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ORIGINAL ARTICLE
Year : 2020  |  Volume : 33  |  Issue : 1  |  Page : 262-266

Determination of oxidative stress in vitiligo by measuring superoxide dismutase levels in vitiliginous skin


1 Department of Dermatology and Andrology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Biochemistry, Faculty of Medicine, Menoufia University, Menoufia, Egypt
3 Department of Dermatology, Sers Ellian General Hospital, Menoufia, Egypt

Date of Submission05-Aug-2018
Date of Decision16-Sep-2018
Date of Acceptance23-Sep-2018
Date of Web Publication25-Mar-2020

Correspondence Address:
Mai A Omar
Sheben El-Kom, Menoufia Governorate
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_246_18

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  Abstract 


Objective
This study was conducted to determine the association between oxidative stress and vitiligo by measuring antioxidant enzyme superoxide dismutase (SOD) in vitiliginous skin.
Background
Vitiligo is an acquired depigmenting disease characterized by milky white patches of skin due to local destruction and loss of the epidermal melanocytes. Complex genetic, immunological, neural and self-destructive mechanisms interplay in its pathogenesis. According to autocytotoxic hypothesis, oxidative stress has been suggested to be the initial pathogenic event in melanocyte degeneration. Oxidative stress is defined as a disruption of the delicate balance between the formation of reactive oxygen species and the antioxidant defense system, The dismutation of superoxide (O2-) by SOD to hydrogen peroxide (H2O2) is generally considered to be the primary antioxidant defense of the body, because this enzyme prevents the further generation of free radicals. SOD exists in virtually every oxygen-respiring organism, and its major function is to catalyze dismutative reaction.
Patients and methods
We determined the activity of SOD in lesional skin only of 20 female patients with vitiligo. The ages of the patients ranged from 18 to 53 years. A total of 17 patients had nonsegmental vitiligo and three had segmental vitiligo, and 10 age-matched and gender-matched apparently healthy volunteers with no past, present or family history of vitiligo, served as a control group. Every case and control was subjected to skin biopsy, and SOD assay of the obtained biopsy was carried out by spectrophotometry.
Results
A significant difference was found between cases and controls with regard to tissue SOD (P = 0.01, <0.05).
Conclusion
There was a significant association between oxidative stress and vitiligo.

Keywords: antioxidants, melanocytes, oxidative stress, superoxide dismutase, vitiligo


How to cite this article:
Bakry O, Elhefnawy S, Seliet E, Omar MA. Determination of oxidative stress in vitiligo by measuring superoxide dismutase levels in vitiliginous skin. Menoufia Med J 2020;33:262-6

How to cite this URL:
Bakry O, Elhefnawy S, Seliet E, Omar MA. Determination of oxidative stress in vitiligo by measuring superoxide dismutase levels in vitiliginous skin. Menoufia Med J [serial online] 2020 [cited 2020 Aug 15];33:262-6. Available from: http://www.mmj.eg.net/text.asp?2020/33/1/262/281279




  Introduction Top


Vitiligo is an acquired depigmenting disease characterized by milky white patches of the skin due to local destruction and loss of the epidermal melanocytes[1]. It occurs with a frequency of 0.1–2% worldwide[2]. Vitiligo can appear at any time, and it significantly impairs the patients' quality of life[3].

The only symptom of vitiligo is the presence of pale patchy areas of depigmented skin, which tend to occur on the extremities. The patches are initially small, but often grow and change shape[1]. When skin lesions occur, they are most prominent on the face, hands, and wrists. The loss of skin pigmentation is particularly noticeable around body orifices, such as the mouth, eyes, nostrils, genitalia, and umbilicus. Some lesions have increased skin pigment around the edges[4].

Although its pathophysiology is still unknown, diverse theories have been proposed, including autoimmune, neural, oxidative stress, apoptosis, and genetic factors[5].

Recent studies have suggested that oxidative stress might play a prominent role in the pathogenesis of vitiligo[6]. Oxidative stress is defined as a disruption of the delicate balance between the formation of reactive oxygen species (ROS) and the antioxidant defense system[7], as patients with vitiligo have an imbalanced redox state of the skin, resulting in the excess production of ROS. These disturbances and ROS accumulation can have toxic effects on all components of the cell (e.g., proteins, lipids) and could potentially result in the destruction of melanocytes[8]. An alteration in the antioxidant pattern, with significantly higher levels of superoxide dismutase (SOD), has been observed in the skin[9], erythrocytes[10], peripheral blood mononuclear cells[11], and serum[12] of vitiligo patients. These findings support the concept of possible systemic oxidative stress in vitiligo.

As there are very few studies showing the extent of oxidative stress at the tissue level, the present study has been undertaken to determine the status of oxidative stress by measuring levels of antioxidant enzymes, that is, SOD in lesional skin of patients with vitiligo and in the skin of normal controls.

This study was conducted to determine the association between oxidative stress and vitiligo by measuring antioxidant enzyme SOD in vitiligenous skin.


