Enzymic and Non-Enzymic Antioxidants in Epidermis and Dermis of Human Skin

A comprehensive comparison of antioxidant defenses in the dermis and epidermis and their response to exposure to ultraviolet (UV) irradiation has not previously been attempted. In this study, enzymic and non-enzymic antioxidants in epidermis and dermis of hairless mice were compared. Enzyme activities are presented both as units/gram of skin and units/milligram of protein; arguments are presented for the superiority of skin wet weight as a reference base. Catalase, glutathione peroxidase, and glutathione reductase (units/gram of skin) were higher in epidermis than dermis by 49%, 86%, and 74%, respectively. Superoxide dismutase did not follow this pattern. Lipophilic antioxidants (alpha-tocopherol, ubiquinol 9, and ubiquinone 9) and hydrophilic antioxidants (ascorbic acid, dehydroascorbic acid, and glutathione) were 24-95% higher in epidermis than in dermis. In contrast, oxidized glutathione was 60% lower in epidermis than in dermis. Mice were irradiated with solar light to examine the response of these cutaneous layers to UV irradiation. After irradiation with 25 J/cm2 (UVA + UVB, from a solar simulator), 10 times the minimum erythemal dose, epidermal and dermal catalase and superoxide dismutase activities were greatly decreased. alpha-Tocopherol, ubiquinol 9, ubiquinone 9, ascorbic acid, dehydroascorbic acid, and reduced glutathione decreased in both epidermis and dermis by 26-93%. Oxidized glutathione showed a slight, non-significant increase. Because the reduction in total ascorbate and catalase was much more severe in epidermis than dermis, it can be concluded that UV light is more damaging to the antioxidant defenses in the epidermis than in the dermis.

A fast single-step lipid extraction procedure and high-performance liquid chromatography with in-line uv and electrochemical detection are used for the simultaneous quantitative determination of tocopherols, ubiquinols, and ubiquinones in blood, plasma, tissue homogenates, and subcellular fractions. The compounds of interest can be quantitatively extracted into hexane from a sodium dodecyl sulfate-treated aqueous homogenate after precipitation of protein by addition of an equal volume of ethanol. alpha-, gamma-, and delta-Tocopherol, ubiquinol 9, ubiquinol 10, and ubiquinones 9 and 10 can be well separated on a reversed phase column. Ubiquinones are detected at 275 nm by the uv detector, and ubiquinols and tocopherols by the electrochemical detector in the oxidative mode. Quantitation is done by comparing chromatographic peak heights to those of a standard solution containing known amounts of tocopherols, ubiquinols 9 and 10, and ubiquinones 9 and 10, analyzed under identical conditions. The high sensitivity of the electrochemical detection allows operation at low potentials (+0.5 V) with low detector response, but high selectivity for the easily oxidizable tocopherols and ubiquinols and decreased baseline noise. The uv detection limits the overall sensitivity of the procedure to 2 pmol ubiquinone, corresponding to 0.1 microM ubiquinone in the lipid extract. The ranges of values obtained for rat and guinea pig tissues, for rat liver mitochondria, and for blood and plasma from rats and humans are given.

Suction blister roofs taken from the involved and uninvolved epidermis of patients with vitiligo showed a consistent reduction in levels of catalase compared to normal healthy controls of matched photo-skin types (Fitzpatrick classification). A decrease in catalase activity is expected to increase the concentration of hydrogen peroxide in the epidermis of these patients. Hydrogen peroxide functions as a reversible inhibitor of human tyrosinase with a KI of 8 X 10(-6) M. Also, hydrogen peroxide undergoes photochemical reduction yielding highly reactive hydroxyl radicals (OH.) and hydroxyl ions (OH-) mainly by the Haber-Weiss reaction. Hydroxyl radicals are capable of bleaching constitutional melanin and cause membrane lysis through lipid peroxidation reactions. Hydroxyl ions increase the pH in the epidermis, and as a consequence glutathione reductase activity is increased in patients with vitiligo compared to controls. Based on these new results, together with the previously reported calcium transport defect, a new hypothesis has been formulated for the pathogenesis of vitiligo.