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Lipid chain dynamics in stratum corneum studied by spin label electron paramagnetic resonance
Chemistry and Physics of Lipids 104 (2000) 101 – 111 www.elsevier.com/locate/chemphyslip
Lipid chain dynamics in stratum corneum studied by spin label electron paramagnetic resonance Antonio Alonso a,*, Nilce C. Meirelles b, Marcel Tabak c b
a Instituto de Fı´sica, Uni6ersidade Federal de Goia´s, Goiaˆnia 74001 -970, GO, Brazil Departamento de Bioquı´mica, Instituto de Biologia, Uni6ersidade Estadual de Campinas, Campinas 13083 -970, SP, Brazil c Instituto de Quı´mica, Uni6ersidade de Sa˜o Paulo, Sa˜o Carlos 13560 -970, SP, Brazil
Received 16 June 1999; received in revised form 6 July 1999; accepted 6 July 1999
Abstract The lipid chain motions in stratum corneum (SC) membranes have been studied through electron paramagnetic resonance (EPR) spectroscopy of stearic acid spin-labeled at the 5th, 12th and 16th carbon atom positions of the acyl chain. Lipids have been extracted from SC with a series of chloroform/methanol mixtures, in order to compare the molecular dynamics and the thermotropic behavior in intact SC, lipid-depleted SC (containing covalently bound lipids of the corneocyte envelope) and dispersion of extracted SC lipids. The segmental motion of 5- and 12-doxylstearic acid (5- and 12-DSA) and the rotational correlation time of 16-doxylstearic acid (16-DSA) showed that the envelope lipids are more rigid and the extracted lipids are more fluid than the lipids of the intact SC over the range of temperature measured. The lower fluidity observed for the corneocyte envelope, that may be caused mainly due to lipid–protein interactions, suggests a major contribution of this lipid domain to the barrier function of SC. Changes in the activation energy for reorientational diffusion of the 16-DSA spin label showed apparent phase transitions around 54°C, for the three SC samples. Some lipid reorganization may occur in SC above 54°C, in agreement with results reported from studies with several other techniques. This reorganization is sensitive to the presence of the extractable intercellular lipids, being different in the lipid-depleted sample as compared to native SC and lipid dispersion. The results contribute to the understanding of alkyl chain packing and mobility in the SC membranes, which are involved in the mechanisms that control the permeability of different compounds through skin, suggesting an important involvement of the envelope in the skin barrier. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Stratum corneum; EPR; Membrane fluidity; Phase transition
1. Introduction
Abbre6iations: EPR, electron paramagnetic resonance; 5DSA, 5-doxylstearic acid; 12-DSA, 12-doxylstearic acid; 16DSA, 16-doxylstearic acid; PBS, phosphate-buffered saline. * Corresponding author. Fax: + 55-62-821-1029.
The principal function of the stratum corneum (SC) is to control transepidermal water loss as well as the diffusion of other substances through the skin. Thin-section transmission microscopy of the SC has shown that the intercellular spaces
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between the corneocytes are partly filled with broad multilamellar sheets (Swartzendruber et al., 1989; Wertz et al., 1989), which are important in regulating the barrier function of the skin (Elias, 1983). Corneocytes are anucleate cells composed of filamentous keratin network containing an insoluble, highly cross-linked peripheral envelope (Elias, 1988). Wertz and Downing (1987) have determined that pig SC contains 2.1% (dry weight) of bound hydroxyceramide, which is just sufficient to provide a monolayer to cover the surface of each corneocyte (Wertz et al., 1989). About half of the hydroxyceramide molecules are bound to the corneocyte envelope by esterification of the vhydroxyl function, and half via one of the hydroxyl groups of sphingosine (Wertz and Downing, 1987). The fraction of chemically unbound lipids in the pig SC that can be removed by exhaustive extraction with chloroform/ methanol mixtures corresponds to about 14% of the tissue dry weight (Swartzendruber et al., 1987) and consists mainly of ceramides, free fatty acids and cholesterol (Gray et al., 1982; Long et al., 1985). White et al. (1988) showed that the lipid structure of the SC of hairless mice is lamellar with a repeat period of 13.1 nm and that the isolated lipids do not produce a single lamellar diffraction pattern. The lipid structure of the SC has since been a subject of intense investigations, using mainly small and wide-angle X-ray (Bouwstra et al., 1992, 1994, 1995; Parrot and Turner, 1993) and infrared spectroscopy (Krill et al., 1992; Ongpipattanakul et al., 1994). The aim of these studies was essentially to understand how these lipids, which are unusual in many respects, exert the selective permeability of the skin. Measurements have been made in human, pig and mouse intact SC and also in many dispersions of isolated lipids in water or buffer solutions. These dispersions have been used as models of the intercellular lamellae found in the SC. However, a consensus about the existent phases or about the precise origin of phase transition temperatures is still missing. Furthermore, a possible lipid exchange between the corneocyte envelope and the intercellular bilayers domains
was not investigated yet. Parrot and Turner (1993) reported that mixtures of cholesterol and ceramides can form a structure of long periodicity (13.5 nm), while more recently McIntosh et al. (1996) have shown that mixtures of specific skin ceramides and appropriate concentrations of cholesterol and fatty acid (similar to those found in SC), can produce a diffraction pattern with a single repeat period of 13.0 nm. It is worth mentioning the recent study (Lopez et al., 1997) of the composition of pig SC showing again the absence of phospholipids and the presence of ceramides and cholesterol as the dominant lipid species (35 and 26.5%, respectively). In previous work (Alonso et al., 1995, 1996), electron paramagnetic resonance (EPR) and fatty acid spin probes were used to evaluate membrane fluidity in intact neo-natal rat SC, and observed that many EPR spectra were able to distinguish two populations of spin labels with very different mobility states. The more fluid lipid domain could be associated with the extracellular bilayers, which can be extracted by organic solvents, while the more rigid one could be associated with the corneocyte envelope, which is associated with the protein, giving a more restricted motion. This suggests that an appreciable part of the fatty acid spin probes could be structured in the corneocyte membrane. Since fatty acids, together with ceramides and cholesterol, are required for permeability barrier homeostasis (Landmann, 1988; Elias and Menon, 1991; Mao-Qiang et al., 1993a,b), it is important to consider the role of both extracellular and envelope membranes, and also their interactions, in the barrier function of the SC. In this work EPR and the spin label method have been used to compare the thermal behavior of lipid fluidity, at pH 7.4, in three types of SC lipid samples: intact SC, lipid-depleted SC (containing only covalently bound lipids) and dispersions of isolated lipids. Fluidity was evaluated at three depths of the membrane alkyl chains in order to monitor lipid–protein interactions and the changes in fluidity following thermally induced phase transitions.
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2. Materials and methods
2.1. SC membranes As in previous reports (Alonso et al., 1995, 1996) SC was obtained from newborn Wistar rats aged less than 24 h. After sacrifice, the skin was excised and fat was removed by rubbing in distilled water. The skin was allowed to stand for 5 min in a desiccator containing 0.5 l of anhydrous ammonium hydroxide and was then allowed to float in distilled water with the epidermis side in contact with the water. After 2 h, the SC was removed to filter paper and then transferred to a Teflon-coated screen, extensively washed with distilled water, and allowed to dry at ambient conditions. This procedure reduced the effect of the remaining epidermal tissues to a minimum. The membranes were stocked in a desiccator containing silica gel under a moderate vacuum until use.
2.2. Extraction of SC lipids Lipids were extracted from rat SC according to the method used by Wertz and Downing (1987) for porcine SC. The sheets of SC ( 80 mg) were incubated for 2 h intervals in each of three successive extraction mixtures (80 ml) of chloroform/ methanol (2:1, 1:1 and 1:2, v/v) and recovered each time by filtration through a 0.25 mm teflon millipore filter (Millipore, Bedford, MA). This extraction procedure was repeated changing the solvents at 1 h intervals followed by an overnight extraction in methanol. All solvent fractions were combined and evaporated under a stream of nitrogen gas. The isolated lipids and the lipid-depleted SC were kept in a desiccator under vacuum until used.
