In-Membrane Enrichment and Peptic Digestion to Facilitate Analysis of Monoclonal Antibody Glycosylation.

The number of therapeutic monoclonal antibodies (mAbs) is growing rapidly due to their widespread use for treating various diseases and health conditions. Assessing the glycosylation profile of mAbs during production is essential to ensuring their safety and efficacy. This research aims to rapidly isolate and digest mAbs for liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification of glycans and monitoring of glycosylation patterns, potentially during manufacturing. Immobilization of an Fc region-specific ligand, oFc20, in a porous membrane enables the enrichment of mAbs from cell culture supernatant and efficient elution with an acidic solution. Subsequent digestion of the mAb eluate occurred in a pepsin-modified membrane within 5 min. The procedure does not require alkylation and desalting, greatly shortening the sample preparation time. Subsequent LC-MS/MS analysis identified 11 major mAb N-glycan proteoforms and assessed the relative peak areas of the glycosylated peptides. This approach is suitable for the glycosylation profiling of various human IgG mAbs, including biosimilars and different IgG subclasses. The total time required for this workflow is less than 2 h, whereas the conventional enzymatic release and labeling of glycans can take much longer. Thus, the integrated membranes are suitable for facilitating the analysis of mAb glycosylation patterns.

The reflux symptom score has good screening value for laryngopharyngeal reflux.

OBJECTIVE: To investigate the consistency between the reflux symptom score (RSS) and the multitemporal salivary pepsin test in screening for laryngopharyngeal reflux (LPR) and the screening value of the RSS for LPR by simultaneously administering daytime multitemporal salivary pepsin test and RSS to patients. METHODS: This was a single-center prospective observational study. All included patients underwent simultaneous daytime multitemporal salivary pepsin testing and RSS. A participant was considered to have LPR when one or more positive salivary pepsin test results or RSS score > 13 were obtained. The consistency between the multitemporal salivary pepsin test and the RSS was compared by the weighted Cohen's kappa statistic. The screening value of the RSS for LPR was investigated by receiver operating characteristic (ROC) analysis. RESULTS: A total of 67 patients were included. The positivity rate of LPR was 71.64% according to the results of the multitemporal salivary pepsin test. According to RSS, the positive rate of LPR was 70.15%. The weighted Kappa value between the multitemporal salivary pepsin test and the RSS was 0.675 (p < 0.001). The area under curve of RSS screening for LPR was 0.843 (p < 0.01), and the sensitivity, specificity, positive predictive value, and negative predictive value of RSS screening for LPR were 89.58%, 78.95%, 91.49%, and 75%, respectively. CONCLUSION: There is a good consistency between the RSS and the multitemporal salivary pepsin test, and the RSS has a good screening value for LPR, which can be applied to screen for LPR in otolaryngologic patients.

Usefulness of pepsin saliva measurement for the detection of primary burning mouth syndrome related to reflux.

OBJECTIVES: To study the diagnostic value of salivary pepsin tests for detecting laryngopharyngeal reflux (LPR) in patients with primary burning mouth syndrome (BMS). METHODS: Patients with BMS and asymptomatic individuals were consecutively recruited from September 2018 to June 2023. Patients underwent hypopharyngeal-esophageal impedance pH-monitoring (HEMII-pH) and saliva collections to measure pepsin. Stomatology evaluation was carried out to exclude other causes of BMS. Oral, pharyngeal and laryngeal signs and symptoms were evaluated with Reflux Sign Assessment (RSA) and Reflux Symptom Score (RSS). Sensitivity, specificity, positive (PPV) and negative (NPV) predictive values of pepsin test were calculated considering the highest values of pepsin tests at ≥ 16, ≥ 36, and ≥ 100 ng/mL cutoffs. Receiver operating characteristic curve (ROC) was evaluated. RESULTS: Forty-nine patients with both BMS and LPR at the HEMII-pH and 21 asymptomatic individuals were recruited. Pepsin test was 83.7%, 79.6%, and 71.4% sensitive at cutoffs ≥ 16, ≥ 36, and ≥ 100 ng/mL, respectively. The ROC analysis reported that a threshold of ≥ 21.5 ng/mL was associated with sensitivity, specificity, PPV and NPV of 81.6%, 81.0%, 90.1% and 65.4%, respectively. The severity score of burning mouth symptom was significantly associated with the saliva pepsin concentration (rs = 0.263; p = 0.029) and the oral RSA (rs = 0.474; p = 0.007). CONCLUSION: Pepsin test is a valuable diagnostic approach for detecting LPR in patients with BMS. Patients with high level of saliva pepsin reported more severe burning mouth symptoms. Future studies are needed to confirm the role of LPR in the primary BMS.

