Article pubs.acs.org/JPCB
Kinetic Evidence Suggests Spinodal Phase Separation in Stratum Corneum Models by IR Spectroscopy Richard Mendelsohn,*,† Ibrahim Selevany,† David J. Moore,† M. Catherine Mack Correa,‡ Guangru Mao,‡ Russel M. Walters,‡ and Carol R. Flach† †
Department of Chemistry, Newark College, Rutgers University, Newark, New Jersey 07102, United States Johnson & Johnson Consumer Companies, Inc., 199 Grandview Road, Skillman, New Jersey 08558, United States
‡
ABSTRACT: Although lipid structure in models for the stratum corneum (SC), the main barrier to skin permeability, has been extensively studied, only limited data are extant concerning the kinetic mechanism for the formation of domains, lamellar phases, and lipid packing motifs. Such information would be of substantial interest in the characterization of the effects of disease states which disrupt the barrier. Kinetic IR spectroscopy measurements probed the temporal sequence of molecular events producing ordered structures in a three-component SC model of equimolar ceramide[NS] (cer[NS]), perdeuterated stearic acid-d35 (SA-d35), and cholesterol. Samples, heated above Tm, were quenched to 31 °C, and then spectra were recorded at ∼15 min intervals for a total of 20−150 h. IR provides unique molecular structure information about headgroup H-bonding, lipid packing, and lipid chain order. The following sequence for phase separation was observed: (1) Formation of ceramide amide H-bonds from disordered forms to ordered structures (0.5−4 h); (2) appearance of ordered ceramide chains with some orthorhombically packed structures (0.5−8 h); and (3) phase separation of large orthorhombic domains of SA-d35 (4−10 h). A spinodal decomposition mechanism, defined by continuous composition changes during the phase separation, suggests a qualitative description for these events.
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INTRODUCTION The primary permeability barrier in human skin resides within the ∼15 μm thick outermost layer, the stratum corneum (SC), which is composed of anucleated corneocytes embedded in a continuous lamellar lipid matrix. Lipid structural organization in the SC has been widely studied with physical methods (e.g., see refs 1−10), and the results have begun to provide a basis for understanding barrier function with the potential for elucidating how skin lipid organization is altered in human disease states. Beginning approximately 20 years ago, smallangle X-ray scattering has been used6,9,11 to identify the existence of two lamellar phases with periodicities of ∼6 and 13 nm, respectively termed the short (SPP) and long (LPP) periodicity phases. In addition to the lamellar organization, the lateral organization of SC lipids has been studied. IR spectroscopy12−17 and X-ray diffraction18−20 measurements on SC and SC lipid models reveal the presence of a substantial fraction of orthorhombic chain packing. Fatty acids are considered to be important for formation of this phase. Thermotropic studies on SC also reveal the occurrence of an orthorhombic → hexagonal packing transition near the physiological temperature of skin. This transition is followed at higher, non physiological temperatures by a hexagonal → disorder transition in the lipid chains.5,12,21,22 Yet, structural studies alone are insufficient to provide a complete description of skin organization/function. Thus, the SC is a dynamic structure, actively responding to the environment, and turns over in an approximately two week © 2014 American Chemical Society
period. Simply stated, SC lipids end up highly ordered in a healthy skin barrier. They probably did not start that way earlier in the temporal sequence, and they can be perturbed from this state. In spite of the need for kinetic studies of SC assembly, investigations of the mechanisms of the development of lamellar phases, lipid packing motifs, and domains are substantially less well elaborated than SC structural studies. Yet such information would be of interest in a variety of areas within biophysics in general, and within skin biophysics in particular. For instance, functions controlled by biomembranes such as lipid or protein transport, intracellular signaling, and regulation of integral membrane proteins have been suggested to involve ordered domains termed “lipid rafts” (e.g., refs 23 and 24). In skin biophysics, the occurrence of ordered lipid domains is a central feature in descriptions of SC lipid organization such as Forslind’s domain mosaic model25 which depicts the barrier lipids as segregated into ordered structures surrounded by grain boundaries formed from lipids in a more fluid (liquid crystalline) state that may provide control of hydration and transport levels. Elucidation at the molecular level of the physical mechanisms and kinetics of lipid domain formation would also help define potential pathways for permeation of hydrophobic species as well as provide clues as to why such a variety of ceramides and additional chemical species are present in the skin barrier. Understanding the Received: January 28, 2014 Revised: April 2, 2014 Published: April 4, 2014 4378
