Home About us Editorial board Ahead of print Current issue Search Archives Submit article Instructions Subscribe Contact Us Login 
An Official Publication of the Indian Association of Oral and Maxillofacial Pathologists


 
  Table of Contents    
ORIGINAL ARTICLE  
Year : 2022  |  Volume : 26  |  Issue : 1  |  Page : 26-30
 

Evaluation of salivary oxidative stress in oral lichen planus using malonaldehyde


1 Department of Oral Pathology and Microbiology, King George's Medical University, Lucknow, Uttar Pradesh, India
2 Department of Oral Pathology and Microbiology, Faculty of Dental Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India
3 Department of Oral Pathology and Microbiology, HKES SN Dental College, Kalaburagi, Karnataka, India
4 Department of Oral Pathology and Microbiology, Sardar Patel Post Graduate Institute of Dental and Medical Sciences, Lucknow, Uttar Pradesh, India

Date of Submission18-Sep-2021
Date of Decision04-Dec-2021
Date of Acceptance13-Jan-2022
Date of Web Publication31-Mar-2022

Correspondence Address:
Fahad M Samadi
Department of Oral Pathology and Microbiology, King George's Medical University, Lucknow, Uttar Pradesh
India
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jomfp.jomfp_333_21

Rights and Permissions

 

   Abstract 


Background: Oral lichen planus is a chronic, mucocutaneous, inflammatory disease, with an unknown etiology. Reactive oxygen species and oxidative damage to the tissues might be the cause. Malonaldehyde (MDA), a low molecular weight end product of lipid peroxidation reaction is a suitable biomarker of endogenous DNA damage. monitoring the oxidant-antioxidant status of saliva may serve as an efficient marker of disease development in oral lichen planus patients.
Aim and Objectives: To evaluate salivary oxidative stress in oral lichen planus subject using MDA and compare it with control subjects. Furthermore, to compare MDA levels in erosive and hypertrophic lichen planus.
Materials and Methods: The current study is case–control study. Unstimulated salivary samples in the morning hours were taken from oral lichen planus subjects (n = 25) and controls subjects without any oral disease (n = 25). The saliva was centrifuged at 900 g for 10 min at a temperature of 4°C. Then, the entire filtrate was transferred to Eppendorf test tubes and frozen at–80°C until analysis. Salivary MDA was done through thiobarbituric acid reactive substance assay as per the protocol laid down by the manufacturer (Sigma Aldrich Lipid Peroxidation Assay Kit).
Results: The data were expressed as the mean ± standard deviation and the statistical analysis was done using Student's t-test using SPSS version 21 IBM software. The salivary level of MDA was significantly higher than that of controls (P < 0.05).
Conclusion: The higher level of MDA in patients with oral lichen planus suggests that free radicals and the resulting oxidative damage may be important in the pathogenesis of oral lichen planus lesions.


Keywords: Malonaldehyde, oral lichen planus, oxidative stress, potentially malignant lesion, saliva


How to cite this article:
Singh S, Singh J, Biradar BC, Sonam M, Chandra S, Samadi FM. Evaluation of salivary oxidative stress in oral lichen planus using malonaldehyde. J Oral Maxillofac Pathol 2022;26:26-30

How to cite this URL:
Singh S, Singh J, Biradar BC, Sonam M, Chandra S, Samadi FM. Evaluation of salivary oxidative stress in oral lichen planus using malonaldehyde. J Oral Maxillofac Pathol [serial online] 2022 [cited 2022 Dec 3];26:26-30. Available from: https://www.jomfp.in/text.asp?2022/26/1/26/341397





   Introduction Top


Lichen planus is a chronic inflammatory condition that may affect the skin, scalp, nails, mucous membranes (especially mouth) and the genitalia.[1] Oral lichen planus is a chronic mucosal condition commonly encountered in clinical dental practice.[2] An overall age-standardized global prevalence is 1.25% (0.96% in men and 1.57% in women) in general population with a malignant transformation rate of 0.4%–1.74%[3]

In recent years, there has been an increasing research interest in the role of oxidative stress in pathogenesis of OLP. Oxidative stress is a disturbance in the equilibrium status of pro-oxidant and anti-oxidant reaction. Activated T-cell release cytokines leading to the attraction of inflammatory cells and the destruction of keratinocytes by cell-mediated cytotoxicity.[4] Cells such as keratinocytes, fibroblasts and inflammatory cells release reactive oxygen species (ROS) may inflict cellular damage by overwhelming the antioxidant defense mechanisms leading to excessive production of ROS.[5]

