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Efficacy of Topically Applied Tocopherols and Tocotrienols in Protection of Murine Skin From Oxidative Damage Induced by UV-Irradiation
Free Radical Biology & Medicine, Vol. 22, No. 5, pp. 761–769, 1997 Copyright q 1997 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/97 $17.00 / .00
PII S0891-5849(96)00346-2
Original Contribution EFFICACY OF TOPICALLY APPLIED TOCOPHEROLS AND TOCOTRIENOLS IN PROTECTION OF MURINE SKIN FROM OXIDATIVE DAMAGE INDUCED BY UV-IRRADIATION Christine Weber, Maurizio Podda, Michalis Rallis, Jens J. Thiele, Maret G. Traber, and Lester Packer Department of Molecular and Cell Biology, University of California-Berkeley, 251 Life Sciences Addition, Berkeley, CA 94720-3200 (Received 15 March 1996; Revised 15 June 1996; Accepted 17 June 1996)
Abstract—To assess the efficacy of various forms of vitamin E in protection of skin from UV-light-induced oxidative stress, vitamin E (tocotrienol-rich fraction of palm oil, TRF) was applied to mouse skin and the contents of antioxidants before and after exposure to UV-light were measured. Four polypropylene plastic rings (1 cm2) were glued onto the animals’ backs, and 20 ml 5% TRF in polyethylene glycol-400 (PEG) was applied to the skin circumscribed by two rings and 20 ml PEG to the other two rings. After 2 h, the skin was washed and half of the sites were exposed to UV-irradiation (2.8 mW/cm2 for 29 mi: 3 MED). TRF treatment (n Å 19 mice) increased mouse skin a-tocopherol 28 { 16-fold, a-tocotrienol 80 { 50-fold, g -tocopherol 130 { 108-fold, and g -tocotrienol 51 { 36-fold. A significantly higher percentage of a-tocopherol was present in the skin as compared with that in the applied TRF. After UV-irradiation, all vitamin E forms decreased significantly (p õ .01), while a larger proportion of the vitamin E remained in PEG-treated (É80%) compared with TRF-treated (É40%) skin. Nonetheless, vitamin E concentrations in irradiated TRF-treated skin were significantly higher than in the nonirradiated PEG-treated (control) skin (p õ .01). Thus, UV-irradiation of skin destroys its antioxidants; however, prior application of TRF to mouse skin results in preservation of vitamin E. Copyright q 1997 Elsevier Science Inc. Keywords—Ubiquinol, Ubiquinone, Hairless mice, Vitamin E, Antioxidants, Skin, Tocopherol, Tocotrienol, Free radicals
antioxidants protect skin against damaging effects from reactive oxygen species.6–8 Among the protective agents are the potent lipid-soluble antioxidants vitamin E and ubiquinol.2,6–8 During oxidative stress caused by prolonged UV-exposure, skin antioxidants are severely diminished, resulting in insufficient protection and cell damage.8–10 Topical administration of antioxidants is one approach to diminishing oxidative injury.11–13 Because vitamin E is the major lipophilic antioxidant of exogenous origin found in tissues, it is an obvious choice for enhancement of antioxidative protection by topical application. Vitamin E is a generic description for all tocopherol and tocotrienol derivatives, which qualitatively exhibit the biological activity of a-tocopherol, and is the collective name for the eight major naturally occurring molecules, four tocopherols and four tocotrienols (Fig. 1). Tocotrienols differ from tocopherols
INTRODUCTION
Skin, the outermost barrier of the body, is exposed to oxidative stress from a variety of environmental insults, including UV-irradiation, ozone, halogenated hydrocarbons, and smoke. This oxidative damage could be an initiator in the pathogenesis of skin cancer and photoaging.1–5 A variety of enzymatic and nonenzymatic Address correspondence to: Lester Packer, Department of Molecular and Cell Biology, 251 LSA, University of California, Berkeley, CA 94720-3200, USA. Present address of Christine Weber: The Technical University of Denmark, Department of Biochemistry and Nutrition, Building 224, 2800 Lyngby, Denmark. Present address of Maurizio Podda: Zentrum der Dermatologie, Klinikum der J. W. Goethe Universita¨t, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany. Present address of Michalis Rallis: University of Athens, School of Pharmacy, Division of Pharmaceutical Technology, 15771 Athens, Greece. 761
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in that they have an isoprenoid instead of a phytyl side chain; the four forms of tocopherols and tocotrienols differ in the number of methyl groups on the chromanol nucleus (a- has three, b- and g- have two, and d- has one). In vitro, the relative order of peroxyl radical scavenging reactivities of a-, b-, g-, and d-tocopherols is 100, 60, 25, 27, respectively.14 a-Tocopherol is generally regarded as the most important lipid-soluble antioxidant in plasma, circulating lipoproteins, and tissues.15–19 g-Tocopherol and the tocotrienols are present in the tissues at much lower concentrations than a-tocopherol.20–23 Earlier reports suggested that in vitro a-tocotrienol has higher antioxidative activity than a-tocopherol against Fe2//ascorbate- and Fe2//NADPH-induced lipid peroxidation in rat liver microsomes.24 Therefore, we hypothesized that tocotrienols might be more effective than tocopherols against UV-induced damage. However, it is not possible to enrich tissues to a large extent with tocotrienols by dietary means,23 probably due to the preferential enrichment of the plasma with a-tocopherol.15 This may be a result of the negative exclusion of all forms of vitamin E in preference to RRR-a-tocopherol by the a-tocopherol transfer protein in the liver.25 Topical application of vitamin E may provide an efficient way of enriching the skin with different forms of vitamin E that have a potentially higher antioxidative activity than a-tocopherol. Therefore, this study was carried out to measure the skin penetration of a mixture of tocopherols and tocotrienols from a tocotrienol-rich palm oil fraction (TRF) and to compare the protection conferred by these various forms of vitamin E against UV-light-induced oxidative stress. MATERIALS AND METHODS
Chemicals All chemicals used were of the highest grade available. TRF was kindly provided by the Palm Oil Research Institute of Malaysia (PORIM, Kuala Lumpur, Malaysia). Tocopherol standards (Covitol) came from Henkel Corporation (LaGrange, IL, USA). Tocotrienols for use as standards were purified from TRF by Dr. Asaf A. Qureshi, University of Wisconsin (Madison, WI, USA). Animals Female hairless mice (8–12 weeks old) were purchased from Charles River Laboratories (Wilmington, MA, USA) and were kept under standard light and temperature conditions. Food (Harlan Teklad Rodent Diet
Fig. 1. Vitamin E structures: a , R1 and R2 Å CH3; b, R1 Å CH3, R2 Å H; g, R1 Å H, R2 Å CH3; d, R1 and R2 Å H.
