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Antioxidant enzymes and lipid peroxidation in the scalp of patients with alopecia areata
Journal of Dermatological Science 29 (2002) 85 – 90 www.elsevier.com/locate/jdermsci
Antioxidant enzymes and lipid peroxidation in the scalp of patients with alopecia areata Ahmet Akar a,*, Ercan Arca a, Hakan Erbil a, Cemal Akay b, Ahmet Sayal b, A. Rıza Gu¨r a b
a Department of Dermatology, GATA School of Medicine, 06018, Ankara, Turkey Department of Pharmaceutical Sciences, GATA School of Medicine, Ankara, Turkey
Received 7 November 2001; received in revised form 13 February 2002; accepted 13 February 2002
Abstract Alopecia areata (AA) is an autoimmune inflammatory disease. However, little is known about the alterations in lipid peroxidation and antioxidant enzymes in the scalp of patients with AA. Therefore, the aim of this study was to investigate the status of oxidative stress in the scalp of patients with AA. We measured the levels of thiobarbituric acid reactive substances (TBARS) as lipid peroxidation status, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) as antioxidant enzymes in the scalp of ten patients with AA and ten control subjects. The levels of TBARS in scalp of patients with AA (3654.1 9 621.2 nmol/g tissue) were significantly higher than those of controls (1210.29 188.8 nmol/g tissue) (P =0.002). The levels of SOD (134.8 923.8 U/g tissue) and GSH-Px (332.7 9 66.2 U/g tissue) in scalp of patients with AA were also significantly higher than those of controls (63.2 9 8.8 U/g tissue, 112.0 918.4 U/g tissue, respectively) (P= 0.019, P= 0.002, respectively). The mean levels of TBARS, SOD and GSH-Px in early phase of disease were increased 2-fold as compared with late phase of the disease. These results indicate that oxidative status is affected in AA. Lipid peroxidation and antioxidant enzymes may be involved in the pathogenesis of AA. Furthermore, we found high SOD and GSH-Px activities in the scalp of patient with AA. These high levels could not protect the patients against the reactive oxygen species, because lipid peroxidation could not be lowered in AA patients. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Alopecia areata; Oxidative stress; Lipid peroxidation; Antioxidant enzyme
1. Introduction Alopecia areata (AA) is a common and sometimes disabling chronic inflammatory disorder of
* Corresponding author. Tel.: + 90-312-3044458; fax: + 90312-3044456. E-mail address: [email protected] (A. Akar).
the hair and nails [1], affecting about 2% of new patients attending dermatological clinics [2]. It ranges in severity from patchy loss of scalp hair to the loss of all scalp hair (alopecia totalis; AT) or all scalp and body hair (alopecia universalis; AU). The etiology and pathogenesis of AA is still unknown. Many factors such as the patient’s genetic constitution, the atopic state, nonspecific immune and organ-specific autoimmune reactions and pos-
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A. Akar et al. / Journal of Dermatological Science 29 (2002) 85–90
sibly emotional stress have been under investigation [3]. AA is characterized by an inflammatory cell infiltrate surrounding the hair follicle. This infiltrate is associated with damage to hair follicle and subsequent change in the normal keratinization [4]. In normal aerobic cells there exists a balance between oxidative damage and antioxidant protection. Inadequate antioxidant protection or excess production of reactive oxygen species (ROS) creates a condition known as oxidative stress, which is thought to play an important role in skin cancers, cutaneous aging and many inflammatory disorders [5–9]. Enzymes including superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were involved in the disposal of superoxide anion and hydrogen peroxide [10]. There are many studies demonstrating an association between the oxidative stress and skin diseases [11–16], but only one study is available in the literature on the antioxidant and lipid peroxidation status of patients with alopecia [17]. Nazirog˘ lu et al. [17] reported that there was a potential role of increased lipid peroxidation and decreased antioxidants in the blood of patients with alopecia. However, no study evaluated antioxidant status in the scalp of the patients with AA. Therefore, the current study was intended to determine the levels of thiobarbituric-acid reactive substances (TBARS) as lipid peroxidation status, SOD and GSH-Px as antioxidant status in the scalp of patients with AA.
2. Patients and methods
2.1. Subjects The study was performed on ten patients with AA (eight male and two female). The mean age of the patients was 23.2 years ranging from 20 to 28 years. Five patients had patchy AA, one had AT, one had AT/AU and three had AU. The last attack of disease was from 2 months to 19 years. The patients had not received any systemic or topical therapy. The control group consisted of ten healthy subjects who undergoing plastic surgery on their scalp matched according to age
and sex. All subjects were Caucasian. After obtaining informed consent, biopsies were taken from the scalps. Biopsies were obtained from sites of maximal disease activity, i.e. the advancing border of active lesions containing exclamationmark hairs in patchy AA patients. In AT, AU or AT/AU patients, biopsies were obtained from the same sites (occipital region of the scalp). This study was approved by local ethical committee. Biopsy specimens were rinsed in saline and were stored − 80 °C until assayed. Scalp biopsies from patients, together with biopsies from controls were studied at same time. Subcutaneous fat was removed with scalpel, and dermis with epidermis was weighed and minced and thoroughly rinsed with physiological saline to wash out traces of blood. And then they were homogenized.
