A novel oxidative stress marker “disulfide-thiol ratio”: new evidence of oxidative
stress in vitiligo
Mehmet Emin YANIK1, Hulya ALBAYRAK1, Gamze ERFAN1, Murat AYDIN2, Ahmet
GUREL3, Ozcan EREL4, Cemile Biçer5, Birol TOPCU6, Feti TULUBAS2, Mustafa
KULAC7
1
Assistant Professor, Namık Kemal University Faculty of Medicine, Department of
Dermatology, Tekirdag, Turkey
2
Associate Professor, Namik Kemal University Faculty of Medicine, Department of
Biochemistry, Tekirdag, Turkey
3
Professor, Namik Kemal University Faculty of Medicine, Department of Biochemistry,
Tekirdag, Turkey
4
Professor, Yildirim Beyazit University Faculty of Medicine, Department of
Biochemistry, Ankara, Turkey
5
Associate Professor, Yildirim Beyazit University Faculty of Medicine, Department of
Biochemistry, Ankara, Turkey
6
Assistant Professor, Namık Kemal University Faculty of Medicine, Department of
Biostatistics, Tekirdag, Turkey
7
Professor, Namik Kemal University Faculty of Medicine, Department of Dermatology,
Tekirdag, Turkey
1
Address for correspondence:
Professor Dr. Mustafa KULAC
Namik Kemal University Medical Faculty, Department of Dermatology
Tekirdag, Turkey
Phone: +90 282 250 52 40
Fax: +90 282 250 99 50
E- mail: [email protected]
2
A novel oxidative stress marker “disulfide-thiol ratio”: new evidence of oxidative
stress in vitiligo
ABSTRACT
Background and aim: Vitiligo is an acquired disease characterized by widespread
depigmented macules and patches over the entire body. Numerous studies have
demonstrated the relationship between vitiligo and oxidative stress. The disulfide-thiol
ratio (DTR) is a new oxidative stress marker in recent years. The aim of this study is to
calculate DTR in vitiligo patients, and to determine whether this marker can be used in
routine clinical applications, making a practical contribution.
Materials and Methods: 65 patients with vitiligo and 42 controls were included in the
present study. The severity of vitiligo was assessed using the “rule of nine”. We measured
serum native thiol (SH), and disulfide (SS) levels for both groups. The DTR was also
calculated.
Results: Serum SH level was found to be lower (p = 0.001) and DTR parameter was
significantly higher (p = 0.01) in vitiligo group. The SS level was found to be similar (p
= 0.590). Disease severity was found to be negatively correlated with serum SH levels,
and positively correlated with DTR.
Conclusions: This study indicates that the thiol-disulfide balance might play a role in the
etiopathogenesis of vitiligo and also serves as further evidence of the relationship between
oxidative stress and vitiligo.
Key Words: Antioxidant, Disulfide, Oxidant, Thiol, Vitiligo
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1. Introduction
Vitiligo is an acquired disease characterized by widespread depigmented macules and
patches over the entire body, which are secondary to the loss of melanocytes (1). The
disease can appear at any age, and has a prevalence of approximately 1% in society (2).
Although the etiology of vitiligo has not been thoroughly explained, there are four
different hypotheses: the autoimmune hypothesis, neural hypothesis, self-destruction
hypothesis, and the oxidation hypothesis (3). Oxidative stress has been a subject of
interest ever since its relationship with vitiligo was first identified, and many oxidative
stress markers have been studied to shed light on etiopathogenesis (4). The effectiveness
of topical and systemic antioxidants has been demonstrated in clinical studies.
The measurement of serum, plasma and tissue levels, or the spectrophotometric
measurement of the enzymatic rates of oxidants in the investigation of oxidative stress,
and antioxidants in the investigation of the antioxidant state, have been used from past to
present. Currently, the calculation of the Disulfide-Thiol balance by the Disulfide/Thiol
ratio (DTR) is considered to be a practical method, which can evaluate the oxidantantioxidant state with a single test, in contrast to other methods that evaluate it separately
in the investigation of the oxidant-antioxidant balance (5).
Thiols known as mercaptans are organic compounds containing a sulfhydryl group (–SH),
which consist of a sulfur and hydrogen atom bonded to a carbon atom (6). Thiols groups
are the important members of the antioxidant system, and eliminate reactive oxygen
species (ROS) and other free radicals through various enzymatic and non-enzymatic
mechanisms (7). In the presence of oxidants, thiols (SH) take part in oxidation reaction,
and end the oxidative reaction by transforming into disulfide (SS) bonds (8). This is a
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two-way reaction: in cases of oxidative stress, the thiol-disulfide balance will be altered
in favor of the latter, and once the oxidative stress is removed, the balance will be restored
and a dynamic thiol-disulfide balance will once again be established (9).
Numerous studies have demonstrated the relationship between vitiligo and oxidative
stress. However, a review of the medical literature indicates that no study related to the
thiol-disulfide balance has so far been carried out. The aim of this study is to calculate
DTR – a new oxidative stress marker – in vitiligo patients, and to determine whether this
marker can be used in routine clinical applications, making a practical contribution.
