ORIGINAL�ARTICLE

ABSTRACT
Objective: The objective of the study was to determine the serum enzymatic antioxidant levels in recurrent 
aphthous stomatitis patients.

Study Design: This was a case control study.

Place and Duration of Study: The study was conducted in biochemistry department of Islamic International 

Medical College- Trust (IIMC-T) Rawalpindi from 15 March 2016 to 14 March 2017.

Materials and Methods: The study included 160 subjects divided into two groups Aphthous group and Non-

Aphthous group. Aphthous group comprised of 70 recurrent aphthous stomatitis  patients with active lesion 

and 90 age and sex matched healthy controls were included in the non-aphthous group .From all the 

participants 2 ml of blood was taken. Serum glutathione peroxidase levels were measured using enzyme linked 

immunosorbent assay technique. This ELISA kit uses Sandwich-ELISA as the method.

Results: The comparison of mean of serum glutathione peroxidase levels between aphthous and non-aphthous 

group was significant (p<o.oo).This study showed that the levels of glutathione peroxidase (a biological stress 

marker) were lower in recurrent aphthous stomatitis patients than in control subjects.
Conclusion: Serum enzymatic antioxidant levels were lower in recurrent aphthous stomatitis patients as 
compared to controls, so oxidative stress plays an important role in the pathogenesis of disease.

Key words: Glutathione peroxidase, Oxidative stress, Recurrent aphthous stomatitis

have  done to find out the causes of recurrent 
aphthous stomatitis  but the exact etiology remains 
unidentified .Potential factors which have been 
identified in the onset of disease include hereditary 
predisposition, local and systemic causes, 
hematological and micronutrient deficiencies, 

11 
emotional  stress and drug allergies. It has been 
postulated that all the predisposing factors for the 
formation of aphthous ulcers act through a common 
pathway that depends on the oxidative stress by 
increasing free radical synthesis. Either the 
enhanced production of reactive oxygen species 
and/or decreased defense by antioxidants result in 
oxidative stress that is lethal as it causes tissue 

12
damage .
Studies suggest that the lack of balance between 
reactive oxygen species and antioxidants have a 
pivotal role in the development and progression of 
inflammatory reactions, so oxidative stress may have 
a crucial role in development of recurrent aphthous 

13
stomatitis as it is inflammatory in nature.  To prevent 
oxidative stress, mammalian cells have developed 
antioxidant defense system including both 
enzymatic and non-enzymatic antioxidants.
Glutathione peroxidase, a selenocysteine containing 

Introduction
Recurrent aphthous stomatitis (RAS) is one of the 
most commonly encountered pathology of oral 

1  
mucosa, affecting nearly 20% of world population.
Aphthous is originated from “aphtha” a Greek word 

2, 3, 4, 5
meaning ulceration.  Recurrent aphthous 
stomatitis is a painful, recurring and distressful 
experience for the patients because it restricts their 

6,7
normal oral activities.  Ulcerations  can be solitary 

8 
or multiple and has a shallow necrotizing center and 
can be classified into three classes depending on 
their type , size , number and their healing time i-e 

9 
minor, major and herpetiform. Minor recurrent 
aphthous stomatitis are the most common type and 

10  
contributes to 80% of total cases. Many studies  

Assessment of Serum Enzymatic Antioxidant Levels in Recurrent 
Aphthous Stomatitis Patients

Correspondence:
Dr. Rabbia Shabbir
Department of Biochemistry
Islamic International Medical College
Riphah International University, Islamabad
E-mail: rabbia.shabbir@gmail.com

Department of Biochemistry 
Islamic International Medical College
Riphah International University, Islamabad

Funding Source: NIL; Conflict of Interest: NIL
Received: April 02, 2017; Revised: Aug 24, 2018
Accepted: Aug 25, 2018