2.3. Preparation of extracted lipid samples Lipid dispersions were prepared at a concentration of 20 mg/ml in phosphate-buffered saline (PBS, 5 mM, pH 7.4) using a procedure similar to that described by Ongpipattanakul et al. (1994). A weighed mass of extracted lipid (40 mg) was placed in a glass tube and 2 ml of buffer was added. The mixture was repeatedly heated to
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70°C under sonication with several cycles of cooling to room temperature between the sonications. This procedure was repeated 3–4 times (20 min) until a homogeneous suspension was obtained.
2.4. Spin-labeled SC samples Spin-labeled derivatives of stearic acids, 5-, 12and 16-doxylstearic acids (5-DSA, 12-DSA and 16-DSA, respectively), were purchased from Aldrich (Milwaukee, WI). These spin labels have a nitroxide radical ring attached at C-5, C-12 and C-16 positions of the acyl chain, respectively. A small aliquot of stock spin label dissolved in ethanol (5 mg/ml) was placed in a glass tube and the solvent was evaporated under a stream of nitrogen gas. Samples containing about 10 mg of intact SC or lipid-depleted SC, cut and ground into very small pieces and suspended in PBS, or an extracted lipid dispersion (in PBS), were added to the spin label thin film at lipid/spin label molar ratios greater than 150 in order to avoid magnetic interactions among the spin labels (Fung and Johnson, 1984). Since the lipid concentrations of extracted unbound lipids and chemically bound lipids for porcine SC were reported by Wertz and Downing (1987) to account for 14.7 and 2.1% of the SC dry weight, respectively, a spin label concentration 7-fold lower for lipid-depleted SC was used compared to intact SC or extracted lipid dispersions. The concentration of spin labels was estimated to be 20 mM for delipidized SC and 140 mM for both intact SC and SC lipid suspensions. After labeling under gentle stirring, the samples were introduced into a capillary tube for EPR measurements. Based on gravimetric measurements, the hydration of tissue samples in the capillary was estimated to be 5897% (w/w). Under these conditions, the SC was considered to be in a fully hydrated state (excess of water).
2.5. EPR measurement conditions A Varian E-9 spectrometer (Varian Associates, Palo Alto, CA) equipped with the rectangular cavity and operating at X-band (9.12 GHz) was utilized in the investigations. The spectral parameters were as follows: microwave power, 20 mW;
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modulation frequency, 100 KHz; modulation amplitude, 2.5 G; magnetic field scan, 100 G; sweep time, 2 min and detector time constant, 0.064 s. The spectra were digitalized by an interfaced computer system (1070 points/spectrum). Temperature was controlled within 0.3°C by a nitrogen steam system from Air Products and Chemicals (Allentown, PA).
2.6. EPR spectra analysis The fluidity of the membrane can be estimated from the 2T parameter as described by Wassal and Stillwell (1990). In the rapid motion regime, as is the case of 16-DSA, the resolution of the outer features of 2T%// becomes very poor. In this case, the calculation of the rotational correlation time, tc, is more appropriate. This empirical parameter describes the rate of motion of the probe as it rotates about its long axis. The EPR parameter tc is also calculated using spectral parameters measured directly in the EPR spectra (Wassal and Stillwell, 1990). The activation energy Ea was also calculated for 16-DSA using the Arrhenius equation: log tc =Ea/2.3RT
cell membrane lipid bilayers and properly distributed throughout the tissues. The proper distribution of spin label was assured by the capacity of samples to get spin labels structured in their lipid bilayers, without allowing magnetic interactions among them (dipole–dipole and exchange interactions). Such magnetic interactions, which cause the broadening of resonance lines and reduce spectral resolution, are largely avoided since the preparation contains 150 or more membrane lipids for each spin label (Fung and Johnson, 1984). The three types of samples analyzed in this work showed EPR spectra with two spectral components corresponding to different states of probe mobility. Both of these components remained resolved for any temperature interval over the whole range (0–90°C) investigated. The low-field spectral features of these two components are indicated by arrows in Fig. 1, for cases in which the coexistence of the two spectral components is more clearly evident. Two-component spectra indicate the presence of at least two lipid domains
(1)
where R is the gas constant and T the absolute temperature. In practice, the numerical value of Ea was determined from the slope of a plot of log tc versus 1/T (Arrhenius plot).
3. Results
3.1. EPR spectra for SC samples The EPR spectra at 38°C for 5-, 12- and 16DSA, incorporated into delipidized SC, intact SC and dispersions of extracted SC lipids are shown in Fig. 1. Due to the large hydrophobicity of these fatty acid spin probes, and to the conditions of the preparations, they were able to penetrate the SC tissue and intercalate with the membrane lipids. The EPR spectra are similar to typical spectra observed in plasma membranes (Griffith and Jost, 1976; Alonso et al., 1995, 1996). The spin labels were adequately incorporated into the
Fig. 1. Electron paramagnetic resonance (EPR) spectra at 38°C of spin labels 5-doxylstearic acid (5-DSA) (a, d, g), 12-doxylstearic acid (12-DSA) (b, e, h) and 16-doxylstearic acid (16-DSA) (c, f, i) in lipid-depleted stratum corneum (SC) (a, b, c), intact SC (d, e, f) and extracted lipid dispersion (g, h, i), suspended in phosphate-buffered saline (PBS) pH 7.4. The arrows mark the field positions where two spectral components (spin labels with different mobility states) are more clearly visible.
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Fig. 2. Electron paramagnetic resonance (EPR) spectra of spin labels 5-doxylstearic acid (5-DSA) (a, b, c), 12-doxylstearic acid (12-DSA) (d, e, f) and 16-doxylstearic acid (16-DSA) (g, h, i) in lipid-depleted stratum corneum (SC) suspended in phosphate-buffered saline (PBS) pH 7.4, at indicated temperatures. The arrows mark the field positions of the two spectral components.
(populations) with different fluidity or molecular order (Griffith and Jost, 1976). In the case of lipid dispersions this can arise from lateral lipid phase separation (Schorn and Marsh, 1996); in intact SC there are at least two lipid domains (envelope and extracellular bilayers) and in the case of lipid depleted samples this can arise from lipid – protein interactions (Griffith and Jost, 1976; Marsh, 1989, 1996) where the lipid chains are immobilized by contact with the hydrophobic surfaces of proteins, the two components corresponding to the ‘boundary’ and ‘bulk’ lipids. In Fig. 2 the EPR spectra for the delipidized samples at temperatures where there is a clear resolution of these two components are presented. For 5-DSA and 12-DSA this occurs around 78°C, as judged by a significant increase of the second low-field resonance line and for 16-DSA this appears to occur around 30 – 42°C. In the spectra from Fig. 1 there is just one apparent component for 5- and 12-DSA and two components for 16-
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DSA. In the time scale of the EPR experiments near the rigid limit, the spectra do not distinguish the two components and the lipid–protein interactions are not clearly visible. This also happens in the limit of fast motions when the spectra have an isotropic appearance even when the interaction with protein is present. This can be observed in the spectra of 16-DSA in lipid-depleted SC presented in Fig. 3. While at 22°C two components are resolved, only one component is observed at 62°C; the spectrum appears isotropic as is the case for the dispersion of extracted lipid where the proteins are absent. Quantification of the fractions between the boundary and bulk lipids has been made by spectral subtraction (Brotherus et al., 1980) and spectral simulation (Ge and Freed, 1999). In both cases these fractions were temperature independent, but the question of how the lipid structure is affected and modulated by membrane proteins is not yet clear (Ge and Freed, 1999).
Fig. 3. Electron paramagnetic resonance (EPR) spectra of 16-doxylstearic acid (16-DSA) in lipid-depleted stratum corneum (SC) (a, b) and in dispersion of SC extracted lipids (c, d) suspended in phosphate-buffered saline (PBS) pH 7.4, at indicated temperatures. The arrows mark the field positions of the two spectral components.
