CED
Experimental dermatology • Original article
Clinical and Experimental Dermatology
Evaluation of antioxidant enzyme activity and antioxidant capacity in patients with newly diagnosed pemphigus vulgaris M. H. Javanbakht,1 M. Djalali,1 M. Daneshpazhooh,2 M. Zarei,1 M. R. Eshraghian,3 H. Derakhshanian1 and C. Chams-Davatchi2 1
Department of Cellular and Molecular Nutrition, School of Nutritional Sciences and Dietetics, Tehran University of Medical Sciences; Tehran,Iran; 2Autoimmune Bullous Diseases Research Center, Department of Dermatology, Razi Hospital, Tehran University of Medical Sciences, Tehran, Iran; and 3Department of Epidemiology and Biostatistics, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran doi:10.1111/ced.12489
Summary
Background. Pemphigus vulgaris (PV) is an autoimmune blistering disorder of the skin and/or mucosa. Increased levels of reactive oxygen species (ROS) were previously reported in PV. Aim. Because oxidative stress has an important role in the inflammatory process, we designed this study to evaluate the antioxidant status in patients with PV and to compare it with that of healthy controls (HCs). Methods. In this case–control study, 43 newly diagnosed patients with PV were compared with 58 HCs. The severity of the disease was estimated according to Harman scores. Erythrocyte glutathione peroxidase (GPx), superoxide dismutase (SOD), CAT and serum malondialdehyde (MDA) activities and total antioxidant capacity were measured. Data were analyzed by independent t-test. Results. Both groups were similar in sex, age and body mass index. Mean duration of disease was 5.6 months. Mean oral and skin severities were 1.79 and 2.3 respectively, based on Harman scores. SOD activity was not significantly different between groups (1003.30 39.96 vs. 1009.76 32.68 U/gHb). Levels were noticeably higher in patients with PV than in HCs for both GPx (52.13 2.85 vs. 36.63 1.49 U/gHb, respectively; P < 0.001) and CAT (205.69 8.10 vs. 130.26 6.80 kU/gHb, respectively; P < 0.001) activities, and CAT activity correlated with disease severity. In addition, patients had lower total antioxidant capacity than controls (3.39 0.06 vs. 3.72 0.09 mmol/L, P = 0.006). There was no noticeable difference in serum MDA between the two groups (P = 0.45). Conclusions. Patients with PV have significantly higher antioxidant enzyme activities and lower total antioxidant capacity compared with HCs. These data indicate the importance of improving antioxidant level in patients with pemphigus.
Introduction The term ‘pemphigus’ represents a group of chronic, autoimmune bullous diseases resulting from autoantibodies aimed at desmosomal proteins. This severe skin Correspondence: Dr Maryam Daneshpazhooh, Department of Dermatology, Razi Hospital, Vahdate_Eslami Square, 11996 Tehran, Iran E-mail: [email protected] Conflict of interest: the authors declare that they have no conflicts of interest. Accepted for publication 28 April 2014
ª 2015 British Association of Dermatologists
disorder is typically characterized by damaging epithelial adhesion molecules, especially desmogleins 1 and 3, which lead to loss of cell–cell contact (acantholysis).1 Pemphigus vulgaris (PV) is the most severe and prevalent type of pemphigus.2 The incidence of PV varies from 0.5 to 4 cases per 100 000 person-years. However, PV is more common in middle-aged patients, and is particularly common in the Mediterranean region, Middle East and India.3,4 The underlying causes of PV are not yet well recognized. However, it seems that genetic factors play a pivotal role, and that
Clinical and Experimental Dermatology
1
Evaluation of antioxidant enzyme activity and antioxidant capacity in PV M. H. Javanbakht et al.
