Introduction
Alopecia areata (AA) is a tissue restricted autoimmune disease that occurs mostly in genetically predisposed individuals 1. It is triggered by several factors. These factors include patients’ genetic constitution, immune factors, and oxidative and emotional stress 2.
Tumor necrosis factor-α (TNF-α) is synthesized and released by immune and nonimmune cells, stimulating intracellular production of mitochondrial reactive oxygen species (ROS) 3. Evidence provides support to the fact that oxidative stress (OST) and apoptosis are closely linked physiological phenomena 4, showing that hair follicle apoptosis is related to oxidants and antioxidants in AA 5. Therefore OST is one of the possible hypotheses involved in the pathogenesis of AA 6.
Selenium (Se) is an essential trace element important in the synthesis of selenoproteins as well as glutathione peroxidase (GSH-Px) and thioredoxin 7 with antioxidant properties essential in the body’s defense mechanisms 8. It has also an immunomodulatory effect 9, mainly by stimulating the high-affinity interleukin 2 receptor on activated T lymphocytes and natural killer cells, leading to their clonal expansion and differentiation into cytotoxic T lymphocytes. Se also prevents OST-induced damage to immune cells 10.
In light of the importance of Se as a trace element and its association with hair fall in rats 11 and AA in humans 12, the aim of the study was to determine the level of serum Se in a group of Egyptian patients with AA in an attempt to explain its importance in the pathogenesis of the disease.
Patients and methods
This case-controlled study included 100 participants; 50 AA patients and 50 healthy volunteers as controls. All participants were Egyptians selected from Dermatology Outpatient Clinics of Kasr Al-Ainy University Hospital and El Houd El Marsoud Public Hospital, Cairo, Egypt (May–December 2009). The Ethics Committee approved the study and a written informed consent was obtained from all participants before enrollment in the study.
Inclusion and exclusion criteria
Patients with localized AA having single, few, or multiple patches only were included in the study. In contrast, AA patients showing spontaneous growth of terminal hair at the time of presentation, using systemic therapy likely to cause hair regrowth one month before presentation, or with any associated diseases that may alter serum Se levels, such as vitiligo or psoriasis, thyroid, hepatic, renal, cardiovascular diseases, diabetes, anemia, or cancer, were excluded. In addition, patients with conditions that affect serum Se levels, such as pregnancy, smoking one month before presentation, and patients less than 18 years or more than 50 years of age were excluded from the study.
Methods
All patients were subjected to full history taking (personal, past history of any illness or medications, family history, and history of present illness).
Clinical assessment of the degree of AA was carried out by dividing the scalp into four quadrants and determining the percentage of scalp hair loss in each quadrant, followed by summation with a maximum score of 100%, according to the Severity of Alopecia Tool (SALT) score 13. Only assessment of scalp hair loss was presented in this study, in accordance with our inclusion criteria (Table 1).
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Table 1: Scalp (S) hair loss (Severity of Alopecia Tool score)
A sample of 5 ml of fasting (6–8 h) venous blood was obtained from each individual and divided into two aliquots; the first aliquot (1 ml) was dispensed into EDTA tubes for hemoglobin determination and the second aliquot of blood was left to clot, centrifuged, and the serum was separated and stored at −20°C for estimation of fasting blood glucose, creatinine, alanine aminotransferase (ALT), aspartate aminotransferase (AST) and thyroid stimulating hormone (TSH) levels, to exclude patients with abnormal results, and Se.
Glucose, ALT and AST, and creatinine levels were determined using the hexokinase method 14, kinetic UV method 15, and Jaffe method 16, respectively, using commercial kits on an Olympus analyser (AU 400; Olympus, Hamburg, Germany). The level of TSH was determined by enzyme immunoassay using kits supplied by IBL International (Hamburg, Germany) 17. Hemoglobin concentration was determined using a cyanmethemoglobin kit supplied by Sigma (St Louis, Missouri, USA) 18.
Determination of serum Se levels was carried out using an atomic absorption spectrometer equipped with a graphite furnace atomizer and an autosampler (GTA-110, Varian SpectrAA 220; Varian, Australia). Analysis of Se involves the vaporization of the treated sample that absorbs energy, emitted from a Se hollow cathode lamp, at a wavelength of 196.3 nm, which is specific to Se and proportional to its concentration. Patient samples and controls were diluted using 0.5% Triton X-100, 0.125% Dow Coming Antifoam-B, and 0.25% ascorbic acid. Ten microliters of palladium solution (1000 mg/l) were used as a matrix modifier. All chemicals were provided by Sigma Chemicals (St Louis, Missouri, USA) 19.
Statistical analysis
The data were analyzed using the SPSS program ( version 9.0; SPSS Inc., Chicago, Illinois, USA) for Microsoft Windows and summarized as mean±SD. The Mann–Whitney U-test was used for analysis of two independent quantitative variables and one-way analysis of variance was used for analysis of more than two quantitative variables. Simple linear correlation analysis (Spearman’s correlation) for quantitative data was also carried out. The r value was considered weak if it was less than 0.25, mild if it was at least 0.25 and less than 0.5, moderate if it was at least 0.5 and less than 0.75, and strong if it was at least 0.75. P-value is considered significant if it is less than 0.05.
