The Extent of Orthorhombic Lipid Phases in the Stratum Corneum Determines the Barrier Efficiency of Human Skin In Vivo

Abstract

full width at 50% peak height hexagonal liquid-crystalline orthorhombic stratum corneum transepidermal water loss TO THE EDITOR The outermost skin layer—the stratum corneum (SC)—constitutes the main barrier for the fluxes of water and chemicals through human skin (Elias, 2004Elias P.M. The epidermal permeability barrier: from the early days at Harvard to emerging concepts.J Invest Dermatol. 2004; 122: xxxvi-xxxixGoogle Scholar). Here, we report on the molecular organization of the SC extracellular lipid matrix and its role in skin barrier efficiency. Using attenuated total internal reflection Fourier-transform infrared spectroscopy and measurements of transepidermal water loss (TEWL) on intact and tape-stripped skin, we demonstrate that highly ordered orthorhombic (OR) lipid phases exist throughout the thickness of human SC in vivo and that their content directly correlates with the TEWL. Our results illustrate the physiological importance of the OR lipid phases in SC as a regulator of the transdermal fluxes. The incomplete understanding of the molecular bases of the skin barrier in healthy skin; the therapeutic relevance of improving the barrier efficiency of diseased, aged, and neonatal skin; and the need for overcoming the skin barrier during transdermal drug delivery have prompted numerous studies of the SC lipid matrix (Harding, 2004Harding C.R. The stratum corneum: structure and function in health and disease.Dermatol Ther. 2004; 17: 6-15Google Scholar; Bouwstra and Ponec, 2006Bouwstra J.A. Ponec M. The skin barrier in healthy and diseased state.Biochim Biophys Acta. 2006; 1758: 2080-2095Google Scholar). Most of the experimental evidence—collected from excised skin (Naik and Guy, 1997Naik A. Guy R.H. Infrared spectroscopic and differential scanning calorimetric investigations of the stratum corneum barrier function.in: Potts R.O. Guy R.H. Mechanisms of Transdermal Drug Delivery. Marcel Dekker, NY1997: 87-162Google Scholar; Babita et al., 2006Babita K. Kumar V. Rana V. Jain S. Tiwary A.K. Thermotropic and spectroscopic behavior of skin: relationship with percutaneous permeation enhancement.Curr Drug Deliv. 2006; 3: 95-113Google Scholar), from SC fragments (Pilgram et al., 1999Pilgram G.S.K. Engelsma-van Pelt A.M. Bouwstra J.A. Koerten H.K. Electron diffraction provides new information on human stratum corneum lipid organization studied in relation to depth and temperature.J Invest Dermatol. 1999; 113: 403-409Google Scholar; Pensack et al., 2006Pensack R.D. Michniak B.B. Moore D.J. Mendelsohn R. Infrared kinetic/structural studies of barrier reformation in intact stratum corneum following thermal perturbation.Appl Spectrosc. 2006; 60: 1399-1404Google Scholar), and from mixtures of SC lipids (Lafleur, 1998Lafleur M. Phase behaviour of model stratum corneum lipid mixtures: an infrared spectroscopy investigation.Can J Chem. 1998; 76: 1501-1511Google Scholar; Moore et al., 2006Moore D.J. Snyder R.G. Rerek M.E. Mendelsohn R. Kinetics of membrane raft formation: fatty acid domains in stratum corneum lipid models.J Phys Chem B. 2006; 110: 2378-2386Google Scholar)—points toward coexistence of OR, hexagonal (HEX), and liquid-crystalline (LIQ) phases. Two recently developed models of the molecular organization—the domain mosaic model (Forslind, 1994Forslind B. A domain mosaic model of the skin barrier.Acta Derm Venereol. 1994; 74: 1-6Google Scholar) and the sandwich model (Bouwstra et al., 2000Bouwstra J.A. Dubbelaar F.E.R. Gooris G.S. Ponec M. The lipid organization in the skin barrier.Arch Dermatol Res. 2000; 208: 23-30Google Scholar)—have proposed the spatial arrangements of these phases. Both models suggest that the most tightly packed, conformationally ordered OR phases regulate the transdermal fluxes: the higher their content, the higher is the barrier efficiency of the SC; this correlation, however, had never been demonstrated in vivo. In this study, we evaluated the barrier efficiency of intact forearm skin in 70 healthy human volunteers of both genders, aged 19 to 62 years, using TEWL, the water flux density describing the passive diffusion of water through the SC (Imhof et al., 2009Imhof R.E. De Jesus M.E.P. Xiao P. Ciortea L.I. Berg E.P. Closed chamber transepidermal water loss measurement: