MATERIALS AND METHODS

Chemicals

All chemicals used were of the highest grade available. - and -tocopherol standards were a kind gift from Henkel (La Grange, IL).

Humans

Permission was granted from the UC Berkeley Committee for the Protection of Human Subjects to obtain human SC using a tape stripping technique before and after UV irradiation; all subjects gave their informed, written consent. Ten healthy volunteers (skin types II and III; mean age 26 3 y) were questioned to ensure that they did not take any medication and had no history of photosensitivity or other current medical problems.

Animals

The animal care, handling, and experimental procedures were carried out as described in the animal use protocol approved by Animal Care and Use Committee of the University of California, Berkeley. Hairless mice (SKH-1, females, 7 wk old, Charles River Laboratories, Wilmington, MA) were kept under standard light and temperature conditions. Food (Harlan Teklad Rodent Diet #1846, WI) and water were provided ad libitum.

Radiation source

Ultraviolet light was generated by an Oriel 1000 W solar UV simulator equipped with a 1000 W ozone free xenon lamp (Oriel, Stratford, CT). The energy output of the solar simulator was measured at the level of the skin surface (beam size of 152 152 mm) using an IL 443 radiometer (International Light, Newburyport, MA) for the UVB portion and a UVX-36 radiometer (UVX-Radiometers, UVP, San Gabriel, CA) for the UVA portion of the solar simulated UV spectrum. The output was 1.85 mW per cm², as measured with the IL 443 radiometer, and 5.16 mW per cm², as measured with the UVX-36-radiometer. For comparison, the irradiances of sunlight measured on a sunny day (July 15 at noon) on the campus of the University of California at Berkeley (latitude 38° N) were 0.5 mW per cm² (IL 443 radiometer) and 2.2 mW per cm² (UVX-36 radiometer), respectively. The solar UV simulator set-up included an in-built UVB/UVA dichroic mirror that allows wavelengths from 280 to 400 nm to pass and greatly reduces the VIS and IR output of the lamp. Furthermore, a UVC blocking filter (Oriel) was used to cut off radiation at wavelengths below 280 nm. The resulting radiation spectrum is called "solar simulated ultraviolet radiation" (SSUV), and is displayed in Figure 1. To obtain a UVA spectrum, the UVC blocking filter was replaced by a UVB/C blocking filter (Oriel), and the 280–400 nm dichroic mirror was kept in place. To obtain the UVB spectrum, the 280–400 nm dichroic mirror was replaced by a 260–320 nm dichroic mirror (Oriel), and the UVC blocking filter was kept in place.

Figure 1.

Emission spectrum of the solar ultraviolet simulator. The solar UV simulator set-up included an in-built UVB/UVA dichroic mirror that allowed UVR from 280 to 400 nm to pass but greatly reduced the VIS and IR output of the lamp, and a UVC blocking filter (Oriel) to cut off radiation at wavelengths below 280 nm. The resulting radiation spectrum was called SSUV.

Full figure and legend (13K)

Human UVR exposure

To estimate the UV sensitivity of each subject (n = 6), nontanned forearm skin was exposed to increasing doses of SSUV. After 24 h, the MED was determined individually by reading of erythema responses. The solar simulator generated 1 MED in 4–6 min, depending on the subject. An area of 5 5 cm of the left upper arm was irradiated with 0.75 MED, whereas the contralateral site on the right upper arm served as control. The total administered doses for 0.75 MED ranged from 1.26 to 1.89 J per cm² (SSUV, sum of UVB and UVA irradiances). The total irradiance was measured by the IL 443 and UVX-36 radiometers and may be an overestimation, due to overlapping sensitivities.

Murine UVR exposure

Prior to the UV exposure or sham irradiation (controls), mice were anesthetized by an intraperitoneal injection of sodium pentobarbital (50 mg per kg body weight, Nembutal, Abott Laboratories, North Chicago, IL). The murine MED, as determined on the back skin of three hairless mice, was obtained in a 3 min exposure to SSUV, equivalent to a total irradiance of 1.26 J per cm². As the mice were all of the same age and skin type, this MED value was used for all mice and was not determined individually.

To evaluate a concentration dependency for SSUV treatment, animals were exposed to 0, 0.75, 1.5, and 9 MED SSUV [each level, n = 3 mice; two groups of mice were used, one group for vitamin E and one group for malondialdehyde (MDA) measurements].

