The nonelectrolyte permeability of planar lipid bilayer membranes

The permeability of lecithin bilayer membranes to nonelectrolytes is in reasonable agreement with Overton's rule. The is, Pd alpha DKhc, where/Pd is the permeability coefficient of a solute through the bilayer, Khc is its hydrocarbon:water partition coefficient, and D is its diffusion coefficient in bulk hydrocarbon. The partition coefficients are by far the major determinants of the relative magnitudes of the permeability coefficients; the diffusion coefficients make only a minor contribution. We note that the recent emphasis on theoretically calculated intramembranous diffusion coefficients (Dm'S) has diverted attention from the experimentally measurable and physiologically relevant permeability coefficients (Pd'S) and has obscured the simplicity and usefulness of Overton's rule.     

Comparative study of molecular dynamics,diffusion, and permeability for ligands in biomembranes of different lipid composition

A comparative study of several model lipid bilayers of different composition, which included analysis of kinetic parameters of model lipid bilayers and permeability of bilayer membranes for small molecules, has been carried out. The conformity of results of numeric experiments to experimental data (structure of membrane lipid bilayers, lateral diffusion coefficients, and relative permeability of biomembranes for ligands) is discussed in the framework of a standard molecular dynamics protocol.     

The passive permeability properties of in vivo perfused rat jejunum

The polarity of the microvillus membrane of rat and rabbit jejunum, determined in vitro by the incremental free energy changes associated with the addition of -CH₂ groups to fatty acids or -OH groups to bile acids, has been found to be more polar than other biological membranes. The reason for the difference in polarity is unexplained but could be due to artifacts caused by the in vitro conditions. In the current studies the apparent permeability coefficients were determined for a homologous series of fatty acids and bile acids in in vivo perfused rat jejunum. The true permeability coefficients were derived by correction for diffusion barrier resistance. The incremental free energy changes associated with the addition of a -CH₂ group to a fatty acid and a -OH group to a bile acid were −619 and +2069 cal/mol, respectively. These values correspond to values determined in other biological membranes such as erythrocytes and adipocytes. Thus, the rat microvillus membrane is more nonpolar than previously observed in vitro. The reason for the discrepancy between the in vivo and in vitro results is most likely due to an underestimation of the permeability coefficients in the in vitro studies.     

Nonelectrolyte diffusion across lipid bilayer systems

The permeability coefficients of a homologous series of amides from formamide through valeramide have been measured in spherical bilayers prepared by the method described by Jung. They do not depend directly on the water:ether partition coefficient which increases regularly with chain length. Instead there is a minimum at acetamide. This has been ascribed to the effect of steric hindrance on diffusion within the bilayer which increases with solute molar volume. This factor is of the same magnitude, though opposite in sign to the effect of lipid solubility, thus accounting for the minimum. The resistance to passage across the interface has been compared to the resistance to diffusion within the membrane. As the solute chain length increases the interface becomes more important, until for valeramide it comprises about 90% of the total resistance. Interface resistance is also important in urea permeation, causing urea to permeate much more slowly than an amide of comparable size, after allowance is made for the difference in the water:ether partition coefficient. Amide permeation coefficients have been compared with relative liposome permeation data measured by the rate of liposome swelling. The ratios of the two measures of permeation vary between 3 and 16 for the homologous amides. The apparent enthalpy of liposome permeation has been measured and found to be in the neighborhood of 12 kcal mol-1 essentially independent of chain length. Comparison of the bilayer permeability coefficients with those of red cells shows that red cell permeation by the lipophilic solutes resembles that of the bilayers, whereas permeation by the hydrophilic solutes differs significantly.     

Relationships between structure and high-throughput screening permeability of peptide derivatives and related compounds with artificial membranes: application to prediction of Caco-2 cell permeability

To evaluate absorption of compounds across the membrane via a transcellular route, the permeability of peptide derivatives and related compounds was measured by the parallel artificial membrane permeation assay (PAMPA). The permeability coefficients by PAMPA were analyzed quantitatively using classical QSAR and Volsurf approaches with the physicochemical parameters. The results from both approaches showed that hydrogen bonding ability of molecules in addition to hydrophobicity at a particular pH were significant in determining variations in PAMPA permeability coefficients. The relationship between Caco-2 cell permeability and artificial lipid membrane permeability was then determined. The compounds were sorted according to their absorption pathway in the plot of the Caco-2 cell and PAMPA permeability coefficients.     

