Oxidative stress and antioxidant markers in patients with alopecia areata: A comparative cross-sectional study

Plain Language Summary

Alopecia areata (AA) is a chronic inflammatory disease. Oxidative stress may contribute to the pathogenesis of this condition. So, we evaluated the serum oxidative stress markers and antioxidant capacity in patients with alopecia areata. This cross-sectional study was performed on 40 patients with alopecia areata and 40 healthy controls. Serum oxidative markers, including advanced glycation end products (AGEs) and advanced oxidation protein products (AOPPs), were measured. Paraoxonase-1 (PON1), lecithin-cholesterol acyltransferase (LCAT) and serum ferric-reducing antioxidant power (FRAP) were determined for antioxidant enzymes in our study. The serum levels of AGEs and AOPPs were significantly higher in patients with alopecia areata, compared to the controls (P < 0.001), whereas the levels of FRAP, PON1, and LCAT were significantly lower in patients with alopecia areata (P < 0.001). According to the results of this study, the oxidant-antioxidant enzymatic system is impaired in alopecia areata due to the increased oxidative products and decreased antioxidant activity.

Introduction

Alopecia areata is a common autoimmune skin disorder, with an overall pooled prevalence of 2.11%.^1,2 Infiltration of T-cell lymphocytes around the hair follicles during the anagen phase plays a pivotal role in this disorder.³ Other triggering factors, including oxidative stress and genetics are also associated with the development of alopecia areata.^4,5 It is known that the skin is continuously exposed to reactive oxygen species, generated by both endogenous and exogenous sources. A balance between oxidative damage and antioxidant protection is necessary for different biological processes.⁶ Also, reactive oxygen species plays a key role in apoptosis for reacting with all macromolecules (e.g., lipids, proteins, and carbohydrates) on the cell membrane.⁷

Paraoxonase-1 and lecithin-cholesterol acyltransferase are enzymes with antioxidant activities.^8-10 The ferric reducing antioxidant power assay is used to measure the antioxidant potential of body samples.¹¹ Also, advanced glycation end products are glycated proteins that lead to the formation of reactive oxygen species through interactions with advanced glycation end product-specific receptors and result in increased oxidative stress.¹² On the other hand, advanced oxidation protein products are oxidized protein products, with a similar structure and biological activity to advanced glycation end products formed during oxidative stress.¹³

The role of products derived from glycosylation, protein oxidation, lecithin-cholesterol acyltransferase and ferric-reducing antioxidant power has been evaluated in a wide range of inflammatory diseases^10,13-15; but limited studies have been done in alopecia areata patients. Protein and glycation end-products are more stable than lipids and may be better markers of oxidative stress.^12,13 Therefore, in this cross-sectional study, we aimed to investigate and compare the levels of oxidative markers (i.e., advanced oxidation protein product and advanced glycation end product) and antioxidants (i.e., paraoxonase-1, lecithin-cholesterol acyltransferase and ferric-reducing antioxidant power) in patients with alopecia areata and healthy controls.

Methods

This cross-sectional study was performed on 40 patients with alopecia areata who were treated in outpatient dermatology clinics of Razi and Imam Komeini Hospitals during 2018-2019. Forty age-, gender- and body mass index (BMI)-matched healthy individuals with aesthetic complaints were also allocated to the control group. An experienced dermatologist confirmed the diagnosis of alopecia areata. The exclusion criteria were as follows: History of inflammatory or autoimmune diseases other than alopecia areata; infection; diabetes mellitus; familial hyperlipidemia; liver or kidney disorders; malignancies; pregnancy or lactation; consumption of antioxidants, vitamins and topical or systemic anti-inflammatory drugs within the past three months; recent history of major surgery; obesity; alcohol or tobacco use; and, professional athlete.

In agreement with the study conducted by Bilgili et al. the sample size was calculated using G*power version 3.1.9.4 (d = 0.64, α = 0.05 and power = 0.8), with an equal allocation ratio for the two-tailed test and assumption of a normal parent distribution. Finally, 40 samples were calculated for each group.⁸ The study was conducted according to the principles outlined in the Declaration of Helsinki and the Medical Research Involving Human Subjects Act.

