Journal of Clinical & Experimental Dermatology Research
Open Access

Low Antioxidant Reserve in First Degree Relatives of Vitiligo Patients

Yogita Rajput^*, Diksha Mahilang, Akanksha Yadav, Kiran Dhruve, Mritunjay Kumar Singh, Vinod Koshley, Jagannath Pal and Pramod Kumar Nigam ^*Correspondence: Yogita Rajput, Department of Multi-Disciplinary Research, Pt. J.N.M Medical College, Raipur, Chhattisgarh, India, Email:

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Introduction

Vitiligo also known as leukoderma is an autoimmune, noncontagious disease in which progressive loss of pigment from skin, overlying hair and oral mucosa consequences from the loss of melanocytes. The aetiology of the disease is still not clear. Many factors have been implicated in the aetiology and pathogenesis of the vitiligo including infections, stress, neural abnormalities, anti-melanocyte antibody, defective melanocyte adhesion and genetic susceptibility. It is the most common pigmentary disorder affecting 0.1%-2% of the world’s population irrespective of race and gender and its incidence ranges from 0.1 to 8.8% in India although the states of Gujrat and Rajasthan have a highest dominance of 8.8%. It can develop at any age but approximately half of all vitiligo patients onset is before the age of 20 years. Though, vitiligo is usually notincapacitating the patients physically, but still it has serious implication in patient’s personal and social life. Vitiligo patients and their family are treated in different way in our community like low esteemed, causing serious psychological trauma and feeling of socially isolation to the patients [1].

Thus according to biochemical hypothesis, melanocyte destruction is due to the accumulation of toxic metabolites from melanogenesis, the breakdown of free radical defense and an excess of hydrogen peroxide. Recent study suggests that oxidative stress as the initial triggering factor in precipitating of vitiligo. Major Oxidative stress markers are Total Antioxidant Capacity (TAC), Superoxide Dismutase (SOD), Catalase (CAT), Glutathione Peroxidase (GPx), Nitric Oxide (NO) synthetase and Malondialdehyde (MDA). Nowadays, in Chattisgarh there is gradual increase in pollutant level because of industrialization, civilization, emission of motor vehicles etc. which are important determinant for triggering the different kinds of stresses including oxidative, immunological and psychological. It causes several adverse effects on health like so many skin problems including vitiligo in Chattisgarh. Besides this, various other geographical factors may also contribute to increase in above factors [2].

There is no study reported exploring antioxidant capacity (TAC) in vitiligo in Indian patients group, correlation with first degree family member. In this study we will evaluate the Total Antioxidants (TAC) reserve rather than individual oxidative stress markers, in progressive vitiligo compare with stable group, subsequently based on the antioxidant level correlation with the clinicopathological parameters, through this pilot study we could find out the vital precipitation factors within the shortest time period. This study may help to formulate personalize treatment strategy in vitiligo patients and also formulate the actual reason of gradual increase in vitiligo patients in Central India [3].

Materials and Methods

Study design

It was a cross sectional and case control study.

Ethical statement

This study was carried out at Department of Skin and Venereal Diseases (VD), B.R.A.M. Hospital and Multi-disciplinary Research Unit (MRU), Pt. Jawaharlal Nehru Memorial Medical College, Raipur, Chhattisgarh, India, following approval of the protocol by the institutional ethical committee, IEC (MC/ Ethics/56). All procedures were carried out in accordance with the Declaration of Helsinki. Human samples were collected following informed written consent from the study subjects [4].

