Bernd Arnold

Quantitative studies on lysolecithin-mediated hemolysis. Use of ether-deoxy lysolecithin analogs with varying aliphatic chain-lengths

The process of lysolecithin-mediated hemolysis has been investigated by use of various ether-deoxy lysolecithin analogs (1-alkyl-propanediol-3-phosphorylcholine) with alkyl residues of 10-22 carbon atoms. Hemolytic activities were defined either as molar amounts to be added for 50% lysis (L50) or as cell-bound amounts at 50% lysis (A50). It was found, that in contrast to L50, A50 values are independent of experimental conditions. Moreover, L50 values primarily reflect the binding affinities, while A50 values give more accurate information on the actual membrane-disturbing potential. The strongest hemolytic C16-lysolecithin analog required 2 - 10(7) or 5 - 10(7) molecules bound per cell for 50% lysis at 0 or 37degrees C, respectively, corresponding to about 10 or 25% of the total membrane phospholipids. Evidence is presented, indicating that (a) lysophosphatides bind to cells below their critical micelle concentration, (b) micelles themselves are not generally necessary for cell lysis. Red cells of different species (man and cattle) as well as at varying temperatures exhibit significantly different sensitivities in terms of L50 and A50 values. These differences, however, depend on the degree of hydrophobicity of the lysolecithins and disappear in the case of lysolipids having C10 or C12 aliphatic residues. The data are in agreement with our hypothesis that cellular sensitivity to lysolecithin lysis may be determined by the degree of segregation of lysolecithin-rich areas within the membrane lipid phase.

Effects of a synthetic lysolecithin analog on the phase transition of mixtures of phosphatidylethanolamine and phosphatidylcholine.

Lysolecithin, i.e. 1-acyl-sn-glycero-3-phosphorylcholine, is a widely distributed surface-active and cytolytic phospholipid [l-3] , which has previously been shown to induce cell fusion [4,5] as well as enhanced immune reactions [6]. Despite various efforts [ l-3,7] , however, the mechanism of action of lysolecithin on natural and model membrane is still poorly understood. So far, all attempts have failed to demonstrate an increased molecular mobility of membrane lipids upon addition of low amounts of lysolecithin, and Klopfenstein et al. [8] have shown that lysolecithin even at a molar ratio higher than 1 : 1 hardly affects the phase transition temperature of dipalmitoyl-lecithin. The present study deals with the effects of a synthetic ether-desoxylysolecithin analog [l] on the miscibility of mixed lipid phases using phosphatidylethanolamine-phosphatidylcholine mixtures and differential scanning calorimetry. purchased from Fluka, Neu-Ulm. Impurities detectable by thin-layer chromatography were below 2%. These lipids were used without further purification. 1 -Hexadecyl-propanediol3-phosphorylcholine (ether-desoxylysolecithin) was synthetized as described elsewhere 111. The lipid mixtures were prepared by dissolving the desired amounts of the dry lipids (30-40 mg) in chloroform. After removal of the solvent by a stream of nitrogen, 25 ml of aqueous 0.05 M Tris-HCI buffer at pH 7.5 were added. The lipids were dispersed ultrasonically at a temperature above the transition temperature of the mixture. The lysolecithin was either mixed with the other lipids in the chloroform phase, or added to the dispersion as aqueous micellar solution. The calorimetric measurements were made using an adiabatic differential scanning calorimeter as described before [9, lo] . The heating rate was 13.5”C/h. To test the reversibility of the system, three runs were made with each lipid mixture.

Distribution and metabolism of synthetic alkyl analogs of lysophosphatidylcholine in mice

Stability, distribution and metabolic fate of alkyl analogs of lysophosphatidylcholine have been studied in mice. Analogs employed were 1-hexadecyl- and 1-dodecyl-propanediol-3-phosphorylcholine (2-deoxy-lysophosphatidylcholine) and rac-1-octadecyl-2-methyl-glycero-3-phosphorylcholine (2-methoxy-lysophosphatidylcholine). It is demonstrated that (1) the half-life of these compounds in the organism was increased to the order of days as compared to minutes or hours for natural lysophosphatidylcholine, (2) the analogs were slowly degraded in vivo with partial incorporation of the hydrophobic part into neutral lipids and phosphatidylcholine. Deoxy analogs were apparently degraded faster than methoxy derivatives, and (3) due to this metabolism, accumulation of these compounds, even after repeated injections, did not occur in mice. Intravenous and intraperitoneal injections led to rapid and complete distribution of the lysolipids with slight enrichment in liver, kidneys and intestine. Subcutaneous or oral application resulted in a similar distribution, however, remarkable depot effects at the sites of administration were observed. Experiments with tumor-bearing mice indicated a reduced metabolic turnover and thus a slight enrichment of ether-lysophosphatidylcholine analogs in tumor tissue.

