Evaluation of Salivary Opiorphin, pH and Flow Rate in Oral Submucous Fibrosis - A Case - Control Observational Study

ABSTRACT

Aim and Objectives:

The aim of the study was to evaluate the salivary pH, flow rate and opiorphin in Oral submucous fibrosis patients and to compare them with healthy individuals.

Materials and Methods:

A comparative study was conducted to evaluate the salivary opiorphin levels, pH and flow rate among 78 patients (39 Oral submucous fibrosis + 39 control). Burning sensation was assessed using VAS score. Opiorphin levels were assessed using Cusabio Human Salivary Opiorphin ELISA kit. Salivary flow rate was assessed using graduated syringes and salivary pH was assessed using Hydrion salivary pH strips. Kolmogorov-Smirnov and Shapiro Wilk test were assessed for normality testing and distribution. Inferential statistics was done by using Unpaired t test/Independent sample t test to compare the mean values of variables included in the study among the OSMF and healthy patients.

Results:

There was statistically significant difference in pH and flow rate with a reduction in the mean pH (p=0.024) and flow rate (p<0.01) among the oral submucous fibrosis patients compared to the control group. A statistically significant increase (p<0.01) in salivary opiorphin levels was noted in the oral submucous fibrosis patients. Majority of the study participants had mild to moderate burning sensation.

Conclusion:

This study was a pilot evaluation of salivary opiorphin in OSMF. Salivary opiorphin could be used as an objective marker for chronic subjective conditions The pre-treatment and post treatment evaluation of salivary opiorphin could provide more insight on its role.

INTRODUCTION

Opiorphin is a newly found endogenous peptide exhibiting pain relieving effects by increasing the action of enkephalins which are endogenous circulating opioid agonists.

Opiorphin is present in biofluids such as saliva, blood, urine, semen, milk, and tears with the highest concentration in human tears and saliva. It plays a salient role in perception of orofacial pain.[1,2]

Orofacial nociception, supported by the trigeminal cranial nerve, is linked with the spinal trigeminal nucleus caudalis which contains a high concentration of μ and δ-opioid receptors that are activated by the enkephalins. The pain perception is via endogenous enkephalin-related activation of mu (μ) and delta (Δ) opioid receptors.[3] The most frequent debilitating symptom that always results in disturbed psychological conduct is pain.[4]

Salivary opiorphin has been found to produce analgesic effects similar to that of morphine by acting on mu and delta receptors. Opioids act on mu (μ), kappa, and delta (Δ) receptors causing adverse effects like physiological dependence, tolerance, and conditioned reward. Opiorphin averts these side effects. Recent research on the role of opiorphin in orofacial nociception and its analgesic effects in various rodent models of pain have proved to be effective.[5,6]

Oral submucous fibrosis has become a major health concern to healthcare providers as young adults are affected and also debilitating, leading to a poor quality of life.[7,8] In the act of ghutka chewing, harmful chemicals are leached into saliva which causes irritation to the mucosa, altering the flow rate and pH.[9-11]

Diagnosis of Oral submucous fibrosis is centered on clinical signs and symptoms.[12] Salivary opiorphin could be a measurable biomarker for objective evaluation of painful conditions of the oral cavity. The literature reviews have shown an increase in salivary opiorphin levels in response to pain and inflammation. To the best of our knowledge, there is no literature suggesting the correlation of salivary pH, flow rate, and opiorphin. A comprehensive history and physical examination are advised as the first stages in reaching an accurate diagnosis.[13,14]

MATERIALS AND METHODS

Ethical consideration

Ethical approval was obtained from Institutional Review Board (IRBNo.SRMDC/IRB/2020/No. 903). Individuals volunteering to participate in the study meeting the criteria of inclusion were recruited in the study following the principles of Helsinki Declaration. The study was carried out from the period of March 2021 to September 2022.

