Vitiligo is an acquired disorder of skin and mucous membranes, which is characterized by persistent well-circumscribed, depigmented macules, and patches caused by selective melanocytes destruction 1–3. It is a multifactorial disorder related to both genetic and nongenetic factors 4. The published prevalence of vitiligo is 0.5–1% 5. All races and both sexes are equally affected 6.
Oxidative stress (OS) theory as a cause of vitiligo suggests that patients have an imbalanced redox state of the skin, resulting in excessive production of reactive oxygen species (ROS). These disturbances can have toxic effects on all components of the cell and could potentially result in destruction of melanocytes, creating the depigmented macules of vitiligo 7. It is well known that ROS are generated in melanocytes during the normal process of melanogenesis. However, its overproduction may damage melanocytes and impair melanocytes’ antioxidant protection systems 8,9.
In 1993, Miller and co-workers created a new test to measure the total antioxidant status, which has been designated as total antioxidant capacity (TAC) 10. The major advantage of this test is that it measures the whole antioxidant capacity of a biological sample and not just a single antioxidant 11.
Several studies have investigated the amounts of ROS and antioxidants in vitiligo. However, measurements of these molecules individually do not reflect the global status of OS. Thus, measurement of TAC using a well-established method evaluates the efficiency of all antioxidants as a whole 12. In addition and to the best of our knowledge, this is the first study that evaluates the tissue levels of TAC in patients with vitiligo.
The aim of the study was to evaluate TAC levels in the serum and tissue of Upper Egyptian patients with vitiligo and to determine the possible correlations of serum and tissue levels of TAC of patients with vitiligo with demographic data and clinical characteristic of the disease itself.
Patients and methods
A total of 48 patients with nonsegmental vitiligo (31 females and 17 males) and 30 age-matched and sex-matched healthy volunteers (17 females and 13 males) were included in this study from the Dermatology Department in Assiut University Hospitals, Assiut, Egypt.
The study was approved by the Research Ethics Committee for Experimental and Clinical Studies at Faculty of Medicine, Assiut University, Assiut, Egypt. Informed consent was obtained from all participants.
The exclusion criteria were as follows:
- Patients and controls who were less than or equal to 16 and more than or equal to 55 years of age.
- Patients and controls on any topical or systemic medications during the past month.
- Patients on phototherapy during the past 3 months.
- Patients and controls with any concomitant systemic or dermatological disease.
- Pregnant females.
- Smokers and alcoholics.
All study participants were subjected to complete history taking and general and dermatologic examinations.
The total body vitiligo area scoring index (VASI) was calculated using the Hamzavi et al.13 formula: VASI=€(hand units in all body sites)×residual depigmentation.
The notion of stable disease is subject to interpretation, and the clinical definition of ‘stability’ for many authors varies greatly. In our study, we identified the stability according to the Vitiligo Global Issues Consensus Conference statements in stability 3. Therefore, we identified any patient who showed neither extension of the existing lesions nor occurrence of new lesions within the past year as a stable case.
Venous blood samples
Overall, 6 ml of venous blood was collected in plain tubes by venipuncture after a rest of 30 min in sitting position by a 25 G needle through the antecubital vein, avoiding hemolysis, and then centrifuged at 1500 rpm for 15 min at room temperature. Sera were divided into 1.5 ml aliquots using sterile plastic transfer pipettes and frozen at −70°C until future analysis.
Three 5 mm punch biopsies were taken from each patient after local anesthesia from lesional, perilesional (5 mm from the peripheral margin), and unaffected skin (10 cm from the peripheral margin). The perilesional biopsy was taken after carefully marking with ink the pigmented side bordering the vitiliginous spot; this facilitated the precise cutting of biopsies, resulting in sections that contain both lesional and nonlesional skin. Moreover, a skin biopsy was taken from the back of each volunteer for cosmetic reasons. Specimens were taken without fixation or preservation. It was washed with ice-cold saline, weight checked, and stored at −70°C. Tissue samples were homogenized by a grinder in 0.1 M sodium phosphate buffer (pH 7.4). The tissue homogenate was centrifuged for 30 min at 1500 rpm. The supernatant was separated and stored in labeled Eppendorf tubes at −20°C until time of assay.
Measurement of total antioxidant capacity
TAC was determined in the serum and in the homogenized tissues using a kit provided by Biodiagnostics, Cairo, Egypt, based on the method of Koracevic et al. 14. This method depends on the reaction of antioxidants in the sample with a defined amount of exogenously provided hydrogen peroxide (H2O2) causing its decomposition. The residual H2O2 is determined by an enzymatic reaction that involves the conversion of 3, 5-dichloro-2-hydroxybenzenesulphonate to a colored product measured colorimetrically at 505 nm. The protein content of homogenate samples was measured using a commercially available kit, and the results of TAC in tissue are calculated according to the total tissue protein level. The measurement unit of TAC in serum is mmol/l and in tissue is mmol/g tissue protein.
The data were tested for normality using the Anderson–Darling test and for homogeneity variances before further statistical analysis. Categorical variables were described as number and percentage, where continuous variables were described as mean and SD. χ2-test and Fisher’s exact test were used to compare between categorical variables, but comparisons between continuous variables were done by t-test and analysis of variance. Pearson’s correlation coefficient (r) was used to assess the association between continuous variables. A P value less than 0.05 was considered statistically significant. All analyses were performed with the IBM SPSS 20.0 software (SPSS Inc., Chicago, Illinois, USA).
