Arsenic-containing hydrocarbons disrupt a model in vitro blood-cerebrospinal fluid barrier.

Lipid-soluble arsenicals, so-called arsenolipids, have gained a lot of attention in the last few years because of their presence in many seafoods and reports showing substantial cytotoxicity emanating from arsenic-containing hydrocarbons (AsHCs), a prominent subgroup of the arsenolipids. More recent in vivo and in vitro studies indicate that some arsenolipids might have adverse effects on brain health. In the present study, we focused on the effects of selected arsenolipids and three representative metabolites on the blood-cerebrospinal fluid barrier (B-CSF-B), a brain-regulating interface. For this purpose, we incubated an in vitro model of the B-CSF-B composed of porcine choroid plexus epithelial cells (PCPECs) with three AsHCs, two arsenic-containing fatty acids (AsFAs) and three representative arsenolipid metabolites (dimethylarsinic acid, thio/oxo-dimethylpropanoic acid) to examine their cytotoxic potential and impact on barrier integrity. The toxic arsenic species arsenite was also tested in this way and served as a reference substance. While AsFAs and the metabolites showed no cytotoxic effects in the conducted assays, AsHCs showed a strong cytotoxicity, being up to 1.5-fold more cytotoxic than arsenite. Analysis of the in vitro B-CSF-B integrity showed a concentration-dependent disruption of the barrier within 72 h. The correlation with the decreased plasma membrane surface area (measured as capacitance) indicates cytotoxic effects. These findings suggest exposure to elevated levels of certain arsenolipids may have detrimental consequences for the central nervous system.

Effects of arsenolipids on in vitro blood-brain barrier model.

Arsenic-containing hydrocarbons (AsHCs), a subgroup of arsenolipids (AsLs) occurring in fish and edible algae, possess a substantial neurotoxic potential in fully differentiated human brain cells. Previous in vivo studies indicating that AsHCs cross the blood-brain barrier of the fruit fly Drosophila melanogaster raised the question whether AsLs could also cross the vertebrate blood-brain barrier (BBB). In the present study, we investigated the impact of several representatives of AsLs (AsHC 332, AsHC 360, AsHC 444, and two arsenic-containing fatty acids, AsFA 362 and AsFA 388) as well as of their metabolites (thio/oxo-dimethylpropionic acid, dimethylarsinic acid) on porcine brain capillary endothelial cells (PBCECs, in vitro model for the blood-brain barrier). AsHCs exerted the strongest cytotoxic effects of all investigated arsenicals as they were up to fivefold more potent than the toxic reference species arsenite (iAsIII). In our in vitro BBB-model, we observed a slight transfer of AsHC 332 across the BBB after 6 h at concentrations that do not affect the barrier integrity. Furthermore, incubation with AsHCs for 72 h led to a disruption of the barrier at sub-cytotoxic concentrations. The subsequent immunocytochemical staining of three tight junction proteins revealed a significant impact on the cell membrane. Because AsHCs enhance the permeability of the in vitro blood-brain barrier, a similar behavior in an in vivo system cannot be excluded. Consequently, AsHCs might facilitate the transfer of accompanying foodborne toxicants into the brain.

Chitosan encapsulation modulates the effect of capsaicin on the tight junctions of MDCK cells.

Capsaicin has known pharmacological effects including the ability to reversibly open cellular tight junctions, among others. The aim of this study was to develop a strategy to enhance the paracellular transport of a substance with low permeability (FITC-dextran) across an epithelial cell monolayer via reversible opening of cellular tight junctions using a nanosystem comprised by capsaicin and of chitosan. We compared the biophysical properties of free capsaicin and capsaicin-loaded chitosan nanocapsules, including their cytotoxicity towards epithelial MDCK-C7 cells and their effect on the integrity of tight junctions, membrane permeability and cellular uptake. The cytotoxic response of MDCK-C7 cells to capsaicin at a concentration of 500 µM, which was evident for the free compound, is not observable following its encapsulation. The interaction between nanocapsules and the tight junctions of MDCK-C7 cells was investigated by impedance spectroscopy, digital holographic microscopy and structured illumination fluorescence microscopy. The nanocapsules modulated the interaction between capsaicin and tight junctions as shown by the different time profile of trans-epithelial electrical resistance and the enhanced permeability of monolayers incubated with FITC-dextran. Structured illumination fluorescence microscopy showed that the nanocapsules were internalized by MDCK-C7 cells. The capsaicin-loaded nanocapsules could be further developed as drug nanocarriers with enhanced epithelial permeability.