It is assumed that markers of oxidative stress are associated with different local oral condition. The level of antioxidant is a potential determinant of susceptibility to be affected by OLP. This suggests that oxidative stress is a major trigger for OLP.[5]

Saliva, as a body fluid in the oral cavity, contacts directly with the local environment. Saliva has numerous benefits, as it is easily accessible, painless and noninvasive, less time-consuming. Furthermore, it can be used for mass screening of large population samples.[6]

Only few studies have been published assessing oxidant-antioxidant status in patients with selected clinical forms of OLP in comparison with healthy individuals without symptoms of the disease.[7],[8],[9] The objective of the current study was to evaluate salivary oxidative stress in Oral Lichen Planus subjects using malonaldehyde (MDA) and compares it with control subjects and evaluate salivary oxidative stress using MDA in sub-groups of oral lichen planus.


   Materials and Methods Top


The current study was a prospective case-control study. Institutional ethical clearance and informed consent from the subject was duly obtained. The patient's demographic profile was noted, brief history was taken including a history of relevant risk factors and habits. Clinical examination was performed and type of lichen planus was recorded. Lichen planus was diagnosed as per the criteria put forth by the World Health Organization with slight modification[10] [Table 1] and [Figure 1].
Table 1: Modified WHO criteria for diagnosing oral lichen planus[10]

Click here to view
Figure 1: Clinical and histopathological picture (×40) of oral lichen planus (Case No: OLP-01)

Click here to view


Subjects were divided into two groups:

  • Group A: Patients with Established cases of oral lichen planus (n = 25)


    • Subgroup A: Erosive oral lichen planus
    • Subgroup B: Reticular oral lichen planus


  • Group B: Age- and sex-matched normal healthy subjects (n = 25) with no oral lesions and no habits.


Subjects with age above 18 years and below 50 years of age of either gender with no systemic illness and cases of control group having no morbidity were included and only established cases of oral lichen planus were included in subject group. Subjects having autoimmune disease or malignancy, history of trauma or surgery, on long-term steroid or other medications, smokers or having periodontitis or diabetes mellitus were excluded from the study.

Procedure

The subjects were asked to rinse their mouth with water before collecting specimen. Un-stimulated salivary samples were taken. The spitting method was used for saliva collection and the total time duration of saliva collection was approximately 5 min. All samples were collected between 8 AM and 11 AM to avoid diurnal variation.

Salivary samples were collected in the morning after 8 h of fasting. The sample was immediately placed into ice and then taken to laboratory. The saliva was centrifuged at 900 g for 10 min at a temperature of 4°C. Then the entire filtrate was transferred to sterile 1.5 ml micro test tubes and adding 15 μl solution of butylated hydroxytoluene (BHT) in ethanol per 1 ml of saliva so as to prevent lipid peroxidation during sample storage and frozen at −80°C until analysis. Salivary MDA was estimated through thiobarbituric acid reactive substance assay (TBARS assay) as per the protocol laid down by the manufacturer (Sigma Aldrich Lipid Peroxidation Assay Kit).

Thiobarbituaric acid (TBA) solution was prepared by reconstituting the given TBA solution with 7.5 mL glacial acetic acid and then adjusted the final volume to 25 mL with water. MDA Standards were prepared by diluting 10 μL of the 4.17 M MDA Standard Solution with 407 μL of water to prepare a 0.1 M MDA Standard Solution. We further diluted 20 μL of the 0.1 M MDA Standard Solution with 980 μL of water to prepare a 2 mM MDA standard. We added 0, 2, 4, 6, 8 and 10 μL of the 2 mM MDA standard solution into separate microcentrifuge tubes, generating 0 (blank), 4, 8, 12, 16 and 20 nmole standards. Water was added to each tube to bring it to the final volume.