Study design and participants

A cross-sectional case control observational study was conducted in the outpatient Department of Oral Medicine and Radiology, SRM Dental College, Ramapuram. The study population included patients reporting to the out-patient Department (OPD) of the college. They were screened along with the recording of their complete dental and medical history. A simple random sampling technique was used in which all the participants who reported with OSMF and a complaint of burning sensation were included in the study. Patients were grouped into two groups, Group I (Oral submucous fibrosis) which included patients within the age group between 20 and 50 years who were clinically diagnosed with Pindborg’s clinical classification of oral submucous fibrosis with no systemic diseases and Group II (Healthy Controls) where patients were age and sex matched. Patients who used opioid analgesics or antibiotics 48 hours prior to saliva collection, systemic conditions, had other conditions that affect salivary flow rate such as xerostomia, diabetes mellitus, and radiotherapy, with complaints of other dental related chronic pain, narcotic drug use, pregnancy and breast feeding, and unwilling to participate in the study were excluded. Patients were explained about the study in both English and vernacular language (Tamil). Written consent was obtained from those patients who were willing to participate in the study.

Methodology

Visual analogue scale (VAS) was used for assessing burning sensation. A scale ranging from 0 (no burning sensation) to 10 (severe burning sensation) was used. Patients were verbally instructed in their local language to use the scale.

Unstimulated whole saliva was collected from all the patients between 10 and 13:00 hours to avoid diurnal variations. Before collection, the participants were requested to rinse with water. The participants were asked to relax, without triggering salivation, comfortably seated with their heads lowered and facing slightly forwards. Then, after swallowing all the saliva present in the mouth, they were directed to permit new saliva in the mouth and to expectorate into a disposable saliva collecting tube. The saliva collected using a disposable 10 mL sterile syringe, preventing contact with the foam. The mean salivary flow rate was determined with the quantity of saliva and the duration of collection. The collected saliva was refrigerated for 1 hour in 4 degrees Celsius. The stored samples were then centrifuged at 4000 rpm for 10 minutes to remove the debris. The supernatants were then transferred into aliquots using 10 mcL pipette. The aliquots were kept in deep freezer at -80 degree Celsius.

The pH of the saliva was determined between 10 and 13:00 hours to avoid diurnal variations. Before collection, the participants were requested to rinse with water. After swallowing all the saliva present in the mouth, the pH strip was placed sublingually and 10 seconds were allowed for color change to occur. Salivary pH was determined by matching the color code chart available in the Hydrion pH strip and the values obtained were recorded.

For the analysis of salivary opiorphin, the samples were thawed at 4 degrees Celsius. As opiorphin is susceptible to the peptidases in the saliva, a protease inhibitor As the stability of opiorphin is susceptible to damage, the samples were treated as suggested by Ozdogan MS et al.[15] before centrifugation. The mixture was centrifuged at 4000 rpm for 10 minutes at 2-8°C and the acid phase with the high molecular weight components were eliminated and the methanol phase with small molecular weight components were collected for analysis.

Statistical analysis

The statistical analysis was performed using the SPSS Software (IBM SPSS Statistics for Windows, Version 26.0, Armonk, NY: IBM Corp. Released 2019). The Normality tests, Kolmogorov–Smirnov, and Shapiro–Wilks tests were used to test for normality distribution of the data. Significance level was fixed as 5% (α =0.05). P value < 0.05 was considered to be statistically significant.

RESULTS

The descriptive statistics of age, pH, and salivary flow rate among the control and the study group was performed. The mean pH and flow rate was higher in control group (6.91,0.42) than the study group (6.49,0.33) [Graphs 1 and 2]. The mean of opiorphin was comparatively more in study group (14.69) than the control group (10.70). The descriptive statistics of VAS score among the study group was also performed which showed that the mean was found to be 3.84 showing that maximum number of patients in study group were having mild-moderate pain according to the mean obtained.

Graph 1.

Mean difference of pH among the control and study group

Graph 2.

Mean difference of flow rate(ml/min) among the control and study group

While obtaining the mean difference of ph among control and study group it was found to be 0.41 and the P value was found to be <0.05 showing statistical significance [Graph 1], the mean difference of flow rate among control and study group it was found to be 0.08 with a P value of <0.01 [Graph 2]. The mean difference of opiorphin was obtained as 3.99 with a P value of <0.01 implying highly statistically significant difference [Graph 3].

Graph 3.

Mean difference of salivary opiorphin(ng/dl) among the control and study group

The influence of pH and salivary flow rate on the amount of opiorphin in control group was assessed. P value was found to be 0.67 which implied that there is no influence of pH on the amount of opiorphin level among the control group.

