pH Transition Point Research Articles

Computational design of non-porous pH-responsive antibody nanoparticles

Programming protein nanomaterials to respond to changes in environmental conditions is a current challenge for protein design and is important for targeted delivery of biologics. Here we describe the design of octahedral non-porous nanoparticles with a targeting antibody on the two-fold symmetry axis, a designed trimer programmed to disassemble below a tunable pH transition point on the three-fold axis, and a designed tetramer on the four-fold symmetry axis. Designed non-covalent interfaces guide cooperative nanoparticle assembly from independently purified components, and a cryo-EM density map closely matches the computational design model. The designed nanoparticles can package protein and nucleic acid payloads, are endocytosed following antibody-mediated targeting of cell surface receptors, and undergo tunable pH-dependent disassembly at pH values ranging between 5.9 and 6.7. The ability to incorporate almost any antibody into a non-porous pH-dependent nanoparticle opens up new routes to antibody-directed targeted delivery.

Polyoxa- and Polyazamacrocycles Incorporating 6,7-Diaminoquinoxaline Moiety: Synthesis and Application as Tunable Optical pH-Indicators in Aqueous Solution.

Synthetic approach to fluorescent polyaza- and polyoxadiazamacrocycles comprising a structural fragment of 6,7-diamino-2,3-diphenylquinoxaline has been elaborated using Pd-catalyzed amination providing target compounds in yields up to 77%. A series of nine novel N- and N,O-containing macrocyclic ligands differing by the number of donor sites and cavity size has been obtained. These compounds possess well-pronounced fluorescent properties with emission maxima in a blue region in aprotic solvents and high quantum yields of fluorescence, while in proton media, fluorescence shifts towards the green region of the spectrum. Using macrocycles 5c and 5e as examples, we have shown that such compounds can serve as dual-channel (colorimetric and fluorimetric) pH indicators in water media, with pH transition point and response being dependent on the macrocycle structure due to different sequences of protonation steps.

3-Aminophenylboronic Acid Conjugation on Responsive Polymer and Gold Nanoparticles for Qualitative Bacterial Detection.

Because of their sensitive and selective responses to a wide variety of analytes, colorimetric sensors have gained widespread acceptance in recent years. Gold nanoparticles (AuNPs) are widely employed in visual sensor strategies due to their high stability and ease of use. Combining AuNPs with a responsive polymer can result in distinct surface plasmon resonance (SPR) changes that can be utilized as colorimetric biosensors. The purpose of this research is to develop a colorimetric-based sensor through the utilization of the optical properties of gold nanoparticles (AuNPs) crosslinked with pH-responsive polymers poly (acrylic acid) (PAA) conjugated to 3-aminophenyl boronic acid (APBA). The polymer (PAA) was synthesized via RAFT polymerization. The inversed Turkevic method was used to produce AuNPs, which were subsequently used in a self-assembly process using poly (acrylic acid)-aminophenyl boronic acid (PAA-APBA) to create the self-assembled AuNPs-APBA-PAA. The particle size, zeta potential, and reversibility of the polymer-modified gold nanoparticles were determined using a transmission electron microscope (TEM), a particle size analyzer (PSA), and an Ultraviolet-Visible spectrophotometer (UV-Vis spectrophotometer). Visual, UV-Vis spectrophotometer and TEM observations confirmed the system's ability to identify bacteria. Statistical analysis was performed using a one-way analysis of variance using Excel software. Using UV-Vis spectrophotometry, the particle size of AuNPs was determined to be 25.7 nm, and the maximum absorbance occurred at 530 nm. AuNPs PAA APBA colloid exhibited an absorbance maximum of 532 nm, a zeta potential of -41.53, and a pH transition point between 4 and 5. At E. coli concentrations of 4.5 x 107 CFU/mL, the color of the system sensors changed from red to blue after 15 hours of incubation, whereas at S. aureus concentrations of 1.2 x 109 CFU/mL, the color changed to purple immediately after mixing. The TEM confirmed that the detection mechanism is based on the boronate-polyol bonding of saccharides on the outer membranes of Escherichia coli and Staphylococcus aureus. The use of APBA in conjunction with pH-responsive PAA polymers containing AuNPs to detect E. coli and S. aureus bacteria induces a maximum wavelength transition, followed by a color change from red to blue. By the process of de-swelling of the responsive polymer, which induces the aggregation of the AuNPs, the established sensor system is able to alter the color. The conjugated polymer and gold nanoparticle-based sensor system demonstrated a promising method for bacterial detection.

