Serum Homocysteine and Total Antioxidant Status in Vitiligo: A Case Control Study in Indian Population.

BACKGROUND: Oxidative stress is considered as an initial pathogenic event in melanocyte destruction. These free radicals are scavenged by antioxidants, whose sum of activity in serum is measured by total antioxidant status (TAS). In addition, homocysteine (Hcy) may mediate melanocyte destruction via increased oxidative damage. However, previous studies investigating these parameters in vitiligo provide equivocal results. AIMS: To study and compare serum Hcy and TAS levels in vitiligo patients with controls and also to correlate these parameters with the various disease characteristics. The present study further looked into any correlation between serum Hcy and TAS in vitiligo.
MATERIALS AND METHODS: A case control study was conducted on 82 vitiligo patients and 83 controls aged 18-45 years after excluding factors which could potentially alter serum Hcy or TAS levels. Disease characteristics were studied and blood samples were obtained for measuring serum Hcy and TAS levels.
RESULTS: TAS levels were lower in vitiligo patients than controls (1.79 ± 0.51 vs. 2.16 ± 0.63 mmol/L; P < 0.001) and had a negative correlation with disease activity (r = -0.410, P < 0.001). However, serum Hcy levels were comparable between vitiligo patients (18.68 ± 9.90 μmol/L) and controls (20.21 ± 13.39 μmol/L) (P = 0.406). No significant correlation was found between serum Hcy and serum TAS levels.
CONCLUSIONS: Serum TAS may be further investigated to establish its role as biomarker for vitiligo since its levels also correlate with disease activity. However, serum Hcy may not be a reliable marker in Indian population probably because of differences in dietary habits.

What was known?

An imbalance in the oxidant–antioxidant mechanism plays an important role in the pathogenesis of vitiligo.

Introduction

Vitiligo is a multifactorial polygenic disorder with a complex pathogenesis. Various theories have been postulated to explain the mechanisms of vitiligo including autoimmune, cytotoxic, biochemical, oxidant–antioxidant, neural, and viral mechanisms for destruction of epidermal melanocytes but they are not fully understood.[1] Several studies validate a possible oxidant stress theory, which suggests that accumulation of free radicals toxic to melanocytes leads to their destruction.[1]

The antioxidant status of serum is well reflected by its antioxidant capacity, which represents a combination of activity of all its antioxidants.[2] However, there are conflicting reports regarding the role of total antioxidant status (TAS) in vitiligo.[345]

It is also postulated that homocysteine (Hcy) may mediate melanocyte destruction via increased oxidative damage, interleukin-6 production and nuclear factor κB activation.[6]

Thus we aimed to study and compare serum Hcy and TAS levels in vitiligo patients with controls besides correlating these parameters with various disease characteristics.

Materials and Methods

This case control study was carried out in the Department of Dermatology in association with the Department of Biochemistry, ESI-PGIMSR, Basaidarapur, New Delhi, India, after the approval of the Institutional Ethics Committee. All clinically diagnosed patients of vitiligo in the age group 18–45 years attending the Outpatient Department were included in the study from November 2011 to August 2012 after obtaining an informed consent. Dietary habits were recorded as vegetarians, eggetarians, and nonvegetarians.

The exclusion criteria were alcohol intake during past 3 months; cigarette smoking; intake of folic acid, vitamins B6 and B12 during past 2 months; intake of antioxidant supplements during past 3 months; intake of drugs known to alter Hcy levels (methotrexate, isoniazid, isotretinoin, cyclosporine, penicillamine, phenytoin, carbamazepine, oral contraceptive pills, hydralazine, L-dopa, procarbazine, acetylcysteine, cholestyramine, and cycloserine). We also excluded pregnant women and those in 1-week postpartum period; known cases of psoriasis, Behcet's disease, systemic lupus erythematosus, cardiovascular disease, chronic kidney disease, diabetes mellitus, hypertension, thyroid disorder, and chronic liver disease, which are associated with deranged serum Hcy levels. In addition, patients with any hematological or biochemical abnormality observed during routine laboratory investigations were also excluded. Of 132 vitiligo patients, 82 were found suitable for the study based on the above-mentioned criteria and were recruited. Similarly, 83 age- and gender-matched patients with dermatoses other than vitiligo were included in the control group.

Detailed history regarding the disease was taken and the activity of the disease was assessed as stable, progressive, or regressive based on the activity of the disease over past 6 months. Vitiligo was clinically defined as localized (focal, segmental, or mucosal), generalized (vitiligo vulgaris, acrofacial vitiligo), or universal.

