BASIC/CLINICAL SCIENCE

Low Glutathione Peroxidase Activity Levels in Patients with Vitiligo Hatem Zedan, Amira Ali Abdel-Motaleb, Nahed Mahmoud Ali Kassem, Heba Ahmed Abdel Hafeez, and Mahmoud Rezk Abdelwhahed Hussein Background: Vitiligo is an idiopathic skin disease characterized by white areas on the skin due to loss of the functional melanocytes, with possible involvement of oxidative stress. Glutathione peroxidase (GPx) is an antioxidant enzyme that protects cells against oxidative damage. Aim: To examine serum GPx levels in patients with vitiligo and to relate the findings to the clinical features. Patients and Methods: The study group included 60 patients with vitiligo and 30 matching healthy controls. GPx activity was evaluated using enzyme-linked immunosorbent assay. Results: We found a significant decrease in serum GPx activity level in the patients with vitiligo compared to the healthy controls (0.29 6 0.14 versus 0.47 6 0.13, p , .001). The levels were significantly low in skin phenotypes III and IV (p , .001). Higher levels were also observed with increasing age ($ 14 years), prolonged disease duration ($ 3 years), and generalized and extensive vitiligo (, 50%). However, these variations were statistically insignificant. Conclusions: Low levels of serum GPx activity, indicative of a disturbed oxidant-antioxidant system, may contribute to the development of vitiligo. Contexte: Le vitiligo est une maladie cutane´e idiopathique, qui se caracte´rise par la pre´sence de plaques blanches sur la peau en raison de la perte des me´lanocytes fonctionnels et du roˆle possible du stress oxydatif. La glutathion-peroxydase (GP) est une enzyme antioxydante, qui prote`ge les cellules du stress oxydatif. Buts: L’e´tude visait a` mesurer le taux se´rique de GP chez des patients atteints de vitiligo et a` e´tablir des liens entre les re´sultats obtenus et les manifestations cliniques. Patients et me´thode: Le groupe a` l’e´tude comptait 60 patients atteints de vitiligo et 30 te´moins apparie´s, en bonne sante´. L’activite´ de la GP a e´te´ mesure´e a` l’aide de la me´thode immunoenzymatique (ELISA). Re´sultats: Une diminution importante du degre´ d’activite´ de la GP se´rique a e´te´ note´e chez les patients atteints de vitiligo comparativement aux te´moins en bonne sante´ (0.29 6 0.14 contre 0.47 6 0.13; p , .001); les degre´s d’activite´ e´taient particulie`rement bas chez les patients au phe´notype cutane´ III ou IV (p , .001). Des degre´s supe´rieurs d’activite´ ont e´te´ enregistre´s chez les patients plus aˆge´s ($ 14 ans) ainsi que dans les cas de dure´e prolonge´e de la maladie ($ 3 ans) et de vitiligo ge´ne´ralise´ et e´tendu (, 50%). Toutefois, les e´carts e´taient statistiquement non significatifs. Conclusions: De faibles degre´s d’activite´ de la GP se´rique, re´ve´lateurs d’un de´se´quilibre entre oxydants et antioxydants, peuvent jouer un roˆle dans l’apparition du vitiligo.

ITILIGO is an acquired, noncontagious, progressive, depigmenting disorder of the skin and mucous membranes. It results from destruction of the melanocytes with loss of melanin pigment production. It has a prevalence ranging from less than 0.1% to greater than 8% worldwide. It is more predominant in women, and about half of all patients develop the disease before 20 years of age.1 Clinically, vitiligo is characterized by the development of white macules.1 The pathogenesis of vitiligo remains unclear; however, possible contributing factors include autoimmune, viral,

V

From the Departments of Dermatology and Venereology and Andrology, Clinical Pathology, and Pathology, Faculties of Medicine, Assuit University, Assuit, Egypt. Address reprint requests to: Mahmoud Rezk Abdelwhahed Hussein, MD, PhD, FRCPath, FRCPC, Faculty of Medicine, Assuit University, Assuit, Egypt; e-mail: [email protected].

