Depletion of Human Stratum Corneum Vitamin E: An Early
and Sensitive In Vivo Marker of UV Induced Photo-Oxidation
Jens J. Thiele, Maret G. Traber, and Lester Packer
Department of Molecular and Cell Biology, University of California, Berkeley, California, U.S.A.
As the outermost barrier of the body, the stratum
corneum (SC) is frequently and directly exposed to a
pro-oxidative environment, including ultraviolet solar
radiation (UVR). Therefore, we hypothesized that the
SC is susceptible to UVR induced depletion of vitamin
E, the major lipophilic antioxidant. To test this, we
investigated (i) the susceptibility of SC tocopherols to
solar simulated UVR in hairless mice, (ii) the baseline
levels and distribution patterns of tocopherols in human
SC, and (iii) the impact of a suberythemogenic dose of
solar simulated UVR on human SC tocopherols. SC
tocopherol levels were measured by high performance
liquid chromotography analysis of SC extracts from tape
strippings. In murine SC, overall tocopherol concentra-
tions were determined, whereas in human SC, 10 consec-
utive layers were analyzed for each individual. The results
L
ocated at the interface between body and environment,
the stratum corneum (SC) is frequently and directly
exposed to a pro-oxidative environment, including
chemical oxidants, air pollutants, and ultraviolet solar
light (Fuchs, 1992; Scharffetter-Kochanek, 1997; Thiele
et al, 1997a). The SC is comprised of a unique two-compartment
system of structural, enucleated cells (corneocytes) embedded in a
lipid enriched intercellular matrix, forming stacks of bilayers that
are rich in ceramides, cholesterol, and free fatty acids (Elias, 1983;
Elias and Feingold, 1992; Mao-Qiang et al, 1996). Diminished
permeability barrier function has been demonstrated after single
exposures to ultraviolet B (UVB; Lamaud and Schalla, 1984;
Miyauchi et al, 1992; Haratake et al, 1997a, b), to ultraviolet A
(UVA), or to combined UVA and UVB (Bronaugh and Stewart,
1985; Bissett et al, 1987); however, the underlying mechanisms of
UVR induced changes in SC barrier function remain unclear.
Although the SC is a major optically protective element of the
epidermis, reflecting approximately 5% of the incident light and
absorbing significant portions of ultraviolet C (UVC; 200–280 nm)
and UVB (280–320 nm) (Megaw and Drake, 1986; Shea and
Parrish, 1991), its antioxidant content and response to UVR has
not yet been investigated.
UVB and UVA irradiation induce the formation of reactive
Manuscript received August 7, 1997; revised October 28, 1997; accepted for
publication January 7, 1998.
Reprint requests to: Dr. Jens Thiele, Heinrich-Heine-Universität, Institut
für Physiologische Chemie I, Postfach 101007, D-40001, Düsseldorf, Germany.
Abbreviations: MDA, malondialdehyde; SC, stratum corneum; SSUV, solar
simulated ultraviolet radiation (280–400 nm).
·
0022-202X/98/$10.50 Copyright © 1998 by The Society for Investigative Dermatology, Inc.
756
on SC tocopherols demonstrated (i) their concentration
dependent depletion by solar simulated UVR in hairless
mice; (ii) a gradient distribution within untreated human
SC, with the lowest levels at the surface (α-tocopherol
6.5 K 1.4 pmol per mg, and γ-tocopherol 2.2 K 1.3 pmol
per mg) and the highest levels in the deepest layers (α-
tocopherol 76 K 12 pmol per mg, and γ-tocopherol
7.9 K 3.7 pmol per mg, n J 10; p < 0.0001); and (iii)
the depletion of tocopherols in human SC by a single
suberythemogenic dose of solar simulated UVR (α-
tocopherol by 45%, and γ-tocopherol by 35% as compared
with controls; n J 6; both p < 0.01). These results
demonstrate that the SC is a remarkably susceptible site
for UVR induced depletion of vitamin E. Key words: α-
tocopherol/antioxidants/γ-tocopherol/oxidative stress/skin/
ubiquinol. J Invest Dermatol 110:756–761, 1998
oxygen species in cell cultures (Wlaschek et al, 1995; Grether-Beck
et al, 1996), skin homogenates (Nishi et al, 1991; Kagan et al, 1992;
Kitazawa et al, 1997), and intact murine and human skin (Jurkiewicz
et al, 1995; Jurkiewicz and Buettner, 1996). Evaluation of the
protective mechanisms of skin have included measurements of
baseline levels of antioxidants in the dermis and epidermis (Shindo
et al, 1993, 1994a), the antioxidant response to UVB and UVA
light in these layers (Fuchs et al, 1989; Shindo et al, 1994b), and
the efficacy of the photoprotective potential of topical antioxidant
supplementation (Werninghaus et al, 1991; Darr et al, 1992; Weber
et al, 1997). UVA and UVB induced damage to cutaneous tissues
is likely mediated, at least in part, by lipid peroxidation (Punnonen
et al, 1991; Shindo et al, 1994b; Basu-Modak et al, 1996). There
is ample evidence that exogenously applied vitamin E, the most
important lipid soluble antioxidant (Traber and Sies, 1996), provides
photoprotective effects in cell culture (Sugiyama et al, 1992; Stewart
et al, 1996) and in hairless mouse skin (Gensler and Magdaleno,
1991; Record et al, 1991; Jurkiewicz et al, 1995; Gensler et al,
1996; Weber et al, 1997; Yuen and Halliday, 1997).
