Healthy skin possesses an antioxidant defense system against oxidative stress.1-4 However, overexposure to ultraviolet radiation (UVR) can overwhelm this defense system’s capacity, leading to skin disorders such as sunburn, photosensitivity reactions or immunologic suppression, as well as photoaging, malignant skin tumors and other long-term sequelae.1,2 Application of topically exogenous antioxidants may prevent or minimize such damage.1,5-8
A previous column by these authors described a rapid, accurate and facile method to quantify the antioxidative capacity of topical formulations in vitro.9 The present article introduces an in vivo model using a photochemiluminescence device and biophysical methods to determine the antioxidative capacity of a topical o/w skin care emulsion with and without vitamin E. The human skin tested was exposed to UVR.
Materials and Methods
Subjects: Ten healthy Caucasians (three male and seven female; mean age 47 ± 10) of the skin types II or III were enrolled.10 The Committee on Human Research at the University of California, San Francisco approved this study.
Test materials: A facial skin care o/w emulsiona was obtained with pH balanced at 5.5% and 22.5% oil content. The emulsion contained water, petrolatum, myreth-3 myristate, glycerin, cetearyl alcohol, tocopheryl acetate, ceteareth-20, dimethicone, sodium PCA, sodium citrate, sodium carbomer, fragrance, benzyl alcohol, methylparaben and propylparaben. The active ingredient in the test emulsion was 2.3% vitamin E as tocopheryl acetate; the vehicle control contained no vitamin E but was otherwise identical. Study design: The study was randomized, double blind and placebo-controlled.
Ultraviolet radiation source: The UV radiation sourceb produced an irradiation of 1.2 mW/cm2 at a distance of 20 cm as measured by a radiometerc. Details of the emission spectrum ranges are described elsewhere.6
Determination of individual minimal erythema dose: The minimal erythema dose (MED) was measured on the flexor aspects of each subject’s forearms. Before day 1 of the study, one forearm of each subject was exposed to a UV light for 30 sec to 3 min to induce the MED. The MED was determined at 24 hr post UV exposure. Details of the method are described elsewhere.6,10
Evaluation of skin response: The following measurements were used to evaluate the UV-induced skin response at baseline, day 2 and day 3 post UV exposures:
• Visual scores (VS) assessment of the test sites were performed by one investigator according to the scale6 with modification: 0 = no redness; 1 = slight redness with a blurred boundary; 2 (= MED) = moderate redness with a sharp boundary; 3 = intense redness; 4 = fiery redness with edema.
• Transepidermal water loss (TEWL) was assessed by a closed evaporimeterd. TEWL documents integrity of stratum corneum (SC) water barrier function and is a sensitive indicator of skin water barrier alteration.11 The measuring principle and standard guidelines are published.12,13 The values of TEWL were expressed as g/m2 per hr.
• Blood flow volume (BFV) was measured instrumentallye to observe the blood flux at test sites. Details and standard guidelines for use were utilized.11,14,15 The values of BFV were expressed as arbitrary units (a.u.).
• Skin color (a* value) was quantified by using a colorimeterf. The a* value represents the color spectrum from total green to pure red and is known to correlate closely with erythema quantification.11,16,17 The values of a* were expressed as a.u.
• Skin capacitance was measured by a corneometerg. Capacitance is a parameter for SC hydration. Details of the methods are described elsewhere.18 The values of capacitance were expressed as a.u.
The measurements were conducted in a room with daily relative humidity (RH) of 58.9 ± 2.0% and temperature of 19.9 ± 0.8°C. Each subject was rested at least 30 min for acclimation before measurements.
Treatment and irradiation procedure: On study day 1, measurements of TEWL, a*, BFV, and skin capacitance were taken at three test sites (1.5 cm2 per site) on the forearm not used to determine each subject’s MED. Following measurements, two coded test emulsions were applied to two pre-designed test sites. After 30 min, these test sites on this forearm were exposed to a UV light to induce the MED. One untreated site (without emulsions) served as a blank control. During UV exposure, forearms were protected by a black cloth everywhere except at the test sites. Visual scoring and instrumental measurements were recorded at 24 hr and 48 hr post UV exposure.