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Fig. 4. Electron paramagnetic resonance (EPR) parameter 2T%// as a function of temperature for spin labels 5-doxylstearic acid (5-DSA) (A), 12-doxylstearic acid (12-DSA) (B) and 16-doxylstearic acid (16-DSA) (C) in lipid-depleted stratum corneum (SC) (squares), intact SC (circles) and SC lipid dispersion (triangles). Mean 9 S.D. (n =3).
3.2. Molecular motion along the fatty acid chain as a function of temperature in the SC samples Fig. 4 shows the dependence of the parameter 2T%// as a function of temperature for 5-, 12- and 16-DSA incorporated into intact SC, delipidized SC and dispersion of extracted SC lipids. Calculation of the order parameter S is limited to spectra in which simultaneous resolutions of T%// and T%Þ occur. As most of the EPR spectra have two components, the T%Þ and T%// parameters can not be accurately measured for both components. For this reason we use only the T%// parameter, which
was sensitive to thermally induced changes in fluidity. The greater the fluidity, the lower the T%//. It is quite important to have in mind that the parameter T%// reflects the maximum experimentally observed nitrogen hyperfine splitting in the EPR spectrum. Thus, the parameter T%// is a static parameter associated with the orientational distribution of spin labels in the membrane, although it is related to the changes in spin labels mobility. T%// reflects the average angle of the axis orientation of the spin label relative to the bilayer normal (Griffith and Jost, 1976; Marsh, 1989). The three spin probes indicated that the membrane of the
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corneocyte lipid envelope is the most rigid and that fluidity is higher for the extracted lipid dispersion than for intact SC. Another parameter, the rotational correlation time, tc, is directly associated with the motional reorientation of the spin label and consequently with the probe mobility in the membrane. The tc values for 16-DSA using the three samples are presented in Fig. 5 as an Arrhenius plot in order to analyze the phase transitions. This probe, located deeper within the membrane, also detected a more rigid membrane for the corneocyte envelope, an intermediate fluidity for intact SC and a higher fluidity for the isolated lipids (see Fig., 1(c), (f) and (i)).
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Table 1 Activation energy for the rotational diffusion of 16-doxylstearic acid (16-DSA) DSA in stratum corneum (SC) membranes: corneocyte envelope (lipid-depleted SC), intact SC and extracted lipid dispersionsa Sample
Temperature range (°C)
DE (Kcal/mol)
Lipid-depleted SC
46–54
9.5 90.3Ab
54–90 42–54 54–90 18–54
5.0 90.9B 5.3 90.5B 9.4 90.7A 7.3 9 0.5C
54–90
9.1 9 0.6A
Intact SC Extracted SC lipids
4.4 9 0.4c 8.3 90.7
The means 9 S.D. were calculated from three independent experiments presented as an average in Fig. 5. b Statistical significance: only the means indicated with different capital letters are statistically different with PB0.01 (Student’s t-test). c Data previously reported for fully hydrated SC (Alonso et al., 1995). a
3.3. Lipid phase transitions assessed by fatty acid chain mobility The curves in Fig. 5 show discrete differences in slope coefficients suggesting the existence of membrane phase transitions. The slope coefficients for the Arrhenius plot give the activation energy for
Fig. 5. Arrhenius plot of the rotational correlation time, tc, of 16-doxylstearic acid (16-DSA) structured in lipid-depleted stratum corneum (SC) (squares), intact SC (circles) and SC lipid dispersion (triangles). Regression analysis was performed considering the following temperature intervals: 46–54 and 54– 90°C for lipid-depleted SC; 42–54 and 54–90°C for intact SC and 18 – 54 and 54 – 90°C for the SC lipid dispersion. Mean9 S.D. (n = 3).
the reorientation of the 16-DSA in the membrane. The calculated activation energies are presented in Table 1. The membrane of the corneocyte envelope shows an apparent phase transition at around 54°C, with its activation energy changing from 9 to 5 Kcal/mol. The activation energies for intact SC and extracted SC lipids were practically the same, changing from 5 to 7 Kcal/mol below 54°C, to 9 Kcal/mol above 54°C. Data obtained previously for intact SC (Alonso et al., 1995) are also included in Table 1. They are, in general, in good agreement with the values obtained in the present work. Despite the fact of the anisotropic motion of 16-DSA in SC (Alonso et al., 1995), it is believed that the present results show an apparent phase transition around 54°C, which is similar to all samples, and the difference in mobility as reflected in tc in the different samples. It is worth mentioning the fact that all plots presented in this work were reversible.
3.4. Temperature dependence of the rotational gradient along the fatty acid chain At high temperatures, the rate of motion is higher (tc B 3×10 − 9 s) and the EPR spectra
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allowed the measurement of the rotational correlation times also for 5- and 12-DSA, in the case of the extracted SC lipids and intact SC. These data for the three spin probes are presented in Fig. 6. For lipid-depleted SC the tc could not be determined accurately for these two probes since two spectral components remain in the EPR spectra even above 70°C; the motion of the probes in this sample is also significantly more restricted as
compared to the other two samples. As the nitroxide moves away from the polar region and approaches the hydrophobic core of the membrane, the rotational motion became less restricted (correlation time shorter). It is possible to note that the rotational gradient is more pronounced for intact SC than the lipid dispersion samples, especially in the region between the C-5 and C-12 atoms of the acyl chains, where the greater differences in tc are observed.
4. Discussion
Fig. 6. Arrhenius plots of the rotational correlation time, tc, of 5-doxylstearic acid (5-DSA) (open square), 12-doxylstearic acid (12-DSA) (open circle) and 16-doxylstearic acid (16-DSA) (triangles) structured in stratum corneum (SC) lipid dispersion (A) and intact SC (B). Mean 9S.D. (n= 3).
This work shows that it is possible to incorporate EPR spin probes directly into SC membranes in intact tissue. Even after exhaustive lipid extraction with a series of chloroform/methanol mixtures, it was possible to intercalate the spin labels with the remaining lipid-depleted SC sample, and to obtain an adequate radical distribution for EPR measurements. In the present work, the fluidity of the SC lipids as a whole was compared with the fluidity of two classes of SC lipids: chemically bound and unbound lipids. Using two EPR parameters (2T%// and tc) and three spin labeled positional isomers, it was shown that lipids covalently bound in SC are more rigid than the lipids in intact SC over the range of temperatures measured. The reverse was observed for the dispersion of extracted lipids that showed a higher fluidity compared to intact SC. This observation is similar to that for several other types of biological membranes in which the extracted lipids are more fluid than the original membrane in the presence of membrane proteins (Griffith and Jost, 1976; Marsh, 1989). A comparison of the results for the temperature dependence of the rotational correlation time for 16-DSA (Fig. 5) with the data from Fourier transform infrared spectroscopy (Golden et al., 1987; Krill et al., 1992; Ongpipattanakul et al., 1994) is quite interesting. While measuring the temperature dependence of the symmetric CH2 stretching frequency for porcine SC hydrated at 75% relative humidity, Ongpipattanakul et al. (1994) observed a general increase in frequency from − 10 up to 100°C, with a particularly sharp increase in rate
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between 60 and 80°C (high slopes) and a low rate of increase between 20 and 60°C (small slopes). Similar results were reported by Krill et al. (1992) for the hairless mouse, except that the temperature range for the smaller slope was observed between 31 and 55°C. Golden et al. (1987) measured the antisymmetric CH2 stretching frequency for porcine SC hydrated at 75% relative humidity, and observed a gradual increase in frequency over the temperature range from 30 to 115°C, with a dramatic increase in rates between 60 and 80°C. The data for the rotational correlation time in neo-natal rat SC (Fig. 5) are in agreement with the above findings. The nitroxide rotation of the 16-DSA probe in intact SC and lipid dispersion was very sensitive to temperature having a higher increase in rates in the temperature range from 54 to 90°C. In the case of lipid depleted samples, the opposite behavior was observed, the increases in rates being lower for temperatures above 54°C. The data depicted in Fig. 5 showed lower tc values for isolated lipid samples than for intact SC samples over all temperature ranges. However, the activation energies for the two samples were essentially the same (Table 1). The state of probe mobility can change through alterations either in the amplitude or frequency of the motion. Hence, since the energy barrier is maintained, the rotational motion of the spin label in intact SC may be lower than in the lipid dispersion due to the smaller motional amplitude in this case. Thus, it is believed that in SC tissue there are some spatial constraints on the rotational motion of 16-DSA that reduce its motional amplitude as compared to its mobility state in extracted lipid dispersions. At temperatures above the phase transition (54°C), lipid-depleted samples showed higher tc values accompanied by a large decrease in activation energy. This fact could be explained by a less densely packed hydrocarbon domain (smaller Ea) together with a lower motion amplitude due to the presence of proteins (greater tc). The presence of two lipid structures (the corneocyte and intercellular bilayers) in the SC, and the complexity of their lipid composition are consistent with the coexistence of several subcell structures. Using X-ray diffraction, White et al.