Study participants

Subjects who attended the Department of Skin and Venereal Diseases (VD), B.R.A.M. Hospital, Pt. J.N.M. Medical College, Raipur, Chhattisgarh, India with white patch had clinical diagnosis of vitiligo and were willing to participate in the study,were referred to Multi-Disciplinary Research Unit (MRU), Pt. Jawaharlal Nehru Memorial Medical College, Raipur, Chhattisgarh, India for enrollment. Normal course of clinical management of the patients was not hampered by the study protocol. Having systemic acute and chronic disease, Undertaken systemic or topical treatment within 3 month, undertaken drugs for any other reason in last one month were excluded from the study. Patients who had active and static vitiligo and also they should be <40 years of age as well as no previous history of systemic, acute and chronic diseases were recruited for the study. Also enrolled the attenders who were first degree family member and age and sex matched healthy volunteer [5].

Sample collection and processing

Relevant history to be taken before collecting samples i.e., occupation, smoking, alcohol consumption, diet, migration (recent/past), chronic systemic diseases, drug history, recent or past infection.

Blood sample collection and storage: Venous blood samples collected from the vitiligo patients as well as paired control and independent healthy volunteers also. After collection of the whole blood, allowed the blood to clot by leaving it undisturbed at room temperature for 15-30 minutes. Centrifuging at 1,000-2,000 × g for 10 minutes in a refrigerated centrifuge and the resulting supernatants serum were kept at -20°C till further processing.

Measurement of Total Antioxidant Capacity (TAC) assay: Total Antioxidant Capacity (TAC) were assessed in stored serum samples by OxiSelectTM Total Antioxidant Capacity (TAC) assay kit (Cell Biolabs, Inc, San Diego, CA) as per the manufacturer’s protocol using Multimode Microplate Reader (TECAN, Mannedorf, Switzerland) [6].

Statistical analysis

In our study group mean and standard deviation were performed in excel sheet. Difference between the observed means in two independent samples and its significance value (P-value) as well as 95% Confidence Interval (CI) were calculated by using software https://www.medcalc.org/calc/comparison_of_means.php. Kolmogorov-Smirnov test of normality was analyzed using social science statistics https://www.socscistatistics.com/tutorials/ttest/ default.aspx. Statistical significance was considered if p ≤ 0.05. The data were further evaluated by one-way Analysis of Variance (ANOVA) accounting for different groups of vitiligo patients and their corresponding paired controls (1° family member) compared with independent healthy control group with post-hoc TukeyKramer comparisons [7].

Results

Demographic profile of vitiligo patients, paired control (1° family member) and independent healthy control

In the present pilot study total 59 vitiligo patients, their corresponding 59 paired control (1° family members) and 32 independent healthy control were recruited. Mean age of vitiligo patients, paired control and independent healthy control were 27.1 ± 18.4, 34.7 ± 11.4 and 27.8 ± 6.6 years respectively. Out of 59 vitiligo patients 18 (31%) were male and 41 (86%) female. Out of 59 patients 1° family members 25 (59%) were male and 24 (41%) female. Out of 34 independent healthy control 24 (71%) were male and 10 (29%) female (Table 1). Majority of vitiligo patients were below 20 years of age group (52.54%, n=31), detail age distribution of the vitligo patients are described in Table 2 [8].

Table 1: Demographic profile of vitiligo case (VP), 1° family member (VH) and Healthy Controls (HC).

Table 2: Age wise distribution of vitiligo patients.

Clinicopathological association with sociodemographic profile of vitiligo patients

The distribution of clinicopathological profile of patients group correlation with social and demographical status of participants were given in Table 3. Out of 59 patients, 33.89% had grouped in progressive vitiligo, followed by 30.20% of stationary vitiligo, 20.33% belongs to resistant vitiligo where as improved patients were only 13.55%. Maximum number of vitiligo patients were belongs to student category 55.93% (33/59) however 22.03% (13/59) participants were engaged in housebound work, 10.16% (6/59) were labour group, 8.47% recorded in office employee but only 3.38% (2/59) farmers were affected. We studied according to the food habit like vegetarian, non-vegetarian and eggetarian in different stage of vitiligo patients like progressive, stationary, improving and resistant, in which higher cases were undertaken the non-vegetarian category which includes fish, chicken and mutton 52.54% (31/59) where most of the participant were in progressive phase 16.95% whereas only 28.81% (17/59) patients had vegetarian dietary food habit and eggetarian patients population were 18.64% [9].

