|Year : 2014 | Volume
| Issue : 4 | Page : 230-236
Estimation of nitric oxide and malondialdehyde in serum and saliva of patients with oral lichen planus
VP Jayasekharan1, R Ramya2, K Rajkumar2, T Dinesh Kumar2, G Nandhini2, S Satish Kumar3
1 Department of Oral and Maxillofacial Pathology and Microbiology, St. Gregorios Dental College, Kothamangalam, Kerala, India
2 Department of Oral and Maxillofacial Pathology and Microbiology, SRM Dental College, SRM University, Chennai, Tamil Nadu, India
3 Department of Oral and Maxillofacial Pathology and Microbiology, Sri Venkateswara Dental College and Hospital, Thalambur, Chennai, Tamil Nadu, India
|Date of Web Publication||20-Nov-2014|
V P Jayasekharan
Department of Oral and Maxillofacial Pathology and Microbiology, St. Gregorios Dental College, Chelad, Kothamangalam - 686 681, Kerala
Aim: The aim of this study is to evaluate the levels of malondialdehyde (MDA) and nitric oxide (NO) in serum and saliva of patients with oral lichen planus (OLP) and to compare the levels. Materials and Methods: The study included 10 cases of OLP patients which formed the study group and 10 cases of healthy individuals, which formed the controls. The saliva sample was diluted with 10 ml of phosphate buffered saline in order to neutralize the pH. After diluting, the mixture was centrifuged for 5 min at 3000 rpm and the supernatant fluid was stored at −80°C until use. Blood was allowed to clot and was centrifuged for 5 min at 3000 rpm. The clear serum was separated and stored at −80°C until analysis. Statistical Analysis: The analysis was done using SPSS software. Mean and standard deviations were analyzed using the independent samples t-test to compare the mean values of NO and MDA assay. Results: The levels of NO and MDA were significantly higher in serum and saliva of cases with lichen planus than in normal and increase in level of NO was significantly higher than the increase in the level of MDA. Conclusion: Salivary levels of NO and MDA were found to be significantly higher than serum levels, which suggested that saliva can be used as an alternate and effective diagnostic tool in evaluating the oxidative stress status of an individual and antioxidants may be used to reverse the oxidative stress status in lichen planus.
Keywords: Free radical, oxidative stress, peroxidation, reactive oxygen species
|How to cite this article:|
Jayasekharan V P, Ramya R, Rajkumar K, Kumar T D, Nandhini G, Kumar S S. Estimation of nitric oxide and malondialdehyde in serum and saliva of patients with oral lichen planus. SRM J Res Dent Sci 2014;5:230-6
|How to cite this URL:|
Jayasekharan V P, Ramya R, Rajkumar K, Kumar T D, Nandhini G, Kumar S S. Estimation of nitric oxide and malondialdehyde in serum and saliva of patients with oral lichen planus. SRM J Res Dent Sci [serial online] 2014 [cited 2018 Nov 14];5:230-6. Available from: http://www.srmjrds.in/text.asp?2014/5/4/230/145123
| Introduction|| |
Oral lichen planus (OLP) is a relatively common disorder of the stratified squamous epithelia. It is a chronic, immunological, muco cutaneous disease with a wide range of clinical manifestations.  The disease has most often been reported in middle-aged patients more commonly in females than males. OLP is also seen in children although rare. The oral mucosa is commonly involved and may be the only site of involvement. 
The basic clinical presentation of this lesion is a white lineal papule that reveals a reticular pattern which is generally asymptomatic. The frequent sites are the buccal mucosa, tongue, gingiva and lips, with a bilateral and symmetrical pattern characteristic of typical
cases.  The clinical forms of OLP are divided into reticular, papular, atrophic and erosive lesions  [Figure 1]. The frequency of malignant transformation ranges from 0% to 5.3% with the highest rate noted in erythematous and erosive lesions. 
Several factors have been proposed for the etiology such as genetic background, dental materials, drugs, infectious agents, autoimmunity, immunodeficiency, food allergies, stress, habits, psychogenic factors, hypertension, diabetes mellitus, virus agents like human papillomaviruses, hepatitis viruses (human herpesvirus-6), herpes simplex viruses-1. ,,,
Oxidative stress and antioxidant defense mechanism may play an important role in the pathology of lichen planus. Oxidative stress results from the metabolic reactions that use oxygen and represents a disturbance in the equilibrium status of pro-oxidant/antioxidant reactions in living organisms.  Increase in oxidative stress and lipid peroxidation together with an imbalance in antioxidant defense system may suggest that reactive oxygen species (ROS) are involved in the pathogenesis of lichen planus. 