In a separate experiment, mice were exposed to a single dose of 2 MED SSUV (6 min exposure, SSUV, 2.52 J per cm², n = 4 mice). To differentiate between the UVB and UVA effects, a second and third group of mice, respectively, were irradiated for the same time as for the 2 MED SSUV exposure (6 min); however, different filters were used to divide the SSUV spectrum into its UVB portion and its UVA portion. For UVB the 280–400 nm dichroic mirror was replaced by a 260–320 nm dichroic mirror, and the UVC filter kept in place, irradiation was for the same time (6 min, irradiance 0.67 J per cm², n = 4); for UVA the UVC filter was replaced by a UVB/C blocking filter, and the 280–400 nm dichroic mirror was kept in place, irradiation was for the same time (6 min, irradiance 1.86 J per cm², n = 4) (see Figure 3). Control mice (n = 12) were exposed to 0 MED (sham irradiation) but treated identically in terms of anesthesia and housing conditions.

Figure 3.

Depletion of murine SC -tocopherol by UVA and UVB. Four groups of mice were used for evaluation of SC tocopherol susceptibility to the UVA and the UVB portion of the SSUV. "Control" mice were sham irradiated (0 J per cm²; n = 12);"SSUV," use of UVC blocking filter and a dichroic mirror that allows wavelengths from 280 to 400 nm to pass, and irradiation with 2 MED, (equivalent to a 6 min exposure to SSUV, irradiance 2.52 J per cm², n = 4);"UVB," the 280–400 nm dichroic mirror was replaced by a 260–320 nm dichroic mirror, and the UVC filter was kept in place, irradiation was for the same time (6 min, irradiance 0.67 J per cm², n = 4);"UVA," the UVC filter was replaced by a UVB/C blocking filter, and the 280–400 nm dichroic mirror was kept in place, irradiation was for the same time (6 min, irradiance 1.86 J per cm², n = 4). ***p < 0.001, *p < 0.05 as compared with control levels.

Full figure and legend (10K)

Tape stripping of human SC

The skin was not prepared in any way before the irradiation or the tape stripping; each subject was told not to use any skin preparations (moisturizers, etc.) on the day of the experiments. Tape stripping was performed 10 min after the irradiation treatment. All human tape strippings were performed on equivalent locations of the upper arm. Samples of human SC were obtained by tape stripping the skin with 5 cm 5 cm pieces of an adhesive tape (Highland 3710, 3M, St. Paul, MN) using a standardized protocol. Tapes strips (5 5 cm) were smoothly adhered onto the skin, flattened equally three times, and removed gently using moderate and even traction. The resultant SC layers adherent to the tapes appeared by light microscopy to be of uniform thickness. Due to the possibility of surface contamination and to remove surface lipids, the first (uppermost) tape stripping was discarded; subsequent tape strippings were used for analyses. The tape strippings were numbered (1–20), and each pair of consecutive tapes (first and second, third and fourth, etc.) was pooled for vitamin E analysis. The amount of SC was determined by the difference in weight before and after application and immediate removal from the skin. In accordance with an earlier report (^{Bommannan et al. 1990}), the weight of SC removed per tape (33 7 g per cm²; n = 10) was independent of tape strip number, and the resultant cumulative SC weight for 20 tape strippings was 660 140 g per cm² (n = 10). Based on the SC weights and the size of the tape, the thickness of SC removed by 20 tape strips was calculated to be 6.6 0.7 m (n = 10), assuming a density of 1 g per cm³ and a uniform coverage of SC on the tape strip as described (^{Kalia et al. 1996}). No significant differences were observed concerning the cumulative weights of the removed SC between the UV treated and the control site (672 91 g per cm² and 660 80 g per cm², respectively; both n = 6).

Tape stripping of murine SC

Tape stripping was performed 10 min after the irradiation treatment. Samples of murine SC were also obtained by tape stripping the skin with 2.5 cm 5 cm pieces of adhesive tape (Supreme Brand, Sekisui TA, Garden Grove, CA). This tape was found most suitable for tape stripping murine skin because its weaker adhesion allowed more homogenous tape stripping of the considerably thinner murine SC. In addition, as opposed to most other adhesive tapes tested, it did not interfere with the electrochemical high performance liquid chromotography (HPLC) detection of vitamin E. The first two tape strippings were discarded, and tape strippings 3–8 were collected and pooled for determination of the vitamin E concentration. Therefore, due to the thin murine SC, only one SC - and one -tocopherol concentration was measured per animal. For estimation of lipid peroxidation, MDA was analyzed in SC adherent to Scotch Superstrength Mailing Tape (3M; 2.5 cm 5 cm) as described previously (^{Thiele et al. 1997c}).