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formation and dissipation kinetics of domains formed from particular ceramide and fatty acid species would aid in defining biological processes such as epidermal desquamation, which includes degradation of layered structures in the intercellular spaces. In addition, Oren et al. showed26 that antimicrobial peptides important in the skin’s ability to repair itself are found within the SC extracellular matrix. The role of enzymes and the dynamics of lipids at the SG−SC interface in controlling in the formation of the barrier has been reviewed by Elias.27 His group also28 investigated the colocalization of phospholipases with polar lipids, suggested to induce fusion of lamellar body discs as a prelude to formation of the SC. The presence of variable lipid domains may also provide one method by which enzyme activity is controlled at different depths within the SC.29,30 Finally, in a medical context, abnormal lamellar structures and disrupted lateral order have been associated with disrupted barrier function in patients with psoriasis, ichthyosis, or atopic dermatitis.31−34 Spinodal decomposition,35 extensively applied to the study of alloy36−38 and polymer phase separation,39−44 may provide a general framework for understanding the formation of complex layered structures, but has not, to our knowledge, been considered in skin biophysics. The closest reported studies with molecules related to those in the SC are vibrational (IR and Raman) spectroscopy,45−49 and small-angle neutron and X-ray scattering50 investigations of binary n-alkane mixtures quenched from melted states. In particular, Snyder and co-workers46−49 tracked the kinetics of demixing by utilizing a variety of IR spectral features to evaluate chain conformational ordering, packing motifs, and domain formation. The dependence of demixing rates on temperature and isotopic composition of alkanes in the C28−C36 length range have been probed. In the SC, at least 11 subclasses of ceramides have been characterized and coexist with relatively large levels of cholesterol, free fatty acids, and relatively minor amounts of cholesterol sulfate, glucosylceramides, and cholesterol esters. The current work is directed toward developing IR spectroscopy for studying lateral phase separation and the early stages of ordered lamellar structure formation in skin lipid models. The complexity of the SC lipid structure will necessitate examination of a large number of skin lipid mixtures over a wide range of compositions and temperatures to evaluate the possible relevance of spinodal mechanisms. For these initial studies, we have selected two previously studied ternary equimolar mixtures, namely ceramide [NS]/ stearic acid/cholesterol and ceramide[AS]/stearic acid/cholesterol (see Figure 1 for structures) for which currently available physical measurements on the same or similar systems51,52 at least begin to define the existence regime for various phases. We are well aware that most studies of phase separation in molecular systems utilize relatively simple binary mixtures whose phase diagrams are or can be readily established. Without such data for the current ternary systems, we must at these early stages forego a quantitative approach, but are guided instead by the criterion of Cahn,53 an initial developer of the theory, who noted explicitly that “To establish that the composition of the phases change continuously during phase separation would be a direct proof” for a spinodal mechanism to be present. The power of IR spectroscopy for these structurally oriented kinetic measurements is clear from the data presented below. The approach directly tracks temporal rearrangements of Hbonds and formation of separate orthorhombic domains from
Figure 1. Chemical structures of skin lipid constituents used in the current work and H-bonded dimer structure with a trans geometry for SA-d35.
the stearic acid and ceramide constituents. The kinetic sequence of events in the early stages of phase separation in each of the aforementioned mixtures is thereby determined. It is also demonstrated that the addition of a single −OH group in going from [NS] → [AS] ceramide introduces substantial differences in the observed kinetics, a finding of consequence for understanding the possible roles of the different ceramide species. We have previously reported preliminary measurements for similar systems54 that focused primarily on SA domain formation. The current work elaborates upon the analysis and introduces a variety of additional IR spectral parameters that provide mechanistic information about the phase separation events.