Reactive oxygen species are short-lived entities that are continuously generated at low levels during the course of normal aerobic metabolism. Free radicals are any atom or molecule that contains one or more unpaired electrons. ROS include singlet oxygen, superoxide anion, nitric oxide (NO), peroxylions. , Oxidative stress drives the production of oxidation products, like 4-hydroxy-2-nonenal or malondialdehyde (MDA) which can denature proteins, alter apoptosis and influence the release of pro-inflammatory mediator.  Free radicals and oxidative stress can cause cell injury and death if the tissue does not respond by making extra antioxidant defenses.  ROS and lipid peroxides may also participate in the pathogenesis of lichen planus and other allergic reactions in the skin. ,
Nitric oxide is a free radical and MDA is a free radical induced lipid peroxidation end product. NO has been reported to be contained in freshly secreted human saliva  and elevated productions of NO have been implicated in the pathogenesis of oral mucosal diseases.  MDA has also been implicated in the pathogenesis of several pathological disorders including OLP. 
As salivary secretions represent the oral microenvironment, estimation of salivary levels of NO and MDA along with serum will reveal the oxidative stress status of OLP more effectively than with estimation in serum alone. Estimation of oxidative stress markers in OLP will help unraveling the pathogenesis, which in turn help improve the therapeutic options in the treatment of OLP.
The present study aims at estimation of NO and MDA in serum and saliva samples of OLP and to compare their levels.
| Materials and methods|| |
The study was approved by the ethical committee of SRM University. The details of the study were explained to all patients, and written informed consent was obtained from the patients and the relatives before entering into the study. Patients for the study were recruited from the outpatient department of SRM Dental College. In this study, a total of 10 cases of OLP and 10 cases of control were taken.
The inclusion criteria for study group included lesion involving bilateral involvement of the posterior buccal mucosa, presence of white striations over the mucosa and histopathologically diagnosed cases of OLP cases, patients of any age and sex and patients from any geographical location. The exclusion criteria included the presence of oral inflammatory conditions such as gingivitis, periodontitis and oral ulcers, medication with immunosuppressive agents and topical medications for the last 2 weeks. Complete case histories, questionnaires, and verbal and written consent were obtained from all patients enrolled in the study. Blood and saliva samples were obtained from all the patients in the two groups. Saliva and serum collected from both the study and control groups were given an identification code from number 1-10. All serum and saliva samples were double blinded in order to avoid bias of values during estimation procedures [Figure 2].
|Figure 2: Saliva and serum sample collection done and identification codes given|
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The subjects in the experimental groups were asked to rinse their mouth with povidone iodine mouth rinse for 2 min that ensured a substantial reduction in bacterial count. The patients were asked to wait for a minute, after which freshly secreted unstimulated saliva, about 1 ml was collected in a sterile container. The sample was then diluted with 10 ml of phosphate buffered saline in order to neutralize the pH. After diluting, the mixture was centrifuged for 5 min at 3000 rpm and the supernatant fluid was stored at −80°C until use.
For collecting serum samples, venous blood was taken from patients and transferred to sterile test tubes. The blood was allowed to clot and was centrifuged for 5 min at 3000 rpm [Figure 3]. The clear serum was separated and stored at −80°C until analysis.
For NO estimation, blood samples were collected and from which serum was separated and stored for further assay. Saliva samples were collected, and the granular part was separated and the aqueous part was stored for the further assay. Serum and saliva samples were thawed to room temperature. 0.5 ml of serum and saliva samples were taken and subjected for NO assay. To this 0.5 ml of Griess reagent was added. The content was mixed thoroughly [Figure 4]. The reaction mixture was measured at 540 nm using Microplate Reader. Standard was prepared at various concentration, which includes 20 μg, 40 μg, 50 μg and 60 μg.
For MDA estimation, serum and saliva samples were obtained, and they were thawed to room temperature. 2.5 ml of trichloroacetic acid was taken and mixed with 0.5 ml of sample (plasma and serum). The contents were mixed well and incubated for 15 min at 90°C. The samples tubes were cooled with cold water. The contents were centrifuged at 3000 rpm for 10 min. 2 ml of supernatant was transferred to a new tube. To this 1 ml 0.675% thiobarbituric acid was added. The tubes were sealed and incubated at 90°C for 15 min. And the content were measured at 586 nm using Microplate Reader [Figure 5].