Characteristics of the study population
The study included 48 patients with nonsegmental vitiligo and 30 age-matched and sex-matched healthy volunteers. The mean age of patients with vitiligo was 32.6±12.4 years and of control cases was 31.9±7.13 years. Overall, 65% of patients were females, and 75% of patients were from rural areas. The mean duration of the disease was 8.21±6.49 years. Moreover, 33% of patients have a positive family history. Vitiligo was unstable in 71% of patients. The most frequent subtype was generalized vitiligo in 60% of patients; followed by the acrofacial type in 40%. The mean VASI score was 5.84±6.57 (Table 1).
Total antioxidant capacity
The mean TAC levels in serum, lesional, perilesional, and unaffected skin of vitiligo patients were 0.98±0.13, 0.8±0.18 mmol/g tissue protein, 0.99±0.33 mmol/g tissue protein, and 1.13±0.33 mmol/g tissue protein, respectively (Table 2, Fig. 1), whereas the mean TAC levels in serum and skin of controls were 1.05±0.06 mmol/l and 1.21±0.28 mmol/g tissue protein, respectively (Table 3, Fig. 2).
The mean serum TAC level was significantly lower (P=0.007) in patients (0.98±0.13) than in controls (1.05±0.06). Moreover, the mean TAC level in the lesional skin (0.8±0.18) was significantly lower (P<0.001) than in the healthy skin of controls (1.21±0.28) (Table 4, Fig. 3).
The TAC level in the lesional skin (0.8±0.18) was highly significantly lower than that in the serum (0.98±0.13, P<0.001), perilesional (0.99±0.33, P<0.001), and unaffected skin (1.13±0.33, P<0.001) of patients (Table 2, Fig. 1). Moreover, it was significantly lower (P<0.001) in perilesional skin (0.99±0.33) than in unaffected skin (1.13±0.33) (Table 2, Fig. 1). The serum TAC level was significantly (P=0.002) much lower (0.98±0.13) than its level in unaffected skin (1.13±0.33) (Table 2, Fig. 1). Interestingly, the same finding was encountered in controls’ serum (1.05±0.06) and skin (1.21±0.28) (P=0.004) (Table 3, Fig. 2).
The mean TAC level was significantly lower in serum (P=0.018), lesional (P=0.038), and unaffected skin (P=0.007) of patients with unstable than that of stable vitiligo (Table 5, Fig. 4).
Regarding clinical type, we did not find any significant difference in TAC levels between generalized and acrofacial types (Table 6).
No significant correlations were detected between any of the demographic data (age, sex, residence, and marital status) and TAC levels.
In this study, we measured TAC levels in serum and tissue of patients with vitiligo including lesional, perilesional, and unaffected skin.
Many studies evaluated the role of OS in the pathogenesis of vitiligo to support the hypothesis that vitiligo can be caused by imbalance in the redox state of the body 7,8,15. Imbalances in the oxidant/antioxidant system, such as the accumulation of H2O2 and low catalase (CAT) levels, have been demonstrated in the epidermis and blood of patients with vitiligo 8. These findings demonstrate impairment in the antioxidant system in vitiligo melanocytes and implicate free radical-mediated damage in melanocytes’ degeneration in vitiligo 7. However, no previous studies evaluated TAC levels in the tissue of different sites of patients with vitiligo in addition to serum.
Our study demonstrated less serum TAC levels in patients with vitiligo than in controls, which means that there is a disturbance in the general redox state of patients with vitiligo. This finding supports the OS as a systemic process that is involved in the pathogenesis of vitiligo. This finding is in accordance with the previous reports that evaluated serum TAC in different countries such as Egypt 16, Turkey 9, and India 7,17.
Beside the serum levels of TAC, we thought that measuring TAC in the skin of patients with vitiligo could be extremely important to give an idea about the local antioxidant state. We measured TAC in lesional, perilesional, and unaffected skin in patients. Moreover, we measured TAC in the skin of controls. In addition, we correlated TAC levels with the demographic and clinical data of the study participants to give a clear understanding of the local TAC state in patients with vitiligo.
We found that the lesional TAC level was lower than that of the serum. This signifies that the antioxidant tissue level has a more significant role in the pathogenesis than the systemic antioxidant status. We also found that the TAC levels in lesional skin were much less than in perilesional and unaffected skin of patients. This means that the more the skin gets affected by the vitiliginous process, the less the level of TAC will be. This further confirms the role of local TAC state in the pathogenesis of vitiligo. So, even though both serum and tissue levels of TAC can be implicated in the pathogenesis of vitiligo, the tissue levels are much more affected, and hence involved in vitiligo pathogenesis.
On the contrary, when we compared TAC levels in serum and unaffected skin, we found that the level was lower in the serum. This finding means that the normal-looking skin has a strong antioxidant status, even stronger than that of the serum. This finding further supports the importance of tissue antioxidant status in the disease process and health. Interestingly, the same finding was also noticed in controls. This means that even in normal persons, the skin TAC levels are much higher than that in the serum.
Also, we found that the TAC levels in all samples were less in unstable than in stable cases. This means that disease progression is accompanied with a profound decrease in TAC levels that reflects the causal relationship of OS with vitiligo.
A limitation of this study is the relatively small sample size. Moreover, because of cosmetic issues, skin biopsies from some patients and controls were not site matched.
Our findings prove that there is a definite affection of the TAC in both serum and tissues of patients with vitiligo. We also established that the local tissue levels of TAC are much more implicated in the disease process. We recommend future studies to evaluate the efficacy of topical antioxidant preparations as a possible effective and safe therapeutic option for this chronic devastating disease. We consider this possible therapeutic effectiveness of topical antioxidants the main clinical implication of our study.
Conflicts of interest
There are no conflicts of interest.
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Keywords:© 2018 Egyptian Women's Dermatologic Society
oxidative stress; total antioxidant capacity; vitiligo