#1846, Madison, WI, USA) and water were provided ad lib. This experiment was carried out five times using four mice per study. Application A 5% w/v mixture containing TRF in polyethylene glycol-400 (PEG; Sigma, St. Louis, MO, USA) was used. Mice were anesthetized by an intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight) and remained anesthetized during the entire experimental period. Four polypropylene plastic rings (1 cm2) were glued onto the animals’ backs, and TRF (20 ml) was applied to the skin circumscribed by two rings and PEG (20 ml) to the other two rings. After 2 h, the excess substance on the treated area was removed as described by Dupuis et al.26 Briefly, the skin was rinsed three times with 300 ml ethanol:water (95:5), then twice with water alone; then the area was dried with a cotton tip. After the rinsing procedure, the position of the application site was marked, the plastic rings were removed, and the mice were exposed to UVirradiation. UV-irradiation The mice were placed under an Oriel 1000-W solar simulator (Oriel, Stratford, CT, USA) with an output of 2.8 mW/cm2 of UVA and UVB light (290–400 nm) and irradiated for 29 min (corresponding to 3 MED) on either the upper or the lower back, while the other part was shielded from UV-light by covering the skin with paper and aluminum foil. After exposure, the mice were killed by neck dislocation, and the skin with subcutaneous fat was excised from the four exposure sites and the samples immediately frozen in liquid nitrogen. The samples were stored up to a week at 0807C. Pre-
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liminary experiments indicated no differences in the concentrations of tocopherols and tocotrienols of skin with or without attached subcutaneous fat.27 Skin extraction Tocopherols and tocotrienols were determined by HPLC-electrochemical detection after hexane extraction, as described.28,29 Briefly, the skin was weighed (É40 mg), ground under liquid nitrogen, homogenized in a Potter-Elvehjem homogenizing tube with 2 ml buffer (10 mM phosphate, 130 mM NaCl, 1 mM EDTA, pH 7.0) and 50 ml BHT (1 mg/ml), and extracted after addition of 1 ml of 0.1 M SDS, 2 ml ethanol, and 2 ml hexane. An appropriate aliquot was used for HPLC analysis. Dietary vitamin E The amount of vitamin E in mouse chow was determined after saponification. Briefly, 1 g of chow was mixed vigorously with 5 ml of 1% ascorbic acid in water. After addition of 10 ml ethanol and 1.5 ml saturated KOH, the samples were saponified at 707C for 30 min, protected from light. After cooling on ice and addition of 5 ml water, the samples were extracted with 10 ml hexane. Then 5 ml of the upper hexane layer was collected and taken to dryness under nitrogen. The residue was diluted 25-fold and 20 ml was injected onto the HPLC. Irradiation of the tocotrienol-rich fraction in vitro The 5% TRF mixture was transferred to a 1-cm2 quartz cuvette and irradiated for 29 min as described above for mice. After irradiation, the samples were diluted 1:1250 in ethanol and analyzed by HPLC for the content of vitamin E. Tocopherol and tocotrienol analysis It was necessary to develop a method for the simultaneous measurement of various vitamin E forms, ubiquinols, and ubiquinones because TRF contains substances that interfere with the measurement of ubiquinol and ubiquinone by our standard procedures; the details of the method are reported elsewhere.27 The HPLC system consisted of a Hewlett Packard 1050 series gradient pump, a SCL-10A Shimadzu System Controller with a SIL-10A autoinjector with sample cooler, a Beckman Ultrasphere ODS C-18 column (4.6 mm i.d., 25 cm, 5 mm particle size) with a Rainin Spheri-5 RP-18 pre-column (5 micron, 30 1 4.6 mm), a Hewlett Packard 1050 diode array detector, and an LC-4B elec-
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trochemical detector from Bioanalytical Systems (BAS, West Lafayette, IN, USA) using a glassy carbon electrode. The detectors were set up in line, the eluent first passing through the diode array detector. The mobile phase consisted of a mixture of A (80:20 v/v methanol:water and 0.2% w/v lithium perchlorate) and B (ethanol, reagent grade, with 0.2% w/v lithium perchlorate) at a flow rate of 1 ml/min. Optimal separation and quantitation of a- and g-tocopherols and a- and g-tocotrienols were obtained using 61% B and 39% A; these were the conditions used for two experiments (n Å 8 mice). Subsequently, optimal conditions using a gradient were achieved for the measurement of ubiquinol and ubiquinone. Here, the initial composition was 61% B and 39% A, after 16 min the mobile phase was changed linearly over 2 min to 100% B, which was continued for 10 min, then was changed linearly over 2 min to the initial composition; total run time was 40 min. The electrochemical detector was operated with a 0.5-V potential, full recorder scale at 50 nA, for quantitation of a- and g-tocopherols, a- and g-tocotrienols, and ubiquinol. Quantitation was carried out by comparison of peak areas to the area of standard curves obtained with authentic compounds. For vitamin E, aand g-tocopherols were used as standards because the chromanol nucleus is the same in a-tocopherol and atocotrienol and in g-tocopherol and g-tocotrienol, respectively. The diode array detector collected spectra at 275 nm for quantitation of ubiquinone-9, which was carried out using peak area comparisons to a standard curve obtained with authentic ubiquinone-9. Phospholipid hydroperoxide quantitation Skin (about 50 mg) was accurately weighed, homogenized with 2 ml of 1:2 methanol:chloroform containing 20 ml BHT (1 mg/ml), then 0.8 ml 0.25 mM NaCl was added and the lower layer was collected and dried under nitrogen. The residue was dissolved in ethanol:methanol (79:21) and injected onto the HPLC system. Lipid hydroperoxides were detected by chemiluminescence as described by Yamamoto et al.30,31 The HPLC setup included in-line UV and chemiluminescence detection. The isocratic mobile phase (70: 25:5 methanol:2-propanol:40 mM NaH2PO4) passed through a Spherisorb S5 NH2 column, then through the UV-detector (206 nm). The effluent was mixed with the chemiluminescence cocktail (1:1 methanol:55 mM potassium tetraborate, pH 10, 177 mg/L isoluminol, 1.5 mg/L microperoxidase) at a postcolumn mixing tee, and the emitted light was measured using a chemiluminescence detector. The data are reported as phos-
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phatidyl choline hydroperoxides/phosphatidyl choline (PCOOH:PC, mmol/mol). These conditions were used for one experiment (n Å 4 mice). Statistical analysis The differences in vitamin E and ubiquinone/ubiquinol contents of the mice were analyzed using twofactor repeated measures ANOVA as all the treatments were given to each individual. The data were transformed using natural logarithms prior to statistical analysis. The vitamin E concentration data from the five experiments are pooled (n Å 19), the ubiquinol/ubiquinone concentrations are from eight mice, and the lipid hydroperoxides are from four mice. The differences between the percentage distribution of vitamin E in the TRF-treated murine skin and in the TRF fraction were investigated using one-way ANOVA. All statistical analysis was carried out using SuperAnova for the Macintosh (Berkeley, CA, USA). A p-value õ .05 was considered statistically significant. RESULTS
Tocopherols and tocotrienols in murine skin The tocopherols and tocotrienols in PEG-treated murine skin were determined to establish the baseline concentrations of vitamin E (Fig. 2, upper panels, A / B). a-Tocopherol concentrations were about 10-fold higher than those of the other vitamin E forms. The presence of tocotrienols in skin, however, was surprising. Therefore, tocotrienols and tocopherols were also measured in skin from three untreated mice and were found at concentrations similar to those in the PEGtreated mouse skin (data not shown). To investigate the origin of the skin tocotrienols, the vitamin E content of mouse chow (diet routinely fed to the mice) was measured. The diet contained a-tocopherol (29.7 { 6.2 mg/kg diet), g-tocopherol (10.3 { 1.1), a-tocotrienol (3.1 { 0.7), and g-tocotrienol (7.4 { 1.7). The distribution of vitamin E forms in chow was different from that in skin of untreated mice—a significantly (p õ .01) lower proportion of a-tocopherol was found in chow (58 { 7%) as compared with skin (89 { 2%); tocotrienols contributed approximately 20% of the total vitamin E in chow (Fig. 3). It is therefore likely that the tocotrienols found in mouse skin arise from the diet, not by contamination from the TRF-treated sites on the mice. Cutaneous absorption of tocopherols and tocotrienols following topical application To evaluate penetration of the different tocopherols and tocotrienols after topical application of TRF, the
Fig. 2. Vitamin E content of murine skin. The upper panels show the concentrations (mean { SD, n Å 19) of (A) a-tocopherol and (B) g-tocopherol, a-tocotrienol, and g-tocotrienol in skin from nonirradiated or UV-irradiated hairless mice with topical application of PEG. The lower panels show the concentrations of (C) a-tocopherol and (D) g-tocopherol, a-tocotrienol, and g-tocotrienol in skin after topical application of TRF to hairless mice before and after UVirradiation. Significant decreases in concentrations of each of the homologues were observed after UV-irradiation in both PEG-treated and TRF-treated skin. By least-squares mean comparisons: PEG vs. PEG / UV for a-tocopherol, p õ .005; for g-tocopherol, p õ .0001; a-tocotrienol, p õ .0001; and g-tocotrienol, p õ .0001. TRF vs. TRF / UV for a-tocopherol, p õ .0001; for g-tocopherol, p õ .0001; a-tocotrienol, p õ .0001; and g-tocotrienol, p õ .0001. PEG vs. TRF for a-tocopherol, p õ .0001; for g-tocopherol, p õ .004; a-tocotrienol, p õ .0001; and g-tocotrienol, p õ .0001. PEG vs. TRF / UV for a-tocopherol, p õ .0001; for g-tocopherol, p õ .0001; a-tocotrienol, p õ .0001; and g-tocotrienol, p õ .0001.