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EXPERIMENTAL SECTION Cholesterol (Sigma Chemical Co., St. Louis, MO) was of stated purity greater than 99%. Chain perdeuterated stearic acid (SAd35), isotopic purity of 99%, was obtained from CDN Isotopes (Pointe Claire, Quebec, Canada). Porcine brain ceramide [NS] (nonhydroxy fatty acid sphingosine ceramide) was purchased from Avanti Polar Lipids, Inc. (Alabaster, AL) and was >99% pure. Ceramide [AS] (α-hydroxy sphingosine ceramide) from bovine brain was purchased from MATREYA, LLC (Pleasant Gap, PA) and was of stated purity greater than 98%. The chain length compositions (in %) of the major lipid components (>7%) of the two ceramides are as follows: Ceramide [NS] C18:0 (11), C22:0 (10), C24:0 (24), C24:1 (31); Ceramide [AS] C18:0 (24), C22:0 (8), C24:0 (35), C24:1 (17). All substances were used without further purification. Equimolar mixtures of the desired ceramide, cholesterol, and SA-d35 were prepared by codissolving the lipids in a chloroform/methanol mixture (9:1 v:v), removing bulk liquid under a gentle stream of N2, and placing the samples under high vacuum for 3 h. The lipids were subsequently hydrated in excess citrate buffer (pH 5.5) by repeated cycles of heating in a water bath (80 °C), cooling and mixing by vortex action. The sample was then sandwiched between ZnSe windows and 4379
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placed in a thermostated IR transmission cell (Harrick Scientific, Ossining, NY). The sample was warmed for one additional hour at temperatures greater that the major order− disorder phase transition ∼(65 °C) and quenched within 5 min to 31 °C (most experiments) or 22 °C as desired. When the required temperature was achieved, spectral acquisition with a Thermo-Nicolet 6700 spectrometer (Thermo Electron Corporation, Madison, WI), equipped with a Mercury−Cadmium− Telluride (MCT) detector, was initiated. Data acquisition continued for the desired period with a spectrum generally being acquired every 15 min for the first 12 h and every 15 or 30 min for the duration of the experiment. Absorbance spectra were generated from 128 coadded, Fourier transformed, interferograms collected at ∼2 cm−1 resolution. Occasional experiments were conducted in which spectra were acquired every 7.5 min. The spectrometer and sample compartments were purged with N2 gas for the duration of the experiment to remove residual water vapor. The cer[NS]-containing ternary mixture was examined in nine separate experiments while the ceramide [AS]-containing sample was examined in three separate experiments. Representative data are shown in the figures. Spectra were processed with GRAMS/32, Thermo Fischer Scientific (baseline correction, spectral subtraction, Fourier deconvolution), ISys 3.1, Malvern Instruments (peak intensity), or software developed at the National Research Council of Canada (peak position). Parameters used for particular operations are noted in the figure captions.
Figure 2. (A) Temporal evolution of the IR CO stretching region in a cer[NS]/cholesterol/SA-d35 equimolar mixture quenched from 65 to 31 °C as described in the text. Top: overlaid spectra acquired every 15 min for the first 3 h immediately following the quench highlighting the evolution of the sharp bands in the ceramide Amide I region. Bottom: overlaid spectra acquired every hour ranging from 3 to 20 h after the quench highlighting the delayed downshift in the stearic acid CO frequency. The arrow indicates increasing time within each spectral set. (B) IR intensities as a function of time for the SA-d35 (1694 cm−1) band (red) and for the cer[NS] Amide I (1644 cm−1) band (black).
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RESULTS A. Phase Separation Events: Headgroup Reorganization, Chain Ordering, and Orthorhombic Phase Formation in Ceramide [NS]/Cholesterol/Stearic Acid-d35. i. Kinetics of Headgroup H-Bond Rearrangements. Figure 2A depicts the temporal evolution of IR spectra post quenching in the spectral region 1510−1760 cm−1. As indicated in the figure, this spectral region encompasses the Amide I (CO stretch) and Amide II (mixed C−N stretch and N−H in-plane bend, ∼1550 cm−1) vibrations of the cer[NS] amide group, as well as the CO stretching mode of SA-d35. The first 3 h of spectral data acquired at 15 min intervals are presented in the top and those acquired from 3 to 20 hours in 1 h intervals are offset and displayed in the bottom of Figure 2A. Significant changes with different time dependencies were noted in each region following the quench. The Amide I contour appears initially as a relatively broad band (halfwidth >50 cm−1) centered at ∼1640 cm−1. Such features in H-bonded systems are common in condensed phase spectra (e.g., thermally denatured proteins) and reflect contours inhomogeneously broadened by a wide distribution of H-bond lengths and geometries to the Amide CO groups immediately following the thermal quenching. As the system evolves temporally, remarkable changes take place from the initial contour. The broad spectral feature is resolved into four relatively sharp features with progressively increasing intensities assigned to three Amide I components at ∼1644 and 1625 cm−1 along with a shoulder at ∼1611 cm−1 and a weak feature of unknown structural origin at 1594 cm−1. A shift in the Amide II contour (∼1550 cm−1) is also evident. The Amide I contour (Figure 2A), at post quenching times longer than a few hours, thus depicts a well-ordered structure, with a much reduced range of H-bond geometries for each of the three identified Amide I sub-bands. The dominant H-bonded Amide I structure at long times presumably arises from the 1644 cm−1
