Aging- and Photoaging-Dependent Changes of Enzymic and Nonenzymic Antioxidants in the Epidermis and Dermis of Human Skin In Vivo

This is a comprehensive study of the changes in major antioxidant enzymes and antioxidant molecules during intrinsic aging and photoaging processes in the epidermis and dermis of human skin in vivo. We show that the activities of superoxide dismutase and glutathione peroxidase are not changed during these processes in human skin in vivo. Interestingly, the activity of catalase was significantly increased in the epidermis of photoaged (163%) and naturally aged (118%) skin (n = 9), but it was significantly lower in the dermis of photoaged (67% of the young skin level) and naturally aged (55%) skin compared with young (n = 7) skin. The activity of glutathione reductase was significantly higher (121%) in naturally aged epidermis. The concentration of α-tocopherol was significantly lower in the epidermis of photoaged (56% of young skin level) and aged (61%) skin, but this was not found to be the case in the dermis. Ascorbic acid levels were lower in both epidermis (69% and 61%) and dermis (63% and 70%) of photoaged and naturally aged skin, respectively. Gluta thione concentrations were also lower. Uric acid did not show any significant changes. Our results suggest that the components of the antioxidant defense system in human skin are probably regulated in a complex manner during the intrinsic aging and photoaging processes. This is a comprehensive study of the changes in major antioxidant enzymes and antioxidant molecules during intrinsic aging and photoaging processes in the epidermis and dermis of human skin in vivo. We show that the activities of superoxide dismutase and glutathione peroxidase are not changed during these processes in human skin in vivo. Interestingly, the activity of catalase was significantly increased in the epidermis of photoaged (163%) and naturally aged (118%) skin (n = 9), but it was significantly lower in the dermis of photoaged (67% of the young skin level) and naturally aged (55%) skin compared with young (n = 7) skin. The activity of glutathione reductase was significantly higher (121%) in naturally aged epidermis. The concentration of α-tocopherol was significantly lower in the epidermis of photoaged (56% of young skin level) and aged (61%) skin, but this was not found to be the case in the dermis. Ascorbic acid levels were lower in both epidermis (69% and 61%) and dermis (63% and 70%) of photoaged and naturally aged skin, respectively. Gluta thione concentrations were also lower. Uric acid did not show any significant changes. Our results suggest that the components of the antioxidant defense system in human skin are probably regulated in a complex manner during the intrinsic aging and photoaging processes. 5,5′-dithiobis-2-nitrobenzoic acid glutathione peroxidase glutathione reductase reduced glutathione oxidized glutathione nitroblue tetrazolium superoxide dismutase The aging process of skin can be attributed to intrinsic aging and photoaging. Damage to human skin due to repeated exposure to ultraviolet (UV) radiation (photoaging) and damage due to the passage of time (chronologic aging) are considered to be distinct entities rather than similar skin aging processes. Clinically, naturally aged skin is smooth, pale, and finely wrinkled. In contrast, photoaged skin is coarsely wrinkled and associated with dyspigmentation and telangiectasia (Lavker, 1979Lavker R.M. Structural alterations in exposed and unexposed aged skin.J Invest Dermatol. 1979; 73: 559-566Crossref Scopus (358) Google Scholar;Lavker and Kligman, 1988Lavker R.M. Kligman A.M. Chronic heliodermatitis: a morphological evaluation of chronic actinic dermal damage with emphasis on the role of mast cells.J Invest Dermatol. 1988; 90: 325-330Abstract Full Text PDF PubMed Google Scholar;Gilchrest, 1989Gilchrest B.A. Skin aging and photoaging: an overview.J Am Acad Dermatol. 1989; 21: 610-613Abstract Full Text PDF PubMed Scopus (373) Google Scholar). Several theoretical mechanisms have been proposed to explain the aging in skin, and the free radical theory (Harman, 1956Harman D. Aging: a theory based on free radical and radiation chemistry.J Gerontol. 1956; 11: 298-300Crossref PubMed Scopus (6036) Google Scholar,Harman, 1981Harman D. The aging process.Proc Natl Acad Sci USA. 1981; 78: 7124-7128Crossref PubMed Scopus (1519) Google Scholar,Harman, 1986Harman D. Free radical theory of aging: role of free radicals in the origination and evolution of life, aging and disease process.in: Johnson J. Walford R. Harman D. Miquel J. Biology of Aging. Liss, New York1986: 3-50Google Scholar) is receiving particular attention because skin is constantly exposed to reactive oxygen species (ROS) from the environment, such as air, solar radiation, ozone, and other air-borne pollutants, or from the normal metabolism, primarily from the mitochondrial respiratory chain wherein excess electrons are donated to molecular oxygen to generate superoxide anions. Accumulated ROS have been