Submicellar complexes may initiate the fungicidal effects of cationic amphiphilic compounds on Candida albicans.

The killing of Candida albicans by a series of amphiphilic quaternary ammonium compounds (QACs) with different hydrocarbon chain lengths was closely related to the binding of the compounds to the cells and damage of the cell membranes. The membrane damage was measured as the level of release of the UV-absorbing material into the medium in which the cells were suspended and as the level of uptake of propidium iodide in individual cells by flow cytometry. It was shown that of the compounds tested, hexadecyltrimethylammonium bromide (cetyltrimethylammonium bromide [CTAB]) bound most efficiently. Tetradecyl betainate chloride (B14), tetradecanoylcholine bromide (C14), tetradecyltrimethylammonium bromide (TTAB), and dodecyltrimethylammonium bromide (DTAB) followed and had declining degrees of binding efficiency. The proportion of CTAB bound was almost total at concentrations up to the critical micelle concentration (CMC) of the compound, whereas that of B14 was somewhat smaller. For the two remaining tetradecyl compounds (C14 and TTAB), still smaller proportions were bound at low concentrations, but the proportions rose disproportionally at increasing concentrations to a distinct maximum at concentrations of 0.2 to 0.5 times the CMC. We propose that interfacial micelle-like aggregates are formed at the cell surface as a step in the binding process. An analogous, but less conspicuous, maximum was seen for DTAB. Thus, great differences in the binding affinity of QACs with different hydrocarbon chains at different concentrations to C. albicans were observed. These differences were related to the CMC of the compound. In contrast, the binding of TTAB to Salmonella typhimurium 395 MS was almost total at low as well as high concentrations until saturation was attained, indicating fundamental differences between binding to the yeast and binding to gram-negative bacteria. The importance of lipid-type complexes or aggregates to the antifungal effect of membrane-active substances are discussed.

Enzymatic hydrolysis of long-chain alkanoylcholines in rat intestinal loops.

BACKGROUND: The hydrolysis of long-chain alkanoylcholines, presumably catalyzed by butyryl-cholinesterase (EC 3.1.1.8), in rat intestinal loops was studied. The substances have earlier been found to be rapidly degraded in vitro. METHODS: Radiolabeled substrates were used, and a radiochromatographic detection method was applied. RESULTS AND CONCLUSION: The long-chain alkanoylcholines were rapidly hydrolyzed. The rates of the reaction and the chain-length dependence were similar to those reported earlier in vitro. At high substrate concentrations the hydrolysis reaction was inhibited. This could be due to conformational changes of the enzyme, caused by the adsorption of the cationic amphiphile, or to a decrease in the free substrate concentration after incorporation of the amphiphilic ester into the lipid layer of the cell membranes. The enzymatic activity towards the substrates in different parts of the rat intestinal tract was also studied and found to be highest in the duodenum.

Long-chain alkanoylcholines, a new category of soft antimicrobial agents that are enzymatically degradable.

A new category of amphiphilic hydrolyzable quaternary ammonium compounds with rapid and high levels of antimicrobial activity was studied. The compounds, alkanoylcholines with hydrocarbon chains of 10 to 14 carbon atoms, are hydrolyzed by butyrylcholine esterase, which is present in human serum and mucosal membranes. The hydrolysis products are common components of human metabolism. Alkanoylcholines were tested and found to be active against gram-negative and gram-positive bacteria as well as yeasts. The microbicidal activities of the alkanoylcholines were comparable to the activities of the stable quaternary ammonium compounds of corresponding chain length and increased with an increasing number of carbon atoms. The compounds were also found to be hydrolyzed by enzymes present in certain microorganisms. The degradation was achieved after reaching the microbicidal effect.

Butyrylcholinesterase activity towards long-chain alkanoylcholines: kinetics and mechanism.

The hydrolysis of long-chain alkanoylcholines catalyzed by butyrylcholinesterase (EC 3.1.1.8) has been studied. Radiolabelled substrates have been used and a radiochromatographic detection method developed earlier has been applied. The long-chain choline esters were found to be excellent substrates for butyrylcholinesterase at low concentrations, with Km values lower than those of short-chain analogues. At higher substrate concentrations, however, the hydrolysis reaction is inhibited, due to the formation of mixed micelles between the amphiphilic substrate and the corresponding alkanoic acid formed in the hydrolysis reaction. The inhibition may also partially be the result of conformational changes of the protein following adsorption of the cationic amphiphile. Critical micelle concentrations (CMC) for the long-chain substrates, as well as for mixed micelles, have been determined.

Liquid chromatographic monitoring of pseudocholinesterase activity: comparison of methods.

In order to study the activity of pseudocholinesterase in vitro, two liquid chromatographic techniques have been developed. One is based on phase transfer-catalyzed (PTC) esterification of the carboxylic acid formed during hydrolysis of the substrate, and the other on the use of a radioisotopically labeled substrate. In both cases, the substrate used was a long-chain choline ester. The PTC method, utilizing (N-9-acridinyl)bromoacetamide as a fluorogenic labeling reagent in an aqueous-organic two-phase system, gives esters with very high fluorescence intensity. The radiochromatographic method makes use of on-line radioactivity monitoring of the substrate and product in order to follow the hydrolysis reaction. In both methods reversed-phase liquid chromatography is used. A method for the synthesis of 3H-labeled choline esters is also described. Both techniques are compared with regard to sensitivity, reproducibility and practical considerations.

N-(9-acridinyl)-bromoacetamide--a powerful reagent for phase-transfer-catalyzed fluorescence labeling of carboxylic acids for liquid chromatography.

Phase-transfer-catalyzed esterification of various carboxylic acids has been carried out on a microanalytical scale with a new reagent, N-(9-acridinyl)-bromoacetamide, in aqueous/organic two-phase systems. The procedure gives esters showing very high fluorescence intensity in acid solution. The large Stokes' shift (greater than 120 nm) means that a low background can be achieved even when using a large emission bandwidth. These derivatives show very good chromatographic behavior on reversed-phase liquid chromatographic columns with the use of aqueous acetonitrile, containing 0.2% of phosphoric acid, as the mobile phase. Detection limits (S/N = 2) as low as 10 fmol can be obtained under optimal conditions.