band, the most intense in this spectral region, assuming the extinction coefficients for each of the aforementioned features to be similar. The CO stretching contour of the SA-d35 initially reveals a main peak near 1710 cm−1 and a shoulder at ∼1734 cm−1 (Figure 2A, top). This contour evolves into a broad single band centered at ∼1694 cm−1 (Figure 2A, bottom). The time evolution of the 1644 and 1694 cm−1 bands, plotted in Figure 2B, demonstrate quite different kinetics. The ordering process for the ceramide Amide CO, which commences close to the quenching time, precedes the stearic acid headgroup structure changes which become significant only at ∼4 h post quenching and diminish in rate after ∼7 h. We note that in repeat experiments a range of delay times (from 4 h, arises from H-bonds in the well-known dimer structure shown in Figure 1. The dimer most likely exhibits the trans geometry also shown in the figure. This structure is common for fatty acid crystals with chain lengths greater than 10 carbons. ii. Kinetics of Ceramide Chain Ordering. In addition to the temporal evolution of the polar headgroup H-bonded structure, insight into the chain behavior of the cer[NS] during the post quench period is revealed through the time-dependence of the asymmetric methylene stretching frequency (υasym(CH2)). The ordering of the chains as deduced from changes in this spectral parameter is shown in Figure 4. The process starts within a few moments of quenching and continues for ∼7−8 h. Several possibilities exist for the origin of the chain ordering. First, the quenching process induces chain ordering from a conformationally disordered state, the remnants of which may be observed, as follows: the CH2 asymmetric stretching frequencies at the earliest times appear at ∼2919.7 cm−1, a frequency which indicates that the chains are already significantly ordered, as the frequency range for this mode typically lies between ∼2916−2917 cm−1 (gel phase) and at ∼2925 cm−1 (liquid crystal, disordered phase). The initial value of ∼2919.7 represents chains that are only slightly disordered. As chain ordering proceeds, the frequency is reduced from
Figure 5. A cartoon depicting the early stages of lamellar phase formation (red, ceramide molecules, two chains; black, stearic acid). Stage 1: Disordered structures during and immediately following quenching. Stage 2: Formation of bilayer structures, some segregation of the SA-d35 with (interlayer) H-bond dimeric structures, and poorly packed cer[NS] with some disordered chain ends. Stage 3: more extensive orthorhombic domains of SA-d35, better packed and chainlength matched ceramide chains (also with some orthorhombic structure). Whether Stage 4 (well separated lamellae) occurs is unknown at present. Cholesterol and water molecules are omitted from this representation. 4381
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structure, some segregation of the SA-d35 with (interlayer) Hbond dimeric structures (discussed above), and poorly packed cer[NS] with some disordered chain ends. At short times post quench (Stage 2), the longer chains of the cer[NS] (depicted in red) extend beyond both the shorter ceramide chains and the SA-d35 chains (depicted in black), or the cholesterol structure (not included in the cartoon), and are presumably not optimally packed. Thus, the protruding ends of the longer chains initially bend toward the lamellar surface. The subsequent chain interaction (Figure 5, Stage 3) is suggested to result from coalescence between the saturated chains on different molecules and results in a reduced fraction of chains with disordered end regions, possibly coupled with interdigitation. Whether Stage 4 (well-separated lamellae) occurs is unknown at present and will require kinetic diffraction studies for detection. A final comment about the cartoon in Figure 5 is that cholesterol (mostly silent in the current IR analysis) and water molecules have been omitted. It is noted that examination of the water O−H stretching modes in our bulk phase samples compared with samples prepared by an alternative method (“spray technique”) reveal the clear presence of bulk phase water in all our current preparations. Although the precise location of water cannot be ascertained in the current experiments, it seems reasonable to assume that the majority of the water resides in the headgroup (interbilayer) region. Experiments quantifying the amount of water in the samples will be presented at a later date. The observed ceramide chain ordering (Figure 4) is accompanied by formation of orthorhombic structures. For this purpose, it is common (for a review, see ref 58) to monitor packing-induced