Inclusion of supine period in short-duration pH monitoring is essential in diagnosis of gastroesophageal reflux disease

Prolonged esophageal pH monitoring is the most accurate method for detecting abnormal gastroesophageal reflux (GER) in patients with gastroesophageal reflux disease (GERD). However, some investigators have found that short-duration postprandial pH monitoring in the upright position is also useful, while others have failed to find such results. Therefore, we have compared a 6-hr period of pH monitoring (3-hr postprandial period after daytime meal and 3-hr supine period) with a total 24-hr period in detecting abnormal gastroesophageal reflux. Sixty-five patients (44 men, mean age 41.3 years) with GERD and 16 healthy volunteers (11 men, mean age 34.3 years) underwent 24-hr pH monitoring according to a standard protocol. Various reflux parameters during 24-hr pH monitoring were compared with reflux parameters during the 6-hr period. Abnormal GER was detected in 56 patients presenting with typical symptoms of GERD (sensitivity 86.2%). These patients could be further divided into upright (N=18), supine (N=15), and combined (N=23) refluxers, depending on the posture in which abnormal reflux occurred. Esophageal pH monitoring during the 3-hr postprandial upright period showed abnormal reflux in only 35 patients (sensitivity 53.8%;P<0.00005, compared with the 24-hr pH monitoring period). Abnormal GER was identified in 13 of 18 upright, 19 of 23 combined, and only one of 15 supine refluxers, as well as in two of nine patients with normal 24-hr pH-metry. However, inclusion of the 3-hr supine monitoring period in the 3-hr postprandial upright period improved detection of abnormal GER to 78.5% (51 patients;P=NS compared with 24-hr pH monitoring period). This was related mainly to improved detection of abnormal GER in supine refluxers (11 of 15; 73.3%). Esophageal acid exposure time correlated significantly with severity of esophagitis only during the total and supine periods of both the 24- and 6-hr periods and not during the upright period. Esophageal acid clearance correlated significantly with increasing grades of esophagitis for the supine and total periods only. We conclude that 3-hr postprandial pH monitoring, as has been conventionally practiced, is not appropriate in the detection of abnormal GER; inclusion of a supine period in the short-duration pH monitoring schedule increases the detection of pathological reflux. We therefore recommend that a supine period should be included in short-duration pH monitoring schedules. We also found that supine reflux was the most important factor in the development of esophagitis.     

Electrochromic shift supports the membrane destabilization model of Tat-mediated transport and shows ion leakage during Sec transport

The mechanism and pore architecture of the Tat complex during transport of folded substrates remain a mystery, partly due to rapid dissociation after translocation. In contrast, the proteinaceous SecY pore is a persistent structure that needs only to undergo conformational shifts between “closed” and “opened” states when translocating unfolded substrate chains. Where the proteinaceous pore model describes the SecY pore well, the toroidal pore model better accounts for the high-energy barrier that must be overcome when transporting a folded substrate through the hydrophobic bilayer in Tat transport. Membrane conductance behavior can, in principle, be used to distinguish between toroidal and proteinaceous pores, as illustrated in the examination of many antimicrobial peptides as well as mitochondrial Bax and Bid. Here, we measure the electrochromic shift (ECS) decay as a proxy for conductance in isolated thylakoids, both during protein transport and with constitutively assembled translocons. We find that membranes with the constitutively assembled Tat complex and those undergoing Tat transport display conductance characteristics similar to those of resting membranes. Membranes undergoing Sec transport and those with the substrate-engaged SecY pore result in significantly more rapid electric field decay. The responsiveness of the ECS signal in membranes with active SecY recalls the steep relationship between applied voltage and conductance in a proteinaceous pore, while the nonaccelerated electric field decay with both Tat transport and the constitutive Tat complex under the same electric field is consistent with the behavior of a toroidal pore.

