Acne vulgaris is one of the common dermatological diseases frequently found in adolescence. Sebaceous hyperplasia, follicular hyperkeratinization, and bacterial hypercolonization, and immune reactions and inflammation may lead to acne vulgaris, which has a quite complex pathogenesis .
Oxygen, which is an important and vital component for the human body, can produce various reactive types (superoxide anion, hydrogen peroxide, and hydroxyl radicals) known as reactive oxygen species (ROS) . Any free radical involving oxygen can be referred to as ROS. Oxygen-centered free radicals contain unpaired electrons in the outer shell. It was suggested that ROS, which may be released from impacted damaged follicular walls, may be the reason for the progress of inflammation in the pathogenesis of the disease . Normally, the production of ROS is slow and they are removed by the antioxidant enzymes existing in the cell. Superoxide dismutase, catalase, glucose-6-phosphate dehydrogenase, and glutathione peroxidase (GSH-PX) are some important antioxidant enzymes . GSH-PX is the general name of an enzyme family with peroxidase activity whose main biological role is to protect the organism from oxidative damage caused by ROS . This antioxidant enzyme consists of the amino acid, glutathione, and the trace element ‘selenium’. The biological functions of GSH-PX are to reduce lipid hydroperoxide to its corresponding alcohol and reduce free hydrogen peroxide to water .
Free radicals have relatively short half-lives and thus the determination of their levels is difficult. Therefore, they can be evaluated indirectly by measurement of the antioxidant enzyme levels, by products of lipid peroxidation such as malondialdehyde (MDA), or by some transition metal levels .
Therefore, the aim of this study was to detect evidence of oxidative damage by ROS precisely in lesions of acne vulgaris as it may be involved in the pathogenesis of acne vulgaris. This was done by studying the tissue levels of GSH-PX, as an antioxidant, and MDA, as a product of lipid peroxidation, in skin lesions of acne vulgaris of different severity compared with healthy controls.
Patients and methods
This study was carried out on a total of 30 patients with acne vulgaris, and 10 age and sex matched healthy controls. All patients were derived from Al Zahraa Outpatient Clinic over a period of 6 months from November 2008 to May 2009.
Their age ranged from 15 to 25 years. They were classified into the following subgroups according to Doshi et al. .
- Ten patients with mild acne vulgaris (two males and eight females).
- Ten patients with moderate acne vulgaris (one male and nine females).
- Five patients with severe acne vulgaris (one male and four females).
- Five patients with very severe acne (nodulocystic acne and acne conglobata) (four males and one female).
All participants were nonsmokers, were not suffering from any coexisting disease, and did not receive any medications or vitamins 2 weeks before the start of the study. A signed informed consent was obtained from each of them. They were subjected to
- (1) Clinical examination to exclude associated diseases;
- (2) Complete dermatological examination;
- (3) Biopsy of acne vulgaris lesion on the face of patients and of normal skin of healthy controls;
- (4) Estimation of GSH-PX enzyme level in the skin biopsy specimens;
- (5) Estimation of MDA level in the skin biopsy specimens.
Sample biopsy and technique
A 3.5 mm skin punch biopsy specimen was taken from an acne vulgaris lesion on the face of a patient and from normal skin of the face of healthy control. Skin biopsy specimens were minced and homogenized in a lysis buffer containing Tris–HCl (10 mmol/l, pH 7.5), NaCl (150 mmol/l), Triton X-100 (1% vand phenylmethylsulfonyl fluoride (10 mmol/l). The homogenized tissues were centrifuged for 45 min at 4°C and 15.000 g and the supernatants were used for biochemical studies .
Determination of malondialdehyde in tissue supernatants
MDA in tissue supernatants was determined according to the method described by Ohkawa et al. . MDA forms a pink color complex with thiobarbituric acid, which is estimated colorimetrically. In brief, 2.5 ml of 20 mg/dl trichloroacetic acid was added to the supernatant and the tube was left to stand for 10 min at room temperature. After centrifugation at 3500 rpm for 10 min, the supernatant was discarded and the precipitate was washed once with 0.05 mol/l sulfuric acid. Thereafter, 2.5 ml of 0.05 mol/l sulfuric acid and 3 ml of thiobarbituric acid were added to this precipitate followed by heating in a boiling water bath and then cooled. The resulting chromogen was extracted with 4.0 ml of n-butyl alcohol by vigorous shaking. Separation of the organic phase was facilitated by centrifugation at 3000 rpm for 10 min and its absorbance was determined at a wavelength of 530 nm. The concentration of MDA tissue was expressed in nanomole/milligram protein. Protein was estimated according to the method described by Lowry et al. .
