Evaluation of Association of Vitamin D in Alopecia Areata: A Case-control Study of 100 Patients in a Tertiary Rural Hospital of Southern India.

BACKGROUND: Alopecia areata is a common autoimmune disorder which is characterized by non-scarring hair loss. Vitamin D plays an important role in immune regulation, cell growth, differentiation, and maintenance of hair cycle. AIMS AND
OBJECTIVES: (1) To evaluate serum vitamin D levels in alopecia areata. (2) To compare serum vitamin D levels in new versus old cases and with respect to severity of alopecia areata.
MATERIALS AND METHODS: A retrospective case-control study with 100 cases of alopecia areata and 100 age- and sex-matched healthy controls was conducted from December 2014 to November 2015. All subjects underwent complete clinical evaluation and serum vitamin D levels.
Results: The mean serum vitamin D level was significantly lower in patients with alopecia areata (18.90 ± 8.32 ng/mL) (64%) as compared to healthy controls (28.21 ± 18.32 ng/mL) (38%) (P < 0.001). The mean serum vitamin D levels was significantly lower in old cases (15.11 ± 4.75 ng/mL) as compared to new cases (20.85 ± 9.09 ng/mL) (P < 0.001). The proportion of subects with vitamin D deficiency was significantly higher among old cases (84.3%) as compared to new cases (53.1%) (P < 0.05). There was a significant inverse correlation between Severity of Alopecia Tool scores and serum vitamin D levels (r = -0.298, P < 0.05).
CONCLUSION: Decreased vitamin D levels were observed in patients with alopecia areata and significant inverse correlation exists between vitamin D levels and duration/severity of the disease. These findings may suggest a causal role of vitamin D deficiency in the pathogenesis and therapeutic role of vitamin D supplementation in the management of alopecia areata.

Introduction

Alopecia areata is a type of non-scarring alopecia involving the hairs of scalp and/or body.[1] It is a common condition, accounting for around 0.7% of new dermatology cases in India.[2] Alopecia areata can be associated with significant psychosocial implications in the patient owing to the cosmetic disfigurement associated with it. It is an autoimmune disease characterized by T-cell (CD4+ and CD8+) infiltrates and cytokine production specifically targeting anagen-stage hair follicles.[3]

Vitamin D functions as a pro-hormone, primarily synthesized in epidermal keratinocytes under the influence of ultraviolet radiation or acquired from diet.[45] It acts as a modulator of immune functions of T lymphocytes and B lymphocytes.[6]

It has been demonstrated that vitamin D receptors are strongly expressed in the key structures of hair follicles and its expression is necessary for the maintenance of normal hair cycle.[7] Hence, we hypothesized that vitamin D deficiency may be a risk factor for development of alopecia areata.j

Materials and Methods

The study was a retrospective case–control study involving 100 cases of alopecia areata and 100 healthy controls conducted from December 2014 to November 2015. The cases and controls were recruited from the outpatient department of dermatology, venereology, and leprology of a medical college and hospital located in rural part of Bengaluru district of Southern India. Ethical clearance for the study was taken from the Institutional Ethics Committee.

All cases and controls were enrolled from February to July, during the months of spring and summer in India. The amount of sun radiations reaching the surface is almost the same in these months.[8]

Patients with alopecia areata belonging to any age group or gender were included. Patients with other types of alopecia (such as tinea capitis, androgenetic alopecia, trichotillomania, scarring alopecia, traction alopecia, telogen effluvium), other coexisting chronic dermatological disease (such as psoriasis, vitiligo), autoimmune or systemic diseases (such as diabetes mellitus, anemia, hypo/hyperthyroidism, systemic lupus erythematosus, rheumatoid arthritis, scleroderma, chronic renal disease, chronic liver disease), and patients who have recieved systemic corticosteroids, immunosuppressive drugs, calcium or vitamin D supplements in the past 4 weeks or phototherapy in the past 3 months were excluded.

Diagnosis of alopecia areata was based on clinical findings. In doubtful cases, histopathological examination (perifollicular lymphocytic infiltration)[1] and trichoscopy (to look for exclamation mark hairs, coudability, yellow dots, black dots, short vellus hairs)[1] were performed. Severity of Alopecia Tool (SALT) score was calculated by dividing the entire scalp into four parts based on the surface area, top (40% – 0.4), posterior (24% – 0.24), right side (18% – 0.18), and left side of scalp (18% – 0.18). The percentage of hair loss in each area was determined independently and was multiplied by the factor mentioned above for each of the four areas and summing the products of each area will give the SALT score and they were divided as S0 (no hair loss), S1 (<25%), S2 (25%–49%), S3 (50%–74%), S4 (75%–99%), and S5 (100%).[9]

Patients were categorized into two subgroups – new cases and old cases. New cases were defined as patients presenting with first episode of alopecia areata and a disease duration lasting less than 6 months. Old cases were defined as patients with first episode of alopecia areata lasting more than 6 months or patients with multiple episodes of alopecia areata.

