Uric Acid: a new antioxidant in patients with pemphigus vulgaris.

BACKGROUND: Increased reactive oxygen species (ROS) and lipid peroxidation are seen in many dermatologic disorders, for example, atopic dermatitis, psoriasis, vitiligo, acne vulgaris, pemphigus vulgaris (PV), lichen planus, and alopecia areata. ROS has an important role in the inflammation process. In PV, increased production of ROS leads to decline of antioxidants in plasma and red blood cells which results in oxidative stress. We aimed to evaluate the level of these antioxidants in PV patients and compare it to the controls.
MATERIALS AND METHODS: Among patients attending the dermatology clinics, 30 patients with PV, who had never been on treatment, were enrolled to the study. The control group consisted of 30 age- and sex-matched healthy non-smoker individuals. Venous blood was collected from the subjects for the evaluation of plasma levels of glutathione peroxidase, vitamin C, selenium, bilirubin, and uric acid.
RESULTS: Age mean and standard deviation of the patients (40.83, 12.74) was comparable to the controls (41.96, 13.08). Mean level of uric acid was significantly lower in PV patients compared to the controls (P = 0.006). Other antioxidants were not different between the two groups. Uric acid of the patients with mucosal involvement was significantly lower than patients with mucocutaneous involvement (P = 0.049). LIMITATIONS: The blood level of other antioxidants (e.g. malondialdehyde) was not evaluated.
CONCLUSIONS: Uric acid as an antioxidant in our study had similar changes to previous studies in the field of other diseases but selenium, bilirubin, and glutathione peroxidase did not differ between patients and controls.

Introduction

Pemphigus vulgaris (PV) is an autoimmune blistering disease of the skin and mucosal membranes.[1-3] Inflammatory pathways lead to neutrophil activation and release of reactive oxygen species (ROS), which mostly implicate their adverse effect through lipid peroxidation, inflammatory cytokines, and the disruption of dermal–epidermal junction in PV.[3-5] Antioxidants are physiological defense mechanisms in alleviating ROS toxicity in human skin problems.[45] The effect of serum/RBC selenium, bilirubin, and uric acid as potential antioxidants were measured in several diseases.[6-15] There have been a few studies considering antioxidants levels in patients with PV.[16-21] We investigated the levels of several antioxidants in PV patients.

Materials and Methods

Among patients attending the dermatology clinics of Shohada-e-Tajrish and Loghman-e-Hakim Hospitals, Tehran, Iran, 30 patients with PV, diagnosed with biopsy and direct immunofluorescence, who had never been on treatment for the disease were enrolled to the study. Exclusion criteria were smoking, alcohol consumption, treatment with oral steroids or any immunosuppressives, history of trauma or surgery in the last month, using selenium or vitamin C containing multivitamins, and any systemic disease other than PV. Thirty age- and sex-matched healthy individuals served as controls. The patients were divided into three groups, namely, cutaneous involvement, mucocutaneous involvement, and mucosal involvement.

Pemphigus vulgaris severity was assessed using autoimmune bullous skin disorder intensity score (ABSIS). This is a novel system for the scoring of bullous disorders, for example, PV.[22] This system considers both the quality and quantity of the lesions on skin and oral mucosa. The extent of skin involvement (skin score) is expressed as a number from 0 to 150. Oral involvement quantity is scored from 0 to 11 (Ms1 score) and the quality from 0 to 45 (Ms2 score). This score does not consider the involvement of genital, nasal, and ocular mucosa. So, we decided to add them to the oral quantity score of ABSIS to make a new total mucosal (TM) score of 0 to 14. This new score can express the extent of mucosal involvement of the whole body.

After taking written consent from the subjects, 10 ml of peripheral venous blood was taken from them to evaluate the plasma levels of GPX, vitamin C, selenium, bilirubin, and uric acid.

Sample GPX activity was assayed by enzymatic colorimetric method (glutathione peroxidase activity kit, Cayman Chemical Company, MI, USA). Coefficient of variation of intra-assay was 2.2%. Sera uric acid and bilirubin (direct and total) were determined by enzymatic and chemical colorimetric methods, respectively (uric acid determination kit and bilirubin determination kit, Pars Azmoun Co., Tehran, Iran). The assay sensitivity and intra-assay coefficient of variation was 0.3 mg/dl and 1.4% for uric acid and 0.07 mg/dl and 2.8% for bilirubin, respectively. Samples’ vitamin C content was also determined by colorimetric method (Vitamin C assay kit, Jaica, Shizouka, Japan). The assay sensitivity was 0.04 mg/dl and its intra-assay coefficient of variation was 5.9%. Sera and red blood cells selenium content determined by graphite furnace atomic absorption spectrometry (GFAAS) method (Atomic absorption spectrometer, Graphite furnace, Shimadsu, AA-680, Japan). The intra-assay coefficient of variation was 4.3%.

Results are expressed as mean (±SD). Independent samples’ t-test was applied to compare the means of continuous variables. When the assumptions of applying t-test for comparing groups were not satisfied, the nonparametric Mann-Whitney U-test was used. Spearman correlation test was used to detect the linear relationship between variables. Statistical analysis was performed using the statistical software SPSS 16.0.0. (SPSS Inc. Chicago, IL, USA). P ≤ 0.05 was considered significant.