2.2. Preparation of tissue homogenate Tissue samples were homogenized in cold KCl solution (1.5%) in a glass homogenizer on ice. Then these samples were centrifuged and supernatant was used for following determination.
2.3. Measurement of tissue TBARS concentration Tissue TBARS concentrations were estimated by method of Ohkawa and Yugi [18]. The supernatant was resuspended in 4 ml water, 0.5 ml glacial acetic acid and 0.5 ml 0.33% aqueous tiobarbituric acid solution. The mixture was heated for 60 min in a boiling water bath. After cooling of the samples, the complex formed by thiobarbituric acid reactant substances was extracted into an n-butanol phase, and the formed chromogen was measured at 532 nm by a spectrophotometer. A standard absorption curve for TBARS was prepared using tetramethoxy propane solution. TBARS level was expressed as U/g tissue.
2.4. Measurement of tissue SOD concentration SOD activity measurement was based on the generation of superoxide radicals produced by xanthine–xanthine oxidase system that reacts with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyl
M M M M M M M M F F
1 2 3 4 5 6 7 8 9 10
22 25 22 23 20 20 21 28 25 26
Age (years)
AT AU AA (20%) AT/AU AA (40%) AU AA (60%) AU AA (40%) AA (40%)
Type of Alopecia Occipital Occipital Vertex Occipital Occipital Occipital Left parietal Occipital Left parietal Frontal
Site of biopsy on the scalp 3 4 7 20 18 19 11 7 4 8
First episode of disease (years) 2 3 3 5 2 19 6 3 1 2
years months months years months years months years year years
Last episode of disease 832 3170 6105 1399 7107 2639 5052 2928 3694 3615
TBARS (nmol/g tissue) 36.52 88.75 235.18 54.23 255.69 89.29 205.31 111.99 150.40 121.20
SOD (U/g tissue)
106 283 551 139 793 158 397 236 342 322
GSH-Px (U/g tissue)
TBARS, thiobarbituric acid reactive substances; SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; percents show hair loss.
Sex
Patient
Table 1 The demographic data and levels of TBARS, SOD and GSH-Px in the scalp of patients with alopecia areata
A. Akar et al. / Journal of Dermatological Science 29 (2002) 85–90 87
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A. Akar et al. / Journal of Dermatological Science 29 (2002) 85–90
tetrazolium chloride to form red formosan dye. The SOD activity is then measured by the degree of inhibition of this reaction. SOD activity was expressed as nmol/g tissue [19].
2.5. Measurement of tissue GSH-Px concentration GSH-Px activity measurement is based on the following principle, GSH-Px catalysis the oxidation of glutathione by tertbutyl hydroperoxide. In the presence of glutathione reductase and reduced nicotinamide adenine dinucletide phosphate (NADPH), the oxidized glutathione is immediately converted to the reduced form with a concomitant oxidation of NADPH to NADP. The decrease in absorbance of NADPH at 340 nm was measured. GSH-Px activity was expressed as U/g tissue [20].
2.6. Statistical analysis All data were given as mean9standard deviation (S.D.). Statistical analysis was done by SPSS (Statistical package for the Social Sciences) statistical program. Tissue levels of TBARS, SOD, and GSH-Px in patients and control subjects were compared by the Mann – Whitney U test. A P value of less than 0.05 was considered to be statistically significant.
3. Results The demographic data and levels of TBARS, SOD and GSH-Px of patients with AA were shown in Table 1. The mean levels of TBARS, GSH-Px and SOD in tissue samples were shown in Table 2. The levels of TBARS in the scalp of patients with AA were significantly higher than those of controls (P =0.002). The levels of SOD and GSHPx in the scalp of patients with AA were also significantly higher than those of controls (P = 0.019, P= 0.002, respectively). The last episode of disease in four patients (Pt. no: 2, 3, 5 and 7) is below 6 months, that is, they are at early phase of disease. The mean levels of TBARS, SOD and GSH-Px in these four patients were 53589 1683 nmol/g tissue, 196.29 74.5 U/g
tissue and 5069 620 U/g tissue, respectively. In other six patients with late phase (long-lasting chronic) of disease, the mean levels of TBARS, SOD and GSH-Px were 25179 1171 nmol/g tissue, 93.99 42.7 U/g tissue and 2179 98 U/g tissue, respectively. The mean levels of TBARS, SOD and GSH-Px in patients with early phase of disease were 2-fold higher than those of patients with late phase.
