Micron-scale assessment of molecular lipid organization in human stratum corneum using microprobe X-ray diffraction

Abstract

This article is available online at http://www.jlr.org complex and specifi c architecture of the inter-corneocyte lipid matrix ( 2 ) composed of a mixture of ceramides, cholesterol, and long-chain fatty acids. Ultrastructural studies, using electron microscopy and diffraction techniques, have revealed that these lipids are organized in stacked bilayers that are predominantly parallel to the skin surface ( 3-5 ). Part of the lipids is covalently bound to the corneocytes whereas another part represents the "free lipids" removable by solvent extraction.X-ray diffraction is an excellent technique for studying the spatial organization of the intercellular matrix because the lipid molecules are highly ordered and oriented in a crystalline structure. This technique has provided a number of important results with regard to the SC structure. In particular, small-angle X-ray scattering (SAXS) gives information on the inter-bilayer distance in the multi-lamellar lipid structures, whereas the wide-angle X-ray scattering (WAXS) data contain information about the in-plane crystalline arrangement of lipids (lattice type).Most of the SAXS studies of human SC fi nd refl ections corresponding to ‫ف‬ 4.5 nm and ‫ف‬ 6.5 nm inter-bilayer spacings ( 4, 6, 7 ). Some studies have also detected a refl ection corresponding to a spacing of 11-13.5 nm ( 6, 8 ), referred to as the "long periodicity phase" ( 6 ). These data indicate that the SC lipid matrix has a complex organization into multiple sublayers or even phase-separated domains with different lamellar repeat distances.Similar complexity is found at the molecular level using WAXS, where two sets of peaks are found, corresponding to orthorhombic (peaks at 0.41 and 0.37 nm) and hexagonal (peak at 0.41 nm) packing of the lipid chains. In addition, a fraction of lipids might exist in a fl uid or amorphous state, which would then contribute to the broad band corresponding to an average distance of 0.46 nm. Abstract Lipid and protein components of the stratum corneum (SC) are organized in complex supramolecular arrangements. Exploring spatial relations between various possible substructures is important for understanding the barrier function of this uppermost layer of epidermis. Here, we report the fi rst study where micro-focus X-ray scattering was used for assessing fi ne structural variations of the human skin barrier with micrometer resolution. We found that the scattering profi les were unchanged when scanning in the direction parallel to the SC surface. Furthermore, small-angle scattering profi les did not change as a function of depth in the SC, confi rming that the lipid lamellar spacings remained the same throughout the SC. However, the wide-angle scattering data showed that the orthorhombic phase was more abundant in the middle layers of the SC, whereas the hexagonal phase dominated in the surface layers both at the external and the lowest part of the SC; i.e., the lipids were most tightly packed in the middle region of the SC. Taken together, our results demonstrate that micro...

Evaluation of the molecular lipid organization in millimeter‐sized stratum corneum by synchrotron X‐ray diffraction

“…5,6,9 Doucet et al estimated that the relative abundance of lipids in organized phases with hexagonal and orthorhombic hydrocarbon packing structures is 20 ± 10% based on WAXD profiles. 13 Hatta et al also concluded that more than 50% of the total intercellular lipids in SC exist in a disorganized liquid state from WAXD measurements on ethanol treated hairless mouse SC. 14 From a solid-state NMR study, Björklund et al proposed that the majority of the intercellular lipids are rigid at 32°C, but they coexist with a small pool of mobile disordered lipids that might be in a disorganized liquid crystalline phase.…”

“…Recently, many authors have proposed from X‐ray diffraction measurements that a disorganized liquid crystalline phase exists in SC . Doucet et al estimated that the relative abundance of lipids in organized phases with hexagonal and orthorhombic hydrocarbon packing structures is 20 ± 10% based on WAXD profiles . Hatta et al also concluded that more than 50% of the total intercellular lipids in SC exist in a disorganized liquid state from WAXD measurements on ethanol treated hairless mouse SC .…”

“…By comparing the diffraction profiles between untreated SC and SC treated with a chloroform/methanol mixture, they have estimated the proportion of the liquid crystalline phase in the intercellular lipid matrix. The proportion of lipids in an ordered phase is only 20 ± 10% . Recently, it has been pointed out that the proportion of the disordered phase is more than 50% .…”

“…The proportion of lipids in an ordered phase is only 20 ± 10%. 13 Recently, it has been pointed out that the proportion of the disordered phase is more than 50%. 14 On the other hand, as strong X-ray diffraction peaks due to the LPP were observed in SAXD measurements in human SC after heat treatment at 120°C, Bouwstra et al concluded that the lamellar phase of LPP has recrystallized.…”

Non-invasive depth profiling of the stratum corneum in vivo using confocal Raman microscopy considering the non-homogeneous distribution of keratin

“…The intercellular lipids (ICL) of the lipid matrix are organized in the form of a classical membrane and their orthorhombic lateral organization represents a highly ordered and very densely packed structure, which is notpermeable for the majority of the non-destructive substances [2][3][4]. The depth distribution of the lateral organization of ICL is non-homogeneous in the SC, showing a prevalence of orthorhombic over hexagonal ordering in the intermediate SC depths measured in vitro [5,6]. The prevalence of the orthorhombic lateral organization in the SC maintains the skin barrier…”