antioxidants present in the TRF-treated and PEGtreated skin from the same mice were compared (Fig. 2: compare A to C, and B to D). TRF treatment resulted in significant increases in vitamin E concentrations; fractional increases were greater in those forms that were present at low initial concentrations. Thus, TRF treatment resulted in a 28 { 16-fold increase in a-tocopherol, a 80 { 50-fold increase in a-tocotrienol, a 130 { 108-fold increase in g-tocopherol, and a 51 { 36-fold increase g-tocotrienol in the skin (Fig. 2, filled bars: compare A to C, and B to D). To evaluate whether the different vitamin E forms penetrated murine skin differently, the percent distribution of each of the vitamin E homologues in the TRF mixture was compared with its percent distribution in skin (Fig. 3). (The percent distribution was calculated for each form as follows: the concentration in PEGtreated skin was subtracted from the concentration found in the TRF-treated skin, then divided by the sum of the skin vitamin E concentrations 1 100.) The per-
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Fig. 3. Distribution of the vitamin E forms in skin, diet, TRF, and skin-absorbed TRF. The percent distributions (mean { SD) of the various vitamin E isomers in untreated (control) mouse skin (n Å 3), diet (n Å 4), TRF mixture (n Å 4), and TRF-treated skin (minus background; n Å 19) are shown. The percent distributions of vitamin E forms in the control skin were significantly different from their percent distribution in the diet; the percent distribution in TRF is significantly different from the forms absorbed into the skin. Comparison Control skin vs. diet Control skin vs. TRF Control skin vs. skin after TRF minus baseline Diet vs. TRF Diet vs. skin after TRF minus baseline TRF vs. skin after TRF minus baseline
% a-Tocopherol
% a-Tocotrienol
% g-Tocopherol
% g-Tocotrienol
0.0001 0.0001 0.0001 0.0002 NS 0.0001
NS 0.0001 0.0001 0.0001 0.0001 0.001
0.0001 0.0005 0.0001 0.0001 0.0001 NS
0.04 0.0001 NS 0.0005 NS 0.0001
cent distribution of vitamin E forms that penetrated the skin (above background concentrations) was significantly different from their distribution in the TRF mixture. Higher percentages of a-tocopherol were found in TRF-treated skin than were present in TRF (p õ .0001), the percentage of g-tocopherol was similar to the TRF mixture, and both a- and g-tocotrienols represented a smaller proportion than they did in the TRF mixture (p õ .001). Effect of UV-irradiation To evaluate the protection by vitamin E against oxidative damage caused by UV-irradiation, the concentrations of the various vitamin E forms present in the
tissue before and after UV-irradiation were measured. After UV-irradiation, the concentrations of all forms of vitamin E in the PEG-treated skin decreased significantly (p õ .01, except p õ .04 for g-tocopherol; Fig. 2, A / B). They also decreased significantly (p õ .001) after UV-irradiation as compared with nonirradiated areas in the TRF-treated skin (Fig. 2, C / D). Notably, after UV-irradiation, the vitamin E concentrations were significantly higher in the TRF-treated irradiated skin than in the PEG-treated nonirradiated skin (p õ .01). The percent vitamin E remaining after UV irradiation is a measure of the susceptibility of the individual tocopherols and tocotrienols to oxidative depletion. In PEG-treated skin, approximately 80% of the different vitamin E forms remained after UV-irradiation; in the
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in the PEG-treated and TRF-treated skin, respectively (Fig. 5). After UV-irradiation, both ubiquinone and total Q decreased significantly (p õ .001) in PEG-treated and TRF-treated skin. Thus, UV-irradiation resulted in statistically significant decreases in ubiquinol, ubiquinone, and total Q that were not prevented by topically applied TRF. Lipid hydroperoxides
Fig. 4. The percent remaining vitamin E after UV-irradiation. For each of the vitamin E forms, the percent remaining was calculated by dividing the concentration remaining after UV-irradiation by the concentration present before UV-irradiation and multiplying by 100 (mean { SD, n Å 3).
TRF-treated skin, approximately 40% remained (Fig. 4). There were no significant differences in the degree of destruction of the various vitamin E homologues, suggesting that they were equally effective. Because the absolute levels of the different vitamin E forms are much lower in the PEG-treated animals, the standard deviation (in %) appears relatively larger in these measurements. To evaluate whether the decrease in vitamin E forms in the TRF-treated samples was due to direct photodestruction of vitamin E, the effect of UV-irradiation of the TRF solution was determined in vitro. The percent remaining after UV-irradiation was 86% for atocopherol, 83% for g-tocopherol, 83% for a-tocotrienol, and 84% for g-tocotrienol. These data suggest that direct photodestruction was similar for all of these vitamin E forms.
The production of lipid hydroperoxides in response to UV-light was measured as a marker of oxidative stress. There were no differences in the PCOOH:PC ratio in the PEG-treated (27 { 20 mmol/mol) and TRFtreated (15 { 10) skin before or after UV-irradiation (PEG-treated 38 { 42 and TRF-treated 45 { 23, respectively). These data demonstrate that statistically significant peroxidative damage to membrane lipids was not apparent immediately after exposure of the skin of hairless mice to 3 MED UV-irradiation. DISCUSSION
Tocotrienols and tocopherols were found in control and PEG-treated murine skin at concentrations ranging from 0.5 to 9 nmol/g. Of mouse tissues, skin is unique in containing appreciable concentrations of tocotrienols; other mouse tissues contain substantially smaller tocotrienol concentrations.27 Hayes et al.23 demonstrated that tocotrienols represent only a small proportion of vitamin E in tissues from hamsters fed toco-
Protection of endogenous antioxidants To evaluate the protective effects of TRF on antioxidants that were not applied topically, ubiquinol and ubiquinone were quantitated. Ubiquinol concentrations were significantly (p õ .002) lower in the TRF-treated skin (1.2 { 0.7) compared with PEG-treated skin (0.8 { 0.6), but these concentrations are low with large variances, so differences may not be physiologically relevant. Following UV-irradiation, ubiquinol concentrations in both PEG-treated and TRF-treated skin decreased fivefold (Fig. 5; p õ .001). Ubiquinone-9 and total Q (ubiquinone-9 / ubiquinol-9) were similar
Fig. 5. Ubiquinol and ubiquinone in murine skin. The concentrations (mean { SD) of ubiquinol, ubiquinone, and total Q in mouse skin after application of PEG or TRF, and exposed or not to UV-light. All parameters decreased significantly after UV-irradiation. For PEG vs. PEG / UV: ubiquinol, p õ .0001; ubiquinone, p õ .0008; total Q, p õ .0004. For TRF vs. TRF / UV: ubiquinol, p õ .0006; ubiquinone, p õ .001; total Q, p õ .0008. There were no significant differences between PEG / UV vs. TRF / UV.
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trienol-enriched diets. The presence of a- and g-tocotrienols in untreated mouse skin was unexpected because (a) the mouse diets were not specially enriched with tocotrienols, and (b) the liver discriminates against tocotrienols in favor of a-tocopherol during repackaging of dietary fats into very low density lipoproteins for secretion into the circulation.15,25 Suarna et al.32 demonstrated that after feeding TRF to rats, all lipoprotein classes contained tocotrienols. Apparently, transfer of tocotrienols to mouse skin must take place following absorption and transport of dietary vitamin E in chylomicrons during postprandial chylomicron clearance and during delivery of tocotrienol-containing lipoproteins.15,25 It is unlikely that these skin tocotrienols were contaminants from the TRF application site because skin from untreated mice (control, Fig. 3) contained concentrations of these vitamin E forms similar to those found in PEG-treated mouse skin. Additionally, the vitamin E forms were distributed differently (a) in the skin, (b) in the diet, and (c) in the TRF mixture. After topical application of TRF, all the vitamin E forms readily penetrated into the skin of hairless mice and were present in concentrations far exceeding the baseline levels (Fig. 2). Norkus et al.33 have also demonstrated that application of a-tocopheryl acetate onto hairless mouse skin results in penetration of high concentrations into the skin. The distribution of the various vitamin E forms in TRF was different from that present in the TRF-enriched mouse skin: a larger fraction of a-tocopherol was absorbed into the skin compared with other forms of vitamin E in TRF (Fig. 3). The percentage of g-tocopherol was similar to TRF; both aand g-tocotrienols represented a smaller proportion than they did in TRF. These data suggest that the isoprenoid tail of the tocotrienols may hinder their penetration into skin. Additionally, the preferential penetration of a-tocopherol into skin suggests that there may be specific mechanisms in skin for enrichment with atocopherol. This discrimination between vitamin E forms, as well as their localization in skin compartments after topical application, merits further investigation. Tocopherols and tocotrienols in murine skin, applied topically or derived from the diet, were significantly depleted by UV-irradiation, indicating a protective antioxidant function (Fig. 2). All vitamin E forms in mouse skin, including small amounts of a- and g-tocotrienols, decreased similarly in response to UV-irradiation, demonstrating that they all afford similar antioxidant protection (Fig. 4). TRF application increased skin concentrations of the various vitamin E homologues, and these remained significantly elevated after exposure to UV-light compared with PEG-treated skin.