4. Discussion To our knowledge, this is the first study investigating lipid peroxidation and primer antioxidant enzymes in scalp tissue of patient with AA. We found that SOD and GSH-Px activities were significantly elevated in the present study, most probably in defense against the excess production of superoxide radical. However, the TBARS levels were still high when compared with control. These results suggest that the increased SOD and GSHPx activities could not cope with elevated superoxide radical, or the increase in TBARS levels might have been originated from other radical(s) which are not metabolized by SOD and GSH-Px. AA is a tissue restricted autoimmune disease [21]. A most characteristic feature of the histopathology of hair follicle in acute AA is the presence of a peribulbar/intrabulbar mononuclear cell infiltrate [22]. Cytokines including interleukin I (IL-I) alpha, IL-I beta and tumor necrosis alpha
Table 2 Mean tissue levels of TBARS, SOD and GSH-Px in controls and patients with alopecia areata
TBARS (nmol/ g tissue) SOD (U/g tissue) GSH-Px (U/g tissue)
Control
Patient
P
1210.2 9188.8
3654.1 9 621.2
P=0.002
63.2 9 8.8
134.8 9 23.8
P= 0.019
112.0 918.4
332.7 966.2
P= 0.002
TBARS, thiobarbituric acid reactive substances; SOD, superoxide dismutase; GSH-Px, glutathione peroxidase.
A. Akar et al. / Journal of Dermatological Science 29 (2002) 85–90
(TNF-a) may play an important part in the pathophysiology of AA [23]. During inflammatory process, TNF-a is synthesized and released into the extracellular milieu by immune and non immune cells. TNF-a stimulates intracellular production of mitochondrial ROS [24]. An increase in ROS production could initiate an increase in the cellular antioxidant defense mechanism, i.e. an increase in SOD and GSH-Px activity [6]. In addition, lipid peroxidation (oxidative stress) has a role in autoimmunity [25]. Furthermore, three distinct patterns of cell degeneration were described including ‘dark cell’ transformation, apoptosis and necrosis at the morphological analysis of hair follicle of AA patients [22]. Moreover, increasing evidences provide support that oxidative stress and apoptosis are closely linked physiological phenomena and are implicated in pathophysiology of some of the chronic diseases including autoimmunity [26]. So hair follicle cells apoptosis may related to oxidants and antioxidants status in AA. Nazirog˘ lu et al. [17] reported that TBARS levels in plasma and erythrocytes were higher in patients with AA than the controls. We determined that TBARS levels in the scalp were significantly higher in patients with AA than in controls. Supporting this study, we have also found increased lipid peroxidation in patients with AA. The mean levels of TBARS in patients with early phase were higher than those of patients with late phase. This result suggests that lipid peroxidation is increased in active AA patients. Moreover, we have found significantly increase in SOD and GSH-Px levels in the scalp of patients with AA compared with controls. However, Nazirog˘ lu et al. [17] observed that activities of GSH-Px in both plasma and erythrocytes were significantly lower in patients with AA than in controls. Our findings are not support their results concerning GSH-Px. In addition, the SOD and GSH-Px activities were increased 2-fold in patients with early phase as compared with late phase. This may be due to increased activity of disease in the early phase. Antioxidant enzyme activities have been reported to vary from one cell type to another. For example, fibroblasts have higher levels of catalase, GSH-Px and SOD activ-
89
ity than keratinocytes. Red blood cells were found to have high levels of catalase activity [6]. We do not exactly know the discrepancies of concerning GSH-Px between skin and blood, but the possible cause of the discrepancies may be related to different tissues analyzed, differences in the employed assays or because of that AA is tissue-specific autoimmune disease. Our results concerning GSH-Px suggest that antioxidant defense including SOD and GSH-Px enzymes is not impaired in AA, unlike to Nazirog˘ lu et al. [17]. Supporting our findings, interestingly, antioxidant enzymes including SOD and GSH-Px activities were found to be elevated in fibroblasts from psoriatic skin [6]. Moreover, increased lipid peroxidation was found in psoriatic erythrocytes [27]. In conclusion, excessive free radical generations can occur in the scalp of the patients with AA, hence lipid peroxidation and antioxidant enzymes may be related to the pathogenesis of AA. Furthermore, we found high SOD and GSH-Px activities in the scalp of patient with AA. These high antioxidant enzymes levels could not protect the patients against the ROS, because lipid peroxidation could not be lowered in AA patients. In addition, lipid peroxidation and antioxidant enzyme activities in early phase of disease were increased 2-fold as compared with late phase of disease.
Acknowledgements We are grateful to Dr O8 zata and Aydin for their review of the manuscript.
References [1] McDonagh AJG, Messenger AG. The pathogenesis of alopecia areata. Dermatol Clin 1996;14:661 – 70. [2] Bodemer C, Peuchmaur M, Fraitaig S, Chatenoud L, Brausse N, Prost Y. The role of cytotoxic T-cells in chronic alopecia areata. J Invest Dermatol 2000;114:112 – 6. [3] Madani S, Shapiro J. Alopecia areata update. J Am Acad Dermatol 2000;42:549 – 66. [4] Duvic M, Nelson A, de Andrade M. The genetics of alopecia areata. Clin Dermatol 2001;19:135 – 9.
