Neil Kitson

The Effect of Ceramide on Phosphatidylcholine Membranes: A Deuterium NMR Study

Biological membranes contain domains having distinct physical properties. We study defined mixtures of phosphoglycerolipids and sphingolipids to ascertain the fundamental interactions governing these lipids in the absence of other cell membrane components. By using (2)H-NMR we have determined the temperature and composition dependencies of membrane structure and phase behavior for aqueous dispersions of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and the ceramide (Cer) N-palmitoyl-sphingosine. It is found that gel and liquid-crystalline phases coexist over a wide range of temperature and composition. Domains of different composition and phase state are present in POPC/Cer membranes at physiological temperature for Cer concentrations exceeding 15 mol %. The acyl chains of liquid crystalline phase POPC are ordered by the presence of Cer. Moreover, Cers chain ordering is greater than that of POPC in the liquid crystalline phase. However, there is no evidence of liquid-liquid phase separation in the liquid crystalline region of the POPC/Cer phase diagram.

Models of stratum corneum intercellular membranes: 2H NMR of macroscopically oriented multilayers

Deuterium NMR was used to characterize model membrane systems approximating the composition of the intercellular lipid lamellae of mammalian stratum corneum (SC). The SC models, equimolar mixtures of ceramide:cholesterol:palmitic acid (CER:CHOL:PA) at pH 5.2, were contrasted with the sphingomyelin:CHOL:PA (SPM:CHOL:PA) system, where the SPM differs from the CER only in the presence of a phosphocholine headgroup. The lipids were prepared both as oriented samples and as multilamellar dispersions, and contained either perdeuterated palmitic acid (PA-d31) or [2,2,3,4,6-2H5]CHOL (CHOL-d5). SPM:CHOL:PA-d31 formed liquid-ordered membranes over a wide range of temperatures, with a maximum order parameter of approximately 0.4 at 50 degrees C for positions C3-C10 (the plateau region). The quadrupolar splitting at C2 was significantly smaller, suggesting an orientational change at this position, possibly because of hydrogen bonding with water and/or other surface components. A comparison of the longitudinal relaxation times obtained at theta = 0 degrees and 90 degrees (where theta is the angle between the normal to the glass plates and the magnetic field) revealed a significant T1Z anisotropy for all positions. In contrast to the behavior observed with the SPM system, lipid mixtures containing CER exhibited a complex polymorphism. Between 20 and 50 degrees C, a significant portion of the entire membrane (as monitored by both PA-d31 and CHOL-d5) was found to exist as a solid phase, with the remainder either a gel or liquid-ordered phase. The proportion of solid decreased as the temperature was increased and disappeared entirely above 50 degrees C. Between 50 and 70 degrees C, the membrane underwent a liquid-ordered to isotropic phase transition. These transitions were reversible but displayed considerable hysteresis, especially the conversion from a fluid phase to solid. The order profiles, relaxation behavior, and angular dependence of these parameters suggest strongly that both the liquid-ordered CER- and SPM-membranes are bilayers. The unusual phase behavior observed for the CER-system, particularly the observation of solid-phase lipid at physiological temperatures, may provide insight into the functioning of the permeability barrier of stratum corneum.

Models of stratum corneum intercellular membranes: the sphingolipid headgroup is a determinant of phase behavior in mixed lipid dispersions.

During formation of the intercellular membranes of mammalian stratum corneum, sphingomyelin and glucosylceramide are converted enzymatically to ceramide. To model in isolation the possible effect of such a lipid modification on the phase behavior of the ensemble, we used proton and deuterium nuclear magnetic resonance to compare an equimolar dispersion of bovine brain sphingomyelin, cholesterol, and perdeuterated palmitic acid (at pH 6.2), with an equivalent dispersion in which bovine brain ceramide was substituted for sphingomyelin. While the sphingomyelin dispersions remain in a homogeneous fluid lamellar phase from 20-75 degrees C under these conditions, those containing ceramide display complex polymorphism.

Phase Behavior of an Equimolar Mixture of N-Palmitoyl-d-erythro-sphingosine, Cholesterol, and Palmitic Acid, a Mixture with Optimized Hydrophobic Matching

The phase behavior and lipid mixing properties of an equimolar mixture of nonhydroxylated palmitoyl ceramide (Cer16), palmitic acid (PA), and cholesterol have been investigated using 2H NMR and vibrational spectroscopy. This mixture is formed by the three main classes of lipids found in the stratum corneum (SC), the top layer of the epidermis, and provides an optimized hydrophobic matching. Therefore, its behavior highlights the role played by hydrophobic matching on the phase behavior of SC lipids. We found that, below 45 degrees C, the mixture is essentially formed of coexisting crystalline domains with a small fraction of lipids (less than 20%) that forms a gel or fluid phase, likely ensuring cohesion between the solid domains. Upon heating, there is the formation of a liquid ordered phase mainly composed of PA and cholesterol, including a small fraction of Cer16. This finding is particularly highlighted by correlation vibrational microspectroscopy that indicates that domains enriched in cholesterol and PA include more disordered Cer16 than those found in the Cer16-rich domains. Solubilization of Cer16 in the fluid phase occurs progressively upon further heating, and this leads to the formation of a nonlamellar self-assembly where the motions are isotropic on the NMR time scale. It is found that the miscibility of Cer16 with cholesterol and PA is more limited than the one previously observed for ceramide III extracted from bovine brain, which is heterogeneous in chain composition and includes, in addition to Cer16, analogous ceramide with longer alkyl chains that are not hydrophobically matched with cholesterol and PA. Therefore, it is inferred that, in SC, the chain heterogeneity is a stronger criteria for lipid miscibility than chain hydrophobic matching.

Fatty Acids Influence “Solid” Phase Formation in Models of Stratum Corneum Intercellular Membranes

Stacked intercellular lipid membranes in the uppermost epidermal layer, the stratum corneum (SC), are responsible for skins barrier function. These membranes are unique in composition, the major lipids being ceramides (Cer), cholesterol, and free fatty acids (FFA) in approximately equimolar proportions. Notably, SC lipids include chains much longer than those of most biological membranes. Previously we showed that Cers small hydrophilic headgroup enabled SC model membranes composed of bovine brain ceramide (BBCer), cholesterol, and palmitic acid in equimolar proportion to solidify at pH 5.2. In order to determine the influence of FFA chain length on the phase behavior of such membranes, we used 2H NMR and FT-IR to study BBCer/cholesterol/FFA dispersions containing linear saturated FFA 14-22 carbons long. Independent of chain length, the solid phase dominated the FFA spectrum at physiological temperature. Upon heating, each dispersion underwent phase transitions to a liquid crystalline phase (only weakly evident for the membrane containing FFA-C22) and then to an isotropic phase. The phase behavior, the lipid mixing properties, and the transition temperatures are shown to depend strongly on FFA chain length. A distribution of FFA chain lengths is found in the SC and could be required for the coexistence of a proportion of solid lipids with some more fluid domains, which is known to be necessary for normal skin barrier function.

Clinical Texting Among Medical Trainees of the University of British Columbia

We believe cellphone text messages are commonly used in medical practice whether in rural or urban settings and that clinical photos are often attached to them. Our interest is the use of this technology to provide dermatology service to rural and remote British Columbia. Concern has been expressed about the security of confidential information and adequacy of privacy protection in such an application. We have found little published information about the extent of texting in rural and remote settings (and none in our jurisdiction) or the number and nature of privacy breaches that have actually occurred as a result. To obtain such information, we first set out to survey medical practitioners about their actual use. The results reported here are from medical trainees enrolled with the University of British Columbia who are in both rural and urban settings.