Comparative Topical Absorption and
Antioxidant Effectiveness of Two Forms of
Coenzyme Q10 after a Single Dose and
after Long-Term Supplementation
in the Skin of Young and Middle-Aged Subjects

Joe Vinson, Sunil Anamandla
Department of Chemistry,
The University of Scranton, Scranton, PA 18510-4626, USA

Abstract
Coenzyme Q10 is an endogenous antioxidant found in the skin along with vitamins A, C and E. Coenzyme Q10 is used increasingly in cosmetic products and is advertised as a skin energizer, protector against skin aging, skin repairer, and an anti-wrinkling agent. As the outermost layer of skin, the stratum corneum is the interface between the body and the environment and requires antioxidants to protect it and the epidermis and dermis below it. Nutrient levels in the stratum corneum correlate with those in the skin. Two forms of coenzyme Q10 (pure coenzyme Q10 and a yeast-based coenzyme Q10) were investigated in a ommercial vehicle. Two groups of subjects were tested aged 21-29 and 51-70, respectively. Coenzyme Q10 absorption in the stratum corneum was determined after a 1-hour application and ethanol extraction. Significantly more yeast coenzyme Q10 was absorbed than the pure coenzyme Q10 and the middle-aged subjects absorbed about twice as much coenzyme Q10 as did the younger subjects. Skin antioxidants were significantly increased by yeast coenzyme Q10 but not by pure coenzyme Q10. Peroxides declined in the stratum corneum after twice daily application of coenzyme Q10 with both forms but the decrease was greater with the yeast form. The older subjects had significantly higher baseline levels of lipid peroxides than did the younger group, indicating an increase in skin oxidative damage with age. The yeast coenzyme Q10 is the superior form of coenzyme Q10 for human skin application.

INTRODUCTION
The ubiquinones or coenzyme Q are a family of lipid-soluble benzoquinones that are widely distributed in living organisms and present in the hydrophobic interior of the phospholipid bilayer of virtually all cellular membranes. The quinone head can alternately assume three different redox states: ubiquinone (Q), the fully oxidized form; ubisemiquinone (•QH), the partially reduced form which is also a free radical; and ubiquinol (QH2), the fully reduced form. The isopropenoid side chain has various lengths (30-50 carbon atoms). The different forms of coenzyme Q are designated by the number of isopropenoid units in the side chain (2-10), with there being 9 or 10 in mammals and 2 in bacteria. Coenzyme Q10 is the human form of coenzyme Q, and it is ubiquitous in human tissue. The level is highest in organs with high rates of metabolism such as the heart and liver [1].

Coenzyme Q can play multifunctional roles in cells, three of which have been well documented. First, the most studied function is that of coenzyme Q in its quinone form. It transfers electrons in the mitochondrial electron transport chain from complexes I and II to complex III, during which protons are passed to the outer mitochondrial compartment thus generating a transmembrane electrochemical gradient [2]. Secondly, it is now well established that the quinol form of coenzyme Q acts as a potent antioxidant in the inner mitochondrial membrane. It inhibits lipid peroxidation either by scavenging free radicals directly or by reducing the α-tocopheroxyl radical to α-tocopherol [3]. Thirdly, it acts as a pro-oxidant in that autoxidation of the semiquinone form is the major intracellular source of superoxide and hydrogen peroxide generation [4]. Thus coenzyme Q can be both an antioxidant and a pro-oxidant.

The skin is the body’s first line of defense against the radicals formed by UVA light from the sun. These radicals cause oxidative damage to lipids, proteins and DNA. The first barrier in the skin is the stratum corneum, which is composed of surface lipids, corneocytes and intercellular lipids. In the skin, the reduced form of coenzyme Q, ubiquinol-10, acts as an antioxidant, with 10-fold higher levels in the epidermis than in the dermis [5]. After UV exposure, mouse skin ubiquinol-9 was depleted more rapidly than α-tocopherol, suggesting that coenzyme Q9 is the first line of defense [6].