changes in the scissoring mode contour at ∼1467 cm−1. However, the presence of overlapped cholesterol vibrations in this spectral region renders the contour too complex to unambiguously resolve. As an alternative approach, the CH2 rocking mode contour is shown in Figure 6A. The main CH2 rocking component at ∼721 cm−1 develops a low intensity shoulder at ∼726 cm−1 beginning at times shortly after quenching. In alkanes and phospholipids the rocking mode splittings are indicative of the presence of orthorhombic perpendicular packing. The same interpretation is suggested here for ceramides. The kinetics of the process is tracked in Figure 6B as shoulder intensity at 726 cm−1. An initial fairly early temporal evolution with a half-life of ∼2 h is followed by a slower process that continues for the duration of the experiment (∼20 h). Thus, Stage 3 (Figure 5) depicts better packed and chain-length matched ceramide chains (some with orthorhombic structure) as well as the formation of extensive large orthorhombic domains of SA-d35 as discussed below. iii. Kinetics of Stearic Acid-d35 Phase Separation. Stage 3 in the kinetic sequence is accompanied by temporal evolution of the splitting of the SA-d35 CD2 scissoring mode contour. Typical experimental data are shown in Figure 7A. The origins of the three components of this contour have been thoroughly analyzed by Snyder and co-workers48,49 and applied to lipid systems.59−61 The central peak near 1088 cm−1 which appears as a single band at short times following the quench, diminishes in intensity at long times, broadens, and develops shoulders and finally two new peaks at 1086 and 1092 cm−1. The 1088 cm−1 feature arises from populations of SA-d35 molecules with either of the following properties:
Figure 6. (A) Temporal evolution of the CH2 rocking region for the sample discussed in the caption to Figure 2. The shoulder that arises temporally at 726 cm−1 is assigned to orthorhombically packed cer[NS] phases. Spectra were baseline corrected between 707 and 732 cm−1. (B) The intensity variation of the 726 cm−1 band as a function of time following the quench (red). Also shown is the intensity variation of the 1085 cm−1 peak (black, measured from baseline corrected (1075−1097 cm−1) deconvolved spectra with γ = 2.2 and 10% Fourier smoothing), the low frequency component of the CD2 contour arising from SA-d35 packed in an orthorhombic phase. The lower X-axis scale is logarithmic.
(a) Molecules that possess either disordered chains or conformationally ordered, hexagonally packed chains. In each instance, these do not undergo the vibrational interchain interactions characteristic of orthorhombically packed chains. (b) Molecules with chains that are orthorhombically packed but are physically isolated from other orthorhombic chains with the same isotopic composition. The frequency difference between the (isolated) CH2 and CD2 scissoring modes (∼380 cm−1) precludes strong interchain vibrational coupling between them. Thus, interactions between isotopically different chains (H, D) in the current case do not contribute to the splitting of the scissoring contour. The observed splitting therefore results only from interactions between perdeuterated SA chains in orthorhombic phases. Beginning at 3−4 h following the quench, the scissoring doublet arising from perdeuterated C18 chains packed in an orthorhombic motif appears with progressively increasing intensity at ∼1086 and 1092 cm−1 (Figure 7B). Prior to revealing discrete new spectral features, the halfwidth of the original scissoring contour increases. Snyder et al.46,48 have analyzed these types of spectral changes during lateral phase separation in mixtures of alkanes of unequal chain length in which one component is deuterated. Two different simulation models have been utilized. Initially,48 it was suggested that the observed splitting between the high and low frequency 4382
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indicating that a similar level of hexagonal or isolated, orthorhombic SA-d35 molecules remain. B. Effects of Quenching Temperature and Ceramide Structure on Phase Separation Kinetics. i. Effect of Temperature Changes. As shown in Figure 8, a reduction in
Figure 7. (A) Temporal evolution of the splitting of the SA-d35 CD2 scissoring mode contour for the sample discussed in the caption to Figure 2. At short times following the quench, a single band arising from ordered, hexagonally packed chains appears as a single feature at 1088 cm−1, while the progressive formation of an orthorhombically packed phase is characterized by a doublet with components at 1086 and 1092 cm−1. (B) Temporal intensity variation of the 1086 cm−1 peak, the low frequency component of the CD2 scissoring contour arising from SA-d35 packed in an orthorhombic phase (blue). Also plotted is the temporal intensity variation of the 1088 cm−1 peak (red), the central component of the CD2 contour arising from SA-d35 packed in a hexagonal phase or in an orthorhombic phase physically isolated from other orthorhombic chains with the same isotopic composition. Intensities are measured from deconvolved spectra (see Figure 6B caption for details).