HPLC analyses

Lipophilic antioxidants were extracted from the tape strips and analyzed by HPLC as described (^{Thiele et al. 1997c}). Briefly, after weighing, tape strips were transferred to a 50 ml centrifuge tube containing 2 ml phosphate buffered saline with 1 mM ethylenediamine tetraacetic acid, 50 l butylated hydroxy toluene (1 mg per ml), 1 ml 0.1 M sodium dodecyl sulfate, and 4 ml ethanol, mixed vigorously, and extracted with 4 ml hexane. Ethanol (2 ml) was added to the hexane to precipitate the glue (from the tape adhesive), which was then removed and discarded. The hexane was taken to dryness under nitrogen; the residue was resuspended in 500 l of ethanol:methanol (1:1). The sample was then injected into the HPLC system (Shimadzu, Kyoto, Japan), consisting of a SCL-10 A system controller, a LC-10AD pump, and a SIL-10 A autoinjector with sample cooler, an Ultrasphere ODS C-18, 5 m particle size column (Beckman, Fullerton, CA) with an a LC-4B amperometric electrochemical detector with a glassy carbon electrode (Bioanalytical Systems, West Lafayette, IN). The mobile phase was methanol:ethanol 1:9 (vol/vol) with 20 mM lithium perchlorate, at a flow rate of 1.2 ml per min. For measurement of ubiquinol-10, - and -tocopherols, the electrochemical detector was operated with a 0.5 V potential and the full recorder scale at 50 nA.

MDA was detected in SC by reacting chloroform extracts from tape strippings with thiobarbituric acid (TBA), separating the TBA reactive substances by HPLC, and quantitating the MDA-TBA adduct by fluorimetric detection as described previously (^{Thiele et al. 1997c}).

Statistical analyses

Statistical analyses were carried out using SuperAnova for the Macintosh (Berkeley, CA). Logarithmic transformation of the mouse data was carried out to normalize variances between treatment groups. The following designs were used: (i) 1-factor ANOVA for comparisons between UV irradiated and control mice, (ii) 1-factor ANOVA with repeated measures designed to analyze the comparisons between successive layers of human SC, (iii) 2-factor ANOVA with repeated measures designed to compare the antioxidant contents in the various layers of human SC, exposed or not exposed to UV irradiation. Least square means comparisons were used to evaluate differences between treatments; p < 0.05 was considered statistically significant. All data shown represent means SD.

RESULTS

UV dose-dependent depletion of tocopherols and increase in MDA formation in murine SC

Baseline concentrations of murine SC -tocopherol were 9.3 1.9 pmol per mg wet weight and of -tocopherol were 3.6 1.4 pmol per mg, which are in accordance with our earlier report (^{Thiele et al. 1997c}). Exposure to a single dose of solar simulated ultraviolet light dose dependently depleted the SC concentrations of -tocopherol, and, less markedly, of -tocopherol. A suberythemogenic dose of 0.75 MED significantly depleted -tocopherol from 9.3 1.9 to 1.4 0.3 (p < 0.0001;Figure 2a), whereas 9 MED were required to significantly deplete -tocopherol from 3.6 1.4 to 1.2 0.2 pmol per mg (p < 0.001;Figure 2b).

Figure 2.

Dose-dependent depletion of murine SC tocopherols and MDA formation by SSUV. After SSUV treatment, SC was obtained by tape stripping and - and -tocopherol, MDA concentrations were analyzed immediately by HPLC as described in Material and Methods. Remarkably, -tocopherol was strongly depleted by 0.75 MED (A), whereas the depletion of -tocopherol (B) and MDA formation (C) reached a significant level only at a very high dose (9 MED) (each UV level, n = 3 mice; two groups of mice were used, one group for vitamin E and one group for MDA measurements). ***p < 0.001, **p < 0.01, *p < 0.05 as compared with control levels (0 MED).

Full figure and legend (19K)

MDA levels in sham irradiated control mice were 4.8 0.5 pmol per mg of SC tissue. The MDA levels after SSUV exposures to 0.75 MED and 1.5 MED were 5.0 0.7 pmol per mg and 5.8 0.8 pmol per mg, respectively. Only after exposure to 9 MED was a significant increase of MDA observed (7.1 0.2; p < 0.01;Figure 2c).