Determination of pepsin in human saliva using pepsin-susceptible peptide reporter and colorimetric dipstick assay: a prospective, cross-sectional study.

A simple and effective pepsin detection assay is reported based on a pepsin-susceptible peptide (PSP) reporter degradation strategy. PSP, which can be specifically cleaved by pepsin, was modified with fluorescein isothiocyanate (FITC) and biotin at the N- and C-terminals to be used as a reporter for colorimetric detection of dipsticks. A universal lateral flow dipstick consisting of a streptavidin test line for biotin binding and a sample pad immobilized with a gold-labeled polyclonal (rabbit) anti-FITC antibody was used to verify PSP-based pepsin detection. When the PSP reporter reacts with pepsin in a tube, it cleaves into two fragments, and the cleaved fragments do not display any color on the test line. Therefore, the higher the concentration of pepsin is, the greater is the decrease in test line intensity (IT-line) and the higher is the control line intensity (IC-line). First, the PSP cleavage and dipstick assay conditions for pepsin detection was optimized. The ratio of color intensity (IT-line/IC-line) of PSP-based dipstick assay showed a linear relationship with log concentration of pepsin ranging between 4 and 500 ng/mL (R2 = 0.98, n = 6), with a limit of detection of 1.4 ng/mL. It also exhibited high specificity and good reproducibility. Finally, pepsin levels were quantified in saliva samples from healthy controls (n = 34) and patients with laryngopharyngeal reflux (LPR, n = 61). Salivary pepsin levels were higher in patients with LPR than in healthy controls. The salivary pepsin levels correlated with those measured using a conventional enzyme-linked immunosorbent assay kit. Therefore, this PSP-based dipstick assay is a convenient tool for assessing salivary pepsin levels.

Portable and Rapid Fluorescence Turn-On Detection of Total Pepsin in Saliva Based on Strong Electrostatic Interactions.

Salivary pepsin has been proposed as a promising diagnostic marker for gastroesophageal reflux disease (GERD). However, the activity of human pepsin is strongly influenced by pH, and the acidic condition (pH ∼ 2) is optimal, which is a contradiction for the pepsin detection kit based on its catalytic activity. Thus, its accurate quantification in saliva (neutral pH) by readily rapid tools with simplicity and low cost is still challenging. Herein, a convenient fluorescence assay has been developed for the rapid detection of pepsin at neutral pH based on its electrostatic interaction with SYBR Green (SG) rather than the bioactivity. At neutral pH, the positively charged SG fluorophore can be effectively adsorbed by the negatively charged pepsin due to the low isoelectric point (pI) and large molecular size of pepsin. Thus, the molecular rotation of SG is limited, and its fluorescence intensity is significantly increased. The strategy was further confirmed to have the same fluorescence response as that of normally active and inactivated pepsin. Due to the unique pI of pepsin, the fluorescence strategy is highly selective for pepsin compared to other proteins. On the basis of this strategy, a smartphone-based fluorescence capture device integrated with a programmed Python program was fabricated for both color recognition and the accurate detection of pepsin within 3 min. Under the optimal conditions, this turn-on sensor allowed for the on-site analysis of pepsin with a detection limit of 0.2 µg/mL. Moreover, this strategy was successfully used to assess salivary pepsin to aid in the noninvasive diagnosis of GERD.

Papain-Based Solubilization of Decellularized Extracellular Matrix for the Preparation of Bioactive, Thermosensitive Pregels.