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(1988) studied the lipid structure of hairless mice SC at 25, 45 and 70°C. The wide-angle patterns at 25°C indicated the presence of a lipid fraction in the liquid state and an additional structure organized as an orthorhombic subcell, while at 45°C, a lipid fraction in the liquid state in equilibrium with lipids in hexagonal subcells was found. At 70°C, only a broad band was observed indicating that all the lipids were in the liquid state, although small-angle X-ray scattering for extracted lipids indicated the hexagonal phase with all alkyl chains in the liquid state. More recently, other studies using the X-ray technique have been performed in human, mouse and pig SC. For human SC, Bouwstra et al. (1992) found a phase transition from an orthorhombic to a hexagonal phase at 40°C. In mouse SC, the orthorhombic subcell disappears at 45°C and at this temperature only hexagonal and liquid lateral packing was observed (Bouwstra et al., 1994). Finally, in pig SC only hexagonal and liquid lateral packing was detected with disappearance of the hexagonal packing in the temperature range of 60–66°C (Bouwstra et al., 1995). Rehfeld et al. (1988), using the EPR technique and the perdeuterated di-tert-butyl nitroxide to study scale from human SC, have identified thermal phase transitions at 34–37 and 50°C which correspond to the phase transitions observed by differential scanning calorimetry. The temperature dependence of the rotational gradient along the fatty acid chain shown in Fig. 6 can provide additional information about the lipid chain dynamics. The rotational gradient in the bilayer region between the 5th and 12th carbon atom is smaller for isolated lipid (Fig. 6A) than for intact SC (Fig. 6B). For the delipidized samples the rotational motion is very restricted, even at high temperatures, making the measurement of tc for 5- and 12-DSA very unreliable. It is well known that in both biological and artificial model membranes, the permeability increases with the increase in fluidity (Fettyplace and Haydon, 1980), suggesting a correlation between membrane transport and lipid fluidity. Golden et al. (1987) measured the activation energy for water flux in porcine SC and found a value of 17 Kcal/mol, suggesting that the water flux in SC is limited by diffusion through the ordered hydrocarbon do-
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mains. So, our data further suggest that the nonlipid depleted corneocyte envelope (in native SC) may have a major role in the physical barrier in SC. In conclusion, the lipids of the corneocyte membrane are assembled in a quite rigid structure, and this low mobility state is caused mainly by their covalent bonding to the envelope proteins and to the strong acyl chain interactions with the hydrophobic surfaces of the proteins. Intact SC and dispersions of extracted SC lipids were considerably more fluid than the envelope membrane, suggesting that the major contribution for the barrier function of SC is provided by the envelope. The reorientational motion of 16-DSA was also able to monitor apparent phase transitions for the intact and extracted SC lipid samples at 54°C. The lipid chain packing detected by the energy barrier for reorientational motion of 16DSA was similar for intact and extracted lipid samples, but since tc values are smaller for extracted lipids, the amplitude of motion is greater for these samples. In addition, it was found that the spin label EPR approach can provide precise evaluations of molecular dynamics and ordering of SC lipids, which also may be used to investigate drug–lipid interaction directly in native SC, as well as different cases of skin diseases and lesions. Acknowledgements This work was supported by Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq; grant process 300908/92-0) and Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP; processes: 97/02431-4 and 95/6177-0). We wish to thank Professor Dr Fernando Pelegrini (Instituto de Fı´sica, Universidade Federal de Goia´s) for his interest in this work. References Alonso, A., Meirelles, N.C., Tabak, M., 1995. Effect of hydration upon the fluidity of intercellular membranes of stratum corneum: an EPR study. Biochim. Biophys. Acta 1237, 6 – 15.
Alonso, A., Meirelles, N.C., Yushmanov, V.I., Tabak, M., 1996. Water increases the fluidity of intercellular membranes of stratum corneum: correlation with water permeability, elastic, and electrical resistance properties. J. Invest. Dermatol. 106, 1058 – 1063. Bouwstra, J.A., Gooris, G.S., Salomons-de Vries, M.A., van der Spek, J.A., Bras, W., 1992. Structure of human stratum corneum as a function of temperature and hydration: a wide-angle X-ray diffraction study. Int. J. Pharm. 84, 205 – 216. Bouwstra, J.A., Gooris, G.S., van der Spek, J.A., Lavrijsen, S., Bras, W., 1994. The lipid and protein structure of mouse stratum corneum: a wide and small angle diffraction study. Biochim. Biophys. Acta 1212, 183 – 192. Bouwstra, J.A., Gooris, G.S., Bras, W., Downing, D.T., 1995. Lipid organization in pig stratum corneum. J. Lipid Res. 36, 685 – 695. Brotherus, J.R., Jost, P.C., Griffith, O.H., Keana, J.F.W., Hokin, L.E., 1980. Charge selectivity at the lipid – protein interface of membranous Na,K-ATPase. Proc. Natl. Acad. Sci. USA 77, 272 – 276. Elias, P.M., 1983. Epidermal lipids, barrier function, and desquamation. J. Invest. Dermatol. 80, 44s – 49s. Elias, P.M., 1988. Structure and function of the stratum corneum barrier. Drug. Dev. Res. 13, 97 – 105. Elias, P.M., Menon, G.K., 1991. Structural and lipid biochemical correlates of the epidermal permeability barrier. Adv. Lipid Res. 24, 1 – 26. Fettyplace, R., Haydon, D.A., 1980. The permeability of lipids membranes. Physiol. Rev. 60, 510 – 550. Fung, L.W.M., Johnson, M.E., 1984. Recent developments in spin label EPR methodology for biomembranes studies. In: Lee, P. (Ed.), Currents Topics in Bioenergetics. Academic Press, New York, pp. 107 – 157. Ge, M., Freed, J.H., 1999. Electron-spin resonance study of aggregation of gramicidin in dipalmitoylphosphatidylcholine bilayers and hydrophobic mismatch. Biophys. J. 76, 264 – 280. Golden, M., Guzek, D.B., Kennedy, A.H., McKie, J.E., Potts, R.O., 1987. Stratum corneum lipid phase transitions and water barrier properties. Biochemistry 26, 2382 – 2388. Gray, G.M., White, R.J., Yardley, H.J., 1982. Lipid composition of the superficial stratum corneum cells of the epidermis. Br. J. Dermatol. 106, 59 – 63. Griffith, O.H., Jost, P.C., 1976. Lipid spin labels in biological membranes. In: Berliner, L.J. (Ed.), Spin Labeling Theory and Applications. Academic Press, New York, pp. 453 – 523. Krill, S.L., Knutson, K., Higuchi, W.I., 1992. The stratum corneum lipid thermotropic phase behavior. Biochim. Biophys. Acta 1112, 281 – 286. Landmann, L., 1988. The epidermal permeability barrier. Anat Embryol. 178, 1 – 13. Long, S.A., Wertz, P.W., Strauss, J.S., Downing, D.T., 1985. Human stratum corneum polar lipids and desquamation. Arch. Dermatol. Res. 277, 284 – 287.