DOI 10.2310/7750.2014.14076 # 2014 Canadian Dermatology Association

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neural, and genetic factors.1,2 Several observations raised the notion that disruption of the oxidant-antioxidant system, with increased production of oxidants due to mitochondrial impairment, is involved in the pathogenesis of vitiligo. Free radicals generated in vitiligo appear to be in part from keratinocytes, melanocytes, and peripheral blood mononuclear cells. Accumulation of free radicals toxic to the melanocytes leads to their destruction and loss of melanin pigment production.3 Glutathione peroxidase (GPx) is the general name of an enzyme family with peroxidase activity. Its main biological role is to protect cells from oxidative damage by reducing lipid hydroperoxides to their corresponding alcohols and reducing free hydrogen peroxide to water. In vitiligo, serum and erythrocyte levels of GPx were examined, and, to date, the results are contrasting. Some studies reported low GPx levels, whereas others indicated either normal or even elevated levels.4–10 Khan and colleagues examined serum levels of malondialdehyde and vitamins E and C, total antioxidant activity, and whole blood levels of superoxide dismutase and GPx in vitiligo patients.5 They found significantly higher levels of malondialdehyde and significantly lower levels of superoxide dismutase, GPx, vitamins C and E, and total antioxidant activity in vitiligo patients compared to healthy controls. Alternatively, some studies reported either normal or increased GPx levels in vitiligo patients.7–9 Ozturk and colleagues reported increased plasma levels of malondialdehyde, glutathione, selenium, hydroxyproline, and GPx activity levels in patients with vitiligo.7 To date, studies about GPx levels in vitiligo are contrasting, and our knowledge about variations in GPx activity levels based on the clinical features (including age, sex, skin phenotype, type, course, and extent) in vitiligo patients is limited. This study tried to address these issues.

Patients and Methods We conducted this study from September 2011 to September 2012 at the Dermatology, Venereology and Andrology and Clinical Pathology departments of Assiut University Hospital, Egypt. The experimental design was approved by the Institutional Ethics and Research Committee of the Faculty of Medicine. Informed consent was obtained from the patients and controls. Exclusion criteria included the use of topical or systemic immunosuppressing therapy, vitamins, and antiinflammatory drugs over the last 4 weeks before the study. Other criteria included a history of smoking, alcoholism, or any debilitating disease (chronic liver, kidney, or cardiovascular disease;

immunosuppressive disease; or malignancy). The study group consisted of 60 patients (39 females and 21 males) with vitiligo and 30 healthy controls (19 females and 11 males). A full clinical history and a physical examination were performed. Patients with vitiligo were divided into several subgroups based on the following criteria: age (37 patients , 14 years old versus 23 patients $ 14 years old), duration of the disease (, 3 years versus $ 3 years), type of vitiligo (localized versus generalized), extent of the disease (using the role of nine), and course of the disease (stationary versus progressive versus regressive). Venous blood (10 mL) was collected in vacutainers without additive, allowed to clot for 30 minutes at room temperature, and centrifuged for 5 minutes to separate the serum. The serum samples were stored at 220uC. Hemolysed samples were excluded. GPx activity levels were measured by enzyme-linked immunosorbent assay (ELISA) using the method of Paglia and Valentine11 and were expressed as mean and standard deviation (SD). The results were analyzed using IBM-SPSS version 20 software (IBM Corp., Armonk, NY), and the level of significance was p , .001.

Results The age of the control group ranged from 7 to 46 years (mean 6 SD of 21.37 6 12.49; 14 of them were , 14 years old versus 16 $ 14 years old). The age of the patients ranged from 6 to 55 years (mean 6 SD of 26.2 6 16.68). We found a significant decrease in GPx activity level in the patients with vitiligo compared to healthy controls (0.29 6 0.14 versus 0.47 6 0.13, p , .001). There was a statistically significant decrease in GPx activity level according to the skin phenotype (skin type III phenotype versus skin type IV or V, p , .001, and skin type IV versus skin type V, p , .001). When we related GPx activity levels to the clinical features (age, sex, course, duration, extent of the disease), they were low among patients with a stationary course, duration , 3 years, and generalized and extensive vitiligo (, 50%). However, these variations were statistically insignificant. Our findings are summarized in Table 1.

