|Year : 2015 | Volume
| Issue : 2 | Page : 76-80
Study of serum levels of Vitamin B12, folic acid, and homocysteine in vitiligo
Soumya Agarwal1, Vibhu Mendiratta1, Ram Chander1, Anju Jain2, Pravesh Yadav1
1 Department of Dermatology and STD, Lady Hardinge Medical College, New Delhi, India
2 Department of Biochemistry, Lady Hardinge Medical College, New Delhi, India
|Date of Web Publication||29-Dec-2015|
KI-94 Kavi Nagar, Ghaziabad - 201 001, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Background: Vitiligo has a complex etiopathogenesis. The role of nutritional factors (including Vitamin B12and folic acid) has been recently proposed in its causation, which needs to be confirmed further. Aims: The study was conducted: (1) To estimate the serum levels of Vitamin B12, folic acid, and homocysteine in patients of vitiligo and control group. (2) To determine the relationship between serum levels of Vitamin B12, folic acid, homocysteine and the extent and activity of vitiligo. Methods: A cross-sectional, observational study consisting of 50 patients with vitiligo, and 35 age and sex matched controls was conducted. Serum homocysteine was estimated by Diazyme homocysteine enzymatic assay kit, and serum Vitamin B12and folic acid levels were estimated by chemiluminescence using the Access Immunoassay System. Results: The mean serum Vitamin B12and folate levels were found to be significantly lower in vitiligo patients than controls (157.18 ± 68.95 pg/mL vs. 306.6 ± 169.73 pg/mL and 4.18 ± 3.55 ng/mL vs. 7.3 ± 3.67 ng/mL, respectively), while serum homocysteine levels were significantly higher in cases (15.39 ± 7.2 μmol/L) as compared to controls (11.88 ± 4.81 μmol/L) (P < 0.05). There was a significant positive correlation of serum homocysteine levels with the duration of disease, Vitiligo Area Scoring Index (score), and type of vitiligo (higher levels in patients with universal vitiligo). Conclusions: Long standing vitiligo (especially universal and generalized variants) may show deranged serum homocysteine, Vitamin B12, and folic acid levels. Supplementation with Vitamin B12/folic acid may have a therapeutic role in improving the treatment outcome.
Keywords: Folate, folic acid, homocysteine, Vitamin B12, vitiligo
|How to cite this article:|
Agarwal S, Mendiratta V, Chander R, Jain A, Yadav P. Study of serum levels of Vitamin B12, folic acid, and homocysteine in vitiligo. Pigment Int 2015;2:76-80
| Introduction|| |
Vitiligo is an acquired, idiopathic disease clinically characterized by circumscribed depigmented patches, with a variable prevalence ranging from 3% to 4% in India. Gujarat in India has recorded the highest prevalence; ~8.8%. Multitude of pathogenetic factors is involved namely genetic, autoimmune, oxidative stress, and nutritional factors. Reduced serum levels of Vitamin B12 and folic acid have been found in patients with vitiligo,, some suggesting clinical improvement after Vitamin B12 and folic acid supplementation., Recently in an Indian study, Singh et al. reported that serum homocysteine level was significantly increased while folic acid and Vitamin B12 levels were significantly decreased in vitiligo group as compared to controls. On the contrary, other studies by Kim et al., Balci et al. did not report any association between Hcy, Vitamin B12/folic acid and vitiligo. Taking into account, the conflicting results of the aforementioned studies, an attempt was made through the following study to find out the exact relationship between serum levels of Vitamin B12, folic acid, homocysteine and vitiligo.
| Methods|| |
This study was conducted in the Department of Dermatology and Biochemistry of a tertiary care hospital as a cross-sectional, observational study during November, 2011 to March, 2013. All clinically diagnosed cases of vitiligo, either untreated or receiving ultraviolet therapy, between 20 and 50 years were included in the study after obtaining written informed consent. A total of 50 cases and 35 age and sex matched healthy controls from the attendants of the study population were recruited in the study. Those who had a history of cigarette smoking; intake of folic acid, Vitamin B6, B12, and hormonal therapy; evidence of any autoimmune disease (thyroid disorder/anemia/diabetes mellitus); diseases known to affect the homocysteine level including hypertension, cardiovascular disease, renal failure, deep venous thrombosis, Behcet's disease, psoriasis; and pregnancy were excluded from the study.
