Evaluation of systemic oxidative stress in patients with melasma

The significance of oxidative stress has been assessed and proven in the etiopathogenesis of many cutaneous disorders, but there are few studies that evaluated the role of only some factors involved in oxidative stress in patients with melasma.

[16] Considering the paucity of literature on the effect of OS on developing melasma and the conflicting results of these few reports, there is a need for further investigations in this field.The present study was designed to examine the role of systemic OS in melasma and to assess the relationship between OS and the severity and extension of this disease.For a comprehensive investigation of the oxidant/antioxidant balance in melasma, the serum levels of superoxide dismutase (SOD), glutathione peroxidase (GPX), catalase (CAT), malondialdehyde (MDA), protein carbonyl (PC), selenium (Se), vitamin E (vit E), and vitamin C (vit C) were measured.

| PATIENTS AND ME THODS
In this case-control study, a total of 50 patients from our outpatient department with a clinical diagnosis of melasma were enrolled as the case group.The control group included 50 healthy volunteers who were matched with the case group in terms of age, sex, and Fitzpatrick skin type.All participants were nonsmokers and had not consumed alcohol, oral contraceptives, folic acid, glutathione, zinc, vitamin E, vitamin C, iron, selenium, multivitamins, or any antioxidants in the last three months.Immunocompromised status, chronic diseases such as liver or kidney disease, diabetes mellitus, active infection or inflammation, other skin disorders, and a history of pregnancy in the last year excluded participation in this study.For patients previously treated for melasma, a washout period of six months was required.
The research protocol was approved by our medical ethics committee.All participants signed an informed consent form after being informed about the study protocol.A single dermatologist reviewed all patients, and their demographic information as well as the extension and severity of melasma were recorded.The extension of the disease was defined as the percentage of the total face area involved, and the severity of the disease was determined using the Melasma Area and Severity Index (MASI) score. 17fteen milliliters of fasting venous blood were collected from all participants and saved in sterile tubes that contained Na 2 EDTA.
Using centrifugation (3000 RPM), serum was recovered after 15 min.The specimens were coded and kept at −70°C under the protection of light to assess serum levels of SOD, GPX, catalase, MDA, PC, Se, vitamin E, and C.
The serum levels of these parameters were compared between the case and control groups.The correlation of melasma extension and MASI score with serum concentrations of measured parameters was also evaluated.

| RE SULTS
Although there was no gender preference for selection, all participants in this study were female.No significant difference was detected between the case and control groups regarding age (36.3 ± 5.5 years in the case group vs. 37.2 ± 4.3 years in the control group) and Fitzpatrick skin type (80% skin type IV in the case group vs. 82% skin type IV in the control group).The age of melasma onset ranged from 21-51 years, with a mean of 32.6 ± 6.7 years.Melasma developed in 22 (44%) patients following pregnancy and in 4 (8%) patients after ingestion of oral contraceptives.Eight (16%) patients identified sun exposure as a precedent to the melasma and in 16 (32%) patients mentioned no trigger could be identified.Melasma covered 6% to 80% of the face (mean, 38.5 ± 18.3%), and the mean MASI score was 18.1 ± 9 with a range of 2.4-34.4(Table 1).
The comparison of the average serum MDA level in the case group (3.08 U/mL) with the control group (2.35 U/mL) revealed a significant difference (p < 0.05).The serum MDA levels had a significant positive correlation with both the MASI score (correlation coefficient, +0.4; p < 0.001) and the extension of the disease (correlation coefficient, +0.3; p < 0.05).
The average serum concentration of vitamin C was 2.16 μg/mL in the case group and 2.57 μg/mL in the controls, with a significant difference between the two groups (p < 0.001).However, it did not have a significant correlation with the extension of the disease or MASI score (Table 2).Age, sex, disease duration, and triggering factors caused no significant difference in the concentrations of measured metabolites.Moreover, there was no significant correlation between the MASI score and age, sex, age of disease onset, disease duration, or triggering factors. in postpubertal women, oral contraceptive consumers, and pregnant women. 3In the current study, a hyperestrogenic state, including pregnancy and ingestion of oral contraceptives, triggered the onset of melasma in more than half of the patients, while Ortonne et al.
observed the development of melasma after taking oral contraceptives in 25% of their patients. 19 recent years, OS has been shown to play a fundamental role in many diseases as well as some cutaneous disorders. 20The production of oxidants, which are involved in cutaneous diseases, occurs via two main pathways: lipid peroxidation and protein peroxidation.
On the other hand, the antioxidant protective system of the skin is divided into two subgroups: enzymatic antioxidants and nonenzymatic antioxidants. 21

| Lipid peroxidation
Lipid peroxidation in the cell membranes has been recognized as the major mechanism of tissue impairment due to OS. Malondialdehyde is an end-product of LPO, which is an indicator of tissue injury mediated by free oxygen radicals. 22,23In this study, a significant increase in serum concentration of MDA was found in the melasma group, which was in a positive correlation with the severity (MASI score) and extension of the disease.The current findings are consistent with the observations of Choubey et al., who found that MDA was higher in the melasma group and correlated with the MASI score. 24Seckin et al. also observed a higher level of serum MDA in melasma patients. 25Nevertheless, to the best of our knowledge, the correlation of melasma extension with the serum level of MDA has not been investigated before.