Intestinal formation of trans-crocetin from saffron extract (Crocus sativus L.) and in vitro permeation through intestinal and blood brain barrier.

AIMS: Extracts of saffron (Crocus sativus L.) have traditionally been used against depressions. Recent preclinical and clinical investigations have rationalized this traditional use. Trans-crocetin, a saffron metabolite originating from the crocin apocarotenoids, has been shown to exert strong NMDA receptor affinity and is thought to be responsible for the CNS activity of saffron. Pharmacokinetic properties of the main constituents from saffron have only been described to a limited extent. Therefore the present in vitro study aimed to determine if crocin-1 and trans-crocetin are able to pass the intestinal barrier and to penetrate the blood brain barrier (BBB). Additionally, the intestinal conversion of glycosylated crocins to the lipophilic crocetin had to be investigated. Experiments with Caco-2 cells and two different porcine BBB systems were conducted. Further on, potential intestinal metabolism of saffron extract was investigated by ex vivo experiments with murine intestine. METHODOLOGY: In vitro Caco-2 monolayer cell culture was used for investigation of intestinal permeation of crocin-1 and trans-crocetin. In vitro models of porcine brain capillary endothelial cells (BCEC) and blood cerebrospinal fluid barrier (BCSFB) were used for monitoring permeation characteristics of trans-crocetin through the blood brain barrier (BBB). Intestine tissue and feces homogenates from mice served for metabolism experiments. RESULTS: Crocin-1, even at high concentrations (1000 µM) does not penetrate Caco-2 monolayers in relevant amounts. In contrast, trans-crocetin permeates in a concentration-independent manner (10-114 µM) the intestinal barrier by transcellular passage with about 32% of the substrate being transported within 2 h and a permeation coefficient of Papp 25.7 × 10(-)(6) ± 6.23 × 10(-)(6) cm/s. Trans-crocetin serves as substrate for pGP efflux pump. Trans-crocetin permeates BBB with a slow but constant velocity over a 29 h period (BCEC system: Papp 1.48 × 10(-)(6) ± 0.12 × 10(-)(6) cm/s; BCSFB system Papp 3.85 × 10(-)(6) ± 0.21 × 10(-)(6) cm/s). Conversion of glycosylated crocins from saffron extract to trans-crocetin occurs mainly by intestinal cells, rather than by microbiological fermentation in the colon. CONCLUSION: The here described in vitro studies have shown that crocins from saffron are probably not bioavailable in the systemic compartment after oral application. On the other side the investigations clearly have pointed out that crocins get hydrolyzed in the intestine to the deglycosylated trans-crocetin, which subsequently is absorbed by passive transcellular diffusion to a high extend and within a short time interval over the intestinal barrier. Crocetin will penetrate in a quite slow process the blood brain barrier to reach the CNS. The intestinal deglycosylation of different crocins in the intestine is mainly due to enzymatic processes in the epithelial cells and only to a very minor extent due to deglycosylation by the fecal microbiome. On the other side the fecal bacteria degrade the apocarotenoid backbone to smaller alkyl units, which do not show any more the typical UV absorbance of crocins. As previous studies have shown strong NMDA receptor affinity and channel opening activity of trans-crocetin the use of saffron for CNS disorders seems to be justified from the pharmacokinetic and pharmacodynamic background.

Exploiting the properties of biomolecules for brain targeting of nanoparticulate systems.

The main obstacle in the treatment of central nervous system diseases is represented by a limited passage of diagnostic and therapeutic agents across the blood-brain barrier, which separates the blood stream from the cerebral parenchyma and maintains the homeostasis of the brain. The growing knowledge about the brain capillary endothelium and the discovery of specific mechanisms for the uptake of substances enables the development of various strategies to enhance the drug delivery rate into the brain. Among the different strategies, nanoparticles are promising candidates for drug delivery to the brain due to their potential in encapsulating drugs and thereby disguising their permeation limiting characteristics. Furthermore a surface functionalization of many nanoparticles can easily be achieved allowing the active targeting of nanoparticles to the brain. For this non-invasive approach, the surface functionalization of nanoparticles with biomolecules has shown promising potential for effective drug delivery to the brain. This review indexes the main classes of biomolecules used for the surface functionalization of nanoparticles and discusses their potential as drug delivery systems for an enhanced passage of diagnostic and therapeutic agents into the brain parenchyma.

