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Abstract
Background: Vitiligo, a multifactorial, depigmented skin disease, is characterised by selective loss of functional melanocytes leading to pigment reduction in the affected areas of the skin.
Aim: We aimed to examine thiol-disulphide homeostasis, IMA, copper, zinc, selenium, vitamin A and vitamin C levels in vitiligo patients. Materials and Methods: The study included 83 vitiligo patients and 72 healthy controls. Copper, zinc, and selenium levels were measured by atomic absorption spectrophotometer; vitamin A and E levels were measured by high-performance liquid chromatography. Ischemia-modified albumin and native/total thiol levels were measured by colourimetric method. Results: Serum native and total thiol levels were significantly lower in vitiligo patients (P < 0.001, for all). Zn levels were significantly higher in vitiligo patients than in the control group (P = 0.004). There was no statistical difference in terms of Cu, Se, vitamin A and vitamin E levels. Conclusions: All thiol-disulphide homeostasis parameters (the most important antioxidant–oxidant system in circulation), trace elements, and vitamins together were evaluated in the present study in vitiligo patients. It can be concluded that vitiligo patients have increased oxidative stress status, and also the increase in the dissemination of the disease also increases the oxidative stress in the body.
Keywords: Oxidative stress, Thiol, Trace elements, Vitamins, Vitiligo
Introduction 
Vitiligo, a multifactorial, depigmented skin disease, is characterised by selective loss of functional melanocytes leading to pigment reduction in the affected areas of the skin. Vitiligo is one of the most common depigmented skin disorders with an estimated prevalence of 0.5–2% of the population in both adults and children worldwide.[1] The characteristic lesion is a completely amelanotic, non-scaly, chalky-white macula with well-defined borders. Vitiligo should not be dismissed as a cosmetic or minor illness, as its effects can be psychologically devastating and often place a significant burden on daily life.[2] Multiple mechanisms have been proposed for melanocyte destruction in vitiligo. These can be listed as genetics, autoimmune responses, oxidative stress, production of inflammatory mediators and melanocyte separation mechanisms.[3]
Oxidative damage and inflammation are thought to play a role in the pathogenesis of vitiligo. It has been reported that melanocytes of vitiligo patients are more sensitive to oxidative damage. Reactive oxygen species (ROS) are released from melanocytes in response to stress.[4] It has also been suggested that this imbalance between pro-oxidants and antioxidants in vitiligo is due to the increased sensitivity of melanocytes to external pro-oxidant stimuli.[5] Thiol-disulphide homeostasis is an indicator of many cellular activities such as antioxidant protection, detoxification, cell growth, and apoptosis.[6] Ischemia-modified albumin (IMA) is formed as a result of the modification of albumin caused by reactive oxygen derivatives formed as a result of ischemia.[7] In addition, it has been reported that the levels of some hormones, enzymes, amino acids and even trace elements and vitamins change simultaneously with the onset of vitiligo.[8] It is known that vitamin A and vitamin C protect membranes from the damaging effects of free radicals and may play a protective role in the development of some autoimmune diseases.[9] Trace elements play an important role in multiple biochemical processes, particularly in immunological and inflammatory reactions, and are therefore thought to play a role in the aetiology of many skin diseases.[10] The pathogenesis of vitiligo disease has not yet been fully elucidated. Oxidative damage is considered to be one of the most important factors.[11],[12]
In this study, it is aimed to examine thiol-disulphide homeostasis and IMA levels as indicators of oxidative damage in vitiligo patients, as well as trace element levels such as copper, zinc, selenium and vitamin A and vitamin C levels as markers of antioxidant status and to evaluate their relationship with the disease.