Results

There were 30 subjects, 22 women (72.33%) and 8 men (26.67%), in each group. The mean ages of patients and control subjects were 40.83 (±12.74) and 41.96 (±13.08), respectively.

In 19 patients (63.33%), PV affected both mucous membranes and the skin. In 8 patients (26.67%) there were only mucous membrane involvement and in 3 patients (10%) the skin was only affected.

Among 27 patients with mucosal and mucocutaneous PV, genital mucosa was involved in 7 (25.9%), ocular mucosa in 8 (29.63%), nasal mucosa in 16 (59.26%), pharyngeal mucosa in 14 (51.8%), and oral mucosa in 25 (92.6%).

The mean duration of the disease was 6.41 (±6.14) months with median of 4.5 months (range 0.5–24 months). In patients with only mucosal lesions, the mean duration of disease was 7.12 (±3.91) months with the median of 7 months (range 2-13 months), and in patients with both mucous membranes and skin involvements it was 5.58 (±5.93) months with median of 4 months (range 0.5-24 months). Mann-Whitney U test showed no significant difference in duration of the disease between these two patients’ groups (P = 0.17).

The median SI score for 22 patients with skin involvement was 4 (range 0.5–105).

There was a statistical significant difference between mean uric acid levels of two groups (P = 0.006), i.e. mean uric acid level was significantly decreased in the patients compared with the controls [Table 1]. No statistical difference was detected between patient and control groups in mean levels of vitamin C, serum selenium, GPX, total bilirubin, direct bilirubin and RBC selenium [Table 1].

Table 1

Mean (standard deviation) of serum antioxidant levels in case and control groups

Using Spearman correlation test, there were no significant differences between skin score (P ≤ 0.09) and the extent of skin involvement (P ≤ 0.10) with antioxidant levels in patients with skin lesions. Also, no statistical significant relationships were observed between Ms1 (P = 0.20), Ms2 (P = 0.10), and TM (P = 0.20) scores with antioxidant levels in patients with mucosal or mucocutaneous involvement [Table 2].

Table 2

Descriptive statistics of ABSIS score of patients with pemphigus vulgaris

Mann-Whitney U-test showed a significant decrease in uric acid levels of patients with mucosal involvement in comparison to patients with mucocutaneous involvement (P = 0.049). No significant differences were observed between other antioxidant levels of these patients [Table 3].

Table 3

Mean (standard deviation) of serum antioxidant levels in patients with mucosal and mucocutaneous involvement

Discussion

Yet there have been two studies discussing the relationship between PV and antioxidants (vitamins and enzymes).[323] Although the pathophysiology of PV is nearly understood and the interaction between desmoglein as autoantigens and autoantibodies is in the prior line of the hypothesis, the role of antioxidants decline and redox state sequencing further oxidative damage in the formation of PV lesions is unknown.[124-26]

Uric acid is found to be a protective antioxidant defense in patients with ischemic heart disease and other fields of surveillance but its role in PV patients is not yet investigated.[7827] Uric acid with antioxidant characteristics was measured for the first time in our study and the meaningful decline in serum levels of uric acid was found in the patients which can verify the antioxidant effect of this factor. GPX in plasma samples did not change significantly in two groups, same as what was seen in the study by Naziroğlu et al.[3] While there is a meaningful drop in serum levels of antioxidant vitamins in the aforementioned study, there was no change detected in serum levels of vitamin C in our study population. Apart from our survey, the study by Naziroğlu is the only study to focus on lipid peroxidation and antioxidant effect of vitamins in PV patients. Although this study supports the hypothesis of imbalance between ROS production and antioxidant defense system through amplifying the role of vitamins, GPX and MDA, our study postulates the effect of uric acid as an antioxidant mediator in PV patients.

In several studies selenium, through its impact on the activity of erythrocyte GPX, is beneficial in establishing the severity of inflammation in different diseases and trauma patients.[1213152829] In the light of the latter hypothesis, the serum and RBC selenium was measured in our study for the first time but no significant change was investigated in patients compared to healthy individuals.

ABSIS system is proposed by Pfütze et al. for a proper scoring of PV.[22] Although comprehensive, this system has some inadequacies in the scoring of mucosal lesions. Oral involvement quantity is portrayed as a number from 0 to 11 according to the 11 sites labeled in the oral cavity. This score does not consider the involvement of genital, nasal, and ocular mucosa. Consequently, we decided to add these three sites to the oral quantity score of ABSIS to make a new TM score of 0 to 14. The new score suggested in our study can improve the accuracy of ABSIS in scoring PV lesions.

In conclusion, uric acid decrease in PV patients emphasizes the underlying hypothesis in the progression of the disease and can highlight the impact of free radicals and leading oxidative damage in PV lesions. Although vitamin C and selenium, as antioxidant elements, did not show any pivotal change in our study, vitamin and enzymatic antioxidants need to be evaluated in forthcoming studies with larger sample size.