Oxidative stress is thought to play a role in the aging process, which is a combination of chronological aging and photoaging [7]. Using human fibroblasts it could be shown that prolonged cell culture mimics chronological aging and that coenzyme Q10 significantly increased cell proliferation [1]. Coenzyme Q10 was also found to increase the life span of the worm C. elegans by reducing oxidative stress, namely by decreasing superoxide [8]. Photoaging is caused by both UVA and UVB. Photoaged skin is characterized by wrinkles and a lack of tensile strength, which is normally provided by the dermis. Hyaluronic acid is lowered during photoaging, which also causes disorganization of the dermal matrix due to the degradation of collagen fibers. Coenzyme Q10 reduced the detrimental effects of UVA on dermal fibroblasts, which maintain the dermal matrix [1]. The level of coenzyme Q10 declines in heart and brain with age [9, 10]. It increases in the stratum corneum from childhood to maturity and then decreases with age [11]. Coenzyme Q10 also decreases in the epidermis in a linear fashion with age [1] and thus the skin is a tissue that would benefit from supplementation.

In order to act as an antioxidant, coenzyme Q10 needs to penetrate into the skin. When a solution of 1% coenzyme Q10 in olive oil was topically applied to rats, coenzyme Q10 was found to reach levels in living rat skin of 8 μg/g after 2 hours and 15 μg/g after 4 hours. There also was a dose-response relationship between the amount of coenzyme Q10 applied and the coenzyme Q10 skin concentration [12]. In porcine skin, coenzyme Q10 administered in ethanol vehicle penetrated into the stratum corneum, with 20% penetrating further into the viable layers of the epidermis and 2% into the dermis [1]. In this study we investigate two forms of coenzyme Q10 to determine which form has the greater absorption and antioxidant efficacy after topical application to young and older humans.

EXPERIMENTAL

Subjects
Eight subjects (4 males and 4 females) aged 21-29 years (average 24 ± 3) and 8 subjects (3 males and 5 females) aged 51-70 years (average 56 ± 7) participated with informed consent. None had any current history of dermatological problems, and they were told to avoid excessive sun exposure during the study. No subjects were taking cholesterol-lowering drugs as determined by a medical history questionnaire.

Skin treatment long-term study study
Five young subjects and 4 middle-aged subjects participated in this study. A 75 mg portion of ointment containing either the yeast or the pure form of coenzyme Q10 (ubiquinone) was smeared on a 25-mm diameter circular area of the inner wrist (randomly chosen) and allowed to absorb for one hour. The stratum corneum coenzyme Q10 of the volunteers was extracted from skin by applying a 25-mm diameter glass cylinder to skin and then rinsing that area three times with 1 mL of anhydrous, non-denatured ethanol [13]. The 3 mL ethanol extract was concentrated 10 times by evaporation and reconstitution with 300 μL of ethanol. This procedure oxidizes ubiquinol to ubiquinone. Extracts were stored at –20°C until assayed.

Skin treatment long-term study
The subjects were given (blinded) two kinds of lotion (coenzyme Q10 and yeast coenzyme Q10), which they applied to the same arm (ventral forearm) in a dose of approximately 75 mg twice every day (morning and evening) for one month using a 1 mL syringe and dispensing 0.1 mL. This applies 75 μg of coenzyme Q10. Extraction was done as described for the absorption study in the morning after the last evening application. No lotion was then applied for 30 days and the extraction was repeated. There was no washout period.

Coenzyme Q10 (oxidized form, ubiquinone) and all reagents were obtained from Sigma Chemical Co., St. Louis, MO, USA. Yeast coenzyme Q10 was 8% coenzyme Q10 by weight in a glycoprotein matrix and was obtained from Phamachem Labs, Kearny, NJ, USA. Jergens Ultra Healing Lotion (Andrew Jergens Co., Cincinnati, OH, USA) was used as the vehicle. This lotion does not contain any added vitamin antioxidants. The ointments with USP coenzyme Q10 and yeast coenzyme Q10 were prepared by weighing. For example 10 mg of coenzyme Q10 was mixed with 10 grams of the lotion or 125 mg of 8% yeast coenzyme Q10 was mixed with 10 grams of the lotion to make a 1 mg coenzyme Q10/g lotion. The lotion was melted in a furnace at 95°C for 2 minutes, uniformly mixed with coenzyme Q10 and sealed during storage.

Assays
Coenzyme Q10 Assay. Ubiquinone was used as the standard. A Hewlett Packard Series 1050 High Performance Liquid Chromatograph was equipped with a Perkin Elmer cartridge C18 column (4.6 mm x 3.5 cm). The mobile phase solvent was 25% isopropanol/75% ethanol and the detector wavelength was 275 nm. The flow rate was 2.0 mL/min.