Figure 8. (A) Effect of quenching temperature on orthorhombic phase formation kinetics of SA-d35, as tracked by the disappearance of the hexagonal marker band at 1088 cm−1 at 22C (red) and at 31C (blue).The lower X-axis scale is logarithmic. Intensities are measured from deconvolved spectra (see Figure 6B caption for details). (B) Effect of quenching temperature on evolution kinetics of H-bond rearrangement of the cer[NS] Amide I peak at 1644 cm−1 at 22C (red) and at 31C (blue). The lower X-axis scale is logarithmic. Intensity measurements were made after the spectra were baseline corrected (1489−1771 cm−1).
components of the contour represents the average number of chains in domains ranging from the smallest domains detectable (∼2−3 chains) to domains of ∼100 molecules in size, which produced the maximal splitting. More recently,46 the band contours have been modeled as linear combinations of deuterated and proteated species in varying mixtures of known concentrations, assuming that the overall IR scissoring contour is described by a domain size distribution function. The splitting (preceded by broadening) observed for SA-d35 in the current experiments increases from the minimum detectable to a maximum of ∼6 cm−1 as noted. This value corresponds closely to that observed52 for pure SA-d35 in an orthorhombic packed state and so suggests, according to Snyder’s first interpretation,48 the presence of large domains with more than 100 chains and a maximum of a few non-SA-d35 chains, since the presence of the latter (i.e., either cholesterol or ceramide) would reduce the splitting from the maximum. Once the maximal splitting is attained following the quench, the 1086 and 1092 cm−1 doublet increases in intensity, without additional frequency shifts. Thus, at times greater than about 4 h, a relatively pure orthorhombic SA-d35 phase has separated laterally, i.e. in an intrabilayer (as deduced from the scissoring band frequencies) and, with the onset of formation of lamellar structures, in an interbilayer manner, as deduced from the frequencies of the SA CO stretching modes. We note that, after ∼30 h, 20% of the central peak intensity is retained
the quenching temperature from 31 to 22 °C significantly slows the kinetics of stearic acid orthorhombic phase formation. The data are presented with a logarithmic time axis for viewing convenience. The rate of orthorhombic phase formation (Figure 8A) is tracked by the disappearance of the hexagonal/disordered marker band at 1088 cm−1. Lowering the quenching temperature to 22 °C slows the ordering kinetics by about an order of magnitude. The cer[NS] headgroup rearrangement from an initially disordered form to an Amide I structure with fixed H-bond geometries and a strong feature at 1644 cm−1 is also slowed to a similar degree (Figure 8B). ii. Effects of Ceramide[NS] → Ceramide[AS] Substitution on Phase Separation Kinetics. The replacement of cer[NS] with cer[AS] significantly changes the kinetics of various steps in the phase separation sequence. Thus, in Figure 9, addition of a single −OH group to the polar region of the ceramide is seen to significantly speed up the changes in the stearic acid Hbonding kinetics. The intensity of the H-bonded SA CO peak at ∼1695 cm−1 in the cer[AS]/SA-d35/cholesterol mixture begins to form immediately upon quenching, and the initial rapid phase of the process is completed after ∼2 h. In contrast, as noted previously, the intensity of the equivalent band in the cer[NS]-containing ternary system depicts a clear initial 4 h lag time followed by a more cooperative transition that spans the time interval ∼4−7 h. 4383
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2), namely the spectral response to alterations in the Hbonding state of the polar headgroups. Significant differences have been noted in Amide I H-bonding patterns between the ternary systems studied here, compared with IR spectra of pure ceramides.65,66 The early steps in ordered lipid lamellae formation observed herein for the first time involve organization of the cer[NS] polar headgroup leading to the formation of three ordered Hbonds with similar kinetics, each characterized by a different Amide I frequency. Without isotopic labeling of the SA acid function along with molecular modeling, it is not possible to definitively assign a particular Amide I mode to a particular Hbonded structure. The observed conformational ordering and orthorhombic phase formation within the cer[NS] chains occurs on a time scale similar to changes in the ceramide polar headgroup region (compare Figures 2B, 4, and 6B). In contrast to the relatively rapid formation of specific Hbonds involving the ceramide, formation of the (predominantly trans) H-bonded dimer in SA (suggested from the frequency shift in the acid carboxyl stretch) commences after a 3−4 h time lag (Figure 2B). SA chain ordering follows a similar time scale. The SA dimer formation along with chain conformational ordering presumably requires a physical separation between the hydrophobic regions in the two molecules so their chains are effectively in separate monolayers and therefore provides clear evidence of the lamellar structure in this system. This process is accompanied temporally by phase separation of the majority of the SA chains into essentially pure orthorhombic domains. B. A Spinodal Mechanism of Phase Separation? Spinodal decomposition provides a framework to be considered as a possible mechanism for the formation of lamellar phases and domains within lamellae in SC models. Homogeneous solutions, such as the one phase regions of solutions