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[5] Lontz W, Sirsjo A, Liu W, Lindberg M, Rollman O, Torma H. Increased mRNA expression of manganese superoxide dismutase in psoriasis skin lesions and in cultured human keratinocytes exposed to IL-1 beta and TNF-alpha. Free Radic Biol Med 1995;18:349 – 55. [6] Therond P, Gerbaud P, Dimon S, Anderson WB, EvainBroin D, Raynaud F. Antioxidant enzymes in psoriatic fibroblasts and erythrocytes. J Invest Dermatol 1996;106:1325 – 8. [7] Niwa Y, Kanoh T, Sakane T, Soh H, Kawai S, Miyachi Y. Detection of enhanced lipid peroxide levels in patients with inflammatory skin diseases. J Clin Biochem Nutr 1987;2:245 – 51. [8] Emerit I. Free radicals and aging of the skin. EXS 1992;62:328 – 41. [9] Miyachi Y. Photoaging from an oxidative standpoint. J Dermatol Sci 1995;9:79 –86. [10] Chiou JF, Hu ML. Elevated lipid peroxidation and disturbed antioxidant enzyme activities in plasma and erythrocytes of patients with uterine cervicitis and myoma. Clin Biochem 1999;32:189 –92. [11] Omata N, Tsukahara H, Ito S, Ohshima Y, Yasutomi M, Yamada A, Jiang M, Hiraoka M, Nambu M, Deguchi Y, Mayumi M. Increased oxidative stress in childhood atopic dermatitis. Life Sci 2001;69:223 –8. [12] Vural P, Canbaz M, Selcuki D. Plasma antioxidant defense in actinic keratosis and basal cell carcinoma. J Eur Acad Dermatol Venereol 1999;13:96 – 101. [13] Yan T, Li S, Jiang X, Oberley LW. Altered levels of primary antioxidant enzymes in progeria skin fibroblasts. Biochem Biophys Res Commun 1999;257:163 –7. [14] Thiele JJ, Schroeter C, Hsieh SN, Podda M, Packer L. The antioxidant network of the stratum corneum. Curr Probl Dermatol 2001;29:26 –42. [15] Beazley WD, Gaze D, Panske A, Panzig E, Schallreuter KU. Serum selenium levels and blood glutathione peroxidase activities in vitiligo. Br J Dermatol 1999;141:301 –3. [16] Giralt M, Cervallo I, Nogues MR, Puerto MA, Ortin F, Argony N, Mallol J. Glutathione, glutathione S-trans-
[17]
[18]
[19]
[20]
[21]
[22] [23]
[24] [25]
[26] [27]
ferase and reactive oxygen species of human scalp sebaceous glands in male pattern baldness. J Invest Dermatol 1996;107:154 – 8. Nazirog˘ lu M, Ko¨ kc¸ am I. Antioxidants and lipid peroxidation status in the blood of patients with alopecia. Cell Biochem Funct 2000;18:169 – 73. Ohkawa H, Ohishi N, Yugi K. Assay for lipid peroxidation in animals tissues by thiobarbuturic acid reactions. Anal Biochem 1979;95:351 – 8. Aydin A, Orhan H, Sayal A, O8 zata M, S¸ ahin G, Is¸imer A. Oxidative stress and nitric oxide related parameters in type II diabetes mellitus: effects of glycemic control. Clin Biochem 2001;34:65 – 70. Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967;70:158 – 9. Kalish RS, Gilhar A. The immunology of alopecia areata and potential application to novel therapies. Dermatologic Therapy 2001;14:322 – 8. Tobin DJ. Morphological analysis of hair follicles in alopecia areata. Microsc Res Tech 1997;38:443 – 51. Philpott MP, Sanders DA, Bowen J, Kealey T. Effects of interleukins, colony-stimulating factor and tumour necrosis factor on human hair follicle growth in vitro: a possible role for interleukin-1 and tumour necrosis factor-alpha in alopecia areata. Br J Dermatol 1996;135:942 – 8. Reid MB, Li YP. Cytokines and oxidative signaling in skeletal muscle. Acta Physiol Scand 2001;171:225 – 32. Khan MF, Wu X, Ansari GA. Anti-malondialdehyde antibodies in MRL +/ + mice treated with trichloroethene and dichloroacetyl chloride: possible role of lipid peroxidation in autoimmunity. Toxicol Appl Pharmacol 2001;170:88 – 92. Kannan K, Jain SK. Oxidative stress and apoptosis. Pathophysiology 2000;7:153 – 63. Kokc¸ am I, Nazirog˘ lu M. Antioxidants and lipid peroxidation status in the blood of patients with psoriasis. Clin Chim Acta 1999;289:23 – 31.