A. Alonso et al. / Chemistry and Physics of Lipids 104 (2000) 101–111 Lopez, O., de la Maza, A., Cordech, L., Parra, J.L., 1997. Study of the composition and structure of pig stratum corneum based on the action of different solubilizing agents. Colloids Surfaces A 123–124, 415–424. Mao-Qiang, M., Feingold, K.R., Elias, P.M., 1993a. Inhibition of cholesterol and sphingolipid synthesis causes paradoxical effects on permeability barrier homeostasis. J. Invest. Dermatol. 101, 190–195. Mao-Qiang, M., Kenneth, R.F., Elias, P.M., 1993b. Exogenous lipids influence permeability barrier recovery in acetone-treated murine skin. Arch. Dermatol. 129, 728–738. Marsh, D., 1989. Experimental methods in spin-label spectral analysis. In: Berliner, L.J. (Ed.), Biological Magnetic Resonance. Plenum, New York, pp. 255–303. Marsh, D., 1996. Membrane assembly studied by spin-label electron spin resonance. Braz. J. Med. Biol. Res. 29, 863– 871. McIntosh, J., Stewart, M.E., Downing, D.T., 1996. X-ray diffraction analysis of isolated skin lipids: reconstitution of intercellular lipid domains. Biochemistry 35, 3649–3653. Ongpipattanakul, B., Francoeur, M.L., Potts, R.O., 1994. Polymorphism in stratum corneum lipids. Biochim. Biophys. Acta 1190, 115–122. Parrot, D.T., Turner, J.E., 1993. Mesophase formation by ceramides and cholesterol. Biochim. Biophys. Acta 1147, 273 – 276. Rehfeld, S.J., Plachy, W.Z., Williams, M., Elias, P.M., 1988. Calorimetry and electron spin resonance examination of lipid phase transition in human stratum corneum: molecular basis for normal cohesion and abnormal desquamation
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in recessive x-linked ichthyosis. J. Invest. Dermatol. 91, 499 – 505. Schorn, K., Marsh, D., 1996. Lipid chain dynamics and molecular location of diacylglycerol in hydrated binary mixtures with phosphatidylcholine: spin label ESR studies. Biochemistry 35, 3831 – 3836. Swartzendruber, D.C., Wertz, P.W., Madison, K.C., Downing, D.T., 1987. Evidence that the corneocyte has a chemically bound lipid envelope. J. Invest. Dermatol. 88, 709 – 713. Swartzendruber, D.C., Wertz, P.W., Kitko, D.J., Madison, K.C., Downing, D.T., 1989. Molecular models of the intercellular lipid lamellae in mammalian stratum corneum. J. Invest. Dermatol. 92, 251 – 257. Wassal, S.R., Stillwell, W., 1990. Interactions of retinoids with phospholipid membranes: electron spin resonance. In: Packer, L. (Ed.), Methods in Enzymology, vol. 189. Academic Press, New York, pp. 383 – 394. Wertz, P.W., Downing, D.T., 1987. Covalently bound v-hydroxyacylsphingosine in the stratum corneum. Biochim. Biophys. Acta 917, 108 – 111. Wertz, P.W., Swartzendruber, D.C., Kitko, D.J., Madison, K.C., Downing, D.T., 1989. The role of the corneocyte lipid envelopes in cohesion of the stratum corneum. J. Invest. Dermatol. 93, 169 – 172. White, S.H., Mirejovsky, D., King, G.I., 1988. Structure of lamellar lipid domains and corneocyte envelopes of murine stratum corneum. An X-ray diffaction study. Biochemistry 27, 3725 – 3732.
Lipid chain dynamics in stratum corneum studied by spin label electron paramagnetic resonance Antonio Alonso a,*, Nilce C. Meirelles b, Marcel Tabak c b
a Instituto de Fı´sica, Uni6ersidade Federal de Goia´s, Goiaˆnia 74001 -970, GO, Brazil Departamento de Bioquı´mica, Instituto de Biologia, Uni6ersidade Estadual de Campinas, Campinas 13083 -970, SP, Brazil c Instituto de Quı´mica, Uni6ersidade de Sa˜o Paulo, Sa˜o Carlos 13560 -970, SP, Brazil
Received 16 June 1999; received in revised form 6 July 1999; accepted 6 July 1999
Abstract The lipid chain motions in stratum corneum (SC) membranes have been studied through electron paramagnetic resonance (EPR) spectroscopy of stearic acid spin-labeled at the 5th, 12th and 16th carbon atom positions of the acyl chain. Lipids have been extracted from SC with a series of chloroform/methanol mixtures, in order to compare the molecular dynamics and the thermotropic behavior in intact SC, lipid-depleted SC (containing covalently bound lipids of the corneocyte envelope) and dispersion of extracted SC lipids. The segmental motion of 5- and 12-doxylstearic acid (5- and 12-DSA) and the rotational correlation time of 16-doxylstearic acid (16-DSA) showed that the envelope lipids are more rigid and the extracted lipids are more fluid than the lipids of the intact SC over the range of temperature measured. The lower fluidity observed for the corneocyte envelope, that may be caused mainly due to lipid–protein interactions, suggests a major contribution of this lipid domain to the barrier function of SC. Changes in the activation energy for reorientational diffusion of the 16-DSA spin label showed apparent phase transitions around 54°C, for the three SC samples. Some lipid reorganization may occur in SC above 54°C, in agreement with results reported from studies with several other techniques. This reorganization is sensitive to the presence of the extractable intercellular lipids, being different in the lipid-depleted sample as compared to native SC and lipid dispersion. The results contribute to the understanding of alkyl chain packing and mobility in the SC membranes, which are involved in the mechanisms that control the permeability of different compounds through skin, suggesting an important involvement of the envelope in the skin barrier. © 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Stratum corneum; EPR; Membrane fluidity; Phase transition
1. Introduction
Abbre6iations: EPR, electron paramagnetic resonance; 5DSA, 5-doxylstearic acid; 12-DSA, 12-doxylstearic acid; 16DSA, 16-doxylstearic acid; PBS, phosphate-buffered saline. * Corresponding author. Fax: + 55-62-821-1029.