Table 3: Clinicopathological association with sociodemographic profile in vitiligo patients.

Total antioxidant reserve in vitiligo patient as well as their 1° degree family members compared with independent healthy control

Total antioxidant reserve was evaluated in vitiligo patients group and also their paired control (1° degree family member) compared with independent healthy control details in Table 4. The mean value as well as standard deviation of Total Antioxidant Capacity (TAC) in vitiligo patients was 0.371 ± 0.107 mM, whereas 0.379 ± 0.124 mM in 1° degree family member but TAC level was high 0.442 ± 0.114 mM in random population. Evaluation of the blood antioxidant capacity of the subjects in relation to random population revealed a significant difference according to student’s t-test (p=0.0034) among vitiligo patients (95% CI: 0.0241 to 0.1179) whereas correlation between 1° family member and random population also observed a significant difference (p=0.0171; 95% CI: -0.1145 to -0.0115), however there was no significance between patients group and 1° family member (paired control) p=0.7082 (Table 5).

Table 4: Total antioxidant reserve in vitiligo patient as well as their 1° degree family members compared with healthy control (mean ± SD, student’s t test, p value and 95% confidence interval).

Table 5: TAC profile of different clinicopathological sub-groups of vitiligo patients.

Evaluation of total TAC profile of different clinicopathological subgroups of vitiligo patients

In this study, the level of TAC in progressive vitiligo group (n=20) were found to be statistically significant as compared with the independent healthy control (P=0.0003) and also shows significance relationship with 1° degree family members with healthy volunteers (P=0.016). Similarly resistant 1o family group also shows significant relation with the healthy control. No significant difference was found between stationary, improving and resistant vitiligo patients with healthy control as well as 1° family members details in Tables 6 and 7.

Comparison of TAC profile between stable and severe viligo patients and compared with independent healthy control

On the basis of severity of vitiligo, subject group is divided into two groups i.e., stable and severe. Stable group belongs to improving as well as stationary group where as severe group includes progressive with resistant. One-way ANOVA analysis revealed that in our study groups severe as well as stable vitiligo patients as well as their corresponding paired control (1° degree family members) are showing significant correlation with independent healthy controls. The f-ratio value is 7.11216 and pvalue is 0.000029. The result is significant at p ≤ 0.05 (Table 6). p-value of distinct comparison of stable vs. severe vitiligo and their relative control (Table 7). VH-stable vs. VP-severe, VP-severe vs. HC and VH-severe vs. HC shows significant association. Supplementary Tables 1 and 2 predict the pair wise comparisons of stable vs. severe vitiligo and their paired control (1° family member) compared with healthy control within one way ANOVA data.

Table 6: TAC profile of stable vs. severe vitiligo patients.

Table 7: p-value of distinct comparison of stable vs. severe vitiligo and their relative control.

Familial aggregation of TAC level with vitiligo patients

Out of 59 paired samples 46 were of 1° relatives (mother: 15, father: 10, son: 6, daughter: 4, sister: 3 and brother: 8) shows non significance correlation (P=0.595) with vitiligo patients and 1° grandfather and rest were non blood relatives (brother in-law, husband, wife) detailed profile in Table 8. Out of 9 blood relatives of VP patients husband: 4, wife 2 and 3 other relative (like brother-in-law, sister-in-law etc.) shows no significance (P=0.644). When we correlated with the antioxidant level of cumulative familial aggregation with patients, it’s again reflects the non-significant association (P=0.708). However we observed highly significant correlation in Vitiligo Patient (VP) vs. independant Healthy Control (HC) and Vitiligo Paired control (VP) vs. Healthy Control (HC) were P=0.0034 and P=0.0171 respectively. As per our patients history, only one patient indicates the familial predisposition rest all the patients having no family history of vitiligo.