possessing a miscibility gap, may develop fluctuations in chemical composition upon supercooling into the spinodal region of the phase diagram. These fluctuations are small at first but grow until domains of equilibrium composition develop. This differs from the alternate phase separation mechanism of nucleation and growth in which the phase separated material has the equilibrium composition required from the phase diagram. The molecules closest structurally to those studied here for which spinodal decomposition has been suggested are binary alkane mixtures differing in chain length and for which one component has been perdeuterated. Gilbert et al.50 have examined binary paraffin mixtures of C30H(D)62 with C36D(H)74 using time-resolved small-angle neutron scattering, as well as binary mixtures of CnH2n+2/C36D74 for 28 ≤ n ≤ 31. A single mixing process resulting in alternating lamellae was suggested. The phase separation time increased as chain length increased and decreased with increasing chain length mismatch. The results were consistent with interplay between C36 conformational defects and screw motion in the individual alkane chains. In the current skin models the formation of ordered H-bonds in ceramide and SA headgroups define additional types of steps in the lateral reorganization. A limitation in the IR studies of alkanes is the inability of the method, due to the lateral nature of the interchain interactions, to reveal the presence of stacked lamellae of a single species at long times. In the current experiments, headgroup CO frequency shifts provide some structural information with regard to lamellar organization. The current study is complicated by several factors compared to alkane mixtures and is probably best viewed as a
Figure 9. Effect of the addition of a single −OH group to the polar region of the ceramide component on the SA-d35 H-bonding kinetics, as tracked by formation of the 1695 cm−1 band in ceramide[AS] (red), compared to cer[NS] (black). Intensity measurements were made after the spectra were baseline corrected (1489−1771 cm−1).
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DISCUSSION A. Sequence of Kinetic Events: A Possible Mechanism for the Onset of Barrier Structure. Lipid organization in human SC and in relevant models for the lipid barrier has been widely studied. In a recent review,10 the authors note the importance of characterizing both the lamellar structure (SPP and LPP phases) as well as the lipid phase behavior parallel to the plane of the lamellae. As is evident from the current experiments, IR spectroscopy is most profitably used to track local component concentrations and structures that characterize the time evolution of microscopic compositional heterogeneity, e.g. domain formation. As noted in the Introduction, structurally oriented studies of the kinetic mechanism of skin barrier formation are sparse. Yet, kinetics plays an essential role in barrier function. Hints as to the formation of metastable phases and the duration of reequilibration kinetics have appeared in the literature. Lafleur62 noted that metastable phases form in an equimolar mixture of bovine brain Type IlI ceramide (aka human type 2, NFA, NS)/ cholesterol/palmitic acid and that the time needed to reform the equilibrium state was “several weeks” at 5 °C and “rapidly” at 40 °C. More recently from this laboratory, Moore et al.52 tracked the kinetics of orthorhombic domain formation in SC lipid models and catalogued kinetic regimes with a power law of the form P = ctα, where P is some (measured) property related to domain size and “c” is a constant. In an application to human SC,63 the partial reformation of the lipid orthorhombic phase was tracked in isolated human SC following heating of the tissue to ∼55 °C and quenching to either 25 or 30 °C. Gorcea et al.64 have most recently used methods similar to those in the current study to qualitatively track lipid domain formation in normal and ceramide deficient SC lipid models. A very small fraction of hexagonally packed (or more disordered) fatty acid was observed in the ceramide deficient models compared to the normal SC model. The biological importance of kinetic behavior has been considered in a recent review by Menon et al.1 in which the dynamic nature of the SC in response to barrier perturbation was demonstrated. These kinetic processes, addressed to date mainly by indirect approaches, appear sufficiently important to justify greater effort toward a more quantitative understanding with more direct physically oriented approaches. IR spectroscopy is particularly effective for the study of chain systems since spectral alterations induced by structural changes are sensitive to both lipid packing and conformational order. An additional element of IR sensitivity is illustrated here (Figure 4384
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three-component SC model of equimolar cer[NS], SA-d35, and cholesterol. IR provides unique molecular structure information about headgroup H-bonding, lipid packing, and chain order. The following temporal sequence for phase separation/lamellar structure formation was observed: 1) reorganization of ceramide amide H-bonds from disordered forms to ordered structures; 2) appearance of ordered ceramide chains with some orthorhombically packed structures; and 3) phase separation of large orthorhombically packed domains of SAd35 with H-bonded dimeric headgroups. A spinodal decomposition mechanism, defined by continuous composition changes during the phase separation, is suggested as a qualitative description for these events.