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The similarity in the degree of depletion of the vitamins in response to an oxidative stress suggests that these vitamin E forms protect similarly against UV-irradiation-induced damage. This observation was also verified by exposing the TRF mixture directly to UV-light. These findings are in contrast to those of Serbinova et al.,24 who reported after in vitro enrichment of microsomal membranes with a-tocopherol or a-tocotrienol that a-tocotrienol has a greater antioxidant activity than does a-tocopherol. In studies of rat and human low density lipoproteins enriched in tocotrienols and oxidized using an azo-initiator to generate a constant rate of peroxyl radicals, Suarna et al.32 reported that the antioxidative activities of a-tocopherol and a-tocotrienol were similar and that the a-isomers had higher activity than did the g-isomers.32 Taken together, these results suggest that depletion of skin vitamin E in vivo using UV-light may be quite different from other oxidizing systems. A larger percentage of the various vitamin E forms remained after UV-irradiation of the PEG-treated compared with the TRF-treated skin (Fig. 4). This implies a greater destruction of the various vitamin E forms in the TRF-treated skin. Whether this is due to increased free radical scavenging remains to be clarified. Localization of the TRF nearer to the upper epidermal layers in the TRF-treated skin could allow increased destruction during UV-irradiation. Alternatively, the TRF vitamin E may have penetrated the lipid components surrounding cells and thus may not have been accessible to aqueous antioxidants that could recycle the vitamin E. Thus, the applied TRF may have a different behavior during UV-exposure than the vitamin E naturally present. It should be emphasized that the skin was washed with ethanol and dried before exposure to UV-light; therefore, the vitamin E forms we have measured are not on the skin surface, but have penetrated into the skin. The lack of detectable formation of lipid hydroperoxides suggests that the applied irradiation was insufficient to cause severe peroxidative damage to membrane lipids, although a significant decrease was seen in the endogenous antioxidant, ubiquinol-9 in response to the irradiation. The applied vitamin E was unable to protect this endogenous antioxidant against UV-induced oxidative injury, although the vitamin E levels in the skin were increased above the endogenous levels, even after UV irradiation. The antioxidative effect of the applied vitamin E was thus limited. Coenzyme Q (ubiquinone/ubiquinol) was chosen as a marker for oxidative damage because ubiquinol is the most labile lipid-soluble antioxidant34–36 and is not present in TRF. Ubiquinol is oxidized prior to a-tocopherol during UV-irradiation of skin and is substan-
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tially depleted before a-tocopherol concentrations are affected.6 We found during this study that TRF contains substances that interfere with the measurement of ubiquinol and ubiquinone in skin by our standard procedures; therefore, we developed a gradient method for measuring ubiquinol and ubiquinone.27 The levels of ubiquinol detected in murine skin are low; nonetheless, after UV-irradiation, ubiquinol, ubiquinone, and total Q all decreased significantly. The disappearance of the ubiquinol that was observed may be due to the direct photodestruction of ubiquinol or to UV-induced oxidative stress, causing decreased capacity in the ubiquinone reducing systems and subsequent further oxidation of the ubisemiquinone radical to nonrecyclable end products, resulting in loss in total Q. The direct photodestruction of ubiquinone is very improbable, as the absorption maximum (275 nm) is below the UVB range (290–320 nm). Direct photodestruction of ubiquinol is possible, since the absorption maximum is 290 nm. However, previous studies in our laboratory have shown that photodestruction accounts for only approximately 30% of the ubiquinol loss during UV-irradiation.37 Regardless of the route of destruction, UV-irradiation caused a loss in the total Q pool, thus depleting the skin of a vital component. A variety of antioxidants, especially vitamin E and ubiquinol, are present in skin. Topical application provides an efficient means of enriching the tissue in protective antioxidants, such as vitamin E. This paper demonstrates that tocopherols and tocotrienols from TRF readily penetrate the skin of hairless mice, are present in levels far above baseline after topical application, and are consumed during UV-light-induced oxidative stress. Acknowledgements — Beth Koh and Kenneth Tsang provided excellent technical assistance. We gratefully acknowledge the efforts of Dr. Asaf A. Qureshi, University of Wisconsin (Madison, WI, USA), who isolated tocotrienols for use as standards for this study. This study was supported by grants from the NIH (CA 47597) and the Palm Oil Research Institute of Malaysia. J.J.T. was supported by a fellowship of the Fritz Thyssen Stiftung, Germany (AZ 21295008).
REFERENCES 1. Perchellet, J. P.; Perchellet, E. M. Antioxidants and multistage carcinogenesis in mouse skin. Free Radic. Biol. Med. 7:377–408; 1989. 2. Nachbar, F.; Korting, H. C. The role of vitamin E in normal and damaged skin. J. Mol. Med. 73:7–17; 1995. 3. Guyton, K. Z.; Bhan, P.; Kuppusamy, P.; Zweier, J. L.; Trush, M. A.; Kensler, T. W. Free radical-derived quinone methide mediates skin tumor promotion by butylated hydroxytoluene hydroperoxide: Expanded role for electrophiles in multistage carcinogenesis. Proc. Natl. Acad. Sci. USA 88:946–950; 1991. 4. Dalle Carbonare, M.; Pathak, M. A. Skin photosensitizing agents and the role of reactive oxygen species in photoaging. J. Photochem. Photobiol. B 14:105–124; 1992.
5. Emerit, I. Free radicals and aging of the skin. Exs. 62:328–341; 1992. 6. Shindo, Y.; Witt, E.; Packer, L. Antioxidant defense mechanisms in murine epidermis and dermis and their responses to ultraviolet light. J. Invest. Dermatol. 100:260–265; 1993. 7. Shindo, Y.; Witt, E.; Han, D.; Tzeng, B.; Aziz, T.; Nguyen, L.; Packer, L. Recovery of antioxidants and reduction in lipid hydroperoxides in murine epidermis and dermis after acute ultraviolet radiation exposure. Photodermatol. Photoimmunol. Photomed. 10:183–191; 1994. 8. Shindo, Y.; Witt, E.; Han, D.; Packer, L. Dose-response effects of acute ultraviolet irradiation on antioxidants and molecular markers of oxidation in murine epidermis and dermis. J. Invest. Dermatol. 102:470–475; 1994. 9. Fuchs, J.; Huflejt, M. E.; Rothfuss, L. M.; Wilson, D. S.; Carcamo, G.; Packer, L. Acute effects of near ultraviolet and visible light on the cutaneous antioxidant defense system. Photochem. Photobiol. 50:739–744; 1989. 10. Fuchs, J.; Huflejt, M. E.; Rothfuss, L. M.; Wilson, D. S.; Carcamo, G.; Packer, L. Impairment of enzymic and nonenzymic antioxidants in skin by UVB irradiation. J. Invest. Dermatol. 93:769–773; 1989. 11. Werninghaus, K.; Handjani, R. M.; Gilchrest, B. A. Protective effect of alpha-tocopherol in carrier liposomes on ultravioletmediated human epidermal cell damage in vitro. Photodermatol. Photoimmunol. Photomed. 8:236–242; 1991. 12. Pauling, L. Effect of ascorbic acid on incidence of spontaneous mammary tumors and UV-light-induced skin tumors in mice. Am. J. Clin. Nutr. 54:1252S–1255S; 1991. 13. Jurkiewicz, B. A.; Bissett, D. L.; Buettner, G. R. Effect of topically applied tocopherol on ultraviolet radiation-mediated free radical damage in skin. J. Invest. Dermatol. 104:484–488; 1995. 14. Burton, G. W.; Ingold, K. U. Autoxidation of biological molecules. I. The antioxidant activity of vitamin E and related chainbreaking phenolic antioxidants in vitro. J. Am. Chem. Soc. 103:6472–6477; 1981. 15. Traber, M. G. Determinants of plasma vitamin E concentrations. Free Radic. Biol. Med. 16:229–239; 1994. 16. Burton, G. W.; Joyce, A.; Ingold, K. U. First proof that vitamin E is major lipid-soluble, chain-breaking antioxidant in human blood plasma. Lancet 8292(ii):3; 1982. 17. Burton, G. W.; Joyce, A.; Ingold, K. U. Is vitamin E the only lipid-soluble, chain-breaking antioxidant in human blood plasma and erythrocyte membranes? Arch. Biochem. Biophys. 221:281– 290; 1983. 18. Kagan, V. E.; Serbinova, E. A.; Forte, T.; Scita, G.; Packer, L. Recycling of vitamin E in human low density lipoproteins. J. Lipid Res. 33:385–397; 1992. 19. Esterbauer, H.; Gebicki, J.; Puhl, H.; Jurgens, G. The role of lipid peroxidation and antioxidants in oxidative modification of LDL. Free Radic. Biol. Med. 13:341–390; 1992. 20. Behrens, W. A.; Madere, R. Mechanisms of absorption, transport and tissue uptake of RRR-a-tocopherol and d-g-tocopherol in the white rat. J. Nutr. 117:1562–1569; 1987. 21. Handelman, G. J.; Epstein, W. L.; Peerson, J.; Spiegelman, D.; Machlin, L. J. Human adipose a-tocopherol and g-tocopherol kinetics during and after 1 y of a-tocopherol supplementation. Am. J. Clin. Nutr. 59:1025–1032; 1994. 22. Peake, I. R.; Bieri, J. G. a- and g-Tocopherols in the rat: In vitro and in vivo tissue uptake and metabolism. J. Nutr. 101:1615– 1622; 1977. 23. Hayes, K. C.; Pronczuk, A.; Liang, J. S. Differences in the plasma transport and tissue concentrations of tocopherols and tocotrienols: Observations in humans and hamsters. Proc. Soc. Exp. Biol. Med. 202:353–359; 1993. 24. Serbinova, E.; Kagan, V.; Han, D.; Packer, L. Free radical recycling and intramembrane mobility in the antioxidant properties of alpha-tocopherol and alpha-tocotrienol. Free Radic. Biol. Med. 10:263–275; 1991. 25. Traber, M. G.; Sies, H. Vitamin E in humans: Demand and delivery. Annu. Rev. Nutr. 16:321–347; 1996. 26. Dupuis, D.; Rougier, A.; Roguet, R.; Lotte, C.; Kalopissis, G. In .