suggested to play important roles in the intrinsic aging and photoaging of human skin in vivo (Kawaguchi et al., 1996Kawaguchi Y. Tanaka H. Okada T. Konishi H. Takahashi M. Ito M. Asai J. The effects of ultraviolet A and reactive oxygen species on the mRNA expression of 72-kDa type IV collagenase and its tissue inhibitor in cultured human dermal fibroblasts.Arch Dermatol Res. 1996; 288: 39-44https://doi.org/10.1007/s004030050020Crossref PubMed Scopus (0) Google Scholar), and ROS has been postulated to be responsible for various cutaneous inflammatory disorders and skin cancers (Cross et al., 1987Cross C.C. Halliwell B. Borish E.T. et al.Davis conference: oxygen radicals and human disease.Ann Intern Med. 1987; 107: 526-545Crossref PubMed Scopus (1495) Google Scholar;Record et al., 1991Record I.R. Dreosti I.E. Konstantinopoulos M. Buckley R.A. The influence of topical and systemic vitamin E on ultraviolet light-induced skin damage in hairless mice.Nutr Cancer. 1991; 16: 219-226Crossref PubMed Scopus (73) Google Scholar). The skin's antioxidant defense system is regulated by an intimately interlinked network, and it is becoming increasingly clear that antioxidants interact in a complex fashion, so that changes in the redox status or concentration of one component may affect many other components of the system (Packer et al., 1979Packer J.E. Slater T.F. Wilson R.L. Direct observation of a free radical interaction between vitamin E and vitamin C.Nature. 1979; 278: 737-738Crossref PubMed Scopus (1145) Google Scholar;Martensson et al., 1991Martensson J. Meister A. Martensson J. Glutathione deficiency decreases tissue ascorbate levels in newborn rats: ascorbate spares glutathione and protects.Proc Natl Acad Sci USA. 1991; 88: 4656-4660Crossref PubMed Scopus (281) Google Scholar;Lopez-Torres et al., 1998Lopez-Torres M. Thiele J.J. Shindo Y. Han D. Packer L. Topical application of α-tocopherol modulates the antioxidant network and diminishes ultraviolet-induced oxidative damage in murine skin.Br J Dermatol. 1998; 138: 207-215Crossref PubMed Scopus (168) Google Scholar). Thus, a comprehensive and integrated antioxidant skin defense mechanism is considered to be crucial for protecting this organ from ROS, and consequently for preventing the aging process of skin. Some researchers have investigated the antioxidant defense mechanisms of human skin in vivo (Shindo et al., 1994aShindo Y. Witt E. Han D. Epstein W. Packer L. Enzymic and non-enzymic antioxidants in epidermis and dermis of human skin.J Invest Dermatol. 1994; 102: 122-124Abstract Full Text PDF PubMed Google Scholar;Podda et al., 1998Podda M. Traber M.G. Weber C. Yan L. Packer L. UV-irradiation depletes antioxidants and causes oxidative damage in a model of human skin.Free Radic Biol Med. 1998; 24: 55-65Crossref PubMed Scopus (202) Google Scholar) and in the aged skin of mice (Lopez-Torres et al., 1994Lopez-Torres M. Shindo Y. Packer L. Effect of age on antioxidants and molecular markers of oxidative damage in murine epidermis and dermis.J Invest Dermatol. 1994; 102: 476-480Abstract Full Text PDF PubMed Google Scholar). To our knowledge, however, the levels of the major enzymic and nonenzymic antioxidants have never been assessed comprehensively during the aging and photoaging processes of human skin in vivo. Here, we report the results of the first comprehensive study upon changes of the major antioxidant enzymes - superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), and glutathione reductase (GR) - and the antioxidant molecules-α-tocopherol, ascorbic acid, uric acid, and glutathione-during the intrinsic aging and photoaging processes of the epidermis and dermis of human skin in vivo. A total of 15 young Koreans (15 men; mean age 22.9 y; age range 19–28 y) and 15 elderly Koreans (10 men and five women; mean age 75.8 y; age range 71–86 y) without current or prior skin diseases provided skin samples. They did not have a recent history of excessive sun exposure. All elderly Koreans had severely photodamaged skin, of more than grade 5 by our photographic grading system (Chung et al., 2001Chung J.H. Lee S.H. Youn C.S. et al.Cutaneous photodamage in Koreans. Influence of sex, sun-exposure, smoking and skin color.Arch Dermatol. 2001; 137: 1043-1051PubMed Google Scholar). One percent lidocaine was injected for the biopsies. Skin samples were obtained from both the forearm and the upper-inner arm of each subject by punch biopsy (8 mm), and immediately frozen in liquid nitrogen and stored at -70°C. All procedures involving human subjects received prior approval from the Seoul National University Institutional Review Board, and all subjects provided written informed consent. To separate the epidermis from the dermis, the defrosted whole skin samples were placed dermis-side down on a Petri dish and heated at 55°C for 2 min; they were then separated gently into epidermis and dermis with a forceps. We confirmed that these conditions did not change either four major antioxidant enzyme activities (SOD, catalase, GPx, and GR) or the oxidation states of antioxidant molecules such as α-tocopherol, ascorbic acid, uric acid, and glutathione (data not shown). The separated epidermis and dermis were immediately frozen in liquid nitrogen, weighed in the frozen state, and used for assays. 