Table 8: Familial aggregation of TAC level with vitiligo patients.

Discussion

Vitiligo is the acquired pigmentary autoimmune disorder of the skin. We conducted a pilot study in vitiligo patient group and compared with the paired control (First degree family members) as well as independent healthy control.

In the present study blood samples of total 59 vitiligo patients, 59 paired control and of 34 independent healthy volunteers as independent control were collected. Average age of the vitiligo patients was observed 27.136 ± 18.453 years. Out of this, mean age group of male was 31.22 ± 18.65 years whereas 25.341 ± 18.305 years in female. Our finding was supported by Singh et al. 13 reported that the total average of the vitiligo patients was 27.35 ± 10.45 years in which mean age of male group was 30.75 ± 15.41 years whereas in female 26.07 ± 14.63 in Punjabian population of India. In the present study group maximum onset of symptom of vitiligo were below age of twenty. Research suggests that the onset of vitiligo symptoms typically occurs before the age of 20 in about 50% of cases. The incidence of vitiligo was higher in female patients group 69.69% (41/59) as compare to male group 30.50% (18/59). This finding is supported with the previous study with a predilection for women being affected two times more often than men where in few reports both genders almost with equal frequency of vitiligo.

Maximum number of vilitgo patients were belongs to student age group 55.93%, followed by house bound occupations 22.03%, labour 10.16%, Office worker 8.47% where as farmers were 3.38%. The occupational profile of vitiligo cases was similar to earlier published studies. Similar finding was observed in the study conducted in South Korea in which the most frequent occupation was house wife (33.0%), followed by office job (30.4%), production (7.4%), student (6.8%). Food habit and lifestyle were important predisposition factors for triggering the oxidative stress. Previous reports suggested that there is a correlation between junk and food with vitiligo. In our study group most of the patients follows the non-vegetarian food habit, same hypothesis establisheded that the incompatible diet (Viruddha ahara) is a potent cause of several diseases especially in present era when faulty dietary practice is in vague. Interestingly we reveal out that most of the patients who belong to progressive vitiligo clinical type having a non-vegetarian dietary habit.

Oxidative stress is one of the key factors in multifactorial pathogenesis of vitiligo. Oxidative stress occurs when there's an imbalance between the production of free radicals (unstable molecules) and the body's ability to neutralize them. In our study population Total Antioxidant Reserve (TAC) in vitliligo petients as well as their 1° family members was lower than independent healthy control. When we segregated the vitiligo patients group into progressive, stationary, improving and resistant, we observed that TAC level in progressive vitiligo as well as corresponding family members was significantly decreased compared with the independent healthy control, suggesting importance of antioxidant reserve for preventing progression of vitiligo (VP vs. HC; P=0.0003 and VH vs. HC; P=0.0022). Carrying out the one-way ANOVA in stable, severe vitiligo group with independent healthy control indicates highly significant association (P=0.000029). Interestingly when we combined the all the spectram of clinical presentation of vitiligo into two group stable (improving and stationary group) and severe (progressive and resistant groups), we observed lower TAC in both severe vitligo as well as 1° family members compared to unrelated HC (VP severe vs. HC ; P=0.0005 and VH severe vs. HC; 0.0003). Previous studies established the fact that oxidative stress plays vital role in the pathogenesis of vitiligo. Interestingly our study suggest that the prevalence of oxidative stress is high not only in severe form of vitiligo patients but also in their family members indicating familial predisposition of oxidative stress in this patient’s group as disease modulating factor. Our findings also supported the previous research work that an enhanced oxidative stress status in the blood of the Egyptian vitiligo population as evidenced by a significant reduction in TAC along with a significant increase in plasma MDA levels of patients compared to controls however similar findings alsoobserved in Indian population. To our knowledge, this is the first report of the anti-oxidant status of the family members of vitiligo patients. At a standstill, bigger sample size are required to establish the correlation. The association of low antioxidant reserve in severe vitiligo along with their family members could be due to common food habit, lifestyle or genetically determined.