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27. 28. 29.
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vivo relationship between horny layer reservoir effect and percutaneous absorption in human and rat. J. Invest. Dermatol. 82:353–356; 1984. Podda, M.; Weber, C.; Traber, M. G.; Packer, L. Simultaneous determination of tissue tocopherols, tocotrienols, ubiquinols and ubiquinones. J. Lipid Res. 37:893–901; 1996. Burton, G. W.; Webb, A.; Ingold, K. U. A mild, rapid, and efficient method of lipid extraction for use in determining vitamin E/lipid ratios. Lipids 20:29–39; 1985. Lang, J. K.; Gohil, K.; Packer, L. Simultaneous determination of tocopherols, ubiquinols, and ubiquinones in blood, plasma, tissue homogenates, and subcellular fractions. Anal. Biochem. 157:106–116; 1986. Yamamoto, Y.; Brodsky, M. H.; Baker, J. C.; Ames, B. N. Detection and characterization of lipid hydroperoxides at picomole levels by high-performance liquid chromatography. Anal. Biochem. 160:7–13; 1987. Yamamoto, Y.; Frei, B.; Ames, B. N. Assay of lipid hydroperoxides using high-performance liquid chromatography with isoluminal chemiluminescence detection. Methods Enzymol. 186:371–380; 1990. Suarna, C.; Hood, R. L.; Dean, R. T.; Stocker, R. Comparative antioxidant activity of tocotrienols and other natural lipid-soluble antioxidants in a homogeneous system, and in rat and human lipoproteins. Biochim. Biophys. Acta 1166:163–170; 1993. Norkus, E. P.; Bryce, G. F.; Bhagavan, H. N. Uptake and bioconversion of alpha-tocopheryl acetate to alpha-tocopherol in skin of hairless mice. Photochem. Photobiol. 57:613–615; 1993. Stocker, R.; Bowry, V. W.; Frei, B. Ubiquinol-10 protects human low density lipoprotein more efficiently against lipid peroxida-
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tion than does alpha-tocopherol. Proc. Natl. Acad. Sci. USA 88:1646–1650; 1991. 35. Niki, E. Chemistry and biochemistry of vitamin E and coenzyme Q as antioxidants. In: Corongiu, F.; Banni, S.; Dessi, M. A.; Rice-Evans, C., eds. Free radicals and antioxidants in nutrition. London: The Richelieu Press Ltd.; 1993:13–25. 36. Kontush, A.; Hu¨bner, C.; Finckh, B.; Kohlschu¨tter, A.; Beisiegel, U. Antioxidative activity of ubiquinol 10 at physiologic concentrations in human low density lipoprotein. Biochim. Biophys. Acta 1258:177–187; 1995. 37. Shindo, Y.; Witt, E.; Han, D.; Packer, L. Dose-response effects of acute ultraviolet irradiation on antioxidants and molecular markers of oxidation in murine epidermis and dermis. J. Invest. Dermatol. 102:470–475; 1994.
ABBREVIATIONS
BHT—butylated hydroxy toluene EDTA—ethylene diamine tetra-acetic acid HPLC-EC—high pressure liquid chromatography with electrochemical detection MED—minimal erythemal dose PEG—polyethylene glycol-400 total Q—sum of ubiquinone and ubiquinol TRF—tocotrienol-rich fraction of palm oil UV—ultraviolet
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Original Contribution EFFICACY OF TOPICALLY APPLIED TOCOPHEROLS AND TOCOTRIENOLS IN PROTECTION OF MURINE SKIN FROM OXIDATIVE DAMAGE INDUCED BY UV-IRRADIATION Christine Weber, Maurizio Podda, Michalis Rallis, Jens J. Thiele, Maret G. Traber, and Lester Packer Department of Molecular and Cell Biology, University of California-Berkeley, 251 Life Sciences Addition, Berkeley, CA 94720-3200 (Received 15 March 1996; Revised 15 June 1996; Accepted 17 June 1996)
Abstract—To assess the efficacy of various forms of vitamin E in protection of skin from UV-light-induced oxidative stress, vitamin E (tocotrienol-rich fraction of palm oil, TRF) was applied to mouse skin and the contents of antioxidants before and after exposure to UV-light were measured. Four polypropylene plastic rings (1 cm2) were glued onto the animals’ backs, and 20 ml 5% TRF in polyethylene glycol-400 (PEG) was applied to the skin circumscribed by two rings and 20 ml PEG to the other two rings. After 2 h, the skin was washed and half of the sites were exposed to UV-irradiation (2.8 mW/cm2 for 29 mi: 3 MED). TRF treatment (n Å 19 mice) increased mouse skin a-tocopherol 28 { 16-fold, a-tocotrienol 80 { 50-fold, g -tocopherol 130 { 108-fold, and g -tocotrienol 51 { 36-fold. A significantly higher percentage of a-tocopherol was present in the skin as compared with that in the applied TRF. After UV-irradiation, all vitamin E forms decreased significantly (p õ .01), while a larger proportion of the vitamin E remained in PEG-treated (É80%) compared with TRF-treated (É40%) skin. Nonetheless, vitamin E concentrations in irradiated TRF-treated skin were significantly higher than in the nonirradiated PEG-treated (control) skin (p õ .01). Thus, UV-irradiation of skin destroys its antioxidants; however, prior application of TRF to mouse skin results in preservation of vitamin E. Copyright q 1997 Elsevier Science Inc. Keywords—Ubiquinol, Ubiquinone, Hairless mice, Vitamin E, Antioxidants, Skin, Tocopherol, Tocotrienol, Free radicals
antioxidants protect skin against damaging effects from reactive oxygen species.6–8 Among the protective agents are the potent lipid-soluble antioxidants vitamin E and ubiquinol.2,6–8 During oxidative stress caused by prolonged UV-exposure, skin antioxidants are severely diminished, resulting in insufficient protection and cell damage.8–10 Topical administration of antioxidants is one approach to diminishing oxidative injury.11–13 Because vitamin E is the major lipophilic antioxidant of exogenous origin found in tissues, it is an obvious choice for enhancement of antioxidative protection by topical application. Vitamin E is a generic description for all tocopherol and tocotrienol derivatives, which qualitatively exhibit the biological activity of a-tocopherol, and is the collective name for the eight major naturally occurring molecules, four tocopherols and four tocotrienols (Fig. 1). Tocotrienols differ from tocopherols
INTRODUCTION
Skin, the outermost barrier of the body, is exposed to oxidative stress from a variety of environmental insults, including UV-irradiation, ozone, halogenated hydrocarbons, and smoke. This oxidative damage could be an initiator in the pathogenesis of skin cancer and photoaging.1–5 A variety of enzymatic and nonenzymatic Address correspondence to: Lester Packer, Department of Molecular and Cell Biology, 251 LSA, University of California, Berkeley, CA 94720-3200, USA. Present address of Christine Weber: The Technical University of Denmark, Department of Biochemistry and Nutrition, Building 224, 2800 Lyngby, Denmark. Present address of Maurizio Podda: Zentrum der Dermatologie, Klinikum der J. W. Goethe Universita¨t, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany. Present address of Michalis Rallis: University of Athens, School of Pharmacy, Division of Pharmaceutical Technology, 15771 Athens, Greece. 761
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in that they have an isoprenoid instead of a phytyl side chain; the four forms of tocopherols and tocotrienols differ in the number of methyl groups on the chromanol nucleus (a- has three, b- and g- have two, and d- has one). In vitro, the relative order of peroxyl radical scavenging reactivities of a-, b-, g-, and d-tocopherols is 100, 60, 25, 27, respectively.14 a-Tocopherol is generally regarded as the most important lipid-soluble antioxidant in plasma, circulating lipoproteins, and tissues.15–19 g-Tocopherol and the tocotrienols are present in the tissues at much lower concentrations than a-tocopherol.20–23 Earlier reports suggested that in vitro a-tocotrienol has higher antioxidative activity than a-tocopherol against Fe2//ascorbate- and Fe2//NADPH-induced lipid peroxidation in rat liver microsomes.24 Therefore, we hypothesized that tocotrienols might be more effective than tocopherols against UV-induced damage. However, it is not possible to enrich tissues to a large extent with tocotrienols by dietary means,23 probably due to the preferential enrichment of the plasma with a-tocopherol.15 This may be a result of the negative exclusion of all forms of vitamin E in preference to RRR-a-tocopherol by the a-tocopherol transfer protein in the liver.25 Topical application of vitamin E may provide an efficient way of enriching the skin with different forms of vitamin E that have a potentially higher antioxidative activity than a-tocopherol. Therefore, this study was carried out to measure the skin penetration of a mixture of tocopherols and tocotrienols from a tocotrienol-rich palm oil fraction (TRF) and to compare the protection conferred by these various forms of vitamin E against UV-light-induced oxidative stress. MATERIALS AND METHODS
Chemicals All chemicals used were of the highest grade available. TRF was kindly provided by the Palm Oil Research Institute of Malaysia (PORIM, Kuala Lumpur, Malaysia). Tocopherol standards (Covitol) came from Henkel Corporation (LaGrange, IL, USA). Tocotrienols for use as standards were purified from TRF by Dr. Asaf A. Qureshi, University of Wisconsin (Madison, WI, USA). Animals Female hairless mice (8–12 weeks old) were purchased from Charles River Laboratories (Wilmington, MA, USA) and were kept under standard light and temperature conditions. Food (Harlan Teklad Rodent Diet
Fig. 1. Vitamin E structures: a , R1 and R2 Å CH3; b, R1 Å CH3, R2 Å H; g, R1 Å H, R2 Å CH3; d, R1 and R2 Å H.
