Recurrent aphthous stomatitis (RAS) is a chronic relapsing inflammatory disorder of the oral mucosa with unknown etiology, characterized by painful ulcerations of various sizes. RAS was reported to affect about 25% of the people in different populations1-4. Trauma, viral or bacterial infections, stress, nutritional deficiencies, systemic diseases, immunological status, or genetic predisposition may elicit the formation of RAS lesions1-6. Since these possible factors may imbalance the oxidant/antioxidant status of the organism6-9, a relationship between systemic oxidative stress (OS) and RAS has been questioned.

OS is the reflection of an imbalance between production of reactive oxygen species (ROS) and the antioxidant mechanisms of the organism that detoxify these radicals. Under physiological circumstances, the production of ROS is not harmful to the organs and is limited by antioxidants. However, if the concentration of ROS exceeds over pathological values, the antioxidants cannot prevent excess cytotoxic effects and oxidative damages to the organisms. There are various antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GSHPx), and catalase (CAT) and as well as non-enzyme antioxidant molecules including glutathione, uric acid, melatonin, and vitamins A, C, E in the organism. In OS, antioxidants are spent for the defense that detoxicifies the oxidant molecules resulting in depletions of antioxidant status10-12. Since the new condition is not balanced, OS affects the entire organism.

In this study, we hypothesized that there is a systemic OS in patients with RAS. Until today, many studies have investigated the amounts of oxidative reactive species, non-enzymatic molecules, and antioxidant enzymes as mentioned above in RAS6,8,9,13-17. However, measurements of these molecules individually do not reflect the global status of OS. Serum (or plasma) concentrations of different oxidant species can be measured in laboratories separately, but such measurements are time-consuming, labour-intensive, costly and require complicated techniques. Since measuring different oxidant molecules separately is impractical and their oxidant effects are additive, the total oxidant status (TOS) sample is extracted instead. In this study, the method determines total oxidative status in relation to cumulative oxidative effects of various oxidants which are generated in biological systems such as hydrogen peroxide, lipid hydroperoxide, protein hydroperoxide, peroxynitrite and other reactive oxygen and nitrogen species18,19. Nevertheless, another recently developed method measuring the total antioxidant status (TAS) provides the evaluation efficiency of all antioxidants20. These measurements show the global effects of the molecules involved in OS conditions rather than individual effects for each oxidant and antioxidant molecular mechanism. To date, there have been no reports investigating the systemic OS from these methods which measures the levels of serum TOS and TAS in RAS.

The paraoxonase 1 (PON1) enzyme is an antioxidant and anti-inflammatory enzyme. The PON1 enzyme has both paraoxonase and arylesterase (ARES) activities. The PON1 ARES activity of the organism protects low-density lipoprotein (LDL) and lipoproteins from oxidation, which are important biomolecules produced during oxidative damages in atherosclerosis21,22. The PON1 enzyme activities have been demonstrated to reduce in metabolic syndrome, atherosclerosis, and diabetes mellitus, in which the OS plays a major role23,24. However, alterations in ARES activities of the patients with RAS have not yet been investigated.

In this study, we determined the status of OS in RAS patients by measuring the levels of TOS, TAS, and oxidative stress index (OSI), and ARES activity.

The study enrolled a total of 44 patients (21 females, 23 males; mean age: 30.5 years; range: 19~55 years) with RAS lesions (Table 1). The patients and healthy controls matched according to age and gender (p-values are 0.825 and 0.389, respectively).

The median duration of RAS attacks of patients was 3.5 years (range: 1.0~30.0 years). The range of frequency of RAS attacks per month was 1 to 6. Twenty-three patients (52.3%) had two attacks per month. The first degree relatives of 12 patients (27.3%) also had medical histories of RAS.

RAS patients had significantly lower TAS levels than control subjects (2.95±0.41 vs. 3.49±0.49; p<0.001). The TOS levels were higher in patients as compared to controls (15.32 [10.83~23.71] vs. 10.58 [7.46~15.08]; p=0.008). The OSI, as calculated by the formula, was significantly higher in RAS patients than the control subjects (0.53 [0.40~0.79] vs. 0.30 [0.23~0.47]; p≤0.001) (Table 2).

The TG amounts were similar between groups (p=0.115); however, HDL levels were lower in RAS patients than control subjects (p<0.001). The ARES activity was lower in patients with RAS than healthy controls (176.43±21.61 vs. 210.47±27.64; p<0.001) (Table 3).