Solubilized, gel-forming decellularized extracellular matrix (dECM) is used in a wide range of basic and translational research and due to its inherent bioactivity can promote structural and functional tissue remodeling. The animal-derived protease pepsin has become the standard proteolytic enzyme for the solubilization of almost all types of collagen-based dECM. In this study, pepsin was compared with papain, α-amylase, and collagenase for their potential to solubilize porcine liver dECM. Maximum preservation of bioactive components and native dECM properties was used as a decisive criterion for further application of the enzymes, with emphasis on minimal destruction of the protein structure and maintained capacity for physical thermogelation at neutral pH. The solubilized dECM digests, and/or their physically gelled hydrogels were characterized for their rheological properties, gelation kinetics, GAG content, proteomic composition, and growth factor profile. This study highlights papain as a plant-derived enzyme that can serve as a cost-effective alternative to animal-derived pepsin for the efficient solubilization of dECM. The resulting homogeneous papain-digested dECM preserved its thermally triggered gelation properties similar to pepsin digests, and the corresponding dECM hydrogels demonstrated their enhanced bioadhesiveness in single-cell force spectroscopy experiments with fibroblasts. The viability and proliferation of human HepaRG cells on dECM gels were similar to those on pure rat tail collagen type I gels. Papain is not only highly effective and economically attractive for dECM solubilization but also particularly interesting when digesting human-tissue-derived dECM for regenerative applications, where animal-derived materials are to be avoided.

Pepsin in saliva for the diagnosis of erosive esophagitis post-sleeve gastrectomy: a prospective observational study.

BACKGROUND: Laparoscopic sleeve gastrectomy (LSG) has become the preferred bariatric procedure in many countries. However, new onset erosive esophagitis (EE) is a major shortcoming. Current recommendation is esophago-gastro-duodenoscopy (EGD) should be performed routinely at 1 year and subsequently every 2-3 years to enable the early detection of Barrett's or esophageal adenocarcinoma. This would put significant strains on resources and costs of bariatric program. Our study assesses the association between and diagnostic value of salivary pepsin concentration and endoscopically proven EE in post-LSG patients as a surrogate for EGD. METHODS: Twenty patients on routine post-LSG endoscopy between June and September 2022 were recruited for this correlational pilot study. Under supervision, fasting and post-prandial saliva sample was collected and analyzed by Peptest lateral flow device. EGD examinations were performed, and patients completed a validated 25-item QoLRAD questionnaire. RESULTS: We found a significant correlation between positive endoscopy findings of EE and salivary pepsin concentrations. The normal group had a lower mean fasting pepsin level (13.13 ng/mL ± 18.97) versus the EE-group (90.55 ng/mL ± 81.28, p = 0.009) and lower mean post-prandial pepsin level (30.50 ng/mL ± 57.72) versus the EE-group (135.09 ng/mL ± 130.17, p = 0.02). The predictive probabilities from the binary regression of fasting and post-prandial pepsin concentrations yield AUC of 0.955 ± 0.044 (95% CI 0.868 to 1.000, p < 0.001). CONCLUSION: Our study distinctively identified salivary pepsin to have excellent sensitivity and negative predictive value in EE, potentially useful to preclude the need for post-LSG EGD in asymptomatic patients with low salivary pepsin.

Diagnostic thresholds and optimal collection protocol of salivary pepsin for gastroesophageal reflux disease.

