Janusz Sarapuk

Hemolysis of erythrocytes and erythrocyte membrane fluidity changes by new lysosomotropic compounds.

This work contains the results of studies on the influence of newly synthesized lysosomotropic substances (lysosomotropes) on human erythrocytes. Six homologous series of the compounds differing in the alkyl chain length and counterions were studied. They were found to hemolyse erythrocytes and to change their osmotic resistance. The observed hemolytic effects were dependent both on the compound’s structure (polar head dimension and alkyl chain length of compound) and its form (the kind of the counterion). In parallel, the influence of lysosomotropes on fluidity of the erythrocyte membrane was studied. Three different fluorescent probes were used; 1,6-diphenyl-1,3,5-hexatriene (DPH), 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene, p-toluenesulfonate (TMA-DPH) and 6-dodecanoyl-2-dimethylaminonaphthalene (laurdan). Their anisotropy (DPH and TMA-DPH) or general polarization (laurdan) values after incorporation into ghost erythrocyte membranes were measured. The results obtained show that fluidity changes accompanied the effects observed in hemolytic experiments both quantitatively and qualitatively.

The role of counterions in the interaction of bifunctional surface active compounds with model membranes.

Interaction of two series of bifunctional surfactants (bromides and chlorides) with red blood cells and planar lipid membranes was studied. The aim of the work was to determine the role of counterions in the mechanism of interaction of bifunctional cationic surfactants with model membranes. In each case bromides influenced model membranes to a greater degree than the corresponding chlorides. The possible explanation of the obtained results is presented. It seems that the greater ability of bromides to destabilize model membranes in comparison with chlorides can be attributed to the greater mobility and the smaller radius of the hydrated bromide ion. This may underlie the greater ease that this anion can modify the surface potential of the lipid bilayer, thus enhancing the interaction of the cationic surfactant with such a modified bilayer.

Role of Hydrophobic and Hydrophilic Interactions of Organotin and Organolead Compounds with Model Lipid Membranes

Abstract The present study was conducted to clarify the mechanism of toxicity of organic compounds using lipid model membranes (liposomes and planar lipid membranes). The compounds studied were trialkyltin and trialkyllead chlorides, dialkyltin dichlorides and some inorganic forms of those metals. Two different (anionic and cationic) detergents were also used in the experiments to change the surface properties of liposomes. As a measure of interaction between the compounds studied and model membranes were the release of liposome bound praseodymium and the change in stability of planar membranes under the influence of those compounds. On the basis of the results obtained it was postulated that the mechanism of interaction between tin-and leadorganics and model lipid membranes is a combination of different factors featuring interacting sides. The most important properties determining the behaviour of organic compounds in the interaction were lipophilicity and polarity of different parts of the organics and the steric arrangement they can take in the medium. On the other hand, the surface potential of the lipid bilayer and the environment of the lipid molecules, that play a significant role in the availability of the lipid bilayer to the organics, were important factors in the interaction.

THE ROLE OF COUNTERIONS IN THE PROTECTIVE ACTION OF SOME ANTIOXIDANTS IN THE PROCESS OF RED CELL OXIDATION

A number of new surfactants with an incorporated antioxidant functional group were synthesized in order to be used as agents protecting biological and/or model membranes against lipid peroxidation. Hydrophobic parts of these amphiphilic antioxidants ensured their incorporation into the membranes studied, viz. pig erythrocytes. Proper concentrations of the antioxidants were used to avoid erythrocyte membrane destruction during the experiments. The work contains the results of studies on the protective effect of two groups of antioxidants. They differed in the chain length incorporated into the membrane hydrophobic part and in the kind of counterion (chloride and bromide). The role of these factors in the protective action of the compounds studied is discussed as well as their practical application.

Stability of Model Membranes in the Presence of Organotin Compounds

The influence of tri- and di-alkyltins (TATs and DATs) as well as di- and triphenyltin compounds (DPhTs and TPhTs) on haemolysis of red blood cells (RBCs) and stability of planar lipid membranes (PLMs) has been studied. The results obtained show that the efficiency of TATs (trimethyl-, triethyl-, tri-n-propyl- and tributyl-tin chlorides) in destroying PLMs did not differ greatly when the compounds were studied in solutions of physiological pH (phosphate buffer, pH 7.4). A decrease in pH to 5.0 caused small changes in the efficiency of the three largest TAT molecules and a significant decrease in the efficiency of trimethyltin chloride. Both haemolytic and PLM experiments showed that the most active TAT was tri-n-propyltin chloride. The destructive action of DAT (dimethyl- and dibutyltin) and DPhT dichlorides was somewhat more differentiated. Dimethyltin dichloride (DMT) interaction with model membranes was a little weaker than that of DPhT and dibutyltin dichlorides and all these compounds influenced the model membranes to a lesser extent than TATs or TPhT. To bring about comparable haemolysis effects the dichlorides had to be used at much greater concentrations than the chlorides. The haemolytic properties of the dichlorides, especially of that of DMT, significantly increased in solution at pH 5.0. TPhT chloride interacted with model membranes similarly to TAT chlorides. Also, no great difference in efficiency of this compound was found for the two buffer solutions used. Copyright

Protective Effect of Quaternary Piperidinium Salts on Lipid Oxidation in the Erythrocyte Membrane

Abstract A new series of amphiphilic compounds with incorporated antioxidant functional group has been investigated. Piperidinium bromides, differing in the alkyl chain length (8, 10, 12, 14 and 16 carbon atoms in the chain) were synthesised to protect biological and/or model membranes against peroxidation and following negative consequences. Their antioxidant activity was studied with erythrocytes subjected to UV radiation. The salts used inhibited lipid oxidation in the erythrocyte membrane. The degree of this inhibition depended on the alkyl chain length of the bromide used and increased with increasing alkyl chain length. A comparison of the results obtained for piperidinium bromides with those obtained for the widely used antioxidant 3,5-di-t-butyl-4-hydroxytoluene (BHT) revealed that only two shortest alkyl chain salts were less efficient than BHT in protecting erythrocyte membranes. A similar comparison with antioxidant efficiency of flavonoids extracted from Rosa rugosa showed that they protected the membranes studied more weakly than the least effective eight-carbon alkyl chain piperidinium bromide. The three compounds of longest alkyl chains were the most active antioxidants. Their activities did not differ significantly.