Serum Enzymatic Antioxidant in Aphthous StomatitisJIIMC 2018 Vol. 13, No.3

133

 
Rabbia Shabbir,  Amena Rahim,  Muhammad Afzal



immunomodulatory agents for past two months. 
Pregnant and lactating mothers were excluded. 
Smokers and alcohol users were excluded. After the 
informed consent of all participants in the study 2 ml 
of venous blood was collected using sterile 
disposable syringe and transferred to serum 
separator tubes. Serum glutathione peroxidase 
levels were measured by Sandwich Enzyme linked 
immunosorbent assay technique (Absorbance Micro 
plate Reader, ELX800, BioTek Instrument, Inc.USA).
The collected data was entered in statistical package 
for social sciences (SPSS) version 21 for analysis. 
Gender was expressed as percentages and 
frequency. Numerical variables like age, glutathione 
peroxidase were expressed as mean and standard 
deviation (±SD). Independent  t –test was used to 
determine the  significant difference of means 
between controls and patients.p values less than 
0.05 were considered as significant.

Results
The Aphthous group had 45 females (64%) and 25 
males (36%) while Non-Aphthous group had 54 
females (60%) and 36 males (40%).The gender 
distribution of the both group I and group II is shown 
in the figure no 1.
Mean age of the Aphthous group was 28.76±9.144 
and of the Non-Aphthous group was 30.53±6.404.  
The comparison of mean ages between the aphthous 
and non-aphthous group was not significant (p>0.05). 
Biochemical findings:
Assessment of oxidative stress marker, Glutathione 
peroxidase:
With regard to oxidant/antioxidant status, mean of 
serum glutathione peroxidase in RAS patients was 
significantly lower (p <o.oo) than that of healthy 
controls, using t-test.

protein is a most potent enzyme in the defense of 
cells against increased reactive oxygen species. It 
utilizes its reduced glutathione form to convert 
hydrogen peroxide and lipid peroxides into 

14
alcohols.  Gpx-1 is found in all cells, present in 
mitochondria, cytosole and in few cases in 
peroxisomes. Studies have shown that it is more 
efficient in getting rid of intracellular hydrogen 
peroxide then catalase in a number of physiological 
situations. Analysis of previous studies indicates that 
for the activity of Gpx-1, selenium (Se) which is a 
trace element is required which is present in the 
form of amino acid Sec. Hence as Gpx-1 
enzymatically detoxifies non-radical hydroperoxides, 
it maintains cellular oxidant/antioxidant status by 
two ways;

a. Via direct removal of hydroperoxides 

b. Secondly through oxidation of reduced form of 
15

glutathione i-e GSH Regarding the insufficient 

studies related to this issue in Pakistan, the 

present study was carried to evaluate the 

possible relation between levels of glutathione 

peroxidase as a biological marker for oxidative 

stress and recurrent aphthous stomatitis.

Materials and Methods
This case-control study was conducted in 
Biochemistry department of Islamic International 
Medical College Trust Rawalpindi in collaboration 
with dental outpatient department of Pakistan 
Ordanance Factories Hospital, Wah Cantt from 15 
March 2016 to 14 March 2017. Convenient non-
probability sampling technique was used .The study 
population comprised of 160 subjects, 70 patients 
and 90 controls .Convenient  non-probability 
sampling technique was used. The institutional 
review board and ethical committee of Riphah 
University (Islamic International Medical College) 
approved the study protocol. The selected patients 
had active lesion of aphthous ulcer and had history of 
recurring oral ulcer attack at least three times a year, 
of any age and gender. For control, healthy 
individuals with no history of any episode of 
recurrent aphthous stomatitis and any systemic 
disease were selected. Patients were excluded if they 
had any history of systemic disease or any other 
concurrent oral lesions. Patients were excluded if 
they were on any therapeutic regimen or 
supplement of iron, multivitamin, steroid or other 

Fig 1: Gender distribu�on of study popula�on

JIIMC 2018 Vol. 13, No.3

134

Serum Enzymatic Antioxidant in Aphthous Stomatitis



In the present study, serum glutathione peroxidase-
1, an antioxidant marker showed significantly lower 
levels in recurrent aphthous stomatitis patients than 
the controls with a p value of zero.
This result was in agreement with the findings of 

12 
Arikan et al   and Cimen et al. In a study conducted 
by Arikan, glutathione peroxidase levels were 
significantly lower in recurrent aphthous stomatitis 
patients as compared to controls .Results of our 
study were in contrast to the findings by Saxena who 
in his study found increased levels of glutathione 
peroxidase-1 in recurrent aphthous stomatitis 