Antioxidant enzymes and lipid peroxidation in the scalp of patients with alopecia areata Ahmet Akar a,*, Ercan Arca a, Hakan Erbil a, Cemal Akay b, Ahmet Sayal b, A. Rıza Gu¨r a b
a Department of Dermatology, GATA School of Medicine, 06018, Ankara, Turkey Department of Pharmaceutical Sciences, GATA School of Medicine, Ankara, Turkey
Received 7 November 2001; received in revised form 13 February 2002; accepted 13 February 2002
Abstract Alopecia areata (AA) is an autoimmune inflammatory disease. However, little is known about the alterations in lipid peroxidation and antioxidant enzymes in the scalp of patients with AA. Therefore, the aim of this study was to investigate the status of oxidative stress in the scalp of patients with AA. We measured the levels of thiobarbituric acid reactive substances (TBARS) as lipid peroxidation status, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) as antioxidant enzymes in the scalp of ten patients with AA and ten control subjects. The levels of TBARS in scalp of patients with AA (3654.1 9 621.2 nmol/g tissue) were significantly higher than those of controls (1210.29 188.8 nmol/g tissue) (P =0.002). The levels of SOD (134.8 923.8 U/g tissue) and GSH-Px (332.7 9 66.2 U/g tissue) in scalp of patients with AA were also significantly higher than those of controls (63.2 9 8.8 U/g tissue, 112.0 918.4 U/g tissue, respectively) (P= 0.019, P= 0.002, respectively). The mean levels of TBARS, SOD and GSH-Px in early phase of disease were increased 2-fold as compared with late phase of the disease. These results indicate that oxidative status is affected in AA. Lipid peroxidation and antioxidant enzymes may be involved in the pathogenesis of AA. Furthermore, we found high SOD and GSH-Px activities in the scalp of patient with AA. These high levels could not protect the patients against the reactive oxygen species, because lipid peroxidation could not be lowered in AA patients. © 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Alopecia areata; Oxidative stress; Lipid peroxidation; Antioxidant enzyme
1. Introduction Alopecia areata (AA) is a common and sometimes disabling chronic inflammatory disorder of
* Corresponding author. Tel.: + 90-312-3044458; fax: + 90312-3044456. E-mail address: [email protected] (A. Akar).
the hair and nails [1], affecting about 2% of new patients attending dermatological clinics [2]. It ranges in severity from patchy loss of scalp hair to the loss of all scalp hair (alopecia totalis; AT) or all scalp and body hair (alopecia universalis; AU). The etiology and pathogenesis of AA is still unknown. Many factors such as the patient’s genetic constitution, the atopic state, nonspecific immune and organ-specific autoimmune reactions and pos-
0923-1811/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 9 2 3 - 1 8 1 1 ( 0 2 ) 0 0 0 1 5 - 4
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A. Akar et al. / Journal of Dermatological Science 29 (2002) 85–90
sibly emotional stress have been under investigation [3]. AA is characterized by an inflammatory cell infiltrate surrounding the hair follicle. This infiltrate is associated with damage to hair follicle and subsequent change in the normal keratinization [4]. In normal aerobic cells there exists a balance between oxidative damage and antioxidant protection. Inadequate antioxidant protection or excess production of reactive oxygen species (ROS) creates a condition known as oxidative stress, which is thought to play an important role in skin cancers, cutaneous aging and many inflammatory disorders [5–9]. Enzymes including superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were involved in the disposal of superoxide anion and hydrogen peroxide [10]. There are many studies demonstrating an association between the oxidative stress and skin diseases [11–16], but only one study is available in the literature on the antioxidant and lipid peroxidation status of patients with alopecia [17]. Nazirog˘ lu et al. [17] reported that there was a potential role of increased lipid peroxidation and decreased antioxidants in the blood of patients with alopecia. However, no study evaluated antioxidant status in the scalp of the patients with AA. Therefore, the current study was intended to determine the levels of thiobarbituric-acid reactive substances (TBARS) as lipid peroxidation status, SOD and GSH-Px as antioxidant status in the scalp of patients with AA.
2. Patients and methods
2.1. Subjects The study was performed on ten patients with AA (eight male and two female). The mean age of the patients was 23.2 years ranging from 20 to 28 years. Five patients had patchy AA, one had AT, one had AT/AU and three had AU. The last attack of disease was from 2 months to 19 years. The patients had not received any systemic or topical therapy. The control group consisted of ten healthy subjects who undergoing plastic surgery on their scalp matched according to age
and sex. All subjects were Caucasian. After obtaining informed consent, biopsies were taken from the scalps. Biopsies were obtained from sites of maximal disease activity, i.e. the advancing border of active lesions containing exclamationmark hairs in patchy AA patients. In AT, AU or AT/AU patients, biopsies were obtained from the same sites (occipital region of the scalp). This study was approved by local ethical committee. Biopsy specimens were rinsed in saline and were stored − 80 °C until assayed. Scalp biopsies from patients, together with biopsies from controls were studied at same time. Subcutaneous fat was removed with scalpel, and dermis with epidermis was weighed and minced and thoroughly rinsed with physiological saline to wash out traces of blood. And then they were homogenized.
