Vitiligo is the most common attained variety of leukoderma, causing major social and emotional hitches in all affected subjects 1. It is presented by disfiguring circumscribed, depigmented macules and patches of skin as a result of melanocyte damage 2. Owing to the multifactorial nature of vitiligo 3, many studies through the years and all over the world have tried to clarify the pathogenesis behind it; nevertheless, the pathogenesis of vitiligo remains an enigma 3–5. Quite a lot of predominant pathogenic theories have been suggested, including autoimmune, cytotoxic, oxidant–antioxidant, and neural mechanisms, in addition to an intrinsic defect of melanocytes 5. Recently, the role of vitamin D3 in the pathomechanism of vitiligo has been suggested, and its relevance in the treatment of vitiligo was reported 6.
Vitamin D, a fat-soluble vitamin, is a secosteroid hormone. In addition to its recognized metabolic function in calcium metabolism and bone health, it has noncalciotropic immunomodulatory character through its wide-ranging sound effects on lymphocytes (T and B), macrophages, and dendritic cells, as all these cells express vitamin D receptors in their nuclei 7,8. A decrease in vitamin D was proposed to act as an environmental prompt and is a subsequent inducer of autoimmunity 7.
Furthermore, it was suggested that vitamin D3 is strongly immunosuppressive, and truncated levels are described in autoimmune disorders including vitiligo. However, the reason of low vitamin D3 level in patients having autoimmune conditions remains to be resolved 9.
Narrow-band ultraviolet B (NBUVB) therapy has been considered as cornerstone in the treatment of vitiligo because of its safety and efficacy. Yet, the mechanism of NBUVB-induced pigmentation is still a substance of discussion and needs to be elucidated. Ultraviolet radiation was reported to have immunomodulatory effects through the stabilization of the local and systemic atypical immune reactions 10. NBUVB induces synthesis of interleukin-1, tumor necrosis factor-α, and leukotriene C-4. These cytokines encourage melanogenesis and melanocyte mitogenesis, in addition to melanocyte migration 6.
The natural ultraviolet B of sunlight (290–320 nm) is responsible for the photochemical transformation of 7-dehydrocholesterol to previtamin D3 in the skin (stratum spinosum and stratum basale), which is the crucial phase of vitamin D3 synthesis and gives rise to more than 90% of the serum vitamin D concentration 8,11. Moreover, molecular studies revealed that vitamin D increases the tyrosinase content of cultured human melanocytes 12, which may propose a potential role of vitamin D3 in modifying melanogenesis. Based on this information, we can hypothesize the valuable role of ultraviolet B phototherapy in vitiligo treatment as an inducer of endogenous production of vitamin D.
Given the deficiency of studies on the role of vitamin D in NBUVB-induced pigmentation in patients with vitiligo, the association of low vitamin D levels with systemic autoimmune diseases including vitiligo 7, the effectiveness of topical vitamin D compounds on repigmentation in vitiligo 13,14, role of natural ultraviolet B on serum 25-hydroxyvitamin D [25(OH)D] concentration 8,12, and the results of the study by Sehrawat et al.6, we aimed to assess serum 25(OH)D levels in patients with vitiligo before and after 12 weeks of NBUVB therapy and correlated it with repigmentation.
Patients and methods
This case–control study was carried out on 50 patients with different degrees of vitiligo severity. They were selected from Dermatology Outpatient Clinic, Faculty of Medicine, Menoufia University Hospital, Egypt. In addition, 25 apparently healthy, vitiligo-free, and age-matched, sex-matched, and Fitzpatrick skin phototype-matched healthy volunteers were included as controls for baseline vitamin D levels. They were recruited during spring from March 2016 to June 2016 to limit the effects of the seasonal variation on vitamin D serum level.
The inclusion criteria included the following:
- Newly diagnosed cases of vitiligo.
- The patient should have stopped treatment for his/her vitiligo 8 weeks before joining the study.
The following were the exclusion criteria:
- Subjects having autoimmmune/inflammatory diseases.
- Subjects with current infection or immunosuppression.
- Patients with liver, kidney, or malabsorption disorders.
- Patients receiving phototherapy or photochemotherapy in the past 6 months.
- The use of any drugs that could alter the outcome of the study such as vitamin D, systemic steroids, weight-loss drugs, and cholesterol-lowering drugs.
- Pregnant or lactating women.
Each individual in the study signed a written consent form. The study protocol was approved by the Research Ethics Committee of Faculty of Medicine, Menoufia University.
All participants in the study were subjected to the following:
- Thorough history taking, stressing on onset, duration, and family history of vitiligo.
- Complete clinical examination:
- General examination to detect any excluding factor.