After a 1 month treatment-free period, 7 mL of fasting blood sample was drawn from patients in both groups for routine hematological and biochemical tests and to measure serum free thyroxine and thyroid-stimulating hormone (TSH), serum Hcy, and serum TAS. Serum was obtained after centrifugation at 3000 g. For Hcy and TAS estimation, 1 mL serum was divided into two aliquots which were immediately stored in screw-capped storage vials at − 20°C until the time of analysis.

TAS levels were determined by the enzymatic colorimetric method based on 2, 2’-azino-di-(3-ethylbenzthiazoline sulfonate]) (TAS-liquid stable kit; Fortress Diagnostics Limited, UK). The results were expressed in mmol/L of trolox equivalent. As per the method, the chromogen in the kit reacts with H2O2 to produce a radical cation which has a relatively stable blue-green color, which is measured at 600 nm. Antioxidants present in the added serum cause suppression of this color production proportional to their concentration.

Serum Hcy was estimated using the Diazyme Hcy enzymatic assay kit (Diazyme Laboratories, Poway, USA) and levels were expressed in μmol/L. In this assay, oxidized Hcy is first reduced to free Hcy which then reacts with a cosubstrate, S-adenosylmethionine in a series of reactions to convert NADH to NAD+. The concentration of Hcy in the sample is indirectly proportional to the amount of NADH converted to NAD+, which is measured at 340 nm.

Statistical evaluation

Statistical evaluation was carried out using SPSS version 20.0 (IBM Corp., NY). The results were expressed as mean ± standard error of mean. Distribution of the continuous variables was determined by the Kolmogorov–Smirnov test. Chi-square test was used to compare differences between the frequencies. To compare the mean values between two groups, unpaired Student's t-test and Mann–Whitney U-test were applied on parametric and nonparametric data, respectively. Spearman's correlation was used to find any significant correlation between clinical as well as biochemical profiles of the cases and the controls. P values < 0.05 were considered statistically significant.

Results

Age and sex profiles of vitiligo patients and controls were comparable (P > 0.05) [Table 1]. The relevant clinical data are summarized in Table 2.

Table 1

Age and sex profiles of cases and controls

Table 2

Clinical data of vitiligo patients

Eighteen vitiligo patients had a history of taking treatment for their disease in the past. Out of these 18 patients, 10 patients had received only topical treatment (topical tacrolimus 0.1% ointment, fluocinolone 0.1% cream), 4 patients had received some systemic treatment-nature of which was not known, 3 patients had received topical PUVAsol, and 1 patient had received narrowband ultraviolet B.

Serum TAS levels ranged from 1.27 to 2.99 mmol/L (mean 1.79 ± 0.51) in vitiligo patients and 1.13–3.03 mmol/L in controls (mean 2.16 ± 0.63). The difference among cases and controls was statistically significant (P < 0.001) [Table 3 and Figure 1a]. The mean serum TAS levels among male and female vitiligo patients were 1.804 ± 0.495 mmol/L and 1.790 ± 0.562 mmol/L, respectively (P = 0.907). Serum TAS levels correlated negatively with disease activity (r = −0.410, P < 0.001) and were found highest in patients with regressive disease (mean 2.08 ± 0.12 mmol/L) and lowest in those with progressive disease (mean 1.60 ± 0.05 mmol/L) [Figure 2a]. Linear logistic regression analysis showed that reduced serum TAS levels was a significant risk factor for vitiligo (<1.30, odds ratio = 2.90, 95% confidence interval 1.55–5.43, P = 0.001). However, there was no significant correlation of serum TAS levels with the disease type (r = −0.167, P = 0.133), duration (r = 0.024, P = 0.832), number of lesions (r = −0.023, P = 0.835), or number of sites involved (r = 0.053, P = 0.639).

Table 3

Specific biochemical parameters in vitiligo patients and controls

Figure 1

(a) Boxplot illustrating serum total antioxidant status levels in cases and controls. Median serum total antioxidant status levels in both groups are indicated (P value for mean total antioxidant status levels in both groups <0.001). (b) Boxplot illustrating serum homocysteine levels in cases and controls. Median serum homocysteine levels in both groups are indicated (P value for mean homocysteine levels in both groups = 0.406)

Figure 2

(a) Bar chart illustrating relationship between mean serum total antioxidant status levels and disease activity. (b) Bar chart illustrating relationship between mean serum homocysteine levels and disease activity

Serum Hcy levels ranged from 6.1 to 54.6 μmol/L in vitiligo patients (mean 18.68 ± 9.90) and 6.3–74.1 μmol/L (mean 20.21 ± 13.39) in the control group [Figure 1b]. However, the difference in cases and controls was not statistically significant (P = 0.406) [Table 3]. Furthermore, the number of patients with elevated serum Hcy levels was comparable in both the groups (P = 0.932). Linear logistic regression analysis revealed that elevated serum Hcy level was not a significant risk factor for the presence of vitiligo (P = 0.932).