Influence of chain ordering on the selectivity of dipalmitoylphosphatidylcholine bilayer membranes for permeant size and shape.

The effects of lipid chain packing and permeant size and shape on permeability across lipid bilayers have been investigated in gel and liquid crystalline dipalmitoylphosphatidylcholine (DPPC) bilayers by a combined NMR line-broadening/dynamic light scattering method using seven short-chain monocarboxylic acids (formic acid, acetic acid, propionic acid, butyric acid, valeric acid, isovaleric acid, and trimethylacetic acid) as permeants. The experimental permeability coefficients are compared with the predictions of a bulk solubility diffusion model in which the bilayer membrane is represented as a slab of bulk hexadecane. Deviations of the observed permeability coefficients (Pm) from the values predicted from solubility diffusion theory (Po) lead to the determination of a correction factor, the permeability decrement f (= Pm/Po), to account for the effects of chain ordering. The natural logarithm of f has been found to correlate linearly with the inverse of the bilayer free surface area with slopes of 25 +/- 2, 36 +/- 3, 45 +/- 8, 32 +/- 12, 33 +/- 4, 49 +/- 12, and 75 +/- 6 A2 for formic acid, acetic acid, propionic acid, butyric acid, valeric acid, isovaleric acid, and trimethylacetic acid, respectively. The slope, which measures the sensitivity of the permeability coefficient of a given permeant to bilayer chain packing, exhibits an excellent linear correlation (r = 0.94) with the minimum cross-sectional area of the permeant and a poor correlation (r = 0.59) with molecular volume, suggesting that in the bilayer interior the permeants prefer to move with their long principal axis along the bilayer normal. Based on these studies, a permeability model combining the effects of bilayer chain packing and permeant size and shape on permeability across lipid membranes is developed.     

Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers

Permeability coefficients across monolayers of the human colon carcinoma cell line Caco-2, cultured on permeable supports, are commonly used to predict the absorption of orally administered drugs and other xenobiotics. This protocol describes our method for the cultivation, characterization and determination of permeability coefficients of xenobiotics (which are, typically, drug-like compounds) in the Caco-2 model. A few modifications that have been introduced over the years are incorporated in the protocol. The method can be used to trace the permeability of a test compound in two directions, from the apical to the basolateral side or vice versa, and both passive and active transport processes can be studied. The permeability assay can be completed within one working day, provided that the Caco-2 monolayers have been cultured and differentiated on the permeable supports 3 weeks in advance.     

Cryoprotectant permeability of aquaporin-3 expressed in Xenopus oocytes

It has been shown that aquaporin-3, a water channel, is expressed in mouse embryos. This type of aquaporin transports not only water but also neutral solutes, including cell-permeating cryoprotectants. Therefore, the expression of this channel may have significant influence on the survival of cryopreserved embryos. However, permeability coefficients of aquaporin-3 to cryoprotectants have not been determined except for glycerol. In addition, permeability coefficients under concentration gradients are important for developing and improving cryopreservation protocols. In this study, we examined the permeability of aquaporin-3 to various cryoprotectants using Xenopus oocytes. The permeability of aquaporin-3 to cryoprotectants was measured by the volume change of aquaporin-3 cRNA-injected oocytes in modified Barth's solution containing either 10% glycerol, 8% ethylene glycol, 10% propylene glycol, 1.5 M acetamide, or 9.5% DMSO (1.51-1.83 Osm/kg) at 25 degrees C. Permeability coefficients of aquaporin-3 for ethylene glycol and propylene glycol were 33.50 and 31.45 x 10(-3) cm/min, respectively, which were as high as the value for glycerol (36.13 x 10(-3) cm/min). These values were much higher than those for water-injected control oocytes (0.04-0.11 x 10(-3) cm/min). On the other hand, the coefficients for acetamide and DMSO were not well determined because the volume data were poorly fitted by the two parameter model, possibly because of membrane damage. To avoid this, the permeability for these cryoprotectants was measured under a low concentration gradient by suspending oocytes in aqueous solutions containing low concentrations of acetamide or DMSO dissolved in water (0.20 Osm/kg). The coefficient for acetamide (24.60 x 10(-3) cm/min) was as high as the coefficients for glycerol, ethylene glycol, and propylene glycol, and was significantly higher than the value for control (6.50 x 10(-3) cm/min). The value for DMSO (6.33 x 10(-3) cm/min) was relatively low, although higher than the value for control (0.79 x 10(-3) cm/min). This is the first reported observation of DMSO transport by aquaporin-3.     