Determination of glutathione peroxidase in tissue supernatants 
Tissue was homogenized in 5–10 ml of cold buffer (i.e. 50 mmol/l Tris–HCl pH 7.5, 5 mmol/l EDTA, 1 mM dithiothreitol) per gram tissue protein. Centrifugation was carried out at 10 000×g for 15 min at 4°C and the supernatant was removed and stored on ice until assay. In brief, 100 μl of assay buffer, 50 μl of cosubstrate mixture, and 20 μl of sample were added wells of a special plate, followed by addition of 20 μl of cumene hydroperoxide to all the wells. The plate was carefully shaken for a few seconds to mix. After the color formed, absorbance was read once every minute at 340 nm using a plate reader to obtain at least five time points.
The statistical software package SPSS version 15 (SPSS Inc., Chicago, Illinois, USA) was used to analyze these data. Data were expressed as mean±standard deviation for quantitative measures and as both number and percentage for categorized data. Student's t test was used to compare between two groups for parametric data and χ2-test was used to compare between two or more groups for categorized data. Spearman's Rank Correlation test was used to study the association between two variables among each studied group. Severity was included as a semiquantitative measure, a score was given for each class (1=for mild, 2=for moderate, 3=severe, and 4=for very severe). A P value of less than or equal to 0.05 was considered significant.
Results of GSH-PX enzyme and MDA levels in skin specimens of acne vulgaris and normal skin of healthy controls showed a high statistically significant decrease in GSH-PX enzyme levels in the patient group compared with the control group (P=0.001), but a high statistically significant increase in MDA levels in the patient group compared with the control group (Table 1). There was a statistically significant positive correlation between MDA and severity of acne vulgaris (r=0.850, P=0.0002) and a statistically significant negative correlation between GSH-PX and severity of acne (r=−0.722, P=0.0006) (Fig. 1). There was a statistically significant negative correlation between MDA and GSH-PX levels in the patient group (r=−0.055, P=0.0007) (Fig. 2).
A statistically significant negative correlation was found between GSH-PX levels and age among both the control (r=−0.149, P=0.0006) and patient groups (r=−0.231, P=0.0002), whereas a statistically significant positive correlation was found between MDA levels and age among both the control (r=0.235, P=0.0005) and the patient (r=0.161, P=0.0004) groups (Table 2). There was a high statistically significant decrease of GSH-PX enzyme level in male patients compared with female patients, however, no significant difference was found in MDA levels with regard to the sex of patients (Table 3).
GSH-PX protects tissues from oxidative damage by reducing lipid hydroxide to corresponding alcohol and reducing hydrogen peroxide to water . MDA is a highly reactive three-carbon dialdehyde produced as a by-product of polyunsaturated fatty acid peroxidation and arachidonic acid metabolism . MDA is one of the most frequently used indicators of lipid peroxidation. On a group basis, data support the fact that MDA is a potential biomarker for oxidative stress .
Acne vulgaris is a chronic inflammatory skin disease of pilosebaceous units . It is mostly characterized by seborrhea, comedones, erythematous papules, and pustules, and less frequently by nodules, deep pustules, or pseudocysts, and in some cases is accompanied by scarring .
To our knowledge, this study was the first to investigate the tissue level of GSH-PX in acne vulgaris lesions.
In this study, there was a high statistically significant increase of GSH-PX enzyme level in patients compared with controls. A statistically significant negative correlation was found between GSH-PX levels and severity of disease. There was a high statistically significant decrease of GSH-PX enzyme level in male patients compared with female patients. There was a statistically significant negative correlation between GSH-PX levels in patient group and the age of the patients.
These results are in agreement with the study carried out by Aybey et al.  who investigated 79 patients (35 females and 44 males) with acne vulgaris and 17 healthy individuals. They reported a significantly lower serum GSH-PX level in patients with acne vulgaris than in controls. In addition, they reported a negative correlation between GSH-PX levels and the disease severity.