Previously, we found the SC to be quite sensitive to ozone
induced oxidative damage in hairless mice. Although we failed to
detect depletion of vitamin E following ozone exposure in whole
murine skin (Thiele et al, 1997b), its depletion was apparent in the
outer epidermis when skin layers where analyzed separately (Thiele
et al, 1997d). Consequently, we measured α- and γ-tocopherols in
murine SC, where tocopherols were most sensitively depleted by
ozone (Thiele et al, 1997c). Therefore, we hypothesized that SC
tocopherols would also be a sensitive target for UVR. To test this
hypothesis, we investigated (i) the susceptibility of SC α- and γ-
tocopherol to solar simulated UV radiation in an animal model
(SKH-1 hairless mouse), (ii) the baseline levels of α- and γ-
VOL. 110, NO. 5 MAY 1998
tocopherol and its distribution gradient in human SC, and (iii) the
impact of a suberythemogenic dose of solar simulated UVR on α-
and γ-tocopherol in human SC.
MATERIALS AND METHODS
Chemicals All chemicals used were of the highest grade available. α- and γ-
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 6 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 3 152 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 Fig 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.
Human UVR exposure To estimate the UV sensitivity of each subject (n 5
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 3 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 5 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 5 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 5 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
DEPLETION OF STRATUM CORNEUM VITAMIN E 757
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.
for the same time (6 min, irradiance 1.86 J per cm2, n 5 4) (see Fig 3).
Control mice (n 5 12) were exposed to 0 MED (sham irradiation) but treated
identically in terms of anesthesia and housing conditions.
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 3 5 cm pieces of an
adhesive tape (Highland 3710, 3M, St. Paul, MN) using a standardized protocol.
Tapes strips (5 3 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 6 7 µg per cm2; n 5 10) was independent
of tape strip number, and the resultant cumulative SC weight for 20 tape
strippings was 660 6 140 µg per cm2 (n 5 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 6 0.7 µm (n 5 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 6 91 µg per cm2 and
660 6 80 µg per cm2, respectively; both n 5 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 3 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 α- and one γ-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 3 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 µl 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
758 THIELE ET AL
Figure 2. Dose-dependent depletion of murine SC tocopherols and
MDA formation by SSUV. After SSUV treatment, SC was obtained by
tape stripping and α- and γ-tocopherol, MDA concentrations were analyzed
immediately by HPLC as described in Material and Methods. Remarkably, α-
tocopherol was strongly depleted by 0.75 MED (A), whereas the depletion of
γ-tocopherol (B) and MDA formation (C) reached a significant level only at a
very high dose (9 MED) (each UV level, n 5 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).
was then removed and discarded. The hexane was taken to dryness under
nitrogen; the residue was resuspended in 500 µl 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 µm 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, α- and γ-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 6 SD.
RESULTS
UV dose-dependent depletion of tocopherols and increase in
MDA formation in murine SC Baseline concentrations of murine
SC α-tocopherol were 9.3 6 1.9 pmol per mg wet weight and of γ-
tocopherol were 3.6 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 α-tocopherol, and, less markedly, of γ-tocopherol.
A suberythemogenic dose of 0.75 MED significantly depleted α-
tocopherol from 9.3 6 1.9 to 1.4 6 0.3 (p , 0.0001; Fig 2A),
whereas 9 MED were required to significantly deplete γ-tocopherol
from 3.6 6 1.4 to 1.2 6 0.2 pmol per mg (p , 0.001; Fig 2B).
MDA levels in sham irradiated control mice were 4.8 6 0.5 pmol
per mg of SC tissue. The MDA levels after SSUV exposures to 0.75
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
Figure 3. Depletion of murine SC α-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 5 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 5 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 5 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 5 4). ***p , 0.001, *p , 0.05 as
compared with control levels.
MED and 1.5 MED were 5.0 6 0.7 pmol per mg and 5.8 6 0.8 pmol
per mg, respectively. Only after exposure to 9 MED was a significant
increase of MDA observed (7.1 6 0.2; p , 0.01; Fig 2C).
Both UVA and UVB deplete α-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 α-tocopherol from 7.7 6 0.9 to 0.5 6 0.4 pmol
per mg (p , 0.0001) and γ-tocopherol from 2.4 6 0.6 to 1.4 6 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, α-tocopherol was depleted to 2.1 6 0.6 pmol per
mg (p , 0.001), whereas UVB irradiation depleted α-tocopherol to
1.5 6 1.0 pmol per mg (p , 0.001). Neither UVA nor UVB alone
had an effect on murine SC γ-tocopherol (Fig 3).