Antioxidant (AOX) Assay [14]
The AOX reagent working solutions were 300 mM acetate buffer (pH 3.6), 10 mM 2,3,6-tripyridyl-s-triazine (TPTZ) in 400 mM HCl, and 20 mM FeCl3. AOX reagent was prepared fresh daily by combining 20 mL of acetate buffer, 0.3 mL of FeCl3 and 0.3mL of TPTZ. A standard solution of 1 mM α-tocopherol in methanol was prepared and standard dilutions from 50 μM to 500 μM were made for the standard curve. Methanol was utilized as the blank. The automated reaction was performed in a temperature-controlled spectrophotometer (Spectronic Genesys 5, Milton Roy, Rochester, NY). A disposable cuvette containing 2.0 mL of the AOX working reagent was placed in the spectrophotometer at a wavelength of 593 nm and constant temperature of 37°C for 6.0 minutes and the absorbance was recorded. 50 μL of blank, standard or sample were added to separate cuvettes that were covered tightly with parafilm and inverted 5 times before they were replaced in the spectrophotometer for 6.0 minutes and the absorbance was recorded again. Each sample was measured in duplicate.

Lipid hydroperoxides and hydrogen peroxide assay [15]
Reagent (1) was 25 mM ammonium iron (II) sulfate in 110 mM perchloric acid. Reagent (2) was 100 mM sorbitol and 125 μM xylenol orange in 110 mM perchloric acid. The working reagent was prepared by mixing 1 volume of Reagent (1) with 100 volumes of Reagent (2). Fresh standards were prepared each day for use from 0.025 M stock solution of H2O2 to yield 1-100 μM standards. Nanopure water was used as the blank. One mL of working reagent was pipetted into each cuvette and 10 μL of blank, standards and samples were added to the appropriate cuvettes. The solutions in the cuvettes were mixed thoroughly and incubated for 30 minutes at room temperature. Absorbance was read at 560 nm.

Statistics
Results are given as mean values and standard errors of the mean. Statistical differences between treatments were evaluated by a paired Student’s t test and between groups by a Student’s t test using Sigma Stat 3.01 for Windows (Systat, Richmond, CA, USA). A p < 0.05 was considered significant.

RESULTS

Figure 1: Percent coenzyme Q₁₀(Q10) absorption in human stratum corneum (SC) after 1 hour of topical application of two different forms of coenzyme Q₁₀to young and middle-aged subjects. Results are reported as the mean±SEM, * p<0.001 vs. younger group, ‡ p<0.05 vs. Q₁₀.

The skin absorption study data is shown in the bar graph of Figure 1. The yeast form had 34% more absorption than pure coenzyme Q10 in the young subjects, and 23% more in the middle-aged subjects. The older subjects absorbed significantly more of the yeast form than pure coenzyme Q10, as did all subjects taken together (p < 0.05). The older subjects absorbed over twice as much coenzyme Q10 as the young subjects, independent of the form, with p < 0.001 in the Student’s test.

Figure 2: Human stratum corneum (SC) antioxidants (nmole units) before and after 1 month of topical application of two forms of coenzyme Q₁₀(Q10) to young and middle-aged subjects. A washout period of 1 month with no treatment followed the coenzyme Q₁₀application. Results are reported as the mean±SEM. * p<0.05 vs. washout, ‡ p<0.01 vs. after coenzyme Q10in the middle-aged group, † p<0.05 vs. before and after washout.

The antioxidants assayed according to the ferric reducing ability of plasma (FRAP) procedure used in this study include small molecules such as vitamin C, tocopherol, uric acid and larger molecules such as albumin and bilirubin. The reduced form of coenzyme Q10, ubiquinol, would also be analyzed by this antioxidant assay. The amounts of antioxidants measured in the stratum corneum are found in Figure 2. The inter-subject variation was large and not all subjects showed an increase after topical application of the antioxidants or a decrease after the washout period. However, six of the eight young subjects had an increase in antioxidants following yeast application and seven of the eight young subjects experienced a decrease in antioxidants after the washout period following yeast application. The young subjects experienced a greater change in stratum corneum antioxidants following application and washout than did the middle-aged subjects, irrespective of the form of coenzyme Q10 applied. In fact, the middle-aged subjects had a small, nonsignificant 2% decrease in skin antioxidants after application of the pure coenzyme Q10 but a 13.3% increase after the yeast form. In middle-aged skin the levels of antioxidants after application of the yeast form were significantly higher than after pure coenzyme Q10 (p < 0.01). Moreover, in the middle-aged group the skin antioxidants were significantly increased after application of the yeast form compared with after the washout period (p < 0.05). Considering all the subjects together, the yeast form caused a significant increase in antioxidants compared with baseline and after washout (p < 0.05). Washout returned the stratum corneum antioxidants to the baseline levels.