Serum Hcy levels showed no significant correlation with the disease activity (r = 0.094, P = 0.400) [Figure 2b], age (r = −0.096, P = 0.393), disease type (r = 0.130, P = 0.245), number of lesions (r = −0.073, P = 0.517), number of sites involved (r = −0.061, P = 0.586), or duration of disease (r = 0.041, P = 0.714). The Hcy levels among male and female vitiligo patients were 19.8 ± 1.5 µmol/L and 16.9 ± 1.5 µmol/L, respectively (P = 0.174). No patient in either group had severe hyperhomocysteinemia (levels > 100 μmol/L).

We observed a positive correlation between serum TAS and serum total protein levels in cases (r = 0.410, P < 0.001) as well as in controls (r = 0.358, P = 0.001). TAS levels also correlated with serum bilirubin in vitiligo patients (r = 0.288, P = 0.009). On the other hand, serum Hcy levels had a positive correlation with serum uric acid in cases (r = 0.255, P = 0.021) as well as in controls (r = 0.076, P = 0.496). Serum TSH levels were shown to correlate with Hcy levels in cases (r = 0.224, P = 0.043) but not in controls (r = 0.023, P = 0.835), however they did not correlate with TAS levels in cases (r = −0.173, P = 0.120) or controls (r = −0.033, P = 0.765).

There was no statistically significant correlation between serum TAS and serum Hcy levels in vitiligo patients (r = −0.180, P = 0.105) or controls (r = 0.103, P = 0.354).

Any past treatment taken for vitiligo did not affect serum Hcy (P = 0.953) or TAS (P = 0.945) levels in this study. There was no significant correlation of dietary habits with serum Hcy (r = −0.049, P = 0.662) or TAS levels (r = 0.158, P = 0.157) in vitiligo patients.

Discussion

The pathogenesis of vitiligo is under debate and has been attributed mainly to autoimmune causes, oxidative stress, and/or sympathetic neurogenic disturbance.[7] However, none of them can fully explain the disease.[8]

Oxidative stress is thought to be the initial pathogenic event in melanocyte destruction[9] and H2O2 accumulation has been observed in the epidermis of active vitiligo lesions.[10] Some studies have also shown that melanogenesis produces significant levels of reactive oxygen species (ROS). This along with other radicals can induce oxidative stress.[11] In addition, an alteration in the antioxidant system, with a significant reduction in catalase activity has been noted in both lesional and nonlesional epidermis of vitiligo patients,[12] in serum,[13] in erythrocytes[14] as well as in melanocytes derived from these patients.[9]

In normal conditions, superoxide dismutase, an antioxidant enzyme, catalyzes the dismutation of superoxide ion (O2•−) into O2 and H2O2 and catalase converts H2O2 to O2 and H2O.[15] In oxidative stress, there is insufficient antioxidant activity characterized by decreased catalase levels[16] leading to excessive accumulation of free radicals, which damage cellular compounds such as protein, carbohydrate, DNA, and lipid.[17]

Therefore, to counteract or scavenge increased levels of O2•−, superoxide dismutase, an antioxidant enzyme, is increased.[16] This finding has been confirmed in a study conducted by Jain et al.[18] in which superoxide dismutase activity in the whole blood sample of vitiligo patients was found to be increased. Hydrogen peroxide, thus produced from O2•−, can readily cross cell membranes, causing much of the damage.[19] Thus, insufficient antioxidant protection or excess production of ROS causes oxidative damage.[4] Furthermore, decrease in the antioxidant enzyme glutathione peroxidase levels has been observed in the sera of vitiligo patients.[20]

There are various mechanisms by which increased oxidative stress leads to melanocyte damage. First, altered redox status has been associated with compromised assembly of the tyrosinase-related protein-1 (TRP-1)/calnexin complex leading to reduced TRP-1 stability with subsequent production of melanin toxic intermediates.[21] Second, some researchers believe that loss of melanocytes in vitiligo is the result of apoptosis.[22] Oxidative stress, which can induce apoptosis by releasing caspase activating cytochrome C from mitochondria,[23] may induce or contribute to apoptosis of melanocytes in vitiligo lesions.