2.2. Preparation of tissue homogenate Tissue samples were homogenized in cold KCl solution (1.5%) in a glass homogenizer on ice. Then these samples were centrifuged and supernatant was used for following determination.
2.3. Measurement of tissue TBARS concentration Tissue TBARS concentrations were estimated by method of Ohkawa and Yugi [18]. The supernatant was resuspended in 4 ml water, 0.5 ml glacial acetic acid and 0.5 ml 0.33% aqueous tiobarbituric acid solution. The mixture was heated for 60 min in a boiling water bath. After cooling of the samples, the complex formed by thiobarbituric acid reactant substances was extracted into an n-butanol phase, and the formed chromogen was measured at 532 nm by a spectrophotometer. A standard absorption curve for TBARS was prepared using tetramethoxy propane solution. TBARS level was expressed as U/g tissue.
2.4. Measurement of tissue SOD concentration SOD activity measurement was based on the generation of superoxide radicals produced by xanthine–xanthine oxidase system that reacts with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyl
M M M M M M M M F F
1 2 3 4 5 6 7 8 9 10
22 25 22 23 20 20 21 28 25 26
Age (years)
AT AU AA (20%) AT/AU AA (40%) AU AA (60%) AU AA (40%) AA (40%)
Type of Alopecia Occipital Occipital Vertex Occipital Occipital Occipital Left parietal Occipital Left parietal Frontal
Site of biopsy on the scalp 3 4 7 20 18 19 11 7 4 8
First episode of disease (years) 2 3 3 5 2 19 6 3 1 2
years months months years months years months years year years
Last episode of disease 832 3170 6105 1399 7107 2639 5052 2928 3694 3615
TBARS (nmol/g tissue) 36.52 88.75 235.18 54.23 255.69 89.29 205.31 111.99 150.40 121.20
SOD (U/g tissue)
106 283 551 139 793 158 397 236 342 322
GSH-Px (U/g tissue)
TBARS, thiobarbituric acid reactive substances; SOD, superoxide dismutase; GSH-Px, glutathione peroxidase; AA, alopecia areata; AT, alopecia totalis; AU, alopecia universalis; percents show hair loss.
Sex
Patient
Table 1 The demographic data and levels of TBARS, SOD and GSH-Px in the scalp of patients with alopecia areata
A. Akar et al. / Journal of Dermatological Science 29 (2002) 85–90 87
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A. Akar et al. / Journal of Dermatological Science 29 (2002) 85–90
tetrazolium chloride to form red formosan dye. The SOD activity is then measured by the degree of inhibition of this reaction. SOD activity was expressed as nmol/g tissue [19].
2.5. Measurement of tissue GSH-Px concentration GSH-Px activity measurement is based on the following principle, GSH-Px catalysis the oxidation of glutathione by tertbutyl hydroperoxide. In the presence of glutathione reductase and reduced nicotinamide adenine dinucletide phosphate (NADPH), the oxidized glutathione is immediately converted to the reduced form with a concomitant oxidation of NADPH to NADP. The decrease in absorbance of NADPH at 340 nm was measured. GSH-Px activity was expressed as U/g tissue [20].
2.6. Statistical analysis All data were given as mean9standard deviation (S.D.). Statistical analysis was done by SPSS (Statistical package for the Social Sciences) statistical program. Tissue levels of TBARS, SOD, and GSH-Px in patients and control subjects were compared by the Mann – Whitney U test. A P value of less than 0.05 was considered to be statistically significant.
3. Results The demographic data and levels of TBARS, SOD and GSH-Px of patients with AA were shown in Table 1. The mean levels of TBARS, GSH-Px and SOD in tissue samples were shown in Table 2. The levels of TBARS in the scalp of patients with AA were significantly higher than those of controls (P =0.002). The levels of SOD and GSHPx in the scalp of patients with AA were also significantly higher than those of controls (P = 0.019, P= 0.002, respectively). The last episode of disease in four patients (Pt. no: 2, 3, 5 and 7) is below 6 months, that is, they are at early phase of disease. The mean levels of TBARS, SOD and GSH-Px in these four patients were 53589 1683 nmol/g tissue, 196.29 74.5 U/g
tissue and 5069 620 U/g tissue, respectively. In other six patients with late phase (long-lasting chronic) of disease, the mean levels of TBARS, SOD and GSH-Px were 25179 1171 nmol/g tissue, 93.99 42.7 U/g tissue and 2179 98 U/g tissue, respectively. The mean levels of TBARS, SOD and GSH-Px in patients with early phase of disease were 2-fold higher than those of patients with late phase.