Briganti and Picardo  suggested that GSH-PX enzyme, which is a part of cell antioxidant defence system, and selenium, the cofactor of this enzyme, may play a role in the etiology and pathogenesis of acne vulgaris. Young et al.  reported that individuals suffering from acne have been shown to have reduced GSH-PX serum levels. After treatment with selenium and vitamin E, GSH-PX levels significantly increase, whereas the severity of acne typically decreases. GSH-PX enzyme and selenium are considered essential in the maintenance of cell integrity and in the arrangement of cell functions by protecting cell membrane lipids, proteins, and nucleic acids from peroxidative damage induced by hydrogen peroxide and lipid peroxide. Hence, they may be effective in the arrangement of mitosis of keratinocytes and it can prevent abnormal keratinization processes that result in keratinocyte proliferation . Therefore, deficiency of GSH-PX in acne vulgaris lesions may be partly responsible for the follicular hyperkeratinization in acne vulgaris.
In this study, a higher level of MDA was observed in the lesions of acne vulgaris patients than in control group. There was a statistically significant positive correlation between MDA levels and the severity of the disease. In addition, there was a high statistically significant correlation between MDA levels and the age in both patients and controls. There was no statistically significant difference in MDA level with regard to sex in the patient group. These findings are in agreement with that of Arican et al.'s  study, which was carried out on 43 patients with acne vulgaris and 46 healthy controls. They reported that MDA level was significantly higher in patients with acne vulgaris compared with controls. In contrast, the level of antioxidants was significantly lower in patients compared with controls. However, their study differed from this study in showing no statistically significant correlation between MDA levels and the severity of the disease. In addition, they reported no statistically significant correlation between MDA levels and either age, sex, or serum antioxidant enzymes levels. This study was in agreement with that of Nielsen et al.  in finding no significant correlation between MDA level and sex of patients and significant correlation between MDA level and age of patients. The relation of GSH-PX and MDA with age and sex has not been understood yet.
High level of MDA in acne vulgaris can be explained on the basis that inflammation seems to be a source of ROS . In acne vulgaris, ROS may be released from impacted damaged follicular walls and the mitochondrial respiratory chain reactions in neutrophils and macrophages . Grange et al.  reported that ROS, and especially superoxide anions O2− were rapidly produced by keratinocytes upon stimulation by Propionibacterium acnes surface proteins. In P. acnes-stimulated keratinocytes, O2− was dismuted by superoxide dismutase to form hydrogen peroxide, which was further detoxified into water by the GSH-PX system. In addition, P. acnes-induced O2− abrogated P. acnes growth and was involved in keratinocyte lysis through the combination of O2− with nitric oxide to form peroxynitrites. With the detected low levels of GSH-PX in acne vulgaris, in this study, there was accumulation of ROS. It is thought to be the reason for the progress of inflammation in the pathogenesis of the disease. ROS also cause the release of some chemotactic factors leading to neutrophil accumulation and this situation causes damage to follicular epithelia after the release of some inflammatory factors such as lysosomal enzymes as a result of phagocytosis .
Retinoic acid derivates, one of the most efficient antiacne drugs, prevent O2− production, interleukin-8 release, and keratinocyte apoptosis, suggesting the relevance of this pathway in acne vulgaris . It is thought to be the reason for the progress of inflammation in the pathogenesis of the disease. ROS also cause the release of some chemotactic factors leading to neutrophil accumulation and this situation causes damage to follicular epithelia after the release of some inflammatory factors such as lysosomal enzymes as a result of phagocytosis . In this study there was a statistically significant negative correlation between MDA and GSH-PX levels in the patient group, which suggests the oxidative stress in the pathogenesis of acne vulgaris.
According to these results, the statistically significant high levels of MDA in lesions of acne vulgaris and its significant positive correlation with the severity of disease reflect the oxidative stress state that can lead to cell injury and progress of inflammation in acne vulgaris. In addition, the statistically significant low levels of GSH-PX enzyme in lesions of acne vulgaris and its negative correlation with severity of the disease attribute the role of oxidative stress in the decrease of the antioxidant, at least, GSH-PX enzyme, as a consequence of its role in the pathogenesis of acne vulgaris.
There is no conflict of interest to declare.
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Keywords:© 2011 Egyptian Women's Dermatologic Society
acne vulgaris; glutathione peroxidase; malondialdehyde; oxidative stress