Partitioning of high-density lipoproteins in charge-sensitive two-phase systems.

Aqueous polymer two-phase systems characterized by a difference in the electrical potential between the upper and the lower phase (charged systems) are useful tools for the detection of changes in the surface charge and hydrophobicity of high-density lipoproteins (HDL). While the large particle size of low-density lipoproteins (LDL) leads to accumulation at the interface, the smaller diameter and the higher surface charge density of the native HDL particles allows partitioning without aggregation at the interface. Charged two-phase systems can be used to check the native state of HDL samples. Moreover, these systems would be suitable for investigating the hydrophobicity and surface charge of modified HDL.

Partitioning of chemically modified low-density lipoprotein in aqueous polymer two-phase systems.

Aqueous polyethylene glycol (PEG)-dextran two-phase systems containing 10 mM Tris.HCl (pH 7.4) were used for the partitioning of chemically modified low-density lipoprotein (LDL). Anionic modification connected with an increase in the negative surface charge of lipoproteins favours the accumulation of modified LDL in the top phase. The partition coefficient increases depending on the extent of modification. Cationic modification yields lower values for the partition coefficient. Positively charged LDL favours a bottom-phase accumulation. With weakly charged and nearly neutral particles, the Van der Waals interaction between polymer and particle preponderates over electrostatic interactions, leading to a favoured accumulation of LDL in the PEG-rich top phase. Results of measurements of the relative electrophoretic mobility and the determination of free amino groups are in agreement with the calculated values of the partition coefficient. Because the partitioning of LDL is accompanied by aggregation at the interface, experimental techniques have to be carefully standardized. Subtle differences in the surface properties of modified LDL can be detected.

Quenching and dequenching of octadecyl Rhodamine B chloride fluorescence in Ca(2+)-induced fusion of phosphatidylserine vesicles: effects of poly(ethylene glycol).

Ca(2+)-induced fusion of SUV and LUV composed of ox brain phosphatidylserine (PS) was studied as a function of temperature and concentration of Ca2+ using octadecyl Rhodamine B chloride (R-18). Ca2+ was added to a 1:1 mixture of labelled (8 mol%) and unlabelled vesicles (assay conditions) or to samples containing only labelled liposomes (control conditions). Both, in SUV and LUV the dependence of differences in fluorescence between assay and control samples on temperature can be divided into three regions. At temperatures lower than 20 degrees C the differences in fluorescence increase only slightly in SUV or remain unchanged in LUV after the addition of Ca2+. At 28 degrees C and higher temperatures the differences of fluorescence intensities increase much more drastically, whereby SUV exhibit higher fusion rates than LUV. Between 20 degrees C and 28 degrees C exists an intermediate region for both SUV and LUV. Here the fluorescence changes continuously from one behaviour to the other independent of the concentration of Ca2+. A drastic quenching of R-18 fluorescence occurs in LUV composed of PS below 10 degrees C, where the lipids are in the gel state. In SUV the fluorescence is only weakly changed in this temperature region. It is assumed that a demixing between dye and phospholipid molecules occurs below phase transition. During fusion the phase transition of PS is shifted from 8-10 degrees C to about 24-28 degrees C as revealed by polarization measurements using diphenylhexatriene. Because the differences in R-18 fluorescence between assay and control samples depend strongly on temperature we assume that the shift in phase transition temperature of PS occurs immediately after the addition of Ca2+ to SUV or LUV. Poly(ethylene glycol) 6000 accelerates fusion in both SUV and LUV under all conditions where a fusion takes place. Further, the threshold concentration of Ca2+ to induce fusion is diminished from about 1 mmol/l without polymers to about 0.5 mmol/l in the presence of 10% (w/v) PEG 6000. The intermediate region of changes in fluorescence properties of R-18 in the Ca(2+)-induced fusion of PS is not changed by PEG.

[In vitro effects of low molecular weight dextran on the surface properties of LDL]. / In-vitro-Effekte von niedermolekularem Dextran auf die Oberflächeneigenschaften von LDL.

The relative number of free amino groups on the surface of low density lipoproteins (LDL) was determined after the incubation with low molecular weight dextran (MW 40,000) in several concentrations by two different assays (fluorescamine, TNBS). A decrease of the detectable free amino groups of 40% was shown by both assays. After the incubation of lysine with dextran these changes could not be observed. These results are explained with the aggregation of LDL particles influenced by dextran.

