Role of oxidative stress in the pathogenesis of vitiligo with special emphasis on the antioxidant action of narrowband ultraviolet B phototherapy

Abstract

Objectives

To evaluate the role of oxidative stress in the pathogenesis of vitiligo and the effect of narrowband (NB) ultraviolet (UV) B phototherapy on oxidative stress markers.

Methods

Patients with vitiligo and healthy control subjects were included in the study. Patients in the vitiligo group were treated with an NB-UVB regimen (3 × weekly for 6 months). Erythrocyte superoxide dismutase activity (SOD), erythrocyte malonyldialdehyde (MDA) and erythrocyte glutathione peroxidase activity (GSH-Px) levels were assessed in all participants at baseline, and after NB-UVB phototherapy in patients with vitiligo.

Results

A total of 24 patients with vitiligo and 27 control subjects were included in the study. Before treatment, erythrocyte MDA levels were significantly higher, and SOD and GSH-Px levels were significantly lower, in patients with vitiligo compared with controls. NB-UVB phototherapy was associated with a significant reduction in MDA levels and a significant increase in GSH-Px levels, compared with baseline, in patients with vitiligo.

Conclusion

NB-UVB phototherapy may relieve oxidative stress in patients with vitiligo by reversing the oxidant–antioxidant imbalance that is considered to play a role in the pathogenesis of this disease.

Introduction

Vitiligo is characterized by the destruction of melanocytes in the skin, resulting in the appearance of well-circumscribed white macules. ¹ The exact pathophysiological mechanism of vitiligo remains elusive, ² which has therefore generated extensive research into melanocyte biology and pigmentation disorders. ³ As a worldwide disease with a prevalence of 2%, vitiligo is considered to be a multifactorial and polygenic disease that is associated with the loss of epidermal melanocytes; the condition is proposed to be underlined by autoimmune, biochemical, oxidant–antioxidant, neural and viral mechanisms, as well as genetic susceptibility. ⁴

While the pathogenesis of vitiligo remains unclear, oxidative stress has been considered to be one of the likely causative factors in the initiation of the white skin patches of vitiligo, ³ based on melanocyte destruction caused by accumulation of toxic free radicals.^4,5

The first report confirming the use of narrowband (NB) ultraviolet (UV) B phototherapy in patients with vitiligo was published in 1997.^6,7 This indicated that a wavelength of around 311 nm had a favourable safety profile and was clinically more effective than full-spectrum UVB. Since then, NB-UVB phototherapy has been used as a first-choice strategy for most vitiligo patients, ⁸ as an alternative to the mainstay of treatment for generalized and focal vitiligo, which utilizes psoralen plus UVA (PUVA) photochemotherapy and topical corticosteroids (such an approach has been used for many years). ⁹

Narrowband-UVB has been reported to be a good wavelength to which vitiligo responds. ¹⁰ Hence, because of the uncertainty concerning the role of oxidative stress in the pathogenesis of vitiligo, as well as the known mechanism of action of NB-UVB on the condition,^1,4,5 the present study was designed to evaluate the role of oxidative stress in the pathogenesis of vitiligo. This was undertaken by investigating serum alterations in oxidant (erythrocyte malonyldialdehyde; MDA) and antioxidant (erythrocyte superoxide dismutase [SOD] and erythrocyte glutathione peroxidase [GSH-Px]) activity markers. Investigation of the effect of NB-UVB phototherapy on oxidative stress markers was the secondary objective of the study.

Methods

Patients

Patients newly diagnosed with generalized vitiligo on admission to the Dermatology Department at Gaziantep University Faculty of Medicine, Gaziantep, Turkey, were recruited sequentially between February 2009 and August 2009. Patients had to be >12 and <68 years of age, with no other skin disease. Control subjects were enrolled from Gaziantep University hospital workers and residency students, as well as volunteers from the town of Gaziantep. Control subjects (who were age- and sex-matched) did not show signs of active infection, were free of any other dermatological disorder, were not using any other medications and did not use alcohol or smoke cigarrettes.

Written informed consent was obtained from every study participant following a detailed explanation of the study objectives and protocol. The study was conducted in accordance with the ethical principles stated in the Declaration of Helsinki and was approved by the Institutional Ethics Committee (19.02.2009/02-2009/32) of Gaziantep University.

Assessments related to vitiligo and NB-UVB therapy

Patient demographics, family history of vitiligo, concomitant autoimmune disorders (such as tiroiditis, pernicious anaemia, alopecia areata, Addison’s disease) and the type of vitiligo were evaluated in the vitiligo group following clinical diagnosis of the disease. Patients’ skin types were assessed with the Fitzpatrick scale, and the distribution and diameter of vitiligo lesions was determined using a Wood’s lamp examination.