NIR‐II pH Sensor with a FRET Adjustable Transition Point for In Situ Dynamic Tumor Microenvironment Visualization

AbstractMonitoring the pH in tumor lesions provides abundant physiological information. However, currently developed pH sensors only achieve sensitive detection in the settled response region around the pH transition point (pHt). To realize tumor pH monitoring with high sensitivity within a wider response region, reported here are serial pHt adjustable sensors (pTAS) that simply regulate the component ratio of second near‐infrared (NIR‐II) emission aza‐BODIPY (NAB) donor and pH sensitive rhodamine‐based pre‐acceptor (NRh) in Förster resonance energy transfer system. Combining the pH response regions of pTAS, a twofold widened pH detection range (6.11–7.22) is obtained compared to the pHt settled sensor (6.38–6.94). With an adjustable pHt, in vivo tumor pH increase and decrease processes could be dynamically visualized through dual‐channel ratiometric bioimaging within the NIR‐II window, with a coefficient of variation under 1 % compared to the standard pH meter.

NIR-II pH Sensor with a FRET Adjustable Transition Point for In Situ Dynamic Tumor Microenvironment Visualization.

Monitoring the pH in tumor lesions provides abundant physiological information. However, currently developed pH sensors only achieve sensitive detection in the settled response region around the pH transition point (pHt ). To realize tumor pH monitoring with high sensitivity within a wider response region, reported here are serial pHt adjustable sensors (pTAS) that simply regulate the component ratio of second near-infrared (NIR-II) emission aza-BODIPY (NAB) donor and pH sensitive rhodamine-based pre-acceptor (NRh) in Förster resonance energy transfer system. Combining the pH response regions of pTAS, a twofold widened pH detection range (6.11-7.22) is obtained compared to the pHt settled sensor (6.38-6.94). With an adjustable pHt , in vivo tumor pH increase and decrease processes could be dynamically visualized through dual-channel ratiometric bioimaging within the NIR-II window, with a coefficient of variation under 1 % compared to the standard pH meter.

Regulating association strength between quaternary ammonium chitosan and sodium alginate via hydration

Studies on interactions between oppositely charged polysaccharides have gathered great interest. We proposed that the association between oppositely charged polymers could be regulated via hydration. A comparison study was carried out by using quaternary chitosan with different counterions(Cl−, Ac−, OH−) and sodium alginate. The results showed that the association between quaternary chitosan with less hydrated counter anion Cl− and sodium alginate was weaker than that between quaternary chitosan with more hydrated counter anion Ac− and sodium alginate. There was a pH transition point of thermal change of association between oppositely charged polymers, as the solution’s pH had more effect on the hydration of polymers than counter ions. Further studies showed that a fraction of Cl− was still attracted by polycation in the complex and competed with the interaction of polyanion after complexation. The competitive combination was critical for the property (such as self healing behavior) of the carbohydrate polymer complex.

PH-responsive Frame-Guided Assembly with hydrophobicity controllable peptide as leading hydrophobic groups

Frame-Guided Assembly (FGA) strategy has been recently reported to prepare vesicles with customized shapes and sizes. However, the effects of the interaction between leading hydrophobic groups (LHGs) and amphiphiles on the thermodynamic and kinetic control of the FGA process haven't been fully understood. In this work, we employed the pH low-insertion peptide (pHLIP) as the LHGs because its interaction with lipids could be finely tuned by pH and investigated the mechanism of FGA in detail. Our study demonstrated the peptide frames could successfully guide the assembly of lipids to form hetero-liposomes below the pH transition point owing to the strong peptide and lipids interaction. The pH-dependent kinetic controlled FGA process was proved and factors affecting the FGA process were also investigated systematically. We believe this pH-responsive FGA strategy improved our knowledge on the mechanism of the FGA and provide inspiration in understanding the sophisticated assembly behavior in life.