Gastroesophageal reflux disease (GERD) is primarily diagnosed based on symptoms and response to a proton-pump inhibitor (PPI) trial. Gold standard testing requires an invasive endoscopic procedure, often with ambulatory pH monitoring. Salivary pepsin is a potential noninvasive modality for GERD diagnosis. This study aimed to assess diagnostic performance of salivary pepsin thresholds for GERD and determine optimal collection protocol of saliva in an external validation cohort. Over 10 months, adults with symptoms of GERD undergoing esophagogastroduodenoscopy with wireless pH-monitoring off PPI were enrolled. Saliva was self-collected by participants over 4 days across three different time points: fasting ante meridiem (AM), post-prandial, and bedtime (PM). Pepsin levels were calculated via Peptest. Pepsin variability and agreement were determined using linear mixed effects models and intraclass correlation. Validation of diagnostic threshold and performance characteristics were evaluated by receiver-operator curve analysis. Twenty participants enrolled in the study; 50% with physiologic acid exposure (acid exposure time < 4% no GERD) and 50% with elevated acid exposure (GERD). Mean pepsin concentrations were significantly lower in the AM (22.6 ± 25.2 ng/mL) compared to post-prandial (44.5 ± 36.7 ng/mL) and PM (55.4 ± 47.0 ng/mL). Agreement between pepsin concentrations across 3 days was substantial for AM samples (kappa 0.61), with lower agreement for post-prandial and PM samples. A single AM pepsin concentration of 25 ng/mL was 67% accurate for GERD with 56% sensitivity and 78% specificity. This validation study highlights fair accuracy and performance characteristics of a single fasting AM salivary pepsin concentration for the diagnosis of GERD.

Saliva pepsin measurements in the detection of gastroesophageal reflux disease in laryngopharyngeal reflux patients: a cohort study.

OBJECTIVE: To study the diagnostic value of salivary pepsin measurement (Peptest) for detecting gastroesophageal reflux disease (GERD) in laryngopharyngeal reflux (LPR) patients. METHODS: Patients with reflux symptoms were consecutively recruited from January 2020 to November 2022. Patients benefited from hypopharyngeal-esophageal impedance-pH monitoring (HEMII-pH), fasting and bedtime saliva collections to measure pepsin. Sensitivity, specificity, positive (PPV) and negative (NPV) predictive values were evaluated for GERD and LPR patients considering the highest values of pepsin tests at ≥ 16, ≥ 75, and ≥ 216 ng/mL cutoffs. The relationship between HEMII-pH, endoscopic and clinical findings, and pepsin measurements was studied. RESULTS: Saliva was collected in 109 LPR patients and 30 individuals with both LPR and GERD. The total number of pharyngeal reflux events was significantly higher in GERD-LPR patients compared with LPR patients (p = 0.008). The mean fasting and bedtime pepsin saliva concentrations were similar between groups. The sensitivity of Peptest in LPR patients was 30.5%, 70.2%, and 84.0% at cutoffs ≥ 16, ≥ 75 and ≥ 216 ng/mL. In GERD-LPR group, Peptest was 80.0%, 70.0%, and 30.0% sensitive. At cutoff 16 ng/mL, Peptest reported PPV of 20.7% and 94.8% in LPR-GERD and LPR groups, respectively. NPV were 73.9% and 8.7% in GERD-LPR and LPR groups, respectively. The consistency analysis between Peptest and HEMII-pH was not significant. Peptest was significantly associated with the number of acid pharyngeal reflux events (rs = 0.182; p = 0.032). CONCLUSION: Pepsin saliva measurements appear to be not a reliable diagnostic tool for the detection of GERD in LPR patients. Future studies are needed to determine the place of Peptest in laryngopharyngeal reflux and gastroesophageal reflux diseases.

SPECIFIC FEATURES OF THE ORAL MICROBIOME IN YOUNG CHILDREN WITH ARYNGOPHARYNGEAL REFLUX AND ITS ROLE THE DEVELOPMENT OF RECURRENT RESPIRATORY DISEASES.