Materials and Methods The study was conducted at Ankara City Hospital Department of Dermatology and Clinical Biochemistry Laboratory for 3 months period. The study included 83 patients who followed up with the diagnosis of vitiligo and 72 controls who were similar in terms of age and gender. The diagnosis of vitiligo was made clinically. The patients with hypopigmented/depigmented macules and patches surrounded by normal skin were evaluated under Wood's lamp. The patients with depigmentation with Wood's lamp examination were included in the study. The subtypes of vitiligo (generalised vitiligo/localised vitiligo) were determined by performing a total skin examination of the patients. The patients with segmental vitiligo were excluded from the study. A detailed history was taken from the patients, and the age at diagnosis of vitiligo and disease duration was determined and recorded. Vitiligo area severity index (VASI) was used to determine the extent of vitiligo disease (the percentage of vitiligo involvement at six body regions multiplied by residual depigmentation). The depigmentation degree was evaluated as 100%, 90%, 75%, 50%, 25% and 10%.[13] The percentage of vitiligo involvement is calculated using the Palmar method. Vitiligo disease activity (VIDA) score was used to evaluate vitiligo disease activity. The VIDA score is a six-point scale that evaluates vitiligo disease activity. VIDA score is evaluated as '+4 = active for the last 6 weeks, +3 = active for the last 3 months, +2 = active for the last 6 months, +1 = active for the last 1 year, 0 = stable for the last 1 year, -1 = stable for the last 1 year', and there is spontaneous repigmentation.[14]
After obtaining informed consent, 10 mL of venous blood was collected aseptically in serum separator blood collection tubes from each subject. Sera were separated after centrifugation and stored at –80°C until analysis. Copper (Cu), zinc (Zn) and selenium (Se) levels were measured by atomic absorption spectrophotometer (AAS) instrument-Shimadzu AA7000 (Kyoto, Japan). Vitamin A and E measurements were done by using high-performance liquid chromatography (HPLC) (Shimadzu, Kyoto, Japan). Serum IMA levels were measured by a colourimetric method developed by Bar-Or et al.[15] Serum native/total thiol levels were measured by Erel and Neşelioğlu method[16] using ADVIA 1800 (Siemens Healthineers, Erlangen, Germany).
The study was approved by the local ethics committee (Reference number: E1-22-2317) in accordance with the 1964 Helsinki Declaration or comparable standards. All participants/parents provided informed consent before inclusion in the study.
Data were analysed using SPSS/IBM for Windows 21.0 (Chicago, IL, USA). Descriptive statistics were expressed as percentage, mean, median, standard deviation and interquartile range (IQR). The conformity of continuous variables to normal distribution was examined using the Shapiro–Wilk test. In cases where parametric test assumptions were met, Student's t-test was used for the difference between the means of two independent groups, and when parametric test assumptions were not met, non-parametric alternatives of these tests and Mann–Whitney U test were used for two groups. Spearman or Pearson tests were used to evaluate the correlation between two numerical values. Categorical data were analysed with the Chi-square test or Fisher's exact test. 95% significance level (or α = 0.05 margin of error) was used to determine the differences in the analyses.
The sample size was calculated according to the prevalence value and the results from similar previous studies. For parameters that were not studied in vitiligo before, previous studies evaluating the same parameters in similar dermatological diseases were used. The sample size was calculated separately for all the parameters, and the result with the largest sample size was used as the final sample size for the study (disulphide, total sample size (patient + control) = 140). Sample sizes were calculated using G power software (Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany).
Results Forty-nine (59%) of the patients were female, and 34 (41%) were male. The mean age of the patients was calculated as 39.9 ± 13.2 years. Forty-eight (57.8%) of the patients had generalised vitiligo, and 35 patients (42.2%) had localised vitiligo. The median VASI score was 4 (IQR = 1.17–10.25), and the median age of onset of vitiligo was 26 (IQR = 13–40). The VIDA score was + 4 in 4% of patients, +3 in 21%, +2 in 22.4%, +1 in 11.8%, 0 in 14.5% and -1 in 26.3% of patients.
Serum selenium, copper, zinc, vitamin A and vitamin E levels of the patient and control groups are given in [Table 1]. There was no statistically significant difference between the patient and control groups in terms of Cu, Se, vitamin A and vitamin E. Zn levels were significantly higher in the patient group than in the control group (P = 0.004).
Table 1: Serum selenium, copper, zinc, vitamin A and vitamin E levels of the patient and control groupsSerum native and total thiol, disulphide and IMA levels of the subjects were presented in [Table 2].
Table 2: Serum native and total thiol, disulphide and IMA levels of the patient and control groupsThe relationship between vitiligo characteristics and serum selenium, copper, zinc, vitamin A and vitamin E levels
Copper, zinc, selenium, vitamin A and vitamin E levels were similar in men and women (P values are 0.580, 0.780, 0.240, 0.660, 0.170, respectively). Copper (P = 0.970, r = -0.04), zinc (P = 0.720, r = -0.04), selenium (P = 0.18, r = 0.17), vitamin A (P = 0.650, r = 0.05) and vitamin E (P = 0.940, r = -0.03) levels were not correlated with age.