PH and exchangeable aluminum are major regulators of microbial energy flow and carbon use efficiency in soil microbial communities

The microbial partitioning of organic carbon (C) into either anabolic (i.e. growth) or catabolic (i.e. respiration) metabolic pathways represents a key process regulating the amount of added C that is retained in soil. The factors regulating C use efficiency (CUE) in agricultural soils, however, remain poorly understood. The aim of this study was to investigate substrate CUE from a wide range of soils (n = 970) and geographical area (200,000 km2) to determine which soil properties most influenced C retention within the microbial community. Using a 14C-labeling approach, we showed that the average CUE across all soils was 0.65 ± 0.003, but that the variation in CUE was relatively high within the sample population (CV 14.9%). Of the major properties measured in our soils, we found that pH and exchangeable aluminum (Al) were highly correlated with CUE. We identified a critical pH transition point at which CUE declined (pH 5.5). This coincided exactly with the point at which Al3+ started to become soluble. In contrast, other soil factors [e.g. total C and nitrogen (N), dissolved organic C (DOC), clay content, available calcium, phosphorus (P) and sulfur (S), total base cations] showed little or no relationship with CUE. We also found no evidence to suggest that nutrient stoichiometry (C:N, C:P and C:S ratios) influenced CUE in these soils. Based on current evidence, we postulate that the decline in microbial CUE at low pH and high Al reflects a greater channeling of C into energy intensive metabolic pathways involved in overcoming H+/Al3+ stress (e.g. cell repair and detoxification). The response may also be associated with shifts in microbial community structure, which are known to be tightly associated with soil pH. We conclude that maintaining agricultural soils above pH 5.5 maximizes microbial energy efficiency.

DNA i-Motifs With Guanidino-i-Clamp Residues: The Counterplay Between Kinetics and Thermodynamics and Implications for the Design of pH Sensors

I-motif structures, adopted by cytosine-rich DNA strands, have attracted considerable interest as possible regulatory elements in genomes. Applied science exploits the advantages of i-motif stabilization under acidic conditions: i-motif-based pH sensors and other biocompatible nanodevices are being developed. Two key characteristics of i-motifs as core elements of nanodevices, i.e., their stability under physiological conditions and folding/unfolding rates, still need to be improved. We have previously reported a phenoxazine derivative (i-clamp) that enhances the thermal stability of the i-motif and shifts the pH transition point closer to physiological values. Here, we performed i-clamp guanidinylation to further explore the prospects of clamp-like modifications in i-motif fine-tuning. Based on molecular modeling data, we concluded that clamp guanidinylation facilitated interstrand interactions in an i-motif core and ultimately stabilized the i-motif structure. We tested the effects of guanidino-i-clamp insertions on the thermal stabilities of genomic and model i-motifs. We also investigated the folding/unfolding kinetics of native and modified i-motifs under moderate, physiologically relevant pH alterations. We demonstrated fast folding/unfolding of native genomic and model i-motifs in response to pH stimuli. This finding supports the concept of i-motifs as possible genomic regulatory elements and encourages the future design of rapid-response pH probes based on such structures. Incorporation of guanidino-i-clamp residues at/near the 5′-terminus of i-motifs dramatically decreased the apparent unfolding rates and increased the thermal stabilities of the structures. This counterplay between the effects of modifications on i-motif stability and their effects on kinetics should be taken into account in the design of pH sensors.

The effect of oral sucralfate on postprandial proximal gastric acid pocket

Background: A postprandial proximal gastric acidpocket (PPGAP) that escapes neutralization by food was demonstrated involunteers and gastroesophageal reflux disease (GERD) patients. It is elusive;however, if this acid layer is morphologically best conceptualized as a realpocket or a film. This study aims to analyze the effect of oral sucralfateadministration on PPGAP to shed some light on PPGAP morphology and treatment. Methods: Twenty-six patients (mean age 51years, 19 females) were studied. A pull-through pH monitoring was performedfrom 5 cm below the lower border of the lower esophageal sphincter (LES) to theactual border in increments of 1 cm, in a fasting state, 10 min after a fattymeal and 10 min after oral sucralfate administration. PPGAP was defined by anacid reading (pH <4) in the proximal stomach between non-acid segmentsdistally (food) and proximally (proximal pH transition point). Standard 24 h pHmonitoring was performed for objective characterization of GERD. The protocolwas approved by local ethics committee. Results: PPGAP was not found in 15 patientsand these were excluded from the post-sucralfate analysis. After sucralfate,PPGAP increased in length in 5/11 (45%) patients; decreased in 2/11 patients(18%). In 3/11 (27%) patients PPGAP disappeared; in 1/11 (9%) PPGAP lengthremained equal. Conclusions: In conclusion,sucralfate altered PPGAP in more than 90% of the patients, supporting thetheory of an acid film. Sucralfate is, however, not an adequate treatment forPPGAP.