Discussion Several previous studies have examined oxidantantioxidant systems, including GPx activity levels in vitiligo, and the results are contrasting.4–10 We speculate that these variations may be due to variations in the methodologies used for analysis of GPX activity among

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Table 1. Glutathione Peroxidase Activity Levels in Patients with Vitiligo and Healthy Controls Characteristic Group Age (yr) $ 14 , 14 Sex Males Females Skin phenotype III IV V Course Progressive Regressive Stationary Duration (yr) ,3 $3 Type of vitiligo Localized Generalized Extent (%) , 20 20–50 . 50

Patients (n)

Controls (n)

p Value

0.29 6 0.14 (60)

0.47 6 0.13 (30)

, .001

0.30 6 0.15 (37) 0.27 6 0.12 (14)

0.52 6 0.16 (16) 0.042 6 0.06 (14)

, .001* , .001*

0.29 6 0.15 (21) 0.29 6 0.14 (39)

0.43 6 0.14 (11) 0.5 6 0.12 (19)

, .001* , .02*

0.15 6 0.06 (18) 0.30 6 0.05 (30) 0.52 6 0.13 (12)

0.36 6 0.05 (10) 0.47 6 0.02 (15) 0.7 6 0.09 (5)

, .001* , .001* .015*

0.29 6 0.14 (48) 0.35 6 0.13 (3) 0.27 6 0.17 (9)

0.47 6 0.13 0.47 6 0.13 0.47 6 0.13

, .001* .136 , .001*

0.25 6 0.11 (21) 0.31 6 0.15 (30)

0.47 6 0.13 0.47 6 0.13

, .001* , .001*

0.3 6 0.15 (20) 0.29 6 0.14 (40)

0.47 6 0.13 0.47 6 0.13

0.3 6 0.13 (44) 0.27 6 0.11 (11) 0.23 6 0.26 (5)

0.47 6 0.13 0.47 6 0.13 0.47 6 0.13

.09 .02* , .001* , .001* .02*

Glutathione peroxidase was expressed as mU/mL. *Significant if less than .05 and highly significant if less than .001.

these studies. Also, the complex interaction of biochemical, environmental, and immunologic events, in a permissive genetic milieu in vitiligo, may account for these contrasting results. There are multiple pathogenic mechanisms for vitiligo, including neuronal, autoimmune, endocrine, reduced melanocyte survival, apoptosis, and oxidative stress.12 It is still possible that the contrasting results may be due to the implication of different etiologic factors. Ines and colleagues examined serum levels of malondialdehyde, selenium, and vitamins E and A and the erythrocyte activities of glutathione peroxidase, superoxide dismutase, and catalase in patients with active vitiligo and patients with stable vitiligo. The authors observed a significantly higher level of serum malondialdehyde and selenium in patients with active disease compared to controls.6 At least one isoform of GPx requires selenium for its activity.13 Also, a significantly higher increase in erythrocyte superoxide dismutase activities was observed in the active vitiligo group, erythrocyte glutathione 146

peroxidase activity was decreased significantly in active disease, and erythrocyte catalase activity and plasma vitamin E and A levels were not different in vitiligo patients compared to controls.6 Alternatively, some studies reported either normal or increased GPx levels in vitiligo patients.7–9 Hazneci and colleagues investigated the erythrocyte and plasma activities of GPx and Cu/Zn superoxide dismutase, plasma nitrite/nitrate levels, and erythrocyte catalase activity in 23 vitiligo patients and 25 controls.9 The erythrocyte superoxide dismutase activity and plasma nitrite/nitrate levels were high in vitiligo patients. Similarly, Yildirim and colleagues examined levels of superoxide dismutase, GPx, malondialdehyde, and nitric oxide in the tissue of 25 patients with generalized vitiligo and 25 healthy controls.8 The levels of superoxide dismutase, GPx, and malondialdehyde in tissue were significantly increased in patients with generalized vitiligo. In accordance with other investigators, we found a significant decrease in GPx activity level in the patients with vitiligo compared to healthy controls.4–6,12 Jalel and