Quantitative studies on lysolecithin mediated hemolysis. Benzylated lysolecithin as a probe to study effects of temperature and red cell species on the hemolytic reaction.

The slow reacting hemolytic lysolecithin analog 1-octadecyl-2-benzyl-glycero-3-phosphorylcholine has been employed for a detailed study of the process of lysolecithin induced hemolysis. Using a radiolabelled analog we found that the different sensitivities of red cells from different species (chicken, man, cattle) are not paralleled by the binding affinities of lysolecithin. Moreover, lysophosphatide binding to the cells is reduced at low temperatures while the hemolytic activity increases. In contrast to continuous changes of lytic activity and binding between 0 and 37 degrees C, the velocity of the hemolytic reaction with human erythrocytes is extraordinarily fast at 10 degrees C. Experiments in sucrose containing buffer indicated principally different lysis mechanisms below and above 15 degrees C. We have further shown that cells loaded sublytically with the lysolecithin at 37 degrees C undergo spontaneous lysis upon cooling to 0 degrees C. The degree of lysis under these conditions, however, is diminished with increasing amounts of cell-bound lysolipid. Determinations of membrane microviscosities by means of fluorescence polarization revealed some qualitative relations between membrane fluidity and sensitivity to lysolecithin. The data are discussed on the basis of recent reports indicating that lysolecithin-distribution in mixed lipid phases may be heterogeneous depending on lipid composition and temperature.

Molecular aggregation of the slow reacting hemolytic lysolecithin analog 1-octadecyl-2-benzyl-glycero-3-phosphorylcholine in aqueous solution.

An attempt has been made to relate the retarded adsoprtion to red cells of the slow reacting hemolytic phosphatide Rac. 1-octadecyl-2-benzyl-glycero-3-phosphorylcholine (benzyl-lysolecithin) to its aggregation status in aqueous solution. Light scattering measurements indicate a critical micelle concentration at 37 degrees of less than 2 X 10(-6) M. The micellar weight as determined by angle dependent light scattering is 6 X 10(7) with about 97 000 molecules per micells. The aggregates, which according to electron-microscopic observations are more similar to lecithin-liposomes than to usual lysolecithin-micelles, undergo a phase transition at 14 degrees from a tightly packed liquid-crystalline state to the more loose structure of a gel phase with increased mobility of the aliphatic chains. The enthalpy of transition is 4.2 kcal/mole. These changes of the micellar structure are reflected in the binding kinetics of benzyl-lysolecithin to red cells in that the binding rate is rather constant below, but strongly increasing above the transition temperature. It is concluded that the unusual micellar structure is responsible for the retarded adsorption of this lysolecithin analog to red cells and that the rate of adsorption is probably determined by the rate of escape of single lysophosphatide molecules from the micelles.

Relation of cytolytic activity of an ether-deoxy lysophosphatidylcholine analog to available membrane surface. Comparison of normal and tumor cells from mice

1-Hexadecylpropanediol-3-phosphorylcholine, an ether-deoxy analog of lysophosphatidylcholine, has been employed to study the sensitivity of various types of mouse cells with respect to changes in membrane permeability induced by lysophosphatidylcholine. Cells used included erythrocytes, thymocytes, spleen cells and macrophage, as well as 4 different tumors (2 lymphomas, 1 Ehrlich acites and 1 methylcholanthren-induced fibrosarcoma). The sensitivity to the lysophosphatide (on a per-cell basis) of the above cell types varied by a factor of 65. When lytic concentrations were related to available membrane surface, this variation was reduced to a factor of 2.5. No principal difference was observed between the sensitivity of normal versus tumor cell membranes with respect to lysophosphatidylcholine lysis. Membrane surface, available for lysophosphatidylcholine, has been estimated from binding equilibria of 14C-labelled deoxy-lysophosphatidylcholine to the cells under standardized conditions. This method is based on the finding that binding equilibria of lysophospholipids to cells are predominantly determined by the available membrane surface.