| Protein peroxidation
The interaction between ROS and proteins results in oxidative damage to amino acids and leads to the generation of protein carbonyl. 26Measurement of the level of PC is a sensitive approach to evaluate oxidative protein damage. 27,28Findings on the role of protein carbonyl are controversial in the literature.Sarkar et al. reported higher concentrations of PC in the melasma group, 5 while Seçkin and coworkers found paradoxically lower levels of PC in the patient group. 25However, in our study, the difference in the level of PC in the case and control groups was not statistically significant.According to these findings, it can be suggested that lipid peroxidation is more important than protein peroxidation in the pathogenesis of melasma.

| Enzymatic antioxidants including superoxide dismutase, glutathione peroxidase, and catalase
SOD is the main enzyme, scavenging free oxygen radicals through the decomposition of superoxide anions into oxygen and H 2 O 2 . 29X is also the main enzyme that neutralizes H 2 O 2 . 30Both GPX and CAT convert H 2 O 2 to oxygen and water. 27,31In the present study, the serum concentrations of enzymatic antioxidants were normal in melasma patients, demonstrating no significant difference from healthy controls.However, Seckin et al. and Choubey et al. noticed significantly higher concentrations of enzymatic antioxidants (in particular SOD and GPX) in the melasma group in comparison with the control group. 24,25Conversely, Sarkar et al. discovered significantly lower concentrations of enzymatic antioxidants (particularly CAT) in melasma patients. 5

| Nonenzymatic antioxidants including selenium (Se), alpha-tocopherol (vitamin E), and ascorbic acid (vitamin C)
Selenium is an essential micronutrient for selenoproteins, including GPX, which contributes to the activation of these enzymes. 32,33tamin C, as another nonenzymatic antioxidant, inhibits pigment production by preventing the formation of nitric oxide, which is a melanocyte-activating molecule; it also inhibits melanin synthesis. 34On the other hand, vitamin E plays a preventive role against Unprotected sun exposure 8 No trigger factor 16 ( LPO. 33,35 It can inhibit tyrosine activity as an active enzyme in melanin synthesis.Moreover, it can decrease pigment. 36Both topical and oral vitamin C and vitamin E have always been among the most common options in the treatment of melasma, without any evidence of their role in the etiopathogenesis. 37,38The present study, as the first in this field, showed significantly lower concentrations of vitamin C in melasma patients, although the serum concentration of vitamin E demonstrated no significant difference.It could be the result of the fact that vitamin C is a water-soluble antioxidant that acts both intracellularly and extracellularly and is involved in the defense against oxidative stress prior to vitamin E, which is a fat-soluble antioxidant. 12,39Selenium is another nonenzymatic antioxidant that was measured in melasma patients for the first time in the current study.However, no significant difference was observed between the groups.
Furthermore, no significant correlation was found between melasma extension or severity and the serum concentration of measured metabolites, except for MDA.
The limitation of our study was that we investigated solely the systemic oxidative stress but not the local status of antioxidants in the skin with melasma.

| CON CLUS ION
The present study showed a greater involvement of nonenzymatic antioxidant defense in the pathogenesis of melasma compared to enzymatic antioxidant defense.Moreover, lipid peroxidation was more remarkable than protein peroxidation.MDA was the most significant

CO N FLI C T O F I NTE R E S T S TATE M E NT
No conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author.The data are not publicly available due to privacy or ethical restrictions.

E TH I C S S TATEM ENT
The Skin Research Center Ethics Committee approved this research.

TA B L E 2
The serum levels of the metabolites in the case and control groups.
IBM SPSS Statistics 24 was used to analyze the data.Independent sample t-test was applied to compare the age and serum concentrations of measured factors in two groups.To evaluate the correlation between serum concentrations of metabolites and the MASI score or extension of the disease, the correlation test and Pearson correlation coefficient were used.If the p-value was less than 0.05, it was considered statistically significant.

TA B L E 1
The demographics and clinical characteristics of the patients with melasma.
indicator of systemic OS in melasma and was correlated with both the severity and extension of the disease.Despite the controversial results of different studies, disrupted oxidant-antioxidant balance in melasma is the common finding in all previous reports as well as study.On the whole, the investigation into the relationship between systemic oxidative stress and melasma can provide new insights into the etiopathogenesis and, consequently, the treatment of this disease.Therefore, further studies are recommended on the indicators of OS in both the sera and skin lesions of the patients to reach a definitive conclusion.Moreover, future clinical trials focusing on the improvement of MASI by antioxidant supplementation to increase MDA would be necessary to confirm the association.AUTH O R CO NTR I B UTI O N SHodaRahimi, Mina Mirnezami, and Aazam Hajihashemi performed the research.Hoda Rahimi, Mina Mirnezami, and Anousha Yazdabadi designed the research study.Mina Mirnezami and Aazam Hajihashemi contributed essential reagents or tools.Anousha Yazdabadi analyzed the data.Hoda Rahimi and Anousha Yazdabadi wrote the paper.