Both UVA and UVB deplete -tocopherol in murine SC

To evaluate whether the destruction of tocopherols in the SC could be attributed to UVA or UVB irradiation, mice were exposed to none, both, or either UVA or UVB light. UVA and UVB irradiation (2 MED) depleted both -tocopherol from 7.7 0.9 to 0.5 0.4 pmol per mg (p < 0.0001) and -tocopherol from 2.4 0.6 to 1.4 0.7 (p < 0.01); however, the effects of either UVA or UVB on the two tocopherols were different. Using filters such that only UVA light irradiated the mice, -tocopherol was depleted to 2.1 0.6 pmol per mg (p < 0.001), whereas UVB irradiation depleted -tocopherol to 1.5 1.0 pmol per mg (p < 0.001). Neither UVA nor UVB alone had an effect on murine SC -tocopherol Figure 3.

Vitamin E concentrations increase with depth of human SC

- and -tocopherol concentrations were determined in 10 sequentially tape stripped layers (two tape strippings combined for each successive pair of 20 strippings) obtained from each of 10 human subjects. In all subjects, the outermost SC layers contained the lowest vitamin E concentrations; the highest concentrations were found in the deepest layer Figure 4. Compared with the outermost SC (6.5 1.4 pmol per mg, tape strippings 1 and 2), the -tocopherol contents were significantly higher in tape strippings 7 and 8 (17 10 pmol per mg, p < 0.005) and in all subsequent layers (p < 0.0001) up to and including the deepest layer (76 12 pmol per mg tape, strippings 19 and 20, p < 0.0001). Thus, -tocopherol concentrations in the deepest layers of SC were 11.7-fold higher than those in the uppermost SC layer.

Figure 4.

Gradients of - and -tocopherol in human SC. Untreated SC of the upper arm of 10 volunteers was sequentially tape stripped and the - and -tocopherol contents determined in 10 layers (each two consecutive tape strips). Tape strippings 1 and 2 represent the surface of the SC, and 19 and 20 the deepest layer. Significant differences between concentrations in single layers and the uppermost layer ("1–2") are indicated by asterisks. **p < 0.01, ***p < 0.001.

Full figure and legend (24K)

-Tocopherol levels were also lowest in the outermost SC (2.2 1.3 pmol per mg, tape strippings 1 and 2), and were significantly higher in tape strippings 7 and 8 (3.7 1.3 pmol per mg, p < 0.03) and in subsequent layers (9 and 10, p < 0.006; 11 and 12, p < 0.01; 13 and 14, p < 0.0002; all others, p < 0.0001) up to and including the deepest layer (7.9 3.7 pmol per mg tape, strippings 19 and 20, p < 0.0001); however, the gradient of -tocopherol was not as steep as that observed for -tocopherol. -Tocopherol concentrations were only 3.6-fold lower in the outermost as compared with the deepest SC layers Figure 4.

Suberythemogenic UV irradiation depletes human SC tocopherols

In six of the 10 human subjects described above, the - and -tocopherol concentrations were measured in tape strippings of both nonexposed (right upper arm, control site) and UV exposed (left upper arm) skin. The nonexposed control SC Figure 5 exhibited similar results as described for - and -tocopherol in Figure 4. The -tocopherol concentrations ranged from 6.7 1.1 pmol per mg in tape strippings 1 and 2 to 77 14 pmol per mg in tape strippings 19 and 20, which reflects a 11.5-fold increase in -tocopherol in the deepest layer. -Tocopherol levels in tape strippings 1 and 2 were 2.8 1.0 pmol per mg and increased 3.4-fold to 8.2 3.0 pmol per mg in tape strippings 19 and 20.

Figure 5.

A single dose of 0.75 SSUV depletes - and -tocopherol in human SC. Upper arm SC of six volunteers was sequentially tape stripped after exposure to 0.75 SSUV (left arm, "0.75 MED"). The contralateral site served as control (right arm, "control"). Significant differences between corresponding SC layers in controls and treated sites are indicated by asterisks. *p < 0.05, **p < 0.01, ***p < 0.001.