Saliva samples were gently mixed with 42 mM sulfuric acid in a microcentrifuge tube. We added phosphotungstic acid solution to it and vortex the solution. The samples were incubated at room temperature for 5 min and then centrifuged the samples at 13,000 rpm for 3 min. In a separate tube, 2 μL of BHT (×100) was added. Then, we re-suspended the pellet on ice with the water-BHT solution and adjusted the volume with water. TBA solution was added to each vial containing standard and sample and incubated at 95°C for 60 min. It was then cooled down to room temperature in an ice bath for 10 min. The resultant solution was matched with the MDA standards and measured absorbance at 532 nm [Figure 2].
Figure 2: Reagents and equipments used in the study

Click here to view


Statistical analysis

Statistical evaluation was done using analysis of variance for scale and ordinal variance. Independent Student's t-test was applied using SPSS version 17 Software (IBM corporation, NY, United States).


   Results Top


A total of 50 subjects participated in the study, of which 25 patients were from the study group (14 males and 11 females) while 25 subjects from the age- and sex-matched the control group (13 males and 12 females). The mean age of the control group was 44.4 ± 5.65 years and that of the control group was 43.12 ± 6.24 years [Figure 3]. In the study group, 11 cases were of erosive lichen planus (Subgroup A) while 14 cases were reticular lichen planus (Subgroup B).
Figure 3: Mean age in case and control

Click here to view


The mean MDA value in the case group was 0.13226 ± 0.108027 nmol/L (mean ± standard deviation) with standard error (SE) of 0.021605. The mean MDA value in the control group was 0.06844 ± 0.040827 nmol/L (with SE of 0.008165. Our study showed higher salivary MDA in cases than in controls and the difference was statistically significant (P < 0.01) [Table 2].
Table 2: Comparison of mean between cases and control

Click here to view


In between subgroups, MDA value of subgroup A 0.400 ± 0.236 nmol/L (SE - 0.071) and in Subgroup B: 0.279 ± 0.221 nmol/L (SE - 0.059). This value was statistically nonsignificant (P = 0.184) [Table 3].
Table 3: Comparison of mean between two sub-groups

Click here to view



   Discussion Top


Lichen planus, first described by Erasmus Wilson in 1869, is a pruritic dermatosis of known etiology that affects the skin and mucous membranes.[11] The exact etiopathogenesis of OLP is unknown, but it is believed to result from an abnormal immune response in which basal epithelial cells are recognized as foreign because of changes in the antigenicity of their cell surface.[12]

The origin of this cellular degeneration can be accredited to subepithelial infiltration of T-lymphocytes that contributes to the local production of cytokines, which in turn can stimulate the production of ROS and cause oxidative damage to the tissues. Reactive oxygen metabolite leads to destruction and damage to cell membranes by lipid peroxidation.[5]

Oxidation of membrane-associated polyunsaturated fatty acids of phospholipids results in lipid peroxidation, which has been considered a foremost contributor of oxidative stress.[13] The end product of lipid peroxidation, MDA is a distinguished marker of free radical-mediated damage and oxidative stress.[14] Saliva is a more striking biological sample for clinical studies on oral diseases due to its diagnostic, prognostic and therapeutic responses.

Sezer et al. reported that there was increased oxidative stress and lipid peroxidation together with an imbalance in the antioxidant defense system in patients with cutaneous lichen planus, suggesting that ROS might be involved in the pathogenesis of lichen planus.[15] Sertan et al., Sezer et al. obtained increased serum MDA levels in oral lichen planus.[15],[16] According to Upadhyay et al., serum levels of MDA were significantly higher in OLP and oral lichenoid reaction than in controls but there was no significant difference in serum MDA levels between oral lichen planus and oral lichenoid reaction patients.[17] Ergun et al. found a positive correlation in serum and salivary MDA levels in OLP patients.[18] Agha-Hosseini et al. found MDA level is higher in saliva samples of OLP cells in comparison to controls similar to our study.[19] Vlková et al. found twice TBARS level in premalignant lesions.[20] Abdolsamadi et al., Rekha et al. and Abbas et al. also found increase in salivary MDA level in in OLP.[8],[21],[22] Mehdipour et al. found MDA levels in OLP and controls to be nonsignificant.[23] The probable reason for varying results could be different geographical location. Although studies have been done on estimation on salivary samples we found only one study, which compared oxidative stress level in two variants of oral lichen planus. The result of Darczuk et al. was inconsistent with our study.[9] The most probable reason being variability in sample size. Most of the studies comply with the finding of our study, that is, increased oxidative stress (MDA levels) in different biological media, i.e., serum, saliva and tissue.