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Hamdaoui examined 60 vitiligo patients and 62 healthy controls.12 All patients with vitiligo showed low levels of total antioxidant status and low GPx activity compared to the control group. Oxidative stress is a well-established triggering factor in the degradation of the melanocyte during the development of vitiligo. During melanin biosynthesis, some intermediates toxic to melanocytes are generated, such as 3,4-dihydroxyphenylalanine, dopachrome, and 5,6-dihydroxyindole.4 The accumulation of these intermediates destroys the melanocytes, resulting in skin depigmentation. In our series, the low GPx levels in patients with vitiligo may be explained by its consumption in neutralizing the increasing levels of the free radicals and hydrogen peroxide. In support, GPx not only degrades hydrogen peroxide but also has the ability to neutralize lipid hydroperoxides.4 Moreover, accumulation of hydrogen peroxide is associated with low blood GPx activity in vitiligo.14 The accumulation of hydrogen peroxide and low catalase levels were recently demonstrated in the epidermis of vitiligo patients.13,15 The pathophysiology of vitiligo is poorly understood. However, oxidative stress is proposed as an important factor in its development. Catalase is the main enzyme responsible for degrading hydrogen peroxide in melanocytes. Low basal levels of catalase activity are associated with the light phenotype in in vitro and ex vivo models. The total melanin concentration is directly correlated to the level of pigmentation; the more the degree of pigmentation increased, the lower the proportion of pheomelanin present. Darkly pigmented melanocytes have two protective mechanisms represented by melanins and catalase activity that counteract the deleterious effects of ultraviolet (UV) radiation. Alternatively, lightly pigmented melanocytes possess lower levels of melanogenic and catalase activity and are therefore more susceptible to accumulate damage after UV exposition.16 In agreement with other groups, we found a statistically significant decrease in GPx activity level according to skin phenotype (skin type III phenotype versus skin type IV or V and skin type IV versus skin type V).17 Briganti and colleagues examined GPx activity level in skin and blood samples from 29 vitiligo patients with different skin phototypes and their matching healthy controls.17 They found a statistically significant decrease in the enzyme activity level in all skin phototypes compared to healthy controls. The authors found a gradual increase in glutathione content from skin type II toward skin type VI. These findings could be explained by the fact that darkly pigmented skin possesses multiple protective antioxidant mechanisms. These include the physical shield of eumelanin that decomposes hydrogen peroxide and the

antioxidant enzyme activity. In contrast, lightly pigmented skin has fewer numbers of melanocytes that contain both pheomelanin and eumelanin. Eumelanin can scavenge the superoxide anion and hydrogen peroxide, whereas pheomelanin can increase the generation of free radicals following UV exposure.16,18,19 When we related GPx activity levels to the clinical features (age, sex, course, duration, extent of the disease), they were low among patients with a stationary course, duration , 3 years, and generalized and extensive vitiligo (, 50%). However, these variations were statistically insignificant. Our findings are in agreement with previous reports.5,20 Taken together, here we report a significant decrease in GPx activity level in patients with vitiligo, especially in skin phenotypes III and IV. These findings suggest that a disturbed oxidant-antioxidant system may contribute to the development of this disease. The underlying mechanisms that contribute to low GPx levels are open for further investigations.

Acknowledgments Financial disclosure of authors and reviewers: None reported.

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Canadian Dermatology Association | Journal of Cutaneous Medicine and Surgery, Vol 19, No 2 (March/April), 2015: pp 144–148

Low glutathione peroxidase activity levels in patients with vitiligo.

Vitiligo is an idiopathic skin disease characterized by white areas on the skin due to loss of the functional melanocytes, with possible involvement o...
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