Vitamin E concentrations increase with depth of human SC α-
and γ-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 (Fig 4). Compared with the outermost SC (6.5 6 1.4 pmol per
mg, tape strippings 1 and 2), the α-tocopherol contents were signific-
antly higher in tape strippings 7 and 8 (17 6 10 pmol per mg,
p , 0.005) and in all subsequent layers (p , 0.0001) up to and
including the deepest layer (76 6 12 pmol per mg tape, strippings 19
and 20, p , 0.0001). Thus, α-tocopherol concentrations in the deepest
layers of SC were 11.7-fold higher than those in the uppermost SC layer.
γ-Tocopherol levels were also lowest in the outermost SC
(2.2 6 1.3 pmol per mg, tape strippings 1 and 2), and were significantly
higher in tape strippings 7 and 8 (3.7 6 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 6 3.7 pmol per mg tape, strippings 19 and 20,
p , 0.0001); however, the gradient of γ-tocopherol was not as steep
as that observed for α-tocopherol. γ-Tocopherol concentrations were
only 3.6-fold lower in the outermost as compared with the deepest
SC layers (Fig 4).
Suberythemogenic UV irradiation depletes human SC
tocopherols In six of the 10 human subjects described above, the
α- and γ-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 (Fig 5) exhibited
similar results as described for α- and γ-tocopherol in Fig 4. The α-
tocopherol concentrations ranged from 6.7 6 1.1 pmol per mg in tape
VOL. 110, NO. 5 MAY 1998
Figure 4. Gradients of α- and γ-tocopherol in human SC. Untreated SC
of the upper arm of 10 volunteers was sequentially tape stripped and the α-
and γ-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.
Figure 5. A single dose of 0.75 SSUV depletes α- and γ-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.
DEPLETION OF STRATUM CORNEUM VITAMIN E 759
strippings 1 and 2 to 77 6 14 pmol per mg in tape strippings 19 and
20, which reflects a 11.5-fold increase in α-tocopherol in the deepest
layer. γ-Tocopherol levels in tape strippings 1 and 2 were 2.8 6 1.0 pmol
per mg and increased 3.4-fold to 8.2 6 3.0 pmol per mg in tape
strippings 19 and 20.
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 α- and the γ-tocopherol
concentrations were depleted in the UV exposed site compared with
the corresponding layer of the control site (Fig 5), these differences
in α-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 γ-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 α-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 6 8 pmol
per mg α-tocopherol, a 55% decrease as compared with the control
site (77 6 15 pmol per mg). Similarly, the γ-tocopherol concentrations
in tape strippings 19 and 20 after UV irradiation were 5.5 6 2.1 pmol
per mg, a 33% decrease as compared with the control site
(8.2 6 3.0 pmol per mg).
The overall human SC concentration of vitamin E in the tape strips
was 33 6 4 pmol per mg for α-tocopherol and 4.8 6 0.8 pmol per
mg for γ-tocopherol. UV light exposure (0.75 MED) depleted SC α-
tocopherol by 45% (to 18 6 6 pmol per mg; p , 0.003) and γ-
tocopherol by 35% (to 3.1 6 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 α-tocopherol by almost
50% (Fig 5), and murine SC α-tocopherol by 85% (Fig 2). Previously,
SSUV doses equivalent to 3 MED or more were necessary to detect
a significant depletion of α-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 α-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 α-tocopherol (Shindo et al, 1994a). Although in SSUV irradiated
murine skin homogenates, ascorbate, the major hydrophilic coantioxid-
ant, can recycle photo-oxidized α-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 α-tocopherol in murine SC was significantly depleted after
suberythemogenic UVR, the lipid peroxidation parameter MDA was
increased only after an unphysiologically high dose (Fig 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
760 THIELE ET AL
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 polyunsat-
urated 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 (Fig 4). In human
epidermis, the ratio of α- to γ-tocopherol is about 10 to one (Shindo
et al, 1994a). This is in accordance with the α- and γ-tocopherol ratio
we have found in the deepest SC layer, with 10-fold higher α-
tocopherol concentration compared with the γ-tocopherol concentra-
tion (Fig 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 α-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 α-tocopherol concentra-
tions 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 α-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 α-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 α-
tocopherol is very susceptible to exposure of ozone, a strong oxidant
and a major air pollutant. Interestingly, in murine SC, α-tocopherol,
as compared with γ-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 α-tocopherol is thought to be higher than that
of γ-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 α-tocopherol (Fig 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 α-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 α-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,
α-tocopherol was reported to act as a human skin penetration enhancer,
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
resulting from its interactions with the gel phase interstitial lipid region
of the SC (Trivedi et al, 1995). In vitro evidence exists that α-tocopherol
modulates the fluidity and permeability of lipid bilayer membranes
(Srivista et al, 1983). Consequently, depletion of inherent SC α-
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 subery-
themogenic 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.
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.
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