While the mean serum zinc value was 1451.4 ± 382.2 in the patients with generalised vitiligo, the mean serum zinc value of the patients with localised vitiligo was 1273.9 ± 276.1. Serum zinc levels were significantly higher in patients with generalised vitiligo (P = 0.049). A statistically significant positive correlation was observed between zinc levels and the dissemination of the disease (VASI score) (P = 0.002, r = 0.35). The patients with generalised and localised vitiligo were similar in terms of serum copper, selenium, vitamin A and vitamin E levels (P values are 0.990, 0.320, 0.160, 0.750, respectively). There was no correlation between the dissemination of the disease (VASI score) and copper (P = 0.200, r = 0.15) and vitamin E (P = 0.590, r = -0.07) levels. A negative correlation was observed between serum selenium (P = 0.009, r = -0.34) and vitamin A levels (P = 0.050, r = -0.22) and the dissemination of the disease (VASI score) [Figure 1].
Figure 1: Correlation between the dissemination of the disease (VASI score) and selenium, copper, zinc, vitamin A and E levelsNo correlation was observed between the levels of copper (P = 0.910, r = -0.01), zinc (P = 0.250, r = -0.13), selenium (P = 0.150, r = 0.19), vitamin A (P = 0.870, r = -0.02) and vitamin E (P = 0.130, r = -0.19) and the age of onset of vitiligo.
No correlation was observed between the levels of copper (P = 0.260, r = -0.14), zinc (P = 0.160, r = -0.16), selenium (P = 0.660, r = -0.06), vitamin A (P = 0.350, r = -0.11), vitamin E (P = 0.18, r = 0.17) and vitiligo disease activity (VIDA score).
The relationship between vitiligo characteristics and serum native and total thiol, disulphide and IMA levels
Serum native and total thiol, disulphide and IMA levels were similar in men and women (P values are 0.430, 0.520, 0.180, 0.110, 0.110, 0.110 and 0.740, respectively). Native thiol (P = 0.160, r = 0.15), total thiol (P = 0.190, r = 0.15), disulphide (P = 0.380, r = 0.09), disulphide/native thiol (P = 0.280, r = -0.12), disulphide/total thiol (P = 0.280, r = -0.12), native thiol/total thiol (P = 0.290, r = 0.12) and IMA (P = 0.260, r = 0.13) levels were not correlated with age.
Native thiol, total thiol, disulphide, disulphide/native thiol, disulphide/total thiol, native thiol/total thiol and IMA levels were similar in the patients with both generalised and localised vitiligo (P values are 0.810, 0.830, 0.880, 0.840, 0.840, 0.840, 0.0920, respectively). There was no correlation between the dissemination of the disease (VASI score) and native thiol (P = 0.700, r = 0.44), total thiol (P = 0.760, r = 0.04), disulphide (P = 0.860, r = -0.02), disulphide/native thiol (P = 0.320, r = -0.11), disulphide/total thiol (P = 0.460, r = -0.08), native thiol/total thiol (P = 0.320, r = 0.11) and IMA (P = 0.490, r = -0.08) levels.
A statistically significant positive correlation was observed between the age of onset of vitiligo and native thiol (P = 0.003, r = 0.33) and total thiol (P = 0.004, r = 0.33). However, there was no correlation between disulphide (P = 0.25, r = 0.13), disulphide/native thiol (P = 0.060, r = -0.22), disulphide/total thiol (P = 0.063, r = -0.23), native thiol/total thiol (P = 0.062, r = 0.21) and IMA (P = 0.13, r = 0.18) levels and the age of the disease onset [Figure 2].
Figure 2: Correlation between disulphide, disulphide/native thiol, disulphide/total thiol, native thiol/total thiol, IMA levels and the age of the disease onsetA negative correlation was observed between disulphide (P = 0.013, r = -0.28) levels and vitiligo disease activity (VIDA score). But no correlation was observed between the levels of native thiol (P = 0.12, r = -0.18), total thiol (P = 0.079, r = -0.21), disulphide/native thiol (P = 0.29, r = -0.12), disulphide/total thiol (P = 0.29, r = -0.12), native thiol/total thiol (P = 0.28, r = 0.13), IMA (P = 0.56, r = -0.07) and vitiligo disease activity (VIDA score).