Determination of Solute Permeability in Chara Internodes by a Turgor Minimum Method : Effects of External pH

The analysis of Sha'afi et al. (Sha'afi, Rich, Mickulecky, Solomon 1970 J Gen Physiol 55: 427-450) for determining solute permeability in red blood cells has been modified and applied to turgid plant cells. Following the addition of permeating solute to the external medium, a biphasic response of cell turgor can be measured with the pressure probe in isolated internodes of Chara corallina. After an initial decrease in turgor due to water flow (water phase), turgor increases due to the uptake of the solute (solute phase) until the original turgor is reattained. From the pressure/time course in the neighborhood of the minimum turgor, the permeability of the osmotic solute can be determined. The data obtained by the minimum method for rapidly permeating (ethanol, methanol) and slowly permeating (formamide, dimethylformamide) solutes are similar to those calculated from the half-time of pressure changes during the solute phase and to data obtained by other workers using radioactive tracers. The methods employing the pressure probe were applied to examine the effect of high pH (up to pH 11) on the membrane permeability. There appeared to be no effect of high pH on the permeability coefficients, reflection coefficients, and hydraulic conductivity.     

A new diffusion chamber system for the determination of drug permeability coefficients across the human intestinal epithelium that are independent of the unstirred water layer.

A new method for determining permeability coefficients, that are independent of the unstirred water layer (UWL), has been developed. The method was used to determine the cellular permeability coefficient of the rapidly absorbed drug testosterone in monolayers of the human intestinal epithelial cell line, Caco-2. Using a new diffusion cell with an effective stirring system based on a gas lift, the cellular permeability coefficient for testosterone was (1.98 +/- 0.13).10(-4) cm/s which is 3.5-times higher than the permeability coefficient obtained in the unstirred system. The thickness of the UWL obtained with the well stirred diffusion cell was 52 +/- 4 microns. This value is much lower than those previously reported in various well stirred in vitro models. The calculated cellular permeability of testosterone was 13-23-times lower than that for an UWL of the same thickness as the epithelial cell (17-30 microns). We conclude that the permeability of the epithelial monolayer must be included in calculations of the thickness of the UWL.     

Permeability studies on red cell membranes of dog, cat, and beef

Water permeability coefficients of dog, cat, and beef red cell membranes have been measured under an osmotic pressure gradient. The measurements employed a rapid reaction stop flow apparatus with which cell shrinking was measured under a relative osmotic pressure gradient of 1.25 to 1.64 times the isosmolar concentration. For the dog red cell the osmotic permeability coefficient is 0.36 cm⁴/(sec, osmol). The water permeability coefficient for the dog red cell under a diffusion gradient was also measured (rate constant = 0.10/msec). The ratio between the two permeabilities was used to calculate an equivalent pore radius of 5.9 A. This value agrees welt with an equivalent pore radius of 6.2 A obtained from reflection coefficients of nonelectrolyte water-soluble molecules, and is consistent with data on the permeability of the dog red cell membrane to glucose. These data provide evidence supporting the existence of equivalent pores in single biological membranes.     

Water permeability of plant cuticular membranes: the effects of humidity and temperature on the permeability of non-isolated cuticles of onion bulb scales

Abstract. Water permeability of cuticular membranes (CM) from the inner bulb scales of Allium cepa has been investigated. CM have a thickness ranging from 0.6 to 1.3 μm. They are composed of a thin (120–200 nm) lamellated cuticle proper and a thicker (300–900 nm) cuticular layer. Permeability coefficients for diffusion of water across these thin membranes are very low (4 × lO⁻¹⁰ms⁻¹⁰). There was no difference in permeability of CM from successive scales of the same onion. Extraction of soluble cuticular lipids (SCL) with chloroform increased permeability by a factor of 1350 to 2050. Preliminary data indicate that only 1 μg cm⁻¹⁰ of SCL are removed by this treatment, hydrocarbons being the main (75%) consistuent. Permeability coefficients of cuticular transpiration were little affected by relative humidity, showing that transport is limited by a hydrophobic barrier that lacks dipoles. However, following extraction, permeability of the membranes depended strongly on humidity due to the presence of polar functional groups in the polymer matrix. Soluble cuticular lipids undergo a phase transition around 47°C. Temperatures higher than that irreversibly increased water permeability.     