However, while individual antioxidant levels have been extensively studied in vitiligo, only a few studies exist in the literature investigating the status of total antioxidant capacity of serum/plasma (TAS) in vitiligo patients and they offer equivocal results. Similarly, association of serum Hcy with vitiligo has been studied worldwide with ambiguous findings. Hence, this study was carried out in an attempt to further study the role of oxidant–antioxidant mechanism in the pathogenesis of vitiligo and to correlate the same with the disease activity.

We observed that serum TAS levels were significantly lower among vitiligo patients and also were inversely related to the activity of the disease. Similar findings have been observed by researchers in different parts of the world.[3424] However, an Indian study reported that individual plasma antioxidant (superoxide dismutase, glutathione reductase, and malondialdehyde) levels, but not total antioxidative potential levels were lower among vitiligo subjects.[25] Interestingly, a study conducted on Fitzpatrick skin type VI population (11 cases, 11 controls) found increased TAS levels in vitiligo patients.[5] This may have probably been due to a phototype-related increase of antioxidant enzyme activities (catalase, superoxide dismutase, and glutathione peroxidase) in vitiligo skin.[26]

In this study, mean serum Hcy levels were found higher than reference standards in both cases and controls. This may be justified partly on the basis of distinct Indian dietary habits and also by genetic polymorphisms. It has been observed that most Indians adhere to a vegetarian diet and the nonvegetarian ones consume a lesser amount of animal-derived protein than their Western counterparts.[27] Refsum[28] reported that cobalamin was found deficient in Indian diet which itself may lead to increased Hcy.[29] Furthermore, serum Hcy levels have been reported to be higher among South Asians[30] and Indians in particular[3132] due to polymorphisms involving methylenetetrahydrofolate reductase (MTHFR) gene.[33]

In the absence of a significant alteration in the levels coupled with a lack of correlation with any of the disease characteristics, this study could not substantiate the role of Hcy in vitiligo. This view is supported by some similar case control studies performed on vitiligo patients,[3435] but not by others.[6293637]

Serum TAS levels positively correlated with serum total protein as well as serum bilirubin levels in both the groups. This is expected since both proteins and bilirubin present in the serum contribute to its antioxidant capacity.[3839] We also observed a correlation between Hcy and uric acid levels in vitiligo patients which may be because of a shared metabolic pathway.[40] Serum Hcy levels were also noted to correlate with TSH levels in vitiligo patients. There are reports in the literature suggesting the same[4142] and the likely reason for this observation is that thyroid hormone deficiency, besides increasing TSH levels, also decreases the levels of enzymes involved in the remethylation pathway of Hcy to methionine and MTHFR.[41]

Few previous studies have failed to establish any correlation between serum Hcy and TAS in diabetics[43] and in healthy individuals.[44] This finding has been reaffirmed in the present study. This is the first study to the best of our knowledge that looks for such correlation in vitiligo patients.

To summarize, TAS levels were found lower in vitiligo patients as compared to controls. This implies that either an increased oxidative stress in vitiligo leads to exhaustion of TAS reserves, or that vitiligo patients have inherently inadequate TAS levels which are unable to effectively neutralize the melanocyte damaging oxidants. Thus, the main limitation with this study is that determining oxidant stress levels in addition could have provided more information about the disturbances in oxidant–antioxidant mechanism in vitiligo. Therefore, further studies investigating the levels of both total oxidant as well as TAS are required. Although the present study could not substantiate the role of increased Hcy levels in vitiligo, one cannot rule out the possibility that this group of patients have a genetic predisposition to develop the disease in the presence of even mild elevations of serum Hcy levels.

Thus we conclude that serum TAS may be further investigated in intervention studies to establish its role as one of the biomarkers for vitiligo because its levels besides being altered also correlate well with disease activity. On the other hand, while Hcy may serve as a disease marker in other parts of the world, it may not be a reliable marker in the Indian population probably because of differences in dietary habits. However, further studies are required to confirm these findings.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

What is new?

Since there is a need for minimally invasive modalities for monitoring/prognostication of vitiligo, serum total antioxidant status levels may be in future used as a biomarker

No correlation was observed between serum homocysteine levels and disease characteristics in vitiligo patients in this study.