demonstration of the feasibility of the approach. The complicating factors are, first, the use of a ternary system with the ceramide component possessing a heterogeneous distribution of alkyl chains. This heterogeneous ceramide species was of course selected to provide a commonly used SC barrier model. Most spinodal experiments on alkanes and polymers involve binary mixtures. The inherent structural complexity of skin models will require extensive experimental work (DSC, IR, etc.) to generate a complete phase diagram from which to define the spinodal regime. Second, the sensitivity of IR to the size of orthorhombic domains that form is limited to 100 chains or so when the splitting of the methylene scissoring contour is used as the spectroscopic probe.48 At that point, the full splitting of the orthorhombic components of the methylene scissoring contour is observed. Thus, detection of the formation of large domains of alternating lamellae lies outside the sensitivity range of the IR technique and must be addressed by scattering measurements. According to Cahn’s stated criteria53 for direct proof of a spinodal mechanism, the compositions of the phases change continuously during phase separation. In several figures6−9 it is evident that the SA phase separates continuously into relatively pure orthorhombically packed domains at ∼3−8 h following the quench. In the early stages of this process, the peak separation between the components of the contour increases to its maximum difference of ∼6 cm−1. Since the splitting is known be sensitive to the number of deuterated chains adjacent to each other, the composition is obviously changing during this stage of the phase separation. These observations are consistent with the Cahn criterion for a spinodal process. A final issue of potential interest to skin biology relevant to the current experiments is the question of why so many ceramide species have evolved and seem to be essential for the correct formation of skin structure. The data in Figure 9 show that addition of a single −OH to the cer[NS] structure has dramatic consequences for the kinetics of barrier formation and likely for the final barrier structure. Several possible causes must be considered. The presence of an additional −OH functional group in Cer[AS] evidently alters the propensity and numbers of H-bonds that form not only to the ceramide headgroup but also to the fatty acid headgroup. This in turn may alter the overall level of SC hydration. Finally, slight differences in chain length distribution (see the Experimental Section) of the acid chain between the two naturally derived ceramides may also influence the kinetics. Further studies are required to delineate the relative importance of each of these factors. However, it seems reasonable even at this early stage to tentatively suggest that each ceramide species will have unique kinetic behavior both for the formation of lamellar structures and for the phase separation processes including the formation of orthorhombic phases. Given that, during the two week SC turnover time between the secretion of lamellar body lipids at the SG-SC border and exfoliation, a proficient barrier is established in healthy skin, the current work begins to suggest models for the complex kinetics and structural rearrangements responsible for competent barrier function.
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AUTHOR INFORMATION
Corresponding Author
*Telephone: 973-353-5613. Fax: 973-353-1264. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS I.S. received student support from Rutgers University. J & J Consumer Companies, Inc. generously provided support for this study. The underpinnings for spectral interpretations were illuminated by discussions with the late R. G. Snyder.
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REFERENCES
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CONCLUSIONS The current experiments provide a framework for investigating the sequence of dynamic events involved in the formation and dissipation of the SC barrier in normal and pathological states. Kinetic IR spectroscopy measurements probed the temporal sequence of molecular events producing ordered structures in a 4385
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