The principal function of the stratum corneum (SC) is to control transepidermal water loss as well as the diffusion of other substances through the skin. Thin-section transmission microscopy of the SC has shown that the intercellular spaces
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between the corneocytes are partly filled with broad multilamellar sheets (Swartzendruber et al., 1989; Wertz et al., 1989), which are important in regulating the barrier function of the skin (Elias, 1983). Corneocytes are anucleate cells composed of filamentous keratin network containing an insoluble, highly cross-linked peripheral envelope (Elias, 1988). Wertz and Downing (1987) have determined that pig SC contains 2.1% (dry weight) of bound hydroxyceramide, which is just sufficient to provide a monolayer to cover the surface of each corneocyte (Wertz et al., 1989). About half of the hydroxyceramide molecules are bound to the corneocyte envelope by esterification of the vhydroxyl function, and half via one of the hydroxyl groups of sphingosine (Wertz and Downing, 1987). The fraction of chemically unbound lipids in the pig SC that can be removed by exhaustive extraction with chloroform/ methanol mixtures corresponds to about 14% of the tissue dry weight (Swartzendruber et al., 1987) and consists mainly of ceramides, free fatty acids and cholesterol (Gray et al., 1982; Long et al., 1985). White et al. (1988) showed that the lipid structure of the SC of hairless mice is lamellar with a repeat period of 13.1 nm and that the isolated lipids do not produce a single lamellar diffraction pattern. The lipid structure of the SC has since been a subject of intense investigations, using mainly small and wide-angle X-ray (Bouwstra et al., 1992, 1994, 1995; Parrot and Turner, 1993) and infrared spectroscopy (Krill et al., 1992; Ongpipattanakul et al., 1994). The aim of these studies was essentially to understand how these lipids, which are unusual in many respects, exert the selective permeability of the skin. Measurements have been made in human, pig and mouse intact SC and also in many dispersions of isolated lipids in water or buffer solutions. These dispersions have been used as models of the intercellular lamellae found in the SC. However, a consensus about the existent phases or about the precise origin of phase transition temperatures is still missing. Furthermore, a possible lipid exchange between the corneocyte envelope and the intercellular bilayers domains
was not investigated yet. Parrot and Turner (1993) reported that mixtures of cholesterol and ceramides can form a structure of long periodicity (13.5 nm), while more recently McIntosh et al. (1996) have shown that mixtures of specific skin ceramides and appropriate concentrations of cholesterol and fatty acid (similar to those found in SC), can produce a diffraction pattern with a single repeat period of 13.0 nm. It is worth mentioning the recent study (Lopez et al., 1997) of the composition of pig SC showing again the absence of phospholipids and the presence of ceramides and cholesterol as the dominant lipid species (35 and 26.5%, respectively). In previous work (Alonso et al., 1995, 1996), electron paramagnetic resonance (EPR) and fatty acid spin probes were used to evaluate membrane fluidity in intact neo-natal rat SC, and observed that many EPR spectra were able to distinguish two populations of spin labels with very different mobility states. The more fluid lipid domain could be associated with the extracellular bilayers, which can be extracted by organic solvents, while the more rigid one could be associated with the corneocyte envelope, which is associated with the protein, giving a more restricted motion. This suggests that an appreciable part of the fatty acid spin probes could be structured in the corneocyte membrane. Since fatty acids, together with ceramides and cholesterol, are required for permeability barrier homeostasis (Landmann, 1988; Elias and Menon, 1991; Mao-Qiang et al., 1993a,b), it is important to consider the role of both extracellular and envelope membranes, and also their interactions, in the barrier function of the SC. In this work EPR and the spin label method have been used to compare the thermal behavior of lipid fluidity, at pH 7.4, in three types of SC lipid samples: intact SC, lipid-depleted SC (containing only covalently bound lipids) and dispersions of isolated lipids. Fluidity was evaluated at three depths of the membrane alkyl chains in order to monitor lipid–protein interactions and the changes in fluidity following thermally induced phase transitions.
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2. Materials and methods
2.1. SC membranes As in previous reports (Alonso et al., 1995, 1996) SC was obtained from newborn Wistar rats aged less than 24 h. After sacrifice, the skin was excised and fat was removed by rubbing in distilled water. The skin was allowed to stand for 5 min in a desiccator containing 0.5 l of anhydrous ammonium hydroxide and was then allowed to float in distilled water with the epidermis side in contact with the water. After 2 h, the SC was removed to filter paper and then transferred to a Teflon-coated screen, extensively washed with distilled water, and allowed to dry at ambient conditions. This procedure reduced the effect of the remaining epidermal tissues to a minimum. The membranes were stocked in a desiccator containing silica gel under a moderate vacuum until use.
2.2. Extraction of SC lipids Lipids were extracted from rat SC according to the method used by Wertz and Downing (1987) for porcine SC. The sheets of SC ( 80 mg) were incubated for 2 h intervals in each of three successive extraction mixtures (80 ml) of chloroform/ methanol (2:1, 1:1 and 1:2, v/v) and recovered each time by filtration through a 0.25 mm teflon millipore filter (Millipore, Bedford, MA). This extraction procedure was repeated changing the solvents at 1 h intervals followed by an overnight extraction in methanol. All solvent fractions were combined and evaporated under a stream of nitrogen gas. The isolated lipids and the lipid-depleted SC were kept in a desiccator under vacuum until used.
2.3. Preparation of extracted lipid samples Lipid dispersions were prepared at a concentration of 20 mg/ml in phosphate-buffered saline (PBS, 5 mM, pH 7.4) using a procedure similar to that described by Ongpipattanakul et al. (1994). A weighed mass of extracted lipid (40 mg) was placed in a glass tube and 2 ml of buffer was added. The mixture was repeatedly heated to
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70°C under sonication with several cycles of cooling to room temperature between the sonications. This procedure was repeated 3–4 times (20 min) until a homogeneous suspension was obtained.
2.4. Spin-labeled SC samples Spin-labeled derivatives of stearic acids, 5-, 12and 16-doxylstearic acids (5-DSA, 12-DSA and 16-DSA, respectively), were purchased from Aldrich (Milwaukee, WI). These spin labels have a nitroxide radical ring attached at C-5, C-12 and C-16 positions of the acyl chain, respectively. A small aliquot of stock spin label dissolved in ethanol (5 mg/ml) was placed in a glass tube and the solvent was evaporated under a stream of nitrogen gas. Samples containing about 10 mg of intact SC or lipid-depleted SC, cut and ground into very small pieces and suspended in PBS, or an extracted lipid dispersion (in PBS), were added to the spin label thin film at lipid/spin label molar ratios greater than 150 in order to avoid magnetic interactions among the spin labels (Fung and Johnson, 1984). Since the lipid concentrations of extracted unbound lipids and chemically bound lipids for porcine SC were reported by Wertz and Downing (1987) to account for 14.7 and 2.1% of the SC dry weight, respectively, a spin label concentration 7-fold lower for lipid-depleted SC was used compared to intact SC or extracted lipid dispersions. The concentration of spin labels was estimated to be 20 mM for delipidized SC and 140 mM for both intact SC and SC lipid suspensions. After labeling under gentle stirring, the samples were introduced into a capillary tube for EPR measurements. Based on gravimetric measurements, the hydration of tissue samples in the capillary was estimated to be 5897% (w/w). Under these conditions, the SC was considered to be in a fully hydrated state (excess of water).
2.5. EPR measurement conditions A Varian E-9 spectrometer (Varian Associates, Palo Alto, CA) equipped with the rectangular cavity and operating at X-band (9.12 GHz) was utilized in the investigations. The spectral parameters were as follows: microwave power, 20 mW;
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modulation frequency, 100 KHz; modulation amplitude, 2.5 G; magnetic field scan, 100 G; sweep time, 2 min and detector time constant, 0.064 s. The spectra were digitalized by an interfaced computer system (1070 points/spectrum). Temperature was controlled within 0.3°C by a nitrogen steam system from Air Products and Chemicals (Allentown, PA).
2.6. EPR spectra analysis The fluidity of the membrane can be estimated from the 2T parameter as described by Wassal and Stillwell (1990). In the rapid motion regime, as is the case of 16-DSA, the resolution of the outer features of 2T%// becomes very poor. In this case, the calculation of the rotational correlation time, tc, is more appropriate. This empirical parameter describes the rate of motion of the probe as it rotates about its long axis. The EPR parameter tc is also calculated using spectral parameters measured directly in the EPR spectra (Wassal and Stillwell, 1990). The activation energy Ea was also calculated for 16-DSA using the Arrhenius equation: log tc =Ea/2.3RT
cell membrane lipid bilayers and properly distributed throughout the tissues. The proper distribution of spin label was assured by the capacity of samples to get spin labels structured in their lipid bilayers, without allowing magnetic interactions among them (dipole–dipole and exchange interactions). Such magnetic interactions, which cause the broadening of resonance lines and reduce spectral resolution, are largely avoided since the preparation contains 150 or more membrane lipids for each spin label (Fung and Johnson, 1984). The three types of samples analyzed in this work showed EPR spectra with two spectral components corresponding to different states of probe mobility. Both of these components remained resolved for any temperature interval over the whole range (0–90°C) investigated. The low-field spectral features of these two components are indicated by arrows in Fig. 1, for cases in which the coexistence of the two spectral components is more clearly evident. Two-component spectra indicate the presence of at least two lipid domains
(1)
where R is the gas constant and T the absolute temperature. In practice, the numerical value of Ea was determined from the slope of a plot of log tc versus 1/T (Arrhenius plot).