Pea protein isolate–high methoxyl pectin soluble complexes for improving pea protein functionality: Effect of pH, biopolymer ratio and concentrations

Recently, there is a strong interest in the incorporation of pea protein as a preferred alternative animal protein into protein fortified beverage because of its cheaper price, more sustainable and gluten free that can be claimed in label. However, poor functionality of pea protein such as acidic solubility and thermal stability has limited their applications in dispersed food systems such as beverage. The aim of this study was to enhance the functionality of pea protein isolate (PPI) by forming soluble complexes with high methoxyl pectin (HMP). The effect of PPI–HMP mixing ratio (1:1–20:1) and PPI concentrations (0.05 and 1.00 wt %) on critical pH transition point (pHs), a key parameter differentiating between soluble and insoluble complexes formation, was investigated using turbidimetric analysis, phase diagram, and ζ–potential. Critical pHs decreased to pH 3.5 as PPI–HMP mixing ratio decreased from 20:1 to 1:1. The ζ–potential results showed a shift in net charge neutrality from pH 4.8 in homogenous PPI solutions to pH 3 for PPI-HPM mixtures with 1:1 ratio. However, HMP induced phase separation of PPI–HMP mixtures at both higher biopolymer concentration (>1.50 wt %) and neutral pH values was observed presumably via a thermal incompatibility mechanism. The pH- percentage solubility of PPI–HMP mixtures at fixed PPI concentration (1.00 wt %) was mixing ratio dependent. When increasing HMP concentration, the pH of minimum protein solubility for biopolymer mixture was shifted towards more acidic condition as compared to PPI alone. Apparent viscosity and thermal denaturation temperature of soluble complexes at fixed PPI concentration (1.00 wt %) slightly increased due to the formation of new net structure in the PPI–HMP systems. The findings derived from this research could provide useful information in the design of pea protein fortified beverage with enhanced pea protein stability.

Short-segment and intrasphincteric gastroesophageal reflux

The traditional gold standard for measuring gastroesophageal acid reflux has been by placing a pH sensor 5 cm proximal to the lower esophageal sphincter. It is known that damage induced by reflux is maximal near to the gastroesophageal junction and this has stimulated interest in determining acid reflux at that site. The extent of esophageal exposure from refluxing gastric acid is inversely related to the distance proximal to the gastroesophageal junction. In addition, the pH transition point from gastric to esophageal pH can be displaced proximally within the lower esophageal sphincter without complete loss of sphincter tone. This intrasphincteric reflux is associated with proximal extension of cardia mucosa because of columnar metaplasia of the most distal esophageal squamous mucosa. The most distal esophageal mucosa is exposed to substantially greater gastric acid refluxate than that recorded at the traditional site 5 cm proximal to the lower esophageal sphincter.

Hiatus hernia in healthy volunteers is associated with intrasphincteric reflux and cardiac mucosal lengthening without traditional reflux

Background and aimsHiatus hernia (HH) is a key mediator of gastro-oesophageal reflux disease but little is known about its significance in the general population. We studied the structure and function...

Side chain impacts on pH- and thermo-responsiveness of tertiary amine functionalized polypeptides

The systemic investigation of the structural impacts of side chains on the pH- and thermo-responsiveness of tertiary amine functionalized poly(l-glutamate)s (TA-PGs) was carried out. The TA-PGs polymers were effectively synthesized by Cu(I)-catalyzed azide-alkyne cycloaddition click reaction of azido tertiary amines with poly(γ-propargyl-l-glutamate) (PPLG). Turbimetric measurements were performed to characterize the pH- and temperature-induced phase transition of TA-PGs in aqueous solution, which suggested a structural dependence of the properties on the N-substituted groups and the “linkers” between 1,2,3-triazole ring and the tertiary amine groups in the side chains. In detail, the pH responsive properties of TA-PGs were basically determined by the hydrophobicity of the N-substituted groups in the side chains and the pH transition point (pHt) decreased as the increasing hydrophobicity of the N-substituted groups, while the temperature-responsiveness of TA-PGs were affected by either the N-substituted groups or the “linkers.” TA-PGs with a moderate N-substituted amine group (e.g., DEA, PR, and PD) or a branched “linker” (e.g., iso-propylene and 2-methylpropylene group) were more likely to express the LCST-type phase transition tuned by pH variation. These structure–property relationships revealed in this study would help to develop the applications of TA-PGs in smart drug delivery systems. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014, 52, 671–679

