S. Witek

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.

Gemini ester quat surfactants and their biological activity

Cationic gemini surfactants are an important class of surface-active compounds that exhibit much higher surface activity than their monomeric counterparts. This type of compound architecture lends itself to the compound being easily adsorbed at interfaces and interacting with the cellular membranes of microorganisms. Conventional cationic surfactants have high chemical stability but poor chemical and biological degradability. One of the main approaches to the design of readily biodegradable and environmentally friendly surfactants involves inserting a bond with limited stability into the surfactant molecule to give a cleavable surfactant. The best-known example of such a compound is the family of ester quats, which are cationic surfactants with a labile ester bond inserted into the molecule. As part of this study, a series of gemini ester quat surfactants were synthesized and assayed for their biological activity. Their hemolytic activity and changes in the fluidity and packing order of the lipid polar heads were used as the measures of their biological activity. A clear correlation between the hemolytic activity of the tested compounds and their alkyl chain length was established. It was found that the compounds with a long hydrocarbon chain showed higher activity. Moreover, the compounds with greater spacing between their alkyl chains were more active. This proves that they incorporate more easily into the lipid bilayer of the erythrocyte membrane and affect its properties to a greater extent. A better understanding of the process of cell lysis by surfactants and of their biological activity may assist in developing surfactants with enhanced selectivity and in widening their range of application.

A comparison of the micellization of selected amphiphilic N,N′-bisdimethyl-1,2-ethanediamine derivatives with some amphiphilic betaine ester derivatives

Abstract Critical micelle concentrations and the changes in enthalpy, entropy, and free energy that accompany micelle formation of selected amphiphilic N,N′ -bisdimethyl-1,2-ethanediamine derivatives (of general formula [C n H 2 n +1 OOCCH 2 (CH 3 ) 2 NCH 2 CH 2 N(CH 3 ) 2 CH 2 COOC n H 2 n +1 ] 2+ · 2Cl − where n = 8, 10, 12, 14, 16) were determined by means of titration calorimetry in an aqueous solution at 298.16 K. It was found that the critical micelle concentrations and thermodynamic data of the studied compounds are close to those of betaine chloride alkyl esters (of general formula [(CH 3 ) 3 NCH 2 COOC n H 2 n +1 ] + · Cl − ) with the alkyl chain longer by four CH 2 groups.

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.

Suppression of radical-induced lipid peroxidation in a model system by alkyl esters of cinnamate quaternary ammonium salts.

Abstract Three new groups of phenolic antioxidants, quaternary ammonium salts with a phenol ring and alkyl chains of different length (pyrrolidine ethyl esters of 3,5-di-t-butyl-4-hydroxydihydrocinnamic acid n-alkoxymethylchlorides (PYE-n) or n-alkylbromides (PYA-n) and 2-dimethylaminoethyl ester n-alkylbromides (PPA-n), were synthesized. Some of them were previously found to efficiently protect yeast cells against oxidants and to inhibit the production of thiobarbituric acid-reactive substances in whole yeast cells and in isolated membrane lipids. The new antioxidants (at 1-100 μm) abolished or diminished peroxidation of oliwe oil emulsions caused by the OH•-producing Fe2+ and RO• and ROO•-producing tertbutylhydroperoxide (TBHP) and the azo compounds 2,2′-azobis-(amidinopropane)dihydronitrile (AAPH) and 1,1′-azobis-(1-cyclohexanecarbonitrile) (ACHN): all present at 10 mᴍ . The efficiency of individual both antioxidants was examined in relation to the type of lipid peroxidation inducer, the site of antioxidant incorporation into the emulsion lipid phase, the length of the alkyl chain, and the maximum concentration of effective antioxidant monomers given by its critical micelle concentration. PYA-n class compounds were highly efficient against all peroxidation inducers and their efficiency did not depend on the position of their molecules in the lipid phase and/or on the aliphatic chain length. In contrast, the efficiency of PYE-n and PPA-n class compounds depended both on the type of oxidant and on the length of their aliphatic chain. Their potency against Fe2+ and ACHN increased with increasing alkyl chain length whereas with AAPH it dropped with increasing alkyl chain length. A similar pattern was found with the action of PYE-n against TBHP whereas in the PPA-n group an extending alkyl chain reduced the anti-TBHP efficiency. These relationships may not be entirely straightforward and other factors (chemical nature of each compound, its possible interaction with fluorescent probes used for diagnostics, etc.) may play a considerable and not yet quite clear role. PPA-n class antioxidants have the lowest critical micelle concentration, which may limit their efficiency. Nevertheless, these phenolic antioxidants can be conveniently employed as highly efficient inhibitors of lipid peroxidation.