The twin arginine translocation pathway is uniquely able to transport fully folded substrates in an ATP-independent manner, relying instead on an electrochemical gradient (i.e., the proton motive force, or pmf) across the transporting membrane. It is crucial to the transport of substrates requiring various cofactors and hetero-oligomeric complexes in prokaryotes and of substrates vital to photosynthetic machinery in thylakoids (1). In plant mitochondria, the Rieske Fe/S protein required for the biogenesis of complex III is transported by the Tat pathway (2–6). It is implicated in both the virulence and antibiotic resistance of various infectious bacteria (7–12) and has been studied for its potential in biotechnology applications (13–15). The uniqueness of Tat functionality and its appearance across the kingdoms of life make it a valuable research target for crop modification, biotechnology, and pathogenesis. Unfortunately, much of the knowledge about its mechanism has been hard won, and the pore structure remains a mystery, likely due to the transient nature of the active complex.The active Tat complex in thylakoids consists of three core subunits, Tha4, Hcf106, and cpTatC, which are homologous to the bacterial TatA, TatB, and TatC, respectively (1, 16). An N-terminal signal peptide with a twin-arginine motif inserts into the cis-side leaflet at the TatBC receptor complex (17–19). Subsequent oligomerization of TatA subunits (20–22) at the TatBC receptor complex results in rings of varying sizes (22, 23), but it is unclear whether these structures represent transport pores. Of particular note is the short TatA transmembrane helix (TMH). Composed of only 16 residues, the solid-state NMR solution suggests that the TMH must tilt and draw a portion of the cis-side amphipathic helix (APH) into the membrane in order to cross the bilayer (24), establishing a resting state of hydrophobic mismatch. During transport, a conformational shift increasing the angle between the TMH and APH results in exacerbated hydrophobic mismatch, as the APH is moved radially away from the center and the TMH is pulled up toward the cis-side in the active state (25, 26). For both native and foreign substrates, the Tat-targeted signal peptide and the pmf are sufficient to cause assembly of the active translocon and achieve transport (27–31). After the translocation event, the complex dissociates into TatA and TatBC components (1, 15, 16) with the exception of some residual TatA bound to the receptor complex in a nonactive state and a spectrum of smaller TatA oligomers (32).Within the thylakoid membrane, it is useful to compare the Tat complex with the general secretory translocon (Sec) because they both function in the same membrane environment (1, 33). Sec translocation first requires recruitment of the substrate to the soluble SecA ATPase to form the substrate–SecA complex, which is then recruited to the SecY pore (1). In the inactive state, the proteinaceous SecY pore prevents ion leakage through a combination of a trans-side plug domain and an internal array of hydrophobic residues (34). Following substrate–SecA docking, a conformational shift in SecY allows substrate movement through the open pore in an ATP-dependent process driven by SecA (35). In the mammalian homolog Sec61, leakage of NAD⁺ ions is recorded during ribosome-bound nascent chain transport in a fluorescence quenching study, suggesting the pore can reach 4 to 6 nm in diameter (36). However, X-ray structures of substrate-fused SecA complexed with SecY (35), conductance studies in ribosome-bound substrates engaged with SecY (37), and SecY plug deletion mutants (38) in Escherichia coli have estimated the open SecY pore diameter to range from 7.3 to 8.8 Å, almost 10-fold lower. This small diameter likely contributes to the restriction of ion movement during Sec transport (39).While the Sec machinery only transports unfolded substrates (40), the Tat pore accommodates substrates ranging from a single unfolded chain in an engineered system (13) to a folded substrate with an average diameter of 70 Å (41). This extended size range raises an interesting question about the pore architecture. In the Sec translocon, X-ray crystallography of the SecY channel in Methanocaldococcus jannaschii (42) and Thermotoga maritima (43) reveals that the SecY pore channel excludes lipids in both the resting state and when engaged with its SecA partner. Further structural work on the E. coli substrate-engaged SecA–SecY complex shows that the SecY channel excludes lipids during transport as well (35). No such structural information about the Tat pore exists, but functional data suggest that TatA plays an important role in the pore (20, 44, 45) and cryogenic electron microscopy structures of TatA oligomers reveal rings of an internal diameter ranging from 30 to 70 Å (23). During transport of the 17-kDa subunit of the oxygen-evolving complex (OE23), the Tat pathway exhibits very low ion leakage (46), estimated to be less than 1 pS. This is despite the exchange of 80,000 protons per substrate (47). Extensive mapping of subunit–subunit and subunit–substrate contacts has revealed no putative plug domain (20, 48–51) that could be compared to that in the SecY protein.Pore architecture can be characterized by membrane conductance behavior. Conductance measured through proteinaceous pores representing the barrel-stave model has a very steep dependence on the voltage applied, whereas conductance in toroidal pores requires a larger voltage to be detected and increases more slowly in response to increasing voltage (52–54). Performing similar experiments on the Tat and Sec translocons would require functional reconstitution of both complexes into an in vitro setting. However, decay of the electrochromic shift (ECS) signal can be used as a measure of ion conductance (46). A transient absorption peak at ∼515 nm arising from carotenoid pigments in response to the native electric field generated by charge separation in the photosynthetic reaction centers (55) can be measured by delivering a single-saturating flash. The decay rate of such a signal is a direct measurement of how quickly the electric field is dissipated by ion movement across the membrane.In the experiments reported herein, ECS signal decay rates revealing the conductance states of resting isolated membranes and those engaged in ongoing transport and in the presence of a constitutively assembled and/or substrate-engaged translocon are used to probe the pore architecture in the Tat and Sec complexes. Increased conductance across the thylakoid membrane is indicated by a more rapidly decaying ECS signal. We find that conductance in thylakoid membranes during Sec-mediated transport and substrate-engaged SecY is consistently higher than that during Tat-mediated transport and with the constitutively assembled Tat complex, respectively, despite the much larger Tat pore required to transport a fully folded substrate. This points to a difference not only in mechanism but in pore architecture between the two. Conductance behavior of membranes undergoing Sec-mediated transport is consistent with that of a proteinaceous pore, while the resistance demonstrated by membranes undergoing Tat-mediated transport is more reminiscent of toroidal pores.     