environmental stimulants also influence the disease incidence. These environmental factors include drugs, chemical compounds, foods, physical agents and stress.5,6 Reactive oxygen species (ROS) are highly reactive molecules that are naturally generated in low concentrations during metabolism, but the amounts of ROS are generally controlled by the body’s antioxidant system.7 Impairment of balance between ROS generation and the antioxidant system (enzymatic and nonenzymatic) results in oxidative stress and cell damage, which is involved in the pathogenesis of many diseases.8,9 Increased oxidative stress has been reported previously in PV.10,11 Naziroglu et al.10 suggested the potential role of increased lipid peroxidation in the pathophysiology of PV and in causing a significant decrease in enzymatic [catalase (CAT) and glutathione peroxidase (GPx)] and nonenzymatic (vitamins A and E) antioxidants. It has also been shown that the oxidative stress index, comprising serum total oxidant capacity, lipid hydroperoxide and TAC, is higher in sera of patients with PV sera compared with that of healthy controls (HCs).11 Some other studies have reported increased nitric oxide as an inflammatory mediator and decreased uric acid as an endogenous protective antioxidant in patients with pemphigus.12,13 Because the current knowledge about the possible role of the oxidant/antioxidant system in pemphigus is limited and insufficient, the aim of the present study was to evaluate the antioxidant status in patients with PV, and compare it with that of HCs.
Methods The research protocol was approved by the ethics committee of Tehran University of Medical Sciences. Written informed consent was obtained from all participants (and in cases where participants were aged < 18 years, from the responsible parent or guardian) before enrolment. Subjects
In this case–control study, we enrolled 43 patients with PV (21 male, 22 female, mean SD age 4.23 11.5 years, range 16–69) and 58 HCs (22 male, 36 female; mean SD age 13.7 10.6 years, range 20–60). All patients were selected from the specialized dermatology departments of Razi Hospital, Tehran University of Medical Sciences. PV was diagnosed based on clinical, histological (suprabasal cleft and acantholysis) and
2
Clinical and Experimental Dermatology
immunological (intercellular deposits of IgG and/or C3) criteria for the disease.14 Patients were newly diagnosed, and had not received corticosteroids or any other systemic treatment for their disease. They were taking no supplements or contraceptive pills. Patients with any hepatic or renal disorders were excluded. HCs were all volunteers with no history of autoimmune or inflammatory disorders. Any potential participants taking supplements, corticosteroids or contraceptives, or with any cardiovascular diseases or diabetes, were excluded. Physical examination
Disease severity was assessed according to Harman scores.15 Patients were examined by a single expert physician in order to minimize observer bias resulting from interindividual variations. The extent of skin and oral mucosa involvement was measured and recorded separately, and scored as a number from 0 to 3. Both patients and controls were interviewed about their medical history and use of drugs and supplements. Their body weight (with minimal clothing) and height (without shoes and in upright position) were measured using a measuring scale (Seca GmbH & co KG, Hamburg, Germany) with an accuracy of 0.1 kg and 5 mm respectively. Body mass index (BMI) was calculated as weight in kg divided by height in cm2. Blood collection
Blood samples were collected from the antecubital vein in the morning after 12–14 h of fasting, and were centrifuged at 1000 g for 10 min at 4 °C. The separated plasma was immediately stored at 80 °C until biochemical analysis. The erythrocytes were washed three times with 0.9% saline, then stored at 80 °C until required. Enzyme activity analysis
CAT activity was tested colourimetrically (cat. no. STA341; Cell Biolabs, Inc. San Diego, CA, USA) according to the manufacturer’s instructions. GPx and superoxide dismutase (SOD) were measured with kits based on enzymatic and colorimetric methods respectively (cat. nos RS505 and SD125, respectively; Randox Labs Ltd. Crumlin, Co. Antrim, UK). Malondialdehyde (MDA; marker of lipid peroxidation) and TAC were assessed in sera samples using ELISA kits (cat. nos STA332, STA310 and STA360, respectively; Cell Biolabs).
ª 2015 British Association of Dermatologists
Evaluation of antioxidant enzyme activity and antioxidant capacity in PV M. H. Javanbakht et al.
Statistical analysis
All data are expressed as mean SD. Normality of data distribution was checked by the Kolmogorov– Smirnov test. Groups were compared with independent sample t-test or v² test for continuous and categorical data, respectively. Correlations were measured using Pearson coefficient or Spearman q, depending on the type of variable. P < 0.05 was considered statistically significant. All statistical analyses was performed using the SPSS software (V11.5, SPSS Inc, Chicago, IL, USA).