Investigation of the permeability and optical clearing ability of different analytes in human normal and cancerous breast tissues by spectral domain OCT

The potential of OCT applied for early breast cancer detection attracted significant efforts. The permeability coefficients and the percentages of OCT signal enhancement for normal and cancerous breast tissues have been investigated from the experiments of 20% glucose, 40% glucose, and 20% mannitol in vitro. Obtained results indicate that the permeability coefficient in breast cancer tissue is prominently larger than that in normal breast tissue, while the optical clearing effect is just the reverse to that for each agent. The results suggest that OCT has the ability to distinguish cancer tissue from different aspect. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Phloem mobility of xenobiotics: tabular review of physicochemical properties governing the output of the Kleier model

The Kleier model of phloem-mobility of xenobiotics combines the intermediate permeability hypothesis with the acid trap mechanism for weak acids. The output of the model is dependent on the lipophilicity of a compound, for which octanol/water partition coefficients (log K_ow) have been used as a measure. The membrane permeability of xenobiotics is predicted from these partition coefficients, and the nature of the sieve tube membranes has been modelled using regressions derived from Nitella or potato permeability data. A wide range of log K_ow values for herbicides, fungicides, insecticides and experimental compounds (400) have been tabulated along with the model output for various membrane parameters. The application of the model is in broad agreement with literature and experimental observations on many of the known phloem mobile herbicides and predicts low phloem mobility for the fungicides and insecticides considered here, again in agreement with the literature. The behaviour of herbicides representative of the main chemical families and modes of action are reviewed, along with examples of the few phloem-mobile fungicides and insecticides identified.Abbreviations K_ow octanol-water partition coefficient - pK_a –log₁₀ acid dissociation constant - C_f Concentration factor - P membrane permeability     

Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells.

Monolayers of a well differentiated human intestinal epithelial cell line, Caco-2, were used as a model to study passive drug absorption across the intestinal epithelium. Absorption rate constants (expressed as apparent permeability coefficients) were determined for 20 drugs and peptides with different structural properties. The permeability coefficients ranged from approximately 5 x 10(-8) to 5 x 10(-5) cm/s. A good correlation was obtained between data on oral absorption in humans and the results in the Caco-2 model. Drugs that are completely absorbed in humans had permeability coefficients greater than 1 x 10(-6) cm/s. Drugs that are absorbed to greater than 1% but less than 100% had permeability coefficients of 0.1-1.0 x 10(-6) cm/s while drugs and peptides that are absorbed to less than 1% had permeability coefficients of less than or equal to 1 x 10(-7) cm/s. The results indicate that Caco-2 monolayers can be used as a model for studies on intestinal drug absorption.     

Effect of grafted polyethylene glycol (PEG) on the size, encapsulation efficiency and permeability of vesicles

Liposomes have been prepared by the vesicle extrusion method (VETs) from mixtures of dipalmitoylphosphatidylcholine (DPPC), phosphatidylinositol (PI) and dipalmitoylphosphatidylethanolamine with covalently linked poly(ethylene glycol) molecular mass 5000 and 2000 (DPPE-PEG 5000 and DPPE-PEG 2000) covering a range of 0-7.5 mole%. The encapsulation of D-glucose has been studied and found to be markedly dependent on the mole% DPPE-PEG. The permeability of the liposomes to D-glucose has been measured both as a function of temperature and liposome composition. The permeability coefficients for D-glucose increase with mole% DPPE-PEG 5000 and with temperature over the range 25-50 degrees C. The activation energies for glucose permeability range from 90 to 23 kJ mol(-1). The decrease in activation energy with increasing temperature is attributed to an increasing number of bilayer defects as the liposome content of PEG-grafted lipid is increased. The dependence of D-glucose encapsulation as a function of PEG-grafted lipid content is discussed in terms of the conformation of the PEG molecules on the inner surface of the bilayer. For liposomes containing DPPE-PEG 5000 the relative percentage encapsulation of glucose, assuming that the PEG surface layer excludes glucose, is comparable to that predicted from the mushroom and brush conformational models.     