Previously it was reported that vitiligo have genetic predisposition. However, in our disease spectrum of vitiligo patients, we could not show any genetic correlation between vitiligo patients and 1° family member. Only one patient had a genetic history, whereas in the rest of the vitiligo group, no family history was reported. There is conflicting evidence of genetic predisposition of vitiligo while some Egyptian study shows genetically non-significant association of vitiligo while the American study observed some significant association (Figures 1 and 2).

Figure 1: Vitiligo patients: A) patches of resistant condition in vitiligo; b) Patches of improving stage in vitiligo; c) Patches of stationary stage in vitiligo; d) Patches of progressive stage in vitiligo.

Figure 2: A linear regression curve of Total Antioxidant Capacity (TAC) assay with a correlation coefficient of 0.999 and regression equation of y=0.790x+0.0.

Limitations

Small sample size; assessments of oxidative stress as well as antioxidant reserves marker substantiate with large sample size in vitiligo patients and association with 1° and 2° degree family members.

Conclusion

Our study suggest that the prevalence of oxidative stress is high not only in severe form of vitiligo patients but also in their family members indicating familial predisposition of oxidative stress in this patient’s group as disease modulating factor. Accordingly oxidative stress should be monitor and treated in vitiligo patients as well as their family members as preventive measure to reduce severe form of vitiligo and many chronic diseases.

References

1. Sehgal VN, Srivastava G. Vitiligo: Compendium of clinico-epidemiological features. Indian J Dermatol Venereol Leprol. 2007;73:149.

   [Crossref] [Google Scholar] [PubMed]

2. Taieb A, Picardo M, Other VETF members. The definition and assessment of vitiligo: A consensus report of the Vitiligo European Task Force. Pigment Cell Res. 2007;20(1):27-35.

   [Crossref] [Google Scholar] [PubMed]

3. Gopal KV, Rao GR, Kumar YH, Rao MA, Vasudev P. Vitiligo: A part of a systemic autoimmune process. Indian J Dermatol Venereol Leprol. 2007;73:162.

   [Crossref] [Google Scholar] [PubMed]

4. Koca R, Armutcu F, Altinyazar HC, Gurel A. Oxidant‐antioxidant enzymes and lipid peroxidation in generalized vitiligo. Clin Exp Dermatol. 2004;29(4):406-409.

   [Crossref] [Google Scholar] [PubMed]

5. Shajil EM, Agrawal D, Vagadia K, Marfatia YS, Begum R. Vitiligo: Clinical profiles in Vadodara, Gujarat. Indian J Dermatol. 2006;51(2):100-104.

   [Google Scholar]

6. Dell'Anna ML, Picardo M. A review and a new hypothesis for non‐immunological pathogenetic mechanisms in vitiligo. Pigment Cell Res. 2006;19(5):406-411.

   [Crossref] [Google Scholar] [PubMed]

7. Schallreuter KU, Moore J, Wood JM, Beazley WD, Peters EM, Marles LK, et al. Epidermal H2O2 accumulation alters tetrahydrobiopterin (6BH4) recycling in vitiligo: Identification of a general mechanism in regulation of all 6BH4-dependent processes?. J Invest Dermatol. 2001;116(1):167-174.

   [Crossref] [Google Scholar] [PubMed]

8. Lodovici M, Bigagli E. Oxidative stress and air pollution exposure. J Toxicol. 2011;2011(1):487074.

   [Crossref] [Google Scholar] [PubMed]

9. Li N, Hao M, Phalen RF, Hinds WC, Nel AE. Particulate air pollutants and asthma: A paradigm for the role of oxidative stress in PM-induced adverse health effects. Clin Immunol. 2003;109(3):250-265.

   [Crossref] [Google Scholar] [PubMed]