Mishra and Maheshwari Uma et al. in their systematic review concluded that different studies suggest an increased oxidative stress and imbalance in the antioxidant defense system in biological fluids of patients with oral lichen planus, thus proving that oxidative stress plays an important role in its pathophysiology.[24]

Rai performed a clinical trial by treating oral lichen planus patients with curcumin and measure oxidative stress in serum and saliva using MDA and found that there is reduction in serum and salivary levels of MDA posttreatment after 7 days as well as after 209 days.[25] Increased oxidative stress and imbalance in the antioxidant defense system in biological fluids of patients with oral lichen planus, proved that oxidative stress plays an important role in its pathophysiology of oral lichen planus.

In our present study, the mean MDA levels in the control population were 0.06 nmol/L. In subjects with oral lichen planus, the mean MDA level was 0.13 nmol/L. Our findings implicate that there is an increase in MDA Levels from control to OLP subjects.


   Conclusion Top


The oxidative stress has a role in etiopathogenesis of oral lichen planus. Although the mean MDA values were higher in erosive variant than reticular variant of OLP, there was no statistical difference between the two variant of oral lichen planus. Antioxidants need to be incorporated as an essential part of treatment protocol to overcome the antioxidant deficiency. Further studies with a large sample size are required for determining its usefulness.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Ismail SB, Kumar SK, Zain RB. Oral lichen planus and lichenoid reactions: Etiopathogenesis, diagnosis, management and malignant transformation. J Oral Sci 2007;49:89-106.  Back to cited text no. 1
    
2.
Edwards PC, Kelsch R. Oral lichen planus: Clinical presentation and management. J Can Dent Assoc 2002;68:494-9.  Back to cited text no. 2
    
3.
McCartan BE, Healy CM. The reported prevalence of oral lichen planus: A review and critique. J Oral Pathol Med 2008;37:447-53.  Back to cited text no. 3
    
4.
Middel P, Lippert U, Hummel KM, Bertsch HP, Artuc M, Schweyer S, et al. Expression of lymphotoxin-alpha by keratinocytes: A further mediator for the lichenoid reaction. Pathobiology 2000;68:291-300.  Back to cited text no. 4
    
5.
Panchal FH, Ray S, Munshi RP, Bhalreo SS, Nayak CS. Alterations in lipid metabolism and antioxidant status in lichen planus. Indian J Dermatol 2015;60:439-44.  Back to cited text no. 5
[PUBMED]  [Full text]  
6.
Malathi N, Mythili S, Vasanthi HR. Salivary diagnostics: A brief review. ISRN Dent 2014;2014:158786.  Back to cited text no. 6
    
7.
Shirzad A, Pouramir M, Seyedmajidi M, Jenabian N, Bijani A, Motallebnejad M. Salivary total antioxidant capacity and lipid peroxidation in patients with erosive oral lichen planus. J Dent Res Dent Clin Dent Prospects 2014;8:35-9.  Back to cited text no. 7
    
8.
Abdolsamadi H, Rafieian N, Goodarzi MT, Feradmal J, Davoodi P, Jazayeri M, et al. Levels of salivary antioxidant vitamins and lipid peroxidation in patients with oral lichen planus and healthy individuals. Chonnam Med J 2014;50:58-62.  Back to cited text no. 8
    
9.
Darczuk D, Krzysciak W, Vyhouskaya P, Kesek B, Galecka-Wanatowicz D, Lipska W, et al. Salivary oxidative status in patients with oral lichen planus. J Physiol Pharmacol 2016;67:885-94.  Back to cited text no. 9
    
10.
Van der Meij EH, van der Waal I. Lack of clinicopathologic correlation in the diagnosis of oral lichen planus based on the presently available diagnostic criteria and suggestions for modifications. J Oral Pathol Med 2003;32:507-12.  Back to cited text no. 10
    
11.
Agarwal R, Saraswat A. Oral lichen planus: An update. Drugs Today (Barc) 2002;38:533-47.  Back to cited text no. 11
    
12.
Patel S, Yeoman CM, Murphy R. Oral lichen planus in childhood: A report of three cases. Int J Paediatr Dent 2005;15:118-22.  Back to cited text no. 12
    