#1846, Madison, WI, USA) and water were provided ad lib. This experiment was carried out five times using four mice per study. Application A 5% w/v mixture containing TRF in polyethylene glycol-400 (PEG; Sigma, St. Louis, MO, USA) was used. Mice were anesthetized by an intraperitoneal injection of sodium pentobarbital (50 mg/kg body weight) and remained anesthetized during the entire experimental period. Four polypropylene plastic rings (1 cm2) were glued onto the animals’ backs, and TRF (20 ml) was applied to the skin circumscribed by two rings and PEG (20 ml) to the other two rings. After 2 h, the excess substance on the treated area was removed as described by Dupuis et al.26 Briefly, the skin was rinsed three times with 300 ml ethanol:water (95:5), then twice with water alone; then the area was dried with a cotton tip. After the rinsing procedure, the position of the application site was marked, the plastic rings were removed, and the mice were exposed to UVirradiation. UV-irradiation The mice were placed under an Oriel 1000-W solar simulator (Oriel, Stratford, CT, USA) with an output of 2.8 mW/cm2 of UVA and UVB light (290–400 nm) and irradiated for 29 min (corresponding to 3 MED) on either the upper or the lower back, while the other part was shielded from UV-light by covering the skin with paper and aluminum foil. After exposure, the mice were killed by neck dislocation, and the skin with subcutaneous fat was excised from the four exposure sites and the samples immediately frozen in liquid nitrogen. The samples were stored up to a week at 0807C. Pre-
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liminary experiments indicated no differences in the concentrations of tocopherols and tocotrienols of skin with or without attached subcutaneous fat.27 Skin extraction Tocopherols and tocotrienols were determined by HPLC-electrochemical detection after hexane extraction, as described.28,29 Briefly, the skin was weighed (É40 mg), ground under liquid nitrogen, homogenized in a Potter-Elvehjem homogenizing tube with 2 ml buffer (10 mM phosphate, 130 mM NaCl, 1 mM EDTA, pH 7.0) and 50 ml BHT (1 mg/ml), and extracted after addition of 1 ml of 0.1 M SDS, 2 ml ethanol, and 2 ml hexane. An appropriate aliquot was used for HPLC analysis. Dietary vitamin E The amount of vitamin E in mouse chow was determined after saponification. Briefly, 1 g of chow was mixed vigorously with 5 ml of 1% ascorbic acid in water. After addition of 10 ml ethanol and 1.5 ml saturated KOH, the samples were saponified at 707C for 30 min, protected from light. After cooling on ice and addition of 5 ml water, the samples were extracted with 10 ml hexane. Then 5 ml of the upper hexane layer was collected and taken to dryness under nitrogen. The residue was diluted 25-fold and 20 ml was injected onto the HPLC. Irradiation of the tocotrienol-rich fraction in vitro The 5% TRF mixture was transferred to a 1-cm2 quartz cuvette and irradiated for 29 min as described above for mice. After irradiation, the samples were diluted 1:1250 in ethanol and analyzed by HPLC for the content of vitamin E. Tocopherol and tocotrienol analysis It was necessary to develop a method for the simultaneous measurement of various vitamin E forms, ubiquinols, and ubiquinones because TRF contains substances that interfere with the measurement of ubiquinol and ubiquinone by our standard procedures; the details of the method are reported elsewhere.27 The HPLC system consisted of a Hewlett Packard 1050 series gradient pump, a SCL-10A Shimadzu System Controller with a SIL-10A autoinjector with sample cooler, a Beckman Ultrasphere ODS C-18 column (4.6 mm i.d., 25 cm, 5 mm particle size) with a Rainin Spheri-5 RP-18 pre-column (5 micron, 30 1 4.6 mm), a Hewlett Packard 1050 diode array detector, and an LC-4B elec-
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trochemical detector from Bioanalytical Systems (BAS, West Lafayette, IN, USA) using a glassy carbon electrode. The detectors were set up in line, the eluent first passing through the diode array detector. The mobile phase consisted of a mixture of A (80:20 v/v methanol:water and 0.2% w/v lithium perchlorate) and B (ethanol, reagent grade, with 0.2% w/v lithium perchlorate) at a flow rate of 1 ml/min. Optimal separation and quantitation of a- and g-tocopherols and a- and g-tocotrienols were obtained using 61% B and 39% A; these were the conditions used for two experiments (n Å 8 mice). Subsequently, optimal conditions using a gradient were achieved for the measurement of ubiquinol and ubiquinone. Here, the initial composition was 61% B and 39% A, after 16 min the mobile phase was changed linearly over 2 min to 100% B, which was continued for 10 min, then was changed linearly over 2 min to the initial composition; total run time was 40 min. The electrochemical detector was operated with a 0.5-V potential, full recorder scale at 50 nA, for quantitation of a- and g-tocopherols, a- and g-tocotrienols, and ubiquinol. Quantitation was carried out by comparison of peak areas to the area of standard curves obtained with authentic compounds. For vitamin E, aand g-tocopherols were used as standards because the chromanol nucleus is the same in a-tocopherol and atocotrienol and in g-tocopherol and g-tocotrienol, respectively. The diode array detector collected spectra at 275 nm for quantitation of ubiquinone-9, which was carried out using peak area comparisons to a standard curve obtained with authentic ubiquinone-9. Phospholipid hydroperoxide quantitation Skin (about 50 mg) was accurately weighed, homogenized with 2 ml of 1:2 methanol:chloroform containing 20 ml BHT (1 mg/ml), then 0.8 ml 0.25 mM NaCl was added and the lower layer was collected and dried under nitrogen. The residue was dissolved in ethanol:methanol (79:21) and injected onto the HPLC system. Lipid hydroperoxides were detected by chemiluminescence as described by Yamamoto et al.30,31 The HPLC setup included in-line UV and chemiluminescence detection. The isocratic mobile phase (70: 25:5 methanol:2-propanol:40 mM NaH2PO4) passed through a Spherisorb S5 NH2 column, then through the UV-detector (206 nm). The effluent was mixed with the chemiluminescence cocktail (1:1 methanol:55 mM potassium tetraborate, pH 10, 177 mg/L isoluminol, 1.5 mg/L microperoxidase) at a postcolumn mixing tee, and the emitted light was measured using a chemiluminescence detector. The data are reported as phos-
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phatidyl choline hydroperoxides/phosphatidyl choline (PCOOH:PC, mmol/mol). These conditions were used for one experiment (n Å 4 mice). Statistical analysis The differences in vitamin E and ubiquinone/ubiquinol contents of the mice were analyzed using twofactor repeated measures ANOVA as all the treatments were given to each individual. The data were transformed using natural logarithms prior to statistical analysis. The vitamin E concentration data from the five experiments are pooled (n Å 19), the ubiquinol/ubiquinone concentrations are from eight mice, and the lipid hydroperoxides are from four mice. The differences between the percentage distribution of vitamin E in the TRF-treated murine skin and in the TRF fraction were investigated using one-way ANOVA. All statistical analysis was carried out using SuperAnova for the Macintosh (Berkeley, CA, USA). A p-value õ .05 was considered statistically significant. RESULTS