Figure 3 : Human stratum corneum (SC) peroxides (pmole x 10 units) before and after 1 month of topical application of two forms of coenzyme Q₁₀(Q10) to young and middle- aged subjects. A washout period of 1 month with no treatment followed the coenzyme Q₁₀application. Results are reported as the mean±SEM, * p<0.01 vs. before, † p<0.05 vs. before and washout.

The results of the lipid hydroperoxides and hydrogen peroxide assay are depicted in Figure 3. The inter-subject variations are very large with this assay. The yeast coenzyme Q10 caused the largest changes in peroxides; for young subjects the level decreased by 61% (p < 0.01), and washout increased the value by 80%. In comparison, pure coenzyme Q10 produced a 33% decrease in peroxides (p > 0.05) and washout increased the value by 37%. There was a significant 53% decrease after topical application of pure coenzyme Q10 in middle-aged subjects (p < 0.01). For middleaged subjects the yeast form produced a 62% decline in peroxides (p < 0.01), and washout increased peroxides by 44%. With all subjects combined, the yeast form produced significant differences after application (p < 0.01) and again after washout (p < 0.05). The pure coenzyme Q10 combined groups also experienced a significant reduction (45%) in peroxides (p < 0.01). Washout did not completely return the levels to those of the baseline; the peroxide levels were always lower than baseline but not significantly different.

DISCUSSION

Although many scientific articles have been written on the medical benefits of oral coenzyme Q10 [16, 17], little has been published on the effects of topical application. However, there are numerous cosmetic formulations containing coenzyme Q10 on the Web, with claims of skin repair and regeneration as well as anti-wrinkling, anti-aging and antioxidant effects. Although a number of articles comparing the human plasma bioavailability of various forms of coenzyme Q10 (powder, gel, emulsified, hydrosoluble) have been published, there have been no published reports comparing the topical effects of different coenzyme Q10 forms. We have shown that significantly more of the yeast form of coenzyme Q10 is absorbed than of the pure coenzyme Q10 after one hour of application in the middle-aged group and in both groups combined. A larger population of younger subjects might have revealed a significant difference between the forms. Twelve hours after the last application (overnight) in the 30-day study no coenzyme Q10 was found in the stratum corneum. This agrees with the results of an earlier study of 60 days’ duration with a 0.05% coenzyme Q10 lotion [18]. Overnight the coenzyme Q10 had moved from the stratum corneum to deeper epidermis layers and to the dermis. Middle-aged subjects absorb more of the lipophilic coenzyme Q10 probably due to differences in hydration of the stratum corneum, which is less for the older subjects [19], and the corneocyte size, which is smaller for older subjects. More benzoic acid, a more hydrophilic molecule than coenzyme Q10, was also absorbed in subjects aged 45-55 years than in those aged 20-30 years [20]. Antioxidants in the skin were measured by the antioxidant (AOX) assay and include ethanol-soluble antioxidant vitamins. Thus the antioxidant assay measured the total low molecular weight antioxidant capacity/defense in the skin. This assay has previously been used for food and beverage antioxidant analysis [21], and for plasma [14]. To our knowledge this is the simplest and cheapest measure of stratum corneum antioxidant capacity. Our results indicate that chronic application of coenzyme Q10 in the yeast form provides significantly more antioxidants to the stratum corneum and surface lipids in both young and middle-aged subjects. Young subjects experienced a greater increase in stratum corneum antioxidants than did the older subjects. In fact the middle-age subjects had no increase at all in skin antioxidants with pure coenzyme Q10. There was no difference in baseline stratum corneum antioxidants between the young and older subjects in the present study. This agreed with the finding of other workers that there is no clear correlation of age with changes in antioxidant concentrations [22].