At day 3, after completing instrumental measurements, each test site was stripped three times with a proprietary adhesive tape disch. These tape discs were quantified for antioxidant capacity using a photochemilluminescence devicej. Details of measuring method and principles of photochemilluminescence analysis are found elsewhere.9,19
Statistical analysis: Statistical analysis was performed using a computer programk. Nonparametric data, such as visual scores, were analyzed by the Friedman Repeated Measures with the ANOVA on ranks. Parametric data such as TEWL, skin capacitance, BFV, a* values, and quantity of antioxidant capacity were analyzed with the One Way Repeated Measures ANOVA. All pairwise multiple comparison procedures were made with Student-Newman-Keuls Method. Statistical significance was accepted at P<0.05.
Results and Discussion
Visual scores: Vitamin E emulsion and vehicle control significantly (P<0.05) suppressed VS as compared to the blank control at day 2 and day 3 post UV exposure. However, vitamin E emulsion showed significantly (P<0.05) lower VS when compared to vehicle control at day 2 and day 3 post UV exposure (Figure 1).
Skin color: Vitamin E emulsion and its vehicle control significantly (P<0.05) diminished a* values when compared to the blank control at day 2 and day 3 post UV exposure (Figure 2). There was no statistical difference between those two emulsions at each time point.
Blood flow volume: At day 2 post UV exposure, only vitamin E emulsion significantly (P<0.05) reduced BFV as compared to the blank control. Vitamin E emulsion and its vehicle control showed significant (P<0.05) reduction of BFV as compared to the blank control at day 3 post UV exposure (Figure 3). There was no statistical difference between those two emulsions at either time point.
Transepidermal water loss: There was no statistical difference among the three tested sites at any time point.
Capacitance: Vitamin E emulsion and its vehicle control significantly (P<0.05) increased skin capacitance as compared to the blank control at day 2 post UV exposure (Figure 4). At day 3 post UV exposure, only the vehicle control showed significantly (P<0.05) increased values of capacitance in comparison with the blank control. There was no statistical difference between those two emulsions at either time point.
Antioxidant capacity analysis: In analyses of the first of the three consecutive strippings, vitamin E emulsion and the blank control showed significantly (P<0.05) higher quantity of antioxidant capacity than the vehicle control. However, there was no statistical difference between stripping 2, 3 or in total strippings.
Exposure to UVR may induce dramatic alternations in skin barrier function; the prime mechanism of UVR-induced damage to cutaneous tissues is thought to be the peroxidation of lipids.2 A single dose of UVR depleted human SC vitamin E by almost 50% and murine SC vitamin E by 85%.20 However, application of topical antioxidants may diminish or minimize such UVR-caused deleterious effects on skin.1,5–8
Vitamin E (tocopheryl acetate) is a lipophilic endogenous antioxidant that provides protection against UV-induced oxidative membrane damage. It is believed that the broad biological activities of vitamin E are due to its ability to inhibit lipid peroxidation and stabilize biological membranes.1,5,6 Vitamin E is a popular component used as an antioxidant in many skin care products to decrease or reverse photoaging and photodamage and this effect has been documented.1,4,8
Healthy human skin has a slightly acid pH and the acidity of the skin maintains antimicrobial activity.21,22 Skin diseases might result if this natural acidic mantle has been disturbed. Particular attention should be paid to this issue when developing skin care products.
The current study tested an emulsion containing two important features: vitamin E and a slightly acidic formulation pH-balanced at 5.5. Previously, these authors demonstrated that this emulsion has superior antioxidant effect over its vehicle control in vitro.9 In the current study, vitamin E emulsion and its vehicle control proved effective in preventing the induction of erythema and reducing inflammation from the UV exposure. Vehicles are well known to influence the hydration of the SC and have some “therapeutic” effects.23 The vehicle control showed partial benefits against UVR. The authors speculate that the vehicle control might have some ability for sunscreening. However, the effect of vitamin E emulsion exceeded the activity of the vehicle control.
Analyzing antioxidant capacity did not show statistical difference except in the first SC stripping. It might be due to the UVR being too aggressive and even in the short period of less than three minutes it depleted the endogenous and exogenous vitamin E in the superficial SC layers. Further experiment is suggested to take stripping into the deeper SC layer in order to explore the time point of UVR exposure affecting depth in SC. Additionally, to decrease UVR exposure—for example, below MED level—is also an interesting study.
Overexposure to UVR may lead to skin disorders. Supplying the topical exogenous antioxidants to the skin may prevent or minimize such damage. Described here was an in vivo model to determine antioxidative capacity of a topical skin care emulsion versus the emulsion’s vehicle on human skin that was exposed to UVR. Results suggest the test emulsion and its vehicle control inhibited the induction of erythema and reduced inflammation caused by the UV exposure.