  Patients and Methods Top


Study population and selection of patients

This study was approved by the ethical committee of Dermatology and Biochemistry Department, Faculty of Medicine, Menoufia University, during the period spanning from October 2016 to June 2017, and informed consent was obtained from every patient and the controls. The study involved 20 female patients with vitiligo and 10 age-matched and gender-matched apparently healthy volunteers with no past, present or family history of vitiligo, as a control group.

Inclusion criteria

Female patients with vitiligo were included in the study. A total of 10 age-matched and gender-matched apparently healthy volunteers with no past, present or family history of vitiligo, were included as a control group.

Exclusion criteria

Patients with dermatological diseases other than vitiligo, systemic autoimmune or endocrine disorders, and the possibility of psychological stress-induced oxidative stress were excluded from the study.

Each of the selected patients was subjected to the following.

Complete history taking including personal, present history including onset, course and duration of the lesion, family history of vitiligo was carried out.

Examination consisted of the following:

  1. General examination
  2. Dermatological examination to determine site, clinical type, presence of leuckotrechia
  3. Assessment of lesion extent by vitiligo area scoring index.


The percentage of vitiligo involvement is calculated in terms of hand units. One hand unit is approximately equivalent to 1% of the total body surface area. The degree of pigmentation is estimated to the nearest of one of the following percentages:

  1. 100%: complete depigmentation, no pigment is present
  2. 90%: specks of pigment present
  3. 75%: depigmented area exceeds the pigmented area
  4. 50%: pigmented and depigmented areas are equal
  5. 25%: pigmented area exceeds depigmented area
  6. 10%: only specks of depigmentation present.


Assessment of disease activity by vitiligo index of disease activity (VIDA) scoring.

The VIDA is a six-point scale for assessing vitiligo activity. Scoring is based on the individual's own opinion of the present disease activity over time. Active vitiligo involves either expansion of existing lesions or appearance of new lesions. Grading is as follows:

  1. VIDA score +4: activity of 6 weeks or less duration
  2. +3: activity of 6 weeks to 3 months
  3. +2: activity of 3–6 months
  4. +1: activity of 6–12 months
  5. 0: stable for 1 year or more
  6. −1: stable with spontaneous repigmentation since 1 year or more. A low VIDA score indicates less activity.


Every case and control was subjected to the following:

  1. Skin biopsy: punch biopsies were taken with 3 mm punch under 2% lignocaine local anesthesia from the center of the vitiliginous macule of 20 patients and from the skin of 10 healthy participants.
  2. SOD assay of the obtained biopsy by spectrophotometry was carried out.


Principle

This assay relies on the ability of the enzyme to inhibit the phenazine methosulphate-mediated reduction of nitroblue tetrazolium dye.

Sample preparation (colorimetric enzymatic method; Biodiagnostic, Giza, Egypt).

Whole tissue:

  1. efore dissection, the tissue was perfused with a PBS solution, pH 7.4, containing 0.16 mg/ml heparin to remove any red blood cells
  2. The tissue was homogenized in 5–10 ml cold buffer (i.e., 100 mm potassium phosphate, pH 7.0, containing 2 mm EDTA) per gram tissue
  3. The tissue was centrifuged at 4000 rpm for 15 min at 4°C
  4. The supernatant was collected; if not assayed immediately, the supernatant was stored at −20°C
  5. A total volume of 0.5 ml of ice-cold extraction reagent to 1.0 ml of supernatant in a glass test tube was added
  6. The sample was vortexed for at least 30 s
  7. The sample was centrifuged at 4000 rpm and 4°C for 10 min
  8. The aqueous upper layer was collected and was kept at 4°C for immediate assay.


Statistical analysis

Data were collected, tabulated, and statistically analyzed using an IBM personal computer with statistical package of the social sciences version 22 (SPSS; SPSS Inc., Chicago, Illinois, USA). The following statistics were applied: descriptive statistics, in which quantitative data were presented in the form of mean, SD, and range, and qualitative data were presented in the form of numbers and percentages and analytical statistics for example, χ2-test, odds ratio, Mann–Whitney test (nonparametric test), Kruskal–Wallis test (nonparametric test) and Pearson correlation (r) (a test used to measure the association between two quantitative variables). A P value of less than 0.05 was considered statistically significant. A P value of less than 0.001 was considered statistically highly significant.