The average net optical denwas calculated by subtracting the average value of sample (A1) from the average value of control (A0) and dividing with the average value of control (A0). Using the obtained absorbance value for each sample, the concentration of NO and MDA in serum and saliva samples were determined. The P value between cases and control in serum and saliva samples of MDA and NO was statistically significant (0.001 and <0.001, respectively).
The data obtained was done using SPSS software by IBM Corportation under IBM Software Group's Business Analytics Portfolio. Mean and standard deviations were calculated for the individual groups. These were then analyzed using the independent samples t-test to compare the mean values of NO and MDA assay.
| Results|| |
Among the 10 patients with OLP, there were six males and four females within the age group of 24-65 years, and there were seven males and three females within the age group of 24-63 years in the control group.
The comparison between the control group and the lichen planus group in serum of MDA assay group revealed that there is an increase in the mean value in lichen planus group. The P value between cases and control in serum samples of MDA assay was 0.001, which was statistically significant [Chart 1] [Additional file 1].The comparison between the control group and the lichen planus group in saliva of MDA assay group revealed that there is an increase in the mean value in lichen planus group. The P value between cases and control in saliva samples of MDA assay was 0.001, which was statistically significant [Chart 2] [Additional file 2].
The comparison between the control group and the lichen planus group in serum of NO assay group revealed that there is an increase in the mean value in lichen planus group. The P value between cases and control in serum samples of NO assay was <0.001, which was statistically significant [Chart 3] [Additional file 3]. The comparison between the control group and the lichen planus group in saliva of NO assay group revealed that there is an increase in the mean value in lichen planus group. The P value between cases and control in saliva samples of NO assay was <0.001, which was statistically significant [Chart 4] [Additional file 4].
Serum and salivary MDA assay values showed an increase in lichen planus in comparison with the control group. Serum and salivary NO assay values also showed an increase in lichen planus in comparison with the control group. The mean serum value of NO is increased in both control and case samples when compared to MDA values. Similarly, the mean saliva values of NO is increased in both control and case samples when compared to MDA values. Interestingly, mean values of NO and MDA in saliva is increased when compared to serum in the study group [Table 1].
|Table 1: Comparison of mean concentration in serum and saliva between groups|
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| Discussion|| |
Lichen planus is a chronic autoimmune, mucocutaneous disease that can affect the oral mucosa, skin, genital mucosa, scalp and nails. The prevalence of OLP in the Indian population is about 2.6%.  It has been pointed out that psychological aspects also contribute to the etiology of OLP. It is also well-known that neuroendocrine substances are elevated in saliva in emotional stress states. Levels of salivary endocrines such as cortisone are high in patients with OLP.
Oral lichen planus may associate with known autoimmune diseases and may itself have an autoimmune pathogenesis.  It has been reported that psychological stress causes the release of NO. Also, diabetes, malignant neoplasms, hepatitis C virus, drugs like nonsteroidal antiinflammatory drugs, penicillamine are considered as vital factors. 
Oxidative stress is caused by an imbalance between the production of reactive oxygen and the ability of the biological system to readily detoxify the reactive intermediates or easily repair the resulting damage. This usually results in the production of free radicals that can damage cell membranes, as well as numerous cellular molecules such as proteins, nucleic acids, amino acids, carbohydrates and vitamins through the production of lipid peroxides.
The origin of cellular degeneration in lichen planus is thought to be the subepithelial infiltration of T-lymphocytes that contributes to the local production of cytokines. These cytokines stimulate the production of ROS and products of lipid peroxidation triggering apoptosis that is a hallmark feature of lichen planus. Oxidative stress also increases the severity of the immunological mechanism in lichen planus strengthening the role of oxidative stress in the pathogenesis of lichen planus.
The enzyme inducible NO synthase (NOS) was originally described in macrophages. It is also known to be expressed by mast cells. The inducible NOS once induced continue to produce NO for a much longer period than either neural or endothelial NOS. Being readily observed in patients with infectious or inflammatory conditions, the expression of inducible NOS and hence NO could be increased in cellular infiltrate seen in OLP when compared with normal mucosa. Further, there was no association between OLP and salivary dysfunction in otherwise healthy patients. ,
In a study conducted by Sunitha and Shanmugam, free radicals including NO and suggested that excess of salivary NO may have pathophysiological implications for OLP.  Ohashi et al. reported that elevated production of salivary NO may have pathophysiological implications for OLP, and it may participate in the generation and advancement of erosive and ulcerative lesions. Aly and Shahin, reported an increase in the levels of NO in serum of patients with lichen planus and pointed to an increase in oxidative stress and an imbalance in the antioxidant defense mechanism in lichen planus. The study analyzed that selective inhibitors of this inducible isoform thus have therapeutic potential in a number of disease states.  Abnormal ratio of retinol to dehydroretinol in lesions of lichen planus was possible due to changes in cutaneous vitamin A metabolism associated with epidermal hyperproliferation and inflammation. 