The P value was found to be 0.84 while assessing the influence of flow rate on amount of opiorphin showing no influence of unstimulated salivary flow rate on the amount of opiorphin level among the control group.

Among the study group, the influence of pH and salivary flow rate on the amount of opiorphin was assessed and the P value was found to be <0.01 in both showing that there is clear influence.

While assessing the difference between Stage I and Stage II of Clinical Classification by Pindborg’s the P value was found to be <0.01 which implies that there is statistically significant difference [Table 1], Stage II oral submucous fibrosis patients had increased levels of salivary opiorphin when compared to Stage I OSMF patients.

Table 1.

This table depicts the difference between Stage I and stage II OSMF patients

Independent Samples Test
	Levene’s Test for Equality of Variances		t-test for Equality of Means
	F	Sig.	t	df	Sig. (2-tailed)	Mean difference	Std. Error difference	95% Confidence Interval of the Difference
								Lower	Upper
STAGES VS OPIORPHIN	3.139	0.085	-6.375	37	0.0001	-1.40595	0.22053	-1.85280	-0.95911

DISCUSSION

Salivary opiorphin levels are seen in higher concentration in saliva and tears followed by other biological fluids like blood, urine, and sweat. Saliva is one of the efficient, non-invasive, simple, cost effective, specimen to be collected.[16]

Adequate salivary flow and saliva content are directly related to health status and are important for maintenance of body functions. Salivary glands, like other human organs such as the kidneys, contain a circadian clock. It has been shown that a decrease in the salivary flow rate results in a decrease in intraoral pH due to reduced intraoral buffering capacity and acid clearance. Salivary pH and flow rate play a vital role in the progression of the disease process. Hence, they were also evaluated along with opiorphin so as to minimize confounding bias. Moreover, in the past several decades, dental researchers reported different aspects of OSMF.[17] Yet, there is no literature in the present scenario of evidence-based dentistry which correlates the role of salivary opiorphin with critical parameters like salivary pH and flow rate in OSMF.

Studies have proved a consistent decrease of opiorphin levels in stimulated saliva. Hence, unstimulated whole saliva was considered in our study.

In the present study, the mean salivary pH and flow rate were reduced in oral submucous fibrosis patients when compared to the healthy subjects.

Prashanth Tamgadge et al. 2020, comparatively evaluated the alteration of salivary pH in areca nut chewers with and without OSMF and healthy subjects. They found that chemicals and metals that leached into the saliva were responsible.[18]

Similar results were also found in Rohit Kumar Sahu et al. 2021. Our results are in line with this study.[19]

The decrease in salivary pH and flow rate in the OSMF group is due to the conversion of lime fro arecoline to arecadine causing atrophy of acinar cells.[20-22]

The enkephalin level during noxious stimuli causes concurrent elevation in the concentration of opiorphin.[23,24]

Boucher et al. reported a variation in the hypothesis and it could be related to the technique of quantification of salivary opiorphin levels.[25]

The salivary opiorphin levels in inferior alveolar nerve block was increased as it requires more than one pick in buccal and lingual sides thereby increasing the patient’s perception of pain.[26]

In our study, we found a steady correlation of salivary pH and flow rate on salivary opiorphin levels. We found that as the pH and flow rate of saliva in study group decreased, the levels of salivary opiorphin increased. These biochemical changes affect salivary homeostasis and are considered as a major cause for hyposalivation in OSMF patients, which in turn is responsible for the development of burning sensation.

We have also found that as the clinical staging of Oral submucous fibrosis increased, the level of salivary opiorphin levels also increased. There was no significant difference in the normal group. As the severity of Oral submucous fibrosis increases, the inflammation and fibrosis also escalated, decreasing the salivary pH and flow rate and thus increasing the amount of salivary opiorphin level.

There are a few limitations to this study. The sample size was less and the subgroup analysis on salivary opiorphin levels based on the Clinical staging of Oral submucous fibrosis in accordance with Pindborg’s classification was not performed.

CONCLUSION

In Oral Submucous Fibrosis, the elevated levels of salivary opiorphin in comparison to healthy patients proves its role in pain and inflammation. The levels of salivary opiorphin were inversely proportion to the salivary pH and flow rate. Further research with the subgrouping in Oral submucous fibrosis patients and corelating the levels of salivary opiorphin at multicentric level could provide improved insight on its role.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.