Enzyme SOD r P CAT r P GPx r P
Disease duration*
Disease severity†
0.03 0.85
0.03 0.86
0.12 0.44
0.33 0.03‡
0.12 0.48
0.04 0.79
CAT, catalase; GPx, glutathione peroxidase; SOD, superoxide dismutase. *Pearson correlation coefficient; †Spearman q; ‡significant.
Results Mean duration of disease group was 5.62 0.17 months. Mean severity score of skin and oral involvement was 2.3 0.15 and 1.79 0.17, respectively. There were no differences in confounding factors such as sex distribution, age, weight, height or BMI between patients and HCs. There was no noticeable difference in serum MDA between the two groups (P = 0.45). However, TAC was conspicuously decreased in the PV group (P < 0.01) (Table 1). Comparing the activity of antioxidant enzymes in erythrocytes of patients and HCs showed markedly higher activity of CAT and GPx in patients with PV (P < 0.001 for both). However, there was no difference in SOD levels between groups (P = 0.9) (Table 1). The correlation between all these parameters with the duration and severity of disease was assessed, and CAT activity was found to be significantly correlated with severity of PV (Table 2).
Discussion Both genetic and environmental factors are involved in pemphigus as in other autoimmune diseases.16
Table 1 Evaluation of antioxidant capacity and antioxidant enzymes. Patients MDA, nmol/mL TAC, mmol/L SOD, U/gHb CAT, kU/gHb GPx, U/gHb
Table 2 Correlation of antioxidant enzyme activity with duration and severity of pemphigus
2.97 3.39 1003.30 205.69 52.13
Controls
0.12 0.06 39.96 8.10 2.85
3.09 3.72 1009.76 130.26 36.63
P
0.09 0.09 32.68 6.80 1.49
0.45 < 0.01* 0.90 < 0.001* < 0.001*
CAT, catalase; GPx, glutathione peroxidase; MDA, malondialdehyde; SOD, superoxide dismutase; TAC, total antioxidant capacity. Data are presented as mean SD. Differences between groups were assessed by independent samples t-test. *Significant.
ª 2015 British Association of Dermatologists
Although antibodies against desmosomal proteins are considered the main pathogenic factor in the development of this inflammatory disease, the role of the oxidant/antioxidant system deserves more research. Because there are few studies discussing the association between pemphigus and antioxidants, and the results of those are inconclusive, we designed and performed this research to evaluate antioxidant status in patients with PV and to compare it with that of HCs. Our findings show that the activity of antioxidant enzymes such as CAT and GPx was significantly increased in patients newly diagnosed with PV, whereas no change was observed in the level of SOD. It is well established that skin has markedly high exposure to ROS. Enzymatic antioxidants such as GPx, CAT and SOD act synergistically to protect skin cells against ROS damage. Thus, any change in the activity of these enzymes might be related to an imbalance in oxidant/antioxidant system (oxidative stress).17 The conspicuous increase in CAT and GPx activity in patients with pemphigus is indicative of higher ROS production and skin injury, followed by an increase in antioxidant enzymes as a protective mechanism. The significant correlation between CAT activity and disease severity highlights the importance of CAT as an indicator of oxidative imbalance. Our data are consistent with those of Abida et al.18 who reported a noticeable rise in CAT activity as a hydrogen peroxide-inactivating enzyme in both subjects with PV and those with pemphigus foliaceus. Those authors concluded that CAT activity was elevated to avoid ROS-induced cellular damage. Increased CAT activity has also been observed in other inflammatory diseases such as rheumatoid arthritis.19 To
Clinical and Experimental Dermatology
3
Evaluation of antioxidant enzyme activity and antioxidant capacity in PV M. H. Javanbakht et al.
