Interfacial properties of avian stratum corneum monolayers investigated by Brewster angle microscopy and vibrational sum frequency generation

https://doi.org/10.1016/j.chemphyslip.2017.08.002Get rights and content

Highlights

  • Structure and organization of lipids in bird skin depends on relative abundance of lipid classes.

  • Stability and rigidity of skin lipids is reflective of the aridity of the environment each species inhabits.

  • Increased ordering of interfacial water correlates to a higher abundance of polar ceramide and cerebroside lipid molecules.

  • Lipid packing and interaction with water molecules may determine the rate of cutaneous water loss in birds.

Abstract

The outermost layer of skin, the stratum corneum (SC), contains a complex mixture of lipids, which controls the rate of cutaneous water loss (CWL) in reptiles, mammals, and birds. However, the molecular structure of SC lipids and how molecular configurations influence CWL is poorly understood. Here, the organization and structure of SC lipids extracted from birds were investigated by means of Langmuir films. Properties of lipids from the SC of arid and semi-arid adapted larks, known to have a low CWL, were compared with lipids extracted from the SC of mesic lark species with higher CWL to gain insight into how structure impacts CWL. Film properties were probed with surface pressure-area isotherms, Brewster angle microscopy (BAM), and vibrational sum frequency generation (VSFG). Results indicate organization and ordering of SC lipids in the arid-adapted hoopoe lark was vastly different from all other species, forming a miscible, rigid monolayer, whereas monolayers from semi-arid and mesic species were immiscible and disordered. Probing of interfacial water structure reveals that film morphology determines organization of water molecules near the monolayer; monolayers with a porous morphology had an increased population of water molecules that are weakly hydrogen-bonded. In general, CWL appears related to the miscibility and ordering of lipid components within the SC, as well as the ability of these lipids to interact with water molecules. From a broader perspective, CWL in larks appears linked to both the SC lipid composition and the aridity of the species’ environment.

Introduction

The largest organ of the vertebrate body, the skin is composed of a dermis and an epidermis, the latter consisting of layers of cells that undergo apoptosis and extrude lipids into the intercellular space as they begin to reach more superficial layers. The stratum corneum (SC), the outer most layer of the epidermis, is composed of flat, dead cells embedded within ordered layers of lipids and is recognized as the physical barrier that limits water loss to the outside environment (Wertz, 2000, Madison, 2003, Wickett and Visscher, 2006, Proksch et al., 2008, Menon et al., 2012). The lipids of the SC are primarily responsible for controlling diffusion of water vapor to the environment, as removal of the SC lipids with organic solvents results in a significant increase in cutaneous water loss (CWL) (Matoltsy et al., 1968, Sweeney and Downing, 1970, Wertz and van den Bergh, 1998). Intercellular lipids of the SC organize into layers called lamellae, composed of either bilayers or trilayers (Forslind, 1994, Bouwstra et al., 2000). It has been suggested that lipid composition influences the organization and structure of the lamellae, and hence water permeation through the SC (Bouwstra et al., 2000, Elias et al., 1981).

The lipids in avian SC consist of cholesterol esters, fatty acid methyl esters (FAMEs), triacylglycerol, free fatty acids (FFAs), cholesterol, ceramides, and cerebrosides (Ro and Williams, 2010, Champagne et al., 2012). The relative proportion of these lipid classes has been shown to influence the rate of CWL in several species (Champagne et al., 2012, Haugen et al., 2003, Muñoz-Garcia and Williams, 2005). Furthermore, CWL and lipid composition in birds correlates strongly with environment. Birds that inhabit areas of low ambient water vapor pressure and high temperature, such as deserts, have evolved lower rates of CWL and higher proportions of more polar ceramides and cerebrosides in their SC. In contrast, species from more mesic areas with high ambient water vapor pressure have a higher fraction of free fatty acids and triacylglycerols and exhibit higher rates of water loss (Champagne et al., 2016, Champagne et al., 2015, Champagne et al., 2012, Tieleman and Williams, 2002). These studies suggest that ceramides and cerebrosides play a key role in the reduction of CWL rates in birds. The ability of these lipids to form strong hydrogen-bonds with adjacent lipids or adjacent water molecules may be a factor influencing water loss (Adams and Allen, 2013, Champagne et al., 2012). Furthermore, the ability of lipid chains to pack tightly together and minimize the presence of gauche conformers may also inhibit water loss (Champagne et al., 2016). While properties such as stability and miscibility of ceramides and cerebrosides have been investigated as part of simple, artificial mixtures (Maggio et al., 1978, Maggio, 1994), the intermolecular interactions between lipids in the SC and how they subsequently regulate CWL are not well understood.