In the patient group, no correlations were observed between OS parameters (TAS, TOS, OSI, ARES activities) and age, gender, duration of disease or frequency of RAS attacks (all p>0.005).

In this study, we determined that there was a systemic OS reflected by an imbalance between the TOS and antioxidant status and lower ARES activities in patients with RAS. The results of serum TOS, TAS, and OSI values demonstrated the global effect of the molecules involving in OS rather than individual effects of each oxidant and antioxidant molecules. To date, many oxidant and/or antioxidant products in patients with RAS have been investigated in recent studies6,8,9,13-17. Several laboratory kits and different methods were used to determine the oxidant reactive species, antioxidant enzymes, and non-enzymatic molecules. In general, studies defined alterations of oxidant and antioxidant status in RAS although there are some conflicting results. Cimen et al.13 demonstrated decreased CAT, GSHPx, and antioxidant potential; and increased malondialdehyde levels in RAS patients. In contrast, Gunduz et al.14 did not find a significant difference between RAS patients and controls with regards to SOD, CAT, nitrite, and nitrate levels. In the study of Karincaoglu et al.15, plasma SOD and CAT were lower and GSHPx was higher in RAS patients than healthy controls; however their salivary levels were opposite to the plasma concentrations. The salivary antioxidant defense seemed to be stimulated against ulcerated lesions. Momen-Beitollahi et al.9 detected similar concentrations of erythrocyte GSHPx and CAT concentrations, salivary and plasma TAS in patients and controls. Only the erythrocyte SOD activity was lower in patients. They concluded that antioxidant system in saliva and plasma might not reflect the TAS of the whole body.

Serum TAS and TOS levels and OSI were evaluated in RAS patients in only one recent study by different methods used in the present study16. The investigators demonstrated that RAS patients had higher TOS and lower antioxidant levels than healthy controls. The results of our study were compatible with these findings. However, the values of these parameters were higher in our study. These differences may be related to the different methods used when measuring the TAS and TOS levels. Since the Erel method19,20 was developed more recently and has been shown to be more specific and sensitive to detect the TAS and TOS levels; higher levels of oxidant and antioxidant molecules might have been measured in the present study. The OSI detected in our study was also higher in RAS patients than in healthy controls, which indicated the exact degrees of imbalance of OS towards the oxidant status. The OSI shows the ratio of TOS and TAS, which is more objective to evaluate the conditions of OS rather than evaluating the TAS and TOS separately25. All results suggested that the release of excessive ROS represents an increase in serum TOS levels in patients with RAS, which affects the entire organism in general. In a recent study, TAS, TOS, and OSI were measured by Erel methods in the saliva of RAS patients and no significant differences were found between patients and healthy controls8. The measurements of same markers in the serum of RAS patients in our study suggest that global serum oxidant and antioxidant molecules and the imbalance between them may be more important than local oxidant and antioxidant markers detected in saliva of RAS patients. Since the patients had minor aphthous lesions, it is interesting that the organism showed an obvious imbalance of OS. Therefore, the micro-environmental tissue damages in small ulcerations may be related to factors causing OS and thus, resulting in RAS formation. Although the peripheral neutrophils of the immune system cells were shown to produce reactive oxygen intermediates6, the origin of OS in RAS still needs further investigations. The total serum oxidants and antioxidants were measured simultaneously, thus, the cause and effect relationship is not certain in the present study. To demonstrate such a relationship between these OS markers, a longitudinal assessment of post-treatment status may be considered after an optimal therapeutic attempt, such as supplementations with antioxidants or treatment with various agents, in order to reduce the level of OS levels. In addition, studies which concurrently compare the circulating levels of OS markers (TAS, TOS, OSI, ARES activity) with tissue levels may help to analyze the details of OS in the pathogenesis of RAS.

In our study, the ARES activity of PON1 enzyme of the RAS patients was found to be lower than healthy individuals. The ARES activity represents one of the antioxidant enzymatic activities of PON1 enzyme, which has an important role in modulating OS and in protection from cardiovascular diseases. This enzyme is located on high density lipoprotein cholesterols (HDL-C) and protects LDL cholesterol from OS by hydrolyzing lipid peroxides26,27. The lower TAS, ARES, HDL-C levels and higher TOS and OSI values provide support that a systemic OS is present in RAS patients. All observations suggest that a systemic OS is responsible for RAS formation as well as affected the entire organism.

In conclusion, systemic OS may have an important role in the etiopathogenesis of RAS. Future studies investigating the origin of OS and the systemic OS effects of the whole organism in RAS patients are needed.