The disease activity was assessed as follows: (1) Stable disease – no change in the vitiligo lesions during the 6 months prior to the study as observed by the patient. (2) Progressive disease – enlargement of already present lesions and/or the appearance of new lesions within the 6 months prior to the study as observed by the patient. (3) Regressive disease – improvement of lesions whether spontaneous or after ultraviolet therapy during the 6 months prior to the study. Extent of vitiligo was measured by the Vitiligo Area Scoring Index (VASI) analogous to the Psoriasis Area Severity Index used for psoriasis. The total body VASI was then calculated using the following formula by considering the contribution of all body regions (possible range, 0–100):
VASI = Σall body sites (hand units) × (residual depigmentation).
The activity of vitiligo was measured by Vitiligo Disease Activity score (VIDA score) [Table 1].
Four milliliter of blood was drawn from the recruited cases and controls. Serum was separated by centrifugation after clotting for 10 min and was immediately stored at −20°C until batch analysis. Serum homocysteine was estimated by an enzymatic method, using Diazyme homocysteine enzymatic assay kit. Vitamin B12 and folic acid levels were estimated by chemiluminescence using the Access Immunoassay System.
The results were analyzed using SPSS software version 20, USA. Mean and standard deviation were calculated for continuous parameters. Unpaired Student's t-test/ANOVA test was used to compare quantitative variables, and Chi-square test was used to compare qualitative variables. P < 0.05 was considered to be statistically significant. The study was approved by the ethical committee of the institute.
| Results|| |
The mean age of patients in the present study was 32.74 ± 10.52 years (M: F = 1:1.63). The mean duration of the disease was 9.0 ± 9.5 years. There was no statistically significant difference in the mean age, sex, religion, and dietary history between the case and control group [Table 2].
VASI score of patients ranged from 0.02 to 99 with a mean score of 16.62. Most of the patients presented with localized disease (VASI score <30).VIDA score of +4 was seen in the majority of the patients (38%), thus showing an active disease in the past 6 weeks in the majority of cases. The disease was progressive in the past 6 months in 68% patients, stable in 22%, and regressive in the remaining 10% patients. The majority of the patients (38%) had involvement of more than 5 body sites. Hands and feet were the most common affected sites (72%) followed by the trunk, face and neck, extremities, and mucosae, respectively. Vitiligo vulgaris was the most common presentation (40%) followed by acrofacial (20%), focal (12%), mucosal (4%), and segmental vitiligo (4%).
The mean serum homocysteine levels in vitiligo patients (15.39 ± 7.2 mol/L) were significantly higher than that of controls (11.88 ± 4.81 μmol/L) (P = 0.02) [Table 3]. The normal range of serum homocysteine was taken as 5 to 15 μmol/L (as mentioned in the Diazyme homocysteine enzymatic assay kit). Serum homocysteine level was increased in 48% patients versus 17.1% controls, and normal in 48% patients versus 77.1% controls (P = 0.007). Serum homocysteine levels showed a highly significant positive correlation with the duration of disease (P = 0.003) and VASI score (P < 0.001) [Table 4]. Serum homocysteine levels were significantly higher in patients with VASI score ≥30 as compared to those with a VASI score of <30 (P = 0.001) [Table 5]. Serum homocysteine levels were significantly higher in patients with universal vitiligo (26.9 ± 6.46 μmol/L) as compared to other clinical types (P = 0.002). Serum homocysteine levels showed an increasing trend with an increase in VIDA score from +1 to +4 (5.57 μmol/L in score +1 vs. 15.44 ± 7.2 μmol/L in score +4), but there was no significant correlation between homocysteine level and the activity of disease.
The mean serum Vitamin B12 levels in vitiligo patients (157.18 ± 68.95 pg/mL) were significantly lower as compared to controls (306.6 ± 169.73 pg/mL) (P < 0.001) [Table 3]. The reference range of normal serum Vitamin B12 level was considered to be 180–914 pg/mL (as mentioned in Access Immunoassay System, Beckman Coulter, REF: 33000, New Delhi). The level was low in 66% patients versus 22.9% controls, and normal in 34% patients versus 74.3% controls (P < 0.001). There was no correlation between serum Vitamin B12 levels and duration, activity (VIDA score), or extent (VASI score) of the disease.