5,5′-Dithiobis-2-nitrobenzoic acid (DTNB), reduced nicotinamide adenine dinucleotide phosphate, oxidized glutathione (GSSG), reduced glutathione (GSH), nitroblue tetrazolium (NBT), triethanolamine, xanthine, butylated hydroxytoluene, α-tocopherol, ascorbic acid, uric acid, metaphosphoric acid, buttermilk xanthine oxidase, and yeast glutathione reductase were purchased from Sigma (St. Louis, MO). Hydrogen peroxide, 2-vinylpyridine, and 2,3-dimercapto-1-propanol were purchased from Aldrich Fine Chemicals (Milwaukee, WI). All other reagents used were of the highest quality generally available. Two biopsied skin samples either from the forearm or the upper-inner arm of each individual were used for the extraction of proteins required for antioxidant enzyme assays. The separated epidermis and dermis were homogenized in 300 µl and 600 µl of buffer A [sodium chloride 130 mM, glucose 5 mM, disodium ethylenediamine tetraacetic acid (EDTA) 1 mM, and sodium phosphate 10 mM, pH 7.0], respectively. The suspension was rotated at 4°C for 10 min, and centrifuged at 14,000g for 10 min. The supernatant was kept at -70°C and used for antioxidant enzyme assays. The activities of SOD (Oberlery and Spitz, 1985Oberlery L.W. Spitz D.R. Quantitation of superoxide dismutase.in: Greenwald R.A. CRC Handbook of Methods for Oxygen Radical Research. CRC Press, Boca Raton, FL1985: 211-220Google Scholar), catalase (Claiborne, 1985Claiborne A. Catalase activity.in: Greenwald R.A. CRC Handbook of Methods for Oxygen Radical Research. CRC Press, Boca Raton, FL1985: 283-284Google Scholar), GPx (Günzler and Floche, 1985Günzler W.A. Floche L. Glutathione peroxidase.in: Greenwald R.A. CRC Handbook of Methods for Oxygen Radical Research. CRC Press, Boca Raton, FL1985: 285-290Google Scholar), and GR (Calberg and Mannervik, 1985Calberg I. Mannervik B. Glutathione reductase.Meth Enzymol. 1985; 113: 484-485Crossref PubMed Scopus (2433) Google Scholar), were assayed spectrophotometrically on a Beckman DU650 spectrophotometer (Beckman Instruments, Fullerton, CA). One enzyme unit is defined as being equivalent to 1 µmol of product formation or 1 µmol of substrate disappearance per min under the defined conditions, with the exception of SOD, in which case 1 unit was defined as the amount of SOD that inhibited the NBT reduction rate by 50% under the given assay conditions. Catalase and SOD activities were measured at room temperature, whereas GPx and GR activities were measured at 37°C. Protein concentrations were determined using the Bio-Rad DC protein assay (Bio-Rad, Hercules, CA) with bovine serum albumin as a reference protein. One biopsied skin sample was used for each antioxidant assay. After separating the epidermis from the dermis, antioxidant molecules were extracted in each extraction buffer. Glutathione was measured using the DTNB-GR recycling assay (Anderson, 1985Anderson M.E. Determination of glutathione and glutathione disulfide in biological samples.Meth Enzymol. 1985; 113: 548-552Crossref PubMed Scopus (2313) Google Scholar). The separated epidermis and dermis were homogenized in 150 µl and 300 µl of ice-cold 3.3% sulfosalicylic acid, 5 mM EDTA, and 1.5 mM butylated hydroxytoluene, respectively. This solution had been previously deoxygenated with nitrogen gas. After homogenization, the samples were immediately centrifuged at 14,000g for 10 min. The supernatant was used to assay total glutathione (GSH + GSSG). For the GSSG assay, 1 µl of 2-vinylpyridine and 3 µl of triethanolamine were added to 50 µl of the above solution. The mixture was incubated for 1 h to derivatize GSH, thereby rendering it inactive during the assay. α-Tocopherol content was determined by high performance liquid chromatography (HPLC) as described byLang et al., 1986Lang J.K. Gohil K. Packer L. Simultaneous determination of tocopherols, ubiquinols, and ubiquinones in blood, plasma, tissue homogenates, and subcellular fractions.Anal Biochem. 1986; 157: 106-116Crossref PubMed Scopus (447) Google Scholar with slight modifications using UV detection. α-Tocopherol from the separated skin samples was extracted in reagent alcohol (ethanol:isopropanol 95:5) and hexane (Lang et al., 1986Lang J.K. Gohil K. Packer L. Simultaneous determination of tocopherols, ubiquinols, and ubiquinones in blood, plasma, tissue homogenates, and subcellular fractions.Anal Biochem. 1986; 157: 106-116Crossref PubMed Scopus (447) Google Scholar). The extracted α-tocopherol from the epidermis and dermis was redissolved in 50 µl and 100 µl of methanol:ethanol (70:30), respectively. Then, a 20 µl aliquot was immediately analyzed by HPLC. Reagent alcohol:methanol (90:10) containing 20 mM LiCl4 was used as the mobile phase. Ascorbic acid (Dhariwal et al., 1991Dhariwal K.R. Hartzell W.O. Levine M. Ascorbic acid and dehydroascorbic acid measurements in human plasma and serum.Am J Clin Nutr. 1991; 54: 712-716Crossref PubMed Scopus (227) Google Scholar) and uric acid (Shindo et al., 1994aShindo Y. Witt E. Han D. Epstein W. Packer L. Enzymic and non-enzymic antioxidants in epidermis and dermis of human skin.J Invest Dermatol. 