OBJECTIVE: The aim: To examine the composition of the oral microbiome in young children with laryngopharyngeal reflux (LPR) and its role the development of recurrent respiratory diseases. PATIENTS AND METHODS: Materials and methods: There were examined 38 children with physiological gastroesophageal reflux (GER), 18 children with LPR who had a medical history of recurrent bronchitis and 17 healthy children (control group). The study included the collection of anamnesis, objective examination. The qualitative and quantitative microbial composition of the upper respiratory tract was performed obtained by oropharyngeal deep swab. Salivary pepsin level and IL-8 were determined by enzyme-linked immunosorbent assay. RESULTS: Results: This research showed significant alterations in the oral microbiome of patients with GER and LPR as compared to healthy control. We found that gram-negative microbiota such as Klebsiella pneumoniae, Escherichia coli, Proteus vulgaris, Proteus spp. and Candida albicans were identified in children with GER and LPR compared to the healthy control. At the same time, the amount of such a representative of the normal microbiome as Streptococcus viridans in children with LPR was sharply reduced. There were established a much higher mean salivary pepsin level of the patients with LPR than in the GER and control group. We found the association between high pepsin levels, saliva IL-8 levels and frequency of respiratory pathology in children with LPR. CONCLUSION: Conclusions: Our study confirms that increased levels of pepsin in saliva are a risk factor for recurrent respiratory diseases in children with LPR.

Rapid and Simple Analysis of the Human Pepsin Secondary Structure Using a Portable Raman Spectrometer.

Human pepsin is a digestive protease that plays an important role in the human digestive system. The secondary structure of human pepsin determines its bioactivity. Therefore, an in-depth understanding of human pepsin secondary structure changes is particularly important for the further improvement of the efficiency of human pepsin biological function. However, the complexity and diversity of the human pepsin secondary structure make its analysis difficult. Herein, a convenient method has been developed to quickly detect the secondary structure of human pepsin using a portable Raman spectrometer. According to the change of surface-enhanced Raman spectroscopy (SERS) signal intensity and activity of human pepsin at different pH values, we analyze the change of the human pepsin secondary structure. The results show that the content of the ß-sheet gradually increased with the increase in the pH in the active range, which is in good agreement with circular dichroism (CD) measurements. The change of the secondary structure improves the sensitivity of human pepsin SERS detection. Meanwhile, human pepsin is a commonly used disease marker for the noninvasive diagnosis of gastroesophageal reflux disease (GERD); the detection limit of human pepsin we obtained is 2 µg/mL by the abovementioned method. The real clinical detection scenario is also simulated by spiking pepsin solution in saliva, and the standard recovery rate is 80.7-92.3%. These results show the great prospect of our method in studying the protein secondary structure and furthermore promote the application of SERS in clinical diagnosis.

Determination of pepsin by capillary electrophoresis using mixed polymer coated capillary with switchable properties toward protein adsorption/desorption.

In this work, a simple online preconcentration method for quantitative detection of pepsin was realized by using the binary mixed polymer brushes coated capillary with switchable properties toward protein adsorption. Firstly, the binary mixed polymer brushes were prepared by grafting poly(2-methyl-2-oxazoline) and poly(4-vinylpyridine) onto the inner wall of the capillary through a polydopamine anchor. Then the coatings were characterized by X-ray photoelectron spectrometer and electroosmotic flow measurement. The results indicated that the composition of coating could be controlled by varying the feed ratio of poly(2-methyl-2-oxazoline) to poly(4-vinylpyridine) and the inner surface charge could be tuned toward the change of pH and ionic strength. The results showed that when the poly(2-methyl-2-oxazoline)/poly(4-vinylpyridine) mass ratio was 80/20, the highest online preconcentration effect was obtained and the sensitivity enhancement factor was 6.3. Moreover, satisfactory sensitivity (limit of detection: 7.5 ng/mL) and good repeatability were obtained with the online preconcentration method. The polymer-coated capillary was still stable for online preconcentration and detection of pepsin after 50 consecutive runs. Last, the proposed method was used successfully to online preconcentrate pepsin in the saliva matrix.

Salivary biomarkers and esophageal disorders.

Saliva is a complex physiologic fluid that contains an abundance of biological analytes, or biomarkers. Recent research has shown that these biomarkers may be able to convey the physiologic health of a person. Work has been done linking derangements in these salivary biomarkers to a wide variety of pathologic disorders ranging from oncologic diseases to atopic conditions. The specific area of interest for this review paper is esophageal disorders. Particularly because the diagnosis and management of esophageal disorders often includes invasive testing such as esophagogastroduodenoscopy, prolonged pH monitoring, and biopsy. The aim of this review will be to explore salivary biomarkers (pepsin, bile, epidermal growth factor, and micro-RNA) that are being studied as they relate specifically to esophageal disorders. Finally, it will explore the benefits of salivary testing and identify areas of possible future research.