The mean serum folate levels in vitiligo patients (4.18 ± 3.55 ng/mL) were significantly lower than that of controls (7.3 ± 3.67 ng/ml) (P < 0.001) [Table 3]. The normal level of serum folic acid was taken as >2.33 ng/mL (as mentioned in Access Immunoassay System, Beckman Coulter, REF: A14208). Serum folate level was decreased in 40% patients, and normal in 60% patients, whereas all the controls showed the normal level (P < 0.001). There was no correlation between serum folate levels and duration, activity (VIDA score), or extent (VASI score) of the disease.
No statistically significant correlation was found between serum homocysteine, Vitamin B12, folate levels with sex, religion, dietary history, and past therapy.
| Discussion|| |
Pathogenesis of vitiligo continues to be under debate and is attributed to the interplay of autoimmunity, oxidative stress, and sympathetic neurogenic disturbance. Despite the documented association of vitiligo with pernicious anemia in the literature, only few studies have evaluated the role of Vitamin B12, folic acid, and homocysteine in vitiligo. In the present study, we attempted to analyze the role of Vitamin B12, folic acid, and homocysteine in the pathogenesis of vitiligo.
Serum homocysteine levels were found to be significantly higher in vitiligo patients as compared to controls. Homocysteine may mediate melanocyte destruction by causing increased oxidative damage, interleukin-6 production, and nuclear factor kappa B activation. The production of toxic reactive species by homocysteine oxidation, namely superoxide anion, hydrogen peroxide and hydroxyl free radicals  together with other biochemical abnormalities of biopterin metabolism, may result in oxidative stress, accumulation of melanocytotoxic compounds, and an inhibition of natural detoxifying processes that may contribute to the destruction of melanocytes in vitiligo skin. Homocysteine also inhibits tyrosinase enzyme which catalyzes the rate-limiting step of melanin biosynthesis, probably by interaction with copper at the active site of the enzyme. Free homocysteine reacts nonenzymatically with the sulfhydryl residues of the body proteins forming adducts with disulfide linkage (thiolation). Extensive thiolation has been found to affect the function of proteins and enzymes. Individuals may show genetic heterogeneity in homocysteine metabolism attributed to mutations in the catalase gene (CAT), and low catalase activity is detected in vitiligo. All these factors might culminate in melanocyte destruction, and failure of melanin production in vitiligo. Our finding was, in concordance with the pilot study, conducted by Shaker and El-Tahlawi who reported that the mean serum homocysteine level in the Egyptian vitiligo patient group was significantly higher than in the control group.
Either a genetic defect in one of the enzymes of homocysteine metabolism or a nutritional deficiency of one or more of the vitamins that participate in homocysteine metabolism can lead to metabolic disruption and potentially to hyperhomocysteinemia. The patients having factors which cause raised homocysteine levels, including impaired renal function, high plasma creatinine, smoking, coffee consumption, alcoholism, and certain drugs (folate antagonists, nitrous oxide, and L-DOPA) were excluded from the study.
In the present study, serum Vitamin B12 and folate levels were noted to be significantly lower in vitiligo patients as compared to controls. Both Vitamin B12 and folic acid are required as cofactors by the enzyme homocysteine methyltransferase for the regeneration of methionine from homocysteine in the activated methyl cycle. Consequently, a nutritional deficiency in either of these two vitamins results in an increase in homocysteine and a decrease in methionine levels in the circulation. Homocysteine levels are determined by serum levels of Vitamin B12 and folic acid. The results obtained were in accordance with a recent Indian study done by Singh et al. who reported that the mean values of Vitamin B12 and folate were significantly decreased in vitiligo group as compared to controls.
There was a significant correlation between duration of the disease and homocysteine levels. It could be concluded that the homocysteine metabolic alterations become more persistent and severe as the disease becomes chronic thus accounting for higher levels in long-standing course of vitiligo. It is postulated that persistent alterations in homocysteine levels may be responsible for greater melanocytic damage resulting in complete depigmentation along with the poor ability for spontaneous repigmentation and response to therapy.
