Polycyclic aromatic hydrocarbons in liquid smoke flavorings obtained from different types of wood. Effect of storage in polyethylene flasks on their concentrations.

Smoke flavorings are widely used as an alternative to the traditional smoking techniques. Smoke generation conditions can determine the level of polycyclic aromatic hydrocarbons (PAHs) in the smoke and, consequently, in these preparations. In this paper, the influence of the wood source on the formation of PAHs is studied. For this purpose, five liquid smoke flavorings, obtained from different types of wood, were used. Sample aliquots, including deuterated internal standards, were subjected to an alkaline treatment, extracted by liquid-liquid partition and cleaned up by means of silica tubes, followed by gas chromatography-mass spectrometry analysis. The results reveal that the flavoring obtained from poplar wood presents the highest number and concentrations of both total and carcinogenic PAHs, even though the levels of these latter are very low. It has also been observed that the storage of smoke flavorings in polyethylene flasks reduces the concentration of some PAHs.

Study of several aspects of a general method for the determination of polycyclic aromatic hydrocarbons in liquid smoke flavourings by gas chromatography-mass spectrometry.

The effectiveness of the steps of a general method, which includes alkaline treatment of the samples, extraction, clean-up and analysis, for the determination of polycyclic aromatic hydrocarbons in liquid smoke flavourings and smoked foods have been studied, by using mixtures of pure polycyclic aromatic hydrocarbons and of smoke flavouring compounds. All these products were applied to samples of liquid smoke flavourings in order to test the effectiveness of each step of the method, as well as to investigate its effect on real samples, and thus obtain a suitable procedure for the study of polycyclic aromatic hydrocarbons in liquid smoke flavourings. The alkaline treatment has been proved to be necessary because of its capability for removing some smoke components. Both cyclohexane and dichloromethane are able to extract polycyclic aromatic hydrocarbons with high recovery percentages; however, cyclohexane is more adequate due to its lower ability to extract other smoke flavourings components. It is important to note that the elution sequence of polycyclic aromatic hydrocarbons during the clean-up step is quite different depending on the solvent used to dissolve the extract, which determines the ability of this process to isolate polycyclic aromatic hydrocarbons for their accurate identification and quantification by gas chromatography-mass spectrometry in selected ion monitoring mode. Finally, the PAHs present in a liquid smoke flavouring and their concentrations were determined; it was observed that, despite the different extraction solvents and clean-up procedures used, the results were very similar.

Determination of polycyclic aromatic hydrocarbons in commercial liquid smoke flavorings of different compositions by gas chromatography-mass spectrometry.

The presence of polycyclic aromatic hydrocarbons (PAHs) in five commercial liquid smoke flavorings, used in the European food industry, was studied. The samples were subjected to an alkaline treatment, extracted with cyclohexane, cleaned up by means of solid-phase extraction tubes, and analyzed by gas chromatography-mass spectrometry. Three different procedures for the cleanup were tested. The results revealed the presence of 34 PAHs, some of them with methyl substituents. In all cases, the concentrations of compounds of low molecular weight were much higher than those of high molecular weight. Relationships between smoke flavoring compositions and PAH levels were also studied. Three of the samples contained high levels of both total and carcinogenic PAHs. Benzo[a]pyrene was also detected in these three samples, but its concentration did not exceed the 10 microg/kg level fixed by the FAO/WHO. Finally, a relation was found, first between the concentrations of total carcinogenic PAHs and benzo[a]pyrene and also between the concentrations of pyrene and benzo[a]pyrene. The latter ratio reveals that pyrene concentration could be very useful in predicting the level of benzo[a]pyrene and, consequently, in estimating the carcinogenicity arising from the presence of benzo[a]pyrene and other carcinogenic PAHs.

Food as a source of polycyclic aromatic carcinogens.