Sleep disordered breathing – a new component of syndrome x?

Sleep disordered breathing (SDB) is a complication of obesity estimated to occur in about 4–6% of overweight individuals. These respiratory disturbances during sleep incorporate a number of conditions including snoring, upper airway resistance syndrome and obstructive sleep apnoea syndrome (OSAS). It is thought that as well as having deleterious effects on sleep quality these conditions may also promote cardiovascular and hormonal changes leading to an elevated blood pressure and an increased incidence of cardiovascular morbidity. Evidence reviewed here points to an alteration in sympathovagal balance, baroreceptor sensitivity, insulin resistance and leptin, growth hormone and lipid levels. Whether these changes are a consequence of the associated obesity or the SDB itself remains to be proven.     

Role of bacterial toxins,bile acids,and free fatty acids in colonic water malabsorption in tropical sprue

Colonic perfusion studies in 10 southern Indian patients with tropical sprue and nine matched healthy adults revealed a defect of water and sodium absorption from the colon in sprue. Heat-labile and heat-stable enterotoxin production was not detected in coliforms cultured from the feces of any of the 19 subjects. The 24-hr fecal bile acid output was increased in patients with sprue, but fecal aqueous bile acid concentrations remained within normal limits, and these did not correlate with defects in colonic water and sodium absorption. Fecal free fatty acid excretion was markedly increased in sprue. There was a negative correlation between fecal excretion of unsaturated free fatty acids and colonic water and sodium absorption.     

