1. ¹Duke University Medical Center, Division of Dermatology, Durham, North Carolina, USA
2. ²North Carolina State University, Raleigh, North Carolina, USA
3. ³Zielinski Research, Vista, California, USA. E-mail: pinne002@mc.duke.edu

TO THE EDITOR

Ubiquinone (Coenzyme Q10) is an important lipophilic antioxidant synthesized by the body and critical for protection of mitochondrial membranes (Crane, 2001; Dallner and Sindelar, 2000). Idebenone is a synthetic derivative of ubiquinone with a shorter carbon side chain and subsequent increased solubility (Wieland et al., 1995). Both have been suggested as topical antioxidant ingredients for the protection of skin from oxidative damage caused by UV irradiation and pollution.

Kinetin (N6-furfuryladenine) is a member of the cytokinin plant growth hormone family. Cytokinins are growth promoters, which positively affect cell number and division rate in both plants and animals (Vesely et al., 1985). In vitro studies have shown that kinetin has antioxidant effects, preventing oxidative damage to DNA (Olsen et al., 1999). Topical kinetin has been shown to improve skin texture and reduce the appearance of fine rhytides in humans (McCullough and Weinstein, 2002) and in animals (Kimura and Doi, 2004).

We have previously reported effective photoprotection properties of an antioxidant solution containing 15% L-ascorbic acid, 1% -tocopherol, and 0.5% ferulic acid (C+E+ferulic acid) (Lin et al., 2005). In this study, we use the same model to evaluate the antioxidant potential of ubiquinone, idebenone, and kinetin by measuring their photoprotective value.

The treatment protocol and experimental design has been published in detail elsewhere (Lin et al., 2003), but will be summarized here in brief. Of each of the following, 250 l (solution) or 250 mg (cream) were applied to 7.5 10 cm patches of pig skin daily for 4 days: a solution containing 15% L-ascorbic acid, 1% -tocopherol, and 0.5% ferulic acid (C+E+ferulic acid), solutions containing 1.0% ubiquinone, 1.0% idebenone, and 0.5% kinetin as well as commercial creams containing 0.1% kinetin (Kinerase, Valeant Pharmaceuticals, Costa Mesa, CA), 1.0% idebenone (Prevage, Allergan Inc., Irvine, CA), 0.5% idebenone (TRUE Youth Revealing Complex, TRUE Cosmetics, San Francisco, CA). A 1000 W solar simulator (Lightning Cure 200, Hamamatsu, Hamamatsu City, Japan) was used to deliver the UV radiation at an intensity of 5 mW/cm² of UVB and approximately 40 mW/cm² of UVA, as measured by a research radiometer (IL1700, International Light, Newburyport, MA). Patches were irradiated with solar-simulated UV as described above in triplicate from 1 to 5 minimal erythema dose (MED) in 1 MED multiples. Evaluation was conducted 24 hours later. All experimental methods were conducted within the guidelines of and with approval by the North Carolina State University Institutional Animal Care and Use Committee. A computerized colorimetry algorithm using digital photographs (Tournas JA and Pinnell SR (2005) A computerized method for skin erythema measurement. J Investig Dermatol 124(S4): A136 (abstract)) and a Microsoft Excel (Microsoft Inc., Redmond, WA) spreadsheet were used to measure and calculate the a^* (redness) values of the experimental spots, as well as to compile the statistics and graph the results. After photography, 8 mm punch biopsy sections were taken of each experimental spot and fixed in formalin. Sections were embedded and sectioned for hematoxylin and eosin (H+E) staining. The H+E-stained sections were then examined for the presence of apoptotic "sunburn cells" (SBCs). The full 8 mm width of each section was counted and the result expressed as SBC density/mm of skin. Microsoft Excel was used to determine the mean and standard deviation of the SBC densities at each UV dosage and to graph the results. Thymine dimer analysis was carried out as in the work of Mitchell et al. (2001). An Olympus BX41 fluorescence microscope with a Q-Fire camera (Olympus America Inc., Melville, NY) was employed to obtain the immunofluorescence images. The results for erythema (a^*) and SBC density are expressed as meanSD (n=6). The P-values were calculated by two-tailed Student's t-test.