Full figure and legend (39K)

Following exposure of the contralateral site to 0.75 MED solar simulated UV radiation, a significant depletion of SC tocopherols was observed. Although it appears that both the - and the -tocopherol concentrations were depleted in the UV exposed site compared with the corresponding layer of the control site Figure 5, these differences in -tocopherol concentrations only reached statistical significance in tape strippings 9 and 10 (p < 0.0007) and in all subjacent layers (each p < 0.0001); and for -tocopherol concentrations in tape strippings 7 and 8 (p < 0.04), 9 and 10 (p < 0.008), 11 and 12 (p < 0.03), 13 and 14 (p < 0.01), 15 and 16 (p < 0.02), 17 and 18 (p < 0.002), and 19 and 20 (p < 0.0002). The largest decrease in -tocopherol in response to UV irradiation was observed in the deepest SC layers (tape strippings 19 and 20), where the UV treated site contained 35 8 pmol per mg -tocopherol, a 55% decrease as compared with the control site (77 15 pmol per mg). Similarly, the -tocopherol concentrations in tape strippings 19 and 20 after UV irradiation were 5.5 2.1 pmol per mg, a 33% decrease as compared with the control site (8.2 3.0 pmol per mg).

The overall human SC concentration of vitamin E in the tape strips was 33 4 pmol per mg for -tocopherol and 4.8 0.8 pmol per mg for -tocopherol. UV light exposure (0.75 MED) depleted SC -tocopherol by 45% (to 18 6 pmol per mg; p < 0.003) and -tocopherol by 35% (to 3.1 1.0; p < 0.001).

DISCUSSION

This study demonstrates that inherent vitamin E in human SC is relatively depleted on the surface and increases with SC depth. Remarkably, a single suberythemogenic dose of solar simulated UV light (SSUV; 0.75 MED) depleted human SC -tocopherol by almost 50% Figure 5, and murine SC -tocopherol by 85% Figure 2. Previously, SSUV doses equivalent to 3 MED or more were necessary to detect a significant depletion of -tocopherol in whole epidermis and dermis (^{Shindo et al. 1993,1994b;Weber et al. 1997}).

The high susceptibility of SC vitamin E to SSUV may be, at least in part, due to a lack of coantioxidants in the SC (^{Packer 1994}). In vitro, ubiquinol-10 protects -tocopherol from photo-oxidation by recycling mechanisms (^{Stoyanovsky et al. 1995}). Previously, we have reported a lack of the lipophilic antioxidant ubiquinol-9 in the SC of hairless mice (^{Thiele et al. 1997c}), as compared with levels found in the whole epidermis and dermis of hairless mice (^{Shindo et al. 1993}). Similarly, no detectable amounts of ubiquinol-10, the human equivalent of this antioxidant, were found in human SC (detection limit: 0.1 pmol), whereas its concentration in full thickness human epidermis is similar to -tocopherol (^{Shindo et al. 1994a}). Although in SSUV irradiated murine skin homogenates, ascorbate, the major hydrophilic coantioxidant, can recycle photo-oxidized -tocopherol (^{Kagan et al. 1992;Kitazawa et al. 1997}), in murine and human SC the levels of ascorbate were very low as compared with epidermal and dermal tissue (Thiele JJ, Traber MG, Packer L, unpublished observations), which is not surprising because the SC is a very hydrophobic tissue. Thus, SC appears to lack any antioxidants that are known to recycle vitamin E, potentially explaining the susceptibility of SC vitamin E to oxidative stress.

Whereas -tocopherol in murine SC was significantly depleted after suberythemogenic UVR, the lipid peroxidation parameter MDA was increased only after an unphysiologically high dose Figure 2c. Similarly, in previous studies on the cutaneous effects of ozone we found that the concentration necessary to detect increased MDA formation in the SC was five times higher than the one needed to deplete vitamin E (^{Thiele et al. 1997c}). It is not clear, however, if SC lipid peroxidation only occurs after exposure to high UV doses, or if the techniques currently available are not sensitive enough to detect more subtle changes in vivo. In vitro studies examining low density lipoproteins oxidation show that lipid peroxidation occurs when vitamin E is almost completely depleted, and that linoleic acid is depleted during this process (^{Esterbauer et al. 1993;Lodge et al. 1995}). Although linoleic acid accounts for only 1.4% of human SC free fatty acids (^{Wertz and Downing 1991}), its presence is critical because essential fatty acid deficiency in rats causes severe barrier abnormalities (^{Hansen and Jensen 1985}). Thus, minor amounts of lipid peroxidation of polyunsaturated fatty acids may not be detectable by current methodologies, yet may have severe pathophysiologic consequences.