Tocopherols and tocotrienols in murine skin The tocopherols and tocotrienols in PEG-treated murine skin were determined to establish the baseline concentrations of vitamin E (Fig. 2, upper panels, A / B). a-Tocopherol concentrations were about 10-fold higher than those of the other vitamin E forms. The presence of tocotrienols in skin, however, was surprising. Therefore, tocotrienols and tocopherols were also measured in skin from three untreated mice and were found at concentrations similar to those in the PEGtreated mouse skin (data not shown). To investigate the origin of the skin tocotrienols, the vitamin E content of mouse chow (diet routinely fed to the mice) was measured. The diet contained a-tocopherol (29.7 { 6.2 mg/kg diet), g-tocopherol (10.3 { 1.1), a-tocotrienol (3.1 { 0.7), and g-tocotrienol (7.4 { 1.7). The distribution of vitamin E forms in chow was different from that in skin of untreated mice—a significantly (p õ .01) lower proportion of a-tocopherol was found in chow (58 { 7%) as compared with skin (89 { 2%); tocotrienols contributed approximately 20% of the total vitamin E in chow (Fig. 3). It is therefore likely that the tocotrienols found in mouse skin arise from the diet, not by contamination from the TRF-treated sites on the mice. Cutaneous absorption of tocopherols and tocotrienols following topical application To evaluate penetration of the different tocopherols and tocotrienols after topical application of TRF, the
Fig. 2. Vitamin E content of murine skin. The upper panels show the concentrations (mean { SD, n Å 19) of (A) a-tocopherol and (B) g-tocopherol, a-tocotrienol, and g-tocotrienol in skin from nonirradiated or UV-irradiated hairless mice with topical application of PEG. The lower panels show the concentrations of (C) a-tocopherol and (D) g-tocopherol, a-tocotrienol, and g-tocotrienol in skin after topical application of TRF to hairless mice before and after UVirradiation. Significant decreases in concentrations of each of the homologues were observed after UV-irradiation in both PEG-treated and TRF-treated skin. By least-squares mean comparisons: PEG vs. PEG / UV for a-tocopherol, p õ .005; for g-tocopherol, p õ .0001; a-tocotrienol, p õ .0001; and g-tocotrienol, p õ .0001. TRF vs. TRF / UV for a-tocopherol, p õ .0001; for g-tocopherol, p õ .0001; a-tocotrienol, p õ .0001; and g-tocotrienol, p õ .0001. PEG vs. TRF for a-tocopherol, p õ .0001; for g-tocopherol, p õ .004; a-tocotrienol, p õ .0001; and g-tocotrienol, p õ .0001. PEG vs. TRF / UV for a-tocopherol, p õ .0001; for g-tocopherol, p õ .0001; a-tocotrienol, p õ .0001; and g-tocotrienol, p õ .0001.
antioxidants present in the TRF-treated and PEGtreated skin from the same mice were compared (Fig. 2: compare A to C, and B to D). TRF treatment resulted in significant increases in vitamin E concentrations; fractional increases were greater in those forms that were present at low initial concentrations. Thus, TRF treatment resulted in a 28 { 16-fold increase in a-tocopherol, a 80 { 50-fold increase in a-tocotrienol, a 130 { 108-fold increase in g-tocopherol, and a 51 { 36-fold increase g-tocotrienol in the skin (Fig. 2, filled bars: compare A to C, and B to D). To evaluate whether the different vitamin E forms penetrated murine skin differently, the percent distribution of each of the vitamin E homologues in the TRF mixture was compared with its percent distribution in skin (Fig. 3). (The percent distribution was calculated for each form as follows: the concentration in PEGtreated skin was subtracted from the concentration found in the TRF-treated skin, then divided by the sum of the skin vitamin E concentrations 1 100.) The per-
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Fig. 3. Distribution of the vitamin E forms in skin, diet, TRF, and skin-absorbed TRF. The percent distributions (mean { SD) of the various vitamin E isomers in untreated (control) mouse skin (n Å 3), diet (n Å 4), TRF mixture (n Å 4), and TRF-treated skin (minus background; n Å 19) are shown. The percent distributions of vitamin E forms in the control skin were significantly different from their percent distribution in the diet; the percent distribution in TRF is significantly different from the forms absorbed into the skin. Comparison Control skin vs. diet Control skin vs. TRF Control skin vs. skin after TRF minus baseline Diet vs. TRF Diet vs. skin after TRF minus baseline TRF vs. skin after TRF minus baseline
% a-Tocopherol
% a-Tocotrienol
% g-Tocopherol
% g-Tocotrienol
0.0001 0.0001 0.0001 0.0002 NS 0.0001
NS 0.0001 0.0001 0.0001 0.0001 0.001
0.0001 0.0005 0.0001 0.0001 0.0001 NS
0.04 0.0001 NS 0.0005 NS 0.0001
cent distribution of vitamin E forms that penetrated the skin (above background concentrations) was significantly different from their distribution in the TRF mixture. Higher percentages of a-tocopherol were found in TRF-treated skin than were present in TRF (p õ .0001), the percentage of g-tocopherol was similar to the TRF mixture, and both a- and g-tocotrienols represented a smaller proportion than they did in the TRF mixture (p õ .001). Effect of UV-irradiation To evaluate the protection by vitamin E against oxidative damage caused by UV-irradiation, the concentrations of the various vitamin E forms present in the
tissue before and after UV-irradiation were measured. After UV-irradiation, the concentrations of all forms of vitamin E in the PEG-treated skin decreased significantly (p õ .01, except p õ .04 for g-tocopherol; Fig. 2, A / B). They also decreased significantly (p õ .001) after UV-irradiation as compared with nonirradiated areas in the TRF-treated skin (Fig. 2, C / D). Notably, after UV-irradiation, the vitamin E concentrations were significantly higher in the TRF-treated irradiated skin than in the PEG-treated nonirradiated skin (p õ .01). The percent vitamin E remaining after UV irradiation is a measure of the susceptibility of the individual tocopherols and tocotrienols to oxidative depletion. In PEG-treated skin, approximately 80% of the different vitamin E forms remained after UV-irradiation; in the
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in the PEG-treated and TRF-treated skin, respectively (Fig. 5). After UV-irradiation, both ubiquinone and total Q decreased significantly (p õ .001) in PEG-treated and TRF-treated skin. Thus, UV-irradiation resulted in statistically significant decreases in ubiquinol, ubiquinone, and total Q that were not prevented by topically applied TRF. Lipid hydroperoxides
Fig. 4. The percent remaining vitamin E after UV-irradiation. For each of the vitamin E forms, the percent remaining was calculated by dividing the concentration remaining after UV-irradiation by the concentration present before UV-irradiation and multiplying by 100 (mean { SD, n Å 3).
TRF-treated skin, approximately 40% remained (Fig. 4). There were no significant differences in the degree of destruction of the various vitamin E homologues, suggesting that they were equally effective. Because the absolute levels of the different vitamin E forms are much lower in the PEG-treated animals, the standard deviation (in %) appears relatively larger in these measurements. To evaluate whether the decrease in vitamin E forms in the TRF-treated samples was due to direct photodestruction of vitamin E, the effect of UV-irradiation of the TRF solution was determined in vitro. The percent remaining after UV-irradiation was 86% for atocopherol, 83% for g-tocopherol, 83% for a-tocotrienol, and 84% for g-tocotrienol. These data suggest that direct photodestruction was similar for all of these vitamin E forms.