our knowledge, there is only one other publication on this subject, which was conducted by Naziroglu et al.10 Their results provided evidence for a potential role in PV of increased MDA as a lipid peroxidation marker and of decreased antioxidant enzymes (including CAT and GPx in red blood cells). According to those authors, inefficiency of the enzymatic antioxidant system may have resulted in enhanced lipid peroxidation. However, in our study, we found that the increased level of antioxidant enzymes compensated for the increased ROS generation. Therefore, no significant change was shown in MDA and lipid peroxidation in patients with PV. This might be due to the short duration of disease, as our patients were all newly diagnosed. In addition, we found that TAC was markedly lower in patients compared with HCs. TAC is an integrated parameter that represents the cumulative action of all known and unknown antioxidants in plasma and body fluids and their synergistic interaction. Therefore, TAC gives insight into the in vivo oxidant/antioxidant balance. It is well recognized that lower TAC levels are associated with greater cell death.20 The significant reduction of TAC in patients with pemphigus might be due to higher ROS production or lower antioxidant food intake. As the role of antioxidant nutrients in fighting against oxidative stress and improving antioxidant capacity is well-established from previous studies,21–23 consumption of diets rich in fruits and vegetables is particularly recommended for patients with pemphigus.
Conclusion Taken together, our findings suggest that oxidative stress plays a pivotal role in the pathogenesis of PV. In our newly diagnosed patients, increased activity of antioxidant enzymes, including CAT and GPx, prevented marked lipid peroxidation. However, the significant decrease in TAC confirms that there is elevated ROS generation and antioxidant depletion in these patients. More studies are needed to elucidate whether improving the antioxidant status of patients with pemphigus could decrease the severity and adverse effects of this life-threatening disease.
Acknowledgements We are grateful to all the study participants. This study was granted by Tehran University of Medical Sciences & Health Services (project no: 6640).
4
Clinical and Experimental Dermatology
What’s already known about this topic? Increased oxidative stress has been reported
previously in PV.
What does this study add? We evaluated the antioxidant enzyme activity
in patients with PV, and compared it with that of HCs. Patients with PV had significantly higher antioxidant enzyme activities and lower TAC compared with HCs.
References 1 Bystryn J-C, Rudolph JL. Pemphigus. Lancet 2005; 366: 61–73. 2 Chams-Davatchi C, Valikhani M, Daneshpazhooh M et al. Pemphigus: analysis of 1209 cases. Int J Dermatol 2005; 44: 470–6. 3 Groves RW. Pemphigus: a brief review. Clin Med 2009; 9: 371–5. 4 Daneshpazhooh M, Chams-Davatchi C, Payandemehr P et al. Spectrum of autoimmune bullous diseases in Iran: a 10-year review. Int J Dermatol 2012; 51: 35–41. 5 Tron F, Gilbert D, Mouquet H et al. Genetic factors in pemphigus. J Autoimmun 2005; 24: 319–28. 6 Ruocco V, Ruocco E. Pemphigus and environmental factors. G Ital Dermatol Venereol 2003; 138: 299–310. 7 Valko M, Leibfritz D, Moncol J et al. Free radicals and antioxidants in normal physiological functions and human disease. Int J Biochem Cell Biol 2007; 39: 44–84. 8 Sies H. Role of reactive oxygen species in biological processes. Klin Wochenschr 1991; 69: 965–8. 9 Apel K, Hirt H. Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 2004; 55: 373–99. 10 Naziro glu M, K€ okcßam I, SßimsßSßek Η, Karakılcßık A. Lipid peroxidation and antioxidants in plasma and red blood cells from patients with pemphigus vulgaris. J Basic Clin Physiol Pharmacol 2003; 14: 31–42. 11 Yesilova Y, Ucmak D, Selek S et al. Oxidative stress index may play a key role in patients with pemphigus vulgaris. J Eur Acad Dermatol Venereol 2013; 27: 465–7. 12 Siebra M, Santos M, Almeida T et al. Evidence for the participation of nitric oxide in pemphigus. Braz J Med Biol Res 2006; 39: 671–5.
ª 2015 British Association of Dermatologists
Evaluation of antioxidant enzyme activity and antioxidant capacity in PV M. H. Javanbakht et al.