Figure 1a shows the erythema response of skin treated with C+E+ferulic acid, 1.0% ubiquinone, 1.0% idebenone, and the 1.0 and 0.5% idebenone creams (Commercial Creams 1 and 2, respectively), and Figure 1b shows the erythema response of skin treated with C+E+ferulic acid, 0.5% kinetin solution, and the 0.1% kinetin cream. It can be seen that while C+E+ferulic acid is protective at 5 MED, ubiquinone, idebenone, and kinetin do not provide protection. The computerized colorimetric measurements show that at all UV dosage levels from 1 to 5 MED, erythema is significantly reduced (P<0.05) when skin is treated with C+E+ferulic acid compared to control, or skin treated with any of the antioxidant solutions or the commercial creams. Additionally, none of the preparations other than C+E+ ferulic acid were significantly different from control at any UV dosage from 1 to 5 MED. Quantitatively, the a^* values for the C+E+ferulic acid-treated area were decreased by 98, 99, 91, 87, and 83% from 1 to 5 MED, respectively.

Figure 1.

Prevention of erythema. (a) Erythema generated in response to UV irradiation from 1 to 5 MED in skin treated with 1% ubiquinone solution, 1% idebenone solution, 1% idebenone cream, and 0.5% idebenone cream, and C+E+ferulic acid. Here it is evident that none of the ubiquinone or idebenone treatments offer more than one- to two-fold protection, while C+E+ferulic acid protects beyond 5 MED. (b) Erythema generated in response to UV irradiation from 1 to 5 MED in skin treated with 0.5% kinetin solution, 0.1% kinetin cream, and C+E+ferulic acid. Neither of the kinetin treatments offer protection, while C+E+ferulic acid protects completely at 5 MED.

Full figure and legend (209K)

Figure 2a and b show the SBC density in skin treated with the test solutions after UV irradiation from 1 to 5 MED. As with the erythema, SBC density was significantly reduced in skin treated with C+E+ferulic acid as compared to control and the other solutions and creams. The 0.5% idebenone cream showed slight benefit in terms of SBC reduction, though it should be noted that this preparation also contains sunscreen in an unknown amount. Quantitatively, the mean SBC reduction afforded by C+E+ferulic acid over control was 86, 93, 95, 92, and 93% from 1 to 5 MED, respectively.

Figure 2.

Prevention of SBC generation. (a) SBCs generated in response to 1–5 MED of UV irradiation in skin treated with antioxidant solutions. ^*P<0.05 versus control, ^**P<0.05 versus all formulations tested. (b) SBCs generated in response to 1–5 MED of UV irradiation in skin treated with 0.5% kinetin solution, 0.1% kinetin cream, and C+E+ferulic acid. ^*P<0.05 vs control and kinetin formulations.

Full figure and legend (92K)

Additional experiments performed but not shown graphically showed that adding 1% idebenone to C+E+ferulic acid did not increase its photoprotective benefit in terms of both erythema and SBCs. Thymine dimer immunohistochemistry also revealed that only C+E+ferulic acid was completely protective at 4 MED. The 0.5% idebenone cream afforded slight protection, and the other preparations were not protective.

The results of this study show that ubiquinone, idebenone, and kinetin offer little to no photoprotective value in comparison to more established therapies. In addition, the slight photoprotective effect seen with commercial creams containing idebenone may be due to the sunscreen ingredients that they contain. Idebenone specifically does not increase the photoprotective value of an established antioxidant combination of C+E+ferulic acid. The results of this study also validate an earlier study showing that C+E+ferulic acid offers eight-fold UV photoprotection to skin (Lin et al., 2005).