Polycyclic aromatic hydrocarbons (PAH) belong to a large chemical family comprising many different compounds with important biological activity in mutagenic and carcinogenic processes. PAH have been detected in both raw and processed foods. The presence of PAH in non-processed foods is associated with environmental pollution from both human and industrial activities, whereas contamination of processed foods can be caused by certain preservation and processing procedures. Both toxicological and epidemiological studies have shown a relation between such compounds and tumor development. The data indicate that PAH must undergo a biotransformation process that causes the formation of biologically active metabolites. In this process, the presence of an enzyme complex that is induced by different xenobiotics is implied, making the toxicity of such compounds hard to predict. As setting a threshold limit below which toxicity could be considered negligible is difficult, the presence of PAH in foodstuffs should be reduced to as low as possible by controlling environmental contamination and all procedures that could cause PAH contamination during food processing, preserving, and packaging.

Real-time measurements of chemically-induced membrane fusion in cell monolayers, using a resonance energy transfer method.

Fusion of mouse melanoma cells grown in monolayers has been directly monitored by fluorescence resonance energy transfer between fluorescein and rhodamine probes attached to octadecanoic acid. Various poly(ethylene glycol)s (PEG), either alone or in combination with amphipathic molecules, have been used as fusogens. Fusion starts at a maximum rate as soon as PEG is removed from the medium and reaches a plateau after 20-30 min. Both the initial rate and extent of fusion have been recorded for each experiment. The extent of fusion shows in general a positive correlation with the initial rate, although PEGs with different molar masses appear to induce fusion at different rates, but to a similar extent. A good correlation has been found between the extent of fusion, as measured by fluorescence, and the 'fusion index' computed from cell and nucleus counting; a calibration curve is provided for the interconversion of both parameters. Optimum fusion values are obtained with 50% (w/v) PEG 1500. The effect of pre-treatments with surfactants (Triton X-100, sodium dodecylsulphate) on PEG-induced fusion has also been tested. Sodium dodecylsulphate, but not Triton, enhances considerably both the rate and extent of cell fusion. The in situ generation of the amphipathic molecule diacylglycerol, through the catalytic activity of a phospholipase C, also enhances significantly the fusion parameters. These results are in good agreement with previous studies based on syncytia counting.

Effective detergent/lipid ratios in the solubilization of phosphatidylcholine vesicles by Triton X-100.

Effective detergent:lipid ratios (i.e. molar ratios in the mixed aggregates, vesicles or micelles) have been estimated for the solubilization of phosphatidylcholine vesicles by Triton X-100. Effective molar ratios are given for both the onset and the completion of bilayer solubilization; small unilamellar, large unilamellar and multilamellar vesicles have been used. Effective detergent:lipid ratios are independent of phospholipid concentration, and their use allows a deeper understanding of membrane-surfactant interactions.

The effects of surfactants on the permeability of isolated perfused fish gills to urea.

1. The diffusional transfer capacity of [14C]urea in isolated perfused trout (Oncorhynchus mykiss) gills in the presence of sodium n-dodecylsulphate (SDS), n-dodecyltrimethylammonium bromide (DTAB) and p-t-octylbenzene oxyethylene10 (Triton X-100) has been measured over a range of surfactant concentrations. 2. Urea has been shown to be transported transcellularly through the respiratory cells of the gill secondary lamellae by passive diffusion. Each surfactant was found to markedly increase the rate of diffusion and the diffusional transfer capacity reached a steady-state at a particular surfactant concentration. 3. The steady state flux was increased by surfactant in the sequence DTAB greater than SDS greater than Triton X-100 and the surfactant concentrations in terms of the critical micelle concentration (CMC) at which the diffusional transfer capacities reached limiting values were 0.92 x CMC (SDS), 0.53 x CMC (DTAB) and 2.5 x CMC (Triton X-100). 4. Compared to interactions between isolated epithelial cells and the surfactants, the rates at which the surfactants changed the urea flux were slow, suggesting that the mucus layer plays a significant role in protecting the epithelial cells of the secondary lamellae from disruption. 5. Relative to the other surfactants, DTAB had the most marked effect on both the rate of flux change and on the magnitude of the change, at concentrations which are low relative to the CMC, suggesting a more specific interaction with the negatively charged mucus layer consistent with the toxic effects of quaternary ammonium compounds on aquatic organisms.

Detergent solubilization of phospholipid vesicle. Effect of electric charge.