Influence of lipid saturation grade and headgroup charge: a refined lung surfactant adsorption model.

Rapid adsorption of surfactant material to the air/liquid interface of the lung is essential for maintaining normal lung function. The detailed mechanism of this process, however, remains unclear. In this study, we elucidate the influence of lipid saturation grade and headgroup charge of surface layer lipids on surfactant protein (SP)-induced vesicle insertion into monolayers spread at the air/water interface of a film balance. We used dipalmitoylphosphatidlycholine (DPPC),1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) as monolayer lipids doped with either hydrophobic surfactant-specific protein SP-B or SP-C (0.2 and 0.4 mol %, respectively). Vesicles consisting of DPPC/DPPG (4:1, mol ratio) were injected into a stirred subphase to quantify adsorption kinetics. Based on kinetic film balance and fluorescence measurements, a refined model describing distinct steps of vesicle adsorption to surfactant monolayers is presented. First, in a protein-independent step, lipids from vesicles bridged to the interfacial film by Ca(2+) ions are inserted into defects of a disordered monolayer at low surface pressures. Second, in a SP-facilitated step, active material insertion involving an SP-B- or SP-C-induced flip-flop of lipids occurs at higher surface pressures. Negatively charged lipids obviously influence the threshold pressures at which this second protein-mediated adsorption mechanism takes place.

Solubility versus electrostatics: what determines lipid/protein interaction in lung surfactant.

Mammalian lung surfactant is a complex lipid/protein mixture covering the alveolar interface and has the crucial function of reducing the surface tension at this boundary to minimal values. Surfactant protein SP-B plays an important role for this purpose and was the focus of many recent studies. However, the specificity of lipid/SP-B interactions is controversial. Since these investigations were accomplished at varying pH conditions (pH 5.5 and 7.0), we studied the specificity of these interactions in a dipalmitoylphosphatidylcholine (DPPC)/dipalmitoylphosphatidylglycerol (DPPG)/SP-B (4:1:0.2 mol %) model system at either pH. Mainly fluorescence microscopy and laterally resolved time-of-flight secondary ion mass spectrometry were used to reveal information about the phase behavior of the lipids and the molecular distribution of SP-B in the lipid mixture. DPPG forms separated condensed domains due to a strong hydrogen-bond network, from which the protein is mainly excluded. Considering the protein as an impurity of the lipid mixture leads to the principle of the zone melting process: an impurity is highly more soluble in a liquid phase than in a solid phase. The phase behavior effect of the lipids mainly outperforms the electrostatic interactions between DPPG and SP-B, leading to a more passively achieved colocalization of DPPC and SP-B.

Lipid specificity of surfactant protein B studied by time-of-flight secondary ion mass spectrometry.

One of the key functions of mammalian pulmonary surfactant is the reduction of surface tension to minimal values. To fulfill this function it is expected to become enriched in dipalmitoylphosphatidylcholine either on its way from the alveolar type II pneumocytes to the air/water interface of the lung or within the surface film during compression and expansion of the alveoli during the breathing cycle. One protein that may play a major role in this enrichment process is the surfactant protein B. The aim of this study was to identify the lipidic interaction partner of this protein. Time-of-flight secondary ion mass spectrometry was used to analyze the lateral distribution of the components in two SP-B-containing model systems. Either native or partly isotopically labeled lipids were analyzed. The results of both setups give strong indications that, at least under the specific conditions of the chosen model systems (e.g., concerning pH and lipid composition), the lipid interacting with surfactant protein B is not phosphatidylglycerol as generally accepted, but dipalmitoylphosphatidylcholine instead.

Lipid-membrane affinity of chimeric metal-binding green fluorescent protein.

The Green Fluorescent Protein (GFP) is a useful marker to trace the expression of cellular proteins. However, little is known about changes in protein interaction properties after fusion to GFP. In this study, we present evidence for a binding affinity of chimeric cadmium-binding green fluorescent proteins to lipid membrane. This affinity has been observed in both cellular membranes and artificial lipid monolayers and bilayers. At the cellular level, the presence of Cd-binding peptide promoted the association of the chimeric GFP onto the lipid membrane, which declined the fluorescence emission of the engineered cells. Binding affinity to lipid membranes was further investigated using artificial lipid bilayers and monolayers. Small amounts of the chimeric GFP were found to incorporate into the lipid vesicles due to the high surface pressure of bilayer lipids. At low interfacial pressure of the lipid monolayer, incorporation of the chimeric Cd-binding GFP onto the lipid monolayer was revealed. From the measured lipid isotherms, we conclude that Cd-binding GFP mediates an increase in membrane fluidity and an expansion of the surface area of the lipid film. This evidence was strongly supported by epifluorescence microscopy, showing that the chimeric Cd-binding GFP preferentially binds to fluid-phase areas and defect parts of the lipid monolayer. All these findings demonstrate the hydrophobicity of the GFP constructs is mainly influenced by the fusion partner. Thus, the example of a metal-binding unit used here shines new light on the biophysical properties of GFP constructs.