  Results Top


The study group included 20 cases of segmental and nonsegmental vitiligo (NSV) (all were female individuals). The ages of the patients ranged from 18 to 53 years (mean age = 31.32 years). The duration of vitiligo ranged from 4 months to 40 years, with a mean duration of 11.3 years. A total of 17 patients had NSV and three had segmental vitiligo (SV). Seven patients had progressive vitiligo and 13 had stable vitiligo. In this study, the levels of SOD in vitiliginous skin of vitiligo patients (9.75 ± 3.81 U/mg of protein) were found to be higher than the levels of SOD in normal skin of controls (5.75 ± 3.22 U/mg of protein). The difference was found to be statistically significant (P = 0.01; <0.05) [Table 1].
Table 1: Comparison between cases and controls with regard to tissue superoxide dismutas

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  Discussion Top


Vitiligo is a common depigmenting skin disorder[13], characterized by acquired, idiopathic, progressive, and circumscribed depigmentation of the skin and hair[14]. The disease is classified according to Taieb and Picardo[15] into four types: NSV, SV, mixed NSV and SV, and unclassifiable types. NSV is divided into subtypes: focal at onset, mucosal, acrofacial, generalized, and universal. It remains unclear what causes damage or death to melanocytes. There are many potential pathophysiological theories involving autoimmune[16], neural, autocytotoxic[17], biochemical[18], viral infection[19], oxidative stress[8], melanocytorrhagy[20], and decreased melanocyte survival hypotheses[21].

Studies have suggested that oxidative stress might play a prominent role in the pathogenesis of vitiligo[22].

Oxidative stress is defined as a disruption of the delicate balance between the formation of ROS and the antioxidant defense system[7], as patients with vitiligo have an imbalanced redox state of the skin, resulting in the excess production of ROS. These disturbances and ROS accumulation can have toxic effects on all components of the cell (e.g., proteins, lipids) and could potentially result in the destruction of melanocytes[8]. These free radicals are scavenged continuously by antioxidant enzymes such as SOD, catalase (CAT), glutathione peroxidase, glutathione reductase, beta carotene, vitamin C, vitamin E, and other trace elements[22].

Epidermal melanocytes are particularly vulnerable to oxidative stress whether exogenous, in the form of physical exposure to ultraviolet radiation and chemicals (e.g., phenolic compounds), or endogenous owing to the pro-oxidant state generated during melanin synthesis, and to the intrinsic antioxidant defenses that are compromised in vitiligo patients[23].

Vitiligo patients are known to have very high levels of hydrogen peroxide (H2O2) and peroxynitrite in their epidermis, concomitant with reduced levels and activity of catalase[24], glutathione peroxidase, and glutathione reductase, which account for sustained high levels of H2O2 in the epidermis[25].

High levels of H2O2 inactivate and reduce the levels of methionine sulfoxide reductase A and B, and thioredoxin/thioredoxin reductase, thus contributing to oxidative stress and melanocyte death in vitiligo[26]. In addition, high levels of H2O2 in the epidermis are found to oxidize adrenocorticotrophic hormone and α–melanocyte-stimulating hormone, both of which have antioxidant and survival effects on human melanocytes[27]. In addition to this, a defective recycling of tetrahydrobiopterin has been reported in the vitiligo epidermis, resulting in oxidative stress[28]. The skin antioxidant defense system can be classified into two major groups: the enzyme group and the low molecular weight antioxidant group. The first group includes SOD, catalase, peroxidase, and some supporting enzymes such as glucose-6-phosphate dehydrogenase and glutathione reductase. The second group includes a large number of compounds capable of preventing oxidative damage by scavenging ROS. They share a similar chemical trait that allows them to donate an electron to ROS. Low molecular weight antioxidants originate from cell synthesis (e.g., reduced glutathione (GSH), nicotinamide adenine dinucleotide (NADH), and carnosine), waste products (e.g., uric acid), and dietary resources (e.g., carotene, polyphenols, and lipoic acid)[29].

The dismutation of superoxide (O2-) by SOD to H2O2 is generally considered to be the primary antioxidant defense of the body, because this enzyme prevents the further generation of free radicals. SOD exists in virtually every oxygen-respiring organism, and its major function is to catalyze dismutative reaction[30].

In the present work, SOD activity was significantly higher in the vitiligo patients than in healthy participants. In agreement with our study, Yildirim et al.[12], Sravani et al.[22], Jain et al.[31], and Ozel Turkcu et al.[32], observed that epidermal SOD activity in vitiligo patients was significantly higher than in healthy controls. However, Passi et al.[33], Picardo et al.[34], and Maresca et al.[6] reported no significant difference. However, Koca et al.[35] reported decreased levels of SOD in vitiligo patients compared with controls.

High SOD might be an adaptation to the increased oxidative stress in these individuals. Presence of high levels of O2- leads to high levels of SOD, followed by high amounts of H2O2. High levels of H2O2 and O2- might result in the destruction of defective melanocytes in vitiligo patients, which already contain low levels of nonenzymatic antioxidants and catalase[10].

Finally, our results clearly support that oxidative stress might be implicated in the pathophysiology of vitiligo, due to alterations in the antioxidant defenses.


  Conclusion Top


From this work, we can conclude that there is impairment in the antioxidant system in vitiligo, leading to free radical-mediated damage to melanocytes, and hence there is a significant association between oxidative stress and vitiligo.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Taieb A, Picardo M. Vitiligo: epidemiology, definitions and classification. In: Taieb A, Picardo M, editors. Vitiligo. 1st ed. Heidelberg: Springer Verlag; 2010. 13–24.  Back to cited text no. 15
    
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