Peroxides in the stratum corneum provide a measure of oxidative damage and stress and consist of lipid hydroperoxides and hydrogen peroxide. Baseline peroxides were significantly higher in the elderly subjects than in the younger subjects with 4,980 and 3,230 pmoles, respectively (p < 0.05). This clearly indicates more oxidative damage to the stratum corneum of elderly subjects. Lipid peroxidation was found to increase with age in human skin [23]. In addition, the same group showed that superoxide dismutase, catalase, and glutathione peroxidase (antioxidant enzymes) decrease with age in skin fibroblasts. Also hydrogen peroxide and DNA damage increase with age in fibroblasts [24]. Our results confirm this finding, as lipid peroxides and the hydrogen peroxide resulting from decreased catalase activity were elevated with age in stratum corneum. Coenzyme Q10 significantly lowered peroxides and the yeast form was more efficacious. Washout did not result in an elevation of stratum corneum peroxides back to baseline levels, indicating a lingering antioxidant effect of topical coenzyme Q10. Hoppe et al. found that topical coenzyme Q10 (0.3%) decreased the level of oxidation in the skin as measured by weak photon emission [1]. Coenzyme Q10 also decreased UVA-induced increases in weak photon emission of skin [25]. Although there are numerous reports in the literature on topical antioxidants and skin protection from UV-induced oxidative stress, this is the first report indicating a decrease in oxidative damage in nonstressed skin as a result of coenzyme Q10 antioxidant application.

CONCLUSION

Antioxidants (especially vitamin E) are routinely added to cosmetic formulations and advertisements tout the benefits. Certainly, human studies have convincingly demonstrated large photo-protective effects of both natural and synthetic antioxidants when topically applied before UV exposure. Oxidative stress has also been linked to skin aging in numerous published studies. We have shown that coenzyme Q10, most effectively in the yeast form, is capable of improving the antioxidant capacity of stratum corneum and decreasing the amount of oxidative damage in nonstressed skin after topical application. We hypothesize that the yeast matrix increases coenzyme Q10 absorption and stabilizes coenzyme Q10. An in vitro stability study on reduced coenzyme Q10 performed in our laboratory showed that the yeast matrix provided increased stability. Since stratum corneum levels correlate strongly with percutaneous absorption [26], our findings indicate that human skin should be correspondingly affected. We also conclude that older subjects have substantially more oxidative damage to skin than younger subjects. This demonstrates the need for topical application of antioxidants throughout the human lifetime.

REFERENCES

[1] Hoppe, U., Bergemann, J., Diembeck,W., Ennen, J., Gohla, S., Harris, I., Jacob, J., Kielholz, J., Mei,W., Pollet, D., Schachtschabel, D., Sauermann, G., Schreiner,V., Stab, F., and Steckel, F., Coenzyme Q10, a cutaneous antioxidant and energizer, Biofactors, 9 (1999) 371-378.

[2] Frei, B., Kim, M.C., and Ames, B.N., Ubiquinol-10 is an effective lipid-soluble antioxidant at physiological concentrations, Proc. Natl. Acad. Sci. USA, 12 (1990) 4879-4883.

[3] Ernster, L. and Dallner, G., Biochemical, physiological and medical aspects of ubiquinone function, Biochim. Biophys. Acta, 1271 (1995) 195-204.

[4] Turrens, J.F., Superoxide production by the mitochondrial respiratory chain, Biosci. Rep., 17 (1997) 3-8.

[5] Shindo, Y., Witt, E., Han, D., Epstein, W., and Packer, L., Enzymic and non-enzymic antioxidants in epidermis and dermis of human skin, Invest. Dermatol., 102 (1994) 122-124.

[6] Shindo, Y., Witt, E., Han, D., and 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 (1994) 470-4759.

[7] Cutler, R.G., Antioxidants, aging, and longevity, in: Pryor, W. (Ed.), Free Radicals in Biology, Academic Press, New York, 1984, vol. 6, pp. 371-428.

[8] Ishii, N., Senoo-Matsuda, N., Miyake, K., Yasuda, K., Ishii, T., Hartman, P. S., and Furukawa, S., Coenzyme Q10 can prolong C. elegans life span by lowering oxidative stress, Mech. Ageing Dev., 125 (2004) 41-46.

[9] Kalen, A., Appelkvist, E. L., and Dallner, G., Age-related changes in the lipid compositions of rat and human tissues, Lipids, 24 (1989) 579-584.

