Ceramides, the major lipid component of the stratum corneum (SC), provide many of its unique physical properties. Surprisingly, only a few biophysical studies of hydrated ceramides have been reported. The current Fourier transform infrared (FTIR) spectroscopy investigation provides the first detailed study of intermolecular and intramolecular chain and headgroup interactions in hydrated non-hydroxy fatty acid (NFA) and hydroxy fatty acid (HFA) ceramides. Information about NFA and HFA ceramide chain subcell structure and conformational order is derived from the temperature dependence of the methylene stretching, scissoring, and rocking mode frequencies. At low temperatures, NFA ceramide is highly ordered and packed in an orthorhombic subcell structure that undergoes a solid−solid phase transition to a conformationally ordered hexagonal phase at 60 °C. A second transition to a conformationally disordered non-bilayer-like phase occurs at 80 °C. The lipid chains of HFA ceramide undergo a single transition from a conformationally ordered, orthorhombic subcell, phase to a conformationally disordered phase at 76 °C. In NFA ceramide the amide I and II modes of the headgroup are each split into two bands, indicating strong intermolecular headgroup coupling between two NFA ceramide headgroups in a factor group perpendicular to the bilayer plane. In contrast, splitting is not observed for either amide mode in HFA ceramide. However, the presence of strong H bonding indicates an interaction between molecules in the bilayer plane. The contrasting behavior of the headgroups in NFA and HFA ceramide suggests that these molecules make distinct contributions to the structural integrity of the stratum corneum. The implications of these findings to the recently proposed domain mosaic model of the stratum corneum lipid barrier are discussed.

IR spectroscopic studies are reported for the phytosphingosine class of ceramides and are compared with two analogous sphingosine ceramides. The phytosphingosine class of molecules, not previously widely investigated by physical techniques, constitutes ∼30% of the total ceramide content of the stratum corneum, the barrier to permeability in skin. The current measurements utilize temperature-controlled horizontal attenuated total reflectance spectroscopy of hydrated films to study H f D exchange in the polar regions of the molecules as well as chain conformational order and packing properties. Analysis of the methylene stretching and scissoring vibrations reveals that the chains of the two phytosphingosine derivatives (ceramides 3 and 7) are much more poorly packed at room temperature than their sphingosine counterparts (ceramides 2 and 5 respectively), despite having order f disorder transitions some 15-20 degrees higher. This unanticipated relative stability of the phytosphingosines is traced to enhanced headgroup H-bonding interactions manifest by lower Amide I and higher Amide II frequencies. Water penetration into the polar regions is monitored by the temperature dependence of the Amide II and O-H/N-H stretching intensities as a function of HfD exchange. Neither ceramide 2 nor 3 exchanges N-H or O-H protons until relatively high temperatures (>65 °C). However, addition of an R-hydroxy group on the fatty acid chain in ceramides 5 or 7 results in exchange events observed at temperatures much closer to physiological. These measurements reveal that the relative contributions of chain packing and H-bonding under physiological conditions differ markedly for the phytosphingosines compared to the sphingosines. The former are characterized by hexagonal chain packing with relatively strong H-bonding; the latter by orthorhombic chain packing and weaker H-bonding. The implications of these molecular structure data for lipid organization in the stratum corneum are briefly discussed.

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