Patients in the vitiligo group were treated with an NB-UVB regimen (3 × weekly for 6 months), using a UVB unit (Waldmann UV7001K; Herbert Waldmann GmbH & Co. KG, Villingen-Schwenningen, Germany), with 24 Philips tubes (310–315 nm; TL01/100 W, Philips, Rosendaal, Holland). Adjustment of the initial dose was made for each patient; accordingly, an initial dose of 0.06 J/cm² in patients with skin type 2 was increased by 10% in each session until a maximum dose of 2 J/cm² was achieved. Likewise, in patients with skin type 3, an initial dose of 0.1 J/cm² was increased by 10% in each session until a maximum dose of 2.5 J/cm² was achieved. Dose was reduced or treatment was withdrawn if there was significant erythema, which was assessed during weekly visits throughout the treatment period. Control subjects were not treated with NB-UVB therapy, nor did they attend further visits.

Biochemical analyses

Blood samples were taken twice from patients with vitiligo (before and after NB-UVB therapy) and once from control subjects, between 08.00 h and 10.00 h on the day of collection. All study participants were asked to fast for ≥12 h before blood samples were taken. A total of 10 ml blood was drawn from each study participant from the cubital vein, and samples were collected into heparinized tubes. Blood samples were stored at −85℃ until analysis.

Blood haemolysate preparation

Collected blood was centrifuged for 10 min at 1660 g at 4℃ and the plasma obtained was removed. The remaining packed red blood cells were washed three times with saline to remove the buffy coat. Haemolysis was performed by pipetting out 1 ml of washed red blood suspension in ice-cold distilled water. Erythrocyte ghosts were sedimented three times in a centrifuge for 5 min at 300 g at 4℃ and supernatant was removed. A mixture, prepared by adding 4.9 ml sterile distilled water to 0.1 ml of the cell content, was kept in the refrigerator at 2–8℃ for 1 h after being vortexed, then sedimented in a centrifuge at 1600 g for 5 min at 4℃. The supernatant was separated out carefully and kept at −85℃ until analysis for erythrocyte SOD, MDA, and GSH-Px levels. Erythrocyte SOD was determined according to the nitroblue tetrazolium reduction method, as previously described. ¹¹ Erythrocyte MDA levels were measured according to the thiobarbituric acid method, ¹² and erythrocyte GSH-Px activity was also measured according to previously described methods. ¹³

Statistical analyses

Data were presented as mean ± SD and/or per cent. Analysis of continuous variables was performed using Student’s t-test or Mann–Whitney U-test, wheareas analysis of categorical variables was performed using χ²-test or Wilcoxon’s signed-rank test. Statistical analyses performed with SPSS® version 13.0 (SPSS Inc., Chicago, IL, USA) for Windows®. P-values < 0.05 were considered to be statistically significant.

Results

A total of 27 patients with vitiligo (14 male) and 27 healthy controls (15 male) were included in the study. Patients with vitiligo were 14–67 years old. Two male patients were excluded due to concomitant disease (hepatitis B and diabetes mellitus) and one female patient was excluded due to pregnancy, yielding a patient population of 24. The vitiligo and control groups were homogenous in terms of age and sex distribution, with equal numbers of female and male patients (Table 1). There were no concomitant autoimmune disorders in the patients with vitiligo. Family history of vitiligo was present in six men and five women.

Table 1.

Patient demographics in healthy control subjects and patients with vitiligo at baseline, in a study that evaluated the role of oxidative stress in the pathogenesis of vitiligo and the effect of narrowband ultraviolet B phototherapy on oxidative stress markers.

Characteristic	Control group n = 27	Vitiligo group n = 24	Total
Age, years	32.2 ± 12.8	34.9 ± 16.7	33.49 ± 13.2
Sex
Female	12 (44.4)	12 (50.0)	24 (100.0)
Male	15 (55.6)	12 (50.0)	27 (100.0)

Data presented as mean ± SD or n (%).

No statistically significant between-group differences (P ≥ 0.05; χ²-test).

Erythrocyte MDA levels in the vitiligo group before treatment were significantly higher compared with levels observed in the control group (P = 0.001; Table 2). When measurements taken in patients before and after treatment were compared, NB-UVB phototherapy was associated with significantly reduced MDA levels (P = 0.001), but these were still markedly higher than the levels obtained in the control group (P = 0.029; Table 2).

Table 2.

Mean erythrocyte superoxide dismutase activity (SOD), erythrocyte malonyldialdehyde (MDA), and erythrocyte glutathione peroxidase activity (GSH-Px) levels in healthy controls, and in patients with vitiligo (before and after 6 months’ narrowband ultraviolet-B therapy given 3 × weekly).

Parameter	Control group n = 27	Vitiligo group pretreatment n = 24	Vitiligo group post-treatment n = 24
MDA, nmol/g Hb	32.57 ± 7.59	43.31 ± 8.62‡	38.30 ± 8.09* §
SOD, U/mg Hb	2.46 ± 0.48	2.19 ± 0.42*	2.20 ± 0.58*
GSH-Px, U/g Hb	18.87 ± 4.42	13.71 ± 3.85‡	15.33 ± 3.25 † ¶

Data presented as mean ± SD.

Hb, haemoglobin.

*P < 0.05, ^†P < 0.01 and ^‡P < 0.001 versus control group. ^¶P < 0.05 and ^§P < 0.001 versus before NV-UVB treatment in vitiligo group (χ²-test).