Predicting blood-brain barrier permeability of drugs: evaluation of different in vitro assays.

The ability of a drug to penetrate the blood-brain barrier (BBB) is essential for its use in the pharmaceutical treatment of CNS disorders. Five different in vitro methods to predict BBB permeability of drugs were compared and evaluated in the present study. All assays were performed with a consistent set of seven compounds and in the same physiological buffer to provide a basis for direct comparison of the results. Octanol-buffer and liposome-buffer partition coefficients were most conveniently obtained but failed to predict BBB permeability for certain drugs. The incorporation of drugs into lipid monolayers at the air-buffer interface was found to be a poor predictor of BBB permeability and was furthermore not considered suitable for screening due to the demanding experimental requirements. Permeability studies using Caco-2 cell monolayers provided a good correlation to an in vitro model of the BBB, which was based on primary cultured porcine brain capillary endothelial cells (PBCEC). However, differences in drug permeability between the intestine and brain derived cells were detected, limiting the advantages of the easy handling of the Caco-2 cell line compared to the more time-consuming primary culture of the BCEC.

Fluorescence light microscopy of pulmonary surfactant at the air-water interface of an air bubble of adjustable size.

The structural dynamics of pulmonary surfactant was studied by epifluorescence light microscopy at the air-water interface of a bubble as a model close to nature for an alveolus. Small unilamellar vesicles of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, a small amount of a fluorescent dipalmitoylphosphatidylcholine-analog, and surfactant-associated protein C were injected into the buffer solution. They aggregated to large clusters in the presence of Ca(2+) and adsorbed from these units to the interface. This gave rise to an interfacial film that eventually became fully condensed with dark, polygonal domains in a fluorescent matrix. When now the bubble size was increased or decreased, respectively, the film expanded or contracted. Upon expansion of the bubble, the dark areas became larger to the debit of the bright matrix and reversed upon contraction. We were able to observe single domains during the whole process. The film remained condensed, even when the interface was increased to twice its original size. From comparison with scanning force microscopy directly at the air-water interface, the fluorescent areas proved to be lipid bilayers associated with the (dark) monolayer. In the lung, such multilayer phase acts as a reservoir that guarantees a full molecular coverage of the alveolar interface during the breathing cycle and provides mechanical stability to the film.

Molecular architecture of the thylakoid membrane: lipid diffusion space for plastoquinone.

We have determined the stoichiometric composition of membrane components (lipids and proteins) in spinach thylakoids and have derived the molecular area occupied by these components. From this analysis, the lipid phase diffusion space, the fraction of lipids located in the first protein solvation shell (boundary lipids), and the plastoquinone (PQ) concentration are derived. On the basis of these stoichiometric data, we have analyzed the motion of PQ between photosystem (PS) II and cytochrome (cyt.) bf complexes in this highly protein obstructed membrane (protein area about 70%) using percolation theory. This analysis reveals an inefficient diffusion process. We propose that distinct structural features of the thylakoid membrane (grana formation, microdomains) could help to minimize these inefficiencies and ensure a non-rate limiting PQ diffusion process. A large amount of published evidence supports the idea that higher protein associations exist, especially in grana thylakoids. From the quantification of the boundary lipid fraction (about 60%), we conclude that protein complexes involved in these associations should be spaced by lipids. Lipid-spaced protein aggregations in thylakoids are qualitatively different to previously characterized associations (multisubunit complexes, supercomplexes). We derive a hierarchy of protein and lipid interactions in the thylakoid membrane.

Scanning force microscopy at the air-water interface of an air bubble coated with pulmonary surfactant.