19
patients.
Selenium deficiency can occur with the decreased 
cellular immunity. By activating T-cell proliferation 
selenium supplement has an ability to markedly 
s t i m u l a t e  t h e  i m m u n e  r e s p o n s e .  
Immunostimulatory role of selenium is supported by 
the study conducted by McKenzie.et.al who 
demonstrated that in the presence of oxidative 
stress selenium supplementation regulates the 
cellular response by rapidly promoting and activating 

14
the cellular antioxidant defense system.
Devasagayam et al postulated that Gpx-1 deficiency 

20
causes free radical accumulation.
Regarding the association of antioxidant and 
recurrent aphthous stomatitis, the disagreement 
amongst different studies may be due to different 
factors including different sample size, genetic 
variation of different population
There is a need to carry out experimental studies to 
find out the causative relationship between 
recurrent aphthous stomatitis and impaired 
oxidant/antioxidant status.
Further prospective studies with larger sample size 
are also required to identify new preventive and 
treatment options in recurrent aphthous stomatitis 
patients.

Conclusion
Glutathione peroxidase levels were significantly 
lower in recurrent aphthous stomatitis patients as 
compared to controls, which suggests there is an 
alteration of oxidant/antioxidant status in RAS 
patients.

REFERENCES
1. Babaee N, Hosseinkazemi H, Pouramir M, Baboli OK, Salehi 

M, Khadir F et al. Salivary oxidant/antioxidant status and 

Discussion
Recurrent aphthous stomatitis or canker sores is an 
inflammatory ulcerative lesion and is one of the most 

1
common pathological disease of oral cavity.
Even though extensive research regarding the 
predisposition of recurrent aphthous stomatitis have 
been done, the definitive cause of RAS remains 
unclear. Moreover directly or indirectly different 
factors involved in the pathogenesis of disease 
results in the loss of balance between oxidant and 
antioxidant system of the body. The present study 
was undertaken to determine serum enzymatic 
enzymatic antioxidant levels in patients with 
recurrent aphthous stomatitis and healthy controls.
Loss of balance among oxidant species and 
antioxidants causes variety of inflammatory oral 
diseases that varies from simple infections to lethal 
cancers.  Recurrent aphthous stomatitis is also a sign 
of this imbalance. In several studies a possible 
relationship between free radical metabolism and 

16
inflammatory reaction have been demonstrated.  

17 
Lih-Brody et al. has found that in mucosa of 
inflammatory bowel disease patients there is an 
imbalance between oxidant/antioxidant statuses. 

15 
Also a study conducted by Kokcam and Naziroglu.
on psoriasis patients demonstrated a possible role of 
increased lipid peroxidation
Oxidative stress increases by the cellular damaging 
cytotoxic effects of reactive oxygen species. 
Nowadays in the maintenance of health and 
prevention from diseases, functional role of 
antioxidants has received huge attention.  
Glutathione peroxidase -1 is the chief antioxidant in 
getting rid of hydrogen peroxide by utilizing reduced 
form of glutathione. Elimination of excessive 
hydrogen peroxide by Gpx-1 is directly related to 
utilization of reduced form of glutathione resulting in 
its decreased levels. So in recurrent aphthous 
stomatitis patients there is a decrease in serum levels 
of Gpx-1.

Table I: Enzyma�c an�oxidant levels of recurrent 
aphthous stoma��s pa�ents and controls

JIIMC 2018 Vol. 13, No.3

135

Serum Enzymatic Antioxidant in Aphthous Stomatitis



JIIMC 2018 Vol. 13, No.3

A. Treatment of recurrent aphthous stomatitis. A literature 
review. Journal of clinical and experimental dentistry. 2014 
(2):e168

12. .Arikan S, Durusoy C, Akalin N, Haberal A, Seckin D. 
Oxidant/antioxidant status in recurrent aphthous 
stomatitis. Oral diseases. 2009; 15 (7):512-5.

13. Najafi S, Tonkaboni A, Mohammadzadeh M. Assessment of 
relation between antioxidants level and clinical 
manifestation in recurrent aphtous stomatitis. Journal of 
Craniomaxillofacial Research. 2015;3(1):157-60.