One of the major presentations of oxidative stress is lipid peroxidation. Peroxidation of lipid-rich membranes alters their fluidity and signaling efficiency, leading to inflammatory changes and to aberrant cell proliferation responses.
Malondialdehyde is the end product of lipid peroxidation and is considered as a good marker of free radical-mediated damage and oxidative stress. It is the principal and most studied product of polyunsaturated fatty acid peroxidation.  MDA can combine with several functional groups on molecules including proteins, lipoproteins, RNA and DNA. Hence, MDA can be considered as a marker, which will eventually reveal free radical mediated oxidative stress in OLP patients.
The antioxidant profile in patients with OLP using serum and salivary samples was evaluated to assess the oxidative stress levels. In the study, the levels of MDA was found to be higher in both serum and saliva samples. Rai and Kharb, estimated the levels of MDA in lichen planus and oral cancer and found out that significantly elevated levels of MDA were observed in both lichen planus and cancer groups when compared to controls.  The study concluded that altered lipid peroxidation in plasma and erythrocytes of oral cancer patients may be related to their compensatory changes in the antioxidants defense system. 
Several studies have taken serum samples as a tool for measuring oxidative stress, and only a few studies have taken saliva samples. Only limited studies exist to compare the oxidative stress status in lichen planus both saliva and serum simultaneously. Therefore, oxidative stress status of patients with OLP was evaluated using both serum and saliva samples and it was necessary to substantiate the role of antioxidants and oxidative stress in OLP. The results showed that there was an increase in the levels of NO and MDA in serum and saliva of patients with OLP when compared with controls.
The increase in the level of NO is more statistically significant than when compared to the levels of MDA. These findings indicate that NO and MDA could be stated to play an important role in the pathogenesis of OLP. This increase of NO can be attributed to the fact that NO is a major form of ROS, and it contributes to inflammatory responses. Increased oxidative modifications point to pathophysiological changes mainly within the basal cell layers of the epidermis and at the dermoepidermal junction. 
The inflammatory cellular infiltrate in lichen planus, which consists mainly of CD4+ lymphocytes, is a well-known source of ROS. ROS in toxic concentrations have been shown to cause damage to endothelial cells with further up-regulation and expression of intercellular adhesion molecule-1 (ICAM-1). ICAM-1 expression is important in facilitating the recruitment of T-lymphocytes at the site of inflammation.  Increase in nitrous oxide is also due to the continuous production of produced NO for much longer period in inflammatory conditions. Saliva is now increasingly used and well validated in diagnosing, monitoring systemic health and disease states and predicting disease progression and detecting biomarkers. The present study has shown a significant increase in levels of NO and MDA in saliva. Interestingly the values are more in saliva when compared with serum that highlights the potential clinical value of saliva as a valid and convenient diagnostic biofluid.
Increased presence of NO in saliva could be due to its production in salivary glands and is regulated by neural mechanisms. Central and peripheral nervous systems have already been shown to regulate the production of NO. Neuroendocrine levels in saliva have been reported to be increased in emotional stress. Psychological stress causes an increase in NO release. NO has cytotoxic effects on the keratinocytes of the oral mucosa increasing the severity of oral mucosal diseases. 
Furthermore, after beta-carotene supplementation, antioxidants were decreased in OLP due to imbalance between the production of ROS, including free radicals and failure of immune system to neutralize them  Salivary antioxidant like uric acid could be considered in the future as useful markers of oxidative stress for elaboration of treatment strategy and monitoring. 
Increase in levels of MDA in saliva occurs in inflammatory states and various oral mucosal diseases like OLP, leukoplakia and oral sub mucous fibrosis. Increased oxidative stress causes increased lipid peroxidation of the cell membrane. Increase in levels of markers of oxidative stress like NO activates a chain of reaction of lipid peroxidation events triggering an increase in levels of MDA.
| Conclusion|| |
Increase in level of NO was significantly higher than the increase in the level of MDA and both the molecules could be stated to play a role in the pathogenesis of OLP. Salivary levels of NO and MDA were found to be significantly higher than serum levels. This suggests that saliva can be used as an alternate and effective diagnostic tool in evaluating the oxidative stress status of an individual. Monitoring the oxidative stress status of lichen planus can be used for therapeutic management. Antioxidants may be used to reverse the oxidative stress status in lichen planus.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]