13 Yousefi M, Rahimi H, Barikbin B et al. Uric acid: a new antioxidant in patients with pemphigus vulgaris. Indian J Dermatol 2011; 56: 278–81. 14 Mihai S, Sitaru C. Immunopathology and molecular diagnosis of autoimmune bullous diseases. J Cell Mol Med 2007; 11: 462–81. 15 Harman K, Seed P, Gratian M et al. The severity of cutaneous and oral pemphigus is related to desmoglein 1 and 3 antibody levels. Br J Dermatol 2001; 144: 775–80. 16 Wang G-Q, Xu H, Wang Y-K et al. Higher prevalence of human herpesvirus 8 DNA sequence and specific IgG antibodies in patients with pemphigus in China. J Am Acad Dermatol 2005; 52: 460–7. 17 Bickers DR, Athar M. Oxidative stress in the pathogenesis of skin disease. J Invest Dermatol 2006; 126: 2565–75. 18 Abida O, Mansour RB, Gargouri B et al. Catalase and lipid peroxidation values in serum of tunisian patients with pemphigus vulgaris and foliaceus. Biol Trace Elem Res 2012; 150: 74–80.
ª 2015 British Association of Dermatologists
19 Biemond P, Swaak AJ, Koster JF. Protective factors against oxygen free radicals and hydrogen peroxide in rheumatoid arthritis synovial fluid. Arthritis Rheum 1984; 27: 760–5. 20 Kusano C, Ferrari B. Total antioxidant capacity: a biomarker in biomedical and nutritional studies. J Cell Mol Biol 2008; 7: 1–15. 21 Ferrari CK. Functional foods, herbs and nutraceuticals: towards biochemical mechanisms of healthy aging. Biogerontology 2004; 5: 275–89. 22 Pitsavos C, Panagiotakos DB, Tzima N et al. Adherence to the Mediterranean diet is associated with total antioxidant capacity in healthy adults: the ATTICA study. Am J Clin Nutr 2005; 82: 694–9. 23 Tyssandier V, Feillet-Coudray C, Caris-Veyrat C et al. Effect of tomato product consumption on the plasma status of antioxidant microconstituents and on the plasma total antioxidant capacity in healthy subjects. J Am Coll Nutr 2004; 23: 148–56.
Clinical and Experimental Dermatology
5
Experimental dermatology • Original article
Clinical and Experimental Dermatology
Evaluation of antioxidant enzyme activity and antioxidant capacity in patients with newly diagnosed pemphigus vulgaris M. H. Javanbakht,1 M. Djalali,1 M. Daneshpazhooh,2 M. Zarei,1 M. R. Eshraghian,3 H. Derakhshanian1 and C. Chams-Davatchi2 1
Department of Cellular and Molecular Nutrition, School of Nutritional Sciences and Dietetics, Tehran University of Medical Sciences; Tehran,Iran; 2Autoimmune Bullous Diseases Research Center, Department of Dermatology, Razi Hospital, Tehran University of Medical Sciences, Tehran, Iran; and 3Department of Epidemiology and Biostatistics, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran doi:10.1111/ced.12489
Summary
Background. Pemphigus vulgaris (PV) is an autoimmune blistering disorder of the skin and/or mucosa. Increased levels of reactive oxygen species (ROS) were previously reported in PV. Aim. Because oxidative stress has an important role in the inflammatory process, we designed this study to evaluate the antioxidant status in patients with PV and to compare it with that of healthy controls (HCs). Methods. In this case–control study, 43 newly diagnosed patients with PV were compared with 58 HCs. The severity of the disease was estimated according to Harman scores. Erythrocyte glutathione peroxidase (GPx), superoxide dismutase (SOD), CAT and serum malondialdehyde (MDA) activities and total antioxidant capacity were measured. Data were analyzed by independent t-test. Results. Both groups were similar in sex, age and body mass index. Mean duration of disease was 5.6 months. Mean oral and skin severities were 1.79 and 2.3 respectively, based on Harman scores. SOD activity was not significantly different between groups (1003.30 39.96 vs. 1009.76 32.68 U/gHb). Levels were noticeably higher in patients with PV than in HCs for both GPx (52.13 2.85 vs. 36.63 1.49 U/gHb, respectively; P < 0.001) and CAT (205.69 8.10 vs. 130.26 6.80 kU/gHb, respectively; P < 0.001) activities, and CAT activity correlated with disease severity. In addition, patients had lower total antioxidant capacity than controls (3.39 0.06 vs. 3.72 0.09 mmol/L, P = 0.006). There was no noticeable difference in serum MDA between the two groups (P = 0.45). Conclusions. Patients with PV have significantly higher antioxidant enzyme activities and lower total antioxidant capacity compared with HCs. These data indicate the importance of improving antioxidant level in patients with pemphigus.