Protection of erythrocytes against organometals-induced hemolysis.

The hemolytic toxicity of tributyllead (TBL) and triphenyllead (TPhL) chlorides and its prevention by dithiotreitol (DTT), diethylenetriaminepentamethylenephosphonic acid pentasodium (PMP) and sodium disulfide (Na2S) was studied. It was found that both TBL and TPhL efficiently hemolyzed pig erythrocytes when used in micromolar concentrations; tributyllead chloride being about twice more efficient than triphenyllead chloride. The hemolytic efficiency of these compounds was blocked by PMP, DTT and Na2S in a concentration-dependent manner. However, significant differences in antihemolytic efficiency of these compounds were found. Namely, DTT and Na2S were very efficiently protecting erythrocytes against the action of organoleads, while the PMP protection was weak. Also, differences between DTT and Na2S protective eficiency were found. They more efficiently prevented erythrocyte hemolysis by TPhL than by TBL. Moreover, erythrocytes were better protected against the action of TBL by Na2S than by DTT. Such differentiation may be connected with possible differences in localization of the organolead compounds and protective agents in the erythrocyte membrane. To check these possibilities a series of experiments was performed using the fluorescence technique and various fluorimetric probes. These measurements enabled to determine fluidity changes induced in erythrocyte membranes by the organoleads and the protective compounds and to formulate some remarks concerning the differences in the mechanism of interaction of the organoleads with these membranes.

Influence of Counterions on the Interaction of Pyridinium Salts with Model Membranes

Abstract The interaction of pyridinium salts (PS) with red blood cells and planar lipid membranes was studied. The aim of the work was to find whether certain cationic surfactant counterion influence its possible biological activity. The counterions studied were Cl- , Br-, I-, ClO4-, BF4- and NO3-. The model membranes used were erythrocyte and planar lipid membranes (BLM). At high concentration the salts caused 100% erythrocyte hemolysis (C 100) or broke BLMs (CC). Both parameters describe mechanical properties of model membranes. It was found that the efficiency of the surfactant to destabilize model membranes depended to some degree on its counterion. In both, erythrocyte and BLM experiments, the highest efficiency was observed for Br-, the lowest for NO3-. The influence of all other anions on surfactant efficiency changed between these two extremities; that of chloride and perchlorate ions was similar. Some differences were found in the case o f BF4- ion. Its influence on hemolytic possibilities of PS was significant while BLM destruction required relatively high concentration of this anion. Apparently, the influence of various anions on the destructive action of PS on the model membrane used may be attributed to different mobilities and radii of hydrated ions and hence, to different possibilities of particular anions to modify the surface potential of model membranes. This can lead to a differentiated interaction of PS with modified bilayers. Moreover, the effect of anions on the water structure must be taken into account. It is important whether the anions can be classified as water ordering kosmotropes that hold the first hydration shell tightly or water disordering chaotropes that hold water molecules in that shell loosely.

Toxicity and model membrane modifying properties of organolead compounds

Department of Botany and Plant Physiology, Agricultural University, Cybulskiego 32, 50–205 Wroclaw,PolandThe influence of trialkylleads on haemolysis ofred blood cells (RBCs), growth of Spirodelaoligorrhiza and stability of planar lipid mem-branes (PLMs) at different pH of solution hasbeen studied. The results obtained show that theefficiency of trialkylleads (methyl-, ethyl-, pro-pyl- and butyl-lead chlorides) in modifying thephysiological and mechanical properties of theobjects studied depended both on pH of solutionand hydrophobicity of the compounds. Namely,it was found that this efficiency increased withpH of solution. The most significant increase wasobserved in PLM experiments. Also, the hydro-phobicity of trialkylleads influenced the proper-ties mentioned. The more hydrophobic a com-pound the greater was its haemolytic toxicity.The same applies to the physiological toxicityof the compounds, whose measure was 50%inhibition of plant growth. Generally, thesequence of modifying possibilities of the com-pounds studied at any pH of the solution was thefollowing:tributyllead >tripropyllead >triethyllead >trimethylleadA possible mechanism of the interaction oforganolead species with model and biologicalmembranes is discussed. Copyright # 2001John Wiley & Sons, Ltd.Keywords: triorganolead chlorides; erythrocytehaemolysis; physiological toxicity; planar lipidmembranes destabilization; interaction mechan-ism.

Antioxidant Protection of Egg Lecithin Liposomes during Sonication

Abstract When model membranes are prepared by ultrasonic treatment of polyunsaturated phospholipids, radical production can induce a partial degradation of the polyunsaturated fatty acyl chains and the formation of lipid hydroperoxides. A suitable antioxidant employed during liposome preparation is able to protect them against lipid peroxidation. This work contains the results of studies on egg lecithin liposomes with incorporated antioxidants that were supposed to play the protective role mentioned. As it has been shown the antioxidant com pounds used ensured a 40-60% , i.e., satisfactory protection of liposomes after 30 min sonication. Possible practical applications are discussed.