The diagnostic value of pepsin concentration in saliva for laryngopharyngeal reflux disease.

OBJECTIVE: To explore the diagnostic efficacy of pepsin concentration in saliva for laryngopharyngeal reflux (LPR) disease. METHODS: In this study, we recruited 40 participants with abnormal sensation of throat into the study who visited our hospital from March 2020 to December 2020. The 24 h multichannel intraluminal impedance and pH monitoring (24 h MII-pH), reflux symptom index (RSI) and reflux finding score (RFS), pepsin concentration in saliva were collected. The Cohen's kappa test and receiver-operating characteristic (ROC) curves were performed to determine and compare the sensitivity and specificity of five diagnostic methods: RSI; RFS, pepsin concentration, RSI + RFS, RSI + RFS + pepsin concentration. RESULTS: The area under the curve (AUC) of RSI, RFS, pepsin concentration, RSI + RFS, RSI + RFS + pepsin concentration were 0.767, 0.733, 0.870, 0.750,0.867, respectively. That is, the pepsin concentration has maximum AUC (the cutoff point is 219.47 (ng/mL); the sensitivity and 1-specificity is 0.300, 0.933, respectively.). The positive predictive value was 90.3% (28/31), and the negative predictive value was 77.8% (7/9). The Cohen's kappa coefficients of the five diagnostic subgroups were: RSI 0.486 (95% CI 0.207-0.764, P = 0.001); RFS 0.333 (95% CI 0.021-0.644, P = 0.032); RSI + RFS: 0.517 (95% CI 0.205-0.829, P = 0.001); pepsin concentration: 0.699 (95% CI 0.379-0.931, P = 0.001); RSI + RFS + pepsin concentration: 0.500 (95% CI 0.181-0.819, P < 0.001). CONCLUSION: The pepsin concentration has the maximum AUC area and highest consistency with the 24 h MII-pH. Therefore, it has certain value in the screening and diagnosis of diseases related to LPR disease.

The effects of Helicobacter pylori eradication therapy on salivary pepsin concentration in patients with laryngopharyngeal reflux.

OBJECTIVE: To investigate the effect of Helicobacter pylori (HP) eradication therapy on salivary pepsin concentration in laryngopharyngeal reflux (LPR) patients with HP infection. MATERIALS AND METHODS: A total of 477 patients with suspected LPR were enrolled from June 2020 to September 2021. Reflux symptom index, reflux finding score, the positive rates and disintegrations per minute values of HP infection detected by 14C urea breath test and salivary pepsin concentrations analyzed using enzyme-linked immunosorbent assay were compared in LPR patients and non-LPR patients with or without HP infection. HP-positive patients were treated with HP eradication therapy while HP-negative patients with PPI therapy. RESULTS: The scores of nagging cough (0.88 vs. 0.50, P = 0.035), erythema or hyperemia (1.93 vs. 1.78, P = 0.035) and vocal fold edema (1.04 vs. 0.85, P = 0.025) were higher in the LPR (+) Hp (+) subgroup than in LPR (+) Hp (-) subgroup. The concentrations of salivary pepsin in the Hp (+) subgroup were higher than in the Hp (-) subgroup either in LPR patients (75.24 ng/ml vs. 61.39 ng/ml, P = 0.005) or the non-LPR patients (78.42 ng/ml vs. 48.96 ng/ml, P = 0.024). Compared to baseline (before treatment), scores of nagging cough (0.35 vs. 0.84, P = 0.019) and erythema or hyperemia (1.50 vs. 1.83, P = 0.039) and the concentrations of salivary pepsin (44.35 ng/ml vs. 74.15 ng/ml, P = 0.017) in LPR patients with HP infection decreased after HP treatment; yet, this was not observed for the LPR patients without HP infection treated with PPI only (P > 0.05). CONCLUSION: HP infection may aggravate the symptoms and signs of LPR patients, partly by increasing their salivary pepsin concentration.