1994; 102: 122-124Abstract Full Text PDF PubMed Google Scholar) were measured by HPLC with UV detection. Trifluoroacetic acid (0.1%) was used as the mobile phase. The separated epidermis and dermis were homogenized in 100 µl and 300 µl respectively of ice-cold 5% metaphosphoric acid and 1.5 mM butylated hydroxytoluene solution, previously deoxygenated with nitrogen gas. After centrifugation at 14,000g for 10 min, a 20 µl aliquot of supernatant was immediately analyzed by HPLC for ascorbic acid and uric acid. Ascorbic acid (2 µM) and uric acid (2 µM) standards were freshly prepared. The concentration of ascorbic acid was determined spectrophotometrically using an extinction coefficient at 265 nm of 14,500 per m per cm. The HPLC system for α-tocopherol, ascorbic acid, and uric acid consisted of a Gilson Model 302 pump with 20 µl injection valve, a Gilson 118 UV/VIS detector, and a Gilson Model 802C manometric module system (Gilson, WI). A Clipeus C18 column, 5 µm, 250 × 4.6 mm (Higgins, CA), was used. Statistical significance was determined by using the Student t test. Results are presented as means ± SEM. All p values quoted are two-tailed and were accepted as significant for p ≤ 0.05. We observed the age-associated changes in four antioxidant enzymes, SOD, catalase, GPx, and GR. For intrinsic aging, the enzyme activities of the skin sample from the sun-protected area (upper-inner arm) of young and old subjects were compared, and similarly for photoaging the activities of skin samples from the sun-exposed area (forearm) were compared (Table I and Figure 1). The activities are reported as units per mg protein.Table IAntioxidant enzyme activities in photoaging and aging human skin in vivoaThe results are the mean ± SEM (young mean age, 21.0 y; old mean age, 76.1 y).YoungbUnits per mg protein. (n = 7)OldbUnits per mg protein. (n = 9)ForearmUpper-inner armForearmUpper-inner armEpidermisDermisEpidermisDermisEpidermisDermisEpidermisDermisSuperoxide dismutase109.7 ± 6.288.2 ± 6.3hEpidermis different from dermis, p < 0.05.105.1 ± 3.380.6 ± 6.6iEpidermis different from dermis, p < 0.01.111.4 ± 5.088.5 ± 6.1iEpidermis different from dermis, p < 0.01.115.7 ± 8.781.2 ± 5.4iEpidermis different from dermis, p < 0.01.Catalase66.8 ± 3.234.6 ± 3.6jEpidermis different from dermis, p < 0.001.82.6 ± 1.6gForearm different from upper-inner arm, p < 0.01.44.1 ± 2.7fForearm different from upper-inner arm, p < 0.05.jEpidermis different from dermis, p < 0.001.108.7 ± 5.1eYoung different from old, p < 0.001.23.0 ± 2.5dYoung different from old, p < 0.01.jEpidermis different from dermis, p < 0.001.97.4 ± 3.7dYoung different from old, p < 0.01.fForearm different from upper-inner arm, p < 0.05.24.3 ± 3.0eYoung different from old, p < 0.001.jEpidermis different from dermis, p < 0.001.Glutathione peroxidase0.02 ± 0.00.03 ± 0.00.02 ± 0.00.0 ± 0.00.02 ± 0.00.02 ± 0.00.0 ± 0.00.0 ± 0.0Glutathione reductase0.03 ± 0.00.01 ± 0.0iEpidermis different from dermis, p < 0.01.0.0 ± 0.00.0 ± 0.0iEpidermis different from dermis, p < 0.01.0.0 ± 0.00.02 ± 0.0jEpidermis different from dermis, p < 0.001.0.04 ± 0.0cYoung different from old, p < 0.05.0.01 ± 0.0jEpidermis different from dermis, p < 0.001.a The results are the mean ± SEM (young mean age, 21.0 y; old mean age, 76.1 y).b Units per mg protein.c Young different from old, p < 0.05.d Young different from old, p < 0.01.e Young different from old, p < 0.001.f Forearm different from upper-inner arm, p < 0.05.g Forearm different from upper-inner arm, p < 0.01.h Epidermis different from dermis, p < 0.05.i Epidermis different from dermis, p < 0.01.j Epidermis different from dermis, p < 0.001. Open table in a new tab The activities of SOD, catalase, and GR were significantly higher in the epidermis than the dermis by an average of 128% (p < 0.05), 190% (p < 0.001), and 236% (p < 0.01), respectively, in young skin, and by 134% (p < 0.01), 436% (p < 0.001), and 238% (p < 0.001) in old skin. The activity of GPx in the epidermis was lower than in the dermis, and was 74% of the dermal level in young skin and 80% of the dermal level in old skin (Table I.). No significant photoaging- or intrinsic aging-related variations were found for SOD and GPx (Table I, Figure 1). Catalase showed significant activity increases, however, in the epidermis of photoaged (forearm) and naturally aged (upper-inner arm) skin (n = 9) compared with those of young skin (n = 7) by 163% (p < 0.001) and 118% (p < 0.01), respectively. In the dermis, catalase showed significantly lower activity in photoaged and naturally aged skin (n = 9) to 67% (p < 0.01) and 55% (p < 0.001) of the young skin levels, respectively (Table I, Figure 1). The activity of GR in the epidermis of naturally aged skin (n = 9) was significantly