In this study, the physical properties of lipids within the SC of four lark species found in regions of different aridity were investigated. At thermoneutral temperatures, the semi-arid adapted black-crowned finchlark (Eremopterix nigriceps) and the extreme arid-adapted hoopoe lark (Alaemon alaudipes), exhibit average water loss rates of 14.0 and 18.5 mgH2Ocm−2day−1, respectively, whereas the horned lark (Eremophila alpestris) and skylark (Alauda arvensis) from mesic regions in Ohio and the Netherlands, respectively, lose water at rates averaging 20.8 and 25.7 mgH2Ocm−2day−1 (Tieleman and Williams, 2002, Haugen et al., 2003, Champagne et al., 2012). Langmuir monolayers of SC lipids extracted from each species were investigated and compared at the air-water interface in an attempt to understand how the structure and organization of these lipid films correlates to CWL rates. Through the use of surface pressure-area isotherms, Brewster angle microscopy, and vibrational sum frequency generation (VSFG) spectroscopy, properties of the SC monolayers were used to gain insight into how SC lipid molecules pack together to form a coherent barrier to CWL, while also assessing the degree to which polar lipids are able to interact with water molecules. Results suggest that the ability of lipids to form ordered structures may impact CWL. SC lipids of arid-adapted hoopoe lark were found to form the most stable and ordered monolayer, whereas lipids of the other three species formed disordered, fluid monolayers. This observation is consistent with the pattern of CWL rates in birds along an aridity gradient; although the black-crowned finchlark has a lower CWL rate than the hoopoe lark at thermoneutral temperatures, the hoopoe lark inhabits more arid environments than the black-crowned finchlark and exhibits an ability to resist water loss even at high temperatures (Philby, 1933, Tieleman and Williams, 2002, Ryan, 2017, de Juana and Suárez, 2017). Additionally, measurement of interfacial water structure reveals that lipid organization appears to strongly influence the way in which water molecules interact with SC lipids.

Section snippets

Materials

Intercellular lipids from lark SC were extracted and quantified in four individuals representing four species (Table 1) by methods that have been previously described (Ro and Williams, 2010, Champagne et al., 2012, Muñoz-Garcia et al., 2006). Although these lipid extracts represent only a single individual from each species, these individuals exemplify the basic lipid composition of conspecifics, as in each case, the three most abundant lipid classes in each individual correspond with the three

Phase behavior of avian SC lipid monolayers

Phase behavior in Π-A isotherms of SC lipid monolayers differed between the four lark species (Fig. 1). All species, however, have a gas-liquid condensed (G-LC) coexistence region at 0 mN/m, as evidenced by BAM. Lipids from black-crowned finchlark transition from the G-LC to the liquid condensed (LC) phase at 80 cm2. The monolayer undergoes a phase transition at ∼25 mN/m, evidenced by the kink in the isotherm, before reaching a maximum surface pressure of 28 mN/m. The hoopoe lark monolayer had a

Summary and conclusions

Here the interfacial properties of SC lipid monolayers from lark species along an aridity gradient were investigated and compared to gain insights into the molecular interactions that influence CWL rates in larks. Results indicate that lipids of more mesic species form fluid monolayers in which components are not miscible and have significant gauche defects in the alkyl chains. In contrast, more arid species did not display similar properties. Lipids from the semi-arid black-crowned finchlark

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

This research was supported under Grant CHE 1111762 (to H.C.A) and Grant IBW- 0212092 (to J.B.W.) from the National Science Foundation. We thank Irene Tieleman and Arne Hegemann for procuring lark specimens.

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      Longer fatty acid carbon chains in sphingolipids lead to stronger Van der Waals forces among molecules, resulting in more ordered lamellae and consequently lower CEWL (Schaefer and Rodelmeier, 1996; Lillywhite, 2006; Muñoz-Garcia et al., 2008). A larger proportion of the more polar sphingolipids yields stronger molecular interactions between lipid head groups and water, and thus leads to a tighter barrier to water vapor molecules (Haugen et al., 2003; Muñoz-Garcia et al., 2008; Adams et al., 2017). Finally, bulky head groups such as the hexose moiety on cerebrosides may not pack closely with other cerebrosides or with other bulky molecules, like cholesterol, because of steric hindrance (Adams and Allen, 2013; Maggio et al., 2006); lipid mixtures with certain proportions of bulky lipids (cerebrosides and cholesterol) will then be immiscible and compromise the water vapor permeability barrier.

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    Present Address: Lehrstuhl für Physikalische Chemie II, Ruhr−UniversitätBochum, 44780 Bochum, Germany

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