In order to explore the effect of electric charge on detergent solubilization of phospholipid bilayers, the interaction of nine electrically charged surfactants with neutral or electrically charged liposomes has been examined. The detergents belonged to the alkyl pyridinium, alkyl trimethylammonium or alkyl sulphate families. Large unilamellar liposomes formed by egg phosphatidylcholine plus or minus stearylamine or dicetyl phosphate were used. Solubilization was assessed as a decrease in light-scattering of the liposome suspensions. The results suggest that electrostatic forces do not play a significant role in the formation of mixed micelles and that hydrophobic interactions are by far the main forces involved in solubilization. In addition, from the study of thirty different liposome-surfactant systems, we have derived a series of empirical rules that may be useful in predicting the behaviour of untested surfactants: (i) the detergent concentration producing the onset of solubilization (Don) decreases as the alkyl chain length increases; the decrease follows a semi-logarithmic pattern in the case of alkyl pyridinium compounds; (ii) for surfactants with critical micellar concentrations (cmc) less than 6 x 10(-3) M, Don. is independent of the nature of the detergent and the bilayer composition; for detergents having cmc greater than 6 x 10(-3) M, Don. increases linearly with the cmc; and (iii) Don. varies linearly with the surfactant concentration that produces maximum solubilization.

Surfactant enhancement of polyethyleneglycol-induced cell fusion.

B16 mouse melanoma cells in monolayers may be satisfactorily fused with 50% PEG 1500. However, pre-treatment with detergents in solution at low concentrations significantly increases PEG fusion, up to 8-fold in some instances, without impairing cell viability. The practical and mechanistical implications of this finding are discussed.

Sublytic and lytic effects of the zwitterionic bile derivative 3-((3-deoxycholamidopropyl)dimethylammonio)-1-propanesulfonate on phosphatidylcholine liposomes.

The effects of the zwitterionic bile derivative 3-((3-deoxycholamidopropyl)dimethyl-ammonio)-1-propanesulfonate (Chaps) on multilamellar phosphatidylcholine liposomes have been characterized. When the surfactant is added to preformed liposome suspensions, equilibrium is attained in less than 6 h. Fifty percent solubilization, as measured by analysis of lipid P in supernatants after solubilization, occurs at a 0.32 lipid/detergent mole ratio for a 1 mM phospholipid concentration. Fifty percent release of entrapped glucose occurs at the same detergent concentration, suggesting that, in this system, no increase in permeability occurs prior to solubilization. A linear relationship is found between phospholipid concentration and amount of surfactant producing 50% solubilization. No lytic effect of Chaps is seen below 2 mM surfactant, this being probably near the critical micellar concentration of the amphiphile under our conditions. In the sublytic range of detergent concentrations, Chaps binds the lipid bilayers with high affinity, so that, at least at 1 mM phospholipid, the amount of free Chaps is negligible; solubilization starts when about two surfactant molecules are incorporated per phospholipid molecule. Differential scanning calorimetry shows that incorporation of Chaps into saturated phosphatidylcholine bilayers, even at concentrations below those producing solubilization, causes a decrease in the Tc gel-to-liquid crystalline main transition temperature of the phospholipid, and a decrease in the transition enthalpy; at the same time, a "shoulder" appears on the low-temperature side of the main endotherm. The ensemble of our data suggests that the behavior of Chaps toward phospholipid bilayers is intermediate between that of the natural bile derivatives and that of some well-known nonionic synthetic surfactants.

Micellar properties of the zwitterionic bile derivative CHAPS.

The critical micellar concentration (cmc) of the zwitterionic bile derivative CHAPS has been measured by fluorescence, light scattering and vapour pressure osmometry methods. The latter technique has been found most convenient and reliable. At 25 degrees C, in pure water, a cmc near 9 mM is found. Temperature decreases only slightly the cmc, from 9 to 7 mM upon heating from 25 to 50 degrees C. pH, in the range between 4 and 11, does not affect the cmc of CHAPS. Ionic strength appears to induce a marked decrease in cmc: measurements in 40 mM KCl give values about one-half those found for CHAPS in pure water.