4. Discussion To our knowledge, this is the first study investigating lipid peroxidation and primer antioxidant enzymes in scalp tissue of patient with AA. We found that SOD and GSH-Px activities were significantly elevated in the present study, most probably in defense against the excess production of superoxide radical. However, the TBARS levels were still high when compared with control. These results suggest that the increased SOD and GSHPx activities could not cope with elevated superoxide radical, or the increase in TBARS levels might have been originated from other radical(s) which are not metabolized by SOD and GSH-Px. AA is a tissue restricted autoimmune disease [21]. A most characteristic feature of the histopathology of hair follicle in acute AA is the presence of a peribulbar/intrabulbar mononuclear cell infiltrate [22]. Cytokines including interleukin I (IL-I) alpha, IL-I beta and tumor necrosis alpha
Table 2 Mean tissue levels of TBARS, SOD and GSH-Px in controls and patients with alopecia areata
TBARS (nmol/ g tissue) SOD (U/g tissue) GSH-Px (U/g tissue)
Control
Patient
P
1210.2 9188.8
3654.1 9 621.2
P=0.002
63.2 9 8.8
134.8 9 23.8
P= 0.019
112.0 918.4
332.7 966.2
P= 0.002
TBARS, thiobarbituric acid reactive substances; SOD, superoxide dismutase; GSH-Px, glutathione peroxidase.
A. Akar et al. / Journal of Dermatological Science 29 (2002) 85–90
(TNF-a) may play an important part in the pathophysiology of AA [23]. During inflammatory process, TNF-a is synthesized and released into the extracellular milieu by immune and non immune cells. TNF-a stimulates intracellular production of mitochondrial ROS [24]. An increase in ROS production could initiate an increase in the cellular antioxidant defense mechanism, i.e. an increase in SOD and GSH-Px activity [6]. In addition, lipid peroxidation (oxidative stress) has a role in autoimmunity [25]. Furthermore, three distinct patterns of cell degeneration were described including ‘dark cell’ transformation, apoptosis and necrosis at the morphological analysis of hair follicle of AA patients [22]. Moreover, increasing evidences provide support that oxidative stress and apoptosis are closely linked physiological phenomena and are implicated in pathophysiology of some of the chronic diseases including autoimmunity [26]. So hair follicle cells apoptosis may related to oxidants and antioxidants status in AA. Nazirog˘ lu et al. [17] reported that TBARS levels in plasma and erythrocytes were higher in patients with AA than the controls. We determined that TBARS levels in the scalp were significantly higher in patients with AA than in controls. Supporting this study, we have also found increased lipid peroxidation in patients with AA. The mean levels of TBARS in patients with early phase were higher than those of patients with late phase. This result suggests that lipid peroxidation is increased in active AA patients. Moreover, we have found significantly increase in SOD and GSH-Px levels in the scalp of patients with AA compared with controls. However, Nazirog˘ lu et al. [17] observed that activities of GSH-Px in both plasma and erythrocytes were significantly lower in patients with AA than in controls. Our findings are not support their results concerning GSH-Px. In addition, the SOD and GSH-Px activities were increased 2-fold in patients with early phase as compared with late phase. This may be due to increased activity of disease in the early phase. Antioxidant enzyme activities have been reported to vary from one cell type to another. For example, fibroblasts have higher levels of catalase, GSH-Px and SOD activ-
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ity than keratinocytes. Red blood cells were found to have high levels of catalase activity [6]. We do not exactly know the discrepancies of concerning GSH-Px between skin and blood, but the possible cause of the discrepancies may be related to different tissues analyzed, differences in the employed assays or because of that AA is tissue-specific autoimmune disease. Our results concerning GSH-Px suggest that antioxidant defense including SOD and GSH-Px enzymes is not impaired in AA, unlike to Nazirog˘ lu et al. [17]. Supporting our findings, interestingly, antioxidant enzymes including SOD and GSH-Px activities were found to be elevated in fibroblasts from psoriatic skin [6]. Moreover, increased lipid peroxidation was found in psoriatic erythrocytes [27]. In conclusion, excessive free radical generations can occur in the scalp of the patients with AA, hence lipid peroxidation and antioxidant enzymes may be related to the pathogenesis of AA. Furthermore, we found high SOD and GSH-Px activities in the scalp of patient with AA. These high antioxidant enzymes levels could not protect the patients against the ROS, because lipid peroxidation could not be lowered in AA patients. In addition, lipid peroxidation and antioxidant enzyme activities in early phase of disease were increased 2-fold as compared with late phase of disease.
Acknowledgements We are grateful to Dr O8 zata and Aydin for their review of the manuscript.
References [1] McDonagh AJG, Messenger AG. The pathogenesis of alopecia areata. Dermatol Clin 1996;14:661 – 70. [2] Bodemer C, Peuchmaur M, Fraitaig S, Chatenoud L, Brausse N, Prost Y. The role of cytotoxic T-cells in chronic alopecia areata. J Invest Dermatol 2000;114:112 – 6. [3] Madani S, Shapiro J. Alopecia areata update. J Am Acad Dermatol 2000;42:549 – 66. [4] Duvic M, Nelson A, de Andrade M. The genetics of alopecia areata. Clin Dermatol 2001;19:135 – 9.