increased by 121% (p < 0.05) compared with that of young skin (n = 7), whereas no significant changes were found in the photoaged epidermis, or the dermis of aged and photoaged skin. To investigate the effect of chronic sun exposure on antioxidant enzyme activities, the ratios of the antioxidant enzyme activities of the forearm to the upper-inner arm samples were compared for young and old volunteers Table II. These ratios did not show any significant differences between young and old skin, with the exception of the catalase activity levels, in which case the forearm to upper-inner arm ratio was higher for both the epidermis and dermis of old skin, although only the epidermal difference was statistically significant (p < 0.05) Table II.Table IIThe ratios of the antioxidant enzyme activities of the forearm to the upper-inner arm in aging humanaThe results are the mean ± SEM (young mean age, 21.0 y; old mean age, 76.1 y).EpidermisbThe ratio of activity of the epidermis of forearm to that of upper-inner arm.DermiscThe ratio of activity of the dermis of forearm to that of upper-inner arm.Young (n = 7)Old (n = 9)Young (n = 7)Old (n = 9)Superoxide dismutase1.1 ± 0.10.1 ± 0.11.1 ± 0.11.1 ± 0.1Catalase0.8 ± 0.01.1 ± 0.1dThe ratio of young different from old, p < 0.05.0.8 ± 0.11.0 ± 0.1Glutathione peroxidase0.1 ± 0.11.1 ± 0.21.0 ± 0.21.1 ± 0.1Glutathione reductase1.0 ± 0.10.9 ± 0.01.0 ± 0.11.0 ± 0.1a The results are the mean ± SEM (young mean age, 21.0 y; old mean age, 76.1 y).b The ratio of activity of the epidermis of forearm to that of upper-inner arm.c The ratio of activity of the dermis of forearm to that of upper-inner arm.d The ratio of young different from old, p < 0.05. Open table in a new tab Changes in the levels of the antioxidant molecules α-tocopherol, ascorbic acid, uric acid, and glutathione were also examined. Basically, the same strategy that was used for the antioxidant enzyme activity analysis was employed. Antioxidant concentrations are reported as nmol per g skin. Nonenzymic antioxidant levels were significantly higher in the epidermis than the dermis. The concentration of the lipophilic antioxidant α-tocopherol was significantly higher in the epidermis than the dermis, by an average of 456% (p < 0.001) in young skin and 303% (p < 0.01) in old skin. The concentrations of the hydrophilic antioxidants ascorbic acid, uric acid, and glutathione were significantly higher in the epidermis, by 227% (p < 0.001), 181% (p < 0.01), and 151% (p < 0.01) for young skin, and by 225% (p < 0.01), 173% (p < 0.01), and 129% (p < 0.05) for old skin Table III, respectively.Table IIIAntioxident molecules concentrations in photoaging and aging human skin in vivoaThe results are the mean ± SEM (young mean age, 21.0 y; old mean age, 76.1 y).Youngbnmol per g skin (Wet weight). (n = 9)Oldbnmol per g skin (Wet weight). (n = 9)ForearmUpper-inner armForearmUpper-inner armEpidermisDermisEpidermisDermisEpidermisDermisEpidermisDermisα-Tocopherol26.3 ± 3.44.2 ± 0.5hEpidermis different from dermis, p < 0.00123.1 ± 2.73.9 ± 00.6hEpidermis different from dermis, p < 0.00114.6 ± 1.9dYoung different from old, p < 0.01.4.9 ± 0.8gEpidermis different from dermis, p < 0.01.13.1 ± 1.3dYoung different from old, p < 0.01.4.6 ± 0.7hEpidermis different from dermis, p < 0.001Ascorbic acid363.7 ± 24.8162.4 ± 18.6hEpidermis different from dermis, p < 0.001354.7 ± 23.5152.0 ± 19.0hEpidermis different from dermis, p < 0.001252.2 ± 19.4dYoung different from old, p < 0.01.102.1 ± 22.8cYoung different from old, p < 0.05.hEpidermis different from dermis, p < 0.001218.5 ± 20.1eYoung different from old, p < 0.001.107.1 ± 10.0cYoung different from old, p < 0.05.Uric acid419.0 ± 21.7193.2 ± 24.1hEpidermis different from dermis, p < 0.001420.5 ± 28.7260.7 ± 31.4gEpidermis different from dermis, p < 0.01.351.1 ± 28.8202.9 ± 36.1gEpidermis different from dermis, p < 0.01.380.6 ± 32.5219.1 ± 29.1gEpidermis different from dermis, p < 0.01.Reduced glutathione237.4 ± 11.5cYoung different from old, p < 0.05.fEpidermis different from dermis, p < 0.05.286.8 ± 17.5hEpidermis different from dermis, p < 0.001427.9 ± 64.9286.8 ± 17.7gEpidermis different from dermis, p < 0.01.307.6 ± 24.6eYoung different from old, p < 0.001.267.7 ± 21.7334.8 ± 46.2521.8 ± 40.7Oxidized glutathione28.7 ± 6.919.2 ± 3.219.5 ± 4.221.7 ± 3.618.8 ± 5.715.8 ± 2.523.9 ± 6.213.1 ± 2.1Total glutathione550.5 ± 43.4306.0 ± 20.1hEpidermis different from dermis, p < 0.001492.3 ± 63.7308.5 ± 19.2gEpidermis different from dermis, p < 0.01.326.4 ± 24.2eYoung different from old, p < 0.001.283.5 ± 22.2358.7 ± 49.1250.5 ± 11.8cYoung different from old, p < 0.05.fEpidermis different from dermis, p < 0.05.GSSG/GSH(%)5.4 ± 1.26.4 ± 0.84.1 ± 1.47.5 ± 1.1fEpidermis different from dermis, p < 0.05.6.7 ± 2.16.2 ± 1.06.7 ± 1.45.5 ± 0.7a The results are the mean ± SEM (young mean age, 21.0 y; old mean age, 76.1 y).b nmol per g skin (Wet weight).c Young different from old, p < 0.05.d Young different from old, p < 0.01.e Young different from old, p < 0.001.f Epidermis different from