Introduction The term ‘pemphigus’ represents a group of chronic, autoimmune bullous diseases resulting from autoantibodies aimed at desmosomal proteins. This severe skin Correspondence: Dr Maryam Daneshpazhooh, Department of Dermatology, Razi Hospital, Vahdate_Eslami Square, 11996 Tehran, Iran E-mail: [email protected] Conflict of interest: the authors declare that they have no conflicts of interest. Accepted for publication 28 April 2014
ª 2015 British Association of Dermatologists
disorder is typically characterized by damaging epithelial adhesion molecules, especially desmogleins 1 and 3, which lead to loss of cell–cell contact (acantholysis).1 Pemphigus vulgaris (PV) is the most severe and prevalent type of pemphigus.2 The incidence of PV varies from 0.5 to 4 cases per 100 000 person-years. However, PV is more common in middle-aged patients, and is particularly common in the Mediterranean region, Middle East and India.3,4 The underlying causes of PV are not yet well recognized. However, it seems that genetic factors play a pivotal role, and that
Clinical and Experimental Dermatology
1
Evaluation of antioxidant enzyme activity and antioxidant capacity in PV M. H. Javanbakht et al.
environmental stimulants also influence the disease incidence. These environmental factors include drugs, chemical compounds, foods, physical agents and stress.5,6 Reactive oxygen species (ROS) are highly reactive molecules that are naturally generated in low concentrations during metabolism, but the amounts of ROS are generally controlled by the body’s antioxidant system.7 Impairment of balance between ROS generation and the antioxidant system (enzymatic and nonenzymatic) results in oxidative stress and cell damage, which is involved in the pathogenesis of many diseases.8,9 Increased oxidative stress has been reported previously in PV.10,11 Naziroglu et al.10 suggested the potential role of increased lipid peroxidation in the pathophysiology of PV and in causing a significant decrease in enzymatic [catalase (CAT) and glutathione peroxidase (GPx)] and nonenzymatic (vitamins A and E) antioxidants. It has also been shown that the oxidative stress index, comprising serum total oxidant capacity, lipid hydroperoxide and TAC, is higher in sera of patients with PV sera compared with that of healthy controls (HCs).11 Some other studies have reported increased nitric oxide as an inflammatory mediator and decreased uric acid as an endogenous protective antioxidant in patients with pemphigus.12,13 Because the current knowledge about the possible role of the oxidant/antioxidant system in pemphigus is limited and insufficient, the aim of the present study was to evaluate the antioxidant status in patients with PV, and compare it with that of HCs.
