. For all the membranes it was found that the elution curves were best described by a homoporous pore volume distribution. It appeared that the bound or non-freezing water in the membranes was at least partly accessible to solutes. The test molecules creatinine and vitamin B 12 both adsorbed to the cellulosic membranes. The adsorption behavior of creatinine was strongly dependent on the NaCl concentration present. The observations could be explained by assuming that cuprophan and RC-HP400A are negatively charged whereas hemophan is positively charged due to the modification with N,N-diethylaminoethyl ether. The net charge of the hemophan is smaller

The feasibility of radiolabeling for human serum albumin (HSA) adsorption studies

Human serum albumin (HSA) was labeled in various ways and with different radioactive labels (Technetium-99m and Iodine-125). Characterization with electrophoresis on polyacryl gel and immunoelectrophoresis did not reveal differences between labeled and nonlabeled HSA. The release of the label from labeled proteins in phosphate buffer (pH 7.4) was studied as a function of time. 125I-labeled proteins were stable and 99mTc-labeled proteins showed different stabilities depending on the labeling method which was used. The adsorption behavior of labeled HSA and HSA onto polystyrene (PS) and silicon rubber (SR) was studied by using two methods. It appeared that all labeled HSA compounds showed a preferential adsorption onto PS (and SR) substrates. The 99mTc-labeled HSA showed a high value of the preferential adsorption factor (φ 1). The φ value for 125I-labeled HSA was about 1.4. It was also shown that φ was dependent on the kind of substrate used. The methods developed to determine preferential adsorption of labeled proteins compared to their nonlabeled analogs are also generally applicable for different types of compounds

Intramolecular crosslinking of poly(vinyl alcohol)

Poly(vinyl alcohol) is crosslinked in dilute solution (c=0.1 wt%) with glutaraldehyde. The reaction product is characterized by viscometry and gel permeation chromatography (g.p.c.). The intrinsic viscosity decreases with increasing degree of crosslinking and does not depend on temperature. G.p.c. reveals that the reaction product is not homogeneous, but consists of a mixture of particles with different sizes, possibly both intra- and intermolecularly crosslinked molecules. The intramolecularly crosslinked molecules are smaller in size than the initial polymer molecules and their size depends on the degree of crosslinking. They possess a narrow particle size distribution even if the initial polymer sample had a broad molecular weight distribution

Characterization of hollow fiber hemodialysis membranes: pore size distribution and performance

The effect of two commonly used sterilization methods for artificial kidneys on the morphology and performance of hollow fiber Hemophan® hemodialysis membranes was studied. A relatively new membrane characterization method, thermoporometry, was used to determine the pore size distributions and porosities of the differently treated membrane samples. The samples used for this study were not treated with a concentrated glycerol solution before sterilization. Hemophan was found to have a pore size distribution with pore radii between 1.5 and 12 nm, the volume porosity was 20%. The sample sterilized with ethylene oxide (EtO) had a volume porosity of 18% which was due to a decrease of the pore volume of the smallest pores. The applied dry steam sterilization treatment resulted in a drastic collapse of the large pores while smaller pores were formed. The calculated porosity was only 10%. The `tortuous capillary pore model¿ was used to predict the performance of the artificial kidneys from the pore size distribution in the membrane material. In vitro dialysis experiments with creatinine and vitamin B12 were carried out to compare the calculated and measured clearance rates. Also the ultrafiltration capacity of the devices was determined. It was found that a reasonable estimation of the ultrafiltration capacity could be made. The calculated clearance rates were systematically underestimated, although the relative dependence of the clearance rates on the applied sterilization methods was approximated reasonably