The main diagnostic method for LPRD that is most commonly practiced clinically is empirical medication with a proton pump inhibitor, although there are other diagnostic methods, such as laryngoscopy examination, 24-hour double pH monitoring, and manometry.15 Thus, although many studies are currently under way to find an objective and noninvasive diagnostic strategy for LPRD, there is still no definitive diagnostic method.23
Recently, the diagnostic value of pepsin has emerged as a noninvasive, low-cost salivary pepsin detection method.8, 19–21,24 Pepsin is a specific biomarker that can detect gastric reflux and saliva, because it is produced only in the stomach.19 After the first use of the salivary pepsin assay to analyze saliva/sputum pepsin in GERD using the fibrinogen digestion method was published, Western blotting and enzyme-linked immunosorbent assay (ELISA) methods were used to detect pepsin.25,26 Furthermore, the rapid lateral flow test was developed to provide rapid analysis and predictive values.26 However, since pepsin can be detected in LPRD as well as in GERD or GERD-related disorders, it might show low sensitivity and specificity values. Therefore, in order to develop a more specific diagnostic tool for LPRD, we focused on the diagnostic feasibility of MMP-7, which was associated with E-cadherin degradation, part of the pathogenesis of LPRD, in our previous study.22
Belafsky et al. developed an index using nine questions to document the severity of LPRD symptoms. 4 The RSI data were derived from personal databases without evidence of LPR, and results were compared before and after PPI prescriptions. The mean pretreatment RSI of LPR patients was significantly higher than that of the control group, and after 6 months of PPI treatment, the mean RSI of LPR patients was close to that of the asymptomatic control group. The indicator is considered abnormal if the RSI score is 13 or higher. In our present study, when we divided a population into a study group and a control group based on a RSI score of 13, the activities of pepsin and MMP-7 showed a significant difference between the two groups.
In the previous studies of an association between RSI score and pepsin in LPRD patients, higher pepsin levels in sputum were associated with higher RSI and RFS scores 27,28. However, considering that pepsin is activated at an acidic condition below pH 4, pepsin levels as well as the presence of acid reflux should be evaluated to diagnose LPRD. Acid reflux can be assessed by 24-hour double pH monitoring, but this diagnostic technique is expensive and invasive, and thus is not used in a real clinical setting. Our previous study demonstrated that the MMP-7 plays an important role in acidic damage to the pharyngeal mucosa in an LPRD in vitro model made by pH 4 acid exposure. Therefore, in this study we proved the correlation with RSI using two main causes, pepsin and acid-induced MMP-7, which cause damage when LPRD occurs.
In addition, to measure the prediction of disease diagnosis, we evaluated clinical sensitivity and specificity using the ROC curve based on the activity of pepsin and MMP-7 in the normal and study groups. When only the activity of pepsin was used as a diagnostic marker, the sensitivity and specificity were 51.43% and 74.68%, which results were similar to those of a previous study 29, in which the presence of pepsin in the saliva of patients with chronic cough and vocal-cord dysfunction induced during LPR was found in 87% of patients with an RSI score of 13 or higher, and in 51% of patients with an RFS score of 7 or higher. However, the sensitivity and specificity of pepsin in saliva were relatively low, at 78% and 53%, respectively. In our present study, when MMP-7 activity was used as a marker, the sensitivity and specificity were 71.43% and 79.75%, and when the two markers, MMP-7 and pepsin, were combined, the sensitivity and specificity increased to 80% and 82.28%, respectively. These results indicate that the disease predictability is better when MMP-7 is used together with pepsin as diagnostic markers. In addition, the presented criterion can be used as a cut-off value to identify the presence or absence of the disease when developed as a diagnostic device in the future.
Since we conducted this study with a group of healthy volunteers classified into a control group and a study group according to the RSI score, there are some limitations in the reliability of the data. In order to assess the diagnostic value of MMP-7, it is necessary to conduct a study using saliva from symptomatic patients diagnosed with LPRD and to evaluate whether the expression of MMP-7 in saliva decreases after LPRD treatment.
In summary, the protein expressions of soluble e-cadherin and MMP-7, as well as the activity of MMP-7 and pepsin, were significantly increased in saliva samples taken immediately after waking up in the morning from the study group with a score of 13 or higher on the RSI. The sensitivity and specificity of MMP-7 for predicting the possibility of an RSI of 13 or higher was higher than that of pepsin. As well, when MMP-7 and pepsin were combined, this sensitivity and specificity increased. Diagnosis using the activity of MMP-7 in saliva is a non-invasive and easy technique and may be an auxiliary tool to diagnose LPRD in the future.