Can systemically generated reactive oxygen species help to monitor disease activity in generalized vitiligo? A pilot study.

BACKGROUND: Generalized vitiligo is a disease with unpredictable bursts of activity, goal of treatment during the active phase being to stabilize the lesions. This emphasizes the need for a prospective marker for monitoring disease activity to help decide the duration of therapy. AIMS AND
OBJECTIVES: In the present study, we examined whether reactive oxygen species (ROS) generated in erythrocytes can be translated into a marker of activity in vitiligo.
MATERIALS AND METHODS: Level of intracellular ROS was measured flow cytometrically in erythrocytes from venous blood of 21 patients with generalized vitiligo and 21 healthy volunteers using the probe dichlorodihydrofluorescein diacetate.
RESULTS: The levels of ROS differed significantly between patients and healthy controls, as well as between active versus stable disease groups. In the active disease group, ROS levels were significantly lower in those being treated with systemic steroids than those that were not. ROS levels poorly correlated with disease duration or body surface area involved.
CONCLUSION: A long-term study based on these findings can be conducted to further validate the potential role of ROS in monitoring disease activity vitiligo.

What was known?

There is documented role of redox imbalance towards local and systemic oxidative stress in the pathogenesis of generalized vitiligo. However, there is a dearth of reports on changes in markers of oxidative stress under therapy.

Introduction

Generalized vitiligo is an acquired skin disorder characterized by loss of pigmentation that results in hypomelanotic macules. Generally, the disease has an unpredictable course where due to unknown triggers, burst of activity ensues with progression of lesions.[1] An active state of vitiligo has been defined as the extension of existing lesion and/or appearance of new lesion within the last 1 year.[2] Reversal to original skin color is difficult and the arrest of spread of the lesion is the clinical goal in the active phase.[1] In this scenario, a prospective marker to assess disease stability becomes important to determine the duration of therapy.

Although exact inciting factors for triggering depigmentation in vitiligo have not been identified, redox imbalance has been implicated in several reports.[345] Some studies have also shown relatively greater levels of markers of oxidative stress in patients with active disease when compared with stable disease – at the tissue level[6] and in monocytes.[7] Changes in antioxidant enzyme levels[8] and glutathione levels[9] in erythrocytes have been reported in patients with vitiligo when compared with controls. However, there is a dearth of reports objectively measuring the effect of therapy on markers of oxidative stress in vitiligo. In this context, we hypothesized that if reactive oxygen species (ROS) generated in red blood cells (RBCs) varied with disease activity, it could translate into a useful tool to monitor activity during treatment. We conducted a pilot study with the objective of determining whether disease activity in generalized vitiligo correlated with the level of ROS in RBCs and how response to treatment modified the same. We also wanted to check the correlation between level of ROS in RBC with disease duration and the involved body surface area (BSA).

Materials and Methods

Patients

We conducted a cross sectional analytical study in patients of generalized vitiligo that came to the dermatology out-patient department of Institute of Postgraduate Medical Education and Research, Kolkata and healthy volunteers. The study was approved by Institutional Ethics Committee. Patients were included regardless of the background treatment they received for vitiligo. However, patients and volunteers on current systemic antioxidant therapy were excluded. Segmental vitiligo patients were excluded. Patients giving the history of or presenting with signs and symptoms suggestive of acute viral, bacterial or parasitic infections within the last 2 weeks were excluded, as ROS levels may increase in these conditions due to activation of the innate immunity. Patients with diabetes mellitus (Types I and II) and autoimmune thyroiditis were excluded based on their medical records and investigations as applicable, as these conditions are known to have high ROS levels. Patients with other autoimmune co morbidities were also excluded based on medical history and records.

After obtaining written informed consent, the dermatologist recorded the history, performed a physical examination and determined vitiligo disease activity (VIDA) score of each patient.[2] Duration of the disease was estimated from history and review of medical records. Percentage of BSA involved was measured by taking the patient's palm as 1% of total BSA.