Clinical photographs of cutaneous lesions were taken for assessment of severity of lesions after taking informed patient consent. The healthy control group consisted of 100 age- and sex-matched individuals without any history of alopecia areata or other autoimmune/systemic diseases. Serum vitamin D was measured through chemiluminescence immunoassay method and graded as follows: deficiency <20 ng/mL; insufficiency 20–29.99 ng/mL; normal >30 ng/mL.

Statistical analysis

The statistical software SPSS 15.0 was used for the analysis of data. Student's (two-tailed, independent) t-test was used to compare the quantitative parameters between two or more groups of patients. Chi-square/Fisher's exact test has been used to compare the categorical variables between two or more groups. Correlation was assessed using Spearman's correlation. P value <0.05 was considered to be significant and P value <0.001 as highly significant.

Results

A total of 100 cases and 100 controls satisfying the inclusion and exclusion criteria were included in the study. Baseline clinical characteristics of cases and controls have been illustrated in Table 1. There were 72 males and 28 females (M:F -2.57:1) in the cases and 58 males and 42 females (1.38:1) in the control group. The age ranged from from 5 to 60 years in cases. The majority of the cases and controls (48) belonged–age group of 21-30 years. The mean age of the cases was 24.52 ± 10.06 years and controls was 28.96 ± 11.49 years. The most commonly involved site was scalp [Table 1].

Table 1

Demographic and clinical data of cases and controls

	Cases (100)	Controls (100)
Sex, n (%) – male	72 (72%)	58 (58%)
Female	28 (28%)	42 (42%)
Male:female	2.57:1	1.38:1
Age (in years), mean age	24.52 ± 10.06	28.96 ± 11.49
Range	5–60	3–72
New cases, n (%)	66 (66%)	-
Old cases, n (%)	34 (34%)	-
Sites – scalp	63	-
Beard	24	-
Scalp + beard	8	-
Scalp + eyebrow	4	-
Saclp + beard + eyebrow	1	-
SALT subclasses, n (%) – S1	75 (99%)	-
S2	-	-
S3	1 (1%)	-
S4	-	-
S5	-	-
Mean serum vitamin D (in mg/mL)	18.90 ± 8.32	28.21 ± 18.32
Serum vitamin D (%) – normal	10	36
Insufficiency	26	26
Deficiency	64	38	P < 0.001

SALT, Severity of Alopecia Tool

The mean serum vitamin D levels of male cases and female cases were 18.38 ± 8.41 and 20.23 ± 8.07 ng/mL, respectively. There was no significant statistical difference in the mean serum vitamin D levels between males and females (P > 0.05). Positive family history was present in 4% of cases.

The mean serum vitamin D level was significantly lower in cases as compared to controls (18.90 ± 8.32 vs 28.21 ± 18.32 ng/mL; P < 0.001). The number of patients with serum vitamin D deficiency was significantly higher among cases than in controls (64 cases vs 38 controls; P < 0.001).

The cases were further divided into new and old cases – 66 new cases and 34 old cases. The mean serum vitamin D level in old cases was significantly lower than that of new cases (15.11 ± 4.75 vs 20.85 ± 9.09 ng/mL; P < 0.001). The number of patients with vitamin D deficiency was significantly higher among old cases ascompared to new cases (29/34 old cases vs 35/66 new cases; P < 0.05).

A total of 76 cases presented with alopecia involving scalp and they were further subclassified from S1 to S5 based on their SALT scores. Almost all the scalp alopecia cases, that is, 75 cases, belonged to subclass 1 (S1). Only one case belonged to subclass 3 (S3). When the serum vitamin D levels were plotted against their SALT score, there was a significant inverse correlation between the two. (r = −0.298; P < 0.05) [Figure 1].

Figure 1

Correlation between serum vitamin D and SALT score

Discussion

Alopecia areata is an autoimmune disease which is characterized by non-scarring hair loss and can affect any hair-bearing area of the body. It is caused by CD4+ and CD8+ T cells targeting the hair follicles as an autoimmune-mediated skin.[10]

Vitamin D is a pro-hormone which is synthesized in the skin and regulates the immune response by controlling T and B lymphocytes. Its deficiency has been established as a risk factor for autoimmune diseases such as systemic lupus erythematosus, psoriasis, vitiligo, rheumatoid arthritis, multiple sclerosis, and inflammatory bowel disease.[1112]

It is hypothesized that the development of hair follicle depends on vitamin D receptor expression which correlates with increased differentiation of follicle keratinocytes.[13] A recent study demonstrated that decreased expression of vitamin D receptor in alopecia areata lesions is related to decreased expression of Wnt signaling pathway, which inhibits proliferation and differentiation of hair follicles and epidermal cells,[14] although the exact mechanism which links serum vitamin D deficiency with alopecia areata is unknown.

Few recent studies have found decreased serum vitamin D levels in patients with alopecia areata.[61315161718] Hence, this study was planned to find out the association between serum vitamin D levels and alopecia areata in South Indian population.

