Penetration of dimyristoylphosphatidylcholine monolayers and bilayers by model beta-blocker agents of varying lipophilicity.

Penetration of model beta-blockers, propranolol, oxprenolol, metaprolol, and nadolol, into dimyristoylphosphatidylcholine (DMPC) monolayers cast on a pH 7.4 phosphate buffer (mu = 0.155 adjusted with NaCl) at 25 degreesC was monitored using a film balance equipped with a Wilhelmy plate for measuring changes in surface pressure. Drug solution (pH 7.4) is injected below the surface of the monolayer. The difference in surface pressure, Delta pi, for each drug concentration added to the monolayer was measured at equilibrium. Delta pi increased with increasing drug concentration. Consistent with the relative lipophilicities, the Delta pi vs drug concentration slopes were as follows: propranolol > metaprolol > oxprenolol > nadolol. The intrinsic surface activity of the beta-blockers was also determined in the absence of the lipid. Differential scanning calorimetry (DSC) measurements were also made on DMPC bilayers in the above buffer. DMPC suspended in buffered drug solutions were scanned over a temperature range of 5 degrees to 40 degreesC at a scan rate of 0.091 degreesC/min. The DSC studies indicate that the DMPC thermotropic phase behavior is modulated by these compounds as follows: propranolol >> metaprolol congruent with oxprenolol > nadolol which agrees with reported partition coefficients as well as the above Delta pi observations. However, an accounting of the intrinsic surface activity of these compounds results in a lower than expected affinity for the DMPC monolayer.

Dioxygen diffusion in the stratum corneum: an EPR spin label study.

The stratum corneum, the outer 10 microns of the skin, serves as a permeability barrier regulating the transport of molecules between the body and the environment. The purpose of this study is to understand this permeability barrier function as it pertains to the diffusion of molecular oxygen. The stratum corneum was investigated with EPR spectroscopy following inoculation with a stearic acid spin probe. The presence of paramagnetic molecular oxygen results in the broadening of the EPR spectral lines of the spin probe. The rate of oxygen diffusion across the stratum corneum, and then the oxygen diffusion coefficient, D(O2), was determined by studying this line-broadening as a function of time. D(O2) in human stratum corneum was found to be 3 x 10(-7) cm2/s at 37 degrees C with an activation energy of approx. 44 kJ/mol. The application of the permeation-enhancing chemicals, DeMSO and DMSO, to the stratum corneum increased D(O2) two- to three-fold.

Dioxygen solubility in aqueous phosphatidylcholine dispersions.

The solubility of molecular oxygen, or dioxygen, in low weight percent (1.5%) sonicated dimyristoylphosphatidylcholine (DMPC) aqueous dispersions saturated with air has been measured as a function of temperature between 10 degrees C and 40 degrees C. A modified Winkler technique was used involving a dual cell coulometric titration with voltammetric endpoint detection in a mixed solvent (methanol/water). The results indicate that dioxygen is approximately four times more soluble in the liquid crystalline bilayers (above 24 degrees C) than in the gel state bilayers (below 24 degrees C). The solubility of dioxygen in the bilayer does not appear to be strongly temperature dependent on either side of the 24 degrees C phase transition. The dioxygen solubility in gel state DMPC is approximately equal to that in water at the same temperature. Our result are contrasted with recent measurements made using EPR spin labels.

Localization of lipid microdomains and thermal phenomena in murine stratum corneum and isolated membrane complexes: an electron spin resonance study.

The intercellular region of the stratum corneum can be isolated in the form of membrane complexes (intercellular lipids and adjacent cornified envelopes) which are devoid of the cytoplasmic components. In this study the temperature induced phase transitions and corresponding lipid domain reorganization in isolated stratum corneum (SC) sheets and SC membrane complexes (SCM) were determined using the electron spin resonance (ESR) spin probe technique. The spin probe perdeuterated di-tert-butyl nitroxide (pdDTBN) in SC and SCM revealed less well-defined physiologic phase transitions in the SCM and a more polar lipid domain in SC. However, the ESR results show the coexistence of highly ordered domains (immobilized spin probe) and less viscous domains in intact SC, which persist in SCM. Above approximately 20 degrees C the spin probe is dissolved in a highly disordered (isotropic) lipid domain in both SC and SCM. In both solvent extracted SC and SCM, the spin probe is dissolved in a highly ordered lipophilic domain associated with the lipids complexed to the corneocyte envelope and exhibiting no thermal transitions between -23 degrees to 60 degrees C. Further, the amount of mobile spin probe is related to the amount of residual lipid. An unexpected finding was the apparent reduction of the spin probe in solvent extracted SCM, suggesting the presence of a previously unrecognized free radical reducing mechanism in these sites. The mobilities of the spin probe when dissolved in model lipids, non-hydroxy, and hydroxy containing ceramides and cholesteryl oleate, differed significantly from those observed in SC or SCM. These studies demonstrate the usefulness of ESR for the localization and characterization of lipid microenvironments in the stratum corneum.