dermis, p < 0.05.g Epidermis different from dermis, p < 0.01.h Epidermis different from dermis, p < 0.001 Open table in a new tab The concentration of α-tocopherol was significantly lower in photoaged and naturally aged epidermis to 56% (p < 0.01) and 61% (p < 0.01) of the young skin levels. No significant difference in the concentration of α-tocopherol, however, was found in the dermis of young and aged skin. The level of ascorbic acid was lower in photoaged and aged epidermis to 69% (p < 0.01) and 61% (p < 0.001) of the corresponding young skin levels, respectively. Photoaged and naturally aged dermis contained 63% (p < 0.05) and 70% (p < 0.05) of the ascorbic acid levels of young dermis. Uric acid did not show any significant changes due to the aging process. The levels of total (GSH + GSSG) glutathione were lower in photoaged and aged epidermis at 59% (p < 0.001) and 73% of the young skin levels, respectively. In photoaged and aged dermis, the total glutathione level was lower at 93% and 81% (p < 0.05) of the young skin levels, respectively. The ratio of GSSG to GSH did not show any difference for young and old epidermis, or dermis (Table III and Figure 2). Antioxidant concentration changes due to chronic sun exposure were investigated by comparing the ratio of the concentration of antioxidant molecules in the forearm skin to that of the inner arm skin for young and old skin. This ratio did not show any significant differences between the young and old volunteers (data not shown). In this study, we demonstrated that the activities of SOD and GPx were not changed during the aging and photoaging processes in human skin in vivo. Interestingly, however, the activity of catalase was significantly higher in the epidermis, whereas it was significantly lower in the dermis, of photoaged and naturally aged human skin in vivo compared with young skin. GR activity was higher in naturally aged epidermis. It was also found that the concentration of the nonenzymic antioxidant α-tocopherol was lower in the epidermis, but not in the dermis, of photoaged and naturally aged skin. Ascorbic acid levels were lower in the epidermis and dermis of photoaged and naturally aged skin. Uric acid levels were similar in the epidermis and dermis, however, during the photoaging and the natural aging processes. The concentration of glutathione was lower in photoaged and naturally aged epidermis and dermis, although statistical significance was only found in the photoaged epidermis and aged dermis. To our knowledge, this is the first comprehensive study of changes in the levels of four major antioxidant enzymes and nonenzymic antioxidants during the photoaging and aging processes of human skin in vivo. Usually, enzymic activity is expressed as units per milligram of protein to determine the degree of purity during separation and purification (Hussain et al., 1995Hussain S. Slikker Jr., W. Ali S.F. Age-related changes in antioxidant enzymes, superoxide dismutase, catalase, glutathione peroxidase and glutathione in different regions of mouse brain.Int J Devl Neurosci. 1995; 13: 811-817Crossref PubMed Scopus (143) Google Scholar;Mo et al., 1995Mo J.Q. Hom D.G. Andersen J.K. Decreases in protective enzymes correlates with increased oxidative damage in the aging mouse brain.Mech Ageing Dev. 1995; 81: 73-82Crossref PubMed Scopus (97) Google Scholar;Pansarasa et al., 1999Pansarasa O. Bertorelli L. Vecchiet J. Felzani G. Marzatico F. Age-dependent changes of antioxidant activities and markers of free radical damage in human skeletal muscle.Free Radic Biol Med. 1999; 27: 617-622https://doi.org/10.1016/s0891-5849(99)00108-2Crossref PubMed Scopus (0) Google Scholar). Some investigators have suggested that units per gram of skin would be more reasonable (Shindo et al., 1993Shindo Y. Witt E. Packer L. Antioxidant defense mechanisms in murine epidermis and dermis and their responses to ultraviolet light.J Invest Dermatol. 1993; 100: 260-265Abstract Full Text PDF PubMed Google Scholar;Shindo et al., 1994aShindo Y. Witt E. Han D. Epstein W. Packer L. Enzymic and non-enzymic antioxidants in epidermis and dermis of human skin.J Invest Dermatol. 1994; 102: 122-124Abstract Full Text PDF PubMed Google Scholar).Lopez-Torres et al., 1994Lopez-Torres M. Shindo Y. Packer L. Effect of age on antioxidants and molecular markers of oxidative damage in murine epidermis and dermis.J Invest Dermatol. 1994; 102: 476-480Abstract Full Text PDF PubMed Google Scholar, however, demonstrated that there was no significant difference between using these two units of measurement, except for GPx when measuring the antioxidant enzyme activities in young and old mice skin. It is also possible that many factors such as biopsy procedures and water accumulation might introduce artifacts into the determined skin weight values, and these factors might cause changes in quoted values (Lopez-Torres et al., 1994Lopez-Torres M. Shindo Y. Packer L. Effect of age on antioxidants and molecular markers of oxidative damage in murine epidermis and dermis.J Invest Dermatol. 