The majority of the cases were males 72%. The male: female ratio of cases in our study was 2.57:1. Similar male predominance ratio was also found by Yilmaz N et al.[13] (2:1), Cerman AA et al.[6] (1.87:1), Attawa EM et al.,[16] (1.87:1) and Nassiri S et al.[19] (2.1:1).

There was no significant difference between mean serum vitamin D levels of male cases and female cases (18.38 ± 8.41 vs 20.23 ± 8.07 ng/mL; P > 0.05). Even Nassiri S et al.[19] and Attawa EM et al.[16] did not find any significant difference between male and females cases.

We found a positive family history of alopecia areata in about 4% cases. However, there was no significant difference in serum vitamin D levels of patients with positive family history as compared to those without any family history of alopecia areata. El-Mongy NN et al.[17] and Attawa EM et al.[16] saw positive family history in 8.6% and 8.7%, respectively, in the cases. Even they did not find any significant difference in serum vitamin D between those with and without family history of alopecia areata.

In our study, the mean serum vitamin D level was significantly lower in cases as compared controls (18.90 ± 8.32 vs 28.21 ± 18.32 ng/mL; P < 0.001). Even Yilmaz N et al.,[13] Cerman AA et al.,[6] Mahamid M et al.,[15] Attawa EM et al.,[16] El-Mongy NN et al.,[17] and Bhat YJ et al.[18] found similar significant lower levels of serum vitamin D in cases as compared to controls. However, the mean serum vitamin D levels of cases in Yilmaz N et al.,[13] Cerman AA et al.,[6] Mahamid M et al.,[15] and Attawa EM et al.[16] were comparatively less when compared to our study. These variations could be due to the studies being conducted in different geographical locations. The amount of radiation reaching the surface of the earth will depend on the latitude of each geographical location which may lead to variation in the amount of serum vitamin D synthesis in the body thus affecting the average serum vitamin D level of the population [Table 2].

Table 2

Comparison of mean serum vitamin D levels and percentage of deficiency of vitamin D to other similar studies

Study	Mean serum vitamin D level (in ng/mL)		Deficiency of vitamin D (in %)
	Cases	Controls	Cases	Controls
Our study	18.90 ± 8.32	28.21 ± 18.32	64	38
Yilmaz N et al.[15]	13.38 ± 7.09	20.51 ± 8.45	85	-
El-Mongy NN et al.[19]	26.6 ± 13.1	33.7 ± 22.3	-	-
Cerman AA et al.[8]	11.84 ± 6.18	23.57 ± 9.03	91	33
Mahamid M et al.[17]	11.32 ± 10.18	21.55 ± 13.62	69	25
Attawa EM et al.[18]	14.13 ± 8.72	22.43 ± 10.94	21.7	13
Bhat YJ et al.[19]	16.6 ± 5.9	25.49 ± 1.02

An analysis of the distribution of serum vitamin D deficiency among cases and controls showed a statistically significant higher percentage of vitamin D deficiency among cases than in controls (64% vs 38%; P < 0.001). The percentage of vitamin D deficiency in southern India was estimated to be about 40%–70%.[20] It is worthwhile to mention that various studies which have estimated vitamin D deficiency in general population have used different cut-offs to define vitamin D deficiency. Some studies have defined values less thn 35 ng/mL as deficiency.[21] Mahamid M et al.,[15] Cerman AA et al.,[6] and Yilmaz N et al.[13] also found that the percentage of vitamin D deficiency was significantly more among alopecia areata cases ascompared to controls.

We found that the mean serum vitamin D level was significantly lower in the old cases as compared to new cases (15.11 ± 4.75 vs 20.85 ± 9.09 ng/mL; P < 0.001). There was significantly higher percentage of patients with vitamin D deficiency among old cases as compared to new cases (85% in old cases vs 53% in new cases; P < 0.05). d'Ovidio R et al.[22] also found lower levels of serum vitamin D in chronic relapsing alopecia areata cases as compared to healthy controls although the difference was not statistically significant.

In addition, we found a statistically significant inverse correlation between SALT score and serum vitamin D levels (P < 0.05). Similarly, Cerman AA et al.,[6] Attawa EM et al.,[16] and Bhat YJ et al.[18] found a significant inverse correlation between SALT scores and serum vitamin D levels.

To our knowledge, this is the first case–control study among South Indian population which has evaluated the role of serum vitamin D in alopecia areata. In addition, our study was unique as we have also investigated the correlation between serum vitamin D levels and duration and severity of disease (SALT score). This association of serum vitamin D levels with respect to alopecia areata, its duration, and severity may suggest a possible role of serum vitamin D deficiency in pathogenesis of alopecia areata. and vitamin D levels as a potential marker for disease duration and severity of alopecia areata. Further studies are required to evaluate the role of supplementing vitamin D in the treatment of alopecia areata.

Some of the limitations of the study were that there was lack of severe cases of alopecia areata and the fact that degree of sunlight exposure and dietary intake of vitamin D can also lead to variation in serum vitamin D levels.

Financial support and sponsorship

None.

Conflicts of interest

There are no conflicts of interest.