Obesity and Waist Belt Distort the Esophagogastric Junction and Induce Intrasphincteric Acid Reflux

Purpose: We examined the esophagogastric junction using novel and high-resolution probes in healthy subjects with and without obesity, and the effects of elevating intra-abdominal pressure with a waist belt. Methods: We recruited 24 subjects: twelve normal and twelve with increased waist circumference, matched for age and gender. A magnet (2 x 1 mm) was endoscopically clipped to the SCJ. Assembly of 3-D Hall-Effect probe, 12-channel pH catheter, and 36-channel 2.7-mm manometer was then passed nasally. A waist belt was applied on a separate study day. All results were in mean. Results: The SCJ (P<0.001) and pH transition-point (P=0.04) were displaced proximally in obese vs. non-obese subjects, but not diaphragm (pLES and PIP). With belt on vs. off, there was proximal displacement of SCJ (P < 0.001), pH transition-point (P<0.001), and also of diaphragm (P<0.001) and LES (upper and lower border, P<0.001 and P=0.002, respectively). The SCJ relative to the diaphragm (pLES and PIP) was more markedly displaced by obesity than by the belt (3.9 vs. 1.9 cm and 3.4 vs. 1.5 cm respectively). The magnitude of proximal migration of SCJ during TLESRs was less in obese vs. non-obese subjects (4.2 vs. 6.8 cm, P=0.04) and belt on vs. off (3.9 vs. 5.5 cm, P=0.01), consistent with its resting position being already proximally displaced. At 5cm above LES and 1-2 cm above upper border LES, the mean % time pH < 4 was minimal (<4%) in all studied groups; however, it was increased (4-30%) in belt on vs. off at 1.3 cm above the SCJ (P=0.02) and in obese subjects with belt on vs. off at 0.5 cm above the SCJ (P=0.02). In obese subjects with belt on vs. off, there was a greater frequency of TLESRs (7.3 vs. 5.0/hour, P=0.001), % TLESRs with reflux (56.4% vs. 32.8%, P=0.01) and a longer acid clearance time (72.3s vs. 23.4s, P=0.01). Conclusion: Our findings indicate that obesity and waist belt cause partial hiatus herniation and intrasphincteric short segment acid reflux in asymptomatic volunteers.Figure

Waist belt and central obesity cause partial hiatus hernia and short-segment acid reflux in asymptomatic volunteers

ObjectiveThere is a high incidence of inflammation and metaplasia at the gastro-oesophageal junction (GOJ) in asymptomatic volunteers. Additionally, the majority of patients with GOJ adenocarcinomas have no history of reflux...

Multicolored pH-tunable and activatable fluorescence nanoplatform responsive to physiologic pH stimuli.

Tunable, ultra-pH responsive fluorescent nanoparticles with multichromatic emissions are highly valuable in a variety of biological studies, such as endocytic trafficking, endosome/lysosome maturation, and pH regulation in subcellular organelles. Small differences (e.g., <1 pH unit) and yet finely regulated physiological pH inside different endocytic compartments present a huge challenge to the design of such a system. Herein, we report a general strategy to produce pH-tunable, highly activatable multicolored fluorescent nanoparticles using commonly available pH-insensitive dyes with emission wavelengths from green to near IR range. The primary driving force of fluorescence activation between the ON (unimer) and OFF (micelle) states is the pH-induced micellization. Among three possible photochemical mechanisms, homo Förster resonance energy transfer (homoFRET)-enhanced decay was found to be the most facile strategy to render ultra-pH response over the H-dimer and photoinduced electron transfer (PeT) mechanisms. Based on this insight, we selected several fluorophores with small Stoke shifts (<40 nm) and established a panel of multicolored nanoparticles with wide emission range (500-820 nm) and different pH transitions. Each nanoparticle maintained the sharp pH response (ON/OFF < 0.25 pH unit) with corresponding pH transition point at pH 5.2, 6.4, 6.9, and 7.2. Incubation of a mixture of multicolored nanoparticles with human H2009 lung cancer cells demonstrated sequential activation of the nanoparticles inside endocytic compartments directly correlating with their pH transitions. This multicolored, pH-tunable nanoplatform offers exciting opportunities for the study of many important cell physiological processes, such as pH regulation and endocytic trafficking of subcellular organelles.