13.
Picardo M, Passi S, Morrone A, Grandinetti M, Di Carlo A, Ippolito F. Antioxidant status in the blood of patients with active vitiligo. Pigment Cell Res 1994;7:110-5.  Back to cited text no. 13
    
14.
Kasperska-Zajac A, Brzoza Z, Rogala B, Polaniak R, Birkner E. Antioxidant enzyme activity and malondialdehyde concentration in the plasma and erythrocytes of patients with urticaria induced by nonsteroidal anti-inflammatory drugs. J Investig Allergol Clin Immunol 2008;18:372-5.  Back to cited text no. 14
    
15.
Sezer E, Ozugurlu F, Ozyurt H, Sahin S, Etikan I. Lipid peroxidation and antioxidant status in lichen planus. Clin Exp Dermatol 2007;32:430-4.  Back to cited text no. 15
    
16.
Sertan E, Sule CT, Warnakulasuriya S, Sevda O, Ayse EO, Duygu O, et al. Evaluation of oxidative stress andantioxidant profile in patients with oral Lichen planus. J Oral Pathol Med 2011;40:286-93.  Back to cited text no. 16
    
17.
Upadhyay RB, Sunitha C, Revathi P, Shenoy G, Prabin M, Mukherjee M. Oxidative stress and antioxidant defence in orallichen planus and oral lichenoid reaction. Scand J Clin Lab Invest 2010;70:225-8.  Back to cited text no. 17
    
18.
Ergun S, Troşala SC, Warnakulasuriya S, Özel S, Önal AE, Ofluoğlu D, et al. Evaluation of oxidative stress and antioxidant profile in patients with oral lichen planus. J Oral Pathol Med 2011;40:286-93.  Back to cited text no. 18
    
19.
Agha-Hosseini F, Dizgah-Mirzaii I, Mikaili S, Abdollahi M. Increased salivary lipid peroxidation in human subjects with oral lichen planus. Int J Dent Hyg 2009;7:246-50.  Back to cited text no. 19
    
20.
Vlková B, Stanko P, Minárik G, Tóthová L, Szemes T, Baňasová L, et al. Salivary markers of oxidative stress in patients with oral premalignant lesions. Arch Oral Biol 2012;57:1651-6.  Back to cited text no. 20
    
21.
Rekha VR, Sunil S, Rathy R. Evaluation of oxidative stress markers in oral lichen planus. J Oral Maxillofac Pathol 2017;21:387-93.  Back to cited text no. 21
[PUBMED]  [Full text]  
22.
Abbas AW, Zaidan TF, Abduldaheem Y, Barrack AA. Assessment of serum and salivary malondialdehyde in patients with oral lichen planus. J Bagh Coll Dent 2014;26:98-102.  Back to cited text no. 22
    
23.
Mehdipour M, Taghavi Zenouz A, Dastanpour S, Bahramian A, Gholizadeh N, Azari-Marhabi S. Evaluation of serum Malondialdehyde level in patients with oral lichen planus. Caspian J Dent Res 2017;6:45-8.  Back to cited text no. 23
    
24.
Mishra SS, Maheshwari Uma TN. Evaluation of oxidative stress in oral lichen planus using malonaldehyde: A systematic review. J Dermatol Dermatol Surg 2014;18:2-7.  Back to cited text no. 24
    
25.
Rai B, Kaur J, Jacobs R, Singh J. Possible action mechanism for curcumin in pre-cancerous lesions based on serum and salivary markers of oxidative stress. J Oral Sci 2010;52:251-6.  Back to cited text no. 25
    


    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

Top
Print this article  Email this article
            

    

 
   Search
 
  
    Similar in PUBMED
    Search Pubmed for
    Search in Google Scholar for
  Related articles
    Article in PDF (1,082 KB)
    Citation Manager
    Access Statistics
    Reader Comments
    Email Alert *
    Add to My List *
* Registration required (free)  


    Abstract
   Introduction
    Materials and Me...
   Results
   Discussion
   Conclusion
    References
    Article Figures
    Article Tables

 Article Access Statistics
    Viewed769    
    Printed12    
    Emailed0    
    PDF Downloaded110    
    Comments [Add]    

Recommend this journal

Journal of Oral and Maxillofacial Pathology | Published by Wolters Kluwer - Medknow
Online since 15th Aug, 2007