2. Materials and Methods
We enrolled consecutive patients admitted to the dermatology outpatient service with a
diagnosis vitiligo. Exclusion criteria included: subjects who were younger than 18 years
old and older than 65 years old, who had another systemic or dermatological disease, and
used any topical or systemic therapies and who did not want to participate in the study.
After exclusions, 65 patients with vitiligo were included in the present study. 42 age and
sex matched healthy subjects with no systemic or dermatological disease consisting of
hospital staffs were recruited as controls. The severity of vitiligo was assessed using the
“rule of nine”.
Blood samples were obtained after an overnight fast. Samples were collected from a
cubital vein into blood tubes, and serum was separated from the cells by centrifugation at
3,000 rpm for 10 min. Samples were stored at -80 ºC until the time of analysis. The
institutional ethics committee approved the study protocol.
2.1 Biochemical procedure
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DTR was determined as described previously.5 Briefly, reducible disulfide bonds were
first reduced to form free functional thiol groups. Unused reductant sodium borohydride
was consumed and removed with formaldehyde, and all thiol groups including reduced
and native ones were detected after reaction with DTNB [5, 5’ -dithiobis- (2-nitrobenzoic)
acid]. Half of the difference between total and native SS provided the dynamic SS
amount. After the determination of native SH and SS amount, DTR was calculated.
2.2 Rule of nine
The “rule of nine” assessment involves the evaluation of the head and neck, each arm,
leg, and the four trunk quadrants for vitiligo patches, each comprising 9% of the total
body surface area with the genitalia comprising 1%. Using the Wood’s lamp examination
of depigmentation of vitiligo patients, the severity of disease is assessed with the rule of
nine, which has been described as subjective and based on visual assessments (10).
2.3 Statistical methods
Data were analyzed using the Statistical Package for the Social Sciences version 18.0
(SPSS Inc., Chicago, IL, USA). Confidence intervals of 95% and a two tailed p-value less
than 0.05 were considered statistically significant for all analyses. All numerical variables
were tested by the Kolmogorov–Smirnov test for normality of distribution. Differences
between groups in terms of age, level of serum SS, SH, and DTR, were tested with Mann
Whitney U tests. Differences between sexes were compared with  2 test. Spearman’s
correlation test was performed for analyzing the correlation coefficients between SS, SH,
DTR and “rule of nine” in vitiligo patients.
3. Results
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The mean age was found to be 38.3 (±12.3) in vitiligo patients, and 39.1 (±10.8) in the
control group. The mean age of the two groups was similar (p = 0.708). The number of
male patients in the vitiligo group was 28 (43%), while the number of female patients was
37 (57%). The control group, on the other hand, consisted of 19 (45%) male and 23 (55%)
female patients. There were no statistically significant differences between the two groups
with respect to gender (χ2 = 0.48, p = 0.826) (Table 1). While Serum SH level (p = 0.001)
and DTR (p = 0.01) were found to be significantly different between the two groups, the
SS level (p = 0.590) was found to be similar (Table 2). The correlation between vitiligo
severity (as calculated according to the “rule of nine”) and biochemical parameters was
also examined. Disease severity was found to be negatively correlated with serum SH
levels (r = 0.286, p = 0.021) (Figure 1), and positively correlated with DTR (r = 0.268, p
= 0.031). (Figure 2) No significant correlation was identified between vitiligo severity
and SS level (r = 0.062, p = 0.62) (Figure 3).
4. Discussion
Vitiligo is a disease for which the pathogenesis has not been clarified. However, a review
of studies that have been published so far reveals that there are four hypotheses regarding
its pathogenesis: the autoimmune hypothesis, the neural hypothesis, the self-destruction
hypothesis and the oxidation hypothesis. In recent years, numerous studies have
demonstrated that oxidative stress plays an important role in the occurrence and
exacerbation of vitiligo (11). As a result of this, antioxidant medications are frequently
used in the treatment of vitiligo, particularly as support treatment (12).
There is no study carried out in the literature regarding the thiol-disulfide balance that
proves the oxidative stress state in vitiligo; however, in general, antioxidant enzymes and
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lipid peroxidation products, which are the indicators of oxidative stress, have been studied
extensively.
The levels of catalase and glutathione peroxidase enzymes, which break down H2O2 and
antioxidant enzymes that remove ROS such as paraoxanase-1 and superoxide dismutase,
and non-enzymatic antioxidants, such as vitamin C, vitamin E and selenium have been
found to be low in the serum and tissue samples of vitiligo patients (11-14).