Serum homocysteine levels showed a significant positive correlation with the VASI score. Moreover, serum homocysteine levels were found to be significantly higher in patients with VASI score ≥30 as compared to those with a VASI score of <30. However, Vitamin B12 and folate levels were not related to the extent of disease. VASI score is a product of bovine serum albumin involvement by vitiligo and the amount of depigmentation. Chronicity of vitiligo is caused by an uninterrupted ongoing melanocytic injury, either because of the natural course of disease or lack of treatment, which results in extensive depigmentation (greater VASI score). Since chronicity of disease is associated with a long-standing insult to melanocytes, both serum homocysteine levels and VASI scores were found to be significantly raised in patients with long-standing disease. In concordance with the present study, El-Dawela and Abou-Elfetouh, and Silverbergand Silverberg  reported a statistically significant association between homocysteine levels and VASI score/extent of disease, but not between folate, or Vitamin B12 levels and the extent of vitiligo. Moreover, Silverbergand Silverbergstrongly recommended the inclusion of homocysteine as a severity marker on the initial examination for vitiligo patients.
The relation between serum homocysteine, Vitamin B12, folate levels, and VIDA score of patients was not statistically significant. This finding was consistent with the studies by Kim et al., and Karadag et al. However, quite a few studies report a significant correlation of serum homocysteine level with the activity of disease, for example those done by Shaker and El-Tahlawi, Singh et al., and El-Dawela and Abou-Elfetouh. It is proposed that homocysteine may be a contributory factor in addition to other factors (genetic susceptibility, autoimmune factors, neural, or melanocytorrhagy) in perpetuating a smoldering course of the disease, rather than acting as a trigger for sudden melanocytic death during periods of disease activity.
No significant association between the homocysteine, folic acid, or Vitamin B12 level and dietary habits of the patients were observed. This is in accordance with previous studies which found no significant difference in serum homocysteine, folic acid, or Vitamin B12 levels with different dietary habits. Dietary habit may influence homocysteine level because Vitamin B12 is mainly present in animal proteins. One of the reasons for almost similar homocysteine, folic acid, or Vitamin B12 levels in all the groups in the present study could be the low frequency of intake of meat by our study subjects.
Homocysteine is widely regarded as a reliable indicator of both functional and intracellular deficiencies of Vitamin B12 and folic acid. It has been established that subtle changes suggestive of a functional intracellular deficiency of Vitamin B12 and folic acid occur in many adult patients in the presence of minimal biochemical changes. In this respect, serum vitamin concentrations have relatively poor sensitivity and specificity in detecting subjects with subtle changes suggestive of vitamin deficiency. This could be the reason for observing a significant correlation of only serum homocysteine levels with duration of disease, VASI score, and type of vitiligo.
Our study despite the small sample size gives some indications for the use of homocysteine as a marker of severity and outcome of vitiligo; however, it needs to be established by larger multicentric studies. Another area to be explored is the therapeutic effect of Vitamin B12 and folic acid supplementation on progression of the disease.
| Conclusion|| |
To conclude, vitiligo is a multifactorial disease, and the plethora of implicated mechanisms that potentially trigger the onset of, and maintain or increase the activity of this disease may show promise for future treatment modalities that might be beneficial for the patients. The improvement of vitiligo following supplementation with Vitamin B12 and folic acid, as reported by few authors  has helped to clarify the complex pathogenesis of the disease to some extent. Vitiligo patients with a chronic and widespread disease should be investigated for serum homocysteine levels for a possible role of raised homocysteine can be postulated in the pathogenesis of vitiligo based on the findings of our study. Moreover, the therapeutic role of supplementation with Vitamin B12/folic acid in vitiligo needs to be studied further.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Dutta AK, Mandal SB. A clinical study of 650 vitiligo cases and their classification. Indian J Dermatol 1969;14:103-11.
Dwivedi M, Laddha NC, Shajil EM, Shah BJ, Begum R. The ACE gene I/D polymorphism is not associated with generalized vitiligo susceptibility in Gujarat population. Pigment Cell Melanoma Res 2008;21:407-8.
Montes LF, Diaz ML, Lajous J, Garcia NJ. Folic acid and Vitamin B12
in vitiligo: A nutritional approach. Cutis 1992;50:39-42.
El-Batawi MM, El-Tawil NE, El-Tawil AE. Serum levels of Vitamin B12
and folic acid in Egyptian patients with vitiligo. Egypt J Dermatol Androl 2001;21:77-80.
Juhlin L, Olsson MJ. Improvement of vitiligo after oral treatment with Vitamin B12
and folic acid and the importance of sun exposure. Acta Derm Venereol 1997;77:460-2.