The clinical information and demographic data of the subjects were documented. The severity of alopecia areata was determined using the Severity of Alopecia Tool.¹⁶ Their BMIs were calculated as weight in kilograms divided by the square of height (kg/m²). A 10-mL venous blood sample was taken from each participant at around 8-9 AM after a 12-hour fasting period to measure the fasting blood sugar level, C-reactive protein level, lipid profile and other laboratory markers. The lipid profile and fasting blood sugar levels were measured using standard enzymatic-colourimetric methods (Pars Azmun Co. Ltd., Tehran, Iran). The serum C-reactive protein level was assessed using a two-site ELISA assay (Diagnostic Biochem, Ontario, Canada).

Additionally, the levels of the advanced glycation end product and advanced oxidation protein product were measured, using the spectrophotometric method (FLUOstar OPTIMA reader, BMG, Germany).¹³ The serum paraoxonase-1 level was also measured, using an automated paraoxonase assay kit (V31137, ZellBio Co., Germany), following a colourimetric method. Also, to determine the lecithin-cholesterol acyltransferase activity, a commercially available kit (Calbiochem Co., USA) was used, based on the fluorometric method, and the results were expressed as the ratio of wavelengths (470 nm/390 nm). Finally, ferric-reducing antioxidant power was measured using spectrophotometry.¹¹

Data analysis was performed using SPSS version 24 (Chicago, IL, USA). For group comparisons, independent sample t -test, Mann-Whitney U test and Chi-square tests were used. Linear regression analysis was also performed to determine the relationships between continuous variables. A P -value of less than 0.05 was considered statistically significant.

Results

This study was conducted on 40 patients with alopecia areata and 40 healthy controls, including 21 (52.5%) females and 19 (47.5%) males in each group. The mean age of the subjects was 29.23 ± 10.69 years in the alopecia areata group and 29.32 ± 11.81 years in the control group (P = 0.96). Other baseline demographics, clinical characteristics and laboratory findings of the patients and healthy controls are presented in Table 1.

The mean serum levels of advanced glycation end products and advanced oxidation protein product were significantly higher in patients with alopecia areata (78.25 ± 4.44 and 182.82 ± 5.99, respectively), compared to the controls (46.48 ± 4.75 and 106.88 ± 7.99, respectively) (P < 0.001). In contrast, the mean serum levels of antioxidant markers, including ferric-reducing antioxidant power, paraoxonase-1 and lecithin-cholesterol acyltransferase, were significantly lower in the alopecia areata group (822.50 ± 49.43, 96.96 ± 10.90 and 34.87 ± 3.91, respectively), compared to the controls (1400 ± 165.74, 206.82 ± 8.62 and 55.46 ± 3.07, respectively) (P < 0.001) [Table 1]. The results showed that the mean fasting blood sugar level was significantly higher in the alopecia areata group, compared to the controls (100.87 ± 15.03 vs. 92.82 ± 11.62; P < 0.001).

There was a significant difference in the advanced glycation end product level between female and male patients with alopecia areata (79.9 ± 3.8 in females vs. 76.42 ± 4.5 in males; P = 0.01). A negative correlation was found between the serum advanced glycation end product and cholesterol levels in the alopecia areata group (r = -0.03, P = 0.04). Statistical analysis based on Spearman’s rho test on the alopecia areata group revealed a positive correlation between the ferric-reducing antioxidant power level and the percentage of hair loss (P = 0.01, r = 0.4), and a negative correlation between ferric-reducing antioxidant power level and C-reactive protein (P = 0.03, r = -0.3). Moreover, the serum triglyceride level was negatively correlated with the paraoxonase-1 activity in this group (P = 0.02, r = -0.35) [Table 2]. The oxidative and antioxidant markers had no significant associations with hair-loss patterns (P > 0.05) [Table 3].