Results
This study included 50 patients with AA: 33 men (66%) and 17 women (34%) with age ranging from 20 to 50 years (mean±SD, 26.8±7.7 years). Fifty normal controls were also included, 20 men (40%) and 30 women (60%) with age ranging from 23 to 50 years (mean±SD, 33.4±8.0 years). The duration of the disease ranged from 0.01 to 4 years (mean±SD, 1.2±1.0 years). Family history of AA was positive in 11 patients (22%).
On comparing serum Se levels (µg/l) of patients (mean±SD, 60.2±8.8) and controls (mean±SD, 77.4±9.6), a significant decrease in Se levels in patients was found (P=0.0001) (Table 2). There was no significant relationship between serum Se levels and sex in both patients (P=0.6) and controls (P=0.6), nor was there a significant relationship between serum Se levels and positive family history of AA in patients (P=0.5).
Table 2: Comparison between serum selenium level of patients and controls
According to the severity of AA (SALT score), on comparing serum Se levels of the three groups, S1 (31 patients, 62%): Se=61.4±7.7 µg/l, S2 (12 patients, 24%): Se=61.4±10.7 µg/l, and S3 (seven patients, 14%): Se=53.0±7.4 µg/l, no statistical significance was found (P=0.06), whereas on comparing serum Se levels of S1 (mild) and S3 (severe) groups, serum Se level of S3 was found to be much lower than that of S1, showing a significant difference (P=0.01; Table 3).
Table 3: Comparison between serum selenium level of patients in relation to severity of disease
No significant linear correlation between serum Se level of patients and duration of the disease (r=−0.1, P=0.4), severity of the disease (r=−0.2, P=0.1), or age of both patients (r=0.1, P=0.4) and controls (r=−0.1, P=0.3) was found.
Discussion
Although AA is considered to have an immunologic origin, the pathogenic mechanisms indicate a rather complex process that sustains an inflammatory reaction, that is, a vicious cycle that leads to hair loss. Keratinocytes release cytokines, which activate endothelial cells that in turn attract T cells and macrophages that release more cytokines such as TNF-α, interleukins, and interferon-γ 20.
It is known that moderate and severe inflammatory reactions lead to a decrease in serum and erythrocyte GSH-Px activities in patients with AA, whereas OST in blood increases in inflammatory reactions 5,6. It is well known that Se is a cofactor of the antioxidant enzyme GSH-Px 21. In the present study, the serum Se levels in patients with AA were significantly lower than in the control group (60.2±8.8 vs. 77.4±9.6 µg/l, P=0.0001). Therefore, low Se levels in the serum of patients with AA can explain, or can be explained by, the inflammatory character of AA. In a similar study, Eken et al.22 reported that elevated risks in AA patients were observed at low levels of blood Se, which might be explained by the anti-inflammatory role of Se 23. Recently, it was reported that serum Se levels were low in patients with inflammatory diseases such as rheumatoid arthritis 24.
Low serum Se levels in AA patients were shown in previous studies on Iranian and Finnish patients 12,25. Furthermore, low plasma Se levels together with decreased GSH-Px activity were found in plasma and erythrocytes of AA patients in several studies 22,25,26.
Whether the observed low serum Se level in AA patients is a primary cause or a secondary phenomenon, this might be explained by the following observations:
First, in the plasma and/or RBCs, there was a decrease in the antioxidant effect, as well as increased levels of lipid peroxidation products, thus shifting the OST/antioxidant system balance in favor of stress, leading to DNA damage or apoptosis of hair follicles 6,27.
Second, in the tissues (scalp), there were increased levels of antioxidants (superoxide dismutase and GSH-Px) as a response to the excessive free radical generation from the increased lipid peroxidation 5, but despite this finding, the available antioxidants could not lower the increased lipid peroxidation products that are presumed to play a role in AA. Therefore, because of the presence of increased ROS in lesions of AA, there is an increased demand for Se in the tissue to form more antioxidants in the lesions. This might lead to the shift of Se from serum to tissue and its consumption; however, because of excess free radical formation in the lesions, the amount of available Se and subsequently the elevated antioxidant levels could not cope with the excess ROS produced. The possibility of the presence of a genetic defect in the antioxidant enzymes that makes them inefficient in combating the OST in AA lesions cannot be ruled out. Accordingly, the decreased Se level in the serum of patients with AA might be, at the end, a result and not a cause.
The current study showed a higher serum Se level in the older age groups of our AA patients, although no significant correlation of Se with age was found. A previous study 28 reported a significant effect of age on whole blood Se level concentrations (P<0.01) being highest in the middle age group that might be related to impaired renal excretion with age 29. Another study 26 observed fluctuation of Se levels in females related to estrogen being highest in the middle age. Only one study showed no age difference 30. Moreover, variations in the levels of serum Se might be influenced by food components – macronutrients and micronutrients and Se content of different soils 10.
A greater decrease in serum Se levels was found in the more severe forms of AA (S3) in comparison to mild form (S1) in this study, which seems logical as extensive lesions need more Se as antioxidants, leading to an increase in the shift from serum to tissues, thus further lowering of the serum level. No difference was noted between male and female patients, adding to the controversy noted as regards the relationship between Se level and sex 31,32.
Conclusion
According to previous results, lower serum Se levels were found in association with AA Egyptian patients, which might be caused by the increased demand for antioxidants to combat the OST and inflammatory process in AA. Whether a cause or effect, controlled studies are recommended to determine the efficacy of Se supplementation in those patients.
Acknowledgements
Conflicts of interest
There are no conflicts of interest.
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