Remarkably, a vitamin E gradient was found in untreated human SC with lowest tocopherol concentrations at the surface and highest tocopherol concentrations in the deepest SC layers Figure 4. In human epidermis, the ratio of - to -tocopherol is about 10 to one (^{Shindo et al. 1994a}). This is in accordance with the - and -tocopherol ratio we have found in the deepest SC layer, with 10-fold higher -tocopherol concentration compared with the -tocopherol concentration Figure 4. The tocopherol gradient in human SC appears to reflect a depletion of tocopherols in surface layers. This seems likely for the following reasons: (i) the irradiance of UVR at the absorption maximum of -tocopherol (around 295 nm) is highest at the outer surface and decreases within the SC (^{Anderson 1983}). (ii) The oxygen partial pressure (pO₂) decreases gradually from the outer to the inner SC layers as a result of the high diffusion resistance (^{Hatcher and Plachy 1993}), and thus is inversely correlated with the -tocopherol concentrations within the SC. Because the presence of oxygen is needed for the induction of UVR induced generation of reactive oxygen intermediates, this might contribute to the increasing -tocopherol depletion towards outer SC layers. (iii) The percutaneous penetration of most molecules, among them noxious, oxidizing xenobiotics, follows a gradient within the SC with highest concentrations in the outer layers (^{Rougier et al. 1990}). Oxidation of -tocopherol by such exogenous oxidants would therefore also be higher in the outer SC. Besides its protection against lipid peroxidation, vitamin E is suggested to stabilize lipid bilayer structures (^{Lucy 1972;Stillwell et al. 1992}), which may also be of relevance for SC lipid bilayers; remarkably, the degree of disorder and the amount of lipids decrease over the outer cell layers of human SC (^{Bommannan et al. 1990}). Thus, low levels of SC vitamin E are associated with a high degree of SC lipid disorder.

Recently, we (^{Thiele et al. 1997c}) demonstrated that murine SC -tocopherol is very susceptible to exposure of ozone, a strong oxidant and a major air pollutant. Interestingly, in murine SC, -tocopherol, as compared with -tocopherol, was more readily depleted by ozone, which is in accordance with the effects of SSUV on murine SC tocopherols demonstrated in this study. Furthermore, the in vivo antioxidant activity of -tocopherol is thought to be higher than that of -tocopherol (^{Kamal-Eldin and Appelqvist 1996}). In theory, the observed SC tocopherol depletion could be caused either directly, by absorption of short wave UVB, and/or indirectly, by excited state singlet oxygen or reactive oxygen intermediates that are generated by photosensitizers upon UV absorption also in the UVA range. Our results indicate that both mechanisms may be relevant, because either UVB or UVA deplete murine SC -tocopherol Figure 3. The absorption maxima of both tocopherols lie close together, which suggests that mechanisms other than direct photodestruction by UV, e.g., free radical scavenging properties, may account for this difference in their susceptibilities to UV mediated depletion.

Pathophysiologically, the findings of this study may have relevance with respect to (i) the optical barrier function of the SC, (ii) the physical barrier function of the SC, and (iii) signaling cascades that are initiated in the SC and extend into adjacent epidermal and possibly dermal layers. Recent evidence suggests that -tocopherol protection against UVB induced lipid peroxidation is a consequence of both its ability to scavenge peroxyl radicals and its UVB absorbance resulting in a sunscreen effect (^{Kramer and Liebler 1997}). Both mechanisms would provide photoprotection in adjacent epidermal layers. Hence, the presence of -tocopherol in the human SC and its distribution gradient appears to be of major relevance, especially because its absorption maximum is very similar to the action spectrum for UVR induced erythema and carcinogenesis of hairless mice with a flat peak at 293 nm (^{Ley et al. 1983}). In addition to its antioxidant properties, -tocopherol was reported to act as a human skin penetration enhancer, resulting from its interactions with the gel phase interstitial lipid region of the SC (^{Trivedi et al. 1995}). In vitro evidence exists that -tocopherol modulates the fluidity and permeability of lipid bilayer membranes (^{Srivista et al. 1983}). Consequently, depletion of inherent SC -tocopherol could affect the barrier function of human SC.

This study demonstrates the specific distribution of inherent vitamin E in the human SC, and its remarkably high susceptibility to suberythemogenic UVR, leading to its depletion prior to visible skin reactions occurring. Thus, vitamin E depletion in the stratum corneum is an early and sensitive in vivo marker of SSUV induced photo-oxidation. Its pathophysiologic relevance, however, needs to be further investigated.

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Acknowledgments

Helpful discussions with Dr. F. Dreher (UCSF) are gratefully acknowledged. N. Espuno and J. H. Choi provided excellent technical assistance. J.J. Thiele (Th 620/1–1) was supported by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft. Research was in part supported by a grant from Unilever Research U.S.A.