Partitioning of hydrophobic probes into lipopolysaccharide bilayers.

Lipophilic solutes permeate rapidly through lipid bilayer membranes. However, the outer membrane of enteric bacteria, which is composed of a lipopolysaccharide monolayer outer leaflet and the glycerophospholipid inner leaflet, shows extremely low permeability to hydrophobic solutes. In order to examine the cause of this exceptionally low permeability, the lipid/water partition behavior of various lipophilic probes was determined by using lipopolysaccharides of various chemotypes and glycerophospholipids. With all probes, under many different conditions, the lipopolysaccharide/water partition coefficients were generally about an order of magnitude smaller than the phospholipid/water partition coefficients, and this result is consistent with the low permeability of the lipopolysaccharide monolayer, and hence the asymmetric bilayer found in the outer membrane. Furthermore, organic polycations significantly increased the partition of N-phenylnaphthylamine into lipopolysaccharides, a result again consistent with the permeability-increasing effect of such cations on intact outer membrane. Very defective, 'deep rough' lipopolysaccharides of chemotypes Rd2, Rd1 and Re, had only slightly (20-75%) higher partition coefficients in comparison with the more complete lipopolysaccharides, and this difference is probably not enough to explain the approximately 100-fold increase in lipophile permeability seen in deep rough strains.

Calorimetric and electron spin resonance examination of lipid phase transitions in human stratum corneum: molecular basis for normal cohesion and abnormal desquamation in recessive X-linked ichthyosis.

Lipids appear to play a critical role as regulators of stratum corneum desquamation. In this study, we observed discrete lipid phase transitions at physiologic temperatures in both normal human scale (NHS) and in lipid extracts of NHS by differential scanning calorimetry. In contrast, such thermal transitions were not observed in recessive x-linked ichthyosis scale (RXLIS). To gain further insight into the molecular basis of the lipid phase transitions in NHS vs. RXLIS, comparable samples were evaluated by electron spin resonance, utilizing the perdeuterated probe, di-t-butyl nitroxide. Upon electron spin resonance analysis, both NHS and RXLIS demonstrated thermal phase transitions in the physiologic range; however, the nature of the lipid environments in each type varied. Whereas the environment of the spin probe was more polar in NHS than in RXLIS, the spin probe partitioned into a more "fluid" environment in RXLIS; i.e., the spin probe was more mobile in RXLIS than in NHS lipid matrices. Because an alteration in the cholesteryl sulfate:cholesterol ratio is the primary lipid abnormality in RXLIS, model cholesterol-fatty acid-cholesteryl sulfate mixtures were prepared in proportion to the lipid composition of NHS and RXLIS. Differences were observed in both thermal transitions and in lipid microenvironments in these mixtures that paralleled those observed in scale samples. Based on these results, a model is proposed that invokes abnormal hydrogen bonding, due to increased cholesteryl sulfate, as the mechanism for the abnormal desquamation in recessive X-linked ichthyosis.

The diffusion-solubility of oxygen in lipid bilayers.

The product, Do alpha, of the oxygen diffusion coefficient, Do, and the oxygen solubility, alpha, is determined in phosphatidylcholine bilayers at temperatures above the lipid phase transitions from ESR spin-exchange measurements. The resulting values of Do alpha are in good agreement with those obtained from fluorescence-quenching experiments. The use of fatty acid spin labels makes it possible to measure Do alpha as a function of the coordinate perpendicular to the bilayer surface. The results indicate that do alpha is a strong function of this coordinate; it is greatest in the bilayer center and least near the bilayer head groups.

The binding isotherms for the interaction of 5-doxyl stearic acid with bovine and human albumin.

Binding isotherms for the interaction of 5-doxyl stearic acid with bovine and human albumin are reported. The critical micelle concentration (CMC) and the limiting solubility of 5-doxyl stearic acid were determined using the electron spin resonance (ESR)-spin label method. The CMC and the limiting solubility of this spin-label stearic acid in saline-phosphate buffer are 3.5 x 10(-5) M and 2 x 10(-4) M, respectively. We found no ESR line width evidence for pre-association of the spin-label stearate below the CMC. Maximum binding of the spin-label stearate to both bovine and human albumin occurs before micelle formation. The binding isotherm for spin-label stearic acid interaction with bovine albumin is in agreement with data obtained by others using [1-(14)C]stearic acid. For human albumin, comparison is difficult since previous data obtained with [1-(14)C]stearic acid vary widely. Comparison of the ESR 2T(||) values (the splitting between low and high field extremes, a measure of the degree of immobilization of protein-bound spin-label stearate) for bovine and human albumin indicates a greater immobilization of the spin-label molecules bound to human albumin. The binding data indicate that complexes are formed with bound spin-label stearate/albumin ratios of at least 18. The computed equilibrium constants for both bovine and human albumin indicate that the first seven spin-label molecules are tightly bound, log K > 5.0. The species predicted to form in solution by these equilibrium constants are reported.