To study the structure-function relationship of pulmonary surfactant under conditions close to nature, molecular films of a model system consisting of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, and surfactant-associated protein C were prepared at the air-water interface of air bubbles about the size of human alveoli (diameter of 100 microm). The high mechanical stability as well as the absence of substantial film flow, inherent to small air bubbles, allowed for scanning force microscopy (SFM) directly at the air-water interface. The SFM topographical structure was correlated to the local distribution of fluorescent-labeled dipalmitoylphosphatidylcholine, as revealed from fluorescence light microscopy of the same bubbles. Although SFM has proven before to be exceptionally well suited to probe the structure of molecular films of pulmonary surfactant, the films so far had to be transferred onto a solid support from the air-water interface of a film balance, where they had been formed. This made them prone to artifacts imposed by the transfer. Moreover, the supported monolayers disallowed the direct observation of the structural dynamics associated with expansion and compression of the films as upon breathing. The current findings are compared in this respect to our earlier findings from films, transferred onto a solid support.

Channel activity of a phytotoxin of Clavibacter michiganense ssp. nebraskense in tethered membranes.

Solid-supported membranes immobilized on gold electrodes were used to detect and characterize the spontaneously inserting anion-selective protein channel (Clavibacter anion channel, CAC) present in the culture fluid of Clavibacter michiganense ssp. nebraskense. Three different membrane systems varying in the composition of the first chemisorbed monolayer were investigated by means of impedance spectroscopy. Conductance changes of the immobilized lipid membranes were sensitively detected after adding the culture fluid of the bacteria to the solid-supported membranes, indicating that the relative change in conductance is largest if the lipid layer is attached to the surface via a flexible lipid anchor. Variation in the d.c. potential revealed that CAC exhibits a voltage dependence in these tethered membranes which can be described by an exponential function in accordance with previous results obtained from patchclamp measurements and impedance analysis. The addition of an inhibitor that selectively blocks anion channels abolished the channel conductance almost completely, indicating that the increased conductivity can be attributed to the specific insertion of the CAC. A linear dependence of the channel conductance on the chloride concentration was found, which was modulated by the charges of the second lipid monolayer. The results demonstrate that tethered lipid membranes on gold surfaces in conjunction with impedance spectroscopy allows one to monitor and characterize water-soluble spontaneously inserting channels, providing an effective means to probe for bacterial toxins.

Porcine Choroid plexus epithelial cells in culture: regulation of barrier properties and transport processes.

The epithelial cells of the choroid plexus are the structural basis of the blood-cerebrospinal fluid (CSF)-barrier. Here we summarise our recent efforts to culture those cells mainly on permeable supports in vitro. Isolated from porcine brains, we report a simple protocol for the primary culture using cytosine arabinoside as an additive that is cytotoxic for other cells except the plexus epithelial cells. Enhanced barrier properties are obtained by withdrawal of serum from the culture medium after confluency is reached. Cells improve their polarity, permeability for hydrophilic substrates is lowered, electrical resistance is increased tenfold, and a pH-gradient is built up across the cell monolayer. Polarised secretion of proteins and most importantly fluid secretion into the apical filter compartment was attained and proven to be dependent on the Na(+),K(+)-ATPase activity. Active transport processes (penicillin G, riboflavin, myo-inositol, ascorbic acid) were studied and clearly showed the involvement of the organic anion transporter. The permeability of the barrier was found to be regulated by cyclic adenosine monophosphate (cAMP). Moreover, we report that cell proliferation and differentiation is controlled by components of the extracellular matrix. The present culture model could now be used as an in vitro system to quantify drug transport across the blood-CSF-barrier.

Lipid polarity in brain capillary endothelial cells.

Brain capillary endothelial cells (BCEC) represent an epithelial like cell type with continuous tight junctions and polar distributed proteins. In this paper we investigated whether cultured BCEC show a polar distribution of membrane lipids as this was demonstrated for many epithelial cell types. Therefore we applied a high yield membrane fractionation method to isolate pure fractions of the apical and the basolateral plasma membrane (PM) domains. Using a set of methods for lipid analysis we were able to determine the total lipid composition of the whole cells and the PM fractions. Both membrane domains showed a unique lipid composition with clear differences to each other and to the whole cell composition. Three lipid species were polar distributed between the two PM domains. Phosphatidylcholine was enriched in the apical membrane whereas sphingomyelin and glucosylceramide were enriched in the basolateral membrane. The possible function of this lipid polarity for the blood-brain barrier mechanism is the generation of a suitable lipid environment for polar distributed membrane proteins and the generation of two PM domains with different biophysical properties and permeabilities.