14. Forgione MA, Weiss N, Heydrick S, Cap A, Klings ES, Bierl C et 
al .Cellular glutathione peroxidase deficiency and 
endothelial dysfunction. American Journal of Physiology-
Heart and Circulatory Physiology. 2002 ;51(4):H1255

15. Lubos E, Loscalzo J, Handy DE. Glutathione peroxidase-1 in 
health and disease: from molecular mechanisms to 
therapeutic opportunities. Antioxidants & redox signaling. 
2011; 15(7):1957-97.

16. Avci E, Akarslan ZZ, Erten H, Coskun-Cevher S. Oxidative 
stress and cellular immunity in patients with recurrent 
aphthous ulcers. Brazilian Journal of Medical and Biological 
Research. 2014; 47(5):355-60.

17. Lih-Brody L, Powell SR, Collier KP, Reddy GM, Cerchia R, 
Kahn E et al . Increased oxidative stress and decreased 
antioxidant defenses in mucosa of inflammatory bowel 
disease. Digestive diseases and sciences. 1996; 
41(10):2078-86.

18. Kökçam İ, Nazıroğlu M. Antioxidants and lipid peroxidation 
status in the blood of patients with psoriasis. ClinicaChimica 
Acta. 1999; 289(1):23-31.

19. Saxena S. Assessment of plasma and salivary antioxidant 
status in patients with recurrent aphthous stomatitis. RSBO 
(Online). 2011 (3):261-5.

20. Devasagayam TP, Tilak JC, Boloor KK, Sane KS, Ghaskadbi SS, 
Lele RD. Free radicals and antioxidants in human health: 
current status and future prospects. Japi. 2004; 52(10):794-
804.

hematological parameters in patients with recurrent 
aphthous stomatitis. Caspian journal of internal medicine. 
2016; 7(1):13.

2. Samet N, Laurent C, Susarla SM, Samet-Rubinsteen N. The 
effect of bee propolis on recurrent aphthous stomatitis: a 
pilot study. Clinical oral investigations. 2007; 11(2):143-7.

3. Azizi A, Shahsiah S, Ahmadi M. Serum vitamin A level in 
patients with recurrent aphthous stomatitis compared with 
healthy individuals. Journal of Islamic Dental Association of 
IRAN (JIDAI). 2012; 24(4):4.

4. Shashy RG, Ridley MB. Aphthous ulcers: a difficult clinical 
entity. American journal of otolaryngology. 2000; 
21(6):389-93.

5. Preeti L, Magesh KT, Rajkumar K, Karthik R. Recurrent 
aphthous stomatitis. Journal of Oral and Maxillofacial 
Pathology. 2011 ;15(3):252.

6. Chavan M, Jain H, Diwan N, Khedkar S, Shete A, Durkar S et 
al.. Recurrent aphthous stomatitis: a review. Journal of Oral 
Pathology & Medicine. 2012; 41(8):577-83.

7. Saral Y, Coskun BK, Ozturk P, Karatas F, Ayar A. Assessment of 
salivary and serum antioxidant vitamins and lipid 
peroxidation in patients with recurrent aphthous 
ulceration. The Tohoku journal of experimental medicine. 
2005; 206(4):305-12.

8. Cağlayan F, Miloglu Ö, Altun O, Erel Ö, Yılmaz AB. Oxidative 
stress and myeloperoxidase levels in saliva of patients with 
recurrent aphthous stomatitis. Oral diseases. 2008; 
14(8):700-4.

9. Bilal N, FatihKarakus M, Varkal MD, Boztepe OF, Bilal B. An 
Assessment of the Levels of Anxiety and Depression in 
Patients with Recurrent Aphthous Stomatitis. Arch 
Otolaryngol Rhinol. 2015; 2 (1): 5001-07.

10. Koybasi S, Parlak AH, Serin E, Yilmaz F, Serin D. Recurrent 
aphthous stomatitis: investigation of possible etiologic 
factors. American journal of otolaryngology. 2006; 
27(4):229-32.

11.  Belenguer-Guallar I, Jiménez-Soriano Y, Claramunt-Lozano 

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