Patients were sub classified into those with active disease (i.e. appearance of new lesions or progression of old lesions within last 1 year) and inactive disease (i.e. no new lesions or progress of older lesions within the last year). Those with active disease were further sub grouped into those who were receiving systemic steroids and those who were either treatment naïve or not on systemic steroids within the last 3 months. In cases where any background treatment was being received, treatment response was noted in terms of progress of lesion in the last month. Those who reported lack of progress in the last month were identified as responders. Heparinized venous blood (1 ml) was then sampled from median cubital vein of the patients.

Measurement of ROS in erythrocytes

Erythrocytes (1 × 106 cells/ml) were loaded with dichlorodihydrofluorescein diacetate (H2 DCFDA, 50 μM) and incubated for 30 minutes at 37°C.[1011] Cells were then washed twice with phosphate buffered saline (0.02 M, pH 7.2). The intracellular fluorescence of RBCs was checked using a flow cytometer (FACS Calibur, Becton Dickenson, San Jose, CA, USA). Fluorescence was measured in the log mode using CellQuest Pro software (BD Biosciences, San Jose, CA, USA) and expressed as geometrical mean fluorescence channel (GMFC). Initially, the RBC population was identified in the flow cytometer using phycoerythrin (PE) labeled anti CD235a and appropriate isotype controls. Eventually, all experiments were performed by applying the same gates. Acquisitions were performed on 10,000 gated events while data analysis was carried out using CellQuest Pro software. All chemicals were procured from Sigma Aldrich, St. Louis, MO, USA, except for PE tagged anti CD235a antibody, which was obtained from BD Biosciences, San Jose, CA, USA.

Statistical analysis

Statistical analyses were performed on GraphPad Prism Version 5.0 (GraphPad Software Inc., San Diego, CA, USA, 2007) and SPSS Version 16.0.1 (SPSS Inc., Chicago, IL, USA, 2007) and P < 0.05 was considered to be significant. Kolmogorov-Smirnov test was used to test the normality of the data (while GMFC was found to be parametric, disease duration and BSA was non-parametric). Therefore, the descriptive results for GMFC have been expressed as mean ± standard deviation while that for the duration of the disease and BSA, median and interquartile range (IQR) have been used. Comparisons between groups were performed by unpaired Student's t-test and Fischer's exact test. Correlations between GMFC and duration and GMFC and BSA were assessed using Spearman's correlation coefficient.

Results

A total of 21 patients with uncomplicated generalized vitiligo were recruited along with 21 age and sex matched healthy controls. Both patients and controls had skin phototypes IV-V (Fitzpatrick classification). Differences in age, sex and skin phototypes were not statistically significant [Table 1]. Median (IQR) duration of disease in patients was 3 years (1.25-11 years) while median (IQR) BSA involved was 3.5% (1.5-7.5%).

Table 1

Demographic, disease and treatment profiles of the study population

Twelve of the patients had active disease, while nine had inactive disease. In the active disease group, six were receiving systemic steroids while three had not received the same in last 3 months. Three in the active disease group were treatment naïve. Those with active disease had VIDA scores +3 and +4 [Figure 1].

Figure 1

Distribution of study subjects in various groups

The GMFC of ROS in RBC from healthy controls (29.71 ± 9.247) differed significantly from patients with vitiligo (55 ± 24.85, P < 0.001). Differences remained significant (P = 0.013) when those with stable disease (42.59 ± 17.73) are compared with healthy individuals. Importantly, the GMFC of those with active disease (64.30 ± 25.97) and those stable differed significantly (P = 0.044). Amongst those with active disease, the difference in GMFC of those on systemic steroids (48.31 ± 12.01) and those not (80.29 ± 26.93) was significant (P = 0.024) [Figure 2]. The responders (n = 6) showed a numerically lower mean GMFC (51.02 ± 10.27) than non-responders (n = 3, 77.92 ± 37.03) [Figure 3]. Again, there was a significant difference (P = 0.014) in GMFC between those with stable disease and the treatment naïve and non-responders taken together (77.58 ± 30.89).