- Dermatological examination to evaluate the vitiligo and to assess its severity based on Vitiligo Area and Severity Index (VASI) score 15.
- Measurement of the serum levels of vitamin D:
- Baseline (week 0) serum 25(OH)D levels were estimated in both patient and control groups. Then, after 12 weeks of NBUVB therapy, 25(OH)D levels were reevaluated in patients with vitiligo. Two milliliters of venous blood was obtained in the morning between 8:00 and 9:00 a.m., under complete aseptic condition. Blood samples were centrifuged at 4000 rpm for 10 min, and the separated sera were stored at −80°C until analysis. Quantitative determination of 25(OH)D was performed by competitive enzyme-linked immunosorbent assay technique using Immunodiagnostic Systems 25(OH)D enzyme immunoassay kit. Quantitative determination of 25(OH)D was performed using reagents supplied by Epitope Diagnostics Inc., San Diego, California, USA. The values were interpreted as follows: less than 25 nmol/l – deficient or very low, 25–74 nmol/l – insufficient or low, and 75–250 nmol/l – sufficient or normal.
- NBUVB phototherapy:
Waldmann ultraviolet 1000 L (TL 01) machine was used for delivery of NBUVB. NBUVB phototherapy was given every other day, three times per week for 12 weeks. During each session, ultraviolet safety glasses for eyes and shields for genital protections were applied. The minimal erythema dose of 0.3 J/cm2 was the starting dose of irradiation 16; the dose was increased by 20% on each consequent appointment till just faint erythema seemed. If any patient developed symptomatic erythema (burning, pain) or blisters, phototherapy was pending till the lesions cured. Then irradiation was started again by a dose of 20% less than that induced erythema or blisters. Then the irradiation dose was increased by 10% on consequent sessions 17. VASI was calculated for every patient by the same physician on the first visit (baseline), and at the end of the therapy 15.
The data were collected, entered, and processed on IBM-PC compatible computer using SPSS software (version 20.0; SPSS Inc., Chicago, Illinois, USA). Student’s t-test and Mann–Whitney U-test were used to compare quantitative data according to its distribution. χ2-Test was used for qualitative data, and Spearman’s correlation to assess correlation. P value of less than 0.05 was considered the cutoff value for significance.
The demographic and clinical data
The present study included 50 patients with vitiligo. There were 18 males and 32 females, with 20–50 years of age (mean 28.76±10.64), median disease duration of 3.5 years (range: 3 months to 20 years), and median affected body surface area of 20% (range: 5–80%). Sixteen (32%) patients were of Fitzpatrick skin phototype III and 34 (68%) patients were Fitzpatrick type IV. Most of our cases (38, 76%) had negative family history. The controls were 25 age-matched (mean age: 28.16±9.84 years), sex-matched (10 males and 15 females), and Fitzpatrick skin phototype-matched [nine (36%) were of phototype III and 16 (64%) were of Fitzpatrick IV] matched vitiligo-free healthy volunteers.
Results before narrow-band ultraviolet B
Baseline serum 25(OH)D levels of studied subjects showed statistically significant difference (P<0.001) between patients with vitiligo and controls, as none of vitiligo cases recorded normal values of serum 25(OH)D. Ten (20%) patients versus 23 healthy controls (92%) had insufficient serum levels of 25(OH)D, and 40 (80%) patients had deficient 25(OH)D levels, whereas control subjects recorded normal level of serum 25(OH)D in two of them, and none of the controls (0%) had deficient 25(OH)D level (Table 1).
Regarding serum 25(OH)D concentrations in the different subgroups of vitiligo cases categorized according to their sex, age, Fitzpatrick skin phototype, and family history of vitiligo, these diverse estimated variables showed that sex and job were the only independent factors affecting serum 25(OH)D values, as significantly lower serum 25(OH)D levels were recorded in female patients and in those with indoor occupation compared with males and those without door occupation (P=0.012 and 0.019, respectively) (Table 2).
Concerning vitiligo severity, the mean VASI score was 2.28±1.28, which was insignificantly correlated with the baseline serum level of 25(OH)D (r=0.154, P=0.462).
Results after narrow-band ultraviolet B
After 12 weeks of phototherapy, clinical improvement – repigmentation – was noticed, and the mean value of the evaluated VASI score showed significant (P<0.001) reduction than its baseline mean value (0.94±0.70 vs. 2.28±1.28, respectively), in addition, after treatment, 25(OH)D serum level (26.69±6.55) also showed significant upgrading (Table 1) (P<0.001). However, comparison and correlation between mean improvement in VASI score and elevated 25(OH)D level at the end of sessions could not reach level of significance (r=0.119, P=0.572).