Discussion

The present results revealed that the mean serum levels of markers of oxidative stress were significantly higher in patients with alopecia areata, compared to the controls, whereas the mean antioxidant activity was significantly lower in the alopecia areata group, compared to the controls. The SALT score and the hair-loss pattern had no significant associations with oxidative and antioxidant markers. Moreover, we found a negative correlation between C-reactive protein and ferric-reducing antioxidant power levels. C-reactive protein (CRP) is an inflammatory mediator and was significantly increased in alopecia areata patients.¹⁷ Although our study did not support this result, the possible role of oxidative stress and decreased antioxidative markers like ferric-reducing antioxidant power with inflammatory biomarkers in the development of alopecia areata. CRP level was not higher in alopecia areata cases. But the possible role of oxidative stress and decreased antioxidative markers like FRAP with inflammatory biomarkers in the development of alopecia areata were indicated.

Generally, it is suggested that oxidative stress plays a critical role in the pathogenesis of autoimmune disorders, including psoriasis,¹⁸ vitiligo¹⁹ and pemphigus vulgaris,²⁰ by causing inflammation, inducing cell apoptosis, and reducing immune tolerance. Although some studies have supported the role of oxidative stress in the pathogenesis of alopecia areata,^21,22 the results are inadequate and conflicting.^23,24

There are also reports of increased lipid peroxidation and decreased activities of antioxidants, such as glutathione, glutathione peroxidase, and superoxide dismutase, in patients with alopecia areata, compared to the controls.²⁵ In this regard, Barky and Bilgili et al. found that the mean total antioxidant capacity in the serum was significantly lower in patients with alopecia areata, compared to the controls.^8,22 Our results also showed that the antioxidant activity was significantly lower in patients with alopecia areata compared to healthy controls. On the other hand, Akar et al. found an increase in the antioxidant activity of superoxide dismutase and glutathione peroxidase in the scalp of patients with alopecia areata, especially in the early stages of the disease, whereas no reduction was observed in the lipid peroxidation rate.²⁴ The results of an Akar study were contrary to our study.

Evidence suggests that paraoxonase-1 is significantly reduced in patients with alopecia areata. This enzyme is majorly influenced by gender, as its activity is higher in females than males.²⁶ However, the present study showed that paraoxonase-1 decrease in patients with alopecia areata but did not confirm any link between paraoxonase-1 activity and gender in alopecia areata patients. It is known that advanced oxidation protein products and advanced glycation end products activate the membrane receptors for the latter to trigger reactive oxygen species generation and oxidative stress, which is involved in inflammatory diseases.^13,14 However, most previous studies have addressed the lipid peroxidation rate as a marker of oxidative stress in alopecia areata.^24,27,28 In this regard, Cwynar et al. reported a statistically non-significant higher plasma level of advanced oxidation protein products in patients with alopecia areata compared to the controls; however.²⁹ However, this difference in our study was statistically significant.

The reduced serum levels of lecithin-cholesterol acyltransferase and ferric-reducing antioxidant power have been reported in a wide range of inflammatory diseases, including psoriasis and chronic kidney disease.^18,30,31 Our results also showed that the serum levels of ferric-reducing antioxidant power and lecithin-cholesterol acyltransferase were significantly lower in patients with alopecia areata, compared to the healthy controls.

Several studies have shown that oxidative stress is involved in the pathogenesis of psoriasis.³² The total oxidant status and malondialdehyde⁴ levels were significantly higher in psoriasis patients, whereas total antioxidant status and catalase levels in these patients were significantly lower than in healthy controls.³³ Based on our study results, the imbalance of oxidative and antioxidant activity may be involved in the pathogenesis of alopecia areata, as it is in psoriasis.

This study had some limitations. First, the sample size was small. Second, most correlations, although significant, were weak and difficult to generalise. Therefore, future research with a larger sample size is necessary to confirm our findings.

We were unable to find any previous reports about serum ferric-reducing antioxidant power, lecithin-cholesterol acyltransferase and advanced glycation end-product levels in patients with alopecia areata. Our results indicated an imbalance of the oxidant-antioxidant enzymatic system in alopecia areata. Therefore, the administration of agents with antioxidant activities may be useful in the management of alopecia areata.