Growth Retarding Effect of N, N-Dimethylmorpholinium Chloride and CCC in Spirodela oligorrhiza

Summary N,N-Dimethylmorpholinium chloride (DMMC) at concentrations of 0.05–0.1 mM markedly reduced the multiplication rate of Spirodela oligorrhiza and increased the chlorophyll and protein contents in plants. Progeny fronds remained attached together in large clumps. Similar effects were produced by 2-chloroethyl-trimethylammonium chloride (CCC), which was proved to be about 5-fold more effective as a growth retardant than DMMC on molar concentration basis. Gibberellic acid and benzylaminopurine partially reduced the visible symptoms of the action of DMMC. GA3 completely reversed the symptoms of the action of CCC. DMMC and CCC at high concentrations inhibited greening of progeny fronds, the effect being of temporary nature.

DL-leucine transport in aSaccharomyces cerevisiae mutant resistant to quaternary ammonium salts

Conclusions1.Quaternary ammonium salts seem to be potent inhibitors of amino acid uptake inS. cerevisiae.2.Inhibition is the strongest in yeast with derepressed transport (growth in Yeast Carbon Base medium with proline as the sole nitrogen source).3.The single-gene resistance to quaternary ammonium salts leads to lowering of transport inhibition.

Inhibition of Lipid Peroxidation in the Erythrocyte Membrane by Quaternary Morpholinium Salts with Antioxidant Function

Abstract The antioxidant activity of five new compounds from a group of quaternary ammonium salts with antioxidant function has been investigated. The effect of the compounds on the degree of lipid oxidation in the erythrocyte membrane subjected to UV radiation was studied. It was found that all the salts used decreased oxidation of the lipids in the erythrocyte membrane. The antioxidant activity of the ammonium salts studied increased with their alkyl chain length. Three compounds with the longest alkyl chains were the most active antioxidants, and their antioxidant properties were comparable to those of a flavonoid extracted from haw thorn. The antioxidant effectiveness of the ten-carbon alkyl chain compound was comparable with that of the known antioxidant 3,5-di-t-butyl-4-hydroxytoluene (BHT). The least effective antioxidant studied proved to be the eight-carbon alkyl chain compound. The effect of these compounds on fluidity of the erythrocyte membrane has been studied, and for all an increase of fluidity of the membrane was observed. The changes in erythrocyte ghosts fluidity depended both on concentration and type of compound. A fluorimetric study also indicated that rigidity of the erythrocyte membrane increased with degree of its oxidation, but with antioxidants present membrane rigidity increased less.

Spontaneous and radical-induced plasma membrane lipid peroxidation in differently oxidant-sensitive yeast species and its suppression by antioxidants

Formation of thiobarbituric acid-reactive substances (TBRS; nmol/mg lipids) indicative of lipid peroxidation was measured in whole cells and in isolated plasma membrane lipids from three yeast species differing in oxidant sensitivity (Schizosaccharomyces pombe, Saccharomyces cerevisiae andRhodotorula glutinis) after exposure to the Fenton reagent, FeII, H2O2,tert-butyl hydroperoxide (TBHP) and azo compounds (AAPH, ACHN). In whole cells, spontaneous TBRS formation rose in the sequenceS. pombeAAPH∼ACHN∼Fe-Fenton>H2O2. This increase need not be solely due to increased lipid peroxidation. In isolated plasma membrane lipids from all three species, the spontaneous TBRS production referred to 1 mg lipids was 9–13-fold higher than in whole cells. InS. pombe lipids, only TBHP increased the TBRS production. In lipids fromS. cerevisiae andR. glutinis, all added oxidants increased the spontaneous TBRS production 2–3 times in the sequence TBHP>ACHN>AAPH>FeII>Fe-Fenton>H2O2. Oxidant-induced TBRS production in both whole cells and isolated membrane lipids was partially suppressed by the lipid peroxidation inhibitors 2,6-di-tert-butyl-4-methylphenol (“butylated hydroxytoluene”; BHT) and the newly synthesized PYA12 compound. Both agents were more effective in isolated lipids than in whole cells and against OH-producing than against ROO-or RO-producing oxidants. Yeast membrane lipids, which are generally poor in polyunsaturated fatty acids, are thus subject to perceptible lipid peroxidation.

Aminoethyl esters of fatty acids as model lysosomotropic substances.

Conclusions1.The activity of the DM-n compounds is chain length and pH dependent; the highest activity being observed for 11–13 carbon atoms chain.2.The highest biological activity was observed at pH above 7, which is characteristic for lysosomotropic agents.3.Hemolytic experiments conformed the lysosomotropic character of DM-n. The free amine (DMF-11) can pass through membranes and at lower pH can interact with the vacuolar membrane.4.In yeast, DM-n mobilized respiration of endogenous substrates.5.In yeast protoplasts treated with DM-n, temporary increase in Ca2+ uptake (characteristic for apoptosis) takes place. This indicates that the compounds have a killing effect.