Singh S, Singh U, Pandey SS. Serum folic acid, Vitamin B12
and homocysteine levels in Indian vitiligo patients. Egypt Dermatol Online J 2012;8:1-7.
Kim SM, Kim YK, Hann SK. Serum levels of folic acid and Vitamin B12
in Korean patients with vitiligo. Yonsei Med J 1999;40:195-8.
Balci DD, Yonden Z, Yenin JZ, Okumus N. Serum homocysteine, folic acid and Vitamin B12
levels in vitiligo. Eur J Dermatol 2009;19:382-3.
Hamzavi I, Jain H, McLean D, Shapiro J, Zeng H, Lui H. Parametric modeling of narrowband UV-B phototherapy for vitiligo using a novel quantitative tool: The vitiligo area scoring index. Arch Dermatol 2004;140:677-83.
Bhatnagar A, Kanwar AJ, Parsad D, De D. Psoralen and ultraviolet A and narrow-band ultraviolet B in inducing stability in vitiligo, assessed by Vitiligo Disease Activity score: An open prospective comparative study. J Eur Acad Dermatol Venereol 2007;21:1381-5.
Taïeb A, Picardo M. Clinical practice. Vitiligo. N Engl J Med 2009;360:160-9.
Guilland JC, Favier A, Potier de Courcy G, Galan P, Hercberg S. Hyperhomocysteinemia: An independent risk factor or a simple marker of vascular disease? 1. Basic data. Pathol Biol (Paris) 2003;51:101-10.
Schallreuter KU, Wood JM, Pittelkow MR, Gütlich M, Lemke KR, Rödl W, et al.
Regulation of melanin biosynthesis in the human epidermis by tetrahydrobiopterin. Science 1994;263:1444-6.
Reish O, Townsend D, Berry SA, Tsai MY, King RA. Tyrosinase inhibition due to interaction of homocysteine with copper: The mechanism for reversible hypopigmentation in homocystinuria due to cystathionine beta-synthase deficiency. Am J Hum Genet 1995;57:127-32.
Lussier-Cacan S, Xhignesse M, Piolot A, Selhub J, Davignon J, Genest J Jr. Plasma total homocysteine in healthy subjects: Sex-specific relation with biological traits. Am J Clin Nutr 1996;64:587-93.
Góth L, Rass P, Páy A. Catalase enzyme mutations and their association with diseases. Mol Diagn 2004;8:141-9.
Shaker OG, El-Tahlawi SM. Is there a relationship between homocysteine and vitiligo? A pilot study. Br J Dermatol 2008;159:720-4.
Mudd SH, Levy HL, Skorby LF. Disorders of transsulfuration. In: Scriver CR, Beudet AL, Sly WS, Valle D, editors. The Metabolic and Molecular Basis of Inherited Disease. 7th
ed. New York: McGraw-Hill; 1995. p. 1279-327.
El-Dawela RE, Abou-Elfetouh S. Relationship between homocysteine, Vitamin B12
, folic acid levels and vitiligo. J Appl Sci Res 2012;8:5528-35.
Silverberg JI, Silverberg NB. Serum homocysteine as a biomarker of vitiligo vulgaris severity: A pilot study. J Am Acad Dermatol 2011;64:445-7.
Karadag AS, Tutal E, Ertugrul DT, Akin KO, Bilgili SG. Serum holotranscobalamine, Vitamin B12
, folic acid and homocysteine levels in patients with vitiligo. Clin Exp Dermatol 2012;37:62-4.
Ambroszkiewicz J, Klemarczyk W, Chelchowska M, Gajewska J, Laskowska-Klita T. Serum homocysteine, folate, Vitamin B12
and total antioxidant status in vegetarian children. Adv Med Sci 2006;51:265-8.
Naurath HJ, Joosten E, Riezler R, Stabler SP, Allen RH, Lindenbaum J. Effects of vitamin B12
, folate, and Vitamin B 6 supplements in elderly people with normal serum vitamin concentrations. Lancet 1995;346:85-9.
Savage DG, Lindenbaum J, Stabler SP, Allen RH. Sensitivity of serum methylmalonic acid and total homocysteine determinations for diagnosing cobalamin and folate deficiencies. Am J Med 1994;96:239-46.
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]
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