The effect of lipid composition and physical state of phospholipid monolayer on the binding and incorporation of a basic amphipathic peptide from the C-terminal region of the HIV envelope protein gp41.

The interaction of a peptide identical to the carboxy terminal region of the envelope glycoprotein gp41(828) of HIV with negatively charged phospholipids in a monolayer was studied by a Wilhelmy film balance. No significant interaction of the peptide with a monolayer composed of pure neutral but a strong affinity to negatively charged phospholipids could be observed. In mixed phospholipid monolayers the binding of the gp41(828) is primarily limited by the amount of acidic phospholipids. The physical state of the monolayer is another important parameter for binding. Clustering of negatively charged phospholipids and the surface pressure are crucial. Ca(2+) ions strongly interfere with the peptide-lipid interaction up to complete abolishment. The effects observed are dependent on the nature of the acidic lipid. Phosphatidylglycerol was found to be more sensitive than phosphatidylserine. The significance of the results for processes like virus assembly and budding will be discussed.

Formation of three-dimensional protein-lipid aggregates in monolayer films induced by surfactant protein B.

This study focuses on the structural organization of surfactant protein B (SP-B) containing lipid monolayers. The artificial system is composed of the saturated phospholipids dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) in a molar ratio of 4:1 with 0.2 mol% SP-B. The different "squeeze-out" structures of SP-B were visualized by scanning probe microscopy and compared with structures formed by SP-C. Particularly, the morphology and material properties of mixed monolayers containing 0.2 mol% SP-B in a wide pressure range of 10 to 54 mN/m were investigated revealing that filamentous domain boundaries occur at intermediate surface pressure (15-30 mN/m), while disc-like protrusions prevail at elevated pressure (50-54 mN/m). In contrast, SP-C containing lipid monolayers exhibit large flat protrusions composed of stacked bilayers in the plateau region (app. 52 mN/m) of the pressure-area isotherm. By using different scanning probe techniques (lateral force microscopy, force modulation, phase imaging) it was shown that SP-B is dissolved in the liquid expanded rather than in the liquid condensed phase of the monolayer. Although artificial, the investigation of this system contributes to further understanding of the function of lung surfactant in the alveolus.

Electromagnetic fields (1.8 GHz) increase the permeability to sucrose of the blood-brain barrier in vitro.

We report an investigation on the influence of high frequency electromagnetic fields (EMF) on the permeability of an in vitro model of the blood-brain barrier (BBB). Our model was a co-culture consisting of rat astrocytes and porcine brain capillary endothelial cells (BCEC). Samples were characterized morphologically by scanning electron microscopy and immunocytochemistry. The BBB phenotype of the BCEC was shown by the presence of zona occludens protein (ZO-1) as a marker for tight junctions and the close contact of the cells together with the absence of intercellular clefts. Permeability measurements using (14)C-sucrose indicated a physiological tightness which correlated with the morphological findings and verified the usefulness of our in vitro model. Samples were exposed to EMF conforming to the GSM1800-standard used in mobile telephones (1.8 GHz). The permeability of the samples was monitored over four days and compared with results of samples that were cultured identically but not exposed to EMF. Exposure to EMF increased permeability for (14)C-sucrose significantly compared to unexposed samples. The underlying pathophysiological mechanism remains to be investigated.

Analysis of lung surfactant model systems with time-of-flight secondary ion mass spectrometry.

An often-used model lung surfactant containing dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), and the surfactant protein C (SP-C) was analyzed as Langmuir-Blodgett film by spatially resolved time-of-flight secondary ion mass spectrometry (TOF-SIMS) to directly visualize the formation and composition of domains. Binary lipid and lipid/SP-C systems were probed for comparison. TOF-SIMS spectra revealed positive secondary ions (SI) characteristic for DPPC and SP-C, but not for DPPG. SI mapping results in images with domain structures in DPPC/DPPG and DPPG/SP-C, but not in DPPC/SP-C films. We are able to distinguish between the fluid and condensed areas probably due to a matrix effect. These findings correspond with other imaging techniques, fluorescence light microscopy (FLM), scanning force microscopy (SFM), and silver decoration. The ternary mixture DPPC/DPPG/SP-C transferred from the collapse region exhibited SP-C-rich domains surrounding pure lipid areas. The results obtained are in full accordance with our earlier SFM picture of layered protrusions that serve as a compressed reservoir for surfactant material during expansion. Our study demonstrates once more that SP-C plays a unique role in the respiration process.