Assessment of Oral Mucosal Integrity Status in Patients with Recurrent Aphthous Stomatitis.

BACKGROUND: Recurrent aphthous stomatitis represents one of the most common oral pathoses with multifactorial etiology. Decrease in mucosal barrier resistance is believed to be one of the etiologies in its pathogenesis. This study was conducted to assess the oral mucosal integrity status by evaluating the salivary albumin level in patients with aphthous stomatitis.
MATERIALS AND METHODS: Thirty patients diagnosed with aphthous stomatitis were selected as case group. Equal number of age- and sex-matched healthy individuals formed the control group. Salivary albumin level was estimated during active and quiescent stage of the disease in both case group and control group. RESULT: Mean salivary albumin level for the case group during the active and quiescent stage was 0.070 g/dL (SD = 0.037) and 0.004 g/dL (SD = 0.007) (SPSS, version 7.0), respectively, and that for the control group was 0.027 g/dL (SD =0.042). Statistically significant difference was found on comparison of the mean salivary albumin level between the case group during active and quiescent stage and the normal controls using Mann-Whitney U test. No statistically significant difference in salivary albumin level was seen between the quiescent stage in case group and the normal controls.
CONCLUSION: Increase in salivary albumin level at the time of disease presentation could be attributed to the leakage of albumin through the ulcerated mucosa. Absence of significant elevation in the salivary albumin level after the resolution of the aphthous ulcer apparently indicates inherently a healthy mucosal barrier in majority of the patients.

INTRODUCTION

Recurrent aphthous stomatitis (RAS) or ulcer is one of the most common oral mucosal pathoses and the reported prevalence in the general population varies from 5% to 66%.[12] There are numerous hypotheses for its pathogenesis with diverse initiating causes, namely allergies, genetic predisposition, hematologic abnormalities, hormonal influences, immunologic abnormalities, infectious agents, trauma, and stress. Grouping all these factors, it has been hypothesized that (i) primary immunodysregulation, (ii) increase in antigenic exposure, and (iii) decrease of the mucosal barrier may play a role in its pathogenesis.[34]

Attempts are being made to establish the role of above-mentioned factors by studying the different salivary markers and utilizing various techniques ranging from simple to more sophisticated ones. It has been hypothesized that whole salivary albumin concentration in the oral cavity might directly reflect the quality of epithelial barrier and it can be used to assess the oral mucosal integrity status.[5678]

With this background, this study was undertaken to evaluate the salivary albumin level in patients with RAS and in normal healthy control subjects using a semi-autoanalyzer.

MATERIALS AND METHODS

In this comparative study, 30 patients diagnosed with RAS formed the case group and equal number of age- and sex-matched healthy individuals without history of RAS formed the control group. Subjects with known systemic diseases, which could affect normal mucosal integrity or salivary characteristics; currently receiving medications for any systemic problems; with severe gingivitis and periodontal problems; and with history of smoking and alcohol were excluded from the study.

The sample size for cases and controls is 30. University ethical committee clearance was obtained for the study. The purpose of the study was explained and a written informed consent was obtained from all the participants. A detailed clinical history for each participant was recorded followed by complete oral examination.

The participants were given the following instructions prior to saliva collection to obtain reliable, unstimulated whole saliva: to refrain from eating and drinking at least 90 min before collection, and to rinse mouth thoroughly with deionized/distilled water prior to the collection to void the mouth of saliva and to avoid swallowing and oral movements during collection. Whole unstimulated saliva (2 mL) was obtained under restful conditions in a sterile graduated container by spitting method.

All collected samples were immediately centrifuged at 2000 rpm for 10 min; salivary supernatant was separated, which was used for estimation of salivary albumin level by bromocresol green method utilizing albumin estimation kit and semi-autoanalyzer.

The albumin concentration level (g/dL) was obtained for the case group during active and quiescent stage, and it was recorded for the control group as well.

RESULTS

A total of 30 patients comprising 3 men (mean age = 27.3 years) and 27 women (mean age = 20.3 years) with RAS of oral mucosa formed the case group, and for each individual case, age- and sex-matched healthy individuals formed the normal control. All the 30 cases were found to have minor type of recurrent aphthous ulcer.

Of the total 30 patients, 11 presented with single ulcer. Of the remaining 19 patients, 12 with 2 ulcers and 7 with 3 ulcers each.

The labial mucosa was found to be the most common site for the lesion [i.e., 25 patients (83.3%)] followed by the alveolar mucosa [i.e., 4 patients (13.3%)], and tongue [1 patient (3.3%)].

The mean salivary albumin level for the case group during the active and quiescent stage of the disease was found to be 0.070 g/dL (SD = 0.037) and 0.004 g/dL (SD = 0.007), respectively. For the control group, the mean salivary albumin level was 0.027 g/dL (SD = 0.042).

Comparison of mean salivary albumin levels between active and quiescent stages in the case group using Mann–Whitney U test is shown in Table 1.

Table 1

Comparison of mean salivary albumin levels between active and quiescent stage in case group using Mann–Whitney U test

Group	Mean	N	Standard deviation	Mean difference	z value	P value
Cases
Active stage	0.070	30	−0.037	0.0658	−4.712	<0.001*
Quiescent stage	0.004	30	0.00733

*Statistically significant.