Methods The research protocol was approved by the ethics committee of Tehran University of Medical Sciences. Written informed consent was obtained from all participants (and in cases where participants were aged < 18 years, from the responsible parent or guardian) before enrolment. Subjects
In this case–control study, we enrolled 43 patients with PV (21 male, 22 female, mean SD age 4.23 11.5 years, range 16–69) and 58 HCs (22 male, 36 female; mean SD age 13.7 10.6 years, range 20–60). All patients were selected from the specialized dermatology departments of Razi Hospital, Tehran University of Medical Sciences. PV was diagnosed based on clinical, histological (suprabasal cleft and acantholysis) and
2
Clinical and Experimental Dermatology
immunological (intercellular deposits of IgG and/or C3) criteria for the disease.14 Patients were newly diagnosed, and had not received corticosteroids or any other systemic treatment for their disease. They were taking no supplements or contraceptive pills. Patients with any hepatic or renal disorders were excluded. HCs were all volunteers with no history of autoimmune or inflammatory disorders. Any potential participants taking supplements, corticosteroids or contraceptives, or with any cardiovascular diseases or diabetes, were excluded. Physical examination
Disease severity was assessed according to Harman scores.15 Patients were examined by a single expert physician in order to minimize observer bias resulting from interindividual variations. The extent of skin and oral mucosa involvement was measured and recorded separately, and scored as a number from 0 to 3. Both patients and controls were interviewed about their medical history and use of drugs and supplements. Their body weight (with minimal clothing) and height (without shoes and in upright position) were measured using a measuring scale (Seca GmbH & co KG, Hamburg, Germany) with an accuracy of 0.1 kg and 5 mm respectively. Body mass index (BMI) was calculated as weight in kg divided by height in cm2. Blood collection
Blood samples were collected from the antecubital vein in the morning after 12–14 h of fasting, and were centrifuged at 1000 g for 10 min at 4 °C. The separated plasma was immediately stored at 80 °C until biochemical analysis. The erythrocytes were washed three times with 0.9% saline, then stored at 80 °C until required. Enzyme activity analysis
CAT activity was tested colourimetrically (cat. no. STA341; Cell Biolabs, Inc. San Diego, CA, USA) according to the manufacturer’s instructions. GPx and superoxide dismutase (SOD) were measured with kits based on enzymatic and colorimetric methods respectively (cat. nos RS505 and SD125, respectively; Randox Labs Ltd. Crumlin, Co. Antrim, UK). Malondialdehyde (MDA; marker of lipid peroxidation) and TAC were assessed in sera samples using ELISA kits (cat. nos STA332, STA310 and STA360, respectively; Cell Biolabs).
ª 2015 British Association of Dermatologists
Evaluation of antioxidant enzyme activity and antioxidant capacity in PV M. H. Javanbakht et al.
Statistical analysis
All data are expressed as mean SD. Normality of data distribution was checked by the Kolmogorov– Smirnov test. Groups were compared with independent sample t-test or v² test for continuous and categorical data, respectively. Correlations were measured using Pearson coefficient or Spearman q, depending on the type of variable. P < 0.05 was considered statistically significant. All statistical analyses was performed using the SPSS software (V11.5, SPSS Inc, Chicago, IL, USA).
Enzyme SOD r P CAT r P GPx r P
Disease duration*
Disease severity†
0.03 0.85
0.03 0.86
0.12 0.44
0.33 0.03‡
0.12 0.48
0.04 0.79
CAT, catalase; GPx, glutathione peroxidase; SOD, superoxide dismutase. *Pearson correlation coefficient; †Spearman q; ‡significant.
Results Mean duration of disease group was 5.62 0.17 months. Mean severity score of skin and oral involvement was 2.3 0.15 and 1.79 0.17, respectively. There were no differences in confounding factors such as sex distribution, age, weight, height or BMI between patients and HCs. There was no noticeable difference in serum MDA between the two groups (P = 0.45). However, TAC was conspicuously decreased in the PV group (P < 0.01) (Table 1). Comparing the activity of antioxidant enzymes in erythrocytes of patients and HCs showed markedly higher activity of CAT and GPx in patients with PV (P < 0.001 for both). However, there was no difference in SOD levels between groups (P = 0.9) (Table 1). The correlation between all these parameters with the duration and severity of disease was assessed, and CAT activity was found to be significantly correlated with severity of PV (Table 2).
Discussion Both genetic and environmental factors are involved in pemphigus as in other autoimmune diseases.16
Table 1 Evaluation of antioxidant capacity and antioxidant enzymes. Patients MDA, nmol/mL TAC, mmol/L SOD, U/gHb CAT, kU/gHb GPx, U/gHb
Table 2 Correlation of antioxidant enzyme activity with duration and severity of pemphigus
2.97 3.39 1003.30 205.69 52.13
Controls
0.12 0.06 39.96 8.10 2.85
3.09 3.72 1009.76 130.26 36.63
P
0.09 0.09 32.68 6.80 1.49
0.45 < 0.01* 0.90 < 0.001* < 0.001*
CAT, catalase; GPx, glutathione peroxidase; MDA, malondialdehyde; SOD, superoxide dismutase; TAC, total antioxidant capacity. Data are presented as mean SD. Differences between groups were assessed by independent samples t-test. *Significant.