Discussion Several studies have been carried out to enlighten the pathogenesis of vitiligo. Different factors are thought to be responsible for the destruction of melanocytes, but the most widely accepted hypothesis blames oxidative stress, genetics, autoimmunity, generation of inflammatory mediators and self-destruction.[1] The role of oxidative stress in the pathophysiology of vitiligo has been reported previously.[17] Oxidative stress is thought to cause some biochemical anomalies resulting from the accumulation of melanocytotoxic substances and inhibition of natural processes of detoxification with the destruction of melanocytes in vitiligo patients.[1],[17],[18]
Free radicals resulting from pathological processes can cause an imbalance in the thiol/disulphide homeostasis in the body. A decreased plasma thiol concentration can indicate an increase in free radical generation and oxidative stress.[4] In our study, native and total thiol levels were significantly lower in vitiligo patients compared with the healthy controls. Disulphide levels were significantly higher in the patient group indicating the increased oxidative stress status in the body. IMA levels also tended to be increased in the patient group as an indicator of ischemia and oxidative damage. When the patients were classified as generalised and localised vitiligo, native and total thiol, disulphide and IMA levels did not differ statistically. As the mentioned parameters show the oxidant–antioxidant status of the body, our findings indicated that oxidative stress is the basic reason of the disease independent from the classification. Several studies support the findings of the present study.[1],[19],[20],[21] We also evaluated the patients according to VASI and VIDA scores, and we found no significant correlation between the scoring systems and native thiol, total thiol, disulphide, disulphide/native thiol, disulphide/total thiol, native thiol/total thiol and IMA levels.
Trace elements are essential for biochemical processes in the body and are involved in many reactions such as keratinisation and melanin formation.[22] Besides Se, Zn and Cu involve in the antioxidant defence system via the destruction of free radicals.[23] In our study, Se levels tended to be lower in vitiligo patients and were similar in both generalised and localised vitiligo patients. The studies showed elevated Se levels in vitiligo patients.[24],[25] Barikbin et al. reported similar Se levels of vitiligo patients and healthy controls[26] supporting the results of our study. We found lower Se levels in the patient group similar to the study of Wacewicz et al.[23] One of the antioxidant enzymes, glutathione peroxidase (GPX), is Se-dependent enzyme and detoxifies organic hydroperoxides in the body. Lower Se levels might indicate reduced GPX activity and increased oxidant and inflammatory status. Se levels were negatively correlated with the VASI score. The increase in the dissemination of the disease was related to increased oxidative stress, and this resulted in lower Se levels. Zn and Cu have roles to protect against reactive oxygen species and promote melanogenesis. The studies reported different results of Zn and Cu levels in vitiligo patients. Some studies showed increased[27] and some showed decreased Zn and Cu levels in vitiligo patients,[28] while some studies found similar Zn and Cu levels in the patient and control group.[29] We found significantly higher Zn levels in vitiligo patients. This might be associated with the increased release of Zn from apoptotic peripheral blood mononuclear cells in vitiligo patients.[23] Also, the patients with generalised vitiligo had higher Zn levels than the patients with localised vitiligo, and there was a positive significant correlation between Zn levels and VASI score. It shows that Zn levels are associated with the dissemination of the disease. Cu levels were lower in the patient group, but the difference was not statistically significant. Cu is a cofactor of tyrosinase and plays a role in the pigmentation process; lower Cu levels could be seen in impaired melanogenesis in vitiligo patients.
Vitamin E, an antioxidant and free radical scavenger, is found in cell membranes and circulating lipoproteins and protects the membranes from the damaging effects of free radicals. Vitamin A, a low molecular weight antioxidant, reduces the damaging effects of oxygen in lipid and non-lipid cellular compartments.[30] There are different antioxidative pathways to eliminate ROS. One of them is some small-molecule antioxidants, such as uric acid, glutathione (GSH), and vitamin E prevents or delays the initiation of various oxidative stress.[31] In our study, vitamin A and vitamin E levels tend to be lower in vitiligo patients. As oxidative damage is higher in the patient group, we thought that vitamin levels were lower due to the consumption against the oxidative status of the patients. Also, vitamin A levels and VASI scores were negatively correlated. Vitamin A levels tend to be lower in generalised vitiligo patients.
One of the limitations of the study was the limited number of oxidant and antioxidant parameters that were included. Also, the relationship between the treatment of the patients and the parameters was not evaluated in detail. In the future, studies can be conducted to enlighten the pathogenesis of the disease by increasing the number of patients and the number of oxidant–antioxidant parameters.
Conclusion As a result, the pathogenesis of vitiligo disease is still unclear. All thiol-disulphide homeostasis parameters (the most important antioxidant–oxidant system in circulation), trace elements, and vitamins together were evaluated in the present study in vitiligo patients. The results showed the relationship between different markers indicating oxidative stress. It can be concluded that vitiligo patients have increased oxidative stress status, and also the increase in the dissemination of the disease also increases the oxidative stress in the body.
Ethical approval
The local ethics committee approved this study (REF number: E1-22-2317).
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References 
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