Platelets modulate the proteolysis of factor VIII:C protein by plasmin

Factor VIII coagulant protein (VIII:C) functions as a critical cofactor with factor IXa, calcium ions, and phospholipid during the activation of factor X. In the course of this reaction, the activity of VIII:C is first increased and then is destroyed by one or more serine proteases that are part of the coagulation sequence. In this study, we have investigated the influence of platelets on the inactivation of VIII:C by plasmin. Platelets were separated from plasma proteins in the presence of granule release inhibitors and were incubated with plasmin and isolated VIII:C or the complex of purified VIII:C/von Willebrand factor (vWF); VIII:C activity and antigen levels were assessed over time. In the presence of platelets, the isolated VIII:C showed an initial increase in VIII:C activity that was not present when platelets were absent, and the VIII:C/vWF showed an increase in VIII:C activity over that seen when platelets were absent. In addition, platelets stabilized VIII:C activity over a one-hour time course when compared with buffer. The VIII:C antigen did not increase and decreased slowly whether platelets were present or absent. Preincubating the platelets with ristocetin, collagen, or plasmin did not alter the results, and experiments using platelets from a patient with severe von Willebrand's disease also showed a pattern similar to that seen with normal platelets. Experiments using fixed platelets or phospholipid vesicles showed that they did not support the activation reaction or delay the inactivation reaction. These studies demonstrate that platelets modulate the activation and inactivation of VIII:C by plasmin, apparently by a mechanism that is independent of the platelet release reaction.     

Aspirin withdrawal in patients treated with ticagrelor presenting with non‐ST elevation myocardial infarction

Essentials

• Strong P2Y₁₂ blockade may cause platelet inhibition that is only minimally enhanced by aspirin.
• We evaluated aspirin withdrawal on platelet reactivity in ticagrelor treated patients.
• Aspirin withdrawal resulted in increased platelet reactivity to arachidonic acid.
• Aspirin withdrawal caused little difference in adenosine diphosphate‐induced platelet aggregation.

Summary

Background

Recent studies have shown that the thromboxane A₂‐dependent pathway is dependent on the ADP–P2Y₁₂ pathway, and that strong P2Y₁₂ receptor blockade alone causes inhibition of platelet aggregation that is minimally enhanced by aspirin. Data from the PLATO trial suggested that, among ticagrelor‐treated patients, high‐dose versus low‐dose (< 100 mg day^?1) aspirin is associated with an increased risk fof ischemic events.

Objectives

To evaluate the impact of aspirin withdrawal on platelet reactivity in acute coronary syndrome (ACS) patients treated with a potent P2Y₁₂ blocker.

Patients/Methods

This was a current prospective, randomized, placebo‐controlled, double‐blind, cross‐over study. The study population comprised 22 consecutive ACS patients who underwent percutaneous coronary intervention and were treated with aspirin (100 mg day^?1) and ticagrelor. Thirty days post‐ACS, open‐label aspirin was stopped, and patients were randomized to either blinded aspirin or placebo for 2 weeks, with each patient crossing over to the other arm for an additional 2 weeks. Platelet reactivity to arachidonic acid and ADP determined with light‐transmission aggregometry (LTA) and VerifyNow was evaluated at baseline, and 2 weeks and 4 weeks later.

Results

Aspirin withdrawal resulted in an increase in arachidonic‐acid induced platelet reactivity as determined with both LTA (77.0% ± 11.3% versus 20.8% ± 4.4%) and VerifyNow (607.7 ± 10.6 aspirin reaction units [ARU] versus 408.5 ± 14.4 ARU). Platelet response to ADP, as determined with both LTA and VerifyNow, did not differ with either aspirin or placebo (32.9% ± 2.6% versus 35.8% ± 3.6%, and 33.5 ± 6.4 P2Y₁₂ reaction units (PRU) versus 29.6 ± 5.7 PRU, respectively).

Conclusions

Aspirin withdrawal early post‐ACS results in increased platelet reactivity in response to arachidonic acid, despite concomitant treatment with the potent P2Y₁₂ blocker ticagrelor.