Diagnostic performance of salivary pepsin for gastroesophageal reflux disease.

Uncertain diagnostic performance has limited clinical adoption of salivary pepsin, a noninvasive diagnostic tool for gastroesophageal reflux disease (GERD). This study aimed to assess diagnostic performance of salivary pepsin, and test validity of thresholds in an external cohort of patients with or without GERD. This two-phase prospective study conducted at two centers enrolled adult asymptomatic volunteers, patients with symptoms of GERD undergoing reflux monitoring, and patients with Barrett's esophagus (BE). Fasting saliva samples were processed for pepsin concentration using Peptest. Phase 1 compared pepsin concentration between No GERD (volunteers/functional heartburn) and GERD (erosive reflux disease/nonerosive reflux disease (NERD)/BE). Phase 2 tested validity of the diagnostic thresholds identified from Phase 1 among external functional heartburn and NERD cohorts. Of 243 enrolled subjects, 156 met inclusion criteria. Phase 1 (n = 114): Pepsin concentrations were significantly higher in GERD (n = 84) versus No GERD (n = 30) (73.8 ng/mL vs. 21.1 ng/mL; P < 0.001). Area under the curve for pepsin concentration was 0.74 (95% CI 0.65, 0.83). A salivary pepsin threshold of 24.9 ng/mL optimized the true negative rate and 100.0 ng/mL optimized the true positive rate. Phase 2 (n = 42): Pepsin concentrations were significantly higher in NERD (n = 22) versus Functional Heartburn (n = 20) (176.0 ng/mL vs. 53.3 ng/mL, P < 0.001). Applying Phase 1 thresholds in this external cohort, salivary pepsin 24.9 ng/mL was 86% sensitive (64%, 97%) and 100.0 ng/mL was 72% specific for distinguishing NERD from functional heartburn. Given modest sensitivity and specificity for GERD, salivary pepsin may have clinical utility as a noninvasive office based diagnostic screening tool for GERD.

Impact of bile acids on the severity of laryngo-pharyngeal reflux.

OBJECTIVES: The primary end point of this study was to evaluate the impact of bile acids on severity of laryngo-pharyngeal reflux (LPR) and the possible correlation with esophagitis and upper airway malignancies. The second end point was to evaluate if salivary bile acids and molecules other than pepsin might serve as diagnostic biomarkers of LPR. DESIGN: Observational prospective comparative study. SETTING: Otorhinolaryngology unit of a tertiary hospital. PARTICIPANTS: Sixty-two consecutive adult outpatients suspected of LPR. MAIN OUTCOME MEASURES: Bile acids, bilirubin and pepsinogen I-II were measured in saliva. Patients underwent pH metry and based on the results of bile acids were subdivided as acid, mixed and alkaline LPR. RESULTS: Significantly higher Reflux Findings Score (RFS) and Reflux Symptoms Index (RSI) were seen in patients with alkaline and mixed LPR compared to acid LPR. Salivary bile acids >1 µmol/L seem to be a reliable indicator of the severity of LPR. Compared to those without, patients with esophagitis or a history of upper airway malignancy have high concentrations of bile acids in saliva. Among the molecules studied, bile acids were the most suitable for diagnosis of LPR, with a sensitivity of 86% and a positive predictive value of 80.7%. CONCLUSIONS: Our data suggest that high concentrations of bile acids are associated with higher values of RSI and RFS in LPR as well as a higher risk of esophagitis and history of upper airway malignancies. We finally observed that bile acids provided the best biometric parameters for diagnosis of LPR among the molecules tested.

[Current possibilities and challenges in the diagnosis of laryngopharyngeal reflux]. / Aktuelle Möglichkeiten und Herausforderungen bei der Diagnostik des laryngopharyngealen Refluxes.