The production of lipid hydroperoxides in response to UV-light was measured as a marker of oxidative stress. There were no differences in the PCOOH:PC ratio in the PEG-treated (27 { 20 mmol/mol) and TRFtreated (15 { 10) skin before or after UV-irradiation (PEG-treated 38 { 42 and TRF-treated 45 { 23, respectively). These data demonstrate that statistically significant peroxidative damage to membrane lipids was not apparent immediately after exposure of the skin of hairless mice to 3 MED UV-irradiation. DISCUSSION
Tocotrienols and tocopherols were found in control and PEG-treated murine skin at concentrations ranging from 0.5 to 9 nmol/g. Of mouse tissues, skin is unique in containing appreciable concentrations of tocotrienols; other mouse tissues contain substantially smaller tocotrienol concentrations.27 Hayes et al.23 demonstrated that tocotrienols represent only a small proportion of vitamin E in tissues from hamsters fed toco-
Protection of endogenous antioxidants To evaluate the protective effects of TRF on antioxidants that were not applied topically, ubiquinol and ubiquinone were quantitated. Ubiquinol concentrations were significantly (p õ .002) lower in the TRF-treated skin (1.2 { 0.7) compared with PEG-treated skin (0.8 { 0.6), but these concentrations are low with large variances, so differences may not be physiologically relevant. Following UV-irradiation, ubiquinol concentrations in both PEG-treated and TRF-treated skin decreased fivefold (Fig. 5; p õ .001). Ubiquinone-9 and total Q (ubiquinone-9 / ubiquinol-9) were similar
Fig. 5. Ubiquinol and ubiquinone in murine skin. The concentrations (mean { SD) of ubiquinol, ubiquinone, and total Q in mouse skin after application of PEG or TRF, and exposed or not to UV-light. All parameters decreased significantly after UV-irradiation. For PEG vs. PEG / UV: ubiquinol, p õ .0001; ubiquinone, p õ .0008; total Q, p õ .0004. For TRF vs. TRF / UV: ubiquinol, p õ .0006; ubiquinone, p õ .001; total Q, p õ .0008. There were no significant differences between PEG / UV vs. TRF / UV.
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trienol-enriched diets. The presence of a- and g-tocotrienols in untreated mouse skin was unexpected because (a) the mouse diets were not specially enriched with tocotrienols, and (b) the liver discriminates against tocotrienols in favor of a-tocopherol during repackaging of dietary fats into very low density lipoproteins for secretion into the circulation.15,25 Suarna et al.32 demonstrated that after feeding TRF to rats, all lipoprotein classes contained tocotrienols. Apparently, transfer of tocotrienols to mouse skin must take place following absorption and transport of dietary vitamin E in chylomicrons during postprandial chylomicron clearance and during delivery of tocotrienol-containing lipoproteins.15,25 It is unlikely that these skin tocotrienols were contaminants from the TRF application site because skin from untreated mice (control, Fig. 3) contained concentrations of these vitamin E forms similar to those found in PEG-treated mouse skin. Additionally, the vitamin E forms were distributed differently (a) in the skin, (b) in the diet, and (c) in the TRF mixture. After topical application of TRF, all the vitamin E forms readily penetrated into the skin of hairless mice and were present in concentrations far exceeding the baseline levels (Fig. 2). Norkus et al.33 have also demonstrated that application of a-tocopheryl acetate onto hairless mouse skin results in penetration of high concentrations into the skin. The distribution of the various vitamin E forms in TRF was different from that present in the TRF-enriched mouse skin: a larger fraction of a-tocopherol was absorbed into the skin compared with other forms of vitamin E in TRF (Fig. 3). The percentage of g-tocopherol was similar to TRF; both aand g-tocotrienols represented a smaller proportion than they did in TRF. These data suggest that the isoprenoid tail of the tocotrienols may hinder their penetration into skin. Additionally, the preferential penetration of a-tocopherol into skin suggests that there may be specific mechanisms in skin for enrichment with atocopherol. This discrimination between vitamin E forms, as well as their localization in skin compartments after topical application, merits further investigation. Tocopherols and tocotrienols in murine skin, applied topically or derived from the diet, were significantly depleted by UV-irradiation, indicating a protective antioxidant function (Fig. 2). All vitamin E forms in mouse skin, including small amounts of a- and g-tocotrienols, decreased similarly in response to UV-irradiation, demonstrating that they all afford similar antioxidant protection (Fig. 4). TRF application increased skin concentrations of the various vitamin E homologues, and these remained significantly elevated after exposure to UV-light compared with PEG-treated skin.
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The similarity in the degree of depletion of the vitamins in response to an oxidative stress suggests that these vitamin E forms protect similarly against UV-irradiation-induced damage. This observation was also verified by exposing the TRF mixture directly to UV-light. These findings are in contrast to those of Serbinova et al.,24 who reported after in vitro enrichment of microsomal membranes with a-tocopherol or a-tocotrienol that a-tocotrienol has a greater antioxidant activity than does a-tocopherol. In studies of rat and human low density lipoproteins enriched in tocotrienols and oxidized using an azo-initiator to generate a constant rate of peroxyl radicals, Suarna et al.32 reported that the antioxidative activities of a-tocopherol and a-tocotrienol were similar and that the a-isomers had higher activity than did the g-isomers.32 Taken together, these results suggest that depletion of skin vitamin E in vivo using UV-light may be quite different from other oxidizing systems. A larger percentage of the various vitamin E forms remained after UV-irradiation of the PEG-treated compared with the TRF-treated skin (Fig. 4). This implies a greater destruction of the various vitamin E forms in the TRF-treated skin. Whether this is due to increased free radical scavenging remains to be clarified. Localization of the TRF nearer to the upper epidermal layers in the TRF-treated skin could allow increased destruction during UV-irradiation. Alternatively, the TRF vitamin E may have penetrated the lipid components surrounding cells and thus may not have been accessible to aqueous antioxidants that could recycle the vitamin E. Thus, the applied TRF may have a different behavior during UV-exposure than the vitamin E naturally present. It should be emphasized that the skin was washed with ethanol and dried before exposure to UV-light; therefore, the vitamin E forms we have measured are not on the skin surface, but have penetrated into the skin. The lack of detectable formation of lipid hydroperoxides suggests that the applied irradiation was insufficient to cause severe peroxidative damage to membrane lipids, although a significant decrease was seen in the endogenous antioxidant, ubiquinol-9 in response to the irradiation. The applied vitamin E was unable to protect this endogenous antioxidant against UV-induced oxidative injury, although the vitamin E levels in the skin were increased above the endogenous levels, even after UV irradiation. The antioxidative effect of the applied vitamin E was thus limited. Coenzyme Q (ubiquinone/ubiquinol) was chosen as a marker for oxidative damage because ubiquinol is the most labile lipid-soluble antioxidant34–36 and is not present in TRF. Ubiquinol is oxidized prior to a-tocopherol during UV-irradiation of skin and is substan-
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tially depleted before a-tocopherol concentrations are affected.6 We found during this study that TRF contains substances that interfere with the measurement of ubiquinol and ubiquinone in skin by our standard procedures; therefore, we developed a gradient method for measuring ubiquinol and ubiquinone.27 The levels of ubiquinol detected in murine skin are low; nonetheless, after UV-irradiation, ubiquinol, ubiquinone, and total Q all decreased significantly. The disappearance of the ubiquinol that was observed may be due to the direct photodestruction of ubiquinol or to UV-induced oxidative stress, causing decreased capacity in the ubiquinone reducing systems and subsequent further oxidation of the ubisemiquinone radical to nonrecyclable end products, resulting in loss in total Q. The direct photodestruction of ubiquinone is very improbable, as the absorption maximum (275 nm) is below the UVB range (290–320 nm). Direct photodestruction of ubiquinol is possible, since the absorption maximum is 290 nm. However, previous studies in our laboratory have shown that photodestruction accounts for only approximately 30% of the ubiquinol loss during UV-irradiation.37 Regardless of the route of destruction, UV-irradiation caused a loss in the total Q pool, thus depleting the skin of a vital component. A variety of antioxidants, especially vitamin E and ubiquinol, are present in skin. Topical application provides an efficient means of enriching the tissue in protective antioxidants, such as vitamin E. This paper demonstrates that tocopherols and tocotrienols from TRF readily penetrate the skin of hairless mice, are present in levels far above baseline after topical application, and are consumed during UV-light-induced oxidative stress. Acknowledgements — Beth Koh and Kenneth Tsang provided excellent technical assistance. We gratefully acknowledge the efforts of Dr. Asaf A. Qureshi, University of Wisconsin (Madison, WI, USA), who isolated tocotrienols for use as standards for this study. This study was supported by grants from the NIH (CA 47597) and the Palm Oil Research Institute of Malaysia. J.J.T. was supported by a fellowship of the Fritz Thyssen Stiftung, Germany (AZ 21295008).
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ABBREVIATIONS
BHT—butylated hydroxy toluene EDTA—ethylene diamine tetra-acetic acid HPLC-EC—high pressure liquid chromatography with electrochemical detection MED—minimal erythemal dose PEG—polyethylene glycol-400 total Q—sum of ubiquinone and ubiquinol TRF—tocotrienol-rich fraction of palm oil UV—ultraviolet
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