An alginate-antacid formulation (Gaviscon Double Action Liquid) can eliminate or displace the postprandial ‘acid pocket’ in symptomatic GERD patients

Recently, an 'acid pocket' has been described in the proximal stomach, particularly evident postprandially in GERD patients, when heartburn is common. By creating a low density gel 'raft' that floats on top of gastric contents, alginate-antacid formulations may neutralise the 'acid pocket'. To assess the ability of a commercial high-concentration alginate-antacid formulation to neutralize and/or displace the acid pocket in GERD patients. The 'acid pocket' was studied in ten symptomatic GERD patients. Measurements were made using concurrent stepwise pH pull-throughs, high resolution manometry and fluoroscopy in a semi-recumbent posture. Each subject was studied in three conditions: fasted, 20 min after consuming a high-fat meal and 20 min later after a 20 mL oral dose of an alginate-antacid formulation (Gaviscon Double Action Liquid, Reckitt Benckiser Healthcare, Hull, UK). The relative position of pH transition points (pH >4) to the EGJ high-pressure zone was analysed. Most patients (8/10) exhibited an acidified segment extending from the proximal stomach into the EGJ when fasted that persisted postprandially. Gaviscon neutralised the acidified segment in six of the eight subjects shifting the pH transition point significantly away from the EGJ. The length and pressure of the EGJ high-pressure zone were minimally affected. Gaviscon can eliminate or displace the 'acid pocket' in GERD patients. Considering that EGJ length was unchanged throughout, this effect was likely attributable to the alginate 'raft' displacing gastric contents away from the EGJ. These findings suggest the alginate-antacid formulation to be an appropriately targeted postprandial GERD therapy.

Characterization of the C-terminal Domain of a Potassium Channel from Streptomyces lividans (KcsA)

KcsA, a potassium channel from Streptomyces lividans, is a good model for probing the general working mechanism of potassium channels. To date, the physiological activator of KcsA is still unknown, but in vitro studies showed that it could be opened by lowering the pH of the cytoplasmic compartment to 4. The C-terminal domain (CTD, residues 112-160) was proposed to be the modulator for this pH-responsive event. Here, we support this proposal by examining the pH profiles of: (a) thermal stability of KcsA with and without its CTD and (b) aggregation properties of a recombinant fragment of CTD. We found that the presence of the CTD weakened and enhanced the stability of KcsA at acidic and basic pH values, respectively. In addition, the CTD fragment oligomerized at basic pH values with a transition profile close to that of channel opening. Our results are consistent with the CTD being a pH modulator. We propose herein a mechanism on how this domain may contribute to the pH-dependent opening of KcsA.

Acidity Surrounding the Squamocolumnar Junction in GERD Patients: "Acid Pocket" Versus "Acid Film"

This study aimed to localize the gastric-to-esophageal pH transition point relative to the squamocolumnar junction (SCJ) and esophagogastric junction (EGJ) high-pressure zone in controls and GERD patients. Ten controls and 10 GERD patients were studied. Subjects had an endoclip placed at the SCJ prior to a pH catheter pull-through (upright and supine) during concurrent fluoroscopy before and after consuming a standardized meal. Six controls and 6 GERD patients also underwent concurrent manometry. The relative positions of the SCJ, EGJ high-pressure zone, and pH transition points were analyzed. Most controls and GERD patients exhibited an unbuffered acidified segment in the proximal stomach postprandially. The proximal pH transition point was confined distal to the SCJ in control subjects, regardless of posture or meal state. GERD patients exhibited a more proximal pH transition point, extending above the SCJ and EGJ high-pressure zone in the supine position, especially postprandially. However, the high-pressure zone was intact. A short segment of unbuffered acidity of unknown volume exists after meals in the proximal stomach. In controls, the unbuffered acidic segment is contained distal to the SCJ while in the GERD patients it extended into and even across the EGJ high-pressure zone. However, this extension through the EGJ in GERD patients occurred in the context of an intact sphincter suggesting that this is best conceptualized as an acid "film" rather than a "pocket." This observation may help explain the propensity of the distal esophageal mucosa to lesions of reflux disease.