A good color matching of the treated area of vitiligo with the surrounding normal skin after NBUVB phototherapy was noticed, as 38 (76%) patients showed the same color of repigmentation in vitiligo patches like that of the adjoining skin, eight (16%) patients developed somewhat darker, and four (8%) patients showed somewhat lighter color in the vitiligo patches after NBUVB sessions. The pattern of repigmentation started mainly perifollicular in 36 (72%) patients, eight (16%) patients showed marginal, and six (12%) had a diffuse kind of repigmentation. Overall, 72% (n=36) of our patients had no adverse effects. Most common adverse effects observed were marked erythema in four (8%) patients and pruritus in eight (16%) patients.
In the present study, highly significant low serum 25(OH)D levels were recorded in patients with vitiligo compared with their controls. In agreement with our results, many authors reported insufficient levels of serum 25(OH)D among patients with vitiligo in their studies in different regions 6,18,19 including Egypt 20. The decreased serum levels of 25(OH)D in vitiligo group of the current study strengthen the role of vitamin D in vitiligo development. As in other autoimmune disorders, deficient serum levels of 25(OH)D in our patients with vitiligo provide additional support to the autoimmune theory of vitiligo and to the noncalciotropic immunomodulatory character of vitamin D 8.
Based on animal studies, it was found that oral vitamin D supplementation could prevent autoimmune diseases, but potential interventional confirmation in humans is quiet deficient 2,21. Consequently, additional studies considering the role of vitamin D supplementation on controlling and modifying clinical aspects of vitiligo or its prevention in susceptible family members are suggested.
On studying the effect of assessed demographic and clinical data of vitiligo on serum 25(OH)D levels, our results revealed that occupation and sex were the only independent factors affecting serum 25(OH)D levels, as those with indoor occupation and female patients had significantly low 25(OH)D levels. This could be explained by the effect of natural ultraviolet-induced vitamin D or could be ascribed to feelings of embarrassment, which can lead to low self-confidence and social loneliness 22. Additionally, it may be related to the less sun exposure in female patients owing to wearing long robes and veil for religious reasons. The reported role of job on serum 25(OH)D levels in the current study was also supported by previous ones 19,20.
This is in accordance with Lim et al.23 and Alnoshan et al.24. Vitamin D was proposed to limit the loss of melanocytes in vitiligo by opposing the local cutaneous immune procedure, oxidative defense, delayed apoptosis, improvement of the peculiar calcium fluxes, and photoprotection of the epidermal melanin unit 13,14. Furthermore, vitamin D analogues control activation, proliferation, and migration of melanocytes, resulting in restoring the melanocyte integrity and the pigmentation pathways, in addition to modulating T cell activation, which is linked with the melanocyte loss and autoimmune damage in vitiligo 13. Moreover, these analogues augment tyrosinase activity 14.
After 12 weeks of NBUVB phototherapy, the results of our study showed that levels of 25(OH)D increased significantly parallel to marked clinical improvement and repigmentation, as VASI score decreased significantly. However, the correlation between the mean improvement in VASI score and increased 25(OH)D could not reach level of significance. This might be attributed to small sample size and short duration of our study. Meanwhile, the assumption of partial responsibility of vitamin D in photo-induced melanogenesis still exists.
The significantly elevated serum 25(OH)D levels in patients with vitiligo after NBUVB were also recorded by Sehrawat et al.6 and Alnoshan et al.24. Additionally, Alnoshan et al.24 reported more significant elevation of serum 25(OH)D levels in their vitiligo cases with longer duration and more NBUVB sessions. Moreover, Sehrawat et al.6 observed that the value of correlation coefficient between serum 25(OH)D and VASI score increased with increase in the cumulative dose of NBUVB. Hence, future longer duration studies were recommended.
As expected, the pattern of repigmentation was mainly perifollicular – in 72% of our cases – indicating that melanin is created by the melanocytes in hair follicles. The reported significant increase in 25(OH)D serum level after NBUVB may act to induce maturation and differentiation of immature melanocytes in the bulge region of hair follicles, with subsequent increase of melanin production 25.
In line with many studies 6,26,27, most of our patients [38 (76%) patients] achieved repigmentation of color matched with their normal skin, eight (16%) patients developed somewhat darker, and four (8%) patients showed somewhat lighter color in the vitiligo patches after NBUVB sessions.
Based on the aforementioned results and discussion, it could be concluded that vitamin D may have an active role, and it contributes in the pathogenesis and development of vitiligo. Furthermore, vitamin D may take part in photo-induced melanogenesis. Yet, the mechanism of NBUVB-induced pigmentation is still far from being clarified and still needs further studies.
Conflicts of interest
There are no conflicts of interest.
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