1994; 102: 476-480Abstract Full Text PDF PubMed Google Scholar). Thus, we expressed enzyme activity in units per milligram of protein. As epidermis is directly exposed to various oxidative stresses from the environment, it is presumed to possess a higher antioxidant defense capacity than dermis, to maintain the redox balance of the skin (Lopez-Torres et al., 1994Lopez-Torres M. Shindo Y. Packer L. Effect of age on antioxidants and molecular markers of oxidative damage in murine epidermis and dermis.J Invest Dermatol. 1994; 102: 476-480Abstract Full Text PDF PubMed Google Scholar;Shindo et al., 1994aShindo Y. Witt E. Han D. Epstein W. Packer L. Enzymic and non-enzymic antioxidants in epidermis and dermis of human skin.J Invest Dermatol. 1994; 102: 122-124Abstract Full Text PDF PubMed Google Scholar). As expected, we observed that the activities of SOD, catalase, and GR were higher in the epidermis than the dermis in both young and aged skin in vivo. The concentrations of the nonenzymic antioxidants α-tocopherol, ascorbic acid, uric acid, and glutathione were also higher in the epidermis than in the dermis of both young and old skin in vivo. Our results are similar to those ofShindo et al., 1994aShindo Y. Witt E. Han D. Epstein W. Packer L. Enzymic and non-enzymic antioxidants in epidermis and dermis of human skin.J Invest Dermatol. 1994; 102: 122-124Abstract Full Text PDF PubMed Google Scholar in terms of the activities of catalase and GR (expressed as units per mg protein), and of the levels of the nonenzymic antioxidants α-tocopherol, ascorbic acid, and glutathione, which were all higher in the epidermis than the dermis. We demonstrated that the antioxidant enzyme catalase is regulated differently in the epidermis and dermis during the photoaging and aging processes of skin. The activity of catalase increased significantly in photoaged and aged epidermis, and increased more in photoaged epidermis than aged epidermis. This increased catalase level in the epidermis during the photoaging process is of interest, as it is known that catalase is destroyed by visible light (Cheng and Packer, 1979Cheng L.Y.L. Packer L. Photodamage to hepatocytes by visible light.FEBS Lett. 1979; 97: 124-128Abstract Full Text PDF PubMed Scopus (41) Google Scholar). It has also been reported that murine epidermal and dermal catalase showed a marked drop in activity after acute UVA and UVB irradiation (Shindo et al., 1994bShindo Y. Witt E. Han D. Epstein W. Packer L. Dose–response effects of acute ultraviolet irradiation on antioxidant and molecular markers of oxidation in murine epidermis and dermis.J Invest Dermatol. 1994; 102: 470-475Abstract Full Text PDF PubMed Google Scholar). The reason for this increased activity of epidermal catalase in photoaged and aged human skin is not clear at this point. It is possible that the induction of catalase in the epidermis may be a defense response to environmental oxidative stress. On the other hand, chronic oxidative stress over a lifetime might stimulate the skin cells to produce certain cytokines that upregulate catalase expression. Further investigations on this topic are being undertaken. In contrast to the epidermis, the activity of catalase decreased significantly in the dermis of photoaged and aged skin, and it tended to decrease less in the photoaged dermis than in the aged dermis. As UVR can reach into the upper dermis, cells in the upper dermis may respond to chronic UV exposure by inducing catalase. Our quantifications of catalase in the dermis are consistent with those ofShindo et al., 1991Shindo Y. Akiyama J. Yamazaki Y. Saito K. Takase Y. Changes in enzyme activities in skin fibroblasts derived from persons of various ages.Exp Gerontol. 1991; 26: 29-35Crossref PubMed Scopus (29) Google Scholar, who compared skin fibroblasts derived from old people with those derived from young people. They also described a remarkable decrease of catalase activity on aging, whereas SOD and GR remained unchanged. The reason for decreased catalase activity in the aged dermis is not clear, though it might be due to the aging process. Our results indicate that catalase activity may be differently regulated in the epidermis and dermis during photoaging and intrinsic aging, and that catalase might be a key enzyme that fulfills an important role in the protection of skin from oxidative damage. The observed effects of aging on antioxidants in human skin in vivo were quite different from those found in murine skin (Lopez-Torres et al., 1994Lopez-Torres M. Shindo Y. Packer L. Effect of age on antioxidants and molecular markers of oxidative damage in murine epidermis and dermis.J Invest Dermatol. 