3. Results
3.1. EPR spectra for SC samples The EPR spectra at 38°C for 5-, 12- and 16DSA, incorporated into delipidized SC, intact SC and dispersions of extracted SC lipids are shown in Fig. 1. Due to the large hydrophobicity of these fatty acid spin probes, and to the conditions of the preparations, they were able to penetrate the SC tissue and intercalate with the membrane lipids. The EPR spectra are similar to typical spectra observed in plasma membranes (Griffith and Jost, 1976; Alonso et al., 1995, 1996). The spin labels were adequately incorporated into the
Fig. 1. Electron paramagnetic resonance (EPR) spectra at 38°C of spin labels 5-doxylstearic acid (5-DSA) (a, d, g), 12-doxylstearic acid (12-DSA) (b, e, h) and 16-doxylstearic acid (16-DSA) (c, f, i) in lipid-depleted stratum corneum (SC) (a, b, c), intact SC (d, e, f) and extracted lipid dispersion (g, h, i), suspended in phosphate-buffered saline (PBS) pH 7.4. The arrows mark the field positions where two spectral components (spin labels with different mobility states) are more clearly visible.
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Fig. 2. Electron paramagnetic resonance (EPR) spectra of spin labels 5-doxylstearic acid (5-DSA) (a, b, c), 12-doxylstearic acid (12-DSA) (d, e, f) and 16-doxylstearic acid (16-DSA) (g, h, i) in lipid-depleted stratum corneum (SC) suspended in phosphate-buffered saline (PBS) pH 7.4, at indicated temperatures. The arrows mark the field positions of the two spectral components.
(populations) with different fluidity or molecular order (Griffith and Jost, 1976). In the case of lipid dispersions this can arise from lateral lipid phase separation (Schorn and Marsh, 1996); in intact SC there are at least two lipid domains (envelope and extracellular bilayers) and in the case of lipid depleted samples this can arise from lipid – protein interactions (Griffith and Jost, 1976; Marsh, 1989, 1996) where the lipid chains are immobilized by contact with the hydrophobic surfaces of proteins, the two components corresponding to the ‘boundary’ and ‘bulk’ lipids. In Fig. 2 the EPR spectra for the delipidized samples at temperatures where there is a clear resolution of these two components are presented. For 5-DSA and 12-DSA this occurs around 78°C, as judged by a significant increase of the second low-field resonance line and for 16-DSA this appears to occur around 30 – 42°C. In the spectra from Fig. 1 there is just one apparent component for 5- and 12-DSA and two components for 16-
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DSA. In the time scale of the EPR experiments near the rigid limit, the spectra do not distinguish the two components and the lipid–protein interactions are not clearly visible. This also happens in the limit of fast motions when the spectra have an isotropic appearance even when the interaction with protein is present. This can be observed in the spectra of 16-DSA in lipid-depleted SC presented in Fig. 3. While at 22°C two components are resolved, only one component is observed at 62°C; the spectrum appears isotropic as is the case for the dispersion of extracted lipid where the proteins are absent. Quantification of the fractions between the boundary and bulk lipids has been made by spectral subtraction (Brotherus et al., 1980) and spectral simulation (Ge and Freed, 1999). In both cases these fractions were temperature independent, but the question of how the lipid structure is affected and modulated by membrane proteins is not yet clear (Ge and Freed, 1999).
Fig. 3. Electron paramagnetic resonance (EPR) spectra of 16-doxylstearic acid (16-DSA) in lipid-depleted stratum corneum (SC) (a, b) and in dispersion of SC extracted lipids (c, d) suspended in phosphate-buffered saline (PBS) pH 7.4, at indicated temperatures. The arrows mark the field positions of the two spectral components.
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Fig. 4. Electron paramagnetic resonance (EPR) parameter 2T%// as a function of temperature for spin labels 5-doxylstearic acid (5-DSA) (A), 12-doxylstearic acid (12-DSA) (B) and 16-doxylstearic acid (16-DSA) (C) in lipid-depleted stratum corneum (SC) (squares), intact SC (circles) and SC lipid dispersion (triangles). Mean 9 S.D. (n =3).
3.2. Molecular motion along the fatty acid chain as a function of temperature in the SC samples Fig. 4 shows the dependence of the parameter 2T%// as a function of temperature for 5-, 12- and 16-DSA incorporated into intact SC, delipidized SC and dispersion of extracted SC lipids. Calculation of the order parameter S is limited to spectra in which simultaneous resolutions of T%// and T%Þ occur. As most of the EPR spectra have two components, the T%Þ and T%// parameters can not be accurately measured for both components. For this reason we use only the T%// parameter, which
was sensitive to thermally induced changes in fluidity. The greater the fluidity, the lower the T%//. It is quite important to have in mind that the parameter T%// reflects the maximum experimentally observed nitrogen hyperfine splitting in the EPR spectrum. Thus, the parameter T%// is a static parameter associated with the orientational distribution of spin labels in the membrane, although it is related to the changes in spin labels mobility. T%// reflects the average angle of the axis orientation of the spin label relative to the bilayer normal (Griffith and Jost, 1976; Marsh, 1989). The three spin probes indicated that the membrane of the
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corneocyte lipid envelope is the most rigid and that fluidity is higher for the extracted lipid dispersion than for intact SC. Another parameter, the rotational correlation time, tc, is directly associated with the motional reorientation of the spin label and consequently with the probe mobility in the membrane. The tc values for 16-DSA using the three samples are presented in Fig. 5 as an Arrhenius plot in order to analyze the phase transitions. This probe, located deeper within the membrane, also detected a more rigid membrane for the corneocyte envelope, an intermediate fluidity for intact SC and a higher fluidity for the isolated lipids (see Fig., 1(c), (f) and (i)).
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Table 1 Activation energy for the rotational diffusion of 16-doxylstearic acid (16-DSA) DSA in stratum corneum (SC) membranes: corneocyte envelope (lipid-depleted SC), intact SC and extracted lipid dispersionsa Sample
Temperature range (°C)
DE (Kcal/mol)
Lipid-depleted SC
46–54
9.5 90.3Ab
54–90 42–54 54–90 18–54
5.0 90.9B 5.3 90.5B 9.4 90.7A 7.3 9 0.5C
54–90
9.1 9 0.6A
Intact SC Extracted SC lipids
4.4 9 0.4c 8.3 90.7
The means 9 S.D. were calculated from three independent experiments presented as an average in Fig. 5. b Statistical significance: only the means indicated with different capital letters are statistically different with PB0.01 (Student’s t-test). c Data previously reported for fully hydrated SC (Alonso et al., 1995). a
3.3. Lipid phase transitions assessed by fatty acid chain mobility The curves in Fig. 5 show discrete differences in slope coefficients suggesting the existence of membrane phase transitions. The slope coefficients for the Arrhenius plot give the activation energy for
Fig. 5. Arrhenius plot of the rotational correlation time, tc, of 16-doxylstearic acid (16-DSA) structured in lipid-depleted stratum corneum (SC) (squares), intact SC (circles) and SC lipid dispersion (triangles). Regression analysis was performed considering the following temperature intervals: 46–54 and 54– 90°C for lipid-depleted SC; 42–54 and 54–90°C for intact SC and 18 – 54 and 54 – 90°C for the SC lipid dispersion. Mean9 S.D. (n = 3).
the reorientation of the 16-DSA in the membrane. The calculated activation energies are presented in Table 1. The membrane of the corneocyte envelope shows an apparent phase transition at around 54°C, with its activation energy changing from 9 to 5 Kcal/mol. The activation energies for intact SC and extracted SC lipids were practically the same, changing from 5 to 7 Kcal/mol below 54°C, to 9 Kcal/mol above 54°C. Data obtained previously for intact SC (Alonso et al., 1995) are also included in Table 1. They are, in general, in good agreement with the values obtained in the present work. Despite the fact of the anisotropic motion of 16-DSA in SC (Alonso et al., 1995), it is believed that the present results show an apparent phase transition around 54°C, which is similar to all samples, and the difference in mobility as reflected in tc in the different samples. It is worth mentioning the fact that all plots presented in this work were reversible.