ª 2015 British Association of Dermatologists
Although antibodies against desmosomal proteins are considered the main pathogenic factor in the development of this inflammatory disease, the role of the oxidant/antioxidant system deserves more research. Because there are few studies discussing the association between pemphigus and antioxidants, and the results of those are inconclusive, we designed and performed this research to evaluate antioxidant status in patients with PV and to compare it with that of HCs. Our findings show that the activity of antioxidant enzymes such as CAT and GPx was significantly increased in patients newly diagnosed with PV, whereas no change was observed in the level of SOD. It is well established that skin has markedly high exposure to ROS. Enzymatic antioxidants such as GPx, CAT and SOD act synergistically to protect skin cells against ROS damage. Thus, any change in the activity of these enzymes might be related to an imbalance in oxidant/antioxidant system (oxidative stress).17 The conspicuous increase in CAT and GPx activity in patients with pemphigus is indicative of higher ROS production and skin injury, followed by an increase in antioxidant enzymes as a protective mechanism. The significant correlation between CAT activity and disease severity highlights the importance of CAT as an indicator of oxidative imbalance. Our data are consistent with those of Abida et al.18 who reported a noticeable rise in CAT activity as a hydrogen peroxide-inactivating enzyme in both subjects with PV and those with pemphigus foliaceus. Those authors concluded that CAT activity was elevated to avoid ROS-induced cellular damage. Increased CAT activity has also been observed in other inflammatory diseases such as rheumatoid arthritis.19 To
Clinical and Experimental Dermatology
3
Evaluation of antioxidant enzyme activity and antioxidant capacity in PV M. H. Javanbakht et al.
our knowledge, there is only one other publication on this subject, which was conducted by Naziroglu et al.10 Their results provided evidence for a potential role in PV of increased MDA as a lipid peroxidation marker and of decreased antioxidant enzymes (including CAT and GPx in red blood cells). According to those authors, inefficiency of the enzymatic antioxidant system may have resulted in enhanced lipid peroxidation. However, in our study, we found that the increased level of antioxidant enzymes compensated for the increased ROS generation. Therefore, no significant change was shown in MDA and lipid peroxidation in patients with PV. This might be due to the short duration of disease, as our patients were all newly diagnosed. In addition, we found that TAC was markedly lower in patients compared with HCs. TAC is an integrated parameter that represents the cumulative action of all known and unknown antioxidants in plasma and body fluids and their synergistic interaction. Therefore, TAC gives insight into the in vivo oxidant/antioxidant balance. It is well recognized that lower TAC levels are associated with greater cell death.20 The significant reduction of TAC in patients with pemphigus might be due to higher ROS production or lower antioxidant food intake. As the role of antioxidant nutrients in fighting against oxidative stress and improving antioxidant capacity is well-established from previous studies,21–23 consumption of diets rich in fruits and vegetables is particularly recommended for patients with pemphigus.
Conclusion Taken together, our findings suggest that oxidative stress plays a pivotal role in the pathogenesis of PV. In our newly diagnosed patients, increased activity of antioxidant enzymes, including CAT and GPx, prevented marked lipid peroxidation. However, the significant decrease in TAC confirms that there is elevated ROS generation and antioxidant depletion in these patients. More studies are needed to elucidate whether improving the antioxidant status of patients with pemphigus could decrease the severity and adverse effects of this life-threatening disease.
Acknowledgements We are grateful to all the study participants. This study was granted by Tehran University of Medical Sciences & Health Services (project no: 6640).
4
Clinical and Experimental Dermatology
What’s already known about this topic? Increased oxidative stress has been reported
previously in PV.
What does this study add? We evaluated the antioxidant enzyme activity
in patients with PV, and compared it with that of HCs. Patients with PV had significantly higher antioxidant enzyme activities and lower TAC compared with HCs.
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Evaluation of antioxidant enzyme activity and antioxidant capacity in PV M. H. Javanbakht et al.
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