Acidic pH is Required for the Multilamellar Assembly of Skin Barrier Lipids in Vitro
Lipid membrane remodeling belongs to the most fundamental processes in the body. The skin barrier lipids, which are ceramide-dominant and highly rigid, must attain an unusual multilamellar nanostructure with long periodicity to restrict water loss and prevent the entry of potentially harmful environmental factors. Our data suggest that the skin “acid mantle”, apart from regulating enzyme activities and keeping away pathogens, may also be a prerequisite for the multilamellar assembly of the skin barrier lipids. Atomic force microscopy on monolayers composed of synthetic or human SC lipids showed multilayer formation (ca 10 nm step height) in an acidic but not neutral environment. X-ray diffraction, Fourier-transform infrared spectroscopy, and permeability studies showed markedly altered lipid nanostructure and increased water loss at neutral compared to acidic pH. These findings are consistent with the altered organization of skin lipids and increased transepidermal water loss in conditions with inadequate skin acidification, e.g., in neonates, elderly, and patients with atopic dermatitis.
Steps to reproduce
Human stratum corneum (SC) lipids Human skin was obtained from female patients who had undergone abdominal plastic surgery and had given their informed consent. The procedure was approved by the Ethics Committee of Sanus First Private Surgical Centre, Czech Republic, and conducted according to the principles of the Declaration of Helsinki. The SC was isolated by trypsin treatment (Kligman and Christophers, 1963), the lipids were extracted using a series of chloroform/methanol mixtures (Bligh and Dyer, 1959), purified by column chromatography by a modified procedure published previously (Pullmannova et al., 2014), and their composition verified using high-performance thin layer chromatography (Vavrova et al., 2014). For FTIR, XRD, and water loss experiments, human SC lipids in hexane/ethanol 96% 2:1 (v/v) were sprayed on glass slides and Nuclepore polycarbonate filters and annealed using a) excess acetate-buffered saline (pH 5.5), b) excess phosphate-buffered saline (pH 7.4), c) water vapor, all at 70°C for 20 min. Langmuir monolayers Lipid monolayers at air-liquid interface were studied using a small Langmuir-Blodgett trough (KSV NIMA, Espoo, Finland). The subphase was either 10 mM acetate-buffered saline (pH 5.5) or 10 mM phosphate-buffered saline (pH 7.4) maintained at 32 °C to mimic the skin surface temperature. Lipids (10 μl, 1 mg/mL in chloroform/methanol 3:1 v/v) were spread at the cleaned air-liquid interface, and the solvents were left to evaporate (20 min) before the lipid film was compressed at 20 mm/min. The surface pressure (π) recorded using a Pt Wilhelmy plate (at least 4 replicates) was plotted against mean molecular area (MMA). The surface compression modulus, which reflects variations of the lipid film in-plane elasticity, was calculated as Cs−1 = −MMA(∂π/∂MMA). Fourier-transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) FTIR and XRD were measured as described previously (Engberg et al., 2020; Pullmannova et al., 2019) . Water loss through human SC lipid films and human SC SC lipid films on support filters or samples of human SC, supported by 0.45 μm Nylon filters (Fisher Scientific EU), were either acidified with acetate-buffered saline (pH 5.5) or neutralized phosphate-buffered saline (pH 7.4). The lipid films or SC were sandwiched between Teflon holders with an available diffusion area of 0.5 cm2 and mounted in Franz diffusion cells. The acceptor part of the Franz cell (approx. 6.5 ml) was filled with either acetate-buffered saline (pH 5.5) for acidified samples or phosphate-buffered saline (pH 7.4) for neutralized samples. The acceptor phase was stirred at 32°C throughout the experiment. The cells were left to equilibrate for 12 h. Water loss was measured using an AquaFlux AF 200 instrument (Biox Systems Ltd, UK), at ambient air humidity of 30-36% and 24-26°C. For details, see the (Supplementary) Methods in the article "Acidic pH is Required for the Multilamellar Assembly of Skin Barrier Lipids in Vitro".