90
A. Akar et al. / Journal of Dermatological Science 29 (2002) 85–90
[5] Lontz W, Sirsjo A, Liu W, Lindberg M, Rollman O, Torma H. Increased mRNA expression of manganese superoxide dismutase in psoriasis skin lesions and in cultured human keratinocytes exposed to IL-1 beta and TNF-alpha. Free Radic Biol Med 1995;18:349 – 55. [6] Therond P, Gerbaud P, Dimon S, Anderson WB, EvainBroin D, Raynaud F. Antioxidant enzymes in psoriatic fibroblasts and erythrocytes. J Invest Dermatol 1996;106:1325 – 8. [7] Niwa Y, Kanoh T, Sakane T, Soh H, Kawai S, Miyachi Y. Detection of enhanced lipid peroxide levels in patients with inflammatory skin diseases. J Clin Biochem Nutr 1987;2:245 – 51. [8] Emerit I. Free radicals and aging of the skin. EXS 1992;62:328 – 41. [9] Miyachi Y. Photoaging from an oxidative standpoint. J Dermatol Sci 1995;9:79 –86. [10] Chiou JF, Hu ML. Elevated lipid peroxidation and disturbed antioxidant enzyme activities in plasma and erythrocytes of patients with uterine cervicitis and myoma. Clin Biochem 1999;32:189 –92. [11] Omata N, Tsukahara H, Ito S, Ohshima Y, Yasutomi M, Yamada A, Jiang M, Hiraoka M, Nambu M, Deguchi Y, Mayumi M. Increased oxidative stress in childhood atopic dermatitis. Life Sci 2001;69:223 –8. [12] Vural P, Canbaz M, Selcuki D. Plasma antioxidant defense in actinic keratosis and basal cell carcinoma. J Eur Acad Dermatol Venereol 1999;13:96 – 101. [13] Yan T, Li S, Jiang X, Oberley LW. Altered levels of primary antioxidant enzymes in progeria skin fibroblasts. Biochem Biophys Res Commun 1999;257:163 –7. [14] Thiele JJ, Schroeter C, Hsieh SN, Podda M, Packer L. The antioxidant network of the stratum corneum. Curr Probl Dermatol 2001;29:26 –42. [15] Beazley WD, Gaze D, Panske A, Panzig E, Schallreuter KU. Serum selenium levels and blood glutathione peroxidase activities in vitiligo. Br J Dermatol 1999;141:301 –3. [16] Giralt M, Cervallo I, Nogues MR, Puerto MA, Ortin F, Argony N, Mallol J. Glutathione, glutathione S-trans-
[17]
[18]
[19]
[20]
[21]
[22] [23]
[24] [25]
[26] [27]
ferase and reactive oxygen species of human scalp sebaceous glands in male pattern baldness. J Invest Dermatol 1996;107:154 – 8. Nazirog˘ lu M, Ko¨ kc¸ am I. Antioxidants and lipid peroxidation status in the blood of patients with alopecia. Cell Biochem Funct 2000;18:169 – 73. Ohkawa H, Ohishi N, Yugi K. Assay for lipid peroxidation in animals tissues by thiobarbuturic acid reactions. Anal Biochem 1979;95:351 – 8. Aydin A, Orhan H, Sayal A, O8 zata M, S¸ ahin G, Is¸imer A. Oxidative stress and nitric oxide related parameters in type II diabetes mellitus: effects of glycemic control. Clin Biochem 2001;34:65 – 70. Paglia DE, Valentine WN. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J Lab Clin Med 1967;70:158 – 9. Kalish RS, Gilhar A. The immunology of alopecia areata and potential application to novel therapies. Dermatologic Therapy 2001;14:322 – 8. Tobin DJ. Morphological analysis of hair follicles in alopecia areata. Microsc Res Tech 1997;38:443 – 51. Philpott MP, Sanders DA, Bowen J, Kealey T. Effects of interleukins, colony-stimulating factor and tumour necrosis factor on human hair follicle growth in vitro: a possible role for interleukin-1 and tumour necrosis factor-alpha in alopecia areata. Br J Dermatol 1996;135:942 – 8. Reid MB, Li YP. Cytokines and oxidative signaling in skeletal muscle. Acta Physiol Scand 2001;171:225 – 32. Khan MF, Wu X, Ansari GA. Anti-malondialdehyde antibodies in MRL +/ + mice treated with trichloroethene and dichloroacetyl chloride: possible role of lipid peroxidation in autoimmunity. Toxicol Appl Pharmacol 2001;170:88 – 92. Kannan K, Jain SK. Oxidative stress and apoptosis. Pathophysiology 2000;7:153 – 63. Kokc¸ am I, Nazirog˘ lu M. Antioxidants and lipid peroxidation status in the blood of patients with psoriasis. Clin Chim Acta 1999;289:23 – 31.