Permeability of barnacle muscle fibers to water and nonelectrolytes

Summary The permeability of isolated muscle fibers of the giant barnacleMegabalanus psittacus to water and nonelectrolytes was studied by determining their reflection and permeability coefficients. Reflection coefficients were obtained by comparing the osmotic fluxes produced by a test molecule to that produced by the impermeant sucrose molecule. Permeability coefficients were determined for measurements of tracer uptake.The results indicate that, in these fibers, nonelectrolyte permeability is closely related to lipid solubility and molecular size.A value of 2.16×10^–12 cm³/sec dyne for the hydraulic conductivity and a value of 10.45×10^–4 cm/sec for³HHO permeability coefficient were obtained.The effect of membrane surface invaginations and clefts on the determination of permeability coefficients is discussed.     

Relationships between structure and high-throughput screening permeability of diverse drugs with artificial membranes: application to prediction of Caco-2 cell permeability

To evaluate the absorption of drugs with diverse structures across a membrane via the transcellular route, their permeability was measured using the parallel artificial membrane permeation assay (PAMPA). The permeability coefficients obtained by PAMPA were analyzed using a classical quantitative structure-activity relationship (QSAR) approach with simple physicochemical parameters and 3D-QSAR, VolSurf. We formulated correlation equations for diverse drugs similar to the equation obtained for peptide-related compounds in our previous study. The hydrogen-bonding ability of molecules, not only the hydrogen-accepting ability but also the hydrogen-donating ability, in addition to hydrophobicity at a particular pH, was significant in determining variations in PAMPA permeability coefficients. Based on this result, an in silico good prediction model for the passive transcellular permeability of diverse structural compounds was obtained. The artificial lipid-membrane permeability coefficients of the drugs, except salicylic acid, were well correlated with the Caco-2 permeability in a previous report suggesting the importance of absorption by the transcellular mechanism for these drugs.     

Effect of the interfacial surfactant layer on oxygen transfer through the oil/water phase boundary in perfluorocarbon emulsions

The effect of interfacial surfactant molecules on oxygen transfer through oil/water phase boundary has been studied in FlurO(2) (TM) emulsions, i.e., perfluorocarbon (PFC) emulsions developed as oxygen carriers in cell culture. Measurements of oxygen permeability were made with a polarographic oxygen electrode in pure PFCs and in emulsions with various PFC volume fractions. Comparison of the experimental results with the theoretically derived values of relative oxygen permeability clearly indicates that the mass transfer resistance caused by the interfacial surfactant layer in PFC emulsions is insignificant. Therefore, oxygen dissolved in the enclosed PFC phase is readily available to cells growing in the aqueous media and FlurO(2) emulsions with very fine emulsion particles (< 0.2 mum) can be used to effectively enhance gas/liquid interfacial oxygen transfer in bioreactors. The inadequacy in describing mass transfer in heterogeneous systems, such as the PFC emulsions, by conventional concentration-based oxygen diffusion coefficients has also been discussed.     

Relationship between structure and permeability of tryptophan derivatives across human intestinal epithelial (Caco-2) cells

L-Trp and its derivatives were used as model compounds to clarify structural factors which influence the intestinal epithelial permeation and metabolism of amino-acid derivatives. Permeability of model compounds through Caco-2 cells was used as an in vitro absorption model for human intestinal epithelial cells. The influence of compound concentration, the effects of various transporter substrates on permeability coefficients, and pH dependency of permeability coefficients were investigated. The transcellular permeability of Trp and Trp-NH2 in the direction from the apical side to the basolateral side, in which nutrients and drugs were ordinarily absorbed, declined with increasing concentration and saturated at more than 1 and 0.4 mM, respectively. The permeability coefficients for N-terminal protected Trp derivatives and Ac-Trp-NH2 showed similar and constant values in both from the apical-to-basolateral and basolateral-to-apical directions. In addition, significant inhibition of the apical-to-basolateral permeation of Trp by Leu and Phe was observed. The permeability coefficient ratio at pH 6.3 to that at pH 7.3 was explained by the ratio of the ionic form to the neutral form of the compounds. Based upon these results and the partition coefficients in the 1-octanol/water system, possible absorption mechanism of Trp and its derivatives across Caco-2 cells was proposed.