Figure 2

Status of reactive oxygen species in erythrocytes from healthy and diseased individuals. (a) A representative profile of the characteristic side and forward scatter of the erythrocyte population gated R1. (b) Histogram showing geometrical mean fluorescence channel (GMFC) of DCF=Dichlorofluorescein in the gated cells from healthy individuals and patients with vitiligo. (c) Mean GMFC of DCF in red blood cells from healthy individuals (bar 1, n = 21), those with inactive disease (bar 2, n = 9) and those with active disease (bar 3, n = 12). *Denotes P < 0.05. Error bars represent standard deviations

Figure 3

Status of reactive oxygen species in erythrocytes in patients with active vitiligo. Mean geometrical mean fluorescence channel of the patients with active disease who are treatment naïve (bar 1, n = 3), non-responders (bar 2, n = 3) and responders (bar 3, n = 6). Error bars represent standard deviations

There was a poor correlation between disease duration and GMFC with Spearman's r = −0.371 (P = 0.097). Again, a poor correlation was found between involved BSA and GMFC with Spearman's r = 0.027 (P = 0.905).

Discussion

According to current treatment guidelines for active vitiligo, systemic steroids are prescribed initially in order to arrest the progression of the lesions and stabilize the disease.[12] In our setting, most of patients with active disease are treated with oral minipulse therapy in the form of 4-5 mg betamethasone tablets twice weekly to start with. After patient reports, the non-progression of disease for some time, stability is assumed, steroid is tapered over 4-5 months and other treatment modalities, including photochemotherapy, are ensued.[121314] While inactivity is defined as lack of progress for at least a year, there is no general consensus as to when to consider the disease to have been stabilized on a short term and when to switch from systemic to local therapy. An objective marker to determine stability will thus be useful.

Reports suggest that although a redox imbalance is apparent in both active and stable vitiligo, it is more prominent in the active phase.[678] Ines et al.[8] and Hazneci et al.[15] have reported levels of various antioxidant enzymes including superoxide dismutase and glutathione peroxidase in patients with active vitiligo differ significantly when compared with controls. Our result suggests that net oxidative stress in RBCs as measured by H2DCFDA may be effective in distinguishing those with active disease from those currently in a stable phase. Again, this parameter varied with therapeutic response. The responders to immunosuppressive therapy amongst those in active disease group had ROS levels closer to levels in stable disease. In contrast, the mean GMFC of the non-responders and treatment naive patients was significantly greater than those with stable disease.

Systemically generated oxidative stress has been positively correlated with duration of various chronic diseases with redox imbalance as etiopathogenic factor.[1617] In this context, another important finding is that ROS level does not correlate well with either disease duration or BSA involved in generalized vitiligo, thus making its case as an activity marker stronger by being less affected by other disease variables. Again, ROS levels vary with age and gender differences. Thus age and sex matched healthy controls were included in our study to be sure that differences in results were not due to demographic confounders.[1819]

From a point of view of therapeutics, our results, as well as those of other groups reporting a correlation of oxidative stress and disease activity[678] suggest that antioxidants might be most effective during the active phase of the disease. Future clinical trials should document the disease activity status of the subjects in the respective treatment arms for better interpretation of results.

ROS can be measured from RBC flow cytometrically using as little as a finger prick sample, without the need for isolation of cells. The logistic simplicity of this method makes it an attractive option for a test to measure systemically generated ROS. However, day-to-day running expenses and technical expertise required for flow cytometry are practical limitations. The intrinsic weakness of ROS as a potential marker, just like ESR or CRP, would be its non-specificity. However, at the same time, baseline pre-treatment ROS levels can be used to monitor the dynamics of an unpredictable disease like vitiligo and objectively quantify treatment response. The limitations of our study was the small sample size, lack of follow-up, lack of paired samples for a before-after analysis and lack of blinding. Furthermore, we could not statistically compare the difference in GMFC between responders and non-responders, since the numbers in the latter group was just three. A long-term prospective study with follow-up of a larger cohort during vitiligo treatment can be planned based on the findings of this pilot project to further investigate the role of ROS generated in RBC as an activity marker in vitiligo.

What is new?

Reactive oxygen species level measured from erythrocytes is greater in active vitiligo than in inactive vitiligo and this parameter is not affected by disease variables such as duration since diagnosis and body surface area involved.

This parameter varies with the response to treatment.

The method we propose is logistically simple and so an attractive option for a test to monitor disease activity in vitiligo.