microclimate, calibration and performance.Int J Cosm Sci. 2009; 31: 97-118Google Scholar). The study protocol was approved by the institutional review board of Firmenich SA and fully conformed with the recommendations of the Declaration of Helsinki. Before participation, the volunteers gave their informed consent in writing. To establish the depth profile of the molecular organization of SC lipids, we used attenuated total internal reflection Fourier-transform infrared measurements of successively deeper SC layers exposed in the course of tape stripping. This spectroscopic technique provides structural information localized in a surface layer of limited thickness (Harrick, 1987Harrick N.J. Internal Reflection Spectroscopy. Harrick Scientific, NY1987: 327Google Scholar); it is uniquely well suited to assess SC in vivo, as it causes minimal perturbation to its native state and is noninvasive (Naik and Guy, 1997Naik A. Guy R.H. Infrared spectroscopic and differential scanning calorimetric investigations of the stratum corneum barrier function.in: Potts R.O. Guy R.H. Mechanisms of Transdermal Drug Delivery. Marcel Dekker, NY1997: 87-162Google Scholar; Mendelsohn et al., 2006Mendelsohn R. Flach C.R. Moore D.J. Determination of molecular conformation and permeation in skin via IR spectroscopy, microscopy, and imaging.Biochim Biophys Acta. 2006; 1758: 923-933Google Scholar). To evaluate the presence and extent of OR phases in the SC, we used the CH2 scissoring bandwidth (full width at 50% peak height, FWHM) calculated from second-derivative attenuated total internal reflection Fourier-transform infrared spectra. We have previously demonstrated that this parameter has a high discriminative power for the type of lateral intermolecular chain packing of lipids in full-thickness mammalian skin (Boncheva et al., 2008Boncheva M. Damien F. Normand V. Molecular organization of the lipid matrix in intact stratum corneum using ATR-FTIR spectroscopy.Biochim Biophys Acta. 2008; 1778: 1344-1355Google Scholar). Figure 1a shows the temperature dependence of FWHM; we have previously shown that the maximal value of 12.0±0.1cm−1 is indicative of lipids organized predominantly in OR lattices and the minimal value of 4.0±0.1cm−1 of purely HEX and/or LIQ phases. We defined a threshold value of 11.3cm−1 (corresponding approximately to the upper eighth of the curve) to distinguish the layers with a prevalent OR organization (denoted as class α) from those with a lower content of OR phases (denoted as class β); the use of these two classes of lateral organization, instead of the exact FWHM values, facilitated the comparison of the molecular organization in lipid ensembles of similar but nonidentical composition (Figure 1b). Figure 1c shows representative examples of the depth profiles of lateral lipid organization. We detected the presence of predominantly OR (class α) lipid phases at all investigated depths, in agreement with previous ex vivo and in vitro studies (Naik and Guy, 1997Naik A. Guy R.H. Infrared spectroscopic and differential scanning calorimetric investigations of the stratum corneum barrier function.in: Potts R.O. Guy R.H. Mechanisms of Transdermal Drug Delivery. Marcel Dekker, NY1997: 87-162Google Scholar; Babita et al., 2006Babita K. Kumar V. Rana V. Jain S. Tiwary A.K. Thermotropic and spectroscopic behavior of skin: relationship with percutaneous permeation enhancement.Curr Drug Deliv. 2006; 3: 95-113Google Scholar; Bouwstra and Ponec, 2006Bouwstra J.A. Ponec M. The skin barrier in healthy and diseased state.Biochim Biophys Acta. 2006; 1758: 2080-2095Google Scholar; Pensack et al., 2006Pensack R.D. Michniak B.B. Moore D.J. Mendelsohn R. Infrared kinetic/structural studies of barrier reformation in intact stratum corneum following thermal perturbation.Appl Spectrosc. 2006; 60: 1399-1404Google Scholar; Boncheva et al., 2008Boncheva M. Damien F. Normand V. Molecular organization of the lipid matrix in intact stratum corneum using ATR-FTIR spectroscopy.Biochim Biophys Acta. 2008; 1778: 1344-1355Google Scholar) and in contrast to the homogeneous gel-phase model of SC lipid organization (Norlen, 2001Norlen L. Skin barrier structure and function: the single gel phase model.J Invest Dermatol. 