As DTR is a new and recent oxidative stress marker, there are increasing numbers of
studies on DTR. A preliminary study carried out by the researchers who developed this
system demonstrated that degenerative conditions and diseases such as pneumonia,
obesity, diabetes, MI, hyperemesis gravidarum and smoking are associated with higher
plasma disulfide levels, while proliferative conditions or diseases such as renal, colon,
lung and urinary bladder cancers and multiple myeloma are associated with lower plasma
disulfide levels. (5, 15-17)
Previous studies evaluating both the tissue and serum levels of glutathione – a major
constituent of the thiol pool – identified lower levels among vitiligo patients compared to
the normal population, which supports the role of oxidative stress in vitiligo (18). In
another study, erythrocyte glutathione levels have been found to be lower in vitiligo
patients (19). In parallel with glutathione levels, glutathione peroxidase activity has been
found to be lower in vitiligo patients (20). Glucose-6-phosphate dehydrogenase enzyme
activity, which plays an important role in the reduction of disulfide bonds and the
reformation of sulfide groups, has also been found to be lower (21). All of these studies
indicate the need to further investigate the relationship between oxidative stress and the
thiol-disulfide balance in vitiligo (18-21). The practical measurement of the DTR will
8
allow the presence of oxidative stress to be identified in patients, thus indicating that these
patients might benefit from antioxidant treatment. With this study we have intended to
investigate this new method in vitiligo patients on whom oxidative stress is the most
prevalent among the dermatological diseases, and on whom topical and systemic
antioxidant support treatments are commonly applied. As a result of this study, we
identified DTR values to be high in vitiligo patients in a way that it supports the presence
of oxidative stress. High DTR values indicate that the balance in the plasma thioldisulfide pool has shifted towards the disulfide side, and that the amount of oxidant
substances that need neutralizing has increased.
In spite of the fact that it has been demonstrated that the systemic oxidant-antioxidant
state is disturbed in both active and stable vitiligo patients, the amount of ROS is higher
and the amount of antioxidant is lower in the serums of active vitiligo patients than those
of stable vitiligo patients (11). In our study, we evaluated the correlation between the
DTR value and disease severity as calculated by the rule of nine with the aim of
investigating whether DTR is associated with the severity of the disease. We identified a
positive correlation between disease severity and DTR. This correlation suggests that, in
vitiligo patients, the level of disease activation can be evaluated solely by looking at the
DTR ratio, without having to make an active- versus -stable distinction (in cases where
oxidative stress is evaluated). We believe that it will be beneficial to conduct further
studies with larger populations to determine whether the thiol-disulfide balance (which
illustrates the DTR level) is correlated to severity of vitiligo or not.
It is currently not possible to clearly determine on an individual basis which mechanism
plays a greater role in the occurrence or exacerbation of vitiligo and in which patients.
The fact that vitiligo patients experience oxidative stress and that this stress is responsible
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for the worsening of the disease is an acknowledged reality, regardless of the theories
proposed to explain the occurrence of vitiligo lesions. If we can determine the vitiligo
patients on whom oxidative stress is prominent, by using a practically applied method,
we will be able to identify patients in whom routine antioxidant treatments might be
beneficial, thus providing a solid scientific basis for the necessity of such treatments.
Furthermore, if the positive correlation we identified between disease severity and DTR
is supported by other studies, this will be a promising development, concerning the
possible use of this ratio by clinicians, as an activation marker.
In conclusion; oxidative stress plays a role in the etiopathogenesis of vitiligo, but in daily
practice, routine measurements are not suitable in technique. The SOD, MDA, GSH-Px,
CAT measurements can be studied manually and this leads differences between the
results. The method used in this study has been standardized and automatized according
to daily use. We believe that DTR ratio is an easy and standardized technique for
measuring the oxidative stress in diagnosis and follow-up of vitiligo patients.
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Table legends
Table 1: Demographic characteristics in the vitiligo patient and control groups
Table 2: Native thiol, Disulfide amounds and Disulfide/Thiol ratio of subjects
Figure legends
Figure 1: The correlation between vitiligo severity and native thiol.
Figure 2: The correlation between vitiligo severity and Disulfide/Thiol ratio.
Figure 3: The correlation between vitiligo severity and disulfide
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Table 1
Age(Year)
Sex
Vitiligo Group (N=65)
Control Group(N=42)
P Value
38,3(±12,3)
39,1(±10,8)
p=0,708
37(57%)
23(55%)
 2 =0,43
Female
Male
28(43%)
19(45%)
P=0,86
Table 1: Demographic characteristics in the vitiligo patient and control groups
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Table 2
Group
SH *
(µmol/L)
Vitiligo
Median(min-
p-
max)
value
326.8(211.7462.4)
15
Control
376(201.5-
0.001
461)
SS †
Vitiligo
(µmol/L)
23.8(12.639.9)
Control
0.590
24.3(9.642.6)
DTR ‡ (%)
Vitiligo
7.08(3.312.5)
Control
0.01
6.37(2.7716.00)
Table 2: Native thiol, Disulfide amounds and Disulfide/Thiol ratio of subjects
* Native thiol, † Disulfide, ‡ Disulfide/Thiol ratio.
Figure 1
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Figure 1: The correlation between vitiligo severity and native thiol.
Figure 2
17
Figure 2: The correlation between vitiligo severity and Disulfide/Thiol ratio.
18
Figure 3
Figure 3: The correlation between vitiligo severity and disulfide
19