Erythrocyte SOD levels in patients with vitiligo before treatment were significantly lower compared with levels in the control group (P = 0.045; Table 2). There was no effect of NB-UVB phototherapy on SOD levels in patients with vitiligo, but values measured after treatment were still markedly lower than those obtained in the control group (P = 0.024; Table 2).

Erythrocyte GSH-Px levels in patients with vitiligo before treatment were significantly lower compared with levels in the control group (P = 0.001; Table 2). NB-UVB phototherapy was associated with significantly increased GSH-Px levels (P = 0.024), which were still markedly lower than the levels obtained in the control group (P = 0.003; Table 2).

Discussion

The precise pathogenesis of vitiligo has not been clarified, despite several proposed theories that implicate the loss of epidermal melanocytes as the main underlying factor in this disorder. ⁴ In this regard, oxidative stress has been suggested as one of the aetiopathogenetic factors of vitiligo disease, triggering melanocyte destruction ¹⁴ resulting from overproduction of reactive oxygen metabolites and/or limited production of antioxidants.^15,16 Hence, patients with vitiligo have been considered to be susceptible to higher levels of oxidative stress than healthy people, due to documented reductions in erythrocyte glutathione levels, which are known to prevent free radical-mediated injury. ¹⁷

Accordingly, the higher levels of MDA – but lower levels of SOD and GSH-Px – seen in patients in this study before NB-UVB treatment seemed to indicate the remarkable imbalance in the oxidant–antioxidant system among patients with vitiligo in favour of oxidant mechanisms, which may play a substantial role in disease pathogenesis.

Indeed, similar increases in oxidative parameters including MDA levels,^15,18,19 as well as depletion in antioxidant markers such as erythrocyte GSH-Px^5,18–23 levels, have also been reported in studies conducted in patients with vitiligo. Data on SOD levels are inconsistent, since both decreases ¹⁵ and increases^18–20,23 in SOD levels have been documented in the clinical course of vitiligo, with changes being linked to variations in the activity and duration of disease, different laboratory analyses ¹⁵ and different amounts of melanin as an antioxidant per se. ²³ In the latter respect, comparison of active versus stabilized vitiligo revealed higher SOD activity in the active form: this was explained by the adaptation to increased oxidative stress in these patients. ¹⁸ In line with the findings of the present study, research conducted in patients with vitiligo in Turkey suggested that the simultaneous reduction in SOD levels and increase in MDA levels be a response to increased superoxide radicals. ¹⁵

Studies concerning alteration in markers of oxidative stress other than MDA, GSH-Px and SOD levels in patients with vitiligo revealed an increase in erythrocyte lipid peroxidation, ¹⁸ serum selenium^21,22 and xanthine oxidase ¹⁵ levels. Other studies have revealed a reduction in serum glutathione levels ²⁰ and erythrocyte glucose-6-phosphate dehydrogenase activity, ²⁰ in people with vitiligo compared with healthy controls.

Phototherapy is used to treat vitiligo in patients who do not respond to more conservative treatments, who have a widespread form of the disease, or who have localized vitiligo that has a substantial impact on health-related quality of life.^4,8 In this context, NB-UVB phototherapy is recommended in preference to oral PUVA because of evidence of greater efficacy and safety, as well as because of the substantial repigmentation rates that are achieved, particularly on the face, trunk and proximal extremities.^1,4,24–26 In this regard, a lower likelihood of erythema and xerosis development, carcinogenic effects and a lack of oral treatment obligation were reported advantages of NB-UVB phototherapy when compared with PUVA. ²⁷

Considering the mechanism of action of phototherapy, while failing to achieve complete normalization of levels of oxidant–antioxidant markers compared with control levels, the use of NB-UVB phototherapy for 6 months in the present study was associated with significant improvement in oxidant–antioxidant imbalance, shown by reduction in MDA levels and an increase in GSH-Px levels.

Addition of oral antioxidants to ongoing therapy has been shown to potentiate the beneficial effects of NB-UVB phototherapy on repigmentation and oxidant–antioxidant imbalance, ²⁸ with oral antioxidants being superior to placebo in induction of repigmentation. Another study found that patients with vitiligo treated with NB-UVB plus oral antioxidants had a significant reduction in plasma MDA levels compared with patients treated with NB-UVB only, as well as a significantly greater improvement in the extent of repigmentation in existing lesions. ⁷

Limitations of the present study include that the control group did not receive NB-UVB treatment; therefore, the effect on changes in oxidative stress markers with NB-UVB treatment in healthy subjects without vitiligo are not known. In addition, it would have been helpful to carry out further haematological assessments on the patients, such as complete blood counts and other analyses, to substantiate the findings of the present paper.

In conclusion, besides the well-known therapeutic effects in the treatment of vitiligo, NB-UVB phototherapy may also relieve oxidative stress by reversing the oxidant–antioxidant imbalance that is believed to play a role in the pathogenesis of vitiligo. In this respect, the addition of a topical and/or systemic antioxidant to a routine NB-UVB phototherapy regimen may potentiate the antioxidant efficacy of phototherapy among vitiligo patients.