Lipid membranes play a crucial role in regulating the body's water balance by adjusting their properties in response to hydration. The intercellular lipid matrix of the stratum corneum (SC), the outermost skin layer, serves as the body's primary defense against environmental factors. Osmolytes, including urocanic acid (UCA) and glycerol, are key components of the natural moisturizing factor that help the SC resist osmotic stress from dry environments. This study examines the effects of UCA and glycerol (each at 5 mol %) on isolated human SC lipids. For this, different techniques were employed, offering complementary information of the system's multiscale characteristics, including humidity-scanning quartz crystal microbalance with dissipation monitoring, infrared spectroscopy, x-ray diffraction, electrical impedance spectroscopy, and studies of water loss and permeability. Our results show that UCA increases water sorption and makes lipid films more liquid-like at high relative humidity, without significantly altering the lipid lamellar structure, chain order, or orthorhombic chain packing. Lipid films containing UCA exhibited higher water loss and significantly higher model drug permeability compared to lipid films without UCA. Further, incorporation of UCA resulted in kinetically faster changes in electrical properties upon contact with aqueous solution compared with control lipids. These observations suggest that UCA reduces lipid cohesion in regions other than the acyl chain-rich leaflets, which may impact SC desquamation. In contrast, glycerol did not influence the hydration or permeability of the SC lipid matrix. However, it increased the proportion of orthorhombic domains at high humidities and slowed the kinetics of the hydration process, as evidenced by slower changes in the dielectric properties of the lipid film. These findings suggest that glycerol enhances lipid cohesion rather than increasing water uptake, which is typically the expected function of humectants. Consequently, UCA and glycerol appear to have distinct roles in maintaining epidermal homeostasis.

• MeSH
• Glycerol * chemistry   pharmacology   MeSH
• Skin metabolism   MeSH
• Urocanic Acid chemistry   pharmacology   metabolism   MeSH
• Humans MeSH
• Lipids chemistry   MeSH
• Permeability MeSH
• Water * chemistry   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Glycerol * MeSH
• Urocanic Acid MeSH
• Lipids MeSH
• Water * MeSH

Omega-O-acyl ceramides such as 32-linoleoyloxydotriacontanoyl sphingosine (Cer[EOS]) are essential components of the lipid skin barrier, which protects our body from excessive water loss and the penetration of unwanted substances. These ceramides drive the lipid assembly to epidermal-specific long periodicity phase (LPP), structurally much different than conventional lipid bilayers. Here, we synthesized Cer[EOS] with selectively deuterated segments of the ultralong N-acyl chain or deuterated or 13C-labeled linoleic acid and studied their molecular behavior in a skin lipid model. Solid-state 2H NMR data revealed surprising molecular dynamics for the ultralong N-acyl chain of Cer[EOS] with increased isotropic motion toward the isotropic ester-bound linoleate. The sphingosine moiety of Cer[EOS] is also highly mobile at skin temperature, in stark contrast to the other LPP components, N-lignoceroyl sphingosine acyl, lignoceric acid, and cholesterol, which are predominantly rigid. The dynamics of the linoleic chain is quantitatively described by distributions of correlation times and using dynamic detector analysis. These NMR results along with neutron diffraction data suggest an LPP structure with alternating fluid (sphingosine chain-rich), rigid (acyl chain-rich), isotropic (linoleate-rich), rigid (acyl-chain rich), and fluid layers (sphingosine chain-rich). Such an arrangement of the skin barrier lipids with rigid layers separated with two different dynamic "fillings" i) agrees well with ultrastructural data, ii) satisfies the need for simultaneous rigidity (to ensure low permeability) and fluidity (to ensure elasticity, accommodate enzymes, or antimicrobial peptides), and iii) offers a straightforward way to remodel the lamellar body lipids into the final lipid barrier.

• Keywords
• NMR spectroscopy, lipid assembly, lipid chain order, long periodicity phase, molecular dynamics, neutron diffraction, stratum corneum models,
• MeSH
• Ceramides chemistry   MeSH
• Epidermis MeSH
• Skin chemistry   MeSH
• Linoleic Acid * MeSH
• Sphingosine analysis   MeSH
• Molecular Dynamics Simulation * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Ceramides MeSH
• Linoleic Acid * MeSH
• Sphingosine MeSH

Epidermal omega-O-acylceramides (ω-O-acylCers) are essential components of a competent skin barrier. These unusual sphingolipids with ultralong N-acyl chains contain linoleic acid esterified to the terminal hydroxyl of the N-acyl, the formation of which requires the transacylase activity of patatin-like phospholipase domain containing 1 (PNPLA1). In ichthyosis with dysfunctional PNPLA1, ω-O-acylCer levels are significantly decreased, and ω-hydroxylated Cers (ω-OHCers) accumulate. Here, we explore the role of the linoleate moiety in ω-O-acylCers in the assembly of the skin lipid barrier. Ultrastructural studies of skin samples from neonatal Pnpla1+/+ and Pnpla1-/- mice showed that the linoleate moiety in ω-O-acylCers is essential for lamellar pairing in lamellar bodies, as well as for stratum corneum lipid assembly into the long periodicity lamellar phase. To further study the molecular details of ω-O-acylCer deficiency on skin barrier lipid assembly, we built in vitro lipid models composed of major stratum corneum lipid subclasses containing either ω-O-acylCer (healthy skin model), ω-OHCer (Pnpla1-/- model), or combination of the two. X-ray diffraction, infrared spectroscopy, and permeability studies indicated that ω-OHCers could not substitute for ω-O-acylCers, although in favorable conditions, they form a medium lamellar phase with a 10.8 nm-repeat distance and permeability barrier properties similar to long periodicity lamellar phase. In the absence of ω-O-acylCers, skin lipids were prone to separation into two phases with diminished barrier properties. The models combining ω-OHCers with ω-O-acylCers indicated that accumulation of ω-OHCers does not prevent ω-O-acylCer-driven lamellar stacking. These data suggest that ω-O-acylCer supplementation may be a viable therapeutic option in patients with PNPLA1 deficiency.

• Keywords
• PNPLA1 deficiency, Skin, acylceramides, barrier function, ceramides, linoleic acid, lipids, model membranes, sphingolipids, stratum corneum,
• MeSH
• Acyltransferases MeSH
• Ceramides * chemistry   MeSH
• Epidermis MeSH
• Ichthyosis MeSH
• Skin * MeSH
• Linoleic Acid MeSH
• Lipase MeSH
• Mice MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Acyltransferases MeSH
• Ceramides * MeSH
• Linoleic Acid MeSH
• Lipase MeSH
• PNPLA1 protein, mouse MeSH Browser