3.4. Temperature dependence of the rotational gradient along the fatty acid chain At high temperatures, the rate of motion is higher (tc B 3×10 − 9 s) and the EPR spectra
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allowed the measurement of the rotational correlation times also for 5- and 12-DSA, in the case of the extracted SC lipids and intact SC. These data for the three spin probes are presented in Fig. 6. For lipid-depleted SC the tc could not be determined accurately for these two probes since two spectral components remain in the EPR spectra even above 70°C; the motion of the probes in this sample is also significantly more restricted as
compared to the other two samples. As the nitroxide moves away from the polar region and approaches the hydrophobic core of the membrane, the rotational motion became less restricted (correlation time shorter). It is possible to note that the rotational gradient is more pronounced for intact SC than the lipid dispersion samples, especially in the region between the C-5 and C-12 atoms of the acyl chains, where the greater differences in tc are observed.
4. Discussion
Fig. 6. Arrhenius plots of the rotational correlation time, tc, of 5-doxylstearic acid (5-DSA) (open square), 12-doxylstearic acid (12-DSA) (open circle) and 16-doxylstearic acid (16-DSA) (triangles) structured in stratum corneum (SC) lipid dispersion (A) and intact SC (B). Mean 9S.D. (n= 3).
This work shows that it is possible to incorporate EPR spin probes directly into SC membranes in intact tissue. Even after exhaustive lipid extraction with a series of chloroform/methanol mixtures, it was possible to intercalate the spin labels with the remaining lipid-depleted SC sample, and to obtain an adequate radical distribution for EPR measurements. In the present work, the fluidity of the SC lipids as a whole was compared with the fluidity of two classes of SC lipids: chemically bound and unbound lipids. Using two EPR parameters (2T%// and tc) and three spin labeled positional isomers, it was shown that lipids covalently bound in SC are more rigid than the lipids in intact SC over the range of temperatures measured. The reverse was observed for the dispersion of extracted lipids that showed a higher fluidity compared to intact SC. This observation is similar to that for several other types of biological membranes in which the extracted lipids are more fluid than the original membrane in the presence of membrane proteins (Griffith and Jost, 1976; Marsh, 1989). A comparison of the results for the temperature dependence of the rotational correlation time for 16-DSA (Fig. 5) with the data from Fourier transform infrared spectroscopy (Golden et al., 1987; Krill et al., 1992; Ongpipattanakul et al., 1994) is quite interesting. While measuring the temperature dependence of the symmetric CH2 stretching frequency for porcine SC hydrated at 75% relative humidity, Ongpipattanakul et al. (1994) observed a general increase in frequency from − 10 up to 100°C, with a particularly sharp increase in rate
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between 60 and 80°C (high slopes) and a low rate of increase between 20 and 60°C (small slopes). Similar results were reported by Krill et al. (1992) for the hairless mouse, except that the temperature range for the smaller slope was observed between 31 and 55°C. Golden et al. (1987) measured the antisymmetric CH2 stretching frequency for porcine SC hydrated at 75% relative humidity, and observed a gradual increase in frequency over the temperature range from 30 to 115°C, with a dramatic increase in rates between 60 and 80°C. The data for the rotational correlation time in neo-natal rat SC (Fig. 5) are in agreement with the above findings. The nitroxide rotation of the 16-DSA probe in intact SC and lipid dispersion was very sensitive to temperature having a higher increase in rates in the temperature range from 54 to 90°C. In the case of lipid depleted samples, the opposite behavior was observed, the increases in rates being lower for temperatures above 54°C. The data depicted in Fig. 5 showed lower tc values for isolated lipid samples than for intact SC samples over all temperature ranges. However, the activation energies for the two samples were essentially the same (Table 1). The state of probe mobility can change through alterations either in the amplitude or frequency of the motion. Hence, since the energy barrier is maintained, the rotational motion of the spin label in intact SC may be lower than in the lipid dispersion due to the smaller motional amplitude in this case. Thus, it is believed that in SC tissue there are some spatial constraints on the rotational motion of 16-DSA that reduce its motional amplitude as compared to its mobility state in extracted lipid dispersions. At temperatures above the phase transition (54°C), lipid-depleted samples showed higher tc values accompanied by a large decrease in activation energy. This fact could be explained by a less densely packed hydrocarbon domain (smaller Ea) together with a lower motion amplitude due to the presence of proteins (greater tc). The presence of two lipid structures (the corneocyte and intercellular bilayers) in the SC, and the complexity of their lipid composition are consistent with the coexistence of several subcell structures. Using X-ray diffraction, White et al.
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(1988) studied the lipid structure of hairless mice SC at 25, 45 and 70°C. The wide-angle patterns at 25°C indicated the presence of a lipid fraction in the liquid state and an additional structure organized as an orthorhombic subcell, while at 45°C, a lipid fraction in the liquid state in equilibrium with lipids in hexagonal subcells was found. At 70°C, only a broad band was observed indicating that all the lipids were in the liquid state, although small-angle X-ray scattering for extracted lipids indicated the hexagonal phase with all alkyl chains in the liquid state. More recently, other studies using the X-ray technique have been performed in human, mouse and pig SC. For human SC, Bouwstra et al. (1992) found a phase transition from an orthorhombic to a hexagonal phase at 40°C. In mouse SC, the orthorhombic subcell disappears at 45°C and at this temperature only hexagonal and liquid lateral packing was observed (Bouwstra et al., 1994). Finally, in pig SC only hexagonal and liquid lateral packing was detected with disappearance of the hexagonal packing in the temperature range of 60–66°C (Bouwstra et al., 1995). Rehfeld et al. (1988), using the EPR technique and the perdeuterated di-tert-butyl nitroxide to study scale from human SC, have identified thermal phase transitions at 34–37 and 50°C which correspond to the phase transitions observed by differential scanning calorimetry. The temperature dependence of the rotational gradient along the fatty acid chain shown in Fig. 6 can provide additional information about the lipid chain dynamics. The rotational gradient in the bilayer region between the 5th and 12th carbon atom is smaller for isolated lipid (Fig. 6A) than for intact SC (Fig. 6B). For the delipidized samples the rotational motion is very restricted, even at high temperatures, making the measurement of tc for 5- and 12-DSA very unreliable. It is well known that in both biological and artificial model membranes, the permeability increases with the increase in fluidity (Fettyplace and Haydon, 1980), suggesting a correlation between membrane transport and lipid fluidity. Golden et al. (1987) measured the activation energy for water flux in porcine SC and found a value of 17 Kcal/mol, suggesting that the water flux in SC is limited by diffusion through the ordered hydrocarbon do-
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mains. So, our data further suggest that the nonlipid depleted corneocyte envelope (in native SC) may have a major role in the physical barrier in SC. In conclusion, the lipids of the corneocyte membrane are assembled in a quite rigid structure, and this low mobility state is caused mainly by their covalent bonding to the envelope proteins and to the strong acyl chain interactions with the hydrophobic surfaces of the proteins. Intact SC and dispersions of extracted SC lipids were considerably more fluid than the envelope membrane, suggesting that the major contribution for the barrier function of SC is provided by the envelope. The reorientational motion of 16-DSA was also able to monitor apparent phase transitions for the intact and extracted SC lipid samples at 54°C. The lipid chain packing detected by the energy barrier for reorientational motion of 16DSA was similar for intact and extracted lipid samples, but since tc values are smaller for extracted lipids, the amplitude of motion is greater for these samples. In addition, it was found that the spin label EPR approach can provide precise evaluations of molecular dynamics and ordering of SC lipids, which also may be used to investigate drug–lipid interaction directly in native SC, as well as different cases of skin diseases and lesions. Acknowledgements This work was supported by Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq; grant process 300908/92-0) and Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo (FAPESP; processes: 97/02431-4 and 95/6177-0). We wish to thank Professor Dr Fernando Pelegrini (Instituto de Fı´sica, Universidade Federal de Goia´s) for his interest in this work. References Alonso, A., Meirelles, N.C., Tabak, M., 1995. Effect of hydration upon the fluidity of intercellular membranes of stratum corneum: an EPR study. Biochim. Biophys. Acta 1237, 6 – 15.
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