Arrangement of Ceramides in the Skin: Sphingosine Chains Localize at a Single Position in Stratum Corneum Lipid Matrix Models

Abstract

Understanding the structure of the stratum corneum (SC) is essential to understand the skin barrier process. The long periodicity phase (LPP) is a unique trilayer lamellar structure located in the SC. Adjustments in the composition of the lipid matrix, as in many skin abnormalities, can have severe effects on the lipid organization and barrier function. Although the location of individual lipid subclasses has been identified, the lipid conformation at these locations remains uncertain. Contrast variation experiments via small-angle neutron diffraction were used to investigate the conformation of ceramide (CER) N-(tetracosanoyl)-sphingosine (NS) within both simplistic and porcine mimicking LPP models. To identify the lipid conformation of the twin chain CER NS, the chains were individually deuterated, and their scattering length profiles were calculated to identify their locations in the LPP unit cell. In the repeating trilayer unit of the LPP, the acyl chain of CER NS was located in the central and outer layers, while the sphingosine chain was located exclusively in the middle of the outer layers. Thus, for the CER NS with the acyl chain in the central layer, this demonstrates an extended conformation. Electron density distribution profiles identified that the lipid structure remains consistent regardless of the lipid's lateral packing phase, this may be partially due to the anchoring of the extended CER NS. The presented results provide a more detailed insight on the internal arrangement of the LPP lipids and how they are expected to be arranged in healthy skin.

Figure 1

Molecular structure of deuterated CERs used in this study. This includes CER NS-d47 where all 47 hydrogen atoms along the acyl chain were replaced with deuterium and CER NS-d7 where the terminal seven hydrogen atoms of the sphingosine chain were replaced with deuterium. The carbon atoms bound to deuterium are highlighted in bold.

Figure 2

Small-angle neutron 1D scattering plots of the fully protiated (A) simple and (B) porcine models when hydrated in 50:50 D₂O/H₂O, measured at a detector position of 13°. All plots were hydrated using 50:50 D₂O/H₂O. The Arabic numbers indicate the various diffraction orders of the LPP; the 7th order diffraction peak is not visible in both spectra, indicating that the scattering intensity of this order is close to zero. The * indicates the position of the crystalline cholesterol peaks. The insert reports the 1st order diffraction peak measured at a detector position of 11.2°.

Figure 3

Water SLD profiles of the (A) simple and (B) porcine models within the LPP. Four areas of greater scattering length density signify a trilayer structure. The position of the CER NS chains in the (C) simple and (D) porcine models including the sphingosine d7 (green) and the acyl chain d47 (red) profiles. CER NS acyl chains are located in both the inner and outer layers of the LPP, while the sphingosine chain extends beyond the inner water region, locating itself in the outer layers.

Figure 4

Comparative SLD profiles from experimental (green) and derived (red) data for the simple lipid model when hydrated with 100:0 and 8:92 D₂O/H₂O. (A) Identified SLD contributions in the LPP unit cell include the inner and outer water boundaries (6.25, 1.9 nm) and the ester group of CER EOS (2.85 nm). (B) Calculated F_n for each of the Bragg peaks and the calculated F_n from the experimental data. (C) Calculated SLD profiles and the SLD profiles calculated from the experimental data.

Figure 5

(A) SAXD peaks of the simple system in the orthorhombic (25 °C, black curve), hexagonal (39 °C, blue curve), hexagonal with a smaller population in the fluid (61 °C, green curve), and mixed hexagonal and fluid (67 °C, red curve) phases. The curves have been stacked, for ease of peak observation. (B) Electron density distribution profiles in the orthorhombic phase (black) and in the fluid with the hexagonal phase (red). The position of the highest electron density intensity describes the position of the lipid headgroups and the boundaries of the trilayer.