The mean albumin level for the case group in active and quiescent stage was 0.070 ± 0.007 and 0.004 ± 0.042 g/dL, respectively. The mean difference was 0.0658 g/dL, which was found to be statistically significant at P value of <0.05 using Mann–Whitney U test.

Comparison of salivary albumin levels between active stages in case group and normal controls using Mann–Whitney U test is shown in Table 2.

Table 2

Comparison of salivary albumin levels between active stages in case group and normal controls using Mann–Whitney U test

Group	Mean	N	Standard deviation	Mean difference	P value
Cases, active stage	0.070	30	0.037	0.0421	0.002*
Controls	0.027	30	0.04273

*Statistically significant.

The mean albumin level for the case group in active stage was 0.070 ± 0.007 g/dL and that for normal controls was 0.027 ± 0.042 g/dL. The mean difference was 0.0421 g/dL, which was found to be statistically significant at P value of <0.05 using Mann–Whitney U test.

Comparison of mean salivary albumin levels between quiescent stages in case group and normal controls using Mann–Whitney U test is shown in Table 3.

Table 3

Comparison of mean salivary albumin levels between quiescent stages in case group and normal controls using Mann–Whitney U test

Group	N	Mean	Standard deviation	Mean difference	z value	P value
Cases, quiescent stage	30	0.004	0.007	−0.022	−1.460	0.144NS
Controls	30	0.027	0.042

NSStatistically not significant.

The mean albumin level for the case group in quiescent stage was 0.004 ± 0.007 g/dL and that for normal controls was 0.027 ± 0.042 g/dL. The mean difference was −0.022 g/dL, which was not statistically significant at P value of <0.05 using Mann–Whitney U test.

DISCUSSION

RAS is a common disease characterized by the development of painful ulcers in the oral mucosa.[12]

Decrease in the oral mucosal barrier has been assumed to a play role in the pathogenesis of RAS along with primary immune dysregulation and antigenic exposure.[34] We hypothesized that compared to other factors such as primary immune dysregulation and increased antigenic exposure, quality of mucosal barrier plays a less significant role in the development of RAS. With this background, this study was conducted to assess the actual role of mucosal barrier resistance in the development of RAS by estimating the albumin level in saliva.

Our study findings showed significant increase in the salivary albumin level in the active stage of the disease, that is, when the ulcers were clinically present in the oral mucosa. This difference was statistically significant when compared to the quiescent stage of the disease, that is, 1 week after the resolution of the ulcers in the same patients and the normal controls. Also, comparison of the salivary albumin level between patient at the time of resolution of the ulcer and the normal controls revealed no significant difference.

Henskens et al.[9] assessed the salivary protein concentration in patients with gingivitis and periodontitis and reported increased salivary albumin in them compared to normal controls.

Shaila et al.[10] and Koss et al.[11] also reported increase in salivary albumin concentration in patients with gingivitis or periodontitis. They had explained that periodontal inflammation accompanied by the degeneration of periodontal cells resulting in defective mucosal barrier allows increased leakage of plasma protein, which leads to increased albumin level in saliva. Because gingivitis and periodontitis can potentially alter the salivary albumin levels, individuals with severe gingivitis and periodontitis were excluded from this study.

Therefore, it is logical to believe that the significant increase in salivary albumin level at the time of active stage of the disease in the patients with RAS is due to increased plasma protein leakage through the ulcerated mucosa. After the healing of ulcers, the oral mucosa in the patients acts as an effective barrier similar to the normal healthy controls.

Also, lack of significant difference in the salivary albumin level between the quiescent stage in the case group and the normal control group can be due to the presence of relatively normal mucosal barrier in the case group after the healing of ulcer and normal controls. This apparently indicates that mucosa is not inherently defective in the patients with RAS in this study.

Martinez et al.[12] analyzed the secretory A immunoglobulin (IgAs) in patients with RAS and demonstrated a significant increase in salivary IgAs in the active stage of RAS compared to quiescent stage, which reflects the increased secondary mucosal immune response to the disease in the active stage and not a primarily weak mucosal barrier, which further supports the findings of this study.

CONCLUSION

Definite increase in the salivary albumin concentration during the active stage of RAS compared to the quiescent stage and the normal controls could be due to the increased leakage of plasma proteins through the ulcerated mucosa. Further, lack of significant elevation in the salivary albumin level after the resolution of the ulcer apparently indicates inherently a healthy mucosal barrier in the patients with RAS. The results of this study support the hypothesis of primary immune dysregulation and increased antigenic exposure playing a major role in the development of RAS compared to inherently defective oral mucosal barrier. However, more studies utilizing larger sample size and other forms of aphthous ulcer, that is, major and herpetiform ulcer, are needed to draw a definite conclusion and more insight into the pathogenesis of RAS.

Financial support and sponsorship

This study got selected for the Indian Council of Medical Research Short-term Studentship (ICMR STS) program. The completed research was approved, and certified by ICMR.

Conflicts of interest

There are no conflicts of interest.