Laryngopharyngeal reflux (LPR) is defined as backflow of gastral or gastroduodenal content into the upper aerodigestive tract and characterized by a variety of unspecific symptoms such as chronic cough, globus sensation, or mucus hypersecretion. Due to the lack of a gold standard and the heterogeneity of studies, the diagnosis of LPR is still problematic and challenging. However, in patients with characteristic symptoms and endoscopic findings, with an increased reflux symptom index, a pathologic reflux finding score (RFS), pathologic 24 h esophageal or oropharyngeal pH monitoring, and without any other underlying condition, the diagnosis of LPR is probable. In the following review, we critically discuss the abovementioned methods as well as more recent tools such as measurements of pepsin concentrations in the saliva for diagnosis of LPR.

The Role of Salivary Pepsin in the Diagnosis of Gastroesophageal Reflux Disease (GERD) Evaluated Using High-Resolution Manometry and 24-Hour Multichannel Intraluminal Impedance-pH Monitoring.

BACKGROUND The Lyon Consensus classification confirms or rules out gastroesophageal reflux disease (GERD). The refractory symptoms of patients with GERD taking proton pump inhibitors (PPIs) are challenging in clinical practice. Salivary pepsin concentration was proposed as a diagnostic biomarker for GERD. We aimed to determine the diagnostic value of salivary pepsin concentration for patients with conclusive GERD, based on the Lyon classification, and the correlation of salivary pepsin concentration with parameters of high-resolution manometry and 24-h multichannel intraluminal impedance-pH in patients with PPI-refractory symptoms. MATERIAL AND METHODS Saliva samples obtained from 130 patients who were suspicious for GERD and had PPI-refractory symptoms were used for pepsin determination using the enzyme-linked immunosorbent assay. All patients underwent upper gastrointestinal endoscopy, high-resolution manometry, 24-h multichannel intraluminal impedance, and pH recording and were classified as conclusive GERD, inconclusive GERD, and evidence against GERD groups according to Lyon classification. RESULTS Salivary pepsin concentration was 8.2 ng/mL (3.8-17.8 ng/mL), 4.0 ng/mL (2.3-6.1 ng/mL), and 2.4 ng/mL (2.2-3.1 ng/mL) in conclusive GERD, inconclusive GERD, and evidence against GERD groups, respectively (P<0.001), and had a negative correlation with distal mean nocturnal baseline impedance and positive correlations with acid exposure time, total number of reflux events, and esophagogastric junction type. The area under the ROC curve of salivary pepsin for conclusive GERD was 0.76 (0.68-0.84), with a sensitivity of 76.36% and a specificity of 63.41% for conclusive GERD diagnosis at a cut-off value of 4.21 ng/mL. CONCLUSIONS Salivary pepsin test had moderate diagnostic value for conclusive GERD by Lyon classification in patients with PPI-refractory symptoms.

Salivary pepsin A detection related to gastro-oesophageal reflux episodes in children undergoing impedance probe monitoring.

AIM: Gastro-oesophageal reflux is routinely diagnosed with invasive intraluminal impedance pH probe monitoring. This study aimed to determine whether gastric pepsin A detected in saliva of children correlates with gastro-oesophageal reflux. METHODS: Patients undergoing probe monitoring were prospectively recruited between 2014 and 2016 at a paediatric hospital. Standard impedance and demographic data were obtained from electronic medical records. Salivary samples were collected during impedance and measured for gastric pepsin A with an enzyme-linked immunosorbent assay. Impedance probe and pepsin data were analysed and compared for correlation. RESULTS: From 52 enrolled subjects, 28 males and 24 females with mean age 8.0 ± 5.9 and range 0.58-18.0 years, 417 salivary samples were collected. Positive pepsin was found in 14% of samples and 48% patients. The sensitivity of pepsin A in predicting an abnormal impedance was 43% and specificity, 50%. Among pepsin A positive samples, 72% corresponded with a gastro-oesophageal reflux episode. Pepsin peak levels significantly correlated with acidic reflux. CONCLUSION: Pepsin A was presented in saliva of children undergoing gastro-oesophageal reflux disease investigation. Positive pepsin A was associated with a gastro-oesophageal reflux episode, and its peak value correlated with acidic reflux. Salivary pepsin as a marker for gastro-oesophageal reflux needs further investigation.