Aggregation of human plasma high density lipoproteins induced by poly(ethylene glycol).

The influence of different surface charge densities (induced by varying pH, addition of positively charged amphiphilic molecules and chemical modification) of high density lipoproteins (isolated by ultracentrifugation) on poly(ethylene glycol) induced aggregation was studied. The effects of different molecular masses of PEG, HDL concentration and the presence of other serum proteins on the PEG mediated aggregation were investigated. The PEG concentration necessary for HDL aggregation is inversely proportional to the used HDL concentration and its molecular weight, and is directly proportional to the presence of other proteins and the magnitude of the negative surface charge density of HDL. The results are in accordance with the predictions of the volume exclusion theory taking into account the influence of repulsive electrostatic forces on the interaction of HDL particles.

Partition of serum lipoproteins in a polyethylene glycol/dextran two-phase system.

Aqueous two-phase systems containing polyethylene glycol (PEG) and dextran as phase forming polymers were used for the partition of unmodified and hypochlorite modified lipoproteins. Low density lipoprotein (LDL) was separated from high density lipoprotein (HDL) by sequential ultracentrifugation from human plasma. In agreement with the higher electrophoretic mobility, high density lipoprotein shows a higher value of the partition coefficient in contrast to low density lipoprotein. An increase in the concentration of chloride ions reduces the enrichment of lipoprotein in the top phase and favours the accumulation of aggregated material at the interface. The partition coefficient strongly depends on the age of the lipoprotein sample. Differences in the value of the partition coefficient could be obtained for the lipoprotein fractions HDL-2 and HDL-3. Hypochlorite modified LDL shows higher values of the partition coefficient due to the higher negative charge of the modified lipoprotein particle.

Modification of low density lipoproteins by sodium hypochlorite.

Human low density lipoproteins (LDL) were incubated with increasing amounts of sodium hypochlorite. A decrease of the number of free amino groups on the LDL surface starts only upon addition of 30-40 moles NaOCl per mole apoB, whereas all detectable SH groups are oxidized after addition of nearly 17-20 moles NaOCl. All hypochlorite-modified LDL samples have a higher electronegative surface charge compared with native LDL as revealed by agarose gel electrophoresis and partition of LDL in an aqueous polyethylene glycol/dextran two-phase system. The more NaOCl is used to alter LDL, the higher is the electrical surface charge. Changes in surface charge are found already at low NaOCl concentrations where no decrease of amino groups is detected. It is assumed that changes in surface charge are caused by the formation of monochloramines and especially at low degrees of modification by a further unknown contribution. An effect on the primary structure of apoB or peroxidation-like changes in NaOCl-altered LDL could not be found under our experimental conditions. The results are discussed with respect to such modifications under in vivo conditions by hypochlorous acid generated in stimulated phagocytosing cells.

Characterization of chemical modifications of surface properties of low density lipoproteins.

Low density lipoproteins (LDL) were modified by incubation with formaldehyde and malondialdehyde and by autoxidation. Different methods were developed to measure alterations of surface properties of LDL. A method based on fluorescamine fluorescence was used to measure changes of free amino groups. All modified LDL samples have a decreased number of amino groups. As shown by gel electrophoresis, the negative surface charge of LDL increases. Aggregation of LDL induced by poly(ethylene glycol) (PEG) is influenced by LDL modification. The PEG 6000 concentration necessary for LDL aggregation is shifted to higher values for modified LDL due to the increased negative surface charge. This result may be of interest for the application of PEG for selective separation of different classes of lipoproteins. The formation of aggregates of LDL induced by dextran sulphate is reduced due to the loss of positive charges of amino groups on the LDL surface.

[Problems of photographic documentation of color patterns in connection with the use of thermography with liquid crystals (author's transl)]. / Probleme der fotografischen Dokumentation der bei Verwendung der Flüssigkeitskristall-Thermographie auftretenden Farbmuster.

Cholesteric liquid crystals show different colors dependent on their temperature. The "thermography with liquid crystals" makes use of this property for the determination of temperature distributions of human skin. The correct photographic documentation of the color patterns is difficult, because the colors depend on the angle of incidence of light and the direction of observation. Further, the quality of the photos is reduced by the direct reflection of light at the surface. Possible geometrical conditions of taking photos are discussed. An optimum arrangement for producing photos without reflections and color changes is proposed.