[10] Soderberg, M., Edlund, C., Kristensson, K., and Dallner, G., Lipid compositions of different regions of the human brain during aging, J. Neurochem., 54 (1990) 415- 423.

[11] Hoppe, U., Bergemann, J., Diembeck, W., Ennen, J., Gohla, S., Harris, I., Jacob, J., Kielholz, J., Mei, W., Pollet, D., Schachtschabel, D., Sauermann, G., Schreiner, V., Stab, F., and Steckel, F., Lipophilic antioxidants in human sebum and aging, Free Radic. Res., 36 (2002) 471-477.

[12] Giovannini, L., Bertelli, A.A., Scalori, V., Dell’Osso, L., Alessandri, M.G., and Mian, M., Skin penetration of coenzyme Q10 in the rat, Int. J. Tissue React., 10 (1988) 103-105.

[13] Pelle, E., Muizzuddin, N., Mammone, T., Marenus, K., and Maes, D., Protection against endogenous and UVB-induced oxidative damage in stratum corneum lipids by an antioxidant-containing cosmetic formulation, Photodermatol. Photoimmunol. Photomed., 15 (1999) 115-119.

[14] Benzie, I.F. and Strain, J.J., The ferric reducing ability of plasma (FRAP) as a measure of »antioxidant power«: the FRAP assay, Biochem., 239 (1996) 70-76.

[15] Gay, C.A. and Gebicki, J.M., Perchloric acid enhances sensitivity and reproducibility of the ferric-xylenol orange peroxide assay, Anal. Biochem., 304 (2002) 42-46.

[16] Tran, M.T., Mitchell, T.M., Kennedy, D.T., and Giles, J.T., Role of coenzyme Q10 in chronic heart failure, angina, and hypertension, Pharmacotherapy, 21 (2001) 797-806.

[17] Horowitz, S., Coenzyme Q10: One antioxidant, many promising applications, Alternative & Complementary Therapies, 9 (2003) 111-116.

[18] Passi, S., De Pita, O., Grandinetti, M., Simotti, C., and Littarru, G.P., The combined use of oral and topical lipophilic antioxidants increases their levels both in sebum and stratum corneum, Biofactors, 18 (2003) 289-297.

[19] Manuskiatti, W., Schwindt, D.A., and Maibach, H.I., Influence of age, anatomic site and race on skin roughness and scaliness, Dermatology, 196 (1998) 401-407.

[20] Rougier, A., Lotte, C., Corcuff, P., and Maibach, H., Relationship between skin permeability and corneocyte size according to anatomic site, age, and sex in man, J. Soc. Cosmet Chem., 39 (1998) 15- 26.

[21] Szeto, Y.T., Tomlinson, B., and Benzie, I.F., Total antioxidant and ascorbic acid content of fresh fruits and vegetables: implications for dietary planning and food preservation, Br. J. Nutr., 87 (2002) 55-59.

[22] Biesalski, H.K., Berneburg, M., Grune, T., Kerscher, M., Krutmann, J., Raab, W., Reimann, J., Reuther, T., Robert, L., and Schwarz, T., Hohenheimer Consensus Talk. Oxidative and premature skin ageing, Exp. Dermatol., 12 (2003) 3-15.

[23] Lu, C.Y., Lee, H.C., Fahn, H.J., and Wei, Y.H., Oxidative damage elicited by imbalance of free radical scavenging enzymes is associated with large-scale mtDNA deletions in aging human skin, Mutat. Res., 423 (1999) 11-21.

[24] Wei, Y.H., Lu, C.Y., Wei, C.Y. Ma, Y.S., and Lee, H.C., Oxidative stress in human aging and mitochondrial disease-consequences of defective mitochondrial respiration and impaired antioxidant enzyme system, Chin. J. Physiol., 44 (2001) 1-11.

[25] Blatt, T., Mundt, C., Mummert, C., Maksiuk, T., Wolber, R., Keyhani, R., Schreiner V., Hoppe, U., Schachtschabel, D.O., and Stab, F., Modulation of oxidative stresses in human aging skin, Z. Gerontol. Geriatr., 32 (1999) 83-88.

[26] Dupuis, D., Rougier, A., Roguet, R., Lotte, C., and Kalopissis, G., In vivo relationship between horny layer reservoir effect and percutaneous absorption in human and rat, J. Invest. Dermatol., 82 (1984) 353-356.