1. DP Steenvoorden and GM van Henegouwen, The use of endogenous antioxidants to improve photoprotection, J Photochem Photobiol B: Biol 41 1–10 (1997)
2. JJ Thiele, Oxidative targets in the stratum corneum: a new basis for antioxidative strategies, Skin Pharmacol Appl Skin Physiol 14 Suppl 1 87–91 (2001)
3. R Kohen, Skin antioxidants: their role in aging and in oxidative stress — new approaches for their evaluation, Biomed Pharmacother 53 181–192 (1999)
4. JJ Thiele, F Dreher and L Packer, Antioxidant defense systems in skin, In: Cosmeceuticals, Drugs vs. Cosmetics, P Elsner and HI Maibach, eds, New York: Marcel Dekker (2000) pp 145–187
5. B Eberlein-Konig, M Placzek and B Przybilla, Protective effect against sunburn of combined systemic ascorbic acid (vitamin C) and d-alpha-tocopherol (vitamin E), J Am Acad Dermatol 38 45–48 (1998)
6. F Dreher, B Gabard, DA Schwindt and HI Maibach, Topical melatonin in combination with vitamins E and C protects skin from ultraviolet-induced erythema: a human study in vivo, Br J Dermatol 139 332–339 (1998)
7. F Stab, R Wolber, T Blatt, R Keyhani and G Sauermann, Topically applied antioxidants in skin protection, Methods Enzymol 319 465–478 (2000)
8. F Dreher and HI Maibach, Protective effects of topical antioxidants in humans, In: Oxidants and Antioxidants in Cutaneous Biology, JJ Thiele and P Elsner, eds, Basel: Karger (2001) pp 157–164
9. H Zhai, MJ Choi, M Arens-Corell, BA Neudecker and HI Maibach, A rapid, accurate, and facile method to quantify the antioxidative capacity of topical formulations, Skin Res Technol 9 254–256 (2003)
10. A Do and J Koo, Initiating narrow-band UVB for the treatment of psoriasis: table 4, how to do MED skin testing, Psoriasis Forum 10 7–11 (2004)
11. KP Wilhelm, C Surber and HI Maibach, Quantification of sodium lauryl sulphate dermatitis in man: comparison of four techniques: skin color reflectance, transepidermal water loss, laser Doppler flow measurement and visual scores, Arch Dermatol Res 281 293–295 (1989)
12. J Pinnagoda, RA Tupker, T Agner and J Serup, Guidelines for transepidermal water loss (TEWL) measurement, Contact Derm 22 164–178 (1990)
13. P Elsner, E Berardesca and HI Maibach, eds, Bioengineering of the skin: water and the stratum corneum, Boca Raton: CRC Press (1994)
14. A Bircher, EM DE Boer, T Agner, JE Wahlberg and J Serup, Guidelines for measurement of cutaneous blood flow by laser Doppler flowmetry, Contact Derm 30 65–72 (1994)
15. E Berardesca, P Elsner and HI Maibach, eds, Bioengineering of the skin: cutaneous blood flow and erythema, Boca Raton: CRC Press (1995)
16. KP Wilhelm and HI Maibach, Skin color reflectance measurement for objective quantification of erythema in human beings, J Am Acad Dermatol 21 1306–1308 (1989)
17. A Fullerton, T Fischer, A Lahti, KP Wilhelm, H Takiwaki and J Serup, Guidelines for measurement of skin colour and erythema, Contact Derm 35 1–10 (1996)
18. H Tagami, M Ohi, K Iwatsuki, Y Kanamaru, M Yamada and B Ichijo, Evaluation of the skin surface hydration in vivo by electrical measurement, J Invest Dermatol 75 500–507 (1980)
19. IN Popov and G Lewin, Photochemiluminescent detection of antiradical activity. IV: Testing of lipid-soluble antioxidants, J Biochem Biophys Methods 111–118 (1996)
20. JJ Thiele, MG Traber and L Packer, Depletion of human stratum corneum vitamin E: an early and sensitive in vivo marker of UV induced photo-oxidation, J Invest Dermatol 110 756–761 (1998)
21. K Chikakane and H Takahashi, Measurement of skin pH and its significance in cutaneous diseases, Clin Dermatol 13 299–306 (1995)
22. F Rippke, V Schreiner and HJ Schwanitz, The acidic milieu of the horny layer: new findings on the physiology and pathophysiology of skin pH, Am J Clin Dermatol 3 261–272 (2002)
23. JW Fluhr and L Rigano, Clinical effects of cosmetic vehicles on skin, J Cosmet Sci 55 189–205 (2004)