1994; 102: 476-480Abstract Full Text PDF PubMed Google Scholar). In the murine epidermis and dermis, no significant differences were found in the activities of SOD, catalase, and GR (expressed as units per mg protein) of young and old animals. Only epidermal GPx showed decreased activity with age. In addition, the hydrophilic (ascorbic acid, glutathione, and uric acid) and lipophilic (α-tocopherol) antioxidants did not change as a function of age in mouse skin. Ascorbic acid levels were lower in both the epidermis and dermis, whereas α-tocopherol was lower in the epidermis but not in the dermis, of both photoaged and aged skin. The reasons for this difference are not known. α-Tocopherol provides protection against oxidative membrane damage caused by various environmental factors, such as UV, presumably by scavenging ROS and free radicals (Burton and Traber, 1990Burton G.W. Traber M.G. Vitamin E: antioxidant activity, biokinetics and bioavailability.Ann Rev Nutr. 1990; 10: 357-382Crossref PubMed Scopus (621) Google Scholar). When skin is exposed to oxidative stress, such as UVR, a large number of tocophenoxy radicals are formed by the oxidation of α-tocopherol, and the antioxidative properties of α-tocopherol are closely linked with its continual regeneration by other antioxidants such as glutathione and ascorbic acid (Vessey, 1993Vessey D.A. The cutaneous antioxidant system.Clin Dermatol. 1993; 8: 81-103Google Scholar). As ascorbate is present in the skin in much higher amounts than α-tocopherol, it acts as a large reservoir of antioxidative potential, which may be delivered more specifically by α-tocopherol (Steenvoorden and Beijersbergen van Henegouwen, 1997Steenvoorden D.P.T. Beijersbergen van Henegouwen G.M.J. The use of endogenous antioxidants to improve photoprotection.Photochem Photobiol. 1997; 41: 1-10Crossref Scopus (174) Google Scholar). It is also possible that α-tocopherol in the dermis can be regenerated by other antioxidants supplied by the blood. It has been demonstrated that, when exposed to UV radiation, levels of α-tocopherol, ascorbate, and glutathione in the skin are depleted (Fuchs et al., 1989Fuchs J. Huflejt M.E. Rothfuss L.M. Wilson D.S. Carcamo G. Packer L. Impairment of enzymic and nonenzymic antioxidants in skin by UVB radiation.J Invest Dermatol. 1989; 93: 769-773Abstract Full Text PDF PubMed Google Scholar;Shindo et al., 1993Shindo Y. Witt E. Packer L. Antioxidant defense mechanisms in murine epidermis and dermis and their responses to ultraviolet light.J Invest Dermatol. 1993; 100: 260-265Abstract Full Text PDF PubMed Google Scholar). As α-tocopherol and ascorbic acid were decreased in both sun-exposed (forearm) and sun-protected (upper-inner arm) skin to a similar extent, other variables, perhaps nutritional factors, rather than chronic UV irradiation seem to play more important roles in the decrease of nonenzymic antioxidants in the skin of the elderly. According to the free radical theory of aging, ROS increases with aging due to the reduced activity of the antioxidant defense enzymes (Harman, 1956Harman D. Aging: a theory based on free radical and radiation chemistry.J Gerontol. 1956; 11: 298-300Crossref PubMed Scopus (6036) Google Scholar,Harman, 1981Harman D. The aging process.Proc Natl Acad Sci USA. 1981; 78: 7124-7128Crossref PubMed Scopus (1519) Google Scholar,Harman, 1986Harman D. Free radical theory of aging: role of free radicals in the origination and evolution of life, aging and disease process.in: Johnson J. Walford R. Harman D. Miquel J. Biology of Aging. Liss, New York1986: 3-50Google Scholar). Our results are compatible with this free radical theory of aging, as the decreased activity of catalase in the dermis, and the decreased levels of nonenzymic antioxidants in epidermis and dermis of photoaged and aged human skin in vivo, are believed to cause an age-associated increase in oxidative stress (ROS) in aged skin. Consequently, ROS, such as hydrogen peroxide, in aged skin may increase and be accumulated; these ROS will affect signaling pathways and finally lead to aged and photoaged skin in vivo (Fisher et al., 1997Fisher G.J. Wang Z. Datta S.C. Varani J. Kang S. Voorhees J.J. Pathophysiology of premature skin aging induced by ultraviolet light.New Engl J Med. 1997; 337: 1419-1428Crossref PubMed Scopus (1081) Google Scholar;Kang et al., 1997Kang S. Fisher G.J. Voorhees J.J. Photoaging and topical tretinoin; therapy, pathogenesis, and prevention.Arch Dermatol. 1997; 133: 1280-1284Crossref PubMed Google Scholar) Figure 3. In our laboratory, higher levels of hydrogen peroxide were found in skin fibroblasts derived from old people than those of younger subjects (personal observation). Thus, the induction and regulation of endogenous antioxidant defense mechanisms may offer a good strategy for the treatment and prevention of aging and photoaging in human skin. This study was supported in part by a grant “Life Phenomena and Function Research Group Program (2000)” from the Ministry of Science and Technology, and a research agreement with the Pacific Corporation. We thank Mi Ran Kwon for her excellent technical assistance. ErrataJournal of Investigative DermatologyVol. 118Issue 4PreviewIn the September 2001 issue of the Journal (117:3, 775) an author was missing from abstract 057 “Functional Analyses of Connexin Mutations in Keratinocytes”. The correct author list is as follows: Full-Text PDF Open Archive