2001; 117: 830-836Google Scholar). Importantly, we found that HEX and/or LIQ phases were also present at all depths; in addition to the two peaks that are characteristic of purely OR phases (centered at ∼1,473 and 1,463cm−1), the scissoring regions of all spectra contained a peak that is characteristic of HEX or LIQ phases (centered at ∼1,468cm−1, Figure 1b). The relative extent of these phases underneath the uppermost SC layers, however, was fairly low, as judged from the positions of the symmetric CH2 stretching modes; in all cases they were located at ∼2,848–2,849cm−1, wavenumbers indicative of ordered lipid phases with a low rotational mobility along the hydrocarbon chains (Lewis and McElhaney, 2002Lewis R.N.A.H. McElhaney R.N. Vibrational spectroscopy of lipids.in: Chalmers J.M. Griffiths P.R. Handbook of Vibrational Spectroscopy. John Wiley & Sons, Chichester, UK2002: 3447-3464Google Scholar). Using these depth profiles and the total thickness of SC (see Supplementary Materials and Methods for details), we calculated the fraction of SC thickness that contained α (that is, predominantly OR) lipid phases. Figure 2 shows the correlation between TEWL and the relative content of α phases in SC. The two parameters correlated strongly (Pearson's correlation coefficient of 0.694, p=2.8e-11). In half of the cases (shown with empty circles), we could only estimate the minimal relative thickness of SC that contains α phases. Considering the typical depth profiles of the lipid phase content, in these cases (for example, profile a in Figure 1c) we still have not reached the phases with the relatively higher disorder that is typical of inner SC layers. Thus, it is possible that in these cases we underestimated the fraction of SC thickness that contained α phases; the horizontal positions of these points could be shifted farther to the right, leading to an additional strengthening of the correlation. The content of α phases was independent of the age and gender of the volunteers, and TEWL did not correlate with the overall thickness of SC (comprising fractions involved in OR, HEX, and LIQ phases). Download .pdf (.11 MB) Help with pdf files Supplementary Materials and Methods Interestingly, the maximal FWHM (FWHMmax) observed for the α phases was inversely proportional to TEWL: volunteers having TEWL values in the ranges of 5–8, 8–10, 10–12, and above 12gm−2 per hour had FWHMmax values of 12.4±0.1, 12.3±0.2, 12.0±0.1, and 11.9±0.2cm−1, respectively. This decrease in FWHMmax might reflect a lower content and/or smaller sizes of the OR domains (Cameron et al., 1981Cameron D.G. Gudgin E.F. Mantsch H.H. Dependence of acyl chain packing of phospholipids on the head group and acyl chain length.Biochemistry. 1981; 20: 4496-4500Google Scholar); either of these effects would lead to an increase in TEWL. It is, however, impossible to calculate the size of the OR domains from the measured FWHM values in human SC; unlike the case of binary systems in which the magnitude of the scissoring split and the size of the OR domains are directly correlated (Snyder et al., 1992Snyder R.G. Goh M.C. Srivatsavoy V.J.P. Strauss H.L. Dorset D.L. Measurement of the growth kinetics of microdomains in binary n-alkane solid solutions by infrared spectroscopy.J Phys Chem. 1992; 96: 10008-10019Google Scholar), the complex composition of the human SC precludes such quantitative interpretation. In conclusion, we have demonstrated the direct correlation that exists between the lateral molecular organization of the SC lipid matrix and the efficiency of the skin barrier: as previously suggested, but to our knowledge never shown in vivo, the higher the extent of purely OR phases, the lower is the inside-out flux of water. Our results lend further support to the domain mosaic and sandwich models of the lipid organization in SC. This work raises several interesting questions relevant to the areas of skin biophysics, dermatology, and transdermal drug delivery, such as the changes in the lipid molecular organization that might be induced by environmental conditions (for example, temperature and humidity) and by topical application of products, the relationship between the inside-out and the outside-in fluxes of water and chemicals through human skin, and the reversibility and time scale of recovery of the molecular organization in SC in vivo after a chemical or environmental insult. We sincerely thank our volunteers for their goodwill and patience. Supplementary material is linked to the online version of the paper at http://www.nature.com/jid